osservatorio erpetologico italiano il ministero dell’ambiente e della tutela del territorio e del mare – direzione per la protezione della natura sponsorizza la stampa del presente numero di acta herpetologica nel quale sono pubblicati aggiornamenti di rilevante interesse per la conoscenza e conservazione dell’erpetofauna italiana. norme per la pubblicazione delle segnalazioni in questa sezione sono pubblicate segnalazioni erpetologiche riguardanti il territorio amministrativo italiano, non ancora incluse nella banca dati della shi e pubblicate nell’atlante nazionale (sindaco et al., 2006). sarà data priorità alle osservazioni riguardanti specie di interesse conservazionistico internazionale (specie incluse negli allegati ii e iv della direttiva europea 92/43/cee “habitat”), nazionale (specie endemiche, rare o al limite di areale) o regionale (nuove segnalazioni per la regione o che definiscono più nel dettaglio in modo significativo, l’areale della specie). saranno inoltre considerate per la pubblicazione segnalazioni di interesse ecologico (ad esempio limiti altitudinali). per quanto riguarda le testuggini del genere testudo, saranno pubblicate solo le segnalazioni di siti in cui è provata la riproduzione. osservazioni opportunamente documentate di popolazioni naturalizzate di specie esotiche (come trachemys sp.) saranno pubblicate. per motivi conservazionistici, le località precise degli avvistamenti non saranno pubblicate. la shi non incoraggia la raccolta di esemplari al solo fine della segnalazione e non pubblicherà segnalazioni di animali sacrificati senza le necessarie autorizzazioni. i segnalatori sono invitati a fotografare gli esemplari in vita, a consegnare quelli trovati morti ad istituzioni pubbliche locali e inviare al presidente della shi le foto e le citazioni dell’istituzione in cui il reperto è conservato. le segnalazioni, che saranno sempre citate col nome dell’osservatore, saranno vagliate dai redattori dell’atlante nazionale, che si avvarrà dei redattori di acta herpetologica e di esperti regionali. la segnalazione potrà eventualmente essere completata con un commento redazionale. indirizzo a cui inviare le segnalazioni (si caldeggia l’invio per posta elettronica) al presidente shi, sebastiano salvidio, dipteris corso europa 26, 16132 genova. e-mail: salvidio@dipteris.unige.it. acta herpetologica 2(1): 65-68, 2007 66 osservatorio erpetologico italiano simbologia utilizzata (possono essere utilizzati più simboli in sequenza) 000001 numero d’ordine della segnalazione +ita prima segnalazione in italia di una specie a distribuzione geografica limitrofa +reg prima segnalazione nella regione di una specie (prime tre lettere della regione) +h-ii nuova segnalazione/sito di specie dell’allegato ii della direttiva habitat +h-iv nuova segnalazione/sito di specie dell’allegato iv della direttiva habitat +h-ii/s riconferma di segnalazione/sito storico (non confermato da almeno 10 anni) di specie dell’allegato ii della direttiva habitat +h-iv/s riconferma di segnalazione/sito storico (non confermato da almeno 10 anni) di specie dell’allegato iv della direttiva habitat +end nuova segnalazione/sito di specie endemica italica in località che ne definiscono meglio l’areale +rar nuova segnalazione/sito di specie endemica rara o al limite di areale +end/s riconferma di segnalazione/sito storico (non confermato da oltre 10 anni) di specie endemica italica +rar/s riconferma di segnalazione/sito storico (non confermato da oltre 10 anni) di specie endemica rara o al limite di areale +tes sito accertato di riproduzione di testuggine del genere testudo +eco significativa segnalazione di interesse ecologico (esempio: limite altitudinale, nuovo ambiente, nuovi aspetti di nicchia trofica o riproduttiva, ecc.) +eso sito accertato di riproduzione di specie esotica osservatorio erpetologico italiano – 2 amphibia a0034 romano antonio +h-ii +end 110.357.0.002.0 salamandrina perspicillata. jenne (rm) aprile 2005. località lungo s.s. 411. a0035 romano antonio +h-ii +end 110.357.0.002.0 salamandrina perspicillata. roccagorda (lt) marzo 2005. località dintorni eremo di s. erasmo, 800 m. a0036 bogaerts sergé, pasmans frank +h-ii +end 110.357.0.002.0 salamandrina perspicillata. san severino lucano (pz) 9 aprile 2004. località dintorni san severino lucano. 67osservatorio erpetologico italiano a0037 romano antonio +h-ii 110.358.0.002.0 triturus carnifex. jenne (rm) aprile 2005. località lungo s.s. 411. a0038 spilinga cristiano, carletti silvia +h-ii 110.358.0.002.0 triturus carnifex. assisi (pg) 3 maggio 2006. località dintorni case il bosco vecchio, sic it5210023. a0039 romano antonio +end 110.358.0.003.0 lissotriton italicus. palazzi (rc) aprile 2004. località dintorni di palazzi. a0040 bodon emanuele, bodon marco +h-ii 110.359.0.006.0 speleomantes strinatii. pieve di teco (im) 30 settembre 2006. località dintorni della grotta du ruchin. a0041 bodon emanuele, bodon marco +end/s 110.366.0.004.0 hyla intermedia. sarzana (sp) 2 giungo 2006. località dintorni di bradiola, 20 m, sic it1343502. a0042 romano antonio +h-iv +end 110.367.0.004.0 rana italica. s. agata di puglia (fg) maggio 2005. località lungo il torrente frugna. a0043 spilinga cristiano, carletti silvia +h-iv +end 110.367.0.004.0 rana italica. assisi (pg) 3 maggio 2006. località dintorni di madonna dei tre fossi, sic it5210022. a0044 bogaerts sergé, pasmans frank +h-iv +end 110.367.0.004.0 rana italica. san severino lucano (pz) 9 aprile 2004. località dintorni san severino lucano. a0045 romano antonio +h-ii + eco 110.365.0.002.0 bufo viridis. vallepietra (rm) maggio 2004. località lungo il fosso fioio 1330 m, limite altitudinale regionale per la specie. 68 osservatorio erpetologico italiano reptilia r0009 fiacchini david, foglia gessica +h-iv 110.393.0.001.0 coronella austriaca. pinzolo (tn) 11 agosto 2006. località vellesinella, 1700 m. r0010 fiacchini david, foglia gessica +h-iv 110.393.0.001.0 coronella austriaca. civita (cs) settembre 2005. località colle s. martino, 1000 m. r0011 fiacchini david, foglia gessica +h-ii 110.369.0.001.0 emys orbicularis. frascineto (cs) settembre 2005. località bosco della fagosa, 1400 m. r0012 oneto fabrizio, ortale stefano, salvidio sebastiano +h-ii 110.379.0.001.0 euleptes europaea. sori (ge) 24 agosto 2006. località dintorni di sussisa 310 m. nuovo sito continentale per la specie. r0013 oneto fabrizio, ottonello dario, salvidio sebastiano +h-ii/s 110.379.0.001.0 euleptes europaea. portovenere (sp) 24 giugno 2006. località isola del tino sic it 1345103 r0014 ottonello dario +rar/s 110.388.0.001.0 timon lepidus. toirano (sv) 24 settembre 2005. località dintorni delle grotte di toirano sic it 1324011. r0015 girani alberto +end 110.391.0.001.0 chalcides chalcides. recco (ge) 1 maggio 2005. località dintorni di megli. r0016 spilinga cristiano, carletti silvia +h-iv 110.394.0.001.0 zamenis longissimus. montecchio (tr) 17 maggio 2006. località dintorni di melezzole, sic it5220008. r0017 spilinga cristiano, carletti silvia +h-ii 110.394.0.002.0 elaphe quatuorlineata. lungano in teverina (tr) 17 maggio 2006. località dintorni di lungano in teverina, sic it5220008. r0018 bogaerts sergé, pasmans frank +h-ii 110.394.0.002.0 elaphe quatuorlineata. rotonda (pz) 9 aprile 2004. località dintorni di rotonda. acta herpetologica 16(1): 59-60, 2021 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-10870 book review: mariella turrini – la tartaruga nella cultura dei popoli – miti, leggende e folklore sebastiano salvidio1,*, massimo delfino2,3 1 dipartimento di scienze della terra, dell’ambiente e della vita (distav), università degli studi di genova, i 16132 genova, italy 2 dipartimento di scienze della terra, università di torino, i 10125 torino, italy 3 institut català de paleontologia miquel crusafont, universitat autònoma de barcelona, cerdanyola del vallès, barcelona, spain *corresponding author. e-mail: sebastiano.salvidio@unige.it the title of this book, written in italian, may be translated as: “turtles in people’s culture – myths, legends and folklore”. the author, mariella turrini, is an italian literature teacher loving animals and travels and this book is clearly the outcome of the cross-over of these two beloved passions. there are nearly no books entirely dedicated to turtles, tortoises and terrapins in culture, art and myth, and therefore this volume seems to fill a real cultural gap. among the exceptions, worth mentioning is “tortoise” by peter young (2003), an interesting small book with a very different structure and with a much narrower scope. remarkable, but focused on a single topic, is also the booklet edited by del cimmuto (2013) that nicely illustrates a thematic collection of art objects and painting portraying turtles, tortoises and terrapins. conversely, mariella turrini’s book is a lengthy, comprehensive work composed by 337 pages of text, 14 pages of notes and 60 illustrations, many of which are original colour artworks and line drawings by the author herself. the long lists of acknowledgments and references testify for a genuine research effort and a solid bibliographic background. this volume has a well-defined structure. there is a short “presentation”, written by franco andreone, herpetologist from the regional museum of natural sciences of torino. then the “introduction” recalls that it was from the latin term testudo that the italian title word “testuggine” was derived. in the latin language, testudo was associated not only with the animal, but also to a stringed musical instrument, obtained from its shell (i.e., the lyra). moreover, the testudo described a roman military tight formation, in which rectangular shields were interlocked around and over soldiers underneath, to protect them completely. after this “introduction”, four short chapters “symbolism”, “etnomedicine”, “christianity” and “medieval age to the renaissance” are dedicated to the historical use, the popular tales, and the ancient legends associated to turtles, terrapins and tortoises, in the mediterranean area. it is noteworthy, that in some of these historical societies the turtles, and their symbolic representations, were often associated with a negative belief, in the sense that these animal were often considered belonging to the “tartarus” the infernal region created by the ancient greek mythology. however, in more recent cultures these animals assumed a much more positive symbolic meaning and, as stated by andreone in the “presentation”, are now the only reptiles generally appreciated by humans. indeed, in many different societies all over the world, these animals are considered harmless, calm, and wise creatures. moreover, they often became associated with personal longevity, character stability and in general with popular wisdom. finally, in many societies turtles and tortoises became also the symbolic representation of human fertility. the book “la tartaruga nella cultura dei popoli”, continues with a complete cultural tour around the world representing the core of the volume both in terms of number of pages and amount of information. indeed, the central and main chapters are dedicated to a well-docu60 s. salvidio, m.delfino mented and in most cases first-hand description of turtles in art, literature, poetry, myth and cultural traditions of “europe”, “africa”, “middle east”, “asia”, “oceania”, north america”, “central america pre-columbian societies” and, in the final chapter “southern america circumcaribbean and sub-andean societies”. it is clear that this book is the result of many years of passionate work and to the many travels all around the globe, completed by the author mirella turrini over the years. all persons attracted to the many humanistic and cultural aspects related to these strange and very peculiar reptiles could be interested in reading this book, although it is written in italian and therefore could have a limited distribution. an english translation, maybe omitting or shortening the purely geographical introductory notes to each country in order to avoid a certain dilution of the turtle topics, would be therefore very welcome. the book “la tartaruga nella cultura dei popoli – miti, leggende e folklore” is edited and available from edizioni belvedere (latina, italy) that with this new publication consolidates furthermore its position as a leading publisher of herpetological books. references del cimmuto, m. (ed.) (2013): tartarughe tra arte e scienza: la “collezione teresita olivares paglione”. quad. mus. univ. “g. d’annunzio” chieti, 2: 1-111. young, p. (2003): tortoise. reaktion books, london. acta herpetologica 4(2): 197-200, 2009 osservatorio erpetologico italiano in questa sezione sono pubblicate segnalazioni erpetologiche riguardanti il territorio amministrativo italiano, non ancora incluse nella banca dati della shi e pubblicate nell’atlante nazionale (sindaco et al., 2006). sarà data priorità alle osservazioni riguardanti specie di interesse conservazionistico internazionale (specie incluse negli allegati ii e iv della direttiva europea 92/43/cee “habitat”), nazionale (specie endemiche, rare o al limite di areale) o regionale (nuove segnalazioni per la regione o che definiscono più nel dettaglio in modo significativo, l’areale della specie). saranno inoltre considerate per la pubblicazione segnalazioni di interesse ecologico (ad esempio limiti altitudinali). per quanto riguarda le testuggini del genere testudo, saranno pubblicate solo le segnalazioni di siti in cui è provata la riproduzione. osservazioni opportunamente documentate di popolazioni naturalizzate di specie esotiche (come trachemys sp.) saranno pubblicate. per motivi conservazionistici, le località precise degli avvistamenti non saranno pubblicate. la shi non incoraggia la raccolta di esemplari al solo fine della segnalazione e non pubblicherà segnalazioni di animali sacrificati senza le necessarie autorizzazioni. i segnalatori sono invitati a fotografare gli esemplari in vita, a consegnare quelli trovati morti ad istituzioni pubbliche locali e inviare al presidente della shi le foto e le citazioni dell’istituzione in cui il reperto è conservato. le segnalazioni, che saranno sempre citate col nome dell’osservatore, saranno vagliate dai redattori dell’atlante nazionale, che si avvarrà dei redattori di acta herpetologica e di esperti regionali. la segnalazione potrà eventualmente essere completata con un commento redazionale. indirizzo a cui inviare le segnalazioni (si caldeggia l’invio per posta elettronica) al presidente shi, edoardo razzetti, università degli studi di pavia, p.zza botta 9, 27100 pavia. e-mail <razzetti@unipv.it> simbologia utilizzata (in ordine possono essere utilizzati più simboli) 000001 numero d’ordine della segnalazione +ita prima segnalazione in italia di una specie a distribuzione geografica limitrofa +reg prima segnalazione nella regione di una specie (prime tre lettere della regione) +h-ii nuova segnalazione/sito di specie dell’allegato ii della direttiva habitat +h-iv nuova segnalazione/sito di specie dell’allegato iv della direttiva habitat +h-ii/s riconferma di segnalazione/sito storico (non confermato da almeno 10 anni) di specie dell’allegato ii della direttiva habitat 198 osservatorio erpetologico italiano +h-iv/s riconferma di segnalazione/sito storico (non confermato da almeno 10 anni) di specie dell’allegato iv della direttiva habitat +end nuova segnalazione/sito di specie endemica italica in località che ne definiscono meglio l’areale +rar nuova segnalazione/sito di specie endemica rara o al limite di areale +end/s riconferma di segnalazione/sito storico (non confermato da oltre 10 anni) di specie endemica italica +rar/s riconferma di segnalazione/sito storico (non confermato da oltre 10 anni) di specie endemica rara o al limite di areale +tes sito accertato di riproduzione di testuggine del genere testudo +eco significativa segnalazione di interesse ecologico (esempio: limite altitudinale, nuovo ambiente, nuovi aspetti di nicchia trofica o riproduttiva ecc.) +eso sito accertato di riproduzione di specie esotica osservatorio erpetologico italiano – 7 amphibia a0069 david fiacchini +eco 110.356.0.003.0 salamandra salamandra. san ginesio (mc), 7 maggio 2007. rio del monte, 1180 m, ruscello in faggeta. parco nazionale dei monti sibillini. a0070 david fiacchini, gessica foglia, cristiano tarsetti +h-ii 110.358.0.002.0 triturus carnifex. fiastra (mc), 1 maggio 2008. località tribbio, 745 m, abbeveratoio. parco nazionale dei monti sibillini. a0071 david fiacchini +h-iv 110.359.0.005.0 speleomantes italicus. san ginesio (mc), 13 aprile 2007. rio del monte, 495 m, sottobosco vegetazione ripariale. parco nazionale dei monti sibillini. a0072 pierangelo crucitti, silvia agabiti rosei +h-ii 110.361.0.001.0 salamandrina perspicillata. monterotondo (rm). 27 settembre 2009. impluvio di fosso del barco, macchia del barco. a0073 luca maurino, silvia alberti, domenico rosselli, stefano doglio +eco 110.359.0.005.0 rana temporaria. pragelato (to). lago fauri, parco naturale regionale della val troncea, quota 2760 m slm. riproduzione accertata ogni anno dal 2005. 199osservatorio erpetologico italiano a0074 matteo elio siesa, roberto ferrari, stefano ravani, roberta salvi. +h-ii 110.367.0.005.0 rana latastei. lago del segrino (co), 17 marzo 2009. a0075 matteo elio siesa, roberta salvi +eco 110.359.0.005.0 rana temporaria. castel del lago, monguzzo (co), 25 marzo 2009. a0076 maurizio valota, mirco cappelli, cristina volontè +eco 110.365.0.001.0 bufo bufo. lentate sul seveso (mi), 19 marzo 2009. laghetto mirabello, parco naturale regionale delle groane. reptilia r0049 david fiacchini +h-ii +eco 110.399.0.004.0 vipera ursinii. acquasanta terme (ap), 3 giugno 2007. monti della laga, 1900 m, prateria primaria. parco nazionale gran sasso monti della laga, sic it5340007 “san gerbone”. r0050 david fiacchini +eco 110.397.0.001.0 natrix maura. castelnovo ne’ monti (re), 12 agosto 2007. località fiume secchia, 465 m. parco nazionale dell’appennino tosco emiliano, sic it4030009 “gessi triassici”. r0051 francesco paolo faraone +end 110.394.0.001.1 zamenis lineatus. terranova di pollino (pz), 12 ottobre 2004, nelle vicinanze del centro abitato, quota 970 m. r0052 gentile francesco ficetola +h-iv/s 110.397.0.003.0 natrix tessellata. bernate ticino (mi). località: lanca di bernate. r0053 carmelo batti +end 110.394.0.001.1 zamenis lineatus. s.alessio siculo (me), 6 maggio 2009. r0054 francesco paolo faraone +h-iv 110.397.0.003.0 natrix tessellata. matera, 16 ottobre 2004, presso l’invaso di san giuliano, quota 102 m. 200 osservatorio erpetologico italiano r0055 fabrizio oneto, dario ottonello +h-ii 110.379.0.001.0 euleptes europea. portovenere (sp), 1 luglio 2009. parco naturale regionale di portovenere. r0056 fabrizio oneto, dario ottonello +h-iv 110.393.0.001.0 coronella austriaca. pigna (im), 17 giugno 2009. parco naturale regionale delle alpi liguri, passo muratone. monitoraggio nazionale dell’erpetofauna alloctona le specie alloctone costituiscono una delle principali minacce per la fauna. nonostante in italia siano presenti numerose specie di erpetofauna alloctona, la loro distribuzione loro distribuzione non è ancora sufficientemente conosciuta, e non è stata finora posta sufficiente attenzione sull’importanza di segnalare tempestivamente novità sulla diffusione di queste specie. la commissione conservazione shi ha quindi deciso di iniziare un monitoraggio nazionale dell’erpetofauna alloctona, per avere un quadro aggiornato della situazione e anche per valutare eventuali cambiamenti nel tempo. chiediamo pertanto aiuto alla rete di erpetologi, per raccogliere segnalazione sulla distribuzione delle specie alloctone. i risultati di questo monitoraggio verranno pubblicati su acta herpetologica, all’interno dell’osservatorio erpetologico italiano, secondo le norme dell’osservatorio. le specie su cui riteniamo di focalizzare l’attenzione sono: rana catesbeiana, xenopus laevis, trachemys scripta e altre testuggini palustri naturalizzate. ovviamente, anche segnalazioni di altre specie di erpetofauna alloctona sono benvenute, ma vi preghiamo di tralasciare l’avvistamento di singoli esemplari. per le testuggini palustri alloctone, sarebbe estremamente interessante (ove possibile) mettere in evidenza le località in cui avviene la riproduzione. le segnalazioni vanno inviate a francesco ficetola, delegato per la commissione conservazione all’indirizzo e-mail: <francesco.ficetola@unimi.it> dati necessari per la segnalazione: • data dell’osservazione, specie, sito (breve descrizione), comune, provincia, località più prossima sull’atlante stradale. se possibile allegare anche: • fotografie, quota, coordinate gps (sistema di coordinate utilizzato), posizione su ctr o su immagine da satellite google earth qualunque nota e commento aggiuntivi sono i benvenuti. acta herpetologica 2006 2 standar karyotypes of two populations of the «scincus scincus» complex from tunisia and morocco (reptilia : scincidae) standard karyotypes of two populations of the scincus scincus complex from tunisia and morocco (reptilia: scincidae) mohsen kalboussi 1, gennaro aprea 2, andrea splendiani 3, massimo giovannotti 3, vincenzo caputo 3,4 1 département des sciences biologique, faculté des sciences de tunis, tunisia 2 dipartimento di biologia evolutiva e comparata, via mezzocannone 8, i-80134 napoli, italy 3 istituto di biologia e genetica, università politecnica delle marche, via brecce bianche, i-60131 ancona, italy; 4 corresponding author. e-mail: v.caputo@univpm.it abstract. a study on chromosomes of two scincus populations attributed to s. scincus and s. albifasciatus was carried out by conventional giemsa staining. both samples showed the same diploid number of 2n = 32 and identical chromosome morphology. therefore, these populations seem to be lacking in chromosome changes that could act as barriers to gene flow. however, because speciation in lizards is not always associated with chromosome repatterning, the taxonomic status of “scincus” and “albifasciatus” forms remains unsettled. keywords. karyotypes, scincus scincus, tunisia; morocco. the scincid lizard genus scincus laurenti, 1768 is a clearly-defined group of species adapted to life in areas of loose, usually wind-blown sand. this taxon is distributed from the western sahara desert and its borders, eastwards to arabia, southern iraq and southwestern iran. north african populations fall into two groups, which may represent largely allopatric species, namely s. scincus (linnaeus, 1758) in the east and s. albifasciatus boulenger, 1890 in the west. however, according to arnold and leviton (1977), evidence for specific status is not conclusive because resting on an unsatisfactory sample base; in addition, as sympatric populations of the two presumed species have not been found so far, it is not possible to evaluate whether gene flow is still acting or not. in the years following arnold and leviton’s review, the taxonomic status of the saharan scincus populations remained unsettled. faunal works and checklists by bons and geniez (1996), schleich et al. (1996), and geniez et al. (2000) variously regarded them as separated species or subspecies of s. scincus, but without further review of evidence. finally, caputo et al. (1994) compared karyotypes of s. scincus from egypt and of s. hemprichii wiegmann, 1837 from the arabian peninsula, calling for a full taxonomic re-evaluation of the group by use of non-morphologic characters like chromosomes and molecular markers. in an attempt to clarify the status of the s. scincus complex in north africa, we have acta herpetologica 1(2): 127-130, 2006 128 m. kalboussi et alii studied the chromosome complements of two populations attributed to the scincus and albifasciatus forms; we have also reviewed and compared the karyological data reported in literature for these palaearctic skinks. the karyotypes analysed come from two adult males and one adult female of scincus albifasciatus laterimaculatus werner, 1914 from south-eastern morocco (hamada du dra, environs of zagora) and from three females of s. scincus cucullatus werner, 1914 from southern tunisia (environs of douz). voucher specimens are deposited in the collection of v. caputo (faculty of sciences, ancona, italy). the animals were injected with colchicine (0.05 mg/ml; 0.01 ml/g body weight) 1 hour before dissection. metaphase plates were obtained from intestine, bone marrow and ovarian or testicular tissue. chromosome number and standard morphology were determined by conventional giemsa staining at ph 7.0. chromosome nomenclature was in accordance with levan et al. (1964). due to the scarcity of metaphases, no banding was possible and we were able to obtain only standard karyotypes. diploid chromosome complements and relative giemsa-stained metaphases are represented in fig. 1. the specimens analysed of the two presumptive species of scincus showed identical 2n = 32 standard karyotypes, in which the first two pairs of metacentric chromosomes were considerably larger than the remaining 14. the latter decreased gradually in size, and therefore it was not possible to distinguish between macroand micro-chromosomes. the chromosomes of the 3rd pair were also metacentric, whereas that of the remaining 13 pairs were subtelocentric. no heteromorphic chromosomes were distinguishable. the karyotype of s. scincus scincus (linnaeus, 1758) from egypt (caputo et al., 1994) is identical to those described in the present paper, whereas that of s. hemprichii fig. 1. karyotypes (right) and giemsa-stained metaphase plates (left) of scincus albifasciatus laterimaculatus from morocco (top) and s. scincus cucullatus from tunisia (bottom). scale bar = 10 μm 129standard karyotypes of two populations of the scincus scincus (branch, 1980) differs in having two additional pairs of small acrocentric chromosomes (2n = 36) (fig. 2). the karyological data presented here and the comparison with those reported in literature evidence a uniform karyotype structure, both in diploid number and in chromosome morphology, between populations attributed to subspecific (cucullatus and scincus, within s. scincus) or specific (s. scincus versus s. albifasciatus) taxa (e.g., schleich et al., 1996; geniez et al., 2000). so, these populations seem to be lacking in chromosome changes that could act as reproductive barriers. however, because speciation in lizards is not always associated with chromosome repatterning (see king, 1981), and considering that the 2n = 32 karyotype is plesiomorphic for skinks (caputo et al., 1994), the lack of karyotypic differentiation does not necessarily indicate that the “scincus” and “albifasciatus” forms belong to the same biological species. therefore, further analyses should be performed in order to clarify the taxonomic status of the scincus scincus complex in north africa. the karyotype of this latter closely resembles that of s. hemprichii (branch, 1980), differing for two less small acrocentric chromosomes. probably, the 2n = 36 karyotype of s. hemprichii represents an apomorphic condition derived by fissions from the complement with 2n = 32 chromosomes, considered the ancestral skink karyotype state (caputo et al., 1994). this hypothesis is also in line with that of arnold and leviton (1977), according to which s. scincus is the most primitive constituent of the genus. the speciation of s. hemprichii and the differentiation of its karyotype would have been promoted by the plio-pleistocene climatic fluctuations (suc, 1984, 1989). indeed, during the most mesic climatic periods, the pluvial phases in the saharan-arabian region would have caused the isolation into a separated arid refugium of a small skink population where the genetic drift favoured the fixation of a new chromosome set. the present limited geographic range of s. hemprichii, marginal to the larger distribution of s. scincus, seems to sustain this hypothesis. fig. 2. karyotype of scincus hemprichii (from branch, 1980). 130 m. kalboussi et alii references arnold, e.n., leviton, a.e. (1977): a revision of the lizard genus scincus (reptilia: scincidae). bull. br. mus. nat. hist. (zoology) 31: 187-248. bons, j., geniez p. (1996): amphibiens et reptiles du maroc (sahara occidental compris). atlas biogéographique. associación herpetológica española, barcelone. branch, w.r. (1980): chromosome morphology os some reptiles from oman and adjacent territories. j. oman stud. spec. rep. 2: 333-345. caputo, v., odierna, g., aprea, g. (1994). a chromosomal study of eumeces and scincus, primitive members of the scincidae (reptilia, squamata). boll. zool. 61: 155-162. geniez, p. mateo, j.a., bons j. (2000): a checklist of amphibians and reptiles of western sahara. herpetozoa 13: 149-163. king, m. (1981): chromosome change and speciation in lizards. in: evolution and speciation, p. 262-285. atchley, w.r., woodruff, d.s., eds. cambridge university press, cambridge mass. levan, a., fredga, k., sandberg, a.a. (1964): nomenclature for centromeric position on chromosome. hereditas 52: 201-220. schleich, h.h., kästle, w., kabish, k. (1996): amphibians and reptiles of north africa. biology, systematics, field guide. koeltz scientific books, koenigstein. suc, j. (1984): origin and evolution of the mediterranean vegetation and climate in europe. nature 307: 429-432. suc, j. (1989): distribution altitudinale et étagement des associations végétales au cénozoïque supérieur dans l’aire ouest-méditerranéenne. bull. soc. geol. france 5: 541-550. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 17(1): 3, 2022 doi: 10.36253/a_h-13069 editorial xi international symposium on the mediterranean lacertid lizards marco mangiacotti1, pietro lo cascio2, claudia corti2, marta biaggini2, miguel angel carretero2, petros lymberakis2 1 editor in chief, acta herpetologica 2 the organising committee of the xi international symposium on the mediterranean lacertid lizards it has been a long time since an editorial opened a new issue of acta herpetologica (it was 2012, volume 7, first issue). the occasion is to inform the readers that the journal of the societas herpetologica italica is pleased to host, from the present issue on (and, possibly, in the coming two), some contributions presented during the last “international symposium on the mediterranean lacertid lizards”, held (on line, sigh!) in lipari last september 2021. the international symposium on the mediterranean lacertid lizards was conceived during the 1st world congress of herpetology held in canterbury (uk) in 1989 to share scientific results, project ideas, conservation initiatives, recruit new enthusiastic students and, overall, enjoying what we are most passionate about studying lacertid lizards in the mediterranean and surrounding areas. the 1st symposium took place in mytilini, lesvos (greece) in 1992. the idea of informally joining scientists, students and conservationists across the mediterranean and beyond in a stimulating ambient was so successfully that it has become an appointment every, more or less, three years: faro (portugal), 1995; čres (croatia), 1998; maó, menorca (spain), 2001; lipari (italy), 2004; mytilíni, lesvos (greece), 2008; palma, mallorca (spain), 2010; koper, (slovenia) 2013; limassol (cyprus), 2016; tel aviv (israel), 2018. the last one has been hold online from the island of lipari (sicily, italy) in 2021 in an unusual form due to pandemic, but sharing the same spirit as always. considering that promoting the study of amphibians and reptiles and the growth of young herpetologists has always been also one of the shi goals, ah has welcomed the request by the organization committee to give the opportunity to publish some of the contributions of the xi symposium (according to the peer-review standard). you will find such contributions, along with the “normal” ones, marked as follows: “presented at the xi international symposium on the mediterranean lacertid lizards, lipari 27-28th september 2021”. enjoy the reading! xi international symposium on the mediterranean lacertid lizards marco mangiacotti1, pietro lo cascio2, claudia corti2, marta biaggini2, miguel angel carretero2, petros lymberakis2 the directional testes asymmetry increases with temperature in seven plateau brown frog (rana kukunoris) populations hai ying li1, man jun shang2, jie guo2, bo jun chen2, peng zhen chen2, tong lei yu1,* influence of tail injury on the development of neotropical elegant treefrog tadpoles ana glaucia da silva martins1,#, raoni rebouças2,3,*,#, isaias santos1, adão henrique rosa domingos1, luís felipe toledo2 the effect of weight and prey species on gut passage time in an endemic gecko quedenfeldtia moerens (chabanaud, 1916) from morocco jalal mouadi1,*, panayiotis pafilis2, abderrafea elbahi3, zahra okba3, hassan elouizgani3, el hassan el mouden4, mohamed aourir1 a contribution to the knowledge on the diet and food preferences of darevskia praticola (reptilia: lacertidae)§ emiliya vacheva*, borislav naumov first report on two loggerhead turtle (caretta caretta) nests in the aeolian archipelago (southern italy) monica francesca blasi1,*, sandra hochscheid2, roberta bardelli3, chiara bruno1, carolina melodia1, perla salzeri1, paolo de rosa4 and paolo madonia5 threatened and extinct amphibians and reptiles in italian natural history collections are useful conservation tools franco andreone1,*, ivano ansaloni2, enrico bellia3, andrea benocci4, carlotta betto5, gabriella bianchi6, giovanni boano7, antonio borzatti de loewenstern8, rino brancato9, nicola bressi10, stefano bulla11, massimo capula12, vincenzo caputo barucchi13, p re-description of external morphology and factors affecting body and tail shape of the stone frog tadpoles’ brena da silva gonçalves1,*, carla. d. hendges2, bruno madalozzo2, tiago g. santos2,3 preliminary data on the diet of chalcides chalcides (squamata: scincidae) from northern italy andrea ciracì1, edoardo razzetti2, maurizio pavesi3, daniele pellitteri-rosa4,* the high diversity and phylogenetic signal of antipredator mechanisms of the horned frog species of proceratophrys miranda-ribeiro, 1920 (amphibia: anura: odontophrynidae) cássio zocca1,2,*, ricardo lourenço-de-moraes3, felipe s. campos4, rodrigo b. ferreira1,2,5 acta herpetologica 4(1): 109-112, 2009 observations of two melanistic smooth snakes (coronella austriaca) from dorset, united kingdom. angelo p. pernetta1,2,3, christopher j. reading1 1 centre for ecology and hydrology, ceh wallingford, benson lane, crowmarsh gifford, wallingford, oxfordshire, ox10 8bb, u.k. 2 school of biological sciences, university of southampton, southampton, so16 7px, u.k. 3 address for correspondence: centre for ecology and hydrology, c/o freshwater biological association, east stoke, wareham, dorset, bh20 6bb, u.k. corresponding author. e-mail: a.p.pernetta@google mail.com abstract. we report the capture of two smooth snakes (coronella austriaca) with melanistic colouration from a site in dorset. these two individuals constitute the second published report of melanism in smooth snakes from the united kingdom. keywords. melanism, smooth snake, united kingdom. melanism is the phenomenon whereby a species has abnormally large amounts of black colouring at the expense of other colours (majerus, 1998). in reptiles this is typically a result of the over-production or dispersion of melanin by melanophores (sherbrooke and frost, 1989). melanism has been described as a common and highly variable phenomenon in snakes (lorioux et al., 2008), which is thought to occur as a result of a trade-off between increased thermoregulatory and reproductive advantages and reduced crypsis (clusella-trullas et al., 2007). melanistic colouration has been observed with varying levels of frequency in a number of european snake species including: coronella austriaca, hierophis gemonensis, h. viridiflavus, natrix natrix, n. maura, platyceps najadum, vipera aspis, v. berus and zamensis longissimus (boulenger, 1913; andrén and nilson, 1981; nilson and andrén, 1981; luiselli, 1995; cattaneo, 2003; zuffi, 2008). we encountered two melanistic smooth snakes during an ongoing mark-recapture study of a population in a commercial forestry area containing a mixture of both broadleaved woodland and coniferous plantations, interspersed with small patches of lowland heathland in dorset, united kindgom. both individuals were found on september 3rd 2008 under artificial refugia (sensu reading, 1997), placed on an area of land that had been recently clear-felled (2004). the vegetation at the site was relatively homogeneous and dominated by molinea caerulea and ulex europeas, with small calluna vulgaris plants growing through. large amounts of deadwood and tree stumps remained on the site. the habitat surrounding the artificial refugia under which the two melanistic specimens were captured appeared to show no obvious differences to that around the other 110 a.p. pernetta and c.j. reading 35 refugia placed at the site. the exact locality has not been disclosed due to the protected status of smooth snakes in britain and in order to prevent unnecessary disturbance to the individuals concerned. the first individual captured was an immature male (fig. 1; snout-vent length, svl = 278 mm, tail = 72 mm, mass = 15 g) that appeared to show partial melanism. the animals dorsal surface was completely black with the exception of a small number of scale rows immediately behind the parietal scales, which where dark brown (fig. 1a). the ventral and subcaudal scales were predominantly black with small amounts of red/orange colouration towards their edges. the second individual was an adult male (fig. 2; svl = 410 mm, tail = 123 mm, mass = 32 g) which was completely melanistic with the exception of a small amount of dark brown colouration towards the edges of it’s infralabial and chin shield scales. both animals were in a healthy condition and were released at the point of capture, following examination. this record constitutes only the second published account of melanism in c. austriaca from the united kingdom, the previous being two specimens collected from a locality near poole over 100 years ago (cambridge, 1894). the only published records of melanistic individuals from other parts of c. austriaca’s distribution appear limited to spain and portugal (boulenger, 1894; hopkins, 1976, meijide et al., 1994; barbadillo et al., 1997). whilst it is unknown whether the cases of melanism reported here have any functional significance, the infrequency with which melanistic specimens of c. austriaca have been recorded suggests it is an uncommon phenomenon. fig. 1. dorsal (a) and ventral (b) views of an immature male coronella austriaca displaying melanistic colouration. 111melanistic smooth snakes acknowledgements this research was carried out under licence from natural england (licence number: 20070553) and financial support was provided by a natural environment research council studentship to a.p.p (ner/s/a/2005/13642).we are grateful to the forestry commission for providing access to the study site. we are grateful to marco a.l. zuffi and luca luiselli for comments on a previous version of the manuscript. references andrén, c., nilson, g. (1981): reproductive success and risk of predation in normal and melanistic color morphs of the adder, vipera berus. biol. j. linn. soc. 15: 235–246. fig. 2. dorsal (a) and ventral (b) views of an adult male coronella austriaca displaying melanistic colouration. 112 a.p. pernetta and c.j. reading barbadillo, l.j., valdermorog, d.g., sánchez-herráiz, m.j. (1997): coronella austriaca melánica depredando sobre lacerta monticola cantabrica en el norte de la península ibérica (burbia, león). bol. asoc. herpetol. esp. 8: 31–33. boulenger, g.a. (1894): on the variations of the smooth snake coronella austriaca. the zoologist 18: 10–15. boulenger, g.a. (1913): the snakes of europe. methusen and co. ltd., london. cambridge, p.o. (1894): reptiles of dorset. proc. dorset. nat. hist. f. cl. 15: 90–102. cattaneo, a. (2003): herpetological notes on the aegean islands of lesvos, chios and samos. boll. mus. civ. stor. nat. venezia. 54: 95–116. clusella-trullas, s., van wyk, j.h., spotila, j.r. (2007): thermal melanism in ectotherms. j. therm. biol. 32: 235–245. hopkins, p.w. (1976): a melanistic spanish smooth snake (coronella a. austriaca). doñana acta vertebr. 3: 93–96. lorioux, s., bonnet, x., brischoux, f., de crignis, m. (2008): is melanism adaptive in sea kraits? amphibia-reptilia 29: 1–5. luiselli, l. (1995): body size, sexual size dimorphism and reproduction in different colour morphs in a population of western whip snakes, coluber viridiflavus. rev. ecol. (terre vie) 50: 365–376. majerus, m.e.n. (1998): melanism: evolution in action. oxford university press, oxford. meijide, m., pérez melero, j.m. (1994): neuvos casos de melanismo en coronella austriaca y natrix natrix (ophidia, colubridae) en el norte de iberia. bol. asoc. herpetol. esp. 5: 33–36. nilson, g., andrén, c. (1981): morphology and taxonomic status of the grass snake, natrix natrix (l) (reptilia, squamata, colubridae) on the island of gotland, sweden. zool. j. linn. soc. 72: 355–368. reading, c.j. (1997): a proposed standard method for surveying reptiles on dry lowland heath. j. appl. ecol. 34: 1057–1069. sherbrooke, w.c., frost, s.k., 1989. integumental chromatophores of a color-change, thermoregulating lizard, phrynosoma modestum (iguanidae; reptilia). am. mus. novit. 2943: 1–14. zuffi, m.a.l. (2008): colour pattern variation in populations of the european whip snake, hierophis viridiflavus: does geography explain everything? amphibia-reptilia 29: 229–233. osservatorio erpetologico italiano in questa sezione sono pubblicate segnalazioni erpetologiche riguardanti il territorio amministrativo italiano, non ancora incluse nella banca dati della shi e pubblicate nell’atlante nazionale (sindaco et al., 2006). sarà data priorità alle osservazioni riguardanti specie di interesse conservazionistico internazionale (specie incluse negli allegati ii e iv della direttiva europea 92/43/cee “habitat”), nazionale (specie endemiche, rare o al limite di areale) o regionale (nuove segnalazioni per la regione o che definiscono più nel dettaglio in modo significativo, l’areale della specie). saranno inoltre considerate per la pubblicazione segnalazioni di interesse ecologico (ad esempio limiti altitudinali). per quanto riguarda le testuggini del genere testudo, saranno pubblicate solo le segnalazioni di siti in cui è provata la riproduzione. osservazioni opportunamente documentate di popolazioni naturalizzate di specie esotiche (come trachemys sp.) saranno pubblicate. per motivi conservazionistici, le località precise degli avvistamenti non saranno pubblicate. la shi non incoraggia la raccolta di esemplari al solo fine della segnalazione e non pubblicherà segnalazioni di animali sacrificati senza le necessarie autorizzazioni. i segnalatori sono invitati a fotografare gli esemplari in vita, a consegnare quelli trovati morti ad istituzioni pubbliche locali e inviare al presidente della shi le foto e le citazioni dell’istituzione in cui il reperto è conservato. le segnalazioni, che saranno sempre citate col nome dell’osservatore, saranno vagliate dai redattori dell’atlante nazionale, che si avvarrà dei redattori di acta herpetologica e di esperti regionali. la segnalazione potrà eventualmente essere completata con un commento redazionale. indirizzo a cui inviare le segnalazioni (si caldeggia l’invio per posta elettronica) al presidente shi, sebastiano salvidio, dipteris corso europa 26, 16132 genova. e-mail: salvidio@dipteris.unige.it. simbologia utilizzata (in ordine possono essere utilizzati più simboli) 000001 numero d’ordine della segnalazione +ita prima segnalazione in italia di una specie a distribuzione geografica limitrofa +reg prima segnalazione nella regione di una specie (prime tre lettere della regione) +h-ii nuova segnalazione/sito di specie dell’allegato ii della direttiva habitat +h-iv nuova segnalazione/sito di specie dell’allegato iv della direttiva habitat +h-ii/s riconferma di segnalazione/sito storico (non confermato da almeno 10 anni) di specie dell’allegato ii della direttiva habitat acta herpetologica 3(2): 185-189, 2008 issn 1827-9643 (online) © 2008 firenze university press 186 osservatorio erpetologico italiano +h-iv/s riconferma di segnalazione/sito storico (non confermato da almeno 10 anni) di specie dell’allegato iv della direttiva habitat +end nuova segnalazione/sito di specie endemica italica in località che ne definiscono meglio l’areale +rar nuova segnalazione/sito di specie endemica rara o al limite di areale +end/s riconferma di segnalazione/sito storico (non confermato da oltre 10 anni) di specie endemica italica +rar/s riconferma di segnalazione/sito storico (non confermato da oltre 10 anni) di specie endemica rara o al limite di areale +tes sito accertato di riproduzione di testuggine del genere testudo +eco significativa segnalazione di interesse ecologico (esempio: limite altitudinale, nuovo ambiente, nuovi aspetti di nicchia trofica o riproduttiva ecc) +eso sito accertato di riproduzione di specie esotica osservatorio erpetologico italiano – 5 amphibia a0059 razzetti edoardo +rar 110.358.0.002.0 triturus carnifex. francavilla in sinni (pz), 13 agosto 2008. località contrada bruscata. a0060 razzetti edoardo + end 110.367.0.004.0 rana italica. san severino lucano (pz), 15 agosto 2008. località mancini, affluente di destra del torrente caramola. a0061 razzetti edoardo +h-ii 110.357.0.001.0 salamandrina terdigitata. san severino lucano (pz), 15 agosto 2008. località mancini, affluente di destra del torrente caramola. a0062 oneto fabrizio +h-ii/s 110.361.0.001.0 salamandrina perspicillata. isoverde (ge), 31 maggio 2008. località gallaneto. a0063 pasmans frank, martel an +rar 110.356.0.003.0 salamandra salamandra. santo stefano in aspromonte (rc), 27 dicembre 2007. località rumia. a0064 pasmans frank, martel an +rar 110.357.0.001.0 salamandrina terdigitata. delianuova (rc), dicembre 2007. 187osservatorio erpetologico italiano a0065 pasmans frank, martel an +rar 110.367.0.004.0 rana italica. delianuova (rc), dicembre 2007. a0066 pasmans frank , crauwels erwin, lafon nathalie, pasmans françois +rar 110.355.0.001.0 euproctus platycephalus. san nicolo gerrei (ca), 12 aprile 2008. a0067 fiorenza tiziano, romano stefano +rar 110.362.0.002.0 discoglossus sardus. porto torres (ss), aprile 2004. località cuile novo. a0068 fiorenza tiziano, romano stefano +rar 110.366.0.003.0 hyla sarda. calangianus (ss), aprile 2004. località luras. reptilia r0036 razzetti edoardo +rar 110.393.0.002.0 coronella girondica (msnpv cr858). ottone (pc), 1 giugno 2008. presso traschio, lungo la statale 45, km 70. r0037 corpo forestale dello stato in servizio nel parco nazionale delle cinque terre +h-iv +rar 110.393.0.001.0 coronella austriaca. vernazza (sp), 26 agosto 2008. strada provinciale 51 bivio per località “piculla”. r0038 razzetti edoardo +h-iv +rar 110.386.0.003.0 lacerta bilineata. san costantino albanese (pz), 13 agosto 2008. località timpa di pietrasasso, quota 1300 m. r0039 razzetti edoardo +h-iv +rar 110.392.1.002.0 hierophis viridiflavus. terranova di pollino (pz), 16 agosto 2008. presso il rifugio catusa, quota 1300 m. r0040 razzetti edoardo +rar 110.386.0.003.0 lacerta bilineata. terranova di pollino (pz), 16 agosto 2008. presso il rifugio catusa, quota 1300 m. r0041 razzetti edoardo +h-iv +rar 110.394.0.001.0 zamenis longissimus. ottone (pc), 1 giugno 2008. località frassi. 188 osservatorio erpetologico italiano r0042 oneto fabrizio, ottonello dario +h-ii +rar 110.379.0.001.0 euleptes europaea. ameglia (sp), 10 giugno 2008. promontorio di montemarcello. r0043 fiacchini david, foglia gessica +h-iv +rar 110.393.0.001.0 coronella austriaca. madonna di campiglio (tn), 11 agosto 2006. parco naturale adamello brenta. val brenta, vallesinella, altezza malga di sopra, quota 1700 m. r0044 fiacchini david, foglia gessica +rar 110.390.0.001.0 zootoca vivipara. 12 agosto 2006. parco naturale adamello brenta. val brenta, carrarecciasentiero n. 323, quota 1200 m. r0045 fiorenza tiziano +rar 110.385.0.001.0 archeolacerta bedriagae. lula (nu), maggio 1997. cima punta catirina, comprensorio del monte albo, quota 990 m. r0046 fiorenza tiziano, romano stefano +h-iv +rar 110.384.0.001.0 algyroides fitzingeri. tempio pausania (ss), aprile 2004. località madonna della neve, quota 1300 m. r0047 fiacchini david, foglia gessica +rar 110.378.0.001.0 hemidactylus turcicus. diamante (cs), 2005. località cirella di diamante. r0048 fiacchini david, foglia gessica +rar 110.391.0.001.0 chalcides chalcides. civita (cs), 2005. località colle della scala, quota 900 m. 189osservatorio erpetologico italiano monitoraggio nazionale dell’erpetofauna alloctona le specie alloctone costituiscono una delle principali minacce per la fauna. nonostante in italia siano presenti numerose specie di erpetofauna alloctona, la loro distribuzione loro distribuzione non è ancora sufficientemente conosciuta, e non è stata finora posta sufficiente attenzione sull’importanza di segnalare tempestivamente novità sulla diffusione di queste specie. la commissione conservazione shi ha quindi deciso di iniziare un monitoraggio nazionale dell’erpetofauna alloctona, per avere un quadro aggiornato della situazione e anche per valutare eventuali cambiamenti nel tempo. chiediamo pertanto aiuto alla rete di erpetologi, per raccogliere segnalazione sulla distribuzione delle specie alloctone. i risultati di questo monitoraggio verranno pubblicati su acta herpetologica, all’interno dell’osservatorio erpetologico italiano, secondo le norme dell’osservatorio. le specie su cui riteniamo di focalizzare l’attenzione sono: rana catesbeiana, xenopus laevis, trachemys scripta e altre testuggini palustri naturalizzate. ovviamente, anche segnalazioni di altre specie di erpetofauna alloctona sono benvenute, ma vi preghiamo di tralasciare l’avvistamento di singoli esemplari. per le testuggini palustri alloctone, sarebbe estremamente interessante (ove possibile) mettere in evidenza le località in cui avviene la riproduzione. le segnalazioni vanno inviate a francesco ficetola, delegato per la commissione conservazione all’indirizzo e-mail: francesco.ficetola@unimi.it. dati necessari per la segnalazione: • data dell’osservazione, specie, sito (breve descrizione), comune, provincia, località più prossima sull’atlante stradale. se possibile allegare anche: • fotografie, quota, coordinate gps (sistema di coordinate utilizzato), posizione su ctr o su immagine da satellite google earth qualunque nota e commento aggiuntivi sono i benvenuti. acta herpetologica 2006 1 rehabilitation of habitat connectivity between two important marsh areas divided by a major road with heavy traffic rehabilitation of habitat connectivity between two important marsh areas divided by a major road with heavy traffic. carlo scoccianti wwf, tuscany, lungarno colombo 44, 50136 florence. e-mail: carlo.scoccianti@inwind.it ‘vignarca’ is a major road between piombino and follonica along the coast in southern tuscany, in the province of livorno. until the 1940s, the area crossed by vignarca was a large marsh, which in turn was the remains of an ancient costal lake (lake piombino). over the years the area was reclaimed, and now only two wetlands remain: one is a typical brackish water marsh (‘orti’) and the other a freshwater marsh (‘bottagone’) with large areas of reedbeds. these marshes are bisected by the vignarca road. since 1991, the marshes have been protected by the wwf by the establishment of the orti-bottagone nature reserve. in 1994, the author began to analyze the impact of the vignarca road traffic on the species that attempted to cross. between 1995 and 1996 this stretch of road was included in a group of roads of tuscany (a total of 55 km) where once a week for one year a census of the remains of vertebrates was taken (scoccianti et al., 2001). in subsequent years many other observations were made in different seasons (data not published). all data collected confirmed the high risk of this road for many species. the impact was particularly high for amphibians: triturus carnifex; triturus vulgaris; bufo bufo; bufo viridis; hyla intermedia and rana synklepton hispanica; and for reptiles: emys orbicularis, lacerta bilineata, podarcis muralis, podarcis sicula, hierophis viridiflavus and natrix natrix. observing where most of the remains of the reptiles were found (generally very close to the edges and much less frequent towards the middle of the road) as well as individuals in activity, clearly indicated that the road edges were preferred areas for thermoregulation activity. in some periods, in particular weather conditions, thousands of individuals were killed. for instance at the end of july 1997, after a night rainstorm, we counted over 6,500 remains of newlyemerged rana synklepton hispanica juveniles on a mere 100 metre stretch of road. similar mass killings of bufo viridis were also noted in subsequent years. even many species of mammals (in particular micromammals) and birds had been found hit by vehicles. in 1998 the administration of the province of livorno began to take an interest in this old road because subsidence caused by heavy truck traffic serving the many steel mills along the coast was increasing risk of its collapse. as a result of a further agreement between wwf and the province of livorno, and within the large-scale project of the overall road reconstruction, the author was given the task of designing an impact mitigation project for the stretch of road which crosses the reserve. acta herpetologica 1: 77-79, 2006 78 c. scoccianti two possible solutions emerged: either to divert the road northwards and build a new road far from the reserve, or build a new road in the same place with new features in several points to offer safe passage to the species (the ‘tunnel’ hypothesis). the first idea was impracticable from the outset because it would have required even more money to expropriate new land and much more time for construction as against the urgency caused by the collapse of the old road. finally, another important fact emerged from the study of the area. besides impeding movements of individual animals between the two marshes, vignarca also acted as an ecological barrier, which had the curious effect of preserving the typical ecological features of both marsh areas. in point of fact, this complex of wetlands is extraordinary varied ecologically because there is both a brackish habitat and a freshwater one (with broad areas of reedbed). many years ago a major power station was built close to the coast which completely isolated the so-called bottagone section of the marsh from the sea. the freshwater springs made it into a wetland with large areas of reedbed (phragmites australis). on the other hand, the so-called orti marsh, despite being further from the sea is linked to it by a canal (the ‘cosimo canal’) and has thus developed features typical of a brackish wetland. the vignarca road is, effectively, the barrier that prevents the mingling of waters of the two marshes. if the two areas were connected, it would lead to salification of the bottagone water as well, with consequent loss of biodiversity, especially of reedbed biocenosis. the results of analysing the various aspects of the problem led to a project for enabling faunal species to cross the road safely while maintaining the two different marsh habitats. to solve this problem, the author, together with the engineering department of the province of livorno, conceived a particularly innovative solution for the entire stretch of road crossing the reserve bisecting the two wetlands. instead of digging several tunnels, as had been done in other mitigation projects (scoccianti, 2001), the road was raised by building a viaduct 215 metres long with nine spans, each of which was some 24 metres long. the piers of the viaduct rise from a bank the height of which was calculated on the basis of flood statistics of the plain over the previous ten years in order to prevent any possibility of the waters mingling even during a flood. this bank was thus determined as 1 mt higher that the average water level of the marshes enabling the preservation of the different original ecological conditions of the freshwater bottagone and the brackish orti (fig. 1). then, the open space needed for the passage of the faunal species was calculated between the top of the bank and the under level of the spans of the viaduct as 1.6 metres. these large spaces provide year-round microclimatic conditions very similar to the outside, which makes it easier for species to cross. moreover the broad field of vision beyond the spans was expected to make it easy for the species to pass under the viaduct. the construction of the viaduct took place in 2003. the work was subsequently completed with 300 metres of drift fence along both sides of the road starting from both ends of the viaduct. the purpose of these drift fences was to prevent amphibians, reptiles and micromammals crossing the road before or after the viaduct and to channel them below it. monitoring the new stretch of road revealed no remains of species of amphibians and reptiles hit by vehicles. at the same time it was observed that many species were using the open space under the spans to go from one marsh to the other and also to find microhabitat for hibernation and aestivation. furthermore, the effect on birds improved significantly. in fact some species can easily pass under the viaduct either by walking (e.g. gallinula chloropus) or flying (e.g. alcedo atthis); at the same time the new road, which is higher 79rehabilitation of habitat connectivity than the old one, gives the other species which fly over the reedbed a better chance of seeing moving obstacles (cars or trucks) in advance and consequently act to avoid collision. furthermore, the 2 metre-high guardrail obliges birds to raise their flight path when they are flying across the road. particular attention was focused on maintaining the reserve biodiversity during the construction works; the project demanded that all the work (demolition of the old road and construction of the new one) be made in the bed of the old road to prevent any even temporary occupation of any part of marsh habitat along the road. references scoccianti, c. (2001): amphibia: aspetti di ecologia della conservazione [amphibia: aspects of conservation ecology]. wwf italia, sezione toscana. editore guido persichino grafica, firenze: xiii+430 pp. scoccianti, c., cigna, p., dondini, g., vergari, s. (2001): studio dell’impatto delle infrastrutture viarie sulla fauna: gli investimenti di vertebrati durante un anno di campionamento di 5 strade in toscana. in: ferri v. (ed.), atti 2° convegno nazionale ‘salvaguardia anfibi’, 15-16 maggio 1997, morbegno (sondrio), italia, rivista di idrobiologia 40 (1): 173-186. fig. 1: the viaduct under construction (2002), seen from the ‘orti’ brackish marsh. piombino, province of livorno, tuscany, italy. diet of the asp viper vipera aspis in woodland habitats of the po plain (nw italy) luca canova, augusto gentilli dipartimento di biologia animale, università di pavia, piazza botta 9, i-27100 pavia. corresponding author. e-mail: canova@unipv.it submitted on 2007, 5th november; revised on 2008, 28th january; accepted on 2008, 31st january. abstract. the diet of asp viper vipera aspis was studied in forested habitat of northern italy the species feeds on a low number of preys, possibly reflecting the reduction of small mammal richness in woodland habitats. keywords. vipera aspis, diet, prey list, northern italy. the diet of the asp viper vipera aspis has been extensively studied all over its distribution range (duguy, 1972; monney, 1990, 1993, 1995). in italy several studies focused on feeding ecology of populations inhabiting mountain and mediterranean habitat (capula and luiselli, 1990; luiselli and agrimi, 1991; capizzi and luiselli, 1996; saviozzi and zuffi, 1997) whereas data from temperate plain areas are scanty or anedoctal (morisi and molinaro, 1980; bruno, 1985). this paucity of information is ultimately due to the 1) strong fragmentation of woodland habitat, 2) a very low population density and 3) a scarce success in ingesta collection (usually less than 30% of captured individuals regurgitate or defecate (gentilli, unpubl. and present study). aim of this note is thereby to improve current knowledge of diet and feeding strategies of asp viper in residual plain populations, as well as to compare diet spectrum and prey availability. our study was carried out from april to september 2006 in two residual woodland areas of po plain (north-western italy). the first area was the bosco fontana nature reserve (45° 12’ 11’’n; 10° 44’ 41’’e), an ancient and mature woodland of 230 ha (hereafter bf). the protected area is uniformly covered by quercus robur and carpinus betulus and a wide prairie is located in the central part of the woodland. the second study area was the baraggia di piano rosa (45° 38’ 47’’n; 8° 28’ 23’’e), a wide protected area characterized by woodlot, hedgerows and cultivated field growing on a acid and dilavated soils (hereafter b). arboreal vegetation is dominated by robinia pseudoacacia, betula pendula and quercus robur; grassy vegetation is mainly constituted by calluna vulgaris and molinia arundinacea. acta herpetologica 3(2): 175-178, 2008 issn 1827-9643 (online) © 2008 firenze university press 176 l. canova and a. gentilli we captured 82 asp viper and 24 meal samples were collected by 24 individuals (13 male, 11 female). vipers were captured by hand along the ecotone woodland-prairie; after capture, vipers were measured, sexed and stored inside cotton bag. prey samples were obtained by forced regurgitation (ingesta) or collected after few hours of captivity (ingesta and faeces). all samples were conserved in ethanole until examination. preys were determined by microscopical analyses of ingesta and faeces contents. methods proposed by debrot et al. (1982), demarinis and agnelli (1993) were adopted for this study; a references collection of mammals hairs was prepared and teerink (1991) was adopted as the identification guide. food availability was estimated on two soil level; firstly we checked for preys availability above ground by disposal of 52 live traps on a standard transect 450 m long (bf) and 36 live traps on a transect 280 m long (b). traps were baited with sunflower seeds and checked at sunset and dawn; trapped individuals were sexed, weighted, marked by toe-clipping and released. we tried to estimate prey availability underground by disposing traps under litter, but this method provided a very poor trap success; data from canova (1992) and nadali (2001), collected in the same habitat or similar wooded habitat, were then adopted to compare diet with underground food availability. on 24 samples only three mammals and one reptilian species were indeed recognized. diet from the two study areas are significantly different (χ2 fisher = 10.4, df = 3, p = 0.004), with microtus savii being the principal prey in bosco fontana and crocidura suaveolens in the baraggia area; rattus sp. and anguis fragilis were occasional preys in our samples (table 1). diet among sexes are not significantly different (χ2 fisher = 4.5, df = 3, p = 0.163) while diet item frequency is significantly different from availability (χ2 = 29.7, d.f. = 5, p < 0.001) and availability differed among sites (χ2 fisher = 8.7, df = 3, p < 0.01). comparison between frequency of mammal preys and available ones showed that apodems agrarius and apodemus sylvaticus are not represented in asp viper diet; only c. suaveolens is preyed in proportion to its availability while m. savii proportion in diet largely overrun its availability (fig. 1). results suggested a diet pattern remarkably restricted, confirming the data reported by capizzi and luiselli (1996) woodland habitats of central italy; on the other hand these data contrast with capula and luiselli 1990 (14 preyed species), luiselli and agrimi (1991) (20 preyed species) and saviozzi and zuffi (1997) (10 preyed species) where diet was considerably wider. the observed differences can reflect the reduction in small mammal richness in mature woodland habitat, as described by gurnell (1985), as well as the effect of a higher table 1. diet of asp viper in the two study areas. bosco fontana baraggia total n % n % n microtus savii 12 75 1 12.5 13 crocidura suaveolens 3 18.75 6 75 9 anguis fragilis 0 0 1 12.5 1 rattus sp. 1 6.25 0 0 1 177diet of vipera aspis habitat heterogeneity in the other study areas. out of general conclusion two additional informations appeared to be of some interest from our study: 1) asp viper diet do not include both the diurnal a. agrarius than the nocturnal a. sylvaticus, two rodents very abundant in our study areas; 2) asp viper feeds on c. suaveolens and largely preferred m. savii, two smaller and less abundant species in forest habitat (canova and fasola, 1991; canova, 1992). more detailed study are clearly needed in order to separate effect of habitat on prey spectrum width and to investigate possible feeding adaptation in local, fragmented populations. acknowledgements thanks are due to lara rondi for her help in sample analyses and to dr. franco mason and carlo bider, directors of bosco fontana and baraggia di piano rosa nature reserve respectively for permission and facilities. this study was carried out with personal fund. references bruno, s. (1985): le vipere d’italia e d’europa, edagricole, bologna. canova, l., fasola, m. (1991): communities of small mammals in six biotopes of northern italy. acta ther. 36: 73-86. canova, l. (1992): distribution and habitat preference of small mammals in a biotope of the north italian plain. boll. zool. 59: 417-421. fig. 1. frequency of mammal preys in diet compared with availability. legend: °° data from present study, ° canova (1992). differences were tested on original frequency by chi-square test and bonferroni confidence intervals: ns=not significant, * p < 0.01, ** p < 0.001. 178 l. canova and a. gentilli capula, m., luiselli, l. (1990): analysis of the gut contents of vipera aspis from an area of central italya (tolfa mountains, latium): a new method to study the terrestrial small mammals. hystrix 2: 101-107. capizzi, d., luiselli, l. (1996): feeding relationships and competitive interactions between phylogenetically unrelated predators (owls and snakes). acta oecol. 17: 265-284. debrot, s., mermod, c., fivaz, g., weber, j.m. (1982). atlas des poils de mammiferes d’europe. ed. inst. zool. univ. neuchatel. demarinis, a.m., agnelli, p. (1993): guide to the microscope analysis of italian mammals hairs: insectivora, rodentia and lagomorpha. boll. zool. 60: 225-232. duguy, r. (1972): notes sur la biologie de vipera aspis dans les pyrénées. rev. ecol. 26: 98-117. gurnell, j. (1985): woodland rodent communities. symp. zool. soc. lond.. 55: 377–411. luiselli, l., agrimi, u. (1991): composition and variation of the diet of vipera aspis francisciredi in relation to age and reproductive age. amphibia-reptilia 12: 137-144. monney, j.c. (1990): régime alimentaire de vipera aspis (ophidia, viperidae) dans le préalpes fribourgeoises (ouest de la suisse). bull. soc. herp. fr. 53: 40-49. monney, j.c. (1993): predation of lizards and frogs by adults vipers, vipera aspis, in the bernese prealpine region (west switzerland). amphibia-reptilia 14: 93-95. monney, j.c. (1995): comparaison du régime alimentair de vipera aspis dans l’oberland bernoise. bull. soc. frib. sc. nat. 84: 105-141. morisi, a., molinaro, e. (1980): su un nuovo caso di viperofagia da parte del biacco, coluber viridiflavus lacépède. riv. piem. st. nat. 1: 201-205. nadali, a. (2001): mammiferi. in: vertebrati di un bosco planiziario padano: bosco della fontana, p. 75-85. longo, l., nadali, a., eds, arcari ed., mantova. prigioni, c., cantini, m., zilio, a. (2001): atlante dei mammiferi della lombardia. regione lombardia e università degli studi di pavia. saviozzi, p., zuffi, m. (1997): an integrated approach to the study of the diet of vipera aspis. herpetol. rev. 28: 23-24. teerink, b.j. (1991): hair of the west european mammals: atlas and identification. 1 western europe, oxford university press, cambridge. discovery of salamandra atra aurorae (trevisan, 1982) on the altopiano di vezzena, trentino (northeastern italy) wouter beukema1, peter brakels2 1 apollo 10, 5591pp heeze, the netherlands. corresponding author. e-mail: wouter.beukema@wur.nl 2 luciastraat 7, 5821cl vierlingsbeek, the netherlands. e-mail: peter2.brakels@wur.nl submitted on 2007, 19th november; revised on 2007, 22th january; accepted 2008, 31st january. abstract. aurora’s alpine salamander is a limited distributed subspecies endemic to the altopiano di asiago, veneto. in the current paper the occurrence of salamandra atra aurorae is described for the altopiano di vezzena, trentino. the aim of this paper is to review the distribution as well as to comment on the conservational status of the subspecies in trentino. keywords. salamandra atra aurorae, first record, trentino, northeastern italy. aurora’s alpine salamander, salamandra atra aurorae (trevisan, 1982) is an endemic terrestrial salamander from the venetian prealps. recent research has identified this subspecies as a relict lineage, which separated from the nominate salamandra atra atra approximately 1 myr ago, and remained isolated in a glacial rifugium (bonato and steinfartz, 2005). although the nominate subspecies occurs at several mountainous locations within trentino, no individuals of s. a. aurorae have been found within the autonomous region (caldonazzi et al., 2002; ommizolo et al., 2002), despite the closeness of the type locality of the latter subspecies to the regional border with veneto. s. a. aurorae is an extremely small-distributed subspecies, occurring at a stretch of approximately 17 kilometres length and several kilometres wide on the altopiano di asiago, veneto (bonato and grossenbacher, 2000). in this article we describe for the first time the presence of this subspecies for trentino province, and present the threats for s. a. aurorae within this region. this research was part of the european community life program, “project sistema aurora” of the university of udine (friuli-venezia giulia). the study area on the altopiano di vezzena can be defined as eastwards from the altopiano di vezzena, to the regional border with the veneto region. the north of this area is bordered by the cima vezzena and cima manderiolo, while the val d’assa acts as a barrier in the south. within this area, from west to east bianco, val pisciavacca, val postesina and the western part of valle sparavieri (costa di sopra), were investigated. the presence of s. a. aurorae was defined by means of ves (visual encounter survey, crump acta herpetologica 3(1): 77-81, 2008 issn 1827-9643 (online) © 2008 firenze university press 78 w. beukema and p. brakels and scott, 1994), using a random walking pattern in the study areas while looking under stones, pieces of bark and logs. each of the four above mentioned localities were visited once during one hour of intensive searching. after discovery of presence, another hour of intensive search was done to locate additional individuals. dorsal photographs were made for individual recognition. 1. bianco (western altopiano di vezzena). this brook is situated on a relative low altitude of 1100 meters, compared with the preferences of alpine salamanders (bonato and fracasso, 2006). 2. val pisciavacca (western altopiano di vezzena). moist mixed forest with dominance of fagus sylvatica, abies alba and picea abies is present in this area, with a comparable undergrowth to that of the habitat at the altopiano di asiago. altitudes are in favour of alpine salamanders. no individuals of s. a. aurorae were located during ves. 3. val postesina (eastern altopiano di vezzena). on the 11th of may 2007, at 09:30 in the morning, an adult male s. a. aurorae (fig. 1) was found under a stone just next to the brook bed, at 1420 m a.s.l. the same individual, identified by its unique colour pattern, was recaptured on 09:27 on the 13th of may under the same shelter. the habitat was characterized by mixed forest of dominating picea abies and abies alba, with a lesser extent of fagus sylvatica. fig. 1. adult male salamandra atra aurorae from val postesina 79discovery of salamandra atra aurorae 4. western valle sparavieri (costa di sopra). at the 22th of may, at 10:00 in the morning a female s. a. aurorae was found under a stone in dense picea abies forest with several fagus sylvatica, on the upper slopes of valle sparavieri, eastwards of malga costa di sotto. several stones and logs were present on the forest floor. although this is the first record for s. a. aurorae in trentino, the occurrence of this subspecies could be expected due to a continuation of the habitat from veneto. up to now, the subspecies was known to be present in valle sparavieri on the regional border (bonato and grossenbacher, 2000), with individuals also dispersed on the western part of the valley (pers. comm.). the discovery of s. a. aurorae in val postesina is however of greater interest since this valley is well within the boundaries of trentino. val d’assa is known to be a natural border for dispersal of s. a. aurorae (bonato and grossenbacher, 2000), while occurrence of this subspecies north or westwards from the study area is not likely, because of altitudinal and climatological differences, not suitable for alpine salamanders (bonato and fracasso, 2006). the elusive behaviour of this subspecies, probably enhanced by the relative dry climatic conditions and the karstic substrate (grossenbacher, 1995), makes it impossible to deny presence in the area between passo vezzena and val postesina. the dominant tree species in the habitat of val postesina is picea abies. while the east slopes of the valley are replanted with p. abies, the west slopes consist of abies alba forest mixed to open grassy spots, almost without surface structure. the dense, planted p. abies forest on the eastern slopes, with little surface structure, and the lack of diurnal shelters and rocky structure on the western slopes creates an unsuitable habitat for the alpine salamanders. both shelters (review in mathis et al., 1995) and sustainable humidity in the structure of the rocky surface (duellman and trueb, 1986) are critical for the survival of salamanders, which need shelters during dry and cold periods, and often a relative high humidity to feed and reproduce. p. abies is not naturally present at the vezzena plateau (gaffa and pedrotti, 1998) and its presence is likely due to replanting for future logging and forest restoration, started after the first world war. therefore, the habitat of s. a. aurorae seems to have changed extensively in the last decades, and only the direct vicinity of the brook provides suitable amount of shelters such as stones, open karstic formations, dead wood and relative high humidity. construction of an unpaved road that winds several times through the brook however also threatens these last refuges, since it makes water flow impossible. at the upper source of val postesina, water is being extracted from the karst, resulting in a reduced water flow (pers. comm.). the final threat is due to logging just north of the suitable habitat, resulting in the subjection of direct sunlight to large areas on the eastern slope and the brook, dehydrating the forest surface and exposing it to the wind (fig. 2). s. a. aurorae seems to be extremely limited within the trentino province, only occurring in the east of the altopiano di vezzena. the geographical barriers mentioned earlier make presence other than the study area highly unfavourable. the survival of the subspecies at least in val postesina seems to be in serious threat due to logging, silvicultural clear cutting of picea abies, and the extraction of water from the karst. these threats are destroying the habitat, the surface structure of the forest and the humidity both in the surface and in the vicinity of the brook. 80 w. beukema and p. brakels ackowledgements we thank marianna bellon and the corpo forestale at levico for arranging the permission to access the forestry roads on the altopiano di vezzena. both authors were supported by an erasmus grant for international studies. kurt grossenbacher and nicola bressi commented on the manuscript. references bonato, l., grossenbacher, k. (2000): on the distribution and chromatic differentation of the alpine salamander salamandra atra laurenti, 1768, between val lagarina and val sugana (venetian prealps): an updated review. herpetozoa 13: 171-180. bonato, l., steinfartz, s. (2005): evolution of the melanistic color in the alpine salamander salamandra atra as revealed by a new subspecies from the venetian prealps. ital. j. zool. 72: 253-260. bonato, l., fracasso, g. (2006): salamandra atra. in: atlante degli anfibi e dei rettili d’italia. atlas of italian amphibians and reptiles, p. 190-195. sindaco, r., razzetti, e., doria, g., bernini f., eds, societas herpetologica italica, edizioni polistampa, firenze. fig. 2. logging and dehydration of the brook in val postesina 81discovery of salamandra atra aurorae caldonazzi, m., pedrini, p., zanghellini, s. (2002): atlante degli anfibi e dei rettili della provincia di trento. 1987-1996 con aggiornamenti al 2001. museo tridentino di scienze naturali, trento. crump, m.l., scott, n.j. jr. (1994): visual encounter surveys. in: measuring and monitoring biological diversity: standard methods for amphibians, p. 84-92. heyer, w.r., donnelly, m.a., mcdiarmid, r.w., hayek, l.c., foster, m.s., eds, smithsonian books, washington d.c. duellman, w.e., trueb, l. (1986): biology of amphibians. mcgraw-hill book co., new york. gaffa, d., pedrotti, f. (1998): fitoclima del trentino-alto adige. studi tren. sci. nat. acta biol. 73: 55-111. grossenbacher, k. (1995): was ist los mit salamandra atra aurorae? elaphe 3: 6-8. mathis, a., jaeger, r.g., keen, w.h., ducey, p.k., walls, s.c., buchanan, b.w. (1995): aggression and territoriality by salamanders and a comparison with the territorial behaviour of frogs. in: amphibian biology, 2: social behaviour, p. 633-676. heatwole, h., sullivan, b., eds, surrey beatty, new south wales, australia. omizzolo, a., lorenzi, p., gianesi g., bruno, s. (2002): appunti sugli anfibi del trentino. ann. mus. civ. rovereto 16 (2000): 157-271. acta herpetologica 4(1): 73-81, 2009 endoscopy of cloaca in 51 emys trinacris (fritz et al., 2005): morphological and diagnostic study filippo spadola1, gianni insacco2 1 facoltà di medicina veterinaria di messina, dipartimento di scienze sperimentali e biotecnologie applicate, polo universitario ss. annunziata i-98128, messina, italy. corresponding author. e-mail: fspadola@unime.it 2 centro regionale recupero fauna selvatica e tartarughe marine fondo siciliano per la natura e museo civico di storia naturale, via generale girlando 2, i-97013 comiso (rg). e-mail: gianni insacco@virgilio.it abstract. in this work the authors are describing cloaca anatomy, pathological findings and endoscopic technique applied to emys trinacris (fritz et al., 2005). the study has been performed on 51 wild subjects of different age and size hosted in a rescue center. cloacoscopy is proposed as a tool for sexing turtles with reduced sexual dimorphism. keywords. reptiles, emys, cloaca, cloacoscopy, endoscopy. introduction variations in cloaca anatomy among chelonian species is poorly documented in literature as well as morpho-functional aspects. in addition, from a medical point of view, the cloaca is likely to be involved in various diseases, such as congenital deformities, microbial and parasitic infections, urolithyasis, neoplasia, foreign bodies, traumas, occlusions, organ prolapse. endoscopy and laparoscopy are well estabilished techniques applied to non conventional species, widley used as a diagnostic tool. moreover, the same instrumentation can be implemented to allow micro invasive surgery and foreign body retrival from different body cavities (divers, 1998; gobel, 1994; jekl, 2007; schildger et al., 1989, 1992; scott, 2003; spadola et al., 2001, 2003, 2008; taylor, 2006). performing a cloacoscopy requires an in-depth knowledge of the anatomy of the cloaca; otherwise the risk of technical mistakes dangerous for the animals, is high (coppoolse et al., 1985, 1986; divers, 1998; jekl, 2007; scott, 2003; spadola et al., 2001, 2003, 2008; smith, 1958; taylor, 2006). the cloaca has a complex structure and is divided in three regions which are termed, progressing cranio-caudally, coprodeum, urodeum and proctodeum. the coprodeum receives the terminal portion of the gut while the urodeum is the portion of the cloaca where the urethra and the vas deferens and the oviducts, in males and in females respectively, open. the proctodeum represents the most caudal por74 f. spadola and g. insacco tion of the cloaca where the can be found the reproductive organs (dulzetto, 1968; miller et al., 2007; spadola et al., 2001, 2003, 2008). our work aims to the description of the cloacal anatomy in emys trinacris, and the cloacoscopic technique, which allows not only to diagnose cloacal pathologies, but also of establishing with certainty gender of animals with a reduced sexual dimorphism. material and methods the study was performed on 51 wild emys trinacris of different age and size, whose cloacal openings were compatible to endoscope diameter and in good apparent general health status assessed by mean of clinical exsamination. in all the specimens cloacoscopy was routinely performed to evaluate the condition of the mucosa, to collect flushing fluids for parasitological and/or bacteriologic determination, to assesed gender in doas subjects were external dimorfism was not evident and pathological changes were recorded. endoscopic exam was performed with a karl storz otoscope with linear vision 0° (8.5 cm lent, 5 mm diameter, serial number 67260a) provided with an instrument channel with an fiber optic light cable. the scope was connected to a camera (telecam dx-ii) and the whole system was connected to a karl storz “tele pack™“ providing light source, camera control and image documentation. for the whole procedure all animals were manually restrained and placed in ventral recumbency. after endoscope introduction a warm saline solution nacl 0.9% at 30 °c added with 2% lidocaine (3 ml/litre) was introduced under visual control to dilatate the cloaca for a wider and clearer vision. distension with fluid was mantained for the whole procedure allowing clearing of cloacal mucosa (spadola et al., 2008). results during cloacoscopy, progressing in a caudo-cranial sense, it was possible to observe in the most caudal portions of the cloaca the pigmented mucosa of proctodeum and in its ventral portion the penis (fig. 1 a, b, c, d) or clitoris (fig. 6 a). advancing more cranially, it was possible to identify on the ventral-lateral wall the wide openings of the two accessory vesicles and, in the middle-ventral portion, the urodeum slit, formed by two muscles which converge dorsally and ventrally to cover the urogenital senum (fig. 2 a, b, c, d; fig. 6 b, c, d). through the thin wall of the accessory vesicles, it was possible to observe some coelomatic organs (testes, ovaries, and intestine) (fig. 3 a, b, c, d; fig. 7 a, b, c, d). in the upper part of the urodeum it is visible a muscular ridge, with horizontal fibres, where the gut void into the cloaca (fig. 4 b, d; fig. 11 a, c). going through the seminal furrow it was possible to reach the inner part of the urogenital senum, where the urethra opening was found in central portion (fig. 4 a, b, c, d). this opening can be entered, allowing visual examination of the bladder wall, which is semi-transparent (fig. 10 a, b, c, d). laterally to the urethral opening the urogenital papillae can be seen. this structure, is small and rod shaped in males while is “cauliflower-shaped” and bigger in females (fig. 5 a, b, c, d; fig. 8 a, b, c, d). on the urogenital papilla, near the ureter opening, was visible the vas deferens opening and the oviduct opening in males and in females, respectively (fig. 5 b, d; fig. 9 a, b, c, d). 75endoscopy of cloaca in emys trinacris fig. 1. fig. 2. 76 f. spadola and g. insacco fig. 3. fig. 4. 77endoscopy of cloaca in emys trinacris fig. 5. fig. 6. 78 f. spadola and g. insacco fig. 7. fig. 8. 79endoscopy of cloaca in emys trinacris fig. 9. fig. 10. 80 f. spadola and g. insacco due to their folding nature, oviducts can be explored during cloacoscopy for a limited lenght, (fig. 9 b, c). after the procedure all animals were kept under observation for 24 hours and then housed in outdoor ponds. no undesired side effect were observed (spadola et al., 2008). three of the 51 examined animals had pathological findings: one had a big bladder stone (spadola et al., 2005) and two showed intracoelomatic neoformations of unknown origin compressing cloacal structures (fig. 11 b, d). discussion further studies are needed to compare cloacal structures of emys trinacris to those of other non european fresh water species. in herpetological medicine is of paramount importance to get a deep knowledge on reptiles’ anatomy and physiology. this can lead to better recognize and treat the specific disease conditions of these peculiar animals. although cloacal pathologies in turtles are easy to find, diagnostic interpretation, should be reserve to specialized professionals. endoscopy is minimally invasive and, thanks to the direct view under magnification and illumination, diagnosis of internal diseases is easily achieved. this technique can also be applied for sex determination in juveniles and in those species where no sexual dimorphism is evident (coppoolse et al., 1985, 1986; divers, 1998; jekl, 2007; kissner et al., 1998; spadola et al., 2001, 2003, 2008; smith, 1958; taylor, 2006). fig. 11. 81endoscopy of cloaca in emys trinacris acknowledgements we wish to thank annalisa zaccaroni, alessia cappello, alessandro frasca, sonia terranova and simona cappello for their cooperation. references coppoolse, k., zwart, j.p. (1985): cloacoscopy in reptiles. vet. q. 7: 3. coppoolse, k., zwart, j.p. (1986): cloacoscopy in reptiles. tijdschr diergeneeskd 207-209. divers, s.j. (1998): an introduction to reptile endoscopy. arav 5th ann. conf. 5: 41-45. dulzetto, f. (1968): anatomia comparata dei vertebrati. ed. calderini. gobel, t. (1994): diagnostic endoscopy in reptiles. verh. ber. erkrg. zootiere 36: 163-167. jekl, v. (2007): cloacoscopy in chelonians – a. valuable diagnostic tool for reproductive tract evaluation. 43rd international symposium on diseases of zoo and wild animals. kissner, k.j., secoy, d.m., forbes, m.r. (1998): sexual dimorphism in size of cloacal glands of the garter snake, thamnophis radix haydeni. j. herpetol. 32: 268-270. miller, j.d., dinkelaker, s.a. (2007): reproductive structures and strategies of turtles. in: biology of turtles, p. 225-278. wyneken, j., godfrey, h., bels, v., eds, crc press boca raton fl. schildger, b.j., wicker, r. (1989): sex determination and clinical examination in reptiles using endoscopy. herp. rev. 20: 9-10. schildger, b.j., wicker, r. (1992): endoscopy in reptiles and amphibians. indications, methods, findings. prakt tierarzt 73: 516-527. scott, j.s. (2003): reptile cloacoscopy. exotic dvm 5: 3. smith, h.m., james, l.f. (1958): the taxonomic significance of cloacal bursae in turtles. trans. kans. acad. sci. 61: 86-96. spadola, f., costa, g., musicò, m., siracusano, l., di blasi, a., cucinotta, g. (2005): calcolosi vescicale in una emys orbicularis (tartaruga palustre europea): terapia endoscopica miniinvasiva. atti xii congresso nazionale s.i.c.v. pisa. 12: 184-186). spadola, f., lentini, s, insacco, g. (2008): studio preliminare sull’anatomia endoscopica della cloaca in emys trinacris (fritz et al., 2005) e possibili applicazioni cliniche. in: herpetologia sardiniae, p. 463-466. corti, c., ed, societas herpetologica italica, edizioni belvedere, latina. spadola, f., musicò, m., interlandi, c., costa, g., cucinotta, g. (2001): preliminary result on clinical application of cloacoscopy. international congress e.s.v.s. 20: 299-304. spadola, f., musicò, m., macrì, f., interlandi, c., costa, g., cucinotta, g. (2003): applicazione clinica della cloacoscopia nei rettili e nei volatili. summa vet. 1: 23-28. taylor, w.m. (2006): endoscopy. in: reptile medicine and surgery, mader, d.r. ed, 2nd ed., p. 549-563. saunders elsevier, st. louis ms. acta herpetologica 2006 2 temperature variation in nests of caiman crocodilus (crocodylia : alligatoridae) temperature variation in nests of caiman crocodilus (crocodylia: alligatoridae) armando h. escobedo galván escuela de ciencias biológicas, universidad nacional, heredia, costa rica. present address: julio cervantes 561 col. san lorenzo oriente c.p. 25060 saltillo, coahuila, méxico. e-mail: elchorvis@gmail. com abstract. caiman crocodilus is a widely distributed species in the neotropics; however, studies of incubation temperatures in wildlife are uncommon. incubation temperatures in four nests of caiman crocodilus were measured with a digital thermometer, in the national wildlife refuge caño negro, costa rica. average temperatures in these four nests (no. 1-4) were 32.13 + 0.92 °c (no. 1), 32.46 + 0.77 °c (no. 2), 33.60 + 0.95 °c (no. 3), and 31.78 + 2.30 °c (no. 4). temperature variations recorded showed higher temperatures than those reported from other studies in caño negro. the temperatures registered in this study will lead to a higher proportion of males within the caiman population, reducing the number of future reproducing females and, therefore, in the population viability. keywords: caiman crocodilus, nest temperatures, costa rica. reptiles have a wide range of sex-determination systems, including genotypic and environmental sex determination. in crocodilians, sex is only determined by incubation temperature (bull and charnov, 1989; thorbjarnarson, 1997), a kind of environmental sex determination. most studies relating incubation with temperature have been carried out in laboratory conditions, because they permit knowledge of the sex ratios under different incubation temperatures (aguilar-miguel et al., 1998). however, these treatments do not necessarily encompass temperature variations that occur in the natural environment. incubation temperature affects the sex ratio of crocodilian species differently. studies of the effect of incubation temperature in crocodilians have been done with alligator mississippiensis (ferguson and joanen, 1982), caiman yacare (campos, 1993; miranda et al., 2002), c. latirostris (piña et al., 2003), crocodylus porosus (magnusson, 1979), c. acutus and c. moreletii (aguilar, 1995). even though caiman crocodilus is a widely distributed species in the neotropics (ross, 1998), studies of incubation temperatures in their natural environment are uncommon (allsteadt, 1994). the most common treatment that has been done in situ in order to measure temperature variations within the nest used mercury thermometers inside the egg cavities. this method only registered temperatures during a limited period of time (hours along some days or weeks) (magnusson et al., 1990; allacta herpetologica 1(2): 131-134, 2006 132 armando h. escobedo galván steadt, 1994; miranda et al., 2002; casas-andreu, 2003) and, it did not evaluate the thermal gradient present inside the egg cavities. due to the lack of information about the variations of temperatures within c. crocodilus nests, the objective of this note is to analyze the daily variations of temperatures within nests of c. crocodilus located in the northern part of costa rica and compare our results with the others existing studies (allsteadt, 1994; junier, 2000). the study was conducted on the national wildlife refuge caño negro, on the frio river, at the northern part of costa rica (10°54’n, 84°47’w). this protected area extends 9,969 ha and is one of the most important wetlands in northern of costa rica. largest population density of c. crocodilus (74.36 ind/km) registered up to now has been reported at this location (cabrera et al., 2003). the nests were located by walking transects in the sites where people had seen caiman nests in previous years. caimans construct mounds of vegetation with leaves, grass, branches and earth in forested areas; mostly in the root area at the base of trees and near water bodies (cintra, 1988; álvarez del toro and sigler, 2001). when nest was discovered, it was opened to determine the presence of the eggs, because in some cases female caimans build up a nest without laying the eggs (álvarez del toro and sigler, 2001). once the eggs were observed at the egg cavity, a digital thermometer (stowaway tidbit temp logger) was placed to register the temperature inside the egg cavity every 10 min, until the eggs hatched. the temperature was registered in four nests; no. 1, no. 2, no. 3 and no. 4 during 32, 31, 20 and, 69 days, respectively. the data were averaged daily, for interpretation. the kruskal-wallis test suggests differences between incubation temperature of the nests (h = 787.266; p < 0.01), with the highest mean temperature being in nest no. 3 (33.60 + 0.95 °c), followed by nest no. 2 (32.46 + 0.77 °c), nest no. 1 (32.13 + 0.92 °c) and nest no. 4 (31.78 + 2.30 °c) (table 1). only one nest (no. 3) indicates significant difference in temperatures between day and night, being higher during the night (student t-test, p < 0.05). temperature variations can be correlated with climatic phenomena. magnusson (1979) observed that the presence of hurricanes affected the temperature within the nests. location of the nests at the refuge can significantly alter temperatures. cintra (1988) mentions that in some cases the nests are not placed at the best sites, because of intraspecific pressure or anthropogenic effects. campos (1993) also observed that the nests that were built on top of organic matter showed higher temperatures than those that were built on top of floating grass. she concluded that nest temperature is determined by nesting habitat but that the effect of habitat depends on weather conditions. table 1. incubation temperature of four nests with mean, standard deviation, minimum and maximum, registered with a digital thermometer in degrees celsius. nest n° mean sd minimum maximum no.1 757 32.13 0.92 25.57 33.39 no.2 735 32.46 0.76 29.11 33.73 no.3 468 33.60 0.95 29.73 35.03 no.4 1654 31.78 2.30 24.82 38.01 133temperature variation in nests of caiman crocodilus allsteadt (1994) and junier (2000) registered a mean of 31.8 °c and 31.6 °c, respectively in c. crocodilus nests in caño negro. even though, there was not a statiscally significant difference between those studies and this research (chi-square test, p > 0.05); there is an increase, in average, of 0.53 °c in incubation temperatures. sometimes crocodilian females built nests on top of old nests, which allows for higher temperatures and also insulates the eggs from low ground temperatures (magnusson et al., 1990). this fact could explain the higher recorded temperatures at the nests in this research. temperature variations registered showed higher variations than those reported in other studies of crocodilians (chabreck, 1973; magnusson, 1979; magnusson et al., 1985; campos, 1993; allsteadt, 1994). the higher temperatures of the nests implicate less reproductive success and decreased numbers of developed embryos but at the same time, hatchlings can be favored by a better growth in length and by a better increase in weight (piña et al., 1997). miranda et al. (2001) observed that hatchlings of caiman nests of temperatures higher than 31 °c presented a better absorption of the yolk and, a faster feeding after hatching. in nest no. 1 there were five post-hatching dead newborns, and no eggs hatched from nest no. 4. we suggest that phenomenon is due to the high and low temperatures registered at those nests, in accordance with the observations of ferguson and joanen (1982). campos (1993) determined that temperatures higher than 31.5 °c within the nest produce a higher number of males. assuming that the critical temperature for the production of clutches with 1:1 sex ratios is about 31.5 °c, the proportion of sexes in the observed nests could be higher than 75% male. this could have repercussions on the number of future reproducing females and, therefore, in the viability of the population. acknowledgements this study was supported by roberto villalobos and josé retana who work at the instituto meteorológico nacional of costa rica. i thank the staff of national wildlife refuge caño negro. i thank marco a.l. zuffi and the reviewers, whose comments helped to improve this note. references aguilar, x. (1995): efecto de la temperatura de incubación sobre la determinación del sexo en crocodylus acutus y c. moreletii. bol. soc. herpetol. mex. 6: 43. aguilar-miguel, x., herrera, j., merchant-larios, h., casas-andreu, g. (1998): efecto de la temperatura de incubación sobre la actividad esteroidogénica en crocodylus acutus y c. moreletii. rev. soc. mex. hist. nat. 48: 95-103. allsteadt, j. (1994): nesting ecology of caiman crocodilus in caño negro, costa rica. j. herpetol. 28: 12-19. álvarez del toro, m., sigler, l. (2001): los crocodylia de méxico. 1a edición. imernar, profepa, méxico. bull, j.j., charnov, e.l. (1989): enigmatic reptilian sex ratios. evolution 43: 1561-1566. 134 armando h. escobedo galván cabrera, j., protti, m., urriola, m., cubero, r. (2003): distribución y abundancia de caiman crocodilus en el refugio nacional de vida silvestre caño negro, costa rica. rev. biol. trop. 51: 571-578. campos, z. (1993): effect of habitat on survival of eggs and sex ratio of hatchlings of caiman crocodilus yacare in the pantanal, brazil. j. herpetol. 27: 127-132. casas-andreu, g. (2003): ecología de la anidación de crocodylus acutus (reptilia: crocodylidae) en la desembocadura del río cuitzmala, jalisco, méxico. acta zool. mex. (n.s.) 89: 111-128. chabreck, r.h. (1973): temperature variation in nests of the american alligator. herpetologica 29: 48-50. cintra, r. (1988): nesting ecology of the paraguayan caiman (caiman yacare) in the brazilian pantanal. j. herpetol. 22: 219-222. ferguson, m.w.j., joanen, t. (1982): temperature of egg incubation determines sex in alligator mississippiensis. nature 296: 850-853. junier, e.f. (2000): análisis de la población de caiman crocodilus en el refugio nacional de vida silvestre caño negro, costa rica. unpublished degree thesis/tesis de licenciatura. universidad nacional, heredia, costa rica. magnusson, w.e. (1979): maintenance of temperature of crocodile nests (reptilia, crocodilidae). j. herpetol. 13: 439-443. magnusson, w.e., lima, a.p., hero, j.m., sanaiotti, t.m., yamakoshi, m. (1990): paleosuchus trigonatus nests: sources of heat and embryo sex ratios. j. herpetol. 24: 397400. magnusson, w.e., lima, a.p., sampaio, r.m. (1985): sources of heat for nests of paleosuchus trigonatus and a review of crocodilian nest temperature. j. herpetol. 19: 199207. miranda, m.p., de moraes, g.v., matines, e.n., pinto, l.c., barbosa, o.r. (2002): thermic variation in incubation and development of pantanal caiman (caiman crocodilus yacare) (daudin, 1802) kept metabolic box. braz. archiv. biol. technol. 45: 333-342. piña, c.i., larriera, a., cabrera, m.r. (2003): effect of temperature on incubation period, sex ratio, hatching success and survivorship in caiman latirostris (crocodylia, alligatoridae). j. herpetol. 37: 199-202. piña, c., donayo, p., barriera, a. (1997): efecto de la temperatura de incubación de huevos de caiman latirostris, sobre diversas variables reproductivas y de crianza, informe de avance. memorias de las 4ta reunión regional del grupo de especialistas de cocodrilos de américa latina y el caribe 4: 137-143. ross, j.p. (1998): crocodiles. status survey and conservation action plan, 2nd edition. uicn/ssc. crocodile specialist group. gland, switzerland and cambridge, london. thorbjarnarson, j. (1997): are crocodilian sex ratios female biased? the data are equivocal. copeia 1997: 451-455. acta herpetologica 2006 2 addenda and errata to «description of a new species of the genus adenomera (amphibia, anura, leptodactylidae) from french guiana.» addenda and errata to “description of a new species of the genus adenomera (amphibia, anura, leptodactylidae) from french guiana.” ariadne angulo 1,2, jean-christophe de massary 3, renaud boistel 4 1 conservación internacional, carrera 13 # 71-41, bogotá, colombia 2 departamento de herpetología, museo de historia natural de san marcos, apartado 14-0434, lima, perú 3 4, rue des tilleuls, 95170 deuil-la-barre, france 4 université paris sud. centre scientifique d’orsay, laboratoire des mécanismes de communication animale, nam cnrs umr 8620. bâtiment 446, 91405 orsay cédex, france in the first issue of acta herpetologica, boistel et al. (2006) described a new species of adenomera (amphibia, anura, leptodactylidae). because of an unfortunate set of events, there are several aspects of this paper that have to be corrected, or elaborated on. we address these issues herein. it is noteworthy to add that since the description of adenomera heyeri, a new article, suggesting a number of taxonomic changes, was published (frost et al., 2006). in this paper it is recommended that adenomera be reconsidered under the genus leptodactylus. this is consistent with the results of other, independent studies (angulo, 2004; kokubum and giaretta, 2005). given the currently available evidence, the new species would be best placed under leptodactylus, its name would thus be leptodactylus heyeri (boistel, et al., 2006), and all other members of adenomera would now be considered leptodactylus. however, in order to facilitate the fluent reading of this note with reference to the original description and minimize confusion, we will refer to the nomenclature used in the original paper. the points that have to be incorporated into the paper or followed up on are as follows: in page 2, paragraph 3, we mention that the advertisement calls of five nominal species of the genus adenomera have been previously described; yet, only four are mentioned (a. hylaedactyla, a. andreae, a. araucaria and a. marmorata). the missing species is adenomera diptyx, whose call was originally published in márquez et al. (1995) as that of adenomera andreae (de la riva et al., 2000). although it was not cited in the text, this last reference was included in the reference list in the description. it is worth mentioning that since the publication of the description of a. heyeri, an additional advertisement call has been described for the group, that of the newly described leptodactylus thomei (almeida and angulo, 2006). in page 3, line 1, remove the word “of ” from the sentence “further analyze of the known vocalizations …”. in page 3, first paragraph under morphological analysis, add “additional materials examined for comparative purposes are deposited at the academy of natural sciences • • • acta herpetologica 1(2): 159-162, 2006 160 a. angulo et alii of philadelphia (ansp), philadelphia, u.s.a. ; museo noel kempff mercado (nka), santa cruz de la sierra, bolivia; museu de ciências e tecnologia da pucrs (mcp), porto alegre, brazil; museu nacional do rio de janeiro (mnrj), rio de janeiro, brazil; national museum of natural history (usnm), washington, d.c., u.s.a.; royal ontario museum (rom), toronto, canada; and zoologische staatssammlung münchen (zsm), münchen, germany.” page 3, morphological analysis: vomerine teeth (vt) are also referred to as vomerine ridges in the species description. page 4, first paragraph under diagnosis: “the new species is distinguished from all other species by its advertisement call and the following combination of characters: (1) two pairs of dorsolateral folds present; (2) smooth skin on lower surface of foot or with a few small white tubercles; (3) throat and belly of males yellow; (4) tarsal fold present and slightly marked.” although the new species may share some of these characters with other species (e.g. presence of two pairs of dorsolateral folds, scant small white tubercles on lower surface of foot, or a weakly marked tarsal fold), this paragraph refers to the use of all of these features combined, rather than taken independently. last three lines of the second paragraph, diagnosis: “adenomera heyeri is distinct from a. hylaedactyla by having the head as wide as long, its snout is, from above, nearly rounded versus subovoid, pulses are absent and note duration is longer”. measurements of the holotype of a. heyeri indicate that head length is 9.6 mm, whereas head width is 9.2 mm. however, head length is not always greater than width in this species, as can be seen from table 3. a. hylaedactyla also has variable head widths and lengths (see heyer, 1973 and angulo et al., 2003). because of these two points, the comparison of head length and head width would not be a useful diagnostic feature of a. heyeri with regards to a. hylaedactyla. in the diagnosis there is no comparison with a. diptyx, which is herein provided: adenomera heyeri differs from adenomera diptyx (boettger, 1885) in having a different advertisement call. a. heyeri also has a distinct tympanum, with a maximum diameter about 2/3 of eye diameter; in a. diptyx the tympanum is half the size of the eye (boettger, 1885). page 4, second line in the description of holotype: “snout from above … head wider than long; …” should read “snout from above … head slightly longer than what it is wide; …”. page 5, table 1: “data for a. heyeri originate from the type specimens (mnhn 1999.8331)” should read “data for a. heyeri originate from the type specimen (mnhn 1999.8331)”. where it reads “asterisks indicate …” should read “asterisk indicates ... two (long distance recordings) specimens.” last three lines of page 5: “… inner and outer metacarpal tubercles large, prominent, outer larger than inner, shape of inner oblong, that of outer long; …” should read “… inner and outer metacarpal tubercles large, prominent, outer larger than inner, shape of inner oblong, that of outer rounded; …” page 6, table 2: – the number of harmonics in a. hylaedactyla reads “2”; it should read “2 to 5”. • • • • • • 161addenda and errata adenomera heyeri add note: “to table 2 the number of harmonics detected will vary as a function of recording distance, as higher frequencies are more readily lost with increased distance.” page 7, coloration in life: in the paragraph “this species can be easily identified by its coloration. the back is … continues along the back tapering, and disappears around the sacral region” there should be a comma between “back” and “tapering”. page 8, secondary sexual characters: remove comma from “males have a vocal apparatus, consisting of …”. page 9, figure 4: removal of the second “of ” in the figure legend: “(c) sonogram with a palette of 12 colors of depicting different intensities”. page 10, advertisement call line 1: – “the call of a. heyeri is distinct from that of the other three species in all parameters (see tables 1 and 2)” should read “the call of a. heyeri differs in mean values from that of the other three species examined in all parameters, although there is some overlap in the ranges of certain features (see tables 1 and 2)”. in addition, the fundamental frequency in adenomera araucaria (1722-3359 hz) overlaps with that of a. heyeri, although they do differ in other important parameters such as 2f0 and amplitude modulation (present in a. araucaria; kwet and angulo, 2002). with regards to adenomera diptyx, the call of this species is much shorter than that of a. heyeri (56.6-88.3 ms vs. 136.87-184.5 ms), with a higher f0 (2180.8-2281.7 hz) and 2f0 (4200-4502.9 hz; márquez et al., 1995). page 10, advertisement call, line 7: “the envelope (fig. 4e) shows one periodical pattern of variation in amplitude with a duration of 13 ms”; where it reads “13 ms” it should be replaced with “12 ms”. page 10, advertisement call, line 8: where it reads “table 2” in the sentence “with regard to spectral features (figs. 4 a, b, c), …” it should read “table 1”. page 10, advertisement call, line 11: where it reads “in all other species of adenomera the dominant frequency is 2f0 …” the word “examined” should be inserted before the word “species”. page 10, advertisement call, line 14: in “notes were found to have a series of six distinguishable harmonics (table 2)”, the words “up to” should be inserted before the word “six”. page 11, discussion, fig. 5: – where it reads “detail view of t-shaped terminal phalange of third finger”, “close up” should replace “detail”. where it reads “detail view of t-shaped terminal phalange of fourth toe”, “close up” should replace “detail”. where it reads “sacral diapohysis …” it should read “sacral diapophysis …” where it reads “fig. 5. x-ray picture of adenomera heyeri, male paratype mnhn 1998.322, with details about the phalanges …” the phrase “showing close up of terminal phalanges” should replace “with details about the phalanges”. page 12, acknowledgements: we would like to express our gratitude to professor jean-pierre gasc (muséum national d’histoire naturelle, paris), who kindly generated the x-ray images; axel kwet, marcos di bernardo, ron heyer, frank glaw, ned gilmore, rossy montaño, ross macculloch and ulisses caramaschi kindly allowed for examination of museum specimens. page 14, appendix 1: – microphone for a. hylaedactyla should read ““sony ecmms907” instead of “sony ecm 307”. svl for a. hylaedactyla should read “23.1” instead of “22.7”. • • • • • • • • • • 162 a. angulo et alii references almeida, a.p., angulo, a. (2006): a new species of leptodactylus (anura: leptodactylidae) from the state of espírito santo, brazil, with remarks on the systematics of associated populations. zootaxa 1334: 1-25. angulo, a. (2004): the evolution of the acoustic communication system in members of the genus adenomera (anura: leptodactylidae): a comparative approach. ph.d. thesis, university of toronto, 232 p. angulo, a., cocroft, r.b., reichle, s. (2003): species identity in the genus adenomera (anura: leptodactylidae) in southeastern peru. herpetologica 59: 490-504. boettger, o. (1885): liste von reptilien und batrachiern aus paraguay. zeitschrift für naturwissenschaft 58: 1-36. boistel, r., massary, j.-c. (de), angulo, a. (2006): description of a new species of the genus adenomera (amphibia, anura, leptodactylidae) from french guiana. acta herpetologica 1: 1-14. de la riva, i., köhler, j., lötters, s., reichle, s. (2000): ten years of research on bolivian amphibians: updated checklist, distribution, taxonomic problems, literature and iconography. revista española de herpetología 14: 19-164. frost, d.r., grant, t., faivovich, j., bain, r.h., haas, a., haddad, c.f.b., de sá, r.o., channing, a., wilkinson, m., donnellan, s.c., raxworthy, c.j., campbell, j.a., blotto, b.l., moler, p., drewes, r.c., nussbaum, r.a., lynch, j.d., green, d.m., wheeler, w.c. (2006): the amphibian tree of life. bulletin of the american museum of natural history 297: 1-370. heyer, w.r. (1973): systematics of the marmoratus group of the frog genus leptodactylus (amphibia, leptodactylidae). contributions in science, natural history museum of los angeles county 251: 1-50. kokubum, m.n.c., giaretta, a.a. (2005): reproductive ecology and behaviour of a species of adenomera (anura, leptodactylinae) with endotrophic tadpoles: systematic implications. journal of natural history 39: 17451758. kwet, a., angulo, a. (2002): a new species of adenomera (anura, leptodactylidae) from the araucaria forest of rio grande do sul (brazil), with comments on the systematic status of southern populations of the genus. alytes 20: 28-43. márquez, r., de la riva, i., bosch, j. (1995): advertisement calls of bolivian leptodactylidae (amphibia, anura). journal of zoology, london 237: 313-336. appendix 2 other specimens examined adenomera andreae type series: brazil: peixeboi: zsm 145/1911/1-4 (four specimens). adenomera araucaria type series: brazil: rio grande do sul: são francisco de paula: potreiro novo: pró-mata pucrs: mcp 2421 (holotype), mcp 1794, mcp 3463, mcp 3672-73, mcp 3677; brazil: rio grande do sul: bom jesus: encruzilhada das antas: mcp 3346. acta herpetologica 16(2): 129-131, 2021 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-10306 sexual size dimorphism in the tail length of the caspian whip snakes, dolichophis caspius (serpentes, colubridae), in south-western hungary györgy dudás1, krisztián frank2,* 1 danube-dráva national park directorate, tettye tér 9, 7625 pécs, hungary 2 szekszárd district office of the government office of tolna county, dr. szentgáli gyula u. 2, 7100 szekszárd, hungary *corresponding author. e-mail: krisz.frank.biol@gmail.com submitted on: 2021, 3rd january; revised on: 2021, 5th may; accepted on: 2021, 11th may editor: marco sannolo abstract. sexual size dimorphism is widespread among snakes and has also been observed in lengths of body appendages such as in tails. males typically possess longer tails than females and this dimorphism in tail length has generally been attributed to the importance of the tail in mating and reproduction. we used body size measurements, snout-vent length (svl) and tail length (tl) as well as a body condition index (bci) as a measure of quality in caspian whip snakes from hungary, in order to shed light on sexual dimorphism patterns. the svl of males (1061 ± 133 mm, n = 25) were significantly longer than that of females (887 ± 208 mm, n = 41). however, the proportion of tl to total length was lower in males than in females (0.257 ± 0.018 and 0.274 ± 0.017, respectively). the bci of females (386 ± 10) was significantly higher than that of males (343 ± 15). females having proportionally longer tails compared to males seems to be the reverse of the usual trend. selective pressures on the tails of female snakes are less obvious, as tail length may be linked to more than one function, and hence be simultaneously subjected to more than one type of selective force. keywords. colubridae, hungary, sexual size dimorphism, tail length. sexual size dimorphism (ssd) is a common phenomenon and has been studied in snakes for decades (klauber, 1943; shine, 1978; king, 1989; shine, 1994). intersexual differences could be attributed to several evolutionary and ecological factors (shine, 1978; king, 1989; luiselli, 1996; sheehy et al., 2016; sivan et al., 2020). dimorphism may result from sexual selection (e.g., male-male combat or female choice), may occur when natural selection favours different traits in females and males, additionally, morphological constraints imposed on members of one sex or another may also result in sexual dimorphism (king, 1989; sivan et al., 2020). though these mechanisms are not mutually exclusive, morphological traits may be simultaneously subjected to more than one type of selective force. tails are important for a variety of functions in sna kes, including locomotion (jay ne and bennett, 1989; shine and shetty, 2001; sheehy et al., 2016), predation (heatwole and davison, 1976) and reproduction (king, 1989; shine et al., 1999; sivan et al., 2020). snakes are sexually dimorphic, and differences in relative tail lengths between sexes have been described in various species (king, 1989; shine et al., 1999). the difference in tail length was initially attributed to structural differences between sexes, as males possess a hemipenis in an elongated pocket at the base of the tail (klauber, 1943). later on, tail length has been shown to be a sexual selective trait, at least by some species, where males with relatively longer tails would have an advantage in reproduction (king, 1989; luiselli, 1996; shine et al., 1999; sivan et al., 2020). this could be because longer tails are advantageous when competing with other males during ball mating (luiselli, 1996; shine et al., 1999), or because the length of 130 k. frank the tail is an index signal by choosing mating partners (sivan et al., 2020). the caspian whip snake, dolichophis caspius, is a large-sized colubrid with a distribution area ranging from the carpathian basin to the west side of the caspian sea and covering most of the balkan peninsula and several neighbouring near east countries (puky et al., 2005). at the north-western edge of its distribution, populations tend to be fragmented and isolated (tóth, 2002; puky et al., 2005). a major part of scientific literature concerning the species deals with its geographic distribution and occurrence data. life-history traits have been studied in the main distributional range in frontier asia and balkans, studies conducted on north-western populations were less prominent. we analysed body size measurements of caspian whip snakes from hungary, in order to shed light on sexual dimorphism patterns. at the north-western edge of its distribution, in south-western hungary, the largest remnant caspian whip snake population is harbouring szársomlyó hill, a strictly protected nature reserve (tóth, 2002; frank et al., 2012). during the period 19982003 road surveys were carried out from april to september. snakes were captured by hand, weighed to the nearest 1 g by a digital balance and measured for snoutvent length (svl) and tail length (tl) to the nearest 1 mm by stretching the animal out along a measuring tape. snakes were probed to determine the sex of the animal. after measuring, snakes were released at the location of capture. recaptures were not included in the statistical analyses. two snakes had damaged tails and were omitted from the analyses. differences in body measurements (svl and tl) and bci between sexes were compared using t-tests. to examine the difference between the two regression estimates of tl on svl in males and females an ancova was used. measurements are presented as means ± se and p < 0.05 was accepted as the level of significance. all statistical analyses were performed with the software past (hammer et al., 2001). average svl of males (n = 25), 1061 ± 133 mm, was significantly longer than that of females (n = 41), 887 ± 208 mm (t = −4.091, p = 0.0001). however, average tl in males, 367 ± 54 mm, and females, 333 ± 72 mm was only marginally different (t = −1.997, p = 0.0501). the regression of tl on svl (fig. 1) was calculated in males as ln(tl) = 1.1734 × ln(svl) – 2.2721, (r2 = 0.597, f = 5.840, p = 0.0002); and in females as ln(tl) = 0.9503 × ln(svl) – 0.6407 (r2 = 0.896, f = 18.339, p = 0.0001). the proportion of tl to total length was lower in males than in females (0.257 ± 0.018 and 0.274 ± 0.017, respectively). both size (as svl) and sex affected tl (f1,64 = 8.129, p = 0.0059). size dimorphism between sexes is widespread among snakes; in a list of 129 species of the family colubridae compiled by shine (1994), males were the larger sex in 24% of species. within the group of longer males, ssd ranged between -0.01 and -0.50 (shine, 1994), the calculated ssd of d. caspius (frank and dudás, 2018) lies in the middle of this range. all 31 colubrids with longer males than females were oviparous (shine, 1994), as is dolichophis. difference in relative tail length between sexes is very widespread in snakes, and relative tail length might be a biologically relevant trait that affects reproduction (king, 1989; shine, 1994; shine et al., 1999; sivan et al., 2020). dimorphism in tl is usually male-biased, i.e. male snakes typically possess longer tails than females. this has generally been attributed to the importance of the tail in mating and reproduction (king, 1989; luiselli, 1996; shine et al., 1999; shine and shatty, 2001; sivan et al., 2020). as pointed out by king (1989), males might benefit from a longer tail because it may provide space for larger hemipenes (“morphological constraint hypothesis”) or because it confers an advantage in mating success (“male mating ability hypothesis”). additionally, females might increase reproductive output due to an increase in body capacity and a secondary reduction of tl (“female reproductive output hypothesis”). females having proportionally longer tails compared to males seems to be the reverse of the usual trend (king, 1989; shine et al., 1999); thus, has been reported substantially less. in a list of 103 colubrid species compiled by king (1989), females had relatively longer tails than males in seven cases (king, 1989). selective pressures on the tails of female snakes are less obvious (shine and shetty, 2001), as tail length may be linked to more than one function, and hence be simultaneously fig. 1. the effect of snout-vent length (svl) on tail length (tl) in female (circles, solid line) and male (triangles, dashed line) freeranging caspian whip snakes, dolichophis caspius. 131sexual size dimorphism in the tail length of the dolichophis caspius subjected to more than one type of selective force. sexual selection of longer tails in females would imply that individuals with longer tails have a higher reproductive output than females with shorter tails. unfortunately, there are no data to confirm this hypothesis in d. caspius, and no finding of selective forces acting on the tails of female snakes has been published in any other species. relative tail length may also be influenced by ecological factors when, for example, males and females use different microhabitats, or have different defensive tactics. arboreal snakes have been shown, in general, to have relatively longer tails than non-climbing species (sheehy et al. 2015), but this trend was not investigated intraspecifically before. besides, it is not likely that female d. caspius are more arboreal than males. however, this is the first study in which the sexual dimorphism in tail length in caspian whip snakes was investigated and as for now the influence of tail length on female reproductive output or any other life-history trait remains unexplained. acknowledgements we are grateful to all volunteers who participated in the fieldwork. this work was supported by birdlife hungary. collecting and measuring the animals was done with permissions from the danube-dráva national park directorate. references frank, k., dudás, gy. (2018): body size and seasonal condition of caspian whip snakes, dolichophis caspius (gmelin, 1789), in southwestern hungary. herpetozoa 30: 131-138. frank, k., majer, j., dudás, gy. (2012): capture-recapture data of large whip snakes dolichophis caspius (gmelin, 1789), in southern transdanubia, hungary. herpetozoa 25: 68-71. hammer, ø., harper, d. a. t., ryan p. d. (2001): past: paleontological statistics software package for education and data analysis. palaeontol. electron. 4: 9. heatwole, h., davison, e. (1976): a review of caudal luring in snakes with notes on its occurrence in the saharan sand viper, cerastes vipera. herpetologica 32: 332-336. jayne, b. c., bennett, a. f. (1989): the effect of tail morphology on locomotor performance of snakes: a comparison of experimental and correlative methods. j. exp. zool. 252: 126-133. king, r. b. (1989): sexual dimorphism in snake tail length: sexual selection, natural selection, or morphological constraint? biol. j. linn. soc. 38: 133-154. klauber, l. m. (1943): tail-length differences in snakes with notes on sexual dimorphism and the coefficient of divergence. bull. zool. soc. san. diego. 18: 1-60. luiselli, l. (1996): individual success in mating balls of the grass snake, natrix natrix: size is important. j. zool. 239: 731-740. puky, m., schád, p., szövényi, g. (2005): magyarország herpetológiai atlasza / herpetological atlas of hungary. varangy akciócsoport egyesület, budapest. sheehy, c. m. iii, albert, j. s., lillywhite, h. b. (2016): the evolution of tail length in snakes associated with different gravitational environments. funct. ecol. 30: 244-254. shine, r. (1978): sexual size dimorphism and male combat in snakes. oecologia 33: 269-278. shine, r. (1994): sexual size dimorphism in snakes revisited. copeia 1994: 326-346. shine, r., olsson, m. m., moore, i. t., lemaster, m. p., mason, r. t. (1999): why do male snakes have longer tails than females? proc. roy. soc. b 266: 2147-2151. shine, r., shetty, s. (2001): the influence of natural selection and sexual selection on the tails of sea-snakes (laticauda colubrina). biol. j. linnean soc. 74: 121129. sivan, j., hadad, s., tesler, i., rosenstrauch, a., degen, a.a., kam, m. (2020): relative tail length correlates with body condition in male but not in female crowned leafnose snakes (lytorhynchus diadema). sci. rep. 10: 4130. tóth, t. (2002): data on the north hungarian records of the large whip snake coluber caspius. herpetozoa 14: 163-167. acta herpetologica vol. 16, n. 2 december 2021 firenze university press a new species of the genus noblella (amphibia: strabomantidae) from ecuador, with new information for noblella worleyae carolina reyes-puig1,2,3,4,*, juan m. guayasamin 2,5 claudia koch6, david brito-zapata1, matthijs hollanders7, melissa costales8, diego f. cisneros-heredia1,2,3 so close so different: what makes the difference? dario ottonello1,7,*, stefania d’angelo2, fabrizio oneto3,6, stefano malavasi1, marco alberto luca zuffi4, filippo spadola5 hematological values of wild caiman latirostris (daudin, 1802) in the atlantic rainforest in pernambuco, brazil luciana c. rameh-de-albuquerque1, alexandre p. zanotti1, denisson s. souza1, george t. diniz2, paulo b. mascarenhas-junior3,4,5,*, ednilza m. santos³, jozelia m. s. correia3 bone histology of broad-snouted caiman caiman latirostris (crocodylia: alligatoridae) as tool for morphophysiological inferences in crocodylia paulo braga mascarenhas-junior1,2,3,6, luis antonio bochetti bassetti4, juliana manso sayão5,6 is the northern spectacled salamander salamandrina perspicillata aposematic? a preliminary test with clay models giacomo barbieri, andrea costa, sebastiano salvidio* sexual size dimorphism in the tail length of the caspian whip snakes, dolichophis caspius (serpentes, colubridae), in south-western hungary györgy dudás1, krisztián frank2* semi-automated photo-identification of bahamian racers (cubophis vudii vudii) sebastian hoefer1,*, andreu rotger2, sophie mills1, nathan j. robinson1,3 distribuzione di coronella girondica (daudin, 1803) in romagna (reptilia: squamata: colubridae) christian pastorelli1, paolo laghi2, luigi melloni3 1via cerchia di s. egidio, 2205, i-47023 cesena (fc), italy. e-mail: pastorellic@libero.it 2museo civico di ecologia e centro visitatori «mirco bravaccini», via alla rocca 21, i-47014 meldola (fc), italy. corresponding author. e-mail: spelerpes@aliceposta.it 3via madonna 16/b, i-48010 bagnara di romagna (ra), italy abstract. the authors report a brief overview of the present knowledges about the distribution of coronella girondica in romagna geographical region, as well as seven unpublished records of occurrence of the species. riassunto. gli autori riportano un breve quadro delle conoscenze attuali sulla distribuzione di coronella girondica nella romagna geografica e segnalano il ritrovamento di nuovi reperti. keywords. coronella girondica, distribution, amphibians, reptiles, romagna geographical region. introduzione il colubro di ricciòli (coronella girondica) è una specie a distribuzione mediterraneo-occidentale, diffusa in africa nord-occidentale ed europa sud-occidentale; in europa è presente la sola sottospecie nominale. in italia la specie risulta più comune lungo il versante tirrenico che su quello adriatico ma il quadro distributivo nel nostro paese è tuttora lacunoso (cfr. lanza, 1993; razzetti e bonini, 2006). nell’ambito di ricerche volte ad incrementare le conoscenze sulla distribuzione dell’ofidiofauna in romagna sono stati rilevati sette nuovi reperti relativi a questo colubride, che vengono qui discussi insieme a una sintesi delle attuali conoscenze sulla sua distribuzione nell’area di studio. materiali e metodi l’area di studio coincide con la romagna delimitata dai suoi confini naturali, così come definiti da zangheri (1966). si tratta di un territorio esteso su una superficie di circa 6.400 km2 che comprende le attuali province di forlì-cesena, rimini e la repubblica di s. marino nella loro totaacta herpetologica 2(2): 121-217, 2007 issn 1827-9643 (online) © 2007 firenze university press 122 c. pastorelli, p. laghi and l. melloni lità, nonché parte delle province di arezzo, ferrara (marginalmente), firenze, bologna, pesarourbino e gran parte della provincia di ravenna. i dati riportati nel presente contributo derivano dall’analisi della letteratura specifica, dalla consultazione della banca dati erpetologica dell’emiliaromagna (cfr. mazzotti and stagni, 1996; mazzotti et al., 1999), depositata presso il museo civico di storia naturale di ferrara (in seguito abbreviata in «bder» per semplicità) e da ricerche sul campo effettuate dagli scriventi. i sopralluoghi, a cadenza irregolare, sono stati condotti in diversi ambienti romagnoli, dal livello del mare al piano montano. i rilievi coprono il periodo compreso tra il 1998 e la primavera del 2006. gli animali sono stati ricercati allo scoperto e all’interno di potenziali rifugi (lastre di arenaria o altre rocce, tronchi d’albero a terra, ecc.), durante il periodo di vita attiva della specie. nonostante i costumi elusivi dell’ofidio rendano difficoltosa l’osservazione di esemplari in attività, solo una minor parte delle nostre osservazioni deriva dalla raccolta di animali investiti dagli autoveicoli sulle strade (cfr. silvano e sindaco, 1998; vanni e nistri, 2006). risultati la tabella 1 riporta le segnalazioni di c. girondica finora note per il territorio della romagna geografica. di seguito si elencano le nuove stazioni di presenza individuate nel corso della ricerca, accompagnate, ove possibile, da brevi descrizioni dell’habitat e delle erpetocenosi rilevate. (i) emilia-romagna, provincia di forlì-cesena, comune di bagno di romagna, località s. paolo, 43°52’28,7’’n-11°57’56,9’’e, 612 m s.l.m. una femmina (lunghezza totale: 50,0 cm, su reperto conservato) investita sulla strada provinciale 26 «passo del carnaio», raccolta il 5.ix.1999, leg. et det. p. laghi e c. pastorelli. la stazione si trova in un impianto di rimboschimento a pinus nigra arn., che si estende tanto a monte quanto a valle della strada, inserito in un’area caratterizzata da un mosaico ambientale con alternanza di calanchi, colture foraggere, querceti mesofili e ornoostrieti (cfr. laghi e pastorelli, 2001). erpetofauna simpatrica: rana italica dubois, 1987, zamenis longissimus (laurenti, 1768), hierophis viridiflavus (lacépède, 1789), anguis fragilis linnaeus, 1758, podarcis muralis (laurenti, 1768), lacerta bilineata daudin, 1802, bufo bufo (linnaeus, 1758) (p. laghi e c. pastorelli, dati inediti). (ii) emilia-romagna, provincia di forlì-cesena, comune di galeata, monte delle forche, località frontina, 44°00’27,3’’n-11°53’45,1’’e, 528 m s.l.m. un maschio (lunghezza totale: 66,5 cm, su reperto conservato) catturato il 30.v.2004, leg. et det. p. laghi e c. pastorelli. documentazione fotografica presso gli autori. l’ambiente circostante la stazione è caratterizzato da un rimboschimento artificiale a pinus nigra su versanti argillosi scoscesi, nella fascia degli orno-ostrieti e querceti xerofili a dominanza di quercus pubescens willd. erpetofauna simpatrica: podarcis muralis, lacerta bilineata (p. laghi e c. pastorelli, dati inediti). (iii) emilia-romagna, provincia di ravenna, comune di brisighella, parco regionale della vena del gesso romagnola, località monte mauro, strada per rio ferrato, 470 m s.l.m., versante sud. un esemplare giovane (lunghezza totale: circa 20 cm) investito, raccolto il 9.iv.2006, leg. et det. l. melloni. l’animale aveva da poco ingollato un maschio di podarcis muralis, fuoriuscito in parte dal corpo del serpente in seguito allo schiacciamento. l’esemplare è stato rinvenuto nel versante sud del parco regionale, sulla strada inghiaiata per rio ferrato, in zona aperta, assolata e calda, caratterizzata da gariga gipsofila con elementi flori123distribuzione di coronella girondica in romagna ta be lla 1 . s eg na la zi on i di c . g ir on di ca p er l a r om ag na g eo gr afi ca . i d at i ch e po rt an o la d ic itu ra « b d er » so no s ta ti d es un ti d al la b an ca d at i er pe to lo gi ca de ll’ em ili ar om ag na – m us eo c iv ic o di s to ri a n at ur al e di f er ra ra [ cf r. m az zo tt i e st ag ni ( 19 96 ) e m az zo tt i et a l. (1 99 9) ]. m z u f = e se m pl ar e co ns er va to pr es so il m us eo z oo lo gi co d el l’u ni ve rs ità d i f ir en ze . d at a lo ca lit à c om un e pr ov in ci a q uo ta (m s .l. m .) fo nt e/ r ile va to re 18 55 im ol a im ol a b o 47 bd er ( le g. m us . c iv . s c. n at . v er on a) 10 .v i.1 98 4 sp in el lo , p og gi o c ar na io sp in el lo fc 75 0 bd er ( le g. c . c ia ni ) 19 89 c a’ n ov et ta fi re nz uo la fi 45 0 b as si , 1 98 9; c fr . m az zo tt i, 19 89 ii i.1 99 1 po zz i z am pi ro li, z at ta gl ia br is ig he lla r a 30 0 bd er ( le g. l . m el lo ni ) 3. iv .1 99 1 z at ta gl ia br is ig he lla r a 30 0 la nd i e t a l., 1 99 1 7. v i.1 99 3 pr em ilc uo re pr em ilc uo re fc 55 0 sc ar av el li, 1 99 4 10 .iv .1 99 4 se gu no c iv ite lla d i r om ag na fc 45 0 bd er ( le g. s . b as si ) v i.1 99 4 v ol tr e, s eg un o c iv ite lla d i r om ag na fc 27 0 bd er ( le g. s . b as si ) 7. v ii .1 99 4 pr ed ap pi o b as sa pr ed ap pi o fc 15 0 bd er ( le g. m . m ila nd ri ) v ii i.1 99 5 is ol a s. s ofi a fc 37 5 bd er ( le g. g . t ed al di ) 4. x .1 99 5 is ol a s. s ofi a fc 35 0 bd er ( le g. g . t ed al di ) 3. v ii .1 99 6 po nt e fa nt el la pr em ilc uo re fc 33 0 bd er ( le g. g . t ed al di ) 3. v ii i.1 99 8 c el le fa en za r a ca . 5 0 le g. g . z in za ni , i n: m el lo ni e g at te lli , 2 00 2 29 .v ii i.2 00 2 c ri ve lla ri , c as a sa ss o, b or go r iv ol a r io lo t er m e r a 30 0 m el lo ni e g at te lli , 2 00 2 5. ix .2 00 5 30 0 m a s d i c as a de lla c ro ce p al az zu ol o su l s en io fi 43 0 s. v an ni ( co m . p er s. ) (l eg . s . v an ni , a . n is tr i, s. c ia nf an el li e e. l or i) — tr a il pa ss o de i t re f ag gi e c av al lin o s. g od en zo fi ca . 8 00 te da ld i, 20 03 ; s . v an ni ( co m . p er s. ) — d in to rn i d i f ir en zu ol a fi re nz uo la fi ca . 4 30 n. 2 78 2 m z u f (l eg . b . l an za ) 124 c. pastorelli, p. laghi and l. melloni stici mediterranei, termofili e xerofili tipici delle bancate gessose. la strada è fiancheggiata da terebinto (pistacia terebinthus l.) e roverella (quercus pubescens) allo stato arbustivo. (iv) toscana, provincia di firenze, palazzuolo sul senio, mulattiera per valdonicacorecchio, m 380 s.l.m.; un esemplare adulto osservato il 25.iv.1998, ai bordi della scarpata su suolo marnoso-arenaceo, con scarsa vegetazione erbacea; obs. et det. l. melloni. (v) toscana, provincia di firenze, palazzuolo sul senio, loc. san ilario, 730 m s.l.m.; un esemplare adulto osservato il 12.v.1999, su suolo marnoso-arenaceo, in ambiente assolato, con radi arbusti di ginestra comune (spartium junceum l.) e roverella; obs. et det. l. melloni. (vi) emilia-romagna, provincia di bologna, casalfiumanese, loc. gesso, 400 m s.l.m., un esemplare adulto osservato il 15.v.2002; obs. et det. umberto fusini (com. pers.). (vii) emilia-romagna, provincia di ravenna, brisighella, loc. monte rontana e monticino, tre adulti investiti osservati negli anni ’90 (sandro bassi, com. pers.; obs. ivano fabbri, det. i. fabbri e s. bassi). discussione c. girondica è un serpente termoxerofilo che frequenta zone a clima mediterraneo o atlantico, prevalentemente fino a 400 m di quota, ove predilige le aree pietrose ad elevata insolazione e dimostra talvolta una certa antropofilia (cfr. razzetti e bonini, 2006). si tratta di un serpente a costumi crepuscolari e notturni, di indole mitissima e molto lento nei movimenti (lanza, 1987). in emilia-romagna la specie, più frequente nei territori orientali, è diffusa nella fascia collinare e submontana, principalmente tra 200 e 400 m, fino a 880 (1086) m s.l.m. e predilige i querceti xerofili e gli orno-ostrieti (cfr. mazzotti et al., 1999; melloni e gattelli, 2002). la sua presenza entro i confini naturali della romagna è stata accertata per le province di bologna, firenze, forlì-cesena e ravenna (tabella 1 e fig. 1), principalmente tra 200 e 500 m di quota (fig. 2). non siamo a conoscenza di dati che documentino la presenza della specie a nord della via emilia e ad est del fiume savio (forlì-cesena), nei territori geograficamente romagnoli delle province di arezzo, pesaro-urbino e rimini (cfr. mazzotti et al., 1999; poggiani e dionisi, 2002; razzetti e bonini, 2006; vanni e nistri, 2006). in romagna sembra prediligere la zona di media e alta collina degli orno-ostrieti e querceti xerofili a dominanza di quercus pubescens willd. e gli arbusteti assolati della bassa collina con elementi floristici mediterranei, termofili e xerofili. alla luce delle considerazioni di cui sopra riteniamo che esistano sul territorio in esame numerosi habitat idonei ad ospitare la specie, la cui distribuzione potrebbe pertanto essere tuttora sottostimata. ringraziamenti si ringraziano per aver fornito dati e/o suggerimenti: umberto fusini di pianoro, stefano mazzotti del museo civico di storia naturale di ferrara, stefano vanni del museo zoologico «la specola» dell’università di firenze, sandro bassi e ivano fabbri di faenza e maurizio sirotti di forlì. 125distribuzione di coronella girondica in romagna fig. 2. distribuzione altitudinale di c. girondica in romagna. fig. 1. distribuzione di c. girondica nella romagna geografica (la base cartografica è tratta da zangheri, 1961, modificata). ■ = dati finora noti; • = nuovi dati risultanti dalla presente ricerca. 126 c. pastorelli, p. laghi and l. melloni bibliografia bassi, s. (1989): un ritrovamento insolito per l’emilia-romagna. natura e montagna 36: 36. laghi, p., pastorelli, c. (2001): le orchidaceae presenti in un rimboschimento a pinus nigra arn., sito nell’appennino romagnolo. jour. eur. orch. 33: 477-482. landi, e., landi, l., melloni, l. (1991): seconda segnalazione di colubro di riccioli (coronella girondica) in romagna (reptilia). naturalia faventina, boll. mus. civ. sc. nat. faenza 1: 45-47. lanza, b. (1987): tutti i serpenti italiani. silva 2: 48-69. lanza, b. (1993): amphibia (generi 355-367), reptilia (generi 368-399). in: checklist delle specie della fauna italiana, fasc. 110: vertebrata, p. 39-44, codice specie: 393.002.0 (p. 44). gandolfi, g., frugis, s., eds, calderini, bologna. mazzotti, s. (1989): nuovi dati e mappe di distribuzione dell’ofidiofauna della romagna. suppl. ric. biol. selvaggina 16: 229-232. mazzotti, s., caramori, g., barbieri, c. (1999): atlante degli anfibi e dei rettili dell’emilia-romagna. quad. staz. ecol. civ. mus. st. nat. ferrara 12: 1-121. mazzotti, s., stagni, g. (1996): gli anfibi e i rettili dell’emilia-romagna (amphibia, reptilia). studi trentini di scienze naturali – acta biologica 71 (1994): 93-95. melloni, l., gattelli, r. (2002): segnalazione faunistica n. 54. coronella girondica (daudin, 1803) (reptilia, squamata, colubridae). quad. studi nat. romagna 17: 122. poggiani, l., dionisi, v., (2002): gli anfibi e i rettili della provincia di pesaro-urbino. provincia di pesaro e urbino, assessorato beni ed attività ambientali, i quaderni dell’ambiente, urbania 12: 1-110. razzetti, e., bonini, l. (2006): coronella girondica (daudin, 1803). colubro di riccioli. in: atlante degli anfibi e dei rettili d’italia/atlas of italian amphibians and reptiles, p. 530-535. sindaco, r., doria, c. razzetti, e., bernini, f., eds, edizioni polistampa, firenze. scaravelli, d. (1994): segnalazioni: zoologia. n. 3 – coronella girondica (daudin, 1803) (reptilia, squamata, colubridae). quad. studi nat. romagna 3: 69-70. silvano, f., sindaco, r. (1998): coronella girondica (daudin, 1803). coronella girondina. in: erpetologia del piemonte e della valle d’aosta. atlante degli anfibi e dei rettili, p. 214-215. andreone, f., sindaco, r., eds, monografie xxvi, museo regionale di scienze naturali, torino. tedaldi, g. (2003): anfibi e rettili nel parco. riconoscimento, distribuzione e note di ecologia sull’erpetofauna dell’area protetta. parco nazionale delle foreste casentinesi, monte falterona, campigna, serie natura, i quaderni del parco, san giustino umbro (pg), 86 pp. tedaldi, g., scaravelli, d. (1994): primo contributo alla conoscenza degli anfibi e dei rettili delle foreste casentinesi. parchi 13: 70-73. vanni, s., nistri, a. (2006): atlante degli anfibi e dei rettili della toscana. regione toscana, firenze 379 pp. zangheri, p. (1961): la provincia di forlì nei suoi aspetti naturali. geografia fisica, clima, geologia fauna e flora, paletnologia (preistoria). saggio di illustrazione naturalistica di una provincia italiana esposta in forma divulgativa. camera di commercio, 127distribuzione di coronella girondica in romagna industria e agricoltura-forlì, ristampa anastatica del 1989, tipografia moderna f.lli zauli, castrocaro terme, forlì, 390 pp. zangheri, p. (1966): repertorio sistematico e topografico della flora e fauna vivente e fossile della romagna. tomo i – regno vegetale. mem. mus. civ. st. nat. verona, f.s. 1, verona: 1-480. amphibians of the aurunci mountains (latium, central italy). checklist and conservation guidelines antonio romano1, gianpaolo montinaro2, marco mattoccia1, valerio sbordoni1 1dipartimento di biologia, università di roma “tor vergata”, via della ricerca scientifica, i-00133 roma, italy. corresponding author. e-mail: antonioromano1@libero.it 2via lanfranco maroi 32, i-00142 roma, italy abstract. the aurunci mounts are among the less investigated areas of latium for herpetological researches. in this study we surveyed 72 potential breeding sites of amphibians within the monti aurunci regional park. fifty-eight spawning sites, and nine amphibian species (64.3% out the 14 amphibian species living in latium region) have been found. green toad and european tree frog were recorded for the first time for the aurunci mounts. reproductive activity was recorded for salamandrina perspicillata, triturus carnifex, lissotriton vulgaris, lissotriton italicus, bufo bufo, pseudopidalea viridis, hyla intermedia, rana italica and rana synklepton hispanica. unexpectedly, no amphibian species has been recorded within the monte redentore (psic it6040027), despite this site was included within the natura 2000 network also basing on the presence of triturus carnifex. keywords. monti aurunci regional park, italy, amphibia, distribution, conservation. introduction the southernmost portion of the latium region is among those areas that are poorly studied by regional herpetological researches (cf. bologna et al., 2000), particularly for that concerning amphibians. the volsci chain is the western portion of this area, which comprises three mainly karstic subgroups: the lepini, ausoni and aurunci mounts. in 1997 a part of the aurunci mounts was acknowledged as a regional park, which overlaps the zps it6040043 (zps area surface: ha 19.374) and four psic: it6040026 (monte petrella), it6040027 (monte redentore), it6040028 (forcelle di campello e fraile) and it6050026 (parete del monte fammera). so far the knowledge on the herpetofauna of the aurunci was only based on a preliminary account by bonifazi and carpaneto (1990), and a few other scattered information acta herpetologica 2(1): 17-25, 2007 18 a. romano et alii are found in other papers concerning wider areas (e.g. bruno, 1973; bologna et al., 2000; corsetti, 2006). in 2004 the “monti aurunci” regional park supported financially both an updated checklist and a breeding sites’ census of the amphibians species. the results of these surveys are hereby reported. material and methods since there is not a clear hydrographical or geological boundary between the ausoni and the aurunci mounts, conventionally the aurunci mounts are considered the mounts at the east of a line connecting fondi-lenola-pico-s.giovanni incarico towns (landi vittorj, 1955). furthermore, the aurunci mounts are delimited northward by the liri river, south-eastward by the garigliano river and southward by the planitial zones before to arrive to the tyrrhenian sea. the aurunci mounts are mainly constituted by limestone. altitude varies from hills to the 1,533 m of monte petrella. main peaks include monte sant’angelo (1,404 m), monte ruazzo (1,314 m), monte revole (1,285 m) and the redentore (1,252 m). the minimum distance from the tyrrhenian sea is about 3 km. due to karstic phenomena, freshwater ecosystems are limited to some minor springs (ephemeral or perennial) and to vernal ponds (sometimes residual pools in stream beds), while running waters, rivers or streams, are almost absent. actually some piedmont perennial springs and rivers run only at low altitudes. other still freshwater ecosystems are artificial stony wells, a very common aquatic typology in this area. on the whole we surveyed 550 km2. field research was carried out mainly from spring to autumn 2003 and it was focused within the park boundaries (almost the third part of the total surveyed area) and more specifically within the psic areas. field researches included (i) the inspection of the sites reported in literature to check both species presence and their breeding activity, (ii) cartographic recognition of further potential aquatic habitats suitable for amphibian populations and the inspection of these sites, (iii) the collection of information from local peoples (mainly from shepherds). since several sites were very close to each other, two or more aquatic habitats less than 50 m apart, and inhabited by the same species, have been considered as a single breeding site. sites were georeferenced and assigned to six different freshwater typologies: springs, drinking places for livestock grazing, ponds, stony wells, lakes or marshes and streams or creeks. sites were also distinguished between breeding sites and presence site (i.e. where spawning activity was not recorded). since variance among altitudes of anurans and urodeles breeding sites was not homogeneous (data not shown), then the non parametric mann-whitney u-test was applied to estimate if any significant difference was detectable between altitudinal preferences showed by anura and urodela. statistical analyses was performed using statistica® ver. 5.0/w statistica package (statsoft inc., usa). finally the sørensen’s coefficient of similarity (hayek, 1994) was carried out among species and sites, using only amphibian breeding sites to detect the affinity among species in their reproductive habitats. amphibian scientific names are here reported following the systematic revision suggested by frost et al. (2006). results ninety-six records of amphibians were collected, and breeding activity was recorded in 58 sites including 98 single spots of aquatic habitats (see material and methods), that is 19amphibians of the aurunci mountains about 80% out of the surveyed potential spawning sites (n = 72). two presence sites were also recorded (one site for green toad and one site for common toad). nine amphibian species were recorded within the aurunci mounts: northern spectacled salamander, salamandrina perspicillata (savi, 1821); italian crested newt, triturus carnifex (laurenti, 1768); smooth newt, lissotriton vulgaris (linnaeus, 1758); italian newt, lissotriton italicus (peracca, 1898); common toad, bufo bufo (linnaeus, 1758); green toad, pseudopidalea viridis (laurenti, 1768); italian tree frog, hyla intermedia (boulenger, 1882); italian stream frog, rana italica (dubois, 1987) and rana synklepton hispanica (including berger’s green frog, rana bergeri [gunther, 1986] and its hybridogenetic hybrid rana kl. hispanica [bonaparte, 1839]). their distribution in the study area is shown in fig. 1 and fig. 2. compared to published data (table 1), the italian newt and the northern spectacled salamander show the highest increments (1600% and 1100% respectively) while the toads show the lowest increments (80% and 100% for common toad and green toad respectively). the mean altitude of records (fig. 3) was 557 ± 218 m (mean ± sd) for urodeles and 314 ± 253 m for anurans. this difference in the altitudinal distribution was highly significant between the two amphibian orders (mann whitney u test = 472, p < 0.0001, n = 65 and n = 31 for urodela and anura respectively; see also fig. 3). sørensen’s coefficients of habitat similarity among amphibian populations of aurunci mts and number of breeding sites for each species are shown in table 1. discussion our surveys increased knowledge on the amphibians’ distribution in the study area (table 1). we surveyed all sites previously reported by bonifazi and carpaneto (1990) and no species loss was recorded in these sites. moreover spawning activity was recorded also in some sites where no amphibian presence were reported by these authors (e.g.: s. maria romana springs, igm 160 iii s.o.). the check list is improved by two new records and thus nine amphibian species have been found in the aurunci mounts, that is 64.3% of the species of latium. the green toad on the aurunci mounts is here reported for the first time (near itri town) since it was previously recorded only in the nearby coastal zones (bonifazi and carpaneto, 1990; bologna, 2000a). however breeding site has been recorded only in the coastal zone. the second new record is hyla intermedia since it was previously reported only in the lepini mts (corsetti, 1994) and in the ausoni mts (bonifazi and carpaneto, 1990; venchi, 2000a). however both green toad and the italian tree frog have not yet been recorded into the monti aurunci regional park. among anurans more records were collected from piedmont areas surrounding the park rather than within zps area (fig. 1), conversely the records of urodeles (fig. 2) were more frequent within the zps area and, therefore, at the medium and high altitudes (fig. 3). the urodeles preferences for highest altitudes it is a datum that agrees to the one reported for wider italian areas (anonymous, 2006). rana synkl. hispanica was the most widespread anuran with the greatest habitat typology differentiation and the highest number of syntopy (table 1). r. italica was found in its typical syntopy with northern spectacled salamander (table 1; cf. corsetti and angelini, 2000). 20 a. romano et alii the abundance of urodeles on the aurunci mts is chiefly due to the widespread presence of newts in the stony wells. indeed these artificial lentic water bodies show the characters of the typical breeding sites for the three newt species, that is large capacity and perennial hydroperiod. more specifically the italian crested newt and the smooth newt are almost exclusive of these artificial aquatic habitats while l. italicus spawns also in vernal ponds and drinking watering places for livestock grazing because this newt is a euriecious species adaptable to very different climatic and hydrobiological conditions (scillitani et al., 2004). the similarity of habitat preferences shown by t. carnifex and l. vulgaris can be observed in table 1 where the sørensen’s index value between these two species is the highest in comparison to other species pairs. this result confirms a trend already observed in other italian regions (e.g. barbieri and cavagnini, 1999; gentilli and scali, 1999). finally, it’s worth mentioning that the breeding site “fontana canale” (1276 m a.s.l.), near the psic monte petrella, is the highest spawning site for l. italicus in the latium region (cf. bologna, 2000b; corsetti et al., 2005; corsetti, 2006) and it is also the fig. 1. distribution of anurans in the aurunci mounts (latium). monti aurunci regional park boundaries (grey line) and psic areas (white areas: 1i= iit6040028; 2i=iit6040027; 3i=iit6040026; 4i=iit6050026) are also showed. crosses = pseudopidalea viridis; triangles = bufo bufo; stars = rana italica; squares = rana synkl. hispanica. rhombuses = hyla intermedia. sites where two or more species are syntopics are indicated by a circle including the symbols of the species. codes of utm maps are also reported. 21amphibians of the aurunci mountains highest breeding site recorded for amphibians on the whole volsci chain so far (cf. corsetti, 1994, 2006). syntopy among t. carnifex, l. italicus and l. vulgaris was previously known only in the site pornito, aurunci mounts (lanza, 1983; bonifazi and carpaneto, 1990), in a site near campobasso, molise region (lanza, 1977) and in a few sites on the ausoni mounts (corsetti, 1999). we recorded the syntopy among these newts in other three sites on the aurunci mounts. two of these sites (piana di s. onofrio and piana dei pozzi) are within the park. s. perspicillata has been found in 12 sites (7 springs, 4 drinking place for livestock, 1 residual pond). some sites typically suitable for salamandrina on the volsci chain (i.e. springs or drinking places for livestock animals) are often occupied exclusively by the italian newt in the aurunci mts. syntopy between northern spectacled salamander and this newt was reported sporadically (e.g. corsetti, 1999). however an aquatic site partitioning between these two species, due probably to food larval competition, cannot be excluded and could be revealed by further studies. fig. 2. distribution of urodeles in the aurunci mounts (latium). monti aurunci regional park boundaries (grey line) and psic areas (white areas: 1i= iit6040028; 2i=iit6040027; 3i=iit6040026; 4i=iit6050026) are also showed. rhombuses = triturus carnifex; squares = lissotriton italicus; triangles = lissotriton vulgaris; stars = salamandrina perspicillata. sites where two or more species are syntopics are indicated by a circle including the symbols of the species. codes of utm maps are also reported. 22 a. romano et alii finally, it is worth mentioning that two other species reported in the neighbourhood have not been recorded in the aurunci mts, namely bombina pachypus (bonaparte, 1838) and rana dalmatina (bonaparte, 1840). in the volsci chain, the first specie is relegated to the northernmost portion of the lepini mts (angelini et al., 2004) and its occurrence on the ausoni and aurunci mts. in few suitable habitats as vernal and residual ponds cannot be ruled out. on the contrary, the absence of r. dalmatina seems more realistic because the surveyed area shows no suitable habitats for this species, namely hygrophilous woods prevalently in planitial zones (venchi, 2000b). conservation notes the psic it6040027 (monte redentore) has been established also because t. carnifex (a species listed in appendix ii of the bern convention and included in dpr n. 357/97: habitat directive, annex ii and iv) was previously reported as resident in this area by c. bagnoli (cf. bonifazi and carpaneto, 1990). it is the only psic of aurunci mts. that includes an amphibian. although a potential suitable aquatic habitat can be found (i.e. a stony tank near the s. michele’s sanctuary) no amphibian appears to live in this area. we argue that this record probably refers to an adjacent locality (pornito) where some stony fig. 3. altitudinal ranges of amphibian species in the aurunci mounts (latium). black bars = urodela; grey bars = anura. codes of species as reported in table 1. number of sites of each species is reported above the bar. mean altitude for each species is indicated by an horizontal segments within the bar. 23amphibians of the aurunci mountains wells are syntopic breeding sites for t. carnifex, l. vulgaris (the second is included in appendix iii of the bern convention) and l. italicus (included in appendix ii of the bern convention and in annex iv of habitat directive). therefore we support the proposal of extending the perimeter of the psic it6040027 to include the locality pornito and, thus, the newt breeding sites above mentioned. on the aurunci mts, amphibians typically breed in stony wells and drinking watering places because these artificial water bodies are the most common aquatic habitats available. a few of these sites are in a state of disrepair with fill-in phenomena and permeability loss because traditional rearing activities (i.e. grazing) are strongly reduced. supporting the traditional methods of rearing appears, therefore, preserving the integrity of these reproductive sites, the best strategy for an appropriate conservation management of the amphibian populations in the aurunci mounts. acknowledgments this research was supported by a grant from the monti aurunci regional park which provided also logistic support. thanks to giorgio bidittu (director of the monti aurunci regional park) who provided useful supports in the research. bruno cari contributed to field research. we are particularly grateful to antonio aniballe (maranola), michele aniballe (maranola) and belmonte palazzo (esperia) who provided relevant information concerning springs and other aquatic sites which are not reported in the i.g.m. maps. stefano de felici kindly provided cartographic supports. thanks to roberto sacchi and marco a.l. zuffi for suggestions improving this manuscript. table 1. sørensen’s coefficients between amphibian populations on the aurunci mounts (latium) and numbers of breeding sites for each species. sp = salamandrina perspicillata; tc = triturus carnifex; lv = lissotriton vulgaris; li = lissotriton italicus; bb = bufo bufo; pv = pseudopidalea viridis; hi = hyla intermedia; ri = rana italica; rsh = rana synklepton hispanica. number of breeding sites for each species is reported in square brackets. number of sites already known in literature is reported in round brackets. sp [12] (1) tc [23] (6) lv [13] (4) li [17] (1) bb [7] (5) pv [1] (1) hi [2] (0) ri [7] (1) tc 0 lv 0 0.686 li 0 0.350 0.200 bb 0.1 0.133 0.190 0 pv 0 0 0 0 0 hi 0 0.083 0 0 0.200 0 ri 0.421 0 0 0.090 0 0 0 rsh [12] (2) 0 0.294 0.400 0.150 0.182 0 0 0.105 24 a. romano et alii references angelini, c., cari., b., mattoccia, m., romano, a. (2004): distribuzione di bombina variegata pachypus (bonaparte, 1838) sui monti lepini (lazio) (amphibia: anura). atti soc. it. sci. nat. museo civ. st. nat. milano 145: 321-328. anonymous (2006): altitudinal distribution. in: atlante degli anfibi e dei rettili d’italia / atlas of italian amphibians and reptiles, p. 142-147. sindaco, r., doria, g. razzetti, e., bernini, f. (eds). societas herpetologica italica, ed. polistampa, firenze. barbieri., f., cavagnini., f. (1999): distribuzione e preferenze ambientali degli anfibi nell’appennino pavese orientale. in: atti ii congresso nazionale s.h.i., p. 97-110. giannotti, f.s., di giovanni, m.v. (eds). rivista di idrobiologia 38 (1/2/3). bologna, m.a. (2000a): bufo viridis (laurenti, 1768). in: anfibi e rettili del lazio, p. 52-53. bologna, m.a., capula, m., carpaneto, g.m. (eds). fratelli palombi editori, roma. bologna, m.a. (2000b): triturus italicus (peracca, 1898). in: anfibi e rettili del lazio, p. 44-45. bologna, m.a., capula, m., carpaneto, g.m. (eds). fratelli palombi editori, roma. bologna, m.a., capula, m., carpaneto, g.m. (eds) (2000): anfibi e rettili del lazio. fratelli palombi editori, roma: 160 p. bonifazi, a., carpaneto, g.m. (1990): indagine preliminare sugli anfibi e sui rettili dei monti ausoni-aurunci (lazio meridionale). centro reg. doc. beni cult. amb. lazio, assessorato cultura reg. lazio, roma: 47 p. bruno, s. (1973): anfibi d’italia: caudata. studi sulla fauna erpetologia italiana – xvii. natura, soc. it., sc. nat., mus. civ. st. nat. acq. civ., milano 64: 209-450. corsetti, l. (1994): anfibi e rettili dei monti lepini. quad. mus. st. nat. patrica (fr) 5: 192 p. corsetti, l. (1999): caratteristiche ambientali dei siti riproduttivi triturus italicus nel lazio (italia centrale). in: atti ii congresso nazionale s.h.i., praia a mare (cs), p. 449455. giannotti, f.s., di giovanni, m.v. (eds). riv. idrobiol. 38 (1/2/3). corsetti, l. (2006): distribuzione e preferenze ambientali degli anfibi urodeli nel lazio meridionale (italia centrale). in: atti v congresso nazionale societas herpetologica italica, calci (pisa), p. 7-18. zuffi, m.a.l. (ed). firenze university press. corsetti, l., angelini, c. (2000): salamandrina terdigitata (lacépède, 1788). in: anfibi e rettili del lazio, p. 38-39. bologna, m.a., capula, m., carpaneto, g.m. (eds). fratelli palombi editori, roma. corsetti, l., ragno, r., romano, a. (2005): triturus italicus (peracca, 1898) in the lepini mountains: the new north western limit of the range. herpetozoa 18: 87-88. frost, d.r., grant, t., faivovich, j., bain, r.h., haas, a., haddad, c.f.b., de sá, r.o., channing, a., wilkinson, m., donnellan, s.c., raxworthy, c.j., campbell, j.a., blotto, b.l., moler, p., drewes, r.c., nussbaum, r.a., lynch, j.d., green, d.m., wheeler, w.c. (2006): the amphibian tree of life. bull. am. mus. nat. hist. 297: 370 p. gentilli, a., scali, s. (1999): analisi della diversità erpetologica in pianura padana. in: atti ii congresso nazionale s.h.i., praia a mare (cs), p. 113-122. giannotti, f.s., di giovanni, m.v. (eds). riv. idrobiol. 38 (1/2/3). hayek, l.a.c. (1994): analysis of amphibian biodiversity data. in: measuring and monitoring biological diversity, standard methods for amphibians, p. 207-269. heyer, 25amphibians of the aurunci mountains r.w., donnelly, m.a., mcdiarmid, r.w., hayek, l.a.c., foster, m.s., eds, smithsonian institution, usa. landi vittorj, c. (1955): appennino centrale. guida dei monti d’italia c.a.i. t.c.i. milano. lanza, b. (1977): simpatry and coexisting in the italian triturus, with notes on the “molge italica molisana” problem (amphibia salamandridae). monit. zool. ital. (n.s.) 11: 113-118. lanza, b. (1983): triturus italicus (peracca). tritone italiano. in: anfibi, rettili (amphibia, reptilia), p. 75-76. collana del progetto finalizzato “promozione della qualità dell’ambiente”. guide per il riconoscimento delle specie animali delle acque interne italiane, 27, cnr, aq/1/205, roma. scillitani, g., scalera, r. carafa, m., tripepi, s. (2004): conservation and biology of triturus italicus in italy (amphibia, salamandridae). in: the conservation status of threatened amphibian and reptile species of italian fauna, p. 45-54. bologna, m.a., la posta, s. (eds). ital. j. zool., 71 (suppl. 1). venchi, a. (2000a): hyla intermedia (boulenger, 1882). in: anfibi e rettili del lazio, p. 54-55. bologna, m.a., capula, m., carpaneto, g.m. (eds), fratelli palombi editori, roma. venchi, a. (2000b): rana dalmatina (bonaparte, 1840). in: anfibi e rettili del lazio, p. 58-59. bologna, m.a., capula, m., carpaneto, g.m. (eds), fratelli palombi editori, roma. acta herpetologica 2006 1 records of «salamandrina perspicillata» (savi, 1821) in the colli albani (latium, central italy), with some ecological notes (urodela, salamandridae) records of salamandrina perspicillata (savi, 1821) in the colli albani (latium, central italy), with some ecological notes (urodela, salamandridae) claudio angelini 1, bruno cari 2, and carlo utzeri 1 1 università la sapienza, dip.to di biologia animale e dell’uomo, viale dell’università 32, 00185 roma, italy; e-mail: (ca) oppela@tin.it, (cu) carlo.utzeri@uniroma1.it 2 piazza pagnoncelli 27, 00049 velletri (roma), italy abstract. the distribution of the northern spectacled salamander in the colli albani (= albani hills), together with some ecological aspects at eight sites are reported. in four sites, oviposition took place between february and april, but in the other four it probably started at least in the first half of december. eggs were deposited into the water of either temporary or perennial spring ponds or inside flooded man-made tuff tunnels. in some sites, which do not undergo summer drought, some larvae surpassed the summer and even the following winter. for one site, length and weight of 52 ovipositing females were recorded and larval development was monitored. a clear relation between larval body size and limb development did not appear. keywords. salamandrina perspicillata, urodela, chorology, phaenology, oviposition, cave-breeding, larval development, larval overwintering introduction the northern spectacled salamander, salamandrina perspicillata (savi, 1821), is an urodele endemic to italy, where it mainly occurs along the tyrrhenian side, between the sea level and over 1500 m a.s.l. in latium region, this species is widely distributed (corsetti & angelini, 2000). although this species was reported from the colli albani by bonaparte (1832-1841), it was rediscovered only recently in this area (angelini & cari, 2004). in this note we report details about the breeding sites of this species and outline some ecological aspects, namely as oviposition period and larval development. acta herpetologica 1: 53-60, 2006 54 c. angelini et alii materials and methods the salamandrina sites herewith concerned are placed within the castelli romani regional park boundaries and its surroundings, which covers most of the colli albani (= albani hills), province of rome. the original phytocenosis of this area is represented by mixed deciduous and beech forests, but at present these are widely replaced by chestnut coppice (bassani, 1980). from june 2001 to june 2003, we searched for the northern spectacled salamander in all available water sites, which were of the following types: lakes, brooks, permanent ponds, spring ponds, ephemeral small ponds, troughs and flooded tuff tunnels. tunnels were excavated, mostly at the beginning of the last century, to obtain sheltered water-bodies in spring spots to facilitate water storage and utilization. all these have muddy bottoms, are several metres long and about 1.2 m wide and 1.2-1.8 m high. searches were carried out at least once a month, but twice or more times a month during the oviposition season. we recorded the eventual presence of eggs, adult and larval individuals at each site. of two populations (palazzolo and belle facce dell’ariano) we photographed the salamander’s ventral pattern, which is unique to each individual and remains invariable all throughout its life (vanni et al., 1991, 1997). the salamanders’ total length (tl) and snout-vent length (svl) were also recorded by means of a ruler, and their body mass was recorded by a 0.05 g division pesola balance. in the northern spectacled salamander, no field marks are available to distinguish the sexes. however all the individuals we sampled were probably females because they either were captured while submerged [according to vanni (1980), lanza (1983), delfino et al. (1984), zuffi (1999), only females submerge] or were actually ovipositing or, following subsequent captures within the same oviposition period, showed a markedly deflated abdomen, which suggested that they had probably laid. from may, 2002, to april, 2003, at the site belle facce dell’ariano, once a month we measured total length and recorded developmental stages of limbs in larval samples. we released both the adults and the larvae at their sites after completing measurements and/or recordings. results we found salamandrina in ten of the 70 water bodies inspected (table 1). in table 1 we have grouped in a single site (pa) three water bodies which are 3-15 m far from each other and are probably inhabited by a single population. the closest and farthest sites are placed as far as 160 (cc-lp) and 10500 m (pa – vi) from each other. all these breeding sites are placed in the remainders of the mixed deciduous forest, except mf, which is placed in a garigue. table 2 shows the measurements of females in pa and bf. at bf, in both 2002 and 2003, larger females oviposited earlier in the season than smaller ones, although the correlation between first oviposition date and individual size (svl) is significant for only the 2003 sample (n = 50, rspearman = –0.41; p < 0.01). at bf the overall larval length ranged between 12-31 mm (average ± se = 17.9 ± 0.2 mm, n = 438). at this site, the recording of developmental stages relative to overall larval length resulted as in table 3. all larvae hatched with non-fingered fore limbs, and some did not develop fingers until 14 mm; in some, balancers were reabsorbed at 13 mm. rudimental hind limbs, which were recorded between 12 and 16 mm, were absent in some 13-mm larvae. three-13 mm larvae (recorded in july and november, 2002, and in january, 2003, respectively) showed fully developed, four fingered hind limbs. larvae with both 55s.perspicillata in the colli albani ta bl e 1. b re ed in g si te s of s al am an dr in a pe rs pi ci lla ta in t he c ol li a lb an i. in t he te m po ra ry s ite s, t he s um m er d ro ug ht la st ed r ou gh ly f ro m ju ly to o ct ob er . o vi po si ti on d at es a re t he e ar lie st a nd la te st r ec or de d, i rr es pe ct iv e to y ea rs [ in b ra ck et s, d at es e st im at ed r ou gh ly o n th e ba si s of t he d ev el op m en ta l s ta ge o f la rv ae [c f. te xt ] an d ti m e re qu ir ed fo r eg g de ve lo pm en t (c or se tt i, 19 99 a) ]. si te co de si te n am e an d co m m on al tit ud e (m a .s .l. ) ty pe w at er p er io d su rf ac e (m 2 ) m ax . d ep th (m ) ov ip os iti on d at es la rv al r ec or di ng d at es m ud dy b ot to m sp ri ng -p on d + pe rm an en t 1. 3 0. 07 pa pa la zz ol o, a lb an o 50 0 m ud dy b ot to m sp ri ng -p on ds in tu nn el + te m po ra ry 0. 02 0. 05 1 fe b – 20 a pr la te a pr – la te a ug tu nn el pe rm an en t >1 9 0. 6 a d a cq ua d el la d on ze lla , v el le tr i 72 0 tu nn el pe rm an en t >1 8 0. 4 26 f eb – 2 m ay la te a pr il w in te r b f b el le f ac ce d el l’a ri an o, r oc ca di p ap a 81 0 tu nn el pe rm an en t 15 0. 6 23 f eb – 2 m ay la te a pr il w in te r c c c ol le d ei c or si , n em i 57 0 m ud dy b ot to m sp ri ng -p on d te m po ra ry 2 0. 1 16 m ar – 2 5 a pr la te a pr il ju l m f m on te d ei f er ra ri , v el le tr i 66 0 ro ck y bo tt om sp ri ng -p on d te m po ra ry 1. 5 0. 1 (m id n ov ) 27 d ec lp le p oz za , n em i 55 0 m ud dy b ot to m sp ri ng -p on d te m po ra ry 1. 5 0. 5 (m id d ec ) 1 fe b m p m on te p es ch io , v el le tr i 80 0 ro ck y bo tt om sp ri ng -p on d te m po ra ry 2 0. 1 (m id d ec ) 23 ja n v i v al le d el l’i nf er no , l ar ia no 44 0 tu nn el te m po ra ry 9. 6 0. 3 (m id d ec ) 20 ja n 56 c. angelini et alii fore and hind limbs fully developed (four fingered) were on average 18 ± 0.2 mm long (i = 13-31; n = 352). starting from august, all larvae, irrespective to size (minimum tl = 14 mm), showed fully developed limbs. fig. 1 shows the growth pattern of the bf larval population, from may, 2002, to april, 2003. in the may sample it is evident the occurrence of two different body-sized larval cohorts, of which the left one probably represents the current year larvae, while the right one probably represents the overwintered larvae from the preceding oviposition season. in the june sample, the number of the latter appears markedly decreased, probably because many had transformed. in the following months, the number of large larvae from the current oviposition season (2002) progressively increased and probably several emerged. at sites herewith concerned we never recorded recently metamorphosed individuals. table 2. measurements of s. perspicillata at two sites in the colli albani in 2002 and 2003. increase in length is relative to year 2003 vs 2002. all measures are given in mm. site tl svl svl increase svl increase % n 11 11 pa x±se 105.2 ± 2.4 40.5 ± 0.5 range 90 114 38 42 n 50 52 14 14 bf x±se 98.5 ± 0.9 38.8 ± 0.3 1.4 ± 0.3 3.8±0.9 range 83 113 33 43 0 4 0 10.8 table 3. measurements of larvae relative to developmental stage. stage tl (mm) average±se n balancers 12 16 14±0.2 31 reabsorbed balancers (≥ 13) 18.2 407 non fingered fore limbs 12 14 12.5±0.2 8 three-fingered fore limbs 13 17 15.3±0.2 50 four-fingered fore limbs 13 > 18±0.1 356 absent hind limbs 12 13 12.5±0.7 2 rudimental hind limbs 12 16 13.7±0.2 28 non fingered hind limbs 15 17 15.4±0.1 20 three-fingered hind limbs 16 17 16.7±0.2 6 four-fingered hind limbs 13 > 18±0.2 352 57s.perspicillata in the colli albani may april march december novemberjuly august februaryoctoberjune januaryseptember n=42, x=14.6ll n=29, x=25.9rr n=56, x=15.2ll n=7, x=26.8rr n=38, x=17.4 n=44, x=17.2 n=29, x=16.6 n=3, x=21.3 n=9, x=21.4 n=62, x=17.9 n=39, x=17.0 n=14, x=18.7n=51, x=17.7 n=14, x=19.5 fig. 1. larval growth at belle facce dell’ariano (samples from may, 2002, to april, 2003). larvae are divided in class sizes (tl) of 1 mm (x axis). in the y axis, values are shown as percent of the monthly sample. for the may and june samples, n and mean values are reported for the two cohorts separately. the very small sample of april is reported in graph for convenience. 58 c. angelini et alii discussion by this note we record eight sites for salamandrina perspicillata in the colli albani, to which angelini & cari (2004) make undetailed reference. bonaparte (1832-1841) reported this species from the same area as “intorno il lago di albano” (around albano lake). to our knowledge, the closest site to albano lake is palazzolo; cc and lp are closer to nemi lake and ad, bf, mf, mp and vi are placed farther than 6.7 kilometres eastward of this lake. therefore the species does occur in the southern zone of the colli albani. still or weakly flowing waters characterise the breeding sites of the norhtern spectacled salamander in the colli albani. in this area, this species is almost exclusive of the relatively undisturbed habitat of the mixed deciduous forest (angelini & cari, 2004; angelini et al., 2004). the chestnut coppice, which represents an altered, man-managed habitat, characterised by periodical wood cutting, transit of heavy vehicles, creation of lumber stock areas and other human activities, could likely bring excessive disturbance to the norhtern spectacled salamander’s potential shelters and/or to breeding-sites. it is of interest the use of the man-made tuff tunnels for oviposition sites [so far, only razzetti et al. (2001) have reported cave-breeding in this species]. surely tunnels do not represent the original habitat of the norhtern spectacled salamander, as do not the troughs built to water livestock, in which this species also lays egg (e. g. corsetti & angelini, 2000). apparently, as concerns to both tunnels and troughs, the man action aimed at transforming the scanty spring water-flows into small, but stable, water bodies, did not create conditions unfavourable to this species. in four sites (pa, ad, bf, cc), oviposition roughly takes place between early february and early may, as is usually reported for the norhtern spectacled salamander (lanza, 1983; zuffi, 1999). but in three (lp, mp, vi), where we recorded larvae as early as 20 january and 1 february (table 1), oviposition should have started at least in the first half of december and in mf at least in the first half of november [corsetti (1999 a) reports for populations from the volsci mountains a period of 34-58 days for eggs to hatch]. so far, autumnal oviposition has been reported only for some populations from the volsci mountain chain (corsetti, 1994 a, b; 1999 a, b; corsetti & angelini, 2000; angelini et al., 2001). larval overwintering, already reported by barbieri & tiso (1993) and angelini et al. (2001), was recorded in both ad and bf. fig. 1 shows that between july and december most larvae were relatively small, even though in this period all had completed their morphological development. it is possible that in the summer months a considerable slowing of growth in body length occurs in (late) larvae, probably caused by high water temperature together with food shortage and low oxygen water content. it will be of interest to check if larvae which do not emerge in late summer undergo emergence in autumn, or in the next winter or spring. according to our sampling, a clear relation does not exist between larval body size and limb development (cf. also antonelli, 1999; angelini, 2000). indeed, at bf, all larvae had in-part-developed fore limbs at hatching, but the development of hind limb rudiments, as well as of three and then four fingers in both fore and hind limbs, apparently occurred at disparate body lengths. such a wide variability contrasts lanza’s (1983) report of successive developmental stages occurring in larvae of regularly increasing size. 59s.perspicillata in the colli albani acknowledgements this research was supported by the castelli romani regional park administration. references angelini ,c. (2000): aspetti della biologia riproduttiva di alcune popolazioni di salamandrina terdigitata (lacépède, 1788) (amphibia urodela) dei monti lepini (lazio, italia centrale). unpublished degree thesis. università di roma “la sapienza”. angelini, c., antonelli, d., utzeri, c. (2001): aspetti della fenologia riproduttiva di salamandrina terdigitata (lacépède, 1788) in italia centrale. pianura 13 (atti 3o congr. societas herpetologica italica): 105-108. angelini, c., cari, b. (2004): the amphibians of the colli albani (latium, central italy): breeding sites and some ecological notes. atti soc .it. sci .nat. 145 (ii): 337-342. angelini, c., della bella, v., cari, b., utzeri, c. (2004): caratterizzazione di siti riproduttivi di anfibi nei colli albani (lazio). riassunti 5° congr. naz. societas herpetologica italica (calci (pisa), 29 settembre-3 ottobre 2004): 22. antonelli, d. (1999): aspetti di ecologia e demografia in una popolazione di salamandrina terdigitata (lacépède, 1788) (amphibia urodela). unpublished degree thesis. università di roma “la sapienza”. barbieri, f., tiso, e. (1993): nuove stazioni di salamandrina terdigitata nell’appennino settentrionale (versante padano). ric. biol. selvaggina (suppl.) 21: 383-385. bassani, p. (1980): la vegetazione. in: un parco naturale regionale nei castelli romani, pp. 33-58. aa.vv., coopsit, velletri. bonaparte, c.l. (1832-1841): iconografia della fauna italica per le quattro classi degli animali vertebrati. 2: amfibi. salviucci, roma. corsetti, l. (1994 a): osservazioni sulla ecologia e biologia riproduttiva di salamandrina terdigitata nei monti lepini (lazio) (amphibia salamandridae). quad. mus.stor. nat.patrica 4: 111-130. corsetti, l. (1994 b): anfibi e rettili dei monti lepini. quad. mus.stor. nat.patrica 5: 1190. corsetti, l. (1999 a): reproductive activity and embryo growth of the spectacled salamander salamandrina terdigitata (lacépède, 1788) in southern latium (central italy). br. herpet. soc. bull. 67: 13-20. corsetti, l. (1999b): habitat e attività riproduttiva in salamandrina terdigitata (lacépède, 1788) nel lazio meridionale (italia centrale). riv. idrobiol. 38: 381-387. corsetti, l., angelini, c. (2000): salamandrina terdigitata (lacépède, 1788). in: anfibi e rettili del lazio, pp. 38-39. bologna m.a., capula m., carpaneto g.m., eds, fratelli palombi, roma. delfino, g., brizzi, r., calloni, c. (1984): lateral line organs in salamandrina terdigitata (lacépède, 1788) (amphibia: urodela). z. mikrosk.-anat. forsch. 98 (2): 161-183. lanza, b. (1983): anfibi, rettili (amphibia, reptilia). consiglio nazionale delle ricerche aq/1/205, verona. 60 c. angelini et alii razzetti, e., bonini, l., barbieri, f. (2001): riproduzione in grotta di salamandra salamandra e salamandrina terdigitata negli appennini settentrionali. pianura 13 (atti 3o congr. societas herpetologica italica): 181-184. vanni, s. (1980): note sulla salamandrina dagli occhiali [salamandrina terdigitata (lacépède, 1788)] in toscana (amphibia salamandridae). atti soc. tosc. sci. nat., mem., ser. b, 87: 135-159. vanni, s., nistri, a., zagaglioni, s. (1991): use of the ventral pattern marking technique in the study of salamandrina terdigitata biology (amphibia, salamandridae). abstr. 6th ordinary general meeting of the societas europaea herpetologica, p. 93. vanni, s., nistri, a., zagaglioni, s. (1997): use of the “pattern mapping” technique to study the biology of salamandrina terdigitata (amphibia caudata salamandridae). atti soc. tosc. sci. nat., mem., ser. b, 103 [1996]: 111-112. zuffi, m.a.l. (1999): salamandrina terdigitata (lacépède, 1788) brillensalamander. in: handbuch der reptilien und amphibien europas. bd. 4/i: schwanzlurche (urodela) i, pp. 229-246. grossenbacher, k., thiesmeier, b., eds, aula-verlag, wiesbaden. acta herpetologica 15(2): 119-123, 2020 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/a_h-10016 does chronic exposure to ammonium during the pre-metamorphic stages promote hindlimb abnormality in anuran metamorphs? a comparison between natural-habitat and agrosystem frogs sonia zambrano-fernández1, francisco javier zamora-camacho2,3,*, pedro aragón2,4 1 universidad de málaga, avda. de cervantes 2, 29016, málaga, spain 2 museo nacional de ciencias naturales, (mncn-csic), c/ josé gutiérrez abascal 2, 28006, madrid, spain 3 department of biological sciences, dartmouth college, 78 college street, 03755 hanover, new hampshire, usa 4universidad complutense de madrid, c/josé antonio novais 2, 2804, madrid, spain *corresponding author. e-mail: zamcam@ugr.es submitted on: 2019, 19th december; revised on: 2020, 6th march; accepted on: 2020, 26th may editor: raoul manenti abstract. despite their detrimental effects on locomotion, prevalence of hindlimb abnormalities is increasing in anuran populations worldwide. among others, agrochemical pollution during the larval stage is a potential cause. however, populations exposed to such a strong selective pressure could evolve resistance. in this work, we examine the potential effects of chronic exposure to ammonium during the pre-metamorphic stages of pelophylax perezi frogs on metamorph hindlimb abnormality prevalence, as compared with unpolluted-water-reared conspecifics. we conducted the experiment on tadpoles either from natural-habitat or from agrosystem parents. we detected no effect of chronic exposure to ammonium on hindlimb abnormality prevalence in frogs from either habitat, which suggests that the lack of effect detected is not related to resistance evolved in agrosystem frogs. keywords. amphibian, anuran, anomaly, malformation, pelophylax perezi. functioning appendages are fundamental for wholeorganism performance of most anurans (johansson et al., 2010; zamora-camacho et al., 2019). hind limb morphology is directly responsible for locomotion of metamorph (zamora-camacho and aragón, 2019a) and adult anurans (zamora-camacho, 2018), either from terrestrial (gomes et al., 2009) or aquatic environments (herrel et al., 2012), regardless of their locomotion mode (enriquez-urzelai et al., 2015). therefore, hindlimb abnormality in this group is likely eradicated by natural selection due to its severe negative effects on locomotion (zamora-camacho and aragón, 2019b). consistently, prevalence of anuran appendage abnormality appears generally below 5% (ouellet, 2000; mester et al., 2015). nonetheless, limb abnormality rates are increasing in anurans worldwide (johnson and bowerman, 2010; laurentino et al., 2016). these include diverse malformations, such as lacking and extra limbs and digits, as well as fused or misshaped limbs (johnson and bowerman, 2010; reeves et al., 2013). limb abnormalities are particularly common in metamorphs (kiesecker, 2002; piha et al., 2006), seemingly because reduced locomotor performance (zamora-camacho and aragón, 2019b) might cause their death shortly after metamorphosis. besides a genetic origin (droin and fischberg, 1980), hindlimb abnormalities in anurans have been related to biotic interactions such as predatory pressure (johnson and bowerman, 2010) or parasite infections (roberts and dickinson, 2012), as well as abiotic factors such as ultra120 sonia zambrano-fernández, francisco javier zamora-camacho, pedro aragón violet-b radiation (pahkala et al., 2001). however, human perturbance frequently provokes these malformations (blaustein and johnson, 2003), which are more common next to roads (reeves et al., 2008) or in agrosystems (ouellet et al., 1997; spolyarich et al., 2011). agrochemicals such as fungicides (bernabo et al., 2016), pesticides (jayawardena et al., 2010), and fertilizers (xu and oldham, 1997) increase limb abnormality prevalence in anurans. the aetiology of these malformations is often multiple (meteyer et al., 2000): trematode infections (haas et al., 2018) and predator attacks (reeves et al., 2010) boost the effects of agrochemicals. albeit, greater selective pressures could also drive the appearance of resistance to the environmental stressors (miaud and merilä, 2001), which could eventually reduce the prevalence of abnormalities in agrosystem populations. metamorph morphology is often related to tadpole growth history (tejedo et al., 2000). in this work, we compare the prevalence of hindlimb abnormality in metamorphs of pelophylax perezi (lópez seoane, 1885) frogs resulting from tadpoles chronically exposed to ammonium contamination with unpolluted-water-reared conspecifics. ammonium is among the most common compounds derived from agricultural fertilizers, with several negative effects on amphibian populations (ortiz et al., 2004). we checked any possible resistance to contamination evolved in agrosystems by applying this treatment to tadpoles from natural habitats and from agrosystems. we expected higher prevalence of hindlimb abnormalities in metamorphs from the ammonium treatment. however, if agrosystem populations have evolved resistance, this effect would be greater in natural-habitat tadpoles. pelophylax perezi is a ranid that occurs naturally throughout the iberian peninsula and southern france (egea-serrano, 2014), in a wide variety of habitats, but always in or not far from waterbodies, either pristine or polluted (egea-serrano, 2014). indeed, it often inhabits human-altered habitats, such as urban or agricultural environments (egea-serrano, 2014). fieldwork was conducted in pristine pinares de cartaya pinus pinea grove and surrounding agrosystems (sw spain, 37º20’ n, 7º09’ w). agrosystems are about 6 km away from pine grove, and consist of a traditional extensive vegetable crop area that has lately transitioned into intensive plantations regularly added fertilizers and at owners’ discretion. in april 2018, 10 adult males and 10 adult females were randomly caught from each habitat. capture was manual, and males were recognized for their greyish forelimb nuptial pads and their vocal sacs in the mouth commissures (egea-serrano, 2014). frogs were pooled separately according to their provenance in two adjacent outdoor semi-natural enclosures with ponds (fig. s1 in supplementary material). ponds were daily checked for the presence of egg masses, which we transferred to the laboratory within 12 hours after they had been laid. in the laboratory, we immediately separated eggs randomly in groups of 15. each group was placed in an aquarium (length×width×height: 38×27×19 cm) with 6 l of untreated spring water. in half of the aquariums, randomly chosen for each egg mass, we added 178.87 mg of 99.7% pure nh4cl, so we obtained a concentration of 10 mg nh4+/l. in a previous study on this species, a concentration of 13.5 mg nh4+/l caused circa 70% mortality rate in a mid-term experiment on larvae of this species from natural habitats (egea-serrano et al., 2009). we chose a concentration slightly lower in order to avoid such mortality rates, while triggering sublethal effects. the other aquaria contained untreated spring water, as a control. thus, we had 15 aquaria with eggs from frogs from each habitat and treatment, totalling 60 aquaria, in a 2 × 2 factorial experimental design. aquaria were kept in shelves in the laboratory until larvae finished metamorphosis. water was completely replaced twice a week, and each time we maintained the treatment and randomly changed the position of each aquaria within the shelves. a window let natural daylight in, permitting adjustment of circadian rhythms. because tadpole diet can affect limb abnormality rates in this species (martínez et al., 1992), all specimens were standardly fed boiled spinach ad libitum. in gosner stage 42, preceding tail resorption (gosner, 1960), tadpoles were transferred to tilted aquaria to allow them to exit the water as metamorphosis ended. some metamorphs presented an abnormality in one of their hindlimbs (fig. 1). abnormal limbs were aberrantly inserted in the pelvis with an approximate angle of 270º with respect to the body axis (fig. 1). moreover, the knee-joints were unable to fold normally in resting position (fig. 1). in all cases, only one hindlimb was abnormal in each individual affected, either the right or the left appendage. we calculated the proportion (number of abnormal metamorphs divided by number of total surviving metamorphs) of abnormal-limbed metamorphs from each aquarium. data met the criteria of homoscedasticity and residual normality (quinn and keough, 2002), so we conducted a two-way anova to test the effects of habitat, treatment, and their interaction on the proportion of abnormal-limbed metamorphs, using the software statistica 8.0. the total numbers and proportions of abnormallimbed metamorphs from each habitat and treatment are in table s1 in supplementary material. the effects of habitat (f1, 56 = 0.026; p = 0.874; fig. 2), treatment (f1, 56 121tadpole exposure to ammonium and froglet hindlimb abnormality = 0.007; p = 0.932: fig. 2), and their interaction (f1, 56 = 0.914; p = 0.343; fig. 2) on the proportion of abnormallimbed metamorphs obtained in each aquarium were non-significant. at the concentration used, chronic exposure to ammonium during the larval stage does not increase the prevalence of hindlimb abnormality in these frogs. however, a subchronic exposure to even lower concentrations of this compound reduces survivorship (egea-serrano et al., 2009) and affects behaviour of p. perezi larvae (egeaserrano et al., 2011). prevalence of limb abnormality in bufo bufo toad metamorphs were higher following an acute exposure to 100 mg/l ammonium nitrate during the larval stage than following a subchronic exposure to 50 and 100 mg/l ammonium nitrate (xu and oldham, 1997). those results could be a consequence of greater mortality of larvae in the subchronic exposure treatment (xu and oldham, 1997), which could mask potential limb abnormalities if future bearers die. chronic exposure to other pollutants, such as mercury in rana sphenocephala frogs (unrine et al., 2004), and nickel, cobalt, or cadmium chlorides in xenopus laevis frogs (plowman et al., 1994), causes malformations in metamorphs. also, subchronic exposure to carbamate and organophosphate pesticides causes malformations in p. perezi (alvarez et al., 1995). juvenile p. perezi from agrosystems are smaller, and show increased limb fluctuant asymmetry, than conspecifics from natural habitats (burghelea et al., 2013). however, we detected no effect of habitat on hindlimb abnormality prevalence on either treatment. aligned with our results, prevalence of limb abnormality was not greater in rana temporaria frogs from agrosystems than from natural habitats (piha et al., 2006). nevertheless, these findings contrast with others that detected increased prevalence of limb abnormality close to agrosystems in several anurans (kiesecker, 2002; guerra and aráoz, 2016). our results do not support the hypothesis of resistance in agrosystem frogs. we obtained an overall prevalence of hindlimb abnormality notably below the 5% detected in other wild amphibian populations (ouellet, 2000; mester et al., 2015). low prevalence in both habitats could be a consequence of the capability of this species to thrive in polluted waters (egea-serrano et al., 2008). acknowledgements fjz-c was partly supported by a juan de la cierva-formación contract by the spanish ministerio de economía, industria y competitividad (mineco). pa was supported by a “ramón y cajal” contract (ryc2011-07670, mineco). this study was in part funded by mineco (project number cgl2014-56416-p). frog capture and management was conducted according to a research permit issued to the authors by the consejería de medio ambiente of the junta de andalucía (reference fig. 2. effects (mean ± 1se) of treatment and habitat on the proportion of abnormal-limbed metamorphs obtained from each aquarium, calculated as the number of abnormal metamorphs divided by the total number of surviving metamorphs. sample sizes indicated represent the number of aquaria in each treatment. fig. 1. pelophylax perezi metamorph affected by the hindlimb abnormality described, with a measuring tape in cm. 122 sonia zambrano-fernández, francisco javier zamora-camacho, pedro aragón sgmn/awg/mgd/mgm gb-270-18) and the bioethics committee of the spanish national research council (reference 720/2018). supplementary material supplementary material associated with this article can be found at < http://www.unipv.it/webshi/appendix > manuscript number 10016. references alvarez, r., honrubia, m.p., 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(2019): the roles of sex and morphology on burrowing depth of iberian spadefoot toads in different biotic and abiotic environments. j. zool. 309: 224-230. acta herpetologica vol. 15, n. 2 december 2020 firenze university press estimating abundance and habitat suitability in a micro-endemic snake: the walser viper gentile francesco ficetola1,2,*, mauro fanelli3, lorenzo garizio3, mattia falaschi1, simone tenan4, samuele ghielmi5, lorenzo laddaga6, michele menegon7,8, massimo delfino3,9. potential effects of climate change on the distribution of invasive bullfrogs lithobates catesbeianus in china li qing peng1, min tang1, jia hong liao1, hai fen qing1, zhen kun zhao1, david a. pike2, wei chen1,* a bibliometric-mapping approach to identifying patterns and trends in amphibian decline research claudio angelini1,*, jon bielby2, corrado costa3 food composition of a breeding population the endemic anatolia newt, neurergus strauchii (steindachner, 1887) (caudata: salamandridae), from bingöl, eastern turkey kerim çiçek1,*, mustafa koyun2, ahmet mermer1, cemal varol tok3 stomach histology of crocodylus siamensis and gavialis gangeticus reveals analogy of archosaur “gizzards”, with implication on crocodylian gastroliths function ryuji takasaki1,2,*, yoshitsugu kobayashi3 does chronic exposure to ammonium during the pre-metamorphic stages promote hindlimb abnormality in anuran metamorphs? a comparison between natural-habitat and agrosystem frogs sonia zambrano-fernández1, francisco javier zamora-camacho2,3,*, pedro aragón2,4 confirming lessona’s brown frogs distribution sketch: rana temporaria is present on turin hills (piedmont, nw italy) davide marino1, angelica crottini2, franco andreone3,* phylogenetic relationships of the italian populations of horseshoe whip snake hemorrhois hippocrepis (serpentes, colubridae) francesco paolo faraone1, raffaella melfi2, matteo riccardo di nicola3, gabriele giacalone4, mario lo valvo5* first karyological analysis of the endemic malagasy phantom gecko matoatoa brevipes (squamata: gekkonidae) marcello mezzasalma1,2,*, fabio m. guarino3, simon p. loader1, gaetano odierna3, jeffrey w. streicher1, natalie cooper1 notes on sexual dimorphism, diet and reproduction of the false coral snake oxyrhopus rhombifer duméril, bibron & duméril, 1854 (dipsadidae: pseudoboini) from coastal plains of subtropical brazil fernando m. quintela1,*, felipe caseiro¹, daniel loebmann¹ acta herpetologica 2(2): 69-77, 2007 issn 1827-9643 (online) © 2007 firenze university press molecular dna variation among triturus vittatus vittatus (urodela) from different breeding sites at the southern limit of its distribution oren pearlson1, 2, 3, gad degani1, 2 1 migal–galilee technology center, p. o. box 831, kiryat shmona 11016, israel. corresponding author. gad degani, e-mail: gad@migal.org.il 2 school of science and technology, tel hai academic college, galilee, israel 3 institute of evolution, faculty of sciences and science education, university of haifa, israel abstract. molecular dna variation among triturus vittatus vittatus (striped newt) from different breeding sites at the southern limit of the species distribution (where environmental conditions are most extreme) was studied by the random amplification of polymorphic dna (rapd) method that has been found to be appropriate for other triturus species. altitudes of the localities ranged between 15-740 m a.s.l. of the 20 primers employed, opa-16 was the only one suitable for t. vittatus, revealing a different band pattern for different populations. genetic similarity was calculated by band sharing, which demonstrated a high similarity among the israeli populations. keywords. genetic polymorphism, xeric habitat, conservation, extreme conditions, triturus vittatus vittatus. introduction the banded newt, triturus vittatus vitattus, is an endangered species in israel (geffen et al., 1987). other species and subspecies of this genus are endangered in other regions of the world. triturus v. vitattus is distributed throughout the western caucasus, turkey, lebanon, syria, israel, iraq and perhaps, jordan (borkin et al., 2003; litvinchuk et al., 2005). the biology and life cycle of triturus vittatus in europe and the mediterranean region have been described by raxworthy (1989) and olgun et al. (1997). until recently, triturus vittatus was thought to have two subspecies: triturus v. vittatus, along the eastern edge of the mediterranean sea from turkey to israel, where it reaches its southern limit, and t. v. ophryticus in the caucasus, east and south of the black sea. based on its trunk vertebrae count, genome size and allozyme data, litvinchuk et al. (2005) suggested that the northern taxon, t. v. ophryticus, is subdivided into two geographic fragments, 70 o. pearlson and g. degani the “western group”, with populations from western anatolian turkey, and the “eastern group”, composed of populations from the remaining region of pontic turkey and the western caucasus. at the southern limit of t. v. vittatus distribution (in israel), environmental conditions are the most extreme. there, limiting factors are likely to be the breeding sites and dryness of the terrestrial habitat. the biology and life cycle of t. v. vittatus in northern israel and the upper galilee have been described by degani and mendelssohn (1983) and degani (1986a, 1996), while a population in central israel has been studied by geffen et al. (1987). triturus v. vittatus inhabit mainly winter pools that typically contain water until the beginning of summer, although occasionally they have water all year-round (degani and kaplan, 1999). like other newts, t. v. vittatus requires water bodies surrounded by an adequate terrestrial habitat to support both life phases. if either habitat is damaged, a population may be unable to survive. zajc and arntzen (2000) utilized fragments of mitochondrial dna to study evolutionary aspects of triturus. their findings support the sister-taxon relationship of t. vulgaris and t. montadoni and the position of t. vittatus within the subgenus, triturus, as a sister taxon of the clade of the large-bodied newts, t. marmoratus and t. cristatus. according to the allozyme analysis of litvinchuk et al. (2005), the genus triturus is not a monophyletic taxon and representatives of the genus neurergus are closely related to t. karelinii. based on these results and reports from earlier studies on mitochondrial dna analyses in newts by other authors, in which it was demonstrated that the genus triturus is paraphyletic, a taxonomic scheme was proposed wherein the genus triturus was divided into four monophyletic genera, triturus s.s., lophinus, mensotriton and ommatotriton. furthermore, the substantial genetic differences that was found between the northern and southern samples of t. vittatus, together with the unique distribution, external morphology and coloration differences of the latter, led them to suggest that the northern populations of t. vittatus be designated a distinct species, t. ophryticus. mikulicek and pialek (2003) used rapd pcr analysis to elucidate the difference between the crested newt species, t. cristatus, t. dobrogicus, and t. carnifex, allowing identification of genetic relatedness among populations of different breeding sites, as an indication of the degree of isolation between the sites, an important aspect for developing management strategies to protect the species. weisrock et al. (2006), in their study, resolved a non-monophyletic history for triturus. they divided triturus species into four main parts: a clade of the t. cristatus species, the t. vulgaris and t. boscai clade, the t. alpestris clade and the t. vittatus clade, whose sister taxon is neurergus. their findings strongly support previous studies, in which the mediterranean island euproctus species, e. montanus (corsica) and e. platycephalus (sardinia), form a strongly supported clade, as well. beebee et al. (1999) used molecular techniques in order to identify three putative newt hybrids of the two parent species, t. vulgaris and t. helveticus. rapd analysis provided straightforward and reliable proof, producing consistent results with specimens from widely separated parts of their geographic ranges. hybrids were identified by amplification of both the mitochondrial cytochrome b and atpase genes, including partial sequencing of the latter. one hybrid had a t. vulgaris mother, while the other two had t. helveticus mothers. much research has focused on the genetic variables of urodela in different geographical regions (e.g. gibbs et al., 1998; steinfartz et al., 2000; lecis and norris, 2004; riberon 71molecular dna variation among triturus vittatus vittatus et al., 2004). however, genetic variation among populations in natural habitats around breeding sites located relatively near each other has received little attention (lecis and norris, 2004). various methods have been employed to study genetic variation in amphibians, including albumins in serum (degani, 1986b), isozymes (veith et al., 1992), rapd pcr (degani et al., 1999; mikulicek and pialek, 2003) and mitochondrial dna sequencing (weisrock et al., 2001). the present study, by means of rapd pcr, examines genetic differences, among t. v. vittatus populations in five different breeding sites in the southern-most region of its distribution, where environmental conditions are most extreme. material and methods study sites nahalit ponds (figs. 1b, 2b): these two ponds are located side by side in cattle grazing land in the upper galilee (longitude 243657, latitude 776401) 665 m a.s.l. the ponds fill up with water fig. 1. the five ponds colonized by newts examined in the study: (a) dovev pond, (b) nahalit pond, (c) matityahu q. pond, (d) amiad water holes, (e) afeka. 72 o. pearlson and g. degani that seeps through chicken farms and cattle pens. the smaller pond holds water from january to may, has an area of ca 50 m2 and a depth in the center of ca 2 m. the other one has an area of ca 1000 m2, a depth in the center of ca 0.8 m and contains water from january to june. both have water plants, such as ranunculus aquatilis, scirpus tuberosus and scirpus tabernae-montanus. adult triturus inhabit the ponds from mid-january until the beginning of april, and larvae were found between mid-march and june, when the pond dries up. dovev pond (figs. 1a, 2a): the pond is a nature reserve site in the upper galilee (longitude 239158, latitude 772801) at an altitude of 740 m a.s.l. the pond is located among apple orchards with water from december to july. it has an area of ca 300 m2, a depth in the center of ca 1.5 m and water plants, such as ranunculus aquatilis, scirpus tuberosus and scirpus tabernae-montanus. adult triturus can be observed from mid-december until the beginning of april, while larvae can be discerned from mid-march until july, when the pond dries up. amiad water hole (figs. 1d, 2d): the water hole is man made and is located in the southern part of the upper galilee (longitude 251721, latitude 757994, altitude: 212 m a.s.l.). the pond is located in cattle grazing land and holds water from december to july. the water hole has a radius of 0.7 m, an area of ca 3 m2, a depth in the center of ca 1.5 m and water plants, such as ranunculus aquatilis. adult triturus can be found in the water from mid-december until the beginning of april, while larvae can be detected only after the water has dried up in july. matityahu quarry pond (figs. 1c, 2c): the pond in the matityahu quarry is located among apple orchards, in the upper galilee (longitude 242783, latitude 774855, altitude: 670 m a.s.l.). the pond contains water from january to june. it is ca 200 m2 and ca 2 m deep in the center. the pond has been dug several times in the last few summers, fills up at the beginning of january and contains water until mid-june. no water plants populate the pond. adult triturus were observed from january until the beginning of april, and larvae were found from mid-march until june, when the pond dries up. afeka pond (figs. 1e, 2e): the pond is located in the central coastal plain, (longitude 182364, latitude 670453) with a mean altitude of 15 m a.s.l. the pond is situated alongside a section of a fig. 2. the five breeding sites of triturus v. vittatus examined in this study: (a) dovev pond, (b) nahalit pond, (c) matityahu q. pond, (d) amiad water holes, (e) afeka. 73molecular dna variation among triturus vittatus vittatus drainage canal. rainwater, draining from adjacent open fields and from the road, usually fills the pool, beginning in december and lasting until mid-march. it is ca 30 m2 in area and ca 0.5 m deep in the center. the vegetation in the pond consists mostly of eleocharis palustris and scirpus marifimus. adult triturus were found in the water from december until the end of february, and larvae were observed from mid-march until mid-april, when the water dries up. dna analyses tissue samples were obtained from triturus larvae and adults. as previously described by degani and mendelssohn (1983), adults migrate to these ponds during the mating season (at the beginning of winter) to breed. there is a large adult population at all five locations (degani and kaplan, 1999). specimens (four individuals per each site) were sampled randomly from the entire area of the water body by hand net. total genomic dna was extracted from tail-clipped tissues of adults or from whole tails of larvae. extraction was carried out by the qiaamp dna mini kit (qiagen, germany), after proteinase k digestion. rapd pcr amplifications were carried out with 10 mer oligonucleotide primers (mikulicek and pialek, 2003) in a 50 µl solution, containing 10 mm tris-hcl, 50 mm kcl, 2.5 mm mgcl2, 1 mm of each dntp, 0.5 µm of primer, 10-500 ng gemonic dna and 2.5 units of taq dna polymerase (promega, usa). rapd fragments were amplified by 45 cycles, including denaturation for 30 s at 94 °c (initial denaturation for 2 min at 94 °c), primer annealing for 1 min at 35 °c and extension for 2 min at 72 °c. the rapd amplification ended with an extension at 72 °c for 7 min. to assess the similarity between individuals, band sharing (bs) of the rapd pcr products was calculated as: bs = 2 × (nab) / (na + nb), where bs = level of band sharing between individuals a and b, nab = number of bands shared by individuals, a and b, na = total number of bands of individual a and nb = total number of bands for individual b (jeffreys and morton, 1987; wetton et al., 1987). the pcr patterns were compared only between samples that had been run on a single gel. differences in bs were examined by the d-test for differences between proportions (parker, 1976). results only one of the 20 primers used in the rapd pcr was revealed to be suitable for the study of dna variation among t. v. vittatus (opa-16; 5’-agccagcgaa, mikulicek and pialek, 2003). other primers either failed to amplify any fragment or only amplified a few fragments, making them inappropriate for a study on variations. in pcrs with the primer opa-16, the number of bands differed among populations from different sites. there were 12-13 identical bands within populations from afeka pond and the amiad waterhole, 13-14 within the populations of dovev pond and matityahu quarry and 16-17 in the population at nahalit pond. twelve bands were common to all of the newts. the newts in nahalit, amiad and dovev had one band each that was not observed in any other population. only three bands, in nahalit, amiad and dovev populations, were not detected in newts from the other ponds. high genetic similarity, calculated by band sharing, was discovered among newts in the ponds at the high altitudes, which were found to have less similarity with the newts from the ponds at lower altitudes (table 1, figs 3, 4). 74 o. pearlson and g. degani table 1. band sharing of newts from studied sites (see methods for details). band sharing (%) nahalit pond amiad waterhole dovev pond afeka pond matityahu quarry 90 96 92 88 nahalit pond — 80 83 80 amiad waterhole — — 88 92 dovev pond — — — 88 fig. 4. band sharing variations among populations (mean + se). fig. 3. rapd pcr results with the opa-16 primer. 75molecular dna variation among triturus vittatus vittatus discussion a low genetic variability was discovered among the different triturus v. vittatus populations from various altitudes and located at short geographical distances, one from the other, in israel. moreover, when these israeli newt populations, which belong to the subspecies triturus v. vittatus, were compared by sequence analysis to the other subspecies t. v. ophryticus (found in european turkey), high genetic variations were observed (veith et al., 2004). mikulicek and pialek (2003) used twenty 10-mer oligonucleotide primers on three crested newt species (triturus cristatus superspecies) and found 19 of them to yield 2 to 22 bands per primer. in the present study, a very high similarity was detected and only one primer out of 20 (mikulicek and pialek, 2003) yielded a reasonable number of bands to show a clear variation among the populations. we believe that the explanation for this result is the very short geographical distances among the populations, together with the fact that all of the habitats were of the unpredictable type, causing the newts to adapt to the wide ecological variations. it has been previously shown that the ecological and biological conditions, suitable for t. v. vitattus, vary, and in unpredictable habitats, such as the rain pools in the southern limit of its distribution, conditions generally correlate with altitude (degani and mendelssohn, 1983; degani, 1986a, 1996; geffen et al., 1987; degani and kaplan, 1999). the effects of habitat and geographic location on genetic variation have been studied in other urodela (steinfartz et al., 2000; lecis and norris, 2004; riberon et al., 2004). much research has been conducted on a range of habitats in widespread locations. however, very few studies have examined genetic variation among different populations at breeding sites, covering a relatively small area, as in the present study. triturus vittatus vittatus is a urodelan, which can be found in different freshwater bodies, such as unpredictable rain pools and springs. water parameters, such as temperature and duration of availability in the winter ponds studied, exert a greater influence on the larvae than does the type of water body. in such ponds, the altitude of the pond influenced the species existing there (degani and kaplan, 1999). the results of the present study are very similar to those of degani et al. (1999), who studied the genetic variation of salamanders from different habitats, using rapd pcr. they discovered a very low genetic variation between two populations from semi-arid habitats (band sharing was 94%), and in contrast, a high genetic variation between populations from semi-arid and humid habitats (85-86% band sharing). their results agree with the present study, in which ecological conditions affected genetic variation. the foremost methods for studying genetic variation are putative proteins (degani, 1986b) and enzymes (veith et al., 1992), differences in dna fingerprints (rapd pcr; degani et al., 1999; mikulicek and pialek, 2003) and sequences of conserved and variable genes (kocher et al., 1989; macgregor et al., 1990; caccone et al., 1997). the methods used in this study support previous findings, demonstrating that rapd pcr is a suitable method for examining small genetic variations among populations, whereas the utilization of cytochrome b sequencing may be applied when there is a greater taxonomic variation, such as between species (steinfartz et al., 2000; lecis and norris, 2004; riberon et al., 2004). although the genetic variation among the triturus vittatus vittatus populations is relatively low, one of the populations, that of nahlit pond, which is the most polluted pond, as mentioned previously, varied more from the other populations. in addition, the 76 o. pearlson and g. degani population of afeka pond, which is located at a relatively low altitude and at a greater distance from the other breeding sites, differed more genetically, as compared to the other populations. further studies are necessary to test various hypotheses that may explain the pattern discerned in the results. references beebee, t.j.c., rowe, g., arntzen, j.w. 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(2000): evolutionary relationships among european newts (genus triturus) as inferred from two mtdna fragments. pflüg. arch. eur. j. physiol. 439: 21-22. the phenology of a rare salamander (salamandra infraimmaculata) in a population breeding under unpredictable ambient conditions: a 25 year study michael r. warburg deparment of biology, technion-israel institute of technology, haifa 32000, israel. e-mail: warburg@ tx.technion.ac.il abstract. this is a long-term study (1974-1999) on the phenology of the rare, xeric-inhabiting salamander salamandra infraimmaculata in a small isolated population during the breeding season near the breeding ponds on mt. carmel. this is a fringe area of the genus’ south-easternmost palaearctic distribution. salamanders were captured during the 25 year long study. the first years up to the 1980s the total number of salamanders increased but during the last years there seems to have been a decline. although this could be a phase in normal population cyclic oscillations nevertheless when compared with long-term data on a european salamandra it does not seem so. the interpretation of the species’ status is dependent on numbers of salamanders captured as well as on the duration of the study. these subjects are reviewed and discussed in this paper. keywords. salamandra, amphibia, urodela, phenology, long-term study, unpredictable climate, population decline. introduction the adult population of salamandra infraimmaculata was studied at the breeding sites on mt carmel during 25 years 1974-1998 (except 1990). this relict population inhabits a fringe habitat on mt carmel at the south-eastern edge of the genus’ range. in israel this salamander is a rare and protected species and lives in only three disjunct northern areas of the country. the main population is located in the mountains of the western and central galilee (degani and warburg, 1978). in addition, two other smaller populations inhabit two disjunct areas: one about 50 km to the north-east (degani and mendelssohn, 1980), and the second about the same distance to the southwest, in the northern portion of mt carmel (warburg, 1986a, 1986b, 1992, 1994). this last population is estimated to be much smaller than that in the galilee mountains (warburg, 1992, 1997). there is no acta herpetologica 2(2): 147-157, 2007 issn 1827-9643 (online) © 2007 firenze university press 148 m.r. warburg apparent reason why salamanders had not extended their distribution into other mountain ranges in the mediterranean region of central israel such as samaria and judea: they are never found there, although palaearctic species of animals (and plants) have succeeded in colonizing this area (yom-tov and tchernov, 1988). the fact that the salamanders are not found south of mt carmel is of interest, and provided the main stimulus for studying this isolated population over such a long period. since it is a fringe population inhabiting an area where conditions are suboptimal to the animals for part of the time, it exhibits special adaptations that are not found in other species in this genus (sharon et al., 1996; warburg, 1997). adult s. infraimmaculata are terrestrial, and only females return to water when mature at the age of 3-4 years, and only for the time needed to lay larvae (see warburg et al., 1978-1979). males usually remain outside the ponds. the objectives of this study were to survey the phenology of a single salamander population over a long period (25 years) in order to assess its status as an endangered species. since it is extremely difficult to make a definite statement about causes that might endanger a species, long-term studies as this are much better qualified to make any suggestions than a 3-4 year standard length study. among several long-term studies (43 research papers) 29 of which concerning urodele species, are listed in appendix 1. there are several advantages in long-term studies. firstly, an entirely different outlook emerges as the research unfolds, and secondly, changes in populations can be followed over a long period enabling a different aspect in evaluating oscillations in population cycles of a long-living urodele. materials and methods the study was carried out south to haifa, at the top of mt carmel located towards its western slopes, about 20 km south of the urban area. the area contains four rock-pools which are important breeding sites for the salamanders in this region. during the 25 years of the study, annual rainfall ranged between 397-1161 mm, being on average 690 mm (table 1); 460 mm (66.6%) fell during the breeding season, in september-january, while 230 mm during the rest of the rainy season. rain during the remainder of winter (end of february) is not of great significance to the breeding pattern of the adults, but for the survival of the larvae, since the larvae need at least 6-8 weeks to metamorphose and they might not be able to complete metamorphosis once the pond water warms up (see cohen et al., 2005, 2006). adult salamanders were captured on stormy winter nights throughout the entire breeding season, i.e. about 10-12 weeks from mid-october to the beginning of january. they were collected, sexed by cloacal examination (warburg et al., 1978, 1979), weighed (mettler balance at + 0.1 g accuracy), measured (to the nearest mm) and photographed, for individual recognition on the basis of dorsal patterns (see warburg, 2006 for details). they were then released to their original collecting site either during the same or on the following night. the number of salamanders active each year during the breeding season at the study site was rather small (< 30) compared to most of other salamander species as well as to numbers of salamandra spp in europe (feldmann, 1987; klewen, 1985). in order to investigate if the numbers of salamanders varied over years or accordingly with yearly rainfall, we used t-test and regression analyses. in this last case, we performed linear, polynomial, and exponential models and we used that with the higher value of r2. 149the phenology of salamandra infraimmaculata results a total of 363 different individual salamanders were hand-captured during 310 visits to the breeding ponds over a period of 25 years, when one year (1990) was not studied table 1. average annual rainfall and number of salamanders captured during the breeding season: september to january. year rainfall (av. ann.) (%) total captured no. visits capts/visits 1974 586 72.5 808 10 5 2 1975 383 59.0 649 7 5 1.4 1976 550 63.1 872 3 3 1 1977 600 82.8 724 8 10 0.8 1978 398 74.2 536 7 21 0.33 1979 642 69.5 923 5 13 0.38 1980 439 70.5 623 11 6 1.83 1981 211 47.8 441 17 14 1.21 1982 600 68.8 872 21 23 0.91 1983 317 61.8 513 21 33 0.63 1984 342 56.1 609 19 17 1.12 1985 369 63.0 586 26 17 1.53 1986 622 79.4 783 29 22 1.32 1987 654 68.8 951 16 9 1.78 1988 339 71.1 477 33 13 2.54 1989 444 60.2 737 21 18 1.17 1990 216 43.6 495 (*) (*) (*) 1991 843 72.6 1161 14 9 1.56 1992 533 72.1 739 10 12 0.83 1993 310 51.5 602 10 4 2.5 1994 611 75.3 811 19 26 0.73 1995 412 66.4 620 8 6 1.33 1996 295 47.9 616 18 8 2.25 1997 477 64.0 745 17 8 2.12 1998 303 76.3 397 13 8 1.62 av 460 66.6% 690 1.23 total 363 (*) courtesy of the israel meteorological services beth dagan. in bold: highest and lowest values 150 m.r. warburg (table 1). the average number of visits to the pond in one year (excluding 1990) is 13.4, ranging between 3 and 33 (there was no correlation between the number of visits and the number of salamanders collected: r2 = 0.34 best fit polynomial regression), and salamanders were captured in 160 visits (51.4 %). altogether the mean number of salamanders captured per visit was 1.23 (table 1). most salamanders were captured between 1983 and 1989, and following 1989 there has been a gradual decline (though non significant r2 = 0.34) in the numbers of salamanders captured (table 1). the highest number of salamanders captured in one visit was 32 specimens in 1988. the captures of salamanders were then calculated for 4-year periods (table 2). the reason for this was in order to compare with the extent of the usual research projects which last up to four years (alford and richards, 1999) although some lasted longer. six such periods fit into the 24 years studied (fig. 1a), most of them differing significantly from each other (table 2). during the first four years (1974-1977), 29 salamanders were captured, while the next four years (1978-1981) 58 salamanders followed by 147 salamanders in the next four years (1982-1985). the last four years of study (1994-1998) 95 salamanders were captured (fig. 1a). after grouping, there appears to be a definite and statistically significant (r2 = 0.85 best fit polynomial regression) decline in numbers of salamanders frequenting this breeding site on mt carmel (fig. 1a). no such difference could be seen in the rainfall pattern when this was arranged for 4-year periods (r2 = 0.04 best fit power regression). if the study would have lasted four years (which is longer than a 3-year average funded research project) e.g. from 1974 to 1976 it could be concluded that the population of adult salamanders is small and declining (table 1). a fourth year did show an increase but the population size never reached its 1974 peak until six years later (1980). from that year onwards the population size increased, reaching a maximum of 32 in 1988 (table 1). in the next 4-year period (1978-1981) the population reached a plateau thereafter starting to increase almost continuously till 1986 when it peaked. if we would have studied this population for a period of three years between 1980-1986 we would conclude that the population is increasing. nevertheless, there does not seem to be a statistically significant relationship between the duration of study and the number of salamanders captured (r2 = 0.34) nor in the rainfall pattern over the study period (r2 = 0.04 best fit power regression). there is no indication that there was indeed any change in the rainfall pattern (table 1), in the hydro period of the ponds, the quality of air, water or soil. moreover, there was no significant relationship between rain and the number of salamanders captured (r2 = 0.0038 best fit power regression). table 2. differences between 4-year periods (t-tests with 6 df ) in captures. 1974-77 1978-81 1982-85 1986-89 1991-94 1978-81 0.329 1982-85 0.001 0.011 1986-89 0.003 0.014 0.531 1991-94 0.014 0.014 0.091 0.072 1995-99 0.041 0.328 0.003 0.036 0.512 151the phenology of salamandra infraimmaculata it would not have been possible to reach the conclusion that there are natural population oscillations unrelated to the amount of annual rainfall unless these long-term observations became available in spite of the fact that no statistically significant relationship could be demonstrated between the duration of the research and the number of salamanders captured. discussion the chances of capturing salamanders increase greatly with the duration of the study period since salamanders are known to return to the breeding sites even after long intervals (warburg, 2006). the reasons why both capture and duration are important for such study are: (i) a specimen may be missed by a few minutes consequently it is not certain fig. 1. phenology of (a) s. infraimmaculata arranged in 4-year periods, and (b) s. terrestris over 21 years study and arranged in 4-year periods; data from feldmann (1987) by permission. 152 m.r. warburg that the salamander did not visit; (ii) if the study would have been shorter, there was the risk to miss salamanders that do not visit the pond every year. s. salamandra in particular seems to be a favorite object for these studies (14 studies see appendix 1). perhaps because they are remarkable in their site fidelity both to the breeding ponds as well as to their winter or summer refuges (feldmann, 1987; warburg, 1996, 2006) and because they are such long-lived animals (see warburg, 2007). thus, many salamanders (50% feldmann, 1971, 63.1% in feldmann, 1978, 70% in the present study and 84% in feldmann and klewen, 1981) returned to their winter refuges for at least six years. the most important point in this study is how to interpret the apparent decline in number of salamanders visiting the ponds on mt carmel. although of statistical significance when analyzed for 4-year periods, this decline could also indicate a low point in long-term population oscillation. similar long-term phenological studies on other urodeles show a variety of patterns: thus, a 6-year study on notophthalmus perstriatus (dodd, 1993), a 7-year study on triturus cristatus (kupfer and kneitz, 2000), and a 16-year study on ambystoma tigrinum, all show a decline in numbers. nevertheless, all these studies may have in fact described a phase in normal population oscillations taking place in urodeles. however, long-term data (21 years) on a s. terrestris population in central europe did not reveal a significant change over the years (feldmann’s data, see fig. 1b). so, we are left with one possibility: a definite decline in numbers of s. infraimmaculata. could such a decline endanger s. infraimmaculata population on mt carmel and cause its extinction? in my opinion it is unlikely since this single population studied here is only one of mt carmel’s salamanders metapopulation which consists of a few, isolated, patch-like populations. moreover, since this rather large salamander (up to 30 cm in length with some females weighing well over 100 g) is capable of moving over long distances and withstanding dehydration (warburg, 1997) it can colonize other ponds (degani et al., 2007). in addition, the female salamander produces annually an average brood size of 100 larvae during at least 16 years as mature female totaling 1600 larvae whence it needs only 2-3 to reach maturity in order to sustain the population. given the long life expectance (20 or more years, see warburg, 2007) and the fact that the female can breed every year (in prep.). references alford, r.a., richards, s.j. 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(1988) a. talpoideum 9 pechmann et al. (2001) a. talpoiderum 12 semlitsch et al. (1993) a. talpoideum 16 semlitsch et al. (1996) a. opacum 16 semlitsch et al. (1996) a. opacum 12 pechmann et al. (1991) a. tigrinum 9 pechmann et al. (2001) a. tigrinum 16 semlitsch et al. (1996) a. maculatum 5 husting (1965) a. maculatum 6 blackwell et al. (2004) a. maculatum 12 brodman (2002) a. jeffersonianum 12 brodman (2002) a. californiense 7 trenham et al. (2000) hynobius nebulosus tokyoensis 7 kusano (1982) desmognathus ochrophaeus 7 tilley (1980) d. quadramaculatus 6 dodd and dorazio (2004) plethodon kentucki 7 marvin (2001) p. jordani 6 dodd and dorazio (2004) p. jordani 7 hairston (1983) p. glutinosus 5 hairston (1983) p. ornata 12 pechmann et al. (1991) p. cinereus 14 jaeger (1980) p. shenandoah 14 jaeger (1980) eurycea quadridigitata 16 semlitsch et al. (1996) e. quadridigittata 6 dodd (1992) e. quadridigittata 9 pechmann et al. (2001) triturus marmoratus 6 diaz-paniagua et al. (1996) t. marmoratus 5 martinez-solano et al. (2003) t. cristatus 6 arntzen and teunis (1993) t. cristatus 6 arntzen (2000) t. cristatus 7 kupfer and kneitz (2000) t. cristatus 20 beebee (1997) t. vulgaris 20 beebee (1997) 157the phenology of salamandra infraimmaculata species years source t. vulgaris 11 gressler et al. (1997) t. helveticus 20 beebee (1997) t. alpestris 5 martinez-solano et al. (2003) t. dobrogicus 11 gressler et al. (1997) t. dobrogicus 5 jehle and hödl (1998) salamandra salamandra 5 martinez-solano et al. (2003) s. salamandra 6 rebelo and leclair (2003) s. salamandra 10 kästle (1986) s. salamandra 20 schmidt et al. (2005) s. terrestris 7 feldmann (1971) s. terrestris 13 feldmann (1978) s. terrestris 21 feldmann (1987) s. terrestris 17 feldmann and klewen (1981) s. terrestris 5 klewen (1985) s. terrestris 6 klewen (1986) s. infraimmaculata 10 warburg (1986a) s. infraimmaculata 11 warburg (1986b) s. infraimmaculata 18 warburg (1994) s. infraimmaculata 25 warburg (present study) notophthalmus viridescens 16 semlitsch et al. (1996) n. viridescens 9 pechmann et al. (2001) n. perstriatus 6 dodd (1992, 1993); dodd and cade (1998) taricha rivularis 7 twitty et al. (1967) appendix 1. continued acta herpetologica 17(1): 21-26, 2022 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-12326 the effect of weight and prey species on gut passage time in an endemic gecko quedenfeldtia moerens (chabanaud, 1916) from morocco jalal mouadi1,*, panayiotis pafilis2, abderrafea elbahi3, zahra okba3, hassan elouizgani3, el hassan el mouden4, mohamed aourir1 1 laboratory “biodiversity and ecosystem functioning”, faculty of sciences, ibn zohr university, agadir, morocco 2 section of zoology and marine biology, department of biology, national and kapodistrian university of athens, panepistimioupolis, 15784 athens, greece 3 laboratory of aquatic systems: marine and continental ecosystems, faculty of sciences, university ibn zohr, agadir, morocco 4 laboratory “water, biodiversity and climatic change”, faculty of sciences semlalia, cadi ayyad university, marrakech, morocco *corresponding author. e-mail: mouadimohamedjalal@gmail.com submitted on: 2021, 21st november; revised on: 2022, 5th april; accepted on: 2022, 8th april editor: andrea costa abstract. gut passage time (gpt), a key factor in digestive procedure, is of pivotal importance for digestion. several parameters may affect gpt, such as temperature, length of gastrointestinal tract and body size. here, we examine the influence of prey weight and prey species on gpt in the endemic diurnal gecko quedenfeldtia moerens, from the anti-atlas mountains in central morocco. we used two prey species, house crickets (acheta domesticus, ad) and mealworms (tenebrio molitor, tm). lizards were fed with the larval stage of tm and nymphs of ad. the influence of prey weight and prey species was tested at a constant temperature. we used three weight classes of each prey species to test the influence of prey weight on gpt. our results showed that prey species affected gpt in a distinct way: mealworms induced a longer gut passage time compared to house crickets. moreover, gpt increased with the increasing weight of prey for both prey species. our finding demonstrates that the effect of prey species and prey weight affect digestion and thus should be better clarified in future studies. keywords. gpt, prey weight, acheta domesticus, tenebrio molitor, quedenfeldtia moerens, morocco. introduction given that digestive activity regulates energy flow to animals, effective digestion is a prerequisite for survival (karasov and douglas, 2013). among the many important parameters that shape the digestive repertoire of animals, the time required for food to pass through the gastrointestinal tract from consumption to defecation, known as gut passage time (gpt) stands out (hume, 1989; van damme et al., 1991). gpt shapes digestive efficiency, as increasing the time food remains in the gastrointestinal tract provides more time for effective digestion (van damme et al., 1991; alexander et al., 2001). gpt may be affected by numerous factors in lizards, among which temperature is maybe the most important. indeed, gpt is temperature-dependent and varies from few hours to several days (christian et al., 1984; karasov et al., 1986), decreasing with increasing temperature (du et al., 2000; pafilis et al., 2016, 2007; sanabria et al., 2020). the reptilian digestive system is characterized by high plasticity and reptiles can control the time food remains in the gut (herrel et al., 2008; sagonas et al., 2015), by elongating the gastrointestinal tract and thus increasing gpt (sagonas et al., 2015; vervust et al., 2010; pafilis et al., 2016). furthermore, the existence of specialized digestive microstructures, such as cecal valves, may prolong the time it takes 22 jalal mouadi et alii for food to pass through and boost gpt (herrel et al., 2008; sagonas et al., 2015). furthermore, gpt may also be influenced by age (karameta et al., 2017a) or tail autotomy aftermaths (sagonas et al., 2021; 2017). here we aim to clarify whether prey characteristics have an effect on gut passage time. to this end, we assessed the impact of prey weight and prey species on the gpt of the atlas day gecko. we expected that the increasing size of a given meal (prey weight) would consequently prolong gpt. we also predicted that different prey species would distinctly affect gpt. materials and methods study species the atlas day gecko (quedenfeldtia moerens) (chabanaud, 1916) is a small diurnal lizard, belonging to the moroccan endemic genus quedenfeldtia of the sphaerodactylidae family. it is widely distributed in the atlas mountains, from 10 to 2,700 m above sea level. the study population originates from the anti-atlas mountains (29°51’n, 09°01’w; 1.900 m a. s. l.). during a field survey in february 2020, we captured, by noose, 12 adult males with snout-vent length (svl) between 40 and 48 mm (mean ± sd = 45.43 ± 1.85) and weight ranging from 2 to 3 grams (mean ± sd = 2.76 ± 0.08). prey species and marking technique captured lizards were transferred to the laboratory and housed individually in transparent plastic terraria (11 x 17 x 7 cm3), with ad-libitum access to water. all lizards were maintained in natural photoperiod and acclimated, for two weeks, inside a temperature-controlled room (25 ± 1°c) (sagonas et al., 2021; 2017). prior to the experiment, we fed the lizards with house crickets (acheta domesticus, ad) nymphs and mealworms (tenebrio molitor, tm) larvae to familiarize them with the specific prey items. both insect species originated from an inhouse breeding colony. to test the effect of prey species on gpt, we selected ad nymphs and tm larvae. in order to evaluate the effect of prey weight on gpt, we categorize three weight classes for each prey species (in total six feeding regimes, three weight classes for tm and three for ad). for tm larvae we distinguish three classes, in ascending order of weight (mean ± sd): l1 (0.013 ± 0.0019 g), l2 (0.033 ± 0.0017 g) and l3 (0.064 ± 0.0036 g). for ad nymphs, the respective weight classes were: n1 (0.032 ± 0.0035 g), n2 (0.065 ± 0.0047 g) and n3 (0.1 ± 0.0057 g). gut passage time prior to the experiment trials, food was withheld from lizards for three days, until no feces were found in the terraria (sagonas et al., 2017). then, we marked the prey items before feeding them to lizards. prey items are typically marked with small pieces of plastic (pvc) that serve as indigestible markers (van damme et al., 1991; pafilis et al., 2007). to avoid possible injuries to gecko’s digestive tracts because of their small body size, we marked prey species using soft thread tied around the abdominal-thorax junction of the insect prey (fig. 1). we fed all 12 lizards with the marked prey. terraria were inspected every hour for the appearance of the marker. after the detection of the marker, we withheld food for another three days. after this period, we repeated trials by feeding lizards with other prey species and weight classes. at the beginning of each trial, we noted the lizard code, the prey species and weight class. gut passage time was determined as the time elapsed from the consumption of the marked prey to the defecation of fecal pellets with the marker (van damme et al., 1991). each lizard was tested six times (with feeding regimes l1, l2, l3, n1, n2 and n3) and respective gpts were recorded. using this specific protocol, all lizards are tested identically, thus minimizing noise associated with individual particularities. statistical analysis we constructed mixed-effects models in r (version 4.1.1; r core team 2021) using the package lme4 (v1.115; bates et al., 2015) with prey species (two levels) and prey weight (continuous covariate) as fixed effect factors (including the interaction between them). to avoid pseudo-replication, we included the lizard’s id as a random effect factor. we used quantile-quantile plots and residual plots to check the models’ assumptions, and we assessed the significance of both comparisons using the anova function in the package car (fox and weisberg, 2019) with type ii sums of squares and the chi-square test statistic. results the marker used in the present study was effective and the thread tied around the prey was easily visible in fecal pellets from both prey species (fig. 1f). before comparing the effect of prey species on gpt, feeding regime to lizards were classed on three weight classes each (table 1). the variance explained by the random effect (lizard id) was nearly equal to 0, which means that the major 23gut passage time in an endemic gecko part observed in our dependent variable was linked to the fixed terms in our model. prey weight significantly affected gpt (χ2 = 54.97, p < 0.01). gpt was negatively correlated to prey weight (r = 0.95): the heavier the prey, the more time food remained in the digestive tract. furthermore, prey species did affect significantly gpt (χ2 = 56.43, p < 0.01): gpt was longer for mealworms than for crickets. finally, the interaction between prey species and their weight was significant and positive (χ2 = 4.49, p = 0.034). this finding indicates that feeding on heavier prey had a stronger effect on gpt for mealworms than for crickets (fig. 2). fig. 1. (a to c): successive photos showing atlas day gecko quedenfeldtia moerens eating a marked mealworm larva. (d and e): the technique used to mark prey (here respectively mealworm larva and house cricket nymph); a thread was tied to prey as shown in photos. (f): fecal pellets where the marker (the thread tied around the prey) was clearly visible. table 1. variation on gut passage time, gpt (mean ± se), as a function of the prey species and prey weight classes in quedenfenldtia moerens. (n: nymph, l: larvae. numbers 1 to 3, designed the weight class of each prey species). prey species/ class mean weight (g) weight range (min-max) gpt (hour) crickets/n1 0.03083 0.024 -0.039 48.20 ± 2.99 crickets/n2 0.0656 0.054 0.074 51.69 ± 2.18 crickets/n3 0.1000 0.090 0.100 70.96 ± 1.50 mealworm/l1 0.0130 0.010 -0.016 51.25 ± 4.07 mealworm/l2 0.0320 0.029-0.036 58.16 ± 1.82 mealworm/l3 0.0650 0.059-0.072 79.14 ± 2.94 fig. 2. variation in the gut passage time (gpt) in hour as a function of increased weight of prey consumed by the atlas day gecko quedenfeldtia moerens, (tm) for mealworm as prey and (ad) for house cricket as prey. solid lines represent the regression line for each prey species predicted by linear mixed model. 24 jalal mouadi et alii discussion digestion, a crucial function for nutrient absorption, rules energy acquisition (karasov et al., 2011). there are many parameters affecting the digestive performance in lizards and, as mentioned above, gut passage time (gpt) is one of them (pafilis et al., 2016; sagonas et al., 2017; karameta et al., 2017b). the latter may be affected by many intrinsic and extrinsic factors such as temperature, length of the gastrointestinal tract, body size and age (van damme et al., 1991, du et al., 2000; pafilis et al., 2016; 2007; sanabria et al., 2020). in this study we found that gpt is also influenced by prey weight and prey species. gut passage time is a general indicator of the digestive process that measures the time that food remains in the gastrointestinal tract, a period which is decisive for digestive efficiency (pafilis et al., 2007). interestingly, though the impact of prey species and prey weight is known to influence overall digestion (johnson and lillywhite, 1979; starck and beese, 2001), the (presumable) impact of the aforementioned prey features on gpt has not been investigated. in this study, we selected two species typically used in captive lizard breeding (tenebrio molitor and acheta domestica) that have been analyzed in previous studies, thus allowing a comparative framework (pafilis et al., 2016; sanabria et al., 2020; miller et al., 2013). according to our results, gpt was significantly longer after the consumption of t. molitor than of a. domestica (table 1). this finding could be attributed to the different energy content and chemical composition of the two prey species. indeed, previous studies focusing on the nutritional composition of invertebrate prey found that tm larvae contained more than double metabolizable energy (2056 kcal/kg) than ad nymphs (949 kcal/ kg) (finke, 2002; 2015). additionally, tm larvae have been reported to be richer in fat and proteins (134 g/kg and 187 g/kg, respectively) than ad nymphs (33 g/kg and 154 g/kg, respectively). in contrast, ad nymphs had higher water content than tm larvae (77.1% vs 61.9%). furthermore, lizards fed with mealworms ingested significantly more energy, had significantly higher food conversion efficiencies, higher daily gains in mass, and greater total growth in mass than lizards fed on crickets (rich and talent, 2008). it seems that the nutrient and energy rich tm meals require more time compared to ad to get effectively absorbed. the observed difference in gpt between the two prey species could be explained by an adaptive strategy of the focal gecko to nutrient and energy rich prey. tm larvae contain more energy than ad nymphs, so lizards increase gpt to maximize the gain of this energy. prey weight also had an impact on gpt. higher prey weights resulted in increased gpt in both cases of the two tested prey species. there was a linear correlation between the prey weight classed and gpt, with l1 and n1 (the lighter weight classes) resulting in lower gpts and l3 and n3 (the heavier weight classes) inducing higher gpts (fig. 2). this should come as no surprise. animals need more time to digest larger meals (karasov and del rio, 2007) and thus the increase of prey weight dictates a considerable prolongation of gpt. the extra time provided by the longer gpts offer gastric enzymes more time to act on ingested food and thus absorb more nutrients and energy (alexander et al., 2001). the gpt values found here are comparable with those reported in previous studies. mean gpt for ad varies between 48.2 and 70.96 hours, while the respective values for tm are somewhat higher (51.25-79.14 hours). gpt may vary with the family: in lacertids, gpt receives values between 36-85 hours (zhang and ji, 2004; vervust et al., 2010; sagonas et al., 2015; pafilis et al., 2016), in cordylids between 20-32 hours (mcconnachie and alexander, 2004), in skinks between 45-74 hours (du et al., 2000) and in agamids between 67-86 hours (karameta et al. 2017a). those differences should be attributed to different body sizes and also distinct phylogenetic histories. more studies on sphaerodactylids will enrich the respective literature and give the opportunity for a comparative approach in saurian digestion. temperature greatly affects digestion and gpt represents no exception (van damme et al., 1991; pafilis et al., 2007). gut passage time decreases with increasing temperature (du et al., 2000; pafilis et al., 2007). our experiment took place exclusively in 25°c, hence we cannot assess the impact of temperature on digestion under different feeding regimes (prey species and weight). in a future study we plan to assess this very important aspect of digestion process. to conclude, this study shows that prey species and weight affected gut passage time in q. moerens. more studies on digestive efficiency, including other variables such as temperature and apparent digestive performance would be valuable. moreover, a comparative study between lizards from different populations will shed more light on the ecology of this moroccan endemic diurnal gecko. acknowledgements the authors thank the “département des eaux et forêts (def-morocco)” for the permit (n° 1795 hceflcd/dlcdpn/dprn/cff) to work on the field. all ani25gut passage time in an endemic gecko mal procedures were in accordance with guidelines of the european council directive (eu2010/63). authors would like to thank dr. marco mangiacotti for providing valuable comments in the early draft of this paper. many thanks are also addressed to the associate editor dr. andrea costa and two anonymous reviewers for their generosity in reviewing the manuscript. we thank dr. john gittings for his assistance in the linguistic revision of the text. references alexander, g.j., van der heever, c., lazenby, s.l. 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(2004): the thermal dependence of food assimilation and locomotor performance in southern grass lizards, takydromus sexlineatus (lacertidae). j. therm. biol. 29: 45-43. xi international symposium on the mediterranean lacertid lizards marco mangiacotti1, pietro lo cascio2, claudia corti2, marta biaggini2, miguel angel carretero2, petros lymberakis2 the directional testes asymmetry increases with temperature in seven plateau brown frog (rana kukunoris) populations hai ying li1, man jun shang2, jie guo2, bo jun chen2, peng zhen chen2, tong lei yu1,* influence of tail injury on the development of neotropical elegant treefrog tadpoles ana glaucia da silva martins1,#, raoni rebouças2,3,*,#, isaias santos1, adão henrique rosa domingos1, luís felipe toledo2 the effect of weight and prey species on gut passage time in an endemic gecko quedenfeldtia moerens (chabanaud, 1916) from morocco jalal mouadi1,*, panayiotis pafilis2, abderrafea elbahi3, zahra okba3, hassan elouizgani3, el hassan el mouden4, mohamed aourir1 a contribution to the knowledge on the diet and food preferences of darevskia praticola (reptilia: lacertidae)§ emiliya vacheva*, borislav naumov first report on two loggerhead turtle (caretta caretta) nests in the aeolian archipelago (southern italy) monica francesca blasi1,*, sandra hochscheid2, roberta bardelli3, chiara bruno1, carolina melodia1, perla salzeri1, paolo de rosa4 and paolo madonia5 threatened and extinct amphibians and reptiles in italian natural history collections are useful conservation tools franco andreone1,*, ivano ansaloni2, enrico bellia3, andrea benocci4, carlotta betto5, gabriella bianchi6, giovanni boano7, antonio borzatti de loewenstern8, rino brancato9, nicola bressi10, stefano bulla11, massimo capula12, vincenzo caputo barucchi13, p re-description of external morphology and factors affecting body and tail shape of the stone frog tadpoles’ brena da silva gonçalves1,*, carla. d. hendges2, bruno madalozzo2, tiago g. santos2,3 preliminary data on the diet of chalcides chalcides (squamata: scincidae) from northern italy andrea ciracì1, edoardo razzetti2, maurizio pavesi3, daniele pellitteri-rosa4,* the high diversity and phylogenetic signal of antipredator mechanisms of the horned frog species of proceratophrys miranda-ribeiro, 1920 (amphibia: anura: odontophrynidae) cássio zocca1,2,*, ricardo lourenço-de-moraes3, felipe s. campos4, rodrigo b. ferreira1,2,5 acta herpetologica 4(2): 125-134, 2009 feeding pattern and use of reproductive habitat of the striped toad rhinella crucifer (anura: bufonidae) from southeastern brazil rodrigo b. ferreira, rogério l. teixeira museu de biologia prof. mello leitão, av. josé ruschi, 4, centro, 29650-000, santa teresa-es – brazil. corresponding author. e-mail: rodrigoecologia@yahoo.com.br abstract. diet composition, foraging mode, and using of reproductive habitat of rhinella crucifer was studied in an artificial pond in espírito santo, brazil. the favored substrate was leaf litter, followed by cyperaceae/poaceae. calling sites, preferred for 23.3 % (n = 7) of the observed toads, were within the water, with only the head not submerged. we analyzed a total of 61 specimens, mainly males (98.5% male and 1.5% female). seven categories of prey were found in the stomach contents: coleoptera, hymenoptera (formicidae), isoptera, lepidoptera, orthoptera, gastropoda (mollusca), opilionida (arachnida). our studies indicate that the diet of rhinella crucifer consists mainly of terrestrial colonial arthropods. formicidae was the predominant food item in frequency of occurrence, number of prey and weight. isoptera and coleoptera were also relevant in terms of weight. neither large ontogenetic dietary nor seasonal shifts were observed in the population studied. our results suggest that no intraspecific food resource partitioning occurs in adult or juveniles. rhinella crucifer adults avoid competition inhabiting different home range habitats and seem to be antspecialist with a wide foraging mode. keywords. amphibian, colonial arthropods, feed, foraging mode, neotropics. introduction rhinella crucifer is found from the northern state of ceará to the state of rio de janeiro, brazil (baldissera et al., 2004; marques et al., 2006), inhabiting both rainforest and disturbed habitats (aquino, 2004). it is the most common toad in some states of brazil (aquino, 2004) whose biology has largely been studied (haddad et al., 1990; baldissera et al., 2004; sabagh and carvalho-e-silva, 2008). many biological aspects, however, remain unstudied. for example, not much is known about the diet, foraging modes and use of reproductive habitat of r. crucifer. these studies are particularly useful in providing practical interpretations of species-specific behavioral observations. toft (1981) and strüssmann et al. (1984) found a positive correlation between diet and foraging mode for anurans. foraging tactics are the central subject of modern behav126 r.b. ferreira and r.l. teixeira ioral ecology. predator animals are known to efficiently capture prey, thus maximizing energy under any environmental conditions (e.g. krebs and davies, 1997). bufonidae have been classified as ant-specialists for most of the authors (toft, 1980; flowers and graves, 1995; isacch and barg, 2002; rosa et al., 2002), although other authors have preferred to classify them as generalists (smith and bragg, 1949; evans and lampo, 1996; grant, 1996; sabagh and carvalho-e-silva, 2008). the use of reproductive habitats by anurans tends to be under specific conditions. anurans differ in habitat use for breeding, calling site, annual reproductive period, daily period of calling activity, and acoustic features of advertisement call, which are interpreted as important isolating mechanisms (e.g. wells, 1977; haddad et al., 1990). even so, rhinella has been often related to natural interspecific hybridization (feeder, 1979; sullivan, 1986; haddad et al., 1990). the purpose of this study is to describe the dietary composition, foraging mode and analyze the use of reproductive habitat of the striped toad, r. crucifer. material and methods study area the studied area was barragem norte (20º45.580’s, 40º34.250’w, 8 m a.s.l.), situated in the city of anchieta, state of espírito santo, southeastern brazil. barragem norte is an artificial lagoon that is covered by extensive vegetation, both inside the lagoon and along its margins. vegetation in the area consisted primarily of typha aff. domingensis (thyphaceae), eleocharis sp. (cyperaceae), nymphaea sp. (nymphaeaceae), lagenocarpus aff. rigidus (cyperaceae), and species of poaceae. the selected study area was approximately 1200 m2. according to köppen-geiger climate classification (1936), the anchieta’s climate belongs to aw tropical type, with high temperatures, rainy summer (december, january, february, and march), and dry winter (june, july, august and september). after 35 years (1971 to 2006) of assessment, the weather station defined an average rainfall of 1000-1150 mm/year and temperature of 23.5 °c (incaper, 2006). the region still has a particular dynamic constrained by the air currents tropical atlântica, which is hot and wet, and the polar atlântica which is dry and cold, acting mainly in the winter season. samples striped toad collections were carried out from november 1999 to september 2000 in barragem norte, with one field visiting each month. specimens were collected manually along random transects of about 100 meters, and along the marginal portions of the ponds, by two people spending five hours a day. toads were sacrificed using ethanol solutions (10%) and transferred to 10% formalin for fixation. to interrupt further digestion of prey items, formalin was also injected intraperitoneally. after a week, the toads were washed and preserved in 70% ethanol. analyses toad snout vent length (svl mm) was measured using vernier calipers (to the nearest 0.01 mm) and weighed using a digital balance (0.1 g precision). toads were then dissected, sex was 127feeding patterns and use of reproductive habitat of rhinella crucifer determined and the stomachs were extracted. stomach contents were spread on petri dishes and analyzed with a stereo-microscope. prey items were identified to order level, counted, and measured; maximum length of prey was measured using vernier calipers (to the nearest 0.01 mm). frequency of occurrence (f), number of prey (n), and wet weight (w; 0.1 mg) were calculated in order to quantify the importance of each prey type. the frequency of occurrence was defined as the number of individuals that had determined item i in the stomachs, divided by the total number of sampled exemplars. predominant hunting method was estimated by divided all prey orders found into ecological guilds, according to prey taxonomic order. the percentage of individuals found in each guild was used for comparison purposes. the relationship between length and mass of preserved specimens was calculated using type iii regression analysis. voucher specimens were deposited in the herpetological collection of the museu de biologia prof. mello leitão (mbml), santa teresa, state of espírito santo, brazil (r. crucifer: mbml 46504652, 4681, 4682). results rhinella crucifer was found from january through september with most individuals collected in may. specimens were frequently collected along the pond margin and occasionally on aquatic vegetation inside the pond, during the months of november, january, march, may, july, and september. urine release and feigning death were the primary defense mechanisms exhibited. vocalizations and amplexus were observed only during winter months (june, july, august, and september). detailed information about calling sites was obtained for 30 individuals. calling sites ranged from 0 to 500 cm from the pond margin with a mean distance of 21.3 ± 263.0 cm. the calling sites above ground elevation from 0 to 10 cm height with an average of 0.3 ± 1.7 cm. leaf litter was the favored substrate for reproductive habitats (14 specimens = 46.7%), followed by cyperaceae/poaceae (8 specimens = 26.7 %). seven specimens (23.3%) of the striped toads chose the water as calling site and one male (3.3 %) was seen calling from wood located in the water. a total of 68 specimens were collected at barragem norte having svl ranging from 54.0-105.2 mm (mean 70.05 mm ± 7.83 mm). the relationship between svl and mass was highly significant (r2 = 0.92; p < 0.01). the collected specimens were heavily biased toward males. only one female was collected and it was the largest specimen. seven categories of prey were identified among the 61 specimens of r. crucifer examined. coleoptera, hymenoptera (formicidae), isoptera, lepidoptera, orthoptera, gastropoda (mollusca), opilionida (arachnida) (table 1). seven specimens (4.76%) had empty stomachs. the diet of r. crucifer consists mainly of terrestrial, colonial arthropods that usually occur on the ground (fig. 1). formicidae was the most predominant items in terms of frequency, number of prey, and weight. also isoptera and coleoptera were relevant in terms of weight. other food items such as lepidoptera larvae, orthoptera, gastropoda, and opilionida were found, but not in large amounts. the diet of r. crucifer varied little in relation to svl classes. the three most important food items (formicidae, coleoptera, and isoptera) occurred at high proportions in almost all svl classes. in the smallest svl class (50.059.9 mm svl), the proportion between formicidae and coleoptera was relatively the same. formicidae had the highest proportion, except for the group with an svl > 80.0 mm, in those classes where isoptera was predominant in relation to weight. 128 r.b. ferreira and r.l. teixeira table 1. summary of the preys found in the stomachs of rhinella crucifer based on 61 specimens; f= frequency; n = number of prey; w= prey weight in mg. legend for guilds: ths: terrestrial, hidden, in, on ground; tac: terrestrial, active, on ground and tc: terrestrial, colonial. guilds f %f n %n w %w insecta coleoptera ths 17 27.9 30 6.1 2054.2 18.2 hymenoptera (formicidae) tc 54 88.5 325 66.2 5802.2 51.5 isoptera tc 9 14.8 129 26.3 2039.2 18.1 lepidoptera larvae ths 1 1.6 1 0.2 484.0 4.3 orthoptera tac 1 1.6 1 0.2 160.7 1.4 insect remains 3 4.9 27.5 0.2 mollusca gastropoda ths 2 3.3 2 0.4 584.1 5.2 arachnida opilionida tac 2 3.3 3 0.6 107.4 1.0 total 491 100.0 11259.3 99.9 fig. 1. percentages of prey items according to ecological prey guilds ingested. legend: terrestrial, active, on ground: orthoptera and opilionida; terrestrial, hidden, in, on surface: coleoptera, gastropoda and lepidoptera; terrestrial, colonial: isoptera and hymenoptera. 129feeding patterns and use of reproductive habitat of rhinella crucifer formicidae were the major prey items ingested in all months except for july, when coleoptera and isoptera were consumed in relatively equal proportions (fig. 2). discussion rhinella crucifer might be classified as a wide forager and an ant-specialist. the classification is justified by having slow-moving locomotion, possessing poisons in the parotid glands, preferencing for small preys, and high frequency and weight of ants founds per stomach. sabagh and carvalho-e-silva (2008) considered this species as generalist since they recorded agile prey, such as cockroaches, crickets, and spiders. sabagh and carvalhoe-silva (2008), however, also found a high proportion of ants within the stomach contents of striped toad, which resulted in a high importance relative index (iri) for this prey. inclusion of mobile preys might be due to nutrients balance in toads diet (clark, 1982), or as response to fluctuations in prey abundance (donnely, 1991). moreover, r. fig. 2. main food items found in the stomach contents of rhinella crucifer according to the collected months and based on the prey wet weight (w), number (n), and the frequency. southeastern brazil.   130 r.b. ferreira and r.l. teixeira crucifer is an active forager. during its locomotion and searching for colonials arthropods, it occasionally encounters and eats other types of prey. sabagh and carvalho-e-silva (2008) suggested that electivity test is necessary to confirm if r. crucifer is an ant-specialist. the positive electivity has been confirmed to bufonidae by toft (1980, 1981), flowers and graves (1995), and others. damasceno (2005) found ants’ positive electivity studying rhinella granulosa (spix, 1824) in the caatinga biome, brazil. isacch and barg (2002) had with the same results for rhinella arenarum (hensel, 1867) and rhinella dorbignyi (duméril and bibron, 1841) in the pampas, argentina. thus, we consider that electivity test is not essential to state that r. crucifer is an ant-specialist. furthermore, colonial arthropods compose approximately 70% of animal biomass in tropical forest (hölldobler and wilson, 1990), being the major food source. clarke (1974) pointed out that formicidae and coleoptera are frequently present in bufonid diet species, the author also stated that could be a consequence of abundance and availability of these arthropods in the soil. ants and several beetle groups (e.g. carabids and harpalids) are unpalatable to many predators due to formic acids and quinones, respectively (zug and zug, 1979). therefore, specialization on those preys might confer certain advantages. predators specialized in eating unpalatable preys decrease food competition with others predators. moreover, thick skin (see brito gitirana and azevedo, 2005) provides more resistance to r. crucifer faces ant bites and stings, allowing them to feed on these insects for longer periods (sabagh and carvalho-e-silva 2008). it is possible that those advantages result from ant-specialist feeding selection. duellman and trueb (1994) suggested that bufonids, within anurans, would be also morphologically constrained to eat small prey (ants). active foraging exposes the frogs to high risks of predation, but most bufonids possesses poisons in parotids glands. thus, they are avoided by natural predator as snakes (lulling, 1971), and are considered toxic to birds and mammals (tokuyama et al., 1969). toft (1980) stated that foraging behavior and anti-predator tactics are complementary and perhaps coevolved. ontogenetic dietary shifts are reported for many anuran species (e.g. woolbright and stewart, 1987; de bruyn et al., 1996; giaretta et al., 1998; ferreira et al., 2007). these shifts allow for intraspecific resource partitioning which facilitates higher population densities due to less intraspecific competition. if formicids are plentiful in the environment, there is probably limited intraspecific competition. nevertheless, our data support the absence of ontogenetic dietary shifts in r. crucifer as suggested also by sabagh (2008). seasonal differences in diet have been reported for many amphibian species, reflecting availability of prey and seasonal differences in prey selection (duellman and trueb, 1994). the large amount of ants found in the diet of r. crucifer suggests that this prey may be available throughout the year, favoring ant-specialists habit. home range appears to differ among adults of r. crucifer, as we never observed two specimens in close proximity to each other, except during amplexus or at calling sites. this behavior may decrease potential interference in the use of the same niche. interactions between ecological and evolutionary mechanisms in space may enhance or diminish the potential for local coexistence (urban and skelly, 2006). many species of anurans migrate to temporary ponds for breeding which increases potential for interspecific interaction (duellman and trueb, 1994). seasonal occurrence of rhinella has been shown in several studies. rossa-ferres and jim (1994) found r. crucifer and r. schnei131feeding patterns and use of reproductive habitat of rhinella crucifer deri at the end of the cold and dry seasons in botucatu, state of são paulo. in the same seasons, pombal (1997) found them in a permanent pond at the serra de paranapiacaba, state of são paulo, and bernarde and anjos (1999) in londrina, state of paraná. teixeira et al. (2007) observed both r. crucifer and r. pombali at three lagoons near anchieta. in april and may, 2006, both species were observed syntopically calling at ponds located in vargem alta and nova lombardia, state of espírito santo (pers. obs.). these species are phylogenetic close and it is possible that they are able to maintain a natural mating. the single captured r. crucifer female was larger than the males. differences in sizes between sexes may be the result of sex-specific growth rates and/or sex-specific longevity (márquez et al., 1997). several studies have reported larger females than males for amphibians. for example lee (2001) found adult females of r. marina significantly larger than adult male. however, the low number of r. crucifer females collected in our study does not allow inferring its analyses. there are two possible explanations for the abundance of males to females: (i) males arrive at the pond (or the vocalization and feeding site) before the females, suggesting that the latter have a different behavior related to their forage and mating activities. if this is the case, our sampling period did not allow opportunities to encounter females. 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(1979): the marine toad, bufo marinus: a natural history resume of native populations. smith. contr. zool. 284: 1–58. acta herpetologica 17(1): 71-76, 2022 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-11386 preliminary data on the diet of chalcides chalcides (squamata: scincidae) from northern italy andrea ciracì1, edoardo razzetti2, maurizio pavesi3, daniele pellitteri-rosa4,* 1 dipartimento di scienze della vita e biologia dei sistemi, università di torino, via accademia albertina 13, i-10124 torino, italy 2 kosmos museo di storia naturale, università di pavia, piazza botta 9-10, i-27100 pavia, italy 3museo di storia naturale, corso venezia 55, i-20121 milano, italy 4 dipartimento di scienze della terra e dell’ambiente, università di pavia, via ferrata 9, i-27100 pavia, italy *corresponding author. e-mail: daniele.pellitterirosa@unipv.it submitted on: 2021, 22nd june; revised on: 2021, 17th november; accepted on: 2022, 11th february editor: marco mangiacotti abstract. the diet in skinks is known mainly for extra-european species, especially from australian ones, where these lizards are represented by a great number of species, while, in comparison, data for species from other continents are scarce. the three-toed skink, chalcides chalcides, is found in a restricted part of northern africa and in italy, where it is distributed almost uniformly throughout the peninsula and on the major islands. although it is well studied for aspects such as morphology and ecology, data concerning trophic preferences are scarce, and available only for the populations of south-central italy. in this note we report preliminary data about the diet of an apennine population of the three-toed skink, chalcides chalcides, at the northern boundary of its distribution area. faecal contents from 20 individuals were collected in june 2015, obtaining an overall sample of 48 prey items. araneae constituted the most preyed taxon (over 40%), followed by hemiptera (35,4%) and other prey taxa (hymenoptera, coleoptera, and dermaptera) in much lower percentages. we found no differences between smaller/younger and larger/older individuals in consumed preys. as well as confirming the general trophic predilection of this skink for spiders, we also found some interesting differences in preyed items with studied populations of south-central italy. keywords. apennines, chalcides chalcides, diet, faecal pellets, northern italy, skink. diet in lizards is a very dynamic component, since it can be variable over time (floyd and jenssen, 1983; dearing and schall, 1992). changes are often seasonal, related to different prey availability and abundance between seasons (durtsche, 1995). lizards diet can also vary among sites (barden and shine, 1994), since prey availability and abundance may vary geographically too. lastly, it may be different between sexes, between adults and juveniles and also among morphs (rocha, 1998; fialho et al., 2000; scali et al., 2016). skinks diet is known mainly for extra-european species, especially from australia (wapstra and swain, 1996; duffield and bull, 1998; clemann et al., 2004; shea, 2006; pavey et al., 2010), where these lizards are represented by a great number of species, while, in comparison, data for species from other continents are scarce. skinks are known to be primarily insectivorous, even though some species may include plants in their diet, as shown in the ocellated skink chalcides ocellatus (kalboussi and nouira, 2004; lo cascio et al., 2008; carretero et al., 2010). the three-toed skink, chalcides chalcides (linnaeus, 1758), is a scincid lizard found in a restricted part of northern africa (ne algeria, mediterranean regions of tunisia and libya), and in italy, where it is distributed almost uniformly throughout the peninsula and on the major islands (caputo et al., 2010). the northern 72 andrea ciracì et alii boundary of its distribution coincides with the northern apennines since the species is almost absent from the po plain, except for few populations near the po delta (caputo et al., 2010). the species shows a snakelike habitus, with reduced tridactyl limbs. the evolution towards limblessness has an adaptive meaning, as suggested by some authors, since it favours the locomotion in grassland habitat (caputo et al., 1995). despite its quite wide range, there is paucity of information regarding some aspects of the biology of the species. this is probably due to its particular lifestyle, and to the consequent elusiveness that makes this reptile difficult to be captured in the field. so far, there is a good amount of information related to morphology and osteology (caputo et al., 1995, 2000; greer et al., 1998; caputo, 2004; guarino, 2010) and to the biology and ecology of the species (orsini and cheylan, 1980; rugiero, 1997; caputo and silvano, 1999; luiselli et al., 2005). on the contrary, data concerning structure and dynamic of the populations are absent, while those concerning trophic preferences are scarce, and available only for the populations of southern-central italy. rugiero (1997) analysed the stomach content of specimens from the surroundings of rome, while caputo (2000) studied the diet composition of a population from molise. the present study aims to collect information about the diet of the three-toed skink in northern italy, analysing the faecal pellets of individuals from a population of northern apennines. all data presented here were collected in june 2015, during the breeding period of the species. we sampled a population located in the so-called hilly area of the “oltrepò pavese”, in the municipality of codevilla (44°57’n, 9°4’e; fig. 1). the site, situated at an altitude of 260 m a.s.l., was characterized by the presence of uncultivated grasslands, surrounded by woodland area represented for the most by quercus pubescens and ostrya carpinifolia. bushy zones of rosa canina and crataegus monogyna were present at some spots inside the grasslands. we caught 20 individuals by hand, searching for them in the grass. each individual was measured using a digital calliper (accuracy ± 0.1 mm) for snout-to-vent length (svl), tail, head size (height, width, and length), weighed by a digital scale (accuracy ± 0.1 g) (table 1), and photographed on the dorsal and ventral pattern. faecal pellets were usually defecated by lizards immediately after capture, although sometimes they were obtained by applying a slight pressure on the belly of each individual, eliciting defecation. pellets were preserved in sterile tubes containing 70% alcohol for subsequent analysis. all individuals in our sample were captured once, as assessed by the manual comparison of both biometric measures and photographic images (dorsal pattern, intersection of head and ventral scales, scars). after each sampling session, all individuals were released at the exact point of capture. it was not possible to attribute sex to captured skinks as this species lacks any external sexual dimorphism, except for very large pregnant females (caputo et al., 2010). in order to tentatively evaluate possible differences in dietary habits between smaller, and consequently younger, individuals and larger/older animals, we separated the 20 skinks into two groups (10 juveniles and 10 adults), based fig. 1. map showing the study site in northern italy (municipality of codevilla, province of pavia). contour lines (elevation a.s.l. in meters) for codevilla municipality are also displayed. table 1. biometric variables of the juvenile and adult three-toed skinks (n = 20) measured in a population of the northern apennines in italy (municipality of codevilla, province of pavia): svl (snout-vent length), ta_l (tail length), tl (total length), hh (head height), hw (head width), hl (head length), w (weight). code capture date svl (mm) ta_l (mm) tl (mm) hh (mm) hw (mm) hl (mm) w (g) cod01 12/6/2015 68.7 70.0 138.7 3.4 4.5 7.4 1.6 cod02 12/6/2015 75.8 76.0 151.8 3.6 4.6 7.9 2.1 cod03 13/6/2015 80.0 81.0 161.0 3.8 4.6 7.6 2.4 cod04 11/6/2015 81.5 85.0 166.5 3.3 4.7 7.9 2.2 cod05 7/6/2015 81.9 84.0 165.9 3.7 5.0 8.0 2.5 cod06 1/6/2015 83.3 91.0 174.3 3.6 4.9 8.4 3.2 cod07 1/6/2015 85.0 91.0 176.0 3.8 4.6 7.8 2.8 cod08 24/6/2015 88.0 60.0 148.0 4.1 4.9 8.5 3.0 cod09 10/6/2015 88.0 95.0 183.0 4.0 4.0 8.6 3.6 cod10 25/6/2015 89.9 96.0 185.9 4.1 4.9 8.6 3.5 cod11 24/6/2015 90.0 32.0 122.0 3.6 4.7 8.3 3.0 cod12 24/6/2015 93.0 98.0 191.0 4.1 4.9 8.9 3.6 cod13 25/6/2015 97.4 98.7 196.1 3.9 4.6 7.8 4.5 cod14 30/6/2015 106.6 119.7 226.3 4.2 5.1 8.9 4.5 cod15 24/6/2015 126.0 139.0 265.0 5.1 6.5 11.7 10.0 cod16 10/6/2015 126.0 138.0 264.0 4.6 5.7 10.2 9.8 cod17 25/6/2015 131.7 129.0 260.7 4.8 5.8 9.4 9.2 cod18 10/6/2015 138.0 138.0 276.0 4.9 5.7 10.3 12.0 cod19 24/6/2015 139.0 145.0 284.0 5.5 6.3 10.5 12.3 cod20 24/6/2015 172.0 93.0 265.0 6.1 6.6 12.2 19.5 73diet of three-toed skink in northern italy on minimum size of adult individuals (svl = 91 mm) as reported in literature (caputo et al., 2010). the analysis of faecal pellets is considered to be fully reliable to describe lizard feeding habits (perez-mellado et al., 2011; civantos et al., 2013; scali et al., 2016). faeces were dissolved in a petri dish to separate all prey items, which were identified by using a stereomicroscope by m.p., expert entomologist of the natural history museum of milan (italy). where possible, prey items were recognized at the family taxonomic level, and were grouped at the order level. however, since some soft diet items (e.g., insect larvae, spiders) might not appear in faecal pellets, we carefully searched for body parts of small and soft-bodied prey taxa that are less likely to be digested (civantos et al., 2013). overall, we obtained 48 prey items from a sample of 20 individual faecal pellets (mean ± se: 2.4 ± 0.3, range: 1-6; 31 from juveniles and 17 from adults). the taxonomic composition of preyed items, with the percentage of contribution of each taxon, is reported in table 2. considering the overall small sample size, differences in prey items frequency between juveniles and adults for each prey taxa were tested using χ2 with monte-carlo simulation (1000000 iterations) to obtain reliable p value (patefield, 1981). the observed frequencies were not significantly different from the expected ones (p = 0.83; fig. 2), indicating that juvenile and adult diets overlapped. in general, the most present taxon in the three toed-skink diet was represented by araneae (juveniles: 14; adults: 7), followed by hemiptera (juveniles: 12; adults: 5). few items were identified as coleoptera (juveniles: 1; adults: 2) and dermaptera (juveniles: 2; adults: 1). it should be stressed that both coleoptera belonging to carabidae and tenebrionidae families and dermaptera are largely nocturnal, nevertheless they are not uncommon in the diet of diurnal lizards (vitt and blackmore, 1991). formicidae (incidentally, consistently wingless insects), again with a quite small sample, represented the only taxon equally preyed (two prey items for both juveniles and adults). ants are mostly diurnal, widespread, abundant, easy-tocatch insects, therefore their relative scarcity in the diet suggests they are not among the preferred prey. the analysis of faecal pellets shows that the most predated invertebrates by the three toed-skink are reprefig. 2. percentages of different taxa of preyed items in chalcides chalcides for juveniles and adults, categorized by svl (< 91 mm: juveniles, n = 10; ≥ 91 mm: adults, n = 10). prey’s legend: ara – araneae; der – dermaptera; hem – hemiptera; col – coleoptera; hym – hymenoptera. table 2. prey items (n = 48) of chalcides chalcides from a site of northern apennines. analyses were based on the faecal pellets of 20 skinks (one pellet for each individual). percentages refer to the number of items for each suborder and order of considered taxa with respect to the total of found items. order suborder family n suborder (%) order (%) araneae labidognatha lycosidae 3 6.25 43.75 labidognatha not determined 18 37.50 dermaptera forficulina anisolabididae 1 2.08 6.25 forficulina not determined 2 4.17 hemiptera heteroptera not determined 6 12.50 12.50 fulgoromorpha issidae 5 10.42 22.92 “homoptera” not determined 6 12.50 coleoptera adephaga carabidae (larvae) 1 2.08 polyphaga elateridae (adult) 1 2.08 6.25 polyphaga tenebrionidae (adult) 1 2.08 hymenoptera apocrita formicidae 4 8.33 8.33 74 andrea ciracì et alii sented by spiders, since they contributed 43.75% of preyed items. the previous studies, conducted on the trophic preferences of this skink in south-central italy, led to the same result, with spiders being the most preyed taxon. in the study of rugiero (1997), based on the analysis of the specimens’ stomach content, spiders contributed 42.45% to the diet composition. caputo (2000) found an even higher contribution, with araneae contributing up to 51.11% to the diet composition. further differences with respect to the previous studies are found in the contribution of the other prey taxa. in our study, hemiptera constituted the second most preyed taxon (35.42%), while both rugiero (1997) and caputo (2000) found this group contributed lower percentages to the diet composition (2.83% and 13.33% respectively). furthermore, we found a higher percentage of formicidae (8.33%) compared to the studies of rugiero (4.71%) and caputo (2.22%, including all hymenoptera). however, percentages of formicidae remain quite low, when their abundance at the soil level is considered. this suggests that formicidae are of quite low value as food for skinks, and only taken as second-choice preys, although not entirely refused. all the other prey taxa we found were present in much lower percentages, such as coleoptera (6.25% considering both adults and larvae), which, on the contrary, contributed in a significant way both in the population of rome (18.86%; rugiero, 1997), and molise (15.56%; caputo, 2000). interestingly, even if with low percentage (6.25%), we firstly detected the presence of dermaptera in the diet of the three-toed skink, not found in the other italian populations. conversely, some taxa were found in south-central italy, but not in northern apennines. for instance, rugiero (1997) found a strong contribution of isopoda (15.09%), which were not found nor in our work, nor in that of caputo (2000). this may be related to a higher aridity of the studied habitats, resulting in a largely nocturnal activity of the quite hygrophilous isopoda, since their abundance in rugiero’s samples indicates they are not counterselected as preys. the latter author found conversely a rather high contribution of orthoptera (15.56%), absent both in the present work and in that of rugiero (1997). gasteropoda, blattodea, diptera were found by rugiero (1997), even though in very small percentages, while myriapoda were found by caputo (2000). none of these taxa were found in our work. however, the differences we found with respect to these studies could be due both to the limited sample sizes in the various surveys and to the fact that none of them considers prey availability. this study allowed not only to give some preliminary insight about the diet of the three-toed skink in northern italy, but also showed no differences in the consumed prey between juveniles and adults. an ontogenetic shift in diet composition, and thus in trophic preferences, has been reported for skinks, but only for extra-european species (hall, 1972; duffield and bull, 1998; shea et al., 2009). it represents a very fascinating topic never investigated before for european skink species, so further studies on this or even other species are needed, possibly taking into account larger sample sizes, in order to perform reliable statistical tests. in conclusion, this study confirms the preference of the three toed-skink for spiders. as hypothesized by caputo (2004), this might be due to the particular structure of the teeth of the species, similar to that of the other smaller species of the genus, characterized by a conical longitudinal section, rendering them particularly suitable for preys with a soft body, such as spiders. moreover, it is not surprising that adult coleoptera are scarce in the faecal pellets of the three toed-skink. coleoptera are usually preyed by larger species of skinks like chalcides ocellatus and chalcides polylepis (bons, 1958; schneider, 1981) which have a stronger bite that easily allows them to crush such hard-bodied preys. the differences we found in the other prey taxa might be due to different factors, such as habitat, climatic conditions or sampling season. this is not uncommon in reptiles that can be at least partially opportunistic in their food choices (manicom and schwarzkopf, 2011). however, our findings put light on a basic ecological aspect of the species in its northernmost distribution area and in a particular habitat, the apennine mountains, never investigated before for skinks. acknowledgements the study was realized in accordance with the national and local legislation and a research permit was granted by regione lombardia, dg ambiente, energia e sviluppo sostenibile rlaoot1_2587 (prot. t1.2015.001437). references barden, g., shine, r. 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(1996): feeding ecology of the tasmanian spotted skink, niveoscincus ocellatus (squamata: scincidae). austral. j. zool. 44: 205-213. xi international symposium on the mediterranean lacertid lizards marco mangiacotti1, pietro lo cascio2, claudia corti2, marta biaggini2, miguel angel carretero2, petros lymberakis2 the directional testes asymmetry increases with temperature in seven plateau brown frog (rana kukunoris) populations hai ying li1, man jun shang2, jie guo2, bo jun chen2, peng zhen chen2, tong lei yu1,* influence of tail injury on the development of neotropical elegant treefrog tadpoles ana glaucia da silva martins1,#, raoni rebouças2,3,*,#, isaias santos1, adão henrique rosa domingos1, luís felipe toledo2 the effect of weight and prey species on gut passage time in an endemic gecko quedenfeldtia moerens (chabanaud, 1916) from morocco jalal mouadi1,*, panayiotis pafilis2, abderrafea elbahi3, zahra okba3, hassan elouizgani3, el hassan el mouden4, mohamed aourir1 a contribution to the knowledge on the diet and food preferences of darevskia praticola (reptilia: lacertidae)§ emiliya vacheva*, borislav naumov first report on two loggerhead turtle (caretta caretta) nests in the aeolian archipelago (southern italy) monica francesca blasi1,*, sandra hochscheid2, roberta bardelli3, chiara bruno1, carolina melodia1, perla salzeri1, paolo de rosa4 and paolo madonia5 threatened and extinct amphibians and reptiles in italian natural history collections are useful conservation tools franco andreone1,*, ivano ansaloni2, enrico bellia3, andrea benocci4, carlotta betto5, gabriella bianchi6, giovanni boano7, antonio borzatti de loewenstern8, rino brancato9, nicola bressi10, stefano bulla11, massimo capula12, vincenzo caputo barucchi13, p re-description of external morphology and factors affecting body and tail shape of the stone frog tadpoles’ brena da silva gonçalves1,*, carla. d. hendges2, bruno madalozzo2, tiago g. santos2,3 preliminary data on the diet of chalcides chalcides (squamata: scincidae) from northern italy andrea ciracì1, edoardo razzetti2, maurizio pavesi3, daniele pellitteri-rosa4,* the high diversity and phylogenetic signal of antipredator mechanisms of the horned frog species of proceratophrys miranda-ribeiro, 1920 (amphibia: anura: odontophrynidae) cássio zocca1,2,*, ricardo lourenço-de-moraes3, felipe s. campos4, rodrigo b. ferreira1,2,5 f1250_acta_herpetologica_2009_2.indd acta herpetologica 4(2): 161-169, 2009 decline of a common reptile: case study of the viperine snake natrix maura in a mediterranean wetland xavier santos, gustavo a. llorente dep. biologia animal, universitat de barcelona, av. diagonal 645, 08028 barcelona, spain. corresponding author. e-mail: xsantos1@ub.edu submitted on: 2009, 10th july; revised on 2009, 27th august; accepted on 2009, 31st august . abstract. the ebro delta is a wetland area in which natural ecosystems have been partially replaced by rice fields. this mixed and productive landscape has allowed the establishment of a rich community of organisms. the viperine snake natrix maura has traditionally been a common and abundant predator because the habitat is favorable and prey availability is high. in june 1995, we conducted a demographic study to evaluate relative densities of snakes in the rice fields. thirteen years later, we repeated the same study in the same area and season. the field work consisted of 29 censuses of one hectare each, and snakes and their potential prey (green frogs and fish) were counted. in 1995, we found 27 snakes (0.93 animals/ha), these occupying 48% of the sites. frogs and fish were observed in 23 of the 29 censuses (79%). in 2008, no snakes were found and frogs and fish appeared in only 11 of the samples (38%). in 2008, we also prospected 20 sites in rice fields located next to the natural lagoons. at these sites, we detected a greater number of snakes (25% of the stations). several factors can explain the clear decline of the n. maura population in the ebro delta rice fields: 1) the transformation and degradation of the habitat; 2) the increase in population densities of natural predators such as herons; 3) the decrease in prey availability; 4) the massive use of pollutants in the rice fields; and 5) snake death on local roads and directly by human persecution. we propose that a combined effect of these factors has caused the alarming decline of this predator. the observation of water snakes in rice fields near natural lagoons indicates that protected natural areas act as natural refuges for fauna with reduced mobility, such as viperine snakes. the recovery of the n. maura population in the rice fields of the ebro delta depends on an integral change in agricultural management, including the reduced use of pollutants, the recovery of snake prey, and the maintenance of favorable habitats to prevent predation. keywords. conservation, habitat loss, mediterranean, pesticides, population decline, snakes. introduction the loss of diversity as a result of habitat degradation has been described in diverse taxa (brooks et al., 2002). decline in reptiles is associated with several factors, such as the 162 x. santos and g.a. llorente loss and degradation of habitats, the introduction of alien species, environmental contamination, disease, unsustainable land use, and global climatic change (gibbons et al., 2000). the detection of population decline in reptiles is difficult because many species register low population densities and there are few long-term studies in natural populations to infer demographic patterns of change or tendencies (gibbons et al., 2000). these drawbacks are even stronger in the case of snakes, for which basic ecological studies are needed to assess their conservation status (seigel, 1993; dodd, 1993). snakes are susceptible to population decline because they have relatively long lives, they are exclusively carnivorous, and they have low reproductive output (scott and seigel, 1992; dodd, 1993; fitzgerald et al., 2004). in addition, these reptiles have high mortality rates as a result of human activities (bonnet et al., 1999; webb and shine, 1998). in summary, several life-history traits make snakes useful indicators of habitat quality (beaupre and douglas, 2009). over centuries, mediterranean ecosystems have been radically altered by human activity, with drastic changes linked to land use (blondel and aronson, 1999). the impact of land use practices on mediterranean biodiversity has been considerable, and 15% of endemic animal and plant species in the mediterranean basin (12% of the reptiles) are under threat of extinction (brooks et al., 2002). declining reptile biodiversity has been reported in agricultural habitats of ne iberian peninsula (ribeiro et al., 2009). however, in this region, the diverse land uses do not affect species to the same extent, as traditional agriculture characterized by a mosaic of cultures and small patches of natural vegetation maintains high indexes of reptile biodiversity (authors, unpublished data). in fact, snakes can change considerably their dietary habits with habitat alteration, either in the tropics (luiselli, 2006) or in the mediterranean basin (capizzi et al., 2008). at the other extreme, the urbanization of the mediterranean coastal belt (greenpeace, 2005) has caused a general decline that affects the entire snake assemblage (santos et al., 2007). natrix maura is a small aquatic snake that forages on fish and amphibians (santos, 2004). it is a common species in the western mediterranean and inhabits natural and artificial water bodies, such as ponds, rivers, streams, dams and marshes (bons and geniez, 1996; naulleau and schätti, 1997; santos et al., 2002; gentilli and scali, 2006). the dietary habits of this water snake and its capacity to colonize new artificial habitats (rugiero et al., 2000; santos, 2004) make it highly adaptable to changes in land use. in addition, n. maura can sometimes show high population densities (up to 4800 adults / ha in the river jalón, alicante, spain, hailey and davies, 1987b) as long as sufficient prey is available (hailey and davies 1987a; santos, 2004). these demographic and ecological trends make this snake a good model to assess the decline of natural populations. the mediterranean wetlands are one of the habitats in which n. maura shows very dense populations. in catalonia (ne iberian peninsula), the ebro delta forms a deltaic platform of 32000 hectares, in which part of the natural vegetation has been replaced by rice fields. the network of canals and rice fields has allowed the development of a dense n. maura population which has grown as a result of the high availability of their preferred prey, namely green frogs and fish (santos et al., 2000). we have evaluated snake-density shifts in the rice fields and discuss the reasons for this change. for this purpose, we conducted censuses in the rice fields during spring 2008, and compared these results with those from censuses made in spring 1995 in the same sites and following the same methodology. 163natrix maura decline in spain material and methods the ebro delta is one of the largest wildlife refuges on the coast of the iberian peninsula. it comprises an extensive surface of wetlands, lagoons and natural vegetation. however, the twentieth century was marked by intense agricultural activity dedicated to rice, which, at that time, accounted for 40% of the delta surface area. far from threatening the wildlife in the delta, rice fields and the network of canals are a good example of how the conservation of the natural patrimony and agricultural activities can be compatible (fasola and ruiz, 1996). the rice field system is especially suitable for certain aquatic species, since the rice cycle modulates their phenology through seasonal variation in food availability (llorente, 1984; ruiz, 1985; fasola and ruiz, 1996). in the ebro delta the rice cycle is seasonal: the canals and rice fields are dry from october to april. in april, the canals overflow and are colonized by diverse aquatic fauna (forés and comín, 1986; gonzález-solís et al., 1996). the rice grows until september, when it is harvested. after that, the fields and canals are drained again. the viperine snake n. maura is one the commonest non-bird predators in the rice fields of the ebro delta. being an aquatic predator, it forages on fish and frogs while juveniles also forage on invertebrates, newborn fish and tadpoles (santos, 2004). n. maura is abundant throughout the ebro delta (llorente et al., 1991). the rice cycle has modified several life-history traits, such as activity (santos and llorente, 2001a), reproduction (santos and llorente, 2001b; santos et al., 2005), diet (santos et al., 2000) and diverse physiological processes (santos and llorente, 2004; santos et al., 2007). although rice fields provide habitat and prey for n. maura, several recent human activities are reducing the quality of this habitat. on the basis of our field work experiment in the ebro delta and our knowledge of this species, we hypothesized that the species is undergoing a population decline in the ebro delta rice fields. to examine this hypothesis and identify the possible causes, we compared the relative abundance of n. maura collected during field censuses made in june 1995 and june 2008. in each period, field work consisted of 29 censuses during which we searched for snakes in the same area and season, and followed the same methodology to compare the relative abundance between years. in addition, in june 2008 we made 20 censuses in rice fields close to lagoons and natural vegetation in order to assess the effect of wetland proximity to rice fields on snake abundance. each census was done by searching for snakes in one hectare. the limits of each square were established by means of aerial photographs in 1995 and gps in 2008. during censuses, we searched for snakes in canals and rice fields, and turned rocks and other materials that often provide refuge to this species. furthermore, we also recorded the number of green frogs and fish, their usual prey (santos et al., 2000). results in 1995, we observed 27 snakes in 48% of the censuses (average 0.9 snakes/census; maximum 6 snakes) whereas in 2008 no snakes were detected in the 29 censuses. this alarming reduction in the number of snakes was statistically significant (χ2 = 18.45, p = 0.00001) and indicates that the population of n. maura has practically disappeared from the rice fields. the only snakes recorded in 2008 were in the rice fields and canals close to the natural lagoons (25% of the censuses). likewise, the abundance of green frogs also declined from censuses of 1995 (79% of censuses) to those of 2008 (38% of censuses; χ2 = 10.24, p = 0.001; table 1). frog counts also fell in rice fields close to the natural lagoons. in contrast, the presence of fish was greater in 2008, especially in rice fields and canals next to the lagoons (table 1). 164 x. santos and g.a. llorente table 1. number and percentage of censuses with presence of viperine snakes, green frogs and fish seen in the censuses done in rice fields at the ebro delta in 1995 and 2008. in 2008, results were separately presented in censuses made near and far from the natural wetlands. presence (%)   censuses n. maura pelophylax perezi fish rice fields in 1995 29 14 (48%) 23 (79%) 2 (7%) rice fields in 2008 29 0 (0%) 11 (38%) 6 (21%) rice fields in 2008 near wetlands 20 5 (25%) 9 (45%) 12 (60%) discussion this study reports the first evidence that n. maura was declining in the ebro delta. in fact, the results of censuses in 2008 indicate that the species is near extinction in the rice fields and canals. however, on the basis of our results made near to the lagoons and natural vegetation, we suspect that these habitats serve as refuges for the species, and probably focus of dispersion for snakes to other delta areas. although the home range of n. maura in the ebro delta is not extensive (1.77 ha, santos and llorente, 1997), snakes can be transported passively by floating on the water of canals, covering great distances in a single day (more than 500 meters, unpublished data of authors). this species can colonize easily new artificial aquatic habitats (rugiero et al., 2000; pleguezuelos and feriche, 2003; santos, 2004). the n. maura decrease could be attributed to the reduced availability of one of its main prey, the green frog pelophylax perezi. the consumption of green frogs is vital for female n. maura to acquire fat reserves (santos and llorente, 2004), and its reduction in the ebro delta may affect the snake reproductive output. however, n. maura is a generalist and opportunistic predator that often consumes alloctonous fish (gutiérrez-estrado and bravo, 1997; santos and llorente, 1998; rugiero et al., 2000; santos, 2004). taking into account the dietary plasticity of snakes with habitat alteration (luiselli, 2006; capizzi et al., 2008), we would not expect n. maura to be susceptible to extinction by green frog rarefaction. it is well possible that both frogs and snakes in the ebro delta declined for some unknown perturbation in the environment. in fact, both species have aquatic habits and therefore the impairment or disturbance of its water habitat could be key factors in their declining (doré, 1989; lizana and barbadillo, 1997; naulleau and schätti, 1997; santos et al., 2002; santos, 2004). thus, other causes that can contribute to the reduction of the snake population can be summarized as follows: 1) bioaccumulation of organochlorine pollutants. the ebro delta ecosystem incorporates great amounts of products derived from the agricultural activity in the delta itself and the industrial activity performed along the river (pastor et al., 2004; piqué et al., 2006). in addition to the use of pesticides during rice culture (fungicides, algaecides and herbicides), there is a massive and diffuse use of pesticides for weed control (mañosa et al., 2001). as predators, snakes accumulate large amounts of chemicals without physi165natrix maura decline in spain ological mechanisms to destroy and remove them (bauerle et al., 1975; stafford et al., 1976). in the ebro delta, high concentrations of organochlorine pollutants have been reported in n. maura muscle (santos et al., 1999). viperine snakes accumulate pesticides in such a way that larger individuals have a greater load than smaller ones (santos et al., 1999). given the massive use of chemicals in the ebro delta and their high persistence, together with the incapacity of snakes to remove these substances from their bodies, we deduce that these chemicals can have a negative effect on its reproduction as observed in other organisms of the delta (mañosa et al., 2001). 2) loss of habitat. there is a progressive transformation of irrigation canals to cement ducts to reduce the loss of water during its transport to the rice fields. in addition, vegetation along the banks of canals is removed by means of herbicides, which are applied for weed control. 3) mechanical arrangement of canals and rice fields. the harvest of natural vegetation along canal margins, which provide refuge to snakes against predators, accidentally kills many snakes (authors, personal observation). 4) predation. among other prey, several herons (e.g., bubulcus ibis, nycticorax nycticorax, ardea purpurea and egretta garzetta) in the ebro delta forage on viperine snakes (ruiz, 1985; martinez et al., 1992; gonzález-martín et al., 1992; gonzálezmartín and gonzález-solís, 1990). heron populations have risen dramatically since 1986 until now (fig. 1). consequently, the predation pressure on snakes and their prey (frogs and fish) has increased, although this has not been corroborated due to the lack of comparative dietary studies of heron populations. 5) road mortality. water snakes are commonly found dead on delta roads (x. santos, pers. obs.). fig. 1. temporal variation of reproductive pairs of heron species in the ebro delta. left y axis: nycticorax nycticorax (solid triangles), ardea purpurea (empty triangles), ardeola ralloides (solid squares) and egretta garzetta (empty circles). right y axis: bubulcus ibis (solid circles). source: ebro delta natural park. 166 x. santos and g.a. llorente 6) direct mortality by people. some people show great antipathy to these inoffensive animals and sometimes kill them during manual work in the rice fields. in the iberian peninsula, local people kill very large female water snakes especially in snake populations with high densities (hailey and davies, 1987b). in summary, here we have measured the reduction of the n. maura population in the ebro delta. diverse factors have contributed to this dramatic decline. some of these directly affect the snake population while others do so indirectly by reducing prey availability or diminishing habitat quality. in order to recover the snake population, extensive changes to habitat management policies are required. during a meeting about the conservation of coastal mediterranean areas twenty seven years ago, ruiz et al. 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(1998): ecological characteristics of a threatened snake species, hoplocephalus bungaroides (snakes, elapidae). anim. conserv. 1: 185-193. acta herpetologica 17(1): 77-83, 2022 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-11945 the high diversity and phylogenetic signal of antipredator mechanisms of the horned frog species of proceratophrys miranda-ribeiro, 1920 (amphibia: anura: odontophrynidae) cássio zocca1,2,*, ricardo lourenço-de-moraes3, felipe s. campos4, rodrigo b. ferreira1,2,5 1 nacional institute of the atlantic forest (inma), av. josé ruschi 4, santa teresa, 29650-000, es, brazil 2 projeto bromélias, instituto de ensino, pesquisa e preservação ambiental marcos daniel (imd), av. eugenio pacheco de queirós s/n, vitória, 29090-160, es, brazil 3 programa de pós-graduação em ecologia e monitoramento ambiental (ppgema), universidade federal da paraíba, av. santa elizabete s/n, rio tinto, 58297-000, pb, brazil 4 nova information management school (nova ims), universidade nova de lisboa, campus de campolide, lisboa, 1070-312, portugal 5 programa de pós-graduação em biologia animal (ppgban), universidade federal do espírito santo (ufes), av. fernando ferrari s/n, vitória, 29075-910, es, brazil *corresponding author. e-mail: zoccabio@hotmail.com submitted on: 2021, 30th august; revised on: 2022, 1st april; accepted on: 2022, 28th april editor: fabio m. guarino abstract. phylogenetic signals indicate the phenotypic similarity of antipredator mechanisms among related species. herein, we assessed the antipredator mechanisms of the horned frog proceratophrys laticeps, compiled a database including closely phylogenetically-related species, and evaluated their phylogenetic signals. our dataset comprises 80 records for 13 species of proceratophrys, totalizing 11 antipredator mechanisms and 15 variations of these mechanisms. six antipredator mechanisms show high similarity in the trees’ roots within proceratophrys (e.g., aggression, aposematism, camouflage, distress call, immobility, and interrupt calling). our observations show the first records of antipredator mechanisms for p. laticeps, and the first report of interrupt calling for proceratophrys genus, contributing to the knowledge on the behavioural ecology of proceratophrys species, addressing new insights for ecological trait evolution by multiple ancestral states of amphibians. keywords. ancestral trait, anurans, brownian motion, defensive strategies, evolution, phylogenetic tree. observations from closely phylogenetically-related species are often statistically non-independent due to common ancestry (felsenstein, 1985; harvey and pagel, 1991). shared history leads to the phenotypic similarity among related species under many evolutionary processes (hansen and martins, 1996). this phylogenetic dependence in the data can be accounted using various special statistical methods developed for phylogenetic data (e.g., felsenstein, 1985; hansen and martins, 1996; rohlf, 2001). phenotypic similarity among related species is known as phylogenetic signal and describes the tendency of a particular characteristic to be conserved (blomberg and garland, 2002). the degree of phylogenetic signal can indicate the weight to which closely related species tend to have similar traits (blomberg et al., 2003). phenotypic traits may depend upon for root of a phylogenetic tree or may converge to their tips (paivone et al., 2010). moreover, the evolution of these characteristics can be explained by brownian motion, a process of random genetic drift at a constant rate of evolution and non-directional selection (diniz-filho and vieira, 1998). 78 cássio zocca et alii predation is probably the most important selective pressure on the evolution of antipredator mechanism diversity in amphibians (brodie et al., 1991; toledo et al., 2007). anurans display 12 antipredator mechanisms and 28 variations that can be displayed into three phases of defence (i.e., avoid detection, prevent attack, and counterattack) to respond to the risks imposed by predators (ferreira et al., 2019). for example, mechanisms such as camouflage and immobility can evade detection by visually oriented predators. display of aposematic colorations, postures, and escape can prevent attacks. lastly, mechanisms such as cloacal discharge, secretion release, aggression, and distress call can be displayed in counterattacks to apprehension by the predator (ferreira et al., 2019). in addition, the sequence and intensity of antipredator mechanisms may be displayed according to the degree of stress imposed by a predator. for example, a single individual can display several antipredator mechanisms during an interaction with a predator (williams et al., 2000; lourenço-de-moraes et al., 2016). the genus of the horned frog proceratophrys miranda-ribeiro, 1920 includes 43 species widely distributed in south america (frost, 2022). they are characterized by the presence of palpebral appendages and cryptic coloration resembling fallen leaves in decomposition (prado and pombal, 2008; toledo and haddad, 2009), favouring the display of camouflage and postures such as stretching limbs to avoid detection and prevent possible attacks (ferreira et al., 2019). in the last decades, studies have recorded the antipredator mechanisms such as camouflage, postures, and aggression for some species of proceratophrys (toledo et al., 2010; 2011; peixoto et al., 2013; mângia and garda, 2015). however, the phylogenetic origin of antipredator mechanisms of proceratophrys genus is still unknown, and a knowledge gap remains with its evolutionary history (lande and arnold, 1983; price and langen, 1992). therefore, herein we evaluated the phylogenetic signal of antipredator mechanisms of proceratophrys species. we hypothesized that antipredator mechanisms of proceratophrys species are purely phylogenetic. we also described the antipredator mechanisms diversity and their variations for p. laticeps (izecksohn and peixoto, 1981), comparing the antipredator mechanisms diversity among congeners. we extracted the records of proceratophrys species from the global database of antipredator mechanisms (see ferreira et al., 2019). we complemented this database with our field observations on p. laticeps. for this, we conducted fieldwork on november 2018 and november 2019 in the estação biologia marinha augusto ruschi (ebmar; 19°58’09”s, 40°08’37”w), located in the district of santa cruz, municipality of aracruz, espírito santo state, south-eastern brazil. we used focal animal sampling (altmann, 1974) and induced the antipredator mechanisms under field conditions using only fingers lightly touching the back, fore and hind limbs, and snout of the frogs, simulating predator attacks (lourenço-demoraes et al., 2016). we followed the classification of antipredator mechanisms proposed by ferreira et al. (2019). after the field observations, the captured males were sacrificed in 3% lidocaine, fixed in 10% formalin, preserved in alcohol 70%, and deposited at the collection of museu de biologia prof. mello leitão (mbml 11562, 11882) from instituto nacional da mata atlântica, municipality of santa teresa, espírito santo state, brazil. for the phylogenetic analysis, we followed the amphibia phylogeny of jetz and pyron (2018) and reconstructed the ancestral character states through maximumlikelihood estimations under stochastic character mapping analysis (simmap; bollback, 2006), using 1.000 simulations for discrete characters based on the matrix data of antipredator mechanisms. we used the d statistic for the phylogenetic signal analysis (fritz and purvis, 2010) to measure phylogenetic signal for discrete attributes. the statistic adds the differences in attributes among sister clades and compares this sum to one generated by the brownian movement. to compare phylogenies, this difference in the sums is divided by subtracting the sum of the differences simulated randomly about the sum by brownian motion using 1.000 simulations. we used the packages “phytools” (revell, 2012) and “caper” (orme et al., 2018) through the r software (r core team, 2017). we recorded two calling males of p. laticeps (svl: 66.1 and 66.7 mm) on the partially-submerged leaf-litter of a swampy forest. when we approached, they displayed interrupt calling, and when we hand-manipulated them, both displayed other six antipredator mechanisms and nine variations: camouflage (variation: background matching [fig. 1a]), immobility, posture (variations: body inflation [fig. 1b], contraction, gland exposure, stretching limbs, death feigning [fig. 1c]), escape (variations: hide, jump away), aggression (variation: kick), and distress call. by adding our field observation on p. laticeps, the final dataset comprises 80 records on antipredator mechanisms for 13 species of proceratophrys (table 1), which represents 33% of the species from the genus. we recorded a total of 11 antipredator mechanisms and 15 variations for species of proceratophrys. the mean of antipredator mechanisms displayed by proceratophrys species was 3.8 (min = 2; max = 7). camouflage was the most displayed antipredator mechanism (n = 13 species; 100%), followed by posture (n = 12 species; 92%), and escape (n = 8 spe79diversity and phylogenetic signal of antipredator mechanisms of proceratophrys cies; 62%). regarding posture, stretching limbs (n = 8 species; 62%), body inflation (n = 7 species; 54%), and death feigning (n = 7 species; 54%) were the most displayed. proceratophrys laticeps (n = 7 mechanisms; 64%) displayed the highest number of antipredator mechanisms, followed by p. boiei (n = 6 mechanisms; 55%), and p. schirchi (n = 6 mechanisms; 55%). proceratophrys schirchi (n = 12 variations; 80%) displayed the highest number of variations, followed by p. boiei (n = 10; 67%). the mechanisms of camouflage, immobility, interrupt calling, aposematism, aggression, and distress calls (values > 0.60) have high phylogenetic structure values (table 2). this result indicates that these mechanisms have high similarity in the trees’ roots within proceratophrys (fig. 2). on the other hand, the mechanisms of charge, warning sound, and poisonous secretion have prevalent brownian origin. this result indicates that these mechanisms can occur randomly at phylogenetic trees’ tips. proceratophrys species display a wide diversity of antipredator mechanisms to avoid detection, prevent attack, and counter-attack. despite species of proceratophrys are frequently sampled, only 13 (33%) species have description of antipredator mechanisms. similarly, only four (40%) species of odontophrynus and 13 (27%) species of physalaemus have been tested but displayed several antipredator mechanisms (n = 11 and 8, respectively) (ferreira et al., 2019). the lack of data on antipredator mechanisms is generalized across anurans, and thus we reinforce fig. 1. antipredator mechanisms displayed by proceratophrys laticeps: a) camouflage of background matching. b) posture of body inflation during hand capture. c) posture of death feigning and body inflation synergistically to distress call. 80 cássio zocca et alii the need to induce antipredator mechanisms for all individuals from most species collected in the field. proceratophrys laticeps displayed high diversity of antipredator mechanisms often in synergy. anurans displaying different synergistic antipredator mechanisms may be more successful against predators (toledo et al., 2007), probably because of higher effectiveness in signal transmission to predators as observed for two species of gastrotheca (lourenço-de-moraes et al., 2016). probably, p. laticeps displays antipredator mechanisms according to researchers’ degree of stress during inductions in the field (see lourenço-de-moraes et al., 2016). despite the high diversity of antipredator mechanisms exhibited, p. laticeps differed from the congeners only by interrupt calling. interrupt calling at predator approach aims to avoid giving predators a cue to the anuran location (ferreira et al., 2019). only 10 anuran species have been recorded interrupt calling, thus this homoplastic mechanism have evolved independently in odontophrynidae (ferreira et al., 2019). the low number of records of interrupt calling is likely a sampling artifact because most researchers do not take notes on frogs that interrupt the calls when approached in the field. camouflage is displayed by all species of proceratophrys studied so far. camouflage is symplesiomorphic in anura (ferreira et al., 2019), and showed high phylogenetic structure in proceratophrys, following a purely phylogenetic model. in fact, camouflage is displayed by most odontophrynids that usually have brown coloration resembling the leaf-litter (sazima, 1978; ferreira et al., 2019). camouflage includes colouring, structural and behavioural adaptations to avoid detection by predators table 1. antipredator mechanisms recorded for proceratophrys species. species antipredator mechanisms ref n bm im ic ah ch be bi ct ge mg re sl df ur hd ja ws ps kc dc p. appendiculata 1 1 1 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 1 p. avelinoi 4 1 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 2 p. boiei 18 1 1 0 1 0 0 1 1 1 1 0 1 1 0 0 1 0 0 1 0 3,4,5,6,7 p. brauni 1 1 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 8,* p. cristiceps 1 1 0 0 0 0 1 1 0 0 1 1 0 0 0 0 1 1 0 0 0 9 p. cururu 1 1 0 0 0 0 0 1 0 1 0 0 0 0 0 1 0 0 1 0 0 6 p. laticeps 7 1 1 1 0 0 0 1 1 1 0 0 1 1 0 1 1 0 0 1 1 7,* p. melanopogon 3 1 0 0 0 0 0 1 0 0 0 0 1 1 0 0 0 0 0 0 0 5,6,10 p. moehringi 3 1 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 11 p. moratoi 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 6,* p. paviotii 5 1 1 0 0 0 0 1 1 1 1 0 1 1 0 0 1 0 0 1 0 7 p. renalis 2 1 0 0 0 0 0 0 1 0 0 0 1 1 0 0 0 0 0 0 0 7,12 p. schirchi 6 1 1 0 0 1 1 1 1 1 0 1 1 1 1 0 1 0 0 1 0 7,13 total 53 13 6 1 2 1 2 7 6 5 3 2 8 7 1 3 6 1 1 4 1 n= number of individuals tested. antipredator mechanisms (variations): bm = camouflage (background matching); im = immobility; ic = interrupt calling; ha = aposematism (hidden); ch = charge; posture (be = body elevation; bi = body inflation; ct = contraction; ge = gland exposure; mg = mouth gape; re = rear elevation; sl = stretching limbs; df = death feigning; ur = unken reflex); escape (hd = hide; ja = jump away); ws = warning sound; ps = secretion (poisonous); kc = aggression (kick); dc = distress call. ref = references: 1 = sazima, 1978; 2 = lourenço-de-moraes and lourenço-de-moraes, 2012; 3 = costa et al., 2009; 4 = toledo and zina, 2004; 5 = toledo et al., 2010; 6 = toledo et al., 2011; 7 = ferreira et al., 2019; 8 = solé, 2003; 9 = mângia and garda, 2015; 10 = moura et al., 2010; 11 = weygoldt, 1986; 12 = peixoto et al., 2013; 13 = mônico et al., 2017; * = present study. table 2. phylogenetic signal of antipredator mechanisms recorded for species of proceratophrys. bold values indicate significant differences. antipredator mechanisms estimated d phylogenetic structure brownian phylogenetic structure aggression 1.753 0.721 0.193 aposematism 8.344 0.695 0.130 camouflage 0.000 1 0.000 charge -1.691 0.229 0.527 distress call 1.732 0.841 0.122 escape 1.199 0.548 0.115 immobility 2.445 0.913 0.071 interrupt calling 8.119 0.669 0.134 secretion -3.581 0.136 0.853 posture 0.767 0.523 0.278 warning sound -4.928 0.243 0.537 81diversity and phylogenetic signal of antipredator mechanisms of proceratophrys (ferreira et al., 2019). in this context, proceratophrys species have morphological adaptations such as supraciliary structures, and a variety of warts and tubercles that likely enhance camouflage (prado and pombal, 2008). after being touched by a predator, proceratophrys species usually display a variety of postures. posture is symplesiomorphic in proceratophrys, showing high phylogenetic structure, being conserved in the genus and in anura. posture was displayed by 12 (30%) species of proceratophrys, and it is the second most displayed antipredator mechanism in the genus. the eight species that displayed stretching limbs may be avoiding detection fig. 2. reconstruction of ancestral state of 11 antipredator mechanisms displayed by 13 species of proceratophrys. a) camouflage, b) immobility, c) interrupt calling, d) aposematism, e) charge, f) posture, g) escape, h) warning sound, i) secretion, j) aggression, k) distress call. black branches = presence of the mechanism; grey branches = absence of the mechanism. 82 cássio zocca et alii by visually oriented predators that forage on the leaf-litter (sazima, 1978). body inflation can fool the predator regarding anuran body size, becoming difficult to being handled and ingested (caro, 2014). death feigning is displayed to resemble a dead organism and generally is displayed after the anuran has jumped away or was handled by a predator (toledo et al., 2011; ferreira et al., 2019). therefore, proceratophrys species likely have success in avoiding predation by displaying postures to intimidate predators, resembling a dead leaf, or making them difficult to be swallowed. we suggest that posture is an effective antipredator mechanism for species of proceratophrys against predators in the leaf-litter. our results suggest that there are antipredator mechanisms with strong phylogenetic signal (camouflage, immobility, distress call, aggression, aposematism, and interrupt calling) in proceratophrys and that their evolution is purely phylogenetic. six antipredator mechanisms displayed by proceratophrys species (i.e., camouflage, immobility, posture, escape, warning sound, and secretion) are plesiomorphic in anura (ferreira et al., 2019), explaining the maintenance of these mechanisms in the genus. in contrast, three antipredator mechanisms displayed by proceratophrys species are homoplastic (i.e., interrupt calling, charge, and distress call), having evolved independently. to conclude, our observations are the first records of antipredator mechanisms for p. laticeps, and the first report of interrupt calling in the genus proceratophrys. also, we showed that several antipredator mechanisms have high phylogenetic signal in this genus. due to limited sample number of both records of different antipredator mechanism and examined species of the genus, our analysis should be treated as a preliminary overview of possibly more complex phylogenetic scenarios of a number of different mechanisms. we suggest also that further studies on this topic should use standardized induction methods and classification system for antipredator mechanisms (see ferreira et al., 2019). acknowledgments we thank estação biologia marinha augusto ruschi (ebmar) for logistical and sampling support during fieldwork. czz and rbf thank conselho nacional de desenvolvimento científico e tecnológico brasil (cnpq 300929/2022-6; 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(1986): beobachtungen zur ökologie und biologie von froschen an einem neotropischen bergbach. zool. jahrb. syst. 113: 429-454. xi international symposium on the mediterranean lacertid lizards marco mangiacotti1, pietro lo cascio2, claudia corti2, marta biaggini2, miguel angel carretero2, petros lymberakis2 the directional testes asymmetry increases with temperature in seven plateau brown frog (rana kukunoris) populations hai ying li1, man jun shang2, jie guo2, bo jun chen2, peng zhen chen2, tong lei yu1,* influence of tail injury on the development of neotropical elegant treefrog tadpoles ana glaucia da silva martins1,#, raoni rebouças2,3,*,#, isaias santos1, adão henrique rosa domingos1, luís felipe toledo2 the effect of weight and prey species on gut passage time in an endemic gecko quedenfeldtia moerens (chabanaud, 1916) from morocco jalal mouadi1,*, panayiotis pafilis2, abderrafea elbahi3, zahra okba3, hassan elouizgani3, el hassan el mouden4, mohamed aourir1 a contribution to the knowledge on the diet and food preferences of darevskia praticola (reptilia: lacertidae)§ emiliya vacheva*, borislav naumov first report on two loggerhead turtle (caretta caretta) nests in the aeolian archipelago (southern italy) monica francesca blasi1,*, sandra hochscheid2, roberta bardelli3, chiara bruno1, carolina melodia1, perla salzeri1, paolo de rosa4 and paolo madonia5 threatened and extinct amphibians and reptiles in italian natural history collections are useful conservation tools franco andreone1,*, ivano ansaloni2, enrico bellia3, andrea benocci4, carlotta betto5, gabriella bianchi6, giovanni boano7, antonio borzatti de loewenstern8, rino brancato9, nicola bressi10, stefano bulla11, massimo capula12, vincenzo caputo barucchi13, p re-description of external morphology and factors affecting body and tail shape of the stone frog tadpoles’ brena da silva gonçalves1,*, carla. d. hendges2, bruno madalozzo2, tiago g. santos2,3 preliminary data on the diet of chalcides chalcides (squamata: scincidae) from northern italy andrea ciracì1, edoardo razzetti2, maurizio pavesi3, daniele pellitteri-rosa4,* the high diversity and phylogenetic signal of antipredator mechanisms of the horned frog species of proceratophrys miranda-ribeiro, 1920 (amphibia: anura: odontophrynidae) cássio zocca1,2,*, ricardo lourenço-de-moraes3, felipe s. campos4, rodrigo b. ferreira1,2,5 osservatorio erpetologico italiano monitoraggio nazionale dell’erpetofauna alloctona le specie alloctone costituiscono una delle principali minacce per la fauna. nonostante in italia siano presenti numerose specie di erpetofauna alloctona, la loro distribuzione non è ancora sufficientemente conosciuta, e non è stata finora posta sufficiente attenzione sull’importanza di segnalare tempestivamente novità sulla diffusione di queste specie. la commissione conservazione shi, nel 2007, ha quindi di iniziare un monitoraggio nazionale dell’erpetofauna alloctona, per avere un quadro aggiornato della situazione e anche per valutare eventuali cambiamenti nel tempo. chiediamo pertanto aiuto alla rete di erpetologi, per raccogliere segnalazione sulla distribuzione delle specie alloctone. i risultati di questo monitoraggio verranno pubblicati su acta herpetologica, all’interno dell’osservatorio erpetologico italiano, secondo le norme dell’osservatorio. le specie su cui riteniamo di focalizzare l’attenzione sono: rana catesbeiana, xenopus laevis, trachemys scripta e altre testuggini palustri naturalizzate. ovviamente, anche segnalazioni di altre specie di erpetofauna alloctona sono benvenute, ma vi preghiamo di tralasciare l’avvistamento di singoli esemplari. per le testuggini palustri alloctone, sarebbe estremamente interessante (ove possibile) mettere in evidenza le località in cui avviene la riproduzione. le segnalazioni vanno inviate a francesco ficetola, delegato per la commissione conservazione all’indirizzo e-mail: francesco.ficetola@unimi.it. dati necessari per la segnalazione: • data dell’osservazione, specie, sito (breve descrizione), comune, provincia, località più prossima sull’atlante stradale. se possibile allegare anche: fotografie, quota, coordinate gps (sistema di coordinate utilizzato), posizione su ctr o su immagine da satellite google earth. di seguito si riportano le segnalazioni pervenute nel corso del 2007. i criteri adottati per la pubblicazione delle segnalazioni pervenute sono stati i seguenti: segnalazioni originali (mai pubblicate precedentemente); per r. catesbeiana i dati di presenza sono in quadrati utm dove la specie non era stata segnalata precedentemente; per trachemys sono riportate solo le segnalazioni di riproduzione accertata in condizioni naturali o seminaturali. acta herpetologica 3(1): 83-84, 2008 issn 1827-9643 (online) © 2008 firenze university press 84 osservatorio erpetologico italiano a0058 gentile francesco ficetola +eso 110.367.0.002.0 rana catesbeiana (riproduzione non accertata). stagni da pesca presso loc. fornace, badia polesine, ro. maggio 2005. r0031 umberto fusini +eso 110.370.0.001.0 trachemys scripta, riproduzione. pianoro, bo. 2004. r0032 luciano poggiani, christian cavalieri e vittorio romeo +eso 110.370.0.001.0 trachemys scripta, riproduzione. “stagno urbani”, 4,5 km dalla foce del f. metauro, fano, pu. 2007. r0033 giuseppe bogliani, diego rubolini +eso 110.370.0.001.0 trachemys scripta, riproduzione. cassinazza, giussago, pv. 2001. r0034 antonio romano +eso 110.370.0.001.0 trachemys scripta, riproduzione. laghetto artificiale “lago delle calle” dei “giardini della landriana”, tor san lorenzo, ardea, rm. 2007. r0035 antonio romano +eso 110.370.0.001.0 trachemys scripta, riproduzione. stagni artificiali, ist. stat. per l’agricoltura, s. benedetto borgo piave, lt. 2007. acta herpetologica 4(1): 15-28, 2009 enzyme polymorphism in indian freshwater soft shell turtle lissemys punctata manoj singh rohilla, rayavarapu jagannadha rao, pramod kumar tiwari centre for genomics, molecular and human genetics, school of studies in zoology, jiwaji university, gwalior 474 011, india. corresponding author. e-mail: pktiwari.ju@gmail.com abstract. genetic diversity among four geographically isolated populations of an indian freshwater turtle, lissemys punctata, was studied using seven metabolically important isozyme/allozymes as genetic markers. a total of twenty-three alleles at fourteen protein-coding loci were identified, six of these loci were monomorphic and the remaining eight were polymorphic. the result suggests that the geographic populations of l. punctata sampled can be characterized by a total genetic diversity of 0.180 (mean ht) with an average of 1.64 alleles per locus. the average proportion of polymorphic loci per population was estimated to be 1.75. the upgma tree of genetic relationship indicated significant differentiation among populations. the results also showed that the geographical and genetic distances are not correlated in these populations of l. punctata. keywords. isozyme, allozyme, genetic diversity, upgma, lissemys punctata. introduction protein based markers, such as isozyme and allozyme, are the potential tools for indirect assessment of genetic diversity in natural populations (lewontin and hubby, 1966). the information obtained from them can serve as probes to detect past, present and possibly future population declines (status), the level of inbreeding and their evolutionary history. natural populations of threatened species are managed to conserve genetic diversity, enhance individual fitness and maintain the evolutionary potential for future adaptations (avise and saunders, 1984; verspoor and hammart, 1991; carmichael et al., 1992; parham et al., 2001). the amount and distribution of genetic variations within and between populations can provide relatively quick and indirect estimate of migration, population subdivisions, and isolation, which can define the appropriate scale for shortand long-term management of wild species in their natural habitat. the estimator most frequently uses the amount of heterozygosity to measure the population-wide genetic diversity. individual heterozygosity describes the proportion of heterozygous loci within the genome of a single individual whereas the average heterozygosity reflects the proportion of heterozygous individuals within a population measured across several loci (hartl and clark, 1997). it is 16 m.s. rohilla, r.j. rao and p.k. tiwari commonly thought that populations that are declining as a consequence of human harvesting and habitat destruction suffer, from low level of genetic variations, which could inhibit their ability to adapt to changing environmental conditions (sattler and ries, 1995). in addition, studies have shown that single (nei et al., 1975) and multiple population bottlenecks (motro and thomson, 1982) can significantly decrease genetic heterozygosity values, and populations that have passed through bottlenecks have shown greatly reduced levels of genetic variations (bonnell and selander, 1974; o’brien et al., 1985). however, some species that have been close to extinction did not show reduced genetic variations (dinerstein and mccraken, 1990), while in other species, such as bandicoot, populations that showed reduced variability have not passed through bottlenecks (sherwin et al., 1991). genetic studies can increase knowledge about the factors that may affect biodiversity, specifically in relation to the loss of genetic diversity and the implications this could have on the persistence of species in their natural habitat. the maintenance of genetic diversity is important because it represents the evolutionary potential of a species (frankham, 1995). to our knowledge, there are very few studies in chelonian, which have investigated the relationship between population structures, sex, age, geographical localities and speciesspecific heterozygosity values (but see, smith et al., 1977; seidel and lucchino, 1981; nevo et al., 1984; bonhomme et al., 1987; georges and adams, 1992). in addition, multi-locus studies in turtles carried out earlier also used electrophoretic techniques but without estimating heterozygosity (vogt and mccoy, 1980; seidel and lucchino, 1981; sites et al., 1981; derr et al., 1987; seidel and adkins, 1987). the present study was directed to understand the genetic characteristics of four geographically isolated populations of lissemys punctata inhabiting different rivers and wetlands of northern and central india. in this study we used the polyacrylamide gel electrophoretic method to estimate the level of heterozygosity in seven metabolically important isozymes/allozymes and compared them with other chelonian species populations as an evidence of genetic divergence or the level of heterozygosity, which may be expected for the geographically distinct natural populations. materials and methods geographical localities and sampling adult individuals of freshwater soft shell turtle l. punctata were collected from various aquatic bodies distantly located to four geographical localities in india, viz., gwalior and chambal (madhya pradesh), bilaspur (chhattisgarh) and allahabad (uttar pradesh), during a period of five years (2002-2007). geographical distances between population sites were taken as euclidean distance and calculated in google earth (version 4.3.7284.3916 beta) using the line function (fig. 1). in gwalior region (26º13’31’’n, 78º11’25’’e), adult turtles were obtained from the local fish markets, collected from nearby water bodies (n = 30). tissues from the turtles sacrificed in the fish markets were also collected for isoform studies. from chambal river (26º41’39’’n, 78º56’24’’e), turtles (n = 30) were collected in a 5 km stretch near barhai, district bhind. from chhattisgarh state, adult individuals (n = 30) were collected from local fish markets in bilaspur (22º03’13’’n, 81º13’35’’e). from ganga river (25º31’21’’n, 81º39’40’’e), a total of 30 individuals were obtained from the local fishermen at niva village (mallahi tolla) near allahabad, uttar pradesh. about 1 ml blood was collected from the femo17enzyme polymorphism in lissemys ral vein of each individual (rohilla et al., 2006, rohilla and tiwari, 2008). all the individuals used for blood collection were active and were left to their respective habitats after blood collection. enzyme extraction and electrophoresis total protein was extracted from rbcs as described by brewer (1970). for the study of tissue specific isoforms, approximately 100 mg tissue was homogenized in 500 μl ice-cold extraction buffer (50mm tris-hcl, ph 7.2, 50% sucrose and 10mm edta) in a microfuge tube using a sterilized glass pestle. the homogenate was centrifuged at 10000 rpm for 30 minutes and the middle layer was transferred to a separate microfuge tube. the middle layer was again centrifuged at 10000 rpm for 30 minutes and finally the supernatant was collected. protein estimation was done by dye binding assay method (bradford, 1976). equal amount of protein samples were loaded on native page in the form of a continuous vertical slab gel (5-7% acrylamide) and elecrophoresed at 140 v in 1x tbe at 4°c to resolve all the isoforms of an enzyme for complete visualization. after electrophoresis the gel was transferred to enzyme staining recipes (shaw and prasad, 1970). statistical analysis allele frequencies at each locus in the four populations of l. punctata were calculated by a simple genotyping method assuming that a protein with the same mobility is controlled by the same allele in different populations and that variations within species were controlled by two or more codominant alleles (buth, 1990; morizot and schmidt, 1990). allelic variants were designated accordfig. 1. geographic locations and distance between the sampling sites in india. 18 m.s. rohilla, r.j. rao and p.k. tiwari ing to their relative mobilities. the most common allele was designated as 100 and the other allele given numbers that indicated their mobility relative to that of the common allele. allele frequency was calculated using the formula: allele frequency = (2ho + he)/2n where ‘ho’ is the observed number of homozygotes for an allele at a given locus, ‘he’, is the number of observed heterozygotes, and ‘n’, is the number of individuals examined (singh and sharma, 1997). the observed individual heterozygosity/locus was calculated using the formula: h = he/n where ‘he’, is the number of heterozygotes in a particular population and ‘n’ is the number of individuals examined (singh and sharma, 1997). the percent polymorphism was calculated as given by singh and sharma (1997). the genetic variations among populations were measured by nei’s (1972) method. the total genetic diversity, dst, was calculated by the formula: dst = total genetic diversity (ht) – diversity within population (hs) the nei’s coefficient of genetic diversity (gst) was calculated using the formula of nei (1973), as gst = dst/ht. the nei’s genetic identity (i) and genetic distance (d) were calculated as described by nei (1972, 1973, 1978). the matrix of distances was converted into a dendrogram using the unweighted pair-group method using arithmetic averages (upgma) (sneath and sokal, 1973). results isozyme and allozyme polymorphisms at different enzyme coding loci specific staining of various enzymes in five turtle tissues, viz., liver, heart, kidney, muscle, rbcs and plasma protein, isolated from 5-10 adult individuals, were carried out to visualize the tissue-specific isoforms and for selection of enzymes for further studies. to avoid animal killing, only rbcs and plasma proteins were used for detailed population-wide genetic analysis of the four geographic populations of l. punctata. (a) esterase (est) the pattern of esterase bands were visualized on the polyacrylamide gels (7%) in all tested tissues viz. muscle, liver, kidney, heart, rbcs and blood plasma. esterase activity was not observed in rbcs. all other examined tissues showed almost similar pattern. a total of three bands, representing esterase activity, were observed. these bands were divided into two zones of activity, which could be related to two enzyme-coding loci, designated as est-1 and est-2. esterase is a monomeric enzyme, made up of single polypeptide chain. hence, in diploid organisms, co-dominant alleles code for two different characters (i.e., two allelomorphs). in turtle populations, the first zone of activity corresponds to est1 locus. a distinct band of enzyme was observed in the individuals of all four populations. this allele was designated as est-1*100 and was monomorphic. the second locus est-2 19enzyme polymorphism in lissemys showed two zones of activity or a total of two bands, both were present in heterozygous individuals and designated as est-2*100 and est-2*90. in homozygous individual only one band was observed, being either est-2*100 or est-2*90. (b) glucose-6-phosphate dehydrogenase (g6pdh) one broad band of activity was observed in all the tissues except blood plasma. high activity was observed in liver, heart, rbcs (blood) and kidney tissues, while it was found low in muscle sample. no allelic variation was observed at this locus, and was designated as g6pdh-1*100. thus, the enzyme (g6pdh) exhibited monomorphism (fig. 2a). (c) lactate dehydrogenase (ldh) the lactate dehydrogenase polymorphism was recorded on 6% polyacrylamide gel. a total of three bands were visualized in all the tissues and plasma of heterozygous individuals. only one broad band was observed in homozygous individuals. the different allozymes fig. 2. electrophoretic patterns of isozyme and allozyme in rbc samples of freshwater turtle l. punctata (n = 7/8). (a) glucose-6 phosphate dehydrogenase (monomorphic), (b) lactate dehydrogenase (polymorphic), (c) malate dehydrogenase (polymorphic), (d) peroxidase (monomorphic), (e) superoxide dismutase (polymorphic) and (f) hemoglobin (polymorphic). 20 m.s. rohilla, r.j. rao and p.k. tiwari of ldh, encoded by ldh-1 locus (homomeric), were designated as ldh-1*100, ldh1*90 and ldh-1*80. high activity of ldh was visualized in heart, muscles, and rbcs samples but it was found low in liver, kidney and plasma (fig. 2b). (d) malate dehydrogenase (mdh) a total of five bands of mdh isozyme were observed, which could be divided into three zones based on their patterns of appearance on polyacrylamide gel. the first zone consisted only of a single band, observed in both heterozygote and homozygote individuals, encoded by mdh-1 locus and designated as mdh-1*100, showed monomorphism. other two loci, mdh-2 and mdh-3 were found polymorphic due to their co-dominant patterns in individuals of the four populations (fig. 2c). at mdh-2 locus, two bands were observed in the second zone of activity, designated as mdh-2*100 and mdh-2*90 in heterozygotes and single band in the homozygote individuals. similarly, mdh-3 locus encoded two bands mdh3*100 and mdh-3*90. in plasma, only one isoform, mdh-1*100, could be recorded. compared to other tissues, mdh 1*100 showed high activity in liver kidney and rbcs. (e) peroxidase (per) the pattern of peroxidase enzyme was visualized on 7% polyacrylamide gel in all the tissues tested, viz., muscle, liver, kidney, heart rbc and blood plasma. a total of three bands, representing peroxidase activity were observed. these bands were divided into two zones of activity on the gel, which could be related to two enzyme-encoding loci. these loci were designated as per-1 and per-2 (fig. 2d). the three isoforms of peroxidase encoded by two different loci were recorded with high activity in liver, heart, kidney, muscle and rbcs, but the blood plasma was found deficient for them. in turtle populations, the first zone of activity corresponding to the locus per-1 revealed only one band, per1*100. the per-2 loci, showed two bands for the enzyme in all the individuals of the four geographic populations and were designated as per-2*100 and per-2*90. both the loci were found monomorphic due to absence of allelic variants in all the individuals of the four geographic populations. (f) superoxide dismutase (sod) the superoxide dismutase was observed (on 6% polyacrylamide) polymorphic in the rbcs of all the individuals of the four geographic populations. a total of three loci sod1, sod-2 and sod-3 encoding five bands in heterozygotes were observed in three distinct zones of activity, and designated as sod-1*100, sod-2*100, sod-2*90, sod-3*100 and sod-3*90 (fig. 2e). sod-1 and sod-3 showed polymorphism, while sod-2 locus encoded only one band, both in homozygotes and heterozygotes. (g) hemoglobin (hb) the pattern of hemoglobin bands was also studied on polyacrylamide gels (5%) in all the five tissues (i.e., liver, kidney muscle, heart and rbcs). the highest activity of this protein was observed in rbc samples but was found poor in liver, muscle, heart and kidney. 21enzyme polymorphism in lissemys a total of three bands, representing hemoglobin activity were observed. these bands were divided into two zones of activity on polyacrylamide gel, hb-1 and hb-2. hb-1 locus, consisted of two bands, identified as the product of two alleles hb-1*100 and hb-1*90. hb-1 locus was observed to be polymorphic due to co-dominant pattern. the locus hb-2 shared only one faint broad band and was observed in all the individuals of the four populations and designated as hb-2*100. it was, therefore, characterized as monomorphic in l. punctata (fig. 2f). genetic variations within and among the populations the genetic diversity within populations was estimated on the basis of the following parameters: (a) percentage of polymorphism, (b) average number of alleles per locus, (c) average heterozygosity/locus, (d) individual heterozygosity/locus and (e) observed heterozygosity/ locus. values of these parameters of all four populations of l. punctata are given in table 1. allelic frequencies became the basis of all the above parameters, which were calculated from the patterns of protein bands observed on native polyacrylamide gels. the products of fourteen protein coding loci were detected, which provided interpretable results for population analysis. all polymorphic loci were noticed to have more than one allele. out of fourteen protein-coding loci evaluated, only seven loci (est2, ldh-1, mdh-2, mdh-3, sod-1, sod-3, and hb-1) were observed polymorphic. all polymorphic loci displayed allozyme-banding patterns consistent with that expected from the known quaternary structure of the proteins. allele frequencies for polymorphic loci were calculated on the basis of gel interpretations (fig. 3). a locus is considered to be polymorphic, if the frequency of most common allele is less then 0.999. the remaining seven loci (est-1, g6pdh, mdh-1, sod-2, per-1, per-2 and hb-2) exhibited monomorphism as no allelic variations were recorded in them in all the four populations of l. punctata. each of the four populations showed different numbers of hetero and homozygous individuals for various enzyme-coding loci. simultaneously, individual heterozygosity per locus for various polymorphic enzymes was also recorded separately across the four populations for comparison. the percent polymorphism observed in four populations was 50%, the average number of alleles per locus was recorded as 1.64 table 1. measures of genetic variations in populations of l. punctata. parameters of variation location gwalior chambal bilaspur allahabad percentage of polymorphic loci 50 50 50 50 average proportion of polymorphic loci 1.75 1.75 1.75 1.75 average number of alleles/locus 1.64 1.64 1.64 1.64 observed heterozygosity/locus 0.242 0.291 0.250 0.225 average heterozygosity/locus 0.252 0.214 0.220 0.234 expected heterozygosity/locus 0.498 0.497 0.494 0.496 22 m.s. rohilla, r.j. rao and p.k. tiwari and the average proportion of polymorphic loci per population was estimated to be 1.75. the observed heterozygosity values in pond populations from gwalior and bilaspur were recorded as 0.242 and 0.250. similarly, in the river populations of chambal and ganga, these values were 0.291 and 0.225, respectively. the average heterozygosity per locus in all the four populations was recorded as 0.252, 0.291, 0.250 and 0.225 for gwalior, chambal, bilaspur and allahabad, respectively (table 1). the nei’s coefficient of genetic diversity (gst) is the quotient of genetic diversity between populations (dst) and total genetic diversity (ht). the nei’s coefficient of genetic diversity (gst) at all polymorphic loci is shown in table 2. the gst values varied from 0.228 to 0.356 under present investigation, while the fit values or index of population differentiation (f-statistics) varied from 0.42 to 1.00 for different polymorphic loci. significant departures (p<0.001) from the hardy-weinberg expectations were encountered at seven loci. fig. 3. bar diagram showing distribution pattern of allele frequencies in four distantly located geographical populations of freshwater turtle l. punctata. table 2. nei’s coefficient of genetic diversity at all polymorphic loci. locus total genetic diversity (ht) average heterozygosity within populations (hs) heterozygosity between populations (dst) nei’s coefficient (gst) est-2 0.748 0.499 0.249 0.332 ldh-1 0.628 0.476 0.152 0.242 mdh-2 0.774 0.498 0.276 0.356 mdh-3 0.774 0.498 0.276 0.356 sod-1 0.757 0.570 0.187 0.247 sod-3 0.757 0.570 0.187 0.247 hb-1 0.605 0.467 0.138 0.228 average value 0.720 0.587 0.209 0.286 23enzyme polymorphism in lissemys phylogenetic analysis nei’s genetic distance calculated for different pair-wise comparisons among four distantly located geographical populations of l. punctata showed lowest distance between bilaspur (pond) and ganga (river, allahabad) populations (d = 0.0012) and highest between gwalior (pond) and chambal population (river) (d = 0.100) (table 3). similarly, chambal and ganga populations appeared more closer, while bilaspur and gwalior are nearest to each other (fig. 4). discussion the average genetic variability of individual turtles (l. punctata) from gwalior, chambal, bilaspur and ganga was found to be very high, similar to that observed for american freshwater turtle species trachemys scripta (nevo et al., 1984). the mean hettable 3. nei’s genetic identity (i) (above diagonal) and genetic distance (d) (below diagonal). population name gwalior chambal bilaspur allahabad gwalior 0.000 0.9042 0.9545 0.9386 chambal 0.1000 0.000 0.9904 0.9960 bilaspur 0.0466 0.0096 0.000 0.9988 ganga 0.0633 0.0040 0.0012 0.000 fig. 4. upgma clustering of genetic distance showing phylogenetic relationship of the four geographical populations of freshwater turtle l. punctata based on allozyme pattern. 24 m.s. rohilla, r.j. rao and p.k. tiwari erozygosity in reptiles is reported to be 0.060 ± 053 (s.e.). the level of heterozygosity (h) in turtles from several localities all around the world is reported to be quite variable, e.g., 0.080-0.138 in sternotherus odoratus (seidel and lucchino, 1981), 019-0.022 in caretta caretta and 0.000-0.135 in chelonia mydas (smith et al., 1977; bonhomme et al., 1987). in l. punctata, the level of average heterozygosity observed in the present study was 0.23 ± 0.008 (s.e.), suggesting that the populations of this species showing higher genetic variability are fitter to adapt to different habitats or sudden environmental changes than those showing lower variability (gillespie and guess, 1978; lewontin et al., 1978; nevo, 1983, 1988; hedrick, 1986). pemberton et al. (1988) and teska et al. (1990) also, correlated the increased heterozygosity with adaptive fitness of individuals from natural populations. we assumed that presence of high heterozygosity in populations of l. punctata could likely be due to wide range of aquatic habitat conditions they are inhabiting (varying biotic and abiotic factors), influencing the animal differentially at various stages of its life cycle. the heterozygosity is reported to vary with age (tinkel and selander, 1973; chesser et al., 1982; scribner et al., 1985; al-hassan et al., 1987), sex (manlove et al., 1975), sex-ratio (simanek, 1978) and or body size (avise and smith, 1974; smith and chesser, 1981; feder et al., 1984). moreover, similar levels of heterogeneity within populations are normally not expected for long-lived vertebrate species, especially when high vagility and extensive interbreeding occur within the population. however, high level of genetic variations may be common within and among the populations of freshwater turtle species, t. scripta (smith and scribner, 1990) and l. punctata (this study). environmental perturbations, which may promote large-scale dispersal, local selection or extreme fluctuations in population size, may prevent genetic equilibrium. further, significant departures (p < 0.001) from hardyweinberg expectations were encountered at seven loci in l. punctata. a hardy-weinberg expectation is an important criterion for inferring the genetic nature of electrophoretic banding variants. several factors may contribute to hardy-weinberg disequilibrium and the ideal hardy-weinberg populations actually do not exist in nature (althukov, 1981). estimates of genetic variability, e.g. percentage of polymorphic loci (p) and heterozygosity (h), are two useful parameters to employ when analyzing the genetic structure of populations. the long-term adaptability of populations, and hence species, is dependent upon sufficient amount of genetic variations, which enables them for continued adaptation to environmental and biotic challenges. as the ultimate goal of conservation is to maintain biological lineages over evolutionary time, a thorough understanding of the extent of genetic variation and diversity in populations is of critical importance in any conservation management plan. however, the reliability of percent polymorphism and heterozygosity are closely correlated with the effective population size, the degree of migration, variability of the environment and the number and choice of loci analysed (simon and archie, 1985; kirpichnikov, 1992). the fit values indicated extensive genetic differentiation in the species at certain loci. these differences may have resulted from different selective forces acting on the gene pools of the species or from stochastic processes. this is the first electrophoretic study of enzymatic and non-enzymatic proteins, which assessed the genetic variations in the indian freshwater turtle l. punctata. this study provides the first account of the genetic analysis of indian freshwater turtle species forming a basis for future population genetic studies involving the species of lissemys and other freshwater turtle species from india. the populations we analysed revealed that the observed high level of genetic variability may be 25enzyme polymorphism in lissemys comparable to those of other endangered turtle species from the same or other river systems and hence, will be highly useful for better management and determination of conservation priorities based on genetic variability. acknowledgements we thankfully acknowledge the department of biotechnology (dbt), govt. of india, new delhi for providing a partial material support for this work through a research project to pkt. the research fellowship to msr by ugc, new delhi under drs-sap programme is gratefully acknowledged. references al-hassan, a.j., webb, c.j., giama, m., miller, p.j. 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(1990): population genetics of the slider turtle. in: life history and ecology of the slider turtle, p. 74-81. gibbons, j.w., ed, smithsonian institution press, washington. 28 m.s. rohilla, r.j. rao and p.k. tiwari smith, m.h., hillestad, o.h., manlove, m.n., straney, d.o., dean, j.m. (1977): management implications of genetic variability in loggerhead and green sea turtle. in: xiiith international congress game biologist, p. 302-312. peterele, j.j., ed, wildlife management institute and the wildlife society, washington. sneath, p.h.a., sokal, r.r. (1973): numerical taxonomy. w.f. freeman, san francisco. teska, w.r. smith, m.h., novak, j.m. (1990): food quality, heterozygosity and fitness correlates in peromyscus polionotus. evolution 44: 1318-1325. tinkel, d.w., selander, r.k. (1973): age dependent allozymic variation in a natural population of lizards. biochem. genet. 8: 231-237. verspoor, e., hammart, j. (1991): introgressive hybridization in fishes: the biochemical evidence. j. fish. biol. 39: 309-334. vogt, r.c., mccoy, c. j. (1980): status of the emydine turtle genera chrysemys and pseudemys. ann. carnegie mus. nat. hist 49: 93-102. acta herpetologica 4(1): 113-115, 2009 release calls of female bombina bombina (anura: bombinatoridae) günter gollmann1,2, andreas benkö1, walter hödl1 1university of vienna, department of evolutionary biology, althanstr. 14, a-1090 wien, austria 2university of vienna, department of freshwater ecology, althanstr. 14, a-1090 wien, austria. corresponding author. e-mail: guenter.gollmann@univie.ac.at abstract. release calls of a female fire-bellied toad, bombina bombina, were recorded in the field while handling the toad. duration and structure of these calls are similar to release calls of male b. bombina. keywords. bombina bombina, female, vocalisation. the vocal repertoire of the fire-bellied toad, bombina bombina (linnaeus, 1761) was described by lörcher (1969) and schneider (2005). these authors distinguished several call types of males (mating call, release call, territorial call, contact call) and stated that females do not emit calls. release calls of females were observed, however, in the related species bombina variegata (savage, 1932; vasara et al., 1991; gollmann and gollmann, 2002) and bombina orientalis (akef and schneider, 1985). release calls in bombina are emitted when an individual is clasped by another toad and are also often produced when toads are gently grasped by a human. here we analyse vocalizations of a female b. bombina which were uttered while the toad was held in the hand immediately after capture in the field. the call series was recorded with an akg 140 d microphone connected to an uher report 4000 tape recorder at an air temperature of 18.5 °c near marchegg (lower austria) on 4th may 1984. the female toad (48 mm snout-vent length) was held at a distance of 10 cm from the microphone. the weak signals of the female are clearly distinguishable from the background sound consisting mainly of advertisement calls of male b. bombina. frequency analysis and spectrograms of eleven calls were generated with the s_tools_stx program package, a development of the acoustics research institute of the austrian academy of sciences (http://www.kfs.oeaw.ac.at). spectrograms were obtained at an fft size of 160 samples, a frequency between 0 and 5000 hz, with analysis bandwidth 440 hz and an amplitude range of -60 db to 30 db. the spectrogram frame had a length of 46 ms with an overlap of 75%. call duration ranged from 35 to 76 ms (mean = 57 ms, ± 13 sd, n = 11). all calls show a slight downward frequency modulation and a harmonic structure with a mean 114 g. gollmann, a. benkö and w. hödl fundamental frequency of 800 hz, with much energy in a second harmonic around 1600 hz and a third harmonic around 2400 hz (fig. 1). the release calls of the female are similar to the release calls of male b. bombina in duration (mean 62 ms, lörcher, 1969) and general structure. fundamental frequency is higher than values reported for males (500 hz, lörcher, 1969; 600 hz, schneider, 2005). harmonic structure and weak frequency modulation match the release calls illustrated by schneider (2005) and those of b. variegata shown by lörcher (1969), whereas the release calls of b. bombina males presented by lörcher (1969) have a higher number of harmonics and upward frequency modulation. this is the first analysis of vocalizations of females in bombina, which so far had only been described verbally. female release calls are known from other families of anurans; usually, their structure is similar to the release calls of males (brzoska et al., 1977), whereas differences in frequency and amplitude are probably caused by sexual dimorphism of the laryngeal apparatus (wells, 2007: 271). release calls of female b. bombina are frequently uttered after capture (e.g., by three of five toads handled for photography on 8th august 2007 in untere lobau, vienna, austria; g.g. unpubl. data). the vocalizations of female fire-bellied toads may have escaped notice of observers in the field because the louder calls of the males usually drown them during behavioural interactions. acknowledgements field work was supported by the austrian research fund fwf (grant 4750 to prof. f. schaller). fig. 1. spectrogram of two release calls of a female bombina bombina (calls four and five out of a series of eleven calls). 115bombina bombina release call references akef, m.s.a., schneider, h. (1985): vocalization, courtship and territoriality in the chinese fire-bellied toad bombina orientalis (anura, discoglossidae). zool. jb. physiol. 89: 119-136. brzoska, j., walkowiak, w., schneider, h. (1977): acoustic communication in the grass frog (rana t. temporaria l.): calls, auditory thresholds and behavioural responses. j. comp. physiol. a 118: 173-186. gollmann, b., gollmann, g. (2002): die gelbbauchunke: von der suhle zur radspur. laurenti verlag, bielefeld. lörcher, k. (1969): vergleichende bio-akustische untersuchungen an der rotund gelbbauchunke, bombina bombina (l.) und bombina v. variegata (l.). oecologia 3: 84124. savage, r.m. (1932): the spawning, voice and sexual behaviour of bombina v. variegata. proc. zool. soc. london 1932: 889-898. schneider, h. (2005): bioakustik der froschlurche: einheimische und verwandte arten. laurenti verlag, bielefeld. vasara, e., sofianidou, t.s., schneider, h. (1991): bioacoustic analysis of the yellow-bellied toad in northern greece (bombina variegata scabra l., anura, discoglossidae). zool. anz. 226: 220-236. wells, k.d. (2007): the ecology and behavior of amphibians. the university of chicago press, chicago. acta herpetologica 2006 2 il rospo smeraldino «bufo viridis» in val d'ossola (amphibia : anura : bufonidae) il rospo smeraldino bufo viridis in val d’ossola (amphibia: anura: bufonidae) sandro zanghellini 1, radames bionda 2, fabio casale 3, alessandro marsilli 1, claudio torboli 1 1 albatros s.r.l., via fiume 20, i-38100 trento. e-mail: info@albatros.tn.it 2 società di scienze naturali del verbano cusio ossola, via beola 18, i-28861 baceno (vb) 3 provincia del verbano cusio ossola, viale dell’industria 25, i-28924 verbania fondotoce (vb) riassunto vengono riportate le osservazioni di rospo smeraldino (bufo viridis) compiute tra il 1998 e il 2004 in val d’ossola (provincia del verbano cusio ossola), un’area nella quale questa specie non era stata mai precedentemente segnalata. abstract. the note refers to records of green toad (bufo viridis) occured between 1998 and 2004 in val d’ossola (province of verbano cusio ossola), an area where this species has never been recorded before. parole chiave. rospo smeraldino, bufo viridis, val d’ossola, nuova località. l’atlante degli anfibi e dei rettili del piemonte e della valle d’aosta (andreone e sindaco, 1998) segnala la presenza del rospo smeraldino in tutte le province piemontesi, limitatamente però ai territori planiziali; il limite distributivo settentrionale della specie viene posto poco a nord della città di novara. dalla data di pubblicazione dell’atlante sono state compiute numerose osservazioni in val d’ossola (provincia del verbano cusio ossola), che riteniamo meritevoli di divulgazione in rapporto alla notevole perifericità delle nuove stazioni rispetto all’areale locale conosciuto. la presenza del rospo smeraldino presso domodossola è stata segnalata per la prima volta da marchesi e rey (2001), che riportano l’osservazione di ovature della specie in una raccolta d’acqua posta su depositi alluvionali (presumibilmente a fregio del fiume toce), avvenuta nel giugno 2001. peraltro, la specie era già stata rilevata nel 1998 (bionda et al., 2002) e, sino al 2004, sono stati raccolti numerosi altri dati di presenza riferiti alle seguenti stazioni: preglia di crevoladossola (292 m), 5 individui osservati con regolarità in un piccolo pozzo in ambiente urbano; masera (290 m), domodossola (265 m), villadossola (232 m), piedimulera (235 m), queste ultime 4 stazioni nell’area golenale del fiume toce. il rospo smeraldino sembra quindi avere una distribuzione piuttosto continua in val d’ossola, per lo meno nel tratto medio. i popolamenti sembrano piuttosto consistenti, dal momento che sono stati più volte acta herpetologica 1(2): 119-120, 2006 120 s. zanghellini et alii uditi contemporaneamente vari maschi in canto. inoltre nel maggio 2003, nell’ambito di un campionamento faunistico svolto con 40 piccole trappole a caduta nell’area golenale del fiume toce presso villadossola, sono stati raccolti 5 esemplari maschi (donati alla collezione del museo regionale di scienze naturali di torino). questo quadro rende sorprendente la totale assenza di dati storici – bibliografici e museali – riferiti alla val d’ossola, un settore geografico peraltro intensamente indagato dai naturalisti del passato (f. andreone, com. pers.). dal momento che il rospo smeraldino non è certo una specie criptica ed elusiva, non pare priva di senso l’ipotesi di una colonizzazione recente della valle da sud, favorita dall’esistenza di ambiti golenali a tratti ancora ben conservati. dal punto di vista della tutela della specie, la presenza di estese superfici di greto e di significative formazioni ripariali lungo il fiume sembra poterne garantire la conservazione nel tempo, anche in rapporto all’esistenza di un’area protetta in qualità di sito di importanza comunitaria (it1140006 greto torrente toce tra domodossola e villadossola). il fattore di minaccia apparentemente più significativo, cioè la carenza di raccolte d’acqua idonee alla riproduzione, potrebbe venire agevolmente contenuto tramite la realizzazione di nuovi invasi, magari proprio nell’ambito degli interventi gestionali dell’area protetta sopra citata. bibliografia andreone, f., sindaco, r. (eds) (1998): erpetologia del piemonte e della valle d’aosta. atlante degli anfibi e dei rettili. monografie xxvi (1998). museo regionale di scienze naturali. torino. bionda, r., casale, f., pompilio, l. (2002): check-list dei vertebrati del verbano cusio ossola aggiornata al dicembre 2001. quad. nat. paes. vco, 1, provincia del vco. verbania. marchesi, p., rey, a. (2001): découverte de la salamandre tachetée (salamandra s. salamandra) au simplon et du crapaud vert (bufo viridis) à domodossola. résumés des communications présentées lors du 8ème colloque herpétologique du centre de coordination pour la protection des amphibiens et des reptiles de suisse (karch). acta herpetologica 18(1): 53-60, 2023 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-14358 assessment of fall season habitat and coverboard use by snakes in a restored tallgrass prairie community carter dollen1,2, tracy j. coleman1,2, travis r. robbins1,2,* 1 glacier creek preserve, university of nebraska omaha, 14810 state street, bennington, nebraska, 68007, usa 2 department of biology, university of nebraska omaha, 6001 dodge street, omaha, nebraska 68182, usa *corresponding author. email: trobbins2@unomaha.edu submitted on: 2023, 3rd february; revised on: 2023, 16th february; accepted on: 2023, 2nd march editor: andrea costa abstract. we assessed habitat use and preference with respect to artificial coverboards for the snake community of a restored tallgrass prairie. coverboards offer herpetofauna protection from predators and space to thermoregulate their body temperature. these covers also create microhabitats that differ from their surrounding habitat. we placed plywood and metal coverboards along a transect that crossed from prairie floodplain into upland prairie. coverboards were checked over a three-week period during the fall season, during morning, afternoon, and dusk. snake species were identified and counted, and ambient temperatures and humidity were checked under each coverboard. we found four snake species across the habitat gradient, common gartersnake (thamnophis sirtalis), plains gartersnake (t. radix), dekay’s brownsnake (storeria dekayi), and western foxsnake (pantherophis ramspotti). species richness was greatest in the floodplain habitat and microhabitat associated with metal coverboards. the floodplain habitat was also the habitat predominantly used by common gartersnake and dekay’s brownsnake. dekay’s brownsnakes, furthermore, preferred utilizing metal coverboards over wood. the composition of snake species we observed suggests that the restoration efforts on this tallgrass prairie system have attracted some grassland snake species, but the possibility of a greater snake community remains. our data suggest that using metal coverboards during the cooler active seasons, such as fall and spring, will increase capture success and more efficiently sample snake communities. studies such as ours to better understand habitat and coverboard use will result in more efficient sampling of herpetofauna for conservation and monitoring efforts. keywords. thamnophis, pantherophis, storeria, microhabitat use, thermal environment, humidity, artificial cover objects, snake ecology. prairie restoration is a common management strategy to increase the biodiversity of an ecosystem, restore native populations and communities, and store large amounts of available carbon (jordon et al., 1988; samson and knopf, 1994; anderson, 2009; guiden et al., 2021). restorations typically include the removal of agriculture plots, replanting native plant species, and relying on the “field of dreams” paradigm that if you build it species native to the area will come (guiden et al., 2021). as such, this paradigm involves building a suitable habitat for organisms in hopes they will find and stay in the restored area. research shows prairie restorations can increase the abundance of animals, including herpetofauna such as snakes (king and vanek, 2020). although not readily observed because of their elusive nature, snakes can reach high abundance and greatly influence natural communities by influencing abundance and behavior of other species (hisaw and gloyd, 1926; kotler et al., 1993; sperry et al., 2008; willson and winne, 2018; king and vanek, 2020). accurately monitoring success of restoration efforts is dif54 carter dollen, tracy j. coleman, travis r. robbins ficult, however, because each restoration effort is unique, including the sampling techniques utilized. sampling herpetofauna in tallgrass environments can be difficult due to the cryptic nature of reptiles and amphibians (fitch, 1987; szaro et al., 1988). visual sampling is a common method for sampling snake species (foster, 2012), however, small, cryptic, or camouflaged species are often difficult to detect in areas of thick vegetation due to their slender bodies (turner, 1977; ward et al., 2017). one efficient form of sampling includes the use of coverboards constructed out of sheets of various heat conducting materials, such as metal, wood, rubber, or asphalt roofing (fitch, 1987; engelstoft and ovaska, 2000). coverboards can significantly increase detection of snakes compared to simple visual surveys (halliday and blouin-demers, 2015). these covers provide attractive areas for snakes and other herpetofauna to seek refuge and an efficient way for scientists to observe, count, or capture snakes (halliday and blouin-demers, 2015). coverboards of different types provide microhabitats of varying temperatures and humidities allowing individuals a choice regarding suitable areas for cover (engelstoft and ovaska, 2000). these microhabitats are impacted by the habitats in which they are placed and seasonality due to falling or rising temperatures. during the cooler months, for instance, snakes may utilize substrates or objects that absorb or retain heat to maintain their optimal body temperature more efficiently (engelstoft and ovaska, 2000). individual preferences for specific microhabitats can arise through various needs associated with age, sex, shedding, food ingestion, circadian rhythms, and reproductive condition (lilywhite, 1987). sampling efficiency associated with coverboard type can therefore vary based on target species, habitat type, season, and interactions among these factors. these relationships mean that the coverboard types used can impact species-specific encounter rates during surveys and biodiversity assessments and can influence the herpetofaunal community composition detected (grant et al., 1992; engelstoft and ovaska, 2000; hampton, 2007). identifying habitat and microhabitat use of snake species in the tallgrass prairies of the great plains of north america has received relatively little attention, despite prairies being the largest vegetative community in north america (samson and knopf, 1994) and the great plains constituting one-third of the united states (deitz, 2022). understanding these preferences can be beneficial to conservation efforts and lead to more accurate and efficient sampling for specific species and communities. in this study, we used metal and plywood coverboards to assess snake habitat and coverboard preference in a restored tallgrass prairie system that includes a habitat gradient from prairie floodplain to upland prairie. we specifically compared the preferences of the snakes for metal or wooden coverboards in association with habitat type, humidity, and ambient temperatures. we sampled snakes within the allwine tract of glacier creek preserve (41.19759n, -96.29893w), a 212 ha (525 acres) preserve that encompasses an entire sub watershed in eastern nebraska, united states. the allwine tract (65 ha; 160 acres) was donated to the university of nebraska at omaha in 1959. in 1970, 57 ha (140 acres) of agricultural land within the allwine tract were seeded with five native prairie grass species and then over-seeded with a diverse mix of local native forbs in the following years. between 2009 and 2019 an additional 147 ha (365 acres) were purchased and added to the preserve, including a mix of agriculture, wetlands, and woodlands. the reconstructed prairie is managed with a 3-year prescribed fire return interval that occurs in mid-spring, where no more than 2 of the 5 units are burned in one year. additional details about the site can be found in bragg et al. (2016), dere et al. (2019), and manning et al. (2022). data was collected during a 3-week period from september 17 to october 4, 2021, during three time blocks of 7:00-9:00, 14:00-16:00, and 18:00-20:00 ct (morning, afternoon, and dusk, respectively). four data collection events occurred during each of the time blocks. we sampled 10 stations that were established in spring of 2018, reflecting 41 months since establishment. sampling efficiency of artificial retreats can increase with time since establishment; however, studies have found that efficiencies reach asymptotic maximums within 12 months, well within our time frame (grant et al., 1992; croak et al, 2010). the stations ran along a north-south transect (800 m) that crossed glacier creek with 5 stations on the south slope and 5 stations on the north slope (104 m average distance between stations; min = 27 m, max = 145 m). each station consisted of two artificial coverboards: a uniformly sized metal (corrugated, galvanized sheet metal) and plywood (12.2 mm or 1/2 inch thickness) coverboard each measuring approximately 122 x 122 cm (l x w) and placed approximately 1.5 m from each other. therefore, there were 20 artificial retreats evenly divided between the two types of material. a kestrel 5000 environmental meter (nielsen-kellerman company) was used to collect relative humidity and temperature data. during each sampling event each board was lifted, and the area underneath scanned to count number of individuals and identify snake species and life stage as either juvenile or adult. the kestrel was then placed under the board to acclimate for 90 seconds. we recorded the relative humidity and temperature from the kestrel and repeated this process for each of the stations 55habitat use of prairie snake community along the transect, whether or not snakes were present. for each station we also measured distance to creek using an aerial map in arc map gis and recorded habitat type (prairie floodplain, floodplain-upland transition zone, and upland prairie). because the transect ran across and perpendicular to the creek, the fl oodplain and transition zone were relatively narrow habitats with four coverboards (two stations) in each habitat and the remaining twelve coverboards (six stations) in the upland prairie. we assessed snake species richness and encounters with respect to multiple covariates and factors such as time of day, date, coverboard type, habitat type, distance to creek, humidity, and temperature. life stages of juvenile and adult were grouped for analyses because of low juvenile numbers. we employed generalized linear models (glm) including covariates and factors as independent variables and species richness (a count of number of species) or species-specifi c encounters (a count of number of individual encounters) as the dependent variable. we tested dependent variables for normality and transformed data when necessary or ran models with the appropriate distribution, such as poisson for count data or non-parametric tests. we also checked for relationships among our independent variables using analyses of variance (anova), pearson or spearman correlations, or linear regressions where appropriate. when variables were related, we chose one to include in our initial model or used residuals from a regression of one variable on the other. temperature and humidity were related, for instance, thus the residuals from a regression of humidity on temperature were used in the initial models. th e date, or sequence of sampling days, did not infl uence species richness and was therefore not included in further models (wald χ2 = 0.423, p = 0.516). because of relationships between some of our independent variables (see results) our initial models included coverboard type, habitat type, and temperature. interactions that were not signifi cant were eliminated from fi nal models. all statistical analyses were conducted with ibm spss for windows, version 29.0. habitat and coverboard type both infl uenced species richness and individual species encounters (table 1, fig. 1 and 2). more species were found in the prairie fl oodplain than in the transition zone or drier upland prairie (fig. 1). we also found more species, almost double the number, underneath the metal coverboards than the wooden coverboards. similar trends were observed at the individual level of species encounters (fig. 2). we encountered significantly more common gartersnakes (th amnophis sirtalis, n = 27) and dekay’s brownsnakes (storeria dekayi, n = 24) in the prairie fl oodplain than the other habitats (fig. 1) and more dekay’s brownsnakes under the metal coverboards (fig. 2). we found too few plains gartersnakes (th amnophis radix, n = 3) to statistically examine their abundances across habitat and coverboard types and no signifi cant patterns in western foxsnakes (pantherophis ramspotti, n = 6, glm all p > 0.4). we caution direct interpretation of our encounters as relative abundances because we did not mark individuals, thus it is possible some were recounted, which could bias the count data. table 1. eff ects of habitat and coverboard type on snake species richness and encounters of individual species in a restored tallgrass prairie. habitats consisted of prairie fl oodplain, fl oodplain to upland transition zone, and upland prairie. coverboard types included plywood and corrugated sheet metal. sampling occurred at glacier creek preserve in bennington, nebraska, across 240 sampling occasions during the fall season. bold text denotes statistical signifi cance at the α = 0.05 level based on generalized linear models. predictors df wald χ 2 p wald χ 2 p wald χ 2 p intercept 1 12.704 < 0.001 18.499 < 0.001 299.111 < 0.001 coverboard type 1 4.229 0.040 2.042 0.153 10.964 0.001 habitat type 2 24.077 < 0.001 16.865 < 0.001 35.427 < 0.001 temperature 1 0.226 0.635 2.727 0.099 2.421 0.120 species richness common gartersnake encounters dekay's brownsnake encounters fig. 1. average species richness and number of encounters for each species (per coverboard) found under coverboards in each habitat type. averages are estimated marginal means based on the generalized linear model for a poisson distribution of count data. associated count totals for common gartersnakes (th amnophis sirtalis) and dekay’s brownsnakes (storeria dekayi) in the upland = 8 and 4, transition = 5 and 1, and floodplain = 15 and 18, respectively. upland translates to prairie upland, transition to upland/fl oodplain transition zone, and floodplain to prairie fl oodplain. a low occurrence of individuals precluded a species-specifi c analysis of plains gartersnakes (th amnophis radix) and pattern detection in western foxsnakes (pantherophis ramspotti). diff erent letters above bars denote signifi cant diff erences between habitats (α = 0.05). error bars are ± 1 se. 56 carter dollen, tracy j. coleman, travis r. robbins relative humidity under the coverboards was not infl uenced by coverboard type (as humidity residuals on temperature; f1, 238 = 3.002, p = 0.084), but it was infl uenced by habitat type (as humidity residuals on temperature; f2, 237 = 8.961, p < 0.001) and negatively related to temperature (f1, 238 = 279.76, p < 0.001, r2 = 0.54). temperature under the coverboards also was not infl uenced by coverboard type (f1, 238 = 0.246, p = 0.620), but was related to time of day (f2, 237 = 207.78, p < 0.001) with aft ernoons being the warmest period (mean ± se for morning = 17.3 ± 0.5° c, aft ernoon = 30.7 ± 0.5° c, dusk = 26.0 ± 0.5° c). th e distance between coverboards and the creek was related to both the relative humidity underneath coverboards (residuals on temperature; spearman’s rho = -0.163, p = 0.012) and to habitat type (kruskal-wallis h = 185.406, df = 2, p = < 0.001). th e results of our study revealed habitat use and coverboard preference of the snake community in a restored tallgrass prairie system during the fall season. we found four snake species at glacier creek preserve including the common gartersnake (th amnophis sirtalis; fig. 3a), plains gartersnake (t. radix), western foxsnake (pantherophis ramspotti; fig. 3b), and dekay’s brownsnake (storeria dekayi). we also observed one lizard species, the northern prairie skink (plestiodon septentrionalis). snake species richness was greatest in the prairie fl oodplain as were snake encounter rates in general (table 1, fig. 1). th e greater overall snake encounters were a result of the signifi cantly higher average number of individuals among common gartersnakes and dekay’s brownsnakes in the fl oodplain compared to the transition areas and upland prairie (fig. 1). more snakes may have occupied the prairie fl oodplain because the proximity of a known hibernaculum used for overwintering (tjc, personal observation; fig. 3). because this study occurred in the fall, snakes may have been moving from the upland areas toward their hibernacula in preparation for winter brumation (mcallister, 2018; bridger and geluso, 2021). using both metal and plywood coverboards provided snakes with an opportunity to choose specifi c microhabitats. a greater number of common gartersnakes and dekay’s brownsnakes chose to settle under metal coverboards than wood, which is consistent with other studies for th amnophis (engelstoft and ovaska, 2000, hampton, 2007) and storeria species (halliday and blouin-demers, 2015). snakes oft en seek materials with higher heat conductivity especially in cooler seasons (hoyer, 1974; fitch, 1987; barker and hobson, 1996; engelstoft and ovaska, 2000). common gartersnakes in british columbia, for instance, preferred metal and asphalt coverboards over wood during fall and spring (engelstoft and ovaska, 2000). coverboards may be used less oft en generally during summer (e.g., mid-august) because associated microhabitat temperatures oft en rise above 40 °c, which is above critical thermal maximum for most reptile species (engelstoft and ovaska, 2000; angilletta, 2009). one might hypothesize that wooden coverboards would be used more during warmer summer months because they maintain both higher humidities and more stable temperatures compared to metal (grant et al., 1992), oft en providing thermal environments similar to ambient conditions (engelstoft and ovaska, 2000). in other nebraska grasslands reptile preference for wooden coverboards has been observed during the warmer months (brown and geluso 2022; approximately 370 km southwest of glacier creek preserve). however, plywood coverboards were not used by reptiles in the british columbia community more oft en in the summer compared to spring and fall (engelstoft and ovaska, 2000). coverboard use may similarly vary with other herpetofauna such as amphibians, where some studies have found more amphibian species under wood coverboards than metal (grant et al., 1992), and others have found no diff erence in amphibian abundance under metal versus wood coverboards (hampton, 2007). during the lower fall season temperatures, the preference of snakes at glacier creek preserve for metal coverboards likely occurred because these spaces heated up more quickly allowing snakes to more effi ciently attain optimal body temperature for various behaviors and bodfig. 2. average species richness and number of encounters for each species (per coverboard) found under wood and metal coverboards. averages are estimated marginal means based on the generalized linear model for a poisson distribution of count data. associated count totals for common gartersnakes (th amnophis sirtalis) and dekay’s brownsnakes (storeria dekayi) found under wood = 10 and 4, and metal = 18 and 19, respectively. a low occurrence of individuals precluded a species-specifi c analysis of plains gartersnakes (th amnophis radix) and pattern detection in western foxsnakes (pantherophis ramspotti). diff erent letters above bars denote signifi cant diff erences between treatments (α = 0.05). error bars are ± 1 se. 57habitat use of prairie snake community ily functions such as foraging and digestion (grant et al., 1992; lillywhite, 1987). relationships among our environmental variables have ecological implications for habitat and coverboard use by snakes. our final model only contained coverboard type, habitat type, and temperature as independent variables and temperature was the only variable that did not directly influence species encounters or richness. interestingly, coverboard type was not directly related to either humidity or temperature in our study, which contrasts with other studies (engelstoft and ovaska, 2000; grant et al., 1992). this is a case where the statistical significance may not match the biological significance as we found increased snake presence under metal coverboards even though we did not observe significantly greater temperatures or relative humidity. the relatively mild fall season climate likely diminished environmental effects of coverboard type that may be more significant during late spring and summer seasons (engelstoft and ovaska, 2000). it may also be a dissociation of measured temperatures at time of capture versus biologically significant temperatures during snake movement and microhabitat choice. our analyses of relationships among environmental variables determined that temperature was related to time of day (afternoons the warmest) and habitat type was related to humidity and distance from the creek. neither relative humidity nor temperature were significant factors in our models of coverboard selection, but our temperature and humidity data were collected at the time of sampling. it is possible that temperature and humidity influence habitat and coverboard use most when the snake first moves to the space, which may occur within a specific window during the day, although we did not observe an effect of time of day on species presence or absence. we do not know when during the day snake activity and coverboard choice occurred, but future studfig. 3. map of glacier creek preserve, a tall grass prairie restoration site located in bennington, nebraska, united states in the heart of the great plains ecosystem of north america. the inset highlights the placement of coverboard stations along the habitat gradient from floodplain to upland prairie. the floodplain is depicted by the lighter beige strip of vegetation (75-300 m) covering both sides of glacier creek. the upland prairie is depicted by the darker tan areas of vegetation moving both north and south of the floodplain, up the slopes (200-600 m). the transition zone consists of the narrow strip (20-30 m) where the floodplain habitat (light beige vegetation) transitions into the upland prairie (darker tan vegetation). 58 carter dollen, tracy j. coleman, travis r. robbins ies examining daily temperature cycles along with snake activity and microhabitat preferences could shed light on this relationship. habitat type, humidity, and distance from creek were all related, with humidity decreasing with distance from creek as habitat changed from the prairie floodplain and transitioned to the upland prairie. the prairie floodplain had the greatest species richness, suggesting that greater humidity and creek proximity could attract more snake species. whether it was the humidity itself or proximity to the creek, however, we cannot discern. because our study was limited to three weeks of a single climatic season, we caution against any definitive conclusion regarding the overall snake community. however, comparing this prairie snake community with assessments of other prairie snake communities within a day’s drive suggests that the glacier creek restoration effort at least partially reflects the “field of dreams” paradigm. the nachusa grassland is a tallgrass prairie system in the state of illinios, 600 km to the east of our restoration site. an extensive survey of the nachusa grassland system detected a snake community nearly identical to ours, including the common gartersnake (thamnophis sirtalis), plains gartersnake (thamnophis radix), and dekay’s brownsnake (storeria dekayi), in addition to the eastern foxsnake (pantherophis vulpinus) rather than our western foxsnake (pantherophis ramspotti; king and vanek, 2020). the konza prairie is another well surveyed tallgrass prairie in the state of kansas, approximately 500 km south of our site with a reported snake community of ten species (wilgers and horne, 2006). the konza prairie snake community overlapped with two of our species, the common gartersnake and dekay’s brownsnake, but contained eight more species, four of which are known to occur in douglas county, nebraska, where glacier creek preserve resides, the gophersnake (pituophis catenifer), eastern racer (coluber constrictor), lined snake (tropidoclonion lineatum), and ring-necked snake (diadophis punctatus; fogell, 2010). together, these comparisons show two snake species common to all three prairies and the possibility of four more species inhabiting glacier creek preserve. our collective knowledge about maintaining, restoring, and monitoring prairie communities is of global importance because grasslands cover one-third of the world’s land area (nunez, 2019). our study is an important first step in the assessment of restoration success with regard to the reptile community of this tallgrass prairie ecosystem in the great plains. our study determined a baseline estimate of the current reptile community (four snake and one lizard species) and the relative efficacy of coverboard type (metal coverboards created a preferred microclimate) in the tallgrass prairie ecosystem. our data suggest that fall surveys should incorporate metal coverboards and focus on the prairie floodplain if the goal is to assess snake diversity. using an array of different coverboard types may be beneficial across seasons because of the different microclimates that they create along with different preferences exhibited by the different species, life stages, or physiological state of individual snakes (halliday and blouin-demers, 2015). although the snake community we recorded was similar to one comparable system, it was different than another. further studies in this system, such as a long-term monitoring program over multiple years and seasons will provide further insight into the restored snake community such as seasonal influence on habitat and coverboard use, and possibly the season in which sampling should be conducted for highest detection rates. acknowledgments we thank t.b. wolfdahlhaus for comments on earlier drafts of the manuscript and personnel at glacier creek preserve including tom bragg and jon soper for logistical support and jacob garabrandt for assistance with placement of the coverboards. we thank barbi hayes, glacier creek preserve, and university of nebraska omaha for providing financial support for this study. the research presented here adhered to guidelines for the use of animals in research and the institutional 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(2006): effects of different burn regimes on tallgrass prairie herpetofaunal species diversity and community composition in the flint hills, kansas. j. herpetol. 40: 73-84. willson, j.d., winne, c.t. (2016): evaluating the functional importance of secretive species: a case study of aquatic snake predators in isolated wetlands. j. zool. 298: 266-273. threats of the emerging pathogen batrachochytrium salamandrivorans (bsal) to italian wild salamander populations lorenzo dondero1, giorgia allaria1, giacomo rosa1, andrea costa1, gentile francesco ficetola2, roberto cogoni3, elena grasselli1, sebastiano salvidio1,* age estimation and body size of the parsley frog, pelodytes caucasicus boulenger, 1896 from lake borçka karagöl, turkey cantekin dursun*, serkan gül, nurhayat özdemir patterns of acoustic phenology in an anuran assemblage of the yungas andean forests of argentina martín boullhesen1,2,*, marcos vaira1, rubén marcos barquez2, mauricio sebastián akmentins1 diet and trophic niche overlap of four syntopic species of physalaemus (anura: leptodactylidae) in southern brazil renata k. farina1, camila f. moser2, stefano scali3, mateus de oliveira4, patrícia witt5, alexandro marques tozetti1,* screening of ophidiomyces ophidiicola in the free-ranging snake community annually harvested for the popular ritual of san domenico e dei serpari (cocullo, aq, italy) daniele marini1,2, ernesto filippi3,*, gianpaolo montinaro4, francesco c. origgi5,6 assessment of fall season habitat and coverboard use by snakes in a restored tallgrass prairie community carter dollen1,2, tracy j. coleman1,2, travis r. robbins1,2,* revisiting the polyploidy in the genus odontophrynus (anura: odontophrynidae) andré luis de souza, mayara aparecida das neves micalichen, roger alves da rocha, rafael bueno noleto* acta herpetologica 4(2): 191-194, 2009 ratsnake response to bottomland flooding: implications for avian nest predation gerardo l.f. carfagno1 , patrick j. weatherhead program in ecology, evolution and conservation biology, university of illinois, 606 e. healey street, champaign, illinois, us-il-61820. 1 present address: department of biology, gettysburg college, 300 n. washington street, gettysburg, pennsylvania us-pa-17325. corresponding author. e-mail: gcarfagn@gettysburg.edu submitted on: 2009, 25th june; revised on 2009, 26st august; accepted on 2009, 28th august. abstract. lower predation has been documented for birds’ nests located over water and a recent study found lower predation on wood duck (aix sponsa) nests in bottomland forest during flooding. in a three-year study we used radio telemetry to determine whether flooding affected use of bottomlands by ratsnakes (elaphe obsoleta) and thus might explain reduced nest predation. of the 22 ratsnakes we tracked, only five used bottomlands and three of them did so exclusively, suggesting a surprising degree of habitat specialization by individual snakes. those individuals regularly moved into flooded forest and their frequency of movement and distance moved appeared unaffected by flooding. although it seems unlikely that ratsnakes leave bottomlands during floods, they may prey more extensively on small mammals that are restricted to trees when the forest is flooded, thereby reducing predation pressure on nesting birds. keywords. elaphe obsoleta, avian nest predation, radiotelemetry. despite the importance of nest predation for birds, ornithologists generally can only infer predator behavior from patterns of predation. a fuller understanding of nest predation requires that we study the predators directly (weatherhead and blouin-demers, 2004). there is considerable evidence that birds reduce nest predation risk by nesting over water (e.g., weatherhead and robertson, 1977; jobin and picman, 1997; sanchez-lafuente et al., 1998; hoover, 2006). also, birds nesting in bottomlands experience less predation when those habitats are flooded (kennamer, 2001; roy neilsen and gates, 2007). the latter result suggests that predators alter their behavior in response to flooding. here we investigate how ratsnakes (elaphe obsoleta) responded to bottomland flooding to determine whether changes in their behavior could account for reduced nest predation. roy neilson and gates (2007) studied wood ducks (aix sponsa) nesting in bottomland and adjacent upland forest in southern illinois, a state located within the ‘midwestern’ region of the usa. in a year of intensive study they found no predation in bottomland nests during a four-week flood, and a retrospective analysis of earlier data revealed lower 192 g.l.f. carfagno and p.j. weatherhead predation during floods for bottomland but not upland nests. ratsnakes and raccoons (procyon lotor) were identified as the two principal nest predators based on direct observation and the state of nests following predation. roy neilson and gates (2007) speculated that lower predation during floods could result from predators moving less, leaving the flooded area, dying, or switching prey. here we use data from a telemetry study of ratsnakes to assess the first three of these hypotheses. ratsnakes exploit a suite of prey (fitch, 1963), with birds an important component of the diet of snakes at our study site (carfagno et al., 2006). coincidentally, our study was also conducted in southern illinois and overlapped temporally with roy neilson and gates’ (2007) study. although respective study sites were in different locations, they were located in adjacent counties within 60 km of each other. we used radio telemetry to study habitat use (carfagno and weatherhead, 2006) and movement (carfagno and weatherhead, 2008) of ratsnakes at the cache river state natural area in johnson county, illinois (37° 23’ n, 88° 54’ w) from 2002 to 2004. similar to the habitat at the wood duck study sites (ryan et al., 1998), our snakes primarily used a mixture of upland and periodically flooding bottomland forests within an agricultural matrix. bottomland forest, including areas classified as bottomland edge (within 15 m of open habitat) comprised approximately 23% of the habitat in our study area. we captured ratsnakes opportunistically and as they emerged from hibernacula. we surgically implanted radio transmitters in 22 ratsnakes and usually relocated them every other day throughout the active season (see carfagno and weatherhead, 2006, 2008 for details). we did not document flooding per se, but recorded whether the habitat was flooded each time we located a snake in the bottomland. although there were permanently flooded forests near our study area, all the bottomlands in our study area flooded only seasonally (usually spring). snake locations were mapped using gps. we calculated frequency of movement as the probability that a snake had moved each time it was relocated and the distance moved as the straight-line distance between consecutive locations. over three field seasons we relocated snakes in bottomlands 420 times. all of these observations came from only five (2 females, 3 males) of the 22 ratsnakes we tracked. furthermore, three of those five individuals used bottomlands exclusively, suggesting some ratsnakes are bottomland specialists. four of those five individuals came from the same hibernaculum. that hibernaculum and the one used by the fifth individual were both located on the edge of bottomland forest, whereas all other ratsnakes we tracked (i.e., those using exclusively upland habitats) hibernated in sites in upland forest. we relocated ratsnakes in flooded areas 24 times. the snakes were always in trees when in flooded habitat. all 24 observations came from the three snakes that used only bottomlands. on nine occasions a snake moved from dry to flooded bottomland, and each time moved back to dry bottomland after remaining in the flooded area for 1 7 relocations. ratsnakes in flooded bottomlands moved slightly more often than when in dry bottomlands (85.3 ± 5.3% vs. 73.1 ± 2.5%) but moved slightly shorter distances (92.2 ± 28.5 m vs. 117.9 ± 7.7 m). at our study site, the spatial distribution of snakes was not found to be related to the distribution of small mammals (carfagno et al., 2006), but nest predation risk did increase when ratsnakes were most active (weatherhead et al., in press). we found a surprising degree of habitat specialization among the ratsnakes we tracked, with only five of 22 individuals using bottomlands at all, and three of those five using bottomlands exclusively. there also appeared to be segregation between hibernation 193nest predation by rat snakes sites of snakes that used upland and bottomland habitats. many of the other ratsnakes we tracked hibernated within several hundred meters of bottomland forest, so their failure to use bottomlands was not a consequence of that habitat being unavailable to them. a consequence of habitat specialization was that the observations relevant to our objectives came from a small number of snakes and thus must be interpreted cautiously. we found no evidence to suggest that flooding altered ratsnake use of bottomlands. snakes moved into flooded areas and appeared to behave similarly in flooded and dry forest in terms of how often and how far they moved. studies in canada have shown that ratsnakes do not spend much time in water, but they do use wetlands and readily cross open water (weatherhead and hoysak, 1989; blouin-demers and weatherhead, 2001). in a study conducted near our site in illinois, hoover (2006) found that apparent snake predation on nests of prothonotary warblers (protonotaria citrea) did not decline with water depth, further supporting the view that water is not a deterrent to ratsnakes. therefore, it seems unlikely that the decline in wood duck nest predation during floods reported by roy neilson and gates (2007) resulted from ratsnakes leaving (or dying in) bottomlands when they flooded, or moving less when in flooded bottomlands. if ratsnakes remain in bottomlands during floods, how can we explain the complete absence of nest predation during a four-week flood reported by roy neilson and gates (2007)? absence of raccoons from flooded areas (cagle, 1949; hoover, 2006) may account for some but not all of the reduced predation. roy neilson and gates (2007) suggested that nest predators might switch to alternative prey during floods (e.g., pehrsson, 1985; summers, 1986), which could apply to ratsnakes. ratsnakes’ prey primarily on small mammals, and in our study area the white-footed mouse (peromyscus leucopus) is the principal mammalian prey (carfagno et al., 2006). in a study in a floodplain in illinois, batzli (1977) found that peromyscus leucopus responded to flooding by moving into trees. with both the mice and snakes restricted to trees during floods, it may be most profitable for the snakes to prey almost exclusively on mice, thereby reducing or eliminating snake predation on birds’ nests. testing this idea will require analyzing the diet of ratsnakes in bottomland forest during flooded and dry conditions. acknowledgements we thank k. bugle, a. hagen, r. kelly, and e. tetauer for assisting with snake fieldwork, j. whittington for assisting with surgeries, the illinois department of natural resources and the u.s. fish and wildlife service for providing support and accommodation, l. luiselli and an anonymous reviewer for comments that improved the manuscript, and the university of illinois and the nature conservancy for financial support. references batzli, g.o. (1977): population dynamics of the white-footed mouse in floodplain and upland forest. am. midl. nat. 97: 18-32. 194 g.l.f. carfagno and p.j. weatherhead blouin-demers, g., weatherhead, p.j. (2001): habitat use by black rat snakes (elaphe obsoleta obsoleta) in fragmented forests. ecology 82: 2882-2896. cagle, f.r. (1949): notes on the raccoon, procyon lotor megalodus lowery. j. mamm. 30: 45-47. carfagno, g.l.f., heske, e.j., weatherhead, p.j. (2006): does mammalian prey abundance explain forest-edge use by snakes? ecoscience 13: 293-297. carfagno, g.l.f., weatherhead, p.j. (2006): intraspecific and interspecific variation in use of forest-edge habitat by snakes. can. j. zool. 84: 440-1452. carfagno, g.l.f., weatherhead, p.j. (2008): energetics and space use: intraspecific and interspecific comparisons of movements and home ranges of two colubrid snakes. j. anim. ecol. 77: 416-424. fitch, h.s. (1963): natural history of the black rat snake (elaphe o. obsoleta) in kansas. copeia 1963: 649-658. hoover, j.p. (2006): water depth influences nest predation for a wetland-dependent bird in fragmented bottomland forests. biol. conserv. 127: 37-45. jobin, b., picman, j. (1997): factors affecting predation on artificial nests in marshes. j. wildl. manage. 61: 792-800. kennamer, r.a. (2001): relating climatological patterns to wetland conditions and wood duck production in the southeastern atlantic coastal plain. wildlife soc. bull. 29: 1186-1192. pehrsson, o. (1985): duckling production of the oldsquaw in relation to spring weather and small-rodent fluctuations. can. j. zool. 64: 1835-1841. roy neilsen, c.l., gates, r.j. (2007): reduced nest predation in cavity-nesting wood ducks during flooding in a bottomland hardwood forest. condor 109: 210-215. ryan, d.c., kawula, r.j., gates r.j. (1998): breeding biology of wood ducks using natural cavities in southern illinois. j. wildlife manage. 62: 112-123. sanchez-lafuente, a.m., alacantara, j.m., romero, m. (1998): nest-site selection and nest predation in the purple swamphen. j. field ornith. 69: 563-576. summers, r.w. (1986): breeding production of dark-bellied brent geese branta bernicla bernicla in relation to lemming cycles. bird study 33: 105-108. weatherhead, p.j., blouin-demers, g. (2004): understanding avian nest predation: why ornithologists should study snakes. j. avian biol. 35: 185-190. weatherhead, p.j., carfagno, g.l.f., sperry, j.h., brawn, j.d., robinson, s.k. (2010): linking snake behavior to nest predation in a midwestern bird community. ecol. appl., in press. weatherhead, p.j., hoysak, d.j. (1989): spatial and activity patterns of black rat snakes (elaphe obsoleta) from radiotelemetry and recapture data. can. j. zool. 67: 463-468. weatherhead, p.j., robertson, r.j. (1977): harem size, territory quality, and reproductive success in the red-winged blackbird (agelaius phoeniceus). can. j. zool. 55: 1261-1267. acta herpetologica 4(1): 1-6, 2009 unexpected phylogeographic affinities of psammodromus algirus from conigli islet (lampedusa) miguel a. carretero1, anna perera1, pietro lo cascio2, claudia corti3, d. james harris1 1 cibio, centro de investigação em biodiversidade e recursos genéticos, campus agrário de vairão, 4485-661 vairão, portugal. corresponding author. e-mail: carretero@mail.icav.up.pt 2 associazione “nesos”, via vittorio emanuele 24, i-98055 lipari (me), italy. 3 museo di storia naturale dell’università di firenze, sezione di zoologia “la specola”, via romana 17, i-50125 firenze, italy. abstract. the only italian population of the lacertid psammodromus algirus is found in conigli islet whereas the species is absent from the nearby island of lampedusa. the phylogeographic relationships of this population were investigated. mitochondrial dna (12s rrna and 16s rrna) fragment sequences were analysed and compared with already published sequences from the whole species range. in all the analyses, the sample from conigli grouped with those from morocco and not with the closer tunisian ones. such surprising result poses serious doubts to the traditional interpretation of the enigmatic distribution pattern of this species in italy suggesting a recent colonisation of the islet from nw africa, probably human-mediated, rather than a land crossing from tunisia during the pleistocene. keywords. phylogeography, psammodromus algirus, lacertidae, conigli islet. introduction the large psammodromus, psammodromus algirus, is a medium-sized lacertid with ground-dwelling habits extensively distributed on both sides of the western mediterranean basin (fig. 1). throughout its range, this generalist species is usually very common (carretero et al., 2002; sindaco, 2006) constrained only by the availability of some plant cover (carretero and llorente, 1997). however, in italy, the species is restricted to the small islet of conigli (lampedusa, pelagian islands), where it occupies an area of 3 ha covered by nitrophilous vegetation. since its discovery (zavattari, 1954), the presence of this population has remained biogeographically unexplained since the islet is separated only by a channel of 30 m width and 1.5 m depth from the main island of lampedusa where the species is completely absent (sindaco, 2006). distance to the closest african population on the tunisian coast is 130 km. recent literature tentatively invokes historical 2 m.a. carretero et alii changes in the landscape and predators due to human introductions to justify the absence from lampedusa (corti and lo cascio, 2002). the species seems, in fact, a poor islet colonizer (carretero et al., 1993), at least in comparison with podarcis sp. (corti et al., 1999; poulakakis et al., 2003; harris et al., 2005). however, phylogeographic evidence indicates that transmarine colonisations in this species are not unrealistic (carranza et al., 2006). using mitochondrial markers, a recent study (carranza et al., 2006) detected strong phylogeographic structure in the species. two clades separated by 3.6 my, one in east iberia and the other in west iberia and north africa, were detected. separation between iberia and north africa (1.9 my) indicates a crossing of the gibraltar strait, after the refilling of the mediterranean at the end of the messinian (5.6 my, hsü et al., 1977). moreover, within north africa, the separation between the populations from morocco and tunisia is estimated to be 1.5 my (carranza et al., 2006). in this context, here we aim to assess the phylogeographic affinities of the conigli population through comparison with the lineages previously described using mtdna sequence data in order to determine the origin of this colonization. material and methods a juvenile specimen of p. algirus was collected from conigli islet (35º30´37’’n, 11º33’30’’e, fig. 1) in september, 2005. once digital photographs were taken and a tail tip was collected, the lizard was released in the site of capture. in the lab, total genomic dna was extracted using standard methods, following harris et al. (1998). polymerase chain reaction primers used in both amplification and sequencing were 12sa and 12sb for the 12s rrna gene and 16sl and 16sh for the 16s rrna gene (kocher et al., 1989). amplification conditions were the same as described by harris et al. (1998). amplified fragments were sequenced using a 310 applied biosystem dna sequencing apparatus following the manufacturer’s protocols. mitochondrial dna sequences were aligned by eye. aligned sequences were 901 base pairs long. genbank accession numbers of new sequences are fj799792 (16s rrna) and fj799793 (12s rrna). the data were imported into paup* v. 4.0b10 (swofford, 2002) for phylogenetic analysis. both neighbour joining (nj) and maximum parsimony (mp) were used to estimate relationships. representatives of all major lineages found by carranza et al. (2006) were included. psammodromus blanci, p. h. hispanicus and p. h. edwarsianus were included as outgroups. for mp a 10 replicate heuristic search was used. in both cases support for nodes was estimated using the bootstrap technique (felsenstein, 1985) with 1000 replicates. additionally relationships were estimated in a bayesian framework, using mrbayes v. 3.1.2 (huelsenback and ronquist, 2001), with 1 × 106 generations using a general-time-reversible model of evolution with a gamma model of among site rate variation and an estimated proportion of invariable sites. after the burning period remaining trees were used to estimate posterior nodal probabilities. results including the outgroups, 31 combined mtdna sequences were analyzed. mp analysis found 5826 equally parsimonious trees of 234 steps. all estimates of relationships differed only at nodes without strong support (fig. 2). all analyses recovered the sample from 3phylogeography of psammodromus algirus from conigli islet conigli islet as a branch within a group otherwise comprised of samples from morocco. relationships between major clades differed slightly from those proposed by carranza et al. (2006) probably due to the weak resolution of these nodes. discussion although only a single specimen was sequenced, the two different mitochondrial markers undoubtedly indicate a phylogeographic affinity with the moroccan populations. such result not only allow ascribing the conigli population to the nominal subspecies (terra typica: ‘mauritania’ linnaeus, 1758) but also poses serious doubts on the traditional interpretation of the enigmatic distribution pattern of p. algirus in the pelagian archipelago. certainly, the bathymetry indicates that lampedusa was connected with tunisia during the last glacial maximum (masseti, 2002) and the small islet of conigli seems to have been united to lampedusa until the roman time (corti and lo cascio, 2002), so that both could have easily been colonised by land from tunisia during the pleistocene. the absence of the species from lampedusa was usually attributed to the degradation of the vegetation by imported herbivores together with the probable increase of predation pressure derived from the introduction of saurophagous snakes of the genera malpolon and macroprotodon (corti and lo cascio, 2002), both absent in conigli. however, in other regions of its distribution range, p. algirus occurs in habitats with low fig. 1. map showing distribution range of p. algirus in shade (after miras et al., 2006). in the square, lampedusa island with conigli islet marked with a circle. 4 m.a. carretero et alii plant cover and coexists with those snakes. conversely, the unexpected phylogeographic results obtained here suggest a more recent colonization from a distant area (nw africa), probably human-mediated. although infrequent, there is at least one case of introduction reported for mallorca, balearic islands (masius, 1999; vicens, 2005). this evidence, does not support the previously accepted theory of extinction, but makes it more feasible that the species had never existed on lampedusa (sindaco, 2006). further analyses of fossil remains could confirm or discard this last hypothesis. in conservation terms, the previously suggested introduction of this species in the main island (padoa-schioppa and massa, 2001) is, hence, not recommended. acknowledgements the lab work was funded by the project poci/bia-bde/55865/2004; m.a.c. and a.p. held the post-doctoral grants sfrh/bpd/27025/2006 and sfrh/bpd/26546/2006, all from fundação para a ciência e a tecnologia, fct (portugal). fieldwork funding and collecting permits were profig. 2. tree derived from nj analysis, based on partial 12s rrna and 16s rrna sequences. sample from conigli islet in bold, all others from carranza et al. (2006). nodes found in the strict consensus of 5826 equally parsimonious trees are indicated with an *. bootstraps from nj and mp are given above, and bayesian probabilities below the nodes. 5phylogeography of psammodromus algirus from conigli islet vided by riserva naturale orientata “isola di lampedusa” (management legambiente). thanks are especially due to giuseppina nicolini (legambiente) for her enthusiastic support and to vasco batista for field assistance. references carranza, s., harris, d.j., arnold, e.n., batista, v., gonzález de la vega, j.p. (2006): phylogeography of the lacertid lizard, psammodromus algirus, in iberia and across the strait of gibraltar. j. biogeog. 33: 1279-1288. carretero, m.a., bosch, m., pedrocchi, v. (1993): nuevos datos herpetológicos de la meda gran (islas medes, girona). bol. asoc. herpetol. esp. 4: 9-11. carretero, m.a., llorente, g.a. (1997): habitat preferences of two sympatric lacertids in the ebro delta (ne spain). in: herpetologia bonnensis, p. 51-62. böhme, w., bischoff, w., zeigler, t., eds, societas europaea herpetologica, bonn. carretero, m.a., montori, a., llorente, g.a., santos, x. (2002): psammodromus algirus. in: atlas y libro rojo de los anfibios y reptiles de españa, p. 259-261. pleguezuelos, j.m., márquez, r., lizana, m., eds, dirección general de conservación de la naturaleza-asociación herpetológica española, madrid. corti, c., lo cascio, p. (2002): anfibi e rettili. in: storia naturale delle isole pelagie, p. 7984. corti, c., lo cascio, p., masseti, m., pasta, s., eds, l’epos, palermo. corti, c., masseti, m., delfino, m., pérez-mellado, v. (1999): man and herpetofauna of the mediterranean islands. rev. esp. herpetol. 13: 83-100. felsenstein, j. (1985): confidence limits on phylogenies: an approach using the bootstrap. evolution 39: 783-791. harris, d.j., arnold, e.n., thomas, r.h. (1998): relationships of the lacertid lizards (reptilia: lacertidae) estimated from mitochondrial dna sequences and morphology. proc. r. soc. b.-biol. sci. lond. 265: 1939-1948. harris, d.j, pinho, c., carretero, m.a., corti, c., böhme, w. (2005): determination of genetic diversity within the insular lizard podarcis tiliguerta using mtdna sequence data, with a reassessment of the phylogeny of podarcis. amphibia-reptilia 26: 401-407. hsü, k.j., montadert, l., bernoulli, d., bianca, c.m., erickson, a., garrison, r.e., kidd, r.b., méliéres, f., müller, c., wright, r. (1977): history of the mediterranean salinity crisis. nature 267: 399-403. huelsenback, j.p., ronquist, f. (2001): mr-bayes: bayesian inference of phylogeny. bioinformatics 17: 754-755. kocher, t.d., thomas, w.k., meyer, a., edwards, s.v., pääbo, s., villablanca, f.x., wilson, a.c. (1989): dynamics of mitochondrial dna evolution in animals: amplification and sequencing with conserved primers. proc. nat. acad. sci. usa 86: 6196-6200. masius, p. (1999): first record of psammodromus algirus on mallorca island. die eidechse 10: 64. masseti, m. (2002): inquadramento, paleontologico, archeozoologico e paleobiogeografico. in: storia naturale delle isole pelagie, p. 27-30. corti, c., masseti, m., pasta, s., eds, l’epos, palermo. 6 m.a. carretero et alii miras, j.a.m., cheylan, m., nouira, m.s., joger, u., sá-sousa, p., pérez-mellado, v. (2006): psammodromus algirus. in: 2007 iucn red list of threatened species. iucn. www. iucnredlist.org. padoa-schioppa, e., massa, r. (2001): possibile effetto della predazione di ofidi sull’abbondanza e sulla taglia media dei sauri di lampedusa. nat. sicil. 25 (suppl.): 99-110. poulakakis, n., lymberakis, p., antoniou, a., chalkia, d., zouros, e., mylonas, m., valakos, e.d. (2003): molecular phylogeny and biogeography of the wall-lizard podarcis ehrardii (squamata: lacertidae). mol. phyl. evol. 28: 38-46. sindaco, r. (2006): psammodromus algirus. in: atlante degli anfibi e dei rettili d’italia / atlas of italian amphians and reptiles, p. 504-507. sindaco r., doria, g., razzetti, e., bernini, f., eds, societas herpetologica italica, polistampa, firenze. swofford, d.l. (2002): paup*: phylogenetic analysis using parsimony (and other methods) 4.0.b10. sinauer associates, sunderland, massachusetts, usa. vicens, p. (2005): sobre la presència de psammodromus algirus linnaeus, 1759 (sauria, reptilia) a mallorca. boll. soc. hist. nat. balears 48: 109-112. zavattari, e. (1954): rinvenimento di psammodromus algirus nell’isola dei conigli di lampedusa. boll. zool. 21: 93-98. acta herpetologica 2006 1 reptiles in the diet of a «oncorhynchus mykiss» (osteichthyes: salmonidae) naturalized population in piedmont (n italy) reptiles in the diet of a oncorhynchus mykiss (osteichthyes: salmonidae) naturalized population in piedmont (n italy) franco bernini 1, alessandro candiotto 1, pietro angelo nardi 1, simone rossi 1, and edoardo razzetti 2 1 dipartimento di biologia animale, università degli studi di pavia, piazza botta 9, i-27100 pavia – email: franco.bernini@unipv.it – 2 sistema museale d’ateneo, sezione museo di storia naturale, università degli studi di pavia, piazza botta 9, i-27100 pavia – e-mail: razzetti@unipv.it abstract. the authors report on predation by salmonid fish on some reptile species in two tributaries of the tanaro river in the alessandria province (nw italy). the remains of podarcis muralis, anguis fragilis and of an undetermined colubrid of the genus natrix were found in bromatological analyses performed on 117 oncorhynchus mykiss specimens. salmonid predation on herpetofauna once again confirms the alimentary opportunism of these fishes; however, predation is an occasional phenomenon and not a threat to the local reptile populations. keywords. predation by fish, salmonidae, oncorhynchus mykiss, anguis fragilis, podarcis muralis. while predation by salmonids and other fish on amphibian populations is already well documented (hecnar & m’closkey, 1997; picariello et al., 1996), information about predation by fish on reptiles is scarce (cf. eder et al., 1988). it therefore seems useful to report observations concerning this phenomenon obtained during a research about on the feeding of onchorhynchus mykiss (walbaum, 1792) in the northern apennines. the study was carried out in two tributaries of the tanaro river, the torrents lemme and gorzente, which are entirely confined to the territory of alessandria province. these water bodies have a relatively small section (width 4-5 m) within the study area, which is located in the mountainous-hilly reach of the tributaries (300-600 m a.s.l.). the plenty of emerging rocks provides a tight link between the aquatic environment and the surrounding landscape. fish communities in the study area feature four species of cyprinids, one gobiid, and consistent salmonid populations following human introductions. in addition to brown trout (salmo trutta) probably of atlantic origin, there are two of the very few populations of rainbow trout (oncorhynchus mykiss) naturalized in europe. bromatological analyses were performed on o. mykiss specimens caught by electrofishing in the rio del molino, an affluent of the gorzente stream and in the lemme acta herpetologica 1: 61-63, 2006 62 f. bernini et alii stream located at molini near fraconalto (alessandria province). the samples were collected from the beginning of february to the end of june 2001 and 2002. seven fish species were found (see table 1). we also observed the herpetofauna present along the banks of the collecting place. andreone & sindaco (1999) report six species of amphibians and twelve species of reptiles for the utm square in which the study area is located (table 2). we observed the following species during fish sampling: bufo bufo, rana temporaria, anguis fragilis, lacerta bilineata, podarcis muralis, natrix natrix and n. tessellata. r. temporaria had not previously been reported in this area; while the n. tessellata observation corroborates a historical record cited in andreone & sindacos’ atlas (1999). a hundred and seventeen specimens of rainbow trout (75 from the lemme torrent and 42 from rio del molino, total length 87-390 mm) were studied. analysis of stomach contents revealed predation of aquatic macroinvertebrates between february and april; whereas in late spring (may and june) diet was mainly based on the organisms living on table 1. fish species found in the lemme stream and in the rio del molino and relative abundance estimates (x=present; xx=abundant¸xxx=dominant). species lemme rio del molino salmo trutta xxx xx oncorhynchus mykiss xxx xxx barbus caninus x barbus plebejus xx leuciscus cephalus xx telestes muticellus xx xx padogobius bonelli x table 2. herpetofauna reported by andreone & sindaco (1999) for the utm square in which the research area is located. amphibians reptiles salamandra salamandra anguis fragilis triturus alpestris lacerta bilineata triturus carnifex podarcis muralis speleomantes strinatii chalcides chalcides bufo bufo coronella austriaca rana dalmatina coronella girondica zamenis longissimus hierophis viridiflavus natrix maura natrix natrix natrix tessellata (historical) vipera aspis atra 63predation by oncorhynchus mykiss on herpetofauna the stream banks and surrounding environments. in addition to the terrestrial and riparian invertebrates which make up most of the diet, some reptiles were noticed: remains of podarcis muralis, anguis fragilis and of an undetermined colubrid of the genus natrix were found in the digestive apparatus of four rainbow trout (see table 3). this finding confirms the already well-known fact (pentelow, 1932; delmastro, 1981) that the diet of salmonids is opportunistic. the presence of reptiles in the diet of o. mykiss was only observed in the summer months, in correspondence with the activity period of these animals. the capture of reptiles by salmonids is an occasional phenomenon, not to be considered a threat to the populations living along the watercourse, unlike their predation on crayfish and amphibians, also noticed during our study. references andreone, f., sindaco, r. (1999): erpetologia del piemonte e della valle d’aosta. atlante degli anfibi e dei rettili. monografia xxvi. torino. museo regionale di scienze naturali. delmastro, g.b. (1981): contributo allo studio dell’alimentazione di salmo gairdneri rich. (osteichthyes, salmonidae). riv. piem. stor. nat. 2: 71-78. eder, j., eder, h., schumacher, a., schumacher, d. (1988): die ringelnatter, natrix n. natrix (linnaeus, 1758) als beute der regenbogenforelle, salmo gairdneri richardson, 1836, und der seeforelle, salmo trutta lacustris linnaeus, 1758. herpetozoa 1(1/2): 69-71. hecnar, s.j., m’closkey, r.t. (1997): the effects of predatory fish on amphibians species richness and distribution. biol. cons. 79: 123-131. pentelow, f.t.k. (1932): the food of the brown trout (salmo trutta l.). j. an. ecol. 1(2): 101-107. picariello, o., scillitani, g., viglietti, s. (1996): prime osservazioni sulla predazione di rana italica dubois, 1987 da parte di salmo trutta l., 1758 nell’appennino campano. studi trent. sci. nat. – acta biol. 71(1994): 197-200. table 3. data on predation by oncorhynchus mykiss on herpetofauna (svl =snout-vent length; tl = total length; w = weight). prey svl of prey (mm) date watercourse tl of predator (mm) w of predator (g) podarcis muralis 38 25/05/01 lemme 250 177 podarcis muralis 02/06/01 rio del molino 179 56 anguis fragilis 02/06/01 rio del molino 189 70 natrix sp. 27/06/01 lemme 244 175 acta herpetologica 2006 2 escape behaviour in the neotropical frog hylodes asper (anura : leptodactylidae) escape behaviour in the neotropical frog hylodes asper (anura: leptodactylidae) claudio e.g. patto 1, marcio r. pie 2 1science department, st francis college, r. belgica 399, são paulo, sp, brazil 01448-030. e-mail: claudiop@stfrancis.com.br 2 departamento de zoologia, universidade federal do paraná, curitiba, pr, brazil. corresponding author. e-mail: pie@ufpr.br abstract. predation is an important selective pressure in natural populations, leading to the bewildering diversity of antipredation strategies found in nature. however, studies focusing on real-time assessment and management of risks by prey are underrepresented in the literature, particularly in the case of anurans. in this study we report on field observations of the escape behavior of the neotropical frog hylodes asper (leptodactylidae). escape distances varied according to the time of the day in both juveniles and adults. moreover, there was a significant influence of age on the escape distance of an individual, with adults escaping at a greater distance than juveniles. keywords. hylodes asper, escape behavior, brazilian rainforest. predation is an important selective pressure in natural populations, what is reflected in adaptations such as crypsis, protective armor, and mimicry (edmunds, 1974; curio, 1976). but even cryptic animals must make day-to-day and even moment-to-moment decisions regarding when and where to be active (lima and dill, 1990; rowe and owings, 1990; pie, 2005). therefore, animals must be able not only to assess the risks associated with interacting with a predator, but also to make behavioral decisions in ways that minimize these risks (rowe and owings, 1990). however, studies focusing on real-time assessment and management of risks by prey are underrepresented in the literature (lima and dill, 1990; kramer and bonenfant, 1997; kavaliers and choleris, 2001; welton et al., 2003). one possible way of addressing this question is to study the factors that influence the maximum distance a prey allows a predator to approach before fleeing, also called escape distance (ed) (rand, 1964). in this study we report on field observations of the escape behavior of the neotropical frog hylodes asper (leptodactylidae), assessing differences in the ed according to age and the time of the day. this study was conducted in a small basin of mountain streams in the brazilian atlantic rainforest. this microbasin comprises approximately 15 ha of forest drained by about 500 m of streams, varying in elevation from 500 to 600 m above sea level in paranapiacaba, municipality of santo andré, state of são paulo, brazil (23o28’s; 45o52’w). hylodes acta herpetologica 1(2): 141-146, 2006 142 c.e.g. patto, m.r. pie asper is a diurnal stream breeder frog (sul = 20 to 50 mm) frequently found in this region, usually perched on river rocks and logs. further details on this species and its habitat can be found in heyer et al. (1990). the protocol for measuring the ed is described as follows: a human observer walked cautiously and at constant speed towards the frog, up to a distance of 30 cm, when the observer attempted to capture it (in case it had not escaped before). the ed was then registered as the smallest distance between the observer and the frog at the moment of the escape. in case the animal did not escape or escaped after being touched the ed was registered as zero. the same observer conducted all the experiments. although the use of a “fake realistic predator” would be ideal, the logistic constraints of getting to the field site would render that alternative impractical. in addition, very little is known about the biology of this species to be able to determine which type of predator would be most realistic. finally, human observers have been successfully used as surrogate predators in a variety of studies (e.g. cooper, 2003; martin et al. 2004, 2005). we carried out these field experiments on two different days, a sunny day (n = 42) and an overcast day (n = 155 observations). observations were conducted along a transect, which was not revisited during the experiments, assuring the independence of the observations. the eds varied significantly across the day, being larger during the morning hours, smaller near midday, and larger again during the afternoon (fig. 1, table 1). this variation was more pronounced during the sunny day, with four-fold variation in mean ed (fig. 1a). this pattern was also present, but less pronounced, in the cloudy day (fig. 1b). there was a significant interaction between time of day and weather (table 1), showing that the daytime curve of escape distance varied with the cloudiness of the day. when age classes were analyzed separately, adults and juveniles differed in their mean ed along the day (tables 2, 3). adults escaped at a greater distance from the experimenter than juveniles. there was no significant interaction between ed for the different age classes and time of the day (table 2), showing that daytime ed curves are similar for both age classes. it is important to note that the time of the day was used as a proxy to environmental variation such as temperature, humidity, and luminosity. we therefore cannot single out temperature as the source of variation, yet this limitation does not invalidate our results, particular with respect to the difference between juveniles and adults. there is a very good visual match between the color pattern of hylodes asper and the rocks from the creeks were it is commonly found (pers. obs.). therefore, crypsis is presumably the first antipredator strategy employed by this frog species. additionally, there is also a backup strategy, which is to flee, jumping from the perching rock into the water. this strategy is efficient if the predator is able to overcome the initial crypsis strategy and detects a perching frog as a potential prey. however, if the frog flees every time it table 1. two-way anova comparing the mean escape distance along the daytime and according to the weather. variable df f p time of day 6 8.739 < 0.001 weather 1 3.246 0.073 time of day x weather 6 3.535 0.002 143escape behaviour in the neotropical frog hylodes asper detects a potential predator, besides the energetic costs of the escape itself and the interruption of other important activities like foraging and searching for mates, this response exposes the frog to the predator for a couple of seconds, and may actually increase the predation risk if it is taken at the wrong moment. the decision of when to shift from one 12 sunny day 08:00 time (h) 3 4 2 1 09:00 10:00 11:00 12:00 13:00 14:00 2 cloudy day time (h) 1.5 1 0.5 15:00 16:0014:0013:0012:0011:0010:0009:0008:0007:0006:00 fig. 1. daily variation in escape distance (ed) in hylodes asper along two different days (means + se). 144 c.e.g. patto, m.r. pie strategy to the other is critical, and factors like the physiological state of the frog must be very important (whiting 2002). the motor performance of ectotherms such as frogs declines with temperature (heckrotte, 1967; bennet, 1980; lillywhite, 1987). hence, at lower temperatures, frogs would tend to have slower escape responses, and therefore a higher probability of capture. in this case, frogs would be expected to be more cautious by fleeing sooner. the variation in ed of h. asper throughout the day and according to the weather detected in the present study is consistent with these predictions. many studies have demonstrated the influence of temperature on the defensive behavior of a variety of ectotherm vertebrates. prior & weatherhead (1994), studying the rattlesnake sistrurus c. catenatus, showed that warmer snakes were more likely to rattle and/or flee than cooler snakes. snakes are also more aggressive when tested at a lower temperature (arnold and bennet, 1984; scheffelin and de queiroz, 1991), also striking with higher velocity, greater accuracy, and less hesitation (rowe and owings, 1990). lizards such as anolis lineatopus also tend to flee sooner as the temperature decreases (rand, 1964). nonetheless, we can not rule out the influence of other factors on the variation in ed, such as an increase in the importance of the perching site to foraging or finding mates during the midday. the most puzzling aspect of our results is the observation that adults tend to escape when the experimenter was at a greater distance than did juveniles. martin et al. (2005) found similar results in a study of the escape behavior of the green frog (rana perezi), with smaller frogs appearing to rely on crypsis more than large frogs by allowing shorter escape distances. if viewed in terms of a trade-off between present and future reproductive success, juveniles have the highest interest in staying alive and therefore should escape sooner. moreover, since males are territorial, there is a clear conflict between territorial defense and avoiding predators; a hiding territorial resident is unable to monitor its tertable 2. two-way anova comparing the mean escape distance along the day and according to the observed age class. variable df f p time of day 9 2.076 0.035 age 1 7.365 0.007 time of day × age 9 1.159 0.320 table 3. variation in mean adult and juvenile ed along the day. hour n adult mean (m) se n juvenile mean (m) se 6-8 17 0.853 0.143 8 0.488 0.173 8-10 17 0.900 0.096 3 0.333 0.192 10-12 22 0.536 0.112 9 0.356 0.119 12-14 21 0.371 0.063 16 0.381 0.095 14-16 24 0.667 0.068 5 0.500 0.224 16-18 7 0.643 0.146 6 0.350 0.143 145escape behaviour in the neotropical frog hylodes asper ritory and to defend it from conspecific intrusions. in this case, we would predict that ed should be smaller in territorial males (e.g. diaz-duarte, 1999). even though females are not territorial in this species (patto and pie, unpublished results), the disruption of mate searching activities should affect ed in the same fashion. why did adults attempt to escape sooner? predators could be more motivated to pursue and attack larger prey, and the observed pattern would be the result of a size-dependent predation strategy. alternatively, predators could be equally motivated to pursue any prey item, but larger prey could be more easily detected than smaller prey. detailed experimental studies are necessary to discriminate between those hypotheses. acknowledgements we thank c.f.b. haddad and d. rosa-feres for valuable discussion. this work was funded by cnpq – conselho nacional de desenvolvimento científico e tecnológico (cegp) and fundação coordenação de aperfeicoamento de pessoal de nível superior – capes (mrp), and was carried out while the authors were part of the graduate program in ecology of the campinas state university. references arnold, s.j., bennett, a.f. (1984): behavioural variation in natural populations. iii: antipredator displays in the garter snake thamnophis radix. anim. behav. 32: 11081118. bennet, a.f. (1980): the thermal dependence of lizard behaviour. anim. behav. 28: 752-762. cooper, w.e. (2003): effect of risk on aspects of escape behavior by a lizard, holbrookia propinqua, in relation to optimal escape theory. ethology 109: 617-626. curio, e. (1976): the ethology of predation. springer-verlag, new york. diaz-duarte, r. (1999): anti-predator behaviour changes following an aggressive encounter in the lizard tropidurus hispidus. proc. r. soc. lond b 266: 2457-2464. edmunds, m. (1974): defense in animals. longman group limited, new york. heckrotte, c. (1967): relations of body temperature, size, and crawling speed of the common garter snake, thamnophis s. sirtalis. copeia 1967: 759-763. heyer, w.r., rand, s.a., cruz, c.a.g., peixoto, o.l., nelson, c.e. (1990): frogs of boraceia. arq. zool., são paulo 31: 231-410. kavaliers, m., choleris, e. (2001): antipredator responses and defensive behavior: ecological and ethological approaches for the neurosciences. neurosci. biobehav. rev. 25: 577-586. kramer, d.l., bonenfant, m. (1997): direction of predator approach and the decision to flee to a refuge. anim behav. 54: 289-295. lillywhite, h.b. (1987): temperature, energetics, and physiological ecology. in: snakes: ecology and evolutionary biology, p. 25-91. siegel, r.a., collins, j.t., novak, s.s., eds, macmillan, new york. 146 c.e.g. patto, m.r. pie lima, s.l., dill, l.m. (1990): behavioral decisions made under the risk of predation: a review and prospectus. can. j. zool. 68: 619-640. martin, j., de neve, l., fargallo, j.a., polo, v., soler, m. (2004): factors affecting the escape behaviour of juvenile chinstrap penguins, pygoscelis antarctica, in response to human disturbance polar biol. 27: 775-781. martin, j., luque-larena, j.j, lopez, p. (2005): factors affecting escape behavior of iberian green frogs (rana perezi). can. j. zool. 83: 1189-1194. pie, m.r. (2005): signal evolution in prey recognition systems. behav. proc. 68: 47-50. rand, a.s. (1964): inverse relationship between temperature and shyness in the lizard anolis lineatopus. ecology 45: 863-864. rowe, m.p., owings, d.h. (1990): probing, assessment and management during interactions between ground squirrels and rattlesnakes. i. risks related to rattlesnake size and body temperature. ethology 86: 237-249. schieffelin, c.d., de queiroz, a. (1991): temperature and defense in the common garter snake: warmer snakes are more aggressive than cold snakes. herpetologica 47: 230237. welton, n.j., mcnamara, j.m., houston, a.i. (2003): assessing predation risk: optimal behaviour and rules of thumb. theor. popul. biol. 64: 417-430. whiting, m.j. (2002): field experiments on intersexual differences in predation risk and escape behaviour in the lizard platysaurus broadleyi. amphibia-reptilia 23: 119-124. rediscovery of pelobates fuscus insubricus in the asti province, north-western italy vincenzo mercurio1, fabrizio li vigni2 1department of ecology and evolution, johann wolfgang goethe university and research institute and natural history museum senckenberg, section herpetology, senckenberganlage 25, d-60325 frankfurt a.m., germany. corresponding author. e-mail: vincenzomercurio@gmx.de 2viale francia 5, i-90146 palermo, italy. e-mail: fabrizio_livigni@hotmail.com abstract. the amphibians of the pond complex “stagni di belangero” in the po plain, asti province, have been studied. the species living in the pond are bufo bufo, bufo viridis, hyla intermedia, pelobates fuscus, rana dalmatina, rana synklepton esculenta, rana cf. kurtmuelleri, triturus carnifex and triturus vulgaris. species composition, migration period of p. fuscus, and biometric data are provided. relevant importance has been given to p. fuscus, since we reconfirm its presence in one of the ponds 13 years after its first finding in the area. this toad is also one of the most threatened species of amphibians in europe and needs particular attention in order to be protected adequately. keywords. pelobates fuscus insubricus, rediscovery, cis stagni di belangero, community composition, migration period. introduction in the last years, the italian subspecies of the spadefoot toad, pelobates fuscus insubricus, has been object of an increasing interest for its natural history, distribution and conservation (andreone and pavignano, 1988; lapini et al., 1993; andreone et al., 1993, 2004; gentilli et al., 1996; mazzotti et al., 2002; scali and gentilli, 2003). this anuran was considered one of the most endangered taxon of all european herpetofauna by bruno et al. (1974), corbett (1989), and andreone and luiselli (2000, 2001): for this reason it was included in annex ii of the european directive 92/43 habitat among the species with priority of conservation (council directive 92/43/eec, 1992). the asti province is characterised by only few recent and scattered data inherent to amphibians and reptiles distribution (gambino et al., 1993; andreone and sindaco, 1998). among the formerly listed species, p. fuscus is of particular interest (gambino et al., 1993). acta herpetologica 2(1): 1-6, 2007 2 v. mercurio and f. li vigni its presence in the “stagni di belangero” was detected on the basis of tadpoles discovery (i. pavignano, pers. comm.), but no voucher specimen is available to confirm the locality record. after a period of 13 years since this finding in the “stagni di belangero”, the current study has been conducted in order to reconfirm the presence of p. fuscus. moreover, we here provide some information regarding the composition of the amphibians community inhabiting the same ponds. material and methods study area the study area is known as “stagni di belangero” (id code it1170003). it is considered as a communitarian important site (cis) and is located on the right shore of the tanaro river, in the “s. marzanotto” alluvial plain, asti province, north-eastern italy (coordinates: 43°78’73’’e, 49°70’66’’n; 120 m a.s.l.). the area is 591 ha in size. it is mainly covered by maize fields, which constitute about the 45% of the land cover, uncultivated fields, some water bodies and aspen plantations, together reaching the 38% of the coverage. the riverbed of the tanaro and the vegetation associated to the humid areas form an additional 10%, represented by populus alba, salix alba and s. caprea. the remaining part of the area is occupied by buildings and roads. the aquatic vegetation is present in almost all the water bodies. it is referred to the alliances of nymphaeion albae and/or hydrocharition, while the terrestrial vegetation is almost entirely represented by a dense bush of amorpha fruticosa and robinia pseudoacacia. wet areas are represented by 20 ponds of anthropic origin of different dimension and by an extensive system of drainage canals. introduced fish species are ictalurus melas, lepomis gibbosus and micropterus salmoides. data collecting opportunistic surveys were carried out from 26 march to 06 may 2004. the observations were aimed to determine spadefoot toad occurrence by means of direct observations during diurnal and nocturnal excursions. from the end of march to the beginning of may 2004, we monitored intensively amphibians in a selected site by means of drift-fence and pitfall traps. the selected site is an ephemeral drainage canal 100 m long, 1-5 m wide, and 10-60 cm deep. other ponds occur all around the canal within a range of 200 m, but these are inhabited by fish. the selected site was partially fenced with 30 pitfalls (25 cm diameter, 25 cm deep) located at a distance of 5 m one from each other. the traps have been checked twice a day, in the morning and in the evening, for a period of permanence on the field variable in function to the presence of the animals in activity. collected toads were marked with single toe-clipping and photographed for dorsal pattern identification. regarding the other species of amphibians the following biometrical measurements were taken: weight (w), snout vent length (svl), and tail length (tl) only for newts. 3pelobates fuscus insubricus rediscovery results pelobates fuscus insubricus during the study period 14 individuals were trapped: 12 adult males and 2 adult females. three males and one female were recaptured, while two other toads were observed out of the pitfall traps. in particular, on 26 march, while digging out to build up the drift-fence, we found an adult burrowed about 20 cm deep underground and 4 m far from the shore of the focal site. the first breeding spadefoot toad was found in water on 2 april, even if no spadefoot toad was directly seen entering the canal. on the contrary, the first toad leaving the pond was observed on 5 april and the last on 30 april. the migration at the “stagni di belangero” is resumed in fig. 1. biometric values (mean ± sd) of the sampled individuals are reported in table 1. amphibians community associated to spadefoot toads the total of amphibians other than spadefoot toads captured in the pitfall traps was 275. following, the species found and in parenthesis the number of individuals: triturus carnifex (130), t. vulgaris meridionalis (6), hyla intermedia (37), bufo viridis and rana dalmatina (12 each), b. bufo (6) and rana synklepton esculenta (72). probably, the low fig. 1. migration from the breeding site of pelobates fuscus insubricus at the study site. 4 v. mercurio and f. li vigni numbers of t. vulgaris, b. bufo, b. viridis and r. dalmatina are related to the late timing of the research rather than reflecting their relative abundance in the area. biometric values (mean ± sd) of the sampled individuals are reported in table 1. discussion this study allowed us to rediscover p. fuscus in the alluvial plane of the tanaro river in the asti surroundings, an area characterised by intensive agricultural exploitation. the sampled population appeared to be small, with a total of 14 specimens intercepted. we ignore if this result reflects a sign of the rarity of the species, or rather if it is a consequence of the study methods or of the meteorological conditions. however, results of a recent study carried out in 33 ponds in scania (south sweden), with the use of hydrophones, analysed some populations of the nominal subspecies and they showed an average of 13 males per pond, with a number of specimens ranging between 1 and 80 (nyström et al., 2002). on the other hand, as for our study, if we take into account (1) that all the trapped individuals were already leaving the breeding site, (2) the bias between the number of males and females (sex ratio = 6), and (3) that the site was only partially fenced, we are inclined to believe that the real population could be bigger than the detected one. we failed to contact the animals during the migration to the breeding site which likely started before 26 march, in spite of the low temperatures and of the absence of rain. indeed, meteorological data inherent to this previous period report low temperatures (0 °c), going from the end of february to 11 march, with snowy precipitations and frost on nights. this harsh period was then followed by an odd and abrupt increase of temperatures up to 13° c in the period comprised between 12 and 16 march (www.meteo.it/almanacco/anno/681_awepson.htm). from examination of still unpublished data upon a population of p. f. insubricus, located in poirino (turin province), about 30 km eastward from our study site, it emerged that the reproductive migration of the observed population started on 15 march (crottini and table 1. biometric values (in mm ± standard deviation): svl, snout-vent length mean; tl, tail length; w, weight; n, number of examined specimens; (m), males; (f), females. species n svl tl w m f m f m f m f p. fuscus 12 2 45.6 ± 2.3 52.3 ± 0.4 13.2 ± 2.1 29.4 ± 0.5 b. viridis 1 2 61.6 67.4 ± 3.7 33.0 35.3 ± 2.5 h. intermedia 6 7 34.05 ± 3.63 40.36 ± 3.84 5.0 ± 0.96 7.55 ± 1.06 r. dalmatina 2 33.62 ± 3.99 4.95 ± 0.07 r. cf. kurtmuelleri 3 1 68.72 ± 10.86 86.0 39.83 ± 14.37 94.0 t. carnifex 1838 70.64 ± 17.13 73.78 ± 22.68 45.99 ± 7.34 50.13 ± 8.39 5.22 ± 1.06 6.06 ± 1.6 t. vulgaris 2 1 39.0 ± 16.69 50.0 33.73 ± 13.11 46.0 2.25 ± 2.33 5.50 5pelobates fuscus insubricus rediscovery andreone, 2006), in coincidence with the temperature increase. from the comparison with these data, we hypothesise a similar timing for the studied population. further investigations are needed to understand if temperature increase could play an important role as it is stated for the spring rain to end the wintering period of p. fuscus (scali & gentilli, 2003). the migration period of our population of p. fuscus comprised between the first and the last intercepted specimen lasted 25 days. basing upon the available comparative data on p. fuscus, a similar migration period is reported by andreone et al. (2004), in a study carried out near ivrea surroundings. here the migration period lasted 18 days, from 17 april to 4 may. on the other side, our result differs significantly from what reported by andreone and pavignano (1988), whose data show a quickly migration of only three days. from such cases, the species turned out to be an explosive breeder but with a reproductive period strongly influenced (that is, prolonged or shortened) by the different climatic conditions. among the detected species, and besides the spadefoot toad, of particular interest it is the probable presence of rana cf. kurtmuelleri. of the nearly 80 “green frogs” observed, some of them presented very large body dimension (a female had a 150 mm svl, and weighed 130 g), whereas all individuals presented a dorsal pattern composed by green blotches on a grey-brown ground color. these characteristics fit with the description of this introduced taxon. the pelobates fuscus insubricus represents a remarkable presence in the stagni di belangero and in the asti province. unfortunately, despite the rediscovery after more than ten years from the first record, and although the area has been included inside “natura 2000 net” (council directive 92/43/eec, 1992), the situation for this population of p. fuscus turns out to be very rough, since the area is heavily affected by anthropogenic disturbance and intensive agricultural exploitation. from all we have hereby exposed, it seems imperative the necessity to carry out further researches about p. fuscus, in order to improve our still scarce knowledge. acknowledgements this research has been possible only thanks to the help and assistance of several persons. vincenzo mercurio wishes to thank: luisa aivano, enrico caprio, luca chiusano, mario cozzo, guido giovara, luigi iguera, nico marinetto, francesco mendola, giancarlo ravetti and angelo rossi. thanks to paolo eusebio bergò and fabio viarengo for the assistance in the field. a special thank goes to piero perosino, for the help during the preliminary phase and the irreplaceable assistance during the field research. last but not least, thanks to franco andreone for improving the first draft of the manuscript and for sharing unpublished data with us. the work was possible due to the agreement and financial support of the ente di gestione dei parchi e delle riserve naturali astigiani. references andreone, f., bergò, e., p., bovero, s., gazzaniga, e. (2004): on the edge of extinction? the spadefoot pelobates fuscus insubricus in the po plain, and a glimpse at its conservation biology. ital. j. zool. 71: 61-72. 6 v. mercurio and f. li vigni andreone, f., fortina, r., chiminello, a. (1993): natural history, ecology and conservation of the italian spadefoot toad, pelobates fuscus insubricus. [storia naturale, ecologia e conservazione del pelobate insubrico, pelobates fuscus insubricus]. zoological society “la torbiera”-scientific reports 2: 1-93. andreone, f., luiselli, l. (2000): the italian batrachofauna and its conservation status: a statistical assessment. biol. conserv. 96: 197-208. andreone, f., luiselli, l. (2001): corrigendum to: “the italian batrachofauna and its conservation status: a statistical assessment”. [biol. conserv 96 (2000) 197: 208]. biol. conserv. 97: 269. andreone, f., pavignano, i. (1988): observations on the breeding migration of pelobates fuscus insubricus cornalia, 1873 at a ditch in north western italy (amphibia, anura, pelobatidae). bull. mus. reg. sci. nat. torino 6: 241-250. andreone, f., sindaco, r. (1998): erpetologia del piemonte e della valle d’aosta, atlante degli anfibi e dei rettili. monogr. mus. reg. sci. nat., torino 26: 1-283. bruno, s., burattini, e., casale, a. (1974): il rospo bruno del cornalia, pelobates fuscus insubricus cornalia 1873 (amphibia, anura, pelobatidae). in: atti iv simposio nazionale sulla conservazione della natura, p. 33-55. vol. iii, bari. corbett, k. (1989): the conservation of european reptiles and amphibians. christopher helm, london. crottini, a., andreone f. (2006): volunteering to monitor an isolated population of pelobates fuscus in nw italy. in: bologna m.a., capula m., carpaneto g.m., luiselli l., marangoni c., venchi a. (eds.): riassunti del 6° congresso nazionale della societas herpetologica italica (roma, 27 settembre 1 ottobre 2006). stilgrafica, roma: 169170. gambino, e., laiolo, p., gallo, m., giacoma, c. (1993): distribuzione degli anfibi in provincia di asti. suppl. ric. biol. selvaggina 21: 693-706. gentilli, a., scali, s., zuffi, m. (1996): confirmation of the presence of pelobates fuscus insubricus in province of varese (amphibia, anura, pelobatidae). natura bresciana, ann. mus. civ. sc. nat., brescia 30 (1994): 259-262. lapini, l., dall’asta, a., richard, j. (1993): pelobates fuscus insubricus cornalia, 1873 (amphibia, salientia, pelobatidae) in north-eastern italy. atti mus. civ. stor. nat. trieste 45: 159-162. mazzotti, s., penazzi, r., lizzio, l. (2002): nuove segnalazioni di pelobates fuscus insubricus cornalia, 1873 nel sistema dei biotopi costieri del ravennate. quaderno di studi e notizie di storia naturale della romagna 17: 91-97. nyström, p., birkedal, l., dahlberg, c., brönmark, c. (2002): the declining spadefoot toad pelobates fuscus: calling site choice and conservation. ecography 25: 488-498. scali, s., gentilli, a. (2003): biology aspects in a population of pelobates fuscus insubricus cornalia, 1873 (anura: pelobatidae). herpetozoa 16: 51-60. book review bartlett and bartlett, 2006. guide and reference to the amphibians of eastern and central north america (north of mexico). bartlett and bartlett, 2006. guide and reference to the crocodilians, turtles and lizards of eastern and central north america (north of mexico). bartlett and bartlett, 2006. guide and reference to the snakes of eastern and central north america (north of mexico). book review: scopes of the volumes. the bartlett and bartlett (2006a, b, c) series of herpetological guides are a recent and informative guide on all the known herpetological species of eastern and central north america (north of mexico), comprising the recent or less recent pest or invasive species (alloctonous taxa). general comments. lacks of scientific descriptor and date of description. bibliografic references lack completely from the main text, limiting the immediacy of any taxonomic or ecological information. habitat pictures are lacking. measures in inches. interesting prize ($ 29.95 each). it has been though more for herpetoculturists than properly for biologists, but it could be a very useful volume(s) in most scientific libraries. bartlett r.d. and bartlett p.p., 2006. guide and reference to the amphibians of eastern and central north america (north of mexico). individual volume report. bartlett and bartlett (2006a) volume has 283 pages, 257 colour photos, several identifying factors, body size, abundance and range in e and cn america, 168 distribution maps, notes on similar species and legal status. bibliografic references lack completely from the main text, limiting immediacy of taxonomic or ecological information. as example: the authors mention that some hybrids could be triploid (p. 125), not explaining the meaning, nor inserting the term in the glossary. some minor flaws: legend on page 133 (fig. 97). glossary is short, but fairly comprehensive. key to faimilies: are simple and intuitive, quite informative. pictures: do not perfectly resemble species, but look quite attractive. photos: are good on average despite many of them reflect of brownish to reddish dominance; on the whole and for many species they offer a valuable range of variability among related taxa. a volume easy in reading and consultation. of interest the complete list of the few introduced amphibian species and comments on them. acta herpetologica 2(2): 159-160, 2007 issn 1827-9643 (online) © 2007 firenze university press 160 book review bartlett r.d. and bartlett p.p., 2006. guide and reference to the crocodilians, turtles and lizards of eastern and central north america (north of mexico) individual volume report. bartlett and bartlett (2006b) volume has 309 pages, 255 colour photos, several identifying factors, body size, abundance and range in e and cn america, 149 distribution maps, notes on similar species and legal status. a short but useful glossary. key to families: are simple and intuitive, and quite informative. pictures and photos: range from good to excellent. habitat pictures are lacking. figure legends are often not enough informative. a volume easy in reading and consultation. of particular interest is the list of the known introduced species and comments on them, their origin and reproduction success, if any. bartlett r.d. and bartlett p.p., 2006. guide and reference to the snakes of eastern and central north america (north of mexico). bartlett and bartlett (2006c) volume has 342 pages, 209 colour photos, several identifying factors, body size, abundance and range in e and cn america, 98 distribution maps, notes on similar species and legal status. glossary is short, and comprehensive. key to families: are simple and intuitive, quite informative. photos: are very good in most cases (some exceptions: photo on page 18), and for some species a good range of variability is shown. maps exhaustive, despite the low scale. a volume easy in reading and consultation. of interest is the complete list of the six introduced snake species and comments on them, their origin and reproduction success, when reported. correspondence: marco a.l. zuffi, 1museo di storia naturale e del territorio, università di pisa, via roma 79, i-56011 calci (pisa), italy. e-mail: marcoz@museo.unipi.it road mortality threatens small northern populations of the european pond turtle, emys orbicularis giedrius trakimas1, jonas sidaravičius2 1 center for ecology and environmental research, vilnius university, m.k. čiurlionio 21/27, lt-03101, vilnius, lithuania. corresponding author. e-mail: giedrius.trakimas@gf.vu.lt 2 veisiejai regional park, santarvės 9, lt-67340, veisiejai, lithuania. submitted on 2008, 2nd january; revised on 2008, 22nd january; accepted on 2008, 20th may. abstract. little is known about road mortality and the effects to european pond turtle emys orbicularis populations at the northern border of its range. survival of the turtle populations in suboptimal conditions depends heavily on longevity, regular annual breeding and relatively large clutch sizes, but additional unnatural mortality could alter their survival rates. loss of only single turtle in majority of northern populations could mean a loss of 3-20% of subpopulation. but due to comparative rarity of the road accidents the effects of individual road mortality to the turtle populations might not be recognized. we discuss possible effects of road–associated mortality, and suggest that precautionary measures as setting of the buffer zones with low road density and possibility of lowering of traffic volume must be considered during the planning of the species conservation actions. keywords. freshwater turtles, conservation, road ecology, small populations, threats. road networks and traffic have diverse and complex ecological effects to animal populations (reviewed in, forman and alexander, 1998; trombulack and frissell, 2000). they fragment habitats and create barriers (mader, 1984; andrews and gibbons, 2005), increase human use of the landscape that leads to negative effects to populations (findlay and houlahan, 1997) and can cause significant mortality of individuals (ashley and robinson, 1996; aresco, 2005). slow-moving species such as ranid frogs, snakes and freshwater turtles are suspected to be especially affected by the road mortality (smith and dodd, 2003). european pond turtle (emys orbicularis linnaeus, 1758) is widely distributed species in europe, but it has become rare in most of the countries where it occurs (fritz and andreas, 2000; ficetola et al., 2004). several anthropogenic threats are supposed to be the cause of this decline. the most frequently cited are habitat loss, poaching for food or commercial purposes and introduction of exotic species (arvy and servan, 1998; cadi and acta herpetologica 3(2): 161-166, 2008 issn 1827-9643 (online) © 2008 firenze university press 162 g. trakimas and j. sidaravičius joly, 2003; kotenko, 2004; puky et al., 2004; rivera and fernandez, 2004). the negative effects of road mortality has been widely described for freshwater turtles in north america (see haxton, 2000; gibbs and shriver, 2002; marchand and litvaitis, 2004; steen et al., 2006), but was rarely mentioned among the e. orbicularis threats in europe. schneeweiss (2002) reported six known road casualties of e. orbicularis that lead to immediate death or animals died from injuries within few days, during period 1980-2000 in brandenburg, germany. these casualties made up 31.6% of all known direct losses caused by human during this period. in south lithuania near veisiejai, three e. orbicularis road kills were observed in 2004-2005 (our data). two killed pond turtles were adults. one of them was male found dead on busy state road no. 134 (average daily traffic: mean ± sd = 1550.7 ± 179.0), another was reported as killed near mikabaliai. the third was a juvenile killed on the gravel road (adt: mean ± sd = 10.7 ± 2.08) 12 meters away from the hatchling site. all three cases appeared in may, in small local populations. in poland, during the long-term field surveys, some adult individuals were reported to be killed on roads (najbar, 2005). most of the e. orbicularis populations distributed nearly northern border of its range are small and scattered (jablonski, 1992; vaičiūnaitė, 1992), sometimes they are considered to be too small to recover themselves (schneeweiss, 2004a) or only single, old individuals occur (balčiauskas et al., 1999). though only a few reports on road mortality of this species in the northern populations are available, careful evaluation is needed in determining whether the road mortality considerably affects present local populations and their future survival. being a semi-aquatic species, e. orbicularis is potentially in danger to the road mortality during its terrestrial activity. females within the nesting season, while searching for suitable nesting site near the road or crossing the roads during migration, could be subjects of increased danger from vehicles and interruption from human. thus, increased sexspecific mortality could alter demographic structure of local populations that was demonstrated with north american freshwater turtle species (steen at al., 2006). though, lack of evidence on altered population structure for northern e. orbicularis populations, data on nesting behaviour show that females perform long distance migrations during the nesting season. in lithuania female turtles move up to 1 km to nesting places (meeske, 1997), in germany up to 1.5 km (schneeweiss et al., 1998), in poland even more than 4 km (jablonsky and jablonska, 1998). when such movements intersect with roads turtles are potentially vulnerable to road associated mortality. additionally, danger form the road-associated mortality could be potentially higher when females choose to nest near the roads, and when the nesting areas are in dense rural or forest road network. in northern populations of e. orbicularis female’s nesting activity (searching of nesting site and construction of the nest) can last several days (meeske and mühlenberg, 2004). they usually start to search for the nesting site between 1700 h and 1800 h, it can last several hours or more than one day, then spend 70-250 min for nest’s construction: for excavation, laying of eggs and closing the nest (meeske, 1997). moreover, sometimes they dig their nests on the field roads (mitrus, 2006; our data). even if females build the nests in the environment with low traffic volume, prolonged staying near the roads during the nesting activities could higher the risk for e. orbicularis females to be disturbed, taken away from the population or killed. similarly, hatchlings from nests neighboring to the roads could be affected by the same threats. understanding that e. orbicularis is not strictly aquatic species (ficetola and de bernardi, 2006) suggests that potential risk from the road mortality previously might be 163road mortality in emys orbicularis underestimated. since both sexes performed frequent overland movements in lithuania (e.g., meeske, 2000), similarly in italy (lebboroni and chelazzi, 1991) together with data on killings of e. orbicularis males in brandenburg (schneeweiss, 2002), where at least two males (30%) were killed, and our data from veisiejai, at least one male found dead on the road, illustrate that not only females could contribute to the road casualties of small northern populations of pond turtles. studies of relict northern populations of e. orbicularis in east germany showed that hatchlings survive hibernation in nests only under favorable weather conditions (schneeweiss and jablonsky, 2000). but longevity of pond turtles and the regular annual clutch size can compensate climate losses (schneeweiss, 2002). nevertheless, complicated natural history, characterized by delayed maturity and high adult survival rates (e.g., mitrus and zemanek, 2004), makes the e. orbicularis extremely vulnerable against direct and indirect human pressure, that caused the collapse of the species population in brandenburg (schneeweiss, 2004b). road mortality is suspected to be one of the factors for decline of freshwater turtles in usa (gibbs and shriver, 2002). turtle demography is unusual to such a degree that slight increases in adult mortality can lead large declines in populations (e.g., congdon et al., 1993). according to brooks et al. (1991) less than 10% annual mortality of matured females may lead to population declines. e. orbicularis populations at the northern edge of distribution are small and scattered. for example in southern lithuania 70% of populations consist under 30 individuals, and 50% have five or less individuals (meeske et al., 2006). for such small local populations, loss of only single turtle of specific age and gender could mean a loss of 3-20% of whole subpopulation, and may shift demographic structure of the population. together with other negative factors as predation and limited recruitment due to the climate losses, this may contribute to the local extinction. the estimation of declines and the detection of threats for small northern populations of e. orbicularis may be difficult without long-term monitoring, thus the effects of individual road mortality might not be recognized, due to comparative rarity of the road accidents. nevertheless, precautionary measures as setting the buffer zones with low road density and possibility of lowering of traffic volume at e. orbicularis habitats must be considered during the planning of conservation actions. acknowledgements we thank jurgis klimas for field assistance, martina anne-claire meeske, sławomir mitrus, norbert schneeweiss and alius ulevičius for their help. gentile francesco ficetola and rimvydas juškaitis provided helpful comments on the manuscript. references andrews, k.m., gibbons, j.w. 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(2006): relative vulnerability of female turtles to road mortality. anim. conserv. 9: 269-273. 166 g. trakimas and j. sidaravičius trombulak, s.c., frissell, c.a. (2000): review of ecological effects of roads on terrestrial and aquatic communities. conserv. biol. 14: 18-30. vaičiūnaitė, r. (1992): balinis vėžlys. in: lietuvos raudonoji knyga, p. 71. balevičius, k., ed., lietuvos respublikos aplinkos apsaugos departamentas, vilnius. © firenze university press www.fupress.com/ah acta herpetologica 5(1): 119-130, 2010 future climate change spells catastrophe for blanchard’s cricket frog, acris blanchardi (amphibia: anura: hylidae) malcolm l. mccallum texas a&m university-texarkana, 2600 robison road, texarkana, texas 75501, usa. e-mail: malcolm.mccallum@herpconbio.org submitted on: 2009, 5th june; revised on 2010, 16th march; accepted on: 2010, 19th april. abstract. climate change may be one of the greatest environmental catastrophes encountered by modern human civilization. the potential influence of this global disaster on wildlife populations is subject to question. i interpolated how seasonal variation in weather patterns influences growth and reproduction in the blanchard’s cricket frog (acris blanchardi). then i extrapolated the influence of future climate conditions on these life history characteristics using fuzzy regression. fuzzy regression was an accurate predictor of growth and reproduction based on the climate conditions present from 1900–2007. it predicted that the climate projections expected for arkansas by 2100 could reduce total reproductive investment in the blanchard’s cricket frog by 33–94%. if these results reflect responses by other poikilotherms, climate change could induce major population declines in many species. because poikilotherms represent the vast majority of vertebrates and significant ecosystem components, it is imperative that we implement strategies to reduce greenhouse gas emissions and circumvent this possible catastrophe. key words. acris blanchardi, amphibians, climate change, fuzzy regression, global warming, growth, reproduction, reproductive investment introduction the impending threat of climate change is a serious concern for the global scientific community (opdam and wascher, 2004; travis, 2000). i am especially concerned about how amphibians respond to this stressor because current amphibian extinction rates are progressing so fast (blaustein et al., 1994; mccallum, 2007; roelants et al., 2007; stuart et al., 2005; wheeler et al. 2002). the potential causes of these declines are numerous and include habitat degradation and loss (brooks et al., 2002), introduced species (adams, 1999), pollution (dunson et al., 1992), contaminants (reeder et al., 1998; relyea, 2005), pathogens (berger et al., 1998; daszak et al., 2002), climate change (pounds and crump, 120 m.l. mccallum 1994; pounds et al., 1999), or interactions among several factors (mccallum and trauth, 2003; pounds et al., 2006; trauth et al., 2006). alteration of precipitation patterns due to climate change may influence many aspects of the biology of an organism. it may partly explain the demise of the golden toad (pounds et al., 1999) and may drive amphibian disease epidemics such as chytrids (pounds et al., 2006). the warming climate may even reduce the intensity of sexual selection, especially by influencing call parameters (gerhardt and mudry, 1980; sullivan, 1982; cocroft and ryan, 1995). it can reduce the overall abundance in anurans (piha et al., 2007). climate associated drought can drive population fluctuations by selecting against specific age classes of rainforest frogs (stewart, 1995) and certainly would positively or negatively influence temperate species as well. in the case of the golden toad and the harlequin frog, the humid climate needed for survival migrated above the mountain leaving no acceptable habitat for these species (pounds et al., 1994). interactions between the increased drought and agriculture-induced landscape homogenization may lead to catastrophic species declines (piha et al., 2007). unfortunately, the complex relationships among climate variables and other stressors (gunn et al., 2004) make it difficult to study their influence on amphibian life history and declines (blaustein and kiesecker, 2002; davidson et al., 2002). we lack a firm understanding of the natural history for many amphibians (bury, 2006; mccallum and mccallum, 2006; trauth, 2006). conservation biologists use the natural history of species as the backbone for orchestrating conservation programs and making management decisions (bury, 2006; greene, 2005; wilcox and possingham, 2002). this makes understanding how climate change may influence natural history characteristics of utmost importance as we plan necessary conservation responses (stenseth and mysterud, 2002; winkler et al., 2002). growth and reproduction are two of the four primary aspects of natural history traits typically related to the health of an organism (the other two are development and behavior; newman, 2001). growth rate and duration play major roles in determining the ultimate body size (werner, 1986) and reproductive output (blueweiss et al., 1978) of an individual. body size and growth are important metabolic determinants of an organism’s overall competitive ability (batzli et al., 1977; blueweiss et al., 1978; werner, 1986) and metabolism in poikilotherms is influenced by ambient temperature (lofts, 1972). generally, larger individuals have fewer predators (werner, 1986; rowe and ludwig, 1991; jung, 1995; laurila and kujasalo, 1999), can compete better for mates (berven, 1981; howard, 1998; smith, 1987), and are more resistant of desiccation (nevo, 1973a; stewart, 1995; wilmer et al., 2000). larger females typically produce larger eggs and egg clutches (darwin, 1874; lofts, 1974; mccallum, 2003), although there are exceptions (shine, 1988). therefore, stressors that suppress growth can affect reproduction and survivorship (batzli et al., 1977; fraser and gilliam, 1992). the systematics of blanchard’s cricket frog has been the subject of much debate (mccallum, 2006; rose et al., 2006; gamble et al., 2008). these frogs frequently occur along stream banks and around springs (trauth et al. 2004), where they are known for their seemingly haphazard jumping patterns when escaping pursuers (mccallum, 1999). blanchard’s cricket frog hibernates terrestrially under rocks in arkansas (mccallum and trauth, 2003b), oklahoma (blair, 1951), louisiana (walker, 1963), and in cracks in the 121future climate change spells catastrophe for blanchard’s cricket frog soil in illinois (gray, 1971). they have a low tolerance for inundation while hibernating (irwin and lee, 1999) and they often die when forced to hibernate in an aquatic laboratory setting (mccallum and trauth, 2003b). calling behavior and immune function are closely tied to temperature (mccallum and trauth, 2007), and we could not maintain these frogs in reproductive condition without orchestrating a combination of cool water and hot air temperatures (mccallum, 2003). they rapidly succumb in captivity to eutrophication arising from warm water (pers. observ.). males often call from inside clusters of emergent grasses near oviposition sites (pers. observ.). oviposition typically occurs in heavily vegetated (e.g., myriophyllum sp., ceratophyllum sp., and algal mats comprised of spirogyra sp. and lemna sp.), shallow water habitats (regan, 1972; mccallum, unpubl. data) that provide ideal habitat for their larvae (johnson 1988). in arkansas, metamorphs emerge from these habitats starting in early july and extending through early winter (mccallum, 2003). they disperse along stream banks and riparian areas, generally avoiding adults possibly due to cannibalism (mccallum et al., 2001). froglets feed on various insects, especially collembola (johnson and christiansen, 1976), with the size of prey generally increasing with body size to encompass small hymenoptera and orthoptera (mccallum, unpubl. data). there is a report of attempted cannibalism by adults on metamorphs (mccallum et al., 2001). body size of blanchard’s cricket frog (acris blanchardi) varies geographically with precipitation levels (nevo, 1973b). males and females reach a minimum adult size in about two months after metamorphosis, but continue to grow until they die, usually within one year post-metamorphosis (mccallum, 2003). males begin calling in early summer, with gravid females entering the chorus largely from late may through june. by september, juveniles of near adult size dominate the population and adults from the previous breeding season are seldom encountered (mccallum, 2003). because the biology of blanchard’s cricket frog is tied to temperature, and its body size correlates regionally with precipitation, and temperature-precipitation regimes influence the biology of other poikilotherms (kristensen et al., 2006; mccallum et al., 2009), i asked if climate change-induced temperature and precipitation flux may influence the growth rates and resulting body sizes of this frog. i report how blanchard’s cricket frog (a. blanchardi), may respond to these predicted changes in temperature and precipitation. i hypothesized that annual changes in seasonal rainfall and temperature may influence growth and reproduction in the a. blanchardi leading to future changes in growth and reproduction. if this were true, i could model interpolated growth and climate data with regression techniques (α = 0.05), and then extrapolate the influence of projected climate change on growth and reproduction using fuzzy regression techniques. if no relationship existed, then no dependable models should be identified, making extrapolation impossible. materials and methods fuzzy mathematics is a conservative, non-subjective generalization of interval analysis that is used for dealing with uncertainty, and requires fewer data than alternative methods like monte carlo simulations (silvert, 1997, 2000; ferson et al., 1999). this method is useful with all kinds of uncertainty, and the subjective interpretations characteristic of monte carlo approaches are unneed122 m.l. mccallum ed. it is based on a consistent axiomatic system that is different from that used in probability theory (ferson et al., 1999). fuzzy mathematics rates data (x-axis) based on degrees of possibility called membership values (y-axis) where y = 0 = lowest possibility and y = 1= highest possibility. if the graphical representation is a triangle, then only one value has the membership value y = 1. if the representation is a trapezoid, then a series of values across the top of the polygon are equally possible and all have membership values y = 1. all other x-values have decreasing membership values (i.e., possibilities) as y approaches zero. fuzzy mathematics is particularly useful where high levels of uncertainty such as ambiguity, non-specificity, discord, and fuzziness exist (klir and yuan 1994). extrapolation of future events is difficult to interpret and plagued by uncertainty. fuzzy mathematics is specifically useful for dealing with this kind of uncertainty and the associated questionable data sets (silvert, 1997, 2000; ferson et al., 1999), making it well suited for analyzing the effects of future climate change on the life history of an organism. i interpolated climate patterns (national oceanic and atmospheric association [noaa], 2000) with reproduction and growth data from a. crepitans (table 1; mccallum, 2003) using multiple and best subsets regression (neter et al., 1996). i used seasonal average temperatures and levels of precipitation because seasonal timing of climate events may be more important than annual variation (arak, 1983; mccallum et al., 2009). then i used fuzzy regression techniques where the variables were fuzzy (taheri, 2003) with previously reported climate change scenarios (u.s. epa, 1998) to extrapolate growth and reproductive outcomes. i constructed fuzzy numbers using the maximum, minimum, and best predictions of climate change in arkansas by 2100 (table 1; u.s. epa, 1998). i substituted fuzzy numbers for the variables in the predictive regression models, then i combined the regression models using substitution to construct more complex regression models that predicted the snout-vent length (svl), body mass (bm), body condition score (bcs), ovarian length (ol), number of mature ova (mo), maximum ova diameter (od), egg volume (ev), and total investment table 1. average seasonal temperature (ºc) and precipitation (cm) data observed from 1901 – 2007 (noaa, 2000), predicted due to climate change by 2100 (u.s. epa 1998), and the associated fuzzy numbers. the mean/best column refers to the average from 1901-2007 or the best estimate from the united kingdom hadley center’s climate model (hadcm2). low mean/best high fuzzy number mean winter temperature 1901-2007 1.28 5.42 9.11 [1.28, 5.42, 9.11] mean spring temperature 1901-2006 13.60 15.74 17.61 [13.60, 15.74, 17.61] mean summer temperature 1901-2006 24.28 26.04 26.33 [24.28, 26.04, 26.33] mean fall temperature 1901-2006 13.50 19.34 19.56 [13.50, 19.34, 19.56] mean winter precipitation 1901-2007 10.26 30.86 58.62 [10.26, 30.86, 58.62] mean spring precipitation 1901-2006 6.21 14.90 26.83 [6.21, 14.90, 26.83] mean summer precipitation 1901-2006 10.64 28.07 44.78 [10.64, 28.07, 44.78] mean fall precipitation 1901-2006 11.61 28.75 58.65 [11.61, 28.75, 58.65] predicted winter temperature 2100 5.97 6.01 8.74 [5.97, 6.01, 8.74] predicted spring temperature 2100 16.32 17.43 18.54 [16.32, 17.43, 18.54] predicted summer temperature 2100 26.60 27.15 29.37 [26.60, 27.15, 29.37] predicted fall temperature 2100 17.14 18.25 19.36 [17.14, 18.25, 19.36] predicted winter precipitation 2100 10.26 30.86 58.62 [10.26, 30.86, 58.62] predicted spring precipitation 2100 39.58 43.35 47.12 [39.58, 43.35, 47.12] predicted summer precipitation 2100 30.73 34.93 39.12 [30.73, 34.93, 39.12] predicted fall precipitation 2100 30.38 33.27 36.16 [30.38, 33.27, 36.16] 123future climate change spells catastrophe for blanchard’s cricket frog in reproduction (ti). i validated these models by comparing observed reproductive and growth data to the average values that were derived from fuzzy regressions using weather data from 1901-2007. then i compared the modeled results from 1901-2007 to the extrapolated results for 2100 to infer the risk of climate change to reproduction in a. blanchardi. results interpolation of climate effects on growth and reproduction resulted in equations 9 – 11 (table 2). many of the equations predicted intervals for life history traits resembled previously reported values (mccallum, 2003). for example, fuzzy climate variables gave accurate fuzzy intervals for svl and bm. bcs was a better predictor of ol than was svl, although svl provided accurate results for the number of mo. bcs and ol that was derived from the bcs (olbcs) gave similar outputs and reflected previous reports for the diameter of the od (mccallum, 2003). ovarian length that was derived from svl (olsvl) was not a good table 2. regression models for growth and reproductive traits in the northern cricket frog (acris crepitans). key: bm = body mass (g), svl = snout-vent length (mm), bcs = body condition score, ol = ovarian length (mm), lo = largest ova diameter (µm), mf = mature follicles (n), vf = vitellogenic follicles (n), wt = winter temperature (ºc), spt = spring temperature (ºc), fr = fall rain (cm), spr = spring rain (cm), sur = summer rain (cm), wr = winter rain (cm). 1. svl♀ vs. ol (r2 = 0.200, df = 3, 57, p = 0.005): ol = 30.8410 – 4.11180 svl + 0.198410 svl2 – 0.0028678 svl3 2. bcs♀ vs. ol (r2 = 0.156, df = 1, 45, p = 0.006): ol = 2.40320 + 77.6546 bcs 3. bcs♀ vs. lo (r2 = 0.311, df = 1, 45, p < 0.001) lo = -179.298 + 14024.0 bcs 4. ol vs. lo (r2 = 0.612, df = 2, 62, p < 0.001): lo = -420.728 + 190.576 ol – 5.15409 ol2 5. svl♀ vs. mf (r2 = 0.273, df = 3, 28, p = 0.028): mf = (1253.58 – 147.312 svl + 5.5347 svl2 – 0.0568778 svl3) (2 ovaries) 6. svl♀ vs. vo (r2 = 0.231, df = 31, p = 0.022): vo = 632.575 – 47.9119 svl + 1.22306 svl2 7. bm♀ (r2 = .840, p < 0.001): bm = 0.193950 – 0.0449324 svl + 0.0029810 svl2 + 0.0000419 svl3 8. bm♂ (r2 = 0.771, p < 0.001): bm = 0.560328 + 0.0213750 svl + 0.0024595 svl2 9. svl♀ vs. wt, spt, sur, and spr (r2 = 0.031, df = 4, 1386, p < 0.001: svl = 33.1 0.835 wt – 0.626 spt + 0.0453 sur – 0.0334 spr 10. bcs♀ vs. wt, fr, wr, spr (r2 = 0.049, df = 4, 1386, p < 0.001: bcs = 0.0850 – 0.00431 (wt) – 0.000298 (fr) + 0.000224 (wr) – 0.000194 (spr). 11. bm♀ vs wt, fr, wr, spr (r2 = 0.035, df = 4, 1386, p < 0.001: bm = 2.00 – 0.109 (wt) – 0.00648 (fr) + 0.0565 (wr) – 0.00604 (spr) 124 m.l. mccallum ta bl e 3. f uz zy e st im at es o f lif e hi st or y ch ar ac te ri st ic s un de r th e cl im at e ob se rv ed f ro m 1 90 120 07 a nd p re di ct ed c ha ng es b y 21 00 . n um be rs i n br ac ke ts a re fu zz y es tim at es r ep re se nt in g th e ve rt ic es o f th ei r tr ia ng ul ar o r tr ap ez oi da l s ha pe . p er ce nt ag es i n pa re nt he se s ar e th e pe rc en ta ge c ha ng e in t he s ta tis tic w ith a m em be rs hi p va lu e = 1. m v 0 i s th e m em be rs hi p va lu e w he n th e lif e hi st or y tr ai t = 0 . li fe h is to ry t ra it m cc al lu m 2 00 3 c al cu la te d 19 01 -2 00 7 pr ed ic te d 21 00 % c ha ng e sv l ♀ ( m m ) 21 .5 3 sd = 1 .8 7 [1 4. 06 , 1 9. 49 , 2 5. 34 ] [1 4. 01 5, 1 7. 30 , 1 8. 35 ] [44 .6 9, 11 .2 3, 3 0. 55 ] b m ♀ ( g) 1. 71 s d = 0 .7 4 [1 .0 4, 2 .8 8, 5 .0 6] [2 .2 7, 2 .6 1, 2 .6 6] [55 .1 0, 9. 23 , 1 54 .3 6] b c s ♀ 0. 05 1 sd = 0 .2 9 [0 .0 25 , 0 .0 57 , 0 .0 88 ] [0 .0 34 , 0 .0 46 8, 0 .0 47 6, 0 .4 95 ] [60 .9 7, 16 .4 8, 1 85 0. 6] o l us in g sv l (m m ) 5. 86 m m s d = 2 .0 [80 .7 9, 4 .8 4, 9 2. 46 ] [23 .3 5, 4 .2 4, 3 2. 12 ] m v 0 > 0 .9 , ( -1 2. 4% ) o l us in g b c s (m m ) 5. 86 m m s d = 2 .0 [4 .3 7, 6 .8 4, 9 .2 3] [5 .0 7, 6 .1 1, 4 0. 80 ] [45 .1 0, 10 .6 9, 8 33 .3 ] lo u si ng b c s (µ m ) 48 0. 9 sd = 2 77 .9 [1 76 .2 3, 6 21 .3 9, 1 05 4. 11 ] [3 02 .1 , 4 89 .4 42 , 4 89 .4 45 , 6 75 5. 8] [71 .3 , 21 .2 , 21 .2 , 3 73 4] lo u si ng o l s v l (µ m ) 48 0. 9 sd = 2 77 .9 [59 87 9, 3 80 .9 2, 1 71 99 .9 ] [10 18 9. 25 , 2 94 .8 3, 5 70 0. 98 ] m v 0 > 0 .9 , ( -1 2. 4% ) lo u si ng o l b c s ( µm ) 48 0. 9 sd = 2 77 .9 [27 .0 0, 6 41 .6 7, 1 23 9. 86 ] [80 36 .3 1, 5 50 .7 9, 5 50 .8 0, 7 22 3. 20 ] m v 0 > 0 .9 , ( -2 2. 6% ) a ve ra ge l o ( µm ) 48 0. 9 sd = 2 77 .9 [19 90 9. 93 ,5 47 .9 9, 64 97 .9 6] {59 74 ,4 45 ,6 56 0] m v 0 > 0 .9 , ( -1 8. 8% ) m f 15 7, s d = 1 7. 87 [46 22 ,1 28 ,5 15 6] [14 28 ,1 34 ,1 79 2] m v 0 > 0 .9 , ( +4 .5 % ) ev b c s ( µm 3 ) -[2 .2 9e 7, 1 .0 1e 9, 4 .9 1e 9] [1 .4 4 e7 , 6 .1 4 e7 , 1 .6 1 e1 1] [99 .7 , 93 .9 , 7 03 0] ev o ls v l (µ m 3 ) -[1. 12 e1 4, 2 .8 9e 7, 2 .6 6e 12 ] [5. 54 e1 1, 1 .3 4e 7, 9 .7 0e 10 ] m v 0 > 0 .9 , ( -5 3. 6% ) ev o lb c s ( µm 3 ) -[1 .0 3e 4, 1 .3 8e 8, 9 .9 8e 8] [2. 72 e1 1, 8 .7 5e 7, 1 .9 7e 11 ] m v 0 > 0 .9 , ( -3 6. 6% ) t i b c s (µ m 3 ) -[2. 27 e1 3, 1 .2 9e 11 , 2 .5 3e 13 ] [ -2 .3 e1 4, 8 .2 3e 09 , 2 .8 9e 14 ] m v 0 > 0 .9 , ( -9 3. 6% ) t i o ls v l (µ m 3 ) -[5. 77 e1 7, 3 .7 0e 09 , 5 .1 8e 17 ] [ -9 .9 3e 14 , 1 .8 0e 09 , 7 .9 1e 14 ] m v 0 > 0 .9 , ( -5 1. 4% ) t i o lb c s (µ m 3 ) -[4. 61 e1 2, 1. 77 e1 0, 5. 15 e1 2] [ -4 .8 7e 14 , 1 .1 7e 10 , 3 .8 8e 14 ] m v 0 > 0 .9 , ( -3 3. 9% ) 125future climate change spells catastrophe for blanchard’s cricket frog predictor of od. some of the fuzzy regression results gave large intervals, but x-values with high membership values (y > 0.5) resembled previous reports (mccallum 2003). these data predict reduced svl, bm, bcs, ol, and od by 2100; whereas, they predict increased mo relative to 1901-2007 predictions (table 3). this culminated in a large reduction in ev (36-94% drop) and ultimately, ti (37-94% drop; table 3). mo, ev, and ti had high membership values greater than 0.9 for x = 0. any time x < 0, it was considered to be biologically zero because no investment existed in that trait. discussion the fuzzy regression produced fuzzy intervals for 1901-2007, which included values similar to actual observations (mccallum, 2003). that finding appears to validate the accuracy of the fuzzy regression approach for predicting climate effects on life history traits. this method provides us with greater confidence in the fuzzy regression forecasts (heshmaty and kandel, 1985) for future climate influences on these life history traits. overall, it revealed that both growth and reproduction in this species could be severely hampered by prospective climate change scenarios. not surprisingly, bcs and olbcs provided similar predictions of od. the small differences between these results may reflect differences in rounding between the calculations for each, or amplification of error in the olbcs results. the olsvl underestimated od relative to actual observations and calculations obtained from bcs and olbcs. this is probably because egg size is generally related to both the body size and the amount of fat stores available for investment in ova (lofts, 1974). the svl tells little about the available fat stores, whereas individuals with sufficient resources for reproduction will generally have higher bcs values (darwin, 1874; lofts, 1974). therefore, the variation between these predictors seems to follow conventional wisdom in that larger females have larger ovaries, hence greater numbers of larger ova (zera and harshman, 2001). although most life history traits decreased under the predicted changes in arkansas climate, mo increased 4.5%. generally, we expect larger females to produce more ova (zera and harshman, 2001). this trade-off between ova size and ova number may be adaptive under high stress conditions. unfortunately, the small increase does not counterbalance the ultimately reduced reproductive investment experienced by frogs in 2100. the frogs are producing a few more eggs, but these are much smaller than ideal. smaller egg size due to climate change is likely to reduce survivorship of larvae and ultimately recruitment (zera and harshman, 2001). these results suggest future concern for populations of a. crepitans. these anurans are adapted to current climate conditions. i thought that increased precipitation would promote this species, but the complex interactions among seasonal perturbations in rainfall and ambient temperature may instead be detrimental. this is a critical point for those studying the influence of climate variables on life histories. timing of climate patterns may be more important than the annual changes involved. summer temperature was not a factor in any model. this may be because predicted increases in summer temperatures are unrelated to periods of dormancy in this species. warmer spring or fall temperatures resulting from climate change would shorten the dormancy period as warmer tempera126 m.l. mccallum tures persist longer into the winter and ensue earlier in the spring. this could lengthen the period of activity for this species and/or increase the metabolic rate of the frogs that do not return from dormancy. this could reduce the bcs as they burn resources due to these periods of warm weather leading to increased metabolism without raised intake of food (lofts, 1972). this could drain resources available for growth and reproduction during the following breeding season. other kinds of stressors such as immune stress are known to similarly reduce reproduction and growth in male a. crepitans (mccallum and trauth, 2007). reduced reproduction and body growth can influence age-specific reproduction, survival rates, population growth rates, effective population sizes, and censused population sizes (soule and mills, 1998), and these factors can feed back on themselves (gilpin and soule, 1986) leading to an extinction vortex. (mills, 2007). therefore, my extrapolations provide evidence that future climate change could seriously impair this species, especially the already declining populations in the northern parts of its range (lehtinen and skinner, 2006). on a broader scale, the results of this study draw concern about the potential for climate change to similarly impact poikilothermic vertebrates, potentially driving them to extinction. amphibians, reptiles, and fishes represent most of the vertebrate species on the planet (wilson, 1992) and are responsible for significant ecosystem services (balvanera et al., 2001; schlaepfer et al., 1999; myers, 1996), form the prey base for many other organisms (blaustein and kiesecker, 2002.), and provide population control of many pest organisms (hirai and matsui, 1999) including vectors of serious human diseases (durvasula et al., 1997). most of these organisms have poor dispersal ability (gibbs, 2004; bohonak, 1999) are unlikely to migrate with moving climates during a mere century, and their physiology is tightly tied to temperature (mccallum and trauth, 2007), thereby amplifying effects of local climate change on life history traits. for this reason, pre-emptive strategies that aim for elimination of greenhouse gasses remain both the best and imperative courses of action for the conservation of amphibians and similar poikilotherms. acknowledgements many thanks to w. meshaka for his valuable input on a previous version of this manuscript. references adams, m.j. 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(2002): abnormalities in the ozark hellbender, cryptobranchus alleganiensis bishop. j. ark. acad. sci. 56: 250-252. the nguru mountains of tanzania, an outstanding hotspot of herpetofaunal diversity michele menegon1,2, nike doggart3, nisha owen4,5 1 sezione di zoologia dei vertebrati, museo tridentino di scienze naturali, via calepina 14, i–38100 trento, italy 2 udzungwa mountains monitoring centre, c/o udzungwa mountains national park, p.o. box 99 mang’ula, tanzania 3 tanzania forest conservation group, p.o. box 23410, dar es salaam, tanzania 4 frontier tanzania forest research program, p.o. box 9473, dar es salaam, tanzania 5 society for environmental exploration/frontier, 50-52 rivington street, london ec2a 3qp submitted on 2008, 7th march; revised on 2008, 22nd august; accepted on 2008, 30th september. abstract. despite the vicinity of a major road, the rainforests of the south nguru mountains in eastern tanzania were virtually unexplored until 2004, particularly from a herpetological point of view. several surveys were conducted between 2004 and 2006 with the aim of providing a comprehensive list of the amphibian and reptile species of this overlooked hotspot of biological diversity. the surveys resulted in this assessment of the herpetofaunal diversity, with 92 species recorded, of which 15 represent new records for this area, and the discovery of 16 species new to science, all of which are likely to be strictly endemic to the nguru mountains. pressure on the forests, particularly the lowland forests, remains high. a conservation planning process is now underway that is attempting to address the loss of these critically important forests. these results, documenting the high species richness and the outstanding number of putative endemics of the forests, strongly highlight the biological importance of the south nguru mountains and place them among the most important sites for the conservation of herpetofauna in africa. keywords. herpetofaunal diversity, new species, endemism, zoogeography, eastern arc mountains. introduction in the last few years increased interest in the forests of the eastern arc mountains has resulted in the biological exploration of several poorly known mountain blocks. previously unsurveyed montane ranges and forest reserves have recently been explored by different acta herpetologica 3(2): 107-127, 2008 issn 1827-9643 (online) © 2008 firenze university press 108 m. menegon, n. doggart and n. owen groups of researchers and faunal lists are being completed. among the ranges explored in the last few years, the south nguru mountains stand out due to their exceptional amphibian and reptile species richness, number of undescribed species and endemism ratio. the present paper outlines the results of several surveys conducted by two different groups of researchers and highlights the richness and uniqueness of the south nguru mountains herpetofauna, furthermore it stresses the need for a rapid taxonomic and conservation assessment of the newly discovered and as yet undescribed species in order to properly assess the biological value of the south nguru mountains forests. the south nguru mountains are part of the eastern arc mountains of tanzania and kenya. this chain of isolated massifs is part of one of the world’s most important biodiversity hotspots (mittermeier et al., 2004). the eastern arc mountain forests are biologically exceptional due to the high concentration of endemic species that have been recorded in the area (howell, 1993; lovett, 1993; burgess et al., 1998; burgess et al., 2007). the biodiversity of the south nguru mountains is less well known than some of the other eastern arc massifs, such as the east usambara and uluguru mountains (burgess et al., 2007). as stated by lovett and thomas (1988) the interest in the south nguru mountains is threefold: they are in the central part of the eastern arc mountain chain and should possess many rare species; the mountains have continuous forest cover from less than 400 m to 2400 m altitude; and are still poorly known biologically. at a national level, the south nguru forests are important as the main catchment area for the wami river which provides water for the town of chalinze, the mtibwa sugar plantation and many villages. locally, the forests are important as a source of wood products, medicinal plants and for their cultural values. most biological research in the south nguru mountains has concentrated on plants. lovett and thomas (1988) surveyed both kanga and nguru south forest reserves providing a brief account of the vegetation. botanical surveys conducted by pócs et al. (1990) in the south nguru mountains provided additional data supplemented by subsequent surveys carried out by lovett and pócs (1993). these surveys indicated a high number of rare and endemic plants in nguru south and kanga forest reserves. pócs (1998) recorded three endemic bryophyte species in the south nguru mountains. results of several bird surveys were published in the past (moreau, 1940; sclater and moreau, 1932-1933; stuart and van der willingen, 1978), but it was not until 1996 that romdal (2001) carried out a systematic ornithological survey covering various altitudes in the ngurus (baker and baker, 2002). until the publication of results of herpetological surveys carried out in the south nguru mountains by emmrich (1994), very little information was available on the herpetofauna. burgess et al. (1998) reported the collection of an undescribed rhampholeon species, subsequently described by mariaux and tilbury (2006). nine species of small mammals (shrews and small rodents), of which three are endemic to the eastern arc mountains, were recorded in the south nguru mountains by stanley et al. (1998). a few further records are available for other mammals. this paper demonstrates that the herpetofaunal value of the south ngurus is exceptional, particularly given that the survey intensity remains low relative to other areas of the eastern arc mountains such as the uluguru and east usambara mountains. 109herpetofauna of nguru mountains study area the south nguru mountains lie between s 05°53’s-s 06°17’ and e 037°27–e 037°45’ in mvomero district, morogoro region, tanzania (fig. 1). nguru south forest reserve (see fig. 2) was gazetted during the german colonial period and covers an area of 18 797 ha with an altitudinal range between 760 and 2400 m. it is contiguous with mkindo forest reserve which covers 5244 ha and extends down to 380 m. the estimated mean rainfall on the eastern slopes of these mountains is around 2000 mm/year increasing up to 3000-4000 mm at 2000 m (lovett and pócs, 1993). kanga forest reserve was gazetted in 1954 and covers an area of 6664 ha with an altitudinal range between 500 and 2019 m. the estimated rainfall is 1500-2000 mm/year (lovett and pócs, 1993). within the south nguru landscape, there are 56 villages. the majority of people in the south nguru landscape depend on agriculture for their livelihood. at the base of the mountains, the mtibwa sugar plantation covers an extensive area. farmers are also involved in smallholder sugar and rice cultivation. at higher altitudes, farmers grow bananas, yams, sweet potatoes, maize and coffee. many farmers also have plots within the nguru south forest reserve where they are growing cardamom and yams. cardamom is one of the main sources of cash income for the communities immediately adjacent to the reserves. cardamom cultivation is now widespread within nguru south forest reserve. fig. 1. location of the sampling sites within the nguru south and kanga forest reserves. 110 m. menegon, n. doggart and n. owen materials and methods between october 2004 and august 2006 a total of 13 sites were surveyed in the south nguru mountain forests, seven sites in nguru south forest reserve and six in kanga forest reserve. two additional sites were surveyed opportunistically in nguru south forest reserve during january 2006 (see table 1). sampling sites were located in submontane and montane forests between 750 and 2200 m asl. further records were obtained during random sampling in the village belt and from local people. sites were sampled by opportunistic search, visual and acoustical monitoring. searches were conducted both during the day and night to sample the highest number of species. sixteen visual encounter survey (ves) quadrats were conducted within a 10x10 m area, requiring one man-hour each per unit. four ves transects of 100 m were also conducted, each requiring four man-hours. another sampling technique used was pitfall trapping with drift fences (heyer et al., 1993). they consisted of three 50 m linear transects each containing eleven 10 litre plastic buckets positioned 5 m apart, sunk into the ground with a plastic sheet ‘drift fence’ stretching between each bucket. each line was placed 50 m apart, encompassing a range of micro-habitats. a total of 264 bucket pitfall trap-nights were conducted in each work unit. specimens have also been sampled opportunistically during forest walks. further records (mainly concerning snakes) were obtained from local people living in the villages at the forest edge. a collection of specimens opportunistically killed by villagers in crop fields surrounding the forest, were identified by the authors and deposited in the collection. the abundance of individuals of different species was also recorded. sampling intensity was comparable in sites 1-4. voucher specimens were collected and, when possible, frog calls were recorded by means of a sony tcm directional microphone and a sharp minidisc or an analogue marantz pmd-222 audiocassette recorder and a sennheiser k6-me66 directional microphone. specimens, fig. 2. the montane rainforest of the nguru south mountains, one of the sampled habitats. 111herpetofauna of nguru mountains photographs and sound recordings were deposited in both the museo tridentino di scienze naturali, trento, italy, and in the collections of the university of dar es salaam, tanzania. the majority of species collected have been identified by the lead author, on the grounds of morphological, molecular and bioacoustic analysis and through comparison with material held in the herpetological collections of the university of dar es salaam, tanzania, the natural sciences museum of trento, italy and the natural history museum, london, uk. molecular analysis were carried out at the centre for alpine ecology, trento, italy; the natural history museum, london, uk and the institute of biogeography, basel, switzerland. identifications or confirmation of identification were also provided by simon loader, institute of biogeography, basel; john poynton, natural history museum, london and van wallach, museum of comparative zoology, harvard university, cambridge, usa. table 1. location and main characteristics of the sampling sites. sampling site utm co-ordinates elevation range m main vegetation type main habitats investigated nguru site 1 ‘maskati’ 37m0333779 9329236 1900-2200 montane and upper montane rain forest open canopy forest, small bogs nguru site 2 ‘pemba’ 37m0336825 9333210 950-1050 submontane forest closed canopy forest, small wetland, ecotone kanga site 3 ‘kanga’ 37m0358812 9336174 750-900 transitional and submontane forest closed canopy forest, kanga site 4 “difinga” 37m0356024 9340888 1000-1300 submontane rain forest closed canopy forest, ft kanga site 5 37m0355845 9340733 1000-1100 submontane rain forest closed canopy forest, ft kanga site 6 37m0358539 9341552 800-950 lowland and submontane forest half-closed, canopy forest ft kanga site 7 37m0356729 9345887 1100-1250 submontane rain forest high dense closedcanopy forest ft kanga site 8 37m0358571 9336368 800-900 lowland and submontane forest half-closed canopy forest ft nguru site 9 37m0333711 9329248 1950-2050 montane and upper montane forest open canopy forest, small bogs ft nguru site 10 37m0333540 9325622 1950-2020 dry upper montane forest open canopy forest, heath ft nguru site 11 37m0334554 9323454 1850-1950 upper montane forest open canopy forest, ft nguru site 12 37m0337300 9322700 1700-1850 submontane and montane forest dense closed-canopy forest ft nguru site 13 37m0361810 9324701 1800-1950 montane forest dense closed-canopy forest ft nguru site 14 37m0335146 9329820 1700 montane forest closed canopy forest ft nguru site 15 37m0336314 9332299 1180 submontane forest open canopy forest village belt (two sites) 1000 – 2000 farmland synanthropic habitats 112 m. menegon, n. doggart and n. owen the nonparametric species richness estimator based on abundance data chao 1, computed along log-linear 95% confidence interval, and the abundance-based coverage estimator ace, were used incorporating abundance data, represented by the number of individuals per species (colwell and coddington, 1994; chao et al., 2000; magurran, 2004). the shared species estimator v (chao et al., 2000) has been used to estimate the number of shared species between sites. analyses were performed using the sofware estimates (magurran, 2004). the species complementarity between sites has been described as an expression of pattern diversity (whittaker, 1972) by means of a braycurtis similarity analysis expressed as a percentage (legendre and legendre, 1998; magurran, 2004). the analysis of similarity has been performed using the software biodiversity pro. only the records from the lead author’s direct collections in sites 1-4 were used in the statistical analysis. amphibian taxonomy follows frost et al., (2006), reptile taxonomy follows spawls et al. (2004), except for colubroidea taxonomy that follows vidal et al. (2007) and lawson et al. (2005) and scincidae taxonomy that follows brandley et al. (2005). results species composition and richness. a total of 41 species of amphibian from 12 families and 51 species of reptile from 11 families were recorded in nguru south forest reserve, kanga forest reserve and farmland adjacent to the two forest reserves during the survey activity. three species previously documented for the area were not found during this study. by combining previous species records with those from the present study, the total number of amphibian and reptile species recorded from the south nguru mountains is 92 species, from 23 families (appendix 1). the majority, 76 species out of a total of 89 species collected during this survey, were recorded from forest, and of these, based on the known information on their ecology, 67 species are considered forest associated based upon available literature (howell, 1993, menegon and salvidio, 2005; burgess et al., 2007). the remaining species were collected from the village belt. biological notes during the surveys we observed the activities of some species whose behaviour is poorly known. these observations are summarised as follows: hoplophryne sp. nov. was active during and immediately after heavy rain in undisturbed forest during the day. at these times, individuals of hoplophryne sp. nov. move around on soft soil and under the leaf litter, they can be detected by searching gently with a stick among the leaf litter. female hyperolius spinigularis were observed returning to their egg clutches and squirting water over the eggs. this was previously observed by stevens (1971), and may be done to avoid desiccation and possibly parasitism of the eggs (j. vonesh, pers. comm.). the geckos urocotyledon wolterstorffii and cnemaspis africana were observed sharing an egg-laying site on dry, sandy soil under a large overhanging rock. two eggs of urocotyledon wolterstorffii and several eggs of cnemaspis africana were found under wood debris. several females cnemaspis africana were probably using the site. one of the eggs of urocotyledon was 12 mm in length. after 16 days in controlled conditions a hatchling 21.25 + 23.37 mm long (head body + tail length) emerged. 113herpetofauna of nguru mountains range extensions of species endemic to the eastern arc mountains the known distributions of 15 species endemic or near endemic to the eastern arc were extended by our findings (see table 2). new species the present study recorded 15 amphibian taxa and one reptile taxon that are sufficiently distinct from the other known taxa that they may be considered new species based upon the available data (see table 3). the discovery of numerous species new to science, table 2. reptile and amphibian species endemic to the eastern arc whose documented ranges have been extended by the present study. species known distribution prior to the present study references for previous records amietophrynus brauni usambara, uluguru and udzungwa mountains channing and howell, 2006 hyperolius spinigularis usambara and uluguru and udzungwa mountains and mulanje mts. malawi channing and howell, 2006 leptopelis vermiculatus usambara, udzungwa and rungwe mountains channing and howell, 2006 leptopelis cf. parkeri usambaras, uluguru and udzungwa mts. channing and howell, 2006 phrynobatrachus uzungwensis nguu, uluguru and udzungwa mountains menegon et al., 2003; channing and howell, 2006 probreviceps macrodactylus macrodactylus* usambara mountains channing and howell, 2006 urocotyledon wolterstorffii east usambara and uluguru mountains spawls et al., 2004 kinyongia oxyrhina uluguru and udzungwa mountains klaver and böhme, 1988; spawls et al., 2002 chamaeleo werneri uluguru and udzungwa mountains spawls et al., 2004 rhinotyphlops gierrai east and west usambara mountains broadley and wallach, 2000 buhoma vauerocegae usambara and uluguru mountains spawls et al., 2004 dipsadoboa werneri usambara mountains and the uzungwa scarp forest reserve in the southern udzungwa mountains rasmussen, 1997; menegon and salvidio, 2005 xyeledontophis uluguruensis uluguru mountains broadley and wallach (2002) elapsoidea nigra north pare, usambara, magrotto and uluguru mountains broadley and howell, 1991; spawls et al. 2004 atheris ceratophorus east usambara, uluguru and udzungwa mountains spawls et al., 2004 * the record of this species from north pare mountains (channing and howell, 2006) is actually due to a misidentification of an undescribed species of callulina. 114 m. menegon, n. doggart and n. owen table 3. reptile and amphibian taxa considered new species based upon the available data. the occurrence and occupancy areas were made without considering elevational constraints, in some cases the area may be an over-estimate. taxa range based on current information estimated maximum area of occupancy estimated maximum extent of occurrence amphibia anura arthroleptidae arthroleptis sp. nov. nguru south and mkindo forest reserves (278 km2) 240.3 km2 240.3 km2 leptopelis sp. nov. kanga forest reserve 66 km2 66 km2 bufonidae nectophrynoides sp. nov. 1 nguru south and mkindo forest reserves 240.3 km2 240.3 km2 nectophrynoides sp. nov. 2 nguru south and mkindo forest reserves 240.3 km2 240.3 km2 nectophrynoides sp. nov. 3 nguru south and mkindo forest reserves 240.3 km2 240.3 km2 nectophrynoides sp. nov. 4 kanga forest reserve 66 km2 66 km2 brevicipitidae callulina sp. nov. 1 nguru south and mkindo forest reserves 240.3 km2 240.3 km2 callulina sp. nov. 2 nguru south and mkindo forest reserves 240.3 km2 240.3 km2 callulina sp. nov. 3 nguru south and mkindo forest reserves 240.3 km2 240.3 km2 callulina sp. nov. 4 kanga forest reserve 66 km2 66 km2 probreviceps sp. nov. nguru south and mkindo forest reserves 240.3 km2 240.3 km2 microhylidae hoplophryne sp. nov. nguru south and mkindo forest reserves 240.3 km2 240.3 km2 petropedetidae petropedetes sp. nov. nguru south, mkindo and kanga forest reserves 306.3 km2 57 km2 gymnophiona caecilidae boulengerula sp. nov. nguru south, mkindo and kanga forest reserves 306.3 km2 57 km2 scolecomorphus sp. nov. nguru south and mkindo forest reserves 278 km2 278 km2 reptilia sauria chamaleonidae rhampholeon sp. nov. nguru south and mkindo forest reserves 240.3 km2 240.3 km2 115herpetofauna of nguru mountains which are likely to be narrowly endemic to the nguru mountains only, is among the most interesting results of this study and make the south nguru mountains one of the most important ranges for herpetofaunal diversity and conservation in africa. the taxonomic status of all the species collected during the surveys has been assessed in the context of a more extensive work on the genera, involving molecular, osteological, bioacoustic and morphological analysis conducted between 2005 and 2008 (loader et al. unpublished; poynton et al. unpublished; menegon et al. unpublished). formal description of some of the unnamed taxa has started. zoogeography as with other eastern arc mountains, the herpetofauna of the south nguru mountains contains a combination of afromontane (eastern arc forests endemic and near endemic species), lowland/coastal and widespread species associated with savannah and woodland (see table 4). while this is a strong indication that the forests of these areas were once connected, more detailed taxonomic work is still needed to fully understand the historical biogeography of the eastern arc mountains. endemism the proportion of amphibian and reptile species from the south nguru mountains that are endemic to these mountains is extremely high. based on the results of this study, 23% (n = 18) of the herpetofauna species are strictly endemic to the south nguru mountains, of which only two were described. a further 33.3 % (n = 26) of the species have ranges restricted to the eastern arc mountains. the percentage of endemic or near endemic species is directly related to elevation, a pattern that has also been documented for other eastern arc forest localities (menegon and salvidio, 2005). based on three collecting localities, endemic species values at different elevation are as follows: kanga, 750 m = 55.1%; pemba, 1000 m = 59.4%; maskati, 2000 m = 72.7%. this highlights the uniqueness of the montane herpetofauna community. species richness and pattern diversity nonparametric estimators were used to estimate the species richness of sites and the number of shared species between sites in order to assess the total richness of the area and the differential diversity across sites and along an altitudinal gradient. in order to avoid biases due to different collecting methods and timing, only records compiled from the lead author’s direct sampling were used to calculate relative abundance and diversity estimates. kanga is estimated to be the richest site (59.26 ± 2.49 ace mean; 54.5 ± 2.79 chao 1 95% ci lower bound), followed by pemba (48.46 ± 1.61 ace mean, 43.05 ± 2.68 chao 1 95% ci lower bound) and maskati (30.73 ± 2.49 ace mean, 26.11 ± 2.79 chao 1 95% ci lower bound), this result is probably due to the low elevation component of the kanga species assemblage, following the assumption that species richness decreases with increas116 m. menegon, n. doggart and n. owen table 4. biogeographic affinities of the recorded species. bibliographic records and species collected by authors were considered. five species not yet identified to species level have not been included in the table, species identified as undescribed are included. nguru south and kanga forest endemics no. of species 18 % on total recorded 23 arthroleptis sp. nov., nectophrynoides sp. nov. 1, nectophrynoides sp. nov. 2, nectophrynoides sp. nov. 3, nectophrynoides sp. nov. 4, leptopelis sp. nov., callulina sp. nov. 1, callulina sp. nov. 2, callulina sp. nov. 3, callulina sp. nov. 4, probreviceps sp. nov., hoplophryne sp. nov., petropedetes sp. nov., scolecomorphus sp. nov., boulengerula sp. nov, rhampholeon acuminatus. rhampholeon sp. nov., kinyongia fisheri eastern arc forest endemics or near-endemics no. of species 26 % on total recorded 33.3 arthroleptis affinis, amietophrynus brauni, afrixalus uluguruensis, hyperolius mitchelli, hyperolius spinigularis, hyperolius sp. (puncticulatus-like), leptopelis cf. parkeri, leptopelis cf. uluguruensis, leptopelis vermiculatus, probreviceps macrodactylus macrodactylus, phrynobatrachus uzungwensis, chamaeleo werneri, chamaeleo deremensis, kinyongia oxyrhina, agama montana, urocotyledon wolterstorffii, scelotes uluguruensis, proscelotes cf. eggeli, leptosiaphos kilimensis, typhlops gierrai, buhoma vauerocegae, rhinotyphlops gierrai, thelotornis usambaricus, crotaphopeltis tornieri, dipsadoboa werneri, elapsoidea nigra, atheris ceratophorus east african coastal mosaic species no. of species 18 % on total recorded 23 arthroleptis xenodactylus, arthroleptis xenodactyloides, afrixalus stuhlmanni complex. afrixalus stuhlmanni stuhlmanni, afrixalus fornasinii*, leptopelis flavomaculatus, gastropholis prasina*, chamaeleo melleri, rieppeleon brevicaudatus, rieppeleon brachyurus, cnemaspis africana, holaspis laevis, melanoseps loveridgei*, aparallactus guentheri, lycophidion capense loveridgei, natriciteres sylvatica, philothamnus macrops, dendroaspis angusticeps. west african forest and savannah species no. of species 2 % on total recorded 2.5 lycophidion meleagre, bitis gabonica east/south african savannah species no. of species 8 % on total recorded 10.2 schismaderma carens, ptychadena anchietae, chiromantis xerampelina xenopus cf. petersii, lygosoma afrum*, rhinotyplops mucruso, aparallactus jacksoni, atractaspis bibroni, philothamnus punctatus, widely distributed savannah species no. of species 15 % on total recorded 19.2 ami e t o p hr y nu s g ut t ura li s , ami e t i a ang o l e n s i s , phrynobatrachus parvulus, chamaeleo dilepis, agama agama, trachylepis varia, trachylepis maculilabris, trachylepis striata, varanus niloticus, typhlops lineolatus, crotaphopeltis hotamboeia, dispholidus typus, lamprophis fuliginosus, prosymna stuhlmanni, philothamnus hoplogaster, bitis arietans. * bibliographic records 117herpetofauna of nguru mountains ing elevation (macarthur, 1972; stevens, 1992). the same pattern of species richness is obtained by considering the simpson index of diversity: simpson mean (d): kanga = 33.43; pemba = 24.93; maskati = 14.29. according to the bray-curtis indices of similarity the site at highest elevation (maskati) shows the greatest difference from other sites (25% of similarity), with medium altitude sites pemba and kanga showing closer faunal similarities (38% of similarity), in spite of the latter two being positioned on two separate massifs (see fig. 4). this pattern of diversity highlights the unique nature of the montane and upper montane forest herpetofauna and demonstrates how altitude influences the composition of assemblages in eastern arc forests. based upon predictions of species richness, estimates suggest an extremely diverse forest associated herpetofauna (possibly > 110 species for the entire landscape). further comparisons with other eastern arc mountains will be necessary to see how significant these estimates are, but it is likely to be higher than most areas based on our current understanding of other eastern arc mountains. species richness comparison the south nguru mountain forests support a rich forest associated herpetofauna of about 67 species. this value is higher than all the considered sites in africa (see table 5) fig. 4. species overlap among sampled sites. the graph shows the high complementarity of the species composition in the sampled sites and how sites at same altitude on different mountain blocks (kanga and pemba) have a more similar species composition than sites at different altitudes on the same mountain block. 118 m. menegon, n. doggart and n. owen and comparable with the one recorded for the virunga national park (de witte, 1941), but in an area about more than 20 times smaller. detailed comparison between sites is complicated by the differences in: total area and elevational ranges of the available sites, and biases in data collecting effort. despite this, a preliminary comparison between sites with similar geographic features places nguru south as being among the richest sites in africa, comparable with the mesoamerican site of monteverde in costa rica. discussion before the surveys described in this paper, the south nguru mountains were believed to have no narrow endemic species (burgess et al., 2007); the results presented in this paper stress the need for a rapid taxonomic assessment of the newly discovered species in order table 5. comparison of the “forest-associated” herpetofauna species richness in some tropical mid-elevation-montane forests of africa, central america, and the philippines (modified from vonesh 1998). site elevation m area (ha) amphibians reptiles total spp /area reference usambara mts., tanzania 600-2286 22100 23 29 52 0.23 howell, 1993; poynton et al., 2007 uzungwa scarp fr, tanzania 600-2000 21000 28 29 57 0.27 menegon and salvidio, 2005 kibale n.p., ugandaa 1400-1550 56000 15 29 44 0.07 vonesh, 1998 bwindi n.p., uganda 1200-2600 31000 20 21 41 0.13 drewes, 1991 virungas n.p., congoc 1300-3000 300000 27 40 67 0.022 dewitte, 1941 korup n.p., cameroond 1080-1768 124000 33 11 44 0.035 lawson, 1993 monteverde, costa ricae 1300-1470 12000 25 36 61 0.5 timmerman, 1981 cuernos de negros, philippine 1050-1350 10000 7 13 20 0.2 brown and alcala, 1961 nguru south, tanzania 750 2200 12000 33 34 67 0.55 this paper a) only kibale species restricted to the forest interior are included; b) species listed as extralimital by drewes and vindum (1991) are excluded; c) only forest species found above 1300 m are included; d) only species found at mt. yuhan (to 1079 m); rumpi hills (1000-1768 m) and nta ali (to 1200 m) are included. lawson (1993) comments that these elevations were under sampled; e only species restricted to timmerman’s (1981) pre-montane and lower montane zones (2-5) are included; f only specimens in submontane and montane forest zones are included from brown and alcala (1961), the source for closed forest area in east usambara and south nguru mountains is newmark (2002) for the uzungwa scarp the closed forest area is assumed to be the same of the gazetted area. 119herpetofauna of nguru mountains to further define the biological value of south nguru mountain forests where 16 out of 18 strictly endemic species have no name or iucn red list assessment. nguru south, kanga and mkindo are categorised as national protection forest reserves. as such they are reserved ‘for the purposes of protection of water sheds, soil conservation and the protection of wild plants’ (government of tanzania, 2002). kanga and nguru south forest reserves are managed directly by the morogoro regional catchment forest office while mkindo is managed jointly by the four surrounding communities and the morogoro regional catchment forest office. the miombo woodland in the lowlands between kanga and nguru south is on village land and does not lie within a protected area. although it is intended that all forest reserves will have a management plan, only mkindo has one at present. however, despite the legal protection afforded by the forest act (2002) enforcement is weak and sporadic. some of the key threats to the herpetofauna of the south ngurus are forest loss and degradation as a result of fire, selective logging, encroachment from agricultural land and the removal of the forest shrub and herb layer for the cultivation of cardamom and yams. many of the species of reptile and amphibian recorded during the survey are associated with forests and several species are associated with the shrub layer of the forest. this includes callulina spp., nectophrynoides spp. and rhampholeon sp. that were only found in the shrub layer of undisturbed forest. while the clearance of the forest is likely to have the most significant negative impact on all forest species in the ngurus, cardamom cultivation is particularly problematic in the forest close to ubiri, mafuta, kwelikwiji, mhonda, digoma, digarama, maskati, pemba and kigugu villages. cardamom cultivation could therefore potentially also have a significant impact, as this agricultural practice will affect many endemic species closely associated with the forest shrub layer. the long term impacts of cardamom cultivation in forest areas are not fully understood, but are likely to impede regeneration of forest plants. the distinct nature of the herpetofauna species assemblages at high altitude and the remarkable number of putative endemics in the south nguru mountains, highlights the conservation importance of the south nguru mountain forests within the eastern arc mountains. the high elevational turnover of species indicates the importance of conserving forest at all altitudes. areas of forest or marginal habitats, even at low elevation, might also be vital in generating high species diversity, and need to be considered in the development of a conservation strategy for the area. differences also exist between assemblages at sites at similar altitudes in adjoining mountain fragments within the south nguru mountain forests indicating the fine scale geographic turnover in the herpetofauna of the area (fig. 3). this suggests that not only is it important to conserve the forest along the elevational gradient but it is also important to conserve fragments in geographically complex terrains, where isolated populations and therefore potentially new species may exist. since 2004, the tanzania forest conservation group (tfcg), a tanzanian non-governmental organisation, has been working with stakeholders to improve conservation planning in the south ngurus. tfcg, through a partnership programme known as pema, has facilitated a dialogue between communities and the government to develop a vision for natural resource management in the south nguru landscape. villagers and government have identified a series of actions required to address the issue of forest loss. this includes a combination of direct forest management activities such as developing and implementing forest management plans and boundary demarcation; and activities aimed at reducing local people’s dependence on unsustainable activities such as the current methods of cultivating 120 m. menegon, n. doggart and n. owen cardamom. the programme represents an opportunity to reverse the current trend of forest loss and degradation. to succeed the programme will need sustained commitment from the government of tanzania, civil society organisations, the local communities and development partners to conserve the unique biodiversity of this area. acknowledgements thanks to abrahaman mndeme, david loserian and ernest benson. moses mwangoka kindly provided information on vegetation of the sampled sites, simon loader provided results of prelimifig. 3. some species recorded during the present study. clockwise: rhampholeon acuminatus; kinyongia fisheri fisheri; dipsadoboa werneri; an undescribed species of callulina; an undescribed species of leptopelis and an undescribed giant species of nectophrynoides. 121herpetofauna of nguru mountains nary molecular investigation on some of the species collected and useful suggestions on an early draft of this manuscript, john poynton confirmed the identification of some of the amphibian specimens, van wallach kindly provided identifications for the typhlopidae, joe beraducci kindly provided data on some snakes species, david moyer, aaron bauer, neil burgess and kim howell helped us in many ways. thanks to an anonymous referee for his valuable comments on our work. finally we would like to thank the government of tanzania, the tanzania commission for science and technology (costech) for providing permission to study reptiles and amphibians of the eastern arc mountains (costech permit no 2004-335-er-98-13 mm), the cites management authority in tanzania and the wildlife division, who issued the necessary permits for the export of the specimens. the surveys were financed by danida through the pema programme and by the museo tridentino di scienze naturali and by the critical ecosystem partnership fund (cepf). references baker, n.e., baker, e.m. 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(1972): evolution and measurement of species diversity. taxon 21: 213251. 124 m. menegon, n. doggart and n. owen appendix 1. check list of the amphibians and reptiles of the nguru south mts. based on findings presented in this paper and bibliographic records. amphibia anura arthroleptidae arthroleptinae arthroleptis affinis ahl, 1939 arthroleptis xenodactyloides (hewitt, 1933) arthroleptis xenodactylus boulenger, 1909 arthroleptis sp. arthroleptis sp. nov. leptopelinae leptopelis sp. nov. leptopelis uluguruensis barbour and loveridge, 1928 leptopelis flavomaculatus (günther, 1864) leptopelis vermiculatus (boulenger, 1909) leptopelis cf. parkeri bufonidae amietophrynus brauni (nieden, 1910) amietophrynus gutturalis (power, 1927) nectophrynoides sp. nov. 1 nectophrynoides sp. nov. 2 nectophrynoides sp. nov. 3 nectophrynoides sp. nov. 4 schismaderma carens (smith, 1848) hyperolidae afrixalus uluguruensis (barbour and loveridge, 1928) afrixalus stuhlmanni stuhlmanni (pfeffer, 1893) afrixalus stuhlmanni complex afrixalus fornasinii (bianconi, 1940) hyperolius mitchelli (loveridge, 1953) hyperolius spinigularis (stevens, 1971) hyperolius sp. (puncticulatus – like) hyperolius sp. brevicipitidae callulina sp. nov. 1 callulina sp. nov. 2 callulina sp. nov. 3 callulina sp. nov. 4 probreviceps macrodactylus macrodactylus nieden, 1926 probreviceps sp. nov. microhylidae hoplophryninae hoplophryne sp. nov. rhacophoridae rhacophorinae chiromantis xerampelina peters, 1854 pyxicephalidae cacosterninae amietia angolensis (bocage, 1866) ptychadenidae ptychadena anchietae (bocage, 1868) 125herpetofauna of nguru mountains petropedetidae petropedetes sp. nov. phrynobatrachidae phrynobatrachus uzungwensis grandison and howell, 1983 phrynobatrachus parvulus (boulenger, 1905) phrynobatrachus sp. pipidae xenopus cf. petersii gymnophiona caecilidae boulengerula sp. nov. scolecomorphus sp. nov. reptilia sauria chamaleonidae chamaeleoninae chamaeleo werneri (tornier, 1899) chamaeleo deremensis matschie, 1892 chameleo melleri gray, 1865 chamaeleo dilepis leach, 1819 kinyongia fischeri (reichenow, 1887) kinyongia oxyrhina (klaver and böhme, 1988) brookesiinae rieppeleon brachyurus (günther, 1893) rieppeleon brevicaudatus (matschie, 1892) rhampholeon acuminatus mariaux and tilbury, 2006 rhampholeon sp. nov. agamidae agama montana barbour and loveridge, 1928 agama agama (linnaeus, 1758) gekkonidae cnemaspis africana werner, 1895 urocotyledon wolterstorffi (tornier, 1900) lacertidae holaspis laevis werner, 1895 gastropholis prasina werner 1904 scincidae lygosominae leptosiaphos kilimensis stejneger 1891 lygosoma afrum peters 1854 trachylepis varia (peters, 1867) trachylepis maculilabris (gray, 1845) trachylepis striata (peters, 1844) scincinae scelotes uluguruensis (barbour and loveridge, 1928) proscelotes cf. eggeli tornier 1902 melanoseps loveridgei brygoo and roux-estève, 1982 varanidae varanus niloticus linnaeus, 1758 serpentes typhlopidae rhinotyphlops mucruso (peters, 1854) 126 m. menegon, n. doggart and n. owen typhlops lineolatus jan, 1864 typhlops gierrai mocquard, 1897 colubridae colubrinae crotaphopeltis tornieri (werner, 1897) crotaphopeltis hotamboeia laurent, 1968 dipsadoboa werneri boulenger, 1897 thelotornis usambaricus broadley, 2001 xyeledontophis uluguruensis broadley and wallach, 2002 dispholidus typus smith, 1829 philothamnus hoplogaster günther, 1863 philothamnus punctatus peters,, 1867 philothamnus macrops (boulenger, 1895) lamprophiidae lamprophinae lamprophis fuliginosus boie, 1827 lycophidion capense loveridgei laurent, 1968 lycophidion meleagre boulenger, 1893 atractaspidinae aparallactus jacksoni günther, 1888 aparallactus guentheri boulenger, 1895 atractaspis bibronii smith,1849 incertae sedis buhoma vauerocegae tornier, 1902 prosymna stuhlmanni (pfeffer, 1893) natricidae natriciteres sylvatica broadley, 1966 elapidae elapinae elapsoidea nigra günther, 1888 dendroaspis angusticeps smith,1849 viperidae viperinae atheris ceratophora werner, 1895 bitis arietans (merrem, 1820) bitis gabonica duméril, bibron and duméril, 1854 appendix 2. voucher specimens collection number amphibia arthroleptis sp.1 – mtsn8135; arthroleptis xenodactyloides – mtsn8529; arthroleptis sp. 2 – mtsn8168; arthroleptis sp. nov – mtsn8142; leptopelis cf. barbouri – mtsn8494; leptopelis uluguruensis – mtsn8227; leptopelis flavomaculatus – mtsn8239, leptopelis vermiculatus – mtsn8230; amietophrynus brauni – mtsn8251; amietophrynus gutturalis – mtsn8355; nectophrynoides tornieri – mtsn8344; nectophrynoides sp. nov. 1 – mtsn8199; nectophrynoides sp. nov. 2 – mtsn8547; nectophrynoides sp. nov. 3 kmh35971; nectophrynoides sp. nov. 4 schismaderma carens – mtsn8231; afrixalus sp. – mtsn8375; afrixalus uluguruensis – mtsn8163; afrixalus stuhlmanni stuhlmanni – mtsn8275; hyperolius mitchelli – mtsn8277; hyperolius spinigularis – mtsn8265; hyperolius puncticulatus mtsn8438; chiromantis xerampelina kmh26922; hyperolius sp. – mtsn8361; cal127herpetofauna of nguru mountains lulina sp. nov. 1 – mtsn8131; callulina sp. nov. 2 – mtsn8138; callulina sp. nov. 3 – mtsn8205; callulina sp. nov 4 – probreviceps sp. nov. kmh26923; mtsn8242; probreviceps macrodactylus macrodactylus – mtsn8507; hoplophryne uluguruensis – mtsn8144, amietia angolensis – mtsn8174; ptychadena anchietae – mtsn8339; petropedetes sp. nov. – mtsn8364; phrynobatrachus uzungwensis – mtsn8371, phrynobatrachus cf. parvulus – mtsn8372; phrynobatrachus sp. – mtsnpending, xenopus cf. petersii – mtsn8249; boulengerula sp. – mtsn8302; scolecomorphus sp. – mtsn8421. reptilia chamaeleo werneri – mtsn8451; chamaeleo deremensis – mtsn8195; chameleo melleri – pictured and released; chamaeleo dilepis pictured and released; bradypodion fischeri mtsn8318; bradypodion oxyrhinum – mtsn8416; rieppeleon brachyurus – mtsn8431; rieppeleon brevicaudatus – mtsn8220; rhampholeon sp. nov. – mtsn pending; rhampholeon sp. – mtsn8539; agama montana – mtsn8186; agama agama – mtsn8346, cnemaspis africana – mtsn8214; urocotyledon wolterstorffi – mtsn8222; holaspis laevis – obs. and pictured; trachylepis varia – obs. and pictured; trachylepis maculilabris – obs. and pictured; trachylepis striata – mtsnpending; proscelotes eggeli kmh26709; scelotes uluguruensis – mtsn8423; varanus niloticus – obs.; rhinotyphlops mucruso – mtsn8335; typhlops gierrai – mtsn8433; thelotornis usambaricus – mtsn8400, buhoma vauerocegae kmh26702; dipsadoboa werneri – mtsn8505; crotaphopeltis tornieri – mtsn8235; crotaphopeltis hotamboeia 8334; dispholidus typus – mtsn8410, lamprophis fuliginosus – mtsn8387; lycophidion capense loveridgei – mtsn8345; lycophidion meleagre – mtsn8414; natriciteres sylvatica – mtsn8439; philothamnus cf. hoplogaster – mtsn8178; philothamnus punctatus – mtsn8190; philothamnus macrops – mtsn8200; prosymna stuhlmanni – mtsn8343; aparallactus jacksoni – mtsn8352; aparallactus guentheri – mtsn8341; atractaspis bibronii – mtsn8354; elapsoidea nigra – mtsn8349; dendroaspis angusticeps – atheris ceratophorus kmh23977; mtsn8206; bitis arietans – mtsn8209. acta herpetologica 17(2): 147-157, 2022 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-12542 molecular analysis of recently introduced populations of the italian wall lizard (podarcis siculus) oleksandra oskyrko1,2,*, lekshmi b. sreelatha1,12,13, iolanda silva-rocha1, tibor sos3,4, sabina e. vlad5,6,7, dan cogălniceanu5,6, florina stănescu6,7,8, tavakkul m. iskenderov9, igor v. doronin10, duje lisičić11, miguel a. carretero1,12,13 1 cibio research centre in biodiversity and genetic resources, inbio, universidade do porto, campus de vairão, 4485-661, vairão, portugal 2 department of zoology, faculty of science, charles university, vinićná 7, 12844, prague, czech republic 3 evolutionary ecology group, hungarian department of biology and ecology, babeș-bolyai university, clinicilor street 5–7, 400006, cluj napoca, romania 4 “milvus group” bird and nature protection association, tîrgu mureș, 540445, romania 5 faculty of natural and agricultural sciences, ovidius university constanţa, aleea universități 1, campus corp b, 900470, constanƫa, romania 6 asociația chelonia românia, 062082, bucharest, romania 7 cedmog center, ovidius university constanța, tomis avenue 145, constanta, romania 8 black sea institute for development and security studies, ovidius university constanța, 900470, constanța, romania 9 institute of zoology, national academy of sciences of azerbaijan, az-1073, baku, republic of azerbaijan 10 zoological institute, russian academy of sciences, universitetskaya nab.,1, 199034, st. petersburg russia 11 division of animal physiology, department of biology, faculty of science, university of zagreb, roosveltov trg 6, hr, 10 000, zagreb, croatia 12 departamento de biologia, faculdade de ciências da universidade do porto, r. campo alegre, s/n, 4169 007, porto, portugal 13 biopolis program in genomics, biodiversity and land planning, cibio, campus de vairão, 4485-661, vairão, portugal *corresponding authors. e-mail: sashaoskirko@gmail.com submitted on: 2022, 2nd february; revised on: 2022, 18th may; accepted on 2022, 24th june editor: simon baeckens abstract. in recent decades, many reptile species have been introduced outside their native ranges, either accidentally through the transportation of goods and materials (e.g., plants, construction materials), but also intentionally through the pet trade. as a paradigmatic example, the italian wall lizard, podarcis siculus, native to the italian peninsula, sicily and the north adriatic coast, has been introduced in several nearby islands since historical times (corsica, sardinia, menorca). besides these regions, scattered populations were later reported from the iberian peninsula, france, switzerland, turkey, greece, the united kingdom and north america. here, we provide molecular evidence regarding the introduction and origin of p. siculus in six new populations outside its native range: romania (bucharest and alba iulia), inland croatia (zagreb and karlovac), italy (lampedusa island) and azerbaijan (baku). phylogenetic analysis suggests that the alba iulia (romania) population originated from a single clade (tuscany), while the population from azerbaijan is admixed including two distinct clades, one similar to those found in sicily and the other present across the tuscany clade. samples from bucharest also have admixed origins in tuscany and the adriatic clades. less surprisingly, samples from zagreb and karlovac are included in the adriatic clade while those from lampedusa originated from sicily. overall, our results further demonstrate that p. siculus is able to establish outside of its native range even under different climatic conditions, not particularly from specific clades or source areas. also, for the first time in this species, our results indicate that repeated human introductions promote lineage admixture and enhance their invasive potential. 148 oleksandra oskyrko et alii keywords. biological invasions, alien species, genetic diversity, human-mediated introductions, lacertidae. introduction long dispersal movements of fauna constitute natural phenomena in the evolution of biological communities (de queiroz 2005). however, the increasing degree of anthropisation and human-mediated transport are leading to an enormous increase in the rates of animal translocations. this has resulted in a major threat of biological invasions the process by which an alien species establishes, expands its geographic range and numbers, and exerts ecological or economic impacts in a new area with negative effects on the native biota (brown et al., 2007). different steps are required for a species to become invasive. the first step is transportation of the species from its native range, then survive both the transportation and the conditions within the new range, and finally, reproduce and spread, threatening native biota (williamson and fitter, 1996). there are currently more than 14,000 alien species recorded in europe (easin, https://easin. jrc.ec.europa.eu/) with more than half originating from outside the territories (roy et al., 2019), and the number of occurrences is rapidly increasing due to new introductions and due to the growing research and awareness on this topic (seebens et al., 2017). evidence of the negative impacts of many alien species (pimentel, 2011), including reptiles and amphibians (shine, 2014; kraus, 2015; measey et al., 2016) is increasing. this has added urgency for achieving a thorough understanding of factors mediating success at different stages of the introduction-naturalization continuum (richardson et al., 2000; blackburn et al., 2011) in order to inform policies and reduce the risk of further invasions. the millennium ecosystem assessment report (2005) showed that invasive alien species are one of the five main drivers of biodiversity loss. there are several mechanisms through which invasive alien species threaten native biodiversity: direct interactions such as predation/herbivory and parasitism, or in direct interactions such as competition for food or other resources, modifications of ecosystems, and introduction of new parasites (hendrix et al., 2008; suarez and tsutsui, 2008; kenis et al., 2009). however, there are alien species which apparently have little or no detectable effects on their new environment (strayer, 2012), leading some authors to consider these effects as positive (sogge et al., 2008; chiba, 2010; schlaepfer et al., 2011), although this view is controversial (simberloff et al., 2012; richardson and ricciardi 2013; cassini, 2020). in most instances the specific source of an introduced population is not known, multiple undocumented introductions are always possible, and putative routes of introduction and transport vectors may not be reliable. information on the origin and introduction pathways is, however, crucial for determining the degree of invasiveness and for implementing appropriate management policies. in this context, molecular markers can help to reconstruct the history of an introduction, identifying the number of native range source populations, their geographic location and extent, and the distribution of variation from these sources in the nonnative range, identifying those where negative effects on native biota are taking place (e.g., kolbe et al. 2004, 2013; fitzpatrick et al. 2012). the italian wall lizard, podarcis siculus (rafinesque, 1810), is one such reptile species that has been widely introduced (kraus, 2009). previous studies have concluded that the pathways by which the species is being introduced are multiple, ranging from the transportation of materials and goods, especially plant materials like olive trees (valdeón et al., 2010; rivera et al., 2011), to the pet trade industry from where individuals escaped or were released (deichsel et al., 2010), to deliberate introductions as a biocontrol agent against pest insects (rocha, 2021). it inhabits a wide range of habitats, from natural areas to agricultural and urban environments, and often uses man-made structures for refuge (capula, 1994; corti, 2006). from its native distribution in the italian peninsula, sicily and the north adriatic coast, this species has been introduced in several other places, such as the tyrrhenian islands, corsica and sardinia, menorca in the balearics (podnar et al., 2005; senczuk et. al., 2017). besides these regions, scattered introduced populations are also known from the iberian peninsula, switzerland, turkey, greece, united kingdom and in the united states (deichsel et al., 2010; schulte and gebhart, 2011; silvarocha et. al., 2012; 2014; kolbe et al., 2013; garin-barrio et al., 2020). in some of these locations, the italian wall lizard has already been demonstrated to be harmful to native species. for example, it outcompetes native podarcis species by being more aggressive (downes and bauwens, 2002) and more adaptable to novel situations (damas-moreira et al., 2019; nicolic et al., 2019), feeding earlier (limnios et al., 2021), eating more and growing faster (damas-moreira et al., 2019) and being less parasitized (tomé et al., 2021), often resulting in spatial exclusion of natives (nevo et al, 1972; ribeiro and sásousa, 2018). it may also hybridize with native, unrelated podarcis species contributing to the dilution of their 149new introduced populations of podarcis siculus genetic identity (capula, 1993, 2002; capula et al., 2002), and may undergo fast phenotypic shifts after introduction suggesting great levels of adaptability (herrel et al., 2008). all this evidence suggests that the italian wall lizard is not only an effective colonizer but also a successful invader. in this context, understanding its colonization patterns provides the basis for delineating early and more effective preventive measures (dorcas et al., 2010). in our study, we provide molecular evidence indicating the origin of p. siculus in six populations outside its native range, from: romania (bucharest and alba iulia; stănescu et al., 2020; iftime and iftime 2021), azerbaijan (baku; iskenderov et al., 2021), lampedusa island (lo valvo and nicolini, 2001) and inland croatia (karlovac and zagreb; d. lisičić unpubl.). we investigated the introduction process of p. siculus by means of mtdna sequences in a phylogeographic framework. clarifying the origin of these alien populations is expected to improve the picture of the colonization pattern revealed by previous studies. we aimed to (i) determine the origin of the introduced populations, (ii) infer the possible colonization routes, and (iii) discuss the management implications from these findings. material and methods the sampling took place in july, august 2020 and july 2021 in romania, and june 2019 in azerbaijan, september 2021 in croatia and september 2005 in lampedusa island. in bucharest, lizards were collected from the rose garden of the university of agriculture and veterinary medicine, while in alba iulia they were collected on the walls of the recently restored alba carolina fortress. in azerbaijan, lizards were found on a private landholding located on the shores of the caspian sea in the village of turkan (administratively included in baku). specimens from karlovac and zagreb in croatia as those in lampedusa town were also collected in urban environments. we collected a total of 16 samples (tail tips) from these six localities (table 1). the tail tips were removed by applying light pressure and were then stored in 96% ethanol. all lizards were released at the capture location. the geographical coordinates were recorded with a handheld gps. the geographic references are given in table 1 and shown in fig. 1. dna extraction was performed using the high-salt method (sambrook et al., 1989). partial sequences of 520 base pairs (bp) of the cytochrome b (cytb) gene were amplified using the primers gludg-l and cb3h from palumbi (1991). amplification of genomic dna began with an initial denaturation for 15 minutes at 94 °c followed by 94 °c for 30 s, annealing at 52 °c for 60 s with 34 cycles, and extension at 72 °c for 60 s. products were visualized with 1.5% agarose gel electrophoresis. the suitable amplicons were sent to external service (beckman coulter genomics) for purification and sequencing. the sequences generated in the present study (genbank accession numbers: on365568-on365583; table 1) were aligned with sequences downloaded from genbank. a total of 277 sequences from the italian peninsula, corsica and sardinia (senczuk et al., 2017), accession numbers: ky064841-ky065117 were downloaded. additionally, 41 published sequences from other introduced populations in eurasia: 33 sequences from the iberian peninsula and menorca (silva-rocha et al., 2012; garin-barrio et al., 2020), accession numbers: jx072938-jx072960, mw192534-mw192543; seven sequences from turkey, greece, and united kingdom (silva-rocha et al., 2014), accession numbers: kp036396-kp036402. the samples from switzerland (schulte and gebhart, 2011) were not included in the analysis because they were not available in genbank but the locations were added to the map according to silva-rocha et al. (2014). one sequence from podarcis melisellensis from genbank was used as an outgroup (accession number ay185057), following silva-rocha et al., (2014). sequences were edited using geneious prime v.2020.1 (https://www.geneious.com). the alignment was performed with mafft v.6 (katoh et al., 2019) and included 330 sequences + one sequence outgroup. the best-fitting model was tim2+i+g using partitionfinder2v. 2.1 (lanfear et al. 2017). a maximum likelihood (ml) tree was constructed using raxml v.7.2 (stamatakis, 2006) with 1000 pseudoreplicates to assess the confidence of branches. bayesian inference (bi) analysis was carried out by mrbayes v.3.2 (huelsenbeck and ronquist, 2001) with 5×107 generations and four chains, and subsampling parameters and trees every 100 generations. finally, 10% of the posterior samples were discarded as burn-in. to inspect the mtdna cytb haplotype diversity, a 95% maximum parsimony haplotype network was constructed using the tcs inference (clement et al., 2000) in popart v.1.7 (leigh & bryant, 2015). the resulting tree was annotated using figtree 1.4.3 (rambaut, 2014). molecular diversity indices, including the number of haplotypes (h), haplotype diversity (h), and nucleotide diversity (π) were evaluated in r 4.2.0 (r core team 2020) using the “pegas” package (paradis, 2010). uncorrected genetic distances (p-distances) within clades of were estimated with paup v.4.0a10 (swofford, 2003). maps were created in qgis 3.10.8 (qgis development team, 2020). 150 oleksandra oskyrko et alii results the final alignment included 331 sequences. the bi consensus tree showed a similar topology to the ml tree. the phylogenetic analysis supported the same topology with five well-supported all clades: s1, s2, s3 a1, a2, a3 and t (appendix 1, fig. s1) following the terminology in senczuk et al. (2017). clades were separated from each other with high support values (1.00-0.98). we identified three clades (s1, s2 and s3) within the siculo-calabrian lineage. the central-northern lineage split approximately into two main groups that for simplicity we refer to as “adriatic” and “tyrrhenian”. the adriatic group also included two clades with a separation of the clades a1, fig. 1. network and maps of the natural distribution and introduced populations of podarcis siculus in eurasia. a. networks of the seven mtdna clades identified by the phylogeny, colours according to senczuk et al. (2017). each circle size is proportional to their frequencies and each filled rectangle represents one substitution. the different colours within each network depict the principal identified clades. the names of the locations of the introduced populations are highlighted in red and the locations from the natural range are highlighted in black; abbreviation: az – azerbaijan, gb great britain, gr – greece, hr – croatia, ib iberian peninsula, it –italy, ro – romania. b. geographic distribution of the mtdna haplotypes in p. siculus and are coloured according to the main haplogroups identified by senczuk et al. (2017). the natural habitat is represented according to crnobrnja-isailović et al. (2009) but modified after senczuk et al. (2017), namely corsica and sardinia are highlighted in gray lines as the introduced area. clade a1 is indicated by the black arrows on the inlay map. c. map of the natural distribution with mtdna haplotypes and introduced populations of lizards in europe and asia. populations of unknown origin are highlighted with white dots. 151new introduced populations of podarcis siculus a2 and a3. the tyrrhenian clade t was also clearly separated from the others. tree presented in appendix 1 (fig. s1). the molecular diversity indices were demonstrated in appendix 1 (table s1). the general sample size (n) is 330 and the introduction sample size (nin) included 57. also the total number of haplotypes (h) is 106. the haplotype diversity (h) and nucleotide diversity (π) were non-significant for cytb but the values were similar to the senczuk et al. (2017). the uncorrected genetic distances among clades are displayed in appendix 1 (table s2). the largest genetic distances are between classes s1 and t (p = 0.0977). clade a2 had a lower distance to clade a1 (p = 0.0146). overall, the six studied populations of the italian wall lizard were assigned to three clades (s3, a2 and t). the phylogenetic analysis suggests that the romanian population from alba iulia originated from the tuscany region (clade t) and was included in the haplogroup together with samples from the giannella and feniglia (italy). the population from bucharest (romania) revealed admixture: one individual was included in the clade t and close with the samples from pian della rasa but the other two belonged to the adriatic clade (clade a2) and included in a large haplogroup with samples from locations such as rosa marina, s. domino, forest umbra, r.n. sale tanagro, melfi and atrium (italy). the population from azerbaijan (baku) was also admixed including two distinct clades, one similar to the clade found in sicily (clade s3). this is a large haplogroup that also includes samples from noto lido, florida, saline di priolo and sorciano (italy). other azerbaijani samples came from pian della rasa (clade t). the sample from italy (lampedusa island) was included in the clade s3 and close with a sample from mazzarino. the croatian samples from zagreb and karlovac came from the same haplogroup with romanian samples from bucharest in clade a2. the previously-studied sequences from alien populations of the italian wall lizard were included in four clades: s3, s2, a2 and t. the tyrrhenian clade t includes the largest number of samples of introduced populations (nin = 28, n = 66, h = 20), mainly distributed across the north-central tyrrhenian coast, included the largest number of samples of introduced populations in eurasia. in addition to romania and azerbaijan, this clade was present in the iberian peninsula (madrid, getaria, cantabria and lisbon) and the united kingdom (buckinghamshire). clade a2 (nin = 13, n = 71, h = 16), ranging across the adriatic coast (excluding clade a1 which is restricted to the curzolan islands, croatia), included two samples from bucharest (romania) and croatia (zagreb and karlovac) as well as the published samples from greece (palaio faliro) and iberian peninsula (bilbao). clade a3 (nin = 0, n = 10, h = 3) includes populations from northern coastal areas of calabria (catena costiera). this clade is more related to the adriatic clade a2, forming part of the central-northern lineage, than to the other clades found in southern calabria. we only failed to find any introduced populations originating from the clade a3. clade s3 is the largest lineage that includes 54 haplogroups (nin = 14, n = 154). within this clade, we identified five alien populations: two new samples from azerbaijan and the island of lampedusa, and also the table 1. novel sequences used in this study and their geographic position. genbank number location country coordinates clade year of the first reportlatitude longitude on365568 bucharest romania 44°470’n 26°065’e a2 2019 on365569 bucharest romania 44°470’n 26°065’e a2 2019 on365570 bucharest romania 44°470’n 26°065’e t 2019 on365571 alba iulia romania 46°067’n 23°566’e t 2019-2020 on365572 alba iulia romania 46°067’n 23°566’e t 2019-2020 on365573 alba iulia romania 46°066’n 23°566’e t 2019-2020 on365574 alba iulia romania 46°066’n 23°566’e t 2019-2020 on365575 alba iulia romania 46°066’n 23°566’e t 2019-2020 on365576 baku azerbaijan 40°363’n 50°212’e s3 2019 on365577 baku azerbaijan 40°363’n 50°212’e t 2019 on365578 baku azerbaijan 40°363’n 50°212’e s3 2019 on365579 karlovac croatia 45°485’n 15°548’e a2 2021 on365580 karlovac croatia 45°485’n 15°548’e a2 2021 on365581 zagreb croatia 45°796’n 15°976’e a2 2021 on365582 zagreb croatia 45°796’n 15°976’e a2 2021 on365583 lampedusa island italy 35°508’n 12°593’e s3 2001 152 oleksandra oskyrko et alii published samples from southern iberia (almería), as well as menorca and turkey (mudanya, güzelyah, iznik). clade s2 (nin = 2, n = 7, h = 7) included only samples from one location (la rioja, north iberia). samples from southern italy (such as gerase, maida and mammola) are also included in this clade. clade s1 (nin = 0, n = 17, h = 3) comprising populations from the southern part of the italian peninsula was more related to clade s2. discussion this study adds new valuable data to the complex picture of the invasion biology of p. siculus, a species with a complex phylogeographic structure, which encompasses multiple lineages across its range (senczuk et al., 2017; fig. s1). the results indicated seven highly supported clades within the eastern three-lined lizard (s1, s2, s3 a1, a2, a3 and t). possibly, the existence of complex topography and multiple refugia across the distribution range of the subspecies have led to the present diversity and distribution pattern of clades (appendix 1, table s2). as such, the studied alien populations not only belonged to different lineages as previously reported for other studies, but also more than one lineage was found in two of the introduced populations (e.g., baku and bucharest; fig. 1). our results support previous claims that the introduced italian wall lizard populations have multiple and even admixed origins within their native range (namely the italian peninsula). in fact, p. siculus may use both vegetation and rocks for foraging, basking and finding shelter (corti, 2006). this probably allows it to be unknowingly transported with construction materials, plants or other materials associated with construction works, agriculture and gardening (silva-rocha et al., 2014). in fact, the habitats of the populations from bucharest, zagreb, karlovac and baku have all undergone gardening and plant importation (iftime and iftime, 2021; iskenderov et al. 2021; d. lisičić unpubl.; fig. 3, b and c). while those in lampedusa have undergone significant reconstruction works during the past years (m. carretero pers. obs., respectively; fig. 3, d). a similar situation occurred in alba iulia (romania), where lizards were found after the reconstruction of the alba carolina fortress (t. sos pers. obs., respectively; fig. 3, a). the distribution of this species to the east is associated with an increase in trade, namely the growth of exports of plants from the mediterranean (kukushkin et al., 2017). p. siculus was also found for the first time in sochi (southern russia, see fig. 1), which is a large port city (tuniyev et al., 2020). the origin of this population is not known today, but the interesting fact is that this population was found simultaneously with the population in baku, azerbaijan (iskenderov et al. 2021). populations from france are also of unknown origin, especially the recent discovery of this species in the gradignan botanical garden, gironde (berroneau et al. 2021, fig. 1). similar pathways of introduction have been reported for the congeneric p. muralis (santos et al., 2019; jablonski et al., 2019) although the more saxicolous habits of this species makes it less suited than p. siculus for using vegetation as an introduction vehicle. because these pathways of introduction are humanmediated, the biogeographic signal was expected to be minimal (helmus et al., 2014). indeed, we found little or no correspondence between the geographical location of the populations and their phylogenetic lineages. the only exceptions were the populations from inland croatia and lampedusa island, which belonged to lineage closest to the native populations (north adriatic and sicily, at a distance of 100-200 km, respectively). the population on fig. 2. representative pictures of podarcis siculus. a. p. siculus from alba iulia (romania); b. p. siculus from bucharest (romania). photos by t. sos. 153new introduced populations of podarcis siculus lampedusa island was discovered in 2001. we collected samples in 2005 and confirmed the existence of this population on the island. populations from croatia (karlovac and zagreb) were found quite recently (in 2021), which requires further research to confirm the successful introduction of populations. other finds from romania and azerbaijan were reported for the first time in 2019-2020. however, lizards were repeatedly observed in the following years in these localities and juveniles were found, which confirms the successful breeding of lizards in new areas (stănescu et al., 2020; iftime and iftime, 2021; iskenderov et al., 2021). in its native range, the italian wall lizard appears to be more thermophilic, but this doesn’t seem to be reflected in the climates prevailing in non-native areas. in particular, several introduced populations (northern iberian peninsula, switzerland, united kingdom, as well as north america, see silva-rocha et al., 2014; here, romania and azerbaijan) clearly occur in non-mediterranean climates, with harsh winters. moreover, there is no apparent correspondence between the lineage subranges, although this should be further explored with modelling evidence (carretero and sillero, 2016). another relevant result, reported here for the first time, is the existence of admixed populations, namely in bucharest and in baku, which were dense although localized. on one hand, this already reveals repeated introductions from different source regions with contrasting climate regimes (tuscany and adriatic in bucharest; tuscany and sicily in baku). on the other hand, potential hybridization between those contacting haplogroups might produce novel phenotypes adapted to local conditions, hence, increasing the invasive potential of the species (kolbe et al., 2007), as it has already been reported for p. muralis (santos et al., 2019; michaelides et al., 2013; while et al., 2015). these phylogenetic outcomes, added to the partial but repeated evidence of functional negative interactions between p. siculus (belonging to multiple lineages and in multiple areas) and native podarcis species, configure an fig. 3. invaded habitats by podarcis siculus in europe and asia: a. alba carolina citadel, alba iulia (romania); b. university of agriculture and veterinary medicine, rose garden, bucharest (romania); c. private garden in baku (azerbaijan); d. the island of lampedusa (italy). photos by t. sos (a, b), t.m. iskenderov (c), m.a. carretero (d). 154 oleksandra oskyrko et alii invasion scenario. the timeframe and spatial scale of such a threat should be uncovered by an assessment of p. siculus at a global level. meanwhile, a principle of caution recommends at least early detection of any new alien population and monitoring of existing ones (carretero and silvarocha, 2015), particularly in potential invasion hubs such as harbours and railways (mollov, 2009; tok et al., 2014). eradication actions should also be considered in the early stages, when the chances of success are higher (e.g., buckinghamshire, south east england hodgkins et al., 2012; athens, greece, adamopoulou and pafilis, 2019). overall, the results obtained here accumulated to the previous evidence strongly suggest that the italian wall lizard p. siculus is an effective invader. its successful acclimatization to environmental conditions different from those prevailing in its original mediterranean range increases the probability of becoming invasive. acknowledgements we are grateful to c. corti, p. lo cascio, o. drăgan and s. t. topliceanu for help during field sampling. the project has been supported by the portuguese foundation for science and technology (fct) project 28014 02/saict/2017. igor v. doronin was supported by the russian science foundation (grant number 22-2400079) and sabina e. vlad was supported by the project antreprenordoc in the framework of human resources development operational programme 20142020, financed from the european social fund (grant number 36355/23.05.2019 hrd op /380/6/13 – smis code: 123847). animals and dna samples were collected with permissions from the comisia de etică a facultăţii de știinţe ale naturii și știinţe agricole in romania and in lampedusa island were provided by riserva naturale orientata “isola di lampedusa” (management legambiente) in 2005; 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(2015): adaptive responses to cool climate promotes persistence of a non-native lizard. proc. r. soc. lond. b 282: 20142638.. acta herpetologica vol. 17, n. 2 december 2022 firenze university press cryptic diversity in pygmy chameleons (chamaeleonidae: rhampholeon) of the eastern arc mountains of tanzania, with description of six new species michele menegon1,2,*, john v. lyakurwa3,4, simon p. loader5, krystal a. tolley6,7 preliminary genetic characterisation of southern smooth snake coronella girondica (serpentes, colubridae) populations in italy, with some considerations on their alpine distribution matteo r. di nicola1, raffaella melfi2, francesco p. faraone3,*, daniel l. n. iversen4, gabriele giacalone5, giovanni paolino1, mario lo valvo6 species diversity and distribution of amphibians and reptiles in sardinia, italy claudia corti1,2,*, marta biaggini1, valeria nulchis2, roberto cogoni2, ilaria maria cossu2, salvatore frau4, manuela mulargia2, enrico lunghi2, lara bassu2. the italian wall lizard, podarcis siculus campestris, unexpected presence on gorgona island (tuscan archipelago) marco a.l. zuffi1,*, alan j. coladonato2, gianluca lombardo3, antonio torroni3, matilde boschetti1, stefano scali4, marco mangiacotti2, roberto sacchi2 molecular analysis of recently introduced populations of the italian wall lizard (podarcis siculus) oleksandra oskyrko1,2,*, lekshmi b. sreelatha1,12,13, iolanda silva-rocha1, tibor sos3,4, sabina e. vlad5,6,7, dan cogălniceanu5,6, florina stănescu6,7,8, tavakkul m. iskenderov9, igor v. doronin10, duje lisičić11, miguel a. carretero1,12,13 sunny-side up: ontogenetic variation in egg mass temperatures of the wood frog rana sylvatica ryan calsbeek*, ava calsbeek, isabel calsbeek ecological niche differentiation in the anatolian rock lizards (genus: anatololacerta) (reptilia: lacertidae) of the anatolian peninsula and aegean islands mehmet kürşat şahin1,*, kamil candan2,3, danae karakasi4, petros lymberakis4, nikos poulakakis4,5,6, yusuf kumlutaş2,3, elif yıldırım2,3, çetin ilgaz2,3 occupancy and probability of detection of the introduced population of eleutherodactylus coqui in turrialba, costa rica jimmy barrantes-madrigal1,*, manuel spínola parallada1, gilbert alvarado 2, víctor j. acostachaves3,4. one site, three species, three stories: syntopy of geckoes euleptes europaea (gené, 1839), hemidactylus turcicus (linnaeus, 1758), tarentola mauritanica (linnaeus, 1758) in a coastal area of southern tuscany (central italy) giacomo radi1,2, marco a.l. zuffi1,* comparative cytogenetics on zamenis lineatus and elaphe quatuorlineata (serpentes: colubridae) marcello mezzasalma1,* , elvira brunelli1, gaetano odierna2, fabio m. guarino2 acta herpetologica 4(1): 117-118, 2009 osservatorio erpetologico italiano in questa sezione sono pubblicate segnalazioni erpetologiche riguardanti il territorio amministrativo italiano, non ancora incluse nella banca dati della shi e pubblicate nell’atlante nazionale (sindaco et al., 2006). sarà data priorità alle osservazioni riguardanti specie di interesse conservazionistico internazionale (specie incluse negli allegati ii e iv della direttiva europea 92/43/cee “habitat”), nazionale (specie endemiche, rare o al limite di areale) o regionale (nuove segnalazioni per la regione o che definiscono più nel dettaglio in modo significativo, l’areale della specie). saranno inoltre considerate per la pubblicazione segnalazioni di interesse ecologico (ad esempio limiti altitudinali). per quanto riguarda le testuggini del genere testudo, saranno pubblicate solo le segnalazioni di siti in cui è provata la riproduzione. osservazioni opportunamente documentate di popolazioni naturalizzate di specie esotiche (come trachemys sp.) saranno pubblicate. per motivi conservazionistici, le località precise degli avvistamenti non saranno pubblicate. la shi non incoraggia la raccolta di esemplari al solo fine della segnalazione e non pubblicherà segnalazioni di animali sacrificati senza le necessarie autorizzazioni. i segnalatori sono invitati a fotografare gli esemplari in vita, a consegnare quelli trovati morti ad istituzioni pubbliche locali e inviare al presidente della shi le foto e le citazioni dell’istituzione in cui il reperto è conservato. le segnalazioni, che saranno sempre citate col nome dell’osservatore, saranno vagliate dai redattori dell’atlante nazionale, che si avvarrà dei redattori di acta herpetologica e di esperti regionali. la segnalazione potrà eventualmente essere completata con un commento redazionale. indirizzo a cui inviare le segnalazioni (si caldeggia l’invio per posta elettronica) a sebastiano salvidio, dipteris corso europa 26, 16132 genova. e-mail: salvidio@ dipteris.unige.it. simbologia utilizzata (in ordine possono essere utilizzati più simboli) 000001 numero d’ordine della segnalazione +ita prima segnalazione in italia di una specie a distribuzione geografica limitrofa +reg prima segnalazione nella regione di una specie (prime tre lettere della regione) +h-ii nuova segnalazione/sito di specie dell’allegato ii della direttiva habitat +h-iv nuova segnalazione/sito di specie dell’allegato iv della direttiva habitat +h-ii/s riconferma di segnalazione/sito storico (non confermato da almeno 10 anni) di specie dell’allegato ii della direttiva habitat 118 osservatorio erpetologico italiano +h-iv/s riconferma di segnalazione/sito storico (non confermato da almeno 10 anni) di specie dell’allegato iv della direttiva habitat +end nuova segnalazione/sito di specie endemica italica in località che ne definiscono meglio l’areale +rar nuova segnalazione/sito di specie endemica rara o al limite di areale +end/s riconferma di segnalazione/sito storico (non confermato da oltre 10 anni) di specie endemica italica +rar/s riconferma di segnalazione/sito storico (non confermato da oltre 10 anni) di specie endemica rara o al limite di areale +tes sito accertato di riproduzione di testuggine del genere testudo +eco significativa segnalazione di interesse ecologico (esempio: limite altitudinale, nuovo ambiente, nuovi aspetti di nicchia trofica o riproduttiva ecc) +eso sito accertato di riproduzione di specie esotica osservatorio erpetologico italiano – 6 – alloctoni simone masin +eso 110.370.0.001.0 trachemys scripta, riproduzione. zona naturalistica laghetti del tolcinasco, pieve emanuele, mi. 2008 nicola nitti +eso 110.370.0.001.0 trachemys scripta, riproduzione. zona lago prichicca, contrada caccamone, castellaneta, ta. 2008 nicola nitti +eso 110.370.0.001.0 trachemys scripta, riproduzione. lago tafuro, zona masseria tafuri, castellaneta, ta. 2008 acta herpetologica 18(1): 45-52, 2023 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-14359 screening of ophidiomyces ophidiicola in the free-ranging snake community annually harvested for the popular ritual of san domenico e dei serpari (cocullo, aq, italy) daniele marini1,2, ernesto filippi3,*, gianpaolo montinaro4, francesco c. origgi5,6 1 department of veterinary medicine, university of perugia, via san costanzo 4, 06126 perugia, italy 2 department of organismal biology, evolutionary biology centre, uppsala university, norbyvägen 18a, 752 36 uppsala, sweden 3 via bagnara calabra 6, 00178 rome, italy 4 rifcon gmbh, goldbeckstraße 13, 69493 hirschberg an der bergstraße, germany 5 institute of animal pathology, department of infectious diseases and pathobiology, vetsuisse faculty, university of bern, länggassstrasse 120, 3012, bern, switzerland 6 institute of infectious animal diseases (iiad), department of veterinary sciences, university of messina, viale giovanni palatucci sn, 98168 messina, italy *corresponding author. email: ernesto.flp@gmail.com submitted on: 2023, 4th february; revised on: 2023, 4th may; accepted on: 2023, 8th may editor: emilio sperone abstract. in the abruzzi village of cocullo (italy), each year, on may 1st, local snake hunters (known as serpari) display colubrids, captured in the wild, to commemorate the ancient ritual of san domenico. the ascomycete ophidiomyces ophidiicola (oo) is the causative agent of ophidiomycosis, an emerging disease with sublethal effects. skin lesions, such as dysecdysis, edematous, crusty or necrotic scales, swellings, nodules, and ulcers, are the most common clinical manifestation of the disease. the pathogen and its associated disease are well characterized in wild snakes in north america, whereas broad screenings of free ranging wild ophidians in europe are rare. in 2019, as part of a multi-year snake health monitoring project, all the cocullo ophidians were carefully examined for integumentary affections and those showing signs consistent with ophidiomycosis were dry swabbed on the skin and on any visible cutaneous lesions with a single applicator. the extracted dna underwent a broad-range panfungal pcr targeting the d1-d2 region, as well as two conventional pcrs specific to the its2 and igs regions of oo dna. twenty-three out of 129 snakes (13/82 elaphe quatuorlineata; 7/31 hierophis viridiflavus; 3/15 zamenis longissumus; 0/1 natrix helvetica) resulted clinically affected, but no specific oo genomic dna was detected by pcr. the cocullo ritual celebration provided a unique opportunity for the first systematic testing of a large sample size of a local snake community for the monitoring of this pathogen in italy. keywords. ophidiomycosis, snake fungal disease, sfd, snakes, health monitoring, cocullo, abruzzi (italy). ophidiomyces ophidiicola (oo) is the etiological agent of ophidiomycosis (also known as snake fungal disease – sfd), a fungal infection of snakes (lorch et al., 2015). this onygenalean fungus is resistant to various physical and chemical agents (allender et al., 2015b), and hibernacula may represent its environmental reservoir (campbell et al. 2021). ophidiomyces infection has been associated with sublethal effects on adults (agugliaro et al., 2020; lind et al. 2018a, b; tetzlaff et al., 2017) and potentially lethal outcomes on newborns (e.g., britton et al., 2019), translating into a potential impact on wild populations’ fitness and a threat to conservation. this emerging 46 daniele marini et alii infectious disease occurs with various cutaneous signs as dysecdysis, desquamation, scales abnormalities (e.g., displacing), local skin thickening, yellowish/brownish crusts, skin ulcerations, swelling and nodules (revised by baker et al., 2019), whereas visceral lesions are less frequently recorded. albeit impacts on different populations seem locally divergent or controversial, oo has been detected in free-ranging ophidians in most part of north america (di nicola et al., 2022). in europe, samples deriving from wild coronella austriaca, hierophis viridiflavus, natrix helvetica, n. maura, n. natrix, n. tessellata, vipera berus, v. nikolskii and zamenis longissimus from uk, czech republic, switzerland, germany, france, austria, hungary, poland, ukraine, or italy tested positive with molecular methods (franklinos et al., 2017; meier et al., 2018; schüler et al., 2022; blanvillain et al., 2022; marini et al., 2023), and a retrospective analysis date back the presence of the fungus in italy and switzerland since 1959 (origgi et al., 2022). in this paper we report the results of an investigation aimed at testing the presence of oo in a snake community from central italy. data were obtained by snakes captured for a religious ritual (the catholic cult of san domenico – of pagan and pre-christian origins) in the village of cocullo (abruzzi). this ceremony takes place in the first days of may, and has remained unchanged for several hundred years. the main feature of this occurrence is the presence of large numbers of wild-caught snakes by local snake hunters (serpari) during the weeks before the events. this ritual is well known and important in abruzzi’s (italy) culture and history, and it is closely dependent on the local ophidiofauna. in recent years, the ceremony has been accompanied by some significant conservation actions by the local authorities of cocullo, due to the increased awareness of the importance of environmental protection, in particular snake conservation. although no decline of cocullo’s snakes’ populations has been anecdotally detected in past years, some effects on the reproductive phenology of e. quatuorlineata and z. longissimus has been observed (filippi and luiselli, 2003) and a few areas surrounding cocullo are characterized by some disturbance factors for ophidiofauna, including a high density of wild boars (regarding this critical issue on snakes see filippi and luiselli, 2002). various species of colubridae are involved during the celebration, in particular elaphe quatuorlineata (pellegrini et al., 2017), one of the largest and more vulnerable species of snakes in mediterranean central italy (filippi and luiselli, 2000; filippi, 2003; capula and filippi, 2011), but also zamenis longissimus and hierophis viridiflavus. snakes are captured by snake hunters from the 19th of march till the 30th of april every year. since 2010, all snakes captured by serpari are assessed during the 2-3 days preceding the event, that has a fixed date on may 1st (overall n = 1300 snakes were registered and checked from 2010 to 2023, filippi and montinaro, in prep.): a scientific committee (composed by ef and gm in collaboration with a veterinarian) records the captured species, biometric data (weight, snout-vent length [svl]), sex, age class (juvenile, subadult, adult), site of capture of the ophidians brought by snake hunters. moreover, pit tags are checked or implanted, and a physical examination is carried out in addition to a swab for bacteriological analyses (processed by izs istituto zooprofilattico sperimentale abruzzo e molise from 2015 – e.g., filippi et al. 2010). then, at the end of the rite, or in any case within seven days, snakes are released by serpari in the same place where they were captured. in 2019, on 29th and 30th of april, we also conducted a focused survey to assess the presence of oo (in 2020 and 2021 the rite did not take place due to the covid-19 pandemic). all snakes underwent an additional physical examination. during this investigation, a particular attention was given to macroscopic clinical signs consistent with ophidiomycosis (fig. 1). based on this, a binary value according to hileman and colleagues (2018) was assigned to each snake: “0” (absence of signs consistent with ophidiomycosis); “1” (presence of signs consistent with ophidiomycosis). the clinical signs from the snakes categorised as “1” (clinically affected) were carefully documented, and the grade of infection severity was retrospectively calculated following the infection severity score (iss) proposed by baker and colleagues (2019) (table 1). a single sterile cotton-tipped applicator with wooden stick (aptaca spa, canelli, italy) was used to (dry) swab each individual belonging to the apparently affected group, and then placed in 20 ml plastic tubes (sarstedt ag & co. kg, nümbrecht, germany). the snakes were swabbed with moderate pressure 10 times along the entire dorsal surface, ventral surface, head, and, additionally, ≥ 10 times on each suspected lesion (see di nicola et al. 2022; marini et al., 2023). in cases in which scales and/or lesions naturally exfoliated during swabbing (i.e., abnormal scales partially detached or scales adjacent to skin lesions) or pieces of exuviae detached (due to dysecdysis), these tissues were stored together with the swab from the same individual in the same 20 ml vial. the tubes were stored at +4 °c until shipment. each snake was handled by the veterinarian carrying out the swab (dm) with new disposable nitrile or latex gloves and all the equipment eventually used (hooks, forceps, scale) was disinfected with 95% denatured ethanol or 5% sodium hypochlorite solution. the dna has been extracted by placing each swab (with or without tissue) in a 1,5 47ophidiomyces screening in cocullo snake community ml safe-seal tube with one aliquot (500 µl) of lysis buffer (0.1 m tris–hcl, ph 8.5, 0.05% tween 20, 0.24 mg/ ml proteinase k), then placed in a heat block first at 60 °c for 1 hour and after 95 °c for 15 minutes. afterward, 10 µl of each lysate were diluted in 90 µl of nuclease free water. a broad-range standard panfungal pcr (amplifying the d1-d2 region of the large subunit [lsu – 28s] of the ribosomal rna [rrna] gene complex – borman et al., 2006) was performed following frankinos et al. (2017). moreover, two different set of primers described by bohuski and colleagues (2015), targeting specific regions of oo genome – its2 (internal transcribed spacer region 2) and igs (intergenic spacer region) within the rrna gene – were employed for conventional pcr assays (origgi et al., 2022). each conventional (qualitative) pcr assay has been run in 30 μl amplification mixture composed of 3 μl of pcr buffer, 0.6 μl dntp mix (10 mm each), 0.75 μl of each primer (100 mm), 0.3 μl of taq polymerase, 2.5 μl of dna template (diluted lysates), 3.8 μl of mgcl2 (25 mm), and 18.3 μl of water (solys biodyne, luzerna chem, lucerne, ch). the reactions were carried out as follows: initial denaturation (95 °c for 3 min) followed by 35 cycles including 30 s at 95 °c (denaturation), 30 s at 52 °c for its and 50°c for igs (annealing), 30 s at 72 °c (elongation). a final extension at 72 °c for 10 min followed. lastly, 5µl of the pcr product were resolved on a 2% agarose gel by electrophoresis and visualized under uv light. a total of 129 snakes (adults, subadults and juveniles) were brought by serpari and examined by the scientific committee. ophidians belonged to the following species: elaphe quatuorlineata (n = 82), hierophis viridiflavus (n = 31); zamenis longissimus (n = 15), natrix helvetica (n = 1). twenty-three snakes out of 129 (17.8%) fig. 1. representative lesions of individuals grouped in category “1” (presence of signs consistent with ophidiomycosis): (a) eq21, elaphe quatuorlineata showing erosions of rostral, right internasal, nasal, preocular, ocular and supraocular scales and displaced dorsal scales; (b) eq46, elaphe quatuorlineata exhibiting multifocal swollen, eroded and hyperaemic (dorsal and ventral) scales associated with crusts and dysecdysis; (c) hv28, hierophis viridiflavus with hyperaemia, erythematous skin, and retained exuvium in the gular region; (d) hv04, hierophis viridiflavus showing wrinkled, depressed and crusty dorsal scales. 48 daniele marini et alii ta bl e 1. i nd iv id ua ls c at eg or is ed a s cl in ic al ly a ffe ct ed ( ca te go ry “ 1” ) an d sa m pl ed a t c oc ul lo ’s fe st iv al i n 20 19 . f or e ac h sn ak e th e ta bl e sh ow s th e id en tifi ca tio n co de , s pe ci es , s ex a nd ag e cl as s, s no ut -v en t le ng th ( sv l) , w ei gh t, ty pe o f co lle ct ed s am pl e, le si on s co re s (t yp e, lo ca tio n, n um be r, co ve ra ge ) an d th e re la tiv e in fe ct io n se ve ri ty s co re ( is s) , a nd t he d es cr ip tio n of g ro ss s ig ns . m : a du lt m al e. f : a du lt fe m al e. s a f: s ub ad ul t f em al e. s : d ry s w ab . t : t is su e. b c s: b od y co nd iti on s co re . id sp ec ie s se x an d ag e sv l (c m ) w ei gh t (g ) sa m pl e ty pe le si on ty pe sc or e le si on lo ca tio n sc or e le si on nu m be r sc or e le si on co ve ra ge sc or e is s (i nf ec tio n se ve ri ty sc or e) g ro ss s ig ns d es cr ip tio n eq 21 el ap he qu at uo rl in ea ta sa f 10 7 30 6 s 3 3 3 3 12 c ru st y er os io ns o f r os tr al , r ig ht in te rn as al , n as al , p re oc ul ar , o cu la r an d su pr ao cu la r sc al es . c ru st y, d ry , d us ty , d is lo ca te d an d (s om et im es ) er od ed sc al es m ul tif oc al ly a lo ng th e do rs um . b ro w ni sh a nd d us ty c ru st o f t he ta il (> 3x 2c m ). h v 04 h ie ro ph is vi ri di fla vu s m 89 26 4 s 2 1 3 3 9 d es qu am at io ns w ith d us ty a sp ec t. c on ca ve , w ri nk le d or c ru st y do rs al s ca le s. tr au m at ic a nd c ru st y le si on s of v en tr al s ca le s. eq 24 el ap he qu at uo rl in ea ta m 13 9 10 86 s 2 2 3 2 9 ec to pi c or c ru st y or d is lo ca te d do rs al a nd ta il sc al es . r et ai ne d sh ed b et w ee n su pr ao cu la r, oc ul ar a nd p os to cu la r sc al es . f oc al d is co lo ra tio ns . l ow b c s. h v 11 h ie ro ph is vi ri di fla vu s m 95 26 0 s 2 2 2 2 8 r et ai ne d sh ed in th e pa ri et al a nd d or sa l s ca le s of h ea d re gi on . c ru st y, d us ty an d/ or r ai se d do rs al s ca le s. l ig ht d eh yd ra tio n. h v 12 h ie ro ph is vi ri di fla vu s m 87 29 6 s 2 2 2 2 8 c oa le sc en t d es qu am at io ns o f h ea d an d tr un k sc al es . c on ca ve o r cr us ty d or sa l sc al es . eq 27 el ap he qu at uo rl in ea ta f 14 2 85 4 s 2 2 2 3 9 bi lo be d lo os e sw el lin g (> 4 x1 ,5 cm p re su m pt iv el y su bc ut an eo us n od ul e) on th e le ft la te ra l r eg io n of th e tr un k. m od er at e nu m be r of e ct op ic a nd di sl oc at ed d or sa l s ca le s. w ri nk le d sc ar b et w ee n ro st ra l a nd in te rn as al s sc al es . z l0 1 z am en is lo ng is si m us m 99 19 4 s 2 3 1 2 8 tu m ef ac tio n on th e le ft si de o f t he u pp er ja w . a t b uc ca l i ns pe ct io n, th e m uc os a w as fo un d hy pe ra em ic , h ae m or rh ag ic a nd s w el le d. z l0 7 z am en is lo ng is si m us m 10 5 39 6 s 2 2 2 2 8 d es qu am at io n w ith a s lig ht ly c ru st y an d w ri nk le d as pe ct o n th e ri gh t l or ea l, pr eo cu la r an d su pr al ab ia l s ca le s. d is lo ca te d, c on ca ve a nd e ct op ic d or sa l sc al es . h v 15 h ie ro ph is vi ri di fla vu s m 96 32 0 s 1 2 2 2 7 a br as io n be tw ee n ro st ra l a nd in te rn as al s sc al es . d is lo ca te d, e ct op ic , w ri nk le d or a bs en t d or sa l s ca le s. eq 39 el ap he qu at uo rl in ea ta f 15 0 10 00 s 2 2 2 1 7 c ru st y le si on o n ri gh t s op ra oc ul ar s ca le . d is lo ca te d do rs al s ca le . eq 40 el ap he qu at uo rl in ea ta m 12 4 68 2 s 1 1 2 2 6 fo ca l d is co lo ra tio ns . c on ca ve d or sa l s ca le s. eq 46 el ap he qu at uo rl in ea ta f 11 2 38 0 s, t 3 1 3 3 10 d ys ec dy si s an d re ta in ed m ou lts in m an y lo ca tio ns . t um ef ac te d, e ro de d an d hy pe ra em ic ( do rs al a nd v en tr al ) sc al es . s ev er al y el lo w is hbr ow ni sh c ru st s le si on s in th e do rs al a nd v en tr al r eg io n of th e tr un k, m ul tif oc al p at te rn . so m e sw ol le n sc al es o r cr us ts u nd er ly in g no du la r fo rm at io ns . l ow b c s an d m us cu la r w ea kn es s. eq 50 el ap he qu at uo rl in ea ta m 11 6 44 4 s 1 2 2 2 7 m ul tif oc al d is co lo ra tio ns . w ri nk le d do rs al s ca le s. i rr eg ul ar c au da l e dg es o f ve nt ra l s ca le s. eq 54 el ap he qu at uo rl in ea ta f 13 8 96 6 s 2 2 2 3 9 c ru st y le si on s on d or sa l s ca le s. c on ca ve a nd d is lo ca te d do rs al s ca le s. er yt he m at ou s an d hy pe ra em ic v en tr al s ca le s. n od ul ar fo rm at io n on th e ta il (> 1 x1 cm ). 49ophidiomyces screening in cocullo snake community id sp ec ie s se x an d ag e sv l (c m ) w ei gh t (g ) sa m pl e ty pe le si on ty pe sc or e le si on lo ca tio n sc or e le si on nu m be r sc or e le si on co ve ra ge sc or e is s (i nf ec tio n se ve ri ty sc or e) g ro ss s ig ns d es cr ip tio n eq 55 el ap he qu at uo rl in ea ta m 11 7 55 6 s 2 1 3 3 9 sc at te re d da rk s ca rs . d is lo ca te d, c ru st y, w ri nk le d or a bs en t d or sa l s ca le s. h v 21 h ie ro ph is vi ri di fla vu s m 96 34 8 s 2 1 3 3 9 br ow ni sh -y el lo w is h m oi st fr es h cr us ts d or sa lly a nd v en tr al ly o n th e ta il. c on ca ve , c ru st y or a bs en t d or sa l s ca le s. h v 23 h ie ro ph is vi ri di fla vu s m 93 28 8 s 3 1 2 2 8 d is lo ca te d an d co nc av e do rs al s ca le s. p at ch es o f d is co lo ra tio ns . t um ef ac tio n (> 1 x1 c m ) on th e le ft si de o f t ai l ( ad ja ce nt e ry th em at ou s an d er os iv e ve nt ra l sc al es ). h v 28 h ie ro ph is vi ri di fla vu s m 10 3 34 2 s, t 2 2 2 3 9 d ys ec dy si s. r et ai ne d ex uv iu m a t l ev el o f s no ut a nd c hi n. h yp er ae m ic a nd er yt he m at ou s sk in in th e gu la r re gi on . d es qu am at io n an d w ri nk lin g of d or sa l sc al es . t w o no du la r fo rm at io ns ( 0. 5x 0. 5 cm ) on th e tr un k. i rr eg ul ar c au da l ed ge s of v en tr al s ca le s. z l1 3 z am en is lo ng is si m us f 79 16 2 s, t 3 1 3 2 9 c ru st y, r ai se d or c on ca ve d or sa l s ca le s. r et ai ne d sh ed . f oc al e ro si ve fr es h le si on o n ve nt ra l s ca le s. m ul tif oc al c ru st s on c au da l t ru nk a nd ta il. eq 58 el ap he qu at uo rl in ea ta m 14 0 10 40 s 2 3 3 2 10 li gh t m ul tif oc al d is co lo ra tio n. c ru st y, d ry o r di sl oc at ed d or sa l s ca le s. th re e cr us ty le si on s on th e ve nt ra l s ca le s (o ne o n th e cl oa ca ). eq 62 el ap he qu at uo rl in ea ta m 13 9 10 88 s 3 1 2 3 9 d ry o r di sl oc at es d or sa l s ca le s. s w el lin g an d hy pe ra em ia o r er os io n an d of ve nt ra l s ca le s cl os e to c lo ac a. o ne n od ul ar fo rm at io n at tr un k le ve l ( 1x 1 cm ), an d on e at ta il le ve l ( 0. 5x 0. 5 cm ). eq 64 el ap he qu at uo rl in ea ta m 13 4 79 0 s 2 1 2 2 7 d is lo ca te d an d ec to pi c do rs al s ca le s. d es qu am at io ns . c ru st y le si on o f t he ta il. eq 69 el ap he qu at uo rl in ea ta m 13 6 85 8 s 2 1 2 2 7 d iff us ed d ar k di sl oc at ed a nd w ri nk le d do rs al s ca le s. th re e no du la r fo rm at io ns o n th e tr un k (< 1 x1 cm ). d oc ke d ta il. 50 daniele marini et alii showed signs consistent with a fungal dermatitis and were assigned to the category “1” (apparently affected): elaphe quatuorlineata (n = 13; 15.8%), hierophis viridiflavus (n = 7; 22.6%); zamenis longissimus (n = 3; 20%). table 1 reports all the clinically affected ophidians and the macroscopic signs that allow ranking these individuals in category “1”, as well as each category calculated for counting the individual iss. among all the individuals, the iss varied between 6 and 12, being 9 the median score. no influence of the species on the iss was found (χ2 = 11.49, p = 0.32, df = 10). cluster analysis of the isss – with and without normalization of the lesion coverage on the individual weight – did not reveal any particular trend or clustering (data not shown). a total of 23 swabs (and 3 tissues linked to one of them – table 1) have been collected. after dna extraction, for every sample (with or without tissue) a conventional pcr was carried out for each of the three targeted region of oo (d1-d2, its2 and igs regions – 23 samples x 3 reactions = 69 results). no product of consistent size was observed on agarose gel from each pcr electrophoresis (0/69). the number of category “1” individuals versus the number of category “0” individuals of each species did not differ statistically (χ2 = 0.89, p = 0.64, df = 2). the presence of clinical signs in adult e. quatuorlineata appeared to be associated with weight (ncat1 = 12, x = 812.00 + 247.01; ncat0 = 63, x = 689.02 + 174.49; t-test = 2.09 p = 0.04) but not with sex (fisher test p = 0.68) and svl (ncat1= 12, x = 131.42 + 10.67; ncat0 =64, x = 127.88; t-test = 0.98, p = 0.33). svl and weight were correlated in both apparently clinically healthy (r61 =0.74, p < 0.01) and clinically affected individuals (r10 =0.92, p < 0.01). no females with clinical signs were observed in h. viridiflavus and, among males, the number of category “1” adults did not appear to be related to svl (ncat1= 7, x = 94.14 + 5.24; ncat0 = 13, x = 91.65 + 6.14; t-test = 0.91, p = 0.38). svl and weight did not positively correlate in clinically affected (r5 =0.62, p > 0.05) while these morphometric parameters were correlated in unaffected individuals (r11 = 0.82, p < 0.05). three adults of z. longissimus (1 female and 2 males) out of 15 were grouped in category “1”. oo genomic dna was not detected in any of our samples (n= 23, observed prevalence 0%, bayesian 95% credible intervals: 0.00 0.14). considering the increasing number of ophidiomycosis reports in europe, a standardised monitoring for snake communities is warranted. to the best of our knowledge, this is the first systematic testing of a large sample size of a local snake community for the monitoring of o. ophidiicola in italy. we investigated only individuals with signs consistent with a fungal dermatitis because the swabs coming from these ophidians are more likely to result (true) positive compared to those showing no lesions (hileman et al. 2018; long et al. 2019). according to our data, none of the species studied shows an obvious high incidence of clinical signs compared to the other species and, within the same species, no particular trends emerged between clinically affected individuals versus clinically not affected ones and parameters as sex, svl and weight. the used iss was a helpful tool to characterize the severity of the infection of each individual, independently of the limitations associated with the lack of positive samples in our study. according to our experience, a normalization of the lesion coverage to the size of the animal is recommended (e.g., weight, surface – see blanvillain et al., 2022). no evidence of the presence of oo dna was revealed by pcr. however, pcr negativity is consistent with either the actual absence of the target dna sequence or its presence under the limit of detection. furthermore, eventual inhibitors could also hamper the pcr results. accordingly, we cannot rule out the occurrence of some false negative. lastly, the snakes were sampled once, and repeated sampling of the same individuals was shown to significantly reduce the probability of a false-negative (hileman et al. 2018). hence, in order to detect eventual false negative and increase sensitivity such screening should be improved by performing multiple re-samplings (e.g., 3-5 swab applicators hileman et al. 2018; marini et al., 2023). also, the use of real-time (quantitative) pcrs instead of conventional (qualitative) ones would improve the detection of false negatives from swab samples (allender et al., 2015a). this fungus might occur in all the temperate regions around the globe (burbrink et al., 2017) and snake susceptibility may vary according to phylogenetic and ecological factors (haynes et al., 2020). the actual natural history of the colonization of this fungus is still unclear. the possible introduction of the fungus into north america by pathogen pollution has been suggested (ladner et al., 2022) along with evidenced of the presence of both the american (switzerland) and the european clade (italy) in the european continent for more than 60 years (origgi et al., 2022). therefore, it is essential to shed lights on the distribution of this fungus in european continent along with its associated (cladespecific). accordingly, it is important to carry out screening in italian territories, implementing what was started in cocullo. on the other hand, the monitoring of the possible presence of oo extends and enriches the health monitoring and conservation actions in place at cocullo since 2010. in particular, to reduce the potential risk of disease and to ensure an excellent standard of handling and keeping of wild ophidians, a vademecum on snakes’ management in terraria has been published and delivered to the serpari. additionally, professional terraria have been allo51ophidiomyces screening in cocullo snake community cated to properly house the snakes every year, and dedicated exhibit with numerous environmental education and training activities concerning snakes are carried out for the thousands of tourists attending the ritual every year. the annual monitoring of the ophidians involved in the cocullo ritual will provide a great opportunity for collecting baseline data critical to assess the population health of the local snake community, which goes beyond the specific oo screening, and which represents a paradigmatic example of how cultural traditions, citizen science and conservation may come all together. acknowledgements handling and capture by serpari, as well as by scientific committee, is allowed under national authorizations [national law dpr 357/97; permit was granted by italian ministry of environment (mattm n. prot. 6265/2017 pnm and n. prot. 24601/2020 pnm). we thank the veterinarians gian lorenzo d’alterio (from 2010 to 2014) and pasqualino piro (from 2014 to date), izs lazio e toscana (i.t. sez. viterbo from 2010 to 2013), izs abruzzo e molise for cooperation in conducting clinical examinations and bacteriological analyses of snake community from rito di san domenico. we are grateful to rifcon gmbh that from 2016 sponsored every year professional terraria to house captured reptiles. we would like to acknowledge the major, the municipality and the people of cocullo to let us keep going these conservation actions and to keep alive this ancient ritual in the respect of nature. we are grateful to ursula sattler (university of bern) for her advice on laboratory diagnostics and to matteo oliveri for his suggestions on the initial research concept. references agugliaro, j., lind, c.m., lorch, j.m., farrell, t.m. 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(2017): snake fungal disease affects behavior of freeranging massasauga rattlesnakes (sistrurus catenatus). herpetol. conserv. bio. 12: 624-634. threats of the emerging pathogen batrachochytrium salamandrivorans (bsal) to italian wild salamander populations lorenzo dondero1, giorgia allaria1, giacomo rosa1, andrea costa1, gentile francesco ficetola2, roberto cogoni3, elena grasselli1, sebastiano salvidio1,* age estimation and body size of the parsley frog, pelodytes caucasicus boulenger, 1896 from lake borçka karagöl, turkey cantekin dursun*, serkan gül, nurhayat özdemir patterns of acoustic phenology in an anuran assemblage of the yungas andean forests of argentina martín boullhesen1,2,*, marcos vaira1, rubén marcos barquez2, mauricio sebastián akmentins1 diet and trophic niche overlap of four syntopic species of physalaemus (anura: leptodactylidae) in southern brazil renata k. farina1, camila f. moser2, stefano scali3, mateus de oliveira4, patrícia witt5, alexandro marques tozetti1,* screening of ophidiomyces ophidiicola in the free-ranging snake community annually harvested for the popular ritual of san domenico e dei serpari (cocullo, aq, italy) daniele marini1,2, ernesto filippi3,*, gianpaolo montinaro4, francesco c. origgi5,6 assessment of fall season habitat and coverboard use by snakes in a restored tallgrass prairie community carter dollen1,2, tracy j. coleman1,2, travis r. robbins1,2,* revisiting the polyploidy in the genus odontophrynus (anura: odontophrynidae) andré luis de souza, mayara aparecida das neves micalichen, roger alves da rocha, rafael bueno noleto* similarities and differences in adult tortoises: a morphological approach and its implication for reproduction and mobility between species marco a. l. zuffi1, annalisa plaitano1 1museo di storia naturale e del territorio, università di pisa, via roma 79, 56011 calci (pisa), italy. corresponding author. e-mail: marcoz@museo.unipi.it abstract. sexes in chelonia display marked differences. sexual size dimorphism (ssd) is important in evolutionary biology. different sexual strategies result in species specific selection. biometric variation in male and female tortoises of two species is studied. eighteen biometrics were measured in 75 museum specimens (20 testudo graeca; 55 t. hermanni). nine of 18 parameters in t. hermanni and two of 18 in t. graeca were sexually dimorphic. multivariate analyses (principal component analysis) highlighted two components, with bridge length the first and anal divergence the second component. the bridge length can be used to separate sexes and species. males of both species were most different, whereas females of two species overlapped in body shape measurements. we hypothesise that female similarity could be a by-product of reproductive biology and sexual selection that optimise individual fitness. keywords. testudo hermanni, testudo graeca, biometry, morphological model, reproductive strategies. introduction the evolution of the complex structures that define extinct and living chelonian species was studied by lee (1996). a progression of anatomical patterns appeared in the evolution of chelonia. physical structures range from complete ossification with a broadly developed carapace (i.e. testudo, geochelone) to reduction in bony shell (i.e. chelydra, apalone), or even to a poorly ossified carapace (i.e. trionyx; ernst et al., 1994). sexual size dimorphism (ssd) (darwin, 1874) is a very often debated aspect in reptilian species, particularly in recent years (berry and shine, 1980; bonnet et al., 2001; olsson et al., 2002; rubolini et al., 2006; zuffi et al., 2006). sexes in chelonia often display marked morphological differences, with the largest dimensions often occurring in females (berry and shine, 1980; ernst and barbour, 1989; ernst et al., 1994). several hypotheses acta herpetologica 2(2): 79-86, 2007 issn 1827-9643 (online) © 2007 firenze university press 80 m.a.l. zuffi and a. plaitano have been advanced to explain the patterns of carapacial, scute or colour variation in turtle sexes. habitat, diet, and habitus provide important correlations with carapace, head and skull shape (e.g. claude et al., 2003, 2004; zuffi et al., 2007), while different degrees of sexual dimorphism were recently found in the european species of the testudo (i.e. t. graeca, t. hermanni and t. marginata) by willemsen and hailey (2003). despite the wide interest in the evolution, selection and adaptive forces that mold carapacial morphology of chelonian species, few papers have dealt with morphology and function (bonnet et al., 2001; claude et al., 2003, 2004; willemsen and hailey, 2003; zuffi et al., 2007). broad patterns have become clearer, but more detailed comparisons at the specific level are needed. as data set for a preliminary comparison to examine details of interspecific sexual dimorphism in tortoises, we selected two similar, palaearctic species of testudo (but see lapparent de broin et al., 2006 and their proposal to change genus into eurotestudo), t. graeca and t. hermanni. in both these species, there are pronounced sexual differences in body size dimensions, and their courtship patterns have been described on average (buskirk et al., 2001; cheylan, 2001), but no real inspection of relationships between morphology and function has been yet performed (but see bonnet et al., 2001 for the testudo horsfieldii). we present a brief study on the sexual dimorphism in t. hermanni and t. graeca, and we examine ssd within a species and between species. our main idea is to test if morphological differences between sexes and between species may be helpful in explaining different evolutionary and reproductive strategies for each species (i.e. reproductive systems, movement). we wish to emphasize that this is a preliminary study and that further analyses might need to be conducted when possible. materials and methods we examined 71 museum specimens (9 males and 9 females of t. graeca; 26 males and 27 females of t. hermanni) in the museum of zoology “la specola” of the university of florence, italy and at the civic museum of natural science “doria” of genoa, italy. we made 18 measurements of shell (soldani, 2000), of the limbs and of the head and tail. measurements were made with a calliper (accuracy ± 0.1 mm) or with a tape measure (all the measurements in millimetres). weights were taken with an analogic dynamometre (accuracy: ± 25 g). for each we record: taxon, sex, age class (adult or juvenile, according to external morphology of plastron and tail length, cheylan, 2001), season, year, locality and region of capture and/or death, and preservation matter (i.e. in liquid, mounted), matter of collecting (alive; dead). animals born and dead in captivity were not considered. abbreviations for morphometrical parameters are: carlen (carapace length from nucal to sopracaudal scute), carwid (carapace width between 6th and 7th marginal scute), plalengmin (minimum length between jugal and anal scute), plalengmax (maximum length between giugal scute and an estreme margin of the anal scute), plawid (piastron width between abdominal and pectoral scutes), brilen (bridge length), carhei (carapace height between plastron base and the 3rd vertebral plate), anadiv (length of anal divergence), latcirc (latitudinal circumference), loncirc (longitudinal circumference), fore (forelimb length), hind (hindleg length), bmass (body mass in grams), healen (head length), heawid (head width), heahei (head height), tailen (total tail length), cloaca (distance between cloaca and tip of the tail). we excluded one juvenile that we could not determine its sex (cheylan, 2001). 81similarities and differences in adult tortoises statistic analyses were performed with spss 13.0 statistical package for windows. all parameters were tested for normality. parameters that were not normally distributed were log transformed prior to analyses. for each of selected morphological variables, we used an analysis of covariance (ancova) to examine dependent variables for the influence of sex, species and their interactions, using the log transformed carapace length as covariate. we, thus, tested the differences between sexes for each species, the differences between males of the two species, and finally between the females of the two species. as all selected variables were highly intercorrelated, we followed up this univariate analysis with a principal component analysis (pca) in order to identify the categorical variables responsible for the variation we measured (see fowler and cohen, 1993). results nine parameters out of 18 in t. hermanni and two out of 18 in t. graeca were sexually dimorphic (see table 1 for details). in particular, males had relatively shorter bridges at any particular carapace length than did females (fig. 1a, b). comparisons of sexes by species were significant (t. hermanni: anova, f1 = 194.653, p < 0.0001, ancova interaction sex × lncarapace length: f2,49 = 281.87, p < 0.0001, radj = 0.917; and t. graeca: anova, f1 = 74.751, p < 0.0001; ancova, f = 43.802, p < 0.0001, interaction sex × lncarapace length f2,15 = 46.538, p < 0.001, radj = 0.843). both species had a marked sexual dimorphism. two parameters were identified by pca as the most important morphometric features of carapace: the bridge length (first component) and the anal divergence (second component, table 2). while females widely overlapped, males were markedly different in morphological space (fig. 2). discussion sexual size dimorphism is very evident in t. hermanni (fig. 2a), but less evident for our much smaller sample of t. graeca (fig. 2b). nonetheless, males attain smaller body sizes than do females. furthermore, we found that the bridge length, a parameter not previously recognized as a sexually dimorphic one (bonnet et al., 2001; claude et al., 2003, 2004; willemsen and hailey, 2003), plays an important role in separating sexes and species. bridge length should be carefully considered in future comparative studies. males of the hermann’s tortoises have a more marked anal divergence and smaller carapace and plastron than do females. in males of the moorish tortoise we observed a marked anal divergence as well, but a larger carapace than females. it is interesting to underline how the bridge length appears to work in defining both sexual and interspecific differences. the bridge length is inversely proportional to the degree of mobility (soldani, 2000). mobile species (i.e. aquatic turtles such as emys, trachemys, lyssemys, pelomedusa and others) have smaller bridges when compared with terrestrial taxa (soldani, 2000; zuffi, unpubl.). besides, within a particular taxon (i.e. genus 82 m.a.l. zuffi and a. plaitano ta bl e 1. m ea n va lu es ( ± st an da rd d ev ia tio n) o f th e 18 m or ph ol og ic al v ar ia bl es m ea su re d in b ot h se xe s of t he t es tu do h er m an ni a nd t . g ra ec a (a n c o va , s ee m et ho ds fo r de ta ils ) (i n bo ld s ig ni fic an t v al ue s) . v ar ia bl e te st ud o he rm an ni df f p te st ud o gr ae ca df f p m al es fe m al es m al es fe m al es ca rl en 12 9. 5± 21 .3 14 5. 9± 34 .7 1, 51 4. 28 0. 04 3 15 5. 09 ±2 4. 22 14 2. 67 ±2 3. 89 1, 16 1. 2 0. 29 ca rw id 10 0. 6± 14 .8 10 9. 9± 24 .3 1, 51 2. 78 2 0. 10 1 11 3. 67 ±1 9. 72 10 4. 67 ±1 7. 02 1, 16 1. 07 4 0. 31 5 pl al en gm in 89 .8 1± 14 .9 1 11 9. 27 ±2 2. 77 1, 50 14 .4 42 <0 .0 00 1 12 9. 76 ±2 3. 74 11 9. 56 ±1 7. 46 1, 14 0. 90 4 0. 35 8 pl al en gm ax 11 0. 18 ±1 7. 52 12 9. 99 ±2 6. 91 1, 50 9. 72 0 0. 00 1 14 1. 98 ±2 5. 46 13 5. 51 ±2 7. 81 1, 14 0. 23 4 0. 63 6 pl aw id 90 .6 8± 13 .2 8 97 .4 7± 16 .6 5 1, 51 2. 61 3 0. 11 2 94 .7 2± 15 .0 8 90 .1 9± 13 .7 9 1, 16 0. 44 3 0. 51 5 br ile n 49 .5 2± 6. 30 64 .0 4± 11 .9 3 1, 50 29 .4 15 <0 .0 00 1 67 .9 9± 11 .8 1 65 .9 4± 8. 89 1, 16 0. 17 2 0. 68 4 ca rh ei 67 .6 2± 10 .3 3 76 .5 1± 14 .2 1 1, 50 6. 63 1 0. 01 3 78 .3 2± 11 .6 5 75 .6 3± 7. 65 1, 15 0. 30 9 0. 58 6 an ad iv 39 .2 9± 9. 16 29 .4 2± 7. 00 1, 50 19 .2 38 <0 .0 00 1 35 .4 4± 8. 52 25 .7 8± 4. 54 1, 14 7. 30 3 0. 01 7 la tc ir c 33 3. 04 ±5 3. 28 36 6. 44 ±7 0. 36 1, 50 3. 68 0. 06 1 38 9. 83 ±6 1. 19 34 7. 51 ±4 8. 84 1, 14 2. 23 0. 15 8 lo nc ir c 27 5. 12 ±4 1. 39 30 0. 41 ±5 6. 00 1, 50 3. 38 4 0. 07 2 31 3. 00 ±4 9. 95 28 4. 57 ±3 2. 58 1, 14 1. 69 2 0. 21 4 fo re 42 .1 2± 7. 74 44 .1 5± 9. 87 1, 28 0. 39 1 0. 53 7 46 .0 7± 8. 24 43 .0 4± 6. 52 1, 14 0. 63 2 0. 44 hi nd 44 .3 1± 8. 41 42 .1 1± 12 .9 0 1, 20 0. 23 5 0. 63 3 45 .1 1± 6. 82 43 .0 1± 11 .2 4 1, 14 0. 21 5 0. 65 bm as s 46 8. 91 ±2 48 .2 5 70 4. 80 ±4 40 .7 1 1, 46 5. 09 6 0. 02 9 67 9. 44 ±5 59 .5 1 31 2. 86 ±1 57 .1 6 1, 14 2. 79 3 0. 11 7 he al en 28 .1 1± 6. 57 28 .7 7± 5. 86 1, 36 0. 10 8 0. 74 4 28 .7 3± 2. 71 26 .4 4± 4. 47 1, 12 1. 34 0. 27 he aw id 19 .3 5± 3. 02 20 .4 7± 2. 58 1, 36 1. 46 7 0. 23 4 19 .4 7± 1. 5 17 .6 9± 1. 71 1, 12 4. 33 7 0. 05 9 he ah ei 18 .6 3± 3. 56 19 .9 0± 2. 89 1, 35 1. 38 5 0. 24 7 16 .8 1± 2. 83 14 .6 6± 2. 64 1, 12 2. 18 4 0. 16 5 ta ile n 49 .5 5± 11 .3 8 31 .9 1± 10 .1 1 1, 47 33 .0 21 <0 .0 00 1 40 .2 0± 11 .2 6 24 .5 7± 4. 04 1, 9 6. 90 4 0. 02 7 cl oa ca 25 .6 1± 6. 77 14 .1 0± 5. 65 1, 34 30 .8 67 <0 .0 00 1 -----83similarities and differences in adult tortoises or family), a bigger and longer bridge is typical of females. females have less space for their limbs than do males (lebboroni and chelazzi, 1991; andreu et al., 2000; corti and zuffi, 2003). in terrestrial tortoises, males must be able to quickly right themselves during malemale interactions for access to the females (bonnet et al., 2001). the righting response is is inversely related to bridge length and positively correlated with space for the legs (soldani, 2000; corti and zuffi, 2003; zuffi, unpubl.). however, a short bridge may also be less effective in protecting males from predation. females apparently sacrifice mobility for protection, thus their relatively long bridges. however, we also found interspecific variation in these sexually dimorphic traits. such differences may be related to differences in species specific courtship behaviours. for instance, male t. hermanni chases the female, bites her hind legs and back of the carapace. he mounts directly on the female carapace, performing such an action for long time before copulation. in contrast, male t. graeca usually courts females with strong fig. 1. morphometric differences in carapace length versus bridge length (log transformed variables) in males and females: (a) in t. hermanni, (b) in t. graeca. table 2. principal component analysis in testudo hermanni and t. graeca (in bold the most informative variables for each component). variable pc1 pc2 brilen 0.982 6.719*10-2 plalengmin 0.967 0.215 plalengmax 0.936 0.281 carhei 0.876 0.384 latcirc 0.867 0.485 loncirc 0.861 0.495 carlen 0.856 0.467 carwid 0.763 0.547 plawid 0.746 0.581 anadiv 0.162 0.973 (a) (b) 84 m.a.l. zuffi and a. plaitano blows from the gular shields on the posterior side of female carapace and with a premounting behaviour before actual copulation. after a typical general scheme, similar to the t. graeca one, the t. hermanni male climbes on the female’s carapace and gives her repeated blows with the great spur of his tail tip. this happens whether the female is stationary or moving. we hypothesize that t. hermanni males need to be more mobile than t. graeca males. our study is a preliminary one and only experimental studies will serve to elucidate the interaction of behavioural and morphological patterns. nonetheless, our finding that sexual dimorphic characters vary interspecifically is important. mobility is a critical skill in tortoises and we show that females have effectively sacrificed this when compared to males. the selection producing such an outcome is little understood. further studies should experimentally examine interspecific variation in these traits by comparing degrees of mobility between males and females and between species of tortoises. acknowledgments we wish to express our gratitude to john l. tucker and to an anonymous referee which with their suggestions and criticism haved greatly improved this ms. fig. 2. principal component analysis of carapacial and sexual differences in t. hermanni and t. graeca. small circles, t. hermanni; large circles, t. graeca; black circles, males; grey circles, females. 85similarities and differences in adult tortoises references andreu, a.c., díaz-paniagua, c., keller, c. (2000): la tortuga mora (t. graeca l.) en doñana. asociación herpetológica española. monog. herpetol. 5: pp. berry, j.f., shine, r. (1980): sexual size dimorphism and sexual selection in turtle (order testudines). oecologia (berl.) 44: 185-191. bonnet, x., lagarde, f., henen, b.t., corbin, j., nagy, k.a., naulleau, g., balhoul, k., chastel, o., legrand, a., cambag, r. (2001): sexual dimorphism in steppe tortoises (testudo horsfieldii): influence of the environment and sexual selection on body shape and mobility. biol. j. linn. soc. 72: 357-372. buskirk, j.r., keller, c., andreu, a.c. (2001): testudo graeca linnaeus, 1758-maurische landschildkröte. in: handbuch der reptilien und amphibien europas, fritz, u., ed, p. 125-177. aula, verlag. cheylan, m. (2001): testudo hermanni gmelin, 1789-griechische landschildkröte. in: handbuch der reptilien und amphibien europas, fritz, u. ed, p. 179-289. aula,verlag. claude, j., paradis, e., tong, h., auffray, j.c. (2003): a geometric morphometric assessment of the effects of the environment and cladogenesis on the evolution of the turtle shell. biol. j. linn. soc. 79: 485-501. claude, j., pritchard, p.c.h., tong, h.j., paradis, e., auffray, j.c. (2004): ecological correlates and evolutionary divergence in the skull of turtles: a geometric morphometric assessment. syst. biol. 53: 933-948. corti, c., zuffi, m.a.l. (2003): aspects of population ecology of testudo hermanni hermanni from asinara island, nw sardinia (italy, western mediterranean sea): preliminary data. amphibia-reptilia 24: 441-447. darwin, c. (1874): the descent of man, and selection in relation to sex. rand, mcnally and co., chicago. ernst, c.h., barbour, r.w. (1989): turtles of the world. smithsonian istitution press, washington and london. ernst, c.h., barbour, r.w., lovich, j.e. (1994): turtles of the united states and canada. smithsonian institution press, washington and london. forsman, a., shine, r. (1995): sexual size dimorphism in relation to frequency of reproduction in turtles (testudines: emydidae). copeia 1995: 727-729. fowler, j., cohen, l. (1993): statistica per ornitologi e naturalisti. muzzio editore, padova. hailey, a., loumbourdis, n.s. (1990): population ecology and conservation of tortoises: demographic aspects of reproduction in testudo hermanni. herpetol. j. 1: 425-434. lapparent de broin, f., bour, r., param, j.f., perälä, j. (2006): eurotestudo, a new genus for the species testudo hermanni gmelin, 1789 (chelonii, testudinidae). c. r. palevol. 5: 803-811. lebboroni, m., chelazzi, g. (1991): activity patterns of emys orbicularis l. (chelonia emydidae) in central italy. ethol. ecol. evol. 3: 257-268. lee, m.s.y. (1996): correlated progression and the origin of turtles. nature 379: 812-815. olsson, m., shine, r., wapstra e., ujvari, b., madsen, m. (2002): sexual dimorphism in lizard body shape: the roles of sexual selection and fecundity selection. evolution 56: 1538-1542. 86 m.a.l. zuffi and a. plaitano rubolini, d., pupin, f., sacchi, r., gentilli, a., zuffi, m.a.l., galeotti, p., saino, n. (2006): sexual dimorphism in digit length ratios in two reptile species. anatom. rec. 288a: 491-497. soldani, a. (2000): il dimorfismo sessuale nei cheloni e ipotesi sul successo riproduttivo. ms. thesis, university of pisa. willemsen, r.e., hailey, a. (2003): sexual dimorphism of body size and shell shape in european tortoises. j. zool., lond. 260: 353-365. zuffi, m.a.l., celani, a., foschi, e., tripepi, s. (2007): geographical patterns of reproductive plasticity in the european pond turtle, emys orbicularis. j. zool, lond. 271: 218224. zuffi, m.a.l., odetti, f., batistoni, r., mancino, g. (2006): morphometrics and genetics in in the study of variability pattern of the european pond turtle, emys orbicularis. ital. j. zool. 73: 363-372. a new finding of salamandra lanzai in the upper sangone valley (nw italy) marks the species’ most disjunct population (amphibia: urodela: salamandridae) giulia tessa1, angelica crottini1, 2, franco andreone1 1museo regionale di scienze naturali, via g. giolitti 36, i-10123 torino, italy. corresponding author. e-mail: franco.andreone@regione.piemonte.it 2università degli studi di milano, dipartimento di biologia, sezione di zoologia e citologia, via celoria 26, i-20133 milano, italy abstract. the presence of salamandra lanzai was confirmed for the upper sangone valley (turin province, nw italy), within the parco naturale orsiera rocciavré. the species attribution was further supported by morphological and genetic (16s) analysis and represents the north-eastern most limit of the species’ distribution. this salamander was so far known only for a few major alpine valleys of italy (po, pellice, and germanasca valleys), and france (guil valley). the new finding is especially interesting since it is separated from its closest known locality by about 15 km. for such a reason this population needs to be carefully managed. keywords. salamandra lanzai, new finding, sangone valley, cottian alps, nw italy. salamandra lanzai is a peculiar alpine urodele: recognised as a distinct species in 1988, it soon became the subject of intensive research activity (e.g., ribéron et al., 1996; andreone and sindaco, 1999) aimed at unveiling its life history requirements and distribution, with important conservation applications. following its discovery, the distribution of s. lanzai initially appeared limited to a few alpine valleys in italian and french cottian alps, roughly around the monviso massif (andreone, 2007), and until recently its known distribution was limited to about 50 observations in france (31 1 × 1 km utm squares, of which 10 confirmed) and 60 in italy (45 1 × 1 km utm squares, of which 31 confirmed) (andreone et al., 2004). in france s. lanzai is currently present exclusively in the guil valley, at an altitude ranging from 1800-2600 m a.s.l., in sites characterised by the presence of alpine prairie (ribéron et al., 1996). in italy it is known from three major valleys: germanasca, po, and pellice valleys, with some further records in the angrogna valley (a secondary valley tributary of the pellice valley) at an altitude ranging 1200-2600 m a.s.l. moreover, a recent report by sindaco (2006) places the meridional limit of this species at oncino (po valley). although its distribution appears restricted to the upper portions of these valleys, there are some unconfirmed reports of alpine salamanders for other north-western parts acta herpetologica 2(1): 53-58, 2007 54 g. tessa et alii of the alps. moreover, most of these reports turned out to be false and likely due to anecdotal findings and incorrect taxonomic determinations (andreone and sindaco, 1999; andreone, 2006, 2007), and there is the perspective that some are indeed true, and the lack of recent confirmed records may be due to a general disappearance of the species. in fact, as pointed out by ribéron (2003), the species was probably more widespread in the past, as corroborated by old french reports of abriès, and the ubaye and agnelle valleys (ribéron, 2003), and by the presence of a preserved specimen from the italian maritime alps, dated from the end of the nineteenth century (andreone and sindaco, 1999; andreone et al., 2004). recently, we obtained information of the possible presence of black salamanders in the upper sangone valley, turin province (e. giuliano, pers. comm.). although these observations were quite circumstantial and made by local rangers they were not accompanied by any photographs or voucher specimens, and therefore remained doubtful for some years. a first visit in the parco naturale orsiera rocciavré (by p. eusebio bergò, v. mercurio, and e. giuliano) did not confirm the records. this was not the case of the summer of 2006, when on the occasion of a further visit to the same site, one of us (gt) found a salamander. the single female was found overnight on 31th july 2006 (h 00.20). the site is located at 2227 m a.s.l., and consists of an alpine meadow with herbaceous cover and rocky debris due to glacial erosion. the area lies in a typical alpine valley, with intense fog formation and heavy and frequent precipitations, due to condensation of wet atmosphere coming from the po plain. these are most likely to be good microclimatic conditions for the species, which is known to require high humidity levels. we do not provide the exact coordinates of this site for conservation reasons, but they will be transmitted to the italian herpetological database to update the species’ distribution pattern. the salamander was measured for its basic parameters: its size of 89 mm snout-vent length and 165 mm total length felt within the typical range of s. lanzai; in addition, we also ascertained the specific identity by comparing the photograph to that of other individuals from the italian and french alps. this showed the species’ distinctive and diagnostic characters, such as the absence of paravertebral glands on the trunk, a massive body with a flattened head, a rounded tail tip, and interdigital webbing (fig. 1). moreover, the tip of the third toe was clipped and stored in pure ethanol for subsequent genetic analysis. the species attribution was then confirmed by the comparison of a fragment of 535 bp of the 16s rrna gene, the suggested standard dna barcoding marker for vertebrate, and hence therefore amphibians (vences et al., 2005). fourteen specimens of s. lanzai sampled in nine different localities (one from sangone valley, six from po valley, three from germanasca valley, two from pellice valley and two from angrogna valley) were also analysed to have a good comparative series. total genomic dna was extracted from the tissue samples using proteinase k digestion (1 mg/ml concentration) followed by a standard salt extraction protocol (bruford et al., 1992). we used the primers 16sa-l 5’-cgcctgtttatcaaaaacat-3’ and 16sb-h 5’-ccggtctgaactcagatcacgt-3’, modified from kocher et al. (1989) and palumbi et al. (1991). pcrs were performed in 25µl reactions using 3µl of genomic dna, 1µl of each 10 pmol primer, 0.5µl of total dntp 10mm in water (promega), 0.1µl of 5u/µl gotaq®, 5µl 5x green gotaq® reaction buffer (promega) and 14.4 µl of water. pcrs were performed using the following conditions: an initial denaturation step at 94 °c for 90 s, 33 cycles of denaturation 55new finding of salamandra lanzai at 94 °c for 45 s, annealing at 55 °c for 45 s and extension at 72 °c for 90 s, and a final extension of 300 s at 72 °c. pcr products were loaded onto 1% agarose gels, stained with ethidium bromide, and visualised on a ‘‘gel doc’’ system (peqlab). if results were satisfying, products were purified using qiaquick spin columns (qiagen). the light strands were sequenced using an abi3730xl by macrogen inc. (sequences genbank accession numbers: ef191028ef191041). sequences were manually edited and aligned using the bioedit sequence alignment editor (version 7.0.5, hall, 1999). genetic distances were computed with mega (version 3.1, kumar et al., 2004) using as reference a homologous sequence of s. atra atra (without a specific locality from “austria”) and a sequence of s. a. aurorae (from bosco del dosso, italy) (sequences kindly provided by m. veith). the uncorrected p-distance, and hence the lowest genetic distance, between s. lanzai and both s. atra subspecies were around 3.2-3.4%. the finding of s. lanzai in the upper sangone valley marks a remarkable extension of the species’ distribution, and it shows that this salamander is not limited to the main alpine valleys (guil valley in france and germanasca, pellice and po valleys in italy), and also gives new light on the species’ conservation, since s. lanzai is considered a “vulnerable” species according to the iucn red list (iucn 2006). in particular, the factors threatening this salamander are limited in their geographical extent, and are mainly due to habitat alteration, tourist pressure, and associated vehicular traffic (andreone et al., fig. 1. the female of salamandra lanzai, photographed in the upper sangone valley. 56 g. tessa et alii 2004). in light of these considerations, the new site of the sangone valley appears quite secure, due to its distance from any close roads, and the fact that it does not host any tourist facilities. furthermore, it is included in a protected area (parco naturale orsiera rocciavré), which potentially represents an advantage in terms of the species’ conservation. the species’ narrow distribution and the fragmented status of its populations are also important conservation factors (andreone et al., 2004). the new finding is particular worth of attention also because it is separate from the closest population (massello valley) by about 14.5 km and by the xerothermic chisone valley, that represents therefore a major fig. 2. the distribution of salamandra lanzai based upon current data. the black circles represent confirmed sites, while the open circles represent unconfirmed sites. (1) indicates the new (confirmed) location in the upper sangone valley, and (2) indicates the (unconfirmed) “fontanette” finding. 57new finding of salamandra lanzai physical barrier (fig. 2). a closest finding was given in the map provided by andreone et al. (2004). anyhow, we do not take into consideration this locality since its coordinates were not clearly ascertained and the toponym (“fontanette”) was not found on cartography. thus we consider this finding as doubtful, pending for further confirm. for these reasons, special attention should be paid to provide effective conservation for this urodele in the upper sangone valley. while the other sites are likely to be in contact and thus still experiencing significant genetic and population exchange, the new site is, at least in the light of current knowledge, almost isolated from the major population nuclei. anyhow, the genetic analyses conducted on the basis of the mitochondrial dna did not exhibit any differences compared with the 13 samples from the other italian sites, suggesting that genetic flow occurred until recently. data formerly presented for cyt b analysis also showed that there is no significant genetic variation between the known populations of s. lanzai, thus suggesting founder effect due to repeated bottlenecks or isolation of small population nuclei due to climatic oscillations during the pleistocene glaciation (ribéron et al., 2002). the new finding considerably widens the species’ known distribution. however, seen that a single individual only was found after a considerable time lapse, the species appears to be quite elusive at this site. this leads us to consider that the distribution of this salamander might be wider than formerly believed, and that therefore the unconfirmed reports from other areas might be valid. for these reasons, further research is urgently needed to clarify whether some of the old and unconfirmed reports are incorrect or simply due to research deficiencies. acknowledgements thanks to all those who helped us with discussions and personal communications: l. tessa, e. giuliano and v. mercurio. p. eusebio bergò assisted with cartography and map preparation. the genetic analyses were conducted in the laboratory of the technical university of braunschweig: thanks to m. vences for his kind hospitality. thanks to m. veith for sharing with us his genetic data. r. berridge corrected the english of a first draft and provided useful suggestions. finally, thanks to r. sindaco, whose comments improved our contribution. references andreone, f. (2006): salamandra di lanza [lanza’s salamander]. in: atlante degli anfibi e dei rettili d’italia, sindaco, r., doria, g., razzetti, e., bernini, f., eds, p. 196-201. firenze, edizioni polistampa. andreone, f. (2007): salamandra lanzai. salamandra di lanza. in: fauna d’italia. amphibia, lanza, b., andreone, f., bologna, m.a., corti, c., razzetti, e., eds. bologna, calderini. (in press) andreone, f., miaud, c., eusebio bergò, p., doglio, s., stocco, p., ribéron, a., gautier, p. (2004): living at high altitude: testing the effects of life history traits upon the conservation of salamandra lanzai (amphibian, salamandridae). ital. j. zool. 71: 35-43. 58 g. tessa et alii andreone, f., sindaco, r. (1999): erpetologia del piemonte e della val d’aosta. atlante degli anfibi e dei rettili. monografie 26. torino, museo regionale di scienze naturali. bruford, m.w., hanotte, o., brookfield, j.f.y., burke, t. (1992): single-locus and multilocus dna fingerprint. in: molecular genetic analysis of populations: a practical approach, hoelzel, a.r., ed., p. 225-270. oxford, irl press. hall, t.a. (1999): bioedit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/nt. nucl. acids symp. ser. 41: 95-98, version 7.0.5 (biological sequence alignment editor for windows 95/98/nt/xp. downloaded on 5 september 2006. iucn (2006): 2006 iucn red list of threatened species. downloaded on 5 september 2006. kocher, t.d., thomas, w.k., meyer, a., edwards, s.v., pääbo, s., villablanca, f.x., wilson, a.c. (1989): dynamics of mitochondrial dna evolution in mammals: amplification and sequencing with conserved primers. proc. natl. acad. sci. u.s.a. 86: 6196-6200. kumar, s., tamura, k., nei, m. (2004): mega3: integrated software for molecular evolutionary genetics analysis and sequence alignment. briefings in bioinformatics 5: 150-163. palumbi, s., martin, a., romano, s., mcmillan, w.o., stice, l., grabowski, g. (1991): the simple fools guide to pcr. version 2.0, dept. zool. and kewalo marine lab., univ. of hawai’i. ribéron, a. (2003): la salamandre de lanza. in: les amphibiens de france, belgique et luxembourg, acemav coll., duguet, r., melki, f. eds, p. 286-289. mèze, parthénope collection. ribéron, a., miaud, c., guyetant, r. (1996): taille, sex-ratio et structure d’âge d’une population de salamandra lanzai (caudata, salamandridae) dans les alpes du sud-est de la france. bull. soc. herp. france 77: 35-45. ribéron, a., sotiriou, e., miaud, c., andreone, f., taberlet, p. (2002): lack of genetic diversity in salamandra lanzai revealed by cytochrome b gene sequences. copeia 2002: 229-232. sindaco, r. (ed.) (2006): segnalazioni faunistiche piemontesi e valdostane. riv. piem. st. nat. 27: 443-459. vences, m., thomas, m., bonett, r. m., vieites, d.r. (2005): deciphering amphibian diversity through dna barcoding: chances and challenges. phil. trans. r. soc. london, ser. b 360: 1859-1868. amphibians of the ausoni mountains (latium, central italy) luigi corsetti1, antonio romano2 1 via adige 45/2, i-04100, latina, italy 2dipartimento di biologia, università di roma “tor vergata”, via della ricerca scientifica, 00133 roma, italy. corresponding author. e-mail: antonioromano71@tele2.it abstract. in this study we searched for amphibians in 89 potential breeding sites within the ausoni mounts, which are among the less investigated areas of latium. sixtynine spawning sites, and eight amphibian species (57.1% of the 14 amphibian species living in latium region) were found. reproductive activity was recorded for salamandrina perspicillata, triturus carnifex, lissotriton vulgaris, lissotriton italicus, bufo bufo, hyla intermedia, rana italica and pelophylax synklepton hispanica. keywords. monti ausoni, latium, amphibia, distribution. introduction despite in latium (central italy) herpetological field researches have been carried out systematically (bologna et al., 2000), there are large disproportion among data from different areas, and the southernmost portion of the region have been investigated only occasionally (cf. bologna et al., 2000; see also anonymous 2006a and maps therein). the volsci mountain chain, the western portion of this area, comprises three karstic subgroups: the lepini, ausoni and aurunci mountains. the information gap on the herpetofauna of two out these chains is now partially filled in by studies on the lepini (corsetti and capula, 1992) and, more recently, on the aurunci mountains (romano et al., 2007). conversely, previous knowledge on the herpetofauna occurring in the ausoni mountains was provided only by a preliminary account (bonifazi and carpaneto, 1990) and few other information scattered in some papers concerning wider areas (e.g. bruno, 1973; bologna et al., 2000; corsetti, 2006). during several years (1998-2007), field activities to update the checklist and to provide a breeding sites’ census of the amphibians species of the ausoni mounts were carried out. the aim of this work is, therefore, to fill in the information gap on the herpetofauna of the ausoni mts and, consequently, to homogenise the herpetological data on the whole volsci chain. acta herpetologica 2(2): 129-137, 2007 issn 1827-9643 (online) © 2007 firenze university press 130 l. corsetti and a. romano material and methods since there is not a clear hydrographical or geological boundary between ausoni and aurunci mounts, conventionally ausoni mounts are considered the mounts at the west of a line connecting fondi-lenola-pico-s. giovanni incarico towns (landi vittorj, 1955). furthermore, ausoni mounts are separated northward from the ernici mounts by the sacco river, north-westward from the lepini mts by the amaseno river and southward by the planitial and coastal zones before to arrive to the tyrrhenian sea. the ausoni mounts are mainly constituted by limestone rocks. altitudes vary from piedmont zones (10 m) to the 1,116 m of monte calvilli. main peaks include monte delle fate (1,090 m), monte calvo (1,038 m), monte chiavino (1,028 m) and cima del nibbio (1,053 m). due to karstic phenomena, natural small still freshwater ecosystems highly outnumber running waters. other still freshwater ecosystems are artificial stony wells, a very common aquatic typology in whole volsci chain. we limited the study area to the one at the north of via appia (statal road 7) because all areas to the south of this road belong to the planitial zone. on the whole we surveyed about 330 km2 (see fig. 1). field researches were carried during 10 years (1998-2007) and included (i) the inspection of the sites reported in literature, (ii) cartographic recognition of further potential aquatic habitats suitable for amphibian populations and the inspection of these sites, (iii) collection of information from local peoples (mainly from shepherds). since several sites were very close to each other, two or more aquatic habitats less than 50 m apart and inhabited by the same species, have been considered as a single breeding site. we adopted, therefore, the same criterion used by romano et al. (2007) for the aurunci mounts. sites were assigned to seven different freshwater typologies: (i) springs, (ii) drinking places for livestock grazing, (iii) natural ponds, (iv) stony wells, (v) caves, (vi) lakes and marshes and (vii) streams and creeks. only breeding sites were considered in this study. since variance among altitudes of anurans and caudatas breeding sites was not homogeneous (data not shown), non parametric mann-whitney u-test was applied to check for significant differences between altitudinal distributions showed by anura and caudata. statistical analyses was performed using statistica® rel. 5.0/w statistica package (statsoft inc., usa). the sørensen’s coefficient of similarity (hayek 1994) was carried out among species and sites, using amphibian breeding sites to detect the affinity among species in their reproductive habitats. amphibian scientific names are here reported following the systematic revision suggested by frost et al. (2006). results and discussion one hundred and thirteen records of amphibians were collected, and breeding activity was recorded in 69 sites, that is 77.5% of the surveyed potential spawning sites (n = 89), including 83 single aquatic habitats (see materials and methods). eight amphibian species were recorded within the ausoni mounts (57.1% of the species inhabiting latium): northern spectacled salamander, salamandrina perspicillata (savi, 1821); italian crested newt, triturus carnifex (laurenti, 1768); smooth newt, lissotriton vulgaris (linnaeus, 1758); italian newt, lissotriton italicus (peracca, 1898); common toad, bufo bufo (linnaeus, 1758); italian tree frog, hyla intermedia (boulanger, 1882); italian stream frog, rana italica (dubois, 1987) and pelophylax synklepton hispanica which includes berger’s green frog, pelophylax bergeri (günther, 1986) and its hybridogenetic hybrid pelophylax kl. hispanica (bonaparte, 1839). their distribution in the study area is shown in fig. 1 and 131amphibians of the ausoni mountains fig. 2. compared to published data (table 1), the smooth newt and the northern spectacled salamander showed the highest increments (900% and 360% respectively). the mean altitude of records (fig. 3) was 461 ± 217 m (average ± sd; n = 65) for caudatas and 295 ± 193 m for anurans (n = 48), and this difference was highly significant (mann whitney u test = 882.5, p < 0.0001; see also fig. 3). excluding the green frog which has been recorded only in three sites, our survey revealed that the amphibian species are widespread in the ausoni mountains, where anurans seem to prefer the southwest side while urodeles the northeast portion (fig. 1 and fig. 2). our check list did not improve that reported by bonifazi and carpaneto (1990), but the knowledge on the amphibians’ distribution in the study area widely increased. we did not find the spring in the locality “madonna della rocca” quoted by bonifazi and carpaneto (1990), but we retain that this site corresponds to a spring called san maufig. 1. distribution of anurans in the ausoni mountains (latium). triangles = bufo bufo; stars = rana italica; squares = pelophylax synkl. hispanica; rhombuses = hyla intermedia. sites where two or more species are syntopics are indicated by a circle including the symbols of the species. 132 l. corsetti and a. romano ro’s spring which is close to the above mentioned locality. excluding this doubtful site, we surveyed all sites previously investigated by bonifazi and carpaneto (1990) and no species loss was recorded. moreover, our observations after the year 2004 (the last observation on april 2007) permit to assert that l. italicus still breeds in “fontana s. stefano” site, although scillitani et al. (2004) suggested that the italian newt become probably extinct in this site. the caudata preferences for highest altitudes we found in this research agree with that reported for other and wider italian areas (anonymous, 2006b) as well for the remaining subgroups of the volsci chain (corsetti, 1994; romano et al., 2007). on the ausoni mts, altitudinal range showed by s. perspicillata, rana synkl. hispanica and h. intermedia is similar in comparison with the analogous range recorded on the aurunci mountains (romano et al., 2007), while for t. carnifex, l. vulgaris and r. italica the altitudinal range is extended remarkably. conversely, on the ausoni mts, the l. italicus and fig. 2. distribution of caudata in the ausoni mountains (latium). rhombuses = triturus carnifex; squares = lissotriton italicus; triangles = lissotriton vulgaris; stars = salamandrina perspicillata. sites where two or more species are syntopics are indicated by a circle including the symbols of the species. 133amphibians of the ausoni mountains b. bufo breed at lower altitudes and show a shorter altitudinal range although the mean altitude of spawning sites is similar to those reported for aurunci mts (cf. romano et al., 2007). caudata were more abundant then anurans, similarly to nearby aurunci mts, although, in the ausoni mts, frogs and toads occurred more frequently than on the aurunci. indeed the ratio between anura records and caudata records on the ausoni is 0.74 while on the aurunci is near one half (0.47). the sørensen’s coefficients of similarity among amphibian populations of ausoni mts, number and typology of breeding sites for each species are shown in table 1. lissotriton italicus is the most widespread amphibian on the area (26 breeding sites) with the highest number of syntopies which are of the same rank then those of green frogs (table 1). the sørensen’s index shows that t. carnifex and l. vulgaris have similar habitat preferences, as reported for other italian areas (e.g. barbieri and cavagnini, 1999; gentilli and scali, 1999) and for the nearby aurunci mountains as well (romano et al., 2007). an higher index value for the sintopy between these two newts was reported for the albani hills, a extinct volcanic complex very close to rome (angelini and cari, 2004). however this high value (the maximum possible for the sørensen’s index) should be compared with caution because only one breeding site for each species (pantani della doganella for both species) was recorded by the authors. sintopy among t. carnifex, l. italicus and l. vulgaris was previously known usually in artificial water bodies (stony wells) of aurunci mountains (lanza, 1983; bonifazi and carpaneto, 1990; romano et al., 2007), in a site near campobasso, molise region (lanza, 1977), in a few site within majella national park, abruzzi (scalera et al., 2006) and in a few sites on the ausoni mountains (corsetti, 1999). so far sintopy among three newts into a natural water body was known only for the ticino lake (scalera table 1. sørensen’s coefficients between amphibian species on the ausoni mounts (latium) and numbers and typology of breeding sites for each species. spx=xsalamandrina perspicillata; tc = triturus carnifex; lvx=xlissotriton vulgaris; lix=xlissotriton italicus; bbx =xbufo bufo; hix=xhyla intermedia; rix =xrana italica; rshx=xrana synklepton hispanica. number of breeding sites for each species is reported in square brackets. number of sites already known in literature is reported in round brackets. breeding site typology for each species on the ausoni mountains is indicated by an alphabetic code following a decreasing order of frequency: c = caves; d = drinking places for livestock grazing; l = lakes and marshes; p = natural ponds; r = stream and creeks; s = springs; w = stony well. sp [14] (3) s, d, p, r, c tc [15] (4) w, p, r lv [10] (1) w, p, r li [26] (7) w, s, p, r, l bb [14] (8) r, p, s, l hi [3] (2) l, p ri [15] (6) r,s,w,p tc 0 lv 0 0.583 li 0.050 0.263 0.111 bb 0 0 0.166 0.200 hi 0 0.111 0 0 0.235 ri 0.345 0 0.080 0.098 0.345 0 rsh [16] (10) w, r, s, p 0 0.194 0.230 0.190 0.333 0.105 0.129 134 l. corsetti and a. romano et al., 2006) and for two sites in the “gola della rossa e di frasassi” regional park, in the marche region (fiacchini, 2003). we found on the ausoni mountains also a natural pond inhabited by the three newt species, which represent the only natural small aquatic body where this sintopy was recorded so far, because the sintopic natural sites recorded previously were marsh, lakes or streams. salamandrina perspicillata have been found in 14 sites, which concentrated along a line connecting monte s. biagio to roccasecca dei volsci towns. this salamander breeds also in an artificial cave, an unusual spawning site typology for this species that was previously reported by razzetti et al. (2001) and angelini and cari (2004). as reported for the albani hills (angelini and cari, 2004) and the neighbour aurunci mountains (romano et al., 2007), in the northern spectacled salamander the sørensen’s index value is generally zero and an habitat partitioning among this salamander and other amphibians is a deep evidence. relevant sintopy was recorded only with rana italica (romano et al., 2007). the occurrence of green toad, pseudepidalea viridis (laurenti, 1768), was reported by bonifazi and carpaneto (1990) and bologna (2000) only in the planitial coastal zones which were, therefore, excluded from our survey. fig. 3. altitudinal ranges of amphibian species in the ausoni mounts (latium). black bars = caudata; grey bars = anura. codes of species as reported in tab. 1. number of sites of each species is reported above the bar. mean altitude for each species is indicated by an horizontal segments within the bar. 135amphibians of the ausoni mountains other anurans which were found on the ausoni mts showed syntopies comparable to the ones reported for the aurunci mts (cf. romano et al., 2007) while, generally, the sørensen’s index values between species are lower then the ones reported by angelini and cari (2004) for the albani hills. other two anurans, bombina pachypus (bonaparte, 1838) and rana dalmatina (bonaparte, 1840) which are widespread in the latium, were not recorded on the ausoni mountains. the occurrence of b. pachypus on the ausoni in few suitable habitats as vernal and residual ponds, cannot be ruled out completely, although also an updated census on the close aurunci mts did not revealed any occurrence of this species (romano et al., 2007). on the volsci chain, therefore, b. pachypus seems relegated to the northernmost portion of the lepini mts (angelini et al., 2004) while the absence of hygrophilous woods, which are suitable habitats for rana dalmatina, probably explains the lacking of this frog. considering the high number of stony wells and through used by amphibians for spawning, the adequate management of these artificial water bodies appears a crucial prerequisite for an effective conservation of the amphibian populations in the ausoni mountains. acknowledgments we are grateful to g. d’onofrio and r. ragno for their contribute in the field activity, and to s. salvidio for its useful suggestions. references angelini, c., cari, b. (2004): the amphibians of the colli albani (latium, central italy): breeding sites and some ecological notes. atti soc. it. sci. nat. mus. civ. st. nat. milano 145: 337-342. angelini, c., cari, b., mattoccia, m., romano, a. (2004): distribuzione di bombina variegata pachypus (bonaparte, 1838) sui monti lepini (lazio) (amphibia: anura). atti soc. it. sci. nat. mus. civ. st. nat. milano 145: 321-328. anonymous (2006a): data gathering and mapping. in: atlante degli anfibi e dei rettili d’italia / atlas of italian amphibians and reptiles, p. 120-135. sindaco, r., doria, g., razzetti, e., bernini, f., eds, societas herpetologica italica, ed. polistampa, firenze. anonymous (2006b): altitudinal distribution. in: atlante degli anfibi e dei rettili d’italia / atlas of italian amphibians and reptiles, p. 142-147. sindaco, r., doria, g. razzetti, e., bernini, f., eds, societas herpetologica italica, ed. polistampa, firenze. barbieri, f., cavagnini, f. (1999): distribuzione e preferenze ambientali degli anfibi nell’appennino pavese orientale. in: atti ii congresso nazionale s.h.i., p. 97-110. giannotti, f.s., di giovanni, m.v., eds, riv. idrobiol. 38. bologna, m.a. (2000): bufo viridis (laurenti, 1768). in: anfibi e rettili del lazio, p. 52-53. bologna, m.a., capula, m., carpaneto, g.m., eds, fratelli palombi editori, roma. bologna, m.a., capula, m., carpaneto, g.m. (2000): anfibi e rettili del lazio. fratelli palombi editori, roma: 160 p. 136 l. corsetti and a. romano bonifazi, a., carpaneto, g.m. (1990): indagine preliminare sugli anfibi e sui rettili dei monti ausoni-aurunci (lazio meridionale). centro reg. doc. beni cult. amb. lazio, assessorato cultura reg. lazio, roma. bruno, s. (1973): anfibi d’italia: caudata. studi sulla fauna erpetologia italiana – xvii. natura, milano 64: 209-450. corsetti, l. (1994): anfibi e rettili dei monti lepini. quad. mus. st. nat. patrica (fr) 5: 1-192. corsetti, l. (1999): caratteristiche ambientali dei siti riproduttivi triturus italicus nel lazio (italia centrale). in: atti ii congresso nazionale s.h.i., praia a mare (cs), p. 449455. giannotti, f.s., di giovanni, m.v., eds, riv. idrobiol. 38. corsetti, l. (2006): distribuzione e preferenze ambientali degli anfibi urodeli nel lazio meridionale (italia centrale). in: atti v congresso nazionale societas herpetologica italica, calci (pisa), p. 7-18. zuffi, m.a.l., ed, firenze university press. corsetti, l., capula, m. (1992): the amphibians and reptiles of the lepini mountains (latium, central italy): checklist and provisional atlas. br. herpetol. bull. 39: 8-16. fiacchini, d. (2003): biotopi d’acqua dolce del parco regionale della gola della rossa e di frasassi (marche): censimento e proposte di gestione. riv. idrobiol. 42: 63-80. frost, d.r., grant, t., faivovich, j., bain, r.h., haas, a., haddad, c.f.b., de sá, r.o., channing, a., wilkinson, m., donnellan, s.c., raxworthy, c.j., campbell, j.a., blotto, b.l., moler, p., drewes, r.c., nussbaum, r.a., lynch, j.d., green, d.m., wheeler, w.c. (2006): the amphibian tree of life. bull. am. mus. nat. hist. 297: 1-370. gentilli, a., scali, s. (1999): analisi della diversità erpetologica in pianura padana. in: atti ii congresso nazionale s.h.i., praia a mare (cs), p. 113-122. giannotti, f.s., di giovanni, m.v., eds, riv. idrobiol. 38. hayek, l.a.c. (1994): analysis of amphibian biodiversity data. in: measuring and monitoring biological diversity, standard methods for amphibians, p. 207-269. heyer, r.w., donnelly, m.a., mcdiarmid, r.w., hayek, l.a.c., foster, m.s., eds, smithsonian institution, usa. landi vittorj, c. (1955): appennino centrale. guida dei monti d’italia c.a.i. – t.c.i., milano. lanza, b. (1977): simpatry and coexisting in the italian triturus, with notes on the “molge italica molisana” problem (amphibia salamandridae). mon. zool. ital. (n.s.) 11: 113-118. lanza, b. (1983): anfibi, rettili (amphibia, reptilia). collana del progetto finalizzato “promozione della qualità dell’ ambiente”. guide per il riconoscimento delle specie animali delle acque interne italiane, 27, cnr, aq/1/205, roma. razzetti, e., bonini, l., barbieri, f. (2001): riproduzione in grotta di salamandra salamandra e salamandrina terdigitata negli appennini settentrionali. in: atti iii congresso nazionale s.h.i., pavia, p. 181-184. pianura 13. romano, a., montinaro, g., mattoccia, m., sbordoni, v. (2007): amphibians of the aurunci mountains (latium, central italy). checklist and conservation guidelines. acta herpetologica 2: 17-25. scalera, r., venchi, a., carafa, m., pellegrini, m., capula, m., bologna, m.a. (2006): amphibians and reptiles of the majella national park (central italy). aldrovandia 2: 31-47. 137amphibians of the ausoni mountains scillitani, g., scalera, r., carafa, m., tripepi, s. (2004): conservation and biology of triturus italicus in italy (amphibia, salamandridae). in: the conservation status of threatened amphibian and reptile species of italian fauna, p. 45-54. bologna, m.a., la posta, s., eds, ital. j. zool. 71 (suppl. 1). contribution to the herpetology of southern libya adel a. ibrahim department of biological and geological sciences, faculty of education at al-arish, suez canal university, north sinai, egypt. e-mail: dolaibrahim@yahoo.com submitted on 2007, 5th september; revised on 2007, 13th november; accepted 2008, 29th january. abstract. the herpetofauna of southwestern libya has been surveyed during 20052006. overall, two amphibian and 18 reptile species were found; of these, 16 reptile species are reported for the first time for morzoq province and one lizard, tarentola mauritanica, for sabha province. keywords. herpetofauna, libya, distribution. introduction previous herpetological activities in libya resulted in the addition of many records to the known herpetofaunal species (e.g., werner, 1909; boulenger, 1914; scortecci, 1935a, b, c, 1937a, b; schnurrenberger, 1958; kramer and schnurrenberger, 1963; schleich, 1987; sindaco, 1995; laurent et al., 1997; frynta et al., 2000; pieh and perälä, 2002; wilms, 2004; ibrahim and ineich, 2005). however, libya is still the least-studied country herpetologically when compared to the adjacent countries in north africa (boulenger, 1891; anderson, 1898; bons, 1958; marx, 1968; blanc, 1978, 1979; blanc and ineich, 1985; le berre, 1989; salvador, 1996; bons et geniez, 1996; schleich et al., 1996; saleh, 1997; baha el din, 2006). most of previous studies were concentrated in the northern libya. on the contrary, the vast area of the southern fezzan desert, in the southwestern part of libya, between morzoq province and ghat, has received little attention regarding the herpetofauna. for example, kramer and schnurrenberger (1963) reported only two snakes from the south (morzoq province) out of 18 species they recorded from libya. in their review, schleich et al. (1996) reported 50 species of amphibians and reptiles from libya including seven species from ghat and one species of toad from morzoq city. frynta et al. (2000) reported two amphibians and 25 reptiles from 30 different localities in libya including one toad and 11 reptiles from sabha and ghat provinces. scortecci (1937b) identified the only toad found in ghat as bufo regularis. however, the identification of this toad is still under debate, since jöger (1981) assumed that this species is bufo xeros, suggesting that the range acta herpetologica 3(1): 35-49, 2008 issn 1827-9643 (online) © 2008 firenze university press 36 a.a. ibrahim of b. regularis extends farther south in africa than the ghat area. the aim of the present study was (1) to enhance knowledge of herpetology in libya by surveying a large sector of unstudied areas in the western part of the south, and (2) to report on the occurrence and taxonomic status of the ghat toad, bufo sp. materials and methods study site the study was conducted in sha’abeyyat morzoq (morzoq province), great socialist people’s libyan arab jamahireyya. the study site covers an area of at least 20,000 km2 from tmessah in the extreme east of the province to tsawah, wadi otba in the extreme west, and from taraghen southward to al-qatroun. fig. 1 shows the map of libya with study sites. more than forty field trips were made in morzoq province from september 2005 through july 2006 (all seasons), covering 20 localities in at least 200 field hours. some localities were visited as many as five times. field trips were launched from taraghen which was surveyed several times during a period of 10 months. field trips were also made to sabha city (sabha province) and ghodwah between taraghen and sabha during this period. during 4-8 july 2006, a field excursion was made to ghat in the extreme southwestern part of the country. fig. 1. main study sites: 1) tsawah; 2) morzoq; 3) taraghen; 4) ghodwah; 5) sabha city; 6) om al-araneb; 7) zowailah; 8) majdoul; 9) tmessah; 10) al-qatroun; 11) ghat; 12) al-berkah. 37contribution to the herpetology of southern libya study sites were chosen to represent different reptile habitats including the sand dunes east of taraghen village; sand steppes belonging to the great sand desert (adhan morzoq) south of taraghen; gravel and red coarse sand areas (which make up a large part of the desert); green farms that occupy up to 28,000 ha in some areas (e.g., om al-araneb and zowailah) and the mountainous limestone plateaus such as majdoul and sandy and rocky areas in the ghat region. urban settlements were checked for reptiles whenever possible. surveys were made during the day and night. range extension from previously known localities was calculated for animals by km as straight-line distances. searches for amphibians were undertaken around water wells, on farms and in places known by local people to have frogs (e.g., in ghat). faunistic information on amphibians and reptiles is shown in appendix 1. field techniques to verify identifications, animals were collected by hand and with rubber bands. other sampling techniques including turning rocks over and excavation of burrows were also used. tracks of animal were considered positive indicators of their existence. a number of specimens were brought alive to the author by students and volunteers. specimens were preserved in 10% formalin or 70% alcohol. voucher specimens were deposited in the collection of the faculty of arts and sciences, sabha university at taraghen (zct), in the paris national museum of natural history (mnhn) and in adel ibrahim collection (aic). ecological notes for each species were recorded whenever possible. results two amphibian species and 18 species of reptiles were recorded from southwestern libya. of these, 16 reptile species are reported for the first time for morzoq province and one lizard, tarentola mauritanica for sabha province. species accounts amphibia: anura: bufonidae green toad, bufo viridis laurenti, 1768. this toad was found in cultivated fields, such as those in om al-araneb that consist of thousands of cultivated acres of edible vegetables, fruits and animal fodder. toads observed in these localities showed a color difference distinct from morzoq area individuals. the former had brighter green spots on the back. toads were generally active throughout the year except for cold days. no hibernation was noted in late autumn (november) and early winter (december). this species was recorded in taraghen by scortecci (1935a) and in cyrenaica (schleich et al., 1996), extending its range by at least 900 km to taraghen. subdesert toad, bufo xeros tandy, tandy, keith and duff-mckay, 1976. this toad was common in ghat, found in pools and puddles on local farms at tunin and nearby the water station at al-berkat. the toads had different colors, ranging between grayish brown, 38 a.a. ibrahim olive and reddish with pairs of identical dark olive patches on the dorsum that were partially bordered with black. all individuals had a light coloredmiddorsal line. tubercles with one or more sharp spines on the upper side of the body and creamy granular venter. the foot was slightly webbed, with well developed anterior and exterior metacarpal tubercles suitable for use in digging. tadpoles of this species were olive brown spotted with black. croaking was heard at a distance, thus the toads could be easily located. however, on some occasions, toads were found in swampy areas with dense vegetation within the farms which made them difficult to catch. toads were commonly seen in highest density just after sunset. during the day, the toads concealed themselves under vegetation that covers most of the pool bottom, and in some cases, it reaches 30 cm above the pool bottom. in addition to their intensive night activity, tadpoles were observed swimming near surface during the day. reptilia: squamata: sauria: agamidae bibron’s agama, agama impalearis boettger, 1874. a single individual was observed on a big boulder in the ghat area through binoculars. the followings characters were noted: upper side reddish brown, head, neck and shoulders with several groups of large spines, no gular pouch. observation suggested that this individual was most likely agama impalearis. bell’s dab-lizard, uromastyx acanthinura merrem, 1820. this species was common in morzoq province, confirming its existence in south libya (see frynta et al., 2000). the species was observed all year around and several individuals were captured during spring and summer but released at the point of capture, including a pregnant female captured in zowailah on 25 june 2006. the range of this species was extended by 700 km from badr to taraghen (ibrahim and ineich, 2005). gekkonidae ragazzi’s fan-footed gecko, ptyodactylus ragazzii anderson, 1889. this gecko was very common in majdoul and ghat. in majdoul, it was observed in residential areas, on building walls, inside houses and around lights. in farms, it was encountered on old ruins and boulders around wells. it became active just before sunset so that several individuals were captured by day. in ghat, this species was the most conspicuous gecko and encountered almost everywhere on building walls at tunin, in ghat new city and on recently constructed buildings. an isolated population inhabited the rest house complexes affiliated with sabha university at ghat. lizards were commonly seen on the walls of buildings and occasionally inside the main entrances and climbing up the higher stories (up to 15 m high). this species was active from sunset to dawn but remained concealed during the day when the air temperature might exceed 40 °c during july. no other scansorial geckos were observed in these places. this gecko appears to be most active during summer. as many as 50 geckos could be counted during one hour of observation in tunin. this gecko was recently reported from the ghat area by frynta et al. (2000), increasing its distributional record by 500 km to majdoul. 39contribution to the herpetology of southern libya petrie’s gecko, stenodactylus petrii anderson, 1896. geckos were only encountered in soft sandy areas. apparently, these geckos commence their activity immediately after sunset. presence of this gecko in taraghen extended its range by 700 km from the badr village (ibrahim and ineich, 2005) and by 900 km from cyrenaica (scortecci, 1935a). elegant gecko, stenodactylus sthenodactylus (lichtenstein, 1823). this was a common cursorial gecko, observed in coarse sand areas in several localities. on 14 may, a female deposited two white shelled eggs in captivity three days after capture. this gecko showed a wide distribution in both morzoq and sabha provinces which was increased by 1000 km from cyrenaica to tsawah (calabresi, 1923; zavattari, 1930; scortecci, 1935a). moorish gecko, tarentola mauritanica (linnaeus, 1758). the species seemed to be uncommon in morzoq province, but more common in sabha city where it was the only scansorial gecko, observed on walls, around lights, even in downtown especially during summer. this gecko was nocturnal, active from sunset to dawn. the distribution range was extended by about 800 km from the known localities in cyrenaica (zavattari, 1930; frynta et. al., 2000). tripoli dwarf gecko, tropiocolotes tripolitanus peters, 1880. the gecko’s local name is “bu kash-shash”. this species was a very common cursorial gecko, found elsewhere in morzoq province on the surface of the sand, under rocks, bricks, tree logs, leaf litter and inside houses. a large number of specimens were observed but not collected. this gecko was frequent in the northern part (werner, 1909; zavattari, 1930; frynta et al., 2000; ibrahim and ineich, 2005) extending the range by at least 700 km to taraghen. in the south, it was recorded from al-fejaj (frynta et al., 2000), extending its range by 250 km southeast to tsawah. lacertidae bosc’s fringe-toed lizard, acanthodactylus boskianus (daudin, 1802). this species was observed on hard terrain with sparse vegetation. it was most active during morning hours between 09:00-11:00 hrs. this species was captured from northern sandy plains in cyrenaica (calabresi, 1923), al-khums (frynta et al., 2000), and badr (ibrahim and ineich, 2005). in the south, it was reported from al-fejaj (frynta et al., 2000), increasing its distribution by 150 km eastward to mosequeen. nidua lizard, acanthodactylus scutellatus (audouin, 1809). individuals were sandy brown, reticulated with conspicuous white and black spots on dorsum; the tail resembles a. longipes, but the dorsal scales of tibia and femoral pores (baha el-din, 1994) follow the typical pattern for scutellatus. only a few lizards were observed during winter and spring, thus it may be active all year around. in badr village, it was also active in sunny days in winter (ibrahim and ineich, 2005). this species was reported from cyrenaica (werner, 1909; calabresi, 1923; zavattari, 1930) increasing its distribution by 950 km to morzoq. 40 a.a. ibrahim red-spotted desert-racer, mesalina rubropunctata (lichtenstein, 1823). a single individual of this species was collected. this lizard was reported from ghat (schleich et al., 1996) and in sinawin at the extreme northwest of libya (frynta et al., 2000), extending its range by 550 and 700 km respectively to zowailah. scincidae ocellated skink, chalcides ocellatus ocellatus (forskål, 1775). this skink was found on farms and in green areas around houses. the known distributional range of c. ocellatus was increased by 900 km from cyrenaica to taraghen (calabresi, 1923; zavattari, 1930). sand fish, scincus scincus scincus (linnaeus, 1758). five specimens of these lizards were captured by pit-fall traps from sand dunes in morzoq province, recording a range extension of 700 km from badr to tsawah (ibrahim and ineich, 2005). varanidae desert monitor, varanus griseus griseus (daudin, 1803). although its conspicuous tracks were often observed in taraghen and its surrounding sand dunes, only one young individual was captured. this species was previously reported from the extreme northeast (zavattari, 1930) and northwest (ibrahim and ineich, 2005) of libya, recording an extension of 900 and 700 km respectively to taraghen. colubridae moila snake, malpolon moilensis (reuss, 1834). an adult female was captured in an old abandoned building at tsawah. this record fills a big gap of the distribution of this snake in the libyan saharan. the range was extended by 750 km southward from tripoli and 1,000 km westward from kufra (kramer and schnurrenberger, 1963). schokari sand snake, psammophis schokari schokari (forskål, 1775). this snake was encountered in sandy areas elsewhere in morzoq province and in ghodwah. it was also captured from cultivated fields. this snake was active throughout the year. p. schokari was previously recorded in zowailah, tmessah, and morzoq (kramer and schnurrenberger, 1963) and in cyrenaica (calabresi, 1923) extending its range by 950 km from morzoq. clifford’s snake, spalerosophis diadema cliffordi (schlegel, 1837). ten individuals were collected from nine localities in morzoq province. of these, six snakes were captured in late autumn (november) and winter (december and january). thus, it seemed active all year around. young individuals first appeared in april and may. in the south, this snake was reported from zowailah (loveridge, 1940), tmessah (schnurrenberger, 1958) and from gabroon (frynta et al., 2000). 41contribution to the herpetology of southern libya viperidae horned viper, cerastes cerastes cerastes (linnaeus, 1758). this was a well-known snake in the south libyan desert. eight individuals were caught. among these, six individuals were captured in december. all vipers were reddish brown with distinct dorsal patterns and had full horns except for a juvenile from majdoul which possessed two rudimentary horns. frynta et al. (2000) reported two almost hornless vipers with no dorsal pattern from al-awaynat, 400 km southwest sabha. the distribution range of this snake was increased from al-awaynat by 450 km to majdoul and by 900 km from benghazi (calabresi, 1923) to haj hjeel. avicenna’s viper, cerastes vipera (linnaeus, 1758). two individuals were captured in sandy areas. the distributional record of this viper is increased by 750 km from badr southward to zowailah (ibrahim and ineich, 2005) and 700 km from the ghat area (scortecci, 1939). discussion all reptiles collected from morzoq province during this study are reported for the first time except for two snakes, psammophis schokari and spalerosophis diadema (kramer and schnurrenberger, 1963). the reason that little attention has been paid to this area is probably due to inaccessibility and lack of logistic facilities that do not encourage researchers to work or to stay long in the area. in addition, the ambient temperature is high throughout most of the year, except for a few weeks in winter. most of herpetological exploration to this country has been conducted in the north rather than in the south (see boulenger, 1914; zavattari, 1922, 1929; calabresi, 1923; scortecci, 1939; schnurrenberger, 1959; schleich, 1984, 1989; frynta et al., 2000; ibrahim and ineich, 2005). the few visits that researchers have made in the far south in the country have been to well-known areas, such as the al-kufrah oasis in the southeast (e.g., peters in rohlfs, 1881; vinciguerra, 1930; scortecci, 1935c); ghat (a tourist destination in the far western south) and to the cities of morzoq and sabha which were former regional capitals (see scortecci, 1935a, 1937a; frynta et al., 2000). this study confirms the occurrence of bufo xeros in the ghat area which is now the only locality known for the species in libya. scortecci (1937b) found b. xeros in great numbers hunting around wells, springs and puddles, but at that time, the species was undescribed. two other frogs were reported from the ghat area, dicroglossus occipitalis from tunin and rana saharica from oasis tin al-kun (scortecci, 1937a). intensive searches in these areas revealed a complete absence of these frogs probably because of the massive environmental changes in the area. the springs and puddles mentioned by scortecci have dried up. it was reported that dicroglossus occipitalis had been introduced to the area to control mosquitoes. however, there are a number of farms in tunin each with a man-made pool in which toads (b. xeros) are encountered. similarly, al-berkah (arabic proper), which means “puddle”, has also been changed as springs and puddles are no longer found, but toads were observed at the water station and on one farm. most of 42 a.a. ibrahim the species recorded in morzoq province were active during the winter except on cold days in contrast to the behavior of the same species in northern libya. for example, the horned vipers and sand vipers that were active in december and january in the south (this study) do not appear in the northwest corner of libya during winter and start their activity in spring (ibrahim and ineich, 2005). schnurrenberger (1959) observed that these vipers were also active in winter and hibernated in january and february except on warm days which could be explained as a function of air temperature. coastal areas of libya have a typical mediterranean climate, and average temperatures ranging from about 30 °c to 8 °c. the south, on the other hand, experiences desert-type climates with summer temperatures soaring over 50 °c and with below-zero nights. in morzoq and sabha provinces, air temperature is generally high most of the year months; winters are mild, with air temperatures ranging from 4 °c to 35 °c. the record of a single specimen of mesalina rubropunctata in zowailah increased the distribution of this lizard largely from previously known records within the country. the rarity of this species in the libyan desert was evident in this study. this is in agreement with schleich et al. (1996) who reported that the population density of this species was very low in libya. frynta et al. (2000) reported on a single specimen from sinawan. likewise, ibrahim and ineich (2005) reported an additional specimen from badr during april. in egypt, this lizard exhibited low encounter rate in the sinai desert, in contrast with other lacertid species (adel ibrahim, unpubl. data). it is believed that activity of this lizard depends – in particular – on the intensity of insolation (schleich et al., 1996). in israel, it emerges in some special weather conditions (y. werner, pers. comm.). the distribution of many species has been greatly affected by the habitat types in the southern libyan sahara. the sand dune community includes acanthodactylus scutellatus, chalcides ocellatus, cerastes vipera, scincus scincus, stenodactylus petrii and varanus griseus, while in firm sand biotope, acanthodactylus boskianus, cerastes cerastes, mesalina rubropunctata, stenodactylus sthenodactylus and tropiocolotes tripolitanus are found. old buildings in tunin usually had a high density of ptyodactylus ragazzii. this gecko moved to the ghat new town with modern rural houses as well as newly established buildings near town, showing a good colonization ability. the prevalence of p. ragazzii in ghat and majdoul is possibly related to the mountainous nature of these locations which may also explain why no scansorial geckos occur in taraghen which is not surrounded by any kind of hills or mountains. moreover, the nearest rocky area to taraghen is about 100 km apart. in addition to their presence in sandy localities, psammophis schokari and spalerosophis diadema were encountered on cultivated farms. the occurrence of these species in green fields was previously reported in sinai (ghobashi et al., 1990) and in the suez canal area (adel ibrahim, unpubl. data). the availability of food such as frogs, rats and even lizards may attract snakes to these places. acknowledgements special thanks and appreciation are due to mr. abid ahmad abid, vice dean of faculty of arts and sciences at taraghen for his continuous assistance and support without which this work would not have been accomplished. i am also grateful to mr. mahdy bakkoury for field assistance 43contribution to the herpetology of southern libya and for the freshmen students of the same school at taraghen, especially, amal sowaileh, haleema omran, rabeea anna’as, jomaa al-mahdi, and abulqassem ibrahim for providing and preserving specimens. i thank dr. annemarie ohler for identification of bufo xeros and dr. ivan ineich for the verification of the identification of some reptiles. references anderson, j. 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(1929): anfibi e rettili della cirenaica raccolti. not. econ. cirenaica. governo della cirenaica. camera di commercio. 5/6: 86-88. zavattari, e. (1930): erpetologia della cirenaica. arch. zool. ital. torino 14: 253-289. 46 a.a. ibrahim a pp en di x 1. f au ni st ic i nf or m at io n on t he h er pe to fa un a re co rd ed i n so ut hw es te rn l ib ya d ur in g 20 05 -2 00 6. m n h n = m us eu m n at io na l d ’h is to ir e n at ur el le , pa ri s; z c t = z oo lo gi ca l c ol le ct io n at t ar ag he n; a ic = a de l i br ah im c ol le ct io n, e gy pt . sp ec ie s na m e n lo ca lit y d at e g az et te er o f s ou th l ib ya n lo ca tio ns v ou ch er s pe ci m en s bu fo v ir id is 4 1 1 3 4 1 1 ta ra gh en , u ni ve rs ity d or m ito ry ta ra gh en , a gr ic ul tu re p ro je ct m or zo q o m a la ra ne b o m a la ra ne b o m a la ra ne b o m a la ra ne b 22 n ov em be r 20 05 1 m ay 2 00 6 24 n ov em be r 20 05 17 d ec em be r 20 05 28 d ec em be r 20 05 24 m ar ch 2 00 6 6 ju ne 2 00 6 25 ° 55 ’ 4 5” n , 1 4° 2 6’ 3 3” e 25 ° 56 ’ 1 3” n , 1 4° 2 8’ 4 0” e 25 ° 55 ’ 2 4” n , 1 3° 5 4’ 5 9” e 26 ° 08 ’ 0 2” n , 1 4° 4 4’ 0 6” e 26 ° 08 ’ 0 2” n , 1 4° 4 4’ 0 6” e 26 ° 08 ’ 0 2” n , 1 4° 4 4’ 0 6” e 26 ° 08 ’ 0 2” n , 1 4° 4 4’ 0 6” e z c t 2 00 5. 58 z c t 2 00 6. 43 z c t 2 00 5. 9 z c t 2 00 5. 21 -2 3 z c t 2 00 5. 37 -4 0 z c t 2 00 6. 24 z c t 2 00 6. 68 bu fo x er os 2 1 a lb er ka h, 8 k m s ou th ea st g ha t tu ni n di st ri ct , g ha t 4 ju ly 2 00 6 5 ju ly 2 00 6 24 ° 52 ’ 1 1” n , 1 0° 1 0’ 2 6” e 24 ° 58 ’ 0 3” n , 1 0° 1 0’ 2 2” e m n h n 2 00 6. 25 33 -2 53 5 a ga m a im pa le ar is 1 2 km fr om th e c ity o f g ha t 6 ju ly 2 00 6 24 ° 58 ’ 0 7” n , 1 0° 1 0’ 0 5” e z c t 2 00 6. 73 u ro m as ty x ac an th in ur a 1 1 1 m aj do ul tm es sa h z ow ai la h 21 d ec em be r 20 05 29 a pr il 20 06 25 ju ne 2 00 6 25 ° 55 ’ 3 7” n , 1 5° 0 7’ 2 0” e 26 ° 23 ’ 2 0” n , 1 5° 4 7’ 4 8” e 26 ° 10 ’ 5 5” n , 1 5° 0 6’ 5 2” e z c t 2 00 5. 31 a ic 2 00 6. 15 50 z c t 2 00 6. 70 pt yo da ct yl us r ag az zi i 2 1 1 1 1 m aj do ul m aj do ul z ow ai la h z ow ai la h g ha t 7 m ay 2 00 6 3 ju ne 2 00 6 23 m ay 2 00 6 18 ju ne 2 00 6 4 ju ly 2 00 6 5° 5 5’ 3 7” n , 1 5° 0 7’ 2 0” e 5° 5 5’ 3 7” n , 1 5° 0 7’ 2 0” e 26 ° 10 ’ 5 5” n , 1 5° 0 6’ 5 2” e 26 ° 10 ’ 5 5” n , 1 5° 0 6’ 5 2” e 24 ° 58 ’ 0 7” n , 1 0° 1 0’ 0 5” e z c t 2 00 6. 47 -4 8 a ic 2 00 6. 15 73 z c t 2 00 6. 63 -6 4 z c t 2 00 6. 69 z c t 2 00 6. 72 st en od ac ty lu s pe tr ii 1 1 10 k m s ou th o f t ar ag he n g ha t 11 m ay 2 00 6 6 ju ly 2 00 6 25 ° 51 ’ 2 9” n , 1 4° 2 5’ 5 0” e 24 ° 58 ’ 0 7” n , 1 0° 1 0’ 0 5” e z c t 2 00 6. 54 z c t 2 00 6. 74 st en od ac ty lu s st he no da ct yl us 2 1 1 1 1 1 1 g ho dw a m aj do ul m or zo q a zzy to un a, 1 5 km e as t o f t ar ag he n ts aw ah ta ra gh en , o m a la ra ne b 26 n ov em be r 20 05 17 d ec em be r 20 05 21 d ec em be r 20 05 2 a pr il 20 06 7 m ay 2 00 6 14 m ay 2 00 6 15 m ay 2 00 6 26 ° 27 ’ 5 9” n , 1 4° 1 8’ 1 7” e 5° 5 5’ 3 7” n , 1 5° 0 7’ 2 0” e 25 ° 55 ’ 2 4” n , 1 3° 5 4’ 5 9” e 25 ° 54 ’ 4 4” n , 1 4° 3 4’ 2 9” e 25 ° 59 ’ 3 5” n , 1 3° 3 1’ 0 0” e 25 ° 59 ’ 3 5” n , 1 3° 3 1’ 0 0” e 26 ° 08 ’ 0 2” n , 1 4° 4 4’ 0 6” e z c t 2 00 5. 11 -1 2 z c t 2 00 5. 24 z c t 2 00 5. 32 z c t 2 00 6. 25 z c t 2 00 6. 49 z c t 2 00 6. 56 z c t 2 00 6. 58 (c on tin ue ) 47contribution to the herpetology of southern libya sp ec ie s na m e n lo ca lit y d at e g az et te er o f s ou th l ib ya n lo ca tio ns v ou ch er s pe ci m en s ta re nt ol a m au ri ta ni ca 1 1 1 o m a la ra ne b o m a la ra ne b sa bh a c ity 6 m ay 2 00 6 10 ju ne 2 00 6 14 o ct ob er 2 00 5 26 ° 08 ’ 0 2” n , 1 4° 4 4’ 0 6” e 26 ° 08 ’ 0 2” n , 1 4° 4 4’ 0 6” e 27 ° 01 ’ 5 9” n , 1 4° 2 6’ 0 0” e z c t 2 00 6. 45 a ic 2 00 6. 15 74 z c t 2 00 5. 01 tr op io co lo te s tr ip ol ita nu s 1 1 4 1 5 1 1 1 1 1 3 5 2 1 5 1 1 h aj h je el g ho dw a g ho dw a ts aw ah ts aw ah ts aw ah m aj do ul m aj do ul m or zo q z ow ai la h a lb ed ai r jiz aw a zzy to un a a zzy to un a ta ra gh en ta ra gh en o m a la ra ne b 28 o ct ob er 2 00 5 29 n ov em be r 20 05 8 m ay 2 00 6 29 n ov em be r 20 05 10 a pr il 20 06 28 m ay 2 00 6 29 n ov em be r 20 05 14 ja nu ar y 20 06 30 n ov em be r 20 05 17 d ec em be r 20 05 17 d ec em be r 20 05 13 ja nu ar y 20 06 8 a pr il 20 06 14 m ay 2 00 6 8 ja nu ar y 20 06 27 ju ne 2 00 6 15 m ay 2 00 6 25 ° 55 ’ 4 7” n , 1 4° 0 2’ 4 0” e 26 ° 27 ’ 5 9” n , 1 4° 1 8’ 1 7” e 26 ° 27 ’ 5 9” n , 1 4° 1 8’ 1 7” e 25 ° 59 ’ 4 6’ ’n , 1 3° 3 0’ 0 3’ ’e 25 ° 59 ’ 4 6’ ’n , 1 3° 3 0’ 0 3’ ’e 25 ° 59 ’ 4 6’ ’n , 1 3° 3 0’ 0 3’ ’e 5° 5 5’ 3 7” n , 1 5° 0 7’ 2 0” e 5° 5 5’ 3 7” n , 1 5° 0 7’ 2 0” e 25 ° 55 ’ 2 4” n , 1 3° 5 4’ 5 9” e 26 ° 10 ’ 5 5” n , 1 5° 0 6’ 5 2” e 26 ° 07 ’ 5 0” n , 1 4° 5 3’ 1 2” e 25 ° 53 ’ 1 2” n , 1 4° 0 9’ 1 5” e 25 ° 54 ’ 4 4” n , 1 4° 3 4’ 2 9” e 25 ° 54 ’ 4 4” n , 1 4° 3 4’ 2 9” e 25 ° 59 ’ 3 5” n , 1 3° 3 1’ 0 0” e 25 ° 59 ’ 3 5” n , 1 3° 3 1’ 0 0” e 26 ° 08 ’ 0 2” n , 1 4° 4 4’ 0 6” e z c t 2 00 5. 02 z c t 2 00 5. 14 a ic 2 00 6. 15 68 -7 1 z c t 2 00 5. 15 z c t 2 00 6. 28 -3 2 z c t 2 00 6. 65 z c t 2 00 5. 16 z c t 2 00 6. 15 -2 1 z c t 2 00 5. 16 z c t 2 00 5. 25 z c t 2 00 5. 26 -2 8 z c t 2 00 6. 09 -1 3 z c t 2 00 6. 26 -2 7 z c t 2 00 6. 57 z c t 2 00 6. 01 -0 5 z c t 2 00 6. 71 z c t 2 00 6. 59 a ca nt ho da ct yl us b os ki an us 1 1 1 1 1 ta ra gh en m os eq ue en m os eq ue en m os eq ue en m or zo q 3 n ov em be r 20 05 10 a pr il 20 05 29 a pr il 20 06 29 m ay 2 00 6 18 m ay 2 00 6 25 ° 59 ’ 3 5” n , 1 3° 3 1’ 0 0” e 26 ° 23 ’ 2 0” n , 1 5° 4 7’ 4 8” e 26 ° 23 ’ 2 0” n , 1 5° 4 7’ 4 8” e 26 ° 23 ’ 2 0” n , 1 5° 4 7’ 4 8” e 25 ° 55 ’ 2 4” n , 1 3° 5 4’ 5 9” e z c t 2 00 5. 04 z c t 2 00 6. 33 z c t 2 00 6. 41 z c t 2 00 6. 66 a ic 2 00 6. 15 72 a ca nt ho da ct yl us s cu te lla tu s 1 1 m or zo q g ho dw ah 28 d ec em be r 20 05 12 m ay 2 00 6 25 ° 55 ’ 2 4” n , 1 3° 5 4’ 5 9” e 26 ° 27 ’ 5 9” n , 1 4° 1 8’ 1 7” e z c t 2 00 5. 41 z c t 2 00 6. 55 m es al in a ru br op un ct at a 1 z ow ai la h 17 d ec em be r 20 05 26 ° 10 ’ 5 5” n , 1 5° 0 6’ 5 2” e z c t 2 00 5. 29 a pp en di x 1. c on tin ue d. (c on tin ue ) 48 a.a. ibrahim sp ec ie s na m e n lo ca lit y d at e g az et te er o f s ou th l ib ya n lo ca tio ns v ou ch er s pe ci m en s c ha lc id es o ce lla tu s 1 1 1 1 1 1 a lq la ib ta ra gh en ta ra gh en d ou ga l g ho dw ah m os eq ue en 28 d ec em be r 20 05 28 d ec em be r 20 05 20 m ar ch 2 00 6 29 d ec em be r 20 05 22 m ay 2 00 6 29 m ay 2 00 6 25 ° 59 ’ 3 5” n , 1 3° 3 1’ 0 0” e 25 ° 59 ’ 3 5” n , 1 3° 3 1’ 0 0” e 26 ° 05 ’ 2 3” n , 1 3° 4 0’ 0 1” e 26 ° 27 ’ 5 9” n , 1 4° 1 8’ 1 7” e 26 ° 23 ’ 2 0” n , 1 5° 4 7’ 4 8” e z c t 2 00 5. 42 z c t 2 00 5. 43 z c t 2 00 6. 23 z c t 2 00 5. 45 z c t 2 00 6. 62 z c t 2 00 6. 67 sc in cu s sc in cu s 1 1 1 3 a lja bb ar tm es sa h tm es sa h a sh -s ho w ai sh 15 a pr il 20 06 29 a pr il 20 06 15 m ay 2 00 5 7 m ay 2 00 6 25 ° 54 ’ 3 0” n , 1 4° 3 3’ 3 8” e 26 ° 23 ’ 2 0” n , 1 5° 4 7’ 4 8” e 26 ° 23 ’ 2 0” n , 1 5° 4 7’ 4 8” e 25 ° 54 ’ 4 ”n , 1 4° 3 4’ 5 0” e z c t 2 00 6. 35 z c t 2 00 6. 42 z c t 2 00 6. 60 z c t 2 00 6. 50 -5 2 va ra nu s gr is eu s 1 a zzy to un a 6 m ay 2 00 6 25 ° 54 ’ 4 4” n , 1 4° 3 4’ 2 9” e z c t 2 00 6. 46 m al po lo n m oi le ns is 1 ts aw ah 17 d ec em be r 20 05 25 ° 59 ’ 4 6’ ’n , 1 3° 3 0’ 0 3’ ’e z c t 2 00 5. 30 ps am m op hi s sc ho ka ri 1 1 1 1 1 m or zo q g ho dw ah m aj do ul o m a la ra ne b a lq at ro un 14 d ec em be r 20 05 2 ja nu ar y 20 06 14 ja nu ar y 20 06 11 ja nu ar y 20 06 4 m ay 2 00 6 25 ° 55 ’ 2 4” n , 1 3° 5 4’ 5 9” e 26 ° 27 ’ 5 9” n , 1 4° 1 8’ 1 7” e 5° 5 5’ 3 7” n , 1 5° 0 7’ 2 0” e 26 ° 08 ’ 0 2” n , 1 4° 4 4’ 0 6” e 24 ° 56 ’ 0 0” n , 1 4° 3 8’ 3 0” e z c t 2 00 5. 20 a ic 2 00 6. 15 43 z c t 2 00 6. 22 z c t 2 00 6. 07 z c t 2 00 6. 44 sp al er os op hi s di ad em a 1 1 1 1 1 1 1 1 1 1 1 1 a lja bb ar h aj h je el z ow ai la h z ow ai la h a zzy to un a ts aw ah m or zo q ta ra gh en m aj do ul g ho dw ah tm es sa h o m a la ra ne b 25 n ov em be r 20 05 28 n ov em be r 20 05 1 d ec em be r 20 05 12 ja nu ar y 20 06 1 d ec em be r 20 05 25 d ec em be r 20 05 28 d ec em be r 20 05 10 a pr il 20 06 10 a pr il 20 06 10 a pr il 20 06 23 a pr il 20 06 15 m ay 2 00 6 25 ° 54 ’ 3 0” n , 1 4° 3 3’ 3 8” e 25 ° 55 ’ 4 7” n , 1 4° 0 2’ 4 0” e 26 ° 10 ’ 5 5” n , 1 5° 0 6’ 5 2” e 26 ° 10 ’ 5 5” n , 1 5° 0 6’ 5 2” e 25 ° 54 ’ 4 4” n , 1 4° 3 4’ 2 9” e 25 ° 59 ’ 4 6’ ’n , 1 3° 3 0’ 0 3’ ’e 25 ° 55 ’ 2 4” n , 1 3° 5 4’ 5 9” e 25 ° 59 ’ 3 5” n , 1 3° 3 1’ 0 0” e 5° 5 5’ 3 7” n , 1 5° 0 7’ 2 0” e 26 ° 27 ’ 5 9” n , 1 4° 1 8’ 1 7” e 26 ° 23 ’ 2 0” n , 1 5° 4 7’ 4 8” e 26 ° 08 ’ 0 2” n , 1 4° 4 4’ 0 6” e z c t 2 00 5. 10 z c t 2 00 5. 13 z c t 2 00 5. 18 z c t 2 00 6. 08 a ic 2 00 5. 15 40 z c t 2 00 5. 36 z c t 2 00 5. 44 z c t 2 00 6. 34 a ic 2 00 6. 15 45 a ic 2 00 6. 15 46 z c t 2 00 6. 36 z c t 2 00 6. 61 a pp en di x 1. c on tin ue d. (c on tin ue ) 49contribution to the herpetology of southern libya sp ec ie s na m e n lo ca lit y d at e g az et te er o f s ou th l ib ya n lo ca tio ns v ou ch er s pe ci m en s c er as te s ce ra st es 1 1 1 2 1 1 1 h aj h je el fo nq ol fo nq ol a zzy to un a m aj do ul m aj do ul g ho dw ah 28 o ct ob er 2 00 5 30 n ov em be r 20 05 9 d ec em be r 20 05 10 , 2 1 d ec em be r 20 05 21 d ec em be r 20 05 7 m ay 2 00 6 29 d ec em be r 20 05 25 ° 55 ’ 4 7” n , 1 4° 0 2’ 4 0” e 25 ° 55 ’ 0 6” n , 1 4° 1 5’ 5 3” e 25 ° 55 ’ 0 6” n , 1 4° 1 5’ 5 3” e 25 ° 54 ’ 4 4” n , 1 4° 3 4’ 2 9” e 5° 5 5’ 3 7” n , 1 5° 0 7’ 2 0” e 5° 5 5’ 3 7” n , 1 5° 0 7’ 2 0” e 26 ° 27 ’ 5 9” n , 1 4° 1 8’ 1 7” e z c t 2 00 5. 03 z c t 2 00 5. 17 z c t 2 00 5. 19 z c t 2 00 5. 18 , 3 3 z c t 2 00 5. 34 -3 5 z c t 2 00 6. 53 z c t 2 00 5. 46 c er as te s vi pe ra 1 1 m a’a fa n, 1 0 so ut h of t ar ag he n z ow ai la h 21 d ec em be r 20 05 8 ja nu ar y 20 06 25 ° 54 ’ 0 1” n , 1 4° 3 4’ 4 5” e 26 ° 10 ’ 5 5” n , 1 5° 0 6’ 5 2” e z c t 2 00 5. 36 z c t 2 00 6. 06 a pp en di x 1. c on tin ue d. acta herpetologica 2006 2 breeding phenology of «bufo viridis» laurenti, 1768 in sicily breeding phenology of bufo viridis laurenti, 1768 in sicily alessandra sicilia 1, francesco lillo 1, bruno zava 2 and franco bernini 3 1 dipartimento di biologia animale, università degli studi di palermo, via archirafi 18, i-90123 palermo, italy. e-mail: ale.sicilia@unipa.it 2 wilderness studi ambientali, via cruillas 27, i-90146 palermo, italy 3 dipartimento di biologia animale, università degli studi di pavia, piazza botta 9, i-27100 pavia, italy abstract. bufo viridis laurenti, 1768 is a common species that inhabits a wide variety of habitats. the different climates characterising its broad range lead to a high degree of variability in its seasonal activity and reproductive cycle. this paper reports some observations carried out on the breeding phenology of this species over a two year period in mediterranean temporary ponds in sicily. the reproductive period of sicilian green toads extends into the autumn months, making it longer than that of other italian populations. this behaviour seems due to the impact of xeric environmental conditions on the seasonal activity of the studied populations. the present study confirms that b. viridis is an opportunistic breeder with a wide margin of variability in annual reproductive cycle patterns, as would be expected of an ecologically variable species. the duration of the reproductive season varied between populations in the same year and between different years for the same population. keywords. amphibian, bufo viridis, phenology, autumn breeding, mediterranean environments, sicily. introduction bufo viridis laurenti, 1768 is a widespread species with a range which extends from eastern france and italy to central asia, including northern africa and numerous mediterranean islands (bologna and giacoma, 2006). because of the high morphological variability of the green toad, several forms, as species and subspecies, have been described within its extensive range (stöck et al., 2001). in italy seems to be present the nominate subspecies (bologna and giacoma, 2006); however, the taxonomical status of the green toad has not so far been clearly assessed, and further investigations are needed (roth, 1986; balletto et al., 2000; stöck et al., 2001). the species inhabits a wide variety of habitats, from mesic to arid, from subtropical to cold temperate, and from below sea level in israel to more than 4000 m a.s.l. in the himaacta herpetologica 1(2): 107-117, 2006 108 a. sicilia et alii layas (dessauer et al., 1975). it is common along coasts, due to its ability to survive and breed in brackish waters (lanza, 1983). its typical reproductive sites are temporary and shallow water bodies (roth, 1997; bologna and giacoma, 2006). the variety of climates covered by its broad range gives rise to high degree of variability in the seasonal activity and reproductive cycle of the green toad (giacoma et al., 2000). breeding periods observed for populations from continental italy and sardinia are generally comprised between the end of february and the end of august, with a maximum length of about three and a half months. a longer reproductive period, extending into the autumn months, was discovered for a population found near palermo (capo gallo, la fossa) in a preliminary study carried over three years from 2000 to 2002 (sicilia, 2006). our paper reports further observations on the reproductive activity and the duration of larval development in the la fossa and ustica island (province of palermo) populations and analyses the differences between these and other italian populations, in order to verify previous observations and to better understand the breeding phenology of b. viridis in sicily. materials and methods study area sicily has a mediterranean climate with mild winters and long, dry summers, characterised by unpredictable rains. the plain areas of the island and some circum-sicilian islands are arid or semiarid in terms of de martonne and gottmann’s aridity index. the west coast and central-southern areas are arid according to lang’s rain factor (duro et al., 1997). its insularity, geographic position, orography, the nature of its soils and environmental degradation preclude sicily having a hydrographic system like that of mainland italy. sicilian freshwater environments are unstable, with wide seasonal oscillations (riggio, 1978) and most natural freshwater ecosystems are temporary. the breeding sites of b. viridis studied in this research are temporary water bodies located in the nw part of the island. la fossa is located in the capo gallo nature reserve, on the coast in the outskirts of palermo (fig. 1). the only reproductive site of b. viridis in la fossa is a small concrete basin with a surface area of about 40 m² and a maximum depth of 80 cm, located about 50 m from the shore-line at sea level (38°12’45”n, 13°17’30”e). the basin is only filled by rainfall and due to underlying calcareous soil, the climate and the presence of a leak, is subject to frequent drying up. the area has an average rainfall of 680 mm per year and an average temperature of 17 °c (zampino et al., 1997a). the vegetation surrounding this site consists of degraded mediterranean scrub. no other amphibian species are present in the area. ustica is a volcanic island about 9 km² wide located in the southern tyrrhenian sea, 60 km north of palermo (fig. 1). precipitation is scarce, with rainfall averaging 320 mm per year. average temperature is 17.1 °c (zampino et al., 1997b). because of the climate and the permeability of the substratum, a permanent surface hydrological network does not exist on the island. occasional rain water concentrates in some less permeable depressions locally called “gorghi” (buffa and di palma, 1990). our observations concern two of these reproductive sites of b. viridis, the only amphibian species present on ustica island. the first, gorgo salato (38°43’07”n, 13°10’35”e), is a temporary pond with unpredictable hydrology, about 150 m² wide and with a maximum depth of 50 cm. it is about 30 m from the shore-line and about 20 m a.s.l. the other, gorgo san bartolicchio (38°42’00”n, 13°10’21”e), located 109 at 100 m a.s.l., is a concrete basin built in the depression of a natural temporary pond. it fills up partially to reach a maximum surface area of about 100 m² and a maximum depth of 60 cm. the landscape surrounding the water bodies consists of cultivations and grassland. the two ponds are about 3 km apart. sampling the data on the breeding phenology of b. viridis populations of la fossa and ustica island are based on field observations carried out from september 2002 to august 2004. samplings were performed twice weekly, once weekly or fortnightly at la fossa, and fortnightly on ustica island. male calling activity, pairs, eggs, embryos and toadlets were recorded. male calling activity and pairs were observed from sunset to 12 pm over a surface area of about 1500 m2 which included the water bodies and their neighbouring area. the presence of eggs and embryos was detected by means of a hand net during the daytime. the presence of toadlets was checked in both diurnal and nocturnal surveys. on some occasions sampling allowed the timing of metamorphosis to be determined. air and water temperatures (°c) were measured at 10 pm on every nocturnal visit, and information on the hydrological phases of the water bodies was recorded. occasional observations were carried out at the reproductive sites of la fossa and coda di volpe (another concrete basin near palermo, at 310 m a.s.l.) during august, september and october 2005 to detect possible spawning events. results variation in maximum depth of the la fossa basin during the two years of this study can be seen in figs. 2 and 3, which show the hydroperiods and frequent drying up of the basin. the peak in depth shown for the second half of may 2004 was due to management of the nature reserve artificially filling up the basin. reproductive period results for la fossa are shown in table 1. eggs and tadpoles often did not complete development because of drying up of the water body. no toadlets were seen during the first year of study, while during the second year they were observed in octofig. 1. study area. 110 a. sicilia et alii ber 2003 and during june, july and august 2004 only thanks to artificial filling of the water body. in the metamorphosis event observed in october 2003, development took 33 days. a spawning event observed in august 2005 was followed by metamorphosis after 27 days. observations from the ustica sites are shown in tables 2 and 3. as in la fossa, reproductive events in gorgo salato were often not successful due to frequent drying up of the pond. sampling on ustica probably was not frequent enough to detect all spawning events and toadlets, however. fig. 2. variation of maximum depth of la fossa basin during the first year of survey. fig. 3. variation of maximum depth of la fossa basin during the second year of survey. 111 table 1. breeding phenology of bufo viridis at la fossa (palermo). sep. 2002 oct. 2002 nov. 2002 dec. 2002 jan. 2003 feb. 2003 mar. 2003 apr. 2003 may 2003 jun. 2003 jul. 2003 aug. 2003 calling activity x x x x pairs x x x x eggs/embryos x x x x toadlets sep. 2003 oct. 2003 nov. 2003 dec. 2003 jan. 2004 feb. 2004 mar. 2004 apr. 2004 may 2004 june 2004 july 2004 aug. 2004 calling activity x x x x x x pairs x x x x x x x eggs/embryos x x x x toadlets x x x x table 2. breeding phenology of bufo viridis at gorgo salato (ustica island, palermo). sep. 2002 oct. 2002 nov. 2002 dec. 2002 jan. 2003 feb. 2003 mar. 2003 apr. 2003 may 2003 jun. 2003 jul. 2003 aug. 2003 calling activity x x x x pairs x x x x x x eggs/embryos x x x x x x x x toadlets x sep. 2003 oct. 2003 nov. 2003 dec. 2003 jan. 2004 feb. 2004 mar. 2004 apr. 2004 may 2004 jun. 2004 jul. 2004 aug. 2004 calling activity x x x x pairs x x x x x x eggs/embryos x x toadlets table 3. breeding phenology of bufo viridis at gorgo san bartolicchio (ustica island, palermo). sep. 2002 oct. 2002 nov. 2002 dec. 2002 jan. 2003 feb. 2003 mar. 2003 apr. 2003 may 2003 jun. 2003 jul. 2003 aug. 2003 calling activity x x pairs x x x x x eggs/embryos x x x x x x x toadlets x x sep. 2003 oct. 2003 nov. 2003 dec. 2003 jan. 2004 feb. 2004 mar. 2004 apr. 2004 may 2004 jun. 2004 jul. 2004 aug. 2004 calling activity x x x x pairs x x x x x x eggs/embryos x x x x x x x toadlets x 112 a. sicilia et alii in all the study sites, although the toads’ breeding activity occurred for several consecutive months, it was not continuous and characterized by many irregularly distanced spawning events. in some cases, the presence of pairs and male calling activity represented only an attempt at reproduction and were not followed by spawning. the minimum air temperatures at which breeding activity was observed around the reproductive site was 7.6 °c at la fossa and 7.4 °c on ustica; while the maximum values recorded during breeding activity were 29.4 °c at la fossa and 23 °c on ustica. the minimum water temperature at which pairs were observed in the ponds was 6.5 °c on ustica. b. viridis eggs were observed in the coda di volpe basin in september and october 2005. discussion lanza (1983) reported that the reproductive season of bufo viridis in italy ranges from march to august. in a more recent review of data from several italian populations, castellano et al. (2000) report that the breeding period starts between the end of february and the end of march depending on the latitude, and lasts from two weeks at amendolea (province of reggio calabria) in calabria to three and a half months at spotorno (province of savona) in liguria. according to these authors, reproductive activity is contingent on a combination of temperature and rainfall. a study carried out on 163 green toad reproductive sites in calabria (s italy) by tripepi et al. (2000) and other information reported for italian populations confirm this cycle of annual breeding activity (vandoni, 1914; delmastro, 1994; aprea, 1996; lapini et al., 1999; bologna, 2000; farinello and del cengio, 2000; caldonazzi et al., 2002; falcioni and poggiani, 2003; furlani and fiacchini, 2003; bologna and giacoma, 2006). only in lombardy reproduction was occasionally observed at the beginning of september: usually it takes place between april and june (bonini and bressi, 2004). the breeding season of the sicilian populations studied is longer than that observed in other italian populations, covering a maximum of seven non-consecutive months at la fossa during the second year of this study, and nine months on ustica island during the first year. reproductive activity generally took place in all months except july, the least rainy month of the year in the areas studied (zampino et al., 1997a, 1997b); spawning events occurred in all months except june and july. this seems in contrast with castellano et al. (2000), according to whom the activity cycle of b. viridis can be divided into three phases: winter latency, the reproductive period and post-reproductive period. the authors maintain that in italian populations, winter latency terminates between the end of february and the end of march, when the reproductive period begins (castellano et al., 2000). studies on several amphibian populations have shown that they can modify their reproductive patterns in mediterranean environmental conditions. in sub-mediterranean areas, triturus carnifex extends the length of its breeding period (cvetkovic et al., 1996). jakob et al. (2003) investigated reproductive strategies in an amphibian community that breeds in hydrologically unpredictable ponds in southern france and found that some species (pelobates cultripes, pelodytes punctatus, hyla meridionalis, rana perezi) exhibit flexibility in timing their breeding periods, a strategy which the authors hypothesize 113 ensures more reliable breeding success. p. cultripes and p. punctatus were found to breed in autumn as well as in spring: autumn reproduction is known of only in mediterranean populations of these species (salvidio and quero, 1987; sindaco and andreone, 1988; diaz-paniagua, 1992; jakob et al., 2003). hyla arborea, rana ridibunda, r. dalmatina and b. viridis in greece exhibit differences compared to the same species in northern europe in several aspects of their reproductive strategies, especially length of breeding season. for example, the spawning season of b. viridis starts at the end of february or in the first week of march and ends at the beginning of july, lasting for 4.5 months, while in the middle european countries the reproductive period shows the shorter length of 3 months (kyriakopoulou-sklavounou, 2000). mediterranean conditions also affect the length of reproductive period in the studied sicilian populations. xeric environments, unpredictable rainfalls and the absence of severe winter temperatures are probably responsible for the extension of the period of breeding activity of the green toad. this adaptation may be very important to the survival of the populations, especially at la fossa and gorgo salato where toads try to exploit every opportunity to attain successful reproduction. indeed, if the reproductive effort were limited to a short period, the risk of failure would be high, as demonstrated by the continual loss of eggs and tadpoles due to the frequent drying up of the water bodies. it is known that amphibians, particularly toads, can successfully inhabit dry environments characterized by the intermittent availability of water. williams (1987) reported general adaptations of amphibians to dry areas to be: the use of temporary water bodies for reproduction, an indefinite breeding season, breeding behaviour cued by rainfall and rapid development of eggs and tadpoles; adaptations all shown by the populations of b. viridis we studied. development from eggs to toadlets takes between one and a half to three months in previously observed italian populations (lanza, 1983; aprea, 1996; lapini et al., 1999; tripepi et al., 1999; farinello and del cengio, 2000; falcioni and poggiani, 2003; furlani and fiacchini, 2003; bonini and bressi, 2004), whereas at la fossa we recorded a time of 27 days. interestingly, a similarly short metamorphosis (21.1 to 31.8 days) was recorded as an adaptation to desert environments in temporary ponds in egypt (hussein and darwish, 2000). however, it is important to note that the rate of larval development can be also affected by other environmental factors, such as temperature, scarce food and high population density (hussein and darwish, 2000). data reported for green toad populations in other dry environments show a shorter breeding season than in the sicilian populations reported here; and there are no records of an autumn reproduction period. in israel, b. viridis spawning mainly occurs from early february to may (blaustein and margalit, 1995; degani and kaplan, 1999). in north africa mating lasts from the end of the winter to may (schleich et al., 1996; hussein and darwish, 2000). mertens (1929) concluded that there were two mating seasons (spring and late summer) on djerba island (central-southern tunisia) (mertens, 1929 in: schleich et al., 1996). in reality, knowledge about the reproductive behaviour of the green toad in dry environments is probably incomplete and further studies are required: in another investigation, one of the authors of this study found tadpoles of b. viridis at stage 25 (according to development stage tables of gosner, 1960) at the end of december 2004 in a sebkha on the extremely arid kerkennah islands (tunisia). observations carried out from 2004 to 2006 on the hydroperiod of this temporary sebkha suggest that the tadpoles observed in 114 a. sicilia et alii 2004 were born from spawnings occurred in a period comprised between the autumn and the beginning of the winter season (a. sicilia, pers. obs.). it seems that not all sicilian populations of b. viridis prolong their breeding season, or at least not every year. in the gorgo di santa rosalia, another b. viridis breeding site located near palermo (at 458 m a.s.l.), the 2000 and 2001 reproductive seasons started at the end of january and lasted 41 and 24 days respectively (sicilia, 2006). this pond is a temporary water body which dries up only during summer months. in the same years spawning events, at la fossa (table 4), were recorded from january to may and during october and november (in 2000); and from february to may (in 2001). it is also worthy of note that the b. viridis reproductive period at la fossa was shorter in 2001 than in other years and there was no autumn reproduction period (sicilia, 2006). evidence of environmental variability also comes from the differences observed in reproductive patterns between sicilian and southern italian populations, e.g. those found in calabria and apulia (tripepi et al., 2000; frisenda, 2002). it is not clear whether this difference is due to lack of data or to different environmental conditions, although the latter is likely, given that in calabria b. viridis prefers medium and large size water bodies such as rivers and lakes (tripepi et al., 2000). to conclude, this study confirms that this taxon is an opportunistic breeder with highly variable annual reproductive cycle patterns. in the populations studied in sicily, the duration of the reproductive season varied between populations in the same year and between different years for the same population. acknowledgements the authors are grateful to the authorities of the natural reserves of capo gallo and ustica. they also wish to thank agostino d’amico, mariagrazia graziano, federico marrone and carlo violani for the help they gave to the carrying out of this research. references aprea, g. 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(1987): the ecology of temporary waters. croom helm, london & sydney. the numerical encoding of scale morphology highly improves photographic identification in lizards roberto sacchi1, stefano scali2, mauro fasola1, paolo galeotti 1 1dipartimento di biologia animale, università di pavia, piazza botta 9, i-27100 pavia, italy. corresponding author. e-mail: roberto.sacchi@unipv.it 2museo civico di storia naturale, c.so venezia 55, i-20121 milano, italy abstract. photographic identification is a promising marking technique alternative to the toe-clipping, but is time consuming, particularly when a large number of individuals is involved. for this reason several authors had frequently preferred the toe-clipping. in this study we analysed the black spot pattern of ventral scales of wall lizards (podarcis muralis) and we showed that photographic identification is an effective method for recognizing individuals, and the error of this technique is much less than that of the toe-clipping arising from natural toe loss. moreover, the numerically encoding of the black spot pattern may radically reduce the time needed to compare the pictures of large samples of individuals, solving one of the more important obstacle against the use of photographic identification. keywords. individual marking, photographic identification, non invasive marking technique, toe-clipping. introduction many types of ecological studies need the unique identification of individuals, which is usually achieved by marking. general methods used for vertebrates such as ringing, tattooing, and banding are difficult to use with reptiles because of their anatomy and skin shedding (spellerberg and prestt, 1978; dunham et al., 1994). up to now the most common method of marking lizards and skinks used has been the toe-clipping, in which a unique combination of digits is removed from each individual (ferner, 1979; hero, 1989; waichman, 1992). recently, the effectiveness of this method has been questioned, since the base assumption that toe-clipping does not influence the survival or behaviours has been shown to be frequently violated (parris and mccarthy, 2001; mccarthy and parris, 2004). indeed, the toe-clipping may cause inflammation, infection of feet and limbs, reduced mobility, increased mortality (bustard, 1968; 1971; clarke, 1972; humphries, 1979; golay acta herpetologica 2(1): 27-35, 2007 28 r. sacchi et alii and durrer, 1994; lemckert, 1996; reaser and dexter, 1996; williamson and bull, 1996; davies and ovaska, 2001; bloch and irschick, 2004), potentially affecting the quality of the data collected during field researches (mccarthy and parris, 2004; bell and pledger, 2005). moreover, natural toe loss is common enough in some species of skinks and lizards to potentially cause difficulties with possible misidentifications of individuals marked by toe-clipping (rand, 1965; schoener and schoener, 1980; middelburg and strijbosch, 1988; hudson, 1996). by contrast, several other studies have found no negative effects of toeclipping (huey et al., 1990; dodd, 1993; van gelder and strijbosch, 1996; hudson, 1996; williamson and bull, 1996; ott and scott, 1999; paulissen and meyer, 2000; borges-landaez and shine, 2003), suggesting that the effects of this technique may vary among species and must be assessed accordingly (funk et al., 2005). irrespectively to the negative effects on individuals, there are also ethical and conservation implications that lead to consider toe-clipping with caution as a marking technique (mccarthy and parris, 2004). in this scenario, several non-invasive marking methods alternative to the toe-clipping have been proposed, such the small passive integrated trasponders (pit, elbin and burger, 1994), the visible implant elastomers (vie, penney et al., 2001), or the freeze-branding (spellerberg and prestt, 1978). the photographic identification is an emergent technique with a promising future for marking lizards, since it is completely harmless, cheap, and allows in theory long time identification of individuals (fox, 1975; gosá, 1987; elbing and rykena, 1996; schmidtloske, 1996; steinicke et al., 2000; perera and perez-mellado, 2004). this approach bases on the identification of regular and individually specific patterns of colour spots or scale shape within well identified body regions of individuals, which do not change over time despite skin moults. for example, the pattern of head scales of the lacerta bilineata is unique within individual, and do not vary over time (fox, 1975; elbing and rykena, 1996), while the scale pattern of the first four rows of the ventrals is suitable for recognizing individual of six species of lacertids (lacerta agilis, l. bilineata, l. viridis, l. perspicillata, zootoca vivipara and podarcis muralis) (steinicke et al., 2000; perera and perezmellado, 2004). colour spot patterns have been also used to individually identify lizards (schmidt-loske, 1996), but spot shape may vary with reproductive condition or age, being less useful for long-term identification (henle et al., 1997). although regular patterns of colour spots and scale shape may supply a useful way to individually recognize lizards, photographic identification is a time consuming technique, particularly when a large number of individuals is involved, since the number of pairedcomparisons for each picture increases exponentially according to the sample size. for this reason, the method must be improved to reduce the time and/or the number of comparisons required for identification. the common wall lizard p. muralis is a good model for testing the effectiveness of photographic identification since it has easily recognizable individual scale shape patterns (schmidt-loske, 1996; steinicke et al., 2000) and shows black spot patterns within ventral scales that are highly variable, particularly among males. in this study we therefore tested the suitability of this ventral pattern of black spots to be used for identifying males, and we proposed a new method for numerically coding the spot pattern in order to minimize the time needed to identify a given individual. 29photographic identification in lizards materials and methods during spring-summer 2004 and 2005 we overall made 235 captures and recaptures of male common wall lizards in an historical garden of cesano maderno (northern italy, 45°38’n – 9°07’e): 41 (35 individuals) were made during the first year and 194 (42 individuals) in the second one. in both years, all lizards were individually marked on the back by a unique combination of coloured inks, photographed ventrally using a nikon coolpix 4300 (resolution 2048 × 1536 pixels), and released. we obtained 6 recaptures (3 individuals) in 2004 and 152 recaptures (33 individuals) in 2005 correctly recognized on the basis of the colour marks on the back; all recaptured lizards were photographed. the ventral pattern of black spots of each lizard was numerically encoded using the following procedure: the shapes of the black spots of the 4 scales of the first 10 ventral rows (overall 40 scales) were classified from 0 to 63 according to the code reported in figure 1, which resumes all possible shapes from an unspotted scale (code = 0) to a completely black scale (code = 63). by this procedure each individual (as well as each picture) was univocally paired to a numerical string of 40 features that could be easily compared with the strings of all other lizards using a worksheet software, such as microsoft excel. we referred to the “code distance” (cd) as the number of differences between the correspondent features of two codes, which therefore varied between 0 (i.e. the codes are the same) and 40 (i.e. all the paired-features of the two codes are different). in order to assess a cd threshold to ascertain if two codes belong to the same lizard or not, the pictures of 10 different males were encoded twice by the same observer and significant differences between the mean cd of each male with all others (cdamong) and the cd of each male with its replicate (cdwithin) were assessed by a one-way anova. then, we used a kernel estimation (bandwidth = 8.40) to compute the probability density curve of the cds and we computed the cd threshold from the graphic. three main sources of error may arise in analysing the pictures: encoding errors, seasonal changes of black spot’s shape, and variability among observers. the first source of error was assessed by verifying that a picture was paired to a unique and repeatable code: the same observer replicated the measures of the cdamong and cdwithin of the previous analysis with a one-day interval, and we evaluated the repeatability of the cd measures (lessels and boag, 1987). in order to analyse the second source of error, we assessed the repeatability of the pictures by encoding two different pictures from 10 males with one-day interval between two successive analysis of the same picture; the observer was the same as previously. finally, in order to assess the effects of the third source of error (variability among the observers), three observers computed the cdamong and cdwithin of the same 10 different males, and significant differences among observers were checked using a mixed analysis of variance where cds were included as the dependent variable, the observer was included as random factor while the type of comparison (among or within individuals) was included as factors; the effect of the interaction between observer and type of comparison was also incorporated into the model. in order to quantify the percentage of error (number of misclassification/number of individuals) of this marking procedure, we compared the numerical codes of all recaptures of colour marked males with the numerical codes of all first captures using a worksheet in excel and we considered as true identifications all pairs of individuals differing less o equal to the cd-threshold. for this analysis we used the sample of 42 first captures and 152 recaptures collected during 2005, and consequently we performed 6384 paired-comparisons. finally, we applied the same procedure to recognize between-year recaptures, comparing all the 35 first captures of males in 2004 with all 194 captures (first captures and recaptures) in 2005; in this case the sample involved 6790 paired comparisons. for this analysis we lacked independent validation (we did not intentionally use toe-clipping, and painting with coloured inks do not persist over successive years), so all pairs of pictures whose codes differed less or equal to the cd-threshold were visually compared using the scale shape patterns (schmidt-loske, 1996; steinicke et al., 2000). all statistics 30 r. sacchi et alii were two-tailed, and were performed with spss 12.0. when necessary, normal distribution and homogeneity of variances was verified. unless otherwise stated, values reported are means ± se. results the cd among different males was on average 35.8 ± 0.6, while the cd between two replicates of the picture of the same individual was 2.9 ± 0.5; this difference was highly significant (f1,18 = 1762, p < 0.0001) and resulted in a cd-threshold of 19 differences (fig. 2). the code of a single picture was highly repeatable (r = 0.98), as well as the pictures of the same individual (r = 0.92). these results suggested that the code coupled with the black spot pattern of the ventral scales univocally identified each male, was unaffected by the quality of the picture, and was univocally recognizable in different pictures of the same individual. the effect of the observer encoding the ventral pattern of black spots was also negligible (fig. 3), since the cdamong was larger than the cdwithin for all observers (f1,2 = 470, p = 0.002), and both -among and -within cds did not differ among them (f2,2 = 2.68, p = 0.27). however, the interaction between the observer and the type of comparison was near to the significant threshold (f2,60 = 2.92, p = 0.06), suggesting that the third observer valued the cdwithin a little higher than that measured by other people, the cdsamong matching perfectly (fig. 3). fig. 1. numerical codes used for encoding of the black spot pattern of ventral scales of wall lizards. 31photographic identification in lizards fig. 2. probability density distribution (kernel procedure) of the cds in the sample of 10 males of wall lizards (see methods for details). fig. 3. cds among individuals and within the replicates of the same individual measured by three different observers. 32 r. sacchi et alii the 96.7% (147 out of 152) of the colour-marked recaptures and 97.0% of the recaptured individuals (32 out of 33 males) were correctly identified basing on the code of the ventral spot pattern. basing on our procedure (cd less or equal to 19), 11 males out of the 35 captures during 2004 were identified as recaptures in 2005, and the visual comparisons of the shape pattern of the first four rows of the ventrals confirmed the identification in 7 cases (64%). all misclassifications had cds varying between 18 and 19, and raised from the higher number of completely unspotted scales that exceeded 50% in all individuals (i.e. the mean number of zeros in the codes of these four misclassified males was on average 22 ± 0.7). discussion this study confirms that photographic identification is a useful marking technique for lizards (steinicke et al., 2000; perera and perez-mellado, 2004), and can be considered an effective alternative to the toe-clipping in ventrally pigmented lizards. indeed, we showed that the mismatching of photographic identification of wall lizards basing on the black ventral spot pattern was very low and much less than the error intrinsic to the toeclipping technique arising from natural toe loss: in our study we failed the recognition of only 4 out 152 recaptures (3.3%) and one out 33 individuals (3.0%). by contrast, hudson (1996) in his study on 12 australian skink species showed that 19% of females (83 out 445) naturally lost toes, and in some populations this feature increased to more than 30%. ontogenic changes of pigmentation occurring in this species (gosà, 1987; henle et al., 1997) did not significantly affected the encoding procedure within a single breeding season, but might reduce it applicability in long term studies. however, the effects of black spot changes might be easily controlled by reducing the time intervals between two successive pictures. the photographic identification technique described in this study allowed also the effective recognition of individuals over successive years, despite the fact that 26% of between-year recaptures resulting from our procedure (4 out of 11) were not confirmed by the direct comparisons of pictures. indeed, all these misclassifications involved males having more than 50% of ventral scales that completely lacked black spots, and an high proportion of zeros obviously increases the probability for given code of matching the codes of other individuals. moreover, in all these cases the cd was very close to the cd threshold for true recognition, suggesting that this nuisance may be easily removed by increasing the number of scale rows to determine. the most important limit to the application of the photographic identification up to now has been the large amount of time needed to compare the pictures of a given sample, which raises exponentially as the number of individuals involved increases. this objection was the main factor that leaded several authors to prefer the toe-clipping as marking technique because it had been considered the most economical and practical method for long term studies among all other current methods (i.e. ott and scott, 1999). in this study we show that the translation of the ventral spot pattern of male wall lizards into a simple numeric code dramatically reduces the time needed to compare a large sample 33photographic identification in lizards of pictures. indeed, we would have performed more than 6000 paired-comparisons for identifying recaptures in the sample of pictures collected during 2005 without this encoding procedure. the time required to encode a picture was less than one minute, while the comparison of codes using a worksheet software is practically instantaneous. this leads us to obtain a complete identification of all individuals of the sample in less than one day. this procedure may be generalized, and other kind of colour spots or scale shape pattern of lizard species may be numeric encoded to immediately identify individuals or, at least, to easily find a very restricted sub-sample of pictures to be visually compared. however, in long term monitoring programs that involve large samples of individuals photographic identification remains a marking technique that does not allow immediate recognition of individuals at the time of their capture, but only after a process of analysis of images. despite this, photographic identification is undoubtedly the less invasive technique available today, and would be preferred for all researches involving the measure of physiological variables, such hormone levels, that greatly varied in response to both stress and injuries. in conclusion, the numeric encoding of individual pattern associated with digital cameras and image processing software radically reduce time consuming of photographic identification, which can be considered a fully alternative method to toe-clipping. acknowledgments we thank adriano trento, roberta talamona, alessandra binda, carlo zucchi, and luca cavigioli for help during field work. we also thank fabio pupin for his helpful suggestions to a previous version of the manuscript. references bell, b.b., pledger, s. (2005): does toe clipping affect the return rates of the terrestrial frog leiopelma pakeka on maud island, new zealand? new zeal. j. zool. 32: 219-220. bloch, n., irschick, d.j. (2004): toe-clipping dramatically reduces clinging performance in a pad-bearing lizard (anolis carolinensis). j. herpetol. 37: 293-298. borges-landaez, p.a., shine, r. (2003): influence of toe-clipping on running speed in eulamprus quoyii, an australian scincid lizard. j. herpetol. 37: 592-595. bustard, r.h. (1968): the ecology of the australian gecko, gehyra variegata, in northern new south wales. j. zool. (london) 154: 113-138. bustard, r.h. (1971): a population study of the eyed gecko, oedura ocellata boulenger, in northern new south wales, australia. copeia 1971: 658-669. clarke, r.d. (1972): the effect of toe-clipping on survival in fowler’s toad (bufo woodhousei fowleri). copeia 1972: 182-185. davis, t.m., ovaska, k. 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(1996): rana pretiosa (spotted frog). toe clipping effects. herpetol. rev. 27: 275-289. schmidt-loske, k. (1996): fotografische identification von podarcis muralis laur., 1768. möglichkeiten und grezen. die eidechse 7: 7-12. schoener, t.w., schoener, a. (1980): ecological and demographic correlates of injury rate in some bahamian anolis lizards. copeia 1980: 839-850. spellerberg, i.f., prestt, i. (1978): marking snakes. in: animal marking: recognition marking of animals in research, p. 133-141. storehouse, b., ed., mc millian press, london. steinicke, h., ulbrich, k., henle, k., grosse, w.r. (2000): eine neue methode zur fotografischen individualidentifikation mittelerupäischer halsbandeidechsen (lacertidae). salamandra 36: 81-88. van gelder, j.j. v., strijbosch, h. (1996): marking amphibians: effects of toe clipping on bufo bufo anura: bufonidae. amphibia-reptilia 17: 169-174. waichman, a.v. 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(1996): population ecology of the common frog crinia signifera: adults and juveniles. wild. res. 23: 249-266. acta herpetologica 18(1): 3-9, 2023 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-13279 threats of the emerging pathogen batrachochytrium salamandrivorans (bsal) to italian wild salamander populations lorenzo dondero1, giorgia allaria1, giacomo rosa1, andrea costa1, gentile francesco ficetola2, roberto cogoni3, elena grasselli1, sebastiano salvidio1,* 1 department of earth, environment and life sciences (distav), university of genova, corso europa 26, i-16132 genova, italy 2 department of environmental science and policy, university of milan, via celoria 10, 20133 milan, italy 3 unione speleologica cagliaritana, via scarlatti 11, i-09045 quartu st elena, italy *corresponding author. email: sebastiano.salvidio@unige.it submitted on: 2022, 23rd june; revised on: 2022, 13th november; accepted on: 2022, 23rd november editor: ilaria bernabò abstract. the salamander killing fungus batrachochytrium salamandrivorans (bsal), recently introduced from asia, is threatening salamander populations in different parts of europe. in fact, this pathogen is rapidly spreading in central europe and has been also introduced into ne spain. of special concern are those regions with an exceptionally high salamander diversity such as italy, where 19 salamander species are present most of which are strictly endemic. in this study, we update the information on the presence of bsal in italian wild salamanders, by adding samples from two presumptive outbreak sites, one on the island of sardinia and one on continental italy (liguria). in addition, we reviewed the potential susceptibility of all the italian salamander species on the basis of laboratory experimental infection trials, or from the tested susceptibility of the phylogenetically nearest species, according to the literature. overall, 15 skin swabs from three species (speleomantes sarrabusensis, speleomantes strinatii and salamandra salamandra) collected in the two presumptive bsal outbreak sites were analysed by quantitative molecular methods, but none gave positive results. the majority of italian salamander species and almost all of the endemic ones showed a high susceptibility to bsal infection. therefore, even if the presence of bsal in italian salamander populations has not been proven yet, the entire salamander fauna is highly threatened and preventive management actions should be taken. the need for strict biosecurity protocols on the international trade of captive or wild amphibians and for the implementation of preventive measures during field activities to limit the introduction and spread of the bsal pathogen is again stressed. keywords. amphibians, biosecurity, chytridiomycosis, conservation, emerging infectious diseases, mitigation, realtime pcr. introduction the chytrid fungi batrachochytrium dendrobatidis (bd) and batrachochytrium salamandrivorans (bsal) are amphibian pathogens that were introduced in europe from east asia, probably through the international trade (o’hanlon et al., 2018; laking et al., 2019). the continuous spreading of these emerging pathogens is now threatening wild amphibian populations in different parts of europe (scheele et al., 2019; bosch et al., 2021). in particular, the salamander killing fungus bsal is a real threat to european salamanders, because many species are extremely susceptible to this infection and, therefore, highly vulnerable (martel et al., 2014). the pathogenic effect of bsal is caused by its zoosporangia that penetrate the skin cells of adult salamanders, breaking innate immunological defences, and producing tissue erosions and deep ulcerations that may disrupt the host infected 4 lorenzo dondero et alii skin respiratory and rehydration functions (martel et al., 2013; grogan et al., 2020). in this way, bsal infections are capable of causing severe illness or even death of the infected amphibian hosts (martel et al., 2013, 2014). in fact, bsal is constantly expanding from the netherlands, where it was first detected and where it caused a population collapse in the local fire salamander salamandra salamandra population (martel et al., 2013). currently, bsal has been confirmed in about 80 localities surrounding bunderbos, in the netherlands, where it was first detected (martel et al., 2013), and also in belgium and germany (e.g., spitzen-van der sluijs, 2016; lötters et al, 2020; schmeller et al., 2020; thein et al., 2020). bsal has also been detected in 2018 in catalonia (northeastern spain), where it has infected the marbled newt triturus marmoratus and caused a local mass mortality event of this species (martel et al., 2020), while its presence in north central spain has not been yet confirmed (bosch et al., 2021). therefore, to date, the presence of bsal in europe has been confirmed in the wild in four countries: the netherlands, belgium, germany and spain. the emergence and the diffusion of b. salamandrivorans through europe, apparently facilitated by humans transferring and introducing in the wild environment captive amphibians, is worrying and requests urgent prevention measures and monitoring actions to prevent further loss of amphibian diversity (thomas et al., 2019). in particular, the high salamander diversity of the southern european peninsulas, such as italy and spain, appears at risk. italy alone hosts 19 species of urodeles (sindaco and razzetti, 2021), many of which are endemic, such as the entire genus salamandrina, the italian newt lissotriton italicus, the sardinian brook newt euproctus platycephalus, and seven species of cave salamanders belonging to the genus speleomantes (sindaco and razzetti, 2021). however, to date, only two studies screened bsal in italian salamanders (grasselli et al., 2019, 2021). these studies analysed by means of real-time pcr (qpcr) 136 skin swabs from 6 species of italian wild salamanders and 53 from non-native individuals that were bred in private collections (grasselli et al., 2019). the results from these studies were that none of the 189 italian salamander swabs gave positive results. however, recent events raised concerns on the possibility of bsal outbreaks in italian populations. in the summer of 2021, a mass mortality of speleomantes sarrabusensis was observed in sardinia by the wildlife photographer emanuele biggi while, more recently (in spring 2022), a living fire salamander bearing skin lesions from liguria was photographed by michael fahrbach and reported to the authors by frank pasmans from ghent university. these observations were compatible with the suspected infection of bsal on italian amphibians but needed robust confirmation by molecular, and histological methods or by both. therefore, the aim of this study was twofold: i) to expand the current knowledge about the presence of bsal in wild salamander populations in italy, adding the molecular data obtained from new skin samples collected from the two bsal-suspected outbreaks and ii) to assess the susceptibility to bsal of all italian salamanders, on the basis of published experimental studies or, in the absence of experimental evidence, from their phylogenetic affinities. this information will be needed to better plan bsal mitigation actions and also to guide strategic conservation and management efforts on the national territory of italy. materials and methods origin of skin swab samples, dna extraction and quantitative pcr we obtained skin swabs from two areas, one in southern sardinia and one in nw italy. in southern sardinia (monte sette fratelli), we sampled a protected underground site that hosts the largest known population of speleomantes sarrabusensis. this site is closed by a gate and only an authorised person has access, however, in this location several dead cave salamanders were observed from july to august 2021, by different herpetologists. skin swabs were collected a few days after the first mortality record (august 2021) from all the living s. sarrabusensis individuals (two individuals), plus one recently dead individual; a fourth swab was obtained from a hyla sarda corpse found at the same site (supplementary material, fig. s1). in the province of genova (liguria, nw italy), we collected twelve swabs in april and may 2022 along a small stream or in an adjacent artificial cave from living fire salamanders (salamandra salamandra) and cave salamanders (speleomantes strinatii) where an individual of salamandra salamandra with suspect skin lesions was photographed (supplementary material, fig. s1). in both sites, all skin swabs were obtained with a standardised protocol used in previous bd and bsal studies on italian salamanders (costa et al., 2021; grasselli et al., 2019, 2021). sterile cotton swabs were rubbed 30 times on the skin of different parts of the salamander’ body and were preserved in individual sterile plastic tubes at 4 °c until extraction (spitzen-van der sluijs et al., 2016). dna was extracted in 200 μl of prepman ultra (thermo fisher scientific technologies, monza, italy). samples were then analysed for bd and bsal dna using a duplex qpcr, targeting the its1 rrna gene of bd and 5.8s rrna gene of bsal, as described by blooi et al. (2013). all samples 5bsal threats to italian salamanders were run in duplicate, together with standard curves obtained from suspensions of known numbers of bd and bsal zoospores (kindly provided by an martel and frank pasmans) in the same plate, and results were expressed in genome equivalents (ges) according to thomas et al. (2018). assessing italian salamanders’ bsal susceptibility the susceptibility of italian salamanders to bsal infection was estimated by collecting all the published data on laboratory experimental infection trials on the same salamander species. in absence of such experimental evidence, the susceptibility was inferred from the phylogenetically nearest species, adopting a precautionary approach and thus selecting the more threatening outcome. species susceptibility was estimated as “high” if the experimental infection had a lethal outcome, “moderate” if the gravity of infection was dose-dependent and “low” if the infected salamander was able to clear infection or remained asymptomatic. moreover, we used the results of beukema et al. (2018, table 2) as an index of potential niche overlap between bsal and italian salamanders. beukema et al. (2018) estimated both native and invasive ecological niches of bsal and of all native european salamanders using three different ordination methods (see material and methods in beukema et al., 2018). the statistical overlap between both bsal native and invasive ecological niches and the current niche of italian salamanders was then calculated (beukema et al., 2018). in the present study, we counted all significant overlaps between italian salamanders and bsal native and invasive niche. therefore, there were several possible outcomes per species, ranging from 0/6 if all overlaps were non-significant, to 6/6 if all the six niches overlapped significantly. this “overlap score” may be used as a gross indication of the environmental compatibility between each italian species and the salamander killing fungus bsal. in this study, we used species nomenclature following the most recent checklist of italian herpetofauna (sindaco and razzetti, 2021), and we revised the available data on the 19 native italian species. we also report information for one subspecies of alpine salamander (salamandra atra aurorae), that is listed as a priority taxon in annex ii of the european union (eu) directive “habitats” 92/43/ cee. according to art. 1 letter (h) of this directive, italy has a particular responsibility for the conservation of this endemic taxon that shows an extremely small distributional range limited to the eastern alps. results dna testing we analysed one skin swab from hyla sarda, three speleomantes sarrabusensis, five speleomantes strinatii and seven salamandra salamandra. none of these swabs gave bd or bsal-positive results. therefore, the possible bsal infection in the sardinian (prevalence 0; 95% confident limits 0.00-0.60), and ligurian (prevalence 0; 95% confident limits 0.00-0.30) sites were not confirmed, but given the very small samples analysed, a high level of uncertainty concerning these results remains. to date, the number of italian wild salamanders screened for bsal is 151, obtained from nine species (table 1). bsal susceptibility ten of the nineteen (53%) salamander species found in italy were infected experimentally in laboratory trials with bsal, thus providing direct evidence of their potential susceptibility to the pathogen (table 2). in the case of the cave salamanders (genus speleomantes), three species were infected in laboratory experiments by martel et al. (2014): s. genei, s. strinatii and s. imperialis. the two former species resulted highly susceptible (i.e., bsal infection was lethal), while the latter was able to clear the infection, thus showing low susceptibility. in this study, following the precautionary approach, we considered all non-tested speleomantes species to be highly susceptible, as already done by gilbert et al. (2020). overall, the majority of italian salamander species, 15 out of 19 (79%), were shown or inferred to possess table 1. italian salamanders tested for batrachochytrium salamandrivorans. a and b indicate presence of samples from the nonconfirmed bsal outbreaks in liguria (2022) and sardinia (2021), respectively. this table is an expanded version of grasselli et al. (2019). species or subspecies sample (n) bd positive bsal positive euproctus platycephalus 3 0 0 ichthyosaura alpestris 76 1 0 lissotriton italicus 22 0 0 salamandra atra aurorae 3 0 0 salamandra salamandraa 11 0 0 salamandrina terdigitata 14 0 0 speleomantes sarrabusensisb 3 0 0 speleomantes strinatiia 5 0 0 triturus carnifex 14 3 0 total 151 4 0 6 lorenzo dondero et alii high susceptibility to bsal infection, while two (11%) had moderate and two (11%) had low susceptibility (table 2). when focusing on endemic salamanders, 10 out of 11 (91%) have, or were inferred to have, high susceptibility, the only exception being s. imperialis (table 2). finally, only four species showed no niche overlap with bsal pathogen. three of them were salamanders found exclusively in the mediterranean bioclimatic region (i.e., lissotriton italicus, speleomantes flavus and speleomantes supramontis), while the fourth one (salamandra lanzai) lives at high altitudes in the alps. on the other hand, four species showed a relatively high overall niche overlap score with bsal: ichthyosaura alpestris, speleomantes ambrosii, speleomantes strinatii and triturus carnifex. the two latter also had a high experimental susceptibility towards the infection with the pathogen (martel et al., 2014). discussion the diffusion of bsal infection is a dramatic threat to salamander diversity in central europe and spain (martel et al., 2014; bosch et al., 2021), and recently two potential outbreak sites have been recorded in italy. however, none of our qpcr samples from these sites provided positive results. clearly, these partial results cannot exclude the presence of bsal-infected individuals in the studied sites or elsewhere, because of the very small sample size tested. the mass mortality event reported in sardinia is particularly concerning, because the cave salamander population lives in a high-altitude protected site, where environmental stressors should be absent. therefore, local authorities in charge of the site management were immediately alerted and the water quality inside the site is monitored to report any further critical condition and to report further changes as well as to mitigate a possible spread of any kind of pathogen, as indicated by thomas et al. (2019). in the ligurian site, no mass mortality was observed, and a monitoring programme is regularly taking place as a preventive mitigation measure (salvidio, unpublished data). our review of species susceptibility already conducted by beukema et al. (2018) and gilbert et al. (2020) shows that the great majority of italian salamanders, and in particular many endemic taxa, are potentially highly vulnerable to bsal infection. indeed, the endemic genus salamandrina and the many species belonging to the genus speleomantes have been experimentally proven highly vulnerable to this infection. in addition, all italtable 2. susceptibility of italian salamander species to bsal infection, according to the literature or inferred from phylogenetic related species. bsal susceptibility was updated from gilbert et al. (2020). the overlap score was calculated from table 2 of beukema et al. (2018). * not reported in beukema et al. (2018). species italian endemic iucn status habitat directive bsal susceptibility comments reference overlap score euproctus platycephalus yes en iv high lethal, laboratory tested martel et al., 2014 1/6 ichthyosaura alpestris no lc -moderate dose-dependent laboratory tested martel et al., 2014 3/6 lissotriton italicus yes lc iv high lethal, laboratory tested martel et al., 2014 0/6 lissotriton vulgaris no lc -moderate dose-dependent, laboratory tested bates et al., 2019 1/6 proteus anguinus no vu ii/iv low asymptomatic, laboratory tested li et al., 2020 2/6 salamandra atra no lc iv high inferred from congeneric species 2/6 salamandra atra aurorae yes ii*/iv high inferred from congeneric species -* salamandra lanzai no vu iv high inferred from congeneric species 0/6 salamandra salamandra no lc -high lethal, laboratory tested martel et al., 2013, 2014 2/6 salamandrina perspicillata yes lc ii/iv high lethal, laboratory tested martel et al., 2014 1/6 salamandrina terdigitata yes lc ii/iv high inferred from congeneric species 1/6 speleomantes ambrosi yes nt ii/iv high inferred from congeneric species 3/6 speleomantes flavus yes vu ii/iv high inferred from congeneric species 0/6 speleomantes genei yes vu ii/iv high lethal, laboratory tested martel et al., 2014 1/6 speleomantes imperialis yes nt ii/iv low clears infection, laboratory tested martel et al., 2014 1/6 speleomantes italicus yes nt ii/iv high inferred from congeneric species martel et al., 2014 1/6 speleomantes sarrabusensis yes vu ii/iv high inferred from congeneric species martel et al., 2014 1/6 speleomantes strinatii no nt ii/iv high lethal, laboratory tested martel et al., 2014 3/6 speleomantes supramontis yes en ii/iv high inferred from congeneric species 0/6 triturus carnifex no lc ii/iv high inferred from congeneric species 3/6 7bsal threats to italian salamanders ian species with few exceptions share a relatively high ecological niche overlap score with bsal (beukema et al., 2018 synthetized in table 2 of this study), suggesting that, even in the mediterranean region, salamanders inhabiting humid and cool microhabitats, such as speleomantes strinatii and triturus carnifex are exposed to a high risk. concerning the eu priority micro-endemic subspecies of alpine salamander, salamandra atra aurorae, beukema et al. (2018) did not calculate niche overlap, while a high susceptibility to bsal was inferred from the experimental data on other salamandra subspecies (gilbert et al., 2010). given the high conservation concern of this taxon possessing a restricted range of occurrence (romano et al., 2018), a demographic monitoring project has been implemented by the local province of trento starting in 2017, while skin swabs have been collected from different sites in august 2022 (romano, pers. com. september 2022). active disease surveillance and large-scale monitoring of populations of the most threatened species should be the ideal preventive strategy, but the costs associated with these activities would be clearly prohibitive (bosch et al., 2021). therefore, a more cost-effective policy should integrate passive reporting and intervention in the cases of confirmed mass mortality events combined with strong preventive measures, as already suggested by thomas et al. (2019). these measures should include testing for bsal throughout the amphibian international commerce to exclude the unintentional introduction of fungal pathogens from captive amphibians legally traded. moreover, controlling and eradicating invasive populations of alien amphibians that could act as bsal intermediate hosts should be considered and realised, when possible. these preventive eradications should be undertaken especially for species that act as intermediate hosts and are associated with the spread of amphibian chytrid pathogens, such as the african clawed frog xenopus laevis and the north american bullfrog lithobates catesbeianus (pasmans et al., 2017). moreover, it is pivotal that professional and amateur herpetologists always implement strict biosecurity measures during field activities, to avoid the spread of the pathogen across distant study areas, and the existence of online information about biosecurity precautions, such as http://bsaleurope.com/, should be better advertised. finally, herpetologists are not the only potential drivers of the spread of these pathogens, because unfortunately they can be spread by any kind of people performing outdoor activities. a broad dissemination of the issues of wildlife pathogens, and of the protocols to limit them, remains a fundamental preventive action (e.g., http://www-9.unipv.it/webshi/images/files/all.%20ii%20 -%20chitridiomicosi.pdf; http://bsaleurope.com/). acknowledgments we thank an martel and frank pasmans for providing dna standards and continuous support to our laboratory, emanuele biggi and alessandro spiga for reporting about the mass mortality in sardinia. permits to swab salamanders were provided by the italian ministry of ecological transition (0039130 of 15/04/2022). the comments and suggestions of giulia tessa and of an anonymous reviewer greatly improved this manuscript. supplementary material supplementary material associated with this article can be found at <http://www-262 9.unipv.it/webshi/ appendix/index.html> manuscript number 13279 references bates, k., shelton, j.m.g., mercier, v.l., hopkins, k.p., harrison, x.a., petrovan, s.o., fisher, m.c. 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(2019) mitigating batrachochytrium salamandrivorans in europe. amphibia-reptilia 40: 265-290. threats of the emerging pathogen batrachochytrium salamandrivorans (bsal) to italian wild salamander populations lorenzo dondero1, giorgia allaria1, giacomo rosa1, andrea costa1, gentile francesco ficetola2, roberto cogoni3, elena grasselli1, sebastiano salvidio1,* age estimation and body size of the parsley frog, pelodytes caucasicus boulenger, 1896 from lake borçka karagöl, turkey cantekin dursun*, serkan gül, nurhayat özdemir patterns of acoustic phenology in an anuran assemblage of the yungas andean forests of argentina martín boullhesen1,2,*, marcos vaira1, rubén marcos barquez2, mauricio sebastián akmentins1 diet and trophic niche overlap of four syntopic species of physalaemus (anura: leptodactylidae) in southern brazil renata k. farina1, camila f. moser2, stefano scali3, mateus de oliveira4, patrícia witt5, alexandro marques tozetti1,* screening of ophidiomyces ophidiicola in the free-ranging snake community annually harvested for the popular ritual of san domenico e dei serpari (cocullo, aq, italy) daniele marini1,2, ernesto filippi3,*, gianpaolo montinaro4, francesco c. origgi5,6 assessment of fall season habitat and coverboard use by snakes in a restored tallgrass prairie community carter dollen1,2, tracy j. coleman1,2, travis r. robbins1,2,* revisiting the polyploidy in the genus odontophrynus (anura: odontophrynidae) andré luis de souza, mayara aparecida das neves micalichen, roger alves da rocha, rafael bueno noleto* acta herpetologica 4(2): 195, 2009 book review stephen j. mullin, richard a. seigel, 2009 (eds). snakes ecology and conservation. a comstock book, cornell university press. book review: “snake: ecology and conservation”, edited by mullin and seigel (2009) is the new edition of previous published volumes snakes: evolutionary biology (1987) and snakes: ecology and behavior (1993). this edition not only represents an updated version of past volumes, but also it does illustrate and discuss new insights and relevant advances into snake life-history traits, such as for instance reproductive biology as a necessary step for conservation purposes. the editors selected modern approaches in snake research, focusing on conservation aims throughout all the chapters’ rationale. the contributing authors are among the world’s top experts in snake ecology and biology: their chapters embraced modern techniques (i.e. from automated telemetry, to cartographic analyses in ecological researches and conservation plans), modern disciplines (i.e., conservation biology) or integrated classical arguments with recent tools and applied techniques. due to my personal feeling in reproductive biology and ecology of snakes, i have been particularly involved in “linking behavioral ecology to conservation objectives” and in “reproductive biology, population viability and options for field management” chapters. thus i am confident that snake experts worldwide could find their preferred and suitable chapter in this book. particularly relevant and stimulating is the chapter on “combating ophiophobia…”: which all not biologists should carefully consider when approaching a snake in the field or considering, and that teachers should comment in primary and secondary schools. three hundred and 65 pages with more than 1.500 literature references (an huge amount!) make this book something special and unique, an outstanding, fundamental tool for snake biologists as well as land managers. despite its scientific accuracy, richness of data presented and new research fields, snake: ecology and conservation is a quite easy to read book. fundamental for everybody wishes “entering” the snake mind and share snake feeling. price of 52.50 usd. correspondence: marco a.l. zuffi 1museo di storia naturale e del territorio, università di pisa, via roma 79, 56011 calci (pisa). e-mail: marcoz@museo.unipi.it acta herpetologica 18(1): 37-43, 2023 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-11294 diet and trophic niche overlap of four syntopic species of physalaemus (anura: leptodactylidae) in southern brazil renata k. farina1, camila f. moser2, stefano scali3, mateus de oliveira4, patrícia witt5, alexandro marques tozetti1,* 1 universidade do vale do rio dos sinos – unisinos, laboratório de ecologia de vertebrados terrestres, são leopoldo, rio grande do sul, brazil 2 universidade federal do pará ufpa, departamento de zoologia, belém, pará, brazil 3 museo civico di storia naturale di milano, corso venezia, 55, i-20121 – milano, italia 4 sintrópica consultoria ambiental, capela de santana, rio grande do sul, brazil 5 universidade do vale do rio dos sinos – unisinos, laboratório de biologia molecular, são leopoldo, rio grande do sul, brazil *corresponding author. email: alexandro.tozetti@gmail.com submitted on: 2022, 8th july; revised on: 2022, 28th august; accepted on: 2023, 18th january editor: dario ottonello abstract. despite the current increase in studies on the diet of neotropical anurans, few of them take a comparative approach between syntopic species. the objective of this study was to compare the diet of four syntopic species of the genus physalaemus. the collections occurred between november 2014 and january 2016 in a preserved area of the subtropical atlantic forest in southern brazil. we compared the gastrointestinal content of 109 individuals distributed in the species physalaemus biligonigerus, p. cuvieri, p. gracilis and p. henselii. we measured the index of relative importance of each prey category and calculated trophic niche breadth using the levins’ index (bsta) and trophic niche overlap using the pianka’s index (ojk). we also applied compositional analysis to evaluate feeding specializations. formicidae was the most important prey category for p. biligonigerus (iri = 88.5%) and p. gracilis (iri = 39.1%). for p. henselii and p. cuvieri, the most important category was isopoda (iri = 51.7% and 57.9%, respectively), followed by formicidae (iri = 34.9% and 24.8%). isopoda was also important in the diet of p. gracilis (iir = 28.6%), followed by araneae (iir = 22.6%). the trophic niche breadth of the four species was narrow, all smaller than 0.32, and the lowest was recorded for p. biligonigerus (0.04). the trophic niche overlap was higher between p. biligonigerus and p. cuvieri (96%), and between p. gracilis and p. henselii (95%). only p. gracilis presented a significant level of feeding specialization. the differences in their diets suggest different uses of the resources, which could relate to different ways of exploring the microhabitat. keywords. foraging, trophic niche, amphibians, behavior. introduction resources can be shared by species in three different dimensions: temporal, trophic and spatial (pianka, 1973). the different ways that species use resources can favor their coexistence and promote a reduction of competition between them (pianka, 1974). these differences can be observed, for example, in the different ways the species use the microhabitat, in the variety and sizes of food items and their period of activity (schoener, 1974; duellman and trueb, 1986). thus, basic data gathering about the ecology of organisms brings a great contribution to the elaboration of hypotheses and premises regarding niche partitioning and general ecology. 38 renata k. farina et alii although the divergence in feeding habits favors the coexistence of sympatric organisms, closely related species tend to use resources in a similar way (pianka, 1973) since they are similar in physiology and often in morphology and behavior as well (heyer et al., 1990). the number of studies on the diet of neotropical anurans has increased in recent years (siqueira et al., 2006, dietl et al., 2009, rodrigues and santos-costa, 2014, moser et al. 2017; oliveira et al. 2017, dias et al. 2018, farina et al. 2018, moser et al. 2019, protázio et al. 2019, oliveira et al. 2021, moser et al. 2022). however, few studies focus on a comparative approach to the species’ diet (oliveira et al., 2015, moser et al., 2017). trophic relationships between species comprise one of the main aspects of their life history (duellman and trueb, 1986; vitt and caldwell, 2009). information on resource partitioning among species can help us understand some parameters about community dynamics such as niche overlap and breadth (lawor, 1980), which are essential data to describe part of their ecological niche (sih and christensen, 2001). the genus physalaemus (leptodactylidae) has about 51species and is widely distributed in the neotropical ecozone (frost et al., 2006). species of this genus are characterized by consuming predominantly ants, beetles and spiders, but having an opportunistic diet pattern (lópez et al., 2003; becker et al., 2007; santana and juncá, 2007; rodrigues and santos-costa, 2014; oliveira et al., 2015; moser et al., 2017; farina et at., 2018). possible variations in diet composition among species are generally associated with variations in prey availability since, in general, anurans have an opportunistic diet (lópez et al., 2003; moser et al., 2017). thus, the study of syntopic populations presents an excellent opportunity to evaluate differences in diet among species that have the same variety and amount of potential prey available. the objective of this study was to compare the diet of four species of physalaemus (p. biligonigerus, p. cuvieri, p. gracilis and p. henselii) that occur in syntopy in a remnant of atlantic forest in southern brazil. material and methods study site the study was carried out in forest habitats inserted in the atlantic forest domain, located in a conservation unit (reserva biológica lami josé lutzenberger rbljl), in porto alegre, state of rio grande do sul (30°14’08”s; 51°05’42”w), southern brazil. the local landscape corresponds to the transition region between forest formations and subtropical restinga, being one of the closest forest remnants to the southern boundary of the atlantic forest (printes, 2002; witt, 2013). the climate of the region is classified as subtropical humid, with an average temperature in the warmer month of 22 ºc and average annual temperature of 18 °c (maluf, 2000). data collection in this study, we explored data from anurans available in herpetological collections. we had the opportunity to access a group of frogs collected accidentally by other colleagues when using pitfall traps (campbell and christman, 1982) for entomological sampling. as traps were constructed with 10-l buckets, they were large enough to capture small frogs. in addition, as the buckets were filled with an ethanol solution (70%), they preserved the gastrointestinal content of frogs. we had access to frogs captured from november 2014 to january 2016 and, according to brazilian laws, since the captures were accidental, no collecting permits were needed. the accidentally caught anurans were donated to the laboratory of ecology of terrestrial vertebrates (levert) by the management team of the biological reserve. in the laboratory, the animals were dissected to remove the gastrointestinal contents (stomach and intestine), which were preserved in 70% ethanol and screened under a stereomicroscope. for each prey category, number, volume and frequency of occurrence were calculated. volume was calculated by estimations of the area (mm²) occupied by each item with a graph paper support attached to the bottom of the petri dish, where we evenly spread each item, maintaining a regular height of 1 mm (hellawell and abel, 1971). in order to calculate each item volume (v), the area value (mm²) was multiplied by its height (1 mm) (oliveira et al., 2015). the set of prey present in each content (individual) was considered as a sample. prey items were identified until the lowest possible taxonomic level based on ribeiro-costa and rocha (2006). we were unable to reach the species level due to the high fragmentation in most of the prey items caused by the digestion process. to improve our data interpretation, we added to our dataset data of physalaemus henselii that had been previously published (farina et al., 2018). we highlight that this non-novel dataset represents 16% of the amount of data of the current study. all analyzed individuals were collected in the same locality with the same trap model and in the same period of the year. data analysis we used the index of relative importance (iri), according to pinkas et al. (1971) to calculate the impor39diet of syntopic species of physalaemus tance of each category of prey using the following equation: iri = (n% + v%) fo%, where n% is the relative abundance of each prey category in the diet, v% is the relative volumetric contribution of the prey in the diet, and fo% is its relative frequency of occurrence in the diet (pinkas et al., 1971; krebs, 1999). th e higher the iri value, the greater is the importance of a given prey category in the diet. we constructed rarefaction curves using past 4.03 soft ware to estimate the sampling representativity of the prey set for each anuran species (sanders, 1968). in this analysis, each gastrointestinal content was considered as a sample. curves were based on jackknife 1 estimator of species richness (burnham and overton, 1978, 1979) which could represent the studied populations. the levin’s standardized niche breadth index (bsta) (krebs, 1999) ranges from 0 to 1 and is calculated according to the following equation: bsta = (b-1) / (n-1), where n is the number of resources registered in the diet (prey categories), and b = 1 / σpi2, p represents the proportion of individuals of a given prey category (i) found in the diet. values near 0 indicate a specialist diet (narrow niche breadth), while values near 1 indicate a generalist diet (wide niche breadth). bsta was used to facilitate comparisons of trophic ecology between species. to analyze the food overlap and/or degree of similarity between species diets, we used the pianka’s trophic niche overlap index (ojk) (pianka, 1973), defi ned by the following equation: where ojk is the niche overlap index between the species j and k; pij is equivalent to the proportion of the resource type i relative to the total of resources used by the species j; pik is the proportion of resource i relative to the total of resources used by the species k; and n is the total number of resource categories used by the species j and k. th is index ranges from 0 to 1 when there is no overlap or a complete overlap between the species diets, respectively (krebs, 1999). for this analysis, we used the program ecosim v1.2d (gotelli and entsminger, 2000). since we have samples from all species from all seasons, comparisons helped us in detect if and how feeding behavior diff er between species. possible prey preferences were evaluated by compositional analysis (aebischer et al., 1993) using the r package ‘adehabitaths’ (calenge, 2013). th is analysis is based on a comparison between the matrix of consumed prey and the mean frequencies of components used by all individuals of the sampled species (sacchi et al., 2013). th e analysis use randomization tests (number of permutations = 500) to assess the signifi cance of the ranking matrices (aebischer et al., 1993). results we analyzed the gastrointestinal contents of 109 individuals (table 1). of these, eight were physalaemus biligonigerus (containing 190 food items distributed in 11 prey categories), nine p. cuvieri (221 items, 13 categories), 74 p. gracilis (529 items, 19 categories) and 18 p. henselii (154 items, 10 categories) (table 1). only one individual of p. cuvieri and 10 of p. gracilis had no gastrointestinal content. even though some species, such as p. biligonigerus (8) and p. cuvieri (9), had a small sample size, we recorded 11 and 13 prey categories for them, respectively. furthermore, an estimate of 9,81 for p. biligonigerus, 8 for p. cuvieri, 74,4 p. gracilis and 18,9 p. henselii was obtained using the jackknife 1 estimator, which indicates that even with a small n (p. biligonigerus and p. cuvieri) the samples were suffi cient to represent the populations, in the same way as for p. henselii. however, the rarefaction curves (fig. 1) did not stabilize, suggesting that, by adding new samples (more frogs), other prey taxa were expected to be recorded. based on this, results must be evaluated with caution. formicidae was the most important prey category for p. biligonigerus (iri = 88.5%) and p. gracilis (iri = 39.1%). for p. henselii and p. cuvieri, the most important category in the diet was isopoda (iri = 51.7% and 57.9%, respectively), followed by formicidae (iri = 34.9% and 24.8%). isopoda was also important in the diet of p. gracilis (iri = 28.6%), followed by araneae (iri = 22.6%) (table 1). th e trophic niche breadth was higher for p. henselii (bsta = 0.32) followed by p. gracilis (bsta = 0.23). th e lowest values of niche breadth were recorded for p. biligonigerus (bsta = 0.04) and p. cuvieri (bsta = 0.13). th e overlap of trophic niche (ojk) was 96% between p. biligonigerus and p. cuvieri, 95% between p. gracilis and p. henselii, 91% between p. cuvieri and p. gracilis, 83% between p. cuvieri and p. henselii, 83% between p. biligonigerus and p. gracilis, and 71% between p. biligonigerus and p. henselii. results from compositional analysis pointed out that only physalaemus gracilis has a certain degree of feeding specialization (lambda = 0.108; p < 0.001; table 1), preying upon araneae, coleoptera larvae, isopoda, formicidae and hymenoptera in a higher frequency than expected by chance. 40 renata k. farina et alii discussion the species of this study exhibited a similar prey composition in their diets. in all of them, formicidae and/or isopoda were the predominant prey categories. the high relevance of ants was also recorded for other populations of physalaemus biligonigerus, p. cuvieri and p. gracilis in studies of restinga habitats in southern brazil (oliveira et al., 2015), atlantic forest biome in northern brazil (santos et al., 2004) and araucaria forest in southern brazil (moser et al., 2017). formicidae had high importance in the diet of p. biligonigerus, reaching iri values of 88.47%, while iri of the second most important prey category was less than 8% (lepidoptera larvae). based on this, we argue that p. biligonigerus is locally a “formicidae specialist”. large consume of formicidae has been previously reported in p. biligonigerus (oliveira et al., 2021) as well in other physalaemus (santana and juncá, 2007). is hard to list environmental components that favored the dominance of ants in contents. on the other hand, high consumption of ants involves physiological adaptations to the digestion due to the presence of formic acid, resulting in a high energy cost (hirai and matsui, 2002). then, the ability to feed on ants would increase the advantages in explore the food resources in the habitat. our sampling design did not make us able for a deeper discuss among diet specialization, niche partitioning and competition. we believe that specialization on ant consumption is a topic that deserves new studies. ants were also important food items for other species of the genus in different brazilian locations, such as the atlantic and amazon forests for p. lisei (moser et al., 2017) and p. ephippifer (rodrigues and santos-costa, 2014), caatinga (protázio et al. 2019; oliveira et al. 2021) and in argentina for p. riograndensis (lópez et al., 2003) and p. albonotatus (falico et al., 2012). in general, ants are considered unpalatable for several predators (hirai and matsui, 2000) but species of physalaemus consume ants with considerable frequency (moser et al., 2017). for some amphibians, the sequestable 1. prey categories found in the gastrointestinal contents of physalaemus biligonigerus, p. cuvieri, p. gracilis and p. henselii. n = number of individuals, v% = total volume (in mm³) occupied by prey category, fo% = frequency of occurrence of prey category, iri% = index of relative importance. prey categories p. biligonigerus (n = 8) p. cuvieri (n = 8) p. gracilis (n = 64) p. henselii (n = 18) n% v% fo% iri% n% v% fo% iri% n% v% fo% iri% n% v% fo% iri% acarina 1.1 0.2 12.5 0.1 2.7 0.5 22.2 0.3 5.7 0.8 18.9 1.6 11.0 2.0 50.0 6.7 amphipoda 1.1 0.7 25.0 0.3 0.0 0.0 0.0 0.0 3.0 0.7 4.1 0.2 0.0 0.0 0.0 0.0 araneae 1.6 1.0 37.5 0.6 5.0 4.1 55.6 2.2 18.0 13.4 55.4 22.6 5.2 3.6 22.2 2.0 blattodea 0.0 0.0 0.0 0.0 1.8 6.1 44.4 8.7 0.6 0.6 4.1 0.1 3.9 8.4 16.7 2.1 coleoptera 2.1 3.0 25.0 0.8 3.6 5.8 44.4 1.8 2.3 4.7 13.5 1.2 5.2 2.0 16.7 1.2 coleoptera (larva) 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.0 8.5 14.9 2.2 0.0 0.0 0.0 0.0 collembola 0.0 0.0 0.0 0.0 1.8 0.8 22.2 0.6 1.0 0.1 2.7 0.0 0.0 0.0 0.0 0.0 dermaptera 0.5 0.1 12.5 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 diptera 1.6 1.5 25.0 0.5 2.3 1.0 22.2 0.7 1.3 0.6 8.1 0.2 2.6 0.5 16.7 0.5 diptera (larva) 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.6 1.2 1.4 0.0 0.0 0.0 0.0 0.0 formicidae 83.7 52.1 100.0 88.5 57.5 15.8 77.8 24.8 32.5 12.0 67.6 39.1 35.1 12.1 72.2 34.9 gastropoda 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 4.2 5.6 18.9 2.4 0.7 0.3 5.6 0.1 hemiptera 0.0 0.0 0.0 0.0 0.5 0.4 11.1 0.0 1.5 1.6 9.5 0.4 0.0 0.0 0.0 0.0 heteroptero 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.0 1.4 0.0 0.0 0.0 0.0 0.0 hymenoptera 0.5 1.0 12.5 0.1 0.5 0.4 11.1 0.0 3.4 1.7 14.9 1.0 2.0 1.4 16.7 0.6 isopoda 1.1 2.2 25.0 0.5 13.1 28.3 66.7 57.9 20.4 29.0 44.6 28.6 33.1 36.7 72.2 51.7 isoptera 0.0 0.0 0.0 0.0 9.5 7.8 11.1 2.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 lepidoptera (larva) 4.7 24.5 37.5 7.4 0.9 0.7 22.2 0.2 0.6 1.1 4.1 0.1 0.0 0.0 0.0 0.0 odonata 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.4 0.6 2.7 0.0 0.0 0.0 0.0 0.0 opilionida 0.0 0.0 0.0 0.0 0.5 0.6 11.1 0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 orthoptera 2.1 2.3 37.5 1.1 0.5 0.1 11.1 0.0 1.0 1.6 6.8 0.2 1.3 0.6 11.1 0.2 pseudoescorpionidae 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.6 0.5 4.1 0.1 0.0 0.0 0.0 0.0 other -11.4 ---27.7 ---15.8 ---32.4 ---  niche breadth 0.04 0.13 0.23 0.32  41diet of syntopic species of physalaemus tration of alkaloids is associated with the consumption of ants (saporito et al., 2004), but no studies prove the ingestion for this purpose by physalaemus. the high consumption of formicidae may be associated with the abundance and displacement capacity of these invertebrates in the environment (baretta, 2007), thus characterizing an opportunistic feeding behavior by the anurans. this behavior may minimize the potential competition between species, favoring their coexistence. also, isopoda was one of the most important prey categories for three of the four species (p. cuvieri, p. gracilis and p. henselii). the relevance of isopoda to the diet of physalaemus species was poorly reported. although the group is part of the genus’ diet (rodrigues and santos-costa, 2014; moser et al., 2017), it is not frequently consumed. in the study by leivas et al. (2018), isopoda were not found in the diet of p. cuvieri, which may be associated with prey availability. we observed feed specialization for two prey groups: ants and isopoda. both of them are social insects. their nests offer a large number of preys which cold leads for their high density in contents of frogs that are able to eat them. the trophic niche breadth for p. biligonigerus, p. cuvieri and p. gracilis was equal to or similar to results found in other studies of the same species (santos et al., 2004, moser et al. 2017). in contrast, for p. henselii (bsta = 0.32, farina et al., 2018), trophic niche breadth was higher than for other species of this study and also of the literature, such as p. gracilis and p. lisei (bsta = 0.15 and bsta = 0.11 respectively, moser et al., 2017), p. ephippifer (bsta = 0.19, rodrigues and santos-costa, 2014) and p. biligonigerus (bsta = 0.04, oliveira et al., 2015), suggesting a more generalist behavior in relation to its congeners (farina et al., 2018). in general, leptodactylids have narrower niches when compared to other families, such as hylids (sabagh et al., 2010; barbosa et al., 2014), revealing a great abundance of local resources that favors coexistence and high trophic niche overlap. despite of little speculative our data suggests a reasonable degree of food specialization in for p. biligonigerus and p. cuvieri. the predominant consumption of ants is one of the reasons for the high trophic niche overlap between the species. the phylogenetic proximity of the species may also relate to their high niche overlap (lourenço et al., 2015), besides indicating a great prey availability in the environment, which is evidenced by the niche breadth. also, species may be foraging in similar places, considering that species that share the same habitat tend to have a similar diet (duellman and trueb 1986, guidali et al., 2000, sabagh et al., 2010). compositional analysis pointed out that differently from other species, p. gracilis exhibited a considerable level of selectivity in its diet, preying on araneae, coleoptera larvae, isopoda, formicidae and hymenoptera more frequently than their expected availability in the habitat. this result brings some light to the relevance of studying syntopic species to elucidate feeding adaptations. acknowledgments: we would like to thank fapergs, capes and cnpq for financial support. we also thank the management team of biological reserve lami josé lutzenberger for the capture and donation of anurans to the terrestrial vertebrate ecology laboratory (levert). since the use of pitfall traps was not aimed at capturing frogs, but soil invertebrates, collecting permits and ethical approval were unnecessary according to the brazilian wildlife regularly agency (icmbio). references aebischer, n.j., robertson, p.a., kenward, r.e. 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(2013): fauna e flora da reserva biológica lami josé lutzenberger. porto alegre: secretaria municipal do meio ambiente. threats of the emerging pathogen batrachochytrium salamandrivorans (bsal) to italian wild salamander populations lorenzo dondero1, giorgia allaria1, giacomo rosa1, andrea costa1, gentile francesco ficetola2, roberto cogoni3, elena grasselli1, sebastiano salvidio1,* age estimation and body size of the parsley frog, pelodytes caucasicus boulenger, 1896 from lake borçka karagöl, turkey cantekin dursun*, serkan gül, nurhayat özdemir patterns of acoustic phenology in an anuran assemblage of the yungas andean forests of argentina martín boullhesen1,2,*, marcos vaira1, rubén marcos barquez2, mauricio sebastián akmentins1 diet and trophic niche overlap of four syntopic species of physalaemus (anura: leptodactylidae) in southern brazil renata k. farina1, camila f. moser2, stefano scali3, mateus de oliveira4, patrícia witt5, alexandro marques tozetti1,* screening of ophidiomyces ophidiicola in the free-ranging snake community annually harvested for the popular ritual of san domenico e dei serpari (cocullo, aq, italy) daniele marini1,2, ernesto filippi3,*, gianpaolo montinaro4, francesco c. origgi5,6 assessment of fall season habitat and coverboard use by snakes in a restored tallgrass prairie community carter dollen1,2, tracy j. coleman1,2, travis r. robbins1,2,* revisiting the polyploidy in the genus odontophrynus (anura: odontophrynidae) andré luis de souza, mayara aparecida das neves micalichen, roger alves da rocha, rafael bueno noleto* acta herpetologica 4(2): 135-142, 2009 structural habitat use by the many-scaled anole, anolis polylepis (squamata: polychrotidae) marco d. barquero, viviana p. arguedas asociación para la conservación y el estudio de la biodiversidad (acebio), casa 15, barrio los abogados, zapote, san josé, costa rica. corresponding author. e-mail: marcodba@hotmail.com, marcobarq@gmail.com. submitted on: 2009, 5th february; revised on 2009, 1st july; accepted on 2009, 4th october. abstract. lizards of the genus anolis are commonly used as models for several ecological studies. nevertheless, some aspects of their ecology have not been studied and information reported previously for several species must be reanalyzed. the aim of this study is to examine the structural habitat use in a population of the many-scaled anole, anolis polylepis, with the purpose of comparing our results with the information reported previously for this species. most of the captured individuals were on stems and we did not find any differences in the structural habitat use among sex/ age classes. we found differences for perch height among individuals shedding their skin regarding those that are not. we also detected differences among our results and the information reported previously for this species. such differences could be due to intrinsic factors of each population, such as the proportion of individuals that were molting their skin in a specific time. more studies with greater sample sizes and to a longer term are required to clearly understand the influence of these factors in the habitat use of a. polylepis and other anoline lizards. keywords. anolis polylepis, lizard, ecology, habitat use, structural habitat. introduction the genus anolis includes species of neotropical, diurnal and mostly arboreal lizards (pough et al., 2001). due to the great diversity presented in the genus and high densities that many species reach in some sites, these lizards are commonly used as models for several ecological studies (losos, 1994; irschick et al., 1997). in this way, it has been possible to identify for many species the relationship between morphological characteristics and habitat use. for example, for the tail, an important organ of balance in these species (ballinger, 1973), it has been observed that individuals with longer tails use narrower perches (irschick et al., 1997). 136 m.d. barquero and v.p. arguedas however, a lot of ecological characteristics remain unknown and information reported previously for several species must be reanalyzed (vitt et al., 2002; vitt and zani, 2005). for many species is still needed to determine if the use of structural habitat (i.e., perch diameter and perch height) varies in time or space and if so, which are possible causes for such variations (rand, 1964; irschick et al., 1997; savage, 2002). it has neither been investigated if structural habitat reported for a particular species is affected by intrinsic factors of the population studied; for example, a high proportion of individuals with malformations or physical damages. in spite of this lack of information, several ecological aspects of anoline lizards have been identified. frequently, it has been reported that intrasexual differences in habitat use are present in anolis species (jenssen et al., 1998; butler et al., 2000; irschick et al., 2005). such differences have been explained as a result of the different roles that each sex has in the environment. in anolis polylepis, a central american polychrotid lizard, adult males use higher perches than adult females because they spend more time defending their territories, so they need to achieve a better visibility of their territory. females and juveniles, on the other hand, dedicate more time than males looking for food, so they use lower and less conspicuous perches to avoid predators (andrews, 1971; hertz, 1974; perry, 1996). it has been demonstrated that in a. polylepis there is a slight change in structural habitat use between seasons (andrews, 1971). males use lower perches during dry season than in the rainy one and both males and females select a greater diversity of perch types during dry season. although some explanations have been proposed, environmental factors that cause such variation are still ignored. the aim of this study is to examine structural habitat use in a population of the many-scaled anole, anolis polylepis, in southwestern costa rica, with the purpose of comparing our results to the information reported previously for this species. also, we will analyze possible factors that affect the use of structural habitat in this species. materials and methods anolis polylepis is a common lizard found in low and middle elevations throughout south pacific of costa rica, usually in habitats with humid and shady conditions (andrews, 1971; socci et al., 2005). this species has a brown dorsal coloration, a medium body size (males bigger than females), and a smooth skin that is molted regularly. males reach sexual maturity at 45 mm of snoutvent length, while females do this at 41 mm (savage, 2002). a. polylepis uses perches up to 3 m above the ground, although individuals are located between 1 and 1.5 m on average (andrews, 1971). study site we carried out this study during part of the rainy season (october and november) of 2006, in the golfito national wildlife refuge (08°38’ n, 83°09’ w), puntarenas province, costa rica. the refuge includes a mountain range that forms a very irregular coast, rains are abundant and constant almost the entire year, and it presents a very humid tropical forest (boza, 1996). we collected data in a secondary forest, with a dense understory dominated by herbaceous plants and some woody bushes, and a relatively closed canopy with some epiphytes. 137structural habitat in anolis polylepis field techniques we used the refuge hiking trails and every day we sampled a different area to avoid registration of the same lizards. we followed the activity cycle of lizards during the morning, from 7:00 to 11:30, and the afternoon, from 2:00 to 5:00, so sampling effort accounted for a total of 7.5 hr/individual/day. we searched for lizards examining all vegetation using the visual encounter survey technique (crump and scott, 1994). we registered the following data for each individual captured: sex/age class (adult male, adult female and juvenile), snout-vent length (svl), and type (stem, tree trunk and leaf ), height and diameter of the perch used in the moment of observation (see rand, 1964). we also registered data that we considered relevant, such as presence of physical damages and ecdysis. statistical analysis. we used the program statistica 7 (statsoft inc., 2004) for all statistical analyses. we did a g test to compare the total number of individuals found on each perch type. since perch height and perch diameter did not fit a normal distribution, we transformed these variables using base-10 logarithms. we used these transformed variables to carry out two-way analysis of variance to compare perch height and perch diameter of individuals on each sex/age class and ecdysis state. since no juveniles with damaged tail were found, we excluded these ones and performed two-way analysis of variance to compare perch height and perch diameter of males and females with and without damages on the tail. for perch diameter, we excluded individuals found on leaves for all tests. results we captured a total of 47 individuals, 66% of which were adult males, 19% adult females and 15% juvenile (table 1). most of these individuals were found on stems than in other perch types (g = 64.68, df = 1, p < 0.001) (table 1). this result contrasts with the information reported previously for a. polylepis, since it has been mentioned that a high proportion of individuals perch on leaves (andrews, 1971; hertz, 1974). also, several authors have reported that adult males of a. polylepis use higher and thicker perches than adult females and juveniles (table 2). however, we did not find differences among sex/age classes for perch height nor for perch diameter (table 3). when we checked individuals for physical damages, only nine table 1. number of adult males, adult females and juveniles of anolis polylepis found on each perch type. perch type sex/age class leaf stem tree trunk total adult males 1 28 2 31 adult females 1 7 1 9 juveniles 0 7 0 7 total 2 42 3 47 138 m.d. barquero and v.p. arguedas table 2. mean perch height and mean perch diameter ± standard deviation reported for sex/age classes of anolis polylepis in diff erent studies. sample size is in parenthesis. adult males adult females juveniles source perch height (cm) 92 (68) 49 (86) andrews, 19711 155 ± 56 (24) 53 ± 46 (18) hertz, 1974 89 ± 61 (32) 38 ± 43 (38) 16 ± 23 (78) perry, 1996 51 ± 29 (27) 31 ± 25 (53) frenkel, unpubl. data 94 ± 51 (31) 63 ± 45 (9) 159 ± 135 (7) th is study perch diameter (cm) 2.65 ± 4.34 (24) 1.24 ± 1.43 (18) hertz, 1974 2 ± 2 (31) 4 ± 3 (9) 2 ± 1 (7) th is study 1 standard deviation not reported. fig. 1. means of perch height (a) and perch diameter (b) for individuals of anolis polylepis with damaged and undamaged tails. lines indicate standard error. 15 a b tail damaged tail undamaged p e rc h h e ig h t (c m ) p e rc h d ia m e te r (c m ) 139structural habitat in anolis polylepis table 3. results obtained using analysis of variance to test the eff ect of factors on perch height and perch diameter of anolis polylepis. eff ect factor with or without ecdysis tail damaged or undamaged1 f df p f df p perch height age/sex classes 0.828 2 0.444 1.430 1 0.240 factor 11.174 1 0.002 0.411 1 0.525 classes*factor 0.597 2 0.555 0.344 1 0.561 perch diameter age/sex classes 0.411 2 0.666 3.114 1 0.087 factor 0.969 1 0.331 0.041 1 0.840 classes*factor 1.075 2 0.351 0.209 1 0.651 1 juveniles are not accounted in this analysis. fig. 2. means of perch height (a) and perch diameter (b) for individuals of anolis polylepis with and without ecdysis. lines indicate standard error. 16 a b without ecdysis with ecdysis p e rc h h e ig h t (c m ) p e rc h d ia m e te r (c m ) 140 m.d. barquero and v.p. arguedas of them presented some kind of damage on the tail. we did not find significant differences for perch height (fig. 1a) or perch diameter (fig. 1b) when we considered sex/age classes, individuals with and without damages on the tail, and the interaction between class and damage on tail (table 3). we also found eight individuals that were shedding their skin. we did not find significant differences for perch height (fig. 2a) when we considered sex/age classes or the interaction between class and presence of ecdysis (table 3). however, we did find that individuals with presence of ecdysis perched higher than those that were not shedding their skin (table 3). for perch diameter (fig. 2b), we did not find significant differences for any of the effects tested (table 3). discussion studies about the ecology of mainland anoline species usually take into account one or just a few populations (talbot, 1979; jenssen et al., 1998; vitt et al., 2005; irschick et al., 2005), and only in some cases results generated have been compared with information reported in other studies (irschick et al., 1997). nevertheless, to achieve an integral knowledge of the ecology of an organism it is necessary to examine all information generated previously and determine if spatial or seasonal variations occur. in this study we compared structural habitat use of one population of a. polylepis with the information reported for populations along the distribution range of this species in costa rica, and we found differences in perch height and perch type. variations in habitat use among populations of different anolis species has been reported previously (pacala and roughgarden, 1982; losos et al., 1993; vitt et al., 2002). explanations that have been mentioned to explain these interpopulational differences are variations in climatic régime, forest structure, interspecific competition, predation, and food availability. however, intrinsic factors of each population could play an equally important role for habitat use of adult males, adult females and juveniles. one of these factors is the proportion of individuals in the population shedding their skin in a given moment. our data suggest that individuals in this stage use higher perches than those that are not. this was particularly notorious for juveniles, which presented a perch height way more above of the mean reported in other studies (table 1). when considering juveniles with and without presence of ecdysis separately, mean perch height was greater for individuals shedding their skin than the one reported in other studies and did not differ for those not shedding (fig. 2a). a possible explanation for these differences involves coloration of this species and light microhabitat differences. individuals of a. polylepis have a brown dorsal coloration which allows them to remain unobserved in low perches near the ground, where intensity of illumination is low. however, shed in these lizards is white, so they become conspicuous close to a dark background. it is possible that, in order to avoid predators, individuals presenting ecdysis tend to use higher perches than those of the rest of the population. by using higher perches, they are located in a brighter environment than if they remained close to the ground, reducing their conspicuousness. although we did not measured environmental illumination for a. polylepis, it has been proved that this factor affects behavior 141structural habitat in anolis polylepis of anolis species (fleishman and persons, 2001; leal and fleishman, 2002; macedonia et al., 2003), so our hypothesis deserves more attention. because the time to complete ecdysis in anoline lizards is short, we could assume that this factor would only affect habitat use in a. polylepis at short term. however, continuous presence of juveniles along the year and a quick growth rate (andrews, 1991) indicates that in this species the proportion of individuals shedding in a given moment could be high (17% in this study). hence, the effect of these individuals on structural habitat of one population of a. polylepis could be high. other intrinsic factors of each population could also affect habitat use of a. polylepis. although our data do not reflect it, individuals with damaged tail could be affected in their movements due to the importance of this organ for keeping balance in these lizards. studies with greater sample sizes, more sampling time, and a larger geographical range will help to elucidate the influence of these and other factors in basic ecological aspects of anoline lizards. however, our data show that structural habitat use in a. polylepis is affected by presence of individuals shedding, generating different information than reported previously for this species. this suggests that intrinsic factors deserve more attention and must be taken into account for ecomorphological studies in anoline lizards. acknowledgements this study was part of a greater investigation carried out with a grant given by the holly hill charitable trust and equipment donation from idea wild. we thank guido saborío, alejandro muñoz and an anonymous reviewer for their suggestions to improve the manuscript. we also thank to those in charge of minae and university of costa rica in golfito for their logistical support. references andrews, r.m. 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(1997): a comparison of evolutionary radiations in mainland and caribbean anolis lizards. ecology 78: 2191-2203. jenssen, t.a., hovde, k.a., taney, k.g. (1998): size-related habitat use by nonbreeding anolis carolinensis lizards. copeia 1998: 774-779. leal, m., fleishman, l.j. (2002): evidence for habitat partitioning based on adaptation to environmental light in a pair of sympatric lizard species. proc. r. soc. lond. b 269: 351-359. losos, j.b. (1994): integrative approaches to evolutionary ecology: anolis lizards as model systems. annu. rev. ecol. evol. syst. 25: 467-493. losos, j.b., marks, j.c., schoener, t.w. (1993): habitat use and ecological interactions of an introduced and a native species of anolis lizard on grand cayman, with a review of the outcomes of anole introductions. oecologia 95: 525-532. macedonia, j.m., echternacht, a.c., walguarnery, j.w. 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(2005): the importance of soil moisture and leaf cover in a female lizard’s (norops polylepis) evaluation of potential oviposition sites. herpetologica 61: 233-240. statistica (data analysis software system), version 7. www.statsoft.com. talbot, j.j. (1979): time budget, niche overlap, interand intraspecific aggression in anolis humilis and a. limifrons from costa rica. copeia 1979: 472-481. vitt, l.j., avila-pires, t.c.s., zani, p.a., espósito, m.c. (2002): life in shade: the ecology of anolis trachyderma (squamata: polychrotidae) in amazonian ecuador and brazil, with comparisons to ecologically similar anoles. copeia 2002: 275-286. vitt, l.j., zani, p.a. (2005): ecology and reproduction of anolis capito in rain forest of southeastern nicaragua. j. herpetol. 39: 36-42. acta herpetologica 4(2): 183-189, 2009 anthropogenic impact or anthropogenic accommodation? distribution range expansion of the common wall lizard (podarcis muralis) by means of artificial habitats in the north-eastern limits of its distribution range iulian gherghel1, alexandru strugariu1, tiberiu c. sahlean2, oana zamfirescu1 1 “al. i. cuza” university, faculty of biology, carol i blvd., no. 20a, r-700506, iasi, romania. corresponding author. e-mail: iuliangherghel@gmail.com 2 university of bucharest, faculty of biology, splaiul independentei blvd., no. 91 – 95, sector 5, r-050095, bucharest, romania. submitted on: 2009, 6th june; revised on 2009, 16st september; accepted on 2009, 25th september. abstract. during 2005-2008, field observations were made on the distribution and habitat occupation by podarcis muralis in the middle bistrita river basin. prior to our study, this lizard was known from only 5 localities in romanian moldavia (bicaz, cheile bicazului, lacu roşu, gherman and dodeni). through the present paper the authors acknowledge the existence of 28 sites populated by p. muralis in moldavia and propose a new model for the range expansion of the species in the area using manmade structures such as road and railway fences and road beds, benefiting from the mild climate provided by the izvorul muntelui barrier lake and forming insular populations to further colonize suitable habitats. keywords. carpathian mountains, romanian moldavia, invasive species, reptiles. the common wall lizard, podarcis muralis (laurenti, 1768), is a relatively widespread reptile species, its range comprising of most of europe: from france and northern spain to romania and northern asia minor (e.g., guillaume, 1997; arnold, 2003). the carpathian mountains represent the north-eastern distribution limit of the species (fuhn and vancea, 1961; iftime, 2005). in romania, the common wall lizard occupies especially the southern and central parts of the carpathians, southern dobrugea, the apuseni mountains and the eastern carpathians (fuhn and vancea, 1961; fuhn, 1969; ghira et al., 2002; iftime, 2005; covaciu-marcov et al., 2006a, 2006b; sahlean et al., 2008; iftime et al., 2008). podarcis muralis is a thermophylous species that prefers habitats composed of abrupt cliffs, debris, stone walls (fuhn and vancea, 1961; iftime, 2005), sometimes being found on railway embankments (covaciu-marcov et al., 2006 b). first reference on the presence of the common wall lizard in moldavia (eastern romania) was made by vancea (1958) at bicaz, later reconfirmed by borcea (1976). fur184 i. ghergel et alii ther records were made by ionescu et al. (1968), fuhn (1969), borcea (1979) and borcea and vancea (1981) at cheile bicazului. after these findings, no new records were made on the distribution or population status of the common wall lizard in romanian moldavia until recent years, when iftime (2005) reconfirms the existence of the species at cheile bicazului. later, gherghel et al. (2008) published a mapping of the herpetofauna of the bistrita river basin and presented 3 new localities for the distribution of the common wall lizard, at lacu roşu, gherman and dodeni and reconfirmed the presence of the species in the localities already present in the literature (cheile bicazului and bicaz). the common wall lizard is considered a species of community interest both in romanian and european legislation, and is listed in the annex 4 of the habitats directive of the european council 92/43/eec and strictly protected in romania by law no. 13/1993, which ratifies the bern convention. in addition, the common wall lizard populations from the middle bistrita river basin are situated at the north-eastern limit of the distribution range of the species, making them a key element for a future conservation plan. our study took place between 2005 and 2008, with the purpose of identification of new populations of the common wall lizard at its north-eastern range limit. the study investigated several potential habitats which were identified through direct field observations, digital elevation terrain models and different geological and topographic maps fig. 1. map of studied area and geographic position of studied sites. 185distribution of podarcis muralis in eastern romania (1:25000). namely, we consider as potential habitats all sites with rocky terrain, rock debris or steppe cliffs, crevices and cover vegetation used for shelter against potential threats, but also sites containing man-made structures known to catalyze the spreading of common wall lizards. the gps coordinates and geographical data were analyzed and verified with diva gis, software also recommended by previous researches (e.g., rödder et al., 2008; rödder 2009). overall, fifty-eight potential sites were identified on an area of 1200 km2, and were investigated repeatedly (10 times for each site), during different seasons between april and october, raising the chances of finding p. muralis populations. as a result of our investigations, 28 sites (48% of all investigated sites) were detected as being inhabited by the common wall lizard (fig. 1, table 1), near the following localities: izvorul muntelui, dodeni, bicaz, capşsa, tarcău, straja, bicazul ardelean, bicaz-chei, gherman, bîrnadu and lacul roşu. these are grouped in two large populations, separated by forests, agricultural fields and meadows, habitats that do not meet the criteria of being favorable for the species (fuhn and vancea, 1961). the subpopulation from cheile bicazului (the southern subpopulation) is distributed over an area that comprises 10 sites (90% satellite rocks of the cheile bicazului geomorphologic formation) which probably represent the initial spread points for the northern subpopulation (further genetic analysis will be needed in order to test this hypothesis) (table 1). the northern subpopulation (around the city of bicaz) is distributed over 18 sites, 94% of which are man-made – railway embankments (59%) and road embankments (41%), only 6% being natural rocky terrain. taking into account the fact that the northern group occupies a wide variety of habitats which have been presented as potentially favoring the extension of this species’ distribution area (e.g., covaciu-marcov et al., 2006; strugariu et al., 2008) but also the fact that a large number of individuals (20 individuals in a discontinuous gradient from west to east) have been found on the railway beds to the east, towards straja (east of this locality not having been found any potential habitats), we could assume that the bicaz population is expanding. the expansion seems to be mediated by elements of anthropogenic origin represented by railway and road beds. the distribution area extension process is on-going in all areas where it lives or has been introduced, being a species with high invasiveness potential (e.g., hedeen and hedeen, 1999; behler and king, 2000). the fact that podarcis muralis is a highly invasive species was demonstrated by the existence and demographic explosion of north american introduced populations (hedeen and hedeen, 1999; behler and king, 2000; deichsel and gist, 2001; deichsel and schweiger, 2004) or by the possible new colonization of habitats using railways towards the metropolitan area of bucharest (strugariu et al., 2008). as with this last case, we consider that common wall lizard populations from the north-eastern distribution range of the species are dynamic, contributing, in the future, to the extension of the species’ distribution range, by colonization of new habitats that meet ecological criteria, but are not yet occupied. this range expansion may be influenced by the local mild microclimate, a tribute to the presence of the izvorul muntelui barrier lake. this has led to a better exposure of rocky habitats, resulting in a better thermic treatment, but at the same time the lake acting as a source of equilibrium, contributing, through summer and multi-annual evaporation, to a temperature rise of approximately 2 oc, compared to the period before the lake, or compared to other areas in the same climate level (ciagloc, 1968). another possibil186 i. ghergel et alii ity, as in other areas of romania, is the involuntary transport of individuals along with calcareous material from rock quarries (covaciu-marcov et al., 2006), these also being present in the study area (cheile bicazului rock quarry). however, this second hypothesis it has not been confirmed in the field: east of the straja rock habitats, road and railway embankments are absent and the animals are not present on the railway beds. this suggests that rock material is improbable to have caused an artificial dispersion of the animals table. 1. descriptive data about studied sites. site altitude coordinates habitat type exposure m a.s.l. n e 1 500 46° 56’ 25’’ 26° 06’ 02’’ rocks e 2 480 46° 56’ 23’’ 26° 06’ 00’’ road fence e 3 475 46° 56’ 17’’ 26° 05’ 54’’ road fence e 4 464 46° 57’ 24’’ 26° 04’ 53’’ road fence s-e 5 404 46° 53’ 59’’ 26° 07’ 07’’ road fence s-e 6 400 46° 53’ 47’’ 26° 07’ 19’’ road fence s 7 405 46° 54’ 20’’ 26° 06’ 25’’ road fence s 8 392 46° 54’ 20’’ 26° 06’ 36’’ road fence s 9 392 46° 53’ 34’’ 26° 08’ 11’’ railway fence s 10 390 46° 53’ 37’’ 26° 08’ 53’’ railway fence s 11 375 46° 53’ 54’’ 26° 09’ 49’’ railway fence s 12 454 46° 54’ 08’’ 26° 04’ 07’’ railway fence s-e 13 457 46° 54’ 05’’ 26° 03’ 60’’ railway fence s-e 14 457 46° 54’ 01’’ 26° 03’ 46’’ railway fence s-e 15 457 46° 53’ 56’’ 26° 03’ 38’’ railway fence s-e 16 460 46° 53’ 56’’ 26° 03’ 27’’ railway fence s-e 17 473 46° 53’ 45” 26° 03’ 08’’ railway fence s-e 18 485 46° 53’ 50’’ 26° 02’ 37’’ railway fence s-e 19 730 46° 50’ 40’’ 25° 54’ 23’’ rocks s 20 620 46° 50’ 05’’ 25° 53’ 07’’ road and railway fance e 21 830 46° 49’ 43’’ 25° 51’ 00’’ rocks s 22 730 46° 49’ 32’’ 25° 51’ 00’’ rocks s 23 670 46° 49’ 30’’ 25° 51’ 03’’ rocks s-e 24 750 46° 49’ 00’’ 25° 49’ 54’’ rocks s-e 25 770 46° 48’ 55’’ 25° 49’ 41’’ rocks s-e 26 820 46° 47’ 60’’ 25° 48’ 12’’ rocks e 27 850 46° 47’ 53’’ 25° 48’ 13’’ rocks e 28 1060 46° 47’19’’ 25° 47’ 11’’ rocks s-e 187distribution of podarcis muralis in eastern romania in the area. by contrast, natural dispersion using road and railway fences or beds has been observed prior to this study in other populations from romanian moldavia (iftime et al., 2008). artificial dissemination is unlikely, as only one road is present in the area that links between a habitat in which the common wall lizard has been confirmed in transylvania (ghira et al., 2002) and moldavia. we hypothesise that the railway beds represent a vector that mediates the extension of the distribution range of p. muralis, as the railways may be used as migration or linkage habitats among rocky areas or man-made structures, forming small, but dynamic populations. this dispersion model, using railway beds, is considered the prior factor for the invasive success of the common wall lizard in north america, the species using anthropogenic structures as a substitute for natural habitats, expanding its distribution range and colonizing new areas (hedeen and hedeen, 1999; deichsel and gist, 2001). range expansion by means of degraded, man-made habitats has been reported for other lizard species (e.g., psammodromus algirus, bauwens et al., 1986; leiocephalus carinatus armouri, smith et al., 2004; hemidactylus turcicus, meshaka et al., 2006), the process being considered as characteristic for invasive species (cohen and carlton, 1998; ward et al., 2005; d’amore et al., 2008; ficetola and padoa-schioppa, 2008). the fact that the common wall lizard produces insular populations is a well known fact and has already been reported elsewhere (e.g., rochelle et al., 1999; crnobrnja-isailovic et al., 2005). in existent populations this is due to habitat fragmentation (crnobrnja-isailovic et al., 2005), the phenomenon being one of the major causes for genetic degradation of common wall lizard populations in particular (e.g., rochelle et al., 1999; crnobrnja-isailovic et al., 2005) and of other species generally (e.g., liolaemus multimaculatus and liolaemus gracilis – vega et al., 2000; nerodia erythrogaster neglecta and nerodia sipedon – roe et al., 2006). in the study area, podarcis muralis coexists with other reptile species – lacerta agilis, zootoca vivipara and coronella austriaca, and different amphibian species – bombina variegata, bufo bufo, lissotriton montandoni, mesotriton alpestris, lissotriton vulgaris (gherghel et al., 2008) found in the springs, streams and marsh areas inside the sites. in the “romanian red book of vertebrates” (iftime, 2005), the common wall lizard is listed as a ‘vulnerable species’, the same source considering the population from cheile bicazului as ‘heavily affected’ because of the activities from the rock quarries. acknowledgements the authors would like to thank dr. alexandru iftime (“grigore antipa” natural history museum, bucharest) for bringing to our attention the existence of the common wall lizard in site no. 1 and for supplying some bibliographic material. references arnold, n.e. (2003): reptiles and amphibians of europe (princeton field guides). princeton university press. 188 i. ghergel et alii bauwens, d., hordies, f., van damme, r., van hecke, a. (1986): notes on distribution and expansion of the range of the lizard psammodromus algirus in northern spain. amphibia-reptilia 7: 389-392. behler, j., king, f. (2000): national audubon society: field guide to reptiles and amphibians. new york: alfred a. knopf inc. borcea, m. (1976): metric and qualitative characteristics variability analysis of lacerta muralis muralis populations from moldavia. stud. cerc. biol., ser. biol. anim. 28: 169-173. (in romanian). borcea, m. (1979): general characterization of the fauna from cheile bicazului-lacu roşu region. ocrot. nat. nemtene: 167-182. (in romanian). borcea, m., vancea, s. (1981): observations regarding the amphibians and reptiles from cheile bicazului-lacu roşu region during 1978-1979. ocrot. nat. 25: 91-95. (in romanian). cohen, a.n., carlton, j.t. (1998): accelerating invasion rate in a highly invaded estuary. science 279: 555-558. covaciu-marcov, s.d., ghira, i., cicort-lucaciu, a.st., sas, i., strugariu, al., bogdan, h.v. (2006a): contributions to knowledge regarding the geographical distribution of the herpetofauna of dobrudja, romania. n. west. j. zool. 2: 88-125. covaciu-marcov, s.d., bogdan, h.v., ferenti, s. (2006b): notes regarding the presence of some podarcis muralis (laurenti 1768) populations on the railroads of western romania. n. west. j. zool. 2: 126-130. crnobrnja-isailovic, j., aleksic, i., bejakovic, d. (2005): fluctuating asymmetry in podarcis muralis populations from southern montenegro: detection of environmental stress in insular populations. amphibia-reptilia 26: 149-158. ciaglic, v. (1968): thermic regime of artificial barrier lake izvorul muntelui-bicaz. stud. biol. geol. geo. res. resort “stejarul”, pângaraţi: 130-152. (in romanian). d’amore, a., hemingway, v., wasson, k. (2009): do a threatened native amphibian and its invasive conqueror differ in response to human alteration of the landscape? biol. invasions (online first) doi:10.1007/s10530-009-9438-z. deichsel, g., gist, d.h. (2001): on the origin of the common wall lizards podarcis muralis (reptilia: lacertidae) in cincinnati, ohio. herp. rev. 32: 230-232. deichsel, g., schweiger, s. (2004): geographic distribution: podarcis muralis. herp. rev. 35: 289-290. ficetola, g.f., padoa-schioppa, e. (2008): human activities alter biogeographical patterns of reptiles on mediterranean islands. global. ecol. biogeogr. 18: 1-9. fuhn, i.e. (1960): the fauna of the people’s republic of romania. vol. xiv, fascicola i. amphibia. ed. acad. r.p.r., bucureşti. (in romanian). fuhn, i.e., vancea, şt. (1961): the fauna of the people’s republic of romania. vol. xiv, fascicola ii. reptilia. ed. acad. r.p.r., bucureşti. (in romanian). fuhn, i.e. (1969): frogs, snakes, lizards. ed. ştiin., bucureşti. (in romanian). gherghel, i., strugariu, a., ghiurca, d., cicort-lucaciu, a.s. (2008): the herpetofauna from the bistrita river basin (romania): geographical distribution. n. west. j. zool. 4: s71s103. ghira, i., venczel, m., covaciu–marcov, s.d., mara, g., ghile, p., hartel, t., torok, z., farkas, l., racz, t., farcas, z., brad, t. (2002): mapping of transylvanian herpetofauna. nymphaea, folia nat. bihariae 29: 145-203. 189distribution of podarcis muralis in eastern romania guillaume, c.-p. (1997): podarcis muralis (laurenti, 1768). in: atlas of amphibians and reptiles in europe, pp. 286-287. gasc, j.-p., cabela, a., crnobrnja-isailovic, j., dolmen, d., grossenbacher, k., haffner, p., lescure, j., martens, h., martinez rica, j.-p., maurin, h., oliveira, m.e., sofianidou, t.s., veith, m., zuiderwijk, a., eds, societas europaea herpetologica and museum national d’histoire naturelle (iegb/spn), paris. hedeen, s.e., hedeen d.l. 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(1999): forest fragmentation: wildlife and management implications, koninkliijke brill nv, leiden, netherlands. roe, j.h., gibson, j., kingsbury, b.a. (2006): beyond the wetland border: estimating the impact of roads for two species of water snakes. biol. conserv. 130: 161-168. rödder, d., solé, m., böhme, w. (2008): predicting the potential distributions of two alien invasive housegeckos (gekkonidae: hemidactylus frenatus, hemidactylus mabouia). n. west. j. zool. 4: 236-246. rödder, d. (2009): ‘sleepless in hawaii’ – does anthropogenic climate change enhance ecological and socioeconomic impacts of the alien invasive eleutherodactylus coqui thomas 1966 (anura: eleutherodactylidae)? n. west. j. zool. 5: 16-25. sahlean, t.c., strugariu, al., zamfirescu, s.r., pavel, a., puscasu, c.m., gherghel, i. 2008. a herpetological hotspot in peril: anthropogenic impact upon the amphibian and reptile populations from the baile herculane tourist resort, romania. herp. rom. 2: 37-46. smith, h.t., engeman, r.m. (2004): a review of the colonization dynamics of the northern curly-tailed lizard (leiocephalus carinatus armouri) in florida. florida field nat. 32: 107-113. strugariu, al., gherghel, i., zamfirescu, s.r. (2008): conquering new ground: on the presence of podarcis muralis (reptilia: lacertidae) in bucharest, the capital city of romania. herp. rom. 2: 47-50. vancea, s. (1958): contributions to the systematics and ecology of the lacertids from r.p.r. iii. the wall lizard (lacerta muralis muralis). stud. cerc. stiint., biol. stiint. agric., acad. r.p.r., 9: 73-84. (in romanian). vega, l.e., bellagamba, p.j., fitzgerald l.a. (2000): long term effects of anthropogenic habitat disturbance on a lizard assemblage inhabiting coastal dunes in argentina. can. j. zool. 78: 1653-1660. ward, d.f., harris, r.j., stanley, m.c. (2005): human-mediated range expansion of argentine ants linepithema humile (hymenoptera: formicidae) in new zeeland. sociobiology 45: 1-7. a review of distribution and conservation status of zamenis situla (linnaeus, 1758) (reptilia: colubridae) in bulgaria borislav naumov1, ljiljana tomović2 1central laboratory of general ecology, bulgarian academy of science, gagarin str. 2, 1113 sofia, bulgaria. e-mail: herpetology_bg@yahoo.com 2institute of zoology, faculty of biology, university of belgrade, studentski trg 16, 11000 belgrade, serbia. corresponding author. e-mail: lili@bf.bio.bg.ac.yu abstract. the leopard snake is one of the rarest snake species in bulgaria. it is included in the red data book of bulgaria as threatened species under high anthropogenic pressure. its distribution range in bulgaria includes only three very distant regions in the southern part of the country. precise literature data about the leopard snake in bulgaria are scarce. the aim of this paper is to present distribution data as well as to review the conservation status of the leopard snake in bulgaria. keywords. leopard snake, distribution, conservation, bulgaria, balkan peninsula. distribution of the leopard snake in bulgaria the leopard snake, zamenis situla (linnaeus, 1758) is distributed mainly in the balkan peninsula (dalmatian coastal region, southern bosnia and montenegro, macedonia, albania, southern bulgaria, european turkey and greece); outside the balkans, this species occurs in southern italy, malta, crete, ionian and aegean islands, western anatolia and in the crimean peninsula (obst et al., 1993; sofianidou, 1997). distribution of the leopard snake in bulgaria includes only three very distant regions in the southern part of the country (fig. 1). it was recorded for the first time in 1898 (buresch and zonkow, 1934). since then, only eleven localities were published. in the black sea coastal region, five localities were published by kovachev (1912), buresch and zonkow (1934), obst (1981), buseke (1982), beyer (2000), moravec and böhme (2003) and naumov (2005) (utm records no. 1-2; fig. 1). the last time when z. situla was recorded in the black sea region was in 2000 (beyer, 2000), at the elenite locality (utm record no. 1). at the same time, this locality is the northernmost in the whole distribution range in bulgaria, but it is still uncertain whether this locality hosts an autochthonous population or not. the locality nesebar (utm record no. 1, fig. 1) is questionacta herpetologica 2(1): 7-10, 2007 8 b. naumov and l. tomović able because the specimen was found inside the town and as naumov (2005) supposes it was probably brought from a different region and escaped from the city. in the rhodopes mountains region, only one specimen was found in asenovgrad (kovachev, 1912) (utm record no. 3; fig. 1). unfortunately, this record was never confirmed after the first finding, and according to beshkov and nanev (2002) this population probably does not exist. in the struma river valley, five localities were recorded by beshkov (1961, 1985), beshkov and dushkov (1981), biserkov (1995) and beshkov and nanev (2002) (utm records no. 5-8, fig. 1). during the last 15 years, the leopard snake was found in a few additional sites in the kresna george (struma river valley) (utm records no. 4-5, fig. 1): gabrovitsa place (220 m a.s.l.), 0.5 km se of the mouth of oshtava river (220 m a.s.l.), 1 km w of stara kresna railway station (450 m a.s.l.), 0.5 km nw of the mouth of rachkin dol river (300 m a.s.l.), and along the railway in the section between kresna inn and vlahi river (200-230 m a.s.l.). published records cover only 0.64 % of the utm-territory of bulgaria. this species inhabits dry, warm, rocky places characterized by well developed micro relief and covered with mediterranean vegetation up to 650 m a.s.l. (beshkov and nanev, 2002). potential suitable habitats of the leopard snake could be found in the wider area of struma river valley including the lower parts of the maleshevska, ograzden, belasica and pirin mountains, as well. new finding sites in the black sea coastal regions (south of burgas in the southern part) and in the east rhodopes mt. bordering region (in the fig. 1. records of zamenis situla in bulgaria (national grid utm 10x10 km reference). 9distribution of the leopard snake in bulgaria easternmost part of the arda river valley) could also be discovered with more detailed survey. conservation status of the leopard snake in bulgaria the leopard snake is considered rare or declining in several parts of its distribution range; the populations are localized and declining in crimea, italy and dalmatia (sofianidou, 1997), while its conservation status is still needed for populations in bosnia, montenegro, macedonia and albania. this species is included in the annex ii/iv of the eu habitats directive, annex ii of the bern convention and treated as dd in the iucn red list of the threatened animals (gasc et al., 1997). in bulgaria, this species is considered as threatened (beshkov, 1985) and thus is included in the red data book and protected by biodiversity protection act of bulgaria (annex ii/iii), since august, 2002. populations are mainly threatened by illegal over-collecting by the amateurs as well as destruction of the habitats. it seems that the populations from the black sea coastal region are upon the highest over collection pressure. the sozopol locality (utm record no. 2) is the most endangered, having in mind that 85 specimens deposed at the museums of bonn and prague “were apparently collected at the same locality just after hibernation in 1981-1983” according to moravec and böhme (2003). in the struma river valley region, the populations from the southern part of the kresna george (utm records no. 4-5) seem to be the most stable, giving the fact that population density is rather high. according to authors’ observations from the last fifteen years in the area, 2-3 specimens can be found per 5-6 km transect along the railway (locality no. 5). unfortunately, the habitats, as well as the populations of amphibians and reptiles in this area, will certainly be under constant threat by the construction of the highway in this region. another conservation problem could be related to reproduction and population growth: average clutch size in bulgarian populations is 2 to 4 eggs (rarely to 5) (beshkov and nanev, 2002) and hence this species has the lowest fecundity of all snake species in bulgaria. suggested conservation measures would have to include the habitats of z. situla into the natura2000 network; strict implementation of the national and international legislative (especially about illegal harvesting) is needed. building of reptile crossing sites could improve conservation of this and all other reptile species, particularly at the main road that passes through the kresna gorge area. merging of habitats of z. situla with existing nature reserve tisata up to the north to kresna inn, could give positive result on population status of this species in the whole region. population studies and detailed distribution data in the southern parts of black sea coastal area are needed for estimation of actual population status of z. situla in sozopol area. acknowledgements l. t. was supported by serbian ministry of science and environment, grant no. 143040 “evolution in heterogeneous environments”. 10 b. naumov and l. tomović references beshkov, v. 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(1997): elaphe situla (linnaeus, 1758). in: atlas of amphibians and reptiles in europe, p. 362-363. gasc, j.-p., cabela, a., crnobrnja-isailović, j., dolmen, d., grossenbacher, k., haffner, p., lescure, j., martens, h., martinez rica, j.p., maurin, h., oliveira, m.e., sofianidou, t.s., veith, m., zuiderwijk, a., eds, societas europaea herpetologica and muséum national d’histoire naturelle, paris. acta herpetologica 4(1): 29-36, 2009 nutritional perfomance of tupinambis merianae lizards fed with corn starch as source of energy harold vega parry, elisa vintiñi, osvaldo e.a. arce, mario e. manes facultad de agronomía y zootecnia, universidad nacional de tucumán, f. ameghino s/n, el manantial, (4105), tucumán, argentina. corresponding author. e-mail: mmanes@faz.unt.edu.ar abstract. efficiency in processing complex carbohydrates as a source of energy was studied in tupinambis merianae lizards. four isoproteic and isoenergetic diets in which different percentages of corn starch substituted fat (0, 10, 20 and 30 dry matter in the diet) were provided. even though consumption was similar in all diets, growth and feeding conversion rates decreased significantly with corn starch supplies of 10% and more. at the end of the trial, pancreatic alpha-amylase activity showed correlated increases, yet these were insufficient to compensate corn starch supplies. results suggest that tupinambis merianae lizards have a restricted omnivorous capacity. therefore, diet formulation for these lizards should exclude high molecular weight carbohydrates. keywords. tupinambis merianae, omnivorous capacity, energy supply, growth, feeding conversion. introduction the genus tupinambis (squamata: teiidae) comprises a group of large size lizards exclusive to the south american plain, east of the andes (presch, 1973). the southernmost species, t. merianae (formerly t. teguixin) and t. rufescens (cei and scolaro, 1982), constituted a traditional resource of aboriginal communities as a source of meat, fat and leather (donadío and gallardo, 1984; norman, 1987). in the last decades, an intense and sustained exploitation of natural populations has taken place to commercialize these species skin, employed in the design of luxury items (fitzgerald et al., 1991). between 1975 and 1986, extraction in argentina was reported to have reached over 16 million skins (chardonnet et al., 2002). this situation had international impact, causing the inclusion of these species in appendix ii of cites. although skin harvest is currently regulated, the authorized extraction quota is still very large: one million skins per year (basso et al., 2005). fortunately, captive breeding programs (mercolli and yanosky, 1990; noriega et al., 1996) supported by a better understanding of these the saurian biology allows foreseeing a more rational use without affecting natural populations. 30 h.v. parry et alii from a nutritional point of view, one of the most significant advances consisted in the design of a hatchery diet, which resulted in growth rates considerably higher than those referred to in previous literature (vega parry and manes, 2000, 2004). this advance was mainly based on the reinterpretation of dietary habits in t. merianae and t. rufescens as essentially carnivorous (vega parry et al., 2000; manes et al., 2007). it is pertinent to note the existence of ontogenetic changes in tupinambis dietary habit associated with changes in tooth structure, but always within a carnivore regime (from insects to more bulky and active preys) (presch, 1974; dessem, 1985). these results do not exclude, however, the possibility of a limited omnivorous capacity in these animals. in fact, a true omnivorous status has been reported for tupinambis by some authors, based on the presence of vegetal material in their digestive tract (milstead, 1961; donadío and gallardo, 1984; mercolli and yanosky, 1994). in this respect, we find it useful from a practical point of view, as of digestive physiology, to analyse the efficiency of these animals in processing complex carbohydrates as a source of energy. this study evaluates growth responses and pancreatic alpha-amylase activity induction in t. merianae lizards fed on corn starch, as a possible alternative integrated diet to improve their production in captivity, thus avoiding the culling of specimens from the wild. materials and methods animals and experimental conditions the study was conducted in spring, during the highest feeding rate period (november-december) using 24 ten-month-old t. merianae juveniles born in captivity and fed on a hatchery diet (vega parry and manes, 2000). these were selected from a population of approximately 200 individuals from 12 different clutches. at this stage the gender of the specimens could not be distinguished. they were treated orally with 0.5 ml/kg of levamisol chlorhydrate to preclude incidental parasites, and were placed separately in outdoor individual cages (0.9 m long × 0.6 m wide × 0.6 m high), supplied with shelter, sunny and shaded spaces, a trough and a drinking dish. troughs consisted of one-opening receptacles having a plastic cover to avoid direct sunlight and food dispersal. diets four diets were compared. one was taken as the control diet (diet a), and it consisted of bovine lean meat and refined fat, supplemented with 5% tricalcium phosphate, 0.75% avian vitamin-mineral supplement (micromix-biofarma), 0.75% common salt and 0.25% butil-hydroxitoluene (bht) on a dry matter basis. the others were prepared by replacing the fat matter in isoenergetic proportions, with 10 (diet b), 20 (diet c) and 30% (diet d) corn starch (lipids: 39.34 kj/g; corn starch: 17.58 kj/g). (hafez and dyer, 1972; inra, 1986). table 1 shows ingredients, protein and energy levels for each diet. feeding process to ensure complete intake of the different diets, a consumption test was performed prior to the experiment, using the control diet (a). from the obtained value (see results), followed the dry matter calculation of the remaining diets (b, c and d) for similar protein and energy delivery. 31nutritional performance in tupinambis merianae food was provided daily at around 1000 h, at the beginning of the diel activity period (emergence from shelters). adjustments to diet followed weight and snout-vent length (svl) increase in each individual, recorded every 5-7 days. growth was estimated in terms of body mass to the nearest 0.1 g, and svl to the nearest 0.1 cm increase. conversion efficiency was calculated as the proportion between body mass gain and dry mass eaten. (staton et al., 1990; manes et al., 2007). experimental design and statistical analysis after 2 days fasting, 24 animals with similar weights were selected. each animal was assigned a diet at random and then was placed in outdoor individual cages, thus conforming 4 groups of 6 individuals. in order to evaluate if initial live weights were similar in every treatment, an analysis of variance was conducted. results showed no significant differences in initial weights among treatments (p = 0.99). since the individuals constituted a random sample and only four diets were tested, a mixed model approach was used in order to analyze data (pinheiro and bates, 2000). the variables evaluated in the fixed part of the model were treatment and concomitant variables, and a random effect was included for the animals. in the case of total weight gain and feed conversion, initial live weights were used as concomitant variables. in the case of total svl gain, initial live weights and initial svl were used as concomitant variables. prior to the analyses, it was verified that a linear relation existed between the concomitant variables and the responses. significant components for the fixed part of the model were chosen by backward elimination. alternative models were compared by means of likelihood ratio tests (pinheiro and bates, 2000). treatments means were compared to the control by means of dunnet’s contrasts. statistical analyses were performed in r (r development core team, 2008). the r package used was nlme (pinheiro et al., 2008). diet chemical and other analysis protein, fat, ash and moisture contents of the diets were determined by wendee´s proximal analysis (aoac, 1980). diet gross energy was calculated using protein, lipid and corn starch energetic values, i.e. 23.65, 39.34 and 17.58 kj/g respectively (hafez and dyer, 1972; inra, 1986). table 1. tupinambis merianae daily supplies per 100 g of live weight. diets (*) meat (g dry matter) / protein (g) (**) fat (g) / gross energy (kj) (***) corn starch (g) / gross energy (kj) total gross energy (kj) energy to protein relationship a (0% starch) 1.39 / 1.15 0.69 / 27.43 — / — 54.62 47.50 b (10% starch) 1.39 / 1.15 0.58 / 23.06 0.25 / 4.39 55.18 47.98 c (20% starch) 1.39 / 1.15 0.46 / 18.29 0.50 / 8.79 54.27 47.19 d (30% starch) 1.39 / 1.15 0.35 / 13.92 0.75 /13.18 54.29 47.20 (*) supplemented with tricalcium phosphate (0.1 g); avian vitamin-mineral supplement (0.015 g); common salt (0.015 g) and butil-hidroxi-toluene (0.005 g). (**) n × 6.25 (correction factor). (***) values correspond to the incorporated fat, plus 4% corresponding to interstitial muscular fat. 32 h.v. parry et alii at the end of the assays, three individuals fed on each diet were euthanized with an overdose (60 mg/kg) of sodium pentobarbital (avma, 2007) injected into the caudal vein. the pancreas was removed and stored at –20 ºc. the enzyme extracts were prepared by homogenizing 150 to 300 mg of pancreatic tissue in 1.5 ml of 0.2 m na-phosphate buffer, ph 7 at 4 ºc. the extracts were centrifuged at 10000 rpm, recovering the supernatant which constituted the crude enzymatic preparation. amylase activity was determined by a kinetic method (gt lab) in diluted samples (1/100). proteins were quantified by the method of bradford (1976). an analysis of variance test was performed on data and dunnet´s post hoc test was applied. analysis was performed in r (r development core team, 2008). results consumption determination the average daily consumption recorded for the 24 individuals, fed on control diet (diet a) during 15 days was 2.5 ± 0.08 g of dry matter per 100 g of bodymass. this value was considered to provide similar protein and energy supplies in the different treatments, without food rejections. growth and feeding responses the 4 diets were supplied during a 30 days-assay in which the animals were effectively fed for 26 days. no food was provided on days when the weather was cold or rainy, since animals remained in the shelters. complete acceptance of food without rejections at the end of each day indicated a similar protein and energy intake in the different diets. the progressive replacement of fat with corn starch in diets resulted in a clear decrease in growth, as reflected both in mass as in svl (table 2). growth decrease was non-significant only for diet a and in terms of svl. correspondingly, feeding conversion rate declined significantly, beginning with a 10% corn starch substitution (table 2). although animals doubled their initial weight, still they did not show evidences of sexual dimorphism. alpha-amylase activity t. merianae juveniles fed on corn starch had their physiologic compensation, increasing their pancreatic alpha amylase activity levels. at the end of the trial, it was observed that activity levels produced by diets with 20 and 30% starch were significantly higher than those resulting from the control diet (50 and 123% respectively, table 3). discussion carbohydrate-free control diet (a) resulted in major growth and best food conversion rate in t. merianae young individuals with respect to other diets with different level of 33nutritional performance in tupinambis merianae carbohydrate. after a 30-day trial experience, individuals supplied with carbohydrate free diet exhibited a 2.2-fold increase in weight and a 1.3-fold increase in lhc (table 2). the replacement in the diet of only 17% of the fat with its energy equivalent in corn starch (diet b) was enough to produce a delay in lizard growth, which intensified with major substitutions. as protein and energy intake was similar in all treatments, and no food rejections were recorded, results can be directly attributed to the difficulty for these lizards to digest starch. the quantity of this ingredient in tupinambis diet is markedly inferior to that used in farm diets for other animals (i.e., diets for poultry, pigs and rabbits), which include a great quantity of cereal flour rich in amylaceous carbohydrates (scott et al., 1973; osman, 1982; inra, 1986). it is also lower when compared with quantities used in rats omnivorous diet models (thornburg et al., 1987) and in human diets (gray, 1992). the restricted use of starch by t. merianae appears to indicate a feature common to all carnivores (pieper and pteffer, 1980; staton et al., 1990; kienzle, 1993a). it has also table 2. growth and feeding conversion of tupinambis merianae juveniles fed on different starch levels during 30 days (mean ± se). item diet a (0% starch) diet b (10% starch) diet c (20% starch) diet d (30% starch) initial live weight (g) 667.03 ± 71.47 657.95 ± 77.63 660.41 ± 80.01 679.73 ± 87.99 final live weight (g) 1463.20 ± 129.79 1363.33 ± 130.81 1285.13 ± 134.20 1214.78 ± 135.53 total weight gain (g) 796.17 ± 58.90 705.38 ± 53.99* 624.72 ± 55.38** 535.05 ± 51.71** initial svl (cm) 26.75 ± 1.11 26.75 ± 0.86 26.33 ± 0.83 26.41 ± 1.24 final svl (cm) 33.83 ± 1.17 32.91 ± 1.15 31.91 ± 1.11 31.75 ± 1.44 total svl gain (cm) 7.08 ± 0.30 6.17 ± 0.54 5.58 ± 0.36** 5.33 ± 0.25** feed conversion: weight gain (g) / ingested dry matter (g) 1.68 ± 0.05 1.46 ± 0.06* 1.24 ± 0.04** 1.02 ± 0.03** (*, **) mean differences of treatments versus control corresponding to dunnet’s test (p < 0.05 and p < 0.01). table 3. tupinambis merianae pancreatic alpha-amylase activity responses to corn starch feeding. diets alpha amylase units / mg pancreatic protein (*) a (0% starch) 582.12 ± 41.21 b (10% starch) 635.53 ± 41.94 c (20% starch) 868.83 ± 48.62 ** d (30% starch) 1273.39 ± 196.56 ** (*) alpha amylase units: amount of enzyme needed to convert 1 μmol of substrate per minute (2-chlorine-4-nitrophenyl-alpha-d-maltotriose). (**) mean differences ± se of treatments versus control corresponding to dunnet’s test (p < 0.01). 34 h.v. parry et alii been suggested that faulty carbohydrate digestion by these animals can indirectly hinder the appropriate assimilation of proteins (kienzle, 1994; refstie et al., 2000; aleixo et al., 2002; zoran, 2002). various physiological studies have considered pancreatic alpha-amylase enzyme in numerous species as indicating omnivorous capacity, on the grounds of its regulation in relation to carbohydrate levels in diets (jain, 1976; brannon, 1990; kienzle, 1993b; topping et al., 1997; chowdhury et al., 2000). the present studies show higher pancreatic alpha-amylase enzyme activity levels in t. merianae, on account of starch contents in diet. this value doubles with the maximum starch supply (diet c, 30% starch), in contrast to a scarce increase in this activity in a strictly carnivorous animal, such as the cat (kienzle, 1993a). nonetheless, the value reported for t. merianae is still notably inferior to the one recorded for various omnivorous species, with increases by up to 500% with comparable energy substitutions (brannon, 1990). thus as in the case of other carnivores, t. merianae exhibits a limited physiologic adaptation to a typically omnivorous diet (skoczylas, 1978; kienzle, 1993a), which is consistent with the reduced length of the large intestine and the lack of structures necessary for fermentative digestion in this species (vega parry et al., 2000). even if the influence of species sexual dimorphism cannot be discarded (donadio and gallardo, 1984; mercolli and yanosky, 1990; fitzgerald et al., 1991; noriega et al., 1996) in the present results, the homogeneity and juvenile status of the studied animals, expressing final weights of 60% of the one necessary to reach sexual maturity (unpubl. data), suggests that this influence is negligible. in conclusion, previous references to tupinambis as an omnivorous species, based on discoveries of vegetal material in the digestive tract of animals living in their natural habitats (milstead, 1961; donadío and gallardo, 1984; mercolli and yanosky, 1994), should be carefully reconsidered. in this sense, the fact that these animals are keen on sweet fruit rich in monosaccharides (donadío and gallardo, 1984), and the fact that a high proportion of them are found as vegetal residues (milstead, 1961; mercolli and yanosky, 1994), seem to point at an opportunistic intake of monosaccharides rather than a real omnivorous capacity in this species. acknowledgements this research was supported by the ciunt (consejo de investigaciones de la universidad nacional de tucumán). we are grateful to adriana manes for the english translation. references aleixo, v.m., cotta, t., logato, p.v.r., gomez de oliveira, a.i., fialo, e.t. 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(2002): the carnivore connection to nutrition in cats. vet med today: timely topics in nutrition. j. am. vet. med. assoc. 221: 1559-1567. acta herpetologica 17(1): 13-20, 2022 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-11453 influence of tail injury on the development of neotropical elegant treefrog tadpoles ana glaucia da silva martins1,#, raoni rebouças2,3,*,#, isaias santos1, adão henrique rosa domingos1, luís felipe toledo2 1 ipbio – instituto de pesquisas da biodiversidade, reserva betary, iporanga, são paulo, brazil 2 laboratório de história natural de anfíbios brasileiros (lahnab), departamento de biologia animal, instituto de biologia, universidade estadual de campinas, cidade universitária zeferino vaz, 13083-970, campinas, são paulo, brazil 3 programa de pós-graduação em biologia animal, instituto de biologia, universidade estadual de campinas, cidade universitária zeferino vaz, 13083-970, campinas, são paulo, brazil *corresponding author. e-mail: raonisreboucas@gmail.com # these authors contributed equally to this work submitted on: 2021, 5th july; revised on: 2021, 1st november; accepted on: 2021, 8th november editor: simon baeckens abstract. anuran larvae in aquatic environments are important prey items for several vertebrate and invertebrate species. besides avoiding predation, there are some strategies that may reduce the physical damage in those tadpoles that survive the predation attempt. for example, the injured tadpole tail can regrow after a predator bite, but few studies have examined the consequences of such injury. we examined the consequences of three levels of injury to the tail and how this influenced development and feeding behavior of tadpoles of the neotropical elegant treefrog, dendropsophus elegans. we collected spawns and kept them in the laboratory until tadpoles reached gosner’s stages 28 to 35. then, they were separated in four experimental groups: individuals with tail trimmed in 30, 50 or 70 % of its length, and a control group, with no tail removing. we counted the days until metamorphosis, calculated the scaled mass index (smi) through weight and length of newly-metamorphosed, and evaluated the feeding frequency to evaluate the influence of tail amputation on them. we found that the time until metamorphosis was positively related to the extent of the amputation, but smi and feeding behavior were not influenced. as the time to metamorphose is related to the survivorship chances of individuals: i.e., if the aquatic environment is with high density of predators, it would be advantageous to rapidly metamorphose out of the water. however, tail injury delays the metamorphose process, which could influence the survival of the individual. keywords. anuran larvae, dendropsophus elegans, atlantic rainforest, tail loss, development, feeding. introduction most anurans present aquatic larval stages and terrestrial post-metamorphic (adult) life stages, and are susceptible to predators of both environments. in this context, several defensive strategies were already reported for tadpoles in face of predators’ attack. for example, tadpoles of pelophylax lessonae can alter their behavior in the presence of dragonfly larvae (van buskirk and arioli, 2002), and tadpoles of dryophites crysoscelis can change the morphology of their tails in order to increase swimming speed, which consequently promotes a higher probability of escaping in a possible attack of predators (mccollum and leimberger, 1997). also, other species rely on visual aspects to avoid predation, such as tadpoles of scinax machadoi, which select background colors to improve 14 ana glaucia da silva martins et alii their camouflage (eterovick et al., 2018; gontijo et al., 2018), pseudacris regilla, which alter their tail color to avoid predator attacks (benard, 2006), and boana semilineata, which uses aposematic coloration to avoid predation (d’heursel and haddad, 1999). hence, other species, such as bufo bufo, rely on chemical defenses to avoid attacks of predators (üveges et al., 2019). anurans are well known to be centralized in trophic webs (blanco-torres et al., 2020) since they are both prey and predators (rebouças et al., 2013; rebouças and solé, 2015). in this way, they evolved several strategies to avoid predation (e.g., lourenço-de-moraes et al., 2016; toledo et al., 2007). in larvae, one of the possible sublethal consequences of a predation attempt is the partial tail loss or injury (morin, 1985; touchon and wojdak, 2014; wilbur and semlitsch, 1990), but the consequences of it to individual survival are very variable. for some species, past evidence suggest that it incurs little cost for tadpoles, since they, after escaping the predation, can regenerate the tail completely (wilbur and semlitsch, 1990). for example, van buskirk et al. (2003) observed that tails may play a role as a lure, in which larger tail fins reduced predations in 16 % of the observations. indeed, although firstly reported that enlarged tail fins enables predator escaping by enabling faster swimming (smith and van buskirk, 1995), posterior studies showed that tadpoles with injured tails did not lost speed in relation to those with an intact tails (van buskirk and mccollum, 2000a). the effect on speed was significant only if large portions of the tails were removed (hoff and wassersug, 2000; van buskirk and mccollum, 2000b). however, for some species tail injuries result in less swimming performance, and consequently a higher predation risk. in dryophytes chrysoscelis, for example, tadpoles with no tail injury presented a survival almost twice as high as those with 75 % of tail loss (semlitsch, 1990). also, in bombina orientalis tadpoles presented less survivorship and longer larval period (parichy and kaplan, 1992). beyond the ecological consequences, tail loss in tadpoles can also present feeding activity modification. theoretically, if individuals need no regenerate tails after a predation attempt, they should acquire more energy through feeding to reach the maximum of tail length as less time as possible, and consequently reach the full swim performance, which is related to tail shape (van buskirk and mccollum, 2000b). however, although modification of feeding behavior is already observed in presence of predators (e.g., feminella and hawkins, 1994; pueta et al., 2016), the effects of tail loss on it, which is the most common consequence of predation attempt, still were not observed. hence, while regenerating the tail, tadpoles are in continuous growth, which per se requires a constant food intake until reach the metamorphosis stage. thus, the tail injury, and an extra acquisition of nutrients during its regeneration, must affect the feedinggrowth-time until metamorphosis balance. it is relevant because tail injuries may impact on the population survivorship coupled with the fact that this species occurs in atlantic rainforest, one of the most diverse and vulnerable environments of the world, where pandemic diseases (carvalho et al., 2017) climatic changes (moura-campos et al., 2021; rebouças et al., 2021), habitat fragmentation (becker et al., 2010; dixo et al., 2009), and introduced predators (da silva et al., 2009; de oliveira et al., 2016; forti et al., 2017) are threatening endemic anurans. therefore, this study evaluates the consequences of tadpole’s tail injuries in a neotropical anuran species, dendropsophus elegans (anura; hylidae), testing the following hypotheses: i) different levels of tail injury result in less healthy newly-metamophosed; ii) different levels of tail injury increase the time to complete metamorphosis; and iii) tail injury reduces foraging activity of tadpoles. materials and methods tadpoles of dendropsophus elegans (fig. 1) were obtained through the maintenance of egg masses collected at reserva betary, iporanga, são paulo, brazil. after hatching, each tadpole was kept in an individual aquarium (40 x 45 x 30 cm), to avoid pseudo-replicates and the influence of one individual in another, maintained at room temperature (25 ºc), and half of the water was replaced twice a week after tadpoles reach the stage 28. we used tadpoles between gosner’s (1960) stages 28 and 36 for the experiments. these stages were chosen because they comprehend most of growth and development of anuran larvae (pfab et al., 2020). environmental conditions of laboratory were constantly monitored and individuals were observed until metamorphosis. thus, our experiment began before hatching and finished after metamorphosis. after the experiment, all individuals were released in the original sampling locality. tadpole development. to evaluate the influence of tail loss in the size and growth of individuals, we selected tadpoles that measured 25 mm of total length. individuals were measured with a digital caliper (to the nearest 0.01 mm) and weighted with a digital scale (to the nearest 0.01 g). we then arranged these tadpoles into four groups, following semlitsch (1990) and figiel jr and semlitsch (1991), representing each of the treatments: i) tadpoles with 30 % of the tail clipped; ii) tadpoles with 50 % of tail clipped; iii) tadpoles with 70 % of tail clipped; 15influence of tail injury on the development of neotropical elegant treefrog tadpoles and iv) tadpoles with intact tails, which was the control group (fig 1). each group contained between 8 and 10 individuals (table 1), which were isolated in each aquarium. tail modifications were performed using a sterilized scalpel blade. individuals in all treatments were equally fed with a standard fish food (extruded aqualine), with 0.1 g every day. individuals were observed until the metamorphosis was completed (complete tail absorption), and snout-vent length (svl) of newly-metamorphosed individuals was measured with the digital caliper and body mass was weighted with the digital scale. body mass and weight were used to calculated the scale mass index (smi), which is and index that can be used as a proxy of animals’ health and fitness (peig and green, 2009). foraging. in order to evaluate the influence of partial tail loss in tadpoles foraging, we performed a second experiment also using 10 individuals measuring 25 mm in total length and between gosner’s (1960) stages 28 and 36. these individuals were separated in two treatments: i) individuals with 70 % of tail amputated; and ii) individuals with intact tails, treated as the control. tadpoles were kept individually in glass jars measuring 6.5 cm in diameter and 6 cm height, with 120 ml of water and 0.1 g of fish food. after two min of acclimation, tadpoles were observed for 12 min. during this time, the feeding frequency was observed in intervals of 20 s, and during each observation we evaluated if were feeding or not. statistical analyses. firstly, we used an analysis of variance (anova) and a student’s t test to evaluate of smi present difference between treatments (tail amputations of 30 %, 50 % and 70 %; and tail amputation per se, respectively). to evaluate the influence of tail injury on smi and on time until metamorphosis, we ran two generalized linear models analyses (glm), both using treatment (30 %, 50 % and 70 % of tail amputations and control, coded as 1, 2, 3 and 0, respectively) as predictive variable, the first one with smi of newly metamorphosed individual as response, and the second with days until metamorphosis as response. both analyses were performed using gaussian family and identity link. additionally, we ran other two glm’s, with the same parameters, to evaluate if smi or days until metamorphosis were influenced by amputation per se (all treatments were classified as “amputated”, for treatments which the tail was clipped, coded as 1, and “intact” for the control treatment, coded as 0). finally, in order to evaluate the fig. 1. an adult individual of dendropsophus elegans (a), tadpoles of control (b) and 50 % of the tail clipped treatments (c), and with regenerated tail (d). 16 ana glaucia da silva martins et alii influence of tail loss in foraging we also used a glm, but with quasipoisson family and logit link, considering “treatment” as predictive variable (control, coded as 0, or amputation, coded as 1), and the feeding frequency as response variable. all models were checked through residuals deviance, and models with more than one predictive variable and collinearity was checked through variance inflation factor (vif) through the “vif ” function of “car” package (fox & weisberg, 2019). we considered levels higher than 4 as an indicator of multicollinearity (hair et al., 2010). hence, as pos hoc tests, we used estimated marginal means to compare groups of tail-trimmed individuals with the control group through the “emmeans” package (lenth, 2020). all analyses were carried out in r 4.1.0 (r core team, 2021) considering a significance level of 5 %. results during the experiment about tadpole development, we recorded the death of four individuals: one from the control group, one from the 50 % amputation group, and two from the 70 % amputation group. all individuals from the treatment groups presented the tail totally regenerated within 12 days after the beginning of the experiment (table 1). we observed tail regeneration in all individuals that had their tail clipped (fig. 2). the average time until metamorphosis (from eggs until newly-metamorphosed) was 87.5 days for the control group (room mean temperature of 26.5 ºc; table 1). we observed no difference between treatment groups (f = 0.91, p = 0.44) or between individuals with tail amputation or not (t = -0.06, p = 0.95). newly metamorphosed individuals presented an average smi of 0.148 ± 0.012, with control group presenting 0.148 ± 0.011, 30 % group presenting 0.143 ± 0.013, 50 % group presenting 0.153 ± 0.012, and 70 % group presenting 0.149 ± 0.014. during foraging experiment, individuals with injured tail were observed feeding in an average of 36.6 ± 25.9 times, while individuals with no tail injuring were observed feeding in an average of 55.1 ± 22.9 times. in our analysis, none of the variables presented vif higher than 4 (svl = 3.02, weight = 3.35, days until metamorphosis = 1.2). we observed no influence of treatment on smi or in weight, but treatment presented a significant influence on time until metamorphosis. amputation per se showed no influence in any of our variables. regarding to foraging, we observed no influence of tail injury on feeding frequency. all model outputs are in table 2 and estimated marginal means in table 3. table 1. time until metamorphosis, snout-vent length (svl) and body mass of newly metamorphosed individuals during experimentation. values presented as mean ± standard deviation (minimum – maximum; number of individuals tested; standard error). treatment time until metamorphosis (days) svl (mm) weight (g) control 29.8 ± 11.5 (18.3 – 41.3; 9; 3.83) 12.06 ± 0.44 (11.62 – 12.49; 9; 0.14) 0.15 ± 0.02 (0.13 – 0.17; 9; 0.007) 30 % 35 ± 10.8 (24.2 – 45.8; 10; 2.98) 11.86 ± 0.46 (11.40 – 12.32; 10; 0.14) 0.14 ± 0.02 (0.12 – 0.16; 10; 0.006) 50 % 32.8 ± 7.1 (25.7 – 39.9; 9; 2.36) 12.13 ± 0.24 (11.90 – 12.37; 9; 0.08) 0.16 ± 0.02 (0.14 – 0.18; 9; 0.007) 70 % 42.0 ± 15.1 (26.9 – 57.1; 8; 5.34) 12.01 ± 0.44 (11.57 – 12.46; 8; 0.14) 0.15 ± 0.02 (0.13 – 0.17; 10; 0.006) fig. 2. days until metamorphosis of dendropsophus elegans tadpoles subjected to four treatments: control (intact tail), and 30, 50 and 70% of tail removal. the top and bottom of the boxes represent the first and last quartiles, the horizontal line within the box represents the median, the whiskers represent the tenth and 90th percentiles. asterisks represent the category of tail amputation that showed significant reduction of time until metamorphosis. 17influence of tail injury on the development of neotropical elegant treefrog tadpoles discussion we showed that although tadpoles reach metamorphosis with the same weight and size in all classes, the time spent until the end of the metamorphosis tends to increase, and it was significantly longer when 70 % of tail is removed. it means that individuals with a severe damage in tail tend to spend more time under larval stage, which can submit individuals that were already threatened by a predator under aquatic predation pressure for a longer time. also, it delays the development of adult life stage, and consequently reproduction can be retarded. therefore, a high predation pressure can influence other life stages of individuals, and in a larger scale, can impair the permanence of a population. we also observed that the feeding frequency was not significantly higher in the group with tail trimmed. some similar results were observed in other experiments involving artificial tail removing in tadpoles of aquarana catesbeiana, where individuals also had a delay in growth and development (wilbur and semlitsch, 1990). a possible explanation for these observed results is that a predation attempt does not result in increasing of uptake but in reallocation of energy, since feeding presented no increasing, and it consequently could cause a delay in development. additional studies are necessary to further elucidate the physiology of this possible energy reallocation and verify this hypothesis. we did not observe influence of tail removal on the smi of newly-metamorphosed individuals, similarly to what was reported for size in osteopilus septentrionalis (koch and wilcoxen, 2019) and hoplobatrachus rugulosus (ding et al., 2014). however, opposing results were found for other species. for example, in bombina orientalis, for which the time until metamorphosis was the same independently of the tail injury extent, newly metamorphosed individuals were smaller than those without tail injury (parichy and kaplan, 1992). likewise, tadpoles with 55 % of the tail removed resulted in smaller newlymetamorphosed individuals in pelobates cultripes (zamora-camacho et al., 2019). besides, such effect lead to a reduction in the jumping performance of post-metamorphic individuals (zamora-camacho and aragón, 2019), which could expose them to higher risk of predation on land. so, these cases highlight a trade-off: tadpoles will either stay longer in the water, exposed for a longer time to aquatic predators but with newly metamorphosed with an ‘ideal’ size, with less exposure to terrestrial predators (semlitsch, 1990; wilbur and semlitsch, 1990), or they could leave the water smaller and with some mobility handicaps, which could limit the exposure to aquatic predators but exposing them more to terrestrial predation table 2. coefficients of generalized linear model analysis, which considers the percentage of tail injury as a predictor of (1) scaled mass index (smi) and (2) days until metamorphosis; tail injury per se as a predictor of (3) smi and (4) days until metamorphosis; and (5) tail injury as a predictor of feeding frequency. all models present degrees of freedom = 35 and significant values are in bold. estimate std error t value p (1) smi ~ % tail injury intercept 0.15 0.004 34.42 <0.001 30% -0.005 0.006 0.79 0.44 50% 0.005 0.006 -0.81 0.42 70% 0.001 0.006 0.25 0.81 (2) days until metamorphosis ~ % tail injury intercept 29.78 2.99 9.95 <0.001 30% 5.22 4.13 1.27 0.21 50% 3 4.23 0.71 0.48 70% 12.22 4.36 2.8 0.008 (3) smi ~ tail injury intercept 0.15 0.004 34.05 <0.001 tail loss 0.0003 0.005 0.05 0.96 (4) days until metamorphosis ~ tail injury intercept 29.79 3.15 9.56 <0.001 tail loss 6.56 3.6 1.82 0.08 (5) feeding frequency ~ tail injury intercept 4.01 0.18 22.56 <0.001 tail loss -0.41 0.28 -1.45 0.17 table 3. summary contrasts of estimated marginal means, used as a pos hoc test to compare groups of different levels of tail injury with the control group. significant value is in bold. estimate std. error p smi ~ % tail injury 30% control -0.005 0.006 0.73 50% control 0.005 0.006 0.75 70% control 0.001 0.006 0.98 days until metamorphosis ~ % tail injury 30% control 5.2 4.13 0.44 50% control 3 4.23 0.79 70% control 12.22 4.36 0.01 smi ~ tail injury injuried control 0.0003 0.005 0.96 days until metamorphosis ~ tail injury injuried control 6.56 3.6 0.07 feeding frequency ~ tail injury injuried control -0.41 0.28 0.15 18 ana glaucia da silva martins et alii in the developmental stage that they are most susceptible to predation (toledo et al. 2007). in d. elegans we observed that the strategy adopted is the first one. tadpoles threatened by a predator spend more time under larval stage, i.e., reduce the growth rhythm, but reach the same size after metamorphosis, and consequently the same smi, than unharmed individuals. we also did not observe change in feeding frequency as a result of tail injury. it probably implies that the tail regeneration was not provided by an extra acquisition of energy – expected by a more frequent feeding. although these stages (stages 28 until 36) are those when generally tadpoles present the most significant growth and energy uptake (pfab et al., 2020), we did not observe any difference when the tail was lost. considering that for some species locomotion is more important than feeding, such as in pleurodema thaul (pueta et al., 2016) and pelophylax lessonae (steiner, 2007), and that tail fins enable fast swimming (smith and van buskirk, 1995), perhaps for d. elegans the regeneration of tail is energetically more important than time until metamorphosis. consequently, there is not an increase in feeding to regenerate the tail, but a reallocation of the energy that otherwise would be used to growth. thus, such observation supports the hypothesis of a probable reallocation of the energy from the regular development/metamorphosis process directed to tail regeneration. however, different results were reported for other species. for example, in ascaphus truei, a simple clue of predators’ presence was enough to modify the foraging in tadpoles, which reduced up to six-folds its foraging activity (feminella and hawkins, 1994). also, similar results were observed for rana sylvatica (fraker, 2010) and rana clamitans (fraker, 2008, 2009). it efforts that more studies are necessary to elucidate this process of energy reallocation during larval stage until metamorphosis. our experiments showed consequences of predatory events in d. elegans tadpoles. tail injury caused by predators can result in several consequences for the individuals, decreasing their survivorship, affecting tadpole morphology (nunes et al., 2010), and swimming speed (figiel jr and semlitsch, 1991). besides, as tadpoles of d. elegans remained more time in the larval stage when the tail was injured, this fact may have several consequences, since evolutionary approach until conservation of native populations. acknowledgements we thank to all volunteers and ipbio – instituto de pesquisas da biodiversidade by its help during the experimentation. the study was conducted with the sampling permits of icmbio (sisbio #24013 and #29484), sisgen (#adea910), and the animal ethics committee approval (ceua #2405-1). this study was supported by coordination for the improvement of higher education personnel (capes finance code 001), são paulo research foundation (fapesp #2016/25358-3; 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(2019): simulated predation pressure in pelobates cultripes tadpoles modulates morphology at the metamorphic stage. curr. zool. 65: 651-656. xi international symposium on the mediterranean lacertid lizards marco mangiacotti1, pietro lo cascio2, claudia corti2, marta biaggini2, miguel angel carretero2, petros lymberakis2 the directional testes asymmetry increases with temperature in seven plateau brown frog (rana kukunoris) populations hai ying li1, man jun shang2, jie guo2, bo jun chen2, peng zhen chen2, tong lei yu1,* influence of tail injury on the development of neotropical elegant treefrog tadpoles ana glaucia da silva martins1,#, raoni rebouças2,3,*,#, isaias santos1, adão henrique rosa domingos1, luís felipe toledo2 the effect of weight and prey species on gut passage time in an endemic gecko quedenfeldtia moerens (chabanaud, 1916) from morocco jalal mouadi1,*, panayiotis pafilis2, abderrafea elbahi3, zahra okba3, hassan elouizgani3, el hassan el mouden4, mohamed aourir1 a contribution to the knowledge on the diet and food preferences of darevskia praticola (reptilia: lacertidae)§ emiliya vacheva*, borislav naumov first report on two loggerhead turtle (caretta caretta) nests in the aeolian archipelago (southern italy) monica francesca blasi1,*, sandra hochscheid2, roberta bardelli3, chiara bruno1, carolina melodia1, perla salzeri1, paolo de rosa4 and paolo madonia5 threatened and extinct amphibians and reptiles in italian natural history collections are useful conservation tools franco andreone1,*, ivano ansaloni2, enrico bellia3, andrea benocci4, carlotta betto5, gabriella bianchi6, giovanni boano7, antonio borzatti de loewenstern8, rino brancato9, nicola bressi10, stefano bulla11, massimo capula12, vincenzo caputo barucchi13, p re-description of external morphology and factors affecting body and tail shape of the stone frog tadpoles’ brena da silva gonçalves1,*, carla. d. hendges2, bruno madalozzo2, tiago g. santos2,3 preliminary data on the diet of chalcides chalcides (squamata: scincidae) from northern italy andrea ciracì1, edoardo razzetti2, maurizio pavesi3, daniele pellitteri-rosa4,* the high diversity and phylogenetic signal of antipredator mechanisms of the horned frog species of proceratophrys miranda-ribeiro, 1920 (amphibia: anura: odontophrynidae) cássio zocca1,2,*, ricardo lourenço-de-moraes3, felipe s. campos4, rodrigo b. ferreira1,2,5 © firenze university press www.fupress.com/ah acta herpetologica 5(1): 13-17, 2010 tarentola and other gekkonid records from djebel ouarkziz (sw morocco) francisco ceacero1,2, enrique garcía-muñoz3,5, luis pedrajas4, ana perera5, miguel a. carretero5 1 departamento de ciencia y tecnología agroforestal, universidad de castilla-la mancha. campus universitario s/n, 02071 albacete, spain. 2 sección de recursos cinegéticos, instituto de desarrollo regional. campus universitario s/n, 02071 albacete, spain 3 departamento de biología animal, biología vegetal y ecología, universidad de jaén. campus de las lagunillas s/n, 23071 jaén, spain. 4 centro de rescate de anfibios y reptiles. c/ real 48, 23680 alcalá la real, spain. 5 cibio, centro de investigação em biodiversidade e recursos genéticos. campus agrário de vairão, 4485-661 vairão, portugal. submitted on: 2009, 31st august; revised on 2010, 8th february; accepted on 2010, 16th march. abstract. tarentola mauritanica pallida was recorded for the first time far inland in the djebel ouarkziz and oued drâa area, in south-western morocco. the taxonomic characters proposed to identify this subspecies, t. m. juliae, and t. boehmei are discussed in view of the specimens observed during this survey. other observations of gekkonid lizards in this area are also reported. keywords. tarentola mauritanica pallida, taxonomy, morocco. introduction south-western morocco harbours a wide diversity of gekkonid lizards including tarentola mauritanica (t. m. pallida and t. m. juliae), t. boehmei, t. annularis annularis, t. ephippiata hoggarensis, t. chazaliae, ptyodactylus oudrii, quedenfeldtia moerens, stenodactylus petrii, s. sthenodactylus sthenodactylus, saurodactylus brosseti, and tropiocolotes algericus (bons and geniez, 1996; geniez et al., 2004; sindaco and jeremčenko, 2008). within this region, djebel ouarkziz and drâa valley are two large geographic areas relevant for the biogeography of some reptile species. djebel ouarkziz is a mountainous region northern to drâa valley roughly oriented from wsw to ene with great daily thermal fluctuations, scarce precipitations, and low vegetation cover (monod, 1958; el gharbaoui, 1987). there, reptiles with both macaronesian and mediterranean affinities are 14 f. ceacero et alii present. even being a low altitude mountain chain (770 m a.s.l.) it constitutes a sizeable barrier to exchanges between northern and southern species (i.e., genus uromastyx, acanthodactylus, and sphenops; mateo et al., 1998; fonseca et al., 2009). drâa basin is a dry region occupied by hamada desert with acacia tortilis raddiana and argania spinosa trees and fagonia sp. and nitraria sp. scrubs in the basin (guinea, 1948; ozenda, 1991; personal observation). a four days survey in the djebel ouarkziz and drâa valley area in the triangle between the cities of assa, zag, and the aouinet torkoz oasis was conducted from 20 to 23 may 2008 and the collected records of gekkonid lizards are here shown. all the specimens were identified through 5th toe lamellae number and characteristics of dorsal tubercles (schleich et al., 1996; geniez et al., 2004; table 1). a t. boehmei sample was confirmed genetically using the 12s rrna fragment following the same primers, methodology and conditions detailed in harris et al. (2004a). a gravid female (specimen #1) and a juvenile which corresponded morphologically to t. m. pallida were found in a ruined adobe house in the crossing of drâa river basin and assa-zag road (locality 1; 28º31’n, 9º24’w, 214 m a.s.l.). an adult male was also found in djebel ouarkziz southern slope near the assa-zag road (specimen #2; locality 2; 28º25’n, 9º25’w, 340 m a.s.l.). additionally, new localities for this species were found in other regions. a gravid female of t. m. juliae was found at kerdous pass, along the road from tiznit to tafraoute (specimen #3; locality 3; 29º33’n, 9º22’w; 1092 m a.s.l.) and one juvenile (specimen #4) and one gravid female (specimen #5) of the same subspecies were found in agadir (locality 4; 30º25’n, 9º37’w; 5 m a.s.l.). t. boehmei was only found in aouinet torkoz oasis (specimens #6 and #7; locality 5; 28º28’n, 9º51’w; 285 m a.s.l.; genbank accession number gu593722). in addition to tarentola, other geckos found in this area during field work were tropiocolotes algericus (28º16’n, 9º20’w, 300 m a.s.l.) and s. s. sthenodactylus (28º35’n, 9º25’w, 299 m a.s.l.; 28º28’n, 9º24’w, 223 m a.s.l.; 28º18’n, 9º21’w; 283 m a.s.l.; both sides of djebel ouarkziz). geniez et al. (2000) indicated that t. m. pallida has not been yet reported in this area but it was expected to occur there. records from drâa river basin and djebel ouarkziz confirm this comment, and significantly increase the distribution of this subspecies to the east (fig. 1). it is possible that the dry basin of the oued drâa allow t. m. pallida to table 1. taxonomic characters of the observed specimens of genus tarentola. the localities of the specimens (see text) are shown in fig. 1. numbers in ‘origin’ column refers to localities in text and figure 1. origin ssp. 5th toe lamellae dorsal tubercles colour #1 oued drâa (1) t. m. pallida 17 keeled without secondary rosette opaque dark grey #2 djebel ouarkziz (2) t. m. pallida 17 keeled without secondary rosette opaque dark grey #3 anti-atlas (3) t. m. juliae 17 keeled with (small) secondary rosette opaque brownish grey #4 agadir (4) t. m. juliae 16 keeled with secondary rosette opaque beige #5 agadir (4) t. m. juliae 17 keeled with secondary rosette opaque beige #6 aouinet torkoz (5) t. boehmei 16 keeled without secondary rosette traslucent rosy #7 aouinet torkoz (5) t. boehmei 16 keeled without secondary rosette traslucent rosy 15tarentola and other gekkonid records from djebel ouarkziz fig. 1. distribution of tarentola mauritanica ssp. (squared grey) and t. boehmei (uniform grey) in morocco. distribution maps were extracted from iucn (2009). numbers correspond to the sampling localities indicated in the text. (▲) indicate proposed sympatric localities for both species. discontinuous lines show the limits among the proposed subspecies of t. mauritanica (mauritanica in the north, juliae in the middle and pallida in the south; joger, 1984; geniez et al., 1999, 2004). fig. 2. characteristics of dorsal tubercles of a) t. m. juliae from agadir (specimen #5), b) t. m. juliae from anti-atlas (specimen #3), c) t. m. pallida from djebel ouarkziz (specimen #2), and d) t. bohemei from aouinet torkoz (specimen #7). 16 f. ceacero et alii expand inland in this northern range of its distribution (in this case more than 100 km), as previously proposed for stenodactylus petrii (herrmann and herrmann, 2003). in south-western morocco, t. mauritanica ranges the atlantic coast, while inland populations are scarce because it is substituted by t. boehmei (bons and geniez, 1996). thus, both species (specifically the subspecies t. m. juliae) where proposed to be vicariant, although they may be sympatric in some places as aoulouz, tafraoute, ouarzazate and aît bekkou (joger, 1984; bons and geniez, 1996; geniez et al., 1999). however, during this survey t. boehmei was only found in aouinet torkoz oasis, and thus, no strict sympatry was detected. both t. boehmei specimens found had 16 lamellae under 5th toe. thus, the proposed characters of a higher number of lamellae in t. boehmei (more than 19 scales following geniez et al., 2004; and 21 to 23 following schleich et al., 1996) than in t. m. pallida (less than 19 scales following geniez et al., 2004; 16 to 20 scales following schleich et al., 1996) does not seem to have taxonomic value. t. mauritanica specimens were morphologically assigned to one of the proposed subspecies juliae or pallida. however, t. m. juliae specimen #3 (from anti-atlas) showed intermediate dorsal tubercles characteristics (fig. 2) between specimen #5 (t. m. juliae from agadir) and specimens #1 and #2 (t. m. pallida from djebel ouarkziz). these findings further confirm previous phylogenetic analyses showing that the mitochondrial lineages in morocco are not in concordance with the current subspecific arrangement (harris et al. 2004 a, b). moreover, none of the t. m. pallida specimens found showed the translucent aspect described by geniez et al. (1999, 2004). nevertheless, probably it was because they all were captured when active during daylight or before dawn. finally, both t. algericus and s. s. sthenodactylus were previously known to occur in djebel ouarkziz area, and these records does not represent a significant increase in their distribution range, although records of s. s. sthenodactylus fulfil a distribution gap in the assa region (bons and geniez, 1996). however, this area still remains poorly known and more fieldwork is needed. acknowledgements authors wish to thank s. fahd, r. sindaco and m.a.l. zuffi, whose comments improved the final manuscript. a. perera is supported by the post-doc grant sfrh/bpd/26546/2006 and lab costs were covered by project ptdc/bia-bde/67678/2006 (to mac) both from fundação para a ciência e tecnologia (fct, portugal). references bons, j., geniez, ph. (1996): anfibios y reptiles de marruecos (incluido el sahara occidental): atlas biogeográfico – amphibiens et reptiles du maroc (sahara occidental compris). asociación herpetológica española, barcelona. el gharbaoui, a. (1987): la grande enciclopédie du maroc. géographie physique et géologie. g.e.i. éditions, rabat. 17tarentola and other gekkonid records from djebel ouarkziz fonseca, m.m., brito, j.c., paulo, o.s., carretero, m.a., harris, d.j. (2009): systematic and phylogeographical assessment of the acanthodactylus erythrurus group (reptilia: lacertidae) based on phylogenetic analyses of mitochondrial and nuclear dna. mol. phylogenet. evol. 51: 131-142. geniez, ph., escatllar, j., crochet, p.a., mateo, j.a., bons, j. (1999): a new form of the genus tarentola (reptilia, gekkonidae) in morocco. herpetozoa 12: 187-194. geniez, ph., mateo, j.a., bons, j. (2000): a checklist of the amphibians and reptiles of western sahara. herpetozoa 13: 149-163. geniez, ph., mateo, j.a., geniez, m., pether, j. (2004): amphibians and reptiles of the western sahara. chimaira, frankfurt am main. guinea, e. (1948): catálogo razonado de las plantas del sáhara español. anales del jardín botánico de madrid 8: 357-442. harris, d.j., batista, v., carretero, m.a., ferrand, n. (2004a): genetic variation in tarentola mauritanica (reptilia: gekkonidae) across the strait of gibraltar derived from mitochondrial and nuclear dna sequences. amphibia-reptilia 25: 451-459. harris, d.j., batista, v., lymberakis, p., carretero, m.a. (2004b): complex estimates of evolutionary relationships in tarentola mauritanica (reptilia: gekkonidae) derived from mitochondrial dna sequences. mol. phylogenet. evol. 30: 855-859. herrmann, p.a., herrmann, h.w. (2003): new records and natural history notes for amphibians and reptiles from southern morocco. herpetol. rev. 34: 76-77. iucn (2009): iucn red list of threatened species. version 2009.2. www.iucnredlist.org. downloaded on 01 february 2010. joger, u. (1986): taxonomische revision der gattung tarentola (reptilia: gekkonidae). zool. beit., bonn. 35: 129-174. mateo, j.a., geniez, ph., lópez-jurado, l.f., bons, j. (1998): chorological analysis and morphological variations of saurians of the genus uromastyx (reptilia, agamidae) in western sahara. description of two new taxa. rev. esp. herpet. 12: 97-109. monod, th. (1958): majâbat al koubrâ. contribution à l’étude de l’empty quarter ouestsaharien. mém. i.f.a.n., 52: 1-407. ozenda, p. (1991): flore et végétation du sahara. editions du cnrs, paris. schleich h.h., kästle, w., kabisch, k. (1996): amphibians and reptiles of north africa. koeltz scientific books, koeningstein. sindaco, r., jeremčenko, v.k. (2008): the reptiles of the western palearctic. 1. annotated checklist and distributional atlas of the turtles, crocodiles, amphisbaenians and lizards of europe, north africa, middle east and central asia. belvedere / societas herpetologica italica, latina. acta herpetologica 2006 2 preliminary data on the biometry and the diet of a microinsular population of podarcis wagleriana (reptilia : lacertidae) preliminary data on the biometry and the diet of a microinsular population of podarcis wagleriana (reptilia: lacertidae) pietro lo cascio 1, salvatore pasta 2 1 associazione nesos, via vittorio emanuele 24, i-98055 lipari (me). e-mail: pietrolocascio@libero.it 2 via salvatore bertini 9, i-90129, palermo. e-mail: salvopasta@alice.it abstract. the results of some investigations on the podarcis wagleriana population of la scuola islet (stagnone lagoon archipelago, w sicily) are here presented. adult mean svl was 69.5 mm and 61.6 mm for males and females respectively. the analysis of faecal pellets showed that the most important prey types were formicidae (32%), coleoptera (15%), and other hymenoptera (13%). the proportion of vegetal matter (occurring in 35% of the examined pellets) was higher than in sicilian populations, suggesting that plant consumption in this micro-insular environment may play a more significant role in the diet of the species. keywords. podarcis wagleriana, ecology, sicily, stagnone islands, micro-insularity. despite of its considerable importance both from zoogeographical and conservational points of view (lo valvo, 1998), present information on the endemic sicilian wall lizard, podarcis wagleriana gistel, 1868, is still fragmentary and almost lacking (böhme, 1986; corti and lo cascio, 2000; capula, 2006). the distribution of this species includes sicily and several circum-sicilian islands (turrisi and vaccaro, 1998; corti et al., 2006). p. wagleriana is sympatric with the italian wall lizard (podarcis sicula) in all its range, but on la scuola, the smaller islet where the species occurs, it is the only lacertid lizard (lo valvo and massa, 1999). in this paper we present and discuss the preliminary data on the biometry and the trophic ecology of this population. la scuola (37°51’76”n-12°27’39”e greenwich) is the smallest islet of the stagnone archipelago, located 1,650 m off the w sicilian coast. it has a surface of 8,100 m2 and a maximum elevation of 3 m a.s.l. the islet is nowadays uninhabited, but it was used during the 20th century as a “lazaretto” (hospital for epidemic illnesses). sandy and rocky seashores are fulfilled with rotten leaves of marine plants, while several chenopod species (such as arthrocnemum macrostachyum and halimione portulacoides) cover the slopes along the perimeter of the islet. on the flat top, lygeum spartum-dominated halo-xerophilous grassland occurs, with few scattered species-poor assemblages in the seagull nesting areas (dominated by chrysanthemum coronarium and galactites tomentosa). apart from p. wagleriana, the ocellated skink (chalcides ocellatus) is the only reptile species so far acta herpetologica 1(2): 147-152, 2006 148 p. lo cascio and s. pasta recorded for the islet (lo valvo and massa, 1999). among the other vertebrates, mediterranean yellow-legged gull (larus cachinnans) has colonised the islet since the 1980s with about 15-20 nesting pairs (lo valvo and massa, 1999); we also observed several marks of rats, probably rattus rattus. since 1984 la scuola has been included as protected area in the riserva naturale orientata “isole dello stagnone di marsala”. the islet was visited in the early june 2004. all the collected lizards were sexed, examined and released in the field. for each lizard, snout-vent length (svl) and tail (if not regenerated) were measured with a “mauser” calliper (0.1 mm accuracy). the faecal pellets were obtained from handled specimens or collected from the substrate in areas where lizards’ activity was previously observed, in order to avoid confusion with those of the sympatric chalcides ocellatus (which pellets are anyway characterised by different shape and size [pers. obs.]). faecal contents were examined in the laboratory under a stereoscope. remains were identified at order or family rank; prey length was obtained measuring the remains with a micrometer eyepiece and calculated by using regression equations (hódar, 1997). when it was possible, plant matter and seeds were identified by comparing them with fresh vegetal samples collected on the islet. statistical analyses were done by using spss 11.5 for windows pc package, with alpha set at 5% and all tests being two-tailed. adult males svl range from 65 to 75 mm (n = 8, mean = 69.50, se 1.50), whereas adult females range from 59 to 64 mm (n = 8, mean = 61.50, se 0.68). as expected, and despite the very small sample size, population body size differed significantly among sexes (oneway anova: f7,8 = 37.39, p = 0.0001), with males bigger than females. tail length range from 123 to 146 mm in males, and from 85 to 123 mm in females. among the measured (n = 16) and the observed individuals (n = 52), only 3 on the whole had the tail broken or regenerated. dorsal pattern did not differ from those of the sicilian populations. males resulted characterised by intensely yellowish (about 45% of the observed specimens) or reddish (about 30%) ventral parts, throats with dense dark spots, and noteworthy lateral maculae. four specimens (3 males and 1 female) with a concolor pattern, that is characterised by the absence of dorsal design, were also observed. all the examined females exhibited marks of copula, and some of these revealed at palpation that were boring oviductal eggs. we examined 20 faecal pellets (8 obtained from handle specimens and 12 collected from the substrate), from which remains of 65 preys were identified at family or order rank (mean = 3.25, sd = 1.67 identifiable food items per pellet). the diet is mainly made up of arthropods, and formicidae (32.3%), coleoptera (15.3%) and other hymenoptera (13.8%) are the most important preys (see table 1). five other taxa were detected in the prey spectrum with percentages of appearance below 10. furthermore, plant consumption seems to represent a remarkable food resource for these lizards. vegetal matter was found in 35% (n = 7) of the examined faecal pellets, with an average volume in percentage of 15.50 ± 5.64 sd per pellet. prey size was determined in 83% (n = 54) of the total sample of consumed items. a size distribution peak results at 3-6 mm (fig. 1), which matches very good with the average estimated length of the majority of formicidae and coleoptera preyed by lizards. concerning the preys from the 8 faecal pellets obtained from handle specimens (3 males and 5 females), there are no significant correlation between svl and mean prey size (r = -0.073, p = 0.86), number of preys (r = -0.515, p = 0.19), and no significant differences among sexes in the number of consumed preys (kruskal-wallis test, h = 2.460, p = 0.11). the food categories frequency did not differ significantly among sexes 149preliminary data on the biometry and the diet of podarcis wagleriana fig. 1. prey size distribution inferred from faecal pellets of podarcis wagleriana. symbols for prey categories: a, 0-3 mm; b, 3.1-6 mm; c, 6.1-9 mm; d, 9.1-12 mm; e, > 12 mm. table 1. podarcis wagleriana dietary composition expressed in terms of number of items (ni), percentage of total (n%), and number (np) of pellets containing that prey type. in the column n% vegetal matter is expressed in terms of average proportion of the total pellet volume occupied by plant matter. prey type ni n% np animals unidentified arthropoda 7 10.7 6 crustacea amphipoda 4 6.1 4 crustacea isopoda 2 3.0 2 araneae 3 4.6 3 heteroptera 3 4.6 2 coleoptera 10 15.3 6 diptera 6 9.2 4 hymenoptera formicidae 21 32.3 11 other hymenoptera 9 13.8 6 plants and seeds vegetal matter – 15.5 7 parietaria seeds 11 – 2 150 p. lo cascio and s. pasta (χ2 test = 6.916, 2×10 contingency table, p = 0.66). anyway, it is interesting to observe that crustacea amphipoda were found only in females’ pellets (3 out of 5), whereas no males’ pellets contained this prey type. as expected, the diet results mainly insectivorous, and the main bulk of prey is constituted by ants, which probably are the most common and abundant arthropods on the islet. mirmecophagy is often frequent in insular populations of the genus podarcis (e.g. ouboter, 1981; quayle, 1983; chondropoulos et al., 1993; pérez-mellado and corti, 1993; adamopoulou et al., 1999; grbac et al., 2001; luiselli et al., 2004; lo cascio et al., 2004) and, even if in smaller percentage, formicidae have been detected among the most important prey-groups of p. wagleriana also in the main island of sicily (sorci, 1990). on the contrary, the diet of the lizards at la scuola includes both terrestrial (coleoptera, formicidae) and flying preys (other hymenoptera, diptera), while in sicily it seems primarily based on terrestrial arthropods (sorci, 1990). on this islet, p. wagleriana seems to use a broader variety of preys than in sicily, consistently with the poor faunal assemblages which presumably occur on this environment. therefore, it can be considered a trophic opportunist and a widely foraging predator (sensu huey and pianka, 1981). amphipoda seem to be preyed almost exclusively by female lizards. these crustaceans have been found occasionally in the diet of coastal or micro-insular podarcis (pérez-mellado and corti, 1993; davenport and dellinger, 1995; adamopoulou and legakis, 2002), even if they did not result exclusive of any specific sex and/or age class among these populations. also, we have seen lizards along the coastal belt, as far as the intertidal zone, catching sand-hoppers and other invertebrates over and into the rotten leaves of marine plants on the shore. most part of them (13 out of 15) were females and only 2 resulted to be subadult males. a possible explanation is that sunny and exposed intertidal zones could represent just “peripherical” part of the lizards’ territories and, thus, exploited mostly by subadults and females. however, if sand-hoppers can be considered as an economically profitable prey-type (due to their abundance, low searching cost, etc.), why adult males do not seem to use this source remains unexplained, and further investigations are needed to confirm the occurrence of different food electivity and/or foraging behaviour within this population. finally, the rather high proportion of vegetal matter found in the pellets deserves special attention. it is well known that in insular environments, insectivorous lizards expand their diet to include plant parts (e.g. sadek, 1981; pérez-mellado and corti, 1993). sorci (1990) has indicated a low percentage (about 5%) of plant matter within the dietary spectrum of this species in sicily. our results suggest that plant consumption is more conspicuous and widespread among the individuals of la scuola population, judging from the high proportion of faecal pellets with vegetal matter. plant parts (even seeds) have a high water and nutritional content and represent an important source of energy (golley, 1961). plant matter in the diet of these lizards seems to confirm the association between (partial) herbivory and insularity, which according to van damme (1999) can be interpreted as adaptation to (i) a reduced arthropod availability and (ii) a low predation pressure. at la scuola, the potential predators can to be represented by the rat and the seagull, although this species is not considered an effective predator of lizards (see araujo et al., 1977). anyway, other traits, such as the low number of regenerated/broken tails (see van damme, 1999), suggest that only a fair predation pressure might occur on this islet. 151preliminary data on the biometry and the diet of podarcis wagleriana acknowledgements we are grateful to mr felice parrinello, who guided us dribbling the posidonia banks of the “stagnone”, and provided useful information about the history of la scuola islet; to dr claudia corti and two anonymous referees, for the critical reviews of the manuscript. we are also indebted to prof. bruno massa, who kindly allowed us to use the stereoscopic equipment at the senfimizo department of the university of palermo. references adamopoulou, c., legakis, a. (2002): diet of a lacertid lizard (podarcis milensis) in an insular dune ecosystem. israel j. zool. 48: 207-219. adamopoulou, c., valakos, e.d., pafilis, p. (1999): summer diet of podarcis milensis, p. gaigeae and p. erhardii (sauria: lacertidae). bonn. zool. beitr. 48(3-4): 275-282. agnesi, v., macaluso, t., orrù, p., ulzega, a. (1993): paleogeografia dell’arcipelago delle egadi (sicilia) nel pleistocene sup.-olocene. naturalista sicil. (4)17(1-2): 3-22. araujo, j., muñoz-cobo, j., purroy, f.j. (1977): las rapaces y aves marinas del archipiélago de cabrera. naturalia hispanica 12: 1-94. böhme, w. (1986): podarcis wagleriana (gistel, 1868) – sizilianische mauereidechse. in: handbuch der reptilien und amphibien europas, bd. 2/ii, echsen (sauria) iii (lacertidae iii: podarcis), p. 377-387. böhme, w., ed, aula-verlag, wiesbaden. capula, m. (2006): podarcis wagleriana gistel, 1868. in: atlante degli anfibi e dei rettili d’italia, p. 494-497. sindaco, r., doria, g., razzetti, e., bernini, f., eds, polistampa, firenze. chondropoulos, b., maragou, p., valakos, e.d. (1993): food consumption of podarcis taurica ionica (lehrs, 1902) in the ionian islands (greece). in: lacertids of the mediterranean region. a biological approach, p. 173-182. valakos, e.d., böhme, w., pérezmellado, v., maragou, p., eds, hellenic zool. soc., athens. corti, c., lo cascio, p. (2000): le ricerche ecologiche sui sauri italiani. in: atti i congresso nazionale s.h.i. (2-6 ottobre 1996), p. 523-529. giacoma, c., ed. museo regionale scienze naturali, torino. corti, c., lo cascio, p., razzetti, e. (2006): erpetofauna delle isole italiane. in: atlante degli anfibi e dei rettili d’italia, p. 613-643. sindaco, r., doria, g., razzetti, e., bernini, f., eds, polistampa, firenze. davenport, j., dellinger, t. (1995): melanism and foraging behaviour in an intertidal population of the madeiran lizard podarcis (= lacerta) dugesii (milne-edwards, 1829). herpetol. j. 5: 200-203. golley, f.b. (1961): energy values of ecological materials. ecology 42: 581-584. grbac, i., leiner, s., perović, f. (2001): thermoregulation and diet composition of insular populations of podarcis melisellensis and lacerta oxycephala. biota 2(suppl.): 38. hódar, j.a. (1997): the use of regression equations for the estimation of prey length and biomass in diet studies of insectivore vertebrates. miscellània zool. 20(2): 1-10. huey, r.b., pianka, e.r. (1981): ecological consequences of foraging mode. ecology 62: 991-999. 152 p. lo cascio and s. pasta lo cascio, p., luiselli, l., corti, c. (2004): preliminary data on the ecology of podarcis filfolensis at lampione islet (pelagie islands, channel of sicily). in: abstracts book 5th intern. symp. lacertids medit. basin, p. 25, corti, c., lo cascio, p., eds, firenze university press, firenze. lo valvo, f. (1998): status e conservazione dell’erpetofauna siciliana. naturalista sicil. (4)22(1-2): 53-71. lo valvo, f., massa, b. (1999): lista commentata dei vertebrati terrestri della riserva naturale orientata “isole dello stagnone” (sicilia). naturalista sicil. (4)23(3-4): 419-466. luiselli, l., capula, m., corti, c., lo cascio, p., pérez-mellado, v. (2004): preliminary data on the feeding ecology of podarcis raffonei (reptilia, lacertidae), a threatened endemic lizard of the aeolian islands (mediterranean sea). in: the biology of lacertid lizards. evolutionary and ecological perspectives, p. 223-229. pérez-mellado, v., riera, n., perera, a., eds, institut menorquí d’etudis, maò. ouboter, p.e. (1981): the ecology of the island-lizard podarcis sicula salfii: correlation of microdistribution with vegetation coverage, thermal environment and food-size. amphibia-reptilia 2: 243-257. pérez-mellado, v., corti, c. (1993): dietary adaptations and herbivory in lacertid lizards of the genus podarcis from western mediterranean islands (reptilia: sauria). bonn. zool. beitr. 44(3-4): 193-220. quayle, a. (1983): notes on the diet of erhard’s wall lizard, podarcis erhardii. brit. j. herpetol. 6: 309-310. sadek, r.a. (1981): the diet of the madeiran lizard lacerta dugesii. zool. j. linn. soc. 73: 313-341. sorci, g. (1990): nicchia trofica di quattro specie di lacertidae in sicilia. naturalista sicil. (4)14 (suppl.): 83-93. turrisi, g.f., vaccaro, a. (1998): contributo alla conoscenza degli anfibi e dei rettili di sicilia. boll. accad. gioenia sci. nat. 30(353): 5-88. van damme, r. (1999): evolution of herbivory in lacertid lizards: effects of insularity and body size. j. herpetol. 33(4): 663-674. acta herpetologica 2006 2 a new finding of «uroplatus alluaudi» in north-eastern madagascar widens considerably its distribution range (reptilia, gekkonidae) a new finding of uroplatus alluaudi in north-eastern madagascar widens considerably its distribution range (reptilia, gekkonidae) franco andreone1, gennaro aprea2 1 museo regionale di scienze naturali, via g. giolitti, 36, i-10123 torino, italy. e-mail: f.andreone@ libero.it 2 università di napoli federico ii, dipartimento di biologia strutturale e funzionale, via cintia, i80126 napoli, italy abstract. the presence of uroplatus alluaudi in ne madagascar (besariaka forest) is here reported. this record enlarges considerably the species’ distribution range, up to now restricted to montagne d’ambre. the new specimen fits totally in morphology, colouration and body scalation with specimens from the type locality. considerations are also provided on its conservation at the light of the updated distribution. keywords. reptilia, gekkonidae, uroplatus, distribution, madagascar. the madagascan endemic genus uroplatus currently includes 12 species of nocturnal and arboreal geckoes widespread in most of the rainy and dry forests of madagascar, typical for their secretive habits and cryptic morphology and colouration (glaw and vences, 1994). indeed, one of the least known species is u. alluaudi. this gecko is peculiar in being featured by a less “extreme” morphology, with a body that is not so flattened as in other species, and with dermal fringes limited to the tail. so far, u. alluaudi was only known from its type locality, montagne d’ambre (glaw and vences, 1994; raxworthy and nussbaum, 1994), while a likely related species, u. malahelo was described from the south of madagascar (nussbaum and raxworthy, 1994). on the occasion of field surveys in north-eastern regions (andreone, 2004), we collected a further individual of u. alluaudi. this represents a remarkable novelty in terms of distribution, since it indicates that the species is not limited to the isolated northern rainforest of montagne d’ambre (fig. 1). the single individual is a male, and was collected by j.e. randrianirina on 28 april 1996 at besariaka forest, campsite 1 (ambinaninimiakamidina), andapa fivondronana, antsiranana (diégo suarez) province, 14°49.30’s, 49°3.25’e, about 940 m a.s.l. besariaka is a classified forest at about 60 km south of andapa, delimited at the north by the reserve spéciale d’anjanaharibe-sud, and southwards by the tsararano chain. the forest is rather degraded, especially in parcels far from streams. acta herpetologica 1(2): 121-125, 2006 122 andreone and aprea this is due to several reasons, among which the use of forest areas for cattle, cutting of trees by villagers, use of path systems, and for hunting (andreone et al., 2000). the individual was found overnight at about 2 m of elevation from the ground (h 19:30). after capture it was anaesthetised with a clorobutanol injection, and fixed in 4% formalin. then it was conserved in 70% ethanol and housed in the herpetological collection of the museo regionale di scienze naturali (torino), under the number mrsn r1630. after about 10 years of conservation this specimen is still in very good conditions, with the tail in good shape and attached to the body (fig. 2). to ascertain and confirm its specific determination we compared it to two u. alluaudi from montagne d’ambre, housed in zoologische staatssammlung münchen: zsm 275/2004 (field number fgzc [f. glaw zoological collection] 528, collected by f. glaw, m. puente, r. randrianiaina and a. razafimanantsoa, 24 february 2004; and zsm 251/2004 (fgzc 490, same collectors, collected 20 february 2004). all the specimens fig. 1. location of besariaka forest (where the new individual of uroplatus alluaudi mrsn r1630 was found) and of other forest sites around andapa, ne madagascar. the arrows indicates the campsite, while the two points on the smaller map of madagascar refer to montagne d’ambre (1) and besariaka (2). 123new finding of uroplatus alluaudi in ne madagascar were measured by the senior author with a hand calliper (precision: 0.1 mm) for standard lengths: snout-vent length (from the tip of the snout to the cloaca); tail length (from the cloaca to the tip of the tail); maximum tail width; head length from the tip of the snout to the jaw articulation; maximum head width; eye diameter, from the snout the nostril, and nostril-eye (table 1). the specific attribution to u. alluaudi for the besariaka specimen is thus justified for the following reasons: (1) the body size, morphology and colouration are very similar in all the three specimens compared, (2) the hemipenial morphology too is almost identical; (3) the besariaka specimen differs from u. malahelo, which has an uniform scalation, in having scattered conical turbercles among the smaller, flat, juxtaposed scales (fig. 3), which is a character diagnostic of u. alluaudi (nussbaum and raxworthy, 1994). furthermore, all the three specimens share an undivided rostral scale, this being a diagnostic character between the species pair u. alluaudi / u. malahelo (undivided scale) and u. fig. 2. dorsal view of two preserved specimens of uroplatus alluaudi. (a) the new individual from besariaka (mrsn r1630), (b) zsm 275/2004 from montagne d’ambre. 124 andreone and aprea guentheri (divided rostral scale), a species that is known from nw madagascar (nussbaum and raxworthy, 1994). the updated distribution of u. alluaudi at the light of the present finding is therefore much wider than formerly believed, and indicates that the species, although likely restricted to northern madagascar, is not a montagne d’ambre endemic. besariaka is about 180 km south of montagne d’ambre (fig. 3). evidently, u. alluaudi is not a common species, although it is likely that its secretive habits plays an important role in the fact that only a few individuals have been collected until now. within the national park of montagne d’ambre u. alluaudi benefits from a certain protection, although in the past a certain number of specimens were possibly captured and exported for the pet-trade. at besariaka the habitat conditions are uncertain, since fig. 3. details of the dorsal skin texture of uroplatus alluaudi. (a) mrsn r1630 (from besariaka), and (b) zsm 275/2004 from montagne d’ambre, both showing the presence of heterogeneous scalation. table 1. morphometric measurements (to 0.1 mm) of the analysed uroplatus alluaudi specimens from besariaka and montagne d’ambre. for abbreviations see the text. mrsn r1630 zsm 275/2004 zsm 251/2004 sex male male male provenance besariaka m. d’ambre m. d’ambre snout-vent length 76.4 77.4 82.2 tail length 37.9 29.0 30.3 tail width 11.4 12.5 10.6 head length 16.1 16.2 16.8 head width 16.7 16.4 15.7 eye diameter 6.6 6.1 7.5 snout-nostril 2.9 3.3 3.3 nostril-eye 7.4 7.9 6.6 125new finding of uroplatus alluaudi in ne madagascar this area, at least at the time during which the specimen was collected, was already highly degraded. regardless, based on the current distribution, we suspect that the species might be also present in the regions between montagne d’ambre and besariaka, such as anjanaharibe-sud, ambolokopatrika, and marojejy. in this case the persistence of major forest blocks and homonymous protected areas would likely warrant its protection. acknowledgements this research was made possible through an agreement with malagasy institutions. the fieldwork was supported in part by grants to f. andreone from the gondwana conservation and research and wwf madagascar, and to g. aprea from the italian ministero dell’università e della ricerca scientifica. thanks for j.e. randrianirina for the information on the collection locality, and to f. glaw for the loan of the two comparative specimens. a. bauer, c.p. blanc, and f. glaw critically reviewed an earlier version of the paper. references andreone, f. (2004): crossroads of herpetological diversity: survey work for an integrated conservation of amphibians and reptiles in northern madagascar. ital. j. zool., suppl 2: 229-235. andreone, f., randrianirina, j.e., jenkins, p.d., aprea, g. (2000): species diversity of amphibia, reptilia and lipotyphla at ambolokopatrika, a rainforest between anjanaharibe-sud and marojejy massifs, ne madagascar. biodiv. conserv. 9: 15871622. glaw, f., vences, m. (1994): a fieldguide to the amphibians and reptiles of madagascar, 2nd edition. vences and glaw, cologne. nussbaum, r.a., raxworthy, c.j. (1994): a new species of uroplatus duméril (reptilia: squamata: gekkonidae) from southern madagascar. copeia 1994: 319-324. raxworthy, c.j., nussbaum, r.a. (1994): a rainforest survey of amphibians, reptiles and small mammals at montagne d’ambre, madagascar. biol. cons. 69: 65-73. ritrovamento di zamenis longissimus (laurenti, 1768) (reptilia, colubridae) sull’isola d’elba (toscana, italia) angelo vaccaro1, giuseppe fabrizio turrisi2 1corso ara di giove 382 int. 26, i-95030, pedara, catania. e-mail: angelovaccaro2@virgilio.it 2dipartimento di biologia animale “marcello la greca”, via androne 81, i-95124 catania, italia. corresponding author. e-mail: turrisifabrizio@yahoo.it abstract. the presence of zamenis longissimus (laurenti, 1768) in elba island (tuscany, italy), previously known by only one ancient and doubtful record, is confirmed. two specimens road killed, were collected in the north-east of the island. the new distributional data is briefly commented in the context of the paleogeographic and historical-environmental background. riassunto. viene confermata la presenza di zamenis longissimus (laurenti, 1768) sull’isola d’elba (toscana, italia), precedentemente nota sulla base di un’unica, antica e dubbia segnalazione. due esemplari, morti su strada, sono stati raccolti nella parte nord-orientale dell’isola. il nuovo dato corologico viene brevemente commentato considerando anche gli aspetti paleogeografico e storico-ambientale. parole chiave. zamenis longissimus, elba, ritrovamento. il saettone comune, zamenis longissimus (laurenti, 1768), è una specie a corotipo sud-europeo, presente in italia in tutte le regioni continentali e peninsulari. il limite meridionale accertato in italia per la specie ricade in abruzzo, e precisamente nella località di rosello, provincia di chieti. in italia meridionale ed in sicilia z. longissimus è vicariato da una specie assai affine, il saettone occhirossi, zamenis lineatus (camerano, 1891) (lenk e joger, 1994; lenk e wüster, 1999; razzetti e zanghellini, 2006). l’area di contatto tra le due specie si trova lungo una linea immaginaria che va dalla provincia di caserta, a ovest, a quella di foggia, a est (razzetti e zanghellini, 2006). per l’isola d’elba sono note quattro specie di ofidi: hierophis viridiflavus (lacépède, 1789), coronella austriaca laurenti, 1768, natrix natrix helvetica (lacépède, 1789) e vipera aspis francisciredi laurenti, 1768. quest’ultimo taxon fu segnalato dell’elba per la prima volta da thiebaut de bernaud nel 1809; natrix natrix helvetica dal giglioli nel 1880; hierophis viridiflavus e coronella austriaca furono indicati da lankes nel 1913 (cfr. lanza, 1996). una quinta specie di ofide, z. longissimus, viene citata da sochurek (1954), sub elaphe longissima (cfr. böhme, 1993). questa segnalazione viene ritenuta non attendibile da mertens (1955), poiché non supportata da alcun reperto. sull’attenacta herpetologica 2(1): 59-63, 2007 60 a. vaccaro and g.f. turrisi dibilità delle segnalazioni del sochurek sussistono molte riserve evidenziate da diversi erpetologi; ad esempio lanza (1996: 48) in una sua annotazione lo definisce addirittura “un dilettante austriaco in fama di grande raccoglitore, ma non di altrettanta affidabilità”. z. longissimus viene indicato dell’elba da bruno (1984, 1998) e da bruno e maugeri (1990), probabilmente sulla base della segnalazione di sochurek (1954). infine, nei recenti atlante degli anfibi e dei rettili d’italia (razzetti e zanghellini, 2006) e atlante degli anfibi e dei rettili della toscana (vanni e nistri, 2006) questa specie viene esclusa dall’erpetofauna dell’elba. nella presente nota si riferisce sul recente ritrovamento di due esemplari di z. longissimus che confermano definitivamente la presenza della specie sull’isola. i due esemplari (figg. 1-3) sono stati rinvenuti morti nella medesima località, lungo una strada del settore nord orientale dell’isola d’elba, a distanza di tre anni l’uno dall’altro, rispettivamente durante la primavera del 2002 e l’estate del 2005. la località di ritrovamento è caratterizzata dalla presenza di densi boschi cedui di quercus ilex l., con presenza di erica arborea l., pistacia lentiscus l., arbutus unedo l. e cistus spp. l’area è inoltre interessata dalla presenza di un torrente a regime idrico stagionale. il primo esemplare (figg. 1-2), una femmina adulta avente lunghezza totale di 113 cm, di cui 18 cm spettanti alla coda, è stato rinvenuto morto lungo una strada in data 8 agosto 2002. dalle condizioni di conservazione è presumibile che la morte sia da far risalire ad almeno 24-36 ore prima. a causa dell’avanzato stato di decomposizione, l’esemplare non è stato conservato in collezione. la colorazione delle parti dorsali è uniformemente brunoolivastra, con poche squame biancastre nella zona mediana; le parti ventrali sono uniformemente giallastro-chiare; il numero delle ventrali è di 228; il numero degli urostegi non è stato rilevato per le pessime condizioni della coda. il secondo esemplare (fig. 3) è un giovane trovato morto lungo una strada il 6 agosto 2005, avente una lunghezza totale di 41,5 cm. dalle condizioni di conservazione è presumibile che la morte sia da far risalire ad almeno 48 ore prima. l’esemplare, nonostante il cattivo stato di conservazione, è stato raccolto e posto nella collezione erpetologica turrisi g.f. e vaccaro a. (catania), in soluzione di etanolo al 70%. sul capo dell’esemplare è ben riconoscibile la tipica ornamentazione dei giovani di z. longissimus, costituita da una stria nera che unisce l’occhio all’ultima squama sopralabiale, da una macchia nerastra suboculare che entra in contatto con il margine labiale e da due macchie bianche semilunari ai lati dell’area nucale. note conclusive da un punto di vista zoogeografico, il popolamento animale dell’isola d’elba ha una notevole affinità con il settore tirrenico dell’italia, segnatamente con la toscana (cfr. mertens, 1955). le ricostruzioni paleogeografiche hanno evidenziato che l’elba, durante il würmiano (22000-18000 anni fa), non era altro che una penisola protesa verso il mar tirreno (masetti e rustioni, 1996). le quattro specie di ofidi accertate per l’elba appartengono ad un contingente faunistico probabilmente migrato dalla vicina toscana, attraverso territori di collegamento (“ponti”) che si costituirono a seguito dei fenomeni di 61ritrovamento di zamenis longissimus sull’isola d’elba figg. 1-2. zamen i s l o n g i s s i m u s (laurenti, 1768), esemplare femmina adulta (lunghezza totale: 113 cm), isola d’elba, 8 agosto 2002. fig. 3. zamenis longissimus (laurenti, 1768), esemplare giovane (lunghezza totale: 41,5 cm), isola d’elba, 6 agosto 2005. 62 a. vaccaro and g.f. turrisi regressione marina, che peraltro interessarono anche altre isole del bacino mediterraneo durante il quaternario. è ben spiegabile, pertanto, anche la presenza di z. longissimus che, al pari delle altre specie di ofidi, avrebbe colonizzato l’elba durante quel periodo geologico. è ipotizzabile che z. longissimus sia divenuto molto raro e localizzato sull’elba a seguito delle vicende storico-ambientali che hanno caratterizzato gli ambienti dell’isola (cfr. del prete e tosi, 1996), segnatamente a causa della drastica riduzione delle originarie cenosi forestali, costituite soprattutto da querceti, nell’ambito delle quali questa specie predilige le aree di radura e le microchiarie. infatti, già al tempo di strabone, nel i sec. d.c., il ferro estratto dalle miniere dell’isola non poteva essere più liquefatto nelle fornaci locali dell’elba, evidentemente per mancanza di materiale legnoso, e pertanto veniva trasportato a tale scopo sul continente (strabone: geografia, l’italia, libri v-vi; introduzione, traduzione e note di a.m. biraschi, biblioteca universale rizzoli). l’attuale vegetazione non sarebbe altro che il risultato di una relativamente recente evoluzione delle garighe e della macchia fino alla ricostituzione di cenosi forestali o prossimo-forestali, che hanno così riconquistato ampi territori, grazie anche alle massicce campagne di rimboschimento intraprese a partire dalla seconda metà del novecento. sui cambiamenti ambientali avvenuti sull’elba esistono comunque pareri discordanti e secondo zecchini (1978), gli etruschi non avrebbero depauperato così pesantemente il patrimonio forestale dell’elba, poiché pare che gli stessi operassero campagne di rimboschimenti dopo il taglio dei boschi. tuttavia, la stessa vicinanza dell’elba al continente non esclude altre ipotesi relativamente alla colonizzazione da parte di z. longissimus. infatti, gli scambi di materiale (ad es. materiale edilizio o alimentare) con la toscana, che avvengono fin da epoca preistorica e che attualmente sono continui e regolari grazie ai collegamenti marittimi, possono aver determinato un trasporto passivo di questo ofide sull’isola. un’ultima ipotesi, da non trascurare, è quella del trasporto attivo da parte di qualche amatore. per verificare quale di queste tre modalità sia quella più accettabile per spiegare la presenza di z. longissimus sull’elba, e quindi se questa specie debba considerarsi indigena dell’isola o meno, sono naturalmente necessarie più approfondite ed organiche ricerche sulla sua diffusione insulare e sull’ecologia, nonché indagini sugli aspetti storico-ambientali dell’elba. in ogni caso il suo ritrovamento sull’elba conferma il dato di sochurek (1954) il quale, a parere di chi scrive, è stato ingiustamente e precipitosamente considerato non attendibile da lanza (1996). ringraziamenti vivi ringraziamenti vanno ad elisabetta spadaro per il supporto logistico e la collaborazione sul campo offerti durante le ricerche svolte sull’isola d’elba. 63ritrovamento di zamenis longissimus sull’isola d’elba bibliografia böhme, w. (1993): elaphe longissima (laurenti, 1768) — askulapnatter. in: handbuch der reptilien und amphibien europas, band 3/i, schlangen (serpentes) i (typhlopidae, boidae, colubridae 1: colubrinae), p. 331-372. böhme, w. ed., aula-verlag, wiesbaden. bruno, s. (1984): guida ai serpenti d’italia. giunti martello, firenze, 192 p. bruno, s. (1998): serpenti. giunti, firenze, 256 p. bruno, s., maugeri, s. (1990): serpenti d’italia e d’europa. editoriale giorgio mondadori, 224 p. del prete, c., tosi, g. (1996): le specie vegetali. in: isola d’elba: geologia, flora, fauna, storia, arte, ambiente, p. 20-29. giubelli, g. ed., pro.gra.ms. italia. lanza, b. (1996): gli anfibi e i rettili. in: isola d’elba: geologia, flora, fauna, storia, arte, ambiente, p. 48-57. giubelli, g. ed., pro.gra.ms. italia. lenk, p., joger, u. (1994): genetic relationship between populations and intraspecific subdivision of elaphe longissima (laurenti, 1768) as suggested by plasma protein electrophoresis and dna fingerprinting. amphibia-reptilia 15: 363-373. lenk, p., wüster, w. (1999): a multivariate approach to the systematics of italian rat snakes of the elaphe longissima complex (reptilia, colubridae): revalidation of camerano’s callopeltis longissimus var. lineata. herpetol. j. 9: 153-162. masetti, m., rustioni, m. (1996): la paleontologia e la paleogeografia. isola d’elba: geologia, flora, fauna, storia, arte, ambiente, p. 16-19. giubelli, g. ed, pro.gra.ms. italia. mertens, r. (1955): die amphibien und reptilien der insel elba. senckenb. biol. 36: 287296. razzetti, e., zanghellini, s. (2006): zamenis longissimus (laurenti, 1768) / zamenis lineatus (camerano, 1891). in: atlante degli anfibi e dei rettili d’italia/ atlas of italian amphibians and reptiles. societas herpetologica italica, pp. 576-583. sindaco, r., doria, g., razzetti, e., bernini, f., eds, edizioni polistampa, firenze. sochurek, e. (1954): amphibienund reptilienleben auf elba. aquaristik-terraristik 1: 213-214. thiébaut de bernaud a.(1808): voyage à l’isle d’elbe, suivi d’une notice sur les autres isles de la mer tyrrhènienne. d. colas & le normant, paris. [in lanza, b. (op. cit.)]. vanni s., nistri a. (2006): atlante degli anfibi e dei rettili della toscana. regione toscana, museo di storia naturale dell’università degli studi di firenze sezione di zoologia “la specola”, 379 p. zecchini, m. (1978): gli etruschi all’isola d’elba. ente valorizzazione elba-portoferraio, 366 p. acta herpetologica 4(2): 119-123, 2009 phylogeographic link between sicilian and corso-sardinian testudo h. hermanni confirmed gabriele giacalone1,2, mario lo valvo1, uwe fritz3 1 laboratorio di zoologia applicata, dipartimento di biologia animale, università degli studi di palermo, via archirafi 18, i-90123 palermo, italy. 2 present address: dipartimento di botanica, università degli studi di catania, via antonino longo 19, i-95125 catania, italy. 3 museum of zoology (museum für tierkunde), senckenberg dresden, a.b. meyer building, königs-brücker landstr. 159, d-01109 dresden, germany. corresponding author. e-mail: uwe.fritz@senckenberg.de. submitted on: 2008, 12th october; revised on 2009, 20th august; accepted on 2009, 31st august. abstract. our study confirms that sicilian and corso-sardinian testudo h. hermanni share a certain mtdna haplotype, while tortoises from peninsular italy harbour slightly different haplotypes. when the fossil record is considered, this striking pattern agrees well with the idea of local extinction in corsica and sardinia and later replacement by tortoises originating elsewhere, either by natural oversea dispersal or translocation by man. keywords. testudo hermanni, phylogeography, corsica, sardinia, sicily. traditionally, the fauna of the islands corsica, sardinia and sicily is considered to constitute the tyrrhenian faunistic province (la greca, 1995). however, while corsica and sardinia are known to share many endemic or closely related taxa, sicily represents a distinct entity (lanza and vanni, 1987; lanza, 1988; cheylan, 1992). most of sicily’s zoogeographic affinities point at the italian peninsula (cheylan, 1992), although some endemic taxa are known (stöck et al., 2008), one of which (bufo siculus) suggests a link to north africa. also other taxa support this relationship between sicily and north africa (la greca, 1990, 1995; cheylan, 1992). fritz et al. (2006) demonstrated that two sicilian testudo hermanni hermanni harboured a mitochondrial haplotype also occurring on corsica and sardinia, an unexpected finding when it is considered that tortoises from the italian peninsula possess distinct haplotypes. in order to find out whether the two individuals studied by fritz et al. (2006) might represent introductions from corsica or sardinia, we obtained blood samples of t. h. hermanni from five other sicilian populations (fig. 1) and sequenced most of the mitochondrial cytochrome b gene (cyt b), the marker used by fritz et al. (2006). in addition, we sequenced part of the faster evolving mitochondrial control region (cr; table 1) from two sicilian, three corsican, and two sardinian tortoises. 120 g. giacalone, m. lo valvo and u. fritz agrigento palermo catania siracusa messina 1 2 3 4 5 6 7 fig. 1. collection sites of sicilian testudo hermanni hermanni samples used in this study and in fritz et al. (2006). site numbers refer to table 1. table 1. geographic origin of testudo hermanni samples used in this study, sicilian samples from fritz et al. (2006) and the studied mtdna fragments. sample numbers refer to the tissue collection of the museum of zoology dresden (mtd t); site numbers, fig. 1. sample site cyt b cr reference 2140 1 – castellammare del golfo (palermo), sicily + fritz et al. (2006) 2141 2 – balestrate (palermo), sicily + fritz et al. (2006) 4078 3 – m. m. belsito (madonie), sicily + this study 4075 4 – collesano-campofelice (madonie), sicily + this study 4077 4 – collesano-campofelice (madonie), sicily + this study 4082 4 – collesano-campofelice (madonie), sicily + this study 4074 5 – caronia (nebrodi), sicily + this study 4079 6 – s. agata di militello (nebrodi), sicily + + this study 4083 7 – augusta (siracusa), sicily + this study 4084 7 – augusta (siracusa), sicily + + this study 4085 7 – augusta (siracusa), sicily + this study 4086 7 – augusta (siracusa), sicily + this study 1921 porto vecchio, corsica + this study 1927 casabianda, corsica + this study 1936 fontane du salario (ajaccio), corsica + this study 1114 porto palmas (sassari), sardinia + + cyt b: fritz et al. (2006), cr: this study 1115 bay of porto ferro (sassari), sardinia + + cyt b: fritz et al. (2006), cr: this study 121phylogeography of testudo hermanni laboratory procedures followed fritz et al. (2006). for amplification and sequencing of an approximately 1000 bp long fragment of cyt b the primers cytbg (spinks et al., 2004), mt-f-na, mt-c-for2, and mt-e-rev (fritz et al., 2006) were used; for an approximately 850 bp long cr fragment, the primers thr-l15569 and phe-h26 (palkovacs et al., 2003). an abi 3130 genetic analyzer was used for automatic sequencing in both directions. obtained sequences were checked by eye and aligned. cyt b sequences were compared with published data (fritz et al., 2006). using tcs 1.21 (clement et al., 2000), a parsimony network was constructed from a 1008 bp long alignment including all 31 previously published western mediterranean t. h. hermanni sequences (fritz et al., 2006) plus our 10 new sequences from sicily (fig. 2). the alignment of the cr sequences comprised 855 bp. all of our 10 sicilian t. h. hermanni harboured the same cyt b haplotype (h5) as the two sicilian tortoises studied by fritz et al. (2006). with respect to the cr, all corsican and sardinian tortoises shared the same haplotype; this haplotype was also found in one sicilian tortoise. the second sicilian individual (mtd t 4079) possessed a similar haplotype differing in only one mutational step (position 815 of our alignment: t instead of a). the two cr haplotypes are deposited under accession numbers fn298446-fn298447 in genbank. compared to the eastern subspecies t. h. boettgeri, the phylogeographic structure of western mediterranean t. h. hermanni is strikingly shallow (fritz et al., 2006). this is unexpected because the fossil record provides evidence for its long presence and wide distribution in the western mediterranean (delfino, 2002; morales pérez and sanchis serra, 2009). amongst others, fossils are known from the middle pleistocene of corsica (hervet, 2000, fig. 2. parsimony network of mtdna haplotypes (cyt b fragment, 1008 bp) of western mediterranean testudo hermanni hermanni, using the dataset of fritz et al. (2006) and 10 new sicilian sequences. haplotype nomenclature follows fritz et al. (2006). symbol size corresponds to haplotype frequency; missing haplotypes, dots. each line between symbols represents one mutation step. the arrow indicates the connection to eastern mediterranean t. h. boettgeri haplotypes. black symbols, peninsular italy and southern france (var); dark grey, sicily; light grey, corsica and sardinia; white, spain (i: introduced populations or populations known to comprise also allochthonous individuals, n: native population). h1: n = 12, h2: n = 1, h3: 7, h4: n = 1, h5: n = 18, h6: n = 1, h7: n = 1. when only western mediterranean haplotypes are considered, h1 is under coalescent theory ancestral to h2-h7 (outgroup probability of h1: 0.3175). 122 g. giacalone, m. lo valvo and u. fritz 2001; hervet and salotti, 2000) and from the plio-pleistocene boundary of sardinia (abbazzi et al., 2004). on the italian peninsula, the oldest findings date back to the pliocene; the oldest fossils from sicily are from the middle pleistocene (delfino, 2002), suggesting that sicily was colonized later than corsica and sardinia. consequently, some extent of genetic differentiation of the island tortoises and a closer relationship of the sicilian and the peninsular italian populations should be expected, but not between sicily and corso-sardinia. the weak genetic differentiation of western mediterranean tortoises might be related to a major climatically caused extinction event some 38000-39500 years ago, wiping out many populations (morales pérez and sanchis serra, 2009). however, while our study confirms that sicilian and corso-sardinian t. h. hermanni are not clearly differentiated in the studied genetic markers, they are slightly distinct from peninsular italian tortoises (and from native spanish tortoises; fig. 2). this pattern supports the survival of several local populations in the western mediterranean, in agreement with fossil evidence (delfino, 2002; morales pérez and sanchis serra, 2009) for instance on the italian peninsula and sicily. furthermore, the genetic identity of corsican, sardinian, and sicilian tortoises agrees also well with the idea of local extinction in corsica and sardinia and later replacement by tortoises originating elsewhere, either by natural oversea dispersal or translocation by man. acknowledgements thanks for introducing gabriele giacalone to lab work go to anna hundsdörfer and anke müller. massimo delfino helped with information about the fossil record. the samples of corsican and sardinian tortoises were provided by marc cheylan and wolfgang wegehaupt. dr. f.p. faraone assisted during field work. references abbazzi, l., angelone, c., arca, m., barisone, g., bedetti, c., delfino, m., kotsakis, t., marcolini, f., palombo, m.r., pavia, m., piras, p., rook, l., torre, d., tuveri, c., valli, a.m.f., wilkens, b. 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(2008): post-messinian evolutionary relationships across the sicilian channel: mitochondrial and nuclear markers link a new green toad from sicily to african relatives. bmc evol. biol. 8: 56. acta herpetologica 2006 1 morphometric differences between extant and extinct italian populations of the adder, «vipera berus» (linnaeus, 1758) morphometric differences between extant and extinct italian populations of the adder, vipera berus (linnaeus, 1758) augusto gentilli 1, stefano scali 2, and roberto sacchi 1 1 laboratorio di eco-etologia, dipartimento di biologia animale, università di pavia, p.zza botta 9, 27100 pavia, italy 2 museo civico di storia naturale c.so venezia 55, 20121 milano, italy vipera berus (linnaeus, 1758) is the terrestrial snake showing the widest distribution in the world, occuring from great britain, france and northern italy to the sakhalin island and north korea (nilson, 1980; saint girons, 1980; nilson et al., 1994; nilson & andrén, 1997a). however, adders do not occur uniformly over their distribution area, but are scattered in several isolated populations (nilson & andrén, 1997a). frequently, ecological traits of borderline and isolated populations differ from those living in the core area of the distribution range of the species, and might be subjected to higher risks of stochastic extinction and higher differentiation rates (mayr, 1970). for example, meadow vipers (vipera ursinii) show a highly fragmented distribution, many of isolated groups being different subspecies (nilson & andrén, 1997b, 2001). in italy vipera berus inhabits the eastern and central alps, occurring in most of the po plain until the nineteenth century (fig. 1, bruno, 1992; societas herpetologica italica, 1996). the extinction of the po plain populations might be caused by land reclamation occurred between the end of 18th century and 1930-1940. the progressive heating occurred over the same period (ghezzi & riva, 1989; camuffo, 1990) might have contributed to the extinction of the po plain adders, favoring more termophilic species, such as v. aspis (saint girons, 1975). po plain adders inhabited only wetlands and marshlands (ninni, 1879; camerano, 1888), while alpine adders regularly frequent dry and rocky habitats, generally above 1000 asl (nilson & andrén, 1997a; lapini et al., 1999; caldonazzi et al., 2002). moreover, any historical or contemporary record of this species in the pre-alpine area is known (fig. 1) (massalongo, 1854, 1859; de betta, 1857, 1874, 1880; lessona, 1877, 1879; bruno, 1992; societas herpetologica italica, 1996), probably because both climatic regime (tommaselli et al., 1973) and habitat features of this area do not favour adders. therefore, po plain populations might have been geographically isolated from the alpine ones. the isolation of the po plain population from the main distribution range of the species leads to hypothize that po plain adders might belong to a different form or subspecies. in this work we investigated this hypothesis, looking for differences in morphometric features between po plain and alpine adders. acta herpetologica 1: 65-71, 2006 66 a. gentilli et alii we collected data for 28 extant and 28 extinct adders from italian natural history museums (n =50, 89.2% of specimens, see appendix 1 for details), and literature (n =6, 10.8% of specimens, camerano 1888; boulenger 1896). all the extinct adders from the po plain preserved in italian museums were considered. since adders are sexually dimorphic (saint girons, 1978; scali & gentilli, 1998), males and females were analysed separately: the po plain group included 15 males and 13 females, while the alpine one included 12 males and 16 females. in order to describe head morphology, for each specimen we considered (see scali & gentilli, 1998 for details): i) the number of postocular scales, ii) the number of front dorsal head scales, iii) the number of subocular scale rows, iv) head length and width (mm), v) the length and width of the right parietal scale (mm), and vi) length and width of the frontal scale (mm). since head width correlated with head length (males: rs = 0.45, n = 22, p = 0.034; females: rs = 0.52, n = 27, p = 0.006), we replaced the head width by the correspondent unstandardized residuals versus head length. since the length and width of frontal and parietal scales were intercorrelated (rs ranging from 0.48 to 0.88 for males and from 0.42 to 0.69 for females), we used a principal component analysis (pca) to reduce them to a “head scale size” component. for both sexes, pca extracted only one component (eigenvalue >1), which accounted for 77.2% and 59.5% of the cumulative variance for males and females respectively, and was positively related to each one of the head scale measures (all variables entered pca with component loadings higher than 0.75 for males fig. 1. distribution of extant (l) and extinct (u) vipera berus in italy; (bruno, 1992; societas herpetologica italica 1996, modified by the authors). 67extant and extint vipera berus and 0.63 for females). the scores of this “head scale size” component for each adder were used to examine if the size of head scales differed between extinct and extant adders. in order to describe body morphology, we considered body length (from the head tip to the vent, mm) and the number of ventral scales. since these variables were highly correlated both in males (rs = 0.60, n = 21, p = 0.004) and females (rs = 0.37, n = 27, p = 0.05), we used a principal component analysis to reduce them to a single “body morphology” component. for both males and females, pca extracted only one component (eigenvalue >1), which accounted for 69.2% and 71.5% of the cumulative variance for males and females respectively, and was positively related both to the body length and to the number of ventral scales (the two variables entered pca with component loading higher than 0.83 for both sexes). the scores of this “body morphology” component for each adder were used to examine if the body size differed between extinct and extant adders. finally, tail features were measured for each specimen using the unstandardized residuals of the tail length versus body length, and the number of subcaudal scales. since these variables were highly correlated (males: rs = 0.46, n = 20, p = 0.039; females: rs = 0.82, n = 27, p<0.001), we used a principal component analysis to reduce them to a single “tail morphology” component. for both males and females, pca extracted only one component (eigenvalue >1), which accounted for 71.3% and 94.2% of the cumulative variance for males and females respectively, and was positively related either to the residuals of tail length as to the number of subcaudal scales (the two variables entered pca with component loadings higher than 0.84 for males and 0.97 for females). the scores of this “tail morphology” component for each adder were used to investigate if tail features differed between extinct and extant adders. we could not collect all measures from each specimens because their non optimal conservation, so sample sizes varied in different analyses (tab. 1). correlation analysis (spearman’s rho coefficient) was used to investigate relationships among variables. we used the bonferroni correction for multiple tests to adjust the observed significance level to the number of comparisons (rice, 1989): in a set of k simultaneous comparisons, a given test was considered to be statistically significant at 0.05 level only if it was significant also at the 0.05/k level. we used discriminant function analysis (dfa, stepwise forward method) using all morphometric measures as independent variables and collecting area (alps vs po plain) as classifying factor, in order to determine i) if po plain (extinct) and alpine (extant) adders differed on the basis of their morphological features, and ii) what morphological features, if any, accounted for such a difference. all analyses were performed using spss rel. 6.1.2 software package (1996). head, body, and tail variables were not intercorrelated after bonferroni correction (k = 28; p < 0.00178 for relationships to be significant). discriminant function analysis for males generated a high significant discriminant function (19.9, p < 0.0001, λ = 0.26), which correctly classified 90% of specimens. the stepwise procedure extracted only two variables: the number of postocular scales and the tail morphology. the males of po plain adders had more postocular and subcaudal scales and longer tails than alpine ones (tab. 1). similarly, dfa for females generated a highly significant discriminant function (27.6, p < 0.0001, λ = 0.15), which correctly classified 95.6% of specimens. the stepwise procedure extracted three variables: the number of postocular scales, the tail morphology, and the 68 a. gentilli et alii ta bl e 1. m or ph om et ri c m ea su re s of p o pl ai n an d al pi ne a dd er s. m al es fe m al es po p la in a lp s po p la in a lp s n m in m ax m ea n sd n m in m ax m ea n sd n m in m ax m ea n sd n m in m ax m ea n sd h ea d m or ph ol og y po st o cu la r sc al es ( n) 11 3 5 3. 7 0. 8 12 2 4 2. 8 0. 6 9 3 5 3. 9 0. 8 15 2 4 3. 2 0. 6 fr on t do rs al h ea d sc al es ( n) 8 7 13 10 .5 2. 2 12 6 22 11 .6 5. 2 11 11 30 17 .6 5. 6 15 5 27 14 .5 5. 0 su bo cu la r ro w s (n ) 11 1 2 1. 2 0. 4 12 1 2 1. 1 0. 3 13 1 2 1. 6 0. 5 16 1 2 1. 2 0. 4 h ea d w id th ( m m ) 10 10 .7 15 .9 13 .6 1. 74 12 9. 2 15 .3 12 .8 1. 78 13 9. 2 19 .0 14 .0 2. 9 14 11 .8 18 .2 14 .9 1. 91 h ea d le ng th ( m m ) 10 19 .3 25 .8 22 .5 1. 91 12 16 .2 29 .2 21 .5 3. 58 13 15 .3 34 .6 24 .1 4. 8 14 18 .1 28 .0 23 .2 2. 44 pa ri et al s ca le le ng th ( m m ) 10 3. 5 6. 2 4. 8 0. 84 12 3. 0 4. 8 3. 9 0. 62 10 2. 6 5. 2 3. 9 0. 8 13 2. 7 5. 5 4. 4 0. 84 pa ri et al s ca le w id th ( m m ) 10 2. 2 3. 3 2. 6 0. 38 12 1. 6 2. 9 2. 2 0. 40 10 1. 6 3. 0 2. 2 0. 4 13 1. 7 3. 3 2. 5 0. 47 fr on ta l s ca le le ng th ( m m ) 10 3. 7 5. 7 4. 6 0. 70 12 2. 4 4. 6 3. 6 0. 71 8 2. 7 4. 5 3. 7 0. 5 15 2. 7 5. 7 4. 6 0. 79 fr on ta l s ca le w id th ( m m ) 10 2. 6 3. 7 3. 3 0. 36 12 1. 8 4. 6 3. 0 0. 78 8 2. 3 3. 7 2. 9 0. 4 15 2. 7 3. 6 3. 1 0. 29 b od y m or ph ol og y b od y le ng th ( m m ) 10 31 1. 4 53 6. 0 43 7. 0 67 .2 7 12 16 6. 8 41 2. 6 32 6. 1 85 .2 3 13 17 3. 7 69 0. 0 42 0. 9 13 3. 8 15 30 6. 9 53 2. 0 43 6. 1 56 .3 3 v en tr al s ca le s (n ) 15 13 6 14 8 14 2. 1 3. 72 12 13 6 14 9 14 1. 0 3. 63 13 14 0 15 4 14 5. 9 4. 6 16 13 8 15 5 14 6. 4 5. 20 ta il m or ph ol og y ta il le ng th ( m m ) 10 44 .0 92 .0 76 .3 15 .7 8 11 30 .0 74 .0 56 .0 15 .3 1 13 28 .0 10 0. 0 56 .6 20 .4 16 34 .0 58 .0 49 .3 7. 07 su bc au da l s ca le s (n ) 14 35 47 41 .5 3. 50 11 33 41 36 .8 2. 66 12 29 42 33 .8 3. 7 16 18 31 26 .8 3. 49 69extant and extint vipera berus number of subocular scale rows. the females of po plain adders had more postocular scales, more subocular scale rows, more subcaudal scales, and tails longer than alpine ones (tab. 1). in conclusion, our results showed morphological differences between po plain and alpine adders, supporting our hypothesis that po plain and alpine populations of this species might be considered two different ecotypes or subspecies. however, our morphological analyses have to be considered only a preliminary step, and genetic analyses are needed to better clarify the sistematic position of po plain adders. further analyses would also check for relationships between po plain adders and v. b. bosniensis, since their distribution range might overlap in the coastal wetlands of north adriatic. acknowledgements we thank the directors and curators of the museums for allowing us to measure the specimens analysed in the present paper. we also thank dr. edoardo razzetti for his useful comments to the manuscript. references boulenger, g.a. (1896): catalogue of the snakes in the british museum (natural history). taylor and francis ltd, london. bruno, s. (1992): repertorio zoogeografico, geonemico, tassonomico, biografico e bibliografico degli studiosi e degli studi di erpetologia italiana. i. serpentes: 1800-1899. atti acc. rov. agiati a. 241 (1991), serie vii 1, b: 5-256. caldonazzi, m., pedrini, p., zanghellini, s. (2002): atlante degli anfibi e dei rettili della provincia di trento (amphibia, reptilia), 1987-1996 con aggiornamenti al 2001. st. trent. sci. nat. acta biol. 77: 1-173. camerano, l. (1888): monografia degli ofidi italiani. parte prima: viperidi. mem. r. accad. sci. torino, serie ii 39: 195-243. camuffo, d. (1990): clima e uomo. garzanti, milano. de betta, e. (1857): erpetologia delle province venete e del tirolo meridionale. atti mem. acc. agr. sci. lett., verona 35: 1-365. de betta, e. (1874): rettili ed anfibi. parte iv. in: fauna d’italia divisa in due branche vertebrati ed invertebrati, p. 1-107. vallardi, milano. de betta, e. (1880): sulla distribuzione geografica dei serpenti velenosi in europa e particolarmente nell’italia. atti r. ist. ven. sci. lett. arti, venezia 6: 359-392. ghezzi, a., riva, i. (1989): il clima del territorio delle province di cremona e mantova. pianura 3: 29-46. lapini, l., dell’asta, a., bressi, n., dolce, s., pellarini, p. (1999): atlante corologico degli anfibi e dei rettili del friuli-venezia giulia. museo friulano di storia naturale, udine. lessona, m. (1877): delle vipere in piemonte. atti r. accad. sci. torino (reprint) 12: 110. 70 a. gentilli et alii lessona, m. (1879): nota intorno al pelias berus in piemonte. atti r. accad. sci. torino (reprint) 14: 1-2. massalongo, a. (1854): saggio di un’erpetologa popolare veronese. tip. giuseppe antonelli, verona. massalongo, a. 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(1997b): vipera ursinii (bonaparte, 1835). in: atlas of amphibians and reptiles in europe, p. 400-401. gasc, j.p., cabela, a., crnobrnja-isailovic, j., haffner, p., lescure, j., martens, h., martínez rica, j.p., maurin, h., oliveira, t., sofianidou, t.s., veith, m., zuiderwijk, a., eds., societas europea herpetologica, muséum national d’histoire naturelle (iegp/spn), paris. nilson, g., andrén, c. (2001): the meadow and steppe vipers of europe and asia – the vipera (acridophaga) ursinii complex. acta zool. hung. 47: 87-267. ninni, a.p. (1879): breve nota intorno al marasso vipera (pelias) berus l. nel veneto. atti soc. ital. sci. nat., milano 22: 175-181. rice, w.r. (1989): analyzing tables of statistical tests. evolution 43: 223-225. saint girons, h. (1975) : coexistence de vipera aspis et de vipera berus en loire-atlantique: un probleme de competition interspecifique. la terre et la vie 29: 590-613. saint girons, h. (1978): morphologie externe comparée et systématique des vipères d’europe (reptilia, viperidae). revue suisse zool. 85: 565-595. saint girons, h. (1980): biogéographie et évolution de vipères européennes. c.r. soc. biogéogr. 496: 146-172. scali, s., gentilli, a. (1998): morphometric analysis, sexual dimorphism and distribution of extinct adders (vipera berus) of the po plane (northern italy). in: current studies in herpetology, p. 391-396. miaud, c., guyétant, r., eds. le bourget du lac, france, s.e.h. societas herpetologica italica (1996): atlante provvisorio degli anfibi e dei rettili italiani. ann. mus. civ. st. nat. «g. doria», genova 91: 95-178. spss (1996): statistical package for social science for windows rel. 6.1. spss inc., chicago. tommaselli, r., balduzzi, a., filipello, s. (1973): carta bioclimatica d’italia. collana verde 33, ministero dell’agricoltura, roma. 71extant and extint vipera berus appendix 1. list of specimens. museum collection code museo civico di storia naturale (milano) 1052; 1529; 1530; 1531; 1532; 1533; 1534; 1535; 1536; 1578a; 1578b; 1591; 1592; 1623; 3286; 3579; 3749; 3758; 3760 museo civico di storia naturale (morbegno) 3; 4; 26; 29; 41; 65; 67; 1f unknown number museo regionale di scienze naturali (torino) r283; r522 museo civico di storia naturale (verona) 866; 867; 868 (2 specimens); 869 (2 specimens); 870; 872; 873a; 873b; 873c; 873d; 887 (2 specimens); 1330 museo zoologico “g. scarpa” (treviso) 2mm unknown numbers museo zoologico dell’università (firenze) 12062; 12101-02 museo zoologico dell’università (pavia) 230/476; 230/477 acta herpetologica 16(2): 89-98, 2021 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-10402 so close so different: what makes the difference? dario ottonello1,7,*, stefania d’angelo2, fabrizio oneto3,6, stefano malavasi1, marco alberto luca zuffi4, filippo spadola5 1 department of environmental sciences, informatics and statistics, cà foscari university of venice, via torino 155, 30172 venezia mestre, italy 2 wwf italia, via po 25/c, 00195 roma, italy 3 centro studi bionaturalistici srl, corso europa 26, 16132 genova, italy 4 museum of natural history, university of pisa, via roma 79, 56011 calci (pisa), italy 5 department of veterinary sciences, university of messina, polo universitario ss. annunziata, 98128 messina, italy 6 università di genova, corso europa 26, 16132 genova, italy 7 arpal ligurian environmental protection agency, via bombrini 8, 16149 genova, italy *corresponding author. e-mail: dario.ottonello@arpal.liguria.it submitted on: 2021, 31st january; revised on: 2021, 5th may; accepted on: 2021, 22nd may editor: emilio sperone abstract. the introduction of alien fish species in wetland ecosystems could have a great impact on freshwater communities and ecological processes. despite fish introduction has been noticed as one of the principal cause of freshwater extinctions, ecosystem processes alteration, and change in aquatic community assemblage, very few data about impact on freshwater reptiles are available. as study model we used two neighbour sub-populations of the endangered sicilian pond turtle, emys trinacris, inhabiting two small, close each other and very similar lakes, except for the presence of allocthonous fish, cyprinus carpio and gambusia hoolbroki in one of the two. the multi-year study allowed highlighting significant differences in abundance, growth and reproductive output between the two freshwater turtle sub-populations, suggesting their influence on phenotypic plasticity of the studied population. these results are discussed in the light of previous evidence about the impact of these alien species on abundance and assemblage of the invertebrate community with an evident impact on niche width, diet composition and therefore energy intake by emys trinacris. these data may provide important information to address management strategies and conservation actions of small wetland areas inhabited by pond turtles, pointing out a threats never highlighted up to now. keywords. emys trinacris, phenotypic plasticity, cyprinus carpio, gambusia hoolbroki, alien fishes impact. introduction phenotypic plasticity is an evolutionary adaptation to environmental variation often used to explain the intraspecific difference observed within various taxa, that could be influenced by local variation in abiotic and biotic factors associated with habitat type (lubcke and wilson, 2007). nowadays, the introduction of alien species is one of the main stressor of freshwater ecosystem biodiversity (genovesi, 2007), and it could have a great impact on freshwater communities causing cascading impacts on food webs and complex interactions among species (ricciardi and macisaac, 2011). in the last decades, many species of fishes have been deliberately introduced worldwide to provide food or sport leisure, but also released from aquaria, bait buckets, and water gardens, as contaminants of fish intended for stocking, or in ballast water (strayer, 2010). despite fish introduction has been noticed as one of the principal cause of freshwater extinctions (dextrase and mandrak, 2006), ecosystem processes alteration (pitcher and hart, 1995) and change in aquatic community assemblage (parkos et al., 90 d. ottonello et alii 2003), very few data about impact on freshwater reptiles are available. within this group, freshwater turtles are known to vary in different natural history traits among conspecific populations both over broad geographic area (lovich et al., 1998; joos et al., 2017) as well as among adjacent sites (tucker at al., 1998). they usually follow a growth pattern that involves rapid growth from hatching to sexual maturity, followed by little or no growth once maturity is attained (e.g., wilbur, 1975; bury, 1979). growth can be influenced both by different ecological factors, such as hatching size, food availability and habitat suitability (mahmoud, 1969; bury, 1979). moreover, different growth patterns can lead to the same size (bury, 1979; andrews, 1982) or to different size at the maturity (stearns and koella, 1986). these differences can have also a direct impact on reproductive output in a taxon where clutch size often increases with maternal body size (zuffi et al., 2004). in particular, ottonello et al. (2017a) showed how the presence of two alien fishes, cyprinus carpio linnaeus, 1758 and gambusia holbrooki girard, 1859, can alter the abundance and the assemblage of the invertebrate community with a considerable impact on niche width and diet composition of an endangered turtle. local food availability and quality may indeed play a key role to regulate feeding strategy with consequences on growth, reproductive output and population density (dunham and gibbons, 1990; parmenter and avery, 1990). for these reasons we have aimed at investigating if the presence of alien fishes can have an impact at local scale on a population of an endangered turtle using as case study two neighbour wild sub-populations under similar climatic and environmental conditions. as model species we selected emys trinacris fritz et al., 2005, the only native freshwater turtle of sicily, a large island off the coast of italy. the sicilian pond turtle lives in wetlands and slowmoving water bodies (e.g., lagoons, deltas, inland waters and mountain lakes) with soft bottoms and abundant aquatic vegetation, especially on the banks, from the sea level up to 1036 m a.s.l. (marrone et al., 2016). however, the presence of the species at about 1250 m a.s.l. was recently observed (f. marrone and r. scardino, pers. obs.). in view of the low number of sicilian indigenous fish (zerunian, 2004) and the absence of specialized aquatic vertebrate predators (aa. vv., 2008), it is likely to assume that emys trinacris is one of the top aquatic predators in many of these environments in sicily, with an opportunistic and generalist pattern oriented mainly towards aquatic invertebrates (ottonello et al., 2017a). because of the introduction of non-native fish species, that are now the most represented non-indigenous taxon occurring in sicily (marrone and naselli-flores, 2015), new dynamics that can significantly alter and threaten the structure of the native biota have been established (naselli-flores and barone, 2012) and top predators are likely to be valuable indicators of ecosystem health (landres et al., 1988). although some data and experimental studies highlighted the poorly competitive abilities of the genus emys against some alien species, like trachemys scripta (cadi and joly, 2003, 2004) and micropterus salmoides lacepède, 1802 (lacomba and sancho, 2004; ayres and cordero, 2007), robust evidence of interactions in the wild is still lacking (but see polo-cavia et al., 2010; lambert et al., 2019). moreover, rakausas et al. (2016), studying the predator-prey interactions between a recent invader in lituania, the chinese sleeper (perccottus glenii dybowski, 1877) and the european pond turtle stated that this fish does not directly contribute to the decline of e. orbicularis because hatchlings turtles are resistant to p. glenii predation and, adults of e. orbicularis consumed juvenile p. glenii, but no data about indirect impacts (e.g., trophic resource competition) have been evaluated. therefore, we assumed that sicilian pond turtles in a habitat without alien fishes would have access to wider food availability than would individuals in a habitat with introduced alien fishes. we therefore hypothesized that this would affect their rates of energy intake and we predicted that turtles in a fish-less habitat would: (1) exhibit different patterns of growth, (2) have higher annual reproductive output, and (3) have higher abundance. to test this hypothesis in the wild, we compared the parameters of two emys trinacris sub-populations inhabiting a fish-inhabited and a fish-less lakes during a multi-year study. the evidence of the negative impact of the presence of introduced fish fauna can provide additional information to further understand the effect of alien species on native fauna as well as promote adequate management plans. materials and methods study area the nature reserve of the “lake preola and gorghi tondi” is a protected area of south-western sicily, established in 1998 and currently managed by the italian association for the world wildlife fund. the nature reserve (medium point at 37°36’36.78”n 12°39’8.19”e) has a surface of 335 hectares and is surrounded by a homogeneous agricultural landscape (vineyards and olive groves). the nature reserve protects four distinct wetlands (pantano murana, lake preola, gorghi altomedio and gorgo basso), originating in a karstic depression. the basins do not have tributaries and the groundwater table is 91so close so different: what makes the difference? located in the calcarenites and is fed by local meteoric recharge (cusimano et al., 2006). lake preola is the widest water body of the area, with a surface of 33 ha approximately, filled with a thin layer of water occasionally deeper than two meters. the ‘‘gorghi’’ are lake environments in a mature euthrophic status, with an average surface area of approximately 3 ha for the lake gorgo basso (altitude: 6 m a.s.l) and 5 ha for the lake gorghi alto-medio (altitude: 3 m a.s.l.), and a maximum depth of 12 m. they are set on sink-holes and they are therefore immediately deep and surrounded by riparian helophytes [phragmites australis (cav.) trin. ex steud., typha latifolia l. and cladium mariscus (l.) pohl], with some peripherals areas with a maximum depth of about 2 m. comparison of water chemistry and benthic samples showed no significant difference between the two lakes and both showed high concentration of lead and arsenic (bellante et al., 2015; arpa sicilia, 2016). the gorghi alto-medio is situated only 200 m away from the gorgo basso, but it is separated by an asphalt road and with a slight difference in elevation. despite their proximity the two sites can be considered inhabited by two distinct sub-populations of emys trinacris. indeed, although no genetic data are available, capture-recapture data showed a very low migration rate between these lakes confirming what observed over the last fourteen years (d’angelo, unpubl. data), when for instance only five individuals migrated between the two sites, three from the gorgo basso to the gorghi alto-medio and two conversely. different non-indigenous species are reported for this territory, like the invasive red swamp crayfish procambarus clarkii girard, 1852 (d’angelo and lo valvo, 2003), and eastern mosquitofish gambusia holbrooki and wild common carp cyprinus carpio (ottonello et al., 2017a). red swamp crayfish is widespread in all the basins without significant difference in abundance and distribution (maccarone et al., 2016), while fishes are limited only to the gorghi alto-medio (ottonello et al., 2017a). in the latter, fish presence affects the food web and the assemblage and abundance of the potential preys of emys trinacris (table 1; ottonello et al. 2017a), in accordance with available results on the negative impacts that allocthonous species produce on local invertebrates communities (cyprinus carpio: parkos et al., 2003; as well as gambusia holbrooki: pyke, 2008). sampling individuals were captured by six fyke nets and 30 baited funnel traps in gorgo basso and gorghi alto-medio located 50 m apart of each other. fieldwork was carried out from 2014 to 2016 with 10 sampling occasions during the active season (march-october). date of capture, sex, age (adult/juvenile), straight carapace length (scl, sliding callipers to the nearest 0.1 mm), width (cw), height (ch) and body mass (bm, digital balance ± 1 g) were recorded on each capture according to a standardized procedure, and only individuals with evident sexual characters were considered as adults (zuffi and gariboldi, 1995). all captured turtles were individually marked by unique notch on their carapace (servan et al., 1986) as a part of a long-term study on the ecology of the species (ottonello et al., 2017b). abundance the abundance of turtles in each site was measured indirectly by means of the catch per unit effort (cpue) method. cpue was calculated for each wetland and sampling occasion with effort measured as trap-day, and the number of traps on a site multiplied by the number of days set. because animals were released after each capture we considered, within the same sampling occasion, only the first event for each individual to avoid pseudoreplication (hurlbert, 1984). we used only data collected in 2015 and 2016 because the sampling was not carried out in gorghi alto-medio in 2014. this method, usually applied in fisheries management (maunder et al., 2006), can be used also for chelonians to infer the relative abundance and compare different groups (selman et al., 2014). size and growth data and sites differences (gorgo basso vs gorghi altomedio) in size and body mass of turtles were firstly checked for normality with shapiro-wilk normality test and then analysed using a student’s t test. females and males were analysed separately due to their difference in size and mass (ottonello et al., 2017b). recaptured animals were excluded from the analysis to avoid pseudoreplication. population structure was analysed using a graphical approach by dividing scl data in size classes of 10 mm. individual variation in growth was assessed by the table 1. comparative descriptive data of the two studied sites. gorgo basso gorghi altomedio surface (ha) 3.1 4.9 open deep water (ha) 2.1 4.4 shallow water dominated by helophytes (ha) 1.0 0.5 maximum depth (m) 11 12 fish absent gambusia holbroki cyprinus carpio invertebrate alien species procambarus clarckii procambarus clarckii temperature (°c)1 27.9 26.6 dissolved oxygen (%sat)1 66.4 77.5 chlorophyll alfa (μg/l)1 0.11 1.36 total phosphorus (μg/l)1 6.9 13.3 total nitrate (mg/l)1 2.21 0.87 n° of invetebrate sampled2 346 103 n° of invertebrate taxa sampled 2 9 6 food niche breadth of e. trinacris2 1.36 0.97 average number of prey per faecal sample of e. trinacris2 15 1 1 arpa sicilia 2016; 2 ottonello et al. 2017a. 92 d. ottonello et alii mean of the relative growth rate (rgr) equation modified from brody (1945) by cox et al. (1991): 𝑅𝑅𝑅𝑅𝑅𝑅 = 𝑙𝑙𝑙𝑙𝑆𝑆𝑆𝑆𝑆𝑆! − ln𝑆𝑆𝑆𝑆𝑆𝑆" 𝑡𝑡! − 𝑡𝑡" where scl1 represents straight carapace length at first capture, scl2 represents straight carapace length at last recapture, and t2 – t1 represents the time interval in months between the two measurements. juveniles at first and last capture were used in both groups (males and females), based on the assumption that early growth trajectories of the sexes do not differ (dunham and gibbons, 1990). moreover, we considered only growing individuals (class 1, 2, 3) excluding aged (class 4) animals (see olivier, 2002 for class definition), to avoid the possible bias due the presence or dominance of no or slow growing old animals in the two areas (bury, 1979). rgr analyses assume that growth is exponential, but this assumption is rarely valid (cox et al., 1991). however, the regression between rgr and scl1 gives an estimate of growth rate at a specific carapace length, allowing direct comparison of growth rates both among individuals with different carapace lengths and among individuals with similar carapace lengths (cox et al. 1991). we tested the effect of the site (gorgo basso vs gorghi alto-medio) on the rgr using the analysis of covariance (ancova), separately for males and females. rgr has been identified as the dependent variable, scl1 as the independent variable and the site as the categorical variable. the growth has been modelled for each site (gorgo basso and gorghi alto-medio) using the von bertalanffy growth model (vbgm), as previously used to estimate size-age relation in different species of chelonians (lovich et al., 1990; litzgus and brooks, 1998; çiçek et al., 2016). the general von bertalanffy equation is: scl = a ∙ (1-be-kt) where scl is straight carapace length, a is asymptotic carapace length, b is a parameter related to length at hatching, e is the base of the natural logarithm, k is the intrinsic growth factor, and t is age in years. since this is a non-linear model for the parameter estimation, non-linear statistic was used, by the estimation of parameters and their confidence interval (95%) with non-linear least-square regression method using the fsa package (ogle, 2012) in r. the function “predict” was used to estimate the age at sexual maturity assuming the scl of the smallest gravid female and of the smallest male showing secondary sex characteristics for each site. the age of individuals was estimated with the count of plastral growth rings (keller et al., 1998), a reliable method for young european pond turtles as showed by çiçek et al. (2016) in a comparative study with skeletochronology. the maximum number of growth rings that was possible to count was nine, after which the plastron abrasion does not allow a reliable estimate of the age of individuals. because no wild new born of sicilian pond turtle were captured, we used data of hatchlings from a local breeding centre – where eggs are incubated naturally – for the estimate of parameter b. juveniles were used in both groups (males and females), based on the assumption that early growth trajectories of the sexes do not differ (dunham and gibbons, 1990). reproductive biology the presence of oviductal eggs was verified by palpation of the inguinal region (zuffi et al., 1999) directly at the capture site. gravid females were transferred temporarily to the veterinary clinic of the university of messina for x-ray examinations. in our experiment the exposure was made at 50 ma, 50 kv, 0.1 min, similar to that used by zuffi et al. (1999) for emys orbicularis (linnaeus, 1758). as these latter authors, we presumed the absence of significant injuries at the gonadal level in our sampled females and in hatchlings (hinton et al., 1997), although we recommended suspending this type of analysis within this population in next years as a precautionary measure. all individuals were released at the point of capture after the end of the procedures. the size of the eggs has been recorded directly from the radiographic images. due to the possibility that the eggs were not placed parallel on the radiographic plane we considered only the minimum diameter (dmin, ± 0.1 mm) in subsequent statistical analyses. the non-parametric mann-whitney u test was used to check any difference in the number of eggs between the two sites (gorgo basso vs gorghi alto-medio). we tested the effect of the site on the number of eggs using the analysis of covariance (ancova) to avoid the “size effect”. the number of eggs has been identified as the dependent variable, scl as the independent covariate and the site as the categorical variable. the data collected in different years were treated uniquely to increase the accuracy of the statistical sample since a no significant inter-annual variability is known for the congeneric emys orbicularis (zuffi and foschi, 2015). only those females with oviductal eggs were considered certainly sexually mature. the reproductive phenology was considered as a whole, merging data of the two groups. all above analyses were carried out with r 3.2.5 (r development core team, 2015). results abundance a total of 1405 captures of 579 different turtles (493 in the gorgo basso, 86 in the gorghi alto-medio) were made in 2015 and 2016. a strong site fidelity was confirmed as only two individuals were captured in both sites, and this happened only once during all sampling occasions. the sub-population of the gorgo basso was more abundant and reached a significant higher cpue than the sub-population of the gorghi alto-medio (mann-whitney u test = 42, n1 = n2 = 6, p < 0.01), respectively with an average of 3.02 and 0.80 turtles/ trap/day. 93so close so different: what makes the difference? size and growth the sub-population of the gorgo basso was characterized by a dominance of turtles (72%) with an scl between 110 and 129.9 mm while the sub-population of gorghi alto-medio is characterized by a dominance of turtles (76%) with an scl between 100 and 119.9 mm (fig. 1). females and males of gorgo basso were heavier (t-testfemales= 8.73, df = 45.17, p < 0.001; t-testmales= -9.60, df = 68.20, p < 0.001) and bigger than individuals of gorghi alto-medio, both in terms of carapace length (t-testfemales= 9.61, df= 41.89, p < 0.001; t-testmales= 10.09, df= 66.31, p < 0.001), width (t-testfemales= 5.50, df= 35.37, p < 0.001; t-testmales= 7.67, df= 77.63, p < 0.001), and height (t-testfemales= 4.22, df= 28.13, p < 0.001; t-testmales= 6.48, df= 65.89, p < 0.001) (table 2). we used data on 79 males (61 from gorgo basso and 18 from gorghi alto-medio) and 41 females (27 from gorgo basso and 14 from gorghi alto-medio) to estimate relative growth rate (rgr). individual variation in growth rate was high (range 0.0 – 0.0176), but with no significant difference between the sexes (t-test = -0.59, p = 0.5564). considering independently the two sites, the models showed for both sexes (models 1 and 3, table 3) that the interaction between the independent variable (scl) and the categorical variable (site) was not significant, indicating that the slopes of fig. 1. structure of sub-populations of emys trinacris inhabiting the gorgo basso (a) and the gorghi alto-medio (b). table 2. mean values of biometric measurements for emys trinacris in the two studied sites. scl = straight carapace length; cw = carapace width; ch = carapace height; bm = body mass; n = number of individuals; s.e. = standard error. biometric measurement gt basso gt alto-medio scl (mm) adult females mean 131.5(n =144) 116.5 (n = 21) s.e. 0.7 0,05 min 110.0 4,42 max 150.9 5,5 scl (mm) adult males mean 120.1 (n = 291) 111.1 (n = 45) s.e. 0.4 0.9 min 101.3 99.7 max 141.1 124.9 cw (mm) adult females mean 101.7 (n = 116) 93.7 (n = 21) s.e. 0.8 1.3 min 77.6 84.0 max 121.3 102.0 cw (mm) adult males mean 94.0 (n = 282) 87.6 (n = 45) s.e. 0.4 0.7 min 69.4 71.4 max 112.0 101.5 ph (mm) adult females mean 50.9 (n = 124) 47.0 (n = 21) s.e. 0.4 0.8 min 38.0 40.2 max 62.6 55.0 ph (mm) adult males mean 42.6 (n = 271) 39.9 (n = 45) s.e. 0.2 0.4 min 25.4 34.6 max 53.5 46.3 bm (g) adult females mean 401.1 (n = 144) 294.7 (n = 21) s.e. 7.7 12.8 min 217.0 221 max 658.0 422 bm (g) adult males mean 281.2 (n = 290) 223.2 (n = 45) s.e. 3.4 5.7 min 143.0 154.0 max 453.0 310.0 94 d. ottonello et alii the regressions were homogeneous. therefore, in both sites, the growth rate is reduced progressively as the individual grows. the other models (models 2 and 4, table 3) showed that the categorical variable (site) has a significant effect on the dependent variable (rgr), due to the significant difference of the intercept regression lines between the two sites. these models showed that individuals, despite having a growth rates that is inversely related to size, display a different growth rate: turtles from the gorgo basso are bigger than those of gorghi alto-medio. the von bertalanffy growth models showed that the sicilian pond turtle of lake preola and gorghi tondi nature reserve appeared to mature sexually at approximately six years for males of gorgo basso (sclmin = 102.8 mm) and of gorghi alto-medio (sclmin = 99.5 mm) and between seven and eight years for females of both sites, with a sclmin of 112.1 mm and 106.2 mm respectively. allometry and reproductive biology a total of 312 captures of 178 different females (152 in the gorgo basso and 26 in the gorghi alto-medio) was made between 2014 and 2016. we collected 28 individuals with detectable eggs, 21 from the gorgo basso and seven from the gorghi alto-medio. reproductive females were found between may and july, with a peak in the first half of june (fig. 2). in this period, the percentage of reproductive females collected over two-weeks interval ranged from 20% to 58.3%. we noticed also a double egg deposition of a female (scl = 132.2 mm) captured on 10 june 2015 with four shelled eggs and on 7 july 2015 with five shelled eggs. the average clutch size was 4.14 ± 0.23 (range 2 – 7, n = 28), with a positive correlation with the mother carapace length (f-statistic = 39.19, df = 27, p < 0.001) and a significant difference between the sites (mannwhitney u test = 132.5, n1 = 21, n2 = 7, p < 0.01). the difference in the clutch size between the sites was significantly independent of the size of the females (table 4). the average clutch size was 4.57 ± 1.07 (range 2 – 7, n = 21) for the sub-population of the gorgo basso and 2.86 ± 0.69 (range 2 – 4, n = 7) for the sub-population of the gorghi alto-medio. the average minimum diameter (dmin) was 19.46 ± 1.21 mm (range 16.0 – 21.5 mm, n = 89), with a not significant difference (t-test = 0.15, df = 24.23, p = 0.8843) between females of gorgo basso (dmin = 19.47 ± 0.94, n = 69) and those of gorghi alto-medio (dmin = 19.42 ± 1.40, n = 20). the average minimum diameter (dmin) was positively correlated with the carapace length of the females (rpearson = 0.70; t-test = 4.45, df = 21, p < 0.001), since the increase of the size allows a greater pelvic aperture width (rpearson = 0.89; t-test= 8.75, df = 21, p < 0.001), that is a mechanical limiting factor for egg size in turtles. table 3. site effect on growth rate. rgr (relative growth rate) = dependent variable, scl1 (first capture straight carapace length) = independent variable, site = categorical variable. f d.f. p m al e model 1 (interaction test) scl1 20.03 1 < 0.001 site 13.17 1 < 0.001 scl1 × site 1.50 1 0.22 model 2 (no interaction) scl1 19.89 1 < 0.001 site 13.08 1 < 0.001 fe m al e model 3 (interaction test) scl1 14.39 1 < 0.001 site 19.84 1 < 0.001 scl1 × site 0.69 1 0.41 model 4 (no interaction) scl1 14.51 1 < 0.001 site 20.00 1 < 0.001 fig. 2. number of females of emys trinacris with and without shelled eggs in the oviduct determined by inguinal palpation. table 4. site effect on number of eggs per female. number of eggs = dependent variable, scl (straight carapace length) = independent variable, site = categorical variable. f d.f. p model 5.11 5 < 0.05 scl 0.81 1 0.38 site 0.48 2 0.63 scl × site 0.55 2 0.59 95so close so different: what makes the difference? discussion the growth of the sicilian pond turtles in the lake preola and gorghi tondi nature reserve followed the general pattern described for other freshwater turtles: juveniles display a fast growth until sexual maturity, followed by a slowdown during ageing and at much larger size (bury, 1979). the population studied seems to fall in the general rule noticed for the genus emys (zuffi et al., 2011) and for many others emydidae (iverson et al., 1993) as a typical southern ecotype with an advance in age at maturity at a smaller body size with respect to the northernmost populations, although regional fluctuations of temperature and precipitation are better predictors of body size than latitudinal temperature clines, especially in females of emys orbicularis (joos et al., 2017). moreover, the average clutch size is lower than that of similar sized population of the related emys orbicularis in italy (zuffi et al., 1999; zuffi et al., 2015), while no substantial variation in the nesting period and in the clutch frequency was noticed. despite these natural history traits, if we analysed separately the two sub-populations, a significant difference is noticeable: turtles in the gorgo basso reached higher body size, produced more eggs per clutch and had higher density than individuals in the gorghi altomedio. excluding both the presence of environmental and genetic effects due to the proximity and similarity of the sites, all these elements suggest the that phenotypic plasticity, driven by a stressor, may explain the observed difference. furthermore, this hypothesis is supported by the different size at maturity reached in the two wetlands, following the pattern “five” described by stearns and koella (1986), where animals, that are forced to grow slowly by an inhibiting factor, mature at a same age, but a smaller size with respect to unstressed con-specific. this type of response is not uncommon in freshwater turtles at a local scale, such as noticed by gibbons et al. (1981) for females of trachemys scripta (thunberg in schoepff, 1792), while at a larger scale it can take over an adaptive process involving environmental and genetic components (bernardo, 1993). indeed, turtles for the most part used energ y resources, depending on age or period of year, for growth, maintenance of baseline metabolism and reproduction (kuchling, 1999). in particular, during the juvenile stage of chelonians, almost all resources are dedicated to rapid growth to reach a body size and a strength of the shell that make them less susceptible to predation (kuchling, 1999) and this is regulated mainly by the temperature and by the availability and quality of trophic resources (kuchling, 1999) as well as by local factors related to the presence of contaminants that can alter the metabolic processes (burger et al., 1998). censi et al. (2013) speculated that smaller body mass of emys trinacris living in lake preola and gorghi tondi nature reserve with respect to another sicilian site can be associated to the supposed reduction of food disposal induced by lanthanide pollution of aquatic environment that influences the growth of aquatic micro-organisms, but no differences was observed within our study sites (d’angelo, unpubl. data). in the same way, there are no substantial differences in the concentration of lead in the sediment an inhibitor of growth in turtles (burger et al. 1998) between the two wetlands (arpa sicilia, 2016). therefore, the differences found could be explained in the light of the alteration of abundance and assemblage of the invertebrate community induced by the presence of alien fishes (ottonello et al., 2017a) possibly exacerbated by the ubiquitous presence of procambarus clarkii. in our opinion these differences in prey availability can definitely alter the energy intake by turtles affecting both the growth of individuals in the two sites, as already observed in other american emydidae (cagle, 1946; macculloch and secoy, 1983; dunham and gibbons, 1990) and in chelydra serpentina linnaeus, 1758 (brown et al., 1994) as well as the abundance (congdon et al., 1986; galbraith et al., 1988). these differences are likely to impact both the genetic variability of the population because males of emys mate preferably with larger females (poschadel et al., 2006), as well as the reproductive output, with females of the gorgo basso that laid twice as many eggs of the gorghi alto-medio. these observations are concordant with the optimal egg size theory, according to which the major variation in the reproductive output due to different environmental conditions is in clutch size rather than in egg size, which has been optimized by natural selection (brockelman, 1975). in our opinion, these results highlighted how the introduction of non-native fish species can affect the ecology of the sicilian pond turtle, also focusing on a often overlooked aspect, the problem of the interactions between alien fishes and turtles, on which data are lacking, in contrast to the more studied competition with non-native freshwater turtles (cadi and joly, 2003; 2004). although further analyses are needed to confirm our finding with other wild populations, we believe that our data may provide important information for the management strategy of the nature reserve and for other similar areas and cases, in order to improve the conservation status of emys trinacris and the quality of habitat in which it lives. 96 d. ottonello et alii acknowledgements we are grateful to nino castelli, michela giribaldo, maurizio marchese, nicola napolitano, gianpiero pace for their help during field surveys and with manuel morici for his help during x-ray laboratory analysis. capture and handling permits were released by the ministero dell’ambiente e della tutela del territorio e del mare (dpn/2012/0003156 on 9.x.2012). d.o. was partially supported by societas europaea herpetologica grant in herpetology 2014. references andreone, f., corti, c., ficetola, g.f., razzetti, e., romano, a., sindaco, r. 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(1999): body size and cluth size in the european pond turtle, emys orbicularis, from central italy. j. zool. 247: 139–143. acta herpetologica vol. 16, n. 2 december 2021 firenze university press a new species of the genus noblella (amphibia: strabomantidae) from ecuador, with new information for noblella worleyae carolina reyes-puig1,2,3,4,*, juan m. guayasamin 2,5 claudia koch6, david brito-zapata1, matthijs hollanders7, melissa costales8, diego f. cisneros-heredia1,2,3 so close so different: what makes the difference? dario ottonello1,7,*, stefania d’angelo2, fabrizio oneto3,6, stefano malavasi1, marco alberto luca zuffi4, filippo spadola5 hematological values of wild caiman latirostris (daudin, 1802) in the atlantic rainforest in pernambuco, brazil luciana c. rameh-de-albuquerque1, alexandre p. zanotti1, denisson s. souza1, george t. diniz2, paulo b. mascarenhas-junior3,4,5,*, ednilza m. santos³, jozelia m. s. correia3 bone histology of broad-snouted caiman caiman latirostris (crocodylia: alligatoridae) as tool for morphophysiological inferences in crocodylia paulo braga mascarenhas-junior1,2,3,6, luis antonio bochetti bassetti4, juliana manso sayão5,6 is the northern spectacled salamander salamandrina perspicillata aposematic? a preliminary test with clay models giacomo barbieri, andrea costa, sebastiano salvidio* sexual size dimorphism in the tail length of the caspian whip snakes, dolichophis caspius (serpentes, colubridae), in south-western hungary györgy dudás1, krisztián frank2* semi-automated photo-identification of bahamian racers (cubophis vudii vudii) sebastian hoefer1,*, andreu rotger2, sophie mills1, nathan j. robinson1,3 an estimate of local population of nyctibatrachus aliciae at two habitat gradients of forest in western ghats sannanegunda venkatarama bhatta krishnamurthy, aall hanumantha manjunatha reddy department of environmental science, kuvempu university, jnana sahyadri, shankaraghatta 577 451, shimoga dist. karnataka, india. correspondending author. e-mail: svkrishnamurthy@yahoo.co.in submitted on 2007, 18th june; revised on 2007, 16th december; accepted 2008, 28th january. abstract. alice’s wrinkled frog (nyctibatrachus aliciae) is an endemic anuran amphibian of the western ghats. the population estimation of this species in native forest and adjoining secondary forest has revealed distinct differences in size. the population size in native forest (n = 554) was large compared to secondary forest (n = 234). the two forest habitats are characterised by air temperature, humidity, light intensity and canopy cover differences. manmade activity and habitat variables likely influenced the reduction of population size in secondary forest. keywords. nyctibatrachus aliciae, population size, india, environmental factors, conservation. alice’s wrinkled frog (nyctibatrachus aliciae inger, shaffer, koshy and bakde, 1984) is an endemic frog of the western ghats, listed as vulnerable of iucn red list categories (anonymous, 2001). this frog inhabits water logged forest floors and streamsides in evergreen and secondary forests at about 900 m a.s.l. within the geographical range of 89°n and 12-13°n (daniels, 1992). the distribution range of this frog is more than 20,000 km2 in restricted patches of western ghats. this frog is known to be threatened by human activites such as deforestation, forest fragmentation and depletion in quality of the habitat (inger et al., 1984). the continued decline of this species has been observed in severely fragmented areas (anonymous, 2001). unfortunately, there are no reliably estimates of population sizes both in different habitat gradients and human affected areas within the forest (manjunatha reddy, 2004). alice’s wrinkled frogs are highly secretive, and confined to debris and organic mulch in forest stream. this frog has strong conformity; adult breed in the same habitat and have very poor dispersal capacity. in the present study, the population of alice’s wrinkled frog was estimated in a native and a secondary forests of kuvempu bioreserve (loc: 13°35’-13°40’n and 75°15’-75°20’e) located in western ghats. the population was estimated using schnabel method of markacta herpetologica 3(1): 51-55, 2008 issn 1827-9643 (online) © 2008 firenze university press 52 s.v. krishnamurthy and a.h. manjunatha reddy recapture as described by sutherland (1997) using knee tagging technique as described by elmberg (1989). silk threads were used to tag the frogs, and in both sites, marked and unmarked frogs were collected for every fortnight. the study was made for a short duration of 8 months of post monsoon (october through january) and premonsoon (february to may), and not made in monsoon (june to september), when abundant water favour dispersal of frogs. data were processed following schnabel method (krebs, 1999). the native forest site (area: 2 ha; altitude: 710 m a.s.l.) is extended on either the banks of a stream within the bioreserve, while the secondary forest site (area: 5 ha; altitude: 650-670 m a.s.l.) is located 1 km downstream to the native site. habitat modifications in this site mainly related to agriculture practices which removed most of native trees and changed the texture of the forest floor. during each survey, important habitat variables viz, air, water, soil temperature, humidity, light intensity, and canopy cover were recorded. air and water temperatures of the habitat were measured using the mercury bulb thermometer (make: jennson, precision 0.1 °c). the soil temperature was recorded using a mercury soil thermometer (make: jennson; graduated to 0.1 °c). an illuminometer (model 5200; kyoritsu, japan) was used to measure the light intensity and values were recorded in lux. using the thermohygro-clock (model j412 – cth, japan), the relative humidity (%) of the habitat was recorded. using a photograph of canopy, the percent coverage of the region was calculated. seventeen field surveys for each study site were conducted early in the morning and four man-hours were spent during each occasion. habitat variables were subjected to anova to find out the significance of differences between the two sites. karl-pearson correlation coefficient was used to check the relationship among the habitat variables. regression analysis was carried out to find out the linearity between proportion of marked frogs in each catch and number of frogs previously marked. all statistics were made using sigmastat (version 3.5) for windows. the characters of the two sites were differentiated based on air, water, soil temperature, humidity, light intensity, and canopy cover recorded during study period. table 1 presents the data on the habitat variables of study sites. table 1. habitat variables (mean ± se) of native and secondary forest sites. values in the parentheses indicate ranges. parameter native forest (n = 17) secondary forest (n = 17) f1, 32 p air temperature (°c) 22.99 ± 0.47 (19-26) 25.31 ± 0.55 (22-30) 10.371 0.003 water temperature (°c) 22.20 ± 0.66 (18-30) 23.18 ± 0.56 (20-26) 1.299 0.264 soil temperature (°c) 22.64 ± 0.36 (21-25) 23.42 ± 0.45 (18-26) 1.832 0.187 humidity (%) 76.88 ± 2.13 (63-90) 69.96 ± 2.80 (44-84) 3.865 0.041 canopy cover (%) 83.53 ± 1.02 (79-92) 80.10 ± 0.37 (78-84) 10.09 0.004 light intensity (lux) 993.42 ± 135.63 (273-1,639) 1,990.88 ± 443.10 (511-7,260) 4.633 0.040 53an estimate of local population of nyctibatrachus aliciae compared to native forest site, average air temperature and light intensity, recorded during the study period, were higher in secondary forest site (table 1). the air temperature of the secondary forest site ranged between 22 and 30 °c against 19° and 26 °c recorded in the native forest site. the light intensity ranged between 511 and 7,260 lux in secondary forest, while in primary forest it ranged between 273 and 1,639 lux and the differences were statistically significant (see table 1 for statistics). similarly, compared to native forest, the humidity and canopy cover were lower in secondary forest and the differences were significant (see table 1 for statistics). air temperature was positively correlated with light intensity (r = 0.51, p = 0.011) and negatively with canopy cover (r = -0.54, p = 0.008). the variation in the canopy thickness and light intensity indicate the changes associated with habitat structure, likely due to human acrivity. population estimate of n. aliciae in two different forest habitats is given in table 2. during the study, a total of 157 frogs from native forest and 97 frogs from secondary forest were collected. out of these, 18% and 25% of the frogs were recaptured respectively. the population estimate value of n. aliciae in the native forest (n = 553.73) was large as compared to secondary forest (n = 234.07). the frequency of marked individuals in the native forest increased linearly with increasing previously marked frogs (fig. 1a; y = 0.077 + 0.001x; r2 = 0.48) indicating that the size of the population was constant. by contrast, in secondary forest (fig. 1b), the non-linearity (y = 0.106 + 0.003x, r2 = 0.124) depicts the violation of assumption generally made for schnabel method of mark-recapture. the non linearity of marc-recapture curve (fig. 1b) indicates disappearance of some frogs from this site over the time of study. since this frog is sensitive to habitat quality, the manmade activities that have changed habitat character could be a factor for high fluctuation in population size. ecologist have used many measures of landscape structure to predict the population dynamic consequences of habitat loss and fragmentation (gustafson and gardner, 1996: with et al., 1997) and microhabitat heterogeneity is known to influence the distribution of frog species (kam and chen, 2000). anthropogenic activities in agriculture and silviculture have negative effect on amphibian populations and influence the abundance and richness (demaynadier and hunter, 1998; ŝireika and staŝaitis, 1999). in the present study, the past anthropogenic activities were evident in the secondary forest (table 1). further, the frog population was low in secondary forest site amounting to only 42.3% of those recorded in native forest site. table 2. population size of alice’s wrinkled frogs (nyctibatrachus aliciae) in kuvempu bioreserve, central western ghats. variables native forest secondary forest number of surveys 17 17 population estimate (size) 553.73 234.07 variance 0.1195 0.699 se 0.000266 0.000007 confidence limit 0.01018-0.0113166 0.010725-0.010758 54 s.v. krishnamurthy and a.h. manjunatha reddy alice’s wrinkled frog, as an endemic amphibian of western ghats, requires specific habitat. manmade activities and conversion of the habitat have rendered direct and indirect effect on habitat quality affecting the population size of this frog. the earlier work (manjunatha reddy, 2004) and present study have revealed the importance of thick canopy cover, low air temperature and light intensity as favourable factor for the occurrence of the species. therefore, it is important to emphasize on these habitat variable for the conservation of species. fig. 1. proportion of marked frogs in native (a) and secondary forest (b) sites. 55an estimate of local population of nyctibatrachus aliciae acknowledgements authors are thankful to two anonymous referees and dr. marco a.l. zuffi, university of pisa for the comments and suggestions on the manuscript. references anonymous (2001): amphibian camp hand book, daptf-sa, zoo-outreach organization, coimbatore. daniels, r.j.r (1992): geographical distribution pattern of amphibians in the western ghats, india. j. biogeography. 19: 521-529. demaynadier, p.g., hundter, m.l., jr. (1998): effects of silvicultural edges on the distribution and abundance of amphibians in maine. conserv. biol. 12: 340-352. elmberg, j. (1989): knee-tagginga new marking technique for amphibians. amphibiareptilia 10: 101-104. gustafson, e.j., gardner., r.h (1996): the effect of landscape heterogeneity on the probability of patch colonization. ecology 77: 94-107. inger, r.f., shaffer, h.b., koshy, m., bakde, r. (1984): a report on a collection of amphibians and reptiles from the ponmudi, kerala, south india. j. bombay nat. hist. soc. 81: 406-427. kam, y-c., chen, t-c. (2000): abundance and movement of a riparian frog (rana swinhoana) in a subtropical forest of guandau stream, taiwan. zool. studies 39: 67-76. krebs, c.j, (1999): ecological methodology. addison-wesley education publishers, inc. manjunatha reddy, a.h. (2004): role of changes in habitat qualities on ecological status of endemic anurans micrixalus saxicola and nyctibatrachus aliceae. ph.d thesis, kuvempu university. ŝireika, e., staŝaitis, j. (1999): abundance and distribution of amphibians in aukŝtaitija national park. acta zool. lituan. biodiversity 9: 91-95. sutherland, w.j (1997): ecological census techniques. cambridge university press, cambridge. with, k.a., gardner, r.h., turnar, m.j. (1997): landscape connectivity and population distributions in heterogeneous landscapes. oikos 78: 151-169. first record of vipera ursinii graeca in albania (reptilia: serpentes, viperidae) zoltán korsós1, zoltán barina2, dániel pifkó2 1 department of zoology, hungarian natural history museum, baross u. 13, h-1088 budapest, hungary. corresponding author. e-mail: korsos@nhmus.hu 2 department of botany, hungarian natural history museum, könyves kálmán krt. 40, h-1087 budapest, hungary. submitted on 2008, 15th january; revised on 2008, 2nd july; accepted on 2008, 18th august. abstract. in the framework of the botanical and zoological expeditions of the hungarian natural history museum to the balkans, a specimen of vipera ursinii graeca was observed in the southern nemerçke mountains of albania. it is the first record of the subspecies outside greece, but zoogeographically it belongs to the northern pindos mountain range. keywords. vipera ursinii graeca, new record, southern albania. herpetofauna of albania may still surprise us with interesting records, although several accounts already exist (werner, 1920; kopstein and wettstein, 1921; karaman, 1939; bruno, 1989; haxhiu, 1998). this is especially true for the southern mountaneous area which is still scarcely visited by naturalists. for several years, the hungarian natural history museum has been organizing regular collecting trips to the balkans, including those remote parts of albania, and accumulates floristic and faunistic information about the “white spots” of the country (farkas and buzás, 1997; fehér et al., 2004). here we present a new record of the rare vipera ursinii graeca nilson et andrén, 1988 which was known to occur only in the pindos range of greece, until now. the meadow or steppe vipers of europe, the vipera (acridophaga) ursinii group is divided into several subspecies (nilson and andrén, 2001) which inhabit mountaneous (v. u. ursinii in france and italy, v. u. macrops in the dinaric and sardo-pindic mountain ranges, v. u. graeca in greece) and lowland (v. u. rakosiensis in hungary, v. u. moldavica in romanian moldova, v. u. renardi in ukraine) grassy habitats. all the populations are nowadays fragmented and isolated, and under serious human (mainly agricultural) pressure, hence the whole species group is listed at the highest priority level of international nature conservation categories (e.g., in the appendices of the bern convention, the acta herpetologica 3(2): 167-173, 2008 issn 1827-9643 (online) © 2008 firenze university press 168 z. korsós, z. barina and d. pifkó eu habitat directive, the natura 2000, the iucn red list, and the cites washington convention; corbett, 1989). the two main groups, the mountain grassland and the lowland steppe subspecies, not only differ in terms of ecological requirements, but morphological and developmental traits as well as differences in reproduction biology were also described (baron et al., 1996; korsós and újvári, 2000; nilson and andrén, 2001; filippi and luiselli, 2002). in 1982, a local and isolated population of small-sized ursinii viper was discovered in the pindos mountains of central greece (dimitropoulos, 1985). nilson and andrén (1988) described it as v. u. graeca, with the diagnostic differences in the low number of subcaudals (18-21 females, 20-27 males), much reduced number of head scales (fewer loreals, only 6-7 supralabials) and posterior dorsal scale rows, midbody scale rows sometimes reduced to 17, and a much reduced colour pattern. morphological analyses were later completed with biochemical differences, too (joger et al., 1992; nilson and andrén, 2001), which showed that graeca can be characterised with a unique serum albumin pattern, and it forms a common evolutionary lineage with macrops. localities in greece were listed as tzoumerka mountain, lakmos or peristeri mountain, and koziakas mountain. all these belong to the pindos range about 1800-2000 m a.s.l., and this can be true for the new locality presented here as well. the new observation of an adult specimen of vipera ursinii graeca was made at a locality situated in southern albania, district of përmet (rrethi i përmetit), nemerçke mountains (mali i nemerçkes). the viper specimen was found and photographed (fig. 1) at the north-eastern slope of mount poliçani, 400 m north-east of peak poliçani (maja e poliçanit, 2138 m), at about 1900 m above the sea level. exact geographical coordinates are not presented because of conservation reasons but they can be obtained from the authors. fig. 1. vipera ursinii graeca on mount poliçani, albania (photo d. pifkó). 169vipera ursinii graeca in albania date of observation is the 23rd of may, 2006, in the late afternoon (around 1600 h), weather conditions were clear, warm and sunny. the habitat was a short-grass montane pasture on the eastern slope of the mountain chain, facing to the east, relatively declivitous (about 10-20 degrees), with steeper neighbouring slopes, on limestone bedrock (fig. 2). exactly at the locality where the specimen was found there were no rocky outcrops, but some smaller pieces appeared a couple of meters away. plant species composition of the grassland included: sesleria (probably tenuifolia) and poa spp., ornithogalum oligophyllum, corydalis solida, scilla bifolia agg., narcissus poeticus, pedicularis sp., and many other herbs not yet in flower. snow also still covered some of the surroundings. the structure of the habitat was tussocky, with characteristic sesleria aff. tenuifolia, and short poa spp. inbetween. although the specimen was not collected for conservation reasons, it is clear from the photograph (taken by the botanist third author of the present paper) that this specimen was v. u. graeca. white labial sutures, low number (6 on one side) of supralabials, fragmented nasal scutes and colour pattern clearly distinguish it from v. u. macrops, the only other alternative (g. nilson, pers. comm.). total length of the specimen was about 60 cm, but no other measurements can be provided because the animal was not handled. the mountainous populations of vipera ursinii in the balkan peninsula have for a long time been assigned to the subspecies macrops méhely, 1911. its distribution range includes part of croatia, bosnia-herzegovina, montenegro, serbia, kosovo, macedonia and northern albania (fig. 3). data of its occurrences have been continuously accumulating (méhely, 1911; werner, 1920; kopstein and wettstein, 1921; fejérváry, 1923; burech and zonkov, 1934; karaman, 1939; radovanović, 1941, 1964; dimovski, 1964; pasuljević, 1968; bruno, 1989; nilson and andrén, 1997; crnobrnja-isailović, 2002; tomović et al., 2004; fig. 2. habitat of albanian v. u. graeca, with the town përmet in the background (photo d. pifkó). 170 z. korsós, z. barina and d. pifkó sterijovski, 2006). populations usually occur above 1000 m a.s.l., up to 2300 m. there are two dubious records in croatia, on the island of krk at sea level (werner, 1895, 1920; schwarz, 1936; karaman, 1939; bruno, 1980), and mt. učka in istria (tvrtković et al., 2006). these records have never been reconfirmed in the field, and v. u. macrops is very rare in croatia (tvrtković et al., 2006). other dubious records of a montane form of v. ursinii in the balkan peninsula are from bulgaria, mt. lülin at 950 m a.s.l. and the region of shumen (question mark in fig. 3; buresch and zonkov, 1934; beschkov, 1973; westerström, 2002; beshkov and nanev, 2002), which are considered by some authors as ssp. rakosiensis (beschkov, 1973; nilson and andrén, 1997). these populations are considered to be extinct (nilson and andrén, 1997; beshkov and nanev, 2002; edgar and bird, 2005). the actual distribution area of v. u. macrops is confined mainly to the mountainous ranges of bosnia-herzegovina and montenegro. here, individuals are relatively frequently observed (radovanović, 1941, 1964; crnobrnja-isailović, 2002; tomović et al., 2004), whereas in the connecting borderzones of extreme south serbia, west kosovo and west macedonia this snake species is very rare (dimovski, 1964; pasuljević, 1968; sterijovski, 2006). in albania, v. u. macrops is recorded in the northern and eastern part of the country, in the mountains of koritnik, korab, shar (or sara, together with kobilitsa) reaching fig. 3. map of the balkan peninsula and italy with the approximate distribution of the mountainous vipera ursinii subspecies. 1 = v. u. ursinii; 2 = v. u. macrops; 3 = v. u. graeca. question marks are unconfirmed records: island krk in croatia, mt. lülin and the region of shumen in bulgaria. star: new record of v. u. graeca in southern albania. 171vipera ursinii graeca in albania over to the neighbouring countries (méhely, 1911; werner, 1920; fejérváry, 1923; burech and zonkov, 1934; pasuljević, 1968; bruno, 1989; haxhiu, 1998; sterijovski, 2006). the new locality in the south is strikingly geographically separated from these mountains, supporting its subspecific difference as well. the habitat of v. u. graeca is very similar to that of macrops: typically it is a rocky, stony subalpine meadow (fig. 2). the difference between the two subspecies seems to be in the altitude above sea level: the range of graeca is about 1750-2000 m, while that of macrops is 1000-2300 m. although the altitude ranges are overlapping, graeca seems to have a narrower distribution range, which is supported by the observation that only it occurs at about 12 sites in southern and central mainland greece only (m. dimaki, pers. comm.). the vegetation in the habitat of our record (mount poliçani, 2138 m a.s.l.) is a short-grass mountain pasture; this is in contrast to the description given by crnobrnjaisailović (2002) and sterijovski (2006), who characterised macrops biotopes as juniperetovaccinetum (bjelasica mountain, eastern montenegro, 1900 m) or calamintho grandiflorae-fagetum (bistra mountain, macedonia, 1650-1900 m), respectively. both are at least partially covered by ligneous bushes (genera juniperus, vaccinium, fagus) whereas these low-grown tree species are totally absent on mount poliçani (fig. 2). in conclusion, our data suggest that there are two different vipera ursinii subspecies in albania: macrops and graeca. their ranges are widely separated in the northern and southern mountain chains of the country; the southeastern, inhabited by graeca, being connected over the greek border to the pindos mountains. because of the single observation, we suggest that more field work is required before any detailed conservation measures can be formulated regarding this endangered member of the vipera ursinii group at the european scale. acknowledgements we are most grateful to dávid murányi (budapest, hungary) for calling our attention to the importance of the observation, to göran nilson (göteborg, sweden) for confirming our identification, to wolfgang böhme (bonn, germany) and balázs farkas (budapest, hungary) for helping with literature, and especially to maria dimaki (athens, greece) for updating our distribution records for greece. references baron, j.-p., ferriere, r., clobert, j., saint girons, h. 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(2006): crvena knjiga vodozemaca i gmazova hrvatske. ministarstvo kulture, državni zavod za zaštitu prirode [red book of the amphibians and reptiles of croatia. ministry of culture, state institute for nature consevation], zagreb, 94 p. werner, f. (1895): beitrage zur kenntnis der reptilien und amphibien von istrien und dalmatien. verh. zool.-bot. ges. wien 41: 751-768. werner, f. (1920): zur kenntnis der reptilienund amphibienfauna albaniens. zool. anz. 51: 21-24. westerström, a. (2002): on the situation of the bulgarian vipera ursinii. in: population and habitat viability assessment (phva) for the hungarian meadow viper (vipera ursinii rakosiensis). workshop report, p. 71. kovács, t., korsós, z., rehák, i., corbett, k., miller, p.s., eds, iucn/ssc conservation breeding specialist group, mn, usa. acta herpetologica 4(1): 83-98, 2009 a misunderstood new gecko of the genus hemidactylus from socotra island, yemen (reptilia: squamata: gekkonidae) roberto sindaco1,5,7, ugo ziliani2, edoardo razzetti3, caterina carugati2, cristina grieco1, fabio pupin4, badr awadh al-aseily6, francesca pella4, mauro fasola4 1 istituto per le piante da legno e l’ambiente, corso casale 476, i-10132 torino, italy. 2 platypus s.r.l., via pedroni 13, i-20161, milano, italy. 3 museo di storia naturale, università degli studi di pavia, piazza botta 9, i-27100 pavia, italy. 4 dipartimento di biologia animale, università degli studi di pavia, piazza botta 9, i-27100 pavia, italy. 5 museo civico di storia naturale, via san francesco di sales 88, i-10022 carmagnola (to), italy. 6 scdp-epa office, hadibo, socotra,yemen. 7 corresponding author. e-mail: rsindaco@gmail.com abstract. a new endemic gecko of the genus hemidactylus is described from socotra island (yemen). it is a rupicolous species characterized by: medium-large size (svl up to 60 mm), back with large trihedral, raised, strongly keeled tubercles intermixed with small granular scales, males with 6-10 preanal pores arranged in two short rows separated by 2-3 scales. in east africa, arabia, the middle east and india the only other tuberculated hemidactylus with preanal pores arranged on two separate rows is the somali h. granchii lanza, 1978, which differs for the comparatively deeper and shorter head, the nostril separated from the first upper labial, less preanal pores, less upper and lower labials, more tubercles at midbody and more lamellae under the inner toe. keywords. socotra, yemen, hemidactylus, new species. introduction the herpetological investigation of socotra started in 1880 with the expedition of isaac bayley balfour; in the following twenty years a few british, german and austrian expeditions collected zoological specimens on the island, including new reptile species that were later described by william thomas blanford (1881), albert günther (1881), wilhelm peters (1882), franz steindachner (1899, 1903) and particularly by george albert boulenger (1899, 1903). the latter author described many species collected during the most important socotran expeditions, such as those of william r. ogilvie-grant and henry o. forbes. moreover, a few new species collected by these explorers passed unnoticed and were described only 90 years later. no other herpetological expedition was carried out until 1953, when george b. popov visited the island and collected reptiles, including some of the undescribed species already 84 r. sindaco et alii collected by ogilvie-grant and forbes. other reptiles were collected in 1956 during the oxford expedition, including a new worm-snake described by hahn (1978). a few more undescribed species were collected in 1967 by kenneth m. guichard, during the middle east command expedition. about 20 years later arnold (1986) described two new semaphore geckoes of the genus pristurus on the basis of old specimens caught during these expeditions and held in the collections of the british museum of natural history (now the natural history museum). for a complete review of the herpetological survey of socotran reptiles and a complete list of the pertinent literature, see the monograph by schätti and desvoignes (1999). finally, during the period of the arab democratic republic of yemen, a few zoological expeditions visited socotra, most of them promoted by german zoologists (cf. wranik 1996, 1998; rösler 1998c; rösler and wranik, 1998, 1999, 2000; schätti and desvoignes 1999). some of this research also resulted in the description of new species: pristurus samhaensis, pristurus obsti and hemidactylus dracaenacolus by rösler and wranik (1999) and mesalina kuri by joger and mayer (2002); other new species have been tentatively identified, but not formally described. among them there is a really misunderstood hemidactylus species that is formally described in this paper. this taxon was recorded for the first time by schätti and desvoignes (1999) (as h. granti), and then also cited by other herpetologists on the basis of a single living specimen that was alternately listed as a slightly different form of h. turcicus or possibly a new unnamed taxon (cf. rösler and wranik, 2003, 2004, 2006). between 2007 and 2009, five scientific surveys were organized by the university of pavia to collect data in the framework of the “socotra conservation and development project”, founded by the italian cooperation – ministry of foreign affairs, and under the auspices of the united nations development programme, with the aim of improving sustainable development and the biodiversity conservation of the socotran archipelago. during these surveys many areas of the island were investigated and a large number of distributional and ecological data for socotran reptiles were collected (sindaco et al. 2008), including many specimens of an undescribed species of the genus hemidactylus, that represents the subject of this paper. materials and methods herpetological data were collected during four field surveys (for a total of 53 days of search effort, by three observers on average). data on reptiles were collected during more than 150 diurnal and nocturnal transects of 30 minutes each, based on the systematic sampling surveys, time-constrained, protocol (heyer et al. 1994). the transects were conducted in all main habitats, distributed over most of the island. moreover, additional opportunistic censuses were conducted to observe as many species as possible in each surveyed locality. during the transects, more than 4000 reptile individuals belonging to all 25 terrestrial reptiles known on the island were recorded. some individuals were collected, measured, photographed and then released. since the taxonomic status of some taxa is still uncertain, and some species are only known on the basis of a very few, often old, specimens, a small number of specimens for each species were collected for further morphological and genetic analyses. to avoid suffering, the collected specimens 85a misunderstood new gecko of the genus hemidactylus were anaesthetized by ether, then fixed in 95° ethanol and thereafter preserved in 75° ethanol. the collected specimens are now deposited in the herpetological collections of the museo civico di storia naturale di carmagnola, torino (mcc), and the museo di storia naturale of the university of pavia (msnpv), italy. the new species was compared to all the other hemidactylus species with similar characteristics living on the socotran archipelago, east africa, the middle east and indian subcontinent. we also checked the collections of several museums (see appendix 1), the original descriptions, and taxonomic reviews (see references). results hemidactylus inintellectus sindaco, ziliani, razzetti, pupin, grieco sp. nov. (figs. 1-4) synonyms hemidactylus granti – schätti and desvoignes (1999: 108-109; fig. 30) hemidactylus aff. turcicus – rösler and wranik (2000: 24; tab. 1) [hemidactylus] turcicus-like – rösler and wranik (in wranik 2003: 133) hemidactylus sp. – rösler and wranik (in wranik 2003: pl. 74 up) hemidactylus sp. b – rösler and wranik (2004: 518; pl. 5, fig. 20) hemidactylus sp. b – rösler and wranik (2006: 127; tab. 1) hemidactylus granti – sindaco et al. (2008: tab. 1) type material holotype – mcc-r1470, adult male, yemen, socotra island, wadi ayhaft (12°36’47”n– 53°57’52”e), about m 200 a.s.l., u. ziliani, e. razzetti, r. sindaco, c. carugati, c. grieco leg., 3.i.2008. paratypes – 15 specimens from socotra island, yemen, collected in the following localities: msnpv-cr866, 1 adult male, wadi ayhaft (12°36’47”n– 53°57’52”e), same data of the holotype. mcc-r1437, msnpv-cr867, 2 adult females, between hadibo and qadub (12°38’22”n – 53°57’45”e), m 80 a.s.l., c. grieco, f. pupin, r. sindaco leg., 27.xii.2008. mcc-r1441, 1 adult female, temedeh area (12°36’41”n – 54°18’12”e), m 10 a.s.l., 29.xii.2008, c. grieco, f. pupin, e. riservato, r. sindaco leg. mcc-r1471 + msnpv-cr868, 2 adult males, wadi kilisan south of afafes m 264 a.s.l. (momi plateau) (12°29’32”n– 54°20’57”e), 7.i.2008, c. grieco, f. pupin, e. razzetti, o. sacchi, r. sindaco, u. ziliani mcc-r1472 (1-4), 1 male, 2 females, 1 immature; msnpv-cr869-872, 1 male, 2 females, 1 immature, dhero area (12°28’59”n – 54° 1’35”e), m 400 a.s.l., 29.xii.2007, c. grieco, f. pupin, r. sindaco, e. riservato, u. ziliani leg. mcc-r1469, 1 adult female, nw foothills of the jebel ma’li, about 6 km sw of qalansiyah (12°39’32”n – 53°26’38”e), m 250, 27.i.2009, d. pellitteri, u. ziliani leg. 86 r. sindaco et alii derivatio nominis the specific epithet inintellectus is a latin adjective meaning “misunderstood”, because the species was observed and/or collected by different authors (including us during the first surveys), but was confused with other taxa or was suspected to be a new taxon, but remained undescribed for about ten years. we suggest “socotran rock gecko” as english name for this species. diagnosis a rather robust hemidactylus (maximum svl: male = 59.5 mm, female = 60.5 mm), easily distinguishable from the others socotran hemidactylus by the following combination of characters: back covered by large, trihedral, strongly keeled tubercles intermixed with a few small, irregular shaped granules (fig. 1d), forming 12-16 (mode 14) irregular transversal rows from axilla to groin (counted along a paravertebral line); nostril in contact with the rostral, the 1st supralabial and 3 postnasals; 6 lamellae under the 1st toe (fig. 1c) and 9-11 (media 10.2 ± 0,54, mode = 10) under the 4th toe (fig. 1e). chin shield subtriangular; first pair of enlarged post-mentals in broad contact each other and with the first and second lower labial; second pair in contact with the second lower labial and usually with small paralabials (fig. 1b). males with 3/3 to 5/5 preanal pores in two short rows, separated medially by 2-3 scales (fig. 1f ); females without pores. pattern with more or less irregular, narrow transversal bands on back and unregenerated tail, sometimes indistinct. ventral part of the tail with transversely enlarged scales intermixed with smaller ones (fig. 1g). description of the holotype head rather depressed (ratio between head length to the mandibular angle and head depth = 2.48), its length 1.55 times its width; snout subacuminate, concave between the nares and the eyes, swollen in front of the eyes, 1.76 times as long (to the anterior margin of the eye, measured in a diagonal line) as the distance between the posterior margin of the eye and the anterior margin of the ear-opening; ear opening elliptical, its major axis subvertical and a little more than 1/3 that of the exposed eye. major diameter of the exposed eye about 1/4 the head length (to the mandibular angle); pupil a vertical slit with lobed margins. rostral subquadrangular, nearly 1.7 wide as high, divided longitudinally for half of its length in the superior part; nostril in contact with the rostral, the 1st supralabial and 3 enlarged postnasals; the post-rostrals separated by a smaller scale by its fellow; 12/11 upper (10/9 before the centre of the eye) and 9/8 lower labials; mental large, subtriangular, longer than the anterior chin-shields; anterior chin-shields broadly in contact along the median line and with the lst sublabial (in narrow contact with second sublabials). a second pair of chin-shields clearly smaller than the first pair touching the 2nd pair of sublabials and 1 rows of paralabials. 87a misunderstood new gecko of the genus hemidactylus fig. 1. morphological characters. a – head from above; b – chin shields arrangement; c – right hand; d – dorsal tubercles; e – left foot; f – anal region; g – ventral side of the tail 88 r. sindaco et alii snout covered by rather irregularly sized, roundish, juxtaposed, more or less convex scales (few of them slightly keeled). posteriorly the scales grade into small, irregular granules in the interorbital region; nape and temporal region with conical or subtrihedral strongly keeled (especially on neck) tubercles. trunk rather depressed, covered dorsally by large, trihedral, strongly keeled and often striated tubercles, arranged in about 12-13 irregular longitudinal rows (6-7 between the hindlimbs), about 14-15 in a straight paravertebral line between axilla and groin; the tubercles are separated by 3-5 small, subimbricate, heterogeneous scales. ventral scales small, flat, smooth, and imbricate, about 40 in a transverse row at midbelly, about 70 between axilla and preanal pores along a line on the middle of the belly; 11 ventral scales in an eye diameter in the middle of the belly, counted longitudinally. throat covered by subimbricate scales (about 25 in an eye diameter in the middle of the throat, counted longitudinally). 10 (5/5) preanal pores arranged in a curved line, interrupted by two scales. antero-dorsal side of forearm, dorsal side of tibia and postero-dorsal side of thigh with very heterogeneous scalation, with large keeled tubercles, the ones of the forearm are smaller than the other ones. limbs rather long: the tip of adpressed hindlimb reaches the elbow of adpressed forelimb. digits free, moderately dilated, slightly webbed at the base with free undilated terminal portion clearly projecting beyond the dilated part (that of 4th toe with 12 scales along the dorsal edge); lamellae beneath the toes from lst to 5th (undivided+divided+entire apical; right / left if different): hands 3+3+1, 1+5+1, 1+6+1/1+5+1, 1+6+1, 2+5+1. feet : 2+3+1, 2+5+1, 1+6+1, 3+6+1, 4+6+1. hemipenial bulges well developed. tail unregenerated, conical, without basal constriction, with a single series of strongly keeled and raised tubercles on the dorsal and lateral sides for each tail segment, 6 near the base and 4 on the distal part; ventral side of the tail with arranged transversely enlarged scales irregularly alternating with un-enlarged ones. measurements. see tab. 1. colouration. in alcohol, greyish with irregular, rather distinct transverse dark bars; tail with 7 distinct dark-light rings; limbs almost uniform. underparts are off-white. variation measurements of the paratypes are given in the tab. 1. there is little variation both in either proportions and scale counts. the ratio head length / head depth ranges from 2.2 to 2.5 (mean 2.4 ± 0.1); ratio head length / head width from 1.4 to 1.5 (mean 1.44 ± 0.05); ratio snout / eye-ear opening distance from 1.5 to 1.8 (mean 1.66 ± 0.08). the ear opening is more or less elongated, its maximum diameter is 0.3-0.4 times the horizontal diameter of the eye (mean 0.32 ± 0.04). the eye is 0.2-0.3 times the length of the head from the tip of the snout to the mandibular angle (mean 0.24 ± 0.01). the arrangement of scales around the nostril is very constant: in all the specimens the 89a misunderstood new gecko of the genus hemidactylus ta b. 1 . m ea su re m en ts a nd s ca le c ou nt s. 1 = s ex / a ge ; 2 = s no ut t o ve nt l en gt h (s v l) ; 3 = t ai l le ng th ( t l) ; th e as te ri sk i nd ic at es t he r eg en er at ed t ai ls ; 4 = h ea d le ng th ( fr om t he t ip o f th e sn ou t to t he m an di bu la r an gl e) ; 5 = h ea d w id th ; 6 = h ea d de pt h; 7 = d is ta nc e be tw ee n th e ti p of t he s no ut a nd t he a nt er io r ed ge o f th e ey e (i n la te ra l vi ew ); 8 = o ri zo nt al e ye d ia m et er ; 9 = d is ta nc e be tw ee n th e po st er io r ed ge o f th e ey e an d th e an te ri or e dg e of t he e ar o pe ni ng ; 10 = m ax im um d ia m et er o f th e ea r op en in g; 1 1 = r at io b et w ee n he ad l en gt h an d he ad d ep th ; 12 = r at io b et w ee n he ad l en gt h an d he ad w id th ; 13 = r at io b et w ee n sn ou t le ng th ( ch ar ac te r 7) a nd t he e ye -e ar d is ta nc e (c ha ra ct er 9 ); 1 4 = r at io b et w ee n di am et er s of t he e ar o pe ni ng a nd e ye ; 1 5 = r at io b et w ee n th e ey e di am et er a nd t he h ea d le ng th ; 16 = n um be r of u pp er l ab ial s (r ig ht / l eft i f di ffe re nt ); 1 7 = n um be r of l ow er l ab ia ls ( ri gh t / le ft i f di ffe re nt ); 1 8 = n um be r of g ul ar ia i n th e ey e di am et er ; 19 = n um be r of ve nt ra li a in th e ey e di am et er ; 20 = lo ng it ud in al ro w s of en la rg ed tu be rc le s at m id tr un k; 21 = n um be r of tu be rc le s be tw ee n hi nd lim bs ; 22 = n um be r of t ub er cl es b et w ee n ax ill a an d gr oi n; 2 327 = s ub di gi ta l la m el la e un de r fi ng er s fr om 1 st t o 5t h (r ig ht / l ef t if d if fe re nt ); 28 -3 2 = su bd ig ita l l am el la e un de r to es fr om 1 st to 5 th ( ri gh t / le ft if di ffe re nt ) – ar e in di ca te d ba sa l u nd iv id ed o ne s + di vi de d + un di vi de d ap ic al la m el la . character mccr1437 msnpvcr867 mccr1441 msnpvcr866 mccr1470 msnpvcr868 mccr1471 msnpvcr871 msnpvcr869 msnpvcr870 mccr1472 mccr1472 mccr1472 msnpvcr872 mccr1472 mccr1469 1 f f f m m m m im m . m f m f f f im m . f 2 57 .0 56 .0 52 .0 52 .5 49 .0 51 .0 56 .0 41 .0 57 .0 56 .0 59 .5 53 .0 51 .0 60 .5 45 .0 52 .0 3 59 .0 * lo st 56 .0 45 .0 * 57 .0 52 .0 52 .0 * lo st 68 .0 tr un ca te d lo st 57 .0 56 .0 lo st 50 .0 58 .0 * 4 18 .2 17 .5 16 .3 16 .8 16 .4 16 .2 17 .6 13 .8 18 .1 17 .2 17 .9 15 .6 15 .1 18 .0 14 .8 16 .0 5 12 .3 12 .4 11 .8 11 .6 10 .6 11 .1 12 .5 9. 2 12 .2 11 .8 12 .8 11 .1 10 .9 12 .5 9. 8 11 .6 6 7. 8 8. 0 7. 0 7. 0 6. 6 6. 8 7. 1 5. 7 7. 1 7. 1 7. 4 6. 8 6. 5 7. 8 6. 0 6. 3 7 7. 8 7. 8 7. 4 7. 2 7. 1 7. 0 7. 8 5. 9 8. 0 7. 5 8. 2 6. 9 6. 8 7. 7 6. 3 6. 8 8 4. 6 4. 5 4. 2 4. 1 4. 1 3. 9 4. 4 3. 6 4. 2 4. 0 4. 4 4. 2 3. 9 4. 8 3. 7 4. 1 9 4. 4 4. 7 4. 4 4. 6 4. 0 4. 2 4. 9 3. 7 4. 8 4. 6 4. 6 3. 8 4. 3 4. 5 3. 9 4. 4 10 1. 4 1. 3 1. 4 1. 2 1. 6 1. 2 1. 2 1. 1 1. 5 1. 1 1. 3 1. 3 1. 6 1. 6 1. 3 1. 5 11 2. 33 2. 19 2. 33 2. 40 2. 48 2. 38 2. 48 2. 42 2. 55 2. 42 2. 42 2. 29 2. 32 2. 31 2. 47 2. 54 12 1. 48 1. 41 1. 38 1. 45 1. 55 1. 46 1. 41 1. 50 1. 48 1. 46 1. 40 1. 41 1. 39 1. 44 1. 51 1. 38 13 1. 67 1. 71 1. 67 1. 55 1. 76 1. 70 1. 54 1. 66 1. 65 1. 55 1. 74 1. 79 1. 61 1. 72 1. 65 1. 53 14 0. 30 0. 28 0. 33 0. 29 0. 38 0. 30 0. 27 0. 30 0. 35 0. 27 0. 29 0. 30 0. 40 0. 33 0. 34 0. 36 15 0. 24 0. 25 0. 25 0. 24 0. 24 0. 23 0. 24 0. 25 0. 22 0. 23 0. 24 0. 26 0. 25 0. 26 0. 24 0. 25 16 11 /1 1 10 /1 1 11 /1 2 13 /1 2 12 /1 1 11 /1 0 12 /1 0 13 /1 1 12 /1 2 12 /1 2 11 /1 1 11 /1 1 12 /1 2 10 /1 1 11 /1 1 11 /1 1 90 r. sindaco et alii character mccr1437 msnpvcr867 mccr1441 msnpvcr866 mccr1470 msnpvcr868 mccr1471 msnpvcr871 msnpvcr869 msnpvcr870 mccr1472 mccr1472 mccr1472 msnpvcr872 mccr1472 mccr1469 17 10 /9 9/ 9 9/ 9 9/ 9 9/ 8 9/ 9 9/ 9 9/ 9 10 /1 0 9/ 9 10 /1 0 9/ 9 10 /1 0 8/ 9 11 /1 0 9/ 8 18 22 24 21 23 25 23 19 24 20 22 21 22 19 18 26 22 19 15 15 13 13 11 11 11 14 -1 5 13 12 10 11 10 11 13 13 20 ~1 2 ~1 2 14 14 12 -1 3 13 -1 4 ~1 2 14 13 -1 4 16 12 13 -1 4 12 12 -1 3 13 -1 4 14 21 56 67 56 6 67 67 56 8 78 8 78 67 56 5 78 78 22 15 12 13 14 14 -1 5 12 -1 3 17 14 13 14 14 15 15 15 -1 6 15 14 -1 5 23 2+ 4+ 1 3+ 3+ 1 2+ 3+ 1 / 3+ 3+ 1 3+ 3+ 1 3+ 3+ 1 3+ 3+ 1 3+ 3+ 1 / 2+ 3+ 1 2+ 3+ 1 / 2+ 4+ 1 3+ 3+ 1 2+ 4+ 1 3+ 3+ 1 / 4+ 3+ 1 3+ 3+ 1 3+ 3+ 1 / 2+ 3+ 1 2+ 3+ 1 / 3+ 3+ 1 3+ 3+ 1 3+ 3+ 1 24 1+ 6+ 1 1+ 6+ 1 / 2+ 5+ 1 1+ 6+ 1 2+ 5+ 1 / 1+ 5+ 1 1+ 5+ 1 1+ 5+ 1 1+ 5+ 1 / 0+ 6+ 1 1+ 5+ 1 2+ 5+ 1 / 1+ 6+ 1 1+ 5+ 1 / 1+ 6+ 1 2+ 5+ 1 2+ 5+ 1 / 1+ 6+ 1 1+ 5+ 1 1+ 5+ 1 0+ 6+ 1 1+ 6+ 1 25 0+ 7+ 1 1+ 6+ 1 1+ 6+ 1 1+ 6+ 1 1+ 6+ 1 / 1+ 5+ 1 1+ 5+ 1 / 0+ 6+ 1 0+ 6+ 1 1+ 6+ 1 / 0+ 6+ 1 1+ 6+ 1 1+ 6+ 1 1+ 6+ 1 / 2+ 5+ 1 1+ 6+ 1 1+ 6+ 1 / 2+ 5+ 1 0+ 5+ 1 / 1+ 5+ 1 0+ 6+ 1 1+ 6+ 1 26 1+ 7+ 1 1+ 6+ 1 2+ 6+ 1 1+ 6+ 2 / 1+ 6+ 1 1+ 6+ 1 1+ 6+ 1 1+ 6+ 1 1+ 6+ 1 2+ 6+ 1 / 1+ 6+ 1 0+ 7+ 1 / 2+ 6+ 1 3+ 5+ 1 / 1+ 6+ 1 2+ 5+ 1 / 1+ 7+ 1 1+ 6+ 1 3+ 5+ 1 / 1+ 5+ 1 1+ 6+ 1 1+ 6+ 1 27 1+ 7+ 1 2+ 6+ 1 2+ 6+ 1 3+ 5+ 1 2+ 5+ 1 3+ 5+ 1 2+ 6+ 1 2+ 6+ 1 / 3+ 5+ 1 2+ 6+ 1 3+ 6+ 1 / 2+ 6+ 1 4+ 5+ 1 3+ 5+ 1 / 2+ 6+ 1 2+ 6+ 1 / 1+ 6+ 1 1+ 6+ 1 / 1+ 5+ 1 2+ 7+ 1 / 1+ 7+ 1 2+ 6+ 1 28 1+ 4+ 1 2+ 3+ 1 2+ 3+ 1 2+ 3+ 1 / 3+ 2+ 1 2+ 3+ 1 2+ 3+ 1 2+ 3+ 1 2+ 3+ 1 1+ 4+ 1 2+ 3+ 1 2+ 3+ 1 / 3+ 2+ 1 3+ 2+ 1 / 2+ 3+ 1 2+ 3+ 1 3+ 2+ 1 / 2+ 3+ 1 1+ 4+ 1 / 2+ 3+ 1 3+ 2+ 1 29 2+ 8+ 1 1+ 6+ 1 1+ 6+ 1 2+ 5+ 1 1+ 6+ 1 2+ 5+ 1 1+ 6+ 1 1+ 6+ 1 1+ 6+ 1 1+ 6+ 1 / 2+ 6+ 1 1+ 6+ 1 1+ 6+ 1 2+ 6+ 1 2+ 5+ 1 2+ 5+ 1 1+ 6+ 1 1+ 6+ 1 30 1+ 7+ 1 2+ 6+ 1 / 1+ 7+ 1 1+ 7+ 1 2+ 6+ 1 1+ 6+ 1 1+ 6+ 1 1+ 6+ 1 1+ 6+ 1 / 0+ 7+ 1 1+ 7+ 1 1+ 7+ 1 2+ 6+ 1 1+ 7+ 1 2+ 6+ 1 1+ 6+ 1 3+ 6+ 1 / 1+ 6+ 1 0+ 7+ 1 31 2+ 8+ 1 2+ 7+ 1 2+ 7+ 1 2+ 7+ 1 3+ 6+ 1 3+ 6+ 1 2+ 7+ 1 / 3+ 6+ 1 3+ 6+ 1 3+ 6+ 1 / 4+ 6+ 1 4+ 6+ 1 2+ 7+ 1 2+ 7+ 1 / 3+ 7+ 1 4+ 6+ 1 2+ 6+ 1 2+ 7+ 1 / 3+ 6+ 1 2+ 7+ 1 32 3+ 7+ 1 / 2+ 8+ 1 3+ 7+ 1 / 4+ 6+ 1 4+ 6+ 1 3+ 6+ 1 / 4+ 6+ 1 4+ 6+ 1 3+ 6+ 1 / 5+ 5+ 1 2+ 7+ 1 / 4+ 6+ 1 3+ 6+ 1 4+ 6+ 1 / 3+ 7+ 1 4+ 6+ 1 / 2+ 6+ 1 5+ 5+ 1 / 3+ 7+ 1 4+ 6+ 1 4+ 6+ 1 4+ 6+ 1 / 4+ 4+ 1 4+ 7+ 1 / 3+ 6+ 1 5+ 6+ 1 / 4+ 7+ 1 91a misunderstood new gecko of the genus hemidactylus nostril is pierced between the rostral, the first upper labial and three nasals; the two upper nasals are separated by a small scale in two specimens only out of 16. the number of upper labials ranges from 10 to 13 (mode 11, mean 11.32 ± 0.79), the lower labials from 8 to 11 (mode 9, mean 9.22 ± 0.66). the arrangement of chin shields is rather constant: the first pair of enlarged postmentals is in broad contact in all specimens; they touch both the first and the second lower labials in 14 specimens at least on one side, but asymmetry is present in four specimens. the second postmentals usually touch the second lower labial, very rarely also the third, and in one case the first (at a single point). the number of gulars in the middle of the throat contained in the eye diameter is from 18 to 26 (mean 21.94 ± 2.24), the ventrals from 10 to 15 (mean 12.64 ± 1.68). the longitudinal rows of enlarged tubercles on back counted across midbody are 12-16 (mode 14), between the hindlimbs 5-8 (mode 6), the number of enlarged tubercles in a paravertebral line from axilla to groin 12-17 (mode 14) although they are very irregular and sometimes difficult to count. the subdigital lamellae under the first toe are 6 in all specimens, under the fourth toe are 9-11 (mean 10.19 ± 0.54, mode 10). in the 6 males studied, the preanal pores are 3+3 in one specimen, 4+4 in two specimens, and 5+5 in three specimens; the two rows of preanal pores are separated by 2 scales in three specimens and by 3 scales in the other three. colouration in life ground color pinkish (sometimes greyish), with more or less distinct transverse, thin, irregular brown bands, often reticulated. in some specimens these bands are interrupted and are replaced by irregular spots. unregenerated tail very distinctly banded, with about seven dark bands alternating with light ground color; the ground color is pinkish in the basal half, whitish in the distal half (figs. 2-3). pupil vertical, black, with slightly lobed shape; iris cream coloured, with a complex pattern of brown reticulated veins (fig. 1a). distribution and habitat besides the localities where the type-specimens have been collected, h. inintellectus was recorded in the following localities: wadi ma’nifoh, m 100-200 a.s.l. (ab. 12°35’n 54°18’e; schätti and desvoignes, 1999); homhil ab. 400 m a.s.l. (ab. 12°32’n-54°27’e; schätti and desvoignes, 1999); diksam 12°29’n-53°59’e, 695 m a.s.l. (rösler and wranik, 2004); jabal falanj near hamero (12°32’n-54°27’e), 324 m a.s.l.; wadi mityaf near afafes (12°29’n-54°23’e), 292 m a.s.l.; firmihin (12°28’n-54°01’e), 610 m a.s.l.; north of shibroh (12°29’n-53°59’e), 100 m a.s.l.; neet, 20 m a.s.l. (12°27’n-53°27’e). as shown in fig. 4, the new species has been observed in different areas of the island, ranging from near the sea level to about 695 m a.s.l.; its occurrence is expected in most of the rocky places of the island, excluding the higher peaks. this species is mainly a rock dwelling gecko. individuals were observed active after dusk, usually climbing on cliffs, deep crevices, large boulders, palm tree trunks, generally 92 r. sindaco et alii fig. 2. immature, socotra, temedeh env., 29.xii.2008. fig. 3. adult, socotra, dhero area, 29.xii.2007. 93a misunderstood new gecko of the genus hemidactylus in well vegetated areas. an individual was also caught on an adenium obesum trunk; the specimen depicted by rösler and wranik (2004) was observed on a dracaena trunk. h. inintellectus was found in syntopy with the following nocturnal reptiles: hemidactylus homoeolepis (91% of sites), haemodracon riebecki (45%), ditypophis vivax (18%), haemodracon trachyrhinus (9%), hemidactylus pumilio (9%). diurnal reptiles found at the same places were pristurus sokotranus (73%), pristurus insignis (55%), trachylepis (?) socotrana (36%), chamaeleo monachus (18%), mesalina balfouri (18%), pristurus guichardi (9%). discussion hemidactylus is one of richest genera of the family gekkonidae in terms of species number, and somalia and its adjoining areas are one of the main centres of speciation, as pointed out by joger (1985): more than 40 species occur, most of them endemic, in somalia, kenya, ethiopia and eritrea (cf. parker, 1942; loveridge, 1947; lanza, 1983; spawls et al., 2002; sindaco et al., 2007). the geographically close socotran archipelago is a center of speciation, with nine species, all but three endemic; most of them somewhat resemble one or the other somalian species (joger, 2000: 341). the island of socotra in particular is inhabited by seven species species of hemidactylus, two of them almost surely introduced by man (h. flaviviridis and h. robustus), and one possibly introduced in arabia from socotra (h. homoeolepis). comparison with other species the new species is easily distinguishable from all other hemidactylus of the socotran archipelago, except from h. oxyrhinus and h. robustus, due to the presence of large, raised fig. 4. distribution of hemidactylus inintellectus. star = locus typicus; squares = examined specimens; triangles = bibliographic data; circles = original observations. localities: 1 wadi ayhaft; 2 betw. hadibo and qadub; 3 wadi ma’nifoh; 4 temedeh; 5 homhil; 6 near hamero; 7 near afafes; 8 south of afafes; 9 dhero; 10 firmihin; 11 diksam; 12 north of shibroh 13 neet; 4 jebel ma’li near qalansiyah. 94 r. sindaco et alii and strongly keeled tubercles on the back. some authors (see synonyms) confused it also with h. granti even if this species lacks the evident large and raised tubercles on the back. it differs from h. oxyrhinus, an endemic species from abd al-kuri island, by the presence of preanal pores in males (males h. oxyrhinus lack preanal pores) and by the presence of small granular scales among the dorsal enlarged tubercles (back covered by large tubercles only, each of them in contact with the others, in h. oxyrhinus). the new species can be distinguished from h. robustus by the obvious larger size (max svl up to 60.5 mm versus 44 mm; grams to 5.8 versus 2.3), by the arrangement of preanal pores in two rows separated by 2-3 scales in h. inintellectus, instead of a single series of 5-9 preanal pores in h. robustus (8 males from socotra). compared with h. robustus, the new species has larger dark spots, often forming some more or less well defined transverse bands on trunk (small scattered spots in robustus), furthermore the unregenerated tails of the new species has few evident dark and light bands (spotted and without well defined bands in h. robustus, at least in adult specimens); the dark stripe across the eye typical for h. robustus is only weakly evident in h. inintellectus. in northeastern africa, arabia and india, the only other hemidactylus that shows raised, strongly keeled enlarged tubercles on the back, and preanal pores arranged in two short rows separated by two scales is the somali h. granchii; this species can be differentiated by the comparatively deeper and shorter head, the nostril not touching the first upper labial, fewer preanal pores (3+2), fewer upper (9/10) and lower (6/7) labials, more tubercles at midbody and more lamellae (7) under the inner toe (lanza, 1978). the following species are morphologically quite similar to h. inintellectus but they show preanal pores arranged on a single row and differ also for other characters. h. macropholis (boulenger, 1896) from ethiopia, somalia and northern kenya, has very enlarged and differently shaped dorsal tubercles mostly on flanks, second pair of post-mentals much smaller, larger size (svl up to more than 80 mm in both sexes), fewer supralabials (mode 9) and infralabials (mode 7), fewer ventrals in an eye diameter (mode 9) and a different colour pattern (lanza, 1978). h. turcicus (linnaeus, 1758), widely distributed along the mediterranean shores is usually smaller, the dorsal tubercles are arranged in regular longitudinal rows, and has a different colour pattern. the postmentals have narrower contact or, very rarely, they are separated by small plates, the number of supralabials (mode = 8) and infralabials (mode 6), is lower as is the number of ventrals in an eye diameter (mode 6) (lanza, 1978). h. sinaitus (boulenger, 1885) (from sudan to northern somalia, and arabia), very similar to h. turcicus, is smaller (maximum svl = 49 mm), has fewer supralabials (mode = 8), infralabials (mode = 7), fewer ventrals in an eye diameter (mode 7) and a different colour pattern (lanza, 1978). h. sinaitus is also very distinctive in having reduced subdigital lamellae counts, and very narrow toe pads as well as uniform subcaudal scales (s. baha el din in litt.). h. yerburii (anderson, 1895) from arabia and northern somalia is a variable species (fritz and schütte, 1987) with a single row of preanal pores, composed by 4-8 pores in some populations, while 11-17 in yemen; the tail is rather strongly depressed; in somalia the first upper labial is excluded from the nostril in about 50% of specimens (lanza, 1978); the specimens studied by lanza show also dorsal tubercles arranged in regular longitudinal rows; the colour pattern is different. 95a misunderstood new gecko of the genus hemidactylus few other hemidactylus species in nort-eastern africa show large triedral tubercles on back and males with preanal pores and can be distinguished by the following characters: h. bavazzanoi lanza 1978, h. barbierii sindaco, razzetti and ziliani, 2007 and h. foudaii baha el din, 2003 are smaller (svl to 44 mm), with very distinctive dorsal patterns that consists of well marked transverse bands, and preanal pores in a uninterrupted row. h. citernii boulenger, 1912, is smaller (svl to 36 mm), and together with h. foudaii, is unique among other east african hemidactylus in being characterized by short free distal joints of the digits of the hand and fewer subdigital lamellae under the first toe (4-5). h. arnoldi lanza, 1978, from northwestern somalia is larger (svl to 82 mm) and has broad dark transverse bars on back; the arrangement of the pores is unknown because any adult male has been discovered until now. h. barodanus boulenger, 1901 is larger (svl about 78 mm), has a pattern of brown dark-edged bands across the body, tail strongly depressed with the outermost row of tubercles forming a sharp ventro-lateral edge proximally, and preanal pores arranged in a single row. h. taylori parker, 1932 (northeastern somalia) has the unregenerated tail root-shaped, with the swollen basal portion marked by a basal constriction. also the only two middle east and indian “tuberculated” species with preanal pores only (see giri and bauer, 2008), h. persicus anderson, 1872 and h. porbandarensis sharma, 1981, are characterized by pores arranged in a single row, as well as h. mindiae from sinai (baha el din, 2005). acknowledgements we wish to address our gratitude to the proges team, charged to design a decision support system for the sustainable management of the socotran archipelago natural resources that involved our team in the field surveys. the “environmental protection authority” (epa) of the ministry of water and environment of the republic of yemen for granting the permission to access to biological resources of yemen (agreement of 29/1/2009). the personnel of the socotra conservation development project (scdp) for supporting the field work: ahmed saeed, abdul raqeeb, ahmed adeeb abdullah. thanks to the friends that helped us in the field work: danilo baratelli, teresa bellamio, eleonora boncompagni, daniele pillitteri, stefania ratano, elisa riservato, and oreste sacchi. lea debernardi and franco bernini provided learned and useful suggestions about the correct meaning of the latin epithet. aaron bauer and sherif baha el din improved the paper with corrections and useful suggestions. references anderson, j. 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(1983): a list of the somali amphibians and reptiles. monit. zool. ital. 8: 193-247. linnaeus, c. (1758): systema naturae per regna tria naturae, secundum classes, ordines, genera, species, cum characteribus, differentiis, synonymis, locis. tomus i. laurentii salvii, holmiae. loveridge, a. (1947): revision of the african lizards of the family gekkonidae. bull. mus. comp. zool. 98: 1-469. parker, h.w. (1932): two collections of reptiles and amphibians from british somaliland. proc. zool. soc. london 1932: 335-367. parker, h.w. (1942): the lizards of british somaliland. bull. mus. comp. zool. 91: 1-101. peters, w.c.h. (1882): die von herrn dr. e. riebeck auf socotra gesammelten reptilien. sitz. gesell. natur. fr. zu berlin 1882: 42-46. rösler, h. (1998c): liste der wahrend eines aufenthaltes vom 03.11.1997 bis 12.11.1997 auf sokotre nachgewiesenen geckos. gekkota 1: 33-35. rösler, h., wranik, w. (1998): beiträge zur herpetologie der republik jemen. 3. geckos des südlichen jemen und der insel sokotra. zool. ab. st. mus. tier. dresden 21: 113-132. rösler, h., wranik, w. (1999): beiträge zur herpetologie der republik jemen. 5. drei neue gecko-arten vom sokotra-archipel (reptilia: sauria: gekkonidae). zool. ab. st. mus. tier. dresden 50: 249-265. rösler, h., wranik, w. (2000): die geckofauna des sokotra-archipels (sauria: gekkonidae). gekkota 2: 20-27. rösler, h., wranik, w. (2003): reptilia (reptiles). in: w., wranik, (ed.), fauna of the socotra archipelago. field guide. pp. 121-151 + pls. 63-92. universität rostock, rostock. rösler, h., wranik, w. (2004): a key and annotated checklist to the reptiles of the socotra archipelago. fauna of saudi arabia 20: 505-534. rösler, h., wranik, w. (2006): the reptiles of the socotra archipelago with special remarks on the slender blind snakes (leptotyphlopidae: leptotyphlops). in: vm. vences, j. köhler, t. ziegler, w. böhme (eds.), herpetologia bonnensis ii. proceedings of the 13th congress of the societas europaea herpetologica, pp. 125-128. societas europaea herpetologica, bonn. schätti, b., desvoignes, a. (1999): the herpetofauna of southern yemen and the sokotra archipelago. instrumenta biodiversitatis iv, muséum d’histoire naturelle, genève. sharma, r. (1981): hemidactylus porbandarensis, a new geckonid lizard from gujarat. bull. zool. surv. india 4: 1-2. sindaco, r., razzetti, e., ziliani, u., wasonga, v., carugati, c., fasola, m. (2007): a new species of hemidactylus from lake turkana, northern kenya (squamata: gekkonidae). acta herpetol. 2: 37-48. sindaco, r., ziliani, u., razzetti, e., pupin, f., carugati, c., fasola, m., grieco, c., pella, f. (2008): the status of knowledge of the herpetofauna of socotra island (yemen). in: 98 r. sindaco et alii c. corti (ed.), herpetologia sardiniae – atti 7° congresso nazionale societas herpetologica italica, oristano, pp. 454-459. societas herpetologica italica / edizioni belvedere, latina. spawls, s., howell, k., drewes, r., ashe, j. (2002): a field guide to the reptiles of east africa. academic press, london and san diego. steindachner, f. (1899): beiricht über eine neue von herrn professor simony wahrend der süd-arabischen expedition in sokotra entdeckte neue sepsina-art, die zugleich einer besonderen subgattung (hakaria) angehort. anz. kaiser. akad. wiss. math.-naturwiss. kl., wien 36: 161-162. steindachner, f. (1903): batrachier und reptilien aus sudarabien und sokotra, gesammelt wahrend der sudarabische expedition der kaiserlichen akademie der wissenschaften. sitz. kaiser. akad. wiss. math.-naturwiss. kl., wien 112: 7-14. wranik, w. (1996): faunistic notes on soqotra island. in: proceedings of the first international symposium on soqotra island: present & future, aden, 1996: 135-198. wranik, w. (1998): contributions to the herpetology of the republic of yemen. 4. sokotra island and southern yemen mainland. zool. ab. st. mus. tier. dresden 21: 163-179. appendix comparative material examined h. angulatus, mcc r0570, mcc r1035 h. bavazzanoi, mzuf 21886 (holotypus) h. barbierii, msnpv-cr849 (holotypus), nmk-l/3054 (paratypus) h. dracaenacolus, 3 specimens (mcc, msnpv) h. flaviviridis, 2 specimens (mcc, msnpv) h. granchii, mzuf 21189 (holotypus), mzuf 21114-18 (paratypi) h. granti, 4 specimens (mcc, msnpv) h. homoeolepis, 20 specimens (mcc, msnpv) h. mabouia, mcc r803, r805, r902, r0903(1-2) h. macropholis, mcc r1224(1-2); 1 es. msnpv h. platycephalus, mcc r1225 h. pumilio, 15 specimens (mcc, msnpv) h. robustus, 16 specimens (mcc, msnpv); mzuf (many specimens) h. yerburii pauciporosus, mzuf 6245 (holotypus) + many additional specimens h. yerburii yerburii, mcc r0814 living on the edge: habitat selection of hierophis viridiflavus stefano scali, marco mangiacotti, anna bonardi museo civico di storia naturale, milano, c.so venezia 55, i-20121 milano (italy). corresponding author. e-mail: stefano.scali@comune.milano.it submitted on 2007, 15th december; revised on 2008, 27th february; accepted on 2008, 1st march. abstract. we analysed habitat choices of h. viridiflavus in a continental area of northern italy and compared our results with those reported from central italy by other authors. we used two different field techniques, visual encounter surveys (ves) and radio tracking (rt), and both pointed out a clear preference for edges, while uniform habitats (like mature woods or meadow) were avoided. the same pattern of habitat use is documented for other whip snakes and can be related to the high thermal quality of edges, although other factors could not be ruled out (e.g., prey/shelters abundance). nevertheless, other researches on mediterranean populations do not show such a preference, suggesting that at lower latitude habitat thermal quality is not the main constraint and h. viridiflavus can behave as a habitat generalist. finally, comparing the two field techniques, we find that ves partially overestimates the importance of edge use, suggesting that caution should be used about its utilization to obtain information in this kind of research. keywords. hierophis viridiflavus, habitat use, radio tracking, visual encounter surveys, latitude effect. introduction habitat selection data are necessary both for wildlife management/conservation policies and for basic ecological knowledge. in the former case, herpetologists focus their efforts mainly on endangered or threatened species in order to define adequate conservation strategies (e.g., weatherhead and charland, 1985; prior and shilton, 1996; nilson et al., 1999; kingsbury and coppola, 2000; webb and shine, 2000; filippi and luiselli, 2003); in the latter case, they obviously prefer species characterized by high-density populations, because they are easily studied (madsen, 1984; luiselli and rugiero, 1990; luiselli et al., 1994; larsson, 1995; luiselli and capizzi, 1997). in this scenario, hierophis viridiflavus (lacépède, 1789) represents an intermediate case: even though it is often very common, particularly in italy (vanni and nistri, 2006), it is protected by european law (included in acta herpetologica 3(2): 85-97, 2008 issn 1827-9643 (online) © 2008 firenze university press 86 s. scali, m. mangiacotti and a. bonardi annex iv of habitat directive 92/43/eec) because of its relatively restricted geographic range (naulleau, 1997). as a typical member of the “whip snakes clan” (sometimes called “racers”), h. viridiflavus is a diurnal, thermophilic, generalist predator (capizzi et al., 1995; rugiero and luiselli, 1995; capula et al., 1997). it can make long-range movements (ciofi and chelazzi, 1991; bonnet and naulleau, 1996a; bonnet et al., 1999) and it frequently lives also near human settlements (luiselli and capizzi, 1997; filippi, 2003). all these features potentially allow it to exploit different habitats, including fragmented and partially anthropic ones. so researchers have both a protected and common snake to study, but despite these favourable condition, habitat preferences of h. viridiflavus had never been investigated in detail and available publications report this kind of ecological information only as marginal note (luiselli and rugiero, 1990; scali and zuffi, 1994; capizzi et al., 1995; capula et al., 1997; luiselli and capizzi, 1997; filippi and luiselli, 2006). the lack of targeted studies prevents from robust conclusions. the first problem concerns the adaptive meaning of habitat selection. previous studies on h. viridiflavus ecology were conducted in mediterranean areas (mainly near rome), characterised by a hot temperate climate (cs), according to the köppen-geiger classification (hufty, 1980), with mean annual temperature (mat) between 14.5 °c and 16.9 °c, and mean temperature in the coldest month (mcm) between 6 °c and 9.9 °c. climatic conditions are very important in the ecology of ectothermic vertebrates, and their variations could heavily influence the activity of a thermophilic species, such as h. viridiflavus, and, as a consequence, influence its habitat choices (shine and madsen, 1996; luiselli and zimmermann, 1997; webb and shine, 1998, 2000; blouin-demers and weatherhead, 2001a, 2002; carfagno and weatherhead, 2006). the italian distribution of h. viridiflavus intersects very different climatic zones: from the temperate subtropical climate (cs) in the southern regions (mat>17 °c; mcm>10 °c) to the temperate cool climate (cf ) in the northern part of the peninsula or in the apennines (6<mat<9.9 °c; -3<mcm<0 °c). so, rising in latitude/ altitude, one may expect a gradient in whip snake preference for habitats that provide the greatest opportunity to reach and maintain optimal body temperatures. a second issue concerns the way habitat selection was assessed in previous studies. habitat use was described as absolute frequencies (capula et al., 1997; filippi and luiselli, 2006), but very few data were reported about habitat availability in those publications. so no deep comparison between use and availability was made and no conclusion about selection can be inferred. finally, the last problem deals with the technique used to obtained field data: previous researches were conducted only using visual encounter surveys (ves); only two papers reported radio-tracking use on this species (ciofi and chelazzi, 1991; 1994), but they were addressed to home-range and homing studies, and they did not deal with habitat selection. applying ves to a fast moving species that often lives in inaccessible habitats, could lead to overor under-estimate of habitat use (kenward, 1987; manly et al., 1993; heyer et al., 1994; bonnet and naulleau, 1996b; whiting et al., 1996). in this case, radio-tracking could be useful to collect more detailed and objective data and it could overcome this kind of problem. the three main aims for our paper are: i) analysis of habitat preferences of h. viridiflavus in relation to habitat availability; ii) comparison of habitat choices of the european 87habitat selection of hierophis viridiflavus whip snake between mediterranean and continental areas; iii) evaluation of the efficacy of visual encounter surveys and radio tracking techniques. materials and methods study area and field techniques we conducted the research in a semi-natural area of the groane regional park, in lombardy (north-western italy; 45°37’n, 9°06’e; mean altitude: 210 m a.s.l.). the study area covers 45 ha and it is surrounded by large urban areas and bordered by a major road to the north. it is a patchwork of different habitat types: open areas, bushes, woods and anthropic structures (a clay quarry and the associated kiln and buildings). it is included in the temperate continental climatic zone (cf ) (9.5<mat<15.0 °c; -1.5<mcm<3.0 °c) (hufty, 1980). to obtain a vegetation map that synthesized structural information from different areas, we chose 52 random points where we recorded the percentage cover of any biological form (raunkiaer, 1934; pignatti, 1997). afterwards, all the sample areas were clumped into five vegetation types using a cluster analysis: mature wood, sparse wood with shrubby layer, brambles, meadow and wetland. we added two habitat types, defined as edge and anthropic structures: the former was a 10 m-wide buffer along habitats interfaces (blouin-demers and weatherhead, 2001b; carfagno and weatherhead, 2006) and the latter included a clay quarry and the kiln. a detailed description of habitat categories is available in table 1. we searched for snakes from march to october, for two years (1998 and 1999), using ves (blomberg and shine, 1996). surveys were conducted three times a week, starting approximately at 0800 h and ending at 1800 h and involving two operators. an overall sample of eleven individuals (7 males and 4 females, see table 2 for details) were radio-tracked, using external transmitters implantation (see ciofi and chelazzi, 1991 for details), modified as described by bonardi et al. (2001). we table 1. description of the seven habitat categories in the study area. habitat type description total area (ha) meadow grassland with rare and sparse shrubs (mainly rosa canina and rubus spp.) 11.19 mature wood wood with closed canopy (robinia pseudacacia, prunus spp.), scarce shrubby layer (<10%) and moderate herbaceous layers (30% on average). 12.61 sparse wood with shrubby layer wood with non-continuous canopy (50% on average), with abundant shrubs coverage (rubus spp. >70% of the surface), and no herbaceous layer. 4.09 brambles thick cluster of brambles (rubus spp.) with no other vegetation layers 0.29 wetland permanent still water. depth from 30 to 130 cm. presence of water-lily, reeds, carex spp. and rush 0.14 anthropic structure the clay quarry, the kiln and their related buildings. all these structures were abandoned and with some pioneer plants. 3.47 edge 10 m-wide buffers along habitats interface. bushy, with several open spots, scarce or absent tree coverage, and often with handmade structures (bricks, ruins, iron bars and other similar materials) 11.61 88 s. scali, m. mangiacotti and a. bonardi used tw4 radio tags (weight 3.0 g; size 22×13×7 mm) and a mariner radar ltd. m57 receiver with a three elements yagi antenna (biotrack, wareham, dorset, uk). transmitters weight never exceeded 3.5% of snake weight. radio tracking was conducted from july to october in 1998 and from march to october in 1999. snake location was determined once in the morning (between 0800 h and 1000 h) and once in the afternoon, about five hours later. data analyses to analyse ves data, we superimposed a grid on the area map, identifying 555 cells of 30×30 m (su = sample unit), being 30 m the mean daily distance moved by radiotelemetered snakes. forty-seven were excluded because less than 30% of their surface belonged to the study area, leading to a sample of 508 sus. we described habitat composition of each su overlaying the vegetation map to the grid, using esri arcview 3.2 gis software. in this way, each su was characterised as the area covered by each habitat type. each cell was also classified as “present” code (psu) if at least one snake was observed in it, otherwise as “absent” code (asu). all the habitat variables were included in the logistic regression (lr) analysis, using the backward stepwise likelihood-ratio method, with the presence/absence of snakes as the dependent variable. model significance was assayed by model chi-square test, while its accuracy was tested by comparing sus values predicted by the model with observed ones (field, 2000). single variable effect on the model was deduced by the regression coefficients (b) sign: a positive b-value means that the probability to find a snake in a given su increased, and vice versa. to assess the contribution of each variable to the model, and so b-values significance, we tested the change in deviance (-2 log likelihood or -2ll) when the estimator was removed (field, 2000). to describe habitat preferences of radio tracked snakes, we used a type iii experimental design, as defined by thomas and taylor (1990), where both the resource use and its availability are identified for each individual. we chose the compositional analysis (ca) to analyse data, overcoming any autocorrelation problem (aebischer et al., 1993; otis and white, 1999; garshelis, 2000; millspaugh and marzluff, 2001). for each snake we calculated the proportional use of each habitat (no. of table 2. characteristics of implanted snakes. id sex body mass (g) svl (cm) year no. of days no. of fixes 12 m 235 95 1998 16 23 13 m 230 96 1998 13 27 14 f 230 90 1998 30 39 15 m 290 89 1999 37 55 16 f 200 82 1999 42 67 18 m 330 88 1999 36 58 27 m 300 93 1999 11 20 28 f 155 79 1999 10 15 30 m 90 64 1999 10 19 32 f 200 83 1999 14 22 33 m 465 101 1999 31 19 89habitat selection of hierophis viridiflavus fixes in habitat i/total no. of fixes). to estimate habitat availability, first we calculated the minimum convex polygon (mcp) for each individual; this home range was increased by a 30 m-wide buffer (mean daily movement calculated for all snakes) to obtain a better approximation of available area. in fact, the mcp is calculated on the basis of most external fixes, so a snake could hypothetically move 30 m daily in any direction starting from one of those points. secondly, we calculated habitat availability for each snake as area covered by ith habitat/total area within extended mcp (emcp) (moore and gillingham, 2006). habitat use data were transformed using arbitrarily the category “mature wood” as reference, accordingly with the formula proposed by aebischer et al. (1993): di = ln(xui/xai) ln(xuj/xaj) where, given a snake, xui is the proportional use of the ith habitat; xai is the available proportion of the ith habitat; xuj is the proportional use of the reference habitat (j); xaj is the available proportion of the reference habitat. when xui was equal to 0, its value was arbitrarily changed to 0.0001 to allow computation (aebischer et al., 1993). for the test of overall selection we calculated wilks’ lambda statistic (λ), using manova techniques, as suggested in millspaugh and marzluff (2001). we used snake id as a fixed factor and five habitats (meadow, mature wood, sparse/shrubby wood, anthropic structures, edge) as dependent variables. brambles and wetland were omitted because they occurred in a small portion of the study area and their proportional use was marginal. to obtain test significance we compared –n × ln × (λ) to χ2 distribution with d-1 degrees of freedom (n is the number of snakes; d is the number of habitat types) (aebischer et al., 1993). to assess which habitats are preferred, we ranked them on the basis of the average di values ( – di) across all the eleven individuals. obviously di is equal to zero for the reference category “mature wood”. to test ranking significance, first we compared – di with the reference value using a one-sample t test to verify if the use of each habitat was significantly different from the use of the reference one. finally, we verified the relative use of each habitat, comparing all pairs of them with a paired t test (aebischer et al., 1993; carfagno and weatherhead, 2006). field techniques comparison we compared ves and radio-racking results to verify their reliability. a problem arose from the non-independence of radio tracking data, so we decided to compare psus obtained simultaneously by visual surveys and radio tracking data (called “cross” psu and coded as “0”) with psus obtained by radio tracking alone (called “tracking” psu and coded as “1”). we then conducted a lr using psu type (cross or track) as the dependent variable and the significant habitat descriptors coming from the previous regression as covariates: if the model is significant, than the two techniques give different results; on the contrary the two methods are equivalent. results habitat selection a total of 172 snakes were observed using ves and a distribution map with regard to the vegetation was drawn (fig. 1). overlapping the 30 × 30m grid to the area map, we obtained 59 psus and 449 asus. the visual comparison (fig. 2) of the mean values of each 90 s. scali, m. mangiacotti and a. bonardi vegetation type among psus, asus and the whole study area shows that puss have larger surfaces covered by edges, sparse wood and anthropic structures than asus and the whole area. before attempting lr we randomly extracted 59 asus to obtain balanced samples. the model built by lr was highly significant (χ2 = 59.707, df = 3, p < 0.001) and included three variables: wood with shrubby layer (b = 2.49 × 10-3; χ2 = 5.988, df = 1, p < 0.05); anthropic structures (b = 1.90 × 10-3; χ2 = 2.894, df = 1, p < 0.10) and edge (b = 7.73 × 10-3; χ2 = 55.826, df = 1, p < 0.001). all of them had a positive effect on the probability of finding snakes in a su (positive b-values), but the most significant one was the edge. the model correctly classified 81.4% of psus and 72.9% of asus, with an overall accuracy of 77.1% and an increase of 27.1% in comparison to the null model. the proportional use of each habitat for each radio tagged snake and the overall habitat availability are summarized in table 3 and in fig. 3. the mean values show a preference for edges, while mature wood, meadow, sparse/shrubby wood and anthropic structure were less used. in particular, it is interesting to note from fig. 3 that mcp-availability is quite different from the total area one, showing that selection may occur also at this level. fig. 1. spatial relations among habitat features and snakes distribution both by ves (filled circles) and rt (open circles). see legend for habitat types and table 1 for their detailed description. 91habitat selection of hierophis viridiflavus data in table 3 were used to calculate the differences in log ratios (di), setting mature wood as the reference category. wetland and brambles were excluded from the analyses because their availability is too scarce (bingham and brennan, 2004). we conducted a manova on the di matrix, and we obtained a significant wilks’ lambda statistic (λ = fig. 3. comparison among use and two different levels of availabilities for the eleven radio tracked snakes. bars represents respectively: percentage coverage of each habitat type in the whole study area (total area availability); mean percentage of habitat availability calculated on the basis of buffered individual mcp (mean individual availability); mean percentage of fixes occurring in each habitat types (mean habitat use). fig. 2. comparison among mean percentages of each habitat types calculated respectively for the whole area (total area), for the absent sus (asu; n = 449) and for the present sus (psu; n = 59). see text for more detailed definitions. 92 s. scali, m. mangiacotti and a. bonardi 5.70 × 10-2; χ2 = 31.441, df = 4, p < 0.001), showing a non-random habitat selection. so, the habitats were ranked on the basis of –di from the least to the most selected as follows: i) sparse wood, ii) meadow, iii) anthropic structures, iv) mature wood, and v) edge. the pairwise comparison among all the habitats gave significant result only for edges, which were preferred over all other categories (all p < 0.01). techniques comparison lr used to discriminate between “cross” and “tracking” psus gave significant result (χ2 = 12.877, df = 3, p < 0.01). among the three previously selected variables, only “edge” had a correspondent b-value significantly different from zero (b = -4.28 × 10-3; χ2 = 9.070, df = 1, p < 0.01). a negative value means that an increase in edge surface produced a decrease in the probability that a psu is a “tracking” unit (coded as “1”). in other words: ves detects that snakes prefer edges to greater degree. the model correctly classified 71.4% of “cross” psus (20 out of 28) and 63.0% of “tracking” psus (10 out of 17). discussion habitat selection our study demonstrates that h. viridiflavus prefers edges and does not appreciate homogeneous habitats, such as meadows and woods. lr extracted three main variables table 3. percentage use and availability of each habitat types for radio-tracked snakes (in parenthesis the sex). individual snake id use/availability (%) meadow mature wood sparse wood/ shrubby layer brambles wetland anthropic structure edge 12 (m) 0.00/12.95 4.35/2.39 4.35/28.84 0.00/0.00 0.00/0.00 0.00/12.45 91.30/43.38 13 (m) 0.00/4.97 0.00/13.12 44.44/25.41 0.00/0.00 0.00/0.00 0.00/0.00 55.56/56.50 14 (f) 5.13/4.67 10.26/46.21 0.00/15.07 0.00/0.00 0.00/0.24 0.00/0.00 84.62/33.81 15 (m) 0.00/10.10 5.46/29.69 3.64/12.93 0.00/0.98 0.00/0.96 0.00/3.33 90.91/42.02 16 (f) 1.47/25.90 0.00/2.17 0.00/4.68 4.41/3.64 0.00/1.65 1.47/7.36 92.64/54.61 18 (m) 0.00/19.55 3.45/2.86 5.17/12.43 3.45/3.07 0.00/1.64 0.00/2.82 87.93/57.63 27 (m) 0.00/15.58 0.00/3.33 0.00/20.47 0.00/1.01 0.00/1.11 0.00/6.00 100.0/52.50 28 (f) 6.67/31.33 0.00/1.47 0.00/5.63 0.00/0.00 0.00/1.70 0.00/2.33 93.33/57.53 30 (m) 10.53/18.17 0.00/0.01 0.00/11.85 0.00/0.94 0.00/2.16 0.00/14.52 89.47/52.35 32 (f) 4.35/0.35 0.00/1.66 0.00/53.38 0.00/0.00 0.00/0.00 4.35/3.26 91.30/41.36 33 (m) 0.00/0.36 15.79/45.66 0.00/19.04 0.00/0.00 0.00/0.00 0.00/0.00 84.21/34.94 93habitat selection of hierophis viridiflavus that directly influence the occurrence probability of the european whip snake: edges, anthropic structures and sparse woods with a shrubby layer. all these variables have a positive effect on the snakes presence. also ca pointed out a clear preference for edges, and a lesser use of all other habitats. the different importance of anthropic structures and sparse/shrubby woods assessed by the two analyses could seem ambiguous, but it could be explained by their different approaches: ca only takes into consideration the occurrence habitat (e.g., the edge) while lr weighs the habitat composition giving indirect information about adjacent habitats that form the edge (e.g., an edge between meadow and mature wood). in our study anthropic structures and sparse/shrubby woods are common elements in defining edges used by snakes (see fig. 1). habitat use in mediterranean populations of h. viridiflavus has both similarities and differences with our results. while the minor utilization of forest and woodland is confirmed, in mediterranean areas snakes show a strong preference for open habitats, represented by grassy pastures and bushlands, often associated with tall grass (capula et al., 1997; filippi and luiselli, 2006; luiselli, 2006). the importance of handmade structures is also pointed out in capula et al. (1997) which reported that over 70% of the observations were done in dry-stone walls and rocky sites surrounded by spiny shrubs. dry-stone walls are not typical of northern italy plains but their role could be performed by other buildings and by ruins such as those associated to the kiln in the present study. these habitats, simulating rocky or semi-natural zones, guarantee the presence of many basking sites, shelters and prey abundance (scali and zuffi, 1994; capizzi et al., 1995; rugiero and luiselli, 1995; webb and shine, 2000). in the meanwhile, they provide low disturbance sites, because the buildings were abandoned, and only small portions of the kiln were occasionally used. under this assumption, our results are similar to those obtained in central italy. the main difference remains the intensive use of grassy pastures in latium, while open and uniform habitats, such as meadows, were not suitable for this species in northern italy. this difference could correspond to a specific selection pattern or to a difference in habitat characterization: the lack of the edge category in the past researches might lead to a subjective assignment of the observations to “pure” habitat types, increasing its importance. analogous problems are reported in carfagno and weatherhead (2006) in their comparative studies of habitat selection of the black rat snake (elaphe obsoleta). the use of edges as habitat category is not usual, but should be more considered by researches, because data from our study and from carfagno and weatherhead (2006) confirm their importance for snakes. according to blouin-demers and weatherhead (2002), edges show the highest thermal quality, because they are located at the interface of cool habitats (e.g., forests) and warm habitats (e.g., grasslands, or other open habitats). thus, edges provide the best opportunities for behavioural thermoregulation of all the habitats, and snakes can invest less in thermoregulation than in areas of low thermal quality. racers are generalist predators, that need a more precise body temperature control to allow greater sprint speeds than more specialized species (shine, 1980; carfagno and weatherhead, 2006). so, it is not surprising that h. viridiflavus prefers edges in colder habitats, where it is more difficult to achieve the preferred body temperature. similar patterns of habitat choice are documented also for coluber constrictor (plummer and congdom, 1994; carfagno and weatherhead, 2006). the differences in the use of open habitats between northern and central italy could be explained considering that mediterranean areas have the optimal thermal characteristics for the thermophilic h. viridiflavus. so, this species can behave as a habitat generalist 94 s. scali, m. mangiacotti and a. bonardi in those areas, avoiding only wetlands and cultivations (filippi and luiselli, 2006) that do not satisfy other ecological needs (e.g., prey or refuge density). on the opposite, in colder areas, the european whip snake could have stronger thermal constraints, that force it to use only optimal habitats, such as edges. techniques comparison the two field techniques (ves and rt) provide comparable results, highlighting the same main habitat choice descriptor (edge). the main difference is that ves partially overestimates the importance of edges that are characterized by discontinuities in vegetation cover which make sightings more likely than in other habitats (kenward, 1987; manly et al., 1993; heyer et al., 1994; bonnet and naulleau, 1996; whiting et al., 1996). this problem suggests caution about ves results. detailed researches on habitat use by colubrid snakes are only available for radio tracking studies (weatherhead and charland, 1985; plummer and congdon, 1994; keller and heske, 2000; blouin-demers and weatherhead, 2001a, b, 2002; rodriguez-robles, 2003; carfagno and weatherhead, 2006), because this technique guarantees a high detail level and a homogeneity of used habitat sampling. snakes have always very cryptic habits, so ves cannot be considered a biasfree research technique for them. the methodological consequence of our data is that ves could be successfully used for habitat selection studies provided that: the target species do not spend most of their time underground or in heavy undergrowth; selected areas can be easily and completely surveyed; the research effort is homogeneous among all the available habitats (manly et al., 1993; garshelis, 2000; millspaugh and marzluff, 2001). acknowledgements the research was partially financed by murst. we would like to thank f. guidali for the logistic support, i. turchini for field assistance, roberto sacchi and david greenberg for their useful suggestions about data analysis and parco groane direction for giving us the permission of study in the park. we are also grateful to xavier bonnet for manuscript improvement. references aebischer, n.j., robertson, p.a., kenward, r.e. 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(1994): radiotelemetric study of activity and movements of racers (coluber constrictor) associated with a carolina bay in south carolina. copeia 1994: 20-26. 97habitat selection of hierophis viridiflavus prior, k.a., shilton, c.m. (1996): post-hibernation mortality in black rat snakes, elaphe o. obsoleta. j. herpetol. 30: 275-278. raunkiaer, c. (1934): the life form of plants and statistical plant geography. clarendon press, oxford. rodriguez-robles, j.a. (2003): home ranges of gopher snakes pituophis catenifer, (colubridae) in central california. copeia 2003: 391-396. rugiero, l., luiselli, l. (1995): food habits of the snake coluber viridiflavus in relation to prey availability. amphibia-reptilia 16: 407-411. scali, s., zuffi, m. (1994): preliminary report on a reptile community ecology in a suburban habitat of northern italy. boll. zool. 61: 73-76. shine, r. (1980): ecology of eastern australian whipsnakes of the genus demansia. j. herpetol. 14: 381-389. shine, r., madsen, t. (1996): is thermoregulation unimportant for most reptiles? an example using water pythons (liasis fuscus) in tropical australia. physiol. zool. 69: 252-269. thomas, d.l., taylor, e.j. (1990): study designs and tests for comparing resource use and availability. j. wildl. manag. 54: 322-330. vanni, s., nistri, a. (2006): hierophis viridiflavus lacépède, 1789. in: atlante degli anfibi e dei rettili d’italia/atlas of italian amphibians and reptiles, p. 544-547. sindaco, r., doria, g., razzetti, e., bernini, f., eds, societas herpetologica italica and edizioni polistampa, firenze. weatherhead, p.j., charland, m.b. (1985): habitat selection in an ontario population of the snake, elaphe obsoleta. j. herpetol. 19: 12-19. webb, j.k., shine, r. (1998): thermoregulation by a nocturnal elapid snake (hoplocephalus bungaroides) in southeastern australia. physiol. zool. 71: 680-692. webb, j.k., shine, r. (2000): paving the way for habitat restoration: can artificial rocks restore degraded habitats of endangered reptiles? biol. cons. 92: 93-99. whiting, m.j., dixon, j.r., greene, b.d. (1996): measuring snake activity patterns: the influence of habitat heterogeneity on catchability. amphibia-reptilia 17: 47-54. acta herpetologica 4(1): 37-46, 2009 modelling a species in expansion at local scale: is hyla meridionalis colonising new areas in salamanca, spain? neftalí sillero centro de investigação em ciências geo-espaciais (cicge), universidade do porto, departamento de matemática aplicada, rua do campo alegre, 687, 4169-007 porto, portugal. e-mail: neftali.pablos@ fc.up.pt abstract. new records of the frog hyla meridionalis have been reported in the province of salamanca (spain). however, it is not clear if those records correspond to an expansion process or to a lack of sampling. thus, the species range was modelled in order to identify which is its potential distribution, which areas are available for expansion, and which variables drive the distribution. ten maxent iterative models were produced at 1×1 km scale. the suitable areas in salamanca occurred only in the south-east part of the province. this area corresponds mainly to the alagón river basin, together with a small part of the tormes river basin. the presence area was 1040 km2, meanwhile the absence one corresponded to 11309 km2. the models indicated a strong relationship with temperature and precipitation factors. predictive models cannot confirm or reject the expansion process of the species. then, h. meridionalis could be in equilibrium (or nearly) with the environment and may not spread further, or in expansion, but the process is almost complete. if the species is not in expansion, new records recently obtained in salamanca could be the consequence of previous lack of sampling. also, the expansion speed could be very low to be detected in human time-life. keywords. hyla meridionalis, spain, salamanca, ecological modelling, gis, areal expansion. introduction new records of the frog hyla meridionalis (anura, hylidae) have been reported recently in the central mountain range of two spanish provinces: salamanca (sillero and carretero, 2007) and madrid (aceituno, 2001; martínez-solano and aceituno, 2001). it is not clear if those records correspond to an expansion process or to a lack of sampling (martínez-solano and fernández-gonzález, 2003; merchán et al., 2004; sillero and carretero, 2007). the species occurs in several countries of north-western africa and southwestern europe (garcía-parís, 1997): morocco, algeria, tunisia, portugal, spain, south and south-eastern france and north-western italy (castanet and guyetant, 1989; bons 38 n. sillero and geniez, 1996; schleich et al., 1996; tejedo and reques, 2002; emanueli and salvidio, 2006; loureiro et al., 2008), as well as madeira, the canary islands and menorca (balearic islands), where it was introduced (pleguezuelos et al., 2002; loureiro et al., 2008). in the iberian peninsula, its range is divided in three isolated nuclei (garcía-parís, 1997; tejedo and reques, 2002): one in the central and south-western parts of the peninsula, one in the north-eastern extreme of spain (catalonia region), and one isolated population in the basque country (northern spain). phylogenetic analysis concluded that the northern iberia and southern france populations come from northern morocco by human introduction, as well as the canary islands (recuero et al., 2007); the southern iberian populations come from southern morocco by natural expansion. recuero et al. (2007) described a biogeographical scenario based on two possible expansion movements. the species expanded naturally from southern to northern morocco, and from there to the iberian peninsula. subsequently, the population of northern morocco became extinct. this area was recolonized with a differentiated population from the south of morocco, and introduced by humans in the mediterranean coast of france, expanding from there eastwards, southwards and northwards. the first record of h. meridionalis in salamanca (spain) was observed by bueno (1991), being the first location north to the central mountain range in the iberian peninsula. posteriorly, merchán et al. (2004) and sillero and carretero (2007) defined the species’ distribution limits, and garcía (2007) found the most northern record. aceituno (2001) and martínez-solano and aceituno (2001) found new populations in the southwestern part of the madrid province. martínez-solano and fernández-gonzález (2003) and merchán et al. (2004) suggested that the species is currently in expansion across the central mountain range, considered as an effective geographic barrier. in salamanca, the species remains apparently isolated from the southern populations (sillero and carretero, 2007). merchán et al. (2004) proposed the global climatic warming as the most probable cause of the expansion, notwithstanding alternative explanations, namely the lack of intense sampling in salamanca. however, martínez-solano and fernández-gonzález (2003) considered madrid a well-sampled area (garcía-parís et al., 1989). ecological modelling (guisan and zimmermann, 2000) is a useful tool for predicting and monitoring the expansion of species (e.g., peterson and vieglais, 2001). pearson (2007) distinguished between potential models and habitat suitability models. potential models are calculated with presence-absence data and predict the species’ realize niche (i.e., those areas that are truly occupied by the species). the model output is the probability of the species to be found in a particular place. however, habitat suitability models are calculated only with presence data and predict the species’ fundamental niche (i.e., the full range of abiotic conditions within which the species is viable). then, the model output is the probability of a particular habitat to be suitable for the species (brito and crespo, 2002). in other words, in potential models is possible to include other factors (history, competition and so on) limiting the species’ distribution (this is obtained using absence data). only with presence data, the predictive model is calculated without any limiting factor, being possible to asses the equilibrium state of the species with the environment (santos et al., 2009). the whole distribution of h. meridionalis has been recently modelled at global scale (10 × 10 km) in two environmental scenarios: in the present and in a near future (sillero, unpubl. data). model results suggested that the species can expand along the mediterranean 39modelling hyla meridionalis expansion basin and central europe (including the british islands), with other areas in asia, australia and south africa. the species’ current potential distribution is very similar to the future one, using the same variables topographical and climatic variables. these global models considered the entire salamanca province as a suitable area. this result depends probably on the model scale (kaliontzopoulou et al., 2008) and it is expected that a more restricted suitable area may be predicted when modelling at a local scale. however, there are not records at 1 × 1 km utm squares or gps resolution for the whole distribution of the species. this situation hampers to calculate predictive models at local scale over larger areas (e.g., iberian peninsula). in fact, gps records only exist for salamanca (sillero and carretero, 2007) and partially for portugal (loureiro et al., 2008). therefore, the hyla meridionalis’ fundamental niche (sensu pearson, 2007) was modelled in the province of salamanca to verify if the species can expand or not, and which factors are driving the expansion. specifically, the following questions were addressed: (1) which is the potential distribution of the species, (2) which areas are available for expansion, (3) which are the variables driving the distribution. material and methods study area the province of salamanca is located in central western spain (fig. 1a). it is mainly a 12500 km2 plain with an average altitude of 700 to 900 m. this plain is disrupted to the south by the mountains of the central mountain range (900-2425 m), and to the north-west by the duero river canyon (750-112 m). the climate is mediterranean, with low precipitation (average of 400 mm throughout the year), and summer drought, and with cool temperatures in winter. these general climate characteristics are modified perceptibly by the relief to the south and north-west. thus, three main ecosystems can be distinguished in the province: 1) the flat area, which has a strong continental climate. it is subdivided into two landscapes considering geological and economical factors, a) the cereal fields to the north-east located over the outer limit of the duero sedimentary basin which provides the deepest soils in the province, and b) the dehesa, principal landscape of salamanca, consisting of large livestock exploitations spotted with more or less dense sets of mediterranean oak trees (quercus ilex) that grow in shallow and stony soils where granite rocks do not outcrop. 2) the arribes canyon, which is the smallest area, with a very abrupt relief and a typical mediterranean microclimate that provides mild temperatures in the winter. 3) the southern mountainous area, which has an average altitude of 900-950 m at its base and reaches 2425 m at the calvitero peak, the summit of the province. the precipitation in the mountain ranges increases with altitude and the average exceeds 700 mm per year. species and environmental data sillero and carretero (2007) collected 51 gps records of h. meridionalis in salamanca, which were used as precise presence records for the models. a total of 22 independent ecogeographical variables (egvs; global spearman’s correlation value lower than 0.7) were used to calculate the models (table 1). all variables were included in the final models. agro-climatic maps from león llamazares (1991) were scanned into images and georeferenced using six points of known coordinates (two middle points and the four map corners) in the arcinfo 9 software (esri, 2000a). then, 40 n. sillero they were automatically vectorized with the arcscan extension (esri, 2000b) and the contour vector lines were interpolated to a raster file of 1 km2 pixel size with a trend surface algorithm, using the contour grid extension (stuckens, 2003) of arcview 3.2a software (esri, 1996). the climatic data cover a period from 1964 to 1990. predictive models the secretive behaviour and activity patterns of h. meridionalis (garcía-paris, 2004) complicate the accurate determination of absences, especially when the species may be in expansion. therefore, a general-purpose machine learning method, maximum entropy (maxent 3.2.1 software version; http://www.cs.princeton.edu/~schapire/maxent), was used to identify the egvs related to the fundamental species niche and to locate the areas of probable occurrence (phillips et al., 2004, 2006; phillips and dudik, 2008). it uses presence-only occurrence data and deals with continuous and categorical egvs. maxent models consistently outperform more established methods, either presence-only methods (bioclim, domain) or presence–absence methods (gam, glm), especially when samples sizes are low (elith et al., 2006; hernandez et al., 2006). maxent selects the model with the highest maximum entropy, i.e. with the most uniform distribution. the expected value of each egv (or its transform and/or interactions) should match its empirical average, i.e. the average value for a set of sample points taken from the species distribution data (phillips et al., 2004, fig. 1. map a shows the location of salamanca (dark grey) in spain (light grey). map b shows the mean of the 10 iterative maxent models of hyla meridionalis. the suitability index increases from white (0) to grey colours (1). 41modelling hyla meridionalis expansion 2006; phillips and dudik, 2008). maxent selects uniformly pseudo-absence data randomly from the background squares. hence, 10 iterative models were run automatically (using the maxent commands) with autofeatures (araújo and new, 2007; martínez-freiría et al., 2008). each egv is weighted by a constant and the estimated probability distribution ranges from 0.0 to 1.0. the program starts with a uniform probability distribution and iteratively alters the gain one weight at a time to maximize the likelihood of the occurrence data set, converging to the optimum probable distribution. the gain is a measure of the likelihood of the samples or how much better the distribution fits the sample points than the uniform distribution does (phillips et al., 2004, 2006; phillips and dudik, 2008). maxent was run randomly selecting 75% (38 records) of the presence records as training data and 25% as test data (12 records). maxent calculates the area under the curve (auc) of the receiver operated characteristics (roc) plots of the 10 iterative models (liu et al., 2005; pearson, 2007). the roc curve describes the relationship between the proportion of observed table 1. description, units, abbreviated name and mean ± standard deviation percentage contribution (highest contributions in bold) of the ecogeographical variables used for calculating the 10 iterative maxent models. all variables were aggregated to a spatial resolution of 1 km2. see methods section for details. ad. = adimensional. variables units abbreviation contribution corine ad. corine 3.68 ± 1.72 variability of precipitation deficit lower than 50 mm in june % def50 jun 12.2 ± 2.5 variability of precipitation deficit lower than 50 mm in october % def50 oct 0.07 ± 0.15 duration of the hot period no. days hot per 19.34 ± 2.25 duration of the cold period no. days cold per 0.36 ± 0.19 duration of the dry period no. days dry per 0.11 ± 0.32 annual real evapotranspiration mm etr 0.35 ± 0.14 annual potential evapotranspiration mm evp 0.06 ± 0.13 date of first frost no. days spring froz 0.15 ± 0.3 date of last frost no. days last froz 0.45 ± 0.44 annual mean temperature ºc annual isot 0.02 ± 0.03 mean temperature in the hot period ºc hot isot 5.72 ± 3.44 annual mean precipitation mm annual isoy 0.19 ± 0.2 winter mean precipitation mm winter isoy 6.66 ± 4.47 autumn mean precipitation mm autumn isoy 3.9 ± 2.4 spring mean precipitation mm spring isoy 0.58 ± 0.38 summer mean precipitation mm summer isoy 1.06 ± 1.47 no. days of august included in the hot period no. days hot august 0 ± 0 no. days of june included in the hot period no. days hot jul 0.84 ± 1.03 no. days of may included in the cold period no. days cold may 44.16 ± 4.19 no. days of october included in the cold period no. days cold oct 0.1 ± 0.1 altitude a.s.l. m altitude 0.01 ± 0.02 42 n. sillero presences correctly predicted (sensitivity) and the proportion of observed absences incorrectly predicted (1 – specificity). therefore, the roc curve of a perfect model follows the left axis and top of the plot, and the curve of a random model follows the 1:1 line. the auc expresses the proportion of the total area of the square defined by the axes of the roc graph. it ranges from 0.5 for random models to 1.0 for perfect models. the importance of each egv for explaining the species distribution was determined by: (1) jack-knife analysis of the average auc data; and (2) average percentage contribution of each egv to the models. for this, maxent excludes a egv in turn and a model was created with the remaining variables; then a model was created using each individual variable. traditionally, a threshold is estimated to reclassify the occurrence map probability as presence/absence areas (brito and crespo, 2002), although this selection is rather subjective (phillips et al., 2004, 2006; phillips and dudik, 2008). maxent offers three different types of thresholds for training data, such as: ‘maximum training sensitivity plus specificity logistic threshold’ and ‘balance training omission, predicted area and threshold value logistic threshold’. some authors considered that the maximum training sensitivity plus specificity threshold does not perform well (jiménez-valverde and lobo, 2007). therefore, the averages of the 10 raw models and the ‘balance training omission, predicted area and threshold value logistic threshold’ were calculated (araújo and new, 2007; martínez-freiría et al., 2008; phillips and dudik, 2008). results the suitable areas in salamanca spread only through the south-east part, whereas the rest of the province appeared as unsuitable (fig. 1). the auc value was very high: 0.9912 ± 0.0008 (range: 0.9899-0.9925). the egvs with the highest contribution to iterative models (table 1) were ‘no. days of may included in the cold period’ (44.16%), ‘duration of the hot period’ (19.34%) and ‘variability of precipitation deficit lower than 50 mm in june’ (12.2%). other variables with a lower contribution (table 1) were ‘winter mean precipitation’ (6.7%), ‘mean temperature in the hot period’ (5.72%), ‘autumn mean precipitation’ (3.9%) and ‘corine’ (3.68%). the remainder variables have a contribution lower than 1 (table 1). the mean variable percentage contribution and auc values of iterative models calculated without one variable were quite similar among variables, both with training and test data, i.e. variable absences did not affect strongly the models (fig. 2). when the models were calculated only with one variable, the contribution varied strongly among egvs, both with training and test data. ‘summer mean precipitation’, ‘mean temperature in the hot period’, ‘no. days of may included in the cold period’ and ‘duration of the hot period’ were the most important variables (fig. 2). finally, the threshold for determining presence/absence areas was 0.0166 ± 0.0011. therefore, the presence (suitable) area was 1040 km2, meanwhile the absence (unsuitable) one corresponded to 11309 km2. discussion suitable areas for h. meridionalis were predicted in the south-western part of salamanca, a mediterranean high plateau between the two highest local central system mountains (hastiala, 1783 m and canchal negro, 2430 m). this area corresponds mainly to the alagón river 43modelling hyla meridionalis expansion basin, together with a small part of the tormes river basin (only with three species records). therefore, the species is currently occupying almost all the suitable area in salamanca: it can only spread further to the north and west. however, these two zones have a low suitability index. thus, h. meridionalis may be in equilibrium in salamanca, or nearly, with the environment. however, predictive models for the global distribution of h. meridionalis confirm that the species can expand along the mediterranean basin and central europe (including the british islands), with other areas in asia, australia and south africa (sillero, unpubl. data). most part of the iberian peninsula is suitable for h. meridionalis, including the province of salamanca. these differences in the results were probably due to the different scale used in both studies. the global study was calculated with a grid of 10 km2, meanwhile the present study used a more precise grid (1 km2). kaliontzopoulou et al. (2008) proved that variables with higher spatial resolutions produce better predictive models. therefore, if the global model uses a higher spatial resolution, might include a smaller potential area. another hypothesis is that the species is not in expansion, but has rather been in equilibrium for a long time. hence, new records in salamanca were observed recently as a consequence of previous lack of sampling (garcía, 2007; sillero and carretero, 2007). martínezsolano and fernández-gonzález (2003) rejected this hypothesis for madrid. nevertheless, the expansion may not be fast enough in order to be noticed in human time-life. it would be necessary to model past environmental conditions in order to identify original suitable areas. if the expansion is not recent and the current suitable areas were occupied many years ago, the current distribution should be very similar to the past one. if the expansion is recent, both suitable areas (past and present) should be different. in that hypothetical case, climatic conditions changed and allowed the species expansion. recuero et al. (2007) cannot determine the date of the expansion process. the models built indicated a strong relationship with temperature and precipitation factors. hence, climatic change might affect the distribution and biogeography of the species, as suggested by merchán et al. (2004). araújo et al. (2006) forecast a general expansion to the north due to global warning. however, future forecasts for the whole global species distribution (calculated with topographical and fig. 2. jack-knife results of the contribution and auc values of each variable from the 10 iterative maxent models for the training and testing records. trwo: training data without one variable; trwi: training data with only one variable; tewo: test data without one variable; tewi: testing data with only one variable; aucwo: auc without one variable; aucwi: auc with only one variable. see more details in the materials and methods section. see abbreviations on table 1. 44 n. sillero climatic variables) were very similar to current potential models (sillero, unpubl. data). the temperature increment could not be enough for producing a expansion movement. in fact, models built for the salamanca province confirmed that absence of ecologically favourable habitats prevented further expansion. recuero et al. (2007) also suggested the presence of h. arborea as a main cause for no further expansion (brito and crespo, 2002). nevertheless, h. meridionalis is syntopic with h. arborea in a large part of its range, and no competition problems have been described until now (garcía-parís, 2004). in summary, h. meridionalis, in salamanca, is in equilibrium (or near) with the environment and only can spread in two small areas located north and west of the salamanca populations. thus, if the species is currently expanding, the process is almost complete. however, predictive models cannot confirm or reject the expansion process of the species. further studies are needed to monitor the h. meridionalis populations in salamanca and madrid, in order to verify the hypothetical expansion process; and for study the influence and competition of h. arborea in the distribution of h. meridionalis. acknowledgements thanks to a. kaliontzopoulou and f. martínez-freiría for improving an earlier version of the manuscript. i am supported by a post-doctoral grant (sfrh/bpd/26666/2006) from fct. this work is dedicated to the memory of daniel omolo. references aceituno, j. 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(2007): mitochondrial differentiation and biogeography of hyla meridionalis (anura: hylidae):an unusual phylogeographical pattern. j. biogeogr. 34: 1207-1219. santos, x., brito, j.c., caro, j., abril, a.j., lorenzo, m., sillero, n., pleguezuelos, j.m. (2009): habitat suitability, threats and conservation of isolated populations of the smooth snake (coronella austriaca) in the southern iberian peninsula. biol. cons. 142: 344-352. schleich, h.h., kästle, w., kabisch, k. (1996): amphibians and reptiles of north africa. koeltz scientific books, koenigstein. sillero, n., carretero, m.a. (2007): a systematic survey on the extralimital populations of hyla meridionalis in salamanca (spain). bol. asoc. herpetol. esp. 18: 59-64. stuckens, j. (2003): contour gridder extension. manual del usuario. tejedo, m., reques, r. (2002): hyla meridionalis. in: atlas de distribución y libro rojo de los anfibios y reptiles de españa, (2ª impresión), p. 117-119. pleguezuelos, j.m., márquez, r., lizana, m., eds, dirección general de conservación de la naturalezaasociación herpetológica española, madrid. longevity and body size in three populations of dyscophus antongilii (microhylidae, dyscophinae), the tomato frog from north-eastern madagascar giulia tessa1, fabio m. guarino2, cristina giacoma3, fabio mattioli4, franco andreone1 1 museo regionale di scienze naturali, sezione di zoologia, via g. giolitti, 36, i-10123 torino, italy. corresponding author. e-mail: franco.andreone@regione.piemonte.it 2 università di napoli federico ii, dipartimento di biologia funzionale e strutturale, via cinthia, i80126 napoli, italy 3 università degli studi di torino, dipartimento di biologia animale e dell’uomo, via a. albertina, 13, i-10123 torino, italy 4 acquario di genova, area porto antico, ponte spinola, i-16128 genova, italy abstract. age profile and body size were studied in three populations of the rare and understudied tomato frog, dyscophus antongilii, from ne madagascar. for each individual, a phalanx was clipped and the bone used for skeletochronology. sexual dimorphism is significantly different between all three populations: females are larger and heavier than males, with males also being distinguishable by a more yellowish throat. age structure analysis was possible on two populations (antara, lampirano). the age within the two populations ranged between 3 and 7 years (mean ± sd = 5.0 ± 0.2) for males, and 3 to 11 years (mean ± sd = 5.8 ± 0.3) for females. longevity was positively correlated to body size and weight within both sexes and populations. sexual maturity was reached between 2 and 3 years, with sexual maturity recorded for males significantly lower than for females. keywords. age profile, dyscophus, madagascar, sexual dimorphism, skeletochronology. the highly diverse madagascan amphibian fauna includes three microhylid subfamilies: cophylinae, scaphiophryininae, and dyscophinae. while cophylinae inhabit rainforests, scaphiophryininae and dyscophinae usually colonise open habitats and can be found in arid and semi-arid areas. the genus dyscophus is represented by three species, of which the tomato frog d. antongilii is one of the best-known frogs from madagascar. this bright, red-orange coloured species, typical of ne madagascar, has a stout body and reaches a size of 105 mm (glaw and vences, 2007). the chromatic attractiveness and peculiar morphology has resulted in the tomato frog becoming one of the preferred targets for the international pet-trade. during the acta herpetologica 2(2): 139-146, 2007 issn 1827-9643 (online) © 2007 firenze university press 140 g. tessa et alii eighties it was exported in high numbers, resulting in its inclusion on cites appendix i (andreone et al., 2006). currently, it is known from two main distribution areas: first, around maroantsetra and second, next to the ambatovaky reserve, which resulted in its classification as “near threatened” (raxworthy et al., 2004; andreone et al., 2005a). like other species of the genus, it breeds in still and slow-moving water, such as swamps, shallow ponds or artificial sewage ditches. during the breeding season the males emit a sound and rhythmic advertisement call that gives the frog its malagasy vernacular name, “sahongongogno”. surprisingly, very little information exists on the biology of d. antongilii and data on its life history are patchy, which mainly refer to a few observations made on captive individuals (pintak, 1987), or reported anecdotally. this is also true for the closely related d. guineti, similar in morphology and life history, yet not listed on cites and exported in high numbers (andreone et al., 2006). considering the importance of life history data, in terms of conservation, this study provides the first data, obtained from skeletochronological methodology (castanet, 1975), and analysis on the age structure of three populations of d. antongilii. age structure was studied as part of a wider conservation project on the species, with an individual’s age estimated by counting the lines of arrested growth (lags). lags result from alternating cold-warm and/or wet-dry seasons indicating yearly rhythms and thus breeding seasons. skeletochronological methods have been used on a variety of species from temperate regions (guarino et al., 1995, 2003) and more recently it was applied to species from tropical and subtropical regions, including madagascar (guarino et al., 1998; andreone et al., 2002, 2005b). three breeding colonies of tomato frogs were used in this study. the first population was studied at maroantsetra (15°25.44’s, 49°40.82’e; elevation ~ 10 m a.s.l.), a small town located on the antongil’s bay, the best-known locality for the species. the breeding site was a small, urban pond used by local people for breeding geese and ducks. the habitat surrounding the pool was represented by ricefields, cultivated fields, meadows and marshes. the other two sites were located more than 100 km south of maroantsetra, close to the ambatovaky special reserve. the antara site (16°53.25’s, 49°10.99’e; 486 m a.s.l.) was situated next to the homonymous river. the frogs were located within a swampy area found within a coffee and vanilla cultivation. the third site was at lampirano (sometimes also quoted as iampirano) (16°49.31’s, 49°11.06’e; 484 m a.s.l.). frogs at this site were found along the sandy banks of a slow running river. at all the sites, the climate was warmhumid with an annual average temperature ranging between 20-30 °c, and a total annual rainfall between 3000 and 4000 mm. at all localities, tomato frogs were searched during the rainy season (january-february 2006), when they were active and in breeding condition. individuals were captured at night with the aid of torches, sexed and measured snout to posterior vent (svl, at the nearest 0.1 mm) with dial callipers and weighed using a spring balance (to the nearest 1 g). males were distinguished from females by their comparatively smaller size, lighter colouration, yellow throat and the presence of deferent ducts that were visible through the belly. the last two phalanges on the second toe of the right foot were removed, for a mark-recapture study, and kept in 90% ethanol for skeletochronology data. they were later decalcified in 5% nitric acid for about 2 h, cross-sectioned at 12 µm with a cryostat and 141longevity and body size in three populations of dyscophus antongilii stained with ehrlich’s haematoxylin for about 10 min (guarino et al., 1995, 1998). sections were observed under a zeiss axioscop light microscope, and images captured with a prog res 3008 colour video camera and ks 300 software interfaced with a personal computer. at maroantsetra, toe samples were collected from 35 frogs (22 females and 13 males), at antara from 37 individuals (30 females and 7 males), and at lampirano from 39 individuals (14 females and 25 males). adult males were significantly smaller than females in all three populations (maroantsetra: males = 61.33 mm ± 5.76 and females = 88.34 ± 5.06, t = 14.01, p < 0.01; antara: 58.79 ± 3.99 vs 80.98 ± 7.31, t = 10.85, p < 0.01; lampirano: 59.62 ± 2.58 vs 81.28 ± 6.23, t = 12.43, p < 0.01). females from maroantsetra were significantly larger than females from antara (t = 4.19; p < 0.01), and lampirano (t = 3.56; p < 0.01). male weight was consistently lower than female weight across all populations: maroantsetra: 28.6 ± 8.5 vs 92.4 ± 14.2, t = 16.59; p < 0.001; antara: 24.6 ± 3.4 vs 58.3 ± 15.1, t = 11.1; p < 0.01; lampirano: 26.9 ± 2.5 vs 53.5 ± 19.8, t = 4.99; p < 0.01. also, females from maroantsetra were consistently heavier in comparison with females from antara (t = 8.32; p < 0.01) and lampirano (t = 6.37; p < 0.01). regarding age structure and determination, the lag counting was possible for antara and lampirano individuals only. in cross sections of their phalanges, a layer of endosteal lamellar bone was present at the edge of medullar cavity, clearly-cut separated from a layer of periosteal bone by a resorption line (fig. 1). lags were visible in both bone layers, but for lag counting only the periosteal lags were used because periosteal bone appeared more developed and less affected by re-adsorption. fig. 1. phalangeal cross-section (× 10), at the diaphyseal level, of dyscophus antongilii from lampirano. female, 817 mm svl, showing 6 lags (arrows), the third of which very faint. eb = endosteal bone; rl = reversal line. 142 g. tessa et alii because all the frogs were collected during the warm season, the outer section of the bone was not externally delimited by a lag. in such cases, the outer margin was interpreted as an indiscernible lag, and the number of years was calculated as the number of visible lags + 1. difficulties usually encountered in skeletochronological studies include double lines, recorded in 6.8% of samples in the studies’ populations. false lines were more frequent, recorded in 23% of samples. lags affected by bone remodelling were identified by osteometrical analysis: the perimeters of the reversal lines of adults were compared with the first visible lag of young specimens. the first inner lag was totally readsorbed in 50% of females and in 34.5% of males, while in only a small percentage of males and females were the first two lags totally resorbed (table 1). cross sections of toes taken from maroantsetra individuals showed homogeneous histological features in both endosteal and periosteal bones. over 51% of the analysed samples did not record any lag, while over 48% of the specimens recorded one to three incomplete or less visible lags. these incomplete lines did not correspond to the typical annual lag nor did they correlate with size (fig. 2). therefore, it was assumed that skeletochronology was not an applicable method for age determination of individuals from maroantsetra. this lack of any discernable lag was considered a consequence of a uniform, warm climate typical of a low-altitude site, where frogs were active all year without any metabolism fluctuations and thus consequent lag formations. differences in the applicability of skeletochronological method between populations of the same species but living under different climatic conditions have been observed in other anurans (morrison et al., 2004), but need further investigation. frogs from the antara and lampirano sites did not differ significantly between svl and lag number (ancova males: f = 0.02, p = 0.90; ancova females: f = 1.59, p = 0.22) (fig. 3). individuals from these populations showed a lag number ranging between 3 and 11 (mean lag number ± sd: males, 5.03 ± 0.99; n = 32; females, 5.81 ± 1.95; n = 42). the oldest specimen recorded was a female with 11 lags (table 2). for this reason data from antara and lampirano sites were pooled into one data set for analysis. differences in the number of lags, svl, weight and age at sexual maturity were not observed between males and females. lag number was significantly related to svl and weight within the two sexes and two populations. table 1. data on intracortical bone remodelling in dyscophus antongilii from antara and lampirano populations. sex number number of resorbed lags 0 1 2 males 32 19 (59.4%) 11 (34.5%) 2 (6.3%) females 42 19 (45.2%) 21 (50.0%) 2 (4.8%) 143longevity and body size in three populations of dyscophus antongilii age at sexual maturity differed significantly between the sexes (2.78 ± 0.20 vs 2.12 ± 0.34, t = 3.582; p < 0.01), with females reaching sexual maturity one year later than males. such a trend has already been observed in another species of malagasy frog, boehmantis microtympanum (guarino et al., 1998). females were known to invest greater enerfig. 2. phalangeal cross-section (× 10), at the diaphyseal level, of dyscophus antongilii from maroantsetra. female, 865 mm svl, showing no lags, and with evident lamellar structure. (× 10). fig. 3. correlation between size and number of lags (l = lampirano, a = antara). 144 g. tessa et alii gy resources into body growth, permitting a higher number of eggs and thus increasing fecundity (woolbright, 1983). individuals recorded at maroantsetra were both larger and heavier than those from the other two sites (fig. 4). such differences could be due to differences in habitats, site elevation and trophic availability. for example, lampirano and antara recorded an elevation around 500 m a.s.l. and variances in climate (temperature, humidity) were likely more variable throughout the year. such variances potentially obligates frogs from these two sites to observe a period of hibernation/aestivation, resulting a regular lag deposition. alternatively, at maroantsetra, the lower elevation site (0-10 m a.s.l.), an increased stability in temperature results in continuous activity of the frogs throughout the year, hence the larger size. furthermore, at maroantsetra the tomato frog colonises ponds within the town that were frequented by domestic fowl and possibly resulting in increased availability of nutrients in the water. regarding conservation concerns, the populations at antara and lampirano (only recently signalled by raxworthy et al., 2004) appear to be potentially more protected from human disturbance via agricultural and grazing practices. however, tomato frogs at maroantsetra were likely to experience greater pressure from such disturbances due to the fact that they are located within the a highly modifiable urban environment. hence this population at maroansetra should be considered at greater potential threat of extirpation. the conservation of this population presents several problems because it would be necessary to restrict the use of these ponds by local people. for these reasons, it would be rectable 2. data on age and size of dyscophus antongilii in the studied populations. values are indicated as mean ± standard deviation; extreme values are between parentheses, and total number of examined phalanges. locality (m a.s.l. ) habitat sex snout-vent length (mm) weight (g) lag count maroantsetra (~10 m) urban site males 61.33 ± 5.76 (52.5-75.1) n = 13 28.6 ± 8.5 (20-47) n = 13 not visible females 88.34 ± 5.06 (76.3-96.6) n = 22 92.4 ± 14.2 (50-110) n = 22 not visible antara (486 m) coffee plantation males 58.79 ± 3.99 (52.1-64.2) n = 7 24.6 ± 3.4 (20-31) n = 7 4.86 ± 0.90 (4-6) n = 7 females 80.98 ± 7.31 (68.2-98.9) n = 30 58.3 ± 15.1 (41-105) n = 30 5.82 ± 2.23 (3-11) n = 28 lampirano (484 m) river males 59.62 ± 2.58 (55.6-64.0) n = 25 26.9 ± 2.5 (22-32) n = 25 5.08 ± 1.04 (3-7) n = 25 females 81.28 ± 6.23 (66.5-91.9) n = 14 53.5 ± 19.8 (26-82) n = 14 5.79 ± 1.31 (4-8) n = 14 145longevity and body size in three populations of dyscophus antongilii ommended that the current, known populations of d. antongilii should be carefully managed, especially considering the species status as an icon of amphibian conservation on madagascar. the age structure and other biological parameters, when correctly collected, would allow the proposition of a greater detailed management plan to ensure the species conservation. acknowledgements the fieldwork was possible upon the help of a. sarovy and j.e. randrianirina. thanks to the malagasy authorities for providing collecting and export permits. a.i. carpenter kindly corrected a first draft of this paper. the study was funded by the zoo zürich, waza, biopat, gondwana conservation and research, and eaza. antongil conservation kindly helped during the permanence at maroantsetra. references andreone, f., cadle, j.e., cox, n., glaw, f., nussbaum, r.a., raxworthy, c.j., stuart, s.n., vallan, d., vences, m. (2005a): species review of amphibian extinction risks in madagascar: conclusions from the global amphibian assessment. conserv. biol. 19: 1790-1802. andreone, f., guarino, f.m., randrianirina, j.e. (2005b): life history traits, age profile and conservation biology of the panther chameleon, furcifer pardalis (cuvier 1829) at nosy be, nw madagascar. trop. zool. 18: 209-225. fig. 4. comparison of size in the three populations of dyscophus antongilii. 146 g. tessa et alii andreone, f., mercurio, v., mattioli, f. (2006): between environmental degradation and international pet trade: conservation strategies for the threatened amphibians of madagascar. natura, 95: 81-96. andreone, f., vences, m., guarino, f.m., randrianirina, j.e. (2002): natural history and larval morphology of boophis occidentalis (anura: mantellidae: boophinae) provide new insights into the phylogeny and adaptive radiation of endemic malagasy frog. j. zool., lond. 257: 425-438. castanet, j. (1975): quelques observations sur la présence et la structure des marques squelettiques de croissance chez les amphibiens. bull. soc. zool. fr. 100: 603-620. glaw, f., vences, m. (2007): a fieldguide to the amphibians and reptile of madagascar. third edition. vences u. glaw verlag gbr, cologne. guarino, f.m., andreone, f., angelini, f. (1998): growth and longevity by skeletochronological analysis in mantidactylus microtympanum, a rain-forest anuran of southern madagascar. copeia 1998: 194-198. guarino, f.m., angelini, f., cammarota, m. (1995): a skeletochronological analysis of three syntopic amphibian species from southern italy. amphibia-reptilia 16: 297302. guarino, f.m., lunardi, s., carlomagno, m., mazzotti, s. (2003): a skeletochronological study of growth, longevity, and age at sexual maturity in a population of rana latastei (amphibia, anura). j. biosc. 28: 775-782. morrison, c., hero, j.m., browning, j. (2004): altitudinal variation in the age at maturity, longevity, and reproductive life span in the anurans in subtropical queensland. herpetologica 60: 34-44. pintak, t. (1987): zur kenntnis des tomatenfrosches dyscophus antongili (grandidier 1877). salamandra 23: 106-121. raxworthy, c., vences, m., andreone, f., nussbaum, r. (2004): dyscophus antongilii. in: iucn 2006. – 2006 iucn red list of threatened species. www.iucnredlist.org. downloaded on 06 june 2007. woolbright, l.l. (1983): sexual selection and size dimorphism in anuran amphibia. am. nat. 121: 110-119. breeding habitat selection of an endangered species in an arid zone: the case of alytes dickhilleni arntzen & garcía-parís, 1995 andrés egea-serrano1, francisco j. oliva-paterna1, miguel tejedo2, mar torralva1 1departamento de zoología y antropología física, facultad de biología, universidad de murcia, 30100 murcia (spain); corresponding author. mail: aegea@um.es 2estación biológica de doñana, consejo superior de investigaciones científicas, avda. m. luisa s/n, 41013 sevilla (spain) abstract. the influence of environmental variables on the selection of a particular water body as breeding habitat by alytes dickhilleni was studied in the southeastern and most arid zone of its distribution range. from november 2002 to october 2003, 50 water bodies were monitored in the south east of the iberian peninsula. environmental data were submitted to a stepwise logistic regression analysis at macrohabitat, water body typology and microhabitat scales in order to establish the main factors influencing the use of a given water body as breeding habitat by this species. statistical analysis showed that the reproduction of alytes dickhilleni is associated with the macrohabitat variable topography, and the water body typology. this species breeds mainly in permanent water bodies located in mountainous topography in the study area. these results should be taken into account when populations of this species are subjected to management and/or recovery programmes in arid areas. keywords. alytes dickhilleni, breeding habitat, iberian peninsula, reproduction, selection. introduction despite demographic trends in amphibian populations may be caused by natural fluctutations (pechmann et al., 1991), a growing number of studies suggests that the worlwide decline of amphibian populations is due to anthropogenic factors (wake, 1991; pechmann and wake, 1997; semlitsch, 2003). the most important factors are overexploitation, habitat loss, disease and climatic change (stuart et al., 2004), as well as complex interactions among these threatening factors, which may act synergistically (gardner, 2001; blaustein and kiesecker, 2002). the response of amphibian populations to the same combination of threatening factors may vary depending on numerous factors such habitat type, life stage acta herpetologica 1(2): 81-94, 2006 82 a. egea-serrano et alii or history of experiencing particular stressors (gardner, 2001; blaustein and kiesecker, 2002). these aspects make it essential to know the ecology and biology of individual amphibian species to stop their decline and for their correct management and conservation (ancona and capietti, 1995). arid regions are characterized by a negative water balance, which creates an unpredictable environmental stress (vidal-abarca et al., 1992). aquatic systems in these regions are subject to natural disturbances, such as drougths and floods, because of their irregular hydrological regimes both on an annual and pluri-annual scale. in the south-east of the iberian peninsula such characteristics are drastic (vidal-abarca et al., 1992). this area, considered as one of the most important areas in the mediterranean region because of the high species diversity and/or endemic amphibians found there (borkin, 1999), represents the southeastern border of the worldwide distribution range of alytes dickhilleni arntzen and garcía-parís, 1995 (garcía-parís and arntzen, 2002). alytes dickhilleni has been categorised as “vulnerable” by the iucn (2004) and in the spanish red book of amphibians and reptiles (pleguezuelos et al., 2002). this conservation status emphasizes the importance of studying biological and ecological characteristics of this species in order to develop proper management strategies. successful propagation of an individual´s genes depends on the selection of breeding habitat, among other factors (duellman and trueb, 1994). nevertheless, although some publications describe the typologies of water bodies used for breeding by alytes dickhilleni in the iberian peninsula, no detailed investigation about breeding habitat selection by this species has been undertaken. therefore, the aim of this study is to establish the main environmental factors that influence the use of a particular water body as breeding habitat by alytes dickhilleni in the most southeastern and most arid zone of its worldwide distribution range. material and methods the study area is located in an eco-geographical sector of the segura river basin (utm 30swh; se iberian peninsula) (vidal-abarca et al., 1990), which extends over an area of about 150 km2. this river basin is included in the most arid zone of the iberian peninsula (vidal-abarca et al., 1987) and, probably, of europe (geiger, 1973). this eco-geographical sector is characterized by 500 mm of annual precipitation, a 4 month negative water balance and hydrological cycles that are severely disturbed by flash floods. it represents the most arid zone in the distribution range of alytes dickhilleni (garcía-parís and arntzen, 2002). the study was carried out from november 2002 to october 2003. during this period a total number of 50 water bodies (fig. 1) were monitored monthly (every two weeks during the breeding season). the different types of methodology used in this study included: dip-net (bradley et al., 1994; babik and rafinski, 2001), visual inspection (babik and rafinski, 2001) and minnow-traps (harrison et al., 1986). the selection of each methodology was decided in situ depending on monitored water body characteristics. however, dip-net and visual inspection were used in all cases. the reproduction of alytes dickhilleni was established by the detection of eggs and/or larvae in the water bodies monitored and their presence/absence was recorded for each sampling site. at each sampling site, environmental variables concerning the main water body features were collected. these variables were classified according to macrohabitat (500 m around sampling site, except altitude) and micro(within sampling site) habitat scales. table 1 shows the variables considered 83breeding habitat selection of alytes dickhilleni at the macrohabitat scale. environmental variables land use cover and topography were determined by visual estimation during the field surveys. altitude (error: ± 5m) was established using a gps receptor garmin® etrex venturetm and lithology was obtained from the available cartography (garcía, 1999). at the microhabitat scale, environmental variables related to water sheet area, aquatic and riparian vegetation, substrate and the physicochemical characteristics of the water were recorded (table 2). at each sampling site, variables for the physicochemical characterization (ph, temperature and conductivity) were measured five times during each visit using an universal pocket meter wtw® multi340i. this measures were taken at surface level (< 15 cm depth) within a 5 h period (1100-1600 h). at the same time, in spring and summer, a water sample of each sampling site was taken and stored at -20 ºc before being analysed by ionic chromatography to determine its ionic concentration. the water sheet area of each sampling site was in situ assigned to one of the classes shown in table 2. in relation to the water body substrate, it was characterised using the methodology proposed by bain (1999), which consists of categorizing the variable and of making at least 10 visual designations at each sampling site. fig. 1. location of all monitored water bodies in the study area. contour lines (m a.s.l.; solid lines) and main water bodies present in this territory (discontinous lines) are also represented. l drinking troughs; p cisterns and ponds; n artificial pools; * streams 84 a. egea-serrano et alii this approach provides three new variables (mean water body substrate, dominant water body substrate and water body substrate heterogeneity). in order to obtain a more comprenhensive environmental characterization of the sampling sites, bain´s methodology was also applied to the variables related to land use and riparian vegetation cover. in addition to the above variables, the typology of the water bodies was considered to be intermediate on a spatial scale. the water body typologies studied included: drinking troughs (lentic permanent artificial small water bodies where cattle drink; although they have vertical walls, medium and small sized stones nearby and inside make them easily accesible to amphibians); cisterns and ponds (lentic permanent artificial but naturalized water bodies used for farming purposes; their intermediate slopes make them accesible to amphibians); artificial pools (lentic permanent medium-sized or large artificial water bodies used for agricultural purposes; their walls are vertical and amphibians usually cannot get out of them); streams (natural headwaters water courses, length < 2 km; totally accessible to amphibians). the presence/absence of reproduction in alytes dickhilleni (dependent variable) and environmental variables (independent variables) were submitted to a stepwise logistic regression analysis (backward method) to establish breeding site selection. this statistical analysis is the most frequently used ecological modelling approach (rushton et al., 2004) and has been successfully used in studies on many amphibian species (vos and stumpel, 1995; hazell et al., 2001; guerry and hunter, 2002; ensabella et al., 2003; jakob et al., 2003; ficetola and de bernardi, 2004; hazell et al., 2004). stepwise logistic regression analysis was carried out independently on all the environmental variables at each spatial scale. before performing the multiple logistic regression analysis, a multiple correspondence analysis was made at macroand microhabitat scales in order to remove any interations between environmental variables which might result in false interactions between dependent and independent variables. only the environmental variables which showed the highest value for one of the dimensions table 1. variables considered at macrohabitat scale to establish breeding habitat preferences by alytes dickhilleni. variable units land use types: (1) forest (dominant species: pinus nigra arnold, quercus rotundifolia lam., juniperus thurifera l., brachypodium retusum (pers.) beauv. and thymus spp. l.); (2) pasture; (3) agricultural: extensive arboreal crop area (species mainly harvested: prunus dulcis (mill) d.a. webb) and extensive herbaceous crop area (species mainly harvested: triticum spp. l., hordeum spp. l.); (4) residential land use cover percentage of each land use type dominant land use dominant land use type measured sensu bain (1999) mean land use mean of land use type measured sensu bain (1999) land use heterogeneity typical deviation value of land use type measured sensu bain (1999) lithology dominant lithology: (1) limestone and compact dolomite; (2) limestone and loam; (3) limestone, loamy limestone and loam; (4) lime conglomerate; (5) loam, clay, limestone and sand; (6) gypsum, loam and clay; (7) loam; (8) gravel and sand; (9) colluvial carbonated blocks; (10) glacis carbonated pebbles; (11) alluvial carbonated rounded pebbles. (garcía, 1999) topography types: (1) steeply sloping; (2) mountainous; (3) intermediate altitude metres above sea level 85breeding habitat selection of alytes dickhilleni extracted by the analysis and the lowest for the rest were included in the multiple logistic regression analysis. of all the variables initially included in the multiple correspondence analysis, these environmental variables can be considered as the main independent environmental variables. to assess the influence of the environmental variables included in the logistic regression model on the reproduction of alytes dickhilleni, deviance values were used. these values are a measure of the fitness of the logistic regression model, so that a low deviance value means high likelihood and, consequently, a good model (silva and barroso, 2004). differences between the null model and amplified model were tested through the pearson chi-square (silva and barroso, 2004). statistical analyses were performed with the spss® statistical package and a significance level of 0.05 was accepted (except for exceptions, where a significance level of 0.10 was accepted). table 2. variables considered at microhabitat scale to establish breeding habitat preferences by alytes dickhilleni. variable units surface of water body m2 of water sheet. classes: (1) 0-5; (2) 5-10; (3) 10-50; (4) > 50 aquatic vegetation aquatic vegetation cover annual average percentage of aquatic vegetation cover aquatic vegetation heterogeneity annual typical deviation value of aquatic vegetation cover riparian vegetation types: (1) absent; (2) herbaceous appearance vegetation (shorter than 10 cm); (3) bushy appearance (taller than 10 cm); (4) mixture of herbaceous and bushy vegetation riparian vegetation cover annual average percentage of riparian vegetation cover dominant riparian vegetation annual dominant riparian vegetation type measured sensu bain (1999) mean riparian vegetation annual mean riparian vegetation type measured sensu bain (1999) riparian vegetation heterogeneity annual typical deviation value of riparian vegetation type measured sensu bain (1999) substrate types: (1) living rock; (2) sand and gravel; (3) mud dominant water body substrate annual dominant water body substrate type measured sensu bain (1999) mean water body substrate annual mean water body substrate type measured sensu bain (1999) water body substrate heterogeneity annual typical deviation value of water body substrate type measured sensu bain (1999) physicochemical characteristics of water temperature annual average water temperature (ºc) temperature heterogeneity annual typical deviation value of water temperature ph annual average water ph ph heterogeneity annual typical deviation value of water ph conductivity annual average water conductivity (ms) conductivity heterogeneity annual typical deviation value of water conductivity spring and summer ion concentration mg/l 86 a. egea-serrano et alii results the reproduction of alytes dickhilleni was confirmed in 62% of the sampling sites. table 3 presents the proportion of these water bodies where companion amphibian species were detected. table 3. proportion of sampling sites (inhabited by alytes dickhilleni, n = 31) where companion species were found. species % of localities salamandra salamandra (linnaeus, 1758) 38.7 pelophylax perezi seoane, 1885 64.5 epidalea calamita (laurenti, 1768) 6.5 bufo bufo (linnaeus, 1758) 9.7 pelodytes punctatus (daudin, 1802) 6.5 discoglossus jeanneae busack, 1986 3.2 table 4. result of multiple correspondence analysis for variables considered at macrohabitat scale (in bold, variables included in multiple logistic regression analysis). variable dimension 1 dimension 2 land use land use cover forest use 0.919 0.916 pinus area 0.245 0.151 quercus area 0.0881 0.246 juniperus area 0.111 0.157 brachypodium and thymus area 0.297 0.525 cattle use 0.404 0.00281 agricultural use 0.862 0.842 extensive arboreal crop area 0.121 0.178 extensive herbaceous crop area 0.838 0.474 residential use 0.335 0.0402 dominant land use 0.464 0.00841 mean land use 0.780 0.854 land use heterogeneity 0.244 0.610 lithology 0.728 0.291 topography 0.666 0.00780 altitude 0.519 0.397 87breeding habitat selection of alytes dickhilleni table 4 shows the scores for each environmental variable in each dimension extracted by the multiple correspondence analysis at macrohabitat scale. topography combined the highest value for dimension 1 (0.666) and the lowest for dimension 2 (0.00780). land use heterogeneity presented the lowest value for dimension 1 (0.244) and the highest for dimension 2 (0.610). only these two macrohabitat variables were included in the multiple logistic regression analysis. the multiple logistic regression analysis revealed topography as the significant variable (α = 10%; p = 0.057) on a macrohabitat scale influencing the selection of breeding habitat by alytes dickhilleni (table 5). fig. 2 shows the significant positive selection this species presents for breeding in sampling sites located in mountainous zones in the study area. in the intermediate spatial scale analysis, the environmental variable typology of water body was included in the logistic regression analysis. the result of this analysis showed the significance of this variable (α = 5%; p = 0.002) in influencing the selection of breeding habitat by alytes dickhilleni in the study area (table 5). the reproduction of this species was confirmed in all the water body categories considered (fig. 3). the results point to a significant negative selection for artificial pools and a positive selection of streams as breeding habitat in the study area. although a high proportion of the sites where the reproduction of the spefig. 2. bar chart showing the distribution of relative frequencies (%) of topography categories for the total number of sampling sites (white bars) and the number of water bodies occupied by alytes dickhilleni (black bars). table 5. result of multiple regression analysis for variables considered at macrohabitat, typology of water body and microhabitat scales (* significant p < 0.1; ** significant p < 0.05). spatial scale deviance significative variables degrees of freedom χ2 value p-value cases correctly classified (%) macrohabitat 60.682 topography 2 5.724 0.057* 70 water body typology 51.802 water body typology 3 14.605 0.002** 70 microhabitat 48.114 4 -6.890 0.142 88 a. egea-serrano et alii cies has been detected correspond to drinking troughs and cisterns and ponds, these typologies are not obviously selected by alytes dickhilleni as breeding habitat. as regards microhabitat scale, table 6 shows the scores for each environmental variable in each dimension extracted by the multiple correspondence analysis at this spatial scale. mean riparian vegetation showed the highest value for dimension 1 (0.718) and the lowest for dimension 2 (0.217). temperature heterogeneity combined the lowest value for dimension 1 (0.169) and the highest value for dimension 2 (0.571). only these two microhabitat variables were included in the multiple logistic regression analysis. none of these variables provided a significant multiple logistic regression model (table 5). discussion as in previous studies on breeding habitat selection by different amphibian species (beebee, 1985; ancona and capietti, 1995; augert and guyétant, 1995; ensabella et al., 2003), a large number of environmental variables was used to characterize the monitored water bodies as fully as possible, due to the difficulty of foreseeing factors that may influence the selection of a certain water body as breeding habitat. according to krawchuk and taylor (2003), a statistical hierarchical approach to data is essential for understanding the responses of species to habitat structure. the statistical analysis presented in this paper avoids mistakes due to interactions between variables at each spatial scale, allowing for the influence of the environmental variables on the reproduction of the species to be ascertained at different scales separately. consequently, the results obtained show the macrohabitat variable topography and the variable water body typology as determining factors in the selection of a given water body by alytes dickhilleni in the study area. fig. 3. bar chart showing the distribution of relative frequencies (%) of the categories of water body typology for the total number of sampling sites (white bars) and the number of water bodies occupied by alytes dickhilleni (black bars). 89breeding habitat selection of alytes dickhilleni at the macrohabitat scale, alytes dickhilleni shows a preference for breeding in water bodies located in mountainous topography. this preference was seen to be significant at a level of 0.10. this positive selection could be explained if it is considered that traditional land uses are almost restricted to mountainous topography, where they allow the existence table 6. result of multiple correspondence analysis for variables considered at microhabitat scale (in bold, variables included in multiple logistic regression analysis). variable dimension 1 dimension 2 surface of water body 0.611 0.224 aquatic vegetation aquatic vegetation cover 0.263 0.393 aquatic vegetation heterogeneity 0.194 0.253 riparian vegetation riparian vegetation cover 0.764 0.280 dominant riparian vegetation 0.476 0.0698 mean riparian vegetation 0.718 0.217 riparian vegetation heterogeneity 0.437 0.286 substrate dominant water body substrate 0.00539 0.0339 mean water body substrate 0.0366 0.0244 water body substrate heterogeneity 0.0161 0.00583 physicochemical characteristics of water temperature 0.0823 0.241 temperature heterogeneity 0.169 0.571 ph 0.129 0.0353 ph heterogeneity 0.0574 0.281 conductivity 0.475 0.193 conductivity heterogeneity 0.0719 0.302 spring and summer ion concentration fluorides 0.0440 0.125 chlorides 0.121 0.132 nitrates 0.130 0.186 phosphates 0.0479 0.261 sulphates 0.308 0.141 sodium 0.369 0.288 potassium 0.255 0.455 magnesium 0.284 0.422 calcium 0.498 0.0706 lithium 0.346 0.139 90 a. egea-serrano et alii of pine, holm-oak, savine and bush areas. these areas are recognized as the environment to which the presence of alytes dickhilleni adult individuals is associated (salvador and garcía-parís, 2001). hence, alytes dickhilleni would breed in available water bodies located near the habitats where the terrestrial phases of this species are lived out, there being no an authentic breeding habitat selection at macrohabitat scale. as regards water body typology, the reproduction of alytes dickhilleni in the study area has been confirmed in water bodies from all the different typologies considered in the present study, which confirms the results presented in previous studies (parís et al., 2002; martínez-solano et al., 2003). this environmental variable influences the reproduction of this species, which shows a positive preference for breeding in streams and a negative preference for artificial pools as breeding habitat. the influence that streams have on the reproduction of alytes dickhilleni could be due to the fact that this typology consists of permanent small headwater watercourses. this would allow the species to finish its long larval development (garcía-parís, 2004), and, additionally, would provide a way of dispersing larvae. the negative selection shown by the study species for breeding in artificial pools could be due to the fact that most of these water bodies in the study area are exposed to agricultural-related activities. such activities include drastic changes in water level and, eventually, total dessication of the water body, cleaning and the addition of chemical products to kill aquatic flora and fauna. as a consequence of these actions, most alytes dickhilleni larvae would die. moreover, these water bodies have vertical walls, which represent an important obstacle for both breeding adults and metamorphic individuals of the species, which simply drown. the results obtained show that alytes dikchilleni shows no obvious preference for drinking troughs or cisterns and ponds as breeding habitat in the study area. nevertheless, a high proportion of the sampling sites where the reproduction of this species has been detected corresponds to these typologies, which represent permanent water bodies where the species can complete its development (garcía-parís, 2004). this suggests the importance of conserving drinking troughs, cisterns and ponds for the conservation of alytes dickhilleni in arid zones, because these water bodies represent a shelter in areas where streams are characterized by disturbances such as droughts and floods (vidal-abarca et al., 1992), which could prevent larval development of this species. at the microhabitat scale, no influence of environmental variables studied on alytes dickhilleni reproduction was detected. this result would suggest the absence of any selection of a particular breeding habitat by alytes dickhilleni at this spatial scale, probably as a result of the reproductive strategy of the species. the genus alytes reproduces on land (márquez, 1992) and male individuals carry the strings of eggs on their hindlimbs (duellman and trueb, 1994). when the larvae begin to hatch, the male toads sit in water and the larvae are released. therefore, alytes dickhilleni does not need a particular type of substrate or vegetation for spawning in contrast to other species of anuran amphibian such as pelodytes punctatus (guyétant et al., 1999) or hyla meridionalis (salvador and garcíaparís, 2001). hence, the detected independence of this species with respect to the microhabitat environmental variables considered suggests that the presence of water in a given water body for a long period of time would be the only condition for reproduction of alytes dickhilleni, so that it could finish its larval phase, as was shown by salvador and garcía-parís (2001). nevertheless, variables such as aquatic vegetation may have impor91breeding habitat selection of alytes dickhilleni tant effects on larval survival, which would affect population recruitment rates and, lastly, breeding habitat selection. so, further studies concerning the growth, development, survival and condition of larvae or metamorphic individuals need to be performed to determine wether there is a real independence of the studied species in relation to microhabitat variables. in short, it has to be noticed that the results obtained point to the great importance of conserving autochthonous vegetation in arid areas where alytes dickhilleni is present for the survival of its populations. traditional farming is still practised in regions where this vegetation is present (pérez and lemeunier, 2003), and so natural water quality and natural fluctuations of the water level in streams are preserved, as well as the presence and maintenance of numerous lentic water bodies (i.e. drinking troughs, cisterns and ponds, …). alytes dickhilleni showed a breeding habitat preference for permanent water bodies. this emphasizes the importance of recovering and conserving traditional farming to ensure the survival of this species, a measure already recognized as one of the most important actions in amphibian conservation (scoccianti, 2001; calhoun and hunter, 2003). so, although further studies, such us those concerning larval survival rates and body condition of metamorphic individuals, must be performed to assess fitness differentials for the studied species in different breeding habitats, these conclusions should be taken into consideration when alytes dickhilleni populations are subjected to management and/or recovery programmmes in arid zones. acknowledgements this research was supported by the environmental service of the autonomous government of murcia, spain. we thank members of group of investigation aquatic vertebrates conservation of the zoology and physical anthropology department of the university of murcia for their help in field sampling. we also thank dr jose francisco calvo and dr jose antonio palazón for statistical assistance, dr rosa gómez for her comments on water body physicochemical characterizations, and philip thomas for the english translation. references ancona, n., capietti, a. 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(1991): declining amphibian populations. science 253: 860. acta herpetologica 17(2): 125-133, 2022 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-13627 species diversity and distribution of amphibians and reptiles in sardinia, italy claudia corti1,2,*, marta biaggini1, valeria nulchis2, roberto cogoni2, ilaria maria cossu2, salvatore frau4, manuela mulargia2, enrico lunghi2, lara bassu2 1 museo di storia naturale dell’università di firenze, museo “la specola”, via romana, 17, 50125 firenze, italia 2 sezione sardegna della societas herpetologica italica “tilighelta” (shisardegna.it) 3 dipartimento di medicina clinica, sanità pubblica, scienze della vita e dell’ambiente (mesva), piazzale salvatore tommasi 1, 67100 coppito, l’aquila, italy 4 viale lungomare156, loc. cala liberotto, 08028 orosei, nuoro, italy *corresponding author. email: claudia.corti@unifi.it submitted on: 2022, 30th august; revised on: 2022, 23rd september; accepted on: 2022, 17th october editor: marco mangiacotti abstract. although distribution databases are a dynamic tool, continuously updated, it is important to take “snapshots” of the species distribution over time to promptly identify potential conservation issues. with this work, we provide an update of the distribution of amphibians and reptiles in sardinia and satellite islands. data derive from both direct field observations (carried out since 2005 until july 2022) and literature, accounting for over 7000 records: 1416 records of 11 species of amphibians and 5600 records of 18 species of reptiles. distribution maps (on 10 × 10 km utm grid) of 29 species are provided in supplementary materials as well as the updated list of the amphibians and reptiles occurring in the circum-sardinian islands. most of the meshes were characterized by the presence of 1-3 amphibian species (73%) and 6-8 or 9-11 reptile species (32% with 6-8 species, 30% with 9-11 species). species abundance was favoured by environmental heterogeneity, and mostly varied in relation to elevation range and edge density. keywords. sardinia, amphibians, reptiles, islands, endemics, micro-insular herpetofauna, distribution maps. introduction sardinia is the second-largest island in the mediterranean and, together with corsica, with which it shares its paleo-origin, it is one of the most relevant biodiversity hotspots in the mediterranean (blondel et al., 2010). due to its long isolation (24-20 mya), the complex geological history, the geographical position, the climatic and historical events, sardinia is home to numerous endemic herpetological species, eight amphibians and five reptiles, some of which derive from ancestors present on the sardinian-corsican microplate before its detachment from the main european plate (alvarez, 1972; lanza, 1983; carmignani et al., 1995, 2001, 2016; corti et al., 1999; speranza et al., 2002; rodríguez et al., 2017). sardinia is home to 11 amphibian and 18 reptile species (shi, 1996; bassu et al., 2010). the current herpetological composition of the island can be mainly referred to a) the messinian salinity crisis which occurred in the miocene (~5 mya) when important climatic variations occurred with consequent impact on flora and fauna, b) sea level oscillations due to the alternation of recurrent glacial and interglacial periods that have repeatedly separated and connected the island with corsica and with the continent, c) the arrival of man (corti et al., 1999; duggen et al., 2003; senczuk et al., 2019). in the “provisional atlas of italian amphibians and reptiles” (atlante provvisorio degli anfibi e rettili italiani, 126 claudia corti et alii as part of the atlas project of the italian society of herpetology, shi, 1996) preliminary distribution maps of the sardinian species were reproduced, subsequently published with some updates in the “atlas of italian amphibians and reptiles” (sindaco et al., 2006). in the last three decades, the scientific interest in sardinian herpetological species has intensified. in addition to some updates on the species distribution, an increasing number of articles have been produced focusing on phylogeography, ecology and conservation which have also contributed to provide data on the distribution of amphibians and reptiles on the island (corti et al., 2000, 2010; vasconcelos et al., 2006; van der meijden et al., 2009; salvi et al., 2010, 2011, 2017; salvi and bombi, 2010; vamberger et al., 2011; de pous et al, 2012; fritz et al., 2012; bombi and vignoli, 2014; biaggini et al., 2016; rodríguez et al., 2017; cossu et al., 2018; ficetola et al., 2018; lunghi et al., 2020; mulargia et al., 2018; sillero et al., 2018; bellati et al., 2019; senczuk et al., 2019; further references are given as supplementary material l1). at the same time, recent paleontological investigations (see zoboli et al., 2019, 2022, and literature therein) are providing interesting baseline information testifying for the presence of taxa that are present in sardinia since a relatively deep past (as green toads, emys orbicularis, testudo hermanni, natrix) or went locally extirpated (as speleomantes, discoglossus, salamandrina, mauremys, giant tortoises, softshell turtles, worm lizards, agamid lizards, timon, vipera) or even globally extinct (‘tomistoma’ calaritanus, trachyaspis lardyi, testudo pecorinii, pleurodiran turtles, sardophis elaphoides). with this work, we aim to provide updated distribution data collected from literature and direct field observations, together with a critical comment on the diversity of the sardinian herpetofauna. although distribution databases are a dynamic tool constantly updated, we still believe it is important to take “snapshots” of the distribution of the various species from time to time to promptly identify potential critical issue and intervene with appropriate conservation measures. a particular focus was also made on the fauna of satellite islands, with an updated list of amphibians and reptiles of the circum-sardinian islands that actively contribute to the herpetological diversity of sardinia. material and methods study site and data source, maps sardinia is located in the western mediterranean and is one of the largest italian regions. the island has an area that slightly exceeds 24,000 km2 and is characterized by a diversified territory consisting of plains, plateaus, hills, and mountains, as well as an extensive and varied geomorphological coastline and numerous satellite islands, islets, and rocks. the data on the distribution of amphibians and reptiles derive from both literature review and direct observations in the field carried out since 2005 until july 2022. surveys have been carried out at different altitudes and visiting different types of natural and anthropogenic habitats, both by day and by night. each data has been georeferenced with a satellite radio navigation device (global positioning system-gps), or has been attributed to a toponym reported by the igm maps (istituto geografico militare). all data are stored in the database of the sardinian section of the italian society of herpetology (shi) “tilighelta”. the dataset was enriched with bibliographic (e.g., corti et al., 2000; bassu et al., 2008, 2010, 2013; salvi & bombi, 2010; de pous et al., 2012; cossu et al., 2018; mulargia et al., 2018) and with museum records (mzuf). for the elaboration of species distribution maps, we used the utm (universal transverse mercator projection, coordinate reference system wgs84 / utm zone 32n) grid (10 × 10 km), dividing the island into 312 meshes. bibliographic data without exact coordinates were reported in the respective utm mesh. each map shows data prior to 2010 and new data recorded from 2010 until july 2022. other categories represented in the maps for some species are: a) doubtful records; b) single sporadic observation, referred to single individuals found out of the species range; c) multiple sporadic observations, when more than one individual was observed simultaneously or over time – out of the species range (e.g., translocated testudo spp.). the species distribution maps (see supplementary material) were produced using qgis 3.14.16-pi (qgis.org, 2022). study species the complete list of amphibian and reptile species inhabiting sardinia is given in table 1, where the endemic species are also indicated; table s1 (supplementary material) reports the updated list of the herpetofauna of the circum-sardinian islands. the species nomenclature follows speybroeck et al. (2020). the presence of zamenis lineatus/longissimus in sardinia is currently debated (razzetti and zanghellini, 2006) and therefore here not reported. introduced species with a relatively wide distribution are reported (e.g., trachemys), while those recorded only through sporadic encounters of single individuals (e.g., mauremys) are not. due to ongoing studies on the presence of different pelophylax species, all the observations related to the species of this genus are reported in a single map. however, in table 1 they are all listed. 127diversity and distribution of sardinian amphibians and reptiles data analyses analyses were performed excluding sporadic observation (mainly related to testudo), doubtful observations, and trachemys spp. as an alien species that in recent times spread on the islands. for each utm mesh, we extrapolated the following environmental variables: number of corine land cover classes, classified at level 3 (nclc3; kosztra et al., 2019); index of environmental heterogeneity increasing with nclc3 and number of land use polygons (heter = nclc3 × n polygons / mesh surface); index of edge density (ed = perimeter/surface calculated on land uses’ polygons; we considered the mean value per mesh); maximum elevation (elev); elevation range (δelev); abundance of wetlands (wet, the relative surface occupied, in a utm mesh, by polygons belonging to the clc classes wetlands and waterbodies). for each utm mesh, we also extrapolated the number of all species (ntot); endemic species (netot, discoglossus sardus, euproctus platycephalus, speleomantes spp., hyla sarda among amphibians; algyroides fitzingeri, archaeolacerta bedriagae, euleptes europaea, natrix helvetica cetti, podarcis tiliguerta among reptiles); amphibian species (namph); endemic amphibian species (neamph); reptile species (nrept); endemic reptile species (nerept). even if the total number of species was correlated with those of amphibians and reptiles (considering all species and the endemic ones; tested with pearson correlation, see results), we performed analyses on all the six categories of species abundance, in order not to miss possible meaningful differences. to test if the species abundance per utm mesh varied depending on the above-listed environmental variables (nclc3, heter, ed, elev, δelev, wet), we used generalized linear models (glz) with, in turn, ntot, netot, namph, neamph, nrept, nerept, as dependent variable, with poisson error distribution. we performed stepwise regression, and we selected the best-fit model according to the akaike information criterion (we selected the models with the lowest aic; burnham and anderson, 2002). results sardinia falls inside 312 grid meshes, seven of which occupied by a very small terrestrial surface (<1 ha to about 37 ha). we analysed 7016 records: 1416 records of 11 species of amphibians and 5600 records of 18 species of reptiles. most of the meshes were characterized by the presence of 1-3 amphibian species (73%; figure 1) and 6-8 or 9-11 reptile species (32% with 6-8 species, 30% with 9-11 species; figure 2). distribution maps (on 10 × 10 km utm grid) of the 29 species are provided as supplementary materials. ntot was correlated with namph (n = 312, r = 0.697, p < 0.001) and nrept (r = 0.961, p < 0.001); netot with neamph (r = 0.772, p < 0.001) and nerept (r = 0.870, p < 0.001). results of the analysis of the pattern of species abundance per utm mesh (model selection and following glzs) are shown in table 2 and 3. ntot decreased in those meshes with higher maximum elevation but, at the same time, was favoured by increastable 1. list of amphibians and reptiles of sardinia. the endemic species are marked as follow: eee = exclusively endemic to sardinia; ee = endemic to sardinia and corsica; e = endemic to the central-western-mediterranean. amphibia euproctus platycephalus (gravenhorst, 1829) eee speleomantes flavus (stefani, 1969) eee speleomantes genei (temminck & schlegel, 1838) eee speleomantes imperialis (stefani, 1969) eee speleomantes sarrabusensis lanza, leo, forti, cimmaruta, caputo & nascetti, 2001 eee speleomantes supramontis (lanza, nascetti & bullini, 1986) eee bufo bufo (linnaeus, 1758) bufotes viridis balearicus (boettger, 1880) discoglossus sardus tschudi, 1837 e hyla sarda (de betta, 1857) e pelophylax bedriagae (camerano, 1882) pelophylax bergeri (günther, 1986) pelophylax kurtmuelleri (gayda, 1940) reptilia emys orbicularis (linnaeus, 1758) trachemys scripta (thunberg in schoepff, 1792) testudo hermanni gmelin, 1789 testudo graeca linnaeus, 1758 testudo marginata schoepff, 1792 euleptes europaea (gené, 1839) e hemidactylus turcicus (linnaeus, 1758) tarentola mauritanica (linnaeus, 1758) algyroides fitzingeri (wiegmann, 1834) ee archaeolacerta bedriagae (camerano, 1885) ee podarcis siculus (rafinesque, 1810) podarcis tiliguerta (gmelin, 1789) ee chalcides chalcides (linnaeus, 1758) chalcides ocellatus (forskål, 1775) hemorrhois hippocrepis (linnaeus, 1758) hierophis viridiflavus (lacépède, 1789) natrix helvetica cetti gené, 1839 ee natrix maura (linnaeus, 1758) 128 claudia corti et alii ing elevation range, number of land uses and edge density. not surprisingly, given the numerical preponderance of reptiles on the total amount of data, the predictors selected when considering all reptiles were the same selected for ntot, with the small difference that, among those with a significant effect, there was the index of heterogeneity instead of nclc3. on the contrary, considering all amphibians, the selected predictors with a signiffig. 1. number of amphibian species in utm 10 × 10 km grid meshes. dots in the map indicate the presence of 1 (white small dot), 2-3 (small dot, in light blue), 4-5 (big dot, in dark blue) species. the pie chart summarizes the number of meshes hosting different ranges of species abundance, including those meshes with no species. fig. 2. number of reptile species in meshes utm 10 × 10 km grid. dots in the map indicate the presence of 1-2 (small white dot), 3-5 (small light green dot), 6-8 (medium light green dot), 9-11 (medium dark green dot), 12-15 (dark green dot) species. the pie chart summarizes the number of meshes hosting different ranges of species abundance, including those meshes with no species. table 2. akaike information criterion (aic) in the selection of the best model explaining the pattern of abundance of all amphibian and reptile species (ntot, namph, nrept) and of only endemic species (netot, neamph, nerept) per utm mesh, considering the following predictors: number of land cover classes (nclc3), index of environmental heterogeneity (heter), index of edge density (ed), maximum elevation (elev), elevation range (δelev), abundance of wetlands (wet); w = akaike weight of the best model; w1/w2 = akaike weight ratios between the first and second ranking models. response variable predictors aic w w1/w2 ntot heter; elev; δelev; nclc3; ed 1667.340 0.326 1.310 n etot heter; δelev; ed 1158.529 0.139 1.014 nrept heter; elev; δelev; nclc3; ed 1525.052 0.315 1.721 nerept heter; δelev; nclc3; ed 955.4630 0.283 2.594 namph heter; δelev; ed; wet 795.2846 0.188 1.141 neamph δelev; ed; wet 800.9570 0.259 1.830 129diversity and distribution of sardinian amphibians and reptiles icant effect on the abundance of species per mesh were δelev, ed, and the relative abundance of wetlands. focusing on endemic species, the abundance of all species was significantly influenced by δelev and ed (heter was selected, but it had no significant effects) (table 2 and 3). nclc3 was added to these predictors when analysing endemic reptiles, and wet when analysing endemic amphibians, but without a significant effect (table 2 and 3). discussion the maps we obtained in this work represent an important improvement on the distribution of amphibians and reptiles in sardinia. compared to previous publications (sindaco et al., 2006; bassu et al., 2008, 2010, 2013), the area surveyed for each species has been widely implemented (percentage increase of utm meshes compared to sindaco et al., 2006: e.g., euproctus platycephalus 142%, speleomantes spp. 0-115%, bufotes viridis balearicus 632%, discoglossus sardus 130%, hyla sarda 379%; euleptes europaea 228%, hemidactylus turcicus 226%, emys orbicularis 518%, testudo hermanni 370%, algyroides fitzingeri 121%, archaeolacerta bedriagae 58%, podarcis tiliguerta 142%, chalcides ocellatus 105%, hierophis viridiflavus 130%, natrix helvetica 54%). by examining the distribution of the single species, it is to be noted that almost all the endemic species are missing in the plains of nurra and campidano (nw and sw sardinia, respectively). only in a few places, some of the endemic species occur in these regions. in particular, the distribution of the endemic lizards algyroides fitzingeri and podarcis tiliguerta very rarely includes wetlands and intensively cultivated plains where they have been observed only in “edge” contexts, while archaeolacerta bedriagae, being a rupicolous species, is found exclusively in rocky habitats, from sea level to high altitudes (sindaco et al., 2010). approximately the same applies to euleptes europaea, a tiny gecko also widely distributed in micro-insular systems. only hyla sarda, among endemic species, being particularly linked to lentic waters, has settled in these two aforementioned plains. among the amphibians and in particular among urodela, the endemic and/or sub-endemic species, such as the endemic sardinian brook newt, euproctus platycephalus, and the cave salamanders, speleomantes flavus, s. genei, s. imperialis, s. sarrabusensis, s. supramontis (the ranges of these last five species do not overlap), are distributed on the main island exclusively in hilly and mountain environments. it is interesting to note that four species of testudines live in sardinia, one emydidae and three testudinidae: the native freshwater european pond terrapin, emys orbicularis and testudo hermanni, whose presence on the island seems to date back to the early pleistocene (biello et al., 2021; zoboli et al., 2022) and, t. graeca and t. marginata. the populations of these last two species settle in distinct areas of the island despite t. marginata, whose large size often makes this species a preferred target of illegal collection and translocation, is the most easily observed in areas far from its primary sardinian range. as for snakes, four species inhabit the island. the distribution of the endemic natrix helvetica cetti (schultze et al. 2020), a relatively elusive subspetable 3. glz testing the effects of the environmental variables selected by models in table 2 on the abundance of all species (ntot, namph, nrept) and of only endemic species (ne, neamph, nerept) per utm mesh. response var. predictors df estimates wald stat. p ntot intercept 1 0.931 57.444 0.000 heter 1 0.006 2.896 0.089 elev 1 -0.001 18.820 0.000 δelev 1 0.001 20.167 0.000 nclc3 1 0.017 5.297 0.021 ed 1 0.018 52.524 0.000 netot intercept 1 -0.222 1.263 0.261 heter 1 -0.000 0.000 0.999 δelev 1 0.001 54.109 0.000 ed 1 0.019 26.551 0.000 nrept intercept 1 0.840 39.018 0.000 heter 1 0.008 4.263 0.039 elev 1 -0.001 20.727 0.000 δelev 1 0.001 14.647 0.000 nclc3 1 0.015 3.089 0.079 ed 1 0.017 38.575 0.000 nerept intercept 1 -0.321 1.852 0.174 heter 1 0.002 0.200 0.654 δelev 1 0.001 24.799 0.000 nclc3 1 -0.033 12.875 0.000 ed 1 0.018 4.563 0.033 namph intercept 1 -0.739 4.879 0.027 heter 1 0.025 2.526 0.112 δelev 1 0.001 17.858 0.000 ed 1 0.0178 7.973 0.005 wet 1 -0.2488 5.259 0.027 neamph intercept 1 -1,238 16.129 0.000 δelev 1 0.018 46.501 0.000 ed 1 0.001 15.233 0.000 wet 1 0.019 0.712 0.399 130 claudia corti et alii cies, follows the distribution pattern of the other endemic taxa, according to his rupicolous habits and avoidance for plains (vanni & cimmaruta, 2010; lunghi et al., 2019). hierophis viridiflavus, is certainly the most widespread snake found on the island whereas hemorrhois hippocrepis, whose presence in the past has been reported in much of south-western sardinia (bruno and hotz, 1976), seems to have restricted its range to such an extent that, in the last decade, it has been reported only for the city of cagliari and its surroundings. contrary to what is known for this species, considered rather xerophilous (zuffi, 2006), the sardinian population of h. hippocrepis lives near wetlands, in agricultural habitats and in urban areas. as for the green frogs, pelophylax spp., further research is needed to draw a clear picture of the distribution of the different taxa on the island, given that p. kurtmuelleri, p. cf. bedriagae and p. bergeri populations have been detected (bellati et al., 2018). the introduced p. kurtmuelleri and p. cf. bedriagae can be considered naturalized following bellati et al. (2019). the latter species is found in both northern and southern sardinia. the settlement of the introduced green frogs may be favoured by vacant niches, even though the particularly dry climate could limit their expansion (bellati et al., 2017, 2018, 2019). when analysing how the number of species varies in relation to several environmental variables, the importance of elevation range and edge density in determining the abundance of herpetofauna species emerges. indeed, increasing the elevation range usually entails a higher habitat diversity, and edge habitats (including, for instance, ecotones and riparian boundaries) are well known key elements for the herpetofauna. the comparison between the factors influencing the abundance of all species and of those influencing the abundance of endemic species only reveals further interesting insights. for instance, the total number of species decreases in mountainous areas (that is those included in meshes with higher maximum elevation), while this was not a limiting factor for endemic species, many of which are also found at high altitudes. when focusing on all amphibians, the relative abundance of wetlands was among the factors influencing species abundance, whereas it was not selected as significant factor when analyzing the endemic species, hyla sarda, discoglossus sardus, and speleomantes spp. indeed, these amphibians often spawn in minor water bodies, not included in the clc classification as “wetlands” and “waterbodies”, or in underground environments as in the case of speleomantes. acknowledgements our thanks go to andrea argiolas, monica aru, stefano bovero, fabio cherchi, ylenia chiari, sergio cossu, corpo forestale della regione sardegna, di vigilanza ambientale, giovanni de falco, michel-jean delaugerre, maria depratis, yuri donno, amedeo fadda, lidia fleba, carmen fresi, antonella gaio, roberta lecis, cristiano liuzzi, pietro lo cascio, simone loi, salvatore manca, gabriele manzottu, marco marrosu, rosalba murgia, mauro murru, sergio nissardi, manuelo olivieri, danilo pisu, massimo putzu, maria grazia satta, daniele seglie, simona serusi, giuseppe sotgiu, giovanna spano, giulia tessa, marco uccheddu, daniel zoboli, carla zucca. special thanks to massimo delfino for revising paleontological considerations, to roberto sacchi and the anonymous referees for their precious suggestions. supplementary material supplementary material associated with this article can be found at <http://www-9.unipv.it/webshi/appendix/ index.html> manuscript number 13627 references alvarez, w. 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(2006): hemorrhois hippocrepis (linnaeus, 1758). in: atlante degli anfibi e dei rettili d’italia/ atlas of italian amphibians and reptiles, pp. 540-543. sindaco, r., doria, g., razzetti e., bernini, f., eds, edizioni polistampa, firenze. acta herpetologica vol. 17, n. 2 december 2022 firenze university press cryptic diversity in pygmy chameleons (chamaeleonidae: rhampholeon) of the eastern arc mountains of tanzania, with description of six new species michele menegon1,2,*, john v. lyakurwa3,4, simon p. loader5, krystal a. tolley6,7 preliminary genetic characterisation of southern smooth snake coronella girondica (serpentes, colubridae) populations in italy, with some considerations on their alpine distribution matteo r. di nicola1, raffaella melfi2, francesco p. faraone3,*, daniel l. n. iversen4, gabriele giacalone5, giovanni paolino1, mario lo valvo6 species diversity and distribution of amphibians and reptiles in sardinia, italy claudia corti1,2,*, marta biaggini1, valeria nulchis2, roberto cogoni2, ilaria maria cossu2, salvatore frau4, manuela mulargia2, enrico lunghi2, lara bassu2. the italian wall lizard, podarcis siculus campestris, unexpected presence on gorgona island (tuscan archipelago) marco a.l. zuffi1,*, alan j. coladonato2, gianluca lombardo3, antonio torroni3, matilde boschetti1, stefano scali4, marco mangiacotti2, roberto sacchi2 molecular analysis of recently introduced populations of the italian wall lizard (podarcis siculus) oleksandra oskyrko1,2,*, lekshmi b. sreelatha1,12,13, iolanda silva-rocha1, tibor sos3,4, sabina e. vlad5,6,7, dan cogălniceanu5,6, florina stănescu6,7,8, tavakkul m. iskenderov9, igor v. doronin10, duje lisičić11, miguel a. carretero1,12,13 sunny-side up: ontogenetic variation in egg mass temperatures of the wood frog rana sylvatica ryan calsbeek*, ava calsbeek, isabel calsbeek ecological niche differentiation in the anatolian rock lizards (genus: anatololacerta) (reptilia: lacertidae) of the anatolian peninsula and aegean islands mehmet kürşat şahin1,*, kamil candan2,3, danae karakasi4, petros lymberakis4, nikos poulakakis4,5,6, yusuf kumlutaş2,3, elif yıldırım2,3, çetin ilgaz2,3 occupancy and probability of detection of the introduced population of eleutherodactylus coqui in turrialba, costa rica jimmy barrantes-madrigal1,*, manuel spínola parallada1, gilbert alvarado 2, víctor j. acostachaves3,4. one site, three species, three stories: syntopy of geckoes euleptes europaea (gené, 1839), hemidactylus turcicus (linnaeus, 1758), tarentola mauritanica (linnaeus, 1758) in a coastal area of southern tuscany (central italy) giacomo radi1,2, marco a.l. zuffi1,* comparative cytogenetics on zamenis lineatus and elaphe quatuorlineata (serpentes: colubridae) marcello mezzasalma1,* , elvira brunelli1, gaetano odierna2, fabio m. guarino2 disappearance of eggs during gestation in a viviparous snake (vipera aspis) detected using non-invasive techniques xavier bonnet1, serge akoka2, richard shine3, léandre pourcelot4 1 cebc, cnrs, 79360, villiers en bois, france. corresponding author. e-mail: bonnet@cebc.cnrs.fr 2 laiem faculté des sciences, nantes cedex 3, france 3 biological sciences a08, university of sydney, nsw, australia 4 université françois rabelais, ufr de médecine, tours, france submitted on 2008, 19th may; revised on 2008, 22nd august; accepted on 2008, 26th august. abstract. the number of eggs released at ovulation may be greater than the number of offspring born, if some of these ovulated eggs and/or embryos disappear during gestation. although this process can potentially exert significant effects on reproductive output, logistical problems have discouraged studies on the disappearance of eggs and embryos in most kinds of vertebrates. nuclear magnetic resonance (nmr) imaging and ultrasound doppler-imaging have not been applied previously to such questions. using these techniques, we monitored changes in the female’s oviduct through gestation in a viviparous snake. we documented a case of disappearance of two ovulated eggs (from a litter of four) in the aspic viper, vipera aspis. the female ovulated four normalsized eggs, two of which contained living embryos when examined by nmr and ultrasound doppler-imaging early in gestation. subsequent nmr imaging midway through gestation showed the same situation, but a third imaging session immediately prior to parturition revealed that the oviducts contained only the two live embryos. the two nonviable eggs had disappeared. the female gave birth to the two live offspring, with no evidence of any additional material. these data thus offer the strongest evidence so far available for egg disappearance (resorption?) during gestation in reptiles. more generally, nmr imaging offers a valuable tool for investigating processes inside the body cavity, where direct observation is otherwise difficult or impossible. the technique does not require sacrifice of the animals, and hence allows dynamic investigations over time. keywords. ultrasonography, gestation, nuclear magnetic resonance imaging, reproduction, reproductive output, snake, ultrasound, vipera. introduction life-history theory suggests that the organisms should divide their available resources between maintenance, growth, storage and reproduction in a way that maximizes total acta herpetologica 3(2): 129-137, 2008 issn 1827-9643 (online) © 2008 firenze university press 130 x. bonnet et alii lifetime reproductive output (e.g., stearns, 1989; roff, 1992). expenditure of resources on reproduction takes many forms in males, but in females of most animal taxa the primary form of maternal investment involves the production of eggs. thus the control of egg production (i.e., the number and size of those eggs) is one of the most immediate targets of natural selection. broadly, such control involves two major physiological mechanisms acting in opposition to each other: recruitment versus regression (follicular atresia, resorption, etc.). a large theoretical, empirical and experimental literature has accumulated on the recruitment of follicles and the maintenance of developing offspring (lack, 1947, 1954; jones, 1978; skinner, 1985; duellman and trueb, 1986; godfray et al., 1991; sinervo and licht, 1991a, b; thibault and levasseur, 1991; jorgensen, 1992; morris, 1992), but egg resorption has received less attention (rosenheim et al., 2000). this dearth of attention partially reflects logistical problems: it is difficult to study phenomena that are hidden within the female’s body. nonetheless, follicular atresia, resorption of ovulated eggs and abortion of embryos have been documented in a wide variety of taxa (edwards, 1954; bell and bohm, 1975; byskov, 1978; wilson, 1985; duellman and trueb, 1986; gosling, 1986; fox and guillette, 1987; thibault and levasseur, 1991), and have been interpreted through both adaptive and non-adaptive explanations. for example, selective resorption of embryos enables females to manipulate sex ratio in mammals (e.g., gosling, 1986; forbes, 1997), and factors such as stress, poor body condition or diseases can induce females to terminate their reproductive effort (boué and boué, 1973; dollander and fenart, 1979; hattel et al., 1998). some species show highly specialized adaptations to reduce egg numbers subsequent to ovulation, including cases of intrauterine cannibalism (oophagy or adelphophagy) in sharks and amphibians (springer, 1948; vilter and vilter, 1960; hourdry and beaumont, 1985). these phenomena are somewhat functionally equivalent to direct resorption, in that they notably (but not exclusively) provide mechanisms by which females can reduce their overall litter size. although both the ultimate selective advantages and the proximate mechanisms underlying post-ovulatory reduction in litter size are undoubtedly complex, technical difficulties preclude studies of this topic in many wild animal species. in most cases, the existence of intrauterine resorption has been inferred from autopsy. for this reason, the most extensive data sets on resorption have come from species that are killed in very large numbers for other reasons (e.g., coypu myocastor coypus: gosling, 1986). such killing clearly cannot be justified on either ethical or ecological grounds for most species of wild animals. furthermore, methods based on autopsy of sacrificed individuals do not allow the investigator to monitor resorption processes dynamically, by following individual females through the gestation process (weintraub et al., 2004). in this paper we present an alternative, non-invasive, method that we have used with snakes, and that could potentially be applied to a wide range of animal species. although scientific interest in reptilian reproduction has increased dramatically in recent decades, a surprisingly high number of basic questions remain to be answered. one such topic is the question of embryonic resorption (blackburn, 1998a). despite frequent anecdotal reports, and occasional claims in the scientific literature, there has been no reliable documentation of ovulated eggs or embryo resorption in any reptile species. intuition and logic suggest that reproducing females might often benefit by resorbing nonviable eggs or embryos. not only may resources be recovered in this way, but females may be 131disappearence of eggs during gestation able to remove dead embryos that would otherwise block the oviduct (blackburn, 1998a, b). such blockages can have fatal consequences for both the female and the more anteriorly-positioned offspring (pers. obs.). the ability to resorb embryos is well-documented in viviparous mammals (e.g., westlin et al., 1995), but anatomical differences between the oviducts of reptiles and mammals may preclude resorption in the former group (blackburn, 1998a, b). obviously, much of the difficulty in evaluating reports of egg/embryo resorption in reptiles (as in other groups) involves the fact that the process (if it occurs) is hidden inside the female’s oviducts. hence, most of the kinds of evidence that have been used to infer resorption are indirect, and open to other interpretations. for example, the production of smaller-than-average nonviable neonates at parturition does not necessarily mean that these offspring were normal-sized at ovulation. ideally, we need to quantify numbers and sizes of “eggs” at the beginning of embryogenesis (i.e., immediately post-ovulation), and then monitor these variables through the period of gestation. imaging techniques developed for medical uses are well-suited to this purpose, and in this paper we describe the application of two such techniques (nuclear magnetic resonance imaging and doppler ultrasounds) to visualizing ovulated (intra-uterine) eggs and developing embryos in a viviparous snake. this technique provides the first direct evidence for the disappearance of ovulated eggs during gestation in any squamate reptile (although we cannot claim any certitude about the mechanisms involved; resorption versus leakage from the oviduct versus expulsion into the peritoneal cavity). materials and methods we used two techniques to examine intra-uterine embryos in gravid snakes. the first of these (nuclear magnetic resonance, or nmr) relies upon the magnetic properties of atomic nuclei when they are exposed to a strong magnetic field. nmr imaging is a non-invasive technique that does not harm the subject, and hence the same animal can be examined on several occasions (lauterbur, 1973). nmr image acquisition was carried out using a biospec bmt 24/40 spectro-imager (brucker) operating at 2.35 tesla and using a 20 cm alderman-grant resonator. animals were not anaesthetized, but were cooled to 15 oc. the snake was gently restrained using a purpose-built device consisting of a hollow foam-plastic tube cut lengthways and positioned on a plexiglas rule; the animal was held in position by velcro strips. the time required to obtain an image was 2 min 34 s. for large snakes, we then moved the animal to another position and repeated the procedure (2 to 4 times depending upon svl), to ensure that the imaging procedure covered the entire abdomen. the second technique (ultrasonography) is based upon detection of movement by red cells in the circulatory system. we performed these tests using a linear array transducer probe with electronic focusing, at a frequency of 10 mhz and connected to a color-coded doppler echograph (esaote au4). with this equipment, it was possible not only to visualize areas of blood flow, but also to record the velocity of that flow pattern using the doppler technique. data in this paper were obtained from three successive imaging sessions with a gravid female aspic viper, vipera aspis. the snake was collected in late april from a large wild population (château d’olonne, vendée) in western central france. she measured 54.5 cm snout-vent length (62 cm total length), and weighed 113.8 g at the time she was first examined. she was maintained in captivity throughout vitellogenesis and gestation, first in an outdoor enclosure (6×3 m) during vitellogenesis and the beginning of gestation (april to late july), and then (just before the second imaging ses132 x. bonnet et alii sion, until the third imaging session and parturition) in a small cage (40×40×40 cm) with the floor covered with a plastic sheet. water was provided ad libitum but the female did not accept any food (mice killed by the snake by provoking the bite to induce feeding behavior), as often observed in pregnant individuals. parturition occurred on 21st august. the cage was regularly inspected (1 to 5 times a day), and we saw no evidence of any expelled ova or other materials (e.g., yolk, membranes, blood, etc. that are very often associated with the expulsion – either of living neonates, stillborn and/or unfertilized eggs) on the floor of the cage at any stage throughout the study. other reproductive females from the same population (n = 10) have been examined during the same sessions, but the disappearance of eggs was detected in only one. our sample size is thus very small, and we have no precise idea about the occurrence egg disappearance in this species. however, the fact that we observed the disappearance during pregnancy of ovulated items shows that this remarkable phenomenon can indeed occur in snakes. furthermore, the main aim of this paper is precisely to present techniques that may be useful to study and quantify such phenomenon. results palpation on 14th may 1996 enabled us to detect five developing follicles. the first imaging session (19th june) took place soon after ovulation, which occurs in the first two weeks of june in western central france (naulleau and bidaut, 1981). nmr revealed the presence of four spherical objects in the oviduct (fig. 1a). these four objects were very similar in size (length 44 to 47 mm; width 16 to 18 mm). their size and location made it obvious that these were oviductal “eggs” (although the oviduct[s] that contained the eggs was not identified). this inference was confirmed by direct visual inspection of the embryos using nmr; as expected, the embryos (including embryonic membranes, etc.) were very small at this early stage of development (9 to 12 mm, within the size range we have observed in autopsied gravid females collected in mid june). only two of the four “eggs” contained discernible embryos. doppler imaging confirmed the presence of viable embryos in these two “eggs”, and the absence of any heartbeat in the other two “eggs”. heartbeats were clearly discernible in the two living embryos, despite their small size. comparisons with maternal heartbeat rates recorded either in the oviductal blood vessels or in the heart showed that the signals from the embryos were not artifacts of signals from the maternal heart; the hearts of the embryos were beating faster than the heart of their mother (75 beats m-1 for the embryos versus 45 beats m-1 for the mother). the cranialcaudal sequence of the four live and dead eggs was as follows: 1 dead egg – 2 living eggs – 1 dead egg. the second imaging session took place 22 days later (10th july), midway through gestation. by this time the two living embryos had increased appreciably in size (19 mm), but no details of their internal anatomy could be clearly resolved (fig. 1b). the two non-viable eggs had decreased only slightly in size (35 to 47 mm in length and 13 to 14 mm width). the third and last imaging session occurred 41 days later, on 21st august, close to the time that we expected parturition. nmr revealed two healthy offspring fully developed, but no other objects within the mother’s oviducts (fig. 1c). note that the resolution of imaging was 2 mm, and that even shriveled eggs would have been detected. possibly due to handling stress, the female gave birth immediately after the conclusion of the nmr session. she produced two healthy neonates, of normal size for this study population (two 133disappearence of eggs during gestation males 7.2 g, 19 cm snout-vent length and 6.2 g, 19 cm), with no evidence of any other materials from the abortive “eggs”. that is, the volume of fluids and membranes expelled with the two live offspring was typical of that usually associated with parturition in this species (pers. obs.). there was no additional yolk or degenerating tissue. palpation confirmed that the oviducts contained no other materials. note that we directly witnessed the birth of the two neonates, and that all material (living snakes and embryonic membranes, etc.) were carefully collected and examined. fig. 1. (a) gravid female aspic viper soon after ovulation (first imaging session). the head of the gravid snake is to the top; only a portion of the abdomen is shown. nmr imaging shows that the oviduct contains four “eggs”, although only three are visible on this slice. only two of these “eggs” contain visible embryos (circular structures within egg # 2 and # 3). (b) the same snake as in figure 1a, 22 days later and one-third of the way through gestation (second imaging session). the two circular structures (containing viable embryos) have increased in size. (c) the same snake as in figs. 1a and 1b, close to parturition (third imaging session). the two embryos in (c) are the two seen in the lower part of (a) and (b); the white area in the top right of the photograph is fat-body material in the upper embryo, and the body of the lower embryo is coiled. 134 x. bonnet et alii discussion our observations clearly indicate that the female viper reduced her litter size at two stages during the reproductive cycle. first, she enlarged five follicles, but ovulated only four of them. this phenomenon (follicular atresia) has been frequently reported in other reptile (see blackburn, 1998a, b; blackburn et al., 1998) and vertebrate species as well (thibault and levasseur, 1991). second, and more important, two of her four normalsized ova failed to develop. these unviable ova somehow disappeared from the oviduct during gestation. follicular atresia must be clearly distinguished from the disappearance of ovulated eggs, because the physiological mechanisms concerned are very different. we do not know if the two eggs that disappeared were unfertilized, or were fertilized and died very early in embryogenesis (e.g., during the first days after ovulation). regardless, the two nonviable “eggs” disappeared from the oviduct some time after the first third of the gestation period (i.e., between our second and third imaging sessions). are there alternative interpretations of our data other than resorption of these eggs? blackburn (1998a) has reviewed the kinds of evidence heretofore available on this topic, and enumerated alternative interpretations for all of them. our data, although preliminary, are more direct and compelling than the kinds of indirect observations previously available. two of the four ova that were ovulated disappeared prior to parturition. the only way that this could happen would be for the embryos to be resorbed via the oviductal wall, or expelled via the cloaca. although expulsion of nonviable embryos and eggs is common in squamates (blackburn, 1998a), we saw no sign of any such material in the female’s cage. more convincingly, one of the nonviable ova was the most anteriorly-placed in the oviduct (fig. 1). in order for this “egg” to be expelled, it would have had to move down past at least one other “egg”. this seems unlikely (given the intimate apposition of maternal and fetal membranes during development: stewart and blackburn, 1988), but is not impossible because extraembryonic membranes do not fully adhere to the uterine lining in viviparous snakes (blackburn, 1998b). nothing remained in the female’s oviducts after parturition; extensive experience with this species indicates that palpation is effective in locating even tiny objects (< 2 g) within the digestive or reproductive tracts. although resorption offers a plausible explanation for our observations, we certainly do not claim certainty in this interpretation. in fact, the main limitation of our study is that the alternative mechanisms that may explain the disappearance of the eggs from oviducts cannot be teased apart. although females of other reptile species have been recorded to consume expelled unfertilized eggs (e.g., even in snakes such as in the epicrates or agkistrodon genus; lourdais et al., 2005), we have never recorded this kind of behavior in very extensive observations of female aspic vipers over several years (direct observation and monitoring of female’s behaviors for several hundreds of parturition; bonnet et al., 2001, 2002). it remains also fully possible that the nonviable eggs degraded slowly over the latter part of gestation, with gradual expulsion of very small amounts of tissue or fluids through the cloaca over the 41 days elapsed between the second and the third session. indeed, such amounts may have been so small as to escape our attention, or accumulation in the oviduct, or even expulsion into the peritoneal cavity (blackburn, 1998a and references therein). a last possibility involves absorption by adjacent normal eggs (blackburn, 1998a), although this is unlikely because the two viable eggs did not increase in size during gestation (that is, they did not incorporate additional yolk). 135disappearence of eggs during gestation overall, the most likely alternative explanations for our observations appear to be as follows: 1) leakage from the oviduct, expulsion into the peritoneal cavity, or 2) resorption by the oviducts (in the sense of blackburn, 1998a). we cannot settle this question with the available data, because the female was not dissected after parturition to confirm the complete absence of embryonic tissue, or to examine anatomical details of the putative “resorption” areas. hence, further investigations are still needed, such as microscopic examination of the uterine cells and tissues likely to accomplish the digestion and absorption. such studies could be conducted on animals killed accidentally or for other purposes, or using sophisticated current techniques (weintraub et al., 2004). despite these caveats, our observations have strong implications for the study of reproduction in reptiles. for example, our data indicate that estimates of reproductive output made at different stages of gestation may yield different results. many authors have based estimates of clutch and litter sizes on the numbers of enlarging (vitellogenic) ovarian follicles, or the numbers of recently-ovulated “eggs” (gregory et al., 1992). if egg/embryo disappearance is common, these figures will over-estimate actual fecundity levels. in the present example, the difference in litter size would be 100% (2 vs. 4 offspring). more generally, our data support the hypothesis that reproducing female reptiles may be able to manipulate their level of reproductive expenditure before, and even after ovulation (blackburn, 1998a). in circumstances in which the “optimal” life-history tactic involves a litter size smaller than the one she currently has, a female may be able to adjust litter size by selective “resorption”. the main result of our study is to encourage the use of the techniques described in this paper. a similar approach, using serial non-invasive magnetic resonance microscopy (but not doppler-ultrasonography) has been successfully employed to study embryo resorption in laboratory mice (weintraub et al., 2004). not only do they offer powerful new opportunities to clarify the dynamics of intrauterine processes, they may also help to identify the cues that stimulate processes such as litter-size reduction. more generally, these methods allow a more direct and reliable comparison between litter sizes at ovulation and at parturition, that sometimes significantly differ in natural conditions as detected on large sample size (bonnet et al., 2001). the techniques may also allow accurate estimation of reproductive condition under circumstances in which existing techniques such as abdominal palpation are unreliable. this may apply, for example, relatively early in gestation (pers. obs.), in large heavy-bodied species in which palpation is ineffective (e.g., nmr revealed > 30 eggs in a large gaboon viper, when palpation revealed none), or in species whose thick abdominal musculature interferes with palpation (e.g., some python species: pers. obs.). imaging techniques also provide accurate data on the timing of events such as ovulation and various stages of embryogenesis, information that may otherwise be difficult to obtain by non-destructive techniques. acknowledgements financial support was provided by the conseil général des deux sèvres, centre national de la recherche scientifique, the faculté de médecine de tours (france) and the australian research council. we thank rex cambag and j.t. wilmslow for participation during the testing of biospec and the doppler apparatus. 136 x. bonnet et alii references bell, w.j., bohm, m.k. 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(2004): prenatal detection of embryo resorption in osteopontin-deficient mice using serial noninvasive magnetic resonance microscopy. pediatr. res. 55: 419-424. westlin, l.m., soley, j.t., van der merwe, n.j., van dyk, y.j. (1995): late fetal development and selective resorption in saccostomus campestris (cricetidae). reprod. fert. devel. 7: 1177-1184. wilson, t.g. (1985): determinants of oocyte degeneration in drosophila melanogaster. j. insect. physiol. 31: 109-117. acta herpetologica 16(2): 109-121, 2021 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-10079 bone histology of broad-snouted caiman caiman latirostris (crocodylia: alligatoridae) as tool for morphophysiological inferences in crocodylia paulo braga mascarenhas-junior1,2,3,6,*, luis antonio bochetti bassetti4, juliana manso sayão5,6 ¹ laboratório interdisciplinar de anfíbios e répteis, universidade federal rural de pernambuco (ufrpe), recife, pernambuco, brazil ² programa de pós-graduação em biologia animal, universidade federal de pernambuco, recife, pernambuco, brazil ³ laboratório de herpetologia, universidade federal de pernambuco (ufpe), recife, pernambuco, brazil 4 laboratório de ecologia isotópica – cena/usp, piracicaba, são paulo, brazil 5 museu nacional do rio de janeiro, universidade federal do rio de janeiro, rio de janeiro, rio de janeiro, brazil 6 centro acadêmico de vitória (cav), universidade federal de pernambuco, vitória de santo antão, pernambuco, brazil *corresponding author. e-mail:paulobragam16@gmail.com submitted on: 2020, 30th november; revised on: 2021, 14th october; accepted on: 2021, 26th october editor: fabio m. guarino abstract. bone histology is an important tool for the interpretation of life patterns in animals of the past and extant fauna. the crocodylians have been studied as important inferential models for morphophysiological characteristics. we aimed to characterize the osteohistology of captive caiman latirostris, identifying its microanatomy related to growth rates, ontogeny, and environmental conditions. we analyzed five pairs of humeri (proximal elements of the appendicular skeleton) and ribs (axial skeleton) of females’ caiman. ribs showed, in general, woven-fibered tissues, with low vascularization and parallel-fibered bone and many resorption and erosion cavities. it presented lines of arrested growth (lags) in three individuals, without skeletochronological compatibility. humeri showed a gradient of woven-fibered to parallel-fibered and lamellar-zonal bone as the individuals aging. we observed compacted coarse cancellous bone (cccb) and a higher number of lags in older specimens. ribs remodel faster than humerus, showing an intra-individual histovariability. the humeri indicated an evident growth pattern with different ontogeny stages and growth rates in different ages. fast-growing tissues are uncommon in crocodylians, but basal metabolism and optimal growth conditions can lead to this. bone histology of c. latirostris shows patterns that can be used as inferential models for extant and extinct groups, but we encourage further studies for a better understanding, under different environmental conditions, such as temperature and food availability. keywords. crocodylians, growth rate, ontogeny, osteohistology. introduction the order crocodylia is a monophyletic group of animals widely distributed in tropical regions, divided into three families: alligatoridae, crocodylidae, and gavialidae (zug, vitt and caldwell, 2001). they have great ecological importance within the ecosystems where they are inserted, as in the control of food chains (fernández-fernández, arias and khazan, 2015) and supply of nutrients from the aquatic environment (fittkau, 1973), besides their economic relevance through meat and skin trade (caldwell, 2017). in south america, broad-snouted caiman (caiman latirostris daudin, 1801) is one of the most abundant species of alligatoridae family, especially in brazilian territory (coutinho et al., 2013). it is considered as a medium-sized crocodylian with records of up to three and a half meters, although free-living specimens hard110 p.b. mascarenhas-junior, l.a. bochetti bassetti, j. manso sayão ly exceed two and a half meters (verdade, larriera and piña, 2010). it is commonly found in lentic freshwater ecosystems, such as dams and ponds (filogonio et al., 2010), and is widely adapted to anthropized environments (mascarenhas júnior, santos and correia, 2018; mascarenhas-junior et al., 2020). it presents opportunistic and generalist feeding behavior, preying from invertebrates to fish, testudines, and small mammals (diefenbach 1988; melo, 2002). its meat and skin have great commercial value, with its rearing being allowed for economic purposes, especially in the closed systems in brazil (farming) and in ranching programs in argentina (coutinho et al., 2013). as it is a well-known species, c. latirostris has an important potential for interpretive and inferential studies, especially in understand past species conditions. knowledge at microscopic level of bone structures of crocodylians is an important tool for biological, behavioral, evolutionary, and ecological inferences through ontogenetic stages (chinsamy, codorniú and chiappe, 2009). due to physiological and morphological similarities between fossil and extant crocodylian taxa, the later are widely used as inferential histological models for past groups (storrs, 1993; andrade and sayão, 2014; woodward, horner and farlow, 2014; sayão et al., 2016, company and pereda-suberbiola, 2017). to understand the microstructural information of a fossilized bone, for example, a series of analyzes of comparative structures in existing organisms through osteohistology are necessary (woodward, horner and farlow, 2014). the histological methods of inference are complementary to the traditional models of morphological descriptions (sayão et al., 2016), as the bone structure consists of biomineralized tissue, formed by deposition of hydroxyapatite and crystalline calcium phosphate, and its inner region consists of bone cells known as osteocytes and various blood and lymph canals (andrade and sayão, 2014). after death, all organic compounds are decomposed, the inorganic part is fossilized, and the shape of microstructures is preserved (ricqlès, horner and padian, 1998), allowing their interpretation even in extinct organisms. however, although we can infer on the natural history of extinct groups with analyzes on extant crocodylians, individual skeletal variations may affect generalizations. factors such as unfamiliarity of environmental influences, bone resorption and remodeling during growth can interfere in a determination of a pattern (hutton, 1986). broad-snouted caiman is one of the extant species of caimaninae subfamily, originally from the cenozoic period (brochu, 2010). osteohistological information serving as inferential tool on the morphology, physiology, and environmental stresses in caimaninae are still incipient, restricted to data of c. yacare (andrade et al., 2018). therefore, we aim to aggregate and refine information on microanatomical bone tendencies in crocodylia by using bone elements from specimens of c. latirostris as a model for deducting structural responses to morphological, physiological, ontogenetic, and environmental questions. material and methods sample study here we used skeletal elements of c. latirostris, kept in captivity and then slaughtered for commercial purposes. they were supplied by the aruman slaughterhouse ltd, located in the municipality of porto feliz, são paulo state, brazil. the slaughterhouse donated bone elements from ten females according to the availability, and we selected pairs from one to six years old (except for five years old specimens), totaling five pairs of different ages. all caimans were slaughtered in the same period and had their body mass weighted at slaughter, ranging from 6.99 kg to 21.35 kg. the individual classification of each specimen follows the control protocols used by the slaughterhouse (table 1). like other crocodylians, c. latirostris exhibits temperaturedependent for sex determination. in captivity, eggs are generally incubated at temperatures ranging from 28 ºc to 34 ºc, to avoid embryonic death. for safety, the hatchery’s incubation chamber in the caiman farm is adjusted to maintain constant temperature of 31 ºc, making 100% of females in its production (parachú-marcó et al., 2017, simoncini et al., 2019). caiman farming in the rearing pens, specimens were fed daily until reach two years old and three times a week at the beginning of the third year of life. pens were formed by water tanks, dry areas, and rooftops, functioning as greenhouses for thermal retention and accelerating the growth of animals. each pen has a total area of 7x5 m, of which 14 m² meters are dry and 21 m² meters are wet, with 80 cm deep pools. the walls measure 1.20 m from the surface of the tanks and the dry floor. abiotic data we obtained the minimum and maximum daily temperatures of porto feliz city during the years that specimens lived in the rearing pens from data obtained from the national meteorological institute (inmet). we categorized temperatures considering each year of the animal’s life as absolute minimum; average minimum; average; average maximum; and absolute average. we note that temperatures tend to be higher in the rearing pens for the use of greenhouses to retain heat and opti111caiman latirostris bone histology mize animal growth, especially in the hottest periods of the year (sarkis-gonçalves et al., 2001). inside greenhouses, the temperature is not constant, forming microclimates in different areas, such as on the dry floor, on the surface, and at the bottom of the water tanks. to determine the temperatures estimated in these three microhabitats, we used the study by fincatti and verdade (2002) as reference for presenting similar rearing pens to those applied by the caiman farm in porto feliz city. we performed temperature estimates for this study based on the difference between the averages measured by fincatti and verdade (2002) in the rearing pens and the approximate local temperature of the external environment, based on data from inmet (2018). the difference between the three microclimates (dry area, tank surface, and tank bottom) and the external environment, considering the physical conditions of the rearing pens, ranges in approximately 8 °c, 10 °c, and 10 °c in the absolute minimum data; 8 °c, 10 °c, and 9 °c in the average minimum data; 9 °c, 4 °c, and 0.1 °c in the maximum averages; and 14 °c, 11 °c, and 0.4 °c in the absolute maximum temperatures (fincatti and verdade, 2002) (available in supplementary material). each same age pair of individuals were subjected to different temperature variations, considering that some of them spent more time in the farm for being older. bone elements we selected 20 bones, one axial (rib) and one appendicular (humerus) element of each specimen, which we removed from the same anatomical position (fig. 1). we measured each bone according to its length, diameter, and the length of the sample used. we defined rib length by the distance between proximal and distal portions. the humeri presented maximum length and diameter ranging between 64.12 mm and 103.34 mm and 7.27 mm and 11.36 mm, respectively. the ribs indicated length ranging between 94.5 mm and 129.82 mm and diameter gradient ranging between 5.24 mm and 8.01 mm. the average length of the sectioned sample were 5.35 mm and 6.08 mm for humerus and rib, respectively (table 1). sample preparation we prepared all the material in the laboratory of paleobiology and microstructures of the academic center of vitória (cav), advanced campus of federal university of pernambuco (ufpe). for preparation of histological slides, we performed thin sections from the medial regions of the bone diaphysis (fig. 1), using standard osteohistological techniques (chinsamy and raath, 1992; lamm, 2013). sampled portions were removed using a dremel 400 rotary tool, embedded in epoxy clear resin resapol t-208 and catalyzed with butanox m50, using a ratio of 1 ml of catalyst for every 100 ml of resin. the samples were adhered to microscopy slides with glue and epoxy araldite hardener. the blocks were roughed and polished using a metallographic polishing machine (arapol vv) with arotec abrasive paper of increasing grit size (grain sizes 60/ p60, 120/p120, table 1. age, weight and morphometric data of the caiman latirostris specimens from the aruman slaughterhouse ltd. used in this study. idnum = identification number; idlabel = identification label; humerus and rib: l = length (mm); d = diameter (mm); s = lenght of the sectioned sample. idnum idlabel age (year) weight (kg) humerus rib l d s l d s 1 3vd4e259 6 15.08 84.07 9.20 6.40 111.65 5.65 6.71 2 3vd4e239 6 21.36 92.11 10.63 6.51 123.57 6.38 6.82 3 5vd2d348 4 19.17 87.54 8.84 6.80 122.84 5.38 7.15 4 5vd6d467 4 12.62 81.98 9.75 6.23 94.50 5.58 6.16 5 6vd3d568 3 10.00 72.56 8.66 4.23 105.62 5.39 6.39 6 6vd3d237 3 10.80 69.14 7.93 4.47 129.82 5.92 6.32 7 7vd5e248 2 8.49 70.55 8.02 4.51 105.43 6.66 5.52 8 7vd5d479 2 7.12 64.12 8.25 5.67 109.05 5.86 6.87 9 8vd7d9 1 6.99 67.49 7.27 3.83 118.90 5.24 5.11 10 8vd2e345 1 7.31 64.85 7.93 4.76 105.91 5.24 4.50 fig. 1. bone elements selected for osteohistological description. section location highlighted between dashed lines. a: humerus; b: rib. 112 p.b. mascarenhas-junior, l.a. bochetti bassetti, j. manso sayão 320/p400, 1200/p2500) until a final thickness of 30 to 60 µm was reached. analysis of histological material we analyzed the bone sections using an axioimager m2 transmitted and polarized light microscope equipped with axiocam digital sight camera. inferences and descriptions followed the study by francillon-vieillot et al. (1990). we selected all the structures of interest observable in sections of the bone cortex. we evaluated humeri together in pairs of the same age for presenting similarities between the observed patterns. ribs were not described in pairs for not presenting microanatomical similarities between same-aged individuals. images of all histological sections are available in the supplementary material. growth curve older specimens presented lines of arrested growth (lags), enabling to estimate the initial growth curves of these animals based on the distances between lines and the medullary cavity. we also calculated the artithmetic means of distances between lags. data analysis for the lack of information on animal size, relationships between specimens were tested according to age, weight, humerus lengths, and rib length. notably, we tested the significance of the bivariate pearson’s correlation coefficient (rp) between all the possible pairs. analyzes were performed using the biostat 5.9.8 software. the level of significance was set 0.05. results we obser ved a signif icant positive correlation between caimans’ weight and humerus length (rp = 0.962; t-test: t = 9.897; df = 8; p <0.001). on the other hand, we did not observe this relationship between weight and rib length (rp = 0.396; t-test: t = 1.221; df = 8; p = 0.257) and between humerus and rib lengths (rp = 0.192; t-test: t = 0.552; df = 8; p = 0.596). humeri one-year-old specimens: we observed a highly vascularized cortex, mainly on endosteal portion, with presence of woven-fibered tissue. anastomosed vascular canals have a reticular pattern, with the presence of simple longitudinal canals and primary osteons distributed throughout the cortex, forming a fibrolamellar bone (fig. 2a). two-year-old specimens: the bone cortex of specimen 8 indicated the presence of fibrolamellar complex. at the endosteal level it shows a woven-fibered bone followed by a lower vascularized transition zone of parallel-fibered tissue towards the subperiosteal region (fig. 2 b). we could observe a similar pattern of decreased vascularization in the medullary-cortex direction in specimen 7, with evidence of secondary lamellar tissue in perimedullary portion (fig. 3 a), woven-fibered and parallel-fibered bone at inner the cortex. simple and anastomosed canals are found inside the cortex, with the presence of primary osteons throughout the cortex and opening towards the periosteum (fig. 2 b). three-year-old specimens: we observed moderate vascularization in the matrix, with mostly simple and few anastomosed canals, presenting longitudinal and reticular patterns and primary osteons (figs. 2 c, 3 b). we noticed an anisotropic tissue in perimedullary portion, indicating secondary remodeling bone with the presence of a compacted coarse cancellous bone (cccb). we also observed parallel-fibered and woven-fibered in medial (fig. 3 b) and parallel-fibered in endosteal portions of the cortex (fig. 2 c). four-year-old specimens: perimedullary portion of both individuals presented remodeling bone, with cccb in specimen 4 (fig. 3 c) and lamellar remodeling tissue in specimen 3 (fig. 3 d), both with mineral erosion cavities, opening to medullary cavity in specimen 4. also, in medullary-periosteum direction, both bones presented a decrease of primary osteons and vascularization, wovenfibered tissue in medial portion of matrix and parallelfibered tissue in periosteal region (fig. 2 d), with an erosion cavity in specimen 3 (figs.2 e,3 d). we observed simple and anastomosed vascular canals pattern in both samples and plexiform canals in specimen 3 (fig. 2 f). the most external portion of both bones presented lags (three in specimen 4, fig. 2 d, and two in specimen 3). six-year-old specimens: both specimens 1 and 2 presented remodeling bone in perimedullary portion (lamellar in specimen 2 and cccb in specimen 1, fig. 3 e). we observed woven-fibered and parallel-fibered tissue (figure 2 g), besides primary osteons throughout the cortex (fig. 2 h), with simple vascular canals and reticular anastomoses within the matrix and low vascularization in periosteal portion. in addition, we observed four lags in specimen 2 (fig. 2 g) and three in specimen 1. ribs specimen 10 (one-year-old): cortex is composed of woven-fibered tissue low vascularized, with few simple 113caiman latirostris bone histology fig. 2. histology under transmitted light of humeri of caiman latirostris specimens in captivity slaughtered in a rearing pen in the municipality of porto feliz, são paulo. the specimens were one year old (a), two years old (b), three years old (c), four years old (d, e, and f), and six years old (g and h) at the time of slaughter. ec: erosion cavity; m: medullary portion; p: periosteal portion; pfb: parallel-fibered bone; po: primary osteon; pvc: plexiform vascular canal; ravc: reticular anastomosing vascular canal; slvc: simple longitudinal vascular canal; wfb: woven-fibered bone. white arrows show lines of arrested growth (lags). 114 p.b. mascarenhas-junior, l.a. bochetti bassetti, j. manso sayão fig. 3. histology under polarized light combined with gypsum filter of humeri of caiman latirostris specimens in captivity slaughtered in a rearing pen in the municipality of porto feliz, são paulo. the specimens were two years old (a), three years old (b), four years old (c and d) and six years old (e). cccb: compacted coarse cancellous bone; ec: erosion cavity; lb: lamellar bone; m: medullary portion; p: periosteal portion; pfb: parallel-fibered bone; ravc: reticular anastomosing vascular canal; wfb: woven-fibered bone. 115caiman latirostris bone histology and anastomosed vascular canals, and few primary osteons. large resorption cavities appear in the endosteal bone. specimen 9 (one-year-old): the tissue is predominantly woven-fibered, with a matrix vascularized mainly through simple canals and presenting primary osteons. within the cortex, we found secondary osteons. resorption cavities are also visible. specimen 8 (two-years-old): woven-fibered bone is observed at endosteal level and parallel-fibered bone evidenced in the periosteal layer, with simple longitudinal vascular canals presenting primary osteons. resorption cavities present mainly within the cortex (fig. 4a). specimen 7 (two-years-old): we found little vascularization in the cortex, presenting simple longitudinal and reticular anastomosing canals with primary osteons. the tissue exhibits woven-fibered pattern and two possible lags in the cortex. specimen 6 (three-years-old): we noticed a large amount of erosion cavities, found in the bone structure until close to the periosteal region. we evidenced woven-fibered tissue in the regions between erosion cavities, comprising most of the tissue and parallel-fibered tissue in the periosteal region. we found little vascularization, with simple longitudinal and reticular anastomosing canals and primary osteon. specimen 5 (three-years-old): we found tissue of woven-fibered type with moderate vascularization, presenting simple longitudinal and reticular anastomosing vascular canals, as well as primary osteons. in the region of the inner cortex, we identified several bone resorption cavities. in one of the periosteal portions, we identified three lags. specimen 4 (four-years-old): we found woven-fibered tissue in this bone with moderate vascularization, presenting simple longitudinal and reticular anastomosing vascular canals. we observed primary osteons and erosion cavities. in addition, it presents four lags in the periosteal region (fig. 4b). specimen 3 (three-years-old): cortex is composed of woven-fibered tissue full of resorption cavities, comprising most of the bone. vascularization is low, composed by primary osteons and simple longitudinal vascular canals. specimen 2 (six-years-old): presence of several bone resorption cavities with woven-fibered bone. the cortex is poorly vascularized, with small amounts of simple longitudinal and reticular anastomosing vascular canals and primary osteons. specimen 1 (six-years-old): primary and secondary osteons compose the cortex. vascularization is low with simple longitudinal and reticular anastomosing canals. bone resorption cavities concentrated endosteal region and woven-fibered tissue is present within the cortex (fig. 4c). distance between lines of arrested growth lags are present only in older specimens. we calculated distances between intervals among lines, as well as the total thickness of the cortex. the distances between the medullary cavity and periosteum ranged between 8,040 μm in specimen 3 and 9,830 μm in specimen 2. the minimum and maximum distances observed between lags ranged from 105 μm to 475 μm. the average spacing between lags was 282.2μm, ranging between 35 μm from the first to the second lags in specimen 1 and 475 μm from the second to the third lags in the same specimen (fig. 5). fig. 4. rib histology under transmitted light of three specimens of caiman latirostris in captivity slaughtered in a rearing pen in the municipality of porto feliz, são paulo. the individuals were two (a), three (b) and four (b) years old at the time of slaughter. ec: erosion cavity; pfb: parallel-fibered bone; po: primary osteon; ravc: reticular anastomosing vascular canal; rc: resorption cavity; slvc: simple longitudinal vascular canal; so: secondary osteon; wfb: woven-fibered bone. white arrows indicate lines of arrested growth (lags). 116 p.b. mascarenhas-junior, l.a. bochetti bassetti, j. manso sayão discussion in general, we observed a fast-growth pattern in ribs than in humeri. younger specimens presented mostly woven-fibered (fast-growing) tissues in the humeri, and during their ontogenetic development, began to form parallel-fibered tissue, indicating growth stabilization. the ribs demonstrated a prevalence of woven-fibered tissue in all ages, with reduced vascularization. this information may be related to a lack of relationship in the growth trend between these bone elements, leading us to believe that the ribs may have a faster growth and remodeling than humeri (enlow and brown, 1958), demonstrating histovariability in axial and appendicular bone elements the same specimen (sayão et al., 2016; sena et al., 2018). this also corroborates what has already been described in alligatoridae crocodylians: alligator and caiman (lee, 2004; woodward, horner and farlow, 2014; andrade et al., 2018). the ribs showed major remodeling in the medullary region, which expanded over a large part of the bone cortex, as already observed in other crocodylomorpha (andrade et al., 2015; sayão et al., 2016; sena et al., 2018). these remodeling processes can directly influence determinations by skeletochronology. specimens 7, 5, and 4 (two, three and four years old respectively) were the only with lags, showing the same number of lines as its age, as found by waskow and mateus (2017). on the other hand, the other ribs showed no evidence of lags, indicating that the bone had possibly been remodeled and the lines lost during the processes of bone absorption and redeposition throughout its development. deficiency to determine ages from axial elements has also been previously reported in the literature in both living (hutton, 1986) and fossil specimens (sayão et al., 2016; sena et al., 2018). for the inconsistency in microanatomical patterns, ribs are not the most suitable elements for skeletochronology (padian, 2011), although having already been used in skeletochronological studies in other groups of archosaurs, especially in fossils, such as dinosauria (gallina, 2012; waskow and sander, 2014; waskow and mateus, 2017; woodruff, fowler and horner, 2017; brum et al., 2021). also, waskow and sander (2014) stated that the posteromedial side of the proximal shaft of the ribs record best growth information in osteohistological observations. it was suggested that in studies without promising results, samples were collected from distal portions of the ribs, not showing lines or other growth marks (waskow and sander, 2014). however, in this study all samples were collected from the proximal portions of ribs (see material and methods), and we identified lags in three of our samples, contradicting this idea, at least for the group under study. we did not identify significant relationship between rib length increase and animals aging, although we found association between the size of the axial element and the presence of lags. the three specimens that presented lags (specimens 4, 5, and 7) had the smallest ribs, being related to bone tissue remodeling, which had not been enough remodeled for the destruction of the lines. the humeri showed tendencies in microanatomy and osteohistology related to animal age. the youngest caimans (10 and 9) presented a homogeneous pattern in tissue composition, being of woven-fibered type. this tissue is quite common in young specimens, with high metabolic rates and rapid growth, presenting great vascularization and primary osteons (huttenlocker, woodward and hall, 2013). the presence of woven-fibered tissue with primary osteons, as already reported in crocodylians, indicate physiological strategies of rapid growth, forming a fibrolamellar complex (ricqlès, 1983; reid, 1984, 1990, 1997; ricqlès, padian and horner, 2001; chinsamy and hillenius, 2004; tumarkin-deratizan, vann and dodson 2007; woodward, horner and farlow, 2014; andrade et al., 2018). this bone composition supports the hypothesis that crocodylians retain an ancestral characteristic of archosauria (cubo et al., 2012), evidenced in young animals. we did not expect the presence of fibrolamellar bones in crocodylians, although it has already been identified in young specimens with optimal grow th conditions in farms, constant feeding, and ideal temperature of approximately 30 ºc to 33 ºc (lang, 1987; woodward, horner and farlow, 2014). during the day, caimans tend to move within the pens, being exposed to the temperature ranges close to the ideal for growth (verdade, 1995). generally, during winter and days of lower temperatures, caimans remain in the dry areas in fig. 5 distance between highlighted lines of arrested growth (lags) in humeri of specimens 1, 2, 3, and 4. 117caiman latirostris bone histology sun periods and in humid areas at night for the different rates of heat accumulation between air and water, as the air is a better heat disperser than water. during summer or in periods with high temperatures, the animals will follow the opposite, preferring wet microenvironments during sun periods and the dry ones at night (verdade et al., 1994). despite lower temperatures, the pens have greenhouses that tend to retain heat, leaving the environment with a higher temperature in relation to the outside (fincatti and verdade, 2002). it is possible that, through thermal stimuli and constant feeding, individuals tend to maintain higher rates of basal metabolism, consequently accelerating their growth with longer lasting strategies of rapid growth. this fact is understandable, since crocodylians spend less energ y producing heat, living with a low metabolic rate. therefore, we assume that the physiological demand for energy, especially glucose, for the growth and maintenance of ectothermic animals is low if compared to that of endothermic carnivores, since there is no need to produce heat from this sugar to maintain temperature body. consequently, a large part of its energy supply is invested in growth and reproduction. this result is obtained rather through predominantly behavioral mechanisms than physiological mechanisms by seeking the ideal temperature range for their metabolic activities, which in general are eight to ten-fold lower than that of endothermic animals, somehow explaining their dependence on room temperature (smith, robertson and davies, 1978; staton, 1988; silva, 2000). specimens 8 and 7, despite being the same age (two years old), presented differences in metabolic growth strategies, observed in the bone. during animal aging, the tendency is the reduction of growth rate, with a transition between tissues that demonstrate fast-growing metabolism strategies (woven-fibered bone) and slow-growing tissues (ricqlès, padian and horner, 2003; huttenlocker, woodward and hall, 2013). with maturation, we observed parallel-fibered tissue, especially in the periosteal region, and secondary remodeling bone, such as cccb and lamellar bone in endosteal portion. there is a gradient associated with age which reflects the start of slow-growth strategy, although without finalization of definitive bone growth, evidenced by the absence of an external fundamental system and the opening of primary osteons in the cortex towards the periosteum (ricqlès et al., 2003; woodward et al., 2011; andrade et al., 2018). the decreasing presence of fibrolamellar complexes implies the reduce of fast-growth rates, a baseline characteristic of tetrapods in general (woodward, horner and farlow, 2014). in some specimens cccb in response of secondarily remodeling processes can be observed, mainly in older individuals or breeding females (hutton, 1986; schweitzer et al., 2007). in female crocodylians, bone remodeling can occur faster than in males due to calcium mobilization for the formation of eggshells (hutton, 1986). the beginning of the reproductive stage in the life of crocodylians is related to their growth, also influenced by temperature, and feeding. females of c. latirostris reaches sexual maturity with approximately 68 centimeters of snout-vent length (leiva et al., 2019) or between four and five years of age in captivity (verdade et al., 2003). in commercial farms, caimans are maintained in greenhouses under high temperatures to reduce their time for slaughter and sexual maturation. we observed a high remodeling bone in females over 10 kg (three years old), which can be an important indicator of reproductive stage beginning. klein, scheyer and tütken (2009) suggest that captive females have great absorption in long bones due to mineral mobilization to form eggshells or due to nutritional deficiencies, and, associated with secondary remodeling bone, the cyclic growth marks may be destroyed. for free-living animals, schweitzer et al. (2007) stated that long bones in wild female alligator mississipiensis do not show major remodeling activities, which may indicate histovariability between wild and captive animals, even in similar ontogenetic stages. as in other reptiles, lags provide clues to both skeletochronology and the growth rates of specimens on an annual basis (castanet et al., 1993; guarino et al., 2020). these thin lines that occur parallel to the periosteum margin (wilson and chin, 2014) are observed in greater quantities with increasing age. according to castanet et al. (1993), the deposition of lags occurs annually in crocodylians, regardless of food, temperature, or photoperiod. tucker (1997) suggested that the appearance of lags is associated with winter periods (food shortages and decreased metabolism). in this study, specimens that were in the first, second, and third years of life did not present evident lines and specimens that were in the fourth and sixth years of life presented underestimated numbers of growth marks regarding age. considering the deposition of one lag per year, we could identify the growth speed of animals that showed these marks. four-year-old specimens had a relatively similar spacing between lags, which may indicate a similar growth rate between the first and second years. the third line of specimen 4 is close to the periosteum, indicating that the animal died before formation of the fourth lag. on the other hand, six-year-olds had different intervals between lines. specimen 2 presented a large 118 p.b. mascarenhas-junior, l.a. bochetti bassetti, j. manso sayão spacing between the first and second lag and between the second and third. between the third and fourth, the distance reduced, leading us to believe that the caiman grew at high rates between the first three years and had a decline in the growth curve in the fourth year. specimen 1 had little spacing between the first and second lag, with a larger distance between the second and third one. it also had a reduction between the third and fourth lines, probably indicating a low growth rate until reaching the third year of life, growing quickly until reaching the fourth year, in which apposition decreased again. both specimens 1 and 2 may have been slaughtered before the formation of the fifth line. these differences in growth patterns can be related both to the intrinsic physiological conditions that influence growth and external factors, such as dominance in the competition for food and the selection of thermal ranges in the pens (hutton, 1986; verdade et al., 1994). scarcity of osteohistological studies conducted with a significant sample of specimens and taxa hinders the confirmation that lags are necessarily formed annually in crocodylians. based on our observations, it is likely that this concept is not consistent with the patterns observed in captive specimens of living taxa or even with those of the genus caiman or of the species c. latirostris. in general, studies that performed osteohistological interpretations in extant crocodylians are restricted to less than ten specimens sampled (schweitzer et al., 2007; woodward, horner and farlow, 2014; andrade et al., 2018), the only exception was a study conducted with 30 different individuals of a. mississipiensis (woodward, horner and farlow, 2011). researches with larger samples are still scarce (hutton, 1986; tumarkin-deratzian, vann and dodson, 2007) and must be incentivized. so far, any other research has been performed with crocodylians from the subfamily caimaninae, which elucidates the incipience of concrete data on the pattern of growth marks in crocodylians. acknowledgements this project has a license from the environmental company of the state of são paulo (cetesb) for the effluent, number 61000099. the aruman slaughterhouse is authorized by the secretariat of environment for the use and management of c. latirostris through license number 000001409, with process number 000762, 2016. all the material used in this research was delivered with the issuance of invoice number 000000097. icmbio authorization was also issued to conduct this work, legally supported under license number 59048-1. the authors would like to thank dr. mariana sena, dr. rafael araújo, esaú victor, and haggy rodrigues for helping with the first processing of material for this research. dr. josé adauto de souza neto for the access to petrographic microscope at laboratório de geoquímica aplicada ao petróleo (lgap) and dr. mariana sena for helping in polarized microscope imaging. to dr. holly woodward (oklahoma state university) and an anonymous reviewer for the suggestions and questions that improved the final version of this manuscript. we would like to express our gratitude to the aruman slaughterhouse, responsible for donating the bone material that enabled this work. this project was partially funded by the national council for scientific and technological development cnpq (proc. n. # 311715/20176 and 314222/2020-0 to jms) and coordination for the improvement of higher education personnel capes (msc fellowship to pbmj). references andrade, r.c.l.p., bantim, r.a.m., lima, f.j., campos, l.s., eleutério, l.h.s., sayão, j.m. 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(2001): herpetology: an introductory biology of amphibians and reptiles. academic press, boston. acta herpetologica vol. 16, n. 2 december 2021 firenze university press a new species of the genus noblella (amphibia: strabomantidae) from ecuador, with new information for noblella worleyae carolina reyes-puig1,2,3,4,*, juan m. guayasamin 2,5 claudia koch6, david brito-zapata1, matthijs hollanders7, melissa costales8, diego f. cisneros-heredia1,2,3 so close so different: what makes the difference? dario ottonello1,7,*, stefania d’angelo2, fabrizio oneto3,6, stefano malavasi1, marco alberto luca zuffi4, filippo spadola5 hematological values of wild caiman latirostris (daudin, 1802) in the atlantic rainforest in pernambuco, brazil luciana c. rameh-de-albuquerque1, alexandre p. zanotti1, denisson s. souza1, george t. diniz2, paulo b. mascarenhas-junior3,4,5,*, ednilza m. santos³, jozelia m. s. correia3 bone histology of broad-snouted caiman caiman latirostris (crocodylia: alligatoridae) as tool for morphophysiological inferences in crocodylia paulo braga mascarenhas-junior1,2,3,6, luis antonio bochetti bassetti4, juliana manso sayão5,6 is the northern spectacled salamander salamandrina perspicillata aposematic? a preliminary test with clay models giacomo barbieri, andrea costa, sebastiano salvidio* sexual size dimorphism in the tail length of the caspian whip snakes, dolichophis caspius (serpentes, colubridae), in south-western hungary györgy dudás1, krisztián frank2* semi-automated photo-identification of bahamian racers (cubophis vudii vudii) sebastian hoefer1,*, andreu rotger2, sophie mills1, nathan j. robinson1,3 acta herpetologica 4(2): 171-175, 2009 first record of salamander predation by a liophis (wagler, 1830) snake in the venezuelan andes luis felipe esqueda1, marco natera-mumaw2, enrique la marca3 1 investigador asociado, colección de anfibios y reptiles, laboratorio de biogeografía, facultad de ciencias forestales y ambientales, universidad de los andes. mérida 5101, venezuela and postgrado de áreas naturales y de conservación de la naturaleza, facultad de ciencias forestales, universidad de chile, santiago de chile, chile. corresponding author. e-mail: luisfesqueda@gmail.com 2 museo de vertebrados, universidad experimental rómulo gallegos, apartado 205, san juan de los morros, guárico 2301, venezuela. e-mail: mnateram@yahoo.com 3 laboratorio de biogeografía, escuela de geografía, facultad de ciencias forestales y ambientales. universidad de los andes, apartado postal 116, mérida 5101-a, venezuela. e-mail: enrique.lamarca @gmail.com submitted on: 2008, 3rd november; revised on 2009, 1st july; accepted on 2009, 26th august. abstract. information available so far is exceedingly meagre about the diet of the snakes included in the genus liophis, one of the most diverse groups that inhabit terrestrial ecosystems of south america. for the first time is documented the predation of a salamander by liophis from venezuela, including a brief overview on the alteration of montane and submontane andean ecosystem and their effect on the natural dynamic. keywords. bolitoglossa guaramacalensis, liophis reginae zweifeli, predation, venezuelan andes. currently, fifty two species of liophis (wagler, 1830), are recognized, among which we can find a wide variety of prey items, that include amphibians (also eggs and tadpoles), invertebrates, lizards, fishes, birds, and small rodents (roze, 1966; duellman, 1978; chippaux, 1986; dixon, 1989; michaud and dixon, 1989; cunha and nascimento, 1993; martins and oliveira, 1999; boos, 2001; dixon and tipton, 2003; savage, 2002; esqueda et al., 2007). although there is a considerable advance on their taxonomy and biogeography, information on their feeding habits is scarce, especially for those species that occur in northern south america. within the material deposited in the biogeography laboratory of the university of los andes (ulabg), we found an adult male of liophis reginae zweifeli roze, 1959 (ulabg 2765), coming from the road potreritos de cendé to miquimú, municipio carache, trujillo state, venezuela, which had in its stomach a partially digested adult specimen of a salamander, which we identified as bolitoglossa guaramacalensis schargel, garcía-pérez, smith (2002) (fig. 1). 172 l.f. esqueda, m. natera-mumaw and e. la marca predation of plethodontids in liophis snakes has been little documented. michaud and dixon (1989) made a list of prey items for twenty species in the genus, finding two of them as potential predators of salamanders: l. reginae (linnaeus, 1758) and l. epinephalus cope, 1862, which suggests that predation on plethodontids in this group is rare or not well documented. they assessed the predation of bolitoglossa altamazonica by l. reginae (cope, 1864), while in l. epinephalus, the species of salamander was not specified. following an ecogeographical approach, it is quite possible that in the first case the appropriate name for the snake species is l. reginae semilineata (wagler, 1824) (see martins and oliveira, 1999: 40-41), while the salamander may well correspond to what is currently known as bolitoglossa paraensis (unterstein, 1930) (see parra-olea et al., 2004). salamanders display an ample repertory of antipredatory strategies, with significant interspecific and/or intraspecific variability as a response to different predators (whitemann and wissinger, 1991; garcía-paris and debam, 1995). b. guaramacalensis prefers more terrestrial places (associated with rocks near streams), than other arboreal species (associated to epiphytic bromeliads). according to schargel et al. (2002), some individuals had a regenerated tail, indicating a potential importance about the autotomy of the tail as an antipredatory behavior. our voucher salamander discovered inside the stomach contents of a l. r. zweifeli snake suggests that it was gobbled head-first and without loosing the tail, suggesting that tail autotomy may not always be used as a defense mechanism (it is likely that this poorly documented behavior is dependent on other variables not even clearly defined). l. reginae has diurnal habits, contrary with the nocturnal activity of bolitoglossa salamanders. a similar case has been documented for bolitoglossa heiroreias greenbaum, 2004, where a specimen was regurgitated by a rhadinaea montecristi mertens, 1952 (greenbaum, 2004), a colubrid with similar habits to l. reginae. the fig. 1. dorsal and ventral views of liophis reginae zweifeli (ulabg 2765). note the partially digested specimen of bolitoglossa guaramacalensis. 173salamander predation by a liophis salamanders in both cases seem to have an arboreal behavior during the night and a terrestrial one by day, the latter most probably associated with salamander refugia). in these cases, perhaps the differential temporal and spatial distribution may help these salamanders to avoid predation by terrestrial and diurnal snake predators. of the six species of salamanders recognized in venezuela, however, only bolitoglossa borburata trapido, 1942, seems to be eminently arboreal (manzanilla et al., 1996; barrios-amorós and fuentes, 1999; schargel et al., 2002; lotzkat, 2007). all other congeners present similar behavior to the one exhibited by b. guaramacalensis, although little is known about other aspects of their natural history. this voucher constitutes the first known instance of predation of bolitoglossa by snakes in venezuela. predation on these vertebrates could likely be occasional in the diet of some terrestrial snakes, but it may be more frequent due to land conversion of natural habitats by antropic effect (agricultural sowing and/or pasture for sowing, deforestation of the forest cover for logging with different purposes, use of not controlled fires, etc.). salamander may then be exposed to more open habitats. according to the level of change, the same could drastically affect the prey-predator dynamics (e.g., several examined specimens of b. guaramacalensis were found in open places during the day, near roads, increasing the likelihood of predation). a recent study on anuran predation considers liophis as prey-specific predators even though, in general, the genus might be considered generalist and/or opportunist, due to its amplitude of prey types (toledo et al., 2006). acknowledgements we are grateful to juan elías garcía for his assistance in the field. to luis felipe toledo and pietro battiston for providing pertinent literature. carlos navas commented on a preliminary version of the manuscript. fig. 2. a living individual of bolitoglossa guaramacalensis. 174 l.f. esqueda, m. natera-mumaw and e. la marca references barrios-amorós, c.l., fuentes, o.r. (1999): bolitoglossa spongai. una nueva especie de salamandra (caudata: plethodontidae) de los andes venezolanos, con comentarios sobre el género en venezuela. acta científic. venez. 19: 9-19. boos, h.e.a (2001): the snakes of trinidad and tobago. texas a&m press, college station. chippaux, j.p. (1986): les serpentes de la guayane francaise. faune tropicale xxvii, parís, francia. cunha, o.r., nascimento, f.p. (1993): ofidios da amazônia. as cobras da regiâo leste do pará. bol. mus. paraense emilio goeldi, ser. zool. 9: 1-191. dixon, j.r. (1989): a key and checklist to the neotropical snake genus liophis with country list and maps. smithsonian herp. inform. serv. 79: 1-28. dixon, j.r., tipton, b.l. (2003): liophis miliaris intermedius (henle and ehrl, 1991) is actually liophis reginae (serpentes: colubridae). j. herpetol. 37: 191. duellman, w.e. (1978): the biology of an equatorial herpetofauna in amazonian ecuador. university of kansas. misc. publ. mus. nat. hist. 65: 1-352. esqueda, l.f., natera, m, la marca, e., ilija-fistar, m. (2007): nueva especie de serpiente (reptilia: colubridae: liophis) de un bosque tropical relictual en el estado barinas, venezuela. herpetotropicos 2: 95-103. garcía-paris, m., debam, s.m. (1995): a novel antipredator mechanism in salamanders: rollings escape in hydromantes platycephalus. j. herpetol. 29: 149-151. greenbaum, e. (2004): a new species of bolitoglossa (amphibia: caudata: plethodontidae) from montane forest in guatemala and el salvador. j. herpetol. 38: 411-421. lotzkat, s. (2007): taxonomie und zoogeographie der herpetofauna des nirgua-massivs, venezuela. unpubl. diploma thesis, department of biological sciences, johann wolfang goethe-univesität frankfurt am main, hesse, germany. manzanilla, j., fernandez-badillo, a., la marca, e., bisbal, f. (1995): fauna del parque nacional henri pittier, venezuela: composición y distribución de los anfibios. acta científic. venez. 46: 294-302. martins, m., oliveira, m.e. (1999): natural history of the snakes in forest of the manaus region, central amazonia, brazil. herpetol. nat. hist. 6 (1998): 78-150. michaud, e.j., dixon, j.r. (1989): prey items of 20 species of the neotropical colubrid snake genus liophis. herpetol. rev. 20: 39-41. parra-olea, g., garcía-parís, m., wake, d.b. (2004): molecular diversification of salamanders of the tropical american genus bolitoglossa (caudata: plethodontidae) and its evolutionary and biogeographical implications. biol. j. linn. soc. 81: 325-346. roze, j. (1966): la taxonomía y zoogeografía de los ofidios en venezuela. universidad central de venezuela. ediciones de la biblioteca. caracas, venezuela. savage, j.m. (2002): the amphibians and reptiles of costa rica: a herpetofauna between two continents, between two seas. the university of chicago press. chicago and london. schargel, w., garcía-pérez, j.e., smith, e.n. (2002): a new species of bolitoglossa (caudata: plethodontidae) from the cordillera de mérida, venezuela. pro. biol. soc. washington 115: 534-542. 175salamander predation by a liophis toledo, l.f., ribeiro, r.s., haddadi, c.f.b. (2006): anurans as prey: an exploratory analysis and size relationships between predator and their prey. j. zool. lond. 271: 170-177. whitemann, h.h., wissinger, s.h. (1991): differences in the antipredator behavior of three pletodontid salamanders to snake attack. j. herpetol. 25: 352-355. material examined liophis reginae zweifeli. road potreritos de cendé-miquimú, municipio carache, trujillo state (ulabg 2765); vigirima, sector el guapo, parque nacional san esteban, municipio guacara, carabobo state (ulabg 4139); la roncona, municipio chiguará, mérida state (ulabg 3772). liophis epinephalus opisthotaenius. road from la grita until crossroads bailadores-pregonero, near hotel la montaña, municipio jáuregui, táchira state (ulabg 2148); la hechicera, municipio libertador, mérida state (ulabg 6699). bolitoglossa guaramacalensis. ulabg 2774. trujillo state, municipio carache, on road potreritos de cendé-miquimú, hacienda el rincón, 2275 m a.s.l. acta herpetologica 18(1): 11-22, 2023 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-11995 age estimation and body size of the parsley frog, pelodytes caucasicus boulenger, 1896 from lake borçka karagöl, turkey cantekin dursun*, serkan gül, nurhayat özdemir department of biology, faculty of arts and sciences, recep tayyip erdogan university, rize, turkey *corresponding author. email: cantekin.dursun@erdogan.edu.tr submitted on: 2021, 31st august; revised on: 2022, 8th april; accepted on: 2022, 13th november editor: raoul manenti abstract. in this study, we described age structure, body size, body mass and the relationships among these parameters for a population of p. caucasicus from lake borçka karagöl, artvin, turkey. the mean svl with standard error was 45.87 mm ± 0.55 (range: 39.98-50.28 mm) and the mean weight with standard error 8.81 g ± 0.39 (range: 6.10-11.47 g) in females whereas 48.16 mm ± 0.45 (range: 43.64-54.78 mm) and 11.32 ± 0.25 (range: 9.56-14.80 g) in males, respectively. we found a significant male-biased difference reflecting sexual dimorphism and statistically significant positive relationships between these variables. according to the results, the age ranged between 2-5 years in females and 2-6 years in males. the mean age distributions significantly differed between the sexes (females: 3.28 years ± 0.19; males: 3.94 years ± 0.20). the mean ages and maximal ages were found identical to the previously reported results from turkey, but the mean ages were higher than in georgian populations. von bertalanffy growth models demonstrated similar curves, and the growth rate was faster up to 3 years in both sexes. to conclude, this study was the first to determine age structure and growth patterns in borçka karagöl population and weight data for p. caucasicus was presented for the first time in the literature. keywords. amphibia, skeletochronology, weight, von bertalanffy, growth rate. introduction the determination of individual age is essential for studies like demographic and life history, including developmental biology and population dynamics of a species. this provides the researchers basic ecological data related to population structure such as sexual maturity time, the age structure, growth rate, and life span. moreover, this is a parameter essential to infer the life history traits of a species and to compare it to other species (de buffrénil et al., 2021; ma et al., 2022). in this context, skeletochronology is an effective and reliable method for estimating the growth and age of many amphibian species as the growth of amphibians is not independent of environmental conditions (guarino et al., 2019; üzüm et al., 2020). in addition, the method can be also applied to animals such as mammals (nacarino-meneses et al., 2016), lizards (beşer et al., 2019), turtles (guarino et al., 2020), and even fossils (de buffrénil et al., 2021). skeletochronology calculates the lines involving the formation of calcium carbonate, called “annual rings” or “growth markers” in bone tissues (castanet, 1994; ma et al., 2022). for this, the diaphyseal region of their long bones, which show weaker vascularity, provides the best result for calculating the age of individuals (castanet et al., 1993). rozenblut and ogielska (2005) showed that lines of arrested growth (lags) are most complete in the middle part of the phalangeal diaphysis in european water frogs and thus pointed out that the middle part of the long bone is optimal for age studies. moreover, the skeletochronology helps to calculate the lifespan of the population in amphibians, explain the sexual size dimorphism, and 12 cantekin dursun, serkan gül, nurhayat özdemir reveal the differences between the sexes in terms of age and size. also, the knowledge based on the skeletochronological studies tends to show population dynamics (peng et al., 2021). pelodytes caucasicus boulenger, 1896, the caucasian parsley frog, is a native species of the caucasus fauna. the species is distributed throughout northwest azerbaijan, georgia (southwest and south ossetia), russia (krasnodar district), and turkey (blacksea region) (zazanashvili et al., 2012; litvinchuk and kidov, 2018; çiçek et al., 2019). the species is considered as near threatened because of natural and anthropogenic pressures (ananjeva et al., 2009; iskanderov, 2009; kaya et al. 2009), so it is recommended that public campaigns should be conducted to raise the awareness for this endemic relict species in the border of georgia and turkey (tarkhnishvili and kaya, 2009). from this aspect, it is important to reveal the population dynamics of p. caucasicus in a new population based on skeletochronology. although the age structure of p. caucasicus was reported by two studies in georgia (gokhelasvili and tarkhnisvili, 1994; chubinishvili et al., 1995), there is only single study in the border of turkey (erişmiş et al., 2009). given the importance of skeletochronological information to better understand the population dynamics, ecological and evolutional processes, we aimed to present the age structure of the borçka karagöl population for the first time and compare the results with the previous studies. we also provided weight data for the first time in this species, as well as assessed the relationship between this trait and svl for age and growth rate. material and methods fieldwork we sampled 50 specimens (18 ♀♀, 32 ♂♂) during the breeding season (2013) from lake borçka karagöl territory, in the vicinity of artvin, turkey (fig. 1). the collected frogs were anesthetized with ms222. snout‐ vent length (svl) was measured at the nearest 0.01 mm using a digital calliper and the weight was noted using the nearest 0.01 g an electronic balance for each sample. to count growth lines, the longest toe of the left hindlimb was clipped and preserved in 70% ethanol. after the sampling procedure, all frogs were released at the site of capture. sex determination was possible due to the presence of secondary sexual characters, such as nuptial pads on the forearm, and presence of vocal sacs in males. the standard skeletochronology procedure (castanet and smirina, 1990) was applied to calculate the number of the lines of arrested growth (lags). after removing soft tissues, the phalanges were washed in tap water, then decalcified in 5% nitric acid for 2 hours. lastly, the samples were washed in distilled water overnight to ensure the removal of nitric acid. the 18 μm cross-sections were obtained from the diaphyseal part of each phalanx using a cryostat (thermo shandon cryotome, germany) and stained with ehrlich haematoxylin approximately for 15 minutes. the stained cross-sections were treated with glycerol to control under a light microscope. the images of selected sections were acquired using olympus bx51 microscope with an integrated camera to estimate the precise age of each specimen. the number of lags was counted by two researchers (s. gül and n. özdemir). the distance between two adjacent lags is a well-known indicator demonstrating individual growth in a given year (kleinenberg and smirina, 1969; kurnaz et al. 2018). following previous studies (özdemir et al., 2012; üzüm et al., 2014), the endosteal resorption was evaluated by comparing the diameters of eroded marrow cavities with the diameters of non-eroded marrow cavities in sections from the youngest specimens. statistical analyses to summarize data and present basic features of the measurements we calculated descriptive statistics using the psych package (revelle, 2019). the normality assumption of the variables was checked using the kolmogorovsmirnov test. since the variables followed a normal distribution (p > 0.05), we used parametric tests in downstream analyses. the homogeneity of variances was compared using levene’s test in the car (fox and weisberg, 2019) package. we utilized the student t-test to compare the mean differences of the variables between males and fig. 1. lake borçka karagöl and surrounding region. 13age structure and body size of pelodytes caucasicus females. thereafter, pearson’s product-moment correlation test was used to estimate the relationships among svl, weight, and age. the relationship of svl and weight was analysed using a linear regression model. additionally, we used an ancova test to explore the patterns of svl and weight between sexes, with age as the covariate. thereafter, posthoc tests were applied using the emmeans package (lenth, 2021) under bonferroni correction (estimated marginal means: aka least-square means or adjusted means). we estimated growth curve models under the typical von bertalanffy’s equation modified by beverton and holt (1957): lt=l∞{1-exp[-k(t-t0)]} where lt is the expected or average length at the time (or age) t, l∞ is the asymptotic average length, k is the so-called brody growth rate coefficient and t0 is a modelling artifact that is said to represent the time or age when the average length was zero. to estimate parameter values and run the analyses fsa (ogle et al., 2021), fsadata (ogle, 2019), fsasim (ogle, 2020) and nlstools (baty et al., 2015) packages were used following the guide “fishr vignette” prepared by derek ogle (2013). to visualize growth curve, we also added a hypothetical individual to the dataset by the reference of erişmiş et al. (2009) under the presented parameters: svl0 at metamorphosis is fixed to mean 20.15 and t0 (age at metamorphosis) is 0.3 year. statistical analyses were carried out using the stats package. data visualization was performed using ggplot2 (wickham, 2016), ggpubr (kassambara, 2020), and ggally (schloerke et al., 2021) packages. all analyses were run in r programming language (r core team, 2020). results we successfully aged 50 individuals using skeletochronology technique. ages ranged between 2-5 years in females, and 2-6 years in males. descriptive statistics of both sexes and age groups are presented in table 1. the variances were found homogenous for svl (f1,48 = 0.2926, p = 0.5911), weight (f1,48 = 1.5006, p = 0.2266) and age (f1,48 = 1.9470, p = 0.1693). according to the student t-test results, statistically significant differences were found between sexes in all variables (fig. 2). males were significantly heavier (t = 5.6509, df = 48, p < 0.001) and larger (t = 3.1545, df = 48, p < 0.01) than females. the mean age was also found male-biased (t = 2.2053, df = 48, p < 0.05). a significantly positive correlation was found between svl and age, svl and weight for both sexes, but the correlation between weight and age was significant only in males. the correlation coefficients and p-values are presented in fig. 3. the constructed linear regression model following the table 1. the summary table of descriptive statistics based on the dataset. variable females males n mean ± se min max n mean ± se min max all specimens svl (mm) 18 45.87 ± 0.55 39.98 50.28 32 48.16 ± 0.45 43.64 54.78 weight (g) 18 8.81 ± 0.39 6.10 11.47 32 11.32 ± 0.25 9.56 14.80 age (years) 18 3.28 ± 0.19 2.00 5.00 32 3.94 ± 0.20 2.00 6.00 2 years specimens svl (mm) 3 43.54 ± 1.83 39.98 46.09 3 44.58 ± 0.49 43.64 45.26 weight (g) 3 7.22 ± 0.55 6.18 8.05 3 10.38 ± 0.22 10.11 10.81 3 years specimens svl (mm) 8 46.07 ± 0.71 43.42 49.26 9 47.18 ± 0.59 44.27 49.72 weight (g) 8 9.55 ± 0.62 6.10 11.47 9 11.12 ± 0.37 10.05 13.28 4 years specimens svl (mm) 6 46.02 ± 0.51 44.20 48.00 9 47.81 ± 0.60 45.08 51.12 weight (g) 6 8.44 ± 0.53 6.70 10.05 9 10.72 ± 0.41 9.56 13.63 5 years specimens svl (mm) 1 50.28 50.28 50.28 9 49.37 ± 0.47 47.13 51.95 weight (g) 1 9.97 9.97 9.97 9 11.68 ± 0.38 10.48 13.90 6 years specimens svl (mm) 2 54.08 ± 0.71 53.37 54.78 weight (g) 2 14.65 ± 0.15 14.50 14.80 14 cantekin dursun, serkan gül, nurhayat özdemir correlations also presented significant equations between svl and weight (males: f1,30 = 42.54, r2 = 0.59, p < 0.001; females: f1,16 = 11.23, r2 = 0.41, p < 0.01). according to the ancova results, the effect of age on the intersexual differences was found significant in svl (f1,47 = 39.8215, p < 0.001) and weight (f1,47 = 8.6144, p < 0.01). post-hoc test indicated that the mean svl score, with age as a covariate, was significantly different between sexes (males: 47.8 ± 0.33; females: 46.6 ± 0.44; p < 0.05). additionally, a statistically significant difference was found in terms of the weight between both sex with the age covariate (males: 11.2 ± 0.25; females: 9.06 ± 0.34; p < 0.001). growth curves estimated by von bertalanffy’s model fit adequately described the relationship between age and svl, and the curves indicated similar shapes in both sexes (fig. 4). the final models were found statistically significant for all parameters (p < 0.01). according to the constructed models, the estimated asymptotic svl was not higher than maximum recorded svl values (males: 54.78 mm; females: 50.28 mm). the growth parameters were presented in table 2. discussion the sexual size dimorphism (ssd) is an observable characteristic in animals, and it is known that the direction of size dimorphism in most of the amphibians is female biased (kupfer, 2007). shine (1979) noted that females are larger than males in 61% of urodeles and 90% of anurans. the female biased ssd is generally explained by the fecundity advantage hypothesis (shine, 1989; andersson, 1994) when the selection is supporting large females due to the larger energy storage capacity for reproduction and more offspring production. however, a male biased ssd is relevant to the dominance in contests of strength, the extent of endurance, mate choice and higher sperm competition success in animal kingdom (hudson and fu, 2013). the male-biased ssd corresponding to 10% amphibian species is especially associated with territoriality behaviours and male-male competitions (nali et al., 2014). the family pelodytidae is including five different species from the single genus pelodytes distributing in western europe especially in the iberian peninsula, caucasia, and north-eastern turkey (amphibiaweb, 2022). the previous studies have reported the female biased sexual size dimorphism in pelodtytes species inhabiting in iberia (talavera, 1990; escoriza, 2017). for instance, esteban et al. (2004) noted the larger average body length in females (43.31 mm) than males (36.32) mm in a northern spain population of pelodytes punctatus species. diaz-rodrigez et al. (2017) have described the presence of four valid species inhabiting along western europe by integrating molecular and morphological data. regardfig. 2. the boxplots are representing the differences between the sexes (a: svl, b: weight, c: age). the associated p-values were shown on the relevant plots. 15age structure and body size of pelodytes caucasicus ing their morphological measurements, all the species (p. punctatus, p. hespericus, p. ibericus and p. atlanticus) were characterized by larger average body length in females. however, our results revealed that unlike iberian species, p. caucasicus males have a larger and heavier body than females. our results are also consistent with the findings presented in previous studies. for example, erişmiş et al. (2009) have comprehensively assessed the age structure and growth in pelodytes caucasicus from uzungöl, turkey and. the mean svl of adult males with sd were reported as 47.16 ± 2.87 mm (n = 44; range 41.4852.58 mm), and significantly smaller in females (45.79 ± 2.29 mm; n = 31; range 40.28-50.62 mm). arıkan et al. (2007) noted the range of svl in sexually mature males between 45.06-52.08 mm, and 46.70-49.62 mm in mature females in uzungöl population. yildirimhan et al. (2009) also recorded the mean svl 50.6 ± 3 mm (range: 43-57 mm) in the population of çaykara, trabzon including 47 males, 7 females. erişmiş et al. (2009) emphasized that the bigger size of older males in their study may deviate the results linked to ssd, but the size differences could also be derived from the biotic or abiotic selective pressures in poikilotherm animals. they validated their findings based on the former studies conducted in climatically different regions in caucasia (gokhelashvili and tarkhnishvili, 1994; chubinishvili et al., 1995) and they suggested male biased ssd is the species characteristic of p. caucasicus. the common ssd pattern in anura is generally female biased (monnet and cherry, 2002). recently, pincheria-donoso et al. (2021) have investigated the ssd of amphibian species in global scale. as a result, they observed 90.8% female biased ssd 7.5% male-biased ssd fig. 3. the matrix is demonstrating the correlation coefficients, scatterplots, and density plots of binary variables. corr values indicate the correlation coefficients (r). the significance level of correlation coefficients was represented with asterisk (*, p < 0.05; **, p < 0.01; ***, p < 0.001). 16 cantekin dursun, serkan gül, nurhayat özdemir and 1.7% monomorphic in anurans. however, han and fu (2013) determined male biased sdd in 19 different anuran families distributing in six distinct continents and along tropical and temperate habitats. moreover, constrained habitats and microhabitats can affect the female size and fecundity, and it can be resulted with more similar body size of both sexes as observed in hylid frogs (silva et al., 2020). from this aspect, it can be suggested that the directional difference of ssd between the iberian species and caucasian p. caucasicus may cause due to different ecological preferences in response to their habitats. the iberian peninsula which has complex orographic structure is surrounded by atlantic ocean and mediterranean sea. the mediterranean coast and surrounding areas show dry and warm/hot summers, and mild and wet winters. the atlantic coasts are characterized by oceanic type of climate, milder but humid winters and cooler/wetter summers, without large temperature variations. lastly, the inner areas which is represented by continental climate type have hot and dry summers, fig. 4. graphical visualization of von bertalanffy’s growth curves and parameter optimization histograms. dotted lines are representing 95% confidence intervals of fitted curves (a: females; b: males). table 2. the constructed final models of von bertalanffy’s growth curves and associated parameters estimated parameters l∞ ci k ci t0 females 47.32 45.93-49.77 0.98 0.48-1.47 -0.56 males 50.38 49.16-52.49 0.73 0.58-0.91 -0.70 17age structure and body size of pelodytes caucasicus and cold and humid winters with large-scale precipitation, but also semi-arid areas extremely low precipitation and very hot temperatures especially in summer season (carvalho et al., 2021) which are corresponding to the distribution of iberian pelodytes taxa (see diaz rodrigez et al., 2017). however, caucasus ecoregion has quietly high mean annual rainfall in the southwestern part over 2000 mm in the coastal area of the black sea. the mean annual temperature in the south caucasus part of the black sea coast around 15 centigrade degree (zazanashvili, 2009). besides, the distribution of p. caucasicus species is restricted to the humid subtropics in caucasia and turkey with colchic vegetation type (tarkhnishvili, 1996; iskanderov, 2009; beşir and gül, 2019). gül (2014) also noted that the species prefers wet and warm microhabitats in turkey. additionally, the amount of precipitation is known as one of the most important factors constructing the distribution of p. caucasicus (lukina and koneva, 1996; litvinchuk and kidov, 2018). pincheria-donoso et al. (2021) proposed that the underlying impact of geographical variation in climatic pressures can shape largescale patterns of ssd in synergy with natural and sexual selection such as intensification of fecundity selection to shorten breeding season in anurans. they also implied that the different selection forms can shaped by macroecological factors climate, geographical gradients and temperature seasonality which are triggering the evolution of life-history traits associated with fecundity. therefore, we suggested that the ecological preferences of p. caucasicus are potential reason causing male-biased ssd comparing to the representatives of iberian peninsula. radojićić et al. (2002) also revealed similar ssd pattern in the bombina species. they noted that bombina bombina showed male-biased ssd while the larger body size of females was observed in b. variegata subspecies, and the differences were associated with reproductive behaviours and possible ecological differences. this pattern can also be observed in pelobates syriacus (bülbül et al., 2020 and references therein), rana arvalis populations (glandt and jehle, 2008) and rana nigrovittata which has similar ecological needs with p. caucasicus such as streams in shaded forest environments (khonsue et al., 2000). on the other hand, the male-male competition was reported in pelodytes species (pargana, 2003; marquez et al., 2004 and unreported mating ball of p. caucasicus observed by s. gül). therefore, it should be also taken into consideration that sexual selection which maximize the fitness in reproductive traits may be an alternative force to describe the ssd pattern in our study as reported in different anuran species e.g., paa spinosa (gen-yu et al., 2010) and hypsiboas atlanticus (camurugi and juncá, 2013). ssd can be driven by life-history traits, so the accuracy of the age determination is critical to estimate these characteristics. the post-metamorphic terrestrial growth is a major part of total growth (over 90%) in amphibians (werner, 1986) and the variation in terrestrial growth rates and age at maturity is playing an important role in the intersexual adult size variability (marangoni et al., 2012). furthermore, the age determination of amphibians yields crucial information on the demographic features such as size at sexual maturity growth, longevity, and growth rate (duellman and trueb, 1994; otero et al., 2017; baraquet et al., 2021). in this study, we found that the age is ranged between 2-6 years. the mean age is 3.28 years in females and 3.94 years in males. our constructed von bertalanffy’s growth curves adequately fitted age/svl relationship. the models demonstrated similar curve shape for both sexes, but the growth coefficient was higher in females. previously, gokhelasvili and tarkhnisvili (1994) studied the age distribution of the reproductive population of p. caucasicus in borjomi canyon during two consecutive years (1992-1993). according to their results, the mean ages were 2.96 and 2.74 for males, 2.74 and 3 for females, respectively. they also reported the dominancy of young specimens in the reproductive portion of the populations and a very-high annual mortality rate index (0.78-0.83). the males reached maximum of 6 years, and 4 years in females, which is in accordance with what gokhelasvili and tarkhnisvili (1994) reported. in the following year, chubinishvili et al. (1995) studied certain aspects of the population ecology of p. caucasicus. they reported sexual maturity between 2-3 years. contrary to their findings, the mean age is found approximately one year above the one measured in the borçka karagöl population. erişmiş et al. (2009) reported the mean age of males and females 3.61 ± 0.9 years and 3.03 ± 0.7 years, respectively and the age structure difference is statistically significant (p < 0.05). the oldest male was 5 years while the female was 4 years. the sexual maturity is reached at two years of age. we also obtained approximate values from the mean svl and age (see table 1). the differences observed in svl and age between sexes were also statistically significant. given the identical results for uzungöl and borçka karagöl populations, we can assume that there is a separation between georgian and turkish populations related to age structure. erişmiş et al (2009) also calculated a survival rate at 0.78 in males and 0.76 in females, and these figures can be taken as a reference to explain the high mortality rate noted by gokhelasvili and tarkhnisvili (1994). based on the literature, the maximum age reported in the genus pelodytes is 10 years for females and 8 years for males in pelodytes punctatus species (esteban et al., 2004). additionally, the mean 18 cantekin dursun, serkan gül, nurhayat özdemir age (burgos: 1.71 years ± 1.41 years, valencia: 3.82 years ± 1.22 years) and mean svl (burgos: 34.36 mm ± 2.22 mm, valencia: 36.41 mm ± 2.50 mm) of males in the study of esteban et al. (2002) were lower than our mean values. the authors noted that males were aged between 1-6 years old, and the age structure was highly skewed corresponding 50% of the sample of males being 1 year old. this situation can cause uncertainties when age/svl relationships in the genus pelodtyes. in this study, we also did not age any individual in 1 year old from lake karagöl population. however, it is known that the low average age and high proportion of younger individuals might be associated with rapid decrease of local population of the species such as rana porosa (misawa et al., 2002) and esteban et al. (2002) pointed that this pattern is more relevant to conservation status due to anthropogenic pressures in the burgos population. according to the growth curves estimation of erişmiş et al. (2009), asymptotic svl and growth coefficient were very similar between the sexes (males: svlmax: 53.42 mm ± 1.01 mm; k = 0.42 ± 0.03; females: svlmax = 52.04 mm ± 0.75 mm; k = 0.38 ± 0.01). in both sexes, the estimated asymptotic svl was slightly higher than the maximum svl record and the age-specific growth rate under the 3 years reported faster than higher ages. on the contrary, the estimated svlmax values were lower, but the estimated k values were higher in our constructed growth model. from this aspect, we think that the more bell-shaped curve of our models is likely due to lack of the data from one-year individuals, and this may affect parameter fitting. the second reason is likely the preferred formula equation differences. the common point of the models is a remarkable decrease of growth rate after 3 years following sexual maturity in both sexes. data on body mass is limited in amphibians, but santini et al. (2018) emphasized that weight data can be used to assess ecological and evolutionary processes such as dispersal distance, reproduction, population abundance and energy intake and it was also highly correlated with svl in various anuran families (bufonidae, hylidae, myobatrachidae, ranidae). moreover, they said that rapid body mass rise is associated with svl rise for terrestrial and semi-aquatic species because allometric relationships between length and mass vary in amphibian species based relevant to the different habitat preferences. lastly, they suggested the geometric similarity hypothesis (hill, 1950) is fitting these assumptions better because if two organisms are geometrically similar their linear dimensions can be made equal by multiplying those of one of them by a constant. in this study, we contributed to the literature by first recorded weight data of p. caucasicus species. in the genus pelodytes the available weight data was presented by esteban et al. (2002) for male p. punctatus specimens (burgos: 2.36 g ± 0.28 g, valencia: 3.71 g ± 0.58 g). contrary to the mean weight in our male specimens, spanish populations have lower values in parallel with mean svl and age differences. the linear regression model and ancova results also indicated the allometric relationship between svl and weight, and the weight differed both between sexes and among age groups. habitat preference of p. caucasicus are terrestrial and freshwater systems (kaya et al., 2009), thus our findings are suggesting the geometric similarity hypothesis and habitat preference effect proposed by santini et al. (2018) to describe the relationship between svl and weight. in addition, xiong et al. (2020) noted that age is affecting the intersexual difference on the weight for the shangcheng stout salamander pachyhynobius shangchengensis. from this aspect, the synchronized rise of svl with aging may contribute to the intersexual difference associated with weight in p. caucasicus species. camurugi and juncá (2013) said that malebiased sexual size dimorphism corresponding to length and weight is unusual pattern and among frogs and even if it is generally described by male-male competition, the reasons are not fully understood yet. they also described the male-biased ssd as synapomorphyc characteristic in hypsiboas punctatus species group. monnet and cherry (2002) indicated that bufo achalensis, rana cascadae, r. nigrovittata species showed male-biased ssd because males were older sex as we determined that the mean age of p. caucasicus males. this pattern is causing due to delayed maturity which is determinant factor when larger body is contributing to mating success and males are not accelerating the growth to breed earlier than females. tarkhnishvili (1993) said that the lower fecundity, smaller eggs, the volume of clutch and smaller body are the differences in spawning mood associated with female body size, and they are causing to rapid maturation for the species including pelodytes caucasicus as a strategy to shorten the period between generations and to increase the number of adult individuals. therefore, we assume that the male biases ssd observed in weight data may also occur in response to the life-history and reproductive traits. to sum up, we determined age structure and growth patterns in borçka karagöl population. future studies can comprehensively investigate the relationship between these variables and ecological conditions, life-history traits, and reproductive characteristics. acknowledgements the study was done with the permission of recep tayyip erdogan university local ethics committee for 19age structure and body size of pelodytes caucasicus animal experiments (2018/1). we also thank dr. tuğba ergül kalaycı for assistance during the laboratory experiments. references afsar, m., afsar, b., ayaz, d., çiçek, k., tok, c. 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(2012): ecoregion conservation plan for the caucasus, revised and updated edition. caucasus biodiversity council, tbilisi. threats of the emerging pathogen batrachochytrium salamandrivorans (bsal) to italian wild salamander populations lorenzo dondero1, giorgia allaria1, giacomo rosa1, andrea costa1, gentile francesco ficetola2, roberto cogoni3, elena grasselli1, sebastiano salvidio1,* age estimation and body size of the parsley frog, pelodytes caucasicus boulenger, 1896 from lake borçka karagöl, turkey cantekin dursun*, serkan gül, nurhayat özdemir patterns of acoustic phenology in an anuran assemblage of the yungas andean forests of argentina martín boullhesen1,2,*, marcos vaira1, rubén marcos barquez2, mauricio sebastián akmentins1 diet and trophic niche overlap of four syntopic species of physalaemus (anura: leptodactylidae) in southern brazil renata k. farina1, camila f. moser2, stefano scali3, mateus de oliveira4, patrícia witt5, alexandro marques tozetti1,* screening of ophidiomyces ophidiicola in the free-ranging snake community annually harvested for the popular ritual of san domenico e dei serpari (cocullo, aq, italy) daniele marini1,2, ernesto filippi3,*, gianpaolo montinaro4, francesco c. origgi5,6 assessment of fall season habitat and coverboard use by snakes in a restored tallgrass prairie community carter dollen1,2, tracy j. coleman1,2, travis r. robbins1,2,* revisiting the polyploidy in the genus odontophrynus (anura: odontophrynidae) andré luis de souza, mayara aparecida das neves micalichen, roger alves da rocha, rafael bueno noleto* acta herpetologica 2006 1 description of a new species of the genus adenomera (amphibia, anura, leptodactylidae) from french guiana 1acta herpetologica 1: 1-14, 2006 description of a new species of the genus adenomera (amphibia, anura, leptodactylidae) from french guiana renaud boistel 1, jean-christophe de massary 2 and ariadne angulo 3,4 1 université paris sud. centre scientifique d’orsay, laboratoire des mécanismes de communication animale, nam cnrs umr 8620. bâtiment 446, 91405 orsay cedex, france. 2 4, rue des tilleuls, 95170 deuil-la-barre, france 3 conservación internacional carrera 13 # 71-41 bogotá, colombia 4 departamento de herpetología, museo de historia natural de san marcos, apartado 14-0434, lima, perú abstract. we describe a new species of the genus adenomera from french guiana. collecting conditions, details about the localities and microhabitats are given. the advertisement call was recorded and is herein analyzed and described. morphological and bioacoustic comparisons are drawn with other species of the genus. the new species is readily distinguished from other taxa by its distinctive coloration pattern, the occurrence of dorsolateral ridges and inguinal glands, and by the longer duration of the notes of its advertisement call. the taxonomy of adenomera is evaluated with regard to current available knowledge. key words. adenomera heyeri n. sp., leptodactylidae, french guiana, morphology, advertisement call introduction the neotropical anuran genus adenomera steindachner, 1867 was resurrected by heyer (1974) for the leptodactylus marmoratus group; this author recognized five species: adenomera andreae (müller, 1923), a. bokermanni (heyer, 1973), a. hylaedactyla (cope, 1868), a. marmorata steindachner, 1867 and a. martinezi (bokermann, 1956). in 1975, heyer (1975) described a sixth species, a. lutzi heyer, 1975. recently, a seventh species, a. diptyx (boettger, 1885), has been resurrected (de la riva, 1996) and a. araucaria kwet and angulo, 2002 is described from southern brazil (kwet and angulo, 2002). to date, eight nominal species are recognized (frost, 2004) , although there is evidence to support a much higher species diversity in this group of frogs (angulo, 2004). a. andreae and a. hylaedactyla, the two nominal species with widespread distributions throughout the amazon basin, have been previously reported in french guiana (heyer, 1973). their calls are frequently heard in rainforest and associated habitats and it is possible to distinguish these 2 r. boistel et alii two species on the basis of their advertisement calls (rodríguez and duellman, 1994). the advertisement call presents a good potential as a means to resolve the systematics of this genus (heyer, 1984; angulo et al, 2003). three specimens of an unknown species of adenomera were found at the field station of saint-eugène, french guiana (fig. 1). moreover, four additional specimens of this new species have been collected at the field station of nouragues, in the same country. herein we describe this new species. the advertisement calls of five nominal species of the genus adenomera, a. hylaedactyla, a. andreae, a. araucaria and a. marmorata have been previously described (heyer, 1973; straughan and heyer, 1976; heyer et al., 1990; márquez et al., 1995; zimmerman and bogart, 1984; kwet and angulo, 2002). in addition to the description of the new spefig. 1. map of french guiana with the two localities known for adenomera heyeri. the asterisk indicates the type-locality (saint-eugène field station), the circle the station of nouragues. 3new species of adenomera cies, we take this opportunity to further analyze of the known vocalizations in order to find discriminating parameters for the genus. materials and methods morphological analysis specimens used in the description of the new species are deposited at the museum national d’histoire naturelle (mnhn), laboratoire de zoologie (reptiles et amphibiens), paris. other museum acronyms follow leviton and gibbs (1988). specimens were fixed in 10 % formalin and preserved in 70 % ethyl alcohol. all measurements were taken after fixation. abbreviations used in the measurements of the seven specimens are el (horizontal eye length), en (distance from anterior edge of eye to nostril), fll (forelimb length, from elbow to base of outer tubercle), ftl (fourth toe length), hl (head length), hw (head width), in (internarial distance), iue (minimum distance between upper eyelids), svl (snout-vent length), td (tympanum diameter), tl (tibia length), tfl (third finger length), and uew (maximum width of upper eyelid). measurements were taken either with a caliper to the nearest 0.1 mm or with a stereomicroscope equipped with an ocular micrometer under magnifications of 6x, 12x, 25x and 50x. we also counted the number of vomerine teeth (vt), both at the left (l) and right (r) side. an x-ray was taken of one paratype in ventral view to examine osteological characters. data acquisition and sound analysis the origin of the data and the recording conditions are summarized in appendix 1. analog signals were digitized using a 16-bit digigram pccard acquisition card at a sampling frequency of 16000 hz. additionally, we sampled at 32000 hz in order to observe harmonic structure. recordings were analyzed with analytical software syntana (aubin, 1994). fast fourier transforms (ffts, window size = 4096 data points, ∆ƒ = 120 hz) were calculated. for the spectrum analysis, we used an fft (window size = 2048 data points, ∆ƒ = 8 hz) at the middle of the note for each selected call of the recorded series. the duration of signals and silences was measured on the oscillogram of all signals. their amplitude envelope and instantaneous frequency function were calculated by means of the hilbert transform (see mbu-nyamsi et al., 1994). we used an envelope (the envelope was digitally filtered using ffts window size = 4096 data points, overlapping 97-100 %, band pass = 0-250 hz) of the signal for the analysis of the structure of am (amplitude modulation) and the instantaneous frequency for analysing the fm (frequency modulation) parameters of the signal. because this method provides the instantaneous frequency, it allows us to obtain fine details not detected by a classic fft as, for instance, the frequency sweeps. the terminology follows boistel and sueur (1997), beeman (1998), gerhardt (1998), and hartmann (1998). we took the following measurements: dsq (duration of sequences), dn (duration of notes), ds (duration of silences), di (duration of intra-signal variation of amplitude), f0 (fundamental frequency), 2f0 (twice the fundamental frequency), fm (frequency modulation) and am (amplitude modulation). we used the nr (note rate, which is the number of notes for each unit of time) and rhythm (ratio of silence and sound duration), according to aubin and brémond (1983). for adenomera araucaria, we used the data on advertisement calls provided by kwet and angulo (2002). 4 r. boistel et alii results adenomera heyeri n. sp. (figs. 2-5) diagnosis the new species is distinguished from all other species by its advertisement call and the following combination of characters: (1) two pairs of dorsolateral folds present; (2) smooth skin on lower surface of foot or with a few small white tubercles; (3) throat and belly of males yellow; (4) tarsal fold present and slightly marked. adenomera heyeri differs from a. martinezi in lacking four longitudinal rows of symmetrically arranged dark spots on its dorsal surface. this news species is distinguished from a. araucaria by its larger size and by its advertisement call. adenomera bokermanni and a. lutzi have a profusion of white-tipped tubercles on the lower surface of the tarsus and the sole of foot, whereas those parts are smooth, or with some very scant, small tubercles in a. heyeri. the vocal slit is elongate, slightly oblique to the jaw or parallel to the jaw in a. marmorata, whereas a. heyeri has small vocal slits, present just behind the angle of the jaw. adenomera heyeri has two pairs of slightly discontinuous parallel folds starting behind the eyes and running toward the posterior part of the back; these are absent in a. andreae; in addition, the signal duration is about 2.5 times longer in a. heyeri than it is in a. andreae (table 1). adenomera heyeri is distinct from a. hylaedactyla by having the head as wide as long, its snout is, from above, nearly rounded versus subovoid, pulses are absent and note duration is longer (tables 1 and 2). holotype. mnhn 1999.8331 (figs. 2, 4), adult male collected at the mnhn field station of saint-eugène (4°51’n; 53°3’w, 65 m elevation), 23 km by air from the newly built petit saut dam, courcibo river, principal tributary of the sinnamary river, french guiana, leg. renaud boistel, 24 april 1998. paratypes. mnhn 1997.2273, adult female, and mnhn 1998.322, adult male, from the type locality, leg jean-christophe de massary, 2 april 1997 and 13 april 1998; mnhn 1999.8301, adult male, and mnhn 1999.8302-8304, three juveniles, from the station of nouragues (4°5’ n, 52°41’ w, 110 m elevation), 8 km n of saut pararé, arataye river, french guiana, leg. renaud boistel, may 1999. description of holotype snout from above nearly rounded, in profile obtuse; canthus rostralis straight, indistinct; loreal region acute, head wider than long; nostrils anterolateral, closer to snout tip than to eyes. interorbital space flat, twice as wide as upper eyelid, slightly longer than the internarial distance; tympanum distinct, its maximum diameter about 2/3 of eye diameter; pupil horizontal and elliptic; vomerine ridges present, in short transverse series l9/r8, separated by a distance smaller than the length of one tooth row; maxillary teeth present; tongue rounded, not emarginated on anterior edge; the elongated vocal slits are present, 5new species of adenomera supratympanic fold distinct, although weakly or barely developed, ranging from eye to angle of jaw. arms short with robust forearms, lacking forearm tubercules (as found in pseudopaludicola); forearms as long as hands; fingers elongate, thin, without dermal fringe or webbing; tips of fingers rounded, flattened, without grooves but with dorso-terminal ridges at the level of digit articulation; finger lengths in increasing order: iv ≤ ii = i < iii; subarticular tubercles well developed, oblong or ovoid; inner and outer metacarpal tubercles large, prominent, outer larger than inner, shape of inner oblong, that of outer long; palmar tubercles absent; supernumerary tubercles distinct, small, rounded; sole of hand table 1. advertisement call measurements for adenomera heyeri, a. andreae, a. hylaedactyla and a. marmorata. data for a. heyeri originate from the type specimens (mnhn 1999.8331). asterisks indicate that the extraction of measurements was not possible in two (long distance recordings) specimen. abbreviations used are: n = number of specimens, dsq = duration of sequences, dn = duration of notes, ds = duration of silences, di = duration of intra-signal variation of amplitude, nr = note rate in number of notes per second (nn/s), f0 = fundamental frequency, 2f0 = twice the fundamental frequency, fm = frequency modulation, am = amplitude modulation. a.heyeri a. andreae a. hylaedactyla a. marmorata n 1 3 1 1 dsq (s) 30.2 39.6 3.7 17.2 dn (ms) 154.2 ± 13.5 (136.87-184.5) n = 17 66.5 ± 10.9 (44.5-85.7) n = 45 49.7 ± 2.5 (45.0-52.7) n = 16 45.9 ± 2.9 (42.2-55.6) n = 16 ds (ms) 1722.4 ± 418.3 (1198.8-2446.1) n = 16 874.8 ± 149.0 (643.3 1264.9) n = 42 193.7 ± 21.5 (162.7-223.8) n = 15 1096.3 ± 154.6 (877.1-1444.7) n = 15 di (ms) 6.8 ± 2.1 (3.9-16.9) n = 351 4.4 ± 0.8 (2.1 7.9) n = 336 8.1 ± 2.3 (5.2 15.4) n = 96 / nr (nn/s) 0.56 1.13 4.32 0.93 rhythm 10.51 11.74 3.66 22.38 f0 (hz) 1856 ± 31 (1815-1878) n = 17 2438 ± 157 (2316-2692) n = 45 2107 ± 19 (2091-2128) n = 16 4790 ± 11 (4782-4808) n = 16 2f0 (hz) 3657 ± 61 (3568-3844) n = 17 4871 ± 359 (4557-5493) n = 45 4341 ± 32 (4282-4407) n = 16 / period of sinusoidal fm (ms) 13.1 ± 2.6 (9.2-25.2) n = 162 11.5 ± 2.7* (6.9-22.6) n = 64 8.1 ± 2.3 (5.2-15.4) n = 96 / linear fm (hz) 399 ± 55 (300-487) n =17 629 ± 105 (451-789) n =45 453 ±2 3 (401 484) n =15 572 ± 66 (463 689) n =16 am (hz) / 210 ± 24 (155.5-278) n =45 124 ± 16 (93.5-144) n =16 / 6 r. boistel et alii smooth. shank longer than thigh and shorter than distance from base of internal metatarsal tubercle to tip of toe iv; relative length of toes, in increasing order: i ≤ v < ii < iii < iv. toe tips bulbous, spatulate, broader than toe width just behind tips; dorso-terminal toe ridges at level of tarsal articulation not developed into fringes; subarticular tubercles well developed, ovoid; outer metatarsal tubercle round, smaller than ovoid, flattened inner tubercle; tarsal fold slightly marked; no metatarsal fold; outer tarsus absent; supernumertable 2. principal parameters characterizing the advertisement call of four adenomera species (see materials and methods for the parameter definitions). a. heyeri a. andreae a. araucaria a. hylaedactyla a. marmorata number of harmonics 2 to 6 1 to 7 3 to 6 2 0 dominant frequency 2f0 2f0 2f0 2f0 f0 fm sinusoidal & linear sinusoidal & linear linear sinusoidal & linear linear nb of oscillations in sinusoidal fm 9,5 4 0 6,2 0 am no yes yes yes no pulses 0 0 0 4 0 tempo slow average slow-average fast average rhythm medium medium strong weak strong fig. 2. overview of the type specimen in its natural habitat; saint-eugène field station, may 1998 (photo r. boistel). 7new species of adenomera ary tubercles on sole of foot hardly distinct, small, rounded. top of head and eyelids, forearms and all the ventral surface smooth. two pairs of slightly discontinuous parallel folds starting behind the eyes and running toward the posterior part of the dorsum. occipital region, dorsum, and dorsal surfaces of forearms, thighs and shanks covered with pustules. upon fixation and preservation both pustules and dermal folds are considerably less marked or lost altogether. two small lumbar glands present. it is possible to observe a small supralabial gland at each side of the jaw. coloration in life this species can be easily identified by its coloration. the back is overall brown but dark markings occur: a mid-dorsal line begins at the snout, is slightly enlarged around the interorbital area, continues along the back tapering, and disappears around the sacral region. two dark stripes (one on either side) with unclear limits, start from the nostrils and continue dorsolaterally following the dermal folds, stopping before the two small, well defined, black lumbar glands. one short dark, oblique lateral band begins at the dermal fold and reaches the inguinal region; one dark stripe starts at the supratympanic fold and disappears on the lower part of the flanks. the background color is light brown. the sides of the head are dark; on each side, a whitish mark starts from under the posterior part of the eye to the insertion of the forelimb; this mark includes the supralabial gland. the lower lip is bordered by dark brown. the gular region is slightly yellowish with small brown spotted marks. the belly and the ventral side of the limbs are uniformly yellowish in life but whitish in preserved specimens. the soles of the feet and the palms of the hands are brown. the iris is bronze with black reticulations; the palpebral membrane is translucent and is bordered in its upper part by a black streak. the forelimbs and hindlimbs are light brown dorsally with, respectively, two and three cross bars. upon preservation, the coloration is overall conserved. measurements of holotype (in mm) snout vent length 22.5, head length 9.6, head width 9.2, interocular distance 2.5, shank length 10.8, length of fourth toe 5.2, eye diameter 2.3, eye-nostril distance 2.0, internarial distance 2.2. fig. 3. left foot (1) and left hand (2) of adenomera heyeri (paratype mnhn 1999.8301). (1) (2) 8 r. boistel et alii variation measurements of seven specimens are given in table 3. according to data currently available, adenomera heyeri can reach a maximum svl of about 26 mm in males and the unique female of our sample measures 23.1 mm. the coloration is similar among all specimens. the gular region is a dull white in the female and is slightly yellowish with small brown spotted marks in the male. the belly and the ventral portions of the upper limbs are uniformly whitish to yellowish in preserved specimens. the dermal folds of some fixed specimens are less marked, and sometimes totally disappear after fixation. juveniles show a slightly different coloration at the level of the upper lip, which is white with brown bands. overall, morphology and coloration are quite uniform. secondary sexual characters males have a vocal apparatus, consisting of a single vocal sac and two small vocal slits present just behind the angle of the jaws; their upper lip is more developed than in females; there are no nuptial pads. the snout from above is nearly rounded to rounded, in profile it is acuminate in our single female and obtuse in males. advertisement call all call data herein refer to the holotype. the advertisement call of adenomera heyeri can be detected by the human ear at over 50-60 metres. temporal features of advertable 3. size measurements on the seven known specimens of adenomera heyeri. the holotype (mnhn 1999.8331) bears an asterisk (see materials and methods for abbreviations of the measurements). 1997.2273 1998.0322 1999.8301 1999.8302 1999.8303 1999.8304 1999.8331* sex f m m j j j m svl 23.1 23.6 25.8 10.0 10.3 10.8 22.5 hl 9.1 9.7 11.0 4.2 4.2 4.6 9.6 hw 10.7 9.8 10.3 4.8 4.8 4.5 9.2 in 2.1 2.7 2.5 1.2 1.1 1.3 2.2 en 1.9 1.9 2.0 0.9 1.0 0.9 2.0 el 2.8 2.6 3.0 1.3 1.3 1.7 2.3 uew 1.5 1.7 1.7 0.9 0.9 1.0 1.5 iue 2.8 2.8 3.3 1.4 1.4 1.7 2.5 td 1.5 1.6 2.0 0.4 0.4 0.5 1.5 fll 5.5 5.9 5.9 2.3 2.2 2.4 5.4 tfl 5.2 5.4 5.4 1.7 5.2 tl 11.2 10.8 10.8 4.5 4.4 5.2 10.8 ftl 10.5 11.3 11.0 4.1 11.2 vt l10/r9 l8/r9 l10/r9 l5/r5 l5/r5 l5/r5 l9/r8 9new species of adenomera a b so lu te a m p li tu d e in s. f r e q . f r e q u e n c y a m p li tu d e a b e d c c b 0 500 1000 1500 2000 2500 3000 3500 1500 2500 3500 4500 5500 6500 2f0 f0 frequency : hz 3f0 13 ms time : msec. 135 ms 12 ms b e b d b a b c 0 db 12 db fig. 4. graphic representation of the advertisement call of adenomera heyeri (holotype mnhn 1999.8331): (a) spectrum, the two peaks correspond to the fundamental frequency (f0) and the first harmonic (2f0); (b) instantaneous frequency by hilbert transform; (c) sonogram with a palette of 12 colors of depicting different intensities, one color represents 3 db, white color is a minimum intensity and gray color on top is maximum intensity; (d) oscillogram; (e) envelope; two periodic variations are detected, 6 and 12 ms. 10 r. boistel et alii tisement calls are given in table 1. the call of a. heyeri is distinct from that of the other three species in all parameters (see tables 1 and 2). the calling sequence is a repetition of identical notes (figs. 4 c, d, e), with each call having an average duration of 154 ms, emitted at a note rate of 0.56 notes/s and a rhythm of 10.51. the duration of silences is about 1722 ms. the durations of notes and silences in a. heyeri are longer than those of the other species examined and its note rate is the slowest; the rhythm is similar to that of a. andreae. the envelope (fig. 4e) shows one periodical pattern of variation in amplitude with a duration of 13 ms. with regard to spectral features (figs. 4 a, b, c), a fast fft indicates that, within the serial harmonic, the average fundamental frequency (f0) is 1856 hz (figs. 4 a, c); this frequency is lower than in the other species dealt with here (table 2). the dominant frequency is located at 3657 hz (2f0). in all other species of adenomera the dominant frequency is 2f0, with the exception of a. marmorata, in which the dominant frequency is f0. the other peaks near the f0 and 2f0 are understood as an effect of frequency modulation (fm). notes were found to have a series of six distinguishable harmonics (table 2). the sonogram (fig. 4 c) and analysis by using the hilbert transform, which gives the instantaneous frequency (fig. 4 b), show a frequency modulation (fm). the fm is linear upward, rising from 3422 to 3821, and with a sinusoidal oscillation repeated 9.5 times in one note; it has an w shaped profile in the dominant frequency (fig. 4 b), differing from a. araucaria and a. marmorata by the absence of a sinusoidal fm. the discontinuities of the frequency curve coincide with the variation of instantaneous amplitude, which has a periodicity of 12 ms (figs. 4 b, e). the call of a. heyeri differs from the calls of a. andreae and a. hylaedactyla by the absence of am. distribution and ecology to date, adenomera heyeri is only known from its type locality and a nearby site (see holotype and paratypes) (fig. 1). few data are available about the species’ ecology. it seems to have a nocturnal activity in terrestrial environments. according to observations made by the first author, males start their calling activity during dusk in the rainy season (april-may). specimens have been found both in moist low elevations and on the summit of small hills (about 120 m elevation), with or without rocks, in drier conditions. though one of us (jcdm) spent about 17 months in the field (i.e. 3 dry seasons and 3 rainy seasons) using pitfall traps continuously, this species was never found during the dry season. moreover, it is interesting to note that all specimens were collected in april or may only. these facts may probably reflect an increase in activity at the onset of the rainy season, possibly for reproductive purposes. in every case, a. heyeri seems to have a “secretive” life, and is therefore difficult to find. etymology this species is dedicated to w. ronald heyer for his important contributions to leptodactylid frog studies of south america and in particular the genus adenomera. 11new species of adenomera discussion systematic issues in adenomera have been difficult to resolve. morphologically, most species are cryptic and heyer (1984) suggested that topotypic advertisement calls could be used to resolve their taxonomy. advertisement call characters in anurans are highly speciesspecific prezygotic isolation mechanisms and as such are excellent indicators of species identity, as accumulated evidence suggests (e.g. heyer et al., 1996). in the case of cryptic species, once it is possible to associate a frog with a particular call, it is also possible to search for correlates between this call and distinctive morphological characters. adenomera heyeri fig. 5. x-ray picture of adenomera heyeri, male paratype mnhn 1998.322, with details about the phalanges of both the right hand and the left foot. note t-shaped tips of phalanges. 12 r. boistel et alii was sufficiently divergent morphologically to immediately suspect it was a new species, and the distinctiveness of its advertisement call corroborated this. however, in other species of adenomera, the morphology is not conspicuously distinct, as in the case of a. andreae and a. hylaedactyla. angulo et al. (2003), using acoustic characters, found that these two adenomera represent four taxa at a locality in amazonian peru. heyer (1974) examined the relationships of frogs of the marmoratus group of leptodactylus, and redefined adenomera as a valid genus with a number of characters distinguishing it from other leptodactylids. although superficially similar to pseudopaludicola, the new species has more attributes in common with adenomera than with any other member of the leptodactylines [characters such as t-shaped terminal phalanges (fig. 5), sternal style and presence of tarsal fold], which is the reason for its placement in this genus. however, there are still a number of characters which are unknown in the new species (e.g. characters of a myological nature, detailed osteological data or reproductive mode). moreover, one character state observed in some specimens of a. heyeri is different from what was previously known for adenomera, i.e. sole of foot smooth. we will not rule out that future analyses of genera may reveal a placement of a. heyeri basal to or out of adenomera. nevertheless, according to the best of our knowledge, it is currently best placed within this genus. acknowledgments rb thanks all the following sponsors: r. berdaa of “société digidigram”; j.-c. roché of the “editions sittelle”; the “société cp france”; the “société fuji-lab”; the “edition nashvert”. the studies led in french guiana by jcdm were supported by “electricité de france (convention edf/mnhn gp 7531); support for aa is acknowledged through operating nserc grant #4946 to g.k. morris. for data on adenomera hylaedactyla, aa thanks inrena (instituto nacional de recursos naturales), peru, for granting research and collecting permits. many thanks to newton college and d. bruggers for providing accommodation and food while conducting research at sachavacayoc centre in the former tambopata candamo reserved zone, southeastern peru. rb and jcdm are very grateful to g. dubost for permission to work at the mnhn saint-eugène field station, french guiana, and the staff of the hydreco laboratory and j.-c. and e. baloup for always assisting us when necessary. also many thanks to p. charles-dominique, p. gaucher, v. hequet and s. lochon, for facilitating the last mission of rb (may 1999) in french guiana. many thanks to the ministère de l’environnement and the préfecture de guyane for delivering collecting permits (respectively 307/96, 98/112/aut and 1021/1d/1b/env) used for the specimens mentioned in this paper. finally, all the authors thank t. aubin, a. dubois, w.r. heyer, i. ineich, j. lescure, and g.k morris for helpful comments in the revision of earlier versions of this paper. references angulo, a. (2004): the evolution of the acoustic communication system in members of the genus adenomera (anura: leptodactylidae): a comparative approach. doctoral thesis, university of toronto, toronto, 232 pp. angulo, a., cocroft, r. b., reichle, s. (2003): species identity in the genus adenomera (anura: leptodactylidae) in southeastern peru. herpetologica 59: 490-504. 13new species of adenomera aubin, t. (1994): syntana: a software for the synthesis and analysis of animal sounds. bioacoustics 6: 80-81. aubin, t., brémond, j. c. (1983): the process of species-specific song recognition in skylark alauda arvensis. an experimental study by means of synthesis. z. tierpsychol. 61: 141-152. beeman, k. (1998): digital signal analysis, editing, and synthesis. in: animal acoustic communication, p. 59-103. hopp, s.l., owren, m.j., evans, c.s., eds. new york, barcelona, budapest, hongkong, london, milan, paris, santa clara, singapore, tokyo: springer-verlag. boistel, r., sueur, j. (1997): comportement sonore de la femelle de platymantis vitiensis (amphibia, anura) en l’absence du mâle. compte rendu de l’académie des sciences, sciences de la vie 320: 933-941. de la riva, i. (1996): the specific name of adenomera (anura: leptodactylidae) in the paraguay river basin. journal of herpetology 30: 56-58. de la riva, i., köhler, j., lötters, s., reichle, s. (2000): ten years of research on bolivian amphibians: updated checklist, distribution, taxonomic problems, literature and iconography. revista española de herpetología 14: 19-64. frost, d. r. (2004): amphibian species of the world: an online reference. version 3.0 (22 august, 2004). electronic database accessible at http://research.amnh.org/herpetol ogy/amphibia/index.php. american museum of natural history, new york, usa. gerhardt, h.c. (1998): acoustic signals of animals: recording, field measurements, analysis and description. in: animal acoustic communication, p. 1-23. hopp, s.l., owren, m.j., evans, c.s., eds. berlin, heidelberg, new york, barcelona, budapest, hongkong, london, milan, paris, santa clara, singapore, tokyo: springer-verlag. hartmann, w.m. (1998): signals sound, and sensation. new york, berlin, heidelberg, barcelona, budapest, hongkong, london, milan, paris, santa clara, singapore, tokyo: springer-verlag. heyer, w.r. (1973): systematics of the marmoratus group of the frog genus leptodactylus (amphibia, leptodactylidae). los angeles county of natural history contributions in science 251: 1-50. heyer, w.r. (1974): relationships of the marmoratus species group (amphibia, leptodactylidae) within the subfamily leptodactylinae. los angeles county of natural history contributions in science 253: 1-46. heyer, w.r. (1975): adenomera lutzi (amphibia: leptodactylidae), a new species of frog from guyana. proceedings of the biological society of washington 88: 315-318. heyer, w.r. (1984): the systematic status of adenomera griseigularis henle, with comments on systematic problems in the genus adenomera (amphibia: leptodactylidae). amphibia-reptilia 5: 97-100. heyer, w.r., rand, a.s., da cruz, c.a.g., peixoto, o.l., nelson, c.e. (1990): frogs of boracéia. arquivos de zoologia 31: 231-410. heyer, w.r., garcía-lopez, j.m., cardoso, a.j. (1996): advertisement call variation in the leptodactylus mystaceus species complex (amphibia: leptodactylidae) with a description of a new sibling species. amphibia-reptilia 17: 7-31. kwet, a., a. angulo (2002): a new species of adenomera (anura, leptodactylidae) from the araucaria forest of rio grande do sul (brazil), with comments on the systematic status of southern populations of the genus. alytes 20: 28-43. 14 r. boistel et alii leviton, a.e., gibbs, r.h. jr. (1988): standards in herpetology and ichthyology. standard symbolic codes for institutional resource in herpetology and ichthyology. supplement no. 1: additions and corrections. copeia 1988: 280-282. márquez, r., de la riva, i., bosch, j. (1995): advertisement calls of bolivian leptodactylidae (amphibia, anura). journal of zoology, london 237: 313-336. mbu-nyamsi, r.g., aubin, t., brémond, j.c. (1994): on the extraction of some time dependent parameters of an acoustic signal by means of the analytic signal concept. its application to the animal sound study. bioacoustics 5: 187-203. rodríguez, l.o., duellman, w.e. (1994): guide to the frogs of the iquitos region, amazonian peru. the university of kansas natural history museum, special publication 22, i-v + 1-80. straughan, i.r., heyer, w.r. (1976): a functional analysis of the mating calls of the neotropical frog genera of the leptodactylus complex (amphibia, leptodactylidae). papéis avulsos de zoologia 29: 221-245. zimmerman, b.l., bogart, j.p. (1984): vocalizations of primary forest frog species in the central amazon. acta amazonica 14: 473-519. appendix 1. summary of the origin and condition of recordings for the different frog species considered. a. andreae a. hylaedactyla a. marmorata a. heyeri data taken by r. boistel a. angulo w.r. heyer r. boistel date 20 april 1998 (18:50-19:20 h) 26 january 1999 (19:11 h) 12 december 1976 24 april 1998 (18:50 h) locality saint-eugène (french guiana) tambopata national reserve (peru) boracéia (brazil) saint eugène (french guiana) tape recorder dat aiwa hds1000 walkman sony d6c uher cr 134 dat aiwa hds1000 microphone b&k 4053 (omni) sony ecm307 (stereo) uher m517 b&k 4053 (omni) distance (cm) 40 and 200-300 15-20 ? 20 and 250-300 specimen released aa 9945 rom 40105 unvouchered holotype mnhn 1999.8331 svl 16 22.7 ? 22.5 t°c 25.5 25.5 19 25 report of a bite by the south american colubrid snake philodryas olfersii latirostris (squamata: colubridae) maría elisa peichoto1, jorge abel céspedez2, juan antonio pascual3 1departamento de bioquímica, facultad de ciencias exactas y naturales y agrimensura (facena), universidad nacional del nordeste (unne), av. libertad 5470, cp. 3400, campus universitario corrientes, argentina 2departamento de biología, facultad de ciencias exactas y naturales y agrimensura (facena), universidad nacional del nordeste (unne), av. libertad 5470, cp. 3400, campus universitario corrientes, argentina. corresponding author. e-mail: cespedez@exa.unne.edu.ar, cespedez2003@yahoo. com.ar 3departamento de clínica médica, hospital “ángela i. de llano”, av. ayacucho 3288, cp. 3400, corrientes, argentina abstract. following the bite of philodryas olfersii latirostris cope, 1862, a 29-year-old male herpetologist developed localized and burning pain, and minimal bleeding from the puncture marks of posterior maxillary teeth, which subsided rapidly. the victim developed no other local signs or symptoms. after few days the victim presented persistent severe rotatory dizziness, nausea, and vomiting. on examination his hearing was normal. neurological exam was otherwise normal. the patient had acute vertiginous symptoms but had no associated neurological signs. computed tomography did not show abnormality. a diagnosis of labyrinthine syndrome was made. it was treated conservatively, and the patient recovered uneventfully. it was assumed as an effect of ophitoxemia. this case may be regarded as an unusual presentation of systemic envenoming following a human bite by philodryas olfersii latirostris. keywords. green snake, philodryas olfersii latirostris, snakebite, argentina, vertigo, labyrinthine syndrome. introduction most snakebites in south america are caused by pit vipers (bothrops, crotalus, lachesis spp.) and coral snakes (micrurus spp.); less serious accidents are caused by colubrids (prado-franceschi and hyslop, 2002). however, some snakes of the family colubridae, usually regarded as not venomous, are responsible by serious and even so fatal accidents (santos-costa and di-bernardo, 2000). rear-fanged species (opisthoglyphous) are responacta herpetologica 2(1): 11-15, 2007 12 m.e. peichoto et alii sible for most colubrid envenomations; the principal genera involved being clelia, helicops, liophis, philodryas, tachymenis, and thamnodynastes (prado-franceschi and hyslop, 2002), and also by phalotris (valls-moraes and lema, 1997). philodryas is a genus of rear-fanged colubrid snakes, which is found in south america, from amazonas to patagonia (assakura et al., 1992, 1994). bites by species of philodryas have been reported from argentina (orduna et al., 1994), brazil (martins, 1916; nickerson and henderson, 1976; silva and buononato, 1983-1984; silveira and nishioka, 1992; bucaretchi et al., 1993; nishioka and silveira, 1994; araújo and dos santos, 1997; ribeiro et al., 1999) and chile (schenone et al., 1954; schenone and reyes, 1965). p. olfersii deserves greatest attention because it has already caused serious accidents, with the patients showing edema, pain, ecchymosis and enlarged axillary lymph nodes (martins, 1916; nickerson and henderson, 1976; silva and buononato, 1983-1984; silveira and nishioka, 1992; bucaretchi et al., 1993; orduna et al., 1994; araújo and dos santos, 1997; ribeiro et al., 1999). systemic envenomation is rare, but ribeiro and coworkers (ribeiro et al., 1999) reported one case of envenomation by p. olfersii in a 2-year-old child which resulted in nausea and vomiting. furthermore, orduna and coworkers (orduna et al., 1994) reported an altered prothrombin clotting time in a patient bitten by p. olfersii. moreover, there is at least one record of a fatal accident caused by p. olfersii (salomão and di-bernardo, 1995). however, none of these authors mention the subspecies involved. on the contrary, kuch (1999) reported a case of a bite by the south american colubrid snake p. olfersii latirostris, which was capable of inflicting even systemic human envenomation with only a very quick defensive bite. we report here the case of a 29-year-old male herpetologist bitten by p. olfersii latirostris, a chacoan subspecies, who presented labyrinthine syndrome few days after the bite. this case may be regarded as an unusual presentation of systemic envenoming by this colubrid snake. although it is difficult to prove that the presentation of labyrinthine syndrome and the snakebite are actually associated, this case would have to be taken into account to alert professionals of the health area about the necessity of attending carefully accidents involving colubrid snakes since very little is known about their venoms and their effects on human victims. case report on 18 november 1997 at 18:30 hr, a 29-year-old male herpetologist was bitten by a male of p. olfersii latirostris, with about 80 cm in total length, while capturing the snake in corrientes city, northeastern argentina. the specimen bit him on ventral region of the elbow of his right arm. the snake chewed several times, and it could be withdrawn only after about one minute because it got into the clothes of the victim. both enlarged rear maxillary teeth deeply penetrated the skin. the victim of the present bite had no known allergies and no history of venomous snakebite or antivenom administration. he had only experienced minor local reactions to previous colubrid snakebites. his last tetanus immunization was one year prior. immediately after the bite, there was a burning pain around the bite marks, which subsided by 22:00 hr. minimal bleeding from the puncture marks of posterior maxillary 13bite by philodryas olfersii latirostris teeth was also presented in the beginning, but stopped very soon. the victim developed no other local signs or symptoms. during the following two days, any sign or symptom of envenomation could be detected. but on 21 november 1997, the victim presented a transient dizziness, which lasted about 5 min. on 24 november 1997, the victim presented again dizziness, but this time it had a more persistent course. so, the victim went to a general hospital. on arrival to the emergency department, he was complaining of severe rotatory dizziness, unsteadiness, nausea, and vomiting. after waking up that morning, he noted that the room was spinning, and he had to hold on to keep from falling after getting out of bed. he vomited several times. he was unable to go out unaccompanied. on examination his hearing was normal. neurological exam was otherwise normal. there was no spontaneous or gaze-evoked nystagmus. his prior medical history was unremarkable for vertigo, systemic illness, trauma, hearing loss, or ear infections. he was not taking any medication. he did not have a recent viral illness (e.g., cold, flu). the patient definitely had acute vertiginous symptoms but had no associated neurological signs. as magnetic resonance imaging (mri) was not available, computed tomography (ct) was carried out which showed no abnormality (data not shown). based on the clinical findings and data from ct, a diagnosis of labyrinthine syndrome was made. thus, dimenhydrinate (50 mg every 6 hr) and deflazacort (6 mg every 12 hr) were prescribed. the next day he was still complaining of intense rotatory vertigo. however, nausea and vomits had completely disappeared. at two weeks follow-up he reported improvement in his symptoms, and his recovery was uneventful. discussion some local symptoms described by kuch (1999) for the envenomation caused by p. olfersii latirostris agree with the observations made in this case: burning pain and minimal bleeding. furthermore, both cases agree in its very quick onset of local symptoms, and the fast subsidence of them. however, the present case differs in that edema, enlargement of axillary lymph nodes and ecchymosis were not observed. there is only one report about the observation of vertigo after a philodryas species bite. in the case of philodryas chamissonis envenomation described by schenone and coworkers (1954), the patient suffered from vertigo, mild headache, and fever, besides characteristic local symptoms. acute onset of vertigo is mostly related to peripheral vestibular disorders (magnusson and karlberg, 2002). the distinction between peripheral and central vertigo usually can be made clinically and guides management decisions (swartz and longwell, 2005). in the case of the victim of this work, the central causes of acute prolonged vertigo were excluded by the absence of both cerebral and cerebellar signs/symptoms, and the lack of visible lesions in the computed tomography scan. thus, labyrinthine syndrome was diagnosed. lesion of the labyrinth is one of the main causes of prolonged rotatory vertigo (kerr, 2005). it was assumed as an effect of ophitoxemia. 14 m.e. peichoto et alii the venom of p. olfersii is highly hemorrhagic, has fibrin(ogen)olytic and edemaforming activities; however, it is devoid of coagulant, procoagulant, phospholipase a2, and platelet aggregating enzymes (assakura et al., 1992). it also has a higher proteolytic activity than that of bothrops spp. (salomão et al., 1990). in the present case, both local as well as systemic damages might have been caused by proteolytic venom components. p. olfersii latirostris is capable of inflicting local and systemic human envenomation (kuch, 1999), and this case may be regarded as an unusual presentation of systemic envenoming by this colubrid snake. although it is difficult to prove that the presentation of labyrinthine syndrome and the snakebite are actually associated, this case would have to be taken into account to alert professionals of the health area about the necessity of attending carefully accidents involving colubrid snakes since very little is known about their venoms and their effects on human victims. acknowledgments the authors would like to thank to dr oscar camacho who provided clinical data about the patient. references araújo, m.e., dos santos, a.c. (1997): cases of human envenoming caused by philodryas olfersii and philodryas patagoniensis (serpentes: colubridae). rev. soc. bras. med. trop. 30: 517-519. assakura, m.t., reichl, a.p., mandelbaum, f.r. (1994): isolation and characterization of five fibrin(ogen)olytic enzymes from the venom of philodryas olfersii (green snake). toxicon 32: 819-831. assakura, m.t., salomão, m.g., puorto, g., mandelbaum, f.r. (1992): hemorrhagic, fibrinogenolytic and edema forming activities of the venom of the colubrid snake philodryas olfersii (green snake). toxicon 30: 427-438. bucaretchi, f., vieira, r.j., fermino, c.a., bavaresco, a.p., fonseca, m.r.c.c., douglas, j.l., zambrone, f.a.d. (1993): acidentes por philodryas olfersii: relato de dois casos. in: resumos do iii congresso latino americano de herpetologia, p. 195. campinas, são paulo. kerr, a.g. (2005): assessment of vertigo. ann. acad. medicine, singapore 34: 285-288. kuch, u. (1999): notes on two cases of human envenomation by the south american colubrid snakes philodryas olfersii latirostris cope, 1862 and philodryas chamissonis (wiegmann, 1834) (squamata: serpentes: colubridae). herpetozoa 12: 11-16. magnusson, m., karlberg, m. (2002): peripheral vestibular disorders with acute onset of vertigo. curr. opin. neurol. 15: 5-10. martins, n. (1916): das opistoglyphas brasileiras e o seu veneno. thesis, faculdade de medicina do rio de janeiro, rio de janeiro. 15bite by philodryas olfersii latirostris nickerson, m.a., henderson, r.w. (1976): a case of envenomation by the south american colubrid, philodryas olfersii. herpetologica 32: 197-198. nishioka s. de a., silveira, p.v.p. (1994): philodryas patagoniensis bite and local envenoming. rev. inst. med. trop., são paulo 36: 279-281. orduna, t.a., martino, o.a.l., bernachea, p., maulen, s. (1994): ophidism produced by snakebite of genus philodryas. prensa méd. argentina 81: 636-638. prado-franceschi j., hyslop, s. (2002): south american colubrid envenomations. toxin reviews 21: 117-158. ribeiro, l.a., puorto, g., jorge, m.t. (1999): bites by the colubrid snake philodryas olfersii: a clinical and epidemiological study of 43 cases. toxicon 37: 943-948. salomão, e.l., di-bernardo, m. (1995): philodryas olfersii: uma cobra comum que mata. caso registrado na área da 8ª delegacia regional da saúde. arquivos da sbz 14/15/16: 21. salomão, m.g., puorto, g., furtado, f., sagawa, p. (1990): philodryas olfersii: morphological, histochemical studies of duvernoy’s gland venom extraction. in: resumos do i simpósio da sociedade brasileira de toxinologia: peçonhas e envenenamento no brasil, p. 8. instituto butantan, são paulo. santos-costa, m.c., di-bernardo, m. (2000): human envenomation by an aglyphous colubrid snake, liophis miliaris (linnaeus, 1758). cuad. herpetol. 14: 153-154. schenone, h., bertín, v., mann, g. (1954): a further case of ophidism. boletín chileno de parasitología. 9: 88-89. schenone, h., reyes, h. (1965): animales ponzoñosos de chile. boletín chileno de parasitología. 20: 104-109. silva, m.v., buononato, m.a. (1983-1984): relato clínico de envenenamento humano por philodryas olfersii. mem. inst. butantan 47/48: 121-126. silveira, p.v., nishioka, s. de a. (1992): non-venomous snake bite and snake bite without envenoming in a brazilian teaching hospital. analysis of 91 cases. rev. inst. med. trop., são paulo 34: 499-503. swartz, r., longwell, p. (2005): treatment of vertigo. am. fam. physician 71: 1115-1122. valls-moraes, f., lema, t. de (1997). envenomation by phalotris trilineatus in rio grande do sul state, brazil: a case report. j. venom. anim. toxins 3: 255. a new species of hemidactylus from lake turkana, northern kenya (squamata: gekkonidae) roberto sindaco1, edoardo razzetti2, ugo ziliani3, victor wasonga4, caterina carugati5, mauro fasola5 1c/o museo civico di storia naturale, via san francesco di sales, i-10022 carmagnola (to), italy 2museo di storia naturale, università degli studi di pavia, piazza botta 9, i-27100 pavia, italy. corresponding author. e-mail: razzetti@unipv.it 3platypus s.r.l., via pedroni 13, i-20161, milano, italy 4department of herpetology, national museums of kenya, museum hills road, p.o. box 40658-00100, nairobi, kenya 5dipartimento di biologia animale, università degli studi di pavia, piazza botta 9, i-27100 pavia, italy abstract. a new species of the genus hemidactylus is described on the basis of two specimens (an adult male and an adult female) collected in 2005 in rocky and sandy habitat of the semiarid climatic region on the eastern shore of lake turkana (kenya). it is a medium-sized hemidactylus (svl from 40 to 50 mm) distinguished from all other species by a unique combination of characters. the back is covered by large, trihedral, strongly keeled tubercles, intermixed with a few small, irregular shaped granules, forming 14 quite regular transverse rows from axilla to groin; nostrils contact the rostral, first supralabial, 1 enlarged internasal and 2-3 postnasals; the dorsal half of the rostral scale is divided longitudinally; there are 6 lamellae under the first toe and 10 under the 4th toe; male with 8 precloacal pores; female without pores. the dorsal colour pattern is very distinctive, consisting of four transverse bands, bordered with dark margins. the types are housed in the herpetological collections of the museo di storia naturale of the university of pavia and in the national museums of kenya (nairobi). riassunto. una nuova specie di hemidactylus dal lago turkana, kenya settentrionale (squamata: gekkonidae). si descrive una nuova specie del genere hemidactylus sulla base di due esemplari (un maschio e una femmina adulti) raccolti nel 2005 sulla sponda orientale del lago turkana (kenya) in ambienti caratterizzati da affioramenti rocciosi e sabbiosi nella fascia climatica semi-arida. si tratta di un hemidactylus di taglia media (lunghezza apice del muso-cloaca da 40 a 50 mm), che si distingue dalle altre specie del genere grazie ad una combinazione di caratteri. l’ornamentazione dorsale è caratteristica e consiste in quattro bande trasversali con margini scuri. sul dorso sono presenti grossi tubercoli triedri fortemente carenati, che formano 14 file trasversali dall’inguine alla base degli arti anteriori, inframezzati da granuli di forma irregolare; le narici sono in contatto con la squama rostrale, con la prima sopralabiale, con una grande internasale e con 2-3 postnasali; la squama rostrale è divisa longitudinalmente per metà della sua lunghezza nella parte superiore; sono presenti 6 lamelle sotto il priacta herpetologica 2(1): 37-48, 2007 38 r. sindaco et alii mo dito del piede e dieci sotto il quarto. il maschio mostra 8 pori precloacali mentre la femmina ne è priva. il materiale tipico è depositato presso il museo di storia naturale dell’università degli studi di pavia e il national museums of kenya di nairobi. keywords. hemidactylus, kenya, lake turkana, new species. introduction hemidactylus is one of most specious genera of the family gekkonidae, that has its main centre of speciation in east africa: somalia, kenya, ethiopia, and eritrea host more than 40 species of hemidactylus, most of which are endemic (cf. parker, 1942; loveridge, 1947; lanza, 1983; spawls et al., 2002; brogard, 2005; largen and spawls, 2006). the two specimens described in the present paper, belonging to an unknown species, were collected during the scientific surveys carried out within the project “conservation of biodiversity and community development of east lake turkana” promoted by pavia university for italian cooperation and with the support of the national museums of kenya and kenya wildlife service. materials and methods we conducted four herpetological field surveys, from 2004 to 2005 (total search effort 53 days of searching with a medium of three observer), throughout all eastern lake turkana, marsabit district, northern kenya, from the ethiopian border to south horr, and from the lake shores eastwards to north horr, about 37°e. although rather varied, the turkana region is typically characterized by scattered low shrub temporary vegetation, and acacia trees emerging from lava barelands, interspersed with dense vegetation of commiphora-acacia mellifera shrub bush, it belongs to the sahel-somalic dominion of the saharo-sahelian region (schnell, 1976). the only areas where the density of fauna seems higher are the dry river beds (lagga) that are narrowly bordered by denser vegetation of shrub and trees and thick undercover. a bimodal and unpredictable distribution of rains (from 200 to 300 mm/year) characterize these semi desert regions (schnell, 1976). the collected specimens are deposited in the herpetological collections of the national museums of kenya in nairobi, and the museo di storia naturale of the university of pavia, italy. the new species was compared to all the other hemidactylus species with similar characteristics and especially to the east african ones. we checked the collections of several museums (see appendix 1), the original descriptions, and taxonomic reviews (see references). 39a new hemidactylus from kenya results hemidactylus barbierii sindaco, razzetti and ziliani, sp. nov. (figs 1-5) type material holotype — adult male msnpv-cr849, kenya, marsabit district, lagga daudi (3°53’08”n – 36°19’02”e), about 17 km south of koobi fora not far from the eastern lake turkana shore, u. ziliani, e. razzetti, r. sindaco, c. carugati leg., 7.vii.2005. paratype — adult female nmk-l/3054, kenya, marsabit district, “petrified forest” (3°40’23”n – 36°19’43”e) about 10 km inland of the eastern lake shore at allya bay, u. ziliani, e. razzetti, r. sindaco, c. carugati leg., 6.vii.2005. derivatio nominis — in memory of our friend francesco barbieri (1944-2001), professor of zoology at the university of pavia, herpetologist and a keen naturalist, one of the founders of the “turkana project” (galeotti, 2002). diagnosis a medium sized hemidactylus (male svl = 40 mm, female svl = 50 mm), with a very distinctive dorsal banded pattern. back covered by large, trihedral, strongly keeled fig. 2. ventral view of hemidactylus barbierii sp. n. right: male (holotype, msnpv-cr849; left: female (paratype, nmkl/3054). fig. 1. dorsal view of hemidactylus barbierii sp. n. right: male (holotype, msnpv-cr849); left: female (paratype, nmk-l/3054). 40 r. sindaco et alii tubercles intermixed with a few small, irregular shaped granules, forming 14 quite regular transverse rows from axilla to groin (counted along a paravertebral line); nostril in contact with the rostral, first supralabial, 1 enlarged internasal (post-rostral) and 2-3 postnasals; rostral scale divided longitudinally for half of its length in the superior part; 6 lamellae under the first toe and 10 under the 4th toe; sole without enlarged scales; male with 8 precloacal pores; female without pores. description of the holotype head rather depressed (ratio of head length to the mandibular angle and head depth = 2.3), length 1.4 times its width; snout subacuminate, concave between the nares and the eyes, swollen in front of the eyes, 1.3 times as long (to the anterior margin of the eye) as the distance between the posterior margin of the eye and the anterior margin of the earopening; ear opening elliptical, its major axis subvertical and slightly more than 1/4 of the exposed eye. major diameter of the exposed eye about 1/5 the head length (to the mandibular angle); pupil a vertical slit with lobed margins. rostral subquadrangular, nearly twice wide as high, divided longitudinally for half of its length in the superior part; nostril in contact with the rostral, first supralabial, 1 enlarged internasal (post-rostral) and 3 postnasals; the uppermost nasal very large and in contact with its fellow; 9/10 (right/left) upper (7/7 before the centre of the eye) and 6/6 lower labials; mental large, subtriangular, approximately as long as the anterior chinshields; anterior chin-shields broadly in contact along the median line and with the first lower labial roughly in contact with the second pair of lower labials. a second pair of chin shields slightly smaller than the first pair (maximum length about 0.74 of the first pair of postmentals), touching the second pair of sublabials and 2 rows of paralabials. snout covered by roundish, juxtaposed, flat or slightly convex scales (keeled on the preocular swelling); these scales vary in size, being smaller centrally, larger peripherally; the largest snout scales are those of the loreal region and preorbital swelling as well as the two behind the supranasals. posteriorly the scales grade into small, juxtaposed granules (about 20 in the interorbital region, in the narrower line between the eyes) mixed on the vertex, nape and temporal region with conical or subtrihedral strongly keeled tubercles clearly larger than the nostril and separated by 1-3 granules. trunk covered dorsally by large, subtrihedral, strongly keeled tubercles, arranged in 14 relatively regular longitudinal rows (6 between the hindlimbs), 14 in a straight paravertebral line between axilla and groin separated by small, subimbricate, smooth, heterogeneous scales. ventral scales large, flat, smooth, and imbricate, about 28 in a transverse row at mid-belly, 43 between axilla and femoral pores along a line on the middle of the belly, fig. 3. gular region of the holotype (right) and paratype (left) of hemidactylus barbierii sp. n. 41a new hemidactylus from kenya 5+2/3 in an eye diameter in the middle of the belly, counted longitudinally. head covered ventrally by juxtaposed or subimbricate granules (about 18 in an eye diameter in the middle of the throat, counted longitudinally). trunk rather depressed; 8 (4/4) precloacal pores forming an obtusely angulated, uninterrupted series. antero-dorsal side of forearm, dorsal side of tibia and postero-dorsal side of thigh with large keeled tubercles, those of the forearm smaller than the other ones. digits free, moderately dilated, slightly webbed at the base with free undilated terminal portion clearly projecting beyond the dilated part (that of fourth toe with 8 scales along the dorsal edge); lamellae beneath the toes from first to fifth (undivided+divided+entire apical): 6 (2+3+1), right 9 (2+6+1) / left 8 (1+6+1), right 9 (1+7+1) / 9 (2+6+1), 10 (3+6+1), right 9 (3+5+1) / 8 left (2+5+1); beneath the fingers: 6 (2+3+1), 7 (1+5+1), 7 (1+5+1), 8 (2+5+1), 6 (2+4+1). adpressed hind limb reaches the elbow of adpressed forelimb. testes well developed. tail lost. measurements. snout-vent = 40 mm; tail lost; head length (to the mandibular angle) = 12.2 mm; head width = 8.5 mm, head depth = 5.4 mm; major (horizontal) diameter of the exposed eye = 2.9 mm; axilla to groin = 16.5 mm; forelimb = 12.5 mm; hind limb = 16.0 mm. colouration. a very distinctive dorsal colour pattern of four complex transverse bands (one on the neck and three between anterior and posterior limbs), each with very dark anterior and posterior margins and less dark inner area; a curved dark line, resembling these margins, bordering the rear of the head continued forwards through the eye to the nostril, canthus rostralis more or less distinctly lighter than the upper parts of the snout; limbs almost patternless. life colouration during daytime: see fig. 4. apparently no change of color was observed between night and daytime. in alcohol the ground colour of back is light roseate-greyish, a little bit darker inside the transverse bands; these bands are delimited by dark brown, irregular and discontinuous margins one-tubercle wide. under parts are off-white. description of the paratype head very similar to that of the holotype, although larger and stouter, a little less depressed (head length / head depth = 1.9), and shorter (length / width = 1.2). 2 (instead of 3) postnasals. enlarged upper labials 7/7 (6/6 before the centre of the eye), enlarged infralabials 6/6. arrangement of the mental scale and chin shields as in the holotype. scales on the upper surface of the head similar to the holotype, those on the upper part of snouth larger than in the male. trunk covered dorsally by tubercles as in the male, but arranged in 16 longitudinal rows (6 between the hindlimbs), 14 between axilla and groin. large ventral scales, flat, smooth, and imbricate, about 32-33 in a transverse row at mid-belly, 49 between axilla and the precloacal scales along a line on the middle of the belly, 7 counted longitudinally in an eye diameter in the middle of the belly; about 18 scales in an eye diameter in the middle of the throat. precloacal pores absent. tip of adpressed hind limb reaches the elbow of adpressed forelimb. 42 r. sindaco et alii measurements. snout-vent = 49.5 mm; tail lost; head length (to the mandibular angle) = 13.6 mm; head width = 11.2 mm; head depth = 7.2 mm; major (horizontal) diameter of the exposed eye = 3.3 mm; axilla to groin = 22 mm; forelimb = 16.0 mm; hind limb = 20.0 mm. subdigital lamellae (first to fifth toes): 6 (1+3+2), 8 (1+6+1), 9 (1+6+2), 10 (1+6+3), 8 (1+5+2). lamellae beneath the fingers: 6 (2+3+1), 7 (1+5+1), 7 (1+5+1), 8 (2+5+1), 7 (1+5+1). colouration. as in the holotype, but more heavily patterned, with dorsal bands better defined; a continuous dark line joins the temporal dark lines across the nape; upper parts of the snout with a “λ” shaped dark arrow bordered, on the canthus rostralis, by a narrow dark stripe. life colouration: see fig. 5. habitat both specimens were collected while active on the ground after sunset. the male was found on the sandy ground of a dry lagga (seasonal stream) with riverine vegetation (fig. 6). the female was collected on rocky ground with scattered vegetation (fig. 7). other reptiles found in the same area where the male was collected were: stenodactylus sthenodactylus, hemidactylus ruspolii, agama rueppelli, agama agama lionotus, psammophis sp.; among amphibians only bufo lughensis and tomopterna cf. cryptotis were found. syntopic reptiles in the paratype collecting locality were: hemidactylus cf. angulatus, h. isolepis and agama agama lionotus. comparison with other species hemidactylus barbierii is closely related to some species of the “arid clade” of hemidactylus sensu carranza and arnold (2006). it belongs to the hemidactylus species characterized by the presence of precloacal pores (lacking femoral pores) in males, and enlarged tubercles on the dorsum. the species, which is most similar to h. barbierii, is the very rare h. bavazzanoi lanza, 1978 (occurring in s somalia and ne kenya), having a similar arrangement of dorsal enlarged tubercles. the main differences are the extremely distinctive pattern, and the sequence of mental scales (in particular the second pair of post-mentals not entering the 3rd infralabials in h. barbierii) and the number of subdigital lamellae beneath the fourth toe (10 in h. barbierii instead of 6-9 in h. bavazzanoi). the recently described h. foudaii baha el din 2003 (baha el din 2003, 2006) from se egypt has a rather similar colour pattern, but without nuchal band; the main differences are the much smaller ventral scales (40-44 across midbelly in h. foudaii instead of 2833) and smaller dorsal tubercles (20 across the back and intermixed with medium-sized granular scales, vs. 14-16 in contact among them in h. barbierii). also h. citernii boulenger, 1912 from nw somalia and n juba province, has large trihedral, strongly keeled tubercles on the back narrowly separated by fine granules; this 43a new hemidactylus from kenya fig. 5. life colouration of the paratype of hemidactylus barbierii sp. n. fig. 4. life colouration of the holotype of hemidactylus barbierii sp. n. 44 r. sindaco et alii taxon differs by the short free distal joints of the digits, the small ventral scales, juxtaposed or subimbricate (strongly imbricate in h. barbierii), and different pattern of the back and limbs. hemidactylus fossatii scortecci 1928, a doubtful taxon from eritrea, differs by having enlarged dorsal tubercles perfectly smooth. h. puccionii calabresi, 1927, from central and se somalia, considered as a synonym of “h. turcicus” s.l. by parker (1942), has the first supralabial excluded from nostril, postmentals separated on median line and the second pair of postmentals smaller; the enlarged dorsal tubercles are only feebly keeled (see also scortecci 1931). all the following species differ from h. barbierii by having the dorsal enlarged tubercles smaller and more widely separated by small granular scales; moreover, most of the individuals of these species, apart from h. sinaitus boulenger, 1885, have the supranasals (internasals) separated by one or more granules. h. arnoldi lanza, 1978 (nw somalia) has an enlarged tubercle on the sole of foot, more rows of dorsal tubercles (27 from axilla to groin), higher number (7-10) of lamellae beneath the first toe, and a different colour pattern. h. barodanus boulenger, 1901 (ethiopia and n somalia) is larger (svl up to 72-78 mm); enlarged tubercles may be large and triedral in some specimens, whereas in other fig. 6. habitat of hemidactylus barbierii sp. n. lagga daudi, marsabit district (3°53’08”n – 36°19’02”e), 7.vii.2005. 45a new hemidactylus from kenya are less than half this size and only feebly keeled (parker, 1942); the internasals are separated by a single granule; the tail is strongly depressed. h. granchii lanza, 1978 (central and se somalia) has the first upper labial widely separated by the nostril, and an inconspicuous colour pattern. h. jubensis boulenger, 1895, is a poorly known species and could be a senior synonym of h. barodanus. the back of this species is covered by round or oval feebly keeled tubercles (instead of strongly keeled tubercles). h. macropholis boulenger, 1896 (ethiopia, somalia, n kenya) has a completely different arrangement of chin-shield, larger size (svl up to more than 80 mm in both sexes), and a different colour pattern; according to lanza (1978: tab. 4) in most of specimens the first chin-shields does not enter the first and the second infralabials (62%) and most of specimens (77%) have separated supranasals (internasals), moreover it has an higher number of lamellae beneath the first toe (7-10 instead of six). h. robustus heyden, 1827 (incl. h. parkeri loveridge, 1936) (egypt to somalia and extreme ne kenya), usually has separated supranasals (76% of individuals), and a different colour pattern. h. sinaitus boulenger, 1885 (from sudan to n somalia, and arabia) has smaller and more widely separated dorsal tubercles, lower number of precloacal pores, and a different colour pattern. fig. 7. habitat of hemidactylus barbierii sp. n. petrified forest, eastern lake turkana, marsabit district, kenya (3°40’23”n – 36°19’43”e), 6.vii.2005. 46 r. sindaco et alii h. taylori parker, 1932 (ne somalia) has supranasals separated and a different colour pattern. h. turcicus (linnaeus, 1758) (widespread in the arabian peninsula and the middle east, and along the mediterranean sea shores, south to northern sudan, and probably replaced further south by h. robustus) has supranasals separated in the 90% of specimens, smaller and more widely separated dorsal tubercles, and a different colour pattern. h. yerburii pauciporosus lanza, 1978 from n somalia (the nominate subspecies occurs in arabia) has a different pattern; the ventral scales are smaller, and the supranasals (internasals) are separated in the 78% of specimens. discussion the horn of africa is the main speciation centre for the genus hemidactylus with more than 40 species widespread mostly in somalia and adjoining kenya, eritrea and ethiopia. the arid eastern shores of lake turkana are the western distributional limit for many somali taxa sensu la greca (1990), and the genus hemidactylus forms the richest reptile genus of this area with six species, the ground dwelling h. barbierii, h. isolepis and h. macropholis, the rock or building climbers h. cf. angulatus and h. ruspolii, and the tree climbers h. platycephalus and h. ruspolii. until recently, the herpetofauna of the eastern lake turkana (west of 37°e), excluding mount kulal, included 27 reptiles species (spawls et al., 2002), a figure now increased to 35 (+30%) by the additional information gathered during the herpetological surveys carried for our “turkana project” (ziliani et al. 2006), and by further unpublished data collected by czech herpetologists. thus the discovery of this new species was not entirely unexpected. the results of these surveys have contributed to enhance the knowledge of the biodiversity of northeastern africa. further efforts should be made to enable further investigation aimed to identify the exact distribution of this new species and its occurrence in such remote areas acknowledgments we wish to thank the italian government, ministero affari esteri e cooperazione italiana, and dr. alfredo guillet, for funding the project “conservation of biodiversity and community development of east lake turkana”, in association with kenya wildlife service and national museums of kenya. gratitude to the community of loyangalani, the staff of the sibiloi national park and of the koobi fora research station, for their contribution and understanding during fieldwork. references baha el din, s.m. (2003): a new species of hemidactylus from egypt. afr. j. herpetol. 52: 39-47. 47a new hemidactylus from kenya baha el din, s.m. (2006): a guide to the reptiles and amphibians of egypt. american university in cairo press, cairo, egypt. boulenger, g.a. (1885): catalogue of the lizards in the british museum (natural history). volume i. geckonidæ, eublepharidæ, uroplatidæ, pygopodidæ, agamidæ. british museum (natural history), london. boulenger, g.a. (1895): esplorazione del giuba e dei suoi affluenti compiuta dal cap. v. bottego durante gli anni 1892-93 sotto gli auspici della società geografica italiana. ann. mus. civ. st. nat., genova 15: 9-17 + pl. i-iv. boulenger, g.a. (1896): a list of the reptiles and batrachians collected by the late prince eugenio ruspoli in somaliland and gallaland in 1893. ann. mus. civ. st. nat. genova 17: 5-16. boulenger, g.a. (1901): a list of the batrachians and reptiles obtained by dr. donaldson smith in somaliland in 1899. proc. zool. soc. london 1901: 47-49 + 1 pl. boulenger, g.a. (1912): list of the reptiles and batrachians. missione per la frontiera italo-etiopica sotto il comando del capitano carlo citerni. ann. mus. civ. st. nat. genova 5: 329-332. brogard, j. (2005): inventaire zoogéographique des reptiles. zoogeographical checklist of reptiles. volume 1, région afrotropicale et région paléartique. afrotropical and paleartic realms. dominique editions, condé sur noireau. calabresi, e. (1927): anfibi e rettili raccolti nella somalia dai proff. g. stefanini e n. puccioni (gennaio-luglio 1924). atti soc. ital. sci. nat. mus. civ. st. nat. milano 67: 1460 + 1 pl. carranza, s., arnold, e.n. (2006): systematics, biogeography and evolution of hemidactylus geckos (reptilia: gekkonidae) elucidated using mitochondrial dna sequences. mol. phylog. evol. 38: 531-545. galeotti, p. (2002). francesco barbieri (voghera, 4 aprile 1944 — pavia, 22 settembre 2001). riv. ital. ornitol. 71: 139-144. la greca, m. (1990): considerazioni sul popolamento animale dell’africa orientale. biogeographia 14 (1988): 541-578. lanza, b. (1978): on some new or interesting east african amphibians and reptiles. monit. zool. ital., n.s. suppl. 14: 229-297. lanza, b. (1983): a list of the somali amphibians and reptiles. monit. zool. ital. 8: 193247. largen, m.j., spawls, s. (2006): lizards of ethiopia (reptilia sauria): an annotated checklist, bibliography, gazetteer and identification key. trop. zool. 19: 21-109. loveridge, a. (1936): new geckos of the genus hemidactylus from zanzibar and manda island. proc. biol. soc. washington 49: 59-62. loveridge, a. (1947): revision of the african lizards of the family gekkonidae. bull. mus. comp. zool. 98: 1-469. parker, h. (1932): two collections of reptiles and amphibians from british somaliland. proc. zool. soc. london 1932: 213-219 + figs 1-10. parker, h. (1942): the lizards of british somaliland. bull. mus. comp. zool. 91: 1-101. schnell, r. (1976): introduction à la phytogéographie des pays tropicaux. volume 3. la flore et la végétation de l’afrique tropicale. 1re partie. bordas, paris. scortecci, g. (1928): una nuova specie di hemidactylus dell’eritrea: hemidactylus fossatii. atti soc. ital. sci. nat. mus. civ. st. nat. milano 67: 33-36 + 1 pl. 48 r. sindaco et alii scortecci, g. (1931): secondo contributo allo studio dei rettili della somalia italiana. atti soc. ital. sci. nat. mus. civ. st. nat. milano 70: 127-152 + 1 pl. spawls, s., howell, k., drewes, r., ashe, j. (2002): a field guide to the reptiles of east africa. academic press, san diego. ziliani, u., sindaco, r., razzetti, e., wasonga, v., modrý, d., necas, p., carugati, c., fasola m. (2006): the herpetofauna of the eastern side of the lake turkana (northern kenya). in: riassunti del 6° congresso nazionale della societas herpetologica italica (roma, 27 settembre – 1 ottobre 2006), p. 196-197. bologna, m.a., capula, m., carpaneto, g.m., luiselli, l., marangoni, c., venchi a., eds stilografica, roma. appendix 1 — materials examined for comparison. abbreviations: mzuf = museo zoologico dell’università di firenze; mcc = museo civico di storia naturale di carmagnola; mzut = museo zoologico dell’università di torino (now in the museo regionale di scienze naturali, torino); msnpv = museo di storia naturale dell’università di pavia; nmk = national museums of kenya (nairobi). h. bavazzanoi. mzuf 21886 (holotypus). h. cf. angulatus. mcc r0570, mcc r1035, msnpv cr647-648, msnpv cr633-636, mzut-r2662, mzut-r3119, mzut-r2393, nmk l/2115/1-4, l/1284/1-2, l/1612, l/1878/1-2, l/1879/1-4, l/2132/1-2, l/2282. h. flaviviridis. mzut-r2660, mzut-r2699. h. frenatus. mzut-r1695, mzut-r2663, mzut-r2669, mzut-r2676, mzut-r2692, mzut-r3071, mzut-r3288. h. granchii. mzuf 21189 (holotypus), mzuf 21114-18 (paratypi). h. mabouia. mcc r803, mcc r805, mcc r902, mcc r0903(1-2), mzut-r2689. h. macropholis. mcc r1224(1-2); nmk l/2587/1-4. h. parkeri. mzuf (many specimens). h. platycephalus. mcc r1225, msnpv cr648. h. ruspolii. msnpv cr680-681, msnpv cr654, msnpv cr639, msnpv cr670, msnpv cr846. h. sinaitus. mzut-r2657. h. turcicus. many specimens in mcc, mzuf, mzut. h. yerburii pauciporosus. mzuf 6245 (holotypus) + many additional specimens. h. yerburii yerburii. mcc r814. f1249_acta_herpetologica_2009_1.indd acta herpetologica 4(1): 99-101, 2009 newborn dicephalic podarcis sicula filippo spadola 1, gianni insacco 2 1 facoltà di medicina veterinaria di messina, dipartimento di scienze sperimentali e biotecnologie applicate, polo universitario ss. annunziata, i-98168 messina. corresponding author. e-mail: fspadola@ unme.it 2 centro regionale recupero fauna selvatica e tartarughe marine, fondo siciliano per la natura e museo civico di storia naturale, via generale girlando 2, i-97013 comiso, ragusa. abstract. the author describes a very rare case of dicephalism in a podarcis sicula found in ragusa (italy). this is the first account in the world of a malformation of this sort in podarcis spp. keywords. podarcis sicula, dicephalism, morphology. dicephalism is a teratological type of anomaly characterised by the presence of two heads. although the existence of malformations of embryonic origin is well documented in many mammals (noden and de lahunta, 1985) and reptiles, very few cases of dicephalism in saurians have been found. a number of cases of dicephalic snakes was found (hoser and harris, 2005; swanson et al., 1997; wallach, 2007), but very little records are presently available in chelonians and saurians (branch, 1982; broadley, 1972; diong et al., 2003; holfert, 1999; matz, 1989). in fact, taking into account only the sub-order sauria, it was possible to find out only three cases: the gekkonid lizard rhacodactylus auriculatus (bavay, 1869, cited in holfert, 1999) and two scincid lizards trachylepis (= mabuya) striata (peters, 1844) and egernia striolata (peters, 1870) described respectively by broadley (1972) and by matz (1989). there is no documented case to date of dicephalism in the lacertidae family, thus making the dicephalic podarcis sicula here described as the first record as far we area aware. the podarcis individual was found in scoglitti, province of ragusa (sicily, italy) in september of 1996. the environment of the location in which it was discovered is typical of the ibleo particularly arid, it consists of fields of gramineae bounded by dry stone walls. podarcis sicula is distributed throughout the entire territory and it can be found everywhere, often alongside with podarcis wagleriana. the individual observed showed the typical olive green and black reticulated colour pattern with the ventral areas a uniform greenish-white, and measured 6.5 cm total length, tail included. it was a small new-born, vital but evidently emaciated. the individual had a second trunk with a second head and two anterior limbs. this type of double foetal monstrosity is defined in teratology as parapagus: conjoned twins joined anterolaterally result from two nearly parallel notochords in close 100 f. spadola and g. insacco proximity. this anomaly represents less than 0.5% of all reported cases of humans conjoined twins (singhal et al., 2006). the specimen is now kept at the natural history museum of comiso (ragusa). acknowledgements we wish to thank the gabinetto provinciale di polizia scientifica di ragusa, salvatore rizza and mario russo.. references branch, w.r. (1982): dicephalic chersina angulata. j. herpetol. assoc. africa 27:12-15. broadley, d.g. (1972): dicephalism in the african striped skink mabuya striata striata (peters). arnoldia 5: 1-2. diong, c.h., tan, l.k.a., leh, c.m.u. (2003): axial bifurcation in a bicephalic chelonia mydas embryo. chel. conserv. biol. 4: 725-727. holfert, t. (1999): doppelköpfigkeit (bicephali) bei rhacodactylus auriculatus (bavay, 1869). elaphe 7: 13. hoser, r., harris, p. (2005): a second case of bicephalism in queensland carpet snakes (morelia spilota mcdowelli) (serpentes: pythonidae). herpetofauna 35: 61. matz, g. (1989): an axial duplication with double body in the lizard egernia striolata (peters). herpetopathologia 1: 57-59. fig. 1. dicephalic newborn of podarcis sicula. 101dicephalism in podarcis sicula noden, d.m., de lahunta, a. (1985): the embryology of domestic animals. developmental mechanisms and malformations. william and wilkins, baltimore. singhal, a., agarwal, g., sharma, s., gupta, a., gupta, d. (2006): parapagus conjoined twins: complicated anatomy precludes separation. j. indian ass. ped. surg. 11: 145-147. swanson, s., van breukelen, f., kreiser, b., chiszar, d., smith, h.m. (1997): a double-bodied midland water snake and additions to literature on ophidian axial bifurcation. bull. chicago herpetol. soc. 32: 80-83. wallach, v. (2007): axial bifurcation and duplication in snakes. part i. a synopsis of authentic and anecdotal cases. bull. maryl. herpetol. soc. 43: 57-95. acta herpetologica 2006 1 primi dati sulla dieta della lucertola ocellata «timon lepidus» (daudin, 1802) in italia primi dati sulla dieta della lucertola ocellata timon lepidus (daudin, 1802) in italia sebastiano salvidio 1, gaia calvi 1, luca lamagni 2, and giulio gardini 1 1 dipartimento per lo studio del territorio e delle sue risorse, dip.te.ris., università degli studi di genova, corso europa 26, i-16132 genova, italia. e-mail: salvidio@dipteris.unige.it 2 via monti 1, i-17100 savona, italia la lucertola ocellata timon lepidus (daudin, 1802) è diffuso nella penisola iberica, nel sud della francia e nella liguria occidentale e centrale (mateo e cheylan, 1997), regione che costituisce il limite orientale della sua distribuzione (camerano, 1885; bruno, 1982; ferri et al., 1991). in italia, l’areale della specie è frammentato (doria e salvidio, 1994) con popolazioni isolate, spesso minacciate dalla continua alterazione degli habitat idonei e in particolare dagli incendi e dalla continua edificazione delle aree costiere (salvidio et al., 2004). l’interesse biogeografico e conservazionistico di queste popolazioni al limite di areale è pertanto evidente, ma i dati sulla biologia di questo lacertide in italia sono ancora scarsi e le informazioni riportate da corti e lo cascio (1999) si riferiscono solo alle popolazioni spagnole e francesi. in questa breve nota sono presentati i risultati di uno studio sull’alimentazione di alcuni esemplari di t. lepidus della liguria occidentale. la ricerca si è svolta da giugno 2000 a settembre 2001 in un sito della piana di albenga (sv) dove la presenza della lucertola ocellata è accertata dal 1999. non sono disponibili dati sull’abbondanza di t. lepidus nell’area di studio; in ogni caso, solo 4 individui diversi sono stati osservati contemporaneamente durante i sopralluoghi (lamagni e salvidio, oss. pers.). è quindi assai probabile che alcuni degli escrementi esaminati siano stati depositati dagli stessi individui. inoltre, poiché i risultati presentati riguardano un solo sito, caratterizzato da un limitato numero di individui, essi non possono essere generalizzati ad altre popolazioni della specie presenti nel nostro paese. nell’area di studio sono presenti altri due lacertidi, il ramarro occidentale lacerta bilineata e la lucertola muraiola podarcis muralis. sono stati pertanto raccolti solo gli escrementi con dimensioni maggiori di 2,5 cm (hódar et al., 1996), escludendo dalle analisi anche gli individui giovani di lucertola ocellata. in laboratorio gli escrementi sono stati ammorbiditi in acqua ed osservati al microscopio binoculare. i resti animali sono stati identificati, utilizzando le collezioni di confronto di uno degli autori (g. g.) e conservati in alcohol. i conteggi delle prede rappresentano il numero minimo di esemplari e alcuni taxa, come i gasteropodi polmonati, sono stati sicuramente sottostimati, in quanto i gusci risultavano fortemente frammentati. i resti vegetali (semi e spighette) sono stati identificati utilizzando collezioni di confronto e berggren (1969). acta herpetologica 1: 73-76, 2006 74 s. salvidio et alii nel corso della ricerca sono stati raccolti 39 escrementi sicuramente attribuiti a t. lepidus: 21 nei mesi primaverili (da aprile a giugno) e 18 in quelli estivi (da luglio a settembre). in totale le prede identificate sono state 249, con una media di 6,4 prede per escremento. i taxa maggiormente rappresentati erano coleotteri, imenotteri, ortotteri e eterotteri che costituivano l’86% delle prede identificate (tabella 1). le dimensioni stimate delle prede variavano da 2 mm (ad es. piccoli formicidi) a circa 60 mm (ad es.. alcuni acrididae e mantidae). negli escrementi non sono stati rinvenuti resti attribuibili a vertebrati. tabella 1. dieta di timon lepidus in un sito della piana di albenga (sv). n = numero di escrementi analizzati. taxa primavera (n = 21) estate (n = 18) totale (n = 39) numero prede numero feci numero prede numero feci numero prede numero feci gastropoda polmonata 11 11 4 4 15 15 crustacea isopoda 8 2 1 1 9 3 aranea araneidae 1 1 1 1 diplopoda julidae 3 2 3 2 mantoidea mantidae 1 1 1 1 orthoptera acrididae 3 3 10 10 13 13 indeterminati 1 1 6 3 7 4 hemiptera coreidae 6 2 6 2 pentastomidae 8 7 4 2 12 9 indeterminati 6 3 6 3 coleoptera buprestidae 1 1 1 1 carabidae 6 6 3 2 8 8 cholevidae 1 1 1 1 coccinellidae 1 1 1 1 2 2 crysomelidae 1 1 1 1 curculionidae 14 10 9 7 23 17 dermestidae 1 1 1 1 elateridae 1 1 3 2 4 3 histeridae 2 1 1 1 3 2 oedemeridae 1 1 1 1 tenebrionidae 1 1 1 1 scarabeidae 25 14 9 5 34 19 indeterminati 5 4 2 2 7 6 hymenoptera apidae 3 3 5 3 6 formicidae 16 6 12 6 12 mutillidae 4 3 7 3 6 vespidae 1 1 16 3 4 indeterminati 5 2 17 8 10 diptera 1 1 1 1 lepidoptera (bruchi) 1 1 1 1 numero totale prede 131 118 249 prede per escremento 6,24 6,56 6,38 75dieta timon lepidus nell’area di studio t. lepidus si ciba prevalentemente di artropodi terrestri o che frequentano le parti aeree di piante erbacee e arbustive. tali osservazioni concordano con quelle riportate per le popolazioni di t. lepidus dell’isola di berlenga in portogallo (vicente et al., 1995) e della spagna (castilla et al., 1991; hernández et al., 1991; hódar et al., 1996) in 16 escrementi, 10 primaverili e 6 estivi, sono stati ritrovati resti di fibre vegetali, spighette e semi, in particolare dei generi carex (in 9), solanum (in 2), prunus e brachypodium (entrambi in un solo escremento). queste osservazioni indicano che i vegetali costituiscono elemento integrante nella dieta di t. lepidus nell’area di studio, come riportato precedentemente da castilla et al. (1991) e hódar et al. (1996) per popolazioni spagnole. ringraziamenti si ringraziano laura cornara e luigi minuto (dip.te.ris., università di genova) per la determinazione dei resti vegetali. bibliografia berggren, g. (1969): atlas of seeds and small fruits of northwest-european plant species with morphological description. part 2. cyperaceae. stockholm, swedish natural science research council. bruno, s. (1982): catalogo sistematico, zoogeografico e geonemico dei lacertidae di corsica, italia e isole maltesi. natura bresciana 19: 39-95. camerano, l. (1885): monografia dei sauri italiani. mem. r. accad. sci. fis. mat. nat. torino, ser. 2 37: 491-591. castilla, a., bauwens d., llorente g. a. (1991): diet composition of the lizard lacerta lepida in central spain. j. herpetol. 25: 30-36. corti, c., lo cascio, p. (1999): i lacertidi italiani. palermo, l’epos. doria, g., salvidio, s. (1994): atlante degli anfibi e rettili della liguria. catalogo dei beni naturali 2. genova, regione liguria. ferri, v., dell’acqua, a., salvidio, s. (1991): distribuzione dei rettili nella fascia costiera della liguria occidentale: i. lacerta l. lepida e malpolon m. monspessulanus. suppl. ric. biol. selvaggina 16: 217-220. hernández a., alegre j., salgado j. m. (1991): ecología trófica de lacerta lepida en la provincia de león, noroeste de españa. amphibia-reptilia 12: 283-292. hódar, j. a., campos, f., rosales, b. a. (1996): trophic ecology of the ocellated lizard lacerta lepida in an arid zone of southern spain: relationships with availability and daily activity of prey. j. arid environm. 33: 95-107. mateo, j. a., cheylan, m. (1997): lacerta lepida daudin, 1802. in: atlas of amphibians and reptiles in europe, p. 246-247. gasc, j.-p., cabela, a., crnobrnja-isailovic, j., dolmen, d., grossenbacher, k., haffner, p., lescure, j., martens, h., martinez rica, j. p., maurin, h., oliveira, m. e., sofianidou, t. s., veith, m., zuiderwijk, a. 76 s. salvidio et alii eds. paris, societas europaea herpetologica, muséum national d’histoire naturelle. salvidio, s., lamagni, l., bombi, p., bologna, m.a. (2004): distribution, ecology and conservation status of the ocellated lizard (timon lepidus) in italy. (reptilia, lacertidae). it. j. zool., 71 (suppl. 1): 125-134. vincente, l.a., araújo, p.r., barbault, r. (1995): ecologie trophique de podarcis bocagei berlengensis et de lacerta lepida (sauria, lacertidae) sur l’île de berlenga (portugal). rev. ecol. 50: 317-351. acta herpetologica 4(2): 143-151, 2009 habitat features and distribution of salamandra salamandra in underground springs raoul manenti1, gentile f. ficetola1,2, barbara bianchi3, fiorenza de bernardi1 1 dipartimento di biologia, università degli studi di milano, via celoria, 26, i-20133 milano, italy. corresponding author. e-mail: raoul.manenti@unimi.it. 2 dipartimento di scienze dell’ambiente e del territorio, università degli studi di milano bicocca, piazza della scienza, 1, i-20126 milano, italy. 3 comitato per la difesa delle bevere, via garibaldi, 10, i-20040 capriano di briosco, briosco (milano), italy. submitted on: 2009, 22nd february; revised on 2009, 15th august; accepted on 2009, 31st august . abstract. subterranean habitats are among the less known terrestrial habitats, but can reveal an unexpected biodiversity, and can play an underestimated role for amphibians. the fire salamander salamandra salamandra is sometimes found in underground environments, but the factors affecting its distribution in subterranean spaces remain substantially unexplored. we repeatedly surveyed some hypogeous springs, such as draining galleries and “bottini” in nw italy, in order to evaluate the relationship between environmental features and distribution of s. salamandra in these underground springs. we performed visual encounter surveys to assess the occurrence of larvae, juveniles or adults in springs. we also recorded four habitat variables: easy of access, isolation, macrobenthos richness and forest cover of the surrounding landscape. we used generalized linear models to evaluate the relationships between habitat features and occurrence of larvae. we observed larvae of s. salamandra in 13 out of 22 springs; their presence was associated to springs with high easy of access and with relatively rich macrobenthos communities. in underground springs, larval development apparently required longer time than in nearby epigeous streams. nevertheless, s. salamandra can attain metamorphosis in this environment. the occurrence of s. salamandra in underground environments was not accidental, but repeated in the time and interesting from an ecological point of view, confirming the high plasticity of the species. keywords. amphibians, cannibalism, cave, draining galleries, ecology, fire salamander, habitat, larval development. introduction in the last years, biospeleology has had a remarkable development. biospeleology has extended its attention not only to cave habitats, but also to interstitials ones, especially ground waters (danielopol and griebler, 2008). an increasing number of studies 144 r. manenti et alii has shown that the subterranean domain, formerly considered as a species-poor environment, often reveals an unexpected biodiversity (botosaneanu and stock, 1997; bottazzi et al., 2008). subterranean communities are composed by both hypogean and epigean taxa (danielopol and griebler, 2008). the occurrence of amphibians in underground habitats is reported in both the herpetological and biospeleological literature (gimenez-lopez and guarner deu, 1982). the life cycle of some taxa is completely (e.g., proteus) or partially (e.g., speleomantes) connected with caves and interstitial habitats. furthermore, several amphibians can use underground habitats even if they do not complete their life cycle there, i.e., they are occasional trogloxenes (romero, 2001; sket, 2008). a number of european amphibians have been more or less regularly found inside caves. multiple mechanisms can explain the underground presence of amphibians: underground spaces can be winter shelters, hiding places during the active season, and feeding habitats (baumgart, 1981; uhrin and lesinsky, 1997). furthermore, caves sometime act as natural traps where amphibians fall or are transported by the water flow (uhrin and lesinsky, 1997). the fire salamander salamandra salamandra has been repeatedly found in underground environments (baumgart, 1981; gimenez-lopez and guarner deu, 1982; uhrin and lesinsky, 1997; razzetti et al., 2001). adults and metamorphs are often observed during latency periods, with fidelity of the individuals to shelter places (feldman, 1967; baungart, 1981). furthermore, some studies reported the presence of larvae in subterranean damp biotopes. gimenez-lopez and guarner deu (1982) point out the finding of a larva in natural cave of catalonia; uhrin and lesinsky (1997) suppose that salamanders reproduce in slovakian underground spaces on the basis of records of larvae in caves and galleries; and veith (1986) supposes that reproduction in underground environments is common in the rhine valley. in italy, several authors have observed larvae of s. salamandra in both natural and artificial underground environments (bressi, 1995; bressi and dolce, 1999; razzetti et al., 2001). nevertheless, data on the occurrence and development of s. salamandra in hypogeous habitats remains anecdotic. furthermore, the factors affecting the distribution of salamanders in subterranean environments are substantially unexplored. recent observations showed that s. salamandra sometimes lays larvae inside the brooks or in the pools of some artificial hypogeous biotopes, such as draining galleries and other subterranean springs in northern italy (manenti, 2007, 2008). the aim of this study was evaluating the distribution of s. salamandra in subterranean springs, to understand the ecological determinants that affect their occurrence, and whether these environments can be a suitable reproductive habitat. materials and methods underground springs considered we considered two typologies of subterranean artificial springs: draining galleries and the so called ‘bottini’. draining galleries (fig. 1a) are characterized by an almost horizontal tunnel that penetrates the side of a slope, to catch the subterranean water of a spring and bring it outward. draining galleries are associated to traditional agriculture, and are found in europe, asia and northern africa (balland, 1992). even if they are less known than other spring typologies, draining 145salamandra salamandra in underground springs galleries are widespread in italy. some of them are very ancient, such as the etruscan spring flow tunnels (caponetti, 2005) or the galleries in the towns of matera and siena (kucher, 2005). they house small brooks and/or water reservoirs. ‘bottini’ (fig. 1b) are small buildings with limited subterranean development and with basins for water collecting. both typologies are frequently obsolete and unused. in most cases, they are hidden and difficult to reach (manenti, 2008). in the study area, draining galleries have a limited width (max 2 m); and a length varying from 5 up of 100 meters. the brooks and the pools within galleries usually have limited depth (average 20 cm). the length of bottini is usually 3-4 m; the average water depth is 41.7 cm. study area and surveys we surveyed 22 subterranean springs in lombardy (northern italy) between the districts of lecco, como, and milan (fig. 2). the study area is comprised in the catchment basins of the lambro, adda and seveso rivers. it is characterized by hilly and mountainous reliefs with a good cover of broadleaved woodlands. in order to identify and reach the springs, we used information available in local studies on troglophyle molluscs and springs features (nardo and guglielmin, 1996; pezzoli, 2007), we collected information from local people and local environmental organisation, and we directly explored the countryside. all springs were permanent or nearly permanent. from september 2007 till january 2009, we surveyed each springs 2-8 (median: 3) times. most of springs were surveyed 2-5 times; two springs with larvae were surveyed 7-8 times, at intervals of one-two months, to better monitor the larval development. the occurrence of larvae in springs was not related to the number of surveys (logistic regression, c21 = 1.05, p = 0.31), indicating that variation in the number of surveys did not bias the results of our analyses. we performed visual encounter surveys to assess the presence and the abundance of s. salamandra larvae, juveniles and adults. the surveyed waterbodies are usually small, always have very clear water, therefore visual census allow a reliable estimation of presence/absence of larvae in the water. when larvae of s. salamandra are present, their per-visit detection probability is > 90% (manenti et al., 2009), therefore with two-three surveys the cumulative probability of undetecting present larvae is < 1%. larvae fig. 1. example of the access tunnel of (a) a draining gallery and (b) a “bottino”. 146 r. manenti et alii were assigned to three development stages on the basis of total length and morphology (jusczcyk and zakrzewski, 1981): 27-45 mm; 46-65 mm; metamorphs with body size > 60 mm. we recorded four environmental variables to describe these subterranean habitats and to evaluate the relationship between habitat features and s. salamandra (manenti et al., 2009): a) easy of access for salamanders, measured using a rank scale (1 = completely closed by doors or other obstacles and apparently inaccessible; 2 = difficult access because of doors or other obstacles; 3 = open and accessible); b) isolation, measured as the distance between each spring and the nearest known laying site of s. salamandra, obtained from previous studies (manenti et al., 2008; ficetola et al., 2009; manenti et al., 2009) or by direct observations during surveys; c) richness of the community of benthonic macro-invertebrates, measured as the number of taxonomic units (see ghetti, 1997; in every spring, we sampled macrobenthos by moving the substrate for 5-10 minutes, and we used a thin-mesh dip net to collect the invertebrates); d) forest cover measured as forest cover percentage within 400 m from each sampling point on the basis of the 1:10000 vector map of lombardy, using the esri arcview 3.2 gis. forests are the major habitat of adults, and forest cover at this scale is strongly associated with populations of s. salamandra (ficetola et al., 2009). statistical analyses we used generalized linear models (glms), assuming binomial error, to evaluate the relationships between habitat features and occurrence of larvae. first, we analyzed all univariate relationships. we also tried to build glms describing the relationships between multiple habitat features and fig. 2. study area. white circles show the location of the draining galleries, while triangles of the “bottini”; grey: hidrographic network. some circles and triangles are partially superimposed, due to geographic proximity. 147salamandra salamandra in underground springs occurrence of s. salamandra, subjected to the constraint that all variables in a given glm must have variance inflation factor ≤ 5 (bowerman and o’connell, 1990). we built glms with all combinations of two or three variables, testing also for interactions. however, none of these glms with multiple variables showed akaike information criterion lower than the best univariate model, probably because of the limited sample size (see burnham and anderson, 2002). therefore, in the results we report the univariate models only. we also calculated nagelkerke’s r2 (r2n) as a measure of the variance explained by glms. if necessary, we transformed variables to improve normality (see table 1). table 1. habitat features of springs with and without larvae, univariate relationships between habitat features and presence / absence of larvae of salamandra salamandra, and comparison with epigeous streams with larvae from the same study area (sites in ficetola et al., 2009). for easy of access, we report the median and the range of variation; for isolation, macrobenthos richness and forest cover, we report mean ± se. r2n: nagelkerke’s r2. presence of larvae c21 p r2n epigeous streams yes (n = 13) no (n = 9) (n = 45) easy of access 3 (2-3) 2 (1-3) 10.57 0.001 0.51 isolation (m)1 475 ± 250 471 ± 292 0.177 0.674 0.01 macrobenthos taxa2 2.38 ± 0.39 1.33 ± 0.62 5.48 0.019 0.30 14.9 ± 1.5 forest cover3 0.66 ± 0.07 0.64 ± 0.09 0.06 0.810 0.00 0.65 ± 0.03 1: log transformed prior to analyze; 2: square-root transformed prior to analyze; 3: square-root arcsine transformed prior to analyze results we observed larvae of s. salamandra in 13 out of 22 springs. furthermore, we found a recently metamorphosed individual (65 mm) in one spring where we did not record larvae. we found adults in five tunnels. we observed recently laid larvae in november 2007, may-april 2008 and december 2008. in nearly all the cases, these larvae overlapped with older larvae previously laid. all the springs with larvae in autumn 2007 received at least a new deposition in spring or autumn 2008. in all springs, we observed multiple development stages during the same visit, indicating the presence of larvae from different clutches. the richness of macrobenthos was usually low. the average number of taxonomic units observed was 1.95 per spring (se = 0.35), and was much lower than observed in epigeous laying streams (manenti et al., 2009; table 1). univariate tests showed that the presence of larvae was associated to the springs with high easy of access and with rich macrobenthos. the relationships with isolation and forest cover were non significant (table 1). easy of access was the variable with the largest explanatory power, and explained 51% of variation of the distribution of s. salamandra larvae (table 1). we observed metamorphs in four springs. furthermore, in all springs with larvae we recorded the presence of late development stages. however, in four sites, the exit of metamorphs from springs seemed to be very difficult. we directly observed episodes of cannibalism in two galleries, in which larvae at late development stages fed on small larvae. 148 r. manenti et alii discussion the occurrence of s. salamandra in the hypogeous springs was not accidental, but repeated in time and interesting from an ecological point of view. all the study springs are underground headwaters. therefore, the presence of larvae can not be explained by drifting or trapping from epigeous environments. instead, the presence of larvae suggests that these headwaters are used by females for laying. furthermore, in all springs with larvae we observed repeated laying during multiple seasons, indicating that laying in these sites was not occasional. easy of access was the variable most strongly related to the presence of larvae. in practice, we observed larvae in most of springs accessible to adults. this suggests that underground springs are selected by females for laying. underground springs have permanent hydroperiod, and can be particularly important in the areas where epigeous streams are temporary. furthermore, we observed a significant association with macrobenthos richness. benthonic invertebrates constitute the major prey items of larvae, and therefore this relationship is not surprising (see manenti et al., 2009). the presence of benthos can be particularly important in these habitats, where invertebrates never reach high densities. it should also be noted that easy of access and macrobenthos richness were positively related (spearman’s correlation, rs = 0.55, p = 0.008), probably because easy of access increases also colonization by insects. therefore, it is possible that these two variables have a synergic effect. nevertheless, the richness of invertebrates remained low in all springs (table 1). this can be a critical factor for the survival and development of larvae in this environment. indeed, the scarcity of shelter and of invertebrates might favours cannibalism. during our surveys, we observed some episodes of cannibalism; furthermore, in several cases we observed larvae with wounds and bite marks in areas without predator insects (see below), suggesting that cannibalism can be frequent. s. salamandra often performs cannibalism; in suboptimal habitats, cannibalism can be the only strategy allowing some larvae (usually the oldest ones) to reach metamorphosis (eitam et al., 2005). previous studies showed that, in epigeous streams, larvae are associated with high forest cover, probably because forests are the major habitat of adults (ficetola et al., 2009; manenti et al., 2009). the lack of a significant relationship (table 1) does not mean that forest presence is unimportant. most of the studied streams are in highly forested landscapes (table 1); the average forest cover across all sites (65%) was similar to the cover in epigeous streams used for laying by salamanders (table 1: ficetola et al., 2009; manenti et al., 2009). therefore, forest presence was not a limiting factor in the landscapes studied. we did not observe a relationship with isolation, and we observed reproductions also in springs very far from the nearest occupied epigeous stream (table 1). amphibians often live in network of metapopulations, and isolation has strong negative effects on the occupancy of wetlands (ficetola and de bernardi, 2004; zanini et al., 2009). however, our knowledge of breeding streams is probably incomplete, and we might have missed nearby sites. amphibians living in cave environments usually have delayed development (clerguegazeau, 1975). we found a similar pattern, with the coexistence of multiple larval stages. our observations suggest that larval development in these caves can require more than eight months. this is more than twice the age at metamorphosis in epigeous streams (usually 3-4 months: nöllert and nöllert, 1992). the slow development rate can be caused by 149salamandra salamandra in underground springs cold temperature, lack of light, and scarcity of food items (e.g., macrobenthos). on the other hand, underground habitats can have advantages, for example because of the constant thermal environment, which allows development also during winter, or the scarcity of predators. for instance, during surveys and macrobenthos samplings, we never observed fish or predatory insects, even those that typically inhabits other s. salamandra breeding sites (e.g., nepa cinerea; larvae of cordulegaster boltoni or other odonata). the observation of metamorphs in several springs confirm that these areas can be suitable habitat for larval development. nevertheless, some of the springs can act as traps. in three sites, the vertical banks did not allow the adults or metamorphs to leave the springs, and we found drown animals. s. salamandra shows high plasticity, has surprising local adaptations, and can perform larval development in habitats ranging from fast running streams to ponds (weitere et al., 2004). the observation of larvae in underground springs is a further confirmation of this plasticity. indeed, the occurrence of s. salamandra in underground, artificial springs is also known for other countries (e.g., switzerland: k. grossenbacher pers. comm.) and similar breeding sites are probably present also in other areas of its distribution range (baumgart, 1981). underground environments are among the less known terrestrial habitats, and can play an underestimated role for amphibians. draining galleries and other underground springs could be very useful to study plasticity and adaptations in s. salamandra. acknowledgements we are grateful to: suor donata, e. pezzoli, l. kalcich, p. pozzoli, f. prada, m. cappelli and d. magni for helping in locating springs and logistic support during surveys. the comments of one anonymous reviewer improved a previous draft of the manuscript. references balland, d. 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(2004): adaptive divergence vs. environmental plasticity: tracing local genetic adaptation of metamorphosis traits in salamanders. mol. ecol. 13: 1665-1677. zanini, f., pellet, j., schmidt, b. (2009): the transferability of distribution models across regions: an amphibian case study. divers. distrib. 15: 469-480. acta herpetologica 2006 2 first record of the four-lined snake elaphe quatuorlineata (lacépède, 1798) in serbia first record of the four-lined snake elaphe quatuorlineata (lacépède, 1789) in serbia nemanja ristić 1, ljiljana tomović 2, rastko ajtić 3, jelka crnobrnja-isailović 4 1balzakova 67, 21000 novi sad, serbia 2institute of zoology, faculty of biology, studentski trg 16, 11000 belgrade, serbia corresponding author. e-mail: lili@bf.bio.bg.ac.yu 3institute for nature conservation of serbia, dr ivana ribara 91, 11070 belgrade, serbia 4 department of evolutionary biology, institute for biological research, bulevar despota stefana 142, 11000 belgrade, serbia abstract. this paper presents the first record of four-lined snake elaphe quatuorlineata in serbia. in serbia and montenegro, according to the literature data, this species was found only in mediterranean and submediterranean montenegro. we present here data about founding site and suggest conservation measures for this species in serbia. keywords. elaphe quatuorlineata, first record, serbia. herpetofauna of serbia generally consists of mediterranean and middle european elements (džukić, 1995). the main mediterranean refugia for amphibian and reptile species are metohia, where climatic influences from the adriatic sea spread through the drim river valley (pasuljević, 1968; džukić and pasuljević, 1979; ajtić and tomović, 2001) and the southernmost part of serbia (i. e. pčinja river valley, crnobrnja-isailović and aleksić, 1999; tomović et al., 2004). pčinja river is the left tributary of vardar river a well known corridor for mediterranean species that spread through the fyr of macedonia to the north (matvejev, 1961; radovanović, 1964; matvejev and puncer, 1989). the occurrence of mediterranean elements have been confirmed for the wide-ranging area in fyr of macedonia: testudo graeca, cyrtopodion kotschyi, algyroides nigropunctatus, lacerta trilineata, podarcis erhardii, pseudopus apodus, typhlops vermicularis, eryx jaculus, platyceps najadum, zamenis situla, elaphe quatuorlineata, malpolon monspessulanus and telescopus fallax (karaman, 1931, 1939; radovanović, 1951; dimovski, 1963, 1966; džukić, 1972; brelih and džukić, 1974). the presence of some mediterranean reptile species in pčinja river valley as the already mentioned taxa: podarcis erhardii, platyceps najadum and testudo graeca (džukić, 1995; crnobrnja-isailović and aleksić, 1999; crnobrnja-isailović et al., 2004; tomović et al., 2004) are further support to the assumptions, based on vegetation data, that strong mediterranean influences spread along the vardar and pčinja river valleys far into the mainland (mišić and dinić, 1970; ranđelović and stamenković, 1974; zlatković and acta herpetologica 1(2): 135-139, 2006 136 n. ristić et alii ranđelović, 2004). potential vegetation units at the pčinja river valley include forests of hungarian oak and turkey oak – quercetum-frainetto-cerris rud. (1940) 1949 s. lat., which here display the influence of the mediterranean, as well as moesian forests of turkey oak – quercetum cerris moesiacum e. vuk. 1966 s. lat. (jovanović et al. 1986), at altitudes of 600-1200 m. general distribution range of the four-lined snake (elaphe quatuorlineata) includes: most of the apennine peninsula, the balkan peninsula – mediterranean parts of slovenia, croatia, montenegro, albania, macedonia, bulgaria, romania; parts of moldavia, ukraine, the russian precaucasus, kazakhstan, turkmenistan and iran (böhme, 1997). but, populations previously regarded as subspecies elaphe quatuorlineata sauromates were recently separated from e. quatuorlineata and raised to species rank (lenk et al., 2001). according to böhme (1997), in serbia and montenegro is present the subspecies elaphe quatuorlineata quatuorlineata. until now, four-lined snake was recorded only in southern montenegro (e.g. boka kotorska, bar, ulcinj, rumija, skadar lake region – radovanović, 1951; džukić, 1972; böhme and szczerbak, 1993; crnobrnja-isailović and džukić, 1997). fig. 1. first record of four-lined snake elaphe quatuorlineata in serbia (national grid utm 10 x 10 km reference). 137first record of elaphe quatuorlineata during a field trip at the pčinja river valley near the macedonian-serbian border (between klenike village and st. prohor pčinjski monastery, n42o38’33”, e21o09’00”, national grid reference, utm 10x10 em79, fig. 1), on august the 11th 2005, one adult male of four-lined snake (elaphe quatuorlineata lacépède, 1789), was caught (by ristić, n.). individual was only photographed and immediately released (fig. 2). founding site is located at approximately 600 m a.s.l. characteristic plant species of this area are quercus pubescens, quercus cerris, carpinus orientalis and juniperus oxycedrus. this individual was found in a rocky microhabitat facing south. syntopic species were: testudo hermanni, lacerta viridis, podarcis erhardii and platyceps najadum. this record extends the distribution range of e. quatuorlineata in this part of the species area, to the north (the closest record is skoplje, over 50 km in a straight line to the fig. 2. adult male of elaphe quatuorlineata from pčinja river region. 138 n. ristić et alii south or approximately 80 km following the mediterranean corridor from pčinja valley to skoplje – karaman, 1939; dimovski, 1966). we suppose that this population is situated at the species distribution edge in the central part of the balkans. although we found only one adult individual, more detailed field research is needed to evaluate the population and conservation status of this species in serbia. having in mind the fact that this is the only founding site of this species in serbia so far, e. quatuorlineata should urgently be added to the list of protected species in national conservation legislative of republic of serbia and to the red data book of serbia and montenegro. the presence of several mediterranean species and very high diversity of amphibians and reptiles (džukić, 1995) are the main arguments that suggest pčinja river region to be included in the list of important herpetological areas in serbia and montenegro and for the balkan peninsula. acknowledgements we are grateful to m. niketić for providing us the map of serbia. references ajtić, r., tomović, lj. (2001): first record of kotschy’s gecko cyrtodactylus kotschyi (steindachner, 1870) (gekkonidae, lacertilia) in fr yugoslavia. arch. biol. sciences, belgrade 53: 23-24. böhme, w. (1997): elaphe quatuorlineata (lacépède, 1789). in: atlas of amphibians and reptiles in europe, p. 358-359. gasc, j.-p., cabela, a., crnobrnja-isailović, j., dolmen, d., grossenbacher, k., haffner, p., lescure, j., martens, h., martinez rica, j. p., maurin, h., oliveira, m.e., sofianidou, t.s., veith, m., & zuiderwijk, a., eds. societas europaea herpetologica and museum national d’histoire naturelle, paris. böhme, w., szczerbak, n.n. (1993): elaphe quatuorlineata (lacépède, 1789) – vierstreifennatter. in: handbuch der reptilien und amphibien europas, vol. 3/i, schlangen 1, p. 373-396. böhme, w., ed, aula-verlag, wiesbaden. brelih, s., džukić, g. (1974): catalogus faunae jugoslaviae. academia scientiarum et artium slovenica, ljubljana. crnobrnja-isailović, j., džukić, g. (1997): raznovrsnost faune vodozemaca i gmizavaca u širem regionu skadarskog jezera i značaj njenog očuvanja. poseban otisak iz zbornika radova “prirodne vrijednosti i zaštita skadraskog jezera”, naučni skupovi, knjiga 44. podgorica, canu. crnobrnja-isailović, j., aleksić, i. (1999): first record of coluber najadum eichwald (1831) in serbia. arch. biol. sciences, belgrade 51: 47-48. crnobrnja-isailović, j., ajtić, r., tomović, lj. (2004): contribution to batrachofauna and herpetofauna of pčinja river in the southern serbia. first symposium of ecologists of the republic of montenegro, book of abstracts, p. 72. 139first record of elaphe quatuorlineata dimovski, а. (1963): herpetofauna na skopska kotlina. i – zoogeografski i ekološki pregled. godišen zbornik prirodno-matematičkog fakulteta, univerziteta u skoplju, skoplje, knjiga 14, biologija 2: 189-221. dimovski, а. (1966): herpetofauna na skopska kotlina. ii faunistički del. godišen zbornik prirodno-matematičkog fakulteta, univerziteta u skoplju, skoplje, knjiga 16, biologija 4: 179-188. džukić, g. (1972): herpetološka zbirka prirodnjačkog muzeja u beogradu. (herpetological collection of the belgrade museum of natural history). glasnik prirodnjačkog muzeja, beograd, ser. b 27: 165-180. džukić, g. (1995): diverzitet vodozemaca (amphibia) i gmizavaca (reptilia) jugoslavije, sa pregledom vrsta od međunarodnog značaja. in: biodiverzitet jugoslavije, p. 449469. stevanović, v., vasić, v., eds, biološki fakultet & ecolibri, beograd. džukić, g., pasuljević, g. (1979): o rasprostranjenju ljuskavog guštera – algyroides nigropunctatus (dumeril et bibron, 1839) reptilia, lacertidae. biosistematika, beograd 5: 61-70. jovanović, b., jovanović, r., župančić, m. (eds) (1986): natural potential vegetation of yugoslavia (commentary to the map 1: 1 000 000). edited for 18th iufro congress yu 86, ljubljana. karaman, s. (1931): zoološke prilike skopske kotline. glasnik skopskog naučnog društva, skoplje, knjiga x, odeljenje prirodnih nauka 4: 1-16. karaman, s. (1939): uber die verbreitung der reptilien in jugoslavien. annales musei serbiae meridionalis, skoplje 1: 1-20. lenk, p., joger, u., wink, m. (2001): phylogenetic relationships among european ratsnakes of the genus elaphe fitzinger based on mitochondrial dna sequence comparisons. amphibia-reptilia 22: 329-339. matvejev, s.d. (1961): biogeografija jugoslavije. naučna knjiga, beograd. matvejev, s.d., puncer, i.j. (1989): karta bioma, predeli jugoslavije i njihova zaštita. glasnik prirodnjačkog muzeja, beograd, posebna izdanja 36: 1-76. mišić, v., dinić, a. (1970): reliktna šumska vegetacija klisure pčinje i kozjaka. archive of biological sciences, belgrade 22: 3-4. pasuljević, g. (1968): prilog poznavanju herpetofaune kosova. zbornik filozofskog fakulteta, priština 1: 61-74. radovanović, m. (1951): vodozemci i gmizavci naše zemlje. naučna knjiga, beograd. radovanović, m. (1964): die verbreitung der amphibien und reptilien in jugoslawien. senckenbergiana. biol., frankfurt a. main 45: 553-561. ranđelović, n., stamenković, v. (1974): flora i vegetacija rujan planine u jugoistočnoj srbiji. leskovački zbornik 24: 375-392. tomović, lj., ajtić, r., đoković, đ., antić, s. (2004): records of testudo graeca ibera pallas, 1814 in serbia and montenegro. herpetozoa 17: 189-191. zlatković, b., ranđelović, v. (2004): records of new species to the flora of serbia. xi optima meeting abstracts, belgrade, serbia and montenegro, 66. acta herpetologica 2006 2 aging «salamandrina perspicillata» (savi, 1821) by skeletochronology aging salamandrina perspicillata (savi, 1821) by skeletochronology stefano bovero 1, claudio angelini 2, carlo utzeri 2 1università di torino, dipartimento di biologia animale e dell’uomo, via accademia albertina 13, 10123 torino, italia; e-mail: stefano.bovero@tin.it 2università la sapienza, dipartimento di biologia animale e dell’uomo, viale dell’università 32, 00185 roma, italia; e-mail: (ca) oppela@tin.it, (cu) carlo.utzeri@uniroma1.it abstract. we assessed age and first reproduction age in salamandrina perspicillata females by means of skeletochronological analysis. as we examined sections from the third toe of the hind limbs, the tecnique herewith introduced is non-letal and compatible with ecological investigations. females reached sexual maturity at four or five years; the oldest female was 12. svl is a reliable body size index for assessing age (r2 = 0.74). keywords. salamandrina perspicillata, skeletochronology. salamandrina (fitzinger, 1826) is a genus unique to peninsular italy. formerly, just s. terdigitata (lacépède, 1788) belonged to this genus, but mattoccia et al. (2005) recently proposed to split it into two species: s. perspicillata (savi, 1821) occurs in central and northern italy and s. terdigitata in southern italy. in terrarium, salamandrina can reach 12 years (rimpp, 1978). only indirect information is available on sexual maturity, based on minimum size of breeders of each population, but minimum body-size can vary strongly among populations (vanni, 1980; angelini et al., 2001, 2006; della rocca et al., 2005; angelini, 2006). we assessed age and first reproduction age by means of skeletochronological analysis. according to halliday and verrell (1988), skeletochronology is a reliable method to assess age in amphibians, mainly in temperate species. using the skeletochronology method for age determination in amphibians has shown how different life-history traits in populations from different altitudes or environmental contexts may be marked by differences in longevity, in age and size at sexual maturity and in the relationships between body size and growth rate (berven, 1982; hemelaar, 1988; caetano and castanet, 1993; diaz-paniagua and mateo, 1999; kutrup et al., 2005). the earliest skeletochronological studies examined skull bones (e.g. senning, 1940). later, sections of humerus and/or femur were used (e.g. smirina and rocek, 1976; francillon, 1979; guarino et al., 1995), but in anurans and larger urodeles the phalanges are now used (e.g. gittins et al., 1982; gibbons and mccarthy 1983; hemelaar, 1985; acker et al., 1986; reading, 1991; flageole and leclair, 1992; acta herpetologica 1(2): 153-158, 2006 154 s. bovero et alii semlitsch et al. 1993; guarino et al., 2003) precluding the need to sacrifice animals and thereby making the technique compatible with mark-recapture investigations. in recent years this non-letal technique has proved to be effective while operating on small or delicate urodeles like triturus helveticus, t. vulgaris and euproctus platycephalus (guyetant et al., 1991; marnell, 1997; bovero et al., 2003). from 2004 to 2005, we studied a population of salamandrina perspicillata which breeds in a spring-fed trough in the monti lepini (latium, central italy) at 816 m a.s.l. as a mean of marking, we took a picture of the ventral pattern (vanni et al., 1997) of 442 ovipositing females. we measured snout-vent length (svl) of 277 females. out of these, we also measured the total length (tl) of 215. measures were taken by a ruler at the nearest mm. we removed the third toe of the hind limb of 33 females, of which we recorded both svl and tl. the removed digits were preserved with 70% alcohol and stored in labelled tubes. the females were realesed at the site. in the laboratory, we removed skin and muscles from the digits and isolated the medial phalangeal bone. this procedure was performed under a stereoscope, while maintaining the digit in a petri-capsula filled with water. we decalcified the bones in 3% nitric acid for 40-50 minutes; after decalcification we put each of them in a glass with tap water. we changed water four times, after 15, 30, 45 and 60 minutes. then the bones were sectioned at 13 μm by a freezing microtome. cross sections were stained in ehrlich’s hematoxylin for 2 minutes. we adopeted a 2 minutes staining time after observing an eccessively dark coloration in the slides stanned for longer times in our rather old hematoxylin preparation. we washed the stained sections with tap water. since the phalanx of s. perspicillata is a very short bone, we could utilise only five to eight sections for the analysis. the best secfig. 1. section of medial phalanx of an 8 years old female s. perspicillata showing lines of arrested growth (numbered from 1 to 8). this female reached the sexual maturity at the age of four years. the first lag was partially eroded. r.b.: redeposited bone; r.l.: resorption line. 155aging salamandrina perspicillata tions were mounted on slides by aquamount (gurr). the sections were then examined under a light microscope and lines of arrested growth (lags) present in the periosteal were counted in order to estimate the individual ages (fig. 1). since in central italy, s. perspicillata may be active from late september to early may (angelini et al., 2001; utzeri et al., 2004; angelini, 2006), while probably estivates in the rest of the year, we assume that each lag represent a one-year growth period. age at the first reproduction was inferred from the first sudden narrowing of the lags, according to caetano and castanet (1993). we compared the slides of humerus, femur and phalanx of five individuals we had found dead: the bones of each individual showed the same number of lags. in the phalanx sections the first lag often appeareds partially eroded by endosteal remodelling. we did not observe any false or double lines. in six out of the 33 females we were unable to read the sections, and in one we could not assess the age at first reproduction. in the field, mean svl (± 1 se) of ovipositing females was 42.0 ± 0.2 mm (n = 277; range 35-50) and mean tl was 107.6 ± 0.6 mm (n = 215; range 85–134). svl size frequency was as fig. 2. measures and age of the 27 females are in table 1. the oldest female (12 years) was 47 mm in svl and 119 mm in tl. the longest female, svl = 50 mm and tl = 133 mm, was 10 years old. using svl as predictor of age, the regression equation for sexual mature females is age = svl(0.5598) – 15.932. females (n = 26) reached sexual maturity at the age of four (80.8%) or five (19.2%) years. we captured two newly breeders: one was four years old and 37 mm in svl, the other was five years old and 36 mm svl. in our sample, females of the same age which bred the first time at four years were larger than those which bred the first time at five, although the difference is not significant (ancova f1,23 = 3.3, p > 0.08). a few populations of salamandrina perspicillata have been studied with regards to individual body size (vanni, 1980; angelini et al., 2001, 2006; della rocca et al., 2005; angelini, 2006). single population mean body size (svl) of adult females ranges from fig. 2. size frequency (svl) of breeder female population at sma. 156 s. bovero et alii 33.6 ± 0.2 to 42.1 ± 0.2 mm (angelini, 2006), and females are larger than males (vanni, 1980). we think that the use of skeletochronology should be useful to understand such both inter-population and inter-sexual variability. acknowledgements. we thank prof. cristina giacoma (dipartimento di biologia animale e dell’uomo, università di torino) for laboratory support. references acker, p.m., kruse, k.c., krehbel, e.b. 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(1991): the relationship between body length, age and sexual maturity in the common toad, bufo bufo. hol. ecol. 14: 245-249. rimpp, k. (1978): salamander und molche. schwanzlurche im terrarium. ulmer, stuttgart. semlitsch, r.d., scott, d.e., pechman, j.h.k., gibbons, j.w. (1993): phenotypic variation in the arrival time of breeding salamanders: individual repeatability and environmental influences. j. anim. ecol. 62: 334-340. 158 s. bovero et alii senning, w.c. (1940): a study of age determination and growth of necturus maculosus based on the parasphenoid bone. am. j. anat. 66: 483-494. smirina, e., rocek, z. (1976): on the possibility of using annual bone layers of alpine newts triturus alpestris (amphibia: urodela) for their age determination. vestný´k ceskoslovenske´ spolecnosti zoologicke´ 40: 232-237. utzeri, c., antonelli, d., angelici, c. (2004): a note on terrestrial activity and feeding in the spectacled salamander, salamandrina terdigitata (urodela, salamandridae). herp. bull. 90: 27-31. vanni, s. (1980): note sulla salamandrina dagli occhiali [salamandrina terdigitata (lacépède, 1788)] in toscana (amphibia salamandridae). atti soc. tosc. sci. nat., mem., ser. b, 87: 135-159. vanni, s., nistri, a., zagaglioni, s. (1997): use of the “pattern mapping” technique to study the biology of salamandrina terdigitata (amphibia caudata salamandridae). atti soc. tosc. sci. nat., mem., ser. b, 103[1996]: 111-112. © firenze university press www.fupress.com/ah acta herpetologica 5(1): 19-22, 2010 predation attempt by oxybelis aeneus (wagler) (mexican vinesnake) on basiliscus plumifrons (cope) paul b.c. grant1, todd r. lewis 2 1 4901 cherry tree bend, victoria b. c., v8y 1si, canada. 2 westfield, 4 worgret road, wareham, dorset, bh20 4pj, uk. corresponding author. e-mail: ecolewis@ gmail.com submitted on: 2009, 31st august; revised on: 2010, 3rd march; accepted on: on2010, 13th march. oxybelis aeneus is a broadly distributed vine snake of the new world (keiser, 1982; savage, 2002). its enormous range stretches from arizona and southern texas, through mexico, honduras, belize, guatemala, nicaragua, costa rica, panama, and into south america where it is found in colombia, ecuador, north-western peru on the pacific versant, and as far south as brazil and bolivia on the atlantic versant (keiser, 1974, 1982; dixon and soini, 1986; keiser, 1991; pérez-santos and moreno, 1991; lehr et al., 2002; savage, 2002; hernandez, 2004; uetz, 2006; cisneros-heredia and touzet, 2007; agfd, 2008). it is also found on trinidad and tobago and other islands such as offshore atolls around belize (campbell, 1998; platt et al., 2002). o. aeneus is easily recognised from other oxybelis sp. vine snakes by its light-grey dorsum and black mouth lining. it is a common colubrid snake within its range, inhabiting open areas, grassland with scrub, forest edges, clearings within forest, abandoned pastures, riparian premontane wet forest, premontane rain forest, in addition to lowland wet and dry forest (franzen, 1996; savage, 2002). it is often encountered among vegetation at 0.3 m to 1.8 m perch heights (franzen, 1996; savage, 2002) and can occur in densities of up to 180 individuals per hectare. it is mildly venomous and possesses enlarged grooved back fangs (savage, 2002; mcconnell, 2008). dietary information for this species is mostly known and its wide prey base includes lizards, amphibians, arboreal mammals, small rodents, small birds and fledglings, insects and also fish (henderson, 1982; campbell, 1998; savage, 2002; hetherington, 2006). studies indicate that lizards, particularly anoles, are important prey for o. aeneus (keiser, 1967; henderson, 1982; wilson and cruz-díaz, 1993; lee, 1996; savage, 2002). o. aeneus has been reported to consume norops rodriguezi, norops bourgeaei, norops uniformis, basiliscus vittatus, iguana iguana, sceloporus sp., and a number of cnemidophorus sp. (campbell, 1998; savage, 2002; diener, 2007). here we document an attempt by an adult o. aeneus to kill a sub-adult basiliscus plumifrons in a lowland wet manicaria forest north of tortuguero, costa rica (myers, 1990; lewis et al., in press). in june 2002 we were photographing a sub-adult b. plumifrons dur20 p.b.c. grant and t.r. lewis ing the day that was basking in a heliconia sp. plant in edge habitat. whilst taking the photo, an adult o. aeneus struck at the lizard from above and within a concealed area of vegetation behind the plant. the snake grasped the individual by the nape (fig. 1). the lizard attempted to escape by dislodging the snake using its legs. however, it ceased struggling after a short duration, possibly from energy expenditure, partial asphyxiation, possible envenomation or some combination of the three. interestingly, the snake held the lizard in its jaws for 4-5 minutes before attempting to swallow. the snake, unable to swallow the lizard due to its large size, ceased consumption and dropped the lizard. after several minutes of subdued, immobile behaviour, the basilisk recovered and escaped into nearby vegetation. if envenomation occurred, it is possible that the lizard was able to overcome the snake’s mild venom from its grooved rear fangs and/or did not receive a lethal dose. the larger oxybelis fulgidus that is found in similar habitats to o. aeneus has been recorded to have a similar diet of birds and lizards but has also taken larger lizard prey such as ctenosaura similis (hayes, 2002; savage, 2002; endo et al., 2007). most snakes are physifig. 1. oxybelis aeneus grasping sub-adult basiliscus plumifrons. photograph by paul grant. 21predation attempt by oxybelis aeneus on basiliscus plumifrons ologically limited in the size of their prey choice by swallowing capability (arnold, 1993) which could explain why the snake terminated consumption of the basilisk on this occasion. acknowledgements we thank the canadian organization for tropical education and rainforest conservation, ministerio de recursos naturales energia y minas, and farnborough college of science and technology for permissions and assistance. references agfd [arizona game and fish department] (2008): oxybelis aeneus. abstract compiled and edited by the heritage data management system, arizona game and fish department, phoenix, arizona. arnold, s.j. (1993): foraging theory and prey-size predator-size relations in snakes. in: snakes: ecology and behaviour, p. 87-115. seigel, r.a., collins, j.t., eds, mcgraw hill, new york. campbell, j.a. (1998): amphibians and reptiles of northern guatemala, the yucatan, and belize. university of oklahoma press, norman. cisneros-heredia, d.f., touzet, j-m. (2007): ecuadorian distribution of snakes of the genera oxybelis (wagler, 1830) and xenoxybelis (machado, 1993). herpetozoa 19: 188189. diener, e. (2007): die erdspitznatter oxybelis aeneus und die lianennatter leptophis mexicanus als prädatoren der schwarzleguane ctenosaura similis und c. bakeri. elaphe 15: 59-62. dixon, j.r., soini, p. (1986): the reptiles of the upper amazon basin, iquitos region, peru. milwaukee public museum, milwaukee. endo, w., amend, m., fleck, l.c. (2007): oxybelis fulgidus prey. herpetol. rev. 38: 209. franzen, m. (1996): ökologische und morphologische aspekte einer costaricanischen population von oxybelis aeneus (wagler, 1824) (serpentes: colubridae). herpetozoa 9: 121-131. hayes, f.e. (2002): predation on birds by snakes in trinidad and tobago. j. trinidad and tobago field nat. club 2002: 59-61. henderson, r.w. (1982): trophic relationships and foraging strategies of some new world tree snakes (leptophis, oxybelis, uromacer). amphibia-reptilia 3: 71-80. hernandez, r.a. (2004): geographic distribution: oxybelis aeneus. herpetol. rev. 35: 84. hetherington, t.e. (2006): oxybelis aeneus diet. herpetol. rev. 37: 94-95. keiser, e.d. jr. (1967): a monographic study of the neotropical vine snake, oxybelis aeneus (wagler). unpublished doctoral dissertation. louisiana state university, louisiana. keiser, e.d. jr. (1974): a systematic study of the neotropical vine snake, oxybelis aeneus (wagler). bull. texas mem. mus. 22: 1-51. 22 p.b.c. grant and t.r. lewis keiser, e.d. jr. (1982): oxybelis aeneus (wagler). cat. amer. amph. and rept. 305: 1-4. keiser, e.d. jr. (1991): bibliography of the genus oxybelis wagler (serpentes: colubridae). smithsonian herpetological information service, washington 86: 1-45. lee, j.c. (1996): the amphibians and reptiles of the yucatán peninsula. cornell university press, new york. lehr, e., kohler, g., streit, b. (2002): die herpetofauna von mittelperu entlang eines transektes von der pazifischen küste bis in die hochanden. faunistische abh. mus. tierkunde, dresden 22: 361-392. lewis, t.r., grant, p.b.c., garcia-quesada, m., ryall, c., laduke, t.c. (in press): a preliminary botanical study of caño palma biological station, tortuguero, costa rica. checklist. mcconnell, g.j. (2008): oxybelis aeneus envenomation. herpetol. rev. 39: 356. myers, r.l. (1990): palm swamps. in: ecosystems of the world, p. 267-278. lugo, a.e., brinson, m., brown, s., eds, elsevier, oxford. pérez-santos, c., moreno, a.g. (1991): serpientes de ecuador. monogr. mus. reg. sci. nat. torino. 11: 1-538. platt, s.g., meerman, j.c., rainwater, t.r. (2002): oxybelis aeneus (wagler): an addition to the herpetofauna of turneffe atoll, belize. herpetol. bull. 82: 30-31. savage, j.m. (2002): the amphibians and reptiles of costa rica. university of chicago press, chicago. uetz, p. (2006): embl reptile database. electronic database accessible at http://www.reptile-database.org (online). embl heidelberg, germany. (accessed: october 2005). wilson, l.d., cruz-díaz, g.a. (1993): the herpetofauna of the cayo cochinos, honduras. herpetol. nat. hist. 1: 13-20. acta herpetologica 17(1): 45-58, 2022 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-12349 threatened and extinct amphibians and reptiles in italian natural history collections are useful conservation tools franco andreone1,*, ivano ansaloni2, enrico bellia3, andrea benocci4, carlotta betto5, gabriella bianchi6, giovanni boano7, antonio borzatti de loewenstern8, rino brancato9, nicola bressi10, stefano bulla11, massimo capula12, vincenzo caputo barucchi13, piero carlino14, umberto chalvien15, marta coloberti16, pierangelo crucitti17, maria c. deflorian18, giuliano doria19, simone farina20, valeria franceschini21, simona guioli22, roberta improta23, luca lapini24, leonardo latella25, giuseppe manganelli26, stefano mazzotti27, marta meneghini28, paola nicolosi20, annamaria nistri29, nicola novarini30, roberta pala1, edoardo razzetti31, giovanni repetto32, roberta salmaso25, guido c. salza33, stefano scali34, giovanni scillitani35, andrea sforzi36, roberto sindaco7, gionata stancher37, maria l. tavano19, marco valle38, giannantonio zanata santi39, marco a. l. zuffi20, giulia tessa1,6 1 museo regionale di scienze naturali, via g. giolitti, 36, i-10123 torino, italy 2 polo museale università di modena e reggio emilia, museo di zoologia e anatomia comparata, via g. campi, 213/d, i-41125 modena, italy 3 museo di zoologia “p. doderlein”, via archirafi, 16, i-90100 palermo, italy 4 museo di storia naturale dell’accademia dei fisiocritici, piazzetta s. gigli, 2, i-53100 siena, italy 5 dipartimento di geoscienze, università di padova, via gradenigo, 6, i-35131 padova, italy 6 museo civico di storia naturale, via cortivacci, 2, i-23017 morbegno, italy 7 museo civico di storia naturale di carmagnola, via san francesco di sales, 188, 10022 carmagnola, italy 8 museo di storia naturale del mediterraneo, via roma, 234, i-57127 livorno, italy 9 museo civico craveri di storia naturale, via f. craveri, 15, i-12042 bra, italy 10 museo civico di storia naturale di trieste, via dei tominz, 4, i-34139 trieste, italy 11 museo di storia naturale, u.o. musei d’ateneo, università di parma, strada l.c. farini, 90, i-43121 parma, italy 12 museo civico di zoologia, via u. aldrovandi, 18, i-00197 roma, italy 13 dipartimento di scienze della vita e dell’ambiente, università politecnica delle marche, via brecce bianche, i-60131 ancona, italy 14 museo di storia naturale del salento, sp calimera borgagne km 1, i-73021 calimera, italy 15 museo civico di storia naturale silvia zenari, via della motta, 16, i-33170 pordenone, italy 16 museo di storia naturale “p. calderini”, palazzo del musei, via calderini, 25, i-13019 varallo sesia, italy 17 società romana di scienze naturali, via fratelli maristi, 43, i-00137 roma, italy 18 muse museo delle scienze, corso del lavoro e della scienza, 3, i-38122 trento, italy 19 museo civico di storia naturale “g. doria”, via brigata liguria, 9, i-16121 genova, italy 20 museo di storia naturale dell’università di pisa, via roma, 79, i-56011 calci, italy 21 collezione di anatomia comparata, sistema museale di ateneo, università di bologna, via f. selmi, 3, i-40121 bologna, italy 22 civico museo di scienze naturali, via a. gramsci, 1, i-27058 voghera, italy 23 centro musei delle scienze naturali e fisiche, università di napoli federico ii, via mezzocannone, 8, i-80134 napoli, italy 24 museo civico di storia naturale, via c. gradenigo sabbadini, 22/32, i-33100 udine, italy 25 museo di storia naturale, lungadige porta vittoria, 9, i-37129 verona, italy 26 università di siena, dipartimento di scienze fisiche, della terra e dell’ambiente, via p.a. mattioli, 4, i-53100 siena, italy 27 museo civico di storia naturale, largo f. vancini, 2, i-44121 ferrara, italy 28 museo di zoologia, università di padova,via g. jappelli, 1/a, i-35121 padova, italy 29 museo di storia naturale, università di firenze, via romana, 17, i-50125 firenze, italy 30 museo di storia naturale di venezia g. ligabue, santa croce, 1730, i-30135 venezia, italy 31 kosmos museo di storia naturale, università degli studi di pavia, piazza botta, 9/10, i-27100 pavia, italy 32 museo civico di storia naturale “f. eusebio”, via vittorio emanuele ii, 19, i-12051 alba, italy 46 franco andreone et alii 33 museo di storia naturale “don bosco”, viale e. thovez, 37, i-10131 torino, italy 34 museo di storia naturale di milano, corso venezia, 55, i-20121 milano, italy 35 università degli studi di bari aldo moro, dipartimento di biologia, via orabona, 4a, i-70125 bari, italy 36 museo di storia naturale della maremma, strada t. corsini, 5, i-58100 grosseto, italy 37 fondazione museo civico di rovereto, largo santa caterina, 41, i-38068 rovereto, italy 38 museo civico di scienze naturali, piazza cittadella, 10, i-24129 bergamo, italy 39 musei del seminario vescovile di treviso, piazzetta benedetto xi, 2, i-31100 treviso, italy *corresponding author. e-mail: franco.andreone@regione.piemonte.it submitted on: 2021, 25th november; revised on: 2022, 24th february; accepted on: 2022, 2nd march editor: andrea villa abstract. natural history museums are irreplaceable tools to study and preserve the biological diversity around the globe and among the primary actors in the recognition of species and the logical repositories for their type specimens. in this paper we surveyed the consistency of the preserved specimens of amphibians and reptiles housed in the major italian scientific collections, and verified the presence of threatened species according to the iucn red list, including the extinct (ex), extinct in the wild (ew), critically endangered (cr), endangered (en), and vulnerable (vu) categories. altogether, we analyzed 39 italian zoological collections. we confirmed the presence of one extinct reptile (chioninia coctei) and five extinct or extinct in the wild amphibian species (atelopus longirostris, nectophrynoides asperginis, pseudophilautus leucorhinus, p. nasutus, and p. variabilis). seven cr amphibians, fourteen cr reptile species and the extinct skink c. coctei are shared by more than one institution. museums which host the highest number of threatened and extinct amphibian species are respectively turin (17 cr and 1 ex), florence (13 cr and 1 ex), and trento (15 cr and 1 ew), while for reptiles the richest museums are those from genoa (15 cr and 1 ex), florence (11 cr and 1 ex), and pisa (7 cr). finally, we discussed the utility of natural history museums and the strategies to follow for the implementation of their functionality. keywords. biodiversity, collections, conservation, herpetology, iucn categories, natural history museums. introduction biodiversity collections hosted in natural history museums, universities and other institutions are important cultural, scientific heritages, irreplaceable resources, and useful tools for a great array of studies to enhance biodiversity understanding and conservation. these include systematic investigations, taxonomic revisions, species descriptions, epidemiology, anatomy, historical reconstructions (e.g., about collectors and the institutions housing their collections, as well as about the history of science in general), but also many more science-outreach purposes, such as educational activities, and exhibitions (bakker et al., 2020). the origin and the history of biological – in particular zoological “voucher specimens” or, simply, “vouchers” (schilthuizen et al., 2015) housed in scientific collections, are rather heterogeneous, since they may result from various activities, e.g., field surveys with collecting, sampling for scientific research, recovery of dead animals (e.g., on roads, from zoological parks, captive breeders, etc.), direct purchases from dealers (for collections increase, exhibits and education), and others (funk et al., 2005). data associated with vouchers may also vary substantially across periods, going from just a rough species determination (as it was often the case in the past, especially for specimens obtained from commercial dealers), to an exhaustive set of information, including place and date of collection, collectors, donors, ecological and behavioral parameters, etc. the different kind of preservation techniques may also affect the degree of scientific, expositive and utility of specimens, going from traditionally naturalized / preserved specimens used for display, to “ad hoc” prepared series for scientific research, accompanied by tissue samples, photographs, parasitological/epidemiological specimens, etc. (lorch et al., 2021). particular and precious vouchers hosted in collections of museums are the so-called «type specimens», i.e., preserved individuals upon which new taxa are described, including, among the others, holotypes, paratypes, lectotypes, and syntypes (dubois, 2017). natural history museums (hereafter nhms or museums) are considered the heirs of the 16th-17th century “wunderkammern”, also known as “cabinets of curiosities” (butler et al., 1998; mccarter et al., 2001). the collections of “mirabilia”, “naturalia” and “exotica” (as natural history objects were sometimes labeled in the 47threatened amphibians and reptiles in italian museums past) were accumulated over the time by naturalists and “savants”. in a first phase of museums’ life, these objects were mainly used to solicit and address the wonders and varieties of nature, at those times still largely unknown. until then, objects were at the same time the origin and the pulsing heart of these collections and early museums, as well as one of the few ways to discover and describe nature (findlen, 1996). nowadays, the ultimate profile of scientific collections can be drawn looking at what happened in medium-large and national museums (suarez and tsutsui, 2004). these institutions, which are true documenting centers and repositories of the world’s geoand biodiversity, manage specimens and associated materials that often represent unique evidence of species distribution and evolution patterns (clemann et al., 2014). the information gathered from extinct species is irreplaceable and lost forever: the international union for conservation of nature (iucn) declared 160 animal and plant species extinct between 2010 and 2019, although recent estimates suggest that this estimate may be as high as one thousand species per year (ceballos et al., 2015; shivanna, 2020). nhms are relevant in preserving specimens and other materials, and this is gradually becoming one of the modern museums’ top finalities. indeed, vouchers of extinct species are useful reservoirs of information, crucial to understand the reasons and history of their extinction. at the same time, the availability of samples belonging to threatened species is an important and irreplaceable source of data for better understanding the conservation threats (e.g., reed and shine, 2002). as reported elsewhere (alberch et al., 1994; bakker et al., 2020), nhms should be primarily considered as research, documentation and divulgation centers, since their role in biodiversity discovery and nature valorization is central, and useful in showing variations in abundance, florae and faunae (ewers-saucedo et al., 2021). natural history and biodiversity museums are also the logical repositories for vouchers of species becoming rarer, and in some cases extinct (buckingham et al., 2021). this allows to keep track of biological changes since preserved specimens are excellent scientific resources representing unique means through which such species can be studied for their morphology, ecology, genetics, and other traits. in addition, they can be used in comparative studies, e.g., with extant species, not only for taxonomic and phylogenetic studies, but also to unveil ecological and life history traits (figueirido and janis, 2011). notwithstanding, while the advancement in our understanding of nature and the drafting of a life catalogue of our planet are still badly needed and considered as a humankind priority mission, in many cases nhms themselves are increasingly facing severe problems of identity and survivorship, due not only to economic reasons but also to a generalized shift and diffused amnesia of their original missions (boero, 2010; andreone et al., 2014, 2022; andreone, 2015; ceríaco et al., 2021). on the other hand, we also believe that it is imperative to reconcile the research/collection and outreach/education components within nhms, and that vouchers and scientific collections can be efficiently used for this aim. in the course of two national projects focused on the valorization of natural history collections – namely “vertex (vertebrata extincta)” and “estinzioni” (extinctions) (nicolosi et al., 2013, 2019) – we evaluated the consistency of vertebrate collections in italian nhms, with the aim to define the conservation status and iucn red list placement of the housed voucher specimens. hopefully, the identification of threatened and extinct species within museum collections is useful to address scientists and the public to understand and contrast the rarefaction and disappearance of our biodiversity, with dedicated temporary expositions, books, postcards, and gadgets. so far, in the present paper we focused our attention on amphibians and non-avian reptiles, two vertebrate groups often treated together in both research and the traditional imagery, which represent a major component of museum collections. here, we also give a general overview of the overall italian herpetological/museological patrimony, about ten years after the first comprehensive work (mazzotti, 2010) and provide indications on their conservation assessment. material and methods we selected the major italian herpetological collections, basing upon data provided by the national association of scientific museums (associazione nazionale dei musei scientifici anms), associated projects, e.g., collmap (vomero, 2013), and previously published contributions (mazzotti, 2010). basing upon feedbacks from curators and referring institutions, we gathered useful information from 39 natural history collections (managed by public museums, universities, and/or a few private bodies), which replied positively to our request and provided relevant data (table 1). a few nhms were excluded since, although known for possessing herpetological specimens, they did not reply, or did not provide sufficiently complete information. the existence of published/unpublished catalogues and/or lists of species/specimens housed in each collection was assured by the relative curator/referent, as well as through bibliography [(carmagnola: boano and del48 franco andreone et alii mastro, 1990; sindaco, 1990); (turin: elter, 1982; gavetti and andreone, 1993; andreone et al., 2007); (domodossola: andreone et al., 2005); (genoa: doria et al., 2002); (milan: leonardi et al., 1995; scali, 1996; blackburn and scali, 2014); (varese: danini and baratelli, 2000); (morbegno: zuffi, 1990); (padua: centis, 2004); (udine: laptable 1. list of the italian natural history museums contributing with collections data (abbreviations of provinces are reported between parentheses; taei = taxonomic auto-evaluation index; total number of species of amphibians and reptiles preserved in the collection is reported). museums accompanied by an asterisk (*) are those having a herpetologist as curator. used acronym and official museum denomination municipality (province) management type taei species number amphibians reptiles to museo regionale di scienze naturali * turin (to) region 3 593 855 to-db museo di storia naturale “don bosco” turin (to) high school 2 12 49 to-c museo civico di storia naturale * carmagnola (to) municipality 4 69 374 cn-a museo civico “federico eusebio” alba (cn) municipality 4 8 9 cn-b museo civico “craveri” di storia naturale bra (cn) municipality 4 10 53 vc-v museo di storia naturale “pietro calderini” varallo sesia (vc) foundation 4 2 20 vco-d civico museo di storia naturale “g. g. galletti” domodossola (vco) municipality 3 28 46 ge museo civico di storia naturale “giacomo doria” * genoa (ge) municipality 4 590 1450 mi museo di storia naturale * milan (mi) municipality 4 116 637 pv museo di storia naturale dell’università * pavia (pv) university 4 100 310 pv-v civico museo di scienze naturali voghera (pv) municipality 4 6 16 bg museo civico di scienze naturali bergamo (bg) municipality 4 28 153 so-m museo civico di storia naturale * morbegno (so) municipality 4 10 16 tv museo zoologico “g. scarpa” treviso (tv) diocese 4 83 285 vr museo di storia naturale * verona (vr) municipality 4 99 263 vi museo naturalistico archeologico di vicenza vicenza (vi) municipality 1 12 23 pd museo di zoologia dell’università padova (pd) university 3 75 115 ve museo di storia naturale “g. ligabue” * venice (ve) foundation 4 77 170 ro fondazione museo civico di rovereto rovereto (tn) foundation 4 11 41 tn muse museo delle scienze * trento (tn) province 4 185 170 pn museo civico di storia naturale pordenone (pn) municipality 3 1 23 ud museo friulano di storia naturale * udine (ud) municipality 4 153 63 ts museo civico di storia naturale * trieste (ts) municipality 4 78 360 pr museo di storia naturale dell’università parma (pr) university 2 17 75 mr museo di zoologia e anatomia comparata dell’università modena (mo) / reggio emilia (re) university 3 37 112 bo collezione di anatomia comparata, sistema museale di ateneo bologna (bo) university 4 4 53 fe museo civico di storia naturale * ferrara (fe) municipality 4 77 95 an-o museo di scienze naturali “luigi paolucci” offagna (an) municipality 3 10 12 fi museo di storia naturale dell’università * florence (fi) university 3 627 1268 pi museo di storia naturale dell’università * calci di pisa (pi) university 2 158 582 gr museo di storia naturale della maremma grosseto (gr) municipality 3 3 14 li museo di storia naturale del mediterraneo livorno (li) province 3 6 31 si museo di storia naturale dell’accademia dei fisiocritici siena (si) association 3 26 95 rm museo civico di zoologia * rome (rm) municipality 4 168 194 rm-s società romana di scienze naturali rome (rm) association 4 43 117 le museo di storia naturale del salento * calimera (le) municipality 4 29 77 na centro museale centro musei delle scienze naturali dell’università naples (na) university 2 35 72 ba museo di zoologia “lidia liaci” dell’università bari (ba) university 4 25 71 pa museo di zoologia “pietro doderlein” dell’università palermo (pa) university 2 32 85 49threatened amphibians and reptiles in italian museums ini, 1984); (trieste: bressi, 1996); (florence: lanza et al., 2005, 2006); (ferrara: mazzotti and miserocchi, 2009, 2010); (rome: capula et al., 2011; crucitti et al., 2017, 2021); (naples: maio et al., 2004)]. we used as taxonomic references “amphibian species of the world” (frost, 2021), “amphibiaweb” (amphibiaweb, 2021), and “the reptile database” (uetz et al., 2021). exhaustiveness and correctness of the reported information is warranted directly by curators of the museums in this study. taken into account that the degree of past and ongoing curatorial activity and taxonomic revisions are rather variable among institutions and since it was impossible to revise all the collections, we asked to provide a “taxonomic autoevaluation index” (taei), as follows: 1 (lowest taxonomic accurateness and/or collection without a proper revision), 2 (collection revised for the 30-40%), 3 (collection revised for the 60-70%), 4 (highest taxonomic accurateness, with both the amphibian and reptile collections fully revised). finally, we carried out a conservation assessment for each species, including attribution of threat categories according to the iucn red list (iucn, 2021). results the examination of the italian herpetological collections produced a catalogue including more than 1400 species and 67 families of amphibians, and more than 2500 species and 80 families of reptiles (respectively 1418 and 2513 species). of the amphibians, 257 (18.1%) belong to a threat category. of these species, 5 (1.9%) are extinct (ex), 47 (18.3%) critically endangered (cr), 100 (38.9%) endangered (en), and 105 (40.9%) vulnerable (vu). regarding reptiles, 210 (8.3%) belong to a threat category: one species (0.5%) is considered ex, 32 (15,2%) cr, 74 (35.2%) en, and 103 (49.1%) vu (fig. 1). museums and collections were quite heterogeneous, varying in size and finalities. overall, the nhms hosting the greatest number of threatened species of amphibians and reptiles (> 50 species of each group) are genoa (174 in total), florence (166 in total), and turin (161 in total) (fig. 2). amphibians hosted in the italian institutions belong to 7 gymnophiona, 52 anura, and 8 urodela families, while reptiles are represented by 13 testudines, one rhyncocephalia, three crocodylia, 38 sauria, and 25 ophidia families (appendix i). species assessed as cr constitute respectively the 0.56% and 0.28% of the amphibian and reptile world fauna, on the basis of the global numbers provided by amphibiaweb (8384 amphibians on 25th october 2021) and reptile database (11570 reptiles on may 2021; uetz et al., 2021). preserved cr amphibians include five urodeles and 46 anurans (table 2). atelopus longirostris, nectophrynoides asperginis, pseudophilautus leucorhinus, p. nasutus, and p. variabilis are currently assessed as ew or ex. about reptiles, we found respectively 32 cr and one ex species, chioninia coctei (table 3). four cr species are loricates, 14 chelonians, 12 saurians, and one snake. fourteen cr reptile species and the extinct c. coctei are shared by more than one collection. nhms hosting the highest number of high threatened and extinct amphibian species are respectively turin (17 cr / 1 ex), florence (13 cr / 1 ex), and trento (15 fig. 1. percentage of threatened species of amphibians (left) and reptiles (right) in the different iucn categories. fig. 2. presence of threatened species in italian museums for amphibians (above) and reptiles (below). museum abbreviation as in table 1. 50 franco andreone et alii cr / 1 ew), while for reptiles are genoa (15 cr / 1 ex), florence (11 cr / 1 ex), and pisa (7 cr). a large part of the cr amphibian and reptile species (26) comes from a single museum/collection (florence). the most shared cr species are, among the amphibians, atelopus ignescens, which is present in four collections, and, among the reptiles, eretmochelys imbricata and gavialis gangeticus, respectively hosted by fourteen and eleven institutions. a particular consideration should be deserved for the case of the axolotl ambystoma mexicanum. this urodele species is present in 16 of the examined collections, and, according to the iucn red list, it should be assessed as cr. anyhow, since it is likely that most of the preserved specimens derivate from captive and laboratory strains and given the impossibility of determining whether these animals (especially the historical ones) introgressed with ambystoma tigrinum (torres-sánchez, 2020), we decided not to consider them within the list of threatened taxa housed in italian museums. discussion extinct and threatened species in scientific collections the presence in natural history collections of amphibian and reptile species included within the iucn’s threatened categories gives the opportunity to unveil aspects otherwise difficult to obtain in the wild. as an example, the availability of a sufficiently large voucher series of some frogs of the genus mantella from madagascar allowed to investigate their fecundity (tessa et al., 2009), age structure (andreone et al., 2011) and, successively, to use these parameters to draw a general model exploitation method (andreone et al., 2021). differently from collecting for food, traditional medicine, fashion market, handicraft production, and other purposes, which are clearly recognized as relevant threats affecting rare and localized species (especially for the high number of traded individuals), a reasoned collecting of scientific vouchers is unlikely to be or become an table 2. list of critically endangered (cr), extinct (ex) and extinct in the wild (ew) amphibian species housed in italian natural history museums and their occurrence in the analysed collections. extinct species are given in bold. museums are reported according to the abbreviations provided in table 1. species iucn family museums hynobius abei cr hynobiidae fi hynobius okiensis cr hynobiidae fi chiropterotriton magnipes cr plethodontidae rm thorius pennatulus cr plethodontidae fi pseudoeurycea goebeli cr plethodontidae ge latonia nigriventer cr alytidae fi arthroleptis nikeae cr arthroleptidae tn atelognathus patagonicus cr batrachylidae fi callulina hanseni cr brevicipitidae tn callulina kanga cr brevicipitidae tn callulina laphami cr brevicipitidae tn callulina meteora cr brevicipitidae tn callulina shengena cr brevicipitidae tn callulina stanleyi cr brevicipitidae tn altiphrynoides osgoodi cr bufonidae fi atelopus boulengeri cr bufonidae to atelopus cruciger cr bufonidae to atelopus ignescens cr bufonidae fi, pd, pi, to atelopus longirostris ex bufonidae fi atelopus varius cr bufonidae pi, to churamiti maridadi cr bufonidae tn incilius cristatus cr bufonidae fi leptophryne cruentata cr bufonidae ge nectophrynoides asperginis ew bufonidae tn nectophrynoides laticeps cr bufonidae tn species iucn family museums nectophrynoides paulae cr bufonidae tn nectophrynoides poyntoni cr bufonidae tn nectophrynoides wendyae cr bufonidae tn werneria mertensiana cr bufonidae tn wolterstorffina parvipalmata cr bufonidae tn craugastor fleischmanni cr craugastoridae to craugastor lineatus cr craugastoridae fi craugastor milesi cr craugastoridae ge hyloxalus vertebralis cr dendrobatidae ge isthmohyla debilis cr hylidae rm hyperolius davenporti cr hyperoliidae tn pleurodema somuncurense cr leptodactylidae fi, to boophis ankarafensis cr mantellidae to boophis tsilomaro cr mantellidae to boophis williamsii cr mantellidae to guibemantis diphonus cr mantellidae to guibemantis punctatus cr mantellidae to mantella milotympanum cr mantellidae to mantidactylus pauliani cr mantellidae to platypelis karenae cr microhylidae to rana holtzi cr ranidae fi, to pseudophilautus leucorhinus ex rhacophoridae ge pseudophilautus nasutus ex rhacophoridae to pseudophilautus variabilis ex rhacophoridae ge, pi rhinoderma rufum cr rhinodermatidae to telmatobius laticeps cr telmatobiidae fi, to 51threatened amphibians and reptiles in italian museums extinction cause (rocha et al., 2014). we stress that specimen collection for scientific purposes is usually limited to a few vouchers, whose capture and collecting need to be authorised by national authorities and regulated by legislation. at the same time, we believe that the collecting activity of vouchers still remains crucial and should be maintained, as stressed by dubois (2003, 2010, 2017), to document not only the presence of rare and threatened species, but also biological parameters and represents an unsurpassed source of scientific data that are only in part exploited. finally, it is often crucial to witness the presence of a species at a confirmed geographic locality, possibly integrated with further evidences, such as edna, acoustic recordings, photographs, and footage. this is particularly important, as the collecting of series of specimens is also useful for conservation purposes, e.g., to identify negative trends in populations, especially in current times which are featured by dramatic changes of climatic and environmental parameters (e.g., hoffmann et al., 2010; hou et al., 2021). threatened and extinct species of amphibians and reptiles in italian collections some of the analysed italian nhms turned out to be especially relevant due to the high number of species table 3. list of critically endangered (cr) and extinct (ex) species of reptiles and natural history museums where they are preserved. extinct species are given in bold. museums are reported according to the abbreviations provided in table 1. species iucn family museums crocodylus intermedius cr crocodylidae pi, pr, rm crocodylus rhombifer cr crocodylidae fi, ge, gr, mi, vco-d, crocodylus siamensis cr crocodylidae fi, ge, mi, na, pi, pv, rm, to, to-db, tv mecistops cataphractus cr crocodylidae pi, to gavialis gangeticus cr gavialidae fi, ge, mi, na, pi, pr, pv, rm, to, to-db, tv eretmochelys imbricata cr cheloniidae bg, br, fi, ge, li, mi, pi, pr, pv, to, tv, ud, vco-d, ve lepidochelys kempii cr cheloniidae bg, ge dermatemys mawii cr dermatemydidae tv batagur baska cr geoemydidae ge batagur dhongoka cr geoemydidae ge batagur kachunga cr geoemydidae ge batagur trivittata cr geoemydidae bg, ge cuora trifasciata cr geoemydidae fi, ud heosemys depressa cr geoemydidae ge astrochelys radiata cr testudinidae fi, mi, na, pi, rm, si, to, ud chelonoidis porteri cr testudinidae rm geochelone platynota cr testudinidae ge psammobates geometricus cr testudinidae fi, pi, pv, rm testudo kleinmanni cr testudinidae ge hemidactylus bouvieri cr gekkonidae cn-b, ge lygodactylus williamsi cr gekkonidae tn conolophus marthae cr iguanidae rm acanthodactylus beershebensis cr lacertidae fi acanthodactylus harranensis cr lacertidae fi, rm-s, to-c eremias pleskei cr lacertidae to-c erythrolamprus cursor cr lacertidae ge gallotia simonyi cr lacertidae ge podarcis raffonei cr lacertidae fe, fi, pa, pd, rm liolaemus rabinoi cr liolaemidae fi chioninia coctei ex scincidae fi, ge, pa, to, tv mabuya mabouya cr scincidae fi, mi pseudoacontias menamainty cr scincidae to spondylurus culebrae cr scincidae ge atheris matildae cr viperidae tn 52 franco andreone et alii and specimens housed in the scientific collections under their care, and on the amount of threatened species. most of the extinct species present in these institutions were likely collected during general collecting activities and/ or obtained in exchange from other scientists/institutions. in the case of the kihansi spray toad nectophrynoides asperginis, a species from tanzania extinct in the wild due to the spread of the chytrid fungus (channing et al., 2006), the specimens were obtained in the context of structured multi-year research (menegon et al., 2004; msuya and mohamed, 2019). the giant cape verde skink chioninia coctei is present in a few italian museums, which are florence, genoa, palermo, turin, and treviso. of special relevance are the live individuals (around forty) imported by the herpetologist mario g. peracca at the end of the 19th century, and currently hosted in turin (andreone and gavetti, 2007, 2010). such a conspicuous purchase was made through an animal dealer, and was accompanied by the concurrent importation of some other rare or iconic live herps, i.e., andrias japonicus, aldabrachelys gigantea, astrochelys radiata, iguana iguana, and sphenodon punctatus (andreone and gavetti, 1998). peracca also made some interesting observations on the skink natural history, and then exchanged some individuals with other naturalists of his time, such as g. scarpa in treviso (andreone et al., 2010). after peracca’s death some of these skinks were donated (as other animals) to the turin museum, which in fact was not the commissioner for the collecting of a rare and threatened species, but just its final repository. with respect to amphibians, the institution hosting the highest number of cr species is the turin museum, with 17 taxa, eight of which originated from the collecting surveys carried out during field-work in madagascar (andreone et al., 2005, 2021), followed by muse museo delle scienze in trento, with 15 species from tanzania and other eastern african countries. further remarkable species available in turin come from latin america, mostly due to the activity of the italo-argentinean herpetologist j. m. cei (cei, 1993). florence and genoa are also the nhms holding the highest number of cr reptile species. this highlights the importance of active research in the constitution of study collections. the cr amphibian species housed in trento originated from systematic field research carried out over the past 20 years in the forests of the eastern afromontane ark (menegon et al., 2008). many natural history museums supported, among their institutional activities, survey works in unexplored or marginally explored areas of our planet. in particular, this was one of the ultimate aims of middle-large museums, where collections were usually regarded as voucher repositories (grimaldi and engel, 2007; engel et al., 2021), much less for smaller museums where the education aspects are usually prioritised. is scientific voucher collecting still a needed practice? in italy, many museums supported collecting activities in the past, but only a few ones pursue research and specimens collecting, especially overseas. in fact, in the 19th century many naturalists gravitating around italian museums were engaged to explore the world and to collect new materials (mazzotti, 2011), such as a. borelli, e. festa, and f. de filippi in turin, l. d’albertis, g. doria, and l. fea in genoa, o. beccari and e. h. giglioli in florence, g. scortecci in milan, o. antinori in perugia, and many others, who mirrored the adventures and travels of victorian naturalists, contributing to discover new species and describing the still unknown world. the beginning of 20th century, however, coincided with a decrease in such activities in most of the italian museums: the systematic and taxonomic zoology and botany that fed those travels were largely left behind, often considered useless and subsidiary to the newly affirmed organismic biology. at the same time, museums were often seen as mere repositories and/or expositive locations, and much less (or no more) as research centers (fischer, 2015). for these reasons too, many ancient collections were neglected and rarely utilised for either scientific or educational purposes (ceríaco et al., 2021). to better frame this situation, it should also be remembered that many italian museums were, and still are, managed by local administrations, such as municipalities, provinces, and regions. this often led to a difficult balance between the expositive/outreach finality and research/collection components, in particular since museums were often nested within culture or education departments and only rarely associated with research and/ or environmental ones. therefore, while research was progressively relegated to a subsidiary activity, most natural history museums acquired a prominent expositive function, sometimes detaching the physiological link between these “souls”. only a few museums appear to have escaped this trend, such as those of turin (andreone, 2013), trento (menegon et al., 2008), verona (latella and zorzin, 2018), and florence (van lien et al., 2014), whose personnel was able to carry out recent oversea research. taking into consideration that many (16) of the analysed italian nhms have herpetologists as curators, it is worth to verify whether their presence is accompanied by a better knowledge of amphibian and reptile taxonomy in their collections. the taei varied from 1 (one collection) to 4 (22 collections), with a mean value of 3.41 + 53threatened amphibians and reptiles in italian museums 0.82, thus indicating that most of these collections were revised recently. this happened mostly for small collections, which were objectively easier to be studied and catalogued, and usually limited to italian/european faunae. since most of these collections are not formed recently, the majority of italian collections are increasingly becoming historical, with recent acquisitions largely due to occasional specimen collections. we consider this a heavy bias, since it means a loss of taxonomic expertise which may have a negative impact on the increase and valorization of scientific collections. andreone et al. (2014), following a proposal by minelli (2013, 2015), suggested that, in absence of a traditional national museum, a “diffuse network” or “metamuseum” could be a solution to manage the italian scientific collections in a joint way, also to share resources and personnel. although little was done to accomplish this proposal so far, this is still an option to be taken into serious consideration together with the possibility of creating a centralized coordination hub. considering the present fragmentation and the scarce connection among museums, it is first of all important that all italian natural history collections are increasingly revised and digitalized by each museum, hopefully using shared protocols. this would be enhanced by the establishment of a national strategy that encompasses the coordination and resource distribution as a priority objective. acknowledgements many friends helped us during the data collection and collection revisions. fa wishes to thank e. gavetti and l. ghiraldi for their assistance with maintaining and organizing the turin herpetological collection. then for useful discussions and practical help over the years he thanks a. angulo, c. barale, s. bovero, m. brocchieri, a. crottini, f. gallo, s. gippoliti, j. luetdke, m. menegon, and v. mercurio. special thanks to the teams which coordinated the vertex and extinction projects. finally, we thank a. kupfer, an anonymous referee for useful comments on a preliminary version of this contribution , and a. gambarelli for useful information. we dedicate this paper and work to the memory of two “giants” of herpetology, j. m. cei and b. lanza. references alberch, p., zavala, l., miles, r. 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(1990): catalogo della collezione erpetologica del museo civico di storia naturale di morbegno (so). il naturalista valtellinese. atti mus. civ. stor. nat. morbegno 1: 61-78. appendix i list of amphibian and reptile families conserved in the italian natural history collections amphibia gymnophiona – caeciliidae, dermophiidae, herpelidae, ichthyophiidae, scolecomorphidae, siphonopidae, typhlonectidae. anura – alsodidae, alytidae, arthroleptidae, ascaphidae, batrachylidae, bombinatoridae, brachycephalidae, craugastoridae, brevicipitidae, bufonidae, calyptocephalellidae, centrolenidae, ceratobatrachidae, ceratophryidae, conrauidae, eleutherodactylidae, cycloramphidae, aromobatidae, dendrobatidae, dicroglossidae, heleophrynidae, hemiphractidae, hemisotidae, hylidae, hylodidae, hyperoliidae, leiopelmatidae, leptodactylidae, mantellidae, megophryidae, micrixalidae, microhylidae, limnodynastidae, myobatrachidae, nyctibatrachidae, odontophrynidae, pelobatidae, pelodryadidae, pelodytidae, petropedetidae, phrynobatrachidae, phyllomedusidae, pipidae, ptychadenidae, pyxicephalidae, ranidae, ranixalidae, rhacophoridae, rhinodermatidae, rhinophrynidae, scaphiopodidae, telmatobiidae. urodela – ambystomatidae, amphiumidae, hynobiidae, plethodontidae, proteidae, rhyacotritonidae, salamandridae, sirenidae. reptilia testudines emydidae, testudinidae, geoemydidae, platysternidae, trionychidae, chelydridae, dermatemydidae, kinosternidae, cheloniidae, dermochelyidae, chelidae, pelomedusidae, podocnemididae. rhynchocephalia – sphenodontidae. crocodylia –  crocodylidae, gavialidae, alligatoridae. sauria – agamidae, chamaeleonidae, corytophanidae, crotaphytidae, dactyloidae, hoplocercidae, iguanidae, leiocephalidae, leiosauridae, liolaemidae, opluridae, phrynosomatidae, polychrotidae, tropiduridae, gekkonidae, carphodactylidae, diplodactylidae, eublepharidae, phyllodactylidae, sphaerodactylidae, pygopodidae, cordylidae, gerrhosauridae, scincidae, xantusiidae, gymnophthalmidae, lacertidae, teiidae, anguidae, diploglossidae, xenosauridae, amphisbaenidae blanidae, rhineuridae, 58 franco andreone et alii trogonophidae, helodermatidae, varanidae, dibamidae. ophidia – acrochordidae, cylindrophiidae, uropeltidae, loxocemidae, pythonidae, xenopeltidae, boidae, colubridae, atractaspididae, cyclocoridae, lamprophiidae, psammophiidae, pseudaspididae, elapidae, anomalepididae, gerrhopilidae, typhlopidae, leptotyphlopidae, xenotyphlopidae, aniliidae, homalopsidae, pareidae, tropidophiidae, viperidae, xenodermidae. acta herpetologica 17(2): 115-124, 2022 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-12556 preliminary genetic characterisation of southern smooth snake coronella girondica (serpentes, colubridae) populations in italy, with some considerations on their alpine distribution matteo r. di nicola1, raffaella melfi2, francesco p. faraone3,*, daniel l.n. iversen4, gabriele giacalone5, giovanni paolino1, mario lo valvo6 1 unit of dermatology, irccs san raffaele hospital, via olgettina 60, 20132 milan, italy 2 dipartimento di scienze e tecnologie biologiche, chimiche e farmaceutiche (stebicef), university of palermo, viale delle scienze ed. 16-17, 90128 palermo, italy 3 viale regione siciliana s.e., 532, 90129 palermo, italy 4 viale giovanni prati, 38066, riva del garda, italy 5 cooperativa silene, via d’ondes reggio, 8/a, 90127 palermo, italy 6 dipartimento scienze e tecnologie biologiche, chimiche e farmaceutiche, university of palermo, via archirafi, 18, 90123 palermo, italy *corresponding author. email: paolofaraone@libero.it submitted on: 2022, 8th january; revised on: 2022, 4th march; accepted on: 2022, 4th may editor: adriana bellati abstract. the southern smooth snake, coronella girondica, is a small-sized colubrid found in northwest africa and southwest europe. mitochondrial dna-based studies showed that the species can be split into five clades: two from northwest africa (one moroccan and one tunisian-algerian) and three from europe (one in the south-west of the iberian peninsula, one in the south-east of spain and one in the rest of the european range). with regards to italy, to date, only two samples have been analysed both from the province of pisa, tuscany, pointing at that fact that genetic characterisation of italian populations is still lacking. accordingly, we have increased the sampling coverage with 19 new samples from northern and central regions of italy, including two populations, apparently disconnected from the rest of the known range, and analysed their phylogenetic relationships using a portion of the mitochondrial cytochrome b gene. our results confirm the general phylogenetic arrangement detected in previous studies; specifically for italian populations, no variability emerged from the apennine populations, and a slight differentiation could be shown for the alpine and subalpine ones. this pattern can be explained assuming past spread and recent isolation of c. girondica relict populations in the alpine region, likely during the last glacial maximum. later, during the holocene, the italian alps and the po plain went through various climatic variations and high anthropization which may have influenced c. girondica distribution through expansion and contraction processes. keywords. coronella girondica, italy, distribution, relict populations. introduction the southern smooth snake coronella girondica (daudin, 1803) is a small-sized colubridae with a mediterranean chorotype (west-mediterranean, sindaco et al., 2013). the species is present in northwest africa (north-central morocco, northern algeria and northwestern tunisia) and southwest europe (portugal, spain, southern france and peninsular italy), where it usually lives in dry and stony mediterranean scrub habitats (sindaco et al., 2013; geniez, 2018; di nicola et al., 2021). 116 matteo r. di nicola et alii with regards to italy, the secretive and mainly crepuscular habits of the species (ferri and morimando, 2004; razzetti and bernini, 2011) hampered the full description of its distribution framework for a long time (razzetti and bonini, 2006) and for some areas its presence has even only been recently confirmed (razzetti et al., 2000; capula et al., 2010; rugiero et al., 2018; di nicola et al., 2020; ferri and soccini, 2020; iversen et al., 2020). the species, originally described by daudin (1803) as coluber girondicus, is currently considered monotypic (razzetti and bernini, 2011; santos and pleguezuelos, 2015; sindaco and razzetti, 2021). subsequently, rhinechis amaliae (boettger, 1881) was described from moroccan specimens, on the basis of a single morphological character relating to the rostral zone (santos and pleguezuelos, 2015). however, further morphological investigations did not provide support for the validity of this taxon (saint girons, 1956; domergue, 1962; santos and pleguezuelos, 2003). more recently, santos et al. (2012a) performed a molecular study across the distribution range of the whole species using analysis of mitochondrial genes and highlighted the presence of three major clades: one from northwest africa, one from the southeast of spain and the latter occurring in the rest part of the european range. according to their calibration, the divergence among the clades occurred around 1.4-2.0 ma, roughly coinciding with the plio-pleistocene transition. within two out of the three major clades, further differentiation was detected into five clades (see santos et al., 2012b): two from northwest africa (one moroccan and one tunisian-algerian) and three from europe (one in the south-west of the iberian peninsula, one in the south-east of spain and one in the rest of europe). in italy, c. girondica is distributed in the north-western part of the country and extends south to the gargano peninsula (northern apulia) and southern lazio, including the region of molise (capula et al., 2010; razzetti and bernini, 2011; rugiero et al., 2018; di nicola et al., 2021). recently, two apparently isolated populations have been found in western lombardy (di nicola et al., 2020) and around lake garda, in an area that is transitional between the trentino-alto adige, veneto and lombardy regions (ferri and soccini, 2020; iversen et al., 2020). these isolated populations are out of the geographical range of the other populations by around 70 km east and 160 km north (fig. 1, 2). with regards to genetic characterisation of the species in italy, only two samples have been analysed, both from the province of pisa (genbank accession numbers jq837570 and jq837571). the present work therefore describes, for the first time, the intraspecific relationships between italian populations of c. girondica including some recently confirmed observations from northern italy (di nicola et al., 2020; ferri and soccini, 2020; iversen et al., 2020). materials and methods given the lack of adequate sample coverage within the italian range of the species, a total of 19 new samples were collected from seven regions of northern and central italy (table 1; fig. 1), including five samples from the isolated populations, respectively western lombardy (n = 1) and trentino-alto adige (n = 4). small tissue fragments (tail tips, pieces of ventral scales) were removed and preserved in ethanol 96% from individuals that were found dead (most of the samples) or live specimens. for live specimens, a non-invasive scale clipping of ventral scales was performed. coronella girondica was sampled by nocturnal active search only at two separate localities of arco (iversen et al., 2020) and somma lombardo (di nicola et al., 2020), while in other cases samples were recovered from dead specimen as roadkills, domestic cats or human persecution (table 1). in order to infer the phylogenetic relationships between italian samples and other populations, we selected the mitochondrial dna cytochrome b (cyt b) gene, a marker often used to infer intra-specific diversity on many vertebrates and invertebrates species, including snakes (carranza et al., 2006; mezzasalma et al., 2015; faraone et al., 2020b). approximately 20 mg of tissue was used to extract total dna as described in tagliavia et al. (2016). genomic dna was used as a template for pcr amplification with primers cb1(f) (5’ccatccaacatctcagcatgatgaaa3’) and cb2(r) (5’ccctcagaatgatatttgtcctca3’) (carranza et al., 1999). dna bands of the expected size (~300 bps) were obtained and then sequenced with the primer cb1(f) (bmr genomics, padua, italy). the resulting sequences were each around 255 nucleotides long and were analysed and manually proof-read with the dna sequencing software chromas v. 2.6.6 (technelysium pty. ltd. 1998, queensland, australia). the coding gene fragments of cyt b were translated into amino acids to assess the lack of premature stop codons. later, using clustal w (larkin et al., 2007) with default parameters, the sequences from italian samples generated in this study were aligned with homologous sequence downloaded from genbank (carranza et al., 2004; santos et al., 2008, 2012a; carvalho et al., 2017). four species belonging to colubridae and psammophiidae families were used as outgroups (carranza et al., 2006; santos et al., 2012a; faraone et al., 2020a, b). 117genetic characterisation of coronella girondica in italy the phylogenetic analysis was performed with maximum likelihood (ml) under the akaike information criterion using the “smart model selection” (sms) (lefort et al., 2017), implemented in phyml v. 3 (guindon et al., 2010). jukes-cantor (jc) (jukes and cantor, 1969) was the most appropriate evolutionary model (-log likelihood value 1283.58), with a 0.32 gamma estimate of invariable sites and a 1.00 discrete approximation of the gamma distribution. the same model was obtained by using both all available sequences and by previously collapsing the sequences into haplotypes. node support was estimated by bootstrap (felsenstein, 1985) with 1,000 replicates and the mega x software (tamura et al., 2021) was used to implement the ml tree. the unrooted minimum spanning network were obtained using the median-joining algorithm (bandelt et al., 1999) implemented in popart (http://popart.otago.ac.nz/) (leigh and bryant, 2015). with the aim of consolidating current knowledge of c. girondica distribution in northern italy, bibliographical data from razzetti and bonini (2006) were recorded. furthermore, unpublished observations from the northern po river area were collected between 2011 and 2021 from authors’ field observations and online records. all observations that were not reported by razzetti and bonini (2006) were considered new and, subsequently, compared with previous unconfirmed findings. in addition, citizen science (see haklay et al., 2021) was also critical to the generation of distributional datasets through collaborative efforts between herpethologists around italy and users of a facebook group managed by two of the authors (mrdn and fpf) “identificazione table 1. italian samples and observation details of coronella girondica used in the present study. the numbers and letters reported respectively in the first and second column are referred to localities shown in fig. 1 and fig. 2. the haplotype code is shown in brackets after the genbank accession number. samples marked with an asterisk were previously published by santos et al. (2012a). fig. 1 fig. 2 year locality n e observer/reference accession number 1 c 2019 arco, trentino alto adige 45.9238° 10.9433° iversen et al., 2020 ok573460 (h2) 2 c 2020 arco, trentino alto adige 45.9238° 10.9433° iversen et al., 2020 ok573463 (h2) 3 c 2020 arco, trentino alto adige 45.9238° 10.9433° iversen et al., 2020 ok573462 (h2) 4 c 2021 arco, trentino alto adige 45.9238° 10.9433° iversen et al., 2020 ok573461 (h2) 5 g 2020 somma lombardo, lombardy 45.6713° 8.6833° di nicola et al., 2020 ok573464 (h2) 6 2020 cassinelle, piedmont 44.5760° 8.5616° cavanna s. pers. obs. ok573465 (h3) 7 2020 isola del cantone, liguria 44.6432° 8.9664° de cresi u. pers. obs. ok573473 (h1) 8 2018 albenga, liguria 44.0970° 8.2129° graglia m. pers. obs. ok573469 (h1) 9 2018 albenga, liguria 44.0970° 8.2129° graglia m. pers. obs. ok573468 (h1) 10 2019 albenga, liguria 44.0970° 8.2129° graglia m. pers. obs. ok573467 (h1) 11 2020 peagna, liguria 44.0989° 8.2013° graglia m. pers. obs. ok573472 (h1) 12 2020 peagna, liguria 44.0989° 8.2013° graglia m. pers. obs. ok573471 (h1) 13 2020 peagna, liguria 44.0989° 8.2013° graglia m. pers. obs. ok573470 (h1) 14 2020 aurigo, liguria 43.9953° 7.9209° fecchio l. pers. obs. ok573466 (h1) 15 2021 vigolzone, emilia romagna 44.9110° 9.6879° gereschi v., mazzotta m. pers. obs. ok573478 (h4) 16 2020 foreste casentinesi, emilia romagna 43.8874° 11.8939° molinari g. pers. obs. ok573475 (h1) 17 2021 foreste casentinesi, emilia romagna 43.8874° 11.8939° molinari g. pers. obs. ok573476 (h1) 18 n/a s. giuliano terme, tuscany* 43.7579° 10.4434° santos et al., 2012a jq837570 (h1) 19 n/a s. giuliano terme, tuscany* 43.7579° 10.4434° santos et al., 2012a jq837571 (h1) 20 2020 piombino, tuscany 42.9268° 10.5310° banchi r. pers. obs. ok573474 (h1) 21 2020 capestrano, abruzzo 42.2867° 13,7942° d’amico m. pers. obs. ok573477 (h1) a 2011 rivoli veronese, veneto 45.5872° 10.8215° campagnari m. pers. obs. a 2013 caprino veronese, veneto 45.5898° 10.8215° campagnari m. pers. obs. b 2020 avio, trentino alto adige 45.7383° 10.9433° secchi m. pers. obs. d 2019 pietramurata, trentino alto adige 46.0268° 10.9420° iversen et al., 2020 e 2020 limone sul garda, lombardy 45.8108° 10.7866° di nicola et al., 2020 f 2020 toscolano maderno, lombardy 45.6666° 10.6166° ferri & soccini, 2020 h 2021 sostegno, piedmont 45.6658° 8.2852° zonari a. pers. obs. i 2015 zubiena, piedmont 45.4833° 8.0333° ciracì a. pers. obs. 118 matteo r. di nicola et alii anfibi e rettili”. the users of the facebook group provided locations for known european amphibians and reptiles and provided further distributional sites and samples for c. girondica found dead in various italian regions. for each observation recorded through social networks, the authors meticulously checked the original files, the species identification and the location provided by the users through field survey of the coordinates. the new observations were mapped using adobe photoshop cc (©19902018 adobe systems incorporated, release 19.1.5), together with those available from recent literature (razzetti and bonini, 2006; di nicola et al., 2020; iversen et al., 2020; ferri and soccini, 2020; di nicola et al., 2021). results overall, 94 sequences of 255 bp total length were analysed including the outgroups and the results confirm the same overall phylogenetic arrangement previously shown by santos et al. (2012a) with five major clades (fig. 3). all the 21 italian samples (genbank accession numbers jq837570, jq837571 and ok573460-78) fall within ‘clade 5’ (sensu santos et al., 2012a) which also includes sequences from france and most of iberian peninsula (fig. 3). however, the monophily of ‘clade 5’ is not adequately supported (bootstrap = 65%) on the basis of the cyt b fragment analysed here and it is considered as an haplogroup. we found four cyt b haplotypes amongst the italian c. girondica populations, differing by 1-2 mutation steps (fig. 1). most of the samples (n: 14) shared the same haplotype (h1), which is also shared with samples from spain (jq837574, jq837587, jq837589, jq837607, jq837610, jq837635) and portugal (jq837569, jq837582, jq837595, jq837634) (fig. 3). in contrast, the sample from emilia romagna (ok573478) share the haplotype h4 with a sample from ciudad real, spain (jq837591). the following two unpublished haplotypes were detected among the northernmost italian samples: h2 shared by all the five samples of the recently confirmed separated localities in western lombardy (ok573464) and trentino-alto adige (ok573460-63) and h3 from southern piedmont (ok573465). h2 and h3 were shown to belong to the same haplogroup (figs. 1, 3). with the exclusion of the observations recently reported in the literature (iversen et al., 2020; di nicola et al., 2020; ferri and soccini, 2020), three new localities (b, i, h) that were not recently confirmed (razzetti and bonini, 2006) have been registered, see table 1 and fig. 2 for details. observation b resulted from a live adult snake observed on a low wall near avio (province of trento, trentino-alto adige) just east of lake garda, as part of a cluster which includes observations recently published by iversen et al. (2020) and ferri and soccini (2020) (table 1, fig. 2). observations i and h corresponded to a live and a road-killed snake respectively and were both recorded in the biella province (piedmont) falling within the geographic range already known in the north-western regions of the alps (razzetti and bonini, 2006) and the point g, a separated locality recently reported in lombardy by di nicola et al. (2020) (fig. 2). discussion the results presented here confirm a low genetic diversity for c. girondica within the cluster ‘clade 5’, as shown by santos et al. (2012a). for this haplogroup, rapid expansion process from south to north has been hypothesised to be likely derived from climatic warming events and followed by a bottleneck effect (santos et al., 2012a). this pattern of recent expansion from the iberian peninfig. 1. minumum spanning network based on the coronella girondica cytochrome b fragment in italy, and geographic distribution of the haplotypes. circle sizes are proportional to haplotype frequency, and each black bars represents a mutational step. samples are numbered as in table 1. the green area in the map represents the approximate range of c. girondica in italy. 119genetic characterisation of coronella girondica in italy sula towards the north-east has also been hypothesised for other snake species such as natrix maura (guicking et al., 2002). the genetic structure that emerges from the italian c. girondica samples shows a lack of mitochondrial variability in the apennine cyt b samples and a slight differentiation in alpine and subalpine samples (fig. 1, 3), which highlight that the endemic haplotypes h2 and h3 belong to the same cluster. this mtdna pattern partially matches with that from other species of the italian herpetofauna (canestrelli et al., 2007; canestrelli and nascetti, 2008; salvi et al., 2013; chiocchio et al., 2021), including the barred grass snake natrix helvetica (schultze et al., 2019) and confirms the margin of the northern apennines as a suture area of the italian peninsula (hewitt, 2011; chiocchio et al., 2021). this is compatible with recent spreading and isolation of c. girondica in northern italy, likely during the last glacial maximums, which allowed a slight haplotype differentiation. subsequently, the italian alps and the po plain went through various climatic fluctuations and extensive human impact during the holocene (colombaroli et al., 2010; nussbaumer et al., 2011; joannin et al., 2013). these factors may have modulated the distribution of c. girondica with expansion and contraction processes. the h4 haplotype, found in a single italian sample (ok573478) collected in emilia romagna (table 1, figs. 1, 3), is identical to jq837591 from ciudad real, central spain (fig. 3). this observation is similar to other cases reported for natrix natrix and n. helvetica (kindler et al., 2017; schultze et al., 2019, 2020) and could be indicative of human translocation events. however, this hypothesis still needs further investigation through testing additional markers. recent italian findings of c. girondica north of the po river can be grouped into two main clusters. a northwestern alpine cluster includes some alpine valleys of northern piedmont and aosta valley, and it is apparently separated from both the neighbouring french and italian populations (razzetti and bonini, 2006; sillero et al., 2014). an eastern alpine cluster is located around lake garda, and falls within the territories of lombardy, trentino-alto adige and veneto (iversen et al., 2020; di nicola et al., 2020; ferri and soccini, 2020). the populations around lake garda have been reported from the literature with many detailed records (de betta, 1857; dalla torre, 1912) and later attested only by a few museum specimens dated up to 1977 (see iversen et al., 2020). other more recent information has been reported by lorenzi and bruno (2006) which, however, do not fig. 2. distribution of coronella girondica in the italian alps north of the po river. red circle are the areas reported up to razzetti and bonini (2006), green landmarks represent the observations published after 2006 (di nicola et al., 2020; iversen et al., 2020; ferri and soccini, 2020), yellow landmarks are the unpublished findings. the details of each point are shown in table 1. 120 matteo r. di nicola et alii fig. 3. maximum likelihood (ml) tree of coronella girondica inferred from the mitochondrial cytochrome b gene, with a detail on the haplotypes of the ‘clade 5’ (sensu santos et al., 2012a) cluster. the haplotypes that occur in italy are marked with the same colors used in fig. 1. the numbers at nodes are ml bootstrap percentages. with the exception of the unpublished samples (see table 1) all the others are taken from literature (carranza et al., 2004, 2006; santos et al., 2008, 2012a, b; carvalho et al., 2017; faraone et al., 2020a, b). 121genetic characterisation of coronella girondica in italy clearly contextualise their personal observations. the scarcity of findings during the twentieth century has led zoologists to consider this population close to extinction (razzetti and bonini, 2006) or probably extinct (razzetti and bernini, 2011). recently, new observations led to the confirmation of the presence of c. girondica in veneto (novarini et al., 2017; iversen et al., 2020), trentino-alto adige (iversen et al., 2020) and lombardy (di nicola et al., 2020; ferri and soccini, 2020). new observations around lake garda largely confirmed previous records provided by dalla torre (1912), including the more recent point “b” (fig. 2), which falls within a locality highlighted by the author between 1896 and 1900. the observations “i” and “h” are located between the northwestern cluster and point “g”, recently reported in lombardy by di nicola et al. (2020) (table 1, fig. 2). point “i” is an unpublished locality, while “h” surprisingly confirms a site with a single specimen (msnm re 1439) preserved in the civic natural history museum of milan (see also andreone and sindaco, 2002), dated back to 1926. the geographic position of points “h” and “i” (fig. 2) suggests that the north-western cluster and point “g” could be connected by small, scattered and low-density populations that may be detected by increasing the environmental monitoring of the species. as indicated by bombi et al. (2009), with the exclusion of the maritime alps and the northern apennines, northern italy has very low suitability values for c. girondica since only a few scattered sub-optimal patches in the area north of the po river have been found. the results obtained here confirm this scenario, since c. girondica has a clearly fragmented distribution in the alpine and subalpine areas. furthermore, this species is mostly found in habitats within sub-mediterranean climates, often characterised by xerophilous faunistic and vegetational communities which are very specific compared to other habitats and surrounding territories (la greca, 1956; gratani and varone, 2003; agabiti et al., 2005). in conclusion, the results generated in this study suggest that the fragmented distribution of c. girondica north of the po river can be mainly attributed to relict populations, based on the following points: (a) the finding of endemic haplotypes, compatible with a recent separation occurred during the last glacial events; (b) the few and scattered observations, highly localised in small patches with xeric mediterranean features, suitable for the species; (c) current records largely match with historical records which, on the other hand, indicate few alpine areas without recent confirmation; (d) the post-glacial history of northern italy is characterised by changes in ecosystems caused by climatic fluctuations and a strong human impact, and this could have caused the expansion and contraction of c. girondica as a thermophilic snake. this hypothesis will be further investigated through more extensive sampling in the field and the analysis of a greater number of loci in order to further detail the genetic structure of the italian c. girondica populations. a greater fieldwork effort will be necessary, especially on the western alps, where our results could indicate a probably incomplete c. girondica distribution, which may be slightly less fragmented than previously thought. acknowledgements the authors would like to thank all the observers reported on table 1 for their valuable contribution; matteo graglia, sergio mezzadri and giuseppe molinari for their precious contribution in the fieldwork, and andrea ciracì for his information. the authors are also grateful to roberta alberigo, federica bracaletta, simona corneti, francesco di toro, eduardo di trapani, lorenzo laddaga, luca miselli, massimo pellegrini, silvia pelti, federico pino, marta teves, marina trevisan for reporting us c. girondica specimens during their hikes. thanks also to giovanni boano, director of the civic natural history museum of carmagnola (turin), and stefano scali from the civic natural history museum of milan. finally, thanks to enrica calò and lesley dhonau for a pre-review of the manuscript and to jean-lou dorne for the proofreading. the animals were handled following ministerial permits: mattm reg. 0083124, 16/10/2020; prot. ispra 46387, 12/10/2020. references agabiti, b., valentinotti, r., salvadori, c. 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(2021): mega11: molecular evolutionary genetics analysis version 11. mol. biol. evol. 38: 3022-3027. acta herpetologica vol. 17, n. 2 december 2022 firenze university press cryptic diversity in pygmy chameleons (chamaeleonidae: rhampholeon) of the eastern arc mountains of tanzania, with description of six new species michele menegon1,2,*, john v. lyakurwa3,4, simon p. loader5, krystal a. tolley6,7 preliminary genetic characterisation of southern smooth snake coronella girondica (serpentes, colubridae) populations in italy, with some considerations on their alpine distribution matteo r. di nicola1, raffaella melfi2, francesco p. faraone3,*, daniel l. n. iversen4, gabriele giacalone5, giovanni paolino1, mario lo valvo6 species diversity and distribution of amphibians and reptiles in sardinia, italy claudia corti1,2,*, marta biaggini1, valeria nulchis2, roberto cogoni2, ilaria maria cossu2, salvatore frau4, manuela mulargia2, enrico lunghi2, lara bassu2. the italian wall lizard, podarcis siculus campestris, unexpected presence on gorgona island (tuscan archipelago) marco a.l. zuffi1,*, alan j. coladonato2, gianluca lombardo3, antonio torroni3, matilde boschetti1, stefano scali4, marco mangiacotti2, roberto sacchi2 molecular analysis of recently introduced populations of the italian wall lizard (podarcis siculus) oleksandra oskyrko1,2,*, lekshmi b. sreelatha1,12,13, iolanda silva-rocha1, tibor sos3,4, sabina e. vlad5,6,7, dan cogălniceanu5,6, florina stănescu6,7,8, tavakkul m. iskenderov9, igor v. doronin10, duje lisičić11, miguel a. carretero1,12,13 sunny-side up: ontogenetic variation in egg mass temperatures of the wood frog rana sylvatica ryan calsbeek*, ava calsbeek, isabel calsbeek ecological niche differentiation in the anatolian rock lizards (genus: anatololacerta) (reptilia: lacertidae) of the anatolian peninsula and aegean islands mehmet kürşat şahin1,*, kamil candan2,3, danae karakasi4, petros lymberakis4, nikos poulakakis4,5,6, yusuf kumlutaş2,3, elif yıldırım2,3, çetin ilgaz2,3 occupancy and probability of detection of the introduced population of eleutherodactylus coqui in turrialba, costa rica jimmy barrantes-madrigal1,*, manuel spínola parallada1, gilbert alvarado 2, víctor j. acostachaves3,4. one site, three species, three stories: syntopy of geckoes euleptes europaea (gené, 1839), hemidactylus turcicus (linnaeus, 1758), tarentola mauritanica (linnaeus, 1758) in a coastal area of southern tuscany (central italy) giacomo radi1,2, marco a.l. zuffi1,* comparative cytogenetics on zamenis lineatus and elaphe quatuorlineata (serpentes: colubridae) marcello mezzasalma1,* , elvira brunelli1, gaetano odierna2, fabio m. guarino2 on the occurrence of the italian aesculapian snake, zamenis lineatus (camerano, 1891), in latium (central italy) luigi corsetti1, antonio romano2 1 via adige 45/2, 04100, latina, italy 2 dipartimento di biologia, università degli studi di roma “tor vergata”, via della ricerca scientifica, i-00133 roma. present address: via creta 6, 04100, latina, italy. corresponding author. e-mail: antonioromano71@tele2.it submitted on 2008, 16th february; revised on 2008, 7th july; accepted on 2008, 18th august. abstract. in this note we report the first detailed notice of the presence of the italian aesculapian snake, zamenis lineatus, in latium region. the localities of morolo (province of frosinone) and latina scalo (province of latina) represent respectively the new northern and the new western limits of the species. the first one is 68 km further north compared to the old one (pannarano, campania region), whereas the western one is 130 km far from the previous one (roccarainola, campania region). keywords. snakes, zamenis lineatus, distribution, central italy. the formerly elaphe longissima laurenti, 1768 has been splitted into two species by lenk and wüster (1999), who used multivariate analyses of external morphological characters and colour pattern. the aesculapian snake, zamenis longissimus (laurenti, 1768), and the italian aesculapian snake, z. lineatus (camerano, 1891), are distinguishable using external morphological characters (lenk and wüster, 1999), molecular data (lenk et al., 2001; utiger et al., 2002), differences in the blood proteins (lenk and joger, 1994), visceral organ topography, osteology and allozymes (helfenberger, 2001). the italian aesculapian snake is endemic to the southern italian peninsula and sicily, and its occurrence in sardinia needs to be confirmed (see razzetti and zanghellini, 2006). lenk and wüster (1999) indicated the northern limit of z. lineatus near pannarano (province of benevento, campania region, southern italy), and they established the southern limit of z. longissimus near rosello (province of chieti, abruzzi, central italy; see also razzetti and zanghellini, 2006). however, recently, capula et al. (2006) revised the distribution of both species in italy considering the campania region as a parapatric contact area and suggesting analogous pattern for apulia region where they recorded z. longissimus for the first time. consequently the distribution range of the aesculapian snake was acta herpetologica 3(2): 179-183, 2008 issn 1827-9643 (online) © 2008 firenze university press 180 l. corsetti and a. romano wider than that previously suggested (lenk and wüster, 1999), and in some areas there appears to be sympatry between the two species. the most evident character with which we can tell the two species apart is their iris colour: reddish in z. lineatus and brown, yellowish or greyish in z. longissimus (schulz, 1995; lenk and wüster, 1999). by analysing our old pictures of aesculapian snakes and by using this diagnostic character, we realized that during our previous field researches on the volsci chain mountains (latium region) we had found both the species (e.g., corsetti and capula, 1992; corsetti, 2006a; corsetti and romano, 2007; romano et al., 2007). the southern parts of this chain comprises three karstic subgroups: the lepini, the ausoni and the aurunci mountains (see corsetti and romano, 2007 and romano et al., 2007, and references therein for detailed information about the boundaries among the three subgroups). while distribution of zamenis longissimus in latium is largely known (e.g., cattaneo and capula, 2000), there are no accurate data on the occurrence of z. lineatus in this region. actually, just a popular book (corsetti, 2006b) and a recent paper (capula et al., 2008) reported some information on the occurrence of z. lineatus in southern latium. corsetti (2006b) reported the occurrence of both z. lineatus and z. longissimus on the lepini mountains, while capula et al. (2008) reported that z. lineatus was present in the ausoni and aurunci, however without providing any detailed locality. here we provide accurate records of the presence of both species in the entire volsci chain. italian aesculapian snakes were observed in 7 locations, two as road kills and the others as living snakes (table 1 and fig. 1). the dead individual from site 3 was collected, stored in ethanol and deposited in the collection of the museo civico di storia naturale di carmagnola, italy (voucher mcc-r1407). it was a male 131.5 cm long, with table 1. records of zamenis lineatus on the volsci chain, latium, central italy. records with “*” were road-killed snakes. the specimen from site 3 was deposited in the collection of the museo civico di storia naturale di carmagnola, italy (voucher mcc-r1407). site code site locality, municipality and province subgroup of the volsci chain mountain altitude (m a.s.l.) utm observation date photo sex 1 morolo, morolo, frosinone lepini 450 ug41 24.05.1981 + male 2 latina scalo, latina, latina lepini 15 ug30 27.09.1987 + juv. 3 camposoriano, terracina, latina ausoni 360 uf58 05.08.2004 + male* 4 la taverna, campodimele, latina aurunci 390 uf87 13.06.2000 + juv.* 5 lombriccio, formia, latina aurunci 330 uf87 15.06.2004 male 6 cerretello, esperia, frosinone aurunci 825 uf98 18.05.2000 + undet. 7 campolaloa, itri, latina aurunci 260 uf66 23.05.2008 + undet. 181occurrence of zamenis lineatus in latium 23 dorsal scales (at mid-body), 235 ventral scales and 72 pairs of subcaudal scales. in the other cases the snakes were always captured, sexed (except two) and photographed (with one exception; see tab.1). all living snakes were immediately released after biometrical recordings. fig. 1. records of zamenis lineatus (stars) on the volsci mountains (latium, central italy). numeric code as reported in table 1. 10x10 km utm grid is also shown. fig. 2. adult of italian aesculapian snake, zamenis lineatus, from ausoni mountains (latium, central italy). this specimen has been observed in the site 7 (see the text). photo courtesy of paolo mazzei. 182 l. corsetti and a. romano the site 1 (utm 33t 0349800, 4610000 wgs84) and 2 (utm 33t 0328700, 4600000 wgs84) here reported are the present northern and western limits of z. lineatus. on the basis of the present data, the species range appears wider than that known up to now (capula et al., 2006; capula et al., 2008). indeed the northern limit extends 68 km further from the previous accurate record (pannarano, campania region; see lenk and wüster, 1999), while the new western limit extends 130 km further from the previous limit (roccarainola, campania region; lenk and wüster, 1999). actually, available information on the findings of italian aesculapian snakes in corsetti (2006b) and in capula et al. (2008) is too vague both for a calculation of their distance from the sites of pannarano and roccarainola reported in lenk and wüster (1999), and for an accurate knowledge of the species distribution. the italian aesculapian snake was recorded in two of the three administrative provinces in which the volsci chain falls, namely the province of latina (lt) and frosinone (fr), while no data are available for the portion of lepini mountains which falls in the province of rome. however, the administrative boundary of the province of rome is only 3.4 km far from the site 1. consequently the occurrence of z. lineatus in the province of rome is very likely. as a consequence of these new findings in latium, the parapatric area between z. longissimus and z. lineatus is wider than the previously known one and the two species are therefore largely overlapped. the recordings of the snakes found in the past in the province of caserta (northern campania) and in the molise region are not attributable to neither of the two species (see razzetti and zanghellini, 2006). furthermore, although z. lineatus (as elaphe longissima romana) was reported for saepinum, in the molise region, by bruno (1973) and by bruno and guacci (1993), the presence of this species in such region has not been reconfirmed by the recent work of capula et al. (2008). however, if we take into consideration capula et al. (2006), that established the new southern limit in apulia, and the data we reported here, we think it is probable that both zamenis lineatus and z. longissimus occur in the province of caserta (northern campania) and in the molise region. acknowledgments we are grateful to carmine esposito and paolo mazzei who provided respectively the data of zamenis lineatus of the sites 6 and 7. we thank fabrizio li vigni and elvira siragusa for the linguistic revision of the note. thanks to edoardo razzetti for the precious suggestions and sebastiano salvidio for the useful comments and critical reading of the draft. many thanks are also due to two anonymous referees whose comments have improved this note. references bruno s. (1973): gli anfibi e i rettili dell’appennino abruzzese con particolare riferimento alle specie del parco nazionale d’abruzzo (studi sulla fauna erpetologica italiana, xiii). lav. soc. ital. biogeogr. 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(2002): molecular systematics and phylogeny of old world and new world ratsnakes, elaphe auct., and related genera (reptilia, squamata, colubridae). russ. j. herp. 9: 105-124. acta herpetologica 16(2): 133-136, 2021 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-11502 semi-automated photo-identification of bahamian racers (cubophis vudii vudii) sebastian hoefer1,*, andreu rotger2, sophie mills1, nathan j. robinson1,3 1 cape eleuthera institute, the cape eleuthera island school, eleuthera, the bahamas 2 animal demography and ecology unit (geda), imedea, csic-uib, 07190 esporles, spain 3 fundación oceanogràfic, oceanogràfic de valencia, 46013 valencia, spain *corresponding author. e-mail: sebastianhoefer@outlook.com submitted on: 2021, 14th july, revised on: 2021, 17th september; accepted on: 2021, 18th september. editor: marco mangiacotti abstract. photo-identification is a non-invasive option for mark-recapture. here, we tested the effectiveness of aphis, a semi-automated photo-identification software, to distinguish between individual bahamian racers (cubophis vudii vudii) on the island of eleuthera, the bahamas. over 10 months, we photographed 50 bahamian racers. we first identified individuals by manually comparing colouration and scale patterns in the pileus and labial regions. next, we used aphis to identify recaptured individuals after manually identifying the locations of intersections of the scales in the pileus and labial regions. in addition, we assessed whether images taken with a hand-held camera or by a smart phone affected the accuracy of aphis. all recaptured snakes were correctly identified using aphis from both camera or phone images as validated by our manually derived results. we conclude that aphis is an effective tool for photo-identification in snakes. keywords. aphis, i³s, mark-recapture, non-invasive, snake, dipsadidae, colubridae, the bahamas. mark-recapture studies require individuals to be reliably identified upon repeat encounters. this enables researchers to monitor specific individuals over time and this can allow for the estimation of ecological relevant information such as growth or survival rates (pradel, 1996; besbeas et al., 2002). in snakes, long-term marking is typically achieved by scale clipping (brown and parker, 1976), branding (winne et al., 2006), passive integrated transponder tags (pit tags) (gibbons and andrews, 2004), and/or visible implant elastomers (vie) (hutchens et al., 2008; major et al., 2020). as each method has different advantages and disadvantages, researchers must critically assess which method is most appropriate for their study. branding and scale clipping is generally inexpensive but can leave lasting physical damage (weary, 1969; brown and parker, 1976). in contrast, pit tags or vie are highly reliable but are associated with considerable financial cost (gibbons and andrews, 2004; major et al., 2020) and can be unsuitable for smaller individuals (gibbons and andrews, 2004). an alternative method that has the benefit of being both non-invasive and relatively inexpensive is the use of photo-identification (sacchi et al., 2016). photo-id has been successfully applied to numerous snake species (e.g., carlström and edelstam, 1946; vaughan, 1999; creer, 2005; bauwens et al., 2018; lunghi et al., 2019). photos can be processed manually but this can be time consuming. in contrast, pattern recognition software offers a fast and robust approach to compare patterns in photos and distinguish between individual animals (sacchi et al., 2010). one such pattern recognition software is the automated photo-identification suite (aphis), developed by moya et al. (2015). aphis enables users to choose between two image matching 134 s. hoefer et alii methods; the image template matching, (itm) a pixelbased colour comparison, and the spots pattern matching (spm) procedure that compares user-defined spot patterns. we selected to use aphis because images can be processed in batches due to the independence of the manual pre-processing and automated photo-matching which allows substantial time saving (see moya et al., 2015). aphis also creates log files that can be used to track the analyses and allow for successive examinations. in addition, aphis offers the use of two image matching methods. initially, we wanted to compare both matching methods but decided to discard the itm method because of quality issues in some of our images and, in addition, we were concerned about potential colour changes of the snakes over the course of the study. so far, aphis has been used to differentiate between individual horseshoe whip snakes (hemorrhois hippocrepis) (rotger et al., 2019) but it has not yet been applied to other snake species. here, we tested the efficacy of the spots pattern matching (spm) procedure implemented in aphis (see moya et al., 2015 for details) to identify individual bahamian racers (cubophis vudii vudii). specifically, we investigated (1) whether aphis was able to accurately identify individuals based on scale patterns in the pileus and labial regions, and (2) if image quality influenced the successful identification. bahamian racers are colubrid snakes endemic to the eastern parts of the great bahama bank (henderson and powell, 2009). these opportunistic snakes feed on a wide variety of vertebrate prey (hoefer et al., 2020; 2021), are diurnally active and frequently encountered around human settlements. on opportunistic encounters with bahamian racers, we observed that head colouration and scalation was notably variable, particularly in the labial and pileus region (fig. 1). thus, we selected these areas for photoid. we did not use colour patterns as there are several examples of ontogenetic colour change in snakes (creer, 2005; lunghi et al., 2019). consequently, we used only head scalation for identification purposes, as it is generally considered to be robust throughout a snakes’ life (bauwens et al., 2018). upon capture, we measured snout-vent length (svl) and tail length (tl) to the nearest millimetre using a flexible measuring tape, weighed the snake to the nearest gram using a weighing scale (dapha dws weighing scale) and determined the sex via probing. using a nikon d3300 dslr camera and sigma 105 mm 1:2.8 dg macro hsm ex lens in combination with an external flash and flash diffuser, we took photos of the pileus and the right and left labial regions. we assumed that both labial regions were identical for the use in aphis and so we combined them when comparing to the pileus region. we initially identified recaptures via visual examination of colour, scale patterns, unique scarring and scale counts. we selected aphis for our recapture matching analyses because pictures can be processed in batches due to the independence of the manual pre-processing and automated photo-matching, which allows for great time savings (moya et al., 2015). in addition, aphis creates log files that can be used to track the analyses and allow for successive examinations. from august 2019 to june 2020, we opportunistically captured a total of 50 bahamian racers including 11 recaptures from five unique individuals. all snakes were found in small shrubs or leaf litter close to walking paths and buildings, which is likely the result of a detection bias due to the opportunistic nature of sampling. for the recaptures, we used 10 pileus, 10 right labial, 9 left labial images. in addition, we used 11 iphone 7 images of a selection of the same recaptures (4 pileus, 4 right labial, 3 left labial) to compare to the dslr photos and assess the usability of phone quality images in aphis. the angle and composition for dslr and phone images were consistent across photos, but overall image quality and resolution differed (dslr: 300 ppi 6000 x 4000 px, iphone 7: 72 ppi 4032 x 3024 px). for each snake, we marked between 35 to 45 reference points on the corners of the scales (fig. 1) and analysed the images using the i3s procedure in an automated process described by moya et al. (2015). we considered a successful match of a recapture when aphis suggested the correct images for an individual within the top 10 candidates (i.e., top 20% of photos) (gatto et al., 2018) and a top match when it was the first suggestion in the list. the candidate list created in aphis is based on a similarity score where images that differ very little, i.e., photos of the same snake, produce a low score and are ranked at the top. images that differ considerably result in a high score and are ranked at the lower end of the list. the similarity score represents the difference between photos in the relative distances of the reference points. when comparing the combined labial and pileus regions, we found that all of the labial and pileus images were correctly identified as recaptures, with 100% (19/19) of the labial photos and 90% (9/10) of the images in the pileus region resulting in a top match, making either area suitable to use in aphis. when only using photos collected via the dslr camera (n = 29) across all head regions combined, recaptures were correctly identified 100% (29/29) of the time and 97% (28/29) were suggested as top matches. images of recaptures taken with an iphone 7 were successfully identified 100% (11/11) of the time when compared to the dslr database photos, with 82% (9/11) of top match sugges135photo-identification of bahamian racers tions. when the top match was incorrectly identified, the difference in score between the incorrect first candidate and the correct candidate was marginal. even when the two images that were compared differed substantially in resolution and composition, aphis was able to successfully match recaptures. in addition, we did not observe any colour or scale pattern changes in neither the labial nor the pileus region of any individual bahamian racer over the course of this study (duration between captures ranging from 6 days to 216 days, figs. s1 and s2). however, 10 months likely only provides a glimpse into the lives of these snakes and, furthermore, all the recaptured individuals were adult snakes and thus ontogenetic changes in colouration or scalation cannot be dismissed. in conclusion, we were able to use aphis to correctly identify and successfully match all our recaptured bahamian racers. the software provided accurate results even when the image quality differed substantially between the photos compared and the recaptures were up to seven months apart. the semi-automated analysis resulted in high matching probability when using images taken from a smartphone, which is likely more accessible in the field than a dedicated camera. the use of pattern recognition software in recapture studies allows for short handling times in the field and only requires taking a photograph. this likely increases the time available for searching and recording new individuals, thus enhancing data collection. particularly for studies on snakes, where detectability of animals can be quite low, maximising the time to find snakes could enable researchers to gather more crucial information for many of these understudied species. even though this study has its limitations due to low sample sizes, we provide fig. 1. photographs of three different bahamian racers: a) – c) shows the labial region; d) – f) shows the pileus region. g) and h) shows an example of the reference points selected within aphis marking the intersection of the scales in the labial and pileus region, respectively. 136 s. hoefer et alii further support for the use of aphis to effectively distinguish between individual animals. acknowledgments we would like to thank the cape eleuthera institute for providing the resources to conduct research in the bahamas. we also thank the many interns, staff, and visitors for reporting live snake sightings and helping with data collection and photos. research was conducted under the cape eleuthera institute research permit number mamr/fis/2/12a/17/17b. supplementary material supplementary material associated with this article can be found at <htttp://www.unipv.it/webshi/appendix> manuscript number 11502. references bauwens, d., claus, k., mergeay, j. 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(2016): digital identification and analysis. in: reptile ecology and conservation. a handbook of techniques, pp. 59-72. dodd, c.k., ed. oxford university press, oxford. sacchi, r., scali, s., pellitteri-rosa, d., pupin, f., gentilli, a., tettamanti, s., cavigioli, l., racina, l., maiocchi, v., galeotti, p., fasola, m. (2010): photographic identification in reptiles: a matter of scales. amphibiareptilia 31: 489-502. silvy, n.j., lopez, r.r., peterson, m.j. (2012): techniques for marking wildlife. in: the wildlife techniques manual, 7th ed., vol. 1, pp. 230-257. silvy, n.j., ed, james hopkins university press, baltimore (us). vaughan, r. (1999): provisional results from study of facial features as a means of individual identification in natrix natrix. herpetol. bulletin 69: 39-46. weary, g.c. (1969): an improved method of marking snakes. copeia 1969: 854-855. winne, c.t., willson, j.d., andrews, k.m. (2006): efficacy of marking snakes with disposable medical cautery units. herpetol. review 37: 52-54. acta herpetologica vol. 16, n. 2 december 2021 firenze university press a new species of the genus noblella (amphibia: strabomantidae) from ecuador, with new information for noblella worleyae carolina reyes-puig1,2,3,4,*, juan m. guayasamin 2,5 claudia koch6, david brito-zapata1, matthijs hollanders7, melissa costales8, diego f. cisneros-heredia1,2,3 so close so different: what makes the difference? dario ottonello1,7,*, stefania d’angelo2, fabrizio oneto3,6, stefano malavasi1, marco alberto luca zuffi4, filippo spadola5 hematological values of wild caiman latirostris (daudin, 1802) in the atlantic rainforest in pernambuco, brazil luciana c. rameh-de-albuquerque1, alexandre p. zanotti1, denisson s. souza1, george t. diniz2, paulo b. mascarenhas-junior3,4,5,*, ednilza m. santos³, jozelia m. s. correia3 bone histology of broad-snouted caiman caiman latirostris (crocodylia: alligatoridae) as tool for morphophysiological inferences in crocodylia paulo braga mascarenhas-junior1,2,3,6, luis antonio bochetti bassetti4, juliana manso sayão5,6 is the northern spectacled salamander salamandrina perspicillata aposematic? a preliminary test with clay models giacomo barbieri, andrea costa, sebastiano salvidio* sexual size dimorphism in the tail length of the caspian whip snakes, dolichophis caspius (serpentes, colubridae), in south-western hungary györgy dudás1, krisztián frank2* semi-automated photo-identification of bahamian racers (cubophis vudii vudii) sebastian hoefer1,*, andreu rotger2, sophie mills1, nathan j. robinson1,3 emergent spatial pattern of herpetofauna in alabama, usa xiongwen chen1, yong wang center for forestry, ecology & wildlife, po box 1927, alabama a & m university, normal, al 35762, usa. corresponding author. e-mail: xiongwen.chen@aamu.edu abstract. analyzing spatial pattern of regional biodiversity and its relationships with environmental factors is important for biodiversity conservation at large scales. the emergent spatial pattern of herpetofauna in alabama is examined by combining thousands of historical records from 132 species of 24 families and environmental conditions. our results indicate that species richness of herpetofauna increases with the increase of latitude, while it decreases with the increase of elevation. a negative spatial association exists between amphibians and reptiles on the scale of 10 km2, but 40% of habitats are still shared by amphibians and reptiles at this scale. the highest species richness of herpetofauna is in the mobile and baldwin counties. power-law relation exists between the county size and the average species richness. total stream length, and road density are highly correlated with species richness at the county level. with the increase of annual precipitation, species richness decreases. species richness is higher in the area with the annual average temperature around 17-18 °c. herpetofaunal diversity in the coosa/tallapoosa river, the alabama river, and the tombigbee river basins is relatively higher than in the perdido river and the escatawpa river basins. the highest species richness exists at the gulf coastal plain, but its species density is the lowest. the highest species richness of herpetofauna exists in the longleaf-slash pine and loblolly-shortleaf pine forests, while lower in oakhickory forest. the emergent spatial pattern may provide important implications for herpetofauna conservation in the face of global climate change and large-scale habitat destruction. the spatial pattern and the possible underlying ecological processes have to be considered for the large scale land zoning and planning. keywords. alabama, amphibians, reptiles, herpetofauna, spatial pattern. introduction herpetofauna plays an important role in both aquatic and terrestrial ecosystems, such as an energetic link in trophics (pough, 1980; whiles et al., 2006). holomuzki et al. (1994) and wissinger et al. (1999) indicated that amphibians may have large impacts on ecosystem structure because they are keystone species in some habitats. regester et al. (2006) acta herpetologica 2(2): 97-115, 2007 issn 1827-9643 (online) © 2007 firenze university press 98 x. chen and y. wang reported that salamander communities transfer an average net flux of 350 g yr-1 in ash-free dry mass into small forest ponds, which is considered equal to or higher than fish or other macroinvertebrates. however, during the recent decades, a decline status for amphibian and reptile species was reported as a global phenomenon (e.g., gibbons et al., 2000; gardner, 2001; stuart et al., 2004). a number of factors were considered to shed doubt on the responsibility for the world-wide decline of amphibians and reptiles, such as physical habitat modification and habitat loss (sjogren, 1991; alford and richards, 1999; chen et al., 2006a), ultraviolet radiation (blaustein et al., 1994; ovaska et al., 1997; crump et al., 1999), chemical pollutions (beebee et al., 1990; lips, 1998; carey et al., 2001; sparling et al., 2001), diseases (laurance et al., 1996), and climate change (pounds and crump, 1994; pounds, 2001; chen et al., 2006b). furthermore, both empirical and theoretical investigations indicated that the extinction of some species will result in the extinction of other species in ecosystems by trophic cascades (jennings et al., 1992; lavin, 1999). due to the global decline of biodiversity and the possible complexity of underlying mechanisms, the previous reductionist approach, which only focuses on a single (or several) species, may not work well to provide general picture about the entire herpetofauna, their suitable habitats and their regional conservation strategies (smallwood et al., 1998; chase et al., 2000; chen et al., 2005). just like the accurate description of the behavior of a single air molecule, may not help to understand the atmospheric dynamics. thus, a topdown approach to evaluate the general regional herpetofauna in their entirety may help us to understand some of their emergent properties. this method may identify the critical resource needs for overall herpetofauna and may lead to infer the important underlying processes for all species (chen et al., 2005). despite the fact that different processes may result in same spatial pattern, analysis of spatial pattern of regional herpetofauna can provide information that can not be obtained from a single species (or several species) approach. the herpetofauna of alabama have been received more attention for its richness and diversity in the usa. several factors interrelated to produce this diversity, such as mild and humid climate, remarkable surface drainage and diverse physiographic subdivisions (mount, 1975). in 1838, holbrook made the earliest significant contribution to alabama herpetofauna by the description of emys (pseudemys) mobilensis at the mobile area. since then numerous investigators contributed significantly about the herpetofaunal species and locality records (e.g. agassiz, 1857; cope, 1880; yarrow, 1882; baur, 1893; brimley, 1904; holt, 1919; dunn, 1920; löding, 1922; blanchard, 1924; haltom, 1931; viosca, 1937; snyder, 1944; grobman, 1950). in 1960s the most significant herpetological event in alabama was the discovery and subsequent description of phaeognathus hubrichti (highton, 1961). in 1970 mount and schwaner published the distribution relationships between natrix rhombifera and n. taxispilota. new locality data for ambystoma texanum and trionyx ferox were published later (scott and johnson, 1972; scott, 1973). however, there is limited study on the quantitative and synergetic analysis of herpetofauna in this region. the study of the spatial characteristics of herpetofauna in alabama based on all historical records of locality can provide some emergent properties of herpetofauna in this area, such as habitat, diversity distribution and gradient, as well as its relation with the environmental condition. the more important information about the strategies of regional herpetofauna conservation can also be inferred. the main aims of this study are (i) to investigate of the 99emergent spatial pattern of herpetofauna in alabama spatial pattern of herpetofauna in alabama; (ii) to examine the relationships between spatial distribution of herpetofaunal diversity and environmental conditions; (iii) to compare the spatial characteristics of herpetofauna with some current theories at a large area; and (iv) to provide implications for large scale conservation. materials and methods study area the study area covers the state of alabama of usa, which is located between the southern foothills of the appalachian mountain range and gulf of mexico (between 31° and 35°n) and includes 67 counties. alabama has warm, humid, subtropical climate. summers are hot and humid with high temperature around 33 °c. in late summer and fall, it is the driest time of the year. winters are typified by series of cold fronts. regional rainfall varies from 150 cm to 162 cm in the north part and 180 cm to 195 cm along the coast (carter and carter, 1984). in alabama, there are five recognized physiographic zones: the highland rim, the cumberland plateau, the alabama valley and ridge, the piedmont upland, and the east gulf coastal plain (fenneman, 1938). alabama’s forests mainly consist of four types – pine, pine-hardwood mixture, bottomland hardwood and upland hardwood. south alabama is abundant in pure stands of pine. from south to north, the type changes to mixed pine-hardwood conditions and then to more complex hardwood forests near tennessee boundary (http://www.forestry.auburn.edu/fpdc/alforest. html, the last visit time is july 31, 2007). in this study, the general forest types in alabama are the longleaf-slash pine, the loblolly-shortleaf pine, the oak-pine, and the oak-hickory. dataset and gis layers the dataset of herpetofauna in alabama is from the book “the reptiles and amphibians of alabama” (mount, 1975), which included thousands of locality records of 132 species in 24 families examined by its author and literature. all the records were digitized by arcgis 9 (esri, 2004). we recognize that this dataset does not represent all species and does not represent the current distribution of species. but it does represent all historical data records in 1970s. this data set represents the time prior to when the most recent major growth in suburban development was initiated, so it can provide a bench marker of spatial pattern of herpetofauna in alabama. the gis data of state boundaries, county boundaries and streams were obtained from alabama state water program (http://www.aces.edu/waterquality, the last visit time is july 31, 2007). the human population data of each county during this study time period (1970s) was from us census bureau. historical roads (including freeway, highway and country way) and other information related with long term average climate condition were from maps provided by the university of alabama (http://alabamamaps.ua.edu, the last visit time is july 31, 2007). spatial association spatial association is the extent that different species (or species groups, here amphibians and reptiles) live together. coomes et al. (1999) suggested the following index to measure spatial association (si): 100 x. chen and y. wang si r n r r n n q ij ij i j ( ) ( )/( / )= ∑ π 2 (1) where nij is the number of grid of species j (e.g. amphibians) within a distance r of species group i (e.g., reptiles). ni and nj are the total numbers of grid covered by species i and j, respectively. q is the total study area. in this study, r is chosen as 10 km. however, with the increase of r the spatial association will be increased. when si > 1, positive association is indicated, whereas si < 1 indicates negative association. spatial overlay ratio chen et al. (2005) suggested spatial overlay ratio to measure the percentage of same location occupied by two or more species groups. it can be estimated by the following simple equation: p ab a b abij = + − (2) where pij is the spatial overlay ratio and ranges from 0 to 1 (or 0-100%). ab is the number of the same locations used by species a and b (here amphibians and reptiles). a and b are the numbers of grids for species a and b, respectively. where pij is the spatial overlay ratio is also scale dependent. within the area of 10 km2 (10×10 km), two locations were considered as spatial overlay. measurement of compositional similarity similarity is used to estimate the similarity of species composition in two locations. the jaccard index of similarity was used in this study (legendre and legendre, 1998): j s s s s ij ij i j = + +/( ) where sij is the number of species common to sampling sites i and j; si is the number of species present on site i but absent on site j; sj is the number of species present on site j but absent on site i. j is in the range of 0-1. data categorization and statistical method in this study, the county size, stream length, and road density were categorized. for county size (m2), they were categorized as 9.1, 9.2…..9.8 with the log10. it is similar for the total stream lengths (m) in all counties, they were classified into 9.8, 9.9….10.4 with its log10. however, the data of road density (km × km-2) in all counties were classified from 2.725 to 3.175 with increment of 0.05. frequency is calculated by the records in the range divided by the total records. the common used least mean square technique with used in regression analysis for the relationships between species richness and environmental factors. 101emergent spatial pattern of herpetofauna in alabama results pattern along latitude, longitude and elevation herpetofaunal and reptile species richness increase with the increase of latitude (utm) (fig. 1a). there exist significant relationships for both herpetofauna and reptiles: herpetofaunal species richness = 70.71 × utm – 162.46 (r2 = 0.81, p < 0.01), and reptile species richness = 86.19 × utm – 258.19 (r2 = 0.81, p < 0.01). species richness pattern along longitude is not significant, but it appears to be a constant of 110. with the increase of elevation, the species richness decreases gradually (fig. 1b). the relationships between species richness and elevation are significant: herpetofaunal species richness = -0.12 × elevation + 121.74 (r2 = 0.89, p < 0.01), reptile species richness = -0.071 × elevation + 73.67 fig. 1. the different patterns of herpetofaunal species richness in alabama along latitude (a) and altitude (b). universal transverse mercator (utm) system was used for latitude and longitude. 102 x. chen and y. wang (r2 = 0.97, p < 0.01), and amphibian species richness = -0.050 × elevation + 48.06 (r2 = 0.66, p < 0.05). there exists negative spatial association (si = -0.32) between amphibians and reptiles on the scale of 10 km2, but the spatial overlay ratio between amphibians and reptiles is about 0.4. this result indicates that although there is a negative spatial association between amphibians and reptiles, there is 40% occasions that they occurred together within the area of 10 km2. species richness at county level because different counties have different integrated environmental conditions and land management policies, there exists uneven spatial distribution in herpetofauna in the counties of alabama (appendix 1a). the highest species richness of herpetofauna is located in the mobile and baldwin counties. the relationship between county size and fig. 2. herpetofaunal species richness increases with total stream length (a) and road density (b) (error bar stands for standard deviation). 103emergent spatial pattern of herpetofauna in alabama species richness is not straightforward. however, if the county sizes are categorized, there is a significant linear relationship between the log value of county size and the average species richness (appendix 1b), average species richness = 123.7 × log(area) 1111.6 (r2 = 0.88, p < 0.01). average species richness increases with increasing county size. on the county level, there exists nonlinear relationship between species richness and its frequency (appendix 1c), frequency (%) = -0.0035 × (species richness)2 + 0.3573 × species richness + 0.6848 (r2 = 0.58, p < 0.05). the relationship between the total human population and species richness is not obvious at county level. after data categorization, there is linear relationship between total stream length (m) and species richness (fig. 2a, species richness = 107.58 × log(total fig. 3. varied relationships between herpetofaunal species richness and annual average precipitation (a) and annual average temperature (b). 104 x. chen and y. wang stream length) – 1032.5, r2 = 0.89, p < 0.01); road density is also correlated significantly with species richness (fig. 2b, species richness = 0.1396 × (road density)5.3898, r2 = 0.73, p < 0.05). climate and herpetofaunal diversity there exists linear relationship between annual average precipitation and species richness (fig. 3a), herpetofauna species richness = -1.015 × precipitation + 250.26 (r2 = 0.66, p < 0.01) and amphibian species richness = -0.89 × precipitation + 171.50 (r2 = 0.86, p < 0.01). with the increase of annual precipitation, total species richness and amphibian richness decreases significantly. the relationship between species richness and annual mean air temperature is significant for amphibians, reptiles and the total (fig. 3b, herpetofauna species richness = -5.41 × (temperature)2 + 191.94 × temperature 1591.8, r2 = 0.67, p < fig. 4. pattern of herpetofaunal species richness at different river basins (a) and the composition similarity with those in tennessee river basin (b). 105emergent spatial pattern of herpetofauna in alabama 0.05; reptile species richness = -3.68 × (temperature)2 + 128.72 × temperature 1059, r2 = 0.60, p < 0.05; amphibian species richness = -1.73 × (temperature)2 + 63.22 × temperature 532.72, r2 = 0.85, p < 0.01). there are higher species richness in the area with the annual average temperature around 17-18 °c. with the annual average temperature is out this threshold, species richness decreases. herpetofaunal diversity in river basins species richness in different river basins was studied because of its relationships with stream length and precipitation. in alabama there are higher herpetofaunal diversity in the coosa/tallapoosa river, the alabama river, and the tombigbee river basins, and less herpetofaunal diversity in the perdido river and escatawpa river basins (fig. 4a). the sipsey river basin has the highest similarity of species compared with the tennessee river basins (fig. 4b), whereas less similarity in the perdido river and the escatawpa river basins. fig. 5. pattern of herpetofaunal species richness in physiographic zones (a) and the species density (b). 106 x. chen and y. wang herpetofaunal diversity in physiographic zones the highest species richness exists at the gulf coastal plain among the five physiographic zones of alabama (fig. 5a), while fewest herpetofaunal species exists at the highland rim. species density is the highest at the piedmount upland and the lowest at the gulf coastal plain (fig. 5b). there are relatively higher species richness and species density at the piedmont upland, the valley and ridge, the cumberland plateau and the highland rim. forest types and herpetofaunal diversity the highest species richness of herpetofauna exists in the longleaf-slash pine and loblolly-shortleaf pine forests, while lower herpetofaunal diversity in the oak-hickory forest (fig. 6). similar pattern exists for amphibians and reptiles, respectively. this means that forests (oak-hickory and oak-pine) in northern alabama have relatively lower herpetofauna diversity than forests (longleaf-slash pine, loblolly-shortleaf pine and oakgun cypress) in southern alabama. discussion the latitude gradient of increasing species richness toward tropical area may be considered as the most fundamental pattern of life on earth (rosenzweig, 1995; willig et al., 2003). willig (2000) suggested that this principle applies to most taxonomic groups in terfig. 6. pattern of herpetofaunal species richness at different forest types. 107emergent spatial pattern of herpetofauna in alabama restrial, freshwater, and marine environments. however, in this study, species richness of herpetofauna increases with the increase of latitude. this result seems to contrast with the global pattern of biodiversity. lyons and willig (1999) suggested that the form of the gradient in biodiversity is likely to be scale dependent. the species richness increases toward equatorial regions if the latitudinal domain is sufficiently extensive (spans more than 15 degrees of latitude) to avoid local variation in geography, climate or edaphic features (willig et al., 2003). alabama only runs across about 4° in latitude. thus, in this study, the latitude pattern of species richness decreases toward equator may be due to local variations, such as geography, habitat, and climate. there is no variation in herpetofaunal diversity along the longitude. the mechanisms may be the same as those for the latitude pattern. herpetofauna is considered sensitive to environmental temperature and moisture conditions. in this study, our result indicated that with the increase of elevation, the species richness decreases. other studies suggested that maximum species richness is at intermediate elevation (li et al., 2003; sanders et al., 2003; fischer and lindenmayer, 2005). the possible reason for maximum species richness at intermediate elevations is that high and low specialists may co-occur in these areas (fischer and lindenmayer, 2005). therefore, the large scale pattern of biodiversity from other research may not apply for the herpetofauna along the latitude and elevation in alabama. species-area relationships (sar), which follow power-law relation, strongly reflect difference in overall diversity and difference between small and large areas (lennon et al., 2001). drakare et al. (2006) conducted a quantitative meta-analysis of 794 sar from a wide span of organisms, habitats and locations and suggested that the average exponent is 0.24 for independent census. they also suggested that spatial scale measured as grain plays a small role for variation in the exponent, and the differences in sar between the aquatic and terrestrial realm are minor. in this study, the exponent of sar is 1.91 (the slope of the fitting line between log (area) and log (species)) which is higher than expected. the possible reason is that the classified data (for area) was used here. the range of the sampled scale can significantly affect the exponent (or slope) of power-law and semi-log sar (drakare et al., 2006). the sar in this study could provide a baseline for the analysis of herpetofaunal diversity in this region or extrapolation of species “carrying capacities”. comparing the variations of sar exponents or frequency distribution may be useful for the local diversity monitoring. applying different sar may improve the management in planning nature reserves and species conservation prioritization (zurlini et al., 2002). human population size is often considered as the driving force for biodiversity loss (e.g. holdren and ehrlich, 1974; thompson and jones, 1999; cincotta et al., 2000). however, the direct relationships between human population size and biodiversity are obscure in this study because indirect effects from increasing human population (such as urban development and land use change) also affect biodiversity change. liu et al. (2003) used the household instead of human population size to study its relationship with biodiversity and indicated that household number and higher per capita resource consumption pose serious challenges to biodiversity conservation. but this information is not available in alabama. there is a high correction between herpetofaunal species richness and the total stream length or road density if aggregated data is used. this result may indicate that most herpetofauna (possible more than 65%) in this area are water related. many amphibians have complex life cycles occupying different habitats, usually aquatic and terrestrial. 108 x. chen and y. wang inger et al. (1986) and ranvestel et al. (2004) indicated that some tadpoles are particularly abundant in ponds and streams. many snake species like to feed on one or more amphibian life stages (jennings et al., 1992). stream and river banks provide important habitats for herpetofauna in alabama. our results also indicate high positive correlation between herpetofaunal diversity and road density. herpetofauna usually suffer from existence and construction of roads (e.g. vos and chardon, 1998; forman et al., 2002). the high correction between species richness and road density may have several meanings: (i) roads might create good habitats for certain herpetofauna before road density reaches certain threshold, such as the threshold of habitat fragmentation; (ii) more observations might occur in the area with roads; and (iii) more roads were constructed in the counties with high species richness. all of them may be possible in this study. herpetofauna are ectotherms, which mean that their body temperature is regulated by ambient temperature. as such, herpetofaunal species may be attracted to warm surfaces of roads and highways where they can raise their body temperature. also, many amphibians depend on seasonal pools along roads for reproductive purposes. during storm events, roadside drainage ditches contribute surface flow of waters used for reproduction (lannoo, 1998; forman et al., 2002). although roads may cause habitat fragmentation, wiegand et al. (2005) studied the effects of habitat loss and fragmentation on population dynamics by computer simulation and indicated that habitat amount accounted for 68% of population variation while fragmentation only accounted for about 13%. they further suggested that study of fragmentation effects requires a good understanding of the biology and habitat use of the species in question. felix et al. (2004) suggested that intermediate disturbances cause the increase of herpetofaunal species richness and abundance. by overlaying the maps of herpetofauna distribution to the long term annual average climate, we found that there are higher species richness in the area with the annual average temperature around 17-18 °c. with the increase of annual precipitation, the total species richness and amphibian richness decrease, but it is not significant for reptiles. both temperature and moisture influence amphibian ecology and physiology because amphibians must maintain moist skin for oxygen and ionic exchange and temperature influences metabolic rates (pounds et al., 1999; alexander and eischeid, 2001). wilson and mccranie (2004) studied the herpetofaunal diversity of cloud forests and indicated that some species only live under the narrow climate condition. it is unsurprising that the changing climate may change species distribution or result into increasing extinction (e.g. gardner, 2001). different diversities of herpetofauna exist among the five forest types of alabama. the highest species richness occurred in the longleaf-slash pine and the loblolly-shortleaf pine forests in comparison with the oak-hickory forest. this result is consistent with the study of loehle et al. (2005) that the pine forest was richer in the total herpetofaunal richness than the pine-hardwood type. however, mitchell et al. (1997) reported that amphibians were more abundant in mature hardwoods than in a white pine plantation. degraaf and rudis (1990) also indicated that northern hardwood and red maple forest supported more species than balsam fir forest. different herpetofaunal richness in different forest types may also reflect site-specific abiotic factors (loehle et al., 2005). lewis et al. (2000) suggested that differences among forest types in herpetofauna in texas were related to differences in moisture availability. fleet and autrey (1999) observed that change in altitude formed a natural moisture gradient across forest types that accounted for the differences 109emergent spatial pattern of herpetofauna in alabama in herpetofaunal richness. it is not clear whether forest type itself directly affect herpetofaunal richness. however, arboreal lizards are associated with structurally complex forests and four parameters of a forest stand (canopy coverage, litter depth, woody plant cover, and large woody debris) explained much of the variation in herpetofaunal species by crosswhite et al. (2004). due to all these biotic and abiotic factors, the distribution of herpetofauna is not uniform among the counties, river basins and geophysical regions of alabama. the baldwin and mobile counties, which only cover about 7% land area of the alabama, host the highest species of herpetofauna. in the five physiographic zones the gulf coastal plain hosts the highest species richness but least species density. fewer herpetofaunal species exist at the piedmont upland, the valley and ridge, the cumberland plateau, and the highland rim, while there are relative higher species richness and species density in those areas. river basins in the middle and south part of alabama (the coosa/tallapoosa, the alabama, and the tombigbee) have higher species richness. special physiographic feature could be important for herpetofauna due to the impact on main ecological processes (wilson and mccranie, 2004), such as in this study the drier cumberland plateau and the valley and ridge physiographic zones occur to the west and a rain shadow exists to the east. analysis of the herpetofauna of central texas revealed that the majority of species (77%) of herpetofauna are limited by the balcones escarpment (smith and buechner, 1947). loehle et al. (2005) indicated that most intensively managed watersheds had higher species richness in herpetofauna than those less intensively managed. they suggested watershed scale forest management (including plantation management) did not affect and even enhanced habitat diversity for herpetofauna. the uniqueness of species and the landscapes in which they live can provide us a good understanding of spatial pattern of herpetofauna. implications for conservation our study may provide a baseline for the spatial pattern of herpetofauna in alabama. the macro pattern identified here may have potential important consequence in the face of global climate change and large-scale habitat destruction. on the large scale, land zoning and planning need to maintain the ecological processes along latitude and elevation which formed the gradient in species richness of herpetofauna. urban development or human settlement should not affect spatial pattern of biodiversity or their major ecological processes. large scale habitat loss and fragmentation should be avoided. current policy regarding land zoning and planning should incorporate large scale (or landscape scale) biodiversity conservation and important threatening processes while understanding of species distribution pattern is a prerequisite (manning et al., 2004). also planning under different scales may produce different outcomes. scheiner and willig (2005) suggested that any environmental factors that affect the number of individuals in an area will increase richness because of three mechanisms (random placement or passive sampling, local extinction, and speciation). spatial heterogeneity in landscapes (e.g. topography, vegetation types, and hydrological regimes) can enhance species richness. the sar is a strong indicator of the sensitivity to the loss of habitat and climate sensitive space. maintaining the integrity of stream and river systems can improve habitats within both the channel and adjacent floodplains. in the areas with high road density, intensive 110 x. chen and y. wang safe passages or crossings should be provided for herpetofauna under or over roadways, especially in areas where roads bisect important habitats or corridors (e.g. roads that parallel water bodies). ecological barriers (such as fences, sheet piles and concrete walls) that run parallel to roads may be constructed to reduce animal mortality and road hazards. acknowledgements we thank dr. luca luiselli, dr. marco a.l. zuffi, dr. roberto sacchi and an anonymous referee for suggestions on the earlier version of the manuscript. this research was partially supported by the school of agricultural and environmental sciences of alabama a & m university, nsf (hrd0420541) and de-fc26-06nt43029-005. references agassiz, l. 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(2002): spatial-accumulation pattern and extinction rates of mediterranean flora as related to species confinement to habitats in preserves and large areas. conserv. biol. 16: 948-963. appendix 1. herpetofauna species richness in different counties. counties with different herpetofauna species richness (a), area-species relationship (b), and richness-frequency relationship (c). 115emergent spatial pattern of herpetofauna in alabama karyotype, chromosome structure, reproductive modalities of three southern eurasian populations of the common lacertid lizard, zootoca vivipara (jacquin, 1787) larissa kupriyanova1, alex kuksin2, gaetano odierna3 1zoological institute of russian academy of sciences, 199034 st. petersburg, russia 2department of zoology of tuvinsky institute copr, siberian department of russian academy of sciences, kizil, russia 3,dipartimento di biologia strutturale e funzionale, university of naples federico ii, i-80124 naples, italy; corresponding author. e-mail: gaetano.odierna@unina.it submitted on 2008, 28th february; revised on 2008, 9th september; accepted on 2008, 16th october. abstract. according to a hypothesis of the evolution of viviparity the lacertid lizard zootoca vivipara, rare relict oviparous populations of the species might occur in southern-eastern part of its distribution area. such a hypothesis has been verified by comparing the karyotype, chromosome structure, and reproductive modality of three populations of south-eastern part of russia, including altai and neighbouring regions, where small territories remained isolated during the pleistocene cooling and where pleistocenic fossils of z. vivipara have been found. the chromosomal study was carried out by conventional staining method and banding methods, namely c-banding and sequential staining of c-banding+ fluorochromes, cma3 and dapi. all studied females displayed viviparous reproductive modality and showed a karyotype of 2n = 35 acrocentric chromosomes, with a z1z2w sex chromosome system. chromosome w was subtelocentric. no inter-population variability on karyotype and heterochromatin distribution and composition was observed. from the obtained data the three studied south-eastern russian viviparous populations belong to the russian viviparous form of z. v. vivipara. keywords. zootoca vivipara, karyotype, heterochromatin, viviparous modality, distribution. introduction the wide-ranged eurasian lacertid species zootoca vivipara (jaquin, 1787) present oviparous and viviparous populations, either showing a high geographical variability in karyotype’s features (chevalier et al., 1979; kupriyanova, 1986, 1990; odierna et al., 1993). along its vast range rather morphologically similar individuals from different populations of z. acta herpetologica 3(2): 99-106, 2008 issn 1827-9643 (online) © 2008 firenze university press 100 l. kupriyanova, a. kuksin and g. odierna vivipara can be recognized on the basis of combined analysis of their karyotypic features and reproductive mode. in western and central europe all so far discovered oviparous and viviparous chromosomal forms appear to have distinct distribution range (parapatric, allopatric and mosaic distributions), some of them inhabiting small areas, others resulting rare within a country and needing protection there (odierna et al., 1993, 1998; kupriyanova et al., 2005a, 2006) (see table 1). recent modern advanced cytogenetical investigations also have given information. it has been shown that oviparous and viviparous populations of different forms are characterized by several chromosome markers (at and gc rich clusters of dna) allowing to (1) identify with precision these forms and subspecies; (2) clarify their distribution; (3) elucidate a possible role of morphological and molecular chromosome change in the processes of subspeciation and form-formation and in evolution of viviparity (odierna et al., 2001, 2004; kupriyanova, 2004; kupriyanova et al., 2005a). concerning z. vivipara populations inhabiting russia, combined data on their reproductive modality, karyotype and chromatinic markers have been obtained mainly in specimens from western and north-western parts of russia: two different chromosomal forms of z. v. vivipara with viviparous mode of reproduction were found (kupriyanova et al. 1995, kupriyanova, 2004; kupriyanova et al., 2007). present paper shows the results of a karyological and reproductive analysis on three previously unstudied z. vivipara populations from southern-central part of european russia, altai and neighbouring regions. data on populations from these areas are of particular interest as may help: to find some additional characters of different forms and subspecies; to clarify the structure and biogeography of the species; to identify the centre(s) of their origin(s) and refugium(a); to give insight on the role of chromosomal changes in the evolution of sex chromosomes and viviparity, in form(s)-formation. furthermore, according to a hypothesis of the evolution of viviparity in this lacertid lizard (heulin et al., 1993), rare relict oviparous populations of the species might occur in southern-eastern part of its distribution areas. in this regard, altai and neighbouring regions are very interesting as they include small territories, with mountain-taiga landscape, which remained isolated during pleistocene cooling (sinizin, 1962), and where pleistocenic fossils of z. vivipara have been found (putieva and chkhikvadze, 1990). materials and methods the karyotypes analysed come from five females and one male of z. vivipara from tambov region, 50 km north of tambov, 53°n, 41°e (southern-central part of european russia) collected in may 2004 (population 1 in fig. 1); from five females and two males of z. vivipara from tuva republic near todga lake, 200 km north-east of kizil, 52°n, 90°e, 2100 m above sea level (southern siberia, asian russia) collected in june 2004 (population 2 in fig. 1); from three females and two males of z. vivipara from the border between tuva and altai regions, 15 km north-west of kara-khol lake, 50°n, 90°e, 2300 m above sea level (southern siberia, asian russia) collected in june 2004 (population 3 in fig. 1). the chromosomes were obtained according to the scraping and air-drying method from intestine, blood and lung tissues (odierna et al., 1993). the specimens were injected with 0.1% phytohemagglutinin m (difco) three times during three weeks (0.08 ml/5 g body weight) and then with 0.05% colchicines (0.1 ml/5 g body weight) 1 hour before sacrificing animal. the slides were stained 101karyotype and chromosome structure in z. vivipara table 1. distribution, taxonomy, reproductive mode and chromosome characters of females from different populations of zootoca vivipara so far known. (ref. 1 = chevalier et al., 1979; 2 = kupriyanova, 1990; 3 = odierna et al., 1999; 4 = odierna et al., 2001; 5 = odierna et al., 2004). populations subspecies reproductive mode female chrom. number female sex chromosome system w chromosome morphology and heterochromatin distribution western and central europe, scandinavia: sweden slovakia western form of z. v. vivipara and z. v. pannonica viviparous 35 z1z2w (ref. 1, 3) eastern europe: russia, estonia, belarus, ukraine, hungary, fennoscandia: finland, sweden. asia russian form of z. v. vivipara and z. v. sachalinensis viviparous 35 z1z2w (ref. 2, 3) central europe: austria (the type locality), austrian pannonian lowland austian form of z. v. vivipara viviparous 35 z1z2w (ref. 2) western europe: western pyrenees, aquitania pyreneean form 1 of z. v. vivipara oviparous 35 z1z2w (ref. 3) eastern pyrenees pyreneean form 2 of z. v. vivipara oviparous 35 z1z2w (ref. 3) southern-central europe: slovenia, northeast italy, southern austria z. v. carniolica oviparous 36 zw (ref. 4) central europe: central hungary (poland (mand), eastern austria hungarian form of z. v. vivipara viviparous 36 zw (ref. 5) 102 l. kupriyanova, a. kuksin and g. odierna for 10 min. with a 5% giemsa solution in ph 7 phosphate buffer. sumner’s indications (1972) were followed for c-banding staining. sequential staining of c-banding and/or alu 1 endonuclease digestion + cma3+dapi were conducted according to odierna et al. (1999). to observe the mode of reproduction three pregnant females from each of studied populations were kept in a terrarium during june and july 2004 up to hatching. the females were reared separately in a plastic terrarium of 30x20x20 cm, equipped with a shelter, dishes of food and water, heated for 6 h/day with a 40w bulb lamp. terraria were checked for clutches four times a day. results and discussion the observations of studied pregnant females of z. vivipara studied in the terrarium and in nature have shown that they were viviparous. independently of provenance males showed a karyotype of 2n = 36 uniarmed acrocentric (a) chromosomes (foundamental number, fn = 36), whereas females had a chromosome set of 2n = 35 acrocentric (a) elements, then possessing a z1z2w sex chromosome system, with w shaped as an uniarmed acrocentric/subtelocentric (a/st) macrochromosome. most of autosomes and w chromosome possessed conspicuous centromeric and tiny telomeric c-bands. additionally, w constantly had a remarkable interstitial c-band , which was alu 1 resistant (fig. 1). after sequential staining of c-banding + dapi + cma3 centromeric c-bands of 14-16 autosomes and w chromosome, as well as the interstitial w heterochromatin, were dapi positive, then at rich (fig. 2), while telomeric c-bands of several autosomes were cma3 positive, then gc rich. however, two of telomeric cma3 positive loci were more intensively stained than others and corresponding to the regions where nors were identifig. 1. distribution of the three studied population of z. v. vivipara: 1, from tambov region; 2, from tuva republic near todga lake; 3, from tuva-altai border, close to kara-khol lake. 103karyotype and chromosome structure in z. vivipara fig 2. c-banded metaphase plate (a) and relative haploid karyotype of a z. v. vivipara female from altaituva regions. fig. 3. c-banding +cma3 (a) and c-banding + dapi (b) metaphase plate of a z. v. vivipara female from altai-tuva regions sequentially stained. the arrows point to chromosomes bearing nors. 104 l. kupriyanova, a. kuksin and g. odierna fied (kupryianova, 1990; odierna et al., 1998, 2001) (fig. 2). no variation was observed in these chromatinic markers within and among the three studied populations. for a comparison detailed data on karyotype and chromatinic markers are known only for populations of z. vivipara inhabiting other regions (see table 1). the comparison shows that specimens here studied showed similarities in their karyotype and chromatinic markers with those of z. vivipara from north-western part of russia. the latter also had viviparous modes of reproduction, allowing to assign the three populations here studied to the viviparous russian form of z. v. vivipara (2n = 35, z1z2w, and w shaped as a/st) (kupriyanova et al., 2005a). this form was for the first time discovered in two populations in asian and european russia based only on the rough chromosome morphology (kupriyanova, 1986). later, it was also found by chromosome banding methods in one locality in the transcarpathian region of ukraine (kupriyanova, 1990), in one locality in western estonia (kupriyanova, 1997), in one locality in eastern hungary (puky et al., 2004), in two localities of eastern finland (kupriyanova et al., 2005b), in one locality in northern sweden (odierna, unpublished evidence) and in two localities in western russia (kupriyanova et al., 2007; kupriyanova and melashchenko, 2008). thus this viviparous russian form of z. v. vivipara has huge distribution area unlike to other discovered viviparous and oviparous forms and subspecies which often have narrow and mosaic distribution ranges. the present combined reproductive and karyological analysis failed to detect relict oviparous populations of z. vivipara in south-eastern russian region. however, further combined reproductive and karyological analyses in specimens of z. vivipara from other areas of altai and neighbouring regions still are needed. acknowledgements research granted by funds from presidium st. petersburg’s scientific centre, the ras; min. nauka (ns-4212.2006.4), and from university of naples federico ii. references chevalier, m., dufaure, j.p., lecher, p. 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(1972): a simple technique for demonstration centromeric heterochromatin. exp. cell. res. 75: 304-208. © firenze university press www.fupress.com/ah acta herpetologica 5(1): 1-11, 2010 a skeletochronological estimation of age structure in a population of the guenther’s frog, hylarana guentheri, from western china cao li1, 2, wen bo liao1, 3, zhi song yang1, cai quan zhou1 1 institute of rare animal and plant, china west normal university, nanchong 637002, china. 2 department of life science and technology, yibing college, yibing 644007, china. 3 corresponding author. e-mail: liaobo_0_0@126.com drcqzhou1@163.com submitted on: 2009, 7th august; revised on: 2010, 11th february; accepted on: 2010, 15th april. abstract. age determination in amphibians is crucial to investigation of life-history traits. in this context, we studied age structure of the guenther’s frog, hylarana guentheri, population from southwestern china located at an altitude of 300 m using skeletochronological method. our results showed that age in adult males ranged from 1 to 4 years, and from 2 to 6 years in adult females. average age in females was significantly older than males in 2008 and 2009. also, females had significantly larger average body size than males in both years. a significant relationship between age and body size within each sex was found in this species for both years. the ancova analysis revealed that there was significant difference in body size between sexes when the effect of age was removed. the von bertalanffy’s model showed that females had larger asymptotic body size than males, but growth rate of females was smaller than males. keywords. skeletochronology, age structure, hylarana guentheri, sexual size dimorphism. introduction mark-recapture studies to obtain data on age, growth and longevity of animals are very time-consuming. an alternative tool to get such data for amphibians is skeletrochronology which is considered the most reliable method of age estimation by counting the lines of arrested growth (lags) recorded in long bones (hemelaar and van gelder, 1980; castanet and smirina, 1990). it has been successfully used on a variety of species from temperate zone (bufo bufo, hemelaar, 1988; rana temporaria, miaud et al., 1999; r. latastei, guarino et al., 2003; r. chensinensis, lu et al., 2006; r. muscosa, matthews and miaud, 2007; r. holtzi, guarino and erismis, 2008; r. ridibunda, kyriakopoulou-sklavounou et al., 2008) and tropical regions (r. nigrovittata, khonsue et al., 2000; limnonectes limnocharis, pancharat2 cao li et alii na and deshpande, 2003). recently, it also has applied to species from subtropical regions (mantidactylus microtympanum, guarino et al., 1998; boophis occidentalis, andreone et al., 2002; r. swinhoana, lai et al., 2005; dyscophus antongilii, tessa et al., 2007; b. andrewsi, liao, 2009; amolops mantzorum, liao and lu, 2010a; hyla annectans chuanxiensis; liao and lu, 2010b; r. limnocharis; liao et al., 2010b). the accurately assessed individual age allowed us greater understanding of life histories in amphibians. the guenther’s frog, hylarana guentheri, is a species endemic to china that is widely distributed in paddyfields. it is commonly the dominant amphibian species even where the amphibian population is declining. this species is a medium-sized frog [adult snout– vent length (svl) ranges from 62 to 80 mm], showing a female-biased size dimorphism. during the breeding period, males actively search for mates and attract them by advertisement calls (fei and ye, 2001). egg-laying extends from march to may, and the guenther’s frog may be classified as a prolonged breeder (sensu wells, 1977). currently, despite breeding ecology, habitat use and morphological traits of the species has been reported (zhao and adler, 1993; fei and ye, 2001), information about demographic data on the species during the breeding period remains largely unknown. in this study, we used phalangeal skeletochronological method to assess individual age, growth and longevity of h. guentheri in two successive years. our aims were 1): to compare the age structure, body size and growth of a h. guentheri population living western china where these demographic parameters are unavailable; 2) to gain insight into factors that affect the differences in body size between males and females. materials and methods the studied population was located in some rice fields of shidong town in anju county (30°23’n, 105°22’e), at an altitude of 300 m a.s.l., in western china. the rice fields are filled with water to a depth of 8-10 cm. the vegetation of the study site is typical eucalyptus (eucalyptus robusta), oriental arborvitae (platycladus orientalis), silvergrass (miscanthus floridulu) and bulrush (phragmites australis). as being a subtropical region, climate in the study area is strongly seasonal. annual average temperature ranges between 16.9-17.2 °c and annual total precipitation is 908-993 mm. monthly average temperature in summer and winter is 38 °c and -3 °c, respectively. frogs hibernate in holes on land, and migrate in march to lay eggs in the rice fields (fei and ye, 2001). adults and juveniles migrate back to the hole for hibernation in november at the end of the active season. at this locality, all frogs were searched with sampling line of 2500 m length and 2 m width in april 2008 and 2009, when they were active and in breeding condition. all observed frogs on the bank of rice fields along the line were caught using a battery flashlight for illumination. each adult specimen was sexed by their secondary sexual characteristics (the nuptial pads on the first finger for male, eggs readily visible by the skin of the abdomen for female). the individuals were classed as juveniles owning to the absence of nuptial pads or of female aspect. body sizes (the snout–vent length: svl) were measured to the nearest 0.02 mm with a caliper. the last two phalanges of the right hind limb were clipped, for a mark-recapture study, and stored in 10% neutral buffered formalin for skeletochronological analysis. all frogs examined were released at the points of capture. a total of 78 adult males, 61 adult females and 35 juveniles were collected during this study. using the mark-recapture data and the lincoln-petersen index (caughley, 1977), we estimate the population size to be about 558 individuals. the standard skeletochronological procedure (e.g., lu et al., 2006; liao, 2009; liao and lu, 2010a) was used to determine age. for each individual digit, we cleaned of surrounding tissues of 3age structure of hylarana guentheri the phalanges, and then put them in 5% nitric acid to decalcify for 48 h. these decalcified digits were stained for 75 min in ehrlich’s haematoxylin. subsequently, these stained bones were dehydrated through successive ethanol stages. phalanges were then processed for paraffin embedding in small blocks. cross-sections (13 μm) thick were obtained by means of rotary microtome, and the phalanx with the smallest medullar cavity were selected and mounted in aqueous synthetic resin. the sections were observed though a light microscope and the best of them were photographed at selected magnifications using a camera lucida. the analysis of lines of arrested growth (lags) was performed by two different persons (w.b. liao and t.l. yu) with previous experience of the technique. as suggested by castanet and smirina (1990), a thin layer of chondroid tissue situated between periosteal and endosteal bones probably are the remnant of the larval cartilage precursor. it is originally described as “wandständiger knorpel” by kastschenko (1881) and has been indentified to define accurately the endosteal resorption in amphibians (rozenblut and ogielska, 2005). sexual size dimorphism was described by sdi index (lovich and gibbons, 1992). the equation form was: sdi  =  (mean length of the larger sex/mean length of the smaller sex) ± 1 (sdi  =  0 when both sexes are of similar size, sdi > 0 when females are larger than males). the relationship between body length and age using inferred data was examined. growth was estimated using von bertalanffy’s (1957) function, a model demonstrated to be suitable for anurans (lu et al., 2006; ma et al., 2009). the equation form was: st  =  smax × (1 e-kt+b), where st is body size at age t, smax is the estimated asymptotic body size, k is a growth coefficient relating to rate of decline in growth as frogs attain the maximum body size, b is a constant. growth rate can be calculated with the following equation: r = ds/dt = k × (smax – st), which is maximal when st is minimal. we assessed interaction between age and sex using general linear models (glms) treating svl as a dependent variable. we used non-parametric tests (mann-whitney u tests) to compare inter-sex differences in body size and age and the spearman correlation to infer a relationship between variables. we also conducted ancovas with age as covariate to see significance of differences in body size between sexes after removing the effects of age. all probabilities were two-tailed, and the level of significance was p < 0.05. the values given are shown as mean ± sd. results lines of arrested growth (lags) were present in cross sections of juvenile and adult phalanges (fig. 1). in juveniles, endosteal resorption was not observed in the samplings (fig. 1a). in the adults, we found that endosteal resorption incompletely eroded the first (innermost) periosteal lags in twelve males and eight females. however, the first lags of three males and two females were completely eroded by endosteal resorption due to the absence of kl, and we added one year as their true age. false lines would result in serious difficulty in interpreting age of individuals, but they did not affect lags counted in the sample. two very close hematoxynophilic lines that were considered as double lags were observed in five males and four females (about 5.8% of the sample). as a rule these lines must be counted as a single true lag in the samplings (fig. 1d). we captured 139 adult frogs (78 males and 61 females) in successive two years resulting in a male-biased sex ratio of 1.28:1. the sex ratio did not differ from 1:1 (χ2  =  2.08, df  =  1, p  =  0.15). a total of 104 individuals (35 juveniles, 45 males and 33 females) were captured and marked by toe-clipping in 2008. we captured 70 individuals (9 juveniles, 41 males and 20 females) and recaptured 2 juveniles, 4 males and 6 females in 2009. frequency of recapture (as an indictor of survival) in males was smaller than females. frequency of recapture of males and females was 8.9% and 18.2%, respectively. 4 cao li et alii adult age ranged from 1 to 4 years in males, and from 2 to 6 years in females (fig. 2). age distributions between males and females did not diff er markedly in 2008 and 2009 (kolmogorov-smirnov test: 2008: d  =  0.58, p  =  0.89; 2009: d  =  0.85, p  =  0.45). females had signifi cantly older average age than males in two years (table 1; mann–whitney u-test: 2008, z = 3.21, p = 0.003; 2009, z = 2.22, p = 0.03). average body size of females was signifi cantly larger than that of males (table 1; mann-whitney u-test: 2008, z  =  6.22, p  <  0.001; 2009, z  =  4.93, p  <  0.001). th ere was a signifi cant diff erence in svl between females and males within each age group (table b kl kl c kl d a kl fig. 1. four selected examples of hematoxylin-stained cross-sections of the longest phalange of hylarana guentheri for two successive years in a subtropical population, western china (a: 1-yr old male; b: 2-yr old female; c: 3-yr old male and d: 4-yr old female, the second lags is double). arrows indicate the lines of arrested growth (lag). kl represents kastschenko line, the interface between the endosteal and periosteal zones. scale bar: 300 μm. 5age structure of hylarana guentheri 2; all p  <  0.05). a non-signifi cant interaction between sex and age (f1,121  =  0.21, p  =  0.81) revealed that relationship between age and size did no diff er apparently among the sexes. th e ancovas analysis showed that the diff erence in body size between sexes remained signifi cant (2008, f1,74  =  49.92, p  <  0.001; 2009, f1,47  =  11.34, p  <  0.002) when the signifi cant eff ect of age was controlled in both years (2008, f1,74  =  28.21, p  <  0.001; 2009, f1,47 = 26.54, p < 0.001). th e spearman correlation showed that signifi cant relationships between age and svl in both years were found within each sex (2008: male, rs = 0.57, n = 42, p < 0.001; female, rs  =  0.56, n  =  32, p  =  0.001; 2009: male, rs  =  0.55, n  =  37, p  <  0.001; female, rs  =  0.72, n  =  10, p  =  0.019). th e sexual dimorphism index (sdi) with body size in 2008 and 2009 was -0.102 and -0.112, respectively. th e von bertalanff y’s model provided a good description for the relationship between body size and age for the populations in either sex [2008, male, st  =  73.5 × (1 e-0.72t-0.99), 0 5 10 15 20 25 1 2 3 4 5 6 0 5 10 15 20 25 1 2 3 4 5 6 n um be r o f i nd iv id ua ls age (yr) 2008 2009 fig. 2. relationship between body size and age classes of hylarana guentheri (male, open bars; female, close bars) for two successive years in a subtropical population, western china. growth curves follow the von bertalanff y model. body size in mm is shown as mean ± sd. 6 cao li et alii r2 = 0.32; female, st = 80.4 × (1 e-0.51t-1.34), r2 = 029; 2009, male, st = 71.9 × (1 e-1.21t-0.11), r2  =  0.64; female, st  =  78.6 × (1 e-0.86t-0.40), r2  =  0.59; fig. 3]. the results showed that females were generally larger than males throughout the life cycle, but growth rate of males was greater than females in the population (2008, male, r  =  3.35; female, r  =  2.11; 2009, male, r = 8.31; female, r = 4.31). discussion this is the first study using skeletochronology to estimate the age structure of a h. guentheri population in western china. stained lags were clearly visible between zones of thicker layers of bone deposited in growth periods (fig. 1). the pattern of lag deposition is considered to be genetically controlled with reinforcement by seasonality such as table 1. body size and age of hylarana guentheri for two successive years in a subtropical population from western china. values in descending order are mean ± sd and sample size. individuals 2008 2009 body size (mm) age (yrs) body size (mm) age (yrs) juvenile 51.8 ± 6.9 (n = 26). 48.9 ± 6.9 (n = 9) male 68.7 ± 3.7 (n = 45) 2.6 ± 0.8 (n = 42) 67.0 ± 3.7 (n = 41) 2.2 ± 0.6 (n = 37) female 75.7 ± 3.8 (n = 33) 3.4 ±1.0 (n = 32) 74.5 ± 4.9 (n = 20) 3.0 ± 1.1 (n = 10) table 2. difference in body size between males and females within each age class group hylarana guentheri in a subtropical population western china. values in descending order are mean ± sd and sample size. age 2008 2009 male female z p male female z p 1 63.2 ± 1.7 (n = 3) 2 67.0 ± 3.7 (n = 23) 72.3 ± 3.0 (n = 5) 2.61 < 0.01 66.1 ± 3.2 (n = 23) 70.2 ± 2.7 (n = 4) 1.95 < 0.05 3 70.4 ± 2.6 (n = 11) 76.0 ± 3.5 (n = 16) 3.65 < 0.001 69.9 ± 3.3 (n = 10) 74.4 ± 1.5 (n = 3) 2.12 < 0.05 4 70.0 ± 1.9 (n = 8) 75.8 ± 2.1 (n = 6) 2.45 < 0.05 72.6 (n = 1) 77.5 ± 7.2 (n = 2) 5 81.1 ± 0.1 (n = 2) 77.2 (n = 1) 6 80.3 (n = 1) 7age structure of hylarana guentheri temperature fl uctuations (alcobendas and castanet, 2000). in our study site, the active period is limited to march-november and growth mark deposit annually in winter. winter growth marks can be signifi cantly expressed in h. guentheri, as previously observed in other species in subtropical regions (guarino et al., 1998; morrison et al., 2004; lai et al., 2005; liao and lu, 2010a). completive lag resorption based on the occurrence of kl was easily identifi able in this species, and it did not complicate age estimation. in some 0 10 20 30 40 50 60 70 80 90 0 1 2 3 4 5 6 7 8 2009 sv l (m m ) 0 10 20 30 40 50 60 70 80 90 0 1 2 3 4 5 6 7 8 2008 age (yr) fig. 3. growth curve of the breeding population of hylarana guentheri from western china (juvenile, open triangles; male, open circles; female, close circles) following the von bertalanff y model for two successive years. 8 cao li et alii temperate amphibians, double lags are the expression of growth interruption twice a year and show a periodic pattern (caetano and castanet, 1993). in our study, hylarana guentheri experienced growth interruption twice a year; the occasionally occurring double lines were easily identified because the annual growth zones were by far broader than the distance between the double lines and thus, did not deteriorate precision of age estimates. in these individuals, false lines differed markedly from lags because they were incomplete and faint hematoxynophilic lines. for many amphibian species it is reported that males reach sexual maturity one year earlier than females (miaud et al., 1999; kyriakopoulou-sklavounou and grumiro, 2002; eaton et al., 2005; liao et al., 2010a). we also found that h. guentheri males reached sexual maturity earlier than females. in the h. guentheri population, longevity of males was shorter than that of females, as observed previously in most anurans (khonsue et al., 2001; ento and matsui, 2002; matthews and miaud, 2007; liao, 2009). the fact that males tended to shorter lifespan than females resulted from the lower survival of males. significant correlation was found between age and svl for both years, as reported in other anurans (hemelarr, 1983; ryser, 1996; lu et al., 2006). there were significant overlaps of the body size distributions between age classes in mature individuals, therefore, body size by h. guentheri was not considered as a reliable predictor of age. this tendency has been pointed out by many previous authors in other amphibians (halliday and verrell, 1988; platz and lathrop, 1992; kutrup et al., 2005). sexual size dimorphism is observed in many amphibians, and females are larger than males (shine, 1979; monnet and cherry, 2002). in h. guentheri, adult males had significantly smaller size than females in two years. larger body size in females has been discussed in relation to the fact that females need more stored energy to allocate much more to gonad and embryo development (shine, 1979; halliday and verrell, 1988). by contrary, males need relatively low investment of energy for reproduction in that spermatogenesis is less costly than vitellogenetic growth of oocytes (jørgensen, 1992). the difference in body size between sexes is also often explained by post-maturation growth rate and growth duration (or age). for h. guentheri, growth rate of males was larger than females, and also grew more quickly towards the asymptotic body size than females. difference in growth rate between sexes would contribute marginally to the observed sexual size dimorphism. amphibians have indeterminate growth (halliday and tejedo, 1995). thus, the earlier age at sexual mature of males in this study resulted in the differences in svl between sexes because males took more energy in reproduction rather than growth. a larger size in females can also be attained thanks to a longer life span and larger mean age in this species, a pattern common among many amphibians (monnet and cherry, 2002; matthews and miaud, 2007), including subtropical species (morrison et al., 2004; lai et al., 2005). moreover, genetic factor may result in difference in body size inter-sex in interand intra-population (berven, 1982; miaud et al., 1999). future studies need to solve the views. acknowledgments we thank liao g.j. and wang z.q. for their assistance in the field and ma x.y. for assistance during the lab work. financial support was provided for the scientific research foundation of chi9age structure of hylarana guentheri na west normal university (09b001) and the scientific research foundation of sichuan provincial education department (09zc010). we declare that all animals used in research should be treated ethically following the all applicable institutional animal care guidelines in china. references alcobendas, m., castanet, j. 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(1957): quantitative laws in metabolism and growth. quart. rev. biol. 32: 217-231. wells, k.d. (1977): the social behaviour of anuran amphibians. anim. behav. 25: 666-693. zhao, e.m., adler, k. (1993): herpetology of china. society for the study of amphibians and reptiles, oxford, ohio. acta herpetologica 4(1): 7-13, 2009 predicting elusiveness: potential distribution model of the southern smooth snake, coronella girondica, in italy pierluigi bombi1, luca luiselli2, massimo capula3, daniele salvi4 1 via maria giudice 23, i-00135 roma, italy. corresponding author. e-mail: bombi@uniroma3.it 2 f.i.z.v. (ecology) and centre of environmental studies ‘demetra s.r.l.’, via olona 7, i-00198 roma, italy. e-mail: lucamlu@tin.it 3 museo civico di zoologia, via ulisse aldrovandi 18, i-00197 roma, italy. e-mail: m.capula@ comune.roma.it 4 dipartimento di biologia ambientale, università roma tre, viale marconi 446, i-00146 roma, italy. e-mail: salvi@uniroma3.it abstract. predictive models of species distribution may be very useful for understanding actual distribution of elusive species, including several snakes. the southern smooth snake (coronella girondica) is likely the most elusive snake species of peninsular italy, and is therefore well-suited for predicting potential distribution studies. in this paper we predict the potential distribution map of this species in italy by using maxent algorithm, that finds the probability distribution of maximum entropy that is constrained by considered ecological parameters. presence data for coronella girondica were gotten from ckmap. the potential distribution model of coronella girondica showed a very good overall performance (auc = 0.959), and indicated that high suitability areas correspond mainly to tyrrhenian north and central regions, including liguria, western emilia-romagna, tuscany, umbria, and northern latium. southern italian regions were clearly unsuitable for this snake species. overall, our study revealed that previous distribution maps indicating the occurrence of coronella girondica in southern italy and sicily were poorly reliable. the conservation and management implications of our study are also addressed. keywords. distribution modelling, elusive species, serpentes, colubridae, italy. introduction analysis of many aspects of ecology and conservation may be helped by the use of predictive models of species distribution (graham et al., 2004), including for instance modelling studies of invasive species spread (e.g., thuiller et al., 2005), impacts of climate change (thomas et al., 2004), and spatial patterns of species diversity (guisan and zimmermann, 2000; graham et al., 2006). modelling potential distribution of a given species based on presence-only data is also a useful tool for predicting new localities of presence for rare and 8 p. bombi et alii threatened species that are hardly found in the wild (godown and peterson, 2000; engler et al., 2004), or for species that are simply very elusive and whose distribution is therefore little known (guisan et al., 2006). the utility of these presence-only modelling methods is that they only require a set of known occurrences together with predictor variables such as topographic, climatic, edaphic, biogeographic and remotely sensed variables (phillips and dudik, 2008). the main advantage of presence-only data methods is that it is not necessary to confirm absence records, a big problem especially in elusive species. several snake species are genuinely elusive, and therefore their distribution may be somewhat underestimated and least known compared to other reptiles. for instance, in recent years it has been demonstrated that the true distribution of the snake hemorrhois hippocrepis is considerably wider in sardinia than previously suspected (e.g., compare maps in razzetti and bonini, 2006 with that in bruno and maugeri, 1977). among the snake species found in the italian peninsula, the southern smooth snake (coronella girondica) is likely the most elusive, and certainly one of the least known in terms of both ecology and distribution. indeed, this snake spends much of the time below-ground or behind flat stones, and is active in the open only at night (agrimi and luiselli, 1994; capula et al., 1995). as a consequence, its local distribution is often very little known (razzetti and bonini, 2006; bologna et al., 2007). coronella girondica is therefore a well-suited species for modelling its niche in order to find new suitable areas, also because there is circumstantial evidence that this species is declining over wide sectors of its current range (bologna et al., 2007). our aim in this paper is to present a model of distribution for coronella girondica in italy, and to discuss the potential importance of distribution maps in terms of ecological and conservation purposes. materials and methods presence data for coronella girondica were gotten from ckmap (stoch, 2000-2005). the ckmap databank represents the largest, most authoritative, and most updated resource of faunistic knowledge in italy, being composed by more than 500000 records regarding approximately 10000 terrestrial and freshwater species (ruffo and stoch, 2005). ckmap data indicates the occurrence of each species within the cells of the universal transverse mercator (utm, 10 × 10 km) grid that intersect the italian territory. the presence data was downscaled to the resolution of environmental predictors (e.g., araújo et al., 2005) and implemented in the modelling procedure. in order to build a reliable model of habitat suitability, one distribution record from north-eastern italy was excluded from the computations, as in recent studies it has been considered to be likely the result of an introduction event (e.g., lapini et al., 1999). potential distribution of coronella girondica in italy was assessed by maxent algorithm (phillips et al., 2004, 2006). in order to estimate the target probability distribution, maxent finds the probability distribution of maximum entropy that is constrained by considered ecological parameters. in the case of modelling ecological niches of species, these constraints consist of the values of those pixels at which the species has been detected (phillips et al., 2004, 2006; peterson et al., 2007). maxent models produce predictions in the form of real numbers between 0 and 100, representing cumulative probabilities of occurrence (phillips et al., 2004, 2006; peterson et al., 2007). maxent can combine predictors to manage over-fitting by regularizing factors (phillips et al., 2006; phillips and dudík, 2008). we performed this algorithm by using the 19 climatic variables (table 1), with a resolution of 30” of geographic degree, provided by worldclim (hijmans et al., 2004). 9modelling distribution of coronella girondica in italy the reliability of the potential distribution model was assessed by area under curve (auc) criterion through a jack-knife procedure. this validation procedure compares the predicted values of habitat suitability assigned to presence and pseudo-absence data in the test subset by producing the receiver operating characteristic (roc) plots (fielding and bell, 1997) and deriving the relative auc value (faraggi and reiser, 2002). the main positive feature of auc consists of being a single threshold-independent measure for model performance (fielding and bell, 1997; manel et al., 2001; allouche et al., 2006; peterson et al., 2007). an auc value can be interpreted as the probability that a presence site, randomly chosen from the dataset, has a higher predicted value than an absence site (elith et al., 2006, phillips et al., 2006). models were run by using maxent 3.1 (phillips et al., 2006), which was also utilized for the jack-knife procedure for the model validation. results the potential distribution model of coronella girondica showed a very good overall performance (auc = 0.959; see fig. 1). the potential distribution map (fig. 2) showed that high suitability areas correspond mainly to tyrrhenian north and central regions, table 1. climatic variables used to elaborate the models var no. description var 1 annual mean temperature var 2 mean diurnal range [mean of monthly (max temp min temp)] var 3 isothermality [(var2 / var7) * 100] var 4 temperature seasonality (standard deviation * 100) var 5 maximum temperature of warmest month var 6 minimum temperature of coldest month var 7 temperature annual range (var5 var6) var 8 mean temperature of wettest quarter var 9 mean temperature of driest quarter var 10 mean temperature of warmest quarter var 11 mean temperature of coldest quarter var 12 annual precipitation var 13 precipitation of wettest month var 14 precipitation of driest month var 15 precipitation seasonality (standard deviation / mean) var 16 precipitation of wettest quarter var 17 precipitation of driest quarter var 18 precipitation of warmest quarter var 19 precipitation of coldest quarter 10 p. bombi et alii including liguria, western emilia-romagna, tuscany, umbria, and northern latium. these regions indeed correspond to the areas with the higher number of known presences (razzetti and bonini, 2006). wide regions in both northern and southern italy appeared completely unsuitable for coronella girondica (fig. 2). discussion the most updated and reliable distribution map for coronella girondica in italy is given in razzetti and bonini (2006). these authors pointed out that several historical records are unreliable, and that the species is likely more common on the tyrrhenian side of italy than on the adriatic side. razzetti and bonini (2006) map was considerably different from previous distribution maps for this species (bruno and maugeri, 1977), where coronella girondica was claimed to be widespread also in the southern regions of italy, i.e. campania, calabria, basilicata, puglia, and sicily. our modelling analysis clearly confirmed the reliability of razzetti and bonini (2006) map compared to bruno and maugeri (1977) map, indicating that the southern italian regions are unsuitable for the presence of this fig. 1. receiver operating characteristic (roc) plots for the potential distribution model of coronella girondica in italy. the “area under roc curve” is the auc value. 11modelling distribution of coronella girondica in italy colubrid species. thus, our study reveals that at least 14 presence records in bruno and maugeri (1977) map are unreliable. on the other hand, the potential distribution map given in this study mirrors widely the map drawn by capula et al. (1995), which was built in consideration of the reproductive ecology characteristics of coronella girondica that were considered inappropriate for the life in the southern italian climates. however, if we compare the map of true distribution given in razzetti and bonini (2006) with the potential distribution map given here, some differences also do exist. indeed, we can stress that the potential range of this snake is much wider in tuscany, umbria and latium than currently demonstrated. for instance, there are very few recent records for presence sites in tuscany, umbria, and latium whereas the distribution suitability map indicated that wide sectors of these regions are very well suited for coronella girondica. we would fig. 2. potential distribution model for coronella girondica. probability of presence increases from white to black. 12 p. bombi et alii therefore urge further accurate research in these regions to improve the knowledge of the local distribution of this species. in conservation terms, it is however necessary to stress that some areas that are depicted as potentially very good for this snake (for instance the areas surrounding rome) are currently strongly altered due to human intervention on the natural habitat. coronella girondica may have been therefore extirpated from these areas, and the remnant populations that are still eventually found should be carefully managed and protected in order to avoid further decline in these potentially important areas for the species. the well-suited areas in tuscany and umbria are likely less exploited than those in latium, suggesting that the relatively few presence records should depend more on unsatisfying field research than on extirpation of the species from potentially good areas. it is, therefore, likely that coronella girondica is not really threatened or in decline in these latter regions, but that its presence may have simply gone undetected in many sites due to its elusiveness accomplished with suboptimal field research by herpetologists. nevertheless these hypotheses need to be confirmed by including human impact variables in the analysis. in conclusion, we suggest that careful field research should be done especially in tuscany and umbria to improve the number of presence data for this species, and that special conservation and management actions should be done in the suitable areas of latium, where the suitability of the areas for coronella girondica is high but where the human intervention on the habitat has been very intense. references agrimi, u., luiselli, l. 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(2005): niche-based modelling as a tool for predicting the risk of alien plant invasions at a global scale. glob. change biol. 11: 2234-2250. phylogenetic relationships of geckos of the genus nactus and their relatives (squamata: gekkonidae) todd r. jackman, aaron m. bauer, eli greenbaum department of biology, villanova university, 800 lancaster ave., villanova, pa 19085, usa. corresponding author. e-mail: todd.jackman@villanova.edu submitted on 2007, 16th june; revised on 2007, 27th november; accepted 2008, 29th january. abstract. we employed nuclear and mitochondrial dna sequence data to investigate relationships within the gekkonid genus nactus and between nactus and other gekkonid genera. nuclear (rag-1, pdc) and mitochondrial (nd2) data provide strong support for conflicting patterns of relationship among bisexual new guinean species of nactus and the unisexual oceanic form n. pelagicus. this may be explained by an ancient mitochondrial introgression event between n. sphaerodactylodes and n. vankampeni, a recent selective sweep of mitochondrial dna throughout n. vankampeni, and gene conflict stemming from the hybrid event that gave rise to n. pelagicus. strong support from all data partitions is obtained for the sister group relationship of nactus to a clade consisting of the australian heteronotia and the southeast asian dixonius. putative synapomorphies of the nactus/heteronotia/dixonius clade include the reduction of the second phalanx of digit iv of the manus and the presence of regular rows of keeled (sometimes multicarinate) dorsal tubercles on the dorsum. nactus and heteronotia both include parthenogenetic species formed via hybridogenesis. this is rare among geckos, and vertebrates in general, and at some level may also be synapomorphic. dixonius is not known to have any all-female species, but “d. siamensis” consists of multiple chromosome “races” that mirror morphologically cryptic, but karyotypically distinct, species in the other two genera. the strong support for the nactus/heteronotia/dixonius clade demonstrates that the leaf-toed digital morphology of dixonius has evolved multiple times within the gekkonidae and suggests that superficial digital morphology may be misleading with respect to gekkonid suprageneric relationships. keywords. gekkonidae, nactus, heteronotia, dixonius, molecular phylogeny, convergence, parthenogenesis, digital morphology. introduction geckos and pygopods comprise the gekkota, a relatively basal lineage among squamates (townsend et al., 2004; vidal and hedges, 2005) and the only major lineage of priacta herpetologica 3(1): 1-18, 2008 issn 1827-9643 (online) © 2008 firenze university press 2 t.r. jackman, a.m. bauer and e. greenbaum marily nocturnal lizards (pianka and vitt, 2003). current estimates of gekkotan diversity recognize approximately 1135 species in 108 genera (kluge, 2001; bauer, 2002; han et al., 2004). the diversity of gekkotans, their great age (165-225 myr; kluge, 1987; vitt et al., 2003; vidal and hedges, 2005; kumazawa, 2007), and broad distribution, as well as their ecological significance and possession of many morphological, physiological, and behavioral autapomorphies (pianka and vitt, 2003) should make them model organisms for study. however, this has been hindered by a lack of a well-corroborated hypothesis of relationship among gekkotan taxa. the composition and interrelationships of higher order gekkotan clades are generally well established (kluge, 1967, 1987; donnellan et al., 1999; harris et al., 1999, 2001; han et al., 2004; townsend et al., 2004), as are species level phylogenies for some of the less diverse basal clades (e.g., pygopodidae – jennings et al., 2003; carphodactylidae – hoskin et al., 2003; schneider, 2004). however, relationships between genera in the most speciose clade of gekkotans, the gekkonidae, have never been resolved. previous morphological studies have recognized the (often digital) autapomorphies of particular genera or clusters of genera, but have been unable to identify relationships between such clusters (loveridge, 1947; russell, 1972, 1976; kluge, 1983). further, extensive homoplasy with respect to digital characters has been both predicted a priori (russell, 1976, 1979) and implied by non-congruence with other data sets (han et al., 2004; gamble et al., 2008). until now mitochondrial dna studies have likewise proved to be incapable of resolving many gekkonid intrageneric relationships as they suffer from both a lack of sampling across all taxa and limitiations in the depth of phylogenetic divergences that can reliably be reconstructed (ota et al., 1999). as part of a broader study of gekkotan relationships we are currently examining both interand intrageneric level relationships among all recognized genera of gekkonids. although several clusters of genera, such as those comprising the “gekko group” and the “pachydactylus group” have received consistent support from both morphological (kluge, 1968; russell, 1972; haacke, 1976; bauer, 1990; kluge and nussbaum, 1995) and molecular studies (han et al., 2004; bauer and lamb, 2005; lamb and bauer, 2006), relationships among most gecko genera remain poorly resolved at best. this is especially true of those taxa that lack the adhesive subdigital mechanism for which many climbing geckos are known. russell (1972, 1979), for example, established many intergeneric morphotypic groupings for “padded” geckos, but included all “padless” forms except cyrtodactylus, stenodactylus and teratoscincus into a single cluster. one padless genus that has received recent taxonomic attention is nactus, a group of ten currently recognized species, as well as an uncertain number of undescribed forms in new guinea (donnellan and moritz, 1995; zug and moon, 1995; zug, 1998; kraus, 2005; rösler et al., 2005). species of nactus are chiefly terrestrial and are represented by bisexual species in the mascarene islands (n. serpensinsula, n. coindemirensis), in northern australia (n. eboracensis, n. galgajuga, n. cheverti), new guinea (n. vankampeni, n. acutus, n. sphaerodactylodes), and the southern solomon islands and northern and central vanuatu (n. multicarinatus). in southern vanuatu, new caledonia, fiji, polynesia, and micronesia a unisexual form, n. pelagicus, occurs (moritz, 1987; zug, 1989; bauer and henle, 1994; zug and moon, 1995; kraus, 2005; rösler et al., 2005). the genus nactus was erected by kluge (1983) to accommodate certain species of geckos that were, at the time, assigned to the large, chiefly tropical asian genus cyrto3phylogenetic relationships of the genus nactus dactylus. kluge divided cyrtodactylus on the basis of the condition of the second ceratobranchial arch. whereas most members of the genus possessed the presumed derived condition of loss of this structure, a small cluster of species retained the primitive state and were thus allocated to kluge’s paraphyletic “ptyodactylini.” in addition to the hyoid arch condition, kluge (1983) considered nactus diagnosable on the basis of several external features including: regular rows of enlarged dorsal tubercles (absent in n. coindemirensis), multicarinate dorsal tubercles and carinate ventral scales (absent in n. galgajuga), and fused nasal bones (kluge, 1983; bullock et al., 1985). among these features, multicarinate tubercles are autapomorphic (kluge, 1983; kraus, 2005), but fused nasals are widespread among diverse genera (bauer, 1990; kluge and nussbaum, 1995). bullock et al. (1985) redefined the genus, adding the lack of a radially directed portion on the ventralmost of the 14 scleral ossicles as a derived character of nactus. kluge (1963) suggested that nactus (then considered part of cyrtodactylus) might be related to the australian heteronotia, but no explicit support for this was provided. russell (1972: 171) subsequently stated “it is clear from the study of digital structure that c. pelagicus should be referred to heteronotia” but no taxonomic action was taken. ulber and gericke (1988) hypothesized that nactus was the sister group of cyrtodactylus plus the genera of palearctic naked-toed geckos (recognized by them as mediodactylus, cyrtopodion, tenuidactylus, agamura, bunopus, alsophylax, carinatogecko and altiphylax) and macey et al. (2000) found weak support for nactus as the sister group to cyrtopodion based on allozymes, but their sampling included only six genera of geckos. greer (1989), on the other hand, considered the affinities of nactus to be unknown and in the absence of any explicit, well-supported higher order patterns of relationship including nactus, kraus (2005) included a diversity of potential outgroups in a phylogenetic analysis of nactus based on 23 morphological characters. he used five different sets of outgroups: 1) “gekkonini” with naked toes – alsophylax, bunopus, cyrtodactylus, cyrtopodion, mediodactylus, tenuidactylus; 2) “ptyodactyini” with naked toes – paroedura [sic!; this genus has distal subdigital scansors], pristurus, quedenfeldtia; 3) “ptyodactylini” with fused nasals – ebenavia, paragehyra, paroedura; 4) austro-papuan gekkonids – cyrtodactylus, gehyra, gekko, hemidactylus, hemiphyllodactylus, heteronotia, lepidodactylus; and 5) a combination of all 17 outgroup genera. kraus (2005) found no evidence for the monophyly of nactus, as the species lacking multicarinate tubercles (n. galgajuga and n. coindemirensis) consistently clustered with outgroup genera or as part of a basal polytomy involving the outgroups and an otherwise monophyletic nactus. although it can be argued that some of the morphological characters employed by kraus (2005) might be prone to homoplasy, at least in part the lack of evidence for the monophyly of nactus may be due to the choice of outgroup taxa. we here present data on both the the interand intrageneric relationships of nactus in order to provide an appropriate basis for future phylogenetic studies within this genus and its nearest relatives and to identify evolutionary questions within this group that will require further study. our findings indicate conflict between nuclear and mitochondrial data sets with respect to intrageneric relationships, but strongly support a pattern of intergeneric relationships that imply that traditional views regarding the phylogenetic value of digital morphology may be misleading. 4 t.r. jackman, a.m. bauer and e. greenbaum material and methods eight samples of nactus representing the parthenogenetic n. pelagicus and four new guinean species were included in a broad multi-locus study of gekkotan relationships (bauer a.m., jackman t.r. and greenbaum e., unpubl. data) that incorporated representatives of 467 species in 97 genera, including all gekkonid genera except one from south america (bogertia) and representatives of the palearctic asian taxa alsophylax, altigecko, siwaligecko and indogecko, all of which are probably members of a larger clade of palearctic bent-toed geckos which was represented by other taxa in our sampling. as a result, a strongly supported clade with long branch length including nactus was identified as a member of a more inclusive group comprising the majority of old world gekkonids exclusive of a small number of chiefly palearctic genera (gamble et al., 2008). for the present study, nactus and the other members of its clade identified in the broader study (heteronotia and dixonius) were treated as ingroup taxa and representatives of two other well-supported lineages in the main gekkonid clade were chosen as outgroups (hemidactylus robustus and gekko gecko) (table 1). results of the broader study of gekkonid relationships will be presented elsewhere. genomic dna was isolated from the tail or liver tissue samples preserved in 95-100% ethanol with the qiagen dneasy tissue kit (valencia, ca, usa). we used double-stranded pcr to amplify 2,971 aligned bases of six mitochondrial (nd2 and five trnas – 1,484 bases) and two nuclear (1,068 bases of rag-1 and 419 bases of pdc [phosducin]) genes using the primers listed in table 2 (see jackman et al., 2007 for details regarding the phosducin gene). all nuclear sequence data was protein coding. amplification of 25 μl pcr reactions were executed on an eppendorf mastercycler gradient thermocycler. amplification of genomic dna began with an initial denaturation for 2 minutes at 95 °c followed by 95 °c for 35 s, annealing at 50 °c for 35 s, and extension at 72 °c for 150 s with 4 s added to the extension per cycle for 32 cycles for mitochondrial dna and 34 cycles for nuclear dna. when needed, annealing temperatures were adjusted to increase or decrease specificity on a case by case basis. products were visualized with 1.5% agarose gel electrophoresis. target products were purified with ampure magnetic bead solution (agencourt bioscience, beverly, ma, usa) and sequenced with either the bigdye® terminator v3.1 cycle sequencing kit (applied biosystems, foster city, ca, usa) or the dyenamic™ et dye terminator kit (ge healthcare, piscataway, nj, usa). sequencing reactions were purified with cleanseq magnetic bead solution (agencourt bioscience, beverly, ma, usa) and analyzed with an abi 3700 automated sequencer. the accuracy of sequences was ensured by incorporating negative controls and sequencing complementary strands. sequences were aligned by eye in the computer program seqman pro (dnastar inc., madison, wi, usa), and all four protein-coding genes were translated to amino acids with macclade (maddison and maddison, 1992) to confirm conservation of the amino acid reading frame and check for premature stop codons. phylogenetic relationships among the samples were assessed with parsimony, likelihood, and bayesian optimality criteria. maximum parsimony (mp) analyses were conducted in paup*4.0b10 (swofford, 2002). the heuristic search algorithm was used with the following conditions: 25 random addition replicates, accelerated character transformation (acctran), tree bisection-reconnection (tbr) branch swapping, zero-length branches collapsed to yield polytomies, and gaps treated as missing data. each base position was treated as an unordered character with four alternate states. we used nonparametric bootstraps (1,000 pseudoreplicates unless stated otherwise) to assess node support in resulting topologies with tbr branch swapping and 5 random addition replicates per pseudoreplicate. strict consensus trees were calculated when several equally parsimonious trees resulted from mp searches. we used the the akaike information criterion (aic) in modeltest 3.06 (posada and crandall, 1998) to find the model of evolution that best fit the data for subsequent maximum likelihood (ml) and bayesian inference (bi) analyses. all genes were pooled to determine the best model for ml analyses, but separate models for each gene were run for bi. separate models for each gene and 5phylogenetic relationships of the genus nactus table 1. list of samples used in this study giving sample locality, museum voucher specimen or collector’s field number, and genbank accession numbers for each gene. collection abbreviations: amb = aaron m. bauer, asw = alison swindle whiting, bpbm = bernice p. bishop museum, cas = california academy of sciences, ff = frank fast, fk = fred kraus, fmnh = field museum of natural history, llg = l. lee grismer. sample museum no. locality genbank accession numbers nd2 rag-1 pdc dixonius siamensis llg 7328 phom aural, pursat province, cambodia eu054299 eu054283 eu054267 dixonius siamensis llg 7378 phom aural, pursat province, cambodia eu054298 eu054282 eu054266 dixonious vietnamensis fmnh 263003 keo seima district, mondolkiri province, cambodia eu054297 eu054281 eu054265 gekko gecko cas 204952 vic. mwe hauk village, ayeyarwardy division, myanmar (16°16’39.2”n, 94°45’37.5”e) eu054288 eu054272 eu054256 hemidactylus robustus fmnh 245519 makran district, gwadar division, baluchistan province, pakistan eu054287 eu054271 eu054255 heteronotia binoei ams 151170 fort grey tip, sturt national park, new south wales, australia eu054301 eu054285 eu054269 heteronotia binoei ams 159893 limestone caves, ashford, new south wales, australia eu054302 eu054286 eu054270 heteronotia planiceps ams 140331 23.3 km nnw of junction of tunnel creek road with great northern hwy., western australia, australia eu054300 eu054284 eu054268 nactus sp. asw 510 tekadu, lakekamu river basin, morobe province, papua new guinea (7°41’s, 135°33’e) eu054292 eu054276 eu054260 nactus sp. asw 666 tekadu, lakekamu river basin, morobe province, papua new guinea (7°41’s, 135°33’e) eu054294 eu054278 eu054262 nactus acutus bpbm 20755 rossel island, louisiade archipelago, milne bay province, papua new guinea eu054289 eu054273 eu054257 nactus pelagicus amb 7287 mt. gouémba, province sud, new caledonia (22°10’00”s, 166°56’27”e) eu054291 eu054275 eu054259 nactus pelagicus ff6 ile des pins, province sud, new caledonia eu054290 eu054274 eu054258 nactus sphaerodactylodes bpbm 20759 sudest island, louisiade archipelago, milne bay province, papua new guinea eu054293 eu054277 eu054261 nactus vankampeni fk 11384 wewak, east sepik province, papua new guinea eu054295 eu054279 eu054263 nactus vankampeni bpbm 18671 mt. shungol, morobe province, papua new guinea eu054296 eu054280 eu054264 6 t.r. jackman, a.m. bauer and e. greenbaum codon position of protein-coding genes were estimated (brandley et al., 2005). ml analyses with empirical base frequencies (obtained in modeltest) were performed in paup* with a neighbor-joining starting tree. as with mp, the nonparametric bootstrap was used to assess the stability of internal nodes in the resulting phylogenies. partitioned bayesian analyses were conducted with mrbayes 3.1 (ronquist and huelsenbeck, 2003) with default priors. analyses were initiated with random starting trees and run for 1,000,000 generations; markov chains were sampled every 1,000 generations. convergence was checked by plotting likelihood scores against generation, and 25 trees were discarded as “burn in.” two separate analyses with two independent chains were executed to check for convergence of log-likelihoods in stationarity (huelsenbeck and ronquist, 2001). maximum likelihood shimodaira-hasegawa (sh) tests (shimodaira and hasegawa, 1999) were used using paup*4.0b10 (swofford, 2002). to compare the nuclear and mitochondrial data sets. parameters for the test were estimated using the alternative topology with a gtr + gamma + invariant model. the test was perfomed using rell bootstrap (one tailed) and 1,000 bootstrap replicates. parsimony estimates of incongruence between data sets were performed using an incongruence-length difference (ild) test (farris et al., 1995) as implemented in in paup*4.0b10 (swofford, 2002) as the partition homogeneity test. for this test, 10,000 replicates of were made to generate the null distribution to test the significance of the observed sum of tree lengths for the data sets. results the combined data set had 2,971 characters and 1,264 variable characters, 904 of which were parsimony-informative. the mitochondrial data set hat 687 informative characters, and the rag1 and pdc had 147 and 70 informative characters respectively. one most parsimonious tree of 2,728 steps was found. the likelihood score of the optimal ml tree was –ln l 16115.19. table 2. primers used in this study. primer gene reference sequence phof2 pdc bauer et al. (2007) 5’-agatgagcatgcaggagtatga-3’ phor1 pdc bauer et al. (2007) 5’-tccacatccacagcaaaaaactcct-3’ l4437b met trna macey et al. (1997) 5’-aagcagttgggcccatacc-3’ l5002 nd2 macey et al. (1997) 5’-aaccaaacccaactacgaaaaat-3’ nd2f101 nd2 greenbaum et al. (2007) 5’-caaacacaaacccgraaaat-3’ nd2r102 nd2 greenbaum et al. (2007) 5’-cagcctaggtgggcgattg-3’ trpr3a trp trna greenbaum et al. (2007) 5’tttagggctttgaaggc-3’ h5934a coi arevalo et al. (1994) 5’agrgtgccaatgtctttgtgrtt-3’ r13 rag-1 groth and barrowclough (1999) 5’tctgaatggaaattcaagctgtt-3’ r18 rag-1 groth and barrowclough (1999) 5’-gatgctgcctcggtcggccaccttt-3’ rag1 f700 rag-1 bauer et al. (2007) 5’-ggagacatggacacaatccatcctac-3’ rag1 r700 rag-1 bauer et al. (2007) 5’-tttgtactgagatggatctttttgca-3’ 7phylogenetic relationships of the genus nactus members of the genus nactus included in our study formed a well-supported monophyletic group in all analyses performed. nactus is the sister group to another well-supported clade including the genera heteronotia and dixonius. each of the three genera, as well as the clade as a whole, and that comprising heteronotia plus dixonius are supported by mp and ml bootstrap values of 100% and bayesian posterior probabilities (pp) of 1.0 (fig. 1). intraspecific relationships within nactus were poorly supported as a result of strong conflict between the data partitions. in a combined analysis of all genes, except for the grouping of nactus sphaerodactylodes and n. vankampeni (pp = 0.97), all branches received weak support in the bayesian analysis (fig. 1). the combined nuclear and mitochondrial mp tree had the same topology as the nd2 tree (undoubtedly reflecting the hemidactylus robus tus pakistan g ekko gecko myanmar dixonius vietnamens is keo seima, cambodia dixonius s iamens is phom aural, cambodia dixonius s iamens is phom aural, cambodia heteronotia planiceps western australia heteronotia binoei sturt national park, nsw, australia heteronotia binoei ashford, nsw, australia nactus acutus rossel island, png nactus s p. tekadu, png nactus s p. tekadu, png nactus pelagicus ile des pins, new caledonia nactus pelagicus mt. gouémba, new caledonia nactus s phaerodactylodes sudest island, png nactus vankampeni wewak, png nactus vankampeni mt. shungol, png0.05 s ubs titutions /s ite figure 1 1.0 100/100 1.0 100/ 1001.0 100/ 100 1.0 1.0 100/ 100 100/ 1001.0 100/ 100 1.0 100/100 0.6 –/– 1.0 100/ 100 0.66 –/– 1.0 100/ 100 0.97 61/52 1.0 100/ 100 fig. 1. bayesian tree with maximum likelihood branch lengths of nactus and its closest relatives based on the combined rag-1, phosducin (pdc) and nd2 data sets. bayesian inference posterior probabilities are shown above the branches and maximum likelihood/maximum parsimony bootstrap values below. the relationships between the species of nactus exactly match those of the mtdna alone, but the low support values between the species reflect a conflict with the nuclear data (see text). 8 t.r. jackman, a.m. bauer and e. greenbaum figure 2 a b nactus acutus rossel island, png nactus s p. tekadu, png nactus s p. tekadu, png nactus pelagicus ile des pins, new caledonia nactus pelagicus mt. gouémba, new caledonia nactus s phaerodactylodes sudest island, png nactus vankampeni wewak, png nactus vankampeni mt. shungol, png 0.1 changes 0.82 1.0 0.98 1.0 1.0 0.98 nactus acutus rossel island, png nactus s phaerodactylodes sudest island, png nactus pelagicus ile des pins, new caledonia nactus pelagicus mt. gouémba, new caledonia nactus s p. tekadu, png nactus s p. tekadu, png nactus vankampeni wewak, png nactus vankampeni mt. shungol, png 0.01 changes 0.91 1.0 1.0 1.0 1.0 0.99 fig. 2. bayesian trees showing hypotheses of relationship within nactus based on a) nd2 mitochondrial dna and b) combined rag-1 and pdc nuclear dna data. values indicated are bayesian inference posterior probabilities. 9phylogenetic relationships of the genus nactus greater number of parsimony informative characters for this gene relative to the nuclear markers), but all interspecific bootstrap values were low. the nuclear data (pdc and rag-1) were combined and the resultant tree (fig. 2b) was compared to the tree based on mitochondrial dna (nd2) alone (fig. 2a). the topologies of the two trees differ significantly with respect to inferred relationships within nactus, and both have relatively high support values for these conflicting relationships. in the mitochondrial tree the clade pelagicus (sphaerodactylodes plus vankampeni) received strong support, whereas in the nuclear tree the relationships: vankampeni (pelagicus plus sp.) were supported by posterior probabilities of 1.0. in addition, although nuclear genes suggest a deep divergence between the two sampled populations of n. vankampeni, they are nearly identical with respect to nd2 sequence. based on the sh test, the nd2 data set rejects the nuclear dna topology as significantly different from the nd2 topology (p = 0.024) and the nuclear data set rejects the nd2 topology as significantly different as well (p = 0.022). in addition, the ild test revealed significant incongruence between the nuclear and mitochondrial data sets (p = 0.045), but no significant conflict between the two nuclear dna data sets (p = 0.344). discussion it is clear from the sh and ild tests that the low support values for relationships in the combined analyses of the genus nactus are the result of character conflict between the nuclear and mitochondrial data sets. although several scenarios may be posited, this strong conflict can be hypothesized to have been generated by three historical events. first, the relationship of n. sphaerodactylodes and n. vankampeni (fig. 2a), which is well supported by mitochondrial dna, can be explained as an ancient mitochondrial introgression event between the two species as has been described recently in phrynosomatid lizards (leaché and mcguire, 2006). second, the nearly identical mtdna but deeply divergent nuclear dna of the two n. vankampeni samples (figs. 2a, 2b) may be the result of a recent selective sweep of mitochondrial dna throughout n. vankampeni. third, the well-supported relationship of n. pelagicus and nactus sp. based on nuclear but not mitochondrial dna may be a consequence of the origin of n. pelagicus from bisexual parental species of two different lineages – the gene conflict representing these disparate lineages. it has been hypothesized that the bisexual species that gave rise to n. pelagicus would have had 2n = 42 and 2n = 28 chromosomes, respectively (moritz, 1987). remaining conflicts in the nuclear and mitochondrial trees are not well supported in one or both trees. increased sampling of the species of nactus may help to further clarify the source of conflict between mitochondrial and nuclear genes in this genus. our analyses of the nuclear data alone are similar to those of kraus (2005) in that n. acutus and n. sphaerodactylodes, which were identical with respect to kraus’s morphological characters, are sister species, albeit with weak support (pp = 0.91). however, kraus (2005) found support for the affinities of this species pair to n. vankampeni. this is inconsistent with our nuclear data, whereas our nd2 and combined analyses support the close relationship of only n. sphaerodacylodes with n. vankampeni. morphological data would link nactus sp. with n. pelagicus, as do the nuclear data. donnellan and moritz (1995) identified a minimum of three bisexual lineages among new 10 t.r. jackman, a.m. bauer and e. greenbaum guinea “n. pelagicus” based on allozyme data. our bisexual “pelagicus” specimens are from tekadu, lakekamu river basin, morobe province, papua new guinea, geographically intermediate between the nearest confirmed localities of all three forms. morphologically, they are most similar to form d, one of four putative taxa identified by rösler et al. (2005). the resolution of specific identity of all of the new guinea bisexual forms is hindered by an incomplete knowledge of the distribution of the different allozyme and morphological forms, and by the complicating fact that several names currently in the synonymy of n. pelagicus may apply to one or more of these (bauer and henle, 1994; donnellan and moritz, 1995; zug and moon, 1995; zug, 1998). george zug (pers. comm.) is currently conducting a comprehensive analysis of new guinea “n. pelagicus,” which include at least five distinct taxa, and specific allocation of our bisexual n. pelagicus will have to wait until this is completed. kraus (2005) found that the species lacking multicarinate tubercles (n. galgajuga and n. coindemirensis) consistently clustered with outgroup genera or as part of a basal polytomy involving his outgroups and an otherwise monophyletic nactus. the polyphyly of the mascarene taxa had earlier been proposed by bullock et al. (1985), who considered n. coindemirensis as basal in the genus, but ulber and gericke (1988) assumed a sister group relationship between mascarene taxa (for which they erected the subgenus mascarenogecko) and pacific nactus, and more recently austin and arnold (2006) have suggested that mascarene nactus are derived from australasia, having dispersed over water via the prevailing currents and winds. kraus’s (2005) placement of n. galgajuga outside of the clade including the other australian species (n. eboracensis, n. cheverti) contradicts the conclusions of zug (1998), who considered the shared smooth subcaudals of all three australian species as evidence of their probable monophyly. our sampling included no australian or mascarene species, however, so we are unable to evaluate kraus’s (2005) placement of the problematic species n. galgajuga and n. coidemeriensis. the inclusion of nactus and heteronotia in a single, well-supported clade (although not as sister taxa) corroborates the work of kluge (1963) and russell (1972), both of whom suggested that the two were closely related. indeed, the external similarity of heteronotia and nactus is evident in the fact that macleay’s (1878) descriptions of the species now assigned to n. eboracensis and n. cheverti placed them in the genus heteronota (subsequently changed to heteronotia owing to its preoccupation). boulenger (1885) subsequently transferred one species (n. cheverti), but not the other, into gymnodactylus (then including taxa now assigned to cyrtodactylus and nactus), despite the great morphological similarity between the two forms (zug, 1998). loveridge (1934) likewise synonymized h. eboracensis with h. binoei. heteronotia currently includes three named and recognized species, h. binoei, h. spelea and h. planiceps (storr, 1989). however, there are three bisexual chromosome races and two genetically diverse parthenogenetic lineages resulting from multiple hybridization events between two of the bisexual races currently subsumed under the name h. binoei (moritz, 1983, 1993; moritz et al., 1989a, b, 1990; moritz and heideman, 1993; strasburg and kearney, 2005; kearney et al., 2006). our two samples are nearly identical to one another and represent the same bisexual cytotype (ea6 fide moritz, 1991). patterns of relationship among the various bisexual and unisexual groups still subsumed within heteronotia binoei are well resolved, with initial diversification of the sexual races estimated 11phylogenetic relationships of the genus nactus to have occurred approximately 6 million years ago and the origin of the two recognized clonal lineages having occurred in the pleistocene, perhaps within the last 300,000 years (strasburg and kearney, 2005; kearney et al., 2006). while the affinities of heteronotia to nactus are not particularly surprising, those of dixonius are. dixonius bauer et al., 1997 was erected to accommodate southeast asian leaf-toed geckos previously assigned to the polyphyletic and nearly cosmopolitan phyllodactylus. four species of dixonius are currently recognized (bauer et al., 2004; das, 2004). dixonius siamensis has the broadest range, occurring from songkhla (7° n), south of the isthmus of kra (taylor, 1963), north to at least chiang mai (19° n) (grossmann et al., 1996; manthey and grossmann, 1997) and from southern myanmar (annandale, 1905a, b) to the lao peoples democratic republic (stuart, 1999) and vietnam (smith, 1935; bourret, 1939; szczerbak and nekrasova, 1994). this broad distribution, along with obvious geographic variation in color pattern (taylor, 1963) suggests that d. siamensis, as presently construed, may actually represent a complex of similar species. this hypothesis is supported by ota et al. (2001), who demonstrated that a minimum of two chromosome forms (2n = 40 and 2n = 42) exist among thai populations of d. siamensis. the remaining species have more limited distributions – d. melanostictus occurs in sara buri and nakhon ratchasima provinces in central thailand (taylor, 1962, 1963; chan-ard et al., 1999), d. hangseesom is known only from kanchanaburi province, western thailand, and d. vietnamensis has been found in southern vietnam and cambodia (bobrov, 1992; das, 2004; stuart et al., 2006). the phylogenetic affinities of dixonius to other gekkonids had not been previously investigated, but allozyme data and morphology did not suggest that it was especially closely related to other clades of leaf-toed geckos (bauer et al., 1997). the possible generic distinctness of the group was first noted by annandale (1905b), who considered the presence of precloacal pores as highly distinctive within phyllodactylus. dixon (1964) subsequently noted that d. siamensis exhibited a reduced manual phalangeal formula of 2:3:4:4:3. russell (1972) demonstrated that there was in fact no phalangeal loss in digit iv of the manus, but identified a unique reduction in size of phalanx ii of this digit. bauer et al. (1997) subsequently diagnosed dixonius relative to other leaf-toed geckos on the basis of these precloacal pore and digital characters, as well as the tuberculate condition of the dorsum and the proximal bifurcation of the hypoischium. in retrospect it is possible to identify known morphological and biological characters that support the monophyly of the nactus/heteronotia/dixonius clade. russell (1972) noted that nactus pelagicus and heteronotia binoei share a nearly identical phalangeal pattern, with a long slender first phalanx and short second and third phalanges. species now allocated to dixonius were distinguished by russell (1972) from all other phyllodactylus by their extremely short second phalanx on digit iv of the manus. the reduced second phalanx in all three forms may be considered a putative synapomorphy of the group as a whole. dorsal scalation serves as another potential feature uniting members of this clade. heteronotia and nactus have the derived condition of multicarinate dorsal tubercles (keeled but unicarinate in dixonius), and all three genera have very regularly arranged rows of dorsal tubercles (lost in some nactus). a third putative synapomorphy is parthenogenesis, or at least the existence of nearly morphologically identical cryptic species with differing chromosomal complements that 12 t.r. jackman, a.m. bauer and e. greenbaum establish the basis for the hybrid origin of such unisexuals. parthenogenesis is rare in nature, with only 0.1% of species exhibiting this reproductive strategy (white, 1978). the infrequency of parthenogenesis in nature is assumed to be related to the deleterious effects of parthenogenesis on fitness (kearney and shine, 2004). all known unisexual vertebrates exhibit a clonal mode of parthenogenesis associated with a hybrid origin (vrijenhoek et al., 1989), which results in high levels of heterozygosity. heterogygosity levels are also increased in the case of allopolyploids (kearney and shine, 2004). it has been suggested that this increased heterozygosity may lead to increased developmental stability and success (vrijenkoek and lerman, 1982; wetherington et al., 1987). specifically, kearney and shine (2004) suggested that parthenogenetic heteronotia binoei might be buffered against effects of temperature during development. it has been suggested that parthenogens of hybrid and polyploidy origin capture and “freeze” the diversity of parental sexual forms and that this, as well as non-additive interactions among the genomes of hybrid polyploids might provide a means of introducing diverse phenotypes that could diversify into and exploit ecological vacuums. kearney (2003) suggested that h. binoei might represent such a case in the australian desert, and one might consider the nocturnal terrestrial niche for lizards in much of the pacific a similar vacuum that may have provided an equivalent opportunity for nactus pelagicus. no parthenogens have been identified within dixonius, but the chromosome variation identified in d. siamensis by ota et al. (2001), sets up the possibility for hybridogenesis. the strong support for the nactus/heteronotia/dixonius clade demonstrates that subdigital scansors, and in particular, the leaf-like terminal scansors of dixonius, have evolved multiple times within the gekkonidae. such scansors occur in a variety of other strongly supported gekkonid clades including the group comprising phyllodactylus, haemodracon, asaccus and their relatives (gamble et al., in press), and at least two separate afro-malagasy clades: uroplatus and its relatives (greenbaum et al., 2007) and paroedura and its relatives (jackman et al., 2008.). russell (1979) highlighted convergence in digital structure between diplodactylid and gekkonid geckos and suggested that similar convergence might occur within gekkonids themselves (russell, 1976). extensive digital variability within single lineages, especially that involving the secondarily derived loss of scansorial morphology, has subsequently been demonstrated in several gekkonid lineages (carillo de espinoza et al., 1990; lamb and bauer, 2006). our results suggest that digital structure may be even more labile than previously proposed and, at least at the superficial level it has been interpreted by most authors (e.g., loveridge, 1947), may be positively misleading with respect to phylogenetic relationships. acknowledgements we are grateful to ross sadlier (australian museum), fred kraus (bishop museum), lee grismer (lasierra university), alison whiting (brigham young university) and allen resetar (field museum of natural history) for providing tissue samples used in this study. the authors were supported by grant deb 0515909 from the national science foundation of the united states. 13phylogenetic relationships of the genus nactus references annandale, n. 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(1995): systematics of the pacific slender-toed geckos, nactus pelagicus complex: oceania, vanuatu, and solomon islands populations. herpetologica 51: 77-90. preferred temperatures of tarentola mauritanica in spring miguel a. carretero cibio, centro de investigação em biodiversidade e recursos genéticos, campus agrário de vairão, 4485-661 vairão, portugal. e-mail: carretero@mail.icav.up.pt submitted on 2007, 28th august; revised on 2007, 26th october; accepted 2008, 30th january. abstract. variation of the preferred temperatures (tp) by the moorish gecko (tarentola mauritanica) in spring was analysed in a population from ne iberia. ten adult specimens were exposed to a thermal gradient and tp was measured at seven time intervals between 16 and 24 h. tp values were low (23.44 ± 0.61 ºc, 20.7-26.9 ºc, mean ± se, range) but geckoes gradually increased tps between late afternoon and early evening. surprisingly, mean, maximum and minimum tps inversely correlated with the body size (svl), but the results of the analyses remained after size correction. compared to a previous study carried out in summer, tps found here were much lower but diel variation persisted. such patterns are more similar to other nocturnal or crepuscular geckonids rather than to the well-studied diurnal lacertids living in the same sites. specifically, tp seems to be more plastic and the relationships with body size are opposite. possible adaptive explanations and guidelines for further work are suggested. keywords. preferred temperatures, thermal ecology, tarentola mauritanica, gekkonidae. introduction in the framework of the lizard studies on thermal ecology, preferred body temperature in the absence of thermoregulatory constraints (tp) constitutes an important trait because it correlates with several physiological optima (huey and bennet, 1987; bauwens et al., 1995; anguilletta et al., 2002). nevertheless, tp may change at both evolutionary and individual scales. on one hand, tp may shift evolutionarily to approach the dominant environmental temperatures but the rate of such adaptive response varies between phylogenetic lineages (hertz et al., 1983; bennet and john-alder, 1986; christian and weavers, 1996; labra, 1998; castilla et al., 1999). on the other hand, individual lizards are also able to adapt their tp in response to temporal variation within their life such as daily and seasonal changes, reproductive condition and feeding status (i.e., van damme et al., 1986; castilla et al., 1999; brown and griffin, 2003; carretero et al., 2005, 2006). acta herpetologica 3(1): 57-64, 2008 issn 1827-9643 (online) © 2008 firenze university press 58 m.a. carretero whereas most of the literature on this topic focuses on diurnal lizards, less attention has being paid to nocturnal species. nevertheless, several studies carried out on crepuscular or nocturnal geckonids indicate that tp within the species level is less rigid than in diurnal lizards (brown, 1996; rock et al., 2000). moreover, individual geckos may also change their tp throughout the day, seasonally or due to pregnancy (refinetti and susalka, 1997; zari, 1999; rock et al., 2000, 2002; anguilletta et al., 2002; hare et al., 2002) although patterns are not common for all the species (anguilletta and werner, 1998). geckoes inhabiting temperate regions are of special interest because they must face stronger thermal constraints than those living in tropical habitats (hitchcock and mcbrayer, 2006). whereas the lizard fauna of the northern side of the mediterranean basin is dominated by lacertids, only four geckonid species, all restricted to the warmer areas, are known to the region (arnold and ovenden, 2002). the moorish gecko, tarentola mauritanica is the most common one in the western mediterranean (martínez-rica, 1997). although it is found on both sides of gibraltar strait, recent phylogeographic evidence (harris et al., 2004a, b) suggests that the species has colonised europe only recently after the glaciations. the moorish gecko is a medium-sized species (45-85 mm, svl), mainly occupying natural and artificial vertical surfaces, displaying both diurnal and nocturnal activity and undergoing a winter diapause in the most continental localities (salvador, 2002; guarino and picariello, 2006). previously, the diel variation of tp in this species was analysed in summer (when geckoes were already involved in reproduction) in a continental population from central spain (gil et al., 1994). the aim of this study was to analyse the intraspecific variation of tp in a population of this species living in milder conditions with full activity but not involved in reproduction. material and methods a total of ten adult t. mauritanica were collected in a thermomediterranean site from ne iberia (bellaterra, cerdanyola del vallès, utm 31t df2395, 150 m altitude) in late april 2000. animals were not sexed because reliable sexing tools (atzori et al., 2007) were not currently available. however, in this period of the year, moorish geckoes were not still involved in reproductive activities and females should not be pregnant (pers. obs.; picariello et al., 1989). geckoes were kept in individual 0.5×0.4×0.3 m housing terraria during less than one week with food (drosophila flies and acheta domestica crickets) and water provided ad libitum and then released back at the site of capture after the experiments. specimens were measured (snout-vent length, svl) to the nearest 0.05 mm with a digital calliper. subsequently, they were individually exposed to a thermal gradient (~15-45 ºc, 0.5×0.5×1.5 m length experimental terrarium) produced by a 100 w reflector bulb fixed 15 cm above the substrate and maintaining natural photoperiod. tp was measured with k-termocouple digital thermometer (digitron 3208k, accuracy 0.01 ºc) by inserting a probe in the cloaca. body temperatures were recorded during a single day at seven consecutive intervals (fig. 1, table 1) between 16 and 24 hours (local time, gmt) when the activity of the species in this season peaks in the field (martínez-rica, 1974). in order to minimise thermal shifts due to stress or contact with the researcher’s hand, no more than 10 seconds mediated between the capture of the gecko in the terrarium and the temperature measurement. values of tp were not transformed since distributions did not deviate from normality (komogorov-smirnov tests, p > 0.05 in all cases), were homoscedastic (univariate levene tests and multivariate box m, p > 0.05) and variances and means were uncorrelated. since measurements 59preferred temperatures of tarentola mauritanica in spring were repeated for the same individual for each interval, an analysis of variance for repeated measures (rmanova) was performed with time as the only independent factor. sphericity assumption was tested prior to the analyses and subsequent corrections were performed in case that assumption was violated (statsoft, 2006). results one of the individuals (no. 4, svl: 73.1 mm) showed an abnormal behaviour, remaining motionless next to the bulb for three hours. because of that, this experiment was interrupted at 21:00 and the specimen was excluded from all the analyses. the tps recorded for this individual were 25.0 ºc (18:00), 25.90 ºc (19:00), 28.0 ºc (20:00) and 28.70 ºc (21:00). for the remaining nine animals, which were normally active, the tp values recorded are shown in table 1, grouped by individual and time interval. the rmanova detected a significant increase in tp throughout the monitoring time (fig. 1, table 2). in fact, the main difference was detected between the measures before sunset, which increased gradually, and those after sunset, which were relatively uniform (fig. 2). fig. 1. temporal variation of the preferred temperatures (tp) of t. mauritanica. the arrow points the sunset. distinctive time measures according to scheffé post-hoc comparisons (p < 0.05) following a significant rmanova (table 2) are indicated in the ordinates. 60 m.a. carretero surprisingly, tp (mean, maximum and minimum of all measures of each individual) was also negatively correlated with body size (fig. 2). because of this, the rmanova was repeated using the residuals of the tp against svl, but results remained identical (table 2). table 1. group n mean se min. max. time 18:00 9 22.32 0.52 20.7 25.3 19:00 9 22.64 0.56 21.2 25.8 20:00 9 23.11 0.59 21.3 26.2 21:00 9 23.17 0.56 21.6 26.2 22:00 9 23.38 0.56 21.6 26.9 23:00 9 23.34 0.53 21.5 26.7 24:00 9 23.40 0.52 21.5 26.6 individual 1 7 21.56 0.11 21.2 21.9 2 7 22.47 0.31 21.7 24.2 3 7 24.39 0.34 23.1 25.4 5 7 22.61 0.33 21.4 23.4 6 7 23.11 0.22 22.1 23.8 7 7 23.90 0.22 23.2 24.7 8 7 26.24 0.21 25.3 26.9 9 7 21.34 0.12 20.7 21.6 10 7 21.84 0.17 21.2 22.4 total* 9 23.05 0.52 21.3 26.2 * calculated over the individual means of the seven time intervals table 2. rmanova tp f df p time (r) 5.96 6, 48 0.0001 mauchley sphericity test, χ2 43.52 20 0.002 greenhouse-geisser epsilon 0.04 hunyh-feldt epsilon 0.05 rmanova tp (svl residual) f df p time (r) 5.96 6, 48 0.0001 61preferred temperatures of tarentola mauritanica in spring discussion the temperatures selected by tarentola mauritanica in the thermogradient corresponded to those typical in other crepuscular geckos in the temperate region. thus, tp values were lower that those of the diurnal lacertid podarcis (hispanica) liolepis, living syntopically in the same locality and time (carretero et al., 2006) and other mediterranean lacertids (bauwens et al., 1995), but still higher that the activity temperatures found in nature (per. obs.). moreover, tps were also much lower than those selected by the continental population in summer (31.56 ºc on average, gil et al., 1994). because there is minimal genetic variation between both populations (harris et al., 2004a, b) and no interpopulational variation in tps has been recorded in other tarentola species (brown, 1996), phylogenetic shift is considered negligible in this case. the lab methodologies between this study and that by gil et al. (1994) were very similar. once comparativeness between both works is granted, differences in tp found are to be attributed to changes in body condition and/or reproductive status. coastal specimens analysed in spring here could be still in low nutritional condition and select for low temperatures (brown and griffin, 2003), whereas continental specimens analysed in summer should have higher fat reserves, but be involved in reproductive activities. authors did not provide information on the reproductive status of the specimens or on female pregnancy. however, in other temperate geckonids, pregnant females are known to select for higher temperatures than non-pregfig. 2. significant relationships between preferred temperatures (mean, maximum and minimum tp) and body size (svl) in t. mauritanica. 62 m.a. carretero nant females and males (rock et al., 2000; hitchcock and mcbrayer, 2006). nevertheless, seasonal acclimatisation also needs to be considered (zari, 1999). whatever the case, the differences between seasons (or reproductive classes) greatly exceed those found in mediterranean lacertids (carretero et al., 2006). huey et al. (1989) suggested that the thermal traits of geckonids are more associated to the temperatures of diurnal shelters than to those of the microhabitats of nocturnal activity. the increase of tp during the final part of the day found here seems to correspond to the gradual selection of warmer microhabitats in the wild when getting dark (martínez-rica, 1974), in order to keep longer foraging activity after sunset (anguilletta et al., 1999). a similar pattern (but a higher level) was also recorded for the continental population in summer (gil et al., 1994). on the other hand, to our knowledge, this is the first evidence of an inverse relationship between tp and body size in geckonids. because of the strict protocol followed here, such differences cannot be attributed to a methodological artefact. in fact, in a previous study on the lacertid podarcis (hispanica) liolepis, similar in size and sympatric with t. mauritanica, the correlation between tp and body size was positive (carretero et al., 2006). usually, small geckos tend to select for similar (anguilletta et al., 1999) or lower (hitchcock and mcbrayer, 2006) temperatures than large ones. different causes have been invoked to explain this pattern including poorer body condition, different foraging and growth rates and avoidance of aggression by large individuals (hitchcock and mcbrayer, 2006). the opposite pattern found here is tentatively interpreted as the result of the monopolisation by bigger animals of the warmer refuges, which would be of higher thermal quality in spring due to moderate diurnal temperatures and restricted crepuscular activity. although the territoriality and interspecific aggressions in this species in not under question (salvador, 2002), this hypothesis needs experimental testing with specimens of different sizes in different seasons. in summary, results indicate that t. mauritanica displays high plasticity in preferred temperatures according to the diel and seasonal variation of its thermal environment and also probably modulated by interspecific interactions and reproductive requirements. acknowledgements i wish to thank anna soler and laia rocavert who helped me in the lab work. thanks are also due to x. espadaler (uab, bellatera) for logistic support.the final part of this research was funded by the projects poci/bia-bde/55865/2004 and poci/bia-bde/56931/2004 and m.a.c. held the post-doctoral contract sfrh/bpd/27025/2006, all from fundação para a ciência e a tecnologia, fct (portugal). collecting permits were provided by the departament de medi ambient of the generalitat de catalunya (spain). references anguilletta, m.j. jr., werner, y.l. (1998): australian geckos do not display diel variation in thermoregulatory behavior. copeia 1998: 736-742. 63preferred temperatures of tarentola mauritanica in spring anguilletta, m.j. jr., montgomery, l.g., werner, y.l. (1999): temperature preference in geckos: diel variation in juveniles and adults. herpetologica 55: 212-222. anguilletta, m.j. jr., niewiarowski, p., navas, c.a. (2002): the evolution of thermal physiology in ectotherms. j. therm. biol. 27: 249-268. arnold, e.n., ovenden, d. (2002): a field guide to the reptiles and amphibians of britain and europe. harpercollins. london. atzori, a., berti, f., cencetti, t., fornasiero, s., tamburini, m., zuffi, m.a.l. (2007): advances in methodologies of sexing and marking less dimorphic gekkonid lizards: the study case of the moorish gecko, tarentola mauritanica. amphibia-reptilia 28: 449-454. bauwens, d., garland, t. jr., castilla, a.m., van damme, r. (1995): evolution of sprint speed in lacertid lizards: morphological, physiological, and behavioral covariation. evolution 49: 848-863. bennet, a.f., john-alder, h. (1986): thermal relations of some australian skinks (sauria: scincidae). copeia 1986: 57-64. brown, r.p. (1996): thermal biology of the gecko tarentola boettgeri: comparisons among populations from different elevations within gran canaria. herpetologica 52: 396-405. brown, r.p., griffin, s. (2003): lower selected body temperatures after food deprivation in the lizard anolis carolinensis. j. therm. biol. 30: 79-83. carretero, m.a.; roig, j.m., llorente, g.a. (2005): variation in preferred body temperature in an oviparous population of lacerta (zootoca) vivipara. herpetol. j. 15: 51-55. carretero, m.a., marcos, e., de prado, p. (2006): intraspecific variation of preferred temperatures in the ne form of podarcis hispanica. in: mainland and insular lacertid lizards: a mediterranean perspective, p. 55-64. corti, c., lo cascio, p., biaggini, m., eds, firenze university press, florence. castilla, a.m., van damme, r., bauwens, d. (1999): field body temperatures, mechanisms of thermoregulation, and evolution of thermal characteristics in lacertid lizards. natura croat. 8: 253-274. christian, k.a., weavers, b.w. (1996): thermoregulation of monitor lizards in australia: an evaluation of methods in thermal biology. ecol. monogr. 66: 139-157. gil, m.j., guerrero, f., pérez-mellado, v. (1994): diel variation in preferred body temperatures of the moorish geckos tarentola mauritanica during summer. herpetol. j. 4: 56-59. guarino, f., picariello, o. (2006): tarentola mauritanica. geco comune, moorish gecko. in: atlante degli anfibi e dei rettili d’italia. atlas of italian amphibians and reptiles. p. 422-425. sindaco, r., doria, g., razzetti, e., bernini, f., eds, polistampa, firenze. hare, k.m., pledger, s., thompson, m.b., miller, j.h.; daugherty, c.h. (2002): daily patterns of metabolic rate among new zealand lizards (reptilia: lacertilia: diplodactylidae and scincidae). physiol. biochem. zool. 79: 745-753. harris, d.j., batista, v., carretero, m.a., ferrand, n. (2004a): genetic variation in tarentola mauritanica (reptilia: gekkonidae) across the strait of gibraltar derived from mitochondrial and nuclear dna sequences. amphibia-reptilia 25: 451-459. harris, d.j., batista, v., lymberakis, p., carretero, m.a. (2004b): complex estimates of evolutionary relationships in tarentola mauritanica derived from mitochondrial dna sequences. mol. phylog. evol. 30: 855-859. hertz, p., huey, r.b., stevenson, r.d. (1983): homage to santa anita: thermal sensitivity of sprint speed in agamid lizards. evolution 37: 1075-1084. 64 m.a. carretero hitchcock, m, mcbrayer, l.d. (2006): thermoregulation in nocturnal ecthotherms: seasonal and intraspecific variation in the mediterranean gecko (hemidactylus turcicus). j. herpetol. 40: 185-195. huey, r.b., bennett, a.f. (1987): phylogenetic studies of coadaptation: preferred temperatures versus optimal performance temperatures of lizards. evolution 41: 1098-1115. huey, r.b., niewiarowski, p.h., kaufmann, j., herron, j.c. (1989): thermal biology of nocturnal echtotherms: is sprint performance of geckos maximal at low body temperatures? physiol. zool. 62: 488-504. labra, a. (1998): selected body temperatures of seven species of chilean liolaemus lizards. rev. chil. hist. nat. 71: 349-358. martínez-rica, j.p. (1974): contribución al estudio de la biología de los gecónidos ibéricos (rept., sauria). publ. centro piren. biol. experim. 5: 1-291. martínez-rica, j.p. (1997): tarentola mauritanica (linnaeus, 1758). in: atlas of amphibians and reptiles in europe. p. 214-215. gasc, j.-p., cabela, a., cronbrnja-isailovic, j., dolman, d., grossenbacher, k., haffner, p., lescure, j., martens, h., martinez rica, j.p., maurin, h., oliveira, m.e., sofianidou, t.s., veith, m., zulderwijk, a. societas europaea herpetológica, muséum national d’histoire naturelle, paris. picariello, o., ciarcia, g., angelici, f. (1989): the annual cycle of the oviduct in tarentola m. mauritanica l. (reptilia, gekkonidae). amphibia-reptilia 10: 371-386. refinetti, r., susalka, s. (1997): circadian rhythm of temperature selection in a nocturnal lizard. physiol. behav. 62: 331-336. rock, j., andrews, r.m., cree, a. (2000): effects of reproductive condition, season, and site on selected temperatures of a viviparous gecko. physiol. biochem. zool. 73: 344-355. rock, j., cree, a., andrews, r.m. (2002): the effect of reproductive condition on thermoregulation in a viviparous gecko from a cool climate. j. therm. biol. 27: 17-27. salvador, a. (2002): salamanquesa común. tarentola mauritanica. in: enciclopedia virtual de los vertebrados españoles, carrascal, l.m., salvador, a., eds, museo nacional de ciencias naturales, madrid. http://www.vertebradosibericos.org/ updated 2007. statsoft, inc. (2006): statistica (data analysis software system), version 7.1. www.statsoft.com. van damme r., bauwens d., verheyen r. (1986): thermoregulatory responses to environmental seasonality by the lizard lacerta vivipara. herpetologica 43: 405-425. zari, t.a. (1999): seasonal acclimatization in metabolic rate of the fan-fingered gecko, ptyodactylus hasselquistii (reptilia: gekkonidae). j. therm. biol. 24: 137-142. © firenze university press www.fupress.com/ah acta herpetologica 5(1): 31-35, 2010 is the european pond turtle emys orbicularis strictly aquatic? – habitats where the turtle lives in central europe sławomir mitrus department of biosystematics, opole university, oleska 22, 45-052 opole-poland. e-mail: slawomir. mitrus@uni.opole.pl submitted on: 2009, 25th june; revised on 2009, 18th december; accepted on 2010, 17th march. abstract. based on ecological characteristics and phylogenetic analysis, it is possible to try to reconstruct the evolution of ecological traits in turtles. however, the european pond turtle is treated by different scientists as aquatic or as semi-aquatic species. the importance of terrestrial behaviour for this species is discussed. keyword. emys orbicularis, habitat, terrestrial behaviour. in their paper, ficetola and de bernardi (2006) stated – in contrast to the ecological character used by stephens and wiens (2003) in their analysis – that the european pond turtle emys orbicularis is a semi-aquatic, not an aquatic species. it is known that terrestrial habitats are important for all freshwater turtles (e.g., because they lay egg on land), and that for protection of the turtles it is important not to overlook the upland environment as it is vital for their survival (semlitsch and bodie, 2003). thus, for the majority of freshwater turtle species, as well for marine ones, the discussion as to whether one is “aquatic” or “semi-aquatic”, seems to be a purely academic discussion about definitions. however, basing on ecological characteristics and phylogenetic analysis, it is possible to try to reconstruct the evolution of ecological traits in emydid turtles (cf. stephens and wiens, 2003), and, for such analysis, information on which ecological group the european pond turtle belongs to, could be essential. i agree with the main conclusion made by ficetola and de bernardi (2006), although some of the information in the article can be disputed. thus, i would like to discuss some cited data about types of habitat used by the turtle. as it may happen that the importance of terrestrial behaviour is different among populations (ficetola and de bernardi, 2006), i have focused this report of mine on data set from central europe, especially from poland. habitats use by the european pond turtle the european pond turtle is only one of the native turtle species living in poland, and in central europe (fritz, 2003). typically, it lives in small bodies of water with muddy bot32 s. mitrus toms, old riverbeds, wetlands and little holes and ponds. natural habitat types inhabited by the turtle in poland are (according to natura 2000 codes; interpretation manual, 2007) (mitrus, 2004; najbar, 2007): – 3150: natural eutrophic lakes with magnopotamion or hydrocharition – type vegetation (the most important habitat for the species in poland), – 3160: natural dystrophic lakes and ponds, – 7140: transition mires and quaking bogs (turtles live in small ponds: natural ones or those left after peat exploitation), – 2330: inland dunes with open corynephorus and agrostis grassland (by the turtle the habitat is used for egg laying). generally, individuals of the species bask and mate in or very close to water (ficetola and de bernardi, 2006). from central europe data about mating is scarce – in the area the turtles mate in shallow water (mitrus and zemanek, 2000; najbar, 2008). however, the problem is how often the turtles use terrestrial areas for different activities, and how far from the water bodies they move. feeding as presented by ficetola and de bernardi (2006), there are only few studies concerning the diet of emys orbicularis. i think that the abundance of terrestrial insects in the diet of the turtle, may represent insufficient evidence that the upland environment can be important for feeding: ottonello et al. (2005) showed that terrestrial arthropods were more abundant in the diet of turtles from canals, than individuals from the marshes. however, the abundance of terrestrial animals in the turtle’s diet i) could be due to turtles hunting activity on land (using terrestrial habitats for feeding), or ii) because terrestrial invertebrates have fallen into the water. the matter may be then influenced by site features and individual population activity. the large abundance of terrestrial invertebrates in the diet of the turtle was also noted by kotenko (2000), and she also stated that in ukraine, the turtle inhabited water bodies located very close to typical terrestrial areas. personally, i observed that in artificial conditions, young european pond turtles sporadically try to hunt crickets (given as food), staying on rocks close to water. it is possible that under natural conditions, the turtle use terrestrial habitats for hunting, but there is insufficient data to conclude that such behaviour is a frequent pattern. overwintering field studies indicate that in central europe the turtle overwinters in water bodies (e.g., najbar, 2008; novotný et al., 2004). adult individuals of the turtle are able to survive winter on land, but most available data about hibernation on land refers to captive individuals (cf. ultsch, 2006). suitable data on the subject are actually needed (e.g., about possibility and frequency under natural condition of an aestivation of the turtle on land). 33is the european pond turtle emys orbicularis strictly aquatic? terrestrial movements in central poland, most of the turtle nests were located less than 150 meters from water bodies, with the largest distance being about 350 m (mitrus, 2006a). in western poland, the distances were 69–85 m (sporadically 150–270 m; najbar and szuszkiewicz, 2007; najbar, 2008), and in slovakia typically 200–800 m (novotny et al., 2004). however, there is problem if we will try to present data on distances of nesting movements. females can not proceed directly from the water to the egg laying area, and at the nesting area turtles hardly ever go straight ahead (e.g., jabłoński and jabłońska, 1998; mitrus, 2006b). there is some information about very long distances of nesting migrations, exceeding 1 km (cf. ficetola and de bernardi, 2006). however, schneeweiss and steinhauer (1998) reported only three females, and only one of them migrated on land at a distance exceeding 1 km (1170 m) [there is a mistake in the references of ficetola and de bernardi (2006)’s paper – an incorrect paper is cited – but i think that the authors have written about data presented in schneeweiss and steinhauer (1998)]. jabłoński and jabłońska (1998) write about a particular 4 km journey, but “mostly through water”. it was reported, from different areas in central europe, that some females probably make the first stage of their nesting movements through water, although precise data are not available (jabłoński and jabłońska, 1998; najbar, 2008; personal observation). longer movements unrelated with egg laying are probably rare. there is information about the migration of turtles, although this is mostly in water areas that have since dried out (e.g., kotenko, 2000), and precise data are very scarce. in lithuania, turtles could migrate between ponds over longer distances using “flooded areas and/or channels as migration routes” (meeske and muhlenberg, 2004). one individual migrated 650 m when one such flooded area “was filled with very little water” (meeske and muhlenberg, 2004). so it is by definition questionable, if it was moving on terrestrial areas or not. in western poland, two males moved 370 m and 450 m respectively from water bodies, to a dry pine forest and back (najbar, 2008). from different areas of europe there are data about land migrations of the turtle, both nesting migrations (e.g., rovero and chelazzi, 1996), and migrations on land not connected with egg laying (e.g., cadi et al., 2008), sometimes exceeding 0.5 km or even 1 km. however, based on available data (and especially on the papers cited by ficetola and de bernardi, 2006), it is difficult to conclude that for the european pond turtle terrestrial movements exceeding 1 km are common. in many cases it is difficult to compare data for different species, presented by different scientists. however, basing on data for emydoidea blandingii and clemmys guttata (joyal et al., 2001), it is logical suppose that emys orbicularis present shorter land migrations that the two species. as ficetola and de bernardi (2006) pointed out in their paper, the european pond turtle is not strictly aquatic. however, stephens and wied (2003) defined an “aquatic” species as those reported to “spend their active season primarily in aquatic habitats, generally leaving the water only to bask, migrate to a new aquatic habitat, or nest”. i do not want do discuss if the definition is the best one available, but based on it, the european pond turtle is – at least in central europe – an aquatic species. whether emys orbicularis should be classified and treated as an aquatic or semi-aquatic species, it would depend on how much 34 s. mitrus time individuals spend on land during migrations not connected with nesting, as well as how often they are overwintering and hunting on land. currently, the available data enable one to say only that the european pond turtle is able to survive the winter on land, and it also feeds on terrestrial invertebrates. the european pond turtle has very wide distribution areas including northwest africa a large part of europe, as well as asia minor, and presently it is divided into many subspecies (fritz, 2003). as the turtles live in different environmental conditions (e.g., in central europe compared to mediterranean areas) it is probable that the terrestrial behaviour may be different among populations (ficetola and de bernardi, 2006) and/or subspecies. thus, for studying the evolution of emydid turtles, comparative analyses of ecology and behaviour of different subspecies and populations of emys orbicularis would be strongly needed. acknowledgements supported by a grant from iceland, liechtenstein and norway through the eea financial mechanism and the norwegian financial mechanism “scientific work finaced from funds for sciences in the years 2008-2011 as a research project”. i thank r. tertil and ian harman, both of letterman, translators & interpreters who provided linguistic corrections. i am very grateful to referees for their comments on previous draft of this paper. references cadi, a., nemoz, m., thienpont, s., joly, p. (2008): annual home range and movement in freshwater turtles: management of the endangered european pond turtle (emys orbicularis). rev. esp. herp. 22: 71-86. ficetola, g.f., de bernardi, f. (2006): is the european “pond” turtle emys orbicularis strictly aquatic and carnivorous? amphibia-reptilia 27: 445-447. fritz, u. (2003): die europäische sumpfschildkröte. bielefeld, laurenti-verlag. interpretation manual of european union habitats (2007): version eur17. european comission, dg environment nature and biodiversity. jabłoński, a., jabłońska, s. (1998): egg-laying in the european pond turtle, emys orbicularis (l.), in łęczyńsko-włodawskie lake district (east poland). in: proceedings of the emys symposium dresden 96. mertensiella 10, p. 141-146. fritz, u., joger, u., podlucky, r., servan, j., buskirk j.r., eds, dght, rheinbah. joyal, l.a., mccollough, m., hunter jr., m.l. (2001): landscape ecology approaches to wetland species conservation: a case study of two turtle species in southern maine. conserv. biol. 15: 1755-1762. kotenko, t.i. (2000): the european pond terrapin (emys orbicularis) in the steppe zone of ukraine. in: die europäische sumpfschildkröte, p. 87-106. hödl, w., rössler, m., eds, land oberösterreich, oö, landesmuseum, linz. meeske, a.-c.m., muhlenberg, m. (2004): space use strategies by a northern population of the european pond turtle, emys orbicularis. biologia 59 (suppl.): 95-101. 35is the european pond turtle emys orbicularis strictly aquatic? mitrus, s. (2004): żółw błotny. in: poradniki ochrony siedlisk i gatunków natura 2000 – podręcznik metodyczny. tom 6. gatunki zwierząt (z wyjątkiem ptaków), p. 309-313. adamski, p., bartel, r., bereszyński, a., kepel, a., witkowski, z., eds, warszawa, ministerstwo środowiska. mitrus, s. (2006a): fidelity to nesting area of the european pond turtle, emys orbicularis (linnaeus, 1758). belg. j. zool. 136: 25-30. mitrus, s. (2006b): spatial distribution of nests of the european pond turtle, emys orbicularis (reptilia: testudines: emydidae), from long-term studies in central poland. zool. abh. (dresden) 55: 95-102. mitrus, s., zemanek, m. (2000): distribution and biology of emys orbicularis (l.) in poland. in: die europäische sumpfschildkröte, p. 107-118. hödl, w., rössler, m., eds, land oberösterreich, oö, landesmuseum, linz. najbar, b. (2007): wyniki monitoringu i nadzoru stanu zachowania żółwia błotnego emys orbicularis w regionie biogeograficznym kontynentalnym. 1220 – żółw błotny, p. 320-324. raport. instytut ochrony przyrody, kraków. najbar, b. (2008): biologia i ochrona żółwia błotnego (emys orbicularis) w zachodniej polsce. oficyna wydaw. uniwersytetu zielonogórskiego, zielona góra. najbar, b., szuszkiewicz, e. (2007): nest-site fidelity of the european pond turtle emys orbicularis (linnaeus, 1758) (testudines: emydidae) in western poland. acta zool. cracov. 50a: 1-8. novotný, m., danko, s., havaš, p. (2004): activity cycle and reproductive characteristics of the european pond turtle (emys orbicularis) in the tajba national nature reserve, slovakia. biologia 59 (suppl.): 113-121. ottonello, d., salvidio, s., rosecchi, e. (2005): feeding habits of the european pond terrapin emys orbicularis in camargue (rhône delta, southern france). amphibia-reptilia 26: 262-265. rovero, f., chelazzi, g. (1996): nesting migrations in a population of the european pond turtle emys orbicularis (l.) (chelonia emydidae) from central italy. ethol., ecol. evol. 8:297-304. schneeweiss, n., steinhauer, c. (1998): habitat use and migrations of a remnant population of the european pond turtle, emys o. orbicularis (linnaeus, 1758), depending on landscape structures in brandenburg, germany. in: proceedings of the emys symposium dresden 96. mertensiella 10, p. 235-243. fritz, u., joger, u., podlucky, r., servan, j., buskirk j.r., eds., dght, rheinbah. semlitsch, r.d., bodie, j.r. (2003): biological criteria for buffer zones around wetlands and riparian habitats for amphibians and reptiles. conserv. biol. 17: 1219-1228. stephens, p.r., wiens, j.j. (2003): ecological diversification and phylogeny of emydid turtles. biol. j. linn. society 79: 577-610. ultsch, g. r. (2006): the ecology of overwintering among turtles: where turtles overwinter and its consequences. biol. rev. 81: 339-367. leech presence on iberian brown frog, rana iberica, (amphibia: anura: ranidae) from north-western spain césar ayres1, julian comesaña iglesias2 1 rede de observación ambiental de galicia (roaga)-cinam, apdo. de correos 127c.p. 36080, lourizán pontevedra-spain. e-mail: cesar@herpetologica.org 2 valladares-pomba, 33, 36315 vigo (pontevedra). spain submitted on 2007, 15th december; revised on 2008, 27th march; accepted on 2008, 28th march. abstract. the authors describe a case of parasitism on rana iberica by two species of leeches, batracobdella sp. and hirudo medicinalis, in a mountainous area of northwestern spain. conservation implications of high parasite load on small and isolated populations are discussed. keywords. amphibia, rana iberica, leech parasitism, spain. leeches are known to parasite many freshwater species including invertebrates (kutschera, 2003), fishes (pearse, 1924), amphibians (fontaneto et al., 1999, romano and di cerbo, 2007), turtles (maccoy et al., 2007; ayres and alvarez, in press), and mammals (davies and macloughlin, 1996). trophic relationships with amphibians are very complex, leeches predate or parasite over all life stages of amphibians, including eggs (romano and di cerbo, 2007), larvae (gunzburger and travis, 2005) and also adults (merilä and sterner, 2002). rana iberica inhabits cold streams, ponds and mountain lakes in the north and west of the iberian peninsula, with some isolated spots in the sistema central mountains (salvador and garcía-parís, 2001). our work reports an episode of parasitism by two species of leeches on r. iberica found in serra do suido mountains, galicia region, north-western spain. the site of collection is located near the top of the mountain at an altitude of 851 m a.s.l. the commonest vegetation is shrubs with dense cover of erica sp. and ulex sp.; arboreal species are restricted to stream and river beds, with betula sp. and salix sp. on 6th november 2007 one male of r. iberica was captured in a small stream in barranqueira de casariños. this stream is used as a water reservoir for free-ranging cattle and horses. due to the severe draught that galicia suffered during 2007 the stream was almost dry, with the exception of some ponds. the individual of r. iberica was captured resting in one shallow pond of the stream, in seemingly poor condition as indicated by the apathetic behaviour. when we examined the frog we found ten leeches attached to its body. most acta herpetologica 3(2): 155-159, 2008 issn 1827-9643 (online) © 2008 firenze university press 156 c. ayres and j. comesaña iglesias of them were attached in the inguinal and axilar area (see fig. 1), but one big leech was attached in the middle of the body. the smallest ones belong to batracobdella sp., which is known to parasite on iberian brown frogs (almaça, 1964; garcía-parís, 1985; galán and fernandez arias, 1993), but the biggest one belongs to hirudo medicinalis, the medicinal leech, a typical parasite of mammals (keim, 1993; davies and macloughlin, 1996). in the iberian peninsula batracobdella sp. is a common ectoparasite of some amphibian species, including discoglossus galganoi and rana iberica (garcía-parís, 1985; galán and fernandez arias, 1993, galán pers. comm.). but to our knowledge there are few reports about amphibian predation by medicinal leeches in the iberian peninsula. as merilä and sterner (2002) stated, medicinal leeches could act as important source of mortality for adult amphibians. in our case, it seems that warm temperatures, 25 ºc max. on 6th november, and low water level, it was the driest autumn in the last fifty years, rise the possibility of dangerous interactions between leeches and amphibians. also recent changes in cattle management, using troughs or watering places during drought, could lead to an increase of leech parasitism on amphibians, due to scarcely presence of mammals. in our study area it seems that some factors are influencing negatively on r. iberica and other amphibians. habitat modification and fragmentation, due to human activity (i.e., road construction, windmills), intentional fires, and cattle pressure (i.e., eutrophication); seem to have a negative effect on amphibian populations (vos and chardon, 1998; ayllon-lopez and dominguez-gonzalo, 2001; hels and buchwald, 2001; jansen and healey, 2003; knutsom et al., 2004; cushman, 2006; nyström et al., 2007). serra do suido mountains suffer intentional fires each year in order to create new pastures for cattle, this problem causes habitat loss and changes in vegetation cover. additionally, construction of windmill parks caused habitat fragmentation, changing natural drainages due to creation of tracks; this problem is more problematic in the last years due to the severe fig. 1. leeches attached to r. iberica. 157leech parasitism in rana iberica draught that galicia region is suffering. all this human pressure limits available habitat for r. iberica, restricting the species to small drainages, with less possibilities for dispersal and colonization of new areas or gene flow between populations. martinez-solano et al. (2005) stated that the combination of both reduced genetic variability and increasing isolation of populations is likely to result in local and regional extinction of populations in sistema central mountains. but maybe leech infestation could also have a negative effect on small isolated populations. fontaneto et al. (1999), merilä and sterner (2002) reported attacks from h. medicinalis, and indirect predation by haemopis sanguisuga, which caused high mortality. also it’s possible to affect indirectly by transmission of blood parasites by non lethal parasitism. another effect that it’s not been evaluated is the possible impact on eggs and larvae by leeches, as is reported by gunzburger and travis (2005) and romano and di cerbo (2007). at a conservational point of view both species, r. iberica and h. medicinalis, are protected under different laws. r. iberica is protected by regional and national laws, catalogued as vu in both red books. medicinal leeches are protected under european laws, including annex v of bern convention, iucn, and also cites. in spain the current situation of the species is unknown, but garcía más and muñoz araujo (2001) stated that this species could potentially occur in almost all iberian peninsula inland waters, but its populations face such threats that its range is drastically decreasing, due to habitat loss and overexploitation. also in r. iberica it’s been reported negative influence from human disturbance (rodriguez-prieto and fernandez-juricic, 2005), as this species show high fidelity to stream beds and low dispersal, effects on the habitat quality could have a higher effect on survival rates. martinez-solano et al. (2003) stated that r. iberica is restricted in its possible expansion to suitable breeding habitat because it breeds almost exclusively in permanent streams. this species also avoid the use of deep ponds or streams inhabited by predatory fishes, which not only reduce amphibian population size but also increase fragmentation and isolation, which could reduce genetic flow or local extinction of small populations. salmonid presence has a strong negative effect on tadpole presence, not only by direct predation but also by altering behaviour due to chemical cues (bosch et al., 2006). in barranqueira de casariños stream some deep ponds are occupied by salmonids, so tadpoles and adults seem to be restricted to shallower marginal areas avoiding central deeper zones with faster current. maybe this behaviour could increase the risk of being parasitized by leeches, as we found most individuals of batracobdella sp. attached under stones in shallow water. it seems that both species, iberian brown frog and medicinal leeches, are suffering dramatic changes in an area that remained quite undisturbed until recent years. these changes made possible more interactions between both species. further monitoring will be necessary to quantify leech infestation and to assess the real impact of leech infestation over mountain populations of amphibians in north-western spain. acknowledgements we would like to thank d. bird, p. galan, and p. garcia for their comments about leeches and amphibians. we also thank i. garcia-mas for his help providing references and identifying leeches. a.r. di cerbo, s. scali and a. romano provided useful comments on an earlier draft of this manuscript. 158 c. ayres and j. comesaña iglesias references almaça, c. (1964): a fauna herpetológica da serra do gerês. naturalia 9: 62-64. ayllón-lópez, e., domínguez-gonzalo, c. (2001): situación actual y problemas de conservación de rana patilarga en la c. a. m. bol. soc. cons. vert. 8-9: 7-15. ayres, c., alvarez, a. (in press): on the presence of placobdella sp. leeches on emys orbicularis. acta biol. univ. daugavpil. 7: in press. bosch, j., rincón, p.a., boyero, l., martínez-solano, i. (2006): effects of introduced salmonids on a montane population of iberian frogs. cons. biol. 20: 180-189. davies, r., macloughlin, n. (1996): the effects of feeding regime on the growth and reproduction of the medicinal leech hirudo medicinalis. freshw. biol. 36: 563-568. cushman, s.a. (2006): effects of habitat loss and fragmentation on amphibians: a review and prospectus. biol. cons. 128: 231-240. fontaneto, d., guidali, f., scali, s. (1999): parasitism and necrophagy of two leeches species on bufo bufo. in: current studies in herpetology, p. 121-124. miaud, c., guietant, r., eds, proc. 9th ogm of societas herpetologica europaea, 25-29 august 1998, societas herpetologica europaea, chambery. galán, p., fernández arias, g. (1993): anfibios e réptiles de galicia. xerais, lugo. garcía más, i., muñoz araujo, b. (2001): hirudo medicinalis linnaeus, 1758. in: los invertebrados no insectos de la “directiva hábitat” en españa, p. 125-130. ramos, m.a., bragado, d., fernández, j., eds, ministerio de medio ambiente – consejo superior de investigaciones científicas, madrid. garcía-parís, m. (1985): los anfibios de españa. ministerio de agricultura, pesca y alimentación, madrid. gunzburger, m.s., travis, j. (2005): critical literature review of the evidence for unpalatabilty of amphibians eggs and larvae. j. herpetol. 39: 547-571. hels, t., buchwald, e. (2001): the effect of road kills on amphibian populations. biol. cons. 99: 331-340. jansen, a., healey, m. (2003): frog communities and wetland condition: relationships with grazing by domestic livestock along an australian floodplain river. biol. cons. 109: 207-219. keim, a. (1993): studies on the host specificity of the medicinal blood leech hirudo medicinalis l. parasitol. res. 79: 251-255. knutsom, m.g., richardson, w.b., reineke, d.m., gray, b.r., parmelee, j.r., shawn, e.w. (2004): agricultural ponds support amphibian populations. ecol. appl. 14: 669-684. kutschera, u. (2003): the feeding strategies of the leech erpobdella octoculata (l.): a laboratory study. intern. rev. hydrobiol. 88: 94-101. martínez-solano, i., bosch, j., garcía-parís, m. (2003): demographic trends and community stability in a montane amphibian assemblage. cons. biol. 17: 238-244. martínez-solano, i., rey, i., garcía-parís, m. (2005): the impact of historical and recent factors on genetic variability in a mountain frog: the case of rana iberica (anura: ranidae). anim. cons. 8: 431-441. mccoy, j.c., failey, e.l., price, s.j., dorcas, m.e. (2007): an assessment of leech parasitism on semi-aquatic turtles in the western piedmont of north carolina. southeast. nat. 6: 191-202. 159leech parasitism in rana iberica merilä, j., sterner, m. (2002): medicinal leeches (hirudo medicinalis) attacking and killing adult amphibians. ann. zool. fennici 39: 343-346. nyström, p., hansson, j., mansson, j., sundstedt, m., reslow, c., broström, a. (2007): a documented amphibian decline over 40 years: possible causes and implications for species recovery. biol. cons. 138: 399-411. pearse, a.s. (1924): the parasites of lake fishes. trans. wisconsin acad. sci. 26: 437-440. rodríguez-prieto, i., fernández-juricic, e. (2005). effects of direct human disturbance on the endemic iberian frog rana iberica at individual and population levels. biol. cons. 123: 1-9. romano, a., di cerbo, a.r. (2007): leech predation on amphibian eggs. acta zool. sin. 53: 750-754. salvador, a., garcía-parís, m. (2001): anfibios españoles. identificación, historia natural y distribución. esfagnos, talavera de la reina. vos, c.c., chardon, j.p. (1998): effects of habitat fragmentation and road density on the distribution pattern of the moor frog rana arvalis. j. appl. ecol. 35: 44-56. acta herpetologica 4(1): 47-56, 2009 the ovipositional behaviour of the endemic whistling lizard calotes liolepis boulenger, 1885 (reptilia: agamidae) in the knuckles forest region of sri lanka d.m.s. suranjan karunarathna1, i. nuwan bandara2, a.w. amila chanaka3 1 iucn – sri lanka country office, no: 53, horton place, colombo 07, sri lanka. corresponding author. e-mail: dmsameera@gmail.com 2 youth exploration society of sri lanka, royal botanic gardens, peradeniya, sri lanka. e-mail: imeshnu1@gmail.com 3 wildlife trust of sri lanka, no. 342, sweet-house building, pitakotte junction, pitakotte, sri lanka. e-mail: boiga_amila@yahoo.com abstract. a mature female calotes liolepis was observed laying eggs on the ground in manigala in the knuckles forest region of sri lanka. this is the first completely described observation of the ovipositioning as well as the captive egg hatching of calotes liolepis. this ovipositional behaviour consisted of the digging of the nest cavity, oviposition, scraping of the soil to bury the eggs, filling of the spaces between the eggs, compression of the soil, and camouflage of the nest. the sizes of three eggs were increased during incubation: day 1 mean = 17.5 mm × 9.2 mm (length × width), and after 70-71 days mean = 21.7 mm × 14.4 mm. three hatchlings were emerged (mean snout-to-vent length = 29.9 mm; tail length = 58.2 mm; head length = 10.2 mm. immediate conservation measures are needed for this endemic and threatened lizard, and the observations related to its egg-laying could be useful in planning and implementing suitable conservation methods. keywords. agamidae, draconinae, calotes, ovipositional behaviour, knuckles, sri lanka. introduction sri lanka and western ghats of india is a biodiversity hotspot, rich in herpetofaunal assemblages because of favorable environmental factors such as high rainfall and humidity, and high density of undergrowth found in this region (bossuyt et al., 2004; de silva, 1996; gunawardene et al., 2007; karunarathna et al., 2006; meegaskumbura et al., 2002; pethiyagoda et al., 2005). there are eighteen species of agamid lizards (family: agamidae) in sri lanka including three endemic genera and 15 (83%) endemic species (bahir and surasingha, 2005; manamendra-arachchi et al., 2006; samarawickrama et al., 2006). the 48 d.m.s. suranjan karunarathna et alii 18 native species belong to the sub-family draconinae and consists of six genera; calotes, ceratophora, cophotis, lyriocephalus, otocryptis and sitana (de silva, 2006; deraniyagala, 1953; macey et al., 2000). the genus calotes consists seven species and five of them are endemic to sri lanka, their are c. ceylonensis, c. desilvai, c. liocephalus, c. liolepis, and c. nigrilabris (das and de silva, 2005; karunarathna and amarasinghe, 2008; manamendraarachchi, 1998), and all of them are nationally threatened (iucn sri lanka and menr 2007). others non-endemic are the c. calotes and c. versicolor respectively. calotes liolepis has been recorded from only a few widely separated localities restricted to the sub-montane forests, mainly in heavily shaded areas of forest in the wet zone and plantations below 1000 m elevation (asela et al., 2007; bahir and maduwage, 2005; manamendra-arachchi and liyanage, 1994). this arboreal species is much slower than other sri lanka agamid lizards, even when tree climbing. its conservation status is vulnerable (iucn sri lanka and menr, 2007). this species is largely arboreal and unusual among agamid lizards due to its habit of uttering a high-pitched whistle when alarmed (das and de silva, 2005). average adult snout-to-vent length (svl) = 72 mm, head length (hl) = 28 mm, tail length (tl) = 190 mm, and axilla-to-groin length (ag) = 37 mm (deraniyagala, 1953). recently, the egg laying behaviour of c. liolepis has been documented by asela et al. (2007), but it lacks many details. in this paper we highlight further details of the ovipositioning behaviour in the wild, as well as the captive incubation and hatching of c. liolepis eggs. materials and methods the length of the enclosure is 300 mm, width 15 mm and height 100 mm. a thermometer and a hygrometer are used to monitor temperature and relative humidity fluctuations. the base of the unit was filled with soil mixed with sand to a depth of nearly 50 mm. pieces of stones, sticks and leaf litters were also provided as hides for hatchlings. the relative humidity varied between about 65% and 75% during incubation. these values may be higher than those found in the open in the wild. the surface soil was generally kept dry but occasionally about 100 ml of tap water was sprayed in to the hatching device to maintain a more natural state. the daily temperature varied between about 1 °c and 2 °c. the above 3 eggs were buried half in the soil and covered with leaf litter. observations of the lizard were made between 1430 and 1730 h from a distance of 2 m. the animal was disturbed two times to take photographs. when disturbed, it expanded its gular sac and looked around for about 5 min (fig. 1). the specimen was examined closely and notes on key characteristics were recorded. all measurements were taken to the nearest 0.1 mm using dial calipers. diagnostic keys in bahir and maduwage (2005), deraniyagala (1953), manamendra-arachchi (1990, 1998), smith (1935) and taylor (1953) were used for species identification. plant nomenclature was based on senaratna (2001) and identified using ashton et al. (1997). in vitro incubation all eggs were removed from the original nest and buried in soil in a glass enclosure that allowed the penetration of light. the incubation container was placed outside the laboratory in a shady environment similar to the original habitat. humidity inside the tank was kept near 70%. eggs were not disturbed during incubation and all were visible to the outside so that all observations during hatching could be made without disturbance. 49ovipositional behaviour of the calotes liolepis in sri lanka results study area and habitat observations were made approximately 1 km from ilukkumbura in manigala in the knuckles forest region (altitude: 800 m) in matale district, central province (7°33’46” n, 80°46’12” e). the ground was covered with 5 cm thick wet leaf litter. the soil was dark and soft with tightly bound particles, and lacked air cavities. there was approximately 75% canopy cover and the undergrowth consisted of shrubs and herbs. the average temperature and humidity were 27 °c and 70%, respectively. on the day of our observations of nesting, the weather was sunny, cloud cover was 6/8, and there was rain prior to the nesting event. the semi-evergreen forests represent the major natural vegetation type in manigala area. the forest consists of three strata; the canopy (20-25 m), sub-canopy (5-10 m) and ground herbaceous vegetation (bambaradeniya and ekanayake, 2003). these forests harbour many large canopy trees such as creteava religiosa, phyllanthus indika, sterculia foetida, bombax ceiba, dimocarpus longan, palaquium hinmolpedda and vitex altissima species and the sub-canopy level consists of breynia vitisidea, miliusa indica, pavetta indica and streblus asper species; ground cover consists of begonia hirtella, carex filicina, carex jakiana, curculio orchioides and procris crenata species (ekanayake and bambaradeniya, 2001). the highland areas of the knuckles forest range is extremely wet throughout the year, with an average annual rainfall > 4000 mm, though the lower fig. 1. during the disturbances it expanded its gular sac and looked around. 50 d.m.s. suranjan karunarathna et alii eastern slopes are much drier. previous surveys have documented eight species of agamid lizards inhabiting the knuckles forest reserve (bambaradeniya and ekanayake, 2003; samarawickrema et al., 2006; de silva et al., 2005; rajapaksha et al., 2006). observations on digging the nest hole a mature female calotes liolepis (svl = 79 mm, hl = 25.1 mm, hw = 14 mm, tl = 177 mm, agl = 51 mm) lying on the ground in the manigala, about 1 km away from the ilukkumbura-manigala nature trail, was observed on 5th april 2008 at about 1430 h. first, the lizard looked around for about 30 min. during this time it repeatedly turned its head approximately 180° fifteen times with moving its body. the lizard then changed its body color to approximate the ground color, probably for camouflage. the female then started digging and scraping the soil with its forelimbs, in the process turning its body clockwise and counterclockwise 24 times. it took ~30 min to make the body pit prior to digging the nest hole. the body pit was 150 mm in diameter. while turning around, the soil was thrown backward using forelimbs and hind limbs. the bottom of the body pit was flat and large enough for the female to lie in while egglaying. after completing the initial pit the lizard started digging the ground with its forelimbs. while digging, it made the margin and inner wall of the nest hole by compressing the soil with the supra-ocular region, snout and anterior half of its lower jaw to avoid collapse. then it stopped digging and looked around for approximately 5 min while repeatedly turning its head 90° six times without moving its body. it continued to dig the hole for another hour, stopping three more times for 5 min per interval. the hole was vertical, 50 mm deep and 35 mm in diameter. during the rest intervals the tail was coiled inside the hole with the head bent at an angle of 90° to look around. laying the eggs after 30 min of digging, the female turned its body 180° clockwise, placing the posterior part of its body at the opening of the hole and the tail coiled at the outer margin of the hole. it then looked around again. the female laid eggs in the hole without lifting its limbs where as limbs placed at the top-opposite side of the body pit by thrusting them to the front (fig. 2). the head and breast were lying very closer to the ground during the whole period. three eggs were laid at a rate of approximately one per three min (fig. 3). the eggs were pure white and elliptical with a thin pliable shell. mean length = 17.5 mm and mean width = 9.2 mm (pooling these data with previous observations of 11 eggs, mean = 17.1 mm × 9.2 mm). after the eggs were laid the female remained motionless for 20 min. burying the eggs and camouflaging the nest following a period of inactivity, the lizard turned 180° clockwise and moved back into the hole for 10 min to pack and place the eggs below ground level using the anterior part of its lower jaw. it then remained motionless for approximately 15 min. it then began dragging soil towards the hole using its forelimbs. the excavated soil was pulled poste51ovipositional behaviour of the calotes liolepis in sri lanka riorly using its hind limbs. after filling approximately 1/3 of the hole in about 5 min, it turned 90° counterclockwise and started pressing the soil with the supra-ocular region, tip of the snout and anterior half of its lower jaw for 30 min. a knocking noise was produced as the lizard hit the substrate 28 times with its lower jaw to compress the soil in the hole. fig. 2. the lizard laying eggs and see tail is curved. fig. 3. the three eggs layed by calotes liolepis in manigala. 52 d.m.s. suranjan karunarathna et alii it then filled another 1/3 of the hole in about 5 min, turned 90° counterclockwise again, and started compressing the soil with the anterior half of its lower jaw for 20 min. finally, the lizard filled last 1/3 of the hole in about 5 min. afterward, it turned 90° counterclockwise again and started pressing the soil with the anterior half of its lower jaw for 10 min, after which the hole was filled to ground level. after looking around, it dragged nearby fallen leaves of dimocarpus longan and palaquium hinmolpedda (family: sapindaceae and sapotaceae) over the nest for camouflage. it remained motionless for 5 min and during this time changed its body color to light greenish brown before running towards the forest. at this time it was caught for measurement and then released. captive eggs hatching notes eggs were removed from the nest cavity and the cavity was examined after the female was released. the bottom of the cavity was conical and the soil soft, dark and wet. the eggs were buried in soil in a screen-topped glass enclosure to incubate. we deposited the eggs in an artificial nest cavity similar in structure to that constructed by the female. the lid of the container was close-fitting to deter predators. after about two days the hatchlings were released to the original habitat. discussion the ovipositional behaviour of this species varies from the ovipositional behaviour of calotes versicolor and c. liocephalus. according to amarasinghe and karunarathna (2007, 2008), c. versicolor places its cloacal aperture over the opening of the hole and lifts its hind limbs while laying its eggs (fig. 4a), c. liocephalus places the posterior part of the body inside the hole while laying eggs (fig. 4b), and c. liolepis places the posterior part of its body at the opening of the hole and the tail was coiled at the outer margin of the hole and then lay eggs to the hole without lifting its hind limbs (fig. 4c). the egg laying of c. ceylonensis is similar to the egg laying of c. liolepis but c. ceylonensis lifts hind limbs then c. liolepis (pradeep and amarasinghe, 2009). the c. versicolor lifts the anterior part of the body with its forelimbs while turning its head to look around and c. liocephalus coils its entire body inside the hole while bending the anterior part of its body to look around but c. liolepis coiled the tail inside the hole with the head bent at an angle of 90° to looking around similar to c. ceylonensis (pradeep and amarasinghe, 2009). calotes versicolor makes a knocking noise while packing and placing the eggs in the hole using its lower jaw (amarasinghe and karunarathna, 2007) while c. liocephalus and c. ceylonensis places them softly without making any noise (amarasinghe and karunarathna, 2008; pradeep and amarasinghe, 2009) and c. liolepis did not show any packing behaviour after laying eggs. however it made a knocking noise while compressing soil in the hole during egg laying. neither c. versicolor nor c. liocephalus marked the body pit to dig the nest hole as c. liolepis and c. ceylonensis (pradeep and amarasinghe, 2009) and also they did not use hind limbs to pull the soil to the hole. calotes versicolor, c. ceylonensis and c. liocephalus threw the soil backward under the body through raised hind limbs whereas c. liolepis threw the 53ovipositional behaviour of the calotes liolepis in sri lanka soil backwards alongside of its body. c. liolepis dug the hole straight inward to the ground while other two species dug the hole into the ground at a 45° angle. we have observed gray hornbills feed on c. liolepis several times. during this ovipositioning we heard a call of gray hornbill but the lizard continued its digging without any change in behaviour. asela et al. (2007) observed three egg laying episodes of c. liolepis from three different conditions between 1300 h and 1540 h in the month of february. we also made the observation during 1430 h and 1730 h in april. these observations suggest that this species prefers to lay eggs at the afternoon during february-april. here we compared egg measurements with the earlier observations by asela et al. (2007). we can conclude that fig. 4. a: calotes versicolor places the cloacal aperture over the opening of the hole while laying eggs; b: calotes liocephalus places the posterior part of the body inside the hole while laying eggs; c: calotes liolepis places the posterior part of its body at the opening of the hole and the tail was coiled at the outer margin while laying eggs (drawings made by a.a. thasun amarasinghe). 54 d.m.s. suranjan karunarathna et alii c. liolepis lays 2-4 eggs during 1300-1730 h in february-april, constructs a hole 30-45 mm diameter (average is 35.75 mm) and hole depth = 38-50 mm (average = 44 mm). the body pit is 140-150 mm diameter (average = 145 mm; table 1). we conclude that there is substantial behavioural consistency in nesting among female c. liolepis. the c. liolepis specimen was observed after egg laying crawling from forest edge to deep forest through dry leaves, which helped it to be camouflaged. the agamid took about 10 min to complete its journey climb the ten-meter sterculia foetida tree. the specimen of c. liolepis was very active when captured, and a bright orange skin tone could be seen between gular scales of throat. additionally, shook its tail slowly and rhythmically. a few diagrams, brief descriptions and notes of c. liolepis are available in popular journals, books and magazines but almost nothing exists on the preand post-mating behaviour, egg laying, captive breeding or hatchlings. immediate conservation measures are needed for this endemic and nationally threatened lizard, and the observations related to its egglaying could be useful in planning and implementing suitable conservation. acknowledgments the authors wish to thank dr. channa bambaradeniya (iucn sri lanka) for the reviewing the manuscript, mr. thasun amarasinghe for marvelous line drawings and valuable help during the preparation of this paper. our heartfelt thanks go to mr. mendis wickramasinghe, mr. bathiya kekulandala, mr. sandun perera, mr. sampath goonatilake, mr. prasanna samarawickrama, mr. naalin perera, mr. sarath ekanayake, mr. vimukthi weeratunga, mr. samantha suranjan and mr. dilup chandranimal (iucn sri lanka) for their kind help during the preparation of this paper. we would also like to thank to mr. kelum manamendra-arachchi and mr. dinesh gabadage (wildlife heritage trust of sri lanka), mr. n. bandara, (hunas falls hotel), course coordinating committee (wildlife trust) and also mr. saman fernando, allowing us to use his camera equipments. finally table 1. ecological factors and egg hole description of calotes liolepis at four sites. delwala, rojersongama 1 and 2, based on asela et al. (2007). ecological data delwala rojersongama 1 rojersongama 2 manigala month february february february april time duration 1300-1400 1400-1500 1500-1540 1430-1730 soil humus wet, dark, soft leaf litter thickness (mm) 40 40 50 canopy cover (%) 75 75 cloud cover 5/8 6/8 6/8 temperature (°c) 27.1 27.0 diameter of the body pit (mm) 140 150 depth of the hole (mm) 40 48 38 50 diameter of the hole (mm) 45 33 30 35 no. of eggs laid 2 4 2 3 55ovipositional behaviour of the calotes liolepis in sri lanka we thank to mr. tiran abeywardena, mr. chamila soysa, mr. toshan peiris, mr. asanka udayakumara, mr. panduka silva, mr. chandana asela, mr. ramyanath sirimanna and mr. devaka jayamanna (yza-young zoologists’ association) for his kind help during the field visit and dr. stefano scali and anonymous reviewers for useful comments. references ashton, m., gunatileke, c.v.s., de zoysa, n., dassanayake, m.d., gunatileke, n, wijesundara, s. 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(1935): the fauna of british india including ceylon and burma, reptilia and amphibia, vol ii sauria. taylor and francis, london. taylor, e.h. (1953): a review of the lizards of ceylon. univ. kansas sci. bull. 35: 1525-1585. acta herpetologica 17(2): 177-186, 2022 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-13209 occupancy and probability of detection of the introduced population of eleutherodactylus coqui in turrialba, costa rica jimmy barrantes-madrigal1,*, manuel spínola parallada1, gilbert alvarado2, víctor j. acosta-chaves3,4 1 instituto internacional en conservación y manejo de vida silvestre, universidad nacional, heredia, costa rica 2 laboratorio de patología experimental y comparada (lapecom), escuela de biología, universidad de costa rica, san josé, costa rica 3 sede del atlántico, universidad de costa rica sede atlántico, cartago, costa rica 4 school for field studies, atenas, costa rica *corresponding author. email: jimmybarrantesm@gmail.com submitted on: 2021, 13th september; revised on: 2022, 7th june; accepted on: 2022, 19th june editor: andrea costa abstract. the puerto rican common coqui frog (eleutherodactylus coqui) has a long history as an invasive species in places such as hawaii. since its introduction in costa rica, scarce information is available to understand why and how the habitat in the turrialba town is suitable for the species. our goal was to analyze the habitat selection of e. coqui to identify if there are key habitat features that explained its success there. we measured 9 site variables that may affect the habitat selection of e. coqui in 92 survey units of 10 m radius distributed over a 500 m radius from its introduction point. we registered the presence/pseudo-absence data of e. coqui and environmental variables in each survey unit during eight surveys. we ran occupancy models to determine the influence of the variables on the habitat selection and to estimate its detection probability. we found that sites near the introduction point, containing abundant vegetation, bromeliads, and palms have a higher probability to be occupied by e. coqui. the habitat selection in costa rica shares characteristics with the populations of puerto rico and hawaii. but, unlike the case in hawaii, in costa rica this species has maintained a limited dispersal because the potentially higher biotic resistance, as well a sedentary behavior. however, the microhabitat conditions used by e. coqui in the study site are common throughout the country. therefore, active management in new populations and environmental education programs to avoid human transportation of the species is critical to reduce its dispersal. keywords. amphibians, conservation, detection probability, invasive species, introduced species, occupancy models. introduction the study of the factors that determine the establishment and dynamics of an exotic species in a new ecosystem is not only a vital component in the development of biological invasion management strategies, but it also provides important information for understanding the processes that take place in natural ecosystems (jiménez-valverde et al., 2011; wan et al., 2019). in most scenarios the introduced species fail to establish or advance beyond the first stages of invasion (zenni and núñez, 2013). however, under the right conditions, these species can colonize and spread over large areas and ecosystems causing severe alterations (mačić, 2018). additionally, in some cases rapid evolutionary processes may occur that favor their adaptation to new conditions (whitney and gabler, 2008; carneiro and lyko, 2020), where characteristics such as behavior, morphological and reproductive traits, and genetic variability of populations of introduced species may differ considerably with respect to the populations in their native range (o’neill et al., 2018). 178 jimmy barrantes-madrigal et alii the common coqui frog (eleutherodactylus coqui, thomas 1966) is a species native from puerto rico with a long history as an invasive species (lowe et al., 2004). in its native habitat e. coqui is one of the most abundant amphibians, and it can be found from the forest floor to the canopy, inhabiting almost all environments (joglar, 1998). it breeds throughout the year (townsend and stewart, 1994). neonates take 8 to 9 months to become sexually mature (towsend and steward, 1994) and lays on average 4-6 clutches of eggs per year, each containing 16-41 eggs per clutch (towsend and stewart, 1994). eggs are generally deposited in covered sites that provide protection from rain and environmental conditions (townsend, 1989; beard and pitt, 2012). egg development is direct (towsend and steward, 1985) and hatch after 14-17 days (towsend and steward, 1994). this anuran was introduced to the hawaiian archipelago in the late 1980s, where in less than 10 years it had spread throughout an extensive area of the archipelago (kraus and campbell, 2002). as in puerto rico, e. coqui populations in hawaii are abundant; it has been reported population densities of up to 91000 individuals per hectare at the archipelago, a number three times higher than the estimates reported in puerto rico (beard et al., 2008). these extreme densities have caused not only ecosystem alterations such as changes in the invertebrate community (choi and beard, 2012), alteration in the nutrient cycle and herbivory regimes (sin et al., 2008), but also social and economic effects due to noise pollution produced by their constant vocalizations and the measures required for its control (beard et al., 2009). in costa rica, the common coqui frog was introduced around 1998 into the city of turrialba (garcía-rodríguez et al., 2010; barrantes-madrigal et al., 2019). unlike its invasion process in hawaii, in the cartago province it has been kept restricted to a few localities for almost two decades: turrialba and juan viñas (barrantes-madrigal et al., 2019). although barrantes-madrigal et al. (2019) provided an update of the invasion status of the species in costa rica, since then have been observed few individuals in san antonio de escazú (san josé province) (https:// www.inaturalist.org/observations/48536340). to continue research on this topic is relevant to understand why this population survived in turrialba, and what implications could it has with the years across the country. although there is much information in the literature about the ecology of e. coqui, this information comes mainly from islands (puerto rico and hawaii) where the ecological conditions are different from the continental neotropical context found in costa rica. the objective of this work is to determine the habitat selection of the common coqui frog (eleutherodactylus coqui) population introduced in the town of turrialba to identify habitat variables that favor its occupation. we predicted that the vegetation structure and the availability of breeding sites would play a relevant role in the selection of the microhabitat of this frog as it has been in its native (townsend, 1989) and exotic range (beard et al., 2003). this research is relevant to understand why this population survived in turrialba, and what implications could it has with the years across the country. materials and methods study area the study was carried out in the city of turrialba, where the initial population of e. coqui was found in costa rica (9°53’42”-9º54’18”n and 83°40’48”83°39’54”e; garcía-rodríguez et al., 2010; fig. 1). turrialba is located on the caribbean slope and belongs to the canton of turrialba, province of cartago. it has an elevation range between 600 and 650 m a.s.l, has a warm and humid climate with an average annual temperature of 22 °c, and, due to its location, it is exposed to humid northeast winds and in certain regions can receive up to 7000 mm of rain (dufour, 1978). the place where e. coqui was first detected is surrounded by an area of heterogeneous composition with residential and commercial areas, including the campus of the university of costa rica (atlantic branch), but also open areas, pastures, plantations, streams, and small patches of secondary forest such as the botanical garden of the centro agronómico tropical de investigación y enseñanza (catie; fig. 2). sampling design we delimited a circular area of 500 m radius (78.5 ha) around the point where this species was first reported (garcía-rodríguez et al., 2010) as the study site. the extension of the sampling area was defined according to a preliminary sampling where we did not find evidence of the presence of the common coqui frog outside the 500 m radius area from the introduction site. we assumed that, since its introduction, the species has had the same probability of dispersal in any direction within the selected area. within this area we delimited three strata: urban, forest and open areas, based on satellite images taken from google earth pro (google, 2016). in each stratum we randomly distributed 29 circular sampling units (su) of 10 m radius (314.1 m2), with a minimum separation of 30 m between each other to capture most of the micro179occupancy and probability of detection of e. coqui in costa rica habitat’s variability in each stratum (fig. 1). we considered 30 m as an adequate distance considering that e. coqui is a sedentary species, with movements of 3 4.5 m on average around its retreat sites (woolbright, 1985). data collection sus were characterized based on nine site covariates distributed in four categories that we considered may influence the habitat selection of e. coqui (table 1). the first category was vegetation, where we estimated volume of vegetation in three vertical strata and tree cover as habitat attributes that could be important in maintaining the environmental requirements of this species (e.g., temperature, humidity), as well as providing foraging sites or perches to vocalize. we registered bromeliads, palms, and leaf litter for their role as possible nesting or refuge sites (stewart and pough, 1983; beard et al., 2003). bromeliads were registered as presence or absence, where we considered less than five bromeliads as an absence. the percentage of palms and leaf litter in the su, as well as the percentage of vegetation mentioned above, were calculated dividing the su into four equal sections by drawing an imaginary line from the central point towards the four cardinal directions, in this way, we visually estimated the percentage of the covariate represented in each section and averaged the result for each su. on the category of water bodies, we quantified the distance to rivers as a measure to analyze the association with gallery forest environments. additionally, to consider the dispersal capability of this species we measured the distance from the site where the first individuals were introduced. both distance measures were calculated using the distance function of the raster package (hijmans, 2016) in the statistical program r v3.3.2 (r core team, 2016). we carried out a minimum of five and a maximum of eight nocturnal surveys (18:00 22:00 h) in each su during october 2016 to february 2017. we implemented a five-minute survey in each su using the visual and fig. 1. point of introduction of eleutherodactylus coqui (red dot) and distribution of sampling units (yellow points) for the analysis of its habitat selection in turrialba, costa rica. 180 jimmy barrantes-madrigal et alii auditory encounter survey technique (crump and scott, 1994) to determine the presence of e. coqui. during each survey, we recorded if there was presence of the species in the sampling units (su). we registered three environmental variables at the beginning of each su survey: relative humidity (hum), air temperature (temp) and the illuminated percentage of the moon (moon). these variables were chosen because there fig. 2. representation of the types of environments contained in the study area for the habitat selection analysis of eleutherodactylus coqui in costa rica. a. forest, b. gardens, c. plantation, d. open area-pasture, e. green areas, f. urban areas. 181occupancy and probability of detection of e. coqui in costa rica is evidence in the literature that they influence the calling activity of e. coqui and other congeners (joglar, 1998; grant et al., 2012). relative humidity and air temperature were quantified using a digital thermo-hygrometer (se = ± 5% and ± 0.1 °c respectively). additionally, the illuminated percentage of the moon was calculated as the percentage corresponding to the lunar phase, where 0% represents new moon and 100% full moon, using a lunar calendar. data analysis we performed a habitat selection analysis using a single-season static occupancy model (mackenzie et al., 2002). these models are especially useful when detection probability is less than 1, as it is expected for most amphibians. first, we standardized all variables (mean = 0, sd = 1) due to their different value scales. we built a global model using the relative humidity, air temperature and the illuminated percentage of the moon as observation variables for the detection history, and vegetation, bromeliads, palms, leaf litter, canopy cover, distance to rivers and distance to origin as site covariates. site and observation covariates were tested to evaluate their correlation, we built a global model with and without each of the correlated variables (pearson |r| < 0.6) and kept those that resulted in the most parsimonious model evaluated by the akaike information criterion (aic; burnham and anderson, 2002). as result, we excluded leaf_litter, veg_low and veg_ high from the global model. we assessed the goodness-offit and overdispersion of the global model with a parametric bootstrap approach based on the χ2 statistic with 1000 bootstrap samples (mackenzie and bailey, 2004). we evaluated all possible combination of the global model and ranked the results by their aic values using the dredge function of the mumin package (barton, 2016). for occupancy and detection probability estimation we used a model averaging over the subset of models with a δaic < 2.0 as all of them were considered robust (weir et al., 2005). finally, we calculated the relative importance of the estimated parameters for the habitat selection analysis using the importance function of the mumln r package (barton, 2016). this function ranks the variable according to the sum of the aic weights in all models where the variable is included over all possible combinations of the global model. models were built using the unmarked package (fiske and chandler, 2011) in the statistical program r v3.3.2 (r core team, 2016). all data and the r code used in the analysis is available as supplementary material. results we detected the presence of e. coqui in 30 of the 92 sus on at least one occasion. the maximum distance from the point of introduction at which the species was recorded was 493 m, near the limit of the study area. a subset of 19 models with different combination of variables resulted with a δaic < 2 (table 2). the estimated c-hat value for site-occupancy model was close to 1 and did not indicate overdispersion or lack of fit (c-hat = 1.08; χ2 = 781.74; p = 0.258). the aic value was lower when we do not use any of the observation-level variables, however temperature and percentage illuminated of the moon were included in the subset of models with table 1. detail of covariables used to analyze the habitat selection of the common coqui frog (eleutherodactylus coqui) in costa rica. covariable id code description vegetation low height vegetation veg_low percentage of the volume between 0 1 m in height within the su occupied by vegetation medium height vegetation veg_med percentage of the volume between 1 2 m in height within the su occupied by vegetation high height vegetation veg_high percentage of the volume between 2 3 m in height within the su occupied by vegetation canopy cover can_cover percentage of canopy cover within the su (measured with a densiometer) retreat sites bromeliads brom number of bromeliads within the su at a height of less than 3 m. leaf litter leaf_litter estimated percentage of leaf litter within the su palms palm percentage of the su volume occupied by vegetation belonging to plants of the arecaceae family water bodies distance to rivers dist_river distance in meters to the closest moving body of water dispersal distance to origin dist_origin distance in meters to the point of introduction of eleutherodactylus coqui in costa rica. 182 jimmy barrantes-madrigal et alii δaic < 2 (table 2). the estimated detection probability using the averaged model was 0.666 (95% ci = 0.596 – 0.736). the variables mid vegetation (veg_med) and distance to origin (dist_origin) stand out as the most influential in the habitat selection of the species (fig. 3). the presence of bromeliads (brom) also obtained a high value (0.60) as did the percentage of palms (palm) (0.57). the other site covariates presented relative importance values lower than 0.36. discussion the distribution of the common coqui frog (eleutherodactylus coqui) in the study area was explained by site features that favor its occupancy. we determined that the vegetation at a height of 1-2 meters, as well as the proximity to the site of introduction, are the site characteristics that best explain the occupation of the species on a microgeographic scale. in puerto rico, individuals of e. coqui have been observed from the ground to the top of the trees (joglar, 1998), however, consistent with our observations, in our study area this species prefers perches with heights of approximately 1 m and has a negative association for higher places (beard et al., 2003). the common coqui uses plants to vocalize and forage, to select low vegetation for that purpose fit with previous habitat description and selection in puerto rico (townsend, 1989). dense and abundant low vegetation cover contributes to maintaining humidity conditions to avoid its desiccation (beard et al., 2009; klawinski et al., 2014). the positive association with the abundance of bromeliads and palms could be explained by the reproductive biology of the frog, because previous research carried out in puerto rico and hawaii highlights the importance of the availability and quality of nesting sites as a limiting factor for the common coqui population, because the hatching success of the spawn is affected by the structure of the selected sites (stewart and pough, 1983; townsend and stewart, 1994; beard et al., 2003). plant species such as cecropia peltata, epiphytic plants as bromeliads and palms (e.g., prestodea montana) are important for the biology of species in puerto rico, especially due to leaf litter produced that could be shelter, nesting site or call perch (townsend, 1989). in turrialba this type of vegetation also occurs everywhere, especially in riparian and secondary forest, but not necessarily in gardens or sidewalks in our study site. however, also into gardens and sidewalks where ornamental introduced palms (e.g., areca sp., wodyetia sp.) or hybiscus sp. bushes are common and frequently pruned to 1-2 m heigh. structurally, our study site provides vegetation requirements that the common coqui required for breeding and shelter, even table 2. first 10 models of the set of models with the best fit (δaic < 2) used in the habitat selection analysis of eleutherodactylus coqui. p: detection probability; psi: selection probability; npar: number of parameters; aic: akaike’s information criterion; δaic: difference with respect to the best model; waic: akaike’s weight. model formula npar aic δaic waic p(.) psi(brom + dist_origin + palm + veg_med ) 5 313,04 0,00 0,106 p(.) psi(brom + dist_origin + veg_med ) 6 313,29 0,25 0,094 p(.) psi(dist_origin + palm + veg_med ) 5 313,47 0,43 0,085 p(.) psi(brom + can_cover + dist_origin + palm + veg_med ) 4 313,58 0,54 0,081 p(.) psi(dist_origin + veg_med ) 7 314,30 1,26 0,056 p(temp) psi( brom + dist_origin + palm + veg_med ) 6 314,36 1,31 0,055 p(temp) psi( brom + dist_origin + veg_med ) 6 314,52 1,48 0,051 p(.) psi(can_cover + dist_origin + palm + veg_med ) 6 314,64 1,60 0,048 p(.) psi(brom + dist_origin + dist_river + palm + veg_med ) 7 314,64 1,60 0,048 p(moon) psi( brom + dist_origin + palm + veg_med ) 6 314,68 1,64 0,047 fig. 3. relative importance of variables in the habitat selection of eleutherodactylus coqui in turrialba, costa rica. dist_origin = distance to origin, veg_med = medium height vegetation, brom = bromeliads, palm = palms, can_cover = canopy cover, dist_river = distance to rivers. 183occupancy and probability of detection of e. coqui in costa rica when leaf litter was not abundant in our study site; the species could be using diff erent types of substrates to lay eggs. we hypothesize that e. coqui uses bromeliads or other epiphytes (e.g., orchids, ferns) frequently found in trees and gardens for this purpose, because it was common to fi nd individuals retreated inside bromeliads (fig. 4) or perching in palm leaves. th e use of bromeliads and epiphytic plants as shelters during the day is well known for the common coqui biology (ovaska, 1992; fogarty and vilella, 2003), as they provide a protected substrate where humidity conditions are maintained (stewart and pought, 1983), and the same conditions required to deposit their eggs (townsend, 1989). although it is common for e. coqui to lay its eggs on the ground or surroundings, this species prefers elevated substrates whenever they are available as it allows it to have greater hatching success and makes it easier for males to access high perches, close to the laying, where they can perform their vocalizations to attract females or defend territories (townsend, 1989). th e detection probability (0.666, 95% ci = 0.596 – 0.736) is similar to values reported in a study from hawaii (0.58 to 0.73; olson et al., 2012). th ese results indicate that, despite being a relatively easy species to detect due to its constant vocalizations, at least three nocturnal surveys (2.73) to each site are required to avoid false negatives in detections of common coqui individuals with a 95% of confi dence. even when none of the quantifi ed environmental variables had a signifi cant infl uence on the detection probability, previous studies indicate that the activity of this species is closely associated with humidity conditions (pough et al., 1983). humidity in turrialba is relatively constant and high across the surveyed months (dufour, 1978). th is lack of variation could be the reason why we did not fi nd a signifi cant infl uence of these variables on the detection probability. th e observed distribution pattern suggests that there is a higher probability of fi nding common coqui individuals near the introduction point (fig. 5). th is same pattern has been observed in hawaii, where their populations are frequently found near points or routes of introduction such as roads or nurseries, and their dispersal throughout the archipelago is mainly due to transport facilitated by humans (rauschert et al., 2017), with the natural dispersal movements being less important during the invasion process (everman and klawinski, 2013). th is anuran is a very sedentary species, its movements at night are generally short and maintains an action range of just a few square meters (woolbright, 1985), limiting its dispersal to more remote areas since its introduction in costa rica. th e limited dispersion documented can be related with the highly heterogeneous matrix with cover that contain potential barriers such as high-speed roads, neighborhoods, or even more complex rainforest fragments. into the jorge de bravo neighborhood and surroundings, the common coqui behaves like strong invader in disturbed areas near the introduction point, but it seems that would be a weak invader outside where natural ecosystems are more dominant because potentially there is more biotic resistance (meyer et al., 2021). th e biodiversity level in the costa rican caribbean is much higher than in islands like puerto rico or hawaii, fig. 4. common coqui frog (eleutherodactylus coqui) found in a bromeliad, turrialba, costa rica. photo by j. barrantes. fig. 5. selection probability of the variables used to analyze the habitat selection of eleutherodactylus coqui in costa rica. 184 jimmy barrantes-madrigal et alii especially vertebrate diversity such amphibian, reptiles (savage, 2002), birds (stiles and skutch, 1989) or bats (laval and rodríguez, 2002) that could be potential competitors or predators for a noisy species of eleutherodactylus. for example, other native amphibians with a similar niche than the common coqui such as tink frog (diasporus diastema), pigmy rain frog (pristimantis ridens), fleischmann’s glass frog (hyalinobatrachium fleischmanni) or green-boned tree frog (scinax elaeochrous) also occur in the study area, including secondary growth, gardens, or perturbed lands (savage, 2002). it is likely that competition, prey abundance, predation and other factors can influence the habitat selection and dispersal of this species. previous work has highlighted that the way in which introduced species interact with native biota at different perturbation levels is an important determinant of their invasion success (shea and chesson, 2002; meyer et al., 2021). further studies are needed in this field to understand the influence of these interactions, both for the target species and for the native species with which it coexists. our study suggests that the habitat selection of the introduced population of eleutherodactylus coqui in costa rica shares characteristics with the populations of puerto rico and hawaii, where low vegetation and refuge sites during the day are decisive. however, unlike the case in hawaii, in costa rica this species has maintained a limited dispersal because the biotic resistance and sedentary behavior discussed previously. therefore, the scenario of a natural dispersion sounds like a less probable one based on what has been recorded in our study site into the turrialba town thought the last 20 years (barrantes-madrigal et al. 2019). moreover, all the populations in turrialba, juan viñas, and potentially escazú, where introduced on purpose or accidentally by humans (barrantes-madrigal et al., 2019). according with our results, the species could potentially colonize areas with open vegetation or crops with small bushes such as parks or sun coffee plantations from lowlands or middle elevations. other species of eleutherodactylus that also succeed in open vegetation are abundant in puerto rican sun coffee plantations (monroe et al. 2017), for example. however, in the other hand, other similar species to the common coqui such as eleutherodactylus planirostris or e. johnstonei has been restricted to a single record or locality, without an important expansion or succeed to stablish new populations (e.g., e. johnstonei; savage, 2002; barquero and araya, 2016). thus, even when an extreme aggressive invasion scenario like the observed in hawaii is unlikely to occur at country scale in costa rica at least soon, because the microhabitat conditions used by e. coqui in the study site are common in other neighboring towns in the lowlands from caribbean or pacific slopes, we consider that rural and peri urban areas with a mixed matrix of agropastoral-urban systems could be more likely to be invade by the common coqui in further years only if transportation by humans continue. anecdotically, during surveys made by barrantesmadrigal et al. (2019), we identified that an important number of people from our study area sympathized with the sound produced by the common coqui, even feeling proud of having the species living in their homes. this can increase the transportation risk of common coqui frogs between people, both intentional and accidental, something that did not happened with other species like e. johnstonei. on the contrary, it was identified that other neighbors from our study area had noise problems due to the extreme local abundance of the frog in their gardens trying to manage the population with invasive and nonfriendly environmental methods but with few succees. we encourage the environmental authorities from minister of environment (minae) to develop an early warning system and apply immediate management measures in new locations where this species is detected to prevent its establishment and spread. additionally, we recommend increasing research and monitoring efforts on the possible negative effects on the ecosystem of the study area and to identify other pathways that could facilitate their dispersal to new regions, mainly those related to movement by humans. our observations could serve as the basis for making microhabitat management decisions in parks or gardens in turrialba where the species represents a nuisance to its inhabitants or a threat to other native species. it would be critical to develop an environmental education program to local people from turrialba or juan viñas to avoid moving the species to new places where biotic resistance could be lesser or environmental conditions could be even more beneficial for the common coqui establishment. acknowledgments we extend our thanks to the rufford foundation for funding this project, as well as idea wild for donating equipment to carry out this investigation. we also thank the universidad de costa rica sede atlántico and the catie botanical garden for allowing us to enter the facilities, and the central conservation area of the national system of conservation areas for the respective research permits. finally, john bohrman and two anonymous reviewers provided comments improving early version of this document. 185occupancy and probability of detection of e. coqui in costa rica supplementary material supplementary material associated with this article can be found at < http://www.unipv.it/webshi/appendix> manuscript number 13209. references barrantes-madrigal, j., parallada, m.s., alvarado, g., chaves, v.j.a. 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(2013): the elephant in the room: the role of failed invasions in understanding invasion biology. oikos 122: 801-815. acta herpetologica vol. 17, n. 2 december 2022 firenze university press cryptic diversity in pygmy chameleons (chamaeleonidae: rhampholeon) of the eastern arc mountains of tanzania, with description of six new species michele menegon1,2,*, john v. lyakurwa3,4, simon p. loader5, krystal a. tolley6,7 preliminary genetic characterisation of southern smooth snake coronella girondica (serpentes, colubridae) populations in italy, with some considerations on their alpine distribution matteo r. di nicola1, raffaella melfi2, francesco p. faraone3,*, daniel l. n. iversen4, gabriele giacalone5, giovanni paolino1, mario lo valvo6 species diversity and distribution of amphibians and reptiles in sardinia, italy claudia corti1,2,*, marta biaggini1, valeria nulchis2, roberto cogoni2, ilaria maria cossu2, salvatore frau4, manuela mulargia2, enrico lunghi2, lara bassu2. the italian wall lizard, podarcis siculus campestris, unexpected presence on gorgona island (tuscan archipelago) marco a.l. zuffi1,*, alan j. coladonato2, gianluca lombardo3, antonio torroni3, matilde boschetti1, stefano scali4, marco mangiacotti2, roberto sacchi2 molecular analysis of recently introduced populations of the italian wall lizard (podarcis siculus) oleksandra oskyrko1,2,*, lekshmi b. sreelatha1,12,13, iolanda silva-rocha1, tibor sos3,4, sabina e. vlad5,6,7, dan cogălniceanu5,6, florina stănescu6,7,8, tavakkul m. iskenderov9, igor v. doronin10, duje lisičić11, miguel a. carretero1,12,13 sunny-side up: ontogenetic variation in egg mass temperatures of the wood frog rana sylvatica ryan calsbeek*, ava calsbeek, isabel calsbeek ecological niche differentiation in the anatolian rock lizards (genus: anatololacerta) (reptilia: lacertidae) of the anatolian peninsula and aegean islands mehmet kürşat şahin1,*, kamil candan2,3, danae karakasi4, petros lymberakis4, nikos poulakakis4,5,6, yusuf kumlutaş2,3, elif yıldırım2,3, çetin ilgaz2,3 occupancy and probability of detection of the introduced population of eleutherodactylus coqui in turrialba, costa rica jimmy barrantes-madrigal1,*, manuel spínola parallada1, gilbert alvarado 2, víctor j. acostachaves3,4. one site, three species, three stories: syntopy of geckoes euleptes europaea (gené, 1839), hemidactylus turcicus (linnaeus, 1758), tarentola mauritanica (linnaeus, 1758) in a coastal area of southern tuscany (central italy) giacomo radi1,2, marco a.l. zuffi1,* comparative cytogenetics on zamenis lineatus and elaphe quatuorlineata (serpentes: colubridae) marcello mezzasalma1,* , elvira brunelli1, gaetano odierna2, fabio m. guarino2 acta herpetologica 17(1): 59-70, 2022 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-11315 re-description of external morphology and factors affecting body and tail shape of the stone frog tadpoles brena da silva gonçalves1,*, carla d. hendges2, bruno madalozzo2, tiago g. santos2,3 1 universidade federal do amazonas, departamento de biologia. avenida rodrigo otávio, 6500, japiim, cep 69077000 manaus, amazonas, brasil 2 programa de pós-graduação em biodiversidade animal, universidade federal de santa maria, cep 97105-900, santa maria, rio grande do sul, brazil 3 universidade federal do pampa campus são gabriel, cep 97307-020, são gabriel, rio grande do sul, brazil *corresponding author. e-mail: brenasgoncalves@outlook.com submitted on: 2021, 8th june; revised on: 2021, 25th october; accepted on: 2022, 1st april editor: raoul manenti abstract. ecological studies testing the preponderance of environmental filters on ontogeny to explain the variation in tadpole morphology are scarce for neotropical anurans. we used tadpoles of the stone frog limnomedusa macroglossa (alsodidae): (1) to assess the variation in body and tail shape; (2) to examine the effect of streamlet depth and allometry on tadpole shape, and (3) to re-describe and compare the tadpole external morphology with closely related species. we obtained the body shape and size from 150 tadpoles. the re-description was based on 57 qualitative and 24 quantitative characters, from 19 tadpoles between stages 30 and 37 and 31 to 37, respectively. allometry was the major factor influencing the lateral view of body shape: smaller tadpoles had round bodies and eyes and nostrils positioned more laterally in comparison with larger ones. thus, the power of ontogenetic variations reported here makes the tadpole developmental “climax” period a questionable concept that deserves additional attention. the depth gradient of streamlets also affected the shape: in shallower environments, the tadpoles presented a decrease in height of the body, fins and tail muscles, and an increase in body width. these results may indicate adaptations allowing better swimming performance in lotic environments with intense water flow. the external morphological characterization of l. macroglossa presented here differed from that previously reported, mainly due to coloration, body shape, nostril, anal tube, tail, shape and position of nostrils and snout. additionally, we presented unknown traits for this species, making comparisons with closely related species within the alsodidae family. keywords. anuran larvae, alsodidae, geometric morphometrics, allometry, streamlet depth. introduction morphology is one of the main factors that influenced, perhaps all, aspects of tadpole biology (mcdiarmid and altig, 1999). for instance, the establishment of a species in a given habitat is largely influenced by morphological features such as type of oral disc, shape of body, fin presence (mcgill et al., 2006; lavorel et al., 2007; queiroz et al., 2015). therefore, morphological traits are a useful character in understanding their phylogenetic, taxonomic, ecomorphological, evolutionary and functional aspects (mcdiarmid and altig, 1999; borteiro and kolenc, 2007; barrasso et al., 2013; pezzuti et al., 2016). one of the principal uses of morphology is to help in the species description. descriptions of the external morphology of south american tadpoles are available at least since 1899, most of them revised by cei (1980). although these early descriptions are a valuable source 60 brena da silva gonçalves et alii of information for several species (the only one for some of them), they usually included on a single or few individuals, without morphometrics and detailed illustrations, thus limiting intra and interspecific morphological comparisons (borteiro and kolenc, 2007), even the taxonomic identification (rojas et al., 2018). not surprisingly, re-descriptions of the morphology of the tadpoles are increasing in the last few years (borteiro and kolenc, 2007; provete et al., 2012; barrasso et al., 2013; iop et al., 2015; pezzuti et al., 2016; rojas et al., 2018). usually based in linear measurements, only a few studies have described the shape of tadpoles obtained with geometric morphometric methods (klingenberg, 2011) as an additional factor to morphological diagnosis (haad et al., 2011; pezzuti et al., 2016). consequently, multivariate measures of size and shape of body structures used as diagnostics characters in tadpoles are still unknown to several species. this is the case of limnomedusa macroglossa (alsodidae) duméril and bibron 1841, in which the larval description is quite brief and based solely on one individual from uruguay (cei, 1980). besides of intraspecific morphological variation, essential measurements of both body and tail structures are missing (e.g., spiracle length, dorsal membrane height, nostril format, anal tube position). moreover, the shape and position of some diagnostic characters in tadpoles (e.g., shape of fins, nostril shape and mouth size) are completely unknown for this species. therefore, a re-description of tadpoles of l. macroglossa is necessary to accurately describe all these features, including intraspecific variation. the genus limnomedusa fitzinger 1834 is monospecific (blotto et al., 2013) and has been included (together with alsodes bell 1843 and eupsophus fitzinger 1843) in the family alsodidae (pyron and wiens, 2011; frost, 2020). the phylogenetic placement of l. macroglossa is historically controversial (frost, 2020). the rapid frog l. macroglossa inhabits rocky streams in southern brazil, uruguay, northeast argentina and northern paraguay (maneyro and carreira, 2012; frost, 2020). the oviposition period of l. macroglossa occurs between september and november, and larval recruitment, from september to february (kaefer et al., 2009). previous studies have found tadpoles in puddles formed on rocks in the stream bed (kwet and lingnau, 2010; maneyro and carreira, 2012) or back waters (kaefer et al., 2009). in this study, we used geometric morphometric procedures to quantify the body and tail shape and to test whether allometry and water depth affect the shape variation of tadpoles. we expect that allometry is not strong and that environmental variables (such as deeper streamlets) are more influential in form, since tadpoles are phenotypically plastic organisms in response to the environment within the developmental “climax” period (grosjean, 2005; xavier jordani et al., 2019. according to the altig and johnston (1989) guild hypothesis’ for tadpoles, lotic forms have more massive tail muscle than lentic forms, and the largest muscles are associated with lowest fins (altig and mcdiarmid, 2006). in fact, rivera-correa and faivovich (2020) described the larvae of hyloscirtus antioquia and showed morphological characters commonly associated with lotic habitats are depressed body, low fins, long tail, well-developed tail musculature, and oral disc with many labial tooth rows. although we expected this general morphological pattern in l. macroglossa, in deeper microhabitat, we also expected to find globular forms and higher fins when compared with shallow habitats where individuals will tend to be more depressed forms and low fins, due to the difference in hydrodynamics present in these environments. additionally, we re-described the external morphology of the tadpole of l. macroglossa, presenting comparisons with closely related species. materials and methods data collection we collected data from 150 tadpoles of l. macrogolossa housed in the herpetological collections of the universidade federal de santa maria, brazil (zufsm). tadpoles were collected in the area de proteção ambiental do ibirapuitã (apa) (30°51’57,41”s; 55°38’59,63”w northernmost limit and 29°57’20,52”s; 55°40’16,80”w southernmost limit), anesthetized with lidocaine 0.1% and fixed in 10% formalin solution. tadpoles were sampled in 13 streamlets during the daytime, using a collecting net with a long handle and a 3 mm metallic mesh (see details in bolzan et al., 2016; fig. 1). the sampling effort consisted by one single full scan along a 100 m section of each streamlet channel. the distance among streamlets varied from 2.97 to 90.36 km (36.79 ± 19.35; mean ± sd). the water depth was measured using a tape measure (five measures along streamlet channels) and varied from 12.4 to 40 cm (18.24 ± 5.27; mean ± sd). tadpole coloring observations were recorded during field activities at apa do ibirapuitã and municipalities of são sepé, santo cristo, and itaara. morphological measures for the larval re-description we based the re-description on 19 tadpoles with developmental stages (gosner, 1960) ranging from 31 to 61morphology and shape of the rapid frog tadpoles’ 37. fifty-seven qualitative (table s1) and 19 quantitative measurements were recorded (table 1; fig. 2), according to lavilla and scrocchi (1986), mcdiarmid and altig (1999) and altig (2007). for the tail length (tl) and body length (bl) measures we used a digital caliper (0.01 mm precision), while the others were recorded under a stereoscopic lens (0.07 mm precision), except for the upper jaw sheath width (ujsw), upper jaw sheath height (ujsh), lower jaw length (llj) and lower jaw height (hlj) measurements, for which we used a lens with 1.5 mm precision. coloration and natural history aspects were described based on field observations. geometric morphometric variation we obtained 2-dimension (2d) images of both leftlateral and dorsal body view from 150 tadpoles of l. macroglossa. by using a geometric morphometric approach, one of us (bsg) digitized 15 landmarks and 2 semilandmarks on lateral, and 9 and 5 in dorsal view to capture the left-lateral and dorsal body shape (fig. 3; table s2). the landmarks and semilandmarks were digitized using tpsdig2 ver. 2.26 (rohlf, 2015). in lateral view, we did not include landmarks in the posterior tip of the tail due to damage, predator marks and deformities observed in some specimens, which should imply errors during the digitization of landmarks or semilandmarks and posterior comparisons. only tadpoles between stages 30 and 37 were included (gosner, 1960), which represents stages within the developmental “climax” period when ontogenetic variation is expected to be low and changes in tadpole’ body parts are expected isometrics (grosjean, 2005). after digitization, the landmark and semilandmarks coordinates of each view were superimposed applying the generalized procrustes analysis (gpa, rohlf and slice, 1990). gpa generates a new set of coordinates, the procrustes coordinates, the tadpole’s body shape fig. 1. distribution of the 13 streamlets in the environmental protect area of the ibirapuitã and surroundings, where tadpoles of limnomedusa macroglossa were collected. the grey area represents boundaries of the apa of the ibirapuitã encompassing four brazilian municipalities in the state of rio grande do sul (rs): alegrete (4), rosário do sul (3), quaraí (2), and santana do livramento (1). 62 brena da silva gonçalves et alii variables. size was obtained as the centroid size; i.e., the square root of the sum of squared distances between each landmark and the configuration centroid (bookstein, 1989). we visualized the shape variation between individuals through a relative warp analysis (rwa, analogous to principal component analysis). to test for allometry, we regressed shape on log-transformed centroid size with a procrustes anova. this analysis was implemented using the function procd.lm in the r package geomorph (adams et al. 2021; r core team 2020). we explored the influence of depth of the streamlets on the shape of tadpoles by implementing a multivariate regression analysis. the geometric morphometric analyses were performed using the tpsrelw and tpsregr, respectively (rohlf, 2015). results shape variation in lateral and dorsal view the first two rwa axes summarized 57.7% of total variation of body shape in lateral view. the tadpoles with mostly positive scores on rw1 (39.64%) exhibited a proportionally smaller and more depressed body, eyes closer to nostrils, and more slender tail (fin and tail muscles), compared to the other tadpoles, with negative scores on rw1. the main variation in rw2 (18.06%) is associated with both fin and body height. on the negative scores, tadpoles have proportionally taller fins (ventral and dorsal) as well as taller and globular bodies in relation to the individuals occupying positive scores (fig. 4a). in dorsal view the first two rwa axes cumulatively explained 58.83% of total variation. the rwa segregated tadpoles proportionally more compressed laterally table 1. quantitative measures (in mm) of 19 individuals l. macroglossa between the stages 31-37, collected in the apa of ibirapuitã, rio grande do sul state, brazil. measures using 0.7 mm increase: bh – body height, bw – body width, bl body length, tl total length, nd – nostril diameter, iod – interorbital distance, nsd – nostril-snout distance, esd – eye-snout distance, ind – internasal distance, sl – spiracle length, wos – width of the opening of the spiracle, sh –spiracle height, tmh – height of the tail musculature, tmw – tail musculature width, dmh – dorsal membrane height, vmh ventral membrane height, hm -height of the mouth, wb width of the mouth. using 1.5 mm increase: ujsw – upper jaw sheath length, ujsh – upper jaw sheath height, llj – lower jaw length and hlj – lower jaw height. average and standard deviation are shown. stage and number of individuals are in main row. measure stage 31, n=2 stage 32, n=2 stage 33, n=2 stage 34, n=1 stage 35, n=4 stage 36, n=7 stage 37, n=1 tl 31.11 ± 0.26 38.21 ± 4.16 34.51 ± 1.63 38.25 42.46 ± 5.77 45.01 ± 3.86 60.22 bl 11.14 ± 0.35 13.38 ± 1.32 11.80 ± 0.09 12.82 16.0 ± 0.61 16.01 ± 1.5 20.43 bw 7.21 ± 0.1 7.9 ± 1.14 7.2 ± 0.1 8.29 10.14 ± 0.78 9.36 ± 1.23 14.43 tmw 2.42 3.01 ± 0.58 2.35 ± 0.3 3.29 4.14 ± 0.81 3.77 ± 0.46 5.86 bh 5.57 ± 0.4 6.29 ± 0.40 5.35 ± 0.3 6.43 8.28 ± 0.45 7.63 ± 0.86 11.14 dmh 2.28 ± 0.4 2.72 ± 0.59 2.14 2.43 2.64 ± 0.14 3.00 ± 0.29 4.43 tmh 2.71 ± 0.2 3.35 ± 0.50 2.71 ± 0.2 3.57 4.14 ± 0.5 3.95 ± 0.35 6.00 vmh 1.57 ± 0.2 1.62 ± 0.32 1.57 ± 0.4 1.43 1.71 ± 0.11 1.77 ± 0.11 2.29 iod 1.64 ± 0.3 1.7 ± 0.42 1.64 ± 0.1 1.86 2.50 ± 0.34 2.18 ± 0.33 2.86 ind 1.71 ± 0.2 1.62 ± 0.32 1.57 ± 0.2 1.71 1.78 ± 0.24 1.87 ± 0.12 1.71 esd 2.64 ± 0.1 2.88 ± 0.96 2.92 ± 0.1 3.00 3.85 ± 0.42 3.75 ± 0.28 5.00 nsd 1.14 1.38 ± 0.26 1.42 1.43 1.89 ± 0.29 1.79 ± 0.21 2.43 ed 1.28 1.31 ± 0.16 1.35 ± 0.1 1.43 1.53 ± 0.07 1.71 ± 0.11 2.14 nd 0.35 ± 0.1 0.36 ± 0.09 0.28 0.43 0.46 ± 0.07 0.40 ± 0.09 0.43 sl 1.42 1.6 ± 0.15 1.21 ± 0.3 1.43 1.75 ± 0.42 1.59 ± 0.30 2.00 sw 0.92 ± 0.1 0.92 ± 0.31 0.85 ± 0.2 0.86 1.28 ± 0.16 1.18 ± 0.22 1.43 wos 0.57 0.55 ± 0.22 0.57 0.71 0.92 ± 0.24 0.73 ± 0.22 1.14 sh 2.64 ± 0.5 2.99 ± 0.41 2.14 ± 0.2 2.43 3.07 ± 0.44 2.85 ± 0.56 4.43 hm 1.52 ± 0.16 1.5 ± 0.30 1.92 ± 0.3 2.71 2.28 ± 0.26 2.59 ± 0.25 2.71 wm 3.14 ± 0.28 3.57 ± 0.2 4.21 ± 0.70 5.14 4.89 ± 0.41 5.30 ± 0.61 5.71 hlj 0.13 0.13 0.16 ± 0.04 0.20 0.20 0.21 ± 0.05 0.27 llj 0.96 ± 0.04 1.06 ± 0.09 0.9 ± 0.33 1.2 1.36 ± 0.34 1.44 ± 0.16 1.67 ujsh 0.26 0.33 0.3 ± 0.04 0.33 0.31 ± 0.03 0.4 ± 0.06 0.53 ujsw 1.23 ± 0.14 1.5 ± 0.23 1.43 ± 0.14 1.33 1.63 ± 0.47 1.73 ± 0.28 2.33 63morphology and shape of the rapid frog tadpoles’ and with eyes and nostrils positioned closer to the edges of the body in the positive scores of rw1 (35.46%). in rw2 (23.37%), at the negative scores, tadpoles had proportionally smaller eyes and nostrils positioned closer to the snout and the body was more globular in the middle third of the tadpole in comparison to those at the positive scores at rw2 (fig. 4b). size showed a weak influence on the body shape of tadpoles (lateral view: r2 = 0.07, f = 11.756, p < 0.05; dorsal view: 0.05% of the variation; f = 1.40; p > 0.05). smaller tadpoles had round bodies, eyes and nostrils positioned more laterally, while larger tadpoles had more oval-shaped bodies with dorsal eyes and nostrils (fig. 5a). water depth influenced the shape variation in both views (lateral: 2.87% of the variation; f = 4.38; p < 0.05; dorsal: 2% of the variation; f = 2.89; p < 0.05). in lateral view, the general height of the body, the ventral and dorsal fins, and the tail muscles increased with water depth, while the position of eyes and nostrils becomes more dorsal. in dorsal view, the body becomes more elongated (fig. 5b). we don’t find correlation between body shape and oxygen dissolved, ph, or temperature (p > 0.05). external morphology re-description the body of l. macroglossa tadpoles is ovoid in dorsal view and depressed globular in lateral view (bh/bw: 0.79), representing one third of the total length (bl/tl: 0.35). the snout is elongated oval in dorsal view and round in lateral view; the small oval nostrils (nd: 0.40 ± 0.08; mean ± sd) with thin edges are dorsolateral and equidistant from the snout and eyes (nsd: 1.67 ± 0.35 and esd: 3.50 ± 0.66). the internasal distance (ind: 1.77 ± 0.19) is smaller than that of the interocular distance (iod: 2.11 ± 0.44). the eyes are dorsal (ed: 1.54 ± 0.24), with dorsolateral orientation. the spiracle is sinistral, long, cylindrical, with a wide opening (wos: 0.74 ± 0.21, free, short and fig. 2. representation of the measures carried out in the larvae of limnomedusa macroglossa used for description of external morphology in dorsal and lateral view (sensu lavilla and scrocchi (1986), mcdiarmid and altig (1999) and altig (2007)): bh = body height, sh = spiracle height, tmh = tail musculature height, vmh = ventral fin height, dmh = dorsal fin height, bl= body length, sl = spiracle length, tl = total length, ed = eye diameter, nd = nostril diameter, ind = internal distance, iod = interorbital distance, esd = eye-snout distance, nsd = nostril-snout distance, wos = spiracle aperture width, sw = spiracle width, bw= body width, tmw = tail musculature width. 64 brena da silva gonçalves et alii posterodorsally oriented tip) (sh: 2.89 ± 0.61). the anal tube is long, connected to the ventral fin, medially positioned, and with a dextral opening. in dorsal view, the width of the tail muscles is greater than one third of body width (tmw: 3.55 ± 0.94, bw: 9.11 ± 1.82), with a gradual tapering, and a wide rounded tip in lateral view. dorsal and ventral fins are low and parallel to the body (dmh: 2.77 ± 0.54, vmh: 1.70 ± 0.22). the dorsal fin gently starts at the junction of the tail and body. the oral disc is proportionally large (wm/bw: 0.50 and hm/bl: 0.14), anteroventral, laterally emarginated, with a broad dorsal gap, a double row of elongated papillae, and dispersed lateroventral and laterodorsal submarginal papillae. the superior jaw has a ventral recess and width four times greater than its height (ujsw/ujsh: 4.56). the lower jaw is v-shaped and wider than the taller (llj/hlj: 6.73). the serrations of the jaws are long with a narrow base. the oral formula is 2(2)/3(1), where p3 is slightly smaller than p2 and p1 (fig. 6). the side-line system is not visible. colouration in live animals, the dorsal region of the body is greybrown, the lateral portion of the body is golden brown, fig. 3. position of the landmarks and semilandmarks on the lateral (a) and dorsal view (b) of the tadpole of limnomedusa macroglossa. landmark and semilandmarks descriptions are in table s2. 65morphology and shape of the rapid frog tadpoles’ with darker shades around the nostrils and eyes. the iris is golden with a vertical pupil. in ventral view, silver pigmentation is observed in the abdominal region, but decreases around the oral disc. tail muscles are yellowish with golden brown pigmentation throughout their length, mainly in the dorsal region. fins are translucent, with fig. 4. scatter plot of rw1 vs. rw2. the deformation grids demonstrate the average deformation of the shape in lateral view (a) and dorsal view (b), at the positive and negative scores of the relative warp axes for tadpoles of limnomedusa macroglossa. 66 brena da silva gonçalves et alii clusters of evenly scattered melanophores. after fixing in 10% formalin, the colour of the body changes to a greyish brown and the eyes become black. fig. 5. shape deformations related to the multivariate regression going from the most extreme negative score (left) to the most positive scores (right). in (a) deformations are from the effect of allometry. in (b) from the depth influence in the lateral and dorsal views of tadpoles of limnomedusa macroglossa. 67morphology and shape of the rapid frog tadpoles’ discussion shape variation in lateral and dorsal view this study provides the first description of the body and tail shape of l. macroglossa tadpoles. the shape variation is mainly explained by allometry for much of the larval period (i.e., between stages 30 and 37). small tadpoles have a rounder body, eyes and nostrils positioned more laterally and, as the body size increases, the tadpole body becomes more oval with eyes and nostrils more dorsal. among the several factors that contribute to the morphological variation in anurans, changes in body size (allometric) associated with development have a strong effect on anatomical forms (e.g., di cerbo and biancardi, 2010; garriga and llorente, 2012; acosta and candioti, 2017). in fact, the effects of allometry on anuran larvae was verified for both external (e.g., di cerbo and biancardi, 2010; garriga and llorente, 2012; acosta and candioti, 2017) and internal larval morphology (e.g., larson, 2002, 2004, 2005; garriga and llorente, 2012), but was expected to be low within the developmental “climax” period, when changes in tadpole’ body parts are expected to be isometrics (grosjean, 2005; xavier jordani et al., 2019). our results also reveal variation in the shape associated with the depth gradient among streams. in lateral view, tadpoles vary in height and body shape (i.e., from depressed to high), position of the eyes and nostrils (close to each other or near the sides of the body), height and width of tail muscles (thin or well developed) and fins (high or low). in dorsal view, the variation observed was associated with body shape (e.g., compressed laterally or globular) and position of eyes and nostrils (e.g., lateral or dorsal). these characteristics show the refined responses of the tadpoles to changes in the habitats, especial the tail and body characteristics (grosjean, 2005; xavier jordani et al., 2019), since morphology should provide an optimal swimming performance in an occupied habitat (pinto and ávila-pires, 2004; marques and nomura, 2015; xavier jordani et al., 2019). we can’t exclude the tadpoles autonomy to access different water depths according to your preferences to abiotic variations on streamlets (warkentin, 1992). according to johnson et al. (2008) and arendt (2010), depth is associated with the selective pressure exerted by the lotic environment on organisms, since the water flow is lower in deeper streams, while in shallow streams, currents are more intense. in the latter, a hydrodynamic body shape is advantageous to minimize drag and allow the animal to move (e.g., increase in tail height and width, reduction in body height, and increase in structures for attachment; arendt, 2010). external morphology re-description. the description presented in our study differs from the previously reported, mainly regarding the following aspects: coloration, body shape, nostrils, anal tube, tail, shape and position of nostrils and snout. in the description by cei (1980), coloration was briefly characterized as “dorsum and tail with dark round spots”, but it does not mention whether this trait was observed in the live specimen or after being fixed. body shape, previously reported as “depressed oval”, differed from that observed in the present study (i.e., ovoid shape in dorsal view and rounded depressed in lateral view). in agreement with the described by cei (1980), the snout of l. macroglossa is round (but only in lateral view), while it has an elongated oval shape in dorsal view. the internasal distance, previously characterized as equidistant from the interocular distance, differs from our findings (i.e., internal distance less than interocular distance). the anal tube, previously described as having a median aperture, differed from that reported here (dextral). besides, we added information for both the shape and position of the anal tube (long and connected to the ventral fin, respectively). the tail muscles are well developed in lateral and dorsal view, as also previously described, gradually tapering into a round and wide tip. fig. 6. tadpole of limnomedusa macroglossa of stage 33: (a) in lateral view (scale 1 cm); (b) in dorsal view (scale 1 cm); (c) oral disc (scale 1mm). drawings by b.s.g. 68 brena da silva gonçalves et alii we added information on the spiracle, which is long cylindrical, ending with a wide opening and free, short, and posterodorsally oriented tip. we described fin shape, emergence angle, and body attachment site, previously unavailable. the pattern of oral formula 2(2)/3(1) agrees with the previously reported, and additional undescribed characters are presented, such as the proportion, position, arrangement, and shape of papillae, as well as oral disc size, and jaw size. for some groups, morphology is widely used as a parameter to reconstruct phylogenetic relationships among species groups (marques and nomura, 2015). thus, future comparisons between l. macroglossa tadpoles and those of the other two genera in the alsodidae family are relevant to understanding the evolution of the group. both aldoses and eupsophus are endemic to beech forests (notophagus spp.) in the patagonian region of chile and argentina (formas and cuevas, 2017; frost, 2020; iucn, 2019) and therefore have a parapatric distribution to l. macroglossa. two modes of obtaining energy have been described for larval development in alsodes and eupsophus: (i) endotrophic tadpoles (i.e., tadpoles obtain energy entirely from maternal energy sources, usually yolk, to become free-living juveniles), and (ii) exotrophic tadpoles (i.e., the energy required for development is ingested by free-living larvae after yolk reserves are depleted; altig and mcdiarmid, 1999). eupsophus larvae differ the most from l. macroglossa tadpoles. according to candioti et al (2011), the eupsophus species is classified as having endotrophic tadpoles that develop in a nest (altig and mcdiarmid, 1999), and are considered uncommon as both eggs and larvae develop in small dark chambers (= burrows) filled or no by water, near streams or flooded areas. on the other hand, alsodes, despite having exotrophic benthic tadpoles (formas and cuevas, 2017) as reported for l. macroglossa, use small waterfilled cavities near streams for larval development (e.g., alsodes vittatus; glime and boelema, 2017) and differ from l. macroglossa tadpoles mainly by the smaller oral disc, a single row of submarginal papillae (also arranged in a single row or clustered in the supra-angular region), smaller eyes and larger fins with a rounded tip. the dissimilarity between l. macroglossa, eupsophus and alsodes tadpoles seem related to the still poorly resolved phylogeny for this group. in fact, the phylogenetic placement of l. macroglossa is historically controversial (frost, 2020). a molecular study did not find a particularly close relationship between limnomedusa and the eupsophus + alsodes clade, but rather a proximity to cycloramphus (cycloramphidae; blotto et al., 2013). recently, sabbag et al. (2018) recovered l. macroglossa as taxon sister of odontophrynidae. thus, according to frost (2020), the inclusion of limnomedusa within alsodidae is provisional and requires further investigation (but see support to alsodidae as a monophyletic group by grant et al., 2017). acknowledgements we are grateful to sisbio-icmbio (instituto chico mendes de conservação da biodiversidade) for providing the collection permit (license #33975/march 2012). we are also grateful to the anonymous reviewers for their comments and suggestions on the manuscript. supplementary material supplementary material associated with this article can be found at <http://www-9.unipv.it/webshi/appendix/ index.html> manuscript number 11315 references acosta, g.n., candioti, f.v. 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(2019): intraspecific and interspecific trait variability in tadpole meta‐communities from the brazilian atlantic rainforest. ecol. evol. 9: 4025-4037. xi international symposium on the mediterranean lacertid lizards marco mangiacotti1, pietro lo cascio2, claudia corti2, marta biaggini2, miguel angel carretero2, petros lymberakis2 the directional testes asymmetry increases with temperature in seven plateau brown frog (rana kukunoris) populations hai ying li1, man jun shang2, jie guo2, bo jun chen2, peng zhen chen2, tong lei yu1,* influence of tail injury on the development of neotropical elegant treefrog tadpoles ana glaucia da silva martins1,#, raoni rebouças2,3,*,#, isaias santos1, adão henrique rosa domingos1, luís felipe toledo2 the effect of weight and prey species on gut passage time in an endemic gecko quedenfeldtia moerens (chabanaud, 1916) from morocco jalal mouadi1,*, panayiotis pafilis2, abderrafea elbahi3, zahra okba3, hassan elouizgani3, el hassan el mouden4, mohamed aourir1 a contribution to the knowledge on the diet and food preferences of darevskia praticola (reptilia: lacertidae)§ emiliya vacheva*, borislav naumov first report on two loggerhead turtle (caretta caretta) nests in the aeolian archipelago (southern italy) monica francesca blasi1,*, sandra hochscheid2, roberta bardelli3, chiara bruno1, carolina melodia1, perla salzeri1, paolo de rosa4 and paolo madonia5 threatened and extinct amphibians and reptiles in italian natural history collections are useful conservation tools franco andreone1,*, ivano ansaloni2, enrico bellia3, andrea benocci4, carlotta betto5, gabriella bianchi6, giovanni boano7, antonio borzatti de loewenstern8, rino brancato9, nicola bressi10, stefano bulla11, massimo capula12, vincenzo caputo barucchi13, p re-description of external morphology and factors affecting body and tail shape of the stone frog tadpoles’ brena da silva gonçalves1,*, carla. d. hendges2, bruno madalozzo2, tiago g. santos2,3 preliminary data on the diet of chalcides chalcides (squamata: scincidae) from northern italy andrea ciracì1, edoardo razzetti2, maurizio pavesi3, daniele pellitteri-rosa4,* the high diversity and phylogenetic signal of antipredator mechanisms of the horned frog species of proceratophrys miranda-ribeiro, 1920 (amphibia: anura: odontophrynidae) cássio zocca1,2,*, ricardo lourenço-de-moraes3, felipe s. campos4, rodrigo b. ferreira1,2,5 amphibian and reptile communities in eleven sites of community importance (sci): relations between sci area, heterogeneity and richness luca canova1, manuela marchesi2 1dipartimento di biologia animale, università di pavia, piazza botta 9, i-27100 pavia, italy. corresponding author. e-mail: canova@unipv.it 2assessorato ambiente, provincia di lodi, via grandi 6, i-26800 lodi, italy abstract. seven species of amphibians and reptiles were observed in eleven sites of community importance (sci) of the lodi province (nw italy). distribution and relative abundance of amphibians appeared more variable than reptiles. some species of conservation concern as r. latastei were influenced by habitat physiognomy, i.e. the surface of wooded areas are important in predict presence and relative abundance of this species. sci with wider surfaces and higher habitat heterogeneity included higher number of species. species richness, here considered as a raw index of biodiversity value and community quality, was significantly related to sci area and habitat heterogeneity; since this significant positive relation is confirmed both for amphibians and reptiles we suggest that, in planning of natural areas, priority must be retained for biotopes able to host the higher number of species. keywords. amphibians, reptiles, sci, richness, habitat heterogeneity, italy. introduction animal communities are the results of two main differing forces; the ecological forces determine the stability of an assemblage, while the evolutive forces determine the pattern of coexistence between different populations (stenseth, 1989). communities parameters such as richness and species diversity are moreover influenced by several factors such as habitat surface and heterogeneity, and by a number of limiting factors, as management at ecosystem level, or vulnerability to allogenic invasions (krebs, 2001). the indipendent analysis of these factors is often very difficult as well as their influence on animal community; scaling problem, for example, is one of the most difficult to resolve in several vertebrate community (webb, 1989). amphibian and, to a lesser extent, reptile communities are known to be vulnerable to several external enviromental factors such as managed alteraacta herpetologica 2(2): 87-96, 2007 issn 1827-9643 (online) © 2007 firenze university press 88 l. canova and m. marchesi tion of primaeval habitats (ryan et al., 2002; ernst et al., 2006), fragmentation (russell et al., 2002), pollution (lebboroni et al., 2006), infections (bosch and martinez-solano, 2006; rachowicz et al., 2006), and to wide-scale global change (pounds et al., 2006). due to this vulnerability, amphibians are considered as a good primer in evaluating habitat quality (rondinini and boitani, 2006). all these factors should be taken into account when amphibian/reptilian community parameters are investigated in protected areas, and when a correct planning should include patches size and habitat heterogeneity, aimed at improving conservation effort and its output (freemark et al., 2006; garcia, 2006). the aim of this study is to: (1) check for presence and abundance of amphibian and reptilian communities in the eleven sites of community importance of the lodi province, (2) describe relationships between community richness, patches size and habitat heterogeneity, and (3) check the suitability of the current asset of sci of the lodi province for amphibian and reptilian conservation. materials and methods the study was carried out from april 6, to july 29, 2003 in the eleven sites of community importance of the lodi province (nw italy, fig. 1, table 1). a systematic sampling system (sss; scott, 1994) was adopted during this study; in each biotope we conducted sistematic research aimed at identifying species, location and their relative abundance. a period of 60 min was adopted as standard effort time and at least a research session was carried out weekly in each biotope from late march to late july. species were identified by means of capture, direct fig 1. the eleven sci surveyed: (1) boschi e lanca di comazzo, (2) garzaia del mortone, (3) bosco del mortone, (4) garzaia della cascina del pioppo, (5) spiagge fluviali di boffalora , (6) lanca di soltarico, (7) la zerbaglia, (8) morta di bertonico, (9) adda morta, (10) bosco valentino, (11) monticchie. 89amphibian and reptile communities in eleven sci observation, collection of dead individuals, shed skins pellets or scats analysys. the total sampling effort was 170 h; sampling effort was equally distributed among biotopes in relation to their area. relative abundance calculated by sss was used in comparative analysis among species abundance. we calculated as richness (r) the number of species recorded during the study period in each biotope and as habitat eterogeneity a shannon-wiener index (h=-σ pi log pi ) where pi is is the proportion of the partial area of the habitat i on the total area of the biotope (ahabitat/abiotope ) and a is the area covered by each habitat and biotope. data analysis were carried out by spss 10.1 package and microsoft excel. relative surface of habitat, coordinates and eu codes for each sci were obtained from the regione lombardia archive data. results communities and local distribution a total of seven species of amphibians (pelophylax synkl. esculenta, pseudoepidalea viridis, rana latastei, bufo bufo, hyla intermedia, triturus carnifex, lissotriton vulgaris) and seven species of reptiles (podarcis muralis, anguis fragilis, lacerta bilineata, hierophis viridiflavus, natrix natrix, natrix tessellata, zamenis longissimus) were recorded in our study areas; taxonomical attribution are from frost et al. (2006). among amphibians p. synkl. esculenta is the most euritopic species and, together with r. latastei, p. viridis and h. intermedia are widely distributed in nearly all the study areas (table 2); this data appeared of good conservation value, since r. latastei is an endemic species of the po plain. the other amphibian species are irregularly distributed and newts were found only in few sites with permanent ponds (table 2). among reptiles p. muralis and n. natrix are the most euritopic species and, together with l. bilineata and h. viridiflavus, are widely distributed, as showed by evennes index table 1. locations, eu code and igm coordinates and surface (in ha) of the 11 sci under study. sci name eu code igm coordinates area (ha) 1 boschi e lanca di comazzo it2090002 45°25’44.36’’n; 9°27’48.69’’e 267 2 garzaia della cascina del pioppo it2090005 45°22’21.05’’n; 9°26’49.85’’e 6 3 bosco del mortone it2090003 45°23’17.17’’n; 9°27’07.99’’e 63 4 garzaia del mortone it2090004 45°23’21.43’’n; 9°26’09.86’’e 35 5 spiagge fluviali di boffalora it2090006 45°21’04.84’’n; 9°28’34.60’’e 172 6 lanca di soltarico it2090007 45°17’17.80’’n; 9°35’04.21’’e 170 7 la zerbaglia it2090008 45°16’41.10’’n; 9°38’25.85’’e 552 8 morta di bertonico it2090009 45°15’17.92’’n; 9°39’45.17’’e 80 9 adda morta it2090011 45°13’17.80’’n; 9°42’09.18’’e 191 10 bosco valentino it2090011 45°12’34.20’’n; 9°45’48.99’’e 48 11 monticchie it2090001 45°08’41.16’’n; 9°39’27.91’’e 238 90 l. canova and m. marchesi ranging form 1 to 0.82 in nearly all the study areas; the other species are irregularly distributed or strongly localized in a single biotope as z. longissimus (table 2). species detectability and abundance differences the frequency of observation/recording was higher from april to june than in july, both for amphibians and reptiles. all amphibians were monthly detected from march to june while detectability of reptiles was slightly lower and not all species were found in the same period; observation rate slightly decreased in the last month of surveys for both taxa. data analysis showed remarkably differences in relative amphibians abundance (fig. 2); the most widespread p. synkl. esculenta is the most abundant specie (mean = 25.5 ind/ h), while abundance of r. latastei, despite its wide local distribution, is constantly less table 2. local distribution of species. evennes index showed how wide species distribution is (index range from 1 for euritopic species to 0 for stenotopic species). 1 b os ch i e la nc a di c om az zo 2 g ar za ia d el m or to ne 3 b os co d el m or to ne 4 g ar za ia d el la c as ci na d el pi op po 5 sp ia gg e flu vi al i d i b off al or a 6 la nc a di s ol ta ri co 7 la z er ba gl ia 8 m or ta d i b er to ni co 9 a dd a m or ta 10 b os co v al en tin o 11 m on tic ch ie ev en ne s pelophylax synkl. esculenta + + + + + + + + + + + 1 rana latastei + + + + + + + + + + 0.91 pseudoepidalea viridis + + + + + + + + + 0.82 bufo bufo + + + + + 0.45 hyla intermedia + + + + + + + 0.64 triturus carnifex + + + 0.27 lissotriton vulgaris + + 0.18 podarcis muralis + + + + + + + + + + + 1 lacerta bilineata + + + + + + + + + + 0.91 anguis fragilis + + + 0.27 natrix natrix + + + + + + + + + + + 1 hierophis viridiflavus + + + + + + + + + 0.82 natrix tassellata + + + + + 0.45 zamenis longissimus + 0.09 91amphibian and reptile communities in eleven sci abundant (mean = 3.18 ind/h). the abundance of the former species is apparently unrelated to habitat physiognomy, while r. latastei abundance increased to increasing of wooded areas (rs = 0.82, n = 10, p = 0.03). abundance pattern showed by h. intermedia and p. viridis, the only other amphibian species for which enough data were collected, is somewhat erratic; p. viridis appeared a localised species with very scarce populations, while h. intermedia is more widely distributed, indipendently of the amount of wooded habitat (rs = 0.44, n = 10, p = 0.20, fig. 2). reptiles abundance appeared less variable; podarcis muralis is the most abundant species in all the habitats and, together with l. bilineata, is widespread in the study area. snakes are widely distributed, showing a very low relative population abundance (fig. 3). relation between richness and habitat area and heterogeneity data showed that pooled species richness and biotope area were significantly and positively related (richness = 0.45 × ln(area) + 0.63, r2 = 0.73, p = 0.004; fig. 4a), and furthermore that a significant relation existed considering richness of each individual group, fig. 2. relative abundance of rana latastei, pelophylax synkl. esculenta, hyla intermedia and pseudoepidalea viridis. 92 l. canova and m. marchesi (i) amphibians (richness = 0.59 × ln(area) + 0.23, r2 = 0.69, p = 0.004), and (ii) reptiles (richness = 0.38 × ln(area) + 0.39, r2 = 0.72, p = 0.003). moreover, we observed a significant relation between pooled species richness and habitat diversity (richness = 0.45 × ln(habitat diversity) + 1.11, r2 = 0.58, p = 0.02), and that such relation was significant, thought with a lower predictive value, when considering separately amphibians (richness = 0.51 × ln(habitat diversity) + 0.38, r2 = 0.52, p = 0.027) and reptiles (richness = 0.34 × ln(habitat diversity) + 0.78, r2 = 0.47, p = 0.032, fig. 4b). discussion following previous research and surveys, the presence of nine amphibians and eleven reptile species had been verified in the southern po plain (lodi province, bernini et al., 2004a). during the current study pelobates fuscus and rana dalmatina were not recorded among amphibians, and emys orbicularis, podarcis sicula, coronella austriaca and vipera aspis among reptiles. in summary, current results showed that nearly 78% of known fig. 3. relative abundance of podarcis muralis, lacerta bilineata, natrix natrix and hierophis viridiflavus. 93amphibian and reptile communities in eleven sci amphibians and 63% of known reptiles are present in the sci. this data can be interpreted in two alternative ways: first, sss method might fails to detect the most elusive species or secondly, sss method might reflect the actual relative abundances of species. the prevalence of unrecorded species among reptiles probably reflected both their local rarity (such as for e. orbicularis; chelazzi et al., 2000; ferri and zuffi, 2004), low detectability (e.g. c. austriaca) as well as patterns of local distribution (p. sicula and v. aspis are known to be present in areas out of the current sci network). species detectability is high during the spring season and showed a slight decrease in july, the warmer month; at our latitude the sss method appeared to be more profitable in early spring than in summer (probably from late winter, i.e. february, for some species as r. latastei and r. dalmatina that are early breeders and this suggestion can be assumed as a methodological improvement at least for the italian northern distributive area of the species under study. detailed analysis on population abundance are clearly precluded by speditive methods adopted during the presents study; only a few species as p. synkl. esculenta and p. muralis fig. 4. relation between richness (r) versus sci area (above) and versus structural diversity (h) of biotopes (below) (data are log transformed). 94 l. canova and m. marchesi are relatively common. relative abundance of other species as p. viridis, appeared lower than expected; in some sectors of the po plain p. viridis is considered a declining species, as previously reported for some european populations (honegger, 1981); the most common threaths to its conservation are identified in the reduction of temporary pond (bonini and bressi, 2004), and this can partially explain the reduction of the species’ population over large areas. we suggest that a general reduction of ponds along the main course of a number of large rivers, and the late egg-laying period (may-june), can heavily influence the reproductive performances of p. viridis in the dry flood plain of the adda river, an habitat dynamics not shared by other amphibian species which reproduced earlier. similar conditions are shared by b. bufo, apparently a species very scarce in our study areas, whose reduction could it be possibly influenced by the delayed effects of agricolture intensive practices (pavignano and giacoma, 1990). the pattern of local distribution of some species, as r. latastei, appeared influenced by habitat physiognomy; the relative abundance of this species is significantly related to the area of wooded patches, a data confirming current informations on its ecology (barbieri and bernini, 2004a); in our study areas r. latastei is abundant in patches of ancient oak woodland (bernini et al., 2004), characterized by high pond availability in early spring; if temporary ponds are available at the start/beginning of breeding season, the species can colonize poplar groves and seminatural wooded habitats. analysis of relation between community parameters and habitat size and physiognomy showed that sci with wider/larger surfaces and higher habitat heterogeneity included an higher number of species. it is well known that biodiversity cannot be expressed only by richness index, since quantitative parameters as abundance and density, and genetic characteristics of populations should be taken into account as well; however richness index can be considered as a raw, sinthetic index of community quality. since the significant positive relation between richness and habitat area, and heterogeneity is confirmed separately both for amphibians and reptiles, we suggest that, in planning of protected areas, priority must be retained for large and heterogeneous biotopes. in this framework, however, a passive protection scheme can be unsuitable to guarantee a safety level of protection for some species; more resources must be devoted in paying attention to both conservation measures and obtained results (watzold and schwerdtner, 2005). despite of the possible failure of sss methodology in elusive species detection, we must take into account the actual function of natura 2000 in protecting local biodiversity. our data did not exclude that the establishment of a continental network of protected areas did not ensure a good level of conservation on local scale, at least for some species of reptiles. in this framework an higher effort in looking for additional spatial and biological indicators is then auspicable (bock et al., 2005; papageorgiou and vogiatzakis, 2006). acknowledgements thanks are due to owners of sci for access permission and facilities and to several students who helped us in data collecting. a. gentilli read a preliminar proof of this paper, providing useful suggestions. data on sci habitat came from the assessorato regionale qualità dell’ambiente, regione lombardia. thanks are due to provincia di lodi, assessorato pianificazione ambientale for support. 95amphibian and reptile communities in eleven sci references barbieri, f., bernini, f. 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(2006): species richness and biodiversity conservation priorities in british columbia, canada. can. j. zool. 84: 20-31. frost, d.r., grant, t., faivovich, j., bain, r.h., haas, a., haddad, c.f.b., de sà, r.o., channing, a., wilkinson, m., donnellan, s.c., raxworthy, c.j., campbell, j.a., blotto, b.l., moler, p., drewes, r.c., nussbaum, r.a., lynch, j.d., green, d.m., wheeler, w.c. (2006): the amphibian tree of life. bull. am. mus. nat. hist. 297: 1-371. garcia, a. (2006): using ecological niche modelling to identify diversity hotspots for the herpetofauna of pacific lowlands and adjacent interior valleys of mexico. biol. cons. 133: 25-46. honegger, r.e. (1981): threatened amphibians and reptiles in europe. in: handbuch der reptilien und amphibien europas, p. 158-159. böhme, w., ed, aula-verlag, wiesbaden. lebboroni, m., ricchiardino, g., bellavita, m., chelazzi, g. (2006): potential use of anurans as indicators of biological quality in upstreams of central italy. amphibiareptilia 27: 73-79. krebs, c.j. (2001): ecology: the experimental analysis of distribution and abundance. benjamin/cummings, philadelphia. 96 l. canova and m. marchesi papageorgiou, k., vogiatzakis, l.n. (2006): nature protection in greece: an appraisal of the factors shaping integrative conservation and policy effectiveness. env. sci. pol. 9: 476-486. pavignano, i., giacoma, c. (1990): a multivariate analysis of amphibian habitat determinants in north western italy. amphibia-reptilia 11: 311-324. pounds, j.a., bustamante, m.r., coloma, l.a., consuegra, j.a., fogden, m.p.l., foster, p.n., la marca, e., masters, k.l., merino-viteri, a., puschendorf, r., ron, s.r., sanchez-azofeifa, g.a., still, c.j., young, b.e. (2006): widespread amphibian extinctions from epidemic disease driven by global warming. nature 439: 161-167. rachowicz, l.j., knapp, r.a., morgan, j.a.t., stice, m.j., vredenburg, v.t., parker, j.m., briggs, c.j. (2006): emerging infectious disease as a proximate cause of amphibian mass mortality. ecology 87: 1671-1683. rondinini, c., boitani, l. (2006): differences in the umbrella effects of african amphibians and mammals based on two estimators of the area of occupancy. cons. biol. 20: 170-179. russell, k.r., hanlin, h.g., wigley, t.b., guynn, d.c. (2002): responses of isolated wetland herpetofauna to upland forest management. j. wild. manag. 66: 603-617. ryan, t.j., philippi, t., leiden, y.a., dorcas, m.e., wigley, t.b., gibbons, j.w. (2002): monitoring herpetofauna in a managed forest landscape: effects of habitat types and census techniques. forest ecol. manag. 167: 83-90. scott, j. (1994): complete species inventories. in: measuring and monitoring biological diversity. standard method for amphibians, p. 78-84. heyer, r.w., donnelly, m.a., mcdiarmid, r.w., hayek, l.a.c., foster, m.s., eds, smithsonian university press, washington. stenseth, n.c. (1989): on the evolutionary ecology of mammalian communities. in: patterns of the structure of mammalian communities, p. 219-229. morris, d.w., abramsky, z., fox, b.j., willig, m.r., eds, texas tech univ. press, lubbock. watzold, f., schwerdtner, k. (2005): why be wasteful when preserving a valuable resource? a review article on the cost-effectiveness of european biodiversity conservation policy. biol. cons. 123: 327-338. webb, s.d. (1989): the fourth dimension in north american terrestrial mammal communities. in patterns of the structure of mammalian communities, p. 181-203. morris, d.w., abramsky, z., fox, b.j., willig, m.r., eds, texas tech. univ. press, lubbock. complete albinism in a podarcis muralis newborn filippo spadola1, francesco di toro2 1facoltà di medicina veterinaria di messina, università degli studi di messina, viale annunziata, i98168 messina, italy. corresponding author. e-mail: filippo.spadola@unime.it 2facoltà di medicina veterinaria, università degli studi di teramo, italy abstract. the authors describe a case of complete albinism in a podarcis muralis newborn, from chieti (abruzzo, central italy) in september 2004. this is the first complete albinism case in a podarcis spp. in the world. keywords. podarcis muralis, albinism, morphology. albinism is a genetic anomaly caused by the absence of the tyrosinase enzyme, which is necessary for the synthesis of melanin (recessive gene), resulting in the absence of pigment in the skin, the iris (red color of the eyes) and the choroid. the albinos (homozygotes) of a visibly weaker constitution, have notably sun-sensitive skin (heliophobic), causing them much difficulty during their growth due to the impossibility of exposure to uv rays. cases of albinism reported for european reptiles are very few. none of them has been recorded in italy regarding lizards, and albinism has been recorded only in a few snakes: the first one dates back to 1878 (pirotta, 1878) and the most recent for the colubrid zamenis longissimus longissimus, which lacked absolutely melanin, and showed the characteristic light skin and red eyes (ferri and bettiga, 1992), and for hierophis viridiflavus (scali, 1992). concerning records of albinism in european lizards, cases of albinism have been reported for timon lepidus (arribas and cliville, 1994), gallotia caesaris gomerae (lópez-jurado and mateo, 1998), euleptes europaea (delaugerre, 1981), anguis fragilis (robert et al., 1965), and podarcis muralis (fontanet and matallanas, 1985). outside of europe, cases of albinism have been found in eumeces fasciatus (brungs et al., 1960), phrynosoma coronatum blainvillii (shaw, 1963), sceloporus undulatus hyacintninus (hensley, 1968), corytophanes hernandezi (pérez-higareda, 1980), tiliqua sp. (o’shea and kent, 1988), ctenosaura similis (mora, 1990), carlia bicarinata (o’shea, 1993), sauromalus obesus (beaman et al., 2003), varanus kingorum (eidenmüllser, 2003). our work reports the first case of complete albinism in a newborn podarcis muralis found in the province of chieti (abruzzo, central italy), in september 2004. it was about 6 cm long and had a light yellow skin over a completely white background and red eyes. it was born in 2004, and seemed undernourished, but not sick at first glance. the site of collection is located in a hilly zone (altitude: 180 m a.s.l.) between the sangro and aventino. the most common vegetation is swamps with an extensive reed thicket with typha laxmanni, epipactis palustris, alnus glutinosa, and quercus peduncolata. acta herpetologica 2(1): 49-51, 2007 50 f. spadola and f. di toro fig. 1. the newborn albino p. muralis found near chieti. fig. 2. dorsal view of the above individual. 51complete albinism in podarcis muralis acknowledgements we wish to thank guido de virgiliis and giovanni vespia for their cooperation. references arribas, o., cliville, s. (1994): albinismo en lacerta lepida daudin, 1802 (reptilia: lacertidae). bol. asoc. herpetol. esp. 5: 20-23. beaman, k.r., toohey, d.r., crandall, g.t. (2003): sauromalus obesus (common chuckwalla). albinism. herpetol. rev. 34: 246. brungs w.a., jr., britt, n.w. (1960): an albino five-lined skink, eumeces fasciatus, linnaeus. copeia 1960: 369-370. delaugerre, m. (1981): cas d’albinisme chez phyllodactylus europaeus gené, 1838: 1er cas signalé dans la famille des gekkonidae (sauria, reptilia). bull. soc. linn. lyon 50: 213-216. eidenmüllser, von b. (2003): haltung und kontinuierliche vermehrung von kings felsenwaran, varanus kingorum (storr, 1980), mit der erstmaligen nachzucht eines albinotischen jungtieres. reptilia 43: 36-40. ferri, v., bettiga, m. (1992): un caso di albinismo nel colubro di esculapio, elaphe l. longissima (laurenti, 1768). il naturalista valtellinese, atti mus. civ. st. nat. morbegno 3: 91-96. fontanet, x., matallanas, j. (1985): nota sobre un cas d’albinisme parcial en podarcis muralis; (laurenti 1768) (sauria, lacertidae). butll. soc. catal. ictiol. herpetol. 11/13: 25-28. hensley, m. (1968): another albino lizard, sceloporus undulatus hyacinthinus (green). j. herpetol. 1: 92-93. lópez-jurado, l.f., mateo, j.a. (1998): albinismo en gallotia caesaris gomerae. bol. asoc. herpetol. esp. 9: 33-34. mora, j.m. (1990): an instance of albinism in the ctenosaur lizard, ctenosaura similis, in costa rica. bull. chicago herpetol. soc. 25: 70. o’shea, m.t. (1993): carlia bicarinata (bicarinate four-fingered skink). partial albinism. herpetol. rev. 24: 59. o’shea, g.m., kent, d.s. (1988): albinism in blue-tongued lizards (scincidae: tiliqua). herpetofauna 18: 3-4. pérez-higareda, g. (1980): albinism in corytophanes hernandezi (lacertilia: iguanidae). bull. maryland herpetol. soc. 16: 97-98. pirotta r. (1878): di alcuni casi di albinismo nei rettili. atti soc. ital. sci. nat. mus. civ. st. nat. milano 21: 448. robert, j., nicolete cl.,, guyard, a. (1965): sur un cas d’albinisme chez l’orvet, anguis fragilis. bull. soc. hist. nat. doubs 67: 27-29. scali, s. (1992): caso di colorazione anomala nel biacco (coluber viridiflavus lacépède, 1789). atti soc. ital. sci. nat., museo civ. stor. nat. milano 133: 294-295. shaw, c.e. (1963): an albino san diego horned lizard (phrynosoma coronatum blainvillii). copeia 1963: 154. acta herpetologica 4(2): 177-182, 2009 a new finding of rhynchocalamus barani, baran’s black-headed dwarf snake (reptilia, colubridae), in the mediterranean region of turkey widens its distribution range aziz avcı1, nazan üzüm1, çetin ilgaz2, kurtuluş olgun1 ¹ adnan menderes university, science and art faculty, department of biology, tr-09010, aydın, turkey. corresponding author. e-mail: ntaskin@adu.edu.tr ² dokuz eylül university, fauna and flora research and application center, 35160, buca-i̇zmir, turkey. submitted on: 2009, 6th june; revised on 2009, 16st september; accepted on 2009, 25th september. abstract. rhynchocalamus barani olgun, avcı, ilgaz, üzüm and yılmaz, 2007 is a small colubrid snake previously known only from the type locality, 34 km east of dört-yol, hatay province from mediterranean region of turkey. a second new locality, yayladağı, hatay, turkey, is reported here in the mediterranean region of turkey in this study. the new specimen corresponds fully in morphology, colouration and body scalation with specimens from the type locality. keywords. reptilia, colubridae, rhynchocalamus barani, distribution, turkey. the genus rhynchocalamus includes three snake species (r. arabicus schmidt 1933, r. melanocephalus [jan 1862] and r. barani olgun, avcı, ilgaz, üzüm and yılmaz, 2007), two of which occur in turkey: r. melanocephalus and r. barani (see franzen and bischoff, 1995; baran and atatür, 1998; olgun et al., 2007; avcı et al., 2008; gruber, 2009). r. barani is an endemic snake to anatolia, firstly described by olgun et al. (2007) on the based on two specimens found 34 km east of dörtyol, hatay province, turkey. this newly described snake species, r. barani, differs from other rhynchocalamus species in having a greater number of dorsalia, fewer ventralia, only one upper labial in contact with the eye and a characteristic color-pattern of the body (olgun et al., 2007; gruber, 2009). during an expedition in 2009 to the mediterranean region of turkey, one specimen of r. barani was collected from yayladağı, hatay, turkey (35° 53’ 338” n; 36° 04’ 396’ ’e) on 22 may 2009 by c. salih demirci at an elevation of approximately 550 m. the specimen is currently deposited in the zoology laboratory of the department of biology at the science and arts faculty, adnan menderes university, turkey. it will later be incorporated into the collection of the zoology department, ege university, turkey (zdeu 36/2009). the specimen was fixed in 96% ethanol then preserved in 70% ethanol as described in başoğlu and baran (1980). the color and pattern characteristics were recorded while the specimen was still 178 a. avcı et alii alive. additionally color slides of living animals were utilized in the study. the ventral plates were counted using the dowling (1951) system. the present paper describes the morphology, color-pattern and body scalation of the single r. barani specimen collected at yayladağı, hatay (fig. 1) and compares it with the individuals described by olgun et al. (2007). the specimen was found under stones on a moist, stony slope with sparse cover of various grasses. the collection area was characterized mainly by tree pinus brutia. the specimen was collected at day time (16:00) at an air temperature of 16 °c. the sympatric herpetofauna comprised only salamandra infraimmaculata martens, 1885. the yayladağ specimen is an adult female with 263.80 and 98.48 mm snout-vent length and tail length, respectively. the head is small, not distinct from the neck, with an oblique shape at the anterior side. the head scales between the rostral and posterior margin of the parietal area are not keeled like in temporals. the rostrum is curved towards the top of the head and intrudes between the internasals; it is bordered by two upper labials, two nasals and two internasals. the nostrils are situated on the contact zone of nasals and loreals on each side. the loreals are in contact with the 1st upper labials at either side. the eyes are small with circular pupils. the 3rd upper labials are in contact with the eyes on each side. four pairs of lower labials touch the anterior chin shields at the left and right side. the scalation characters are summarized and compared with those of the dörtyol specimens (olgun et al., 2007) in table 1. the yayladağ specimen has remarkable differences in terms of temporals and ventrals. it has more temporals and fewer ventrals compared to values that was given in olgun et al. (2007). for general aspect, the color-pattern and head scalation of the new specimen are shown in fig. 2a-d. the basic color of the head (from tip of the rostral to posterior marfig. 1. distribution of rhynchocalamus barani in turkey, showing the known distribution according to literature, with star for the new locality. 1: 34 km e of dörtyol, hatay (olgun et al., 2007), 2: yayladağ, hatay. 179new finding of rhynchocalamus barani ta bl e 1. p ho lid os is c ha ra ct er s an d m or ph ol og ic al m ea su re m en ts o f th e th re e sp ec im en s of r hy nc ho ca la m us b ar an i o lg un e t al , 2 00 7 kn ow n fr om t ur ke y. z d eu – z oo lo gy d ep ar tm en t o f e ge u ni ve rs ity . [ se x, p o – p re oc ul ar , l o – l or ea ls , p t o – p os to cu la rs , t – t em po ra ls , p t – p os te m po ra ls , u l u pp er la bi al s, l l lo w er la bi al s, g si g ul ar s ca le s in a r ow b et w ee n po st er io r in fr am ax ill ar s, d st d or sa ls p lu s te m po ra ls s ca le s su rr ou nd in g th e po st er io r m ar gi n of th e pa ri et al s, v – v en tr al s, d sm d or sa l s ca le r ow s at m id -b od y, d sn d or sa l s ca le r ow s on n ec k on e he ad -l en gt h be hi nd h ea d, d sa d or sa l s ca le r ow s on e he ad -l en gt h an te ri or to a na l, sc su bc au da ls , r h r os tr al h ei gh t, rw r os tr al w id th , i n i nt er -n os tr il di st an ce , d e d ia m et er o f e ye s, s w s up ra oc ul ar s w id th , f w f ro nt al w id th , f l fr on ta l l en gt h, a il a nt er io r in fr am ax ill ar le ng th , p il p os te ri or in fr am ax ill ar le ng th , i n s in te rna sa l t ri an gu la r (d ) or t ra pe zo id s ha pe d (t ), sl i/ sl pf s ut ur e le ng th o f i nt er na sa l m uc h sh or te r (-) , s ho rt er ( -) , e qu al ( =) o r lo ng er ( +) t ha n pr ef ro nt al s ut ur e, p l/ r fl p ar ie ta ls sh or te r (t ), eq ua l ( n ) or lo ng er ( ¤ ) th an t he d is ta nc e fr om p os te ri or t ip o f r os tr al to t he p os te ri or t ip o f f ro nt al , p ll a p ai rs o f l ow er la bi al s in c on ta ct w ith an te ri or c hi n sh ie ld s, p l pi le us le ng th , p w p ile us w id th , h h h ea d he ig ht , s v l sn ou tve nt le ng th , t l ta il le ng th ( a ll m ea su re m en ts a re in m m )] . z d eu .1 22 /2 00 61 (h ol ot yp e) z d eu .1 22 /2 00 62 (p ar at yp e) z d eu .3 6/ 20 09 z d eu .1 22 /2 00 61 (h ol ot yp e) z d eu .1 22 /2 00 62 (p ar at yp e) z d eu .3 6/ 20 09 se x fe m al e m al e m al e in 3. 16 3. 04 3. 14 po 1/ 1 1/ 1 1/ 1 d e 1. 78 1. 84 1. 74 lo 1/ 2 a bs en t 1/ 1 sw 1. 50 1. 20 1. 34 pt o 2/ 2 2/ 2 2/ 2 fw 2. 60 2. 52 2. 62 t 1/ 1 1/ 1 2/ 3 fl 3. 10 3. 10 3. 12 pt 2/ 2 2/ 2 2/ 2 a il 2. 42 2. 64 2. 62 u l 5/ 5 5/ 5 5/ 5 pi l 1. 54 2. 02 2. 00 ll 7/ 8 7/ 7 7/ 8 in s t t t g si 1 2 2 sl i/ sl pf + (= ) (= ) d st 13 11 12 pl /r fl t ( 3. 54 -4 .6 4) t ( 3. 60 -4 .4 2) t ( 3. 50 -4 .5 2) v 17 3 16 3 15 6 pl la 3/ 4 3/ 3 3/ 3 d sm 17 17 17 pl 9. 00 8. 22 8. 72 d sn 17 17 17 pw 4. 48 4. 30 4. 44 d sa 17 17 17 h h 4. 04 3. 70 3. 80 sc 65 /6 5+ 1 74 /7 4+ 1 68 /6 8+ 1 sv l 31 2. 98 25 2. 20 26 3. 80 r h 2. 06 1. 64 1. 66 t l 89 .6 0 90 .0 4 98 .4 8 rw 2. 66 2. 48 2. 46 180 a. avcı et alii gin of the parietals) is ash-gray. this basic color extends to the first upper labial plate at the flanks of the head. a narrow black blotch extends from the lower part of the eyes up to the contact of the 3rd and 4th upper labials. the 2nd, 3/4 of 3rd, 4/5 of 4th and 2/3 of 5th upper labials are white. other lower labials are also white except for the last lower labial which is black. the narrow black neck band extends along the upper part of the head, from the margin of the parietals to the top of the posterior margin of the temporals; in the gular region of the head the band is in contact with dorsals over a width of six scales. this band does not touch ventrals at the lower part of the head. the length of the head fig. 2. general and head aspects of rhynchocalamus barani specimen captured from yayladağ, hatay, turkey (zdeu. 36/2009) (a-d). 181new finding of rhynchocalamus barani patterns (from the tip of the rostrum), proportional to snout-vent length is 4.8 mm. temporals are mostly white (ca. 75%) or otherwise black. the basic color of the dorsum is reddish brown without spots. the lower part of the head and ventral, separated by the black head band, are white without spots, except the connection site of the anterior and posterior inframaxillars. there is a small black spot at this site. the lower part of the tail is white, including the tail tip. the new locality record extends the known range of the species about 110 km to the southwest as measured from the type locality. r. barani has been known only from the type locality up to now (olgun et al., 2007). however, the new locality (yayladağ) indicates a much wider distribution of the species within the mediterranean region of turkey and possibly northwestern syria. the anatolian diagonal is an important barrier affecting the distribution of reptilian species such as apathya cappadocica (wertner 1902), anatololacerta danfordi (günther 1876) and eirenis jan 1863 species (nilson et al., 1990; sindaco et al., 2000). the anatolian diagonal is a mountain range that extends from the region of bayburt-gümüşhane and the province of erzurum in the northeast towards the provinces of kayseri and kahramanmaraş (anti-taurus) in the southwest. the range splits here and one branch continues further southwest towards bolkar mountain in the cilician taurus. the other runs more southwards through the province hatay towards the direction of the lebanon mountains (nilson et al., 1990). amanos mountain is situated southwards of the anatolian diagonal and its altitudinal range extends from 1500 to 2600 m. we hypothesise that amanos mountain could be a barrier that prevents the extension of the distribution of r. barani to the west anatolia. acknowledgements this work forms part of a project (project no. tbag-108t162) supported by tübi̇tak (the scientific and technical research council of turkey). we are most grateful to salih demirci for obtaining specimen. we thank to dr. suat ateşlier, from adnan menderes university for help while taking the head photos. we are also grateful to m. stackhowitsch from vienna university, for english corrections on an earlier manuscript version. the authors wish to acknowledge the use of maptool program for analysis and graphics in this paper. maptool is a product of seaturtle.org (information is available at www.seaturtle.org). references avcı, a., dinçaslan, y.e., ilgaz, ç., üzüm, n. (2008): contributions to the distribution and morphology of rhynchocalamus melanocephalus melanocephalus (jan 1862) (reptilia, colubridae) in turkey. north-western j. zool. 4: 161-166. baran, i̇., atatür, m.k. (1998): turkish herpetofauna (amphibians & reptiles). republic of turkish ministry of environment, ankara, turkey. başoğlu, m., baran, i̇. (1980): türkiye sürüngenleri kısım ii. yılanlar. ege üniversitesi fen fakültesi kitaplar serisi, bornova, i̇zmir, turkey. dowling, h.g. (1951): a proposed standard of counting ventrals in snakes. br. j. herpetol. 1: 97-99. gruber, u. (2009): die schlangen europas. alle arten europas und des mittelmeerraums. kosmos, stutgart. franzen, m., bischoff, w. (1995): erstnachweis von rhynchocalamus melanocephalus melanocephalus für die turkei. salamandra 31: 107-122. olgun, k., avcı, a., ilgaz, ç., üzüm, n., yılmaz, c. (2007): a new species of rhynchocalamus (reptilia: serpentes: colubridae) from turkey. zootaxa 1399: 57-68. nilson, g., andrén, c., flärdh, b. (1990): vipera albizona, a new mountain viper from central turkey, with comments on isolating effects of the anatolian “diagonal”. amphibia-reptilia 11: 284-294. sindaco, r., venchi, a., carpaneto, g.m., bologna, m.a. (2000): the reptiles of anatolia: a checklist and zoogeographical analysis. biogeographia 21: 441-554. acta herpetologica 16(1): 37-44, 2021 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-10418 sex chromosome diversification in the smooth snake coronella austriaca (reptilia, serpentes) marcello mezzasalma1,2,*, gaetano odierna1 1 department of biology, university of naples federico ii, via cinthia 26, 80126, naples, italy 2 cibio, centro de investigação em biodiversidade e recursos genéticos, inbio, universidade do porto, campus agrário de vairão, rua padre armando quintas, no 7, 4485-661 vairão, portugal (current address) *corresponding author. e-mail: m.mezzasalma@gmail.com submitted on: 2021, 13th february; revised on: 2021, 16th april; accepted on: 2021, 25th april editor: fabio m. guarino abstract. the smooth snake coronella austriaca is a widespread palearctic colubrid species. the species has been the subject of several molecular and phylogeographic studies which highlighted the occurrence of distinct genetic lineages in different areas of the species distribution, but scarce cytogenetic data are currently available on the species. in this paper we present a molecular and karyological study performed with several banding, staining methods and norfish on samples of c. austriaca from different geographical areas (italy and greece) of the species distribution. the molecular and phylogenetic analysis unambiguously placed the studied samples in different clades with a clear geographical pattern. the karyotype of the two female samples studied was composed of 2n = 36 chromosomes with 16 macroand 20 microchromosomes and a mix of plesiomorphic and derivate chromosome features. all macrochromosomes were biarmed with the exception of pair 5 that was telocentric. nors were detected on a microchromosome pair. in both females, the pair 4 was heteromorphic (and completely heterochromatic after c-banding in the italian female), representing the first report of a zz/zw sex chromosome system with female heterogamety in c. austriaca. in addition, the w chromosome showed a different morphology between the two female studied (submetacentric and subtelocentric), highlighting the occurrence of a chromosomal diversification among distinct geographical areas of the species distribution and further supporting that the species contains different diverging evolutionary clades. keywords. karyotype, heterochromatin, fish, sex chromosomes, squamates, snakes. introduction the smooth snake coronella austriaca laurenti, 1768, is a small sized ovoviviparous colubrid with a widespread distribution across the western palearctic (strijbosch, 1997). the species occurs in western, central and southern europe reaching as far as the ural mountains and the caspian sea, up to northern iran and western kazakhstan. coronella austriaca is absent from european and mediterranean islands, except for southern england, sicily, the island of elba and the island of krk (engelmann, 1993; strijbosch, 1997; 2006). in its large distribution range, the species have a relatively uniform morphology and only two subspecies are currently recognised: c. a. acutirostris malkmus, 1995, from the north-western iberian peninsula and the nominal subspecies c. a. austriaca which occupies the rest of the geographical range of the species. a third subspecies, c. a. fitzingeri (bonaparte, 1840) was formerly described from southern italy and sicily, but it has been recently synonymised with the nominal subspecies (speybroeck et al., 2016). the species has been the object of several molecular and phylogeographic studies (santos et al., 2008; llorente et al., 2012; galarza et al., 2015; jablonsky, 2019) and rep38 m. mezzasalma, g. odierna resents an ideal candidate to assess patterns of intraspecific genetic diversity among different populations as well as the occurrence of different refugial areas across its distribution range. these studies evidenced that c. austriaca comprises several distinct clades showing a complex haplotype structure and deep genetic divergence. these evidences do not reflect the current taxonomy of the species and a revision is probably required to better describe its molecular and geographic intraspecific diversity. on the other hand, the available chromosome data on c. austriaca are dated and refer to the studies by matthey (1931) and kobel (1967) that described the karyotype of two males from switzerland, but no information is currently available from other, geographically and genetically distinct clades of the species. the karyotype described by matthey (1931) and kobel (1967) was composed of 2n = 36, with 16 macrochromosomes and 20 microchromosomes. all the macrochromosomes were biarmed, excluding the elements of 5th pair that were telocentric. furthermore, these studies were performed only with standard staining methods. no information is currently available on the sex chromosome system, location of nucleolar organizer regions (nors) or other chromosome markers which would be useful for karyotype comparison among different population of the species or with other phylogenetically related colubrid species. in fact, cytogenetic inferences, especially when linked to molecular data in a phylogenetic perspective, are useful tools to detect plesioand apomorphic states and to reconstruct evolutionary trends in the studied species (odierna et al., 1987; mezzasalma et al., 2013, 2017 a, 2017b; fuller et al., 2018). in this work we present the results of a karyological study performed on smooth snake samples belonging to different populations and geographical areas of the species distribution (italy and greece) using several standard staining and banding methods and molecular cytogenetics. the aim of this study was to evidence the possible presence of a chromosome sex determination system, the occurrence and location of heterochromatic regions and of nors. we also performed a molecular and a phylogenetic analysis on the studied samples, adding our data to those available from the literature, in order to evaluate their genetic diversity and place our chromosome data in a phylogenetic context. material and methods sampling we studied preserved tissue and cell suspensions of two samples of c. austriaca obtained from existing collections and no aminal was collected during the realization of this study. studied samples include one female from italy (picentini mountains, avellino), and one female from greece (peloponnese) both hosted at the dipartimento di biologia evolutiva e comparata, università degli studi di napoli federico ii, since 1972 (sample numbers ca0201ca0202). these two samples were both used in a preliminary molecular analysis and in the karyolgical study as described below. the samples used in this study were already used in previous analyses (mezzasalma et al., 2014, 2016a). molecular and phylogenetic analysis in order to properly identify the studied samples, assess their genetic diversity and establish the taxonomic affinities with other available sequences on the species, a molecular analysis was performed using a fragment of the mitochondrial cytochrome b (cytb) gene. this mtdna gene was chosen considering the number of available sequences for several populations of c. austriaca (llorente et al., 2012; santos et al., 2008, 2012; galarza et al., 2015; jablonsky et al., 2019). genomic dna was extracted from both samples using the standard phenol-chloroform method by sambrook et al. (1989) and then used in the pcr amplification of a fragment of about 600 bp of the cytb. the primers used were forward: 5’ aacttcggatccatactactaa 3’ and reverse: 3’ taaagatgttaggggtgaatga 5’, and the pcr parameters those reported by mezzasalama et al. (2015a). pcr products were sequenced on an automated sequencer abi 377 (applied biosystems, foster city, ca, usa) using bigdye terminator v3.1 (abi). sequences were blasted in genbank and chromatograms were checked and edited using chromas lite 2.1.1 and bioedit 7.2.6.1 (hall, 1999). the best-fitting substitution model (gtr+i+g) was chosen using jmodeltest 2.1.7 (darriba et al., 2012), under the corrected akaike information criterion (aicc). in the phylogenetic analysis we used the two newly determined sequences (accession numbers: mw861682-mw861683) and available homologous sequences from jablonski et al. (2019), downloaded from genbank, choosing when possible, the longest available sequences for each major clade (see fig. 1). as outgroup we used an available homologous sequence of c. girondica (an: af471088). phylogenetic analysis with bayesian inference (bi) was performed using mrbayes 3.2.7 (ronquist et al., 2012), with two parallel runs of 8 000 000 generations and four incrementally heated markov chains (using default heating values), with a burn-in of 25% and sampling the chains every 500 generations. a 50% majority-rule consensus tree was retrieved from the post-burn-in samples and chain convergence was checked with convergence diagnostic values (average standard deviation of split frequencies >> 0.01, potential scale reduction factor). chromosome analysis chromosomes were obtained from tissue samples and cell suspensions using the air-drying method, as described in mez39karyological diversification in the smooth snake zasalma et al. (2019). the chromosome analysis was performed with traditional staining (5% giemsa solution at ph 7 for 10 min) and several chromosome staining and banding techniques: quinacrine staining, chromomycin a3-methyl green staining (cma/mg) (mezzasalma et al., 2015b), c-banding (sumner, 1972), and sequential c-banding + cma + dapi (sidhom et al. 2020a, b). karyotype reconstruction was performed after scoring at least five metaphase plate from each sample studied. nucleolus organizing regions (nors) were located following the ag-nor banding protocol reported by howell and black (1980) and fluorescence in situ hybridization (nor-fish) as described in sidhom et al. (2020a), using as probe the pcramplified and biotinylated 18s rrna gene of the gekkonid tarentola mauritanica. the detection of fish signals was carried out using extravidin fitc (sigma aldrich), counterstained with propidium iodide (pi). metaphase plates were detected and recorded using an epifluorescent microscope (axioscope zeiss) equipped with an image analysis system. results molecular and phylogenetic analysis a a fragment of about 500 bp of the cytb was successfully amplified and sequenced from the two original samples, respectively from italy and greece (see sampling), used in the cytogenetic analysis. no interruptions of the reading frame were detected in either sequences. nucleotide identity samples was about 99% between the newly sequenced sample from italy (monti picentini) and an available sequence from italy (an: mh382919), and 100% between the newly determined sequence from greece and an available sequence from greece (an: eu022647). in the phylogenetic analysis, the final cytb alignment contained 39 sequences and 1031 nucleotide positions. the resulting tree (fig. 1) retrieved all the main clades and a topology similar to that reported by jablonski et al. (2019). the relative evolutionary relationships among the main clades mostly reflect an east-west fig. 1. phylogenetic analysis with bayesian inference (bi) of cytb sequences (up to 1031 bp) of c. austriaca. numbers at nodes represent posterior support values. clades denomination follows jabloski et al. (2019). red circles = original samples used in the cytogenetic analysis. 40 m. mezzasalma, g. odierna scenario of geographical diversification by distance. our tree retrieved, four major clades, comprising several smaller subclades: iranian 1, sicilian + iberian 1-3 + western 1-2, anatolian 1-2 +balkan + central european, and iranian 2 + transcaucasian + eastern, respectively (fig. 1). in our tree, the easternmost major clade (excluding the only sample of the iranian 1 clade), comprising the iranian 2 + transcaucasian + eastern subclades, is the outgroup of all the remaining clades. the two original samples from italy and greece clustered unambiguously within the western 2 and balkan clades, respectively (fig. 1). statistical support values were generally high at terminal nodes while varied from high to low at deeper nodes (fig. 1). chromosomal analysis the italian and greek samples of c. austriaca showed a very similar karyotype composed of 2n = 36 chromosomes, with 16 macrochromosomes and 20 microchromosomes (fig. 2). among macrochromosomes, pairs 1, 3, 6 and 8 were metacentric, pairs 2 and 7 were submetacentric, and pair 5 was telocentric. the pair 4 was heteromorphic, carrying the female sex chromosomes, zw. in particular, the chromosome z was always metacentric while the w resulted subtelocentric in the female from picentini mountains (italy) (fig. 2a) and submetacentric in the female from peloponnese (greece) (fig. 2b). other chromosome stainings and banding techniques were performed only on the italian sample of c. austriaca, as quality and quantity of metaphase plates of the greek individual were adequate just for its karyotype description. quinacrine stained evenly both macroand microchromosomes (fig. 3a), while cma/mg evidenced two microchromosomes and the telomeric regions of all macrochromosomes and the telomeric regions almost all microchromosomes (fig. 3b). a microchromosome pair resulted specifically marked by both ag-nor staining and nor-fish, (fig. 3c and d). after c-banding, heterochromatic bands were hardly visible, if not absent, on most macroand microchromosomes, with the exclusion of the w chromosome, which was completely heterochromatic (fig. 4a), but not evidenced with cma or dapi (fig. 4b, c). discussion our phylogenetic inference produced similar results to those obtained by jablonski et al. (2019), retrieving all the 14 clades previously described. the main differences concern the position of one of the major clades (comprising iranian 2 + transcaucasian + eastern subfig. 2. giemsa stained karyotypes of c. austriaca from italy (picentini mountains) (a) and greece (peleponnese) (b). the frame includes the zw sex chromosome pair. fig. 3. metaphase plates of c. austriaca stained with quinacrine (a), cma/mg (b), ag-nor staining (c) and nor-fish banding (d). arrows point at a microchrosome pair evidenced with cma/mg, ag-nor stainingand nor-fish. scale bar applies all images. 41karyological diversification in the smooth snake clades) which is not involved in a basal polytomy with the other major clades but appear to be the outgroup of all the remaining clades. this results further supports the hypothesis of an east-west geographical diversification process in c. austriaca. similar east-west differentiation processes are also shared by different european and palearctic reptile taxa, including distinct major snake lineages of asiatic origin (see, e.g., utiger et al., 2002; nagy et al., 2004; mezzasalma et al. 2015a, 2018). however, the phylogenetic position and the evolutionary relationships of some clades (such as the iranian 1 clade) remain to be better determined and this hypothesis should be further tested. in fact, as already highlighted by jablonski et al. (2019), the uncertain phylogenetic position of some subclades as well as the low statistical support values at some nodes is probably due to a number of missing haplotypes and a more inclusive sampling with the addition of more molecular markers is probably required to better assess the evolutionary relationship of some subclades. the karyological formula of the two females of c. austriaca here studied is consistent with that previously described by mattey (1931) and kobel (1967) for two males from switzerland. however, the comparison among the pair 4 of the different karyotypes allowed us to detect for the first time the zz/zw sex chromosome system in c. austriaca. in particular, the metacentric element of the 4th pair was identified as the z sex chromosome and its heteromorphic counterpart as the w chromosome. in addition, the w chromosome showed a different morphology among the two studied females, suggesting the occurrence of a chromosomal diversification among distinct molecular clades. overall, the karyotype of c. austriaca shows a mixture of plesiomorphic and derivate chromosomal characters. in particular, the chromosomal characters that can be considered plesiomorphic in colubrids include the karyological formula, the microchromosomal localization of the nors, and the pair 4 representing the zz/zw sex chromosomes. a chromosome complement composed of 2n = 36 chromosomes with 16 macroand 20 microchromosomes is displayed by both primitive (henophidia) and advanced (caenophidia) snake lineages and is supposed to represent the ancestral snake karyotype (gorman and gress, 1970; singh, 1972;, 1986, oguiura et al., 2009; mezzasalma et al., 2014, 2016b, 2019). concerning the localization of nors, the methods here used show their presence on two microchromosomes. ag-nor banding suggests that both loci of nors are active. in fact, ag binds to proteins essential to nucleolar structure and therefore to the transcriptional activity of ribosomal cistrons during the previous interphase (howell, 1977; jiménez et al., 1988). the occurrence of nors on a microchromosome pair is not unusual among snakes, being exhibited in representatives of various families, including colubridae (olmo and signorino, 2006), and it is considered a primitive condition in squamata (porter et al., 1991; aprea et al., 2006). derivate chromosome characters in the karyotype of c. austriaca can be considered the morphology of the pair 5 (telocentric in all the studied samples), and the occurrence of an heteromorphic, completely heterochromatic w sex chromosome, which is also morphologically differentiated betwenn the two studied samples from different geographic regions. in fact, in the putative ancestral snake karyotype of 2n = 36 all the macrochromosome pairs are biarmed (metaor submetacenfig. 4. metaphase plates of c. austriaca sequentially stained with c-banding + giemsa (a),+cma (b), and +dapi (c). arrows point at the w sex chromosome. scale bar applies to all images. 42 m. mezzasalma, g. odierna tric), though a telocentric morphology of the pair 5 has been observed in different colubrid species belonging to independent evolutionary lineages, such as different species of the genera elaphe and hierophis (see also singh, 1972; kobel 1967; mezzasalma et al., 2015b). according to singh (1972), because the fifth pair is biarmed in most of the other colubrids, a simple pericentric inversions can be assumed to explain the different morphology of this pair. the zz/zw sex determination system was supposed to be a plesiomorphic state in snakes (mengden, 1981; matsubara et al., 2016), however recent evidences suggest that different sex chromosome systems evolved multiple times, independently, in different snake lineages, including species with either female (zz/zw) or male (xx/xy) heterogamety along with a discrete number of species with undifferentiated sex chromosomes (gamble at al., 2017; mezzasalma et al., 2019). this makes the suborder serpentes, and more in general the whole order squamata, which also includes various taxa with temperature-dependent sex determination (gamble, 2010; gamble et al., 2017; pallotta et al. 2017; alam et al., 2018), a unique study system to analyze the evolution and diversification of different mechanisms of sex determination. nevertheless, in the family colubridae, the fourth macrochromosome pair is usually composed of the zw elements: the z is metacentric and conserved in most species, while the w is often heteromorphic compared to the z and largely heterochromatic (see e.g., mengden, 1981; mezzasalma et al., 2015a; rovatsos et al., 2015; matsubara et al., 2016). in c. austriaca, the studied greek and italian samples display a different morphology of the w chromosome, resulting submetacentric and subtelocentric, respectively. in the studied italian female, the w chromosome is completely heterochromatic but not evidenced with fluorochromes (dapi and cma3). the lack of data on the chromatin composition and distribution of the w chromosome from the greek female here studied, does not allow us to establish if the differences between the w chromosomes of the italian and the greek sample also concern the heterochromatin pattern. nevertheless, the different w morphology here found among greek and italian samples of c. austriaca highlight the occurrence of a karyological diversification among different clades of the species (balkan and western 2, see fig. 1 and jablonski et al. 2019), further supporting that the species contains different diverging evolutionary lineages. from a biogeographic point of view, as already documented for other palearctic reptiles, the quaternary climatic oscillations had an important role in shaping the current diversity of extant species, mainly through the contraction and re-expansion of their distribution ranges and the isolation of populations in different “refugia within refugia” (see, e.g., ursenbacher et al., 2008; gvoždík et al., 2010; kindler et al., 2013; mezzasalma et al., 2018). unstable climatic conditions, fragmentation of the distribution range, small population size and isolation in distinct glacial refugia are also particularly favorable conditions for the fixation of chromosome mutations (see also mezzasalma et al., 2015a, 2017a for similar examples in different amphibian and reptile species), which may happen in different populations independently. in conclusion, this paper provides the first record of a zz/zw sex chromosome system in c. austriaca, with the occurrence of different morphologies of the w chromosome in different clades (western and balkan). more inclusive molecular and cytogenetic data from other areas of the wide distribution of c. austriaca would be useful to characterize the chromosome variability of different molecular clades the european smooth snake, helping to better assess their taxonomy. aknowledgements this research would not have been possible without the collaboration of the former dipartimento di biologia evolutiva e comparata of the università degli studi di napoli federico ii which hosted several preserved cell suspensions amphibians and reptiles and provided us with the study samples. references alam, s.m.i., sarre, s.d., gleeson, d., georges, a., ezaz.t. 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(2002): molecular systematics and phylogeny of old and new world ratsnakes, elaphe auct., and related genera (reptilia, squamata, colubridae). russ. j. herpetol. 9: 105-124. acta herpetologica 2006 1 nuove segnalazioni di «zootoca vivipara» jaquin e di «vipera berus» linnaeus, in piemonte, italia nord-occidentale (novitates herpetologicae pedemontanae ii) nuove segnalazioni di zootoca vivipara jaquin e di vipera berus linnaeus, in piemonte, italia nord-occidentale (novitates herpetologicae pedemontanae ii) samuele ghielmi 1, paolo eusebio bergò 2, franco andreone 2. 1 civico museo insubrico di storia naturale, piazza giovanni xxiii 4, 21056 induno olona (va). 2 museo regionale di scienze naturali, via giolitti 36, 10123 torino. abstract. we provide new distribution data about zootoca vivipara and vipera berus in piedmont (nw italy). the former species has been reported for: bognanco (vb), sessera (bi), mastallone (vc), rimella (vc), and strona (vb) valleys. the adder, on the other hand, has been observed in sorba (vc) and strona (vb) valleys. comments on these new records are also provided. riassunto. vengono forniti nuovi dati distributivi di zootoca vivipara e di vipera berus in piemonte (italia nordoccidentale). la prima viene segnalata nelle valli bognanco (vb), sessera (bi), mastallone (vc), rimella (vc) e strona (vb). il marasso viene invece segnalato per le valli sorba (vc) e strona (vb). i nuovi dati distributivi vengono commentati brevemente. la distribuzione dell’erpetofauna in piemonte (italia nord-occidentale) è stata oggetto di numerosi studi che hanno visto una loro sintesi organica operata da andreone & sindaco (1999), i quali hanno evidenziato la mancata o scarsa copertura dei distretti settentrionali della regione. nell’intento di acquisire nuove conoscenze sulla distribuzione dell’erpetofauna di tale area si è ritenuto fornire nuove indicazioni sulla presenza di due taxa particolarmente rari o difficilmente rilevabili in natura: la lucertola vivipara (zootoca vivipara) e il marasso (vipera berus). zootoca vivipara è una specie ad ampia distribuzione euroasiatica. in italia è presente con apparente continuità sulle alpi centro-orientali, mentre risulta più frammentata su quelle centro-occidentali. sono peraltro note alcune stazioni isolate e relitte nella pianura padano-veneta (salmaso e osella, 1989; richard e semenzato, 1992; baratelli e ghielmi, 1994; s.h.i., 1996; lapini et al., 1999). all’interno dell’areale italiano sono presenti due sottospecie caratterizzate da una differente modalità di riproduzione: z. v. vivipara, con modalità di riproduzione vivipara, che occupa la porzione alpina dell’areale e z. v. carniolica, con modalità di riproduzione ovipara, la quale invece occupa la porzione padana, prealpina e, parzialmente, alpina dell’areale (mayer et al., 2000; ghielmi et al., 2004). in piemonte la specie è conosciuta per quattro aree distinte e apparentemente isolate tra loro: (1) l’alta valle d’ossola (valle formazza, alpe devero), (2) il massiccio del monte mottaacta herpetologica 1: 29-36, 2006 30 s. ghielmi et alii rone a ridosso del lago maggiore, (3) le prealpi biellesi (valli sessera e di oropa) e (4) il massiccio del monviso con alcune valli limitrofe (valli troncea, germanasca, pellice e varaita) (andreone e sindaco, 1999; sindaco et al., 2002). pregresse indagini molecolari su alcune di queste popolazioni hanno consentito di identificare due diversi aplotipi per z. v. carniolica nell’area in esame: l’aplotipo os5 per il monte mottarone e os4 per la valle di oropa (surget-groba et al., 2002) (per i codici degli aplotipi vedi surget-groba et al., 2002; per i dati genetici originali surget-groba, com. pers.). vipera berus presenta un areale che ricalca in buona misura quello della lucertola vivipara. in piemonte questa specie risulta confermata sulle prealpi biellesi (valli elvo, di oropa, cervo e sessera) e, storicamente, anche nelle zone contigue dell’alta valle sesia (capra, 1954; pascutto e ghielmetti 1997; ferri e sindaco, 1999; danini e baratelli, 2000) oltre che in una località ad ovest di torino (monasterolo) con una popolazione di incerta origine, forse introdotta (chiariglione, 1994). rimangono invece in attesa di conferma i dati di presenza della specie per il parco nazionale della val grande (ferri e sindaco, 1999). il presente contributo si basa su dati originali raccolti durante escursioni effettuate tra gli anni 1995 e 2003. per ogni stazione di osservazione sono stati rilevati il numero e, quando possibile, il sesso e lo stadio di sviluppo degli individui osservati, quota, esposizione e coordinate geografiche (rilevate con gps o calcolate su carta topografica) oltre ad alcune note ambientali. in un caso sono inoltre state considerate testimonianze di persone residenti nei luoghi di rilevamento, suffragate da prove attendibili (animali conservati o rilievi fotografici). la revisione della collezione erpetologica del museo di storia naturale di domodossola ha consentito il rinvenimento di alcuni reperti storici ed inediti di zootoca vivipara (andreone et al., 2005). nell’intento poi di contribuire ad un’attribuzione sottospecifica delle popolazioni piemontesi di z. vivipara si è proceduto, sugli animali catturati (indicati nel testo con un asterisco), ad un prelievo tissutale (apice della coda), fissato in alcool e destinato ad analisi genetiche. sempre per questa specie si è provveduto a mantenere in terrario tre ff gravide (indicate nel testo con due asterischi) di differenti località al fine di osservarne la modalità di riproduzione. due esemplari di vipera berus rinvenuti morti fanno ora parte della collezione del museo regionale di scienze naturali di torino (mrsn r1982, r2106). una femmina di lucertola vivipara della valle germanasca e un maschio della valle sorba, nato in cattività durante le osservazioni sulla modalità di riproduzione della specie e deceduto accidentalmente, sono stati fissati in alcool e conservati nella stessa collezione (mrsn r2104, r2105). tutti gli altri individui catturati o nati in cattività sono stati rilasciati nei luoghi di provenienza. le analisi molecolari condotte sui campioni di zootoca vivipara raccolti nel corso del presente studio hanno permesso di stabilire l’aplotipo di appartenenza delle varie popolazioni, consentendo inoltre una loro attribuzione sottospecifica, come riassunto in tab. 1. inoltre come atteso dai dati molecolari, le due ff gravide di zootoca vivipara provenienti dalla valle sorba e dalla valle strona, stabulate in terrario, hanno deposto rispettivamente 3 e 9 uova con guscio bianco e completamente calcificato, schiusesi in 33 e 25 giorni di incubazione a temperatura ambiente. nelle popolazioni di valle sorba e di valle mastallone è stato riscontrato l’aplotipo os4 già noto per la popolazione di oropa, mentre in quelle di valle strona e valle di rimella, peraltro adiacenti tra loro, è risultato presente l’aplotipo os5 precedentemen31nuove segnalazioni di zootoca e di vipera te individuato sul massiccio del mottarone. in una località della valle sessera (bocchetta luvera) è invece emerso un nuovo aplotipo prossimo a quello della popolazione della palude brabbia (va, lombardia; surget-groba, com. pers.). per quanto riguarda la terza femmina gravida stabulata in terrario e proveniente dalla valle germanasca (to), da un sito già noto per la specie (sindaco, 1999), sia l’osservazione di un “parto” viviparo (9 uova costituite da sottili membrane trasparenti contenenti piccoli completamente formati e schiusesi nell’arco di un giorno dalla deposizione), sia i dati genetici confermano l’attribuzione di questa popolazione al gruppo viviparo occidentale attualmente ascritto alla ssp. vivipara; alla stessa sottospecie appartengono anche i reperti della collezione erpetologica del museo di domodossola, tra i quali è presente una femmina dalla quale sono state estratte 8 uova non calcificate e contenenti embrioni completamente sviluppati. l’elenco delle valli e delle aree ove le specie sono state rinvenute è riportato di seguito ed è riassunto in fig. 1. valle bognanco (vb) per questa località si riportano i dati emersi dall’esame del catalogo e della collezione del museo di domodossola. zootoca vivipara – data di raccolta non nota, 3 mm, 3 ff e 8 uova non calcificate estratte da una femmina, contenenti embrioni completamente sviluppati, località s. lorenzo di bognanco, valle bognanco (numero di catalogo msnd rso12, g. bazzetta leg.). valle sessera (bi) osservazioni effettuate in due distinte località della valle: (1) lungo il sentiero che dall’alpe campo della quara sale alla colma del balmello e (2) lungo il sentiero n. 9 che da bocchetto luvera conduce a bocchetto sessera alle pendici della rocca d’argimonia. zootoca vivipara 1/10/2002 (peb, obs.) f subadulta*, nei pressi di bocchetto luvera, 1400 m s.l.m., esposizione n, 45°40’22’’n, 08°06’35’’e; 4/05/2003 (sg, peb, obs.), 3 mm adulti* all’interno di piccola depressione umida nei pressi dell’alpe campo della quara, 1455 m s.l.m., esposizione s-so, 45°41’18’’n, 08°02’44’’e; 29/05/2003 (sg, peb, obs.), f adulta*, nei pressi di alpe campo della quara, 1450, esposizione so, 45°41’18’’n, 08°02’44’’e; 1 juv.* lungo il versante xerico soprastante l’alpe campo della quara, 1750 m s.l.m., esposizione s, 45°41’30’’n, 08°02’42’’e.; f gravida* (senile) in tratto di versante posto fra l’alpe campo della quara e la colma del balmello, 1830 m s.l.m., in sinistra idrografica rispetto al corso del torrente sessera, esposizione so, 45°41’38’’n, 08°02’46’’e. valle del torrente mastallone (vc) l’area indagata è quella lungo il sentiero che dal borgo s. maria porta al passo di alpe selle. zootoca vivipara 23/06/02 (sg, peb, obs.), m subadulto* nei pressi di alpe baranca, 1600 m s.l.m., su muretto a secco di sostegno al sentiero, esposizione ne, 45°55’28’’n, 08°06’26’’e. tabella 1: sintesi delle osservazioni raccolte per l’attribuzione sottospecifica di zootoca vivipara nelle popolazioni indagate. località attribuzione sottospecifica aplotipo n uova data deposizione data schiusa valle sorba z. v. carniolica os4 3 17/07/2001 19/08/2001 valle mastallone z. v. carniolica os4 valle strona z. v. carniolica os5 9 18/06/2002 12/07/2002 valle rimella z. v. carniolica os5 valle sessera z. v. carniolica nuovo aplotipo valle bognanco z. v. vivipara non analizzato 8 (vedi testo) valle germanasca z. v. vivipara viviparo occidentale 9 11/07/2003 12/07/2003 32 s. ghielmi et alii vallone di rimella (vc) il sito di rilevamento ricade nel bacino idrografico del torrente mastallone (affluente di sinistra del fiume sesia), in prossimità del crinale che divide il vallone di rimella dall’adiacente valle strona. zootoca vivipara 14/08/2002 (sg, p. ghielmi, obs.), m adulto*, tra alpi pianello e bocchetta di rimella (o di campello), 1880 m s.l.m., esposizione s-e, 45°54’55’’n; 08°13’25’’e. valle sorba (vc) le osservazioni sono state effettuate lungo il sentiero che dal borgo di rassa (917 m) sale all’alpe il dosso (1400 m) e riguardano 56 individui di lucertola vivipara, tra cui diverse femmine gravide, rilevati in poco più di un’ora. abbiamo ritenuto opportuno riportare la quota e il luogo di osservazione soltanto per gli individui catturati e sottoposti a prelievo di tessuto per le indagini genetiche: zootoca vivipara 08/07/2001 (sg, d. bisoni, obs.), 1 subadulto*, località alpe sorba, 1160 m s.l.m., esposizione e-se, 45°45’08’’n; 08°00’12’’e; 3 ff adulte*, m adulto*, in una radura lungo il sentiero compresa tra 1240 e 260 m s.l.m., esposizione e-se, 45°44’41’’n; 07°59’46’’e; f adulta gravida** (m subadulto ottenuto dalla deposizione in cattività di questa femmina e deceduto accidentalmente, è conservato col numero di catalogo mrsn r2105), località alpe il dosso, 1395 m s.l.m., esposizione e-se, 45°44’28’’n; 07°59’27’’e. vipera berus 18/08/2001 (peb, fa, det.) 1 esemplare adulto e 1 juv., catturati nei dintorni dell’alpe sorba da l. simone qui residente e conservati in alcool denaturato. per questi esemplari, catturati intorno al 1990, è certa la provenienza dalla valle sorba anche se non è stato possibile risalire con precisione alla quota e al luogo di cattura. due degli autori (peb e fa) hanno provveduto a fotografare i reperti. valle strona (vc) osservazioni effettuate su vari sentieri che dalla frazione campello monti (1305 m s.l.m.) raggiungono le cime e i passi che delimitano la testata della valle strona; la quota massima da noi raggiunta è quella di 2100 m s.l.m. riferita al lago capezzone posto sotto l’omonima cima. zootoca vivipara 02/06/2002 (sg, obs.), 2 ff adulte gravide (di cui una **), 1 individuo juv. sul bordo del sentiero che da campello monti conduce al l. capezzone, quota compresa tra 1510 fig. 1. nuove segnalazioni di zootoca vivipara e di vipera berus in piemonte. 1, z. v. vivipara, valle bognanco; 2, z. v. carniolica, bocchetta luvera valle sessera; 3, z. v. carniolica, valle sessera; 4, z. v. carniolica, valle di rimella; 5, z. v. carniolica, valle mastallone; 6, z. v. carniolica, valle sorba; 7, vipera berus, valle sorba; 8, z. v. carniolica, valle strona; 9, vipera berus, valle strona. 33nuove segnalazioni di zootoca e di vipera m e 1540 m s.l.m., esposizione se, 45°56’13’’n; 08°13’35’’e; f adulta sul ciglio del sentiero che da campello monti conduce al lago capezzone, 1620 m s.l.m., esposizione se, 45°56’16’’n; 08°13’32’’e; 14/08/2002 (sg, obs.), m adulto* a margine del sentiero nei pressi di alpe calzino, esposizione ene, 1900 m s.l.m., 45°55’25’’n; 08°13’29’’e; f adulta* a margine del sentiero poco sopra alpe del vecchio. esposizione ne, 1520m s.l.m., 45°55’58’’n; 08°13’51’’e.; 27/04/2003 (sg, obs.) m ad., sul sentiero tra alpe fornale di sopra e alpe fornale di sotto, 1720 m s.l.m., esposizione e, 45°56’52’’n; fig. 2. esemplare di vipera berus della valle strona (foto p. eusebio bergò, 30/07/2001). 34 s. ghielmi et alii 08°13’34’’e. vipera berus 23/07/1995 (sg, obs.), 1 individuo adulto, sul sentiero che da campello monti conduce al lago capezzone, 2000 m s.l.m. esposizione e, 45°56’13’’n; 08°12’44’’e; 30/07/2001 (sg, peb, leg.) un esemplare rinvenuto ucciso all’interno di una piccola torbiera poco sopra alpe capezzone, 1850 m s.l.m., esposizione e-ne, 45°55’50’’n; 8°13’26’’e (mrsn r1982); 1 f, sul sentiero per lago capezzone, presso un impluvio con piccolo scolo d’acqua, 1620 m s.l.m., esposizione se, 45°56’21’’n; 8°13’14’’e; 14/08/2002 (sg, p. ghielmi, leg.), 1 juv. (neonato), trovato schiacciato sul sentiero nei pressi di alpe capezzone, 1800 m s.l.m., esposizione e, 45°56’06’’n; 08°13’12’’e (mrsn r2106); 27/04/2003 (sg, obs.), m adulto, sul sentiero tra alpe fornale di sopra e alpe fornale di sotto, 1690 m s.l.m., esposizione e, c.g. 45°56’52’’n; 08°13’34’’e; 12/08/2003 (sg, obs), 1 individuo adulto, sul sentiero che percorre il vallone a ovest di monte prevor, 1600 m s.l.m., esposizione so, 45°56’23’’n; 08°13’42’’e. per quanto riguarda zootoca vivipara la presenza dello stesso aplotipo nelle popolazioni di oropa e valle sorba da una parte e in quella di valle mastallone dall’altra, fa ragionevolmente pensare ad un flusso genico tuttora in atto o interrotto in tempi recenti tra queste popolazioni: questo fatto ci permette di ipotizzare l’esistenza di ulteriori nuclei di lucertola vivipara negli ambienti idonei dei versanti settentrionali ed occidentali della valle sesia. al momento quindi per z. v. carniolica si delineerebbe in piemonte una distribuzione incentrata sulle prealpi e alpi biellesi, vercellesi e del medio verbano e disgiunta rispetto alle popolazioni della ssp. vivipara che rimane invece relegata nella media e alta val d’ossola e nelle alpi cozie, in continuità rispettivamente con le popolazioni svizzere e francesi della specie. l’attuale distribuzione di v. berus in piemonte risulta isolata dall’areale principale della specie, dal momento che nell’adiacente territorio elvetico questa vipera manca nel canton vallese meridionale (sinistra orografica del fiume rodano) e nel canton ticino occidentale (destra orografica del fiume ticino). a tale proposito monney (in hofer et al., 2001) ipotizza una competizione tra vipera berus e v. aspis che avrebbe visto il prevalere di quest’ultima sulla prima, oppure l’eventualità che la specie non abbia mai raggiunto tali distretti, provenendo da est, per l’interposizione di una barriera biogeografica. merita un cenno il fatto che, come già osservato dal capra (1954), su alcuni esemplari dell’alto biellese, anche due dei sei individui da noi rilevati in valle strona presentavano una livrea che, in luogo della ordinaria banda dorsale a zig-zag continua, era caratterizzata da una losanga ampiamente frammentata e irregolare (fig. 2). un unico esemplare melanico, raccolto in valle elvo da c. grassi (1600 m s.l.m.) il 13/09/1970 è attualmente conservato nella collezione erpetologica del museo regionale di scienze naturali di torino (reperto privo di numero di catalogo), mentre nessuno degli individui da noi osservati mostrava simili caratteristiche. ringraziamenti un ringraziamento particolare va agli amici y. surget-groba e b. heulin (università di rennes, bretagna, francia) per la disponibilità nell’esecuzione delle analisi genetiche. s. scali ed un revisore anonimo hanno riletto una prima versione del presente articolo. 35nuove segnalazioni di zootoca e di vipera bibliografia andreone, f., gavetti e., volorio, p. (2005): gli anfibi e i rettili del museo di storia naturale “g. g. galletti” di domodossola: catalogo sistematico, con note storiche e riflessioni sul valore scientifico delle collezioni naturalistiche minori in italia. boll. mus. reg. sci. nat. torino, 23 (1), in stampa. andreone, f., sindaco, r. (editors), (1999): erpetologia del piemonte e della valle d’aosta. atlante degli anfibi e dei rettili. monografie. museo regionale di scienze naturali, torino 26 (1998): 1-283. baratelli, d., ghielmi, s. (1994): conferma della presenza di lacerta (zootoca) vivipara jaquin nella palude brabbia (lombardia, varese). boll. soc. tic. sci. nat. (lugano), 82 (1): 121-126. capra, f. (1954): la vipera berus in piemonte. ann. mus. civ. st. nat. genova 66: 301-312 + 2 tavv. chiariglione, a. (1994): le valli di lanzo. guida naturalistica. cierre edizioni, verona: 1287 pp. danini, g., baratelli, d. (1996): catalogo della collezione erpetologica del museo insubrico di storia naturale di induno olona (va). cristina giacoma (editor), 2000. atti i congresso nazionale della societas herpetologica italica (torino, 1996). museo regionale di scienze naturali, torino: 49-55 ferri, v., sindaco, r. (1999): vipera berus (linneus, 1758). in: erpetologia del piemonte e della valle d’aosta. atlante degli anfibi e dei rettili, p. 228-229. andreone f., sindaco r., eds. monografie. museo regionale di scienze naturali, torino 26. ghielmi, s., giovine, g., menegon, m., lapini, l., surget-groba, y., heulin, b. (2004): le attuali conoscenze sulla distribuzione di zootoca vivipara carniolica, mayer, böhme, tiedeman, bischoff, 2000 in italia (reptilia: lacertidae). v° congresso nazionale della societas herpetologica italica, 29 settembre-3 ottobre 2004, (riassunti): 29-30. hofer, u., monney, j.c., dusej, g.; con contributi di guisan a., fossati a., kessler e., jacquat m. s., honegger r.e., baur b. (2001): i rettili della svizzera. birkhauser, basel; boston; berlin: 1-202 pp. lapini, l., dall’asta, a., bressi, n., dolce, s., pellarini, p. (1999): atlante corologico degli anfibi e dei rettili del friuli-venezia giulia. edizioni del museo friulano di storia naturale, 43: 1-149. mayer, w., böhme, w., tiedemann, f., bischoff, w. (2000): on oviparous populations of zootoca vivipara (jaquin, 1787) in south-eastern central europe and their phylogenetic relationship to neighbouring viviparous and south-west european oviparous populations. herpetozoa 13 (1/2): 59-69. pascutto, t., ghielmetti, e. (1997): osservazioni sull’ornamentazione della vipera aspis (l.1758) e della vipera berus (l. 1758) dell’alta valsessera, in: studi e ricerche sull’alta valsessera. docbi-centro studi biellesi: pp. 117-126. richard, j., semenzato, m. (1992): nuovi rinvenimenti di bombina variegata (linnaeus, 1758) e lacerta (zootoca) vivipara jaquin, 1787 nella pianura veneta. atti soc. it. sci. nat. e mus. civ. st. nat., milano 132 (1991): 181-191. salmaso, r., osella, g. (1989): studi sulla palude del busatello (veneto lombardia) 27. l’erpetofauna. mem. mus. civ. st. nat., verona (ii serie), sez. biol. 7: 237257 36 s. ghielmi et alii sindaco, r. (1999): zootoca vivipara jaquin, 1787 – in: erpetologia del piemonte e della valle d’aosta. atlante degli anfibi e dei rettili, p. 206-207. andreone f., sindaco r., eds. monografie. museo regionale di scienze naturali, torino 26. sindaco, r., biggi, e., boano, g., delmastro, g.b. (2002): novitates herpetologicae pedemontanae. i. (amphibia, reptilia). riv. piem. st. nat. 23: 195-206 surget-groba, y., heulin, b., ghielmi, s., guillaume, c.-p., vogrin, n. (2002): phylogeography and conservation of the populations of zootoca vivipara carniolica. biol. cons. 106: 365-372 acta herpetologica 4(1): 103-107, 2009 effects of light and group size on the activity of wood frog tadpoles (rana sylvatica) and their response to a shadow stimulus katherine v. mcclure, jordan w. mora, geoffrey r. smith department of biology, denison university, granville, ohio 43023 usa. corresponding author. e-mail: smithg@denison.edu abstract. tadpoles are known to behaviorally respond to cues from aquatic predators. however, there are several additional factors that might affect tadpole behavior. we examined the influence of light conditions and group size on the activity of wood frog (rana sylvatica) tadpoles and their response to a simulated non-aquatic predator (i.e., a shadow stimulus). activity levels of undisturbed wood frog tadpoles were higher in larger groups (15 tadpoles) than in the smaller groups (5 tadpoles). activity following exposure to a simulated aerial predator (i.e., a shadow stimulus) was also higher in the larger groups of tadpoles than in the smaller groups. light conditions did not influence activity level in undisturbed tadpoles, but did affect the response to the shadow stimulus, with the greatest responses being observed under bright light conditions. our results suggest that the factors influencing tadpole activity can include a diverse range of factors and cues, including lighting conditions and group size. keywords. rana sylvatica, antipredator response, environmental effects. several studies have examined the activity levels of tadpoles in response to predators (anholt et al., 2000; eidietis, 2005; relyea, 2005; smith et al., 2008; smith and awan, 2009), with many finding that tadpoles are generally less active when predation risk is high (relyea, 2004; eidietis, 2005). however, there are several potential factors that might affect tadpole behavior in addition to cues from aquatic predators. for example, visual cues from non-aquatic predators (e.g., shadows) may represent an immediate threat to tadpoles and lighting conditions and group size may mediate perceived risk (e.g., group size dilutes predation risk, see spieler, 2005 for example in tadpoles) or the ability to actually perceive predation risk. one might expect non-aquatic or terrestrial predators to induce a startle or flight response as opposed to freezing or reduction in activity levels. similarly, shadows or visual stimuli indicate a predator is in the immediate vicinity whereas chemical cues indicate the presence of predators, with relatively little precision either spatially or temporally. thus, a flight response might be expected to a shadow stimulus rather than a decrease in activity. we examined the influence of light conditions and group size on the activity of wood frog (rana sylvatica) tadpoles, and their response to a simulated non-aquatic predator (i.e., a shadow stimulus). 104 k.v. mcclure, j.w. mora and g.r. smith multiple wood frog egg masses were collected from a local pond and hatched in the laboratory. tadpoles were gosner stage 26 (gosner, 1960) when used in our experimental trials. for each trial, 5 (low group size) or 15 (high group size) tadpoles were placed in plastic containers (33 × 17 × 11 cm) containing 4 l of aged tapwater. no individual tadpole was used in more than 1 trial. each treatment combination was replicated 12 times. trials were conducted from 1300 h to 1700 h. trials were conducted under low light (0.35 µmol photons s-1 m-2), medium light (2.90 µmol photons s-1 m-2), or high light (213.0 µmol photons s-1 m-2) conditions. these light conditions were chosen to provide a range of illumination likely to occur throughout the day, including dawn and dusk, when visually oriented terrestrial predators (e.g., birds) are active. lighting conditions were created by manipulating overhead lighting, and using a 100 w standard light bulb suspended at a constant height over the experimental container. after a 15 minute acclimation period, undisturbed tadpole activity was determined as the proportion of tadpoles moving (i.e., a scan sample of activity; lehner, 1996). a cut-out of an aerial predator was then passed over the container at a constant speed and tadpole activity immediately determined by a scan-sample of the proportion of tadpoles active. we used two-way anovas on the arcsine-square root transformed proportional data for undisturbed activity and the shadow-stimulated activity with group size and lighting condition as independent variables. we used fisher’s protected least squares difference post-hoc tests to further explore significant effects when appropriate. undisturbed activity levels were higher in the high density tadpole groups than in the low density groups (fig. 1a; f1,66 = 6.74, p = 0.012). light conditions had no effect on undisturbed activity levels (fig. 1a; f2,66 = 1.16, p = 0.32). the interaction between light conditions and group size was not significant (f2,66 = 0.23, p = 0.80). activity in response to the shadow stimulus was higher in the high density groups of tadpoles compared to the low density groups (fig. 1b; f1,66 = 6.46, p = 0.013). activity induced by the shadow stimulus was highest under bright light conditions (fig. 1b; f2,66 = 4.27, p = 0.018; fisher’s plsd: high vs. medium: p = 0.019; high vs. low: p = 0.010; low vs. medium: p = 0.80). the interaction between light conditions and group size was not significant (f2,66 = 1.88, p = 0.16). in the absence of any predator cues, activity levels of the wood frog tadpoles were higher in the larger groups than in the smaller groups. in addition, activity in response to the shadow stimulus was higher in the larger groups of tadpoles than in the smaller groups of tadpoles. rot-nikcevic et al. (2006) found that activity of r. sylvatica tadpoles increases in the presence of higher numbers of conspecifics, either real or simulated using mirrors (see also relyea, 2002). however, awan and smith (2007) found no effect of group size on wood frog tadpole activity level, although they tested groups of up to 8 tadpoles. it thus appears that wood frogs in general increase activity with tadpole density, with one or two exceptions. such a response may reflect lower perceived predation risk in larger groups (e.g., peacor, 2003) or a response to increased numbers of conspecifics (i.e., potential competitors (e.g., relyea, 2002). the higher activity in response to the shadow stimulus in larger groups may reflect the startle response of one or a few individual tadpoles being transmitted to others in the group, suggesting a potential benefit to larger tadpole group sizes (e.g., the confusion effect, miller, 1992; krakauer, 1995; krause and ruxton, 2002). light conditions did not influence activity level in the undisturbed tadpoles, but did affect the response of tadpoles to the shadow stimulus, with the greatest responses being 105response to shadow by wood frog tadpoles observed under bright light conditions. increased responses to predators in brighter conditions likely result from an increase in the ability of the tadpoles to visually perceive the predator. previous studies on tadpole activity suggest light conditions can influence activity patterns in tadpoles. bufo rufus tadpoles are inactive at night, but when a light is shined on them they become active (eterovick and sazima, 1999). xenopus laevis tadpoles alter their behavior as lighting conditions change (jamieson and roberts, 2000). activity level of bufo americanus tadpoles increases as light increases, and decreases on overcast days (beiswenger, 1977). similarly, activity of bufo bufo tadpoles is concentrated during the day (griffiths et al., 1988). in contrast, activity of bullfrog (rana catesbeiana) tadpoles is highest at dusk or at night (smith et al., 2007). in addition, lighting conditions may affect how tadpoles respond to predators. for example, fraker (2008) found that green frog (rana clamitans) tadpoles reduced their activity more in the presence of a predator during daylight hours than at other times of the day. in conclusion, our results suggest that the factors influencing tadpole activity can include a diverse range of factors and cues. in particular, group size can influence undisturbed activity and activity levels following a visual stimulus. lighting conditions can influence activity in response to a shadow stimulus. fig. 1. the effect of lighting conditions and group size on a) the activity level under undisturbed conditions, and b) the responses of wood frog (r. sylvatica) tadpoles to a shadow stimulus. means are given ± 1 se. 106 k.v. mcclure, j.w. mora and g.r. smith acknowledgments this experiment was conducted under approval of the denison university institutional animal care and use committee. references anholt, b.r., werner, e., skelly, d.k. (2000): effect of food and predators on the activity of four larval ranid frogs. ecology 81: 3509-3521. awan, a.r., smith, g.r. (2007): the effect of group size on the responses of wood frog tadpoles to fish. am. midl. nat. 158: 79-84. beiswenger, r.e. (1977): diel patterns of aggregative behavior in tadpoles of bufo americanus, in relation to light and temperature. ecology 58: 98-108. eidietis, l. (2005): size-related performance variation in the wood frog (rana sylvatica) tadpole tactile-stimulated startle response. can. j. zool. 83: 1117-1127. eterovick, p.c., sazima, i. (1999): description of the tadpole of bufo rufus with notes on aggregative behavior. j. herpetol. 33: 711-713. fraker, m.e. (2008): the influence of the circadian rhythm of green frog (rana clamitans) tadpoles on their antipredator behavior and the strength of the nonlethal effects of predators. am. nat. 171: 545-552. gosner, k.l. (1960): a simplified table for staging anuran embryos and larvae with notes on identification. herpetologica 16: 183-190. griffiths, r.a., getliff, j.m., mylotte, v.j. (1988): diel patterns of activity and vertical migration in tadpoles of the common toad, bufo bufo. herpetol. j. 1: 223-226. jamieson, d., roberts, a. (2000): repsonses of young xenopus laevis tadpoles to light dimming: possible roles for the pineal eye. j. exp. biol. 203: 1857-1867. krakauer, d.c. (1995): groups confuse predators by exploiting perceptual bottlenecks: a connectionist mode of the confusion effect. behav. ecol. sociobiol. 36: 421-429. krause, j., ruxton, g.d. (2002): living in groups. oxford university press, oxford. lehner, p.n. (1996): handbook of ethological methods, 2nd edition. cambridge university press, cambridge. miller, r.c. (1922): the significance of the gregarious habit. ecology 3: 122-126. peacor, s.d. (2003): phenotypic modifications to conspecific density arising from predator risk assessment. oikos 100: 409-415. relyea, r.a. (2002): competition-induced plasticity in tadpoles: consequences, cues, and connections to predator-induced plasticity. ecol. monogr. 72: 523-540. relyea, r.a. (2004): fine-tuned phenotypes: tadpole plasticity under 16 combinations of predators and competitors. ecology 85: 172-179. relyea, r.a. (2005): the heritability of inducible defenses in tadpoles. j. evol. biol. 18: 856-866. rot-nikcevic, i., taylor, c.n., wassersug, r.j. (2006): the role of images of conspecifics as visual cues in the development and behavior of larval anurans. behav. ecol. sociobiol. 60: 19-25. 107response to shadow by wood frog tadpoles smith, g.r., awan, a.r. (2009): the roles of predator identity and group size in the antipredator responses of american toad (bufo americanus) and bullfrog (rana catesbeiana) tadpoles to different predators. behaviour 146: 225-243. smith, g.r., burgett, a.a., sparks, k.a., temple, k.g., winter, k.e. (2007): temporal patterns in bullfrog (rana catesbeiana) tadpole activity: a mesocosm experiment on the effects of density and bluegill sunfish (lepomis macrochirus) presence. herpetol. j. 17: 199-203. smith, g.r., burgett, a.a., temple, k.g., sparks, k.a., winter, k.e. (2008): the ability of three species of tadpoles to differentiate among potential fish predators. ethology 114: 701-710. spieler, m. (2005): can aggregative behaviour of phrynomantis microps tadpoles reduce predation risk. herpetol. j. 15: 153-157. acta herpetologica 17(1): 37-43, 2022 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-10188 first report on two loggerhead turtle (caretta caretta) nests in the aeolian archipelago (southern italy) monica francesca blasi1,*, sandra hochscheid2, roberta bardelli3, chiara bruno1, carolina melodia1, perla salzeri1, paolo de rosa4, paolo madonia5 1 filicudi wildlife conservation, location stimpagnato filicudi, 98055 lipari (me), italy 2 marine turtle research group, department of marine animal conservation and public engagement, stazione zoologica anton dohrn, via nuova macello 16, 80055, portici, italy 3 department of earth and marine science, university of palermo, via archirafi, 22, 90123, palermo, italy 4 attivastromboli, via marina, 98050 stromboli (me), italy 5 istituto nazionale di geofisica e vulcanologia, sezione di catania-oe, piazza roma 2, 95125 catania, italy *corresponding author. e-mail: blasimf@yahoo.com submitted on: 2021, 11th january; revised on: 2021, 14th june; accepted on: 2022, 2nd january editor: emilio sperone abstract. the aeolian archipelago (southern tyrrhenian sea, italy) hosts important foraging/overwintering habitats for mediterranean loggerhead sea turtles (caretta caretta), although nesting sites have never been documented. this study reports the data of two nesting events occurred in summer 2019 at stromboli and lipari islands. a hatchling success of 20.69 % (18 hatchlings from 87 eggs) was recorded at stromboli, while a complete hatchling unsuccess characterised the lipari nest, where 111 eggs were deposited. data acquired during the monitoring of the nests suggest that combined factors, mainly temperature, beach morphology, and sand composition, could be the causes for the low success of these nesting events. keywords. anoxic conditions, lipari, mediterranean sea, stromboli, temperature. introduction the aeolian archipelago (sicily, italy), composed of 7 islands and located in the southern tyrrhenian sea (italy) (fig. 1), is of volcanic origin with both extensive neritic and oceanic habitats within short distances (favalli et al., 2005), which provide optimal foraging and overwintering grounds for both immature and adult loggerhead turtles (blasi et al., 2016; blasi and mattei, 2017; blasi et al., 2018) and fall within the historical nesting range of loggerhead turtle, although the 1960s quotations were not supported by documented data (mingozzi et al., 2007). italy hosts regular nesting events along the ionic coasts of the southern calabria and in the pelagian islands (linosa and lampedusa; mingozzi et al., 2008). irregular nesting events are also reported on the coasts of sicily, sardinia, apulia and the ionic coasts of basilicata and calabria. however, in recent years, a significant increase in the numbers of nests along the italian coasts has been reported, with 30-40 nests estimated per year up to 70 nests recorded in 2018, through a survey carried out on the ionian coasts of calabria, facing the messina strait (mingozzi et al., 2007) and in sicily (casale et al., 2012). in sicily there are numerous suitable coasts for loggerhead turtle nesting; nesting events are also occasionally reported by tourists or local people. for example, in 2011, seven nests were reported along the coasts close to palermo and on the southern sicily (casale et al., 2012). even though many potential nesting sites are not ade38 monica francesca blasi et alii guatelly monitored and consequently the actual nesting level and distribution in several areas remain partially unknown. here we report data on two nesting events by loggerhead turtles at stromboli and lipari islands (aeolian archipelago) in summer 2019. our data represent the first official documentation showing that the aeolian archipelago could host irregular nesting events, and suggest a higher monitoring and conservation efforts to increase the chance of positive hatchings of these sites. materials and methods nesting sites two loggerhead turtle nests were surveyed and monitored during summer 2019 at stromboli and lipari islands, respectively. these two islands are stratovolcanoes, built by alternating hard lava flows and pyroclastic deposits of different sizes (ashes, pumices, scoriae, lithic fragments, and volcanic bombs). stromboli, characterized by a continuous volcanic activity during the last 2,000 years, has a basaltic nature, with abundance of dark fe-mg minerals (rosi et al., 2013). its activity generated beaches composed of large (meters) basaltic rock blocks mixed to abrasive textures of sand and pebbles, with sizes up to few tens of centimetres. the dark colour of this substrate encourages the adsorption of the thermal infrared solar radiation, with surface daytime summer temperatures that can exceed 50 °c. the volcanic products of lipari island are more acidic, i.e., with major abundances of whitish silica. solid wastes of pumice extraction from a close coastal quarry, presently inactive, are transported to the beach (anzidei et al., 2017). furthermore, during the last years, artificial replenishments of the beach were carried out, causing dramatic changes in its original morphology and lithological nature. the first loggerhead turtle nested on the 21st of june at 7:35 am (gmt+1) at scari beach, in the north-east of stromboli island (15.2428°e, 38.8034°n wgs84), about 1 km n of the main harbour and 14 m from the seashore (fig.1). the site is characterized by a berm composed of large-sized pebbles, with a slope of 16.9°, located seaward of a dumping site. an anchor buoy field is present in its immediate neighbourhood and a sailing boat (about 50’ long) was stranded in front of the nest few days after its emplacement, and stayed there for the whole incubation period. the nesting event was reported by two tourists and lasted about 2 hours. the second loggerhead turtle nested on the 5th of july at 9:00 pm (gmt+1) in the heavily urbanised canneto beach, in the north-east of lipari island (14.9617°e, 38.4937°n wgs84), at its inner end (fig. 1). the turtle was disturbed by people with light and noise and consequently multiple nesting attempts were made before the final site was chosen. facilities and roads run along the coast and, during the summer season, many tourists overcrowd the beaches all day long for the presence of bathhouses. a runoff channel is located in the proximity of the nesting site. nests monitoring and data collection standard fences were constructed to protect both sites immediately after the nesting events (nooren and claridge, 2002). a 24-hour monitoring was provided for each nest by filicudi wildlife conservation volounteers during the whole incubation period, for checking sand temperatures and preventing predator/human intrusions. two onset usb data loggers, equipped with 12-bit temperature smart sensors, were provided by the istituto nazionale di geofisica e vulcanologia (ingv); temperature sensors were buried at 10 and 40 cm depths in the proximity of the nests, and acquired data every 30 minutes in order to provide non-aliased hourly data. a viaradio remote reolink ip rcl-410 w (4.0 mp) night camera was installed at stromboli (courtesy of attiva stomboli association). the duration of incubation period for both nests was predicted using the recorded mean temperature during the middle third of the incubation period as reported by kaska et al. (1998) and other authors (reid, 2005; reid et al., 2009; houghton and hays, 2001; godley et al., 2001). the hatchling phase was continuously monitored with at least two operators for each nest, equipped with red filter headlights. during the hatchling phase at stromboli, date and time of the emerging events were recorded and body size measurements (scl, scw, spl, spw, and weight) of hatchlings were collected, with a calibre and a scale, following standard protocols (bolten et al., 1993; nooren and claridge, 2002). dead hatchlings were stored in tubes provided with 70% ethanol solution, encoded with id number, date, and time of collection. at the end of each emergence event, the hatchlings were left free inside the fenced area for a few minutes, to allow the imprinting process. afterwards, considering that lights near to the beach and ostacles on the sand are a considerable risk to the hatchlings (demetropoulos and hadjighristophorous, 1995), they were placed in a basket filled with sand, which was transported offshore by boat, and then released directly into the sea. as a matter of fact the light might attract them in the wrong direction or slow down the race to the sea extanding the period hatchlings remain on land. moreover, obsta39loggerhead sea turtle nests in the aeolian islands cles on the sand surface and in front of nest could also may extend the time to arrive at sea for hatchlings on the beaches. finally, the longer hatchlings reaminding on land the higher is the risk of predation as well (demetropoulos and hadjighristophorous, 1995). the features of the beach and the set of circustances did not allow to let the hatchlings spontaneously reach the sea. the beach was characterized of large rocks, and pieces of bamboo cane. furthermore, in front of the nest, there is an area where many sailing boats are in the harbour and during the night, a sailboat ran around on the beach. at the end of the last hatching phase, fixed at 72 hours fig. 1. on the top, location of study area and loggerhead turtle nests. on the lower left (a), particular of the scari-stromboli nest with in evidence (1) the dumping site, (2) the buoy field, and (3) the sailing boat stranded on the beach. on the lower right (b), particular of the canneto-lipari nest with in evidence (1) the cemented road (2) the runoff channel and (3) the boat storage. the nest removable protective coverages were indicated for both nests. 40 monica francesca blasi et alii (demetropoulos and hadjighristophorous, 1995) from the last emergence event, the nest was excavated. after excavation, unhatched eggs were counted, weighed, and the developmental stage of embryos assessed according to standard classification (miller, 1985), using the methodology stated by kobayashi et al. (2017). three embryogenetic classes were used in this study: a) ≤ 21st stages, b) between 22nd-29th stages, and c) ≥ 30th stages, which included pipping and not emerged hatchlings (for detailed embryonic stage description see miller, 1985). finally, each unhatched/hatched egg/embryo was stored in a plastic bag, encoded with an id number, time, and date of collection, and immediately frozen at -20 °c. core drill samples for sand analyses and nest measurements were taken, including nest minimum and maximum depths and distance from the sea. results temperature monitoring temperature data acquired at 10 cm and 40 cm depths are reported for both nests (fig. 2a). in the stromboli nest the temperature at 10 cm depth was over the upper threshold for ideal egg maturation (32 °c) during the majority of the study period. in particular, higher values were reported on the 11th and 12th of july and since the 17th of july, every afternoon. on the other hand, temperatures < 26 °c were never recorded, with the exception of the 17th of july. at 40 cm the temperature regime was more stable, with a daily oscillation not higher than 1 °c, one order of magnitude less than that one observed at 10 cm. an approximately 0.5 °c temperature passing of the maximum threshold was observed in the period between the 29th of july and the 4th of august, after which temperature oscillations remained permanently below this limit. in addition, 2 peaks over the maximum threshold of temperature were recorded on the 15th and the 16th of july respectively, as a consequence of a rainfall event of 1 mm. the minimum temperatures recorded at 40 cm depth were over the lower threshold (26 °c) during the entire period, except for a few days in the middle of july. temperatures at lipari showed lower variations (fig. 2b). the maximum daily oscillation was < 5 °c, at the end of august and at 10 cm depth, and a few decimals of °c at 40 cm. values over the upper threshold were recorded only after the 17th of august at 10 cm depth. the lower temperature values recorded were between 29 and 31.5 °c at 40 cm depth, and between 27 and 30 °c at 10 cm. hatchlings at stromboli 87 eggs were found in the nest (table 1). the nest had a width of 15 cm (maximum distance between two eggs) and a depth of 18 cm and 33 cm to top and bottom of the eggs chamber, respectively. the incubation period ranged 46 days in stromboli with a hatchling duration of 46-51 days. particularly, 3 hatchling events were recorded and 2 excavations performed for a total of 18 emerged hatchlings (20.69%): – on the 6th of august, between 9:00 pm and 11:46 pm, with 6 emerging hatchlings, – on the 7th of august, at 2:30 pm, with a single emerging hatchling immediately dead, probably for the high surface temperature (about 39 °c): – on the 8th of august, between 5:56 pm and 8:25 pm, with 8 emerging hatchlings, one of which died shortly after; – on the 10th of august, at 11:00 pm, after two days since the last emersion, we cautiously excavated the most surficial portion of the nest, finding other two alive hatchlings stuck between basaltic stones; – on the 11th of august, at 11:00 pm, after another day without any activity, we continued the excavation discovering the last alive hatchling blocked in the sand. the remaining 69 eggs (79.31%) hosted unhatched embryos at ≤ 21st embryonic stages (table 1). the lipari nest was excavated at the 54th day of incubation, on the 28th of august at 8:03 pm, since no emergences had occurred several days after the predicted incubation period (i.e., 45-50) (kaska et al., 1998; reid, 2005; godley et al. 2001), finding 111 eggs with unhatched embryos. twenty-one eggs (18.92%) contained embryos at ≤ 21st developmental stages, 70 eggs (63.06%) had embryos at 22nd-29th stages, and 16 eggs (14.41%) had embryos at ≥ 30th stages (table 1), 11 of them (9.91%) had pipped (stage 31a) (table 1). additionally, 4 not-emerged hatchlings (stage 31b) were found dead at the upper part of the nest (3.60%). the nest had a width of 19 cm (maximum distance between two eggs) and a depth of 16 cm and 35 cm to top and bottom of the eggs chamber, respectively. average body size measurements of hatchlings for both nests are reported in table 2. discussion this study is the first quantitative documentation of two nesting events of loggerhead turtle in the aeolian archipelago, with 18 hatchlings (20.69%) from 87 41loggerhead sea turtle nests in the aeolian islands eggs at stromboli and no successfully released hatchlings at lipari (111 eggs). both nests were laid during the seasonal period of maximum frequency for the species in italy and in the mediterranean sea (giacoma et al., 2011). similarly, the incubation period and the clutch size fall within the normal range for the species (giacoma et al., 2011). different reasons could be at the base of the scarce hatchling success at stromboli and its total unsuccess at lipari. in the case of stromboli, the high temperatures recorded inside the nest, due to the color and composition of sand, could have been the main reason of the low percentage of hatchlings. the nest showed temperatures at 10 cm in depth over the upper threshold for ideal egg maturation (32 °c), with particular reference to the later incubation period. conversely, temperatures were never below the lower threshold. studies on nests with similar temperature ranges report on a low emergence success (chu et al., 2008; read et al., 2012), especially during the fig. 2. nest temperature at 10 cm and 40 cm depths recorded at stromboli (a) and lipari (b) during the nesting periods. ideal temperature range for egg maturation, hatchlings and rainfall events are also reported for stromboli. 42 monica francesca blasi et alii last days of incubation (matsuzawa et al., 2002; maulany et al., 2012). finally, the presence of basaltic products mixed to sand in the nests could have influenced escape success (i.e., 3 hatchling events and 3 blocked hatchlings) and duration (from 46-51 days) of hatchling phase (foley et al., 2006). at lipari nest temperature was always within the thresholds for ideal egg maturation, so different reasons should be invoked for explaining the complete hatchling unsuccess. possible explanations could be found in the absorption of parassites/contaminants from the material constituting the partially artificial beach (alava et al., 2006), fauvored by a rainfall event during the middle third of the incubation period (foley et al., 2006), or in anoxic conditions (margaritoulis, 2005; lolavar and wyneken, 2015) due to the presence of the very fine particulate created by the mechanical crushing of the pumice. from this study, we have learned that the aeolian archipelago may ideally host irregular nesting sites for loggerhead turtles. a higher monitoring and conservation effort is recommended for these sites to increase the chance of positive hatchings. acknowledgements we thank all the people and institutions that cooperated for protecting the nests and gave material and moral support to this study: among these, the coast guard and the municipality of lipari, sonia d’ambra and franco zurro for the logistic support, the aeolian islands preservation foundation for the economic support, aldo and miriam for their report of the turtle nesting at stromboli, the volunteers and students of filicudi wildlife conservation, enpa lipari, blu bar of canneto, and many others. permits to monitor, collect and manipulate turtles during the study period were provided by both the italian ministry of environment (for stazione zoologica anton dohrn: prot. n° 0024471 del 22-11-2016. for filicudi wildlife conservation: prot n° 0011903 del 01-062016) and the dipartimento regionale dello sviluppo rurale e territoriale of the sicilian region (servizio 3 u.o 1, d.d.g. n° 00115 del 20/02/2020; 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(2016): interaction of loggerhead turtles (caretta caretta) with traditional fish aggregating devices (fads) in the mediterranean sea. herpetol. conserv. bio. 11: 386-401. blasi, m.f., tomassini, l., gelippi, m., careddu, g., insacco, g., polunin, n.v.c. (2018): assessing resource use patterns of mediterranean loggerhead sea turtles caretta caretta (linnaeus, 1758) through stable isotope analysis. eur. zool. j. 85: 71-87. bolten, a.b., martins, h.r., bjorndal, k.a., gordon, j. (1993): size distribution of pelagic-stage loggerhead table 1. percentage of hatchlings and embryos at different development stages (miller, 1985) for stromboli and lipari nests. stromboli (%) (n=87) lipari (%) (n=111) hatchlings successfully released 18.40 0 pre-emergence death 0 3.60 post-emergence death 2.29 0 embryos ≤ 21 79.31 18.92 22-29 0 63.06 ≥ 30 0 14.41 table 2. average morphometric data for stromboli and lipari hatchlings. scl = straight carapace length; scw = straight carapace width; spl = straight plastron length; spw = straight plastron width (bolten et al., 1993). morphometrics data mean (±sd) stromboli mean (±sd) lipari scl (mm) 39.57 ± 3.73 37.26 ± 2.64 scw (mm) 30.31 ± 4.35 27.48 ± 3.43 spl (mm) 30.32 ± 5.19 26.96 ± 6.19 spw (mm) 29.41 ± 4.11 23.36 ± 1.23 weight (gr) 14.28 ± 1.27 11.6 ± 2.79 43loggerhead sea turtle nests in the aeolian islands sea turtle (caretta caretta) in the waters around the azores and madeira. arquipel. cienc. biol. mar. 11: 49-54. casale, p., palilla, g., salemi, a., napoli, a., prinzi, m., genco, l., bonaviri, d., mastrogiacomo, a., oliverio, m., lo valvo, m. 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(1998): natural temperature regimes for loggerhead and green turtle nests in the eastern mediterranean. can. j. zool. 76: 723-729. kobayashi, s., wada, m., fujimoto, r., kumazawa, y., arai, k., watanabe, g., saito, t. (2017): the effects of nest incubation temperature on embryos and hatchlings of the loggerhead sea turtle: implications of sex difference for survival rates during early life stages. j. exp. mar. biol. ecol. 486: 274-281. lolavar, a., wyneken, j. (2015): effect of rainfall on loggerhead turtle nest temperatures, sand temperatures and hatchling sex. endanger. species res. 28: 235-247. margaritoulis, d., argano, r., baran, i., bentivenga, f., bradai, m.n., camiñas, j. a., casale, p., de metrio, g., demetropoulos, a., gerosa, g., godley, b. j., haddoud, d. a., houghton, j., laurent, l., lazar, b. (2003): loggerhead turtles in the mediterranean sea: present knowledge and conservation perspective. in: loggerhead sea turtle, pp. 175-198. bolten, a.b., eds, smithsonian institution press, washington d.c., usa. margaritoulis, d. (2005): nesting activity and reproductive output of loggerhead sea turtles, caretta caretta, over 19 seasons (1984-2002) at laganas bay, zakynthos, greece: the largest rookery in the mediterranean. chelonian conserv. biol. 4: 916-929. matsuzawa, y., sato, k., sakamoto, w., bjorndal, k.a. (2002): seasonal fluctuations in sand temperature: effects on the incubation period and mortality of loggerhead sea turtle (caretta caretta) pre-emergent hatchlings in minabe, japan. mar. biol. 140: 639-646. maulany, r.i., booth, d.t., baxter, g.s. (2012): emergence success and sex ratio of natural and relocated nests of olive ridley turtles from alas purwo national park, east java, indonesia. copeia. 4: 738-747. miller, j.d. (1985): embryology of marine turtles. in: biology of the reptilia, pp. 269-328. gans, c., billett, f., maderson, p.f.a., eds, john wiley & sons, new york. mingozzi, t., masciari, g., paolillo, g., pisani, b., russo, m., massolo, a. (2007): discovery of a regular nesting area of loggerhead turtle caretta caretta in southern italy: a new perspective for national conservation. biodivers. conserv. 16: 3519-3541. nooren, h., claridge, g. (2002): guidelines for turtle hatchery management, turtle foundation, hauptstr. 1, d-82541, ammerland, germany. read, t., booth, d.t. limpus, c.j. (2012): effect of nest temperature on hatchling phenotype of loggerhead turtles (caretta caretta) from two south pacific rookeries, mon repos and la roche percée. aust. j. zool. 60: 402-411. reid, k.a. (2005): incubation conditions of the loggerhead sea turtle caretta caretta in kyparissia bay, western peloponnesus, greece. doctoral dissertation. university of aberdeen. reid, k.a., margaritoulis, d., speakman, j.r. (2009): incubation temperature and energy expenditure during development in loggerhead sea turtle embryos. j. exp. mar. biol. ecol. 378: 62-68. rosi, m., pistolesi, m., bertagnini, a., landi, p., pompilio, m., di roberto, a. (2013): stromboli volcano, aeolian islands (italy): present eruptive activity and hazards. geol. soc. lond. mem. 37: 473-490. xi international symposium on the mediterranean lacertid lizards marco mangiacotti1, pietro lo cascio2, claudia corti2, marta biaggini2, miguel angel carretero2, petros lymberakis2 the directional testes asymmetry increases with temperature in seven plateau brown frog (rana kukunoris) populations hai ying li1, man jun shang2, jie guo2, bo jun chen2, peng zhen chen2, tong lei yu1,* influence of tail injury on the development of neotropical elegant treefrog tadpoles ana glaucia da silva martins1,#, raoni rebouças2,3,*,#, isaias santos1, adão henrique rosa domingos1, luís felipe toledo2 the effect of weight and prey species on gut passage time in an endemic gecko quedenfeldtia moerens (chabanaud, 1916) from morocco jalal mouadi1,*, panayiotis pafilis2, abderrafea elbahi3, zahra okba3, hassan elouizgani3, el hassan el mouden4, mohamed aourir1 a contribution to the knowledge on the diet and food preferences of darevskia praticola (reptilia: lacertidae)§ emiliya vacheva*, borislav naumov first report on two loggerhead turtle (caretta caretta) nests in the aeolian archipelago (southern italy) monica francesca blasi1,*, sandra hochscheid2, roberta bardelli3, chiara bruno1, carolina melodia1, perla salzeri1, paolo de rosa4 and paolo madonia5 threatened and extinct amphibians and reptiles in italian natural history collections are useful conservation tools franco andreone1,*, ivano ansaloni2, enrico bellia3, andrea benocci4, carlotta betto5, gabriella bianchi6, giovanni boano7, antonio borzatti de loewenstern8, rino brancato9, nicola bressi10, stefano bulla11, massimo capula12, vincenzo caputo barucchi13, p re-description of external morphology and factors affecting body and tail shape of the stone frog tadpoles’ brena da silva gonçalves1,*, carla. d. hendges2, bruno madalozzo2, tiago g. santos2,3 preliminary data on the diet of chalcides chalcides (squamata: scincidae) from northern italy andrea ciracì1, edoardo razzetti2, maurizio pavesi3, daniele pellitteri-rosa4,* the high diversity and phylogenetic signal of antipredator mechanisms of the horned frog species of proceratophrys miranda-ribeiro, 1920 (amphibia: anura: odontophrynidae) cássio zocca1,2,*, ricardo lourenço-de-moraes3, felipe s. campos4, rodrigo b. ferreira1,2,5 standard karyotype and nucleolus organizer region of neotropical blindsnake typhlops brongersmianus (serpentes: typhlopidae) josé augusto ruiz garcía, alejandra hernando laboratorio de herpetología, fac. de ciencias exactas y naturales y agrimensura, universidad nacional del nordeste, av. libertad 5450 w 3404 aas, (3400) corrientes, argentina. corresponding author. e-mail: ahernando@infovia.com.ar abstract. the karyotype of typhlops brongersmianus is reported on the basis of specimens from north-eastern argentina. the conventional giemsa staining showed that the species has 2n = 34 chromosomes, including 8 pairs of macrochromosomes and 9 pairs of microchromosomes. ag-nor staining revealed the nors location on a pair of macrochromosomes. the chromosome number and karyotypic morphology are similar to those of neotropical typhlopid previously karyotyped. keywords. cytogenetics, scolecophidia, typhlops brongersmianus, northeastern argentina. among serpentes, two major lineages are recognized, alethinophidia and scolecophidia. alethinophidia include most of the extant snakes while scolecophidia is considered a basal clade that comprises 305 species of small burrowing snakes with shiny scales, reduced eyes and traces of the pelvis girdle in most taxa (pough et al., 2005). this group, commonly known as blindsnakes, is found on all continents except antarctica and comprises three families: typhlopidae, anomalepididae and leptotyphlopidae. despite their diversity, karyological knowledge of scolecophidia lineage is still very limited. according to olmo and signorino (2005) chromosome data are based on non differentially stained karyotypes for seven species that constitutes about 2% of the total blindsnakes species currently recognized (olmo, 2005). in this study, we describe the karyotype and the location of ag-nors of neotropical typhlops brongersmianus based on specimens collected from northeastern argentina. six specimens of t. brongersmianus (four adult males and two females) were analyzed. voucher specimens are deposited in the herpetological collection of the universidad nacional del nordeste (unnec) under the following catalog numbers: unnec 08648 (f) from pampa del indio (26°03’s, 59°55’w); unnec 08178 (m) from napenay (26º44’s, 60°37’w); unnec 08099, unnec 08848 (mm) from corrientes (27°28’s, 58°51’w); unnec 08512 (f) from san cosme (27°22’s, 58°31’w); unnec 08812 (m) from paso de la patria (27°19’s, 58°35’w). acta herpetologica 2(2): 117-120, 2007 issn 1827-9643 (online) © 2007 firenze university press 118 j.a. ruiz garcia and a. hernando animals were injected with 0.10.5 ml of 0.1% of colchicine solution 4 h before dissection. the chromosomes were obtained from intestinal epithelium and testes squash as described by kezer and sessions (1979). the preparations were stained with giemsa solution at ph 7.0. the nucleolar organizer regions (nors) were detected by silver nitrate staining (howell and black, 1980). the measurements of chromosomes arms were made on ten metaphase plates. macrochromosomes were classified according to sessions (1996). the diploid chromosome number of typhlops brongersmianus was 2n = 34 (16 m + 18 m) with clear demarcation between microand macrochromosomes (fig. 1). the macrochromosomes pairs could be divided into two groups by size. the first group contained large chromosomes (pair 1-3) which were metacentric. pairs 4-8 composed the second group with metacentric and submetacentric chromosomes (table 1). several microchromosomes showed metacentric morphology. in diplotene cells of males were observed 8 macrobivalents and 9 microbivalents (fig. 1b). heteromorphic sex chromosomes were not distinguished. the ag-nors were always located at the distal region of the long arm of the third macrochromosome pair (fig. 1c). to date about 10% of snakes have been karyotyped mostly using conventional staining methods (olmo, 2005). the aletinophidia lineage exhibits high chromosome variability, especially colubroidea clade. diploid number range from 2n = 24 to 2n = 52 and 52 different karyotypes were described (olmo, 2005; olmo and signorino, 2005). among scolecophidia lineage, no anomalepididae was analyzed chromosomically while one leptotyphlopidae and five bisexual and one unisexual typhlopidae species were studied (wynn et al., 1987; das and ota, 1998; olmo and signorino, 2005). three diploid chromosome number (2n = 32, 34 and 36) and five different karyotypes are known (table 2). the highest diploid number is found in leptotyphlops phillipsi (2n = 36) whereas two typhloid species of rhinotyphlops analyzed share the same chromosome number (2n = 32) differing r. schlegelii from r. simonii by the macrochromosome morphologies. only three species of the large pantropical blindsnake genus typhlops have been investigated chromosomally (olmo and signorino, 2005). there are intrageneric differences in diploid, macrochromosome and microchromosomes numbers and macrochromosome morphologies. t. jamaicensis and t. richardi from central america were both reported to have 2n = 34 (16 m + 18 m) (wynn et al., 1987) and the karyotypes are comparable to our sample of t. brongersmianus. the karyotype of the old world species t. punctatus (2n = 32) has fig. 1. a) giemsa-stained karyotype of typhlops brongersmianus (2n = 34, 16 m + 18 m) (unnec 08648, f). b) male meiosis in t. brongersmianus showing 17 bivalents. c) stained metaphase of t. brongersmianus showing the two macrochromosome bearing nors. a b c 119standard kariotype and nucleolus organizer region of neotropical blindsnake distinctive macrochromosome and microchromosome numbers and differ from the one shared by the neotropical species. the unisexual ramphotyphlops braminus has been proposed to be triploid (2n = 3x = 42) (wynn et al., 1987; das and ota, 1998). in snakes only few banding studies exist. among alethinophidia lineage nors location for about 93 species has been studied (olmo and signorino, 2005). except in natricinae and several species of colubrinae (colubridae subfamilies), snakes belonging to boidae, colubridae and viperidae families exhibit a single pair of nor-bearing microchromosomes (moreno et al., 1987; porter et al., 1991, 1994; camper and hanks, 1995; aprea et al., 2006). it has been assumed that these microchromosomes are homologous representing microchromosomal nors a primitive condition among snakes (camper and hanks, 1995). in scolecophidia we document nors location by ag-staining method. t. brongersmianus presents ag-nors at the peritelomeric region on the long arm of chromosome 3. the same position of nors was observed in t. vermicularis (16 m + 16 m) (g. odierna, pers. comm.). further analyses using various banding techniques for karyotype characterization of scolecophia blindsnakes are necessary to establish a pattern on chromosome evolution in this lineage. table 1. quantitative characteristics of typhlops brongersmianus chromosomes. l.r. (%): percentage length of chromosome pair over total genome length. c.i.: centromeric index chromosome 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 l.r. 21.3 18.3 13.8 7.0 6.2 5.5 4.7 4.2 2.6 2.5 2.3 2.2 2.1 2 1.9 1.8 1.7 c.i. 0.42 0.42 0.43 0.39 0.31 0.32 0.33 0.30 ---------table 2. diploid number (2n) and karyotype description (i, ii, iii) of species of scolecophidia cytogenetically studied. karyotype description = i: total number of biarmed macrochromosomes, ii: total number of uniarmed macrochromosomes, iii: total number of microchromosomes. families species 2n: karyotype description references leptotyphlopidae leptotyphlops phillipsi 36: 10, 6, 20 werner (1959)* typhlopidae rhinotyphlops schlegelii 32: 14, 2, 16 fischman et al. (1972)* rhinotyphlops simonii 32: 10, 6, 16 werner (1959)* typhlops jamaicensis 34: 16, 0, 18 wynn et al. (1987) typhlops richardi 34: 16, 0, 18 wynn et al. (1987) typhlops brongersmianus 34: 16, 0, 18 this study typhlops punctatus 32: 20, 0, 12 de smet (1978)* ramphotyphlops braminus 3n: 42: 21, 0 wynn et al. (1987); das and ota (1998) *references were extracted from olmo and signorino (2005) 120 j.a. ruiz garcia and a. hernando acknowledgements funds for the laboratory work were provided by the secretaría general de ciencia y técnica de la universidad nacional del nordeste (argentina). references aprea, g., gentilli, a., zuffi, m.a.l., odierna, g. (2006): the karyology of vipera aspis, v. atra, v. hugyi, and cerastes vipera. amphibia-reptilia 27: 113-119. camper, j.d., hanks, b. (1995): variation in the nucleolus organizer region among new world snakes. j. herpetol. 29: 468-471. das, i., ota, h. (1998): a checklist of chromosome numbers of south asian reptiles. hamadryad 23: 179-193. howell, w.m., black, d.a. (1980): controlled silver-staining of nucleolus organizer regions with a protective colloidal developer: a 1step method. experientia 36: 1014-1015. kezer, j., sessions, s.k. (1979): chromosome variation in the plethodontid salamander, aneides ferreus. chromosoma 71: 65-80. moreno, r., navarro, j., iturra, p., veloso, a. (1987): the karyotype of philodryas chamissonis (colubridae). identification of nucleolar organizer regions (nor) and sex chromosomes by banding methods. brazil. j. genetics 10: 497-506. olmo, e. (2005): rate of chromosome changes and speciation in reptiles. genetica 125: 185-203. olmo, e., signorino, g. (2005): chromorep: a reptile chromosomes database. internet references: http://193.206.118. 100/professori/chromorep.pdf, 15.09.06. porter, c.a., haiduk, m.w., de queiroz, k. (1994): evolution and phylogenetic significance of ribosomal gene location in chromosomes of squamate reptiles: systematic and evolutionary implications. copeia 1994: 302-313. porter, c.a., hamilton, m.j., sites, jr., j.w., baker, r.j. (1991): location of ribosomal dna in chromosomes of squamate reptiles: systematic and evolutionary implications. herpetologica 47: 271 -280. pough, f.h., janis, c.m., heiser, j.b. (2005): vertebrate life. pearson prentice hall, new jersey, eeuu. sessions, s.k. (1996): chromosomes: molecular. in molecular systematics, p. 121-168. hillis, d., moritz, c., mable, b., eds, sinauer associates, sunderland, ma. wynn, a.h., cole, ch.j., gardner, a.l. (1987): apparent triploidy in the unisexual brahminy blind snake, ramphotyphlops braminus. am. mus. novitates 2868: 1-7. acta herpetologica 2006 2 discussion of the origin and evolution of the oral apparatus of anuran tadpoles discussions of the origin and evolution of the oral apparatus of anuran tadpoles ronald altig department of biological sciences, mississippi state university, mississippi state, ms, usa 39762. email: raltig@biology.msstate.edu abstract. because studies of the oral apparatus of anuran tadpoles seem to be mired at the descriptive stage, a discussion based mostly on developmental concepts is presented as a primer to studies of developmental genetics and evolution. an oral configuration common among many ecomorphological guilds of anuran tadpoles is used as a morphological standard without ascribing evolutionary significance. comments on the patterns of modifications of the jaw sheaths and labial teeth and other morphological patterns are presented. keywords. tadpole, mouthparts, development, evolution, systematics. introduction many morphological patterns in the oral structures common to specific taxa (e.g., wide dorsal and ventral gaps in the marginal papillae of bufo) are recognized, but the description of the unusual tadpole of phyllodytes gyrinaethes (peixoto et al., 2003) shows that we do not yet fully understand tadpole morphological diversity and that generalizations can be misleading; other known phyllodytes tadpoles are quite typical. likewise, the development, genetic control, and evolution of these structures are understood in basic terms or often not at all. studies of the anatomy of the jaw and associated muscular and their functions (e.g., gradwell, 1972; wassersug and hoff, 1979; larson, 2002; haas, 2003) are available, but studies of the actual mouthparts seem to be inconveniently shackled at the descriptive level of research. our understanding is further obscured by a confusing interplay of several poorly known areas: variations from microand macrogeography (e.g., savage, 1960; gollman and gollman, 1996), ontogeny, development, and various forms of plasticity (e.g., relyea and auld, 2005). this exploratory discussion does not present a unifying hypothesis, and i do not assert that the various ideas as necessarily correct; the intention was to stimulate new questions in a poorly studied field. the oral terminology of altig and mcdiarmid (1999a) was folacta herpetologica 1(2): 95-105, 2006 96 ronald altig lowed, and four assumptions are made. (1) the tadpole stage is primitive within anura. (2) the primitive tadpole had some kind of an oral apparatus. (3) many changes in the oral disc represent adaptive morphologies associated with specific modes of feeding constrained by a large but poorly understood phylogenetic component. (4) the unique jaw complex (gradwell, 1972; svenson and haas, 2005) was present in protoanuran larvae. the oral apparatus of a typical, exotrophic, pond tadpole (fig. 1a) includes an oral disc composed of an upper labium with free lateral edges and two tooth rows, a larger lower fig. 1. (a) a typical oral apparatus of a pond tadpole (modified from altig and mcdiarmid, 1999a; notations: a-1-2 = anterior tooth rows 1 and 2; p-1-3 = posterior tooth rows 1-3; ljs = lower jaw sheath; ujs = upper jaw sheath), the (b) oral disc of hyla chrysoscelis at gosner (1960) stages 21 showing the stomodeum and ciliated epidermal cells throughout the presumptive area of oral disc formation, (c) unusual. transient spike-like jaw sheaths of heleophryne (modified from visser, 1985), (d) somewhat similar jaw structures that persist throughout larval life in mantidactylus lugubris, and unusual labial “teeth” in (e) m. lugubris (from rows on lower labium in panel d), (f) phyllodytes gyrinaethes, and (g) hoplobatrachus rugulosus (also note biserial construct) 97evolution of tadpole mouthparts labium with free marginal edges and three tooth rows, unmodified jaw sheaths, a wide dorsal gap in the marginal papillae, and submarginal papillae laterally and ventrolaterally. we have little idea of the characters of the primitive tadpole, and this oral configuration is used as a comparative standard without implying that it is primitive (e.g., cannatella, 1999). less plastic structures than mouthparts probably will prove to be the most phylogenetically informative; nonetheless an understanding of the evolution of mouthparts would contribute immensely to understanding anuran larvae and this complex suite of oral characters. while acknowledging the issues concerning the use of ontogenetic data in phylogenetic inference (e.g., krause, 1988; mabee, 1989), those points where developmental data appear to give the only available signals are pointed out. haas (2003) presented a recent phylogenetic analysis based on larval characters. pervasive models involving larval characters will be devised only after we understand the developmental mechanisms and homologies of larval features and can interpret observed patterns in a phylogenetic framework. after presenting ideas on the origins of the tadpole oral apparatus, i consider other patterns of modification of the oral features. i attempt to describe evolutionary trajectories within lineages that support the argument that larval traits have phylogenetic value. a proper analysis of the interactions between the gathering (oral apparatus) and filtering (buccopharyngeal structures) components would be informative, but the utility of this poorly understood link cannot be fully explored until we understand the evolution and function of the mouthparts. changes in the oral apparatus as a consequence of the evolution of the “tadpole stage” and the highly specialized anatomy and life history of anurans are discussed. origins of the oral apparatus this discussion focuses on oral morphology exclusive of the jaws and musculature. is the face of an oral disc homologous with surfaces that surround the mouths of other vertebrates or is it derived from a reflexed surface as in extremely pouted lips? developmental patterns (e.g., thibaudeau and altig, 1988) show that the oral disc initially is defined by an approximately circular furrow that forms peripherally to the stomodeum; the incipient tooth rows are dorsal and ventral to the stomodeum within the boundaries of the presumptive labia. the presence of ciliated epidermal cells characteristic of larval epidermis on the presumptive face of the disc (fig. 1b) signals that the disc face is homologous with the general surfaces surrounding the mouth and that it is not a reflexed surface. this unusual growth pattern whereby the structure is delimited from, instead of growing out of, a local surface seems to resemble the growth pattern that produces the nose star of the star-nosed mole (condylura cristata; see catania et al., 1999). intrinsic musculature of the oral disc is unknown, and extrinsic musculature originates on meckel’s cartilage and inserts at various sites, especially the bases of tooth ridges, within the oral disc (gradwell, 1972; carr and altig, 1991). keratinized structures frequently occur in association with various feeding structures of chordates (e.g., cyclostomes, some teleosts, some larviform salamanders, some reptiles, and birds), and the propensity for keratinized tissues to occur on various surfaces suggests a conserved genetic background of a labile mechanism. most tadpoles have the suprarostral and infrarostral cartilages covered by keratinized sheaths, and transverse rows of 98 ronald altig labial teeth occur on each labium. the dense, pyncnotic tissue that is continually renewed mitotically adds strength and protection from abrasion during feeding activities. comparative histological studies likely would show differences in the formation, maintenance, and structure of these keratinized structures. in general, keratinized oral structures occur in organisms that lack calcified teeth. keratin is lighter, less brittle, and more resilient than bone. it is probably cheaper to produce by organisms that sometimes discard these structures at some point, and it is readily developed by epidermal tissues. keratinized structures may develop in response to abrasion; for example, keratin sometimes appears on nonworking surfaces of tadpole mouthparts where other keratinized structures make repeated contact (e.g., ventrolateral to robust lower jaw sheaths of the rana pipiens group, personal observations; kassina senegalensis, noble, 1926). if such a mechanism initially produced keratinization on the jaw cartilages of tadpoles, selection might have resulted in the various shapes to improve specific functions and have given rise to the present ecological and morphological diversity (altig and mcdiarmid, 1999b). typical jaw sheaths vary in every dimension, and further examinations of the cross-sectional shapes and the back surfaces (altig and mcdiarmid, 1999a) likely would reveal insights as to the function and interaction of these structures. noteworthy developmental information probably could be gleaned by observing the development of jaw sheaths in tadpoles having atypical jaws (e.g., all stages of lepidobatrachus [ruibal and thomas, 1988], young heleophryne [fig. 1c; visser, 1985], otophryne [wassersug and pyburn, 1987], and mantidactylus lugubris (fig. 1d). these various spikelike structures may be hypertrophied serrations with the sheath lacking or minimally developed and thus may be further evidence of a developmental relationship between jaw sheaths, papillae, tooth ridges and labial teeth (thibaudeau and altig, 1988). two arrangements seem possible for the original arrangement of labial teeth or their ancestral morphologies. there may have been a burr-like surface produced by rather rigidly attached, tooth-like projections over the entire face of the oral disc, or the projections may have originated in crudely arranged transverse rows but not on tooth ridges. manipulations of embryos and later stages suggest that all surfaces of the face of the oral disc are totitpotent for tooth formation throughout most of larval life (e.g., thibaudeau and altig, 1988; d. drake, unpublished data), and a multiserial, burr-like surface of firmly attached teeth is approximated in the distal rows of ascaphus truei. the original spinules likely had short, weakly spatulate heads; cusps were small to lacking, and replacement teeth were absent. teeth were attached strongly to the disc tissue and curved toward the mouth on each labium. changes in orientation other than minimal modifications caused by changes in the shape of the entire disc during feeding strokes were minor. by interacting with substrate irregularities and the tangled array of periphyton, such a burred surface might have had the initial function of stabilizing the oral disc while the jaw sheaths worked as the primary food removal surfaces. why were teeth eventually arranged in linear transverse rows? transverse tooth rows with the teeth facing the mouth seems intuitive based on the directions and presume functions of either removing or conveying materials toward the mouth or stabilizing the oral disc via a gripping action. a gripping or rasping surface that moves longitudinally would not function well if the effective structures also were arranged longitudinally. even though a snail radula moves differently than a tadpole’s oral disc, the power strokes basi99evolution of tadpole mouthparts cally are longitudinal in both cases, and radular teeth also are either arranged transversely or at a shearing angle to the direction of travel. arrangement of spinules in rows with no particular alignment of teeth among rows may have been mechanically more effective and less subject to impediment or fouling with the available muscular forces. there were no grinding/crushing actions involved, so alignment of teeth between rows or special surfaces would not be required. why are the tooth rows on the tops of tooth ridges? tooth rows positioned at the tops of ridges probably served two purposes. tooth ridges vary in height and flexibility among taxa (e.g., shorter and less flexible in stream forms; taller and flexible in pond forms) but the ability to change the orientation and operation of the teeth at different parts of the operating cycle of the disc relative to the substrate would enhance their functions. emlet (1991) analyzed an amazing parallel case. ciliate protozoans have parallel rows of cilia positioned on ridges, and the movements of the cilia either move the organism forward or move water over the animal. because they are positioned on ridges, the cilia move more fluid with a greater displacement per stroke than cilia on flat surfaces. during feeding actions, the rotation of the tips of labial teeth distally from the mouth at maximum disc excursion is caused by extrinsic musculature and tensions in the tooth ridges caused changes in disc shape. if all rows respond with simultaneous changes in orientation and shape during a feeding cycle, fluid-borne particles should be moved toward the mouth more effectively than if the teeth were on a flat surface. tooth ridges and the connective tissues in their bases (carr and altig, 1991) also add “corrugations” on the face of the disc which keep the disc from buckling uncontrollably; this mechanism would enhance feeding activities in rasping tadpoles and substrate adhesion in fast-water forms. teeth are reoriented by extrinsic muscles when the disc opens but return to their resting positions by elastic recoil of the connective tissues in the ridges. wear of keratinized mouthparts likely is a viable means to study function and warrants further attention in both ecological and developmental aspects. wear can be assessed only in scanning electron microscope images, but the incorporation of generalities derived from studies of the wear of mechanical abrasive tools surely would improve our understanding of the morphological and ecological aspects of tooth function. extreme diversity in all aspects of labial teeth (e.g., tooth size, shape and cusping pattern; tooth density; number of replacement teeth; number and disposition of rows) suggests differential feeding functions. correlations in tooth characteristics across taxa and ecological groups seem apparent (altig and johnston, 1989). however, even though factors associated with feeding during competitive interactions abound (alford, 1999), specific actions or harvesting abilities provided by a given morphology have not been verified. as an extreme, it is curious that tadpoles that have lost all keratinized mouthparts from infections of chytrid fungi are not emaciated even though their growth trajectories lag that of intact controls (parris, 2004). do these individuals change the content and site of feeding, or in fact are the keratinized structures not that influential on feeding? if the latter were true, is the diversity of keratinized oral structures somehow pleiotropic to some other feature(s)? the fact that the first teeth to erupt ontogenetically have a simpler morphology than subsequent generations (m. penuel-matthews, unpublished data) of teeth may involve a phylogenetic signal. thus, it remains unclear whether the primary function of labial teeth concerns food harvesting in general, a specific form or part of food harvest100 ronald altig ing, or stabilization of the tadpole while the jaw sheaths act as the primary food-removal structures. there is probably a wide latitude of functions that might be called into play only when crucial conditions demand interspecific differences in feeding ecology; that is, competition based on differential feeding abilities may occur only under specific and extreme conditions. also, with the addition of more information on the feeding biology of tadpoles, we may find that a typical pond tadpole performs mostly as a carnivore (schiesari, 2004) and perhaps employs three different feeding modes under specific circumstances. perhaps the jaws function most effectively when a tadpole harvests a reasonably tall overstory without producing a particulate suspension (e.g., fibrous periphyton, 1-2 mm; personal observations), and the labial teeth would serve primarily in stabilization in this case. when a tadpole feeds on a very thin layer (e.g., diatom films), perhaps the labial teeth function most effectively, and at least part of the material is rendered as a suspension that is then captured by the buccopharyngeal structures. the third scenario involves the capture of naturally suspended particles (e.g., seale, 1982) mostly by actions of the food traps. if the labial teeth initially provided stabilization, one has to ask why the extreme diversity in tooth morphology occurs, but one has to assume that our present concepts of tooth diversity somehow includes ideas of the origins of these structures. the data on labial teeth are limited to snapshots of individuals, and additional data on intraand inter-row and ontogenetic variations across many taxa, stages, and sites would be helpful. searches for patterns and correlations among these data would promote informative ideas, and one has to consider that variations in tooth morphology may occur by genetic mechanisms other than selection for a given function. in any case, an explanation of the large diversity of labial tooth shapes and cusping patterns and the transverse topography of labial tooth rows requires further study. there are “teeth” with aberrant morphologies and development that suggest that they may not be homologous with typical labial teeth. the tadpole of phyllodytes gyrinaethes (peixoto et al., 2003) has crowded rows of spade-shaped structures (fig. 1f) that lie tightly flat against the local surface. the tadpole of mantidactylus lugubris has conical, toothlike structures in three transverse rows on only the lower labium (glaw and vences, 1992; fig. 1e). scanning electron microscopy shows that these structures are not typical teeth. these pigmented, pointed cones stand upon but not embedded in what appear to be flattopped tooth ridges, and the heads are simple cones. one has to ask if they are ancestral to, or derived from, typical labial teeth or are they merely labial tooth analogs? if the latter were true, then the mechanisms that produced “labial teeth” likely differed. the presence of these seemingly abrasive structures only on the posterior labium again indicates that this surface is functionally more important than those on the upper labium. however, if posterior tooth rows were lost in the common ancestor of the group to which m. lugubris belongs, then selection may have promoted modification of submarginal oral papillae to take on the functional role of tooth rows on the posterior labium. most other members of mantidactylus in the lugubris group have typical teeth, although they are sometimes weakly developed. the ranid hoplobatrachus rugulosus (fig. 1g; chou and lin, 1997) is the third example of aberrant “teeth” and perhaps the second nonhomologous case, after bombinatorids and discoglossids, in which each tooth ridge has two lines of teeth (= biserial). this seemingly is the only case of biserial tooth structures in advanced frogs, but the occasional occurrence of sections of biserial teeth in species that normally have unise101evolution of tadpole mouthparts rial tooth rows shows that the developmental potential likely occurs throughout anura. also, the development of these large spiked teeth is entirely different than typical teeth (r. altig, unpublished data). the intriguing fact that this configuration occurs within just one genus of ranids suggest that simple and labile genetic mechanisms control tooth morphology. perhaps these structures are in fact modifications of typical teeth; compaction of the mitotic bed that produces labial teeth toward the surface of the tooth ridge (i.e., seemingly a developmental truncation) would seem to be a primary prerequisite. do biserial tooth rows represent any indication of being derived from the burred surface suggested above? were the typical uniserial tooth rows seen in most extant tadpoles derived from the biserial condition either by fusion or the loss of some of the transverse, multilineal arrays? the alternative, of fission of uniserial rows to produce a biserial condition cannot be ignored a priori, and early embryological examinations of tooth and tooth ridge formation in representative taxa would likely indicate which case is likely. the second of two scenarios mentioned above provides that teeth appeared in rows a priori and perhaps, if these teeth had replacement series in the subsurface tissue, these rows were on ridges merely because there had to be a place to house the mitotic beds that produce the teeth. recall that the entire face of the oral disc appears to be potent for tooth formation and that tooth ridges and submarginal and marginal papillae may be homologs (thibaudeau and altig, 1988). melanic pigmentation provides strength to structures, and labial teeth precursors may have been modified from papillae. oral apparatus changes the paucity of descriptive information coupled with a lack of understanding of developmental mechanisms forces us to make predictions based mostly on fully developed structures. heterochronic alterations of developmental trajectories are likely common genetic mechanisms of modification, and it seems that metamorphic reductions occur in reverse order of formation (thibaudeau and altig, 1988). phylogenetic losses seem generally to follow the same sequence as metamorphic losses, but the mechanism of formation and comparisons of interspecific differences in row lengths, spacing, and gaps within and among parts of the oral disc needs attention. parts of the presumed inductive events that produce an oral disc and the tooth ridges can be disrupted so that a disc with marginal papillae forms without tooth ridges and therefore, tooth rows (g. thibaudeau, unpublished data). the generalized sequence of formation of oral structures for rana sphenocephala proposed by thibaudeau and altig (1988) is noted: oral disc, jaw sheaths, marginal papillae, labial tooth ridges, labial teeth, and submarginal papillae. the ventrolateral margins of the lower labium are the first tissues of the oral disc to materialize from the surrounding body surfaces. nascent marginal papillae subsequently appear in these same areas before they do on other margins of the disc, and they are the last to atrophy during metamorphosis. most of the marginal papillae of the tadpoles of bufo involve only these papillae. the corners of the mouth of a tadpole do not extend backwards to form a frog mouth until this tissue atrophies. the mechanisms that account for the formation of marginal and submarginal papillae are unknown, although apoptosis surely is an important mechanism (e.g., glückmann, 102 ronald altig 1951; hammar and mottet, 1971). additional data are needed to determine the function and what controls the size, therefore number, of papillae/distance. these variables likely reflect an interplay of evolutionary history and functional demands (e.g., stream vs pond dwellers). a number of taxa have transverse rows of submarginal papillae distal to both upper and lower tooth rows that resemble either nascent or vestigial tooth ridges. the significance of the common pattern of submarginal papillae clustered in the lateral and ventrolateral parts of the disc is not known. vestigial, presumably not rudiments, tooth ridges occur in some members of the hyla leucophyllata (dendropsophus leucophyllatus of faivovich et al., 2005) group (thibaudeau and altig, 1988) and some microhylids (e.g., nelsonophryne, donnelly et al., 1990; otophryne, wassersug and pyburn, 1987) with reduced oral discs and no labial teeth. in the latter cases, these toothless ridges add credibility to the idea that the highly modified mouthparts of microhylids are derived from a more typical morphology and credence to the pattern of ontogenetic gain and loss of tooth rows that is mimicked phylogenetically. that is, the formation of tooth ridges is independent of, and occurs ontogenetically prior to, the presence of mitotic beds for tooth formation in the ridges. the potential to form teeth is likely not lost from the genome, but the regulation of the developmental program forbids the expression of this activity. last, the idea that tooth ridges and at least submarginal papillae, which can form labial teeth at times, are homologs is supported. because teeth seem to erupt on a ridge in a medial-to-lateral progression (e.g., tubbs et al., 1993), i interpret a row that is shorter than expected as a developmental truncation. likewise, the lack of row p-3 in a species (e.g., pseudacris crucifer) or group (e.g., bufo debilis group) whose close relatives typically have three posterior rows is also interpreted as developmental truncations. the configuration of the m. mandibulolabialis, an extrinsic oral disc muscle that originates on meckel’s cartilage and inserts at various places within the labia (gradwell, 1972; carr and altig, 1991) also signals developmental truncations. the inferior slip of this muscle inserts within the lower labium and terminates where the tooth ridge for row p-1 would be expected if tooth rows were present in tadpoles of afrixalus brachycnemis, hyla ebraccata (dendropsophus ebraccatus) and hyalinobatrachium eurygnathum (carr and altig, 1991). the position and vestigial nature of this muscle suggest that the ancestors of these frogs had teeth, that toothlessness was derived by developmental truncation, and that p-1 was the last tooth row lost on the lower labium. the position and size of the stomodeum within the presumptive field of the oral apparatus may be another marker. that is, might changes in the pattern of eruption of the stomodeum in the oral disc field influence the oral structures? for example, a change dorsally could produce a more terminal oral disc, and if the stomodeum changed in either actual size or developmental influence within the field that forms the oral disc, then the eventual size of the disc and the geography of its parts would differ. tracking marked cells by timelapse photography would reveal these patterns. data from hybridizations could provide a partial surrogate for molecular information on tooth-row formation, but no one has produced the more informative f2 generation. hybrids from reciprocal crosses between rana cascadae [ltrf 3(2-3)/4] and r. pretiosa [2(2)/3(1)] had a ltrf of 2(2)/4(1) (haertel and storm, 1970). this hybrid with a ltrf of pretiosa on the upper labium and cascadae on the lower labial suggests a different genetic 103evolution of tadpole mouthparts control for tooth-row formation on each labium. also, the size of the gap in a-2 and the presence of a gap in p-1 are pretiosa traits. the absence of jaw sheaths and presence of labial teeth in tadpoles of heleophryne superficially confutes the developmental sequence of appearance of oral structures outlined above, but knowing the developmental pattern (visser, 1985) resolves the seeming contradiction. a few, isolated “fangs” presumed to be derived from jaw serrations form at the time and at the sites where jaw sheaths would be expected and then disappear. even so, the absence of both sheaths confounds discussions of feeding in these suctorial tadpoles. these tadpoles and those of ascaphus are superficially similar in morphology and habitat, and the large number of tooth rows made of very small, closely-spaced teeth in heleophryne may be somehow compensatory for the absence of jaw sheaths. even so, one must assume that the details of their feeding activities differ considerably. conclusions many morphological variations coupled with meager developmental data were used to develop speculative hypotheses on the evolution of tadpole mouthparts. large amounts of descriptive (i.e., morphology and development coupled with descriptions of taxa to understand total diversity) and experimental (e.g., embryological manipulations and molecular genetics) research will be required before specific hypotheses concerning such a diverse and plastic series of structures can be formulated. essays, such as svenson and haas (2005), are valuable bridges between herpetological and molecular perspectives. researchers will benefit initially by figuring out a way to view the entire apparatus as a series of component parts and how they interact developmentally. convergences surely have been common; major events (e.g., gains or losses of various components, which likely reflect heterochronic changes) likely have occurred multiple times throughout anura, and this repetitiveness suggests reasonably simple genetic mechanisms have been conserved. gene regulation rather than mutations or changes via selection is likely the more common developmental phenomenon. other intraand interfamilial convergences, such as umbelliform and suctorial oral discs and belly suckers (= gastromyzophory), are additional circumstances that need further study. the following quote is a pertinent research insight for the complex subject of this discussion. “if you are a researcher you are trying to figure out what the question is as well as what the answer is. you want to find the question that is sufficiently easy that you might be able to answer it, and sufficiently hard that the answer is interesting. you spend a lot of time thinking and you spend a lot of time floundering around (e. witten, physicist, princeton university; 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(1979): a comparative study of the buccal pumping mechanism of tadpoles. biol. j. linnean soc. 12: 225-259. wassersug, r.j., pyburn, w.f. (1987): the biology of the pe-ret’ toad, otophryne robusta (microhylidae), with special consideration of its fossorial larvae and systematic relationships. zool. j. linnean soc. 91: 137-169. acta herpetologica 4(2): 153-160, 2009 distribution of tadpoles of large wrinkled frog nyctibatrachus major in central western ghats: influence of habitat variables kalamanji govindaiah girish, sannanegunda venkatarama bhatta krishnamurthy department of environmental science, kuvempu university, jnana sahyadri, pin 577 451, shimoga dist. karnataka, india. corresponding author. e-mail: svkrishnamurthy@gmail.com submitted on: 2009, 15th may; revised on 2009, 2nd july; accepted on 2009, 2nd october. abstract. the relationship between habitat variables and the distribution and abundance of adults and tadpoles of nyctibatrachus major (large wrinkled frog) in 35 forest streams in central western ghats is detailed in this paper. tadpoles were not equally distributed among these streams. adult frogs and tadpoles were absent from 19 streams. in the remaining 16 streams, adults were found throughout the study period but the density of tadpoles varied considerably. analysis of habitat variables at streams showed significant relationship with canopy cover over the streams, presence of leaf litter and high relative humidity on the occurrence of tadpoles. reduction in canopy cover increases light level and air and water temperature of the streams discouraging the occurrence of adult frogs and tadpoles. however, canopy cover in the study area is frequently altered by agriculture related human activities including removal of trees for fuel and timber, pruning of green leaves and twigs for making manure and conversion of forest into commercial plantations. the results suggest that disturbances to forest canopy near streams could have deleterious effects on the occurrence and distribution of tadpoles. keywords. tadpole, nyctibatrachus major, habitat variables, western ghats, india. introduction understanding the relationships between animal distribution and habitat characteristics plays an important role in designing and developing conservation strategies for threatened species (boyd et al., 2008). habitats of most of the endemic anuran amphibians of western ghats are being altered or destroyed by different human activities. despite the distribution of amphibians and their habitat features have been documented in the western ghats (krishnamurthy, 2003), little is known about the factors influencing the distribution of tadpoles. hence we detail the distribution of tadpoles of the large wrin154 k.g. girish and s.v.b. krishnamurthy kled frog (nyctibatrachus major) and factors influencing their distribution in evergreen forest patches of central western ghats. n. major is an endemic frog (iucn: vulnerable; anonymous, 2001) that inhabits forest streams with adults generally breeding in the same habitat. recently, the habitat of the species has been being shrinking due to the extraction of timber, fuel wood, and organic mulch, which cause reduction of canopy and conversion to agricultural land (krishnamurthy, 1997). in addition, these activities can also cause fluctuations in environmental variables of natural habitats of n. major. we conducted this study in order to investigate the influence of the habitat structure on the distribution of n. major tadpoles. materials and methods from july 2006 to june 2007, we surveyed 35 streams in central western ghats (13˚ 35’ 14˚ 11’ n and 74˚ 49’ – 75˚ 37’ e; altitude 577 – 780 m a.s.l.), which were categorized as “streams with tadpoles” (swt) and “streams without tadpoles” (swot) according to the outcome of surveys. in swt, tadpoles were sampled in four 1 × 1 m squares randomly selected within a 10 × 10 m area, it also randomly selected along the stream. since none of the swt was wider than 3 m, a 10 × 10 m area was chosen that encompassed both the banks across the stream. tadpoles were sampled using hand nets and were identified following the description of pillai (1978), whereas adults were caught by hand. tadpole density in 1 × 1 m sampling squares was calculated following sutherland (2000), and for each stream we computed a mean tadpole density by averaging the values obtained for the corresponding four 1 × 1 m squares. while the density of adult in each 10 × 10 m area is taken directly for all calculation. for each of the 35 streams we measured the following environmental variables: the air, water and soil temperatures (using a mercury bulb thermometer, precision 0.1 °c), the water ph (using a portable ph probe, hach), the light level (using a lux meter, kyoritsu, model 5200), the humidity (using a thermo-hygro clock, j412-cth, japan), the canopy cover (% above the sampling area using photographic images), the litter thickness, stream width and water depth (using a measuring tape graduated in millimeters), and the tree density. this last variable was recorded on either bank of the stream within an area of 10 × 10 m following the method proposed by cox (1981), and only trees above 15 cm girth at breast height (gbh) were considered. in order to detect the environmental variables best predicting the presence or absence of n. major tadpoles, we used a simple one-way anova to compare habitat variables between swt and swot streams. the relationship between the habitat variables and the density of tadpoles in swt were examined using a pearson correlation. finally, a stepwise multiple regression analysis was carried out to obtain a model on the selection of different habitat variables for the distribution of adults and tadpoles. we used spss version 12.0 for windows for all statistical analyses. results overall, we found 213 tadpoles and 106 adult frogs in 16 out of the 35 streams surveyed. air, water and soil temperatures, light intensity and depth of the water column were found to be higher in swot than swt, whereas the opposite was found for the canopy cover, tree density, litter thickness and humidity (see table 1 for statistics). both stream width and ph did not differ between swt and swot (table 1). 155distribution of tadpoles: influence of habitat variables the density of n. major tadpoles in the 16 swts ranged from 2 to 5 tadpoles/m2, being on average 3.8 ± 0.7 tadpoles/m2. all swts also harbored adult frogs (table 2), whose density was on average 6.6 ± 2.3 frogs/10m2, ranging from 3 to 12. both adult frog and tadpole densities varied among the 16 swt. the correlation analysis showed that the densities of both adults and tadpoles significantly increased according to canopy cover, tree density, litter thickness and humidity, whereas decreased with all other variables (table 3). it is very obvious that as tree density increases, canopy cover and leaf litter thickness also increase (r = 0.61, p = 0.018 and r = 0.69, p = 0.012 respectively), and thick forest cover supports higher humidity. canopy cover, tree density, leaf litter thickness and humidity are positively related to frog density, frogs were confined to undisturbed forest streams. although these four parameters support tadpole density, tadpole abundance was greater at 60-80% canopy cover with 15-20 trees per 10 m2, medium litter thickness (2-4 cm), and high humidity of 89-92%, respectively (fig. 1). however, the result of multiple regression analysis have revealed influence of water and soil temperature for the adult density (adult density = -3.184-3.662 × [water temperature] + 4.113 × [soil temperature]), while for tadpole density have developed a model table 1. habitat variables (mean ± sd) of streams with (swt) and without (swot) tadpoles of nyctibatrachus major. values in parenthesis denote the range. habitat variables swt (mean ± sd) (n = 16) swot (mean ± sd) (n = 19) f33 p air temperature (oc) 22.62 ± 0.96 (21.08-24.90) 24.61 ± 1.83 (21.25-27.68) 15.468 0.0001 water temperature (oc) 22.30 ± 0.56 (21.50-23.68) 23.22 ± 0.84 (21.90-25.08) 14.024 0.001 soil temperature (oc) 22.22 ± 0.37 (21.78-23.15) 23.15 ± 1.36 (20.51-27.03) 6.978 0.013 luminosity (lux) 1061.64 ± 665.42 (277.5-3212.5) 6714.34 ± 7518.62 (1075-33000) 8.942 0.005 canopy cover (%) 70.94 ± 7.18 (57.5-80.0) 42.76 ± 18.23 (7.5-72.5) 33.675 0.0001 tree density (no./10m2) 18.00 ± 4.93 (10-26) 9.53 ± 5.99 (2-22) 20.362 0.0001 leaf litter thickness (cm) 2.77 ± 1.96 (0.23-7.93) 1.09 ± 0.91 (0.13-3.23) 11.171 0.002 stream width (m) 1.16 ± 0.46 0.59-2.38) 1.13 ± 0.72 (0.40-3.43) 0.018 0.893 water depth (cm) 7.34 ± 2.09 (3.53-10.63) 11.95 ± 8.56 (2.38-32.75) 4.414 0.043 water ph 6.85 ± 0.24 (6.5-7.25) 6.82 ± 0.46 (6.0-7.50) 0.060 0.807 humidity (%) 89.20 ± 3.06 (84.0-97.25) 79.51 ± 7.52 (67.75-89.75) 23.219 <0.0001 156 k.g. girish and s.v.b. krishnamurthy table 2. distribution of adult individuals and tadpoles of n. major in sixteen streams at the study area. stream number latitude – longitude altitude (m a.s.l.) density of adult frogs (mean n/10m2 ± sd) density of tadpoles (mean n/m2 ± sd) 1 13° 51’ 41.0” n 75° 03’ 12.2” e 580 11.7± 2.1 4.7 ± 0.6 4 13° 43’ 04.8” n 75° 00’ 03.6” e 581 7.6 ± 1.1 3.5 ± 0.6 5 13° 43’ 04.3” n 74° 59’ 52.3” e 577 8.2 ± 1.3 4.5 ± 0.6 8 13° 55’ 38.5” n 75° 07’ 44.0” e 623 4.8 ± 2.3 2.8 ± 0.5 9 14° 09’ 11.3” n 74° 49’ 05.8” e 611 3.4 ± 1.6 3.3 ± 0.2 10 14° 09’ 55.0” n 74° 49’ 01.1” e 619 4.3 ± 1.1 4.0 ± 0.8 15 13° 44’ 07.8” n 75° 00’ 45.5” e 586 8.1 ± 2.0 3.7 ± 0.6 16 13° 47’ 05.9” n 75° 00’ 17.9” e 620 7.3 ± 1.2 4.8 ± 0.5 17 13° 55’ 50.4” n 75° 08’ 05.8” e 646 4.3 ± 1.2 3.5 ± 0.6 23 13° 36’ 13.6” n 75°18’ 07.6” e 655 6.2 ± 1.3 3.8 ± 1.0 24 13° 38’ 08.5” n 75°17’ 51.7” e 650 7.1 ± 2.0 4.3 ± 1.0 25 13° 35’ 55.5” n 75°19’ 31.5” e 717 5.7 ± 1.2 3.0 ± 0.8 26 13° 36’ 55.4” n 75°19’ 29.3” e 732 5.8 ± 3.0 4.0 ± 0.8 27 13° 36’ 57.2” n 75°19’ 30.8” e 730 4.2 ± 2.3 2.5 ± 0.7 28 13° 36’ 33.9” n 75°19’ 35.1” e 732 8.3 ± 1.7 4.8 ± 1.0 29 13° 36’ 19.8” n 75°19’ 06.2” e 692 9.1 ± 1.9 4.0 ± 0.8 table 3. influence of habitat variables (as indicated by pearson correlation coefficient) on adult individuals and tadpoles of nyctibatrachus major. habitat variables adult frogs tadpoles r p r p air temperature (oc) -0.49 0.003 -0.55 0.001 water temperature (oc) -0.60 0.0001 -0.57 0.001 soil temperature (oc) -0.36 0.037 -0.41 0.016 luminosity (lux) -0.41 0.015 -0.45 0.007 canopy cover (%) 0.65 0.0001 0.69 0.0001 tree density (n/10m2) 0.54 0.001 0.58 0.0001 leaf litter thickness (cm) 0.47 0.004 0.47 0.004 water depth (cm) -0.32 0.064 -0.34 0.044 humidity (%) 0.57 0.0001 0.62 0.0001 157distribution of tadpoles: infl uence of habitat variables (tadpole density = 5.223 + 0.06474 × [canopy cover] 0.1040 × [tree density] 0.1274 × [leaf litter thickness] 0.04241 × [humidity]) that is not statistically signifi cant (r2 = 0.21, p = 0.58). discussion a comparison of the habitat variables of sites that shelter and do not shelter n. major within a locality was used to decipher the habitat preferences. with the increase of light intensity, air, water and soil temperatures gradually raised. probably for this reason, streams which run at open sites on forest borders do not shelter adult individuals of n. major or their tadpoles. th is could be also a clear indication of sensitivity of the species to these variables. nyctibatrachus major occurs at places with a thick canopy, high tree density, deep leaf litter and high humidity. in earlier studies of gururaja et al. (2003), it was found that adult individuals of this species require low air and water temperature. th is fig. 1. frequency distribution of tadpoles of nyctibatrachus major against canopy cover (a), tree density (b), leaf litter thickness (c) and humidity (d). 16 fig. 1 a c b d 158 k.g. girish and s.v.b. krishnamurthy study shows that, in addition to adult distribution, the tadpoles of n. major also require a narrow range of habitat variables. modification of the landscape generally alters the spatial structure of habitats and affects the distribution of organisms (weins et al., 1993). habitat preservation and protection are important steps in maintaining amphibian populations. baseline information on species distribution, abundance, and habitat requirements are needed, especially in the case of poorly known and/or threatened species to clarify the extent and pattern of population declines (parris, 2002). analysis of habitat variables can help to elucidate the distribution, habitat requirements and preferences of a particular amphibian species. nyctibatrachus major is a threatened endemic frog confined to the native forest streams of western ghats. this frog forages and breeds in a very narrow range of microhabitats, whose availability may influence on the survival, reproduction and viability of its populations. the influence of habitat variables on the distribution of tadpoles of n. major may help to conserve the species population in the local scale. various herpetological researchers conducted studies on habitat quality, habitat requirement, distribution and factors influencing different species of frogs and salamanders (hollis, 1995; gillespie and hollis, 1996; welsh and lind, 1996; grover, 1998; harper and guynn, 1999; wilkins and peterson, 2000; parris, 2001; lecis and norris, 2004; casatti et al., 2006; muenz et al., 2006). the distribution and abundance of the cascade tree frog litoria pearsoniana was found to be greatly influenced by stream size and mesic mid-storey vegetation in the riparian zone (parris, 2001). in brazil, stream volume, arboreal vegetation cover at stream margins and microhabitat diversity were shown to influence the distribution of adult frogs and tadpoles (eterovick and barata, 2006). canopy cover and leaf litter in bodies of water have influenced the abundance and species richness of frogs and performance of tadpoles (binckley and resetarits, 2007; williams et al., 2008). in the present study, the tadpoles of n. major were found to be abundant in streams with thick canopy cover. populations of n. major could be threatened by a number of factors, including extensive anthropogenic pressures such as litter and mulch collection, timber extraction, stream modification, construction of check dams, deforestation. these man-made activities are known to reduce the population size of a congeneric species (n. aliciae) in western ghats (krishnamurthy and reddy, 2008). increased human activities (e.g. collection of timber wood, fuel wood and organic mulch, pruning of green leaves) in the forest areas of central western ghats alter light, temperature and moisture regimes (krishnamurthy, 1996). these activities might gradually change the structure and composition of mid and understory vegetation, resulting in more open canopies in the forest, possibly increasing fluctuations in habitat variables of stream. acknowledgements we thank dr. geoffrey r. smith, department of biology, dension university, oh, usa for his constructive comments and suggestions on this paper. help rendered by mr. k.n. shankaranarayana bhat and mr. amaresh bhat during the field work is gratefully acknowledged. we are also thankful to two anonymous referees for their suggestions. 159distribution of tadpoles: influence of habitat variables references anonymous (2001): amphibian camp hand book, daptf-sa, zoo-outreach organization, coimbatore. binckley, c.a., resetarits, w.j. 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(1996): habitat correlates of the southern torrent salamander, rhyacotriton variegatus (caudata: rhyacotritonidae) in northwestern california. j. herpetol. 30: 385-398. wilkins, r.n., peterson, n.p. (2000): factors related to amphibian occurrence and abundance in headwater streams draining in second growth douglas-fir forests in southwestern washington. forest ecol. manage. 139: 79-91. williams, b.k., rittenhouse, t.a.g, semlitsch, r.d. (2008): leaf litter input mediates tadpole performance across forest canopy treatments. oecologia 155: 377–384. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 6(1): 11-14, 2011 extreme feeding behaviours in the italian wall lizard, podarcis siculus massimo capula1, gaetano aloise2 1 museo civico di zoologia, via u. aldrovandi 18, 00197 roma, italy. corresponding author. e-mail: massimo.capula@comune.roma.it 2 museo di storia naturale della calabria e orto botanico, università della calabria, via p. bucci sn, 87036 rende (cosenza), italy. e-mail: aloise@unical.it submitted on: 2010, 10th september; revised on: 2011, 1st february; accepted on: 2011, 2nd february. abstract. in the present paper the occurrence of cannibalism, unusual predation on small reptiles [hemidactylus turcicus (reptilia, gekkonidae)], and foraging on small mammal carrion [suncus etruscus (mammalia, soricidae)] by p. siculus is reported. keywords. podarcis siculus, feeding behaviour, predation, italy. podarcis siculus (rafinesque-schmaltz, 1810) is a lacertid lizard occurring in italy and in the northwestern balkan peninsula (corti and lo cascio, 2002; corti, 2006). this lizard is an opportunistic species characterized by broad ecological tolerance and high spreading capacity (nevo et al., 1972; gorman et al., 1975). podarcis siculus can be considered as an active forager and a generalist predator (kabisch and engelmann, 1969; pérez-mellado and corti, 1993). it preys upon a wide variety of invertebrates, mainly on arthropods (arachnidae, insects larvae, diptera, coleoptera, heteroptera, hymenoptera, orthoptera, gastropoda; see e.g. capula et al., 1993; rugiero, 1994; corti and lo cascio, 2002; bonacci et al., 2008; corti et al., in press), but occasionally small vertebrates can be also preyed (sorci, 1990; sicilia et al., 2001). its feeding behaviour seems to be opportunistic, as indicated by the consumption of different preys in different habitats and/or geographic areas: e.g. the dominant preys are diptera and isopoda on the formica di burano islet (italy); spiders and coleoptera on menorca, (balearic islands); larvae of insects and ants on the sparviero islet (italy) (pérez-mellado and corti, 1993); coleoptera on sicily (sorci, 1990); ants, afids and vegetable matter on the vivaro di nerano islet (italy) (ouboter, 1981); diptera and larvae of lepidoptera in a urban park (rome, italy) (capula et al., 1993); isopoda in a coastal dune habitat of central italy (rugiero, 1994). although being a typical insectivorous lacertid lizard, p. siculus can also feed on a significant amount of plant matter on some mediterranean islands (see e.g. ouboter, 1981; pérez-mellado and corti, 1993; cooper and vitt, 2002; herrel et al, 2008). this behaviour is shared with several other species of the family 12 m. capula and g. aloise lacertidae occurring on mediterranean islands (capula and luiselli, 1994; pérez-mellado and traveset, 1999; van damme, 1999). cannibalism seems to be rare and uncommon (e.g. ouboter, 1981; rugiero, 1994; henle, 1988; burke and mercurio, 2002). as to cases of predation on small vertebrates, the only available literature data refer to the predation of two small individuals discoglossus pictus (sicilia et al., 2001), and to undeterminate vertebrate preys observed in the faecal pellets of adult p. siculus (sorci, 1990). in the present paper the occurrence of cannibalism and unusual predation on small vertebrates by p. siculus is reported. in the first case it was possible to observe a large male p. siculus preying upon a juvenile of the same species. this case of cannibalism was observed on 26 september, 2006, at 0930 h a.m., on the side of a dry wall close to the small village fiumefreddo bruzio (province of cosenza, calabria, southern italy). the male p. siculus captured the juvenile by hardly biting on the hips of the small lizard. in the second case it was possible to record a female p. siculus preying upon a juvenile hemidactylus turcicus (reptilia, gekkonidae). predation was observed on 20 august 2003, at 0200 h p.m., on the wall of an old building in the historical centre of fiumefreddo bruzio village (province of cosenza, calabria, southern italy). it was also possible to take a picture of this unusual case of predation (see fig. 1). this is the first documented case of predation on a gekkonid lizard by p. siculus. in the third case, a large male p. siculus was observed and photographed feeding upon a carrion of an adult suncus etruscus (mam   fig. 1. female podarcis siculus preying upon a juvenile hemidactylus turcicus (fiumefreddo bruzio, province of cosenza, calabria, southern italy). photo by gaetano aloise. 13extreme feeding behaviours in the italian wall lizard, podarcis siculus malia, soricidae) (see fig. 2). foraging on carrion was recorded on 16 june 2004, at 1030 h a.m., on one side of a dry wall surrounding a private house garden in the village of torchiara (province of salerno, campania, southern italy). this is the first documented case of foraging on small mammal carrion by p. siculus. acknowledgments the authors are gratefully indebted to bruno cignini (roma) for providing useful information and photographic material. references bonacci, t., aloise, g., brandmayr, p., zetto brandmayr, t., capula, m., (2008): testing the predatory behavior of podarcis sicula (reptilia: lacertidae) towards aposematic and non-aposematic prey. amphibia-reptilia 29: 449-453. burke, r.l., mercurio, r.j. (2002): food habits of a new york population of italian wall lizards, podarcis sicula (reptilia, lacertidae). am. midl. nat. 147: 368-375. capula, m., luiselli, l. (1994): resource partitioning in a mediterranean lizard community. boll. zool. 61: 173-177. capula, m., luiselli, l., rugiero, l. (1993): comparative ecology in sympatric podarcis muralis and p. sicula (reptilia: lacertidae) from the historical centre of rome: what about competition and niche segregation in an urban habitat? boll. zool. 60: 287-291.   fig. 2. male podarcis siculus feeding upon a carrion of an adult suncus etruscus (torchiara, province of salerno, campania, southern italy). photo by bruno cignini. 14 m. capula and g. aloise cooper, w.e. jr, vitt, l.j. (2002): distribution, extent, and evolution of plant consumption by lizards. j. zool., lond. 257: 487-517. corti, c. (2006): podarcis sicula (rafinesque, 1810) italian wall lizard. in: atlante degli anfibi e dei rettili d’italia, p. 486-489. sindaco, r., doria, g., razzetti, e., bernini, f., eds, polistampa, firenze. corti, c., lo cascio, p. (2002): the lizards of italy and adjacent areas. chimaira verlag, frankfurt am main. corti, c., biaggini, m., capula, m., in press. podarcis siculus (rafinesque-schmaltz, 1810). in: fauna d’italia. reptilia, vol xlv. capula, m., luiselli, l., razzetti, e., sindaco r., eds, edizioni calderini de il sole 24 ore editoria specializzata s.r.l., bologna. gorman, g.c., soulé, m., yang, s.y., nevo, e. (1975): evolutionary genetics of insular adriatic lizards. evolution 29: 52-71. henle, k. (1988): dynamics and ecology of three yugoslavian populations of the italian wall lizard (podarcis sicula campestris de betta) (reptilia: lacertidae). zoolog. anz. 220: 33-48. herrel, a, huyghe, k, vanhooydonck, b., backeljau, t., breugelmans, k., grbac, i., van damme, r., irschick, d.j. (2008): rapid large-scale evolutionary divergence in morphology and performance associated with exploitation of a different dietary resource. proc. nat. acad. sci., u.s.a. 105: 4792-4795. kabisch, k., engelmann, k.e. (1969): zur nahrung von lacerta muralis (laurenti) in ostbulgarien. zool. abh. 30: 89-92. nevo, e., gorman, g.c., soulé, m., yang, s.y., clover, r.,. jovanović, v. (1972): competitive exclusion between insular lacerta species (sauria, lacertidae). notes on experimental introductions. oecologia 10: 183-190. ouboter, p. e. (1981): the ecology of the island-lizard podarcis sicula salfi: correlations of microdistribution with vegetation coverage, thermal environment and food-size. amphibia-reptilia 2: 243-257. pérez-mellado, v., corti, c. (1993): dietary adaptations and herbivory in lacertid lizards of the genus podarcis from western mediterranean islands (reptilia: sauria). bonner zool. beitr. 44: 193-220. pérez-mellado, v., traveset, a. (1999): relationships between plants and mediterranean lizards. nat. croat. 8: 275-285. rugiero, l. (1994): food habits of the ruin lizard, podarcis sicula (rafinesque-schmaltz, 1810), from a coastal dune in central italy. herpetozoa 7: 71-73. sicilia, a., violani, c., zava, b. (2001): predazione di podarcis sicula su discoglossus pictus. atti 3° congresso nazionale della societas herpetologica italica (pavia, 14-16 settembre 2000), pianura 13: 283-284. sorci, g. (1990): nicchia trofica di quattro specie di lacertidae in sicilia. naturalista siciliano 14 (suppl.): 83-93. van damme, r. (1999): evolution of herbivory in lacertid lizards: effects of insularity and body size. j. herpetol. 33: 663-674. bbib67 ole_link1 ole_link2 bbib28 ole_link5 ole_link6 ole_link7 ole_link8 _goback ole_link1 ole_link2 ole_link3 ole_link4 ole_link1 ole_link2 ole_link19 ole_link20 ole_link21 ole_link29 ole_link3 ole_link4 ole_link5 ole_link31 ole_link14 ole_link15 ole_link12 ole_link13 ole_link16 ole_link17 ole_link22 ole_link23 ole_link24 ole_link8 ole_link9 ole_link10 ole_link11 ole_link18 ole_link27 ole_link28 ole_link25 ole_link26 ole_link6 ole_link7 ole_link34 ole_link37 ole_link38 acta herpetologica vol. 6, n. 1 june 2011 firenze university press widespread bacterial infection affecting rana temporaria tadpoles in mountain areas rocco tiberti extreme feeding behaviours in the italian wall lizard, podarcis siculus massimo capula1, gaetano aloise2 lissotriton vulgaris paedomorphs in south-western romania: a consequence of a human modified habitat? severus d. covaciu-marcov*, istvan sas, alfred ş. cicort-lucaciu, horia v. bogdan body size and reproductive characteristics of paedomorphic and metamorphic individuals of the northern banded newt (ommatotriton ophryticus) eyup başkale1, ferah sayım2 , uğur kaya2 genetic characterization of over hundred years old caretta caretta specimens from italian and maltese museums luisa garofalo1, john j. borg2, rossella carlini3, luca mizzan4, nicola novarini4, giovanni scillitani5, andrea novelletto1 the phylogenetic position of lygodactylus angularis and the utility of using the 16s rdna gene for delimiting species in lygodactylus (squamata, gekkonidae) riccardo castiglia*, flavia annesi localization of glucagon and insulin cells and its variation with respect to physiological events in eutropis carinata vidya. r. chandavar1, prakash. r. naik2* the balearic herpetofauna: a species update and a review on the evidence samuel pinya1, miguel a. carretero2 effects of mosquitofish (gambusia affinis) cues on wood frog (lithobates sylvaticus) tadpole activity katherine f. buttermore, paige n. litkenhaus, danielle c. torpey, geoffrey r. smith*, jessica e. rettig food composition of uludağ frog, rana macrocnemis boulenger, 1885 in uludağ (bursa, turkey) kerim çiçek preliminary results on tail energetics in the moorish gecko, tarentola mauritanica tommaso cencetti1,2, piera poli3, marcello mele3, marco a.l. zuffi1 climate change and peripheral populations: predictions for a relict mediterranean viper josé c. brito1, soumia fahd 2, fernando martínez-freiría1, pedro tarroso1, said larbes3, juan m. pleguezuelos4, xavier santos5 assessing the status of amphibian breeding sites in italy: a national survey societas herpetologica italica* osservatorio erpetologico italiano acta herpetologica journal of the societas herpetologica italica acta herpetologica rivista della societas herpetologica italica simple method of blood sampling from indian freshwater turtles for genetic studies manoj singh rohilla, pramod kumar tiwari school of studies in zoology, jiwaji university, gwalior 474 011, india. corresponding author. e-mail: pktiwari.ju@gmail.com submitted on 2007, 12th november; revised on 2007, 17th december; accepted 2008, 31th january. abstract. biological material is now commonly employed during the characterization of species. superficial blood vessels are the most appropriate sites in reptiles for blood sampling. in this study we describe a method of blood collection from various species of indian freshwater turtles for genetic characterization and discuss its advantages over the previously described techniques. the method proved most suitable, easy, and less harmful. the quantity of blood samples obtained is enough for different applications such as karyotyping, cytological examinations of cell types, isozyme analysis from rbcs and plasma, dna isolation for pcr-rapd and mitochondrial gene specific amplification. we conclude that the femoral vein is probably the most suitable site for blood collection from indian freshwater turtles for various experimental procedure and methodologies related to genetic characterization. keywords. femoral vein, blood sampling, indian freshwater turtles. genetic characterization of endangered species is essential for proper genetic conservation and management in their respective habitat. studies on various aspects of genome characterization require blood or tissue samples. for this purpose, sampling from substantial number of individuals is required, but the classical methods of hematological, biochemical (e.g., allozyme) cytogenetic, or dna analysis use materials from blood cells, spleen, liver, muscle, kidney, intestine or cell cultures of heart and skin fibroblast (baker et al., 1971; bickham, 1975; frye, 1991; mader, 2000). all the above methods involve killing or surgery, and this is generally not feasible in the case of endangered species, where the source as well as number of animals, both is limited. under such conditions, the development of new and simple methods becomes highly desirable. thus, we have optimized a safe, sterile and efficient blood sampling method, which is equally applicable to a variety of reptilian species, including indian freshwater chelonian species. a variety of sites in the chelonian body have been used to obtain blood, including heart, veins (e.g., jugular, brachial, ventral coccygeal or scapular), brachial artery, orbitacta herpetologica 3(1): 65-69, 2008 issn 1827-9643 (online) © 2008 firenze university press 66 m. singh rohilla and p. kumar tiwari al sinus, cervical sinus, sub-carapacial venipuncture site and trimmed toe-nails, (gandal, 1958; dessauer, 1970; mcdonald, 1976; maxwell, 1979; owens and ruiz, 1980; taylor and jacobson, 1981; stephens and creekmore, 1983; avery and vitt, 1984; jacobson, 1987; ulsh et al., 2000; hernandez-divers et al., 2002; rogers and booth, 2004; gregory and gabriel, 2006). each sampling method has certain advantages and disadvantages. some methods have even higher risk of infection (gandal, 1958; nicole et al., 2007). in turtles and tortoises, orbital sinus sampling method has been used for collecting small volumes of blood through capillary tubes (nagy and medica, 1986). this method, however, results in the dilution of blood sample with extra cellular fluids and secretions, which may alter the composition of plasma and affect percent volume of cellular components. since lymphatics are well developed in chelonian forelimbs (ottaviani and tazzi, 1977), obtaining blood samples from veins and arteries may result in hemodilution with lymph. sub-carapacial venipuncture site is located at the angle where cervical vertebrae join the shell and is formed by the junction of the common intercostals and the caudal cervical branch of the external jugular veins. blood is readily obtained from a post occipital venous plexus, that is located dorsally to the cervical vertebrae, behind the occipital protuberance of the skull of tortoises (gottdenker and jacobson, 1995). most of the published techniques have been applied in adult turtles and hatchlings of long-tailed species such as graptemys geographica, trachemys scripta and marine turtle species (gregory and gabriel, 2006; wibbels et al., 1998; bennett, 1986; ulsh et al., 2000). however, the tail is very short in indian freshwater turtle species like asperidetes gangeticus, geoclemys hamiltoni, kachuga sp., lissemys punctata, and thus, blood collection from the coccygeal vein is very difficult. the tail cannot be held firmly by any recommended device as described for trachemys scripta (ulsh et al., 2000). in addition, the amount of blood obtained from such species is very low, not enough for lymphocyte separation and culture for cytogenetic characterization. we collected blood samples from juveniles (less than 20 g body mass) and adults (weighing up to 5 kg) of both, hard shell k. dhongoka (n = 5) and g. hamiltoni (n = 1) and soft shell turtle species l. punctata (n = 10) and a. gangeticus (n = 1). adult individuals of soft shell turtles were collected from ponds and dams located in the gwalior-chambal region with the help of local fishermen and also purchased from local fish markets. the animals were reared in an artificial pond of dimension 5×2×1 m near the animal house of the department of zoology. animals were fed on freshwater frozen fishes, live earthworms, snails, and aquatic weeds. the animal care procedures followed in this study were as recommended by ethical committee of jiwaji university and as per guide lines of the forest department of madhya pradesh government (rohilla et al., 2006). the hind limbs were cleaned with distilled water-soaked cotton and after complete drying swabbed twice with absolute ethanol. the femoral vein site was identified for accurate vein puncture for collection of blood. it is located in the midventral part of the femur above the knee joint. a small muscle depression appears which was identified as the correct site for venipuncture (fig. 1). before inserting the needle in the skin, 50-100 μl of anticoagulant was taken into the syringe. the plunger of the syringe was withdrawn gently (up to the 0.1 to 0.3 ml mark) to create a vacuum for easy withdrawal of blood. a 2-6 mm deep insertion of the needle is required, which, however, depends on the musculature and age of the animal, generally being 2-3mm in hatchling or juvenile. the correct insertion 67simple method of blood sampling from indian freshwater turtles for genetic studies of needle requires a 30o-40° angle downward, between the thigh and the needle. if blood does not appear in the syringe immediately, the syringe is gently rotated and the angle is slowly decreased until blood enters into the syringe. from a healthy 5 kg animal, we could easily withdraw about 4-5 ml blood through the femoral vein with the help of 5 ml syringe equipped with 25g, 5/8-inch needle pre-flushed with sodium heparin, but keeping 250 μl heparin saved in the syringe to prevent immediate clotting. similarly, about 0.2-0.3 ml of blood was collected from one month old juveniles of three kachuga species with the use of a 13 mm insulin or tuberculin syringe. after blood collection the skin was again swabbed with absolute ethanol and medicated with betadine solution (tincture iodine) or sofaramycin cream to prevent microbial infection. finally the turtle was left on the sand to recover for about one to two hours before transferring to water. none of the adults, hatchling and juvenile turtles used to collect blood showed any sign of diseases or health related problems, even after three months. our procedure on soft shell turtle species of lissemys and kachuga allows to collect enough blood for cytology (see mader, 2000), genetic characterization, including identification of cell types for cell culture, gene-specific pcr amplification, rapd fingerprinting and allozyme analyses from rbcs or whole blood. this method appeared more appropriate, effective and easy with several advantages. the femoral vein is located in the peripheral part of the body, hence, there is no chance of injury to vital organs of the body and fig. 1. position and site (femoral vein) of blood collection from the hind leg of adult freshwater soft shell turtle l. punctata (inset: enlarged view to locate the exact position). 68 m. singh rohilla and p. kumar tiwari sufficient amount of blood can easily be withdrawn from both large and small-sized animals. the method demonstrated an easy mode of holding the animal, expected to be less painful, does not require anesthesia and provides sterile conditions. this method can be adopted for several other reptilians also with some modifications (see frye, 1991). on a wild lizard, varanus bengalensis, it proved to be equally effective (our unpublished observation). however, this technique may not be equally applicable for indian tortoise species (i.e., geochelone sp.) due to hard scales on the legs, which can damage the tip of the needle and unlike freshwater turtles, the leg protrusion is difficult to obtain easily. acknowledgements we gratefully acknowledge the department of science & technology (dst), and university grants commission (ugc), govt. of india, new delhi for providing financial support through a research project to pkt and drs-sap programme to school of studies in zoology, jiwaji university. references avery, h.w., vitt, l.j. (1984): how to get blood from a turtle. copeia 1984: 209-210. baker, r.j., bull, j.j., mengden, g.a. (1971): chromosomes of elaphe subocularis (reptilia: serpentes) with the description of an in vivo technique for preparation of snake chromosomes. experientia 27: 1228-1229. bennett, j.m. (1986): a method for sampling blood from hatchling loggerhead turtles. herpetol. rev. 17: 43. bickham, j.w. (1975): a cytosystematic study of turtles in the genera clemmys, mauremys and sacalia. herpetologica 31: 198-204. dessauer, h. (1970): blood chemistry of reptiles physiological and evolutionary aspects. in: biology of the reptilia, p. 1-72. gans, c., parsons, t.s., eds, academic press, new york. frye, f. l. (1991): hematology as applied to clinical reptile medicine. in: biomedical and surgical aspect of captive reptile husbandry, p. 325. frye, f. l. ed, malabar, krieger publishing co., florida. gandal, c.p. (1958): cardiac punctures in anesthetized turtles. zoologica 43: 93-94. gottdenker, n.l., jacobson, e.r. (1995): effect of venipuncture sites on hematologic and clinical biochemical values in desert tortoises (gopherus agassizii). amer. j. vet. res. 56: 19-21. gregory, b.c.v., gabriel, b.d. (2006): an improved blood sampling technique for hatchling emydid turtles. herpetol. rev. 37: 318-319. hernandez-divers, s.m., hernandez-divers, j.s., wyneken, j. (2002): angiographic, anatomic, and clinical technique descriptions of a subcarapacial venipuncture site for chelonians. j. herp. med. surg. 12: 32-37. jacobson, e.r. (1987): reptiles small animal practice. in: veterinary clinics of north america, p. 1203-1225. harkness, j., ed, saunders, philadelphia. 69simple method of blood sampling from indian freshwater turtles for genetic studies mader, d. r. (2000): normal hematology of reptiles. in: veterinary hematology, p. 11261132. feldman, b. f., zinkl, j. g., jain, n. c., eds, lippincott williams and wilkins, philadelphia. maxwell, j.h. (1979): anesthesia and surgery. in: turtles perspectives and research, p. 127152. harless, m., morlock, h., eds, john wiley and sons, new york. mcdonald, h.s. (1976): methods for the physiological study of reptiles. in: biology of the reptilia, p. 19-125. gans, c., dawson w.r., eds, academic press, new york. nagy, k., medica, p.a. (1986): physiological ecology of desert tortoises in southern nevada. herpetologica 42: 73-92. nicole, i.s., alleman, r.a., harr k.e. (2007): circulating inflammatory cells. in: infectious diseases and pathology of reptiles, p. 167-169. jacobson, e.r., ed, crc press, new york. ottaviani, g., tazzi, a. (1977): the lymphatic system. in: biology of the reptilia, p. 315462. gans, c., parsons, t.s., eds, academic press, new york. owens, d.w., ruiz, g.j. (1980): new methods of obtaining blood and cerebrospinal fluid from marine turtles. herpetologica 36: 17-20. rogers, k.d., booth, d.t. (2004): a method of sampling blood from australian freshwater turtles. wildl. res. 31: 93-95. rohilla, m. s., rao, r. j., tiwari, p. k. (2006): use of peripheral blood lymphocyte culture in the karyological analysis of indian freshwater turtles, lissemys punctata and geoclemys hamiltoni. curr. sci. 90: 1130-1134. stephens, g.a., creekmore, j.s. (1983): blood collection by cardiac puncture in conscious turtles. copeia 1983: 522-523. taylor, r.w., jacobson, e.r. (1981): hematology and serum chemistry of the gopher tortoise, gopherus polyphemus. comp. biochem. physiol. 72: 425-428. ulsh, b.a., congdon, j.d., hinton, t.g., whicker, f.w., bedford, j.s. (2000): culture methods for turtle lymphocyte. met. cell sci. 22: 285-297. wibbels, t., hanson, j., balazs, g., hillis-starr, z.m., phillips, b. (1998): blood sampling techniques for hatchling cheloniid sea turtles. herpetol. rev. 29: 218-220. negative density dependence of sympatric hinge-back tortoises (kinixys erosa and k. homeana) in west africa luca luiselli1, francesco m. angelici2, lorenzo rugiero3, godfrey c. akani4, edem a. eniang5, nic pacini6, edoardo politano1 1 f.i.z.v. (ecology) and centre of environmental studies “demetra” s.r.l., via olona 7, i-00198 rome, italy. corresponding author. e-mail: lucamlu@tin.it 2 f.i.z.v. (mammalogy), via cleonia 30, i-00152 rome, italy. 3 f.i.z.v. (herpetology), via domenico cimarosa 13, i-00198 rome, italy. 4 department of applied and environmental biology, rivers state university of science and technology, p.m.b. 5080, port harcourt, rivers state, nigeria. 5 department of forestry and wildlife, university of uyo, akwa-ibom state, nigeria. 6 via burali 96, i-04020 itri (latina), italy. submitted on 2007, 2nd september; revised on 2007, 4th december; accepted 2008, 3rd february. abstract. a series of 59 transect surveys was conducted in selected wet forest habitats, along the coast of west africa, to estimate the density distribution of african hinge-back tortoises (kinixys homeana and k. erosa). line transect data were fed into a simple model to derive a detection function. the parameters estimated by the model produced an elaborate characterisation of tortoise distribution, which proved to be useful in the formulation of hypotheses about tortoise densities. line transect data were analysed by distance, with a series of key and the series adjustment: the uniform function, the 1-parameter half-normal function, and the 2-parameter hazard-rate function were considered as key functions; the cosine series, simple polynomials, and hermite polynomials were considered as series expansions. the detection function was estimated separately for kinixys homeana and k. erosa, and for transects grouped for each study area by considering all the combinations of the above key functions and series expansions. the akaike information criterion (aic) was computed for each candidate model and used for model selection. the best model of the detection function, for both the tortoise species was the uniform function with no series expansion. model results indicated that the density of the two species was inversely related at the local scale, and complementary across the region; such that the density of one species increases from west to east while the other one declines. overall, the comparison of density estimates between the two tortoises is consistent with a former hypothesis suggesting inter-specific competition and consequent resource partitioning. other causes may contribute to explain the observed patterns, including the low productivity of rainforest habitats and long-term human perturbation. keywords. conservation, distance sampling, ecology, kinixys, population size, west africa. acta herpetologica 3(1): 19-33, 2008 issn 1827-9643 (online) © 2008 firenze university press 20 l. luiselli et alii introduction in reptilian guilds of species, resource partitioning may occur along one or more of three niche dimensions: the spatial niche, the trophic niche, and the temporal niche (e.g., pianka, 1973, 1986; toft, 1985). recent reviews highlighted that resource partitioning patterns in consequence of strong interspecific competition is the rule in some groups of reptiles, especially in tropical regions (i.e., snakes, see luiselli, 2006a; 2008a, 2008b), but not in terrestrial turtles where the potential for interspecific competition seems to be low (luiselli, 2006b). the niche dimension to be partitioned by sympatric reptiles tends to vary according to the phylogeny of the groups: instead of the majority of snake communities that partition the trophic dimension (e.g., luiselli, 2006a), lizards and terrestrial turtles generally partition the spatial dimension (e.g., pianka, 1986 for lizards, and luiselli, 2006b for turtles). in the guinea-congo rainforests there are two pairs of terrestrial reptiles, one of snakes (the vipers bitis gabonica (duméril, bibron and duméril, 1854) and bitis nasicornis (shaw, 1802)) and one of turtles (the hinge-back tortoises kinixys erosa (schweigger, 1812) and kinixys homeana (bell, 1827)) that may represent ecological enigmas because they are ecologically quite similar in many respects and are also morphologically extremely similar, nonetheless they are usually syntopic even at microhabitat scale, and have a wide greatly overlapping distribution across the continent (e.g., pritchard, 1979; ernst and barbour, 1989; chippaux, 2006). how is possible that these species sharing broad ecological similarities are able to coexist across such a large region despite the predictions of general ecological niche theory, has been a debated question for reptile ecologists since long time (e.g., see schmidt, 1919 and later studies). recent research has demonstrated that the intensity of interspecific competition in both these sets of species increases with rainforest alteration (luiselli, 2006c), and that the overall response is very similar between coexisting vipers and coexisting tortoises (luiselli, 2006c). however, modelling analyses also showed that sympatric vipers show an inverse density relationships; i.e. the rainforest productivity does not seem to support abundant populations of these species when sympatric, consequently at least one of the two species is always rare when the other is locally abundant (luiselli, 2006d). this pattern could also be explained by subtle microhabitat differences that, however, are not easily observable and therefore do not allow a discrimination between the microhabitat types frequented by the two species. considering that: (i) the hinge-back tortoises seem to experience similar ecological patterns as the two bitis vipers (i.e. broad coexistence in the guinea-congo rainforest region despite their nearly identical habits, ecology and morphology, see ernst and barbour, 1989; lawson, 2000, 2001, 2006); and that (ii) the response to forest habitat alteration is almost identical for the two pairs of species (i.e., increase in potential competition intensity, luiselli, 2006c), it is likely that the kinixys case study may share other common patterns with the bitis one. the central question examined in this paper is whether the two tortoise species, which overlap broadly in their niche (ernst and barbour, 1989; luiselli, 2003a; lawson, 2006) show negative density dependence in their joint occurrence. negative density dependence, such that when one species is abundant the other is rare, may be caused by a variety of processes including competition, differences in habitat requirements, and asymmetric pre21negative density dependence of kinixys erosa and k. homeana dation rates (density dependent predation). our goal here is not to test the competition hypothesis directly but to determine whether the density of the tortoises shows negative density dependence at a large scale in west africa. the topic of the paper is relevant to community ecology theory and the processes that regulate biodiversity (schoener, 1982; simberloff and dayan, 1991). in addition, one of the tortoise species, k. homeana, is on the iucn red list (2006) as vulnerable species and information about the processes that influence its density would be valuable to conservationists and natural resource managers in the region. the density parameters of k. erosa and k. homeana were collected in wet rainforest habitat sites selected across a wide geographical region including ghana, benin, and nigeria, by means of independent line-transect surveys. line-transect surveys are a popular field method for assessing the density of free-ranging populations and have been utilised with a wide variety of different species (eberhardt, 1978, 1979; gaillard et al., 1993 and references therein). the complete theory of this method – proposed by leopold in 1933 – was just recently reformulated (burnham et al., 1980) and revisited in combination with novel statistical approaches (buckland and elston, 1993; buckland et al., 1993, 2001), such as model selection theory (akaike, 1985) and occupancy modelling analysis (mackenzie et al., 2002, 2004), to become a statistically sophisticated tool for quantitative assessment (e.g., katsanevakis, 2006; luiselli, 2006a). density profiles generated by line transect surveys are easily standardised in the field and therefore highly valuable for interregional comparisons, and therefore used for the present study. materials and methods study areas line-transect surveys were conducted in several areas of ghana, benin and nigeria, by applying exactly the same field and modelling procedures (see below). the selected sites included the tropical forest-grassy vegetation mosaic surrounding koforidua and accra in ghana, the swamps and wet forests of porto novo and cotonou in benin, and the swamp-forest habitats of lagos-lekki, ibadan, edo, bayelsa, rivers, akwa-ibom, and cross river states in nigeria. in our analyses, we distinguished ‘western nigeria’ including all transects situated west of the niger river main axis, from ‘eastern nigeria’ including transects situated east of the river. this distinction reflects a recognised biogeographical separation, reflected by documented differences in local faunal and floral endemisms (kingdon, 1990). detailed description of these forest areas are available respectively in werre (1991), politano (1998), and in luiselli (2005a). in general, we selected study areas that were similar each other in terms of main vegetation characteristics (rainforest), although possibly at different stages of maturity depending on the availability of mature rainforest patches in the various regions. these rainforest patches were generally interspersed among plantations and deforested areas, and/or (especially in southern nigeria) were characterized by the mosaics of swamps produced by floods from river niger drainage. common tree species at most areas were lophira alata, pycnanthus angolensis, sacoglottis gabonensis, uapaca spp., hallea ledermannii, albizia adianthifolia, irvingia gabonensis, klainedoxa gabonensis, treculia africana, ficus vogeliana, and elaeis guineensis, and the understorey is often dominated by rattans. average tree height (20-25 m) was relatively low for rain forest at all sites, and emergents, which can reach heights of 35-40 m, generally occurred at low densities. the low abundance of emergents was caused by high exploitation of these forest sites by people. 22 l. luiselli et alii protocol tortoise presence and density were recorded along 59 line transects, each 5,000 m long and 20 m wide, surveyed during different seasons, from 2003 to 2005. the transects were totally independent from each other, however their vegetation structure, species composition, and successional development stage were comparable, and represented the most adequate habitat type for tortoises in each study region. transects were selected after pilot surveys conducted in the years 2001 and 2002, that were performed to determine the spatial character (uniform, gradient, clustered, etc.) of tortoise abundance in each habitat. this was done to insure that the samples were unbiased, as non-uniform object distributions, particularly loosely clumped distributions, may bias population estimates (buckland et al., 2001). thus, we selected only transects where the tortoise distribution appeared relatively uniform and the detectability apparently comparable (luiselli, 2005a). however, it is not necessary that the animals are randomly or uniformly distributed, whereas it is critical that the line is placed randomly with respect to the distribution of animals. given the random line placement, it can safely be assumed that animals are uniformly distributed with respect to perpendicular distance from the line (buckland et al., 2001). during each survey, transects were covered during different hours of the day (see below for details) by walking consistently in the same direction. all the kinixys encountered during transect were captured; the specific site of capture (including the shortest perpendicular distance from the transect), the time, and the habitat type were carefully recorded. the captured tortoises were individually marked by notching a plate of their carapace, and measured while recording plastron length (svl) and weight. modelling density density estimates were generated by ‘distance sampling analysis’. the data were elaborated with distance 5.0 (buckland et al., 2001; thomas et al., 2007), a dedicated software, utilised with free-ranging animal populations (e.g., see katsanevakis, 2006). distance produces a detection function g(x) describing the probability of detecting an object (a tortoise species in our study case) located at distance x from the line transect under survey (see the key and the series adjustment framework described in buckland et al., 2001). the relationship between g(x) and p – the proportion of kinixys individuals in area a that was actually detected – can be expressed as: p = w-1∫ g(x)dx where 2w is the width of the transect, while d represents the density of kinixys tortoises within a surface a; d = n (a × p)-1= n (2wl × p)-1 l: transect length; n: number of detected individuals. the detection function g(x) was modelled in the general form: g(x) = key(x)[1 + series(x)] × {key(0)[1 + series(0)]}-1 where key(x) is the key function and series(x) is a series expansion used to adjust the key function. the uniform function, the 1-parameter half-normal function, and the 2-parameter hazard-rate 23negative density dependence of kinixys erosa and k. homeana function were considered as key functions; the cosine series, simple polynomials, and hermite polynomials were considered as series expansions (buckland et al., 2001). the detection function was estimated separately for kinixys homeana and k. erosa and for the transects grouped for each study area (e.g., koforidua, accra, lagos, etc.), by considering all the combinations of the above key functions and series expansions. the akaike information criterion (aic) was used for model selection (akaike, 1985) and computed for each candidate model. the effective strip width (esw) was estimated for each transect by distance from the sample data. esw varied slightly between transects, making the intertransect comparisons feasible. in any cases, values of the effective detection radius (denoted edr in the following text) are reported where pertinent in the tables included in this article. given the generally low abundance of tortoises in the field (luiselli, 2005a, 2006b), in most cases we detected small number of individuals along each transect. distance data were not combined with mark-and-recapture data (alpizar’s method, see alpizar-jara and pollock, 1996) as there were too few recapture instances to build a consistent database. all other statistics were computed by means of spss (version 11.0) pc package, with all tests being two-tailed and α set at 5%. in order to correlate density estimates of one species against the other, we used the modelled density values from the better 45 transects, according to aic scores. results modelling density overall, the two species occurred syntopically in 49 out of 59 transects (83%), while in 10 transects only one of the two species was seen. in ghana, we conducted six independent line transects, capturing and marking 8 k. homeana and 21 k. erosa (table 1). because of the small sample of k. homeana observed, the statistical power of the estimate relative to this species was less than the one relative to k. erosa (see aic values in table 1). however, there is no doubt that the small sample size of k. homeana genuinely reflected true rarity (hence, reduced density) of the species in this geographic region. in order to increase sample size and improve the power of the analysis, further density estimates were obtained by pooling: (i) the three transects at koforidua and the three at accra (table 1), and (ii) the six ghana transects (column ‘overall’ in table 1). modelled estimates indicated that: (i) the density of k. erosa was higher than that of k. homeana at all sites, and (ii) the density of both species was higher at korofidua than at accra. in benin, six independent transects across two distinct areas inhabited by sympatric kinixys were surveyed with the capture of a total of 16 k. homeana and 9 k. erosa (table 2). model results revealed a considerable degree of variability in tortoise densities between transects, but in general there was a higher density for k. homeana than k. erosa (table 2). after pooling the transects relative to the two main areas surveyed (cotonou and porto novo), it emerged that there were no significant differences between species abundances at porto novo, whereas at cotonou limited sample size prevented any reliable density calculation. in nigeria (west of the niger), three line transects were surveyed in the coastal forests of lagos-lekki, 3 in the forest around ibadan, and 3 in the flooded forests of edo 24 l. luiselli et alii state, with a total of 23 k. homeana and 11 k. erosa being captured (table 3). results of the modelled densities for both species are given in table 4. the modelled density of k. homeana was constantly higher than that of k. erosa at all sites, although the density difference between species was not high. furthermore, the density of k. homeana was much higher at edo state than at the other transects, whereas there was no significant inter-site difference in the density of k. erosa. in nigeria (east of the niger), density estimates were generated for 38 independent transects surveyed in bayelsa, rivers, akwa-ibom, and cross river states. detailed examination of the data transect-by-transect is not given here for brevity; the data pooled for each state surveyed are given in table 4. in total, 97 k. homeana and 60 k. erosa were captable 1. results of kinixys surveys conducted during six transects in ghana. distances from the transect (m) are presented as mean (± sd). ‘overall’ refers to estimates generated by pooling all transects in the analysis. empty cells indicate transects where density parameters could not be reliably calculated (too few data points). symbols: kof = koforidua; acc = accra coastal forest surroundings; no. obs. ind. = number of observed individuals; k = number of parameters; esw/edr = effective strip width/detection radius; d = individuals density per hectare; d (lcl) = lower density confidence limits ; and d (ucl) = upper density confidence limits ; d (cv) = density coefficient of variation. species kof1 kof2 kof3 kof (pooled) acc1 acc2 acc3 acc (pooled) overall k. homeana no. obs. ind. 2 1 3 2 1 1 0 8 distance±sd. 3.7±4.6 0 3.3±4.0 3.6±2.2 0 3 1.5±2.1 -aic 9.78 14.48 26.39 6.39 33.22 k 1 1 2 1 2 edr 7.0 8.0 3.59 3.0 3.31 d 0.286 0.375 0.558 0.222 0.403 d (lcl) 0.024 0.132 0.030 0.133 d (ucl) 5.875 2.349 1.646 1.222 d (cv) 0.711 0.630 0.671 0.535 k. erosa no. obs. ind. 3 3 4 2 3 6 21 distance±sd. 0.7±0.6 4.3±3.5 3.8±4.3 3.0±3.4 7.0±5.6 6.0±1.7 4.3±5.2 5.3±4.3 -aic 2.0 14.48 20.30 44.24 11.59 13.68 32.49 58.52 104.64 k 1 1 1 2 1 1 1 1 2 edr 1.0 8.0 8.29 3.82 11.0 7.0 8.88 9.50 5.58 d 3.0 0.375 0.482 0.872 0.182 0.429 0.675 0.386 0.627 d (lcl) 0.221 0.022 0.082 0.374 0.030 0.213 0.136 0.364 d (ucl) 40.76 6.263 2.851 2.036 6.023 2.141 1.100 1.079 d (cv) 0.667 0.731 0.605 0.388 0.677 0.472 0.400 0.265 25negative density dependence of kinixys erosa and k. homeana tured. the highest modelled densities were reached in bayelsa state for k. homeana and in cross river state for k. erosa. density relationships between species and among sites there was a significant negative correlation between the modelled density of k. homeana and that of k. erosa (rs = -0.313, n = 45, p < 0.03; fig. 1). the modelled density of k. homeana was not significantly related to longitude from west to east (i.e., from koforidua to cross river state transects) (rs = 0.150, n = 11, p = 0.663); the same held also for k. erosa (rs = -0.191, n = 11, p = 0.352). however, the sign of the correlation coefficient was opposite for the two species, suggesting that the density of the two species tended to decrease geographically along a west-to-east gradient in k. homeana, the opposite trend being observed in k. erosa. table 2. results of kinixys surveys conducted during six transects in benin. distances from the transect (m) are presented as mean (± sd). ‘overall’ refers to estimates generated by pooling all transects in the analysis. empty cells indicate transects where density parameters could not be reliably calculated (too few data points). symbols: cot = cotonou; por = porto novo; other symbols are as in table 1. species cot1 cot2 cot3 cot (pooled) por1 por2 por3 por (pooled) overall k. homeana no. obs. ind. 4 2 3 1 4 2 16 distance±s.d. 1.2±1.5 3.5±4.9 1.0±0 1.7±2.2 4 2.3±2.9 3.0±4.2 2.7±2.7 -aic 10.79 9.78 2.0 31.32 16.31 9.17 27.08 58.79 k 1 1 1 2 1 1 1 2 edr 3.0 7.0 1.0 2.32 5.6 6.0 6.0 2.92 d 1.333 0.286 3.0 1.293 0.250 0.720 0.333 0.389 0.914 d (lcl) 0.205 0.237 0.587 0.115 0.117 0.478 d (ucl) 8.672 38.01 2.847 4.522 1.291 1.745 d (cv) 0.643 0.645 0.358 0.629 0.482 0.316 k. erosa no. obs. ind. 2 1 2 0 3 1 9 distance±s.d. 3.5±3.5 3 2.5±3.5 3.0±2.5 -2.7±3.0 1 2.2±2.6 -aic 9.17 8.44 12.75 16.22 34.25 k 1 1 1 1 1 edr 6.0 0.400 6.0 5.02 6.0 d 0.333 0.500 0.266 0.250 d (lcl) 0.031 0.033 0.109 d (ucl) 8.050 2.143 0.573 d (cv) 0.719 0.745 0.395 26 l. luiselli et alii ta bl e 3. e st im at ed d en si tie s of k in ix ys t or to is es a lo ng n in e lin etr an se ct s in w es te rn n ig er ia . e m pt y ce lls i nd ic at e tr an se ct s w he re d en si ty p ar am et er s co ul d no t b e re lia bl y ca lc ul at ed ( to o fe w d at a po in ts ). sy m bo ls : l a g = l ag os ; i ba = i ba da n; e d o = e do s ta te ; o th er s ym bo ls a re a s in t ab le 1 . sp ec ie s la g 1 la g 2 la g 3 la g (p o o le d ) ib a 1 ib a 2 ib a 3 ib a (p o o le d ) ed o 1 ed o 2 ed o 3 ed o (p o o le d ) o v er a ll k . h om ea na n o. o bs . i nd . 3 0 4 7 2 2 3 7 5 1 3 9 23 d is ta nc e± s. d . 0. 3± 0. 6 1. 5± 1. 3 0. 8± 1. 6 0. 5± 0 0. 5± 0. 7 3. 3± 3. 2 1. 4± 1. 5 0. 2± 0. 4 0 1 .3 ±0 .6 0. 8± 0. 5 -a ic 2. 0 10 .7 9 16 .6 0 -0 .7 7 2. 00 13 .6 8 24 .4 0 1. 58 8. 44 6. 16 12 .6 9 55 .6 8 k 1 1 1 1 1 1 2 1 1 1 1 2 ed r 1. 0 3. 0 2. 11 0. 50 1. 00 7. 00 2. 22 0. 74 5. 0 2. 0 1. 3 1. 73 d 3. 0 1. 33 3 1. 10 2 4. 00 0 2. 00 0. 42 9 1. 04 9 6. 78 6 0. 40 0 1. 50 0 2. 37 1 1. 48 0 d ( lc l) 0. 19 4 0. 19 9 0. 20 6 0. 02 7 0. 31 0 1. 69 6 0. 08 2 0. 68 3 0. 89 9 d ( u c l) 4. 6 8. 94 4 5. 90 0 6. 88 8 3. 54 9 27 .1 54 27 .3 00 8. 23 5 2. 43 6 d ( c v ) 0. 70 7 0. 65 6 0. 56 8 0. 71 9 0. 52 4 0. 53 2 0. 75 9 0. 45 0 0. 31 6 k . e ro sa n o. o bs . i nd . 0 1 2 3 1 1 1 3 2 2 1 5 11 d is ta nc e± s. d . 1. 80 2. 0± 1. 4 1. 9± 0. 8 0 4. 2 6. 4 3. 3± 3. 6 1. 0± 1. 4 4. 5± 2. 1 5 3. 5± 2. 7 -a ic -6. 39 8. 59 12 .7 5 4. 77 9. 17 19 .9 2 41 .4 2 k 1 1 1 1 1 1 1 ed r 3. 0 3. 0 6. 0 2. 0 6. 0 6. 0 6. 0 d 0. 50 0 0. 66 7 0. 33 3 0. 25 0 0. 14 3 0. 16 7 1. 00 0 0. 33 3 0. 25 0 0. 27 8 0. 20 4 d ( lc l) 0. 05 1 0. 02 7 0. 09 9 0. 10 3 d ( u c l) 2. 17 0 1. 02 7 0. 78 3 0. 40 2 d ( c v ) 0. 74 3 0. 44 2 0. 83 7 0. 86 4 0. 43 5 0. 33 0 27negative density dependence of kinixys erosa and k. homeana discussion in a recent review of terrestrial turtle communities worldwide, it has been noticed that the potential for interspecific competition should be minor for these reptiles, with much of their species diversity reflecting geographic replacement of one species by another (luiselli, 2006b). however, the hinge-back tortoises (k. erosa and k. homeana) are clearly among the few candidates of their group to show community-organised assemblages because of their nearly identical distribution range at the continental scale (ernst and barbour, 1989, iverson, 1992), and their strong interspecific similarities in: (i) habitat requirements (ernst and barbour, 1989; lawson, 2006), (ii) home range and spacing behaviour (lawson, 2006), (iii) diet composition (luiselli, 2003a), (iv) temporal activity (lawson, 2006, luiselli, 2003a), and (v) thermal ecology (luiselli, 2005b); thus indicating very high realised niche overlap (sensu pianka, 1986). when high overlap in realised niche is found, table 4. results of kinixys surveys conducted during 38 transects in eastern nigeria. columns contain estimates generated by pooling all transects conducted in a given state. symbols: bay = bayelsa state (n = 11 transects); riv = rivers state (n = 9); akw = akwa-ibom state (n = 8); cro = cross river state (n = 10). distances from the transect (m) are presented as mean (±sd). species akw bay riv cro k. homeana no. obs. ind. 21 39 23 14 distance±s.d. 2.7±2.0 1.7±1.6 1.5±1.3 1.8±1.6 aic 80.41 115.29 62.96 47.22 k 1 2 1 1 edr 5.16 2.20 2.78 3.29 d 0.533 1.611 0.920 0.425 d (lcl) 0.322 1.027 0.543 0.239 d (ucl) 0.884 2.525 1.558 0.756 d (cv) 0.248 0.226 0.256 0.280 k. erosa no. obs. ind. 10 13 8 29 distance±s.d. 1.6±0.9 2.0±1.7 3.0±2.7 1.6±1.6 aic 23.97 40.52 34.46 88.69 k 1 1 1 2 edr 3.0 3.24 5.73 2.13 d 0.417 0.336 0.155 1.236 d (lcl) 0.165 0.175 0.068 0.707 d (ucl) 1.054 0.646 0.353 2.158 d (cv) 0.460 0.318 0.401 0.274 28 l. luiselli et alii theory predicts that competition intensity should be strong and one of the potential competitors should begin to decline and eventually even disappear from the site (schoener, 1974, 1982, 1983). in this paper we did not test directly the competition hypothesis, but we provided the first data on the density of these two species at a large geographical scale. our data showed that the two species were found coexisting in over 80% of the surveyed transects, suggesting that disappearance of one of the two species does not occur in the wet forest habitats surveyed. on the other hand, there was a negative relationship between the densities of the two species. for instance, in ghana we observed the lowest modelled density of k. homeana and contextually one of the highest modelled densities of k. erosa; similarly, in western nigeria, the highest density of k. homeana was observed next to one of the lowest densities for k. erosa. however, careful inspection of the results tables for the pooled transects reveals that the confidence intervals of the modelled densities for the two tortoise species overlap considerably, indicating that for most of the sites the populations are in fact not statistically different in abundance. only in eastern nigeria do the statistical results suggest that the populations of the two species differ very much in abundance, with k. homeana being the dominant species. this pattern may apparently contradict the overfig. 1. relationships between the modelled density of kinixys homeana and k. erosa, considering the best 45 independent transects selected by their relative aic score. for statistical details, see the text. density of kinixys homeana 76543210-1 d e n s it y o f k in ix y s e r o s a 3.5 3.0 2.5 2.0 1.5 1.0 .5 0.0 -.5 29negative density dependence of kinixys erosa and k. homeana all evidence that the density of the two tortoises is negatively related, but it seems clear that the confounding pattern is generated merely by the low abundance exhibited by tortoises at the local scale of the single transects, thus producing problems in the modelling of the estimate confidence intervals. the problem of the low abundance of tortoises is not easily solved in population density studies with these ectotherms, because this low density is indeed a generalized pattern of tortoise populations worldwide (luiselli, 2006b), with only a minor number of exceptions being known (e.g., niederberger and seidel, 1999; hailey and willemsen, 2000). based on data presented elsewhere (food habits, habitats; luiselli, 2006b, 2006c), we suggest that the negative density dependence between kinixys populations can be explained by natural community processes, i.e.: (1) interspecific competition and consequent resource partitioning between forest tortoises (see also luiselli, 2006c); (2) low rain-forest productivity hindering the establishment of abundant populations of the two tortoise species when sympatric; and (3) the two species selecting different microhabitats which are not evenly distributed along transects. concerning point (2), the same was also supposed for the coexistence of bitis gabonica and b. nasicornis in the coastal rainforests of nigeria (luiselli, 2006d). concerning point (3), this is compatible with the opinion expressed by early authors suggesting that k. erosa was linked to somewhat wetter microhabitats than k. homeana (ernst and barbour, 1989), although this suggestion could not be demonstrated during recent field studies (lawson, 2006; luiselli et al., 2006). overall, this study also indicates that both kinixys tortoises exhibit low population densities throughout the west african range studied, despite our selection of apparently suitable habitats. the methodology used in the present paper has not applied previously with kinixys, and thus our results could not be reliably compared with previous literature data on density patterns of the same genus (e.g., coulson and hailey, 2001; luiselli, 2003b). nonetheless, it should be remarked that the average densities detected for the two species during the various transects surveyed (ranging in most cases from 0.2 to 2.3 individuals × ha-1, with peaks of around 7 individuals × ha-1) were similar to those recorded by capturemark-recapture experiments conducted with the nigerian k. homeana (ranging from 0.2 to 2.8), the nigerian k. erosa (from 0.1 to 1.4 individuals × ha-1), the nigerian kinixys belliana nogueyi (from 0.1 to 2.7 individuals × ha-1) (luiselli, 2003b, 2006b), and the zimbabwean kinixys spekii (0.2 individuals × ha-1; coulson and hailey, 2001). the low densities we observed, can therefore be considered as the common density values characterising the distribution of these tortoises within their current african range. more in general, this study confirms that land tortoises tend to exhibit low population densities, as already stated by luiselli (2006b) in a global review of tortoise populations (for a few exceptions, see hailey et al., 1988; niederberger and seidel, 1999; hailey and willemsen, 2000). however, it is also necessary to point out the limits of the method used. indeed, distance sampling is very good for getting density data from several areas within a short time-span (as done in this paper), but not really very precise for density estimations when the number of transect replicates and the density of the animals is not high. in our study case, density estimates, particularly of kinixys homeana in ghana and benin, are therefore not particularly accurate, as the method would have required more observations and lines to correctly asses the precision of parameters and sampling variance (buckland et al., 2001). in general, it is recommended to use more than 20 lines to correctly estimate vari30 l. luiselli et alii ance in sample size, while the minimum number of observations should be related to the number of parameters of the chosen model and to the needs for stabilizing error estimate (buckland et al., 2001). as a general rule, we suggest to use usual capture-mark-recapture procedures to estimate population sizes and density of tortoise species rather than distance estimations, also because it is somewhat difficult to evaluate the independence of the various observations. in conclusion, this survey established that the two hinge-back tortoises, although effectively coexisting also at a local scale in most forest transects along the coast of west africa, tend to have a complementary distribution, with the population density of the one species roughly increasing from west to east and the reverse being true for the other species, and exhibit an inverse relationship in terms of population density at the local scale. we infer that the observed effects can be explained by interspecific competition with density regulation at each site being maintained by resource availability and, possibly at the local scale, by historical effects which could be implicated in the dominance of the one species on the other. for instance, in sites where both species are actively hunted by people (for instance in the niger delta in southern nigeria; see luiselli, 2003b), small-sized individuals tend to achieve a higher life expectancy, whereas large ones are removed (lawson, 2000). this process could favour the dominance of the smaller sized k. homeana over the larger k. erosa, despite and beyond natural competition. a clear understanding of the community dynamics of these sympatric species still requires much research (for instance, by applying removal experiments, etc.); the data presented here provides an encouraging basis for further research addressing the population biology of these tortoises. aknowledgements the study was financially supported by aquater s.p.a., associazione altair, chelonian research foundation (linnaeus funds, 1999 to 2002), conservation international, institute demetra, oceano s.r.l., snamprogetti s.p.a., and turtle conservation fund (2004, 2005), and logistically supported by nigerian agip oil company and other companies of the e.n.i. group, and by ‘seniores italia’. the federal departments of forestry authorized to conduct the field study and to handle tortoises in the field, dr bill branch and an anonymous referee improved an early draft. references akaike, h. 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(1991): a survey of the taylor creek forest area, rivers state, nigeria. shell petroleum development company of nigeria, ltd. and the nigerian conservation foundation, port harcourt. acta herpetologica 17(1): 27-36, 2022 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-12179 a contribution to the knowledge on the diet and food preferences of darevskia praticola (reptilia: lacertidae)§ emiliya vacheva*, borislav naumov institute of biodiversity and ecosystem research, bulgarian academy of sciences, gagarin street 2, 1113 sofia, bulgaria § presented at the xi international symposium on the mediterranean lacertid lizards, lipari 27-28th september 2021 *corresponding author. e-mail: emilia.vacheva@gmail.com submitted on: 2021, 15th october; revised on: 2022, 6th january; accepted on: 2022, 22nd march guest editors: claudia corti, marta biaggini abstract. the meadow lizard (darevskia praticola s.l.) is one of the more poorly-studied lizard species in europe, and no detailed data on its diet is available. we investigated a total of 180 faecal samples of d. praticola s.l. from two locations in bulgaria, and conducted a comparison between sex and age groups (adult males, adult females, and immatures). in addition, the correlations between the consumed prey and the available resources were also analysed. food selectivity was analysed by comparing the faecal samples with pit-fall trap samples on the basis of abundance of prey items from particular operational taxonomic units (otus). results indicate that the diet of the meadow lizard contains mainly arthropods (insects and spiders) and the most abundant prey items belong to araneae, auchenorrhyncha, and coleoptera. according to the used electivity indices none of the otus are highly preferred by d. praticola s.l., but formicidae are the most avoided otu for all sex/age groups. differences in food preferences can be found between adults and immatures, while differences among males and females seem to be insignificant. the lack of clear differentiation between males and females could be a result of their similar size and locomotor ability. in conclusion, our results reveal that darevskia praticola s.l. is a generalist and it shows no food specialization due to its narrow spatial niche. keywords. faecal samples, keratophagy, trophic niche, saurophagy, sauria. introduction dietary studies play a key role in understanding lizard ecology and knowledge on feeding ecology is of crucial importance in establishing the interactions among species. european lizards from the family lacertidae feed on a wide variety of arthropods and therefore they could be considered generalist predators which do not exhibit well-defined patterns of prey selection. nevertheless, there are data which show that (at least) some lacertid species have precisely defined patterns of food selection (e.g., díaz, 1995; carretero, 2004). in the last decades, numerous studies on food preferences and trophic ecology of lacertids were conducted (arnold, 1987; carretero and llorente, 1993; capula and luiselli, 1994; pérezmellado et al., 2011; crovetto and salvidio, 2013; mamou et al., 2016, 2019), although there are still many gaps in dietary research in some species and/or regions. the meadow lizard (darevskia praticola s.l.) occurs only in se europe and its distribution is limited to parts of ne serbia, s romania, bulgaria, ne greece, european turkey, sw russia, and nw georgia (sillero et al., 2014). the taxonomic status of the meadow lizard populations from the balkans is still not fully clarified (doronin and ljubisavljevic, 2014; freitas et al., 2016; saberi-pirooz et al., 2018). moreover, d. praticola is one of the leaststudied european lizard species in regards of ecology and especially trophic niche. in bulgaria, this species has a 28 emiliya vacheva, borislav naumov widely but very sporadic distribution, from the sea level up to about 1100 m a.s.l., yet is missing from the southwestern part of the country (stojanov et al., 2011). stugren (1984) summarised all available data for d. praticola and found that quantitative analyses of the food composition were lacking. some data on the diet are presented for the eastern (sub)species (e.g. terentyev and chernov, 1949; bannikov et al., 1971; 1977), but the trophic spectrum of d. praticola s.l. from the balkans and adjacent areas remains unstudied. the aim of the present study was to document the diet and feeding preferences of d. praticola s.l. in bulgaria, including possible intraspecific variation. in that sense, the following work hypotheses were formulated: 1) considering what is found in other lacertids, immatures should be unable to eat large prey items, therefore their trophic spectrum should be narrower than that of adults; 2) considering what is found in regard to microhabitat choice and sexual size dimorphism of darevskia praticola s.l., there should be no substantial differences between sexes in their food preferences. material and methods study area for the sampling, we chose two sites in western bulgaria: the first site was situated at the east coast of the ogosta reservoir, 3.5 km from the town of montana (43.3739° n, 23.2086° e, 180-240 m a.s.l.), and the second was situated in the sredna gora mts., near gabrovitsa village (42.2602° n, 23.9208° e, 430-570 m a.s.l.). according to “world-clim v.2” (fick and hijmans, 2017) the annual mean temperature is 11.3 °c for ogosta and 10.6 °c for gabrovitsa, and the annual precipitation is respectively 624 and 568 mm (the values are extracted from the respective freely available gis-layers with original resolution ≈ 1 km2 cell). more detailed descriptions of the studied sites are given by vacheva et al. (2020). sampling for the purpose of the study, we used a faecal samples analysis: a non-invasive method which, despite of some limitations (e.g., impossibility for prey recognition in such taxonomic level as by direct analysis of the stomach content), provides adequate results in dietary studies (bombi and bologna, 2002; luiselli et al., 2011; pérezmellado et al., 2011). lizards were captured in 2013, 2014 and 2016 in ogosta and in 2017 and 2018 for gabrovitsa. a total of 53 field days were conducted, as follows: ogosta – 28 days and gabrovitsa – 25 days. lizards were captured by hand and were measured (snout-vent length, svl) with a transparent ruler to the nearest 1 mm. for each captured lizard, sex and age class were recorded. age was not determined directly but estimated from body size and sexual secondary characters, so two age groups were defined: adults (svl > 45 mm) and immatures (svl between 24-44 mm). all of the captured lizards were placed separately in plastic boxes until defecation and after that, released at the site of capture. faecal samples from each lizard were preserved in separate test tubes with ethanol for further examination under stereoscopic microscope (magnification 10-40x). invertebrate remnants were identified to the lowest possible systematic level (in most cases to the level of order). collected invertebrates (both from the faecal and trap samples) were categorized with regards to their hardness (hard, intermediate, soft) and evasiveness (sedentary, intermediate, evasive) in accordance with verwaijen et al. (2002) and vanhooydonck et al. (2007). food resources were evaluated by pit-fall traps, which is a widely used method in similar studies (see vacheva and naumov, 2020 and references therein). a total of 24 pit-fall traps were exposed (10 meters apart) in four different habitat types (river bed, meadow, deciduous forest and the ecotone between the meadow and the forest). this was done only in gabrovitsa for 23 and 17 days in spring, and 16 and 23 days in summer for 2017 and 2018 respectively. collected invertebrates were identified to the lowest possible taxonomic level. we use the term “operational taxonomic unit” (abbreviated as otu) instead of the term “taxon” for the invertebrates from both feacal samples and traps, because here the individual taxa are considered without taking into account their rank. statistics taxonomic diversity in the diet of d. praticola s.l. was analysed by rényi’s index family (diversity profiles), which is considered one of the most useful methods for ordering communities according to their diversity (see tóthmérész, 1995). the significance of differences in diversity between the separate samples (adult males, adult females, and immatures) was assessed by a permutation test, based on the diversity indices of shannon (h) and simpson (1-d). food selectivity was analysed by comparing the faecal samples with trap samples on the basis of abundance of individuals from particular otus (standardized toward total number of individuals in the sample). the electivity was described by the indices of ivlev (e) and vanderploeg and scavia (e*) (see ivlev, 1961 and vander29diet of darevskia praticola ploeg and scavia, 1979, respectively). both indices take values from -1 to +1, where the positive values indicate that the respective component is preferred, and the negative – it is avoided (for a detailed review of the electivity indices see lechowicz, 1982). spearman’s rank correlation coefficient was used to test for correlation between abundance and frequency of the prey items. a chi-square test was used for the comparison between sexes and between age groups, regarding the categories of evasiveness and hardness of the nutritional components. calculations of the diversity indices, as well as statistical tests, were done using past 3.21 (hammer et al., 2001). the electivity indices were calculated in microsoft excel (2010) after manual input of the respective formulas. results a total of 180 faecal samples from d. praticola s.l. were collected – 31 from ogosta and 149 from gabrovitsa. among them, 136 from adults (70 male and 66 female) and 44 immatures. the distribution of the material from the faecal samples of darevskia praticola from ogosta and gabrovitsa is presented in appendices 1 and 2. the identifiable invertebrate remnants could be attributed to 622 individual specimens: 100 from ogosta and 522 from gabrovitsa (appendix 3). the average number of invertebrates found in the faecal pellets was 3.46 (3.23 for ogosta and 3.74 for gabrovitsa), and the maximum was 15. a total of 23 otus were identified in the faecal samples, and most of them were the same for both study sites. in gabrovitsa more otus were observed – 22, in contrast to ogosta where only 15 otus were observed (appendices 1 and 2). dermaptera, formicidae, gastropoda, mecoptera, myriapoda, pseudoscorpiones, and scorpiones were recovered only from gabrovitsa, while hemiptera were found only in the samples from ogosta. among all of the otus, the most abundant and frequent for ogosta were araneae and coleoptera, as well as blattodea but only by frequency of occurrence, while in gabrovitsa predominant by both number and frequency were araneae, auchenorrhyncha, and insecta indet. (fig. 1). the correlation between abundance and frequency of occurrence of otus for the two sites was positive and with very high level of statistical significance (table 1). the total number of otus registered in the pitfall traps at gabrovitsa was 25. most abundant otus were formicidae, aranea, and coleoptera (appendix 3). according to the electivity indices, none of the otus were highly preferred by d. praticolа s.l. (table 2). the highest values for both indices were observed for blattodea for males, for insecta larvae for females and for hymenoptera (excl. formicidae) in immatures. on the other hand, with lowest values of the indices (close to -1) were formicidae for all of the three sex/age groups (the index values for immatures are not presented in the table). according to the rényi’s profiles (fig. 2) the highest diversity of the diet was observed in males, and likewise diversity in the diet of adults was higher than in immatures. statistically significant differences between adult males and immatures were established for the sample from gabrovitsa with respect to the shannon index (table 3); the number of registered otus in adults was 22, while for immatures this was only 13. while the total number of otus was lower for ogosta, this could be due to the lower sample size, and a total of 13 otus were observed in adults, compared to only 9 in immatures. otus presented only in adults were acari, dermaptera, gastropoda, hemiptera, heteroptera, isopoda, mecoptera, myriapoda, pseudoscorpiones and scorpiones. none of these otus were present in immatures only. the total number of observed otus in males was 21, while in females this was 19; for ogosta there were 11 and 8 otus respectively, and for gabroivitsa, where the sample size was larger – 20 and 18 respectively. otus observed only in males were isopoda, hemiptera, dermaptera, and scorpiones, while in females lepidoptera, mecoptera, and myriapoda, but represented by single items. regarding the evasiveness of the prey, the highest values in faecal samples at both sample sites were sedentary prey items, and in terms of hardness, soft prey items were consumed more often (table 4). the results of chi square test did not show statistically significant difference between all age/sex groups, in regards to neither evasiveness nor hardness of the prey items (table 5). in addition, parts of ingested tails and finger were discovered in the faecal samples. cases of saurophagy were established in two adult males and one female from gabrovitsa, i.e., in 2.01% of the samples from gabrovitsa and 1.66% of total sample size. keratophagy (the consumption of shed skin) was observed in two adults – a male and a female, or 1.34% of the samples from gabrovitsa and 1.11% from the total sample size. non-organic matter (grit) was recorded in three individuals, and plant matter was recorded in nine adults (five males and four females), which presents 5% from the total sample size (appendix 2). discussion our results suggest that d. praticola s.l. feeds mainly on arthropods, like many other lacertids, with insects 30 emiliya vacheva, borislav naumov being the predominant group – more than 60% of the total items recovered from the faecal pellets. seven groups (araneae, auchenorrhyncha, coleoptera, insecta indet., formicidae, and other hymentoptera) composed more than 70% of the consumed prey, and among them the most abundant food source were spiders (more than 30%.) we also recorded otus that could be described as “dangerous prey”, such as dermaptera, myriapоda, and scorpiones, which were present with single individuals and in adults only. in the available literature there are no detailed data about diet and food preferences in d. praticola s.l. in a few sources, a brief description of the most common prey was provided: terentyev and chernov (1949) state that the food of darevskia praticola s.l. consists mainly of beetles (about 50%), orthopterans, arachnids, and dipterans. bannikov et al. (1971) assign small insects, spiders, earthworms, molluscs and other invertebrates as prey to the meadow lizards, specifying that among insects, small beetles, ants, orthopterans, leafhoppers, caterpillars, earwigs, aphids, as well as woodlice were the most consumed (bannikov et al., 1977). the above mentioned has been confirmed by other authors (e.g., orlova and tertyshnikov, 1979; tertyshnikov, 2002) and some anecdotal fig. 1. percentage of the invertebrates by otus according to: total number of specimens in the faecal samples of d. praticola (n); number of faecal samples in which the otu occurs (fr); total number of specimens, collected by pitfall traps (tr). otus are in descending order according to the values of n. 31diet of darevskia praticola data are reported in stugren, 1984. bischoff (1976) points out that the meadow lizard, like its relatives, preys on all edible invertebrates that can be overwhelmed. differences in food preferences were found between adult males and immatures, and the diet of adults in general was more diverse than that of immatures. it could be due to size limitation of the immatures (i.e., impossibility to consume large invertebrates), but also could be (at least partially) a result of the bias in terms of sample size differences (much smaller in immatures). in adults, the established lack of clear differentiation between males and females could be a result of their similar size and locomotor ability. in term of evasiveness of the prey, less mobile prey categories were predominant, which can be explained by the fact, that the meadow lizard is ground-dwelling and comparatively slow-moving species (arnold, 1987). regarding the hardness of the prey, predominant were soft prey categories. in view of the relatively small head size in comparison to body size (personal data), d. praticola s.l. probably avoids highly chitinized invertebrates. cannibalism and saurophagy in general, has been observed more often in island populations, where it could be caused by high lizard density and scarce food fig. 2. diversity profiles of the diet in males (m), females (f), and immatures (sub) of d. praticola according to the abundance of otus in the faecal samples from ogosta (a) and gabrovitsa (b). table 1. correlation (rho) between abundance and frequency of otus in the faecal samples of male (m), female (f) and immature (sub) darevskia praticola, and its statistical significance (p). rho p ogosta m 0.84 0.0023 f 0.77 0.0476 sub 0.89 0.0179 gabrovitsa m 0.97 0.0000 f 0.92 0.0000 sub 0.83 0.0014 table 2. list of the most abundant (r > 5%) otus from the faecal samples of males (m), females (f), and immatures (sub) of darevskia praticola according to the electivity indices of ivlev (e) and vanderploeg & scavia (e*); r = percentage in the faecal samples, p = percentage in the pitfall traps.   otu r p e e* m blattodea 5.19% 0.55% 0.8084 0.3087   hymenoptera (ef) 5.19% 2.17% 0.4103 -0.3516   araneae 27.36% 18.39% 0.1961 -0.5402   auchenorrhyncha 8.49% 7.14% 0.0865 -0.6147   coleoptera 5.19% 12.81% -0.4234 -0.8497   formicidae 12.74% 36.60% -0.4837 -0.8695 f insecta (larvae) 6.02% 1.22% 0.6630 0.0611   araneae 31.94% 18.39% 0.2694 -0.4307   auchenorrhyncha 12.04% 7.14% 0.2555 -0.4428   coleoptera 9.72% 12.81% -0.1369 -0.7038   formicidae 7.87% 36.60% -0.6461 -0.9061 sub hymenoptera (ef) 6.38% 2.17% 0.4927 0.3822   auchenorrhyncha 17.02% 7.14% 0.4091 0.2891   araneae 30.85% 18.39% 0.2532 0.1213   coleoptera 8.51% 12.81% -0.2016 -0.3286 32 emiliya vacheva, borislav naumov resource (pérez-mellado and corti, 1993; castilla and van damme, 1996; cooper et al., 2015), while it is rare in continental populations (simović and marković, 2013). cases of saurophagy were more frequent in males, as males often display more aggressive behaviour to other conspecifics (castilla, 1995), and the presence in females mentioned here is interesting. on the base of the pholidosis, we determined that the remnants of the consumed lizard parts in the faecal samples of d. praticola s.l. belong to representatives of lacertidae family, but because of the presence of two other syntopic lacertids in gabrovitsa (lacerta viridis (laurenti, 1768) and podarcis muralis (laurenti, 1768)), we can suggest only saurophagy, as far as there are no direct evidence for cannibalism. until now, cases of (partial) saurophagy, were not established for darevskia praticola s.l., and this is the first observation to our knowledge. the only known record of saurophagy in another member of the darevskia genus was mentioned for an adult female darevskia brauneri (méhely, 1909) that fed on a juvenile lacerta agilis (golynsky and doronin, 2014). another interesting feeding behaviour, keratophagy (the ingestion of shed skin), was observed for the first time in d. praticola s.l. keratophagy was previously known for only four lacertids (mitchell et al., 2006 and references therein) but in more detailed dietary study it was recorded for the viviparous lizard zootoca vivipara (lichtenstein, 1823) (see vacheva, 2018; vacheva and naumov, 2020), where keratophagy was present in more than 9% of the samples, as well as in two other lacertids (podarcis muralis, and lacerta viridis), hence this event seems to be more common than previously thought and probably has more complex and important evolutionary significance. the meadow lizard is a species with very limited spatial niche and it is a typical forest inhabitant, strongly associated with deciduous forests (mostly oak forests) table 3. diversity indices of the diet in males (m), females (f), and immatures (sub) of darevskia praticola, and the statistical significance of the differences between them (permutation p). index value permutation p ogosta gabrovitsa ogosta gabrovitsa simpson 1-d m 0.82 0.86 m vs. f 0.29 0.36 f 0.78 0.82 m vs. sub 0.07 0.19 sub 0.75 0.80 f vs. sub 0.42 0.72 shannon h m 1.95 2.37 m vs. f 0.17 0.14 f 1.76 2.16 m vs. sub 0.09 0.002 sub 1.70 1.95 f vs. sub 0.71 0.1 table 4. division of the invertebrates per categories of evasiveness (e1, e2, and e3) and hardness (h1, h2, and h3) as a percentage of all of the identified invertebrates in the faecal samples of male (m), female (f), and immature (sub) darevskia praticola. ogosta gabrovitsa m f sub m f sub evasiveness e1 44.68% 48.00% 55.00% 53.68% 60.82% 63.29% e2 25.53% 24.00% 10.00% 24.21% 22.16% 15.19% e3 29.79% 28.00% 35.00% 22.11% 17.01% 21.52% hardness h1 59.57% 68.00% 80.00% 60.53% 56.70% 54.43% h2 8.51% 0.00% 0.00% 3.68% 3.61% 2.53% h3 31.91% 32.00% 20.00% 35.79% 39.69% 43.04% table 5. chi-square test for the differences between male, female, and immature darevskia praticola in regards to evasiveness and hardness of the prey items. ogosta gabrovitsa evasiveness χ2 2.14 4.73 df 4 4 p 0.71 0.32 hardness χ2 5.49 1.53 df 4 4 p 0.24 0.82 33diet of darevskia praticola (vacheva et al., 2020). as an active and effective thermoregulator, with a preferred temperature close to the lower limit of mean body temperatures in comparison to other european lacertids (ćorović and crnobrnjaisailović, 2018), it has to choose suitable thermal microhabitats. in that sense, the observed low food specialization can be explained mainly by the narrow spatial niche and the species can be categorized as a generalist in regards to its prey choice. acknowledgements the authors thank emilia zafiraki, irina lazarkevich, steliyana popova, vladislav vergilov and nikola stanchev for their help during the fieldwork, rostislav bekchiev, georgi hristov and nikolay simov for the help with the entomological material, simeon lukanov for the valuable suggestions and language corrections on the manuscript, and dr. anthony herrel and anonymous reviewer for the helpful comments. handling of animals was in accordance with ministry of environment and water permits № 520/23.04.2013 and № 656/08.12.2015. references arnold, e.n. 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(2002): relationships between head size, bite force, prey handling efficiency and diet in two sympatric lacertid lizards. funct. ecol. 16: 842-850. 35diet of darevskia praticola appendix 1 distribution of the material from the faecal samples of darevskia praticola from ogosta per otu (m, f, and sub: males, females and immatures; evas.: evasiveness category, e1, e2, and e3 refer to sedentary, intermediate, and evasive, respectively; hard.: hardness category, h1, h2, and h3 refer to soft, intermediate, and hard, respectively; n: number of identified specimens; fr: number of the faecal samples in which otu occurs; ef: except formicidae).  otu evas. hard. m f sub total n fr. n fr. n fr. n fr. acari e1 h1 1 1 1 1 0 0 2 2 araneae e1 h1 15 12 9 5 9 6 33 23 arthropoda (indet.) 0 0 0 0 1 1 1 1 auchenorrhyncha e1 h3 0 0 0 0 1 1 1 1 blattodea e3 h1 2 2 3 1 3 3 8 6 coleoptera e2 h3 12 8 6 4 2 2 20 14 diptera e3 h1 6 2 1 1 2 1 9 4 hemiptera (indet.) 2 2 0 0 0 0 2 2 hymenoptera (ef) e3 h3 3 1 2 2 1 1 6 4 insecta (indet.) 4 3 0 0 0 0 4 3 insecta (larvae) e1 h1 0 0 2 1 0 0 2 1 isopoda e1 h2 1 1 0 0 0 0 1 1 lepidoptera e3 h1 0 0 1 1 1 1 2 2 opiliones e1 h1 4 3 0 0 1 1 5 4 orthoptera e3 h2 4 4 0 0 0 0 4 4 appendix 2 distribution of the material from the faecal samples of darevskia praticola from gabrovitsa per otu (m, f and sub: males, females and immatures; evas.: evasiveness category, e1, e2, and e3 refer to sedentary, intermediate, and evasive, respectively; hard.: hardness category, h1, h2, and h3 refer to soft, intermediate, and hard, respectively; n: number of identified specimens; fr: number of the faecal samples in which otu occurs; ef: except formicidae opi: other prey items).  otu evas. hard. m f sub total n fr. n fr. n fr. n fr. acari e1 h1 7 4 0 0 0 0 7 4 araneae e1 h1 58 41 69 46 29 20 156 107 arthropoda (indet.) 2 2 1 1 1 1 4 4 auchenorrhyncha e1 h3 18 17 26 22 16 13 60 52 blattodea e3 h1 11 9 7 7 2 2 20 18 coleoptera e2 h3 11 11 21 16 8 7 40 34 dermaptera e2 h1 3 3 0 0 0 0 3 3 diptera e3 h1 8 7 5 4 3 3 16 14 formicidae e2 h3 27 15 17 8 4 4 48 27 gastropoda e1 h3 1 1 3 2 0 0 4 3 heteroptera e2 h1 5 4 3 1 0 0 8 5 hymenoptera (ef) e3 h3 11 8 10 5 6 2 27 15 insecta (indet.) 20 19 21 20 14 12 55 51 insecta (larvae) e1 h1 9 8 13 13 2 2 24 23 isopoda e1 h2 2 1 0 0 0 0 2 1 lepidoptera e3 h1 7 8 4 4 4 4 15 16 mecoptera e2 h1 0 0 1 1 0 0 1 1 myriapoda e2 h1 0 0 1 1 0 0 1 1 36 emiliya vacheva, borislav naumov  otu evas. hard. m f sub total n fr. n fr. n fr. n fr. opiliones e1 h1 4 4 6 5 3 3 13 12 orthoptera e3 h2 5 5 7 7 2 2 14 14 pseudoscorpiones e1 h1 2 2 1 1 0 0 3 3 scorpiones e1 h1 1 1 0 0 0 0 1 1 opi (cannibalism) 1 1 1 3 opi (keratophagy) 1 1 0 2 opi (grit) 3 0 0 3 opi (plant material) 5 4 0 9 appendix 3 distribution of the material from the pitfall traps in gabrovitsa per otu (evas.: evasiveness category, e1, e2, and e3 refer to sedentary, intermediate, and evasive, respectively; hard.: hardness category, h1, h2, and h3 refer to soft, intermediate, and hard, respectively; ef: except formicidae). otu evas. hard. n acari e1 h1 832 aphidoidea e1 h1 22 araneae e1 h1 1839 archaeognatha e1 h1 9 auchenorryncha e1 h3 714 blattodea e3 h1 55 coleoptera e2 h3 1281 collembola e1 h1 83 dermaptera e2 h1 4 diptera e3 h1 586 formicidae e2 h3 3661 gastropoda e1 h3 16 heteroptera e2 h1 81 hymenoptera (ef) e3 h3 217 insecta (larvae) e1 h1 122 isopoda e1 h2 57 lepidoptera e3 h1 27 mecoptera e2 h1 1 myriapoda e2 h1 42 neuroptera e2 h1 3 oligochaeta e1 h1 10 opiliones e2 h1 168 orthoptera e3 h2 168 pseudoscorpiones e1 h1 2 trichoptera e3 h1 2 xi international symposium on the mediterranean lacertid lizards marco mangiacotti1, pietro lo cascio2, claudia corti2, marta biaggini2, miguel angel carretero2, petros lymberakis2 the directional testes asymmetry increases with temperature in seven plateau brown frog (rana kukunoris) populations hai ying li1, man jun shang2, jie guo2, bo jun chen2, peng zhen chen2, tong lei yu1,* influence of tail injury on the development of neotropical elegant treefrog tadpoles ana glaucia da silva martins1,#, raoni rebouças2,3,*,#, isaias santos1, adão henrique rosa domingos1, luís felipe toledo2 the effect of weight and prey species on gut passage time in an endemic gecko quedenfeldtia moerens (chabanaud, 1916) from morocco jalal mouadi1,*, panayiotis pafilis2, abderrafea elbahi3, zahra okba3, hassan elouizgani3, el hassan el mouden4, mohamed aourir1 a contribution to the knowledge on the diet and food preferences of darevskia praticola (reptilia: lacertidae)§ emiliya vacheva*, borislav naumov first report on two loggerhead turtle (caretta caretta) nests in the aeolian archipelago (southern italy) monica francesca blasi1,*, sandra hochscheid2, roberta bardelli3, chiara bruno1, carolina melodia1, perla salzeri1, paolo de rosa4 and paolo madonia5 threatened and extinct amphibians and reptiles in italian natural history collections are useful conservation tools franco andreone1,*, ivano ansaloni2, enrico bellia3, andrea benocci4, carlotta betto5, gabriella bianchi6, giovanni boano7, antonio borzatti de loewenstern8, rino brancato9, nicola bressi10, stefano bulla11, massimo capula12, vincenzo caputo barucchi13, p re-description of external morphology and factors affecting body and tail shape of the stone frog tadpoles’ brena da silva gonçalves1,*, carla. d. hendges2, bruno madalozzo2, tiago g. santos2,3 preliminary data on the diet of chalcides chalcides (squamata: scincidae) from northern italy andrea ciracì1, edoardo razzetti2, maurizio pavesi3, daniele pellitteri-rosa4,* the high diversity and phylogenetic signal of antipredator mechanisms of the horned frog species of proceratophrys miranda-ribeiro, 1920 (amphibia: anura: odontophrynidae) cássio zocca1,2,*, ricardo lourenço-de-moraes3, felipe s. campos4, rodrigo b. ferreira1,2,5 © firenze university press www.fupress.com/ah acta herpetologica 5(1): 91-101, 2010 amphibians of the simbruini mountains (latium, central italy) pierangelo crucitti, davide brocchieri, federica emiliani, marcello malori, sebana pernice, luca tringali, carlo welby società romana di scienze naturali, srsn, ente di ricerca pura via fratelli maristi 43, i-00137 roma, italy. corresponding author. e-mail: info@srsn.it submitted on: 2009, 8th june; revised on: 2009, 17th november; accepted on: 2010, 4th june. abstract. little attention has been paid to the herpetological fauna of the simbruini mountains regional park, latium (central italy). in this study, we surveyed 50 sites in the course of about ten years of field research, especially during the period 2005-2008. nine amphibian species, four caudata and five anura, 60.0% out of the 15 amphibian species so far observed in latium, were discovered in the protected area: salamandra salamandra, salamandrina perspicillata, lissotriton vulgaris, triturus carnifex, bombina pachypus, bufo balearicus, bufo bufo, rana dalmatina, rana italica. physiography of sites has been detailed together with potential threatening patterns. for each species the following topics have been discussed; ecology of sites, altitudinal distribution, phenology, sintopy. salamandra salamandra and bombina pachypus are at higher risk. the importance of the maintenance of artificial/natural water bodies for the conservation management of amphibian population of this territory is discussed. keywords. monti simbruini regional park, italy, amphibia, distribution, conservation. introduction the simbruini mountains, recently acknowledged as an italian regional park (the simbruini mountains natural regional park, pnrms), is one of the largest protected areas of central italy and preserves an high biodiversity of both animals and plants (e.g., 1508 species and subspecies of vascular plants among which 18.3% are rare or extremely rare in the territory of latium (attorre et al., 2000). nevertheless, this chain of the central apennines is among those many areas that are still poorly studied by herpetological researches. so far, the knowledge on the amphibians of the simbruini was based on few scattered information found in other papers concerning wider areas (e.g., bologna et al., 2000, 2007). we have been aimed at improving the knowledge of the herpetofauna distributive patterns of this area, with special emphasis to conservation and protection measures in decreasing populations and species. 92 p. crucitti et alii materials and methods the surface area of the pnrms extends for over 30,000 ha with northwest-southeast orientation according to the apennine ridge, and is delimited north-eastward by the boundaries with the abruzzi region.the chain of simbruini (main peak, monte viglio 2156 m) is mainly constituted by limestone, and is considered the central ring of the sheltered system of latium apennine, joined with the bordering lucretili and ernici mountains and directly linked with the urban and suburban area of rome. due to its remarkable orography, the annual thermal range is noticeable, ranging from -9.5 and -19.5°c in the coldest months (january / february) to 31.5-39.5°c in the hottest one (july); the pluviometric range is as much wide with yearly rainfall between 775.1 and 2376.0 mm. among habitats, mesoxerophilous grassland, green pasture of middle and high mountains are prevailing, like uncultivated and mowing fields as much as the decayed areas on decalcified soils of karstic plateau; shrubs with genista sp. are predominant. pure or mixed formation of fagus sylvatica and quercus ilex are prevailing among mature woods. field research was carried out from spring 1999 to spring 2008 during day and night shift. as a whole, we dedicated to field surveys 66 days. yearly distribution of days was as follows: 4 (1999), 2 (2000), 4 (2005), 10 (2006), 25 (2007), 21 (2008); monthly distribution of the inspections to the sites was as follows (months of the year in roman characters): 1 (i), 2 (ii), 11 (iii), 22 (iv), 6 (v), 7 (vi), 6 (vii), 5 (viii), 4 (ix), 1 (x), 1 (xi). field research included: inspection of one site reported in literature (romano et al., 2003) to check for species and breeding activities; cartographic recognition of further potential aquatic habitats, suitable for amphibian populations, and the inspection of these sites; the collection of information from local people, mainly park-keepers and shepherds. we have examined 101 sites within the pnrms boundaries, 50 of which suitable to amphibians according to the presence of at least one among the following features: larvae, sub-adults, adults, breeding and spawning activity. we considered as a single site all those biotopes very close to each other; consequently, 54 biotopes were numbered out of the 50 monitored sites. all sites were georeferenced and assigned to six following different freshwater typologies: river, stream or creek (c), springwatering trough (f), artificial or natural cave (g), madicolous habitat (rock surface moistened by flowing water, overgrown with mosses) (p), lake and pond (s), and “volubro”, i.e. artificial or semi-artificial catch basin exploited by livestock breeder without water turnover (v). sites were also distinguished between breeding and presence sites. all sites are listed in table 1 and represented in fig. 1. table 1. list of sites with their freshwater typology. site locality name, a.s.l. (m) type 1 fonte martino, cervara di roma (rm), 938 m f 2 volubro di camposecco, camerata nuova (rm), 1320 m v 3 cimitero di cervara, cervara di roma (rm), 1053 m v 4 volubro di pozzo verardi, camerata nuova (rm), 1053 m v 5 colle frassigno, forestal refuge, fondi di jenne (rm), 1346 m v 6 stadio di cervara, cervara di roma (rm), 1217 m v 7 campaegli, cervara di roma (rm), 1423 m v 8 campaegli, cervara di roma (rm), 1411 m v 9 campo buffone, campo dell’osso, subiaco (rm) 1444 m v 10 campitelle, fosso fioio, vallepietra (rm), 1355 m p 93amphibians of the simbruini mountains (latium, central italy) site locality name, a.s.l. (m) type 11 campitelle, fosso fioio, vallepietra (rm), 1347 m v 12 monte calvo, subiaco (rm), 1415 m v 13 campominio, campo dell’osso, subiaco (rm), 1590 m v 14 canali, sp subiaco-livata 44/b km 6.900 subiaco (rm), 1058 m f,g 15 ponte del tartaro, cimitero di vallepietra, vallepietra (rm), 813 m g 16 campo ceraso, filettino (fr), 1552 m v 17 fosse di livata, livata sp 44/b, subiaco (rm), 1245 m v 18 fonte della radica o roglioso, filettino (fr), 1135 m p 19 fiume aniene, laghetto di san benedetto, subiaco (rm), 349 m c 20 fonte della moscosa, filettino (fr), 1410 m f 21 fontana della scrofa, m.te pratiglio, jenne (rm), 1253 m v 22.a. san giovanni dell’acqua, jenne (rm), 778 m f 22.b. san giovanni dell’acqua, jenne (rm), 773 m g 23. volubro nuovo, monte pratiglio, jenne (rm), 1405 m v 24.a. sp 45/a subiaco-jenne km 7.200, jenne (rm), 838 m f 24.b. sp 45/a subiaco-jenne km 7.200, jenne (rm), 830 m f 25 sp 45/a subiaco-jenne km 7.200, jenne (rm), 821 m f 26 fosso di acqua corore, filettino (fr), 970 m p 27 san giovanni dell’aniene, subiaco (rm), 512 m s 28 fonte canali, jenne (rm), 1230 m v 29 fosso di acqua corore, fiumata, filettino (fr), 963 m c 30 monte porcaro, parking area, sp 45/a, jenne (rm), 907 m f 31 fiumata, filettino (fr), 907 m f 32 fiume simbrivio, ex ponte castello, vallepietra (rm), 643 m p 33 fiumata, filettino (fr), 949 m c 34 mola vecchia, jenne (rm), 527 m g 35 fontana fossatello, ex ponte castello, vallepietra (rm), 749 m f 36 campanile del diavolo, filettino (fr), 1067 m p 37 sentiero sotto municipio, jenne (rm), 804 m f 38 fontanile delle sette cannelle, trevi nel lazio (fr), 612 m p 39 colle dei porcili, la cimata, jenne (rm), 915 m f 40 grotte di san matteo, trevi nel lazio (fr), 652 m p 41 collerello, fiume aniene, trevi nel lazio (fr), 789 m c 42 le fontane, trevi nel lazio (fr), 905 m f 43 fonte del cardellino, trevi nel lazio (fr), 450 m g,p 44 fiume aniene, sp 29/c km 0.500, trevi nel lazio (fr), 556 m p 45 fonte suria, trevi nel lazio (fr), 761 m g,p 94 p. crucitti et alii site locality name, a.s.l. (m) type 46 laghetto del pertuso, trevi nel lazio (fr), 693 m s 47 fonte suria, trevi nel lazio (fr), 731 m f 48 comunacque, fiume aniene, trevi nel lazio (fr), 560 m g 49 caprareccia, fiume aniene, trevi nel lazio (fr), 500 m p 50 ponte delle tartare, fiume aniene, trevi nel lazio (fr), 629 m p results the sørensen’s coefficient of similarity (hayek, 1994) was calculated among species and sites, using only amphibians breeding sites to detect the affinity among species in their reproductive habitats. amphibians scientific names are here reported following the systematic revision suggested by frost et al. (2006), lanza et al. (2007, 2009), and stöck et al. (2008). statistical analysis was performed using on-line clustering calculator package (web site: www2.biology.ualberta.ca/jbrzusto/cluster.php). fig. 1. the distribution of surveyed sites in the study area, the simbruini mountains regional park (bold squares; utm 5 × 5 km) and their position in latium, central italy. low square; the position of latium in the italian peninsula. 95amphibians of the simbruini mountains (latium, central italy) overall, 80 records of amphibians were collected and breeding activity was assessed in 38 sites (sites of reproductive activity scar 76% out of total sites). the most widespread species, occurring in high frequency were salamandrina perspicillata, triturus carnifex, bufo bufo, and rana italica, all present in more than the 50% of the monitored sites (table 2). table. 2. list of species and collecting localities; numbers of localities refer to table 1; the frequency is calculated as the percentage of place of presence by respect to the sampled localities. species localities frequency salamandra salamandra (linnaeus, 1758) 26 2% salamandrina perspicillata (savi, 1821) 13, 22, 24a, 24b, 27, 38, 40, 42, 43, 45, 47. 62% lissotriton vulgaris (linnaeus, 1758) 3, 10, 21, 27, 28 16% triturus carnifex (laurenti, 1768) 3, 4, 5, 6, 8, 10, 11, 12, 15, 16, 17, 21, 22, 23, 24a, 28, 35, 49 60% bombina pachypus (bonaparte, 1838) 35 8% bufo bufo (linnaeus, 1758) 2, 3, 6, 8, 11, 12, 13, 14, 16, 17, 20, 21, 22, 23, 25, 27, 28, 30, 32, 33, 35, 36, 37, 39, 41, 44, 46, 49, 50 76% bufo balearicus boettger, 1880 7, 9 4% rana dalmatina bonaparte 22, 27, 43 6% rana italica dubois, 1987 1, 13, 14, 18, 19, 22, 24b, 27, 29, 31, 33, 34, 41, 43, 45, 46, 48 60% italian and smooth newts were mainly found in “volubri” (80% and 78% of sites respectively), located in small valley or high pastures along the border of dry grassland and beech-wood. the common toad mainly frequented the “volubri” (37% of sites), spring-watering trough (24%) and ponds near river (17%), whereas 13 sites are inside or at the edge of oak-woods, 9 inside beech-woods, 5 in moist meadow at the border of river beds, and two inside a pinus nigra reforestation. the two sites of the italian green toad were a “volubro” in a meadow and grazing-land close to a suburban area where the species was already observed by park-keepers (l. songini, in verbis, 2008), and a meadow with grazing-land near a beech wood (fosso fioio) just pointed out by romano et al. (2003). the first one represents the absolute height record for this species, 1411 m a.s.l. the agile frog was found in three sites only: an underground tunnel with flowing water and slimy-clayed substratum, a pond in a meadow nourished by waterfall in travertine rock and an artificial cave with a stream of water and “pearls of cave” substratum. the italian stream frog mainly frequented limestone caves or galleries (47% of sites), whereas other sites were streams, small lakes, spring watering-troughs and madicolous habitats; nine sites are located inside or at the edge of mixed oak woods, five inside beech-woods, and two are riverine habitat and one is in pinus nigra reforestation. finally, it is worth mentioning that other taxa reported in the neighbourhood have not been recorded in the simbruini mountains, namely the green frogs, pelophylax bergeri / p. klepton hispanicus (bonaparte, 1839) and pelophylax lessonae (camerano, 1882) 96 p. crucitti et alii fig. 2. altitudinal ranges of amphibians in the simbruini mountains regional park; mean altitude of six species is indicated by values within each bars; for three species, single data are reported (circles and numbers). ss, salamandra salamandra; sp, salamandrina perspicillata; lv, lissotriton vulgaris; tc, triturus carnifex; bp, bombina pachypus; bb, bufo bufo; bl, bufo balearicus; rd, rana dalmatina; ri, rana italica. fig. 3. phenology of amphibians observed in water biotopes of the simbruini mountains regional park. eggs, empty circles; sub-adults, squares; adults, full circles. abbreviations of species as in fig. 2. 97amphibians of the simbruini mountains (latium, central italy) and especially the italian tree frog hyla intermedia boulenger 1882, whose occurrence on the altipiano di arcinazzo at the south-western border of the pnmrs was already noticed (f. bubbico, d. cicuzza, iv.1999, pers. com.). 1,0 0,9 0,8 0,7 0,6 0,5 0,4 0,0 ss bl bp ri sp rd lv tc bb fig. 4. sintopy of amphibians observed in the simbruini mountains regional park; for details see the text. abbreviations of species as in fig. 2. 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0,0 7 26 13,22 27 14,46 31,33 4,5 10,21 2,20 35 6,8 3,28 24,42 45,48 15 25,30 11,12 47 37,39 16,17 41,44 23 49 fig. 5. sites of amphibians reproductive activity in the simbruini mountains regional park; for details see the text. abbreviations of species as in fig. 2. 98 p. crucitti et alii for some species, altitudinal distribution as min.-max. values (m a.s.l.) were detailed: s. perspicillata, 450-1058; l. vulgaris, 512-1347; t. carnifex, 500-1590; b. bufo, 500-1590; r. italica, 349-1135. range of altitudinal records was 450-1590 for caudata and 349-1590 for anura (fig. 2). t. carnifex, b. bufo and r. italica were found in water during the whole year (fig. 3). in 27 sites (54% out of sites) only one species was found, while in 14 sites (28%) two species were found: eight sites with t. carnifex and b. bufo, four with b. bufo and r. italica; in seven sites (14%), three species were found: l. vulgaris, t. carnifex and b. bufo were observed together in three sites. in two sites only, sintopy of five species was observed, though not in a single inspection exclusively. discussion our surveys increased the knowledge on the amphibians’ distribution in the study area. nine amphibians species, four caudata and five anura, that is 60.0% of the species of latium, were recorded within the hinterland and isolated area of the simbruini mountains. in accordance with the adequate biological conditions of water, as suggested by the presence of certain invertebrate taxa (mancini and arcà, 2000), amphibians are represented by many species, some of which are locally (s. perspicillata) or generally (t. carnifex, b. bufo) abundant. for the whole territory of latium, the ratio between caudata and anura species (r) is quoted as 6 : 9 (bologna et al., 2000) and in many, highland and large, protected areas, r ≤ 1, i.e. balanced or hardly favourable to anura: pnrms, 4 : 5; lepini mountains, 4 : 4 (corsetti, 1994; bologna et al., 2007); ausoni mountains, 4 : 4 (corsetti and romano, 2007); aurunci mountains, 4 : 5 (romano et al., 2007); for lowland and small protected areas, r << 1, i.e. heavily favourable to anura; “nomentum” and “macchia di gattaceca and macchia del barco”, 1 : 5 1 : 6 (crucitti et al., 2004, 2009 and unpublished data). uncommon levels of species richness i.e. sinthopic presence of five species, were observed in the south-western belt of the area, conversely to the harshness conditions of the inner and higher territories; all that in accordance with observations made in other areas of central apennine, e.g. the majella national park (scalera et al., 2006). height distribution of caudata and anura largely overlap, however the range of records for caudata is restricted to the highest belt, a datum that agrees to the one reported for wider italian areas (romano et al., 2007). the abundance of caudata in the simbruini mountains is chiefly due to the widespread presence of newts, especially t. carnifex, in the “volubri” and the italian spectacled salamander, s. perspicillata, in spring-watering trough. the district of simbruini mountains represents a refugial area for s. salamandra (linnaeus, 1758), a strongly localised species in the whole territory of latium (bologna et al., 2000, caldonazzi et al., 2007), as much as b. pachypus a species in dramatic decline in most of the italian territory (guarino et al., 2007). b. balearicus is mainly localised to piedmont areas (balletto et al., 2007) as much as r. dalmatina which is especially found in hygrophilous woods of planitial zones; besides, the agile frog is not widespread in central italy (bernini et al., 2007). l. vulgaris, though eurytopic species, is mostly found in planitial and hilly areas and rarely at higher altitude (razzetti et al., 2007). the relative abundance of s. perspicillata populations in some hilly areas, as appointed by recent herpetological atlas for the italian territory (bologna et al., 2000, 2007; sindaco et al., 2006; 99amphibians of the simbruini mountains (latium, central italy) see also romano et al., 2009), is corroborated. inside the pnrms territory, natural or artificial lakes and ponds are uncommon, conversely to extensive fluvial habitat such as the high watercourse of the river aniene and its main tributary (river simbrivio). in this last environments, it is possible to check the presence of b. bufo along the perifluvial vegetation belt; inside the river bed, where waters show a laminar flow allowing the development of a thick periphyton and macrofite, there are favourable conditions for the presence of s. perspicillata, b. bufo and r. italica. widespread water point are “volubri” and springwatering trough; in the first, the fauna is represented by the association t. carnifex-b. bufo; in the second, by s. perspicillata, also sinthopic with the previous one and with r. italica. artificial cave, especially near the entrance, together with underground tunnel, house r. italica adults and offspring and, rarely, r. dalmatina. thirteen out of 50 sites (26% of total sites) are strongly threatened, particularly the spring-watering trough of rural farm in which the periodic cleaning, especially if carried out inadequately, arousing the disappearance of the whole communities (cf. scoccianti, 2001). sites characterised by an elevated number of species (sites 22, 27) are considered strategic for the purpose of conservation. nevertheless, the species considered in this paper are strictly protected inside the territory of pnrms and formally protected inside the whole territory of latium (regional law 18/1988). in the light of these data, the importance of artificial/natural water bodies for the appropriate conservation management of the amphibian population in the simbruini mountains regional park cannot be underestimated. acknowledgments this research was supported by a grant from the ente parco monti simbruini according to the protocol marked between this institution and the società romana di scienze naturali on 19/iv/2006 in the park centre of jenne (rome). thanks to maurizio fontana (director of the pnrms), cosimo marco calò, paolo gramiccia and luigi russo; moreover, maria paola fratticci, luca tarquini, giuseppe campanella, emiliano de santis, alberto dominici, ilaria guj, alberto pelliccia, leonardo pucci and leonardo songini. some people of the srsn staff contributed to field research; marco andreini, francesco bubbico, sergio buccedi, luca cavalletti, fabrizio ceci, daniele cicuzza, angelo coccaro, maria nausica fravili, giovanni rotella. thanks to antonio romano, roberto sacchi and marco a. l. zuffi for suggestions and comments that improved a previous draft of this manuscript. references angelini, c., vanni, s., vignoli, l. 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(2009): distribution and morphological characterization of the endemic italian salamanders salamandrina perspicillata (savi, 1821) and s. terdigitata (bonnaterre, 1789) (caudata: salamandridae). ital. j. zool., first published on: 10 november 2009 (ifirst): 1-11. scalera, r., venchi, a., carafa, m., pellegrini, m., capula, m., bologna, m.a. (2006): amphibians and reptiles of the majella national park (central italy). aldrovandia 2: 31-47. scoccianti, c. (2001): amphibia: aspetti di ecologia della conservazione. [amphibia: aspects of conservation ecology], wwf italia, sezione toscana, editore guido persichino grafica, firenze. sindaco, r., doria, g., razzetti, e., bernini, f. (2006): atlante degli anfibi e dei rettili d’italia/atlas of italian amphibians and reptiles, societas herpetologica italica, edizioni polistampa, firenze. stöck, m., sicilia, a., belfiore, n.m., buckley, d., lo brutto, s., lo valvo, m., arculeo, m. (2008): post messinian evolutionary relationships across the sicilian channel: mitochondrial and nuclear markers link a new green toad from sicily to african relatives bmc evol. biol. 8 (56) (http://www.biomedcentral.com/1471-2148/8/56). egg predators of an endemic italian salamander, salamandrina perspicillata (savi, 1821) antonio romano1, cristiano spilinga2, 5, francesca montioni3, david fiacchini4, bernardino ragni5 1 università degli studi di roma “tor vergata”, via della ricerca scientifica 1, i-00133 rome, italy. corresponding author. e-mail: antonioromano71@tele2.it 2 studio naturalistico hyla, via sette martiri 2, i-06069 tuoro sul trasimeno (pg). e-mail: info@studionaturalisticohyla.it 3 località pietreto 3, i-06060 paciano (pg), italy. e-mail: booboo3@libero.it 4 via brancasecca, 11, i-60010 ostra vetere (an), italy. e-mail: david.fiacchini@libero.it 5 dipartimento di biologia cellulare e ambientale, università degli studi di perugia, via elce di sotto, i-0612 perugia, italy. e-mail: lynx@unipg.it submitted on 2007, 3rd october; revised on 2007, 25th november; accepted 2008, 30th january. abstract. we report new aquatic predators feeding on northern spectacled salamander eggs, salamandrina perspicillata, an endemic italian species. eggs were preyed upon by the leech, trocheta bykowskii, and the trichopteran larvae of potamophylax cingulatus and halesus appenninus. riassunto. in torrenti dell’umbria e delle marche è stata osservata la predazione di uova di salamandrina perspicillata da parte di una specie di sanguisuga, trocheta bykowskii e da larve di tricotteri appartenenti alle specie potamophylax cingulatus e halesus appenninus. keywords. salamandrina perspicillata, egg predation, leeches, trichopterans, urodela, italy. among oviparous species that do not guard their eggs during incubation several environmental factors often cause high embryonic mortality: sinking into mud that may reduce respiration is among the commonest natural environmental risks for amphibian eggs (miaud, 1994; griffiths, 1995). egg predation is the other main cause of amphibian embryo mortality (e.g., miaud, 1993), particularly for eggs that are preyed upon by a wide range of specialized as well as occasional egg-consumers (see romano and di cerbo, 2007, and references therein). in this work we report new invertebrate predators feeding on northern spectacled salamander eggs, salamandrina perspicillata (savi, 1821). acta herpetologica 3(1): 71-75, 2008 issn 1827-9643 (online) © 2008 firenze university press 72 a. romano et alii during 2005-2007, we surveyed 24 and 44 breeding sites of salamandrina in umbria and marche regions respectively. aquatic typologies of these breeding sites in marche were reported in fiacchini and di martino (2007), while the sites monitored in umbria were slow running streams (n = 15) and drinking throughs (n = 9). one specimen of each kind of predator was collected during predation activity, stored in ethanol, and determined in the laboratory by taxonomic specialists. eggs of this endemic italian salamander were observed to be preyed upon by a macrophagous leech trocheta bykowskii gedroyc 1913, and by trichopteran larvae of potamophylax cingulatus gambaricus (malicky, 1971) and halesus appenninus moretti and spinelli batta 1979. all these three invertebrate species are widespread in both marche and umbria. the leech and the former caddisfly have been collected in a slow running stream in umbria (see spilinga et al., 2006 for a detailed description of this site), the latter trichopteran species was detected both in marche (three slow running streams in serra san quirico, genga and sassoferrato municipalities, ancona province) and in umbria region (in the same site above mentioned). for species with complex life cycles such as amphibians, early stages (i.e., eggs and larvae) represent the most vulnerable phases (e.g., alford, 1999). if larvae may develop antipredator strategies (alford, 1999), eggs are more exposed to predation and other potential risks, due to their immobility. significant reduction in reproductive success due to predation on amphibian eggs has been shown by several studies (henrikson, 1990; axelsson et al., 1997; richter, 2000) and, in many cases, predation upon eggs can lead to 100% embryo mortality (e.g., petranka and kennedy, 1999). although some amphibians have also developed antipredator strategies to protect their eggs as wrapping behaviour of females during oviposition (orizaola and brana, 2003), unpalatibility of eggs (gunzburger and travis, 2005), earlier hatching of the embryos (e.g., warkentin, 1995; vonesh, 2000; chivers et al., 2001) or delayed hatching (e.g., sih and moore, 1993; laurila et al., 2002; schalk et al., 2002). feeding behaviour of trichopteran larvae on amphibian eggs is mainly reported for american species (richter, 2000; gunzburger and travis, 2005, and references therein). for that concerns european species, data on egg predation by caddisfly larvae were scarcely available and refer mainly to anurans (di cerbo and ferri, 1996; majecki and majecka, 1998). similarly, s. perspicillata is the only european urodele for which eggs predation by leeches was recorded so far, while predation on european anurans was reported several times (see romano and di cerbo, 2007 for a review). previous studies reported egg predation on northern spectacled salamander by another species of caddisfly plectronemia conspersa (curtis, 1834) (vignoli et al., 2001), and leech haemopis sanguisuga (linnaeus, 1758) (romano and di cerbo, 2007). also barbieri (2001) suggested the salamandrina eggs were preyed by trichopterans, even if species were not mentioned. furthermore, a record on salamandrina eggs predation has regarded rana italica tadpoles dubois 1987 (laghi and pastorelli, 2006). amphibian eggs are not usual preys for leeches and trichopterans, and might be used as alternative food resource when preferred preys are scarcely available, as suggested by gunzburger and travis (2005). however egg predation by trocheta bykowskii, potamophylax cingulatus gambaricus and halesus appenninus was observed several times during the study period, and, therefore, we can not considered it as occasional behaviour. furthermore, the fact that h. appenninus was observed to prey on salamandrina eggs both in 73egg predators of salamandrina perspicillata marche and umbria region, could suggest a widespread opportunistic feeding behaviour by this caddisfly where the two species are sintopic. egg predation by trocheta bykowskii was observed in 1.5% of the surveyed sites where salamandrina spawned and predation by potamophylax cingulatus gambaricus and by halesus appenninus was recorded in 1.5% and 5.9% of the surveyed sites respectively (n = 68). however it is worth to mention there was a different research efforts among different sites in the surveyed areas and predation was recorded only in the sites where a long term monitoring was applied. it is plausible, therefore, that these prey-predator relationships are spreader then here estimated. eggs predation in salamandrina by other vertebrata was previously reported only once (laghi and pastorelli, 2006), even though among vertebrates a lot of species have been reported to feed on eggs of many amphibian species (see for references cicortlucaciu et al., 2005; orizaola and brana, 2006; romano and di cerbo, 2007). by contrast, eggs of northern spectacled salamander highly suffer predation by invertebrates such as trichopterans larvae and leeches are (vignoli et al., 2001; romano and di cerbo, 2007; present paper), and salamandrina seems to be the most preyed species by these invertebrate taxa of egg consumers (gunzburger and travis, 2005; romano and di cerbo, 2007). females of salamandrina perspicillata lay about 20-50 eggs, in groups of 5-15 each time, singularly disposed and never in clutches (lanza, 1983; corsetti, 1999) and attach eggs to a wide set of submerged substrates (stones, steams, dead leaves, vertical walls of drinking through and so on, zuffi, 1999; della rocca et al., 2005). salamandrina females never guard their own eggs, and never provide any form of protection as it occurs in many other species of urodela. for example, newts carefully wrap their eggs in leaves of aquatic plants usually folding a part of the leaf over the egg with the back feet for protection (e.g. diaz-paniagua, 1989; duellman and trueb, 1994; griffiths, 1995; miaud 1995). therefore, the high number of invertebrate predators reported for the salamandrina perspicillata eggs could also reflect the absence of any egg protection, peculiar to this species. acknowledgements we thank fernanda cianficconi and barbara todini for taxonomic determination of caddisfly larvae and alessandro minelli for determination of the leech. we thank also sebastiano salvidio for its useful suggestions. thanks to the referees anna rita di cerbo and marco a.l. zuffi who hardly worked to improve the quality of this papers. references alford, r.a. 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(1999): salamandrina terdigitata (lacépède, 1788) – brillensalamander. in: handbuch der reptilien und amphibien europas. band 4/1. schwanzlurche (urodela) i, p. 229-246. grossenbacher, k., thiesmeir, b., eds, aula-verlag, wiesbaden. © firenze university press www.fupress.com/ah acta herpetologica 5(1): 23-29, 2010 age and growth of the sand lizards (lacerta agilis) from a high alpine population of north-western italy fabio m. guarino1, ivan di già2, roberto sindaco3 1 dipartimento di biologia strutturale e funzionale, università di napoli federico ii, via cinthia, i-80126, napoli, italy. corresponding author. e-mail: fabio.guarino@unina.it 2 via latina 126, i-10093, collegno (to), italy. 3 i.p.l.a. istituto per le piante da legno e l’ambiente, c.so casale 476, i-10132, torino, italy. submitted on: 2009, 12th november; revised on 2010, 21th march; accepted on 2010, 7th april. abstract. we studied growth and longevity of lacerta agilis from a sample (34 adults and 2 small-sized juveniles) of a population living at high altitude in north-western italy using skeletochronological method. snout vent length (svl) mean of males did not significantly differ from that of females although the latter were in average bigger (svl ± sd, males: 69.3 ± 7.1 mm, n  =  11; females: 73.9 ± 9.7 mm, n  =  22; mannwhitney u-test, u  =  1.76, p  =  0.077). age ranged from 2 to 4 years (mean age ± sd  =  2.3 ± 0.2) in males and from 2 to 3 years in females (mean age ± sd  =  2.59 ± 0.5 years). age mean did not significantly differ between the sexes (mann-whitney u-test, u  =  1.35, p  =  0.174). the two juveniles were 30 and 32 mm in svl and both were 1-2 months old. in both sexes, a significant positive correlation between svl and age was recorded although weakly significant for males (spearman’s correlation coefficient, males: rs  =  0.70, p  =  0.05; females: rs  =  0.75, p < 0.001). von bertalanffy growth curves well fitted to the relationships between age and svl and showed a different profile between males (asymptotics size, svlmax  =  81.9 mm; growth coefficient, k  =  0.63) and females (svlmax  =  100 mm; k  =  0.40). results indicate that individuals of l. agilis studied by us are short-living when compared with other populations of the same species. keywords. growth, longevity, skeletochronology, lacerta agilis, italy, alps. introduction lacerta agilis is one of the most widely distributed palaearctic lizards, being found from the pyrenees, central france and england in the west to central asia in the east (sindaco and jeremčenko, 2008). in italy, the species seems to be quite rare; in fact, so far it has been found only in small areas of south-western piedmont and north-eastern friulivenezia-giulia (lapini and sindaco, 2006). 24 f.m. guarino, i. di già and r. sindaco populations living in high altitude habitats in the south-western alps, between italy and france, are isolated from the main range of the species, where lizards live mostly at much lower altitudes. unfortunately, data on the biology of sand lizard in italy are still largely incomplete (di già and sindaco, 2004; lapini and dall’asta, 2004) and this represents a serious gap in terms of conservation of the species which appears as severely declining in a number of countries at the north-western part of its range (edgar and bird, 2006). this study aimed to know longevity and growth rates of l. agilis from a south-western alpine population using skeletochronological method. materials and methods animals the sample investigated consisted of 36 animals (11 males, 23 females and 2 small-sized juveniles) captured by hand in july-early september 2001. in the field, each lizard was measured for snout-vent length (svl, to the nearest 1 mm) and toe clipped at level of the second phalanx of the fourth, third or second toe of the right forelimb to mark individually the specimens. the cut toe was immediately fixed in 70° ethanol and stored until the time of skeletochronological analysis. after measurements and toe-clipping, each lizard was photographed and released in the same place where captured. no lizards were sacrificed for this study. study area the studied population is located in the surroundings of the small village of ferrere (argentera municipality, cuneo province, piedmont region), between 1790 and 1890 m a.s.l. as in the other known localities of western italian alps, the vegetation is a typical alpine pasture habitat on calcareous substrate, on terraced slopes mostly facing either east or south, with only a few scattered small trees or bushes. at the study site l. agilis prefers the edge of stone heaps and dry-stone walls, hidden among the residual patches of higher vegetation of rather overgrazed pastures, in particular urtica dioica and nepeta nepetella (di già and sindaco, 2004). laboratory technique skeletochronology was performed according to a standard protocol used for other species of reptiles (andreone and guarino, 2003; guarino et al., 2004). after removal of soft tissues, phalanx was decalcified with 3% nitric acid for a time ranging between 1 and 2 hours depending on bone size, rinsed in tap water and cross sectioned using a cryostat. diaphyseal cross sections was stained with mayer’s haemalum (25 min) and mounted in aqueous resin. periosteal lines of arrested growth (lags) were independently counted by two researchers using a light microscope equipped with an image analyser. two representative sections with the smallest medullar cavity were acquired and transformed as digital photo. images were optimized with respect to contrast and brightness using adobe photoshop version 6.0 to facilitate the distinction of the periosteal lags from the surrounding bone matrix. possible under estimation of lag number due to endosteal resorption, which may cause the complete loss of the inner (perimedullar) lags, was evaluated by osteometric analysis (guarino et al., 25skeletochronology of lacerta agilis 2003, 2004). the perimeter of reversal line (rl), which is the boundary between periosteal and endosteal bone, and that of the bone outer margin (bom) of the small sized individuals, was measured and compared with the perimeter of rl and bom of animals with one or more lag. since we cut the second phalanx of the third toe from the forelimb of the two small sized lizard, the comparisons was possible only with males and females from which was cut the same type of skeletal element. growth patterns were estimated according to von bertalanffy’s model, which previous studies have shown to be the model that better fits the relation between body size and age for most saurians (james, 1991; wapstra et al., 2001; roitberg and smirina, 2006). the general form of the von bertalanffy equation used is: lt = a × (1-e-k (t-t0)) where lt is the svl (mm) at time t, a is the estimated average maximum svl that can be reached (or mean asymptotic svl), e is the base of the natural logarithm, k is the growth rate (the speed at which asymptotic svl is attained), and t0 is the age at hatching, which is the starting point of the growth interval under study. we assumed as size at hatching (lt0) the mean value provided by in den bosh and bout (1998) because of the lack of incontrovertible data on the size at hatching of the studied population, due to the very low number of young collected during the field study. the parameters a (asymptotic svl) and k, and their asymptotic confidence intervals, were estimated using a non-linear regression procedure by means of the growth ii software (henderson and seaby, 2006). growth curves were considered to be significantly different if the 95% confidence intervals did not overlap (james, 1991; wapstra et al., 2001). all numerical data were analyzed by non parametric tests which have been proved to be effective in the case of small samples of data. in particular, mann-whitney u test was used for the comparison of means. spearman’s rank order correlation coefficient was used for estimating the relationship between age and svl. a probability level of p < 0.05 was considered significant. results females were bigger in average and maximum svl than males but svl mean did not significantly differ between the sexes (svl ± sd, males: 69.3 ± 7.1 mm, n  =  11, range: 60-81 mm; females: 73.9 ± 9.7 mm, n  =  22, range: 50-90; mann-whitney u-test, u  =  81, p  =  0.101). the two juveniles were 30 and 32 mm long. all phalangeal cross sections showed well-defined lines of arrested growth (lags) (fig. 1a-c). as in other species of temperate areas, we assumed that only one lag is formed during each hibernating period, that for the analyzed lizards ranges from late september to advanced may. therefore we considered the number of observed lags, plus that of completely reabsorbed lags, if any, equivalent to the individual’s age. both juveniles showed one visible hematoxynophilic line (fig. 1a), very close to the marrow cavity and partially destroyed, here considered as hatching line therefore, the two juveniles were estimated as few months old. based on osteometric analysis, in two males (18.2%) and 4 females (13.6%), respectively, the innermost lags were estimated to be completely removed by endosteal remodelling. maximum age was 4 years for males and 3 years for females (fig. 2). the mean age ± sd was 2.4 ± 0.7 years in males (n  =  11; range: 2-4 years) and 2.5 ± 0.5 years in females (n  =  22 range, 2-3 years). age mean did not significantly differ between the sexes (mannwhitney u-test, u = 80, p = 0.161). in both sexes, a positive correlation between svl and age was recorded, but it was not significant for males (spearman’s correlation coefficient, males, rs = 0.59, p = 0.06; females, rs = 0.65, p < 0.001). growth pattern estimated by von bertalanffy’s showed a well fit to the relation between age and svl (fig. 2). for both sexes, the estimated asymptotic svl was slightly 26 f.m. guarino, i. di già and r. sindaco fig. 1. representative cross-sections of phalanges of lacerta agilis. (a) individual 32 mm svl with no line of arrested growth (lag). (b) female 73 mm svl with 1 visible lag plus one situated at the outer margin of the periosteal bone. (c) male 81 svl with 4 lags. arrows indicate lags. abbreviations: hl = hatching line; rl = reversal line. all figures are at the same magnification. fig. 2. the von bertalanffy growth curves for males (solid square, solid line) and females (open circle, dot line) of l. agilis. arrow shows svl mean of the lizards at hatching as reported by in den bosch and (1988). double arrow shows svl of the two juveniles sampled in this study. growth parameters are given in the text. 27skeletochronology of lacerta agilis higher than the maximum svl recorded (svlasym ± ci, males: 81.9 ± 1.65 mm; females: 100 ± 4.9 mm). growth coefficient was higher in males than in females (k ± ci, males: 0.63 ± 0.04; females: 0.40 ± 0.03). growth curve of males was significantly different from that of females. discussion among reptiles body length, growth rates, age at maturity and longevity can widely vary between populations of the same species. as a rule, individuals from high-elevation sites and northern latitude live longer than those from low-elevations sites and southern latitudes (wapstra et al., 2001; roitberg and smirina, 2006). thermal constraints may provide a possible explanation for this trend. in fact, the shorter activity period in cooler climate, due to unfavourable conditions of temperature and food availability, should reduce the risk of predation (seurs, 2005; roitberg and smirina, 2006). in addition, some authors also suggested that in cooler areas the lower predation pressure could also be due to the presence of a reduced number of predator species (roitberg and smirina, 2006). therefore, it is interesting that longevity of l. agilis recorded in this study (4 years for males and 3 for females) is lower than those reported for other populations of the same species. in fact, in l. agilis living in daghestan (russia), at different altitudes, maximum longevity ascertained was 6 years for males and 5 years for females in the population from lowland and submontane regions (until 600 m. a.s.l.); 7 years for males and 6 years for females in the population from highlands (starting from 960 m. a.s.l) (roitberg and smirina, 2006). in l. agilis living along the western coast of sweden, maximum longevity was 11 years for males and 12 for females (olsson and shine, 1996). it is surprising that the studied population is so short living and the reason for this is not yet well understood. although data about predation on the lizards under study are not available, predators nor potential competitors were not observed at high concentrations during the two years of field survey. the only lizard specialist is the smooth snake (coronella austriaca), found only once during the study; an occasional predator is also the asp viper (vipera aspis), rare in the area. among birds, observed predators were the kestrel (falco tinnunculus) and the red-backed shrike (lanius collurio), both very widespread on the whole range of l. agilis and apparently not more numerous at the study site than in other similar places of the western italian alps. studies on other western alpine populations of l. agilis are needed to understand if the short longevity of the studied sample is due to a local conditions or is the rule in these isolated populations. the two small sized juveniles (30 and 32 mm in svl) examined in this study were younger than 1 year (0 year old). this is highly plausible taking into account that: a) in this species, the mean svl ± sd at hatching is 24.6 ± 5.1 according to in den bosch and bout (1998); b) at the sampling site the hatchings usually occur between advanced julyearly august; c) activity period during which the lizards may forage and growth is very short (late may to end of august-early september); d) the two juveniles were sampled at the end of august 2001. as in other saurian species (galan, 1999; wapstra et al., 2001), also in l. agilis growth rates are very high before the attainment of sexual maturity and decline with age. a recur28 f.m. guarino, i. di già and r. sindaco rent adaptive explanation of this growth pattern is that the animals use energy mainly for somatic growth before sexual maturity. in males of l. agilis studied by us a marked increase in body length is observed between the first and the second hibernation (second year of life) and in most of females between the second and third hibernation (third year of life). these findings would suggest that males of l. agilis from north-western italy attain maturity after the second year of life, while females after the third year of life, like in other mountain population of this species (roitberg and smirina, 2006). the growth trajectories are differ between sexes. the lower value of k in the von bertalanffy equation in females suggests that they attain the asymptotic body length slower than males. this finding is similar to that from swedish populations but different from that from russian ones. the diversity of growth pattern of the different populations of l. agilis could be due to different selective pressure in the different populations so far studied. to conclude, the studied population is very peculiar because of its short-living individuals: this is maybe symptomatic of a ecological problem in the studied area, although causes are still unknown. if the study of other close ranging populations will confirm than other western alpine l. agilis are short lived too, then the explanation will be found in some ecological adaptations, if not it will be searched on man induced problems. in the latter case, it will be very important to identify and remove the causes: in fact, in italy l. agilis is a very rare reptile of national conservation concern, at the edge of its range, and the studied area was established as a site of the natura 2000 network of european protected areas also owing to its presence. acnowledgements the field work was conducted in the framework of the convention between the italian ministry of the environment and the unione zoologica italiana “the conservation status of threatened amphibians and reptiles species of italian fauna” (see bologna, 2004). we thanks dr. lisa signorile for reviewing the english version of the manuscript. references andreone, f., guarino, f.m. (2003): giant and long-lived: age structure in macroscincus coctei, an extinct skink from cape verde. amphibia-reptilia 4: 459-470. bologna, m. (2004): introduction: a monitoring project on threatened italian amphibians and reptiles. ital. j. zool. 71 (suppl. 1): 3-8. di già, i., sindaco, r. (2004): the distribution and status of lacerta agilis in piedmont (nw italy). ital. j. zool. 71 (suppl. 1): 117-119. edgar, p., bird, d.r. (2006): a plan for the conservation of the sand lizard (lacerta agilis) in northwest europe. convention on the conservation of european wildlife and natural habitats, standing committee 26th meeting strasbourg, 27-30 november 2006. galan, p. (1999): demography and population dynamics of the lacertid lizard “podarcis bocagei” in north-west spain. j. zool., lond. 249: 203-218. 29skeletochronology of lacerta agilis guarino, f.m., di maio, a., caputo, v. (2004): age estimation by phalangeal skeletochronology of caretta caretta from mediterranean sea. ital. j. zool. 71 (suppl 2): 175-180. guarino, f.m., lunardi, s., carlomagno, m., mazzotti, s. (2003): a skeletochronological study of growth, longevity and age at sexual maturity in a population of rana latastei boulenger, 1879 (amphibia, anura). j. biosci. 28: 775-782. henderson, p.a,. seaby, r.m. (2006). growth ii, pisces conservation ltd., lymington, england. james, c.d. (1991): growth rates and ages at maturity of sympatric scincid lizards (ctenotus) in central australia. j. herpetol. 25: 284-295. in den bosch, h.a.j., bout, r.g. (1998): relationships between maternal size, egg size, clutch size, and hatchling size in european lacertid lizards. j. herpetol. 32: 410-417. lapini, l., dall’asta, a. (2004): lacerta agilis in north-eastern italy. ital. j. zool. 71 (suppl.1): 121-124. lapini, l., sindaco, r. (2006): lacerta agilis. in: sindaco, r., doria, g., razzetti, e., bernini, f. eds, atlante degli anfibi e dei rettili d’italia/atlas of italian amphibians and reptiles. societas herpetologica italica, edizioni polistampa, firenze, p. 448-453. olsson, m., shine, r. (1996): does reproductive success increase with age or with size in species with indeterminate growth? a case study using sand lizards (lacerta agilis). oecologia 105: 175-178. roitberg, e.s., smirina, e.m. (2006): age, body size and growth of lacerta agilis boemica and l. agilis strigata: a comparative study of two closely related lizard species based on skeletochronology. herpetol. j. 16: 133-148. seurs, m.w (2005): geographic variation in the life history of the sagebrush lizard: the role of thermal constraints on activity. oecologia 143: 25-36. sindaco, r., jeremčenko, v.k. (2008): the reptiles of the western palearctic. 1. annotated checklist and distributional atlas of the turtles, crocodiles, amphisbaenians and lizards of europe, north africa, middle east and central asia. monografie della societas herpetologica italica. i. edizioni belvedere, latina (italy). wapstra, e., swan, r., o’reilly, j.m. (2001): geographic variation in age and size at maturity in a small australian viviparous skink. copeia 2001: 646-655. © firenze university press www.fupress.com/ah acta herpetologica 5(2): 255-258, 2010 first report of freshwater leech helobdella stagnalis (rhyncobdellida: glossiphoniidae) as a parasite of an anuran amphibian rocco tiberti*, augusto gentilli dipartimento di biologia animale, università di pavia, via ferrata, 1, i-27100, pavia, italy. * corresponding author. e-mail: fitibert@inwind.it submitted on: 2010, 19th may; revised on: 2010, 31th august; accepted on: 2010, 25th october. abstract. the authors describe the first case of parasitism on anuran amphibian, rana temporaria, by the freshwater leech helobdella stagnalis, in a mountainous area of northwestern italy. the presence of skin abrasions and haemorrhages attributable to leech attack discards the hypothesis of a simple phoretic relationship between leech and frog. keywords. helobdella stagnatilis, leech, parasite, anuran, rana temporaria. helobdella stagnalis (linnaeus, 1758) is an aquatic predator which preferentially feed on immature stages of freshwater arthropods, annelids and snails (sawyer, 1986). even if it has been reported as an occasional parasite of poikilothermic vertebrates (malek and mccallister, 1984), there are few data supporting this contention: reports of h. stagnalis as a parasite of fish are rare (mishra and chubb, 1969; malek and mccallister, 1984) and walton (1964) did not include h. stagnalis in the summary of host-parasite associations involving amphibians. the only description of h. stagnalis as an amphibian parasite dates back to ‘90s, when it was found from an urodele host, the tiger salamander (ambystoma tigrinum) from a population of indiana (u.s.a.) (platt et al., 1993); as a matter of fact, minelli (1977) described h. stagnalis as an occasional parasite of amphibian, but he did not support this assertion with any reference or observed data. this paper describes the second report of h. stagnalis from an amphibian host and the first from an anuran host: the common frog (rana temporaria), from mountain ponds in northern italy. frogs were collected in may, 2005, from three permanent pastureland ponds filled by melting snow and rain (table 1). the ponds lie on a mountain pastureland in mount guglielmo massif (southern italian alps – valtrompia – municipality of pezzaze) (table 1). frogs were collected in water during the breeding season: just one parasited frog was collected for anatomopathological analysis and delivered alive to the izs (istituto zoopro256 r. tiberti and a. gentilli filattico sperimentale) of brescia; 14 other individuals were captured, observed and immediately released. leeches have been kept in 70° ethanol and readily identified as h. stagnalis thanks to the presence of a chitinized nuchal scute, which is characteristic of this species (sawyer, 1986) (fig. 1). a summary of the observations on collected frogs is presented in table 2. frog 1 (table 2) was the only collected frog: it was under a strong leech attack and it showed several dark globular clusters under the skin and in the foot webs; laboratory analysis confirmed the presence of h. stagnalis and reported the presence of skin abrasions and haemorrhages attributable to leech presence. the dark globular clusters were identified as melanophore accumulations coupled with local dermic inflammation. this kind of hyperplasia, or cell proliferation, is usually a non-specific finding and can occur at sites of previous cutaneous injury or infection (pessier, 2007); for this reason we suggest a causal relationship between leech attack and melanophore globular clusters. in the table 1. main geographical and morphological data of pond-1, pond-2 and pond-3; a: area; d: maximum depth. lat. long. alt. (m a.s.l.) a (m2) d (m) pond-1 45°45’43’’ n 10°11’59’’ e 1373 1125 1,1 pond-2 45°45’35’’ n 10°11’56’’ e 1408 890 1,7 pond-3 45°45’59’’ n 10°11’85’’ e 1321 485 0.6 fig. 1. helobdella stagnalis from an anuran host on a 5 mm grid. 257first report of helobdella stagnalis as a parasite of an anuran amphibian former observation of leeches parasitism on a. tigrinum (platt et al., 1993) skin abrasions were absent as well as melanophore accumulations. therefore, our observations demonstrate without doubt an actual parasitism from leech on r. temporaria and not only a simple phoretic relationship between leech and frogs. moreover, on the 25th of may, the latest part of breeding period, the observed frogs were almost not interested by the presence of leeches, but they still showed melanophore accumulations (table 2). we consider the melanophores accumulations as a mark of previous leech attack and the observed lack of h. stagnalis as a consequence of more terrestrial habits in post breeding frogs. acknowledgements we are particularly grateful to prof. alessandro minelli who checked our determination on helobdella stagnalis and to gev-valle trompia for their kind help in data collection. sample examination would not have been possible without the contribution of the izs (istituto zooprofilarttico sperimentale) of brescia. references malek, m., mccallister, g. (1984): incidence of leech helobdella stagnalis on the colorado river in west central colorado. great basin nat. 44: 361-362. minelli, a. (1977): irudinei (hirudinea). guide per il riconoscimento delle specie animali delle acque interne italiane. cnr aq/i/2, roma. table 2. summary of the observations on frogs collected in mount guglielmo area. pond date h. stagnalis presence frog 1 2 may 5th, 2005 y some tens of parasites on belly, throat, anterior and posterior legs; presence of dark globular clusters frog 2 2 may 5th, 2005 y some tens of parasites on belly, throat, anterior and posterior legs. frog 3 2 may 5th, 2005 y some tens of parasites on belly, throat, anterior and posterior legs. frog 4 3 may 5th, 2005 y less than ten parasites on belly and posterior legs frog 5 1 may 5th, 2005 y some tens of parasites on belly, throat, anterior and posterior legs. frog 6 1 may 5th, 2005 y some tens of parasites on belly, throat, anterior and posterior legs. frog 7 1 may 5th, 2005 n absence of parasites frog 8 1 may 5th, 2005 n absence of parasites frog 9 1 may 5th, 2005 n absence of parasites frog 10 3 may 25th, 2005 n absence of parasites; presence of dark globular clusters frog 11 3 may 25th, 2005 n absence of parasites; presence of dark globular clusters frog 12 3 may 25th, 2005 n absence of parasites; presence of dark globular clusters frog 13 3 may 25th, 2005 n absence of parasites; presence of dark globular clusters frog 14 3 may 25th, 2005 n absence of parasites frog 15 2 may 25th, 2005 y just one parasite on the posterior legs 258 r. tiberti and a. gentilli mishra, t.n., chubb, j.c. (1969): the parasite fauna of the fish of the shropshire union canal, cheshire. proc. zool. soc. lond. 157: 213-224. pessier, a.p. (2007): cytologic diagnosis of disease in amphibians. veterinary clinics of north america. exot. anim. pract. 10: 187-206. platt, t.r., sever, d.m., gonzalez, v.l (1993): first report of the predaceous leech helobdella stagnalis (rhynchobdellida: glossiphoniidae) as a parasite of an amphibian, ambystoma tigrinum (amphibia: caudata). am. midl. nat. 129: 208-210. sawyer, r.t. (1986): feeding biology, ecology and systematics. in: leech biology and behaviour, vol. ii, p. 419-793. clarendon press, oxford. walton, a.c. (1964): parasites of amphibia. wildlife disease. contribution no. 39. knox college, galesburg, illinois. acta herpetologica 18(1): 23-36, 2023 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-14050 patterns of acoustic phenology in an anuran assemblage of the yungas andean forests of argentina martín boullhesen1,2,*, marcos vaira1, rubén marcos barquez2, mauricio sebastián akmentins1 1 instituto de ecorregiones andinas (inecoa), conicet – unju. san salvador de jujuy, argentina 2 argentina b instituto de investigaciones de biodiversidad argentina (pidba), facultad de ciencias naturales e instituto miguel lillo, san miguel de tucumán, argentina *corresponing author. email: m.boullhesen@conicet.gov.ar submitted on: 2022, 7th december; revised on: 2023, 21st april; accepted on: 2023, 27th april editor: mattia falaschi abstract. breeding seasons in anurans are usually noticed by their advertisement calls, which stand as the main signal emitted by males during their adult life. these calls are species-specific signals with multiple information and can be used to monitor anuran populations over extended time periods. applying a passive acoustic monitoring method (pam), we described the acoustic breeding phenology of an anuran assemblage along an altitudinal elevation range in the yungas andean forests of argentina. in addition, we propose a new classification scheme for their acoustic phenological strategies, based on the male’s calling records throughout an entire year. also, we assessed the temporal and spectral niche overlap by the anuran species recorded. the assemblage was active throughout the entire year, with a higher concentration of calls recorded during the spring-summer season. we describe five distinct acoustic breeding strategies based on the calling patterns of the recorded species. temporal niche overlap was higher in the springsummer season and in the lowest study site. the use of a pam as a tool to monitor the advertisement calls in anurans communities could be a reliable technique to obtain different information about the species’ acoustic phenology and the temporal use of the acoustic communication channel. keywords. acoustic breeding strategies, acoustic phenology, advertisement calls, anurans, calling guilds, passive acoustic monitoring. introduction advertisement calls are the main signals emitted by anurans during the breeding season (wells, 2007). these species-specific features are mainly expressed by males, with some exceptions in females (e.g., the bullfrog lithobates catesbeianus) of certain species, and voiceless males like rhinella gallardoi (emerson and boyd, 1999; carrizo, 1992). advertisement calls are considered key factors for mate selection (márquez and verrell, 1991; gerhardt and huber, 2002; wells and schwartz, 2007) and are used by receivers according to their temporal and spectral characteristics, both for identification and location of their potential pairs, as well as to recognize the quality of the emitter (gerhardt and schwartz, 2001; mason, 2007). thus, these signals are used in mate choice behaviour, transmitting several messages simultaneously (candolin, 2003). in addition, due to their “species-specific” nature, these are used as a taxonomic character for species description and identification (köhler et al., 2017). thus, advertisement calls have been widely used in long-term monitoring programs (bridges and dorcas, 2000; dorcas et al., 2009; llusia, 2013; márquez et al., 2014; measey et al., 2016). the surveying of advertisement calls has proven to be a useful technique for detection and monitoring of anuran species at large spatial scales (de solla et 24 martín boullhesen et alii al., 2006), as well as for assessing population changes over time (buckley and beebee, 2004; pieterson et al., 2006). these kinds of surveys can also provide valuable information about threatened species that breed in narrow time windows or display sporadic calling activity (williams et al., 2013; willacy et al., 2015; akmentins and boullhesen, 2020). the breeding phenology of anurans, monitored through their advertisement calls, can be explored at different time scales, mainly in regions where seasonality shapes the extent, the start, and ending of their breeding activity (weir and mossman, 2005). thereby, different species may have distinct daily and seasonal calling patterns (cook et al., 2011; yoo et al., 2012). studying these calling patterns, can serve as a tool to define the core calling periods of the species recorded (lemckert and mahony, 2008), for which it is considered of utmost importance to increase the detection probability in anuran survey programs.  the use of automated recording devices for passive acoustic monitoring techniques (pam) can add valuable information such as the acoustic breeding patterns, as several species of anurans may breed simultaneously in different reproductive sites (nelson and garcia, 2017; duarte et al., 2019; ulloa et al., 2019; pérez-granados et al., 2020). amphibians are suffering alterations in their phenological patterns because of the increasingly frequent extreme climatic events caused by the global climate crisis (lanno and stiles, 2020). this is particularly concerning for the species distributed throughout the biodiversity hotspot of the tropical andes of south america (myers et al., 2000), which are severely affected by the consequences of climate change (burrowes, 2008). in addition, in times of the global diversity crisis, amphibians are considered the most affected groups among terrestrial vertebrates (stuart et al., 2004; iucn, 2023). based on the temporal pattern of their breeding phenology, anurans have been historically classified into two discrete groups, as proposed by wells (1977): explosive breeders and prolonged breeders. although there is a behavioural continuum between these two extremes (wells, 1977; 2007). several works focused on better describing the breeding acoustic strategies that can be found in anuran species (chen et al., 2023; donnelly and guyer, 1994; forti et al., 2022; huang et al., 2001; prado et al., 2005; prasad et al., 2022; bertoluci and rodrigues, 2002) but there is still missing information mainly for neotropical anurans. continuous monitoring of anuran advertisement calls along different spatial-temporal scales, can provide valuable information about the different breeding strategies displayed by each species within an assemblage (moreira et al., 2007). the acoustic niche hypothesis (krause, 1987) proposes that each individual elaborating messages through sound in each environment will present a partition in its spectral and temporal features, to avoid being masked by others (krause, 1993). in species-rich assemblages from different clades, different strategies are expected to be present to avoid the competition in the acoustic communication channel (bertolucci and rodrigues, 2002; herrick et al., 2018; klump and gerhardt, 1992). one way to minimize competition for the acoustic space is made effectively by segregating the niche in its temporal and spectral dimensions (both and grant, 2012; sinsch et al., 2012; guerra et al., 2020, lima et al., 2019). the temporal segregation of the advertisement calls can be fundamental for the constitution of large anuran assemblages, mainly in breeding areas where several species vocalize simultaneously (drewry and rand, 1983; schwartz and wells, 1983; bertolucci and rodrigues, 2002; duarte et al., 2019).  the yungas ecoregion is one of the most biodiverse environments in argentina (brown et al., 2006) harbouring up to 40 species of anurans (modified from lavilla and heatwole, 2010). these subtropical montane forests are characterized by a steeped altitudinal gradient described by phytogeographic stratums (grau and brown, 2000). the anuran assemblages that inhabit the subtropical montane forest of the parque nacional calilegua (pnc) within the yungas ecoregion, was reported to present a wide range of temporal and spatial breeding patterns ranging from opportunistic to prolonged breeders (vaira, 2002). however, these records of breeding activity were obtained based on regular monthly surveys lasting from 3 to 5 days carried out through active searches by visual and acoustic sampling (vaira, 2002). the anuran assemblage of the pnc is composed by a few endemic species to the yungas ecoregion and by numerous species with a wide distribution in argentina and other south american countries (lavilla, 2001; vaira et al., 2017). in addition, a low number of studies aimed to describe and understand the complete acoustic breeding phenology over an entire year in neotropical anurans (bertoluci and rodrigues, 2002; prado et al., 2005; saenz et al., 2006). therefore, there are still wide gaps of information about the daily and annual patterns of calling activity and the breeding acoustic phenology of the anuran species that conforms different assemblages inhabiting the yungas forests.  a recent study suggests the use of pam as an effective technique to monitor the species of anurans in the yungas forests (boullhesen et al., 2021). in addition, with the implementation of a pam program, researchers were able to discover hidden behavioural insights of phenological activity, such as the nocturnal calling activity of a 25patterns of acoustic phenology in anurans of the yungas frog species inhabiting the yungas forests, that was previously considered as a strictly diurnal species (pereyra et al., 2016). in this study, we describe the daily and annual patterns of calling activity of the anuran assemblages inhabiting the yungas andean forests using pam. furthermore, we propose a new classification scheme for the acoustic breeding strategies used by different species of neotropical anurans. also, we aim to explore the occupation level of the acoustic communication channel and to determine the  temporal overlap of the acoustic niche of this subtropical anuran assemblage. materials and methods study sites and data collection a pam was carried out for 12 months (from september 2017 to august 2018) along an altitudinal gradient in the pnc, being the most representative portion of the yungas ecoregion in jujuy province, argentina. the pnc harbours the phytogeographic strata described for the yungas forests as well as different environments where anurans breed (vaira, 2002). the study was carried out in the three main forest types described for the ecoregion: premontane lowland forests (400 700 m a.s.l.) which is a semideciduous environment with a marked seasonality, the understory consists of dense bushes, herbs, ferns and lianas; lower montane forests (700 1300 m a.s.l.) dominated by evergreen trees such as juglans australis, cedrella balansae and enterolobium contortisiliquum; the upper temperate montane forests (1500 – 2500 m a.s.l.) which is a primary old forest dominated by trees from myrtaceae family (grau and brown, 2000). the two upland forest types have markedly more moisture than the lower forest. three automated recording units song meter 4 (wildlife acoustics inc., concord, massachusetts), one per site were installed and programmed to record 3 minutes per hour (24/7) (shirose et al., 1997; márquez et al., 2014). recordings were done in mono channel using in-built low noise microphones and stored in 32gb sdxc flash cards in .wav format. the recorders were placed at 1.5 m above ground in three sites: premontane forest (pf) (23°45’16.84”s; 64°50’59.35”w, 650 m a.s.l.), at the edge of a permanent pond with an approximate area of 1114 m2. the lower montane forest (lmf) (23°41’36.84”s; 64°52’5.04”w, 1125 m a.s.l.), at the edge of a permanent stream and in upper montane forest (umf) (23°40’28.56”s; 64°53’44.15”w, 1750 m a.s.l.), attached to a tree near temporary ponds. recorders were visited monthly to data download and battery replace. these locations are representative to the breeding areas used by the anuran assemblage of the region (vaira, 2002). data analysis 13,485 recordings were listened corresponding to one day per week (= 224.75 hours) from the three study sites together throughout a year-round monitoring. recordings were inspected manually by a trained specialist in anurans call recognition of yungas forests (mb) in raven pro 1.5 (bioacoustics research program, 2014) using a window type = hann, dft size = 512 samples, and overlap = 50%. for the general description of the acoustic phenology of the anuran species recorded, the monitored year was divided in two seasons (six months each) marked by the regional climate as follows: a) spring-summer season, corresponding to the period from september 2017 to march 2018. b) fall-winter season, corresponding to the period from march 2018 to september 2018. to describe the annual acoustic phenology, we used the classification of core calling periods proposed by lemckert and mahony (2008). this considers the core calling period for each species as the time-period containing > 90 % of the calling events. for these classifications we considered the species with a total of ≥ 50 calling events records only. with the data of the calling events per-day recorded throughout the year-round study we conducted a bottom-up hierarchical cluster analysis using vegan and cluster r packages. for this analysis euclidean distance and complete method were employed after correlation checking of the cophenetic distance obtained with the original data used (>0.90). this analysis was implemented in free software r. to describe the daily and annual vocal activity of each species we adapted the classification proposed by bridges and dorcas (2000) as follows: 0 = no male vocalizing; 1 = one male vocalizing; 2 = multiple males vocalizing with the possibility of occasionally distinguishing single calls; 3 = multiple males vocalizing but unable to distinguish single calls. we considered each advertisement call detected from a recording as a “calling event”, since we could not assign a distinct call to an individual. circular statistics was employed to describe and analyse the daily calling patterns (jammalamadaka et al., 2001; pewsey et al., 2013). rayleigh test was applied to explore whether the population of circular data, from which a sample is drawn, differs from randomness (wilkie, 1983). to explore temporal niche overlap in calling activity, we computed the pianka (pianka, 1973) and czekanowski indices (feinsinger et al., 1981) using the timeoverlap program (castro-arellano et al., 2010; guerra, 2020). the czekanowski index or proportional similarity index 26 martín boullhesen et alii varies between 1 (widest amplitude of the niche where the population exploits the resources in proportion to their availability) and 0 (where the population specializes in the rarest state of a resource and skip the other items).  whereas the pianka index can return values less than zero (allowing for a coexistence between species) or greater than one (promoting a competition between species). to characterize the use of the acoustic communication channel in anurans recorded, the methodology proposed by emmrich et al. (2020) was used according to their acoustic calls features where: guild a = unmodulated simple call guild b = modulated simple call guild c = unmodulated pulsed call guild d = modulated pulsed call guild e = unmodulated pulsed multi-note call guild f = uniform modulated pulsed multi-note call guild g = non-modulated different multi-notes call guild h = modulated different multi-notes call these guilds groups where previously visualized using clean calls from each species recorded with the seewave package in r using a hanning type windows, 75 % overlap and 1024 sample size (fig. s1). results annual calling periods a total of 3318 calling events of anuran species were recorded in the three study sites within the calilegua national park (fig. 1). we detected calling activity throughout the entire year (fig. 1), with a minimum of one species vocalizing in the driest months of july and august and a maximum of 16 species vocalizing simultaneously during december (fig. 1).  most of the calls were recorded during the hottest and rainy period (spring-summer seasons), with peaks of calling activity during november, december, and january (tables 1 and 2). boana riojana was the only species recorded throughout the year-round monitored and presented high records in the fall-winter period (fig. 1). meanwhile, the rest of the species from hylidae family were recorded calling over the spring-summer period (fig. 1-2). most of the species from leptodactylidae family where recorded calling at spring-summer periods, with the summer period having the highest core calling periods. however, pleurodema borellii presented peaks of calling activity in the fall-winter period (fig. 1). the two toads of the genus rhinella were registered calling during the early springsummer period. meanwhile melanophryniscus rubriventris was recorded vocalising in the spring-summer, but was also detected in the early dry-cold fall-winter period (fig. 1). the direct development-frogs from strabomantidae family presented calling records concentrated in the spring-summer period, mainly during the summer season (fig. 1-2). the only arboreal specie from phyllomedusidae family (p. boliviana) presented calling records concentrated in the summer season (fig. 1-2). acoustic breeding strategies the cluster analysis revealed five different acoustic breeding strategies (fig. 3): continuous breeders, prolonged non-seasonal breeders, prolonged seasonal breeders, prolonged non-regular breeders, and sporadic seasonal breeders. a continuous breeding strategy was found in males of boana riojana, who vocalized during all months of the year. a prolonged non-seasonal breeding pattern was observed in pleurodema borelli, which started calling in spring-summer and continued through the fall-winter period. prolonged seasonal breeders were clustered into two subgroups: prolonged-regular breeders called evenly spaced throughout the breeding season (spring summer period), presenting many calling events records; prolonged non-regular breeders had a calling activity spaced throughout the summer season. sporadic seasonal breeders were found in nine species belonging to five different families, which called sporadically during the springsummer period only (fig. 3). daily calling patterns species of the family bufonidae presented highly contrasting daily calling patterns. species of the genus rhinella presented a mainly crepuscular and nocturnal calling pattern; r. arenarum presented a peak of activity at 20:00 h, while r. diptycha vocalised mainly between 20:00 h and 21.00 h (rayleigh = 0.77; p = 0.0002; rayleigh = 0.93; p = 0.0001) (table 1, fig. 4). meanwhile, melanophryniscus rubriventris was mainly a diurnal species, with peaks of vocal activity at 06:00 h and 18:00 h (rayleigh = 0.52; p <0.0001 (table 1, fig. 4). the species of the family hylidae at the pf site recorded a mainly crepuscular-nocturnal vocal activity, although sporadic calling activity was detected during the day. boana riojana was recorded at pf and at the lmf sites, at the pf site presented a mainly nocturnal calling activity, with peaks of vocal activity at 01:00 h and 03:00 h; meanwhile, at the lmf site, with a higher number of records, presented a mainly nocturnal calling pattern, 27patterns of acoustic phenology in anurans of the yungas but also with diurnal vocalizations (table 1, rayleigh = 0.8; p < 0.0001). dendropsophus nanus presented a crepuscular-nocturnal vocal activity, with separated activity peaks at 20:00 h, 21:00 h, and 05:00 h (rayleigh = 0.65; p <0.0001). scinax fuscovarius presented a calling pattern mainly crepuscular and nocturnal, with a peak of records at 22:00 h and 06:00 h, but also presented vocalizations during the day at 14:00 and 17:00 h (rayleigh = 0.42, p <0. 0001). by the other hand, s. nasicus presented a more sporadic and nocturnal vocal activity, with peaks at 22:00 h and 23:00 h (rayleigh = 0.8, p = 0.005). trachycephalus typhonius had a mainly crepuscular activity, with a peak of activity at 20:00 h, and was also recorded calling during the day at 14:00 h (rayleigh = 0.59, p = 0.007). fig. 1. total number of calling events recorded for anuran species in the parque nacional calilegua spanning the three sites together. grey squares indicate calling activity. dark squares indicate core calling periods. fig. 2. records of calling activity in the anuran assemblage of the parque nacional calilegua. black cells = maximum call events registered (3). grey cells = medium calling events registered (2). light cells = minimum callings events registered (1). pf = premontane forest; lmf = lower montane forest; ump = upper montane forest. 28 martín boullhesen et alii fig. 3. dendrogram showing the bottom-up hierarchical cluster analysis in the species recorded at the parque nacional calilegua. colours represent the five acoustic breeding strategies obtained. table 1. circular statistics for the anuran species recorded in the study sites. mu = trigonometric moment; rho = length; sd = standard deviation; cos = cosine; sin = sine; p = order; n = number of calling events; rayleigh = rayleigh uniformity test; p value = confidence value. species mu sd rho cos sin p n rayleigh test p value boana riojana (pf) 6.08º 0.66º 0.38 0.38 -0.008 2 100 0.8 <0.0001 b. riojana (lmf) 6.25º 0.98º 0.15 0.15 -0.01 2 337 0.6 <0.0001 dendropsophus nanus 0.51º 0.92º 0.15 0.02 0.15 2 167 0.65 <0.0001 leptodactylus apepyta 4.13º 0.74º 0.33 0.29 0.15 2 50 0.75 <0.0001 l. macrosternum 5.14º 1.01º 0.17 0.17 0.02 2 127 0.59 <0.0001 l. elenae 6.49º 0.86º 0.14 0.14 -0.01 2 130 0.68 <0.0001 l. fuscus 8.89º 0.89º 0.21 0.15 -0.14 2 59 0.66 <0.0001 l. gracilis 6.9º 0.85º 0.08 0.08 -0.02 2 13 0.69 0.0009 l. latinasus -0.4º 0.77º 0.74 -0.07 0.73 2 115 0.74 <0.0001 melanophryniscus rubriventris 12.43º 1.13º 0.05 -0.006 -0.05 2 67 0.52 <0.001 physalaemus cuqui 5.77º 0.98º 0.02 0.02 0.001 2 212 0.61 <0.0001 phyllomedusa boliviana 5.52º 0.64º 0.4 0.39 0.04 2 107 0.81 <0.0001 pleurodema borellii 10.6º 1.35º 0.089 0.031 -0.08 2 96 0.39 <0.0001 oreobates barituensis 13.85º 1.16º 0.18 -0.08 -0.016 2 31 0.5 0.0002 o. berdemenos 15.39º 1.53º 0.07 -0.05 -0.04 2 167 0.3 <0.001 rhinella arenarum 4.22º 0.71º 0.43 0.39 0.19 2 12 0.77 0.0002 r. diptycha 5.93º 0.35º 0.77 0.77 0.01 2 6 0.93 0.001 scinax fuscovarius 1.13º 1.3º 0.2 0.06 0.19 2 77 0.42 <0.0001 s. nasicus 4.23º 0.65º 0.6 0.54 0.27 2 7 0.8 0.005 trachycephalus typhonius 5.38º 1.01º 0.079 0.078 0.012 2 13 0.59 0.007 29patterns of acoustic phenology in anurans of the yungas fig. 4. rose diagrams showing daily calling activity of recorded species in the study sites. dotted lines = kernel data distribution. strabomantidae 30 martín boullhesen et alii the species of the family strabomantidae presented a daily vocal pattern but was mainly crepuscular-nocturnal. oreobates barituensis presented a sporadic pattern during the day with records of calls from 17:00 h to 20:00 h, with a peak of vocal activity at 05:00 h (rayleigh = 0.5; p <0.0002) (table 1, fig. 4). oreobates berdemenos presented markedly crepuscular-nocturnal vocal activity with a peak of vocal activity between 05:00 h and 06:00 h but was recorded vocalizing continuously during the 24 hs of the day (rayleigh = 0.3; p <0.0001). species of the family leptodactylidae presented a mostly crepuscular-nocturnal daily vocal pattern, but records of calling activity were also detected during the day for some species. leptodactylus apepyta began to vocalize at 19:00 h in the evening and peaked at 20:00 h and at 21:00 h (rayleigh = 0.75; p <0.0001). leptodactylus macrosternum presented a mainly nocturnal calling activity with a peak between 24:00 and 01:00 h but also was recorded calling during the daytime, frequently between 06:00 h and 08:00 h (rayleigh = 0.59; p <0. 0001). leptodactylus elenae called mainly at night, presenting peaks of calling activity at 22:00 h and at 01:00 h (rayleigh = 0.68; p <0.0001). leptodatylus fuscus presented a mainly nocturnal vocal activity, with sporadic peaks at 21:00 h and between 05:00 h and 06:00 h, with sporadic records during the daytime (rayleigh = 0.66, p <0.0001). leptodactylus gracilis was recorded even more sporadically, mainly at twilight, with a peak at 20:00 h (rayleigh = 0.69; p = 0.0009). leptodactylus latinasus presented a twilight-nocturnal vocal activity, beginning to call at 19:00 h, with extensive records during the last hours of the night and a peak of calling activity at 20:00 h (rayleigh = 0.74; p <0.0001) (table 1, fig. 4). phyllomedusa boliviana, an arboreal species, presented a strictly nocturnal vocal activity, with records of calls at 21:00 h and at 02:00 h, and a peak of calling at 22:00 h (rayleigh = 0.81; p <0.0001) (table 1, fig. 4). temporal niche overlap the annual temporal niche overlap, and the springsummer season overlap of the recorded species, was moderately high at pf site according to pianka’s index. according to the czekanowski’s index, the use of the temporal acoustic space was equitable (czekanowski ~ 0.5) in the species assemblage recorded at the same site, both for the entire year and during the highest records of vocal activity in spring-summer season (table 2). meanwhile, the temporal niche overlap was lower in the lmf and umf sites (table 2), according to the pianka’s index (table 2). in addition, the time niche overlap during the fall-winter season was moderate at lmf, according to the pianka’s index (table 2). according to the czekanowski’s index, a temporal niche overlap was found to be greater than that expected by chance (coincident activity) in the spring-summer period in the species that vocalize at the lmf site (table 2). calling guilds six different calling guilds were recorded based on their advertisement calls features (table 3; fig. s1). leptodactylus apepyta, was the only representative of the calling guild “a”; l. elenae, l. fuscus, l. latinasus, physalaemus cuqui and trachycephalus typhonius, were framed within the calling guild “b”. rhinella arenarum and r. dypticha were part of the guild “d”. oreobates barituensis table 2. temporal acoustic niche overlap recorded in the study sites. czekanowski index (0-1); pianka index (0-1). site period czekanowski p-value pianka p-value pf annual 0.51 < 0.001 0.62 < 0.001 lmf annual 0.26 0.003 0.37 0.005 umf annual 0.38 1.12 0.42 0.041 pf spring-summer 0.51 < 0.001 0.61 < 0.001 lmf spring-summer 0.31 0.003 0.4 0.007 umf spring-summer 0.38 1.12 0.42 0.041 pf fall-winter 0.12 0.13 0.21 0.1 lmf fall-winter 0.42 0.051 0.59 0.054 umf fall-winter table 3. calling guilds recorded along an altitude gradient within the parque nacional calilegua, argentina: permanent pond in the premontane forest (pf, 650 m a.s.l.), permanent stream in lower montane forest (lmf, 1125 m a.s.l.), and upper montane forest (umf, 1750 m a.s.l.). total number of species recorded n = 19. guild a = unmodulated simple call; guild b = modulated simple call; guild c = unmodulated pulsed call; guild d = modulated pulsed call; guild e = unmodulated pulsed multi-note call; guild f = uniform modulated pulsed multi-note call; guild g = nonmodulated different multi-notes call; guild h = modulated different multi-notes call. guilds n° of recorded species percent pf lmf umf a 1 5.26 % 1 b 5 26.31 % 5 c d 2 10.52 % 2 e 1 5.26 % 1 1 f 8 42.10 % 7 2 1 g 2 10.52 % 1 1 h 31patterns of acoustic phenology in anurans of the yungas was the only representative of the calling guild “e”. boana riojana, dendropsophus nanus, l. gracilis, oreobates berdemenos, phyllomedusa boliviana, pleurodema borellii, scinax fuscovarius, s. nasicus were part of the largest recorded group, the calling guild “f”. leptodactylus macrosternum and melanophryniscus rubriventris formed the calling guild “g”. discussion in this study, the calling phenology of anuran species was described at a fine timescale (hourly and daily) and quantitatively assessed along an altitudinal gradient in three andean montane forest ecosystems of the yungas ecoregion, within the pnc. this level of detail enabled us to propose a new classification scheme of acoustic breeding strategies according to the time spent by each species calling throughout a year. previous breeding strategies classification schemes were used to describe the breeding patterns of anuran species in the parque nacional calilegua, following duellman and trueb (1986). following this previous classification scheme only two breeding strategies were recorded for the entire species assemblage in this protected area (vaira, 2002). prado et al. (2005) described three reproductive activity patterns for a diverse anuran assemblage in a floodplain in the pantanal region of brazil. with the use of automatic recorders saenz et al. (2006) were able to classify the anuran breeding activity patterns in: explosive breeders, winter breeders, summer breeders and continuous breeders depending on the core calling period recorded. kopp et al. (2010) identified up to four distinct anuran reproductive patterns recorded by 13 visual encounter surveys in the cerrado of brazil. in this study, with the implementation of a pam over an entire year we were able to identify five distinct breeding acoustic patterns and provide a novel, standardized classification framework, so that it can be tested in other ecosystems. the great majority of the species recorded in this study showed a markedly seasonal breeding acoustic pattern, with their core calling periods centred during the spring-summer season in agreement with the warm and rainy season. these records were also reported by a previous study conducted on the same assemblage 20 years ago (vaira, 2002). however, we now provide a detailed acoustic breeding phenology of the anuran species inhabiting three phytogeographical strata of the yungas andean forest. the marked seasonal acoustic pattern might be related to the breeding behaviour present in these species that mainly use lentic waterbodies for their reproduction, with free-living tadpoles (vaira et al., 2002; pereyra et al., 2018; boullhesen et al., 2019). strictly sporadic seasonal breeders were found in the families bufonidae, leptodactylidae, and strabomantidae, with one to four calling events per month. the calling activity recorded for sporadic-breeding species in a tropical forest of guyana lasted between one and three consecutive nights (gottsberger and gruber, 2004). the records of calls from explosive breeders were in coincidence with the warm and rainy period of the study. the calling activity of explosive breeders has been documented to correlate positively with temperature and rainfall cues in tropical anuran assemblages that use ephemeral ponds (ulloa et al., 2019). similarly, another study described the explosive breeders to call in the rainy season in the pantanal of brazil (prado et al., 2005). prolonged seasonal breeders were the most representative groups in the study area. in another study along an altitudinal gradient of brazil this reproductive pattern was also predominant among anuran species whose calling activity was recorded for 6 months (arzabe, 1999). these temporal reproductive patterns agreed with others already reported for the yungas forests (vaira, 2002; akmentins et al., 2015; pereyra et al., 2018). the species of the family leptodactylidae are mainly prolonged breeders, calling throughout the night and with sporadic calling activity during the day in representatives of the genus: leptodactylus, physalaemus, and pleurodema (vaira, 2002; camurugi et al., 2017; boullhesen et al., 2019). however, it should be noted that certain species of the family such as leptodactylus fuscus were characterized as mainly nocturnal in other ecoregions (lucas et al., 2008; guerra et al., 2020), although they showed an extended pattern of calling activity in daylight time recorded by a pam in the yungas ecoregion (boullhesen et al., 2019). our work provides novel information about the acoustic phenology patterns of a recently described species, l. apepyta, calling in the summer season showing a crepuscular-nocturnal activity where males vocalize outside subterranean nests that they built near the edge of temporary ponds (schneider et al., 2019). the tree frog boana riojana was the only continuous breeder recorded in the study area calling throughout  every month of the year. duellman (1970) suggested that the hylids of central america reproduce continuously throughout the year if the environmental conditions are favourable. in addition, these records may, in turn, be linked to the characteristics of the monitored site. for example, the site lmf is characterized by abundant vegetation (evergreen forest) and a permanent mountain stream, providing optimal conditions for b. riojana to vocalise and breed throughout the entire year. in other 32 martín boullhesen et alii species of this genus, a continuous breeding strategy has been suggested to correlate with adult male gravid and post metamorphic females’ abundance recorded throughout a year of survey (hiert and moura, 2010). previous research suggested that the members of the family hylidae have a crepuscular-nocturnal vocal activity (guerra et al., 2020). however, our pam study showed that b. riojana also calls during daylight, thus suggesting a more plastic calling behaviour in this species. there are reports of diurnal movement in species of the genus boana in temperate forests of brazil (de oliveira et al., 2016). therefore, it is interesting to explore the vocal repertoire of b. riojana, to determine if these daytime vocalizations correspond to advertisements calls or other types of vocalizations, such as territorial interactions or rain calls (toledo et al., 2015).  the anuran assemblage recorded in this study presented a slight temporal acoustic niche overlap (time spent calling), mainly in the site with the highest diversity of species (pf). in addition, the temporal overlap of advertisement calls detected during the spring-summer period in the lmf site is remarkable. these results are in contrast with those reported for congeneric syntopic species that reproduce in permanent water bodies in the cerrado savanna of brazil (guerra et al., 2020). the high diversity of species that compose the assemblages of the yungas forests (lavilla and heatwole, 2010; vaira et al., 2017), suggests that in places where several species reproduce simultaneously, the greatest diversity of calling patterns should be found to avoid being masked by others (bertoluci and rodrigues, 2002; herrick et al., 2018; klump and gerhardt, 1992). the classification of advertisement calls of anurans, according to their spectral parameters in guilds, enables the comparison between groups of species to be faster and easier (emmrich et al., 2020). this variety of acoustic strategies can be seen reflected in the calling guilds diversity detected in this study, mainly in the assemblage belonging to the pf site, with a total of five calling guilds recorded. nevertheless, the calling guild with modulated pulsed multi-note calls was the most representative of the study, and unlike other guilds detected where there is a strong phylogenetic signal, this guild is made up of species from five different families. this observed pattern may conform with the public information theory where anuran species with different phylogenetic relatedness elaborate advertisement calls with similar acoustics features (danchin et al, 2004; goodale et al., 2010; sugai et al., 2021). in this sense, the anuran species belonging to different lineages could be using the same venue for information to determine the optimal conditions to breed. thus, enabling an inadvertent social information resource available and driving the species to use similar acoustic traits (danching et al., 2004; goodale et al., 2010).  the pam employed in this study allowed us to describe in detail the acoustic reproductive patterns in an anuran assemblage in the mountain forests of the southern yungas of north-western argentina. this information made it possible to extend the previous knowledge regarding the calling activity of the species, contributing quantitatively to a better knowledge of a key aspect in the life history for each species recorded (calling activity), and to add new data for several secretive species. in addition, it was possible to better characterize the variety of calling breeding strategies of the species recorded and, describe a standardized classification scheme to be tested in other ecosystems employing pam method. in addition, we were able to access precise information on the temporal daily and seasonal acoustic distribution of the recorded anuran assemblage. this data is valuable and may be of great importance for implementing monitoring programs of anuran diversity in the andean regions of south america in the current context of climate crisis. acknowledgments we thank dirección regional noroeste of the administración de parques nacionales (apn drnoa) for providing mb with the research permits in the calilegua national park (118/2017 rnv. 1). we thank the rufford foundation for awarding mb a rufford small grant (project id22246-1). we thank conicet for a full scholarship awarded to mb. the present work was partially supported by pio conicet 094 and pue inecoa 22920170100027co. supplementary material supplementary material associated with this article can be found at <http://www-9.unipv.it/webshi/appendix/ index.html> manuscript number 14050. references akmentins, m.s., pereyra, l.c., sanabria, e.a., vaira, m. 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(2012): abiotic effects on calling phenology of three frog species in korea. anim. cells syst. 16: 260-267. threats of the emerging pathogen batrachochytrium salamandrivorans (bsal) to italian wild salamander populations lorenzo dondero1, giorgia allaria1, giacomo rosa1, andrea costa1, gentile francesco ficetola2, roberto cogoni3, elena grasselli1, sebastiano salvidio1,* age estimation and body size of the parsley frog, pelodytes caucasicus boulenger, 1896 from lake borçka karagöl, turkey cantekin dursun*, serkan gül, nurhayat özdemir patterns of acoustic phenology in an anuran assemblage of the yungas andean forests of argentina martín boullhesen1,2,*, marcos vaira1, rubén marcos barquez2, mauricio sebastián akmentins1 diet and trophic niche overlap of four syntopic species of physalaemus (anura: leptodactylidae) in southern brazil renata k. farina1, camila f. moser2, stefano scali3, mateus de oliveira4, patrícia witt5, alexandro marques tozetti1,* screening of ophidiomyces ophidiicola in the free-ranging snake community annually harvested for the popular ritual of san domenico e dei serpari (cocullo, aq, italy) daniele marini1,2, ernesto filippi3,*, gianpaolo montinaro4, francesco c. origgi5,6 assessment of fall season habitat and coverboard use by snakes in a restored tallgrass prairie community carter dollen1,2, tracy j. coleman1,2, travis r. robbins1,2,* revisiting the polyploidy in the genus odontophrynus (anura: odontophrynidae) andré luis de souza, mayara aparecida das neves micalichen, roger alves da rocha, rafael bueno noleto* issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 6(2): 223-227, 2011 tail tip removal for tissue sampling has no short-term effects on microhabitat selection by podarcis bocagei, but induced autotomy does enrique garcía-muñoz1,2, francisco ceacero3, luis pedrajas4, antigoni kaliontzopoulou1, miguel á. carretero1 1 cibio-up, centro de investigação em biodiversidade e recursos genéticos. campus agrário de vairão, 4485-661 vairão, portugal. 2 departamento de biología animal, biología vegetal y ecología. campus de las lagunillas s/n. universidad de jaén. e-23071 jaén, spain. corresponding author. e-mail: engamu@gmail.com 3 departamento de ciencia y tecnología agroforestal y genética, etsia, uclm, campus universitario s/n, 02071 albacete, spain. 4 centro de rescate de anfibios y reptiles, plaza arcipreste de hita 1, 23680 alcalá la real, spain. submitted on: 2011, 4th october; revised on: 2011, 30th november; accepted on: 2011, 22nd december. abstract. tail tip removal is a common method for obtaining tissue samples for genetics and other studies on lizards. this study evaluates the effect of tail tip autotomy on microhabitat selection in the lacertid podarcis bocagei. different-length tail fragments were experimentally removed from lizards of a small population. forcing lizards to autotomise small tail tips (<1 cm) did not affect microhabitat selection. in contrast, a significant negative effect was observed in those lizards which underwent induced autotomy of the entire tail (> 5 cm). after autotomy these lizards were observed to favour more closed habitats, where predator avoidance is expected to be more efficient, although of potentially lower thermal quality. keywords. podarcis, tip removal; tissue sampling. material for genetic studies in lizards and for physiological and ecotoxicological investigations is usually obtained from tail-tip removal. many lizard species have the ability to autotomize their tails when seized by predators (arnold, 1984, 1988; bellairs et al., 1985), which increases the probability of escape and survival, but may in turn entail long-term costs (wilson, 1992; downes and shine, 2001; niewiarowski et al., 1997). since arnold’s (1984) comprehensive review of reptile caudal autotomy as a defensive behaviour over 20 years ago, our understanding of the costs associated with tail loss has increased remarkably (bateman and fleming, 2009). lizards are known to significantly modify their behaviour in response to tail loss. many of these changes are a by-product of decreased locomotor performance due to the absence of tail (e.g. chapple and swain, 2002). however, 224 e. garcía-muñoz et alii altered behaviour has also been recorded without a concomitant measurement of reduced locomotory ability (reviewed in bateman and fleming, 2009). although arnold (1988) noted that tailless lizards are faced with an increased risk of predation through not having a tail to lose to a subsequent predator, few quantitative data on the behavioural responses to caudal autotomy were available at the time. lizards may change their habitat selection post-autotomy, due to locomotor restrictions, energetic requirements and/or the lack or reduced efficiency of this important antipredatory mechanism. for example, due to compromised mobility and balance, tailless lizards may use different habitats from tailed ones (ballinger, 1973). from a physiological perspective, lizards may select open areas which are more favourable for thermoregulation (martín and salvador, 1992), presumably to speed up regeneration and recovery processes. however, lizards may also have to use sub-optimal habitats (martín and salvador, 1993), particularly those areas with greater cover, as a mechanism for reducing conflicts with conspecifics and exposure to predators (martín and salvador, 1992; cooper, 2007). these observations are mainly based on studies focusing on natural autotomy of the entire tail or experiments simulating it. however, energetic and behavioural costs generated by tail tip removal cannot be assumed to be insignificant without previous investigation. in this context, as indicated above, behavioural changes in microhabitat use can be indicative of costs associated to overall locomotor performance, physiology and predation risk (clause and capaldi, 2006). we studied a population of podarcis bocagei, a lacertid lizard endemic to the northwestern iberian peninsula to evaluate the effect of forced tail-tip removal for tissue sampling in relation to microhabitat use. the sampling site (gião, nw portugal 41.318°n; 8.676°w) was a small, physically restricted area comprising granite walls where lizards were easy to capture. the surrounding landscape consisted of an agroenvironment dominated by corn fields, patched with eucalypts (eucaliptus globulus) and maritime pines (pinus pinaster) and disseminated houses. we began sampling activities 30 min after the sun reached the rock walls. a total of 27 individuals grouped into three age/sex classes (9 juveniles, 9 adult females and 9 adult males; table 1) with intact tails, were collected in july 2009. our field experiment consisted of two steps. in the first step lizards were marked and underwent three different tail manipulative treatments 0: no manipulation; 1: induced autotomy of the entire tail; 2: removal of a small tail tip (induced by hand; < 1 cm; cordero et al., 1998). afterwards, lizards were uniquely marked with a dorsal number painted with a marker pen to facilitate behavioural observations of habitat use and then released in the same microhabitat of capture. microhabitat was categorised into four classes based on the percentage of vegetation cover 1: totally covered (> 75% of vegetation); 2: partially covered (50-75%); 3: partially uncovered (25-50%); 4: totally uncovered (<25%). in the second step, one week later and at the same time and weather conditions than in the first survey, lizards were visually identified and the microhabitat at first sight during each transect was recorded. transects were repeated four times (≈ one survey per hour) on the same side of the granite wall. a general linear model (glm) was performed in order to evaluate the effect of tail manipulation and age/sex (class) on microhabitat selection. a post-hoc duncan test was used to detect significant differences of tail manipulations and differences in habitat used by class. autotomy of a small segment of the tail (<1 cm) had no detectable effects on microhabitat selection, but autotomy of the entire tail had (tables 1 and 2). this pattern was 225tail tip removal has no effect on habitat selection by podarcis bocagei independent from class, as the class*treatment interaction term had no significant effect on microhabitat selection (table 2). results showed that independently of the class all individual that we induced autotomy selected more cover microhabitats. in addition, results showed intrinsic differences before tail manipulation in microhabitat use among the three class groups studied. at our study site, males selected more open microhabitats, juveniles used microhabitats with higher cover, with potentially lower thermal advantages and females displayed an intermediate microhabitat selection. the differences observed among the three class groups in terms of microhabitat selection are in accordance with previous observations on this species (galán, 1994). results suggest that collecting a small piece of tail (<1 cm), as usually carried out for genetic, physiological and ecotoxicological studies, has a negligible effect on the lizards’ table 1. differences in microhabitat selection [1: total cover (> 75% of vegetation); 2: partial cover (5075%); 3: mostly uncovered (25-50%); 4: totally uncovered (<25%)] before and after tail manipulation (treatment) among the three age/sex (class) studied (m, adult males; f, adult females; j, juvenile). code class svl treatment before after microhabitat microhabitat #1 m 56.11 tail tip 3 4 #2 m 44.75 tail tip 3 3 #3 m 57.14 no treatment 3 3 #4 m 50.24 no treatment 4 4 #5 m 50.76 no treatment 4 4 #6 m 50.23 autotomy 4 2 #7 m 49.71 autotomy 4 2 #8 m 49.19 autotomy 3 1 #9 m 44.37 tail tip 4 4 #10 f 54.23 tail tip 4 3 #11 f 48.77 tail tip 3 2 #12 f 46.90 tail tip 2 2 #13 f 41.04 no treatment 3 3 #14 f 43.55 no treatment 3 3 #15 f 46.06 autotomy 2 1 #16 f 46.64 autotomy 3 1 #17 f 41.32 autotomy 3 1 #18 f 40.75 no treatment 3 2 #19 j 38.17 autotomy 3 1 #20 j 35.26 tail tip 1 1 #21 j 32.35 autotomy 1 1 #22 j 31.04 autotomy 1 1 #23 j 30.38 tail tip 1 3 #24 j 32.8 tail tip 1 3 #25 j 30.11 no treatment 3 3 #26 j 31.72 no treatment 2 2 #27 j 31.87 no treatment 2 2 226 e. garcía-muñoz et alii behaviour in terms of microhabitat selection as compared to the effect observed due to forced tail autotomy. however, further studies are necessary to determine the maximum length of tail that may be amputated without producing behavioural responses in lizard microhabitat selection. lin and ji (2005) found that locomotor performance in takydromus septentrionalis, a very long-tailed, grass runner lacertid, was almost unaffected by tail loss until at least more than 71% of the tail length was experimentally removed. the effects of tail autotomy on lizard survival and behaviour are well documented (for reviews see: arnold, 1984, 1988; maginnis, 2006, bateman and fleming 2009, clause and capaldi, 2006). although tail autotomy is a common phenomenon in lizards, the highest rates have been associated to increased exposure to inefficient predation (medel et al., 1998, bateman and fleming, 2011). significantly, bateman and fleming (2011) showed that the frequency of regenerated tails in brown anoles was dependent of the behaviour of both the predator and the lizard. they provided empirical support for the hypothesis that predator efficiency, and not necessarily the number of predators, is the mechanism through which selection may act to retain tail autotomy as a defensive trait. the `proportion of regenerated tails of adult p. bocagei species varied from 54.55% to 80.95% in different localities from coastal n portugal (carretero, unpublished). in the study area, the rate was 65%, hence falling within commonly found interval. thus, the proximity of houses in the study areas likely favouring the presence of domestic cats as inefficient predator (according to bateman and fleming, 2011) did not severely altered inefficient predation pressure. microhabitat studies in lacertids have demonstrated behavioural changes in individuals as a consequence of tail autotomy (martín and salvador, 1992, 1993). tailless lizards may become more cryptic, use different substrates, and shift to different environments. thus, autotomy is expected to have relevant consequences on individual fitness (arnold 1988; mcconnachie and whiting, 2003) in terms of costs associated with locomotion, tail regeneration, and, as shown here, on microhabitat selection. however, our study demonstrate that collecting a small piece of tail (<1 cm) does not result on short-term effects on microhabitat selection by p. bocagei, while forced caudal autotomy occurring at the extreme base of the tail may greatly reduce fitness of lizards. table 2. results of general linear model (glm) performed to evaluate the effect of tail manipulation and age and sex class (class) in microhabitat selection before and after (time) tail amputation or autotomy treatments.   ss df ms f p intercept 337.500 1 337.500 569.531 < 0.001 class 20.333 2 10.167 17.156 < 0.001 treatment 9.333 2 4.667 7.875 < 0.01 class*treatment 0.667 4 0.167 0.281 > 0.05 error 10.667 18 0.593 time 2.241 1 2.241 10.083 < 0.01 time*class 2.926 2 1.463 6.583 < 0.01 time*treatment 7.704 2 3.852 17.333 < 0.001 time*class*treatment 1.630 4 0.407 1.833 > 0.05 error 4.000 18 0.222     227tail tip removal has no effect on habitat selection by podarcis bocagei references arnold, e.n. (1984): evolutionary aspects of tail shedding in lizards and their relatives. j. nat. hist. 18: 127-169. arnold, e.n. (1988): caudal autotomy as a defence. in: biology of the reptiles, p 235-273. gans, c., huey, r., eds, alan r. liss, new york. ballinger, r.e. (1973): experimental evidence of the tail as a balancing organ in the lizard anolis carolinensis. herpetologica 29: 65-66. bateman, p.w., fleming, p.a. (2009): to cut a long tail short: a review of lizard caudal autotomy studies carried out over the last 20 years. j. zool. 277: 1-14. bateman, p.w., fleming, p.a. (2011): frequency of tail loss reflects variation in predation levels, predator efficiency, and the behaviour of three populations of brown anoles. biol. j. linn. soc. 103: 648-656 bellairs, a.d.a., bryant, s.v. (1985): autotomy and regeneration in reptiles. in: biology of the reptiles, p. 301-410. gans, c., billet f., eds, alan r. liss, new york. chapple, d.g., swain, r. (2002): effect of caudal autotomy on locomotor performance in a viviparous skink, niveoscincus metallicus. funct. ecol. 16: 817-825. clause, a.r., capaldi, e.a. (2006): caudal autotomy and regeneration in lizards. j. exp. biol. 305(a): 965-973. cooper, w.e. jr. (2007): compensatory changes in escape and refuge use following autotomy in the lizard sceloporus virgatus. can. j. zool. 85: 99-107. cordero, p. j., salvador, a., veiga, j.p. (1998): a method of dna sampling in lizards with tail autotomy. herpetol. rev. 29: 23-25. downes, s.j., shine, r. (2001): why does tail loss increase a lizard’s later vulnerability to snake predators? ecology 82: 1293-1303. galán, p. (1994): selección del microhábitat en una población de podarcis bocagei del noroeste ibérico. doñana, acta vert. 21:153-168. lin, z.h., ji, x. (2005): partial tail loss has no severe effects on energy stores and locomotor performance in a lacertid lizard, takydromus septentrionalis. j. comp. physiol. b 175: 567-573. maginnis, t.l. (2006): the costs of autotomy and regeneration in animals: a review and framework for future research. behav. ecol. 17: 857-872. martín, j., salvador, a. (1992): tail loss consequences on habitat use by the iberian rock lizard, lacerta monticola. oikos 65: 328-333. martín, j., salvador, a. (1993): tail loss and foraging tactics of the iberian rock-lizard, lacerta monticola. oikos 66: 318-324. mcconnachie, s., whiting, m. (2003): costs associated with tail autotomy in an ambush foraging lizard, cordylus melanotus melanotus. afr. zool. 38: 57-65. medel, r.g., jimenez, j.e., fox, s.f., jaksic, f.m. (1988): experimental evidence that high population frequencies of lizard tail autotomy indicate inefficient predation. oikos 53: 321-324. niewiarowski, p.h., congdon, j.d., dunham, a.e., vitt, l.j., tinkle, d.w. (1997): tales of lizard tails: effects of tail autotomy on subsequent survival and growth of free-ranging hatchling uta stansburiana. can. j. zool. 75: 542-548. wilson, b.s. (1992): tail injuries increase the risk of mortality in free-living lizards (uta stansburiana). oecologia 92: 145-152. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 6(2): 161-168, 2011 orientation and directional escape by blanchard’s cricket frog, acris blanchardi (amphibia: anura: hylidae), in response to a human predator malcolm l. mccallum 117 linda lane, texarkana, tx 75501, usa. e-mail: malcolm.mccallum@herpconbio.org submitted on: 2011, 5th february; revised on: 2011, 16th june; accepted on: 2011, 23rd june. abstract. i investigated the seemingly haphazard jumping pattern of blanchard’s cricket frog (acris blanchardi) to determine if it was random or patterned. i approached frogs from the front (cranial), back (caudal), and side (lateral) simulating an attacking predator. on average, frogs jumped away at 135.73° (se = 2.31°) from the direction of attack. i did not distinguish between right and left directions in this study. there were significant differences in the angle of escape among the three attack directions (f2,87 = 17.64, p < 0.001). there were no significant differences in escape angle between frogs that i approached from the front (mean = 130.4°, se = 2.59°) or the side (mean = 126.3°, se = 3.75°, tukey interval: -0.261, 0.626). the angle of escape was not uniform and directional escape was around 120° across all tests. frontal and side approaches led to escape angles near 120° but attacks from the rear resulted in two modes of escape, at about 180° and 120° from the angle of approach. these frogs have a possible blind spot in the rear of their field of vision that might explain this bimodal escape pattern in rear attacks. the tendency to jump away at an angle rather than strait away from the predator likely represents as an evolutionary compromise between an attempt to maximize angular and linear displacement from the attacking predator. this optimal strategy may demonstrate a fitness and survival advantage. keywords. acris blanchardi, blanchard’s cricket frog, predator-prey, evolutionarily stable strategies. introduction anuran jumping patterns are important to investigate in order to understand the evolutionary forces that led to, and have maintained this behavior’s prevalence in anurans. previous studies on anuran jumping have focused on the jump length (stokely and berberian, 1953; gans and rosenberg, 1966; rand and rand, 1966; zug, 1978), the jump angle above the substrate (gans and rosenberg, 1966), the endurance of the behavior (rand, 162 malcolm l. mccallum 1966) especially under environmental stress (beuchat et al., 1984), and the physiological effects of jumping (miller et al., 1993; lutz and rome, 1994). understanding the nature of this behavior is also important from a conservation perspective in that any environmental stressor (e.g. contaminants that retard reaction time) could reduce the effectiveness of escape behavior, hence demonstrating key linkages between evolutionary biology, natural history and conservation (bury, 2006; mccallum and mccallum, 2006). the objective of this study was to investigate how the direction of predator approach can influence the direction of escape in acris blanchardi (harper, 1947). acris blanchardi is a small, semelparous (mccallum et al., 2011), non-arboreal tree frog known for its confusing taxonomy (mccallum, 2003; mccallum and trauth, 2006; gamble et al., 2008). like many amphibians (mccallum, 1999a; trauth et al., 2000; wheeler et al., 2002; mccallum, 2007) a. blanchardi is a conservation concern in many parts of its range (reeder et al., 2005; lehtinen and skinner, 2006). it has a low stress threshold which can stimulate declines (mccallum and trauth, 2007), a wide range of limb and other abnormalities are becoming more common in otherwise healthy populations (mccallum and trauth, 2003), and it is at a probable risk due to climate change (mccallum, 2010). its erratic style of escape, jumping long bounds (zug, 1978) in a seemingly random, zigzag pattern away from the on-looker (conant, 1986) escaping to refuges where it remains motionless (mccallum, 1999b) is of interest. this behavior is a possible reason for its success along many streams, ponds, and lakes in north america because the combined behavior in a group of frogs is very confusing to an onlooker who attempts to catch them (tyning, 1990). i hypothesized that blanchard’s cricket frogs escape in a patterned response. if this is true, i expected frogs to display a common angle of escape regardless of the angle of approach. if the escape angles were uniformly distributed, then the frogs escape behavior is random materials and methods all frogs originated from white oak park lake (normal, mclean co., illinois, u.s.a.). i introduced each of 90 a. blanchardi (estimated svl = 2.0-2.7 cm) into a circular arena with a diameter of 60 cm and a vertical wall height of 10 cm. recent studies suggest that a height of 20 cm might be better (royan et al., 2010), but the implementation of my study predates these later published investigations so we were not privy to these later studies. each frog was tested once without replacement. because the size of a frog may influence aspects of its jumping (rand and rand, 1966; tejedo et al., 2000), i balanced the distribution of body sizes among the treatment groups. i hung an incandescent lamp above the arena to counteract the possibility of a sun-compass in this species (ferguson, 1967). i marked the floor of the arena in light pencil at 0°, 30°, 45°, 60°, 90°, 120°, 135°, 150°, and 180° on both sides of the median. for the purposes of data recording, analysis and graphical presentation, i measured all jump angles between 0-180° regardless if the prey jumped to the left or the right of the predator (e.g. i recorded a jump angle of 270° as 90°). i conducted tests during the day (~13:00-15:00 hr) when this diurnal species is normally actively foraging (mccallum, unpubl. data). upon introduction into the arena, i covered each frog an inverted 1 l opaque plastic drinking cup that had a 1 cm hole in the bottom to observe the specimen. after the subject settled down (ca. 2-3 min), or became alert if it was death feigning (mccallum, 1999a), i positioned myself at the appropriate angle (either head on [n = 35], from behind [n = 26], or laterally [n = 29]) as an 163directional escape in acris attacking predator. so, although the arena was in a fixed position, the position of the frog under the cup was random. by coincidence, this led to an effect similar to rotating the arena to avoid possible magnetic compass effects (royan, et al., 2010). at this time i lifted the cup and reached for the frog with my right hand to simulate an approaching predator (the releaser) to induce the escape response. i recorded the direction of each leap as degrees from the angle of attack. i calculated descriptive statistics with oriana 2.0 (kovach computing services, anglesey, wales), and i used a one-way anova to compare the three approach angles using minitab 13.30 (minitab, inc., state college, pennsylvania, usa). i used oriana 2.0 to calculate rayleigh’s uniformity test to test the null hypothesis that the data from each direction of approach were uniformly distributed, and the v-test to test uniform escape angles against the mean escape angle for each direction of attack. i hand calculated chi square to compare the number of frogs that turned before jumping at each angle of attack. i used α = 0.05 to assign significance for all inferential statistics. results on average, frogs jumped away at 135.73° (se = 2.31°) from the direction of attack (fig. 1a). there were significant differences in the angle of escape among the three attack directions (f2,87 = 17.64, p < 0.001). there were no significant difference in escape angle between frogs that i approached from the front (fig. 1b, mean = 130.4°, se = 2.59°) or the side (fig. 1c, mean = 126.3°, se = 3.75°, tukey interval: -0.261, 0.626); whereas, the rear approach resulted in different angles compared to attacks from the front (tukey interval: 0.470, 1.38) and side (tukey interval: 0.633, 1.58). the angle of escape was not uniform when approached from the rear (z = 22.83, p << 0.001), front (z = 32.58, p < 0.001), or the side (z = 25.61, p < 0.001). when approached from the rear (fig. 1d), frogs escaped at a more obtuse angle (mean = 153.9°, se = 4.05°; v153.9° = 0.937, p < 0.001) than either from the front (mean = 130.4°, se = 2.59; v130.4° = 0.965, p < 0.001) or side (mean = 126.3°, se = 3.75; v126.3° = 0.940, p < 0.001). however, rear attacks led to a bimodal escape pattern in which most frogs jumped away at either ~130° (50% [13/26]) or ~180° (34.6% [9/26]) and the balance jumping somewhere in between these two modes. the angle of approach influenced if a frog repositioned itself prior to jumping (χ2 = 3.69, df = 2, p < 0.001). frogs turned more frequently before jumping (χ2 = 14.86, df = 1, p = 0.0001) when approached from the front (97%, n = 34) than from the side (21%, n = 29). they also turned more frequently (χ2 = 5.32, df =1, p = 0.021) when approached from the side (21%, n = 29) than when approached from the rear (0%, n = 26). discussion there is a strong tendency for a. blanchardi to make its initial jump from a pursuer at approximately 135° from the angle of attack, especially when approached from the front or the side. this may be advantageous when pursued by a predator such as a snake. if a frog jumps away at 90° from an on-coming predator, it would maximize displacement from the direction of attack on its first leap (fig. 2a). this may maximize the likelihood that a lunging predator would lose track of the frog during pursuit and its lunge would carry the 164 malcolm l. mccallum on-comer away from the prospective prey. if the frog jumped at 180° from the angle of attack (fig. 2b), it would sacrifice displacement and remain in the general line of sight of the lunging predator. furthermore, the lunge would carry the aggressor toward the frog, thus sacrificing maximum distance from the predator. if a frog jumps at 120-150° from the lunging predator, it optimizes displacement and distance from the predator, especially after a second jump using the same patterned response. the second jump on any of these attack directions is important to explain why frogs should not jump 180° from the angle of approach. a first jump at either 90° or 135° followed by any angular jump will make it very likely that the predator loses the predator among vegetation and other frogs. in fact, these frogs metamorphose in explosive numbers where the populations are strong (pers. obs.). metamorph abundances reaching 45 frogs/m2 are common in some areas of the ozarks (unpubl. data, 2000-2003) and i recall similarly abundant juveniles in some populations in central and southwestern illinois from 1980-1999. where large numbers of closely aggregated frogs occur, two leaps may be sufficient to confuse a potential a. b. c. d. 60 60 60 60 50 50 50 50 40 40 40 40 30 30 30 30 20 20 20 20 10 10 10 10 0 90 180 270 30 30 30 30 25 25 25 25 20 20 20 20 15 15 15 15 10 10 10 10 5 5 5 5 0 90 180 270 15 15 15 15 12.5 12.5 12.5 12.5 10 10 10 10 7.5 7.5 7.5 7.5 5 5 5 5 2.5 2.5 2.5 2.5 0 90 180 270 15 15 15 15 12.5 12.5 12.5 12.5 10 10 10 10 7.5 7.5 7.5 7.5 5 5 5 5 2.5 2.5 2.5 2.5 0 90 180 270 fig. 1. the average angle of escape by blanchard’s cricket frogs (acris blanchardi) when approached by a predator from (a) all three directions combined, (b) the front, (c) the side, (d) behind. the bars indicate the number of frogs jumping at the designated angle from the predator’s approach. in all cases, the predator is approaching from 0°. no distinction is made between jumps to the left or right. 165directional escape in acris fig. 2. potential adaptive significance for blanchard’s cricket frog’s (acris blanchardi) that escape at 90° versus 180°or 135°. a = 1st jump, b = 2nd jump, c = compromise between distance and displacement. fig. 3. a blanchard’s cricket frog (acris blanchardi) viewed from above. notice that the eyes are tilted slightly forward to potentially provide better binocular vision while leaving a potential blind spot behind. 166 malcolm l. mccallum predator, especially as neighboring frogs get disturbed during the encounter. in fact, my personal experience is that if a group of a. blanchardi starts jumping it creates sufficient confusion for the collector that catching an individual frog by hand is near impossible if you did not successful on the first try. even where not abundant, a second jump could be sufficient for an individual to find an abode in which to hide from an aggressor. in fact, a. blanchardi often occurs along the banks of creeks, although they sometimes venture far from such habitats (gray, 1983; pers. observ.). after a few jumps, especially if they reach an aquatic habitat, the frog will either lie quietly on the substrate (mccallum, 1999b) where it often blends in with the mud (gray, 1984), or it floats motionless, suspended in vegetation (mccallum, 1999b) where its color stripe provides a cryptic disguise (gray, 1984; pers. observ.). these behavioral observations seem to support the adaptive significance of patterned jumping in this frog. when approached from behind, blanchard’s cricket frog jumps at a much more obtuse angle than at either other angle of attack. this may stem from its ocular anatomy as revealed by the tendency to turn prior to jumping when approached at angles other than behind. when i approached subjects from behind the frogs never turned and jumped at 130-180° from the angle of attack. about one third of the escaping frogs jumped at 180° from my approach. the eyes of cricket frogs appear to be positioned to improve binocular vision (fig. 3). this may create a blind spot behind the frog (fig. 3). if a predator approaches a cricket frog from behind, it may get much closer before the frog detects the pursuer. this is supported by the fact that none of the frogs in this study turned prior to jumping, and that frogs approached from the front and the side frequently turned. predators approaching from the front or side are clearly in the frog’s field of view. this allows a frog to detect an aggressor earlier, turn in preparation for jumping at an optimum escape angle and in an anatomical conformation that likely provides maximum thrust from both rear legs. predators approaching from behind may not be detected so readily, providing less time to prepare for escape, thus leading to less ideal escape angles relative to the aggressor and reduced thrust. because frogs attacked from behind did not turn and had less ideal escape angles, combined with their ocular anatomy suggests these frogs have a blind spot that causes the altered jumping pattern associated with a rear attack. further, it is a less ideal escape angle if as is the case in other species (royan et al. 2010) jump distance is negatively correlated with angle of escape. by turning, the frog can jump further in its initial leap, a clear advantage in escaping an approaching predator. other frogs vary in their angular escape from predators (royan et al., 2010). mannophryne trinitatis does not demonstrate angular escape preference when approached from the front. however, when approached from the right or left they jump at 90-135° and when attacked from the back they jumped at 135-180° away from the predatory stimulus. hypsiboas geographicus demonstrates confusing patterns of stimuli response that are difficult to interpret (royan et al., 2010). at some ages this species jumps in patterns similar to that of m. trinitatis and a. blanchardi; whereas, other ages do not exhibit preferred escape trajectories. trachycephalus venulosus also demonstrates preferred escape trajectories (royan et al., 2010). when it is attacked from the front it does not escape in a preferred direction. however, when an attacker approaches from behind, the frog jumps directly forward and away from the attacker. when approached from the right or left, this species also jumps at an optimizing angle from the predator’s approach. it appears that the response patterns dem167directional escape in acris onstrated by a. blanchardi may be more widely distributed among anurans and represent a tactic that may have evolved independently in many other anuran lines. an early source of error to this study was this species apparent attraction to the sound of running water. during the initial experiment, the frogs generally jumped toward the aquarium present in the room, regardless of the direction they faced or from which the attacker approached. upon turning off the aquarium bubbler, they ceased responding to it. these data were discarded and not used in this study, but this observation suggests that patterned jumping is subject to additional outside influences not addressed in our study and that these frogs may phonotactically respond to moving water. acknowledgements i thank lauren brown for encouraging and advising me during this undergraduate research, stanley trauth for editorial comments, and dave germano for discussions on the use of oriana software. references beuchat, c.a., pough, f.h., stewart, m.m. 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(2000): breeding mortality in the wood frog, rana sylvatica (anura: ranidae), from northcentral arkansas. j. ark. acad. sci. 54: 154-156. tyning, t. (1990): a guide to amphibians and reptiles. little brown and company. boston, ma. wheeler, b.a., mccallum, m.l., trauth, s.e. (2002): abnormalities in the ozark hellbender, cryptobranchus alleganiensis bishopi. j. ark. acad. sci. 56: 250-252. zug, g.r. (1978): anuran locomotion-structure and function, 2: jumping performance of semiaquatic, terrestrial, and arboreal frogs. smithsonian contrib. zool. 276: iv, 1-31. acta herpetologica 17(2): 135-145, 2022 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-12388 the italian wall lizard, podarcis siculus campestris, unexpected presence on gorgona island (tuscan archipelago) marco a.l. zuffi1,*, alan j. coladonato2, gianluca lombardo3, antonio torroni3, matilde boschetti1, stefano scali4, marco mangiacotti2, roberto sacchi2 1 museo di storia naturale, università di pisa, via roma 79, i-56011 calci (pisa), italy 2 dipartimento di scienze della terra e dell’ambiente, viale taramelli 24 i-27100 pavia, italy 3 dipartimento di biologia e biotecnologie “lazzaro spallanzani”, via ferrata 9, i-27100 pavia, italy 4 museo civico di storia naturale, corso venezia 55, i-20100 milano, italy *corresponding author. e-mail address: marco.zuffi@unipi.it submitted on: 2021, 4th december; revised on: 2022, 17th february; accepted on 2022, 8th may editor: enrico lunghi abstract. we here report the unexpected presence of the italian wall lizard (podarcis siculus campestris) on gorgona island, in the parco nazionale arcipelago toscano (tuscan archipelago, tyrrhenian sea, tuscany, central italy). field observations were carried out in 2020 confirming its presence on the island, where it had never been reported before. we recorded 37 gps points of the species in three major areas of gorgona (with 50 lizard records) and about 180 visual counts regarding all age classes (newborns, juveniles and adults). the species was found in the urban area (site of state prison) and in two grassy and bushed areas, around and along olive tree plantations. seven individuals were captured and their tails were used to assess the sequence variation of the mitochondrial cyb gene. biometrical parameters were also evaluated for six of these individuals. we detected three distinct cyb haplotypes that were compared to podarcis siculus cyb sequences available in public databases. they resulted identical or phylogenetically closest to those found in mainland tuscany. one haplotype, found in three specimens, was identical to one previously detected at orti bottagone (wwf oasis in piombino), while the other two haplotypes were most similar to haplotypes reported in the giannella peninsula and pisa, respectively. keywords. introduced species, podarcis siculus campestris, gorgona island, tuscan archipelago, mtdna cyb sequences. introduction the italian wall lizard podarcis siculus (rafinesqueschmaltz 1810) is a mediterranean species endemic to the italian peninsula, sardinia, sicily, corsica, coastal slovenia and croatia, and the majority of small islets of the adriatic and tyrrhenian seas (corti, 2006). it is also present, as an allochthonous taxon, in several european and non-european countries (crnobrnja isailovic et al., 2009; corti et al., 2011; silva-rocha et al., 2012, 2014; adamopoulou, 2015; mizsei et al., 2016; ribeiro and sásousa, 2018; clemens and allain, 2021). according to crnobrnja isailovic et al. (2009) “generally it is an invasive that can displace native populations of other species in its invasive range (the southern part of its range and in the areas where it has been introduced)”. recent data on introduced populations found that the species shows a marked resilience (burke et al., 2002), and efficient adaptation patterns (kapsalas et al., 2016). in some cases, the species showed clear-cut ecological plasticity in adapting to a new environment, changing some anatomical and physiological traits (herrel et al., 2008). locally, 136 marco a.l. zuffi et alii the species is supposed to drive the extinction of indigenous lizards (ribeiro and sá-sousa, 2018). as far as we were aware, eradication projects proved successful results only in greece (adamopolou and pafilis, 2019). thus, p. siculus still represents worldwide a serious threat to autochthonous species, particularly due to the facility with which it can be transferred from its native area into a new environment (adamopoulou, 2015; silva-rocha et al., 2012; mizsei et al., 2016; clemens and allain, 2021). in italy, it is widespread from the north to the south, being common in coastal and hilly areas of northern and central italy, while in the south it can reach higher altitudes (corti et al., 2011). recent unpublished data report new introduced podarcis siculus individuals in some areas in northern italy (province of trento), via olive trees transfer from central italy (k. tabarelli de fatis pers. comm.). the species is naturally present in tuscany and in its insular environments, as reported by the latest regional atlas (vanni and nistri, 2006). regarding the seven larger islands of the tuscan archipelago, it has been reported as naturally present in capraia, elba (with three small populations), montecristo, giglio and giannutri, and it was likely introduced in pianosa (vanni and nistri, 2006). however, the species was never reported from gorgona island. previous repeated survey sessions, whose results were published in 2006 and in 2011 (corti, 2006; corti et al., 2011) did not find the species on the island, thus suggesting that this unexpected presence should be a very recent introduction. from a phylogeographic and phylogenetic point of view, several papers have evaluated the distribution and the genetic variation of this taxon (podnar et al., 2005; senczuk et al., 2017), also when regarding allochthonous populations (silva-rocha et al., 2012, 2014). considering the above scenario regarding the dispersal ability of the species in novel places and its ecological plasticity, we have aimed at assessing the population distribution of p. siculus campestris on gorgona, within the framework of a larger project granted by the “parco nazionale arcipelago toscano” on habitats directive species occurring in the tuscan archipelago, and the mitochondrial dna (mtdna) variation (cyb gene) of some individuals from the island, with the overarching goal to obtain preliminary results concerning the geographical origin of the female founders. materials and methods study area and sampling the study area is gorgona island (43.429008°n, 9.899226°e), the northernmost island of the tuscan archipelago, about 34 km westward from the italian coast. it is a rocky island with a very small surface (2.1 km2) and a perimeter of about 7 km (fig. 1a). the herpetological assemblage of the island lacks amphibians, due to the absence of freshwater areas, while known reptile species are the wall lizard (podarcis muralis), the moorish gecko (tarentola mauritanica), the turkish gecko (hemidactylus turcicus) and the whip snake (hierophis viridiflavus) (vanni and nistri, 2006), species that are still present and abundant according to recent unpublished surveys (c. corti, pers. comm.; m.a.l. zuffi and m. boschetti pers. obs.). sampling surveys were limited to podarcis species and were carried out from mid to end of summer 2020, on three different occasions, on the 29th of july, 29th and 30th of september. we selected three different transects, a = 1,678 m, b = 2,073 m, and c = 2,240 m (fig. 1b). according to the recommendation concerning the monitoring of relative small-sized lizards (sacchi and scali, 2016; sindaco et al., 2016), we adopted a visual census, that shall be repeated in the following years to establish the relative abundance of the target species and other reptile species for the above-mentioned project. we counted all the lizards observed along each transect and, every 10-12 m, we marked the lizards presence with a gpsmap® 62 series gps. we therefore provided i) a distribution mediated by the gps recording and ii) an overall count of observed animals along the whole transect. according to the capture feasibility, in some areas along the different transects, we also captured seven individuals of p. siculus by noosing. from the captured individuals (six out of seven, one escaped before measurements) we recorded multiple morphometrics and body size (body mass, snout to vent length, svl, head length, width and height, as in kaliontzopoulou et al., 2007; table 1), determined sex and ontogenetic stage (male, female, juvenile) and collected the tail tip to assess the variation of the mitochondrial cyb gene. from its external appearance, the species shows the typical continental p. siculus campestris dorsal pattern, with two green parietal bands (fig. 2). being on this island allochthonous and likely invasive, after the measurements the captured individuals were transferred to the museum lab, maintained alive in terraria for further analyses and comparisons, waiting for the ministry’s approval for euthanasia. samples analysed for cyb gene variation seven p. siculus specimens collected in gorgona were analysed for the sequence variation of the cyb gene at the university of pavia. dna was extracted from either tails or tail re-growths stored in 95% ethanol. the majority of the cyb gene (at least 924 bp, from np 14,357 to np 15,280) was determined for all specimens. 137allochtonous podarcis siculus on gorgona island at the time when our analyses were performed, there were 532 p. siculus cyb sequences in genbank. only 394 of these, whose cyb sequence covered the 764 bp between np 14,417 and 15,180 (akopyan et al., 2017; buglione et al., 2019; deichsel et al., 2010; garcia-porta and irisarri, 2019; kolbe et al., 2013; podnar et al., 2004, 2005, 2007, 2009; senczuk et al., 2017, 2018; taverne et al., 2020), were employed for comparisons along with the p. siculus reference sequence (psrs) (nc_011609). detailed information on the overall 402 samples (7 from this study and 394 as reference) is provided in table s1. dna extraction genomic dna was extracted via the reliaprep™ (promega madison, wi, usa) gdna tissue kit, using the standard protocol for mouse tail. roughly, 0.5-1 cm of the tail was cut into smaller parts using a scalpel and homogenised in a 2 ml test tube. we added to the samples 100 μl of tail lysis buffer (tla) and 20 μl of proteinase k (20 mg/ml), vortexed and incubated at 56 °c overnight. then we added 300 μl of cell lysis buffer (cld) and 20 μl rnase a, vortexed and incubated (56 °c) until clear. dna was then purified using a standard phenol/chloroform method. purified genomic dnas were eluted into promega elution buffer. cyb sequencing and data analysis the seven samples were sanger sequenced. pcrs were carried out in 50 µl reactions with a standard reaction mix containing 1x buffer (1.5 mm mgcl2), 0.2 mm of each dntp, 2 u of gotaq g2 polymerase (promega), 0.3 µm of each primer (cytf and h15425 by senczuk et al., 2017) and ~100 ng of dna template, using the following pcr protocol: 95 °c (2 min); 10 cycles at 95 °c (30 s), 52 °c (30 s), 72 °c (2 min); 25 cycles at 95 °c (30 s), 50 °c (30 s), 72 °c (2 min) and a final extension at 72 °c (10 min). pcr products were visualised on a 1% agarose gel and amplicons were sequenced with standard dideoxy sequencing with primers cytf and h15425 using dye terminator chemistry (applied biosystems) and following the manufacturer’s protocol. sequences were output in for and rev files in .ab1 format, cleaned by hand to remove ambiguous tails, aligned to psrs (nc_011609) and exported to the standard fasta format. phylogenetic analyses and age estimates of mtdna haplogroups a maximum likelihood (ml) tree was built with the software megax using the gtr model (8γ distributed categories) with 1,000 bootstraps (extensive spr method). it encompassed 402 cyb sequences (our fig. 1. a. tuscan archipelago islands with the gorgona island, marked with a white circle. b. distribution of selected transects (a, b, c) on the gorgona island. c. distribution of allochtonous podarcis siculus (marked with white circles) and the congeneric p. muralis (other unmarked waypoints). figures 1a and b are modified from google earth. fig. 2. adult male of podarcis siculus, showing the typical “campestris” pattern (picture taken on gorgona). 138 marco a.l. zuffi et alii seven sequences, 394 from genbank plus the reference sequence) and was rooted with the corresponding cyb sequence from p. muralis (nc_011607) using geneious 8.1.5 (biomatters; kearse et al., 2012). bayesian estimations were performed using beast 2.6.0 (bouckaert et al., 2019) under the hky substitution model (gammadistributed rates plus invariant sites) with a relaxed clock (log normal). the clock value of 1 × 10-8 base substitution per nucleotide per year (2% divergence rate myr-1), was entered as prior. the chain length was established at 50,000,000 iterations, with samples drawn every 1,000 markov chain monte carlo (mcmc) steps after a discarded burn-in of 5,000,000 steps. results transects we visually counted 180 podarcis siculus and, among them, we recorded 37 gps points corresponding to 50 individuals (10 adult males, 30 adult females and 10 juveniles). we counted more than 400 p. muralis and, among them, we recorded 92 gps points corresponding to 74 individuals (20 adult males, 11 adult females and 43 juveniles) (fig. 1c). we captured seven italian wall lizards in three different areas of the island (all variables recorded in table 1), from which a small piece of the tail tip was obtained and preserved in 95% etoh. almost all the observed p. siculus were distributed along the transects characterized by open and sunny areas (fig. 1b), while only three individuals were found in the urban context of the island (transect c). specifically, only some individuals were found along transect a, three only in transect c and almost all the other lizards in transect b. this latter transect is characterized by an abundant olive tree plantation, whose establishment is relatively recent (from 1999 to 2015). on the contrary, p. muralis was common and widespread on the island (see fig. 1c), being relatively scarce only along transect b, especially where the habitat is much open, sunny and cultivated. podarcis siculus cyb sequences we sequenced 924 bp of the cyb gene from the seven tails collected from gorgona island. we detected three haplotypes (hd = 0.714 ± 0.127) and a total of seven variable sites (table 2). on average 3.43 ± 1.08 nucleotide differences were found between any two sequences and the average nucleotide diversity (π) was 0.373% (± 0.066%). when considering all available cyb sequences (n = 402), we detected 229 haplotypes (hd = 0.994 ± 0.001) with 217 variable sites. on average 35.48 ± 1.14 nucleotide differences were found between any two sequences and π was 5.59% (± 0.06%). phylogeny of podarcis siculus cyb sequences an initial phylogenetic survey by senczuk et al., (2017) encompassing 277 mtdna cyb sequences revealed three major haplogroups present throughout the species’ distribution range. they were named a for adriatic, t for tyrrhenian and s for sicily. the addition of our seven samples from gorgona together with 118 additional sequences from genbank (podnar et al., 2004, 2005, 2007; mayer et al., 2010; kolbe et al., 2013; akopyan et al., 2017; senczuk et al., 2018; buglione et al., 2019; garcia-porta and irisarri, 2019; taverne et al., 2020) provided a more in-depth resolution of the species phylogeny (fig. 3). all samples fall within haplogroups, a, t and s whose founding nodes were dated, through bayesian estimates, to 2,602 ± 426, 1,925 ± 3,259 and 4,709 ± 620 thousand years ago (kya), respectively. haplogroups a and t are sister clades whose ancestral at node is dated at 3,909 ± 534 kya. the p. siculus ancestral mitogenome (psam) was estimated at 6,150 ± 735 kya. table 1. biometry of podarcis siculus samples from gorgona. bmass = body mass (g); svl = snout to vent length; h_l = head length; h_w = head width; h_h = head height (all length in mm). sample id sex age site transect bmass svl h_l h_w h_h gorg01 male adult torre vecchia a 7.1 65.0 17.5 9.9 7.9 gorg02 male juvenile capanne b 4.0 58.0 14.5 8.4 6.3 gorg03 male adult village c 8.1 71.0 17.6 10.0 8.2 gorg04 male adult capanne b 8.2 71.0 17.5 10.1 8.2 gorg05 female adult capanne b 3.1 55.5 12.7 7.5 5.4 gorg06 female adult village c 4.5 55.5 13.3 7.4 5.8 gorg07 female adult torre vecchia a ----------139allochtonous podarcis siculus on gorgona island haplogroup a (n = 112), representative of individuals with adriatic origins, was indeed mainly sampled around the adriatic basin (croatia and italy), but also in umbria, lazio, campania and calabria. it harbours the lowest intra-clade nucleotide diversity (1.565 ± 0.125 %) (table 3) and is composed of three major sub-haplogroups: a1, a2 and a3. haplogroup a1 (n = 9) encompasses only croatian individuals and is the youngest (359 ± 133 kya). haplogroup a2 is the most represented (n = 101) and the oldest (909 ± 164 kya). it encompasses samples from the italian adriatic coast, but also from calabria, campania, lazio and lombardia. haplogroup a3 (n = 11) (807 ± 219 kya) includes mainly individuals from calabria, but also one each from campania and emilia-romagna. haplogroup t (n = 97) is representative of individuals with tyrrhenian origins (toscana, umbria and lazio), but also from emilia-romagna. it is composed of two major sub-haplogroups, which were renamed from the original study (senczuk et al., 2017) to t1 and t2 given their major split, which was not considered previously. haplogroup t1 (n = 45), dated at 990 ± 224 kya, is mainly toscana-specific with a couple of individuals collected in umbria. it is further sub-divided into haplogroups t1a and t1b (ta and tb, respectively, in senczuk et al., 2017) though t1a only encompasses one individual. haplogroup t1b (535 ± 131 kya) was found to include all seven sequences from this study (fig. 4). samples gorg 03, 05 and 06 from gorgona island share the same haplotype (n. 1 in table 2) with jx186543 (kolbe et al., 2013) from orti bottagone (wwf oasis in piombino). gorg 02 harbours a novel haplotype (n. 2), though similar to those detected in samples from the giannella peninsula (ky065091-ky065095) (senczuk et al., 2017). finally, gorg 01, 04 and 07 harbour the same novel haplotype (n. 3), which appears to be closest related to jx186545 from pisa (kolbe et al., 2013) according to the haplotype network (fig. 5). thus, in all cases the closest cyb sequences were found in mainland toscana. haplogroup t2 (n = 52; 791 ± 184 kya) mainly encompasses individuals from central italy. it is composed of two major sub-haplogroups t2c and t2d (tc and td, respectively, in senczuk et al., 2017). haplogroup t2c (444 kya ± 110 kya) is found in emilia-romagna, toscana, umbria and lazio, while haplogroup t2d is younger (246 ± 73 kya) and appears to be lazio-specific. haplogroup s (n = 193), the most represented and with the largest intra-haplogroup nucleotide diversity (3.185 ± 0.245 %) (table 4), is representative of individuals with a sicilian origin. it is composed of three main sub-haplogroups: s1, s2 and s3. haplogroup s1 (2,170 ± 403 kya) is mainly found in calabria, while haplogroup s2 (404 ± 146 kya) appears to be calabria-specific. finally, haplogroup s3, which is the most divergent (1,423 ± 230 kya), is almost completely endemic to sicilia and subdivided into 13 sub-haplogroups (s3a-s3m). discussion our survey on gorgona island reports for the first time the occurrence of the italian wall lizard, which has probably been accidentally introduced to the island during the last few years. the species is now markedly widespread, despite never being found prior to our survey (corti, 2006; corti et al., 2011). the last survey on lizards was carried out at the beginning of 2000 (c. corti, pers. comm.), and no evidence of podarcis siculus occurrence was reported. therefore, the time gap between the last and the current survey is well-defined. nevertheless, we cannot table 2. nucleotide substitutions identified in the three p. siculus cyb haplotypes from gorgona. haplotype sample a mutations relative to the reference sequence (nc011609) genbank accession numbernp 14,436 np 14,607 np 14,985 np 15,027 np 15,042 np 15,063 np 15,255 b reference (nc011609) t c a t c a t 1 gorg03 c . g . . c . om925988 1 gorg05 om925989 1 gorg06 om925990 2 gorg02 . . g c . t . om925991 3 gorg01 c t . . t t c om925992 3 gorg04 om925993 3 gorg07 om925994 a 924 bp (from np 14,357 to np 15,280) of the cyb gene were sequenced for all samples. b this nucleotide position was not included in phylogenetic analyses because outside of the sequence range available for most of the cyb sequences from genbank. 140 marco a.l. zuffi et alii fig. 3. bayesian inference phylogeny of podarcis siculus cyb sequences. this tree was obtained via the bayesian method. it encompasses 402 partial cyb sequences (764 bp, nps 14417-15180) and was rooted using podarcis muralis (nc_011607). the time scale is in thousands of years ago (kya). coloured bars indicate haplogroup/sub-haplogroup affiliation, following colour scheme and nomenclature from senczuk et al., (2017). new sub-haplogroups are indicated by an asterisk. a2a was removed due missing regions within the sequence. 141allochtonous podarcis siculus on gorgona island state precisely when this species reached gorgona island. it is worth underscoring that olive trees and grapevines have been transplanted on the island in the last two decades for agricultural purposes, according to regional and eu projects. thus, passive transport with plants is a possible scenario, as recently reported in the uk (clemens and allain, 2021). passive transportation of animals, and especially reptiles, has been documented worldwide (burke et al., 2002; silva-rocha et al., 2014; adamopoulou, 2015; mizsei et al., 2016; d’amico et al., 2018; ribeiro and sá-sousa, 2018; clemens and allain, 2021). in addition, on gorgona island, cattle, horses and sheep have increased in number and much more fodder is imported from the mainland, via boats from piombino harbour. most reptile invaders have a survivorship rate usually at about 10% of the total (ferreira et al., 2012), supporting the idea that new colonizers frequently survive the introduction and may be more competitive than resident species (mangiacotti et al., 2013; kapsalas et al., 2016; ribeiro and sá-sousa, 2018; damas-moreira et al., 2020). in particular, the conclusion of detwiler and criscione (2014) “invasive metapopulation has rapidly reached the establishment stage as indicated by relatively constant effective sizes and migration rates among introduced subpopulations”, appears to fit very well with the high number of adult and juvenile p. siculus that we observed on gorgona island. colonization times and population structure of introduced species are often underestimated and genetic data of insular populations may provide correct information on the original distribution of analysed species (silvarocha et al., 2019). importantly, some research underlined the different invasion origins (toscana, sardegna, calabria, sicilia, silva-rocha et al., 2012), and possible times of introduction, ranging from the middle age for the balearic islands to the first half of the xx century for the almeria and cantabrian populations, or even more recently (silva-rocha et al., 2012). our data indicate the arrival of the lizards on gorgona island from the area occurring between pisa and orbetello, particularly because of the overlap (or close relationship) of the three haplotypes observed on the island with those previously reported in a wide area of the coast of toscana. to explain the detection of three distinct cyb haplotypes, at least three unrelated female founders from the mainland have to be postulated, individuals that most likely reached gorgona island through distinct introduction events. different and not related introduction events, table 3. nucleotide diversity (%) within and between p. siculus cyb sequences belonging to different haplogroups and from different geographic areas. intra-group nucleotide diversities (π) are on the diagonal. haplogroup a n = 112 haplogroup t n = 97 haplogroup s n = 193 haplogroup a 1.565 ± 0.125 5.586 ± 0.148 7.283 ± 0.157 haplogroup t --2.154 ± 0.037 8.093 ± 0.147 haplogroup s ----3.185 ± 0.245 fig. 4. maximum likelihood phylogeny of haplogroup t1 sequences. this tree is a subset of the one in figure s1, encompassing only haplogroup t1 sequences. numbers at nodes indicate the bootsrap values. asterisks indicate samples from gorgona island and arrows indicate their closest related relative. 142 marco a.l. zuffi et alii fig. 5. phylogeny of the 293 haplotypes found in the 402 p. siculus cyb sequences. partial cyb sequences are subdivided into main haplogroups (senczuk et al., 2017). it was constructed using fitchi (matschiner m. (2015), https://evoinformatics.group/fitchi.html). sizes of circles are proportional to the number of cyb sequences, with the smallest circle (for each panel) representing one individual. dots on branches represent intermediate haplotypes and ‘@’ is the reference sequence. gorgona island samples are highlighted with an asterisk. 143allochtonous podarcis siculus on gorgona island are the unique logical explanation for having found the three distinct cyb haplotypes, similarly to what has been found in the iberian peninsula (e.g., silva-rocha et al., 2012) and in some other countries, where pathways and origins have been determined (silva-rocha et al., 2014). these introductions were accidental and the lizards possibly arrived with olive trees and other plants (clemens and allain, 2021), rather than together with the fodder for domestic animals or using man-made objects, confirming the high invasive potential of the species (silvarocha et al., 2014; clemens and allain, 2021). further surveys and molecular analyses are required to understand i) the number of colonization events and ii) if other founder haplotypes are present. finally, it could be important to monitor and study ecological and behavioural patterns of gorgona island population(s) with respect to those living on the continent, to assess how p. siculus interacts with the locally adapted p. muralis, and to evaluate if the eradication of this allochthonous species from gorgona island should be performed. experiments on competitive interactions (i.e., chemical avoidance, territorial behaviours, food preference) and biometric analyses of head shape and body size between the two podarcis species may give useful insights into the ecological plasticity of both the residential and the alien lizard. acknowledgements we are indebted to parco nazionale arcipelago toscano for permission entering the protected area and to the amministrazione penitenziaria in livorno for logistics and support on the island; to c. corti for having provided unpublished information on data on previous monitoring on the island. molecular analyses received support from prin2017 2017cwhlhy (to a.t.) and from dipartimenti di eccellenza program (2018–2022) – dipartiemto di biologia e biotecnologia “l. spallanzani’’ university of pavia (to a.t.). capture and handling permissions were issued by ministero ambiente (prot. 0008139, 9 april 2019, for the 2019-2021 period, to mal zuffi) and for allochthonous species eradication, as in the decreto legislativo 15 dicembre 2017 n. 230. supplementary material supplementary material associated with this article can be found at <http://www-9.unipv.it/webshi/appendix/ index.html> manuscript number 12388 references adamopoulou, c. 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(2006): atlante degli anfibi e dei rettili della toscana. edizioni regione toscana, firenze. acta herpetologica vol. 17, n. 2 december 2022 firenze university press cryptic diversity in pygmy chameleons (chamaeleonidae: rhampholeon) of the eastern arc mountains of tanzania, with description of six new species michele menegon1,2,*, john v. lyakurwa3,4, simon p. loader5, krystal a. tolley6,7 preliminary genetic characterisation of southern smooth snake coronella girondica (serpentes, colubridae) populations in italy, with some considerations on their alpine distribution matteo r. di nicola1, raffaella melfi2, francesco p. faraone3,*, daniel l. n. iversen4, gabriele giacalone5, giovanni paolino1, mario lo valvo6 species diversity and distribution of amphibians and reptiles in sardinia, italy claudia corti1,2,*, marta biaggini1, valeria nulchis2, roberto cogoni2, ilaria maria cossu2, salvatore frau4, manuela mulargia2, enrico lunghi2, lara bassu2. the italian wall lizard, podarcis siculus campestris, unexpected presence on gorgona island (tuscan archipelago) marco a.l. zuffi1,*, alan j. coladonato2, gianluca lombardo3, antonio torroni3, matilde boschetti1, stefano scali4, marco mangiacotti2, roberto sacchi2 molecular analysis of recently introduced populations of the italian wall lizard (podarcis siculus) oleksandra oskyrko1,2,*, lekshmi b. sreelatha1,12,13, iolanda silva-rocha1, tibor sos3,4, sabina e. vlad5,6,7, dan cogălniceanu5,6, florina stănescu6,7,8, tavakkul m. iskenderov9, igor v. doronin10, duje lisičić11, miguel a. carretero1,12,13 sunny-side up: ontogenetic variation in egg mass temperatures of the wood frog rana sylvatica ryan calsbeek*, ava calsbeek, isabel calsbeek ecological niche differentiation in the anatolian rock lizards (genus: anatololacerta) (reptilia: lacertidae) of the anatolian peninsula and aegean islands mehmet kürşat şahin1,*, kamil candan2,3, danae karakasi4, petros lymberakis4, nikos poulakakis4,5,6, yusuf kumlutaş2,3, elif yıldırım2,3, çetin ilgaz2,3 occupancy and probability of detection of the introduced population of eleutherodactylus coqui in turrialba, costa rica jimmy barrantes-madrigal1,*, manuel spínola parallada1, gilbert alvarado 2, víctor j. acostachaves3,4. one site, three species, three stories: syntopy of geckoes euleptes europaea (gené, 1839), hemidactylus turcicus (linnaeus, 1758), tarentola mauritanica (linnaeus, 1758) in a coastal area of southern tuscany (central italy) giacomo radi1,2, marco a.l. zuffi1,* comparative cytogenetics on zamenis lineatus and elaphe quatuorlineata (serpentes: colubridae) marcello mezzasalma1,* , elvira brunelli1, gaetano odierna2, fabio m. guarino2 acta herpetologica 17(2): 187-195, 2022 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-11547 one site, three species, three stories: syntopy of geckoes euleptes europaea (gené, 1839), hemidactylus turcicus (linnaeus, 1758), tarentola mauritanica (linnaeus, 1758) in a coastal area of southern tuscany (central italy) giacomo radi1,2, marco a.l. zuffi1,* 1 museum natural history, university of pisa, via roma 79, i-56011 calci (pisa) 2 loc. biagioni 7, 58020 scarlino (grosseto) *corresponding author. email: marco.zuffi@unipi.it submitted on: 2021, 30th july; revised on: 2022, 10th june; accepted on: 2022, 30th june editor: fabio m. guarino abstract. ecological aspects of syntopic geckoes were rarely addressed in the mediterranean basin. we reported basic information on habitat use, and activity patterns of three species found in syntopy in cala violina site (divided in three subareas), a highly touristic beach located in southern tuscany, central italy, during 2009-2010. the most abundant species at first capture is hemidactylus turcicus (94 individuals), while tarentola mauritanica and euleptes europaea are less represented (28 animals in both cases). total captures and recaptures were 175. sex ratio did not differ from 1:1 in all the species, nor sexes of adults did differ in size. ambient temperatures did not differ in t. mauritanica and e. europaea, while were different in h. turcicus. despite the humidity of capture sites did not vary among species, we recorded the highest number of e. europaea at 95% and h. turcicus at 62% humidity. wind influenced negatively t. mauritanica and h. turcicus presence, not on e. europaea. higher observation rate took place between 21:00 and 22:00. after 23:00, only euleptes was active. height from the ground was different only in h. turcicus. general linear models showed that interaction substrate-height at capture was important for euleptes, not for the other two species. along the area, e. europaea was more concentrated in the northern patch, while t. mauritanica and h. turcicus distributed more homogeneously. we suggest limitation of human presence for conservation purposes. keywords. syntopy, geckoes, tarentola, hemidactylus, euleptes, central italy. introduction the study of the ecology of the italian reptile species is particularly advanced for some groups, especially for tortoises (e.g., chelazzi and carlà, 1986; rugiero and luiselli, 2006), pond turtles (e.g., rovero and chelazzi, 1996; lebboroni and chelazzi, 1998; zuffi et al., 2004, 2007), as well as for snakes (e.g., luiselli et al., 1996; zuffi, 2008; zuffi et al. 2009; scali et al., 2011) and lizards (e.g., perezmellado and corti, 1993; sacchi et al., 2007; salvidio and oneto, 2008; bombi et al., 2009; zuffi et al., 2011, 2012). most information about italian lacertilia sensu lato has been provided in atlases and distributive maps (vanni and nistri 2006; corti et al., 2011), despite quite anecdotal and descriptive. on the contrary, complete and scrutinized data concern phylogeographic and taxonomic features (harris et al., 1998; oliverio et al., 1998; gamble et al., 2008), and, partially, ecological-behavioural features (vervust et al., 2007; biaggini et al., 2009; marsili et al., 2009; sacchi et al., 2015; scali et al., 2016). however, research considering comparative aspects in different reptile species are relatively limited (i.e., capula, luiselli and 188 giacomo radi, marco a.l. zuffi rugiero, 1993; capula and luiselli, 1994; carvalho jr et al., 2008; gordon et al., 2010; maura et al., 2011; simbula et al., 2019) and further studies are strongly needed. there are four gecko species in italy (corti et al., 2011): tarentola mauritanica and hemidactylus turcicus, distributed in most of the mediterranean coastal environments, and euleptes europaea and mediodactylus kotschyi more localised, in western mediterranean italy and in south-eastern italy (apulia), respectively. although the distribution of tarentola, hemidactylus and euleptes is to some extent overlapped in north-western italy, namely in coastal tuscany (vanni and nistri, 2006), the only site where the three species actually occur in sympatry and in syntopy is in southern tuscany, in the municipality of scarlino, province of grosseto (radi, 2013). on average, available data on euleptes europaea refer to a few sites only in liguria (tinetto, tino, genoa), sardinia (sassari, gallura) and tuscany (castiglione della pescaia and the tuscan archipelago) and regard morphology and population dynamics (salvidio and delaugerre, 2003; salvidio and oneto, 2008; salvidio et al., 2011, for a review). ecological data on hemidactylus turcicus and tarentola mauritanica of italy are quite descriptive (capula and luiselli, 1994; luiselli and capizzi, 1999; aprea et al., 2011; zuffi et al., 2011) with the exception of a few studies on sympatric geckoes in italy and croatia (lisicic et al., 2012; simbula et al., 2019). our research, as far as we are aware, is likely the first one aimed at comparing three gekkota species in sympatry and syntopy, and analysing and comparing biometrical features, population structure and ecology patterns in a quite unique zoogeographic context. material and methods study area study area is in cala violina, municipality of scarlino (province of grosseto), which is a highly frequented touristic place during summer. this area extends for 1000 × 300 m and falls within the “monte d’alma” 108 sir (sito di interesse regionale, regional interest site; it51a0008), and psic (sito di interesse comunitario, eu interest site; natura 2000 it51a0008) and the a.n.p.i.l. “costiere di scarlino” (area naturale protetta di interesse locale, protected natural area of local interest) (42.856850°n, 10.774386°e) (fig. 1). we have focused the field activity on the maximum area extension, which is about 670 m long sector; the area is characterized by a central sandy part. proceeding towards the far ends of the promontories, that are quite high and boulder-like, the sandy part gradually changes into coarse soiled sandstone cliffs. going down from the shore to the mediterranean scrub it is possible to find dissolved bedrock and sandstone slopes, transitional environments in which geckoes live. climate is mediterranean, with average rainfall of 600-800 mm during winter, and average temperatures of 14 °c (selvi and stefanini, 2005). specifically, in follonica, the closest meteorological station to the study area, average rainfall and temperatures are 655.2 mm and 15.7 °c respectively (barazzuoli et al., 1993). sampling and measurements sampling was carried out with censuses during two annual sessions, in 2009, from 23rd july to 22nd november and in 2010, from 07th april to 30th august. we did 20 sampling days in 2009 (18 out of 20 during the night) and 22 in 2010 (20 out of 22 during the night) for 42 sampling days. each sampling lasted five hours on average for a total of 210 hours of field night sampling. sampling occurred from 20:00 to 02:00 solar hour to avoid touristic disturbance and to match species’ activity. the area is naturally divided in three sectors t1, north, ca 220 m long t2, central, ca 70 m long, and t3 south, ca 350 m long, by two forest tracks (the first 24 m wide, the second 4 m wide) loading to the beach from the forest, for a total of 670 m transect length (fig. 2). we have considered the three sectors as a unique survey area. we captured geckoes by hand, or with a noose on a long stick, and placed in cotton sacks before data recording. at each capture, we registered solar time, substrate type (sandstone, loose ground, boulders, sand, vegetation), ambient temperature, humidity (thermofig. 1. satellite picture (source: google earth) of follonica gulf delimited by piombino promontory (li) to the north, and by punta ala promontory (gr) to the south. cala violina is pointed by a white pin. 189three geckoes syntopy on coastal tuscany hygrometer hanna, hi9565, precision 0.1 °c, 0.1% humidity), wind (with the empirical beaufort scale), animal position (height from the ground, distance from transect starting point). morphological data were snout to vent length, tail length, head length, width and height, eye diameter, distance between eye and nostril, internarial distance, interorbital distance. we also determined size and sex class, as described in full by atzori et al. (2007). we therefore considered males and females, juveniles (medium size, unsexable) and new-borns (very small size, unsexable). geckoes were marked with acrylic water pens for short term recognition and with a cut of coded sub-digital scales (see atzori et al., 2007) for a long-term recognition. we did not apply sub-digital marking to the new-borns of all the three species due to their markedly small size, and to the adults of the european leaf-toad gecko, due to the extremely reduced size of lamellae and very thin fingers. we assessed female reproductive status by manual palpation and, in some cases, using a direct light placed on the female vent, to detect eggs for transparency. in the whole period, we captured 175 geckoes: 110 hemidactylus, 34 euleptes and 31 tarentola. we excluded recaptures and visual encounters from this research. we therefore analyzed 159 unique gecko records, 100 of which were hemidactylus (59 in 2009, 41 in 2010), 31 were euleptes (21 in 2009, 10 in 2010), and 28 tarentola (14 in 2009, 14 in 2010). we defined three age classes: males, females, and juveniles. we considered juveniles and new-borns together as juvenile category. we tested sex-ratio differences within and among species using a log-linear model (with binomial distribution). we tested differences in size and biometry (all variables were normally distributed, kolmogorov-smirnov test, p > 0.05) with a general linear model (multivariate glm, with species and sex as fixed factors and their interaction). we applied this analysis only to the adults. given all environmental variables, we used a multivariate glm to test if species do differ in some way between years and among them. in addition, to describe ecological relationships among gecko species, we applied a principal component analysis (varimax procedure, eigenvalue ≥ 1, rotated matrix), to all environmental variables and two biometrical features (svl, body mass), extracting the most correlated variables within each main component. therefore, we were able to describe the components driving the average ecology of the three species of geckos. to analyse the spatial distribution of captured and observed geckoes, we normalized the three subsectors, creating a unique transect. we also considered the width of the two tracks used to reach the beach: 26 and 4 metres, respectively. temperature, humidity, wind presence and hourly distribution of captures were not normally distributed, and we therefore considered them in glm and pca analyses. we carried out univariate and multivariate analyses with spss 20.0 release. results we sampled 31 euleptes (17 males, eight females and six juveniles), 100 hemidactylus (35 males, 36 females and 29 juveniles, and 28 tarentola (nine males, seven females and 12 juveniles). sex ratio of adults (juveniles were excluded) did not differ from 1:1 in each of the three species (wald test = 0.785, df = 1, p = 0.376). the three species are markedly different for all considered biometric features (table 1). euleptes is the smallest, hemidactylus is intermediate and tarentola the largest (all with p values < 0.0001, but inter-nasal p = 0.015). they do not show sexual difference (p values from 0.093 for head width to 0.663 for svl) nor sex × species interaction (p values from 0.130 for head width to 0.695 for inter-orbital). all statistics are reported in supplementary table 1. ecological variables recorded at each gecko capture (table 2) showed differences in most cases: multivariate glm showed a marked difference between 2009 and 2010 for wind (f2,131 = 5.807, p = 0.017) and humidity (f2,131 = 36.593, p < 0.0001). species differed in site position (f2,131 = 4.011, p = 0.020), height from the ground (f2,131 = 11.670, p < 0.0001) and hour of capture (f2,131 = fig. 2. satellite image (google earth) of cala violina. white lines show three transects (t1, t2, t3), a1 and a2 indicate access to the beach. 190 giacomo radi, marco a.l. zuffi 8.587, p < 0.0001). year × species interaction was significant only for humidity (f2,131 = 3.781, p < 0.025). pca (sampling adequacy = 0.551; sphericity bartlett test = 424.286, p < 0.0001) extracted four main components, explaining about 68% of total variance (table 3). the rotated matrix showed the first component describing species and body size, the second describing period, site position, ground type and height from the ground, the third describing wind and humidity, the fourth describing temperature and ground type (table 4). the distribution of the three species of geckos according to four components are shown in figures 3-5. figure 3 shows that smaller geckoes, as euleptes and smaller hemidactylus and tarentola, tend to be distributed to the northern part of the study area. figure 4 shows that euleptes is associated to low or no wind but with high humidity, while the other two species are more associated to a relative absence of humidity. figure 5 shows that table 1. average in mm and grams ± 1 sd of selected variables for each gecko species. variable taxon average sd head length euleptes 10.230 0.312 hemidactylus 13.996 0.173 tarentola 16.419 0.367 head width euleptes 6.809 0.191 hemidactylus 9.251 0.106 tarentola 11.767 0.224 head height euleptes 3.471 0.126 hemidactylus 5.561 0.070 tarentola 6.965 0.149 eye diameter euleptes 2.036 0.059 hemidactylus 2.701 0.033 tarentola 3.012 0.069 nostril eye euleptes 2.795 0.087 hemidactylus 3.695 0.048 tarentola 5.010 0.102 inter-nasal euleptes 1.735 0.052 hemidactylus 1.863 0.029 tarentola 1.964 0.061 inter-orbital euleptes 4.567 0.132 hemidactylus 4.702 0.073 tarentola 6.630 0.155 bmass euleptes 1.292 0.235 hemidactylus 3.152 0.130 tarentola 5.208 0.276 svl euleptes 38.939 1.086 hemidactylus 50.534 0.601 tarentola 55.377 1.276 table 2. variability of ecological variables recorded at gecko capture. wind in m/sec, umidity in %, site position and hmslm in m, hour as in hours (solar time). variable species average ± sd sample wind euleptes hemidactylus tarentola 0.28 ± 0.45 0.22 ± 0.45 0.16 ± 0.37 29 77 25 umidity euleptes hemidactylus tarentola 77.38 ± 17.70 69.66 ± 18.17 72.20 ± 20.60 29 77 25 site position euleptes hemidactylus tarentola 157.21 ± 45.14 119.65 ± 69.67 119.24 ± 60.10 29 77 25 hmslm euleptes hemidactylus tarentola 2.74 ± 1.15 1.61 ± 1.04 1.72 ± 1.05 29 77 25 hour euleptes hemidactylus tarentola 17:55 ± 08:13 21:22 ± 00:32 21:14 ± 00:17 29 77 25 table 3. first four principal components explaining about 68% of total variance. component eigenvalues weights of rotated factors total % variance % cumulated total % variance % cumulated 1 2.653 26.533 26.533 2.405 24.048 24.048 2 1.687 16.873 43.407 1.705 17.053 41.101 3 1.359 13.592 56.999 1.377 13.767 54.868 4 1.105 11.052 68.051 1.318 13.183 68.051 table 4. rotated component matrix and main contribution of each variable (in bold) to each component. variable component 1 2 3 4 species 0.679 0.157 0.074 -0.270 month -0.202 0.622 0.206 -0.008 wind -0.179 0.365 -0.771 -0.087 t° 0.124 -0.041 -0.032 0.853 umidity -0.225 0.264 0.817 -0.187 siteposition -0.228 -0.717 0.208 -0.099 ground -0.026 0.518 -0.073 0.586 hmslm -,484 -0.548 0.077 0.078 bmass 0.898 -0.022 -0.049 0.199 svl 0.844 -0.076 -0.089 0.276 191three geckoes syntopy on coastal tuscany euleptes and hemidactylus are active at lower temperatures and on similar substrate ground than tarentola. multiple histograms show the occurrence of captures of the three species along the normalized transect (670 m long). red bars indicate and delimitate the accesses to cala violina (fig. 6). all the species showed a different distribution, tarentola and hemidactylus were quite homogeneous, while euleptes was more concentrated in the first patch of the area (transect 1). spatial distribution was not normal (kolmogorov smirnov z = 2.149, p < 0.001), and the observed differences in spatial distribution were significant (p = 0.007). discussion the gecko community in this area shows a sex-ratio not differing from the expected 1:1, for all the species considered. average body size of the leaf-toad gecko and of the turkish gecko falls within the range of the species in italy (corti et al., 2011), while the moorish gecko results smaller in size with respect to available data (tuscany: atzori et al., 2007). in our studied sample, we did not find any significant sexual difference within each species. this matter may occur in some other populations of euleptes (delaugerre, 1985), likely due to the biotic capacity of the site (salvidio et al., 2011). hemidactylus males generally present much larger heads than the females, while size is similar for the two sexes (corti et al., 2011). in tarentola, sexual dimorphism is significantly marked for some features: larger eye diameter and bigger head in males (more voluminous head than equal sized females) and, same svl, lighter body mass in females (zuffi et al., 2011). in our study area, the difference between males and females may be underestimated because the number of the two sexes of juvenile age, or as young adults, probably does not present yet the strongly different sexual dimorphism when considering adults. interestingly, as pointed out by simbula et al. (2019) where diurnal vs nocturnal tarentola were considered, sexual dimorphism was not significant, a similar matter as occurred in our population. furthermore, nocturfig. 3. geckos distribution along the component 1-component 2 relationship. fig. 4. geckos distribution along the component 1-component 3 relationship. fig. 5. geckos distribution along the component 1-component 4 relationship. fig. 6. capture frequency distribution of target species in the study area. red lines represent the borders of forest tracks to the beach. 192 giacomo radi, marco a.l. zuffi nal individuals attained a smaller body size than diurnal ones (simbula et al., 2019), suggesting an analogous pattern also in our sample. body size did differ for almost all parameters among the species. we have recorded, on average, a different association of species as regards site position, height from the ground and hour of observation. between the two years humidity and wind were different, with a significant interaction among species and year for humidity only. to date, ecology studies on these three species of geckoes are relatively scarce, covering trophic ecology (capula and luiselli, 1994; luiselli and capizzi, 1999; hòdar et al., 2006), or underlining competition for spatial niches in sympatric populations of hemidactylus and tarentola in croatia (lisicic et al., 2012), and on lizard and geckoes’ community in central italy (simbula et al., 2019). overall, our research is among the very few field works on species assemblages and, actually, it is the first work on comparative ecology of these three species of geckoes in condition of syntopy. tarentola seems to prevail in the northern portion of the area, while hemidactylus is more common in the northern and southern portion, as supported by glm and chi square analyses. according to our data, wind and humidity had more effects on tarentola and on hemidactylus respectively (table 2), the species are differently distributed in the area (e.g., site position) and placed at different heights from the ground, euleptes being relatively higher than the other two species, with a different hour distribution of observation. however, in accordance with data and analyses, the three species seem to occupy and frequent the area not in a markedly different way. pca results showed a different distribution of age classes (as size component) relatively to the site position especially for hemidactylus and tarentola (fig. 3) and a relatively importance of wind, humidity, temperature and ground type for geckoes observation (figures 4-5). the whole scenario is in accordance with previous ecological observations (see for instance delaugerre, 1984; simbula et al., 2019). the similar occurrence of species along the transect despite the slight, significant, differences among them in many ecological parameters, resembles the pattern found and underlined by simbula et al. (2019). specifically, the “the observed overlap in spatial resource use was higher than expected by chance, thus showing a shared resource use instead of a partitioning pattern” (simbula et al., 2019). for what we know so far, cala violina is the only syntopy area for euleptes europaea, hemidactylus turcicus and tarentola mauritanica, and it arises a pivotal importance for the conservation of the three species. on average, we must stress that human presence in this area, as a marked touristic presence for most of the geckoes active season, is on one side undoubtedly a risk for the three species. on the other side, according to recorded data, it is not possible yet to assert if studied populations of the three species have suffered numerical losses because of direct anthropic actions (killings, capture, and removal) or indirect (alteration or damage to habitat, increment of tourism), because there are not yet studies underlining the risk factors. locally, arsons, inappropriate woodcutting and touristic impacts are the strongest risk factors in the study area. the touristic activity on the seaside probably plays a negative role on daily activity of tarentola mauritanica. in fact, the study area is characterised by intensive daily seaside activity in the spring-summer seasons (from june to september). cala violina is featured in s.i.r. (site of regional interest) 108 “monte d’alma” (it51a0008), in homonym ps.i.c. (cod. nature 2000 it51a0008), and in a.n.p.i.l. (area naturale protetta di interesse locale, “protected natural area of local interest”) “costiere di scarlino”, but the actual presence of euleptes europaea justifies the opportunity to propose the realization of a biotope. the “rete ecologica regionale” (ecological regional network), together with the “piano territoriale di coordinamento” (ptc, “territorial coordinational plan) applicable in the grosseto province, represent two essential instruments enforcing the importance of environmental connectivity and natural resources protection. biotopes, in fact, are an innovative aspect in the field of planning management, to define further restrictions congruent to the latest acts of territory planning. in a conservation and management planning for this area, priority should be given to intervention finalized to protect slopes and cliffs in which these geckoes live and spent most of their life history traits. protective intervention for slopes and cliffs is desirable to avoid further human disturbance, and particularly to avoid damages to natural fissures and crevices (abandon of garbage and other objects in fissures, destruction of portions of loose soil by sun beds, chairs and other), likely used for egg deposition, and surely for daily hideaway. these protective measures could be made up by wood barriers adequately distant from sides (0.5 – 1 m), which do not consist of a landscape obstacle, and with explicative posters making visitors aware of the site peculiarities, fauna richness and of all the protection measures. besides, it would be appropriate to evaluate through a dedicated study how much touristic impact influences these geckoes and other herpetofauna activity in the area, and to limit, if necessary, the daily access to cala violina with a maximum number of people per day (a maximum of 700/day since the last year; p. biagini pers. comm). measure of human disturbance on a lizard community has been recently carried out in spain on a population of 193three geckoes syntopy on coastal tuscany wall lizard (podarcis muralis), in a strongly touristic area (amo, lopez and martin, 2006), where the authors pointed out that tourism had harmful effects on physical condition and relations host-parasite in this reptile. therefore, it would be desirable to verify the actual situation in cala violina, because of this and other studies (e.g., attum et al., 2006; french et al., 2008). acknowledgements we thanks all friends and colleagues, which supported and helped in the field: giovanni bencini, marco porciani, nicola destefano, giuliano franchi, matteo bencini, lorenzo saccucci, emanuele biggi, flavio lo scalzo, pietro giovacchini, fausto corsi, marco dragonetti, sara costa, marco balzarini, elisa riservato, roberto sacchi, roberto sindaco, anna rita di cerbo. thanks to comune di scarlino and complesso agricolo forestale regionale “bandite di scarlino” (dr. patrizio biagini) for permissions to enter the protected area and to capture and handle the protected species. supplementary material supplementary material associated with this article can be found at <http://www-9.unipv.it/webshi/appendix/ index.html> manuscript number 11547 references amo, l., lopez, p., martin, j. 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(2011): sexual size and shape dimorphism in the moorish gecko (tarentola mauritanica, gekkota, phyllodactylidae). north-west. j. zool. 7: 189-197. acta herpetologica vol. 17, n. 2 december 2022 firenze university press cryptic diversity in pygmy chameleons (chamaeleonidae: rhampholeon) of the eastern arc mountains of tanzania, with description of six new species michele menegon1,2,*, john v. lyakurwa3,4, simon p. loader5, krystal a. tolley6,7 preliminary genetic characterisation of southern smooth snake coronella girondica (serpentes, colubridae) populations in italy, with some considerations on their alpine distribution matteo r. di nicola1, raffaella melfi2, francesco p. faraone3,*, daniel l. n. iversen4, gabriele giacalone5, giovanni paolino1, mario lo valvo6 species diversity and distribution of amphibians and reptiles in sardinia, italy claudia corti1,2,*, marta biaggini1, valeria nulchis2, roberto cogoni2, ilaria maria cossu2, salvatore frau4, manuela mulargia2, enrico lunghi2, lara bassu2. the italian wall lizard, podarcis siculus campestris, unexpected presence on gorgona island (tuscan archipelago) marco a.l. zuffi1,*, alan j. coladonato2, gianluca lombardo3, antonio torroni3, matilde boschetti1, stefano scali4, marco mangiacotti2, roberto sacchi2 molecular analysis of recently introduced populations of the italian wall lizard (podarcis siculus) oleksandra oskyrko1,2,*, lekshmi b. sreelatha1,12,13, iolanda silva-rocha1, tibor sos3,4, sabina e. vlad5,6,7, dan cogălniceanu5,6, florina stănescu6,7,8, tavakkul m. iskenderov9, igor v. doronin10, duje lisičić11, miguel a. carretero1,12,13 sunny-side up: ontogenetic variation in egg mass temperatures of the wood frog rana sylvatica ryan calsbeek*, ava calsbeek, isabel calsbeek ecological niche differentiation in the anatolian rock lizards (genus: anatololacerta) (reptilia: lacertidae) of the anatolian peninsula and aegean islands mehmet kürşat şahin1,*, kamil candan2,3, danae karakasi4, petros lymberakis4, nikos poulakakis4,5,6, yusuf kumlutaş2,3, elif yıldırım2,3, çetin ilgaz2,3 occupancy and probability of detection of the introduced population of eleutherodactylus coqui in turrialba, costa rica jimmy barrantes-madrigal1,*, manuel spínola parallada1, gilbert alvarado 2, víctor j. acostachaves3,4. one site, three species, three stories: syntopy of geckoes euleptes europaea (gené, 1839), hemidactylus turcicus (linnaeus, 1758), tarentola mauritanica (linnaeus, 1758) in a coastal area of southern tuscany (central italy) giacomo radi1,2, marco a.l. zuffi1,* comparative cytogenetics on zamenis lineatus and elaphe quatuorlineata (serpentes: colubridae) marcello mezzasalma1,* , elvira brunelli1, gaetano odierna2, fabio m. guarino2 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 6(1): 35-45, 2011 the phylogenetic position of lygodactylus angularis and the utility of using the 16s rdna gene for delimiting species in lygodactylus (squamata, gekkonidae) riccardo castiglia*, flavia annesi dipartimento di biologia e biotecnologie “charles darwin”, università “la sapienza” di roma, via borelli 50, i-00161 rome, italy. *corresponding author. e-mail: castiglia@uniroma1.it submitted on: 2010, 27th december; revised on: 2011, 25th march ; accepted on: 2011, 27th april . abstract. the african genus lygodactylus gray, is composed of roughly 60 species of diurnal geckos that inhabit tropical and temperate africa, madagascar, and south america. in this study, we assessed the phylogenetic position of l. angularis, for which molecular data were so far lacking, by means of sequence analysis of the mitochondrial 16s rdna gene. we also compared intraspecific vs. interspecific genetic divergences using an extended data set (34 species, 153 sequences), to determine whether a fragment of this gene can be useful for species identification and to reveal the possible existence of new cryptic species in the genus. the analysis placed l. angularis in a monophyletic group together with members of “fischeri” and “picturatus” groups. nevertheless, the independence of the “angularis” lineage is supported by the high genetic divergence. comparison of intraspecific vs. interspecific genetic distances highlights that, assuming an equal molecular rate of evolution among the studied species for the used gene, the threshold value useful for recognising a candidate new species can be tentatively placed at 7%. we identified four species that showed an intraspecific divergence higher than, or close to, the 7% threshold: l. capensis (8.7%), l. gutturalis (9.3%), l. madagascariensis (6.5%) and l. picturatus (8.1%). moreover, two species, l. mombasicus and l. verticillatus, are paraphyletic in terms of gene genealogy. thus, the study shows that a short fragment of the 16s rdna gene can be an informative tool for species-level taxonomy in the genus lygodactylus. keyword. african reptiles, biodiversity, dna barcoding, genetic divergence, sister species, taxonomy. introduction the genus lygodactylus gray, 1864 encompasses about 60 species of diurnal geckos that inhabit tropical and temperate africa, madagascar, and south america (spawls et al., 36 r. castiglia and f. annesi 2002). lygodactylus has traditionally been divided into different “species groups” based on phenotypic characteristics (loveridge, 1947; pasteur, 1965; jacobsen, 1992; röll, 2000, 2004; puente et al., 2009). the phylogenetic relationships among species from madagascar have been assessed by puente et al. (2005) using a mitochondrial gene. recently, röll et al. (2010) provided a robust phylogeny of 28 species by using both mitochondrial and nuclear genes. this phylogeny mainly confirmed the monophyly of many of the species groups, previously identified on the base of morphology, as well as provided evidence for at least two additional lineages. moreover, this study identified some divergent taxa which possibly belonged to additional undescribed species. in fact, the taxonomy of lygodactylus at a species level is still uncertain, with some species known only from the type locality. furthermore, the earlier identification of many species was based on scale features only and, consequently, due to their small sizes, the identification during field work is extremely difficult (spawls et al., 2002). a recent approach to studying species limits involves the use of a single short fragment of dna that can help in the identification of an organism by its assignment to a previously described species (dna barcoding). in the same way, it is possible to predict and describe new taxa using dna (dna taxonomy) (blaxter, 2004). moreover, this approach allows inclusion of additional information in a taxonomy based on morphological characters only (padial et al., 2010). new candidate species can be identified on the basis of 1) elevated intraspecific genetic distances, similar to the ones found among different species and 2) the presence of paraphyletic species that suggests a need of taxonomic revision. yet, this approach has some limitation, as in case of incomplete gene sorting, hybridization, different rates of molecular evolution at the same marker in a group of species or accidental amplification of nuclear mitochondrial pseudogenes (frézal and leblois, 2008). different genes are currently used as dna barcodes in animals (e.g., cytochrome oxidase subunit i – coi, 16s rdna – 16s, cytochrome b cytb). evidence suggests that no “universal” gene exists. to be useful for delimiting species, the gene must have one principal characteristic that is to exhibit limited overlap between intraspecific and interspecific divergence. for example, an accurate comparison of performances of coi and 16s in amphibians showed that 16s is well suited to distinguish between intraspecific and interspecific divergence, whereas, in birds, the best results were obtained using coi (vences et al., 2005; aliabadian et al., 2009). in this study, we first analyse the phylogenetic position of l. angularis, a member of the “angularis” species group together with l. grzimeki bannikov, using a mitochondrial molecular marker. at present, there are no other molecular data on the members of this species group neither to confirm that they both belong to a separate lineage, nor to place them in a phylogenetic context. besides, lygodactylus seems to be an appropriate genus for testing the utility of a single dna fragment as a dna barcode, because of the problems in identification and unstable taxonomy. a recent paper by chiari et al. (2009), concerning species from madagascar, highlighted that two of the eight analysed species (l. madagascariensis and l. tolampyae) showed a very high intraspecific genetic divergence using a fragment of the 16s rdna. consequently, in this paper, we extend the record with a dataset formed by a higher number of species in order to determine whether the same genetic marker can be useful for species identification. finally, we reveal the possible presence of new cryptic species in lygodactylus by providing evidence of genetic divergence and species paraphyly. 37dna taxonomy in lygodactylus materials and methods we utilised almost all the available 16s rdna sequences from lygodactylus downloaded from genbank (updated on february 2011; 34 species, 153 sequences; table 1). sequences from specimens with uncertain taxonomical attribution were excluded. sequences from additional specitable 1. species, number of localities and number of individuals used for the comparative analysis done on the 16s rrna gene fragment. lygodactylus sp. 1 and sp.2 are undescribed species (röll et al. 2010). data from chiari et al. (2009), röll et al. (2010), puente et al. (2005) and rocha et al. (2009) and present data. species number of localities number of specimens l. angularis günther (1893) 1 1 l. arnoulti pasteur (1964) 1 5 l. bivittis (peters, 1883) 1 4 l. blancae pasteur (1995) 1 2 l. bradfieldi hewitt (1932) 2 3 l. capensis (smith, 1849) 6 9 l. chobiensis fitzsimons 1932 1 2 l. conraui tornier (1902) 1 2 l. gravis pasteur (1964) 1 2 l. grotei sternfeld (1911) 1 2 l. guibei pasteur (1964) 2 3 l. gutturalis bocage (1873) 2 5 l. keniensis parker (1936) 1 3 l. kimhowelli pasteur (1995) 1 4 l. laterimaculatus pasteur 1964 1 3 l. lawrencei hewitt (1926) 3 3 l. madagascariensis (boettger, 1881) 3 6 l. miops günther (1891) 1 4 l. mirabilis pasteur (1962) 1 31 l. mombasicus loveridge (1935) 2 4 l. montanus pasteur (1964) 1 2 l. pauliani pasteur and blanc (1991) 1 1 l. picturatus (peters, 1868) 2 4 l. pictus (peters, 1883) 3 12 l. rarus pasteur and blanc (1973) 1 2 l. stevensoni hewitt (1926) 1 2 l. thomensis (peters, 1880) 1 1 l. tolampyae (grandidier, 1872) 3 5 l. tuberosus mertens (1965 1 11 l. verticillatus mocquard (1895) 2 5 l. williamsi loveridge (1952) 1 2 lygodactylus sp. 1 1 4 lygodactylus sp. 2 1 1 38 r. castiglia and f. annesi mens belonging to four species were added to the dataset (l. mombasicus, nairobi, kenya, 01°16’s 36°49’e; l. picturatus, morogoro, tanzania, 06°49’s 37°40’e ; l. capensis, mutanda, zambia, 12°22’s 26°16’e; l. angularis, mbeya, tanzania, 12°33’s 25°41’e; accession numbers hq87245963). the new 16s rrna gene sequences were obtained from tissues fixed in ethanol 80%. dna was extracted using the qiamp tissue extraction kit (qiagen). the primers 16sa-l (light chain; 59-cgc ctg ttt atc aaa aac at-39) and 16sb-h (heavy chain; 59-ccg gtc tga act cag atc acg t-39) were used to amplify a section of the mitochondrial 16s ribosomal rna gene (palumbi et al. 1991). the pcr cycling procedure was performed as follows: 34 cycles of denaturation for 90 sec at 95 °c, primer annealing for 60 sec at 50 °c, and extension for 90 sec at 72 °c. all sequences were aligned with muscle, using default settings, and then adjusted manually. sites including gaps and hypervariable regions, identified by visual inspection of the alignment, were removed. the final alignment was 432bp long. a preliminary nj tree was built with this dataset, using kimura 2-parameter distances (k2p) and 10000 bootstrap replicates generated by mega 4 (kumar et al., 2008). we calculated the intraspecific distance for each species and for each species with 2 or more populations separately. average k2p distances were computed based on pairwise comparisons of all sequences for each of these species. a correlation was made between number of localities and average intraspecific genetic distance. information about the number of populations were obtained from the published articles. for interspecific distances, we calculated mean pairwise k2p distance among all pairs of species and, separately, between pairs of sister species. sister species were identified according to our phylogenetic tree (see below) and by referring to the tree proposed by röll et al. (2010). to study the phylogenetic position of l. angularis, the dataset was pruned to reduce the computation time of analyses, and only two or three sequences were retained for each species (80 sequences, 432bp, from 35 species). when possible, we kept sequences from different populations for each species. phylogenetic relationships were assessed by bayesian inference (bi), unweighted maximum parsimony (mp) and maximum likelihood (ml). we used the same outgroups used by röll et al. (2010), i.e., phelsuma standingi and rhoptropella ocellata, since they are the closest relatives of lygodactylus (austin et al., 2004). the appropriate model of substitution was chosen using the model test 3.7 program (posada and crandall, 1998). models of evolution, which provide the best approximation to the data, were chosen for subsequent analysis according to the akaike information criterion (aic). the chosen model was the general time reversible (gtr) model with rate variation among sites (+g), a proportion of invariable sites i=0.2038 and a gamma distribution shape parameter of 0.3846. the models and parameters were used for ml trees in phyml (guindon and gascuel, 2003). maximum parsimony trees were obtained with paup 4.0b10 (swofford, 2000) using a heuristic search and tree –bisection– reconnection and random addition of sequences. the robustness of the nodes was assessed using the bootstrap with 1000 replicates for mp and 500 replicates for ml. for the bi we constructed the phylogeny using the software mrbayes v. 3.1.2 (huelsenbeck and ronquist, 2001) using the same model as in the ml analysis. two independent markov chain monte carlo analyses were run. we used 1 million generations, four chains and a burn-in of 10% of the generated tree. based on our phylogenetic results, the paraphyly of l. mombasicus and l. verticillatus was further investigated using a reduced dataset comprising sequences of species from the same species group. for l. mombasicus the dataset was composed of twenty-two sequences (482bp) belonging to seven species. for l. verticillatus the dataset was composed of twenty sequences (482bp) belonging to seven species. in this procedure, longer alignments could be used, since the internal hypervariable region is less extended when close species are compared, which offers greater power for phylogenetic resolution. phylogenetic relationships were then assessed by bi, mp and ml. 39dna taxonomy in lygodactylus results phylogenetic position of l. angularis. the phylogenetic analysis of the pruned dataset (432 bp, 80 sequences from 35 species) does not generally conflict with a previous phylogeny obtained by multigene analysis (röll et al., 2010) and exhibits deep, weakly supported branches connecting well-supported species groups. the only species group that is not supported by present analysis is the “pictus-mirabilis” group, which is split into three lineages (not shown). the analysis places l. angularis in a moderately supported clade, which includes species of the “picturatus” and “fischeri” groups. (fig. 1a). within this clade, the species of the “fischeri” group and of the “picturatus” group are well supported. however, basal relationships within this clade are not resolved. instances of paraphyletic species. our phylogenetic analysis identified two putative instances of paraphyletic species (l. mombasicus and l. verticillatus). the paraphyly was further analysed with a dataset including species related to either of them. for the “picturatus” group, the dataset retrieved a topology (fig. 1a) similar to the one obtained by röll et al. (2010) that also included nuclear data. however, haplotype lyg1 from l. mombasicus has a basal position with respect to a cluster formed by the other sequences belonging to l. mombasicus and l. kimhowelli. this topology is well supported by all the methods used (fig. 1a). for l. verticillatus, the paraphyly is straightforward, since two distinct clusters within l. verticillatus have been found (min-max, 8.8-9.1% sequence divergence). one of the two clusters has high sequence similarity with the specimens identified as l. heterurus (2.3% sequence divergence) (fig. 1b). intraspecific vs. interspecific genetic distance. figure 2a shows the distribution of interspecific and intraspecific average pairwise genetic divergence. the two distributions overlap. the amount of overlap is between 4.9% and 9%. for intraspecific divergence, the range is from 0 to 9% (mean 1.8%; s.e. 2.6; n = 30), and four out of 30 values lie in the overlapping interval. interspecific distances range from 4.9 to 37% (mean 24.2%; s.e. 6.0%), and eight out of 595 values lie in the overlapping interval. for interspecific pairwise divergence, we eliminated two pairs of close species that showed paraphyly (see above). the intraspecific genetic distance is strongly correlated with the number of localities (r = 0.78; p<<0.0001). accordingly, the level of overlap between intraand interspecific divergences can be inflated by the fact that we underestimated the intraspecific divergence: in many species, only one or a few populations were available for the analysis. for this reason we compared (fig. 2b) the distribution of intraspecific genetic distances for species including more than two populations with the distribution of genetic divergences between pairs of sister species. we used pairs of sister species in order to capture the divergence of recently emerged species. the intraspecific distances for species with more than 2 populations range between 0.6% to 9% (mean 4.4%, s. d. 2.8, n=11). the distribution of genetic divergences between pairs of sister species ranges between 4.9 and 15.7% (mean 10.5%, s.e. 4.0% n = 8). the two distributions overlap between 5% and 9%. the species with high average intraspecific genetic divergences are l. capensis (8.7%), l. gutturalis (9.3%), l. madagascariensis (6.5%), l. tolampyae (5.3%) and l. picturatus (8.1%). the sister species with the lowest intraspecific divergence are l. arnoulti and l. pauliani (4.9%). 40 r. castiglia and f. annesi  fig. 1. bayesian trees of a) the clade including the “picturatus” group, the “fischeri” group and l. angularis and b) the haplotypes belonging to l. verticillatus and l. heterurus. the haplotypes analysed are in bold. numbers at the node correspond to the bayesian posterior probabilities, the percentage of 1000 bootstrap replicates for maximum parsimony and over 500 replicates for maximum likelihood respectively. 41dna taxonomy in lygodactylus discussion phylogenetic position of l. angularis. even if the basal nodes of the tree are not supported, the phylogenetic position of l. angularis is well-supported in our tree, which was built with a single mtdna gene. this species resulted in a monophyletic group togeth  fig. 2. pairwise k2p pairwise distances in the 16s gene in lygodactylus. a) black bars are comparisons among conspecific sequences (left axis); grey bars represent comparisons among different species (right axis). in (b), black bars refer to comparisons of conspecific specimens for species with two or more populations. gray bars refer to pairs of sister species only. 42 r. castiglia and f. annesi er with members of “fischeri” and “picturatus” groups. the clustering of the members of these two species groups was previously supported by roll et al. (2010). all these species share some scale characters, such as an undivided mental scale in most species. this character is also present in l. angularis (loveridge, 1947), in accordance with the presently determined phylogenetic position of this species. the “fischeri” group contains west african species, and the “picturatus” group consists of mainly east african species. l. angularis shares a similar geographic distribution in eastern africa with the member of the “picturatus” group. moreover, l. angularis is also large-sized, as the species of the “picturatus” group, whereas geckos of the “fischeri” group are small and slender. another character that may be in common between members of the “picturatus” group and l. angularis, is the presence of sexual dichromatism with coloured males. in fact, l. angularis males have rose pink ventral surface, while females are entirely lemon yellow. nevertheless, the independence of the “angularis” lineage from the species of “picturatus” and “fischeri” groups is supported by the high genetic divergence (14-20%). interand intraspecific genetic divergence. the distribution of intraspecific and interspecific genetic divergence shows an overlap between 5% and 9%. the distribution of the interspecific divergence represents probably a good approximation of the real differences between species. however, we found that intraspecific divergence depends strongly on the number of localities sampled. for this reason, we suggest that the intraspecific diversity might be underestimated. despite this limitation, the threshold values useful for recognising a “good” species, according to our data, can be tentatively placed at 7% (k2p distance). in fact, only a couple of species shows an interspecific genetic divergence below this value (l. arnoulti and l. pauliani, 4.9% genetic divergence). owing to scarcity of data on intraspecific variation, we cannot say whether the use of a lower threshold (5%) would produce an excessive number of false positives. additional intraspecific data are needed to resolve this problem and ultimately to support a threshold shift from 7% to 5%. we identified four species that showed an intraspecific divergence higher than, or close to, the 7% threshold: l. capensis (8.7%), l. gutturalis (9.3%), l. madagascariensis (6.5%) and l. picturatus (8.1%). present data confirm and extend previous results obtained with the same mtdna marker on eight species of lygodactylus, among which l. madagascariensis and l. tolampyae showed high intraspecific divergence (4.3% and 9.1%) (chiari et al., 2009). comparing the present and the previously published data, one can see that the differences in values of genetic divergences are most likely due to the different number of individuals (for in l. tolampyae) and the different length of the sequence used. in fact, the 16s rdna gene has conserved regions, but also hypervariable ones. consequently, the length of the fragment used for analysis affects easily the calculation of divergence. for a barcoding approach, obviously, the interand intraspecific divergence should be only compared by means of the same dataset. the species with high intraspecific divergences, l. gutturalis and l. capensis, are widely distributed. the divergent sequences belong to distant populations (guinea bissau and uganda for l. gutturalis; zambia, namibia, and south africa for l. capensis). additional nuclear markers and morphological characteristics should be used to infer the taxonomic status in these cases. the same concern arises for l. picturatus, a species in south-eastern kenya and eastern tanzania. the specimen of from central tanzania studied here shows a 43dna taxonomy in lygodactylus high divergence and results basal to the other sequences from the populations of the kenyan and tanzanian coasts. current information on l. madagascariensis, distributed in the northwestern part of madagascar, is too scant to allow any tentative discussion of species limits. two instances of paraphyletic species. we identified two paraphyletic species in terms of gene genealogy, namely l. mombasicus and l. verticillatus. in fact, the haplotype of l. mombasicus from kenya, here analysed, is basal to other sequences from l. mombasicus and l. kimhowelli. according to röll et al. (2010), l. kimhowelli and l. mombasicus are very similar morphologically. their colour patterns differ, but they share all scale characters and the pattern of conspicuous black markings on head and neck (röll, 2003). genetic divergence between haplotypes of l. mombasicus and l. kimhowelli is low (2.6-3.1%), whereas the haplotype from kenya diverges by 6% with respect to both of them. this value is only slightly lower than the threshold values identified above. it is evident that an accurate morphological and molecular study of specimens belonging to l. mombasicus and l. kimhowelli is needed before any conclusions regarding species status can be drawn. another species found to be paraphyletic is l. verticillatus. röll et al. (2010) concluded that l. verticillatus and l. heterurus, small geckos that are very similar in morphology, are also quite similar genetically. for this reason, the authors proposed conspecificity of the two taxa. our analysis includes two additional sequences (puente et al., 2005) and reveals a more complex pattern. in fact, these sequences are the sister group of the other sequences belonging to l. verticillatus and l. heterurus, with a very high genetic divergence of 9%. this value is higher than the 7% threshold proposed above and suggests that l. verticillatus and l. heterurus are two different sister species. conclusion this study shows that a short fragment of the 16s rdna gene may be an informative tool for species-level taxonomy in the genus lygodactylus. however, other genes should be tested, since no comparative results with other molecular markers are reported in this paper. future studies should be planned to obtain a more accurate picture of intraspecific divergence for the studied gene, as well as for other molecular markers, and to include samples from underrepresented african regions. finally, additional nuclear markers and other kind of data (for example, behavioural) should be analysed in those instance, where high intraspecific divergences were confirmed. acknowledgements we wish to express our gratitude to georges pasteur, who kindly identified the specimens studied in this paper and ekaterina gornung for critical reading of the manuscript. we also thank lucia salis for help in laboratory. two anonymous referees provided very helpful comments to the manuscript. this work was supported by funds “università” (grants to r.c.). 44 r. castiglia and f. annesi references aliabadian, m., kaboli, m., nijman, v., vences, m. 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(2005): deciphering amphibian diversity through dna barcoding: chances and challenges. phil. trans. r. soc. b 360: 1859-1868. bbib67 ole_link1 ole_link2 bbib28 ole_link5 ole_link6 ole_link7 ole_link8 _goback ole_link1 ole_link2 ole_link3 ole_link4 ole_link1 ole_link2 ole_link19 ole_link20 ole_link21 ole_link29 ole_link3 ole_link4 ole_link5 ole_link31 ole_link14 ole_link15 ole_link12 ole_link13 ole_link16 ole_link17 ole_link22 ole_link23 ole_link24 ole_link8 ole_link9 ole_link10 ole_link11 ole_link18 ole_link27 ole_link28 ole_link25 ole_link26 ole_link6 ole_link7 ole_link34 ole_link37 ole_link38 acta herpetologica vol. 6, n. 1 june 2011 firenze university press widespread bacterial infection affecting rana temporaria tadpoles in mountain areas rocco tiberti extreme feeding behaviours in the italian wall lizard, podarcis siculus massimo capula1, gaetano aloise2 lissotriton vulgaris paedomorphs in south-western romania: a consequence of a human modified habitat? severus d. covaciu-marcov*, istvan sas, alfred ş. cicort-lucaciu, horia v. bogdan body size and reproductive characteristics of paedomorphic and metamorphic individuals of the northern banded newt (ommatotriton ophryticus) eyup başkale1, ferah sayım2 , uğur kaya2 genetic characterization of over hundred years old caretta caretta specimens from italian and maltese museums luisa garofalo1, john j. borg2, rossella carlini3, luca mizzan4, nicola novarini4, giovanni scillitani5, andrea novelletto1 the phylogenetic position of lygodactylus angularis and the utility of using the 16s rdna gene for delimiting species in lygodactylus (squamata, gekkonidae) riccardo castiglia*, flavia annesi localization of glucagon and insulin cells and its variation with respect to physiological events in eutropis carinata vidya. r. chandavar1, prakash. r. naik2* the balearic herpetofauna: a species update and a review on the evidence samuel pinya1, miguel a. carretero2 effects of mosquitofish (gambusia affinis) cues on wood frog (lithobates sylvaticus) tadpole activity katherine f. buttermore, paige n. litkenhaus, danielle c. torpey, geoffrey r. smith*, jessica e. rettig food composition of uludağ frog, rana macrocnemis boulenger, 1885 in uludağ (bursa, turkey) kerim çiçek preliminary results on tail energetics in the moorish gecko, tarentola mauritanica tommaso cencetti1,2, piera poli3, marcello mele3, marco a.l. zuffi1 climate change and peripheral populations: predictions for a relict mediterranean viper josé c. brito1, soumia fahd 2, fernando martínez-freiría1, pedro tarroso1, said larbes3, juan m. pleguezuelos4, xavier santos5 assessing the status of amphibian breeding sites in italy: a national survey societas herpetologica italica* osservatorio erpetologico italiano acta herpetologica journal of the societas herpetologica italica acta herpetologica rivista della societas herpetologica italica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 6(2): 261-266, 2011 absence of invasive chytrid fungus (batrachochytrium dendrobatidis) in native fijian ground frog (platymantis vitiana) populations on viwa-tailevu, fiji islands edward narayan1*, frank molinia2, jean-marc hero1 1 environmental futures centre, school of environment, griffith university, gold coast campus, qld 4222, brisbane, australia. *corresponding author. e-mail: e.narayan@griffith.edu.au 2 landcare research, private bag 92170, auckland 1142, new zealand. submitted on: 2011, 24th may; revised on 2011, 16th june; accepted on 2011, 17th june. abstract. we report on the first survey of chytridiomycosis (batrachochytrium dendrobatidisbd) in the endangered fijian ground frog (platymantis vitiana) population on viwa-tailevu, fiji islands. this fungal pathogen has been implicated as the primary cause of amphibian declines worldwide. few cases have been reported from tropical asia however it was recently documented in 4 species of frogs in indonesia. two hundred individual frogs were swabbed from 5 different sites on viwa island. swabs were tested to quantify the number of bd zoospore equivalents using real-time polymerase chain reaction (qpcr) technique. we found zero (%) prevalence of bd in ground frogs. the lack of bd may be due to 1) hot weather all year round inhibiting the spread of bd, 2) bd may be absent from viwa island due to a lack of amphibian introductions (not introduced or importation of exotic frogs such as rana catesbeiana, or xenopus spp or pet trade spp) or 3) the lack of introduction by human vectors due to the geographic isolation, and low visitation of non-local people into the island. while it is difficult to test these hypotheses, a precautionary approach would suggest an effective quarantine is required to protect fiji’s endemic frogs from future disease outbreak. conservation effort and research is needed at international level to assist the fiji government in monitoring and protecting their unique endemic amphibians from outbreaks of b. dendrobatidis. keywords. platymantis vitiana, chytridiomycosis, infection, conservation, biosecurity, bd. chytridiomycosis is the only explanation, for which supporting evidence is available, for the global ‘‘enigmatic’’ declines and disappearances of frog populations and species (skerratt et al., 2007; fig 1: bd global mapping project www.bd-maps.net). the high prevalence of bd fungus amongst native frog species in australia and new zealand (skerratt 262 edward narayan, frank molinia and jean-marc hero et al., 2007; bishop et al., 2009) in all habitats suggest that it could be widespread in the pacific islands region. fig. 1. worldwide distribution of batrachochytrium dendrobatidis (bd), the amphibian chytrid fungus. the image is a screenshot from www.spatialepidemiology.net/bd-maps/. viwa island is located on latitude (-18.0986) and longitude (178.6615). of the currently recognized 29 families of frogs in the world, 10 are located in the pacific islands. fiji island has eastern most distribution of frogs in the southwest pacific, with two extant native frog species belonging to the genus platymantis (fijian ground frog, p. vitiana and fijian tree frog, p. vitiensis). the platymantines belong to the largest anuran family (ceratobatrachidae), and are endemic to the philippines, papua new guinea, palau, the moluccas (eastern indonesia), new britain and admiralty island, the solomon islands, and fiji. the fijian ground frog has disappeared from natural habitats on several islands in fiji, and the discovery of bd fungus in native frog populations in fiji islands could provide the major piece of the conservation puzzle solving the cause of these declines. thus the aim of the current study was to determine the prevalence of bd fungus amongst a native frog population on viwa (fiji) island. the fiji islands (latitude 18o00’ south and longitude 175o00’ east) are a biodiversity hot spot of the south pacific archipelago that historically included 59 species of breeding land birds (46% endemic), 20 breeding seabird species, 3 species of amphibians, 26 species of reptiles (40% endemic), and six species of bats (pernetta and watling, 1978). the fijian island of viwa-tailevu is being proposed as a potential biodiversity hotspot and tourist destination because much of its flora and fauna, including many endemic species, remains intact. recent conservation action on this island included eradication of small mammalian introduced predators including the polynesian rats rattus exulans and pigs sus scrofa, and europeans introduced cats felis catus and dogs canis familiaris (morley, 2006). community based conservation actions for increasing the reproductive success of the ground 263absence of chytrid fungus in the fijian ground frog frogs were also achieved (see: ruffordsmallgrants.org/rsg/projects/edward_narayan). viwa island is also free of the invasive small indian mongoose herpestes javanicus. viwa, a 60 ha island situated 900 m east off the coast of fiji’s mainland, tailevu, viti levu, is the smallest of the five islands in fiji with extant populations of the fijian ground frog. the fijian ground frog (fig. 2) is currently listed as endangered (en b1 + 2c) under the current iucn criteria (zug et al., 2004). the fijian ground frog has a small population surviving within remaining forested habitats as well as agricultural sites on viwa. the fijian ground frog mainly faces threats from habitat destruction and predation from nonnative cane toad (rhinella marina). there is no previous report of bd fungus within wild fijian frog populations. furthermore, if this small island frog population is free of bd then this naïve population could be used as a reserve population for future captive breeding programmes without risk of translocating diseased animals into captivity. fig. 2. adult female fijian ground frog (snout-vent length = 75 mm) from viwa (fiji) islands. field sampling was conducted near ponds within, forested landscapes and agricultural areas on viwa island (60 ha island present 900 m east off the coast of mainland viti levu. frogs along 100 m transects (n = 5) within each habitat and 20 frogs per transect were caught. field work was done during the coldest months in fiji (april to july) because of high intensity of bd first reported elsewhere during cold periods (kriger and hero, 2007a). frogs were captured using clean, unused 20 25 cm plastic bags. each caught frog 264 edward narayan, frank molinia and jean-marc hero was sampled for bd by firmly running a cotton swab (medical wire & equipment, mw 100-100; 10 times over each of the following locations: (i) the frog’s dorsal surface; (ii) the frog’s sides, from groin to armpit; (iii) the ventral surface; and (iv) the undersides of the thighs. additionally, five outward strokes of the swab were employed on the undersides of each frog’s feet, for a total of 70 strokes. swabbing was done based on methods of kriger et al. (2006c). swabs were replaced in their original container (a plastic tube), stored on ice in a cooler until return from the field, and frozen at -20°c. to ensure that no frogs are inadvertently sampled twice, sampling of frogs will not commence until all frogs at given section of each transect were caught, and no further sampling took place at that section of transect after frogs were released. swabs were analysed for the presence of bd using quantitative (real-time) polymerase chain reaction techniques (qpcr) described by boyle et al. (2004), and employing the changes described by kriger et al. (2006c). thus, all samples that tested positive in the initial singlicate qpcr assay were re-analysed using a triplicate assay and a full set of bd standards, in order to confirm the initial result and accurately quantify the number of bd zoospores present. results of real time prc analysis of bd swabs from 200 ground frogs were all negative. baseline data for islands, even when negative, is highly valuable. the baseline study such as is described here showed no presence of bd (garcia et al., 2007) until the pathogen emerged on montserrat island in 2009, extirpating the mountain chicken frog (leptodactylus fallax), the largest caribbean amphibian species. in february 2009, dead and dying frogs were found at several sites on montserrat, prompting an evacuation of 40 apparently healthy l. fallax to three different institutions for establishment of a captive breeding program (garcía et al., 2009). several hypotheses could explain the lack of bd in fijian ground frogs on viwa island: 1. that bd fungus has possibly not yet reached or emerged on the fiji islands; 2. that bd fungus has been introduced to fiji but the high temperatures all year round inhibit the likelihood of bd surviving and infecting amphibians (such as thermal tolerance, kriger and hero, 2007a); 3. viwa island is physically isolated from the mainland and bd has not been able to colonise the island; 4. the fgf’s on viwa have been exposed to bd but the entirely terrestrial breeding nature of ground frogs may enable these frogs to reduced exposure and hence susceptibility to this aquatic pathogen. low prevalence has been recorded in some terrestrial breeding species such as microhylids (kriger and hero, 2006a) and the pouched frog (assa darlingtoni) record in kriger and hero (2007b) but not others i.e. the eleutherodactylids in central and south america (beard and o’neill, 2005); 5. the fgf on viwa have been exposed to bd but they have skin defences that prevent bd infection (woodhams et al. 2003; woodhams and alford, 2005). this is unlikely as most species that are resilient still have bd positive individuals within the population – however no clinical sign of disease (parker et al., 2002). many other anuran species are resilient to the pathogen and either do not become susceptible to it or have some level of population-level resistance (e.g. rana catesbeiana, xenopus leavis) [fisher et al. 2009], l. wilcoxi (kriger and hero, 2006b). species that are non-susceptible to bd are presumably either protected by poor abiotic growing conditions for bd (piotrowski et 265absence of chytrid fungus in the fijian ground frog al., 2004), immunological response (ramsey et al., 2010) low virulence of bd strain, or bacterial epibiotic symbionts (harris et al. 2009). furthermore, the pacific islands are within the ecological niche occupied by bd. therefore, it is unlikely that these environments are too hot for bd to persist (lotters et al., 2010). in summary, the results suggests that bd has either not yet emerged on viwa, fiji islands or that some other ecological factor is preventing bd from fully invading fijian ground frogs. more data is needed from frogs living on other islands around fiji to fully assess the threat of bd to native fijian frogs. although bd appears widely distributed throughout australia and new zealand, the evidence from this study suggests that bd has not yet reached amphibian populations in pacific islands. thus far, no positive bd results have been reported from fijian tree frogs and native frogs from neighboring islands such as solomon islands and papua new guinea (dahl et al., unpublished data), but that may change as the disease emerges or as conditions change (e.g., climate change, general biodiversity loss). funds are urgently required to assist long-term amphibian population monitoring throughout pacific islands in order to detect any future amphibian population declines. acknowledgements this field research was co-funded by the rufford small grants foundation (rsg) and the critical ecosystems partnership funds (cepf). references beard, k.h., o’neill, e.m. (2005): infection of an invasive frog eleutherodactylus coqui by the chytrid fungus batrachochytrium dendrobatidis in hawaii. biol. conserv. 126: 591-595. bishop, p.j., speare, r., poulter, r., butler, m., speare, b.j., hyatt, a., olsen, v., haigh, a. (2009): elimination of the amphibian chytrid fungus batrachochytrium dendrobatidis by archey’s frog leiopelma archeyi. dis. aquat. org. 84: 9-15. boyle, d.g., boyle, d.b., olsen, v., morgan, j.a.t., hyatt, a.d. (2004): rapid quantitative detection of chytridiomycosis (batrachochytrium dendrobatidis) in amphibian samples using real-time taqman pcr assay. dis. aquat. org. 60: 141-148. fisher, m.c., garner, t.w.j., walker, s.f. (2009): global emergence of batrachochytrium dendrobatidis and amphibian chytridiomycosis in space, time, and host. ann. rev. microbiol. 63: 291-310. harris, r.n., brucker, r.m., walke, j.b., becker, m.h., schwantes, c.r., et al. (2009): skin microbes on frogs prevent morbidity and mortality caused by a lethal skin fungus. isme j. 3: 818-824. garcía, g., cunningham, a.a., horton, d.l., garner, t.w.j., hyatt, a., hengstberger, s., lopez, j., ogrodowczyk, a., fenton, c., fa., j.e. (2007): mountain chickens lepto266 edward narayan, frank molinia and jean-marc hero dactylus fallax and sympatric amphibians appear to be disease free on montserrat. oryx 41: 398-401. garcía, g., lopez, j., fa, j.e., and gray, g.a.l. (2009): chytrid fungus strikes mountain chickens in montserrat. oryx 43: 323-328. kriger, k.m., hero, j.m. (2007a): large-scale seasonal variation in the prevalence and severity of chytridiomycosis. j. zool. 271: 352-359. kriger, k.m., hero, j-m. (2007b): the chytrid fungus batrachochytrium dendrobatidis is non-randomly distributed across amphibian breeding habitats. divers. distrib. 13: 781-788. kriger k.m., hero, j.m., (2006a): cophixalus ornatus (ornate nursery frog). chytridiomycosis. herpetol. rev. 37: 443. kriger, k.m., hero, j.-m. (2006b): survivorship in wild frogs infected with chytridiomycosis. ecohealth 3: 171-177. kriger, k.m., hines, h.b., hyatt, a.d., boyle, d.g., hero, j-m. (2006c): techniques for detecting chytridiomycosis in wild frogs: comparing histology with real-time taqman pcr. dis. aquat. org. 71: 141-148. lotters, s., kielgast, j., bielby, j., schmidtlein, s., bosch, j., veith, m., walker s.f., fisher, m.c., rodder, d. (2010): the link between rapid enigmatic amphibian decline and the globally emerging chytrid fungus. ecohealth 6: 358-372. morley, c.g. 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(2007): the spread of chytridiomycosis has caused the rapid global decline and extinction of frogs. ecohealth 4: 125-134. woodhams, d.c., alford, r.a. (2005): ecology of chytridiomycosis in rainforest stream frog assemblages of tropical queensland. conserv. biol. 19: 1449-1459. woodhams, d.c., alford, r.a., marantelli, g. (2003): emerging disease of amphibians cured by elevated body temperature. dis. aquat. org. 55: 65-67. zug, g., watling, d., morrison, c. (2004): platymantis vitianus. in: iucn 2010. iucn red list of threatened species. version 2010.4. <www.iucnredlist.org>. downloaded on 15 june 2011. acta herpetologica 17(2): 197-203, 2022 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-13467 comparative cytogenetics on zamenis lineatus and elaphe quatuorlineata (serpentes: colubridae) marcello mezzasalma1,*, elvira brunelli1, gaetano odierna2, fabio m. guarino2 1 department of biology, ecology and earth science, university of calabria, via p. bucci 4/b, 87036 rende, italy 2 department of biology, university of naples federico ii, via cinthia 26, 80126, naples, italy *corresponding author. email: m.mezzasalma@gmail.com submitted on: 2022, 28th july; revised on: 2022, 21st october; accepted on: 2022, 5th november editor: andrea villa abstract. because of their peculiar genomic and chromosomal characteristics, reptiles are extraordinary model organisms to study karyotype and sex chromosome evolution, but despite the growing interest in their evolutionary cytogenetics, only a small fraction of species have a known karyotype. we performed a comparative cytogenetic analysis on elaphe quatuorlineata and zamenis lineatus, using classic and molecular techniques. we provide the karyotype of these two species and an assessment of their chromosomal features. chromosome analysis was performed with standard karyotyping, c-banding, sequential c-banding + cma3 + dapi and ag-nor staining. on e. quatuorlineata, we also performed cma3-methyl green staining and fluorescence in situ hybridization mapping nor loci (nor-fish). elaphe quatuorlineata and z. lineatus show a very similar karyotype of 2n = 36, with 8 macroand 10 microchromosome pairs, but differ in the morphology of the pair 8, which resulted submetacentric in the former and metacentric in the latter species. by comparing our data to those available from the literature on congeneric species, we analysed the occurrence of primitive and derivate chromosomal characters and provide cytotaxonomic insights, which further support the species status of z. lineatus. in both species, the 4th pair was identified as the sex chromosome pair (zz/zw) and nors were localized on a microchromosome pair. we finally highlight in both genera elaphe and zamenis different stages of heterochromatinization of the w chromosome, in agreement with the progressive diversification model of sex chromosome as already shown in different reptile taxa. keywords. chromosome, evolution, karyotype, nors, squamates, snakes. introduction classic cytogenetic through differential staining and banding of chromosomes permits to describe and compare karyotypes, whereas the molecular cytogenetic approach, employing fluorescence in situ hybridization (fish) with specific probes, allows the detection of particular sequences present in genomes (matsuda et al., 2005; dumas and sineo, 2014), including repetitive dna sequences (scardino et al., 2020a). among those repetitive dna elements are the ribosomal dna (rdna), encoding rrna. these elements have been successfully used as markers for comparative cytogenetic studies and phylogenetic analyses. the rdna is organized into 2 families: 5s (minor) and 45s (major) rdna. the latter comprises the genes for 18s, 5.8s, and 28s rrna and is located in the so-called nucleolus organizer regions (nors). the nors can be identified either by silver staining, which detects only transcriptionally active loci, or more accurately, by fish, which permits the identification of both active and inactive nors. the location of the rdna loci in the karyotype may show a species-specific pattern, so rdna loci are often used for complex karyotype characterizations (scardino et al., 2020a). indeed, compara198 marcello mezzasalma et alii tive chromosome analyses can be useful to identify plesiomorphic and apomorphic states and the occurrence of different evolutionary lineages (deakin and ezaz, 2014; damas et al., 2018; scardino et al., 2020b). chromosome rearrangements may precede or follow molecular evolution, directly promoting cladogenesis or resulting from phylogenetic diversification (noor et al., 2001; rieseberg, 2001). in either case, they represent discrete evolutionary markers able to detect different evolutionary trends or apomorphisms in the taxa studied (dobigny et al., 2004; olmo, 2008; dumas et al., 2015). squamate reptiles, due to their peculiar genomic and chromosomal characteristics, are exceptional model organisms in the study of karyotype evolution and sex chromosome diversification of vertebrates (olmo, 2008; alam et al., 2018). squamates display a remarkable variability in chromosome number and morphology, number and location of different chromosome markers and the occurrence of environmental genetic sex determination, with the independent evolution of simple and multiple sex chromosome systems with either male or female heterogamety (olmo, 2008; pallotta et al., 2017; deakin and ezaz, 2019; sidhom et al., 2020; mezzasalma et al., 2021 a). the cytogenetic approach used for the study of chromosome rearrangements and different morphologies and/ or levels of heterochromatinization of the heteromorphic sex chromosomes have been previously used in different phylogenetically closely-related european squamate taxa such as the snakes of the genus hierophis (fitzinger, 1843), anguis fragilis linnaeus, 1758, and a. veronensis pollini, 1818, and geographically distinct populations of coronella austriaca laurenti, 1768 (mezzasalma et al., 2013, 2015, 2018b; mezzasalma and odierna, 2021). however, despite the growing interest in the evolutionary cytogenetics of squamate reptiles, only a small fraction of the described squamate species have a known karyotype (olmo and signorino, 2006; mezzasalma et al., 2021), leaving most of their chromosomal diversity still unexplored. this is also true for some peculiar mediterranean reptile species such as the european four-lined snake elaphe quatuorlineata (bonnaterre, 1790) and the italian aesculapian snake zamenis lineatus (camerano, 1891). in this work, we performed a comparative cytogenetic analysis on e. quatuorlineata and z. lineatus, using a combination of standard staining and banding techniques. we provide the first karyotype description of these two species and an assessment of their chromosomal features. by comparing our data to those available from the literature on phylogenetically closely-related species, we evidence and discuss the occurrence and distribution of primitive and derivate chromosomal characters in the species studied and provide cytotaxonomic insights, which support the species status of z. lineatus. we also highlight that both genera elaphe and zamenis show progressive evolutionary stages of the w chromosome, supporting the heterochromatinization model of sex chromosome diversification (see e.g., mezzasalma et al., 2021). material and methods we analysed two samples (one male and one female) of z. lineatus and one female of e quatuorlineata from piedimonte matese, campania, italy. specimens were anesthetized on ice and, after taking a 0.5 ml of blood aliquot from the caudal vein, they were released in the capture site. chromosomes were obtained from blood cultures following odierna et al. (2004). namely, blood aliquots were incubated for four days at 30 °c in 5 ml of lymphocyte medium culture (3.8 ml of dmem, 0.5 ml sterile distilled water, 0.5 newborn calf serum, 0.1 ml antibiotics, 0.1 ml pha). chromosome harvesting was performed by adding 0.1 ml of colcemid (10 μg/ml) and two hours later the cells were collected by centrifugation (1000 rpm/min), incubated for 30 min in 5 ml of hypotonic solution (kcl 0.075 m) and fixed in methanolacetic liquid (methyl alcohol + acetic acid, 3:1). slides were prepared using the air-drying method, as described in mezzasalma et al. (2019). the cytogenetic analysis was performed with traditional karyotyping (5% giemsa solution at ph 7 for 10 min) and additional chromosome staining and banding techniques; in particular, c-banding was performed following sumner (1972) and sequential c-banding + cma3 + dapi according to sidhom et al. (2020), which highligh cg and at-rich regions, respectively. nucleolus organizing regions (nors) were identified following the ag-nor staining method described by howell and black (1980). given quantity and quality of metaphase plates, on e. quatuorlineata we also performed chromomycin a3-methyl green staining (cma/mg) (a staining method usefulf to hightlight cg-rich chromosome regions) as described by sahar and latt (1980) and fluorescence in situ hybridization (nor-fish) following mezzasalma et al. (2018a), using as probe the pcramplified and biotinylated 18s rrna gene of the gekkonid tarentola mauritanica (linnaeus, 1758). in brief, after denaturation in 70% formamide and 2x ssc for 2 min at 80 °c, slides were incubated overnight at 40 °c with the hybridization mixture (10 ng/ml biotinylated 16 dutp probe 0.1 µm/ml escherichia coli dna in 50% formamide and 2x ssc). after washing in 2x ssc, cytochemical detection was performed using 5 µm/ml fitc-conjugated extravidin (sigma) in 4x ssc + 1% bsa + 0.1% 199karyology of european colubrids tween 20, ph 7. after washing three times in 4x ssc and 0.1% tween 20 for 10 min at 42 °c, the detection of fish signals was performed with extravidin fitc (sigma aldrich) counterstained with propidium iodide (pi) (200 ng/ml) in 2x ssc, ph 7, for 2 min at room temperature. metaphase plates were scored and recorded with an optical and an epifluorescent microscope (axioscope zeiss) equipped with an image analysis system. karyotype reconstruction was performed after scoring at least five metaphase plates from each sample studied and chromosomes were classified according to levan et al. (1964). results the karyotypes of e. quatuorlineata and z. lineatus are both composed of 2n = 36 chromosomes, with 8 macrochromosome pairs and 10 microchromosome pairs (fig. 1a, 2a). the two species also show the same chromosome morphology with the exception of the chromosome pair 8, which resulted submetacentric in e quatorlineata and metacentric in z. lineatus (table 1, fig. 2a). arm number (an) resulted = 50 in both colubrids. morphometric parameters of each macrochromosome pair of both species studied are reported in table 1. c-banding and ag-nor revealed in both species the occurrence of nor loci on a microchromosome pair, as confirmed by nor-fish in e. quatuorlineata (fig. 1b-d). c-banding showed heterochromatin content on autosomes, mostly concerning telomeric and centromeric regions in both e. quatuorlineata (fig. 1e-g) and z. lineatus (fig. 2b-d). furthermore, in the female samples of both species, one element of the 4th macrochromosome pair resulted to be largely heterochromatic, allowing us to identify this pair as a homomorphic zw sex chromosome pair (fig. 1e-g, 2b-d). this w chromosome resulted highly positive with both cma3 and dapi in e. quatuorlineata (fig. 1e-g), whereas it was clearly evident with dapi and less evident with cma3 in z. lineatus (fig. 2b-d). fig. 1. karyotype and metaphase plates of e. quatuorlineata stained with giemsa (a), ag-nor (b), cma3/mg (c), nor-fish (d), c-banding + giemsa (e), + cma3 (f), + dapi (g). * = loci of nors. fig. 2. karyotype and metaphase plates of z. lineatus stained with giemsa (a), ag-nor (b), c-banding + cma3 (c), + dapi (d). * = loci of nors. table 1. chromosome morphometric parameters. chr. = chromosome number; rl = relative length (chromosome length/total karyotype length*100); ci = centromeric index (short arm length/ chromosome length*100); m = metacentric; sm = submetacentric; t = telocentric. chr. elaphe quatuorlineata zamenis lineatus rl ci rl ci 1 19.4 ± 0.8 44.9 ± 3.4 (m) 19.3 ± 1.0 48.4 ± 3.4 (m) 2 16.5 ± 0.5 34.8 ± 4.0 (sm) 16.1 ± 0.7 35.2 ± 4.0 (sm) 3 11.1 ± 0.5 48.4± 3.8 (m) 10.8 ± 0.4 43.2 ± 3,8 (m) 4(z) 7.7 ± 0.6 45.9 ± 2.8 (m) 8.3 ± 0.6 45.4 ± 2.8 (m) 4(w) 7.6 ± 0.4 45.1 ± 3.4 (m) 8.4 ± 0,3 44.9 ± 3.4 (m) 5 7.5 ± 0.7 48.3 ± 4.3 (m) 7.9 ± 0.5 42.8 ± 4.3 (m) 6 6.0 ± 0.4 0.0 ± 3.0 (t) 7.1 ± 0.6 0.0 ± 3.0 (t) 7 5.6 ± 0.7 36.0 ± 3.1 (sm) 6.3 ± 0.5 36.3 ± 3.1(sm) 8 5.3 ± 0.4 36.9 ± 3.3 (sm) 4.4 ± 0.6 40.1 ± 3.3 (m) 9-18 20.6 ± 1.1 19.8 ± 1.3 200 marcello mezzasalma et alii discussion our results show that the karyotypes of e. quatuorlineata and z. lineatus have the same diploid number (2n = 36) and a similar general structure, but a different morphology of chromosome pair 8. in order to highlight the occurrence of simplesiomorphic, sinapomorphic and apomorphic states and add data for the reconstruction of the chromosomal evolution in the genera elaphe and zamenis, we compared the newly generated karyotypes to those available from the literature on congeneric species as well as with that of the hypothesized ancestral snake karyotype (ask) (see kobel, 1967; bianchi et al., 1969; singh, 1972; itoh et al., 1970; de smet, 1978; augstenová et al., 2017; rovatsos et al., 2018; cole and hardy, 2019) (fig. 3). this comparison permits to show that e. quatuorlineata and z. lineatus, as well as most congeneric species with a known karyotype, have different chromosomal characters which are considered simplesiomorphisms and found in the hypothesized ask (see kobel, 1967; bianchi et al., 1969; singh, 1972; itoh et al., 1970; de smet, 1978; augstenová et al., 2017; rovatsos et al., 2018; cole and hardy, 2019; this paper). these shared ancestral characters include: diploid number, number of macroand microchromosome pairs, the general morphology of several macrochromosome pairs and the localization of nor loci on a microchromosome pair (see also cole and hardy, 2019). all this evidence permits to confirm that elaphe and zamenis are karyologically very conservative, but for the morphology and sequence content of the w chromosome, which are variable among different taxa (see also augstenová et al., 2017; cole and hardy, 2019; mezzasalma and odierna, 2021). nevertheless, the karyotypes of e. quatuorlineata and z. lineatus also possess some peculiar derivate features, which characterize their respective karyotype from those of phylogenetically related species. in elaphe, autosomal fig. 3. original karyograms of z. lineatus and e. quatuorlineata compared with the ancestral snake karyotype (ask) and available literature data on congeneric species (kobel, 1967; de smet, 1978; itoh et al., 1970; augstenová et al., 2017; rovatsos et al., 2018; cole and hardy, 2019; mezzasalma and odierna, 2021). sc = secondary constriction, (i) = chromosome inversion. red chromosomes = nor-bearing pair. black regions/chromosomes = heterochromatin. red arrows indicate progressive steps of sex chromosome diversification. 201karyology of european colubrids rearrangements from the hypothesized ancestral snake karyotype involve a putative inversion of the 8th pair in e. quatuorlineata and in e. bimaculata that can be considered a sinapomorphism, and in the second species, also an inversion of the 7th pair, as previously showed (see fig. 3) (itoh et al., 1970; rovatsos et al., 2018), which can be considered an apomorphism. furthermore a translocation of loci of nors on the 2nd chromosome pair in e. climacophora and e. quadrivirgata, evidenced by a secondary constriction (see itoh et al., 1970), represent a further apomorphism, which is not present in the species here analyzed. zamenis lineatus shows a metacentric 8th chromosome pair, which probably originated by means of a pericentromeric inversion as previously showed also in z. situla (augstenová et al. 2017), thus representing a sinapomorphism linking the two species. it should also be noted that in the previously described karyotypes of z. longissimus (kobel 1967; de smet, 1978), a different macrochromosome number (8 and 9, respectively) is reported, without any changes in the total chromosome count (2n = 36). the additional macrochromosome pair reported by de smet (1978) is probably due to the amplification of nor-linked heterocromatin of the nor microchromosomes bearing pair, but more focused analyses are needed to confirm the occurrence of intraspecific chromosomal variability in z. longissimus. progressive steps of the configuration of the heterogametic w chromosome are important events in reptiles and are clearly visible in many species, supporting the general heterochromatinization hypothesis of sex chromosome diversification (augstenová et al., 2017; alam et al., 2018; cole and hardy 2019; mezzasalma et al., 2020). in fact, it is widely accepted that heteromorphic sex chromosome pairs begin their morphological and molecular diversification starting from a homomorphic state (gamble et al., 2014; mezzasalma et al., 2021). from this condition, two alternative pathways are known to potentially lead to a fully differentiated sex chromosome pair: a progressive heterochromatinization of the heterogametic chromosome or the insurgence of an inversion in the homomorphic proto-w chromosome (wright et al., 2016; natri et al., 2019; mezzasalma et al. 2021). in either cases, the progressive diversification of the w element eventually leads to its evolutionary isolation (loss of recombination) and degeneration, finally reaching the size of a microchromosome (marshall graves, 2016; mezzasalma et al., 2016; wright et al., 2016). progressive steps of the configuration of the heterogametic w chromosome are visible in the species here analysed and in the phylogenetically closely-related taxa elaphe and zamenis (fig. 3) (see also kobel, 1967; de smet, 1978; itoh et al. 1970; augstenová et al. 2017; rovatsos et al., 2018; mezzasalma and odierna 2021). in particular, the w chromosome appears at a relatively earlier stage of diversification in e. quatuorlineata, in which it resulted largely heterochromatic, but homomorphic to the z (this paper). more advanced diversification stages are represented by the w elements of e. bimaculata and e. climacophora, in which the morphology of the w chromosomes progressively diverged from the z, reaching a telocentric configuration in e. quadrivirgata (fig. 3) (see also itoh et al., 1970; rovatsos et al., 2018). the w chromosome is homomorphic but largely heterochomatic in z. lineatus, representing an initial diversification step from the z element (this paper). a progressive addition of heterochromatin may produce a heterogametic chromosome, which appears sensibily larger than the z: a condition similar to that reported in z. situla (augstenová et al., 2017) (fig. 3). furthermore, it is possible to highlight that the differences in the morphology of the w chromosome and of the 8th and 9th chromosome pairs found between z. lineatus and z. longissimus (kobel 1967; de smet, 1978; this paper) are in agreement with their species status, originally proposed using a combination of morphological and molecular data (lenk and wüster, 1999; utiger et al., 2002). this evidence underlines that in squamate reptiles the cytotaxonomic approach is a useful tool for characterizing closely-related lineages as already shown in other squamate taxa (mezzasalma et al., 2013, 2015, 2018b; mezzasalma and odierna, 2021). acknowledgements sampling was carried out under the authorization of the 01/06/2000 n. scn/2d/2000/9213 from the italian ministry of environment. references alam, s.m., sarre, s.d., gleeson, d., georges, a., ezaz, t. 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(2016): how to make a sex chromosome. nat. commun. 7: 12087. acta herpetologica vol. 17, n. 2 december 2022 firenze university press cryptic diversity in pygmy chameleons (chamaeleonidae: rhampholeon) of the eastern arc mountains of tanzania, with description of six new species michele menegon1,2,*, john v. lyakurwa3,4, simon p. loader5, krystal a. tolley6,7 preliminary genetic characterisation of southern smooth snake coronella girondica (serpentes, colubridae) populations in italy, with some considerations on their alpine distribution matteo r. di nicola1, raffaella melfi2, francesco p. faraone3,*, daniel l. n. iversen4, gabriele giacalone5, giovanni paolino1, mario lo valvo6 species diversity and distribution of amphibians and reptiles in sardinia, italy claudia corti1,2,*, marta biaggini1, valeria nulchis2, roberto cogoni2, ilaria maria cossu2, salvatore frau4, manuela mulargia2, enrico lunghi2, lara bassu2. the italian wall lizard, podarcis siculus campestris, unexpected presence on gorgona island (tuscan archipelago) marco a.l. zuffi1,*, alan j. coladonato2, gianluca lombardo3, antonio torroni3, matilde boschetti1, stefano scali4, marco mangiacotti2, roberto sacchi2 molecular analysis of recently introduced populations of the italian wall lizard (podarcis siculus) oleksandra oskyrko1,2,*, lekshmi b. sreelatha1,12,13, iolanda silva-rocha1, tibor sos3,4, sabina e. vlad5,6,7, dan cogălniceanu5,6, florina stănescu6,7,8, tavakkul m. iskenderov9, igor v. doronin10, duje lisičić11, miguel a. carretero1,12,13 sunny-side up: ontogenetic variation in egg mass temperatures of the wood frog rana sylvatica ryan calsbeek*, ava calsbeek, isabel calsbeek ecological niche differentiation in the anatolian rock lizards (genus: anatololacerta) (reptilia: lacertidae) of the anatolian peninsula and aegean islands mehmet kürşat şahin1,*, kamil candan2,3, danae karakasi4, petros lymberakis4, nikos poulakakis4,5,6, yusuf kumlutaş2,3, elif yıldırım2,3, çetin ilgaz2,3 occupancy and probability of detection of the introduced population of eleutherodactylus coqui in turrialba, costa rica jimmy barrantes-madrigal1,*, manuel spínola parallada1, gilbert alvarado 2, víctor j. acostachaves3,4. one site, three species, three stories: syntopy of geckoes euleptes europaea (gené, 1839), hemidactylus turcicus (linnaeus, 1758), tarentola mauritanica (linnaeus, 1758) in a coastal area of southern tuscany (central italy) giacomo radi1,2, marco a.l. zuffi1,* comparative cytogenetics on zamenis lineatus and elaphe quatuorlineata (serpentes: colubridae) marcello mezzasalma1,* , elvira brunelli1, gaetano odierna2, fabio m. guarino2 chemical skin defence in the eastern fire-bellied toad bombina orientalis: an ultrastructural approach to the mechanism of poison gland rehabilitation after discharge sara quagliata1, cecilia malentacchi2, filippo giachi1, giovanni delfino1* 1dipartimento di biologia evoluzionistica dell’università, via romana 17 i-50125 firenze, italy. *corresponding author. e-mail: giovanni.delfino@unifi.it 2dipartimento di fisiopatologia clinica dell’università, viale pieraccini 6 i-50139 firenze, italy submitted on 2008, 17th march; revised on 2008, 25th july; accepted on 2008, 26th august. abstract. type i serous glands in the skin of the eastern yellow-bellied toad bombina orientalis released their product massively after 10-3 m nor-adrenalin (na) stimulation, mimicking orthosympathetic control on poison emission in chemical skin defence. features of cutaneous glands involved in this bulk discharge were observed under light and electron microscopes. furthermore, restoration of depleted glands was followed after 1, 2 and 3 weeks, and compared with serous biosynthesis during larval gland development. bulk discharge was caused by contraction of myoepithelial cells (mecs) encircling the secretory units. mec compression dramatically affected the secretory unit, but parts of this syncytial cytoplasm were saved from degeneration and cooperated in gland renewal with stem cells from the gland neck. these adenoblasts underwent proliferation and secretory cytodifferentiation, until merging with the syncytium. cytoplasm that had resumed secretory activity showed the features typical of larval gland development: the endoplasmic reticulum (rer) cisterns were aligned in close parallel arrangement and golgi stacks released minute type i granules. secretory rehabilitation led to increasing amounts of granule content. in the meantime, rough cisterns decreased in number and assumed the less ordered pattern described in control specimens. data collected in the present study revealed that chemical skin defence in anurans is a multi-factorial mechanism involving specific activities: mechanical from mecs, biosynthetic from secretory syncytium and proliferative from intercalated stem cells. keywords. serous glands, anuran skin, secretory release, ultrastructure. introduction pioneering light microscope (lm) studies on the serous (poison or granulous) cutaneous glands in yellow bellied toads of the genus bombina (formerly bombinator) provided acta herpetologica 3(2): 139-153, 2008 issn 1827-9643 (online) © 2008 firenze university press 140 s. quagliata et alii evidence on the features of their serous product and the syncytial structure of their secretory units (faraggiana, 1937, 1939; bertossi, 1937). the secretory product consists either of small (average diameter 2 μm) or large (10 μm) granules contained in the secretory syncytium of type i or, respectively, ii glands. these glands perform a functional cycles consistent with a defence role (faraggiana, 1939; delfino, 1978, 1980): smooth muscle cells (myoepithelial cells or mecs) ensheathing the secretory unit contract, causing granules to be discharged towards the skin surface through the intraepidermal duct. since secretory discharge involves both granules and syncytial cytoplasm, it is referred to as holocriny (faraggiana, 1938b). serous gland holocriny requires a turnover mechanism based on proliferation and secretory differentiation of the stem cell pool in the “neck” (faraggiana, 1939), a regenerative region intercalated between the secretory unit and duct. investigations into several frog and toad species revealed that this renewal mechanism is a shared functional trait within the anuran order (faraggiana, 1938a, 1939). lm and transmission electron microscope (tem) investigations subsequently confirmed these results, while providing ultrastructural details of the cytodifferentiation processes of intercalated tract cells (delfino, 1978, 1980). furthermore, tem analysis revealed that the manufacture of serous product does not actually involve any degenerative processes –the damage observed merely derives from not-adequate fixation methods and/or compression by mecs. therefore, the concept of “bulk discharge” has been introduced to describe the main functional trait of these glands: secretory release from anuran poison glands is a massive process affecting secretory granules as well as the syncytium storing them (delfino et al., 1996). bulk discharge has been investigated in several hylid species, with observations extending to the serous product collected after release; this material contained nuclei and complements of the rough endoplasmic reticulum (rer), along with integral secretory granules (melis et al., 2000; delfino et al., 2002, 2006; nosi et al., 2002), thus mec compression, whilst indeed damaging the syncytial cytoplasm, left the secretory product intact. whereas the peculiar features of secretory discharge from anuran cutaneous serous glands is widely confirmed in current literature, further investigation is required to clarify their restoration mechanism. indeed, along with stem cells, secretory rehabilitation may also involve residual portions of the pre-existing syncytium (neuwirth et al., 1979; delfino, 1980). to evaluate how the secretory syncytium and stem cells contribute to serous gland restoration, we carried out an experimental study on bombina orientalis, following the early, intermediate and ultimate steps of the renewing process. our research, which also involves a comparison between adult glands during restoration and larval glands during development, aims to complete previous studies on chemical skin defence in this species (delfino et al., 1990; sanna et al., 1993). materials and methods specimens and pharmacological treatment we procured adult specimens (8) and tadpoles (12 specimens, ontogenetic range 34-43, according to gosner, 1960) of the eastern red-bellied toad bombina orientalis (boulenger, 1890) from authorized dealers and acclimatised them for three weeks to laboratory condi141ultrastructure of poison glands in bombina orientalis tions in the dipartimento di biologia evoluzionistica (università di firenze, italy). during this period adults were fed on meal worms (larval tenebrio molitor) and kept in 20 °c water under a natural light cycle. larval specimens were fed on boiled spinach. before pharmacological treatment and/or sacrifice, adult and larval specimens were kept at 4 °c to reduce response to external stimulation. this procedure, which avoided use of anaesthetics in pharmacological tests, minimised stress and pain in the animals during manipulation. to observe control glands, and glands immediately after stimulation, we removed twelve skin strips of small surface area (25-36 mm2) from the backs of two toads (killed with 0.2% chlorobutanol) and pooled them together. half of the pooled samples were immersed in amphibian ringer containing 10-3 m nor-adrenalin (na), and their responses checked under a stereomicroscope (slm, delfino, 1980), until secretory discharge ceased (usually, within 15-20 min at room temperature). the control skin samples (six) from the same pool were immersed in the ringer solution, observed with the slm for 20 min, and processed in the same manner as the experimental and larval strips for microscopic observation (see below). skin strips collected from larval specimens were 16 mm2 wide. to follow gland regeneration, we injected 10-3 m na (1 ml) into the dorsal lymph sacs of 6 animals and photographed their responses to the pharmacological treatment with a coolpix 4500 digital camera. after treatment, we kept the toads under the same acclimatization conditions described above for one, two and three weeks. two specimens were sacrificed at the end of each interval, and 25-36 mm2 skin strips (4 each toad) removed from dorsal areas 5 mm away from the injection site. preparation for microscopic analysis skin strips from experimental (0, 1, 2, 3 weeks) and control specimens as well as tadpoles, were treated for prefixation (3 h, 4 °c) with a glutaraldehyde-paraformaldehyde mixture in 0.1 m, ph 7.0 cacodylate (karnovsky, 1965), and then washed in this buffer solution. the skin samples were then reduced into 4 mm2 surface area strips, and postfixed (1.5 h) in oso4 (1% in cacodylate). after rinsing in the same buffer, the samples were dehydrated in graded ethanol, soaked in propylene oxide and infiltrated in epon 812. after 72 h polymerisation, epon blocks were cut with a nova lkb ultramicrotome into 1-1.5 µm semithin sections and yellow, interference colour ultrathin sections. semithin sections were stained with buffered (1% borax) toluidine blue, and lm informative pictures were collected with the coolpix camera adapted to a leitz dialux lm. ultrathin sections, collected on 300 mesh copper grids, were electron-dense stained with a saturated, hydroalcoholic solution of uranyl acetate, followed by a lead citrate alkaline solution (2 mg/ml). tem observations were performed at 80 kv with a philips m300 transmission electron microscope (laboratorio di botanica generale, università di firenze). results macroscopic (slm) observations after na injection, the toads assumed a resting posture or only moved slowly, but in no case displayed the typical unkenreflex. within five minutes, a foamy product was dis142 s. quagliata et alii charged onto the skin surface and was later detected on the floor of the pot in which toads had placed. the serous product had obviously condensed on this substrate and formed yellowish bloblets (fig. 1). secretory discharge ceased within twenty minutes. microscopic analysis since consistent findings revealed that nor-adrenalin stimulation only affected serous type i glands, type ii and mucous glands are described only for general comparison. the serous cutaneous glands in control animals exhibited the usual lm features, including the spheroid (fig. 2a) or ellipsoidal shape of the syncytial secretory unit (fig. 2b) with a peripheral row of nuclei and thin sheath of myoepithelial cells. mucous glands consisted of discrete, secretory cells in various phases of activity, as suggested by the varying height and density of their cytoplasms (fig. 2a). type i and ii serous secretory units contained remarkable amounts of granules that occupied the syncytial compartment almost entirely, whereas the lumen was extremely reduced or totally missing (fig. 2a, b). ultrastructural analysis of type i serous units revealed scanty and slender rer cisterns and small golgi stacks at the boundary between myoepithelium and secretory syncytium, along with rod-shaped mitochondria (fig. 2c). myoepithelial cells were spindle-like in shape and contained a myofilament apparatus with homogeneous density (fig. 2d). thin nerve endings appeared in the interstice between contractile and secretory compartments: they held dense-cored synaptic vesicles and established neuro-contractile contacts with myoepithelial cells (fig. 2d). this association between axonal endings and myoepithelial cells represents the neuro-contractile apparatus responsible for regulating secretory discharge from serous glands in anuran skin defence responses. type i serous glands in skin specimens fixed immediately after pharmacological treatment showed the typical histological features of myoepithelium contraction and secretory fig. 1. (slm) secretory product released after na injection. 143ultrastructure of poison glands in bombina orientalis release. these morphological traits included thickened myoepithelial sheaths (fig. 3a, b), while secretory discharge patterns ranged from fully depleted glands (fig. 3a) to others still containing secretory products (fig. 3b). totally discharged glands resembled empty ice-pouches in shape, with remarkably thickened mecs around an extremely reduced cavity (fig. 3a). this hollow is not a lumen but a residue of the compartment that in control glands once held the secretory unit cytoplasm. indeed, it contained sparse remnants of the syncytium, including degenerating nuclei. glands with residual granules still somewhat resembled the control glands, although the syncytium nuclei were crowded centrally in the secretory unit (fig. 3b). compared with type i glands, type ii and mucous glands were not affected by pharmacological treatments and closely resembled their control counterparts (fig. 3c, d). tem observation of type i glands disclosed in detail ultrastructural features brought about by pharmacological stimulation. in smooth muscle cells the peripheral cytoplasm bulged locally towards the secretory compartment (fig. 4a) and fig. 2. control specimen (lm: a, b and tem: c, d): a) contiguous type i and mucous glands, characterised by small secretory granules in the syncityum and variable staining intensity of mucocytes, respecitvely. the polygonal profiles (arrowheads) are mucous cells in tangential section. b) type ii gland with large granules of varying densities. c) biosynthesis apparatus and mitochondria in type i secretory syncytium. d) same as above: nerve ending in the interstice between myoepithelium and secretory syncytium. notice synaptic vesicles and neurotubules (large and thin arrows, respectively). dc = duct, ds = desmosome, g = golgi stack, i = interstice, m = mucous gland, mec = myoepithelial cell, n = gland neck, rer = rough endoplasmic reticulum. 144 s. quagliata et alii there were comb-like profiles on the, opposite, dermal side (fig. 4b). the bulges had a transparent sarcoplasm (fig. 4a) since myofilaments were concentrated in the inner cell regions, forming thick bands (fig. 4a, b). in glands that were not fully depleted, remants of syncytium contained closed membrane profiles, which were never observed in control specimens. the cytoplasm at the boundary with the contractile cells held small vesicular structures (diameter range: 0.2-1 μm) with a light inner compartment (fig. 4a), whereas in the central regions of the residual syncytium larger vesicles (2-6 μm) were prominent, with a denser content that resembled the cytoplasm background (fig. 4c). in the first week after treatment, type i glands were engaged in early phases of renewal activity, that involved both stem cells in the gland neck (intercalated tract) and residual portions of the secretory syncytium. as revealed by lm analysis, undifferentiated cells in the intercalated tract underwent mitotic processes and migrated toward the secretory compartment (fig. 5a). under the tem, their light background cytoplasm, clearly distinguishable from the denser cytoplasma of the contiguous syncytium (fig. 5b), was rich in free ribosfig. 3. treated specimens immediately after na injection (lm). a) and b) type i glands which underwent full and partial depletion, respectively. notice thickened mecs (opposite arrowheads) and cytoplasm waste (forked arrow) in a, residual granules (arrows) and central nuclei in b. c) and d) type ii gland and mucous gland, respectively, resembling control specimens (compare with 2a and b). notice in d variable secretory features in mucocytes. 145ultrastructure of poison glands in bombina orientalis omes and contained peculiar mitochondria that were ring-shaped in section and possessed tubular christae (fig. 5c). before undergoing secretory cytodifferentiation and merging into the syncytium, the adenoblasts derived from stem cell proliferation, could still be recognised on account of their high nucleo-plasmatic ratio and structureless cytoplasm (fig. 5d). the still thick myoepithelium encircled the residual secretory syncytium, that had resumed an obvious functional polarization, as suggested by the peripheral row of nuclei in semithin sections (fig. 6a). in glands undergoing renewal, the peripheral cytoplasm of the secretory unit was denser and contained minute opaque granules, whereas the inner cytoplasm was lighter with typical type i granules (fig. 6a, b). although relative amounts of inner granules and peripheral cytoplasm might vary, depending on the level of the section and/or phase of gland restoration, the centripetal, functional polarization of the secretory unit was a consistent feature. the biosynthesis machinery included flat cisterns of rough endoplasmic reticulum (rer), arranged in a closely parallel pattern and interspersed among secretory granules (fig. 6c). minute stacks of golgi saccules (dictyosomes) were also observed, with flat saccules of the trans face involved in condensing tiny secretory granules (fig. 6d). mitochondria were abundant in this phase, and were often ring shaped in section (fig. 6e), closely resembling those described in proliferating neck cells. after two weeks, an increase of type i product throughout the secretory syncytium was detected under the lm, and suggested that serous gland rehabilitation had reached intermediate steps. the degree of granule accumulation varied considerably: they were relatively dispersed when newly synthesized (fig. 7a) or occupied the entire syncytium when new and pre-existing products coexisted (fig. 7b). single centrioles in the syncytial cytoplasm were sometimes observed under the tem (fig. 7c), as possible remnants of the mitotic processes described in the first week. the close parallel orientation of rough cisterns was virtually lost (fig. 7d), whereas granulogenesis still proceeded in golgi areas fig. 4. same as above (tem). ultrastructural patterns of type i secretory syncytia and myoepithelia. a) mec cytoplasm form electron transparent bulges towards the secretory syncytium (arrowheads), where two distinctive regions are obvious: an outer one with minute vesicles, and an inner one with a moderately dense background. b) comb-like mec profile on the dermal side, and thick bands of myofilaments (arrows). c) detail of the boundary zones between the two syncytium regions, notice wide and minute vesicular profiles (large and small arrows, respectively). 146 s. quagliata et alii (fig. 7e). when glands in early and intermediate steps of renewal activity were compared with larval glands in pre-metamorphic stages), rer and golgi apparatuses exhibited similar features (fig. 7f, g) despite the different conditions that drove the processes, experimental vs. ontogenetic. as a slight difference, rer profiles in developing glands included small rough membrane vesicles and moniliform complements (fig. 7f). as observed under lm, secretory product accumulation proceeded in the final stage of gland renewal, at the third week after discharge, which involved both fully depleted glands (fig. 8a) and others that underwent partial depletion (fig. 8b). as a rule, newly formed granules were larger than in the early steps of functional rehabilitation, possibly due to reciprocal, serial confluences (fig. 8a, b). in both cases, the myoepithelia were relaxed, although some glands still maintained an irregular shape (fig. 8a). ultrastructural changes in the biosynthesis apparatus led these glands to closely resemble control glands in ultrastructural features of their secretory units. there were fewer rer cisterns which were located at the boundary between secretory unit and myoepithelium. most rough profiles were found in the perinuclear cytoplasm, approximately parallel to the secretory-contractile interface (fig. 8c, d). the golgi apparatus maintained its supra-nuclear location and was involved in the usual condensation activity (fig. 8c), producing granules that underwent serial merging processes (fig. 8e). fig. 5. early steps of gland renewal a week after na injection (lm: a and tem: b, c, d). a) mitotic processes (arrows) involving cells from neck region. b) detail of the previous gland, compare the secretory syncytium (right) with cell involved in mitosis (left). c) peculiar mitochondria in this cell, compare with 6e. d) cell originated by mitotic process (opposite arrowheads) within the syncytium. 147ultrastructure of poison glands in bombina orientalis discussion chemical skin defence in extant anurans is a complex survival device, based on biosynthesis, storage and release of noxious (toxic/repellent) molecules, and dependent on an efficient turnover activity. serous glands provide all these functions on account of their complex morpho-functional architecture, including the syncytial secretory unit (poison production and accumulation), neuro-muscular apparatus (secretory discharge) and intercalated tract (regenerative activity). our results confirm previous studies on the release mechanism (holmes et al., 1977; holmes and balls, 1978; delfino et al., 1982): poison discharge from anuran skin glands is an adrenergic mechanism, i.e., mediated by the orthosympathetic nervous system. this is also suggested by the occurrence of dense-cored synaptic vesicles in effectory nerve endings (dockray and hopkins, 1975; delfino, 1979; delfino et al., 1990; delfino, 1991; delfino et al., 1992, 1995a, b, 1998a, b, 1999a, b; melis et al., 2000; arifulova et al., 2007), complemented by hystochemical data (sjoberg and flock, 1976), and therefore it pertains to the repertory of fight or flight responses. studies on the yellow bellied toads of the genus bombina suggest that this mechanism is highly discriminatory since it only involves type i glands, whereas electric stimulation is effective on both serous gland types (faraggiana, 1939; delfino, 1978, 1980). pharmacological reproduction of the orthosympathetic mechanism in living bombina specimens allowed selective stimulation of type fig. 6. same as above (lm: a, b, tem: c, d, e). a ) and b) renewing glands with different amounts of pre-existing secretory material (large arrows), along with newly synthesized granules (small arrows). notice thickened myoepithelial cells (opposite arrowheads). c) rer cisterns arranged in parallel; opposite arrowheads point to perigranular compartment enlargements. d) granulogenesis patterns in golgian area. e) peculiar mitochondria in restored secretory syncytium: compare with 5c. g = golgi stack. 148 s. quagliata et alii i serous glands (delfino, 1980; delfino et al., 1982), and collection of adequate amounts of poison to assay its biological properties (barberio et al., 1987; mastromei et al., 1991; balboni et al., 1992; sanna et al., 1993). the na treatments could be repeated after some weeks on account of the regenerative processes we have described in this study. secretory unit rehabilitation requires coordinated activities in the intercalated tract and residual syncytium. stem cells proliferate and merge together in the common cytofig. 7. intermediate steps of gland renewal two weeks after na injection (lm: a , b and tem: c, d, e) and development of larval glands (stages 36-38, tem: f, g). a), b) these type i glands have been sectioned along the larger longitudinal diameter as demonstrated by occurrence of gland neck in both. the gland on the left was completely depleted: it holds newly formed secretory products (arrows) throughout the syncytial cytoplasm. in the gland on the right, old and new granules co-exist (large and small arrows respectively). notice thick myoepithelia in type i glands (opposite arrowheads), and glands of the mucous and type ii, serous type (right part of b) resembling control specimens. c) centriole in the secretory syncytium, possibly residual from mitotic processes. d) these rer cisterns display a less ordered arrangement compared with 6c; arrowhead points to an area enlarged in e. e) detail of d, showing a golgi stack. f, g) during ontogenesis, rer and golgi apparatus patterns exhibit features resembling those of gland renewal. g = golgi stack, m = mucous gland, n = neck. 149ultrastructure of poison glands in bombina orientalis plasm, as confirmed by the occurrence of similar mitochondria with exclusive features in both discrete cells and the syncytium. three-dimensionally, these organelles are tube or cup-shaped, a morphological adaptation which increases the surface area involved in exchange between the cytoplasm and inner compartments. the proliferation-differentiation-merging sequence we observed repeats the same phases described during ontogenesis, when adenoblasts from the presumptive gland neck contribute to the gradual increase in size of the secretory unit (delfino et al., 1988, 1993, 1994, 2001a; terreni et al., 2003). since this sequence has also been described in single neck cells under resting conditions (delfino et al., 1990, 1992), it appears to be a constitutive process of anuran serous glands. early steps in functional rehabilitation involve enhanced biosynthesis processes as suggested by numerous rer cisterns with the typical parallel arrangement already described in type i serous gland buds of bombina pachypus (delfino, 1977a, b). three weeks after stimulation, the rer machinery had virtually resumed the traits described in control glands (delfino et al., 1990), with scanty, peripheral cisterns. confirming previous studies (delfino, 1980; melis et al., 2000; delfino et al., 2006), stimulated mecs resemble smooth muscle fibres undergoing contraction (wagenwoort and dingemans, 1985; kargacin and fay, 1987). their compression caused substantial damage to the syncytium, similar to the effects of manual squeezing (toledo et al., 1992). despite these degenerative changes, which also involved loss of the plasmalemma encirfig. 8. treated specimen three weeks after na injection (lm: a, b and tem: c, d, e). a) and b) notice increased amounts of secretory granules in these glands that correspond to glands in 7a and b. serous products include newly formed granules involved in reciprocal merging processes (small arrows) and pre-existing granules (large arrow). myoepithelial cells (opposite arrowheads) appear to be relaxed. c) rer cisterns are scanty and reduced to the periphery of the syncytium; notice a golgi stack. d) rer profiles beneath the nuclear level. e) merging between newly formed granules (arrowheads). g = golgi stack, n = neck. 150 s. quagliata et alii cling the exiguous lumen, the residual secretory syncytium resumed its activity. it should be recalled that secretory granules are provided with limiting membranes, as emphasised by the distinctive halos encircling them (delfino et al., 2001b). furthermore, these perigranular compartments are constantly involved in reciprocal merging processes (delfino, 1991), resulting in a somewhat continuous, membrane bounded compartment extending throughout the syncytial cytoplasm. when mecs force secretory granules towards the duct lumen, a continuation of the external environment, part of this inner membrane system is maintained, and guarantees isolation of the cytoplasm compartment. the occurrence of peculiar vesicular profiles in residual syncytia suggests that membrane patches may fuse together, resembling an unusual, short-cut secretory process (neuwirth et al., 1979). on the other hand, membrane fusion may contribute to restoring the plasmalemma, when it occurs at the boundary with the external environment. the ultrastructural evidence collected in this experimental study on b. orientalis serous glands stresses their functional traits, coherent with chemical skin defence against predators. although mec contraction seems to be an all-or-nothing event, poison discharge from a relatively wide skin area is modulated by several factors, both exogenous and endogenous 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(1938a): ricerche istologiche sulle ghiandole cutanee granulose degli anfibi anuri. i. bufo vulgaris e bufo viridis. archo ital. anat. embriol. 39: 327-376. faraggiana, r. (1938b): la struttura sinciziale e il meccanismo di secrezione delle ghiandole cutanee granulose di anfibi anuri. monit. zool. ital. 49: 105-108. faraggiana, r. (1939): ricerche istologiche sulle ghiandole cutanee granulose degli anfibi anuri. ii. rana esculenta, rana agilis e bombinator pachypus. archo ital. anat. embriol. 41: 390-410. gosner, k.l. (1960): a simplified table for staging anuran embryos and larvae with notes of identification. herpetologica 16: 183-190. holmes, c., balls, m. (1978): in vitro studies on the control of myoepithelial cell contraction in the granular glands of xenopus laevis skin. gen. comp. endocrinol. 36: 255-263. holmes, c.h., moondi, p.s., rao, r.r., balls, m. (1977): in vitro studies on the effects on granular gland secretion in xenopus laevis skin of stimulation and blockade of and adrenoceptors of myoepithelial cells. cell biol. int. rep. 1: 263-270. kargacin, g.j., fay, f.s. (1987): physiological and structural properties of saponin-skinned single smooth muscle cells. j. gen. physiol. 90: 49-73. karnovsky, m.j. (1965): a formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. j. cell biol. 27: 137a. mastromei, g., barberio, c., pistolesi, s., delfino, g. (1991): a bactericidal protein in bombina variegata pachypus skin venom. toxicon 29: 321–328. melis, g., brizzi, r., delfino, g. (2000): serous cutaneous glands in the cuban tree frog hyla septentrionalis: an ultrastructural study on secretory release. in: atti i congresso nazionale della societas herpetologica italica (torino 1996), p. 211-217. giacoma, c., ed, boll. mus. reg. sci. nat. torino, torino. neuwirth, m., daly, j.w., myers, c.w., tice, l.w. (1979): morphology of the granular secretory glands in skin of poison-dart frogs (dendrobatidae). tissue cell 11: 755-771. 153ultrastructure of poison glands in bombina orientalis nosi, d., terreni, a., alvarez, b.b., delfino, g. (2002): serous gland polymorphism in the skin of phyllomedusa hypochondrialis azurea (anura, hylidae): response by different gland types to norepinephrine stimulation. zoomorphology 121: 139 – 148. sanna, a., bernabei, p.a., brunelli, t., rossi ferrini, p., delfino, g. (1993): the cutaneous venom of bombina orientalis: cytotoxic effects on the human hl 60 cell line and a comparison with bombina variegata. j. nat. toxins 2: 161–173. sjöberg, e., flock, å. (1976): innervation of skin glands in the frog. cell. tissue res. 172: 81-91. terreni, a., nosi, d., greven, h., delfino, g. (2003): development of serous cutaneous glands in scinax nasica (anura, hylidae): patterns of poison biosynthesis and maturation in comparison with larval glands in specimens of other families. tissue cell 35: 274-287. toledo, r.c., jared, c., brunner, j.a. (1992): morphology of the large granular alveoli of the parotid glands in toad (bufo ictericus) before and after compression. toxicon 30: 745-753. wagenvoort, c.a., dingemans, k.p. (1985): pulmonary vascular smooth muscle and its interaction with endothelium. morphologic consideration. chest 88: 200-202. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 6(1): 15-18, 2011 lissotriton vulgaris paedomorphs in south-western romania: a consequence of a human modified habitat? severus d. covaciu-marcov*, istvan sas, alfred ş. cicort-lucaciu, horia v. bogdan university of oradea, faculty of sciences, department of biology; universităţii str.1, oradea 410087, romania. *corresponding author. e-mail: scovaciu@uoradea.ro submitted on: 2010, 10th march; revised on:2011, 2nd february; accepted on: 2011, 2nd february. abstract. a lissotriton vulgaris paedomorph population was identified for the first time ever in south-western romania. the newts inhabiting a permanent but artificial habitat, surrounded by agricultural fields. keywords. paedomorphosis, altered habitat, lissotriton vulgaris, sw romania. paedomorphosis, the phenomenon in which larval characteristics are retained trough sexual maturity, is widely spread amongst urodela (denoël et al., 2005a). there are several mechanisms that tie the occurrence of paedomorphosis to certain local environment conditions (semlitsch, 1987; semlitsch et al., 1990; ryan and semlitsch, 2003; denoël et al., 2005a) and it is usually expected in situation where the aquatic environment is more favorable than the terrestrial habitat (whiteman, 1994). prior to this study, it was only rarely encountered in romania, and only for one species: lissotriton vulgaris (fuhn, 1960, 1963; covaciu-marcov and cicort-lucaciu, 2007). the aim of this paper is to provide evidence for the first known population of paedomorph l. vulgaris in south-western romania. the candidate paedomorph population is found in the south-western part of romania (44º26’54,49’’n / 22º43’01,51’’e), in mehedinţi county, near the village of scăpău (fig. 1a). the habitat is represented by an artificial ditch, with concrete sides, with the water level being about 1,5 m lower than that of the surrounding lands (fig. 1b). the channel stretches for several kilometers, is tens of years old, and is indicative of wide-spread regional agricultural modifications. the water level in the ditch is constant year-round at 60-70 cm but can reach up to 1m in depth. aquatic vegetation in the ditch is dense and consists of several aquatic or amphibious plants and algae, with reeds forming compact bands in some locations. paedomorphs were captured in a 100 m stretch of the ditch. we sampled this population in 2007 and 2008 and observed several adults with gills and numerous larvae. in 2007 we captured a gilled female but did not consider this an 16 s.d. covaciu-marcov, i. sas, a.ş. cicort-lucaciu and h.v. bogdan important find as paedomorphosis was induced by similar conditions in western romania in the same year (covaciu-marcov and cicort-lucaciu, 2007). on 25 april 2008, we captured another gilled female paedomorph. we than decided to investigate the habitat more thoroughly. we used round nets assembled on 2 m long metal rods, utilized from the shore or in the water. along those 100 m of the ditch had worked three people about three hours, making several hundred dragging. we managed to capture five gilled females and two gilled males, but no metamorphs. on subsequent visit in july 2008 we managed to capture over 40 larvae and another paedomorph female. the breeding population at this location appears to be made entirely of gilled adults. both sexes of paedomorphs have normal dimensions for adults (total length of 7.0-8.2 cm). their gills are well developed, with the longest pair reaching 6.5 mm, longer than in other similar cases (litvinchuk, 2001). the body colour and markings of these paedomorphs are the same as the adults of both sexes (fig. 2). the males presented sexual characteristics, including the swollen cloacae, spots and the side and a well developed dorsal crest. the larvae captured on the 10th of july were different sizes, between 2 and 4 cm, indicating that they belong to different spawn and period of egg lying. newts are generally negatively affected by the presence of fish in their habitats (joly et al., 2001; hartel and öllerer, 2009). in the case of paedomorphs several populations have disappeared because of fish introduction (denoël et al., 2005b, 2009). in the scăpău channel there are at least 5 different species of fish (lepomis gibossus, carassius carassius, misgurnus fosillis, rodeus sericeus, cobitis taenia) and numerous odonata and dytiscidae larvae. it is likely that the newts minimize predation by using microhabitats within aquatic vegetation (denoël and andreone, 2003). the appearance of this phenomenon at scăpău is likely to be linked with local habitat characteristics. near scăpău there is little precipitation and newts are generally quite rare in the region (covaciu-marcov et al., 2009). in this context, the permanent channel, with its vegetation, offers one of the few suitable habitats in this region. however, this ditch is fig. 1. the geographical position (a) of the studied habitat (b) near scăpău locality (south-western romania). a b 17lissotriton vulgaris paedomorphs in south-western romania situated among heavily modified and degraded terrain that has been used for agriculture. the fields around the channel are represented either by cattle pastures or by fields cultivated with various cereal crops. the occurrence of the l. vulgaris paedomorph population from scăpău is possibly a consequence of antropogenic activities that have destroyed the surrounding terrestrial habitat. acknowledgements we thank dr. thomas luhring, whose comments on an earlier version of the manuscript have essentially contributed to improve its quality. references covaciu-marcov, s.d, cicort-lucaciu, a.ş. (2007): notes on the presence of facultative paedomorphosis in the smooth newt lissotriton vulgaris (linnaeus, 1758) in western romania. north-west. j. zool. 3: 53-57. fig. 2. paedomorph individuals of l. vulgaris from the studied habitat (left: male, right: female). 18 s.d. covaciu-marcov, i. sas, a.ş. cicort-lucaciu and h.v. bogdan covaciu-marcov, s.d., cicort-lucaciu, a.ş., gaceu, o., sas, i., ferenţi, s., bogdan, h.v. (2009): the herpetofauna of the south-western part of mehedinţi county, romania. north-west. j. zool. 5: 142-164. denoël, m., andreone, f. (2003): trophic habits and aquatic microhabitat use in gilled immature, paedomorphic and metamorphic alpine newts (triturus alpestris apuanus) in a pond in central italy. belg. j. zool. 133: 95-102. denoël, m., džukić, g., kalezić, m. (2005b): effect of widespread fish introductions on paedomorphic newts in europe. conserv. biol. 19: 162-170. denoël, m., ficetola, g. f., ćirović, r., radovanić, d., džukić, g., kalezić, m., vukov, t. (2009): a multi-scale approach to facultative paedomorphosis of european newts (salamandridae) in the montenegrin karst: distribution pattern, environmental variables, and conservation. biol. conserv. 142: 509-517. denoël, m., joly, p., whiteman, h.h. (2005a): evolutionary ecology of facultative paedomorphosis in newts and salamanders. biol. rev. 80: 663-671. fuhn, i. (1960): amphibia, “fauna r.p.r.”, vol. xiv, fasc. i.. academiei r.p.r. publishing bucharest. (in romanian) fuhn, i. (1963): sur un nouveau cas de néoténie en masse du triton vulgaire (triturus v. vulgaris l.). acta soc. zool. bohem. 27: 62-69. hartel, t., őllerer, k. (2009): local turnover and factors influencing the persistence of amphibians in permanent ponds from the saxon landscape of transylvania. northwest. j. zool. 5: 40-52. joly, p., miaud, c., lehmann, a., grolet, o. (2001): habitat matrix effects on pond occupancy in newts. conserv. biol. 15: 239-248. litvinchuk, s.n. (2001): first record of paedomorphosis for the smooth newt (triturus vulgaris) from ukraine. russ. j. herpetol. 8: 77-78. ryan, t.j., semlitsch, r. d. (2003): growth and the expression of alternative life cycles in the salamander ambystoma talpoideum (caudata: ambystomatidae). biol. j. linn. soc. 80: 639-646. semlitsch, r.d. (1987): paedomorphosis in ambystoma talpoideum: effects of density, food and pond drying. ecology 68: 994-1002. semlitsch, r.d., harris, r.n., wilbur, h.m. (1990): paedomorphosis in ambystoma talpoideum: maintenance of population variation and alternative life-history pathways. evolution 44: 1604-1613. whiteman h. h. (1994): evolution of facultative paedomorphosis in salamanders. q. rev. biol. 69: 205-221. bbib67 ole_link1 ole_link2 bbib28 ole_link5 ole_link6 ole_link7 ole_link8 _goback ole_link1 ole_link2 ole_link3 ole_link4 ole_link1 ole_link2 ole_link19 ole_link20 ole_link21 ole_link29 ole_link3 ole_link4 ole_link5 ole_link31 ole_link14 ole_link15 ole_link12 ole_link13 ole_link16 ole_link17 ole_link22 ole_link23 ole_link24 ole_link8 ole_link9 ole_link10 ole_link11 ole_link18 ole_link27 ole_link28 ole_link25 ole_link26 ole_link6 ole_link7 ole_link34 ole_link37 ole_link38 acta herpetologica vol. 6, n. 1 june 2011 firenze university press widespread bacterial infection affecting rana temporaria tadpoles in mountain areas rocco tiberti extreme feeding behaviours in the italian wall lizard, podarcis siculus massimo capula1, gaetano aloise2 lissotriton vulgaris paedomorphs in south-western romania: a consequence of a human modified habitat? severus d. covaciu-marcov*, istvan sas, alfred ş. cicort-lucaciu, horia v. bogdan body size and reproductive characteristics of paedomorphic and metamorphic individuals of the northern banded newt (ommatotriton ophryticus) eyup başkale1, ferah sayım2 , uğur kaya2 genetic characterization of over hundred years old caretta caretta specimens from italian and maltese museums luisa garofalo1, john j. borg2, rossella carlini3, luca mizzan4, nicola novarini4, giovanni scillitani5, andrea novelletto1 the phylogenetic position of lygodactylus angularis and the utility of using the 16s rdna gene for delimiting species in lygodactylus (squamata, gekkonidae) riccardo castiglia*, flavia annesi localization of glucagon and insulin cells and its variation with respect to physiological events in eutropis carinata vidya. r. chandavar1, prakash. r. naik2* the balearic herpetofauna: a species update and a review on the evidence samuel pinya1, miguel a. carretero2 effects of mosquitofish (gambusia affinis) cues on wood frog (lithobates sylvaticus) tadpole activity katherine f. buttermore, paige n. litkenhaus, danielle c. torpey, geoffrey r. smith*, jessica e. rettig food composition of uludağ frog, rana macrocnemis boulenger, 1885 in uludağ (bursa, turkey) kerim çiçek preliminary results on tail energetics in the moorish gecko, tarentola mauritanica tommaso cencetti1,2, piera poli3, marcello mele3, marco a.l. zuffi1 climate change and peripheral populations: predictions for a relict mediterranean viper josé c. brito1, soumia fahd 2, fernando martínez-freiría1, pedro tarroso1, said larbes3, juan m. pleguezuelos4, xavier santos5 assessing the status of amphibian breeding sites in italy: a national survey societas herpetologica italica* osservatorio erpetologico italiano acta herpetologica journal of the societas herpetologica italica acta herpetologica rivista della societas herpetologica italica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 6(2): 297-302, 2011 batrachochytrium dendrobatidis in amphibians from the po river delta, northern italy gentile francesco ficetola1, alice valentini2, claude miaud2, andrea noferini3, stefano mazzotti4, tony dejean2,5 1 dipartimento di scienze dell’ambiente e del territorio, università di milano-bicocca. piazza della scienza 1, 20126 milano italy. corresponding author. e-mail: francesco.ficetola@unimib.it 2 laboratoire d’ecologie alpine, umr cnrs 5553, université de savoie, 73376 le bourget-du-lac cedex, france. 3 parco regionale del delta del po emilia-romagna. via mazzini 200, 44022 comacchio (fe), italy. 3 museo civico di storia naturale di ferrara, via f. de pisis 24, 44121 ferrara, italy. 5 parc naturel régional périgord-limousin, la barde, 24450 la coquille, france. submitted on: 2011, 30th august; revised on: 2011, 7th november; accepted on: 2011, 11th november. abstract. batrachochytrium dendrobatidis is a pathogen infecting amphibians at the global scale and causing their decline, but knowledge of the distribution of this pathogen is far from complete. we sampled amphibians from three species (hyla intermedia, rana dalmatina and pelophylax synklepton esculentus) to evaluate whether b. dendrobatidis infects amphibians in the po river delta natural park, northern italy. we detected the pathogen in one population of p. sk. esculentus (prevalence: 0.33). these findings expand the known distribution of b. dendrobatidis in italy and add further concern to the conservation of amphibians in this area. keywords. amphibian decline, chytridiomycosis, emerging infectious disease, management, prevalence, pool frogs. the chytrid fungus, batrachochytrium dendrobatidis (hereafter bd), is the agent of chytridiomycosis, an emerging infectious disease causing amphibian decline and extinctions at the global scale (e.g., berger et al., 1998; fisher et al., 2009). the study of distribution, prevalence and impact of bd is becoming a central theme of amphibian conservation: information on bd distribution can be used to better understand the causes of amphibian decline and eventually to set up management and prevention protocols (andreone et al., 2008; fisher et al., 2009). however, in several areas of the world the knowledge of bd distribution remains limited. for instance, only a few studies investigated the occurrence and the impact of bd in italy. bd has been detected in populations of the yellow-bellied toad (bombina pachypus) in the northern apennines (stagni et al., 2004), in pool frogs (pelophylax synklepton esculentus) from piedmont and central italy 298 g.f. ficetola et alii (simoncelli et al., 2005; di rosa et al., 2007), in populations of the frogs rana latastei and lithobates catesbeianus from piedmont (garner et al., 2004; garner et al., 2006) and in the endemic painted frog (discoglossus sardus) and sardinian newt (euproctus platicephalus) in sardinia (bovero et al., 2008; bielby et al., 2009). furthermore, bd is probably a cause of the decline of b. pachypus, e. platicephalus and d. sardus (stagni et al., 2004; bovero et al., 2008; bielby et al., 2009). nevertheless, information on the presence (or absence) of bd from large portions of italy is extremely limited, and further data are required for a more complete assessment of the distribution of bd; these data may also have important consequences for the conservation and management of amphibian populations. the aim of this study was to evaluate whether bd infects amphibians in the po river delta. this area is particularly interesting for the study of bd because it includes some of the most important wetland systems of italy and hosts a rich herpetofauna (mazzotti, 2007; mazzotti et al., 2007). on the other hand, the po river delta is one of the first areas of italy that has been invaded the american bullfrog l. catesbeianus (albertini and lanza, 1987; ficetola et al., 2010), and this alien species is probably implicated in the global spread of bd (garner et al., 2006). in may 2010, we sampled amphibians in three areas of the po river delta regional park (table 1). one area (bosco mesola integral natural state reserve) is located in the north of the park, while the other two are nearby the town of ravenna, in the central part of the park. amphibians where captured by hand or using small nets. the complete body was comprehensively swabbed using fine-tip swabs (medical wire & equipment co. mw 100–100). the underside of the legs, feet and drink patch was swabbed 3 to 5 five time following the protocol of hyatt et al. (2007). all individuals were released in the environment immediately after swabbing. swab samples were stored frozen until dna extraction. dna extraction and real-time pcr were performed using the protocol by boyle et al. (2004)slightly modified. swab was cut and a 2 mm portion was put in 60 μl of prepman ultra (applied biosystem) with 30 to 40 mg of 0.5 mm diameter glass beads (scientific industries, inc.). the samples were homogenised for 45 s at 30 m/s in a qiagen tissuelyser ii (retsch technology gmbh) and centrifuged for 30 s at 14000 g. the homogenisation and centrifugation was repeated. samples were then incubated at 100°c for 10 min, cooled for 2 min, then centrifuged at 14000 g for 3 min. all the liquid was collected and stored at -20°c. the dna extracted was diluted 10 times for subsequent real-time quantitative pcr assay. real-time quantitative pcr reactions were performed in a iq5 system (biorad) following the protocol described by boyle et al. (2004), but using iq supermix (biorad) instead of taqman master mix. both samples and the negative controls were run in duplicates. pcr standtable 1. sampling locations, amphibian species and prevalence of b. dendrobatidis. locality lat. long. species n prevalence 95%ci bosco mesola (fe) 44.83°-44.85°n 12.25°-12.26°e rana dalmatina 2 0 0-0.667 bosco mesola (fe) 44.83°n 12.26°e pelophylax sk. esculentus 3 0.33 0.039-0.823 ravenna 44.58°n 12.27°e pelophylax sk. esculentus 27 0 0-0.088 bardello (ra) 44.53°n 12.24°e hyla intermedia 28 0 0-0.085 299batrachochytrium dendrobatidis in amphibians in northern italy ards were obtained using the protocol described by boyle et al. (2004). the isolates were acquired from infected alytes obstetricans collected in spain and cultured at the imperial college of london (fisher et al., 2009). infection data were submitted to the bd global mapping project (www.bd-maps.net) we then used the bayesian equal-tailed jeffreys prior intervals to estimate 95% confidence intervals (ci) of bd prevalence in collected samples (brown et al., 2001). jeffreys intervals have been shown to perform well in estimating binomial ci under a variety of conditions (brown et al., 2001). we obtained samples from 60 individuals from 3 species: italian treefrog (hyla intermedia), agile frog (rana dalmatina) and pool frogs (p. sk. esculentus) (table 1). one adult pool frog from bosco mesola was positive to bd; the amount of bd dna in the positive frog was 0.24 genome equivalents. the detection of bd infection in amphibians from the po river delta adds evidences to the presence of this pathogen in multiple areas of mainland italy, and improves our knowledge of its distribution. bd prevalence in our populations was probably not high, as we detected only one positive sample. on the other hand, in the bosco mesola area we captured a small number of individuals, therefore the confidence intervals associated to the prevalence in this area remain wide (table 1). in this area, further sampling is required to assess the actual prevalence of bd in pool frogs and in other amphibians. furthermore, sampling should cover multiple seasons, because the prevalence and intensity of infection are strongly affected by climatic conditions and may vary seasonally (pullen et al., 2010; savage et al., 2011). it should be noted that the only positive sample was from p. sk. esculentus. this result was analogous to the findings of federici et al. (2008) from piedmont (north-western italy): out of 10 species analyzed, they detected bd in pool frogs only. as this species may coexist with bd without suffering a direct decline, it might act as a reservoir for the pathogen, spreading it to other species (simoncelli et al., 2005; di rosa et al., 2007). the po river delta hosts important populations of amphibians (mazzotti et al., 2007), but unfortunately these are threatened by multiple factors, including the salinization of breeding wetlands, the isolation of wetlands, and alien invasive species (ficetola et al., 2004; mazzotti, 2007). as a consequences, some threatened amphibians, such as the spadefoot toad pelobates fuscus and the italian agile frog r. latastei are nearly extinct in the area (mazzotti, 2007). the presence of bd is a further threat to amphibians, and increases the complexity of management. for instance, captive breeding and translocation programs are ongoing in the study area for the conservation of several amphibians (costa and gattelli, in press). the potential impact of bd should be taken into account during these actions, because translocating animals may increase the spread of the pathogen. it would be particularly interesting testing whether the declining p. fuscus and r. latastei are infected by bd. furthermore, a more systematic application of precautionary measures to avoid the dissemination of these emerging diseases through human activities is highly desirable (speare et al., 2004; dejan et al., 2010; phillott et al., 2010). to date, information on the spread of bd in italian amphibians remains limited. it is thus important to expand the sampling of this pathogen and to evaluate whether it is implicated in the decline of other italian amphibians (bonardi et al., 2011). 300 g.f. ficetola et alii aknowledgements we thank gregoire noirjean (pnrpl) for help in the field. this article was written as part of a research partnership on amphibian disease between the parc naturel régional périgord-limousin, the university of savoie, the university of milano-bicocca and the parco regionale del delta del po emilia-romagna. funding was provided by the european union (feder limousin), the conseil régional du limousin and the association nationale de la recherche et de la technologie (anr) and eu program biodiversa (race). gff was funded by a by a scholarship of the university of milano bicocca. the parco regionale del delta del po emilia-romagna provided the authorization to perform sampling. references albertini, g., lanza, b. (1987): rana catesbeiana shaw, 1802 in italy. alytes 6: 117-129. andreone, f., carpenter, a.i., cox, n., du preez, l., freeman, k., furrer, s., garcia, g., glaw, f., glos, j., knox, d., köhler, j., mendelson iii, j.r., mercurio, v., mittermeier, r.a., moore, r.d., rabibisoa, n.h.c., randriamahazo, h., randrianasolo, h., raminosoa, n.r., ramilijaona, o.r., raxworthy, c.j., vallan, d., vences, m., vieites, d.r., weldon, c. (2008): the challenge of conserving amphibian megadiversity in madagascar. plos biol. 6: e118. berger, l., speare, r., daszak, p., green, d.e., cunningham, a.a., gogging, c.l., slocombe, r., ragan, m.a., hyatt, a.d., mcdonald, k.a., hines, h.b., lips, k.r., marantelli, g., parkes, h. (1998): chytridiomycosis causes amphibian mortality associated with population declines in the rain forests of australia and central america. proc. natl. acad. sci. usa 95: 9031-9036. bielby, j., bovero, s., sotgiu, g., tessa, g., favelli, m., angelini, c., doglio, s., clare, f.c., gazzaniga, e., lapietra, f., garner, t.w.j. (2009): fatal chytridiomycosis in the tyrrhenian painted frog. ecohealth 6: 27-32. bonardi, a., manenti, r., corbetta, a., ferri, v., fiacchini, d., giovine, g., macchi, s., romanazzi, e., soccini, c., bottoni, l., padoa schioppa, e., ficetola, g.f. (2011): usefulness of volunteer data to measure the large scale decline of “common” toad populations. biol. conserv. 114: 2328-2334. bovero, s., sotgiu, g., angelini, c., doglio, s., gazzaniga, e., cunningham, a.a., garner, t.w.j. (2008): detection of chytridiomycosis caused by batrachochytrium dendrobatidis in the endangered sardinian newt (euproctus platycephalus) in southern sardinia, italy. j. wildlife dis. 44: 712-715. boyle, d.g., boyle, d.b., olsen, v., morgan, j.a.t., hyatt, a.d. (2004): rapid quantitative detection of chytridiomycosis (batrachochytrium dendrobatidis) in amphibian samples using real-time taqman pcr assay. dis. aquat. organ. 60: 141-148. brown, l.d., cai, t.t., dasgupta, a. (2001): interval estimation for a binomial proportion. stat. sci. 16: 101-117. costa, m., gattelli, r. (in press): riequilibrio delle cenosi faunistiche nella provincia di ravenna: il progetto ri.v.i.v.ro’. in: proceedings of the fourth conference “safeguard amphibians”. ferri, v., ed, cremona, italy, monografie di pianura. 301batrachochytrium dendrobatidis in amphibians in northern italy dejan, t., miaud, c., schmeller, d. (2010): protocole d’hygiène pour limiter la dissémination de la chytridiomycose lors d’interventions sur le terrain. bull. soc. herpetol. france 133: 1-4. di rosa, i., simoncelli, f., fagotti, a., pascolini, r. (2007): the proximate cause of frog declines? nature 447: e4-e5. federici, s., clemenzi, s., favelli, m., tessa, g., andreone, f., casiraghi, m., crottini, a. (2008): identification of the pathogen batrachochytrium dendrobatidis in amphibian populations of a plain area in the northwest of italy. herpetol. notes 1: 33-37. ficetola, g.f., maiorano, l., falcucci, a., dendoncker, n., boitani, l., padoa-schioppa, e., miaud, c., thuiller, w. (2010): knowing the past to predict the future: landuse change and the distribution of invasive bullfrogs. global change biol. 16: 528-537. ficetola, g.f., padoa-schioppa, e., monti, a., massa, r., de bernardi, f., bottoni, l. (2004): the importance of aquatic and terrestrial habitat for the european pond turtle (emys orbicularis): implications for conservation planning and management. can. j. zool. 82: 1704-1712. fisher, m.c., garner, t.w.j. walker, s.f. (2009): global emergence of batrachochytrium dendrobatidis and amphibian chytridiomycosis in space, time, and host. ann. rev. microbiol. 63: 291-310. garner, t.w.j., pearman, p.b., cunningham, a.a. fisher, m.c. (2004): population genetics and disease threats across the entire range of rana latastei. in: v° congresso nazionale della societas herpetologica italica, 29 settembre-3 ottobre 2004. abstract book, p. 62. zuffi, m.a.l., ed, calci (pisa). garner, t.w.j., perkins, m.w., govindarajulu, p., seglie, d., walker, s., cunningham, a.a., fisher, m.c. (2006): the emerging amphibian pathogen batrachochytrium dendrobatidis globally infects introduced populations of the north american bullfrog, rana catesbeiana. biol. letters 2: 455-459. hyatt, a.d., boyle, d.g., olsen, v., boyle, d.b., berger, l., obendorf, d., dalton, a., kriger, k., hero, m., hines, h., phillott, r., campbell, r., marantelli, g., gleason, f. colling, a. (2007): diagnostic assays and sampling protocols for the detection of batrachochytrium dendrobatidis. dis. aquat. organ. 73: 175-192. mazzotti, s. (2007): linee guida per la gestione e la conservazione dell’erpetofauna del parco del delta del po. quad. staz. ecol. civ. mus. st. nat. ferrara 17: 135-141. mazzotti, s., mantovani, s., penazzi, r., cavalieri d’oro, a., gentile, v., rossini, m., lizzio, l., rizzati, e., frasson, f., mingozzi, v., noferini, a. (2007): le comunità degli anfibi del parco del delta del po. quaderni della stazione di ecologia del civico museo di storia naturale di ferrara 17: 49-58. phillott, a.d., speare, r., hines, h.b., skerratt, l.f., meyer, e., mcdonald, k.r., cashins, s.d., mendez, d., berger, l. (2010): minimising exposure of amphibians to pathogens during field studies. dis. aquat. organ. 92: 175-185. pullen, k.d., best, a.m., ware, j.l. (2010): amphibian pathogen batrachochytrium dendrobatidis prevalence is correlated with season and not urbanization in central virginia. dis. aquat. organ. 91: 9-16. savage, a.e., sredl, m.j., zamudio, k.r. (2011): disease dynamics vary spatially and temporally in a north american amphibian. biol. conserv. 144: 1910-1915. 302 g.f. ficetola et alii simoncelli, f., fagotti, a., dall’olio, r., vagnetti, d., pascolino, r., di rosa, i. (2005): evidence of batrachochytrium dendrobatidis infection in water frogs of the rana esculenta complex in central italy. ecohealth 2: 307-312. speare, r., berger, l., skerratt, l.f., alford, r.a., mendez, d., cashins, s.d., kenyon, n., hauselberger, k., rowley, j.j.l. (2004): hygiene protocol for handling amphibians in field studies. james cook university, amphibian diseases group. url : http://www. jcu.edu.au/school/phtm/phtm/frogs/field-hygiene.doc, townsville, australia. stagni, g., dall’olio, r., fusini, u., mazzotti, s., scoccianti, c., serra, a. (2004): declining populations of appennine yellow-bellied toad bombina pachypus in the northern apennines (italy): is batrachochytrium dendrobatidis the main cause? ital. j. zool. 71 (suppl. 2): 151-154. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 7(1): 171-174, 2012 scavenging in the genus natrix cesar ayres ashega. barcelona 86, 6ºc. 36211, vigo (pontevedra), spain. e-mail: cesar@herpetologica.org submitted on: 2011, 17th december; revided on: 2012, 30th january; accepted on: 2012, 15th february. abstract. scavenging is reported as an unusual behaviour of snakes. however, it is likely more common than is supposed. here i report the use of dead newts as prey source by water snakes of the genus natrix at a dam in north-western spain. juveniles and adults viperine snakes (natrix maura), and also an adult grass snake (natrix natrix) were found feeding on newt carcasses. keywords. scravenging, natrix, spain. scavenging in wild snakes has been widely ignored or is barely known, even though there are many references describing this behaviour (see references in devault and krochmal, 2002). from older reports (cowles, 1946) to the most recent ones (platt and rainwater, 2011; lillywhite and brischoux, 2011) the number of species observed scavenging has increased, involving pitvipers (lillywhite et al., 2008) and piscivorous species more frequently than other species (devault and krochmal, 2002). the use of carrion as a food resource could be difficult to evaluate using traditional methods, as stomach contents analysis, unless prey items present signs of decomposition or contain fly larvae. most of the references originates from accidental observations, some from road-killed preys (mora, 1999; phelps, 2006; ventura, 2012). it has been also related with cannibalistic behaviour in some cases (lillywhite, 1982). during the monitoring of a mass-mortality of amphibians in a small reservoir close to pontevedra (42.29º n, 8.36º w spain), i have observed several scavenging events by water snakes (genus natrix). the first one was detected on april 26th 2011 (ayres, 2012); a juvenile viperine snake (natrix maura) was detected while trying to ingest a dead individual of bosca´s newt (lissotriton boscai). on may 23th and 30th 2011 four n. maura individuals were found dead without symptoms of predation or illness, three juveniles and one adult, all of them had ingested dead l. boscai with symptoms of decomposition. on may 2nd a grass snake (natrix natrix) was detected on the shore of the reservoir lake, over dried aquatic vegetation. the snake was actively searching under the vegetation by tongue flicking and quick head movements. a couple of minutes later the head of 172 c. ayres the n. natrix disappeared under the vegetation, and subsequently reappeared with a dead newt in its mouth (fig. 1). this behaviour is consistent with previous findings of sazima and strüssman (1990) and devalt and krochmal (2002). these authors suggest that carrion consumption could be influenced by behavioural processes. species that use chemical cues to find their prey and semi-aquatic or aquatic species scavenge more frequently than species that used visual cues. sazima and strüssmann (1990) suggested that carrion aggregation by water currents increases the probability of detection, also influenced by chemical gradients that give more directional information in water. importance of chemical cues in the behaviour of scavenging has been studied in detail on the brown tree snake (boiga irregularis) by shivik and clark (1997) and shivik (1998, 1999). these authors demonstrated that b. irregularis is able to find carrion using only chemical cues, but the brown tree snake needs a combination of chemical and visual cues to capture live prey. consumption of carrion was described for n. natrix by poschadel and kirschey (2002). scavenging was not described for n. maura, although hailey and davies (1986) reported that exploratory activities and cruising could be use to find dead preys. but recent findings (ayres, 2012) confirm that both species can use carrion as a food resource. even more, juvenile viperine snakes have been observed to feed on caned sardines used as bait (ayres, pers. obs.). as a conclusion, it seems that scavenging behaviour in snakes is more common than it was supposed previously, just often overlooked or simply not observed. some species fig. 1. adult grass snake with a dead newt in its mouth. 173scavenging in the genus natrix use carrion consumption as an adaptation to a specific habitat (i.e., insular isolation; lillywhite et al., 2002, 2008). carrion consumption by florida cottonmouths (agkistrodon piscivorus conanti) living in seahorse key has been studied in detail. this snake population depends mostly on fish that are dropped or regurgitated by colonial wading birds that nest on the island (lillywhite et al., 2002, 2008). these authors also suggested that it could be part of an “island syndrome” which implies behavioral and physiological modifications as a response to resource limitations on islands. other species could use carrion as an opportunistic food resource, like the described episode. the absence of more reports could be due to the inconspicuous life of many snake species, including rarely seen feeding behaviour. acknowledgements konrad mebert and an anonymous reviewer provided valuable comments and suggestions that improved this note. references ayres, c. (2012): natrix maura (viperine snake): diet. herp. rev. (in press) cowles, r. b. (1946): carrion eating by a snake. herpetologica 3: 121-122. devault, t.l., krochmal, a.r. (2002): scavenging by snakes: an examination of the literature. herpetologica 58: 429-436. hailey, a., davies, p.m.c. (1986): diet and foraging behaviour of  natrix maura.  herpetol. j. 1: 53-61. lillywhite, h. (1982): cannibalistic carrion ingestion by the rattlesnake, crotalus viridis. j. herpetol. 16: 95. lillywhite, h.b., sheehy iii, c.m., mccue, m. (2002): scavenging behaviors of cottonmouth snakes at island bird rookeries. herp. rev. 33: 259-261. lillywhite, h.b., sheehy iii, c.m., zaidan iii, f. (2008): pitviper scavenging at the intertidal zone: an evolutionary scenario for invasion of the sea. bioscience 58: 947-955. lillywhite, h.b., brischoux, f. (2011): is it better in the moonlight? nocturnal activity of insular cottonmouth snakes increases with lunar light levels. j. zool. lond.: doi 10.1111/j.1469-7998.2011.00866.x mora, j.m. (1999): leptodeira annulata (culebra destenida, banded cat-eyed snake): diet. herp. rev. 30: 102. phelps, t. (2006): naja nivea (linnaeus, 1758) cape cobra. scavenging. african herp news 40: 24. platt, s.g., rainwater, t.r. (2011): an observation of scavenging by crotalus molossus (baird and girard, 1853). j. kansas herpetol. 37: 8-9. poschadel, j., kirschey, t. (2002): aasfressen bei der ringelnatter (natrix n. natrix).    z. feldherpetol. 9: 223-226. sazima, i., strussmann, c. (1990): necrofagia em serpentes brasileiras: exemplos e previsoes. rev. bras. biol. 50: 463-468. 174 c. ayres shivik, j. a. (1998): brown tree snake response to visual and olfactory cues. j. wildlife manage. 62: 105-111. shivik, j.a. (1999): carrion, context, and lure development: the relative importance of sensory modalities to foraging brown treesnakes (boiga irregularis). ph.d. dissertation, colorado state university, fort collins, colorado, u.s.a. shivik, j.a., clark, l. (1997): carrion seeking in brown tree snakes: importance of olfactory and visual cues. j. exp. zool. 279: 549-553. ventura, f. (2012): comportamiento carroñero en malpolon monspessulanus. bol. asoc. esp. 23 (in press). issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 7(1): 41-47, 2012 eryx jaculus (reptilia, boidae) north of danube: a road-killed specimen from romania severus d. covaciu-marcov¹,*, sára ferenţi¹,², alfred s. cicort-lucaciu¹, istván sas¹ 1 university of oradea, faculty of sciences, department of biology; universităţii str. 1, oradea 410087, romania. * corresponding author, e-mail: scovaciumarcov@yahoo.com 2 “babeş-bolyai” university, faculty of biology and geology, department of biology, gheorghe bilaşcu (republicii) str. 44, cluj-napoca 400015, romania submitted on: 2011, 22nd october; revised on 2011, 6th november; accepted on 2011, 14th november. abstract. in september 2011 we identified a killed specimen of eryx jaculus on the road between the towns of turnu măgurele and corabia, in southern romania. this seems to be the first record of the species north of the danube. although surprising, the identification of e. jaculus in the area is interpreted in the light of the presence of other species of herpetofauna in the danube floodplain with similar ecological requirements, reaching here their northern distribution limit. survival of the species appears to be favoured by the existence of some protected areas in the region. keywords. eryx jaculus, romania, first record, distribution, roadkill. the javelin sand boa, eryx jaculus in romania is a species of community interest that requires strict protection (o.u.g. 57/2007). nevertheless, it is the only species of romanian herpetofauna which has not been included in any protected natural area (iojă et al., 2010). this is due to the great rarity of the species in the country as it has only been recorded in four localities (krecsák and iftime, 2006). these localities are situated south of danube, in dobruja, most of the records being dated (see in: krecsák and iftime, 2006). e. jaculus is a rare species in europe as well, being vulnerable especially at its northern distribution limit (teynié, 1997), located in romania. south of romania, in bulgaria, the species is also rare and has a scattered distribution (naumov, 2006). given the available data on the species distribution in the country, its identification in the fall of 2011 in an area where it has not been reported before is quite surprising. in the evening of 4th september 2011 we found an individual of e. jaculus (fig. 1a, b, c) killed by the road traffic on the route that connects the towns of turnu măgurele and corabia. the finding was accidental as it happened while we were studying the impact of road traffic on the large whip snake, dolichophis caspius. the individual was discovered around 42 s.d. covaciu-marcov, s. ferenţi, a.s. cicort-lucaciu and i. sas 19:00, near the boundary among teleorman and olt counties, between islaz and gârcov villages. the area is located west of olt river, close to the point where it flows into the danube (fig 2). the snake was dead for approximately 24 hours, being in a relatively good condition. it was crossing the road from south to north when it was run over by a single car, near the verge of the road. it displayed the species features (fuhn and vancea, 1961) and had a total length of 439.67 mm from which 36.98 mm being its tail. fig. 1. eryx jaculus from southern romania (a – general view of the road kill specimen; b – details of the head; c – details of the tail) to: romania (a: general view of the road kill specimen; b: details of the head; c: details of the tail). a b c 43road killed eryx jaculus from romania the finding locality is nearly 300 km to the west than those previously known in romania, and, more important, it is the first record of the species outside of dobruja. judging by its distribution in europe (teynié, 1997), this seems to be the first record of e. jaculus north of the danube. its identification in the danube floodplain raises the issue of its actual distribution in romania and, in a broader sense, at its northern range limit. it has been recently pointed out that the areas from dobruja in which the species was recorded in the past are not necessarily the most favourable climatically for it (gherghel et al., 2009a). our finding seems to be located in an even more unfavourable area in terms of climate than those from dobruja (gherghel et al., 2009a). thus, the territory of the country which would be climatically suitable for e. jaculus, has broadened to the whole dobruja, danube floodplain and eastern bărăgan plain, areas where also the soil meets the species requirements. it seems, however, very difficult to identify the species due to its nocturnal lifestyle (fuhn and vancea, 1961), not being previously reported in danube floodplain although some extensive herpetological studies in the area were conducted (iftime, 2005a; iftime and iftime, 2007; török, 2001). though it seems possible that e. jaculus is presently extinct in romania (iftime, 2001), it appears that the species actually occupies a larger area than it was considered in the past. although surprising, the fact can be logically argued by the present record and by the distribution of other species of herpetofauna with similar climatic requirements which are also at their northern distribution limit, but which are more easily to observe due to their lifestyle. this is the case of the eastern spadefoot toad, pelobates syriacus (džukić et al., 2008) and of the large whip snake, dolichophis caspius (covaciu-marcov and david, 2010; sahlean et al., 2010; ferenti et al., 2011). both spefig. 2. new record of eryx jaculus in romania (filled circle). previous records from dobrogea after krecsák and iftime 2006 (squares). 44 s.d. covaciu-marcov, s. ferenţi, a.s. cicort-lucaciu and i. sas cies reach their northern distribution limit in romania, occurring mainly in dobruja and in danube floodplain. the fact that we identified only one individual can raise the problem of its origin. there are known cases when reptiles were transported by human in new areas (e.g. caputo et al., 1997; wiles, 2000; buden et al., 2001; baldo et al., 2008). in other cases they spread along some communication roads, especially railroads (covaciu-marcov et al., 2006; gherghel et al., 2009b). however, we do not consider that the identified e. jaculus individual has arrived accidentally in the zone, but it belongs to a population present there. firstly, southern to danube in bulgaria there is a relative large neighbouring zone where the species was identified, even if it seems to be isolated in the present from other populations (teynié, 1997; biserkov, 2007). minor changes in danube riverbed probably allowed its distribution northward. on the other hand, the region where e. jaculus was identified does not seem to confirm the hypotheses of its accidental introduction. this is a rural, relatively poor region, in which probably there are no activities that allow the introduction of the species. anyway, the problem could be solved properly only by later studies. the area in which e. jaculus was identified, is surrounded by agricultural fields (fig. 3). situated at the limit between the danube floodplain and the higher terraces which border it northward, it seems that at least the sectors north of the road are almost completely degraded and used for agriculture. but south of the road, near the danube, it appears that there are large areas with sandy soils. according to literature data, the species is related to such areas (fuhn and vancea, 1961; iftime, 2005b). thus, the population is probably fig. 3. the area where the road-killed erix jaculus was discovered. 45road killed eryx jaculus from romania located in that sector but some individuals can move on certain distances, this being also the case of the one found by us. the areas at the limit between the danube floodplain and the higher terraces from plain are important for other species of snakes as well, such as the large whip snake (covaciu-marcov and david, 2010; ferenti et al., 2011). the finding of e. jaculus was accidental, road mortality having a great impact on snakes (e.g. ciesiołkiewicz et al., 2006; roe et al., 2006; harris et al., 2010; tok et al., 2011), with nocturnal species being frequent victims (das et al., 2007). this discovery must be followed by detailed studies in all areas with favourable habitats from the region and with similar climatic conditions as that in which the species was found. the new locality could also solve the problem concerning the absence of the species from protected areas in romania (iojă et al., 2010), in close vicinity of the road where the dead sand boa was found being two protected natural areas (corabia-turnu magurele, confluence olt-danube). the moving direction of the identified snake allows us to assume that this is present in one of these protected areas. their herpetofauna should be therefore investigated in detail, being necessary the accurate establishment of the species distribution. besides, immediate measures have to be taken on the protection of e. jaculus in the region, the species being under high anthropogenic pressure in other areas (naumov, 2006). acknowledgements our study was supported by freies europa weltanschauung foundation, to which we thank in this manner. custodian of some protected natural areas from romania, freies europa weltanschauung foundation encourages activities dedicated to the investigation of the biodiversity from romania. references baldo, d., borteiro, c., brusquetti, f., garcia, j.e., prigioni, c. 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(2000): recent records of reptiles and amphibians accidentally transported to guam, mariana islands. micronesica 32: 285-287. © firenze university press www.fupress.com/ah acta herpetologica 5(2): 223-231, 2010 morphometric study on tadpoles of bombina variegata (linnaeus, 1758) (anura; bombinatoridae) anna rita di cerbo*, carlo m. biancardi centro studi faunistica dei vertebrati, società italiana di scienze naturali, c.so venezia 55, i-20121, milano, italy. *correspondig author. e.mail: bombinatoridae@gmail.com submitted on: 2009, 23th november; revised on 2010, 10th october; accepted on 2010, 12th november. abstract. the tadpoles of yellow-bellied toad (bombina variegata) can be easily recognized from other italian anuran species, except those of b. pachypus (though the two congeneric species are allopatric). in this paper we report morphometric data on b. variegata tadpoles from a lombard population living near a torrent at 450 m a.s.l. on a sample of 264 tadpoles (stages 19-44, according to gosner, 1960) we measured the following five variables: snout-vent length, tail length, maximum tail height, total length and weight. we found a slight allometric relationship between snout-vent length and tail length, while, as expected, the weight is nearly proportional to the cube of linear measures. according to literature data, our results point to highly constant proportions during the development phases up to prometamorphic stages. the ratio between snout-vent length and tail length was about 0.75 during the whole growing phase, while from stage 42 the proportion increases as the resorption of the tail starts. keywords. tadpole morphology, yellow-bellied toad, bombina variegata. larval stages of amphibians are well studied, even if inadequate morphological descriptions are common (mcdiarmid and altig, 1999). according to morphotype classification of orton (1957), bombina tadpoles belongs to type 3 (jaw sheaths, labial teeth, medial spiracle), and is included within the following ecomorphological categories: exotroph, lenthic, benthic (mcdiarmid and altig, 1999). differently from adults, which are considered among the most primitive anurans (griffith, 1963; noble, 1922), phylogeny aspects of larval morphology are not yet totally clarified (orton, 1957; sokol, 1975, 1977; cannatella, 1999; haas, 2003; pugener et al., 2003). a description of early development stages of b. orientalis and a study on differentiation of cutaneous granular glands in tadpoles of b. pachypus were respectively reported in prema (1981) and delfino (1977). besides, bonacci et al. (2008) recently described morphology and development of the oral disc of the latter species. regarding yellow-bellied toad, old morphological studies on tadpoles have included both b. variegata and b. pachypus (boulenger, 1910; lanza, 1983), since the latter form was only 224 a.r. di cerbo and c.m. biancardi recently considered a valid species (nascetti et al., 1982, lanza and vanni, 1991; lanza and corti, 1993). so far, we think better to reconsider the morphometric features of yellowbellied toad tadpoles, in order to reassess the mean measures. the tadpoles of bombina variegata can be easily recognized from other italian anuran species, except those of b. pachypus. however, the two species are allopatric occurring only north and south of the po river, respectively. within a lombard population living in a lotic habitat at 450 m a.s.l. (albino, 45°45’ n, 9°47’ e), a total of 264 tadpoles of yellow bellied toad were captured and measured during monthly survey (june to august) from 1994 to 2000. the study area includes a torrent section formed by a main river-bed and a series of pools more or less connected or completely isolated from the stream. it comprehends also the surrounding woody area formed by a phytocoenosis of querco-carpinetum with a recent introduction of the allochthonous pinus strobus. climatically, the area is characterized by a temperate cool climate (cfa) according to köppen-geiger climate classification (peel et al., 2007). monthly mean air and water temperatures of the study area were reported in di cerbo and biancardi (2004). however, the water temperature at pools is subject to variations due to sun exposure, pond size and connection to the stream. overall, an increasing trend of water temperatures was observed along the season (june, mean: 19.03 °c and range: 11.4 – 32.0; july, mean: 20.4 °c and range: 13.5-34.0; august, mean: 21.3 °c; range: 15.6-30.0). invertebrate (dragonfly nymphs, dytiscidae, heteroptera, trichoptera and leeches) and vertebrate predators (s. salamandra larvae, natrix natrix) of eggs and tadpoles, together with larval competitors (bufo bufo, rana temporaria) were recorded in the pools occupied by b. variegata. the tadpoles were collected with a hand net and immediately put in a transparent small basin. they were dried briefly on a piece of paper towel and then separately weighed (wgt) with an electronic balance (tanita, mod. 1479, ± 0.1 g precision). however the weights of the tadpoles up to stage 25 are missing, as well as centigrams differences in early stages (many individuals in class 0.1; 0.2 g etc.). after that, they were put on a graph paper sheet and measured using callipers (0.1 mm accuracy). the tadpoles were released just after measurement. we collected the following biometrical measures according to grosjean (2005): total length (tl), as straight line distance measured from the tip of the snout to the tip of the tail, snout-vent length (svl), as straight line from the tip of the snout to the opening of the vent tube, tail length (vt), as straight line from the opening of the vent tube to the absolute tail tip, maximum tail height (ht), as greatest vertical distance of the tail muscle plus both fins (fig. 1). the statistical analyses were performed using the software spss ver. 17.0 . otherwise stated, reported values are means and standard deviations. we considered the stages 19-44, according to the classical gosner staging series (gosner 1960). our sample includes almost the whole development categories of amphibian larvae, and we divided them in: embryo (19-22), hatchling (23-25), tadpole (26-38), prometamorph (39-41) and metamorph (42-44). the descriptive statistics of the sample are shown in table 1. ratio between the svl and vt ranged between 0.40 – 1.14, but its variance was very low and the modal value was 0.75, which means that, svl was about ¾ of vt. previous reference values were 0.66 (boulenger, 1910) and 0.66-0.8 (lanza, 1983). vt/ht ratio ranged from 1.82 to 3.25. tail was in average about twice longer than height. distributions of svl and vt per development category (fig. 2). 225morphometry of bombina variegata tadpoles as expected, all morphological variables are highly correlated (table 2). the body weight scales with approximately the third power of linear measures. the ratio between tail length and height (vt/ht) does not significantly change in the different stages (anova: f = 0.52; df = 2, 57; p = 0.59). we particularly investigated the relationship between svl, which is usually considered the best predictor of body size, and vt for its implication in the dramatically changes occurring during the metamorphic stages. the fig. 1. tadpole of bombina variegata. biometric measures. fig. 2. variation of svl and vt of b. variegata tadpoles according to growing stage. 226 a.r. di cerbo and c.m. biancardi ta bl e 1. d es cr ip tiv e st at is tic s of th e sa m pl e (v ar ia bl es e xp la in ed in th e te xt ). em br yo ( 19 -2 2) h at ch lin g (2 325 ) ta dp ol e (2 638 ) pr om et am or ph ( 39 -4 1) m et am or ph ( 42 -4 4) n m in -m ax m ea n± sd n m in -m ax m ea n± sd n m in -m ax m ea n± sd n m in -m ax m ea n± sd n m in -m ax m ea n± sd sv l (m m ) 10 3. 0 4. 5 3. 8 ± 0. 5 96 3. 5 7. 0 5. 7 ± 0. 8 75 4. 6 14 .0 9. 2 ± 2. 6 48 12 .0 2 1. 0 17 .2 ± 2 .2 35 15 .0 2 1. 0 17 .4 ± 1 .9 v t ( m m ) 10 3. 5 7. 0 4. 7 ± 1. 2 96 6. 0 12 .8 7. 8 ± 1. 0 75 8. 5 19 .0 13 .0 ± 2 .6 47 12 .0 3 2. 2 22 .8 ± 4 .4 34 13 .5 2 5. 0 19 .0 ± 2 .4 t l (m m ) 10 7. 0 11 .0 8. 5 ± 1. 4 96 10 .5 1 8. 0 13 .5 ± 1 .5 75 15 .5 3 2. 0 22 .2 ± 3 .8 47 24 .0 5 2. 0 40 .0 ± 6 .3 34 32 .0 4 2. 0 36 .3 ± 3 .4 h t ( m m )   1 9 9 32 4 7 5. 3 ± 0. 8 14 6 12 8. 8 ± 1. 8 14 6 11 8. 7 ± 1. 8 w g t ( g)   2 0. 1 0. 1 68 0. 1 0. 6 0. 2 ± 0. 1 48 0. 2 2. 1 1. 1 ± 0. 4 35 0. 5 1. 3 1. 0 ± 0. 2 sv l/ v t 10 0. 57 1 .1 4 0. 84 ± 0 .2 2 96 0. 36 0 .9 2 0. 74 ± 0 .1 0 75 0. 34 1 .0 9 0. 73 ± 0 .2 2 47 0. 61 1 .0 0. 77 ± 0 .0 9 34 0. 64 1 .5 2 0. 93 ± 0 .1 4 v t /h t     1 0. 89 0. 89 32 1. 16 3 .2 5 2. 30 ± 0 .3 9 14 1. 91 3 .0 3 2. 35 ± 0 .3 5 14 1. 82 2 .8 3 2. 21 ± 0 .3 3 227morphometry of bombina variegata tadpoles ratio between svl and vt is almost constant from embryo until 39-41 stages, while is significantly higher at metamorphic stages (fig. 3): at these stages the growth stops and starts the resorption of the tail. an anova confirmed the significant differences comparing the developmental category 42-44 with all the others (anova: f = 11.50; df = 4, 257; p < 0.001), while all the comparisons among the other stages (bonferroni post hoc test) are not significant at α = 0.5. tadpole development process provides for three distinct periods (etkin, 1968): premetamorphic (growth of feeding tadpoles; gosner stage 25-35), prometamorphosis (growth and differentiation of hind limbs; gosner 36-41), metamorphic climax (forelimb development and tail resorption; gosner 42-46). all these modifications are under control of thyroid hormone (troncale et al., 2007). most growth of a tadpole occurs during table 2. pearson correlation coefficients among morphological variable of b. variegata tadpoles. tl svl vt ht wgt tl 1.000 .967 .977 .895 .963 svl .967 1.000 .890 .851 .971 vt .977 .890 1.000 .888 .912 ht .895 .851 .888 1.000 .863 wgt .963 .971 .912 .863 1.000 fig. 3. svl/vt ratio of b. variegata tadpoles at different growing stages. 228 a.r. di cerbo and c.m. biancardi the exponential phase of a sigmoid curve and is quite isometric. this period of maximum growth and minimal development is followed by periods of significant development and little growth (altig and mcdiarmid, 1999). in fact our results show a linear relationship between snout-vent length and tail length up to the metamorphic climax (vt = 1.03 × svl + 1.71, r2 = 0.87; f = 968.74; df = 1, 226; p < 0.001). this trend can be appreciated comparing the distribution of svl and vt at different stages (fig. 2: 19-22 to 39-41). when tadpoles at gosner 42-44 are included in the regression analysis, a slight allometric relationship best fit the data. the allometric growth of body and tail, confirmed also by the presented analysis of variance, can be described by the power equation vt = 1.59 × svl 0.89 (r2 = 0.92; f = 2671.51; df = 1, 240; p < 0.001) which underlines that vt is proportional to svl0.89. in the linear relationship between log-transformed svl and vt, displayed in fig. 4, the residuals of a type-ii linear regression are almost homogeneously distributed. this confirms the goodness of the power relationship between body and tail lengths, while other non-linear relationships do not fit the allometric model. our results clearly show the growth pattern of b. variegata tadpoles at different stages. being collected in different periods and years, our data reflect the growth capacity of this species during each development stage, giving reference measures for body and tail length. previous studies on amphibian larvae have shown that geographic and ecological parameters such as latitude, temperatures, density of population or presence/absence of predators, habitat type, water quality, food abundance and quality could influence the growth rate of tadpoles and the measures at metamorphosis (parichy and kaplan, 1992; ultsch et al., 1999; van burkirk, 2000; olsson and uller, 2002; di cerbo and biancardi, 2004). besides, they could even determine intraspecific variability in anuran morpholfig. 4. allometric relation between svl and vt of b. variegata tadpoles. both axes in logarithmic scale. 229morphometry of bombina variegata tadpoles ogy (smith-gill and berven, 1979; hanken and hall, 1984; smirnov, 1992; strauss and altig, 1992). for instance, recent studies emphasized the effects of the amphibian pathogen batrachochytrium dendrobatidis on anuran larvae. this fungus can cause mouthpart deformities, variations in feeding kinematics, than a compromise on the feeding efficiency of tadpoles and a smaller size in infected individuals (venesky et al., 2010). kaplan and phillips (2006) reported that development of b. orientalis tadpoles at higher temperatures increased both length and height of the tail, but was associated with decreased svl. the tail shape can influence the swimming performance of tadpoles and their vulnerability to predators (chovanec, 1992). recent researches have documented predator-induced polyphenism in tadpole tail shape (hoff and wassersug, 2000; 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(2002): does differential susceptibility to predation in tadpoles stabilize the bombina hybrid zone?. ecology 83: 1648-1659. acta herpetologica 17(2): 165-175, 2022 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-13089 ecological niche differentiation in the anatolian rock lizards (genus: anatololacerta) (reptilia: lacertidae) of the anatolian peninsula and aegean islands mehmet kürşat şahin1,*, kamil candan2,3, danae karakasi4, petros lymberakis4, nikos poulakakis4,5,6, yusuf kumlutaş2,3, elif yıldırım2,3, çetin ilgaz2,3 1 department of biology, kamil özdag faculty of science, karamanoglu mehmetbey university, karaman, turkey 2 department of biology, faculty of science, dokuz eylül university, buca, i̇zmir, turkey 3 research and application center for fauna flora, dokuz eylül university, buca, i̇zmir, turkey 4 natural history museum of crete, school of sciences and engineering, university of crete, knossos av, 71409 irakleio, greece 5 department of biology, school of sciences and engineering, university of crete, vassilika vouton, 70013, irakleio, greece 6 institute of molecular biology and biotechnology (imbb), foundation for research and technology – hellas (forth), n. plastira 100, vassilika vouton, gr-70013 irakleio, greece *corresponding author. email: yasambilimci.kursat@gmail.com submitted on: 2022, 9th may; revised on: 2022, 8th june; accepted on: 2022, 27th june editor: mattia falaschi abstract. the genus anatololacerta is distributed in the eastern mediterranean region including asia minor and some east aegean islands. recent phylogenetic studies suggested that this genus displayed cryptic diversity and was divided into five species: a. anatolica, a. pelasgiana, a. ibrahimi, a. finikensis and a. danfordi. the ecological niche differentiations of these species have not been studied so far. our aims for this study were to predict the potential suitable habitats for the species nested in genus anatololacerta, and to examine the niche overlaps and differentiations via identity and background tests. the occurrence data were obtained from literature and our own field surveys. occurrence records were rarefied and assessed in a 30 arc-second resolution layer, compatible with several bioclimatic and topographic variables. species distribution analyses were performed using maximum entropy approach and pairwise niche comparisons were evaluated by identity and background tests. our results demonstrated that the species delimitation among this genus was not only affected by geographic isolation but also that precipitation and temperature influenced the habitat suitability for these species. predicted suitability usually well matched the actual species distributions. moreover, the niche overlap (identity test) analyses verified that allopatric anatololacerta species show clear ecological differentiations. however, a niche overlap between parapatric species a. pelasgiana and a. finikensis, was confirmed by identity and background tests. it has been suggested that these parapatric species could be more affected by microclimatological parameters than the others. the results of our study are in agreement with the latest phylogenetic study within this genus. keywords. squamata, anatololacerta, niche overlap, precipitation, temperature, maxent, anatolia. introduction ecological factors, e.g., climatic factors, significantly affect the distribution of organisms and may lead to formation of new species (zhao et al., 2019). each species often has unique ecological niche characteristics and as a result of it, ecological needs differ even for sympatric or sister species (soberon and peterson, 2005). quantifying 166 mehmet k. şahin et alii and visualizing the effects of spatial and temporal ecological patterns on speciation processes have contributed to our knowledge of interactions between species and their environments (jezkova and wiens, 2018; kurnaz et al., 2019; şahin et al., 2021). in the last two decades, ecological niche modeling (enm) was frequently used to better understand these processes. enm is a method used to predict the habitat suitability of species across space by using occurrence records and bioclimatic and topographic variables (barve et al., 2011; kass et al., 2018; hosseinian yousefkhani et al., 2019). moreover, enm is a very beneficial approach to better understand aspects of conservation, ecology, distribution, and evolutionary history of the species (guisan and zimmermann, 2000; araújo et al., 2006; phillips et al., 2006). latest tool developments in modeling studies such as enmtools (warren et al., 2021), enmeval (muscarella et al., 2014) and kuenm (cobos et al., 2019) provide frameworks able not only to generate maps but also to assess the niche overlap and the possible degree of differentiation among multiple species. the complex geological history of western asia has shaped the anatolian peninsula, the caucasus mountains, and the iranian steppes, resulting in high variations in vegetation covers and topographic patterns in these regions (rajabizadeh et al., 2016). in addition, many environmental dynamics, like atmospheric concentrations of greenhouse gases, precipitation and temperature fluctuations, alterations in land use and cover have influence on ecosystem and biodiversity structure in mediterranean basin (klausmeyer and shaw, 2009). despite some parts of the anatolian peninsula and its close areas have been studied in terms of enm species distribution analysis for several herptile species (gül et al., 2015, 2016, 2018; hosseinian yousefkhani et al., 2016, 2019; heidari, 2019; candan et al., 2021; kurnaz and şahin, 2021a), the western part of the peninsula and/or with aegean islands and cyprus has still been less represented (kıraç et al., 2022). the herpetofauna in the anatolian peninsula and aegean islands is rich (180 species) (kurnaz, 2020; baran et al., 2021; yaşar et al., 2021), almost as the 60 % of whole european continent (301 species) (speybroeck et al., 2020). besides, recent discoveries of the new species have been making the herpetofauna richer (tuniyev et al., 2018; jablonski et al., 2019; yılmaz et al., 2021; kurnaz and şahin, 2021b; arribas et al., 2022; kurnaz et al., 2022). however, even though this region has been investigated in several biogeographic or phylogeographic studies (kornilios et al., 2012; skourtanioti et al., 2016; kotsakiozi et al., 2018; bozkurt and olgun, 2020), the effects of environmental conditions on the distribution of reptile species or subpopulations are being studied only in the last decade (fattahi et al., 2014; gül et al., 2015; hosseinian yousefkhani et al., 2019; kurnaz and hosseinian yousefkhani, 2020, 2021). anatololacerta arnold, arribas & carranza, 2007 is an eastern mediterranean lacertid genus that is distributed along the western and southern parts of anatolia and some aegean islands (karakasi et al., 2021). however, taxonomic debates on some populations of this genus have been historically controversial. species of this genus represent an example of cryptic diversity (bellati et al., 2015; candan et al., 2016), a common phenomenon among lacertids (kaliontzopoulou et al., 2012; barata et al., 2012; tamar et al., 2015; freitas et al., 2016; psonis et al., 2017; šmíd et al., 2017; mendes et al., 2018). the recent study on the phylogenetic relationships of anatololacerta clades (karakasi et al., 2021) classified them into five species: i) anatololacerta anatolica (werner, 1900) distributed in northwestern anatolia, ikaria and samos islands ii) anatololacerta pelasgiana (mertens, 1959) in southwestern anatolia, symi and rodos islands iii) anatololacerta finikensis (eiselt & schmidtler, 1987) in western part of mediterranean region and psomi island iv) anatololacerta ibrahimi (eiselt & schmidtler, 1987) central part of mediterranean region v) anatololacerta danfordi (günther, 1876) in eastern mediterranean region. therefore, the cryptic diversity within this genus inspired us to test if the species delimitations can be affected by bioclimatological and/or topographic factors. that’s why the objectives of the present study are i) to predict highly suitable areas for each anatololacerta species distribution and determine which environmental factors are important; ii) to measure and compare the niche divergence within the genus anatololacerta, as a case study for cryptic species. materials and methods study area and input data this study was conducted within 25-37° east longitude and 34.5-41° north latitude, covering the western and southern parts of anatolia and aegean islands (fig. 1). a total of 159 occurrence data (31 for a. anatolica, 46 for a. pelasgiana, 22 for a. finikensis, 37 for a. ibrahimi, and 23 for a. danfordi) were obtained from field surveys and literature (eiselt and schmidtler, 1986; mulder, 1995; baran and kumlutaş, 1999; kumlutaş et al., 2015; yakın and tok, 2015; beşer, 2015; bellati et al., 2015; candan et al., 2016; sarıkaya et al., 2017; beşer et al., 2020; karakasi et al., 2021). the raw input data for localities are given in table s1. data for these species were error-checked and improved to meet appropriate standards for ecological 167ecological niche differentiation in the anatolian rock lizards niche modeling in two steps. firstly, georeferenced data were checked for error and data consistency for geographic coordinates (chapman, 2005). secondly, in order to avoid spatial sampling biases and misinterpretation of the habitat suitability analysis and niche overlap tests, the occurrence records for each species were spatially rarefied with keeping one locality in each 2 km by sdm toolbox 2.0 (brown, 2014). nineteen bioclimatic and one topographic variables were downloaded from worldclim version 2.1 (fick and hijmans, 2017). the bioclimatic data were generated from global esri grids for current conditions (~19702000). additionally, three topographic variables were obtained from the studies of gavashelishvili and tarkhnishvili (2016), and gavashelishvili et al. (2018). all these environmental variables were at 30 arc-second resolution (~1 km) (table s2) and each layer was clipped for the study area in arcgis 10.6.1 (esri, california, ca, usa) for the whole study area. pearson correlations between variables were calculated in r v4.1.3 (r core team, 2020) and highly correlated variables were eliminated (r ≥ |0.8|) (fig. s1). ecological niche modeling due to its robustness and dependence on presence and pseudo-absence data, maximum entropy approach was used for niche modelling. the maximum entropy algorithm, which generates the probability of presence of a given species that varies between 0 to 1, provides predictions from presence and pseudo-absence data (phillips et al., 2009). a total of 2000 background points for each species were randomly sampled across the study area. the potential habitat suitability was modeled by using the kuenm package in r for the implementation of maxfig. 1. species occurrence records for genus anatololacerta in the anatolian peninsula and aegean islands. 168 mehmet k. şahin et alii ent 3.4.1 (phillips et al., 2017; cobos et al., 2019). to create the models for each anatololacerta species, 80 % of the occurrences were used for the creation of candidate models and the remaining 20 % for independent presence as test data. the bioclimatic and topographic envelopes, derived from environmental variables, were constructed as set for each species (table 1). model selection to optimize model complexity for all 5 species, 31 combinations of maxent’s 5 feature classes [hinge (h), threshold (t), product (p), quadratic (q) and linear (l)] along with 17 regulation multiplier values (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 8, 10) were evaluated. using these combinations allowed us an optimal approach for generating diverse candidate models in order to select the models that explain our data best (muscarella et al., 2014; cobos et al., 2019). after that, candidate models were evaluated and best models were selected using not only auc values (with the highest values), but also akaike information criterion corrected for small sample sizes (aicc) (with the lowest values) (hurvich and tsai, 1989). significance tests were performed using partial roc (peterson et al., 2008), and predictive power with a 5% omission rate (anderson et al., 2003). model auc scores are evaluated as follows: auc = 0.5: a performance equivalent to random, auc > 0.7: useful performance, auc > 0.8: good performance, auc > 0.9: excellent (manel et al., 2001). finally, all model inputs were transformed into binary predictions using minimum training presence as the threshold to distinguish unsuitable from suitable areas (pearson et al., 2007; rodríguez-ruiz et al., 2020). niche equivalency in order to assess the niche overlap among anatololacerta species, enmtools (warren et al., 2021) was applied to calculate schoener’s d (schoener, 1968) and hellinger’s-based i (warren et al., 2008) niche similarity metrics for niche overlap test due to their simplicity, long usage time and effective method to measure niche similarities (warren et al., 2008). these indices ranged from 0 (no overlap) to 1 (identical niches). the significance of the niche difference was assessed by pooling the occurrences from each taxon, and generating 100 pseudo-replicates. afterwards, one-sided test and an α level of 0.05 was applied to compare the true calculated overlap to the null distribution of niche overlap. this means that the enm of two species is not equivalent when the overlap value is smaller than 5% of the null distribution. background test background test was conducted in order to determine the differential availability of habitat for examined species (warren et al., 2008). the running conditions of the background test were similar to the niche overlap tests (identity test). results ecological niche models and the contribution of environmental variables on the basis of minimum training presence threshold, ecological niche modeling predictions for each anatololacerta species were reliable enough to result in realistic maps, and these predictions were separately conducted for each species with lowest aicc values. a total of 4 bioclimatic and 2 topographic variables contributed to map the predicted distribution of each species (table 1). the ultimate models were selected based on the lowest aicc from evaluation metric results (table 2). the maxent models demonstrated a significant ability to generate ecological niche models for anatololacerta species with average test auc of models as follows: 0.818 ± 0.093 for a. anatolica, 0.818 ± 0.083 for a. pelasgiana, 0.927 ± 0.045 for a. finikensis, 0.830 ± 0.083 for a. ibrahimi and 0.895 ± 0.104 for a. danfordi. based on these results, most of the suitable predicted areas were relatively wider than the present distributions of each species. the potential distribution of all anatololacerta species are shown in fig. 2 a-e. although the bioclimatic and topographic variables that contributed to species distribution were the same, their contribution percentiles were different. the distribution of a. anatolica is highly associated with the temperature annual range bio 7 (35.4 %), while that of a. danfordi with the mean leaf area index table 1. percentage contribution of the environmental layers used in species distribution modeling of anatololacerta species. species bio 3 bio 5 bio 7 bio 17 river_dist lai a. anatolica 3.1 11.1 35.4 29.5 5.9 15.1 a. danfordi 13.5 1.5 17.8 19.9 3.6 43.7 a. finikensis 19.7 27.3 5.5 35.7 6.5 5.3 a. ibrahimi 0.9 8 7.2 36.6 36.9 10.4 a. pelasgiana 7.3 26 19.6 23 10.7 13.3 169ecological niche differentiation in the anatolian rock lizards (43.7 %), and that of a. finikensis with the precipitation of driest quarter bio 17 (35.7 %). the distribution of the remaining two species was highly determined by the environmental variables as follows: distance to the river (36.9 %) for a. ibrahimi and maximum temperature of the warmest period bio 5 (26 %) for a. pelasgiana. table 2. summary statistics for the best models selected for species distribution maps of anatololacerta species via kuenm package. aicc: a corrected aic score, used for a small sample size by increasing the cost for each parameter; waicc: the model weight is the relative likelihood for each model, divided by the total relative likelihood for all models that were considered; δaicc: the difference between the model with the lowest score (the “best” model) and the aicc score for each model; auc: area under the curve is a measure of the accuracy of the model; mean auc ratio ≥1.00, p<0.05 means predictions are significantly better than a random model. species best maxent features aicc waicc δaicc auc mean auc ratio a. anatolica hinge 765.536 0.191 0.134 0.818 ± 0.093 1.672 (p = 0.03) a. danfordi threshold 583.369 0.584 1.881 0.895 ± 0.104 1.000 (p = 0.02) a. finikensis quadratic 394.171 0.182 0.395 0.927 ± 0.045 1.619 (p = 0.03) a. ibrahimi threshold 908.851 1.000 0.143 0.830 ± 0.083 1.353 (p = 0.01) a. pelasgiana product 993.837 1.000 0.111 0.818 ± 0.083 1.720 (p = 0.02) fig. 2. habitat suitability predictions of a. a. anatolica, b. a. danfordi, c. a. ibrahimi, d. a. finikensis, e. a. pelasgiana in the anatolian peninsula and aegean islands (warmer colors refer to high suitability). 170 mehmet k. şahin et alii on the other hand, even though enm in geographic space was generally suited to determine geographic isolation between the cryptic species, discussions on species delimitation have been continuing for the last two decades (raxworthy et al., 2007; fišer et al., 2018). therefore, over-predicted areas were discarded from our final discussion. niche overlap tests the measured niche overlaps among all species are presented in table 3. the null hypothesis of niche overlap between anatololacerta species (except pelasgiana vs finikensis) were rejected because empirical values for schoener’s d and hellinger’s based i test statistics were significantly different than the null distribution of overlap test for each species comparisons (fig. s2 a-j) (t test, df = 99, p < 0.05). in other words, the ecological niche models of most of these species were nonequivalent. background test for the parapatric a. pelasgiana and a. finikensis confirmed the niche overlap between these species in terms of global bioclimatic and selected topographic variables (fig. s2 k). on the other hand, background tests for species that represent allopatric diversification patterns demonstrated that empirical values for schoener’s d and hellinger’s based i test statistics did not significantly differ from the null distribution (fig. s2 l-t). discussion enm on environmental layers has revealed not only additional insights into evolutionary lineages (rissler and apodaca, 2007) but also niche distinctiveness of species (nakazato et al., 2010). climatic niche has a remarkable effect on the area where species occur and each species requires a unique niche according to its ecological needs (gewin, 2006; rissler and apodaca, 2007; gül, 2019). there has been no study on the ecological niche of all anatolian rock lizards so far. in this study, we have modeled environmental niches of all anatololacerta species on the anatolian peninsula and the aegean islands. our results suggested that the niche divergence among the genus was confirmed for allopatric species (fig. s2 a-j). however, the results for a. pelasgiana and a. finikensis showed that there is a niche overlap between these species (fig. s2 k). the present study showed the differentiation in the requirements of the ecological conditions among the anatololacerta species (fig. 2 a-e, table 1). in other words, we assessed ecological niche differentiation to examine the phylogenetic-based taxonomic outputs for this genus. the speciation process within this genus was so far explained only by the geological factors and physical barriers (schmidtler, 1998; bellati et al., 2015). based on the given results, we could assume that the differentiation within the genus and their present allopatric distribution on the anatolian peninsula and the aegean islands is associated with ecological factors as well. the bioclimatological and topographical conditions provided remarkable contributions to the genetic diversity among this genus in terms of allopatric speciation. even though enm for each anatololacerta species were generated using the same bioclimatic and topographic layers, the contribution percentiles of these variables were different. for instance, the dominant contributing variable for each anatololacerta species is different: temperature annual table 3. niche overlap analyses among anatololacerta species in the anatolian peninsula and aegean islands. comparisons anatololacerta sp. measured niche overlap identity test background test** schoener’s d hellinger’s based i schoener’s d hellinger’s based i schoener’s d hellinger’s based i pelasgiana vs. anatolica 0.418 0.712 0.679* 0.896* 0.362 0.648 pelasgiana vs. danfordi 0.529 0.803 0.639* 0.867* 0.307 0.586 pelasgiana vs. finikensis 0.666 0.888 0.633 0.862* 0.342 0.610 pelasgiana vs. ibrahimi 0.641 0.879 0.693* 0.910* 0.346 0.623 anatolica vs. danfordi 0.389 0.669 0.693* 0.909* 0.347 0.625 anatolica vs. finikensis 0.341 0.688 0.592* 0.851* 0.328 0.606 anatolica vs. ibrahimi 0.453 0.740 0.646* 0.883* 0.305 0.577 danfordi vs. finikensis 0.472 0.748 0.582* 0.835* 0.292 0.569 danfordi vs. ibrahimi 0.593 0.847 0.649* 0.884* 0.294 0.574 finikensis vs. ibrahimi 0.547 0.822 0.655* 0.895* 0.313 0.559 * identity test showed significant niche differentiation (all p-value < 0.05). ** no background test showed significant overlap (all p-values > 0.05). 171ecological niche differentiation in the anatolian rock lizards range for a. anatolica, mean leaf area index for a. danfordi, distance to river for a. ibrahimi, precipitation of driest quarter for a. finikensis, and maximum temperature of the warmest period for a. pelasgiana. on the other hand, allopatric speciation dynamics were not only supported climatologically but also geographically (eiselt and schmidtler, 1986; bellati et al., 2015). for example, separation between a. anatolica and the rest of the genus was highly related to the occurrence of the great menderes river. additionally, a. danfordi was isolated from the rest of the genus by central taurus mountains and located in the eastern part of mediterranean region. lastly, a. ibrahimi was only distributed in the northern and southern slopes of central taurus mountains. when it comes to parapatric speciation, the niche overlap case, that was demonstrated in the comparison between a. pelasgiana and a. finikensis, was needed to be discussed in another way, because distribution of both species was limited to only southwestern anatolia and some aegean islands. the actual utilization of the niche is significantly influenced by ecological interactions of various sorts. thus, it could be helpful to use data on different selective regimes to examine the speciation dynamics of these parapatric species (gavrilets et al., 2000; mammola et al., 2018). in order to discuss these speciation dynamics among this genus, it might be also beneficial to have evaluations on climate based historical perspective. karakasi et al. (2021) revealed that the first split in anatololacerta occurred in early pleistocene approximately 1.62 mya with the separation of a. anatolica and the recent one was between a. pelasgiana and a. ibrahimi (0.56 mya). the latter split matches the mindel glacial period. according to the literature, 16 glacial periods have occurred during the last 2.4 million years in the pleistocene (webb and bartlein, 1992; hewitt 1996, 2000). moreover, it was highly thought that the last four glacial periods in pleistocene had a remarkable impact on faunal composition of anatolia and related areas (çıplak, 2004). fluctuations in the temperature during these periods not only affected the movements of old anatolian populations (çıplak, 2004) but also shaped the vegetation dynamics with important changes (jiménez-moreno et al., 2015). in addition, the precipitation and temperature dynamics for a long time might have an impact on the vegetation patterns along western and southern parts of anatolian peninsula (şahin et al., 2021). on the other hand, the comparisons among allopatric anatololacerta species revealed that, while their niches are not more similar than expected by chance (fig. s2 a-j), their niches are not equivalent (fig. s2 l-u). studies on allopatric neurergus species in anatolia (gül, 2019), speciation dynamics of endemic lizards in madagascar (nunes et al., 2022) and diversification of shrews in island dispersal events (esselstyn et al., 2011) demonstrated that differences between their climatic niches are compatible with the abiotic environmental conditions between the geographical regions where allopatric species have been inhabiting. in fact, although this situation shows that allopatric anatololacerta species living in the same geography have different niche requirements, the isolation areas between the inhabiting zones do not have an effect on the differentiation of environmental characteristics. if species fit to particular climatic conditions (or various local conditions), it brings to niche differentiation because of the unique adaptations needed to survive and breed (nakazato et al., 2010). in the present study, ecological niche divergence has been inferred to display ecological speciation of species with allopatric distributions. our results are compatible with the taxonomic suggestion of the work of karakasi et al. 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(2019): climatic niche comparison across a cryptic species complex. peerj 7: e7042. acta herpetologica vol. 17, n. 2 december 2022 firenze university press cryptic diversity in pygmy chameleons (chamaeleonidae: rhampholeon) of the eastern arc mountains of tanzania, with description of six new species michele menegon1,2,*, john v. lyakurwa3,4, simon p. loader5, krystal a. tolley6,7 preliminary genetic characterisation of southern smooth snake coronella girondica (serpentes, colubridae) populations in italy, with some considerations on their alpine distribution matteo r. di nicola1, raffaella melfi2, francesco p. faraone3,*, daniel l. n. iversen4, gabriele giacalone5, giovanni paolino1, mario lo valvo6 species diversity and distribution of amphibians and reptiles in sardinia, italy claudia corti1,2,*, marta biaggini1, valeria nulchis2, roberto cogoni2, ilaria maria cossu2, salvatore frau4, manuela mulargia2, enrico lunghi2, lara bassu2. the italian wall lizard, podarcis siculus campestris, unexpected presence on gorgona island (tuscan archipelago) marco a.l. zuffi1,*, alan j. coladonato2, gianluca lombardo3, antonio torroni3, matilde boschetti1, stefano scali4, marco mangiacotti2, roberto sacchi2 molecular analysis of recently introduced populations of the italian wall lizard (podarcis siculus) oleksandra oskyrko1,2,*, lekshmi b. sreelatha1,12,13, iolanda silva-rocha1, tibor sos3,4, sabina e. vlad5,6,7, dan cogălniceanu5,6, florina stănescu6,7,8, tavakkul m. iskenderov9, igor v. doronin10, duje lisičić11, miguel a. carretero1,12,13 sunny-side up: ontogenetic variation in egg mass temperatures of the wood frog rana sylvatica ryan calsbeek*, ava calsbeek, isabel calsbeek ecological niche differentiation in the anatolian rock lizards (genus: anatololacerta) (reptilia: lacertidae) of the anatolian peninsula and aegean islands mehmet kürşat şahin1,*, kamil candan2,3, danae karakasi4, petros lymberakis4, nikos poulakakis4,5,6, yusuf kumlutaş2,3, elif yıldırım2,3, çetin ilgaz2,3 occupancy and probability of detection of the introduced population of eleutherodactylus coqui in turrialba, costa rica jimmy barrantes-madrigal1,*, manuel spínola parallada1, gilbert alvarado 2, víctor j. acostachaves3,4. one site, three species, three stories: syntopy of geckoes euleptes europaea (gené, 1839), hemidactylus turcicus (linnaeus, 1758), tarentola mauritanica (linnaeus, 1758) in a coastal area of southern tuscany (central italy) giacomo radi1,2, marco a.l. zuffi1,* comparative cytogenetics on zamenis lineatus and elaphe quatuorlineata (serpentes: colubridae) marcello mezzasalma1,* , elvira brunelli1, gaetano odierna2, fabio m. guarino2 © firenze university press www.fupress.com/ah acta herpetologica 5(1): 103-106, 2010 an attempted predation on a four-lined snake elaphe quatuorlineata (lacépède, 1789) by a common buzzard buteo buteo (linnaeus, 1758) gaetano aloise1, antonio mazzei2, pietro brandmayr2 1 museo di storia naturale della calabria e orto botanico, università della calabria, via p. bucci, s.n., i-87036 rende (cs), italy. corresponding author. e-mail: aloise@unical.it 2 dipartimento di ecologia, università della calabria, via p. bucci, s.n., i-87036 rende (cs), italy. e-mail: antonio.mazzei@unical.it; brandmay@unical.it submitted on: 2009, 11th december; revised: 2010, 18 january; accepted on 2010, 26th february. abstract. the authors report on a case of attempted predation on a sub-adult fourlined snake by a common buzzard observed in calabria region (southern italy). keywords. elaphe quatuorlineata, buteo buteo, predation, calabria, italy. the four-lined snak e elaphe quatuorlineata (lacépède, 1789) is the largest european snake, with a maximum length recorded as 250 cm, but adults are usually 100-150 cm long. in the calabria region, nevertheless, specimens of over 190 cm size had been observed (maximum size: male, may 1999, falconara albanese, c.da vassallo, cosenza, 665 m a.s.l. 195 cm. g. aloise, unpublished data). its distribution area covers large part of italy, the coastal lines of the balkan peninsula and related islands, albania, bulgaria, greece and several greek islands. the species is usually preyed by wild boar sus scrofa, badger meles meles, fox vulpes vulpes, dog and some falconiforms (especially, short-toed eagles circateus gallicus and common buzzards buteo buteo, cattaneo and carpaneto, 2000). this note reports an attempted predation on a sub-adult four-lined snake by a common buzzard. the common buzzard feeds on a wide range of preys, mainly rodents, but also on other vertebrates and invertebrates of appropriate size. its diet reflects underlying differences in prey-availability (cramp and simmons, 1980). the diet shows seasonal and geographical variations, with a greater consumption of reptiles and insects in the southern part of its area. (glutz von blotzeim et al., 1971; cramp and simmons, 1980). in central and southern italy, during the favourable seasons, the common buzzard preys mainly reptiles (> 50%) and, among them, snakes are usually prevalent (see moltoni, 1937; lovari, 1975; massa, 1981; manzi and pellegrini, 1989). however, the common buzzard generally prefers the small sized snakes (about 50 g average weight, especially hierophis, 104 g. aloise, a. mazzei and p. brandmayr elaphe, natrix and vipera) that are avoided by short toed eagles. this last species, in fact, is specialised in capturing ophidia and weigh twice the common buzzard, preferring preys weighing more than 100g (petretti, 2008). on 13th june 2009, at about 06:00 p.m., in cerenzia (province of crotone, calabria region, southern italy), on a 450 m a.s.l. unlevelled road crossing a pasture on clay soils, an attempted predation on a four-lined snake by a common buzzard was observed and documented. when i found the common buzzard, it was immobilized on its back by the coils of the snake (fig. 1). for some minutes, the fighting animals seemed not to notice the presence of the human observers within a walking distance. only when the animals were disturbed by the means of a stick , the snake distracted its attention from the buzzard towards the new “danger” (fig. 2 a). during the fighting, which lasted about 15 minutes, the snake continued to subdue the raptor, striking it with several bites to its wings and its head (fig. 2 b-d), before releasing its hold, because of a second human interference. the observation seems to confirm the substantial protection that a large size offer the four lined snake, when preyed by the common buzzard, that, obviously, can prey only young smaller-size specimens of this species. fig. 1. a common buzzard immobilized by a four-lined snake. dates and location in the text (photo: a. mazzei). 105an attempted predation on a four-lined snake by a common buzzard acknowledgments mariella vercillo has improved the english of the manuscript. references cattaneo, a., carpaneto, g.m. (2000): elaphe quatuorlineata (lacépède, 1789). in: anfibi e rettili del lazio, pp. 98-99, bologna, m.a., capula, m., carpaneto, g.m., eds, fratelli palombi editori, roma. cramp, s., simmons, k.e.l. (1980): handbook of the birds of europe, the middle east and north africa. vol. ii. oxford university press, oxford. glutz von blotzheim, u.n., bauer, k.m., bezzel, e. (1971): handbuch der vögel mitteleuropas. band 4. falconiformes. akademische verlagsgessellschaft, wiesbaden. fig. 2. the sequence of the attempted predation on a subadult four-lined snake by a common buzzard. see text (photo: a. mazzei). 106 g. aloise, a. mazzei and p. brandmayr lovari, s. (1975): the feeding habits of four raptors in central italy. raptor research, 8: 45-57. manzi, a., pellegrini m. (1989): dati sulla biologia riproduttiva della poiana buteo buteo in un’area della fascia collinare abruzzese. avocetta 13: 109-114. massa, b. (1981): le regime alimentaire de quatorze especes de rapaces en sicilie. rapaces méditerranées, annales du c.r.o.p., aix en provence 1: 119-129. moltoni, e. (1937): osservazioni bromatologiche sugli uccelli rapaci italiani (continuazione e fine). riv. ital. ornitol. 7: 13-33; 61-119. petretti, f. (2008): l’aquila dei serpenti. pandion edizioni, roma. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 7(1): 81-90, 2012 a scanning electron microscopic study of the surface morphology of nuptial pads in male amphibians (genus: bombina, pelophylax, rana) pasqualina kyriakopoulou-sklavounou*, efthymia papaevangelou, nikolaos kladisios department of zoology, school of biology, faculty of sciences, aristotle university of thessaloniki, gr-54124 thessaloniki, greece.*correspondence author. e-mail: kyriakop@bio.auth.gr submitted on: 2011, 6th june; revised on: 2011, 27th november; accepted on: 2012, 21st march. abstract. the fine structure of nuptial pad surface of the anuran amphibians bombina variegata, pelophylax epeiroticus, pelophylax ridibundus and rana dalmatina, was examined by scanning electron microscopy. nuptial pads are cutaneous secondary sexual characters of males that appear during the breeding season and disappear afterwards following an annual cycle. in males of p. epeiroticus, p. ridibundus and r. dalmatina, nuptial pads were observed on the ventrolateral aspect of the first digit (the thumb) as darkish and remarkably keratinized papillae. in males of b. variegata nuptial pads were almost black and very visible on the thumb, the second and the third digit of the front legs. they also extended on the ventral surface of the forearms. under scanning electron microscope numerous small papillae were observed rising above pad’s surface. in p. epeiroticus, p. ridibundus and r. dalmatina, these papillae were almost rounded at the base while at the dome shaped top they had many microprocesses organised in groups, thus assuming the shape of a “flower” which differed slightly among these three ranid species. in b. variegata the protuberances were conical with heavily keratinized hooks without microprocesses. our results show that surface morphology of nuptial pads is unique for each species and could be considered as a species-specific character. keywords. sem, nuptial pads, bombina variegata, pelophylax epeiroticus, pelophylax ridibundus, rana dalmatina. introduction nuptial pads, also called thumb pads or nuptial excrescences, are cutaneous male secondary sexual characters of anurans. they appear during the breeding season as dark and strongly keratinized protruberances or papillae on the ventrolateral surface of the first dig82 p. kyriakopoulou-sklavounou, e. papaevangelou and n. kladisios it or the first three digits and also on the inside of the forearms and disappear afterward. they consist of thickened epidermis and dermis. the external dark layer of the epidermis is thickened to form a horny covering that may be simply rugose or modified into cones or spines. clusters of large mucous glands occupy the pad dermis their ducts reaching the pad surface. these patterns are confirmed both under light and transmission electron microscopes (parakkal and ellis, 1963; kurabuchi, 1993, 1994; brizzi et al., 2003). nuptial pads are thought to facilitate the male’s grip on the female during amplexus. furthermore, the surface micro-sculptures provide the friction necessary for clasping the smooth body of the female (lofts, 1974; duellman and trueb, 1994; wells, 2007). the development and maintenance of these structures is seasonal and regulated by androgenic hormones (parakkal and ellis, 1963; lofts, 1974; zamachowski and zysk, 1978; guarino and bellini, 1993; thomas et al., 1993; epstein and blackburn, 1997; van wyk et al., 2003; kaptan and murathanoglu, 2008). studies on the 3-dimensional structure of the surface architecture of nuptial pads are limited and these mainly concern species from japan and italy (kurabuchi, 1993, 1994; brizzi et al., 2003). in the present study light and scanning electron microscopy was employed to investigate the fine structure of nuptial pads of some common anurans in greece. the investigation aim at revealing potential interspecific differences. for this purpose four species were selected: the closely related water frogs pelophylax epeiroticus and pelophylax ridibundus and the brown frog rana dalmatina, from the family ranidae; the yellow-bellied toad bombina variegata was also studied which belongs to the distant and more primitive family of bombinatoridae. materials and methods mature males of four anuran species were used in this study. all specimens were collected during the breeding season march and april of 2006-2007. two males of bombina variegata with a snout-vent length 56.7 mm and 42.1 mm, two males of rana dalmatina with a snout-vent length 57.2 mm and 62.7 mm were collected from pertouli (altitude 900 meters, longitude 21o27’51” e, latitude 39o32’19” n). two males pelophylax epeiroticus (formerly known as rana epeirotica), with a snout-vent length 76 mm and 84.2 mm were collected from lake of ioannina (altitude 480 meters, longitude 20o50’56” e, latitude 39o40’3” n). two males of pelophylax ridibundus (formerly rana ridibunda) with a snout-vent length 82.5 mm and 64.2 mm were collected from lake kerkini (altitude 52 meters, longitude 23o15’4” e, latitude 41o15’42” n). also one or two female individuals from each species were collected in order to point out sex-linked differences. preparation of tissues for scanning electron microscope (sem) frogs were transported alive to laboratory and within few hours were prepared for examination. after being sacrificed in 0.1-0.5 % solutions of ms 222 the first digits (the thumbs) or the entire hands with well-developed nuptial pads were excised and fixed in glutaraldehylde 3 % for 24 hours. after they were dehydrated in serial ethanol, they were dried using the critical point method with liquid co2 and coated with gold in a sputtering system. also the first digits of females were prepared on the same manner. finally, materials were examined with a scanning electron microscope jeol (jsm-840a). 83scanning electron microscopic study in male amphibians preparation of tissues for light microscope (lm) for the histological examination nuptial pads were removed and fixed in formaldehyde solution 4%. after dehydration with ethanol solutions (70%, 90%) and clearing with xylene, the thumb pads were embedded in paraffin, serially sectioned at 7 μm and stained with harris haematoxylineosin (he). sections were examined in a zeis axioskop and photographed with a digital camera olympus c-5060 adjusted to microscope. results gross anatomy observation (not shown) of the three ranid species p. epeiroticus p. ridibundus and r. dalmatina revealed that they are cutaneous papillae located on the first digits (thumbs) of the forelimbs and are extended from the ventrolateral base of the thumb to near the digital tip. in b. variegata, nuptial pads are visible on the 1st, 2nd and 3rd digits of the forelimbs, also extending on the ventral surface of the forearms. the pad regions were darkish in r. dalmatina, dark grey in p. epeiroticus, p. ridibundus and almost black in b. variegata. light microscopy (lm) sections of the nuptial pads of the four studied species observed under lm revealed the basic structure of the skin of anuran amphibians (fig. 1a-f). they consisted of a thickened epidermis with several layers of cells and a dermis that contained linear clusters (rows) of large mucous glands opening on the pad surface. however, several differences were detectable among the four studied species. in r. dalmatina the outer skin surface shows conical hill-like protruberances with obvious keratinised layers (fig. 1a-b) whereas in p. epeiroticus and p. ridibundus the protruberances are slightly rounded (fig. 1c-d). in b. variegata the epidermis is modified into spines with remarkable amounts of melanin (fig. 1 e-f). differences also appeared in the size and shape of the mucous glands that were large and elongated orthogonal to the skin surface in the three species of ranids (fig. 1a, d). they were ellipsoidal in b. variegata the major axis parallel to the skin surface (fig. 1e). scanning electron microscopy (sem) under sem, the region of nuptial pads in the three species of ranids, the region of nuptial pads was easily distinguishable, even at low magnification, from the ordinary epidermis by the occurrence of numerous protuberances or papillae (fig. 2, 1a-3a). in contrast to males the epidermis on the digits of females was smooth. rana dalmatina observation from above of papillae of nuptial pads in r. dalmatina showed that they were cylinder-shaped at base with a dome-like at top or apex (fig.2, 1b-1d). more specifi84 p. kyriakopoulou-sklavounou, e. papaevangelou and n. kladisios cally several groups of microprocesses were detected on the top of the papilla, whereas the lower microprocesses were arranged in concentric circles round the apex. fig. 1. cross-sections of nuptial pads observed under lm. a-b. rana dalmatina a. epidermis and underlying elongated mucous glands. ×100. b. epidermis with obvious keratinised layers ×400. c-d. epidermis and underlying mucous glands of pelophylax epeiroticus and pelophylax ridibundus. ×400. e-f. bombina variegata. e. epidermis and underlying ellipsoid mucous glands. ×100. f. epidermis with dark pigmented spines. ×400. 85scanning electron microscopic study in male amphibians pelophylax epeiroticus. in p. epeiroticus the papillae were cylinder-shaped with a rounded or semi-spherical top (fig. 2, 2b-2d). the arrangement of groups of microprocesses was different from that of r. dalmatina. in p. epeiroticus two main groups were seen on the top of the papilla whereas the contiguous groups exhibited a spiral course (fig. 2, 2c). pelophylax ridibundus in p. ridibundus the shape of papillae was cylindrical at base and spherical at the top (fig. 2, 3b-3d). specifically, four or five smaller groups of microprocesses that are arranged in a cycle surround the central large group (fig. 2, 3c). higher magnification of the top showed the numerous microprocesses with flattened tips that differed from those of p. epeiroticus and r. dalmatina (fig. 2, 1d). fig. 2. sem micrographs of nuptial pads of the ranid species rana dalmatina (1a-1d), pelophylax epeiroticus (2a-2d), and pelophylax ridibundus (3a-3d) at four different magnifications. 1a-3a: first digit of a male with numerous papillae (scale bar = 2 mm). 1b-3b: numerous papillae cylinder-shaped at base with a dome-like top (scale bar = 200 μm). 1c-3c: the pattern of papillae and the arrangement of microprocesses (scale bar = 100 μm). arrows show the gland pores of the skin distributed among the papillae. 1d-3d: the flattened tips of the microprocesses (scale bar = 10 μm, 1d and 5 μm 2d, 3d). 1a 1b 1c 1d 86 p. kyriakopoulou-sklavounou, e. papaevangelou and n. kladisios bombina variegata distribution and shape of papillae in the nuptial pads of b. variegata were different. in this species we observed numerous hook-like or rose-spiny projections with a remarkably keratinization not only at the first finger but also in the second and partially the third digits and the ventral surface of the forearms (fig. 3a). the papillae were cylindrical at the base and tapered upward to a point forming a hook or spine strongly keratinized (fig. 3, b, c, d). discussion as expected, the results of this study showed that scanning electron microscopy is a powerful tool that provides important details of the microstructure of the surface morphology of nuptial pads of frogs that could not be detected under light microscope. crosssections of thumb pads observed under lm are in good agreement with previous histological studies concerning the species rana dalmatina, rana esculenta and rana perezi (guarino and bellini, 1993; brizzi et al., 2003), but they revealed the inner texture of the fig. 2. continued. 2a 2b 2c 2d 87scanning electron microscopic study in male amphibians skin (including mucous glands in the spongy dermis) without many details of the external surface. the results of sem revealed remarkable differences in the surface morphology of the nuptial pads in the four species. furthermore, slight differences existed between the water habit frogs p. epeiroticus and p. ridibundus, which are considered as very closely related by schneider et al. (1984). it should be noticed that among the three ranid species, the observed differences between the surfaces of the nuptial pads mainly consisted of the arrangement and the number of groups of microprocesses on top of the papillae: pelophylax epeiroticus shows two central groups of microprocesses and a spiral arrangement, p. ridibundus has a clear central group surrounded by four to five smaller clusters, whereas in rana dalmatina, there is no clear central grouping but several small groups of microprocesses in concentric circles. on the other hand, a completely different pattern, with epidermal keratin hooks, was found in b. variegata. these results suggest that surface morphology of nuptial pads may be species-specific in anurans. there are not many sem studies on the surface morphology of nuptial pads of anurans for comparisons. moreover, the few existing concern some hylid, ranid and rhacophorid frog species of japan (kurabuchi 1993, 1994). more recently brizzi et al. (2003) published a sem study for the european species rana perezi (now pelophylax perezi). in this species, there are five to six groups of microprocesses arranged spirally around the top fig. 2. continued. 3a 3b 3c 3d 88 p. kyriakopoulou-sklavounou, e. papaevangelou and n. kladisios of the papillae. this morphology appears more similar to that of p. epeiroticus and differs considerably from those of p. ridibundus and rana dalmatina. regarding the japanese species, kurabuchi (1993) reported that r. nigromaculata (now pelophylax nigromaculatus) and r. brevipoda porosa (now pelophylax porosus), are similar in shape, size and colour. they have overlapping ranges and produce frequent natural hybrids, whereas they differ only on the morphology of the nuptial pads. this observation suggests that these species are actually different and stress the taxonomic value of the pattern of external morphology of nuptial pads. nuptial pads are associated with amplexus, helping the male hold onto the female, and preventing the female from escaping, while they may play a role in male-male combat (duellman and trueb, 1994; wells, 2007). the correlation between the type of amplexus (axillary or inguinal) and the 3-dimensional architecture of the nuptial pad surface has been also discussed by kurabuchi (1993). axillary amplexus is common in ranids, while inguinal amplexus is encountered in xenopus laevis. the nuptial pads of the latter species show numerous hook-like or rose-spiny projections with heavy keratinisation of surface cells. these differences in nuptial pad morphology were supported by our findings. the nuptial pads of the three ranid species were similar to that of the japanese ranids, which fig. 3. sem micrographs of nuptial pads of bombina variegata. a: forelimb of a male showing the nuptial pads at the first three digits (scale bar = 2 mm). b-c: the form of the spines at different magnifications (scale bar = 200 μm and 100 μm). d: the form of the top of a spine (scale bar = 20 μm). 89scanning electron microscopic study in male amphibians all use an axillary amplexus. in contrast, the pattern of b. variegata was similar to that of x. laevis, which both use an inguinal amplexus. moreover, it has been reported that well-developed nuptial pads are associated with breeding in water, while less-developed pads are found in terrestrial or land breeders (wells, 2007). all four species of the present study breed in water and had accordingly well-developed nuptial pads. the results of the present study support previous inferences of taxonomic and functional importance of the nuptial pads. given their significance in anuran behaviour and evolution, we believe that further studies on more species may contribute to elucidate such aspects. acknowledgements the authors thank prof. sklavounos s.a., and assist. prof. pavlidou e. from the sem laboratory of aristotle university of thessaloniki, for their valuable contribution to this investigation. references brizzi, r., delfino, g., jantra, s. (2003): an overview of the breeding glands. ιn: jamieson gm, editor, reproductive biology and phylogeny of anura, p 253-317. science publishers inc., enfield, nh, usa. duellman, w.e., trueb, l. (1994): biology of amphibians. new york:mcgraw-hill. epstein, m.s., blackburn, d.g. (1997): histology and histochemistry of androgen-stimulated nuptial pads in the leopard frog, rana pipiens, with notes on nuptial gland evolution. can. j. zool. 74: 472-477. guarino, f.m., bellini, e. (1993): reproductive activity and plasma androgen concentrations in the male of rana dalmatina. boll. zool. 60: 281-286. kaptan, e., murathanoglou, o. (2008): annual morphological cycles of testis and thump pad of the male frog (rana ridibunda). the anatomical record 291: 1106-1114. kurabuchi, s. (1993): fine structure of nuptial pad surface of male ranid frogs. tissue and cell 25: 589-598. kurabuchi, s. (1994): fine structures on the surface of nuptial pads of male hylid and rhacophorid frogs. j. morphol. 219: 173-182. lofts, b. (1974): physiology of the amphibia. academic press. new york and london. parrakal, p.f., ellis, r.a. (1963): a cytochemical and electron microscopic study of the thumb pad in rana pipiens. exp. cell res. 32: 280-288. schneider, h., sofianidou, t.s., kyriakopoulou-sklavounou p. (1984): bioacoustic and morphometric studies in the water frogs (genus rana) of lake ioannina in greece, and description of a new species (anura, amphibia). z. zool. syst. evolutionsforsch. 22: 349-366. thomas, e.o., tsang, l., licht, p. (1993): comparative histochemistry of the sexually dimorphic skin glands of anuaran amphibians. copeia 1993: 133-143. 90 p. kyriakopoulou-sklavounou, e. papaevangelou and n. kladisios van wyk, j.h., pool, e.j, leslie, a.j. (2003): the effects of anti-androgenic and estrogenic disrupting contaminants on breeding gland (nuptial pad) morphology, plasma testosterone levels, and plasma vitellogenin levels in male xenopus laevis (african clawed frog). arch. environ. contam. toxicol. 44: 247-256. wells, k.d. (2007): the ecology and behavior of amphibians. the university of chicago press usa. zamachowski, w., zysk, a. (1978): morphological and histological changes in the testes and nuptial pads of the water frog, rana esculenta l., during the annual cycle. acta biol. cracov. 21: 69-81. © firenze university press www.fupress.com/ah acta herpetologica 5(2): 131-142, 2010 growth patterns and reproductive strategies in the lizard, calotes versicolor raised in captivity bhagyarekha n. pandav, bhagyashri a. shanbhag*, srinivas k. saidapur department of zoology, karnatak university, dharwad – 580 003, india. *corresponding author. e-mail: bhagyashrishanbhag@gmail.com submitted on: 2009, 4th february; revised on: 2009, 22th july; accepted on: 2010, 16th march. abstract. the paper describes the growth patterns and breeding strategies in the lizard, calotes versicolor in captivity. the lizards were raised in laboratory from hatching. it was observed that these lizards attain sexual maturity at 7.42 ± 0.5 months of age. growth rate in males is always higher than in females and sexual size dimorphism in c. versicolor is due to the difference in growth rate between the sexes. the study revealed that some females reproduce in the first year at smaller body size (snout-vent length-svl ~ 8 cm) with small clutch size while some skip reproduction in the first year, grow larger (svl ~11.5 cm) and reproduce in second year with larger clutch size, yet others reproduce in first year, skip reproduction in second year and reproduce in the third year and some skip reproduction in the first 2 years of life and reproduce in third year attaining larger body size (svl >13.6 cm) with greater clutch size. thus, females exhibit different strategies of trade-off between growth and reproduction. in addition, the study showed that c. versicolor may lay as many as 3 clutches of eggs in a breeding season. thus, the study provides information on growth, age at sexual maturity, relationship between growth and reproduction and reproductive strategies exhibited by the lizard in captivity. keywords. growth, breeding, reproductive episodes, calotes versicolor. introduction growth pattern is a key aspect in the life history of any species (andrews, 1982; sinervo and aldoph, 1989; roff, 1992; arendt, 1997) and reproduction is the most costly event in an animal’s life. body size and growth rate are particularly important life history traits because of their relation to reproductive output, longevity, age at first reproduction, and so on (andrews, 1982; bauwens and verheyen, 1985; ferguson and talent, 1993; clobert et al., 1998; lorenzon et al, 1999; bronikowski, 2000). knowledge of growth rates can also help in determining the size at sexual maturity and the maximum size attained (andrews, 1982; bronikowski, 2000; seminoff et al., 2002). 132 b.n. pandav, b.a. shanbhag and s.k. saidapur most studies on growth rates in lizards are mainly based on mark and recapture surveys (bellairs, 1969; jenssen and andrews, 1984; shine, 1988; james, 1991; sugg et al., 1995; allan et al., 2006) or on frequency distribution of individuals in a population collected at different points of time in a year (andrews, 1976; ferguson and brockman, 1980; bishop and echternacht, 2003). surprisingly, a few studies are devoted to understand the relationship between investments in somatic growth and reproduction in reptiles (towns, 1975). studies on captive breeding of the giant day gecko (demeter and birchard, 1994), snakes (shiryaev et al., 2007), turtles (wood and wood, 1980; perez et al., 2004), crocodiles and gharial in india (whitaker and basu, 1983), do not touch upon the relationship between growth and reproduction. the studies on growth patterns in captive lizards (e.g., phrynosoma ditmarsi montanucci, 1988; p. madagascariensis demeter and birchard, 1994; sceloporus undulatus haenel and john–alder, 2002) report the growth up to sexual maturity and in relation with reproductive events like clutch size frequency of clutches in a single breeding season, etc. thus, the relationship between investments in somatic growth versus reproduction in lizards is poorly understood. calotes versicolor is a seasonal breeder. the dorsal body color of the lizard is lightbrown greyish, transverse spots on back and sides (tikader and sharma, 1992). both the sexes develop red color on antero-dorsal region during breeding season (pandav et al., 2007). it has an extended breeding season in southern india (may to october). the adult female lizard measures from 8–14.5 cm in snout vent length (svl). it is a multi-clutched lizard with clutch size varying from 7–33 eggs (shanbhag et al., 2000). hence, it forms a good model to study the energy allocation (trade-off ) between growth and reproduction. the present study deals with growth pattern in relation to sex, age at first maturity, and reproductive strategies in c. versicolor raised in captivity from hatching. materials and methods we collected c. versicolor from dharwad city (15°17’n, 75°3’e), karnataka, india in mayjune 2003. six females in early gestation were identified by the presence of eggs that felt soft during palpation of abdomen and these were maintained in outdoor terraria at department of zoology, karnatak university, dharwad. upon appearance of a single crease on the lateral side of the abdominal wall close to the hind limbs, the oviductal eggs in these lizards were deemed to have reached stage 27–28 embryos (shanbhag et al., 2003). the eggs with oviductal embryos at stage 27–28 were stripped from these lizards, since the oviposition in c. versicolor takes place at embryonic stage 27–28 (muthukarruppan et al., 1970) and were incubated in the laboratory. each gravid lizard yielded 20 ± 3 eggs (range 17–22, n = 6) and a total of 124 eggs were collected. the eggs were incubated in laboratory (at ambient temperature 27.71 ± 0.29 °c and moisture 40–45%) and hatchlings were obtained. as soon as hatchlings emerged from the eggs (n = 114), they were placed in the glass terraria (30×30×25 cm) with wire mesh at the top for ventilation. up to 10 hatchlings were reared per terrarium and 12 terraria were used for rearing hatchlings. when lizards attained two months, they were maintained in terraria (90×90×60 cm) made up of wire mesh on all sides except bottom. the lizards were kept in 1:2 male/female ratio in captivity as the males are generally seen with more than one female in the wild during breeding season. the sex of the lizards was identified at hatching by protrusion of hemipenis (harlow, 1996). thus, each terrarium housed 6 individuals (2 males, 4 133growth patterns and reproductive strategies in the lizard females) and was exposed to natural sunlight. when lizards grew six months old, they were maintained in outdoor terraria of size 150×120×120 cm with wire mesh on top and two sides, and other two sides of transparent acrylic plates. they were provided with soil-sand mixed bed, large dried twigs for climbing, the potted plants, hiding places with broken earthen pots, and a water bowl. the newborn hatchlings were fed daily with termites and early instar silkworm larvae. juveniles and subadults were fed with the silkworms, small cockroaches and grasshoppers on alternate day. adult lizards were likewise fed on alternate day with silkworms, silk-moths, cockroaches, and grasshoppers and other insects caught in swapping of insect nets. a total of 122 hatchlings (10 hatchlings obtained from wild caught lizards, that survived for more than 5 months, and 112 hatchlings obtained from 9 clutches of 7 captive born lizards) were used to study the growth patterns in this species. the snout-vent length (svl), head length (hl), head width (hw), and head depth (hd) to the nearest mm were recorded at hatching. the hatchlings were marked by thermocol beads tied loosely on the posterior part of the trunk. subsequently when hatchlings were one month old they were permanently marked by unique toe clipping. svl and head size (hl, hw, and hd) of these lizards were recorded at monthly intervals. statistical analysis in order to assess the variation in growth, the slopes for svl of individual lizard were derived by taking observations of svl in 5 consecutive months at a time moving from hatching to 4th month (h to 4), from 1 to 5, from 2 to 6, and so on till one year (12th month) by using time as the independent variable. the slopes obtained were compared by global f test to know the fluctuations in the growth pattern with respect to month and age (rohatgi, 1976). significance level was checked at 0.05. the lizards that survived for one year or more (n = 14) were considered for statistical analysis to have longitudinal data though 46% (n = 50) lizards born in captivity survived up to 7 months. to know whether there is a monthly variation in svl and growth rate of male and female lizards (within a sex), if any, the data were analyzed by friedman rank analysis. variations in monthly growth in svl, and head size (length, width, and depth) at a given age between the sexes were analyzed by mann-whitney u test. data are represented as mean ± se. to know the relationship between growth patterns and temperature, if any, the data was analyzed by linear regression. results the eggs from natural nests hatched on 80 ± 2 days. all eggs in a clutch hatched within three days. the hatching success was higher than 95% in a clutch and 88% of these hatchlings survived at the end of the first month, while 63% of them survived at the end of the 3rd month. out of these 46% lizards survived up to the 7th month. seven females and seven male lizards survived for 1 year and 2 females survived for 2 years and 8 months. overall, survival rate at the end of the first year was 13%. when lizards were 6 months old, both the sexes developed black patches ventro-laterally in the neck region, a sign of onset of sexual maturity (pandav et al., 2007). the males exhibited red colored hues on the head and gular area. the lizards began exhibiting courtship behavior when they were between 7–8 months. svl of these males was 9.4 ± 0.02 cm (n =17) and that of females was 8.3 ± 0.13 cm (n = 33). the mated females were soon gravid and laid eggs in captivity (fig. 1). 134 b.n. pandav, b.a. shanbhag and s.k. saidapur among the captive born lizards that survived for a year or more, 6 of them (l1 to l5 and l8) bred in the 1st year between the age of 7-8 months (first breeding season after their hatching), 1 lizard (l6) skipped breeding in the 1st year and bred in the 2nd year at an age of 21 months and another lizard (l7) skipped breeding in the first two years and fig. 1. calotes versicolor laying eggs in captivity. fig. 2. changes in the snout to vent length (svl) in female c. versicolor that bred in first year and those that skipped breeding in the first or/and second year. arrowheads indicate the month when the lizard oviposited. note that the variations in growth pattern of lizards reflect a trade-off between growth and reproduction. h = month at hatching (october). 135growth patterns and reproductive strategies in the lizard bred in the 3rd year at the age of 32 months. two lizards laid 2 clutches in first breeding season, the interval between two clutches was 25 days for one lizard and 45 days for the other. lizard (l8) laid 3 clutches in first year (fig. 2), not included in analysis as it died at the age of 10 months), laid second clutch of eggs after 25 days after ovipositing first clutch and third after 51 days of ovipositing second clutch not included in analysis as it died at the age of 10 months. this lizard died while ovipositing the third clutch. the lizards that bred and laid eggs within the first year, the clutch size ranged from 11–15. however, the lizards that skipped the breeding in the first year or two years the clutch size was 20–21. pattern of growth changes in svl of male and female c. versicolor for a period of one year is shown in table 1. svl of both the sexes was comparable up to 4 months. however, the males grew larger than the females from 5th month onwards (table 1, 2). the female svl increased by 5–7 mm/month during the initial 4 months. the growth in svl increased rapidly by 8–9 mm/month in the next 3 months (march to may), prior to onset of breeding season (f7 = 83.83, p = 0.000). the growth in svl was significantly low during 8 to 12 months of age (june to october) (table 1, 3). in males, svl increased by 3–12 mm/month during the first 4 months. the svl increased rapidly (12–13 mm) in the 5th month to attain a size of 73.14 mm ± 0.9 in the 6th (april) month (table 1). the growth in svl of males was low when the lizards were 8 to 9 months (june to july) coinciding with the breeding activity (f7 = 22.39, p = 0.021). an increase in svl was seen in 10th to12th (august to october) months (table 1). table 1. shows the snout-vent length (svl), head length (hl), head width (hw), and head depth (hd) in c. versicolor from hatching up to one year of age h = month at hatching (october); n =14 (females n = 7; males n = 7). months svl (mm ± se) hl (mm ± se) hw (mm ± se) hd (mm ± se) female male female male female male female male h 26.87 ± 0.7 25.53 ± 0.7 9.54 ± 0.4 9.27 ± 0.1 5.97 ± 0.1 5.81 ± 0.1 5.23 ± 0.2 5.07 ± 0.1 1 33.02 ± 1.2 37.36 ± 0.6 10.98 ± 0.3 11.36 ± 0.3 6.77 ± 0.1 6.9 ± 0.1 5.76 ± 0.15 6.02 ± 0.1 2 38.38 ± 1.1 40.52 ± 0.6 13.16 ± 0.4 12.86 ± 0.2 7.45 ± 0.1 7.56 ± 0.1 6.75 ± 0.07 6.8 ± 0.1 3 42.97 ± 0.5 43.77 ± 0.8 14.62 ± 0.4 14.35 ± 0.6 8.09 ± 0.2 8.49 ± 0.2 7.28 ± 0.1 7.35 ± 0.2 4 48.96 ± 0.7 48.18 ± 1.4 15.63 ± 0.3 18.38 ± 1.2 8.64 ± 0.1 10.61 ± 0.7 7.75 ± 0.2 9.13 ± 0.6 5 52.85 ± 0.3 60.88 ± 2 18.06 ± 0.4 22.91 ± 0.8 9.58 ± 0.1 12.25 ± 0.5 8.57 ± 0.2 10.71 ± 0.4 6 60.49 ± 1.2 73.14 ± 0.9 20.01 ± 0.7 25.6 ± 0.8 10.49 ± 0.2 13.57 ± 0.3 9.61 ± 0.3 11.91 ± 0.2 7 69.87 ± 3 90.59 ± 1.4 22.33 ± 0.5 27.7 ± 0.4 11.91 ± 0.3 15.39 ± 0.3 10.57 ± 0.3 12.99 ± 0.2 8 81.48 ± 2.8 92.79 ± 1.3 23.89 ± 0.2 30.52 ± 0.6 13.54 ± 0.2 17.99 ± 0.8 12.3 ± 0.4 14.29 ± 0.2 9 86.98 ± 2.7 94.89 ± 1.6 25.39 ± 0.6 32.66 ± 0.8 15.57 ± 0.8 19.46 ± 0.9 12.99 ± 0.5 16 ± 0.4 10 90.72 ± 3 105.2 ± 2.0 27.88 ± 1.2 33.65 ± 1.4 16.31 ± 0.5 21.37 ± 0.9 13.98 ± 0.6 16.7 ± 0.5 11 102.95 ± 2.2 113.02 ± 2.9 27.96 ± 0.9 37.5 ± 1.04 16.72 ± 0.8 23.5 ± 0.5 14.7 ± 0.5 18.5 ± 0.5 12 108.25 ± 2.2 120.32 ± 2.8 32.13 ± 0.8 40 ± 0 21.17 ± 1.2 27.2 ± 0.9 16.04 ± 0.4 20.1 ± 0.5 136 b.n. pandav, b.a. shanbhag and s.k. saidapur there was no significant difference in hl and hw between the sexes during the first 3 months. however, hl and hw were significantly larger in males than in females from 4th month onwards (table 1, 2). also, a significant difference in the hd was observed in lizards of 5 months or older, the head depth being greater in males than females (table 1, 2). linear regression analysis revealed that ambient temperature does not have any influence of temperature on growth patterns (r2 = 0.070, p = 0.696). growth, age at first reproduction and reproductive strategies exhibited by female individual growth in svl of 7 female lizards that were born, bred and survived for a year or more in captivity is depicted in fig. 2. the lizards l1 to l5 attained sexual maturity at the age of 7.42 ± 0.5 months and reproduced in the first breeding season (june-august, 2004) at 7.5–9.8 cm svl (fig. 2). the clutch size these lizards ranged from 11 to 15. among these 4 individuals, l4 lizard also bred in the second year (june-july, 2005) at the age of 20 months (clutch size 20). however, this lizard died due to oviductal infection. svl of l1 to l3 was more than that of l4 at the onset of subsequent breeding season (fig. 2). the lizard (l5) laid 2 clutches of eggs at an interval of 45 days between june-august (2004) at 9.2 cm svl, and clutch size was 11and 15. this lizard attained the size of 10.9 cm at the end of the first year. it skipped reproduction in the second year and attained a size of 13.3 cm at the end of second year of age (june, 2005) (fig. 2). it bred in the 3rd year at the age of 30 months. table 2. shows u and p-values from mann whitney u test for monthly variations in snout vent length (svl), head length (hl), head width (hw), and head depth (hd) between male and female c. versicolor from hatchling until one year of age (n = 14). months svl hl hw hd u p u p u p u p h 15 0.224 15 0.224 19 0.482 19 0.482 1 7 0.025 17 0.337 20 0.565 16 0.276 2 17 0.337 21 0.654 19 0.482 22.5 0.798 3 20 0.565 21 0.654 16 0.276 22 0.749 4 17 0.338 9 0.048 8 0.035 13 0.141 5 1 0.003 1 0.003 2 0.004 2 0.004 6 0 0.002 0 0.002 0 0.002 1 0.003 7 0 0.002 0 0.002 0 0.002 0 0.002 8 0.5 0.002 0 0.002 0 0.002 4 0.008 9 7 0.025 0 0.004 0 0.027 2 0.01 10 0 0.002 3 0.038 0 0.008 3 0.038 11 5.5 0.015 0 0.014 0 0.013 0 0.013 12 5.5 0.015 0 0.007 0.5 0.019 0 0.013 h = month at hatching (october). significant values are given in bold. 137growth patterns and reproductive strategies in the lizard table 3. shows f and pvalues from global f test for changes in growth in snout vent length (svl) in c. versicolor from hatching until a period of one year (n = 14). months f – value p – value males (n = 7) females (n = 7) males females h to 4 0.35 1.00 >0.05 >0.05 1 to 5 0.66 1.25 >0.05 >0.05 2 to 6 0.32 0.28 >0.05 >0.05 3 to 7 0.22 0.90 >0.05 >0.05 4 to 8 0.28 0.68 >0.05 >0.05 5 to 9 0.74 0.02 >0.05 >0.05 6 to 10 1.62 0.91 >0.05 >0.05 7 to 11 3.19 5.87 <0.05 <0.05 8 to 12 1.34 6.55 >0.05 <0.05 h = month at hatching (october) the lizard l6 did not breed in the first year (2004) and grew up to 11.1 cm. this lizard reproduced for the first time at age of 21 months (june 2005) at svl of 11.7 cm and laid 20 eggs (fig. 2). the lizard (l7) that did not breed in the first year attained a size of 11.5 cm and was the largest among the females at the end of the first year. this lizard did not show any breeding activity in the 2nd year also. the lizard showed 3–5 mm/month growth throughout the second year and attained the size 13.4 cm at the end of the 2nd year. it reproduced in the 3rd year, at the age of 32 months (june, 2006) and at size of 13.7 cm and developed a clutch of 21 eggs. however, it died before oviposition due to some infection in the digestive tract. the lizards (l1 to l5) that bred in the first year showed almost negligible growth in svl during reproductive period and attained svl of 9.15 ± 0.84 cm at the age of one year. in contrast, the lizards (l6 and l7) that did not reproduce in the first year grew in their svl and attained size of 10.3 and 11.5 cm at the age of one year. the lizards (l5 and l7) that did not reproduce in the second year also showed growth in their svl and attained size of >13 cm at the age of 2 years. in general, growth in svl was low during the breeding and also in the 2nd and 3rd year of their life when compared to that in the first year (fig. 2). discussion the present study provides the first information on the age at maturity, growth, and breeding frequency in captivity in an arboreal agamid lizard the c. versicolor from the tropical region of south india. the earlier reports suggested that c. versicolor attains maturity in 9–12 months (asana, 1931) and lays more than one clutch (shanbhag, 2002). the present findings showed that some members in a population of c. versicolor may attain sexual maturity as early as seven months of age and breed in first year while others may breed in 2nd or 138 b.n. pandav, b.a. shanbhag and s.k. saidapur 3rd year. the earlier assumption that c. versicolor is a multi-clutched lizard (shanbhag, 2002) is confirmed by the present study. one of the lizards developed three clutches of eggs during the same breeding season. thus, the present study showed that if food supply is uninterrupted, c. versicolor may produce as many as 3 clutches in the first year itself. it is well known that the growth rate in herpets changes between cool and warm seasons in temperate habitats (bjorndal et al., 1998; wapstra et al., 2001), with arrest of growth during cooler seasons. however, present study revealed that growth is low, but not arrested in c. versicolor during winter. statistical analysis also showed that temperature per se has no significant influence on growth rate. it may be because winter is not so severe in tropical part of india unlike in temperate areas. further, the growth rate though low continues even after attaining sexual maturity in c. versicolor as in crotalus horridus atricaudatus, leiolopisma suteri and s. undulatus (towns, 1975; shine and iverson, 1995; haenel and john–alder, 2002). there are several reports comparing growth rates between the sexes in lizards based on the wild populations and mark-recapture studies (bellairs, 1969; jenssen and andrews, 1984; shine, 1988; james, 1991; sugg et al., 1995; allan et al., 2006). the males of agama agama, phrynocephalus interscapularis, anolis opalinus and cophosaurus texanus scitulus grow larger than the females. the females exhibit slow growth rate as they invest more energy in reproduction (bellairs, 1969; jenssen and andrews, 1984; shine, 1988; sugg et al., 1995). the study by james (1991) showed that the female of the genus ctenotus grows slower than the male but reaches the larger asymptotic body size than its male counterparts in most species. males and females of common wall lizard, podarcis muralis reach similar snout–vent lengths, but males have relatively longer tails and heavier in body mass (allan et al., 2006). a study involving demographic analysis of growth rate in field and also in controlled laboratory condition on eastern fence lizard, sceloporus undulatus reported that in natural environment females grow faster than their male counterparts before the end of the first year (before sexual maturity) while in laboratory there was no sex specific difference in growth until maturity (haenel and john– adler, 2002). the present study shows that svl growth is more pronounced in males than in females before attaining sexual maturity in captive c. versicolor. hence, males attain slightly larger size than the females at first maturity. further, males are also larger than females at one year of age. these observations suggest that males are larger in size than the females at comparable age. sexual size dimorphism (ssd) has been reported in several reptiles by either collecting data on wild caught species or by mark – recapture studies (berry and shine, 1980; gibbons and lovich, 1990; forsman and shine, 1995; brana, 1996; huang, 1996; fernandez and rivera, 2001; webb et al., 2002; pinto et al., 2005). however, studies on ssd in reptiles by long term growth studies in laboratory are scarce (haenel and john-alder, 2002). the differential growth rate between the sexes prior to sexual maturity seems to lead to ssd in tropidurus torquatus (pinto et al., 2005). an earlier study by radder et al (2001) reported that svl biased ssd does not occur in c. versicolor. conclusions of radder et al (2001) were based on wild caught lizards and they did not consider the age of the lizards. but the present study showed a significant difference in svl between the sexes from 5 months of age and also after maturity in the same age group suggesting ssd in svl. however, ssd observed in relative head size in the present study is in conform139growth patterns and reproductive strategies in the lizard ity with the findings of radder et al (2001). the larger body and head sizes in males may help to overpower its rival and get access to the female similar to reported in eumeces laticeps, niveoscincus microlepidotus, and lacerta vivipara (cooper and vitt, 1993; olsson et al., 2002; hofmann and henle, 2006). during breeding season, male-male combat is often seen in c. versicolor possibly over a female. during such combats we have seen that males virtually stand on their hind legs and tail and blow each other with heads. two important life history traits in lizards are the size at first maturity and fecundity. a trade–off between the body size and age at reproduction is reported in s. undulatus (ferguson and talent, 1993) and microlophus delaninis (jordan and snell, 2002). also, the clutch size is reported to be positively correlated to the body size in lizard species (ferguson and talent, 1993; blanckenhorn, 2000; shanbhag et al., 2000; radder and shanbhag, 2003, 2004). thus, larger body size is related to the fecundity/fitness of the female. however, attainment of a larger body size delays reproduction and is often accompanied by a lower probability of survival to the next breeding season. the present study revealed a plasticity in size and age at maturity operating among a population of c. versicolor. some females may mature at smaller size with small clutch size as early as at seven months of age like the lizard l3 in the present study while others may skip reproduction at first breeding season after their birth, grow larger in size, reproduce in second year and thus increase fecundity with greater clutch size (lizard l6). yet, others (lizard l5) reproduce in the first year and skip reproduction in the second year and reproduce in the third year with even greater clutch size. also, some females do not reproduce in first two years, grow in size and reproduce in the third year with highest fecundity (lizard l7). thus, a tradeoff is seen between age at sexual maturity, body size, and fecundity in c. versicolor. the present study shows that differential growth patterns in female c. versicolor lead to the variations in key life history traits such as age at maturity, body size and annual and lifetime reproductive output of individuals in a population. though our sample size of lizards beyond one year was small, the data highlight different adaptive strategies (growth and reproduction) operating in a population of c. versicolor. acknowledgements the work is supported by a grant (no.sr/so/as-35/2003) from dst, new delhi awarded to bas and partly supported by ugc-sap ii-drs grant, new delhi. bnp is grateful to ugc, new delhi for 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(1980): captive breeding of the green sea turtle (chelonia mydas). proceedings of the annual meeting world mariculture society. 8: 533–541. acta herpetologica 4(1): 57-71, 2009 a comparative study of contractility of the heart ventricle in some ectothermic vertebrates sergey kharin, dmitry shmakov laboratory of cardiac physiology, institute of physiology of the komi science centre, ural branch of the russian academy of sciences, 50 pervomayskaya st., syktyvkar, komi republic, 167000, russia. corresponding author. e-mail: s.kharin@physiol.komisc.ru (s.kharin@mail.ru) abstract. the purpose of this study was to analyze contractility of the heart ventricle in selected reptilian and amphibian species having the same ventricular excitation pattern. systolic time intervals and indices of contractility of the heart ventricle were measured in anaesthetized frogs, snakes, and tortoises by use of polycardiography. the electromechanical delay was significantly shorter in tortoises compared with the other two species. the isovolumetric contraction time in frogs was approximately twofold longer than in reptiles. the pre-ejection period was the longest in frogs and the shortest in tortoises, whereas snakes were intermediate. the ejection time was slightly longer in tortoises compared with the other two species. the greatest isovolumetric contraction index and the smallest myocardial tension index corresponded to the frog and tortoise heart ventricle, respectively. the intrasystolic index in tortoises was significantly greater than in frogs, whereas quite similar to that in snakes. the frog ventricle had lower contractility compared with the reptilian one. although ventricular contractility tended to be lower in snakes compared with tortoises, this difference was not statistically significant. possible causes for these differences are discussed. we suppose a large variety in ventricular contractility among amphibian and reptilian species having the same ventricular activation pattern. this variety may be conditioned by heart anatomy, intracardiac shunting, lifestyles, and habitats. it can only be hypothesized that on the average, ventricular contractility is higher in reptiles compared with amphibians and in chelonians compared with snakes. keywords. heart function, cardiac performance, amphibians, reptiles, frogs, snakes, tortoises. introduction the heart fulfils the pump function and pushes blood into the arterial system providing blood circulation. this is due to myocardium contraction that results from its electrical excitation. four types of ventricular myocardial activation were developed as a result of the heart evolution in vertebrates (roshchevsky and shmakov, 2003). however, the question, how different excitation patterns relate to cardiac performance, has not been addressed yet. 58 s. kharin and d. shmakov amphibians and reptiles are characterized by the same ventricular activation pattern, which presents the successive character of excitation spreading from endocardium to epicardium and from the base to the apex of the heart ventricle (shmakov and roshchevsky, 1982; shmakov and abrosimova, 1989; roshchevsky and shmakov, 2003; azarov et al., 2007). this pattern differs from ventricular excitation patterns in fishes and homoeothermic vertebrates (roshchevsky and shmakov, 2003). at the same time, it is not known whether contractility of the heart ventricle differs between vertebrate species. little information is available concerning contractility of the heart ventricle in reptiles and amphibians (furnival et al., 1973; sham et al., 1989). systolic time intervals of the heart ventricle have not been measured in ectothermic vertebrates, although some attempts have been done for frogs (shelton and jones, 1965a, b) and snakes (johansen, 1959; johansen and holl, 1960). the purpose of the study reported here was to analyze by measurements of systolic time intervals, how ectothermic vertebrates with the same ventricular excitation pattern differ in contractility of the heart ventricle. in addition, a comparison of ventricular contractility of three-chambered reptilian hearts was made. the frog rana temporaria was chosen as a widely-distributed amphibian species, the snakes natrix natrix and n. tessellata as typical ophidian species, and the central asian tortoise testudo horsfieldii was selected as a representative of chelonians. materials and methods animals, anesthesia, and surgical procedures the investigation conformed with the guide for the care and use of laboratory animals published by the us national institutes of health (nih publication no. 85-23, revised 1996). experiments were carried out on anuran amphibians r. temporaria (n = 6), grass snakes n natrix and dice snakes n. tessellata (n = 4), and tortoises t. (agrionemys) horsfieldii (n = 6). the frogs were collected in summer in the central regions of the european part of russia and stored at 0-4 ºc till the use in experiments. the reptiles were caught in the moscow region (snakes) of russia and in kazakhstan (tortoises) a few weeks before the experiments began. all animals were obtained from commercial suppliers. after transportation to our laboratory (a few days before the experiments), animals were kept in a terrarium at 20-25 ºc and supplied with food and tap water. the experiments were performed from january to may (frogs), in july (snakes, tortoises) and november (snakes). frogs and tortoises varied in weight from 96 to 116 g (mean ± sd: 102 ± 10 g) and from 80 to 138 g (108 ± 20 g), respectively. snakes varied in length from 62 to 83 cm (70 ± 9 cm) and in weight from 38 to 102 g (62 ± 28 g, p < 0.05 compared with the other two species). the experiments were performed at a room temperature (18-22ºc). frogs were anaesthetized by immersion in a 40% ethanol solution. snakes and tortoises were anaesthetized by an intra-abdominal injection of xylazine (40 μg/g) and sodium thiopental (50 μg/g). in case of need, ether was used for additional anesthesia. each frog was placed ventral side up, the heart was exposed by cutting of clavicles and coracoids and removing of the sternum. each reptile was placed ventral side up, tracheotomized, and mechanically ventilated. mechanical lung ventilation was performed to avoid hypoxia, resulted from the depressant action of sodium thiopental on respiration. the heart was exposed by a longitudinal midline incision of the ventral side of the body in snakes and by removing of the plastron in tortoises. at the end of the experiment, each animal was euthanized. frogs were euthanized by decapitation followed by pithing. reptiles were euthanized with an overdose of sodium thiopental (100 59heart contractility in ectothermic vertebrates μg/g, intra-abdominally), which caused cardiac arrest (diagnosed from an ecg). after euthanasia, the heart was removed and the ventricle was weighed in each animals examined. electrocardiographic and apexcardiographic recordings in each anaesthetized animal, polycardiographic tracings were obtained by means of a computer system (poly-spectrum-eps, neurosoft, russia) that had a speed of 50 mm/s and an accuracy of 24 bits (fig. 1). the polycardiogram consisted of a standard bipolar lead ecg and apexcardiogram that were recorded synchronously during 30 to 60 s. the electrocardiographic channel of the polygraph had a bandwidth and sampling rate of 0.5 to 75 hz and 1000 hz, respectively. to obtain a standard bipolar lead ecg, needle electrodes were attached intramuscularly in a modified einthoven lead system. in frogs and tortoises, the red and yellow electrodes were attached to the proximal aspect of the right and left forelimb, respectively; the green electrode was attached to the proximal aspect of the left hind limb; the ground electrode was placed on the right hind limb. in snakes, the placement of electrodes relatively to the position of the heart was the same as in frogs. the position of the heart in snakes was defined by inspection and palpation of the heart beat. the three standard bipolar leads were i, ii, and iii as derived from einthoven’s triangle: for the lead i, the negative component was the red electrode and the positive component was the yellow electrode; for the lead ii, the negative component was the red electrode and the positive component was the green electrode; for the lead iii, the negative component was the yellow electrode and the positive component was the green electrode. the ecg was standardized so that 20 mm was equivalent to 1 mv. fig. 1. representative polycardiographic tracing obtained from a snake, indicating the simultaneous recording of a standard bipolar lead ii ecg (“red electrode – green electrode”) and apexcardiogram (acg). electrocardiographic intervals are marked in the ecg: the p wave (p), qrs complex (qrs), t wave (t), and q-t interval (q-t). the ejection peak (e) and the d point are indicated on the acg. systolic time intervals are marked: the electromechanical delay (q-s1), isovolumetric contraction time (ict), pre-ejection period (pep), and ejection time (et). paper speed = 50 mm/s; 2 cm = 1 mv. 60 s. kharin and d. shmakov to obtain apexcardiographic recordings, a tensometric pulse wave transducer (neurosoft, russia) (frequency range, 0.3 to 200 hz; sensitivity, 100 mv/pa; and time constant, 0.6 s) was fixed near the heart ventricle. the position of the transducer was adjusted to the apex of the heart ventricle and was modified to improve unsatisfactory tracings, if any. the sampling rate and sensitivity of the apexcardiographic channel of the polygraph were 1000 hz and 0.3 to 1.5 86 mv/mm, respectively. data analysis, calculations and statistics the ventricle index was calculated as ventricle weight / body weight. heart rate was determined from measurement of the r-r interval in the limb lead ii ecg. the durations of the qrs complex and q-t interval were measured. the electromechanical delay (emd) was measured from the onset of the qrs complex in the limb lead ii ecg to the onset of the upstroke in the apexcardiographic tracing (fig. 1). the isovolumetric contraction time (ict) was measured from the onset of the upstroke to the ejection peak in the apexcardiogram. the pre-ejection period (pep) was calculated as emd plus ict. the ejection time (et) was measured from the ejection peak to the d point indicated on the apexcardiogram. the duration of mechanical systole (sm) was computed as ict plus et. the duration of electromechanical systole (so) was calculated as emd plus sm. the duration of diastole was calculated by subtracting so from the r-r interval. on the basis of the measurements, indices of ventricular contractility were defined. the myocardial tension index was defined as pep/so. this index reflects the time a heart needs to prepare for blood ejection (i.e., unproductive expenditure of contraction time). the myocardial tension index decreases when systolic function improves. the pep-to-et ratio, which is a commonly used index of ventricular contractility and performance, was determined. the pep-to-et ratio decreases when systolic function improves, whereas reduced ventricular contractility is reflected by an increase in the pep-to-et ratio. the intrasystolic index was calculated as et/sm. this index reflects the time a heart needs to eject blood (i.e., productive expenditure of contraction time). the intrasystolic index increases when systolic function improves. the isovolumetric contraction index was computed as ict/pep. reduced ventricular contractility is reflected by an increase in ict/pep. the duration of cardiac output ejection was defined as a product of et and heart rate. all values for each animal were averaged on the basis of measurements from 5 consecutive cardiac cycles. statistical analysis was performed using descriptive statistics. data are presented as mean ± sd. differences were considered to be significant at p < 0.05. results heart ventricle weight and electrocardiographic parameters the mean values of heart ventricle weight were found to be similar in frogs (216 ± 53 mg), snakes (271 ± 126 mg), and tortoises (231 ± 46 mg). the ventricle index in snakes (4.39 ± 0.52 mg/g) was twofold greater (p < 0.001) than in frogs (2.10 ± 0.32 mg/g) and tortoises (2.12 ± 0.12 mg/g). the durations of the qrs complex, r-r interval, and q-t interval were summarized (table 1). there were no significant differences between the three species in the r-r interval, which was slightly longer in frogs and shorter in snakes. the duration of the qrs complex was more than 1.5-fold greater in snakes compared with the other two species. in comparison with frogs and tortoises, snakes had intermediate values of the q-t interval. 61heart contractility in ectothermic vertebrates table 1. electrocardiographic intervals and heart rate for the species studied. values are mean ± sd. ranges of values are given in round brackets. * p < 0.05 vs. snakes. parameters frog n = 6 snake n = 4 tortoise n = 6 r-r interval, ms 2138 ± 782 (1386-3638) 1776 ± 161 (1591-1974) 1934 ± 420 (1573-2678) heart rate, min-1 30 ± 9 (16-44) 34 ± 2 (30-38) 32 ± 6 (22-38) qrs complex, ms 100 ± 22 * (80-140) 169 ± 16 (150-190) 107 ± 32 * (55-155) q-t interval, ms 1016 ± 169 (805-1225) 1112 ± 34 (1068-1148) 1195 ± 154 (980-1450) table 2. systolic time intervals, their proportions in the cardiac cycle, and indices of ventricular contractility for the species studied. values are mean ± sd. proportions of systolic time intervals of the cardiac cycle are given in round brackets. r-r, the r-r interval of ecg; emd, the electromechanical delay; ict, the isovolumetric contraction time; pep, the pre-ejection period; et, the ejection time; sm, mechanical systole; so, electromechanical systole; do, diastole; mti, the myocardial tension index; isi, the intrasystolic index; ici, the isovolumetric contraction index; pep/et, the pep-to-et ratio; tco, the duration of cardiac output ejection. * p < 0.05 vs. tortoises. † p < 0.05 vs. frogs. parameters frog n = 6 snake n = 4 tortoise n = 6 r-r, ms (%) 2138 ± 782 (100) 1776 ± 161 (100) 1934 ± 420 (100) emd, ms (%) 117 ± 31 * (5.9 ± 0.8) 143 ± 29 * (8.0 ± 0.9 * †) 90 ± 19 (4.8 ± 0.4) ict, ms (%) 209 ± 103 (10.1 ± 2.0 *) 111 ± 10 † (6.3 ± 0.5 *) 91 ± 39 † (4.7 ± 0.7) pep, ms (%) 327 ± 131 * (16.0 ± 2.7 *) 254 ± 33 * (14.3 ± 1.1 *) 181 ± 55 (9.5 ± 1.0) et, ms (%) 777 ± 170 (39.4 ± 5.7) 762 ± 207 (42.5 ± 5.6) 829 ± 277 (43.1 ± 4.4) sm, ms (%) 982 ± 150 (49.3 ± 5.4) 875 ± 201 (48.9 ± 5.1) 919 ± 261 (47.8 ± 4.0) so, ms (%) 1100 ± 163 (55.3 ± 5.9) 1018 ± 216 (56.9 ± 5.3) 1010 ± 257 (52.6 ± 3.8) do, ms (%) 1038 ± 749 (44.7 ± 6.0) 758 ± 100 (46.3 ± 3.1) 925 ± 294 (47.4 ± 3.8) mti 0.296 ± 0.115 0.255 ± 0.045 0.191 ± 0.081 † isi 0.791 ± 0.112 0.865 ± 0.031 0.892 ± 0.066 † ici 0.616 ± 0.082 0.440 ± 0.052 † 0.490 ± 0.072 † pep/et 0.45 ± 0.26 0.35 ± 0.08 0.24 ± 0.14 tco, s 24 ± 8 25 ± 5 26 ± 7 62 s. kharin and d. shmakov systolic time intervals and indices of ventricular contractility the values of systolic time intervals were summarized (table 2). the differences in the duration of the r-r interval were accounted for to a greater extent by the duration of diastole and to a lesser extent by the duration of systole. the ejection time was quite similar in the studied animals, although being slightly longer in tortoises compared with the other two species. the differences in the pre-ejection period and the duration of its constituent parts were found to be significant between the animals examined. the electromechanical delay in tortoises was shorter than in the other two species. the isovolumetric contraction time in frogs was approximately twofold longer than in both reptiles. as a consequence, the longest pre-ejection period was in frogs, whereas the shortest one was in tortoises. the significant differences in the electromechanical delay, isovolumetric contraction time, and pre-ejection period were also found to be between the animals, when assessing proportions of various phases of the cardiac cycle (table 2). the proportion of the isovolumetric contraction time was significantly greater in frogs compared with tortoises, whereas a less difference was found to be between snakes and tortoises. the proportion of the electromechanical delay was significantly greater in snakes compared with the other two species. as a result, the proportion of the pre-ejection period was found to be quite similar in snakes and frogs but significantly higher in these species compared with tortoises. proportions of the ejection time, mechanical systole, electromechanical systole, as well as diastole, did not differ between the species examined. the values of indices of ventricular contractility were summarized (table 2). the greatest isovolumetric contraction index was found to be in frogs. the smallest myocardial tension index was observed in tortoises. the value of the intrasystolic index in tortoises was significantly greater than in frogs and quite similar to that in snakes. the variety in pep-to-et ratios among the species was considerable but not statistically significant. discussion ventricle index ventricle weights were comparable in all species studied. at the same time, the ventricle index was similar in tortoises and frogs, whereas it was significantly greater in snakes compared with the other two species. these differences are probably conditioned by lifestyle, with active species having a greater ventricle index. the heart index varies in a wide range among anuran amphibians, chelonians, and snakes (crile and quiring, 1940; wilber, 1962; wang et al., 2002; vinogradov and anatskaya, 2006). although the heart index on the average is comparable between these ectotherms, it is higher in snakes. this is in consistent agreement with our data. however, there is some disagreement between our data and other findings (vinogradov and anatskaya, 2006). in natrix snakes, the ventricle index in the present study is 2.2-fold higher than the heart index reported for the other study (vinogradov and anatskaya, 2006). this disagreement is likely attributable to the significant difference between snakes from the two studies in body weight. 63heart contractility in ectothermic vertebrates electrocardiographic data the values of electrocardiographic intervals in frogs reported in our study are approximately twofold higher than those of other studies, which were performed at a similar temperature (shmakov and abrosimova, 1989; cakir and strauch, 2005). on the other hand, our data are in consistent agreement with findings in toads (chapovetsky and katz, 2003), the mean duration of the qrs complex reported in our study for tortoises is similar to that in turtles (wilber, 1962). on the other hand, our data differ from other findings in tortoises of the same species (shmakov and roshchevsky, 1982; roshchevsky and shmakov, 2003). the difference between study results is likely attributable to differing temperatures. the duration of the qrs complex reported in the present study is twoto threefold greater than the duration of the ventricular excitation process evaluated by use of intracardiac electrography at a higher temperature (shmakov and roshchevsky, 1982; roshchevsky and shmakov, 2003). in another study, the duration of depolarization of the ventricular epicardial surface in chelonians (pseudemys and testudo) during lung ventilation (190-210 ms) (burggren, 1978) is greater than the duration of the qrs complex in tortoises in our study. the difference between study results is likely attributable to differing ventricle sizes. heart sizes in tortoises in our study were approximately twofold less than those in the other study (burggren, 1978). the mean duration of the qrs complex reported in our study for snakes is 2.4-fold greater than that reported in the other study for ophidian species (mullen, 1967). the other study used a protocol that involved a wide temperature range (mullen, 1967). the values of the duration of the q-t interval in reptiles in our study are within a range of values reported by other researches for chelonian and ophidian species (wilber, 1962; mullen, 1967). the differences between study results in electrocardiographic data may be attributable to differing experimental conditions, such as anesthesia, temperature, and techniques, as well as interspecies differences in cardiovascular parameters, temperature and seasonal changes in cardiac activity in ectothermic vertebrates (wilber, 1962; risher and claussen, 1987; rocha and branco, 1997, 1998; chapovetsky and katz, 2003). ventricular function in amphibians and reptiles is known to be influenced by lung ventilation (burggren, 1978; segura et al., 1981). probably, age and sex have an influence on electrical activity of the heart and, therefore, on electrocardiographic parameters in ectothermic animals. it should be mentioned that the duration of electrical (excitation and recovery) and thereof mechanical (contraction and relaxation) processes in the hearts of ectothermic vertebrates depends on heart size. this also contributes to both intraand inter-observer variability of results. systolic time intervals little information is available concerning the phase structure of the cardiac cycle in amphibians (shelton and jones, 1965a, b) and reptiles (johansen, 1959; johansen and holl, 1960). in the present study, an attempt was made to obtain information on systolic time intervals and indices of contractility of the heart ventricle in frogs rana temporaria, snakes natrix, and tortoises testudo horsfieldii. the values of the duration of ventricular systole reported in our study for snakes are significantly greater than those of another study (johansen, 1959). the values of the dura64 s. kharin and d. shmakov tion of the ejection period reported by other researchers for snakes (n. natrix and vipera berus, johansen and holl, 1960) are less than those of our study. one might assume that the differences between study results are most likely attributable to differing heart rates, because the ejection time and duration of ventricular systole are correlated with heart rate in endothermic vertebrates (goch, 1981; kharin and shmakov, 2006) with various patterns of ventricular excitation. however, an additional investigation is required to confirm this assumption for ectothermic vertebrates. the values of the isovolumetric contraction time reported in our study for snakes are in consistent agreement with other findings in snakes n. natrix (johansen, 1959). on the other hand, our data differ from values reported for the duration of isovolumetric contraction in snakes n. natrix and vipera berus) in another study, in which the isovolumetric contraction was defined by use of cineradiography (johansen and holl, 1960). the value of the isovolumetric contraction time in frogs reported in our study is 2.5fold higher than that reported by other researches (shelton and jones, 1965a). this difference between study results may be attributable to interspecies differences, as well as differing experimental conditions (anesthesia and methods) and heart sizes. the isovolumetric contraction time should be less in a small heart ventricle compared with a large one. frogs used in our study and another one (shelton and jones, 1965a) differed significantly in body weights; therefore, one might assume that heart sizes also differed. it should also be noted that in ectothermic vertebrates, the isovolumetric contraction time is possibly correlated with heart rate. if that’s the case, this relation may contribute to the difference between study results in isovolumetric contraction times. our data differ significantly from values reported for heart rate in frogs and snakes in other studies (shelton and jones, 1965a; johansen, 1959; johansen and holl, 1960). however, an additional investigation is required to confirm this assumption, because there is a disagreement among data on correlation between the isovolumetric contraction time and heart rate reported for endothermic vertebrates (goch, 1981; kharin and shmakov, 2006) with different patterns of ventricular excitation. we found the significant difference between the species in the electromechanical delay, isovolumetric contraction time, and pre-ejection period. the similarity of other systolic time intervals (i.e., the ejection time, mechanical systole, and electromechanical systole), as well as diastole, in the three species may be attributable to the similar length of the cardiac cycle. the electromechanical delay and qrs complex were found to be longer in snakes compared with the other two species. the proportion of the cardiac cycle that was comprised of the electromechanical delay was also greater in snakes. these findings are quite appropriate to the greater ventricle index in snakes in our study, because a large heart needs more time to be excited. during the isovolumetric contraction of a heart ventricle, intraventricular pressure rises to the pressure level in the aortic arches. we found the isovolumetric contraction time to be significantly less in both reptiles compared with frogs. taking into account this fact, one might assume that the frog ventricle developed higher pressure than the reptilian one, whereas a similar pressure level was developed by the heart ventricle in snakes and tortoises. however, systemic blood pressure in frogs r. temporaria (shelton and jones, 1965a, 1968) is approximately twofold lower than in snakes natrix (johansen, 1959; kharin, s.n., unpubl.). in comparison with the frogs and snakes, tortoises t. graeca (shelton and burggren, 1976), 65heart contractility in ectothermic vertebrates closely related to tortoises t. horsfieldii, have intermediate values for systemic blood pressure. thus, it is most likely, that among the species studied, the frog ventricle developed the lowest pressure for the longest period. snakes and tortoises in our study had comparable values for the isovolumetric contraction time, although there was a significant difference between these reptilian species in the proportion of the cardiac cycle that was comprised of the isovolumetric contraction time. this difference was probably related to the greater ventricle index in snakes compared with tortoises. thus, one might assume that in comparison with the tortoise heart ventricle, the snake one developed higher pressure for the longer period. this assumption is in consistent agreement with an influence of gravity on cardiovascular parameters in snakes (seymour, 1987; young et al., 1994; seymour and arndt, 2004). intraventricular and systemic blood pressures are quite comparable both among anuran amphibians (vogt, 1941; shelton and jones, 1965a, 1965b, 1968; furnival et al., 1973; segura et al., 1981; sham et al., 1989; burggren et al., 1992; michalicek and campbell, 1993; west and smits, 1994; west et al., 1998; rocha and branco, 1997, 1998; andersen et al., 2003) and chelonians (steggerda and essex, 1957; woolley, 1959; wilber, 1962; white and ross, 1966; shelton and burggren, 1976; stephens et al., 1983; comeau and hicks, 1994; hicks and comeau, 1994; hicks et al., 1996; crossley et al., 1998; hicks and farrell, 2000; overgaard et al., 2002). at the same time, arterial blood pressure can differ sevenfold in various ophidian species (johansen, 1959; lillywhite and seymour, 1978; lillywhite and pough, 1983; seymour, 1987; stinner and ely, 1993; wang et al., 2001, 2003; seymour and arndt, 2004). in snakes, there is also a large intraspecies variety in arterial blood pressures (johansen, 1959; lillywhite and seymour, 1978; stinner and ely, 1993), which is probably conditioned by a wide diversity of habitats (lillywhite and pough, 1983; seymour and arndt, 2004) and activity levels (stinner and ely, 1993; wang et al., 2001), as well as a dependence of blood pressure on body weight (seymour, 1987), temperature (lillywhite and seymour, 1978; stinner, 1987), and stress (stinner, 1987; stinner and ely, 1993). on the average, blood pressure in turtles (steggerda and essex, 1957; woolley, 1959; wilber, 1962; white and ross, 1966; shelton and burggren, 1976; stephens et al., 1983; comeau and hicks, 1994; hicks and comeau, 1994; hicks et al., 1996; crossley et al., 1998; hicks and farrell, 2000; overgaard et al., 2002) is quite comparable with that in aquatic snakes (lillywhite and pough, 1983; seymour and arndt, 2004) and anuran amphibians, whereas blood pressure in tortoises (shelton and burggren, 1976) seems to be close to terrestrial snakes (johansen, 1959; lillywhite and seymour, 1978; wang et al., 2000, 2001, 2002, 2003; seymour and arndt, 2004; galli et al., 2005; skals et al., 2005; zaar et al., 2007). thus, regarding blood pressure levels, one may assume that the species used in our study are typical representatives of anuran amphibians, tortoises, and terrestrial snakes. the isovolumetric contraction phase is followed by a period, during which blood is ejected from the heart ventricle into the arterial system. the duration of the ejection period is characterized by the ejection time. the ejection time has been found in the present study not to differ significantly between the species examined that is likely attributable to a similarity of stroke volumes. it might be supposed, because ventricle weights are comparable among the three species. there is a large variety in stroke volumes and stroke indices (the ratio stroke volume / body weight) among anurans (shelton and jones, 1965a; west and smits, 1994; andersen et al., 2003), chelonians (shelton and burggren, 1976; comeau 66 s. kharin and d. shmakov and hicks, 1994; hicks et al., 1996; crossley et al., 1998; overgaard et al., 2002) and snakes (stinner, 1987; secor et al., 2000; wang et al., 2000, 2002; galli et al., 2005; skals et al., 2005). these differences are apparently conditioned by body size, lifestyle, and habitat. on the average, however, the stroke index in chelonians is comparable with that in frogs that is in consistent agreement with our findings regarding the ventricle index, which is similar in tortoises and frogs but slightly less in snakes. the minor difference between the two reptilian species in the ejection time was observed in our study. the mean value of the ejection time in snakes was slightly less than that in tortoises. this finding is in consistent agreement with data of other researches, which found differences among reptilian species in the rate of rise of contraction in cardiac tissue (galli et al., 2006). in this study, the rate of rise of contraction in ventricular tissue in snakes was higher than in turtles. indices of ventricular contractility in the present study, minor differences between snakes and tortoises in ventricular contractility were determined, when assessing indices of ventricular contractility. this finding is in accordance with the fact that various reptilian species are characterized by the same pattern of ventricular activation (shmakov and roshchevsky, 1982; roshchevsky and shmakov, 2003). nevertheless, ventricular contractility tended to be lower in snakes compared with tortoises. first, anesthesia, which was used in our study, might contribute to the difference between snakes and tortoises in ventricular contractility. second, the mentioned tendency might be attributable to the longer pre-ejection period of the snake heart ventricle in combination with the greater ventricle index and arterial pressure, which may be due to an influence of the activity level (stinner and ely, 1993; secor et al., 2000; wang et al., 2001) and gravity (seymour, 1987; young et al., 1994; seymour and arndt, 2004) on cardiovascular physiology in snakes. it should be mentioned that an ophidian heart taken as a whole does not conform well the principle of laplace (seymour, 1987), although force production of a part of the snake heart ventricle depends to a greater degree on thickness of the ventricular wall rather than on intrinsic properties of cardiac tissue (zaar et al., 2007). in general, peculiarities of heart anatomy and intracardiac blood shunting (johansen and holl, 1960; robb, 1967; snyder et al., 1999; victor et al., 1999; sklansky et al., 2001; hicks, 2002; chetboul et al., 2004; schilliger et al., 2006), as well as lifestyle, may cause differences among reptilian species in ventricular contractility. in the study reported here, systolic time intervals and ventricular contractility were assessed in anaesthetized animals. anesthesia influences cardiac activity. barbiturates have the cardiac depressant action (list et al., 1972; manders and vatner, 1976; komai and rusy, 1984). in spite of the fact that we used sodium thiopental for reptile anesthesia, we found ventricular contractility to be higher in both reptiles compared with frogs. in comparison with tortoises and frogs, snakes had intermediate values of indices of ventricular contractility. the large difference between snakes and frogs in ventricular contractility was generally not statistically significant. the larger difference between frogs and tortoises in ventricular contractility was statistically significant. these differences in ventricular contractility cannot be explained by ventricular excitation patterns or heart rates, because reptiles (shmakov and roshchevsky, 1982; roshchevsky and shmakov, 2003) and amphibians (shmakov and abrosimova, 1989; roshchevsky and shmakov, 2003) have the same pat67heart contractility in ectothermic vertebrates tern of ventricular excitation, and heart rates have been observed in the present study to be similar in the animals examined. more possible explanations of higher ventricular contractility in both reptiles compared with frogs are peculiarities of heart anatomy and intracardiac blood shunting (johansen and holl, 1960; robb, 1967; snyder et al., 1999; victor et al., 1999; sklansky et al., 2001; hicks, 2002; chetboul et al., 2004; schilliger et al., 2006). in summary, contractility of the heart ventricle was higher in tortoises t. horsfieldii compared with anuran amphibians r. temporaria, whereas snakes natrix were intermediate. these differences were likely conditioned by heart anatomy, intracardiac blood shunting, and lifestyle; anesthesia might also cause some differences in systolic time intervals and ventricular contractility. it should also be noted that our study included a small number of animals, as well as nonstandard methods for the cardiac evaluation of ectothermic vertebrates; therefore, our data are inappropriate to be used as reference values. however, we attempted to perform a comparative evaluation of cardiac performance in three species of ectothermic vertebrates with attention to the similarity between their ventricular excitation patterns. taking into account a large variety among amphibian and reptilian species in lifestyles, habitats, intracardiac blood shunting, and heart anatomy, we suppose large differences in ventricular contractility among ectothermic vertebrates having the same ventricular excitation pattern; therefore transferring the results of our study to all amphibians and reptiles should be undertaken with caution. however, it can only be hypothesized that on the average, ventricular contractility is higher in reptiles compared with amphibians and in chelonians compared with snakes. acknowledgements the present study was supported by the russian science support foundation and the ural branch of the russian academy of sciences (the program of support for basic research performed in the ural branch of ras in association with the far eastern branch of ras). references andersen, j.b., hedrick, m.s., wang, t. 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(2007): contractile properties of the functionally divided python heart: two sides of the same matter. comp. biochem. physiol. a mol. integr. physiol. 146: 163-173. acta herpetologica 16(2): 123-128, 2021 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-10229 is the northern spectacled salamander salamandrina perspicillata aposematic? a preliminary test with clay models giacomo barbieri, andrea costa, sebastiano salvidio* distav, university of genova, corso europa 26, i-16132 genova, italy *corresponding author. e-mail: sebastiano.salvidio@unige.it submitted on: 2020, 29th december; revised on: 2021, 11th may; accepted on: 2021, 3rd october editor: dario ottonello abstract. aposematism is a visual communication system in which bright and contrasted coloured prey warn predators about their unprofitability. the northern spectacled salamander salamandrina perspicillata, a small terrestrial salamander endemic to italy, displays a uniform dark dorsal colouration and a contrasted ventral side in which a bright red colour is displayed by coiling the tail over the body. in amphibians, this behaviour, known as “unkenreflex”, is usually considered to be aposematic. in this study, we used realistic plasticine replicas to test this aposematic hypothesis in the northern spectacled salamander. of the 199 clay models placed in a natural habitat, 165 (83%) were recovered and 39 (24%) showed some sign of predation. the head of the models was more attacked than expected by chance (p = 0.042), suggesting that potential predators were perceiving models as real prey. however, there were no differences in the proportion of dorsal (18/83 = 22%), and ventral (21/82 = 26%) models attacked by predators. therefore, contrary to expectations our experiment did not support the aposematic hypothesis. however, predation experiments with clay models have limitations and our results should be considered as preliminary, deserving further research to better understand the northern spectacled salamander prey-predator system. keywords. animal replicas, plasticine, predation, salamander, unkenreflex. many animal taxa evolved conspicuous colourations associated with different mechanisms of antipredator defence such as claws, beaks, teeth, spines, stings and a variety of toxic compounds that are actively inoculated or passively delivered to predators (stevens, 2013; caro and allen, 2017). this visual communication system is known as aposematism (steven, 2013), a widespread phenomenon that independently evolved many times in different amphibians lineages (wells, 2007). aposematic colourations are usually associated with a variety of toxic compounds that are produced or sequestered and stored in specialised glandular skin glands (e.g., wells, 2007; rojas, 2017; demori et al., 2019). several species of aquatic and terrestrial salamanders exhibit a variety of combinations of red, orange yellow or white marks usually displayed on brown or black backgrounds. in salamanders, conspicuous colourations are typically assumed to act as aposematic warning anti-predatory signals (e.g., wells, 2007; lüddecke et al., 2018). in fact, several alkaloids (e.g., tetradoxins) and other toxic compounds are isolated from the skin of newts and salamanders, reinforcing the assumption that these contrasted colourations are associated with unpalatability (e.g., yotsu-yamashita et al., 2007, 2017; preissler et al., 2019). usually, bright colour patterns are usually displayed on the dorsal surface of amphibians. however, some salamanders are dorsally cryptic while possessing aposematic colourations on the underside. these species display their ventral bright colours by exposing the venter by coiling laterally (e.g., the asian newt paramesotriton deloustali) or coiling the tail above the body (e.g., the north american newt taricha rivularis and the alpine 124 g. barbieri, a. costa, s. salvidio newt ichthyosaura alpestris) (see fig. 14.30 in wells, 2007). this latter anti-predatory behaviour is known as “unkenreflex”, first described in the fire-bellied toad bombina bombina (hinsche, 1926). this peculiar behaviour, i.e. exposing bright ventral colouration by arching the body, is displayed also by the two species of spectacled salamander belonging to the genus salamandrina fitzinger, 1826: s. perspicillata and s. terdigitata (angelini et al., 2007). however, lanza (1967) casted doubts on the aposematic function of unkenreflex in salamandrina, because this genus does not possess the parotoid glands typical of many toxic salamanders, while utzeri et al. (2005) presented some anecdotal evidence for unpalatibility. therefore, the function of the black, white and red coloured ventral side and of the red tail of salamandrina remains uncertain, although no alternative explanation has been proposed. to better understand if the ventral colouration of s. perspicillata could represent an aposematic signal, we made a predation experiment by using of clay replicas representing the dorsal and ventral patterns of the focal species. experiments using this technique are non-invasive and easy to perform in natural habitats, but problems and limitations should be also taken into consideration (bateman et al., 2017; rössler et al., 2018). in the present study, clay models were exposed in the species’ natural habitat and predation rates on different model types compared. if the aposematic hypothesis holds true, we expected that models with a conspicuous red colouration typical of the body underside and tail would be attacked less frequently than those bearing a dark dorsal appearance. salamandrina perspicillata (savi, 1821), the northern spectacled salamander, is endemic to central and northern italy (angelini et al., 2007). this species is found from the sea level up to about 1900 m, in mediterranean vegetation areas and in humid broadleaf woodlands (angelini et al., 2007; romano et al., 2009). adults usually range from 70 to 100 mm in total length and are fully terrestrial, with the exception of gravid females that enter water bodies for spawning (angelini et al., 2007). the dorsal colouration is characterised by a very dark or black dorsal pattern with, during the terrestrial phase, a matt appearance. on the salamander’ head the presence of a characteristic whitish or yellowish mark between the eyes suggested the common species name “spectacled” (fig. 1). the ventral surface displays a conspicuous combination of black and white in its central and anterior part, while the posterior part of the abdomen, cloaca and tail, and the inferior part of all four limbs is brightly coloured in red (angelini et al., 2007; fig. 1). when disturbed, s. perspicillata sometimes displays its conspicuous ventral colouration by coiling its tail above the body in an unkenreflex posture (lanza, 1967; angelini et al., 2007). the northern spectacled salamander’ replicas were made from plasticine, a soft material prepared from clay, wax and oil. clay models are malleable, retain predatory marks (kuchta, 2005; salvidio et al., 2017) and have been used to analyse predation in relation to colour polymorphism and aposematism in amphibians (e.g., kraemer et al., 2016; paluh et al., 2014). clay models were obtained from a 3d printer template. we imported a photographic image of s. perspicillata in the software “rhinoceros”. the template was printed in pla (a plastic material) with the “wanhao duplicator 6” printer (supplementary material fig. s1). models, possessing a total length of 79 mm, were hand painted with acrylic water-soluble red, black and white colours (maimeri polycolor # 220, 530 and 021, respectively). the model head, torso and tail surfaces were measure by imagej software (schneider et al., 2012) giving these relative proportions: head (17.35%), torso (27,33%) and tail (55.32%). the experiment took place, from october 26th to november 1st 2020, in north-western italy, province of genova, in a mixed humid deciduous forest at about 900 m a.s.l. (44°34’00”n; 9°08’10”e). in this area the northern spectacled salamander is widespread along forest streams and in the leaf litter. during autumn, northern spectacled salamanders can be found active on the forest floor also during daytime (salvidio et al., 2012). models showing the ventral or dorsal coloration were alternatively placed every 2 m on stones, fallen branches and moss. after 6 days, models were examined in the field with the aid of a ×20 geologist lens and removed. clay models were scored as attacked, if they showed evident predatory marks on any body part, excluding the limbs. in many cases, predators could be identified as mammals, if tooth marks were evident, or as birds, if v-shaped peck marks were observed (fig. 1). in some cases, however, the predator could not be identified. predations on dorsal and ventral-coloured models were compared by means of fisher exact test or χ2, while absolute frequencies of attacks on the different body parts were compared to the expected frequencies, proportional to the relative surfaces of the model parts, by means of a χ2 goodness-of fit test (zar, 2005). we set the significance level at α = 0.05 and all tests were performed with past 4.03 software (hammer et al., 2001). of the 199 models placed on the forest floor (99 dorsal and 100 ventral), 165 (83%) were recovered (82 dorsal and 83 red). the total number of models attacked was 39 (24%), 18 dorsal and 21 ventral models (table 1). head, torso and tail of the dorsal and ventral-coloured models 125is the northern spectacled salamander salamandrina perspicillata aposematic? received a similar number of attacks (χ2 = 3.25, df = 2, p = 0.172). overall, there was a tendency that more attacks were aiming to the head than expected by chance (16 observed versus 9.19 expected attacks; fig. 2), this difference being statistically significant (head vs torso + tail: goodness-of-fit χ2 = 4.147, df = 1, p = 0.042). dorsal and ventral-coloured models were attacked with similar frequencies: 18 dorsal and 21 ventral models (fisher exact test, p = 0.715; table 1). in our experiment the models’ head was attacked more than expected by chance. therefore, potential predators were perceiving the salamander replicas as real prey, suggesting that our experimental setting was successful. contrary to expectations in case of aposematic colouration, clay models painted with conspicuous colours and representing the contrasted ventral side of the northern spectacled salamander were attacked with similar frequencies as clay models displaying the dorsal colouration. this finding may be surprising, given that to date no alternative hypothesis to aposematism has been discussed in detail for this species. however, utzeri et al. (2005) described the occurrence a b c d e f 1 fig. 1. dorsal (a) and ventral (b) view of live norther spectacled salamander salamandrina persicillata; dorsal (c) and ventral (d) view of clay models displayed in the field; e) clay model showing a bird v-shaped beak mark on the tail; f) clay model showing mammal teeth marks on the torso. 126 g. barbieri, a. costa, s. salvidio of salamanders showing their ventral coloration in the field, during male-male contests and possibly during sexual encounters. moreover, a possible deimatic function for salamandrina unkenreflex was suggested during the review process by both reviewers. deimatism is a behaviour by a sender that produces a sudden change in shape, colour or emits noises. this unexpected behaviour of the prey may cause hesitation or recoil in the predator (umbers et al. 2017). unlike aposematism, deimatism does not require predator learned aversion and may be unrelated to unpalatability or armed defence. these alternative hypotheses should deserve attention in future studies on the colourations of the spectacled salamanders. in any case, predations trials using motionless animal replicas have several shortcomings and should be always interpreted with caution (bateman et al., 2017; rössler et al., 2018; costa et al., 2020). actually, studies using clay modes in well-established prey-predator systems, as in the amazonian poisonous frog adelphobates galactonotus (rojas et al., 2015) and the brazilian venomous coral snake micrurus corallinus (banci et al., 2020), were not able to validate the aposematic function of bright and conspicuous colourations. in fact, many predators use prey movements to search, spot and select their preferred prey items (paluh et al., 2014). therefore, it is possible that movement is playing a relevant role in the prey-predator system involving the norther spectacled salamander, and that motionless models were not recognised as aposematic by predators. for example, red models of the dendrobatid poison frog oophaga pumilio equipped with a moving mechanism were attacked less frequently than moving brown models, suggesting that in this species aposematic warning signals need to be broadcasted through an association of colour and behaviour to become effective (paluh et al., 2014). indeed, combined factors may increase the specific recognition of visual signals as aposematic and reinforce learning abilities of predators (stevens, 2013). unfortunately, we do not have enough information about the prey-predator system of the norther spectacled salamander in its natural habitat and, in particular, if the main predators are birds or mammals (angelini et al., 2007). however, starting from our results, it could be possible to perform new and more accurate experiments to tests for aposematism (i.e., with moving models and displaying the replicas in different positions), or for alternative explanations such as deimatism or intra-specific communication, that could explain the significance of the bright ventral colouration and of the peculiar behaviour of s. perspicillata known as unkenreflex. acknowledgments we are grateful to luigi cuneo (3d ink point genova, affiliated to 3d ink) for the aid with 3d template and print, to giacomo bruni and a second anonymous reviewer for suggesting the deimatic defensive behaviour hypothesis, during the review process. table 1. distribution of attacks and supposed predators on the different body parts of salamandrina perspicillata clay models. body part attacked/predator model black red all head/bird 2 0 2 head/mammal 4 3 7 head/unidentified 0 0 0 torso/bird 0 0 0 torso/mammal 2 3 5 torso/unidentified 1 0 1 tail/bird 2 2 4 tail/mammal 4 8 12 tail/unidentified 1 0 1 entire model/bird 0 0 0 entire model /mammal 2 5 7 entire model/unidentified 0 0 0 intact models 64 62 126 missing models 17 17 34 total 99 100 199 fig. 2. percentages of expected and observed attacks on different body parts of clay models of the northern spectacled salamander salamandrina persicillata. expected percentages are proportional to the relative surfaces of the model parts (see text). 127is the northern spectacled salamander salamandrina perspicillata aposematic? supplementary material supplementary material associated with this article can be found at <htttp://www.unipv.it/webshi/appendix> manuscript number 10229 references angelini, c., vanni, s., vignoli, l. 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(2014): biostatistical analysis, 5th ed. pearson education limited, london. acta herpetologica vol. 16, n. 2 december 2021 firenze university press a new species of the genus noblella (amphibia: strabomantidae) from ecuador, with new information for noblella worleyae carolina reyes-puig1,2,3,4,*, juan m. guayasamin 2,5 claudia koch6, david brito-zapata1, matthijs hollanders7, melissa costales8, diego f. cisneros-heredia1,2,3 so close so different: what makes the difference? dario ottonello1,7,*, stefania d’angelo2, fabrizio oneto3,6, stefano malavasi1, marco alberto luca zuffi4, filippo spadola5 hematological values of wild caiman latirostris (daudin, 1802) in the atlantic rainforest in pernambuco, brazil luciana c. rameh-de-albuquerque1, alexandre p. zanotti1, denisson s. souza1, george t. diniz2, paulo b. mascarenhas-junior3,4,5,*, ednilza m. santos³, jozelia m. s. correia3 bone histology of broad-snouted caiman caiman latirostris (crocodylia: alligatoridae) as tool for morphophysiological inferences in crocodylia paulo braga mascarenhas-junior1,2,3,6, luis antonio bochetti bassetti4, juliana manso sayão5,6 is the northern spectacled salamander salamandrina perspicillata aposematic? a preliminary test with clay models giacomo barbieri, andrea costa, sebastiano salvidio* sexual size dimorphism in the tail length of the caspian whip snakes, dolichophis caspius (serpentes, colubridae), in south-western hungary györgy dudás1, krisztián frank2* semi-automated photo-identification of bahamian racers (cubophis vudii vudii) sebastian hoefer1,*, andreu rotger2, sophie mills1, nathan j. robinson1,3 acta herpetologica 16(2): 99-108, 2021 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-11339 hematological values of wild caiman latirostris (daudin, 1802) in the atlantic rainforest in pernambuco, brazil luciana c. rameh-de-albuquerque1, alexandre p. zanotti1, denisson s. souza1, george t. diniz2, paulo b. mascarenhas-junior3,4,5,*, ednilza m. santos³, jozelia m.s. correia3 1 parque estadual de dois irmãos, recife, pernambuco 52171-011, brazil 2 fiocruz, fundação oswaldo cruz instituto ageu magalhães, recife, pernambuco 50670-420, brazil 3 laboratório interdisciplinar de anfíbios e répteis da universidade federal rural de pernambuco, recife, pernambuco 52171-900, brazil 4 programa de pós-graduação em biologia animal da universidade federal de pernambuco, recife, pernambuco 50670-901, brazil 5 centro universitário brasileiro, unibra, recife, pernambuco 50050-230, brazil *corresponding author. e-mail: paulobragam16@gmail.com submitted on: 2021, 14th june; revised on: 2021, 18th october; accepted on: 2021, 29th october editor: daniele pellitteri-rosa abstract. hematological studies in crocodilians are important tools in the evolutionary diagnosis and control of sicknesses, such as anaemia, malnutrition, dehydration, inflammation, and parasitism, among others. we aimed to obtain reference intervals for the hemogram of caiman latirostris in wild populations that inhabit recife’s metropolitan region, pernambuco. we obtained blood samples from 42 caimans, from different sexes (22 males and 20 females) and ages classes (eight hatchlings, 24 subadults and 10 adults) in two areas of atlantic rainforest domain. we found that hematological parameters were included within the reference intervals for other crocodilian species. it was possible to observe differences between the areas for the mean corpuscular volume values, suggesting a possible difference between adult and juvenile individuals in the two study areas. when comparing sexes, there was no significant difference between the study parameters, but it was possible to observe differences in the mean corpuscular volume, mean corpuscular hemoglobin and hemoglobin in the estação ecológica de tapacurá region. although small differences have been observed between the two populations, we can infer that the hematological parameters are similar. we can use this information to evaluate animal’s health in nature and for comparations with captive individuals, allowing the establishment of ideal maintenance conditions and assisting in the identification of possible pathologies. keywords. broad-snouted caiman, crocodilians, hematimetric indices, hematology. introduction the broad-snouted caiman (caiman latirostris) occurs in rivers, mangroves and flooded areas in argentina, bolivia, brazil, paraguay, and uruguay. in brazil, this species is found in the cerrado, caatinga, atlantic forest and pampas biomes, from the coastal region of rio grande do norte, distributed through the basins of the rivers são francisco and paraná/paraguay to rio grande do sul (coutinho et al., 2013). in the wild, these caimans can live in large aggregations or in small groups and are always present in the mangrove areas of rivers, lagoons, wetlands, and lakes (filogonio et al., 2010). this species, classified as least concern (lc), presents a large geographical area of distribution and an apparent ability to colonise anthropic environments (júnior et al., 2018). however, anthropic pressure caused by the constant growth of human populations which, to a certain extent, triggers the destruction of their habitat, mainly through the drainage of water bodies, deforestation, pollution, 100 l.c. rameh-de-albuquerque et alii intensive pesticide use, as well as illegal hunting in certain regions. these impacts can affect connectivity and consequently gene flow between populations on a micro and macrogeographic scale, with such processes resulting in the decline of populations (coutinho et al, 2013; bassetti and verdade, 2014). reference hematological and blood biochemistry values are necessary for detecting the effects of environmental, infectious, parasitic, or toxicological stress in these animals, providing information on their health and therefore, can be used as a rapid diagnostic tool (heatley and russel, 2019). hematological studies in wild or captive broad-snouted caimans have a scientific, educative and production improvement outcomes, and can be applied to conservation, reproduction, and reintroduction projects (barboza et al., 2006; adelakun et al., 2017). blood analysis is a relatively non-invasive method which can provide important clinical data as well as information about the physiological conditions and health of animals (padilha et al., 2011; adelakun et al., 2017). the hemogram is comprised of the determination of the total erythrocyte count, hematocrit, hemoglobin concentration and hematological indices such as the mean corpuscular volume, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration (saggese, 2009; heatley and russel, 2019). the erythrogram data serve to aid in the evolutionary diagnosis, characterisation, and control of sicknesses, such as anaemia, malnutrition, dehydration, inflammation, and parasitism among others (barboza et al., 2007; saggese, 2009; heatley and russel, 2019). the clinical interpretation of the leukogram is challenging for many reasons, primarily because of the strict reference intervals, including the total leukocyte count and smear cell percentages which are not available for all species of healthy reptiles (campbell, 2006; saggese, 2009; zayas et al., 2011). normal hematological values for reptiles, including crocodilians, when determined by different laboratories, demonstrate ample inter and intraspecific variation due to the differences in blood sample collection, handling of the specimen, analysis techniques, physiological state of the reptiles, age, sex, nutrition, population dynamics, environmental conditions and use of anaesthetics (stacy and whitaker, 2000; campbell, 2006; saggese, 2009; zayas et al., 2011). reference intervals can be found for several reptile species, including crocodilians (stacy and whitaker, 2000; padilha et al., 2011; zayas et al., 2011). since the mean hematological parameter values can vary between species, the reference values obtained for healthy animals can serve as a guide for dealing with sick animals (stacy and whitaker, 2000). lastly, the effects on the leukocyte response to bacterial, fungal, viral, and parasitic infections or stress agents have been minimally investigated for this species (heatley and russel, 2019). thus, when referring to a set of reference values, it is important to determine the conditions in which the blood samples were obtained. understanding and recognising possible variations in hematological results for reptiles contributes to a critical interpretation of the published reference values and their clinical significance. thus, the aim of this study was to describe the morphological characteristics of the peripheral blood of c. latirostris and establish erythrogram reference indices for this species in wild protected areas in recife’s metropolitan region. material and methods study area between 2014 and 2015, we collected samples in water bodies located in the municipalities of recife and são lourenço da mata, both belonging to the recife’s metropolitan region (rmr), the main socio-economic centre of the state of pernambuco, brazil. the region is dominated by atlantic forest and the climate is tropical and humid, with autumn-winter rains (alvares et al., 2013). furthermore, the area has a large water network (carvalho, 2004) with the capibaribe river, and great influence on rmr, occupying an area of 7,545.88 km2, which represents 7.6% of pernambuco (apac, 2020). in the municipality of são lourenço da mata, we collected samples in the estação ecológica de tapacurá (eet) reservoir (8°2’s, 35°11’e), a lentic environment in a rural area, formed by the damming of the tapacurá river. the water body is approximately 7.5 km2 and is surrounded by sugar cane matrices (almeida and oliveira, 2009), open areas, agricultural areas, livestock, and riverside communities (mascarenhas-junior et al., 2020). it also has 5.35 km2 of forest fragments, of which 1.72 km2 belong to conservation units of integral protection (mata do camucim, mata do toró, mata do outeiro de pedro) (cprh, 2020) (fig. 1). in the reservoir areas, there are also constant fishing activities, with recurring recordings of bycatch of local fauna (mascarenhas-junior et al., 2018; santos et al., 2020). approximately 25 km from tapacurá, we also collected samples from caimans located in the parque estadual de dois irmãos (pedi) (8°0’s, 34º56’e), in the municipality of recife, capital of pernambuco. the area comprises approximately 11.6 km2 of protected forests (mata de dois irmãos, 3.8 km2; fazenda brejo dos macacos, 7.8 km2) and in these fragments there is approximately 1.4 km2 of water bodies forming the prata microbasin, the açude do meio (~0.024 km2), açude do prata (~0.025 km2), açude de dentro (~0.015 km2) and açude de dois irmãos (~0.135 km2). despite this area is in an urban environment with anthropic pressures, the presence of a mature marginal forest aids in the maintenance of the water quality (lima, 2004). in this study, we only accessed the açude de dentro and açude de dois irmãos. 101caiman latirostris hematological parameters data collection captures were performed bimonthly between 2014 and 2015, with active captures during the nocturnal period and with the aid of aluminium boats. the individuals were identified in the environment through the reflection of eyeballs (tapetum lucidum) from the interception of a beam of concentrated light using a spotlight (magnusson, 1995). the captures were performed manually or with the use of cables snares connected to a telescopic pole (up to 5 m). additionally, aquatic funnel traps with baits to attract the caimans were used. all physical postcapture restraint was performed using adhesive tapes. the caimans were evaluated in terms of trauma, body scars and the absence of clinical signs and symptoms of diseases. following this evaluation, blood samples were collected through supravertebral occipital venous sinus punctures using 20-gauge needles without anticoagulants and disposable needles. immediately following the collection of samples, blood smears were prepared, air dried and stained with modified may-grünwaldgiemsa colouration (rosenfeld, 1947). a small volume of blood (0.5 ml) was collected in a microtube containing heparin for the determination of hematological parameters. the total hemocyte count (thc), total leukocyte count (tlc) and total thrombocyte count (ttc) were performed manually in a neubauer chamber after 10 ml of blood being diluted in 2.0 ml of natt and herrick solution. the hematocrit (ht) was determined using the microhematocrit method, through the centrifugation of microcapillaries at 10000 rpm (coles, 1986) and the hemoglobin concentration (hb) was determined using the cyanmethemoglobin method, mixing 20 ml of blood with 2.5 ml of drabkin solution (labtest diagnostica®). through conventional calculations, hematometric indices of mean corpuscular volume (mcv), mean corpuscular hemoglobin (mch), mean corpuscular hemoglobin concentration (mchc) were calculated. to count the differential leukocytes, a total of 100 cells were counted using a microscope under immersion (1000x). after the collection of the blood material, the individuals were measured, weighed, sexed, and marked. the size class was determined through the snout-vent length (svl), following the proposal by leiva et al. (2019): class i or hatchlings (< 25cm); class ii or subadults (25 cm to 67.9 cm); class iii (68 cm to 99.9 cm) and class iv (over 99 cm) or adults. the weight was determined using scales with a limit of 40 kg. furthermore, sex was determined for larger individuals using a cloacal palpation technique (yanosky, 1990) and, in smaller caimans, surgical tweezers were inserted into the cloaca to separate the edges for fig. 1. recife’s metropolitan region (rmr) with tapacurá reservoir and parque estadual de dois irmãos highlighted in white lines. 102 l.c. rameh-de-albuquerque et alii the observation of the genitalia (webb et al., 1984). lastly, all the individuals were marked with cuts in the scales of the single and double crests and with the subcutaneous implantation of microchips. at the end of data collection, caimans were released back into the water bodies from where they were captured. data analysis to test the normality and homogeneity of the variables included in the study, we used shapiro wilk and bartlett tests, respectively. for the analyses of the quantitative variables, we used a bartlett test for homogeneity and the shapiro wilk test for the normality of the variables included in the study. the mean differences for the independent variables were evaluated using an anova followed by tukey’s post-hoc test when observing the presumption of homogeneity. otherwise, kruskal-wallis tests were used followed by the post-hoc test using fisher’s criteria of the lowest significant difference (lsd). a student t test was also used to observe when there was a presumption of normality, and when there was not, a mannwhitney test was used to evaluate the medians of the variables included in the study. we used categorical analyses for the comparative analysis between the qualitative variables, such as pearson’s χ2 test andm when necessary, fisher’s exact test. all the conclusions were taken at a significance level of 5%. the r software (r core team, 2021) was used for the evaluation of the study results. results cells morphological aspects the erythrocytes presented an elliptical shape, with abundant cytoplasm, acidophilic, pinkish in colour, occupying approximately 80% of the cell. the nucleus presented condensed chromatin, basophilia, predominantly elliptical and occupies a central position in the cell. larger ery throcy tes with spherical nuclei and more rounded forms were also occasionally found (fig. 2a). no intracellular inclusions or hemoparasites were observed. the thrombocytes were predominantly elliptical with abundant cytoplasm, hyaline, also presenting a less elongated shape with little cytoplasm. eventually, azurophilic granules were observed in the cytoplasm. the predominantly elliptical nucleus can present morphological variations, with chamfers, indentation or grooves, violet in colour with a central location (fig. 2a, 2f and 2h). the lymphocytes presented a large variation in terms of size and shape. the presence of irregularly shaped or spherical cells was common. the cytoplasm is scarce, basophilic, with azurophilic granules, commonly exhibiting cytoplasmic projections (fig. 2a, 2b and 2g). the monocytes presented a spherical to oval shape, often presenting an irregular outline (fig. 2b, 2c and 2i). the pale to moderately basophilic cytoplasm may contain cytoplasmic vacuoles of varying sizes. the nucleus may be spherical, oval or even u-shaped, generally with irregular outlines, occupying an off-centre position. the eosinophils generally have a spherical shape. the cytoplasm is abundant and homogenously filled by acidophilic granules which are compact, spherical, oval or slightly elongated, pinkish in colour with a relatively homogenous colour. the nucleus is violet in colour and is generally spherical or lenticular and is in an off-centre position and may often be bilobulated (fig. 2d and 2f). the heterophils are large and are spherical, oval or irregular. the cytoplasm is generally abundant, full of compact granules whose acidophilia varies in its intensity, depending on the granule aspect and is of a dark pink or salmon colour. in terms of their morphology, the granules have varying aspects, both in terms of their colour intensity and their shape, where they can be spherical, fusiform with intense colouration or even stick, drumstick or oval shaped. there is a clear predominance of fusiforms compared to the others. the spherical nucleus is located off-centre or peripherally (fig. 2d, 2e and 2g). the basophils have a spherical shape and are smaller when compared to the other granulated leukocytes. the cytoplasm presents strongly basophilic spherical granfig. 2. photomicrographs of caiman latirostris blood cells coloured with modified may-grunwald-giemsa (rosenfield, 1947) (magnification 1000x). (a) mature erythrocytes in high quantity, lymphocytes (arrow), thrombocytes (head of arrow); (b) monocytes (arrow), lymphocyte (head of arrow); (c) monocyte; (d) eosinophil (arrow), heterophil (head of arrow); (e) heterophil; (f ) eosinophil; (g) heterophile (arrow), thrombocytes (head of arrow), lymphocyte (outlined arrow); (h) thrombocytes; (i) monocytes with vacuoles. 103caiman latirostris hematological parameters ules of varying sizes and when located on top of the nucleus it can be impossible to distinguish between their outlines. general parameters of caimans blood samples from 42 caimans were collected, with 22 from pedi and 20 from eet. the sex proportions of the captured individuals were 22 males (pedi = 12, eet = 10, 52.38%), 18 females (pedi = 10, eet = 8, 42.86%) and 2 undetermined individuals (4.76%). from the total number of animals, 10 were adults (pedi = 3, eet = 7, 23.9%), eight hatchlings (pedi = 5, eet = 3, 19.0%) and 24 subadults (pedi = 14, eet = 10, 57.1%). between the two locations it was possible to observe differences in the capture of different age classes, with a greater number of adults in the eet and more subadults in pedi. the biometric and hematological parameters collected for caiman latirostris in the two locations are presented in table 1. the svl varied from 17.30 cm to 100.04 cm and the weight varied from 114.2 g to 51,380 g. it was possible to observe differences in the hematological parameters for the different areas for the values of mcv (p = 0.04), thrombocytes (p ≤ 0.01), monocytes (p = 0.01), basophils (p = 0.01) and eosinophils (p = 0.02; table 1). the hematological data and their reference intervals for the different age groups are established in table 2 and table 3. the mcv values did not indicate difference between adult and subadult individuals (p = 0.06). in the comparisons between the areas, differences in the values for hatchling thrombocytes (p = 0.04) and adult eosinophils (p = 0.03) were identified (table 2 and table 3). for the comparison between the sexes in the samples, there was no significant difference between the hematological parameters, with the only significant difference being for tl (p = 0.04). however, when performing this evaluation between individuals of the same area, it was possible to observe differences in the red blood cells (p = 0.02), mcv (p < 0.01), mch (p = 0.01) and monocytes (p = 0.01) in tapacurá and for the hemoglobin values in the pedi (p = 0.02) with higher values for red blood cells in females. the other parameters, mcv, mch, monocytes and hemoglobin, were higher for males (table 4 and table 5). discussion in general, the hematological parameters found for caiman latirostris in wild environments in the state of pernambuco fall within the reference intervals for other species of crocodilians (stacy and whitaker, 2000; padiltable 1. hematological reference values for wild caiman latirostris and differences between the two study areas (pernambuco, brazil). sd (standard deviation), p (p value), svl (snout-vent length), tl (total length), tec (total erythrocyte count), tlc (total leukocyte count), hb (hemoglobin), ht (hematocrit), mvc (mean corpuscular volume), mch (mean corpuscular hemoglobin), mchc (mean corpuscular hemoglobin concentration), ttc (total thrombocyte count). parameter pedi (n = 22) eet (n = 20) p mean sd range mean sd range svl (cm) 47.2 36.14 17.3-100 58.88 25.5 17.8-100.04 0.09 tl (cm) 81.77 41.62 36-209 106.31 60.64 28-211 0.30 weight (g) 5287 11934 125-51380 8140 12030 114.2-38300 0.42 tec (103 cells/mm3) 178.86 73.58 30-355 219.75 63.58 120-345 0.06 tlc (103 cells/mm3) 5.23 2.87 0.75-11.75 3.84 2.01 1.25-8.25 0.07 hb (g/dl) 6.85 0.82 5.2-8.0 7.22 0.6 5.8-8 0.11 ht (%) 20.18 6.13 3-29 21.5 3.61 15-29 0.40 mcv (fl) 1.53 1.18 0.76-5.45 1.02 0.18 0.75-1.42 0.04 mch (pg) 0.5 0.4 0.22-2 0.35 0.09 0.22-0.59 0.15 mchc (%) 42.66 40.91 25-223.3 33.9 4.33 22.3-41.76 0.67 ttc (103 cells/mm3) 2.98 1.94 0.5-8.37 4.5 1.59 1.5-7 0.01 lymphocytes (%) 54.32 12.37 33-81 51.5 12.95 31-77 0.48 monocytes (%) 5.18 2.72 1.0-9.0 3 2.18 0-9 0.01 basophils (%) 2.73 1.75 0-5 1.4 1.79 0-8 0.01 heterophils (%) 34.82 12.94 6.0-60.0 41.8 13.83 9.0-63.0 0.10 eosinophils (%) 2.95 1.73 0-6 1.8 1.4 0-5 0.02 104 l.c. rameh-de-albuquerque et alii ha et al., 2011; zayas et al., 2011; bassetti and verdade, 2014). some factors that may have affected the results obtained in previous studies may be related to the methodology used, the environmental conditions and the diet of the population sampled. among the hematological parameters, it was possible to observe differences between the areas in the mcv values. these values suggest a possible difference between adult individuals and subadults between the two study areas. the causes of physiological changes in table 2. hematological reference values for different age groups of wild caiman latirostris (parque estadual de dois irmãos, pernambuco, brazil). sd (standard deviation), p (p value), svl (snout-vent length), tec (total erythrocyte count), tlc (total leukocyte count), hb (hemoglobin), ht (hematocrit), mvc (mean corpuscular volume), mch (mean corpuscular hemoglobin), mchc (mean corpuscular hemoglobin concentration), ttc (total thrombocyte count). parameter adults (n = 3) hatchlings (n = 5) subadults (n = 14) p mean sd range mean sd range mean sd range svl (cm) 99.13 62.39 70-109 22.17 6.48 17.3-21.0 44.25 4.83 32.4-50.0 <0.01 weight (g) 23553 21138 3834-51380 330 370.63 125-1140 1799 965.94 166-3210 <0.01 tec (103 cells/mm3) 171.25 101.77 30-255 175.71 26.21 150-210 183.64 88.29 55-355 0.28 tlc (103 cells/mm3) 5.87 3.05 1.5-8.5 4.86 1.04 3.5-6.5 5.23 3.69 0.7-11.75 0.34 hb (g/dl) 6.45 0.7 6-7.5 7.11 0.73 5.8-8 6.84 0.91 5.2-8 0.44 ht (%) 18.5 3.42 14-22 21.57 4.72 12-26 19.91 7.71 47178 0.80 mcv (fl) 1.88 1.87 0.76-4.67 1.24 0.28 0.80-1.73 1.58 1.31 0.82-5.45 0.22 mch (pg) 0.73 0.85 0.26-2 0.41 0.03 0.37-0.46 0.48 0.3 0.22-1.21 0.16 mchc (%) 35.45 5.74 30.5-42.86 34.21 6.8 26.54-48.34 50.66 57.78 25-223 0.86 ttc (103 cells/mm3) 2.75 2.01 0.75-5.5 2.54 0.82 1-3.5 3.35 2.45 0.5-8.37 0.04 lymphocytes (%) 43.25 8.88 35-55 59.29 8.12 48-68 55.18 13.82 33-81 0.22 monocytes (%) 5.25 3.5 1-9 4.57 2.94 2-9 5.55 2.5 2-9 0.07 basophils (%) 3.25 2.22 0-5 2.29 1.5 0-5 2.82 1.83 0-5 0.11 heterophils (%) 44 7.12 38-52 30.43 9.25 18-45 34.27 15.41 6-60 0.28 eosinophils (%) 4.25 0.96 3-5 3.43 1.62 2-6 2.18 1.72 0-4 0.03 table 3. hematological reference values for different age groups of wild caiman latirostris (estação ecológica de tapacurá, pernambuco, brazil). sd (standard deviation), p (p value), svl (snout-vent length), tec (total erythrocyte count), tlc (total leukocyte count), hb (hemoglobin), ht (hematocrit), mvc (mean corpuscular volume), mch (mean corpuscular hemoglobin), mchc (mean corpuscular hemoglobin concentration), ttc (total thrombocyte count). parameter adults (n = 7) hatchlings (n = 3) subadults (n = 10) p mean sd range mean sd range mean sd range svl (cm) 74.71 18.39 70-100.4 41.1 35.12 17.8-24.0 44.24 14.97 59.0-67.5 <0.01 weight (g) 15495 13641 1015-38300 709.73 827.8 114-216 829 512.35 540-1740 <0.01 tec (103 cells/mm3) 223.64 47.17 130-290 246.25 70.28 185-345 190 89.93 120-345 0.28 tlc (103 cells/mm3) 4.34 1.92 1.75-7.75 3.19 0.24 3-3.5 3.25 2.9 1.25-8.25 0.34 hb (g/dl) 7.19 0.65 5.8-8 7.4 0.54 6.9-8 7.12 0.61 6.2-7.8 0.44 ht (%) 21.45 3.24 15-26 23.25 4.35 19-29 20.2 4.02 16-26 0.80 mcv (fl) 0.98 0.15 0.75-1.26 0.96 0.08 0.84-1.02 1.15 0.25 0.75-1.42 0.22 mch (pg) 0.33 0.05 0.27-0.45 0.31 0.06 0.22-0.37 9.42 0.13 0.22-0.59 0.16 mchc (%) 33.91 3.37 28.33-33.33 31.33 6.16 22.33-36.31 35.92 4.51 30-41.76 0.86 ttc (103 cells/mm3) 4.02 1.5 1.5-6 6.12 0.77 5.25-7 4.25 1.61 1.75-5.75 0.04 lymphocytes (%) 54.09 13.68 37-77 53.5 14.84 32-66 44.2 8.64 31-55 0.22 monocytes (%) 2.18 1.54 0-5 4.25 1.5 3-6 3.8 3.27 1-9 0.07 basophils (%) 1 1.1 0-3 1 0 1 2.6 3.13 0-8 0.11 heterophils (%) 40.36 15.59 9-59 39 16.06 30-63 47.2 7.6 35-54 0.28 eosinophils (%) 1.45 0.82 0-3 2.25 2.22 0-5 2.2 1.79 0-4 0.03 105caiman latirostris hematological parameters erythrogram parameters for reptiles are numerous. with increasing age, the total erythrocyte count, the mcv, mch, mchc, hematocrit and hemoglobin, tend to increase (heatley and russel, 2019). a relative increase in the total erythrocyte count, hemoglobin and/or hematocrit occurs in males of some table 4. hematological reference values for male and female wild caiman latirostris (parque estadual de dois irmãos, pernambuco, brazil). sd (standard deviation), p (p value), svl (snout-vent lenght), tl (total length), tec (total erythrocyte count), tlc (total leukocyte count), hb (hemoglobin), ht (hematocrit), mvc (mean corpuscular volume), mch (mean corpuscular hemoglobin), mchc (mean corpuscular hemoglobin concentration), ttc (total thrombocyte count). parameter males (n=12) females (n=10) p mean sd range mean sd range svl (cm) 37.77 25.43 17.3-100 49.51 13.3 42-86.50 0.07 tl (cm) 74.21 51.07 36-209 90.85 26.18 75.5-163 0.11 weight (g) 5944 14631 125-51380 4498 8332 166-28000 0.22 tec (103 cells/mm3) 203.33 39.9 150-255 149.5 94.5 30-355 0.12 tlc (103 cells/mm3) 5.77 2.3 2.75-11 4.57 3.45 0.75-11.7 0.36 hb (g/dl) 7.21 0.69 5.8-8 6.43 0.78 5.2-8 0.02 ht (%) 22.25 4.22 12-28 17.7 7.3 3-29 0.10 mcv (fl) 1.12 0.27 0.76-1.73 2.01 1.64 0.82-5.45 0.12 mch (pg) 0.36 0.07 0.26-0.46 0.67 0.56 0.22-2 0.21 mchc (%) 33.3 5.6 25.71-48.34 53.9 60.09 25-223 0.62 ttc (103 cells/mm3) 2.77 1.13 1-5.75 3.23 2.67 0.5-8.37 0.69 lymphocytes (%) 56.17 12.38 38-81 52.1 12.64 33-73 0.46 monocytes (%) 5.08 3 1-9 5.3 2.5 2-9 0.85 basophils (%) 2.67 1.56 0-5 2.8 2.04 0-5 0.87 heterophils (%) 32.92 13.06 6-52 37.1 13.09 17-60 0.46 eosinophils (%) 3.16 1.99 0-6 2.7 1.42 0-4 0.53 table 5. hematological reference values for male and female wild caiman latirostris (estação ecológica de tapacurá, pernambuco, brazil). sd (standard deviation), p (p value), svl (snout-vent lenght), tl (total length), tec (total erythrocyte count), tlc (total leukocyte count), hb (hemoglobin), ht (hematocrit), mvc (mean corpuscular volume), mch (mean corpuscular hemoglobin), mchc (mean corpuscular hemoglobin concentration), ttc (total thrombocyte count). parameter males (n=10) females (n=8) p mean sd interval mean sd interval svl (cm) 67.73 27.31 28.5-100.04 59.75 14.14 41-73.5 0.49 tl (cm) 104.33 78.14 28-211 124.3 25.24 82-147 0.50 weight (g) 12032 15975 540-38300 5584 4447 1015-11260 0.57 tec (103 cells/mm3) 192.5 67.17 120-345 259.37 46.17 195-345 0.02 tlc (103 cells/mm3) 3.72 2.34 1.25-8.25 4.15 1.9 1.75-7.75 0.67 hb (g/dl) 7.04 0.69 5.8-8 7.5 0.44 7-8 0.11 ht (%) 20.8 4.29 15-29 22.75 2.87 18-26 0.27 mcv (fl) 1.12 0.17 0.84-1.42 0.89 0.11 0.75-1.02 0.00 mch (pg) 0.39 0.1 0.22-0.59 0.29 0.04 0.22-0.35 0.01 mchc (%) 34.21 5.54 22.33-41.76 33.28 3.16 30-40 0.66 ttc (103 cells/mm3) 4.62 1.66 1.75-7 4.09 1.67 1.5-6 0.51 lymphocytes (%) 47.1 10.88 31-66 58.75 12.52 44-77 0.06 monocytes (%) 3.9 2.28 1-9 1.5 1.07 0-3 0.01 basophils (%) 1.8 2.3 0-8 1.25 1.2 0-3 0.49 heterophils (%) 45 10.87 30-59 36.25 15.56 9-54 0.20 eosinophils (%) 2.2 1.69 0-5 2.25 0.89 0-3 0.15 106 l.c. rameh-de-albuquerque et alii reptile species. however, many species may not correspond to this expectation (heatley and russel, 2019). in this study, most parameters did not present significant differences between males and females, but we observed differences in the mcv, mch and hemoglobin values, which were higher in males, whereas the total erythrocyte count was higher in females in the eet region. the main aims of the blood smear readings include the differentiation of cell types, the evaluation of cell morphology, the observation of anomalies in cell morphology and the observation of cell inclusions or extracellular anomalies, such as hemoparasites (heatley and russel, 2019). in this study, no intracellular inclusions nor hemoparasites were found. this study corroborates the findings of basset (2016) for c. latirostris and moura et al. (1999) for caiman yacare, in relation to the types of cells found in peripheral blood. the leukocytes were classified as lymphocytes, azurophilic monocytes, eosinophils, heterophils and basophils. for many authors, azurophils are a variation of monocytes, as can be observed (zayas et al., 2011), for the same species, whereas other researchers recommend that these cells could be counted separately in snakes but should be grouped for other reptile species (moura et al., 1999). in this study all the cells were grouped as as monocytes, and we observed we observed differences in the number of monocytes between areas, with the pedi presenting higher values compared to the eet. likewise, when comparing sexes within the same area, males in the eet presented higher values than females. in relation to the morphological characteristics of the other leukocytes, this study corroborates the findings described for other species of crocodilians (moura et al., 1999; stacy and whitaker, 2000; padilha et al., 2011; zayas et al., 2011). the reptile heterophile has larger cytoplasmic granules, despite being fewer in number, compared to lizards and snakes (clever and quaglia, 2009; sacchi et al., 2011). the lymphocytes are the most common leukocytes, accounting for up to 80% of the differential count in healthy reptiles (heatley and russel, 2019). in this study the lymphocytes were the most numerous leukocytes, followed by heterophils, monocytes, eosinophils, and basophils, which differs from the results found by zayas et al. (2011), for the same species. they observed a higher number of lymphocytes followed by heterophils. however, they found more basophils compared to monocytes and eosinophils. stress factors may be responsible for the increase in total leukocyte count and differential count; however, the extent of these changes has not been comprehensively investigated in reptiles. with a few exceptions, we lack fundamental knowledge on the timing of inflammation and associated cellular responses in reptiles (heatley and russel, 2019). although we attempted, in this study, to relate the differences found between ages, sex and sample areas, additional factors should be considered, as the influence of environmental parameters such as climate, seasonal period, nutritional state and population dynamics (moura et al., 1999; stacy and whitaker, 2000; heatley and russel, 2019). this study corroborates the idea presented by basset (2016) where the results obtained from experiments involving blood hematology and biochemistry parameters should always be considered as a diagnostic tool for an animal’s or even of a population’s state of health. as affirmed by stacy and whitaker (2000) in their study on crocodylus palustris, the differences found may not be a true reflection of the differences between species, therefore making it difficult to come to any firm conclusion about the biological significance of these differences. however, it is still worth considering this data when interpreting the blood parameters of one species, since these are the best data available currently. acknowledgments we would like to thank the pedi management and the eet manager and crew for making field trips and sample collections possible. we also thank the fundação de amparo a ciência no estado de pernambuco (facepe) for funding’s (apq 0245/2.04-15), the sistema de autorização e informação em biodiversidade (sisbio) for the sampling license (39929-2) and ethics and animal use committee from universidade federal rural de pernambuco (ceuaufrpe) for authorizing the execution of the project (license number 068/2014). references adelakun, k.m., kehinde, a.s., olaoye, o., ihidero, a.a., dalha, a. 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(2011): hematology and blood biochemistry of young healthy broad-snouted caimans (caiman latirostris). j. herp. 45: 516-524. acta herpetologica vol. 16, n. 2 december 2021 firenze university press a new species of the genus noblella (amphibia: strabomantidae) from ecuador, with new information for noblella worleyae carolina reyes-puig1,2,3,4,*, juan m. guayasamin 2,5 claudia koch6, david brito-zapata1, matthijs hollanders7, melissa costales8, diego f. cisneros-heredia1,2,3 so close so different: what makes the difference? dario ottonello1,7,*, stefania d’angelo2, fabrizio oneto3,6, stefano malavasi1, marco alberto luca zuffi4, filippo spadola5 hematological values of wild caiman latirostris (daudin, 1802) in the atlantic rainforest in pernambuco, brazil luciana c. rameh-de-albuquerque1, alexandre p. zanotti1, denisson s. souza1, george t. diniz2, paulo b. mascarenhas-junior3,4,5,*, ednilza m. santos³, jozelia m. s. correia3 bone histology of broad-snouted caiman caiman latirostris (crocodylia: alligatoridae) as tool for morphophysiological inferences in crocodylia paulo braga mascarenhas-junior1,2,3,6, luis antonio bochetti bassetti4, juliana manso sayão5,6 is the northern spectacled salamander salamandrina perspicillata aposematic? a preliminary test with clay models giacomo barbieri, andrea costa, sebastiano salvidio* sexual size dimorphism in the tail length of the caspian whip snakes, dolichophis caspius (serpentes, colubridae), in south-western hungary györgy dudás1, krisztián frank2* semi-automated photo-identification of bahamian racers (cubophis vudii vudii) sebastian hoefer1,*, andreu rotger2, sophie mills1, nathan j. robinson1,3 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 6(1): 87-99, 2011 food composition of uludağ frog, rana macrocnemis boulenger, 1885 in uludağ (bursa, turkey) kerim çiçek ege university, faculty of science, biology department, zoology section, 35100, bornova, izmir, turkey. e-mail: kerim.cicek@ege.edu.tr or kerim.cicek@hotmail.com submitted on: 2011, 24th january; revised on: 2011, 25th may; accepted on: 2011, 27th may. abstract. feeding habit and food preferences of uludağ frog, rana macrocnemis were studied in 2006 and 2007 in uludağ (bursa, turkey). stomach contents of 165 (87 males, 58 females, 20 juveniles) individuals were analyzed and a total of 2,129 prey items were determined. it was found that the species fed mainly on a variety of invertebrates and especially on insects (96.5%). the most frequently consumed prey items were coleoptera (62.8%), diptera (14.4%), and hymenoptera (9.8%). there was no significant sexand age-dependent difference in the feeding regime. it appears that the species is feeding less in the breeding period and more in the post-breeding period. it was also evident that there was an increase in the consumption of coleoptera depending on the elevation. keywords. rana macrocnemis, food composition, uludağ, turkey. introduction amphibians are among the indispensable elements of the ecosystem as they are a bridge for energy flow between invertebrates and higher trophic levels (burton and likens, 1975). determination and understanding of their role on this trophic network is an essential step for understanding the amphibian ecology (e.g. duellmand and trueb, 1986; cogălniceanu et al., 2000), and to evaluate the quality of their occupied habitats (e.g. kovács et al., 2007; lima et al., 2010). the uludağ frog, r. macrocnemis, has a quite broad distribution area of 891,072 km2 (kuzmin et al., 2008). the species is widely distributed in the forest and subalpine belt of the caucasus and the adjacent territories of turkey and iran (başoğlu and özeti, 1973; tarkhnishvili and gokhelashvili, 1999), and its vertical distribution in anatolia is from the sea level up to 2,600 m (veith et al., 2003). the species is included in lc category in the iucn red list, and it is reported that its populations tend to decrease (kuzmin et al., 88 k. çiçek 2008). although there have been numerous studies on its food composition in caucasus (e.g. khonyakina, 1973; velia, 1977; tertyshnikov et al., 1979; ushakov and tusnolobova, 1986; kuzmin and tarkhnishvili, 1997; meschersky, 1997), there are limited studies on the anatolian population (uğurtaş et al., 2004). uludağ (bursa, turkey) is located in the east of lake uluabat and in the south of the gulf of gemlik. it is bordered by nilüfer tributary in the west and south and by bursa and inegöl plains in the north and east (eken et al., 2006). it has an area of 136,480 ha, and it has an elevation of 130-2,543 m. the uludağ population of rana macrocnemis is particularly exposed to anthropogenic pressure (çiçek, 2009). the objective of the present study is to obtained detailed information on the food habits of the uludağ frog, rana macrocnemis inhabiting uludağ (bursa, turkey), depending on season and elevation. materials and methods the study was conducted at four stations in uludağ. kirazlıyayla (40°07’210’’n, 29°05’259’’e, 1476 m a.s.l.) and sarıalan (40°07’964’’n, 29°06’753’’e, 1617 m a.s.l.) (fig. 1a) are located in the fir forest (abies bornmuelleriana). in both stations, r. macrocnemis breeds in temporary ponds which size ranges 10–40 m2. after the breeding period, the individuals spend their time on the shore of brooks near 1–100 m from ponds. lake kilimli (40°04’627’’n, 29°13’293’’e, 2,275 m a.s.l.) (fig. 1b) and lake kara (40°04’491’’n, 29°13’761’’e, 2,214 m a.s.l.) stations are located in the subalpine belt of uludağ. these lakes are permanent and glacial lakes. however, water level decreases 1–3 m from shore during the summer months. field surveys were conducted at night (19.00–03.00) between april 2006 and september 2007. to be able to compare their food contents, the populations were classified as forest (kirazlıyayla and sarıalan) and subalpine belt (lakes kilimli and kara) according to their location and as breeding [from the beginning of april to the end of june (çiçek, 2009)] and post-breeding (from the beginning of july to the end of september) according to season. during the study, four samplings were made each year, two in the breeding period and two in the post-breeding period. first of all, the sex of the captured individuals was determined, and snout-urostyle length (sul, mm) was measured with a digital calliper. within an hour following capture, individuals were anaesthetized in a 1% solution of ms-222 (methane tricaine sulfonate) in the field and their stomach contents were extracted by forced regurgitation with forceps (hirai and matsui, 2000). the stomach contents were fig. 1. habitat types of rana macrocnemis at uludağ (bursa, turkey). a: sarıalan, b: lake kilimli. a b 89food composition of uludağ frog, rana macrocnemis boulenger, 1885 preserved in 70% ethanol for later analysis. after these procedures, frogs were released on the spot where they had been captured. at least two days before each sampling, two-liter volume pitfall traps were placed at the study stations; they were filled with 40% ethylene glycol (cogălniceanu et al., 2000); and placed 1–5 m around the all stations in order to compare stomach contents with prey availability. number of pitfall traps changed between four to eight according to the size of sampling station. in addition, sampling was also carried out on flying and diving invertebrates using a trap and a dip net, respectively, during each sampling. obtained items were determined to the order level and compared to prey items in the stomach contents. the prey items were identified to the lowest possible taxon. vegetal materials, sand and little pebbles were also encountered in the food content. however, these materials were most likely ingested accidentally during foraging and we did not consider them as food. the food contents were assessed with respect to numeric proportion (n%) (the number of a particular prey item in all preys, n%) and frequency of occurrence (the frequency of frog stomachs containing a particular prey type, f%). trophic niche overlap between sexes and habitat types were measured using pianka’s index (o, pianka, 1973):   ∑∑ ∑ = n i ik n i ij n i ik jk pp pp o ij 22 where pij= proportion of prey item i of the stomach contents used by species j; pik= proportion of prey item i of the total resources in the environment k. this index varies between 0 (no similarity) and 1 (total similarity). for comparison, food-niche breadth between sexes and habitat types was determined using shannon’s index (h, shannon, 1948):   ∑−= i ii pph' ln where pi is proportion of prey item i found in stomach contents (krebs, 1989). all niche calculations were done using the “ecosim 700” program (gotelli and entsminger, 2010). in order to determine food preference, we used the vanderploeg and scavia’s (1979) relativized electivity index (e*):   ∑= i iiiii prprw //)/(   )]/1(/()]/1([* nwnwe ii +−= where ri = proportion of prey item i in the stomach content; pi = proportion of the prey item i in the environment; wi= selectivity coefficient of prey item i; and n = category number of prey items. e* ranges between -1 (avoidance) and 1 (preferences). the index was used to compare the potential surrounding prey items having fallen into the traps and the prey items detected in the stomach contents of the individuals. the t-test, kruskal-wallis test and mann-whitney u test were used to compare sexes; statistical analyses were performed using spss 10.0; and the alpha level was set at 0.05. in the results section, the mean values are given with their standard deviations. 90 k. çiçek results during the study, 165 (87 males, 58 females, 20 juveniles) individuals of r. macrocnemis from uludağ (bursa, turkey) were examined. the average snout-urostyle length was 27.8 (sd = 7.65, range= 17.7–43.7) mm for juveniles, 59.2 (5.40, 42 –69.9) mm for males, and 59.7 (5.76, 48.3–72.7) mm for females. females are slightly larger than males (sul values), but no statistically significant difference was observed between sexes in terms of their sizes (t-test, t = 0.548, p ≤ 0.585). besides there is variation in sul among individuals depending on elevation. the greatest values were observed in subalpine population (table 1). we found 2,129 prey items in the stomach contents of 165 individuals, (177 in juveniles, 1,192 in males, and 760 in females), with body lengths ranging from 1 to 130 mm, resulting in a median number of 9 (range= 1-60) prey items. the number of median prey items was 6.5 (2–37) in juveniles, 8.5 (1–60) in males and 10.0 (2–54) in females. no statistically significant difference was detected between sexes considering the number of prey items in the stomach (kruskal-wallis test, x2= 5.45, p ≤ 0.06). juveniles generally consume smaller items (1–25 mm) and prey upon less preys than adults (table 2). the individuals consumed 668 prey items in the breeding period (from the beginning of april to the end of june) and 1,461 prey items in the post-breeding period (from the beginning of july to the end of september). the median number of prey items in males was 8.0 during the breeding period, while this value rose to 16.0 in the post-breeding period (table 2). the median number of prey items in females was 6.0 in the breeding period, whereas it rose to 18.0 in the post-breeding period. in juveniles, the median numbers of prey items were 5.5 and 13.5 by period, respectively. in the post-breeding period, a partial increase was observed in the feeding rates of individuals (mann whitney u test, z = 8.43, p < 0.0001). some 653 prey items were observed in the stomachs of 90 (42 males, 33 females, 15 juveniles) individuals in the forest population, while 1,476 prey items were observed in the stomachs of 75 (45 males, 25 females, 5 juveniles) individuals in the population of the subalpine belt. generally, the median number of prey items in the forest population was 6.5 (1-30), while it was found as 17.0 (1-60) in the population of the subalpine belt (mann whitney u test, z= 7.95, p < 0.0001). a higher number of prey items were observed in the stomachs of individuals inhabiting the subalpine belt. table 1. measures on snout-urostyle length (sul, in mm) of r. macrocnemis, according to sex, age class and habitat type. habitat sul (mm) juveniles males females fir forest mean±sd median range 27.6 ± 8.69 26.7 17.7-43.7 55.3 ± 4.24 55.6 42.9-61.42 56.3 ± 3.84 56.1 48.30-64.89 subalpine belt mean±sd median range 28.1 ± 3.68 28.9 21.8-72.7 62.1 ± 3.66 62.5 56.5-69.9 64.1 ± 4.86 64.7 54.7-72.7 overall mean±sd median range 27.8 ± 7.65 28.6 17.7-43.7 59.2 ± 5.40 59.5 42.9-69.9 59.7 ± 5.76 59.2 48.3-72.7 t test t = 0.16, p = 0.877 t = 8.55, p < 0.0001 t = 6.57, p < 0.0001 91food composition of uludağ frog, rana macrocnemis boulenger, 1885 a total of 2,129 prey items were found to belong to eight classes, 17 orders and 37 families in the stomach contents of 165 individuals (table 3). the prey groups included the classes arachnida (araneae), chilopoda (lithobiomorpha), diplopoda (julida), insecta (odonata, plecoptera, heteroptera, homoptera, hymenoptera, coleoptera, diptera, tricoptera, lepidoptera, and colembolla), gastropoda (basommatophora), oligocheta (haplotaxida), malacostraca (isopoda), and amphibia (anura). insects form the highest number of prey groups (97%) and ten orders were identified within the class. among them, the largest groups by numeric proportion (n%) found in the stomach contents were coleoptera (62.8%), diptera (14.4%), and hymenoptera (9.8%), respectively. the largest rate by frequency of occurrence also belonged to these groups: coleoptera (84.2%), diptera (44.2%), and hymenoptera (32.1%). a total of 1,073 (n%= 50.4%, f%= 49.7%) terrestrial and 1,056 (49.6%, 50.3%) aquatic prey items were discovered in the stomach contents of all individuals. in addition, 1,388 (65.2%, 65.5%) adult and 742 (34.8%, 34.5%) larval preys were found within the class insecta. within the forest population, the most frequently encountered prey orders in the food content were coleoptera (n%= 47.8, f%=76.7), diptera (17.9%, 28.9%), and hymenoptera (11.5%, 22.2%). the prey orders most consumed by the population of the subalpine belt were coleoptera (69.5%, 93.3%), diptera (12.8%, 62.7%), and hymenoptera (9.0%, 44.0%) (table 3). as it is seen, the order coleoptera is the most preferred prey group in both the subalpine and forest populations. especially the aquatic preys (959, 45.0% of total preys) were consumed at a higher rate by the subalpine population. nevertheless, only 97 (n%= 4.5%) of the aquatic preys were found in the forest population. the forest population consumed 53 (n%= 2.5) larval prey items, while the subalpine population consumed 689 (32.4%) larval prey items. the prey groups most consumed during both breeding [coleoptera (n = 330, n% = 49.4%), diptera (120, 18.0%), and hymenoptera (64, 9.6%)] and postbreeding periods [coleoptera (1,008, 69.0%), diptera (186, 12.7%), and hymenoptera (144, 9.9%)] were the same. table 2. number of prey recorded in stomach contents of r. macrocnemis according to sex, age class, habitat type and season. period fir forest subalpine belt overall juveniles males females juveniles males females juveniles males females breeding mean±sd median range 5.6±2.47 5.5 2-11 7.8±5.54 8.0 2-30 6.2±3.83 5.0 1-19 10.8±8.35 8.0 3-22 11.5±5.32 11.5 6-17 5.6±2.47 5.5 2-11 8.2±5.88 8.0 2-30 6.7±4.29 6.0 1-19 postbreeding mean±sd median range 9.0 14.0±2.16 13.5 12-17 10.0±2.83 10.0 8-12 17.8±11.72 15.0 6-37 19.6±11.90 16.5 5-54 23.9±15.32 19.0 6-60 16.3±11.01 13.5 6-37 19.1±11.49 16.0 5-54 22.7±15.16 18.0 6-60 overall mean±sd median range 5.9±2.53 6.0 2-11 8.4±5.60 8.0 2-30 6.4±3.85 6.0 1-19 17.8±11.7 15.0 6-37 18.6±11.8 16.0 3-54 22.0±14.9 18.0 6-60 8.8±7.86 6.5 2-37 13.7±10.63 8.5 1-60 13.1±12.72 10.0 2-54 kruskalwallis test x2= 3.689, p= 0.158 x2= 4.41, p= 0.08 x2= 4.41, p= 0.08 92 k. çiçek ta bl e 3. f oo d co m po si tio n of 1 65 ( 87 m al es 5 8 fe m al es , a nd 2 0 ju ve ni le s) u lu da ğ fr og , r an a m ac ro cn em is a cc or di ng t o se x, a ge c la ss a nd h ab ita t ty pe . n (% ): n um er ic p ro po rt io n, f( % ): fr eq ue nc y of o cc ur re nc e. pr ey ta xa fi r fo re st (4 2 m al es , 3 3 fe m al es , a nd 1 5 ju ve ni le s) su ba lp in e (4 5 m al es , 2 5 fe m al es , 5 ju ve ni le s) o ve ra ll j m f n (n % ) f ( f% ) j m f n (n % ) f ( f% ) n (n % ) f ( f% ) a r a c h n id a 18 .2 6. 5 2. 8 45 ( 6. 9) 9 (1 0. 0) 0. 4 0. 9 8 (0 .5 ) 3 (4 .0 ) 53 ( 2. 5) 12 ( 7. 3) a r a n ea 18 .2 6. 5 2. 8 45 ( 6. 9) 9 (1 0. 0) 0. 4 0. 9 8 (0 .5 ) 3 (4 .0 ) 53 ( 2. 5) 12 ( 7. 3) a ge le ni da e 4. 5 0. 8 0. 9 9 (1 .4 ) 6 (6 .7 ) 0. 2 1 (0 .1 ) 1 (1 .3 ) 10 ( 0. 5) 7 (4 .2 ) c yb ae id ae a 0. 6 2 (0 .3 ) 2 (2 .2 ) 0. 2 1 (0 .1 ) 1 (1 .3 ) 3 (0 .1 ) 3 (1 .8 ) l yc os id ae 2. 3 1. 4 1. 9 11 ( 1. 7) 6 (6 .7 ) 0. 4 3 (0 .2 ) 2 (2 .7 ) 14 ( 0. 7) 8 (4 .8 ) c h il o po d a , l ith ob io m or ph a 1. 1 0. 28 0. 9 4 (0 .6 ) 3 (3 .3 ) 0. 7 4 (0 .3 ) 2 (2 .7 ) 8 (0 .4 ) 5 (3 .0 ) l ith ob iid ae , l ith ob iu s sp . 1. 1 0. 28 0. 9 4 (0 .6 ) 3 (3 .3 ) 0. 7 4 (0 .3 ) 2 (2 .7 ) 8 (0 .4 ) 5 (3 .0 ) d ip lo po d a , j ul id a 0. 2 2 (0 .1 ) 1 (1 .3 ) 2 (0 .1 ) 1 (0 .6 ) in se c ta 80 .7 92 .9 96 .2 60 3 (9 2. 3) 89 ( 98 .9 ) 10 0 99 .5 98 .4 14 63 ( 99 .1 ) 74 ( 98 .7 ) 20 66 ( 97 ) 16 3 (9 8. 8) t er re st ri al la rv ae 1. 7 0. 5 7 (1 .1 ) 4 (4 .4 ) 1. 7 3. 1 31 ( 2. 1) 7 (9 .3 ) 38 ( 1. 8) 11 ( 6. 7) o d o n a ta 0. 3 0. 9 3 (0 .5 ) 2 (2 .2 ) 1. 3 11 ( 0. 7) 11 ( 14 .7 ) 14 ( 0. 7) 13 ( 7. 9) pl ec o pt er a 1. 1 4 (0 .6 ) 2 (2 .2 ) 0. 1 0. 7 5 (0 .3 ) 2 (2 .7 ) 9 (0 .4 ) 4 (2 .4 ) h et er o pt er a 1. 1 1. 9 8 (1 .2 ) 2 (2 .2 ) 1. 1 2. 7 24 ( 1. 6) 10 ( 13 .3 ) 32 ( 1. 5) 12 ( 7. 3) c or ix id ae , c or ix a sp .a 0. 4 2 (0 .1 ) 1 (1 .3 ) 2 (0 .1 ) 1 (0 .6 ) g er ri da e, g er ri s sp .a 1. 9 4 (0 .6 ) 1 (1 .1 ) 1 (1 .3 ) 4 (0 .2 ) 1 (0 .6 ) l yg ae id ae 0. 5 1. 3 11 ( 0. 7) 5 (6 .7 ) 11 ( 0. 5) 5 (3 .0 ) n ot on ec tid ae , n ot on ec ta s p. a 0. 4 2 (0 .1 ) 1 (1 .3 ) 2 (0 .1 ) 1 (0 .6 ) p en ta to m id ae , p en ta to m a sp . 1. 1 4 (0 .6 ) 2 (2 .2 ) 0. 6 0. 7 9 (0 .6 ) 5 (6 .7 ) 13 ( 0. 6) 7 (4 .2 ) h o m o pt er a 0. 5 1 (0 .1 ) 1 (1 .1 ) 0. 1 1 (0 .1 ) 1 (1 .3 ) 2 (0 .1 ) 2 (1 .2 ) c ic ad el lid ae , c ic ad a sp . 0. 5 1 (0 .1 ) 1 (1 .1 ) 0. 1 1 (0 .1 ) 1 (1 .3 ) 2 (0 .1 ) 2 (1 .2 ) h y m en o pt er a 6. 8 15 .2 7. 1 75 ( 11 .5 ) 20 ( 22 .2 ) 8. 99 6. 8 12 .4 13 3 (9 .0 ) 33 ( 44 .0 ) 20 8 (9 .8 ) 53 ( 32 .1 ) f or m ic id ae 5. 7 13 .8 7. 1 69 ( 10 .6 ) 21 ( 23 .3 ) 8. 99 3. 2 7. 3 75 ( 5. 1) 23 ( 30 .7 ) 14 4 (6 .8 ) 44 ( 26 .7 ) i ch ne um on id ae 0. 6 2 (0 .3 ) 1 (1 .1 ) 0. 7 0. 2 7 (0 .5 ) 4 (5 .3 ) 9 (0 .4 ) 5 (3 .0 ) p om pi lid ae 0. 3 1 (0 .1 ) 1 (1 .1 ) 0. 9 0. 5 11 ( 0. 7) 3 (4 .0 ) 12 ( 0. 6) 4 (2 .4 ) 93food composition of uludağ frog, rana macrocnemis boulenger, 1885 pr ey ta xa fi r fo re st (4 2 m al es , 3 3 fe m al es , a nd 1 5 ju ve ni le s) su ba lp in e (4 5 m al es , 2 5 fe m al es , 5 ju ve ni le s) o ve ra ll j m f n (n % ) f ( f% ) j m f n (n % ) f ( f% ) n (n % ) f ( f% ) s ph ec id ae 0. 6 1. 5 13 ( 0. 9) 4 (5 .3 ) 13 ( 0. 6) 4 (2 .4 ) v es pi da e 0. 3 1 (0 .1 ) 1 (1 .1 ) 0. 1 1. 1 7 (0 .5 ) 4 (5 .3 ) 8 (0 .4 ) 5 (3 .0 ) c o le o pt er a 28 .4 44 .9 60 .7 31 2 (4 7. 8) 69 ( 76 .7 ) 84 .2 7 71 .6 63 .9 10 26 ( 69 .5 ) 70 ( 93 .3 ) 13 38 ( 62 .8 ) 13 9 (8 4. 2) c an th ar id ae 0. 1 1 (0 .1 ) 1 (1 .3 ) 1 (< 0. 1) 1 (0 .6 ) c ar ab id ae 18 .2 24 .9 34 .6 17 7 (2 7. 1) 52 ( 57 .8 ) 5. 62 7. 4 6. 4 10 2 (6 .9 ) 38 ( 50 .7 ) 27 9 (1 3. 1) 90 ( 54 .5 ) c er am by ci da e 1. 7 5. 2 17 ( 2. 6) 7 (7 .8 ) 0. 6 1. 1 11 ( 0. 7) 6 (8 .0 ) 28 ( 1. 3) 13 ( 7. 9) c oc ci ne lli da e, c oc ci ne lla s p. 0. 6 0. 9 4 (0 .6 ) 3 (3 .3 ) 2. 25 0. 9 1. 1 16 ( 1. 1) 6 (8 .0 ) 20 ( 0. 9) 9 (5 .5 ) c ur cu lio ni da e 2. 3 1. 1 6 (0 .9 ) 2 (2 .2 ) 0. 00 4. 2 4. 2 58 ( 3. 9) 12 ( 16 .0 ) 64 ( 3. 0) 14 ( 8. 5) d yt is ci da e, a ga bu s sp . a 1. 1 6. 2 17 ( 2. 6) 5 (5 .6 ) 2. 2 12 .5 9. 1 15 7 (1 0. 6) 40 ( 53 .3 ) 17 4 (8 .2 ) 45 ( 27 .3 ) d yt is ci da e, la rv ae a 7. 9 7. 1 43 ( 6. 6) 15 ( 16 .7 ) 74 .2 44 .3 40 .1 65 7 (4 4. 5) 28 ( 37 .3 ) 70 0 (3 2. 9) 43 ( 26 .1 ) e la te ri da e 0. 6 2 (0 .3 ) 1 (1 .1 ) 0. 4 2 (0 .1 ) 1 (1 .3 ) 4 (0 .2 ) 2 (1 .2 ) h yd ro ph ili da e, e nc hr us s pa 0. 2 1 (0 .1 ) 1 (1 .3 ) 1 (< 0. 1) 1 (0 .6 ) s ca ra ba ei da e 0. 9 2 (0 .3 ) 1 (1 .1 ) 2 (0 .1 ) 1 (0 .6 ) s ta ph yl in id ae 3. 4 0. 6 5 (0 .8 ) 2 (2 .2 ) 0. 6 0. 4 7 (0 .5 ) 4 (5 .3 ) 12 ( 0. 6) 6 (3 .6 ) t en eb ri on id ae 2. 3 4. 5 0. 9 20 ( 3. 1) 8 (8 .9 ) 0. 9 1. 1 14 ( 0. 9) 4 (5 .3 ) 34 ( 1. 6) 12 ( 7. 3) d ip t er a 23 .9 16 .7 17 .5 11 7 (1 7. 9) 26 ( 28 .9 ) 6. 74 13 .8 12 .2 18 9 (1 2. 8) 47 ( 62 .7 ) 30 6 (1 4. 4) 73 ( 44 .2 ) a si lid ae 2. 3 2. 3 0. 5 11 ( 1. 7) 5 (5 .6 ) 0. 2 2 (0 .1 ) 1 (1 .3 ) 13 ( 0. 6) 6 (3 .6 ) c ul ic id ae , a ed es s p. a 6. 8 2. 3 1. 4 17 ( 2. 6) 6 (6 .7 ) 10 .0 7. 6 12 6 (8 .5 ) 31 ( 41 .3 ) 14 3 (6 .7 ) 37 ( 22 .4 ) m us ci da e 3. 4 0. 6 0. 5 6 (0 .9 ) 4 (4 .4 ) 1. 1 1. 1 15 ( 1. 0) 5 (6 .7 ) 21 ( 1. 0) 9 (5 .5 ) s yr ph id ae 4. 5 16 ( 2. 4) 2 (2 .2 ) 0. 4 2 (0 .1 ) 1 (1 .3 ) 18 ( 0. 8) 3 (1 .8 ) t ab an id ae 6. 8 1. 7 2. 4 17 ( 2. 6) 6 (6 .7 ) 2. 0 11 ( 0. 7) 2 (2 .7 ) 28 ( 1. 3) 8 (4 .8 ) t r ic o pt er a 6. 8 8. 8 4. 3 46 ( 7. 0) 9 (1 0. 0) 1. 5 2. 5 27 ( 1. 8) 5 (6 .7 ) 73 ( 3. 4) 14 ( 8. 5) l im ne ph ili da e, a du lt 6. 8 8. 2 4. 3 44 ( 6. 7) 8 (8 .9 ) 1. 4 2. 5 26 ( 1. 8) 4 (5 .3 ) 70 ( 3. 3) 12 ( 7. 3) l im ne ph ili da e, la rv ae a 0. 6 2 (0 .3 ) 1 (1 .1 ) 0. 1 1 (0 .1 ) 1 (1 .3 ) 3 (0 .1 ) 2 (1 .2 ) le pi d o pt er a 11 .4 1. 7 1. 9 20 ( 3. 1) 6 (6 .7 ) 1. 1 0. 5 12 ( 0. 8) 3 (4 .0 ) 32 ( 1. 5) 9 (5 .5 ) n oc tu id ae 3. 4 1. 7 9 (1 .4 ) 3 (3 .3 ) 0. 4 3 (0 .2 ) 1 (1 .3 ) 12 ( 0. 6) 4 (2 .4 ) 94 k. çiçek pr ey ta xa fi r fo re st (4 2 m al es , 3 3 fe m al es , a nd 1 5 ju ve ni le s) su ba lp in e (4 5 m al es , 2 5 fe m al es , 5 ju ve ni le s) o ve ra ll j m f n (n % ) f ( f% ) j m f n (n % ) f ( f% ) n (n % ) f ( f% ) c o le m b o ll a 0. 2 2 (0 .1 ) 1 (1 .3 ) 2 (0 .1 ) 1 (0 .6 ) g a st r o po d a 0. 3 1 (0 .1 ) 1 (1 .1 ) 1 (< 0. 1) 1 (0 .6 ) p la no rb id ae , p la no rb is s p. a 0. 3 1 (0 .1 ) 1 (1 .1 ) 1 (< 0. 1) 1 (0 .6 ) o li g o c h et a 1. 1 1. 1 0. 5 6 (0 .9 ) 5 (5 .6 ) 0. 2 1 (0 .1 ) 1 (1 .3 ) 7 (0 .3 ) 6 (3 .6 ) l um br ic id ae , l um br ic us s p. a 1. 1 1. 1 0. 5 6 (0 .9 ) 5 (5 .6 ) 0. 2 1 (0 .1 ) 1 (1 .3 ) 7 (0 .3 ) 6 (3 .6 ) m a la c o st r a c a 2. 3 2 (0 .3 ) 1 (1 .1 ) 2 (0 .1 ) 1 (0 .6 ) o ni sc id ae , o ni sc us s p. 2. 3 2 (0 .3 ) 1 (1 .1 ) 2 (0 .1 ) 1 (0 .6 ) a m ph ib ia 0. 5 1 (0 .1 ) 1 (1 .1 ) 0. 1 1 (0 .1 ) 1 (1 .3 ) 2 (0 .1 ) 2 (1 .2 ) r an id ae r an a m ac ro cn em is a 0. 5 1 (0 .1 ) 1 (1 .1 ) 1 (< 0. 1) 1 (0 .6 ) r . m ac ro cn em is ta dp ol ea 0. 1 1 (0 .1 ) 1 (1 .3 ) 1 (< 0. 1) 1 (0 .6 ) to ta l n um be r of p re y ite m 88 35 3 21 1 89 83 9 54 9 21 29 sh an no n’ s in de x h ’ 2. 24 2. 37 2. 08 2. 03 2. 14 2. 24 a: a qu at ic a nd s em ia qu at ic p re ys . 95food composition of uludağ frog, rana macrocnemis boulenger, 1885 according to the pianka’s niche overlap index (o), food compositions of sexes are mostly similar (om, f= 0.99, om, j= 0.95, of, j= 0.94) (fig. 2). in forest (om, f= 0.99, om, j= 0.95, of, j= 0.94) and subalpine (om, f= 0.99, om, j= 0.92, of, j= 0.93) populations as well, the food contents considerably overlapped within their respective populations. nevertheless, there are differences between forest and subalpine populations in terms of food composition (oforest, subalpine= 0.55). when considering forest and subalpine populations (shannon’s index, h), we recorded a close food niche breadth between the sexes (hforest m = 2.37, hforest f = 2.08, hforest j = 2.24; hsubalpine m = 2.14, hsubalpine f = 2.24, hsubalpine j = 2.03). furthermore, depending on elevation, the individuals inhabiting the forest area had a partially wider food niche breadth (hforest= 2.34, hsubalpine=2.16). according to the results of vanderploeg and scavia’s electivity index (e*), e* value ranged from -0.71 to 0.96: coleoptera (e*= 0.96), arenea (0.91), and terrestrial insect larvae (0.88) had the highest values (table 4). this result also indicates that the food contents of the species vary partially according to the availability of the surrounding prey items. discussion our study revealed that uludağ frog, r. macrocnemis, feeds largely on various invertebrates and predominantly on the insecta. the food content consists mainly of coleoptera, diptera and hymenoptera. in previous studies on the species, arachnida (araneae), opiliones, chilopoda, diplopoda, insecta, gastropoda, oligocheta, malacostraca (isopoda) and amphibia groups were determined in the food content (khonyakina, 1973; velia, 1977; tertyshnikov et al., 1979; ushakov and tusnolobova, 1986; kuzmin and tarkhnishvili, 1997; meschersky, 1997; kuzmin, 1999; tarkhnishvili and gokhelashvili, 1999; uğurtaş et al., 2004). fig. 2. cladogram for food niche similarity (o) between sexes. 96 k. çiçek it was found that in caucasian populations, the food content of the species consisted largely of coleoptera (30–46%), predominantly the carabidae family (10.0–54.2%) (khonyakina, 1973; velia, 1977; tertyshnikov et al., 1979; ushakov and tusnolobova, 1986; kuzmin and tarkhnishvili, 1997; meschersky, 1997; kuzmin, 1999; tarkhnishvili and gokhelashvili, 1999). the other important prey groups that were observed were diptera and araneae (kuzmin, 1999; tarkhnishvili and gokhelashvili, 1999). furthermore, in the study performed in uludağ, uğurtaş et al. (2004) encountered preys included in the insecta group at the rate of 68.0% in the food content of the species and stated that coleoptera (36.1%), plecoptera (19.2%), and diptera (22.1%) were the most significant prey groups. this result also supports the previous studies. nevertheless, it is noteworthy that the rate of aquatic preys was small in previous studies (e.g. kuzmin, 1999; tarkhnishvili and gokhelashvili, 1999; uğurtaş et al., 2004). in this study, it was found that the species feeds on both aquatic and terrestrial prey items. therefore, this shows that it forages both in water and on land. especially the individuals in the subalpine belt feed on aquatic preys at a higher rate. it is striking that the species observed in the food of the species consist largely of nocturnal prey items. it was also previously reported that the night activity of the species is higher (ushakov and tusnolobova, 1986; çiçek, 2009). insects, and particularly coleoptera, also have a significant place in the food of brown frogs such as r. arvalis (aszalós et al., 2005; dobenkov et al., 2005; sas et al., 2005, stoyanova and mollov, 2008), r. dalmatina (aszalós et al., 2005; kovács et al., 2010) and r. temporaria (houston, 1973; drobenkov et al., 2005, stoyanova and mollov, 2008). on the table 4. percentages of potential preys in the environments and vanderploeg and scavia’s electivity index (e*) for prey taxa. prey taxa prey abundance e* fir forest subalpine belt overall fir forest subalpine belt overall aranea 2.62 1.28 1.92 0.951 0.743 0.913 chilopoda 1.36 1.05 1.20 0.744 0.609 0.684 diplopoda 0.97 0.83 0.90 0.448 0.282 terrestial larvae 2.70 1.35 2.00 0.713 0.923 0.877 odonata 3.00 0.69 1.80 0.395 0.891 0.723 plecoptera 3.23 2.42 2.81 0.482 0.383 0.439 heteroptera 6.74 7.73 7.26 0.464 0.542 0.558 homoptera 7.20 6.14 6.65 -0.515 -0.700 -0.612 hymenoptera 12.59 15.48 14.09 0.864 0.806 0.844 coleoptera 20.91 25.06 23.07 0.943 0.956 0.958 diptera 18.63 21.97 20.37 0.871 0.806 0.846 tricoptera 6.67 5.96 6.30 0.882 0.662 0.805 lepidoptera 5.90 4.54 5.20 0.773 0.482 0.662 colembolla 2.54 1.56 2.03 0.162 -0.119 gastropoda 1.63 1.61 1.62 0.171 -0.340 oligocheta 2.36 1.39 1.86 0.708 -0.125 0.501 malacostraca 0.94 0.93 0.94 0.662 0.262 97food composition of uludağ frog, rana macrocnemis boulenger, 1885 other hand, flying insects play a noticeable role in the feeding of r. temporaria (houston, 1973; drobenkov et al., 2005). besides the invertebrate prey items, one tadpole and one juvenile r. macrocnemis were also observed in the food content. it was also previously reported that the species displays cannibalistic behavior (meschersky, 1997; kuzmin, 1999; tarkhnishvili and gokhelashvili, 1999; uğurtaş et al., 2004). as competition for food among individuals increases, cannibalism is a mechanism that increases the survival rate (polis, 1981). it was also previously reported that the food compositions of ranids are associated with the surrounding prey items (hirai and matsui, 2000, 2001; sas et al., 2005). according to the obtained results, the food habit of the species varies largely by the abundance of the surrounding prey items. however, it does not totally depend on abundance. particularly in the breeding period, a quite high number of culicid larvae are available in kirazlıyayla and sarıalan. nevertheless, this is barely reflected on the food content. it was also previously reported that no complete relationship was available between food composition of amphibians and the surrounding prey availability (e.g. cogălniceanu et al., 2000). generally, no difference was observed among the food contents of females, males and juveniles. the overlapping of the food composition indicates that it does not vary by sex and age and that individuals use the same microhabitat in order to forage (e.g. hirai and matsui, 2000, 2001; parker and goldstein, 2004). however, differences in terms of abundance of prey items in the forest and subalpine areas also affect the feeding regimes of individuals. widely-foraging predators encounter and consume mostly non-moving types of prey items (pianka, 1966). the availability of generally slow-moving prey items in the food content of r. macrocnemis shows that the species is an opportunistic widely-foraging predator, like many ranids (e.g. duellman and trueb, 1986; cogălniceanu et al., 2000; parker and goldstein, 2004; sas et al., 2005). in conclusion, the food habit of r. macrocnemis generally varies by the availability of surrounding prey items (table 4), and it is an opportunistic widely-foraging predator, the food of which consists largely of coleoptera (mainly carabidae and dytiscidae), diptera and hymenoptera (mainly formicidae). acknowledgements this study financially supported by tubi̇tak [project number: 105t336], ebi̇ltem [2007bi̇l015] and research fund accountancy of ege university [2006fen015]. i am indebted these establishments for financial support. i thank to d. ayaz and s.k. aserim for helping field studies, i. mollov for reviewing english style. references amphibiaweb, (2009): information on amphibian biology and conservation. <http:// amphibiaweb.org/>. downloaded on 9 september 2009. aszalós, l., bogdan, h., kovács, é.h. & peter, v.i. 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(in russian). bbib67 ole_link1 ole_link2 bbib28 ole_link5 ole_link6 ole_link7 ole_link8 _goback ole_link1 ole_link2 ole_link3 ole_link4 ole_link1 ole_link2 ole_link19 ole_link20 ole_link21 ole_link29 ole_link3 ole_link4 ole_link5 ole_link31 ole_link14 ole_link15 ole_link12 ole_link13 ole_link16 ole_link17 ole_link22 ole_link23 ole_link24 ole_link8 ole_link9 ole_link10 ole_link11 ole_link18 ole_link27 ole_link28 ole_link25 ole_link26 ole_link6 ole_link7 ole_link34 ole_link37 ole_link38 acta herpetologica vol. 6, n. 1 june 2011 firenze university press widespread bacterial infection affecting rana temporaria tadpoles in mountain areas rocco tiberti extreme feeding behaviours in the italian wall lizard, podarcis siculus massimo capula1, gaetano aloise2 lissotriton vulgaris paedomorphs in south-western romania: a consequence of a human modified habitat? severus d. covaciu-marcov*, istvan sas, alfred ş. cicort-lucaciu, horia v. bogdan body size and reproductive characteristics of paedomorphic and metamorphic individuals of the northern banded newt (ommatotriton ophryticus) eyup başkale1, ferah sayım2 , uğur kaya2 genetic characterization of over hundred years old caretta caretta specimens from italian and maltese museums luisa garofalo1, john j. borg2, rossella carlini3, luca mizzan4, nicola novarini4, giovanni scillitani5, andrea novelletto1 the phylogenetic position of lygodactylus angularis and the utility of using the 16s rdna gene for delimiting species in lygodactylus (squamata, gekkonidae) riccardo castiglia*, flavia annesi localization of glucagon and insulin cells and its variation with respect to physiological events in eutropis carinata vidya. r. chandavar1, prakash. r. naik2* the balearic herpetofauna: a species update and a review on the evidence samuel pinya1, miguel a. carretero2 effects of mosquitofish (gambusia affinis) cues on wood frog (lithobates sylvaticus) tadpole activity katherine f. buttermore, paige n. litkenhaus, danielle c. torpey, geoffrey r. smith*, jessica e. rettig food composition of uludağ frog, rana macrocnemis boulenger, 1885 in uludağ (bursa, turkey) kerim çiçek preliminary results on tail energetics in the moorish gecko, tarentola mauritanica tommaso cencetti1,2, piera poli3, marcello mele3, marco a.l. zuffi1 climate change and peripheral populations: predictions for a relict mediterranean viper josé c. brito1, soumia fahd 2, fernando martínez-freiría1, pedro tarroso1, said larbes3, juan m. pleguezuelos4, xavier santos5 assessing the status of amphibian breeding sites in italy: a national survey societas herpetologica italica* osservatorio erpetologico italiano acta herpetologica journal of the societas herpetologica italica acta herpetologica rivista della societas herpetologica italica acta herpetologica 2006 1 habitat characteristics of nesting areas and of predated nests in a mediterranean population of the european pond turtle, «emys orbicularis galloitalica» habitat characteristics of nesting areas and of predated nests in a mediterranean population of the european pond turtle, emys orbicularis galloitalica marco a.l. zuffi 1, laura rovina 1,2 1 museo di storia naturale e del territorio, università·di pisa, via roma 79, 56011 calci (pisa)-italy; e-mail: marcoz@museo.unipi.it 2 present address: lungarno galilei 14, 56125 pisa-italy abstract. one of the largest population of emys orbicularis galloitalica of central italy inhabits the canal system wet areas within a natural protected park. features of nesting habitats, nest structure, and predation patterns of 209 nests of a large population of the european pond turtle are here presented and analysed. nest sites were characterised by sunny bushy areas in strip habitat, digged along north-south oriented canals, on average with about 26% of the area covered by vegetation, less than one meter distant from 30 cm height bushes, at about 11 m from water and at about 13 m distance from wooded areas, 28 m away from a road. principal component and discriminant analyses were used on 20 selected variables in order to reduce the number of physical variables, and indicate that canal border, strip habitat, and canal orientation are grouping variables, that correctly classified 41.6%, 66.5%, and 100 % respectively of nest presence. keywords. mediterranean emys orbicularis; nesting habitat; nest predation. introduction the study of life-history traits in chelonians has longly stimulated the interest of herpetologists (ernst and barbour, 1989; iverson et al., 1993; ernst et al., 1994; forsman and shine, 1995). research, which has been focused on clutch size-maternal body size relations and latitudinal variation of reproductive traits on most of the freshwater turtles, has paid special attention to frequency of reproduction, relative clutch mass, egg mass and egg size. the nesting activity represents the most important and necessary behavioral pattern between egg production and reproductive success. nesting activity covers both the choice of suitable areas, the physical burrowing of the nest chamber, and egg deposition. acta herpetologica 1: 37-51, 2006 38 m.a.l. zuffi and l. rovina in freshwater turtles of the family emydidae, the largest family of chelonians (ernst and barbour, 1989), the nest differs between genera and species, mainly in average width and depth: the smaller the species, the smaller the clutch (iverson et al., 1993), and accordingly, the smaller the nest, which hosts from 3 to 19 eggs (emys orbicularis: jablonski and jablonska, 1998), depending on latitude and maternal body size (iverson et al., 1993). the european pond turtle, emys orbicularis, ranges from portugal in the west to the area of the aral sea (kazakhstan) in the east, from northern germany and denmark to southern italy, sicily and northern africa and south-eastwards from turkey to the eastern caucasus republics and the southern shore of the caspian sea (podloucki, 1997; kuzmin, 2002; fritz, 2004). populations of e. orbicularis are mainly distributed along coastal areas and across many internal plain areas of the region. populations of e. orbicularis in italy live mainly in marshy lands, humid areas, ponds, large rivers of both coastal and plain habitats, but also in mountainous areas (zuffi, 2000). they are found in two main types of habitat: the “pond” system, consisting of one or more natural, shallow bodies of water (ponds and marshy areas) with abundant water and riparian vegetation in forested areas, and in open areas, and the “canal” habitat (lebboroni and chelazzi, 1998), which is characterised by artificial canals of drainage waters, generally in open or marginal areas. furthermore, it is also possible to find pond turtles in altered habitats, like in old caves, abandoned fisheries, springs and sources. it is often very hard for researchers to locate intact nests, but on average, it has been found that selected habitats for nesting are close to, or not very far from, the water, where e. orbicularis females usually live (rovero and chelazzi, 1996; schneeweiss and steinhauer, 1998; schneeweiss et al., 1998; kotenko, 2000). in some cases, females can migrate to considerable distances from basking areas to nest, and come back after deposition (rovero and chelazzi, 1996; schneeweiss, 1998; chelazzi et al., 2000). information about nest size and nest architecture, as well as nesting habitats, is to date either anecdotal or fairly descriptive, even if it agrees with similar data available for most regions of europe. in central italy are reported first data on a small mountainous population, but with raw data on high predation rate and apparently small clutch size (e.g. 3-4 eggs per average clutch) (rovero and chelazzi, 1996), and other preliminary data on a large populations of plain italy with larger clutch size (e.g. 5-6 eggs per average clutch), and high predation rate (zuffi et al., 1999; gianatti et al., 2000). also in most of the known populations of central and northeastern europe, available data on nesting habitats or on nest features mainly refer to small data set. nevertheless, some interesting data emerge from these reports. in poland and in germany it has been mainly referred to reproductive output and on reproductive success, with females emys orbicularis that lay larger clutch size (e.g. 9 to 12 eggs per average clutch) than the studied mediterranean populations (jablonski and jablonska, 1998; mitrus and zemanek, 1998; schneeweiss et al., 1998). similar data has been reported for ukraine (kotenko, 2000), with high clutch size and high predation estimation. basic description of nest chamber of intact nest in germany is given by andreas and paul (1998). the presence of potential predators, like mammals or birds (chelazzi et al., 2000; rössler, 2000a; zuffi, 2000), is, on average, common in natural and semi-natural habitats, and it is assumed that predators may damage 75-95% of intact nests (rovero and chelazzi, 1996; zuffi and odetti, 1998; zuffi et al., 1999; kotenko, 2000; rössler, 2000a), but further specific analyses are still lacking. man and his associated activities may represent a significant additional source 39habitat of nesting and predated nests in emys of disturbance and injury, reducing, modifying, polluting or destroying suitable habitats (cheylan and poitevin, 1998; najbar and maciantowicz, 2000). we then considered that the analysis of predated nest characteristics could be likely a direct estimator of suitable nesting features in an area, being predation on the nest a constant in all the populations studied up to now. this research has been planned to underline the pattern for selection of suitable nesting areas comparing most of the european pond turtle populations, using this study data set and discussing them with available data from literature. we selected the canal system because it is more abundant in natural protected areas of italy than the pond system (lebboroni and zuffi, unpubl.). more specifically, our work has been aimed at verifying if i) there is a significant habitat preference (i.e. a choice between n-s or ew oriented canals) during the nesting activity ; ii) if the frame of nesting ground is randomly selected among different types; iii) if there is any role of water proximity to the nest. we also tried to investigate all tracks and signs around and on nests and eggs in order to identifying predator species. our contribution will show one of the largest available data set on nesting habitats, nest structure, and predation on nests of the european pond turtle in central italy (emys orbicularis galloitalica, fritz, 1995). such a set of information may certainly help in describing the actual role of artificial rectilinear water systems of protected areas in italy, especially in the light of management and protection of these environments. materials and methods study site the study was conducted from 1998 to 1999 in the u.s. army “camp darby” area, approximately 10 km2, about 10 km south west of pisa and 4 km east and inland from the ligurian sea coast. this site (fig. 1) is included in the regional “parco naturale di migliarino, s. rossore, massaciuccoli”, in western tuscany, central italy (43° 39’ 48” n, 10° 16’ 06” e), one of the largest protected coastal areas of italy. the site is connected to the coast, and is characterised by an evident alteration of past centuries, with introduced pine woods (pinus pinea), and rectilinear drainage canals. the terrestrial vegetation is mature mediterranean woodland (pinus pinea, p. pinaster, quercus ilex), while that around canals and in water is typical of wet areas. on average, trees that naturally fall or die are maintained in situ, with the exception of those that interrupt any military road within the us army area. natural vegetation on river banks is here reduced to a minimum due to excessive grazing of hundreds of fallow deers, dama dama, introduced around the ‘70ies. no drainage or management of canals have been yet observed. each canal, pond or marshy area was recorded and classified, and its width and length and the depth of the water were recorded. population features during present research we considered 256 adult females, according to external sexual characteristics. we considered standard measurements of carapace and body mass with accuracy of ± 1 mm and ± 1 g respectively (see zuffi et al., 1999). each marked female has been also provided with numbered white labels on both sides of carapace for recognition at distance. 40 m.a.l. zuffi and l. rovina nest identification nests were visually searched, along canals where the presence of adult females was usually common. additional random routes were added searching for nests in both favourable, even with scarcity of turtles, as well unfavourable, as wooded areas or open areas close to dried canals. intact nests were occasionally found within one to two days after deposition, and were externally characterised by the soft and humid, plain, slightly encircled sandy area, that females handled with their feet. due to small sample size, intact nests were not considered in this paper. then we refer to predated nests only. they appear as small holes in the ground, often irregularly shaped, with a sort of asymmetric opening that brings to the bottom of the nest chamber; in most cases predated eggs lay in the immediate neighbouring of the nest. maximum width, depth, and length of chamber pavement, in addition to the number of predated as well intact eggs were counted. predated eggs may have resulted broken in the middle or at one cap, or divided in two or even more parts. only pairs of half eggs were considered as single eggs. results obtained from the morphological analysis of predated eggs (zuffi and rovina, unpubl. data) were used for data interpretation. fig. 1. map of camp darby. letters represent canal and road numbers and numbers represent intersection with canals and roads. canals as dots on thin line; roads as thin line; area margins as thick line. 41habitat of nesting and predated nests in emys environmental variables nest position with respect to main environmental features, as canal border, bush, tree, or proximal nest was considered. this basic data set was necessary in order to establish a first, fairly complete information data set on habitat characteristics for future comparisons. due to environmental complexity, we measured many variables directly in the field, while others, as i.e. distance to the nearest road, were calculated using local topographic maps. the following data were recorded for each nest as: distance to the nearest water site (dnw); distance to the nearest tree (dnt); distance to the nearest wood margin (dnwd); distance to the nearest road (dnr); distance to the nearest path (dnp); distance to the nearest vegetation > 15 cm height (dnv); height of the nearest vegetation (hnv); mean height vegetation in an area of 9 m² around the nest (mhv); percent cover of vegetation in the area of 9 m² around the nest (pcv): we divided this area into a 0,5 x 0,5 grid, then we counted and summed the squares containing any vegetation > 15 cm height (table 1). soil type (st): we considered three categories: i) sandy soil, ii) compact soil, iii) stony soil. canal orientation (co): north-south oriented, east-west oriented. canal (c): distance to the nearest canal. canal vegetation (cv): type of vegetation of the nearest canal; we considered four vegetation characteristics: i) no vegetation, ii) bank vegetation (rush), iii) bank vegetation and “sparse” emergency reeds (where the distance between two adjacent reeds was greater than 20 cm), iv) bank vegetation and “thick” emergency reeds (where inter-reed distance was less than 10 cm) as in di trani and zuffi (1997). ground slope (gs): slope of nest ground with respect of the horizontal ground. type of vegetation in the 9 m² around the nest (tvc), with four different categories, that are i) grass (height < 15 cm), ii) short bush (height between 15 and 30 cm), iii) high bush (height > 30 cm), and iv) shrub. light intensity (li): we coded four categories, i) shade, ii) half shade, iii) sunlight, iv) full sunlight. canal margin (cm): northern, southern, eastern and western margin. habitat (h): we classified nest sites as being in strip or non strip habitats; strip habitats were 1-7 m wide and > 15 m long, included for example roadsides; and non strip habitats were all open areas (congdon et al., 1983). rubble (i.e. short cuts of building remains) cover (rc): presence of rubble in the 9 m² around the nest, we identified three categories i) no rubble, ii) intermediate cover (< 50%), and iii) high cover (> 50%) (table 2). statistical procedures all considered parameters were tested for normality and processed accordingly with parametric or non parametric statistics. comparison of observed differences of a given nesting area feature, table 1. frequency of quantitative variables nest characteristics means ± 1 sd min. max. n size distance to nearest water (dnw) (m) 11.53 ± 20.97 1.5 200 209 distance to nearest tree (dnt) (m) 11.47 ± 10.83 1.5 95 209 distance to nearest wood (dnwd) (m) 12.83 ± 11.04 1.5 95 209 distance to nearest road (dnr) (m) 28.08 ± 28.84 0.15 100 131 distance to nearest path (dnp) (m) 4.05 ± 5.70 0 40 209 distance to nearest vegetation (dnv) (m) 0.70 ± 0.70 0 3 179 distance to nearest nest (dnn) (m) 8.79 ± 8.81 0.7 40 154 height of nearest vegetation (hnv) (m) 0.61 ± 0.37 0.10 2 179 mean eight vegetation (mhv) (m) 0.85 ± 0.40 0.10 2 179 percent cover vegetation (pcv) 25.90% ± 8.81 0% 90% 209 42 m.a.l. zuffi and l. rovina table 2. means values of qualitative variables variable n soil type (st) 209 sandy soil 119 earthy soil 50 stony soil 40 canal orientation (co) 209 north-south 155 east-west 54 canal vegetation (cv) 209 no vegetation 0 banks vegetation 41 banks vegetation and sparse reeds 85 banks vegetation and tick reeds 83 ground slope (gs) 209 slope 62 flat 147 type vegetation cover (tvc) 209 grass (h< 15 cm) 30 short bush (15 < h < 30 cm) 4 high bush (h> 30 cm) 139 shrubs 36 light intensity (li) 209 shade 34 half shade 29 sunlight 101 full sunlight 45 canal margin (cm) 209 northern margin 12 southern margin 42 eastern margin 61 western margin 94 habitats (h) 209 strip habitats 127 non strip habitats 82 rubble (r) 209 no rubble 153 rubble cover < 50% 25 rubble cover > 50 % 31 43habitat of nesting and predated nests in emys for instance the distribution of nests between the opposite sides of a canal, has been processed with χ² with yate’s correction, or with mann-whitney u test. in order to reduce the number of variables for nest characteristics, and with the aim at classifying most of the canals and habitats, with a reduced number variables, two multivariate methods were used: factor analysis (principal component analysis: pca; lenk and wüster, 1999) and the discriminating analysis (da) (disi, 1987; marnell, 1998). pca was run after varimax rotation of all considered characters. factor extraction was performed on data with eigenvalues larger than one, and it was used to verify whether environmental variables did really contribute to the description of nesting habitats. da was used to verify which variable actually had a role in the discrimination (marnell, 1998). this analysis was carried out using stepwise method. significance level was set at α = 0.05. statistical analyses were performed with spss 6.1.2 for windows 95. results female population features on average, females of this area displayed secondary sexual characteristics at 99-100 mm carapace length, and have been then considered adult. the first reproductive female was captured at 112 mm carapace length (zuffi and odetti, 1998; rovina, 1999; zuffi et al., 1999). adult females measured (n = 256; average ± 1 sd) 133.1 ± 10.2 mm carapace length (range 100-155 mm), and 54 ± 4.5 mm carapace height (range 41-68 mm), and weighed 425.14 ± 88.88 g (range 160-660). clutch size ranged from four to nine eggs (43 clutches from 24 gravid females and 19 intact nests; mean = 5.72 ± 1.28, mode = 5, coefficient of variation = 22.4 %; zuffi et al., 1999). even if home-range estimation was not considered as a priority in our study, females seemed to move only slightly within their main area along a same canal: most marked females were observed in the same area during the whole active season. nevertheless we could not exclude a priori any long term movement as found in other populations (lebboroni and chelazzi, 1991; rovero and chelazzi, 1996; schneeweiss and steinhauer, 1998). predated nests predated nests averaged 120 ± 40 mm width (range 50-360 mm, n = 202), 100 ± 20 mm depth (range 50-170 mm, n = 202), and 120 ± 40 mm length (range 40-360 mm, n = 202). the average estimation of nest clutch size was 3.19 ± 1.83 eggs (range 0-8, n = 184), suggesting that a significant part of layed eggs has been removed by the predator far from the nest. we found infact that main predators’ signs on eggs belonged at least to birds (garrulus glandarius, corvus corone), rodents (rattus and microtus), ungulates (sus scrofa) and carnivores (mustela nivalis, vulpes vulpes), that likely bring elsewhere part of their preys (rovina and zuffi, submitted). average untransformed data were as follows for predated nests: distance to the nearest water site was within 6 m for 50% nests and within 21 m for 90% nests; distance to the nearest tree ranged from 1.5 to 95 m and was within 9 m for about 50% nests, while was within 22 m for 90% nests; distance to the nearest wood margin ranged from 1.5 to 95 m and was within 10 m for 50% and within 22 m for 44 m.a.l. zuffi and l. rovina 90 nests; 56.9 % nests were in sandy soil, while 19% was in stony soil and 24.1% was in compact soil. 0 20 40 60 80 100 120 140 160 180 ns canal ew canal ns ew fig. 2. frequency of distribution of predated nests in north-south and east-west canals. ordinate represents the amount of canal types expressed in meters x 100 and the absolute frequency of predated nests per canal type. table 3. rotated factor matrix. variable factor 1 factor 2 factor 3 factor 4 factor 5 factor 6 factor 7 dnw 0.12986 0.01539 0.56873 0.09502 0.30620 0.07942 0.24861 dnt 0.11258 0.03699 0.15997 0.89306 0.16347 0.09049 0.01861 dnwd 0.36089 0.07443 0.02411 0.81579 0.01816 0.09754 0.00432 dnr 0.69397 0.46543 0.20733 0.15110 0.01313 0.11262 0.12452 dnp 0.08890 0.46508 0.57768 0.10282 0.41495 0.23140 0.08526 dnv 0.30537 0.01293 0.65348 0.10864 0.06269 0.39255 0.16482 dnn 0.10598 0.14878 0.05425 0.00418 0.02834 0.05544 0.92211 hnv 0.02064 0.11103 0.06020 0.04647 0.08784 0.85896 0.04054 mhv 0.23494 0.01828 0.45860 0.09256 0.10921 0.60604 0.01107 pcv 0.43277 0.10599 0.70344 0.22658 0.00628 0.13036 0.01396 st 0.09329 0.75347 0.21962 0.17612 0.41997 0.04997 0.00423 co 0.87488 0.10252 0.26392 0.13181 0.06116 0.08135 0.02468 c 0.60259 0.00674 0.36267 0.07800 0.41865 0.03436 0.2721 cv 0.60691 0.01574 0.06575 0.25394 0.55083 0.27381 0.09033 gs 0.21356 0.37964 0.08653 0.29337 0.17184 0.32119 0.31668 tvc 0.46389 0.19009 0.39893 0.25394 0.22485 0.28793 0.14575 li 0.15798 0.23932 0.10552 0.09225 0.75292 0.11839 0.01650 cm 0.89630 0.06403 0.04918 0.24970 0.11478 0.04407 0.01916 h 0.04343 0.88961 0.01546 0.10650 0.08742 0.04235 0.09267 r 0.27494 0.64362 0.03085 0.04051 0.42897 0.08943 0.10738 45habitat of nesting and predated nests in emys canals canals are of a same type, that is rectilinear artificial type, and the main difference between them regards their distributive patterns: type 1) are mainly north-south (ns) oriented; type 2) are mainly east-west (es) oriented. overall length of ns oriented canals with water is 5300 m and ew oriented canals with water is 9500 m. we recorded 209 predated nests, 155 of them along ns canals and 54 along ew canals (fig. 2). the difference of number of nests and their relative distribution versus availability of two canal types, is highly significant (χ² with yate’s correction = 132.09, 1 df, p < 0.001). nests of ns oriented canals were distributed on western (n = 94) and eastern (n = 61) sides; nests of ew oriented canals were distributed on northern (n = 12) and southern (n = 42) sides. the difference in nest distribution between sides of ns and ew oriented canals is significant (χ² with yate’s correction = 15.57, p < 0.01; χ² with yate’s correction = 6.61, p < 0.05, respectively), clearly indicating a marked preference for warmer sun exposed areas. habitat and canal classification pca analysis found that habitat, canal margin, average height of the closest bush, distance to the nearest tree, percentage of vegetation coverage, light intensity and inter nest distance, among other parameters, explained more than 70 % of the observed variability of table 4. eigenvalues and variance of the overall factors. factor eigenvalue pct of var. cum. pct 1 6.1464 30.7 30.7 2 2.33321 11.7 42.4 3 1.73466 8.7 51.5 4 1.41025 7.1 58.1 5 1.24647 6.2 64.4 6 1.06170 5.3 69.7 7 1.02837 5.1 74.8 8 0.95043 4.8 79.6 9 0.71653 3.6 83.1 10 0.67844 3.4 86.5 11 0.48190 2.4 88.9 12 0.44274 2.2 91.2 13 0.36934 1.8 93.0 14 0.35278 1.8 94.8 15 0.27894 1.4 96.2 16 0.23978 1.2 97.4 17 0.23471 1.2 98.5 18 0.14861 0.7 99.3 19 0.10231 0.5 99.8 20 0.04247 0.2 100.0 46 m.a.l. zuffi and l. rovina nesting area feature. the rotated factor matrix (table 3) indicated that nests were positively selected for sunny bushy areas, with proximity to moderately high bush, distant from the wood, relatively distant from the road, and scarce in habitats with grounds covered by rubble. discriminant analysis were carried out using the seven most informative variables (see table 4), and considering habitat (h), canal orientation (co) and canal margin (cm) as grouping variables. habitat correctly classified 66.5 % of considered cases (n = 139 out of 209; after function 0, wilks’ λ = 0.799178, χ2 = 29.254, 3 df, p = 0.00001). canal orientation correctly classified 100 % of considered cases (n = 209 out 209; after function 0, wilks’ λ = 0.213841, χ2 = 201.299, 3 df, p = 0.00001). canal margin classified 41.6 % of considered cases (n = 87 on 209; after function 0, wilks’ λ = 0.649133, χ2 = 55.743, 12 df, p = 0.00001). east west canals the average distance to the nearest path (dnp) is greater in northern than southern margins of ew canals (mann-whitney z test = -2.79, p < 0.01); the average height of the closest bush (hnv) is significantly greater in northern than southern margins of ew canals (mann-whitney z test = -2.65, p < 0.01). the average bush coverage (mhv) is significantly wider in northern than southern margins of ew canals (mann-whitney z test = -2.13, p < 0.05); the percentage of vegetation coverage (pcv) is significantly greater in northern than southern margins of ew canals (mann-whitney z test = -2.33, p < 0.05). st: stony soil is more abundant, and sandy soil is less abundant on southern than northern canal borders (χ2 = 7.96, 2 df, p < 0.05). cv: bank vegetation and sparse emergent reeds (category 3) are less common than expected, while bank vegetation only (category 2) is more abundant than expected on northern margins (χ2 = 6.1, 2 df, p < 0.05). tvc: high bush is more distributed on northern than southern margins, and shrubs are less abundant than on northern margins (χ2 = 21.35, 3 df, p < 0.01). li: sun light is more intense than expected on northern margins, and less intense than expected on southern margins; full sunlight is more evident on southern margins (χ2 = 15.76, 3 df, p < 0.01). h: strip habitat is more frequent on southern margins, and non strip habitat is more frequent on northern margins (χ2 = 4.36, 1 df, p < 0.05). north south canals the distance from a road (dnr) is greater in western than in eastern margins of ns oriented canals (mann-whitney z test = -2.87, p < 0.01); the average distance to the nearest path (dnp) is lower in western than in eastern margins of ns oriented canals (mannwhitney z test = -3.5, p < 0.01). the inter nest distance (dnn) is lower in western than in eastern margins of ns oriented canals (mann-whitney z test = -2.12, p < 0.05); the average height of the closest bush (hnv) is significantly greater in western than eastern margins of ns oriented canals (mann-whitney z test = -2.18, p < 0.05). the percentage of coverage (pcv) is significantly greater in western than eastern margins of ns oriented canals (mannwhitney z test = -2.77, p < 0.01). st: stony soil is less abundant on western margins, than on eastern margins; earthy soil is less abundant than expected on eastern margins (χ2 = 26.3, 47habitat of nesting and predated nests in emys 2 df, p < 0.01). cv: bank vegetation and thick emergent reeds (canal vegetation, category 4) is less abundant on eastern margins than on western margins; bank vegetation and sparse emergent reeds (canal vegetation, category 3) is less frequent than expected on western margins than on eastern margins (χ2 = 12.39, 2 df, p < 0.01). tvc: grass is less distributed on western margins than on eastern margins (χ2 = 15.14, 3 df, p < 0.01). light intensity (li): was not different between eastern and western margins (χ2 = 4.89, 3 df, not significant). h: strip habitat is more frequent on eastern margins, than on western margins (χ2 = 7.83, 1 df, p < 0.01). rc: high cover rubble (category 3) is more frequent on eastern than western margins (χ2 = 19.99, 2 df, p < 0.01). comparison between ns and sw canals. the occurrence of several considered parameters (e.g., dnw, dnr, dnp, hnv, mhv, pcv) in ns canals was significantly greater than that observed in ew canals (mann-whitney z test, p ranging from < 0.05 to < 0.001); only dnv was significantly lower in ns than in ew canals (mann-whitney z test = -3.84, p < 0.001). st: stony soil is less abundant on ns oriented canal than on ew canals, and earthy soil is less frequent on ew oriented canals (χ2 = 29.89, 2 df, p < 0.01). cv: category four (bank vegetation and thick emergent reeds) is less frequent on ew canals as well as category two is more frequent on ew oriented canals (χ2 = 51.67, 2 df, p < 0.01). tvc: high bush is less frequent than expected on ew oriented canals; shrub is less abundant on ns oriented canals (χ2 = 48.9, 3 df, p < 0.01). li: sunlight is more abundant, but full sunlight is less abundant on ew canals (χ2 = 12.07, 3 df, p < 0.01). rc: category one is less abundant, and category three is more abundant on ew canals (χ2 = 20.04, 2 df, p < 0.01). discussion our work allowed us to construct, probably for the first time with this wide number of variables, the main largest available database of physical characteristics that describe nesting habitats and predated nests of the european pond turtle. these nesting habitats describe with particular accuracy the canal habitat, one of the two main habitat systems recorded for emys orbicularis in italy (lebboroni and chelazzi, 1998). because of the relatively common occurrence of canal habitat in several parts of the european distribution area of the species (austria: rössler, 2000b; france: servan, 2000; hungary: farkas, 2000; italy: lebboroni and chelazzi, 1998; ukraina: kotenko, 2000), our information set arises to a particular relevance. our data set confirms the few scattered information available up to date on nesting habitats, and on nest size (rovero and chelazzi, 1996; lebboroni and chelazzi, 1998; zuffi et al., 1999; ballasina and lopez-nunes, 2000; gianatti et al., 2000; rössler, 2000c; but see also andreas, 2000). a specific interest on the role of reproductive migrations, in the light of the spatial organisation of movement, has been recently increased (lebboroni and chelazzi, 1991, 1998; rovero and chelazzi, 1996; schneweiss, 1998), adding further information on the use and the characteristics of nesting areas. it has also been reported that fidelity to the nesting sites is a frequent pattern, with preferred migration routes (rovero and chelazzi, 1996; schneeweiss et al., 1998). this implies a specific pattern of habitat use following the preferred paths. 48 m.a.l. zuffi and l. rovina canal habitats north-south oriented were the most important habitat feature regarding nesting areas for emys orbicularis. particularly important were the western sides of northsouth canals and southern sides of east-west canals (see gianatti et al., 2000). there is evidence that nests were positively selected in sunny bushy areas, in proximity to moderately high bush structure, distant from the wood, relatively distant from the road, but were scarce in habitats with grounds covered by rubble. the high occurrence of predation on nests reflects a typical, natural condition of the life history traits of most of the studied fresh water turtles (ernst and barbour, 1989). nevertheless, in emys orbicularis, observation of juveniles is often scarce or not common; this fact has been assumed to be the result of sampling bias and low recruitment rate or due to environmental pollution or habitat alteration (devaux and bley, 1998; keller et al., 1998). large pond turtle populations do not seem to suffer even of an intense predatory activity on nests, while small, scattered, and sparse populations, may be endangered by both nest predation, and by habitat alteration (andreas and paul, 1998; cheylan and poitevin, 1998; devaux and bley, 1998; jablonski and jablonska, 1998; mascort, 1998; schneeweiss, 1998; mitrus and zemanek, 2000). in most of these cases, the protection of nests against predators has been claimed by several authors as a necessary procedure towards the protection and management of wild populations (see mitrus and zemaneck, 1998; mitrus, 2000; rössler, 2000c). furthermore, habitat protection should be primarily considered especially in all those cases where human action has been markedly negative. we now suggest that the habitats to be protected should cover both primary aquatic habitats, with a special emphasis to nursery sites (lebboroni and chelazzi, 1998), and to routes of migration to and from nesting areas (schneweiss, 1998; rössler, 2000), as well as to and from nursery areas. it also emerges that most nature parks and protected areas may arise to the natural role of core of production of both turtle eggs and hatchlings: the higher presence of nests and eggs, in areas with high turtle frequency, should stimulate the idea of an import/ export trade of eggs and hatchlings to those areas where living specimens of the same taxon are scarce. on average, the searching for a nesting area, could be easily performed following a relatively small number of radiotracked females, captured prior to their prereproductive migration (usually from may to july), collecting the layed eggs and store them in incubatory chambers; alternatively or in parallel, several reproductive females carrying detectable eggs (zuffi et al., 1999), can be brought to individual terraria, where they can lay their eggs, and then to put the layed eggs into incubatory chambers. any future management plan, should be mostly tested in protected areas, in order to assess any possible problems due to technical procedures; in protected areas, it should be possible to increase the experimental didactic information to local people. the system could be then exported to non protected, or altered areas. acknowledgements we are very grateful to francesca odetti, maria elena fabbri for continuos help in the field; piero bellinvia, italian army commander, for permission enter in camp darby area; stefano maes49habitat of nesting and predated nests in emys trelli, president and to all the staff of the nature regional park migliarino san rossore massaciuccoli. all friends and colleagues that furthermore helped during some of the field stages of this research are also acknowledged. at least, but not last two anonymous referees that have greatly improved a previous draft to the ms. literature cited andreas, b. 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(1998): reproductive ecology data of the european pond turtle (emys o. orbicularis) in brandenburg, northeast germany. mertensiella 10: 227-234. 51habitat of nesting and predated nests in emys schneeweiss, n., steinhauer, c. (1998): habitat use and migrations of a remnant population of the european pond turtle, emys o. orbicularis (linnaeus, 1758), depending on landscape structures in brandenburg, germany. mertensiella 10: 235-243. servan, j. (2000): die “brenne” in mittelfrankreich: land der 1.000 teiche und 50.000 sumpfschildkröten emys orbicularis (l.). stapfia 69: 205-210. zuffi, m.a.l. (2000): biology of the conservation of the european pond turtle, emys orbicularis, of italy. stapfia 69: 219-228. zuffi, m.a.l., odetti, f. (1998): double egg deposition in the european pond turtle, emys orbicularis, from central italy. ital. j. zool. 65: 187-189. zuffi, m.a.l., odetti, f., meozzi, p. (1999): body-size and clutch-size in the european pond turtle, emys orbicularis, from central italy. j. zool., lond. 247: 139-143. issn 1826-0373 (print) © firenze university press issn 1970-9498 (online) www.fupress.com/ah acta herpetologica 6(1): 127-129, 2011 osservatorio erpetologico italiano in questa sezione sono pubblicate segnalazioni erpetologiche riguardanti il territorio amministrativo italiano, non ancora incluse nella banca dati della shi e pubblicate nell’atlante nazionale (sindaco et al., 2006). sarà data priorità alle osservazioni riguardanti specie di interesse conservazionistico internazionale (specie incluse negli allegati ii e iv della direttiva europea 92/43/cee “habitat”), nazionale (specie endemiche, rare o al limite di areale) o regionale (nuove segnalazioni per la regione o che definiscono più nel dettaglio in modo significativo, l’areale della specie). saranno inoltre considerate per la pubblicazione segnalazioni di interesse ecologico (ad esempio limiti altitudinali). per quanto riguarda le testuggini del genere testudo, saranno pubblicate solo le segnalazioni di siti in cui è provata la riproduzione. osservazioni opportunamente documentate di popolazioni naturalizzate di specie esotiche (come trachemys sp.) saranno pubblicate. per motivi conservazionistici, le località precise degli avvistamenti non saranno pubblicate. la shi non incoraggia la raccolta di esemplari al solo fine della segnalazione e non pubblicherà segnalazioni di animali sacrificati senza le necessarie autorizzazioni. i segnalatori sono invitati a fotografare gli esemplari in vita, a consegnare quelli trovati morti ad istituzioni pubbliche locali e inviare al presidente della shi le foto e le citazioni dell’istituzione in cui il reperto è conservato. le segnalazioni, che saranno sempre citate col nome dell’osservatore, saranno vagliate dai redattori dell’atlante nazionale, che si avvarrà dei redattori di acta herpetologica e di esperti regionali. la segnalazione potrà eventualmente essere completata con un commento redazionale. indirizzo a cui inviare le segnalazioni (si caldeggia l’invio per posta elettronica) al presidente shi, edoardo razzetti, università degli studi di pavia, p.zza botta 9, 27100 pavia. e-mail: razzetti@unipv.it simbologia utilizzata (in ordine possono essere utilizzati più simboli) 000001 numero d’ordine della segnalazione +ita prima segnalazione in italia di una specie a distribuzione geografica limitrofa +reg prima segnalazione nella regione di una specie (prime tre lettere della regione) +h-ii nuova segnalazione/sito di specie dell’allegato ii della direttiva habitat 128 osservatorio erpetologico italiano +h-iv nuova segnalazione/sito di specie dell’allegato iv della direttiva habitat +h-ii/s riconferma di segnalazione/sito storico (non confermato da almeno 10 anni) di specie dell’allegato ii della direttiva habitat +h-iv/s riconferma di segnalazione/sito storico (non confermato da almeno 10 anni) di specie dell’allegato iv della direttiva habitat +end nuova segnalazione/sito di specie endemica italica in località che ne definiscono meglio l’areale +rar nuova segnalazione/sito di specie endemica rara o al limite di areale +end/s riconferma di segnalazione/sito storico (non confermato da oltre 10 anni) di specie endemica italica +rar/s riconferma di segnalazione/sito storico (non confermato da oltre 10 anni) di specie endemica rara o al limite di areale +tes sito accertato di riproduzione di testuggine del genere testudo +eco significativa segnalazione di interesse ecologico (esempio: limite altitudinale, nuovo ambiente, nuovi aspetti di nicchia trofica o riproduttiva ecc.) +eso sito accertato di riproduzione di specie esotica osservatorio erpetologico italiano – 8 amphibia a0077 roberto cobianchi +eso 110.367.0.002.0 rana catesbeiana. s.giovanni in persiceto (bo), 2 giugno 2009. oasi di manzolino tivoli. a0078 mauro della toffola +eso 110.367.0.002.0 rana catesbeiana. livorno ferraris (vc), 19 marzo 2010. reptilia r0050 enrico romanazzi, silvia bertollo, massimo semenzato +eso 110.370.0.001.0 trachemys scripta. tombolo (pd), 2009. sic palude di onara. riproduzione accertata. r0051 lorenzo stefani +eso 110.370.0.001.0 trachemys scripta. vicenza, osservazioni ripetute dal 1999 al 2009. oasi wwf degli stagni di casale “a. carta”. riproduzione accertata. 129osservatorio erpetologico italiano r0052 cristiano liuzzi +eso 110.370.0.001.0 trachemys scripta. matera, 26 giugno 2005. diga di san giuliano. riproduzione probabile. monitoraggio nazionale dell’erpetofauna alloctona le specie alloctone costituiscono una delle principali minacce per la fauna. nonostante in italia siano presenti numerose specie di erpetofauna alloctona, la loro distribuzione loro distribuzione non è ancora sufficientemente conosciuta, e non è stata finora posta sufficiente attenzione sull’importanza di segnalare tempestivamente novità sulla diffusione di queste specie. la commissione conservazione shi ha quindi deciso di iniziare un monitoraggio nazionale dell’erpetofauna alloctona, per avere un quadro aggiornato della situazione e anche per valutare eventuali cambiamenti nel tempo. chiediamo pertanto aiuto alla rete di erpetologi, per raccogliere segnalazione sulla distribuzione delle specie alloctone. i risultati di questo monitoraggio verranno pubblicati su acta herpetologica, all’interno dell’osservatorio erpetologico italiano, secondo le norme dell’osservatorio. le specie su cui riteniamo di focalizzare l’attenzione sono: rana catesbeiana, xenopus laevis, trachemys scripta e altre testuggini palustri naturalizzate. ovviamente, anche segnalazioni di altre specie di erpetofauna alloctona sono benvenute, ma vi preghiamo di tralasciare l’avvistamento di singoli esemplari. per le testuggini palustri alloctone, sarebbe estremamente interessante (ove possibile) mettere in evidenza le località in cui avviene la riproduzione. le segnalazioni vanno inviate a francesco ficetola, delegato per la commissione conservazione all’indirizzo e-mail: francesco.ficetola@unimi.it dati necessari per la segnalazione: • data dell’osservazione, specie, sito (breve descrizione), comune, provincia, località più prossima sull’atlante stradale. se possibile allegare anche: • fotografie, quota, coordinate gps (sistema di coordinate utilizzato), posizione su ctr o su immagine da satellite google earth qualunque nota e commento aggiuntivi sono i benvenuti. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 7(1): 1-12, 2012 the gulf of manfredonia: a new neritic foraging area for loggerhead sea turtles in the adriatic sea paolo casale1,2, giovanni simone3,4, ciro conoscitore3, michele conoscitore3, pasquale salvemini5 1department of biology and biotechnologies “charles darwin”, university of rome “la sapienza”, viale dell’università 32, 00185 roma, italy. corresponding author. e-mail: paolo.casale@uniroma1.it 2wwf italy, via po 25c, 00198 roma, italy 3associazione “centro cultura del mare” manfredonia, via gargano 42, 71043 manfredonia, italy 4lega navale italiana sezione di manfredonia, banchina di tramontana, 71043 manfredonia, italy 5sea turtle rescue centre wwf, via g. puccini 16, 70056 molfetta, italy submitted on 2011, 24th october; revised on 2012, 26th january; accepted on 2012, 27th february. abstract. the adriatic sea is an important foraging area for the loggerhead sea turtle, caretta caretta, but neritic habitats for this species along the italian coast were identified in the northern shallow area only. the gulf of manfredonia is a relatively wide shallow area in the south-west adriatic and its features and preliminary information make it a potential foraging ground for turtles. in order to assess sea turtle occurrence in the area, we monitored seven bottom trawlers based in the port of manfredonia during the period oct 2010 – jul 2011 through a voluntary logbook programme, resulting in a total of 62 turtle captures during 617 fishing days. since a turtle capture represents a rare event during such sampling, data were analysed by a zero-inflated poisson (zip) model. results indicate that: (i) the gulf is a neritic foraging ground for loggerhead turtles which occur there with a relatively high density comparable to other mediterranean foraging grounds, (ii) it is frequented by a wide range of size classes, including small juveniles as well as adults, (iii) the highest occurrence is during the period jun-dec, (iv) over 1700 turtle captures occur in the gulf annually. preliminary findings about recaptured individuals suggest that part of the turtles are resident in the area. the peculiar features of the gulf of manfredonia and the collaboration of the fishing fleet, make it a valuable index site for studying current trends of sea turtle populations at sea as well as other aspects of sea turtle biology and conservation. keywords. sea turtle, trawling, bycatch, neritic habitat, mediterranean. introduction the adriatic sea has been recently identified as one of the most important areas for sea turtles in the mediterranean, notably loggerhead turtles, caretta caretta, while the 2 p. casale et al. other two sea turtle species frequenting the mediterranean, the leatherback, dermochelys coriacea, and the green turtle, chelonia mydas, occur in relatively low numbers in the adriatic (casale et al., 2003; lazar et al., 2004a; lazar et al., 2008). the adriatic is clearly an important foraging ground for loggerhead turtles of all life stages, as indicated by several studies. first, high numbers of turtles are incidentally caught by fishing gears (lazar and tvrtković, 1995; casale et al., 2004). second, tag recoveries and satellite tracking of adults tagged while breeding in greece showed that the adriatic is one of the few foraging grounds for adult loggerheads from greek rookeries (margaritoulis et al., 2003a; lazar et al., 2004b; zbinden et al., 2008; hays et al., 2010; schofield et al., 2010; zbinden et al., 2011) and a few turtles from turkey and cyprus were also found (lazar et al., 2004b). third, medium-long term permanence of juvenile loggerhead turtles in the area has been shown by tag returns (casale et al., 2007b). fourth, foraging in the adriatic has been specifically assessed (lazar et al., 2002; lazar et al., 2011a). fifth, the adriatic and in particular the southern part, is an important developmental area for loggerhead turtles in the first years of life, probably hatched in greece (casale et al., 2010). loggerhead sea turtles are listed as endangered in the iucn red list of threatened species (iucn, 2011). the main identified threats at sea in the mediterranean are incidental catch in fishing gear, collision with boats, and intentional killing (tomás et al., 2008; casale et al., 2010; casale, 2011) that appear to increase overall mortality (casale et al., 2007c; casale et al., 2010) and as a whole represent a high level of threat (wallace et al., 2011). there is growing evidence of several anthropogenic threats in the adriatic: the aforementioned incidental catch and also collision with boats, debris ingestion and pollutants (affronte and scaravelli, 2001; franzellitti et al., 2004; casale et al., 2010; lazar and gračan, 2011; lazar et al., 2011b). given the scale of movements and the different habitats experienced during their lives, information about habitat use and most frequented areas is key for planning sea turtle conservation (hamann et al., 2010). as a general tendency, small juveniles feed on preys in the epipelagic zone, usually in oceanic areas (i.e., out of the continental shelf ) and as they grow they tend to frequent more neritic habitats (i.e., on the continental shelf ), where they feed on benthic preys (musick and limpus, 1997; bolten, 2003; casale et al., 2008). so far, neritic habitats for loggerhead turtles in the adriatic were identified in the northern wide shallow area while the southern adriatic, with deeper waters, was thought to be frequented by smaller juveniles (casale et al., 2004; casale et al., 2010). however, recent anecdotal information suggested that the wide shallow area of the gulf of manfredonia, in the southern adriatic, might host a neritic foraging area for high numbers of turtles. this study aims to provide a preliminary assessment of the occurrence of loggerhead sea turtles in the gulf of manfredonia, in the context of the current knowledge about the distribution of the species in the adriatic sea. materials and methods the incidental catch by fishing gear (or bycatch) is commonly used as an index of turtle abundance (e.g. casale et al., 2004) and with appropriate units of fishing effort it allows comparison among 3sea turtles in the gulf of manfredonia different areas. in neritic areas turtles are particularly subject to capture by bottom trawls, since this fishing gear captures all animals at sea bottom, including turtles when they feed on benthic preys. in the 10-months period from 1 october 2010 to 31 july 2011 the turtle bycatch of seven bottom trawlers based in the port of manfredonia (italy) (fig. 1) was monitored through a voluntary logbook programme similar to previous studies (e.g., carreras et al., 2004; casale et al., 2007a; cambiè, 2011), i.e. fishermen periodically reported the number of fishing days and the number of turtles caught. this period represents a full fishing year because of the fishing closure in august-september, however in 2011 fishing effort was reduced in the period jan-mar due to strikes and this period was excluded from the following analysis. vessel length ranged between 14.7 and 16.8 m (mean: 15.6 m; sd: 0.9 m; n = 7), headrope length (the upper of the two horizontal ropes of the trawl net’s opening) was ≈ 40 m, the typical tow duration was 1-1.5 hrs, with 5-7 hauls per day, and speed ranged between 2.9 and 4 knots (data provided by the fishermen). fishermen were also interviewed about the perceived trend of turtle catch categorized into three cases: increasing, stable, decreasing. a sam   fig. 1. study area of the gulf of the manfredonia with 10 m isobaths. 4 p. casale et al. ple of 121 turtles captured by the monitored or other bottom trawlers based at the manfredonia port were landed and measured (curved carapace length notch to tip, ccln-t) (bolten, 1999) and the species determined. before release turtles were tagged on both the front flippers with inconel tags style 681 (national band and tag, newport, ky, usa), stamped with an alphanumeric code and a return address, in order to assess multiple captures. catch data were analysed with a zero-inflated poisson (zip) model, an extension of generalized linear models (glm) (lambert, 1992) already used in a similar study (cambiè, 2011). in sea turtle bycatch data, captures typically comprise a small part of the units of fishing effort (days, in the present study) and the majority of units have zero values (no turtle catch) while just a few have nonzero values (turtle catch). in such cases, the frequency of zeros is higher than that expected in a poisson or a negative binomial distribution and zero-inflated models are preferred since they separately calculate the probability of being in a “perfect state” (zero catch) and in a “imperfect state” (including zero and non-zero values). the model was run with the pscl package for zip analysis (zeileis et al., 2008; jackman, 2011) in the r environment (r development core team, 2009). the model provided catch rates (crs) in the form of turtles per day per vessel. for comparison with other studies, other two crs were calculated: turtles per month per vessel, and turtles per year per vessel. the total number of turtles caught by the manfredonia trawling fishery in the period monitored by this study was estimated from the total fleet of 204 trawlers (source: coast guard of manfredonia) that are of similar size of monitored vessels. fishermen from 11 trawlers, including the monitored ones, were interviewed about turtle trends. results a total of 671 fishing days were monitored and 62 turtle captures recorded (table 1). all the landed individuals in the framework of a wider project including more vessels than those monitored in this study, were loggerhead sea turtles, caretta caretta, and ranged from 28.5 to 82 cm ccl (mean: 56.1 cm; sd: 11.4 cm; n = 121) (fig. 2). turtle size was not significantly different among months of capture (kruskal-wallis test; h = 12.1; p = 0.21; n = 121). two turtles were re-captured (previously captured by trawlers in the study area and tagged) during the study period: turtle 1713a was captured the first time on 30 sep 2010 and the second time on 15 dec 2010, while turtle 1731a was captured on 4 nov 2010 and 22 apr 2011, respectively. table 1. monitored fishing days of bottom trawlers in the gulf of manfredonia and by-caught turtles. monitored days n turtles range turtles/vessel day oct 107 16 0-2 nov 97 12 0-1 dec 87 12 0-2 apr 90 3 0-1 may 96 1 0-1 jun 97 7 0-2 jul 97 11 0-2 total 671 62 5sea turtles in the gulf of manfredonia the value of the dispersion parameter of the overall zip model (φ) was close to 1 (φ = 0.99), indicating that the model fitted the data quite well. the logistic component of the model gave a probability p = 0.46 of zero turtle by-catch and therefore a probability 1 p = 0.54 that sea turtle by-catch followed a poisson distribution with mean (λ) = 0.17, yielding a mean value of 0.09 (± 0.01 se) turtles day-1 vessel-1. since a difference by month was detected (kruskal-wallis test; h = 17.7; p < 0.01; n = 671), in contrast with vessels (kruskal-wallis test; h = 1.0; ns; n = 671), another zip model was run on data aggregated by month in order to calculate mean cr and se for each month. monthly crs ranged between 0.01 and 0.15 turtles day-1 vessel-1, with higher crs in oct-dec and junjul than in apr-may (fig. 3; table 2). considering the average fishing days per month as 13.1 (mean of the fishing days per month per vessel), crs ranged from 0.13 and 1.96 turtles month-1.   0 10 20 30 40 50 25 35 45 55 65 75 85 ccl (cm) n um be r o f t ur tle s fig. 2. frequency distribution of curved carapace lengths (ccl) of 121 loggerhead turtles incidentally caught by bottom trawlers in the gulf of manfredonia. table 2. results of the zip model based on turtle by-catch data (response variable) with pair-wise comparison with only one covariate (month) from seven bottom trawlers fishing in the gulf of manfredonia. logistic regression part poisson regression part factors coefficient z-value p-value coefficient z-value p-value apr vs jun 8.84 0.04 ns 2.25 1.98 0.05 apr vs nov -7.39 0.00 ns 1.31 2.01 0.04 apr vs oct 7.43 0.04 ns 2.10 2.32 0.02 jun vs may -7.36 -0.03 ns -3.41 -2.22 0.03 jul vs may -7.92 -0.01 ns -2.92 -2.06 0.04 nov vs may 0.83 0.00 ns -2.47 -2.37 0.02 6 p. casale et al. the overall mean cr was 8.6 (sd: 2.4) turtles yr-1 vessel-1. if this catch rate is applied to the 204 bottom trawlers based at the port of manfredonia, the total catch by the local trawl fleet would be 1749 (sd: 484) turtles yr-1, which concerns only the months adequately monitored in this study and therefore represents an underestimation of the actual annual catch. most fishermen (10 out of 11 interviewed boats or 90.9%) declared that turtle catch is increasing in the study area while one fisherman declared that the trend is stable. discussion present results provide the first evidence of the importance of the gulf of manfredonia as a neritic foraging ground for the loggerhead sea turtle in the mediterranean. comparison with other mediterranean areas (table 3) indicates that turtle density in the study area is one of the highest known so far. moreover, the re-capture of two turtles in the same area after 2-5 months suggests that part of the turtles are resident in the area and not just migrating along the adriatic coasts (schofield et al., 2010). the highest crs were observed in the months jun-jul and oct-dec, suggesting higher turtle occurrence during these months and during the fishing closure in aug-sep, then during the period apr-may. such a seasonal variation of turtle occurrence in the study area may be due to several factors, but a major one is probably temperature, that drops in the range 9-15°c during jan-apr in the shallow waters of the gulf of manfredonia, rising to about 18°c in may and over 23°c in june (source: monthly sst contours by maptool, www.seaturtle.org, derived from noaa goes daily sst satellite data). for comparison, lower turtle occurrence during winter was explained by low temperatures in the north adriatic (<1112°c; lazar et al., 2003) and in the ligurian sea (12-13°c; lauriano et al., 2011).   0.00 0.05 0.10 0.15 0.20 oct nov dec jan feb mar apr may jun jul c at ch ra te (t ur tle s / d ay v es se l) fig. 3. mean and se sea turtle catch rates (turtle per day per vessel) by bottom trawlers in the gulf of manfredonia from october 2010 to july 2011. 7sea turtles in the gulf of manfredonia the gulf of manfredonia is frequented by loggerhead turtles of a wide range of sizes. bottom trawlers capture turtles while they are at the sea bottom, probably feeding on benthic preys, and the capture of very small individuals (min: 28.5 cm ccl) further supports the recent “opportunistic model” in which loggerhead turtles take advantage of the peculiar oceanographic features of the mediterranean to start feeding on benthic preys earlier (casale et al., 2008) than they do in other areas like in the atlantic ocean (bolten, 2003). the south adriatic and ionian seas are developmental areas for juveniles in the first years of life (casale et al., 2010) which predominantly freed upon pelagic preys, but present results indicate that they take the opportunity of shallow areas like the gulf of manfredonia and possibly others to feed on benthic preys as well. the proportion of turtles > 70 cm ccl, representing the adult size range for mediterranean loggerheads (margaritoulis et al., 2003a; casale et al., 2005), indicates that the gulf is also an inter-breeding foraging area for adults. actually, the proportion of turtles in this size range (11.6%; n = 121) is not lower than the one observed in the wider neritic area of the north adriatic (6.6%; n = 61) (casale et al., 2004), that is well known as a foraging ground for adults breeding in zakynthos (greece) (schofield et al., 2010; zbinden et al., 2011). while the mediterranean is frequented by loggerhead turtles belonging to two regional management units (wallace et al., 2010), available data indicate that the adriatic is a foraging ground for the mediterranean loggerheads only, at least from rookeries in greece, turkey and cyprus (lazar et al., 2004b; giovannotti et al., 2010), therefore these, in particular greece, are the most likely rookeries of origin for the turtles foraging in the gulf of manfredonia. since the period jan-mar was excluded from the analysis due to strikes which reduced the fishing effort, probably the annual catch rate per vessel and consequently the table 3. sea turtle catch rates per vessel, by bottom trawling in the mediterranean. country area turtle year-1 turtle month-1 turtle day-1 source italy gulf of manfredonia 8.6 0.13-1.96 0.01-0.15 present study algeria 1.41 (laurent, 1990) croatia 3-10 (lazar and tvrtković, 1995) egypt 1.72 (nada and casale, 2011) france mainland 0-3 (laurent, 1991) greece thracian sea (aegean) 0.0625 (margaritoulis et al., 2003b) italy north-west adriatic 0.052-0.438 (vallini et al., 2003; casale et al., 2004) italy central med lampedusa 0.376 (casale et al., 2007a) italy central med other 38-161 (casale et al., 2007a) spain north 0.07 (alvarez de quevedo et al., 2006) spain balearic 0.018 (carreras et al., 2004) tunisia gulf of gabes 0.121 (jribi et al., 2007) turkey south 8.5-11 (oruç et al., 1997; oruç, 2001) 8 p. casale et al. total number of capture events per year by the large trawl fishing fleet of manfredonia is higher than 1700. the level of associate mortality needs proper investigation, but it is probably low due to the short tow duration, which is the main parameter affecting mortality caused by forced apnoea (sasso and epperly, 2006). in the north adriatic, a mortality rate of 0.6% was observed from trawlers with a mean tow duration of 78 min (casale et al., 2004), comparable to the one adopted by the manfredonia trawlers, while a specific study in the gulf of mexico reported very low mortality from tow durations below 60 min (sasso and epperly, 2006). according to fishermen, turtle occurrence is increasing in the gulf of manfredonia, in contrast to negative trends reported by fishermen from other mediterranean areas, such as the balearic islands (spain), egypt and the area between sicily and tunisia (carreras et al., 2004; casale et al., 2007a; nada and casale, 2011). trends of sea turtle populations are very difficult to obtain, especially at sea. the most used index of turtle abundance is the number of nests, but it has intrinsic fluctuations and it is an index of current adult female abundance, not of the current population which is mostly made by juveniles. current population trends will be evident at nesting sites only after a long time, due to the long maturation of loggerhead turtles that in the mediterranean takes about 15-28 years (casale et al., 2009; casale et al., 2011). the major nesting site from which most of the turtles foraging in the study area probably originate is zakynthos (greece), where no clear nesting trend has been reported so far (margaritoulis, 2005). the trend reported by fishermen in the present study either may reflect the current trend of the population that will be observed on zakynthos in the future or may be the combination of trends and change of foraging area by different sub-populations or could be due to a misperception by the fishermen. this can only be assessed by a medium-long term monitoring project using catch rates as an index and by assessing the origin of turtles through genetic markers. the peculiar features of the gulf of manfredonia, i.e. a relatively small area frequented by high numbers of turtles of a wide range of size (age) classes and accessible through the collaboration of a large fleet of trawlers with several hundreds of captures per year, make it a valuable index site for studying current trends of sea turtles population at sea as well as other aspects of sea turtle biology and conservation. acknowledgements we thank all the fishermen who collaborated to this study and to the wwf sea turtle project. figure 1 was prepared with the program maptool (seaturtle.org, www.seaturtle.org). we also thank dr g. cambiè and an anonymous reviewer for constructive suggestions on a first version of the manuscript. references affronte, m., scaravelli, d. 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(2008): important areas at sea for adult loggerhead sea turtles in the mediterranean sea: satellite tracking corroborates findings from potentially biased sources. mar. biol. 153: 899-906. zbinden, j.a., bearhop, s., bradshaw, p., gill, b., margaritoulis, d., newton, j., godley, b.j. (2011): migratory dichotomy and associated phenotypic variation in marine turtles revealed by satellite tracking and stable isotope analysis. mar. ecol.-prog. ser. 421: 291-302. zeileis, a., kleiber, c., jackman, s. (2008): regression models for count data in r. journal of statistical software 27(8). url http://www.jstatsoft.org/v27/i08/. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 6(2): 169-174, 2011 ontogenetic pattern change in amphibians: the case of salamandra corsica wouter beukema itc, faculty of geo-information science and earth observation, university of twente, hengelosestraat 99, enschede, the netherlands. e-mail: wouter.beukema@gmail.com submitted on: 2011, 27th february; revised on: 26th july; accepted on: 16th september. abstract. ontogenetic, post-metamorphic pattern development is a rarely studied topic in amphibian science. as there are indications that the pattern of salamandra corsica might expand over time, digital image analyses were applied in order to measure several phenotypical variables which were related to the snout vent length. results show a significant increase of patches which change to irregular shapes while svl increases. digital image analysis is identified as a suitable tool to explore pattern shape and change in general, while the documented pattern development in s. corsica might be one of the first quantified cases of post-metamorphic ontogenetic pattern change in amphibians. keywords. salamandra corsica, digital image analysis, ontogeny, colour pattern development. coloration in the amphibian skin is composed of structural different xanthopores, iridophores and melanophores, which usually develop during the larval stage (pederzoli et al., 2003; vitt and caldwell, 2009), after which colour patterns typically stabilize shortly after metamorphosis (e.g. klewen, 1991). however, there are several reports of ontogenetic pattern change, i.e. post-metamorphic change in the colour pattern over time in several species of amphibians (e.g. lantz, 1953; bogaerts, 2002). while few in number, the majority of experimental pattern development research has taken place during the early 20th century and was mostly based on the relation between background colour and amount of dorsal and ventral patches in members of the genera salamandra and triturus (kammerer, 1913; herbst, 1924; lantz, 1953). likely due to the rarity of such studies and the fact that most were based on anecdotal evidence, ontogenetic pattern change was not mentioned in several recent reviews on amphibian biology (duellman and trueb, 1994; wells, 2007; vitt and caldwell, 2009). recent developments in digital image analyses of amphibian phenotypes (e.g. davis and maerz, 2007; vörös et al., 2007) could be especially useful in exploring and quantifying ontogenetic pattern change. these analyses are increasingly used to measure a vari170 w. beukema ety of morphological and phenotypical features in amphibians such as coloration, pattern and shape (e.g. relyea, 2004; davis and maerz, 2007; vörös et al., 2007) which can be applied for multiple purposes including evolutionary research and systematics (e.g. davis and grayson, 2007; vörös et al., 2007). their use permits the quantification of phenotype traits, such as amphibian colour patterns, which have been used widely in taxonomy, but traditionally often have been based on anecdotal evidence only. members of the genus salamandra occur over most of europe and the mediterranean area, ranging southand eastwards into north africa and the near east (review in thiesmeier, 2004). while a high number of (subspecific) taxa have been described during the last century especially within salamandra salamandra, the general taxonomy of the genus is unresolved (steinfartz et al., 2000). traditionally, the majority of taxa belonging to the ‘fire salamanders’ (as opposed to the ‘alpine salamanders’) have been described based on their diagnostic yellow colour pattern composed of xanthopores (eiselt, 1958). however, data regarding colour patterns is often restricted to the type series of the taxon originating from a single locality, while only limited research has been performed concerning intraspecific pattern colour variation within salamandra (but see e.g. bosch and lópezbueis, 1994; bogaerts, 2002; donaire barroso et al., 2009). there are indications of ontogenetic pattern change in several salamandra taxa (eiselt, 1958; mutz, 1992; bogaerts, 2002). contrasting statements exist on the pattern of one of these taxa, salamandra corsica. savi (1838) described the species as being characterized by only sparse yellow coloration, while eiselt (1958) suspected the yellow pattern to expand and merge during development after metamorphosis. consequently, qualitative analysis of the pattern of s. corsica might both provide an example of rarely studied pattern change, while resolving pending issues on the morphology of the species. in order to gather data on dorsal pattern of s. corsica, fieldwork was conducted in forêt de vizzavona (haute-corse, 42°06’n, 9°07’e), corsica at approximately 1200m altitude, from the 28th of april until the 2nd of may 2005. the study site is a fagus sylvatica forest on a granite slope with a thick leaf litter layer and conclusively little undergrowth apart from several asplenium species and cyclamen hederifolium. a small brook crosses the area which is used for reproduction by s. corsica. dorsal photos were made with a nikon coolpix e4600 of a total of 35 individuals which were immediately released after measuring their snout vent length in mm (svl; the distance from the tip of the snout to the posterior margin of the cloaca). due to undulations of the limbs and tail, images of individual s. corsica were cropped in adobe photoshop cs2 to include only the dorsum and head defined as the region of interest (roi). the noise filters ‘despeckle’ (detects the edges in an image and blurs all of the selection except those edges) and ‘median’ (within the radius of a pixel those pixels with similar brightness are selected while discarding pixels that differ too much from adjacent pixels; the centre pixel is replaced with the median brightness value of the searched pixels) were used to facilitate subsequent selection of the roi against heterogeneous backgrounds. using the ‘magic wand tool’ with a tolerance of 50, yellow patches (defined as areas of xanthopores) on the dorsum and head, as well as lateral patches continuing onto the dorsum were selected and saved as a separate image (fig. 1). pixel surfaces of the roi, area of yellow patches, perimeter of yellow patches and circularity (ci) were calculated with the freeware imagej (ferreira and rasband, 2011). the area of yellow patches was converted to a percentage of the roi area. patch circularity was defined as 171ontogenetic pattern change in amphibians ci = 4π(area/perimeter2) in which a ci of 1 represents a perfect circle while 0 is an infinitely elongated polygon. furthermore, the number of yellow patches was visually summed. statistics were computed in spss16. a shapiro-wilk test was used to investigate normality. subsequently, spearman’s rank correlation test was used to examine the strength of relations between svl, ci, number of patches and area of patches. significant relations between variables were tested with a p < 0.05. descriptive statistics on the measured variables (n = 35) are given in table 1. svl displayed a strong correlation with circularity (spearman’s rs = -0.889, p < 0.0001) and the number of patches (rs = 0.783, p < 0.0001). additionally, circularity was strongly correlated with the number of patches (rs = -0.864, p < 0.0001). although a weak correlation was recovered between svl and the area of patches (rs = 0.254) the relation proved to be insignificant (p = 0.141). table 1. descriptive statistics on the phenotypical variables derived from digital image analyses and the snout vent length of thirty-five salamandra corsica. variable mean minimum maximum sd circularity (index) 0.0000046 0.0000023 0.0000086 0.0000017 area of patches (%) 29.73 15.68 49.78 10.04 number of patches 19.54 11 35 7.04 svl (mm) 74.71 32.43 100.01 21.83 the current results show a significant positive relationship between svl and number of yellow patches, and a negative relationship between svl and patch circularity. while fig. 1. example of unprocessed images (a: juvenile salamandra corsica, c: adult salamandra corsica) to processed images showing the selected patch area on the region of interest of the same individuals (b,d). 172 w. beukema the area of yellow patches does show a weak relation with svl, this relationship is not significant. this could be an effect of low sample size. on the other hand, the relative area of yellow patches might increase only slightly or remain approximately equal when patches ontogenetically change to a more irregular shape. visual inspection of the photographed individuals indeed showed several cases of merged patches on the head (see also fig. 1) as described by eiselt (1958), but the general pattern seems to be highly inconsistent with considerable variation in patch number and area of yellow patches in adults (fig. 2). therefore, as displayed by the highly significant relationships ontogenetic pattern development is present in s. corsica, but is characterised by substantial intraspecific variation. bogaerts (2002) provided photographical evidence on the ontogenetic pattern development in a small sample of south-iberian s. s. crespoi and s. s. gallaica over the course of a fig. 2. correlations between snout vent length and number of patches (a) and circularity (b). 173ontogenetic pattern change in amphibians few years, while noting that moroccan s. algira tingitana seems to show ontogenetic reduction of xanthopores. a similar tendency of iridophore decrease was suspected by beukema et al. (2009) for a population of anatolian lyciasalamandra luschani finikensis. in addition to its relevance for general morphology and systematics, ontogenetic pattern change can be also an important factor within long term population studies based on individual recognition by means of colour pattern. such studies have been applied on different subspecies of s. salamandra (e.g. kopp-hamberger, 1998; carafa and biondi, 2004), without taking significant changes in pattern into account. errors could potentially arise when recognition of prior identified individuals fails due to post-metamorphic development, similar to a recent case described for the viperid vipera ursinii (tomović et al., 2008). conclusively, the currently documented pattern development in s. corsica might be one of the first quantified cases of post-metamorphic pattern change in amphibians. while according to literature ontogenetic pattern change appears to occur rarely, it is at least present in several taxa within the salamandridae, and presents an interesting topic for future research. acknowledgements jan beukema and mark bakkers are thanked for assistance in the field. sergé bogaerts provided valuable discussion on pattern development among salamandra taxa. references beukema, w., de pous, p., brakels, p. 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(2009): herpetology: an introductory biology of amphibians and reptiles. third edition. burlington, massachusetts. vörös, j., szalay, f., barabás, l. (2007): a new method for quantitative pattern analysis applied to two european bombina species. herpetol. j. 17: 97-103. wells, k. (2007): the ecology and behaviour of amphibians. the university of chicago press, chicago. acta herpetologica 17(1): 5-11, 2022 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-12187 the directional testes asymmetry increases with temperature in seven plateau brown frog (rana kukunoris) populations hai ying li1, man jun shang2, jie guo2, bo jun chen2, peng zhen chen2, tong lei yu1,* 1 college of life science, xinyang normal university, sd 464000, china 2 college of international education, xinyang normal university, sd 464000, china *corresponding author. e-mail: yutonglei_00000@163.com submitted on: 2021, 4th november; revised on: 2022, 10th february; accepted on: 2022, 11th february editor: wei chen abstract. environmental stress is generally regarded as an important evolutionary force for promoting the differentiation of shape, structure and function of animal organs closely related to survival and reproduction. geographical variation of temperature and corresponding change in intensity of male-male competition might drive inter-population differences in directional testes asymmetry (dta). here, we investigated inter-population variation in dta of the brown frog (rana kukunoris) at seven different altitudes on the eastern tibetan plateau. we found that the size of right testes increased with temperature, but not left testes. we also found that male age, body mass or body condition, and testis mass had not effect on dta, suggesting that heavier or older r. kukunoris males or those with larger testes had not stronger dta. the operational sex ratio did not affect dta, but there was a positive correlation between dta and temperature, suggesting that differences in the length of activity period and resources availability across locations may affect the energy budget of this frog, resulting in a gradual change in reproduction energy parallel to increasing temperature. keywords. environmental factor, testes asymmetry, body condition, age, the brown frog. introduction environmental stress (e.g., resource availability, competition, or temperature) has generally been an important evolutionary force for promoting the differentiation of life-history traits (blanckenhorn and demont, 2004; liao et al., 2015). for example, variation in energy acquisition under great differences in environmental conditions and environmental stress might lead to differences in the size, structure and function of animal organs and tissues among populations (jönsson et al., 2009; chen et al., 2011). in most species of birds, the left testis mass is larger than the right one (e.g., friedmann, 1927; møller, 1994; rising, 1996; jamieson et al., 2007; iddriss et al., 2018; sara et al,. 2019). møller (1994) hypothesized that the increase in size of the right testis is only to compensate for a reduced function of the left one, and thus, the degree of directional testes asymmetry (dta) in testis size is a measure of male body condition. moreover, the degree of dta is correlated with age in that the older males in a population because they could allocate more energy to reproduction than younger individuals (birkhead et al., 1997; graves, 2004). thus, there is correlation between possibilities for energy acquisition and male quality, thus the energy acquisition might have a potential impact on the degree of (dta). in recent years, although the testis asymmetry has been proved in some anuran species (zhou et al., 2011; liu et al., 2011, 2012; mi et al., 2012; yu and guo, 2015; wu and liao, 2017), there only few studies have focused on exploring geographical variation in dta (hettyey et al., 2005). 6 hai ying li et alii sperm competition may be attributed to drive the evolution of directional testes asymmetry. males owning large testis mass indicate experiencing strong sperm competition when the male/female sex ratios was highly male-biased (gage, 1994; pitcher et al., 2005; soulsbury, 2010; zeng et al., 2014). for instance, compared to control treatments, males increased investment in testis mass, ejaculates or accessory glands when they lived at large population density or high male/female sex ratios (gage, 1995; tan et al., 2004; ramm and stockley, 2009). in this case, two larger testes may be more effective in increasing overall larger testes size because it might be very costly to produce directional asymmetry (møller, 1994). however, although relevant substantial data were collected, an understanding of the causes for geographical variation in dta remains ambiguous and contentious. hence, independent datasets, especially on different populations within species, that do help us to have a better understanding of the general geographical patterns of variation in male quality, age and dta. in this study, we explore the occurrence of dta in all study populations of the brown frog (rana kukunoris), as well as the association between the degree of dta and the body condition or age. this species is endemic to the eastern tibetan plateau, inhabits open alpine marshes, and their habitats are located from 2200 to 4400 m in altitude. r. kukunoris deposits larger energy reserves in fat bodies and liver, but pre-hibernation energy stores decrease with increasing altitude (chen et al., 2013). further, degree of dta was not related to altitude or body size across three populations of r. kukunoris (chen et al., 2014). however, we expected a positive correlation between degree of dta and temperature as a consequence of decreasing developmental stress (sensu møller, 1994; hettyey et al., 2005). then, we expected degree of dta increased with body condition or age. finally, we expected degree of dta decreased with increasing operational sex ratio (osr, the ratio of the sexually competing males to fertilisable females in a breeding aggregation at a given time)in response to high sperm competition level. material and methods study site and sample collection we collected rana kukunoris individuals from seven populations (elevations ranging from 2506 to 3478 m, fig. 1) along the eastern tibetan plateau, china. we randomly collected 10–53 individuals by hand in the medium spawning period from late-march to mid-april in 2012 at each site. in this study, all individuals were sampled at the same time in their breeding cycle. then, these frogs were identified as adult males if they displayed nuptial pads on the fore digits, others as females. all adult females examined were released to the original spawning sites. the population-specific osr was calculated as the number of males to the number of fertilizable females in a breeding aggregation at a given time (3-5 days; mai et al., 2017). in this case, osr was used to estimate under the specific assumption that they could reflect the average number of males mating with each female. all captured males were brought to our field laboratory close to breeding sites. at room temperature, they were put into individual plastic opaque containers (diameter = 16.75 cm), filled 2 cm deep with fresh water. then, the snout-vent length (svl, to the nearest 0.1 mm) was measured with a vernier caliper (lxz919160, shenzhen luxianzi technology co., ltd.), and body mass (to the nearest 0.01 g) was weighed with an electric balance (sl202n, shanghai minqiao precision scientific instrument co., ltd.). a plastic bucket was prepared with the tms (tricaine methane sulfonate, cas: 886-86-2, purity: > 97.0%, sigma-aldrich) at 2g/l in 5000 ml fresh water aerated for at least 48 h for dechlorination and oxygenation (paduano et al., 2013). then, every five frogs were put into this plastic bucket containing the 5000 ml anesthetic bath. the degree of sedation was assessed through testing of the limb retraction reflex in response to gentle pinching of a toe. once anesthesia was achieved, adult males were euthanized by two-pithing and dissected (liao et al., 2016). both testes were removed and then weighed them to the nearest 0.1 mg with an electronic balance (cav264c, ohaus instrument (shanghai) co., ltd.). following the protocol of hettyey et al. (2005) and chen et al. (2014), directional testes asymmetry (dta) was calculated with the following equation: dta = right testis mass left testis mass. age determination we removed the longest phalange of the left hindfoot of male adults in each population and preserved in 10% aqueous solution of formaldehyde. following the protocol of ma et al. (2009), we produced histological sections of the frog phalanges and determined age by counting the number of lines of arrested growth (lag) in the sections. numerous studies have confirmed that improved method of paraffin section and ehrlich’s haematoxylin stain display seasonal growth of amphibian species (e.g., yu and lu, 2013; yu et al., 2019; yu et al., 2021). 7the directional testes asymmetry increases with temperature environmental factor collection the annual mean temperature did not decrease significantly with elevation (spearman’s correlation: r = −0.643, p = 0.119), and latitude (r = 0.607, p = 0.148). thus, we used annual mean temperature as environmental factor in this study. we downloaded temperature data from worldclim (http://www.worldclim.org; hijmans et al., 2005). worldclim data were for the period of 1950– 2000 at a resolution database of 0.167° × 0.167° grid cells. statistical analyses we used one-way anova to test whether the male size, age, the left or right testis mass and dta differed among populations. the body condition was measured using residuals of body mass regressed against svl. then, we performed a linear mixed models (lmms) to test variation in the left or right testis mass, where the left or right testis mass as the dependent variable, body condition, osr, temperature and age as fixed effects, population as a random effect. to test whether degree of dta covaried with the population-specific osr as proxies of sperm competition levels, we also performed linear mixed models (lmms) to test variation in degree of dta among populations, where body condition, osr, temperature and age as fixed effects, population as a random effect. in the subsequent analyses of reproductive traits against sperm competition levels (osr) the temperature remained in a simplified models as a covariate, but not include body condition. prior to analyses, we logtransformed body mass, the left or right testis mass and operational sex ratio of each population to better attain normality and enhance homogeneity of variance. spss 20.0 (spss inc., chicago, illinois, usa) was used for all analyses. fig. 1. topographic map showing the location of the 7 rana kukunoris study populations in the eastern tibetan plateau. 8 hai ying li et alii results mean values of male size, age, left or right testis mass, and degree of dta differed significantly between populations (table 1). the lmms showed that the right testes was not correlated with the osr (t = -2.105, p = 0.158), but increased with temperature (t = 3.284, p = 0.042, table 1, fig. 2), body condition (t = 5.447, p < 0.001) and age (t = 3.235, p = 0.001) when controlling for population (random effect: z = 0.537, p = 0.591). similarly, the left testes was not correlated with the osr (t = -0.262, p = 0.809) and temperature (t = 0.643, p = 0.558, fig. 2), but increased with body condition (t = 6.361, p < 0.001) and age (t = 5.564, p < 0.001) when controlling for population (random effect: z = 1.077, p = 0.281). the degree of dta was not significant correlated with age (t = -1.540, p =0.125), body mass (t = 0.788, p = 0.432) and testis mass (t = 0.107, p = 0.915), when controlling for population (random effect: z = 1.057 p = 0.290). the lmms showed that the degree of dta was not correlated with the osr (t = -1.623, p = 0.248), body table 1. comparisons of svl, age, body mass, testis mass and directional testes asymmetry of rana kukunoris from seven altitudes in the east tibet plateau, china. values represent mean ± se for each measure; n = number of individuals. population altitude (m) latitude (degrees) annual mean temperature (°c) n osr svl (mm) body mass (g) age (years) right testis mass (mg) left testis mass (mg) directional testes asymmetry dapuzi’cun 2297 36.65 5.30 17 2.43 54.70 ± 0.90 17.38 ± 0.86 3.06 ± 0.13 14.26 ± 1.08 11.51 ± 1.32 2.76 ± 0.87 shiya’zhuang 2594 36.68 3.50 39 2.79 51.27 ± 0.58 18.35 ± 0.62 3.13 ± 0.08 14.32 ± 0.76 14.05 ± 0.76 0.27 ± 0.50 damoshi’cun 2789 36.49 0.20 53 1.23 50.25 ± 0.58 15.39 ± 0.69 2.81 ± 0.10 10.92 ± 0.72 10.97 ± 0.73 -0.05 ± 0.54 zecha’zhan 3049 34.49 1.50 19 1.90 54.57 ± 0.59 18.23 ± 0.79 3.32 ± 0.13 12.53 ± 1.24 14.26 ± 1.21 -1.74 ± 0.85 shibadao’wan 3060 34.47 1.40 11 1.67 51.23 ± 0.90 14.74 ± 0.72 3.45 ± 0.16 8.79 ± 1.09 9.05 ± 0.99 -0.26 ± 0.52 shilin’ zhan 3233 34.37 1.00 24 2.20 47.40 ± 0.70 12.46 ± 0.41 2.96 ± 0.14 8.07 ± 0.64 8.42 ± 0.54 -0.35 ± 0.45 guoguo’ri 3441 34.29 0.80 10 1.48 51.74 ± 0.98 17.56 ± 0.94 3.20 ± 0.13 12.07 ± 0.99 11.64 ± 0.79 0.43 ± 1.01 f 6.44 7.64 5.41 6.44 5.41 2.91 p < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 0.01 fig. 2. relationship between annual mean temperature and left testis mass (open circles) and right testis mass (solid circles) in 7 rana kukunoris populations. solid lines: the right testes and p < 0.05; dashed line: the left testes and p > 0.05. p values were computed using linear mixed models. each dot represents the residual value for a given individual corrected for the effect of operational sex ratio, body condition and age. fig. 3. relationship between annual mean temperature and directional testes asymmetry in 7 rana kukunoris populations. each dot represents the residual value for a given individual corrected for the effect of operational sex ratio, body condition and age. 9the directional testes asymmetry increases with temperature condition (t = -0.108, p = 0.914) and age (t = -1.369, p = 0.173), but increased with temperature (t = 2.899, p = 0.053; fig. 3) when controlling for population (random effect: z = 0.280, p = 0.780). meanwhile, in a simplified model controlling only for population (z = 0.253, p = 0.800), age (t = -1.377, p = 0.171) and temperature (t = 2.952, p = 0.050), dta did not increase with the osr (t = -1.676, p = 0.244). furthermore, the right testis was significantly heavier than the left testis in only one of seven studied populations (5.3°c, paired test: t = 3.165, df =16, p = 0.006; other population, p = 0.055-0.928, table 1) where the temperature was warmest among all the studied locations. discussion dta is common in male animals (møller, 1994; simmons and kotiaho, 2002). specially, this phenomenon has been proved in some anurans, including rana temporaria (hettyey et al., 2005), rhacophorus omeimontis (mi et al., 2012) and rana nigromaculata (zhou et al., 2011). the degree of dta may be used to reveal individual quality (møller, 1994; simmons and kotiaho, 2002) because some studies found that there were positive correlations between dta and body condition (e.g., møller, 1994; hettyey et al., 2005). however, other studies (e.g., rana omeimontis, mi et al., 2012; hylarana guenther, liu et al., 2011; rana kukunoris, chen et al., 2014) do not show this pattern. in this study, our results showed that dta did not co-vary with male condition or body mass, suggesting individuals with good condition or heaver did not tend to have a larger degree of dta. thus, the degree of dta as a good measure of male body condition remains ambiguous and contentious in male animals (birkhead et al., 1997; kimball et al., 1997; kempenaers et al., 2002; wu and liao, 2017). environmental stresses may drive the evolution of testis asymmetry (møller, 1994). although directional asymmetry in testis size has been observed in some anurans within a population (mi et al., 2012; zhou et al., 2011; yu and guo, 2015), most studies so far have found the level of dta was not correlated with latitude or altitude in spite of large differences in environmental conditions and environmental stress (hettyey et al., 2005; chen et al., 2014; zhang et al., 2018). however, our results support this hypothesis as the degree of dta covaried with temperature in the study populations of rana kukunoris. furthermore, the right testis was significantly heavier than the left testis in the populations with locations of warmest temperature. one possible explanation is that adaptive asymmetry might be difficult to evolve. for example, the development of testes asymmetry is adaptive, but very costly because reduction or loss of an organ on one side of the body could commonly not be compensated, leading to physiological or morphological handicaps. males living in warm regions could allocate more energy to reproduction, in that they increase resource availability in relative longer the length of the activity seasons than those living in cold regions. this suggests that low environmental stresses (e.g., warm temperature) provide the opportunity for males to increase in size of the right testis, thus compensate for a reduced function of the left one. in addition, we found no evidence for a geographical trend in genetic stress, and this result was consistent with other anurans species (palo et al., 2003; hettyey et al., 2005). previous studies have shown that dta covaries with age (e.g., birkhead et al., 1997; graves, 2004; liu et al., 2012), suggesting that males with a larger degree of dta have a longer life span. however, the degree of dta was not positively correlated with age in current study. this result was consistent with liu et al. (2011) and zhou et al. (2011), suggesting that older males did not indicate a higher degree of dta than younger males. an explanation for this phenomenon was males have already reached complete reproductive maturity during the first breeding year (liu et al., 2011). sperm competition is also attributed to drive the evolution of ejaculate quality in a wide range of taxa, in that higher levels of sperm competition tend to result in larger testes (møller, 1991; møller and briskie, 1995). assuming that it is costly to have high the level of dta, thus two large testes might be advantageous to increase testes size. however, we found annual mean temperature did not effect on testis mass (yu, unpublished data). moreover, our results also showed that degree of dta did not decrease with the osr, suggesting that male–male competition did not lead to an increase in levels of sperm competition. in conclusion, we did not find evidence for the suggestion that dta is related to male condition, age and osr, although there is a significant directional testes asymmetry in r. kukunoris. we found a positive correlation between the level of dta and temperature, suggesting that differences in the length of activity period and resources availability across locations may affect the energy budget of this frog, resulting in a gradual change in reproduction energy parallel to increasing temperature. acknowledgements we are very grateful to the staff of the gahai-zecha national nature reserve for assistance in the field. 10 hai ying li et alii the gahai-zecha national nature reserve management bureau approved this project (approval number ghzcrmb/03-212014), and gave permission for fieldwork. handling and processing of frogs followed approved protocols from the animal scientifc procedures act 1988 by the state department of china. all experiments involving the sacrifice of live animals were approved by the animal ethics committee at xinyang normal university. the study was funded by emergency management program of national natural science foundation of china (grant no. 31741019). references birkhead, t.r., buchanan, k.l., devoogd, t.j., pellatt, e.j., szèkely, t., catchpole, c.k. 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(2011): testis asymmetry in the dark-spotted frog rana nigromaculata. herpetol. j. 21: 181-185. xi international symposium on the mediterranean lacertid lizards marco mangiacotti1, pietro lo cascio2, claudia corti2, marta biaggini2, miguel angel carretero2, petros lymberakis2 the directional testes asymmetry increases with temperature in seven plateau brown frog (rana kukunoris) populations hai ying li1, man jun shang2, jie guo2, bo jun chen2, peng zhen chen2, tong lei yu1,* influence of tail injury on the development of neotropical elegant treefrog tadpoles ana glaucia da silva martins1,#, raoni rebouças2,3,*,#, isaias santos1, adão henrique rosa domingos1, luís felipe toledo2 the effect of weight and prey species on gut passage time in an endemic gecko quedenfeldtia moerens (chabanaud, 1916) from morocco jalal mouadi1,*, panayiotis pafilis2, abderrafea elbahi3, zahra okba3, hassan elouizgani3, el hassan el mouden4, mohamed aourir1 a contribution to the knowledge on the diet and food preferences of darevskia praticola (reptilia: lacertidae)§ emiliya vacheva*, borislav naumov first report on two loggerhead turtle (caretta caretta) nests in the aeolian archipelago (southern italy) monica francesca blasi1,*, sandra hochscheid2, roberta bardelli3, chiara bruno1, carolina melodia1, perla salzeri1, paolo de rosa4 and paolo madonia5 threatened and extinct amphibians and reptiles in italian natural history collections are useful conservation tools franco andreone1,*, ivano ansaloni2, enrico bellia3, andrea benocci4, carlotta betto5, gabriella bianchi6, giovanni boano7, antonio borzatti de loewenstern8, rino brancato9, nicola bressi10, stefano bulla11, massimo capula12, vincenzo caputo barucchi13, p re-description of external morphology and factors affecting body and tail shape of the stone frog tadpoles’ brena da silva gonçalves1,*, carla. d. hendges2, bruno madalozzo2, tiago g. santos2,3 preliminary data on the diet of chalcides chalcides (squamata: scincidae) from northern italy andrea ciracì1, edoardo razzetti2, maurizio pavesi3, daniele pellitteri-rosa4,* the high diversity and phylogenetic signal of antipredator mechanisms of the horned frog species of proceratophrys miranda-ribeiro, 1920 (amphibia: anura: odontophrynidae) cássio zocca1,2,*, ricardo lourenço-de-moraes3, felipe s. campos4, rodrigo b. ferreira1,2,5 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 6(2): 229-236, 2011 medical cautery units as a permanent and non-invasive method of marking lizards anna ekner1, zofia sajkowska1, krzysztof dudek1, piotr tryjanowski2 1 department of behavioural ecology, adam mickiewicz university in poznań, 61-614 poznań, poland. corresponding author. e-mail: anna.ekner@gmail.com 2 institute of zoology, poznań university of life sciences, wojska polskiego 71 c, 60-625 poznań, poland. submitted on: 2011, 10th march; revised on 2011, 17th october; accepted on 2011, 20h october. abstract. the identification of previously captured individuals is essential for a wide variety of ecological and behavioural studies. a lot of different methods are used for marking lizards, however they have many drawbacks. in presented study we used heat-branding method, using pen-like medical cautery units, previously employed to successfully mark other lizard species and snakes. the technique is permanent, readable and harmless for lizards, as well quick and easy. in 2009 we marked 111 individuals of sand lizard, lacerta agilis. next year we caught 88 lizards, 17 of them were re-captured. among these re-captured lizards, five were caught after 26.8 (± 16.3) days (means in the same year) and 12 after 308.8 (± 64.3) days (means in the next year). recaptured individuals were still unambiguously recognisable. keywords. branding, sand lizard, less harmful method, resistant, lacerta agilis. introduction many studies require individuals to be marked for identification. accurate marking may be of great importance in ecological and behavioural studies, such as the determination of growth rates, movement patterns, reproductive histories, and other individual attributes that are critical to understanding species demography and population ecology (fitch, 1987; gaisler and chytil, 2002; venkatarama et al., 2008; wanger et al., 2008; angelini et al., 2010). marking methods should be permanent, readable, as well as not harmful to the marked individuals (murray and fuller, 2000). lizards are usually marked by toe-clipping (ikeuchi et al., 2005; ferner, 2007). however, toe-clipping has drawbacks for reptiles and may negatively affect survival, mate acquisition, performance (bustard, 1971; blocha and irschick, 2005; schmidt and schwar230 a. ekner et alii zkopf, 2010) and endurance (schmidt and schwarzkopf, 2010). in addition, toe-clipping is painful and possibly stressful to the animals. moreover, lizards sometimes naturally lose their toes (hudson, 1996; ribeiro and sousa, 2006) which can cause incorrect identification of the animal. toe-clipping, as well as other methods like clipping scales (atzori, 2007), may induce infection (weary, 1969). sometimes microchips called passive integrated transponders (pit tags) are used to uniquely mark lizards, but they are very expensive (winne et al., 2006), not suitable for small vertebrates (gibbons and andrews, 2004) and may induce more stress than toe-clipping (langkilde and shine, 2006). in some studies less harmful techniques are used, such as tagging lizards with bee marking kits or paint (johnson, 2005). however, these methods are not permanent (simon and bissinger, 1983; todd, 2005) and are only useful in short-term studies, mainly because lizards will lose such marks when they shed their skin or if they suffer skin abrasion against surfaces. ribeiro and sousa (2006) introduced lizard banding, using elastic hair fitted to the lizard’s neck. this inexpensive method allows marking of lizards of small to moderate body size, causes no injury and does not appear to strongly influence lizard movements. however, animals have to have a head much larger than their neck diameter and coarse scales. moreover, any conspicuous external markers may affect the lizard’s traits, such as camouflage and hiding from predators. such methods may also be unsuitable for studies of social status or mate choice if the focal species use colour for communication (ribeiro and sousa, 2006). alternatively, lizards can be individually marked by freeze-branding (lewke and stroud, 1974) or heat-branding (ehmann, 2000). however, these methods traditionally have been difficult to apply in the field, because they required specialized equipment (e.g., liquid nitrogen) or an electrical current (winne et al., 2006), and were considered time-consuming. in the current study we used and discuss of potential pro and cons of a heat-branding method, using medical cautery unit. it was previously successfully utilized in multi-year snakes research (seminatrix pygaea, agkistrodon piscivorus, coluber constrictor, nerodia spp., crotalus horridus, cemophora coccinea, lampropeltis triangulum, tantilla coronata; winne et al., 2006). snakes were individually marked, by branding 1-3 ventral scales anterior to the anal plate and extending the mark diagonally onto adjoining lateral scales. medical cautery units were also successfully used for podarcis sicula lizards marking (vervust and van damme, 2009). in this case a first mark was cauterized on one of the first ventral scales on the right side of the lizard body, creating a reference point. following marks were burned into other ventral scales and were given names reflecting their position with regard to the first mark. the lizards were kept for 48 hours after marking, and during that time researchers did not notice any obvious negative effects of the medical cautery unit on the animals. materials and methods the study was carried out in april september 2009 and april june 2010. sand lizards (lacerta agilis) were caught one day in april, june and september 2009, three days in may 2009, one day in march, may and june 2010, and three days in april 2010. the study area were close to the barycz valley in western poland (51°34’n, 17°40’e, elevation 110-170m) and included gravel pits, meadows, forest edges and were surrounded with extensive used farmlands (for details see ekner et al., 2008). liz231a permanent and non-invasive method of marking lizards ards were captured using landing fishnets or by hand, then aged (adult, subadult and juvenile), sexed and measured (snout to vent length, svl). we individually marked lizards by heat-branding them with “aaron medical change-a-tip cautery units” (http://www.boviemed.com). medical cautery units are pen-like appliances with a hot wire at the end and are available in two temperature classes (1204 °c and 704 °c). we used the low-temperature class. we held a lizard in one hand and used the other hand to mark the lizard with the cautery unit (fig. 1). the procedure took about 10 seconds. we marked lizards with cauterized dots on the ventral surface of the lower jaw. we assigned appropriate digits to mental and submandibular scales, to form a unique numerical code (fig. 2). fig. 1. procedure of marking lizards using the medical cautery unit. lizards are held in one hand and marks are left on mental and submandibular scales using the cautery unit held in the other hand. fig. 2. a numerical code created on ventral part of the lizard’s head. the code is created by dots, left on the particular scales using a medical cautery unit. 232 a. ekner et alii we noted any aberrations in the arrangement of scales (fig. 3). lizard visible on the photo has only eight scales, hence, counting in sequence, it may have the number “467/8”. however, it is only suggestion and because first two scales on the left side looks like one but double, the specimen could have a number “456/7 with double first scale”. the details depend on the researcher, however the numbers should be assign consistently. only numbers with ascending digits were used, e.g. so that 12 and 21 are not mixed up, because each combination of the digits is used only once in the marked population. lizards normally have nine mental and submandibular scales, hence the method provides at least 511 possible number of combinations, in case of 1-9 of dots cauterized. the total number of possible codes that can be marked using two dots, three dots or four dots is 36, 84, 126, respectively. however, these are minimum numbers of combinations, because additional possibility is when considerate any aberrations in the arrangement of scales, mentioned above (fig. 3). through the paper the results are shown as mean ± sd, or as a mean with 95% confidential limits, calculated by an excel macro for presence-absence data results during april-september 2009 we marked 111 l. agilis specimens. in march-june 2010 we caught 88 lizards in the area. at this time, 71 of the lizards were captured for the first time (80.7%, 95% cl: 70.9-88.3), and 17 were re-captured (19.3%, 95% cl: 1.7-29.1). five of 17 re-captured specimens were caught once again in the same year after 26.8 (± 16.3) days from the first catch, others 12 were caught next year after 308.8 (± 64.3) days. all of the two groups of re-captured lizards were still unambiguously recognisable. however, fig. 3. examples of the lizards marked using a medical cautery unit. we noticed each modification in the arrangement of scales. on the photograph specimens nr: “467/8” or “456/7 with double first scale”. 233a permanent and non-invasive method of marking lizards fig. 4. the marks left on lizard scales re-captured after about one month (lizard no 156). fig. 5. the marks left on lizard scales re-captured after about one year (lizard no 56). 234 a. ekner et alii the marks left on lizard bodies re-caught after about one month (fig. 4) were completely different from the marks left on lizards re-caught after about one year (fig. 5). after one month marks on the scales are visible as a dots and are similar to those seeing immediately after the marking. whereas, after a year marks look like cracks in the scales. discussion almost 17% (12 of 71) of the marked lizards were re-captured after about one year. such small number of re-caught animals can be caused by very big population living in a study area (more than 350). we successfully used a medical cautery unit to mark sand lizard l. agilis. after an average of one year marks were still well-defined. after examination, using a magnifying glass, we didn’t notice any disappearance traits of the marks. the marks left on lizard bodies re-caught after about one year were different from the marks left on lizards re-caught after about one month and shortly after a branding. however, that marks were looking almost the same as those presented in other lizard’s study (vervust and van damme, 2009). we didn’t have an opportunity to re-visit the branded lizards later, however vervust and van damme (2009) report that marks remained clearly visible over a period of at least 485 days during which the lizards resided in their natural habitat. moreover, the marking procedure has proved to be effective for other reptiles (snakes) for more than three years (winne et al., 2006). in that study marks were readable even as the snakes have tripled in length and increased many-fold in mass. there are a lot of advantages of using this method. medical cautery units are small, field-portable and inexpensive (about 30 usd, price in summer of 2010). this method is capable of being quickly and easily used in the field without any preliminary training and is not affected by the researcher skills. the short time (about 10 seconds) and the simplicity of the procedure likely make marking less stressful for lizards than many other methods. research with less disturbance is especially important in studies investigating patterns of behaviour (johnson, 2005). in our study animals didn’t show any visible reaction to cauterizing, hence we think that this technique is relatively painless for lizards. moreover, previous study showed the marking method does not have any obvious negative effects on reptiles (winne et al., 2006; vervust and van damme, 2009). the method provides at least 511 possible number combinations, using up to nine marks per lizard. however, if study required to mark more lizards, dots could be cauterized on scales on other areas of the body, e.g. ventral scales, where even only four dots allow to marked as much as 197709 individuals (vervust and van damme, 2009). it is a precise and easily visible way to identify particular individuals. if dots are left on the mental and submandibular scales or other ventral scales, marks are hardly visible for researches, but not conspicuous to predators or other individuals and hence probably do not affect survival or behaviour (ribeiro and sousa, 2006). we recommended medical cautery units for lizard study, both in the wild and in laboratory conditions. we suggested to use the method for thick-skinned species, however to be honest we are not sure if it would be appropriate for reptiles with fragile and thin skin, like gekkonidae, phyllodactylidae or sphaerodactylidae. 235a permanent and non-invasive method of marking lizards acknowledgements we would like to thank all those who helped us in the field. t. sparks critically read the previous versions of the manuscript. this work was financially supported by a scientific grant of the ministry of science and higher education of poland no. n n 303 317 433 and scientific grant of the ministry of science and higher education of poland no. n n 304 381 338. lizard capture was carried out according to polish law and the ethical commission for study on animals (lke 12/2007). references angelini, c., antonelli, d., utzeri, c. (2010): capture-mark-recapture analysis reveals survival correlates in salamandrina perspicillata (savi, 1821). amphibia-reptilia 31: 21-26. atzori, a., berti, f., cencetti, t., fornasiero, s., tamburini, m., zuffi, m.a.l. (2007): advances in methodologies of sexing and marking less dimorphic gekkonid lizards: the study case of the moorish gecko, tarentola mauritanica. amphibia-reptilia 28: 449-454. blocha, n., irschick, d.j. (2005): toe-clipping dramatically reduces clinging performance in a pad-bearing lizard (anolis carolinensis). j. herpetol. 39: 288-293. bustard, h.r. (1971): a population study of the eyed gecko, oedura ocellata boulenger, in northern new south wales, australia. copeia 1971: 659-669. ehmann, h. (2000): microbranding: a low impact permanent marking technique for small reptiles and frogs as an alternative to toe clipping. anzccart news 13: 6-7. ekner, a., majlath, i., majlathova, v., hromada, m., bona, m., antczak, m., bogaczyk, m., tryjanowski, p. (2008): densities and morphology of two co-existing lizard species (lacerta agilis and zootoca vivipara) in extensively used farmland in poland. folia biol. 56: 3-4. ferner, j.w. (2007): a review of marking and individual recognition techniques for amphibians and reptiles. herpetological circulars 35. society for the study of amphibians and reptiles, salt lake city. fitch, h.s. (1987): collecting and life history techniques. in: snakes: ecology and evolutionary biology, p. 143-164. seigel, r.a., collins, j.t., novak, s.s., eds, macmillan publishing company, new york. gaisler, j., chytil, j. (2002): mark-recapture results and changes in bat abundance at the cave of na turoldu, czech republic. folia zool. 51: 1-10. gibbons, j.w., andrews, k.m. (2004): pit tagging: simple technology at its best. bioscience 54: 447-454. hudson, s. (1996): natural toe loss in southeastern australian skinks: implications for marking lizards by toe-clipping. j. herpetol. 30: 106-110. ikeuchi, i., mori, a., hasegawa, m. (2005): natural history of phelsuma madagascariensis kochi from a dry forest in madagascar. amphibia-reptilia 26: 475-483. johnson, m.a. (2005): a new method of temporarily marking lizards. herp. rev. 36: 277-279. langkilde, t., shine, r. (2006): how much stress do researchers inflict on their study animals? a case study using a scincid lizard, eulamprus heatwolei. j. exp. biol. 209: 1035-1043. 236 a. ekner et alii lewke, r.r., stroud, r.k. (1974): freeze branding as a method of marking snakes. copeia 1974: 997-1000. murray, d.l., fuller, m.r. (2000): a critical review of the effects of marking on the biology of vertebrates. in: research techniques in animal ecology: controversies and consequences, p. 15-64. boitani, l., fuller, t.k., eds, columbia university press, new york. ribeiro, l.b., sousa, b.m. (2006): elastic hair bands: an effective marking technique for lizards in mark-recapture studies. herp. rev. 37: 434-435. schmidt, k., schwarzkopf, l. (2010): visible implant elastomer tagging and toe clipping: effects of marking on locomotor performance of frogs and skinks. herpetol. j. 20: 99-105. simon, c.a., bissinger, b.e. (1983): paint marking lizards: does the color affect survivorship? j. herpetol. 17: 184-186. todd, a.c. (2005): the social mating system of hoplodactylus maculatus. new zealand j. zool. 32: 251-262. venkatarama, s., krishnamurthy, b., hanumantha, a., reddy, m. (2008): an estimate of local population of nyctibatrachus aliciae at two habitat gradients of forest in western ghats. acta herpetol. 3: 51-55. vervust, b., van damme, r. (2009): marking lizards by heat branding. herpetol. rev. 40: 173-174. wanger, t.c., motzke, i., furrer, s.c., brook, b.w., gruber, b. (2008): how to monitor elusive lizards: comparison of capture–recapture methods on giant day geckos (gekkonidae, phelsuma madagascariensis grandis) in the masoala rainforest exhibit, zurich zool. ecol. res. 24: 345-353. weary, g.c. (1969): an improved method of marking snakes. copeia 1969: 854-855. winne, c.t., willson, j.d., andrews, k.m., reed, r.n. (2006): efficacy of marking snakes with disposable medical cautery units. herpetol. rev. 37: 52-54. © firenze university press www.fupress.com/ah acta herpetologica 5(2): 259-263, 2010 two new types of noose for capturing herps enrique garcía-muñoz1, 2, neftalí sillero3 1 cibio, centro de investigação em biodiversidade e recursos genéticos, campus agrário de vairão, p-4485-661, vairão, portugal. corresponding author. e-mail: engamu@gmail.com 2 departamento de biología animal, biología vegetal y ecología. campus de las lagunillas s/n. universidad de jaén. e-23071, jaén, spain. 3 centro de investigação em ciências geo-espaciais (cicge); universidade do porto, faculdade de ciências, rua do campo alegre, 687, p-4169-007, porto, portugal. e-mail: neftali.pablos@fc.up.pt submitted on 2010, 20th july; revised on 2010, 27th october; accepted on 2010, 15th november. abstract. we present two new types of noose for capturing herps, which allow adapting the size of the loop to the size of prey rapidly. we use a plastic ring to make the loop: the thread moves perfectly without any impediment or friction, and remains in good conditions for a longer time. the plastic ring can be made during fieldwork. both new types of noose increase capture effectiveness, as it is possible to noose different types of preys with a simple and rapid adaptation of the loop. we have tested this methodology in different species of lacertids, some snakes under rocks, and also some amphibians. keywords. capture herps, loop, lizard, pastic ring. multiple factors affect capture success, including animal body size, trap avoidance behaviour, and weather. many techniques exist for sampling a vast array of lizard species, including noosing poles, grab sticks, scoop buckets, sticky sticks, rubber bands, sticky traps, and habitat traps (bertram and cogger, 1971; bauer and sadlier, 1992; witz, 1996; durtsche, 1996; downes and porges, 1998; whiting, 1998; hamilton et al., 2007). one of the methods employed to catch small lizards is the noose (see among others: vanhooydonck and damme, 2003; li et al., 2009; marsili et al., 2009). however, the size of the noose involves the size of prey capture, so the noose must be constantly adapted to the size of the dam at the time of capture (author´s pers. obs.), which may mean the flight of animals. we present here two different variants of noose (figs. 1 and 2). both methodologies could tailor the size of the loop to the size of prey rapidly. the innovation of these methodologies is the use of a plastic ring (from an ink cartridge pen, such as a bic pen) to make the loop. this plastic ring allows a perfect movement of the thread without any impediment or friction. furthermore, the thread remains in good conditions for a longer time. finally, the plastic ring can be made, in situ, at the moment during fieldwork: the 260 e. garcía-muñoz and n. sillero fig. 1. description of the way to make the noose type 1. materials: wire (≈ 0.5 mm ø), thread, and a plastic ring (≈1 mm ø). methods: form a loop of thread over the end of the rod (a; i), roll backward (ii) and pass the end of the thread inside loop and pull (b; i). pass this end of the thread (b; i) through the ring (c; ii) (diameter of the ring is greater than the end of the rod) and make a knot (iii). to form this noose, tie two knots. th e fi rst knot is shaded. th e second knot is pulled fi rmly against the fi rst knot. cut the excess thread (iv). pass the end of the rod through the ring, and pull the ring to get the position l1 (e.g. for capturing podarcis sp), or pass the ring until position l2 (e.g., for capturing tarentola sp). 261two new types of noose for capturing herps fig. 2. description of the way to make the noose type 2. materials: wire (≈ 0.5 mm ø), dental thread, a plastic ring (≈1 mm ø), and adhesive tape. methods: do a loop with the dental thread on the plastic ring (i; a), and pass the free extreme through the interior of the ring (see fig.1; iii). put two pieces of adhesive tape around the stick (i; b) and the dental thread (this should be parallel to the stick), one piece on the stick extreme, and the other one a little lower (≈ 5 cm between adhesive tape). tie the dental thread to the stick in order to leave the extreme of the thread opposite to the adhesive tape. in this way, it is not possible to untie the thread if this is pulled off the stick. th e dental thread can move freely through the adhesive tapes, allowing to use a small loop (e.g., for capturing podarcis sp.; l1) or a big loop (e.g., for capturing agama sp.; l2). 262 e. garcía-muñoz and n. sillero plastic ring can be replaced very quickly and easily. the first type of noose (fig. 1) allows modifying the size and type of the loop. this methodology allows for a typical loop (fig.1 l1) with a small opening, to catch species like podarcis sp., and a second position (fig.1 l2) with a wider loop, and one of their rigid parts, making it easier to capture species like tarentola sp., snakes under the rock or amphibians under water. the second type of noose (fig. 2) allows modifying the size of the loop moving the thread along the stick. these new methodologies increase capture effectiveness, as we can noose, with a simple and rapid adaptation of loop, different types of preys. both types of noose are more resistant to loose the thread, when the animal is big. however, if the diameter of the plastic ring is larger than one millimeter, the prey can escape very easily. in any case, during fieldwork tests, we did not have a higher proportion of animal releasing from noose in comparison with other ways of making the loop. we have tested this methodology in different species of lacertids (acanthodactylus aureus, a. boskianus, a. busacki, a. erythrurus, agama bibronii, algyroides marchi, darevskia armeniaca, d. dahli, d. nairensis, d. portschinskii, d. raddei, d. rostombekovi, d. unisexualis, d. valentini, lacerta agilis, l. media, l. strigata, l. schreiberi, laudakia caucasia, podarcis carbonelli¸ p. hispanicus, p. vaucheri, p. bocagei, psammodromus algirus, ps. hispanicus, quedenfeltia moerens, q. trachyblepharus, tarentola mauritanica, t. boehmei, timon lepidus, t. pater, trachylepis septemtaeniata), some snakes under rocks (hemorrhois hippocrepis, malpolon monspessulanus) and also some amphibians (hyla meridionalis and pelophylax saharica). this paper aims to share a change into a technique used in herpetological studies, and consequently to facilitate the subsequent field sampling. acknowledgements field work was carried out with the support of the projects ptdc/bia-bde/67678/2006 and ptdc/bia-bec/101256/2008 funded by “fundação para a ciência e a tecnologia” (portugal) and under the permit n° 14 heceflcd/dlcdpn/cff of the “haut commissariat aux eaux et forêts et à la lutte contre la désertification” (morocco). our thanks go to the consejería de medio ambiente (junta de andalucía) for permission to the lizards surveys. references bauer, a.m., sadlier, r.a. (1992): the use of mouse glue traps to capture lizards. herp. rev. 23: 112-113. bertram, b.p. , cogger, h.g. (1971): a noosing gun for live captures of small lizards. copeia 1971: 371-373. downes, s., porges, p. (1998): sticky traps: an effective way to capture small terrestrial lizards. herp. rev. 29: 94. durtsche, r.d. (1996): a capture technique for small, smooth-scaled lizards. herp. rev., 27: 12-13. hamilton, a.m., klein, e.r., eckstut, m.e., hartfield, e.e. (2007): a simple, inexpensive method to capture arboreal lizards. herp. conserv. 2: 164-167. 263two new types of noose for capturing herps li, h., qu, y., hu, r., ji, x. (2009): evolution of viviparity in cold-climate lizards: testing the maternal manipulation hypothesis. evol. ecol. 23: 777-790. marsili, l., casini, s., mori, g., ancora, s., bianchi, n., d’agostino, a., ferraro, m., fossi, m.c. (2009): the italian wall lizard (podarcis sicula) as a bioindicator of oil field activity. sci. tot. environ. 407: 3597-3604. vanhooydonck, b., van damme, r. (2003): relationships between locomotor performance, microhabitat use and antipredator behaviour in lacertid lizards. funct. ecol. 17: 160-169. whiting, m.j. (1998): increasing lizard capture success using baited glue traps. herp. rev. 29: 31. witz, b.w. (1996): a new device for capturing small and medium-sized lizards by hand. herp. rev. 27: 130-131. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 7(1): 49-55, 2012 updated distribution of hybrids between lissotriton vulgaris and lissotriton montandoni (amphibia: caudata: salamandridae) in romania iulian gherghel1,*, alexandru strugariu1, ioana-madalina ambrosă2, ștefan r. zamfirescu1 1 faculty of biology, “alexandru ioan cuza” university, carol i blvd., no. 20a, 700505, iaşi, romania. *corresponding author. e-mail: iuliangherghel@gmail.com 2 faculty of psychology and education sciences, “alexandru ioan cuza” university, toma cozma str. no 3, 700554, iaşi, romania submitted on: 2011, 17th november; revised on: 2012, 29th january; accepted on: 2012, 29th january. abstract. lissotriton montandoni is an endemic newt species found only in the carpathian mountains and lives in sympatry with lissotriton vulgaris in many aquatic habitats from the entire range of the former species in the carpathian and sudetes mountains or in the hilly areas from the subcarpathians. these two species usually generate hybrids where their parapatric ranges meet, especially along rivers that flow from the inside of the carpathians, where valleys are used as ecological corridors by l. vulgaris. we surveyed several regions of the eastern carpathian mountains between 2008 and 2011 and found 11 new populations of newts where hybrids between the two mentioned species were present. all new records of l. montandoni x l. vulgaris were described in the eastern part of the eastern carpathians, in neamț county, a region known also from previous literature to be a ‘hot spot’ for hybrids between these two species. the present paper also presents an updated review of the distribution of lissotriton hybrids in romania. keywords. amphibians, montandon’s newt, smooth newt, hybridization, carpathians. lissotriton montandoni and lissotriton vulgaris are two genetically related species with similar sexual behaviour (belyaev, 1981; pecio and rafinski, 1985; rafinski and arntzen, 1987; arntzen and sparreboom, 1989) and they usually generate hybrids where the parapatric ranges of both species meet (zavadil et al., 2003). lissotriton montandoni is an endemic newt species found only in the carpathian and sudetes mountains and lives in sympatry with l. vulgaris in many aquatic habitats from the entire range of l. montandoni, especially along small rivers in the mountains or in the hilly areas from the subcarpathians (e.g. fuhn, 1963; şova, 1973; szymura, 1974; fuhn et al., 1976). 50 i. gherghel et al. many authors have indicated the presence of hybrids in areas where the ranges of these species overlap: ukraine (hofmann, 1908; kushniruk, 1963), poland (szeligamierzeyewski and ulasiewicz, 1931; juszczyk and swierad, 1984; rafinski, 1985, 1988; pecio and rafinski, 1985; rafinski and pecio, 1989; babik et al., 2003), the czech republic (rehák, 1993; šálek, 1993; kotlík et al. 1997; kotlík and zavadil, 1999; zavadil et al., 2003, 2004; mikulicek and zavadil, 2008). in romania, hybrids between these two species were first reported by fuhn (1963), fuhn et al. (1976), iftime (2004) (transylvanian alps) and by gherghel et al. (2008) (eastern carpathian mountains). in this study, we present an updated distribution of hybrids between l. montandoni and l. vulgaris in romania, based on the previous literature and several new, previously unpublished records. field surveys were conducted between the years 2008 and 2011 in the eastern carpathian mountains, with emphasis on neamț county, where several hybrid populations have been previously reported (gherghel et al., 2008). we captured newts from various aquatic habitats using drag nets. each newt individual was photographed using a nikon l100 digital camera for future analysis of hybrid colouration and, subsequently, released in the original habitat. the morphological characteristics used for identifications of hybrids are those previously described by kotlik and zavadil (1999), iftime (2004) and mikulicek and zavadil (2008) (table 1). throughout our survey, we found hybrids between l. vulgaris and l. montandoni in 11 new locations (table 2). all the sites with hybrids were located in the contact zone between the carpathians and the subcarpathians, areas where all breeding ponds were used by both parental species. gherghel et al. (2008) found the hybrids at the contact of two biogeographic regions: continental (the subcarpathians) and alpine (the carpathian table 1. comparison of morphological characters used for determination of animals. morphological feature lissotriton vulgaris lissotriton montandoni dorsal crest high and spotted very low and without spots crest denticulation denticulated not dorsolateral folds absent present lateral stripe on the lower tail margin pale blue cream coloured spots on the belly present absent spots on the throat present absent colour of the cloaca the same as the surrounding skin including spots black tail filament absent present colour on the flanks wide gold stripe between the orange ventral and brownish dorsal coloration the orange ventral colour merges smoothly into dorsal coloration webbing on the hind limbs present absent colour of palms of the hind limbs the same as the surrounding skin, often with spots gray to black and without spots 51distribution of lissotriton hybrids in romania fig. 1. habitat overview of breeding ponds of lissotriton hybrids in the eastern carpathian mountains; in the left photo (a) is a fresh snow-melt temporary pond, in the right photo (b) is a roadside ditched with water used by hybrids for reproduction. fig. 2. updated distribution of l. montandoni x l. vulgaris hybrids in romania.   52 i. gherghel et al. mountains). the characteristics of the breading ponds where we observed l. vulgaris x l. montandoni hybrids are similar to those found by fuhn et al. (1976), iftime (2004) and gherghel et al. (2008) and can be described as temporary and permanent ponds formed by rainwater or snow-melt (fig. 1a) or by overflowing mountain rivers. other important habitats used by newts for reproduction are roadside puddles or roadside ditches (fig. 1b). as observed by iftime (2004), hybrids are generally present mostly in human disturbed areas, where the anthropogenic impact (like deforestation, heavy road traffic, near localities) is greater than inside forests. almost all records of l. montandoni x l. vulgaris in romania were found in the eastern part of the eastern carpathians and in the extreme southern range of l. montandoni. until now, no hybrids between these species have been recorded in northern and western slopes of the eastern part of eastern carpathians (fig. 2, fig. 3, table 2). acknowledgements we want to use this opportunity to express our gratitude to dr. krystyna nadachowsha brzyska who read the article in its preliminary version and offered valuable comments for its improvement. a part of this study was financed through cncsis –uefiscsu, project pnii – idei 2098 no.1041/2009. fig. 3. some individuals of l. montandoni x l. vulgaris founded in negritești, neamț county. 53distribution of lissotriton hybrids in romania references arntzen, j.w., sparreboom, m. (1989): a phylogeny of the old world newts, genus triturus: biochemical and behavioural data. j. zool. 219: 645-664. babik, w., szymura, j.m., rafinski, j. (2003): nuclear markers, mitochondrial dna and male secondary sexual traits variation in a newt hybrid zone (triturus vulgaris x t. montandoni). mol. ecol. 12: 1913–1930. belyaev, a.a. (1981): on application of some characters of sexual behavior to solution of problems of systematics and evolution of animals (on an example of representatives of the genus triturus: urodela, salamandroidea). priroda i musei riga 1: 32-52. cogălniceanu, d. 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(1997): allozyme evidence for hybridization between triturus vulgaris (linnaeus, 1758) and triturus montandoni (boulenger, 1880) (amphibia: caudata) in the czech republic. preliminary results. in: herpetologia bonnensis, pp. 195-198.w. bohme, w. bischoff and t. ziegler, eds, she, bonn. kotlik, p., zavadil, v. (1999): natural hybrids between the newts triturus montandoni and t. vulgaris: morphological and allozyme data evidence of recombination between parental genomes. folia zool. 48: 211–218. kushniruk, v.a. (1963): on biology of the carpathian newt triturus montandoni boulenger, 1880. zoo. zhurnal 22: 300-302. mikulicek, p., zavadil, v. (2008): molecular and morphological evidence of hybridization between newts triturus vulgaris and t. montandoni (caudata: salamandridae) in slovakia. biologia 63: 127-131. pecio, a., rafinski, j. (1985): sexual behaviour of the montandon’s newt, triturus montandoni (boulenger) (caudata: salamandridae). amphibia-reptilia 6: 11-22. rafinski, j. (1985): natural hybridization between triturus vulgaris and t. montandoni. in: third ord. gen. meet. soc. europ. herpetol., p. 94-95. rocek, z., ed., prague. rafinski, j. (1988): zroznicowanie fenetyczne i fylogeneza gatunkow rodzaju triturus (amphibia: urodela: salamandridae). rozpravy habilitacyjne, nr 146, uniwersitet jagiellonski, krakow. 86 pp. rafinski, j., arntzen, j.w. (1987): biochemical systematics of the old world newts, genus triturus: allozyme data. herpetologica 43: 446-457. rafinski, j., pecio, a. (1989): craniometric studies on the species of the genus triturus rafinesque, 1815 (amphibia, salamandridae).folia biol. 37: 131–150. rehak, i. (1993): colek karpatsky (triturus montandoni) na morave a jeho kfizeni s colkem obecnym (t. vulgaris). akvarium terrarium 36: 32-34. šalek, p. (1993): čolek karpatsky triturus montandoni boulenger, 1880 – novy člen jihomoravske fauny. ochrana přirody 48: 237. şova, c. (1973): contributions to the ecology of amphibians (ord. caudata, genus triturus) from the sereth river basin. univ. bucharest, fac. biol., stud, si comun., muz. ştiinţ. natur. bacau. 6: 1-286. szeliga-mierzeyewski, w., ulasiewicz, w. (1931): plazy i gady pow. molodeczanskiego. triton intermedius nov. for. die reptilien und lurche des kreises molodeczno (wojew. wilno). triton intermedius nov. for. trav. soc. sci. class math. nat. 6: 17–24. 55distribution of lissotriton hybrids in romania szymura, j.m. (1974): a competitive situation in the larvae of four sympatric species of newts (triturus cristatus, t. alpestris, t. montandoni and t. vulgaris) living in poland. acta biol. cracov. 17: 235-262. zavadil, v., belansky, p., kautman, j. (2004): vyznamna reprodukčni lokalita obojživelniků na uzemi horne oravy v silnemohroženi. acta rer. natur. mus. slov. (bratisl.) 50: 67–70. zavadil, v., pialek, j., dandova, r. (2003): triturus montandoni (boulenger, 1880) – karpatenmolch. in: hanbuch der reptilienund amphibien europas, band 4/iia, schwanzlurche (urodela) iia, salamandridae ii: triturus 1, pp. 657–706. grossenbacher k. and thiesmeier b., eds, aula verlag, wiesbaden. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 6(2): 267-274, 2011 haematology and serum biochemical parameters in freeranging african side neck turtle (pelusios sinuatus) in ibadan, nigeria a.o. omonona1, s.g. olukole2, f.a. fushe1 1 department of fisheries and wildlife management, faculty of agriculture and forestry, university of ibadan, ibadan, nigeria. 2 department of veterinary anatomy, faculty of veterinary medicine, university of ibadan, nigeria. corresponding author. e-mail: deborolukole@yahoo.com submitted on: 2010, 10th november; revised on 2011, 18th april; accepted on 2011, 05th july. abstract. the haematology and serum biochemical parameters in free-ranging african side neck turtle (pelusios sinuatus) in ibadan, nigeria was carried out with the view of establishing baseline blood health indices of this species and generating data which could be useful in the comparative physiology of turtles. a total of sixty free ranging turtles comprising juveniles and adults of both sexes were used for the study. the mean values for the rbc, pcv, hb, mcv, mch and wbc counts observed in male juvenile were significantly higher (p < 0.05) than those of the females. nevertheless, in adult turtles, the mean values for the rbc, pcv, hb, mcv, mch and wbc counts observed in females were significantly higher (p < 0.05) than those of the males. similarly, in juvenile turtles, the absolute heterophil, lymphocyte and monocyte counts in females were relatively higher (p < 0.05) than that of male while in adult turtles there were no significant differences (p < 0.05) in these parameters between the males and females. there were no significant difference (p < 0.05) in the values for the total protein, albumin, globulin, creatinine, serum glutamic-oxalacetic transaminase (sgot), serum glutamic-pyruvic transaminase (sgpt) and blood urea nitrogen in the males and females of both the juvenile and adult turtles. the outcome of this study presents baseline data on the haematology and serum biochemical parameters in free-ranging african side neck turtle (pelusios sinuatus) in ibadan, nigeria, which could also serve as a template for the comparative physiology of fresh and sea turtles. keywords. heamatological parameters, serum, pelusios sinuatus, reptilia. introduction the african side neck turtle (pelusios sinuatus), usually found in freshwater habitats, from rivers and lakes to ephemeral ponds being widely distributed in africa, madagascar 268 a.o. omonona, s.g. olukole and f.a. fushe and the seychelles islands belongs to the family pelomedusidae, and is the largest species of its genus (carapace length up to 55 cm), with the females larger than the males (anderson, 1995; broadley and boycott, 2009). this turtle is small to medium in size, with relatively extensive plastron that may have a hinge present between the pectoral and abdominal scutes (olukole et al., 2010). the neural series is highly variable (four to eight), and the pleural bones almost meet the midline posterior to the neurals. a pair of mesoplastral bones is present between the hyoand hypoplasta in contact (boycott and bourquin, 2000). blood is a special type of connective tissue composed of formed elements (erythrocytes, leukocytes and platelets) in a fluid matrix. plasma is the fluid portion, called serum when depleted of fibrinogen (bacha and bacha, 2000). reptiles had been reported to constitute a heterogeneous group among vertebrates in terms of their blood cell morphology, and demonstrated considerable variations among orders, even within the same family members. different blood cells of reptiles had identified: erythrocytes, leukocytes (lymphocyte, monocyte, heterophile [heterophile], eosinophile and basophile) and thrombocytes (arikan and cicek, 2010). blood analysis is a useful tool for the diagnosis and health monitoring of animals, as well as to distinguish pathogenic processes from those that might be purely physiological (christopher et al., 1999). haematologic and biochemical parameters knowledge of freeranging freshwater turtles is important for assessing and managing their populations (nagy and medica, 1986). blood is composed of cells and plasma, serving such functions as respiratory, excretory, nutritive, thermal regulation of the body, protective, and regulatory. a number of studies had been reported hematological values in turtles, mainly marine (bolten et al., 1992) and terrestrial turtles (dickinson et al., 2002; christopher et al., 1999; diaz-figueroa, 2005), and very few in freshwater turtles (brenner et al., 2002). limited research studies had been reported in the african side neck turtle; conservation, nutrition and history of migration (broadley and boycott, 2009); morphometry of the external body anatomy (olukole et al., 2010). no information exists on the hematology and blood biochemistry of the african side neck turtle. this study, the first of its kind, was therefore designed to investigate into the hematological and plasma biochemical parameters in free-ranging adult and juvenile african side neck turtles in ibadan, nigeria, with the view of establishing baseline blood health indices of this species. materials and methods experimental animals a total of sixty african side neck turtles picked up between april and june 2010 (rainy season) from various river banks in ibadan, nigeria, were used for the study. these comprised 30 juveniles (15 males, 15 females) and 30 adults (15 males, 15 females). the animals were housed at the animal house of the faculty of the veterinary medicine, university of ibadan and were stabilized for 72 hours prior to standard body measurement and collection of blood samples. they were fed with corn pap ad libitum, fresh water was also provided for the animals. standard body parameters were all determined using a draper® 115 mm vernier caliper and metric tape. the body weight of the animals was taken with the aid of a microvar® weighing balance. the curved and straight carapace 269haematology in african side neck turtle lengths of the turtles were measured after they were physically examined and found to be free from any external injury or adhesion. blood sample collection and analyses blood (2-4 ml) was collected from the right subclavian vein between the neck and foreflipper with the use of an 18gauge, 3.8-cm needle and a 5-ml syringe coated with sodium heparin. the blood samples collected from the animals were put into clean test tubes containing edta. the haematological parameters were determined as described by zaias (2000). drops of whole blood were used to fill some heparinised microhematocrit capillary tubes to determine packed cell volume (pcv), and hemoglobin (hb). whole blood was also used to make three air dried blood smears. the smears were stained with wright’s stain and examined for red blood cell (rbc), white blood cell (wbc), differential wbcs (lymphocytes, heterophil, monocytes) and platelet estimate, while mean cell haemoglobin (mch), mean cell haemoglobin concentration (mchc) and mean cell volume (mcv) were calculated. blood samples were also collected for biochemical analysis, centrifuged at 3000 rpm for ten minutes to isolate the serum. total protein, albumin, globulin, creatinine, serum aspartate aminotransferase (ast), serum alanine aminotransferase (alt) and blood urea nitrogen were determined by use of automated analysers as described by dickson et al. (2002). data analysis all data were expressed as means and standard error of means, comparison was by the student t test using the graphpad prism version 4.00 for windows, graphpad software. significance was reported at p < 0.05. results the mean values for the haematological parameters of juvenile male and female african side-neck turtle (pelusios sinuatus) are given in table 1. the average body weights for the juvenile and adult turtles used for the study were 0.31 ± 0.01 kg and 1.8 ± 0.32 kg, respectively. the dimensions for the curved carapace lengths adult turtle were 18 ± 1.43 cm and 21.45 ± 1.34 cm for male and female respectively while those of the juvenile turtles were 10 ± 0.93 cm and 12.21 ± 0.86 cm for male and female respectively. there was a positive correlation (r = 0.746, p < 0.01) between the weights of the turtles and their curved carapace lengths. the mean values for the rbc, pcv, hb, mcv, mch and wbc counts observed in male juvenile were significantly higher (p < 0.05) than their female counterparts. nevertheless, in adult turtles, the mean values for the rbc, pcv, hb, mcv, mch and wbc counts observed in females were significantly higher (p < 0.05, t-test) than those of the males. similarly, in juvenile turtles, the absolute heterophil, lymphocyte and monocyte counts in females were relatively higher (p < 0.05) than that of male while in adult turtles there were no significant difference (p < 0.05) in these parameters between the males and females (table 1). there were no significant difference (p < 0.05) in the values for the total protein, albumin, globulin, creatinine, serum alt, and blood urea nitrogen bun in the males and females of both the juvenile and adult turtles (table 2). 270 a.o. omonona, s.g. olukole and f.a. fushe discussion haematological and biochemical parameters are useful tools in measuring the physiological status of turtles because they may provide information for diagnosis and prognosis of diseases (whiting et al., 2007; oliveira-junior et al., 2009). moreover, such tools have been used as physiological disturbance indicators of diseases, stress or exposition to contaminants, as well as to assess degrees of dehydration (peterson, 2002; christopher et al., 2003; tavares-dias et al., 2009). the turtles used for the study can be said to represent a normal state of physiology since their external body (carapace, plastron, head, neck, tail and limbs) were free from wounds and or adhesions. the strong positive correlation observed between the weights of the turtles and their curved carapace lengths in this table 1. the mean and sem values for the hematological parameters of juvenile and adult african side neck turtle (pelusios sinuatus). parameters juvenile male juvenile female adult male adult female pcv (%) 31.0 ± 6.32 a 25.5 ± 2.88 b 22.1 ± 3.93 b 32.0 ± 4.20 a hb (g/l) 13.24 ± 2.14 a 8.25 ± 0.97 b 7.27 ± 1.32 b 10.5 ± 1.39 a rbc (x1012/l)) 15.84 ± 2.05 a 9.54 ± 3.04 b 10.84 ± 3.61 b 16.6 ± 4.66 a wbc (x109/l) 17.98 ± 4.17 a 9.06 ± 3.74 b 10.38 ± 3.01 b 20.5 ± 5.34 a platelets (x103/l) 10.4 ± 3.50 13.13 ± 4.20 11.28 ± 1.98 9.71 ± 3.19 mcv (fl) 25.6 ± 5.86 a 19.3 ± 4.23 b 32.1 ± 4.50 b 44.0 ± 5.34 c mch (pg) 26.4 ± 4.22 a 18.13 ± 3.66 b 18.0 ± 6.88 b 27.3 ± 5.53 a mchc (g/l) 33.0 ± 0.70 32.5 ± 0.76 30.57 ± 0.58 34.9 ± 0.38 lymphocyte (%) 68.8 ± 8.26 a 57.25 ± 7.06 b 33.57 ± 3.58 c 31.9 ± 3.38 c heterophil (%) 27.4 ± 7.47 a 20.5 ± 7.01 b 34.30 ± 7.32 c 33.6 ± 7.74 c monocyte (%) 2.0 ± 0.02 1.00 ± 0.01 0.57 ± 0.01 0.28 ± 0.04 means with different superscripts within rows are significantly different at p < 0.05. table 2. the mean and sem values for the serum biochemical parameters of juvenile and adult african side neck turtle (pelusios sinuatus). parameters juvenile male (n = 15) juvenile female (n = 15) adult male (n = 15) adult female (n = 15) total protein (g /dl) 4.54 ± 0.39 3.70 ± 0.67 3.99 ± 0 .36 4.17 ± 0.37 albumin (g/dl) 1.18 ± 0.13 1.07 ± 0.71 1.13 ± 0.08 1.10 ± 0.10 globulin (g/dl) 3.36 ± 0.30 2.63 ± 0.61 2.86 ± 0.41 3.07 ± 0.35 creatinine (u/dl) 1.13 ± 0.18 1.17 ± 0.05 1.19 ± 0.13 1.22 ± 0.14 sgot (u/l) 33.20 ± 6.44 29.25 ± 5.20 31.14 ± 8.21 23.00 ± 6.50 sgpt (u/l) 32.40 ± 5.37 30.14 ± 4.34 34.86 ± 6.74 33.43 ± 6.99 bun (g/dl) 1.66 ± 0.61 1.93 ± 0.6 1.66 ± 0.61 1.97 ± 0.84 271haematology in african side neck turtle study is in agreement with previous reports (olukole et al., 2010; boycott and bourquin, 2000). also, the significant differences observed between the curved carapace lengths of the male and female turtles (adult and juvenile) used for the study is in line with the existing body of literature on the family pelomedusidae to which the african side neck turtle belongs (anderson, 1995; broadley and boycott, 2009). the haematocrit values observed in the study were similar to those observed in freshwater turtles, such as trachemys scripta elegans and chrysemys picta, which were described by moon and foerster (2001). sea turtles have a higher average haematocrit value, which may reflect a physiological adaptation to the environment (moon and foerster, 2001). moreover, the values found for total haemoglobin were similar to those described in the literature for chelonia mydas and chelonoidis chilensis (wood and ebanks, 1984; troiano and silva, 1998). nevertheless, the significantly higher values observed for the rbc, pcv, hb, mcv, mch and wbc counts in adult female turtles when compared to those of the males used for the study, shows a variance with previous reports on fresh water turtles emys orbicularis and mauremys rivulata from turkey (yilmaz and tosunoglu, 2010). this difference could be species specific, but then the presence of great variation among turtle species in terms of erythrocyte count had been reported by previous researchers (hutchison and szarski 1965; duguy, 1970; yilmaz and tosunoglu, 2010). unlike the values for the rbc, pcv, hb, mcv, mch and wbc counts in the adult turtles used for the study, the male juvenile turtles showed a significantly higher values to those of the females. the absolute leukocyte count observed in the study was higher than that reported in geoffroy’s side-necked turtle (phrynops geoffroanus testudines) as reported by zago et al., 2010. nevertheless, the erythrocyte and leukocyte counts obtained in the study are similar to that reported in the in the wild and captive central american river turtles (dermatemys mawii). hematological values in wild d. mawii had been reported to be different to those of other chelonians, such as the green turtle, chelonia mydas (bolten and bjorndal, 1992), the california desert tortoise, gopherus agassizii (christopher et al., 1999). also, in the present study the absolute heterophil, lymphocyte, monocyte counts in male juvenile african side neck turtle were higher than that of the female, though without any significant difference (p < 0.05). also, there were no significant differences in total protein, albumin, globulin, creatinine, serum ast, serum alt and bun for both sexes of juvenile and adult african side neck turtles. this corresponds to the findings of yilmaz and tosunoglu (2010) on fresh water turtles emys orbicularis and mauremys rivulata. according to metin et al. (2008), there is no difference between females and males in terms of glucose, triglyceride, urea and total protein in mauremys caspica. the mean plasma albumin concentration values of 1.18 ± 0.13 g/dl, 1.07 ± 0.71 g/dl , 1.13 ± 0.08 g/dl and 1.10 ± 0.10 g/dl obtained in this study for juvenile male, juvenile female, adult male and adult female turtles respectively are similar to the report of rangel-mendoza et al., 2009, in the wild and captive central american river turtles (dermatemys mawii). nevertheless, plasma albumin concentrations of the turtles used for this study were higher than those reported for loggerhead sea turtles (0.6 ± 0.8 g/dl) (bolten, et al., 1992). however, the mean values observed for total protein for all the turtles used for the study are similar to the values reported in loggerhead sea turtle (gicking et al., 2004) and in leatherback sea turtles dermochelys coriacea (deem et al., 2003). the serum globulin concentration (2.63-3.36 272 a.o. omonona, s.g. olukole and f.a. fushe g/dl) obtained in the study is similar to the range (2.3-4.4 g/dl) reported in the loggerhead sea turtles, caretta caretta (gicking et al., 2004). nevertheless, the absence of significant differences in serum globulin concentration of the juvenile and adult turtles of both sexes is at variance to previous report in the loggerhead sea turtles, caretta caretta (gicking et al., 2004). the serum bun range (1.66-1.97 g/dl) obtained for the turtles used in the study is similar to that reported by deem et al. (2003) in leatherback sea turtles (dermochelys coriacea). findings of this study presnt baseline data on the haematology and serum biochemical parameters in free-ranging african side neck turtle (pelusios sinuatus) in ibadan, nigeria. it also provides a template for the comparative physiology of fresh and sea turtles. references anderson, n.b. (1995): life history notes: pelusios sinuatus: reproduction. african herp news 23: 49. arikan, h., cicek, k. (2010): morphology of peripheral blood cells from various species of turkish herpetofauna. acta herpetol. 5: 179-198. bolten, a.b., bjorndal, k.a. (1992): blood profiles for a wild population of green turtles (chelonia mydas) in the southern bahamas: size specific and sex-specific relationships. j. wild. dis. 28: 407–413. bolten, a.b., jacobson, e.r., bjorndal, k.a. (1992): effects of anticoagulant and auto analyzer on blood biochemical values of loggerhead sea turtle. amer. j. vet. res. 53: 2224-2227. boycott, r.c. (2001): the terrapins and tortoises (chelonia: pelomedusidae and testudinidae) of swaziland, durb. mus. novt. 26: 25-37. boycott, r.c., bourquin, o. (2000): the southern african tortoise book: a guide to southern african tortoise, terrapins and turtles. o bourquin, hilton, kwazulunatal, south africa. brenner, d., lewbart, g., stebbins, m., herman, d.w. (2002): health survey of wild and captive bog turtles (clemmys muhlenbergii) in north carolina and virginia. j. zoo wild. med. 33: 311–316. broadley, d.g., boycott, r.c. (2009): pelusios sinuatus (smith 1838) –serrated hinged terrapin. conservation biology of freshwater turtles and tortoises, chel. res. monogr. 5: 036.1-036.5. christopher, m.m., berry, k.h., wallis, i.r., nagy, k.a., henen, b.t., peterson, c.c. (1999): reference intervals and physiologic alterations in hematologic and biochemical values of free-ranging desert tortoises in the mojave desert. j. wildl. dis. 35: 212–238. christopher, m.m., berry, k.h., b.t. henen, b.t., nagy, k.a. (2003): clinical disease and laboratory abnormalities in free-ranging desert tortoises in california (1990-1995). j. wildl. dis. 39: 35-56. deem, s.l., starr, l., norton, t.m., karesh, w.b. (2003): sea turtle health assessment programme in the caribbean and atlantic. proc. 22nd annu. symp. sea turtle biol. conserv. noaa-tm-nmfs-sefsc-503. 273haematology in african side neck turtle diaz-figueroa, o. (2005): characterizing the health status of the louisiana gopher tortoise gopherus polyphemus). thesis of master of science. louisiana state university and gricultural and mechanical college, philadelphia, u.s.a. dickinson, v.m., jarchow, j.l., trueblood, m.h. (2002): haematology and plazma biochemistry reference range values for free-ranging desert tortoises in arizona. j. wildl. dis. 38: 143-153. duguy, r. (1970): numbers of blood cells and their variation. in: biology of reptilia, pp. 93-109, gans, c., parsons, t.s., eds, academic press, london and new york. gicking, j.c., allen, m. foley, kendal e. harr, m.s., rose e. raskin, elliott jacobson (2004): plasma protein electrophoresis of the atlantic loggerhead sea turtle, caretta caretta. anesth. analg. surg. 14: 13-18. hutchison, h.v., szarski, h. (1965): number of erythrocytes in some amphibians and reptiles. copeia 3: 373-375. metin, k., basnmoglu koca, y., kargnn knral, f., koca, s., türkozan, o. (2008): blood cell morphology and plasma biochemistry of captive mauremys caspica (gmelin,1774) and mauremys rivulata (valenciennes, 1833). act. veterin. brno 77: 163-174. meyer, d., harvey, j. (1998): veterinary laboratory medicine: interpretation and diagnosis, 2nd edition, wb saunders co, philadelphia. moon, p.f., foerster, s.h. (2001): zoological restraint and anesthesia: reptiles, aquatic turtles (chelonians): 2001. international veterinary information service. available at [http://www.ivis.org]. accessed august 2010. nagy, k.a., medica, p.a. (1986): physiological ecology of desert tortoises in southern nevada. herpetol. 42: 73–92. oliveira-junior, a.a., tavares-dias, m., marcon, j.l. (2009): biochemical and hematological reference ranges for amazon freshwater turtle, podocnemis expansa (reptilia: pelomedusidae), with morphologic assessment of blood cells. res. vet. sci. 86: 146-151. olukole, s.g., aina, o.o., okusanya, b.o. (2010): morphometric analysis of the external body anatomy of the african side neck turtle. proc. 30th annu. symp. sea turtle biol. conserv. 265. peterson, c.c. (2002): temporal, population, and sexual variation in hematocrit of free-living desert tortoises: correlational tests of causal hypotheses. can. j. zool. 80: 461-470. reece, w.o. (1997): physiolog of domestic animals. 2nd ed. baltimore, lippincott williams & wilkins. rosenthal, k.l. (2000): avian protein disorders. in: laboratory medicine: avian and exotic pets, pp. 171-173, fudge, a.m., ed, saunders, philadelphia. tatum, l.m., zaias, j., mealey b.k., cray, c., bossart, g.d. (2000): protein electrophoresis as a diagnostic and prognostic tool in reptile medicine. j. zoo. wildl. med. 31: 497-502. tavares-dias, m., oliveira-junior, a.a., marcon j.l. (2008): methodological limitations of counting total leukocytes and thrombocytes in reptiles (amazon turtle, podocnemis expansa): an analysis and discussion. act. amaz. 38: 351-356. tosunoglu, m., tok, c.v., gül, ç. (2005): hematological values in hermann’s tortoise (testudo hermanni) and spur-thigted tortoise (testudo graeca) from thrace region (turkey). int. j. zool. res. 1: 11-14. troiano, j.c., silva, m.c. (1998): valores hematológicos de referencia en tortuga terrestre argentina (chelonoidis chilensis chilensis). analect. vet. 18: 47-51. 274 a.o. omonona, s.g. olukole and f.a. fushe whiting, s.d., guinea, m.l., limpus, c.j. (2007): blood chemistry reference values for two ecologically distinct populations of foraging green turtles, eastern indian ocean. comp. clin. pathol. 16: 109-118. wood, f.e., ebanks, g.k. (1984): blood cytology and haematology of the green sea turtle, chelonia mydas. herpetologica 40: 331-336 zago, c.e.s., ferrarezi, a.l., vizotto, l.d., oliveira, c., cabral, s.r.p., taboga, s.r., bonilla-rodriguez, g.o., venancio, l.p.r., bonini-domingos,c.r. (2010): hemoglobin polymorphism and hematological profile of geoffroy’s side-necked turtle (phrynops geoffroanus, testudines) in the northwestern region of são paulo state, brazil. genet. molecul. res. 9: 721-726. yilmaz, n., tosunoglu, m. (2010): haematology and some plasma biochemistry values of free-living freshwater turtles (emys orbicularis and mauremys rivulata) from turkey. north-west. j. zool. 6 : 107-117. zaias, j., fox, w.p., cray, c., altman, n.h. (2000): hematologic, plasma protein, and biochemical profiles of brown pelicans (pelecanus occidentalis). amer. j. vet. res. 61: 771-774. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 7(1): 175-180, 2012 new data on the distribution of darevskia pontica (lantz and cyrén, 1919) (reptilia: lacertidae) in romania: filling a significant gap tibor sos1,*, attila kecskés1, zsolt hegyeli1, bela marosi2 1milvus group bird and nature protection association, crinului 22, 540343, târgu mureş, romania. *corresponding author. e-mail: tibor.sos@gmail.com 2molecular biology center, interdisciplinary research institute on bio-nano-sciences, babeş-bolyai university cluj-napoca, treboniu laurian 42, 400271, cluj-napoca, romania submitted on: 2011, 27th december; revised on: 2012, 29th february; accepted on: 2012, 21st march. abstract. the distribution of the meadow lizard, darevskia pontica, in romania is still inadequately documented. in the light of new distribution data reported here and gleaned from the literature, the species is more widely distributed in the country. the distribution seems to be continuous in southern romania, even if fragmented and associated with extant woodland patches. the present distribution pattern could be the result of extensive deforestation process in the area, which isolated this forest lizard to remnant patches, as already indicated in the literature. keywords. distribution, darevskia pontica, southern romania. the western meadow lizard, darevskia pontica (lantz and cyrén, 1919) ranges into the balkan peninsula to romania and the black sea basin of western caucasus (sindaco and jeremcenko, 2008; tuniyev et al., 2011). in south-eastern europe the western meadow lizard inhabits romania, serbia, bulgaria, turkey-in-europe and greece (sindaco and jeremcenko, 2008). generally, it is considered a species with a patchy distribution, associated with broad-leaved woodlands exposed to the influence of a sub-mediterranean climate (stugren, 1984). in romania its distribution was and still is considered to be restricted to the south-western and south-eastern parts of the country (ljubisavljević et al., 2006). this picture of the distribution is not quite accurate even in the light of distribution data from the 20th century (fig. 1a). the species was first reported from banat (méhely 1895 a,b), muntenia (kiriţescu, 1901), transylvania (fejérváry-lángh, 1943) and from dobrudja (fuhn and hârşu, 1962). the best-known area where the species has a continuous distribution is the lower part of cerna valley, in the banat mountains (fuhn and vancea, 1961; covaciu-marcov et al., 2009 a; fig. 1a). the presence of the species in the poiana ruscă mountains (fig. 1a) 176 t. sos et al. enlarged the known distribution area to the north in the banat (fejérváry-lángh, 1943). later new localities were reported here (bogdan et al., 2011) and also in the north-eastern limits of the same mountains (ghira, 1994). in the oltenia plain and getic tableland the known distribution area was extended to the jiu valley by cruce (1971), and the gaps in the distribution were filled up considerably by lazăr et al. (2005) and covaciu-marcov et al. (2009 a; fig. 1a). here the lizard is associated with the fragmented broad-leaved woodlands, thus displaying a patchy distribution. recently, two new isolated records related to the contact zone between the meridional carpathians and the getic sub-carpathians have extended the south-western distribution of the species to the east (fig. 1a). iftime and iftime (2006) found the species in the olt river valley, in cozia massif. covaciu-marcov et al. (2009 b), recorded this lizard in the lower course of jiu river gorge, and filled the gaps: (i) between the northern occurrence of the species in transylvania and the banat; (ii) between the cozia massif and the area of the banat mountains; (iii) between the jiu river valley and the danube valley (fig. 1a). another occurrence of the species was identified in the vicinity of bucharest in the romanian plain (fuhn and vancea, 1961; fig. 1a.) and close to the river danube (kiriţescu, 1930). in 2010 a new observation enlarged the species distribution area to the north-east in the buzău subcarpathians region, about 60 km from the nearest population in ilfov county (gherghel et al., 2011; fig. 1a). the relatively late confirmation of the species’ occurrence in southern dobrudjan tableland (fuhn and hârşu, 1962; fig. 1a) and the assumption that the lizard could be more widespread in the area were later confirmed (andrei, 2002, covaciu-marcov et al., 2008; fig. 1a). fig. 1. a. distribution of d. pontica in romania: half-filled dots, data from present study; arrow, the new occurrence data from teleorman county; white dots, localities from literature (méhely, 1895 a,b, 1903; schreiber, 1912; méhely, 1918; kiriţescu, 1930; fejérváry-lángh, 1943; fuhn and vancea, 1961; stugren, 1961; fuhn and hârşu, 1962; fuhn, 1969; cruce, 1971;fuhn, 1974; stroescu 1982; ghira, 1994; iftime, 2001; andrei, 2002; iftime, 2005; lazăr et al., 2005; iftime and iftime, 2006; covaciu-marcov et al., 2008; iftime and iftime, 2008; iftime et al., 2008; covaciu-marcov et al., 2009a, b; bogdan et al., 2011; gherghel et al., 2011). the circles represent the distribution areas of the species as discussed in the paper: 1, poiana ruscă mountains; 2, cerna valley and banat mountains; 3, the contact zone between the meridional carpathians and the getic sub-carpathians; 4, getic tableland and oltenia plain, 5, romanian plain; 6, buzău subcarpathians; 7, southern dobrudjan tableland. b. subadult from the new locality (satu vechi, teleorman county). 177distribution of darevskia pontica our new distribution data, reported here, are dispersed across the banat, oltenia and muntenia regions (fig. 1a). the populations from dobraia and driştie (caraş-severin county), şviniţa hamlet and petriş (mehedinţi county) are inside the conventional distribution area of the species. the species appears in the habitat types generally described for it, i.e. deciduous forests (mainly oaks) with scattered trees and warm clearings, generally in the adjacent areas of river valleys or small streams. contrary to these data, an occurrence in the vedea river valley, in the romanian plain, is biogeographically important (fig. 1a, half-filled dot with arrow). our own record from the vicinity of satu vechi (teleorman county) connects the south-eastern distribution area (the closest known locality to the west it is more than 90 km away in the jiu river valley) and the distribution of the species from the environs of bucharest (the closest known locality to the east it is more than 90 km away near the argeş river valley). near satu vechi we found two subadult lizards (fig. 1b). the habitat was unusually outside of the forested area, in a sparse scrub belt along a small stream called burdea. the lizard specimens were found in a dry microhabitat, under the cover of the scrub belt, which was formed by small willows (salix spp.) and hawthorn (crataegus monogyna), with a smaller quantity of white poplar (populus alba) and dogwood (cornus sanguinea). the shrub belt with its woody vegetation probably provides the connection between two small forest patches (to the west and east) through agricultural land. at the discovery site, the scrub belt was more accentuated on the left bank of the stream, while the right bank was characterized by herbaceous vegetation with teasel (dipsacus spp.), as well as a few salix and crataegus, with marsh vegetation in a few spots. this humid meadow was formed recently from an abandoned agricultural field. the nearby oak-hornbeam forest of pădurea muți (30 ha) lies on the floodplain of the burdea stream, about 100–150 m north-west from the discovery site. the trees are a mix of quercus sp., carpinus betulus, tilia sp. and populus alba. between the forest and the discovery place stretches a country road, and on weekends and public holidays the forest suffers some human disturbance. the whole burdea stream with the scrub belt along its course lies in contact with other small forest patches to the north and with a large riparian and oak-hornbeam forest south of the lizard discovery site. covaciu-marcov et al. (2009 a), described a similar habitat type at scăpău in the blahniţa valley (mehedinţi county), but far (at least 20 km) from forested areas. here the species was found to live in a narrow grassy vegetation girdle bordering some of the permanent canals on the plain. while according to covaciu-marcov et al. (2009 a) the meadow lizard in the blahniţa valley was forced into this humid habitat due to the deforestation of the area, near the satu vechi locality the presence of adjacent forested habitats prove some ability of the species to colonize even in non-wooded areas. in conclusion, according to our new data and the data from the literature (see the tendency in fig. 2) the distribution of the meadow lizard in romania is far from being known. the distribution of the species could indeed be continuous in southern romania, even if fragmented and connected to extant woodland patches. gherghel et al. (2011) suggested that until recently the species has inhabited the whole southern romania, as this region has been mostly covered by forest. the increasing deforestation process, mainly at the end of the 17th century, has isolated the species into the remaining forest patches. this hypothesis could be true for the population from the romanian lowlands, but not for the sub-carpathians, where extensive forested areas still exist (but see gherghel et al., 2011). 178 t. sos et al. according to the old and recent distribution data, the species had another possibility to enlarge and achieve its actual range in addition to following the southern edge of the carpathians and the wooded areas. the species could have reached the warm sides of the carpathian areas after spreading along the more or less forested and warmer river valleys of the area, even through small non-wooded zones, as indicated above. acknowledgments we wish to thank andrás istván csathó, istván kovács, hana latková and emilie dauphaus, who helped us during our fieldwork and who provided some of the occurrence data. we are grateful to john akeroyd (fundatia adept), whose comments much improved the content and language of the paper. fig. 2. the number of localities of d. pontica in romania, distributed in different time periods from the first report of the species. for the descriptions of the zones see fig. 1a. 179distribution of darevskia pontica references andrei, m.d. 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(2011): systematic and geographical variability of meadow lizard, darevskia praticola (reptilia: sauria) in the caucasus. russ. j. herp. 18: 295316. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 6(1): 47-57, 2011 localization of glucagon and insulin cells and its variation with respect to physiological events in eutropis carinata vidya. r. chandavar1, prakash. r. naik2* 1 yuvaraja’s college, university of mysore. mysore570006. india. 2 endocrinology laboratory, department of studies in zoology, university of mysore, manasagangorti, mysore 570006, india. *corresponding author. e-mail: vidyachandavar@yahoo.co.uk submitted on: 2010, 23th december; revised on 2011, 2nd february; accepted on 2011, 31th may. abstract. the aim of the present investigation was to localize glucagon and insulin immunoreactive (ir) cells of pancreas during annual seasonal cycle of reproduction and to find out whether they had any effect on the regulation of plasma glucose level in the skink eutropis carinata. immunolocalized pancreatic cells revealed significantly different mean numbers in different periods of reproduction. the numbers of glucagon-ir and insulin-ir cells were highest in recrudescent period which was corresponded with low plasma glucose level. unlike other lizards the arrangement of insulin cells in the central core and glucagon cells at the periphery was absent instead glucagon-ir and insulin-ir cells were paracrine in arrangement. among the two immunoreactive cells glucagon-ir cells were predominant. morphological differences between two cell types were observed by electron microscopy after staining with uranyl acetate and lead citrate. plasma glucose showed cyclic change being highest during reproductive period. keywords. glucagon-ir, insulin-ir, lizard, plasma glucose, reproductive cycle. introduction eutropis (mabuya) carinata is an insectivorous skink (lizard), inhabitant of south asia. it runs swiftly, usually basks during winter and shifts to shady area in summer. elsalhy and grimelius (1981) made histological and immunohistochemical investigation of endocrine pancreas of the grass lizard, mabuya quinquetaenia-ta, and that of the desert lizard, uromastyx aegyptia. rhoten and hall (1982) examined the differentiation of islets of langerhans in the lizard anolis carolinensis. the endocrine pancreas of the lizard, podarcis hispanica, consists of single scattered cells or small groups of two to five cells forming islet-like structures (lopez et al., 1988) and that of podarcis s. sicula is concentrated more in the splenic than in duodenal region and never formed large clusters (putti mailto:vidyachandavar@yahoo.co.uk http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22el-salhy%20m%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22el-salhy%20m%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22grimelius%20l%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22rhoten%20wb%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22hall%20ce%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus 48 v.r. chandavar and p.r. naik et al., 1991). the comparative morphology of islets of langerhans in 11 species of lacertids demonstrated the central core of b cells and a cells at the periphery with the predominance of earlier-cells (putti et al., 1992). della rosa and putti (1995) reported the distribution and frequency of different endocrine cells in the lacertid pancreas. ku and lee (2004) studied the regional distribution and frequency of the pancreatic endocrine cells in the splenic lobe of the grass lizard, takydromus wolteri, by immunohistochemistry. most of the earlier studies on endocrine pancreas of different lizards were confined to identifying different cell types by histology, immunocytochemistry and electron microscopy where as the present investigation was under taken to localize glucagon and insulin cells of pancreas during annual seasonal cycle of reproduction and to find out whether they have any effect on the regulation of plasma glucose level in the skink e. carinata. materials and methods animals we collected e. carinata from manasagangotri campus, mysore (latitude 12°18’n; longitude, 76°42’e; altitude, 777 m asl). they were maintained in the reptile house in the open and fed silk moth (bombyx mori) ad-libitum. “guidelines for care and use of animals in scientific research” were followed (anonymous, 2000). experimental protocols were approved from institutional animal ethics committee (iaec). the animals were studied in the annual seasonal cycle of reproduction (2007 to 2009) which is distinguished into three separate periods namely regenerative, reproductive and recrudescent. ten adults of e. carinata weighing 12-30 g were utilized in each period irrespective of the sex. as the animals were collected from the field it was not possible to determine their age, hence measurements were taken. the animals were injected with sodium pentobarbital (50 mg/kg body weight) intra peritoneal for recording their body length and weight and were sacrificed. the length of the animal was measured from tip of the snout to tip of the tail. the pancreas was freed, its length measured and weighed and fixed in bouin-hollande sublimate solution for 18-20 h and processed for light microscopy and immunocytochemistry. plasma glucose simultaneously, blood samples from carotid artery were collected and centrifuged at 4 °c and 10,000 rpm for 10 minutes. the separated plasma of ten active animals (10 consecutive samples from each animal) was immediately used for estimation of glucose by enzyme glucose oxidase method of trinder (1969) as described earlier (chandavar and naik, 2004; 2008). histology paraffin embedded pancreas from each of the animals was sectioned at 4-5 µm in series. ten to 15 sections were mounted on a slide and every second slide was used for light microscopy. chrome alum hematoxylin and phloxin (chp) staining method (gomori, 1941) was employed for light microscopy. sections were treated with acidified kmno4 and subsequently decolourised with sodium bisulphite, stained with hematoxyline, differentiated in 1% acidified water, counter stained in phloxin for few minutes and mordant in phosphotungstic acid. chp stained sections were used 49localization of glucagone in eutropis carinata for islet measurement at their longest axis at 400×. the size of islet was calculated by random selection of 100 observations in each period (weesner, 1960; ku and lee, 2004), using software image pro express, version 5.1. immunocytochemistry all the chemicals used in immunocytochemistry were purchased from sigma-aldrich, usa. every third slide was used for glucagon cell localization and the fourth slide having sections of the same islet was used for insulin cell localization. they were immunolocalized by the extravidinbiotin peroxidase method after yang et al. (1999) and as per the instruction manual provided with the kit. the paraffin embedded sections were deparaffinised processed through grades of alcohol, washed in running water, pretreated with 3% h2o2 in methanol, rinsed with phosphate buffered saline (pbs, ph 7.6) and non-specific reactive sites blocked with 5% normal goat serum. they were then incubated for 1 h at 37 °c in a humidified chamber with the respective primary monoclonal mouse antibody (porcine glucagon was used as immunogen, product no g2654; diluted 1:2000 and human insulin was used as immunogen, product no i2018; diluted 1:1000). the sections were carefully washed 10 to 15 times with pbs and incubated for 30 min with biotinylated goat anti-mouse immunoglobulin secondary antibody and extravidin-peroxidase (mouse extravidin peroxidase staining kit stock no. extra-2, sigma), each diluted 1:20. pbs with 5% normal goat serum was used as diluent. the peroxidase activity was demonstrated using 0.7 mg/ml 3,3’-diaminobenzidine tetrahydrochloride (dab) in 0.17 mg/ml urea hydrogen peroxide and 0.06 m tris buffer for 1-3 min. to show that the labeling is specifically due to the primary antibody, the primary antibody is replaced with similarly diluted normal serum from the same species, keeping all the other steps the same in controls (burry, 2000). another control for specificity which included omission of primary monoclonal antibody and parallel incubation with antibody reabsorbed with excess of respective antigen. no immunostaining was obtained in the controls. this further confirms that the immunolocalization has taken place in islets only. pancreatic sections containing islets were observed throughout the pancreas. immunoreactive cell count was done by random selection of 100 sections in every period. glucagon-ir and insulinir cell counting was done separately by using software image pro express, version 5.1.total number of glucagon-ir and insulin-ir cells of all ten animals in every period was considered as 100 percent and quantitative analysis in terms of percentage of glucagon-ir and insulin-ir cells was calculated. digital photographs were taken using olympus b × 60. liver histochemistry specimens autopsied to collect blood and pancreases were also used for histochemical localization of liver glycogen. liver was fixed in rossman’s fixative and then processed, sectioned at 9 -10 µm and stained following periodic acid-schiff (pas) technique of hotchkiss (1968). the pas positive masses localized in the cytoplasm was taken into consideration for qualitative analysis of glycogen. electron microscopy pancreases of 0.5 mm3 were fixed for 24 h at 4 °c in 3% glutaraldehyde in 0.1 m phosphate (ph 7.2-7.4), then post fixed in 1% buffered osmium tetroxide, en-bloc stained with 2% uranyl acetate in 95% ethanol and embedded in araldite-cx resin after polymerizing it at 60 °c for 48 h. ultrathin sections were obtained with lkb ultracut microtome, stained with uranyl acetate fallowed by lead citrate, and examined by fm jeol, em, electron microscope (johannessen, 1978). 50 v.r. chandavar and p.r. naik statistical analysis the measurements were expressed as mean ± sd for islet size (in mm); cell count (number) and plasma glucose level (mg/dl) during different periods was carried out using analysis of variance (anova). wherever the anova values (f) were found to be significant, duncan’s multiple range test (dmrt) was applied. results initiation of gonad activity occurred during august-september (table 1) in both the sexes of e. carinata and this duration is designated as regenerative period, which corresponded with late monsoon. peak of gonad activity was observed during october-december, during which the animals exhibit well developed ova/ testis. this is referred to as reproductive period. testis in males become smaller; oviduct of females had either fully mature eggs in them which was about to lay or they had no eggs with reduced oviducts in recrudescent period which fall in the months of january-july. different periods of reproductive cycles were assigned by careful observation of the status of the gonad during two successive cycles of reproduction. weight of the animals varied with the season, being highest in regenerative period (table 2). the mean pancreas weight was least in this period while the length of the pancreas was moderate. the reproductive animals weighed minimum, having a higher pancreas weight. during recrudescence, the animals weighed moderate. significant difference existed between periods with respect to weight of the animal (table 2). the mean weight of pancreas during reproductive period was higher and differed significantly from the other two periods. the pancreas on an average measured 2.97 ± 1.03 cm in length and weighed 0.18 ± 0.16 g. animal length and pancreas length did not differ significantly between the periods. the islets in e. carinata were irregular without connective tissue capsule. they stained as dark blue clumps of cells surrounded by lighter stained exocrine pancreas in chp method, unlike in laboratory mammals (rat). conspicuous variations in staining property of islets between e. carinata and mammals reveal that the cytoplasmic granules of glucagon and insulin cells differ from that of mammals.the insulin cells stained darker, round or elongated with central nuclei but the glucagon cells were not evident in chp method. hence, immunocytochemistry was carried out to localize both the cell types. the immunoreactive cells were located in the exocrine pancreas as solitary or two to three cell clusters or as islet throughout the gland (fig.1, a to f). smaller clusters were not evident in chp method. larger islets exhibit capillary spaces in them and were oriented towards the blood vessel, indicating their endocrine property. this was further confirmed by electron microscopy (em) (fig. 2, a and b).the endocrine pancreatic cells were found to be distributed along the capillaries. morphological differences between two cell types were observed. under em the glucagon cells were oval in shape. their nuclei were placed away from the centre with or without eccentric nucleoli. cytoplasm showed the presence of dense granules. the secretory granules were round, electron dense and devoid of electron lucent space (fig. 2b). insulin were elongated or oval in shape with central round nuclei. secretory granules were uniformly distributed throughout the cytoplasm. the granules were characteristically 51localization of glucagone in eutropis carinata membrane bound, filled with dense, rectangular or polymorphic matrix. a distinct electron lucent space between membrane and matrix was prominent (fig. 2b). there was no significant change in the distribution of islets throughout the pancreas in both the sexes. during regenerative period, the islets measured 0.22 ± 0.09 mm with lesser count of both glucagon-ir (2645 ± 32) and insulin-ir cells (2164 ± 24). the plasma glucose was moderate. the regenerative animals exhibited very few pas positive masses in their liver samples (table 2). the animals were found basking during reproductive period and it corresponded with winter. the pancreas showed the presence of islets in smaller clumps. on an average, the islet measured 0.41 ± 0.24 mm. glucagon-ir (6624 ± 35) and insulin-ir cell (5425 ± 36) number was higher than that during regenerative period. the plasma glucose recorded the highest value. there was no localization of pas positive masses in liver sections in this period (table 2). the maximum number of glucagon-ir (9351 ± 36) and insulin-ir (7651 ± 40) cells was evident during recrudescence. the size of the islet measured 0.69 ± 0.26 mm. the table 1. annual seasonal cycle and reproductive events in e. carinata. reproductive periods regenerative reproductive recrudescent gonad activity initiation peak regression month aug-sept oct-dec jan-july season late monsoon winter summer/monsoon temperature: max 31 ± 2 °c 28 ± 0.5 °c 37 ± 2 °c min 19 ± 0.5 °c 13 ± 2.1 °c 18 ± 2 °c note: specimens were collected at different periods of the year. autopsy was carried out in the above mentioned months and temperature was recorded during those months. table 2. different periods of reproduction and morphometric measurements in e. carinata. reproductive periods statistics regenerative reproductive recrudescent parameters (mean ± sd) (mean ± sd) (mean ± sd) f-value p-value animal weight (g) 31.26 ± 5.10 17.4 ± 4.85 22 ± 3.76 23.494 < 0.001 animal length (cm) 26.80 ± 3.32 27.5 ± 1.58 25.1 ± 1.65 2.817 0.077 pancreas weight (g) 0.039 ±.016 0.46 ± 0.44 0.052 ± 0.008 8.865 0.001 pancreas length (cm) 3.48 ± 1.3 2.75 ± 1.31 2.7 ± 0.54 1.545 0.232 plasma glucose (mg%) 205 ± 57 233 ± 47 164 ± 25 17.645 < 0.001 abdominal fat +++ -/+ ++ liver glycogen ++ -/+ +++ note: mean with same letters is not significantly different from each other. sd, standard deviation; +++, maximum; ++, moderate; -/+, minimum. 52 v.r. chandavar and p.r. naik   fig. 1. left top (a). pancreas of regenerative period showing very few glucagon-immunoreactive cells appearing dark (arrows) between acini (ex), blood capillary (c) is also seen. × 200. right top (b). succeeding section of regenerative period showing very few insulinimmunoreactive cells appearing dark (arrows) between acini (ex) and near the blood capillary (c). × 200. middle left (c). pancreas of reproductive period showing glucagon-immunoreactive cells in larger group as dark mass (arrows) between acini (ex) and blood capillary (c). × 200. middle right (d). succeeding section of reproductive period showing darkly stained larger group of insulin-immunoreactive cells (arrows) than the earlier period between acini (ex). × 200. bottom left (e). pancreas of recrudescent period localized for glucagonimmunoreactive cells which appear as dark mass between acini (ex). × 200. bottom right (f). succeeding section of recrudescent period showing darkly stained insulinimmunoreactive cells (arrow) between acini (ex). × 200. all the above photographs are the representatives of each period. 53localization of glucagone in eutropis carinata   fig. 2. (a) electron micrograph of islet region showing glucagon (glu) cell with basal nucleus (n) and insulin (ins) cell with central nucleus (n) and eccentric nucleolus (nu). the endocrine cells were surrounded by the blood capillary (c ). × 5400. (b) electron micrograph of an enlarged region of above islet showing glucagon (glu) and insulin (ins) cell with nucleus (n) and nucleolus (nu) × 10000. secretory granules of glucagon cells were electron dense (white arrow) with closely fitting membrane. secretory granules in insulin cell were prominent with electron lucent space (black arrow) between membrane and matrix. 54 v.r. chandavar and p.r. naik plasma glucose was lowest of all the periods. liver sections of recrudescent period reveled intensely stained pas positive masses. the mean cell count for both-ir cells during recrudescence was highest and differed significantly from regenerative and reproductive periods. immunolocalized pancreatic cells revealed significantly different mean numbers of localized cells in different phases of reproduction being highest in recrudescence. but proportion of glucagon-ir (55%) and insulin-ir (45%) cells remained similar in all periods with paracrine arrangement being adjacent to one another. this was also evident under electron microscopic studies (fig. 2a). significant difference in mean plasma glucose level between periods was observed being highest in reproductive period. mean size of the islet also showed significant variation between periods being highest in recrudescent period. discussion the food consumed by the animals during regenerative period was converted and stored as reserve food. conversion of food into fat and muscle mass rendered the animals to weigh higher. reproductive period appears to be energetically expensive and are found to be reliant on stored energy in e. carinata. therefore the animals of this period had no abdominal fat and glycogen mass in their liver. absence of reserve food (glycogen and fat) may be due to higher cell count of glucagon-ir and insulin-ir cells in comparison to regenerative period. the food consumed by the animals was mainly utilized for the development and maturity of gonads. the reserved food was found diverted to reproduction instead of constructing the body mass, and the animals, on an average weighed the least of all the periods though they were gravid. decreased liver glycogen may be one of the contributing factors for increasing the plasma glucose during reproductive period which is accompanied by increased glucagon cells rendering glycogenolysis compared to regenerative period. the phosphorolysis of glycogen is mainly mediated by glucagon (bollen et al., 1998). glucagon-ir and insulin-ir cells increased from reproductive period to recrudescent period. the increase in number of both cell types may be due to stimulus of higher plasma glucose of reproductive period. increased insulin cells of this period than the preceding periods contributed gradually in building up of liver glycogen and this accounted for decrease in glucose output. the conversion of glucose into glycogen in liver and availability of glucose to peripheral tissues accompanied by endocrine cells resulted in building up of muscle mass. increased insulin-ir cells facilitated glucose uptake by peripheral tissue as well as anabolic effect on liver to construct glycogen. as a result the animals weighed more than those in reproductive period with least plasma glucose. the regulation of hepatic glucose metabolism has a key role in whole-body energy metabolism, as the liver is able to store and to produce glucose (foufelle and ferré, 2002). glycogen is stored as a reserve of glucose in liver for extra hepatic tissues. another glycogenic stimulus for the liver is insulin. as glucagon-ir cells were more numerous than insulin-ir cells, stored energy in the form of abdominal fat were utilized. eutropis carinata exhibits annual cycle of energy storage in the form of abdominal fat. due to cyclic change in plasma glucose, liver glycogen, abdominal fat, glucagon-ir http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22foufelle%20f%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22ferr%c3%a9%20p%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract 55localization of glucagone in eutropis carinata and insulin-ir cell count, e. carinata exhibited significant difference in body weight and pancreas weight and not in their lengths, which usually remains static for a species. in the present investigation monoclonal antibodies were used to localize glucagon-ir and insulin-ir cells. use of monoclonal antibodies is the most reliable method for localizing glucagon and insulin cells in pancreas. the most abundant endocrine cell type was glucagon-ir cells (55%). in most lacertids studied, the central core consisted of b cells and a-cells at the periphery, with predominance of b-cells (putti et al., 1992; el-salhy et al., 1983). in the pancreas of the reptilian species, insulin-ir cells were present as solitary cells or in groups. they were located in the central core of the pancreatic islets and the most predominant cell type (perez-tomas et al., 1989; morescalchi et al., 1997). in the present study of e. carinata, very few glucagon-ir and insulin-ir cells were scattered or present in smaller groups, particularly in regenerative period. in larger islets, glucagon-ir and insulin-ir cells were found scattered throughout the islet. this was further confirmed by em. insulin containing b cells or glucagon containing a cells were not clustered in the islet as in other lizards (el-sahly et al., 1983; perez-tomas et al., 1989; putti et al., 1992; morescalchi et al., 1997), but showed paracrine association with one another. paracrine interactions might have increased both cell types in number form regenerative to reproductive and from reproductive to recrudescent period. among the two cell types glucagon-ir cells were predominant (55%) in all the periods. in this respect, e. carinata appears to be unique. the normal fasting blood glucose level of five lizard species namely eumeces obsolecte, e. skiltonianus, e. fasciatces, a. carrolinensis, and sceloporus accidentalis ranged 74.0113.9 mg% as against post-pranadial blood glucose level of 142.5-219.7 mg% (miller and wurster, 1956). the highest mean plasma glucose in e. carinata was 233 mg%. this indicates that the lizards are capable of tolerating values exceeding 200 mg/dl. in the present investigation it was found that physiological events were accompanied by substantial fluctuations in plasma glucose. in e. carinata, the study leads us to suggest that glucagon and insulin cells could regulate fat, glycogen and glucose metabolism in paracrine manner on reproductive events. acknowledgments the first author acknowledges the university of mysore for the award of teacher fellowship, neuropathology department of national institute of mental health and neuro science, bangalore for providing the facility to carry out electron microscopy and the ugc for the grants provided to carry out immunocytochemical studies. references anonymous (2000): guidelines for care and use of animals in scientific research. indian national science academy, new delhi. bollen, m., keppens, s., stalmans, w. 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(1999): immunocytochemical characterization of the pancreatic islet cells of the nile tilapia (oreochromis niloticus). gen. comp. endocr. 114: 47-56. http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22rhoten%20wb%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22hall%20ce%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus file:///users/riccardo/documents/editoria/fup/riviste/acta%20herpetologica/f1619_acta_herpetologica_2011_1/02_editing/javascript:al_get(this,%20'jour',%20'am%20j%20anat.'); bbib67 ole_link1 ole_link2 bbib28 ole_link5 ole_link6 ole_link7 ole_link8 _goback ole_link1 ole_link2 ole_link3 ole_link4 ole_link1 ole_link2 ole_link19 ole_link20 ole_link21 ole_link29 ole_link3 ole_link4 ole_link5 ole_link31 ole_link14 ole_link15 ole_link12 ole_link13 ole_link16 ole_link17 ole_link22 ole_link23 ole_link24 ole_link8 ole_link9 ole_link10 ole_link11 ole_link18 ole_link27 ole_link28 ole_link25 ole_link26 ole_link6 ole_link7 ole_link34 ole_link37 ole_link38 acta herpetologica vol. 6, n. 1 june 2011 firenze university press widespread bacterial infection affecting rana temporaria tadpoles in mountain areas rocco tiberti extreme feeding behaviours in the italian wall lizard, podarcis siculus massimo capula1, gaetano aloise2 lissotriton vulgaris paedomorphs in south-western romania: a consequence of a human modified habitat? severus d. covaciu-marcov*, istvan sas, alfred ş. cicort-lucaciu, horia v. bogdan body size and reproductive characteristics of paedomorphic and metamorphic individuals of the northern banded newt (ommatotriton ophryticus) eyup başkale1, ferah sayım2 , uğur kaya2 genetic characterization of over hundred years old caretta caretta specimens from italian and maltese museums luisa garofalo1, john j. borg2, rossella carlini3, luca mizzan4, nicola novarini4, giovanni scillitani5, andrea novelletto1 the phylogenetic position of lygodactylus angularis and the utility of using the 16s rdna gene for delimiting species in lygodactylus (squamata, gekkonidae) riccardo castiglia*, flavia annesi localization of glucagon and insulin cells and its variation with respect to physiological events in eutropis carinata vidya. r. chandavar1, prakash. r. naik2* the balearic herpetofauna: a species update and a review on the evidence samuel pinya1, miguel a. carretero2 effects of mosquitofish (gambusia affinis) cues on wood frog (lithobates sylvaticus) tadpole activity katherine f. buttermore, paige n. litkenhaus, danielle c. torpey, geoffrey r. smith*, jessica e. rettig food composition of uludağ frog, rana macrocnemis boulenger, 1885 in uludağ (bursa, turkey) kerim çiçek preliminary results on tail energetics in the moorish gecko, tarentola mauritanica tommaso cencetti1,2, piera poli3, marcello mele3, marco a.l. zuffi1 climate change and peripheral populations: predictions for a relict mediterranean viper josé c. brito1, soumia fahd 2, fernando martínez-freiría1, pedro tarroso1, said larbes3, juan m. pleguezuelos4, xavier santos5 assessing the status of amphibian breeding sites in italy: a national survey societas herpetologica italica* osservatorio erpetologico italiano acta herpetologica journal of the societas herpetologica italica acta herpetologica rivista della societas herpetologica italica © firenze university press www.fupress.com/ah acta herpetologica 5(1): 107-112, 2010 chromatic variation in populations of xenodon merremi (serpentes: dipsadidae) in paraguay pier cacciali departamento de paleontología, facultad de ciencias, universidad de la república. iguá 4225 cp 11400, montevideo, uruguay. instituto de investigación biológica del paraguay, del escudo 2044, asunción, paraguay. e-mail: pier_cacciali@yahoo.com submitted on: 2009, 10th june; revised on 2010, 25th march; accepted on 2010, 9th april. abstract. xenodon merremi is a polychromatic species distributed in south america. among its wide range of color patterns, the most common pattern resembles a pitviper of the genus bothrops. in this work is recorded the different patterns found in paraguayan populations of x. merremi. four patterns can be observed: marked pattern, slightly marked pattern, smooth, and banded pattern. the marked pattern is the most common pattern (mimetic with pitvipers), and all juveniles bear this coloration. only adults show variation in their coloration. keywords. xenodon merremi, paraguay, chromatic variation. xenodon merremi is a colubrid snake widely distributed from ecuador and the guianas, to northern and central argentina, and uruguay (cerreira et al., 2005; tipton, 2005). throughout its distribution, x. merremi occurs in both natural and anthropogenic areas (cabrera, 2004; scrocchi et al., 2006) and feeds largely on toads (carreira, 2002). several works have made reference to the variable polychromatic coloration of x. merremi (under the genus waglerophis) even in local areas (giraudo, 2001; cabrera, 2004; scrocchi et al., 2006). the typical coloration of x. merremi is a pattern with semicircular brownish lateral marks with wavy edges and skirted by white thin margins, this pattern being mimetic with pitvipers of the genus bothrops (giraudo, 2001; cabrera, 2004; carreira et al., 2005; scrocchi et al., 2006). additionally, brodie and brodie (2004) also pointed out that the species can show coloration mimetic of coral snakes (see plate 1338 in campbell and lamar, 2004). included in the range of variation reported in x. merremi are specimens with black marks (not brownish); specimens with the body completely yellowish, brown, or even almost completely blackish; as well as a specimen with partial albinism (scrocchi et al., 2006). giraudo (2001) identified six different patterns: a (uniformly immaculate), b (with rings on the back), c (with small semicircular marks on the sides, with a vertebral stripe), 108 p. cacciali d (the typical pattern with smooth marks), e (the typical pattern with clear center marks), and f (with one undulated stripe on each side). giraudo (2001) stated that there is no concordance between coloration variation and distribution, but there is an ontogenetic pattern: juveniles are always marked with patterns d or e), whilst adults are variable. ontogenetic changes were also recorded by norman (1994). in this work is presented a summary of the coloration patterns recorded in paraguay, together with the frequency of each pattern. paraguay with an area of 406,752 km2 is located in the center of south america, and divided approximately in half by the paraguay river. western paraguay (commonly referred to as “the chaco”) makes up 60% of the territory, whereas the remaining 40% is represented by the eastern or oriental region. biogeographically, paraguay is divided into seven ecoregions (fig. 1). x. merremi is distributed almost throughout the whole country. for this study, a sample of 60 paraguayan specimens (18 juveniles and 42 adults, appendix 1) preserved in the museo nacional de historia natural del paraguay (mnhnp) was analysed. specimens shorter than 25 cm were considered juveniles. four different patterns were recognized in this sample: marked (normal pattern), slightly marked, smooth, and banded. all juveniles analysed were of the normal marked pattern, only adults exhibited variation. there follows a brief definition of each of the observed patterns, with comments on their frequency in the sample. fig. 1. ecoregional distribution of paraguay. 109chromatic variation in populations of xenodon merremi in paraguay marked pattern: this is the “typical” pattern of the species, with series of marks on the sides (fig. 2a). marks can reach the vertebral zone, joining with its couple of the other side. although different color patterns were observed, all are here grouped as “marked” individuals. all juveniles were shown to exhibit this pattern in addition to 22 of the adults (52.4% of the adults in the sample). no difference was observed between specimens from the western and oriental regions (fig. 3a). slightly marked pattern: “slightly-marked” individuals tend to lose the body markings (fig. 2b), though typically traces of marks remain near the vertebral zone. eleven adults bore this pattern corresponding to 26.2% of the sample. no juveniles in the sample exhibited this pattern. specimens with “slightly marked pattern” are present in a wide variety of environments, and have been recorded from all the paraguayan ecoregions except for the pantanal (fig. 3b). smooth: specimens with smooth coloration, lack contrasting pattern (fig. 2c) and are usually uniformly yellowish or brownish. six adults were recorded with this coloration, representing 14.3% of the adults examined. specimens with smooth coloration were collected from the dry chaco and wet chaco, and were recorded in the oriental region only in the departments of central and ñeembucú, where the predominant habitat is similar to that of wet chaco (fig. 3c). fig. 2. body color patterns of paraguayan populations of xenodon merremi. a: marked pattern, b: slightly marked pattern, c: smooth coloration, and d: banded pattern. 110 p. cacciali banded pattern: the “banded pattern” consists of a series of black bands or rings along the body, against a grey background (fig. 2d). specimen mnhnp 2601 additionally shows vertebral constriction in some bands, and is quite similar in shape to the typical “marked” pattern. only three adults in the sample showed this pattern, corresponding to 7.1% of the total adults. the three specimens were collected from three widely-dispersed localities: cerro corá national park (cerrado ecoregion, departamento amambay), colonia walter insfrán (alto paraná atlantic forest ecoregion, departamento caaguazú), and the surroundings of loma plata (dry chaco ecoregion, departamento boquerón) (fig. 3d). fig. 3. distribution of examined specimens, according to the color pattern. black dots are adults, and open squares are juveniles. note that there are juveniles only in “a”. a: marked pattern, b: slightly marked pattern, c: smooth coloration, and d: banded pattern. 111chromatic variation in populations of xenodon merremi in paraguay results show that like other previous works (norman, 1994; giraudo, 2001), juvenile coloration is always consisting in well defined marks, whereas adults can show different patterns of coloration. this results confirms that coloration probably change ontogenetically in this species. the commonest adult pattern in the paraguayan sample was the marked pattern, which accounted for 52.7% of the adult specimens. giraudo (2001) defined two different kinds of “normal” patterns, to differentiate them from the other marked patterns. nevertheless, in paraguay, the marked specimens only exhibit the normal marked coloration (fig. 2a), and not the wide range described by giraudo (2001, fig. 42). these findings suggest that more than half of all paraguayan specimens retain juvenile coloration into adulthood. in the dry chaco, it is possible to find all the different patterns. probably also in the wet chaco; but examined specimens with banded pattern was not presents in the wet chaco. an important fact is that in the cerrado, occur three of the four different patterns, being absent only the smooth coloration. in the table 1, is presented a list of number of individuals of each color pattern in each ecoregion. any specimen came from pantanal. more sampling effort is required to get a clearer picture of the distributional patterns of the different color types. given the small sample size the current observed distribution of the pattern types in paraguay may be a result of different sampling efforts in different locations. bearing this mind it should be noted that the smooth type could yet prove to have a wider distribution in paraguay than it currently appears. acknowledgements thanks to martha motte (mnhnp) for allowing access to the specimens under her care. to norman scott for providing information of united states museums records. to david gill and paul smith for corrections to the english version. also to three referees whose suggestions helped improve the work. to mónica rumbo for the patience and help. this work was done within the framework of the thesis project “biogeography of the reptiles of paraguay” that is being carried out by the author. table 1. patterns frequencies in each ecoregion. pa (pantanal), dc (dry chaco), wc (wet chaco), ce (cerrado), af (alto paraná atlantic forest), cp (central paraguay), mg (mesopotamian grasslands). there are 12 additional sampled specimens without specific locality data. pa dc wc ce af cp mg total pattern a 0 8 3 1 7 10 3 32 pattern b 0 3 0 1 2 2 0 8 pattern c 0 3 1 0 0 1 0 5 pattern d 0 1 0 1 1 0 0 3 total 0 15 4 3 10 13 3 48 112 p. cacciali references brodie iii, e.d., brodie jr., e.d. (2004): venomous snake mimicry. in: the venomous reptiles of the western hemisphere, p. 617-633. campbell, j.a., lamar, w.w., eds, cornell university, new york. cabrera, m.r. (2004): las serpientes de argentina central. publicaciones de la universidad nacional de córdoba, córdoba. campbell, j.a., lamar, w.w. (2004): the venomous reptiles of the western hemisphere. cornell university, new york. carreira, s. (2002): alimentación de los ofidios de uruguay. monografías de herpetología 6: 1-126. carreira, s., meneghel, m., achaval, f. (2005): reptiles de uruguay. universidad de la república, montevideo. giraudo, a. (2001): serpientes de la selva paranaense y del chaco húmedo. literature of latin américa, buenos aires. norman, d. (1994): anfibios y reptiles del chaco paraguayo, tomo i. san josé, costa rica. scrocchi, g., moreta, j.c., kretzschmar, s. (2006): serpientes del noroeste argentino. fundación miguel lillo, tucumán. tipton, b. (2005): snakes of the americas, checklist and lexicon. krieger publishing company, florida. appendix 1 examined specimens a. marked pattern (including juveniles): paraguay (mnhnp 2611, 9256, 9429, 9431, 9432, 9516, 9554, 9583). alto paraguay: fortín madrejón (mnhnp 2657). amambay: parque nacional cerro corá (mnhnp 2660). boquerón: 15.6 km s of filadelfia (mnhnp 10033); 26 km n of filadelfia (mnhnp 9736); parque nacional teniente enciso (mnhnp 6524); filadelfia (mnhnp 2610, 8495); neuland (mnhnp 9992); route ix, km 519 (mnhnp 6825). caazapá: parabel (mnhnp 8808). central: asunción (mnhnp 3180, 6520); guarambaré (mnhnp 2664); luque (mnhnp 6288-90); san lorenzo (mnhnp 7461, 9939). concepción: horqueta (mnhnp 2609). cordillera: altos (mnhnp 10309). itapúa: isla yacyretá (mnhnp 9568). misiones: yabebyry (mnhnp 3779-80). ñeembucú: estancia yacaré (mnhnp 4556, 6679). paraguari: acahay (mnhnp 3540); coronel c. barrientos (mnhnp 3781); parque nacional ybycui (mnhnp 2661-3); yaguarón (mnhnp 2658). presidente hayes: estancia bella vista (mnhnp 10610). b. slightly marked pattern: paraguay (mnhnp 9456, 9593, 9594). alto paraguay: fortín madrejón (mnhnp 2679). amambay: parque nacional cerro corá (mnhnp 9193). boquerón: parque nacional teniente enciso (mnhnp 2656); filadelfia (mnhnp 3839); central: luque (mnhnp 6286); san lorenzo (mnhnp 9172). guairá: 13 km w of villarrica (mnhnp 6518). itapúa: coast of the paraná (mnhnp 4213). c. smooth coloration: paraguay (mnhnp 9566). alto paraguay: estancia tres marías (mnhnp 9197); laguna león (mnhnp 11041). boquerón: route ix, km 508 (mnhnp 2655); central: san lorenzo (mnhnp 7053). ñeembucú: estancia yacaré (mnhnp 4585). d. banded pattern: amambay: parque nacional cerro corá (mnhnp 2659). boquerón: 9 km s of loma plata (mnhnp 9996). caaguazú: colonia walter insfrán (mnhnp 2601). issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 7(2): 347-353, 2012 differences in habitat use of two sympatric species of ameiva in east costa rica esther sebastián-gonzález1, ramón gómez2 1 departamento de ecologia. universidade de são paulo. corresponding author. e-mail: esebastian@ umh.es 2 ptda. de la solana 6473. e-46870 ontinyent (spain) submitted on: 2012, 3rd march; revised on: 2012, 21st november; accepted on: 2012, 23rd november. abstract. identifying the differences in habitat use for sympatric species is important for understanding the species preferences and the limits of population distribution. we studied the differences in the habitat use of two understudied sympatric species of ameiva (a. festiva and a. quadrilineata) in a natural reserve of the caribbean coast of coast rica. ameiva quadrilineata showed a more restrictive habitat use pattern than a. festiva. a. quadrilineata’s smaller body size may be one of the factors limiting its habitat range. both species showed higher density in regenerated forests, while a. quadrilineata was never found in swamp forests. the air temperature and the meteorological condition at the moment of the survey also influenced the occurrence of the a. quadrilineata, while the juveniles of a. festiva were only affected by the meteorological condition. none of the studied variables seemed to affect the occurrence of a. festiva adults. the results of this study can be useful to evaluate possible changes in the species distribution patterns as a consequence of direct (i.e., deforestation) or indirect (i.e., climate change) human activities in the distribution area of these species. keywords. habitat use, activity pattern, glmms, costa rica, ameiva. understanding which factors influence habitat use patterns may be of major importance to evaluate the threatened status of the species and to develop management strategies. it is especially urgent nowadays because of the difficulty of separating human impacts from natural stochastic events (pechmann et al., 1991). environmental conditions may change due to human-induced global climate change (sinervo et al., 2010) and there is a need to identify the species’ habitat requirements to develop management strategies accordingly. moreover, the patterns of habitat use in reptiles are very linked to the species-specific thermal restrictions. lizard species may differ in their ability to use habitats with different solar exposure by having different morphology, physiology or behaviour (pianka and vitt, 2003). for example, lizards foraging in shaded areas rapidly cool as a consequence of low thermal inertia and they need to newly expose directly to the sun to 348 e. sebastián-gonzález and r. gómez raise their body temperature. consequently, it is important to combine data on patterns of habitat use with the thermal information to develop more accurate management strategies for reptiles. in tropical areas there is a lack of scientific information about the basic ecology of many species (peres, 2005). the lack of funding, or the difficulties in the accessibility to the natural areas are some of the causes of this information deficiency (sodhi, 2008). in the tropical pacuare reserve (caribbean coast of costa rica, 10°13’n, 83°15’w; maximum altitude of 1 m a.s.l.) two species of lizards from the genus ameiva (a. festiva and a. quadrilineata, teiidae) have been identified (abella et al., 2008). the populations of these two species overlap in their distribution, but previous studies have identified differences in their specific habitat (hillman, 1969). the information related to these two species is dated and not abundant, referring basically to their temperature preferences (hirth, 1964), ecology (hirth, 1963; hilman 1969; vitt and zani, 1996) and reproduction (smith, 1968). specifically, hillman (1969) found that a. quadrilineata occurred with a higher frequency (74% of occurrence) at low vegetation with a high percent of insolated substrate, while a. festiva was found foraging both in the edge and inside the forest and with a lower insolation (60% of occurrence). however, the available information on their occurrence in different habitat types still has many gaps. we know from previous studies that, for species with similar thermal preferences and tolerance limits, larger lizards will loss heat with a slower rate than the smaller ones, permitting longer times without a direct exposure to sun (asplund, 1974; stevenson, 1985). we also know that a. quadrilineata is slightly smaller than a. festiva, so its ability to stay away from sunny areas might be smaller, but their thermal preferences and tolerance limits are similar (hillman, 1969). thus, the aims of this study are: (i) to evaluate the habitat use and activity patterns of two sympatric species of ameiva; (ii) to qualitatively assess if the possible differences in habitat use agree with the differences in thermal inertia between the two species due to changes in body size. we hypothesized that the habitat requirements of a. quadrilineata may be more limited than those of a. festiva. we randomly selected eight transects (100 m length) following the approach outlined by heyer et al. (1994). we selected two transects by each of the four main habitat types. the “sandy forest” is formed by all the vegetation related to the sandy beach, including several plants adapted to the high salinity and the marine influence. all plants are relatively small (not taller than 3 m) and the soil is formed by volcanic grey sand. the “regenerated forest” includes re-grown forests in old open pasture previously used for cattle ranching, and abandoned since 1989. it is composed by many species of medium-small trees (diameter less than 30 cm), such as zygia longifolia, or prioria copaifera. the forest composition of the “secondary forest” is similar to the one in the “regenerated forest”, but the trees are older (the diameter can be more than 30 cm for some trees) and the forest is more mature. the trees and shrubs density is higher and there is, in general, less light reaching the soil. finally, the “swamp forest” is located close to a small pond with permanent water, and soils surrounding the pond retain water for long periods of time each year. the vegetation is dense, dominated by raphia taedigera and there are other species such as zygia longifolia, and erythrina cochleata. the area floods easily when the rain is strong. the surveys were performed at three different times of the day: in the morning (5:30-7:30), midday (12:00-14:00) and evening (16:00-18:00), but the same transect was 349habitat use of two sympatric lizards only surveyed once a day. the meteorological condition (sunny, partially cloudy, cloudy, raining), the air temperature (in celsius degrees), the relative humidity, and the precipitation (raining, last rain less than one hour ago, between one and five hours ago, between five and ten hours ago, between ten and 24 and more than 24 h ago) were identified. we used a digital thermometer (precision ± 1 ºc) for measuring the temperature and a hygrometer (precision ± 1%) for relative humidity. each transect was walked slowly, fitting the total survey time to 15 minutes. the number of times each transect was walked varied between 12 and 22 times (average 16.2 times). the occurrence of individuals of a. festiva and a. quadrilineata was recorded, distinguishing between adults and juveniles. we distinguished juveniles and adults depending on the body size. juveniles of a. festiva were shorter than 7.8 cm while juveniles for a. quadrilineata were shorter than 6.2 cm (savage, 2002). we could not effectively identify males from females and therefore sexual differences were not analysed. at the detection points we measured some microhabitat variables: the total height from the soil layer; the litter height; the distance between the soil and the location of the individual at the dead leaves. we also measured the exposure degree of the individual to the sun (shade, filtered sun or sun), the air temperature, and the relative humidity. generalized linear mixed models (glmms) with the r 2.1.1 software (r development core team, http://www.r-project.org) were used to relate the abundance of both species in each transect to environmental and habitat variables. we calculated the abundance as the numbers of ameiva individuals of each species per transect, and it was used as the dependent variable for the models. because the activity and abundance of the species can change depending on the time of the day, we included this variable as a covariate. moreover, as we surveyed the same transects several times, we also included transect as a random variable. we constructed one multivariate model to assess the effect of the environmental variables, and one univariate model to evaluate the habitat preferences. we built four separate models to evaluate the differences between species and ages: one model for adults and one for juveniles for each of the two species. glmms permit the use of suitable error distributions, and some of the limitations of conventional regression models were avoided. we used the poisson distribution as error function and the glmer function from the lme4 package. for the multivariate model, we used a backward removal procedure to obtain a final model containing only significant factors that significantly improved the fit of the model by more than 1% of the explained deviance (see santoul et al., 2004 for a similar approach). finally, we used non-parametric tests to analyse the differences between the species and between adults and juveniles at the microhabitat level. we observed a total of 190 individuals. from these individuals, 155 were a. quadrilineata (70 adults and 85 juveniles), and 35 were a. festiva (17 adults and 18 juveniles) in 132 transects (36 in regeneration forest, 39 in secondary forests, 26 in swamp forests and 32 in sandy areas). the final models explaining the environmental factors affecting abundance along transects were similar for both species (table 1). juveniles of a. festiva were affected by the meteorological condition, while juveniles of a. quadrilineata were also affected by the temperature. only the adults of a. quadrilineata were significantly affected by the temperature. moreover, the best time of the day for surveying was at midday for both species (fig. 1). previous studies have associated the activity of a. festiva with the time of day 350 e. sebastián-gonzález and r. gómez when temperatures and sun exposure are maximum, both of which appear necessary for attainment of high activity body temperatures (vitt and zani, 1996). the insolation and the precipitation did not affect the occurrence of any of the species. hirth (1963) affirmed that the activity of teiidae almost comes to a stop when clouds hide the sun. ameiva individuals were found with a higher frequency in sunny days in the study area. nevertheless, they were also observed active in partially cloudy, and even in cloudy days. a. festiva had already been observed in these meteorological conditions (vitt and zani, 1996), but no records had been found before for a. quadrilineata (hirth, 1963; hillman, 1969). the habitat use patterns differed between a. festiva and a. quadrilineata, and also between adults and juveniles. a. quadrilineata was distributed differentially in the four habitats, while a. festiva did not show any pattern (table 1 and fig. 2). when lizards forage in the shade, their body temperatures drop to their lower limit, since the temperatures of the shade (i.e., air cold and substrate cold) normally falls below the lowest recorded fig. 1. histograms comparing the percentage of the observations at each time of the day (a) and for each meteorological condition (b) for both studied species. table 1. summary of the results from the two final set of glmms for the two species and for each of the two considered stage (adult, juvenile). model 1 stays for glmm built using environmental variables as predictors; in model 2 the only predictor is the habitat type. time of the day was included as a covariate while survey was included as a random variable. coefficients (coeff ) and associated p-values (p) are shown for the variables included in the final models. temp: air temperature; mc: meteorological condition. species model stage variable coeff. p a quadrilineata 1 adult temp 0.569 <0.001 juvenile temp 0.521 <0.001 mc 0.645 0.035 a. festiva 1 adult temp 0.209 0.013 juvenile mc -0.976 0.028 a. quadrilineata 2 adult habitat -1.197 0.006 juvenile habitat -0.244 0.244 a. festiva 2 adult habitat -0.297 0.192 juvenile habitat -0.253 0.658 351habitat use of two sympatric lizards body temperature (huey and tewksbury, 2009). to raise their body temperature again, the lizards may seek direct exposure to sunlight. as the body size of a. quadrilineata is small, its cooling rate may be higher than the one of a. festiva (it needs about three more minutes than a. quadrilineata for its body temperature to decrease six degrees; hillman, 1969), and this may be influencing the differences in habitat use found for the two species. vegetation cover also directly or indirectly influences substrate and habitat temperatures. this agrees with the finding that the occurrence of a. quadrilineata in dense vegetated areas was less frequent and it was never found in habitats without areas with direct sun exposure (hirth, 1963; hillman, 1969). ameiva festiva showed a different pattern occurring in all habitat types without presenting significant habitat preferences. the larger body size of this species allowed it to stay longer periods of time away from the direct exposure to the sun (hillman, 1969; vitt and zani, 1996), and it was often detected both in secondary and swamp forests. its ability to shuttle between sun and shade helps this species to maintain the body temperature at high constant level (see van berkum, 1986 for a similar behaviour in anolis species). it is important to notice than other variables, such as differences in the behaviour and differences in their physiology may also be influencing their dissimilar ability to use the studied habitat types and further studies are required in order to confirm our hypotheses. ameiva juveniles did not show strong differences in habitat use with respect to adults (fig. 2). even, the density of a. quadrilineata juveniles at the sandy forest (the area with the highest insolation degree) was higher than for the adults. moreover, juveniles of a. festiva presented high densities in swamp and regeneration forests. the small number of observations for a. festiva can bias the results for this species. at the microhabitat level, significant differences between species emerged in the temperature (mann-whitney u-test, u = 2064, p = 0.027) and relative humidity (u = 3453, p = 0.012) when both species were found. as a. quadrilineata is smaller, it is more affected by changes in environmental temperature and insolation (hillman, 1969) and its activity is related to a combination of adequate temperatures with other environmental variables such as the meteorological condition (i.e., sunny days). the observed temperature ranges for a. festiva were broader than those observed in previous studies in a population in fig. 2. histogram representing the percentage of observations found at the four habitat types for adults and juveniles of the two studied species. 352 e. sebastián-gonzález and r. gómez southeastern nicaragua (between 27-37 in our study and 26-32 in vitt and zani, 1996). ameiva festiva was found at higher relative humidity than a. quadrilineata (peak of 82% vs. 68%). this can be related to differences in skin water loss between species, but experimental studies are required to confirm the existence of these differences. we did not find differences in the soil and death litter height where the individuals of the two species were found. moreover, we also compared the temperature and relative humidity between adults and juveniles. we found differences for a. quadrilineata (mann-whitney u-test: temperature, u = 2354, p = 0.026; relative humidity, u = 3693, p = 0.010), but not for a. festiva. the temperature of a. quadrilineata varied from 34.2 to 33.4 °c (adults-juveniles) and the relative humidity from 69.3% to 72.5%. to sum up, we have shown differences in the habitat selection and in environmental variables determining the occurrence of a. festiva and a. quadrilineata in our study-area. these results can be useful to evaluate possible changes in the species distribution patterns as a consequence of direct or indirect human activities in the distribution area of these species. because lizards have in general very strict thermal requirements, climate change is one of the most important human activities that can affect ameivas’ distribution (huey et al., 2009). if the temperature in an area changes, the species will move to other areas, adjust to the new environment, or get extinct (sinervo et al., 2010). moreover, climate change can also benefit some reptile species if the area with the preferred temperature is increased. anyway, a more detailed knowledge on the habitat and thermal preferences of the species can be used to model possible changes in the distribution and in the behaviour of the focal species, and the consequences of global warming on the population. acknowledgements we are grateful to the pacuare reserve for giving us the chance to perform this study. we thank i. abella and m. lópez for their help in designing the sampling and collecting the data, and e. graciá and a. camacho for their comments on an earlier version of this manuscript. the research assistants from the reserve made the data sampling easier. e. sebastián-gonzález benefits from a fapesp research grant. the comments from two anonymous reviewers improved the quality of the article. references abella, i., gómez, r., lópez, m. (2008): annotated amphibian and reptiles check-list of pacuare nature reserve, costa rica. bol. asoc. herpetol. esp. 19: 64-67. asplund, k.k. (1974): body size and habitat utilization in whiptail lizards (cnemidophorus). copeia 1974: 695-703. heyer, w.r., donnelly, m.a., mcdiarmid, r.w, hayek, l.c., foster, m.s. (1994): measuring and monitoring biological diversity: standard methods for amphibians. smithsonian institution press, washington, dc. hillman, p.e. (1969): habitat specificity in three sympatric species of ameiva (reptilia: teiidae). ecology 50: 476-481. 353habitat use of two sympatric lizards hirth, h.f. (1963): the ecology of two lizards on a tropical beach. ecol. monogr. 33: 83-112. hirth, h.f. (1964): temperature preferences of five species of neotropical lizards. herpetologica 20: 273-276. huey, r.b.,tewksbury j.j. (2009): can behavior douse the fire of climate warming? p. natl. acad. sci. usa 106: 3647-3648. huey, r.b., deutsch, c.a., tewksbury, j.j., vitt, l.j., hertz, p.e., álvarez-pérez, h.j., garland, t. jr. (2009): why tropical forest lizards are vulnerable to climate warming? proc. r. soc. lond. b 276: 1936-1948. pechmann, j.h.k., scott, d.e., semlitsch, r.d., caldwell, j.p., vitt, l.j., gibbons, j.w. (1991): declining amphibian populations: the problem of separating human impacts from natural fluctuations. science 253: 892-895. peres, c.a. (2005): why we need megareserves in amazonia. conserv. biol. 19: 728-733. pianka, e.r., vitt, l.j. (2003): lizards windows to the evolution of diversity. university of califormia press, berkeley. santoul, f., figuerola j., green a.j. (2004): importance of gravel pits for the conservation of waterbirds in the garonne river floodplain (southwest france). biodivers. conserv. 13: 1231-1243. savage, j.m. (2002): the amphibians and reptiles of costa rica. university of chicago press, chicago. sinervo, b., méndez-de-la-cruz, f.r., miles, d.b., heulin, b., bastiaans, e., villagrán-santa cruz, m., lara-reendiz, r., martínez-méndez, n., calderón-espinosa, m.l., mezalázaro, r.n., gadsden, h., avila, l.j., morando, m., de la riva, i., sepúlveda, p.v., rocha, c.f.d., ibargüengoytía, n.r., aguilar puntriano, c., massot, m., lepetz, v., oksanen, t.a., chapple, d.g., bauer, a.m., branch, w.r., clobert, j., sites, j.w. jr. (2010): erosion of lizard diversity by climate change and altered thermal niches. science 328: 894-899. smith, r.e. (1968): studies on reproduction in costa rican ameiva festiva and ameiva quadrilineata (sauria: teiidae). copeia 1968: 236-239. sodhi, n.s. (2008): invited views in basic and applied ecology tropical biodiversity loss and people a brief review. basic appl. ecol. 9: 93-99. stevenson, r.d. (1985): body size and limits to the daily range of body temperature in terrestrial ectotherms. am. nat. 125: 102-117. van berkum, f.h. (1986): evolutionary patterns of the thermal sensitivity of sprint speed in anolis lizards. evolution 40: 594-604. vitt, l., zani, p. (1996): ecology of the lizard ameiva festiva (teiidae) in southeastern nicaragua. j. herpetol. 30: 110-117. acta herpetologica vol. 7, n. 2 december 2012 firenze university press advertisement call of species of the genus frostius cannatella 1986 (anura: bufonidae) flora a. juncá1, david l. röhr2, ricardo lourenço-de-moraes3, flávio j. m. santos1, airan s. protázio1, ednei a. mercês1, mirco solé4 amphibians in southern apennine: distribution, ecology and conservation notes in the “appennino lucano, val d’agri e lagonegrese” national park (southern italy). antonio romano1,*, remo bartolomei1, antonio luca conte1, egidio fulco2 the significance of using satellite imagery data only in ecological niche modelling of iberian herps neftalí sillero1, josé c. brito2, santiago martín-alfageme3, eduardo garcía-meléndez4, a.g. toxopeus5, andrew skidmore5 reproductive strategy of male and female eastern spiny lizards sceloporus spinosus (squamata: phrynosomatidae) from a region of the chihuahuan desert, méxico aurelio ramírez-bautista1,*, barry p. stephenson2, xóchitl hernández-ibarra1, uriel hernández-salinas1, raciel cruz-elizalde1, abraham lozano1, and geoffrey r. smith3 morphological variability of the hermann’s tortoise (testudo hermanni) in the central balkans katarina ljubisavljević1, georg džukić1, tanja d. vukov1, miloš l. kalezić1,2 the usefulness of mesocosms for ecotoxicity testing with lacertid lizards maria josé amaral1,2,*, rita c. bicho1, miguel a. carretero2, juan c. sanchez-hernandez3, augusto m. r. faustino4, amadeu m. v. m. soares1, reinier m. mann1,5 does acclimation at higher temperatures affect the locomotor performance of one of the southernmost reptiles in the world? jimena b. fernández*, nora r. ibargüengoytía advertisement call of scinax littoralis and s. angrensis (amphibia: anura: hylidae), with notes on the reproductive activity of s. littoralis michel v. garey1, thais r. n. costa2, andré m. x. de lima2, luís f. toledo3, marília t. hartmann4 book review: marine s. arakelyan, felix d. danielyan, claudia corti, roberto sindaco, alan e. leviton 2011. herpetofauna of armenia and nagorno-karabakh. society for the study of amphibians and reptiles stefano scali book review: rafaqat masroor 2012. a contribution to the herpetofauna of northern pakistan stefano scali evidence of high longevity in an island lacertid, teira dugesii (milne-edwards, 1829). first data on wild specimens. j. jesus first assessment of the endoparasitic nematode fauna of four psammophilous species of tropiduridae (squamata: iguania) endemic to north-eastern brazil markus lambertz1,*, tiana kohlsdorf2, steven f. perry1, robson waldemar ávila3, reinaldo josé da silva4 rediscovery and redescription of the holotype of lygosoma vittigerum (= lipinia vittigera) boulenger, 1894 yannick bucklitsch1, peter geissler1, timo hartmann1, giuliano doria2 , andré koch1,* reproductive phenology of the tomato frog, dyscophus antongili, in an urban pond of madagascar’s east coast ori segev1,*, franco andreone2, roberta pala2, giulia tessa2, miguel vences1 range extension of the critically endangered true poison-dart frog, phyllobates terribilis (anura: dendrobatidae), in western colombia roberto márquez1,*, germán corredor2, carlos galvis3 daniel góez2, & adolfo amézquita1 differences in habitat use of two sympatric species of ameiva in east costa rica esther sebastián-gonzález1, ramón gómez2 acta herpetologica journal of the societas herpetologica italica acta herpetologica 2006 1 a new narrow-mouthed frog of the genus paradoxophyla (microhylidae: scaphiophryninae) from masoala rainforest, northeastern madagascar a new narrow-mouthed frog of the genus paradoxophyla (microhylidae: scaphiophryninae) from masoala rainforest, northeastern madagascar franco andreone 1, gennaro aprea 2, gaetano odierna 2, and miguel vences 3 1 museo regionale di scienze naturali, via g. giolitti, 36, i-10123 torino, italy corresponding author. e-mail:f.andreone@libero.it 2 università di napoli federico ii, dipartimento di biologia strutturale e funzionale, via cinthia, napoli, italy 3 institute for biodiversity and ecosystem dynamics, zoological museum, mauritskade 61, 1092 ad amsterdam, the netherlands [current address: zoological institute, technical university of braunschweig, spielmanstr. 8, 38106 braunschweig, germany] abstract. a new microhylid frog of the genus paradoxophyla is described from the rainforests of northeastern madagascar (masoala). paradoxophyla tiarano n. sp. was found in still waters within rainforest. in external morphology it is similar to p. palmata, hitherto the only known species belonging to this genus, but it differs by a much less extended foot webbing. we also provide a description of the probable tadpole of the species, which is a specialized suspension feeder of the typical microhylid type. the new species also differs from p. palmata in karyology and mitochondrial dna sequences. paradoxophyla tiarano n. sp. is currently known from masoala, but we suspect that its distribution might extend over a wider area in northeastern madagascar. key words. amphibia, anura, microhylidae, paradoxophyla, new species, madagascar. introduction most of the amphibians of madagascar, currently estimated to consist of over 220 species (andreone et al., 2005), breed in or along forest streams or are independent from water. in contrast, a few species prefer stagnant pools for reproduction. these frogs are usually linked to desert and savannah habitats where streams and ponds are rare, and they exhibit highly seasonal breeding habits depending on the availability of free water. the few pond breeders occurring in rainforest belong to the genera scaphiophryne, dyscophus and boophis (vences et al., 2002 b, 2003). however, these species appear much less diverse compared to their rainforest acta herpetologica 1: 15-27, 2006 16 f. andreone et alii relatives that have stream-breeding and other derived reproductive modes (i.e., some clades of mantidactylus, boophis, and cophyline microhylids; vences et al., 2002 a). all pond-breeding madagascan frogs go to water only (or mainly) during the rainy season, and during the rest of the year they carry out a terrestrial or arboreal life. one genus, paradoxophyla blommers-schlösser & blanc, 1991, is an exception in two respects. this genus, so far represented by the single species paradoxophyla palmata (guibé, 1974), is the only deep madagascan lineage restricted to rainforests and at the same time specialized to pond breeding. it is a pond breeding species with a hydrodynamic and flattened body, small eyes, and extensive foot webbing that resembles that of the african xenopus and hymenochirus and suggests a strongly aquatic preference. however, this small microhylid, included in the subfamily scaphiophryninae (blommers-schlösser & blanc, 1991), is probably fully aquatic only during its relatively explosive reproduction and is not found in water except when breeding (glaw & vences, 1994). in the course of a study at sites in the masoala forest, northeastern madagascar, we found a paradoxophyla with much less extensive webbing that also was different from p. palmata in molecular and karyological characters. we here describe it and its probable tadpole and provide data on its mitochondrial and karyological differentiation. materials and methods capture, preservation, and drawing techniques frogs and tadpoles were captured in a pool close to a forest stream. after photography, they were euthanized with chlorobutanol, fixed in 4% formalin or 90% ethanol, and preserved in 70% ethanol. voucher specimens were deposited in the museo regionale di scienze naturali, torino under the acronym mrsn. drawings were made by the senior author from original photographs along with comparisons with the preserved adults and tadpoles. morphometric measurements measurements were made with calipers (precision: 0.1 mm): svl (snout-vent length), hw (head width), hl (head length, from the maxillary commissure to the snout tip), ed (horizontal eye diameter), end (eye-nostril distance), nsd (nostril-snout tip distance), nnd (inter-narial distance), td (horizontal tympanum diameter), hal (hand length, from the carpal-metacarpal articulations to the tip of the longest finger), forl (forelimb length, from the axilla to the tip of the longest finger), hil (hind limb length, from the cloaca to the tip of the longest toe), fol (foot length, from the tarsal-metatarsal articulations to the tip of the longest toe), fotl (foot length including tarsus, from the tibiotarsal articulation to the tip of the longest toe), and tibl (tibia length). the toe webbing formula is given as in blommers-schlösser & blanc (1991) and glaw & vences (1994). for the tadpoles, we measured the following parameters: bl (body length), tal (tail length), tmh (tail muscle height), th (tail height including the dorsal and ventral fins), and hw (head width). comparative examined specimens we compared the adult specimens of the new species with the holotype and paratype of paradoxophyla palmata from ambana (south-eastern madagascar), and with a further series from 17a new paradoxophyla from ne madagascar eastern-central madagascar (andasibe and ranomafana). tadpoles were compared with the description given by blommers-schlösser & blanc (1991). used acronyms were as follows: mrsn, museo regionale di scienze naturali, torino; mnhn, muséum national d’histoire naturelle, paris; zfmk, zoologisches forschungsintitut und museum alexander könig, bonn; zsm, zoologische staatssammlung, münchen. dna analysis muscle tissue samples taken from freshly killed specimens were preserved in 98% ethanol. dna was extracted with different standard protocols and a fragment of the mitochondrial 16s rrna gene was amplified with the primers 16sa-l and 16sb-h of palumbi et al. (1991). after purification with qiaquick kits (qiagen), the fragments were resolved on an automated dna sequencers (abi 377 and abi 3100). sequences were validated and aligned with the software sequence navigator (applied biosystems) and deposited in genbank (accession numbers of newly obtained sequences ay834186ay834198) the alignment required inclusion of gaps to account for indels in only a few cases in one hypervariable region. data analysis was carried out with paup, version 4b10 (swofford, 2002). because of the low number of informative sites in scaphiophryne (vences et al. 2004), we refrained from performing phylogenetic analyses and report genetic divergences among sequences only. exploratory maximum parsimony and maximum likelihood analyses in all cases recovered paradoxophyla as highly supported monophyletic group, with the northeastern specimen placed as a sister to a clade containing the eastern specimens. altogether 103 sites were parsimony informative and 416 were constant. karyology the chromosomes were obtained from intestine, spleen, gonads and lungs with the air-drying method of odierna et al. (2001). we compared the karyology of one adult specimen of the new species from masoala (mrsn a2525) and one adult p. palmata from andasibe (mrsn a2518). besides conventional staining (5% giemsa at ph 7), the following techniques were applied: (1) ag-nor banding of the nucleolar organizer regions following howell and black (1980); (2) the c+g specific fluorochrome chromomycin a3 (cma3) /methylic green staining according to sahar & latt (1980) with exposure to the non-fluorescent dye, methyl green reduced to a few seconds; and (3) c-banding according to sumner (1972) by incubating the slides for 5 min at 45°c in ba(oh)2. good results were achieved by staining, either separately or sequentially, with cma3 and dapi after hydrolysis in ba(oh)2 (odierna et al., 2001). from each taxon, at least four giemsa-stained metaphases and two metaphases stained with each of the banding methods were studied. results description of paradoxophyla tiarano sp. nov. (figures 1-6) type series holotype mrsn a2498 (male) and paratypes mrsn a2499 and a2525, adult male and adult female, all from masoala peninsula, campsite 5 (locally known as menamalo18 f. andreone et alii na), antalaha fivondronana, antsiranana faritany (diégo suarez province), 15°22.87’s, 49°59.27’e, 780 m, leg. f. andreone, 10.xii.1999. additional material mrsn a2522, 70 tadpoles, with the same locality and collector. they are assigned to p. tiarano because due to its similarity to the tadpole of p. palmata, as shown by blommers-schlösser & blanc (1991) (see below). furthermore, the tadpoles show a typical microhylid morphology, which in the study area is not known to be shared by other frog species. diagnosis an aquatic paradoxophyla characterized by a small-medium size, dorso-ventrally flattened body, a sharply pointed and longish snout, small and circular eyes, fingers without evident expansions, toes slightly enlarged, absence of hand webbing and foot webbing nearly absent. fig. 1. paradoxophyla tiarano. the paratype mrsn a2499 in nature. menamalona forest, masoala peninsula, ne madagascar. 19a new paradoxophyla from ne madagascar ta bl e 1. m or ph om et ri c m ea su re m en ts ( to 0 .1 m m ) of s pe ci m en s of p ar ad ox op hy la t ia ra no s p. n ov . a nd p ar ad ox op hy la p al m at a. m = m al e, f = f em al e, h t = ho lo ty pe , p t = p ar at yp e. f or o th er a bb re vi at io ns s ee t he te xt . m us eu m a cr on ym a nd n um be r sp ec ie s pr ov en ie nc e se x r an k sv l h w h l ed en d n sd n n d h a l fo r l fo l t ib l m r sn a 24 98 p. ti ar an o m as oa la m h t 17 .5 5. 9 5. 5 1. 5 1. 8 1. 9 1. 6 4. 2 9. 7 12 .8 8. 8 m r sn a 24 99 p. ti ar an o m as oa la f pt 28 .1 9. 6 7. 9 1. 7 2. 1 2. 2 2. 3 7. 1 13 .2 20 .0 9. 9 m r sn a 25 25 p. ti ar an o m as oa la f pt 30 .7 8. 5 8. 3 1. 9 2. 2 1. 9 2. 3 6. 8 14 .5 19 .9 13 .9 m n h n 1 97 311 46 p. p al m at a a m ba na m h t 19 .9 6. 6 5. 8 1. 7 1. 9 1. 4 1. 8 6. 1 9. 6 11 .3 11 .9 m n h n 1 97 311 47 p. p al m at a a m ba na m pt 19 .5 6. 5 5. 8 1. 9 2. 1 1. 6 1. 9 6. 0 9. 8 11 .9 11 .7 m r sn a 25 18 p. p al m at a a nd as ib e m 22 .1 8. 3 6. 7 1. 7 2. 3 1. 5 1. 9 6. 3 12 .3 14 .7 11 .2 z fm k 5 27 63 p. p al m at a a nd as ib e m 20 .2 6. 9 7. 5 1. 7 1. 9 1. 3 1. 8 4. 5 11 .5 14 .5 12 .2 z fm k 5 27 64 p. p al m at a a nd as ib e m 19 .2 6. 8 6. 1 1. 6 2. 2 1. 5 1. 8 5. 1 11 .7 13 .9 10 .4 z fm k 6 00 09 p. p al m at a a nd as ib e m 21 .5 8. 4 7. 3 1. 6 2. 5 1. 6 2. 1 4. 6 11 .9 15 .9 11 .7 z sm 7 92 /2 00 3 p. p al m at a r an om af an a m 19 .6 6. 5 6. 4 1. 8 2. 1 1. 7 1. 7 6. 5 12 .4 16 .4 11 .9 m r sn a 25 27 p. p al m at a a nd as ib e f 24 .7 8. 1 7. 3 1. 8 2. 4 1. 5 2. 1 5. 6 13 .7 19 .8 15 .6 z fm k 5 27 61 p. p al m at a a nd as ib e f 22 .1 8. 7 8. 4 1. 7 1. 3 1. 5 1. 6 5. 5 13 .2 16 .8 12 .9 z fm k 5 27 62 p. p al m at a a nd as ib e f 25 .6 8. 3 7. 8 2. 5 2. 2 1. 1 1. 6 6. 3 13 .9 20 .8 13 .8 z fm k 6 22 16 p. p al m at a a nd as ib e f 22 .9 7. 1 7. 1 1. 9 2. 0 1. 7 1. 6 5. 6 10 .8 16 .6 12 .2 20 f. andreone et alii morphology measurements of the three adult specimen are given in table i. head almost as long as wide; snout longish, protruding beyond margin of lip; pointed in dorsal view and in lateral profile; end bigger than ed; end 27–33% of hl; eye small in size; ed 22–27% fig. 2. paradoxophyla tiarano. drawing of the male holotype (mrsn a2498), dorsal side (a), and ventral side (b) svl = 17.5 mm. fig. 3. paradoxophyla tiarano. drawing of the female paratype (mrsn a2499), dorsal side (a), ventral side (b). svl = 28.1 mm. a b a b 21a new paradoxophyla from ne madagascar of hl; upper eye lid smooth. top of head flat; canthus rostralis indistinct; internarial area not depressed; nostril circular; protruding laterally at a point above margin of lower jaw. slight supratympanic fold usually visible, tympanum not visible. choanae relatively small, round, separated, partially obscured by palatal shelf of maxillary arch. vomerine teeth present and posterior to choanae. tongue trapezoid in shape with rounded edges, widest at the free margin, no groove or notch, free behind for about two-thirds of its length. pupil circular. skin on dorsum of head, body, and limbs smooth in the female, and with rounded warts and tubercles in the male. ventral surfaces smooth. cloacal opening elliptical, unmodified. fingers moderate in length, with small, moderately sized discs; disc on first digit smaller than others: 1<2=4<3; subarticular tubercles flattened, ovoidal, not elevated; supernumerary tubercles absent; palmar tubercle semidistinct, not very elevated. the 3rd finger is thicker than the other three in both sexes. no webbing between digits of hands. feet without outer metatarsal tubercle; inner metatarsal tubercle present, although weakly evident. small enlarged pads on digits and toes, ovoid in shape. the feet have only traces of web. toe length: 1<2<5<3<4. colouration and variation after about five years of preservation, the type specimens show a colouration that is quite similar to that observed in life (fig. 1). the dorsal colour shades from light brown to tan, with small and irregular markings. legs have darker transverse bands. the belly is whitish in its central part, with a darker network and spots, which in some cases assume the aspect of a reticulation. the throat is darker than the surrounding areas. the two females (mrsn a2499 and a2525) are larger than male (mrsn a2498). tadpoles tadpoles assigned to p. tiarano correspond to the microhylid type 1, suspension feeder, sensu altig & johnston (1989), altig & mcdiarmid (1999), and mcdiarmid & altig (1999). they have a roundish body, a little bit longer than wide. the snout is flattened and quite horizontal, subcircular when seen from above, and rounded in profile. the eyes are distant and project dorsolaterally. the location of the closed external nares are dorsal, closer to snout tip than to eyes, and positioned in a slight light-coloured furrow. the ventral fin extends parallel to the tail musculature, the dorsal fin has a maximum height at about three-fourth to a third of the tail. the dorsal fin originates at the tail-body junction, and the ventral fin originates at the posteroventral terminus of the body. the spiracle opens ventrally and has a fringed border. the mouth is wide, with the marginal edges linearly arranged. the lower jaw projects upwards. the mouth is therefore visible in dorsal view. at stage 38 morphometric measurements are (data for a sample from mrsn a2618, given as means ± standard deviation): body length = 11.2 ± 1.4 mm (n = 20, range: 9.013.9), tal = 16.2 ± 2.3 mm (n = 17; 12.0-21.1); tmh = 2.6 ± 0.3 mm (n = 19; 2.0-3.5), th = 6.1 ± 0.9 mm (n = 15; 4.3-7.9), hw = 8.5 ± 0.8 mm (n = 20; 7.00-9.80). in life, the body of the tadpoles is transparent and translucent without a distinct colour, although the muscular part of the tail and the dorsal wall of the body are light brownish, shading to 22 f. andreone et alii pinkish (fig. 4). the belly is whitish, while the blackish intestines are seen through the ventral, lateral and dorsal body walls. the dorsal and ventral fins are transparent, but the margins are more opaque than the rest. in preservative the specimens maintained their natural colour pattern. etymology the specific epithet “tiarano” (pronounced: tee-how-row-noo) is a malagasy term composed by two words: “tia”, which means “love” and “to love,” “to like,” and “rano,” meaning “water.” the name means “that one loving the water,” and is used as a noun in apposition, underlining the aquatic habits of this frog. fig. 4. tadpole assigned to paradoxophyla tiarano derived from some individuals of the sampe mrsn a2166, dorsal (a), ventral (b), lateral (c) view. total length of about 28 mm. a b c 23a new paradoxophyla from ne madagascar natural history and distribution the adults and tadpoles were collected in a small pool (about 3.0 x 1.5 m; 1 m deep) a short distance from a forest stream. we believe that the adults carry out an aquatic life at least during the wet season (from november to april). tadpoles were found in december suggest that reproduction likely occurred some days or weeks before. we suppose that the distribution extends at least over most of the masoala rainforest, at least at low and midelevations. morphological comparisons with other species paradoxophyla tiarano appears externally similar to p. palmata. of course, comparisons are limited by the small number of available specimens. the new species has a body size 17-30 mm long, while the measured specimens of p. palmata ranged 19-26 mm. when compared to specimens of p. palmata, the male of p. tiarano appears a little bit wartier and with a somewhat darker colouration. the most important distinction between the two species is that in p. palmata the feet are extensively webbed, while the foot webbing is lacking or scarcely present in the new species. the foot webbing in p. palmata is usually given as “total” (see guibé, 1974; blommers-schlösser & blanc, 1991; glaw & vences, 1994). our comparative analyses of the specimens from ambana extended types), ranomafana and andasibe confirmed the webbing character and revealed the following webbing formula: table 2. genetic differentiation among species of paradoxophyla, scaphiophryne and dyscophus. the table shows pairwise divergences among sequences (535 base pairs) of the mitochondrial 16s rrna gene. above diagonal are total number of substitutions, below diagonal are uncorrected p-distances. the shaded parts of the matrix show intrageneric comparisons. 1 2 3 4 5 6 7 8 9 10 11 12 13 1 paradoxophyla tiarano sp. n. 41 41 56 53 53 66 56 56 54 78 78 81 2 p. palmata (andranomena) 7.8 5 60 61 57 67 65 62 58 74 74 74 3 p. palmata (ranomafana) 7.8 1.0 60 58 58 70 65 60 55 73 73 75 4 scaphiophryne marmorata 10.7 11.5 11.5 17 19 31 21 18 15 65 65 66 5 scaphiophryne boribory 10.1 11.6 11.1 3.2 21 30 20 13 9 63 63 63 6 scaphiophryne brevis 10.1 10.9 11.1 3.6 4.0 27 26 18 21 62 62 67 7 scaphiophryne calcarata 12.6 12.8 13.4 5.9 5.7 5.1 29 24 27 68 68 72 8 scaphiophryne gottlebei 10.7 12.4 12.4 4.0 3.8 5.0 5.5 15 19 72 72 72 9 scaphiophryne spinosa 10.7 11.8 11.5 3.4 2.5 3.4 4.6 2.9 12 65 65 67 10 scaphiophryne madagascariensis 10.3 11.1 10.5 2.9 1.7 4.0 5.2 3.6 2.3 65 65 65 11 dyscophus antongili 14.9 14.1 14.0 12.4 12.0 11.8 13.0 13.7 12.4 12.4 0 14 12 dyscophus guineti 14.9 14.1 14.0 12.4 12.0 11.8 13.0 13.7 12.4 12.4 0 14 13 dyscophus insularis 15.5 14.2 14.4 12.6 12.0 12.8 13.8 13.8 12.8 12.4 2.6 2.6 24 f. andreone et alii 1(0), 2i(1), 2e(0), 3i(1), 3e(1), 4i(1.5), 4e(2), 5(0). in p. tiarano the foot has only traces of webbing, both in the examined male or females (see fig. 5 for a better comprehension of this character). fig. 5. particular of the foot in paradoxophyla palmata (zfmk 60009) and p. tiarano (mrsn a2499). note the state of the webbing, present and complete in p. palmata (left), and rudimentary to absent in p. tiarano (right). fig. 6. karyotypes of p. palmata (a) and p. tiarano. (c), and relative metaphasic plates, with c-banding (b and d). the square borders include the pairs with nors. 25a new paradoxophyla from ne madagascar mitochondrial differentiation in the mitochondrial 16s rrna gene, the uncorrected pairwise dna sequence divergence of the paradoxophyla tiarano sequence to those of p. palmata from east-central and southeastern madagascar (7.8%, corresponding to 41 mutations) is about twice that observed between many species of scaphiophryne (tab. 2). such high divergences have so far not been found within species of malagasy amphibians, and the molecular data therefore corroborate the hypothesis of reproductive isolation and species status of paradoxophyla tiarano. karyological comparison both the species show a karyotype of 2n = 26 chromosomes, all biarmed and with the first five pairs much larger than the remaining 6-13 pairs. the pairs 2, 3, 10 and 11 are submetacentric, and the others are metacentric (fig. 6 a, c). loci of nors in both the species are close to the centromere on the short arm of the 2nd chromosome pair (fig. 6 a, c). the chromosomal formula and nor location are basal, as is typical for the subfamily scaphiopryninae and were found in 8 out of the 9 scaphiophryne species (unpublished data). in the anurans it is common to observe the nor location shared in several groups of closely related species (mahony & robison 1986, aprea et al. 2004). the two paradoxophyla species differ distinctly for the distribution of the heterochromatic material (fig. 6 b, d). in p. palmata centromeric c-bands are visible only on the centromeric regions of the 2nd chromosome pair, corresponding to the nor-associated heterochromatin. in p. palmata all the chromosomes have abundant telomeric heterochromatin, while in paradoxophyla tiarano the telomeric heterochromatin is scarce. the centromeric heterochromatin, beside the nucleolus-associated material in the 2nd pair, is present in all the chromosomes of 6-13 pairs. justification for the new species the difference in webbing between the two paradoxophyla species is remarkably diagnostic and indicates a substantial morphological split. furthermore, the dna differentiation of the northern paradoxophyla (7.8%, 41 mutations) to those from central, eastern and southeastern madagascar is about twice as great as that between many species of scaphiophryne (tab. 2). concerning the karyological aspects, there is a remarkable repartition of the heterochromatic component. the heterochromatin is mainly constituted by families of highly repeated dna. such a fraction of dna rapidly accumulates mutations and diversifies quickly during the evolutionary speciation processes (singer, 1982). c-banding staining carried out in different amphibian species, including the congeneric ones, shows karyotypes almost identical in number, shape and size. in all cases, the different species, and also the closely-related ones, showed differences in the distribution and/or composition of the cbands (morescalchi, 1983). thus, the differences found in the c-banding pattern between the two paradoxophyla species appear to be taxonomically relevant and support the morphological and molecular conclusion of distinctness of p. tiarano. 26 f. andreone et alii discussion the discovery of a new paradoxophyla in northeastern madagascar confirms that the herpetofauna of this area is still incompletely known and home of peculiar endemics (andreone, 2004). the application of two non-morphological techniques (karyology and mitochondrial dna analysis) also detected a relevant differentiation between the populations from northeastern and eastern madagascar. these differences are at the same level of those observed in other well-differentiated and undisputed species of microhylids, such as scaphiophryne and dyscophus (see tab. 2). this confirms the validity of these methods that have been used in previous taxonomic surveys of madagascan frogs (e.g., andreone et al., 2003). the morphology and colouration of the two paradoxophyla species appear similar and rather conservative, and the new species does not exhibit important and diagnostic morphological differences except for the webbing. we lack call recordings of the new species. although living within rainforest, paradoxophyla palmata shows an explosive reproductive behaviour, and we predict that this might also apply to p. tiarano. such a breeding strategy is typical for frogs from ephemeral waters in xeric areas, although it is also exhibited by species which frequent seasonal habitats. in general, these species are characterised by a great number of eggs. it might be questioned whether the explosive reproductive habitats is a plesiomorphic character or a phylogenetic apomorphy (as considered by blommers-schlösser & blanc, 1993). the habitat choice in paradoxophyla is considered as an adaptation to temporary pools in the forest. however, because the sister group of scaphiophrynines is not reliably known (van der meijden et al., 2004), phylogenetic data are currently insufficient and premature to speculate whether the presence of paradoxophyla may be a secondary adaptation. in terms of conservation, p. tiarano as well as p. palmata have so far only been found in aquatic environments within or very close to quite unaltered rainforests. therefore, they appear to be critically tied to mature rainforests and to high humidity levels, especially outside of the breeding season. given the fast deforestation rate in madagascar, the safeguard of as many fragments as possible of pristine rainforests is almost certainly the most effective strategy to conserve madagascar’s peculiar amphibian fauna. the presence of a large national park at masoala (angap, 2003) is indeed a favourable factor for the conservation of p. tiarano and the many described or yet undiscovered frogs inhabiting this large forest block. acknowledgements the research of f. andreone was made in cooperation between the mrsn and the parc botanique et zoologique de tsimbazaza, antananarivo, and supported by the wildlife conservation society, world wide fund for nature, gondwana conservation and research, and nando peretti foundation. we are grateful to j. e. randrianirina for field assistance, and to the malagasy authorities for collection and export permits. thanks also to w. böhme, a. dubois, f. glaw and a. ohler, who enabled us to study comparative material held in their care. e. olmo and an anonymous referee improved a first draft of this paper. r. altig contributed with very useful advice and corrections of the english style and information on the tadpole of paradoxophyla. 27a new paradoxophyla from ne madagascar literature cited altig, r. johnston g. r. 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(1983): i cromosomi nell’evoluzione e nella speciazione. il caso degli anfibi. ix seminario sulla evoluzione biologica e i grandi problemi della biologia. atti accad. naz. lincei 64: 63-109. odierna, g., vences, m., aprea, g., lötters, s., andreone, f. (2001): chromosome data for malagasy poison frogs (amphibia: ranidae: mantella) and their bearing on taxonomy and phylogeny. zool. sci. 18: 505-514. palumbi, s. r., martin, a., romano, s., mcmillan, w. o., stice, l., grabowski, g. (1991): the simple fool’s guide to pcr, version 2.0. privately published document compiled by s. palumbi. dept. zoology, univ. hawaii, honolulu. sahar, e., latt, s. a., (1980): energy transfer and binding competition between dyes used to enhance staining differentiation in metaphase chromosomes. chromosoma 79:1-28. singer, m.f. (1982): highly repeated sequences in mammalian genomes. int. rev. cytol. 76: 63-112. 28 f. andreone et alii swofford, d. l. (2002): paup*. phylogenetic analysis using parsimony (* and other methods), version 4. sinauer associates, sunderland, massachusets. van der meijden, a., vences, m., meyer, a. (2004): novel phylogenetic relationships of the enigmatic brevicipitine and scaphiophrynine toads as revealed by sequences from the nuclear rag-1 gene. proc. roy. soc. lond. b (suppl.) 271: 378-381. vences, m., andreone, f., glaw, f., kosuch j., meyer, a., schaefer, c., veith, m. (2002a): exploring the potential of life-history key innovation: brook breeding in the radiation of the malagasy treefrog genus boophis. mol. ecol. 11: 1453-1463. vences, m., aprea, g., capriglione, t., andreone, f., odierna, g. (2002b): ancient tetraploidy and slow molecular evolution in scaphiophryne: ecological correlates of speciation mode in malagasy relict amphibians. chromos. res. 10 (2):127-136. vences, m., raxworthy, c.j., nussbaum, r.a., glaw, f. (2003): a revision of the scaphiophryne marmorata complex of marbled toads from madagascar, including the description of a new species. herpetol.j. 13: 69-79. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 7(2): 203-219, 2012 amphibians in southern apennine: distribution, ecology and conservation notes in the “appennino lucano, val d’agri e lagonegrese” national park (southern italy) antonio romano1,*, remo bartolomei1, antonio luca conte1, egidio fulco2 1 wwf italia, via po 25/c, 80136 roma, italy. *corresponding author. e-mail: antonioromano71@ gmail.com 2 studio naturalistico milvus, via f.lli perito snc, 85010 pignola submitted on: 2012, 17th january; revised on: 2012, 6th march; accepted on: 2012, 23rd april. abstract. italy is the european country with the highest amphibian richness and endemism. however distributional data from some southern italy areas are scanty, in particularly for the basilicata region. in this study, we present the results of field and bibliographic survey on the amphibians of the “appennino lucano, val d’agri e lagonegrese” national park (almost 70,000 ha). we recorded breeding activity of 12 amphibian species in 307 sites, for a total of 493 records. for some endemic species we provide new ecological data, such as new altitudinal limit (salamandrina terdigitata) or expansion of the annual activity cycle (bombina pachypus). indices of diffusion, density and rarity were applied to test the status of each species in the park. correspondence analyses showed a clear aquatic habitat partitioning between anurans and urodelans and, concerning the latter, between newts and salamanders, newts being strictly dependent on artificial water bodies. our results support the growing idea, recently formalized by the iucn, that maintaining and restoring artificial water bodies may be fundamental for an appropriate conservation management of amphibian communities in mediterranean rural landscapes. keywords. amphibians, artificial water bodies, basilicata, correspondence analyses, distribution, habitat partitioning. introduction amphibians are the vertebrate group experiencing the steeper global population decline and species loss (stuart et al., 2004; gascon et al., 2007). baseline data are urgently needed about causes and magnitude of species loss and population declines for effective biodiversity monitoring programs. italy has the highest amphibian biodiversity in europe hosting 43 species, of which 15 endemics. southern italy and sardinia are the major italian hotspots of 204 antonio romano et al. herpetological endemicity (sindaco et al., 2006), even if they were the less investigated areas during the herpetological surveys that leaded to the publication of the italian atlas (sindaco et al., 2006). in this context, basilicata is the least investigated region of southern italy, and probably of the entire country (see figures 3.4, 3.5 and 3.6 in sindaco et al., 2006). in december 2007, the youngest italian national park, “parco nazionale dell’appennino lucano, val d’agri e lagonegrese” (pnalval, fig.1a) was established in basilicata. this protected area lies between 15,584° and 16,152° n 40,454° and 40,273° e covering 68.996 hectares. the park covers 26 utm 10x10 km squares, but 11 of them just marginally (i.e., less than 10% of utm square included in pnalval; fig.1a), and comprises 12 sites of community importance (sci). the altitude ranges from about 290 m a.s.l. to 2005 m (monte papa, province of potenza). the low altitude areas are covered by mesophilous and thermophilous mixed-oak woods (quercus cerris, q. pubescens, q. frainetto, fraxinus ornus, carpinus orientalis) while beech (fagus sylvatica) forests, interspersed with manmade grassland and pastures, are dominant on the mountain tops. along streams, rivers and valley bottoms the riparian vegetation is dominated by poplars (populus nigra, p. alba), alders (alnus glutinosa, a. cordata) and willows (salix alba, s. purpurea). typical mediterranean marquis vegetation dominated by the holm oak (quercus ilex) and arbutus unedo, pistacia lentiscus, phillyrea latifoglia occurs in the “murgia san lorenzo”, the easternmost portion of the park (aita et al., 1974; corbetta and pirone, 1996; fascetti, 1996; costantini et al., 2006). the more widespread lithological substrate is given by carbonate units, that gives rise to karstic phenomena, and in particular to underground waterflows. therefore surface hydrology is reduced and characterized by temporary running and still waters. man-made artificial water reservoirs and water bodies are also present. since there were no baseline data on amphibians, in 2011 a project on their distribution and conservation status was funded by the park administration. the specific objectives of this study were to describe the amphibian richness and distribution, their use of aquatic habitats and to asses the main potential threats for conservation. material and methods data collection the presence of preserved specimens from basilicata was verified in the collection catalogues of the following italian natural history museums: museo civico di storia naturale of carmagnola (turin), museo civico di storia naturale of genoa, museo civico di zoologia of rome, museo di storia naturale of naples, museo civico di storia naturale of trieste, museo di storia naturale di florence, museo naturalistico of alburni mountains (corleto monforte, salerno). we also checked in the distribution database available in the ckmap 5.3.8 software of the italian ministry of environment, land and sea (stoch, 2000-2005). field surveys were preceded by a careful analysis of the maps produced by the istituto geografico militare (i.g.m, 1:25000), in which specific cartographic symbols refer to different typologies of water bodies (springs, running waters, tanks, drinking-troughs, wells, lakes, marshes; see capello, 1968). moreover, many hydronyms (in italian or in local dialect), allow accurate identification of many wetlands. i.g.m. cartographic recognition were implemented by digital orthophotos (scaled aerial photographs) obtained from of the italian environmental ministry (http://www.pcn.minambi205amphibians of the appennino lucano national park fig. 1. a. location of the “appennino lucano, val d’agri e lagonegrese” national park, southern italy (pnalval, grey area), and distribution of amphibians historical data (see appendix 1 for the correspondence between sites (numerical codes) and species). b-h. distribution of anura in the pnalval. utm grid (10x10 km) with the alphanumeric code of squares is also shown.   synkl. 206 antonio romano et al. ente.it/gn/). field surveys were carried out systematically from february to september 2011 (about 5 field sampling days every 7 days for a total of 120 field days) although scattered observations were also obtained from january to october 2009-2010. utm squares included for more than 10% in the park were visited 2-10 times. sampling effort per square was roughly proportional to the number of water points individuated by the cartographic recognition. as a general rule, amphibian low-altitude populations breed early than higher-altitude ones (wells, 2007 and reference therein; lanza et al., 2007). consequently, sampling efforts were concentrated in late winter and early spring in lowland sites, and in late spring and summer at higher altitudes. to evaluate species presence, aquatic sites were sampled by twenty dip-nettings, including blind ones (i.e., without previous visual detection of amphibians), although sampling effort was proportional to the complexity or morphology of the habitat s (i.e., deep water, dense aquatic vegetation). road casualties and terrestrial shelters in proximity of aquatic sites were always checked. calling surveys (cs) started 30-min after sunset and were usually completed by 01:00 h (dorcas et al., 2009). call survey lasted five minutes, with three minutes of preadaptation before the beginning. when hyla intermedia and pelophylax synkl. hispanicus were not detected within five min, we used an acoustic digital (mp3 format) playback stimulus, rate 1 call/20 sec, over an additional 3-minute period to induce call frogs. each calling species was given a ranked score, known as index of calling surveys (ics; as for the north american amphibian monitoring program; see mossman et al., 1998; weir and mossman, 2005): 0 = no anurans calling; 1 = not overlapping individual calls; 2 = calls overlapping, but individuals still distinguishable; 3 = numerous anurans heard, chorus constant and calls overlapping. in few cases we performed visual encounter surveys (ves, following heyer et al., 1994). because ves and other unrepeated visual contacts are not appropriate method for determining population densities we did not perform demographic analyses. however for populations who had high numbers of individuals (n > 20) detected during one single survey, we have noted their number to provide a minimum size of the those populations. moreover, since rana dalmatina is an explosive breeder (sofianidou and kyriakopoulou-sklavounou, 1983; guarino and bellini, 1993) and each female lays a single egg mass per season, usually fixed to the substrate (nollert and nollert, 1992), we used the counts of rana dalmatina egg mass as proxies of the minimum female population size (counting were performed in a unique sampling session), according to the methods proposed by griffiths and raper (1994) and grossenbacher et al. (2002). among the r. dalmatina breeding sites we found, we chose those where populations seemed particularly large and the site typology allowed easy location of the egg masses (i.e., shallow clear waters). aquatic sites were assigned, considering their origin, to two main categories (i.e., artificial and natural) each including four different typologies: (i) tanks: concrete quadrangular tanks used for agricultural purposes, (ii) drinking-troughs for livestock grazing, (iii) wells: circular stony wells; (iv) artificial ponds; (v) lakes: lakes and marshes; (vi) running waters: rivers, streams and creeks; (vii) springs, (viii) natural ponds: natural ponds, pools and puddles. because several sites were very close to each other, two or more aquatic habitats less than 50 meters apart belonging to the same category (e.g., well, pond and so on) were considered as a single breeding site, following the criterion used by romano et al. (2007, 2010). phenological data on the aquatic activity of each species were recorded. however on the basis of data recorded on the field, additional information was inferred (for example, if eggs in a advanced stage of development were found at the beginning of a given month, we assumed adults were in water in the previous month, see table 1). data analysis two indices describing species diffusion (w: wide; m: medium; l: limited) and density (c: common; f: frequent; r: rare) are provided; they are obtained from the graphs of the relationship 207amphibians of the appennino lucano national park between the coverage (%) of the utm grid (10x10 km) and the mean number of observations for a square occupied by each species, according to the method proposed by doria and salvidio (1994) and already used in other herpetological studies (turrisi and vaccaro, 2004). furthermore, for each species we calculated an index of specific rarity expressed as percentage: isr = (1-n/n)*100 (gheu and gheu, 1980), where n is the number of utm squares occupied by a given species and n is the total number of utm squares. we used as n only utm squares occupied by more than 10% of the park area (i.e., n = 23). isr ranges from 0 (very common species) to nearly 100 (very rare species). to identify associations of aquatic habitats with amphibian presence/absence we used a correspondence analysis (coa). differences in number of amphibian species among the different habitat typologies were examined using kruskal-wallis (kw) non-parametric analysis of variance. coa and kw were performed as in the statistical package past (hammer et al., 2001). finally, to determine if our effort provided an exhaustive representation of the amphibian assemblage in the different freshwater typologies, the non-parametric estimator of species richness chao2 (chao, 1987), that controls for the effect of sampling effort (walther and morand 1998; hortal et al., 2006), was implemented with program estimates version 7.5.2 (colwell, 2004). results the literature reported only eight species within the park boundaries (see appendix 1): salamandrina terdigitata (bonnaterre, 1789), triturus carnifex (laurenti, 1768), lissotriton italicus (peracca, 1898), bombina pachypus (bonaparte, 1838), bufo bufo (linnaeus, 1758), hyla intermedia boulenger, 1882, rana italica dubois, 1987, pelophylax synkl. hispanicus (bonaparte, 1839). the water frog synklepton is formed by two entities which are considered both at species rank (but see also canestrelli and nascetti, 2008): the parental species, p. bergeri (gunther, 1985) and its hemiclonal hybrid, the klepton p. kl. hispanicus. all these species presented a extremely localized distribution (they were found mainly in laudemio lake or on monte sirino; see fig. 1 and appendix 1). our investigations confirmed the occurrence of these species and provided first data on the presence of other 4 species: salamandra salamandra (linnaeus, 1758), bufo balearicus boettger, 1880 and rana dalmatina fitzinger in bonaparte, 1838. almost five hundred amphibian records were collected during the field survey (493); breeding activity was recorded in 307 sites from 23 utm squares, corresponding to 80% of the surveyed potential spawning sites. species distributions and number of breeding sites of each species in the pnalval are given in fig. 1 and fig. 2, while fig. 3 shows the altitudinal range, the aquatic site preferences and the index of species rarity (isr) of each species. indices of relative diffusion and density (fig. 4b) showed that different species ranged from “widespread” to “very common” (upper right corner of fig.4b and lower values of isr in fig.3), while some species had “limited diffusion” (lower left corner of the fig. 4b and higher value of isr, fig. 3). aquatic phenology is summarized in table 1: activity ranged from january to october with marked differences among species. the index of calling surveys (ics), a very rough estimate of population size, was applied on some anuran species and provided the following values: bombina pachypus, bufo balearicus, pelophylax synkl. hispanicus (ics from 1 to 2); hyla intermedia (ics from 1 to 3). estimates of minimum female population size of rana dalmatina in three sites were 42, 58 and 85 females. 208 antonio romano et al. water bodies of different typology hosted different number of species: tanks (n = 38, mean of species richness ± sd = 1.74 ± 0.98), drinking-troughs (n = 53, 1.49 ± 1.77), wells (n = 5, 1.60 ± 0.55), artificial ponds (n = 30, 2.23 ± 1.25), lakes (n = 16, 2.62 ± 1.86), running waters (n = 112, 1.32 ± 0.67), springs (n = 11, 2.36 ± 1.28), natural ponds (n = 46, 1.54 ± 0.78). the number of species among these height habitat typologies was significantly different (kw, h = 26.97, p < 0.001). cumulated species richness values summed across all water bodies of a given typology ranged from 3 to 10 (fig. 4a). two different levels of species richness among habitats could be detected. a low level grouped artificial ponds and wells while an intermediary-high level grouped the remaining typologies, where running waters and drinking-troughs emerged as the richest typologies (fig. 4a). estimated fig. 2. distribution of urodela in the “appennino lucano, val d’agri e lagonegrese” national park (southern italy). utm grid (10x10 km) with the alphanumeric code of squares is also shown.   209amphibians of the appennino lucano national park and observed cumulated numbers of species were concordant and, for some habitat, the observed species slightly outnumbered of the median of the estimated ones (fig. 4a). this suggests that observed richness provides a reliable estimate of the actual richness. fig. 3 altitudinal ranges (box plots; altitudinal level on the right vertical axe), habitat partitioning (histograms, number of sites on the left vertical axe) and index of species rarity (isr, black dots, values on the left vertical axe) of the amphibians in the “appennino lucano, val d’agri e lagonegrese” national park (southern italy). boxes represent the 25-75 percent quartiles of altitudinal range and the median altitude is shown with a horizontal line inside the box; the minimal and maximal values are shown with short horizontal lines (whiskers). horizontal arrows indicate the altitudinal limit of the park. asterisk indicates the species for which the maximum altitude record in the park is also the upper limit for the species in its whole distribution range. in the isr, low value indicates common species, high value the rare ones. salsal = salamandra salamandra; salter = salamandrina terdigitata; tricar = triturus carnifex; lisita = lissotriton italicus; bompac = bombina pachypus; bufbuf = bufo bufo; bufbal = bufo balearicus; hylint = hyla intermedia; randal = rana dalmatina; ranita = rana italica; pelshis = pelophylax synkl. hispanicus.   210 antonio romano et al. fig. 4. a. comparison of amphibian species richness values among the aquatic habitat typologies in “appennino lucano, val d’agri e lagonegrese” national park (southern italy), based on empirical data (field richness, black dots) and estimated richness values (chao2 estimator, box plot; see text for details). for each aquatic site typology the 25-75 percent quartiles are drawn using a box and the median value is shown with a horizontal line inside the box; the minimal and maximal values are shown with short horizontal lines (whiskers). b. relationship between percentage of amphibian species occurrence in utm square grids (10x10 km) and mean number of observations per utm square. black dots: urodelan species; grey dots: anuran species. codes of species are as reported in fig. 3.   211amphibians of the appennino lucano national park table 1. aquatic phenology of amphibian species in the “appennino lucano, val d’agri e lagonegrese” national park (southern italy). for newts which carefully wrap their eggs in leaves of aquatic plants making them very difficult to see, the egg stage was not recorded. neotenic stage of newts, living the whole year in water body, was not considered. black color: recorded data. grey color: inferred data. species stage months j f m a m j j a s o n d salamandra salamandra adult larvae salamandrina terdigitata adult egg larvae triturus carnifex adult eggs larvae lissotriton italicus adults eggs larvae bufo bufo adult eggs larvae bufo balearicus adult eggs larvae bombina pachypus adult eggs larvae hyla intermedia adult eggs larvae rana dalmatina adult eggs larvae rana italica adult eggs larvae pelophylax synkl. hispanicus adult eggs 212 antonio romano et al. on the two dimensional coa scatter plot (fig. 5) the first two axes explained 80% of the variation in amphibian species composition among habitats. as expected there was a high associations between species and aquatic site categories. urodelans are clearly separated in two groups, the newts (left lower quadrant) associated to artificial habitats and the salamanders (sensu titus and larson, 1995; right upper quadrant) associated to natural running waters. also anurans shows two groups distributed along the first axis, with species associated to lentic waters (left upper quadrant) and others to running waters (right upper quadrant). bombina pachypus and rana italica shows an intermediate association with artificial water bodies and natural ones. fig. 5. correspondence analysis (coa) scatter plot illustrating variations of species distribution with aquatic breeding sites. triangles: artificial water bodies; squares: natural water bodies; black dots: urodelan species; grey dots: anuran species. the percentages of variation explained by each axis are given in round brackets. codes of species are as reported in fig. 3.   213amphibians of the appennino lucano national park discussion distribution species distribution data, together with information on their ecology and environmental contexts, are essential to investigate global population declines and to assess the threat status of a given taxon (see the criteria used by the iucn for the species assessment process). the pnalval has a rich amphibian fauna because all those expected to be present (12 species), on the bases of ecology and biogeography, were found during the intensive survey. the results give insights on distribution pattern and aquatic habitat partitioning of amphibians (fig. 4a). within the pnalval, two species are rare, t. carnifex with medium diffusion (figs. 3, 4b) and b. balearicus with a limited diffusion (figs. 1d, 3, 4b), possibly because of the rarity of clay or sandy soils at high altitude (sindaco et al., 2006). some other species, even if with limited diffusion, are locally frequent (e.g., b. pachypus and s. salamandra). the distributive gap of s. salamandra, s. terdigitata and b. pachypus (in the northern part of the park, figs. 2a, 2b, 1b) could be partially filled by further research, because many suitable habitats still have to be intensively sampled in the park. moreover these are species that probably have low probability of being detected when breeding at low densities (tanadini and schmidt, 2011) about the hypothetical presence of the alpine newt ichthyosaura alpestris (laurenti, 1768) in basilicata, its presence in mount sirino lakes, that fall within the boundaries of the pnalval, was reported in the past (bruno, 1996), but the species is now considered absent from the region (sindaco et al., 2006). the alpine newt has a very sparse distribution in southern apennine (sindaco et al., 2006) with relict populations in calabria (60 km southward from mount sirino) and latium (330 km northward from m. sirino). the populations from calabria occur in small glacial lakes, ecologically similar to those in monte sirino. however in these lakes we found other amphibians species but the alpine newt. consequently, the possible occurrence of a relict population of alpine newt on the sirino massif, in our opinion, have to be almost certainty excluded ecology considering the diversity of breeding sites and their altitudinal range (fig. 3), l. italicus, b. bufo and r. italica can be classified as euryecious species, while s. salamandra is a strictly stenoecious species. the remaining species are moderately euryecious, sometime showing opposite trends, in particular in the increased or decreased frequency related to altitude. as expected, there is a strong association between species and aquatic site categories. clearly, the first axis of the coa scatter plot (fig. 5) represents differences in waterflow intensity while the second axis roughly expresses differences in size and possibly in temperature of water bodies. true salamanders (sensu titus and larson, 1995) with stream-type larvae (griffiths 1996, pough et al. 2001) were strictly associated to natural sites characterized by running waters, where also the two anurans bufo bufo and rana italica often spawn. newts (sensu titus and larson, 1995) with pond-type larvae, were found in strict association with man-made aquatic sites characterized by low intensity of 214 antonio romano et al. water flow. artificial aquatic sites also play a role, in addition to natural lentic waters, as breeding habitat for bombina pachypus (figs. 3, 5). other anurans are strictly associated with lentic natural waters (figs. 3, 5). during this study, we found the highest breeding site (1525 m a.s.l.) known for s. terdigitata. the biological cycle and the activity patterns of s. terdigitata are poorly known and are often derived from those of the sister species s. perspicillata (angelini et al., 2007). in pnalval salamandrina terdigitata breeds in different categories of sites, both natural and artificial (fig. 3, 6). the earliest spawning was recorded in early march 2011 and females entered in water in the late february (table 1). however the breeding peak was in may and decreased until the june, when it apparently stopped. abundant populations, with dozens of females simultaneously in water for spawning, were recorded in spring in the catchment of the agri river, in small mediterranean streams. bombina pachypus, that in basilicata reaches its altitudinal limit (1710 m a.s.l. in this study, 1930 m a.s.l. by talarico et al., 2004) is active from april to october (sindaco et al., 2006). however in other sites at low altitude, b. pachypus was found in water earlier, already at the beginning of march (table 3) but possibly also in february as observed for other populations near the park boundaries (ef unpublished data). bufo balearicus has been found only along the agri river, but populations were relatively large (ics = 2) and produced thousands of tadpoles and hundreds of neo-metamorphosed in late summer. triturus carnifex had a medium diffusion (fig. 4b) but homogenous distribution (fig. 2d). almost all of its aquatic habitats were near-permanent (sensu boulton and brock, 1999) or permanent and moderately deep (1-3 m) or deep (depth > 3 m) artificial sites (figs. 3, 6). our data only partially agree with the findings of andreone and marconi (2006), who considered plains and moderately elevated areas as the most suitable habitats for this species. however their results are primarily mainly on observation in central and northern italy. the elevation range of the t. carnifex breeding sites in pnalval (fig. 3), showed that in southern italy this species prefer hilly and mountainous areas as already stated (talarico et al., 2004; romano et al., 2010). in the whole, we suppose that the occurrence of many ecologically diverse aquatic sites and the wide altitudinal range (300-1400 m a.s.l.) in utm squares we85 and we95 can be responsible of the concentration of amphibian findings in these utm (fig. 1b, 2b), also of stenozonal and moderately euryzonal species. conservation number of syntopic species and species richness among the different breeding habitat types showed a reverse pattern. lakes and artificial ponds had the highest level of syntopy but they hosted a restricted variety of species. conversely, running waters and drinkingtroughs, with a low degree of syntopy, were the habitats hosting the highest amphibian biodiversity. how and where to invest limited resources to achieve effective conservation of the biodiversity is often a difficult but obligatory choice. knowing when which habitat provide a differential contribution to the maintenance of biodiversity is an essential information for implementing effective conservation strategies. in general, animal conservation may be regarded at two different levels: conservation of rare species at local level and conservation of populations that deserve particular 215amphibians of the appennino lucano national park attention due to the situation of their species at global level (wells et al., 2010). these two levels may overlap or not, being both taxonand local distribution-dependent. the familiar slogan “think globally, act locally”, that has been used in various contexts, fits perfectly with an appropriate strategy for the amphibian conservation (gascon et al., 2007) that, to be effective, must take place synergistically at all levels from local to global (andreone, 2008). in pnalval the area with the highest amphibian richness is the upper and middle section of the agri valley. this area maintains a continuous and well preserved forest cover, that has not been exploited recently and may be considered the most relevant for amphibian conservation. however some parts of the valley are experiencing desertification and land abandonment, and the potential effects of global warming now threaten other parts of the park (geeson et al., 2002) which are highly exposed to desertification risk (povellato and ferraretto, 2005; brandt and geeson, 2011). although bufo balearicus is not an endangered species in the core of its italian distribution range (it is considered as least concern by the iucn; sindaco et al., 2009), it is the rarest specie in the study area and the preservation of these populations plays a important role in the conservation of amphibians biodiversity for this protected area. conversely, bombina pachypus, with relatively healthy populations in the park, is considered endangered by the iucn at species level (andreone et al., 2009), with dramatic declines in many areas and populations (guarino et al., 2007 and references therein). therefore conservation of pnalval populations transcends the local interest and assumes a key role in the conservation of the species at a global level. the conservation goals could be better achieved through conservation projects such as creating ecological corridors (even in areas outside the park) and new potential breeding sites. in fact, the protection, restoration and increase of aquatic sites is an essential tool in the amphibians conservation strategy. in the pnalval almost all amphibians use traditional artificial aquatic habitats as breeding sites (fig. 3). for some species (t. carnifex, l. italicus and b. pachypus), the knowledge of the association with man-made aquatic sites (fig. 5) is a basic tool for any sustainable and effective conservation strategy planning. in italy, rural artificial water sites are often neglected (see also romano et al., 2010) in the context of the abandonment of agricultural land and of traditional silvo-pastoral practices in european mountainous areas (torta et al., 2004 and references therein; denoël and ficetola, 2008; curado et al., 2011). although the basilicata region retains a widespread agricultural and silvo-pastoral vocation (fucella et al., 2010; psr, 2007), about 20% of the artificial or semi-natural water bodies we censused were destroyed or abandoned, and therefore unusable for amphibians reproduction. therefore, restoring these sites will contribute to maintain the traditional rural landscape, will give socio-economic benefits to local stakeholders and will also enhance the conservation status of a rich amphibian fauna. indeed, the mediterranean basin, and in particular in southern italy, preservation and management of artificial aquatic sites used for rural purposes is a prerequisite for effective conservation measures of amphibian populations (temple and cox, 2009 and reference therein). we provide here strong evidence in support the hypothesis that conservation of biodiversity in mediterranean must necessarily pass through the proper planning of rural development and landscape management. 216 antonio romano et al. acknowledgements the research was supported by a grant from “parco nazionale dell’appennino lucano, val d’agri e lagonegrese”, through an agreement between the park and wwf (d.d. 351 of 22 november 2010). the project has been carried out under the patronage of societas herpetologica italica. drawings of s. salamandra and l. italicus were kindly provided by luigi corsetti. that of s. terdigitata is original by ar, all other species drawings are of d.w. ovenden which were published on the field guide “reptile and amphibians of britain and europe” (harpercollins). annamaria nistri kindly provided data on specimens preserved in the museo zoologico de la specola (university of firenze). caterina coppola, silvia sgrosso, giuseppe priore provided useful data on breeding sites and contributed to the field researches. s. sgrosso and g. priore also provided kindly hospitality to ar. we are indebted with sebastiano salvidio for the critical reading and the improvement of the ms. supplementary material supplementary material associated with this article can be found at: <www.unipv.it/webshi/ appendix>. manuscript number 10503: appendix 1. references aita, l., corbetta f., orsino f. 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(2004). status and conservation of herpetofauna from the iblean area (south eastern sicily). ital. j. zool. 71 (suppl. 2): 185-189. walther, b.a., morand, s. (1998): comparative performance of species richness estimation methods. parasitology 116: 395-405. weir, l.a., mossman, m.j. (2005): north american amphibian monitoring program (naamp). in: amphibian declines: conservation status of united states species, pp. 307–313. lanoo, m.j., ed, university of california press, berkeley. wells, k.d. (2007): the ecology and behavior of amphibians. chicago university press, chicago. wells, j.v., robertson, b., rosenberg, k.v., mehlman, d.w. (2010): global versus local conservation focus of u.s. state agency endangered bird species lists. plos one 5: e8608. doi:10.1371/journal.pone.0008608 acta herpetologica vol. 7, n. 2 december 2012 firenze university press advertisement call of species of the genus frostius cannatella 1986 (anura: bufonidae) flora a. juncá1, david l. röhr2, ricardo lourenço-de-moraes3, flávio j. m. santos1, airan s. protázio1, ednei a. mercês1, mirco solé4 amphibians in southern apennine: distribution, ecology and conservation notes in the “appennino lucano, val d’agri e lagonegrese” national park (southern italy). antonio romano1,*, remo bartolomei1, antonio luca conte1, egidio fulco2 the significance of using satellite imagery data only in ecological niche modelling of iberian herps neftalí sillero1, josé c. brito2, santiago martín-alfageme3, eduardo garcía-meléndez4, a.g. toxopeus5, andrew skidmore5 reproductive strategy of male and female eastern spiny lizards sceloporus spinosus (squamata: phrynosomatidae) from a region of the chihuahuan desert, méxico aurelio ramírez-bautista1,*, barry p. stephenson2, xóchitl hernández-ibarra1, uriel hernández-salinas1, raciel cruz-elizalde1, abraham lozano1, and geoffrey r. smith3 morphological variability of the hermann’s tortoise (testudo hermanni) in the central balkans katarina ljubisavljević1, georg džukić1, tanja d. vukov1, miloš l. kalezić1,2 the usefulness of mesocosms for ecotoxicity testing with lacertid lizards maria josé amaral1,2,*, rita c. bicho1, miguel a. carretero2, juan c. sanchez-hernandez3, augusto m. r. faustino4, amadeu m. v. m. soares1, reinier m. mann1,5 does acclimation at higher temperatures affect the locomotor performance of one of the southernmost reptiles in the world? jimena b. fernández*, nora r. ibargüengoytía advertisement call of scinax littoralis and s. angrensis (amphibia: anura: hylidae), with notes on the reproductive activity of s. littoralis michel v. garey1, thais r. n. costa2, andré m. x. de lima2, luís f. toledo3, marília t. hartmann4 book review: marine s. arakelyan, felix d. danielyan, claudia corti, roberto sindaco, alan e. leviton 2011. herpetofauna of armenia and nagorno-karabakh. society for the study of amphibians and reptiles stefano scali book review: rafaqat masroor 2012. a contribution to the herpetofauna of northern pakistan stefano scali evidence of high longevity in an island lacertid, teira dugesii (milne-edwards, 1829). first data on wild specimens. j. jesus first assessment of the endoparasitic nematode fauna of four psammophilous species of tropiduridae (squamata: iguania) endemic to north-eastern brazil markus lambertz1,*, tiana kohlsdorf2, steven f. perry1, robson waldemar ávila3, reinaldo josé da silva4 rediscovery and redescription of the holotype of lygosoma vittigerum (= lipinia vittigera) boulenger, 1894 yannick bucklitsch1, peter geissler1, timo hartmann1, giuliano doria2 , andré koch1,* reproductive phenology of the tomato frog, dyscophus antongili, in an urban pond of madagascar’s east coast ori segev1,*, franco andreone2, roberta pala2, giulia tessa2, miguel vences1 range extension of the critically endangered true poison-dart frog, phyllobates terribilis (anura: dendrobatidae), in western colombia roberto márquez1,*, germán corredor2, carlos galvis3 daniel góez2, & adolfo amézquita1 differences in habitat use of two sympatric species of ameiva in east costa rica esther sebastián-gonzález1, ramón gómez2 acta herpetologica journal of the societas herpetologica italica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 6(1): 19-25, 2011 body size and reproductive characteristics of paedomorphic and metamorphic individuals of the northern banded newt (ommatotriton ophryticus) eyup başkale1, ferah sayım2, uğur kaya2 1 pamukkale university, faculty of science & arts, department of biology, 20017 denizli, turkey. corresponding author. email: ebaskale@pau.edu.tr 2 ege university, science faculty, section of biology, department of zoology, bornova-i̇zmir, 35100 turkey. submitted on: 2010, 10th july; revised on: 2011, 9th march; accepted on: 2011, 21st march. abstract. paedomorphs and metamorphs of the northern banded newt (ommatotriton ophryticus) were compared with respect to body size and some reproductive traits. paedomorphic females are smaller than metamorphic individuals of the same sex. both morphs have a similar reproduction pathway in terms of egg number and incubation period, while hatching success and egg diameter appear to be different between morphs. survival rates of two morphs were significantly different and calculated as 66% for paedomorphs, and 81% for metamorphs. on the other hand, both forms shared a common allometric slope of the svl vs. number of eggs and egg diameter, and larger females tend to produce more and larger eggs. keywords. facultative paedomorphosis, reproductive output, fecundity, body size. in many urodele species, including european newts (especially in mesotriton sp., lissotriton spp.) there are populations with alternative life-history pathways: metamorphosis vs. paedomorphosis (e.g. duellman and trueb, 1994). most larvae transform into immature individuals which remain more or less terrestrial before reaching sexual maturity; however, some larvae attain sexual maturity with larval morphology. such a discrete life-history polymorphism has both genetic and environmental components, and is usually viewed as an adaptation to contrasted environments in time and space (e.g. denoël et al., 2005). environmental conditions causing paedomorphosis are multifactorial and vary among species, but are often related to unsuitable terrestrial conditions, such that an aquatic lifestyle would be more beneficial. paedomorphic forms of northern banded newt ommatotriton ophryticus (berthold, 1846) were described in 2008 by kaya et al. (2008) near karasu in the vicinity of sakarya, turkey. this species ranges from the western caucasus in southern russia and georgia, mailto:ebaskale@pau.edu.tr 20 e. başkale, f. sayım and u. kaya through northwestern armenia and northern turkey west to the bosphorus strait. it has been recorded from near sea level (tabbaria lake) up to around 2,750m asl (in turkey). it is found in coniferous, mixed and deciduous forests up to sub-alpine meadows. reproduction of this species occurs in lakes, ponds, large puddles, drainage canals, roadside ditches in meadows, slow-flowing streams and stream pools in open areas near or within forests. o. ophryticus is listed as near threatened (nt) by iucn (2009). for conserving any target species, firstly, we have to understand its life-history. although, breeding biology is an important part of life-histories, it has not been well documented for o. ophryticus until now. for this reason, we aimed to describe some aspects of the reproductive biology of paedomorphic and metamorphic o. ophryticus specimens by quantifying clutch size, egg size, hatching success and female size-reproductive output relationships. location of the study site and its characteristics were described in kaya et al. (2008). to collect paedomorphic individuals, the field studies were conducted between late february and early march. these months were previously reported as the breeding season of o. ophryticus (andren, 1997; kutrup et al., 2005). both forms were monitored for three years (2007-2009), and each year they were sampled at least three times. individuals were captured by dip nets. even though, a total of 146 individuals were captured in 2007, there were no paedomorphic individuals. in 2008, 191 individuals were captured of which seven paedomorphic specimens, while in 2009 of 280 captured individuals six were paedomorphic individuals. capture probability of paedomorphic individuals was estimated as 0 in 2007, 0.04 in 2008 and 0.02 in 2009 breeding seasons for this population. all captured paedomorphic individuals and randomly selected metamorphic newts were transported alive to our amphibia laboratory in covered plastic boxes. to compare reproductive output, 21 pairs of metamorphic females and males (16 pairs in 2008 and five pairs in 2009) randomly selected, were transported to our laboratory. the temperature of the laboratory was maintained at a constant temperature of 20±1 oc with a light/dark schedule of 14/10 hours. each paedomorphic and metamorphic female was weighed with an ufo tech precision balance and their snout-vent lengths (svl) were measured with digital calipers following başoğlu and özeti (1973). then we placed them with a metamorphic male in separate 20 liter glass aquariums and some small-leaved aquatic plants were provided for egg deposition. newts were fed three times a week with tubifex worms and earthworms. we changed the water of each aquarium every other day. paedomorphs and metamorphs in captivity displayed courtship behaviour and then breeding activity. the metamorphic and paedomorphic forms generally started to lay eggs on the 3rd day of captivity. eggs were collected and counted four times a day, over the course of the entire oviposition period. as many species of newts and salamanders are especially fond of consuming their own eggs (dasgupta, 1996; kuzmin, 1991), we moved and tried to rear these eggs separately from the parents’ aquaria. eggs which were transferred in a separate container were checked three or four times a day. instead of the evaporating water during the incubation period, clean water was added to aquaria. newly hatched larvae were counted and moved in a separate aquarium, and the experiment was lasted. the mean ovum diameter was calculated by measuring 20 randomly chosen eggs (in 1st cleavages) from each individual under a stereomicroscope with an ocular micrometer. hatching success (survival rate) was defined as the number of hatched larvae divided by 21reproductive characteristics of the northern banded newt the number of eggs and multiplied by 100. confidence interval of survival rates was calculated as ci = 2 sqrt [q(1–q)/n], where q = 1-sr, sr is the survival rate and n is number of eggs at the beginning of the experiment (miaud, 1994). body size measurements and all reproductive output parameters were normally distributed (kolmogorov–smirnov d test, all p > 0.05), thus allowing comparisons using parametric tests. firstly, to explore the level of variation among females within morphs, analyses of one-way anova were performed using spss 13.0 version for windows. to analyze the relationships between female body size and egg size, we used standardized major axis (sma) analysis (warton et al., 2006). the mean of egg diameter for each female was regressed to the svl in sma analysis. sma analysis provide a better estimate of the line summarizing the relationship between two variables (i.e., the main axis along which two variables are correlated) to that of ordinary linear regression, because the residual variance is minimized in both “x” and “y” dimensions. the analysis also determined the differences between the slopes obtained for each species or for each maternal tree, so that a significant p indicated differences between the slopes of the groups studied. we used the free software statistics package smart vers. 2.0 (falster et al. 2006). there was a strong positive correlation between female svl and body mass in paedomorphic (pearson’s, n = 13; r2 = 0.820; p < 0.01), and metamorphic forms (pearson’s, n = 21; r2 = 0.600; p < 0.01). paedomorphic females were significantly smaller and lighter than metamorphic females (table 1 and fig. 1). body size can be varied between paedomorphs and metamorphs depending on the population source (denoël et al., 2009). for example, in some populations of newts (smooth, palmate and alpine newt) paedomorphic forms can be smaller than metamorphs or vice versa as well as both forms have approximately same size (denoël et al., 2009; kalezić et al., 1996). the most notable results of our study that the mean number of eggs of the paedomorphs lesser than metamorphs, although, there are not significant differences the number of eggs between paedomorphs and metamorphs. however, egg diameters showed statistically difference between paedomorphs and metamorphs (table 1 and fig. 1). generally, fecundity parameters and mating success are related to body size (semlitsch, 1985), but the relation appears to be different in different cases. paedomorphic females produced lesser eggs than metamorphic counterparts (semlitsch, 1985; kalezić et al., 1996), but, table 1. descriptive statistics of female size and reproductive outputs shown in two forms of o. ophryticus. results of one way anova indicate the differences between paeodomorphic and metamorphic forms. form n min max mean std. dev. df f sig. svl (mm) p 13 31.88 43.08 37.47 3.495 1 117.82 < 0.0001 m 21 44.96 64.34 52.68 4.232 body mass (g) p 13 1.7 3.2 2.35 0.427 1 21.67 < 0.0001 m 21 2.4 6.4 3.70 0.985 number of eggs p 13 16 189 83.46 55.611 1 3.59 0.067 m 21 25 296 122.90 60.965 egg diameter (mm) p 116 0.60 0.98 0.78 0.068 1 314.41 < 0.0001 m 105 0.79 1.02 0.92 0.049 22 e. başkale, f. sayım and u. kaya in some cases, the reverse situation also exists or they produced approximately the same number of eggs (semlitsch, 1985; kalezić et al., 1996; rot-nikcevic, et al., 2000). totals of 1085 eggs of paedomorphic forms and 2581 eggs of metamorphic forms were layed, 717 and 2092 larvae of which developed, respectively. photographs of an egg and a resulting larva are shown in fig. 2. on the side, survival rates of 0.66 (66%), ci 0.63-0.69 for paedomorphs, and 0.81 (81%), ci 0.80-0.83 for metamorphs. these results indicate that survival rates of two morphs are significantly different each other. the growth and development rates of amphibians are influenced by a number of intrinsic (body size or egg size and yolk reserves) and extrinsic factors (temperature, density and competition, food supply and quality, predation, breeding habitat and inhibitory compounds) (crump, 1974, 1984; kuramoto, 1975; berven et al., 1979; kaplan, 1980; berven and chadra, 1988; duelman and trueb 1994). most likely, low survival rate in paedomorphic forms is underlined by intrinsic factors mostly because of constant laboratory conditions. the linear regression analysis indicated that female svl showed a strong positive relationship with egg diameter (metamorphs: r2 = 0.858; p = 0.024, paedomorphs: r2 = 0.823; p = 0.013), as well as with the number of eggs (metamorphs: r2 = 0.446; f = 15.232; p = 0.001, paedomorphs: r2 = 0.653; f = 20.713; p = 0.001). sma analysis showed that both forms shared a common allometric slope of the svl vs number of eggs and egg diameter. pairwise combinations of svl and number of eggs/egg diameters showed significant differences in elevation shift (table 2). consequently, our results indicate that larger individuals of both forms tend to produce more and larger eggs. similarly, furtula et al. (2008) found conformable relationships between the female svl and vitellus volume in four crested newt species. in this sense, larger females produce larger eggs, and they have more yolk for larval development and/or thicker mucoid capsules which provide better protection (semlitsch, l985; semlitsch and gibbons, 1990; kalezić et al., 1994). for this reason, female body size and/or egg size can affect the low survival rates in paedomorphic forms in contrast to metamorphic counterparts.   (a) s v l (m m ) 31 41 51 61 71 paedomorphs metamorphs (b)b od y m as s (g ) 1,7 2,7 3,7 4,7 5,7 6,7 paedomorphs metamorphs (c)n um be r of e gg s 0 50 100 150 200 250 300 paedomorphs metamorphs (d)e gg d ia m et er ( m m ) 0,59 0,69 0,79 0,89 0,99 1,09 1,19 paedomorphs metamorphs fig. 1. variations of svl (a) and body mass (b), number of eggs (c) and egg diameter (d) in females of paedomorphic and metamorphic forms. 23reproductive characteristics of the northern banded newt acknowledgements we thank dr. milos kalezić for his valuable comments on the manuscript, and also i̇. ethem çevik and şamil yıldırım for assisting during field studies. the permissions for field work and handling of the newts were issued by the animal ethics committee of faculty of medicine, ege university.  fig. 2. the eggs: fertilized (a), cleaved (b), and a resulting larva of paedomorphic form (c). table 2. results of standardized major axis (sma) regression analysis for all pairwise combinations of snout-vent length (svl) and reproduction traits of both forms. trait pair (x and y) forms n r2 p slope intercept slope homo geneity (p) shift in elevation (p) shift along slope (p) svl and egg number p 13 0.592 0.002 0.1286 1.333 0.877 0.000 < 0.0001 m 21 0.187 0.050 0.1340 1.454 svl and egg diameter p 6 0.342 0.223 18.47 4.288 0.409 0.004 0.009 m 5 0.062 0.687 34.57 2.589 24 e. başkale, f. sayım and u. kaya references andren, c. 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(2006): bivariate line-fitting methods for allometry. biol. rev. 81: 259–291. bbib67 ole_link1 ole_link2 bbib28 ole_link5 ole_link6 ole_link7 ole_link8 _goback ole_link1 ole_link2 ole_link3 ole_link4 ole_link1 ole_link2 ole_link19 ole_link20 ole_link21 ole_link29 ole_link3 ole_link4 ole_link5 ole_link31 ole_link14 ole_link15 ole_link12 ole_link13 ole_link16 ole_link17 ole_link22 ole_link23 ole_link24 ole_link8 ole_link9 ole_link10 ole_link11 ole_link18 ole_link27 ole_link28 ole_link25 ole_link26 ole_link6 ole_link7 ole_link34 ole_link37 ole_link38 acta herpetologica vol. 6, n. 1 june 2011 firenze university press widespread bacterial infection affecting rana temporaria tadpoles in mountain areas rocco tiberti extreme feeding behaviours in the italian wall lizard, podarcis siculus massimo capula1, gaetano aloise2 lissotriton vulgaris paedomorphs in south-western romania: a consequence of a human modified habitat? severus d. covaciu-marcov*, istvan sas, alfred ş. cicort-lucaciu, horia v. bogdan body size and reproductive characteristics of paedomorphic and metamorphic individuals of the northern banded newt (ommatotriton ophryticus) eyup başkale1, ferah sayım2 , uğur kaya2 genetic characterization of over hundred years old caretta caretta specimens from italian and maltese museums luisa garofalo1, john j. borg2, rossella carlini3, luca mizzan4, nicola novarini4, giovanni scillitani5, andrea novelletto1 the phylogenetic position of lygodactylus angularis and the utility of using the 16s rdna gene for delimiting species in lygodactylus (squamata, gekkonidae) riccardo castiglia*, flavia annesi localization of glucagon and insulin cells and its variation with respect to physiological events in eutropis carinata vidya. r. chandavar1, prakash. r. naik2* the balearic herpetofauna: a species update and a review on the evidence samuel pinya1, miguel a. carretero2 effects of mosquitofish (gambusia affinis) cues on wood frog (lithobates sylvaticus) tadpole activity katherine f. buttermore, paige n. litkenhaus, danielle c. torpey, geoffrey r. smith*, jessica e. rettig food composition of uludağ frog, rana macrocnemis boulenger, 1885 in uludağ (bursa, turkey) kerim çiçek preliminary results on tail energetics in the moorish gecko, tarentola mauritanica tommaso cencetti1,2, piera poli3, marcello mele3, marco a.l. zuffi1 climate change and peripheral populations: predictions for a relict mediterranean viper josé c. brito1, soumia fahd 2, fernando martínez-freiría1, pedro tarroso1, said larbes3, juan m. pleguezuelos4, xavier santos5 assessing the status of amphibian breeding sites in italy: a national survey societas herpetologica italica* osservatorio erpetologico italiano acta herpetologica journal of the societas herpetologica italica acta herpetologica rivista della societas herpetologica italica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 7(2): 315-323, 2012 first assessment of the endoparasitic nematode fauna of four psammophilous species of tropiduridae (squamata: iguania) endemic to north-eastern brazil markus lambertz1,*, tiana kohlsdorf2, steven f. perry1, robson waldemar ávila3, reinaldo josé da silva4 1 institut für zoologie, rheinische friedrich-wilhelms-universität bonn, poppelsdorfer schloss, 53115 bonn, germany. *corresponding author. e-mail: lambertz@uni-bonn.de 2 departamento de biologia, faculdade de filosofia, ciências e letras de ribeirão preto, universidade de são paulo, avenida bandeirantes 3900, bairro monte alegre, 14040-901, ribeirão preto, sp, brazil 3 universidade regional do cairi, centro de ciências biológicas e da saúde, departamento de ciências biológicas, campus do pimenta, rua cel. antonio luiz, 1161, bairro do pimenta, 63105-100, crato, ce, brazil 4 unesp universidade estadual paulista, campus de botucatu, instituto de biociências, departamento de parasitologia, distrito de rubião júnior, 18618-970, botucatu, sp, brazil submitted on: 2012, 24th may; revised on: 2012, 15th october; accepted on: 2012, 15th october. abstract. tropiduridae (squamata: iguania) is a lizard taxon widely distributed in the neotropics. among its representatives, some species are classified as generalists regarding habitat usage. others exhibit a very restricted and probably relict distribution, and are strongly associated with predominantly sandy and dry habitats. within this rather ecologically similar than phylogenetically closely related group we examined specimens of eurolophosaurus amathites, e. divaricatus, tropidurus hygomi, t. psammonastes for endoparasites. in all four species examined we recorded parasitic nematodes (nemathelminthes: nematoda). at least three nematode species were recovered: parapharyngodon sp., physaloptera lutzi and strongyluris oscari, with ph. lutzi being the most abundant parasite encountered in all lizard species examined. in spite of the hosts’ habitat specialization, these parasites are also found frequently in non-psammophilous tropidurid species as well as in other squamates. individual species richness per lizard was low, with usually just one species parasitizing at a time. these are the first parasites registered for these tropidurids and constitute a total of six new host records. keywords. caatinga, psammophily, eurolophosaurus, nematoda, neotropics, tropidurus. complete characterization of the ecological affinities of a species requires the analysis of a variety of aspects, ranging from physiological, through environmental to behavioral 316 markus lambertz et al. traits that are often interconnected. among these various factors the parasites of a given species also constitute an important ecological data point (poulin, 1999), as they can provide basic faunistic information about parasite-host relationships. however, especially in the case of neotropical squamates even such basic parasitological data are often lacking (salgado-maldonado et al., 2000). tropidurid lizards (squamata: tropiduridae) are found throughout most of south america and the vast majority were long lumped in the genus tropidurus wied-neuwied, 1824 (frost, 1992; frost et al., 2001). studies by rodrigues (1988) revealed that the brazilian distribution patterns of this former tropidurus-complex, fall into four ecological (not phylogenetic) groups: (1) species with very wide-ranging distributions associated with open savanna-like formations, (2) species associated with small, isolated, forested regions, (3) species associated with mountain ranges, and (4) species with very limited or disjunct distributions, strongly associated with sandy soils. species that inhabit similar environments are likely to experience similar ecological phenomena, including host-parasite interactions. in the latter context we focus the present study fig. 1. photographs of living representatives of the four tropidurid species examined (specimens not collected): eurolophosaurus amathites at gameleira de assuruá, gentio do ouro (a), e. divaricatus at ibiraba, barra municipality (b), tropidurus hygomi at praia do forte, mata de são joao municipality (c), t. psammonastes at ibiraba, barra municipality (d). all species are found in localities in the state of bahia, brazil. photographs by a. camacho (a,c) and m. lambertz (b,d). 317nematodes of four psammophilous tropidurids from ne brazil on endoparasites in four tropidurid species of the “sandy habitat” ecological group (fig. 1). tropidurus hygomi reinhardt and lütken, 1861 exhibits a disjunct distribution along a few coastal dunes in the states of bahia and sergipe (fig. 2). virtually nothing is known about the ecology of this microendemic species, and in particular, information about nutritional aspects are lacking. the fragmentary information regarding microhabitat usage, soils and ambient vegetation is summarized by vanzolini and gomes (1979) and martins et al. (2010). the other three species studied here are endemic to a small dune field located on both sides of the middle portion of the são francisco river in north-western bahia (fig. 2). these relict habitats within the caatinga biome are populated by a large number of endemic lizards and snakes, and some general information is summarized by rodrigues (1996) and lambertz (2010). eurolophosaurus amathites (rodrigues, 1984) is geographically isolated from the remaining two species on the dunes at the eastern bank of the são francisco river and fig. 2. map of bahia and sergipe in northeastern brazil showing the occurrence of the examined species of tropiduridae. 1: eurolophosaurus amathites, 2: e. divaricatus, 3: tropidurus hygomi, 4: t. psammonastes. two species, e. divaricatus and t. psammonastes, occur sympatric and are geographically isolated from e. amathites by the são francisco river. one species, t. hygomi, occurs with a disjunct distribution close to the atlantic coast in more than 500 km from the other populations. distributional data after rodrigues (1988). 318 markus lambertz et al. also for this species there are virtually no ecological data available. eurolophosaurus divaricatus (rodrigues, 1986) and t. psammonastes rodrigues, kasahara and yonenaga-yassuda, 1988, on the other hand, sympatrically inhabit the dunes at the western bank of the são francisco river. rocha and rodrigues (2005) compared microhabitat usage and diet of these two species and found that t. psammonastes shows a positive electivity for shaded and protected areas and mainly feeds on ants, while e. divaricatus shows a negative electivity for shaded and protected areas and feeds mainly on flowers. endoparasites have been reported for some representatives of tropiduridae (vicente et al., 1993; fontes et al., 2003; silva and kohlsdorf, 2003; bursey and goldberg, 2004; bursey et al., 2005; pérez et al., 2007; almeida et al., 2009; bursey and goldberg, 2009; goldberg et al., 2009; ávila and silva, 2010; ávila et al., 2010, 2011, 2012; pereira et al., 2012). however, to date there is no report on any of the psammophilous species mentioned above and the data basis for lizards of the caatinga biome in general is extremely poor (ávila et al., 2012). the primary purpose of the present study is to contribute to the knowledge of the helminth fauna of north-eastern brazil and to provide the first parasitological data especially for these relict tropidurids. twenty-four lizards (six adults of each species) from the coleção herpetológica de ribeirão preto (chrp, departamento de biologia, ffclrp, universidade de são paulo, ribeirão preto, são paulo state, brazil) were examined for endoparasites. all representatives were collected in locations in the state of bahia, brazil (e. amathites: october 2009, cacimbas, 11°07’s, 42°44’w, chrp 267, 269-273; e. divaricatus: june 2010, ibiraba, 11o01’s, 43o08’w, chrp 297, 300, 306, 314-316; t. hygomi: june 2002, salvador, 12°55’s, 38°20’w, chrp 473, 475, 476, 479-481; t. psammonastes: june 2010, ibiraba, 11°01’s, 43o08’w, chrp 320-325). except for two e. divaricatus, all lizards examined were male. the specimens were originally collected for ecophysiological studies, after which they were killed by an overdosed intramuscular injection of ketamine and subsequently fixed according to standard procedures (e.g., simmons, 2002) and maintained in 70% ethanol. in the present study, snout-vent length (svl) was measured with calipers (to the nearest 0.1 mm) (table 1), the specimens were opened ventrally, and the coelomic cavity was examined for parasites both with the naked eye and using a dissecting microscope. the viscera were then removed, the gastrointestinal tract opened longitudinally and examined for parasites as described above. parasites were removed, transferred to separate small, capped vials, labeled according to the location in which they were found (coelomic cavity, stomach, intestines), and stored in 70% ethanol. nematodes were cleared with lactophenol and examined using the leica qwin lite 3.1 computerized system. voucher helminth specimens are deposited in the coleção helmintológica do instituto de biociências de botucatu (chibb, universidade estadual paulista, botucatu, são paulo state, brazil). five parameters regarding parasite infections studied here, individually for each lizard species, are (1) prevalence (ratio of hosts infected with a given parasite species in relation to total number of potential hosts examined), (2) mean intensity of infection (average number of each parasite species per infected host), (3) range of intensity (lowest and highest numbers of parasites encountered in infected hosts), (4) range of parasite abundance (lowest and highest numbers of parasites encountered in all potential hosts) and (5) mean parasite abundance (ratio of the total number of a given parasite in relation to total number of potential hosts examined). all parameters are defined according to bush et al. (1997). 319nematodes of four psammophilous tropidurids from ne brazil ta bl e 1. b as ic d at a on t he li za rd s ex am in ed a nd p ar as ito lo gi ca l d at a fo r th e m os t fr eq ue nt ly e nc ou nt er ed s pe ci es o f pa ra si te , p hy sa lo pt er a lu tz i, as w el l a s th e re m ai ni ng n em at od es . li za rd h os ts n sv l [m m ] ph ys al op te ra lu tz i pr ev al en ce [ % ] r an ge o f in te ns ity o f in fe ct io n m ea n in te ns ity of in fe ct io n ± sd r an ge o f pa ra si te ab un da nc e m ea n pa ra si te ab un da nc e ± sd si te (s ) of in fe ct io n vo uc he rs eu ro lo ph os au ru s am at hi te s 6 60 .5 -7 5 33 .3 12 1. 5 ± 0. 71 02 0. 5 ± 0. 84 st om ac h c h ib b 5 07 1, 50 73 eu ro lo ph os au ru s di va ri ca tu s 6 66 .5 -9 1. 5 66 .7 214 5. 5 ± 5. 74 014 3. 67 ± 5 .2 8 st om ac h c h ib b 5 07 450 77 tr op id ur us h yg om i 6 60 -6 9. 9 10 0 16 2. 67 ± 1 .9 7 16 2. 67 ± 1 .9 7 st om ac h, co el om ic c av ity c h ib b 5 07 850 81 , 5 08 350 85 tr op id ur us ps am m on as te s 6 89 .5 -9 7. 5 66 .7 16 2. 25 ± 2 .5 06 1. 5 ± 2. 26 st om ac h, up pe rm os t in te st in e c h ib b 5 08 650 90 o th er n em at od es e nc ou nt er ed pa ra ph ar yn go do n sp . ( in e. a m at hi te s on ly ) 50 12 1. 33 ± 0 .5 8 02 0. 67 ± 0 .8 2 in te st in e c h ib b 5 06 9, 50 70 , 5 07 2 st ro ng yl ur is o sc ar i ( in t . hy go m i o nl y) 16 .6 7 2 2 02 0. 33 ± 0 .8 2 in te st in e (t er m in al re gi on ) c h ib b 5 08 2 320 markus lambertz et al. endoparasitic nematodes were observed in all four lizard species examined. in total, 56 nematodes belonging to at least three different species were found: parapharyngodon sp. (oxyuroidea: pharyngodonidae), physaloptera lutzi cristofaro, 1976 (spiruroidea: physalopteridae) and strongyluris oscari travassos, 1923 (oxyuroidea: heterakidae). physaloptera lutzi was found in specimens of all four lizard species examined. in t. hygomi it exhibited the highest prevalence (= 100 %) and in e. divaricatus exhibited the highest intensity of infection (= 14). the detailed results and voucher numbers for ph. lutzi are summarized in table 1. further unidentifiable (larval) specimens belonging to parapharyngodon chatterji, 1933 were encountered in e. amathites only. strongyluris oscari was found exclusively in one specimen of t. hygomi. refer to table 1 for details on these nematodes as well. except for one specimen of e. amathites, which harbored parapharyngodon sp. and ph. lutzi simultaneously, all other lizards were infected by just one species of parasite at a time. the present study suggests that the psammophilous tropidurids from the brazilian dunes do indeed host similar species of endoparasitic nematodes, with ph. lutzi as the most frequently encountered and most abundant species. moreover, based on our data it appears that compared with other closely related lizards (e.g., fontes et al., 2003; ávila and silva, 2010), the infestation per individual in all of the psammophilous species is limited to a small number of parasitic species at a time, usually one. large sample sizes, as in the study of fontes et al. (2003), were not possible for these rare tropidurids and consequently statistically supported generalizations on the population level cannot be made. nevertheless, the present study resulted in a total of six new host records, and this general knowledge about parasite-host relationships is an important contribution to our better understanding the ecological affinities of these species. physaloptera lutzi has been recorded for the first time parasitizing e. amathites, e. divaricatus, t. hygomi as well as t. psammonastes. strongyluris oscari has been recorded for the first time parasitizing t. hygomi, and we recorded for the first time a representative of parapharyngodon in e. amathites. the nematode species reported here are frequently encountered parasitizing other tropidurid species as well as other squamates that do not inhabit extreme habitats, and appear to be widely distributed generalists. the evolutionary history of these host-parasite associations therefore could mirror the assumed relict distribution of these psammophilous lizards, and thus the initial infections could date back to phylogenetically earlier events with only very tolerant (mainly regarding temperature and aridity) parasitic elements still present. nevertheless, studying the actual phylogenetic coupling of parasitehost interactions is a highly complex issue, which requires a special experimental design (e.g., regenfuss, 1978). while the present study was not designed specifically to address this evolutionary topic, our findings may serve as a starting point for further such parasitological studies on tropidurid lizards. especially the frequent infestation with ph. lutzi could be useful in a molecular phylogeographic analysis that individually compares haplotypes of parasites and hosts. one good possibility for this application could be in the clarification of controversially discussed host phylogeny, such as in eurolophosaurus (compare rodrigues, 1986; frost et al. 2001; passoni et al., 2008). the report on several gutted specimens of t. hygomi published by vanzolini and gomes (1979) in part was based on material they had received from unnamed helminthologists, but we were unable to find any reference to a parasitological study on this 321nematodes of four psammophilous tropidurids from ne brazil species in spite of our intensive literature surveys. thus, all of the parasite-host interactions described above are new host records. it is further relevant to point out that the one specimen of ph. lutzi found in the coelomic cavity of a t. hygomi specimen is interpreted as an artifact, as this parasite is known to infect the gastrointestinal tract of a variety of lizards (e.g. ávila and silva, 2010). furthermore, various species of physaloptera rudolphi, 1819 are known to attach themselves to the gastric mucosa of their host without feeding on it and remain within the digestive system (anderson, 2000), but there is only limited information on species in reptiles, and apparently none on ph. lutzi. we recorded specimens of this species actually piercing the wall of the digestive tract in other specimens of t. hygomi, where about one third of the nematode extended freely into the coelomic cavity while the rest remained within the organ. we therefore assume that this particular “coelomic” individual had successfully escaped from its natural habitat, most likely after the death of its host. in conclusion, psammophilous lizards appear to share a similar, non-diverse assemblage of endoparasitic nematode species and the associated parasites are apparently limited to a few common generalists. understanding how the hosts’ evolutionary history couples with these parasites may further elucidate various aspects of the lizards’ biology including their phylogenetic relationships. acknowledgements we thank fabio c. de barros and felipe a.m. zampieri for collecting most of the lizards examined. collecting and handling permits were kindly provided by the instituto brasileiro do meio ambiente e dos recursos naturais renováveis (ibama, permit numbers 23033-2 to melissa b. closel and 022/02-ran to tk). parts of this study were financed by the fundação de amparo à pesquisa do estado de são paulo (fapesp 2005/60140-4 to tk and fapesp 2006/56962-5 to rjds) and the programa de pos-graduação em biologia comparada (ffclrp universidade de são paulo). we acknowledge agustín camacho for kindly providing the photos for figures 1a and 1c. two anonymous reviewers are thanked for their valuable suggestions on an earlier version of the manuscript. references almeida, w.o., ribeiro, s.c., santana, g.g., vieira, w.l.s., anjos, l.a., sales, d.l. 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(2010): kommentierte liste der squamaten reptilien des sanddünengebietes am mittleren rio são francisco (bahia, brasilien) unter besonderer berücksichtigung endemischer faunenelemente. ophidia 4: 2-17. martins, k.v., dos reis dias, e.j., da rocha, c.f.d. (2010): ecologia e conservação do lagarto endêmico tropidurus hygomi (sauria: tropiduridae) nas restingas do litoral norte da bahia, brasil. biotemas 23: 71-75. passoni, j.c., benozzati, m.l., rodrigues, m.t. (2008): phylogeny, species limits, and biogeography of the brazilian lizards of the genus eurolophosaurus (squamata: tropiduridae) as inferred from mitochondrial dna sequences. mol. phylogenet. evol. 46: 403-414. pereira, f.b., sousa, b.m., de souza lima, s. (2012): helminth community structure of tropidurus torquatus (squamata: tropiduridae) in a rocky outcrop area of minas gerais state, southeastern brazil. j. parasitol. 98: 6-10. pérez z., j., balta, k., salizar, p., sánchez, l. 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(1984): uma nova espécie brasileira de tropidurus com crista dorsal (sauria, iguanidae). pap. avulsos zool. (são paulo) 35: 169-175. rodrigues, m.t. (1986): um novo tropidurus com crista dorsal do brasil, com comentários sobre suas relações, distribuição e origem (sauria, iguanidae). pap. avulsos zool. (são paulo) 36: 171-179. rodrigues, m.t. (1988): distribution of lizards of the genus tropidurus in brazil (sauria, iguanidae). in: procedings of a workshop on neotropical distribution patterns, pp. 305315. heyer, w.r., vanzolini, p.e. eds, academia brasileira de ciências, rio de janeiro. rodrigues, m.t. (1996): lizards, snakes, and amphisbaenians from the quaternary sand dunes of the middle rio são francisco, bahia, brazil. j. herpetol. 30: 513-523. rodrigues, m.t., kasahara, s., yonenaga-yassuda, y. (1988): tropidurus psammonastes: uma nova espécie do grupo torquatus com notas sobre seu cariótipo e distribuição (sauria, iguanidae). pap. avulsos zool. 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(são paulo) 32: 243-259. vicente, j.j., de oliveira rodrigues, h., gomes, d.c., pinto, r.m. (1993): nematóides do brasil. parte iii: nematóides de reptéis. rev. bras. zool. 10: 19-168. wied-neuwied, m. (1824): verzeichnis der amphibien, welche im zweyten bande der naturgeschichte brasiliens von prinz max von neuwied werden beschrieben werden (nach merrems versuch eines systems der amphibien). isis von oken 14: 661-673. acta herpetologica vol. 7, n. 2 december 2012 firenze university press advertisement call of species of the genus frostius cannatella 1986 (anura: bufonidae) flora a. juncá1, david l. röhr2, ricardo lourenço-de-moraes3, flávio j. m. santos1, airan s. protázio1, ednei a. mercês1, mirco solé4 amphibians in southern apennine: distribution, ecology and conservation notes in the “appennino lucano, val d’agri e lagonegrese” national park (southern italy). antonio romano1,*, remo bartolomei1, antonio luca conte1, egidio fulco2 the significance of using satellite imagery data only in ecological niche modelling of iberian herps neftalí sillero1, josé c. brito2, santiago martín-alfageme3, eduardo garcía-meléndez4, a.g. toxopeus5, andrew skidmore5 reproductive strategy of male and female eastern spiny lizards sceloporus spinosus (squamata: phrynosomatidae) from a region of the chihuahuan desert, méxico aurelio ramírez-bautista1,*, barry p. stephenson2, xóchitl hernández-ibarra1, uriel hernández-salinas1, raciel cruz-elizalde1, abraham lozano1, and geoffrey r. smith3 morphological variability of the hermann’s tortoise (testudo hermanni) in the central balkans katarina ljubisavljević1, georg džukić1, tanja d. vukov1, miloš l. kalezić1,2 the usefulness of mesocosms for ecotoxicity testing with lacertid lizards maria josé amaral1,2,*, rita c. bicho1, miguel a. carretero2, juan c. sanchez-hernandez3, augusto m. r. faustino4, amadeu m. v. m. soares1, reinier m. mann1,5 does acclimation at higher temperatures affect the locomotor performance of one of the southernmost reptiles in the world? jimena b. fernández*, nora r. ibargüengoytía advertisement call of scinax littoralis and s. angrensis (amphibia: anura: hylidae), with notes on the reproductive activity of s. littoralis michel v. garey1, thais r. n. costa2, andré m. x. de lima2, luís f. toledo3, marília t. hartmann4 book review: marine s. arakelyan, felix d. danielyan, claudia corti, roberto sindaco, alan e. leviton 2011. herpetofauna of armenia and nagorno-karabakh. society for the study of amphibians and reptiles stefano scali book review: rafaqat masroor 2012. a contribution to the herpetofauna of northern pakistan stefano scali evidence of high longevity in an island lacertid, teira dugesii (milne-edwards, 1829). first data on wild specimens. j. jesus first assessment of the endoparasitic nematode fauna of four psammophilous species of tropiduridae (squamata: iguania) endemic to north-eastern brazil markus lambertz1,*, tiana kohlsdorf2, steven f. perry1, robson waldemar ávila3, reinaldo josé da silva4 rediscovery and redescription of the holotype of lygosoma vittigerum (= lipinia vittigera) boulenger, 1894 yannick bucklitsch1, peter geissler1, timo hartmann1, giuliano doria2 , andré koch1,* reproductive phenology of the tomato frog, dyscophus antongili, in an urban pond of madagascar’s east coast ori segev1,*, franco andreone2, roberta pala2, giulia tessa2, miguel vences1 range extension of the critically endangered true poison-dart frog, phyllobates terribilis (anura: dendrobatidae), in western colombia roberto márquez1,*, germán corredor2, carlos galvis3 daniel góez2, & adolfo amézquita1 differences in habitat use of two sympatric species of ameiva in east costa rica esther sebastián-gonzález1, ramón gómez2 acta herpetologica journal of the societas herpetologica italica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 6(1): 101-103, 2011 preliminary results on tail energetics in the moorish gecko, tarentola mauritanica tommaso cencetti1,2, piera poli3, marcello mele3, marco a.l. zuffi1 1 museo di storia naturale e del territorio, università di pisa, via roma 79, 56011 calci, pisa, italy 2 via di montesenario 1723/a, 50036 vaglia, firenze, italy. corresponding author. e-mail: tommaso cencetti@yahoo.it 3 dipartimento di agronomia e gestione dell’agroecosistema, università di pisa, via san michele degli scalzi 2, 56124 pisa, italy. submitted on: 2011,14th february; revised on 2011, 18th march; accepted on 2011, 2nd june. abstract. the amount of lipids, proteins, ashes and water in original versus regenerated tails in tarentola mauritanica shows differences, especially on the lipid fraction, supporting adaptive function hypotheses during reproduction of regenerated tails in geckos. keywords. tail autotomy, energetic components, moorish gecko, tarentola mauritanica. the tail in vertebrates has the basic function to help the animal’s movement during swimming (e.g. in fish, newts, marine snakes, crocodiles and in marine mammals), during running, jumping and climbing (many lizards and most terrestrial mammals) or flying (birds and bats). in some reptiles, however, especially in lizards, the tail has a double, integrated function: i) balancing during movement and ii) acting as predator escape device. tail loss may happen during a predation attempt or as a result of intraspecific interactions. in both cases, tail loss could affect many other features of a lizard’s ecology and biology, such as locomotory performances (bateman et al., 2009), the total amount of energetic reserves or courtship ability. in fact, lizards may lose a substantial amount of lipids and proteins along with the tail loss, and further energies are needed to regenerate the appendage as to previous anatomical and physiological status. in addition to several papers inspecting both the adaptive and functional meaning of tail autotomy (e.g. perez-mellado et al., 1997), since more than 40 years analytical data set on tail energetics have been collected on several species of lizards of different families, especially in the gekkonid genera coleonyx and hemidactylus, and in the skinks eumeces and ctenotus. as far as we are aware, no european species has been still considered (see table 1). we performed chemical analysis of integer and regenerated tails of the moorish gecko, tarentola mauritanica, to test for any differences in the content of water, lipids, proteins and ashes between the two tail types. more specifically, we are aimed at underlining 102 t. cencetti, p. poli, m. mele and m.a.l. zuffi if energetic contents of tarentola tails may be different according to the systematic position and relationship of the reference taxa. we analyzed a total of 31 tails, 16 original and 15 regenerated, equally distributed between adult males (six vs. four), adult females (three vs. five) and juveniles (five vs. three), plus five belonging to adult geckos whose sex was not recognizable. all samples were freshly weighted. tail fragment autotomized at 2nd or 3rd segment represents on average 9% of total body mass (5.5% in juveniles), disregarding of tail being original or regenerated (t-test on arcsine transformed value = 0.987, 30 df, p = 0.342). regenerated tails slightly differ in shape, being shorter and bulkier than the original ones, even not significantly (t-test = 0.144, 30 df, p = 0.888), thus resulting in a sort of carrot-shape element. the subsequent processing follows a procedure recently applied on snakes, described in zuffi et al. (2010). for the nutrient analysis following water extraction (proteins, fat and table 1. available data on chemical and energetics in lizard species. “o” means original, “r” regenerated. species tail n calories lipids proteins ashes water coleonyx variegatus (vitt, 1977) o 3 5.79 34.9 9.01 ± 2.06 25.7 r 19 6.24 24.9 6.52 ± 0.67 25.7 coleonyx brevis (dial and fitzpatrick, 1981) o 8 6.04 ±0.10 . . 8.21 ± 1.10 71.9 ±1.61 eumeces gilberti (vitt, 1977) o 3 5.12 46.9 15.86 ± 0,48 71.0 r 14 6.59 58.0 7.69 ± 0.45 52.9 gerrhonotus multicarinatus (vitt, 1977) o 3 5.11 10.5 23.88 ±0.51 71.1 r 18 6.41 30.3 5.95 ± 0.63 63.6 podarcis erhardii (simou et al., 2006) o 9 33 ± 18 30 ± 5 r 14 40 ± 12 28 ±5 phyllodactylus marmoratus (daniels, 1984) o 7 32.4 ± 8.7 hemidactylus mabouia (meyer, 2002) o 14 65¹ r 18 50¹ ¹ mg/g of tissue. table 2. energetic components in tarentola mauritanica tails. all components are expressed as percentage of dry weight, except water (percentage of wet weight), and calories (per mg of dry weight). tail type original regenerated proteins 69.23 56.72 lipids 14.29 27.34 ashes 13.10 11.65 h₂o 66.0 ± 10.0.4 67.7 ± 8.3 kcal/g 4.04 5.18 103tail energetics in moorish geckos ashes), due to the minimum weight analyzable (3.5 grams of dried tails), we had to pool all samples in just two groups: regenerated and original, as in table 2. our analyses show a decreased amount of proteins and ashes in regenerated tails, while the lipid fraction is much higher than in original tails; no difference at all in water content, other than for juveniles (data not shown). the whole pattern resembles that one found in other lizard species and the positive variation of lipids in regenerated tails is in line with several other lizard species, irrespective of their taxonomic position (see table 1), but is much more pronounced than in the majority of them, especially more than in other geckos. in fact, lipid contents in regenerated tails of t. mauritanica is almost double than in original tails. the overall energetic estimation does not differ from the patterns found in other studied taxa (see table 1). a careful comparison, albeit preliminary, between literature data and present study seems hazardous: data on proteins in most species and families are in fact lacking, and data set is very variable, referring to taxa belonging to different taxonomic positions (e.g. scincidae, gekkonidae), and showing contrasting life-history traits (e.g. terrestrial vs. arboreal species), that could influence energetic gain patterns. despite that we are referring to preliminary data, we are confident they should be considered as valuable, due to the evident data scarcity and complete analyses on this topic since the last 20 years. we furthermore underline the need for a deeper investigation with larger samples, sexual differences in energy allocation processes and the adaptive meaning of these mechanisms, and on multiple species. references bateman, p.w., fleming, p.a. (2009): to cut a long tail short: a review of lizard caudal autotomy studies carried out over the last 20 years. j. zool., lond. 277: 1–14. daniels, c. b. (1984): the importance of caudal lipids in the gecko phyllodactylus marmoratus. herpetologica 40: 337–44. dial, b.e., fitzpatrick, l.c. (1981): the energetic costs of tail autotomy to reproduction in the lizard coleonyx brevis (sauria: gekkonidae). oecologia 51: 310–317. meyer, v., preest, m.r., lochetto, s.m. (2002): physiology of original and regenerated lizard tails. herpetologica 58: 75–86. perez-mellado, v., corti, c., lo cascio, p. (1997): tail autotomy and extinction in mediterranean lizards. a preliminary study of continental and insular populations. j. zool., lond. 243: 533–541. simou, c., pafilis, p., skella, a., kourkopuli, a., valakos, e., d. (2008): physiology of original and regenerated tails in aegean wall lizard (podarcis erhardii). copeia 3: 504– 509. vitt, j.l., congdon, j.d., dickson, n.a. (1977): adaptive strategies and energetic of tail autotomy in lizards. ecology 58: 326–337. zuffi, m.a.l., fornasiero, s., picchiotti, r., poli, p., mele, m. (2010): adaptive significance of food income in european snakes: body size is related to prey energetics. biol. j. linn. soc. 100: 307–317. bbib67 ole_link1 ole_link2 bbib28 ole_link5 ole_link6 ole_link7 ole_link8 _goback ole_link1 ole_link2 ole_link3 ole_link4 ole_link1 ole_link2 ole_link19 ole_link20 ole_link21 ole_link29 ole_link3 ole_link4 ole_link5 ole_link31 ole_link14 ole_link15 ole_link12 ole_link13 ole_link16 ole_link17 ole_link22 ole_link23 ole_link24 ole_link8 ole_link9 ole_link10 ole_link11 ole_link18 ole_link27 ole_link28 ole_link25 ole_link26 ole_link6 ole_link7 ole_link34 ole_link37 ole_link38 acta herpetologica vol. 6, n. 1 june 2011 firenze university press widespread bacterial infection affecting rana temporaria tadpoles in mountain areas rocco tiberti extreme feeding behaviours in the italian wall lizard, podarcis siculus massimo capula1, gaetano aloise2 lissotriton vulgaris paedomorphs in south-western romania: a consequence of a human modified habitat? severus d. covaciu-marcov*, istvan sas, alfred ş. cicort-lucaciu, horia v. bogdan body size and reproductive characteristics of paedomorphic and metamorphic individuals of the northern banded newt (ommatotriton ophryticus) eyup başkale1, ferah sayım2 , uğur kaya2 genetic characterization of over hundred years old caretta caretta specimens from italian and maltese museums luisa garofalo1, john j. borg2, rossella carlini3, luca mizzan4, nicola novarini4, giovanni scillitani5, andrea novelletto1 the phylogenetic position of lygodactylus angularis and the utility of using the 16s rdna gene for delimiting species in lygodactylus (squamata, gekkonidae) riccardo castiglia*, flavia annesi localization of glucagon and insulin cells and its variation with respect to physiological events in eutropis carinata vidya. r. chandavar1, prakash. r. naik2* the balearic herpetofauna: a species update and a review on the evidence samuel pinya1, miguel a. carretero2 effects of mosquitofish (gambusia affinis) cues on wood frog (lithobates sylvaticus) tadpole activity katherine f. buttermore, paige n. litkenhaus, danielle c. torpey, geoffrey r. smith*, jessica e. rettig food composition of uludağ frog, rana macrocnemis boulenger, 1885 in uludağ (bursa, turkey) kerim çiçek preliminary results on tail energetics in the moorish gecko, tarentola mauritanica tommaso cencetti1,2, piera poli3, marcello mele3, marco a.l. zuffi1 climate change and peripheral populations: predictions for a relict mediterranean viper josé c. brito1, soumia fahd 2, fernando martínez-freiría1, pedro tarroso1, said larbes3, juan m. pleguezuelos4, xavier santos5 assessing the status of amphibian breeding sites in italy: a national survey societas herpetologica italica* osservatorio erpetologico italiano acta herpetologica journal of the societas herpetologica italica acta herpetologica rivista della societas herpetologica italica acta herpetologica 17(2): 159-164, 2022 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-12660 sunny-side up: ontogenetic variation in egg mass temperatures of the wood frog rana sylvatica ryan calsbeek*, ava calsbeek, isabel calsbeek department of biological sciences, dartmouth college, hanover, new hampshire 03755 usa *corresponding author. e-mail: ryan.calsbeek@dartmouth.edu submitted on: 2022, 24th january; revised on: 25th may 2022; accepted on: 26th may 2022 editor: raoni rebouças abstract. the efficacy of most biological processes is temperature dependent and, within physiological limits, on average, warmer is better. this axiom of biology has led to a wide range of adaptations for dealing with temperatures that are outside of an organism’s preferred temperature. many pond-breeding amphibians lay their eggs during early spring, when water temperatures are near freezing. communal nest-site selection has been proposed as a mechanism to increase developmental temperatures, and temperatures near the center of egg-mass aggregations are elevated relative to egg-masses on the aggregation’s periphery. it is unclear whether this spatial variation in temperature is due to concentration of metabolic heat, absorption of solar radiation, or both. here, we explore finer scale spatial variation within egg masses of the wood frog rana sylvatica, one of the earliest amphibians to breed during the north american spring. we compared peripheral and core temperatures of egg masses that were exposed either to 1) ambient sunlight from above, or 2) sunlight reflected by a mirror from below. we found that differences between core and peripheral temperatures were higher in the control than in the mirror treatment, but core and peripheral temperatures were statistically indistinguishable in both cases. moreover, the difference in peripheral and internal temperatures increased significantly over the course of development. however, these trends were only significant in ambient sunlight and actually decreased in the mirror group. our results suggest that the benefits of communal nesting are also experienced by individual egg masses, albeit to a lesser extent. in addition, the lack of effect in shaded egg masses suggests that the thermal advantage is tied to sun exposure and not due to concentration of metabolic heat. keywords. anurans, egg-mass aggregations, developmental temperatures. introduction a central tenet of biology is that all physiological processes are temperature dependent (huey, 1991). this is because enzymes that regulate physiology have specific temperature ranges over which they can operate (knies et al., 2009). the vast majority of these thermal-performance ranges are left-skewed and show improving physiological performance with increasing temperature, until some threshold ‘optimal temperature’ (topt) is reached, at which point physiological performance drops off rapidly. the generality of this pattern underlies the adage that, within physiological limits, hotter is better (frazier et al., 2006; angilletta et al., 2010). organisms in environments characterized by extreme temperatures face unique challenges around life-history and reproduction, since sub-optimal environmental conditions should constrain physiological performance (skelly, 2004; tryjanowski et al., 2006; benard 2015). these challenges are especially prevalent for ectotherms (huey and tewksbury, 2009), which depend on environmental sources of warmth to maintain metabolic activity. temperatures that exceed a critical threshold (e.g., ctmax) may be lethal and during hot periods 160 ryan calsbeek, ava calsbeek, isabel calsbeek ectotherms may require shaded habitat or underground refugia to behaviorally thermoregulate (díaz-ricaurte et al., 2022; sinervo et al., 2010). high temperatures may lead to dehydration which itself can change thermoregulatory dynamics (guevara-molina et al., 2020). many ectotherms, including insects, reptiles and amphibians, buffer themselves from the negative consequences of sub-optimal temperatures by estivating or entering diapause (blanckenhorn and fairbairn, 1995; ellner et al., 1998; storey and storey, 2012) during colder periods of the year. nevertheless, these same organisms may often face extremely cold conditions that occur at the peak of their reproductive activity in early spring (costanzo and lee, 1993). many pond breeding amphibians emerge from winter hibernacula as soon as temperatures exceed freezing (waldman, 1962; herreid and kinney, 1967). the first amphibians to emerge at the end of north american winters often breed while ponds are still ice-covered, and females lay eggs in water that is very close to its freezing point (costanzo and lee, 1993). aggregating egg masses in communal nest sites is one potential adaptation to cold. previous studies have shown that egg masses at the center of these communal aggregations are warmer compared to those on the periphery (savage, 1961; hassinger, 1970). warmer temperatures should be advantageous to developing embryos, since warmer temperatures speed development and tadpoles that reach metamorphosis more quickly will have a higher probability of escaping vernal pools before they dry (goldstein et al., 2017). other work (arrighi et al., 2013) has shown that the influence of diel fluctuations in temperature may be as (or more) important as that of average temperature on developmental rate. in addition, amphibian eggs often have higher concentrations of melanin in their dorsal hemisphere, which may serve as both protection from ultraviolet radiation and a means of absorbing and retaining heat (licht, 2003). although a handful of studies have documented a thermal advantage to aggregated egg masses (waldman, 1982; skelly, 2004), the source of thermal variation (e.g., solar vs. metabolic) remains unclear as does the degree to which this thermal advantage occurs at smaller spatial scales (i.e., within individual egg masses). moreover, all of these studies have been conducted as point estimates in time and so we currently have no information about how developmental progression impacts temperature of the egg mass itself. developmental stage may be important for impacting both the generation of metabolic heat as well as greater thermal absorption by larger embryos. finally, it is possible that egg mass aggregations are not at all adaptive but are the result of space constraints within a pond, aggregation due to wind currents, or other neutral exaplanations. here we build on previous work to address these shortcomings. the wood frog, rana sylvatica, is among the earliest amphibians to breed in north america, emerging from winter hibernacula as soon as temperatures exceed freezing (slough and mennell, 2006). wood frogs at our study populations near norwich vermont, usa, elevation ca. 300m; 43.7153°n, 72.3079°w (wgs 84 web mercator), typically emerge during the end of march-early april (brady et al., 2019; goedert and calsbeek, 2019). males and females arrive at breeding ponds and breed explosively (swierk et al., 2014), most oviposition occurring within a few days of arrival, and eggs are often laid while ponds are still partially covered in ice. these life history traits make wood frogs especially relevant for understanding the effects of temperature on embryo development. first, we test whether individual egg masses also exhibit warmer internal compared to peripheral temperatures. next, we experimentally test the hypothesis that egg masses warm by absorbing radiant heat from above (i.e., via the pigmented dorsal surface) more efficiently than from below. lastly, we provide temperature measurements over the time-frame from oviposition to hatching to assess the degree to which development itself may influence temperature variation (e.g., by metabolic warming) within egg masses methods we collected 10 wood-frog egg masses from a single pond within 36 hours of oviposition in april of 2021. egg masses were collected by hand and carefully transferred to a plastic holding tank along with ~6l of unfiltered water from the same pond. we assessed gosner’s stage (gosner, 1960), recording the average the developmental stage of five embryos scored under a microscope for each egg mass. all embryos were at gosner’s stage 10 at the start of the experiment. developmental stage was thereafter scored by visual inspection to minimize disturbance over the course of the experiment. egg masses were sectioned into two groups of ca. 75 embryos each (i.e., 150 eggs total from each egg mass) and these two sections of egg mass were then split randomly (by coin toss) into the two groups, such that ~75 individuals from of each of the 10 egg masses were represented in both groups. each set of 75 embryos in the first group were placed in separate 500 ml cylindrical plastic containers (10 cm diameter, 10 cm deep), covered with an opaque lid and placed outdoors on wire shelving that was suspended approximately 30 cm above a mirror. the mirror group was designed to 161variation in egg mass temperatures of the wood frog reverse the direction of sun exposure from the melanic dorsal to the white ventral side of the embryo. each group of 75 embryos in the second group were likewise placed in individual 500 ml plastic containers but were covered with a clear plastic lid and placed on wire shelving over a dark green substrate that was covered with leaf litter from the adjacent forest. the two groups were placed in the same outdoor location with no canopy cover. photoperiod during the experiment was approximately 14:10 (l:d). all sections of egg mass were suspended in approximately 450 ml of pond water, which was changed once, seven days into the experiment. every one to two days we chose half of the ten containers in each group at random for temperature measurements. we recorded air temperatures using an outdoor accu-rite™ thermometer, and egg-mass/water temperature using a digital thermometer (thermoworks model rt600c-n) on the periphery of the egg mass and inserted into the center of the egg mass at the same water depth (~2 cm). we recorded air temperatures outside of the containers, and water temperatures at the time of each measurement. we monitored rates of development by recording changes in gosner stage (gosner, 1960) during each temperature recording. temperature measurements continued until hatching or 14 days, at which point the experiment was ended and the hatchlings and a few unfertilized eggs were transferred to larger volume holding tanks. in total we recorded 110 temperatures during 11 of the 14 days. to minimize the problem of pseudo-replication, we calculated the mean temperature for each group on each day and used these mean values as our unit of observation in all analyses: 11 averages per group, 22 total observations. all data met the criteria for parametric statistics (normality, homoscedasticity, and independence; sokal and rohlf, 1995) based on testing in jmp pro v.16. we tested for relationships between time elapsed since the start of the experiment (days) and air/water temperature using simple linear regression. we tested for a difference in peripheral temperature and core temperature between groups using anova with 1 and 19 degrees of freedom. we tested for a difference in temperature from the center and periphery of the egg mass by subtracting the latter from the former and then using these difference values as the dependent variable using anova with 1 and 19 degrees of freedom. we included gosner’s stage and tested for an interaction between gosner’s stage and experimental group to test whether there was a stage specific difference in temperature between groups in an anova with 1 and 18 degrees of freedom. all tests were twotailed and were conducted in jmp pro v.16 (sas institute, cary, north carolina, usa 1989-2022). results owing to the vicissitudes of spring temperatures during 2021 (typical of the ne united states), there was no significant correlation between measurement date and either air temperature (r2 = 0.03, t20 = 0.74, p = 0.47) or water temperature (r2 = 0.07, t20 = 1.23, p = 0.23). mean temperature at the periphery of the egg mass section was not significantly different between the two groups (x ± se: control = 13.13 ± 1.09 °c, mirror = 12.45 ± 1.05 °c; anova: f1,19 = 0.04, p = 0.80, effect of gosner’s stage p = 0.23; fig. 1a). mean temperature at the center of the egg mass section was likewise not significantly different (control = 13.21 ± 2.47 °c, mirror = 12.32 ± 2.36 °c; anova: f1,19 = 0.87, p = 0.79, effect of gosner’s stage p = 0.21; fig. 1b). however, the differences between core and peripheral temperatures were significantly larger in the control than in the mirror group (0.39 ± 0.08 vs. fig. 1. temperatures did not differ at the periphery of the wood frog egg mass (a), nor at the core of the egg mass (b) in either group. however, the difference in core and peripheral temperatures (that is, comparing means in panels a and b was significantly higher (* t20 = 2.29, p = 0.03) for the control group compared to the mirror group (c). histograms bars show mean values ± one standard error. 162 ryan calsbeek, ava calsbeek, isabel calsbeek 0.18 ± 0.05 °c respectively, anova: f1,19 = 6.44, p = 0.02, eff ect of gosner’s stage p = 0.03; fig. 1c). moreover, this diff erence increased over the course of development resulting in a signifi cant group × gosner’s stage interaction (anova: f1,18 = 6.42, p = 0.04; fig. 2). hatching success at the conclusion of the experiment did not differ between groups; nearly all containers showed 100% hatching success. whereas developmental stage and hatching in the mirror group lagged the control group by one day, developmental stage did not vary with position in egg mass section in either group. nor did hatching dates vary within groups. given this lack of variation, these results were not analyzed statistically. discussion th e challenges associated with life in cold climates select for a variety of adaptations that facilitate heat retention and speed development (skelly 2004, sparks et al. 2006, benard 2015). ectotherms, which rely on external sources of heat to sustain metabolic processes, exhibit patterns in nature that may represent a limited set of strategies to maximize heat retention. concentrating melanin and other dark pigments in the dorsal hemisphere of amphibian eggs may enhance thermal absorption within individual egg masses (licht 2003). likewise, communal nest sites, in which aggregations of egg masses are formed, may concentrate temperatures on their interior (hassinger 1970). we have shown that both of these patterns occur not just in aggregate, but also at the smaller spatial scales of individual portions of egg masses. eggs in our control group experienced significantly warmer temperatures at the core of the egg mass compared to their peripheral counterparts but the same was not true in the mirror group. th is suggests that the darker dorsal side of the developing embryo may briefl y enhance thermoregulation during development compared to the white ventral side of the embryo. th is eff ect is likely to be short-lived in nature since the dark pigment quickly subsumes the entire embryo. wood frog embryos appear to maintain this thermal advantage even following disturbance suffi cient to re-orient the embryos in their horizontal plane. we included the mirror group in our study to account for the rotational behavior of fl ipped embryos. it is worth noting that embryo rotation is likely a result of diff erences in density and not a response to light, since all embryos retained their normal orientation in both the mirror and control group. th e diff erence between internal and peripheral temperatures increased signifi cantly over the course of development in our control group but not in the mirror group. this further supports a role for the pigmentation of embryos in enhancing thermoregulation (clusella-trullas et. al., 2007, 2009; stuart-fox et. al., 2019). th e initial diff erence between dorsal and ventral pigment persists up to about the 13th gosner’s stage in wood frogs (gosner, 1960), which occurs about one week aft er oviposition. aft er stage 13, the embryo enters gastrulation and the distribution of pigment is more evenly distributed throughout the entire embryo (altig, 1972). as diff erentiation proceeds, the surface area to volume ratio of the embryo increases dramatically and the uniformly dark body acts as a heat sink. th e fact that this ontogenetic shift was absent in the mirror group suggests that despite our attempts to maintain similar degrees of light exposure in the two groups, a mirror may have been insuffi cient to match the thermal energy absorbed in the control group. an alternative hypothesis is that the diff erence in peripheral and core temperatures arises due either all, or in part, to metabolic heat production. th ere are at least three reasons to think that this may not be true: fi rst, ectotherms fig. 2. top. th e diff erence in temperature between core and periphery increased throughout embryonic development of wood frogs (rana sylvatica) for the control group but not in the mirror group. data points show mean values ± one standard error. bottom. picture of the study species. 163variation in egg mass temperatures of the wood frog produce negligible amounts of metabolic heat (andrade et al., 2015). second, we see no reason why differences in metabolism should have arisen between groups. third, egg masses held in a dark, temperature-controlled room as part of a separate experiment (calsbeek, unpublished) showed no variation in temperature between the center and periphery of the egg mass. given the rapid loss of polarity in the pigmentation of embryos, any thermal advantage to the dorsal orientation of the pigmented embryo should be short-lived (e.g., a few days). combined with the lack of an effect in the mirror group, we suggest that our results are consistent with a brief thermal advantage to an egg that rests sunny-side up (i.e., darker side dorsal), followed by a rapidly increasing thermal advantage associated with shifts in the surface area to volume ratio of the developing anuran embryo. understanding the importance of incubation temperature for amphibians with different oviposition behaviors (e.g., communal versus solitary nesting) could prove important for understanding the potential impacts of climate warming. future work should include tests for the joint roles of nesting behavior and incubation temperatures in tropical species, since temperatures in the tropics are both warmer and less variable than in the temperate zone (sinervo et. al., 2010). these differences in thermal regime suggest that tropical ectotherms may be especially vulnerable to climate warming, since even subtle changes in temperature could surpass thermal maxima (huey and tewksbury, 2009). as such, small differences in temperature within egg masses could have important implications for rates of development and/or survival for tropical amphibians. acknowledgments we thank madilyn gamble, ridhi chandarana and two anonymous referees for helpful comments that improved this experiment. rc was supported by an award from the national science foundation nsf deb1655092. all research was conducted with permission from the vermont department of fish and wildlife and iacuc protocol 00002097. references adrade, d.v., gavira, r.s.b., tattersall, g.j. 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(1982): adaptive significance of communal oviposition in wood frogs (rana sylvatica). behav. ecol. sociobiol. 10: 169-174. acta herpetologica vol. 17, n. 2 december 2022 firenze university press cryptic diversity in pygmy chameleons (chamaeleonidae: rhampholeon) of the eastern arc mountains of tanzania, with description of six new species michele menegon1,2,*, john v. lyakurwa3,4, simon p. loader5, krystal a. tolley6,7 preliminary genetic characterisation of southern smooth snake coronella girondica (serpentes, colubridae) populations in italy, with some considerations on their alpine distribution matteo r. di nicola1, raffaella melfi2, francesco p. faraone3,*, daniel l. n. iversen4, gabriele giacalone5, giovanni paolino1, mario lo valvo6 species diversity and distribution of amphibians and reptiles in sardinia, italy claudia corti1,2,*, marta biaggini1, valeria nulchis2, roberto cogoni2, ilaria maria cossu2, salvatore frau4, manuela mulargia2, enrico lunghi2, lara bassu2. the italian wall lizard, podarcis siculus campestris, unexpected presence on gorgona island (tuscan archipelago) marco a.l. zuffi1,*, alan j. coladonato2, gianluca lombardo3, antonio torroni3, matilde boschetti1, stefano scali4, marco mangiacotti2, roberto sacchi2 molecular analysis of recently introduced populations of the italian wall lizard (podarcis siculus) oleksandra oskyrko1,2,*, lekshmi b. sreelatha1,12,13, iolanda silva-rocha1, tibor sos3,4, sabina e. vlad5,6,7, dan cogălniceanu5,6, florina stănescu6,7,8, tavakkul m. iskenderov9, igor v. doronin10, duje lisičić11, miguel a. carretero1,12,13 sunny-side up: ontogenetic variation in egg mass temperatures of the wood frog rana sylvatica ryan calsbeek*, ava calsbeek, isabel calsbeek ecological niche differentiation in the anatolian rock lizards (genus: anatololacerta) (reptilia: lacertidae) of the anatolian peninsula and aegean islands mehmet kürşat şahin1,*, kamil candan2,3, danae karakasi4, petros lymberakis4, nikos poulakakis4,5,6, yusuf kumlutaş2,3, elif yıldırım2,3, çetin ilgaz2,3 occupancy and probability of detection of the introduced population of eleutherodactylus coqui in turrialba, costa rica jimmy barrantes-madrigal1,*, manuel spínola parallada1, gilbert alvarado 2, víctor j. acostachaves3,4. one site, three species, three stories: syntopy of geckoes euleptes europaea (gené, 1839), hemidactylus turcicus (linnaeus, 1758), tarentola mauritanica (linnaeus, 1758) in a coastal area of southern tuscany (central italy) giacomo radi1,2, marco a.l. zuffi1,* comparative cytogenetics on zamenis lineatus and elaphe quatuorlineata (serpentes: colubridae) marcello mezzasalma1,* , elvira brunelli1, gaetano odierna2, fabio m. guarino2 acta herpetologica 18(1): 61-67, 2023 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-12539 revisiting the polyploidy in the genus odontophrynus (anura: odontophrynidae) andré luis de souza, mayara aparecida das neves micalichen, roger alves da rocha, rafael bueno noleto* departamento de ciências biológicas, universidade estadual do paraná, 84600-185, união da vitória, paraná, brazil *corresponding author. e-mail: rafael.noleto@unespar.edu.br submitted on: 2022, 4th january; revised on: 2022, 6th august; accepted on: 2022, 23rd october editor: marcello mezzasalma abstract. the genus odontophrynus, composed of ten species, is found in practically the entire south of south america. odontophrynus americanus was the first vertebrate registered to present natural polyploidy, considering that most individuals have 2n = 4x = 44 chromosomes, although having 2n = 2x = 22 chromosomes is considered the ancestral condition for all genera of the family odontophrynidae. the present study aimed to analyze the karyotype of o. americanus, providing a detailed and comparative description of conventional chromosomal markers, with focus on a possible diploidization process operating in this polyploid genome. the individuals were collected in a fragment of atlantic forest in the south-central region of paraná state, brazil. the analyzed individuals presented the tetraploid pattern, with biarmed chromosomes. the c-banding showed heterochromatic regions restricted to centromeres and telomeres. among homologous chromosomes of the same quartet, small differences were observed in morphology, possibly the result of differentiation after the polyploidization event. finally, the 45s rdna (nucleolar organizer regions) was mapped in the short arm of quartet 11, showing the nucleolus organizing regions active in the four homologous chromosomes. this genome, although structurally polyploid, may be undergoing a process of diploidization, by becoming functionally equivalent to a diploid genome, via chromosomal rearrangements, epigenetic mechanisms, and/or repetitive dna dynamics. keywords. amphibian, diploidization, heterochromatin, rdna. according to frost (2023), the family odontophrynidae currently contains 55 species distributed in three genera macrogenioglottus carvalho, 1946, odontophrynus reinhardt and lütken, 1862, and proceratophrys miranda-ribeiro, 1920. earlier phylogenies validate the monophyly of the family, as well as that macrogenioglottus and odontophrynus are sister taxa (pyron and wiens, 2011; feng et al., 2017). the genus odontophrynus is composed of eleven species widely distributed in southern and eastern south america. odontophrynus americanus (duméril and bibron, 1841), a small fossorial anuran with no apparent sexual dimorphism (quiroga et al., 2015), has the greatest distribution, its range extends to central and southern argentina, southern paraguay, southern brazil, and uruguay (frost, 2023). odontophrynus americanus was the first case of natural polyploidy found in vertebrates (beçak et al., 1966). the odontophrynus americanus species group is a complex of morphologically indistinguishable diploid and tetraploid species. it includes currently four diploid species: o. cordobae martino and sinsch, 2002, o. juquinha rocha, sena, pezzuti, leite, svartman, rosset, baldo, and garcia, 2017, o. lavillai cei, 1985 and o. maisuma rosset, 2008 with 2n = 2x = 22 chromosomes, and one widely distributed tetraploid species (o. americanus) with 2n = 4x = 44 chromosomes (beçak et al., 1966; ruiz et 62 andré luis de souza et alii al., 1981; martino and sinsch, 2002; rosset et al., 2006; rosset, 2008). martino et al. (2019) established the existence of cryptic diversity and overestimation of species richness by combining molecular, morphological, and bioacoustic data. populations known as o. americanus comprise at least three species. polyploidy plays an important role in speciation and evolution in anurans, with about 50 polyploid species described in several families (bogart, 1980; mable et al., 2011; evans et al., 2012; schmid et al., 2015). polyploids originate by autopolyploidization (intraspecies wholegenome duplication) or allopolyploidization (associated with interspecific hybridization). thus, individuals with an autotetraploid genome can originate by fusion of unreduced (i.e., diploids) gametes, or by suppression of the first mitotic division in fertilized eggs (schmid et al., 2015). in recently evolved autopolyploids, the homologous chromosomes of a quartet are expected to exhibit identical chromosome banding patterns in somatic metaphases, leading to the multivalent formation during the first meiotic division. on the other hand, in an allopolyploid genome, if there are differences among the karyotypes of the parental species, the banding techniques or the genomic in situ hybridization (gish) allow chromosomes from parental species to be distinguished (schwarzacher et al., 1989), which will form bivalent configurations in meiosis (schmid et al., 2015). in this study, the structure of polyploid karyotype o. americanus from a southern brazilian population is described and subjected to comparative analysis in order to add new data regarding the speculated species complex. additionally, the data are placed in an evolutionary context, thus contributing to a better understanding of the evolutionary scenario concerning ploidy levels in this group. cytogenetic analyses were carried out on six juveniles of o. americanus collected in união da vitória, paraná state, brazil (26º13’48”s and 51º05’09”w). chromosome preparations were performed directly from bone marrow, according to baldissera et al. (1993). briefly, the animals received intraperitoneal injection of aqueous solution of colchicine (0.01 ml/g body weight) 1% per 6 h, and then subjected to deep sedation euthanasia by dermal absorption of lidocaine 5% pomade, following the recommendations of the ethical committee in animal use from universidade estadual do paraná. conventional staining was performed using 5% giemsa in sodium-phosphate buffer (ph 7.0, for 10 min). detection of the constitutive heterochromatin was accomplished according to sumner (1972). silver staining technique (ag-nor detection) was carried out according to howell and black (1980). the mitotic metaphases were analyzed under a carl zeiss axiolab a1 microscope equipped with the software zen lite and a zeiss axiocam icc1 camera with a resolution of 1.4 megapixels (carl zeiss, oberkochen, germany). chromosomes were classified based on the centromeric index according to green and sessions (1991) and were arranged in decreasing size. the specimens of o. americanus showed a karyotype of 2n = 4x = 44 chromosomes, distributed in eight metacentric quartets (1, 5–11) and three submetacentric quartets (2–4), thus presenting a fundamental number (fn) = 88 (fig. 1). there was no variation among the specimens karyotyped. exclusively between homologous chromosomes of quartets 2, 3, and 4, small differences were observed in terms of chromosomal morphology, which often made it difficult to organize these quartets. the centromeric indexes were established confirming the morphology discrepancies between homologs of the same quartet (fig. 1). according to the relative size of the chromosomes, the species has a karyotype with four different sizes of chromosomes: one large quartet (1), fig 1. giemsa-stained karyotype of o. americanus. highlighted the ag-nors site localized on the quartet 11. ci: centromeric index; ct: chromosome type; m: metacentric; sm: submetacentric; st: subtelocentric. bar = 10 µm. 63incipient diploidization process in odontophrynus three medium quartets (2–4), four small (5–8), and three very small (9–11). nucleolus organizer regions (nors) were observed on the short arm of quartet 11. such regions are coincident with secondary constrictions (fig. 1). a nor size heteromorphism between homologous chromosomes of the quartet was frequently observed. the c-banding showed the presence of constitutive heterochromatin in the centromeric and telomeric regions of almost all quartets (absence of centromeric bands in quartets 8 and 9), and coincident with ag-nor staining (quartet 11) (fig. 2). the family odontophrynidae was first established as a tribe within the (then) huge family leptodactylidae (lynch, 1971). the karyotype with 2n = 2x = 22 chromosomes is considered the ancestral condition, given its high frequency in all three genera. this characteristic karyotype is believed to have arisen from the differentiation of the primitive chromosome number of 2n = 26 chromosomes present in the family leptodactylidae, followed by centric fusions (bogart, 1973). karyotype descriptions of the genus odontophrynus reveal so far a very similar and conserved karyotype, which is composed exclusively of biarmed chromosomes, reflecting in fundamental numbers always twice the 2n, with some constant pairs in morphology between the species (table 1). these small variations are a consequence of chromosomal rearrangements that only modify the chromosome morphology, such as pericentric inversions, although the centromere repositioning, which alters the chromosome morphology without any accompanying chromosomal rearrangements (rocchi et al., 2012), could be an alternative pathway leading to chromosomal remodeling. a special interest has been devoted to the study of the occurrence of diploid (2n = 2x = 22) and tetraploid (2n = 4x = 44) constitutions in the o. americanus species group (see table 1). in this sense, several studies have indicated that it could consist of a complex of species (rosset et al., 2006; lanzone et al., 2008; cianciarullo et al., 2019; martino et al., 2019) and thus, the o. americanus listed with 22 chromosomes are expected to probably be other distinct species. the difficulty in organizing some quartets (i.e., 2–4) in conventional staining, due to small differences in the position of centromeres, may represent a prognosis for an incipient process of diploidization, as observed in other populations (ruiz et al., 1981; schmid et al., 1985). a structural heterogeneity must be created between homologous of quartets in the polyploid karyotype, which can originate even from small rearrangements such as pericentric inversions (ohno, 1970; ohno, 1974). therefore, the differences within the quartets in question can be interpreted as post-polyploid events, indicating a diploidization process operating in this polyploid genome (ohno, 1970; schmid et al., 1985; beçak, 2014). the variation of nors location in species of odontophrynus is the result of translocations (beçak and beçak, 1974) and/or transposable elements-mediated transpositions events (gray, 2000; mandrioli, 2000), which switched these ribosomal genes to other pairs promoting karyotype diversification. the karyotype with nors on pair 11 is considered as the plesiomorphic condition, found in diploid species from three species groups of odontophrynus, as well as in most individuals studied from tetraploid populations of o. americanus (see table 1). a size heteromorphism between homologous was frequently observed. the presence of nor-associated heterochromatin demonstrated that this heteromorphism between homologous of quartet 11 comprises both functional and structural aspects. this condition may have facilitated breaks and transpositions of rrna genes to other sites in different species and populations of odontophrynus (wiley et al. 1989; carvalho et al. 2014). fig 2. c-banding karyotype of o. americanus. bar = 10 µm. 64 andré luis de souza et alii heteromorphic nors could also be related to differences in genetic activity (amaro-ghilardi et al., 2008). in fact, in polyploids, while the number of 45s rdna citrons is proportional to the degree of ploidy, gene expression may be equivalent to a diploid genome (schmidtke and engel, 1976). epigenetic mechanisms are responsible for modulating gene expression through chemical modifications of histones, via methylation, acetylation, and/or phosphorylation (furey and sethupathy, 2013). equalization of gene activity between 2x and 4x species could be at the transcriptional level, probably by rdna methylation (hashimshony et al., 2003). indeed, ruiz and brison (1989) found high levels of methylation of ribosomal genes in tetraploid genomes of o. americanus. it has been validated by cianciarullo et al. (2000), who found only 25–30% more ribosomes in o. americanus tetraploid than do 2n cells. therefore, polyploid genomes may become functionally diploid throughout evolution (schmid et al., 2015). the presence of constitutive heterochromatin on centromeric and telomeric regions is an expected pattern in odontophrynus. the eventual variation involves the additional presence of interstitial bands that characterize some species/populations (see table 1). the variation in the distribution pattern of constitutive heterochromatin is generally associated with the dynamics of different classes of repetitive dna. heterochromatin is normally rich in repetitive sequences, which can have important functions in speciation and/or adaptation, as they are less subject to selective pressures, favoring the accumulation of differences throughout the evolutionary process (martins, 2007; böhne et al., 2008). in conclusion, the intraand interpopulation chromosomal variability in odontophrynus is a consequence of its wide geographic distribution throughout south america. regarding polyploidy within the group, its origin via autopolyploidization seems to be the most accepted, mainly due to the presence of multivalents at meiosis (beçak et al., 1966; schmid et al., 1985; lanzone et al., 2008). however, multivalent formation can also be observed in some allopolyploids, because the structure of chromosomes from different species (i.e., homeologous) can be sufficiently conserved to permit multivalent associations. autopolyploids, on the other hand, might also have mechanisms that prevent multivalent contable 1. summary of the chromosome findings of the species of odontophrynus: diploid number (2n), centromeric heterochromatin (ⓒ), telomeric heterochromatin (ⓣ), interstitial heterochromatin ⓘ, nucleolus organizer region (nor), short arm (p), long arm (q), fundamental number (fn), *artificial hybrid. species group species locality 2n ploidy level c-banding nor fn reference o. americanus o. americanus brazil 22 2x 4p 44 ruiz et al. (1981) argentina 22 2x ⓒⓣⓘ 4p 44 ruiz et al. (1981) brazil 22 2x ⓒⓣⓘ 4p, 11p 44 ruiz et al. (1981) argentina 33 3x 66 grenat et al. (2018) brazil 44 4x 11p 88 beçak et al. (1966) argentina 44 4x 11q 88 bogart (1967) uruguay 44 4x 4p 88 ruiz et al. (1981) argentina 44 4x 88 grenat et al. (2018) brazil 44 4x ⓒⓣⓘ 11p 88 ruiz et al. (1981) uruguay 44 4x ⓒⓣⓘ 4p, 11p 88 ruiz et al. (1981) argentina 44 4x ⓒⓣⓘ 11p 88 schmid et al. (1985) brazil 44 4x ⓒⓣ 11p 88 present study uruguay 66* 6x 11p 132 ruiz et al. (1981) o. cordobae argentina 22 2x 1144 martino and sinsch (2002) argentina 22 2x 4p 44 salas and martino (2007) o. juquinha brazil 22 2x 4p 44 rocha et al. (2017) o. lavillai argentina 22 2x 4p 44 rosset et al. (2006) o. maisuma uruguay 22 2x 4p 44 rosset (2008) uruguay 22 2x ⓒⓘ 4p 44 borteiro et al. (2010) o. reigi argentina, brazil, paraguay 22 2x 4p 44 rosset et al. (2021) o. cultripes o. cultripes brazil 22 2x ⓒⓣⓘ 11p 44 ruiz and beçak (1976) o. carvalhoi brazil 22 2x ⓒⓣⓘ 8p 44 ruiz et al. (1981) o. occidentalis o. occidentalis argentina 22 2x ⓒⓣⓘ 9q, 11p 44 ruiz et al. (1981) 65incipient diploidization process in odontophrynus figuration and thus form bivalents (gregory and mable, 2005). therefore, distinguishing between autoand allopolyploidization is difficult, since the scenario possibly involves a combination of both mechanisms. the disjunct tetraploid populations are closely associated with several diploid species, which suggests that polyploidy has multiple origins, with putative older lineages accumulating more chromosomal changes within the homologous quartets. the evidence suggests that the benefits of polyploidization are stabilized by epigenetic mechanisms, small structural rearrangements, and repetitive dna dynamics, which lead the tetraploid genomes to become functionally diploid (diploidization). given this scenario, the analysis throughout the chromosomal mapping of repetitive elements represents a crucial tool for clarifying the dynamic processes concerned with the karyotype diversification in odontoprhynus species, especially in this group with the uncertain taxonomic assignment. acknowledgments we would like to express our thanks to two anonymous reviewers for helpful comments that improved the manuscript. the authors are grateful to the sistema de autorização e informação em biodiversidade (sisbio) (63336-1) for authorizing the capture of specimens and ethical committee in animal use of the universidade estadual do paraná for authorizing the execution of the project (process ceua 2021/001). besides, we are grateful to flávia thaís carneiro for proofreading the manuscript. this work was supported by fundação araucária for funding (grant number 073/2018). references amaro-ghilardi, r.c., silva, m.j.d.j., rodrigues, m.t., yonenaga-yassuda, y. 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(1972): a simple technique for demonstrating centromeric heterochromatin. exp. cell res. 75: 304-306. wiley j.e., little m.l., romano m.a., blount d.a., cline, g.r. (1989): polymorphism in the location of the 18s and 28s rrna genes on the chromosomes of the diploid-tetraploid tree frogs hyla chrysoscelis and h. versicolor. chromosoma 97: 481-487. threats of the emerging pathogen batrachochytrium salamandrivorans (bsal) to italian wild salamander populations lorenzo dondero1, giorgia allaria1, giacomo rosa1, andrea costa1, gentile francesco ficetola2, roberto cogoni3, elena grasselli1, sebastiano salvidio1,* age estimation and body size of the parsley frog, pelodytes caucasicus boulenger, 1896 from lake borçka karagöl, turkey cantekin dursun*, serkan gül, nurhayat özdemir patterns of acoustic phenology in an anuran assemblage of the yungas andean forests of argentina martín boullhesen1,2,*, marcos vaira1, rubén marcos barquez2, mauricio sebastián akmentins1 diet and trophic niche overlap of four syntopic species of physalaemus (anura: leptodactylidae) in southern brazil renata k. farina1, camila f. moser2, stefano scali3, mateus de oliveira4, patrícia witt5, alexandro marques tozetti1,* screening of ophidiomyces ophidiicola in the free-ranging snake community annually harvested for the popular ritual of san domenico e dei serpari (cocullo, aq, italy) daniele marini1,2, ernesto filippi3,*, gianpaolo montinaro4, francesco c. origgi5,6 assessment of fall season habitat and coverboard use by snakes in a restored tallgrass prairie community carter dollen1,2, tracy j. coleman1,2, travis r. robbins1,2,* revisiting the polyploidy in the genus odontophrynus (anura: odontophrynidae) andré luis de souza, mayara aparecida das neves micalichen, roger alves da rocha, rafael bueno noleto* issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 7(1): 119-138, 2012 skeletal development and adult osteology of hypsiboas pulchellus (anura: hylidae) julio m. hoyos1, marcelo r. sánchez-villagra2, alfredo a. carlini3, christian mitgutsch2,* 1 pontificia universidad javeriana, facultad de ciencias, departamento de biología, unidad de ecología y sistemática, a.a 56710, bogotá, d.c., colombia. 2 paläontologisches institut und museum, universität zürich, karl schmid-strasse 4, ch-8006 zürich, switzerland. current address: laboratory for evolutionary morphology, riken center for developmental biology, 2-2-3 minatojima-minamimachi, chuo-ku, kobe, 650-0047 hyoko, japan. *corresponding author. e-mail: christian.mitgutsch@gmail.com. 3 paleontología de vertebrados, museo de la plata, paseo del bosque s/n (b1900fwa), la plata, buenos aires, argentina. submitted on: 2011, 7th july; revised on: 2012, 18th april; accepted on: 2012, 27th april. abstract. osteological and skeletal characters have long been proven to be particularly informative in taxonomic and systematic research. furthermore, ossification sequences are assumed to be a potential tool to investigate developmental states and developmental modes of fossil and extant skeletal specimens. herein, we provide a detailed account on adult osteology and skeletogenesis in the montevideo treefrog, hypsiboas pulchellus (anura: hylidae) based on evaluation of a series of cleared and stained specimens. a consensus sequence of ossification, i.e., the order of appearance of mineralized elements until early metamorphosis could be determined as (parasphenoid, presacral vertebrae i-vii, frontoparietal, exoccipital) – transverse processes of presacral vertebrae i-viii – sacral vertebra – (humerus, radioulna, ilium, femur, tibiofibula, scapula) – (cleithrum, clavicle, coracoids, metacarpals, tarsals, metatarsals, phalanges, hypochord) – (prootic, angulosplenial, dentary, maxilla, premaxilla, squamosal). comparing the state of mineralized elements in individual specimens, a number of skeletal elements, including the exoccipital, frontoparietal, parasphenoid and prootic, as well as elements of the shoulder and pelvic girdles, and the phalanges, were found to vary intraspecifically regarding the relative time of their ossification within the ossification sequence. keywords. anuran anatomy, comparative developmental biology, hylidae, hypsiboas pulchellus, sequence of ossification, skeletogenesis 120 j.m. hoyos et al. introduction studies of osteology and skeletal development in anuran amphibians have a long legacy in biological research; the relative ease of access to embryos and a stunning diversity of life histories render anurans to be particularly suitable targets for comparative studies in skeletal and skeletogenic evolution. in particular, understanding the evolution of skeletal repatterning in metamorphosing species is a subject worth of investigation (e.g., duellman and trueb, 1994; gurdon and hopwood, 2000; callery, 2006; crump, 2009). detailed descriptions of anuran skeletons are available from a wide range of taxa, both extinct and extant. summaries of osteological patterns and interpretations of taxonomic distributions of osteological characters can be found in trueb (1973, 1993), báez (2000), rage and roček (2000), roček (2000, 2003), clarke (2007); examples of in-depth descriptions and discussions of anuran chondrocrania in gaupp (1896), ramaswami (1944), van eeden (1951), de sá and trueb (1991), haas (1995), de sá and hill (1998), larson and de sá (1998), swart and de sá (1999), larson (2002), and larson et al. (2003). particularly, the cranial development of frogs has been discussed in connection with a variety of topics, including conceptual issues such as head segmentation (vogt, 1842; stöhr, 1881) and life history evolution including traits and topics such as direct development, metamorphosis, or miniaturization (e.g., trueb and alberch, 1985; hanken et al., 1992; rose and reiss, 1993; yeh, 2002; maglia et al., 2007; kerney et al., 2007, 2010). in the context of vertebrate skeletogenesis, special focus has been given to the evolution of ossification sequences, i.e., accounts on the relative order in which bones ossify in a given taxon. heterochronies within ossification sequences have been shown to be potentially informative about life history traits (weisbecker et al., 2008). furthermore, ossification sequences could enlighten the life histories of fossil embryonic specimens using an extant phylogenetic bracket (e.g., balanoff and rowe, 2007). great effort has been put into establishing ossification sequences in a considerable variety of species from a wide range of vertebrate taxa. tools to analyze ossification sequences and temporal sequences of other developmental events have been developed and applied in a variety of studies (e.g., nunn and smith 1998; richardson et al., 2001; jeffery et al. 2002a, b, 2005; koenemann and schram, 2002; poe and wake, 2004; schulmeister and wheeler, 2004; harrison and larsson, 2008; germain and laurin, 2009). from several taxa however, increasing evidence has been accumulated that intraspecific variation in ossification sequences might be considerable and that ossification sequences potentially evolve relatively quickly (hanken and hall, 1984; dunlap and sanchiz, 1996; mabee and trendler, 1996; mabee et al., 2000; bininda-emonds et al., 2003; moore and townsend, jr., 2003; sheil and greenbaum, 2005; maxwell, 2008; weisbecker and mitgutsch, 2010; mitgutsch et al., 2011). as a consequence, while ossification data are available from a broad taxonomic range, the scarce availability of taxonomically densely sampled datasets makes closer investigations of such sets highly desirable. de sá and trueb (1991) stated that for no more than 0.6% of all described anuran species ossification sequences have been reported. since then, several ossification sequences for neobatrachians in general and for hylids in particular have been published, but many more new anuran species have also been described. sheil and alamillo (2005) emphasized that very few osteological or developmental descriptions of hylids exist, giv121skeletogenesis and osteology of hypsiboas pulchellus en the total number of hylid species described (almost 900). besides, some of the studies in which ossification sequences were reported, do not provide information on postcranial ossification although a number of studies did cover descriptions of whole ossification sequence (de sá, 1988; haas, 1996; maglia and púgener, 1998; púgener and maglia, 1997; dunlap and sanchiz, 1996; wiens 1989; haas, 1999; wild, 1999; sheil and alamillo, 2005; trueb et al., 2000). within hylids, either cranial or postcranial ossification sequences are available for a number of species, namely hypsiboas lanciformis (de sá 1988), pseudacris triseriata (stokely and list 1954), p. regilla (gaudin 1973), phyllomedusa vaillanti (sheil and alamillo 2005) smilisca baudinii, triprion petasatus, and osteopilus septentrionalis (trueb 1970); for a summarized comparison of the cranial ossification patterns in these species see weisbecker and mitgutsch (2010). in the present study, we describe the development and the adult osteology of both the cranial and postcranial skeleton of a hylid frog species, the montevideo treefrog, hypsiboas pulchellus. furthermore, we provide information about intraspecific variation in the relative order of the onset of ossification in individual skeletal elements. materials and methods specimens of hypsiboas pulchellus (duméril and bibron, 1841) were collected from the wild near la plata, argentina. the animals were anesthetized and fixed in 4% buffered formaldehyde. after fixation, the frogs were cleared and double stained for the visualization of mineralized bone and cartilage with alcian blue and alizarin red following standard laboratory protocols (dingerkus and uhler, 1977). prior to documentation, selected specimens were partially dissected to allow for visualization of individual parts of their skeletons. to study intraspecific variation in ossification orders, ontogenetic states-tables were filed as suggested by mitgutsch et al. (2011). the numbers of specimens sharing one ontogenetic state are shown in fig. 8. skeletal specimens examined were: one adult male specimen, one freshly metamorphosized froglet and 32 tadpoles of gosner-stages (gs; gosner, 1960) gs43/44 (2 individuals), gs42 (3), gs41 (5), gs39/40 (4), gs 37 (1), gs36 (3), gs35 (2), gs34 (3), gs33 (2), gs31 (2), and gs30 and younger (5). anatomical nomenclature herein is largely on the basis of haas (1999) and sheil and alamillo (2005); for further discussions see trueb (1973) and roček (2003). results adult osteology cranium (fig. 1a-d) the septomaxillae are paired bones located dorsally to the vomers, within the nasal capsules and embedded in the nasal cartilages. they are situated posterolateral to the alary cartilages. each septomaxilla has three processes: anterolateral, dorsomedial and posterior. the dorsomedial and the posterior processes are longer than the anterolateral process. the septomaxillae are medially separated. 122 j.m. hoyos et al. the nasals are paired bones, have a crescent shape, lying in an oblique position. they are separated medially; the septum nasi is not covered by them. the posterior margin of each nasal invests (or overlaps) the anterior margin of the sphenethmoid. the maxillar process is large, articulating with the pars facialis of the maxilla. they do not have a rostral process; they are not in contact with the premaxilla. the frontoparietals are paired and narrow bones separated by a large gap. their medial borders are concave, the lateral borders convex. the anterior ends overlap with the dorsolateral and the posterior edge of the sphenethmoid. posterolaterally, each bone invests both the epiotic eminence (= eminentia epiotica) of the prootic and the anterior end of the exoccipital bone. the parasphenoid is a flat, in ventral view t-shaped appearing bone, the long bar pointing anteriorly. the cultriform process is pointed at the anterior end, wide in the middle and constricted posteriorly. the parasphenoid alae are distally truncated, slightly inclined, extending posterolaterally to invest the lateral border of the otic capsules (prootic and exoccipital). the posteromedial process reaches the ventral margin of the foramen magnum. the vomers are paired and dentigerous (six teeth). they are medially articulated to each other and are posteriorly either articulated or fused to the proximal end of the palatine bones; the vomers are not articulated to the pars facialis of the maxilla. the vomers have four processes: the odontophore (dentigerous process) is slightly inclined and posterior to the palatine. the preand postchoanal processes are very short, placed anterior and postefig. 1. skeletal elements of the adult cranium. a) dorsal, b) lateral, c) dorsal view; d) ventral view on jaw and maxilla. abbreviations: asp, angulosplenial; cpr, coronoid processus; den, dentary; ee, epiotic eminence; exo, exoccipital; fp, frontoparietal; fpf, frontoparietal fenestrum; mm, mentomeckelian; mx, maxilla; nas, nasal; npal, neopalatine; papm, pars alaris premaxillaris; pfpm, pars facialis premaxillaris; pmx, premaxilla; pr, prootic; ps, parasphenoid; pt, pterygoid; qj, quadratojugal; sph, sphenetmoid; sq, squamosal; ts, tectum synoticum; v, vomer. scale 1 mm. 123skeletogenesis and osteology of hypsiboas pulchellus rior to the odontophorous process. the vomers have a free and truncated anterior processus. posteriorly, the vomers are fused to the anterior border of the sphenethmoid. the premaxillae are upside down t-shaped, paired bones consisting of three parts: pars alaris, p. palatina, and p. dentalis. the pars alaris projects posterodorsally; the dorsal ends of the partes alares have a notch. the pars dentalis is the base of the “t” bearing the teeth. the partes dentales are separated by a narrow space between them. in our largest specimen we found 15 teeth in each of the partes dentales. the posterolateral element of the pars dentalis (or pars facialis sensu sheil et al., 2005) slightly overlaps with the anterolateral end of the pars facialis of the maxilla. each pars palatina bears triangular, prominent posteromedial processes, slightly widened posterolaterally. these processes contribute to the articulation of the premaxillae along the medial borders of its posteriomedial process. the pars palatina is as long as the pars dentalis. the posterior (distal) end is in contact with the pars palatina of the maxilla and is slightly widened. the medial part is concave, the proximal ones triangular. the premaxillar pars palatina is not articulated to the pars palatina of the maxilla. the maxillae are composed of three parts: facialis, palatina, and dentalis. the pars dentalis of each maxilla bears nearly 60 teeth in our largest specimen. the anterior end of the pars dentalis overlaps the posterior end of the pars dentalis of the premaxilla. the maxilla extends posteriorly, overlapping laterally the first third of the quadratojugal. the pars facialis is much higher than the pars dentalis in the preorbital region, diminishing in height towards the orbital region. the pars facialis projects onto the planum antorbitale and articulates with the maxillary processes of the nasals. the pars palatina does not articulate with the pars palatina of the premaxilla. suspensorium the squamosals are well ossified and triradiate: the pars zygomatica has a mediolaterally flat anterior end; pars otica has an otic plate, and the ventral (pterygoid) ramus that invests in both the external part of the quadratojugal and the distal end of the posterior arm of the pterygoid bone. the pars pterygoidea supports the tympanic annulus, and its proximal end is wider than the distal end. the posterior border of the proximal end is concave, while the distal end is flat; its maxillar slope is about 45°. the pars otica is short and wide and unornamented, bearing an otic plate which overlaps the crista parotica. the zygomatic ramus is twice as long as the otic ramus, without including the otic plate. the pterygoid is well ossified and edentate. it has three rami arranged in a y-shape: the anterior ramus articulates to the dorsal surface of the pars palatine of the maxilla at the level of the orbit, and just to the proximal end of the maxilla. the medial ramus articulates with the ventrolateral margin of the prootic-exoccipital. the posterior ramus invests the point of articulation of the ventral arm of the squamosal, the quadratojugal and the articular process of the angulosplenial. this ramus runs parallel and very close to the ventral ramus of the squamosal. the quadratojugals are short bones that are positioned posterior to the maxillae. the anterior portion of the quadratojugal invests the posterior and external surface of the maxilla; it articulates with the pars articularis of the angulosplenial and with the inner surface of the distal end of the pterygoid branch ventral to the squamosal. the anterior and dorsal end is dagger-shaped, whereas the posteroventral end forms a cup-like base. 124 j.m. hoyos et al. the articular region of the palatoquadrate is ossified forming the quadrate bone and fused to the quadratojugal. the mandibles are comprised of the mentomeckelian, dentary, and angulosplenial bones. they are well ossified and edentate. the mentomeckelian bones are located in the anterior part of the mandible and separated from their antimere by a symphyseal space. they are fused to the dentaries. each dentary invests laterally and externally more than one half of both the angulosplenial bone and the meckel´s cartilage. it forms the anterior half of the mandibular arch (half the length of the mandible) with a pointed posterior end. the angulosplenials extend from the posterior end of the dentaries, to its articulation with the quadratojugal by the articular process. in the prearticular region, the angulosplenial bears a medial and posterior well-developed dorsomedial coronoid process. meckel´s cartilage is found between the dentary and the angulosplenial, extending from the distal end of the mentomeckelian bones and to four-fifths the length of the angulosplenial. the prootics and exoccipitals are fused synostotically. the exoccipitals form the posterior end of the neurocranium; they are also a part of the dorsal surface of the skull joining the frontoparietals, and most of the margin of the foramen magnum. they form the ossified and rounded exoccipital condyles too. the anterior margin of the exoccipital is part of the posterior part of the parietal fenestrum. the prootics are partially invested by the posterior end of the frontoparietals. the crista parotica is in contact with the otic plate of the squamosal; it bears posterolateral processes with cartilaginous terminal ends. the palatines (for discussion on nomenclature see de sá and trueb, 1991; trueb, 1993) are medially articulated to the vomers and to the sphenethmoid. the palatines have a distal double articulation to the maxilla: with the pars facialis and with the anterior end of the pars palatina, but do not articulate with the distal end of the anterior ramus of the pterygoid bone. it invests ventrally and posteriorly the cartilaginous planum antorbitale. the sphenethmoid is an unpaired bone that forms the anterior part of the braincase. it is visible from dorsal between the frontoparietals and nasals, and from ventral between the vomers and the parasphenoid bone. the dorsal and posterolateral margins of the sphenethmoid are partly covered by the frontoparietals. the nasals overlap the anterior borders of the sphenethmoid. ventrally, the sphenethmoid is a short bone extending over the anterior one third of the skull floor. ventrally and posteriorly, the sphenethmoid reaches the anterior end of the cultriform process of the parasphenoid; the anterior border of the sphenethmoid is dorsal to the dentigerous processes of the vomers. the annulus tympanicus of the plectral (auditory) apparatus (fig. 2) is dorsally incomplete. the partes media et interna plectri are ossified, and fused synostotically to each other; the pars externa plectri is cartilaginous, distally enlarged, and approximately two thirds of the length of the stapes (partes interna and media plectri). the pars externa plectri has an angle of nearly 45° to the pars media plectri. the central corpus (=hyoid plate) of the hyobranchial skeleton (fig. 3) is partially ossified. the width of the hyoid plate is about three times the anteroposterior length. the u-shaped hyoglossal sinus is deep and wide. the cartilaginous hyalia (=hyales sensu maglia et al., 2007) extend posterolaterally and recurve to articulate with the otic capsule. there are no anterolateral processes. the posterolateral processes are very short compared to the posteromedial processes, with pointed posterior ends. the posteromedial process125skeletogenesis and osteology of hypsiboas pulchellus es are well ossified, with their distal and proximal ends dilated, approximately equal in width. the proximal ends are clearly separated by the posterior border of the hyoid plate. a parahyoid bone is absent. the laryngeal apparatus occupies most of the space between the posteromedial processes of the hyoid apparatus. the arytenoid cartilages are hemispherical, medially separated, with medioventral faces concave and separated. the cricoid rings are triangular, and bear bronchial processes that extend posteroventrally from the anterolateral margins of this ring. their distal ends are not curved medially. postcranium the vertebral column comprises eight procoelous presacral vertebrae, a sacrum and the urostyle. the urostyle forms a bicondylar articulation with the sacrum. vertebrae are not fused, neural spines are minute, and presacral vertebra viii is not fused to the sacrum. the presacral vertebra i is type i (lynch, 1971), having widely separated atlantal cotyles (fig. 4). the relative lengths of the transverse processes of the presacral vertebrae are: sacral diapophyses > iii > iv > viii > ii ≥ v ≥ vi ≥ vii. the transverse processes of presacral vertebrae ii, vii, and viii are oriented anteriorly, whereas those of vertebrae iii, iv, and v are oriented posteriorly, and those of vertebra vi are perpendicular to the axis of the vertefig. 3. hyobranchial apparatus (male). abbreviations: c, corpus (hyoid plate); hy, hyoid; plpr, posterolateral processus; pmpr, posteriomedial processus. scale 1mm; blue: cartilage, red: mineralized bone. fig. 2. auditory apparatus, abbreviations: op, operculum; pep, pars externa plectra; pip, pars interna plectra; pmp, pars media plectra; tmp, tympanic. scale 1mm; blue: cartilage, red: mineralized bone. 126 j.m. hoyos et al. bral column. the sacral diapophyses are broadly expanded and oriented laterally from the midline of the body. the transverse processes of vertebra ii are more robust than those of other vertebrae. their lateral margins are expanded and slightly cartilaginous. the urostyle is rounded with a spinal process (urostyle crista); its sacral articulation is bicondylar. the neural spine of the axis is projected over the posterior border of the atlas. the pectoral girdle (fig. 5) is arciferal. the epicoracoids are ossified and broadly overlapping. the epicoracoids are connected to each other at the epicoracoid bridge, at the base of the omosternum. the procoracoid is ossified; it is continuous with the epicoracoid, bordering distally about one third of the posterior edge of the clavicles. the omosternum has two elements: the distal element, cartilaginous and expanded, and the proximal one (stylet), calcified and laterally cartilaginous. the sternum consists of one element; proximally, it is slightly calcified, distally it is cartilaginous and notched. the pectoral fenestrum is ovoid and long. the clavicles are ossified, deeply concave anteriorly and separated medially by the epicoracoid bridge. distally close to the articulation with the pars acromialis (lateral end), they are much more expanded than at the proximal end. the clavicles are also articulated with the coracoids. the anterior and posterior margins of the coracoids are concave. the sternal (proximal) ends of the coracoids are wider than the glenoid (proximal) ends. the coracoids do not articulate with the sternum, but with the epicoracoid. the scapulae are well ossified, expanded at the ends and proximally bicapitate: their partes acromiales articulate with the clavicles, and their partes glenoidales articulate with both the coracoids and the clavicles. the scapula is nearly as long as the clavicle. the anterior margin is concave, as the proximal posterior margin, and two thirds of the distal posterior margin is convex. the distal end of the scapula articulates with the ossified suprascapula which is distally expanded and exhibits anteriorly a hooked process (processus suprascapularis, fig. 5c). the posterior border of the suprascapula is weakly ossified. the cleithrum is well ossified, occupying nearly one third of the suprascapula. at the anterior and medial borders there is a curved process which is mostly cartilaginous but mineralized at the base. the forelimb has proximally a well ossified humerus with a prominent crista ventralis (=deltoid crest sensu trueb and báez, 2006), extending over nearly the proximal two thirds of the shaft of the humerus. the proximal (glenoid) and distal (olecranon sensu sheil and alamillo, 2005; eminentia capita sensu gaupp, 1896) heads are well ossified. distally the humerus bears a short and prominent crista lateralis which extends over the distal one fourth of the anterodorsal border of the shaft of the humerus. the radioulna (for discussion of the nomenclature see maglia and púgener, 1998; maglia et al., 2007) is well ossified. although radius and ulna are fused at the midline, a distinct and incomplete sulcus intermedius (sensu sheil and alamillo, 2005; groove sensu maglia and púgener, 1998) is fig. 4. adult vertebrae, dorsal view; trpr, transversal processus. scale 1 mm. 127skeletogenesis and osteology of hypsiboas pulchellus present. the sulcus is deeper at the distal half than at the proximal half. the radioulna is about three-fourth of the length of the humerus. it has a well-developed olecranon (=ulnar) process articulating with the distal head of the humerus. the manus (fig. 6a) has six ossified carpal elements: ulnare, radiale, distal carpal 5-4-3, distal carpal 2, element y and proximal prepollex. the relative size of the carpal elements is: carpal 5-4-3 > ulnare > radiale > element y > carpal 2 > proximal prepollex. the distal carpal 5-4-3 is articulated with all carpal elements, except the proximal prepollex, and it is articulated also to the proximal head of the metacarpals iii, iv, and v. the prepollex has two elements, the proximal element being shorter than the distal, claw-shaped one. both are completely ossified. the relative lengths of the metacarpals are iv > iii > v > ii. the relative lengths of the digits are iv > v > iii > ii, and the phalangeal formula is 2-2-3-3. small, flat, and slightly calcified intercalary elements are articulated between the overlapping ventral and distal end of the penultimate phalange, and the dorsal and proximal end of the ultimate phalange. the distal phalanges are bottle-shaped. the proximal and distal ends, except those of the ultimate phalanges, are covered by calcified and fibrous tissue. no sesamoid elements were observed. the elements of the pelvic girdle (fig. 7) are ossified, except the slightly fig. 5. (a) ventral and (b) lateral view of the pectoral girdle skeleton, (c) dorsal view suprascapula. abbreviations: cla, clavicle; clt, cleithrum; crc, coracoid; epc, epicoracoid; epcb, epicoracoid bridge; hum, humerus; omo, omosternum; pac, pars acromialis; prsu, suprascapular process; scp, scapula; sscp, suprascapula. scale 1mm; blue: cartilage, red: mineralized bone; epc, crc depicted partly “semintransparent” to demonstrate the outline of both elements. 128 j.m. hoyos et al. cartilaginous medial symphysis between each pubis. the ilia do not have ilial crests. posteriorly, the ilia have a dorsal protuberance (=ilial protuberance sensu maglia et al., 2007). the internal margins of the ilia are v-shaped in dorsal view. medially the three elements are fused. the ischia are fused with the pubes. the anterior end is articulated with the lateroventral surface of the corresponding sacral diapophysis. the articulation extends over nearly one third of the length of the ilial shaft. the ischium bears an interischiadic crest which is located posterodorsal to the acetabular area, well developed and posteriorly flattened. the demarcation between the ischia and the pubes, and the pubes and the ilia are clearly visible. the articulation between the ilia and the ischia is also clearly differentiated. the articulation of the ilia and the sacral diapophyses is articulation type i (emerson, 1979). the femur of the hindlimb is well ossified; the ventral and the posterior borders have a femoral crest along the posterior to the proximal end. this crest runs along one-seventh of the length of the femur. proximally, the femur is fig. 6. (a) right hand in dorsal view, (b) left foot in dorsal (top) and ventral (bottom) view, not to scale. abbreviations: c, carpals; dpp, distal prepollex; dp1, distal prehallux 1; dp2, distal prehallux 2; fib, fibulare; mp, manual phalanges; pp, prepollex; pph, pedal phalanges, r, radius; ra, radiale; t, tarsals; tib, tibiale; u, ulna; ul, ulnare; y, element y. fig. 7. pelvic skeleton, abbreviations: sd, sacral diapophysis; ur, urostyle. scale 1 mm. 129skeletogenesis and osteology of hypsiboas pulchellus slightly curved anteriorly. the tibiofibula is approximately equal in length to the femur. it is compressed and narrowed at the midshaft; the distal and proximal ends are equal in size, with grooves just proximally and distally. the tibiale and fibulare of the pes (fig. 6b) are nearly equal in length, and are fused distally and proximally. they are almost two-thirds as long as the tibiofibula. the relative sizes of the tarsal elements are: y > 3 = 4 > 2. the fibulare articulates directly with metatarsals iii, iv, and v. the tarsal 2 articulates with the metatarsal i, and the tarsal 3-4 articulates with the metatarsals ii and iii. the element y is ventral to the tarsal 1, and is articulated to the tibiale. the prehallux has four elements: the prehallux and three distal prehallical elements. the prehallux is ossified; the three distal prehallical elements are calcified and smaller than the prehallux. relative lengths of the metatarsals are: iv > v > iii > ii > i. the relative lengths of the digits are: iv > iii = v > ii > i. the phalangeal formula (from digit i to v) is 2-2-3-4-3. the intercalary elements are small, slightly calcified, between the ventral end of the penultimate phalange and the dorsal end of the distal phalange. the distal phalange is bottle-shaped. no sesamoid elements could be demonstrated. larval osteology the skeletal morphology of juvenile individuals is representatively described taking into account several cleared and stained specimens. the onset of ossification of the individual skeletal elements is shown in fig. 8. cranium in individuals at gs39/40 (fig. 9), the cartilago labialis superior (sensu haas, 1999); suprarostral cartilage (sensu maglia and púgener, 1998) of the chondrocranium is a discontinuous plate divided in a central corpus (corpus rostrale) and a posterior and dorsolaterally expanded ala (pars alaris haas, 1999); the two parts are articulated. the suprarostral cartilages lack foramina but bear both notches on their ventral margins and small posterior processes. each ala is posteriorly enlarged, and is dorsolateral and ventrally expanded. they are not fused but articulated to the cornu trabeculae. each ala has three processes, two of which are articulated to the cornu trabeculae and one with the corpores suprarostrales. the dorsal posterior process (processus dorsalis) extends dorsad to the cartilage of meckel. there are no fenestrations between corpus and ala. an adrostral cartilage is absent. the admandibular cartilages are present on the anteroventral margin of meckel’s cartilage. meckel’s cartilages are l-shaped structures, each projecting anteromedially and slightly dorsally to articulate with the infrarostral cartilage. their proximal ends are articulated with the partes alares and with the corpus suprarostralis. the angle of divergence is nearly 25°. the cornua trabeculae form the planum ethmoidale; they are relatively short (one fifth of the length of the chondrocranium), taking into account a subterminal mouth, with a process for the joint of the ligamentum quadratoethmoidale. at the anterior end they support the suprarostral cartilages, and articulate laterally with the partes alares. ventrally they are joined at the ethmoidal plate. the ethmoidal plate is formed at the posterior end of the cornua trabeculae. there is an association between the tectum nasi and the commissura quadratocranialis. the tectum nasi is formed by cartilage posterior to the choanae, dorsal to the ethmoidal plate. the basis cranii (basal plate) forms 130 j.m. hoyos et al. fig. 8. ontogenetic states regarding the presence of ossified skeletal elements in the postcranium (top) and cranium (bottom) of hypsiboas pulchellus. the table heads show the skeletal elements that have been found mineralized in at least one specimen followed by the number of states in which the elements were found present in brackets. the table lists all combinations (states) of mineralized bones that have been observed in at least one specimen (gray: mineralized element present, white: mineralized element not present). the states are named with the total number of mineralized elements, with an additional letter if different combinations were found for the same number of elements and the number of individuals in which the state was observed in brackets. from left to right: bones found mineralized in most individuals (presumably elements to mineralize earliest) to bones found mineralized in the least individuals. from top to bottom: states showing the most mineralized elements (presumably exhibited by the most advanced individuals) to states showing the least numbers of mineralized elements. 131skeletogenesis and osteology of hypsiboas pulchellus the floor of the braincase. it has neither a basicranial fenestra nor craniopalatal foramina. the otic capsules are ventrally perforated by a big fenestra ovalis, which is obscured by the cartilaginous operculum. the posterolateral border of the basis cranii shows a posterior process anterior to the otic capsule. the palatoquadrate is not articulated with the anterior part of the basis cranii with its great ala. there are three connections to the neurocranium: the commissura quadratocranialis anterior, the processus ascendens, and the otic connection. it is articulated to the commissura quadratocranialis anterior. this part of the palatoquadrate is the processus muscularis quadrati; the lateral side of the palatoquadrate forms the arcus subocularis. ventral to the processus muscularis quadrati, the hyoquadrate process is located. the posterior end is articulated to the pila antotica by the ascending process of the palatoquadrate. the latter articulates with the pila antotica and with the ventral process of the tectum synoticum, anterior to the otic capsule. the processus muscularis quadrati is articulated to the commissura quadratocranialis. the palatoquadrate curvature and the processus anterolateralis of the crista parotica are in contact. at this stage, both the processus anterolateralis and the posterolateralis are elongated. the cartilaginous hyobranchial skeleton bears the copula i and ii (sensu maglia and púgener, 1998; sheil and alamillo, 2005; basihyal and basibranchial respectively sensu haas, 1999), the pars reuniens, the paired ceratohyalia, four pairs of ceratobranchials, and two hypobranchials plates (sensu maglia and púgener, 1998; sheil and alamillo, 2005; planum hypobranchiale sensu haas, 1999). the ceratohyalia are the biggest cartilages in the hyobranchial apparatus. each ceratohyal possesses an anterior process (=processus anterior sensu haas, 1999), and a well-developed anterolateral process (=processus anterolateralis sensu haas, 1999; hyoquadrate process sensu maglia et al., 1998). this process is longer than the processus anterioris. the processus lateralis points posterolaterally. the large posterior process (=processus posterior sensu haas, 1999) is nearly in central contact with fig. 9. (a) dorsal view and (b) ventral view of a tadpole (gs39-40) chondrocranium, (c) hyobranchial apparatus of a tadpole (gs39-40). abbreviations: as, arcus subocularis; bbr, basibranchial; cb, ceratobranchials; cli, cartilago labialis inferior; cls, cartilago labialis superior; cm, meckel’s cartilage; cmqc, commissura quadratocranialis; cot, commissura terminalis; cs, corpus suprarostralis; ct, cornu trabeculae; con, condylus articularis; hpqr, hyoquadrate processus; ns, nasal septum; of, optical foramen; ot, otic capsule; pa, processus antorbitalis; pal, processus anterolateralis; palt, processus anterolateralis; pf, prootic foramen; phb, planum hypobranchiale; pla, processus lateralis of certatobranchial; pmq, processus muscularis quadrati; ppl, processus posterolateralis; ppo, processus posterioris of ceratohyal; pq, palatoquadrate; pqe, processus quadratoetmoidalis; pre, pars reunion; ps, parasphenoid; sp, spiculae; tm, taenia tecti medialis; ts, tectum synoticum. scale 1mm; blue: cartilage, red: mineralized bone. 132 j.m. hoyos et al. the hypobranchial plate (=planum hypobranchiale sensu haas, 1999). the pars reuniens is poorly chondrified, interconnects the ceratohyalia (=ceratohyals), and is articulated with the hypobranchial plates posteriorly. the hypobranchial plates are triangular, and articulate with each other along the medial margins; posterolaterally they articulate with the ceratobranchials, but are continuous with ceratobranchial i. the ceratobranchialia articulate distally via the commissurae terminalis. the width of the ceratobranchials is irregular; the ceratobranchials iii and iv are the widest, whereas ceratobranchials i and ii are the thinnest. both ceratobranchials and each hypobranchial plate bear spiculae. those in the hypobranchial plate are located at the end of the fusion of the hypobranchial with the ceratobranchial i. postcranium in the youngest specimen investigated (gs 25), no postcranial skeletal elements could be found. at gs 28-31, in the axial skeleton, cartilaginous primordia of vertebral centra were present, anteriorly more advanced in development than posterior. small rib primordia laterally to the neural arches of vertebrae ii and iii were present at gs 33 specimens, in which also sacral and postsacral vertebrae had begun to develop. at gs 34, traverse processes of vertebrae ii to iv were present, although less pronounced in vertebra iv. ventral ossifications in vertebrae i-iv could be observed in gs 36 specimens (hypoapophyses); a cartilaginous primordium of the urostyle (or the postsacral vertebra) was also present at this stage. ossifications in the neural arches were found at gs 37, with the dorsal processes still cartilaginous, at the same time the transverse processes of vertebrae ii-v were present. a cartilaginous primordium of the sacral diapophysis was developed at gs 39-40. an ossified hypochord and ossifications in postsacral vertebrae were not found before gs 41. at gs 42, at least primordial of transverse processes could be found up to vertebrae viii, postsacral vertebrae were ossified by then. vertebrae became discernible as procoelic. chondral primordia of the forelimb elements humerus, radius, radiale, and ulnare were present at gs 34; the anterior radiale (=r’ sensu fabrezi, 1993), carpals 5-4, and metacarpals iv-v could be found at gs 35. at gs 36, radiale (r’ and r) and the primordial of the proximal phalanges of finger iv and v were present. at gs 37, when the longbones humerus, radius, and ulna had begun to ossify in the centers and at their ends, the cartilaginous carpals 5-4-3 and 2 and the proximal element of the prepollex were visible, the phalangeal formula of the hand was 1-2-3-3. element y (sensu fabrezi, 1993) was found at gs 39/40. at gs 42, radius and ulna were nearly totally fused and the phalangeal formula was as in adults. the cartilaginous hindlimb elements femur, tibia, fibula, tibiale, and fibulare were present at gs 34, at gs 35 metatarsals iii, iv, and v were also found, at gs 36 the phalangeal formula was 1-1-1-2-2 and element y was visible. at gs 37, at the beginning of the ossification of several longbones, the distal tarsals 1, 2, and 3 (or 2-3) and all metatarsals were discernible, and the phalangel formula was 1-1-2-3-2. the three elements of the pelvic girdle developed at gs 34; at the same stage the shoulder girdle was composed by cartilaginous procoracoids, coracoids, scapulae, and suprascapulae. at gs 37 a weakly developed epicoracoid was present and the scapula began to ossify. clavicle and cleithrum could only be found from gs 42 onward. 133skeletogenesis and osteology of hypsiboas pulchellus discussion in this study, we described the osteology and aspects of skeletal development of the hylid frog hypsiboas pulchellus. regarding adult osteology, in hypsiboas pulchellus, the one aspect to highlight is the presence of a process in the suprascapula (herein processus suprascapularis) (fig. 5c). this structure has not been demonstrated in any other anuran species to exhibit this specific shape. maglia et al. (2007) described a similar process in a hylid, acris crepitans. however, this process is morphologically different because is exhibits a more triangular shape (“anteriorly-projecting triangular process”, maglia et al., 2007: 212). regarding skeletal development, general patterns reported previously for anuran and hylid skeletogenesis can be confirmed for h. pulchellus. banbury and maglia (2006) divided cranial development into five phases: 1) postembryonic (gs 30-33), 2) premetamorphic (gs 34-39), 3) early metamorphic (gs 39-42), 4) late metamorphic (gs 42-45), and 5) postmetamorphic. following this schema in studying the relative timing of the onset of ossification (ossification sequence), the postembryonic stages comprise the onset of the ossification of the parasphenoid, presacral vertebrae i-vii, frontoparietal and exoccipital. during the premetamorphic phase, the onset of the ossification of the transverse processes of presacral vertebrae i-viii, sacral vertebra ix, humerus, radioulna, ilium, femur, tibiofibula, and scapula occurs. in the early metamorphic phase the cleithrum, clavicle, coracoids, metacarpals, tarsals, metatarsals, phalanges, hypochord, and prootic begin to ossify. in the late metamorphic phase the angular, dentary, maxilla, premaxilla and squamosal ossify. in the postmetamorphic phase the sequence of ossification ends, remaining as such in adults. to assess intraspecific/individual variability in the relative orders of onset of ossification in individual skeletal elements (ossification sequences), ideally in vivo observations would be necessary. since however, the absolute number of ossified elements can be used as a measure of the developmental progress – transient elements removed – ossification sequences are in contrast to temporal orders of other developmental character sets relatively easy to reconstruct from preserved specimens without additional age information. we found intraspecific variation in both cranial and postcranial ossification sequences. cranially, with one exception (the missing prootic in cranial state 08, fig. 8), changes in order took place between elements that where temporally neighbored within ossification sequences, within this sample between exoccipital, parasphenoid, and frontoparietal. these elements have been reported to be among the first cranial elements to ossify in a variety of anurans (hanken and hall, 1988; wiens, 1989; dunlap and sanchiz, 1996; púgener and maglia, 1997; haas, 1999; wild, 1999; trueb et al. 2000; banbury and maglia, 2006). within anurans, changes in the relative order of ossification of these elements have been reported before for the tailed frog, ascaphus truei (moore and townsend jr., 2003), and are evident for the hyperossified frog, pyxicephalus adspersus (haas, 1999; sheil, 1999). in the postcranium, elements found involved in changes in the sequence of ossification were the sacrum, scapula, ilium, urostyle, and phalanges of hands and feet; the variability in relative onset time of ossification in the latter had previously been reported for a number of other tetrapod taxa including birds, turtles, and mammals (rieppel, 1993; sheil and greenbaum, 2005; maxwell, 2008; sánchez-villagra et al., 2009; wilson et al., 2010; mitgutsch et al., 2011). intraspecific variability, as known from osteological and other morphological data, should thus also for ossification 134 j.m. hoyos et al. sequences be carefully accounted for in comparative studies. research on a variety of aspects regarding the intraspecific variation found in ossification sequences such as temporal distribution or taxonomic differences will be additional important steps in understanding the evolution of skeletal development. acknowledgements we thank the pontificia universidad javeriana (jmh) and the paläontologisches institut und museum, universität zürich, zürich, switzerland for support. we also thank catalina mantilla for preparing the final drawings and two anonymous reviewers for helpful comments on an earlier version of this manuscript. references báez, a.m. (2000): tertiary anura of south america. in: amphibian biology, v. 4: palaeontology, p. 1388–1401. heatwole, h., carroll, r.l. eds, chipping norton: surrey beatty & sons. balanoff, a.m., rowe, t. 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(2002): the evolution of development: two portraits of skull ossification in pipoid frogs. evolution 56: 2484–2498. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 6(2): 303-313, 2011 dynamics of ovarian follicles in tupinambis merianae lizards m. valeria garcía valdez1, silvia chamut1, gonzalo valdez jaen, osvaldo e. a. arce1, mario e. manes1,* 1 facultad de agronomía y zootecnia, universidad nacional de tucumán, f. ameghino s/n, el manantial, (4105), tucumán, argentina. *corresponding autor. e-mail: mmanes@faz.unt.edu.ar submitted on: 2011, 10th march; revised on: 2011, 29th august; accepted on: 2011, 7th october. abstract. follicular dynamics of tupinambis merianae lizards was analyzed by means of ultrasound examination and radio frequency identification, during three consecutive reproductive periods, both in reproduction and in sexual isolation. series of follicular development which ended in ovulation, and series of follicular development without ovulation, followed by a regression process, were observed, as well as a combination of both. ovulation was found to be strongly dependent on mating consummation. in 34% of the cases in which follicular development ended with ovulation, and hence oviposition, follicles showed a steady and uniform growth up to the periovulatory period, also acquiring a mild echogenicity. in the absence of ovulation (66% of cases), follicular development showed two patterns: a short and a long-term anovulatory follicular cycle. in the first pattern, follicles showed limited growth and a non-echoic aspect, which suggests a previtellogenic condition. in the second pattern, follicles exhibited a growth and echogenicity similar to periovulatory ones, presumably attaining a vitellogenic stage. in addition, follicular development in reproducing females was superior to that of sexually isolated females. follicular development, ovulation, and follicular regression appeared to constitute generalized events affecting most, if not all, of the recruited follicles. keywords. tupinambis, follicular dynamics, sexual interaction, induced ovulation, follicular regression. introduction reptiles exhibit a variety of reproductive strategies as response to different environmental conditions. in species with seasonal reproduction, gonadal cycle phases (recrudescence, climax, and gonadal quiescence) appear temporarily organized according to their thermic and energy demands, and possibly their duration (saint girons, 1984; 1985). 304 m.v. garcía valdez et alii consequently, differentiated gamete maturation requirements lead to gonadal cycle dissociation between sexes, a widely reported event in reptile reproduction (moore and lindzey, 1992; whittier and tokarz, 1992). therefore, apart from adequately adjusting to physical and climatic conditions, reptiles also use socio-sexual signals to coordinate their reproductive activity with that of individuals of the opposite sex. this type of intersexual regulation is mentioned by numerous reports of gonadal recrudescence induction, and concomitant hormonal changes (crews and garrick, 1980; garstka et al., 1985; mendoça and crews, 1990; summers et al., 1995; shanbhag et al., 2002). the all-female parthenogenetic lizard cnemidophorus uniparens constitutes an interesting evolutive phenomenon, since gonadal recrudescence is actually promoted by pseudo-male individuals (crews et al., 1986). furthermore, recent studies revealed cases of mating-induced ovulation in the loggerhead sea turtle, caretta caretta (manire et al., 2008) and possibly in the brown tree snake, boiga irregularis (mathies et al., 2004), as seen in mammals having reflex ovulation. the south american oviparous lizards tupinambis rufescens and t. merianae (presch, 1973) which live in template and subtropical climates, show a lethargy period of approximately 6 months, which restricts main reproductive events (mating, oviposition, and incubation) to spring and early summer (donadío and gallardo, 1984; mercolli and yanosky, 1990; noriega et al., 1996). studies on these species have also shown that gonadal activity is asyncronic between sexes. in fact, whereas the highest testicular activity coincides with mating events (noriega et al., 2002), follicular development is only completed approximately 20 days after mating (manes et al., 2007). moreover, the short and intense vitellogenesis observed at the postnuptial stage pointed towards mating as the stimulus that triggers vitellogenesis and subsequent ovulation (manes et al., 2007). in this article, we analyzed the follicular dynamics of tupinambis merianae lizards by means of ultrasound examination and identification of individuals through microchips. animals in reproduction or sexually isolated were studied comparatively, in order to evaluate the influence of sexual interactions on follicular development and ovulation. materials and methods animals and study conditions the study was carried out using adult specimens of tupinambis merianae, raised in the experimental hatchery of facultad de agronomía y zootecnia of universidad nacional de tucumán. female specimens had a 35 cm snout-vent length or more; male specimens were 39 cm or more. besides their adult size, selected females have oviposited at least once. the study was conducted during 2007-08, 2008-09 and 2009-10 reproductive seasons, in el manantial, province of tucumán, north of argentina. this site has a warm temperate climate with dry season in the cold period. the animals were kept in open-air enclosures, provided with shelters and shades. the enclosures were surrounded by 1.2 m tall masonry walls, and allowed each individual an area of 2 m2. they were fed ad libitum on a hatchery diet (vega parry and manes, 2000). 305dynamics of ovarian follicles in tupinambis merianae lizards in order to facilitate individual follow up, each specimen was implanted with a subcutaneous microchip of 11.5 x 2.1 mm (id-100a microtransponder, trovan electronic identification devices ltd), on the left abdominal flank. experimental design to determine the influence of sexual interactions on follicular dynamics, the animals were subjected to two experimental conditions: under reproductive conditions and in sexual isolation. breeding groups were formed with three to five females and one male. sexually isolated groups were formed with three to five females in absence of male. new groups were created for each reproductive season, randomly redistributing the individuals. to verify reproductive cycle events in the reproductive group (courtship, mating, oviposition, incubation, reproductive quiescence) (noriega et al., 1996; manes et al., 2007), the male specimens were placed daily with the females between 9 and 12 am, and their activities were checked by an observer. to allow comparisons, sexually isolated females examination was synchronized with that of females in reproductive conditions. ultrasound studies the ovaries were examined with an ultrasound scanner berger lc 2010, with a micro convex probe of 5/7 mhz. ultrasonographic observations were made every 7 to 14 days, since their departure from hibernation until oviposition. these were resumed after the incubation period, at the reproductive quiescense. images of representative follicles in both ovaries were recorded both, on paper and digitally. statistical analyses the statistical modeling was carried out by using the statistical software r (r development core team, 2009), packages nlme (pinheiro and bates, 2000; pinheiro et al., 2009) and lattice (sarkar, 2008; 2009). the mixed models methodology approach was applied to the data (pinheiro and bates, 2000; vonesh and chinchilli, 1997). data was considered to be generated from a factorial experiment with repeated measures in time. factor under analyses were: sexual interaction (in reproduction or in sexual isolation), follicular development (with ovulation, without ovulation: short and long term anovulatory cycles) and days after hibernation emergence. data for oviposition were analyzed separately. the minimal adequate model (crawley, 2007), i.e. a model with all significant effects, was reached after backward selection of variables. comparisons of models with alternative fixed parts and the same random structure were accomplished by means of likelihood ratio tests and the akaike information (aic) and bayesian criterions (bic). estimation procedure was maximum likelihood (ml). estimations for comparison of models with the same fixed part and alternative random structure were accomplished by means of restricted maximum likelihood (reml). the significance of the fixed effects was tested by means of conditional f tests (pinhero and bates, 2000). the maximum of each curve was obtained setting the first derivative to zero. the growth rate in the initial increasing growth phase was obtained evaluating the first derivative in the point corresponding the half the time necessary to reach the maximum follicular size. in the case of oviposition, a straight line model was fitted so the growth rate was just the estimated slope parameter. 306 m.v. garcía valdez et alii results twenty female specimens were observed, throughout three consecutive reproductive cycles. six individuals where studied throughout one reproductive cycle, seven were observed during two cycles, and seven during three cycles, making a total of 41 cases. one of these developed an ovarian tumor (not described in this study). ultrasound examinations were performed from the end of hibernation (september) until reproductive quiescence (february). at the end of hibernation, ovaries appeared consistently undeveloped, as two small elongated formations, without detectable follicles. afterwards, follicles started to grow, and they could be detected when they reached 3 mm in diameter. ultrasound examinations revealed uniform follicular growth in both ovaries, after which follicles were either ovulated or went into regression. follicular development with ovulation thirty four percent of the observed follicular cycles (14/41) ended in ovulation followed by oviposition (fig. 1). lack of oviductal egg retention in this species, makes oviposition a safe indicator of ovulation (unpublished observations). in this variant, the follicles showed a steady, uniform growth throughout approximately 50 days (between 35 and 68 days). few days before ovulation, follicles of mated females reached an average diameter of 2.66 (± 0.16) cm, against 2.33 cm of follicular diameter of the only female, which happened to ovulate in the sexually isolated group. during development, follicles acquired a smooth echogenicity, irregularly distributed throughout the follicular mass (fig. 2). since follicular discharge was massive, there were no remaining grown follicles in the ovaries at the end of ovulation. in a few cases, we observed small non-identifiable postovulatory formations, ephemeral and anechogenic, which ranged between 5 and 9 mm in diameter. we also succeded in observing oviductal eggs which appeared slightly elongated and aligned, although ultrasonography did not allow estimating their number, nor distinguishing whether they were shelled or unshelled. follicular development without ovulation in the remaining 66% of cases (27/41), follicles also showed a noticeable growth, but unlike what was noticed in the previous situation, failed to ovulate, and underwent an extensive involution process. according their duration, we recognized two anovulatory cycle patterns (fig. 3): short term anovulatory cycle follicles involved in this cycle grew poorly, reaching an average diameter of 1.05 (± 0.06) and 0.91 (± 0.2) cm in reproducing females and in sexually isolated ones, respectively (fig. 3). subsequently, they underwent regression, turning undetectable 80 days after 307dynamics of ovarian follicles in tupinambis merianae lizards the end of hibernation. throughout the entire process, follicles had a cystic appearance, and were characteristically anechogenic (fig. 4). long term anovulatory cycle average follicular size in this cycle almost equaled that of periovulatory follicles: 2.58 (± 0.16) and 2.32 (± 0.3) cm in diameter, in reproducing females and in sexually isolated ones, respectively (fig. 3). the follicles as those destined to ovulation acquired echogenicity mainly concentrated at the follicular cortex (fig. 5). fig. 1. tupinambis merianae follicular development followed by ovulation. () reproductively active females; () the only case of ovulation among sexually isolated females. fig. 2. tupinambis merianae preovulatory follicles showing a mild irregular echogenicity. 308 m.v. garcía valdez et alii fig. 3. tupinambis merianae anovulatory follicular cycles. short term cycle: () reproductively active females; () sexually isolated females. long term cycle: () reproductively active females; () sexually isolated females. fig. 4. maximally grown tupinambis merianae follicles from a short term anovulatory follicular cycle. observe their anechogenic aspect. the subsequent involution period was longer, so it was possible to find follicles 200 days after the end of hibernation. involution in this case was also characterized by an intensified echogenicity of the whole follicle. the involution process in both anovulatory cycles, seemed to affect the whole of recruited follicles from both ovaries. mating took place between 2 and 4 weeks after the end of hibernation, involving females with varying degrees of follicular development (between 0.9 and 2.8 cm in diameter). the average time between mating and oviposition was 22 days, ranging from 7 to 33 days. 309dynamics of ovarian follicles in tupinambis merianae lizards effects of sexual interactions on follicular behavior ovulation rate, based on ovipositions, was considerably higher in sexually interacting females as compared to those sexually isolated: 45% (13 out of 29) versus 8% (1 out of 12) (table 1). ovulation frequency within the reproducing group was clearly related to mating consummation (table 1): verified mated females (21) presented 13 ovipositions, whereas nonmated ones (8) had none. curiously, the only case of ovulation without mating involved a sexually isolated female (table 1). in absence of ovulation, the two anovulatory follicular cycles were observed (table 1), without either being favored by mating. fig. 5. maximally grown tupinambis merianae follicles from a long term anovulatory follicular cycle. observe the echogenic aspect of the follicular cortex. fig. 6. male effect on tupinambis merianae follicular growth. (—) follicular development with ovulation, r2= 0.92; anovulatory follicular cycles (r2= 0.86): short term cycle: () reproductively active females; () sexually isolated females. long term cycle: () reproductively active females; () sexually isolated females. 310 m.v. garcía valdez et alii follicular growth was also superior in reproducing females compared to sexually isolated ones (fig. 6, table 2). such extra follicular growth derived from male presence rather than mating occurrence, since no differences were observed between mated and unmated females. in addition, sexually isolated females exhibited sexual behaviors similar to the reproducing ones. among females that joined reproductive groups during two or three consecutive years (14), we found some individuals that exhibited recurrence of oviposition cycles, some others, recurrence of anovulatory cycles, and finally others, combining both cases. discussion follicular growth in tupinambis merianae began at the end of hibernation (september), leading either to ovulation or involution. growth and ovulation processes appear to be generalized events, affecting both ovaries, and all or most recruited follicles in each reproductive cycle. this seem suitable for a once-a-year clutch production with high number of eggs (donadío and gallardo, 1984; mercolli and yanosky, 1990; noriega et al., 1996), which can, in turn, be associated to a compact reproductive schedule for a limited table 1. mating incidence on tupinambis merianae ovulation/oviposition. experimental condition ovulation / oviposition follicular involution reproductively active females 29 mated 21 13 8 unmated 8 8 sexually isolated females 12 1 11(*) (*) including a female which developed an ovarian tumor. table 2. effect of the male presence on tupinambis merianae follicular growth parameters. experimental condition follicular cycle type maximum follicular diameter (cm) time to maximum follicular diameter (days) growth (cm/day) reproductively active females with ovulation 2.83 (± 0.59) 84 0.032 short term anovulatory follicular cycle 0.998 (± 0.04) 30 0.0086* long term anovulatory follicular cycle 2.347 (± 0.03) 91 0.026* sexually isolated females short term anovulatory follicular cycle 0.7998 (± 0.04) 52 0.014* long term anovulatory cycle follicular cycle 2.046 (± 0.06) 81 0.023* * estimated during the increasing phase of the slopes. 311dynamics of ovarian follicles in tupinambis merianae lizards activity period in subtropical and temperate climates (donadío and gallardo, 1984; mercolli and yanosky, 1990; noriega et al., 1996). the strong correspondence between mating and oviposition frequency seems to indicate a mating increased level of stimuli required for the follicular cycle to end up with ovulation. however, not all matings led successfully to ovulation. mating-induced ovulation is a widespread phenomenon in mammals, but has very few examples among reptiles (manire et al., 2008). however, it may be more usual than expected, though masked by an interval between mating and ovulation, as seen in reptilian dissociated gonadal cycles (moore and lindzey, 1992; whittier and tokarz, 1992). such asynchronicity would also require sperm retention in female genital ducts, which is a common reptilian strategy (cuellar, 1966; gist and jones, 1987; sever and hamlett, 2002). a sperm retention mechanism in tupinambis merianae females was proposed to overcome the gap of about 20 days between copulation and ovulation (manes et al., 2007 and present results). we believe that such sperm retention does not extend beyond one reproductive period, since animals that copulated without ovipositing in a cycle, did not oviposit in the following cycle either, when regrouped into sexually isolated lots. in addition, the extra follicular growth shown by females kept in reproduction as compared to sexually isolated ones, reveals that the sole male presence without mating induces a certain degree of gonadal recrudescense, as previously observed in other reptiles (crews and garrick, 1980; garstka at al., 1985; mendoça and crews, 1990; summers et al., 1995; shanbhag et al., 2002). likewise, cases of spontaneous vitellogenesis and ovulation may reflect endocrine stimulation levels, similar to those achieved through sexual interactions. in this sense, we can not rule out pheromones interacting between reproducing and sexually isolated individuals, as suggested by sexual behaviors in isolated females. the absence of ovulation in tupinambis merianae was clearly related to an extensive follicular atresia. considering its general physiological role as regulator of the oocyte number to ovulate, massive follicular atresia in this species looks more like the sign of a failure in follicle maturation or ovulation. follicular atresia in short term anovulatory cycles, presumably involving previtellogenic follicles, could represent a timely departure of the follicular cycle to prevent a great vitellogenic effort considering the huge periovulatory ovarian mass (about 400 grams, manes et al., 2007). on the contrary, follicular atresia in long term anovulatory cycle would be a rather late step back, when the vitellogenic process has already taken place. as indicated by tumor formations (apichela et al., 2002 and present study), ovarian recovery does not always end up with satisfactory results. as reviewed by saidapur (1978), an endocrine production by atretic follicles cannot be dismissed either. apart from the two general types of follicular involution described here, a previous sexual isolation essay (apichela et al., 2002), showed the release of large follicular aggregates in the coelomic cavity. this phenomenon may represent a different type of “bursting atresia”, as referred by saidapur (1978) for some reptiles and birds. finally, from the present results, we can conclude that tupinambis merianae lizards are able to oviposition every year, although for still unknown reasons, anovulatory cycles may appear. 312 m.v. garcía valdez et alii acknowledgments this study was funded by the consejo de investigaciones de la universidad nacional de tucumán (ciunt). we thank verónica garcía valdez and adriana manes for the english translation references apichela s., campos casal f., manes m.e. 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(2000): alimentación de lagartos overos tupinambis merianae con subproductos avícolas. rev. argentina prod. anim. 20: 135–143. vonesh, e., chinchilli, v. (1997): linear and nonlinear models for the analysis of repeated measurements. dekker, new york. whittier, j.m., tokarz, r.r. (1992): physiological regulation of sexual behavior in female reptiles. in: biology of the reptilia, vol 18, p. 24-69. gans, c., crews, d., eds, the university of chicago press, chicago. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 6(2): 131-136, 2011 site fidelity in the sichuan torrent frog (amolops mantzorum) in a montane region in western china wen bo liao1, 2 1 key  laboratory  of  southwest  china  wildlife  resources  conservation ministry  of  education,  china west normal university, nanchong, 637009, p. r. china. e-mail: liaobo_0_0@126.com 2 institute of rare animals and plants, china west normal university, nanchong 637009, p. r. china. submitted on: 2010, 23rd december; revised on: 2011, 8th november; accepted on: 2011, 5th december. abstract. i used mark-recapture technique to estimate site fidelity in a subtropical high-elevation torrent frog (amolops mantzorum) during the breeding season in fengtongzhai national nature reserve in western china. i captured, measured, and individually marked 30 males and 15 females in 20 may 2007. i recorded each individual’s initial positions using a global positioning system (gps). for each night from 21 may to 10 june 2007, i recaptured the marked individuals and recorded capture points. the results showed that 16 males and 4 females were never recaptured in the field experiment. most of the remaining individuals were recaptured only one time. males and female were recaptured more than 2 and 8 times, respectively. males and females were recaptured from subsequent positions as far apart as 55 m and 30 m, as close as 0.2 m and 0.1 m. average neighbor distances on successive capture positions of males recaptured was 10.1 m, and that of females was 4.2 m, suggesting that there were significant difference in site fidelity between females and males. however, there was not significant average activity distance between the sexes. for females, small average activity areas were 10.9 ± 14.9 m2. keywords. site fidelity, amolops mantzorum, mark-recapture. many anuran species exhibit territoriality, and the resource and predation defended varies with the behavioral strategy exhibited by an individual (wells, 1977; mathis et al., 1995; bevier, 2006). the prolonged breeders may establish territories that include highquality oviposition sites and low predator risks in breeding season (wells, 1978). resident male for several nights or weeks defend against conspecific intruders, a pattern that may increase terrestrial male’s success in attracting females who lay eggs in his territory (wells, 1978). territories established by females can provide for high-quality oviposition environment and abundant food resources (wells, 1977). the genus amolops consists of 18 ranid frogs adapted to running steam habitats in subtropical mountain regions in western china (zhao and adler, 1993; liu et al., 2000). 132 w.b. liao the sichuan torrent frog amolops mantzorum occurs in the montane regions of western sichuan, southwestern yunnan and southern gansu, where it is found over a broad geographical area in terms of both latitude (between 23°57’n and 33°55’n) and elevation (1,000-3,800 m). the frogs prefer to stay on the surface of big rocks in the stream in night during the breeding season (fei and meng, 2005). breeding season ranges from early may until late october, and frogs can be considered a prolonged breeder. in recent years, preliminary information on age structure and egg size of sichuan torrent frog in the wild has been published (fei and meng, 2005; liao and lu, 2010a, b). so far, information about site fidelity during the breeding period is unavailable. in this study, i used mark-recapture method to measure the time recaptured of individuals and compare difference in site fidelity between males and females. the study area is distributed in a mainly subtropical evergreen broadleaf forest in western china (30º33’n, 102º56’e, 1,700 m a.s.l.). it has the annual mean temperature ranging from 5.9 to 7.2 and the annual total precipitation ranging from 700 to 1,300 mm from 1976 to 2000. the vegetation covering the area is characterized by yushania brevipaniculata, tetracentro sinense, bashania faberi, tsuga chinensis (liao and lu, 2010b). the study site is a stream reach located in dengchigou protection station of fengtongzhai national nature reserve. the stream reach dimension (length) and widen where the study has been performed is 1000 and 20 meters, respectively. i performed twenty-two sampling dates for 20 consecutive nights in spring 2007 (20 may-10 june). i captured a. mantzorum individuals by hand in night with the use of a 12 v flashlight when they stayed on emerged rocks in the stream. in the first sampling session i individually marked, in the field, each frog captured by clipping different toes of four limbs, i distinguished the sex (by nuptial pads on the first finger for male, eggs readily visible by the skin of the abdomen for female), and i measured the body length (from snout to vent, svl) using a caliper to the nearest 0.1 mm. i recorded the gps coordinates at the point of capture for each individual when the distance between successive individual captures was above 2 meters. we used tape measure to measure the distance of two successive points if it was less than two meters. in the subsequent sampling sessions i searched for individuals marked in the first session and i recorded each time, with the gps, their position. in each sampling session, frogs were immediately released at the capture site after recording the coordinates of their position. i calculated average distances between individuals in each of 20 nights and average distances between successive individual captures using range v software. i used arcview gis 10.0 to calculate the activity distance and area for each male and female. all statistical analyses were conducted using spss version 15.0 software (statistical product and service solutions company, chicago). i applied the welch ‘s t-test to test differences in body size, activity distances and neighbor distances on successive capture points between males and females. spearman correlation (rs) was used for relationships between average neighbor distances on successive capture positions and the frog body size. the sampling efforts were comparable for all sampling dates. the values given are reported as mean ± sd and all statistical tests were two-tailed. i captured and marked total of 30 males and 15 females in the first sampling. average svl differed significantly between males (53.9 ± 2.2 mm, range = 50.9-59.1 mm, n = 30) and females (67.8 ± 2.3 mm, range 62.9-71.1 mm, n = 15; welch’s t-test: t = 19.68, 133site fidelity in the sichuan torrent frog p < 0.001). eleven females and fourteen males were recaptured at least once. recaptured females (svl = 68.0 ± 2.5 mm, range = 62.9-71.1 mm) were significantly larger than males (svl = 54.2 ± 2.0 mm, range = 52.1-59.1 mm; welch’s t-test: t = 3.98, p < 0.001). there were significant differences in the average of number of times i recaptured males and females (fig. 1; welch’s t-test: t = 3.03, p = 0.006). i captured males and females an average of 1.1 ± 0.4 times and 2.8 ± 2.0 times, respectively. of these recaptured individuals, 25 individuals (55.6%) were captured 1–8 times. males and females were recaptured more than 2 and 8 times. recapture rates in males and female were 46.7% and 73.3%. average distances between successive individual captures in males (10.1 ± 14.2 m, range = 0.2-30.0 m, n = 16) were significantly longer than those of females (4.2 ± 6.3 m, 10.1 ± 14.2 m, range = 0.2-30.0 m, n = 31; welch’s t-test: t = 3.12, p = 0.002). there was significant correlation between body size and average distances between successive individual captures in females (fig. 2; spearman correlation analysis: rs = 0.77, n = 11, p = 0.005), but not in males (rs = -0.26, n = 14, p = 0.36). average activity distances did not differ significantly between males and females (males, 11.6 ± 15.1 m; females, 1.9 ± 11.3 m; welch’s t-test: z = 0.84, p = 0.41). because all males recaptured did not exceed 2 numbers of times, i can not compute their activity areas. for females, average activity areas was 10.9 ± 14.9 m2 (n = 5), ranging from 0.04 to 34.50 m2. like most anurans (monnet and cherry, 2002), my results indicated that females had significant larger body size than males. the sexual size dimorphism in this frog may be attributed to the fact that natural selection favors females that have larger svl to improve offspring size or fecundity (kupfer, 2007). in my study, i found that toe-clipping did not fig. 1. the difference in average times between males and females recaptured of the sichuan torrent frog (amolops mantzorum) in a montane region in western china. recaptured rate is shown as mean ± sd. 0 0.5 1 1.5 2 2.5 3 3.5 males females sexes a ve ra ge t im e s in d iv id u al s re ca p tu re d 134 w.b. liao affect the survival and recapture rate, similar result has been reported in the frogs (liao and lu, 2010b). similar to male mink frog rana septentrionalis (bevier et al., 2006), male sichuan torrent frog (amolops mantzorum) was rarely recaptured at the same site, and exhibited relatively large nearest-neighbor distances on consecutive nights. this pattern suggests that males do not maintain long-term discrete territories to defend areas with resources important to females in a prolonged breeding period. in contrast, in may other frog species, males exhibit intense site fidelity, also for prolonged periods (wells, 1977; judge and brooks, 2001; gordon, 2004; reviewed in wells, 2007). in contrast, in males of a. mantzorum i found rarely site fidelity since nearly half the marked male individuals were observed only once. compared to a. mantzorum males, females had relatively higher recapture rates. differences in the capture rates between males and females may be due to behavioral differences related to the sex (i.e. different dispersion ability or similar behaviors involving moves). females from my results had a smaller average distances between successive capture points than males, showing stronger site fidelity. in summarizing the reason that a. mantzorum females have small average distances between successive capture points, we make two possible points. firstly, females returned to the same general site on consecutive nights in order to increase their chances of attracting and mating with a male due to a female-bias sex ratio in the site. secondly, the small distances between rock-sites stopped on successive capture points suggested that females need defend high-quality areas with abundant food resources important to oviposition. my data revealed that female body sizes in a. mantzorum were positively correlated with average neighbor distances between successive capture points. we speculated that fig. 2. the relationship between body size and average neighbor distances between successive capture positions of the sichuan torrent frog (amolops mantzorum) (male, open bars; female, close bars) in a montane region in western china. body size in mm is shown as mean ± sd. average distances between successive captures (m) b od y si ze ( m m ) 45 50 55 60 65 70 75 -2 3 8 13 18 23 28 33 135site fidelity in the sichuan torrent frog larger females need larger area to obtain more food resources in reproduction than smaller ones maybe because they being larger are simply capable to cover larger distances. however, there was not significant correlation between male body size and average distances between successive individual captures, suggesting that both larger and small males do not defend areas with resources for females, but only search their chances to mate them. this is similar to previous study for site fidelity in r. septentrionalis (bevier et al., 2006). in my study, females had minimal average activity areas of 10.9 m2 which also indicated their site fidelity. similar results occurred in other anurans species. for example, rana clamitans males defend relatively small areas that are up to 2.4 m2 in dense vegetation and up to 9 m2 in more open areas (wells, 1977). male territory size is approximately 3 m2 in rana virgatipes (given, 1988). however, i did not obtain activity areas in males due to small samplings and shorter period studied. therefore, deep study for male activity areas need be conducted in future. acknowledgements i thank tong lei yu, ben qing yang and jian cheng for assistance during the filed work. financial support is provided by the foundation of key laboratory of southwest china wildlife resources conservation (ministry of education) education), china west normal university, p. r. china (xnyb01-3) and national sciences foundation of china (31101366). references bee, m.a. (2002): territorial male bullfrogs (rana catesbeiana) do not assess fighting ability based on size-related variation in acoustic signals. behav. ecol. 13: 109-124. berven, k.a. (1982): the genetic basis of altitudinal variation in the wood frog rana sylvatica. i. an experimental analysis of life history traits. evolution 36: 962-983. bevier, c.r., tierney, d.c., henderson, l.e., reid, h.e. (2006): chorus attendance and site fidelity in the mink frog, rana septentrionalis: are males territorial? j. herpetol. 40: 160-164. fei, l., meng, x.l. (2005): a handbook of recognizing amphibians in china. china. forestry publishing house, beijing, people’s republic of china. given, m.f. (1988): territoriality and aggressive interactions of male carpenter frogs, rana virgatipes. copeia 1988: 411-421. gordon, n.m. (2004): the effect of supplemental feeding on the territorial behavior of the green frog (rana clamitans). amphibia-reptilia 25: 55-62. judge, k.a., brooks, r.j. (2001): chorus participation by male bullfrogs, rana catesbeiana: a test of the energetic constraint hypothesis. anim. behav. 62: 849-861. kupfer, a. (2007): size dimorphism in amphibians: an overview. in: sex, size and gender roles: evolutionary studies of sexual size dimorphism, p. 50-60. fairbairn, d.j., blanckenhorn, w.u., szekely, t., eds, oxford university press, new york; and wells, 2007: the ecology and behavior of amphibians.university of chicago press, chicago, usa. 136 w.b. liao liao, w.b., lu, x. (2010a): age and growth of a subtropical high-elevation torrent frog, amolops mantzorum, in western china. j. herpetol. 44: 172-176. liao, w.b., lu, x. (2010b): a skeletochronlogical estimation of age and body size by the sichuan torrent frog (amolops mantzorum) between two populations at different altitudes. anim. biol. 20: 479-489. liu, w.z., yang, d., ferraris, t.c., matsui, m. (2000): a new species of stream-breeding frog from western yunnan, china (anura: ranidae). copeia 2000: 536-541. mathis, a., jaeger, r.g., keen, w.ó., ducey, p.k., walls, s.c., buchanan, b.w. (1995): aggression and territoriality by salamanders and a comparison with the territorial behavior of frogs. in: amphibian biology. vol ii. social behavior, p. 633–676. heatwole, h., sullivan, k.b., eds, surrey beatty and sons, chipping norton, new south wales, australia. monnet, j.m., cherry, m.i. (2002): sexual size dimorphism in anurans. proc. r. soc. lond. b biol. sci. 269: 2301-2307. trivers, r.l. (1974): parent–offspring conflict. am. zool. 14: 249-264. wells, k.d. (1977): territoriality and male mating success in the green frog (rana clamitans). ecology 58: 750-762. wells, k.d. (1978): territoriality in the green frog (rana clamitans): vocalizations and agonistic behavior. anim. behav. 26: 1051-1063. wells, k.d. (2007): the ecology and behavior of amphibians. chicago, il: university of chicago press. zhao, e., adler, k. (1993): herpetology of china. society for the study of amphibians and reptiles, oxford, ohio. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 8(1): 69-73, 2013 comparative cytogenetics of two species of ground skinks: scincella assata and s. cherriei (squamata: scincidae: lygosominae) from chiapas, mexico riccardo castiglia1,*, alexandra m.r. bezerra2, oscar flores-villela3, flavia annesi1, antonio muñoz4, ekaterina gornung1 1 dipartimento di biologia e biotecnologie “charles darwin”, università di roma ‘‘la sapienza’’, via a. borelli 50, 00161 roma, italy. *corresponding author. e-mail: castiglia@uniroma1.it 2 departamento de zoologia, universidade de brasília, icb, asa norte, cep 70910-900, brasília, df, brazil 3 museo de zoología, facultad de ciencias, universidad nacional autónoma de méxico, a.p. 70-399, méxico d.f. 04510 4 el colegio de la frontera sur san cristóbal de las casas carr. panamericana y av. periférico sur s/n 29290 san cristóbal de las casas, chiapas, méxico submitted on: 2012, 10th september; revised on: 2013, 5th april; accepted on: 2013, 13th may. abstract. standard karyotypes of two species of the genus scincella, s. assata and s. cherriei, both from chiapas state, mexico, were described for the first time. the diploid chromosome number was 28 in s. assata, whereas 30 in s. cherriei. the karyotypes of the two species, while differing in the number of microchromosomes, 14-15 in s. assata and 16-17 in s. cherriei, share four pairs of large metacentric, two pairs of medium-sized metacentric, and one particular pair (number 7) of chromosomes. female s. assata carries chromosome pair 7 composed of two identical mediumsized subtelocentric chromosomes. this chromosome pair is heteromorphic in males of both species, i.e., one component of the pair is similar to the homomorphic chromosomes 7 of the s. assata female, while the other is nearly one-half the size of its counterpart and resembles a microchromosome. the homology of such externally different elements is deducted from the presence of an asymmetric bivalent in spermatocytes at diplotene-diakinesis. female s. cherriei was not available. we suspect that the two scincella species possess an xy sex determination system, as previously reported for the north american congeneric species, s. lateralis. key words. mexican reptiles, sex chromosomes, sphenomorphus. lizards of the scincid genus scincella are small semifossorial ground skinks. the genus was originally established to accommodate species occurring almost exclusively in central, east, and southeast asia (greer, 1974; ouboter, 1986), but a number of species are currently recognized in the new world (garcía-vázquez and feria-ortiz, 2006; garcía-vázquez et al., 2010; uetz, 2012), which were thought to have resulted from a single dispersal event across the bering land bridge during the miocene (honda et al., 2003; macey et al., 2006). at present, mexican herpetofauna includes seven scincella species, which had formerly been regarded as the new world members of sphenomorphus, but reassigned to scincella on the basis of molecular phylogenetic analyses (honda et al., 2003; linkem et al., 2011). the two sister species scincella assata (cope, 1864) and s. cherriei (cope, 1893) belong to this group. chromosomal data are known for only one of the total of 35 currently recognized species of the genus scincella the north-american s. lateralis (say, 1823), in which a 2n = 30/29 karyotype and a male heterogamety with intraspecific variation were reported (wright, 1973; 70 riccardo castiglia et al. hedin et al., 1990). karyological data are also available for four species of sphenomorphus “sensu lato”, a paraphyletic assemblage, which currently includes 122 species widely distributed in asia and australia (uetz, 2012). in this latter grouping, the three karyotyped species, the asian sph. indicus (gray, 1853) and sph. incognitus (thompson, 1912) (ota and lue, 1994) and the australian eulamprus (sphenomorphus) tympanum (lönnberg and andersson, 1915) (wright, 1973), have mainly 2n = 30 karyotype, but, in sph. indicus, 2n = 28 (makino and momma, 1949; yang et al., 1989) and 2n = 26 (guo and dong, 1988) karyotypes were also described in the earlier reports. in these species, no sex chromosome heteromorphisms have been asserted. here, we describe karyotypes of scincella assata and s. cherriei and provide comparative cytogenetic analysis of these and related taxa. specimens were identified on the basis of cephalic lepidosis and body coloration (cope, 1864; stuart, 1940; kohler, 2008). one male (rcmx 85) and two females (rcmx 86, rcmx 92) identified as s. assata were collected in the leaf litter along a dry small stream in a dry tropical forest of the “la sepultura” biosphere reserve, chiapas state, mexico (16°20’42” n, 93°50’26” w), and two males (rcmx 219, rcmx 235) identified as s. cherriei, were collected in the evergreen tropical forest of the “montes azules” biosphere reserve, selva lacandona, chiapas state, mexico (16°06’44’’n, 90°56’26’’w) (figure 1). vouchers are deposited at the herpetological collection of the comparative anatomy laboratory, “charles darwin” department of biology and biotechnologies, “la sapienza” university of rome, italy, and in the museo de zoologia “alfonso l. herrera”, universidad nacional autónoma de méxico, mexico city. for chromosomal analysis, femurs and vertebral column were removed from each animal one hour after an intra-peritoneal injection of a 0.1% solution of vinblastine sulphate (lilly); also, testes were taken from the males. the samples were processed following the procedure described in castiglia et al. (2010). for each specimen, about 10 metaphases were photographed after conventional giemsa staining. the diploid numbers of chromosomes of s. assata and s. cherriei were found to be, respectively, 2n = 28 and 2n = 30. the karyotypes of both species are composed of four pairs of large and two pairs of medium-sized metacentric chromosomes (pairs 1-6), as well as one or two small to medium-sized subtelocentric chromosomes (pair 7), and a series of microchromosomes (figure 2). in s. assata, females possess two apparently identical subtelocentric chromosomes for pair 7 and 14 microchromosomes (figure 2a), while the male shows only one chrofigure 1. above, scincella assata from “la sepultura” biosphere reserve (chiapas, mexico) (photo a.m.r. bezerra). below, s. cherriei from “montes azules” biosphere reserve, selva lacandona (chiapas, mexico) (photo r. castiglia). figure 2. karyotypes of (a) female (rcmx 92) and (b) male (rcmx 85) of scincella assata (2n = 28) and of (c) male (rcmx 235) of s. cherriei (2n = 30). diplotene-diakinesis bivalents of the males of (d) s. assata (rcmx 85) and (e) s. cherriei (rcmx 219). note heteromorphic chromosomes 7 and corresponding asymmetric bivalents. bar shows 10 µm. 71cytogenetics of skinks mosome 7 and 15 microchromosomes (figure 2b). of these microchromosomes of the male s. assata, the largest one does not pair with either of the remainder. this feature of male karyotype of s. assata is shared with that of s. cherriei, although they differ from each other by the total number of microchromosomes, 15 in the former and 17 in the latter (figure 2c). the unpaired microchromosome in each male karyotype may be the counterpart of the single subtelocentric chromosome 7, although the former is nearly one-half in size of the latter. in the analyses of male meiosis at diplotene-diakinesis for both species, we identified four large and two medium-sized bivalents plus one small asymmetric bivalent, as well as seven (s. assata) or eight (s. cherriei) micro-bivalents (figure 2d and e). generally, karyotypes of the scincid lizards are composed of 2n = 26, 28, 30, or 32 chromosomes that form three distinct size-groups (large and small biarmed macrochromosomes, and a series of microchromosomes), with the exception of the eutropis [formerly mabuya] macularia species complex, which possesses 2n = 34 or 38 chromosomes forming a continuous series (ota et al., 1996; 2001). most scincid karyotypes so far studied can be assigned to one of the two main groups: the first group, represented by most species of the subfamily lygogominae and a number of species of the subfamily scincinae (sensu greer, 1970), is distinguished by four pairs of distinctly large biarmed chromosomes, while the second group, accommodating some other scincine species, is characterized by no more than two pairs of distinctly large metacentric chromosomes (giovannotti et al., 2009). for instance, two asian lygosomine species, sphenomorphus indicus and sph. incognitus, possess four pairs of large and three pairs of distinctly smaller metacentric macrochromosomes and 8 pairs of microchromosomes (ota and lue, 1994). the presently studied karyotypes of s. assata and s. cherriei both conform to the arrangement typical of scincid karyotype with four pairs of large biarmed chromosomes. the difference in diploid numbers between the two sister species is undoubtedly due to a lack of one pair of microchromosomes in s. assata. reduction in the number of microchromosome pairs seems to have frequently occurred in reptiles. microchromosomes may fuse together, or be converted into macrochromosomes by addition of heterochromatin, or be even translocated onto a macrochromosomal pair, as was reported for the australian skink lampropholis delicata (donnellan, 1991). it was not possible to specify the pair of microchromosomes to which the difference in chromosome numbers between s. assata and s. cherriei could be attributed, since the morphology of microchromosomes was resolved, to some extent, only in the metaphase chromosomes of s. cherriei. actually, there are a few data reporting morphology of microchromosomes, e.g., castiglia et al. (2006) and the recent analyses of slightly extended dapi stained chromosomes carried out in several skink species including two lygosomines, lepidothyris fernandi (2n = 30) and trachylepis quinquetaeniata (2n = 32) (giovannotti et al. 2009). however, in the congeneric species scincella lateralis, which is the closest relative to the clade of the presently studied species, this task has been computed by synaptonemal complex (sc) karyotype analysis that revealed the exact position of all centromeres, thus recognizing one metacentric and seven acrocentric small microchromosomal bivalents, in addition to four large and two medium-sized metacentric bivalents (hedin et al., 1990). this pattern corresponds very well with the one presently observed in s. cherriei. scincella assata diverges for the diploid number of chromosomes not only from the congeneric species, s. cherriei and s. lateralis, but also from the other species of the sphenomorphus group (greer, 1979; honda et al., 2000; reeder, 2003), matching only one karyotype reported for a population of sph. indicus from sichuan, china (guo and dong, 1988; yang et al., 1989) and for one of tropidophorus (ota et al., 1991), a genus generally assigned to the sphenomorphus group (e.g., honda et al., 2000). if 2n = 30 is considered the ancestral chromosome number (honda et al., 2000) in the sphenomorphus group, reduction of the diploid chromosome number observed in s. assata and sph. indicus most probably has independent origin, as the two species are phylogenetically distant (linkem et al., 2011). finally, males of both species studied here have remarkably heteromorphic chromosome pair 7, which is unequivocally homomorphic in females of s. assata (females of s. cherriei were not available to us: see above), suggesting the presence of an xy sex chromosome system as in another new world congener, s. lateralis (wright, 1973). the present hypothesis is worth investigating, considering that the male represents the heterogametic sex in the skinks where heteromorphic sex chromosomes occur (olmo and signorino, 2005). further studies, examining more specimens of the two species, female s. cherriei in particular, are desirable to verify this idea. acknowledgments we are grateful to julia carabias, javier de la maza, rodrigo león, diego noriega, and to all the staff of the estación chajul biological research station for their precious help during the permanence of r.c. and o.f.v. in chiapas. thanks are extended to the two anonymous reviewers. this work has been supported by funds received from d.g.a.p.a., u.n.a.m. for sabbatical leave of o.f.v. funds received from the university 72 riccardo castiglia et al. of rome “la sapienza” (progetti di ricerca di università) supported r.c. secretariade medio ambiente y recursos naturales (semarnat) granted licenses (faut-0015) for specimen collection. amrb received a postdoctoral fellowship from cnpq. references castiglia, r., corti, m., annesi, f. 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(1989): karyotypic studies of sphenomorphus indicus (scincidae) and takydromus septentrionalis (lacertidae). chin. herpetol. res. 2: 55-59. acta herpetologica vol. 8, n. 1 june 2013 firenze university press the oogenic cycle of the caspian bent-toed gecko, cyrtopodion caspium (squamata: gekkonidae) in iran vida hojati1*, kazem parivar2, eskandar rastegar-pouyani3, abdolhossein shiravi1 altitudinal effects on life history parameters in populations of liolaemus pictus argentinus (sauria: liolaemidae) joel antú gutiérrez1,*, carla piantoni2, nora r. ibargüengoytía1,3 recent cryptic extinction of squamate reptiles on yoronjima island of the ryukyu archipelago, japan, inferred from garbage dump remains yasuyuki nakamura1,*, akio takahashi2, hidetoshi ota3 trophic niche and feeding biology of the italian wall lizard, podarcis siculus campestris (de betta, 1857) along western mediterranean coast marco a.l. zuffi*, chiara giannelli novel, non-invasive method for distinguishing the individuals of the fire salamander (salamandra salamandra) in capture-mark-recapture studies goran šukalo1,*, sonja đorđević2, dragojla golub1, dejan dmitrović1, ljiljana tomović2,3 going out tonight? when insular hierophis viridiflavus breaks the whip snakes rules delaugerre michel-jean first evidence of a paedomorphic population of the smooth newt (lissotriton vulgaris) in the czech republic václav gvoždík1,2, veronika javůrková4, oldřich kopecký5,* no detection of chytrid in first systematic screening of bombina variegata pachypus (anura: bombinatoridae) in liguria, northern italy stefano canessa1,*, an martel2, frank pasmans2 status of the european pond turtle, emys orbicularis (reptilia: testudines: emydidae) in vorarlberg, austria andreas kleewein1, günther wöss2 comparative cytogenetics of two species of ground skinks: scincella assata and s. cherriei (squamata: scincidae: lygosominae) from chiapas, mexico riccardo castiglia1,*, alexandra m.r. bezerra2, oscar flores-villela3, flavia annesi1, antonio muñoz4, ekaterina gornung1 polydactyly in the tyrrhenian wall lizard (podarcis tiliguerta) antigoni kaliontzopoulou1,*, daniele salvi1, verónica gomes1, joão p. m. c. maia1,2,3, panagiotis kaliontzopoulos4 book review: roberto sindaco, alberto venchi, cristina grieco. the reptiles of the western palearctic. 2. annotated checklist and distributional atlas of the snakes of europe, north africa, middle east and central asia, with an update to the vol. 1. edoardo razzetti acta herpetologica journal of the societas herpetologica italica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 8(2): 93-97, 2013 intraspecific variation in erythrocyte sizes among populations of hypsiboas cordobae (anura: hylidae) mariana baraquet1,2,*, pablo r. grenat1,2, nancy e. salas1, adolfo l. martino1 1 ecología, departamento de ciencias naturales, facultad de ciencias exactas, físico-químicas y naturales, universidad nacional de río cuarto. ruta nacional n° 36 km 601, (x5804bya) río cuarto, argentina. *corresponding author. e-mail: mbaraquet@exa.unrc. edu.ar 2 conicet fellowships submitted on 2013, 14th june; revised on 2013, 2nd august; accepted on 2013, 3rd october. abstract. we studied the morphology and size of erythrocytes of h. cordobae, and analysed the geographic variation of this character along the distribution of the species, in relation to the latitudinal and altitudinal distances. erythrocyte shape of the h. cordobae is ellipsoidal and the nuclei are also ellipsoidal and centrally oriented. erythrocyte and nuclear size showed significant differences among populations, with the highest mean size corresponding to the population of achiras (low altitude site) and the lowest mean size to los linderos (high altitude site). there was no significant relationship between the latitude of each population and the both erythrocyte and nuclear size. the altitudinal variation in erythrocyte cell size may be attributable to the surface available for gas exchange; a small erythrocyte offers a possibility of greater rate of exchange than a larger one. our results are consistent with studies of other amphibians, where intraspecific comparisons of populations at different altitudes show that individuals at higher altitudes are characterized by smaller erythrocytes. keywords. hypsiboas cordobae, erythrocyte and nuclear size, geographic variation. the description of the anuran amphibian hematology is insufficient, although this is a diverse group of vertebrates (cabagna et al., 2011). the majority of the references to hematology in different species of anurans have been limited to blood cell counts (martínez et al., 1985; arıkan, 1990; arserim and mermer, 2008; dönmez et al., 2009). however, there are also some studies on erythrocyte sizes of several amphibian species (hartman and lessler, 1964; matson, 1990; atatür et al., 1998, 1999, 2001; wojtaszek and adamowicz, 2003; zhelev et al., 2006; gao et al., 2007; grenat et al., 2009a, b; arıkan et al., 2010). some investigators have stressed that erythrocyte size in amphibians may be used to ploidy determination, because blood cells of amphibians conserve their nucleus and the erythrocyte size is correlated with the dna content (stöck and grosse, 1997; schröer and greven, 1998; atatür et al., 1999; martino and sinsch, 2002; rosset et al., 2006; gao et al., 2007; grenat et al., 2009a, b; valetti et al., 2009). this method is simple, rapid and minimally invasive (grenat et al., 2009a). in this paper, we studied six populations of a single species, in which ploidy level is the same (baraquet et al., 2013). it is well-known that in amphibians there is an extensive range in the erythrocyte size. morphology and size of erythrocytes have shown great inter-specific and even intra-specific variations (arıkan and çiçek, 2010). furthermore in comparison with other organisms, amphibian red blood cells tend to be larger (duellman and trueb 1994; gregory, 2001; campbell, 2004). this relationship between erythrocyte size and the level of ploidy has also been discussed on the basis of differences in metabolic rates between different groups of vertebrates (gregory, 2000), because the size and shape of red blood cells give 94 mariana baraquet et al. an indication of the surface available for the exchange of gases in respiratory functions (hartman and lessler, 1964; sevinç et al., 2000). the availability of oxygen limits the metabolic potential and, therefore, the behaviour of animals in a particular environment. thus, the adaptation to an environment depends on the development of suitable mechanisms to overcome these limitations. so, it is not strange that these adaptations in amphibians influence the properties of blood and parameters that most affect this tissue (martínez et al., 1985). several studies have demonstrated that variations in erythrocyte counts and size are correlated with metabolic activity of the animal, indicating that the more active species have smaller erythrocytes while those with less oxygen consumption have bigger ones (evans, 1939; smith, 1925; szarski, 1970, 1976). the distribution of the species under study, hypsiboas cordobae (barrio 1965), is restricted to córdoba and san luis provinces, argentina (barrio, 1965; cei, 1980; gallardo, 1974, 1987; di tada, et al. 1996; faivovich, et al. 2004). this restricted distribution and a broad altitudinal range, together with the reported iucn status (i.e., data deficient), make this species an interesting research model. although, various hematological studies were carried out on many anuran species, information is not available for h. cordobae. here, we examine the morphology and size of erythrocyte of h. cordobae and report their geographic variation along a latitudinal and altitudinal gradient in cordoba and san luis provinces, argentina. a total of 66 adult individuals of h. cordobae (57 ♂♂ and 9 ♀♀) were collected from six localities of cordoba and san luis provinces (argentina), between september 2006 and may 2011. the study area covers a latitudinal gradient across an area of approximately 20 000 km2, with an altitudinal range between 800 m and 2300 m in elevation. the sampled localities were: achiras (n = 10, 808 m a.s.l., 33º 09’s, 64ºw), las guindas (n = 21, 930 m a.s.l., 32º s, 64º w), la carolina (n = 15,1634 m a.s.l., 32º 48’s, 66º 05’w), los tabaquillos (n = 9, 2107 m a.s.l., 32º 23’s, 64º 55’w), pampa de achala (n = 6, 2150 m a.s.l., 31º 49’s, 64º 51’w), los linderos (n = 5, 2310 m a.s.l., 32º 00’s, 64º 56’w). the blood samples were obtained by angularis vein puncture (nöller, 1959). smears of fresh blood were airdried and stained with a 10% solution of giemsa for 5 min. slides were observed by using a microscope carl zeiss trinocular primo star (pack 5), photographed with a canon power shot g10 digital camera and processed using the image software axiovision 4.8. the photographs were used to record the erythrocyte measurement by adobe® photoshop® 9.0. on each blood smear, length (l) and width (w) of forty randomly chosen erythrocytes and their respective nuclei were measured. erythrocyte and nuclear areas were calculated assuming an ellipsoid shape according to formula l*w*π/4. we calculated mean, standard deviation and maximum and minimum values for each variable. since these variables had a normal distribution (shapiro-wilks test, p > 0.05), differences between males and females were compared by t-tests and inter-population comparisons by analyses of variance (anova). if the anova revealed significant differences among populations, pairwise tukey’s hsd tests were used to determine which groups differed significantly from one another. pearson correlation coefficient (r) was used to measure association of erythrocyte and nuclear size with latitude and altitude of the population studied to investigate geographic variation. mean values of each individual were used and all data were processed using statgraphics plus 5.0. because no sex differences were found for any of the size variables (t tests, p > 0.05 in all cases), data from both sexes were pooled. the mean erythrocyte and nuclear length, width, area and length/width ratio for each population sampled of the h. cordobae are given in table 1. the characteristic erythrocyte shape of the h. cordobae was ellipsoidal (l/w = 1.51). nuclei were also ellipsoidal (l/w = 1.79) and centrally located. in the population studied, erythrocyte lengths and sizes varied between 21.14 μm and 23.66 μm and 230.56 μm2 and 280.72 μm2, respectively. the longest erythrocytes were observed in the population from las guindas. the largest erythrocyte areas were observed in the population from achiras while the shortest and the smallest erythrocytes were observed in los linderos. in terms of l/w ratio, the most ellipsoidal cells were those of la carolina and las guindas populations while the least ellipsoidal ones were observed in los tabaquillos (table 1). the longest and the largest nuclei were observed in achiras while the shortest and the smallest nuclei were measured in the population from los linderos. the most ellipsoidal nuclei were observed in la carolina and the least ellipsoidal ones were found in pampa de achala (table 1). erythrocyte and nuclear size showed significant differences among populations (anovas: f = 2.88, p = 0.02; f = 3.70, p ≤ 0.01, respectively). pairwise test showed that erythrocyte and nuclear sizes of achiras and los linderos populations differed significantly (tukey’s hsd tests, p < 0.05, in both cases). in these populations we found the extreme erythrocyte and nuclear sizes: the largest size in achiras and the smallest in los linderos. 95intraspecific variation in erythrocyte sizes among populations of hypsiboas cordobae pearson correlation tests indicated there was not a significant relationship between latitude of each population and size of both erythrocyte and nuclei (r = 0.12, p = 0.81; r = 0.40, p = 0.43). conversely, correlations showed a negative significant relationship between altitude and size of both erythrocyte and nuclei (r = -0.82, p = 0.04; r = -0.91, p = 0.01, respectively). erythrocyte and nuclear size decreased significantly with increasing altitude of h. cordobae populations (figure 1). in the present paper, we have analysed the spatial pattern of erythrocyte size variation along the distribution of h. cordobae. the univariate analyses revealed significant differences among populations. there are many ways in which erythrocyte size is of relevance to organism biology; larger erythrocytes contain more hemoglobin (gregory, 2001). one of the most important functions of erythrocytes is to carry oxygen and carbon dioxide. the erythrocyte size and shape are indicators of the area available for gas exchange in respiratory function. therefore, small erythrocyte offers a possibility of greater rate of exchange than a larger one (hartman and leesler, 1964; martinez et al., 1985; sevinç et al., 2000; wojtaszek and adamowicz, 2003). consequently, at altitude where there are lower levels of oxygen available smaller erythrocytes should be selected. indeed, our results showed a negative relationship among the altitude and size of both erythrocyte and nuclei in the six populations studied. moreover, achiras and los linderos populations showed the erythrocyte and nuclear sizes values extremes. besides, the study of erythrocytes in different species provides an interesting comparison of the erythrotable 1. erythrocyte data in six populations of h. cordobae. n: sample size, l: erythrocyte length, w: erythrocyte width, a: erythrocyte size, l/w: erythrocyte ratios of length/width, l: nuclei length, w: nuclei width, a: nuclei size, l/w: nuclei ratios of length/width (means ± standard deviations). population n l (μm) w (μm) a (μm2) l/w l (μm) w (μm) a (μm2) l/w achiras (808 m a.s.l.) 10 23.42 ± 1.60 15.18 ± 1.55 280.72 ± 45.40 1.55 ± 0.10 9.99 ± 0.85 6.00 ± 1.12 47.72 ± 12.99 1.69 ± 0.18 las guindas (930 m a.s.l.) 21 23.66 ± 1.14 15.06 ± 1.05 280.58 ± 28.22 1.58 ± 0.10 9.92 ± 0.83 5.55 ± 0.64 43.57 ± 7.91 1.80 ± 0.14 la carolina (1634 m a.s.l.) 15 22.93 ± 1.01 14.62 ± 1.23 263.83 ± 30.05 1.58 ± 0.11 9.66 ± 0.60 5.09 ± 0.42 38.63 ± 4.20 1.91 ± 0.17 los tabaquillos (2107 m a.s.l.) 9 21.62 ± 0.67 15.35 ± 0.61 260.91 ± 15.99 1.41 ± 0.05 9.44 ± 0.54 5.19 ± 0.40 38.51 ± 4.41 1.83 ± 0.14 pampa de achala (2150 m a.s.l.) 6 22.41 ± 0.52 15.04 ± 1.31 265.39 ± 27.32 1.50 ± 0.11 9.12 ± 0.84 5.44 ± 0.26 39.02 ± 4.11 1.68 ± 0.17 los linderos (2310 m a.s.l.) 5 21.14 ± 0.80 13.85 ± 0.94 230.56 ± 20.44 1.53 ± 0.10 9.01 ± 0.30 4.77 ± 0.07 33.69 ± 1.37 1.89 ± 0.06 h. cordobae (range) 66 22.54 ± 0.99 (21.14-23.62) 14.94 ± 0.62 (13.85-15.59) 265.40 ± 19.00 (230.56-280.72) 1.51 ± 0.07 (1.41-1.57) 9.54 ± 0.41 (9.01-9.99) 5.33 ± 0.42 (4.77-6.00) 40.86 ± 5.60 (33.69-47.72) 1.79 ± 0.10 (1.66-1.90) fig. 1. correlation of erythrocyte (a) and nuclear (b) size with altitude of h. cordobae. 96 mariana baraquet et al. cyte size in relation to activity and habitat (hartman and lessler, 1964). in amphibians, erythrocyte size has long been known to correlate negatively with metabolic rates (smith, 1925; vernberg, 1955; monnickendam and balls, 1973). small erythrocytes improve the uptake of oxygen joined to a high number of red blood cells; this allows the organism to adapt to environments with low oxygen pressures (hutchison et al., 1976). this relationship stems from the fact that larger surface-area-to volume ratios in smaller cells allow for more efficient exchange of oxygen. this idea is exemplified in intraspecific comparisons of amphibians at different altitudes, where animals at higher latitudes have smaller erythrocytes (ruiz et al. 1983; arıkan, 1989; weber, 2007), presumably to maximize cellular efficiency of oxygen transport and exchange in a low oxygen environment. our results are in agreement with this, strongly suggesting a negative correlation between altitude and erythrocyte size. however, further studies about metabolic rate and oxygen consumption would be required to analyse the causes of erythrocyte size variation in populations of h. cordobae living at different altitudes. acknowledgements the first authors thank the national scientific and technical research council (conicet) for support and two anonymous reviewers for improving the ms. the secretary of science and technology of national university of río cuarto (secytunrc) provided funds by grant ppi 18c/350. we thank j. valetti for their help in the field and sample. our study was authorized by cordoba environmental agency (a.c.a.s.e.). references arıkan, h. 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(2006): a study of some metric parameters of the erythrocytes in rana ridibunda (amphibia: anura) derived from an area of highly developed chemical industry. acta zool. bulgar. 58: 235-244. acta herpetologica vol. 8, n. 1 june 2013 firenze university press journal of the societas herpetologica italica acta herpetologica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 8(1): 47-52, 2013 going out tonight? when insular hierophis viridiflavus breaks the whip snakes rules delaugerre michel-jean conservatoire du littoral, 3, rue luce de casabianca f20200 bastia, france. e-mail: m.delaugerre@conservatoire-du-littoral.fr submitted on: 2012, 11th november; revised on: 2013, 20th january; accepted on: 2012, 5 th february. abstract. hierophis viridiflavus has a strong diurnal rhythm as demonstrated by many field studies. it belongs to the “whip snakes” characterized by slender bodies, large eyes, high speed, saurophagy and diurnality. on giraglia island (corsica) the snakes do forage also nightly. this unexpected shift in the circadian rhythm might be related to a local adaptation to trophic requirements. keywords. activity pattern shift, microinsular adaptation, corsica, mediterranean, nocturnal. in insular contexts, characterized by genetic isolation and peculiar ecological constraints, animals often display geographic variations affecting their morphology such as body size and proportions, pigmentation, pattern (case, 1978; lomolino, 2005; meiri, 2007; pafilis et al., 2009; novosolov et al., 2012); biology and ecology such as diet and foraging modes, ecological interactions, micro habitat selection and many other features of their life history traits and behaviour (pérez-mellado and corti, 1993; traveset and sáez, 1997; van damme, 1999; zuffi, 2001; filippi et al., 2003; olesen and valido, 2004; herrel et al., 2008; delaugerre et al. 2012). according to shine (1980) “whip snakes” are conspicuous elements of the terrestrial snake fauna in most parts of the world. these phylogenetically unrelated taxa (families colubridae and elapidae), characterized by convergent evolution, display morphological, ecological and behavioural traits such as slender bodies, long tails, large eyes, alertness, diurnality, terrestriality, saurophagy, oviparity and high speed. a broad ecological study of the western whip snake hierophis viridiflavus (lacépède, 1789) in central italy (capula et al., 1997) partially corroborated shine’s (1980) views. we report and discuss preliminary data regarding the micro insular activity pattern shift of the western whip snake, occurring on giraglia island. giraglia island (43°01’30”n; 9°24’24”e; 10 hectares, 65 m a.s.l., distance from the main island 1.4 km) is located at the northernmost point of corsica (fig. 1). it was severed from the coast by sea level rise some 5000 years ago (lambeck and bard, 2000). the dense and low vegetation is dominated by allium commutatum and halimione portulacoides (with a total of 60 vascular plants, rivière et al., 2012). it hosts two geckos euleptes europaea and tarentola mauritanica, one lacertid podarcis tiliguerta pardii and one colubrid snake hierophis viridiflavus (lanza and brizzi, 1974). except for t. mauritanica, the giraglia herpetofauna is supposed to be native. the island is rat (and mammal) free. nesting birds are calonectris diomedea, larus michaellis (since the 90’s), phalacrocorax aristotelis desmarestii, apus pallidus and columba livia; falco tinnunculus and falco peregrinus often visit the island. giraglia has hosted a small human settlement (2-5 men and few cattle) since the end of the 16th century until the end of the 20th century when the lighthouse has been automated. included in a site of community importance (eu), the island is a protected area. the public access is forbidden, except for the lighthouse maintenance and biodiversity monitoring. nocturnal investigations (initially focussed on gecko’s monitoring) were performed on 12 september 2000, on 5 and 9 august 2012 and on 6 october 2012. 48 delaugerre michel-jean in august 2012 additional data (table 1) were gathered by naturalists monitoring cory’s shearwater. the moonlight was very bright in 2000 and sampling occurred during all moon phases in 2012. while rocky outcrops were thoroughly investigated with head lamp, western whip snakes were seen, foraging from 19:30 h to 2:00 h. they were actively crawling on rocks, on footpaths and on vegetation, tongue flicking while slowly exploring the substrate surfaces and crevices. two snakes were seen in 2000 and seventeen in 2012. in 2000 both were sub-adults; in 2012 all age classes were represented, with a majority of young and youngsters (table 1). one adult was also observed inactive under exsiccated plants. during the last fifteen years, many italian and french publications have been devoted to various aspects of the biology and ecology of h. viridiflavus. these studies have been performed at different latitudes, from vendée on the french atlantic coast, switzerland, to northern and central italy. in all these papers the activity pattern of this snake is considered as diurnal. capula et al. (1997) assessed a bimodal (and lower) daily activity in summer. lelièvre et al. (2010, 2011) confirmed the diurnal habits of this thermophilous species, even radio tracked animals were only recorded during the daytime, so did ciofi and chelazzi, (1991) and scali et al. (2008). ciofi and chelazzi (1994), who recorded activity rhythms 24 h long, also stated that “coluber viridiflavus was definitely diurnal; the primary and secondary shelters were used overnight…” the recent synthesis (vanni and nistri, 2006; vanni and zuffi, 2010) asserted its diurnality; only (santos et al., 2010) stated “sin embargo, no es extraño observar actividad crepuscular en los meses más calurosos”(however, it is not unusual to see crepuscular activity in the warmer months). although no nocturnal habits were documented in literature, could these studies, performed mainly by “diurnal herpetologists”, with visual encounters and some radio tracking performed only in the daytime, have missed a point? our field data have been investigated. intensive nocturnal searches (224 hrs) were performed in corsica (and sardinia), for geckos in rocky habitats and, to a lesser extent, for amphibians in wetlands. those investigations occurred in many localities of corsica and also on the satellite islands where the density of the western whip snake is very high, as on lavezzu island (table 2). except for giraglia, only one observation of true nocturnal activity has been recorded (bonifacio, 13 june 1983, 21:30 h, dark moon, air temperature 20.8 °c, juvenile snout vent length 33 cm, tail 8 cm). a crepuscular sighting of a young animal has also been recorded on 16 july 2006 (cape corse, moulin mattei). the nocturnal activity of h. viridiflavus does not occur or is very rare. this snake appears to have a strong rhythm for a specific diel activity pattern despite seasonal changes in temperature (gibbons and semlitsch, 1987) and latitudinal variations. among western european snakes (corti et al, 2010; salvador and marco, 2010), most diurnal species (16) are variable; they adjust their circadian rhythm to season and environmental temperature with crepuscular or nocturnal foraging in sumfigure 1. the giraglia island seen from the north-east. © f. rombaldi/assoc. finocchiarola. 49night activity in hierophis viridiflavus mer, whereas other species are strictly diurnal (eight) or nocturnal (two). these later species (including h. viridiflavus) having supposedly an activity pattern “genetically determined to the extent that their response to the light-dark is endogenous and invariable” (gibbons and semlitsch, 1987). the nocturnal activity reported on giraglia island is an outstanding exception. there, young and also adult snakes (corsican whip snakes are small sized according to cheylan, 1992 and vanni and zuffi, 2010) forage also nightly, and not only during the hottest months. this enlargement of the activity spectrum isn’t related to the moon phase, nor to the light intensity. although these preliminary data will need further study, they raise an array of questions. taking into account, at a broad level, the value of a phylogenic perspective in understanding patterns of evolution in behavior, morphology and physiology (autum et al, 2002) and also the suggested possibility that nocturnality might be the primitive condition of squamates (sites et al., 2011), we consider that the observed enlargement of the temporal niche probably results of a rather recent local insular microevolution. hypotheses for possible advantages of such nocturnal shift may be related to: 1) avoidance of diurnal predators; 2) avoidance of high temperatures; 3) easier acquirement of preferred/available food at night; or combination of these factors (crawford, 1934; gibbons and semlitsch, 1987). 1) avoidance of diurnal predators. falco tinnunculus is well known to predate young and subadult snakes. but empirical observations (to be confirmed) suggest that whip snakes of all age classes are also pretty active in the daytime. furthermore, during the larus michahellis nesting season, the f. tinnunculus avoids the island. nevertheless if a strong interaction between whipsnakes and this raptor is not likely to occur nowadays, we cannot discard its occurrence during the past history of the island. 2) avoidance of high temperatures. h viridiflavus is a thermophilous species and the island is complex enough (rocky outcrops, soil, low but thick vegetation) to provide natural shelters with medium temperatures. 3) easier acquirement of prey. the prey items available are mostly podarcis tiliguerta lizards (high density), the nocturnal gecko euleptes europaea, invertebrates and presumably passerine birds in spring migration. should the microinsular limitation of prey type diversity lead to increase the intraspecific competition for food between young and adult snakes and thus favor an enlargement of the temporal niche for the younger snakes? (see luiselli, 2006 for tropical vipers in a context of habitat alteration). but these intraspecific interactions are less likely to occur with high density (= availability) of the main food resource (lizards). an alternative hypothesis would be a local adaptation to a peculiar behavior of the prey. on giraglia island, podarcis lizards are often resting by table 1. nocturnal activity of hierophis viridiflavus sighted (or captured) on giraglia island on 2012 (5 and 9 august and 6 october m. delaugerre; 12 to 22 august a. prudor and n. el ksabi). in august night air temperature range 23-25 °c; relative hygrometry 73-82%; in october night air temperature range 18-19 °c; relative hygrometry 68-83%. date universal time micro habitat approximative age class svl (cm) tail (cm) weight (g) body temperature (°c) 05/08/12 21:43 lighthouse pavement juvenile 26.0 8 4.2 05/08/12 1:10 rocks juvenile 05/08/12 1:40 rocks adult 69.0 24 09/08/12 20:07 footpath adult 71.0 26 09/08/12 21:12 stone wall sub adult 55.6 20 38 12/08/12 19:40 stone wall sub adult 13/08/12 20:30 vegetation sub adult 14/08/12 23:49 lighthouse pavement adult 72.5 23.5 15/08/12 0:00 lighthouse pavement juvenile 15/08/12 22:15 footpath juvenile 17/08/12 19:30 footpath juvenile 19/08/12 20:00 vegetation juvenile 19/08/12 20:40 vegetation juvenile 21/08/12 23:15 footpath sub adult 06/10/12 18:50 stone wall sub adult 44.0 14.0 17 28.0 06/10/12 18:45 rocks juvenile 29.0 9.5 6.5 25.8 06/10/12 23:25 footpath sub adult 50.5 19.5 24 26.0 50 delaugerre michel-jean table 2. nocturnal investigations performed in corsica (and sardinia) from 1980 to 2012. sighting per unit effort (spue) = n / (to)100; where n = number of h. viridiflavus sighted; t = duration of searches in minutes; o = number of observers. observers: if n = 1= m-j. delaugerre and m. biaggini (caprera 2012, lavezzu 2010, 2011, 2012), ch.-h. bianconi (gargalu 1985); c. corti (caprera 2012, lavezzu 2010, 2011, 2012); f. grita (lavezzu 2010, 2012), p. lo cascio (caprera 2012, lavezzu 2010, 2012). some of the giraglia data of tab 1 are not reported here because spue could not be calculated. locality year month n minutes prospection n obs. n minutes observation snakes sighted spue corsican satellite islands giraglia island (n corsica) 2000 9 360 1 360 2 0.556 giraglia island (corsica) 2012 8 406 1 406 5 1.232 giraglia island (corsica) 2012 10 260 1 260 3 1.154 lavezzu island (s corsica) 1982 5 300 1 300 0 0.000 lavezzu island (s corsica) 1984 9 120 1 120 0 0.000 lavezzu island (s corsica) 2010 6 275 2 550 0 0.000 lavezzu island (s corsica) 2011 6 427 1 427 0 0.000 lavezzu island (s corsica) 2012 6 180 4 720 0 0.000 mezzumare island (w corsica) 2011 6 180 1 180 0 0.000 mezzumare island (w corsica) 2012 8 213 1 213 0 0.000 gargalu island (w corsica) 1985 4 530 2 1060 0 0.000 gargalu island (w corsica) 1990 7 505 1 505 0 0.000 corsica main island trinité (bonifacio, s) 1983 6 300 1 300 1 0.333 acciola (sartene, sw) 1980 5 60 1 60 0 0.000 conca-senetosa (sw) 1986 8 180 1 180 0 0.000 pianottoli (s) 1986 8 120 1 120 0 0.000 galeria (w) 1981 5 60 1 60 0 0.000 galeria (w) 1983 4 60 1 60 0 0.000 galeria (w) 1982 5 60 1 60 0 0.000 galeria (w) 1985 4 180 1 180 0 0.000 galeria (w) 1985 7 180 1 180 0 0.000 galeria (w) 1986 4 130 1 130 0 0.000 scandola (w) 1982 5 2105 1 2105 0 0.000 scandola (w) 1982 6 1465 1 1465 0 0.000 scandola (w) 1983 4 595 1 595 0 0.000 scandola (w) 1983 6 490 1 490 0 0.000 scandola (w) 1985 7 100 1 100 0 0.000 villanova (ajaccio, w) 1981 5 810 1 810 0 0.000 villanova (ajaccio, w) 1981 6 125 1 125 0.000 piana (w) 1981 5 60 1 60 0 0.000 m.on forestière lumio (1000m) 1981 5 60 1 60 0 0.000 a serra (calvi, w) 1981 5 60 1 60 0 0.000 lucciana (ne) 1985 4 60 1 60 0 0.000 capandula (cap corse, n) 2012 8 270 1 270 0 0.000 sardinia itiri (sardinia) 1982 6 90 1 90 0 0.000 caprera island (maddalena, n) 2012 5 240 3 720 0 0.000 sum in minutes 11,616 13,441 sum in hours 224 51night activity in hierophis viridiflavus night in very exposed spots, not hidden in refuges, while e. europaea, a slow moving gecko, forages on rock surfaces. snakes might have learned a way to easily pick some preys by night. if so, it would mean an important ecologic change in an evolutionary perspective: the quick pursuit foraging strategy (capula et al., 1997), relying on diurnal vision, being replaced by a slow search conducted by nocturnal vision or/and by the use of chemosensory cues. chemical scents would be used for the localization of the prey, as males are able to do for trailing and sexually discriminate conspecifics (fornasiero et al., 2007). in snakes, saurophagy is compatible with nocturnality, for instance in coronella and macroprotodon (cheylan, 1986). as stated by shine (1980): “apart from demansia, the only australian elapids that definitely are known to feed primarily on lizards (> 70% of the diet) are small fossorial nocturnal species… all of these fossorial saurophagous snakes capture their prey at night when the lizards are inactive (an african elapid… forages in the same way…). prey items probably are located by scent… this foraging strategy is fundamentally different from that of the whipsnakes, which locate their prey items visually, during the day, and capture them by direct chasing.” the expression of adaptative “inventiveness” is highly stimulated in microinsular context, is that the reason why the giraglia island’ h. viridiflavus have broken the whip snakes rules? acknowledgments the access to the island was permitted by the préfecture de haute-corse and the “phares et balises” authorities. collection permits were issued by the dreal corse. thanks to aurélien prudor and nory el ksaby for their interest and valuable help in the field. thanks to claudia corti for the experienced comments on the manuscript and to the editor and the reviewers. references autumn, k., ryan, m. j., wake, d. b. 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(2001): diet and morphometrics of coluber (=hierophis) viridiflavus on the island of montecristo (tyrrhenian sea, italy). herpetol. j. 11: 123-125. acta herpetologica vol. 8, n. 1 june 2013 firenze university press the oogenic cycle of the caspian bent-toed gecko, cyrtopodion caspium (squamata: gekkonidae) in iran vida hojati1*, kazem parivar2, eskandar rastegar-pouyani3, abdolhossein shiravi1 altitudinal effects on life history parameters in populations of liolaemus pictus argentinus (sauria: liolaemidae) joel antú gutiérrez1,*, carla piantoni2, nora r. ibargüengoytía1,3 recent cryptic extinction of squamate reptiles on yoronjima island of the ryukyu archipelago, japan, inferred from garbage dump remains yasuyuki nakamura1,*, akio takahashi2, hidetoshi ota3 trophic niche and feeding biology of the italian wall lizard, podarcis siculus campestris (de betta, 1857) along western mediterranean coast marco a.l. zuffi*, chiara giannelli novel, non-invasive method for distinguishing the individuals of the fire salamander (salamandra salamandra) in capture-mark-recapture studies goran šukalo1,*, sonja đorđević2, dragojla golub1, dejan dmitrović1, ljiljana tomović2,3 going out tonight? when insular hierophis viridiflavus breaks the whip snakes rules delaugerre michel-jean first evidence of a paedomorphic population of the smooth newt (lissotriton vulgaris) in the czech republic václav gvoždík1,2, veronika javůrková4, oldřich kopecký5,* no detection of chytrid in first systematic screening of bombina variegata pachypus (anura: bombinatoridae) in liguria, northern italy stefano canessa1,*, an martel2, frank pasmans2 status of the european pond turtle, emys orbicularis (reptilia: testudines: emydidae) in vorarlberg, austria andreas kleewein1, günther wöss2 comparative cytogenetics of two species of ground skinks: scincella assata and s. cherriei (squamata: scincidae: lygosominae) from chiapas, mexico riccardo castiglia1,*, alexandra m.r. bezerra2, oscar flores-villela3, flavia annesi1, antonio muñoz4, ekaterina gornung1 polydactyly in the tyrrhenian wall lizard (podarcis tiliguerta) antigoni kaliontzopoulou1,*, daniele salvi1, verónica gomes1, joão p. m. c. maia1,2,3, panagiotis kaliontzopoulos4 book review: roberto sindaco, alberto venchi, cristina grieco. the reptiles of the western palearctic. 2. annotated checklist and distributional atlas of the snakes of europe, north africa, middle east and central asia, with an update to the vol. 1. edoardo razzetti acta herpetologica journal of the societas herpetologica italica © firenze university press www.fupress.com/ah acta herpetologica 5(2): 179-198, 2010 morphology of peripheral blood cells from various species of turkish herpetofauna hüseyin arıkan, kerim çiçek* ege university, faculty of science, biology department, zoology section, 35100, bornova, izmir-turkey. *corresponding author. e-mail: kerim.cicek@ege.edu.tr submitted on: 2010, 15th june; revised on: 2010, 6th october; accepted on: 2010, 27th october. abstract. in this study, measurements of morphological and size parameters of peripheral blood cells (erythrocyte, leucocyte, thrombocyte) on blood smear preparation devices stained with wright’s stain were given for 87 species from turkish herpetofauna (19 amphibian species including 7 urodeles and 12 anurans as well as 68 reptile species including 4 turtles, 30 lizards and 34 snakes). it was determined that erythrocyte and nucleus sizes showed great variations among the species of herpetofauna and even among the preparations of the same species; the largest blood cells (erythrocyte, leucocyte, thrombocyte) were found in urodeles; aquatic and semiaquatic species had larger erythrocytes than terrestrials, and the largest erythrocytes were in turtles among the reptile species examined. lymphocytes were determined as the predominant cells among the blood leucocytes in blood smears of all the examined species. keywords. amphibians, reptiles, blood smears, blood cell morphology. introduction blood analyses are useful, widely used tools that aid in the diagnosis and monitoring of animal health and disease and in the differentiation of physiologic processes (christopher et al., 1999). these techniques are used with several wildlife species, especially for threatened or endangered populations, and help to indicate ecosystem health (deem et al., 2006). however, much of our knowledge regarding vertebrate blood and blood cells is based on mammalian references (claver and quaglia, 2009). the studies of nonmammalian vertebrate blood is relatively new (e.g., canfield, 1998; mader, 2000; campbell, 2004; allander and fry, 2008), and blood cell morphology of reptiles still completely unknown (e.g., frye, 1991; mader, 2000; campbell, 2004; strik et al., 2007, sykes and klaphake, 2008). the studies on the comparative morphologies of the peripheral blood cells in different amphibians and reptiles mainly concentrate on seasonal and sexual variations of counts (vernberg, 1955; altman and dittmer, 1961; foxon, 1964; hutchison and szaski, 1965; 180 h. arıkan and k. çiçek dessaurer, 1970; duguy, 1970; jerrett and mays, 1973; alleman et al., 1999; wojtaszek and adamowicz, 2003; solís et al., 2007) and sizes of blood cells (erythrocyte, leucocyte, thrombocyte) (gulliver, 1875; wintrobe, 1933; hartman and lessler, 1964; szarski, 1968; saint girons and saint girons, 1969; saint girons, 1970; wojtaszek et al., 1997; harr et al., 2001; knotková et al., 2002; salakij et al., 2002), and blood parasites (espinosa-avilés et al., 2008; roca and galdón, 2010). there are several review on morphology of blood cells in amphibians and reptiles (e.g., hutchison and szaski, 1965; hartman and lessler, 1964; szarski and czopek, 1966; canfield, 1998; mader, 2000; campbell, 2004; allander and fry, 2008, sykes and klaphake, 2008). since 1989, there have been several studies on morphology of peripheral blood cells in turkish amphibians (e.g., arıkan, 1989; atatür et al., 1998, 1999; arıkan et al., 2001, 2003a, 2003b, 2010; gül and tok, 2009) and reptiles (e.g., arıkan et al., 2004, 2009a, 2009b; atatür et al., 2001; sevinç et al., 2000; uğurtaş et al., 2003). the objective of the present study is to obtain detailed information on morphology and size of peripheral blood cell in 87 amphibian and reptile species in turkey comparatively, and the results were discussed with literature. material and methods individuals of 87 species belonging to sexually-mature amphibians and reptiles were collected from anatolian and thracian parts of turkey (table 1). the field studies were carried out in aprilmay for amphibians and in april-june for reptiles. the individuals were primarily collected on several herpetofaunal trips of previous studies or projects performed between 1989 and 2009. blood samples were obtained from heart ventriculus of amphibians via heparinized glass capillaries, from caudal vein of turtles via heparinized injector (hutchison and szarski, 1965; szarski and czopek, 1966), and from postorbital sinuses of lizard and snake individual via heparinized glass capillaries according to maclean et al. (1973). after obtaining blood samples in reptiles, they were released to their natural environments. for each individual, approximately 4-5 blood smears were prepared and stained with wright’s stain for the measurements of morphology and size parameters of blood cells. blood cells were table 1. collecting localities of 87 species from turkish herpetofauna [n: number of individuals]. species n locality latitude longitude urodela lissotriton vulgaris 5 bornova – izmir 38.472031 27.262555 lyciasalamandra atifi 4 alanya – antalya 36.594658 32.123258 mertensiella caucasica 3 akçaabat – trabzon 41.000657 39.569261 neurergus strauchii 10 yam village – bitlis 38.375950 42.091056 ommatotriton vittatus 5 mezitli – mersin 36.857369 34.397636 salamandra infraimmaculata 4 harbiye – antakya (hatay) 36.138361 36.143428 triturus karelinii 4 kalecik ankara 40.097222 33.408333 (continued). 181blood cell morphology of turkish herpetofauna species n locality latitude longitude anura pelophylax bedriagae 5 bornova-izmir 38.472031 27.262555 pelophylax caralitanus 10 beyşehir – konya 37.676389 31.726111 rana dalmatina 4 belgrad forest istanbul 41.194309 28.951383 rana holtzi 5 mountain bolkar – niğde 37.438813 34.604279 rana macrocnemis 6 mountain uludağ – bursa 40.072066 29.216721 bufo bufo 7 marmaris – muğla 36.863411 28.275040 pseudepidalea variabilis 10 sülüklüpınar – adana 37.040136 37.040136 pelobates syriacus 8 seydişehir – konya 37.423871 31.850475 pelodytes caucasicus 10 uzungöl – trabzon 40.622109 40.285267 bombina bombina 5 büyükdöllük – edirne 41.760133 26.603753 hyla arborea 5 fethiye – muğla 36.651389 29.123056 hyla savignyi 6 alanyalı village – mersin 37.094734 34.501284 testudines emys orbicularis 10 lake yayla – denizli 38.059675 28.778826 mauremys caspica 8 nusaybin – mardin 37.066667 41.216667 mauremys rivulata 3 northern cyprus 35.237616 33.471477 testudo graeca 8 izmir 38.418850 27.128720 lacertilia ablepharus chernovi 4 çamardı – niğde 37.832029 34.986486 chalcides ocellatus 6 finike – antalya 36.300827 30.144497 eumeces schneideri 5 meke saltern – konya 37.682887 33.636460 ophiomorus punctatissimus 4 kaş – antalya 36.204441 29.638982 trachylepis aurata 4 karapınar – konya 37.714821 33.552237 trachylepis vittata 4 kırobası – mersin 36.722014 33.909358 acanthodactylus boskianus 4 adana 36.999996 35.321314 acanthodactylus harranensis 7 şanlıurfa 37.149994 38.799857 anatololacerta danfordi 4 çamlıyayla mersin 37.170139 34.608260 apathya cappadocica 8 ulukışla – niğde 38.055150 34.310216 darevskia praticola 4 kırklareli 41.733333 27.216667 darevskia uzzelli 5 kars 40.592680 43.077692 darevskia valentini 12 ardahan 41.110477 42.702174 lacerta pamphylica 3 mut – mersin 36.644337 33.435555 lacerta trilineata 7 çamlıyayla – mersin 37.170139 34.608260 lacerta viridis 4 hendek – adapazarı (sakarya) 40.805100 30.749291 mesalina brevirostris 2 akçakale – şanlıurfa 36.711006 38.947988 ophisops elegans 6 mut – mersin 36.644337 33.435555 parvilacerta parva 2 çamardı – niğde 37.832029 34.986486 podarcis muralis 2 kırklareli 41.733333 27.216667 podarcis siculus 2 istanbul 41.005270 28.976960 timon princes 2 eruh – siirt 37.750000 42.183333 eublepharis angramainyu 3 birecik – şanlıurfa 37.025002 37.976955 table 1. (continued). (continued). 182 h. arıkan and k. çiçek species n locality latitude longitude cyrtopodion heterocercum 2 mardin 37.301906 40.730414 cyrtopodion scabrum 2 şanlıurfa 37.120305 38.784801 hemidactylus turcicus 3 northern cyprus 35.237616 33.471477 laudakia stellio 2 mut – mersin 36.644337 33.435555 trapelus lessonae 2 birecik – şanlıurfa 37.025002 37.976955 chamaeleo chamaeleon 2 northern cyprus 35.237616 33.471477 varanus griseus 1 viranşehir – şanlıurfa 37.178374 39.761510 serpentes leptotyphlops macrorhynchus 2 birecik – şanlıurfa 37.025002 37.976955 typhlops vermicularis 5 mut – mersin 36.644337 33.435555 eryx jaculus 2 mut – mersin 36.644337 33.435555 dolichophis caspius 2 ulukışla – niğde 38.055150 34.310216 dolichophis jugularis 2 mut – mersin 36.644337 33.435555 dolichophis schmidti 2 suruç – şanlıurfa 36.974652 38.424516 eirenis barani 1 kahramanmaraş 37.583309 36.933403 eirenis coronella 2 birecik – şanlıurfa 37.025002 37.976955 eirenis decemlineatus 1 diyarbakır 37.914409 40.230624 eirenis eiselti 2 diyarbakır 37.914409 40.230624 eirenis levantinus 1 samandağ – antakya (hatay) 36.082392 35.999324 eirenis modestus 2 çamlıyayla mersin 37.170139 34.608260 eirenis punctatolineatus 2 eruh – siirt 37.750000 42.183333 eirenis rothii 2 küplüce – kilis 36.757230 37.237016 hemorrhois nummifer 2 mut – mersin 36.644337 33.435555 hemorrhois ravergieri 1 sakçagözü gaziantep 36.715370 37.117360 malpolon monspessulanus 2 çiğli – i̇zmir 38.499432 27.038216 natrix natrix 4 mut – mersin 36.644337 33.435555 natrix tessellata 4 beyşehir – konya 37.676389 31.726111 platyceps collaris 1 midyat – mardin 37.416667 41.369719 platyceps najadum 1 mut – mersin 36.644337 33.435555 platyceps ventromaculatus 1 harran – şanlıurfa 36.866667 39.033331 rhynchocalamus melanocephalus 1 antakya (hatay) 36.401829 36.349788 spalerosophis diadema 2 birecik – şanlıurfa 37.025002 37.976955 telescopus fallax 2 northern cyprus 35.237616 33.471477 telescopus nigriceps 2 kilis 36.718399 37.121220 zamenis hohenackeri 2 antakya (hatay) 36.401829 36.349788 zamenis longissimus 1 zonguldak 41.456406 31.798752 macrovipera lebetina 2 dikmen – northern cyprus 35.268159 33.324760 montivipera albizona 1 mountain balık – kahramanmaraş 37.516405 36.449976 montivipera wagneri 2 karakurt – kars 40.169027 42.605943 montivipera xanthina 2 gümüldür – izmir 38.076415 27.022031 vipera eriwanensis 2 ardahan 41.110477 42.702174 walterinnesia morgani 1 tek tek mountains – şanlıurfa 36.812953 39.252838 table 1. (continued). 183blood cell morphology of turkish herpetofauna measured using a mob-1-15× micrometrical ocular. lengths (l) and widths (w) of 40 randomly chosen erythrocytes as well as nuclear lengths (nl) and nuclear widths (nw) were measured for each blood smear. erythrocyte sizes (es) and their nuclei sizes (ns) were computed from es= lwπ/4 and ns= nlnwπ/4. cells and nuclear shapes were compared with l/w and nl/nw ratios, and nucleus/cytoplasm with ns/es ratio. in addition, from the blood smears of each species, measurements of leucocytes (lymphocytes, monocytes, heterophils, eosinophils, basophils) and thrombocytes (tl, tw) were also taken to determine their sizes. the photomicrographs of the blood cells were taken with olympus bx51-altra 20 soft imaging system. correlation between body size and erythrocyte size were analyzed by non-parametric kendall τ test. results characteristic erythrocyte shape of amphibians and reptiles we analyses is oval, similar to that of vertebrate fish and birds. except for montivipera xanthina, the erythrocytes of the examined species have a somewhat ellipsoidal nucleus, uniformly located in the centre of the cell (fig. 1a). however, in m. xanthina, the irregularly shaped nuclei were determined in erythrocytes (fig. 1l). on smears stained with wright’s stain, the cytoplasms were light yellowish pink and the chromophilic nuclei were dark purplish blue. the blood smears of the examined species demonstrated interspecific and even intraspecific variations in terms of the lengths, widths and sizes of the erythrocytes and nuclei. the erythrocyte measurements (lengths and widths), sizes, l/w ratios, nuclear measurements and nucleocytoplasmic ratios are given in table 2. among the amphibian and reptile species of turkish herpetofauna, the largest erythrocyte was observed in urodele species (fig. 1a). mean length, width and size of erythrocytes in urodeles ranged respectively between 28.06 μm-33.28 μm, 16.63 μm-20.13 μm and 367.05 μm2-523.44 μm2. in addition, l/w ratio, mean lucleus length, mean nucleus width, mean nucleus size, nl/nw ratio and nucleocytopasmic ratio (ns/es) were found to change between 1.63-1.80, 13.86 μm-16.86 μm, 8.53 μm-10.46 μm, 92.85 μm2-138.51 μm2,1.56-1.69 and 0.22-0.34, respectively. the biggest erythrocytes and nuclei were observed in salamandra infraimmaculata and the smallest in ommatotriton vittatus. similarly; the most strongly ellipsoidal erythrocytes and nuclei were observed in mertensiella caucasica and the least ellipsoidal in triturus karelinii. and, the shortest nucleus was observed in lissotriton vulgaris, and the least ellipsoidal nuclei in neurergus strauchii (table 2). anurans were determined to have smaller erythrocytes and nuclei than urodeles (fig. 1b, c). mean erythrocyte length, ranged between 15.29 μm-24.36 μm, erythrocyte width 9.68 μm-15.05 μm, erythrocyte size 116.42 μm2-276.62 μm2, l/w ratio 1.63-2.35, mean nucleus length 6.21 μm-9.59 μm, nucleus width 3.47 μm-5.03 μm, nucleus size 18.13 μm236.66 μm2, nl/nw ratio 1.61-2.35 and nucleocytoplasmic ratio 0.10-0.14. among the anuran species examined, the largest and the most strongly ellipsoidal erythrocytes were observed in aquatic pelophylax caralitanus (fig. 1c) and the smallest erythrocytes in terrestrial pelodytes caucasicus (fig. 1c). the least ellipsoidal cells were found in pseudepidalea varibilis and the largest nuclei in bombina bombina; in addition, the shortest nuclei in pelobates syriacus, the most strongly ellipsoidal nuclei in hyla arborea, and the least ellipsoidal nuclei in rana dalmatina (table 2). 184 h. arıkan and k. çiçek of the reptiles, the largest erythrocytes were observed in turtles. and among the turtles, the largest erythrocytes were observed in aquatic species (e.g. 200.67 μm2 in emys orbicularis), and the smallest erythrocytes in a terrestrial species testudo greaca as 163.81 μm2 (fig. 1d, e). in turtles; mean erythrocyte length ranged between 17.35 μm-19.99 μm, erythrocyte width 11.90 μm-12.76 μm, erythrocyte size 163.81 μm2-200.67 μm2, l/w ratio 1.47-1.61, mean nucleus length 6.09 μm-7.15 μm, nucleus width 4.91 μm-6.31 μm, nucleus size 23.60 μm2-35.64 μm2, nl/nw ratio 1.14-1.25 and nucleocytoplasmic ratio 0.15-0.20 (table 3). in this regard; the longest, widest and largest erythrocytes were observed in e. orbicularis; the most strongly ellipsoidal erythrocytes and the least ellipsoidal nuclei in mauremys caspica. in addition, the smallest and the least ellipsoidal cells were found in t. graeca and the longest, widest and largest nuclei in m. caspica. however, the shortest, narrowest and the most strongly ellipsoidal nuclei were determined in t. graeca; the biggest nuclefig. 1. photomicrographs of erythrocytes of some species belong to turkish herpetofauna. a: o. vittatus, b: p. caralitanus, c: p. caucasicus, d: e. orbicularis, e: t. graeca, f: o. elegans, g: m. brevirostris, h: a. danfordi, i: l. trilineata, j: l. macrorhynchus, k: h. ravergieri, l: m. xanthina. horizontal bar: 20 μm. 185blood cell morphology of turkish herpetofauna ta bl e 2. th e er yt hr oc yt e an d th ei r nu cl ei m ea su re m en ts ( ± w ith t he ir s ta nd ar d er ro rs ) es ta bl is he d in t he p er ip he ra l b lo od s of 1 9 am ph ib ia n sp ec ie s be lo ng t o 7 fa m ili es f ro m t ur ke y [l : e ry th ro cy te le ng th , w : e ry th ro cy te w id th , e s: e ry th ro cy te s iz e, n l: n uc le us le ng th , n w : n uc le us w id th , n s: n uc le us s iz e; n s/ es : n uc le oc yt op la sm ic r at io ]. sp ec ie s er yt hr oc yt es n uc le i l (μ m ) w ( μm ) l/ w es ( μm 2 ) n l (μ m ) n w ( μm ) n l/ n w n s (μ m 2 ) n s/ es u ro de la sa la m an dr id ae li ss ot ri to n vu lg ar is 30 .0 2± 0. 16 17 .8 1± 0. 08 1. 69 ±0 .0 1 41 9. 44 ±3 .1 1 13 .8 6± 0. 13 8. 53 ±0 .0 7 1. 62 ±0 .0 2 92 .8 5± 0. 68 0. 22 ±0 .0 02 ly ci as al am an dr a at ifi 33 .2 8± 0. 17 19 .4 4± 0. 09 1. 73 ±0 .0 2 50 7. 54 ±3 .3 5 14 .9 9± 0. 11 9. 44 ±0 .0 9 1. 59 ±0 .0 2 11 1. 14 ±0 .6 4 0. 22 ±0 .0 02 m er te si el la c au ca si ca 31 .6 9± 0. 29 17 .6 9± 0. 29 1. 80 ±0 .0 2 44 0. 44 ±5 .7 9 16 .6 4± 0. 16 9. 84 ±0 .0 9 1. 69 ±0 .0 2 12 8. 60 ±0 .9 2 0. 29 ±0 .0 02 n eu re rg us s tr au ch ii 31 .2 0± 0. 21 18 .9 3± 0. 08 1. 65 ±0 .0 1 46 3. 82 ±4 .3 5 15 .4 5± 0. 07 9. 88 ±0 .0 3 1. 56 ±0 .0 3 12 0. 10 ±0 .7 7 0. 26 ±0 .0 02 o m m at ot ri to n vi tt at us 28 .0 6± 0. 16 16 .6 3± 0. 12 1. 70 ±0 .0 1 36 7. 05 ±3 .8 2 16 .0 3± 0. 14 9. 86 ±0 .0 9 1. 63 ±0 .0 2 12 4. 14 ±0 .7 0 0. 34 ±0 .0 02 sa la m an dr a in fr ai m m ac ul at a 33 .1 0± 0. 20 20 .1 3± 0. 13 1. 65 ±0 .0 2 52 3. 44 ±4 .7 9 16 .8 6± 0. 19 10 .4 6± 0. 11 1. 61 ±0 .0 2 13 8. 51 ±0 .8 4 0. 27 ±0 .0 02 tr itu ru s ka re lin ii 29 .5 0± 0. 16 18 .1 4± 0. 09 1. 63 ±0 .0 1 42 0. 37 ±2 .9 6 14 .9 8± 0. 11 9. 44 ±0 .0 7 1. 59 ±0 .0 3 11 1. 06 ±0 .7 4 0. 26 ±0 .0 02 a nu ra r an id ae pe lo ph yl ax b ed ri ag ae 23 .1 4± 0. 22 13 .1 0± 0. 09 1. 61 ±0 .0 3 26 5. 63 ±2 .5 3 8. 51 ±0 .1 1 5. 01 ±0 .0 1 1. 58 ±0 .0 3 38 .3 4± 0. 74 0. 14 ±0 .0 03 pe lo ph yl ax c ar al ita nu s 24 .3 6± 0. 23 14 .4 6± 0. 11 1. 69 ±0 .0 1 27 6. 62 ±3 .8 6 8. 13 ±0 .1 3 5. 03 ±0 .0 3 1. 61 ±0 .0 2 32 .1 5± 0. 62 0. 12 ±0 .0 01 r an a da lm at in a 19 .9 9± 0. 24 12 .1 1± 0. 11 1. 65 ±0 .0 2 19 0. 47 ±3 .5 4 8. 78 ±0 .0 9 5. 59 ±0 .0 2 1. 57 ±0 .0 2 38 .4 8± 0. 42 0. 20 ±0 .0 01 r an a ho ltz i 19 .1 0± 0. 12 12 .8 0± 0. 06 1. 64 ±0 .1 0 19 2. 81 ±1 .8 3 7. 84 ±0 .0 4 4. 13 ±0 .0 4 1. 93 ±0 .0 2 25 .4 6± 0. 27 0. 13 ±0 .0 02 r an a m ac ro cn em is 20 .5 5± 0. 12 13 .4 6± 0. 05 1. 54 ±0 .0 1 21 7. 68 ±1 .7 6 8. 66 ±0 .0 6 4. 14 ±0 .0 4 2. 14 ±0 .0 3 28 .0 3± 0. 28 0. 13 ±0 .0 02 bu fo ni da e bu fo b uf o 20 .8 5± 0. 10 13 .4 5± 0. 07 1. 55 ±0 .0 1 22 1. 22 ±1 .9 0 7. 81 ±0 .1 0 4. 34 ±0 .1 0 1. 86 ±0 .0 5 26 .6 0± 0. 63 0. 12 ±0 .0 02 ps eu de pi da le a vi ri di s 17 .8 6± 0. 07 12 .7 1± 0. 04 1. 38 ±0 .0 1 17 9. 18 ±0 .9 6 6. 25 ±0 .1 3 3. 72 ±0 .0 4 1. 96 ±0 .0 5 18 .1 3± 0. 56 0. 11 ±0 .0 02 pe lo ba tid ae pe lo ba te s sy ri ac us 17 .5 6± 0. 08 11 .7 0± 0. 07 1. 50 ±0 .0 1 16 1. 85 ±1 .3 1 6. 63 ±0 .0 9 3. 47 ±0 .0 4 1. 96 ±0 .0 5 18 .1 3± 0. 56 0. 11 ±0 .0 02 pe lo di tid ae pe lo dy te s ca uc as ic us 17 .5 6± 0. 08 9. 68 ±0 .0 9 1. 58 ±0 .0 1 11 6. 42 ±2 .0 7 6. 21 ±0 .0 7 3. 81 ±0 .0 5 1. 63 ±0 .0 2 18 .7 1± 0. 31 0. 16 ±0 .0 02 b om bi na to ri da e bo m bi na b om bi na 21 .8 0± 0. 12 15 .0 5± 0. 08 1. 45 ±0 .0 2 25 8. 14 ±2 .3 6 9. 59 ±0 .1 6 4. 88 ±0 .0 6 1. 98 ±0 .0 4 36 .6 6± 0. 82 0. 14 ±0 .0 02 h yl id ae h yl a ar bo re a 19 .8 0± 0. 10 12 .8 9± 0. 06 1. 54 ±0 .0 1 20 0. 33 ±1 .6 6 7. 94 ±0 .1 0 3. 50 ±0 .0 8 2. 35 ±0 .0 7 21 .8 8± 0. 61 0. 11 ±0 .0 01 h yl a sa vi gn yi 18 .6 3± 0. 18 12 .4 1± 0. 08 1. 50 ±0 .0 2 18 1. 44 ±1 .9 8 7. 09 ±0 .0 9 3. 97 ±0 .0 8 1. 82 ±0 .0 4 22 .3 0± 0. 55 0. 12 ±0 .0 02 186 h. arıkan and k. çiçek ta bl e 3. th e er yt hr oc yt e an d th ei r nu cl ei m ea su re m en ts ( ± w ith t he ir s ta nd ar d er ro rs ) es ta bl is he d in t he p er ip he ra l bl oo ds o f 4 tu rt le s sp ec ie s be lo ng t o 3 fa m ili es , 3 0 liz ar d sp ec ie s be lo ng to 7 fa m ili es , a nd 3 4 sn ak e sp ec ie s be lo ng to 6 fa m ili es fr om t ur ke y. sp ec ie s er yt hr oc yt es n uc le i l (μ m ) w ( μm ) l/ w es ( μm 2 ) n l (μ m ) n w ( μm ) n l/ n w n s (μ m 2 ) n s/ es te st ud in es em yd id ae em ys o rb ic ul ar is 19 .9 9± 0. 11 12 .7 6± 0. 09 1. 58 ±0 .0 1 20 0. 67 ±1 .8 8 7. 15 ±0 .0 5 6. 26 ±0 .1 9 1. 19 ±0 .0 1 35 .3 7± 1. 10 0. 18 ±0 .0 1 g eo em yd id ae m au re m ys c as pi ca 18 .9 9± 0. 09 11 .9 0± 0. 07 1. 61 ±0 .0 1 17 7. 64 ±1 .4 2 7. 15 ±0 .0 5 6. 31 ±0 .0 4 1. 14 ±0 .0 1 35 .6 4± 0. 41 0. 20 ±0 .0 1 m au re m ys r iv ul at a 19 .0 2± 0. 12 12 .1 9± 0. 08 1. 57 ±0 .0 1 18 2. 74 ±1 .9 7 6. 72 ±0 .0 4 5. 89 ±0 .0 4 1. 15 ±0 .0 1 31 .2 2± 0. 35 0. 18 ±0 .0 1 te st ud in id ae te st ud o gr ae ca 17 .3 5± 0. 14 11 .9 6± 0. 11 1. 47 ±0 .0 1 16 3. 81 ±2 .3 4 6. 09 ±0 .0 5 4. 91 ±0 .0 4 1. 25 ±0 .0 1 23 .6 0± 0. 34 0. 15 ±0 .0 1 sq ua m at a, s au ri a sc in ci da e a bl ep ha ru s ch er no vi 14 .1 3± 0. 07 7. 58 ±0 .0 3 1. 87 ±0 .0 1 84 .1 2± 0. 57 6. 12 ±0 .0 5 2. 50 ±0 .0 0 2. 45 ±0 .0 2 12 .0 1± 0. 10 0. 14 ±0 .0 01 c ha lc id es o ce lla tu s 14 .6 8± 0. 06 7. 92 ±0 .0 4 1. 86 ±0 .0 1 91 .3 3± 0. 61 5. 15 ±0 .0 3 2. 64 ±0 .0 3 1. 98 ±0 .0 2 10 .7 0± 0. 13 0. 12 ±0 .0 01 eu m ec es s ch ne id er i 15 .1 7± 0. 06 7. 74 ±0 .0 4 1. 97 ±0 .0 1 92 .3 1± 0. 56 7. 11 ±0 .0 4 2. 54 ±0 .0 2 2. 81 ±0 .0 2 14 .2 0± 0. 13 0. 15 ±0 .0 01 o ph io m or us p un ct at is si m us 15 .1 4± 0. 06 7. 73 ±0 .0 4 1. 96 ±0 .0 1 92 .0 8± 0. 64 6. 05 ±0 .0 5 2. 68 ±0 .0 4 2. 30 ±0 .0 3 12 .7 0± 0. 21 0. 14 ±0 .0 02 tr ac hy le pi s au ra ta 14 .2 7± 0. 08 7. 56 ±0 .0 2 1. 90 ±0 .0 1 84 .8 8± 0. 54 5. 06 ±0 .0 2 2. 52 ±0 .0 1 2. 01 ±0 .0 1 10 .0 2± 0. 07 0. 12 ±0 .0 01 tr ac hy le pi s vi tt at a 14 .1 4± 0. 08 7. 55 ±0 .0 2 1. 87 ±0 .0 1 83 .7 7± 0. 53 6. 14 ±0 .0 5 2. 50 ±0 .0 0 2. 46 ±0 .0 2 12 .0 6± 0. 10 0. 14 ±0 .0 01 la ce rt id ae a ca nt ho da ct yl us b os ki an us 14 .2 2± 0. 98 7. 92 ±0 .4 1 1. 80 ±0 .1 4 88 .4 5± 8. 37 6. 24 ±0 .5 5 4. 02 ±0 .1 2 1. 56 ±0 .1 5 19 .6 9± 1. 70 0. 23 ±0 .0 2 a ca nt ho da ct yl us h ar ra ne ns is 15 .4 6± 1. 24 8. 56 ±0 .5 9 1. 81 ±0 .1 3 10 4. 22 ±1 3. 53 6. 59 ±0 .5 0 4. 07 ±0 .1 3 1. 62 ±0 .1 5 21 .0 2± 1. 49 0. 21 ±0 .0 2 a na to lo la ce rt a da nf or di 14 .1 4± 1. 17 9. 09 ±0 .5 1 1. 56 ±0 .1 1 10 1. 13 ±1 2. 10 6. 73 ±0 .6 1 4. 41 ±0 .1 3 1. 53 ±0 .1 4 23 .3 2± 2. 30 0. 23 ±0 .0 3 a pa th ya c ap pa do ci ca 13 .4 2± 0. 90 7. 94 ±0 .4 7 1. 69 ±0 .1 4 83 .7 3± 8. 13 6. 33 ±0 .4 6 4. 11 ±0 .1 3 1. 54 ±0 .1 3 20 .4 2± 1. 58 0. 25 ±0 .0 2 (c on tin ue d) . 187blood cell morphology of turkish herpetofauna sp ec ie s er yt hr oc yt es n uc le i l (μ m ) w ( μm ) l/ w es ( μm 2 ) n l (μ m ) n w ( μm ) n l/ n w n s (μ m 2 ) n s/ es d ar ev sk ia p ra tic ol a 13 .0 8± 0. 97 8. 01 ±0 .3 8 1. 64 ±0 .1 3 82 .3 4± 7. 78 6. 19 ±0 .4 3 4. 31 ±0 .1 2 1. 44 ±0 .0 2 20 .9 3± 1. 46 0. 26 ±0 .0 2 d ar ev sk ia u zz el li 13 .6 5± 0. 96 7. 84 ±0 .4 8 1. 74 ±0 .1 2 84 .2 2± 9. 33 5. 98 ±0 .3 6 4. 34 ±0 .1 4 1. 38 ±0 .1 1 20 .3 6± 1. 22 0. 25 ±0 .0 3 d ar ev sk ia v al en tin i 13 .3 2± 0. 93 7. 73 ±0 .5 7 1. 73 ±0 .1 4 80 .9 7± 9. 47 6. 13 ±0 .4 8 4. 28 ±0 .1 6 1. 43 ±0 .1 0 20 .6 3± 2. 03 0. 26 ±0 .0 3 la ce rt a pa m ph yl ic a 15 .6 1± 1. 00 7. 89 ±0 .5 2 1. 99 ±0 .1 6 96 .7 7± 9. 91 6. 33 ±0 .4 1 4. 23 ±0 .1 1 1. 49 ±0 .0 9 21 .0 1± 1. 61 0. 22 ±0 .0 2 la ce rt a tr ili ne at a 14 .3 9± 1. 01 7. 63 ±0 .4 9 1. 89 ±0 .1 2 86 .3 1± 10 .2 2 6. 93 ±0 .5 2 3. 93 ±0 .1 1 1. 77 ±0 .1 6 21 .3 8± 1. 56 0. 25 ±0 .0 2 la ce rt a vi ri di s 14 .9 4± 1. 04 8. 16 ±0 .5 6 1. 83 ±0 .1 0 96 .0 3± 11 .7 8 6. 64 ±0 .5 2 4. 36 ±0 .1 3 1. 53 ±0 .1 4 22 .6 8± 1. 73 0. 24 ±0 .0 2 m es al in a br ev ir os tr is 14 .0 6± 0. 91 8. 07 ±0 .4 2 1. 75 ±0 .1 4 89 .0 9± 7. 52 6. 46 ±0 .4 9 3. 84 ±0 .1 5 1. 69 ±0 .1 7 19 .4 8± 1. 34 0. 22 ±0 .0 2 o ph is op s el eg an s 12 .4 3± 0. 65 7. 51 ±0 .2 5 1. 66 ±0 .0 9 73 .2 7± 4. 88 6. 51 ±0 .3 4 3. 84 ±0 .1 5 1. 70 ±0 .1 1 19 .6 3± 1. 22 0. 27 ±0 .0 2 pa rv ila ce rt a pa rv a 13 .6 3± 0. 86 8. 01 ±0 .4 4 1. 70 ±0 .1 2 85 .8 0± 8. 39 6. 12 ±0 .4 8 3. 98 ±0 .1 0 1. 54 ±0 .1 2 19 .1 5± 1. 67 0. 22 ±0 .0 2 po da rc is m ur al is 13 .9 3± 0. 95 8. 43 ±0 .5 9 1. 66 ±0 .1 1 92 .4 6± 11 .3 2 6. 36 ±0 .5 4 4. 35 ±0 .1 2 1. 46 ±0 .1 2 21 .7 4± 2. 06 0. 24 ±0 .0 2 po da rc is s ic ul us 13 .8 9± 0. 94 8. 10 ±0 .3 5 1. 74 ±0 .1 2 87 .4 1± 7. 85 6. 59 ±0 .5 1 4. 19 ±0 .1 2 1. 57 ±0 .1 3 21 .6 9± 1. 82 0. 25 ±0 .0 2 ti m on p ri nc ep s 14 .9 8± 1. 14 8. 43 ±0 .4 7 1. 78 ±0 .1 4 99 .2 7± 10 .8 3 6. 04 ±0 .5 3 3. 99 ±0 .1 1 1. 52 ±0 .1 5 18 .8 9± 1. 67 0. 19 ±0 .0 2 eu pl eb ha ri da e eu bl ep ha ri s an gr am ai ny u 16 .5 7± 0. 17 8. 93 ±0 .0 8 1. 86 ±0 .0 2 11 6. 29 ±1 .9 5 7. 38 ±0 .0 7 4. 38 ±0 .0 2 1. 69 ±0 .0 2 25 .3 5± 0. 24 0. 22 ±0 .0 1 g ek ko ni da e c yr to po di on h et er oc er cu m 16 .1 7± 0. 20 8. 81 ±0 .0 6 1. 84 ±0 .0 3 11 1. 77 ±1 .5 7 7. 57 ±0 .0 8 4. 59 ±0 .0 2 1. 65 ±0 .0 2 27 .2 7± 0. 33 0. 24 ±0 .0 1 c yr to po di on s ca br um 14 .8 3± 0. 10 8. 34 ±0 .0 7 1. 78 ±0 .0 1 97 .1 3± 1. 26 7. 08 ±0 .0 6 4. 38 ±0 .0 2 1. 62 ±0 .0 1 24 .3 4± 0. 24 0. 25 ±0 .0 1 h em id ac ty lu s tu rc ic us 16 .5 6± 0. 21 8. 91 ±0 .0 6 1. 86 ±0 .0 2 11 5. 89 ±1 .9 3 7. 44 ±0 .0 9 4. 40 ±0 .0 2 1. 69 ±0 .0 2 25 .7 1± 0. 32 0. 22 ±0 .0 1 a ga m id ae la ud ak ia s te lli o 16 .8 5± 0. 18 9. 12 ±0 .0 6 1. 85 ±0 .0 2 12 0. 71 ±1 .7 1 7. 84 ±0 .0 8 4. 40 ±0 .0 2 1. 79 ±0 .0 2 27 .0 8± 0. 29 0. 23 ±0 .0 1 tr ap el us le ss on ae 14 .7 5± 0. 16 8. 69 ±0 .0 8 1. 70 ±0 .0 2 10 0. 78 ±1 .6 7 6. 91 ±0 .0 6 4. 58 ±0 .0 2 1. 51 ±0 .0 1 24 .8 3± 0. 25 0. 25 ±0 .0 1 ta bl e 3. ( co nt in ue d) . (c on tin ue d) . 188 h. arıkan and k. çiçek sp ec ie s er yt hr oc yt es n uc le i l (μ m ) w ( μm ) l/ w es ( μm 2 ) n l (μ m ) n w ( μm ) n l/ n w n s (μ m 2 ) n s/ es c ha m ae le on id ae c ha m ae le o ch am ae le on 15 .9 7± 0. 16 9. 75 ±0 .0 8 1. 64 ±0 .0 2 12 2. 34 ±1 .8 1 7. 72 ±0 .0 9 4. 85 ±0 .0 3 1. 59 ±0 .0 2 29 .3 7± 0. 35 0. 24 ±0 .0 1 v ar an id ae va ra nu s gr is eu s 16 .2 4± 0. 13 10 .2 1± 0. 09 1. 59 ±0 .0 1 13 0. 33 ±1 .9 9 7. 09 ±0 .0 7 4. 69 ±0 .0 3 1. 51 ±0 .0 2 26 .1 2± 0. 34 0. 20 ±0 .0 1 sq ua m at a, o ph id ia le pt ot yp hl op id ae le pt ot yp hl op s m ac ro rh yn ch us 15 .8 6± 0. 11 9. 29 ±0 .0 8 1. 71 ±0 .0 2 11 5. 75 ±1 .4 5 7. 33 ±0 .0 8 4. 45 ±0 .0 2 1. 65 ±0 .0 2 25 .5 8± 0. 31 0. 22 ±0 .0 1 ty ph lo pi da e ty ph lo ps v er m ic ul ar is 16 .5 7± 0. 17 9. 13 ±0 .0 6 1. 82 ±0 .0 2 11 8. 76 ±1 .6 0 7. 27 ±0 .0 8 4. 54 ±0 .0 2 1. 60 ±0 .0 2 25 .9 3± 0. 29 0. 22 ±0 .0 1 b oi da e er yx ja cu lu s 16 .3 6± 0. 19 8. 77 ±0 .0 7 1. 87 ±0 .0 2 11 2. 83 ±2 .0 1 7. 16 ±0 .0 9 4. 39 ±0 .0 2 1. 63 ±0 .0 2 24 .6 7± 0. 33 0. 22 ±0 .0 2 c ol ub ri da e d ol ic ho ph is c as pi us 14 .9 1± 0. 16 7. 64 ±0 .1 2 1. 96 ±0 .0 2 89 .8 8± 2. 21 10 .0 1± 0. 08 4. 84 ±0 .0 4 2. 07 ±0 .0 2 38 .0 8± 0. 52 0. 43 ±0 .0 1 d ol ic ho ph is ju gu la ri s 16 .2 9± 0. 18 7. 48 ±0 .0 7 2. 18 ±0 .0 2 95 .8 1± 1. 69 10 .5 7± 0. 06 4. 98 ±0 .0 4 2. 13 ±0 .0 2 41 .2 7± 0. 37 0. 44 ±0 .0 1 d ol ic ho ph is s ch m id ti 16 .2 1± 0. 14 9. 88 ±0 .0 7 1. 64 ±0 .0 1 12 5. 82 ±1 .7 1 7. 67 ±0 .0 8 4. 27 ±0 .0 2 1. 80 ±0 .0 2 25 .7 0± 0. 31 0. 20 ±0 .0 1 ei re ni s ba ra ni 16 .1 8± 0. 12 9. 68 ±0 .0 5 1. 67 ±0 .0 1 12 2. 98 ±1 .2 7 7. 94 ±0 .0 8 4. 54 ±0 .0 2 1. 75 ±0 .0 2 28 .3 2± 0. 29 0. 23 ±0 .0 1 ei re ni s co ro ne lla 16 .5 9± 0. 23 10 .2 2± 0. 11 1. 63 ±0 .0 2 13 3. 52 ±2 .8 1 7. 43 ±0 .0 9 4. 58 ±0 .0 2 1. 62 ±0 .0 2 26 .6 6± 0. 34 0. 20 ±0 .0 1 ei re ni s de ce m lin ea tu s 14 .7 5± 0. 14 10 .0 3± 0. 07 1. 47 ±0 .0 1 11 6. 25 ±1 .5 5 7. 68 ±0 .1 0 4. 50 ±0 .0 3 1. 71 ±0 .0 3 27 .1 2± 0. 37 0. 23 ±0 .0 1 ei re ni s ei se lti 14 .1 3± 0. 13 9. 62 ±0 .0 7 1. 47 ±0 .0 1 10 6. 84 ±1 .6 5 7. 29 ±0 .0 5 4. 54 ±0 .0 2 1. 60 ±0 .0 1 26 .0 0± 0. 22 0. 25 ±0 .0 1 ei re ni s le va nt in us 16 .6 0± 0. 15 10 .0 4± 0. 09 1. 66 ±0 .0 2 13 0. 84 ±1 .7 7 8. 12 ±0 .0 8 4. 47 ±0 .0 2 1. 82 ±0 .0 2 28 .4 7± 0. 29 0. 22 ±0 .0 1 ei re ni s m od es tu s 14 .4 7± 0. 15 7. 45 ±0 .0 8 1. 95 ±0 .0 2 84 .7 8± 1. 53 10 .0 5± 0. 06 4. 92 ±0 .0 7 2. 06 ±0 .0 2 38 .8 0± 0. 55 0. 46 ±0 .0 1 ei re ni s pu nc ta to lin ea tu s 16 .2 2 ±0 .1 5 9. 58 ±0 .0 7 1. 70 ±0 .0 1 12 2. 07 ±1 .7 8 7. 63 ±0 .0 8 4. 52 ±0 .0 3 1. 69 ±0 .0 2 27 .0 6± 0. 31 0. 22 ±0 .0 1 ta bl e 3. ( co nt in ue d) . (c on tin ue d) . 189blood cell morphology of turkish herpetofauna sp ec ie s er yt hr oc yt es n uc le i l (μ m ) w ( μm ) l/ w es ( μm 2 ) n l (μ m ) n w ( μm ) n l/ n w n s (μ m 2 ) n s/ es ei re ni s ro th ii 14 .7 7± 0. 15 8. 73 ±0 .0 6 1. 69 ±0 .0 2 10 1. 24 ±1 .4 4 7. 84 ±0 .1 2 4. 14 ±0 .0 2 1. 90 ±0 .0 3 25 .4 5± 0. 38 0. 25 ±0 .0 1 h em or rh oi s nu m m ife r 15 .6 1± 0. 10 9. 33 ±0 .0 4 1. 68 ±0 .0 1 11 4. 30 ±0 .9 0 6. 92 ±0 .0 8 4. 52 ±0 .0 1 1. 53 ±0 .0 2 24 .5 3± 0. 24 0. 21 ±0 .0 1 c ol ub ri da e h em or rh oi s ra ve rg ie ri 14 .7 6± 0. 16 9. 95 ±0 .1 1 1. 49 ±0 .0 2 11 5. 38 ±1 .9 1 7. 49 ±0 .0 9 4. 91 ±0 .0 4 1. 53 ±0 .0 2 28 .9 0± 0. 50 0. 25 ±0 .0 1 m al po lo n m on sp es su la nu s 15 .2 4± 0. 13 11 .1 6± 0. 09 1. 37 ±0 .0 1 13 3. 60 ±1 .7 7 7. 42 ±0 .0 9 4. 84 ±0 .0 3 1. 54 ±0 .0 2 28 .1 7± 0. 38 0. 21 ±0 .0 2 n at ri x na tr ix 16 .8 7± 0. 18 10 .1 5± 0. 08 1. 67 ±0 .0 2 13 4. 46 ±1 .6 6 7. 95 ±0 .0 7 4. 56 ±0 .0 2 1. 74 ±0 .0 2 28 .4 9± 0. 30 0. 21 ±0 .0 1 n at ri x te ss el la ta 15 .9 8± 0. 21 7. 92 ±0 .0 9 2. 02 ±0 .0 3 99 .6 1± 2. 13 10 .2 1± 0. 09 5. 04 ±0 .0 4 2. 03 ±0 .0 2 40 .4 6± 0. 61 0. 41 ±0 .0 1 pl at yc ep s co lla ri s 14 .4 0± 0. 12 10 .0 4± 0. 08 1. 44 ±0 .0 1 11 3. 63 ±1 .5 4 7. 29 ±0 .0 7 4. 62 ±0 .0 2 1. 58 ±0 .0 2 26 .4 2± 0. 26 0. 23 ±0 .0 2 pl at yc ep s na ja du m 15 .4 7± 0. 14 10 .2 3± 0. 13 1. 52 ±0 .0 1 12 4. 50 ±2 .4 1 8. 44 ±0 .2 3 5. 03 ±0 .0 5 1. 69 ±0 .0 5 33 .3 2± 0. 97 0. 27 ±0 .0 1 pl at yc ep s ve nt ro m ac ul at us 15 .9 4± 0. 17 10 .6 7± 0. 11 1. 50 ±0 .0 2 13 3. 60 ±2 .1 4 6. 91 ±0 .0 6 4. 49 ±0 .0 3 1. 54 ±0 .0 2 24 .3 3± 0. 22 0. 18 ±0 .0 1 r hy nc ho ca la m us m el an oc ep ha lu s 17 .9 6± 0. 20 9. 85 ±0 .0 7 1. 83 ±0 .0 2 13 8. 88 ±1 .9 0 7. 95 ±0 .0 8 4. 47 ±0 .0 2 1. 78 ±0 .0 2 27 .8 8± 0. 28 0. 20 ±0 .0 1 sp al er os op hi s di ad em a 15 .7 4± 0. 18 9. 52 ±0 .1 3 1. 66 ±0 .0 2 11 8. 10 ±2 .6 9 6. 81 ±0 .1 0 4. 67 ±0 .0 2 1. 46 ±0 .0 2 24 .9 8± 0. 42 0. 21 ±0 .0 2 te le sc op us fa lla x 18 .3 3± 0. 23 10 .3 3± 0. 10 1. 78 ±0 .0 2 14 8. 80 ±2 .5 7 7. 53 ±0 .0 6 5. 06 ±0 .0 4 1. 49 ±0 .0 2 29 .8 7± 0. 34 0. 20 ±0 .0 2 te le sc op us n ig ri ce ps 18 .5 5± 0. 20 10 .4 3± 0. 11 1. 78 ±0 .0 2 15 2. 14 ±2 .7 9 7. 96 ±0 .1 0 4. 60 ±0 .0 2 1. 73 ±0 .0 2 28 .7 3± 0. 37 0. 19 ±0 .0 2 z am en is h oh en ac ke ri 17 .6 6± 0. 24 9. 91 ±0 .0 9 1. 79 ±0 .0 3 13 7. 55 ±2 .5 1 8. 49 ±0 .1 3 4. 44 ±0 .0 2 1. 92 ±0 .0 3 29 .5 3± 0. 41 0. 22 ±0 .0 1 z am en is lo ng is si m us 12 .7 1± 0. 15 7. 38 ±0 .0 7 1. 72 ±0 .0 2 73 .8 3± 1. 38 6. 61 ±0 .0 8 4. 56 ±0 .0 3 1. 45 ±0 .0 2 23 .6 3± 0. 31 0. 32 ±0 .0 1 v ip er id ae m ac ro vi pe ra le be tin a 17 .2 1± 0. 25 9. 83 ±0 .1 0 1. 75 ±0 .0 2 13 3. 11 ±2 .6 1 6. 68 ±0 .1 2 4. 74 ±0 .0 5 1. 41 ±0 .0 3 24 .8 7± 0. 51 0. 19 ±0 .0 3 m on tiv ip er a al bi zo na 17 .1 6± 0. 26 9. 67 ±0 .1 3 1. 78 ±0 .0 3 13 0. 72 ±3 .3 1 7. 39 ±0 .1 4 4. 36 ±0 .0 5 1. 70 ±0 .0 1 25 .3 2± 0. 62 0. 20 ±0 .0 1 m on tiv ip er a w ag ne ri 17 .6 3± 0. 20 7. 62 ±0 .1 0 2. 32 ±0 .0 3 10 5. 71 ±2 .2 2 10 .6 1± 0. 07 4. 70 ±0 .0 5 2. 27 ±0 .0 2 39 .1 9± 0. 55 0. 38 ±0 .0 1 m on tiv ip er a xa nt hi na 17 .0 8± 0. 16 7. 20 ±0 .1 0 2. 38 ±0 .0 3 96 .7 8± 2. 08 v ip er a er iw an en si s 16 .9 8± 0. 17 7. 58 ±0 .0 8 2. 25 ±0 .0 3 10 1. 16 ±1 .6 5 10 .5 8± 0. 06 4. 91 ±0 .0 4 2. 16 ±0 .0 2 40 .7 7± 0. 45 0. 41 ±0 .0 1 el ap id ae w al te ri nn es ia m or ga ni 16 .2 0± 0. 15 10 .1 4± 0. 10 1. 60 ±0 .0 2 12 9. 12 ±2 .0 5 7. 53 ±0 .0 8 4. 82 ±0 .0 4 1. 57 ±0 .0 2 28 .5 2± 0. 41 0. 22 ±0 .0 1 ta bl e 3. ( co nt in ue d) . 190 h. arıkan and k. çiçek ocytoplasmic ratio in m. caspica and the smallest in t. graeca. nuclei were found more spherical in turtles than amphibians. in the lizard species examined; mean length, width and size of erythrocytes ranged respectively between 12.43 μm-16.85 μm, 7.51 μm-10.21 μm and 73.27 μm2-130.33 μm2; on the other hand, l/w ratio between 1.56-1.99 (fig. 1f, g, h, i). in this regard, the longest erythrocytes were observed in laudakia stellio; the widest and largest in varanus griseus; the shortest, narrowest and smallest in ophisops elegans (fig. 1f). and in terms of l/w ratios, the most strongly ellipsoidal cells were determined in lacerta pamhylica and the least ellipsoidal cells in anatololacerta danfordi (table 3). the longest nuclei were found in l. stellio, the widest and largest in chamaeleo chamaeleon, the shortest and smallest in trachlepis aurata; the narrowest in ablepharus chernovi and trachlepis vittata. considering nl/nw ratios, the most strongly ellipsoidal nuclei were found in eumeces schneideri, and the least ellipsoidal in darevskia uzzelli. the highest nucleocytoplasmic ratio was determined in o. elegans, and the smallest in t. aurata and chalcides ocellatus (table 3). in the snake species examined; mean length, width and size of erythrocytes ranged respectively between 14.13 μm-18.55 μm, 7.20 μm-11.16 μm and 84.78 μm2-152.14 μm2; and l/w ratio between 1.37-2.38 (fig. 1j, k, l). in this regard, the longest and largest erythrocytes were observed in telescopus nigriceps; the widest in malpolon monspessulanus; the shortest in eirenis eiselti; the narrowest in m. xanthina and the smallest in eirenis modestus. in terms of l/w ratio; the most strongly ellipsoidal cells were found in m. xanthina, and the least ellipsoidal in m. monspessulanus (table 3). because of the irregular nuclei shapes of erythrocytes in m. xanthina, measurements of nuclei were not given in table 3. among the examined species, the longest nuclei were observed in montivipera wagneri; the widest in telescopus fallax; the largest in dolichopis jugularis; the shortest in macrovipera lebetina; the narrowest in eirenis rothi, and the smallest in platyceps ventromaculatus. in terms of nl/nw ratio; the most strongly ellipsoidal nuclei were determined in m. wagneri; the least ellipsoidal in m. lebetina; the highest nucleocytoplasmic ratio in vipera eriwanensis, and the smallest in zamenis longissimus (table 3). according to the data obtained in the study, there was no correlation between body size and their erythrocytes size (kendall τ test, r = 0.024, p≤ 0.845). regarding leucocytes, both small and large lymphocytes were observed as the dominant cells in blood smears of all species in herpetofauna. lymphocytes and monocytes were formed by 80% in leucocytes of the examined species. in small lymphocytes, chromophilic nuclei almost filled the whole cell. cytoplasm was pushed to a small zone (fig. 2a). the biggest mean diameter of small lymphocytes was observed in urodeles (14.92 µm), and the smallest (7.79 µm) in lizards (table 4). spherical nuclei were more chromophilic in large lymphocytes, and localized in a certain cell zone. cytoplasm covered larger area than small lymphocytes and was stained a pale blue, and nuclei was stained a purplish blue with wright’s stain (fig. 2b). the biggest mean diameter in large lymphocytes was observed in urodeles (20.73 µm), and the smallest (11.63 µm) in lizards (table 4). monocytes were similar to large lymphocytes; however, could easily be differentiated by kidney shaped nuclei. cytoplasm was stained a light gray, and the nuclei was stained a dark purplish blue with wright’s stain (fig. 2c, d). the biggest mean diameter in monocytes was observed in urodeles (21.00 µm), and the smallest (12.20 µm) in turtles (table 4). no monocyte was observed in m. lebetina (a snake species). 191blood cell morphology of turkish herpetofauna the biggest mean diameter in heterophils of granulocytes, spherical cells, was observed in urodeles ((22.78 µm), and the smallest (11.49 µm) in turtles (table 4). their cytoplasms were stained a light blue, and the nuclei, consisting of 2-3 lobes, was stained a red to brown with wright’s stain (fig. 2c, e, f). the granules are eosinophilic, elongated, or spindle shaped and could be numerous. cytoplasms of eosinophils were stained a light yellowish color with wright’s stain. since nucleus was masked by the large and bright red granules in cytoplasm, its shape couldn’t be fully distinguished (fig. 2g, h). the biggest mean diameter in eosinophils was observed in urodeles (21.13 µm), and the smallest in lizards (table 4). no eosinophil was observed in w. morgani. fig. 2. photomicrographs of leucocytes and thrombocytes of some species belong to turkish herpetofauna. a: small lymphocyte (l. trilineata); b: large lymphocyte (z. hohenackeri); c: monocyte and heterophile (o. elegans); d: monocyte (p. najadum), e: heterophile (n. strauchi), f: heterophile (e. modestus), g: eosinophile (p. najadum), h: eosinophile (p. caralitanus), i: basophile (a. cappadocica), j: basophile (s. diadema), k: a group of thrombocytes (o. elegans), l: a group of thrombocytes (p. najadum). horizontal bar: 20 μm. 192 h. arıkan and k. çiçek the biggest mean diameter determined in basophils which was smaller than other granulocytic cells was observed in urodeles (19.41 µm), and the smallest (9.99 µm) in lizards (table 4). their cytoplasms were filled with black granules, and the nucleus was masked by granules just like in the eosinophils (fig. 2i, j). no basophile was observed in w. morgani. thrombocytes were observed as spindle shaped in some species (fig. 2l), and as nearly spheroidal in others (fig. 2k). chromophilic nuclei were found to fill nearly the whole cell. the longest and largest thrombocytes were observed in urodeles (tl = 24.13 µm, tw = 12.88 µm), and the shortest and narrowest in lizards (tl = 7.13 µm, tw = 5.02 µm, table 4). discussion and conclusions as stated in literature (wintrobe, 1933; foxon, 1964; hartman and lessler, 1964; kuramoto, 1981; claver and quaglia, 2009), findings of the study clearly demonstrated that urodeles had the biggest blood cells (erythrocyte, leucocyte, thrombocyte) among the amphibians and reptiles of herpetofauna, and blood smears displayed considerable interspecific and even intraspecific variations in terms of cell sizes (fig. 1, 2; table 2, 3). no important difference was observed between both the erythrocyte and nucleus sizes of urodele and anuran species of the examined amphibians. however, it is impossible to attribute these differences to the correlation regarding body weight and size, defined by vernberg (1955). more probably, these differences were derived from various environmental conditions (e.g. temperature, air pressure) (ruiz et al. 1983, 1989) and/or various activity levels (e.g. healthy, breeding, hibernating, foraging, and daily activity) (e.g. wojtaszek et al., 1997; campbell, 2004; allander and fry, 2008, sykes and klaphake, 2008), for erythrocytes were found larger in aquatic species than terrestrials, and smaller in more active species. this view is compatible with the conclusions of haden (1940), altman and dittmer (1961), harris (1963), atatür et al. (1998, 1999) and gül and tok (2009). table 4. leucocytes and thrombocytes size (± with their standard errors) in the peripheral bloods of turkish amphibians and reptiles [tl: thrombocyte length, tw: thrombocyte width]. lymphocyte (small) (μm) lymphocyte (large) (μm) monocyte (μm) heterophile (μm) eosinophil (μm) basophil (μm) thrombocytes tl (μm) tw (μm) amphibia urodela 14.92±0.20 20.73±0.48 21.0±0.18 22.78±0.14 21.13±0.18 19.41±0.67 24.13±0.64 12.88±0.23 anura 9.68±0.14 12.46±0.23 14.75±0.34 13.63±0.19 11.61±0.16 11.77±0.20 8.82±0.18 5.93±0.12 reptilia testudines 8.52±0.16 11.91±0.18 12.20±0.14 11.49±0.71 11.80±0.32 10.73±0.10 13.68±0.35 6.27±0.25 squamata sauria 7.79±0.13 11.63±0.25 13.52±0.28 12.39±0.21 10.47±0.14 9.99±0.10 7.13±0.16 5.02±0.06 ophidia 7.99±0.11 12.48±0.20 12.85±0.12 11.87±0.31 11.00±0.12 10.52±0.08 10.17±0.42 5.95±0.13 193blood cell morphology of turkish herpetofauna l/w ratio ranged between 1.63-1.80 in urodeles, and 1.38-1.69 in anurans (table 2); consequently, erythrocyte shape was more ellipsoidal in urodeles than anurans. nl/nw ratio ranged between 1.55-1.69 in urodeles, and 1.57-2.35 in anurans (table 2); that is, contrary to the situation in l/w, anurans were found to have more ellipsoidal nucleus than urodeles, which was compatible with the findings of kuramoto (1981). nucleocytoplasmic ratio ranged between 0.22-0.34 in urodeles, and 0.10-0.16 in anurans (table 2); that is, anurans had wider cytoplasmic surface area than urodeles in terms of the nuclear surface area in erythrocytes. therefore, erythrocytes in anurans were more convenient for gas exchange than urodeles. wintrobe (1933) stated that erythrocyte size reflected the place of a species in the evolutionary scale where the lower vertebrate and the species which were unsuccessful from evolutionary aspect had larger and nucleated erythrocytes; on the other hand, the higher vertebrates had small and enucleated erythrocytes. from this respect, reptiles are regarded as intermediate between amphibians and birds (szarski and czopek, 1966; szarski, 1968). results of the study indicate that erythrocyte size reflected the place of a species in the evolutionary scale in higher taxa. different researchers (hartman and lessler, 1964; szarski and czopek, 1966; saint girons and saint girons, 1969; saint girons, 1970; arıkan et al., 2004; frye, 1991; mader, 2000; campbell, 2004; strik et al., 2007; sykes and klaphake, 2008; arıkan et al., 2009a, b; claver and quaglia, 2009) reported that reptiles constitute a heterogeneous group among vertebrates in terms of their blood cell morphology, and demonstrated considerable variations among orders, even within the same family members. among reptiles, the largest erythrocytes were observed in sphenodon punctatus, in turtles and crocodiles; and the smallest in lacertid lizards (hartman and lessler, 1964; saint girons and saint girons, 1969; saint girons, 1970; sevinç et al., 2000). among the turtle species examined, aquatic ones had larger erythrocytes and nuclei than terrestrial t. graeca (table 3). aquatic species had more ellipsoidal erythrocytes than t. graeca regarding l/w ratio; however, t. graeca had more ellipsoidal nuclei than aquatic species regarding nl/nw ratio (table 3). this confirms the findings of uğurtaş et al. (2003). nucleocytoplasmic ratio was found smaller in t. graeca than aquatic species (table 3). consequently, it can be concluded that t. graeca had more convenient erythrocytes for gas exchange than aquatic ones. among the 30 lizard species examined, the largest erythrocytes were observed in v. griseus and the smallest in o. elegans. erythrocyte size demonstrated great variations among the families, and in some cases even within the species of the same family, which we believe were caused by different activity levels (e.g., healthy, breeding, hibernating, foraging, and other daily activities). regarding l/w ratio, the most ellipsoidal erythrocytes were observed in l. pamhylica, and the least or nearly spheroidal ones in a. danfordi. regarding nl/nw ratio, scincid lizards had more ellipsoidal nucleus than others (table 3). generally, there was a positive correlation between erythrocyte and nucleus sizes in lizards. nucleocytoplasmic ratio ranged between 0.12-0.15 in scincidae family, and 0.190.27 in others (table 3). in this regard, we could deduce that scincidae had more convenient erythrocytes for gas exchange than other lizards. saint girons and saint girons (1969) reported that except for typhlops vermicularis with their relatively small erythrocytes and large nuclei, the snakes formed a homoge194 h. arıkan and k. çiçek nous group regarding their erythrocyte sizes. however, in this study, great variations were found among families, and even within the same family members regarding their erythrocyte sizes. among the 34 species examined, the largest erythrocytes were observed in t. nigriceps, and the smallest in z. longissimus. regarding l/w ratio, the most ellipsoidal erythrocytes were in m. xanthina, and the least ellipsoidal or nearly spheroidal ones in m. monspessulanus. results of the study indicated that small sized species (e.g., t. vermicularis’s mean size is 25 cm) don’t have smaller erythrocytes than the biger ones (e.g., z. longissimus’s mean size is 150 cm). some researchers (gulliver, 1875; saint girons and saint girons, 1969; arıkan et al., 2004) reported the presence of somewhat irregular nuclei in the erythrocytes of viperid and elapid species. similar results were found especially in m. xanthina. regarding nl/ nw ratio, the most ellipsoidal nuclei were observed in m. wagneri, and the least ellipsoidal in m. lebetina. contrary to lizards, there was no correlation between the erythrocyte and nuclei sizes in snakes. regarding nucleocytoplasmic ratio, snakes formed a heterogeneous group, and this ratio ranged between 0.19 and 0.46. lymphocytes are generally dominant leucocytes in amphibians and reptiles (frye, 1991; mader, 2000; campbell, 2004; strik et al., 2007; allander and fry, 2008; sykes and klaphake, 2008). saint girons (1970) and arıkan et al. (2004, 2009a) reported that small and large lymphocytes were the dominant cells in blood smears of different reptile species, and the nuclei were not easily be distinguished, for they were masked by dense granulations in the cytoplasms of both eosinophils and basophils. in this study investigating both amphibian and reptile species of herpetofauna; the largest leucocytes were found in urodeles (table 4); small and large lymphocytes were the dominant cells in the blood smears; and the shapes of the nuclei were not distinguished because of the dense granulations in the cytoplasms of both the eosinophils and basophils, which were all compatible with the literature (e.g., claver and quaglia, 2009). though monocytes, heterophils, and eosinophils were present in amphibians and reptiles (allander and fry, 2008; sykes and klaphake, 2008), cannon et al. (1996) reported that the heterophils were not observed in cyrtopodion scabrum. besides, eosinophils were observed in crocodilia and chelonia, but their existence in squamata is controversial (claver and quaglia 2009). even inside a genus of snakes, eosinophils were found in some species and not found in some others (alleman et al., 1992; troiano et al., 1997). number and kind of leucocytes could be change environmental and physiological activity (allander and fry, 2008; sykes and klaphake, 2008). however, blood cells of reptiles still completely unknown (e.g., frye, 1991; mader, 2000; campbell, 2004; strik et al., 2007), five types of leucocytes are observed (lymphocyte, monocyte, heterophile [neutrophile], eosinophile and basophile) (sykes and klaphake, 2008). thrombocytes were defined by some researchers as spindle shaped cells with centrally localized extremely chromophilic nuclei (saint girons, 1970; canfield and shea, 1988; arıkan et al., 2004, 2009a; allander and fry, 2008; sykes and klaphake, 2008). spindleshaped thrombocytes were observed in some species of herpetofauna, and nearly spheroidal ones were found in some other species. in this study, the largest thrombocytes were observed in amphibians and the smallest in lizards (table 4). in conclusion, the findings of the study presented basic data comprising cytomorphological structure of peripheral blood cells (table 2, 3, 4) of some turkish amphibians and reptiles. according to the results, morphology and size of erythrocytes have showed 195blood cell morphology of turkish herpetofauna great variations among species and even among the preparations of the same species. the largest blood cells were found in urodeles and aquatic and semiaquatic species had larger erythrocytes than terrestrials; in addition, the largest erythrocytes were in turtles among the reptile species examined. lymphocytes were predominant cells among leucocytes in blood smears of the species. references allander, m.c., fry, m.m. 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(1997): circulating blood parameters of the water frog, rana esculenta l. at pre-wintering stage. zool. pol. 1: 117-126. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 7(2): 355, 2012 book review: marine s. arakelyan, felix d. danielyan, claudia corti, roberto sindaco, alan e. leviton 2011. herpetofauna of armenia and nagorno-karabakh. society for the study of amphibians and reptiles stefano scali museo civico di storia naturale di milano, corso venezia 55, i-2012 milano. e-mail: stefano.scali@ comune.milano.it “herpetofauna of armenia and nagorno-karabakh”, written by arakelyan, danielyan, corti, sindaco and leviton (2011) is an original contribution to the knowledge of amphibians and reptiles of a very poorly known but biogeographical relevant area. the authors are among the most important experts of this interesting region and they resumed all the available data on amphibians and reptiles collected during many years of research. the book includes a geographic, ecological and zoogeographic framework of the two nations. furthermore, a paleontological record and a historical overview of armenian herpetological studies complete the introductory part of the volume. the book covers 17 families (5 of amphibians and 12 of reptiles) and 59 species of which 11 are endemic to the armenian plateau and lesser caucasus. identification keys to all the genera and species are also provided and they prove to be very useful, in particular for some similar species, such as those belonging to the parthenogenetic genus darevskia. the special part describes all the known species, including english vernacular name, synonyms, type locality, taxonomy, general and local distribution, morphological description, karyotype, ecology, and behavior. the species accounts are completed by conservation status information for armenia and nagorno-karabakh and by a short bibliography, whereas a complete literature is present at the end of the book. fifteen plates with small but exhaustive pictures illustrate all the species and their distribution in the study area. seventeen more plates with 92 photos show the main habitats occurring in this region. a total of 154 pages and 152 pictures, with more than 500 bibliographic references make this book a fundamental instrument for researchers interested in caucasian fauna, and give a great contribution the knowledge of the whole area. the book can be purchased at the cost of $ 40.00 at the publisher’s website (http://www.ssarherps.org/). acta herpetologica vol. 7, n. 2 december 2012 firenze university press advertisement call of species of the genus frostius cannatella 1986 (anura: bufonidae) flora a. juncá1, david l. röhr2, ricardo lourenço-de-moraes3, flávio j. m. santos1, airan s. protázio1, ednei a. mercês1, mirco solé4 amphibians in southern apennine: distribution, ecology and conservation notes in the “appennino lucano, val d’agri e lagonegrese” national park (southern italy). antonio romano1,*, remo bartolomei1, antonio luca conte1, egidio fulco2 the significance of using satellite imagery data only in ecological niche modelling of iberian herps neftalí sillero1, josé c. brito2, santiago martín-alfageme3, eduardo garcía-meléndez4, a.g. toxopeus5, andrew skidmore5 reproductive strategy of male and female eastern spiny lizards sceloporus spinosus (squamata: phrynosomatidae) from a region of the chihuahuan desert, méxico aurelio ramírez-bautista1,*, barry p. stephenson2, xóchitl hernández-ibarra1, uriel hernández-salinas1, raciel cruz-elizalde1, abraham lozano1, and geoffrey r. smith3 morphological variability of the hermann’s tortoise (testudo hermanni) in the central balkans katarina ljubisavljević1, georg džukić1, tanja d. vukov1, miloš l. kalezić1,2 the usefulness of mesocosms for ecotoxicity testing with lacertid lizards maria josé amaral1,2,*, rita c. bicho1, miguel a. carretero2, juan c. sanchez-hernandez3, augusto m. r. faustino4, amadeu m. v. m. soares1, reinier m. mann1,5 does acclimation at higher temperatures affect the locomotor performance of one of the southernmost reptiles in the world? jimena b. fernández*, nora r. ibargüengoytía advertisement call of scinax littoralis and s. angrensis (amphibia: anura: hylidae), with notes on the reproductive activity of s. littoralis michel v. garey1, thais r. n. costa2, andré m. x. de lima2, luís f. toledo3, marília t. hartmann4 book review: marine s. arakelyan, felix d. danielyan, claudia corti, roberto sindaco, alan e. leviton 2011. herpetofauna of armenia and nagorno-karabakh. society for the study of amphibians and reptiles stefano scali book review: rafaqat masroor 2012. a contribution to the herpetofauna of northern pakistan stefano scali evidence of high longevity in an island lacertid, teira dugesii (milne-edwards, 1829). first data on wild specimens. j. jesus first assessment of the endoparasitic nematode fauna of four psammophilous species of tropiduridae (squamata: iguania) endemic to north-eastern brazil markus lambertz1,*, tiana kohlsdorf2, steven f. perry1, robson waldemar ávila3, reinaldo josé da silva4 rediscovery and redescription of the holotype of lygosoma vittigerum (= lipinia vittigera) boulenger, 1894 yannick bucklitsch1, peter geissler1, timo hartmann1, giuliano doria2 , andré koch1,* reproductive phenology of the tomato frog, dyscophus antongili, in an urban pond of madagascar’s east coast ori segev1,*, franco andreone2, roberta pala2, giulia tessa2, miguel vences1 range extension of the critically endangered true poison-dart frog, phyllobates terribilis (anura: dendrobatidae), in western colombia roberto márquez1,*, germán corredor2, carlos galvis3 daniel góez2, & adolfo amézquita1 differences in habitat use of two sympatric species of ameiva in east costa rica esther sebastián-gonzález1, ramón gómez2 acta herpetologica journal of the societas herpetologica italica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 7(1): 13-21, 2012 preliminary analysis of dorsal pattern variation and sexual dimorphism in montivipera latifii (mertens, darevsky and klemmer, 1967) (ophidia: viperidae) mehdi rajabizadeh1,*, ali yazdanpanah1, sylvain ursenbacher2 1 department of biodiversity, international center for science, high technology & environmental sciences. kerman (iran). corresponding author. e-mail: khosro.rajabizadeh@gmail.com 2 department of environmental sciences, section of conservation biology, university of basel, st. johanns-vorstadt 10, ch-4056 basel (switzerland) submitted on: 2011, 20th july; revised on: 2011, 14th october; accepted on: 2011, 2nd december. abstract. in this study, sexual dimorphism and dorsal patterns were investigated in latifi’s viper (montivipera latifii) from iran. sexual dimorphism was evaluated in 13 males and 15 females using 12 morphological characteristics. despite the low sample size, the results showed that both sexes significantly differ in the number of subcaudal scales, the number of outer circumocular scales and tail length. in a limited area, the lar national park, three different dorsal patterns were observed (n=26 specimens): about 50% displayed a complete zigzag dorsal pattern, 15% of the individuals displayed a striped dorsal pattern, and about 35% had an incomplete zigzag dorsal pattern. these findings confirmed partially results from former published studies. finally, we hypothesised that the four pattern described in m. latifii could be a combination of only two genetically define dorsal marks. keywords. montivipera latifii, dorsal pattern, sexual dimorphism, genetic basis. introduction montivipera latifii (mertens et al., 1967) is one of the four endemic vipers of iran (rastegar-pouyani et al., 2008) and is a member of the m. raddei group. a particularity of this species is a high level of polymorphism of the dorsal pattern. so far, four different patterns have been identified in m. latifii. most of the specimens present a darker vertebral line connecting with a varying number of alternating or opposing blotches that, together, represent an intensive and well-developed zigzag pattern (bare; fig. 1c). very occasionally, the dorsal pattern can consist of a one-to-two scales width, straight vertebral stripe, without additional blotches (stripy; fig. 1a). occasionally, the central vertebral line 14 m. rajabizadeh, a. yazdanpanah and s. ursenbacher is missing and only the alternating blotches remain, resulting in a diffuse spotty or blotchy pattern (spotted; fig. 1b). mertens et al. (1967) reported a unique specimen with no pattern (plain). such kinds of patterns seem to be extremely rare in m. latifii because, to the best of our knowledge, no other mentions of them have been recorded. in a study carried out by andrén and nilson (1979) on the variability of the dorsal pattern in m. latifii in lar valley, no specimen with a completely uniform colour was observed, whereas 59% of the individuals presented a well-developed zigzag pattern, 33% had spots or a blotchy pattern and only 8% had a vertebral stripe. additional detailed information about the morphological description and diagnosis of this species have been given in the following publications: mertens et al. (1967), andrén and nilson (1979), nilson and andrén (1986), latifi (2000), mallow et al. (2003). current knowledge of the distribution of m. latifii suggests that this species is restricted to a small area in the central alborz mountain range (fig. 2; rajabizadeh, 2008; behruz et al., 2009). consequently, this species has been listed in iucn red list as “endangered” (http://www.iucnredlist.org). sexual dimorphism (sd), defined as the phenotypic difference between males and females of a species, is a frequent phenomenon in animals including reptiles (andersson, 1994). stuart-fox and ord (2004) regrouped three main forms of sd in reptiles; sexual size dimorphism (ssd), ornamentation dimorphism, and coloration dimorphism. sexual size dimorphism (ssd) describes the situation where the two sexes differ in certain morphological traits, especially body length (svl). in about two-thirds of snake species, females grow larger than males (shine, 1998). in addition, significant dimorphism has been documented regarding relative head size, head shape and tail size (shine, 1993). furthermore, a general trend towards more ventrals in females and more subcaudals in males has also been documented (e.g. pope, 1935; de silva, 1969). although snakes do not show as vivid colours as many sexually dichromatic lizard species, sex differences in colour are more common among snakes than has generally been appreciated (shine, 1993). although a large dorsal pattern variation was observed in m. latifii, practically no information on intrapopulation variation is available. the aims of this study were to i) evaluate the proportions of different dorsal patterns in the lar valley (iran) and ii) to assess morphological differentiation between both sexes at a larger scale. finally, different scenarios regarding the possible genetic aspects of the different patterns of m. latifii will be discussed. materials and methods to study dorsal pattern variation in the lar valley populations, we gathered data from 26 specimens collected between 2006 and 2009 in surveys conducted by m. rajabizadeh and r. behruz and pictures taken by amateur iranian photographers from “gozal darre” and alarm in lar valley. for this study, all samples collected in the lar river valley and side branches of the lar national park were regarded as belonging to the lar valley study area (fig. 2). the direct observations and pictures allowed the dorsal pattern to be evaluated and classified into four categories (stripy, spotted, bare and plain; see above for a complete description and fig. 1), as recognized by nilson and andrén (1986). to study sexual dimorphism in m. latifii, a total of 28 specimens including 15 females and 13 males were examined. the sampling regrouped five specimens from central albert (collection of the 15morphology variation in montivipera latifii fig. 1. the three differently coloured patterns of montivipera latifii observed in lar national park (iran). a: stripy; b: spotted (incomplete zigzag); and c: bare (complete zigzag). 16 m. rajabizadeh, a. yazdanpanah and s. ursenbacher tehran university zoological museum), five specimens from lar valley (mr personal collection) and 18 specimens collected by m. latifi (razi institute collection); the exact collection locality of these specimens was unknown. eight meristic and three morphometric characters were selected for this study (see table 1 for further details). the morphometric characters were measured to the nearest 0.1 mm with a vernier calliper. the proportions of the head and the tail sizes were calculated in order to remove the impact of individual size (strongly correlated with age) on the analyses. in order to study the sexual dimorphism, an anova test was conducted for each meristic and morphometric character. in addition a principle component analysis (pca) was carried out to determine the principal characters that distinguished between the sexes. for this analysis, all morphological variables were standardized to avoid bias. all statistical analyses were performed using the spss statistical package (version 15; spss inc., chicago). results 1. dorsal pattern about 50% of the m. latifii from lar national park population had a complete zigzag dorsal pattern, whereas the stripy dorsal pattern was detected in about 15% of the individuals and about 35% had an incomplete zigzag dorsal pattern (table 2). no individual with a “plain” pattern was observed in the lar valley. fig. 2. distribution of montivipera latifii and the sampling locations in lar national park, iran. 17morphology variation in montivipera latifii 2. sexual dimorphism significant morphological differences were found for the number of subcaudal scales, the number of outer circumocular scales and the length of the tail of m. latifii (table 3). additionally, the proportion of the tail was marginally significant (p = 0.053). intersexual comparisons were made using a pca. the first principal component accounted for 34.5% of the total variation, whereas the first three principal components regrouped 62.2% of the total variation (table 4). the number of subcaudal scales and the tail length proportions had a high impact on the first axis, whereas the second principal component axis (14.7% of the variability) was mainly composed of the number of outer circumocular scales, the number of inner circumocular scales and the total number of intercanthal + intersupraocular scales. table 1. list of characters used in the morphological examination of montivipera latifii specimens. ven number of ventral scales (following dowlin, 1951) scd number of subcaudal scales incansup number of intercanthal + intersupraocular scales spl* number of supralabial scales ifl* number of infralabial scales incir* number of inner circumocular scales outcir1* number of outer circumocular scales (round eye and supraocular) outcir2* number of outer circumocular scales, round eye (following nilson and andrén, 1986) svl (mm) snout vent length: from tip of snout to vent tl (mm) tail length: from vent to tip of tail hl (mm) head length: from tip of rostral to end of lower jaw hw (mm) head width at the widest point ltl*100 ratio of tail length on total length * 100 lhl*100 ratio of head length on total length * 100 hl.hw ratio of head length on head width * sum of left and right scales used in the analysis. table 2. proportions of the different dorsal patterns of montivipera latifii observed in lar national park (iran). dorsal pattern number % stripy 4 15.4% spotted (incomplete zigzag) 9 34.6% bare (complete zigzag) 13 50% total 26 100% 18 m. rajabizadeh, a. yazdanpanah and s. ursenbacher table 3. evaluation of the morphometric and meristic characters measured for 28 montivipera latifii individuals, including mean, standard error, minimum and maximum values. significant differences between the sexes (*) were tested using anova. males (n=13) females (n=15) df f sig. mean ± se range mean ± se range ven 165.30 ± 0.71 160 169 164.26 ± 0.86 157 – 169 1 0.833 0.370 scd 36.48 ± 0.33 34 38 32.40 ± 0.74 29 – 39 1 22.640 <0.001* incansup 38.69 ± 1.36 30 48 39.66 ± 1.00 30 – 47 1 0.342 0.564 spl 18.61 ± 0.18 18 20 18.80 ± 0.29 18 – 22 1 0.263 0.612 ifl 23.23 ± 0.69 15 25 23.73 ± 0.25 22 – 26 1 0.512 0.480 incir 26.38 ± 0.50 24 30 27.26 ± 0.58 24 – 31 1 1.281 0.268 outcir1 35.61 ± 0.69 32 41 37.53 ± 0.44 34 – 40 1 5.702 0.024* outcir2 27.92 ± 0.47 26 32 29.00 ± 0.37 27 – 31 1 3.235 0.084 svl 526/38±23.48 397-658 469.40±29.16 168-637 1 2.221 0.148 tl 53.14±2.00 38.70-64.0 44.24±2.64 17-58.0 1 6.788 0.015* hl 25.85±0.78 20.50-30.0 23.72±1.11 11.70-28.40 1 2.293 0.142 hw 18.05±0.54 14.80-20.60 16.51±0.97 7.30-22.0 1 1.729 0.200 ltl*100 9.09 ± 0.15 7.97 9.95 8.66 ± 0.14 7.74 9.72 1 4.127 0.053* lhl*100 4.49 ± 0.77 3.85 – 4/91 4.73 ± 0.14 4.09 6.32 1 2.034 0.166 hl.hw 1.43 ± 0.02 1.28 1.53 1.59 ± 0.12 1.24 3.14 1 1.392 0.249 table 4. scores of the first three main components (pc1 to pc3) of the principal component analysis for morphological and meristic measurements of 28 montivipera latifii individuals. component variable pc1 pc2 pc3 zscore(ven) 0.386 -0.116 0.063 zscore(scd) 0.705 -0.113 -0.297 zscore(incansup) 0.262 0.540 -0.109 zscore(sup) -0.097 0.026 0.730 zscore(ifl) 0.049 -0.349 0.570 zscore(incir) -0.009 0.508 -0.119 zscore(outcir1) -0.338 0.803 0.223 zscore(outcir2) -0.279 0.806 0.275 zscore(ltl*100) -0.104 0.213 -0.854 zscore(lhl*100) -0.858 0.047 -0.174 zscore(hl.hw) -0.302 -0.280 -0.147 eigenvalue 5.18 2.21 1.94 percent variability (%) 34.54 14.73 12.93 cumulative percent (%) 34.54 49.28 62.21 19morphology variation in montivipera latifii discussion although the number of m. latifii individuals examined was limited, the preliminary results are in agreement with the general pattern of intersexual differences in snakes. indeed, a higher number of subcaudal scales and a longer tail length were observed in males in comparison to females, confirming the previous observation of nilson and andrén (1986). the higher number of subcaudal scales in males is often associated with a proportionally longer tail due to the occurrence of the two hemipenises. based on the observations made in lar valley, it was found that the plain coloration is absent or very rare in m. latifii. a large proportion of the individuals had a normal bare pattern with a complete zigzag, whereas the stripy pattern, although not very frequent, was regularly observed. a previous study by andrén and nilson (1979) provided similar proportions of the different dorsal patterns, based on newborns from two females. the occurrence of the plain pattern seems to be very rare and, to the best of our knowledge, only one observation was reported. probably, it could correspond to an anecdotal observation of a complete lack of dorsal pattern, as reported in vipera aspis (mebert et al., 2011) and v. seoanei (brodmann, 1987). former studies on dorsal patterns of m. latifii reported four distinct dorsal patterns (nilson and andrén, 1986). it can be hypothesised that the most common pattern, bare, may be a combination of stripy and spotted patterns. in such circumstances, the species would only present three distinct dorsal patterns (spotted, stripy and plain), while the bare pattern would be a combination of the two other patterns (spotted + stripy). thus, the dorsal pattern could be limited to the occurrence or the lack of two parameters, a dorsal line (stripy) and a dotted pattern (spotted). the lack of both patterns would result in the plain pattern, whereas the occurrence of both would provide a bare pattern. such hypotheses need however confirmation. two main drivers of dorsal pattern variation in montivipera latifii may be hypothesised: a genetic and/or an ecological basis. the present study, based on random observations of m. latifii in nature, was not sufficient to allow us to speculate about the genetic basis of the different dorsal patterns in this species. however, we can propose a scenario whereby the two distinct dorsal patterns of m. latifii, the incomplete zigzag pattern (spotted) and stripy pattern (stripy), are controlled by two genes. the complete zigzag pattern, which is combination of these two basic patterns, is observed when both genes are expressed. if we considering that the population in lar valley is in hardy-weinberg equilibrium, gene a for the spotted characteristic (a is dominant over a) and gene b for the stripy characteristic (again, b is dominant over b), the gene combinations aabb, aabb, aabb and aabb would provide a bare pattern, aabb and aabb a stripy pattern, and aabb and aabb a spotted pattern, whereas the combination aabb would result in the lack of a dorsal pattern (plain). using this hypothesis, the proportions of each pattern observed in lar valley can allow a rough estimation of the frequency of both genes (a=0.40 and b=0.58). in snakes, the dorsal pattern is often related to a defence strategy. for instance, the zigzag pattern in european vipers has been shown to act as an aposematic signal for birds (wüster et al., 2004; niskanen and mappes, 2005). in addition, stripy patterns are thought to confuse predators over the direction and speed of movement of the snake by making it difficult to visually focus on any specific point along the animal (wolf and werner, 1994). 20 m. rajabizadeh, a. yazdanpanah and s. ursenbacher comparing the environment of the different species of the genus, m. latifii can be found at altitudes higher than 2400 m in central alborz, which is among the highest of the elevated habitats (nilson and andrén, 1986). montivipera latifii is also found in one of the most sparsely vegetated habitats within the genus. consequently, the lack of vegetation may have selected for different dorsal patterns. however, this hypothesis should be investigated, for instance, by studying the predation rates of the different patterns by analysing the survival rate of snakes with both patterns (via capture-mark-recapture methods) or by experiments using plasticine snakes. acknowledgements we are grateful to abbas zare, head of the poisonous animals unit, razi vaccine and serum institute, for his cooperation. also, we thank our colleagues h. gudarzi, y. werner, r. behruz, h. parsa, h. salehi, n. kian and h. rostami for their kind cooperation. references andersson, m. (1994): sexual selection. princeton university press, princeton. andrén, c., nilson, g. (1979): vipera latifii (reptilia, serpentes, viperidae) an endangered viper from lar valley, iran, and remarks on the systematic herpetofauna. j. herpetol. 13: 335-341. behruz, r., rajabizadeh, m., kaboli, m. (2009): investigation of the destruction of the habitat of montivipera latifii (mertens, darevsky and klemmer,1967) (reptilia: viperidae) in central alborz, iran. in: abstract of 15th european congress of herpetology. kusadasi. turkey. brodmann, p. (2007): die giftschlangen europas und die gattung vipera in afrika und asien. kümmerly + frey, bern. de silva, p.h.d.h. (1969): taxonomic studies on ceylon snakes of the family colubridae, spolia zeylanica 31: 431-546. latifi, m. (2000): snakes of iran, 3th edition. department of the environment. tehran [in persian]. mallow, d., ludwig, d., nilson, g. 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(2008): conservation of vipera latifii in central alborz. first international congress documenting, analyzing and managing biodiversity in the middle east. agaba, jordan. rastegar-pouyani, n., kami, h.g., rajabizadeh, m., shafiei, s., anderson, s.c. (2008): annotated checklist of amphibians and reptiles of iran. iran. j. anim. biosyst. 4: 43-66. shine, r. (1993): sexual dimorphism in snakes. in: snakes – ecology and behavior, p. 49-86. seigel, r.a., collins, j.t., eds, mcgraw-hill, new york. shine, r. (1998): sexual size dimorphism and male combat in snakes. oecologica, berlin 33: 269-278. stuart-fox, d.m., ord, t.j. (2004): sexual selection, natural selection and the evolution of dimorphic coloration and ornamentation in agamid lizards. proc. r. soc. lond. b-biol.sci. 271: 2249-2255. wolf, m., werner, y.l. (1994): the stripped colour pattern and striped/non-striped polymorphism in snakes (reptilia: ophidia). biol. rev. 69: 500-610. wüster, w., allum, c.s.e., bjargardottir, i.b., bailey, k.l., dawson, k.j., guenioui, j., lewis, j., mcgurk, j., moore, a.g., niskanen, m., pollard, c.p. (2004): do aposematism and batesian mimicry require bright colours? a test, using european viper markings. proc. r. soc. lond. b-biol. sci. 271: 2495-2499. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 7(1): 91-103, 2012 chemosensory responses to chemical and visual stimuli in five species of colubrid snakes anthony j. saviola1, valerie j. mckenzie2, david chiszar3 1 school of biological sciences, university of northern colorado, 501 20th street, cb 92, greeley, co 80639 usa. corresponding author. e-mail: anthony.saviola@unco.edu 2 department of ecology and evolutionary biology, university of colorado, ramaley n122, ucb 334, boulder, co 80309 usa 3 department of psychology, cb 345, university of colorado, boulder, colorado 80309 usa submitted on: 2011, 20th may; revised on 2011, 27th february; accepted on 2011, 29th march. abstract. snakes utilize chemical and visual stimuli during predation, however the emphasis on these cues and which cues are used to initiate predation varies among species. for example, rattlesnakes using the ambush strategy rely on chemical cues to locate an ambush station, then visual and thermal cues to initiate envenomating strikes, then chemical cues again to track prey. by contrast, many natricine snakes use chemical cues to initiate predation, increasing the rate of tongue flicking regardless of whether visual cues are present. the present study examined the individual and interactive effects of chemical and visual stimuli of prey on the predatory behavior of five snake taxa representing three feeding guilds. bull snakes (pituophis catenifer), eastern corn snakes (pantherophis guttatus), and midland rat snakes (scotophis spiloides) have a diet primarily consisting of mammals; western fox snakes (mintonius vulpina) prey primarily on bird eggs; and common kingsnakes (lampropeltis getula) prey equally on mammals and reptiles. three patterns of response to chemical and visual stimuli of the test prey (mus musculus) were observed. mammal specialists responded to chemical cues. fox snakes responded to visual cues, but not to chemical cues. kingsnakes exhibited increased rates of tongue flicking in response to both chemical and visual stimuli. this study suggests correlations between the evolution of prey preference, foraging ecology and the utilization of chemical or visual stimuli by snakes. keywords. chemical cues, predatory behavior, tongue flicking, visual cues, vomeronasal chemoreception introduction most squamates have three chemosensory systems: the nasal olfactory system, the vomeronasal system, and taste buds (schwenk, 1995). chemoreception in snakes is known 92 a.j. saviola, v.j. mckenzie and d. chiszar to mediate numerous behaviors, including mate selection, exploratory behavior, predator identification, prey choice, and prey location (kubie et al., 1978; weldon and burghardt, 1979; chiszar and scudder, 1980; clark 2004; saviola et al., in press). snakes typically recognize potential prey either by chemical cues produced by exocrine glands and other sources (chiszar et al., 1990; duvall et al., 1990), or by thermal and visual cues (herzog and burghardt, 1974; chiszar et al., 1981; hennessy and owings, 1988). natricine snakes, for example, respond to visual stimuli with orientation and investigation, however further behaviors such as increased rates of tongue flicking, trailing, and striking are initiated by the presence of olfactory cues (burghardt, 1969, 1970; chiszar et al., 1981). midland rat snakes (scotophis spiloides) utilize the behavior of avian provisioning as a visual stimulus for locating bird nests in a natural setting (eichholz and koenig, 1992; neal et al., 1993; mullin and cooper, 1998). however, crotalid species utilize chemical cues in ambush site selection, yet visual-thermal cues initiate the strike, then chemical cues are again used to relocate the envenomated prey (dullemeijer, 1961; chiszar et al., 1992; kardong, 1992; clark, 2004). broad headed snakes (hoplocephalus bungaroides) select ambush sites based on both visual and chemical stimuli (du et al., 2009). clearly, the pattern of response to stimuli arising from prey varies among snake taxa. tongue flicking in snakes is a stimulus-seeking behavior, and is the main process for delivering volatile and non-volatile cues to the vomeronasal organs (halpern, 1992; cooper, 1994), which mediates definitive analysis of chemical information (cowles and phelan, 1958; halpern, 1992). tongue flicking is activated by detection of volatile chemical cues by the nasal olfactory system, and by visual or thermal stimulation (burghardt 1970; chiszar et al., 1981; burghardt and denny, 1983; saviola et al., 2011); therefore, the rate of tongue flicking can be used as a convenient measure of a snake’s response to any or all of these stimuli. chiszar et al. (1981) showed, during presentation of prey, that garter snakes responded with an increased rate of tongue flicking to chemical cues irrespective of whether visual stimuli were present or absent, whereas rattlesnakes did not respond significantly to chemical stimuli and showed a greater rate of tongue flicking to visual-thermal stimuli relative to garter snakes. further, burghardt and denny (1983) showed that garter snakes selected moving prey when chemical cues were present compared to moving prey without chemical cues. feeding experience may alter chemosensory responses in snakes (burghardt, 1993), and cooper (2008) showed that evolutionary changes in chemosensory responsiveness to chemical cues from prey is correlated with dietary change. relatively few studies examining chemosensory responses in snakes combine chemical stimuli with the presence of visual stimuli, or examine chemosensory responses derived from visual cues alone. in addition, interactions between these two stimuli are infrequently reported, however there is accumulating information demonstrating the interactive effects of chemical and visual cues stimulating vomeronasal chemoreception in a few species of snakes (terrick et al. 1995; shivik, 1998). the goal of the present study was to test the hypothesis that snake species with different diets will show different responses to prey-derived cues, favoring those most likely to allow detection and capture of preferred prey. we tested responses to chemical and visual stimuli in snake taxa that feed primarily on either mammals (eastern corn snake, pantherophis guttatus, midland rat snake, scotophis spiloides, and bull snake, pituophis catenifer), bird eggs (western fox snake, mintonius vulpina), or equally on several different prey types (common kingsnake, lampropeltis getula) (rod93chemosensory responses of colubrid snakes ríguez-robles and dejesús-escobar, 1999; nomenclature from collins and taggart, 2009). we measured the rate of tongue flicking as well as time spent investigating the stimuli as indicators of predatory behavior. materials and methods six l. getula, nine p. guttatus, five s. spiloides, four m. vulpina, and seven p. catenifer were subjects. the snakes were all wild-caught adults, and had been in captivity for at least two years, housed individually in glass terraria (62x32x32 cm) containing paper floor covers, hide boxes, water bowls, and rocks to assist with shedding. the laboratory was maintained at 26-30 °c and photoperiod was automatically controlled on a 12:12 light:dark cycle. prey used for the behavioral experiments were mice (mus musculus) provided by the department of molecular, cellular, and developmental biology, university of colorado at boulder. snakes were fed euthanized laboratory mice weekly prior to and during the study period, and feeding during the experiment occurred several hours after tests. successive tests on the same snakes were always separated by at least one week. all snakes always attacked prey immediately during feeding sessions, hence we considered them to be equally responsive to prey. stimuli were presented to snakes using plexiglass boxes (10x10x10 cm) of four types: clear without perforations, permitting only visual cues to be presented to the snake; clear with perforations on each side, allowing presentation of both visual and chemical cues; black with no perforations, preventing both visual and chemical cues, and black with perforations, allowing presentation of chemical but not visual cues (chiszar et al., 1981, 2009; saviola et al., 2011). all perforations were circular, two mm in diameter, therefore large enough to permit passage of volatile chemicals, but not large enough to permit visual examination of the interior of the box. all boxes were washed using quatricide-pv®, a commercial disinfectant and deodorant to remove any traces of chemical cues between trials. each snake was tested with each of the four boxes, and a single adult male mouse, 17-20 grams, was placed inside the boxes. as an additional control, we also observed each snake with a black, unperforated box without a mouse. the five conditions were offered to each snake in random order, and the length of each trial was 10 min. a box was placed into the snake’s terrarium, and all tongue flicks emitted and seconds spent investigating the stimulus box were recorded every minute for the duration of the trial. we also recorded a third operationally distinct dependent variable, the number of tongue flicks aimed at the stimulus box. correlations between all pairs of dependent variables were calculated with the result that the third variable was highly related to those already mentioned (r = 0.75 and r = 0.88, respectively). not surprisingly, the outcomes of inferential test (see below) were not different for this dependent variable relative to the outcomes for the other two, especially time spent investigating the stimulus boxes. hence, we omit further treatment of the third dependent variable. incidentally, the correlation between the first and second dependent variables was r = 0.65. though significant, this correlation was low enough to permit different, inferential outcomes, and this in fact occurred in a few cases. since it was obvious which condition was being tested (i.e. clear box vs. a black box, etc), we were unable to record the dependent variables blind to the conditions. data for each species were analyzed first by 5 (species) x 5 (conditions) x 10 (minutes) anovas completed on total tongue flicks and number of seconds at the box.   then data for each species were analyzed separately using analyses of variance (anova) and newman-kuels post-hoc tests. two types of anovas were used. first, a one-way anova with repeated measures was applied to each of the five conditions (four conditions containing a live mouse and the additional control of the black, unperforated box without a mouse) for each species. second, 2x2 anovas treated presence vs. absence of chemical and visual cues as orthogonal factors, using the four relevant condi94 a.j. saviola, v.j. mckenzie and d. chiszar tions. in the 2x2 anovas, all interactions involving subjects were pooled, giving rise to a composite error term with greater power than each of the individual interactions (hicks, 1964). results tongue flicks mean number of tongue flicks (± sem) emitted during the 10 min trials are shown in figure 1. for all conditions and species, a 5 (species) x 5 (conditions) x 10 (min) anova treated species as a between-subjects factor and conditions and minutes as repeatedmeasures factors. all main effects were significant (species: f = 7.69, df = 4,26, p < 0.01; conditions: f = 8.05, df = 4, 104, p < 0.01; minutes: f = 7.32, df = 9, 234, p < 0.01), but no interaction was significant. however, the species x conditions interaction was close (f = 2.00 df = 16, 104, p < 0.05), but then corrected by the greenhouse and geisser (greenhouse and geisser, 1959; see also winer, 1971) method, the df were reduced to 4, 26, and the f ratio was then only marginal (0.10 > p > 0.05). no other interaction came close to being significant before or after the greenhouse and geisser (1959) correction. this is particularly interesting in the case of the robust main effect of minutes. the mean number of tongue flicks declined over minutes starting at 96.8 on min 1 (across all species and conditions) and ending at 65.5 on minute 10, but the magnitude of the decline did not vary with species or conditions. in other words, the rate of habituation (or sensory adaptation) was essentially constant, regardless of the starting level. it must be kept in mind, however, that the rate on minute 10 was rather high, indicating that the snakes were still interested in the stimuli. whether, the rate of tongue flicking after minute 10 would continue to decline at a constant rate is conjectural. data from each species were subjected to additional anovas in order to follow up the 5x5x10 anova. first, the data from each species were subjected to one-way repeated measures anovas to verify the conditions main effect. second, the data from each species were subjected to 2x2 repeated-measures anovas to assess the effects of absence vs. presence of visual and chemical cues. all f ratios from these anovas are shown in table 1. results of newman-keuls post-hoc comparisons are shown by superscripts in fig. 1. four one-way anovas revealed significant main effect of conditions; for bull snakes the f ratio for the conditions main effect fell slightly short such that 0.10 > p > 0.05. thus, one or more significant differences probably existed among conditions within each species (see fig. 1). for all species except kingsnakes the mean for the additional control (empty unperforated black box) was not significantly different from the mean for the condition where a mouse was in the unperforated black box, implying that all snakes except kingsnakes failed to detect the mouse in the latter condition. because kingsnakes appeared to detect the mouse when no chemical or visual cues were explicitly provided, we executed the 2x2 anova on the kingsnake data twice, first we used the data from the control in which a mouse was present in the unperforated black box, then we substituted data from the additional control in a second 2x2 anova. the purpose of this procedure was to remove all possible prey-derived cues, in case kingsnakes had heightened sensitivity (especially to chemical cues) that other species lack. if such was the case, then the addi95chemosensory responses of colubrid snakes fig. 1. mean number of tongue flicks (those directed at the stimulus as well as any others) for the duration of the trial (± sem) from the five species tested. dissimilar letters above histogram bars indicate significant differences between responses; same letters indicate no significant differences. the additional control refers to the treatment with an unperforated empty black box. table 1. anova outcomes for each species for mean numbers of tongue flicks shown in figure 1. the second 2x2 anova results for l. getula substitutes the additional control (unperforated empty black box) for the standard control (unperforated black box containing a mouse). species one-way anova 2x2 anova chemical cues visual cues interaction f df f f f df p. guttatus 13.69* (4, 32) 20.43** 1.03 10.79** (1, 24) p. catenifer 2.67+ (4, 24) 3.44* 0.00 2.80 (1, 18) m. vulpinus 4.03* (4, 12) 0.08 9.18* 3.48+ (1, 9) s. spiloides 3.17* (4, 16) 13.76** 0.00 1.16 (1, 12) l. getula 3.38* (4, 20) 0.19 0.90 1.11 (1, 15) 6.07* 0.84 6.64* (1, 15) + .10 > p > .05; *p < 0.05; **p < 0.01 96 a.j. saviola, v.j. mckenzie and d. chiszar tional control would be more appropriate for kingsnakes than the standard control. outcomes of both 2x2 anovas are shown in table 1. two patters of response are visible in table 1. corn snakes, bull snakes, rat snakes, and kingsnakes, (second anova) exhibited significant effects of chemical cues, no effects of visual cues, and corn and kingsnakes also exhibited a significant interaction. these four species responded to visual cues slightly more when chemical cues were absent but not when chemical cues were present. for corn snakes and kingsnakes, this circumstance was quite obvious and it was also clear the presence of chemical cues generated a stronger response than visual cues. hence, for these four species, chemical cues were of primary importance and visual cues were at most secondary. fox snakes, by contrast, revealed a significant effect of visual cues, no effect of chemical cues and a marginal interaction, reflecting use of chemical cues when visual cues were absent but not when visual cues were present. number of seconds spent investigating stimuli figure 2 shows the mean number of seconds spent investigating the stimulus boxes for all species and conditions. a 5 (species) x 5 (conditions) x 10 (min) anova revealed a strong main effect of conditions (f = 18.24, df = 4, 104, p < 0.01) but no main effect of species or minutes. one interaction survived the greenhouse and geisser (1959) correction, species x condition (f = 3.40, df = 16, 104, p < 0.01; when corrected, the df drops to 4, 26, and this caused a drop in p < 0.05). one-way anovas (table 2) revealed significant main effects of conditions in all species, except rat snakes where the effect was marginal (0.10 > p > 0.05). hence, there likely were significant differences among treatment means in all species. subsequently 2x2 anovas verified significant effects of chemical cues in corn snakes, rat snakes, bull snakes, and kingsnakes (second anova). fox snakes did not respond to chemical cues but exhibited a strong effect of visual cues. bull snakes also showed a significant effect of visual cues, and kingsnakes showed a marginal effect (second anova). corn snakes also showed a marginal interaction. bull snakes, corn snakes, rat snakes, and kingsnakes showed strong effects of chemical cues and a small effect of visual cues when chemical cues were not present. hence, chemical cues were of primary importance for these four taxa and visual cues were, again, at most secondary. for fox snakes, on the other hand, visual cues were of primary importance and chemical cues added little or nothing to the response. the species x conditions interactions detected by the 5x5x10 anovas (marginal for tongue flicking, significant for time spent investigating the boxes) are explained by the differential responses shown by fox snakes versus the other four taxa. the fact that the two dependent variables gave different anova outcomes is important. it tells us that tongue flicking is readily elicited and runs its course more-or-less independently of other factors. number of seconds spent investigating the boxes appears to be a more discriminating dependent variable that reveals whether or not the stimulus is strong enough to command attention. that is, a snake may be tongue flicking (searching) but this does not necessarily imply that the snake is aware of the presence of a mouse. a lot of time spent at the box, on the other hand, may imply that the snake is aware of the prey.   97chemosensory responses of colubrid snakes fig. 2. mean number of seconds spent investigating the stimulus boxes (± sem) by the five species tested. dissimilar letters above histogram bars indicate significant differences between responses; same letters indicate no significant differences. table 2. anova outcomes for each species for means shown in figure 2. species one-way anova 2x2 anova chemical cues visual cues interaction f df f f f df p. guttatus 4.16** (4, 32) 4.67* 4.40* 3.22+ (1, 24) s. spiloides 2.58+ (4, 16) 8.95* 2.20 0.37 (1, 12) m. vulpinus 5.39* (4, 12) 0.29 13.93** 0.02 (1, 9) p. catenifer 4.45** (4, 24) 11.06** 0.02 1.66 (1, 18) l. getula 3.37* (4, 20) 0.44 0.10 0.10 (1, 15) 5.06* 3.57+ 1.60 (1, 15) + .10 > p > .05; *p < 0.05; **p < 0.01 98 a.j. saviola, v.j. mckenzie and d. chiszar discussion an unexpected outcome of this study was the finding that kingsnakes detected the mouse inside the unperforated black box whereas none of the other taxa appeared to do so. this could have been based on greater sensitivity by kingsnakes to tactile or thermal cues. it is also possible that kingsnakes were able to detect small traces of chemical cues in the mouse-present control condition whereas the other taxa lacked this keener sensitivity. whatever the reason, it appears that kingsnakes have a sensory ability that the other snakes lack. aside from this aspect of the results, we saw two patterns of behavior. for all taxa except fox snakes, chemical cues were utilized and visual cues played only a secondary role, if any in our study. the second pattern was observed in fox snakes where visual cues invariably exerted a significant main effect on both dependent variables, with chemical cues having no significance. natural selection can be expected to influence snakes to respond to those stimuli that are most likely to lead to capture of preferred prey (tinbergen, 1951; cooper, 2008). in other words, the evolution of prey-type preferences and foraging ecology could also determine differential responses to chemical and visual stimuli. bull snakes, corn snakes, and rat snakes emphasize mammals in their diet, fox snakes primarily utilize birds eggs, and kingsnakes equally take several types of prey, ranging from mammals and snakes to lizards and squamate eggs (mullin, 1998; rodríguez-robles and dejesús-escobar, 1999). therefore, our study encompasses two or three distinct snake feeding guilds. a strong response from snakes to rodent chemical cues may arise due to the numerous exocrine glands of mammals associated with their heavy reliance upon pheromonal cues during social and reproductive behaviors (mateo and johnston, 2000). therefore, snakes specializing on rodent prey may have taken advantage of the sensory channel most valuable to their prey and, hence, of stimuli most available to the predators. while chemical cues are critical for prey recognition in some snakes, visual cues are key to capturing prey in other snake species (ford and burghardt, 1993; saviola et al., 2011). consistent with the results from the present study, burghardt and abeshaheen (1971) found that neonate fox snakes did not strike at cotton-swabs soaked in prey extract, but did respond with strikes to a wide variety of visual stimuli. the greater rate of tongue flicking in the presence of visual cues observed here is also consistent with the fact that fox snakes prey on ground-nesting birds (rodríguez-robles and dejesús-escobar, 1999), and that foraging snakes may locate nests by responding to the visual cues of provisioning birds (porter and czaplicki, 1977). although rodents leave odor trails that can be utilized by predators (chiszar et al., 1992; clark, 2004), birds flying to and from nesting sites leave little, if any, chemical trails on the ground. distant visual cues associated with arboreal nest cavities have been shown to be crucial in the foraging behavior of gray rat snakes (pantherophis obsoleta) (mullin and cooper, 1998), although chemical cues appear to be more important as the snake gets close to the prey. the role of visual cues during predation has apparently increased in several thamnophis species that respond to aquatic prey outside of tongue flicking range (drummond, 1985). while chemical cues may be used when the snake is relatively close to the source, initial predation responses and the activity of searching are likely based primarily on visual cues from the prey. 99chemosensory responses of colubrid snakes lampropeltis getula appears to be a generalist preying on ectotherm and endotherm prey with similar frequency (rodríguez-robles and dejesús-escobar, 1999). such a highly varied diet may be the reason why these latter snakes showed strong rates of response to all conditions (original control box containing a live mouse as well as visual and chemical cues from prey). perhaps these snakes attend to any disturbance or to all stimuli available to them, with no preference for one modality over another. thus, all stimulus conditions always elicited investigation, which would make sense if the highly varied diet results in equal attention to all sensory modalities. the apparent uniformity of response across stimulus dimensions in kingsnakes is consistent with the results from several earlier studies with this species. neonatal kingsnakes did not respond differentially when offered various prey extracts (brock and meyers, 1979), and williams and brisbin (1978) found that extracts of mouse, snake, chick, and amphibians did not elicit significantly different rates of tongue flicking in kingsnakes fed exclusively on mice for 2-10 years. foraging behavior and diel activity patterns of prey will affect the foraging behavior of predators. active tongue flicking to detect prey is thought to be absent in ambush foragers (except during prior location of a favorable station), as this behavior may cause the predator to be detected by the prey (cooper, 1994, 1995). in contrast, tongue flicking (to detect volatile and non-volatile cues) in active foragers would cause little if any problems since the predators are already engaged in locomotion and other obvious movements (cooper, 1995). foraging underground or during the night, on the other hand, will provide few if any visual stimuli, therefore blind, fossorial, crepuscular or nocturnal snakes rely on chemical cues, whereas diurnal species foraging above ground may tend to utilize visual cues. for instance, several studies have indicated that blind snakes follow trails of commonly consumed ant prey, however ignore trails of non-consumed prey (watkins et al., 1969; gehlbach et al., 1971; webb and shine, 1992), further emphasizing the importance of chemical stimuli in predation. although primarily diurnal, bull snakes capture their prey by actively searching underground tunnels and other retreats of prey, or by pursuing small mammals while they rest at night (rodríguez-robles, 2002). the snakes in the present study were all tested with laboratory mice (mus musculus), and thus the responses to chemical stimuli by these snakes could be specific to this prey species (known to be particularly odoriferous by comparison with deer mice, peromyscus maniculatus) (duvall et al., 1990). however, clark (2004) found that crotalus horridus, born and raised in captivity and never having encountered natural prey items, still showed a greater response to chemical cues from their natural prey than to chemical cues from closely related species (see also burghardt, 1969). hence odoriferousness of m. musculus may not have biased results for snakes involved in the present study. several studies have examined the correlation between diet and chemosensory responses of squamates to chemical cues (e.g. burghardt, 1967; gove and burghardt, 1975; cooper et al., 2000; cooper, 1995, 2008), and such responses have evolved to coincide with dietary shifts (cooper, 1997, 2008; saviola et al., in press). although some studies have demonstrated the importance of visual cues in initiating vomeronasal chemoreception (ford and burghardt, 1993; drummond 1985; saviola et al., 2011), others fail to emphasize the combination. recently, working with mangrove saltmarsh snakes (nerodia clarkii compressicauda), hansknecht and burghardt (2010) showed that that there was decreased lingual luring and predatory attacks in the presence of a single prey cue, however the combination of chemical and visual cues increased both of these behaviors to a 100 a.j. saviola, v.j. mckenzie and d. chiszar greater extent. our results indicate that even in the presence of visual stimuli, 4 of the 5 species tested demonstrated significant rates of tongue flicking to chemical cues, and visual cues were of secondary importance in our study. therefore, it may be suggested that even in the presence of visual stimuli, for many snakes, chemical cues play primary importance in initiating predation. aknowledgments we thank b. demmig-adams and a. cruz for endless help during this project and many others. we would also like to thank m.t. bealor and s.p. mackessy for their helpful feedback. finally, we would like to thank g. ackerman, e. arcibal, l. benedict, a. gandara, a. hill, c. meeks, and s. wagner for their support and assistance. this research was carried out under animal protocol #0907-sav-01 approved by the institutional animal care and use committee of the university of colorado at boulder. references burghardt, g.m. 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(1971): statistical principles in experimental design. mcgraw-hill, new york. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 8(1): 9-17, 2013 altitudinal effects on life history parameters in populations of liolaemus pictus argentinus (sauria: liolaemidae) joel antú gutiérrez1,*, carla piantoni2, nora r. ibargüengoytía1,3 1 departamento de zoología del centro regional universitario bariloche, unidad postal universidad nacional del comahue, quintral 1250, san carlos de bariloche, río negro 8400, argentina 2 departamento de fisiologia, instituto de biociências, universidade de são paulo, rua do matão tr. 14 no. 321, cep 05508-900 são paulo, sp, brazil. corresponding author. e-mail: carla.piantoni@gmail.com 3 inibioma-conicet, universidad nacional del comahue, quintral 1250, san carlos de bariloche, río negro 8400, argentina submitted on 2012, 15th may; revised on 2013, 1st march; accepted on 2013, 1st march. abstract. we used skeletochronology to assess the age structure, body size and sexual maturity in two populations of liolaemus pictus argentinus from san carlos de bariloche, argentina. the species occupies a wide altitudinal range within the patagonian lake district which enabled us to choose populations from two climatic extremes: 771 m a.s.l. and 1615-1769 m a.s.l. age of sexual maturity in both populations of l. p. argentinus is achieved with a minimum body size of 49 mm. however, at the high-altitude site, lizards matured between the ages of three to six years and had a lifespan of eight years limiting some individual’s reproductive life to only two years. lizards from the low-altitude site achieved maturity at the age of four and lived until the age of nine years old. despite the environmental variations between sites populations’ growth curves’ patterns were similar represented by a rapid initial growth rate of 10.3 mm/year in youngest juvenile which slowed considerably to 4.9 mm/year after attaining sexual maturity, as energy is reallocated towards reproduction, to finally grow at a rate of 0.1 mm/year in the oldest adults. present results show intraspecific differences in l. pictus, whether it results from adaptive polymorphism or physiological plasticity remains uncertain. keywords. age estimation, altitudinal range, patagonia, populations, skeletochronology. introduction lizards require an external source of heat to initiate activities in order to obtain resources to grow, reproduce and maintain their physiological functions. the allocation of energy to each of these three processes decreases the amount of energy available for the other two. a disproportionate allocation to only one or two of these functions can affect individual fitness in a given environment (kublcka and kratochvil, 2009). therefore, an optimal allocation will depend on complex trade-offs among resource availability, physiological and behavioral traits and the environmental conditions (sears and angilletta, 2004). for example, individual’s maximal growth can be affected when resources, such as food or water, diminish or when predators, which may limit foraging, become more numerous (sears and angilletta, 2004). on the other hand, fast growth reduces the cost of maintenance, and can be enhanced by a more efficient digestion (sears and angilletta, 2004), as well as by longer activity hours (shine, 2005). besides, faster growing lizards are more exposed to predators and can result in higher mortality than slower growing lizards (sears and angilletta, 2004). in particular, the short activity seasons in the * we remember here our friend joel antú gutierrez who passed away during the revision of the manuscript, for his comradeship and passion for science. 10 joel antú gutiérrez et al. cold-temperate lands of patagonia, impose severe tradeoffs among thermoregulation, reproduction and foraging, since energy storage and a rapid growth of newborns becomes necessary to afford the brumation period and next spring reproduction (ibargüengoytía and cussac, 1996, 2002; boretto and ibargüengoytía, 2009). in addition, differences in the spectrum of variation of the life-history traits associated to growth (e.g., growth rates, longevity, age at sexual maturity, and fecundity) between and within populations can vary with daily thermal conditions and the length of the activity season (sinervo and adolph, 1989; niewiarowski and roosenburg 1993; wapstra et al., 2001). for lizards, the extent of daily and seasonal duration of activity can be more relevant than the variation in body temperatures (tb) that can be experienced during activity (sears and angilletta, 2004; angilletta, 2009). for example, differences in the activity regime can happen, as in sceloporus merriami populations, along an altitudinal gradient (smith and ballinger, 2001), and lizards that have longer periods of activity are expected to grow quicker and to reach sexual maturity sooner than lizards that have a shorter activity period (adolph and porter, 1993; niewiarowski, 2001). likewise, intraand inter-specific differences in daily metabolic rates were found in two species of the genus pristidactylus that inhabit different thermal areas, being higher in the forest than in the sunnier and warmer shrublands areas (labra and rosenmann, 1992). the activity and brumation periods of lizards from temperate regions like patagonia are defined by the strong seasonality between winter and summer. moreover this pattern generates cyclical growth, characterized by an alternation between fast growth in spring-summer and slow growth in autumn-winter (leclair and castanet, 1987; wake and castanet, 1995). previous studies confirmed that femurs of phymaturus tenebrosus (piantoni et al., 2006a) and the geckonid homonota darwini (piantoni et al., 2006b; kubisch et al., 2012) exhibited growth marks associated to periods of high and low growth rates. the same pattern was found in liolaemus multicolor and l. irregularis (valdecantos et al., 2007) from the highlands of salta, argentina, and in the amphibian pleurodema thaul (iturra-cid et al., 2010) from central and southern chile. here we compare growth, longevity, and fecundity of two populations of liolaemus pictus argentinus, located at approximately the same latitude and longitude (41°15’s, 71°17’w) but at different altitudes: 771 m a.s.l. in melipal beach, and 1615-1769 m a.s.l. in the challhuaco mountain. the following questions were addressed: (1) is there an intra-specific variation in bone growth patterns related to altitude? (2) do growth related life history traits vary in relation to habitat air temperature (ta) in l. p. argentinus? results are discussed based on the differences in the length of the daily and seasonal duration of activity and in the efficiency of thermoregulation observed in previous studies on the same populations (gutiérrez et al., 2010). material and methods study area two populations of liolaemus pictus argentinus were sampled at two sites in northwestern patagonia, argentina: a) 13 juveniles, seven males and 11 adult females were captured in the area of challhuaco mountain, in the surroundings of san carlos de bariloche, province of río negro, (“high-altitude site”, 41°15’57.9”s, 71°17’57.4”w; 1615-1769 m a.s.l.); and b) eight juveniles, 10 males and 11 adult females were collected in melipal beach by the lake nahuel huapi (“low-altitude site”, 41°07’41.53”s, 71°20’44.87”w; 771 m a.s.l.). lizards were captured during activity season 2005 to 2007. field work was carried out with authorization from the wild life service of the province of río negro and national park service. capture and autopsy specimens were captured by hand or noose, then transported to the department of zoology laboratory at the centro regional universitario bariloche, universidad nacional de comahue, san carlos de bariloche, argentina. for histological studies the specimens were euthanized with an overdose of thiopental sodium, fixed in a bouin solution for 24 hours, and finally transferred to a solution of 70% ethanol. lizards’ snout-vent length (svl) was measured using a caliper (svl ± 0.02 mm). histological technique one femur of each specimen was removed for decalcification. the pieces were left in 5-7% nitric acid; the smaller pieces for 5 h and the larger ones for 17 h. bones were then dehydrated through a series of increasing concentrations of ethanol solutions, cleared with toluene, and then embedded in paraplast for 24 h at 60 °c. cross-sections at mid-diaphyseal level were stained with hematoxylin and eosin, according to martoja and martoja pierson (1970). determination of growth rate, age, and longevity the histological preparations were analyzed and photographed using an olympus bx40 microscope equipped with a pro-series high performance ccd camera. digital images were taken at different magnifications (×40, ×100 and ×200) and measurements made using an image-pro plus analyzer. the ten best sections of each bone were selected to estimate the following variables proposed by leclair and castanet (1987): the 11skeletochronology of liolaemus pictus argentinus minimum and maximum radius from the center of the medullar cavity (1) minimum and maximum diaphyseal diameter, (2) estival-layers or ring thickness, and (3) the number of lines of arrested growth (lags) which corresponds to the winter growth. the average of the minimum and maximum radius of each variable was calculated to minimize the asymmetry of the bone sections and medullar cavity. when endosteal resorption had taken place, estimated age was calculated using the size of the first growth marks of juveniles in order to back-calculate the number of rings removed following piantoni et al. (2006a). in those cases the number of reabsorbed rings was added to the number of observed rings. to determine the relationship between individual svl and the number of rings, the best fitted curve using the highest r2 was selected using table curve 2d software. growth rates were calculated as the derivative of the curve of svl vs. age. specific growth rates were obtained by dividing the growth rates by the svl of each estimated age group. relationship between growth and reproduction to estimate fecundity the following variables were taken into consideration: (1) period between age at maturity and longevity, (2) litter size, and (3) frequency of reproduction according to ibargüengoytía and cussac (1996). for comparative purposes the fecundity of other reptiles was calculated extracting bibliographical data of age at sexual maturity, longevity, litter size and frequency of reproduction (table 1). results body size the populations did not show differences in body size (mann-whitney, t1, 31+29 = 950, p = 0.384; rangehighaltitude = 23.5 to 62.1 mm; median svlhigh-altitude = 50.8 mm; rangelow-altitude = 24.8 to 64.3 mm; median svllow-altitude = 56.3 mm; table 2). bone growth patterns both populations exhibited similar bone growth patterns of lamellar bone. most of the mid-diaphyses examined showed periosteal bone with an abundant number of osteocytes and a larger medullar cavity diameter was observed in older specimens. lines of arrested growth (lags), which in l. p. argentinus correspond to the winter period, were strongly stained by hematoxylin and appeared between lighter and thicker growth zones which correspond to summer growth periods (fig. 1). bone resorption and estimated individual age medullar radius showed a significant increment with the svl in femurs of both populations (regression, fhighaltitude 1, 30 = 40.39, p < 0.001; flow-altitude 1, 28 = 9.38, p < 0.05). medullar resorption in both populations removed from one to four rings in juveniles and from two to six in adults (fig. 2, table 2). the estimated age, adjusted for reabsorbed rings, ranged from one to six years for juveniles at high-altitude. adults, at this site, ranged from three to eight years old in females and from four to eight years old in males (fig. 3a). at the low-altitude site juveniles’ age ranged from one to four years, and in adults from four to seven years in females and from four to nine years in males (fig. 3b). the relationship between estimated age and svl in both populations fitted a sigmoid regression (fhigh-altitude 3,30 = 28.45, r2 = 0.76, p < 0.0001; flow-altitude 3,28 = 29.26, r2 = 0.78, p < 0.0001; fig. 4a). growth rates and reproductive life specific growth rates of juveniles and adults were significantly different within populations (high-altitude site: mann-whitney, t = 283.0, p = 0.003, n = 31; lowtable 1. data obtained from bibliography and adapted for comparative purposes. sexual maturity, longevity and reproductive frequency are expressed in years. species geographic coordinates and altitude (m a.s.l.) sexual maturity longevity reproductive frequency litter size fecundity holbrookia maculata a 41°34’n, 99°43’w; 1170 0.5 4 0.5 4.5 31.7 homonota darwini b, c 41°0.8’s, 71°0.8’w; 930 3 9 2 1 4 hoplodactylus duvauceli d 41°0.6’s, 174°25’e; 0 7 36 2 2 29 woodworthia “otago/southland” (formerly hoplodactylus maculatus) d, f, g 45°28´s, 170°28’e; 300–700 4 36 2 1.7 22.4 phymaturus tenebrosusg i, j 41°0.8’s, 71°0.8’w; 575–1230 7 16 2 2 9 ajones and ballinger, 1987; bkubisch et al., 2012; cibargüengoytía and casalins, 2007; dcree, 1994; fcree and guillette, 1995; ghare and cree, 2005; ; hibargüengoytía, 2004; ipiantoni et al., 2006b. 12 joel antú gutiérrez et al. altitude site mann-whitney, t = 199, p < 0.001, n = 29; table 3; fig. 4b). no differences were found between the specific growth rates of male and female adults in either population (high-altitude site: mann-whitney, t = 50.5, p = 0.152, n = 18; low-altitude site: mann-whitney, t = 89 p = 0.138, n = 21; table 3). the specific growth rates of juveniles and adults from high and low-altitudes were also compared and showed no significant differences (mann-whitney, tjuveniles = 105, p = 0.229, n = 21; tadults = 347.0, p = 0.723, n = 39, table 3). considering the youngest and the oldest adults of both populations, sexual maturity was attained at the age of three and the maximum longevity was nine years. populations did not show differences in the number of years during which they are reproductively active. the reproductive life, considering the youngest adult male or female, was of five years. females reproductive cycle in l. p. argentinus can be biennial or triennial (reproduction occurs every two or three years) with a mean litter size of 4.5 offspring (ibargüengoytía and casalins, 2007). hence, the annual reproductive output results in 1.8 and a mean fertility of 9 offspring. discussion during the activity season, in the nothofagus forest near the summit of challhuaco mountain, the monthly mean air and median operative temperatures (ta = 22.8 ± 1.0 ºc; te = 24.9 ºc) resulted overall lower than at the melipal beach (ta = 23.2 ± 0.9 ºc; te = 31.3 ºc; gutierrez et al., 2010). possibly due to a greater radiation index, from mid-november to mid-january, liolaemus pictus argentinus table 2. comparison between juveniles, females, and males of liolaemus pictus argentinus populations from challhuaco mountain and melipal beach concerning snout-vent length (svl; mm), marrow radius (mra; mm), number of reabsorbed rings (rr), measured rings (mr), and estimated age (years). challhuaco mountain (high-altitude site) melipal beach (low-altitude site) svl mra rr mr age svl mra rr mr age juveniles 23.5 0.11 0 0 1 24.8 0.14 0 1 1 24.7 0.15 0 0 1 27.2 0.14 0 2 2 25.5 0.13 0 0 1 28.7 0.18 1 2 3 26 0.13 0 0 1 29.6 0.13 0 2 2 30.5 0.15 0 1 1 30.9 0.12 0 2 2 34.9 0.2 2 2 4 31 0.15 0 2 2 40.4 0.17 1 4 5 43.1 0.22 2 2 4 42.4 0.18 1 2 3 44.4 0.16 1 3 4 42.9 0.14 0 3 3 44.3 0.15 0 4 4 46.8 0.15 0 4 4 48.7 0.2 2 4 6 48.8 0.18 1 2 3 females 50.4 0.19 1 2 3 51.7 0.18 2 3 5 50.6 0.19 1 2 3 52.3 0.14 0 5 5 50.8 0.15 0 3 3 54.6 0.2 2 3 5 53 0.18 1 2 3 55.8 0.21 2 3 5 55.4 0.2 2 3 5 56.3 0.18 2 4 6 54.8 0.17 1 3 4 57 0.19 2 4 6 56.5 0.21 3 3 6 57.3 0.2 2 2 4 60.5 0.22 3 2 5 57.4 0.17 1 3 4 60.7 0.22 3 5 8 57.7 0.19 2 2 4 60.8 0.21 3 2 5 57.8 0.18 2 5 7 62.1 0.23 3 4 7 64.3 0.14 0 6 6 males 51.4 0.2 2 3 5 49.8 0.14 0 4 4 56 0.21 3 5 8 54.7 0.17 1 5 6 58.7 0.25 4 3 7 57.8 0.18 2 4 6 60.2 0.19 1 3 4 58.9 0.15 0 5 5 60.8 0.16 1 5 6 60.8 0.21 2 5 7 60.9 0.23 3 3 6 60.2 0.16 0 7 7 62 0.19 1 4 5 60.1 0.16 1 6 7 61.4 0.21 2 2 4 61.9 0.19 2 4 6 63.2 0.28 6 3 9 fig. 1. diaphysial cross-section of three (a), five (b) and seven (c) year-old specimens of liolaemus pictus argentinus. arrows indicate the lines of arrested growth (lags). sm: first lag after attaining sexual maturity. scale bars: 100 mm. 13skeletochronology of liolaemus pictus argentinus experienced warmer tas at high-altitude (gutierrez et al., 2010), while at the beach ta was buffered by the effect of the lake (koeppen, 1948). nevertheless, the winter snow, hence brumation, lasted one month more in the mountains than at the beach reducing the lizards’ activity season at the high-altitude site (september, gutierrez et al., 2010). at high-altitude, l. p. argentinus behaved as a moderate thermoregulators (sensu hertz et al., 1993), compensating the fewer warm microenvironments, and the shorter activity season showing low body temperatures (tb = 28.97 ± 0.8 °c), while at the low altitude site, in a more benign environment, they behaved as thermoconformers, achieving a mean tb of 32.67 ± 0.9 °c (gutiérrez et al., 2010). these intra-specific differences among different thermal-ecological environments could have been driven by local selection on fitness through the phenotypes or could also be due to behavioral plasticity associated to its thermal physiology. behavioral plasticity may counteract the effects of the harsh environmental conditions at high-altitudes, and partially explain the similarities in the growth patterns and svl of the studied populations. pincheira-donoso and tregenza (2011) discussed how a stronger fecundity selection can direct relative adjustments for optimization of fecundity in species of liolaemus from cold areas. they also suggest that variation in ta along a geographical gradient alone would poorly explain the real outcome of fecundity selection on reproductive maximization. in support of their conclusions, sexual maturity of l. p. argentinus at the high-altitude site was attained earlier in life compensating the shorter lifespan and resulting in the same fecundity output than at the low-altitude. further tests are needed to determine the real cause of these inter-populational differences. as other ectotherms, growth of l. p. argentinus is indeterminate, considerably different in juveniles and adults, and can be assessed by studying the femur’s cross sections where growth bands become progressively thinfig. 2. estimates of medullar resorption. the lines of lineal regression of the observed medullar radius (arrow and dashed line) and the radius of growth rings (continuous lines) vs. snout-vent length (svl; mm) in liolaemus pictus argentinus at the high (a) and low (b) altitude sites. medullar resorption can be calculated placing the vertical line on a particular svl and the horizontal line on the intersection between the vertical line and the medullar radius regression line. the number of reabsorbed rings would correspond to the number of regression lines under the medullar radius line for that size (methodology used in piantoni et al., 2006a,b). fig. 3. growth lines of juveniles (black triangles), females (black dots), and males (empty dots) at the high (a) and low (b) altitude sites (the curve and 95% confidence intervals are indicated). the discontinued line indicates the maximum juvenile size of liolaemus pictus argentinus according to ibargüengoytía and cussac (1996). 14 joel antú gutiérrez et al. ner as the animal grows, especially after reaching sexual maturity. this growth pattern that relates age with svl can be represented by sigmoidal (pielou, 1977; castanet et al., 1988; measey, 2001; bruce et al., 2002), logarithmic (valdecantos et al., 2007) or logistic (mouden et al., 1999) curves. growth of l. p. argentinus was better modeled by a sigmoidal curve characterized by a greater growth rate in juveniles and an asymptotic phase in adults related to a typical re-allocation of the energy from growth to reproduction (castanet et al., 1988; zug and glor, 1998; mouden et al., 1999; wapstra et al., 2001; roitberg and smirina, 2006; guarino et al., 2010). the sigmoidal curve has also characterized the growth of p. tenebrosus (piantoni et al., 2006a), while the best fitted curve for the growth in other liolemids, l. irregularis, and l. multicolor, was represented by logarithmic equation (valedecantos et al., 2007). individuals living in warmer areas are predicted to mature earlier, whereas co-specifics from colder environments can sometimes delay maturity, investing available energy into present growth and future reproduction (roitberg and smirina, 2006). based on our results and in agreement with ibargüengoytía and cussac (1996), sexual maturity in l. p. argentinus appeared to be body size dependant. accordingly, a great heterogeneity in age was observed for the same body size (fig. 3) particularly in juveniles at the high-altitude site. however, in contrast with our prediction, l. p. argentinus at higher altitudes achieved sexual maturity earlier, at the age of three years in females and at four to six years in males (fig. 3a), but longevity in both sexes was of eight years. on the other hand, at low-altitude both, males and females, reached sexual maturity at the age of four years, and longevity was extended to nine years (fig. 3b). sexual maturity brings along secondary sexual characters and sometimes sexual dimorphism in svl, which may result in larger and more successful males (devender, 1978; anderson and vitt, 1990; verrastro, 2004), but most importantly, larger females that could produce larger and/or more numerous litters (tracy, 1999; smith, 2002; pincheira-donoso and tregenza, 2011). this could be an important adaptive trait especially in cold-temperate environments where higher thermal inertia relative to size can determine survival, mostly in newborn. these secondary sexual characters and behavior can determine the amount of energy that will be allocated to reproduction which can vary within species and populations (olsson et al., 2002). growth will depend on the remaining table 3. comparison between juveniles, females and males of liolaemus pictus argentinus at the highand the low-altitude sites with respect to: mean snout-vent length (svl) and svl range (mm), estimated age as number and range of rings (years), growth rate range [(mm x year)-1], and mean specific growth rate and specific growth rate range [mm x (year x mm)-1]. populations challhuaco mountain (high-altitude site) melipal beach (low-altitude site) sex and reproductive state (n) juveniles (13) females (11) males (7) total adults (18) juveniles (8) females (11) males (10) total adults (21) svl (mean; range; mm) 36.9; 23.5-48.8 55.9; 50.4-62.1 58.6; 51.4-62 48.5; 23.5-62.1 32.5; 24.8-44.4 56.6; 37.5-47.1 59; 36.2-44.1 57.7; 49.8-64.3 estimated age (mean; range; years) 2.8; 1-6 4.7; 3-8 5.8; 4-8 5.1; 3-8 2.5; 1-4 5.2; 4-7 6.1; 4-9 5.6; 4-9 growth rate range (mm x year-1 ) 8.395-1.825 4.952-0.880 3.621-0.880 4.9520.880 10.2794.948 7.996-0.823 7.996-0.128 7.9960.128 specific growth rate {mean and range; [mm x (year x mm)-1]} 0.179; 0.3570.0375 0.0593; 0.09830.0145 0.0357; 0.06020.0157 0.0501; 0.08380.0983 0.260; 0.358-0.180 0.0784; 0.1400.0142 0.0512; 0.1600.00202 0.0654; 0.1600.00202 15skeletochronology of liolaemus pictus argentinus energy and therefore is expected to vary as well. sexual dimorphism with respect to svl and growth rates were absent in both highand low-altitude populations. this is also a common pattern in southern south american squamates like p. tenebrosus (piantoni et al., 2006a), h. darwini (piantoni et al., 2006b; kubisch et al., 2012), l. irregularis, l. multicolor (valdecantos and lobo, 2007), iguana iguana (zug and rand, 1987) and tupinambis rufescens (fitzgerald et al., 1993). the relationship between growth and elevation in l. p. argentinus could be explained by differences in the hours of activity. high-altitude stands for shorter time of activity (gutierrez et al., 2010) which restricts foraging, hence energy intake (sears and angilletta, 2004; shine, 2005). this has a negative effect on certain traits associated to growth: in the mountains, juveniles usually hatch or are born later and go into hibernation earlier than those in lower altitudes (roitberg and smirina, 2006; gutiérrez et al., 2010). an earlier achievement of sexual maturity in the high-altitude population of l. p. argentinus can compensate for the shorter lifespan and ensure enough reproductive years, similar to the ones in the low-altitude site. similarly to l. p. argentinus female growth rates of h. darwini and p. tenebrosus are also greater during the juvenile stage (piantoni et al., 2006a,b; kubisch et al., 2012) which allows them to reach the minimum body size for reproduction and attain sexual maturity earlier, and in this way extend their reproductive life (piantoni et al., 2006a). this characteristic of life-history is especially advantageous in patagonia where lizards face periods of restrictive growth due to hibernation (ibargüengoytía and casalins, 2007). in comparison to most lizards l. p. argentinus exhibits low growth rates, a reduced reproductive life and, as a consequence, low fertility, but compensates with early sexual maturity in females. this pattern is commonly found in other patagonian species although sexual maturity tends to be reached later. females of p. tenebrosus achieve late sexual maturity at the age of seven years old and males at the age of nine years old. fecundity in this lizard is reduced to nine offspring, possibly due to its prolonged reproductive cycle (piantoni et al., 2006a). despite the differences observed in the thermal microenvironments, both populations of l. p. argentinus exhibited a reproductive life-span of five years. considering that the l. p. argentinus females reproduce every two or three years (ibargüengoytía and cussac, 1996) the total reproductive output (five years) would be among the lowest reported for lizards (table 1). in conclusion we agree with ferguson and brockman (1980) and sinervo and adolph (1989) in that both environmental and body temperatures (gutiérrez et al., 2010) as well as effectiveness in thermoregulation, are fundamental factors affecting metabolism and growth, and this can determine the time of sexual maturity and life-span. thermal constrains in cold-temperate environments like patagonia restrict some aspects of the life history of l. p. argentinus, such as growth, sexual maturity, reproductive frequency and fertility, when compared, in a general context, to lizards from temperate or tropical climates (ibargüengoytía and casalins, 2007). acknowledgments in memory of our friend and colleague, joel, we dedicate this work to his wife florencia cuassolo and son lautaro gutierrez. references adolph, s.c., porter, w. 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(1998): estimates of age and growth in a population of green sea turtes (chelonia mydas) from the indian river lagoon system, florida: a skeletochronological analysis. can. j. zool. 76: 1497-1506. acta herpetologica vol. 8, n. 1 june 2013 firenze university press the oogenic cycle of the caspian bent-toed gecko, cyrtopodion caspium (squamata: gekkonidae) in iran vida hojati1*, kazem parivar2, eskandar rastegar-pouyani3, abdolhossein shiravi1 altitudinal effects on life history parameters in populations of liolaemus pictus argentinus (sauria: liolaemidae) joel antú gutiérrez1,*, carla piantoni2, nora r. ibargüengoytía1,3 recent cryptic extinction of squamate reptiles on yoronjima island of the ryukyu archipelago, japan, inferred from garbage dump remains yasuyuki nakamura1,*, akio takahashi2, hidetoshi ota3 trophic niche and feeding biology of the italian wall lizard, podarcis siculus campestris (de betta, 1857) along western mediterranean coast marco a.l. zuffi*, chiara giannelli novel, non-invasive method for distinguishing the individuals of the fire salamander (salamandra salamandra) in capture-mark-recapture studies goran šukalo1,*, sonja đorđević2, dragojla golub1, dejan dmitrović1, ljiljana tomović2,3 going out tonight? when insular hierophis viridiflavus breaks the whip snakes rules delaugerre michel-jean first evidence of a paedomorphic population of the smooth newt (lissotriton vulgaris) in the czech republic václav gvoždík1,2, veronika javůrková4, oldřich kopecký5,* no detection of chytrid in first systematic screening of bombina variegata pachypus (anura: bombinatoridae) in liguria, northern italy stefano canessa1,*, an martel2, frank pasmans2 status of the european pond turtle, emys orbicularis (reptilia: testudines: emydidae) in vorarlberg, austria andreas kleewein1, günther wöss2 comparative cytogenetics of two species of ground skinks: scincella assata and s. cherriei (squamata: scincidae: lygosominae) from chiapas, mexico riccardo castiglia1,*, alexandra m.r. bezerra2, oscar flores-villela3, flavia annesi1, antonio muñoz4, ekaterina gornung1 polydactyly in the tyrrhenian wall lizard (podarcis tiliguerta) antigoni kaliontzopoulou1,*, daniele salvi1, verónica gomes1, joão p. m. c. maia1,2,3, panagiotis kaliontzopoulos4 book review: roberto sindaco, alberto venchi, cristina grieco. the reptiles of the western palearctic. 2. annotated checklist and distributional atlas of the snakes of europe, north africa, middle east and central asia, with an update to the vol. 1. edoardo razzetti acta herpetologica journal of the societas herpetologica italica © firenze university press www.fupress.com/ah acta herpetologica 5(2): 143-150, 2010 longand short-term impact of temperature on snake detection in the wild: further evidence from the snake hemorrhois hippocrepis francisco j. zamora-camacho1,*, gregorio moreno-rueda2, juan m. pleguezuelos1 1 departamento de biología animal, facultad de ciencias, universidad de granada, e-18071, granada, spain. *corresponding author. e-mail: chioglossa@hotmail.com 2 estación experimental de zonas áridas (csic), la cañada de san urbano, ctra. sacramento s/n, e-04120, almería, spain. submitted on: 2010, 25th february; revised on: 2010, 15th, august; accepted on: 2010, 22th, october. abstract. global change is causing an average temperature increment which affects several aspects of organisms’ biology, especially in ectotherms. nevertheless, there is still scant knowledge about how this change is affecting reptiles. this paper shows that, the higher average temperature in a year, the more individuals of the snake hemorrhois hippocrepis are found in the field, because temperature increases the snakes’ activity. furthermore, the quantity of snakes found was also correlated with the temperature of the previous years. our results suggest that environmental temperature increases the population size of this species, which could benefit from the temperature increment caused by climatic change. however, we did not find an increase in population size with the advance of years, suggesting that other factors have negatively impacted on this species, balancing the effect of increasing temperature. keywords. climate, hemorrhois hippocrepis, population dynamics, temperature, spain, global change. human activities are causing important changes in our planet, including a generalized average temperature increment (ipcc, 2007). the biology of organisms, especially ectotherms, depends on environmental temperature, and therefore many species are showing alterations in phenology, distribution, morphology, and population dynamics in response to those changes (reviews in hughes, 2000; walther et al., 2002; parmesan and yohe, 2003; root et al., 2003; parmesan, 2006; weatherhead and madsen, 2009). some species will not be capable of responding adaptively to climatic change, so there will be an increasing number of extinctions (thomas et al., 2004). in reptiles, climatic change is considered to be an important threat increasing the risk of extinction (pounds et al., 1999; gibbons et al., 2000; araújo et al., 2006; whitfield et al., 2007; reading et al., 2010; 144 f.j. zamora-camacho, g. moreno-rueda and j.m. pleguezuelos sinervo et al., 2010). however, in temperate regions, an increase in environmental temperature could also enlarge the snake’s available time for feeding (peterson et al., 1993), body growth (lindell, 1997), breeding success (chamaillé-jammes et al., 2006), and survival (altwegg et al., 2005), which could lead to increased population sizes. in fact, a previous study showed that the number of montpellier snake (malpolon monspessulanus) detected in the field in south-eastern iberian peninsula increased with the average temperature of the previous years, suggesting a positive effect of temperature on its population dynamics (moreno-rueda and pleguezuelos, 2007). therefore, the overriding impact of climate change of everything related to natural history of the snakes requires much more attention than has been apparent (seigel and mullin, 2009). in this work, we extend the previous investigation, examining the effect of temperature on the field detection of another mediterranean snake: the horseshoe snake (hemorrhois hippocrepis). hemorrhois hippocrepis is a thermophilic colubrid distributed over north western africa and the southern two thirds of the iberian peninsula, being found in lowto midlands of south-eastern spain, where the data for this article were collected (38º30’-37º15’n; 5º30’-2º30’w). the data used here were restricted to an altitudinal range of 0-1000 m a.s.l., extending over thermomediterranean and lower mesomediterranean bioclimatic stages, which this species mainly inhabits (pleguezuelos and feriche, 2002). these data were taken between 1980 and 2008, as a part of a long-term study on mediterranean snakes biology (feriche et al., 2008). during this period, the sampling effort was constant (about four sampling hours each work day, three days a month, every month, randomly encompassing the whole sampling area). only individuals active (not those found in their refuge), or recently killed by traffic (time of death estimated as less than 24 h) were recorded. we assumed that snakes killed on roads were active at the moment when the accident happened. in fact, there is a positive correlation between the number of road-killed snakes and the number of alive-detected snakes every year (moreno-rueda and pleguezuelos, 2007). for each year we recorded the total number of snakes detected, and the average altitude (m a.s.l.) of records. furthermore, data on the average total precipitation (mm) and temperature (ºc) in the study area were taken from the national meteorology institute. meteorological stations (n = 98) used to gather these data were evenly distributed over the sampling area. because of the elusive nature of snakes, as well as their patchy distributions, low population densities, and solitary behaviour, finding them has a strong stochastic character (fitch, 1987), which makes long-term monitoring programmes difficult, and capturerecapture studies almost an utopia (seigel and mullin, 2009). therefore, the sampling error in this study, referring to snakes’ field abundance, may be relatively high. this error diminishes the statistical power of our tests, making it more difficult to find significant effects, and thus the conclusions have to be more conservative (yezerinac et al., 1992). we correlated yearly average temperatures and total precipitation with the number of snakes detected; correlations with the meteorological variables of the preceding year were also performed. the yearly number of snakes detected was log-transformed, and all variables approximated to a normal distribution according to the shapiro-wilk test (p > 0.15). therefore, parametric statistics were used, namely pearson’s product-moment correlation (quinn and keough, 2002). in order to test for a possible independent effect of each meteorological variable over the detected snakes’ abundance, a multiple regression was used, estimating the partial correlations. 145impact of temperature on hemorrhois hippocrepis during the study period, local temperature increased an average of 0.015 °c per year (moreno-rueda et al., 2009). we found 337 h. hippocrepis in the restricted study area, with an average of 11.6 snakes per year (s.e. = 1.59, range 1-36, n = 29 years). the number of snakes found did not vary over the years (r = 0.07, p = 0.70, n = 29 years). the same happened with average altitude of the records (r = -0.07, p = 0.73, n = 29 years), suggesting that average altitude did not affect our results. the number of snakes found was significantly correlated with the average temperature of the current year (r = 0.43, p = 0.02, n = 28; fig. 1a), and tended to be correlated with temperature of the preceding year (r = 0.36, p = 0.06, n = 28; fig. 1b) as well as two years before (r = 0.32, p = 0.10, n = 27 years; fig. 1c). precipitation of a given year or of the preceding year was not related to the number of records of this species (r = -0.01, p = 0.95, and r = 0.25, p = 0.20, respectively; n = 28 years for both cases). temperature or precipitation of a specific year were not correlated with temperature or precipitation of the preceding year (r = -0.02, p = 0.91, and r = 0.05, p = 0.79, respectively; n = 27 years for both cases), suggesting an absence of temporal autocorrelation. the multiple-regression analysis showed an independent significant effect of average temperature of the sampling year (β = 0.44, t22 = 2.77, p = 0.01), the previous year (β = 0.38, t22 = 2.34, p = 0.03), and two years before (β = 0.34, t22 = 2.09, p < 0.05) on the number of individuals detected (r2 = 0.43, f3, 22 = 5.61, p = 0.005). there was no significant effect of precipitation when it was included in the model (β = -0.05, t21 = 0.30, p = 0.77), and the r2 value increased by only 0.002. lastly, we found a strong positive correlation between the number of h. hippocrepis and m. monspessulanus specimens detected in the field (from data in moreno-rueda and pleguezuelos, 2007; r = 0.72, p < 0.001, n = 26 years; fig. 2). species responses to global warming include short-term effects on populations (e.g., changes in abundance), but also long-term effects (e.g., shifts in species distribution; weatherhead and madsen, 2009). this study shows that the number of h. hippocrepis found in the field increased with average temperature of the current year. this result is not surprising, since the higher the temperature, the more active snakes are (nelson and gregory, 2000; pough et al., 2004; moreno-rueda et al., 2009), particularly this species, considered the most thermophilous iberian snake (feriche, 2004). it bears noting that the preceding years’ temperature also affected the number of snakes detected in the current year. the same result was found in m. monspessulanus (moreno-rueda and pleguezuelos, 2007), and the number of records of both species in the last 25 years was strongly correlated. both results suggest that high temperatures increases the survival and/or breeding success of both species, fostering a higher population size (and thus more detected individuals) the next year. there may be several causes for this (peterson et al., 1993). as temperature of a given year is higher, snakes have more time available for foraging and basking (sinervo and adolph, 1994), which might diminish their mortality rate for different reasons: less torpor (bennett, 1980), which diminishes predation risk (goode and duvall, 1989), and improved feeding ability (greenwald, 1974), which increases immune capacity (french et al., 2007) and the quantity of resources to survive the winter (naya et al., 2008). in fact, several studies in temperate regions have shown that as temperature increases, reptile survival rates also increase (altwegg et al., 2005; chamaillé-jammes et al., 2006; but see sinervo et al., 2010). reproductive rates also rise with temperature (lourdais et al., 2002), and a higher temperature also boosts juvenile growth rate (sinervo 146 f.j. zamora-camacho, g. moreno-rueda and j.m. pleguezuelos a) b) c) fig. 1. relationship between average temperature of the current year (a), the previous year (b), and two years before (c), with the number of horseshoe whip snake (hemorrhois hippocrepis) detected in the fi eld in se iberian peninsula. for the number of specimens, raw data are showed, although the analyses were performed with log-transformed data. 147impact of temperature on hemorrhois hippocrepis and adolph, 1989). moreover, the two studied species (h. hippocrepis, m. monspessulanus) share a north african origin (carranza et al., 2006), and are the only two european continental snakes that exhibit a vernal spermatogenetic cycle (pleguezuelos and feriche 1999; feriche et al., 2008). th e vernal spermatogenic cycle has strong thermal requirements that constrain its maintenance and distribution to regions with long and overall, warm springs (saint girons, 1982). th e fact that the number of individuals found for h. hippocrepis and m. monspessulanus are mediated by temperature, and the number of individuals recorded for both species are strongly correlated, even in spite of the altitudinal ranges of the populations studied were rather diff erent, suggests that more general conclusions can be proposed concerning the eff ect of a rising temperatures on mediterranean snake-population dynamics. similarly, weatherhead et al. (2002) found that population dynamics of two north-american populations of the black rat snake (elaphe obsoleta) were strongly correlated, probably because of a climatic eff ect (see also reading et al., 2010). th us, in general, it may be expected that higher temperatures will be benefi cial for mediterranean snake populations, except if other factors counteract these eff ects, as has been found in france for the ocellated lizard (timon lepidus; cheylan and grillet, 2005). in fact, in the present study, the density of snakes did not increase over the years, suggesting that other unknown factors off set the benefi ts of higher temperatures, such as deleterious eff ects on metabolism or prey availability. in conclusion, our study suggests that the rising temperature favours an increase in the population sizes of mediterranean snakes, as deduced by the number of individuals observed in the fi eld. we encourage other snake biologists to use their longterm data sets to identify population trends, because these trends may be associated with changes in climate (wheatherhead and madsen, 2009). fig. 2. relationship between the number of horseshoe whip snake (hemorrhois hippocrepis) and montpellier snake (malpolon monspessulanus) detected in the fi eld in the se iberian peninsula during 25 years (1980-2005 period). raw data are showed, although the analysis was performed with log-transformed data. 148 f.j. zamora-camacho, g. moreno-rueda and j.m. pleguezuelos acknowledgements we are grateful to the people that altruistically collaborated in sampling, and very especially to esmeralda alaminos. fjz-c was supported by a grant of initiation to the research by the university of granada, and another grant by the spanish government. david nesbitt and two anonymous referees improved the manuscript. references altwegg, r., dummermuth, s., anholt, b.r., flatt, t. 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(1992): measurement error and morphometric studies: statistical power and observer experience. syst. biol. 41: 471-482. © firenze university press www.fupress.com/ah acta herpetologica 5(2): 233-244, 2010 amphibians of the “cilento e vallo di diano” national park (campania, southern italy): updated check list, distribution and conservation notes antonio romano1, nicola ventre2, laura de riso3, camillo pignataro4, cristiano spilinga1 1 studio naturalistico associato hyla, via della pace 4, 06069 tuoro sul trasimeno (pg), italy. corresponding author. e-mail antonioromano71@gmail.com 2 via rimembranza 40, i-84075 stio (sa), italy. 3 ente parco nazionale del cilento e vallo di diano, piazza santa caterina 8, i-84078 vallo della lucania (sa), italy 4 museo naturalistico dei monti alburni, via forese, i-84020 corleto monforte (sa), italy submitted on: 2010, 10th april; revised on: 2010, 3rd august; accepted on: 2010, 22th october. abstract. in this study, we present the results of our field and bibliographic survey on the amphibians of the “cilento and vallo di diano” national park (southern italy). two hundred and thirty three spawning sites (167 original and 66 derived from literature), and 11 amphibian species were found. reproductive activity was recorded for salamandra salamandra, salamandrina terdigitata, triturus carnifex, lissotriton italicus, bufo bufo, hyla intermedia, rana italica, rana dalmatina and pelophylax synkl. hispanicus. the distribution record of many species is widely improved with respect to bibliographic data. our results also suggested that preservation and restoration of small aquatic sites, in particular of the artificial ones, such as stony wells and drinking-troughs, are fundamental for an appropriate conservation management of amphibians in the “cilento and vallo di diano” national park. keywords. amphibian distribution, cilento, vallo di diano, artificial aquatic site. introduction the “cilento e vallo di diano” national park (cvdnp) is situated in the province of salerno, campania region, southern italy (fig. 1). it lies between 40°00’ and 40°30’n and 14°50’ and 15°40’e making up for a total area of 181.048 ha, being the second widest italian national park and including 80 municipalities. the park falls in 36 utm 10x10 km squares (in 10 of them just marginally). the altitude ranges from sea level to the top of the cervati mountains (1898 m asl). the park’s first aim is preserving the traditional 234 a. romano et alii combination of cultural and natural landscapes as well as the unique plant diversity, characteristic for the region. on 1998, unesco inserted the cvdnp in the list of humanity heritage world sites. with the exception of some scattered information, previous organic studies on the herpetofauna of areas corresponding to cvdnp were provided by caputo et al. (1985) for the northernmost portion of the park, i.e. the alburni massif, and by talenti (1988) and by caputo and guarino (1992) for the remaining areas. in 2007, the cvdnp financially supported an updated checklist and a breeding sites’ census of amphibians’ species. the results of these surveys are hereby reported. material and methods study area. in the park two main domains can be distinguished (fig. 1): the cilento and vallo di diano area (cvd) and the northern part of the park that is the alburni massif (alb). these two parts are divided by sele, ripiti, tammaro and calore rivers. two main litologies are the mesozoic and cenozoic flysch, referred to as the “cilento flysch”, and the triassic-miocenic carbonate units, in the mountainous internal complex (alburno-cervati) (critelli, 1999). cvd is characterised by running waters and still waters (natural or artificial). conversely, due to karstic phenomena, natural small still freshwater ecosystems highly outnumber running waters on the alb. the park includes coastal areas, with a mediterranean climate and mediterranean bush vegetation, and inner areas, with mountainous climate influences, presenting extensive forests and a cooler and more humid climate. olive and chestnut are largely cultivated in the region. methods. field surveys were carried out systematically from march 2008 to june 2009 (230 hours of field research) and included (i) the inspection of some sites reported in literature, (ii) cartographic recognition of further potential aquatic habitats suitable for amphibian populations and the fig. 1. location of the study area, the “cilento e vallo di diano” national park (grey area). in the park, two main domains can be distinguished: the cilento area (light grey) and the alburni massif (dark grey), which are divided by hydrographic boundaries. 235amphibians of the “cilento e vallo di diano” national park inspection of these sites, (iii) collection of information from local peoples (mainly from shepherds). furthermore some of us (nv and cp), who collected data since many years in this area, provided further information. to evaluate the presence of amphibians, aquatic sites were both visually screened and sampled by performing about twenty dip-nettings, including blind ones (i.e., without previous visual detection of amphibians), although sampling effort was proportional to the complexity of habitats. audio strip transects (after dark) and checking under stones, which could be used as terrestrial shelters around the aquatic sites, were also performed. field researches were focused on the utm squares where data were scanty. sites were assigned to six different freshwater typologies: (i) lakes and marshes (ii) ponds (iii) running waters (rivers, streams and creeks); (iv) drinking-trough for livestock grazing; (v) circular wells, (vi) cement quadrangular tanks (i.e., “peschiere”). since several sites were very close to each other, two or more aquatic habitats less than 50 meters apart, inhabited by the same species, and belonging to the same category (e.g., well, pond) have been considered as a single breeding site. we adopted, therefore, the same criterion used by romano et al. (2007) and corsetti and romano (2007). among species and sites, the sørensen’s coefficient of similarity (hayek et al., 1994), which ranges between 0 (two given species never inhabit the same site) and 1 (two species are always syntopic), was calculated to detect affinities among species in their reproductive habitats. we compared the available bibliographic data (costa, 1874; caputo et al., 1985; talenti, 1988; caputo and guarino, 1992) and the data available in the ckmap 5.3.8 software (stoch, 2000-2005) to avoid sites overestimation. because cvd and alb are geomorphologically well separated (see above), they were commonly treated as separate unit in naturalistic contributes, in particular before the founding of the park (e.g., caputo et al., 1985; de filippo et al., 1985; caputo and guarino, 1992; nazzaro et al., 1996). in the discussion section we often considered, to allow comparisons with previous studies, the traditional subdivision in these two main domains results we found 11 species in the cvdnp: salamandra salamandra (linnaeus, 1758), salamandrina terdigitata (bonnaterre, 1789), triturus carnifex (laurenti, 1768), lissotriton italicus (peracca, 1898), bombina pachypus (bonaparte, 1838), bufo bufo (linnaeus, 1758), hyla intermedia boulenger, 1882, rana dalmatina fitzinger in bonaparte, 1838, rana italica dubois, 1987, pelophylax synkl. hispanicus (bonaparte, 1839). the synklpeton is formed by two entities which are considered both at species rank: the parental species, p. bergeri (gunther, 1985) and its hemiclonal hybrid, the kpleton p. kl. hispanicus. two hundred and thirteen seven records of amphibians were collected, and breeding activity was recorded in 167 sites, corresponding to 75% of the surveyed potential spawning sites (table 1). a lot of sites included in the ckmap (stoch, 2000-2005) were previously reported in caputo et al. (1985) and in caputo and guarino (1992). about 30% of selected bibliographic sites were not localisable because they were reported without necessary details. in the whole, literature data reported 12 species in the cvdnp (66 sites with a total of 156 records, table 1) because also the occurrence of bufo balearicus boettger, 1880 (as bufo viridis) was recorded (but this datum should be considered erroneous, see discussion). species were recorded in 24 utm squares (ve95, we04, we05, we14, we15, we16, we17, we23, we24, we25, we26, we27, we28, we32, we33, we34, we35, we36, we37, we38, we44, we45, we46, we54; in italic are reported squares where amphibians were recorded for the first time). 236 a. romano et alii species distributions in the cvdnp are shown in fig. 2 and fig. 3. compared to published data (table 1), new records for hyla intermedia, salamandrina terdigitata and rana italica, outnumber significantly the bibliographic records (767%, 260% and 250% respectively) while the lowest increments are for bombina pachypus (50%) and rana dalmatina (71%). in fig. 4 is shown the altitudinal range for each species. fig. 5 shows the aquatic site preferences of amphibians. sørensen’s coefficients of similarity among amphibian populations of the park are shown in table 2. discussion check list and distribution our survey, although did not improve the species check list obtained pooling the data of caputo et al. (1985) and caputo and guarino (1992), greatly improved the knowledge of amphibian distribution in a wide territory (about 181,000 ha) of southern italy, which is among the less investigated areas of italy (cf. sindaco et al., 2006). furthermore our work revealed that some errors were included in the atlas of italian herpetofauna (sindaco et al., 2006) and in the herpetological section of ckmap (stoch, 2000-2005), because they were built on the same data matrix which contained the original errors (r. sindaco in litt.). table 1. number of records of amphibian species in the parco nazionale del cilento e vallo di diano (southern italy) as reported in the literature and as resulting from this work. comparison between old data and new records of species in the two main domains of the park (alb = alburni massif; cvd= cilento and vallo di diano) is also shown. x = erroneous and doubtful data. species bibliographic sites original sites tot n cvd alb n cvd alb urodela salamandra salamandra (linnaeus, 1758) 7 + 9 + + 16 salamandrina terdigitata (bonnaterre, 1789) 5 + 13 + + 18 triturus carnifex (laurenti, 1768) 15 + + 12 + + 27 lissotriton italicus (peracca, 1898) 36 + + 69 + + 105 anura bombina pachypus (bonaparte, 1838) 18 + + 9 + + 28 bufo bufo (linnaeus, 1758) 26 + + 19 + + 45 hyla intermedia boulanger, 1882 3 + 23 + + 26 rana dalmatina fitzinger in bonaparte, 1838 7 + + 5 + + 12 rana italica dubois, 1987 26 + + 60 + + 86 pelophylax. synkl. hispanicus bonaparte, 1839 12 + + 18 + + 30 bufo balearicus x tot 155 237 393 237amphibians of the “cilento e vallo di diano” national park salamandra salamandra and salamandrina terdigitata were considered absent from the alburni massif by caputo et al. (1985), because suitable habitats were not available in the area. however, s. salamandra was reported for a site in the alb by bruno (1973). we found both species on the alburni mountains (fig. 2). furthermore, salamandrina cannot be considered as rare in the area as it is widespread in the whole park. both species breed often in streams, but occasionally also in drinking-troughs (fig. 5). we observed individuals of salamandra breeding from may and july, while salamandrina was observed to breed from april to june. triturus carnifex is dishomogeneously distributed in the park, where the species is strictly associated to old stony well on the alburni massif. it may be considered the rarest specie for the cvd (where it breed also in a drinking-trough, see fig. 5), being relegated to the peripheral north-eastern areas (fig. 2). this high habitat selectivity causes the lowest number of syntopies (4 syntopies, tab. 2), which are the half in comparison with the ones for other amphibian species (that reached 8 or 9 syntopies). all breeding habitats were found to be permanent and moderately deep (one site was 1 m in depth) or deep water bodies (depth > 3 m). this datum agrees with the preferred habitat for the species as described by andreone and marconi (2006). conversely, the elevation of the breeding sites we found in the park (the lowest sites is at about 550 m a.s.l., see fig. 4) disagrees fig. 2. distribution of caudate in the “cilento e vallo di diano” national park (southern italy). stars = sites from literature; dots = new records; crosses: erroneous data from literature (see text for additional information). utm grid (10x10 km) is also shown and representative codes of utm map are reported on the map in the up left corner. 238 a. romano et alii with the data presented by andreone and marconi (2006), who considered as favourite habitats the plains and the moderately elevated areas. bruno (1973) reported the species also for a site which falls in the centre of the cvd (alento river, near cicerale town), but our research did not confirm this finding. lissotriton italicus is the commonest amphibian in the park, where presents a uniform distribution and breeds in all aquatic site typologies (fig. 5). our observation confirms the high plasticity of the species, which is adaptable to very different climatic and hydrobiological conditions (scillitani et al., 2004). reproductive activity of italian newt in drinking-troughs on the alb is more frequent than as considered by caputo et al. (1985). larval overwintering and neoteny was also recorded and adult newts may be found in water in every months with a peak of presence between february and november. the highest sørensen’s coefficient showed between t. carnifex and l. italicus agrees to that reported for other italian areas where both species occur (romano et al., 2007). although hyla intermedia was recorded previously (just for one site) in the western foothill sector of alb (caputo et al., 1985), the site was outside of the park boundary. our surveys showed that the species is widely distributed both in the alb and in the cvd (fig. 3). we also observed that the italian tree frog breeds in all aquatic typologies, but prefers residual ponds near running waters and artificial habitats such as “peschiere” and wells (fig. 5). tree frog calls have been recorded from march to october. it is interesting to point fig. 3. distribution of anurans in the “cilento e vallo di diano” national park (southern italy). stars = sites from literature; dots = new records. utm grid (10x10 km) is also shown and representative codes of utm map are reported on the map in the up left corner. 239amphibians of the “cilento e vallo di diano” national park fig. 4. altitudinal ranges of amphibian species in the “cilento e vallo di diano” national park (southern italy). black bars = caudata; grey bars = anura. codes of species as reported in tab. 2. the mean altitude of distribution for each species is indicated by a horizontal segment within the bar. histogram was built pooling original and bibliographic records. fig. 5. habitat partitioning among amphibian species in the “cilento e vallo di diano” national park (southern italy). codes of species are as reported in tab. 2. histogram was built pooling original and bibliographic records. 240 a. romano et alii out that also this species, which traditionally considered a typical for marsh and ponds, according to our data often uses artificial water bodies as breeding habitat rana italica is the commonest anuran in the park, with a rather uniform distribution, and it is consistently more spread in the alb than previously supposed (fig. 3). the typical breeding sites in cvdnp are running waters but the apennine frog spawns also in drinking-troughs and, occasionally, in oxygenated ponds and wells (fig. 5). spawning activity was recorded from late february to june. bufo bufo presents rather uniform distribution and shows a great habitat typology differentiation (fig. 5). spawning was recorded from february to may. rana dalmatina and bombina pachypus showed the lowest increment of new records in comparison to the other species. the low number of new records of agile frog could be interpreted as confirmation that this species is not very common in the park (cf. bibliographic sites and new records in fig. 3). instead, apennine yellow-bellied toad was greatly widespread in the past (cf. bibliographic sites and new records in fig. 3). twenty five years ago this species was considered the commonest anurans on alb (caputo et al., 1985), and to be very common in the inner areas of cvd (talenti, 1988). long term observations by two of us (cp and nv) confirmed that the species disappeared from many sites in the last two decades and also that the number of observable individuals, of each population, decreased dramatically. the low increment of new records and long term observations, both on the distribution and on the size populations, agree with the negative trend reported for the species which has declined in almost all of its range (cf. andreone et al., 2008). finally, the occurrence of the green toad in the park has to be corroborated by further observations. suitable habitat for the species can be found, in particular, on the sandy coastal zones and near estuarine areas of cvd, although our research, also in those areas, did not produce any finding. table 2. sørensen’s coefficients among amphibian species in the “cilento e vallo di diano” national park (southern italy). salsal=salamandra salamandra; salter= salamandrina terdigitata; tricar= triturus carnifex; lisita= lissotriton italicus; bompac=bombina pachypus; bufbuf=bufo bufo; hylint= hyla intermedia; randal=rana dalmatina; ranita=rana italica; pelshsp= pelophylax synkl. hispanicus. coefficients were calculated pooling original and bibliographic records. salsal salter tricar lisita bompac bufbuf hylint randal ranita salsal salter 0.133 tricar 0 0 lisita 0.017 0.033 0.33 bompac 0.045 0.043 0.29 0.18 bufbuf 0.098 0.127 0.06 0.2 0.219 hylint 0.095 0.045 0.08 0.122 0.055 0.113 randal 0.143 0.133 0 0.103 0.15 0.105 0.211 ranita 0.098 0.192 0 0.178 0.158 0.29 0.054 0.102 pelshis 0.087 0.083 0 0.119 0.103 0.267 0.179 0.095 0.189 241amphibians of the “cilento e vallo di diano” national park erroneous data there are two main errors in the literature that should be discussed to avoid further propagation of errors. the occurrence of the green toad, bufo balearicus (as b. viridis), in the cvdnp was reported near sicignano town, on the alb, by caputo et al. (1985) not as a direct observation. this datum was reported also in ckmap (stoch, 2000-2005) and in the italian atlas (sindaco et al., 2006) where in addition another record was reported for the alb area (in corleto monforte town). the latter record was attributed to one of the authors of this paper (cp) who, however, observed, in corleto monforte, only the common toad and not the green toad. thus the datum reported in ckmap and in the italian atlas should be considered erroneous, while the datum from sicignano should be considered doubtful. in fact, this datum is generic (only the municipality is reported but not the locality), sicignano is exactly on the border of the park (the record could falls outside of the park) and, finally, because the species was not recorded in our systematic field research and in that of caputo et al. (1985). other problematic data are the ones concerning triturus carnifex. once again, in stoch (2000-2005) and in sindaco et al. (2006) the same an erroneous datum was reported. italian crested newt was reported in ckmap and in the italian atlas, for the cvd area, in four utm meshes (we26, we33, we34, and we54) and it seems to be relatively widespread both in alb and in cvd (fig. 2). however, the sites of the four meshes are the same site (i.e., pozzi monaci, near casalletto spartano town) and, moreover, this site falls in another mesh (we55). conservation notes undoubtedly, the “cilento e vallo di diano” national park represents a protected area of notable importance for the protection of amphibians. more than 50% of the species we found in the park are italian endemics. out of the 11 species, three (s. terdigitata, t. carnifex and b. pachypus) are included in the annex ii and iv of “habitat” directive 92/43/ cee and the other species (excluding b. bufo and s. salamandra), in the annex iv. furthermore, the park is a wide area where many breeding sites of a species considered as endangered by the iucn, i.e. b. pachypus, occur. we considered two areas particularly worth of consideration for their herpetofaunal biodiversity. they were proposed as “area di rilevanza erpetologica nazionale” (aren, i.e., area of herpetological importance at national level) and approved, on 2009, by the council directive and conservation commission of societas herpetologica italica. in the first aren (code ita063cam002), in the cvd, s. terdigitata, l. italicus, b. pachypus, b. bufo, h. intermedia, r. dalmatina and r. italica breed. in the second aren (ita064cam003), on the alburni massif, t. carnifex, l. italicus, b. pachypus, h. intermedia and p. synkl. hispanicus spawn. the second aren, interesting also on the historical point of view, was mentioned, with a list of amphibian species that could be found, by achille costa (1874). in both aren many reptiles also occur (see the official web site http:// www-3.unipv.it/webshi/conserv/areeril.htm for further details on these aren). in the cvdnp all amphibian species use traditional artificial aquatic habitats as breeding sites (fig. 5). for some of them, which deserve particular attention due to their 242 a. romano et alii endemicity and/or to their habitat fragmentation (andreone and luiselli, 2000; sindaco, 2000), artificial water bodies represent the exclusive breeding sites (for t. carnifex, see fig. 5), the preferred breeding sites (for l. italicus and b. pachypus, fig. 5) or a significant portion of the spawning sites (for s. terdigitata, and h. intermedia). the abandonment of agricultural land, traditional silvo-pastoral and cultivation practices is a common and widespread phenomenon in the mountainous regions of italy, which is linked to the large-scale socio-economic transformations that affected the whole country in the last century (see torta et al., 2004 and references therein). traditional artificial water bodies are also affected by this phenomenon. the main problems related to the conservation of these amphibian habitats, as emerged in our study, are (i) fill-in phenomena of the aquatic sites, (ii) their disrepair and consequent permeability loss, (iii) aggressive mechanical and chemical cleaning, in particular of drinking-troughs, (iv) tapping of wells for safety measures (in particular of abandoned or less used wells), (v) unsuitability of many artificial water bodies to act as amphibian breeding site because they have some “architectural fences” that obstruct the entry in and/or the exit from the water (vi) introduction of alien species (mainly goldfishes, carassius auratus) to “adorn” the aquatic sites. the importance of traditional artificial water bodies for amphibians is highlighted by several studies in different countries (e.g., contreras et al., 2009; knutson et al., 2004; romano et al., 2007) and, in particular, in the mediterranean, as stressed in the updated iucn european red list of amphibians (temple and cox, 2009). for this reason the park supported the draft of a guideline for the appropriate management of artificial water bodies (romano, 2009), which is a fundamental prerequisite for appropriate conservation strategies and for preservation of amphibian biodiversity. furthermore, the park is also the leading exponent of a regional-european project, which aims is to restore or to build small artificial water bodies (i.e. drinking-throughs and wells), using the methods of the rural architecture, for the landscape valorisation and biodiversity conservation (rural development programmes, second pillar, art. 36-51, axis 2, measure code: 216) acknowledgments the research was supported by a grant from “parco nazionale del cilento e vallo di diano” (d.d. 59124/12/2007). the project has been carried out under the patronage of societas herpetologica italica. sergé bogaerts, filomena carpino, romina fusillo, arnaldo iudice, manlio marcelli, frank pasmans, lorenzo pecoraro and paolo varruzza provided useful data on breeding sites. we are especially grateful to sebastiano salvidio for his contribute in the field researches. we are indebted to ylenia chiari for the linguistic revision and for critical reading of the ms. references andreone, f., corti, c., sindaco, r., romano, a., giachi, f., vanni, s., delfino, g. (2008): bombina pachypus. in: iucn 2009. iucn red list of threatened species. version 2009.2. www.iucnredlist.org. downloaded on 10 december 2009. 243amphibians of the “cilento e vallo di diano” national park andreone, f., luiselli, l. (2000): the italian batrachofauna and its conservation status: a statistical assessment. biol. cons. 96: 197-208 andreone, f., marconi, m. (2006): triturus carnifex (laurenti, 1768). in: atlante degli anfibi e dei rettili d’italia / atlas of italian amphibians and reptiles, p. 220-225. sindaco, r., doria, g., razzetti, e., bernini, f., eds, societas herpetologica italica, edizioni polistampa, firenze. bruno, s. (1973): anfibi d’italia: caudata. studi sulla fauna erpetologia italiana – xvii. natura, soc. it., sc. nat., museo civ. st. nat. e acquario civ., milano 64: 209-450. caputo, v., kalby, m., de filippo, g. 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(2004): consequences of rural abandonment in a northern apennines landscape (tuscany, italy). in: recent dynamics of the mediterranean vegetation and landscape, p. 157-165. mazzoleni, s., di pasquale, g., mulligan, m., di martino, p., rego, f., eds, wiley, london. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 8(2): 123-128, 2013 comparative ecophysiology of two sympatric lizards. laying the groundwork for mechanistic distribution models enrique garcía-muñoz1,2,3*, miguel angel carretero1 1 cibio, centro de investigação em biodiversidade e recursos genéticos, universidade do porto, campus agrário de vairão, 4485-661 vairão, portugal. *corresponding author. e-mail: engamu@gmail.com 2 cesam, centro de estudos de ambiente o do mar, universidade de aveiro, campus universitário de santiago, 3810-193 aveiro, portugal 3 departamento de biología animal, biología vegetal y ecología. campus de las lagunillas, s/n. universidad de jaén. 23071 jaén, spain submitted on 2013, 26th july; revised on 2013, 3rd december; accepted on 4th december. abstract. distribution modelling usually makes inferences correlating species presence and environmental variables but does not take biotic relations into account. alternative approaches based on a mechanistic understanding of biological processes are now being applied. regarding lacertid lizards, physiological traits such as preferred body temperature (tp) are well known to correlate with several physiological optima. much less is known about their water ecology although body temperature and evaporative water loss (wl) may trade-off. two saxicolous lacertids, algyroides marchi and podarcis hispanica ss are sympatric in the subbetic mountains (se spain) were they can be found in syntopy. previous distribution modelling indicates the first species is associated with mountains, low temperatures; high precipitation and forest cover whereas the second one is more generalistic. here, we perform two ecophysiological tests with both species: a tp experiment in thermal gradient and a wl experiment in sealed chambers. although both species attained similar body temperatures, a. marchi lost more water and more uniformly in time than p. hispanica ss that displayed an apparent response to dehydration. these results suggest that water loss rather temperature is crucial to explain the distribution patterns of a. marchi in relation to p. hispanica ss, the former risking dehydration in dry areas no matter what temperature is. ecophysiological traits represent a promising tool to build future mechanistic models for (lacertid) lizards. additionally, the implications for their biogeography and conservation are discussed. keywords. water loss, preferred body temperature, mechanistic models, algyroides marchi, podarcis. introduction thermal ecology is central in ecophysiological studies on lizards, and lacertids are not an exception to this rule (castilla et al., 1999). past decades gave rise to a plethora of detailed studies describing the proximate mechanisms linking temperature to physiology, life history, and behaviour (angilletta, 2010). preferred body temperature in the absence of thermoregulatory constraints (tp) constitutes an important trait in lizard studies correlating with several physiological optima (huey and bennet, 1987; bauwens et al., 1995; angilletta et al., 2002; carretero, 2008a). in contrast, lizard water ecology has been neglected. although protocols for testing water loss rate (wl) already exist (lillywhite, 2006) and even when morphological proxis have been found (caslbeek et al., 2006) no comparative results are really available. nevertheless, preliminary evidence suggests that body temperature and evaporative water loss may trade-off (bruce, 1990; bowker, 1993). those species capable to reduce wl while remaining mobile have the best opportunities for a precise control of temperature (tracy, 1976; buttemer, 1990). an animal with low wl may move to regulate its temperature precisely without experiencing the cooling and dehydrating effects of evaporation (tracy and christian, 2005). nonetheless, both types of variables (tp and wl) are essential to develop mechanistic models of potential distribution of species (adolph and porter, 1993; fei et al., 124 enrique garcía-muñoz, miguel angel carretero 2012). species distribution modeling is currently dominated by correlation analyses. that is, to infer the factors restricting the range of an organism, its presence is correlated to the environmental variables prevailing in its range. this procedure produces good results (peterson and kitchell, 2001; martínez-freiria et al., 2008; santos et al., 2009), especially when the species have low dispersal abilities (sillero et al., 2009). however, other external variables can be involved namely, biotic factors which can keep species absent from environmentally suitable areas (kearney and porter, 2004). among these factors, competitive interactions between ecologically similar species may restrict species ranges (begon, 2006). even if species interactions can be modeled based on correlation approaches (costa et al., 2008), the determination of physiological traits may provide solid evidence (porter et al., 2002,). podarcis and algyroides are two mediterranean lacertid genera with highly contrasting richness and distribution patterns. while 21 species of podarcis wall lizards are currently recognized, most of them with broad continuous distributions (arnold et al., 2007), algyroides consist of only four species with separate relict ranges (harris et al., 1999). among them, algyroides marchi is the most saxicolous species restricted to a very small range in the subbetic mountains, se spain (carretero et al., 2010). this species may be found in sympatry with podarcis hispanica ss a member of the p. hispanica species complex (carretero, 2008b; kaliontzopoulou et al., 2011, 2012), which also inhabits rocky habitats. correlative distribution models (rubio and carrascal, 1994; sillero et al., 2009; carretero et al., 2010) indicate that, at large scale, the presence of a. marchi depends on mountains, low temperatures, high precipitation and abundant forest cover, whereas, at small scale, it is constrained by terrain roughness and closure. although both species can be found in syntopy (carretero et al., 2010), the general trend is that a. marchi is restricted to the more humid spots, on the contrary p. hispanica ss inhabits drier and warmer rocky habitats, therefore we can say that p. hispanica ss is more tolerant than a. marchi. to elucidate to what extent this is due to ecophysiological differences or to (mutual) exclusion, we here analyse tp and wl experimentally in both species to determine whether these traits 1) differ intrinsically between them; and 2) are affected by intraspecific and interspecific interference. materials and methods five adult males of each species were collected in a syntopic area (rambla los vaquerizos, 38º3´n, 2º29´e). p. hispanica ss was assessed following kaliounzoupoulou et al. (2011) where this samples were genetically analyzed. all individuals were captured by hand or noose (garcía-muñoz and sillero, 2010) in july 2010, and brought to the laboratory. individuals were marked using a marker pen and kept in individual terraria for a total of eight days with food (tenebrio molitor larvae and grasshoppers) and water provided ad libitum and under light (14 h light – 10 h darkness) and temperature (23.3 ºc ± 1 ºc) conditions similar to the natural regime. each lizard was subjected to two experiments (see below). after each set of experiments, lizards were supplied with water and food ad libitum until they recovered the initial weight, and at the end of all experiments lizards were released in the capture site. lizards were exposed to a classic tp experimental design of thermal gradient (~25-45 ºc, 0.3 × 0.4 × 1.0 m length experimental terrarium, veríssimo and carretero, 2009) produced by a 100 w infrared reflector bulb fixed 15 cm above the substrate and maintaining natural photoperiod. the walls of the terrarium were covered with white panels to prevent external stimuli influencing the lizard’s thermoregulatory behaviour. tp was measured with k-thermocouple digital thermometer (hibok 14; manufactured by hibok, precision 0.01 ºc) by inserting a probe in the cloaca every hour during 10 hours (from 9:00 to 18:00 h local time). snout ventral length (svl) of each lizard was also registered to the nearest 0.01 mm using a digital calliper (model: mitutoyo digimatic caliper). evaporative water loss experiment were developed using sealed chambers (~25 ºc controlled temperature chamber, ~30-35 %rh generated by 100 g of silica gel). wl was measured with a precision balance (precision 0.0001 mg; cpa model 224s, sartorius bohemia, new york). initial weight (w0) at the start of the evaporative water loss experiment was recorded. lizards were individually placed in transparent plastic boxes covered with a perforated lid and floor placed inside another box with 5 g of silica gel at the bottom that allowed free air exchange between the individual box and sealed chambers. five small boxes with lizards were placed in a sealed chamber at the same time. lizards inside the boxes were removed from the chamber hourly, weighed together with the box on the precision balance and put back into the chamber, over a period of 12 hours. the whole operation did not take longer that 20 sec. the sealed chambers were maintained in obscurity and an undisturbed room in order to avoid stressing the animals. values of tp (untransformed) did not deviate from normality (shapiro-wilk’s test, p > 0.05 in all cases), were homoscedastic (univariate levene’s tests and multivariate box m, p > 0.05 in all cases) and variances and means were uncorrelated. repeat measures (rm) anova, and rmancovas (with logsvl; and with logsvl and logw0 (initial weight) as covariables) were performed in order to evaluate differences in tp (hourly tp; ºc) between both species (sp; a. marchi and p. hispanica s.s.) a rmanova, and two rmancovas (with logsvl, and with logsvl and initial weight, logw0 as covariables) were performed to evaluate differences on accumulative water loss (wl = log {[(wo-wn)/wo]*100}). a post-hoc duncan´s test was performed after rmanova and rmancova in order to detect differences (with and without body size/mass effects) in wl between species. in addition, pearson correlation and linear least-squares were performed (for both species) in order to determine if there 125comparative ecophysiology of two sympatric lizards is a relationship between tp (mean tp for the 10 h, ºc) and total wl (for the total accumulative water loss 12 h; wl = log (w8-w20)). results lizards of both species showed similar preferred body temperatures (algyroides marchi, mean ± sd tp = 31.5 ± 0.5; p. hispanica ss, mean ± sd tp = 31.8 ± 1.5) and repeated measures an(c)ova showed no statistical differences, also after size-mass correction (rmanova, f9, 72 = 1.702, p = 0.104; rmancova, f9, 63 = 0.394, p = 0.933, logsvl = 3.830; rmancova, f9, 54 = 0.293, p = 0.973, logsvl = 3.830, logw0 = 0.776). in water loss experiments, a. marchi showed the highest wl rates, reaching at the end of the experiment a water loss of approximately 10% of the initial weight. in contrast, p. hispanica ss showed much lower rates, especially during the first hours, final water loss attaining 3% of the initial weight. in addition, both species showed different wl rates though time (time*sp, rmanova, fig. 1) and post-hoc analyses revealed that both species responded in different ways to wl in the first hours (duncan test: wl1-4, p = 0.037; wl1-5, p = 0.035). these differences between both species were not svlor weight dependent, and these interactions remained significant (time*sp; rmancovas, table 2) after size/body mass was accounted for. in addition, another post-hoc test was performed after including covariables to test if the differences between both species were svlor weight dependent or intrinsic (duncan test: cov logsvl, wl1-3, p = fig. 1. cumulative water loss in both species tested (a. marchi; p. hispanica ss), different whiskers denote different cumulative water loss intervals and their 95% ic, wl = log{[(w0-wn)/w0]*100}, for both species in an experiment conducted during 12 hours. fig. 2. relationship among preferred temperature (mean; log transformed) and total water loss (log transformed) in both species 126 enrique garcía-muñoz, miguel angel carretero 0.046; wl1-4, p = 0.021; wl1-5, p = 0.020; wl1-6, p = 0.029; duncan test: covw0, wl1-4, p = 0.029, wl1-5, p = 0.027, wl1-6, p = 0.039). regression between tp (mean tp 10 hours, ºc) and wl (12 hours, total accumulative water loss) varied between species. while in a. marchi no significant relations were detected, p. hispanica ss showed an inverse relationship between tp and wl (fig. 2). discussion our results indicate that both sympatric lizards differ in some ecophysiological traits. concerning the thermal traits, both species appeared to have similar preferred temperatures which remained not significant after variation in body mass/size was accounted for. as other lacertid lizards, a. marchi and p. hispanica ss kept their body temperatures within a narrow range when free of thermal constraints (reviewed in castilla et al., 1999; carretero et al., 2005; carretero at al., 2006; veríssimo and carretero, 2009). it is well documented that behavioural adjustments are the primary means by which lizards buffer fluctuations in ambient heat loads to maintain their tp within the range that is conducive to optimal performance (castilla et al., 1999). regarding the water ecophysiology, much less is known in lizards in general, and only two very recent studies analysed lacertid species (carneiro et al., in press; osojnik et al., 2013). nevertheless, physiological studies have provided abundant information on osmoregulation and on mechanisms of water conservation of other lizards (minnich, 1982; nagy, 1982). thus, the resistance to water loss reflects the combined effect of two integumentary components on the rate of water loss. the first one is a structural component, including differences in skin micro-structures and lipid content. the second component is more dynamic, representing physiological, vasomotor responses to short-term variations in the environment. this physiological response enables better and more immediate control of wl (eynan and dmi´el, 1993). our results not only show intraspecific differences in the magnitude of absolute water loss rate but also provide evidence for a dissimilar pattern of water loss throughout a normal diel activity period. under the same conditions, a. marchi lost more water and at a more even rate than p. hispanica ss. in fact, p. hispanica ss displayed an apparent response to dehydration within the first four hours while a. marchi did not. in addition, results for p. hispanica ss supported a trade-off between tp and wl at the individual level while those for a. marchi were inconclusive. future studies should evaluate such a relationship in others species. altogether, our ecophysiological results suggest that water should be more important than temperature to explain the distribution patterns of a. marchi vs. p. hispanica ss, the former risking dehydration in dry areas whatever the temperature. this is accordance with high resolution distribution models (sillero et al., 2009; carretero et al., 2010) indicating that a. marchi is restricted to mountains, high precipitation and good forest cover at a large scale and rugged and steep terrains at a small scale. these humid environments (allow to minimise lizard evaporative loss) are scarce in mediterranean areas, which are characterised by midday and aestival draught, particularly in se iberia. hence, it is not surprising that a. marchi displays such a restricted distribution. also, taking into account the episodes of aridification that the whole mediterranean basin suffered periodically since the pliocene (cavazza and wezel, 2003; jiménez-moreno et al., 2010), it would be expectable that a. marchi became gradually replaced by p. hispanica ss in this region. going further, it is tempting to associate the climate transition, from subtropical to mediterranean, since the miocene to the spread and diversification of podarcis sp. (carretero, 2008b) in contrast to the retraction of algyroides ssp. in southern europe (harris et al., 1999). this, however, needs confirmation by further comparative studies with other species of both genera. last but not least, if the scenarios for climate change (ipcc, 2007) are confirmed, the vulnerability of a. marchi to dehydration and competitive displacement by sympatric p. hispanica ss put the former species at a serious extinction risk, in fact higher than suggested by correlation models (carvalho et al., 2010). overall, ecophysiological traits like preferred temperatures and water loss rates represent a promising tool to build future mechanistic models for lacertids and likely for other lizard groups. acknowledgements the junta de andalucía provided permits for sampling (sgyb/foa/afr). funding for research was provided by a ptdc/bia–bec/101256/2008 project awardfrom fundaçao para a ciência e a tecnologia (fct, portugal). eg-m was supported by a postdoctoral grant from fct (sfrh/ bpd/72806/2010). references adolph, s.c., porter, w.p. 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(2009): preferred temperatures of podarcis vaucheri from morocco: intraspecific variation and interspecific comparisons. amphibia-reptilia 30: 17-23. acta herpetologica vol. 8, n. 1 june 2013 firenze university press journal of the societas herpetologica italica acta herpetologica © firenze university press www.fupress.com/ah acta herpetologica 5(1): 37-62, 2010 nomenclatural availability of the names applied to “varieties” of the green toad (bufo viridis subgroup) in the italian territory, with emphasis on the variety lineata of ninni (anura: bufonidae) nicola novarini1, lucio bonato2 1 museo di storia naturale di venezia, santa croce 1730, i-30125, venezia, italy. corresponding author. e-mail: nicola.novarini@fmcvenezia.it 2 dipartimento di biologia, università di padova, via ugo bassi 58b, i-35131, padova, italy. e-mail: lucio.bonato@unipd.it submitted on: 2009, 24th august; revised on 2010, 25th march; accepted on 2010, 26th march. abstract. recent molecular investigations on eurasian green toads led to the recognition of distinct lineages and to the establishment of new taxa within the former bufo viridis; as a consequence, significant range-wide nomenclatural changes have been proposed, although some uncertainties remained on the available names applicable within the italian territory. in order to contribute to clarify the matter, we evaluated, under the provisions of the international code of zoological nomenclature, the nomenclatural availability of all the names that have been applied to infrasubspecific entities of the bufo viridis subgroup within the italian territory. we also provided a historical overview of the usage of all these names, as well as detailed information on the original material upon which the variety lineata of a.p. ninni was established. our analysis supports the view that only the names crucigera eichwald, 1831 and balearica boettger, 1880 are available, the former being however junior synonym of b. viridis laurenti, 1768, whereas the names acutirostris and obtusirostris of lessona, lineata of ninni, concolor and maculata of camerano, and nardoi of paolucci, fuhn and bruno are all not available. keywords. bufo viridis subgroup, green toads, italy, nomenclature, taxonomy, availability, infrasubspecific names, variety. introduction on-going molecular investigations on eurasian green toads (bufo viridis laurenti, 1768 subgroup; sensu stöck et al., 2001, 2006) are greatly contributing to reveal phylogenetic diversity and phylogeographic structure within this anuran group (balletto et al., 2000, 2007; stöck et al., 2005, 2006, 2008b; batista et al., 2006). the consequent recognition of 38 n. novarini and l. bonato distinct lineages within b. viridis has led to the establishment of new taxa and therefore to the introduction of new names or the resurrection of old, neglected synonyms (stöck et al., 2005, 2006, 2008a, b; balletto et al., 2007). as a result, significant range-wide nomenclatural changes appeared recently in the taxonomy of this widespread species-group. in the mean time, a further source of nomenclatural “destabilization” was introduced, at genus level, by the comprehensive amphibian phylogeny of frost et al. (2006), who transferred the bufo viridis subgroup to the newly established genus pseudepidalea frost, grant, faivovich, bain, haas, haddad, de sá, channing, wilkinson, donnellan, raxworthy, campbell, blotto, moler, drewes, nussbaum, lynch, green and wheeler, 2006. claimed flaws in the analysis (e.g.: wiens, 2007), however, may allow the adoption of only part of their nomenclatural revision, suggesting instead the possible placement of the green toads within the genus epidalea cope, 1864 (speybroeck and crochet, 2007). these new generic combinations, although promptly adopted by some authors, have been regarded by others at least as premature, if not truly unjustified (lanza et al., 2007a; vences, 2007; stöck et al., 2008b), whereas some advocated their usage at subgenus rank (smith and chiszar, 2006 [fide frost, 2009]; duda, 2008). within this framework, recent investigations on green toad populations from the italian region led to the recognition of at least four lineages deserving the status of distinct species, although different research groups achieved partially different results, thus proposing different nomenclatural changes. particularly, both stöck et al. (2006, 2008b) and balletto et al. (2000, 2007) distinguished independently a previously unrecognized taxon (mainly widespread in the italian peninsula, the po plain, corsica and sardinia) from bufo viridis laurenti, 1768 (sensu stricto) (inhabiting most of europe and reaching italy in the north-east only). due to partial differences in their samples and analyses, however, two alternative names were proposed for the same species, namely bufo balearicus by stöck et al. (2006, 2008b), from bufo viridis balearicus originally introduced by boettger (1880) as a variety, and bufo lineatus by balletto et al. (2007), from the variety name lineata established by ninni (1879). razzetti (2008) discussed in detail several apparent homonyms and synonyms of these names, including the previous usage of the binomen bufo lineatus by daudin (1802) and donoso-barros (1972), applied to taxa unrelated to the b. viridis subgroup.  razzetti (2008) concluded that either lineatus or balearicus should be properly applied to the species inhabiting most of the italian region, the correct choice being dependent upon the definitive identification of the taxon living in venice and the surrounding plain (venetia region, ne italy), from where the lineata variety was originally described. notwithstanding taxonomic uncertainties, both names have been hitherto considered “available” in the sense established by the international code of zoological nomenclature (international commission of zoological nomenclature, 1999), hereafter iczn, even though the matter – at best of our knowledge – has not been explicitly addressed or discussed. more generally, out of the many names coined in the past for varieties and other apparently infrasubspecific entities of green toads from the italian region, only few of them have been explicitly evaluated for their nomenclatural availability, namely concolor and maculata of camerano (1883) (stöck et al., 2008b) and nardoi of paolucci et al. (1999) (razzetti, 2008). the aim of the present paper, therefore, is to contribute an evaluation of the nomenclatural availability of the names applied to putative infrasubspecific entities of the bufo viridis subgroup within the italian region, with emphasis on the name lineata. instrumen39nomenclature of italian bufo viridis tal to this evaluation, we provide a historical overview of the usage of all these names, as well as detailed information on the original material upon which the variety lineata was established. to provide the reader with the appropriate background for a thorough evaluation of the nomenclatural availability of each name, we believe useful to include, as footnotes, the most significant excerpts of all relevant publications in their original language, together with an english version (our translations). any taxonomic issue is out of our scope. nevertheless, we hope that the present nomenclatural analysis will be useful in supporting the taxonomic changes required by recent systematic advances, contributed by phylogeographic investigations in progress, within the b. viridis subgroup. discussion “varieties” of the green toad in the italian territory: a historical overview several previous authors working on green toads in italy acknowledged great variability in morphology and colouration but failed to detect any consistent geographical pattern for such variation that could be suggestive of distinct phyletic lineages, a concept that was stressed explicitly by camerano (1882, 1883). nevertheless, over the past 150 years, several varieties and other apparently infrasubspecific entities were identified, and often named, within the italian region. the first variety identified in italy was bufo viridis var. calamita laur., reported by jan (1857) from lombardy and by nardo (1860) from the “province venete” (venetian provinces, roughly corresponding to present-day venetia and friuli, ne italy), in both cases as bare list entries without comments. the name calamita was first introduced by laurenti (1768) at species rank and indeed bufo calamita laurenti, 1768 is presently recognised as a distinct species, inhabiting western and central europe. however, around the middle of the 19th century, following dumeril and bibron (1841), calamita was regarded as a mere striped phenotype of bufo viridis by several authors (see also: de betta, 1857; nardo, 1874; camerano, 1883). misdirected by the work of jan (1857), nardo (1860) and others, de betta (1874) included the species b. calamita within the fauna of venetia, though possibly limited to the alps. a few years later, lessona (1877) described in great detail the morphology and colour of the anurans of piedmont, recognizing many “forme” (forms) and “varietà” (varieties), among which several forms and varieties of green toads (as bufo viridis) were reported based on jaw morphology, wart arrangement and colour pattern. however, these three character sets were considered independently and different forms or varieties were identified with respect to one set at a time. particularly, based on jaw morphology, lessona (1877) distinguished and named two forms of b. viridis, “forma acutirostris” and “forma obtusirostris” respectively1, apparently following var. acutirostris and var. obtusirostris coined by fatio (1872) for rana temporaria linnaeus, 1758. it is worth noting 1 “anche qui possiamo stabilire negli individui adulti una forma acutirostris ed una forma obtusirostris.” [= here also, we can distinguish, among adults, an acutirostris form and an obtusirostris form.] (lessona, 1877, p. 1087). 40 n. novarini and l. bonato that these same two terms were applied, across the paper (lessona, 1877), to alternative phenotypes of several species, often using a trinomial arrangement (e.g.: rana esculenta acutirostris and rana esculenta obtusirostris, rana temporaria acutirostris and rana temporaria obtusirostris, bufo vulgaris acutirostris and bufo vulgaris obtusirostris), although the explicit trinomial form was never used with b. viridis. in addition, morphologically distinguished phenotypes were always referred to as “forme”, whereas those based on colouration or wart arrangement were called “varietà”, a linguistic distinction which is consistent throughout the whole paper. the distinction acutirostris vs. obtusirostris does not seem to relate consistently to the same factor of variation across species, in some being related either to sex or to growth stage, in others to geographical provenance, but sometimes simply indicating a general dimorphism. the latter seems the case with respect to the green toad, since none of the two forms was indicated as limited to a single sex, to a particular growth stage or to any locality or area. in the same publication, lessona (1877) described also two sets of “varietà” of the green toad, one based on wart size, shape, number and arrangement, and the other on colour pattern. the first set included four varieties, which were not named but simply labelled with the letters “a” through “d”2. two of these varieties were reported as associated to sex and/or age, whereas none of the four was reported as exclusive of particular localities or areas, neither occurring alone in any place. with respect to colour and spot arrangement, lessona (1877) described instead seven varieties, again unnamed and simply listed in alphabetical order from “a” to “g”3, clear2 “possiamo per rapporto alle verruche stabilire le varietà seguenti: || a) pelle liscia; qualche verruca nella regione lombare; parti inferiori granulose; non rara. || b) verruche numerose, ma non molto cospicue, alcune disposte in modo da formare due linee che partendo un po’ sotto alle ghiandole auricolari si dirigono in basso e si continuano sui fianchi (tavola v fig. 36). | questa varietà non è molto comune in piemonte; ne ho trovato individui nei contorni di saluzzo, di torino, di vigevano. || c) il dorso ed i fianchi cosparsi di verruche di grandezza fra loro pressoché eguali munite di una piccola spina nera nel mezzo. comune, specialmente nei maschi. || d) verruche numerose e cospicue; granulazioni della parte inferiore molto sviluppate; le zampe anteriori e le posteriori pure coperte di grosse e numerose verruche; non rara nei maschi vecchi.” [= with respect to warts, we can distinguish the following varieties: || a) smooth skin; some warts in the lumbar region; granulation on the ventral side; not rare. || b) many warts, but not very conspicuous; some of them arranged in two lines that, starting slightly below parotoid glands, point ventrally and continue along the flanks (table v fig. 36). | this variety in not very common in piedmont; i found specimens around saluzzo, turin, and vigevano. || c) back and flanks covered with scattered warts of almost equal size provided with a small black spine in the middle. common, especially among males. || d) many conspicuous warts; well developed granulation on the ventral side; forelegs as well as hind legs covered with many large warts; not rare among old males.] (lessona, 1877, p. 1089). 3 “possiamo negli adulti, avuto riguardo ai colori ed alla disposizione delle macchie, stabilire le varietà seguenti: || a) tinta fondamentale grigia, più o meno intensa a seconda delle stagioni; macchie verdi non molto numerose e cospicue, numerosi i punticini rossi sparsi su tutto il corpo. | questa varietà non è rara specialmente fra i maschi dei contorni di saluzzo, cuneo, vigevano, mondovì. || b) le macchie sopra la regione della nuca incontrantisi in modo da formare grossolanamente una croce di sant’andrea; colore fondamentale tendente al rosso. | questa varietà è molto affine a quella che l’eichwald descrisse col nome di bufo crucigera; non molto comune in piemonte: ne trovai alcuni individui nei contorni di vigevano e torino. || c) color fondamentale giallognolo più o meno intenso, più o meno puro secondo le stagioni, macchie di color verde grandi e numerose disposte regolarmente: verruche più o meno numerose; comunissima in tutto il piemonte sia fra i maschi sia fra le femmine. || d) color fondamentale bruno grigio uniforme; nessuna macchia sul dorso, sui fianchi, sulle zampe anteriori e sulle coscie; poche o poco distinte sulle gambe posteriori; non rara fra i maschi specialmente dei contorni di torino (rivoli, venaria reale). || e) color fondamentale come nella varietà precedente; macchie piccole e numerosissime; non rara in tutto il piemonte. || f) color fondamentale grigio o giallognolo; macchie numerose; presenza di una linea dorsale chiara, meno ampia e meno regolare che non quella del bufo calamita. 41nomenclature of italian bufo viridis ly unrelated to the previous wart-based ones. these varieties were defined explicitly for adult specimens only, some of them were reported as associated with sex or particularly frequent in some places, whereas multiple varieties were reported from single localities. lessona (1877) made his observations from either living or preserved individuals, some of which were illustrated by l. camerano in the plates accompanying the article; unfortunately, throughout the paper, lessona did not say a word about the fate of the specimens he had meticulously described. in 1879, a.p.  ninni published a paper on the green toads (as bufo viridis) of venetia, in which, acknowledging the existence of lessona’s dorsally striped variety, he suggested that the claimed presence of bufo calamita among venetian amphibians had been due to a mistake of nardo (1860), who misidentified local specimens of this variety of green toad as “b. viridis var. calamita” (ninni, 1879). in a footnote to the paper, ninni (1879) named this striped variety of green toad “lineata”. ninni’s (1879) article comprises three well distinct sections. two of them are announced yet in the title: 1) “on the supposed existence of bufo calamita laur. in venetia (…)” and 2) “(…) on a distinctive habit of the green toad”, whereas the third one is the final “note” dealing with the donation of his collection to the museum of venice, but also introducing for the first time the name “lineata” 4. in the first part, ninni’s (1879) aim is to report, explicitly and exclusively, on the putative presence of b. calamita in venetia, claimed by nardo (1860) and later acknowledged by de betta (1874) and others, and to rectify such a misunderstanding. here, the author, after summarizing the controversy, introduced the variety “f” of lessona (1877), quoting literally the diagnosis and comments (i.e.: in piedmont, espenon rara in piemonte specialmente fra i maschi. || g) color fondamentale grigio, o giallognolo; macchie cospicue e disposte in modo da formare due linee non interrotte sul dorso; verruche disperse in due linee regolari sui fianchi. | questa varietà non è molto comune in piemonte; fino ad ora non ne ho trovato che qualche individuo nei contorni di torino, di vigevano e di saluzzo.” [= in adults, with respect to the colour and arrangement of spots, we can establish the following varieties: || a) grey background, more or less intense according to the season; few, conspicuous green spots; many small red dots scattered on the whole body. | this variety is not rare, especially among males in the surroundings of saluzzo, cuneo, vigevano, mondovì. || b) spots on the nuchal region meeting each other so as to shepe roughly a st. andrew cross; reddish background. | this variety is very close to that described by eichwald as bufo crucigera; not very common in piedmont; i found some specimens in the vicinity of vigevano and turin. || c) yellowish background, more or less deep, more or less pure according to seasons; numerous, large green spots, evenly arranged: more or less numerous warts; very common in the whole piedmont among both males and females. || d) evenly grey-brown background; no spots on the back, the sides, the forelimbs and the thighs; few or undefined spots on the hind limbs; not rare among males, especially in the surroundings of turin (rivoli, venaria reale). || e) background like in the previous variety; small, very numerous spots; not rare in the whole piedmont. || f) grey or yellowish background; numerous spots; presence of a light dorsal line, less wide and less regular than that of bufo calamita. not rare in piedmont especially among males. || g) grey background, or yellowish; conspicuous spots arranged so as to shape two unbroken lines on the back; warts scattered along two regular rows along the flanks. | this variety is not very common in piedmont; so far, i found only some individual in the surroundings of turin, vigevano and saluzzo.] (lessona, 1877, p. 1089-1090). 4 “nota. gli esemplari di bufo viridis della varietà ch’io chiamerò lineata, nonché i girini dei quali feci menzione più sopra, si trovano nella collezione zoologica del civico museo di venezia. nel museo anzidetto io depositai una raccolta completa di tutti i rettili ed anfibi del veneto comprese le specie più rare, tra le quali l’unico e rarissimo esemplare di chelonia mydas preso vicino a venezia.” [= note. the specimens of bufo viridis of the variety that i will name lineata, as well as the tadpoles mentioned above, are in the zoological collection of the civic museum of venice. in the above mentioned museum i deposited a complete collection of all reptiles and amphibians of venetia including the rarest species, among which is the single and very rare specimen of chelonia mydas caught near venice.] (ninni, 1879, p. 973). 42 n. novarini and l. bonato cially among males) of the latter author5. in the following lines, ninni stated explicitly that the same (lessona’s) variety was rather common also in venetia, especially among young individuals, as he could find it in the city of venice, as well as in the provinces of padua and treviso. nowhere, in this section, ninni made any comment, explicit or implicit, on the taxonomic status of this variety, and no statement by the author can be interpreted as possibly disagreeing with lessona’s original view, other than including venetia into the variety distribution range. it is also reported here the collecting of several young, 30 mm-long specimens bearing the line described by lessona, while catching green toad tadpoles. in the second part of the paper, ninni (1879) dealt only with the ecology and habits of green toads, reporting about the dens dug by these amphibians in the sand dunes of the lido di venezia island, on their food and on some usage in the venetian traditional medicine. nowhere, in this “ecological” section, the author mentions the variety he had just discussed, but reference is made only to “bufo viridis” in general. the third part, a tail-end note, simply reports the donation to the museum of venice, together with a full collection of amphibians and reptiles from venetia, of “the specimens of bufo viridis of the variety that i will name lineata [the 30 mm-long specimens mentioned in the first section], as well as the tadpoles that i mentioned above [also in the first part]” (ninni, 1879). according to ninni’s own words then, the tadpoles, although collected together with striped young toads, are not included as lineata, a name that the author appears to reserve exclusively to those individuals visibly bearing the character at hand (i.e.: the dorsal stripe), obviously absent on tadpoles. therefore, in our view, the content of ninni (1879) is suggesting unambiguously, although not explicitly, the value of mere individual phenotype of the var. lineata. nevertheless, a look to ninni’s usage of the term “varietà” in other contexts, as well as to his attitude toward the application of taxonomic ranks (which included the use of species, subspecies and variety as well distinct entities), may be helpful for better understanding his taxonomic philosophy. actually, whenever ninni had been dealing with biological entities that did not appear to him as clearly separated from the “tipo” (the “type” 5 “il prof. lessona descrisse una varietà del bufo viridis, che avrebbe i seguenti caratteri: color fondamentale grigio o giallognolo, macchie numerose, presenza di una linea dorsale chiara, meno ampia e meno regolare che non quella del bufo calamita. non rara in piemonte specialmente nei maschi (2). | questa varietà che, a primo aspetto, potrebbe essere scambiata col bufo calamita (3), e che non fu notata dai naturalisti veneti, è abbastanza comune tra noi specialmente nei giovani individui. io la trovai nella stessa città dì venezia, ed anche nello scorso giugno pescando, per oggetto di studio, girini di rospo smeraldino, molti dei quali avevano già compiuta la loro metamorfosi (1), raccolsi parecchi esemplari giovani (lungh. corp. mm. 30 circa) con la riga descritta dei prof. lessona, ed altri pure di simili potei vederne in epoche diverse nelle provincia di padova e di treviso. | non vi ha dubbio per me, che gli esemplari dati dal nardo come bufo calamita (= var. calamita) appartengono invece alla varietà da me indicata, per cui resta, io credo, spiegata la illegittima comparsa del bufo calamita negli elenchi degli anuri del veneto.” [= prof. lessona described a variety of bufo viridis, which bore the following characters: grey or yellowish background, numerous spots, presence of a light dorsal line less wide and less regular than that of bufo calamita. not rare in piedmont especially in males (2). | this variety that, at first glance, could be mistaken for bufo calamita (3), and which has not been noticed by venetian naturalists, is quite common around us especially in young individuals. i found it in the same city of venice, and even last june, while fishing tadpoles of green toad, for scientific purposes, most of which had already completed their metamorphosis (1), i collected many young specimens (body length ca. 30 mm) with the line described by prof. lessona, and i could see other similar ones as well, at different times, in the provinces of padua and treviso. | it is doubtless to me that the specimens identified by nardo as bufo calamita (= var. calamita) belong instead to the variety indicated by myself; therefore, i think, it is so explained the incorrect appearance of bufo calamita within the lists of anurans of venetia.] (ninni, 1879, p. 970-971). 43nomenclature of italian bufo viridis form), he always referred to them as “forme” (forms) and/or “varietà” (varieties). this is the case, for instance, with tinca italica and t. chrysitis, which he claimed as simple varieties of tinca tinca (linnaeus, 1758) (as tinca vulgaris) due to the existence of uninterrupted series of intermediate forms (ninni, 1863); with a newly established variety of natrix natrix (linnaeus, 1758) (as tropidonotus natrix), which he named concolor but that he reported as connected to the “tipo” by an uninterrupted series of intermediate phenotypes and also did not differ from it in pholidosis or body shape (ninni, 1880); with some varieties of rattus rattus (linnaeus, 1758) (as mus rattus), including the newly named variety intermedius (ninni, 1882). he also used the term “varietà” while reporting single peculiar zoological specimens, as a single light-coloured specimen of marten (ninni, 1864) and a single large specimen of the bat nyctalus lasiopterus (schreber, 1780) (as vesperugo noctula) (ninni, 1883). in addition, criticism was always raised by ninni towards those authors who attempted to establish species or subspecies in absence of well supported evidences, as demonstrated by his argument with e. de betta. in fact, in the course of a lively academic debate on italian brown frogs (rana spp.), de betta (1885) proposed subspecific rank for rana agilis thomas, 1855 (= r. dalmatina fitzinger in bonaparte, 1838) and rana latastei boulenger, 1879, due to the supposed existence of many intermediate forms between them and rana temporaria linnaeus, 1758. to this argument ninni (1885) replied rejecting the subspecific rank for these two taxa, based on an explicit conceptual difference between subspecies and local or individual mutations. moreover, the following year, he published two other papers on amphibians: a short paper on “triton cristatus, laur. s.sp. karelinii” (= present-day triturus carnifex (laurenti, 1768), non triturus karelinii (strauch, 1870)) (ninni, 1886b), where the taxon was clearly indicated as subspecies, and a paper on venetian anurans (ninni, 1886a), where lineata was still regarded as a variety and was listed together with two other varieties of bufo viridis described by camerano (1883). following ninni’s argument about nardo’s misidentification, de betta (1883, 1885) acknowledged the existence of a striped form of green toad (as b. viridis), definitively rejecting the presence of b. calamita in venetia. meanwhile, camerano (1882) published a paper on the variability of rana esculenta linnaeus, 1758 and bufo viridis, where several varieties of the former species were described, including one named “lessonae”, now pelophylax lessonae (camerano, 1882). in the paper section on bufo viridis, camerano (1882) stated explicitly  that, after examining specimens from different italian regions (including piedmont, lombardy, venetia, tuscany, sicily, sardinia) and other areas in south-eastern europe and the middle east, he could not single out any defined subspecies6. consistently with his opinion on the lack of any taxonomically meaningful pattern of variation in green toads, while discussing colour pattern, he described four “sub-variétés” (sub-varieties), labelled “a” through “d”7. these 6 “le bufo viridis est, ainsi que la rana esculenta, très variable dans sa forme; mais s’après l’examen des exemplaires de plusieurs localités que j’ai pu observer in piémont, lombardie, vénétie, toscane, sicile, sardaigne (en italie), grèce, syrie, tiflis, erivan, je n’ai pu conclure à quelques séparations de sous-espèce un peu marquées.” [= bufo viridis is, as much as rana esculenta, very variable in shape, however, after the examination of specimens from several localities, that i could observe in piedmont, lombardy, tuscany, sicily, sardinia (within italy), greece, syria, georgia, armenia, i could not come to the distinction of any subspecies pronounced enough] (camerano, 1882, p. 690). 7 “la coloration dans cette espèce est très variable. je ne parle ici que de variétés qui proviennent des localités en dehors du piémont, car le professeur lessonna [sic], dans l’ouvrage que j’ai cité, a déjà traité des 44 n. novarini and l. bonato sub-varieties were referred only to sardinia (a and b) and the middle east (a through d), whereas for piedmont the author explicitly addressed the reader to lessona (1877), without further discussing the rest of italy. no one of the four sub-varieties was reported as bearing a dorsal-lined pattern. the same author, in his influential monograph of italian anuran amphibians (camerano, 1883)8, published a revised, more detailed treatment of the variability of green toads (as b. viridis), integrating the contributions of previous authors with his own observations. there, he finally described and named four “varietà”, all based on colour pattern only9. variétés du piémont. | sous-variété a. parties supérieures claires (animaux dans l’alcool) avec des taches plus ou moins nombreuses d’une couleur verdâtre sombre, arrondies, isolées; parties inférieures avec quelques taches brunes (perse, sardaigne). | sous-variété b. parties supérieures claires, taches olivâtres, sombres, évidentes et confluentes entre elles. un nombre plus ou moins grand de tubercules sur le dos; sur les flancs et sur les extrémités (sardaigne, syrie). | sous -variété c. parties supérieures d’une couleur brune, olivâtre obscur, taches très peu évidentes; parties inférieures avec des petites taches brunes (perse). | sous-variété d. parties supérieures brunes uniformément, taches tout à fait invisibles; parties inférieures sans taches (perse, erivan).” [= the colouration in this species is very variable. i do not report here about any variety but those coming from outside piedmont, because professor lessona, in the work i formerly quoted, treated already the varieties of piedmont. | sub-variety a – light upper sides (animals in alcohol) with more or less numerous spots of greenish colour, rounded and isolate; ventral side with some brown dot (persia, syria). | sub-variety b – light upper sides, olive spots, dark, evident and merging into each other. a more or less large number of warts on the back, the sides and the limbs (sardinia, syria). | sub-variety c – upper sides of a dark brown-olive colour, spots poorly discernible; underside with small brown spots (persia) | sub-variety d – upper sides even brown, spots completely invisible; lower parts without spots (persia, armenia)] (camerano, 1882, p. 692). 8 camerano’s “monografia degli anfibi anuri italiani”, first read at the academy of sciences of turin in 1882, is often quoted as published either in 1883 (e.g., de betta, 1885; ninni, 1886a; boulenger, 1898; sindaco et al., 2006) or in 1884 (e.g., gavetti and andreone, 1993; andreone and sindaco, 1999; bonato et al., 2007; razzetti, 2008; stöck et al., 2008a,b). this is because the work was published as both a stand-alone monograph in 1883 (page numbering: 1-100) and a journal article in 1884 (in the “memorie della reale accademia delle scienze di torino”, ser. ii, vol. xxxv: 187-287). camerano himself quotes this work as published in 1883 in several following papers (camerano, 1884, 1885, 1891), stating explicitly “uscita nel 1883” [= published in 1883] in one of them (camerano, 1884, p.  3 [= 1885, p.  405]: another paper published twice in subsequent years). in fact, the “memorie” were published usually at the end of each academic year (encompassing part of two calendar years), as a collection of the essays read at the academy during that academic year (occasionally including essays from previous ones), which, however, had been already printed individually at the time of the reading (e. borgi, pers. comm.). so, since “1883” appears the proper year of publication of the first available print of camerano’s anuran monograph, we recommend using this print when dealing with issues relevant for nomenclature and taxonomy. 9 “la colorazione del bufo viridis è molto variabile, e non ho trovato nessuna varietà locale ben caratterizzabile. [...] rispetto alla colorazione io credo si possano stabilire le seguenti varietà principali: || var. maculata. – parti superiori con macchie più o meno cospicue, più o meno numerose a contorni netti e di forma più o meno allungata o rotondeggiante; le macchie sono disposte irregolarmente. questa varietà è assai frequente in tutte le località italiane; talvolta alcune delle macchie del dorso si uniscono per qualche tratto e pare tendano a formare come delle fascie longitudinali (qualche individuo di modica-sicilia). || var. crucigera. | sin. bufo crucigera. eichw. zool. spec. ross. et polon., p. 167, 3 γ. | il colore fondamentale tende al rosso, le macchie della nuca si incontrano in modo da formare grossolanamente una croce di s. andrea. | questa varietà è rara in italia, io non l’ho osservata che in piemonte (1). || var. lineata. | sin. bufo viridis var. lineata. a. ninni. sulla supposta esistenza del bufo calamita nel veneto, ecc., atti inst. venet., ser. v, vol. v. | bufo viridis var. calamita (2). nardo, prospetti sistemat. degli anim. prov. ven., atti r. inst. venet., sor. iii, vol. v. p. 605. | bufo viridis var. f. lessona. anfibii del piemonte, atti acc. lincei, ser. iii, vol. i. | macchie delle parti superiori più o meno numerose, rotondeggianti e talvolta più o meno confluenti: una linea dorsale mediana longitudinale più o meno ampia e più o meno regolare va dall’apice del muso all’ano; talvolta questa linea è interrotta da qualche macchia rotondeggiante. | questa varietà che ha fatto credere a vari autori l’esistenza del vero bufo calamita in varie località italiane, si trova frequente in piemonte, nel veneto, in lombardia. gli esemplari più belli io li ho avuti da catania. || var. concolor. | sin. bufo viridis var. d. lessona, op. cit. | color fondamentale bruno grigio uniforme; nessuna macchia 45nomenclature of italian bufo viridis of these four varieties, only one was first established and named in this paper (camerano, 1883), the “var. maculata”, which was generically reported as very common everywhere in italy, the single locality of modica (sicily) being cited only as the origin of some specimens bearing a peculiar colour pattern. according to tortonese (1942), specimens from different italian localities (marcellise (venetia), florence, modica (sicily), sardinia and corsica) in the collections of the “reale museo zoologico” of turin were all attributed by camerano to the variety maculata, although elter (1982) and gavetti and andreone (1993) mentioned only three or four specimens labelled as “maculata” (all from sardinia). recently, however, the locality of modica has been regarded as the single locality from where the variety maculata had been described (balletto et al., 2007; frost, 2009), which appears unjustified. following the resemblance first noticed by lessona (1877), a second variety was identified by camerano (1883) with the “var. crucigera” of eichwald (1831), which the author observed only in piedmont and considered rare within italy. bufo variabilis var. crucigera was described by eichwald (1831) upon specimens from astrakhan (southern european russia) (see also: kuzmin, 1999; stöck et al., 2001); later on, eichwald himself cited it in a binomen within a simple list of taxa (eichwald, 1834, p.  31: “bufo cruciger, m.”; with the genus name oddly written in lower case), but in a further publication he reported it again unambiguously as a variety (eichwald, 1840, p.  127: “observavi astrachani varietatem crucigeram” in the main text, and “var. bufo cruciger” in an associated footnote). although several subsequent authors cited this variety in binominal form, as either “bufo crucigera” or “bufo cruciger” (e.g.: schreiber, 1875, 1912; lessona, 1877; camerano, 1883; bedriaga, 1890; nikolskii, 1918 [1963]), none seemed to foster in any way, explicitly or implicitly, species rank for this variety, crucigera being always reported explicitly as variety in the text, and as junior synonym of bufo viridis (as such or as bufo variabilis) in the synonymy lists. thus, occasional binominal arrangement for crucigera seems due to misreading or misinterpretation of eichwald’s (1831, 1834, 1840) works (see above) and not to any authors’ choice to properly treat this name at species rank. more recently, kuzmin (1999) listed it also among the synonyms of bufo viridis viridis laurenti, 1768, as “bufo cruciger eichwald, 1831” (page 251) and “bufo variabilis crucigera eichwald, 1831” (p. 255), a conclusion acknowledged by stöck et al. (2001). sul dorso, sui fianchi, sulle zampe anteriori e sulle coscie, poche e poco distinte sulle gambe posteriori. non ho osservato questa varietà, fino ad ora, che in piemonte.” [= the colouration of bufo viridis is very variable, and i did not find any well distinguishable local variety. [...] with respect to coloration, i think the following main varieties might be established: || var. maculata. – upper sides with spots more or less conspicuous, more or less numerous with distinct borders and more or less elongate or roundish in shape; spots are arranged irregularly. this variety is very common in all italian localities; at times, some dorsal spots become partially coalescent, so that they appear forming longitudinal bands (some specimens from modica-sicilia). || var. crucigera. | sin. bufo crucigera. eichw. zool. spec. ross. et polon., p. 167, 3 γ. | the background is reddish, the nuchal spots meet so as to shape roughly a st. andrew’s cross. | this variety is rare in italy, i could not observe it but in piedmont (1). || var. lineata. | sin. bufo viridis var. lineata. a. ninni. sulla supposta esistenza del bufo calamita nel veneto, etc., atti inst. venet., ser. v, vol. v. | bufo viridis var. calamita (2). nardo, prospetti sistemat. degli anim. prov. ven., atti r. inst. venet., sor. iii, vol. v. p. 605. | bufo viridis var. f. lessona. anfibii del piemonte, atti acc. lincei, ser. iii, vol. i. | spots of the dorsal parts more or less numerous, roundish and sometimes more or less coalescent: a mid-dorsal longitudinal line, more or less wide and more or less regular, runs from the tip of the snout to the anus; sometimes this line is broken off by some roundish spot. | this variety, which led some authors to believe the existence of the true bufo calamita in various italian localities, it is frequently found in piedmont, in venetia, in lombardy. i had the finest specimens from catania. || var. concolor. | sin. bufo viridis var. d. lessona, op. cit. | background uniformly grey-brown; no spots on the back, flanks, forelimbs and thighs, few and little marked [spots] on the hind limbs. so far, i could not find this variety but in piedmont.] (camerano, 1883, p. 49-50). 46 n. novarini and l. bonato the third variety recognized by camerano (1883) was the “var. lineata” of ninni (1879), which he reported as common in piedmont, venetia and lombardy, but evidently considered present also in other regions, as he explicitly stated to have received his finest specimens from catania (sicily). lastly, camerano (1883) newly introduced the name “var. concolor” for the variety “d” of lessona (1877) (doubtlessly the colour-based variety “d”, not the wart-based one), which he reported from piedmont only. likely due to the authorship of the description, bedriaga (1890) erroneously credited to lessona the name concolor. later on, nikolskii (1918 [1963]) cited this variety in a trinomen, “camerano’s bufo viridis concolor”, probably misquoting camerano (1883) and only to remark that it “is nothing but a specimen of the usual b. viridis without spots” (see below). according to camerano (1883, p.  18), all specimens listed in the paper, numbering more than 1200 individuals and including 59 green toads, were deposited in the italian vertebrates collection of the “r. museo zoologico” of turin. apparently, before the second world war, most of those green toads were still recognizable within the collection of italian amphibians and reptiles of the museum, as reported in a catalogue by tortonese (1942). unfortunately, the bombing of the museum in 1942, as well as some periods of missing cares, produced several damages to the fluid collections and many specimens were destroyed or had to be discarded (tortonese, 1957). today in fact, italian specimens of “bufo viridis” from the original collection of camerano do not seem to exist anymore in the present-day regional museum of natural sciences in turin, or either they cannot be confidently located, to the possible exception of some samples from sardinia (elter, 1982; gavetti and andreone, 1993; balletto et al., 2007; stöck et al., 2008b; f. andreone, pers. comm.). acknowledging the conclusions of camerano (1883), a subsequent check-list of venetian anurans compiled by ninni (1886a) mentioned three “varietà” of green toad (as b. viridis), namely maculuta [sic], lineata and concolor (thus extending to venetia the range of the latter variety). for the description of all anuran varieties reported in this paper, including lineata, the author explicitly referred the reader to the works of lessona and camerano (ninni, 1886a). during the 20th century, attention was paid only rarely to these varieties. most surprisingly, in a paper on the morphological variability of bufo viridis and other mediterranean toads, amazingly rich of morphometric data, camerano (1904) himself did not mention at all the four varieties he had described two decades earlier. in the second edition of his herpetologia europaea, however, schreiber (1912) reported a short diagnosis for the “typus” of bufo viridis, followed by four varieties, unnamed and listed in alphabetical order (“a” through “d”), each distinguished upon colour pattern or morphological traits10. particularly, varieties “a”, “b” and “c” were clearly derived from three of the varieties reported by camerano (1883), namely crucigera, lineata and concolor 10 typus: supra sordide grisescens vel albidus, maculis viridibus lemniscatis variegatus || var. a) maculis obscuris in cervicibus decussatim confluentibus. | bufo crucigera. eichw. zoo1. spec. ross. et polon. pag. 167, 3, γ (1831). || var. b) dorso linea vertebrali flavescente. | bufo viridis var. lineata ninni sulla susp.  esist. d. bufo calam. n. veneto. atti inst. venet. ser. v vol. v. – bufo viridis var. calamita nardo prospetti sistem. d. anim. prov. ven. atti inst. ven. ser. iii, vol. v, pag. 605. – bufo viridis var. f. lessona anf. d. piem. atti acc. lin. ser. iii, vol. i. || var. c) supra griseo-fuscescens, concolor. bufo viridis var. d. lessona l. c. – bufo viridis var. concolor camerano anf. an. ital. pag. 50 (1883). || var. d) membrana natatoria plantarum distinctissima, fere integra. bufo variabilis var. balearica. boettg. zoolog. anzeig. nr. 72 (1880). (schreiber, 1912; p. 218). 47nomenclature of italian bufo viridis (maculata, possibly identified with the “typus”, was not mentioned); varieties “b” and “c” were reported as exclusive of northern italy, whereas “a” was referred to the whole southeastern europe (schreiber, 1912; p.  220). the fourth variety (“d”) was identified with the “var. balearica” of boettger (1880). the same are also listed in the systematic synopsis at the end of the volume as “v. cruciger eichw.”, “v. lineatus ninni”, “v. concolor cam.” and “v. balearicus boettg.” (schreiber, 1912; p.  915), but are again recalled through alphabetical letters only in the next year’s supplement (schreiber, 1913; p. 12). the varieties of camerano (1883) were mentioned also by vandoni (1914), who erroneously credited all four to the first author, still as mere chromatic varieties and without making reference to their geographical distribution11. instead, a green toad specimen was reported by paolucci (1915) as “bufo maculata” in the catalogue of his herpetological collection (n.  7), now in the “museo paolucci” of offagna (ancona). however, the original jar label of this specimen reports “bufo viridis var. maculata | trasimeno” (v. caputo, pers. comm.), suggesting, together with other mistakes (e.g.: many species names are wrongly credited to linnaeus), that the name was reported erroneously in the paper by the author or the printer (paolucci, 1915). nikolskii (1918 [1963]), in his “comparative notes” on green toads12, stated explicitly that “there are no varieties of the green toad” and, among others, regarded “camerano’s bufo viridis concolor” as mere unspotted individuals of the proper b. viridis. the same author, however, seems somehow to acknowledge taxonomic status to “bufo viridis balearicus boettg.”, as based upon precise anatomical features. oddly, in the “notes”, the varieties just mentioned are reported as trinomia, whereas a third one (crucigera) is cited as “bufo crucigera”. the synonymy list of the same paper, however, reports the latter as “bufo viridis var. crucigera” and does not mention at all the other two varieties (nikolskii, 1918 [1963]; p. 73). later on, mertens and wermuth (1960) listed “1831 bufo variabilis var. crucigera eichwald” (terra typica: streets of the city of astrakhan), “1879 bufo viridis var. lineata 11 “esse possono essere distinte e isolate (var. maculata camer.), qualche volta confluenti, talora disposte in modo da formare sulla regione nuco-scapolare una croce (var. crucigera camer.) delle punteggiature rosse sono sparse sui fianchi e sulle cosce; tanto queste che le macchie verdi possono mancare od essere appena accennate (var. concolor camer.). [...] in certi individui si può notare una sottile striscia gialla decorrente lungo la colonna vertebrale (var. lineata camer.). questo carattere ha fatto credere a molti che gli individui italiani appartenessero alla specie calamita laur: questa in realtà è assai affine alla forma presente, ma in italia non è ancora stata rinvenuta.” [= they [the spots] may be distinct and isolated (var. maculata camer.), sometimes coalescent, sometimes arranged so as to shape a cross on the nuchal-scapular region (var. crucigera camer.). some red dots are scattered on the flanks and the thighs; these dots as well as the green spots can be absent or barely distinguishable (var. concolor camer.). [...] in some specimens a yellow narrow stripe can be seen running along the vertebral column (var. lineata camer.). this character induced many authors to believe that the italian specimens bearing it were belonging to the species calamita laur: actually, this [latter species] is very similar to the present form, but it has not been found yet in italy.] (vandoni, 1914, p. 73). 12 comparative notes. despite its wide distribution, there are no varieties of the green toad. it is true that some zoologists have tried to establish the existence of such varieties, but the only differences found in the specimens taken were in the colour. however, the colour of this toad varies to such a degree that one can hardly find even two specimens of the same colour. pallas, with good reason, called it bufo variabilis. under the name bufo crucigera eichwald describes specimens in which the spots of the back are shaped like a st. andrew’s cross. merrem* described specimens, with rose coloured spots on the back, which he termed bufo roseus. camerano’s** bufo viridis concolor is nothing but a specimen of the usual b. viridis without spots, while only b. viridis balearicus boettg.*** is clearly characterised in that the toes of the forelegs are clearly webbed. (nikolskii, 1918 [1963], p. 77). 48 n. novarini and l. bonato ninni” (terra typica: venetia) and “1883 bufo viridis var. concolor camerano” (terra typica: piedmont) only as synonyms of bufo viridis viridis; like schreiber (1912), they did not mention the var. maculata. more recently, paolucci et al. (1999) quoted the varieties maculata and concolor for abruzzo (central italy) from “altobello [1930]”, an unpublished catalogue of g. altobello’s amphibian collection (bruno and guacci, 1993; c. guacci, pers. comm.). then, inexplicably, after reporting literally camerano’s descriptions for the two above-mentioned varieties, the authors claimed camerano (1883) to have left a “nomenclatural gap” by not providing a name for the variety he had referred to both nardo’s “var. calamita” and lessona’s “var. f” 13. while not mentioning at all the name lineata of ninni (1879), clearly acknowledged by camerano (1883) for the dorsally striped variety, these authors, or more likely s. bruno alone (see also: razzetti and sindaco, 2006; razzetti, 2008), suggested instead the new name “var. nardoi”, dedicated to g.d. nardo. the status of this latter variety has been discussed by razzetti (2008), who recognized its unavailability. however, he erroneously related “nardoi” to bufo siculus stöck, sicilia, belfiore, buckley, lo brutto, lo valvo and arculeo, 2008, whereas the name was clearly proposed as a substitute for the italy-wide distributed variety lineata. lastly, stöck et al. (2006) identified significant molecular divergence between a taxon inhabiting the italian peninsula south of the po basin, a small part of sicily, corsica and 13 “a questo punto è forse doverosa la seguente precisazione per colmare un vuoto nomenclatoriale lasciato, inspiegabilmente, dallo zoologo piemontese [l. camerano, cit., 233-234, nota (2) di 233]: | “bufo viridis var. calamita (2). nardo, prospetti sistemat. degli anim. prov. ven., atti r. inst. venet., ser. iii, vol. v, p. 605. | “bufo viridis var. f. lessona, anfibii del piemonte, atti acc. lincei, ser. iii, vol. i. | “macchie delle parti superiori più o meno numerose, rotondeggianti e talvolta più o meno confluenti: una linea dorsale mediana longitudinale più o meno ampia e più o meno regolare va dall’apice del muso all’ano; talvolta questa linea è interrotta da qualche macchia rotondeggiante. | “questa varietà ha fatto credere a vari autori l’esistenza del vero bufo calamita in varie località italiane, si trova frequente in piemonte, nel veneto, in lombardia. gli esemplari più belli io li ho avuti da catania”. | inoltre, sempre nella nota (2), l. camerano precisa: “è molto probabile, come dice il ninni nell’opera sopra menzionata [“sulla supposta esistenza del bufo calamità nel veneto, ecc., atti inst. venet, ser. v, vol. v], che il nardo, abbia considerato come appartenente alla var. calamita (così allora si considerava il bufo calamita propriamente detto) gli esemplari del veneto aventi una linea dorsale longitudinale chiara. non possiamo, per ragioni di chiarezza, conservare alla varietà in questione del bufo viridis il nome del nardo”. | di conseguenza: dal momento che l. camerano ha chiamato concolor la var. d del lessona (*1877) perché, dopo le sue giuste riserve in nota (2), non ha dato una denominazione, nomenclatoriamente attendibile, anche alla var. f del lessona? a nostro avviso, fermo restando i presenti quali parametri della questione, riteniamo che il suo appellativo storiograficamente più appropriato possa essere quella di bufo viridis varietà nardoi.” [= at this point, perhaps, the following clarification is needed to fill a nomenclatural gap left, inexplicably, by the piedmontese zoologist [l. camerano, cit., 233-234, note (2) of 233]: | “bufo viridis var. calamita (2). nardo, prospetti sistemat. degli anim. prov. ven., atti r. inst. venet., ser. iii, vol. v, p.  605. | “bufo viridis var. f. lessona, anfibii del piemonte, atti acc. lincei, ser. iii, vol. i. | “spots of the dorsal parts more or less numerous, roundish and sometimes more or less coalescent: a mid-dorsal longitudinal line, more or less wide and more or less regular, runs from the tip of the snout to the anus; sometimes this line is broken off by some roundish spot. | “this variety led some authors to believe the existence of the true bufo calamita in various italian localities, it is found frequently in piedmont, in venetia, in lombardy. i had the finest specimens from catania.” | moreover, still in the note (2), l. camerano clarify: “it is very likely, as ninni says in the above mentioned publication [“sulla supposta esistenza del bufo calamita nel veneto, ecc., atti inst. venet, ser. v, vol. v”], that nardo has regarded the venetian specimens bearing a light longitudinal dorsal line as belonging to the var. calamita (so it was considered, at that time, the proper bufo calamita). for the sake of clarity, we cannot keep the name of nardo for the variety of bufo viridis in question”. | therefore: since l. camerano called concolor the variety d of lessona (*1877), why, after expressing his proper reservation in the note (2), did he not give a nomenclaturally reliable name to lessona’s variety f as well? in our view, we think that the name historiographically most appropriate could be that of bufo viridis variety nardoi.] (paolucci et al., 1999, p. 30). 49nomenclature of italian bufo viridis sardinia, as well as the balearic islands, and other green toads (as bufo viridis viridis), which were recognized only in north-east italy (padua and trieste) and further north and east in europe. the clustering of ne-italian specimens (not including samples from venice, however) with bufo viridis led stöck et al. (2006, 2008b) to discard the name lineata of ninni (1879) for the italian green toad, which they considered junior synonym of bufo viridis viridis laurenti, 1768. instead, since their samples from the rest of italy (to the exception of most of sicily) clustered with those from the balearic islands, they proposed for the italian species the name bufo balearicus boettger, 1880, from bufo viridis balearicus, the subspecies currently recognized in the balearic islands (pons and palmer, 1996; garcía-parís et al., 2004; pleguezuelos et al., 2004). this latter taxon, originally described by boettger (1880) as “bufo variabilis pall. var. balearica” upon specimens from mallorca and minorca, and for which a lectotype was selected by mertens (1967), was raised to subspecies rank by hemmer et al. (1981) on the basis of serological and bioacoustical results. however, since the latter authors did not provide a detailed taxonomic analysis and the validity of the variety had been questioned by vidal-celma (1965) and vidal (1966) on morphological ground, several subsequent authors regarded this subspecies as doubtful (e.g.: roth, in gasc et al., 2004; lanza et al., 2006; bologna and giacoma, in sindaco et al., 2006; balletto et al., 2007). it is worth noting, however, that the sample of “bufo viridis viridis” that vidal (1966) compared to his balearic specimens included also individuals from sardinia and venice. in addition to stöck et al. (2006, 2008b), strong phylogenetic affinities of the ssp.  balearicus with the green toads of corsica and sardinia were also reported by hemmer et al. (1981), who also proposed a bronze-age human introduction of green toads into the balearic islands from the tyrrhenian islands, and batista et al. (2006). about the same time, the name lineata was resurrected and elevated to species rank by balletto et al. (2007), as “bufo lineatus ninni, 1879”. this name was adopted by the authors following the claimed results of molecular analyses, so far unpublished, that revealed the occurrence in most of italy, including sardinia and corsica, of a taxon different from bufo viridis laurenti, 1768, inhabiting most of europe and that they restricted, within italy, to friuli-venezia giulia only (balletto et al., 2000, 2007). although balletto et al. (2007) did not specified whether or not the molecular evidences they quoted (“cervella et al., unpublished” and “lattes et al., unpublished”: p.  297, 300) included in fact specimens from venice or its surroundings, samples from the nearby island of pellestrina were used at least for some analyses (e. balletto, pers. comm.; also suggested by previous papers: castellano and giacoma, 1998; balletto et al., 2000). on a pure nomenclatural ground, balletto et al. (2007) apparently took for granted that the name lineata was available since its original introduction by ninni (1879), possibly due to its recurrent presence in the synonymy lists of many papers (e.g.: mertens and wermuth, 1960), as did likely lanza et al. (2007b) as well (but see also lanza et al., 2009: p.  394). in fact, they labelled the species name as “bufo lineatus ninni, 1879 status novus” (balletto et al., 2007, p.  299), did not provide any comment on its nomenclatural status and identified the main locality cited by ninni (1879) as the type locality of the taxon, although in generic terms14. despite the explicit indication of a type locality and the possible indication of a deposi14 “locus typicus: “vicino a venezia” (in museo civico di storia naturale di venezia).” [= type locality: “near venice” (in the civic museum of natural history of venice)] (balletto et al., 2007, p. 299). 50 n. novarini and l. bonato tory for the type material, however, balletto et al. (2007) did not make explicit reference to a holotype or syntypes, neither in general to ninni’s original specimens (that they did not check), so that their species account stands just as a taxonomical change of status and cannot be considered a valid description of a new taxon, according to the iczn. nomenclatural availability of names calamita – originally coined by laurenti (1768, pp.  27 and 119) as “bufo calamita”. a clearly available name under the provisions of the iczn, however coined for a species belonging to a fully different lineage and that entered the present context due to misidentification. crucigera – originally coined by eichwald (1831, p.  167) as “var. γ. crucigera” of bufo variabilis (formerly regarded as junior synonym of b. viridis, but see stöck et al., 2006, 2008a). available under the provisions of the iczn, as it was introduced following a binomen as a “variety”, the author did not expressly give it otherwise infrasubspecific rank, neither the content of the work unambiguously reveal infrasubspecific status (iczn: 45.6.4), and it was introduced accompanied by a description (iczn: 12.1). type material: not stated. type locality: the city of astrakhan, russia (eichwald, 1831, 1840; mertens and wermuth, 1960; kuzmin, 1999; frost, 2009). acutirostris – originally coined by lessona (1877, pp.  1087) as “forma acutirostris” of bufo viridis. not available under the provisions of the iczn, as it was introduced for an entity whose infrasubspecific status is unambiguously revealed by the content of the work (iczn: 45.5, 45.6.1, 45.6.4) and – at the best of our knowledge – it was neither adopted as the valid name of a species-group taxon or treated as a senior homonym before 1985 (iczn: 45.6.4.1). evidence for infrasubspecific status from the content of the original publication (lessona, 1877): i) acutirostris is not indicated as restricted to localities or areas; ii) acutirostris is distinguished based on a single character only, whereas different varieties are distinguished in the same species following two other fully independent classifications based on different, unrelated characters; iii) the same name acutirostris is used in many other species to distinguish a form based on the same character; iv) among the other species in which a homonymous form is distinguished, that form is sometimes associated to sex or growth stage, in no case clearly suggesting a possible subspecific status. obtusirostris – originally coined by lessona (1877, pp.  1087) as “forma obtusirostris” of bufo viridis. not available under the provisions of the iczn, as it was introduced for an entity whose infrasubspecific status is unambiguously revealed by the content of the work (iczn: 45.5, 45.6.1, 45.6.4) and – at the best of our knowledge – it was neither adopted as the valid name of a species-group taxon or treated as a senior homonym before 1985 (iczn: 45.6.4.1). evidence for infrasubspecific status from the content of the original publication (lessona, 1877): the same listed for acutirostris (see above). lineata – originally coined by ninni (1879, p.  973) as “varietà […] lineata” of bufo viridis. not available under the provisions of the iczn, as it was introduced for an entity whose infrasubspecific status is unambiguously revealed by the content of the work (iczn: 45.5, 45.6.1, 45.6.4) and – at the best of our knowledge – it was neither adopted as the valid name of a species-group taxon or treated as a senior homonym before 1985 (iczn: 45.6.4.1). evidence for infrasubspecific status from the content of the original publication 51nomenclature of italian bufo viridis (ninni, 1879): i) lineata was explicitly coined for one of the alternative chromatic phenotypes previously described by lessona (1877), who did not name them and treated them as merely infrasubspecific entities without any reasonable doubt; ii) no consideration of taxonomic relevance is made by ninni other than providing a name for lineata and extending its range to venetia; iii) lineata is not reported as restricted to localities or areas, neither to be present as the exclusive variety in some localities or areas; iv) lineata is reported with different frequency among different subsets of the population related to sex and growth stage, tadpoles being especially not reported as lineata. the elevation to species rank by balletto et al. (2007) did not confer availability to the name (iczn: 45.5.1, 16.1, recommendation 16a) neither it has to be taken as the introduction of an available new name, as it was not accompanied by the explicit fixation of a holotype or syntypes (iczn: 16.4.1, 72.3). balearica – originally coined by boettger (1880, p. 642) as “var. balearica” of bufo variabilis (formerly regarded as junior synonym of b. viridis, but see stöck et al., 2006, 2008a). available under the provisions of the iczn, as it has been introduced following a binomen as a “variety”, the author did not expressly give it otherwise infrasubspecific rank (other than as above), neither the content of the work unambiguously reveal infrasubspecific status (iczn: 45.6.4), and it has been established accompanied by a description (iczn: 12.1). moreover, should balearica be demonstrated as originally established for an infrasubspecific entity, it is nevertheless to be considered subspecific from its original publication as it has been adopted as a valid subspecies before 1985 (iczn: 45.6.4.1). the availability of balearica was already recognized by stöck et al. (2006, 2008b) and razzetti (2008). type material: lectotype: smf 3722 (formerly: 1297, 1a), paralectotype: smf 3726 (mertens, 1967; l. acker, pers. comm.), in the senckenberg forschungsinstitut und naturmuseum (frankfurt, germany). type locality: majorca and minorca, balearic islands (boettger, 1880), restricted to palma (majorca, balearic islands, spain) by lectotype designation (mertens, 1967). fig. 1. specimens of the green toad upon which ninni (1879) established the variety lineata, from the collections of the museum of natural history of venice (italy): (a) msnve-848, with label “b. viridis | v. lineata nin. | venezia vii 1879”; (b) msnve-952, with label “bufo viridis | var. lineata. venezia.” (photo: n. novarini). 52 n. novarini and l. bonato maculata – originally coined by camerano (1883, p.  49) as “var. maculata” of bufo viridis. not available under the provisions of the iczn, as it was introduced for an entity whose infrasubspecific status is unambiguously revealed by the content of the work (iczn: 45.5, 45.6.1, 45.6.4) and – at the best of our knowledge – it was neither adopted as the valid name of a species-group taxon or treated as a senior homonym before 1985 (iczn: 45.6.4.1). evidence for infrasubspecific status from the content of the original publication (camerano, 1883): i) the “varietà” is considered an infrasubspecific rank by the author, as clarified by the taxonomic treatment of other species, for which he distinguished between “sottospecie” (subspecies) and “varietà” (variety), sometimes clearly citing a variety hierarchically under a subspecies; ii) maculata is not reported as restricted to localities or areas, neither to be present as the exclusive variety in any locality or area. moreover, should maculata be deemed specific or subspecific from its original publication, it would become a primary junior homonym of bufo maculatus (hallowell 1854), being permanently invalid (iczn: 57.2). the unavailability of the name has been already recognized by stöck et al. (2008b, add. file 2) and registered by razzetti (2008). concolor – originally coined by camerano (1883, p.  50) as “var. concolor” of bufo viridis. not available under the provisions of the iczn, as it was introduced for an entity whose infrasubspecific status is unambiguously revealed by the content of the work (iczn: 45.5, 45.6.1, 45.6.4) and – at the best of our knowledge – it was neither adopted as the valid name of a species-group taxon or treated as a senior homonym before 1985 (iczn: 45.6.4.1). evidence for infrasubspecific status from the content of the original publication (camerano, 1883): i) the “varietà” is considered an infrasubspecific rank by the author, as clarified by the taxonomic treatment of other species, for which he distinguished between “sottospecie” (subspecies) and “varietà” (variety), sometimes explicitly citing a variety hierarchically under a subspecies; ii) concolor is explicitly considered identical to one of the alternative chromatic phenotypes previously described by lessona (1877), who did not name them and treated them as merely infrasubspecific entities without any reasonable doubt. the unavailability of the name concolor has been already recognized by stöck et al. (2008b, add. file 2). nardoi – originally introduced in paolucci et al. (1999, p.  30) as “varietà nardoi” of bufo viridis. not available under the provisions of the iczn, as it was introduced after 1960 for an infrasubspecific entity (iczn: 45.5, 45.6.3). the unavailability of the name has been already recognized and discussed by razzetti (2008). the original specimens of bufo viridis var. lineata ninni (1879) stated that he had found green toad specimens bearing a dorsal line inside the city of venice and, while collecting tadpoles in june 1879, apparently in the same place, he obtained some young metamorphosed individuals bearing the same pattern, about 3 cm long. upon these specimens he established the variety lineata. he also stated to have observed specimens of this variety in the provinces of padua and treviso as well, but did not mention any collecting from these localities. all the collected specimens belonging to this variety were donated by ninni himself to the civic museum of venice, as part of a larger herpetological collection including the above-mentioned tadpoles and a specimen of chelonia mydas caught near venice. 53nomenclature of italian bufo viridis the herpetological collection of a. p.  ninni still exists in the present museum of natural history of venice (msnve), which inherited the zoological collections of the former “museo civico e raccolta correr”. ninni’s collection, which was donated to that museum in several subsequent lots, was ordered and catalogued first by the contemporary naturalfig. 2. green toads (msnve-19319) from one of the extant populations inhabiting the city of venice, in the outdoor spaces of “the venice biennale” (specimens found dead near breeding site, april 2008) (photo: n. novarini). 54 n. novarini and l. bonato ist giuseppe scarpa of treviso, at the end of the 19th century, then by a. p.  ninni’s son emilio ninni, who reorganized his father’s collections after the foundation of the msnve in 1923, and possibly again by other museum technicians and curators at later times (levi-morenos, 1897; scarpa, 1897; anon., 1930; novarini, in press). in the present-day fluid collection of the museum, all what remains of the original material upon which ninni described the variety lineata can be confidently recognized in five specimens. these are all metamorphosed juveniles (total length = 26.9-30.3 mm), and are preserved in two small jars identified as msnve-848 and msnve-952 respectively. msnve-848 contains 3 specimens and is labelled “b. viridis | v. lineata nin. | venezia vii 1879” (fig. 1a), whereas msnve-952 contains 2 specimens and is labelled “bufo viridis | var. lineata. venezia” (fig. 1b). all five specimens are sufficiently well preserved to be fig. 3. specimen of green toad found in the small urban wood “bosco dell’osellino” in mestre, about 6 km nw of venice, in spring 2008 (photo: n. novarini). arrows point at a well visible, very thin dorsal line. 55nomenclature of italian bufo viridis unambiguously recognizable as green toads of the bufo viridis subgroup, although they are largely discoloured and their dorsal pattern is barely distinguishable. even though the two jars are not identified by any of the labels usually attached to ninni’s specimens (i.e.: bearing “raccolta a.p. ninni”, in print) and their labels have different origin, both jars can be assigned confidently to ninni’s herpetological collection, as the original material upon which the variety lineata was established, on the basis of the following evidences: i) after a comparison of the label handwritings with documents signed by a.p.  ninni held in the museum library, the label of msnve-848 (the most informative one, bearing also the date of collection) can be confidently attributed to a.p.  ninni, whereas the other label may have been compiled later by his son e. ninni; ii) both labels report explicitly the variety name “lineata”, whereas no other specimens currently present in the collections of the museum, at either adult, young or larval stage, bear the same name; iii) both labels report “venezia” as collection locality, which is consistent with ninni (1879); iv) the body length of all specimens is congruent with that reported by ninni (1879) for the specimens he collected; v) no other specimens compatible with the information given by ninni (1879) are present in the museum of venice. a minor discrepancy can be noticed, however, between the date of collection written on one of the jar labels (the other bearing no date), july 1879, and that reported by ninni (1879) in the paper, june 1879. both having been written by the collector himself, we cannot rule out that the apparent difference may be due to a lapsus calami in the label or to a typographical error in the paper. in a following paper, ninni (1886a) reported also the collection of tadpoles of b. viridis (unspecified variety), at different developmental stages, from the lido di venezia island on june 28th 1879. this might imply that the specimens mentioned in ninni (1879) may have been actually collected in the neighbouring island of lido, instead of venice, although it is far more probable that the author had been collecting in both islands (just a few kilometres apart) during that june. a few jars containing tadpoles of b. viridis, apparently from the 19th century, are actually present in the museum of venice but, bearing no data at all, they cannot be reliably identified as those collected by ninni in june 1879, neither they can be confidently assigned to ninni’s collection in general. it is also worth noting that no samples referred to b. viridis var. lineata are present in other collections to which a.p.  ninni is known, or suspected, to have contributed samples (novarini, in press), including the collection of g. scarpa in the “museo g. scarpa” of treviso (pers. obs.; also g. zanata, pers. comm.), despite the close friendship and documented exchanges of specimens between the two naturalists (scarpa, 1882; carraro, 1933), the collection of e. de betta in the “museo civico di storia naturale” in verona (maucci, 1971; r. salmaso, pers. comm.) and that of l. camerano in the “museo regionale di scienze naturali” in turin (tortonese, 1942; elter, 1982; gavetti and andreone, 1993; f. andreone, pers. comm.), both frequently engaged in sample exchanges with ninni as well, neither among the collections of the natural history museums of vienna (h. grillitsch, pers. comm.), pavia (e. razzetti, pers. comm.), genoa (doria et al., 2002), domodossola (largely contributed by camerano; andreone et al., 2005) and padua (b. centis, unpubl.). recently, the geographical provenance of the specimens upon which ninni established the variety lineata has been sometimes reported as “vicino a venezia” (near venice) (balletto et al., 2007; frost, 2009). the use of quotation marks seems to imply a literal cita56 n. novarini and l. bonato tion, possibly from ninni (1879) where, however, the author expressly indicated the city of venice, not its vicinity, as the collecting place, which is also confirmed by the jars labels of the putative voucher specimens. actually, the exact wording “vicino a venezia” does appear in the relevant footnote of ninni (1879), however, it is not referred to specimens of green toads but to the mentioned individual of chelonia mydas. at present, within the present-day historical centre of venice, at least two populations of green toads still survive, but at the end of the 19th century the city was less urbanized and the species may have been more widespread. the two extant populations are located one in the gardens of “the venice biennale” and the other in the nearby island of giudecca (novarini, 2005); venice itself being a system of islands, both populations appear fairly isolated. several other populations are present in adjacent islands, still within the present borders of the venice municipality, e.g.: sant’erasmo, murano, mazzorbetto, lido di venezia and pellestrina, as well as in the neighbouring mainland of mestre and marghera (novarini, 2005, 2006). mid-dorsally striped specimens can still be found in many of these populations, together with non-striped individuals. in general, when present, the stripe appears more often the result of the arrangement of the green spots, which tend to merge lengthwise into two paramedian bands leaving a light vertebral area in between (fig. 2), rather than a true stripe. nevertheless, some individuals actually show a poorly defined mid-dorsal stripe, sometimes just a very thin line (fig. 3), slightly lighter than the background, apparently unrelated to green spots arrangement and that appears more evident especially during the aquatic phase (pers. obs.). conclusions among the names so far applied to varieties, or other infrasubspecific entities, of green toads of the bufo viridis subgroup inhabiting the italian territory, our evaluation of relevant published works revealed that only crucigera, coined by eichwald (1831), and balearica coined by boettger (1880), are available names under the provisions of the iczn. however, crucigera eichwald, 1831, which was established for specimens from the northwestern coast of the caspian sea (type locality: the city of astrakhan) and was later applied by camerano (1883), at clearly infrasubspecific rank, to indicate an apparent chromatic phenotype of some italian populations, is now recognized as junior subjective synonym of bufo viridis laurenti, 1768 (kuzmin 1999, stöck et al. 2001, frost, 2009). instead, balearica boettger, 1880, which was already in use for the balearic subspecies of b. viridis (type locality: palma, majorca), has been recently identified as the valid name for the newly detected species b. balearicus (stöck et al., 2006, 2008a, b). conversely, the names acutirostris and obtusirostris introduced by lessona (1877), lineata introduced by ninni (1879), concolor and maculata introduced by camerano (1883) and nardoi introduced by paolucci et al. (1999) are all not available within the meaning of the iczn. therefore, irrespective of any taxonomic opinion, the recently proposed adoption of the name lineata of ninni (1879) for a newly recognized species appears inappropriate on a pure nomenclatural ground. 57nomenclature of italian bufo viridis acknowledgments we are deeply indebted with the following colleagues who discussed with us the nomenclatural issues presented above. we are especially grateful to a. minelli (università di padova and international commission of zoological nomenclature), e. razzetti (museo di storia naturale, università di pavia) and m. stöck (université de lausanne) for their thorough analysis and helpful comments on an earlier version of our manuscript. c. giacoma (università di torino), e. balletto (università di torino) and m.a. bologna (università roma tre) also discussed with us upon some of the issues addressed in this contribution. l. acker (senckenberg forschungsinstitut und naturmuseum, frankfurt am main), f. andreone (museo regionale di scienze naturali, torino), v. caputo (università politecnica delle marche, ancona), h. grillitsch (naturhistorisches museum wien), e. razzetti (museo di storia naturale, università di pavia), r. salmaso (museo civico di storia naturale, verona) and g. zanata (museo zoologico “g. scarpa”, treviso) kindly provided us with useful information about the herpetological collections in their institutions. e. borgi (accademia delle scienze, torino), c. guacci (baranello, campobasso) and g. masato (museo di storia naturale di venezia) provided valuable help with bibliographic information. finally, we are also grateful to the internet archive (http://www.archive.org/) and its contributing institutions, that proved a great source of ancient publications otherwise difficult to reach. references andreone, f., gavetti, e., volorio, p.  (2005): gli anfibi e i rettili del museo di storia naturale “g. g. galletti” di domodossola: catalogo sistematico con note storiche e riflessioni sul valore scientifico delle collezioni naturalistiche minori in italia. boll. mus. reg. sci. nat. torino 23: 343-379. andreone, f., sindaco, r. 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(2007): the amphibian tree of life [book review]. q. rev. biol. 82: 55-56. © firenze university press www.fupress.com/ah acta herpetologica 5(1): 113-117, 2010 first record of kemp’s ridley sea turtle, lepidochelys kempii (garman, 1880) (cheloniidae), from the italian waters (mediterranean sea) gianni insacco1, filippo spadola2 1centro regionale recupero fauna selvatica e tartarughe marine fondo siciliano per la natura e museo civico di storia naturale, via generale girlando 2, i-97013 comiso (rg), italy. e-mail: gianniinsacco @virgilio.it 2facoltà di medicina veterinaria di messina, dipartimento di scienze sperimentali e biotecnologie applicate, polo universitario ss. annunziata i, i-98128, messina, italy. corresponding author. e-mail: fspadola@unime.it submitted on: 2009, 2nd december; revised on 2010, 22nd january; accepted on 2010, 15th april. abstract. in this work the authors describe the first case of lepidochelys kempii stranding (garman, 1880) happened in italian waters (sicily, messina) and considered to be the fifth in the entire mediterranean sea. a young individual was recovered with a longline hook in its oesophageal. keywords. lepidochelys kempii, kemp’s ridley turtle, italian waters, mediterranean sea. lepidochelys kempii lives in tropical and subtropical seas in western atlantic ocean. in spite of its recent increase in population, it is still regarded as one of the most threatened sea turtle species worldwide (iucn, 2009; márquez, 2001; márquez et al., 2005; tewg, 2000). the nesting areas are almost entirely concentrated in a single location within the gulf of mexico, a few miles southward the tropic of cancer, and especially in the regions of tamaulipas and veracruz in mexico (heppell et al., 2005; márquez, 1994, 2001; márquez et al., 2005; oliver and pigno, 2005; tewg, 2000; tomás et al., 2003a). after the nesting, both adult and juvenile turtles usually head northwards, particularly for the north-western atlantic coast of the united states, considered to be the most important feeding and nursery area (heppell et al., 2005; márquez, 1994, 2001; márquez et al., 2005; tewg, 2000). incidental scattering of lepidochelys kempii recorded in north african and north europe coasts can be caused by the gulf stream system (gyory et al., 2008; márquez, 1994; márquez et al., 2005; pascual, 1985). lepidochelys kempii strandings records are extremely rare in the mediterranean sea. brongersma and carr (1983) made the first identification of kemp’s ridley turtle on a turtle 114 g. insacco and f. spadola that had been seized off the island of malta in october 1929 (brongersma and carr, 1983) (fig. 1, table 1). the second record of lepidochelys kempii in the mediterranean sea occurred in france at valras-plage (hérault) (oliver and pigno, 2005) (fig. 1, table 1). the capture of a kemp’s ridley turtle was then reported in tabarca (spain) in october 2001 between the island and the beaches of santa pola (alicante) (tomás et al., 2003b) (fig. 1, table 1). the fourth most recent record dates back to july 2006 and occurred in spain, nearby the turia river in the south of valencia harbour (tomás and raga, 2007) (fig. 1, table 1). on august 19, 2009 in capo peloro messina (italy) was found stranded sea turtles by the coast guard (38°16’00”n 15°39’08”e) (fig. 1, table 1). the turtle was taken into the regional wildlife recovery and sea turtles center of the sicily wildlife fund comiso (ragusa) for appropriate care. operators immediately noticed turtle phenotypic differences and concluded that it was a young subject of lepidochelys kempii after a careful analysis based upon biometric parameters (scl 27.8 cm; scw 25.2 cm; tscl 37.5 cm; hw 5.50 cm; weight 2.85 kg). it was the fifth record in the mediterranean sea and the first in fig. 1. locality records for the kemps’ ridley turtle. 115first record of kemp’s ridley sea turtle from the italian waters italy. the inspection evidenced: white bright green skin, triangular head with 2 pairs of prefrontal scales (fig. 2 b, c), front legs with 2 pairs of nails (fig. 2 d), the shell showed spherical shape (fig. 2 a) and consisted of 5 dorsal shields (fig. 2 a), 5 costal pairs (fig. 2 a), 12 marginal pairs (fig. 2 a), 4 inframarginal pairs (fig. 3) and 4 pairs of rathke scent glands pores for each inframarginal shield (fig. 3) (márquez, 1990). during the clinical examination, the kemp’s ridley turtle showed a 3 cm longline hook in the oesophagus. diagnosis suggested the execution of an esophagotomy in order to extract the foreign body. the operation was successful and the animal is finally on the way to recovery, waiting to be returned to wildlife. mediterranean records showed that it’s a juvenile (table 1) undoubtedly coming from the atlantic ocean, thereby confirming the theories related to the gulf stream system (márquez, 1994; gyory et al., 2008). we suspect, with tomás and raga (2007) that many fig. 2. lepidochelys kempii specimen (see text for explanation to figure). 116 g. insacco and f. spadola operators, particularly if non-experts, are likely to mistake young lepidochelys kempii turtles for young caretta caretta subjects, thereby distorting recent case histories in the mediterranean sea. a more significant presence of kemp’s ridley turtles in the mediterranean is therefore to be regarded as quite probable. acknowledgements special thanks to vincenzo inclimona, sonia terranova and simona cappello (crrfstm comiso), deborah ricciardi and adriana nunnari (m.a.n. messina) and the coast guard of messina for their collaboration during recovering, care and rehabilitation, and to adriana de pasquale (dip. scisba – unime) for linguistic advice. references brongersma, l.d., carr, a.f. (1983): lepidochelys kempii (garman) from malta. proc. konink. nederl. akad. wetenschap. ser.c 86: 445-454. gyory, j., mariano, a.j., ryan, e.h. (2008): the gulf stream ocean surface currents. http://oceancurrents.rsmas.miami.edu/atlantic/gulf-stream.html fig. 3. position of the rathke scent glands pores 117first record of kemp’s ridley sea turtle from the italian waters heppell, s.s., crouse, d.t., crowder, l.b., epperly, s.p., gabriel, w., henwood, t., márquez, m.r., thompson, n.b. (2005): a population model to estimate recovery time, population size, and management impacts on kemp’s ridley sea turtles. chelon. conserv. biol. 4: 767-773. iucn, international union for conservation of nature and natural resources (2009): the iucn red list of threatened species. http://www.iucnredlist.org/ márquez, r.m. (1990): fao species catalogue sea turtles of the world. fao fish. synop. 125: 38-43. márquez, r.m. (1994): synopsis of the biological data on the kemp’s ridley turtle lepidochelys kempi (garman 1880). noaa tech. memo. nmfs-sefcsc-343. márquez, m.r. (2001): status and distribution of the kemp’s ridley turtle, lepidochelys kempii, in the wider caribbean region. in: eckert, k.l., abreu grobois, f.a., eds, proceedings of the regional meeting: “marine turtle conservation in the wider caribbean region: a dialogue for effective regional management,” santo domingo, 16-18 november 1999. widecast, iucn-mtsg, wwf & unep-cep., pp. 51-46. márquez, m.r., burchfield, p.m., diaz, j., sanchez, m., carrasco, m., jimenez, c., leo, a., bravo, r., pena, j. (2005): status of the kemp’s ridley sea turtle, lepidochelys kempii. chelon. conserv. biol. 4: 761-766. oliver, g., pigno, a. (2005): première observation d’une tortue de kemp, lepidochelys kempii (garman, 1880), (reptilia, chelonii, cheloniidae) sur les côtes françaises de méditerranée. bull. soc. herp. fr. 116: 31-38. pascual, x. (1985): contributions on the study of sea turtles from the coasts of spain 1. distribution. mist. zool. 9: 287-294. tewg, turtle expert working group. (2000): assessment update for the kemp’s ridley and loggerhead sea turtle populations in the western north atlantic. u.s. dep. commer. noaa tech. mem. nmfs-sefsc-444, 115 pp. tomás, j., fernández, m., raga, j.a., (2003): sea turtles in spanish mediterranean waters: surprises in 2001. m.t.n. 101: 1-3. tomás, j., formia, a., aznar, f.j., raga, j.a. (2003b): a long journey: one kemp’s ridley sea turtle (lepidochelys kempii) visits the mediterranean sea. in seminoff, a.j. , ed, proceedings of the twenty-second annual symposium on sea turtle biology and conservation: 296-297. tomás, j., formia, a., fernández, m., raga, j.a. (2003a): occurrence and genetic analysis of a kemp’s ridley sea turtle (lepidochelys kempii) in the mediterranean sea. sci. mar. 67: 367-369. tomás, j., raga, j.a. (2007): occurrence of kemp’s ridley sea turtle (lepidochelys kempii) in the mediterranean. j.m.b.a. – biodiver. rec. 2: 1-3. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 7(2): 263-280, 2012 the usefulness of mesocosms for ecotoxicity testing with lacertid lizards maria josé amaral1,2,*, rita c. bicho1, miguel a. carretero2, juan c. sanchezhernandez3, augusto m.r. faustino4, amadeu m.v.m. soares1, reinier m. mann1,5 1cesam & departmento de biologia, universidade de aveiro, 3810-193 aveiro, portugal. *corresponding author. e-mail: mjamaral@ua.pt 2cibio – centro de investigação em biodiversidade e recursos genéticos, universidade do porto, campus agrário de vairão, 4485-661 vairão, portugal 3laboratorio de ecotoxicología, facultad de ciencias del medio ambiente y bioquímica, universidad de castilla-la mancha, avda. carlos iii, 45071 toledo, spain 4departamento de patologia e imunologia molecular, icbas, universidade do porto, 4050-123 porto, portugal 5centre for environmental sustainability, university of technology sydney, nsw 2007, australia submitted on 2012, 7th may; revised on 2012, 15th october; accepted on 2012, 22nd october. abstract. mesocosms (i.e., outdoor, man-made representations of natural ecosystems) have seldom been used to study the impact of contaminants on terrestrial ecosystems. however, mesocosms can be a useful tool to provide a link between field and laboratory studies. we exposed juvenile lacertid lizards for a period of over one year to pesticides (herbicides and insecticides) in mesocosm enclosures with the intention of validating field observations obtained in a previous study that examined the effects of corn pesticides in podarcis bocagei. our treatments replicated field conditions and consisted of a control, an herbicides only treatment (alachlor, terbuthylazine, mesotrione and glyphosate) and an herbicides and insecticide treatment (including chlorpyrifos). we used a multi-biomarker approach that examined parameters at an individual and sub-individual level, including growth, locomotor performance, standard metabolic rate, biomarkers of oxidative stress, esterases and liver histopathologies. although mortality over the course of the exposures was high (over 60%), surviving individuals prospered relatively well in the mesocosms and displayed a broad range of natural behaviours. the low numbers of replicate animals compromised many of the statistical comparisons, but in general, surviving lizards exposed to pesticides in mesocosm enclosures for over one year, thrived, and displayed few effects of pesticide exposure. despite the difficulties, this work acts as an important stepping-stone for future ecotoxicology studies using lizards. keywords. pesticides, reptiles, biomarkers, terrestrial ecotoxicology, alachlor, chlorpyrifos. 264 maria josé amaral et al. introduction laboratory toxicity testing using single species is by far the most commonly employed approach for establishing the toxicity of chemicals to both aquatic and terrestrial organisms. laboratory studies are relatively inexpensive and allow a high degree of control over various exposure parameters and interactions, like dose, test duration or temperature. however, their simplistic approach brings several limitations (reviewed by banks and stark, 1998). for example, terrestrial organisms such as lizards are not continuously in contact with the exposure media, and simulation of toxicant exposure in the laboratory that would simulate real-world conditions is a difficult task. at the other end of the broad spectrum of studies encompassed by the discipline of ecotoxicology, field studies are crucial to understand sitespecific questions. however, field studies are difficult to replicate and interpret because of the complexity and variability of natural habitats, involving multiple, frequently synergistic, causative factors (van den brink et al., 2005). furthermore, the effects of a contaminant in natural situations can be altered or masked by the presence of biotic (competition, predation) or abiotic (temperature, humidity, rainfall) factors, or by interaction with other contaminants (reviewed by banks and stark, 1998). in this context, mesocosms can provide a bridge between field studies and overly contrived laboratory manipulations, bringing both environmental realism and control, and serving as a validation tool for both types of studies. mesocosms have been defined as outside enclosures with a semi-controlled ecosystem that provide an environment that can be replicated, with some degree of realism (odum, 1984; van den brink et al., 2005). organisms are contained to ensure re-sampling but are subject to natural conditions, species interactions, etc. most importantly, contaminated and uncontaminated environments can exist in the same place, and experience exactly the same climatic and geochemical conditions. mesocosm enclosures have been used regularly in aquatic environments, for example to study the responses of amphibians to contaminants (rowe and dunson, 1994). in the terrestrial environment however, they have been used to a far lesser extent (boone and james, 2005). still, they can be useful to examine the effects of pesticides on terrestrial vertebrates because the entire terrestrial environment can be contaminated, thereby replicating a realistic exposure scenario. in the case of reptiles, mesocosms have rarely been employed to study the impact of contaminants (e.g., alexander et al., 2002; guirlet and das, 2012). however, european lacertid lizards have been proposed as potential model species for reptile ecotoxicology (e.g., mann et al., 2006; marsili et al., 2009) and are ideal candidates for mesocosm studies. two characteristics of lacertid lizards that make them well suited for mesocosm studies are their small size and small home-range. recently, we completed a field study that examined various population indices and biomarkers of pesticide exposure and effect among several sub-populations of podarcis bocagei living in field margins of corn pastures where several pesticides were annually applied, or within agricultural fields characterized by an absence of pesticide use (amaral et al., 2012a, b). these studies provided evidence for an adverse effect of pesticide exposure at the individual level, although lizard populations seemed to persist in the exposed fields despite evidence for toxic effects. the goal of the present study was to expose p. bocagei lizards to the same cocktail of pesticides found in our previous field surveys and thereby reinforce the findings of those studies. 265mesocosms in reptile ecotoxicology materials and methods animals podarcis bocagei is a small insectivorous lacertid endemic to the north-western iberian peninsula that occupies a variety of habitats, from wide open forests with brushwood to coastal dunes and agricultural areas (galán, 1986). hatchlings were reared from eggs laid in the lab by females collected in coastal dune system of mindelo (vila do conde) on the north-western coast of portugal during april-may 2009. gravid females were maintained in pairs in terraria (40 x 20 x 25 cm), in a climate room (22 ± 1 ºc) with food (mealworms, tenebrio molitor larvae) and water provided ad libitum. snout-vent length of each female was measured when they were brought into the lab. each terrarium contained a terracotta vase (diameter = 16 cm) that provided refuge and a basking location, and a 25  w incandescent lamp that created a thermal gradient in the terrarium (25–35 ºc; 8 h day-1). lighting was provided by natural sunlight, fluorescent lighting (2 x 40 w) and a high pressure sodium lamp (400 w, 7000-12 000 lx) for 12  h day-1. every night females were kept in individual boxes filled partially with clean commercial soil where they could lay eggs. the boxes containing newly laid eggs were placed in an incubator at 33  ºc until hatching. after hatching, juvenile lizards from the same clutch were kept together in similar conditions to those of females until the last hatchling attained one month of age. hatchlings were fed mealworms, isopods (porcellionides pruinosus) and fruit flies (drosophila melanogaster). hatchlings used for the experiment had an average age of 65.5 days (32-88 days, minimum and maximum) and size of 27.6 mm (snout-vent length, 23.7-32.8 mm, minimum and maximum). study design our experiment was conducted from september 2009 to november 2010 in the grounds of the university of aveiro (80 km south of the collection point), encompassing the first year of development of the hatchlings, including a hibernation period. in early 2009, we constructed 12 enclosures (100 x 150 x 100 cm), with four replicates per treatment: control (ctr), herbicides (herb), herbicides + insecticide (herb+ins). the mesocosms were constructed from transparent acrylic (4 mm) joined at each corner with stainless steel brackets, and buried 50 cm in soil to prevent lizard escape by burrowing (fig. 1a). the bottom of each enclosure was filled with a layer of sand and soil from the same location to ensure adequate drainage. this was then capped with an upper layer of commercial soil (siro® universal subtract). in the spring of 2009, turnip (brassica rapa) was planted in all mesocosms and spontaneous vegetation was allowed to emerge (fig. 1b). each mesocosm was furnished with three terracotta building bricks with multiple cavities to provide refuge; five plastic tubes dug into the soil (up to 0.15 m) to simulate burrows and a water and food dish. the buried tubes provided an insulated refuge to facilitate survival over winter. a net (mesh size 10 mm) covered each mesocosm to prevent the entrance of predators (e.g., birds, cats). we used the same pesticides as those routinely applied in the corn-growing regions of northwestern portugal (amaral et al., 2012b) and they were applied at the same application rates as recommended for corn agriculture. in the herb treatment, we applied four herbicides with a 2 l handheld sprayer: controller-t® (336  g l-1 alachlor and 144  g l-1 terbuthylazine sapec, 7  l ha-1), callisto® (100 g l-1 mesotrione syngenta, 1.5  l ha-1) and roundup® (360 g l-1 glyphosate monsanto, 4 l ha-1). in the herb+ins treatment, we applied ciclone 5g® in granular formulation (5% p/p chlorpyrifos sapec, 60kg ha-1) in addition to the herbicides. pesticides were applied during june of 2009 and april 2010 in all mesocosms (except ctr enclosures). three hatchlings, individually marked by toe clipping, were randomly assigned to each mesocosm and treatment group and introduced to the enclosures in september 2009 (day 0), approximately three months after the initial pesticide application in june 2009. animals were recovered from 266 maria josé amaral et al. the mesocosms at two interim time-points. the first was in march 2010 (day 190), one month before a second pesticide application, and the second in april 2010 (day 220) shortly after pesticide application. the experiment was concluded in october 2010 (day 413) (fig. 2). lizards could eat wild prey or mealworms provided twice a week. provided mealworms were placed during four days in a portion of soil collected from control or treated enclosures in day 0 or after pesticide application in 2010. at each of the two interim time points, lizards were assessed for growth and locomotor performance. at the end of the experiment, lizards were measured, rendered slightly torpid by cooling, sacrificed by decapitation, and dissected. blood was immediately collected with a micropipette from the exposed trunk, centrifuged for 10 min at 800 rpm and 4  ºc to obtain plasma, which was frozen in liquid nitrogen. intestine, two thirds of the liver and brain were similarly frozen in liquid nitrogen and stored at -80  ºc prior to biochemical assays. the remaining third of the liver was fixed in davidson´s solution for 24  h, washed in distilled water and stored in 70% ethanol until histopathological analysis. all procedures in this study complied with portuguese animal ethics guidelines as stipulated by direcção geral de veterinária and instituto da conservação da natureza e biodiversidade. pesticide concentrations in soil soil samples were analyzed for the applied pesticides on four occasions: at the beginning of the experiment, on days 190 and 220 and at the end. at the beginning of the experiment we collected one sample per enclosure. in the subsequent samplings we decided to collect three replicate soil samples per enclosure to account for variability within enclosures. the location of the sampling points in each enclosure was randomly selected from a 9 square grid. samples were preserved in clean, new, 200  ml glass vials, covered with parafilm® and sent to marchwood scientific services (southampton, uk) for quechers chemical analysis (anastassiades et al., 2003). growth and body size snout-vent length (svl) and body mass were measured (to the nearest 0.01  mm and 0.1 g, respectively) on four occasions: day 0, 190, 220 and 413. mean growth rates (svl; cm day-1) between each period were calculated for each treatment. fig. 1. a) scheme of the mesocosm. b) photo of one of the enclosures in spring 2010, before pesticide application.   267mesocosms in reptile ecotoxicology locomotor performance locomotor performance was evaluated twice, before and after pesticide application in 2010 (day 190 and 220) by measuring lizard maximum sprint speed on a cork substrate (2  ×  0.1  m sprint track). each lizard was fasted for 48  h before the start of the experiment, weighed, warmed to its optimal temperature of 33  ºc (amaral, unpublished data) and raced three times with at least one hour rest interval between trials. lizards were hand-chased through the track and trials were recorded with a video camera (sony/dcr-hc46 – 25 fps). following holem et al. (2008), mean maximum speed (mms) was calculated as the average of the fastest 0.10 m interval in each of the three replicate sprints, and maximum speed (ms) as the fastest 0.10 m interval in any of the trials. trials in which lizards did not run continuously were repeated. standard metabolic rate standard metabolic rate (smr), the metabolic rate of an inactive lizard, was measured at the end of the experiment (day 413). lizards were fasted for 2 days (water provided) before the measurements, as a post-absorptive metabolism is a prerequisite condition for smr measurements (bennet and dawson, 1976). lizards were placed in covered acrylic tubes (230 cm3), connected to 12 channels of a respirometer (analytical developments co. ltd, adc-225-mk3, hoddesdon, england) housed in a constant temperature room (22 ±  1 °c). an open flow system was employed with a flow rate of 250 ml min-1. flow rate and co2 production were recorded at 30 min intervals during the night, when the animals are inactive and assumed to be in a resting state. lizards were weighed and randomly assigned to one of two test groups (it was not possible to run all animals at the same time) and a test channel. the lowest 50% of measurements for each individual, corresponding to 18 sequential measurements of co2 production representing 9 h during the night were averaged and used to calculate the molar quantity of oxygen used by each individual lizard (assuming a respiration quotient of 1:1). a first run with empty channels and one empty channel during each run served to calibrate the apparatus. biomarkers of oxidative stress antioxidant enzymes were determined in brain, intestine and liver. glutathione s-transferase (gst, ec 2.5.1.18) activity was determined spectrophotometrically according to habig et al. (1974). specific activity was expressed as mu/mg protein using a millimolar extinction coefficient of 9.6 fig. 2. timeline (days) of study.   268 maria josé amaral et al. mm-1 cm-1. glutathione reductase (gr, ec 1.6.4.2) activity was measured following the method described in ramos-martinez et al. (1983). the decrease in absorbance at 340  nm due to nadph oxidation by reduced glutathione was measured for 1 min, and a millimolar extinction coefficient of -6.22 mm-1 cm-1 was used for the specific activity calculations. all kinetics were carried out at room temperature (20-22 ºc) and blanks (reaction mixture free of sample) were periodically checked for non-enzymatic reaction and enzyme activity was then corrected. protein concentrations were determined by the bradford method (bradford, 1976) using bovine serum albumin (bsa) as a standard. concentrations of reduced glutathione and oxidized glutathione were fluorimetrically determined according to the method of hissin and hilf (1976). reduced glutathione was determined by incubation of the deproteinized samples in the presence of 1  mg ml-1 ortho-phthalaldehyde (opa) in na-phosphate (0.1 m)ethylenediaminetetraacetic acid (edta, 5 mm) buffer (ph 8.0). determination of the oxidized glutathione concentrations required a preliminary incubation step with n-ethylmaleimide to prevent glutathione oxidation, and 1 n naoh was used instead of the phosphate-edta buffer. in both methods, the reaction mixture was incubated for 15 min at 20-22  ºc and the fluorescence was read at excitation wavelength (ex) =  350  nm and emission wavelength (em)  =  420 nm. quantification was performed using a set of external standards of reduced (3.27 to 327 nmol ml-1) and oxidized (1.62 to 82 nmol ml-1) glutathione, which were prepared in the same manner as the samples. lipid peroxidation was included as a measure of oxidative damage. the method described in ohkawa (1979) was used to estimate lipid peroxidation through the formation of thiobarbituric acid reactive substances (tbars). lipid peroxidation was expressed as nmol tbars mg protein-1 using a millimolar extinction coefficient of 156 mm-1 cm-1. esterases carboxylesterase (cbe) activity was measured in the intestine, liver and plasma using two substrates: α-naphthyl acetate (α-na) and the 4-nitrophenyl valerate (4-npv). hydrolysis of α-na was performed following the method by gomori and chessick (1953) as adapted by bunyan et al. (1968), by which the formation of α-naphthol occurs in a reaction medium that contains 25 mm tris-hcl (ph 7.6), 2 mm α-na and the sample. the hydrolytic reaction was stopped after 10 min by addition of 2.5% (w/v) sds and subsequently 0.1% fast red itr in 2.5% triton x-100. the solutions were allowed to stand for 30 min in the dark and the absorbance of the naphthol–fast red itr complex was read at 530 nm (ε=33.225×103 m−1 cm−1). hydrolysis of 4-npv by cbe activity was measured according the method by carr and chambers (1991). the reaction mixture contained 1 mm 4-npv, 50 mm tris–hcl (ph 7.5) and the sample. after 15 min, the reaction was stopped by adding a solution of 2% (w/v) sds and 2% (w/v) tris base. the 4-nitrophenolate liberated was read at 405 nm and quantified by a calibration curve (5–100 μm). cholinesterase activity was determined using the substrates acetylthiocholine iodide (acsch) and s-butyrylthiocholine iodide (busch) as described by ellman et al (1961). specific che activity was expressed as mu mg-1 of protein using a molar extinction coefficient of 14.15 x 103 m-1 cm-1 (eyer et al., 2003). liver histopathology samples were embedded in paraffin and 2 µm sections cut on a rotary microtome (leica rm 2035). tissues were stained with haematoxylin and eosin (h&e) and examined under a light microscope (olympus bx51) equipped with an olympus camera. liver sections were also stained with masson’s trichrome to assess liver fibrosis; with periodic acid-schiff (pas) for the presence of lipid or sugar accumulation, and with perls’ prussian blue to verify iron pigmentation (bancroft and stevens, 2002). one representative section per sample was scanned at 400x magnification. this gen269mesocosms in reptile ecotoxicology erally involved an examination of a minimum of 25 fields of view. the incidence of histopathological changes was classified according to a semi-quantitative scoring system: 0 – normal tissue, 1 changes in less than 50% of the section, 2 changes in more than 50% of the section. statistical analyses all randomizations were performed using the function rand of excel. the results obtained from the treatment groups were compared to the control using analysis of variance (anova), repeated measures analysis of variance or/and analysis of co-variance (ancova) followed by dunnett’s or duncan’s multiple comparison post-hoc tests. treatment was the dependent variable, and svl or body mass were used when appropriate as covariates to check for significant interactions. differences in the incidence of histological changes between treatments were compared with the control using fisher’s exact tests (zar, 1996). differences in svl were compared using student’s t-test or the non-parametric mann-whitney test when data were not normal. statistical analyses were performed using statistica 7 (hill and lewicki, 2006), for windows. values of p < 0.05 were considered significant. results and discussion lizard survivorship lizard survivorship was poor in all treatment groups. of the 36 original lizards, 30% did not survive the winter and less than 40% survived during the entire period, with similar levels of mortality occurring among all groups and mesocosms. mortality was not related to size of the lizards, both in the first interval (mann-whitney u = 312.5, p = 0.99, n = 36) and in the last interval (student’s t = -0.7, p = 0.46, n = 23). in the second interval, only two lizards did not survive. these were two of the smallest animals but an accurate statistical comparison is not possible because of the small sample size. high mortality rates after hibernation for oviparous species were described for several lizards (pike et al., 2008), including lacertids – 92% of males and 86% of females (civantos and forsman, 2000), and in particular among p. bocagei juveniles – 52% (galán, 1999, 2004). despite the high mortality rates, the remaining lizards prospered relatively well in the mesocosms, and displayed a broad range of natural behaviours. at the end of the exposure period, we recovered the following individuals from each treatment: ctr – 3; herb – 4; herb+ins – 6. the small number of individuals per treatment prevented us from using the enclosures as replicates and all the statistical analyses are based on individual responses. we were also unable to test sex-related variations in our dependent variables. nevertheless, as we were dealing with juveniles and sub-adults we can expect that sex differences will not be overly relevant. levels of pesticides in mesocosm soil active ingredients of the applied pesticides were found in varying concentrations during the experimental period (fig. 3). pesticide levels in control soil were always below 270 maria josé amaral et al. quantification limits. as the study involved manual spraying of small volumes of herbicides, some discrepancy between and within mesocosms was expected. at day 0, more than three months after the initial application of pesticides, there were residues of all applied fig. 3. average pesticide content of soils (µg kg soil-1) in the three treatment groups, ctr, herb and herb+ins. data points represent the mean of the mean values for each enclosure of the three replicate sampling points. error bars represent standard error). limit of detection 0.5 µg kg-1.   271mesocosms in reptile ecotoxicology herbicides in the herb and herb+ins mesocosms, with alachlor occurring at the highest concentration. in the herb+ins treatment there was also a high concentration of chlorpyrifos. at day 190, 10 months after the first pesticide application, all the concentrations had decreased and only alachlor and chlorpyrifos were detectable (in herb and herb+ins, respectively). half-lives for these products in soil are of 5-42 days for alachlor (mackay et al., 2006) and 30-60 days for chlorpyrifos incorporated in soil (racke et al., 1996). after the re-application at day 220, pesticide concentrations reached their maximum for the experiment, and again decreased by the end of the study (day 413), with the exception of alachlor in the herb+ins treated mesocosms. the high mean concentration for alachlor was influenced by a single replicate soil sample in one of the enclosures. these high variations between or within enclosures may be the result of slightly different microclimatic conditions inside each mesocosm (e.g., shading). the half-life of a chemical has been shown to depend on a number of factors, including climate and soil characteristics (gevao et al., 2000). in particular, soil microorganisms (bacteria and fungi) primarily degraded alachlor through conjugation with glutathione s-transferases (gst) (reviewed by sette et al., 2004). soil microorganism activity is dependent on soil temperatures; thus, it can be expected that if that area of the enclosure was shaded for longer periods, soil temperature was likely to be lower and the rate of alachlor degradation would be lower when compared with the other sampling points in the same enclosure or other mesocosms. we can expect that lizards in our mesocosm will be exposed to these products by dermal exposure and ingestion of contaminated food or soil. the turnover of insects and other invertebrates in the enclosures is expected to be high, which could decrease the rate of exposure through trophic transfer. however, the inclusion of pre-contaminated mealworms within the experimental design ensured that the lizards were exposed to some contaminated prey items. variation in life-traits galán (1997) demonstrated for p. bocagei that the size of the mother influences the number of eggs and hatchlings but not their size at hatching. our results also found no correlation between the size of the mother and the size of individual hatchlings at the beginning of the experiment. this, together with the random distribution of individuals in treatment groups guaranteed that variations in body size could not be attributed to variations at hatching, and more specifically, to the size of the mothers. lizard body size increased in all treatments, achieving sizes equivalent to those found in nature (galán, 1997; carretero et al., 2006). at the end of the experiment, all surviving individuals had attained adult size (fig. 4a). growth, expressed as change in body size, is an integrative process that can be related with several population level parameters and influence population structure and dynamics (gaston et al., 2001), and growth rate can be interpreted as an index of individual fitness. failure to grow and mature within a specified time would indicate that animals were unable to obtain and assimilate the resources necessary for maintenance as well as allocate energy for growth, storage of energetic reserves or reproduction (brown et al., 1992; civantos and forsman, 2000; mitchelmore et al., 2006). this would be especially critical before hibernation, as it is likely the main determinant of juvenile survival (civantos and forsman, 2000). 272 maria josé amaral et al. in the present study, individuals displayed a typical pattern of growth for lacertids, characterized by rapid growth during the juvenile period (dependent on environmental conditions) and decline with age, with the growth curve approaching an asymptote (stamps and andrews, 1992). growth-rates varied significantly with time and treatment (fig. 4b, repeated measures anova treatment x time: f4,20 = 4.9, p = 0.01; number of live animals in each sampling period: 13-36). as expected, growth rates were low in the first interval (september 2009 to march 2010), which encompassed the winter hibernation, increased in the period from march to april, and then decreased in the final interval (april to november 2010) when animals were reaching adult size. during march and april 2010, lizards in the herb+ins treatment grew significantly faster than ctr individuals (duncan’s pos-hoc p < 0.01, n = 23). the effects of pesticides on lizard growth have received little attention. in sceloporus lizards repeatedly exposed to malathion, no direct effects on growth were observed (holem et al., 2008). with regard to the lizards in our study, we cannot rule out the possibility that exposure to pesticides could have influenced growth, particularly in light of the increased oxygen requirements observed in field animals from areas exposed to pesticides (amaral et al., 2012a). however, we can also attribute this increase in growth to an indirect effect of the pesticides on plant cover. the herbicide application decreased plant cover in the enclosures, and we can speculate that prey fig. 4. a. mean snout-vent length (mm) of podarcis bocagei lizards raised in ctr, herb and herb+ins mesocosms. measures were taken when lizards were first introduced in the mesocosm (day 0), and at three subsequent sampling dates (day 190, 220, 433). mean ± standard error. the n value varied over time because of lizard mortality. b. mean growth rate (mm day-1) of p.  bocagei lizards raised in ctr, herb and herb+ins mesocosms over the three time intervals (days 0-190, 190-220, 220-433). n varied along the experiment: 36 – 25 – 23 – 13.   273mesocosms in reptile ecotoxicology items would be more visible to the lizards and/or that the lizards would have increased basking opportunities and may have reached optimal temperatures for hunting more quickly. the insecticide, on the other hand, could increase food availability for lizards by debilitating prey items. therefore animals could boost their feeding activity and thus increase their growth rate. sub-lethal effects mean maximum sprint speed (mms) and maximum sprint speed (ms) were correlated in the two intermediate periods (p < 0.001). therefore, we chose to use only ms in the subsequent analyses. maximum sprint speed did not vary significantly between treatments and was not affected by recent exposure to pesticides (repeated measures anova treatment x exposure timing: f2,20 = 0.09, p = 0.91; treatment: f2,20 = 2.5, p = 0.11; exposure timing: f1,20 = 0.2, p = 0.65, n = 25). ctr lizards achieved slightly higher velocities (march 1.8 ± 0.53 cm s-1; april 1.8 ± 0.98 cm s-1, mean ± sd), followed by herb+ins lizards (march 1.4 ± 0.42 cm s-1; april 1.6 ± 0.45 cm s-1, mean ± sd) and herb lizards (march 1.2 ± 0.43 cm s-1; april 1.3 ± 0.39 cm s-1, mean ± sd). similar locomotor performance assays were performed using lizards collected from pesticide exposed corn fields (amaral et al., 2012a), and among those animals we also did not detect any difference in locomotor performance after exposure to a cocktail of herbicides. in light of previous failures to correlate changes in locomotor performance with exposure to carbamate and organophosphorus insecticides (holem et al., 2006; durant et al., 2007), the data presented here and in our previous studies (amaral et al., 2012a, 2012c), suggest that these kinds of performance assays are not sufficiently sensitive to evaluate the consequences of pesticide exposure. in our previous field studies we demonstrated increased metabolic demand among individuals sampled from pesticide-treated fields. this was manifested as increased smr (amaral et al., 2012a). in the present study, we did not find a similar trend. at the end of the exposure period (5 months after the second pesticide application), lizards exposed to pesticides in mesocosm enclosures had similar smr to control animals when corrected for body mass (fig. 5, ancova treatment: f2,9 =  0.10, p  =  0.90, n = 13). however, these results might be masked by the number of individuals and by the pooling of genders. we measured biomarkers of oxidative stress following pesticide exposure (table 1). glutathione s-transferase (gst) activity in intestine differed significantly between ctr lizards and the pesticide treated lizards (anova treatment: f2,10 = 6.5, p = 0.02, n = 13). no differences were found in gst activity in the other tissues. gsts have an important role in contaminant clearing by conjugating contaminants or their metabolites with glutathione, creating a conjugate metabolite that can be readily excreted. therefore, we would expect an increase of gst activity in both of our treatments rather than the observed decrease. the mean concentration of reduced glutathione in the intestine of lizards in the herb treatment was significantly lower than in ctr individuals (dunnett’s post hoc test: p < 0.01). no statistically significant differences were found in the reduced glutathione content of brain and liver or for oxidized glutathione in any of the tissues. we would expect that a reduction in the reduced glutathione concentration would be accompanied by an increase of the oxidized glutathione, which did not occur. other parameters of oxidative stress such as glutathione reductase (gr) activity and lipid peroxidation showed no signif274 maria josé amaral et al. icant variation between ctr and the two pesticide treatments. pesticides have been shown to enhance the production of reactive oxygen species and, as a consequence, trigger the molecular and enzymatic mechanisms that counteract oxidative stress. alterations in glutathiones and/or in the activity of glutathione-related antioxidative enzymes have been regarded as an indication of oxidative stress (costantini and verhulst, 2009; koivula and eeva, 2010). the fact that none of the other variables measured showed any variation in this study, together with the results obtained in the field study seem to hamper their utility as biomarkers of contaminant exposure in reptiles. in a previous laboratory study, we found that lizards exposed to realistic oral doses of chlorpyrifos displayed reduced b-esterases activities (amaral et al., 2012c). cholinesterases and carboxylesterases are inhibited by organophosphorus and carbamate insecticides, and have been extensively used as biomarkers of pesticide exposure for aquatic and terrestrial organisms (sanchez-hernandez, 2007). there was no difference in cholinesterase activities between ctr lizards and pesticide exposed lizards, irrespective of the tissue type (table 1). lizards from both herb and herb+ins treatments showed lower intestinal carboxylesterase (cbe) activity (4-npv substrate) when compared to the control (dunnett’s post hoc test: p < 0.05). there were also differences in intestinal cbe when the α-na substrate was used, and in liver cbe with both substrates, but these differences were not significant because of the large variability within treatment groups, and likely related to the fig. 5. a. mean ln-transformed oxygen consumption rates (µmoles/hour) for podarcis bocagei lizards raised in ctr, herb and herb+ins mesocosms at the end of the exposure period plotted against ln-transformed body mass (g). all measures were taken from post-absorptive lizards at 22  ºc (n = 13). encircled points represent females. b. mean oxygen consumption rates for p.  bocagei lizards raised in ctr, herb and herb+ins mesocosm at the end of the exposure period. bars represent means + standard error (n = 13).   275mesocosms in reptile ecotoxicology ta bl e 1. m ea n (± s ta nd ar d de vi at io n) a ct iv iti es o f gl ut at hi on ede pe nd en t an tio xi da nt e nz ym es , g lu ta th io ne c on ce nt ra tio ns a nd a ct iv iti es o f es te ra se s in b ra in , in te st in e an d liv er o f p od ar ci s bo ca ge i f ro m c on tr ol ( c tr , n = 3 ), he rb ic id e tr ea tm en t ( h er b, n = 4 ) an d he rb ic id e+ in se ct ic id e tr ea tm en t ( h er b+ in s, n = 6) . bi om ar ke r br ai n in te st in e li ve r c tr h er b h er b+ in s c tr h er b h er b+ in s c tr h er b h er b+ in s g st ( m u m g pr ot ei n1 ) 18 .3 ±1 .9 19 .5 ±8 .0 35 .0 ±2 2. 6 68 .5 ±2 .8 37 .9 ±8 .0 * 49 .7 ±1 6. 2* 77 8. 4± 27 4. 5 35 9. 7± 16 0. 8 43 6. 9± 28 5. 5 g r ( m u m g pr ot ei n1 ) 4. 4± 1. 5 6. 0± 2. 4 3. 6± 1. 4 20 .9 ±1 0. 1 13 .3 ±9 .5 13 .3 ±5 .1 g sh ( nm ol m g pr ot ei n1 ) 20 .3 ±2 .3 23 .0 ±5 .5 39 .5 ±3 1. 2 2. 4± 0. 3 1. 7± 0. 3* 2. 5± 1. 1 18 .4 ±1 1. 2 11 .1 ±5 .5 13 .3 ±2 .3 g ss g ( nm ol m g pr ot ei n1 ) 5. 6± 0. 9 5. 8± 2. 1 10 .3 ±7 .8 2. 3± 0. 5 1. 6± 0. 02 2. 5± 1. 1 11 .8 ±5 .5 7. 4± 2. 4 7. 5± 4. 0 lp o ( nm ol t ba r s m g pr ot ei n1 ) 0. 4± 0. 03 0. 5± 0. 2 0. 8± 0. 4 0. 5± 0. 1 0. 5± 0. 2 0. 4± 0 .2 1. 5± 0. 6 0. 8± 0. 3 1. 1± 0. 2 c be α -n a( m u m g pr ot ei n1 ) 41 .7 ±1 1. 2 28 .8 ±1 3. 2 27 .7 ±9 .4 10 7. 5± 62 .3 67 .1 ±3 6. 1 74 .5 ±1 0. 4 c be 4 -n pv ( u m g pr ot ei n1 ) 40 .3 ±8 .7 20 .3 ±1 2. 9* 22 .5 ±7 .3 * 82 .6 ±5 7. 9 51 .0 ±1 3. 7 45 .6 ±1 6. 1 a c he ( m u m g pr ot ei n1 ) 53 .1 ±8 .3 51 .5 ±2 5. 9 51 .5 ±2 8. 9 g st – g lu ta th io ne s -t ra ns fe ra se ; g r – g lu ta th io ne r ed uc ta se ; g sh – r ed uc ed g lu ta th io ne ; g ss g – o xi di ze d gl ut at hi on e; lp o – li pi d pe ro xi da ti on ; t ba r s th io ba rb itu ri c ac id r ea ct iv e su bs ta nc es ; c be – c ar bo xy le st er as e; a c he – a ce ty lc ho lin es te ra se . *s ta tis tic al d iff er en ce b et w ee n tr ea te d an d co nt ro l a ni m al s (d un ne tt’ s po st h oc te st : p < 0 .0 5) . 276 maria josé amaral et al. low number of individuals. cbe are implicated in pesticide detoxification pathways and have been shown to be more sensitive than ches in several laboratory and field studies (wheelock et al., 2008). sanchez-hernandez et al. (2009) described higher basal levels of cbe in the anterior intestine of lumbricus terrestris earthworms compared to other tissues, and hypothesized that the alimentary canal could have an important role in pesticide detoxification, thereby reducing the uptake of pesticides. in laboratory experiment with chlorpyrifos, cbe also reacted more strongly than cholinesterases and were strongly inhibited in the gut (amaral et al., 2012c). liver mass did not change between individuals exposed or not exposed to pesticides (ancova treatment: f2,9 =  1.2, p  =  0.35, n = 13). there was no statistically significant difference in the occurrence of histological changes in individuals exposed to herb and herb+ins treatments when compared to ctr (fisher exact tests: p > 0.33). however, where those changes were evident, they were manifested as increased prevalence in the treated lizards. in general, individuals from herb and herb+ins treatment groups displayed higher accumulation of iron pigments, severe liver congestion and increased hepatocyte vacuolation and degeneration. these results, despite the lack of significant differences, suggest that pesticide exposed lizards are under stress, and tend to be in agreement fig. 6. representative liver section of podarcis bocagei lizard raised in herb treatment mesocosm, stained with periodic acid-schiff glycogen vacuoles (pas positive) are circled; the black arrow indicates round shaped lipid vacuoles and the round-headed black arrow represents other vacuoles.   277mesocosms in reptile ecotoxicology with observations in our previous field study: animals from locations where pesticides were in use, displayed increased hepatocyte degeneration and accumulation of iron pigments (amaral et al., 2012a). interestingly, and contrary to what was observed with field collected animals, hepatocyte vacuolation was more evident in animals from both pesticide treated groups. in the field study, increased hepatocyte vacuolation was related with an accumulation of lipids and glycogen (pas positive) in reference animals (amaral et al., 2012a). in the case of the mesocosm animals, there were three types of vacuoles, the pas positive glycogen vacuoles, the perfectly round shaped lipid vacuoles and translucent vacuoles that failed to stain for fat or glycogen (fig. 6). hepatocyte vacuolation is not characteristic of any particular disease and can be related with several disturbances, including toxicosis (myers and mcgavin, 2007). conclusions to our knowledge, this is the first published study that has used a mesocosm approach to examine the effects of agricultural pesticides in a reptile. the approach might be used in the future to validate field observations and provide a bridge between highly controlled laboratory studies and ecological studies. in our study, although there were high rates of mortality, surviving p. bocagei lizards that were either exposed or not exposed to pesticides in mesocosm enclosures for over one year thrived and reached adult size. minor differences in growth rates can be attributed to an indirect effect of the applied pesticides on vegetation coverage and food availability. our results are in general concordance with those obtained in our previous experiments with animals exposed in field conditions – lizards tend to show some individual symptoms of pesticide exposure, but in general seem able to compensate. however, the low number of individuals that survived in our study prevented robust data comparisons. therefore, low survival needs to be accounted for in future studies with lacertid lizards. a greater number of individuals in the beginning of the experiment are clearly necessary to compensate for mortality and also to compensate for the fact that sex might influence biomarker results. the main obstacle to achieving large numbers of animals in each mesocosm was the intensive effort required to locating and capturing adequate numbers of gravid females within a relatively short mating season, which would need to be surmounted in future experiments. despite the difficulties encountered, we believe mesocosms would provide a useful tool for ecotoxicology studies with lacertid lizards. acknowledgements we appreciate the assistance of a.r. agra for mesocosm construction and cibio members in animal collection. all animals were collected under a permit issued by the instituto da conservação da natureza e biodiversidade. this research was supported by feder through compete-programa operacional factores de competitividade, and by national funding through fct-fundação para a ciência e tecnologia, within the research project lab-pet – lacertid lizards as bioindicators of pesticide exposure and toxicity in intensive market garden agriculture (fct ptdc/ amb/64497/2006) and through a fct phd grant to m. j. amaral (sfrh/bd/31470/2006). 278 maria josé amaral et al. references alexander, g.j., horne, d., hanrahan, s.a. 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(1996): biostatistical analysis. prentice hall, upper saddle river, usa. acta herpetologica vol. 7, n. 2 december 2012 firenze university press advertisement call of species of the genus frostius cannatella 1986 (anura: bufonidae) flora a. juncá1, david l. röhr2, ricardo lourenço-de-moraes3, flávio j. m. santos1, airan s. protázio1, ednei a. mercês1, mirco solé4 amphibians in southern apennine: distribution, ecology and conservation notes in the “appennino lucano, val d’agri e lagonegrese” national park (southern italy). antonio romano1,*, remo bartolomei1, antonio luca conte1, egidio fulco2 the significance of using satellite imagery data only in ecological niche modelling of iberian herps neftalí sillero1, josé c. brito2, santiago martín-alfageme3, eduardo garcía-meléndez4, a.g. toxopeus5, andrew skidmore5 reproductive strategy of male and female eastern spiny lizards sceloporus spinosus (squamata: phrynosomatidae) from a region of the chihuahuan desert, méxico aurelio ramírez-bautista1,*, barry p. stephenson2, xóchitl hernández-ibarra1, uriel hernández-salinas1, raciel cruz-elizalde1, abraham lozano1, and geoffrey r. smith3 morphological variability of the hermann’s tortoise (testudo hermanni) in the central balkans katarina ljubisavljević1, georg džukić1, tanja d. vukov1, miloš l. kalezić1,2 the usefulness of mesocosms for ecotoxicity testing with lacertid lizards maria josé amaral1,2,*, rita c. bicho1, miguel a. carretero2, juan c. sanchez-hernandez3, augusto m. r. faustino4, amadeu m. v. m. soares1, reinier m. mann1,5 does acclimation at higher temperatures affect the locomotor performance of one of the southernmost reptiles in the world? jimena b. fernández*, nora r. ibargüengoytía advertisement call of scinax littoralis and s. angrensis (amphibia: anura: hylidae), with notes on the reproductive activity of s. littoralis michel v. garey1, thais r. n. costa2, andré m. x. de lima2, luís f. toledo3, marília t. hartmann4 book review: marine s. arakelyan, felix d. danielyan, claudia corti, roberto sindaco, alan e. leviton 2011. herpetofauna of armenia and nagorno-karabakh. society for the study of amphibians and reptiles stefano scali book review: rafaqat masroor 2012. a contribution to the herpetofauna of northern pakistan stefano scali evidence of high longevity in an island lacertid, teira dugesii (milne-edwards, 1829). first data on wild specimens. j. jesus first assessment of the endoparasitic nematode fauna of four psammophilous species of tropiduridae (squamata: iguania) endemic to north-eastern brazil markus lambertz1,*, tiana kohlsdorf2, steven f. perry1, robson waldemar ávila3, reinaldo josé da silva4 rediscovery and redescription of the holotype of lygosoma vittigerum (= lipinia vittigera) boulenger, 1894 yannick bucklitsch1, peter geissler1, timo hartmann1, giuliano doria2 , andré koch1,* reproductive phenology of the tomato frog, dyscophus antongili, in an urban pond of madagascar’s east coast ori segev1,*, franco andreone2, roberta pala2, giulia tessa2, miguel vences1 range extension of the critically endangered true poison-dart frog, phyllobates terribilis (anura: dendrobatidae), in western colombia roberto márquez1,*, germán corredor2, carlos galvis3 daniel góez2, & adolfo amézquita1 differences in habitat use of two sympatric species of ameiva in east costa rica esther sebastián-gonzález1, ramón gómez2 acta herpetologica journal of the societas herpetologica italica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 6(2): 237-245, 2011 a skeletochronological estimate of age in a population of the siberian wood frog, rana amurensis, from northeastern china wen bo liao1, 2 1 key laboratory of southwest china wildlife resources conservation (ministry of education), china west normal university, nanchong, 637009, p. r. china. e-mail: liaobo_0_0@126.com 2 institute of rare animals and plants, china west normal university, nanchong 637009, p. r. china. submitted on: 2011, 18th april; revised on: 2011, 16th september; accepted on: 2011, 23rd september. abstract. i used skeletochronology to estimate age structure of the siberian wood frog, rana amurensis, from northeastern china. average age did not differ significantly between males and females. age at sexual maturity in both males and females was 1 year. for both sexes the maximum age observed was 4 years. average body size differed significantly between the sexes, with males being larger than females. a significantly and positively correlation between body size and age was found within each sex in the population. when the effect of age was controlled, males also had larger size than females, suggesting that sexual selection for larger males might improve male mating success. keywords. siberian wood frog, skeletochronology, age, body size. introduction the siberian wood frog, rana amurensis, is widely distributed in west and east siberia, the russian far east, korea, northern and central mongolia, and northeastern china (terent’ev and chernov, 1965; kuzmin, 1999; zhao and adler, 1993). according to iucn’s evaluation r. amurensis cannot be regarded as a declining species (kuzmin et al., 2008). this species occurs most frequently in open, wet places such as wet meadows, swamps, overgrown shores of lakes, riverbanks, and open areas in forests with abundant vegetation and arboreal debris (zhao and zhao, 1994). breeding activity occurs in shallow lakes, ponds, large puddles and swamps with stagnant water (fei, 1999). hibernation mainly occurs from early october to april-may (zhao and adler, 1993). reproduction takes place from march-april (usually may elsewhere), whereas in cold northern areas the breeding season may extend until mid-july (fei, 1999). so far, information about demographic data on the species remains largely unknown. 238 wen bo liao seasonal climate variation affects the physiological activities of amphibians (i.e. bone growth) in temperate regions, resulting in formation of line of arrested growth (lag) (smirina, 1972). skeletochronology, i.e., recording the periods of arrested growth in long bones like the phalanges (castanet & smirina, 1990), is an effective tool and has been widely used to evaluate age and growth of amphibian populations in natural conditions. this method has also applied to many anurans species from widespread regions (temperate, hemelaar, 1988; miaud et al., 1999; lu et al., 2006; matthews and miaud, 2007; guarino and erismis, 2008; ma et al., 2009; tropical, khonsue et al., 2000; pancharatna and deshpande, 2003; subtropical, lai et al., 2005; liao and lu, 2010a, b, c; liao et al., 2011; li et al., 2010). here, i used skeletochronology to assess individual age, growth and longevity the siberian wood frog, rana amurensis, from northeastern china where these demographic parameters were unavailable. my aims were to compare age structure, body size and growth between males and females and gain insight into the mechanisms determining sexual size dimorphism. material and methods the population was located in a lake of shamajie town in zalantun city (47°58’n, 122°46’e), at an altitude of 300 m a.s.l., in inner mongolia autonomous region of northeastern china, where all individuals hibernated in holes at the bottom of the lake in groups. being in a temperate region, the study area has a strongly seasonal climate. monthly average temperature ranges between -25.7 °c and 22.8 °c. annual average temperature is 2.4 °c and annual total precipitation is 480 mm. the typical vegetation around the lake includes zhang child pine, pinus sylvestris, spruce, picea asperata, mountain willow, populus davidiana, yuehua tree, betula dahurica and black birch, b. pendula. all frogs were captured in holes of the lake from mid-december 2009 to early january 2010 when they were in hibernation. adult specimens were sexed by their secondary sexual characteristics (nuptial pads in male, evidently swollen belly due to egg masses in female). in the absence of nuptial pads and swollen belly individuals were classed as juveniles. body size was measured using a caliper to the nearest 0.1 mm. the third phalanx of longest toe of the right hind limb was clipped, and stored in 10% neutral buffered formalin for skeletochronology. in order to minimize disturbance of frogs during hibernation, we quickly collected data and released the individuals at the points of capture. each individual digit was cleaned of surrounding tissues of the phalanx, and then put in 5% nitric acid to decalcify for 48 h. decalcified digits were stained for 200 min in harris’s haematoxylin. subsequently, stained bones were dehydrated through successive ethanol stages of 70, 80, 95, and 100% for approximately 1 h in each concentration. phalanges were then processed for paraffin embedding in small blocks. cross-sections (13 μm) thick were obtained by means of rotary microtome, and the phalanx with the smallest medullar cavity was selected and mounted on glass slides. i observed the sections though a light microscope and photographed the best of them using a motic ba300 digital camera mounted on a moticam2006 light microscope at × 400 magnifications. the analysis of lines of arrested growth (lags) was performed by two persons (wb liao and zp mi) with previous experience of the technique. i confirmed the endosteal resorption of lags in this species based on the presence of the kastschenko line (kl; the interface between the endosteal and periosteal zones; rozenblut and ogielska 2005). sexual size dimorphism was described by sdi index (lovich and gibbons, 1992). the equation form was: sdi = (mean length of the larger sex/mean length of the smaller sex) ±1 (sdi = 0 when both sexes are of similar size, sdi > 0 when females are larger than males, sdi < 0 when males are larger than females). 239age and body size in rana amurensis i assessed interaction between age and sex using general linear models (glms) treating svl as a dependent variable, and age and sex as fixed factors. i used student’s t-test to compare differences in body size and age between males and females and kolmogorov-smirnov test to identify age structure between the sexes. the relationship between body size and age was analyzed using linear regression. i also conducted ancovas treating svl as a dependent variable, with age as covariate to see significance of differences in body size between sexes when the effects of age were controlled. all values given are shown as mean ± sd, and the level of significance was p < 0.05. results i captured 92 individuals (45 males, 46 females and 1 juvenile) in this population. of 91 adult specimens, all individuals exhibited clear lines of arrested growth (lags) in their cross sections phalanges (fig. 1). an endosteal resorption of lag that completely eroded the first (innermost) periosteal lag was observed in one female and one year was fig. 1. four hematoxylin-stained cross sections of the phalangeal bone (a: a 1-yr old male; b: a 2-yr old female; c: a 3-yr old female; d: a 4-yr old male) in adult wood frog, rana amurensis, from northeastern china. arrows indicate the lines of arrested growth (lag). kl represents kastschenko line, the interface between the endosteal and periosteal zones. scale bar: 150 μm. kl kl kl kl a b c d 240 wen bo liao added as her true age. very close space of hematoxylinophilic lines (double lags) and false lines were not observed. i found some individuals that had reached sexual maturity before entering hibernation in the year they metamorphosed. the smallest sexually mature males had a mean svl of 42.5 mm. the smallest gravid females had a mean svl of 39.5 mm. i found only one immature individual that was 1 year old and had svl of 29.8 mm, far away from the average minimum for sexually mature 1-year-old frogs (42.5 mm). adult age ranged from 1 to 4 years in both males and females (fig. 2). age distribution did not differ significantly between males and females (kolmogorov-smirnov test: d = 0.35, p = 1.00). average age did not differ significantly between males and females (males, 1.8 ± 1.0 yr; females, 1.6 ± 0.8 mm; student’s t-tests: t = 1.04, p = 0.30). there was significant difference in average body size between males and females, with males having larger size than females (males, 45.9 ± 5.8 mm; females, 41.5 ± 5.3 mm; student’s t-tests: t = 3.79, p < 0.001). the sexual dimorphism index (sdi) with body size was –0.106. there was also a significant difference in body size between females and males in both 1 and 2 age groups (table 1; all p < 0.05). the ancovas analysis revealed that the difference in body size between the sexes remained significant (f1, 91 = 18.28, r2 = 0.57, p < 0.001) when the effect of age was controlled (f1, 91 = 89.18, p < 0.001). average body size in r. amurensis males was 60.6 ± 2.9 mm and that of females was 67.7 ± 4.6 mm. linear regression analysis showed that significant relationships between age and body size were found within each sex (fig. 3; males, body size = 4.45 age + 38.12, f1, 44 = 49.92, r = 0.73, p < 0.001; females, body size = 4.61 age + 34.32, f1, 45 = 38.00, r = 0.68, p < 0.001). the ancova analysis revealed a non-significant interaction between sex and age (f1, 91 = 1.28, p = 0.21), and age-size relationships between the sexes do not differ in slope, suggesting that males and females have a similar growth pattern. fig. 2. adult age structure (male, open bars; female, close bars) in the siberian wood frog, rana amurensis, from northeastern china. 0 5 10 15 20 25 30 1 2 3 4 age (yr) nu m be r o f i nd ivi du al s 241age and body size in rana amurensis table 1. difference in body size (svl, mm) within each age group in the the siberian wood frog, rana amurensis, from northeastern china. values in descending order are mean ± sd, range and sample size. age class females males z p 1 39.5 ± 3.9 30.3-47.6 n = 27 42.5 ± 4.0 31.3-47.7 n = 24 2.83 0.005 2 41.7 ± 2.5 37.7-46.0 n = 13 46.9 ± 4.3 38.7-52.3 n = 11 2.75 0.006 3 48.8.0 ± 5.2 42.3-55.0 n = 5 52.0 ± 4.0 46.7-59.1 n = 7 0.73 0.47 4 58.4 n = 1 55.2 ± 3.9 51.2-58.9 n = 3 20 25 30 35 40 45 50 55 60 65 0 1 2 3 4 5 bo dy s ize (m m ) age (yr) fig. 3. the significant positively correlation between age and body size (male, open circles; female, close circles) in the siberian wood frog, rana amurensis, from northeastern china. discussion. skeletochronology has been widely used to estimate age in amphibians (castanet, 2002), and it can also estimate accurately age structure in r. amurensis. for temperate species, growth-marker formation is thought to be a result of seasonal climate changes because the changes will affect bone growth, resulting in an active growth period during the warm season followed by reduced or arrested bone growth during the cold sea242 wen bo liao son (smirina, 1972). the narrow hematoxylinophilic line, called line of arrested growth (lags) in bone sections of r. amurensis are likely formed as a consequence of its prolonged period of hibernation. age at maturity was estimated at 1 year in males and females, suggesting that individuals would spend more time in reproduction. this pattern is similar to the short-living anuran species (r. chensinensis, lu et al., 2006, r. epeirotica, tsiora and kyriakopoulou-sklavounou, 2002, r. nigromaculata, liao et al., 2010, r. limnocharis, liao et al., 2011, hyla annectans chuanxiensis, liao and lu, 2010b; bufo andrewsi, liao and lu, 2011a). however, some studies suggest that males reach sexual maturity one year earlier than females (r. temporaria, miaud et al., 1999, amolops mantzorum, liao and lu, 2010a, bufo andrewsi, liao, 2009). in the present study, the maximum age was estimated at 4 years within each sex. contrary to this, the maximum age was determined as 5-11 years old in other regions (kuzmin, 1999; zhao and adler, 1993; chen and lu, 2011). i found illegal collections actually existing from studied locality. moreover, mass mortality in hibernacula may occur in study population due to climate condition history (kuzmin and maslova, 2003). sexual size dimorphism is observed in amphibians (shine, 1979), and females are often larger than males in most anurans (monnet and cherry, 2002). in this study, i found that r. amurensis males have larger body size than females. this is in agreement with previous studies for difference in body size between sexes in the caudata triturus vulgaris (halliday and verrell, 1988), ommatotriton ophryticus (kutrup et al., 2005) and mertensiella caucasica (üzüm, 2009). several studies on anurans suggest that sexual size dimorphism results from differences in age structures and growth rates before attainment of maturity between the sexes (khonsue et al., 2001; monnet and cherry, 2002; lu et al., 2006). for r. amurensis, ancova revealed age being a major factor affecting sexual size dimorphism because of males having older age than females. however, growth rate did not affect sexual differences in body size due to a similar growth rate for both sexes. moreover, sexual selection for body size is one such factor where increased male body size is an important determinant of male mating success (üzüm, 2009). as in most anurans (ryser, 1996; lu et al., 2006; liao and lu, 2010a; liao et al., 2010; liao and lu, 2011b), the age of adult frogs is positively correlated with their size within each sex. however, in other anurans a positive correlation may be true for only one sex (gibbons and mccarthy, 1984; leclair and castanet, 1987; cherry and francillon, 1992). age, body size and growth rate changes with altitude or latitude in most anurans, individuals from high-altitude or latitude populations having later age at sexual mature, longer longevity, slower growth rate and larger body size than low-altitude or latitude populations due to lower temperature and smaller food availability in high altitude or latitude (miaud et al., 1999; lu et al., 2006; matthews and miaud, 2007; liao and lu, 2010a). for r. amurensis, age at sexual mature, longevity and body size will increase with increasing altitude or latitude (ishchenko, 1996; zhao and adler, 1993; kuzmin et al., 2008). chen and lu (2011) found that the maximum age of males and females at the 900-m population was 6 and 7 years, and 5 and 7 years at the 500-m population. this pattern suggests that lower temperature and smaller food availability from cold region may increase longevity and size. however, age at first reproduction in r. amurensis was 2 years for males and 3 years for females in both populations at different altitude. the finding may be related to small samplings in both populations. although this species has been threatened by general habitat loss (such as the construction of dams on large rivers) and the drainage and pollution of breeding pools 243age and body size in rana amurensis (kuzmin et al., 2008), no historical information is available about r. amurensis population in northeastern china. the 2-3 year as the dominant class in r. amurensis is consistent with that displayed by two populations in northeastern china (chen and lu, 2011). moreover, there is significant over harvesting of this species for food, especially in northeastern china where illegal collection has increased since the 2000s. in this population, i did not find older and larger individuals. therefore, conservation attention should be taken to restore the habitats and reduce human disturbance. this study provides the baseline information on demography in r. amurensis and will be useful for their conservation. acknowledgements i thank long jin, sang lin lou, bing yao chen and zhi ping mi for assistance during the filed and laboratory work. financial support is provided by the foundation of key laboratory of southwest china wildlife resources conservation (ministry of education), china west normal university, p. r. china (xnyb01-3), the scientific research foundation of china west normal university (09b001, 10a004) and national sciences foundation of china (31101366). references castanet j. 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(2009): a skeletochronological study of age, growth and longevity in a population of the caucasian salamander, mertensiella caucasica (waga 1876) (caudata: salamandridae) from turkey. north west. j. zool. 5: 74-84. zhao e., adler k. (1993): herpetology of china. society for the study of amphibians and reptiles, oxford, ohio. zhao e., zhao h. (1994): chinese herpetological literature: catalogue and indices. chengdu university of science and technology, chengdu. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 7(2): 325-329, 2012 rediscovery and redescription of the holotype of lygosoma vittigerum (= lipinia vittigera) boulenger, 1894 yannick bucklitsch1, peter geissler1, timo hartmann1, giuliano doria2 , andré koch1,* 1 zoologisches forschungsmuseum alexander koenig, section of herpetology & leibniz institute of terrestrial biodiversity research, adenauerallee 160, d-53113 bonn, germany. *corresponding author. e-mail: andrepascalkoch@web.de 2 museo civico di storia naturale “giacomo doria”, via brigata liguria 9, i-16121 genova, italy submitted on: 2012, 4th june, revised on 2012, 2nd october; accepted on 2012, 8th october. abstract. we report about the rediscovery of the holotype of the southeast asian striped skink lipinia vittigera and provide a detailed redescription together with photographs and drawings. the species was first described by george albert boulenger in 1894 as lygosoma vittigerum based on a specimen collected by elio modigliani on the island of sereinu (= sipura), west of sumatra. the original type specimen was considered to be lost for more than a century and was recently rediscovered in the museo civico di storia naturale “giacomo doria” (msng) in genova, italy. keywords. holotype, rediscovery, re-description, lipinia, scincidae, squamata, indonesia. in 1894, the little striped skink lipinia vittigera was described as lygosoma vittigerum by george albert boulenger (1858-1937) of the british museum of natural history based on a voucher specimen collected by elio modigliani (1860-1932), an italian anthropologist-zoologist, on the island of sereinu (= sipura), west of sumatra (boulenger, 1894; see also fig. 2). this conspicuous but rather rare lipinia is a widespread southeast asian species that ranges from southern myanmar through thailand and cambodia to vietnam and the malaysian peninsula, finally reaching sumatra, borneo, and the mentawai archipelago (das and austin, 2007; grossmann, 2010; grismer, 2011). currently, two subspecies of this mainly arboreal skink are recognized. these are the nominotypic l. vittigera vittigera inhabiting most of the species range and l. vittigera microcercum (boettger, 1901), which is restricted to southern vietnam, laos and thailand (smith, 1935; nguyen et al., 2009; teynié and david, 2010). the subspecific status of l. vittigera in cambodia is not yet documented (stuart, 2006; grismer et al., 2008). in addition, smith (1922) described the subspecies l. vittigera kronfanum from langbian plateau, “south annam”, today south326 yannick bucklitsch et al. ern vietnam, which turned out to be a synonym of l. vittigera microcercum (smith, 1935). however, das (2010) erroneously treated l. v. kronfanum as a valid subspecies. in a recent attempt to determine some lipinia skinks, one of us (ak) was looking for the original type specimen of l. vittigera. as most of modigliani’s collections are housed in the museo civico di storia naturale “giacomo doria” (msng) in genova, italy (van steenis-kruseman, 1950), ak consulted the type catalogue about reptiles published by capocaccia (1961). therein, however, no type specimen of l. vittigera was mentioned. likewise, an inquiry to the natural history museum (london) returned without success (c. mccarthy pers. comm.), but with the advice to contact the genova museum directly. finally, the missing holotype (msng 55855) of boulenger’s (1894) taxon could be located in the msng collection by gd after more than a century (see also das and greer, 2002). an earlier identification of the original type specimen was hampered, because when l. vittigera was described the holotype was not labelled and a catalogue number was not assigned to it. during a flood in 1970, that damaged many collections of the natural history museum in genova, the jar with the type specimen was broken. so only recently it was possible to associate some original labels (fig. 1b) with the type material (fig. 1a). beside the information of the original labels the rediscovered specimen itself (msng 55855) agrees with the original specimen designated as type by boulenger (1894) in the following morphological details: snout-vent length (svl) 37 mm (boulenger, 1894) vs. 36.9 mm (our measurements); head length from tip of snout to posterior margin of parietal (hl) 10 mm (boulenger, 1894) vs. 10.1 mm (our measurements); head width at the widest point of temporal region (hw) 5 mm (boulenger, 1894) vs. 4.8 mm (our measurements); largest part of tail absent. although no illustration of the original type specimen was provided by boulenger (1894), this individual combination of characters allowed us to assign the re-discovered specimen to the type description. in order to support future taxonomic investigations in the genus lipinia, we provide an extended redescription, photographs and drawings of the rediscovered holotype specimen. fig. 1. holotype of lipinia vittigera (boulenger, 1894), dorsal view (a) and original labels (b). photos by g. doria. 327rediscovery and redescription holotype lygosoma for the redescription the original holotype specimen of lygosoma vittigerum (msng 55855, figs. 1-2) was borrowed from the msng. the morphological investigations were performed using a detailed morphological protocol as used by nguyen et al. (2011). measurements were taken with a digital caliper to the nearest 0.1 mm. the following abbreviations are used: end = distance from anterior corner of eye to posterior border of nostril; el = eye length (distance between anterior and posterior corners of eyelid); fll = forelimb length (from anterior junction of forelimb and body wall to the tip of fourth finger, with the limb held at right angles to the body); hll = hind-limb length (anterior junction of hind limb and body wall to the tip of fourth toe, with the limb held at right angles to the body); hh = head height (at the point of maximum head height); sl = snout length (from anterior corner of eye to tip of snout); stl = distance from snout to anterior border of tympanum; sfll = snout–forelimb length (from tip of snout to anterior junction of forelimb and body wall, with the limb held at right angles to the body); tal = tail length; trunkl = trunk length (from posterior end of the forelimb to the anterior part of the hind-limb); tyd = maximum diameter of tympanum. scalation. paravertebral scales: number of scales in a line from posterior edge of parietals to dorsal point opposite posterior margin of the medial precloacals; ventral scale rows: number of scales from first gular to anterior margin of precloacals. bilateral scale counts are given as left/right. generic allocation of lipinia vittigera (boulenger, 1894) lygosoma vittigerum boulenger, 1894 was allocated to the genus lipinia by greer (1974). the genus lipinia belongs to the sphenomorphus group of lygosomine skinks fig. 2. holotype of lipinia vittigera (boulenger, 1894), head dorsal view (a, c) and head portray (b, d). photos by a. koch, drawings by t. hartmann. 328 yannick bucklitsch et al. (greer, 1979) and has the following derived characters: small body size (svl ≤ 58 mm); lower eyelid with a clear window; auricular lobules absent; body scales smooth; midbody scale rows ≤ 28; basal subdigital lamellae expanded; postorbital absent; vomers fused; pterygoid teeth absent; dorsal color pattern compromising a pale (rarely dark) mid-dorsal stripe at least anteriorly; visceral fat bodies absent; brood size two or one. the genus currently contains 27 different species and ranges from continental southeast asia and the philippines through the indo-australian archipelago to new guinea, the solomon islands and the republic of belau. re-description of the holotype of lipinia vittigera (boulenger, 1894) l. vittigera is a small skink (svl: 36.9 mm) and shows a slender appearance. the holotype has lost its tail, the right hemipenis is partly everted. measurements in mm: svl = 36.9; tal (largest part of tail missing) = 4.5; trunkl = 17.1; hl = 10.1; hw = 4.8; hh = 3.4; sl = 4.1; stl = 9.5; sfll = 14.7; end = 3.0; el = 1.5; tyd = 0.4; fll = 12.5. colour pattern: the holotype exhibits two dorsal dark brown stripes starting above the eye and one pale stripe starting from the tip of the nose. the flanks are dotted with brownish spots. the limbs show brown spots. dark spots are also visible at the side of the neck. the snout is obtuse from above but rather pointed at the lateral view. the rostral is wider than long. the prefrontals contact each other medially. the frontal is narrowing posterior. four supraoculars, first three in contact with frontal. the frontoparietals are in contact with each other, with the frontal, and with supraoculars three and four. the interparietal and parietals are distinct; parietals contact each other posteriorly; 3/4 nuchals. the nasal is in contact with the rostral and the first supralabial; the postnasal and the supranasal are not present; 2/2 loreals; two preoculars; one presubocular; seven supraciliaries; one primary temporal; two secondary temporals; seven supralabials, five and six below the eye; two postsupralabials; lower eyelid with a clear window; external ear opening present; 7/7 infralabials. the mental is rounded anteriorly, wider than long; the postmental is in contact with the first infralabial, first pair of chin shields, and anterior portion of second infralabial; three pairs of chin shields, first pair in contact medially; second pair separated by one scale, third pair separated by three scales; the chin shields are in contact with the infralabials; 30 midbody scale rows; 56 paravertebral scales, the dorsal scales are larger than the ventral scales; 58 ventral scale rows from first gular to anterior margin of precloacals; four precloacals; 15/16 subdigital lamellae on fourth finger, 25/25 subdigital lamellae on fourth toe. acknowledgements we are grateful to colin mccarthy (formerly bmnh) for information about lipinia type material under his care. in addition, we would like to thank two anonymous reviewers for their valuable comments on an earlier draft. 329rediscovery and redescription holotype lygosoma references boettger, o. (1901): aufzählung einer liste von reptilien und batrachiern aus annam. ber. senckenb. naturf. ges. 1901: 45-53. boulenger, g.a. (1894): a list of reptiles and batrachians collected by dr. e. modigliani on sereinu (sipora), mentawei islands. ann. mus. civ. st. nat. genova, series 2, 14: 613-618. capocaccia, l. (1961): catalogo dei tipi di rettili del museo civico di storia naturale di genova. ann. mus. civ. st. nat. genova 72: 86-111. das, i. (2010): a field guide to the reptiles of south-east asia. new holland publishers, london. das, i., austin, c.c. (2007): new species of lipinia (squamata: scincidae) from borneo, revealed by molecular and morphological data. j. herpetol. 41: 61-71. das, i., greer, a.e. (2002): lipinia nitens (peters, 1871): discovery of a second specimen and a rediscription of the holotype. raffles bull. zool. 50: 483-485. greer, a.e. (1974): the generic relationships of the scincid lizard genus leiolopisma and its relatives. austr. j. zool. (suppl.). 31: 1-67. greer, a.e. (1979): a phylogenetic subdivision of australian skinks. rec. austr. mus. 32: 339-371. grismer, l.l. (2011): lizards of peninsular malaysia, singapore and their adjacent archipelagos. edition chimaira, frankfurt. grismer, l.l., neang, t., thou, c., grismer, j.l. (2008): checklist of the amphibians and reptiles of the cardamom region of southwestern cambodia. camb. j. nat. hist. 1: 12-28. grossmann, w. (2010): lipinia vittigera (boulenger, 1894). sauria 32: 1-2. günther, a. (1873): notes on some reptiles and batrachians obtained by dr. adolf bernhard meyer in celebes and the philippine islands. proc. zool. soc. london 1873: 165-172. nguyen, v.s., ho, t.c., nguyen, q.t. (2009): herpetofauna of vietnam. edition chimaira, frankfurt. nguyen, t.q., schmitz, a., nguyen, t.t., orlov, n.l, böhme, w., ziegler, t. (2011): review of the genus sphenomorphus fitzinger, 1843 (squamata: sauria: scincidae) in vietnam, with description of a new species from northern vietnam and southern china and the first record of sphenomorphus mimicus taylor, 1962 from vietnam. j. herpetol. 45: 145-154. smith, m. a. (1922): notes on reptiles and batrachians from siam and indo-china. j. nat. hist. soc. siam 4: 203-214. smith, m.a. (1935): the fauna of british india, including ceylon and burma. reptiles and amphibia, vol. ii. sauria. taylor and francis, london. van steenis-kruseman, m.j. (1950): malaysian plant collectors and collections. flora malesiana 1: 1-639. stuart, b.l., sok, k., neang, t. (2006): a collection of amphibians and reptiles from hilly eastern cambodia. raffl. bull. zool. 54: 129-155. taylor, e.h. (1917): snakes and lizards known from negros, with descriptions of new species and subspecies. philipp. j. sci. 12: 353-381. teynié, a., david, p. (2010): voyages naturalists au laos. les reptiles. revoir editions, chamalières. acta herpetologica vol. 7, n. 2 december 2012 firenze university press advertisement call of species of the genus frostius cannatella 1986 (anura: bufonidae) flora a. juncá1, david l. röhr2, ricardo lourenço-de-moraes3, flávio j. m. santos1, airan s. protázio1, ednei a. mercês1, mirco solé4 amphibians in southern apennine: distribution, ecology and conservation notes in the “appennino lucano, val d’agri e lagonegrese” national park (southern italy). antonio romano1,*, remo bartolomei1, antonio luca conte1, egidio fulco2 the significance of using satellite imagery data only in ecological niche modelling of iberian herps neftalí sillero1, josé c. brito2, santiago martín-alfageme3, eduardo garcía-meléndez4, a.g. toxopeus5, andrew skidmore5 reproductive strategy of male and female eastern spiny lizards sceloporus spinosus (squamata: phrynosomatidae) from a region of the chihuahuan desert, méxico aurelio ramírez-bautista1,*, barry p. stephenson2, xóchitl hernández-ibarra1, uriel hernández-salinas1, raciel cruz-elizalde1, abraham lozano1, and geoffrey r. smith3 morphological variability of the hermann’s tortoise (testudo hermanni) in the central balkans katarina ljubisavljević1, georg džukić1, tanja d. vukov1, miloš l. kalezić1,2 the usefulness of mesocosms for ecotoxicity testing with lacertid lizards maria josé amaral1,2,*, rita c. bicho1, miguel a. carretero2, juan c. sanchez-hernandez3, augusto m. r. faustino4, amadeu m. v. m. soares1, reinier m. mann1,5 does acclimation at higher temperatures affect the locomotor performance of one of the southernmost reptiles in the world? jimena b. fernández*, nora r. ibargüengoytía advertisement call of scinax littoralis and s. angrensis (amphibia: anura: hylidae), with notes on the reproductive activity of s. littoralis michel v. garey1, thais r. n. costa2, andré m. x. de lima2, luís f. toledo3, marília t. hartmann4 book review: marine s. arakelyan, felix d. danielyan, claudia corti, roberto sindaco, alan e. leviton 2011. herpetofauna of armenia and nagorno-karabakh. society for the study of amphibians and reptiles stefano scali book review: rafaqat masroor 2012. a contribution to the herpetofauna of northern pakistan stefano scali evidence of high longevity in an island lacertid, teira dugesii (milne-edwards, 1829). first data on wild specimens. j. jesus first assessment of the endoparasitic nematode fauna of four psammophilous species of tropiduridae (squamata: iguania) endemic to north-eastern brazil markus lambertz1,*, tiana kohlsdorf2, steven f. perry1, robson waldemar ávila3, reinaldo josé da silva4 rediscovery and redescription of the holotype of lygosoma vittigerum (= lipinia vittigera) boulenger, 1894 yannick bucklitsch1, peter geissler1, timo hartmann1, giuliano doria2 , andré koch1,* reproductive phenology of the tomato frog, dyscophus antongili, in an urban pond of madagascar’s east coast ori segev1,*, franco andreone2, roberta pala2, giulia tessa2, miguel vences1 range extension of the critically endangered true poison-dart frog, phyllobates terribilis (anura: dendrobatidae), in western colombia roberto márquez1,*, germán corredor2, carlos galvis3 daniel góez2, & adolfo amézquita1 differences in habitat use of two sympatric species of ameiva in east costa rica esther sebastián-gonzález1, ramón gómez2 acta herpetologica journal of the societas herpetologica italica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 8(2): 153-157, 2013 reproductive output of the brown frog rana kukunoris at high altitude of the tibetan plateau wei chen1,*,#, li zhao1,#, ying wang2, hua li3, dujun he2, lina ren2, zhonghai tang2, xin lu4 1ecological security and protection key laboratory of sichuan province, mianyang normal university, mianyang, 621000, china. *corresponding author. e-mail: wchen1949@gmail.com. #co-first authors 2college of life science and biotechnology, mianyang normal university, mianyang, 621000, china 3management bureau of zoige wetland national nature reserve, zoige 624500, china 4department of zoology, college of life sciences, wuhan university, wuhan, 430072, china submitted on 2013, 24th april; revised on 2013, 31st october; accepted on 2013, 31st october. abstract. rana kukunoris is endemic to the eastern tibetan plateau and its breeding ecology was not known so far. in this study, we investigated the reproductive output patterns of the species during the breeding periods of 2012 and 2013. our results showed that clutch size and total clutch volume increased with maternal body size. however, no significant correlation between egg number and egg size was observed, which suggested that the tradeoff between offspring number and size in this species was lack. keywords. clutch size, egg size, clutch volume, rana kukunoris, trade-off, high altitude. offspring number and size can determine simultaneously the fitness of animals (bernardo, 1996; räsänen et al., 2005; marangoni and tejedo, 2008; marangoni et al., 2008). accordingly, female amphibians usually show a trade-off between offspring number and size when resources available for reproductive production are limited (jørgensen, 1981; berven, 1988; sinervo and licht, 1991; lips, 2001; lüddecke, 2002). given limited clutch holding capacity (jørgensen, 1981; berven, 1982; cummins, 1986; ryser, 1988; elmberg, 1991; donnelly, 1999; lüddecke, 2002; castellano et al., 2004), and fluctuating environments between years, female reproductive output is usually changeable (wells, 2007). suprisingly, studies on anurans reproductive output have focused on lowaltitude native species (morrison and hero, 2003; wells, 2007), virtually no data exist on the trade-off pattern of reproductive output in anurans from high-altitude zone, especially from tibetan plateau. rana kukunoris is endemic to the eastern tibetan plateau (chen et al., 2011), and the species is an explosive breeder with a pure capital breeding strategy, allocating to reproduction the reserves stored in previous years (lu et al., 2008; chen and lu, 2011; chen et al., 2011). female fecundity is known to be positively related to body size (lu et al., 2008) and males prefer to mate with larger females (chen and lu, 2011). the breeding biology of r. kukunoris has been generally described in some studies concerning the population age structure (li and li, 1991; chen et al., 2011), hibernation habitat use (liu and shi, 2000), seasonal patterns of body condition (dai et al., 2004), post-breeding habitat selection (dai et al., 2005) and prehibernation energy storage (chen et al., 2013b). little is known about r. kukunoris maternal investment, although the species seems to be a good model to study the tradeoffs in reproductive output of females at high altitudes due to large clutch sizes and no parental care (wells, 2007). in this study, we investigated in detail the breeding ecology of r. kukunoris, with the aim to investigate its maternal investment, such as the trade-off between egg number and egg size, and the influence of body size on clutch parameters. 154 wei chen et al. fieldwork was conducted in zoige county, sichuan from march to april 2012 and 2013. being r. kukunoris a capital breeder, the climatic patterns of the year preceding the reproduction are important, so we collected the climatic data of 2011 and 2012 at study site located 20 km away from city of zoige. annual average temperature of 2011 and 2012 at this site was 2.3°c and 2.5°c, respectively, and annual total precipitation was 702.8 mm and 686.6 mm (data from the weather station of zoige county, fig. 1). the study site (located at 103.004°e and 33.557°n; 3500 m a.s.l.) was a spawning pond with an area of 60 m2 and water depth of 10-40 cm. vegetation covering the pond consisted of short terrestrial and aquatic grasses. before the start of breeding season, we randomly hand-collected 50 frog pairs (26 pairs in 2012 and 24 pairs in 2013, table 1) at the spawning pond of r. kukunoris. almost 1,000 frogs come at this breeding site every year, therefore the probability of recapturing the same individuals in two consecutive years was very low. after capture, individuals were temporarily held in pails (40 cm in width × 60 cm in height) containing pond water and transported to the field laboratory (50 m away from the study site). after finishing their egg-laying, we measured snout-vent length of females with a ruler to the nearest 0.1cm. in addition, we also counted egg number and photographed the eggs (10-50 eggs per female) with a scale ruler using a canon camera with macro lens (eos 550) in order to measure egg size. all frogs and clutches were then released again into the spawning pond. in the laboratory, egg size (10-30 eggs randomly selected from the pictures) was measured to the nearest 0.01 mm, and averaged (minimum and maximum diameter for each egg) to obtain mean egg diameter. egg size was always measured at gosner stage 9-10 (gosner, 1960) and all measurements were obtained by the same person. clutch volume was defined by the cube of egg dimensions. pooled data from both years were analyzed by pearson correlation to explore the relationship between clutch size and egg size. to control for the effect of body size, partial correlation was performed with clutch size and egg size as dependent variables and body size as controlling variable. we then investigated the yearly variation in clutch parameters (clutch size, egg size and clutch volume) and the relationship between these parameters and body size by using general linear models (glms), with clutch parameter (clutch size, egg size or clutch volume) as dependent variable, year as fixed factor and body size as covariate. all variables were log10-transformed to approximate normality and enhance homogeneity of variances before the analysis. all statistical tests were performed with spss 16.0 statistical software. r. kukunoris breeding began when average daily temperature was above 0°c, and amplexing females began to lay egg on april 4, 2012 and march 17, 2013. the breeding period lasted about four weeks with a peak period of seven days. the number of eggs per clutch ranged from 160 to 1285 (n = 50), average egg diameters of different clutches from 1.72 mm to 1.88 mm (n = 50) and total clutch volume from 2516.1 mm3 to 4086.5 mm3 (table 1). there was no significant correlation between clutch size and egg size (r = –0.14, n = 50, p = 0.332, fig. 2). this held true even when the effect of body size was removed (partial correlation: r = –0.061, n = 47, p = 0.675). we found significant correlations between body size and clutch size as well as between body size and total clutch volume, but not for body size and egg size (table 2). interannual variation in the clutch parameters was low and did not show significant differences. no significant interactive effects between year and body size on parameters were found (table 2). generally, our results showed that large r. kukunoris lay larger clutches than smaller individuals, but egg size only exhibited little variation among the female individuals with different sizes. we also found low variation in clutch size, egg size and clutch volume between 2012 and 2013. fig. 1. monthly mean air temperatures and monthly mean rainfall of 2011 (hollow point) and 2012 (solid point) at the study site from which rana kukunoris were collected (data are from the weather station of zoige county). 155reproductive output of the high-altitude frog rana kukunoris life history theory emphasizes that animals should optimally partition limited resources between growth and reproduction throughout life (roff, 2002). so larger female amphibians have large clutch size and high clutch volume (duellman and trueb, 1986; roff, 1992; castellano et al., 2004; dziminski and alford, 2005). similar to the growth pattern of most amphibians, growth of female r. kukunoris fitted the von bertalanffy’s model. there was a rapid somatic growth for the earlier time and slower after sexual maturity (chen et al., 2011). this implied that more energy would be devoted to reproduction with increasing maternal age (jørgensen, 1992; czarnoleski and kozlowski, 1998), ultimately resulting in the body size-specific reproductive output. this also explains the significant differences in clutch size. large females often invest more energy into egg growth, so it is common pattern that there is a positive but weak correlation between egg size and body size of female in many amphibians (jørgensen, 1981; deullman and tureb, 1986; roff, 1992; castellano et al., 2004; dziminski and alford, 2005). in contradiction with this pattern, in r. kukunoris, large females did not lay larger eggs. our result was similar to those from several other investigators (tejado, 1992; castellano et al., 2004; dziminski and alford, 2005; chen et al., 2013a), who found that larger female allocated their main investment in larger clutch sizes rather than larger eggs (jørgensen, 1981). usually, a negative correlation between clutch size and egg size is a common pattern observed in amphibians (duellman and trueb, 1986), although this trade-off may exhibit considerable variation among different species (positive correlation: rana ridibunda kyriakopoulou-sklavounou and loumbourdis, 1990; negative: rana temporaria cummins, 1986; rana sylvatica berven, 1988; hyla labialis lüddecke, 2002, rana dalmatina and r. temporaria weddeling et al., 2005 and no correlation: bufo calamita tejado, 1992 ; bufo viridis castellano et al., 2004). our result showed there was no correlation between these two life-history traits of r. kukunoris. this can be explained by the fact that limited physical clutch holding capacity can’t increase simultaneously the clutch size and egg size (jørgensen, 1981) or that the frogs in the study population were below maximum physical constraint and egg size was mainly determined by factors such as energy availability during vitellogenesis (kaplan and salthe, 1979). previous studies from temperate-zone amphibians with capital breeding strategy showed that they exhibtable 1. year, samples size (n), means, standard deviations (sd) and minimum and maximum of body size, clutch size, egg size and clutch volume of rana kukunoris in a population from tibetan plateau. varables year n mean sd minimum maximum body size (cm) 2012 26 4.5 0.6 3.6 5.8 2013 24 5.0 0.5 4.0 5.9 clutch size 2012 26 481.4 179 160 870 2013 24 641.9 268 265 1285 egg size (mm) 2012 26 1.85 0.04 1.72 1.88 2013 24 1.85 0.03 1.79 1.88 clutch volume (mm3) 2012 26 1580.6 569.4 530.4 2516.1 2013 24 2118.6 857.8 862.8 4086.5 fig. 2. scatter plot of clutch size and egg size in rana kukunoris in a population from tibetan plateau. table 2. glms showing the effect of year, body size and interaction between year and body size on clutch size, egg size and clutch volume of rana kukunoris in a population from tibetan plateau. parameter df f p clutch size (n = 50) intercept 1 19.328 < 0.001 year 1 0.961 0.332 body size 1 28.420 < 0.001 year×body size 1 0.912 0.345 egg size (n = 50) intercept 1 365.640 < 0.001 year 1 3.208 0.080 body size 1 2.999 0.090 year×body size 1 2.976 0.091 clutch volume (n = 50) intercept 1 43.206 < 0.001 year 1 1.618 0.210 body size 1 25.653 < 0.001 year×body size 1 1.526 0.223 156 wei chen et al. ited year to year variation in both clutch size and egg size (cummins, 1986; berven, 1988; kaplan and king, 1997). dissimilar to this pattern, r. kukunoris with capital breeding strategy showed only low yearly variation in these two life-history traits during 2012 and 2013. this may be the result of very similar climate conditions (temperature and rainfall) in the preceding years 2011 and 2012, so food availability and the nutritional condition of females could be more or less similar. acknowledgements we thank dr. david shackleton for their comments on the early draft of the manuscript and thank dr. rocco tiberti for valuable suggestion to improve the manuscript. financial support was provided by the scientific research foundation of mianyang normal university (no. 2011a17 and no. qd2012a13), youth foundation of sichuan provincial department of education (no. 11zb138). all field and laboratory work was performed under licenses from the wildlife protection law of china. references bernardo, j. 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(2013b): do anurans living in higher altitudes have higher prehibernation energy storage? investigations from a high-altitude frog. herpetol. j. 23: 45-49. chen, w., yu, t.l., lu, x. (2011): age and body size of rana kukunoris, a high-elevation frog native to the tibetan plateau. herpetol. j. 21: 149-151. cummins, c.p. (1986): temporal and spatial variation in egg size and fecundity in rana temporaria. j. anim. ecol. 55: 303-316. czarnoleski, m., kozlowski, j. (1998): do bertalanffy’s growth curves result from optimal resource allocation. ecol. lett. 1: 5-7. dai, q., dai j.h., zhang, m., zhang, j.d., li, c., liu, b., liu, z.j., wang, y.z. (2005): terrestrial habitat selection of three amphibians (rana kukunoris, nanorana pleskei and bufo minshanicus) during summerautumn in zoige wetland national nature reserve. zool. res. sinica 26: 263-271. dai, q., zhang, m., li, c., zhang, j.d., wang, y.z. (2004): study on relative fatness of rana kukunoris in zoige wetland in sichuan. sichuan j. zool. 24: 351-354. donnelly, m.a. (1999): reproductive phenology of eleutherodactylus bransfordii in northeastern costa rica. j. herpetol. 33: 624-631. duellman, w.e., trueb, d.l. (1986): biology of amphibians. mcgraw-hill inc, new york. dziminski, m.a., alford, r.a. (2005): patterns and fitness consequences of intraclutch variation in egg provisioning in tropical australian frogs. oecologia 146: 98-109. elmberg, j. (1991): ovarian cyclicity and fecundity in boreal common frogs rana temporaria l. along a climatic gradient. funct. ecol. 5: 340-350. gosner, k.l (1960): a simplified table for staging anuran embryos and larvae with notes on identification. herpetologica 16: 183-190. jørgensen, c.b. (1981): ovarian cycle in a temperate zone frog, rana temporaria, with special reference to factors determining number and size of eggs. j. zool. 195: 449-458. jørgensen, c.b. (1992): growth and reproduction. in: environmental physiology of the amphibians. feder, m.e., burggren, w.w., eds, university chicago press, chicago, illionis. kaplan, r.h., king, e.g. (1997): egg size is a developmentally plastic trait: evidence from long-term studies in the frog bombina orientalis. herpetologica 53: 149-165. kaplan, r.h., salthe, s.n. (1979): the allometry of reproduction: an empirical view in salamanders. am. nat. 113: 671-689. kyriakopoulou-sklavounou, p., loumbourdis, n. (1990): annual ovarian cycle in the frog, rana ridibunda, in northern greece. j. herpetol. 24: 185-191. li, l.x., li, d.h. (1991): a preliminary analysis on the age composition of rana chensinensis kukunoris in alpine meadow, northern qinghai. alpine meadow ecosystems 3: 209-212. 157reproductive output of the high-altitude frog rana kukunoris lips, k.r. (2001): reproductive trade-offs and bet-hedging in haly calypsa, a neotropical treefrog. oecologia 128: 509-518. liu, h., shi, h.y. (2000): hibernating habitats of rana kukunoris in the zoige wetlands. sichuan j. zool. 19: 68-69. lu, x., zeng, x.h., du, b., nie, c. (2008): reproductive ecology of rana kukunoris nikolskii, 1918, a high-elevation frog native to the tibetan plateau. herpetozoa 21: 67-77. lüddecke, h. (2002). variation and trade-off in reproductive output of the andean frog hyla labialis. oecologia 130: 403-410. marangoni, f., tejedo, m., gomez-mestre, i. (2008). extreme reduction in body size and reproductive output associated with sandy substrates in two anuran species. amphibia-reptilia 29: 541-553. marangoni, f.,tejedo, m. (2008). variation in body size and metamorphic traits of iberian spadefoot toads over a short geographic distance. j. zool. 275: 97-105 morrison, c., hero, j.m. (2003): geographic variation in life-history characteristics of amphibians: a review. j. anim. ecol. 72: 270-279. räsänen, k., laurila, a., merilä, j. (2005): maternal investment in egg size: environmentand populationspecific effects on offspring performance. oecologia 142: 546-553. roff, d.a. (1992): the evolution of life histories. chapman and hall, new york. roff, d.a. (2002): life history evolution. sinauer associates, sunderland, ma. ryser, j. (1988): clutch parameters in a swiss population of rana temporaria. herpetol. j. 1: 310-311. sinervo, b., licht, p. (1991): proximate constraints on the evolution of egg size, number, and total clutch mass in lizards. science 252: 1300-1301. tejado, m. (1992): absence of the trade-off between the size and number of offspring in the natterjack toad (bufo calamita). oecologia 90: 294-296. weddeling, k., bosbach, g., hatchel, m., sander, u., schmidt, p., tarkhnishvili, d. (2005): egg size versus clutch size: variation and trade-offs in reproductive output of rana dalmatina and rana temporaria in a pond near bonn (germany). in: herpetologia petropolitana. proc. of the 12th ordinary general meeting of the societas europaea herpetologica, august 12–16, 2003, pp. 238-240. ananjeva, n., tsinenko, o., eds, societas europaea herpetologica, st. petersbourg. wells, k.d. (2007): the ecology and behavior of amphibians. the university of chicago press, chicago and london. acta herpetologica vol. 8, n. 1 june 2013 firenze university press journal of the societas herpetologica italica acta herpetologica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 6(2): 315-321, 2011 new distribution and genetic data extend the ranges of the spectacled salamanders, genus salamandrina, in the apulia region (south italy) cristiano liuzzi1, fabio mastropasqua1, daniele salvi2 1 societas herpetologica italica – sezione puglia, via polignano 36, 70014 conversano (ba), italy. 2 cibio, centro de investigação em biodiversidade e recursos genéticos, campus agrário de vairão, 4485-661 vairão, portugal. corresponding author. e-mail: danielesalvi.bio@gmail.com submitted on: 2011, 11th october; revised on: 2011, 24th october; accepted on: 2011, 24th october. abstract. additional data on the distribution of the genus salamandrina in the apulia region (southern italy) are provided. based on fieldwork carried out from may to august 2011 in two new localities, volturara appula (foggia province) and spinazzola (barletta province), the presence of salamandrina species was recorded. results from the genetic analyses of the 12s rrna gene fragment from six individuals demonstrated that s. perspicillata occurs in volturara appula while s. terdigitata in the spinazzola locality. the latter species is reported for the first time for the apulia region. these new distribution data represent considerable range extensions for the salamandrina species, indicating that more surveys are needed to complement the existing knowledge on their distribution as well as of the herpetofauna from the apulia region. the conservation implications of our findings are also discussed. keywords. salamandrina perspicillata, salamandrina terdigitata, distribution, 12s rrna, genetic diagnosis, apulia, italy. introduction the genus salamandrina, endemic to the italian peninsula, includes two vicariant species, the northern spectacled salamander s. perspicillata (savi, 1821) and the southern spectacled salamander s. terdigitata (bonnaterre, 1789). while the genetic distinction of the two species is obvious both at the mitochondrial (nascetti et al., 2005; mattoccia et al., 2005) and at the nuclear level (nascetti et al., 2005; canestrelli et al., 2006), no morphological traits allow a clear distinction between the two species (angelini et al., 2007). although some differences in size and coloration would allow a tentative distinction between adults of s. terdigitata and s. perspicillata (romano et al., 2009), it is not possible to identify neither young individuals nor larvae based exclusively on their morphology. 316 f. spadola and g. insaccoc. liuzzi, f. mastropasqua and d. salvi as a consequences of the difficulty of identifying the two species on the base of their morphology, the knowledge of their distribution ranges is currently still coarse in areas where the two species’ ranges meet. genetic data from the previously referenced published studies indicated that s. terdigitata occurs in the southern italian regions of calabria, basilicata and southern campania, while s. perspicillata is distributed from the northern districts of campania and apulia regions to the northern apennines (piemonte region). the sympatry between s. terdigitata and s. perspicillata has been recorded in a small area of the campania region for which recent genetic studies suggested the occurrence of hybridization between the two species (hauswaldt et al., 2011; mattoccia et al., 2011). another candidate area of sympatry between the two species could be in the northern part of the apulia region, which is quite close to the north-eastern limit of the s. terdigitata’s range, but where only one record of s. perspicillata is known (romano et al., 2009). with the aim of filling the gap of knowledge on the distribution of the genus salamandrina in the apulia region, we carried out an extensive field survey in two different districts at the border between the apulia region and the regions molise, campania and basilicata. the results of the genetic analyses carried out for the identification of the specimens found provided new distribution data for the genus salamandrina in the apulia region. materials and methods extensive field surveys were carried out in two geographical districts of the apulia region: the dauno sub-apennine, at the border with the campania region, and the north-western murgia, at the border with the basilicata region. using igm and orthophotos maps numerous springs and streams were identified and then inspected in areas potentially suitable for the salamandrina species. surveys were carried out between may and august at fortnightly intervals. we surveyed both larvae, by inspecting water bodies by means of nets, and adults, which were actively sought in the wooded areas adjacent to water. the urodele larvae found were collected, photographed, assigned to the genus salamandrina using the identification keys provided by lanza et al. (2007) and immediately released on site. all the equipment was disinfected after each sampling session, following the requirements of the conservation commission of the societas herpetologica italica (available at http://www-3.unipv.it/ webshi). the salamandrina individuals sampled were assigned either to s. perspicillata or to s. terdigitata based on genetic diagnoses. total genomic dna was extracted from tail tips from six specimens (four individuals from volturara appula and two from spinazzola), following the standard saline method (sambrook et al., 1989). a fragment of the mitochondrial 12s rrna gene was amplified by pcr using primers and conditions as in salvi et al. (2011). pcr products were purified and sequenced by an external service (macrogen korea). multiple 12s rrna sequences alignment was performed by clustal w (thompson et al., 1994) including ten sequences from s. perspicillata and s. terdigitata and one from lissotriton vulgaris downloaded from genbank (accession numbers reported in fig. 3). a maximum likelihood (ml) phylogenetic analysis was carried out in mega 5 (tamura et al., 2011) employing the tamura’s 3-parameter model of evolution (tamura, 1992) selected by mega 5 under the bayesian information criterion. node support (bp) was calculated over 1000 bootstraps replicates. 317incomplete albinism in discoglossus pictusnew ranges of the spectacled salamanders results field surveys the presence of salamandrina larvae was found at two sites about 100 km apart (fig. 1). in both cases, animal at a larval stage were found. adults were not encountered neither in aquatic nor in terrestrial environments. the first site where the animals were found is located in the sub-apennine, in the municipality of volturara appula (foggia province, fg) about 7 km from the border with campania region. spectacled salamanders larvae (developmental stage 42-43; harrison, 1969) were found in an artificial reservoir fed by a natural perennial spring located about 800 m asl, within a mixed mesophilous forest. the second site is located in the territory of spinazzola (barletta province, at). at this fig. 1. distribution ranges of the spectacled salamanders, genus salamandrina: light grey, range of the northern spectacled salamander s. perspicillata; dark grey, range of the southern spectacled salamander s. terdigitata. the boxed area is described in fig. 2. 0 250 km area described in fig. 2 salamandrina terdigitata salamandrina perspicillata regional administrative boundaries 318 f. spadola and g. insaccoc. liuzzi, f. mastropasqua and d. salvi site, the presence of salamandrina larvae (developmental stage 42-43; harrison, 1969) was recorded in a perennial stream in a narrow valley with a stand of turkey oak (quercus cerris) located at around 400 m asl. in this site larvae were found in water from early june through to the end of july, while during a survey on the 29th of august, no larvae were found, indicating that metamorphosis presumably takes place before the end of august. the temperature and ph of the water in the period investigated remained fairly stable, at respectively 15 ± 0.5 °c and 8.3 ± 0.2, respectively. additionally, at both sites the presence of both larvae and adults of rana italica was recorded. genetic analyses dna sequences of the 12s rrna gene fragment resulted in an alignment of 367 base pairs (bp) among which 58 sites were variable. sequences from the six larvae of salamandrina sampled (genbank accession numbers he610671-he610676) collapsed in two haplotypes, one present in salamanders from volturara appula and the other one occurring in salamanders from spinazzola. the sequence divergence (uncorrected p-distance) between these two haplotypes was equal to 3%. the maximum likelihood phylogenetic tree (fig. 3) clearly showed that spectacled salamander from volturara appula clustered with s. perspicillata, while those from spinazzola are included in the clade of s. terdigitata with high bootstrap support (bp ≥ 98). fig. 2. map of the study area. the new distribution sites of volturara appula and spinazzola are indicated by square and triangle symbols respectively. the putative boundary area between s. perspicillata and s. terdigitata is defined according to romano et al. (2009). boundary between s. perspicillata and s. terdigitata spinazzola volturara appula salamandrina sp. study area regional boundaries 0 25 km 319incomplete albinism in discoglossus pictusnew ranges of the spectacled salamanders discussion this study provided two new distribution records for the salamandrina genus in the apulia region. based on genetic diagnoses, the salamandrina population breeding at the volturara appula was assigned to s. perspicillata, while the population breeding at the site of spinazzola was identified as s. terdigitata. volturara appula is adjacent to the only known location for s. perspicillata in apulia, which is san marco la catola in foggia province (romano et al., 2009). on the other hand, the s. terdigitata population recorded in the site of spinazzola represents the first sighting fig. 3. maximum likelihood phylogenetic tree based on12s rrna sequences depicting the relationships between salamandrina haplotypes identified by previous studies (nascetti et al., 2005; mattoccia et al., 2005; zhang et al., 2008) and those from the six individuals (in bold) sampled in the volturara appula and spinazzola populations (apulia region, south italy). lissotriton vulgaris (genbank accession number tvu04704) was used as outgroup (not shown). bootstrap support values over 1000 replicates are indicated above the nodes. s. perspicillata (latina, ay898731) s. perspicillata (rome, ay898729 ) s. perspicillata (volturara-01) s. perspicillata (volturara-02) s. perspicillata (volturara-03) s. perspicillata (volturara-04) s. perspicillata (terni, ay898728) s. perspicillata (eu880332) s. perspicillata (tolfa, ay928620) s. perspicillata (genoa, ay898727) s. perspicillata (latina, ay898730) s. terdigitata (reggio calabria, ay898733) s. terdigitata (spinazzola-01) s. terdigitata (spinazzola-02) s. terdigitata (taverna, ay928621) s. terdigitata (potenza, ay898732) 100 98 0.01 320 f. spadola and g. insaccoc. liuzzi, f. mastropasqua and d. salvi of this species for the apulia region and is rather distant from any other known site where the genus salamandrina has been recorded. the closest known sites of s. terdigitata are two localities in the basilicata region about 45 km far from spinazzola: one near rionero in vulture and the other near accettura (potenza province, pz; romano et al., 2009). the nearest record of s. perspicillata relative to spinazzola is even further away of about 90 km (san bartolomeo in galdo, benevento province, bn, campania region; romano et al., 2009). based on the results of this study, the range of both s. perspicillata and s. terdigitata was extended eastwards in the apulia region. no evidence of sympatry between these two species was found based on our surveys. s. perspicillata is currently known to occur in two apulian localities, while the site of spinazzola represents the only known locality of s. terdigitata in the apulia region. this finding is worthy of attention, since the spinazzola site is discontinuous with any geographical and ecological areas potentially suitable for the species. indeed, with the exception of a few regimented channels, which dry out for most of the year, the surroundings of the site are dominated by cereal and forage crops. from the results of this preliminary study, the following conclusions can be drawn: i) it is plausible that the western border of the apulia region is the south-eastern outpost for both species of the genus salamandrina; ii) s. perspicillata and s. terdigitata have an allopatric distribution in their eastern range limit. forthcoming studies should focus on the northwestern district of apulia and on areas not surveyed in this study in order to conclusively validate the allopatry between these two species and to definitely define their eastern range limits. finally, knowledge of the fauna in the apulia region, such as that reported herein, is an important first step to ensure that these areas currently not subject to any kind of protection may enter into a functional network of protected areas. indeed, among other animals, many species of amphibians such as salamandrina, triturus carnifex, rana italica and bombina pachypus, which are identified by european community lists as deserving conservation priority, have in the apulia region the limit of their range. acknowledgements we thank antonio romano for his helpful suggestions to an early version of this manuscript. salamanders were captured under permits from the italian ministry of environment dpn/2009/0005106. ds is supported by the fct post-doctoral grant sfrh/bpd/66592/2009 and by the synthesys project http://www.synthesys.info/ which is financed by european community research infrastructure action under the fp7 capacities programme at the museo museo nacional de ciencias naturales of madrid (csic). references angelini, c., vanni, s., vignoli, l. 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(2005): the spectacled salamanders, salamandrina terdigitata (lacépède, 1788) and s. perspicillata (savi, 1821): genetic differentiation and evolutionary history. rend. fis. acc. lincei 16: 159-169. romano, a., mattoccia, m., marta, s., bogaerts, s., pasmans, f., sbordoni, v. (2009): distribution and morphological characterization of the endemic italian salamanders salamandrina perspicillata (savi, 1821) and s. terdigitata (bonnaterre, 1789) (caudata: salamandridae). ital. j. zool. 76: 422-432. salvi, d., harris, d.j., perera, a., bologna, m.a., carretero, m.a. (2011): preliminary survey on genetic variation within the pygmy algyroides, algyroides fitzingeri, across corsica and sardinia. amphibia-reptilia 32: 281-286. sambrook, j., fritsch, e.f., maniatis, t. (1989): molecular cloning: a laboratory manual, second ed. cold spring harbor press, new york. tamura, k. (1992): estimation of the number of nucleotide substitutions when there are strong transition-transversion and g + c-content biases. mol. biol. evol. 9: 678-687. tamura, k., peterson, d., peterson, n., stecher, g., nei, m., kumar, s. (2011): mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. molecular biology and evolution doi: 10.1093/molbev/msr121. thompson, j.d, higgins, d.g, gibson, t.j. (1994): clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. nucleic acids res. 22: 4673-4680. zhang, p., papenfuss, t.j., wake, m.h., qu, l., wake, d.b. (2008): phylogeny and biogeography of the family salamandridae (amphibia: caudata) inferred from complete mitochondrial genomes. mol. phylogenet. evol. 49: 586-597. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 7(2): 357, 2012 book review: rafaqat masroor 2012. a contribution to the herpetofauna of northern pakistan stefano scali museo civico di storia naturale di milano, corso venezia 55, i-2012 milano. e-mail: stefano.scali@ comune.milano.it “a contribution to the herpetofauna of northern pakistan”, written by rafaqat masroor (2012) is a contribution to the knowledge of amphibians and reptiles of a restricted area in northern pakistan. the authors is in charge of the herpetological collections of the pakistan museum of natural history in islamabad and he was trained by khalid javed baig, who founded the collections at the museum and was one of the most important herpetologists in pakistan. the book’s introduction briefly describes pakistan’s geography and macroecology and gives more detailed information about margalla hills national park, the area covered by the present study. the park encompasses about 174 km² in the northern surroundings of islamabad and is considered particularly interesting due to its biological diversity and its role as transitional area between two different zoogeographic regions. a checklist of the 42 species of amphibians and reptiles (belonging to 16 families) and identification keys to all the species complete the introductive part of the book. the special part describes all the known species, including english vernacular name, diagnostic features, description, habits, habitats, distribution, conservation and remarks. all the species accounts are accompanied by one or more color pictures and a color distribution map of the species in pakistan. a comprehensive table of habitats recorded for each species in the study area, a short chapter about conservation problems of herpetofauna in the margalla hills national park, a rich glossary, and a complete checklist of amphibians and reptiles of pakistan integrate book text. a total of 217 pages and 106 pictures, with more than 260 bibliographic references complete this book that gives an important contribution to the knowledge of an interesting, even if rather small, region. the book can be purchased at the cost of $ 45.00 at the publisher’s website (http://www.ssarherps.org/). acta herpetologica vol. 7, n. 2 december 2012 firenze university press advertisement call of species of the genus frostius cannatella 1986 (anura: bufonidae) flora a. juncá1, david l. röhr2, ricardo lourenço-de-moraes3, flávio j. m. santos1, airan s. protázio1, ednei a. mercês1, mirco solé4 amphibians in southern apennine: distribution, ecology and conservation notes in the “appennino lucano, val d’agri e lagonegrese” national park (southern italy). antonio romano1,*, remo bartolomei1, antonio luca conte1, egidio fulco2 the significance of using satellite imagery data only in ecological niche modelling of iberian herps neftalí sillero1, josé c. brito2, santiago martín-alfageme3, eduardo garcía-meléndez4, a.g. toxopeus5, andrew skidmore5 reproductive strategy of male and female eastern spiny lizards sceloporus spinosus (squamata: phrynosomatidae) from a region of the chihuahuan desert, méxico aurelio ramírez-bautista1,*, barry p. stephenson2, xóchitl hernández-ibarra1, uriel hernández-salinas1, raciel cruz-elizalde1, abraham lozano1, and geoffrey r. smith3 morphological variability of the hermann’s tortoise (testudo hermanni) in the central balkans katarina ljubisavljević1, georg džukić1, tanja d. vukov1, miloš l. kalezić1,2 the usefulness of mesocosms for ecotoxicity testing with lacertid lizards maria josé amaral1,2,*, rita c. bicho1, miguel a. carretero2, juan c. sanchez-hernandez3, augusto m. r. faustino4, amadeu m. v. m. soares1, reinier m. mann1,5 does acclimation at higher temperatures affect the locomotor performance of one of the southernmost reptiles in the world? jimena b. fernández*, nora r. ibargüengoytía advertisement call of scinax littoralis and s. angrensis (amphibia: anura: hylidae), with notes on the reproductive activity of s. littoralis michel v. garey1, thais r. n. costa2, andré m. x. de lima2, luís f. toledo3, marília t. hartmann4 book review: marine s. arakelyan, felix d. danielyan, claudia corti, roberto sindaco, alan e. leviton 2011. herpetofauna of armenia and nagorno-karabakh. society for the study of amphibians and reptiles stefano scali book review: rafaqat masroor 2012. a contribution to the herpetofauna of northern pakistan stefano scali evidence of high longevity in an island lacertid, teira dugesii (milne-edwards, 1829). first data on wild specimens. j. jesus first assessment of the endoparasitic nematode fauna of four psammophilous species of tropiduridae (squamata: iguania) endemic to north-eastern brazil markus lambertz1,*, tiana kohlsdorf2, steven f. perry1, robson waldemar ávila3, reinaldo josé da silva4 rediscovery and redescription of the holotype of lygosoma vittigerum (= lipinia vittigera) boulenger, 1894 yannick bucklitsch1, peter geissler1, timo hartmann1, giuliano doria2 , andré koch1,* reproductive phenology of the tomato frog, dyscophus antongili, in an urban pond of madagascar’s east coast ori segev1,*, franco andreone2, roberta pala2, giulia tessa2, miguel vences1 range extension of the critically endangered true poison-dart frog, phyllobates terribilis (anura: dendrobatidae), in western colombia roberto márquez1,*, germán corredor2, carlos galvis3 daniel góez2, & adolfo amézquita1 differences in habitat use of two sympatric species of ameiva in east costa rica esther sebastián-gonzález1, ramón gómez2 acta herpetologica journal of the societas herpetologica italica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 7(1): 181-188, 2012 exceptional sea turtle nest records in 2011 suggest an underestimated nesting potential in sicily (italy) paolo casale1,2, giuseppe palilla3, alessandro salemi3, angelo napoli4, maurizio prinzi5, laura genco6, davide bonaviri6, angela mastrogiacomo2, marco oliverio2, mario lo valvo7 1 wwf italy, via po 25c, 00198 roma, italy. *corresponding author e-mail: paolo.casale@uniroma1.it 2 department of biology and biotechnologies “charles darwin”, university of rome “la sapienza”, viale dell’università 32, 00185 roma, italy 3 riserva naturale orientata torre salsa, via roma 156/d, 92010 siculiana, italy 4 wwfsez. di menfi, via garibaldi, cort. 28/7 92013 menfi, italy 5 wwf palermo, via caltanissetta 2/b, 90141 palermo, italy 6 riserva naturale orientata capo rama, via rimembranze 18, 90049 terrasini (pa), italy 7 dipartimento biol. ambientale e biodiversità, university of palermo, via archirafi 18, 90123 palermo, italy sumbitted on: 2012, 21st march; revised on: 2012, 3rd may; accepted on: 2012, 9th may. abstract. we report seven nesting events by loggerhead sea turtles in sicily (italy) in 2011. in comparison to past records, this number is relatively high and may be at least in part due to an awareness campaign carried out in 2011 to solicit such reports. this suggests that sicily may host a much higher nesting activity than previously thought and higher monitoring effort is recommended, especially in certain coastal tracts. sand temperatures and incubation periods indicate that the beaches monitored so far in the southern coast are not optimal for development, resulting in low hatching success, and produce a majority of males. five 2011 nests and two past nests from the same area had mtdna haplotype cc-a2.1, the most common in the mediterranean. keywords. caretta caretta, sea turtle, distribution, nesting, mediterranean. introduction loggerhead sea turtles (caretta caretta) are listed as endangered in the iucn red list of threatened species (iucn, 2011) and the main identified threats in the mediterranean are destruction or disturbance at reproductive habitats (casale and margaritoulis, 2010), incidental catch in fishing gear, collision with boats, and intentional killing (tomás et al., 2008; casale et al., 2010; casale, 2011) and as a whole represent a high level of threat 182 p. casale et al. (wallace et al., 2011). the mediterranean hosts a loggerhead population that exhibits limited gene flow with those in the atlantic and represents a regional management unit for conservation (wallace et al., 2010). nowadays, major nesting sites are in greece, turkey, libya and cyprus, with minor sites or scattered nesting in several other countries in the eastern basin, including italy (casale and margaritoulis, 2010). since the beginning of sea turtle surveys in the mediterranean it was evident that italy did not host major nesting sites (argano and baldari, 1983; argano, 1992) and a recent review suggested 30-40 nests per year in italy (mingozzi et al., 2007) as compared with over 7200 in the whole of the mediterranean (casale and margaritoulis, 2010). on the other hand, actual nesting level and distribution in italy is not well understood yet. many potential nesting sites have not been adequately monitored, as demonstrated by the case of south calabria, where the most important nesting area in italy known so far was discovered only when a proper monitoring programme was launched (mingozzi et al., 2007). in sicily there are potentially suitable coasts for sea turtle nesting, and nests have occasionally been reported there by tourists or local people (mingozzi et al., 2007; genco et al., 2008). the aim of this study was to increase available data and have clues on potential nesting sites in sicily. to this aim, in 2011 an awareness campaign was launched in order to solicit reports by coastal people and tourists, and data about nesting activity and egg incubation were collected. results are here reported and discussed in terms of the potential importance of sicily as a nesting area of loggerhead sea turtles. materials and methods in this study, information on sea turtle tracks or sea turtles found while nesting was received from people frequenting the beaches in the night or early in the morning, possibly a consequence of the 2011 wwf’s awareness campaign aimed to solicit such reports. date of nesting was conventionally assigned as the day after the night when the clutch was laid. digital thermometers were placed at about 1 m distance (parallel to the seashore) from six of the seven identified nests, with probes buried at a depth of 40 cm, the approximate depth of the middle of loggerhead nests in zakynthos, greece (39 cm; zbinden et al., 2006) and cyprus (39.8 cm; godley et al., 2001). minimum and maximum temperatures, memorized by the thermometers, were recorded once a day and the mean was assumed to be the mean of the day (godfrey and mrosovsky, 1994). this was also validated with temperatures recorded every hour during three days at four nests (a, b, c, d). most nests were continuously monitored for all the study period, and hatchling emergence was recorded. carapace length and weight of a sub-sample of hatchlings from three nests were measured. samples of dead hatchlings or embryos from nests a, d, e, f, g, and from two past nests (2005, giallonardo; 2008, siculiana marina) (fig. 1) were analyzed for mtdna haplotype. duplication (i.e. clutches laid by the same female) cannot be excluded. a stretch of ≈ 800 bp of the mitochondrial control region was pcr amplified with primers lcm15382+ and h950(abreu-grobois et al., 2006). sequences were then compared with the haplotypes known for the species, available at the archie carr center for sea turtle research web site (university of florida, usa; http://accstr. ufl.edu/cclongmtdna.html). 183sea turtle nesting potential in sicily results a total of seven sea turtle nests were reported to our project in the summer 2011 (table 1) (fig. 1). clutches c and f were laid by the same female, identified through a flipper tag. nests b and f did not result in any hatch and were dug 79 and 104 days after nesting respectively. no evident embryos were present in eggs of nest b, suggesting no fertilization, while almost completely developed embryos were present in nest f. minimum incubation period (defined as the period between nesting and first hatchling emergence) of the other five nests ranged from 65 to 79 days (mean: 70.0 ± 5.4 sd; n = 5). hatchlings did not emerge simultaneously and emergence took from 3 to 10 days. total number of eggs ranged from 80 to 119 (mean: 97.9 ± 12.5 sd; n = 7) and hatching success (proportion of hatched eggs) ranged from 0 to 81.3% (mean: 27.4 ± 32.5 sd; n = 7). mean sand temperatures at a depth of 40 cm near the six monitored nests ranged from 23.2 to 28.9 °c (table 2), with daily values ranging from 19.8 to 30.5 °c (fig. 2). all the seven clutches analyzed for mtdna haplotype had the same haplotype cc-a2.1. discussion the number of sea turtle nests reviewed for the main island of sicily in 2011 is very high if compared with the total of 30 nest records reviewed for the period 1965-2009 fig. 1. sicily island (italy). places where nests were found in 2011 are shown. the circle shows the coastal tract including siculiana marina, giallonardo and punta grande where a minimum of seven nests were found in the past (see text). the inset map shows the study area within the mediterranean. 184 p. casale et al. (mingozzi et al., 2007; mingozzi, 2010). this may reflect an actual increase of nesting activity in the area or it may be due to a higher reporting rate by tourists and local people or a combination of the two. the fact that in 2011 an awareness campaign specifically focused on this aspect was launched suggests that reporting rate is an important and highly variable factor. it is likely that nesting activity in sicily is greatly under-reported, and further awareness campaigns can help increasing nest records and identifying nesting sites. during 2011, four nests were found on the same beach (giallonardo). since loggerhead turtles lay multiple clutches at an interval of about two weeks, with a minimum of 10 days recorded in the mediterranean (margaritoulis et al., 2003), dates of nesting indicate that more than one female nested on this beach, and one female nested both on this and on another beach about six km away (punta grande). in the tract of coast (approximately 10 km long) including siculiana marina, giallonardo and punta grande, four nests were recorded in the period 1995-1999 (mingozzi et al., 2007), two nests at giallonardo in 2005 (galia et al., 2006), one nest at siculiana marina and multiple tracks and hatchlings at giallonardo in 2008 (d. bonaviri, g. palilla, pers. comm.), and five nests in 2011 (present results). this suggests that this tract of coast might be a nesting area for a group of adult loggerhead females, not necessarily with a strict fidelity to a specific beach. another potential nesting area is the tract of coast near menfi, where three nests were recorded in the period 1993-2003 (mingozzi et al., 2007), one in 2006 (a. napoli, pers. comm.) and one in 2011 (present results). the nest in palermo was the first ever recorded in the northern coast of sicily, although several nest records are known from other tyrrhenian coasts of italy (mingozzi et al., 2007). fig. 2. temperatures of the sand at 40 cm depth and at 1 m distance from nests (a to f). 185sea turtle nesting potential in sicily ta bl e 1. in cu ba tio n an d ha tc hi ng s uc ce ss d at a of s ev en lo gg er he ad s ea tu rt le n es ts in s ic ily in 2 01 1. n es t pl ac e n es tin g fi rs t e m er ge nc e m in in cu ba tio n pe ri od ( da ys ) em er ge nc e (d ay s) n e gg s n h at ch ed e gg s h at ch in g su cc es s (% ) d ea d ha tc hl in gs a g ia llo na rd o (a g ) 26 /0 6/ 11 13 /0 9/ 11 79 5 80 11 13 .8 2 b pa le rm o (p a ) 11 /0 7/ 11 no t h at ch ed 90 0 0. 0 c pu nt a g ra nd e (a g ) 13 /0 7/ 11 16 /0 9/ 11 65 no t a va ila bl e 93 12 12 .9 0 d g ia llo na rd o (a g ) 15 /0 7/ 11 20 /0 9/ 11 67 8 10 7 70 65 .4 0 e g ia llo na rd o (a g ) 02 /0 8/ 11 10 /1 0/ 11 69 10 96 78 81 .3 5 f g ia llo na rd o (a g ) 06 /0 8/ 11 no t h at ch ed 10 0 0 0. 0 g po rt o pa lo d i m en fi (a g ) 09 /0 8/ 11 18 /1 0/ 11 70 3 11 9 22 18 .5 1 ta bl e 2. h at ch lin gs m ea su re m en ts a nd s an d te m pe ra tu re s fo r si x lo gg er he ad s ea tu rt le n es ts in s ic ily in 2 01 1. n es t te m pe ra tu re s h at ch lin gs m ea n ± sd ( ra ng e; n ) m on ito re d pe ri od (d ay s) c ov er ag e of th e in c. pe ri od ( % ) m ea n ± sd ( ra ng e; n ) (° c ) c ar ap ac e le ng th ( m m ) w ei gh t ( g) a 29 ju l 1 3 se p 58 .2 26 .5 ± 0 .5 ( 24 .4 -2 7. 3; n = 3 5) 40 .8 ± 0 .9 ( 39 .3 -4 2. 5; n = 1 9) 15 .7 ± 0 .9 ( 14 .0 -1 7. 0; n = 1 9) b 22 ju l 2 8 se p 28 .9 ± 1 .5 ( 24 .0 -3 0. 5; n = 6 9) c 29 ju l 2 4 se p 87 .7 27 .3 ± 0 .8 ( 25 .6 -2 8. 3; n = 2 2) d 29 ju l 2 0 se p 79 .1 26 .6 ± 0 .6 ( 24 .4 -2 7. 4; n = 4 1) 40 .5 ± 1 .4 ( 39 .0 -4 3. 5; n = 1 0) 15 .0 ± 1 .6 ( 11 .0 -1 6. 0; n = 1 0) e 3 a ug 1 0 o ct 98 .6 26 .1 ± 1 .0 ( 22 .9 -2 7. 3; n = 4 7) 41 .3 ± 0 .8 ( 39 .5 -4 2. 5; n = 1 3) 16 .6 ± 0 .9 ( 15 .0 -1 8. 0; n = 1 3) f 24 s ep 2 0 o ct 23 .2 ± 2 .1 ( 19 .8 -2 6. 2; n = 1 5) 186 p. casale et al. overall, hatching success was very low in 2011 nests, and also in one monitored nest in 2005 (25.8%; n = 66; galia et al., 2006). only two nests (d, e) had a hatching success close to the one observed in zakynthos island (71.5%), a major loggerhead sea turtle nesting site in the mediterranean (margaritoulis, 2005). in other italian nesting sites, the mean hatching success was 86% (calabria) (mingozzi et al., 2007) and 81.5% (lampedusa) (prazzi et al., 2010). the high variability within the same beach suggests that environmental factors varying within the beach may be involved. in general, the beaches of giallonardo and punta grande had rather cold sand temperatures, which may extend the incubation period till the autumn, with high risk of further drops of temperature. for comparison, the average incubation duration in the mediterranean is less than 60 days (margaritoulis et al., 2003). in other italian nesting sites, the mean incubation duration was 46 days (calabria) (mingozzi et al., 2007), 67 days (lampedusa) (prazzi et al., 2010) and 47 days (linosa) (corti et al., 2011). temperature has also an effect on sex determination, with more males being produced below 29.3 °c (pivotal temperature) and above 52.6 days of incubation period (pivotal incubation duration) (mrosovsky et al., 2002). the observed sand temperatures and incubation durations suggest that a majority of males are produced by the beaches in the southern coast of sicily, at least those monitored so far. in the mediterranean, current knowledge about sex ratio of hatchlings and juveniles is somehow contrasting, with more male juveniles than expected from hatchling sex ratio in monitored nesting sites (casale et al., 2006). therefore, male-producing areas, even if scattered over long coastal tracts, may contribute to a better understanding of the sex ratio patterns and may also represent important areas in future scenarios of climate change, with increased temperatures and consequently increased female production (hawkes et al., 2009). cold and variable temperature regimes are probably also the reason of the asynchronous emergence (houghton and hays, 2001), i.e. hatchlings emerging in small groups and not altogether. the only mtdna haplotype observed in the analyzed clutches (cc-a2.1) is prevalent in the main nesting area in italy (calabria), in the major mediterranean rookeries, and is also common in the atlantic (garofalo et al., 2009; monzon-arguello et al., 2010). as a result of this study, we recommend higher monitoring efforts in sicily in order to identify individual nesting events and nesting sites, by means of both direct monitoring of specific coastal tracts and citizen science initiatives through awareness campaigns. characterization of thermal features of beaches may also help identifying potential nesting sites and further genetic surveys can help understanding the link with other nesting areas. ackowledgements we thank f. adragna, s. anzà, l. gagliano, g. culmone, d. freggi, g. insacco, f. licata, s. mineo, s. garofalo, “associazione archelon”, guardie venatorie volontarie wwf nucleo di palermo, and “lido italia” (palermo) for their helpful collaboration and capitaneria di porto di palermo, comune di menfi, ripartizione faunistico-venatoria di agrigento and palermo for their support. fig. 1 was prepared with maptool (seaturtle.org). finally, we thank two anonymous reviewers for their helpful comments. 187sea turtle nesting potential in sicily references abreu-grobois, a., horrocks, j., formia, a., dutton, p., leroux, r., vélez zuazo, j., soares, l., meylan, p. 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(2006): metabolic heating in mediterranean loggerhead sea turtle clutches. j. exp. mar. biol. ecol. 334: 151-157. acta herpetologica 16(2): 63-87, 2021 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-10742 a new species of the genus noblella (amphibia: strabomantidae) from ecuador, with new information for noblella worleyae carolina reyes-puig1,2,3,4,*, juan m. guayasamin2,5, claudia koch6, david brito-zapata1, matthijs hollanders7, melissa costales8, diego f. cisneros-heredia1,2,3 1 universidad san francisco de quito usfq, colegio de ciencias biológicas & ambientales cociba & instituto de diversidad biológica tropical ibiotrop, museo de zoología/ laboratorio de zoología terrestre, campus cumbayá, quito, ecuador 2 universidad san francisco de quito usfq, colegio de ciencias biológicas y ambientales cociba, instituto biosfera, campus cumbayá, quito, ecuador 3 instituto nacional de biodiversidad inabio, unidad de investigación, quito, ecuador 4 cibio/inbio, centro de investigação em biodiversidade e recursos genéticos da universidade do porto, instituto de ciências agrárias de vairão, r. padre armando quintas, 4485-661 vairão, portugal 5 universidad san francisco de quito usfq, colegio de ciencias biológicas y ambientales cociba, laboratorio de biología evolutiva, campus cumbayá, quito, ecuador 6 zoologisches forschungsmuseum alexander koenig zfmk, leibniz-institut zur analyse des biodiversitätswandels, bonn, germany 7 southern cross university, school of environment, science and engineering, lismore, australia 8 university of new brunswick, department of biology, fredericton, canada *corresponding autor. e-mail: creyesp@usfq.edu.ec submitted on: 2021, 31st march; revised on: 2021, 22nd july; accepted on: 2021, 23rd july guest editor: aaron m. bauer abstract. we describe a new species of terrestrial-breeding frog of the genus noblella from the northwestern slopes of the andes of ecuador, in the province of pichincha, ecuador, and report a new locality for the recently described n. worleyae. we include a detailed description of the osteology of both species and discuss their phylogenetic relationships. the new species is differentiated from other species of noblella by having discs of fingers rounded, without papillae; distal phalanges only slightly t-shaped; toes slightly expanded and rounded distally, without papillae; dorsum uniform brown with irregular suprainguinal dark brown marks; venter yellowish cream, ventral surfaces of legs and thighs reddish to brownish cream; and dark brown throat. the new locality for n. worleyae is located in los cedros reserve, an area highly threatened by mining. we highlight the importance of protecting endemic species of small vertebrates in northwestern ecuador. keywords. frog, los cedros biological reserve, endemism, imbabura, mindo, pichincha, phylogeny. introduction the amphibian diversity in the tropical andes is outstanding (duellman, 1988; myers et al., 2000; hutter et al., 2013, 2017). each year, several species are described from montane forests of this biodiversity hotspot (e.g., rojas-runjaic et al., 2018; guayasamin et al., 2019; paez and ron, 2019; reyes-puig et al., 2019b; santa-cruz et al., 2019; yanez-muñoz et al., 2019; acevedo et al., 2020; ospina-sarria et al., 2020; lehr et al., 2021). most described species from ecuador belong to the hyper-diverse genus pristimantis (paez and ron, 2019; reyes-puig et al., 2020a), but diversity in other anuran taxa has also increased considerably (e.g., osornophryne, hyloscirtus, noblella, centrolenidae; mueses-cisneros et al., 2010; cisneros-heredia and gluesenkamp, 64 c. reyes-puig et alii 2010; yánez-muñoz et al., 2010a; páez-moscoso and guayasamin, 2012; almendáriz et al., 2014; guayasamin et al., 2017a, 2019; reyes-puig et al., 2019c). terrestrial-breeding frogs of the genus noblella barbour 1930 are minute-size anurans (svl < 22 mm), morphologically differentiated by having terminal discs on digits not or barely expanded, discs and circumferential grooves present distally (except in n. duellmani), terminal phalanges narrowly t-shaped, pointed tips of at least toes iii‒iv, and an inner tarsal tubercle (de la riva et al., 2008; hedges et al., 2008; duellman and lehr, 2009). however, phylogenetic relationships of noblella are not fully resolved and its monophyly is uncertain (de la riva et al., 2017; santa-cruz et al., 2019). as currently defined, noblella includes 16 species, fourteen distributed in the andes of ecuador, peru, and bolivia, and two (n. losamigos and n. myrmecoides) in the amazonian lowlands from southeastern colombia, ecuador, peru, bolivia, and western brazil (frost, 2021). during the last 15 years, the number of species in the genus has doubled; and four new species have been described since 2019 (catenazzi and ttito, 2019; reyespuig et al., 2019c, 2020b; santa-cruz et al., 2019). currently, the total number of species of the genus noblella is 16, distributed in ten species in peru, seven in ecuador, three in bolivia, and one in colombia and brazil (frost, 2021). andean species of the genus noblella show a high level of endemicity, with very restricted distributions. while some species of noblella may apparently be able to survive in environments modified by humans (e.g., n. duellmani, n. losamigos, n. lochites, n. naturetrekii; duellman and lehr, 2009; reyes-puig et al., 2019c; santa-cruz et al., 2019); most species (e.g., n. coloma, n. heyeri, n. personina, n. pygmaea; lynch, 1986; guayasamin and terán-valdez, 2009; harvey et al., 2013) seem to depend on undisturbed forest. three species of noblella have been described from western ecuador, all from mature mountain forests: noblella heyeri (lynch, 1986) occurs in southwestern ecuador and extreme northwestern peru; noblella coloma guayasamin and terán-valdez, 2009 is known from its type locality and surroundings (rio guajalito and chiriboga area; ron et al., 2019); and noblella worleyae, a recently described species is known just from seven specimens, all found in mature forest in the río manduriacu reserve, province of imbabura, ecuador (reyespuig et al., 2020b). while the ecuadorian andes have suffered serious habitat destruction and fragmentation caused by expansion of deforestation, agriculture, mining, among others (castellanos et al., 2011; roy et al., 2018; guayasamin et al., 2019; lessmann et al., 2019; ortega et al., 2021), there are still some areas with mature forests that have not been exploited due to their complex topography, difficult access, private protection, or preservation for touristic activities. unfortunately, all such sites are under strong anthropogenic pressure, including mining concessions and the expansion of agricultural boundaries, among others (cuesta et al., 2017; roy et al., 2018; guayasamin et al., 2019; ortega et al., 2021). these privileged areas have proven to keep an extremely high cryptic diversity of small vertebrates and contain the last remnant populations of numerous threatened species (cisnerosheredia and yanez-muñoz, 2010; reyes-puig et al., 2010, 2019a, 2019b; yánez-muñoz et al., 2010b, 2018; guayasamin et al., 2018, 2019, 2020; sánchez-nivicela et al., 2018; barrio-amorós et al., 2020). during the last five years, we have carried out surveys on the western slopes of the andes in the provinces of imbabura and pichincha, ecuador. as a result of this continuous effort, we found a new species of leaf-litter frog of the genus noblella, which we describe herein based on a combination of morphological, molecular, and osteological features. we also document new information on distribution, external morphology and osteology for the recently described noblella worleyae, information that was not described in detail in the original description. we also include intraspecific variation that will allow complete full with members of the same genus in the future. materials and methods taxonomy we followed the family taxonomy proposed by heinicke et al. (2018) and, also we revised de la riva et al. (2017) and barrietos et al. (2021). for identifying species, we assumed the unified species concept (de queiroz, 2005, 2007). information for species comparisons was extracted from the original descriptions and cited once at the beginning of the comparison. study area and fieldwork over the last three years (i.e., 2018–2020), we have carried out field surveys at several localities in montane forests of northwestern ecuador, mainly in the provinces of imbabura and pichincha. specimens of two different species of noblella were found in mindo (province of pichincha) and los cedros biological reserve (province of imbabura). mindo is a small town renowned for its adventure and nature-based touristic activities; thus the area has numerous reserves that protect cloud forests (arteaga-navarro et al., 2013). los cedros biological reserve is a protected area that contains 6,879 hectares 65a new species of the genus noblella from ecuador of premontane humid tropical forest and cloud mountain forest. this reserve is located south of the cotocachi-cayapas ecological reserve (state protected area), and is also recognized by its endemic microfauna (hutter and guayasamin, 2015). collected specimens were euthanized with benzocaine, fixed in 8% formalin, and preserved in 75% ethanol. liver and leg muscle tissue samples were collected from all individuals prior to preservation. tissues were preserved in 95% ethanol and stored at -20°c at the laboratorio de biología evolutiva usfq. specimens were deposited in the museo de zoología, universidad san francisco de quito, ecuador (zsfq). dna extraction, amplification, and sequencing we obtained new dna sequences of noblella sp. nov. (zsfq 050–051). dna was extracted from muscle or liver tissue following the protocol by peñafiel et al. (2019). standard polymerase chain reaction (pcr) was performed to amplify a fragment of the mitochondrial gene 16s rrna, using a combination of the following primers: 16l10, 16h36e, 16l34, 16h47 (heinicke et al., 2007). amplicons were sequenced in both directions by the macrogen sequencing team (macrogen inc., seoul, korea). the new sequences were assembled and edited with geneious 7.1.7 (genematters corp). after assemblage, the sequences were combined with sequences from genbank for all species of noblella and representatives of the genera within the terrarana clade (sensu hedges et al., 2008), including barycholos heyer 1969, bryophryne hedges, duellman & heinicke 2008, craugastor cope 1862, haddadus hedges, duellman & heinicke 2008, holoaden miranda-ribeiro 1920, ischnocnema reinhardt & lutken 1862, lynchius hedges, duellman & heinicke 2008, niceforonia goin & cochran 1963, oreobates jiménez de la espada 1872, qosqophryne catenazzi, mamani, lehr, von may 2020, phrynopus peters 2873, pristimantis jiménez de la espada 1870, psychrophrynella hedges, duellman & heinicke 2008, and microkayla de la riva, chaparro, castroviejo-fisher, padial 2017. genbank codes are shown in our inferred phylogenetic tree (fig. 1). phylogenetic analyses phylogenetic relationships were inferred using maximum likelihood as the optimality criterion. the final matrix, with 52 terminals, was aligned with mafft v.7 (multiple alignment program for amino acid or nucleotide sequences: http:// mafft.cbrc.jp/alignment/software/), with the q-ins-i strategy. macclade 4.07 (maddison and maddison, 2005) was used to visualize the alignment, which contained a total of 492 bp. phylogenetic analyses were performed under the ml criteria in garli 2.01 (genetic algorithm for rapid likelihood inference; zwickl, 2006) for the mitochondrial gene 16s. garli uses a genetic algorithm that finds the tree topology, branch lengths, and model parameters that maximize lnl simultaneously (zwickl, 2006). individual solutions were selected after 10,000 generations with no significant improvement in likelihood, with the significant topological improvement level set at 0.01. then, the final solution was selected when the total improvement in likelihood score was lower than 0.05, compared to the last solution obtained. default values were used for other garli settings, as per recommendations of the developer (zwickl, 2006). bootstrap support was assessed via 1,000 pseudoreplicates under the same settings used in tree search. pairwise genetic distances between species (uncorrected-p) for gene 16s were calculated with paup 4a (swofford et al., 1996). external morphology diagnosis and description of the new species follow formats proposed by duellman and lehr (2009) and lynch and duellman (1997). for comparisons, we examined specimens of other species of noblella (see appendix i). we followed the sequence of characters proposed by guayasamin and terán-valdez (2009). we measured preserved specimens using digital calipers to the nearest 0.01 mm. these measurements are: snout to vent length (svl), from the tip of the snout to the cloaca; head length (hl), measured from tip of snout to anterior edge of tympanum; head width (hw), measured at midorbital region; horizontal diameter of the eye (ed); eye–nostril distance (en), from anterior ocular angle to posterior edge of nostril; horizontal diameter of tympanum (td); minimum interorbital distance (miod); minimum eyelid width (mwe); hand length (lh), from posterior edge of palmar tubercle to tip of third digit; shank length (ls), from the tip of the ankle to the knee; and foot length (lf), from posterior edge of external metatarsal tubercle to tip of toe iv. we determined sexual maturity by the presence of vocal slits or extended vocal sacs in males and by the presence of eggs or convoluted oviducts in females. detailed illustrations of the head, hands and feet were done with adobe indesign ©. osteology osteological descriptions were based on one specimen of the new species (zsfq 050) and one of noblella worleyae (mzuti 1709). both specimens were scanned using a highresolution micro-computed tomography (micro-ct) desktop device (bruker skyscan 1173, kontich, belgium) at the zoologisches forschungsmuseum alexander koenig (zfmk, bonn, germany). to avoid movements during scanning, specimens were placed in a small plastic container and mounted with styrofoam. acquisition parameters comprised: an x-ray beam (source voltage 43 kv and current 114 µa) without the use of a filter; 800 projections of 500 ms exposure time each with a frame averaging of 5 recorded over a 180° continuous rotation (rotation steps of 0.3 degrees), resulting in a scan duration of 49 min; a magnification setup generating data with an isotropic voxel size of 19.16 µm (mzuti 1709) and 14.55 µm (zsfq 050), respectively. the ct-dataset was reconstructed with n-recon software (bruker microct, kontich, belgium) and rendered in three dimensions using ctvox for windows 64 bits version 2.6 (bruker microct, kontich, belgium). osteological 66 c. reyes-puig et alii terminology follows trueb (1973), duellman and trueb (1994), fabrezi and alberch (1996), guayasamin and terán-valdez (2009), scherz et al. (2017), and suwannapoom et al. (2018). cartilage structures were omitted from the osteological descriptions because micro-ct does not render cartilage. results phylogenetic relationships and genetic distances (fig. 1) the inferred phylogeny shows that the new species described herein is part of a clade composed of taxa distributed along the western slopes of the ecuadorian andes. this clade is composed by the new species noblella sp. nov., noblella coloma guayasamin and terán-valdez, 2009, and n. worleyae reyes-puig, maynard, trageser, vieira, hamilton, lynch, culebras, kohn, brito and guayasamin, 2020. uncorrected p genetic distances are as follow: n. coloma (qcaz 40579) and the new species (zsfq 050) = 5.1%; n. coloma (qcaz 40579) and n. worleyae (zsfq 550–551) = 8.3%; n. worleyae (zsfq 550–551) and the new species (zsfq 050) = 1.2%. generic placement we place the new species in the genus noblella based on morphological and molecular evidence (fig. 1). morphologically, we assign the new species to the genus noblella, as defined by hedges et al. (2008), based on possession of the following traits: head not wider than body; cranial crests absent; tympanic membrane differentiated (undifferentiated in n. duellmani, n. naturetrekii and n. madreselva); dentigerous processes of vomers absent; terminal discs on digits not or barely expanded; discs and circumferential grooves present distally (absent in n. duellmani); terminal phalanges narrowly t-shaped; finger i shorter than, or equal in length to, finger ii; finger iv containing two phalanges in noblella carrascoicola (de la riva and köhler, 1998), n. lochites (lynch, 1976b), n. losamigos (santa-cruz et al., 2019), n. myrmecoides (lynch, 1976b), n. naturetrekii (reyes-puig et al., 2019c), and n. ritarasquinae (köhler, 2000) and three phalanges in n. coloma (guayasamin and terán-valdez, 2009), n. duellmani (lehr, aguilar, and lundberg, 2004), n. heyeri (lynch, 1986), n. sp. nov., n. lynchi (duellman, 1991), n. madreselva (catenazzi, uscapi, and von may, 2015), n. personina (harvey, almendáriz, brito-m., and batallas-r., 2013), n. peruviana (noble, 1921), n. pygmaea (lehr and catenazzi, 2009), and n. thiuni (catenazzi and ttito, 2019); toe iii shorter than toe v (except in n. naturetrekii and n. worleyae); tips of at least toes iii–iv acuminate; subarticular tubercles not protruding; dorsum pustulate or shagreen; venter smooth; svl less than 22 mm. systematic accounts noblella mindo new species. noblella coloma arteaga et al. (2013). figs. 2–8 lsid urn:lsid: lsid:zoobank.org:act:3b7741ef-bf264589-b231-73f198aa1218 proposed standard english name. mindo leaf frog proposed standard spanish name. rana noble de mindo holotype. zsfq 050 (fig. 2–6), adult female, collected in el cinto, 11 km e from mindo town, mindo (0.09022°s, 78.818581°w; 1,673 m; fig. 2), province of pichincha, república del ecuador, by melissa costales, matthijs hollanders and emilia peñaherrera on 08 july 2017. paratypes (2 females, 2 males). adult males (zsfq 049, 051) and adult females (zsfq 304–305) collected at the type locality (same data as holotype), by melissa costales on 04 october 2015. etymology the specific name “mindo” is a word of unknown meaning in panzaleo, an extinct pre-columbian language of northern ecuador (jijón y caamaño 1940). it is used as a noun in apposition, and alludes to the valley of mindo, where the type locality of the new species is located. the remnant forests of this emblematic valley protect several species of endemic amphibians and reptiles such as pristimantis mindo, noblella mindo, and anolis proboscis. diagnosis noblella mindo sp. nov. (figs. 3–8) presents the following characteristics: (1) skin of dorsum finely shagreen, skin on venter smooth, discoidal fold slightly defined, discoidal and thoracic folds absent; (2) tympanic annulus and membrane visible externally, supratympanic fold inconspicuous (figs. 3, 4); (3) snout short (eye-to-nostril distance 57% of eye diameter), rounded in dorsal and lateral views (fig. 3); (4) eyelids without tubercles; (5) dentigerous processes of vomers absent; (6) vocal slits and sac present, nuptial pads not visible; (7) fingers not expanded distally, finger tips rounded, without papillae (fig. 3); finger i shorter than finger ii (fig. 3); (8) distal phalanges slightly t-shaped, phalangeal formula of hands: 2-2-3-3 (fig. 7); (9) supernumerary palmar tubercles present (slightly visible) mostly at the base 67a new species of the genus noblella from ecuador of the digits, ulnar tubercles diminutive and rounded, subarticular tubercles rounded; circumferential grooves absent; (10) one tarsal tubercle elongated and subconical (fig. 3); two prominent metatarsal tubercles (inner tubercle 3–4 times size of external); toes slightly expanded and rounded distally, without papillae; (11) toe v shorter than toe iii, supernumerary plantar tubercles absent, distal portions of circumferential grooves not visible; (12) phalangeal formula of feet: 2-2-3-4-3 (fig. 7); (13) in life, uniform brown dorsum, cream middorsal, longitudinal line distinct and present in all individuals, dark brown suprainguinal marks, white rictal gland, noblella thiuni (mk072732, corbidi 18723) noblella madreselva (mn056356, corbidi 15770) noblella madreselva (mn064565, corbidi 15769) psychrophrynella usurpator (ky652662, ac186-09) psychrophrynella usurpator (ku884559, corbidi 16495) psychrophrynella glauca (mg837565, corbidi 18729) noblella losamigos (mn100040, —) noblella losamigos (mn366392, mvz 292687) noblella losamigos (ky652644, rvm3-12) noblella pygmaea (ky652645, musm 24536) noblella sp (ky652646, musm 27582) bryophryne bustamantei (kt276293, mhnc6019) bryophryne cophites (ef493537, ku173497) bryophryne phuyuhampatu (mf419259, —) bryophryne tocra (mf186398, mubi5419) barycholos pulcher (eu186709, ku 217781) barycholos ternetzi (ku495150, malcx17p10) noblella coloma (mt710932, qcaz 40579) noblella coloma (mt710931, qcaz 32702) noblella mindo sp. nov. (mt710934, zsfq-050) noblella mindo sp. nov. (mt710935, zsfq-051) noblella worleyae (mt710935, zsfq 551) noblella worleyae (mt710934, zsfq 550) noblella worleyae (mt710933, zsfq 2502) noblella lochites (eu186699, ku177356) noblella personina (mk838465, qcaz 58818) noblella heyeri (jx267463, qcaz 31471) noblella myermecoides (jx267464, qcaz 40180) noblella naturetrekii (mk838462, dhmecn 13307) ischnocnema bilineata (jx267324, mnrj 46476) holoaden bradei (ef493366, usnm 207945) holoaden luederwaldti (eu186710, mzusp 131872) ischnocnema colibri (mh538418, cfbh_41810) ischnocnema parnaso (mh538421, cfbh 41812) niceforonia brunnea (ef493357, ku178258) niceforonia peraccai (ef493710, ku178266) niceforonia dolops (ef493394, —) lynchius parkeri (mk423937, qcaz 61015) lynchius simmonsi (jf810005, qcaz 41640) oreobates barituensis (jf810000, mcn1360) oreobates quixensis (qcaz 31186, jf810003) phrynopus barthlenae (am039653, —) phrynopus inti (mf651906, ummz 245218) phrynopus kauneorum (am039655, —) phrynopus sp (am039660, —) phrynopus pesantesi (am039656, —) phrynopus mariellaleo ( mh538299, corbidi 11658) pristimantis cedros (kt210170, mzuti 1710) pristimantis ornatissimus (ku574613, mzuti 4329) pristimantis subsigillatus (ku999217, mzuti 1999) haddadus aramunha (kf740845, ufba 283) haddadus binotatus (kf740846, usp/tcx51st09) craugastor aenigmaticus (mk211617, ucr 22737) craugastor tarahumaraensis (eu186702, —) craugastor_longirostris (ef493395, ku 177803) 0.05 length units m i c r o k a y l a + q o s q o p h r y n e c l a d e “n o b le lla ” s o u th e rn c la d e n o b le lla n o rth e rn c la d e 5 8 9 5 9 8 5 8 7 4 7 6 1 0 0 5 9 9 3 9 9 1 0 0 9 5 7 4 8 0 9 6 9 8 8 4 5 3 7 0 6 8 5 0 7 8 1 0 0 6 8 7 6 5 6 9 5 7 8 6 7 6 8 1 0 0 fig. 1. phylogeny of noblella (light gray boxes) showing the relationships of n. mindo sp. nov. the phylogeny was inferred based on mitochondrial (16s) dna sequences (16s; 52 terminals, 492 bp) and under the maximum likelihood criterion. for each individual, museum catalog number or, if unavailable, genbank accession number is shown. 68 c. reyes-puig et alii flanks with dark brown band narrowing towards groin, dotted with white, light groin, with low concentration of melanophores, dark brown throat, chest and ventral surfaces of arms with a white cross formed by a longitudinal, fine line running from chin to chest crossing a similar line departing from midventral surface of each forelimb, yellowish-cream venter, reddish-copper iris with minute turquoise scattered dots (fig. 4); (14) svl in adult males 16.5–17.0 mm (mean 16.7 mm, n = 2), svl in adult females 18.3–19.5 mm (mean 19.0 mm, n = 3). comparisons (fig. 6, table 1) noblella mindo sp. nov. differs from its congeners by the presence of rounded fingertips, without papillae; distal phalanges slightly t-shaped; toes slightly expanded and rounded distally, without papillae; dorsum uniform brown with middorsal cream line, suprainguinal marks dark brown, rictal gland white, light groin, throat and chest dark brown with white cross, and venter yellowish cream. noblella mindo sp. nov. is most similar and closely related to n. coloma and n. worleyae (fig. 1), but they differ as follows (characters of n. mindo sp. nov. in parentheses): noblella coloma has all finger tips acuminate (all rounded), dark middorsal line (light), dark rictal gland (white), orange to reddish-venter (yellowishcream), dark groin (light), uniform dark brown throat, chest and ventral surfaces of arms (dark brown with white cross), ulnar tubercles absent (diminutive and rounded), and smaller body size of 16.0 mm svl in adult female (18.3–19.5 mm svl in adult females); the new species (characters of n. mindo sp. nov. in parentheses) is distinguished from noblella worleyae has finger tips slightly acuminate on fingers i and iv and acuminate on fingers ii and iii (rounded), t-shaped distal phalanges (slightly t-shaped), prootic and exoccipital fused to form otoccipital (separated), sphenethmoid well-ossified and ventrally fused at midline (moderately ossified, ventrally fused at midline in posterior half and separated in anterior half). for more comparison’s information see table 1. noblella mindo sp. nov. has three phalanges on finger iv like n. duellmani (lehr, aguilar and ludenberg, 2004), n. heyeri (lynch, 1986), n. lynchi (duellman, 1991), n. madreselva (catenazzi, uscapi and von may, 2015), n. personina (harvey, almendáriz, brito, and batallas, 2013), n. peruviana (noble, 1921), n. pygmaea (lehr and catenazzi, 2009), and n. thiuni (catenazzi and ttito, 2019), but they differ as follows (characters of n. mindo sp. nov. in parentheses): noblella duellmani has dorsal skin pustular (finely shagreen), tympanum membrane and annulus absent (present), upper eyelid bearing small tubercles (absent), ulnar tubercles coalesced into low folds (diminutive and round, not forming a fold), outer edge of tarsus bearing row of low and elongate tubercles (absent), tips of fingers i-ii slightly expanded and tips of fingers iii–iv slightly acuminate (all finger tips rounded), venter brown with tan mottling (yellowish-cream), and larger body size of 20.0 mm svl in adult female (18.3–19.5 mm svl in adult females); noblella heyeri has dorsal skin weakly pustulate (finely shagreen), snout subacuminate in dorsal view (round), ulnar tubercles distinct and round (diminutive, round), toe tips slightly acuminate (round), venter brown with cream fleck (yellowish-cream), and smaller body size of 13.1–15.9 mm svl in adult females (18.3–19.5 mm svl in adult females); noblella lynchi has dorsal skin pustular (finely shagreen), snout subacuminate in dorsal view (round), ulnar tubercles coalesced into low folds (diminutive and rounded, not forming a fold), outer edge of tarsus bearing row of low and elongate tubercles (absent), toe tips weakly acuminate (round), and venter brown with fine cream flecks (yellowish cream); noblella madreselva has dorsal skin with small tubercles (finely shagreen), tympanic membrane not differentiated and tympanic annulus barely visible below skin (well-differentiated), upper eyelid with minute tubercles (absent), toe tips weakly acuminate (rounded), venter black with large and irregularly shaped white mark (yellowishcream), and smaller body size of 17.6 mm svl in adult female (18.3–19.5 mm svl in adult females); noblella fig. 2. distribution of noblella mindo sp. nov and n. worleyae in ecuador. 69a new species of the genus noblella from ecuador personina has dorsal skin smooth with pustules (finely shagreen), snout subtruncate in profile (round), finger and toe tips acuminate with papillae (round, lacking papillae), venter white (yellowish-cream), and smaller body size of 15.6–17.9 mm svl in adult females (18.3– 19.5 mm svl in adult females); noblella peruviana has tympanic membrane not differentiated (differentiated), toe tips slightly acuminate (round), and venter tan (yellowish cream); noblella pygmaea has dorsal skin tubercular (finely shagreen), thoracic fold present (absent), dorsolateral fold on anterior half of body present (absent), upper eyelid bearing small tubercles (absent), minute tubercle on heel present (absent), toe tips pointed (round), venter pale grayish brown (yellowish-cream), and smaller body size of 11.3–12.4 mm svl in adult females (18.3–19.5 mm svl in adult females); noblella thiuni has thin dorsolateral folds visible on anterior half of body (absent), tympanic membrane not differentiated (differentiated), fingertips bulbous (round), ulnar tubercles absent (present, diminutive and round), venter copper reddish with a profusion of silvery spots (yellowish cream), and smaller body size of 11.0 mm svl in male (16.5–17 mm in adult females). noblella carrascoicola (de la riva and köhler, 1998), n. lochites (lynch, 1976b), n. losamigos (santa-cruz et al., 2019), n. myrmecoides (lynch, 1976b), n. naturetrekii (reyes-puig et al., 2019c), and n. ritarasquinae (köhler, 2000) are easily differentiated from n. mindo sp. nov. by having two phalanges on finger iv instead of three. description of the holotype adult female (zsfq 050); head narrower than body, its length 40.8% of svl; head longer than wide; head width 31.1% of svl; snout round in dorsal and lateral fig. 3. noblella mindo sp. preserved holotype, zsfq 050, adult female, svl = 18.3 mm. (a) foot in ventral view. (b) hand in ventral view. (c) head in dorsal view. (d) head in lateral view. illustrations by carolina reyes-puig. fig. 4. color patterns of noblella mindo sp. nov. in life. (a, c) dorso-lateral and ventral patterns of holotype, zsfq 050, adult female, svl = 18.3 mm. (b, d) dorsolateral and ventral patterns of paratype, zsfq 051, adult male, svl = 16.9 mm. photos by matthijs hollanders. 70 c. reyes-puig et alii views; canthus rostralis straight, slightly concave in profile; loreal region slightly concave; upper eyelid 45.6% of interorbital distance; eye-nostril distance 54.8% of eye diameter; tympanum visible externally, tympanic membrane differentiated from surrounding skin; supratympanic fold indistinct. dentigerous processes of vomers absent and vomerine teeth absent; choanae laterally oriented; tongue longer than wide, elongated, partially notched posteriorly. skin of dorsum finely shagreen, evident tubercles absent; skin on flanks smooth; venter smooth; discoidal fold slightly visible, dorsolateral folds and thoracic folds absent; diminutive rounded ulnar tubercles; palmar tubercle oval, about 2 times the size of the thenar tubercle; supernumerary palmar tubercles present, mainly at the base of the digits; proximal subarticular tubercles prominent, rounded; phalangeal formula 2-2-3-3; fingers not expanded distally, finger tips rounded, circumferential grooves absent; relative lengths of fingers: i < ii < iv < iii; forearm lacking evident tubercles. hindlimb lengths moderate, tibia length 49.3% of svl; foot length 46.1% of svl; dorsal surfaces of hindlimbs shagreen; tubercles on the heel absent; one prominent elongated tarsal tubercle on ventral surface of tarsus; two metatarsal tubercles, inner elongated conspicuous, outer subconical; proximal and distal subarticular tubercles well-defined; supernumerary tubercles absent. toes slightly expanded and rounded distally; distal portions of circumferential grooves not visible; phalangeal formula 2-2-3-4-3; relative lengths of toes: i < ii < v < iii < iv. measurements of holotype (in mm) svl= 18.3, hl= 7.5, hw= 5.7, ed= 2.4, en= 1.3, mwe= 1.5, td= 0.9, miod= 3.4, lh= 3.4, ls= 9.0, lf= 8.4. for measurements of the type series (mm) see table 2. color of holotype in life (fig. 4) dorsum brown, grayish brown towards the flanks; well-defined cream middorsal stripe, extending from interparietal region to cloaca and continuing along posterior surfaces of hindlimb. loreal region black, extending as homogeneous dark band to upper insertion of arm and into body flanks, narrowing towards groin and limited dorsally with a lighter brown line; flanks strongly light flecked; groin dark. rictal gland white. venter and ventral surfaces of hindlimbs yellowish cream; throat dark brown with large irregular yellowish cream marks and fig. 5. color variation of preserved noblella mindo sp. nov. in (a–e) dorsal and (f–j) ventral views: (a, f) zsfq 305, paratype, adult female, svl = 19.5 mm; (b, g) zsfq 304, paratype, adult female, svl = 19.2 mm; (c, h) zsfq 050, holotype, adult female, svl = 18.3 mm; (d, i) zsfq 051, paratype, adult male, svl = 17.0 mm; (e, j) zsfq 049, paratype, adult male, svl = 16.5 mm. photos by david brito-zapata and carolina reyes-puig. 71a new species of the genus noblella from ecuador medium longitudinal line. forelimbs ventrally yellowish cream with dark brown marks, dorsally light brown with dark brown marks; iris reddish copper with minute scattered turquoise dots. hindlimbs like dorsum. color of holotype in ethanol (fig. 5) dorsum brown, darker towards middorsum, welldefined middorsal line cream, extending from interparietal region to cloaca where stripe continues along posterior surface of thighs and pes. dorsal surfaces of forelimbs brown with black spots. labial bars absent; rictal gland light brown. loreal region black, extending as homogeneous dark band to upper insertion of arm and into body flanks, narrowing towards groin; flanks strongly light flecked; groin dark. dorsal surfaces of hindlimbs lighter brown than dorsum to cream with dark fleck and spots. throat dark brown with cream irregular marks and medium longitudinal stripe. chest, venter and ventral surfaces of thigh and crus cream. variation of color patterns and external morphology (figs. 4–5) adult females zsfq 050, 304–305 and the adult male zsfq 051 exhibit a cream middorsal stripe extending from interparietal region to cloaca; stripe of zsfq 305 is thinner and faintly defined. dark suprainguinal marks are faint in zsfq 049. throat, chest and ventral surfaces of forelimbs are dark brown with a white cross formed by a longitudinal, fine line running from chin to chest, crossing a similar line departing from midventral fig. 6. ventral views of (a–d) hands and (e–h) feet from three species of noblella: (a, e) noblella coloma, extracted from guayasamin and terán-valdez (2009); (b, f) n. worleyae (holotype); (c, g) n. worleyae (zsfq 3851); (d, h) n. mindo sp. nov. (holotype). scale bars = 1 mm. illustrations by carolina reyes-puig. 72 c. reyes-puig et alii ta bl e 1. m ai n di ag no st ic c ha ra ct er s of th re e sp ec ie s of n ob le lla fr om n or th w es te rn e cu ad or . sp ec ie s c ha ra ct er s so ur ce fi ng er ti ps to e tip s d is ta l ph al an ge s to es pa pi lla e u ln ar tu be rc le s v en te r an d th ro at co lo ra tio n pr oo tic an d ex oc ci pi ta ls ph en et hm oi d le ng th o f tr an sv er se pr oc es se s of pr es ac ra ls n eu ra l a rc h of pr es ac ra ls n ob le lla co lo m a a cu m in at e sl ig ht ly e xp an de d an d ac um in at e di st al ly tsh ap ed a bs en t a bs en t v en te r or an ge w ith m in ut e w hi te a nd b ro w n sp ot s se pa ra te d w el los si fie d, ve nt ra lly no t f us ed a t m id lin e ii , v ii i< v – v ii <i v <i ii pr es ac ra ls ii i– v w ith ra is ed m ed ia l ri dg e te rá nv al de z an d g ua ya sa m in , 20 09 n . w or le ya e sl ig ht ly ac um in at e on f in ge rs i an d iv a nd ac um in at e on fi ng er s ii a nd ii i sl ig ht ly e xp an de d an d sl ig ht ly a cu m in at e on t oe s i an d v, a nd cu sp id at e tip s on t oe s ii –i v tsh ap ed a bs en t pr es en t v en te r ye llo w is h cr ea m w ith m in ut e sp ec kl in g; th ro at w ith ir re gu la r br ow n m ar ks to ho m og en eo us ly b ro w n fu se d to fo rm ot oc ci pi ta l w el los si fie d, ve nt ra lly fu se d at m id lin e ii <v – v ii i< iv <i ii w ith r ai se d m ed ia l r id ge in al l p re sa cr al s r ey es -p ui g et al ., 20 20 an d th is p ap er n . m in do sp . n ov . r ou nd ed sl ig ht ly e xp an de d an d ro un de d di st al ly sl ig ht ly tsh ap ed a bs en t pr es en t th ro at , c he st a nd v en tr al su rf ac es o f a rm s da rk br ow n w ith a w hi te c ro ss , ve nt er y el lo w is h cr ea m se pa ra te d m od er at el y os si fie d, ve nt ra lly fu se d at m id lin e in po st er io r ha lf an d se pa ra te d in a nt er io r ha lf v –v ii i< ii – iv <i ii pr es ac ra ls ii i– v ii i w ith ra is ed m ed ia l ri dg e th is p ap er 73a new species of the genus noblella from ecuador surface of each forelimb (zsfq 050, 304, 774), longitudinal line on throat of zsfq 773 is faint, while cross is almost unnoticeable in holotype due to extensive light color of throat. venter and ventral surfaces of hindlimbs are cream (zsfq 305), dirty cream with diffused irregular brown marks (zsfq 304), or pinkish cream (zsfq 049–051). the throat in females is cream (zsfq 304– 305) or pinkish cream (zsfq 050) with large irregular dark brown marks; meanwhile in males it is homogenously dark brown with a slightly defined medium longitudinal stripe (zsfq 049, 051). osteology osteological description of noblella mindo sp. nov. is based on micro-ct images of the adult female holotype (zsfq 050). details of skull morphology and osteological aspects of hand and foot are presented in fig. 7 and main skeletal features are shown in fig. 8. skull (fig. 7) skull slightly longer than wide; widest part is at about where quadratojugal meets maxilla and is 89% of skull length. rostrum short; distance from anterior edge of frontoparietals to anterior face of premaxilla is 16% of skull length. at level of midorbit, braincase is about 38% of maximum skull width. braincase combines well and poorly ossified elements. frontoparietals are welldeveloped bones, distinctly longer than broad, slightly narrower anteriorly than posteriorly; narrowly separated along most of their length and only fused in anterior region. boarder between frontoparietals and prootics not well-resolved in micro-ct scan. ventrally, prootics in contact with parasphenoid alae. prootics well-separated from each other. exoccipitals approximate one another ventromedially and dorsomedially but still clearly separated with about same distance ventrally and dorsally; separated from frontoparietals. anterolaterally, frontoparietals in contact with sphenethmoid. sphenethmoid ventrally at midline separated in anterior half and fused in posterior half; posterior margin does not reach midpoint of orbit and is broadly separated from prootic and in ventral contact with parasphenoid. cultriform process of parasphenoid well-ossified posteriorly, thinning anteriorly, and about 28% width of braincase at mid-orbit. lateral margins of process approximately parallel. parasphenoid alae long but poorly ossified at their lateral ends. neopalatines very thin and long, articulate with sphenethmoid and approximate but not contact maxilla. columella (or stapes) large and well-ossified. due to tiny size and fine structure, septomaxilla is not well-resolved in micro-ct scan. dorsal investing bones moderately developed. nasals thin and broadly separated from one another, posteriorly in contact with anterior end of frontoparietals and posterolaterally in thin contact with maxilla. they curve ventrally towards their lateral edges. small prevomers broadly separated from one another medially, their anterior edge almost contacts a long and thin posterior projecting ramus of septomaxilla. maxillary arcade bears many small, poorly resolved teeth on premaxillae and maxillae. premaxillae separated medially, and their anterodorsal alary processes rise divergent from midline but still distinctly separated from nasals. premaxilla and maxilla in lateral contact, with anterior edge of maxilla slightly overlapping lateral edge of premaxilla. pars palatina of premaxilla broad, with two well-defined processes: medial process thin and acuminate, running about parallel to its counterpart, being distinctly separated from it; lateral process about the same length, but slightly broader, especially at its truncate posterior ending. maxilla long, its posterior end acuminate and in contact with quadratojugal. triradiate pterygoid bears a long, curved anterior ramus oriented anterolaterally toward maxilla, with which it articulates at ventral boarder slightly anterior to midline of orbit. posterior ramus of pterygoid about same length as medial ramus and both about half length of anterior ramus; however, posterior ramus more robust than other two. edge of medial ramus overlaps lateral edge of prootic. quadratojugal slender, almost straight and articulating anteriorly with maxilla and posterodorsally with ventral ramus of squamosal. squamosal t-shaped, with a long laminar otic ramus; zygomatic ramus much shorter and slender; ventral ramus about same length as otic ramus, laminar and broad, increasing in width ventrally. mandible slim and edentate. mentomeckelians small, medialtable 2. measurements (in mm) of type series of noblella mindo sp. nov. ranges followed by mean and standard deviation in parentheses. characters noblella mindo sp. nov. females (n = 3) males (n = 2) svl 18.3–19.5 (19.0 ± 0.6) 16.5–17.0 (16.7 ± 0.3) hl 7.1–7.5 (7.2 ± 0.2) 6.2–7.0 (6.6 ± 0.6) hw 5.7–6.7 (6.3 ± 0.5) 5.37–5.4 (5.38 ± 0.02) ed 2.2–2.5 (2.4 ± 0.2) 1.9–2.0 (1.95 ± 0.1) en 1.26–1.29 (1.28 ± 0.01) 1.21–1.22 (1.215 ± 0.01) mwe 1.2–1.5 (1.3 ± 0.2) 1.1–1.2 (1.15 ± 0.01) td 0.8–1.2 (0.9 ± 0.2) 0.78–0.82 (0.8 ± 0.02) miod 3.3–3.4 (3.3 ± 0.1) 3.0–3.4 (3.2 ± 0.3) lh 3.5–3.9 (3.7 ± 0.2) 3.0–3.2 (3.1 ± 0.2) ls 9.0–9.3 (9.1 ± 0.2) 7.7–8.0 (7.8 ± 0.2) lf 8.4–9.0 (8.6 ± 0.4) 7.3–7.5 (7.4 ± 0.1) 74 c. reyes-puig et alii ly, and laterally slightly broadened, and separated medially by a narrow gap. dentary long and thin, reaching to about anterior corner of orbit; it is posteriorly acuminate and overlapping angulospenial, seeming to be in contact with this bone for about the posterior half of its length; anteroventrally it contacts mentomeckelian bones. angulosplenial long and arcuate. coronoid process is a moderately long and slightly raised ridge. the only ossified portions of hyoid apparatus are two posteromedial processes, which are anteriorly slightly and posteriorly moderately expanded, approaching each other at anterior ends but being still moderately separated. postcranium (fig. 8) eight presacral vertebrae. all presacrals non-imbricate. presacral i longer than posterior vertebrae. all except presacral i bear well-developed diapophyses. transverse processes of presacrals v–viii similar in size, being the shortest and thinnest, those of presacrals ii and iv also about similar in size and being slightly larger, and those of presacral iii being the longest and widest of all transverse processes. transverse processes of presacrals ii and viii have slightly anterolateral orientation, those of presacral iii are laterally oriented and the others are slightly posterolaterally oriented. neural arch of presacrals iii–viii bears a raised medial ridge. sacrum bears slightly expanded diapophyses. urostyle long, slender, slightly shorter than presacral portion of vertebral column and bearing a well-pronounced dorsal ridge along most of its length, beginning at its anterior end. the bone has a bicondylar articulation with the sacrum. pectoral girdle with well-ossified coracoids, clavicles, scapulae and cleithra. suprascapular and sternum unossified and not visible in micro-ct scan, and omosternum hardly visible. clavicles long and slim, oriented anteromedially, slightly curved, with medial tips touching each other. laterally, clavicles firmly articulating with scapulae. coracoids stout and glenoidal and sternal ends about equally expanded. anterior edges of coracoids slightly curved, posterior edges almost straight. medial tips of coracoids broadly separated from another. scapula long, with a prominent pars acromialis not separated from pars glenoidalis. cleithrum long, broader, and thicker at scapular boarder, thinning posteriorly. in pelvic girdle, long, slender iliac shafts bearing conspicuous dorsolateral ridges along most of their length, except anteriormost region. ilia are fused posteriorly with ischium and pubis. ischium stout, whereas pubis is thin and blade-like. manus and pes (fig. 7) all phalanges are ossified with a phalangeal formula for fingers and toes: 2-2-3-3 and 2-2-3-4-3, respectively. order of finger length: i < ii < iv < iii, and that of toes: i < ii < v < iii < iv. distal knobs present on terminal phalanges of all fingers and toes. terminal phalanges of all toes and fingers narrower than penultimate phalanges of all toes and fingers, respectively. carpus and tarsus not well-resolved in micro-ct scan. however, carpus seems to be composed of a radiale, ulnare, element y, ossified prepollex element, carpal 2 and a large post-axial element probably representing a fusion of carpals 3–5. tarsus seems to be composed of two tarsal elements: tarsal 1 and tarsal 2 + 3, with latter being distinctly larger than tarsal 1. a moderately large centrale and small ossified prehallux are also present. in ventral view, three sesamoids of subequal sizes are overlaying proximal end of metatarsals iv–v, a further smaller sesamoid is overlaying parts of tarsal 1. distribution and natural history noblella mindo sp. nov. is only known from el cinto (0.09022°s, 78.81858°w; 1,673 m), mindo, province of pichincha, ecuador (fig. 2). noblella mindo sp. nov. inhabits secondary cloud forests, with the presence of palmito (bactris gasipaes) plantations and trees that have emerged after the massive logging of forests in the area. these forests have a high humidity index, dense leaf litter layer, and abundant epiphytes. it has a restricted distribution; sampling activities were carried out in a range up to 3km around the type locality, and no individuals nor calls of n. mindo sp. nov. were recorded. the gecko lepidoblepharis conolepis was found in sympatry. the locality is surrounded by livestock areas and within the type locality forest, there are trails used by farmers to move their livestock. the population of noblella mindo sp. nov. could be impacted if livestock activity or deforestion expands. three individuals (zsfq 049–051) were found active during the day between 10:00 and 11:00 am; all frogs were on the ground in a 2-meter depth hole. noblella worleyae reyes-puig, maynard, trageser, vieira, hamilton, lynch, culebras, kohn, brito, and guayasamin 2020: new locality figs. 2, 9–12 new records (3 females, 1 male). all individuals were collected at different localities inside los cedros biological reserve: zsfq 3851 (figs. 9–10), adult female and zfsq 3852, adult male, collected at 0.31501°n, 78.77943°w, 1,612 m (wgs84; fig. 2), garcía moreno, cotacachi, province of imbabura, by david brito-zapata and martín obando on 26 october 2019. mzuti 1708, adult female, collected at 0.31125°n, 78.78095°w, 1,417 m, by giusseppe gagliardi and jmg on 13 march 75a new species of the genus noblella from ecuador fig. 7. details of (a–d) skull morphology and osteological aspects of (e–f) hand and (g–h) foot of noblella mindo sp. nov., zsfq 050, holotype, adult female. the skull is shown in (a) dorsal, (b) ventral, (c) frontal, and (d) lateral views. alary p = alary process, angspl = angulosplenial, col = columella, dent = dentary, fpar = frontoparietal, max = maxilla, mmk = mentomeckelian bone, nas = nasal, npl = neopalatine, occ con = occipital condyle, otoc = otoccipital (fused prootic and exoccipital), pmax = premaxilla, prsph = parasphenoid, prvom = prevomer, pter =pterygoid, qj = quadratojugal, smax = septomaxilla, spheth = sphenethmoid, sq = squamosal. the right hand is shown in (e) dorsal, and (f) palmar aspects; and the left foot in (g) dorsal, and (h) plantar aspects. digits numbered i–v. antbr = os antebrachii (radius + ulna), carp d = carpale distale, cent = centrale, fib = fibulare, mtc = metacarpalia, mtt = metatarsalia, ph d i–iv = finger phalanges f1–f4, ph d i–v = toe phalanges f1–f5, prhl =prehallux, prpl = prepollex, rad = radius, tar d = tarsale distale, tib = tibiale, uln = ulnare. images prepared by claudia koch. 76 c. reyes-puig et alii 2012; mzuti 1709, adult female, collected at 0.3184°n, 78.7837°w, 1,790 m, by jaime culebras and jmg on 15 march 2012. diagnosis specimens collected in los cedros reserve show morphological characters described for noblella worleyae (reyes-puig et al., 2020; figs. 6, 9–10) as follow (variation from original description in bold): (1) skin of dorsum finely shagreen, skin on venter smooth; (2) tympanic annulus and membrane visible externally, supratympanic fold inconspicuous; (3) snout, rounded in dorsal and lateral views; (4) eyelids without tubercles; (5) dentigerous processes of vomers absent; (6) vocal slits and vocal sac present, nuptial pads not visible; (7) fingers not expanded or slightly expanded distally, tips of fingers i and iv rounded and tips of fingers ii and iii slightly acuminate (originally described as tips of fingers i and iv slightly acuminate, fingers ii and iii acuminate), without papillae (fig. 6), finger i shorter than finger ii,(8); distal phalanges t-shaped (originally described as slightly t-shaped), phalangeal formula of hands: 2-2-3-3 (fig. 11); (9) supernumerary palmar tubercles few but present, ulnar tubercles diminutive and round (decreased by preservation effects), subarticular tubercles rounded, discs lacking circumferential grooves; (10) one tarsal tubercle, elongated and subconical, two metatarsal tubercles (inner tubercle 2 times size of external); toes slightly expanded distally and rounded on toes i and v, cuspidate tips on toes ii–iv, papillae present on toes ii–iv (fig. 6); (11) toe v shorter than toe iii distal portions of circumferential grooves present on toes ii–v, supernumerary tubercles absent (12) phalangeal formula of feet: 2-2-3-43 (fig. 11); (13) in life, dorsum brown, with two suprainfig. 8. osteology of noblella mindo sp. nov., zsfq 050, holotype, adult female. the full skeleton is shown in (a) dorsal, (b) ventral, and (c) lateral views. antbr = os antebrachii (radius + ulna), clav = clavicle, cle = cleithrum, cor = coracoid bone, crur = os cruris (tibia + fibula), fem = femoral bone, fib = fibulare, hm = humeral bone, il = ilium, isch = ischium, pr p-m = processus postero-medialis, prsac v = presacral vertebrae, pub = pubis, sac v = sacral vertebra, sc = scapula, ur = urostyle, tib = tibiale. images prepared by claudia koch. 77a new species of the genus noblella from ecuador guinal dark brown marks; middorsal, longitudinal line faint cream; rictal gland dark brown; flanks with dark brown band narrowing towards groin and with clusters of turquoise specks towards ventral side; groin dark, with high concentration of melanophores; throat, chest and ventral surfaces of arms dark brown; venter yellowish cream, with brownish-orange tones on ventral surfaces of legs and thighs, iris reddish copper; (14) svl in one adult male 16.1 mm; in adult females 19.1–20.4 mm (mean 19.8 mm, n = 3) (range originally described 18.1–19.1 mm). variation of color patterns and external morphology (figs. 9–10) specimens of noblella worleyae from los cedros reserve vary from brown (mzuti 1708–1709) to dark brown (zsfq 3851–3852). in preservative, specimen zsfq 3852 has a grayish-brown dorsal coloration. black suprainguinal spots vary in size and may be diffused but are always present. all specimens exhibit a faint, cream middorsal stripe extending from the head to cloaca, but only in one specimen (mzuti 1709) this line continues onto posterior surfaces of thigh, disappears in crus, and reappears in posterior surfaces of pes. specimens mzuti 1708 and zsfq 3852 have a homogenously dark brown throat like the holotype, but in mzuti 1709 the throat is brown with scattered irregular cream marks. ventral surfaces of thighs and crus of zsfq 3852 retain pinkish-cream color in preservative. male zsfq 3852 exhibits an evident discoidal fold. osteology description osteological description of noblella worleyae is based on micro-ct images of an adult female (mzuti 1709). details of skull morphology and osteological aspects of hand and foot are presented in fig. 11 and main skeletal features are shown in fig. 12. fig. 9. color pattern of noblella worleyae in life: (a, c) dorso-lateral and ventral patterns of zsfq 3851, adult female, svl = 19.1 mm; (b, d) dorsal and ventral patterns of zsfq 3852, adult male, svl = 16.1 mm. photos by david brito-zapata. 78 c. reyes-puig et alii skull (fig. 11) skull almost as wide as long; widest part is at about where quadratojugal meets maxilla and is 97% of skull length. rostrum short; distance from anterior edge of frontoparietals to anterior face of premaxilla is 18% of skull length. at level of midorbit, braincase is about 34% of maximum skull width. braincase combines welland poorly ossified elements. prootic and exoccipital seem to be fused to form otoccipital. frontoparietals are welldeveloped bones, distinctly longer than broad, slightly narrower anteriorly than posteriorly; narrowly separated along most of their length and only fused in anterior region. posterior portion of braincase seems to be fully enclosed by partial fusion of frontoparietals with otoccipitals. however, there might still exist some traces of boarders between bones, but these parts are not wellresolved in micro-ct scans. ventrally, otoccipitals are in contact with parasphenoid alae. prootic part of otoccipitals are well-separated from each other. exoccipital parts approximate one another ventromedially and dorsomedially but are still clearly separated with a broader ventral than dorsal gap between them. anterolaterally, frontoparietals are in contact with sphenethmoid. sphenethmoid is well-ossified and ventrally fused at midline; posterior margin almost reaches midpoint of orbit but is still broadly separated from prootic part of otoccipitals and is in ventral contact with cultriform process of parasphenoid. cultriform process of parasphenoid is well-ossified posteriorly, thinning anteriorly, and about 31% of width of braincase at mid-orbit. lateral margins of process are approximately parallel. parasphenoid alae are long and well-ossified. neopalatines are very thin and long, articulating with sphenethmoid dorsomedially and maxilla laterally. columella (or stapes) is large and well-ossified. because of tiny size and fine structure, septomaxilla is not well-resolved in micro-ct scan. dorsal investing bones are moderately developed. nasals are thin and broadly separated from one another, posteriorly in contact with anterior end of frontoparietals and posterolaterally in very thin contact with maxilla. they curve ventrally towards their lateral edges. small prevomers are broadly separated from one another medially, their anterior edge approximates a long and thin posterior projecting ramus of septomaxfig. 10. color variation of preserved noblella worleyae in (a–d) dorsal and (e–h) ventral views: (a, e) mzuti 1708, adult female, svl = 20.4 mm; (b, f) mzuti 1709, adult female, svl = 19.9 mm; (c, g) zsfq 3851, adult female, svl = 19.1 mm; (d, h) zsfq 3852, adult male, svl = 16.1 mm. photos by david brito-zapata and carolina reyes-puig. 79a new species of the genus noblella from ecuador fig. 11. details of (a–d) skull morphology and osteological aspects of (e–f) hand and (g–h) foot of noblella worleyae, mzuti 1709. the skull is shown in (a) dorsal, (b) ventral, (c) frontal, and (d) lateral views. alary p = alary process, angspl = angulosplenial, col = columella, dent = dentary, fpar = frontoparietal, max = maxilla, mmk = mentomeckelian bone, nas = nasal, npl = neopalatine, occ con = occipital condyle, otoc = otoccipital (fused prootic and exoccipital), pmax = premaxilla, prsph = parasphenoid, prvom = prevomer, pter =pterygoid, qj = quadratojugal, smax = septomaxilla, spheth = sphenethmoid, sq = squamosal. the right hand is shown in (e) dorsal, and (f) palmar aspects; and the left foot in (g) dorsal, and (h) plantar aspects. digits numbered i–v. antbr = os antebrachii (radius + ulna), carp d = carpale distale, cent = centrale, fib = fibulare, mtc = metacarpalia, mtt = metatarsalia, ph d i–iv = finger phalanges f1–f4, ph d i–v = toe phalanges f1–f5, prhl =prehallux, prpl = prepollex, rad = radius, tar d = tarsale distale, tib = tibiale, uln = ulnare. images prepared by claudia koch. 80 c. reyes-puig et alii illa. maxillary arcade bears many small, poorly resolved teeth on premaxillae and maxillae. premaxillae are separated medially, and their anterodorsal alary processes rise divergent from midline but are still distinctly separated from nasals. premaxilla and maxilla are in lateral contact, with anterior edge of maxilla slightly overlapping lateral edge of premaxilla. pars palatina of premaxilla is broad, with two well-defined processes: medial process acuminate, and runs about parallel to its counterpart, being distinctly separated from it; lateral process is slightly shorter and broader. maxilla is long, its posterior end acuminate and in contact with quadratojugals. triradiate pterygoid bears a long, curved anterior ramus that is oriented anterolaterally towards maxilla, with which it articulates at ventral boarder anterior to midline of orbit. posterior ramus of pterygoid is about same length as medial ramus and both are about half length of anterior ramus; however, posterior ramus is more robust than the other two. edge of medial ramus overlaps lateral edge of prootic part of otoccipital. quadratojugal is slender, slightly curved and articulates anteriorly with maxilla and posterodorsally with ventral ramus of squamosal. squamosal is t-shaped with long laminar otic ramus; zygomatic ramus is much shorter and more slender; ventral ramus is about same length as otic ramus, laminar and broad, increasing in width ventrally. mandible is slim and edentate. mentomeckelians are small, medially and laterally slightly broadened, and medially contacting each other. dentary is long and thin, reaching to about anterior corner of orbit; posteriorly acuminate and overlapping angulospenial, seeming to be in contact with this bone for most of its length, except the most anterior part; anteroventrally it contacts mentomeckelian bones. angulosplenial is long and arcufig. 12. osteology of noblella worleyae, mzuti 1709, adult female. the full skeleton is shown in (a) dorsal, (b) ventral, and (c) lateral views. antbr = os antebrachii (radius + ulna), clav = clavicle, cle = cleithrum, cor = coracoid bone, crur = os cruris (tibia + fibula), fem = femoral bone, fib = fibulare, hm = humeral bone, il = ilium, isch = ischium, pr p-m = processus postero-medialis, prsac v = presacral vertebrae, pub = pubis, sac v = sacral vertebra, sc = scapula, ur = urostyle, tib = tibiale. images prepared by claudia koch. 81a new species of the genus noblella from ecuador ate. coronoid process is a moderately long and strongly raised ridge. the only ossified portions of hyoid apparatus are two posteromedial processes, which are anteriorly slightly more expanded than posteriorly, approaching each other at anterior ends but being still distinctly separated. postcranium (fig. 12) eight presacral vertebrae. all presacrals are nonimbricate. first presacral vertebra is longer than posterior vertebrae. all except presacral i bear well-developed diapophyses. transverse processes of presacrals v–viii similar in size and being the thinnest and second shortest, those of presacral ii being the shortest, and those of presacral iii being the longest and widest of all transverse processes. transverse processes of presacrals ii and viii have slightly anterolateral orientation, those of presacrals iii and vii are laterally oriented and others are slightly posterolaterally oriented. neural arch of all presacrals bears a raised medial ridge. sacrum bears slightly expanded diapophyses. urostyle is long, slender, about similar in length as presacral portion of vertebral column and bearing a well-pronounced dorsal ridge along about two-thirds of its length, beginning at its anterior end, with a lateral foramen in anterior region. the bone has a bicondylar articulation with the sacrum. pectoral girdle with well-ossified coracoids, clavicles, scapulae and cleithra. suprascapular, omosternum, and sternum unossified and not visible in micro-ct scan. clavicles are long and slim, oriented anteromedially, slightly curved, with medial tips approaching but not touching each other. laterally, clavicles firmly articulating with scapulae. coracoids are stout and glenoidal and sternal ends are about equally expanded. anterior edges of coracoids are curved, the posterior edges are almost straight. medial tips of coracoids are broadly separated from another. scapula is long with a prominent pars acromialis that is not separated from pars glenoidalis. cleithrum is long, broader and thicker at scapular boarder, thinning posteriorly. in pelvic girdle, long, slender iliac shafts bear conspicuous dorsolateral ridges along most of their length, except the anterior most region. ilia is fused posterirly with ischium and pubis. ischium is stout, whereas pubis is thinner and blade-like. manus and pes (fig. 11) all phalanges are ossified, with phalangeal formula for fingers and toes: 2-2-3-3 and 2-2-3-4-3, respectively. order of finger length: i < ii < iv < iii, and that of toes: i < ii < v < iii < iv. distal knobs seem to be absent on finger i but are present on terminal phalanges of all other fingers and toes. nevertheless, they are not wellresolved in micro-ct scans and are sensitive to thresholds used during reconstruction. terminal phalanges of all toes and fingers are narrower than penultimate phalanges of all toes and fingers, respectively. carpus and tarsus are not well-resolved in micro-ct scan. however, carpus seems to be composed of a radiale, ulnare, element y, ossified prepollex element, carpal 2 and a large post-axial element probably representing a fusion of carpals 3–5. tarsus seems to be composed of two tarsal elements: tarsal 1 and tarsal 2 + 3, with latter being larger than tarsal 1. a moderately large centrale and small ossified prehallux are also present. in ventral view, three sesamoids of subequal sizes overlaying proximal end of metatarsals ii–iv, a further smaller sesamoid overlaying parts of tarsal 1 and proximal end of metatarsal i. natural history we report a new locality for noblella worleyae: los cedros biological reserve, province of imbabura, ecuador (fig. 2), at approximately 8.4 km in a straight line between the type locality of n. worleyae (i.e., manduriacu reserve). the individuals were found at several point-localities between 1,417–1,790 m of elevation. the reserve presents an important track of mature low montane evergreen forest. all specimens were collected at night between 7:00 and 11:50 pm and were found on the ground, in areas covered with abundant leaf litter. individuals appeared inactive, because they were located by movement only when litter was removed. syntopic species were pristimantis mutabilis (guayasamin et al., 2017b), p. crenunguis, (lynch, 1976a) and alopoglossus viridiceps (torres-carvajal and lobos, 2014). noblella worleyae seems to be a rare species at los cedros biological reserve. only two individuals were found in two different surveys with known sampling effort. the first sampling was carried out between 22–25 august 2019, with five people for approximately nine hours per day. the second sampling was carried out between 26–30 october 2019, with two people for about nine hours per day. individuals were found in the lower forest stratum, camouflaged extremely well in leaflitter and having an evasive behavior, similar to other noblella species (reyes-puig et al., 2019c). discussion our phylogenetic analyses (fig. 1) agree with previous studies that have shown that southern species of noblella (n. losamigos, n. madreselva, n. pygmaea, n. thiuni) are more closely related to species of psychrophrynella, rather than to northern species of noblel82 c. reyes-puig et alii la (n. coloma, n. heyeri, n. lochites, n. mindo sp. nov., n. myrmecoides, n. personina, n. worleyae) (reyes-puig et al., 2019c, 2020; santa-cruz et al., 2019). noblella peruviana is the type species of the genus, which, based on geography, is most likely part of the southern clade. however, since there are no sequences of n. peruviana, we refrain from proposing a new generic arrangement. species richness of the genus noblella has increased dramatically over the last decade (frost, 2020). about a decade ago, only three species of noblella were known in ecuador and no species had been described from the northwestern slopes of the andes of ecuador (cisnerosheredia and reynolds, 2007). nowadays, there are eight species of noblella reported from ecuador, including three from the western ecuadorian andes (n. coloma, n. mindo sp. nov., and n. worleyae) that form a distinct clade among the northern noblella (fig 1). the diversity of the genus noblella has also increased in the peruvian andes, where in recent years three species been described, forming a clade with the previously described psychrophrynella and noblella (catenazzi et al., 2015; santa cruz et al., 2019; catenazzi and ttito, 2019). our new records of noblella worleyae from los cedros reserve add important intraspecific variation to the original description, in terms of its body size, coloration and shape of tips of the digits. in particular, variation in the fingertips was found, with tips of fingers i and iv varying from slightly acuminate (original description) to rounded (data presented herein), and tips of fingers ii and iii from acuminate (original description) to slightly acuminate (data presented herein). we also strengthen the original publication (reyes-puig et al., 2020) with a detailed description of the osteology of the species. diversification in noblella seems be related with the linearity of the andes, with allopatric and parapatric populations being separated by ecological and geographic barriers. in ecuador, all species of noblella are allopatric and most of them are restricted to very specific geographic areas. noblella mindo sp. nov. occurs in low montane forest in the valley of mindo, in the nambillo river watershed, western slopes of the pichincha massif, northwestern andes, at 1,673 m; while n. worleyae inhabits low montane forest in the manduriacu-los cedros watersheds, southern slopes of the toisan massif; and n. coloma is restricted to the cloud forests of the río guajalito watershed, western slopes of the atacazo volcano. noblella worleyae is separated from n. coloma and n. mindo sp. nov. by the guayllabamba river valley (fig. 2), an important biogeographic barrier, especially for frog species with low vagility (hillman et al., 2014). although the valley of mindo (type locality of n. mindo sp. nov.) and the valley of guajalito (type locality of n. coloma) are ca. 20 km apart in straight line, they are in different watersheds, separated by the nambillo river and complex orogeny caused by the pichincha massif and the atacazo volcano. all species currently recognized under the genus noblella are miniaturized frogs, among the smallest neotropical vertebrates (duellman and lehr, 2009). they are cryptic and adapted to live amidst or under leaf litter in forests, where they are often overlooked by amphibian visual surveys, being easier to locate by their calls or through pitfall traps (reyes-puig et al., 2019c). although some species may be abundant locally, most species appear to have low densities. for example, yearly surveys between 2000–2012 at the type locality of n. coloma produced only three records, while up to 20 individuals of n. lochites were found in 2014 at a single locality in the province of zamora-chinchipe (d. f. cisneros-heredia pers. obs.). most remnants of mature forests in the andes of ecuador are nowadays either inside public or private protected areas or persist due to their inaccessibility. private conservation initiatives have become extremely important in ecuador (betancourt et al., 2018; guayasamin et al., 2018, 2019; reyes-puig et al., 2019a, 2019b, 2019c), where public protected areas do not cover all critically important regions for biodiversity (lessmann et al., 2014; cuesta et al., 2017; reyes-puig et al., 2017). unfortunately, habitat loss due to unsustainable expansion of the agricultural frontier, mining and infrastructure projects have placed a heavy burden on several private reserves (roy et al., 2018; guayasamin et al., 2019). in the early 2000s, the region of mindo was heavily threatened by the construction of an oil pipeline (oleoducto de crudos pesados ocp). fortunately, local, national and international protests managed to promote some actions to mitigate the largest impacts of the pipeline development. eventually mindo has transformed into one of the most popular ecotourism destinations in northwestern ecuador, allowing several private protected areas to preserve large tracks of mature forest (welford and yarbrough, 2015). unfortunately, twenty years later, history repeats itself, now at los cedros reserve, but this time with mining concessions, exploration and exploitation (roy et al., 2018). thus, biodiversity conservation is facing an uncertain future. the increasing descriptions of new species within the ecuadorian territory have a practical application in the conservation of biodiversity. by highlighting the presence of new vertebrates with restricted distributions in the andes, the visualization of this unique biodiversity is indisputable. 83a new species of the genus noblella from ecuador acknowledgements we conducted this research under research permits and agreement for genetic resources access (mae-dnbcm-2018-0106, 019-2018-ic-fau-dnb/mae) issued by ministerio del ambiente del ecuador. we carried out this study following the guidelines for treatment and management of live amphibians and reptiles in field and laboratory investigations (beaupre et al., 2004), recommended by the american society of ichthyologists and herpetologists, the herpetologists’ league and the society for the study of amphibians and reptiles. this study was developed as part of “proyecto descubre napo”, an initiative of universidad san francisco de quito in association with wildlife conservation society, and funded by the gordon and betty moore foundation, as part of the project “wcs consolidating conservation of critical landscapes (mosaics) in the andes”. we express our gratitude to the following people for their support: ana nicole acosta-vásconez, susana cárdenas, mariela domínguez, jonathan guillemot, andrés leónreyes, emilia peñaherrera, carolina proaño, robert p. reynolds, alejandra robledo, ana sevilla, rebecca zug. work at los cedros biological reserve was developed as part of project “muestreo de grupos ecológicos clave”, research program “evaluación biológica rápida del corredor norte de la reserva de mashpi”, a joint initiative of fundación futuro and universidad san francisco de quito usfq (instituto ecolap, museo de zoología, instituto ibiotrop). this research was supported by universidad san francisco de quito usfq through research funds for the museo de zoología & laboratorio de zoología terrestre, instituto de diversidad biológica y tropical ibiotrop granted to dfch; usfq collaboration grants and cociba grants (project hubi id 34, 39, 48, 1057, 7703, 12268, 13524) granted to dfch and crp; and collaboration grants and cociba grants (project hubi id 5521, 5467, 5447, 11164, 16871) granted by usfq to jmg. work by dfch was supported by programa “becas de excelencia”, secretaría de educación superior, ciencia, tecnología e innovación (senescyt), ecuador; the smithsonian women’s committee, the 2002 research training program, national museum of natural history, smithsonian institution; maría elena heredia and laura heredia. we thank the inédita program from the ecuadorian science agency senescyt (respuestas a la crisis de biodiversidad: la descripción de especies como herramienta de conservación; inedita pic-20-ine-usfq-001) that funded the molecular component of this study. we are grateful 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[garli: genetic algorithm for rapid likelihood inference. available for download at: http://www.bio. utexas.edu/faculty/antisense/garli/garli.html. appendix i examined specimens. noblella lochites. ecuador, napo: zsfq 346, archidona, reserva narupa, 1176 m; zsfq 347, reserva narupa, 1152 m; zsfq 348, reserva narupa, 1167 m; zamora chinchipe: zsfq 1119, yantzaza, concesión la zarza, 1385 m; zsfq 1124, concesión la zarza, 1357 m; zsfq 1186, zsfq 1187, zsfq 1188, yantzaza, río blanco, 1654 m; zsfq 1188, río blanco, 1830 m. noblella cf. lochites. ecuador, zamora chinchipe: zsfq 3262 – 326, yantzaza, estación experimental el padmi unl, 775 m. noblella myrmecoides: ecuador, napo: zsfq 670, mera, parque nacional llanganates, 1325 m; zsfq 671, parque nacional llanganates, 1352 m; zsfq 672, parque nacional llanganates, 1327 m. noblella cf. myrmecoides. ecuador, tungurahua: zsfq 1341, río negro, reserva río zuñag, 1269 m. noblella coloma. ecuador: pichincha: qcaz 7277, 7412, 8701, 11614, 26307, 32702, reserva ecológica río guajalito; 1800–2000 m. noblella heyeri. ecuador, loja: qcaz 31470, 31471, 31473, loja–zamora road; 2385 m, qcaz 22501, zamora-huaico; 2000 m. noblella worleyae. ecuador, imbabura: zsfq 345, 550, 551, 552, 2502, 2503, 2504, reserva manduriacu, 1184–1597 m. acta herpetologica vol. 16, n. 2 december 2021 firenze university press a new species of the genus noblella (amphibia: strabomantidae) from ecuador, with new information for noblella worleyae carolina reyes-puig1,2,3,4,*, juan m. guayasamin 2,5 claudia koch6, david brito-zapata1, matthijs hollanders7, melissa costales8, diego f. cisneros-heredia1,2,3 so close so different: what makes the difference? dario ottonello1,7,*, stefania d’angelo2, fabrizio oneto3,6, stefano malavasi1, marco alberto luca zuffi4, filippo spadola5 hematological values of wild caiman latirostris (daudin, 1802) in the atlantic rainforest in pernambuco, brazil luciana c. rameh-de-albuquerque1, alexandre p. zanotti1, denisson s. souza1, george t. diniz2, paulo b. mascarenhas-junior3,4,5,*, ednilza m. santos³, jozelia m. s. correia3 bone histology of broad-snouted caiman caiman latirostris (crocodylia: alligatoridae) as tool for morphophysiological inferences in crocodylia paulo braga mascarenhas-junior1,2,3,6, luis antonio bochetti bassetti4, juliana manso sayão5,6 is the northern spectacled salamander salamandrina perspicillata aposematic? a preliminary test with clay models giacomo barbieri, andrea costa, sebastiano salvidio* sexual size dimorphism in the tail length of the caspian whip snakes, dolichophis caspius (serpentes, colubridae), in south-western hungary györgy dudás1, krisztián frank2* semi-automated photo-identification of bahamian racers (cubophis vudii vudii) sebastian hoefer1,*, andreu rotger2, sophie mills1, nathan j. robinson1,3 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 6(1): 27-34, 2011 genetic characterization of over hundred years old caretta caretta specimens from italian and maltese museums luisa garofalo1, john j. borg2, rossella carlini3, luca mizzan4, nicola novarini4, giovanni scillitani5, andrea novelletto1 1 dipartimento di biologia, università di roma “tor vergata”, via della ricerca scientifica, i-00133, roma, italy. corresponding author. e-mail: luisa.garofalo@uniroma2.it, novelletto@bio.uniroma2.it 2 national museum of natural history, vilhena palace, st. publius square, rbt-12, mdina, malta. e-mail: john.j.borg@gov.mt 3 museo civico di zoologia, via ulisse aldrovandi 18, i-00197, roma, italy. e-mail: rossella.carlini@ comune.roma.it 4 museo di storia naturale di venezia, santa croce 1730, i-30125, venezia, italy. e-mail: luca.mizzan@comune.venezia.it, nicola.novarini@fmcvenezia.it 5 dipartimento di biologia, sezione di biologia animale ed ambientale, laboratorio di istologia ed anatomia comparata, università degli studi di bari aldo moro, via orabona 4/a, i-70126, bari, italy. e-mail:g.scillitani@biologia.uniba.it submitted on: 2010,5th november; revised on: 2011, 14th april; accepted on: 2011, 20th april. abstract. museum collections have proven to be a useful source of samples for the reconstruction of evolutionary history and phylogeography of many taxa. this study was aimed at assessing the success rate in a genetic analysis of historical material, in order to explore the feasibility and eventually begin the diachronic description of caretta caretta stocks in italian and maltese coastal waters. the endangered status of the species and the difficulty to study it in the wild make its common occurrence in italian museum collections a valuable resource. we used minimally invasive methods to collect biological material from specimens dating from the end of the 19th century to 2003, belonging to four museums. as a control for amplification success and absence of cross-contamination, four dinucleotide microsatellite loci of different average length (cc7, cc141, cm72 and cm84) were typed. all individuals with two or more successfully amplified microsatellites (36%) displayed distinct genotypes, thus excluding contamination as a major flaw in the data. we then targeted 380 bp of the mtdna control region, which allows comparisons with many living populations worldwide and represents the optimal marker for the philopatric behaviour of this species. all individuals but 2 were successfully sequenced. haplotype cc-a2 was found in 68 individuals, whereas cc-a1 and cc-a3 were found only in one tyrrhenian and one s-adriatic specimens, respectively. this study demonstrates that genetic analysis of marine turtles from museum specimens is feasible. data generated from cohorts of several generations ago are potentially useful for research and dissemination purposes. 28 l. garofalo, j.j. borg, r. carlini, l. mizzan, n. novarini, g. scillitani and a. novelletto keywords. historical collections, museum specimens, marine turtles, caretta caretta, molecular markers, mtdna, microsatellites. introduction museum collections have proven a useful source of samples for the reconstruction of the evolutionary history and phylogeography of many taxa (wandeler et al., 2007). so far genetic analysis of museum specimens of turtles has been scanty (e.g. parham et al., 2006; feldman & parham, 2004; parham et al., 2004; austin et al., 2003), yet revealing relevant hidden quotas of diversity (poulakakis et al., 2008). studies on marine turtle specimens from museum collections in italy have never been conducted before, despite the availability of many chelonian collections (e.g. mizzan, 1994; mazzotti, 2010). genotyping of specimens from museums and other natural history collections is exposed to low success rate and a high risk of contamination, mainly due to low nondegraded dna yields (taberlet and luikart, 1999; wandeler et al., 2007). time elapsed since preservation (specimen age) is not the only important factor affecting dna yield and quality. different preservation techniques may negatively influence the possibility to extract, amplify and sequence dna. in addition, nucleotide misincorporation during amplification of damaged dna or allelic dropout in microsatellite analysis can lead to the overor under-estimation of genetic diversity in past populations, respectively (wandeler et al., 2007). however, providing that these factors are kept under control, the sampling of museum-preserved material has the advantages of being promptly available and of reducing the needs for tissues from living individuals. this may be particularly valuable for species that are both endangered and difficult to study in the field. we address here the most common marine turtle in the mediterranean, caretta caretta, which is widely represented in italian museum collections. this species is suffering a dramatic reduction in the size of its foraging and reproductive stocks (current iucn classification: endangered). thus, genetic analysis of museum samples may be important to catch changes in the pattern of diversity across generations, associated with population fluctuations and/or environmental shifts. we then aimed at assessing the success rate in a genetic analysis of historical c. caretta material through minimally invasive sampling methods, and eventually commence the description of the diachronic composition of c. caretta stocks in italian and maltese coastal waters. materials and methods the samples contributed by different museums, partitioned according to their geographic origin and preservation period, are summarized in table 1. the preserved starting material for dna preparation consisted of dried bones (skulls, vertebrae, carapaces and nails), specimens in ethanol and ethnographic artifacts (painted carapaces). each specimen was labeled with the museum’s catalogue number and/or cites id. only one source 29genetic characterization of caretta caretta from museums of material was used for each specimen. sample collection and subsequent molecular analyses were carried out in controlled conditions. for dried material (including ethnographic artifacts), powder was obtained by means of minimally invasive drilling (drill bit diameter = 6 mm) on the ventral side of carapace or from hidden sites of bones and skulls, after discarding the superficial part; holes were later sealed with bee wax. for internal parts, remains of meninges and spinal cord were collected by gentle grasping the inner side of neural arches of neck and trunk vertebrae of preserved skeletons, carapaces and skulls. for liquid-preserved material a few mm3 tissue biopsy was obtained from the front flipper with a sterile blazer, after discarding the superficial part (epidermis) (table 2). while the majority of specimens could be measured and assigned to three major age classes, information on sex was available only if recorded at the time of collection or for large, stuffed and mounted whole animals (table 3). no more than 10 samples were extracted simultaneously (nucleospin tissue kit, macherey nagel gmbh, duren, germany), to decrease the risk of cross-contamination. as a control for amplification success and absence of cross-contamination, typing of dnas at four dinucleotide microsatellite loci of different average length (cc7, cc141, cm72 and cm84, in order of increasing molecular weight) (carreras et al., 2007, and conditions reported therein) was performed. this choice was dictated by the high polymorphic content of the loci and the possibility of comparing our results with literature data. a fragment of 380 bp of the mitochondrial (mtdna) control region, obtained using the primers tcr5 and tcr6 (norman et al. 1994) was amplified (annealing temp. 52°c; 32-36 cycles) and sequenced. a mtdna fragment 815 bp long was sequenced from a single sample carrying haplotype cc-a1, in the conditions described by garofalo et al. (2009). in all cases sequencing was carried out with standard protocols for an abi3100 avant automated sequencer on both strands, with the same primers used for pcr. electropherograms were visually inspected and sequences aligned with the bioedit software (hall, 1999). mitochondrial dna haplotype nomenclature follows that reported by the archie carr center for sea turtle research (http://accstr.ufl.edu/ccmtdna.html). in each lot of samples subjected to pcr, negative controls were performed. pcr and sequencing of the same sample was performed twice (cooper and poinar, 2000), by the same researcher. results sample composition the sample series reported here represent the totality of individuals available in the four museum collections analyzed (listed in table 1) up to 2009. most of the samples had been collected within the last 50 years (table 1), whereas 12.5% (9/72) are older than 1900. the majority of specimens consisted of dried-preserved parts (table 2), i.e. entire skeletons or isolated carapaces and crania. the large osteological collections of rome and venice contributed the majority of samples, leading likely to an overrepresentation of individuals from the tyrrhenian and n-adriatic seas in the overall series (table 1). the proportion of juveniles and adults is close to 50:50 in all geographic samples. subadults were found only among tyrrhenian specimens. in addition, while an even proportion of sexes was found among the n-adriatic specimens, the tyrrhenian adults of known sex were all females (table 3). 30 l. garofalo, j.j. borg, r. carlini, l. mizzan, n. novarini, g. scillitani and a. novelletto control amplifications. loci cc7, cc141, cm72 and cm84 were amplifiable in 35, 29, 23 and 16 individuals, respectively (table 4). as expected for fragmented dna, longer loci displayed a reduced amplification rate [called “molecular behavior” in cooper and poinar, 2000]. overall, 26 out of 72 samples could be amplified for at least two microsatellites, with replicable results. tables 1 and 2 show the success rate in amplifying microsatellite loci according to different variables. recent samples (1950-2003) produced good results in about 50% of instances, i.e. slightly better than average. within this category six samples were older than 10 years. moreover, amplification of dna from material dating back to before 1950 was also possible. as to preservation methods, fluid-preserved specitable 1. sampling coverage and results according to the date of collection. each entry reports the number of individuals successfully amplified at least at two microsatellite loci out of the total sampled from each institution. source institution codes are: mczrm (museo civico di zoologia, rome), msnve (museo di storia naturale di venezia, venice), mzuba (museo di zoologia, università di bari), nmnhm (national museum of natural history, mdina, malta), n.a. = not assigned. source geographic origin before 1900 1900 -1950 1950 2003 n. a. total mczrm tyrrhenian 2 / 8 10 / 29 2 / 4 14 / 41 msnve n-adriatic 1 / 3 4 / 8 2 / 10 7 / 21 mzuba s-adriatic 0 / 1 3 / 4 3 / 5 nmnhm malta 2 / 5 2 / 5 table 2. amplification success relative to preservation method of the original samples, shown as the number of individuals successfully amplified (at least at two microsatellite loci) out of all available individuals for each method. groups dried bonesa fluid specimens internal parts tyrrhenian 14 / 41 n-adriatic 5 / 19 2 / 2 s-adriatic 2 / 3 1 / 2 malta 2 / 4 0 / 1 a: includes ethnographic artifacts. table 3. number of samples analyzed for each location and life stage (n.a. = not assigned). individuals were considered juveniles if ccl < 50 cm, subadults if between 50 and 60 cm, and adults if > 60 cm. only adults can be externally sexed. sampling location n° of samples life stage sex juveniles subadults adults n. a. f m tyrrhenian 41 15 11 15 7 n-adriatic 21 9 8 4 2 2 s-adriatic 5 2 1 2 malta 5 2 3 31genetic characterization of caretta caretta from museums mens too yielded an adequate dna quantity and quality for genetic analysis. however, bone is usually the preferred material for this kind of analysis, due to the high degree of conservation and the lower risk of external contamination. all individuals with two or more successfully amplified microsatellites displayed distinct genotypes. this excluded contamination as a major flaw in the data. allele ranges at all the four str loci (table 4) fitted those of mediterranean nesting populations (carreras et al., 2007) and do not support a relevant presence of atlantic alleles (reported in moore and ball, 2002). as to a more precise discrimination among different sources in the mediterranean, this is currently not possible due to the lack of geographically-confined allele sizes (carreras et al., 2007). genetics. mtdna. pcr amplification of the mtdna control region was attempted in all samples, in view of the larger representation of this target in cellular material (mulligan, 2005). in no instances negative controls produced amplified material at the level of sensitivity of agarose gels. overall 70 individuals, i.e. all but two (one from venice [1988] and one from bari [muzac collection, 1742]), were successfully sequenced for the 380 bp in mtdna in a single pcr reaction. sequencing of both strands produced traces overlapping for 350 positions. in all cases a perfect overlapping was obtained, showing that electropherogram interpretation was unambiguous. in all cases the results of two independent dna extracts of the same specimen or pcr reactions of the same extract produced identical results. haplotype cc-a2 was found in 68 individuals, whereas cc-a1 and cc-a3 were found only in one tyrrhenian (juvenile, 2002) and one s-adriatic (juvenile, 1980’s) specimens, respectively. haplotype cc-a3 differs from cc-a2 by a single nucleotide substitution out of the 380 bp segment scored, while haplotype cc-a1 is at 35 mutational steps from cc-a2. in order to further confirm the reliability of this latter result, the cc-a1 sample was amplified and sequenced also for a 815 bp fragment, flanking the previous one on both sides. the resulting sequence is identical to the cc-a1 subtype cc-a1.1. the fact that variation was observed only at positions already known to vary in mediterranean and atlantic haplotypes shows that pcr and sequencing did not introduce artifacts in our dna series as far as mtdna is concerned. table 4. allelic range at each microsatellite locus by location. each entry reports the minimum and maximum allele size in bp (above) and the number of individuals successfully amplified (below). groups cc7 cc141 cm72 cm84 tyrrhenian 167-203 n = 17 187-207 n = 14 223-249 n = 12 313-323 n = 10 n-adriatic 163-197 n = 10 197-203 n = 7 223-247 n = 7 313-323 n = 3 s-adriatic 171-193 n = 4 183-203 n = 4 223-231 n = 2 315-323 n = 1 malta 171-191 n = 4 187-207 n = 4 223-241 n = 2 313-323 n = 2 32 l. garofalo, j.j. borg, r. carlini, l. mizzan, n. novarini, g. scillitani and a. novelletto discussion almost all individuals from our comprehensive series could be sequenced at the mtdna control region, and the success rate of microsatellites genotyping was inversely related to the length of the targeted fragment. these results demonstrate that, adopting good laboratory practices and proper techniques, dna contamination of old samples can be largely avoided, producing reliable genotyping results in roughly 1/3 of samples up to 50 years old. the higher rate of mtdna genotyping, though somewhat expected, awaits further confirmation by resampling of the same specimens and testing in an independent laboratory, to fulfill the recommended guidelines (willerslev and cooper, 2005). we nevertheless show that genetic analysis of marine turtle museum specimens is feasible and can potentially provide data on cohorts of several generations ago. one consequence of the complex migratory pattern of marine turtles is that many different variables may affect the genetic composition of a population stock and samples drawn from it. by controlling for the original provenance of each museum specimen (e.g. strandings, bycatch), our series can be compared with modern samples of similar origin. in interpreting our results the strong philopatric behaviour of females must be taken into account. under these circumstances mtdna becomes, by and large, a marker for the geographic origin of the maternal 50% of an individual’s genome. conversely, the origin of the paternal component is uncertain, due to the loose philopatry of males to breeding grounds. our overall data indicate that most individuals collected along italian coasts can be traced to a generic maternal “mediterranean” source, given that the most common mtdna haplotype in our series is cc-a2, also shared by all mediterranean nesting populations. the finding of a single carrier of haplotype cc-a1, observed only in atlantic nesting colonies, testifies the presence of sporadic juveniles from this ocean in central and western mediterranean feeding aggregates. the individual carrying haplotype cc-a3 probably came from turkish nesting sites, where this haplotype is common. similar frequencies for both haplotypes have been reported by maffucci et al. (2006). all individuals from malta showed haplotype cc-a2. given that these specimens dated back to the 1970-1980’s, they can potentially represent the remnants of the last generation born in the island (a recent report of breeding in malta, sometime in the 1960s, was reported by deidun and schembri, 2005). in this case, this nesting colony as well would have hosted the haplotype shared by all mediterranean rookeries. several authors alerted on the limitations of molecular analyses of material with low dna content (cooper and poinar, 2000; pääbo et al., 2004; taberlet and luikart, 1999; willerslev and cooper, 2005). however, they concluded that dna from historical specimens adds an important temporal dimension to the genetic study of endangered species. studies of this kind remain pivotal in conservation genetics, for their power in revealing changes in population size and structure, phylogenetic relationships and to define evolutionary significant (and management) units (wandeler et al., 2007). if extended to additional series (for a comprehensive description of italian collections see mazzotti, 2010) the combined use of genetic data and information on place and date of collection, sex, life stage and cause of death, can produce valuable knowledge on a species that is currently endangered, and is also difficult to study in the wild. 33genetic characterization of caretta caretta from museums the present work is potentially relevant for the knowledge of the biology of c. caretta. in fact, the phylogeographic information here obtained can be easily incorporated into an “identity card” of the preserved specimens, that will augment their value in case of a re-examination for research purposes. moreover, the display of the same card in exhibits will also serve dissemination purposes, by rising public interest towards genetics and conservation issues, and promoting museum collections as irreplaceable banks of the world’s biodiversity. acknowledgements work supported by “tor vergata” university of rome (rsa funds) and miur (“iniziative a favore della diffusione della cultura scientifica”). we are grateful to s. angilletti, t. fattizzo, p. reggiani, l. rositani, f. trimigliozzi, p. ventrella, c. vianello and all the scientific staff of the civic museum of zoology in rome for their collaboration during sample collection. we would like to thank also dr. gabrielle zammit for the help in the establishment of the collaboration with the maltese institutions. an anonymous referee highly improved our ms draft. references austin, j.j., arnold, e.n., bour, r. 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(2005): ancient dna. proc. r. soc. b 272: 3-16. bbib67 ole_link1 ole_link2 bbib28 ole_link5 ole_link6 ole_link7 ole_link8 _goback ole_link1 ole_link2 ole_link3 ole_link4 ole_link1 ole_link2 ole_link19 ole_link20 ole_link21 ole_link29 ole_link3 ole_link4 ole_link5 ole_link31 ole_link14 ole_link15 ole_link12 ole_link13 ole_link16 ole_link17 ole_link22 ole_link23 ole_link24 ole_link8 ole_link9 ole_link10 ole_link11 ole_link18 ole_link27 ole_link28 ole_link25 ole_link26 ole_link6 ole_link7 ole_link34 ole_link37 ole_link38 acta herpetologica vol. 6, n. 1 june 2011 firenze university press widespread bacterial infection affecting rana temporaria tadpoles in mountain areas rocco tiberti extreme feeding behaviours in the italian wall lizard, podarcis siculus massimo capula1, gaetano aloise2 lissotriton vulgaris paedomorphs in south-western romania: a consequence of a human modified habitat? severus d. covaciu-marcov*, istvan sas, alfred ş. cicort-lucaciu, horia v. bogdan body size and reproductive characteristics of paedomorphic and metamorphic individuals of the northern banded newt (ommatotriton ophryticus) eyup başkale1, ferah sayım2 , uğur kaya2 genetic characterization of over hundred years old caretta caretta specimens from italian and maltese museums luisa garofalo1, john j. borg2, rossella carlini3, luca mizzan4, nicola novarini4, giovanni scillitani5, andrea novelletto1 the phylogenetic position of lygodactylus angularis and the utility of using the 16s rdna gene for delimiting species in lygodactylus (squamata, gekkonidae) riccardo castiglia*, flavia annesi localization of glucagon and insulin cells and its variation with respect to physiological events in eutropis carinata vidya. r. chandavar1, prakash. r. naik2* the balearic herpetofauna: a species update and a review on the evidence samuel pinya1, miguel a. carretero2 effects of mosquitofish (gambusia affinis) cues on wood frog (lithobates sylvaticus) tadpole activity katherine f. buttermore, paige n. litkenhaus, danielle c. torpey, geoffrey r. smith*, jessica e. rettig food composition of uludağ frog, rana macrocnemis boulenger, 1885 in uludağ (bursa, turkey) kerim çiçek preliminary results on tail energetics in the moorish gecko, tarentola mauritanica tommaso cencetti1,2, piera poli3, marcello mele3, marco a.l. zuffi1 climate change and peripheral populations: predictions for a relict mediterranean viper josé c. brito1, soumia fahd 2, fernando martínez-freiría1, pedro tarroso1, said larbes3, juan m. pleguezuelos4, xavier santos5 assessing the status of amphibian breeding sites in italy: a national survey societas herpetologica italica* osservatorio erpetologico italiano acta herpetologica journal of the societas herpetologica italica acta herpetologica rivista della societas herpetologica italica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 7(1): 57-68, 2012 altitudinal variation in body size in the rice frog (rana limnocharis) in southwestern china yan hong liu1,2, yu zeng1,2, wen bo liao1,2,*, cai quan zhou1,2,*, zhi ping mi1,2, min mao1,2, lin chen1,2 1key laboratory of southwest china wildlife resources conservation (ministry of education), china west normal university, nanchong, 637009, p. r. china. *corresponding authors. e-mail: liaobo_0_0@126.com; drcqzhou1@163.com 2institute of rare animals and plants, china west normal university, nanchong 637009, p. r. china. submitted on: 2010, 23rd december; revised on 2011, 30th may; accepted on 2012, 3rd february. abstract. bergmann’s rule states that, within species of endotherms, individuals tend to be larger in cooler environments, and has been reputed to apply to some ectotherms including amphibians. however, the validity of the rule has been debated, questioning whether bergmann’s clines are generally present in anurans. in the view, we studied altitudinal variation in body size in the rice frog (rana limnocharis) among populations from sichuan province located at three altitudes to find if there exist any differences in a relatively small altitudinal range (290-375 m). the results showed that individuals from higher altitudes tended to be larger in body size than lower altitudes, which was consistent with bergmann’s cline. moreover, when the effect of age was removed, variation in body size of the frogs across altitudes still remained. our findings suggested that age affected the pattern of variation in body size across the altitudinal cline, we also discussed that factors other than age also contributed to size differences among populations. keywords. altitudinal gradient; age; bergmann’s cline; body size; rice frog (rana limnocharis). introduction large-scale variation patterns of body sizes, which crucially contributes to the life history differentiation among populations (sambucetti et al., 2006), are central themes in evolutionary ecology. the body size of animals increases from warm to cool climates (bergmann, 1847; mayr, 1956). this trend, known as bergmann’s rule, which is currently defined as a within species or among closely related species, holds for body size variation in the majority of endothermic vertebrates (ashton, 2002; blackburn and hawkins, 58 y.h. liu et al. 2004) and the mechanism underlying may be understood with regarding to heat conservation benefit enjoyed by bigger individuals in cooler climates (walters and hassall, 2006). among ectotherms, however, no consistent clines are apparent, with some taxa following the rule (ashton, 2002, 2004; blanckenhorn and demont, 2004; atkinson and sibly, 1997; olalla-tárraga and rodríguez, 2007; ficetola et al., 2010; liao and lu, 2011a; meiri, 2011), others not following it (laugen et al., 2005; romano and ficetola, 2010; watt et al., 2010; liao and lu, 2011b), and many conversing it (mousseau, 1997; ashton and feldman, 2003; liao et al., 2010; partridge and coyne, 1997; walters and hassall, 2006). among various studied species, amphibians are particularly interesting (ashton, 2002; laugen at al., 2005; olalla-tárraga and rodríguez, 2007; adams and church, 2008). however, empirical evidence for the prevalence of bergmann’s clines in amphibians is still controversial. altitude as one geographical gradient that create environmental variation mainly in temperature and season time length may influence life histories of ectothermic organisms. numerous studies have indicated that altitudinal comparisons in age, growth and body size are particularly useful (berven, 1982; blanckenhorn and demont, 2004; lu et al., 2006; liao and lu, 2010a; liao et al., 2010, 2011). especially sampling a relatively small altitudinal range can allow us to estimate variation in age, growth and body size more accurately due to the influences of environmental factors on breeding time and developmental patterns (duellman and trueb, 1994). the rice frog (rana limnocharis) is a common species, widely distributed in southeastern asian (liu, 1950; zhao and adler, 1993). in china it is distributed in a wide range area in terms of altitudes (2-2,000 m) (fei and ye, 2001; liao et al., 2011). this species is classified as a prolonged breeder due to egg-laying lasting from early april to mid-september (wells, 1977). although population dynamic, age structure, breeding ecology, habitat use and morphological and genetic divergences in different populations have reported in recent years (fei and ye, 2001; zhou, 2001; wang et al., 2006; liao et al., 2011), no information about variation in body size of the species along a relatively small altitudinal range is available. here, we described the altitudinal variation in body size of three rana limnocharis populations from different elevation sites (290 to 375 m a.s.l.) in sichuan, china. our aims were to: (1) test whether age and body size vary with altitude, and any observed changes follow the pattern displayed by other amphibians; (2) confirm whether body size and age differ significantly between the sexes; (3) test whether altitude and sex affect body size. materials and methods in this study, three rana limnocharis populations were collected from yonghong, shengzhong and gaoping sites in may 2010, at different altitudes: 375 m, 364 m and 290 m, respectively. altitude, latitude, longitude and annual average temperature of the three sites and the number of individuals captured were shown in table 1. annual mean air temperatures in the three sites decrease significantly with altitude (pearson’s correlation coefficient: r = -0.999, n = 3, p = 0.024). the three studied populations situated in nanchong city occurs in paddyfields with approximately 150 m length and 50 m width. the vegetations at the sampling sites are characterized by silvergrass (miscanthus floridulus), common bread wheat (triticum aestivum), bulrush (phragmites australis), eucalyptus (eucalyptus robusta) and oriental arborvitae (platycladus orientalis). 59altitudinal variation in rana limnocharis we caught all individuals by hand at night using a flashlight. we confirmed the sex of each individual by direct observation of the secondary sexual characteristics (i.e., the vocal sacs in adult males and the ova in adult females). we measured body size (the snout–vent length: svl) of all individuals using a vernier caliper with an accuracy of 0.1 mm. we removed the second phalange of the hind limb longest finger and stored them in 10% neutral buffered formalin for skeletochronology. some individuals were subsequently released at the point of capture and the others were taken in lab to study sperm morphology. individual age was estimated by skeletochronology (see castanet and smirina, 1990; liao and lu, 2010b; li et al., 2010). we removed the skin and muscle tissues of each digit, and decalcified the remaining bones in 5% nitric acid for 48 h. then, we washed them in running tap water for 24 h. we stained the decalcified digits for 150 min in harris’ haematoxylin and rinsed with distilled water. subsequently, we dehydrated these stained bones through successive ethanol stages for 1 h in each concentration. tissues were embedded in small paraffin blocks. we cross-sectioned the diaphyseal region of each phalanx at a thickness of 8 μm and selected the smallest medullar cavity of the sections to examine lags with a leitz dialux 40 microscope, and photographed the best sections using a motic ba300 digital camera mounted on a moticam2006 light microscope at × 400 magnifications. as suggested by the authors (lu et al., 2006; liao et al., 2011; liao 2011), we assumed that each lag corresponds to an annual arrest of individual growth owing to consideration in the microclimatic parameters of the sampling area where frogs regularly overwintered from december to march. all fingers were collected after two months of emergence from hibernation, so we did not count the outer margin of the bone as an additional lag. endosteal resorption of long bones starts from the inner surface of the bone, enlarging the marrow cavities and eroding a portion of lags when frogs have completed their hibernation (rozenblut and ogielska, 2005). following the protocol of rozenblut and ogielska (2005), we confirmed the first lag endosteal resorption of bone based on the occurrence of the kastschenko line (kl; the interface between the endosteal and periosteal zones). we applied student’s t-test to test differences in body size and age between males and females within each population when the statistic samples match the assumption for parametric statistic. the correlation between body size and age within each population for each sex was evaluated using linear regression. for each sex, differences in average body size among populations were tested using general linear models (glms) with the helmert contrast treating svl as a dependent variable and population as a fixed factor. we performed bonferroni post hoc multiple comparisons (fisher’s lsd) to evaluate differences between pairs of populations when there was a significant difference among populations in body size. general linear models were then used with age as covariate to see whether differences in body size among populations still remained after removing the effects of age. differences in mean adult age among populations were also tested using general linear models and bonferroni post hoc multiple comparisons. all probabilities were two-tailed, and the significant level was set at a = 0.05. means were given ± sd. all statistical testes were performed using spss software version 13.0. table 1. resources, altitudes and locations of rana limnocharis sampled from three localities in southwestern china. altitude (m) sites north latitude east longitude annual average temperature (°c) no. of males no. of females 375 yonghong 31°26’ 105°45’ 16.9 15 20 364 shengzhong 31°29’ 105°45’ 17.0 55 10 290 gaoping 30°48’ 106°06’ 17.5 26 12 60 y.h. liu et al. results we captured 141 individuals in the three populations. a series of narrow concentric hematoxylinophilic rings or lines separated by wider layers of paler background with sparsely distributed osteocytes was observed in phalanges for 138 frogs (96 males and 42 females) of 97.9% (fig. 1). two males and one female were damaged or had inadequate bone sections that did not allow for proper analysis. false and double lines were rarely observed and were not considered as true lags in all samplings. endosteal resorption affected only the first lag and it was rarely observed in all individuals based on the kastschenko line. there was not significant difference in average age between the sexes for all population (table 2; fig. 2). age at sexual maturity was 1 year in males and females. maximum longevity was 4 years for both males and females (fig. 2). in terms of age composition, dominant age was 1 and 2 yrs in males, while it was 2 and 3 yrs in females (fig. 2). average body size differed significantly between males and females at the 375-m and 364-m sites, but not at the 290-m site (table 2). within each age group, females had larger body size than males at the 375-m and 364-m sites, while body sizes in males were larger than females at the 290-m site (table 3). there was positive correlation between body size and age for only one sex within each population (fig. 3; linear regression: 375-m site: males, svl = -0.53 age + 40.50, f1,14 = 0.84, r = 0.25, p = 0.38; females, svl = 2.37 age + 37.57, f1,19 = 5.09, r = 0.47, p = 0.04; 364-m site: males, svl = 0.93 age +37.27, f1,54 = 8.97, r = 0.38, p = 0.004; females, svl = 2.59 age +39.55, f1,9 = 1.30, r = 0.37, p = 0.29; 290-m site: males, svl = 1.15 age + 34.68, f1,25 = 3.97, r = 0.38, p = 0.06; females, svl = 5.72 age + 22.97, f1,11 = 55.12, r = 0.92, p < 0.001). fig. 1. two examples (a: a 1-yr old male, b: a 3-yr old female) of hematoxylin-stained cross-sections of the phalangeal bone of rana limnocharis from both high and low altitudes in southwestern china. arrows indicate the lines of arrested growth (lag). kl represents resorption line, the division line between endosteal and periosteal zones. scale bar: 100 μm. 61altitudinal variation in rana limnocharis average age did not vary significantly across populations in males (f2, 93 = 2.611, p = 0.079) and females (f2, 39 = 2.04, p = 0.14). however, frogs only from 375-m site were significantly older than ones from 364-m site in males (p = 0.03). there was significant difference in adult average svl among populations within each sex (males, f2, 93 = 12.08, p < 0.001; females, f2, 39 = 18.36, p < 0.001). males from the 290-m site were significantly smaller than males from two other sites (post hoc test, p < 0.001), and the latter populations were similar in mean body size (p = 0.43). in females, table 2. differences in mean body size (mm) and age (years) between the sexes for rana limnocharis populations using student’s t-test in three different altitudes. values in descending order are mean ± sd, with sample sizes in parentheses. altitude (m) 375 364 290 body size (mm) male 39.32 ± 1.46 (n = 15) 38.88 ± 1.59 (n = 55) 36.84 ± 2.63 (n = 26) female 43.51 ± 4.18 (n = 20) 44.74 ± 4.62 (n = 10) 34.41 ± 5.30 (n = 12) t 3.70 7.46 1.91 p 0.001 0.000 0.07 age (years) male 2.20 ± 0.68 (n = 15) 1.73 ± 0.65 (n = 55) 1.88 ± 0.86 (n = 26) female 2.50 ± 0.83 (n = 20) 2.00 ± 0.67 (n = 10) 2.00 ± 0.85 (n = 12) t 1.15 1.21 0.38 p 0.26 0.23 0.70 table 3. body size in relation to age in rana limnocharis from three different populations in southwestern china. per each row, values are: mean ± sd, range and sample size. age 375-m site 364-m site 290-m site male female male female male female 1 39.1 ± 0.1 39.0-39.2 2 39.2 ± 3.2 36.9-41.5 2 38.3 ± 1.4 35.1-40.6 21 39.8 ± 9.9 32.7-46.8 2 35.8 ± 3.1 29.7 -39.0 9 29.5 ± 2.2 26.4 31.5 4 2 39.9 ± 1.6 36.6-42.3 8 43.0 ± 2.9 37.3-46.5 8 39.0 ± 1.5 36.1-42.0 28 46.3 ± 2.3 43.0-48.5 6 37.1 ± 2.3 32.6-40.8 13 32.7 ± 1.7 30.7-34.7 4 3 38.6 ± 1.3 37.3-39.8 5 43.7 ± 5.0 34.9-49.6 8 40.4 ± 1.9 37.4-42.2 6 45.0 ± 1.9 43.6-46.3 2 36.4 ± 0.3 36.2-36.6 2 41.0 ± 1.5 39.7-43.1 4 4 48.2 ± 1.1 47.4-48.9 2 40.1 ± 1.1 39.3-40.9 2 62 y.h. liu et al. animals inhabiting the 290-m site were also significantly smaller than ones from two other sites (post hoc test, p < 0.001), while the latter were statistically equal (p = 0.50). after controlling for the effect of age (male, f1, 92 = 9.62, p = 0.003; females, f1, 38 = 21.64, p < 0.001), differences in body size among populations still remained (male, f2, 92 = 13.41, p < 0.001; females, f2, 38 = 23.22, p < 0.001). fig. 2. age composition of rana limnocharis sampled from three localities with different altitudes in southwestern china. open bars, males; shaded bars, females. 63altitudinal variation in rana limnocharis discussion the use of phalangeal growth marks can assess individual age of the subtropical frog r. limnocharis (liao et al., 2011). in our study, we found that scheletochronology works well in the species. moreover, false and double lines did not affect the estimate of age for most individuals. fig. 3. the relationship between age and body size in rana limnocharis (males, open circles; females, closed circles) for three elevation-populations in southwestern china 64 y.h. liu et al. in most anurans, females have larger body size than males, showing sexual size dimorphism. this may arise because of age differences, or from differences in growth rates within each sex (monnet and cherry, 2002). in this study, there was no significant difference in average age between sexes for r. limnocharis, suggesting that age was not the cause of sexual size dimorphism. liao et al. (2011) found that growth rates in r. limnocharis did not differ between reproductive females and males, suggesting that growth rate also do not result in difference in body size between the sexes. it is possible that larger svl of females is caused by natural selection to improve fecundity or offspring size in 375-m and 364-m populations, because a large number of eggs might increase the number of offspring reaching maturity, and larvae which hatched from large eggs can have faster development or better survival than those hatched from small eggs (ficetola and de bernardi, 2009; ficetola et al., 2010). the different pattern in the 290-m population may be related to small sample size. in this study, variation in body size of three r. limnocharis populations inhibiting different altitudes showed that individuals from higher altitudes were larger than those from lower altitude. the same results are reported in earlier studies for r. limnocharis from two relatively larger altitudinal gradient populations (310-800 m) (liao et al., 2011). these observations support bergmann’s rule in r. limnocharis. similar results were reached in the earlier studies on r. chensinensis from three different-altitudes along a montane river (lu et al., 2006; ma et al., 2009). a review of body size variation for amphibians using meta-analytical techniques have indicated that 23 of 34 species conform to the pattern expected under bergmann’s rule, whereas 11 of 34 species exhibit a converse of bergmann’s cline (ashton, 2002). however, a recent meta-analysis of adams and church (2008) questioned the generality of bergmann’s rule, since they found that body size was significantly related to the mean annual temperature in only 10 out of 40 plethodon salamanders, three negatively and seven positively, i.e., only three species exhibits a pattern consistent with this prediction. previous studies have indicated that three proximate factors including egg size, size at metamorphosis and age affect body size of adult anurans (lu et al., 2006; ma et al., 2009; liao and lu, 2010a, c). in amphibians, large eggs often produced by anurans living in colder conditions may lead to large metamorphs and in turn large adults (liao and lu, 2011). furthermore, allocating limited resources into bigger offspring allows them to survive harsh environments (kaplan and king, 1997; roff, 2002; morrison and hero, 2003; dziminski and roberts, 2006). in our study, we did not compare egg size variation and size at metamorphosis along the altitudinal gradients. therefore, future studies will have to assess the contribution of eggs size and size at metamorphosis to adult size. in this study, average age of r. limnocharis did not change consistently with altitude in males and females, but age affected significantly variation in the mean body size of adults. nevertheless, when the influence of age was taken into account, individuals from the high-altitude sites remained larger than the low-altitude sites. the findings suggested that there were factors other than age also contributed to size differences among populations. similar results are reported in earlier studies for r. limnocharis from two different sites (liao et al., 2011) and for r. chensinensis from three different altitudes along a montane river (lu et al., 2006; ma et al., 2009). except for the egg size and age factors, we hypothesized four possible reasons to explain a larger body size at high elevations in this species. firstly, it is important to note 65altitudinal variation in rana limnocharis heat conservation, the mechanism originally proposed by bergmann. although some authors declared that this mechanism can not explain trends for bergmann’s rule in ectotherms (reviewed by blackburn et al., 1999), it has been suggested that consistency with bergmann’s rule is still a thermoregulatory adaptation in salamanders (spotila, 1972; heath, 1975; bernardo, 1993; ficetola et al., 2010) and anurans (morrison and hero, 2003; olallatárraga and rodríguez, 2007). it is reported that several amphibians are able to maintain a body temperature consistently above the environmental temperature by using behavioral mechanisms such as basking and selection of warm patches (heath, 1975; zug et al., 2001; ficetola et al., 2010), since thermoregulators have advantages of heat gain by increasing body size during thermoregulation in cool climates (meiri and dayan, 2003). secondly, ficetola et al. (2010) found that populations with larger body size are associated with high primary productivity. thus, when fecundity increases more with size in the cold environment than it does in the warm environment, a larger adult size may be adaptive (arendt, 2011). thirdly, fasting endurance has also been proposed to explain that larger organisms tend to have larger fat reserves and can therefore better survive lean times cooler climates (cushman et al., 1993; ashton and feldman, 2003). finally, previous studies indicated that body size in amphibians might also be more strongly tied to moisture than to temperature because of the need to keep the skin moist to allow respiration (duellman and trueb, 1994; ashton, 2002). in r. limnocharis, differences in precipitation are significant among these populations (nanchong: 1020 mm, suining: 927 mm, lingguan: 1317 mm), so moisture may be a further cause of variation in body size across altitudes. acknowledgments we thank long jin and sang lin lou for assistance during the field and laboratory work. financial support is provided by the foundation of key laboratory of southwest china wildlife resources conservation (ministry of education), china west normal university, p. r. china (xnyb01-3). references adams, d.c., church, j.o. 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(2001): herpetology. academic press, san diego. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 8(1): 75-78, 2013 polydactyly in the tyrrhenian wall lizard (podarcis tiliguerta) antigoni kaliontzopoulou1,*, daniele salvi1, verónica gomes1, joão p.m.c. maia1,2,3, panagiotis kaliontzopoulos4 1 cibio, centro de investigação em biodiversidade e recursos genéticos, campus agrário de vairão, 4485-661, vairão, portugal. *corresponding author. e-mail: antigoni@cibio.up.pt 2 departamento de biologia, faculdade de ciências, universidade do porto, rua do campo alegre fc4 4169-007 porto, portugal 3 institut de biologia evolutiva (csic-upf), passeig marítim de la barceloneta, 37-49, 08003, barcelona, spain 4 florinis 54, vrilissia, greece submitted on: 2012, 30th november; revised on: 2013, 23rd april; accepted on: 2013, 28 th april. abstract. polydactyly is a fairly frequent phenomenon in tetrapod populations, but it is relatively rare in reptiles. here we report the occurrence of polydactyly in a random sample of the tyrrhenian wall lizard (podarcis tiliguerta) from sardinia. in the locality of siniscola (ne sardinia), we found two polydactylous female lizards, one of which showed polydactyly in one and the other in both hind limbs. this observation constitutes, to the best of our knowledge, the highest frequency of polydactyly ever reported in a single lizard population (4.54%). while providing a direct explanation for polydactyly is complicated, the genetic data available show that the two polydactylous individuals are not direct siblings, excluding the hypothesis of direct maternal inheritance of this condition. keywords. polydactyly, skeletal abnormalities, tyrrhenian wall lizard. polydactyly is an interesting phenomenon that frequently occurs in tetrapod populations but has never been evolutionarily truly re-established in any species. strikingly, while a reduction in the number of digits has repeatedly occurred in several instances throughout tetrapod evolution, this condition has never been reversed (lande, 1978; tabin, 1992; galis et al., 2001). this is intriguing, as a selective advantage of increased digit number exists under several evolutionary situations, enhancing for instance swimming, digging or grasping (galis et al., 2001). however, this evolutionary advantage is always attained by extra digit-like structures that are in reality never “true” digits, but are rather modified wrist bones or extra phalanges (caroll, 1997). the lack of polydactyly in evolution is not due to lack of heritable variation for that trait, as it is a condition very frequently encountered in many tetrapod taxa. for instance, the presence of an additional finger or toe is the most common anomaly at birth in humans (castilla et al., 1996). polydactyly is also quite frequent in other mammal species (brignolo et al., 2002; chapman, 2006; moore et al., 2007; gugolek et al., 2011), as well as birds (fox, 1989; huang et al., 2006; sakai, 2006), and particularly amphibians (mizgireuv et al., 1984; johnson et al., 1999; vorobyeva, 1999; kiesecker, 2002; taylor et al., 2005; piha et al., 2006). it is less frequent in reptiles, with a single known case in chelonians (martínez-silvestre et al., 1998), and a few rare cases reported in lizards (bauer et al., 2009). while sampling a population of podarcis tiliguerta (gmelin, 1789) near siniscola (40.48° n, 9.78° e, datum wgs1984; fig. 1), sardinia (italy), we found two female lizards with postaxial polydactyly. the sampling site was located in a natural area characterised by low mediterranean maqui vegetation and very low anthropogenic disturbance. the first lizard was 54 mm in snout vent length (svl) and exhibited six digits in each of the hind feet (fig. 2ai and 2aii). on the right foot the polydactyly cannot easily be described without radiography, but it seems to result from the duplication of digit iii, while 76 antigoni kaliontzopoulou et al. on the left foot it is clearly a duplication of digit i. the second polydactylous lizard was 52 mm in svl and presented six toes on the left hind limb, which also seems to be the result of a metatarsal duplication of digit iii (fig. 1b). unfortunately, the animals in question were released before noting this condition and could not be examined radiographically to identify the osteological basis of the polydactyly. however, at least in two of the three cases (those in fig. 1aii and 1b), the polydactyly appears to involve a normal number of metatarsals but an abnormal number of digits (brachydactyly sensu bauer et al., 2009), as the supernumerary digits are clearly stemmed at a position posterior to the metatarsals. both polydactylous females were adults, and their body size was within the normal range of the population (47-59 mm for the female individuals sampled in siniscola). they both seemed in normal physical condition, and only presented a few red mite parasites (as seen in fig. 2b), as was also the case for most of the individuals examined from the same population. both presented copulation marks, indicating that they had mated recently, and the larger one was gravid when captured. available dna sequence data for the mitochondrial 12s rrna gene obtained following the procedure described in salvi et al. (2011), showed that the two polydactylous individuals present similar, but different haplotypes (accession numbers hf948026 and hf948027). given the matrilineal inheritance of the mitochondrial genome, the occurrence of different haplotypes in the two polydactylous individuals indicates that they are not siblings from a single clutch. two aspects are worth noting in the case of polydactyly reported herein. first, this is the third case of polydactyly reported in wall lizards podarcis (after p. pityusensis by carretero et al., 1995 and p. muralis by lazić and crnobrnja-isailović, 2012). given the low number of reported cases in lizards, totalling seven that we are aware of (i.e. carretero et al., 1995; cuadrado, 1996; pelegrin, 2007; bauer et al., 2009; minoli et al., 2009; lazić and crnobrnja-isailović, 2012; megía, 2012; but see also norval et al., 2009), this means that a very high frequency of reported cases is observed in the genus podarcis (representing almost half of the existing observations). while this may be the result of a reporting bias, it may also indicate that this condition is for some unknown reason more frequent in podarcis wall lizards. similarly high frequencies are only known from chameleons, where polydactyly seems to be quite frequent and has been observed in several populations of the same species (cuadrado, 1996). second, this is the first time that two polydactylous individuals have been encountered in a random lizard sample. in fact, this is the highest reported frequency of polydactyly in a lizard population, since the two observations come from a total, random sample, of 44 individuals (4.54%). this is remarkably high for lizards, where reported frequencies range between 0.2 and 0.6% (bauer et al., 2009). according to genetic data, a direct sibling association could not be established between the two individuals. consequently, while a genetic cause underlying this observation of polydactyly cannot be excluded at the population level, the high frequency reported here does not seem to be the outcome of the reproduction of a single pair of lizards. the causes of polydactyly are difficult to decipher. mutations in genes involved in digit development and specification (such as the hedgehog, bmp, fgf and hox gene families) are known to cause polydactyly in mammals, birds, and amphibians (zákány et al., 1997; villagómez and alonso, 1998; yokoyama et al., 1998; kraus et al., 2001; huang et al., 2006). polydactyly is also   figure 1. geographic location of the studied population of podarcis tiliguerta from siniscola in sardinia (italy). 77polydactyly in podarcis tiliguerta known to be frequently associated to chromosomal trisomies, at least in mammals (pugsley, 1985; brignolo et al., 2002; moore et al., 2007). in amphibians, polydactyly has been associated with infection from some parasites (jonhson et al., 1999; kiesecker, 2002) or viruses (borkin and pikulik, 1986), as well as environmental contamination due to the use of pesticides (mizgireuv et al., 1984; diana and beasley, 1998; kiesecker, 2002; taylor et al., 2005; piha et al., 2006). in reptiles, the endogenous (genetic) and exogenous (environmental) causes of polydactyly are not fully understood, yet the case study reported herein and previous observations suggest that lizards of the genus podarcis would be a promising model organism for investigating the occurrence of polydactyly and its proximate determinants. acknowledgments the italian ministry of environment issued permits, dpn-2009-0005106, for the capture of the lizards. ak and ds were supported by post-doctoral grants (sfrh/ bpd/68493/2010 and sfrh/bpd/66592/2009 respectively) and jpmcm by a pre-doctoral grant (sfrh/bd/74305/2010), all by fundação para a ciência e tecnologia (fct, portugal). support was also provided by fct project ptdc/biabec/101256/2008. references bauer, a.m., hathaway, s.a., fisher, r.n. 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(1997): regulation of number and size of digits by posterior hox genes: a dose-dependent mechanism with potential evolutionary implications. p. natl. acad. sci. usa 94: 13695-13700. acta herpetologica vol. 8, n. 1 june 2013 firenze university press the oogenic cycle of the caspian bent-toed gecko, cyrtopodion caspium (squamata: gekkonidae) in iran vida hojati1*, kazem parivar2, eskandar rastegar-pouyani3, abdolhossein shiravi1 altitudinal effects on life history parameters in populations of liolaemus pictus argentinus (sauria: liolaemidae) joel antú gutiérrez1,*, carla piantoni2, nora r. ibargüengoytía1,3 recent cryptic extinction of squamate reptiles on yoronjima island of the ryukyu archipelago, japan, inferred from garbage dump remains yasuyuki nakamura1,*, akio takahashi2, hidetoshi ota3 trophic niche and feeding biology of the italian wall lizard, podarcis siculus campestris (de betta, 1857) along western mediterranean coast marco a.l. zuffi*, chiara giannelli novel, non-invasive method for distinguishing the individuals of the fire salamander (salamandra salamandra) in capture-mark-recapture studies goran šukalo1,*, sonja đorđević2, dragojla golub1, dejan dmitrović1, ljiljana tomović2,3 going out tonight? when insular hierophis viridiflavus breaks the whip snakes rules delaugerre michel-jean first evidence of a paedomorphic population of the smooth newt (lissotriton vulgaris) in the czech republic václav gvoždík1,2, veronika javůrková4, oldřich kopecký5,* no detection of chytrid in first systematic screening of bombina variegata pachypus (anura: bombinatoridae) in liguria, northern italy stefano canessa1,*, an martel2, frank pasmans2 status of the european pond turtle, emys orbicularis (reptilia: testudines: emydidae) in vorarlberg, austria andreas kleewein1, günther wöss2 comparative cytogenetics of two species of ground skinks: scincella assata and s. cherriei (squamata: scincidae: lygosominae) from chiapas, mexico riccardo castiglia1,*, alexandra m.r. bezerra2, oscar flores-villela3, flavia annesi1, antonio muñoz4, ekaterina gornung1 polydactyly in the tyrrhenian wall lizard (podarcis tiliguerta) antigoni kaliontzopoulou1,*, daniele salvi1, verónica gomes1, joão p. m. c. maia1,2,3, panagiotis kaliontzopoulos4 book review: roberto sindaco, alberto venchi, cristina grieco. the reptiles of the western palearctic. 2. annotated checklist and distributional atlas of the snakes of europe, north africa, middle east and central asia, with an update to the vol. 1. edoardo razzetti acta herpetologica journal of the societas herpetologica italica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 7(1): 23-28, 2012 on the distribution of uromastyx alfredschmidti wilms and böhme, 2000 (squamata: agamidae: uromastycinae) roberto sindaco1, thomas m. wilms2, alberto venchi1 1 c/o museo civico di storia naturale, via s. francesco di sales 88, i-10022 carmagnola (to), italy. corresponding author. e-mail: rsindaco@gmail.com 2 zoologischer garten frankfurt, bernhard-grzimek-allee 1, d-60316 frankfurt am main, germany submitted on: 2011, 19th april; revised on 2011, 2nd november; accepted on 15th november. abstract. the article reports on two new findings of uromastyx alfredschmidti wilms and böhme, 2000 that allow for a more precise definition of its distribution range and, consequently, a new conservation assessment in compliance with iucn parameters. following review of available museum specimens, the hoggar mts. (algeria), should not to be considered within the natural range of the species. nomenclatorial clarifications on existing literature are also provided. keywords. uromastyx alfredschmidti, distribution, conservation status. the first record of the genus uromastyx in sw libya was reported by scortecci (1937: 176), who stated that “uromastyx acanthinurus non è presente affatto nei dintorni di gat e delle altre oasi del territorio, ma negli uiadin a prode rocciose quali lo iseien [e10°19’ n24°48’], lo ertà a occidente di feuat [n24°58’ e10°05’], presente e abbondante sull’akakus e sui tassili, dove l’ho rinvenuto fino a 1500 metri di altezza.” [= uromastyx acanthinurus is absent from gat and other surrounding oases, but occurs in the rocky wadies such as isein, ertà west of feuat, occurring and abundant in the akakus and the tassili, where i found it up to 1500 m a.s.l.]. apparently, there have been no further records of uromastyx from sw libya since frynta et al. (2000: 21) that reported a damaged specimen from “akakus mts” at n24°41’ e10°38’, 790 m a.s.l., found dead on 4-5 october 1999, doubtfully identified it as u. acanthinura. since this locality is very close to the place where the senior author observed u. alfredschmidti in 2008, the specimen likely belongs to this latter species, because sympatry between u. alfredschmidti and u. acanthinura has never been observed as yet, and the range of the latter does not extend to the central sahara. wilms and böhme (2000) described a new species, uromastyx alfredschmidti, belonging to the u. acanthinura complex from central sahara, on the basis of five 24 r. sindaco, t. wilms and a. venchi museum specimens. the only precise locality known by the authors was the type locality: “tassili n’ajjer, tamrit plateau (1600 m) [n24°38’ e9°40’], ca. 30 km nord-östlich [= north-east] von djanet”. the four paratypes came from vague localities: “hoggar”, “tassili n’ajjer” and “sahara”. moreover a libyan record from “südlicher akakus” [= southern akakus] is reported in the map by wilms and böhme (2000), on the basis on a photographic record by heiner rohrwick (fig. 1). during the study of the herpetological collection of the rome university “la sapienza” (now deposited in the museum of zoology of rome municipality, mczrv), two authors (av and rs) found an adult specimen of an indeterminated uromastyx (mzur r/380, provisional collection number) collected by giuseppe scortecci in october 1936 in “fezzan occidentale, tassili” [= western fezzan, tassili] (fig. 2). this specimen was identified as u. alfredschmidti (identification confirmed by tw) and it is the first libyan museum specimen belonging to this species. on 22 april 2008, an adult specimen of uromastyx alfredschmidti was found in the wadi imlal (n24° 46’ 47.5” e10° 38’ 45.3”), akakus mts, sw libya, 814 m a.s.l., at about 14:00h by cristina grieco, claudia corti and riccardo saccardi and the senior author (rs). the locality is close to wadi iseien, where scortecci (1937) reported u. acanthinura. the specimen was observed eating small leaves of a thorny shrub of acacia sp., and when disturbed, it took refuge under a big stone. it was captured, photographed and then fig. 1. uromastyx alfredschmidti from southern akakus (photo by heiner rohrwick, without date). 25uromastyx alfredschmidti distribution released at the site of capture. a photograph of this specimen was published by sindaco and jeremčenko (2008: 432). up to now u. alfredschmidti was quoted from less than a dozen of localities, many of which are vague (see table 1); since precise records exist in these areas, only these have been mapped (fig. 3). the precise localities fall in a small area, about 100 km wide, between algerian tassili and libyan akakus. from the area of the tassili du hoggar in southern algeria there are only two known museum specimens (mhnp 9905, mhnp 1961.261) with vague localities (“algeria, hoggar”). one of the authors (tw) examined more than 50 uromastyx from southern algeria, western libya, northern mali and northern niger, deposited in 14 european museum collections (the natural history museum, london (bmnh), gothenburg museum of natural history (gnhm), hessisches landesmuseum darmstadt (hlmd), muséum d’histoire naturelle, genève (mhng), muséum national d’histoire naturelle, paris (mnhp), museum für tierkunde, dresden (mtkd), museo zoologico de „la specola“, firenze (mzuf), zoological museum of rome (mzur/mczrv), naturhistorisches museum wien (nmw), naturmuseum und forschungsinstitut senckenberg, frankfurt a.m. (smf), zoologisches forschungsmuseum a. koenig, bonn (zfmk), museum für naturkunde, humbold-universität, berlin (zmb), zoologisches institut und zoologisches museum der universität hamburg (zmh), and zoologische staatssammlung münchen (zsm). all specimens from the tassili du hoggar (but the above mentioned two doubtful specimens) were found to belong either to uromastyx geyri müller 1922 or u. dispar maliensis joger and lambert 1996. the occurrence of the species in the hoggar appears therefore more than doubtful, and we consider the algerian tassili du hoggar as not lying within the natural distribution range of uromastyx alfredschmidti. fig. 2. uromastyx alfredschmidti. mzur r/380. libya, “fezzan occidentale, tassili”, giuseppe scortecci leg., october 1936.   26 r. sindaco, t. wilms and a. venchi on the basis of our new findings, the u. acanthinurus by scortecci (1937) is added in the chresonymy of u. alfredschmidti, as well as, tentatively, the u. acanthinura by frynta et al. (2000). furthermore, following the removal of the uncertain and inaccurate records from the known range, the distribution of u. alfredschmidti turns out to be restricted only to a small area between algerian tassili n’ajjer and libyan akakus, thus being much smaller than previously thought. this species is assessed as near threatened in the iucn red list of threatened species (version 2010.4.) because its range was not much greater than 20,000 km² and it is overharvested, making it close to qualifying for vulnerable (joger and böhme 2006). this assessment should be reconsidered in light of the new interpretations presented in the present paper, since the new extent of occurrency (as defined by iucn 2001) is reduced to about 1,640 kmq, the known locations are less than 10 and a continuing decline of the quality of its habitat is inferred. these criteria better fit with the iucn threat category vulnerable according to the iucn criteria b2ab(iii). in conclusion, the new findings allow to define more precisely the distribution range of u. alfredschmidti and to assess more correctly the species’ conservation status. table 1. known records of u. alfredschmidti. map number locality lat lon precision notes reference 1 tassili n’ajjer, tamrit plateau 24,63 9,67 2 type locality wilms and böhme, 2000 2 ertà [uadi] w of feuat 24,96 10,08 3 scortecci, 1937 3 akakus” at n24°41’ e10°38’ 24,68 10,63 2 frynta et al., 2000 4 iseien [uadi] 24,80 10,66 3 scortecci, 1937 5 “wadi imlal” 24,76 10,63 1 present paper 6 sefar, tassili 24,66 9,74 3 photographic record wilms, 2001 7 djanet, 40 km ne 24,79 9,76 3 photographic record antonini, 2004 “akakus” 0 see records 3 and 4 scortecci, 1937 “südlicher akakus” 0 see record 5; photographic record wilms and böhme, 2000 “tassili n’ajjer” 0 see record 1 wilms and böhme, 2000 “sahara” 0 vague wilms and böhme, 2000 “fezzan occidentale, tassili” 0 see record 1; mzur r/380 present paper ? “hoggar” 0 doubtful wilms and böhme, 2000 precision: 0 = generic, not mapped; 1 = g.p.s. point; 2 = coordinates provided by authors; 3 = coordinates from gazetteers or maps. 27uromastyx alfredschmidti distribution references antonini, o. (2004): les fouette-queues – lézards du genre uromastyx. animalia éditons, 96 pp. frynta, d., kratochvíl, l., moravec, j., benda, p., dandová, r., kaftan, m., klosová, k., mikulová, p., nová p., schwarzová l. (2000): amphibians and reptiles recently recorded in libya. acta soc. zool. bohem. 64: 17-26. iucn. (2001). iucn red list categories and criteria: version 3.1. iucn species survival commission. iucn, gland, switzerland and cambridge, uk. ii + 30 pp. joger, u., böhme w. (2006): uromastyx alfredschmidti. in: iucn 2010. iucn red list of threatened species. version 2010.4. <www.iucnredlist.org>. [downloaded  on 05 march 2011].   fig. 3. distribution of uromastyx alfredschmidti. 28 r. sindaco, t. wilms and a. venchi scortecci, g. (1937): relazione preliminare di un viaggio nel fezzan sud occidentale e sui tassili. atti soc. it. sci. nat., mus. civ. st. nat. milano 76: 105-194. sindaco r., jeremčenko v.k. (2008): the reptiles of the western palearctic. 1. annotated checklist and distributional atlas of the turtles, crocodiles, amphisbaenians and lizards of europe, north africa, middle east and central asia. monografie della societas herpetologica italica i. edizioni belvedere, latina (italy); 580 pp. wilms, t. (2001): dornschwanzagamen, lebensweise, pflege, zucht.herpeton-verlag, 144 pp. wilms t., böhme w. (2000): revision der uromastyx acanthinura artengruppe, mit beschreibung einer neuen art aus der zentralsahara. zool. abhand. staat. mus. tierk. dresden 51: 73-104. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 6(2): 275-288, 2011 morphological variation of ridged frogs of the taita hills, kenya rossa ng’endo1,*, eunice kairu1, zipporah osiemo2, callistus ogol1 1department of zoological sciences, kenyatta university, p.o. box 43844, nairobi, kenya. *corresponding author. e-mail: rossangendo@yahoo.com 2department of zoology faculty of science, jomo kenyatta university of agriculture and technology, p.o box 62000-00200, nairobi-kenya. submitted on: 2010, 5th october; revised on: 2011, 20th july; accepted on: 2011, 2nd december. abstract. comparing of morphological character variation within taxa continues to play an important role in improving species inventories. using morphometrical and non-meristic morphological adult characters, the diversity of the genus ptychadena in taita hills was studied. comparative material from elsewhere was not used, and therefore species names were only provisionally allocated to the taxa identified. available names were discussed on the basis of comparisons with morphological data from other regions. the results revealed that female species are larger in size than males. two species were identified and for each a standardized diagnosis of 32 characters is provided. comparison of results with morphological data from related studies done elsewhere reveals that certain characters are of critical importance in differentiating the two ptychadena species. the power of these morphological characters is discussed, especially for the background of rapid and easy identification of ptychadena species in the field for conservation purposes. keywords. diversity, morphological characters, ptychadena, taita hills, kenya. introduction african herpetofauna is quite diverse in terms of species richness. however, geographical sampling and taxonomic inventories are inadequate and most countries are poorly investigated (poynton, 1999). this study focused on the species diversity of ridged frogs, genus ptychadena boulenger, 1917 (ptychadenidae), in taita hills. these anurans are common across sub-saharan africa, madagascar and some smaller oceanic islands. currently, 49 species are recognized (frost, 2008), which often occur syntopically (rödel, 2002; channing and howell, 2006). 276 r. ng’endo et alii generally, the female species in this genus are larger in size in relation to the males, with most species sharing several morphological characteristics (channing and howell, 2006; bwong et al., 2009). there have been several efforts towards an understanding of ptychadena systematics (guibé and lamotte, 1957, 1958, 1960; lamotte, 1967; perret, 1979, 1981, 1987, 1994, 1996; amiet, 1989; poynton and broadly, 1985; largen, 1997, 2000). however, the genus can not be considered as well understood as still the majority of the suggested species are improperly defined. standardized diagnostic schemes have been applied by few previous researchers but only for a limited number of species (guibé and lamotte, 1957; perret, 1979; poynton and broadly, 1985). as a result, several of the suggested taxa are difficult to distinguish and their geographic ranges remain unknown. moreover, there are only a few comparative studies, and therefore it is difficult to follow the placement of certain nominal species into the synonymy of others (bwong et al., 2009). while the genus ptychadena in part has already been studied in other regions of africa (channing, 2001; rödel, 2002; channing and howell, 2006), it is apparent that the study of east african ptychadena lags behind. so far, the study on the genus has mainly been based on different morphological characters and in merely part vocalization (perret, 1996; rödel, 2002; bwong et al., 2009). findings from these studies reveal that most species look quite similar in appearance and cryptic diversity occurs in some species. therefore, more research is necessary in order to obtain suitable characters to differentiate species. this would also be a step towards reviewing the status of east african ptychadena (bwong et al., 2009). this study aimed at determining the diversity of the genus ptychadena in taita hills using morphological characters, and to provide standardized diagnostic schemes for the species identified. since this study has neither included specimens from elsewhere nor type material, names are only provisionally applied. the results from this study were further compared with findings from other regions where more or less same studies have been conducted, in expectance of comprehensive revisionary action on ridged frogs. materials and methods taita hills lie in south eastern kenya (3°15’s, 3°30’s) and (38°15’e, 38°30’e) in taita taveta district of coast province (fig. 1), and form part of the eastern arc mountains (bennun and njoroge, 1999). they are at an elevation of 1350–2228 m a.s.l. the climate of the area is under the influence of the innertropical convergence zone, with a bimodal rainfall pattern. the long rains occur in march to june and the short rains in october to december, peaking in april and november respectively. annual rainfall ranges between 600 mm to 1329 mm per year (bennun and njoroge, 1999). these hills are well known for remarkable fauna and flora with high levels of specific and generic endemism (eaws, 2005). therefore, our study on ptychadena species is of special significance, since the hills are bio-geographically outstanding – i.e. listed as one of the conservation hot spots worldwide and displays a high degree of endemism, even in amphibians (myers et al., 2000; measey, 2004; measey and malonza, 2006). opportunistic field sampling, both by day and night (heyer et al., 1994) was carried out in the field between november 2005 and march 2006. specimens were collected from 18 different sites in water paddies located at periphery of the different forest blocks, fixed in formalin and pre277morphological variation of ridged frogs served in 70% methanol. specimens were assigned field numbers and preliminarily identified into two groups based on body colorations and other easily observable non-meristic morphological characters that are distinct. a total of 113 adult ptychadena specimens were used for the analysis, 20 of which had been earlier collections from other researchers (appendix 1). all the samples were deposited at national museums of kenya (nmk)-department of herpetology, and further used for morphometrical and non-meristic morphological analysis. sexes were determined through presence (males) versus absence (females) of lateral vocal openings. following guibé and lamotte, (1957), 1958, 1960), lamotte (1967), perret (1979, 1981, 1987, 1994, 1996), amiet (1989) largen (1997, 2000), poynton and broadley (1985), rödel (2002), channing and howell (2006), 10 morphometrical and 22 non-meristic morphological characters of adults were identified to be useful in defining and diagnosing ptychadena taxa. using dial callipers,10 body measurements were taken to the nearest 0.1 mm: (1) horizontal eye diameter (ed); (2) distance from anterior corner of eye to posterior nostril (en); (3) foot length from the proximal edge of the heel to the tip of toe iv (fl); (4) head length from angle of jaw to tip of snout (hl); (5) head width at broadest (hw); (6) inter-narial distance (id); (7) distance from tip of snout to anterior fig. 1. map showing the location of taita hills and position of different hill blocks. the specimens were collected from water bodies at forest floors in 18 different sites represented by the names. (map modified from bennun and njoroge, 1999). 278 r. ng’endo et alii corner of nostril (sn); (8) snout-vent length (svl); (9) horizontal tympanum diameter (td); (10) tibia length (tl). fifteen non-meristic morphological features were coded as present or absent: (11) dark bands on posterior face of femur; (12) dark mottling on posterior face of thigh; (13) green or light brown median dorsal band; (14) light bands on posterior face of femur; (15) light tibial line; (16) outermost dorsal ridge coloured whitish; (17) outer metatarsal tubercle; (18) pale triangle on dorsal snout; (19) ridges on lateral sides of body; (20) ridges on the legs; (21) row of tubercles on metatarsus; (22) warts on lateral sides; (23) warts on legs; (24) whitish ring (at least in part) around tympanum; (25) whitish spots on lower lip. four other non-meristic morphological characters were coded as follows: if dark bands on femurs present, are they continuous or discontinuous from knee to knee (in species definitions below added to (11) if applicable); if outer metatarsal tubercle present is it smaller or larger than inner (in species definitions below added to (17) if applicable); (26) canthus rostralis from eye to nostril concave versus straight; (27) nostrils visible versus invisible from above. also studied was (28) if the external vocal openings in males are situated above, below or at level of arm insertion: counted were (29) the number of tubercles on toe iv, (30) the number of dorsal ridges plus (31) the number of incomplete dorsal ridges, and (32) the foot webbing pattern following the manner described by glaw and vences (1994). diagnoses of species from the taita hills covered 32 aspects using characters (1) to (32) listed above. both variable and non-variable characters were considered in order to provide a standardised diagnostic scheme applicable to the entire genus ptychadena. information summarized by frost (2008) was used for the discussion of available names. to compare the variation in morphometry within and between the two species, a student t-test was used. further, cluster analysis for the 10 morphometrics and five of the 15 present or absence morphological characters (green or light brown median dorsal band, light tibial line, pale triangle on dorsal snout, ridges on the legs and whitish ring at least in part around tympanum). the five present or absent characters were selected on the basis that a character in consideration is only present in species 1 and not in species 2 and vice versa. prior to analysis, the combined morphometrics and the morphological data were standardized through log transformation (anderson et al., 2006). a dissimilarity distance matrix was generated, from which cluster representations of the 113 specimens were constructed. all the analyses were carried out using packages stats and gclus, implemented in r (r development core team, 2010). results a standard diagnoses of morphometric and non-meristic morphological characters for each identified species: species 1 and species 2 (l=length: measurements are mean ± sd, followed by the range in mm) resulted in to two groupings i.e. species 1 and species 2 described below using 32 characters. species 1 “anchietae” diagnosis: (1) ed 4.5 ± 0.6 (5.5–2.6); (2) en 3.1 ± 0.5 (4.2–2.1); (3) fl 32.6 ± 5.3 (42.0–23.4); (4) hl 12.7 ± 1.7(17.3–9.2); (5) hw 11.4 ± 1.7 (14.8– 8.5); (6) id 3.1 ± 0.4 (3.9–2.0); (7) sn 3.1 ± 0.5(4.2–2.3); (8) svl 39.8 ± 6.2 (52.4–27.8, females > males); (9) td 3.1 ± 0.5 (2.8–3.9); (10) tl 20.6 ± 3.3 (27.9–14.1); (11) dark bands on posterior femur present, discontinuous from knee to knee; (12) dark mottling on posterior femur absent; (13) light bands on posterior femur present; (14) light or green medial dorsal band absent; (15) canthus rostalis straight; (16) nostril visible from above; (17) pale triangle on snout present; (18) 6–8 dorsal ridges; (19) 2 incomplete ridges; (20) ridges on legs present; (21) ridges on lateral side of body absent; (22) outer dorsal ridge whitish; (23) tibia line not present; (24) outer metatarsal tubercle absent; (25) rows of 279morphological variation of ridged frogs tubercles absent; (26) warts on legs absent; (27) whitish ring around tympanum present; (28) whitish spots on lower lip present; (29) vocal openings in males positioned below arm insertion; (30) 3 tubercles on toe iv; (31) 1–2 phalanges on toe iv free of web; (32) foot webbing formula 1e (0) 2i/e (0) 3i/e(0) 4i/e(1) 5i(0). species 2 “mascareniensis” diagnosis: (1) ed 4.6 ± 0.5 (5.6–2.7); (2) en 3.1 ± 0.5 (4.2– 2.0); (3) fl 34.5 ± 6.4 (48.2–21.3); (4) hl 13.5 ± 2.5 (18.5–8.2); (5) hw 11.8 ± 1.8 (15.1– 7.7); (6) id 2.8 ± 0.5 (4.2–1.7; (7) sn 2.9 ± 0.5 (4.2–1.3); (8) svl 43.3 ± 7.4 (57.0–26.4, females > males); (9) td 3.4 ± 0.6 (4.7–2.0 ); (10) tl 19.9 ± 4.0 (29.3–10.3);). (11) dark and (12) light bands on posterior femur present, continuous or discontinuous from knee to knee; (13) dark mottling on posterior femur absent; (14) canthus rostalis straight; (15) nostril visible from above; (16) pale triangle on snout absent; (17) medial dorsal band present; (18) lateral ridges absent; (19) 6–8 dorsal ridges; (20) if 8, two are short dorsolateral ridges only; (21) outer dorsal ridges whitish; (22) ridges on legs absent; (23) outer metatarsal tubercle absent; (24) row of tubercles absent; (25) tibia line present; (26) warts on lateral sides present or absent; (27) warts on legs absent; (28) whitish spots on lower lips present; (29) vocal openings in males above arm insertion; (30) 3 tubercles on toe 1v; (31) 2 phalanges on toe 1v free of webbing; (32) foot webbing formula 1e (1) 2i/e (1-2) 3i/e (1.5-2.5) 4i/e (2-3) 5i (1-2). there were a total of 28 females and 24 males of species 1 “anchietae”; and 29 females and 32 males of species 2 “mascareniensis”. some male specimens of species 2 “mascareniensis” showed peculiar aspects in that they lacked certain characters i.e. tibia line and medial dorsal band (ref. numbers: rnm001//a5035/3; rnm002//a5035/2; rnm001//a5035/6; krnm061//x; krnm062//x; krnm065//x from collection sites wundanyi posta farml for the first three and shate area for the last three specimens. two female specimens (ref. sl390//a/5034/5; sl395//a/5055/12 collected from wundanyi posta farml also showed a similar phenomenon. comparisons between morphometry of species 1 “anchietae” female and males revealed high variation in parameters such as en, fl, hw, sn td and svl (p < 0.01). species 2 “mascareniensis” females and males showed high significant differences in all parameters (p < 0.01) except for ed. there was no significant variation in all the parameters (p = 0.05) between species 1 and 2 females, except for hl that showed a significant variation (p < 0.05). whereas there was high variation in parameters such as id, sn, and tl between species 1 and 2 males (p < 0.01), the other parameters did not significantly vary (p = 0.05). species 1 and 2 females showed high significant differences in parameters such as fl, hw, id, sn, td, and tl (p < 0.01); significant variation in hl and svl (p < 0.05), and no significant difference in ed (p = 0.05). on the other hand, species 1 males and species 2 females highly varied in all parameters (p < 0.01) except for id and sn that were not significantly variable (p = 0.05). cluster representation performed for the 10 morphometrics alone reveal an overlap between the two species (fig. 2). in contrast, a cluster representation considering five non-meristic morphological characters (fig. 3) support two distinct clusters of species 1 “anchietae” and 2 “mascareniensis”. cluster representation of a combination of morphometrics and the five non-meristic morphological characters again advocated for two distinct clusters i.e. of species 1 and 2 (fig. 4), and the clustering of individual species seems to be influence by sex differences for the two species to some extent. 280 r. ng’endo et alii discussion our results depict that two ptychadena species syntopically occur in taita hills. species 1 is referable to p. anchietae (bocage, 1868 “1867”) as it was called by schick et al. (2005). originally described from angola, this ridged frog is suggested to display a distribution from southern africa north to eritrea (channing and howell, 2006). on the other hand, species 2 “mascareniensis” is an unidentified taxon in the species complex behind the name p. mascareniensis (duméril and bibron, 1841). fig. 2. a representation of 113 specimens in the genus ptychadena belonging to species 1 “anchietae” (taxa in grey) and species 2”mascareniensis” (taxa in black) using 10 morphometric characters. two distinct clusters are not formed and the two species are seen to overlap. the letters f and m after the underscore in taxa code number represent sexes for the specimens of the two species respectively. 281morphological variation of ridged frogs the standard diagnoses of the two species shows that the females are generally larger than their male counterparts, notable either by mere looks at the two species and even as revealed by the morphometrics, with high variation in size notable between the different sexes of species 1 and 2. the two species share and also differ in certain morphological characters. we noted that lateral warts were present in some specimens of species 2 “mascareniensis” and absent in others. species 2 “mascareniensis” has a tendency to show variation within its members in non-meristic morphological characters (bwong et al., 2009). the absence of certain characters e.g. tibia line in some specimens in species 2 fig. 3. a representation of 113 specimens of the frog genus ptychadena for species 1 “anchietae”(taxa in grey) and species 2 “mascareniensis”(taxa in black) using five present /absent characters. two distinct clusters are evident, an indication that morphological characters can effectively discriminate the two species. the letters f and m after the underscore in taxa code number represent sexes for the specimens of the two species respectively. 282 r. ng’endo et alii “mascareniensis” that occur in same collection sites may be attributed to breeding between species lacking this characteristic. this aspect is however subject to further research and therefore not featured in the discussion below. variation in non-meristic morphological characters between different species plays an important role in entirely discriminating them. discrimination between species 1“anchietae” and species 2 “mascareniensis” was mainly achieved on the basis of presence or absence of the morphological character in consideration. ridges on legs, pale triangle fig. 4. a representation of 113 specimens of the frog genus ptychadena for species 1 “anchietae”(taxa in grey) and species 2 “mascareniensis”(taxa in black) using a combination of 10 morphometric characters and five present /absent characters. two distinct clusters are evident, an indication that a combination of morphometric and non-meristic morphological characters can effectively discriminate the two species. the letter f and m after the underscore in taxa code number represent sexes for the specimens of the two species respectively. 283morphological variation of ridged frogs on the snout and a white ringed tympanum were present only in species 1“anchietae”, whereas tibia line and medial dorsal band were only present in species 2 “mascareniensis”, except for some few specimens (see results) . these characters were noted to be the most informative present or absent non-morphometric characters in discriminating species 1 ”anchietae” and 2 “mascareniensis”. this is also confirmed by several studies that aimed at discriminating the two species (stewart, 1967; channing, 2001; channing and howell, 2006; bwong et al., 2009). clustering using morphometry alone was not informative in discriminating the two species, an indication that some of their body proportions are more or less the same between the two species. on the other hand, non-morphometrical morphology and a combined analysis using morphometry and non-morphometrical morphology proved reliable in discriminating the two. for the two species, the differences between sexes tend to influence the clustering of the individual species in the cladogram, notable by presence of same sex specimens in some clades. this may be largely influenced by the morphometric characters. a recent study by bwong et al. (2009) effectively used combined analysis on this genus but also noted that when a large group of species are in consideration, nonmorphometrical morphology or combining morphometry and non-meristic characters may be ineffective in species discrimination, thus calling for inclusion of other tools such as vocalization and genetic markers. snout-vent length is a widely used measure in most morphometric studies on the genus ptychadena and other anurans (stewart, 1967; channing and howell, 2006; bwong et al., 2009) and can be readily used for a comparative survey. based on this study, species 1 “anchietae” and 2 “mascareniensis” had svls of between 28 mm to 52 mm and 27 mm to 57 mm respectively. channing and howell (2006) described p. anchietae (species 1) as a medium sized frog, with svl for males up to 51 mm and females up to 62 mm. on the other hand the females of p. mascareniensis (species 2) have svl of 65 mm and males 53 mm, and their internarial distance is equal to the nostril-snout distance. recent studies in kakamega forest (bwong et al., 2009) have indicated that the svl of species 1 “anchietae” range from 22.9 to 60.9 mm, with the females being larger than males while that of species 2 “mascareniensis” range from, 34.1 to 61.1 mm, with the females being larger than the males. in as much as there is variation on what is the minimum or maximum svl in different studies, it is clear that all the svl values discussed above from different studies are within the same range. in differentiating the two ptychadena species, foot webbing was also informative in that the phalanges in toe 1v free of web were 4ie (1) in species 1 ”anchietae” and 4ie (2) in species 2 “mascareniensis”; where (i) represent the internal and (e) the external part of toe 1v free of webbing. the overall foot web formula for species 1 is (1e (0) 2i/e (0) 3i/e (0) 4i/e (1) 5i (0), which vary from that of species 2; 1e (1) 2i/e (1-2) 3i/e (1.5-2.5) 4i/e (2-3) 5i (1-2), thus differentiating the two species. similar foot webbing patterns were also noted in the two species from kakamega forest (bwong et al., 2009) and malawi (stewart, 1967). this shows that foot webbing offers an important character in discriminating not only the two species studied here, but also other species within the genus ptychadena and amphibians as a whole (stewart, 1967). this study is unique in that, it is the first of its kind in taita hills, and provides a vital basis for future monitoring of ptychadena species. the results depict that two spe284 r. ng’endo et alii cies of ptychadena occur in taita hills. our results further propose that morphometrics and non-meristic morphological characters are helpful in species identification and especially when used in combination. important to mention here is that the possibility of even more species in genus ptychadena occurring in taita hills cannot be ruled out. further research has been done to establish the overall diversity of amphibians in these hills, with some species presumed to also belong to the genus ptychadena being discovered at the lowlands. these include p. schillukorum & p. mossambica, occurring at the bases of sagalla hill and mt. kasigau (p. malonza, pers. comm.). however, these have not been subjected to a thorough identification process as is species 1”anchietae” and 2 “mascareniensis” because they are only a few in number. as shown above, for the identification – not definition – of species, a combination of morphometrical and non-morphometrical morphology characters are suitable. we are optimistic that the existing gaps can be filled because ptychadena species display various external features which should allow for proper diagnoses when applied in a standardized way. standardised schemes may be especially important as a background of rapid and easy field identifications for conservation purposes in regions where this genus is not yet studied. acknowledgements we are grateful to the staff of the department of herpetology of national museums of kenya for logistic support especially p. malonza and b. bwong and wilson. also to thank is the forest department in taita hills for allowing us to conduct the research, not forgetting the anonymous referees for their positive comments aimed towards improving this work. this study was funded by katholischer akademischier ausländerdienst, germany (kaad), in collaboration with the biodiversity monitering transect analysis in africa (biota e08), part of the biolog campaign under the federal ministry of education and research (germany), to whom we are thankful. references anderson, m.j., ellingsen, k.e., mcardle, b.h. 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(1967): amphibians of malawi. albany. state university of new york press. appendix 1 113 frog species from the genus ptychadena used for the analysis in this study. letters x represent missing information, yet to be allocated. more details on collectors, collection dates and a complete numbering can be obtained from the national museums of kenya, department of herpetology. locality field number nmk catalogue number supposed name collectors kasigau x y/3026/1 p. anchietae kasigau x y/3026/2 p. anchietae mbololo x a/3549/1 p. anchietae mbololo x a/3549/2 p. anchietae mbololo x a/3549/3 p. anchietae mbololo x a/3549/4 p. anchietae mbololo x a/3549/5 p. anchietae mbololo x a/3549/6 p. anchietae fururu x y3250 p. anchietae kasigau x y3340/2 p. anchietae kasigau x y3340/2 p. anchietae mbololo x a/3696/1 p. anchietae mbololo x a/3696/2 p. anchietae chomboke area ysl201 a/5029/1 p. anchietae wilson ngarenyi dam ysl206 a/5029/3 p. anchietae wilson mchome area ysl203 a/5029/7 p. anchietae wilson chombeke area ysl066 a/5029/10 p. anchietae wilson kasigau x y/3125 p. anchietae p. malonza mbololo x y/3696/3 p. anchietae p. malonza mbololo x y/3696/4 p. anchietae p. malonza irido rnm10 a/5024/1 p. anchietae wilson irido rnm011 a/5024/2 p. anchietae wilson irido rnm012 a/5024/3 p. anchietae wilson irido rnm013 a/5024/4 p. anchietae wilson wasinyi sl398 a/5028/1 p. anchietae wilson wasinyi sl387 a/5028/2 p. anchietae wilson 287morphological variation of ridged frogs locality field number nmk catalogue number supposed name collectors wasinyi sl389 a/5029/1 p. anchietae wilson wundanyi stadium sl361 a/5034/1 p. anchietae r. ng’endo wundanyi stadium sl362 x p. anchietae r. ng’endo wundanyi stadium sl363 a/5034/13 p. anchietae r. ng’endo wundanyi stadium sl364 a/5034/7 p. anchietae r. ng’endo wundanyi stadium sl381 x p. anchietae r. ng’endo wundanyi stadium sl382 a/5034/2 p. anchietae r. ng’endo wundanyi stadium sl284 a/5034/4 p. anchietae r. ng’endo wundanyi stadium sl380 a/5034/5 p. anchietae r. ng’endo wundanyiposta stadium rnm007 a/5027/4 p. anchietae r. ng’endo wundanyiposta stadium rnm008 a/5027/2 p. anchietae r. ng’endo wundanyiposta stadium rnm009 a/5027/3 p. anchietae r. ng’endo serienyi sl354 x p. anchietae r. ng’endo serienyi sl385 x p. anchietae r. ng’endo mwatate marshes sl351 a/5031/5 p. anchietae r. ng’endo mwatate marshes sl352 a/5031/1 p. anchietae r. ng’endo mwatate marshes sl353 a/5031/3 p. anchietae r. ng’endo mwatate marshes sl356 a/5031/4 p. anchietae r. ng’endo mwatate marshes sl357 a/5031/2 p. anchietae r. ng’endo mwatate marshes sl358 a/5032 p. anchietae r. ng’endo mwatate marshes sl368 a/5025/1 p. anchietae r. ng’endo karangar.falls rnm014 a/5030/1 p. anchietae wilson karangar.falls rnm015 a/5030/3 p. anchietae wilson karangar.falls rnm017 a/5030/2 p. anchietae wilson wund.opp. bank rnm153 x p. anchietae r. ng’endo wund. opp. bank rnm036 a/5027/1 p. anchietae r. ng’endo wundanyi posta farml rnm001 a/5035/3 p. mascareniensis r. ng’endo wundanyi posta farml rnm002 a/5035/2 p. mascareniensis r. ng’endo wundanyi posta farml rnm003 a/5035/6 p. mascareniensis r. ng’endo wundanyi posta farml rnm004 a/5035/4 p. mascareniensis r. ng’endo wundanyi posta farml rnm005 a/5035/1 p. mascareniensis r. ng’endo wundanyi posta farml rnm006 a/5035/5 p. mascareniensis r. ng’endo wundanyi stadium tren sl396 x p. mascareniensis r. ng’endo wundanyi posta farml sl390 a/5034/5 p. mascareniensis r. ng’endo wundanyi posta farml sl391 a/5038/8 p. mascareniensis wilson wundanyi posta farml sl393 a/5035/7 p. mascareniensis wilson wundanyi posta farml sl394 y/5038/9 p. mascareniensis wilson wundanyi posta farml sl395 a/5035/12 p. mascareniensis wilson mwatate dam sl355 a/5031/5 p. mascareniensis p. malonza mwatate dam sl359 a/5026/2 p. mascareniensis r. ng’endo mwatate dam sl360 a/5026/4 p. mascareniensis r. ng’endo mwatate dam sl365 y/5026/3 p. mascareniensis wilson mwatate dam sl366 a/5026/1 p. mascareniensis wilson 288 r. ng’endo et alii locality field number nmk catalogue number supposed name collectors mwatate dam sl367 a/5026/6 p. mascareniensis wilson irido sl369 a/5022/4 p. mascareniensis wilson wundanyi stadium sl370 a/5036/11 p. mascareniensis wilson wundanyi stadium sl371 a/5036/5 p. mascareniensis wilson wundanyi stadium sl372 a/5036/1 p. mascareniensis wilson wundanyi stadium sl373 a/5036/12 p. mascareniensis r. ng’endo wundanyi stadium sl374 a/5036/7 p. mascareniensis r. ng’endo wundanyi stadium sl375 a/5036/8 p. mascareniensis r. ng’endo wundanyi stadium sl376 a/5036/3 p. mascareniensis r. ng’endo wundanyi stadium sl377 a/5036/6 p. mascareniensis r. ng’endo wundanyi stadium sl378 a/5036/4 p. mascareniensis r. ng’endo wundanyi stadium sl379 a/5036/10 p. mascareniensis r. ng’endo wundanyi stadium sl383 x p. mascareniensis r. ng’endo wundanyi stadium sl399 a/5036/13 p. mascareniensis r. ng’endo wundanyi stadium sl397 a/5036/14 p. mascareniensis r. ng’endo taita hills jm01873 x p. mascareniensis p. malonza taita hills ak/893 x p. mascareniensis p. malonza taita hills ak892 x p. mascareniensis p. malonza taita hills rnm030 a/5033/3 p. mascareniensis wilson taita hills rnm024 a/5033/2 p. mascareniensis wilson taita hills sl388 x p. mascareniensis wilson wund. opp. bank sl368 x p. mascareniensis wilson wundanyi field rnm072 a/5036/2 p. mascareniensis wilson wasinyi rnm078 a/5036/4 p. mascareniensis wilson wund.opp. bank sl392 a/5035/10 p. mascareniensis wilson wund.opp. bank rnm122 a/5035/4 p. mascareniensis wilson irido rnm048 a/5022/1 p. mascareniensis wilson shate area krnm049 a/5022/2 p. mascareniensis r. ng’endo mchome area krnm050 a5022/3 p. mascareniensis r. ng’endo shate area krnm051 x p. mascareniensis r. ng’endo mchome area krnm052 x p. mascareniensis r. ng’endo ngalenyi dam krnm053 x p. mascareniensis r. ng’endo mchome area krnm054 x p. mascareniensis r. ng’endo mchome area krnm055 x p. mascareniensis r. ng’endo mchome area krnm056 x p. mascareniensis r. ng’endo mchome area krnm057 x p. mascareniensis r. ng’endo madoghodo area krnm058 x p. mascareniensis wilson shate area krnm059 x p. mascareniensis wilson shate area krnm060 x p. mascareniensis wilson shate area krnm061 x p. mascareniensis wilson shate area krnm062 x p. mascareniensis wilson tambaru area krnm063 x p. mascareniensis wilson tambaru area krnm064 x p. mascareniensis wilson shate area krnm065 x p. mascareniensis wilson © firenze university press www.fupress.com/ah acta herpetologica 5(2): 265-273, 2010 sightings and successful reproduction of allochthonous reptiles in calabria emilio sperone1, antonio crescente1, elvira brunelli1, giuseppe paolillo2, sandro tripepi1 1 department of ecology, university of calabria, via p. bucci, i-87036, rende (cs), italy. corresponding author. e-mail: sperone@unical.it 2 wwf calabria, via popilia 42, i-89900, vibo valentia (vv), italy. submitted on: 2010, 16th july; revised on: 2010, 8th september; accepted on: 2010, 20th september. abstract. this paper reports information about the presence of three allochthonous reptiles species in calabria: testudo marginata, trachemys scripta elegans and chamaeleo chamaeleon. the first one was found in three sites located in the catena costiera massif and in the crati valley (northern calabria). the slider turtle was found in seven different sites throughout all the region. it massively colonised the angitola artificial lake: here, this turtle lives in natural conditions and its reproduction was confirmed by the presence of nests, eggs and hatchlings. c. chamaeleon is present in sandy coastal habitats near palmi and gioia tauro (southern calabria). from a conservationistic point of view, serious damages to autochtonous species could be caused by the spreading of t. scripta elegans: this species has already determined the local extinction of angitola’s emys orbicularis populations. keywords. allochthonous reptiles, calabria, testudo marginata, trachemys scripta elegans, chamaeleo chamaeleon. introduction animals or plants that have been intentionally or accidentally transported by man outside their biogeographic area are considered as introduced, non-native, allochthonous, exotic or alien species. an introduced species turns into an invasive species when, once overcome the acclimation period, it can spread in the new environment and causes ecological and economical injuries (pimentel et al., 2000). the introduction of a growing number of species outside their native range is one of the main causes of biodiversity loss and species extinction: in fact, exotics may displace and reduce populations of native species (holland, 1993; vitousek et al., 1996; kupferberg, 1997; wilson, 1997; hoddle, 2004) and can influence the way by which they use habitat resources (herlbold and moyle, 1986; williamson, 1996). 266 e. sperone et alii the principal factors behind species introduction, whether intentional or unintentional, are to be found not only in tourism and international trade, but also in the pet market and into a series of productive activities such as agriculture and breeding for food and industrial purposes (howarth, 1991; hill and greathead, 2000; henneman and memmott, 2001; hoddle, 2004; gherardi et al., 2008). the ecological implications of these invasions are of primary importance and concern the entire biosphere, from aquatic to land habitats. a first and valuable tool for managing invasive species of conservation importance is the knowledge of their presence and localization in the territory and the confirmation of their successful reproduction in natural conditions. for example, in italy as far as mammals are concerned, there are 15 non-native species, the majority being rodents, and 110 nonnative species of birds (andreotti et al., 2002). at least, six reptiles and one amphibian have been introduced (sindaco et al., 2006). moreover, the outcome of an introduction is usually unpredictable unless demography, resource utilisation and biotic relationships have been carefully investigated (lodge, 1993; kareiva, 1996; shigesada and kawasaki, 1997; cadi and joly, 2004). the aim of this paper is to provide data on the presence of allochthonous reptiles in calabria, the southernmost region of the italian peninsula, and to assess their successful reproduction. in fact, observations related to this topic, especially to trachemys scripta elegans reproduction, are scant for southern italy (ficetola et al., 2009). materials and methods data collection included field researches and the collection of information from local peoples, mainly from farmers, shepherds and foresters. field researches were performed during years from 1998 to 2008, above all in spring and summer when species are more active. a total of 269 sampling sites were investigated along all the region. on average, each site was visited three times (range 1-7). data collected were registered in the herpetological database of the department of ecology, university of calabria (rossi et al., 1991). verification of red-eared terrapin reproduction in the angitola lake was performed during 2007 and 2008, by visual census. the reproductive success was considered as “confirmed” if nests with eggs and hatchlings were observed. cartographical analysis was carried out employing the gis vector cartographic software map-info. results twelve records of allochthonous reptiles were collected, for an amount of three species: testudo marginata schoepff, 1792, trachemys scripta elegans (schoepff, 1792) and chamaeleo chamaeleon (linnaeus, 1758). their distribution in the study area is showed in fig. 1. the presence of t. marginata (fig. 2 a) was confirmed in three sites located in the catena costiera massif and in the crati valley with an elevation from 160 to about 800 m a.s.l. all our observations referred to adult specimens observed in the wild. no nests nor newborns were observed. the slider turtle t. scripta elegans (fig. 2 b) was found in seven different sites with an elevation from 0 to 50 m a. s. l.; the individuals observed near brancaleone (rc) represent the southernmost record known for the italian peninsula. data collected in the field 267sightings and reproduction of allochthonous reptiles in calabria showed that the species has massively colonised the angitola artificial lake (vv): here, during 2007 and 2008, we observed that this turtle lives in natural conditions and it is well naturalized. in this site, the reproduction of the species was widely confirmed. eighty nests were discovered during a systematic search around the lake perimeter (fig. 2 c). clutch size ranged from one to ten eggs for nests (average 3.73 eggs for nest). newborns were observed in water. c. chamaeleon (fig. 2 d) was found in two sites characterised by coastal scrub habitats or pine forests near palmi and gioia tauro (rc). all our observations referred to mature specimens. in table 1 locality, elevation and habitat description for each site of presence of allochthonous reptiles are reported. fig. 1. distribution map of sightings of allochthonous reptiles in calabria. 268 e. sperone et alii table 1. features of the finding sites where allochthonous reptiles were found species finding site habitat elevation year c. chamaeleon palmi coastal pine forest 5 m a.s.l. 1994 c. chamaeleon gioia tauro coastal scrub 10 m a.s.l. 2000 c. chamaeleon palmi coastal pine forest 5 m a.s.l. 2002 c. chamaeleon palmi coastal pine forest 5 m a.s.l. 2007 t. marginata s. vincenzo lacosta uncultivated area 800 m a.s.l. 1997 t. marginata rose scrub 800 m a.s.l. 1999 t. marginata tarsia uncultivated area 160 m a.s.l. 2007 t. scripta angitola lake 70 m a.s.l. 2007 t. scripta paola stream 10 m a.s.l. 1999 t. scripta nasari pond 50 m a.s.l. 2001 t. scripta crotone pond 27 m a.s.l. 2003 t. scripta brancaleone pond 50 m a.s.l. 2005 t. scripta lamezia terme pond 100 m a.s.l. 2005 t. scripta castiglione cosentino pond 250 m a.s.l. 2007 fig. 2. allochthonous reptiles of calabria: testudo marginata (a), trachemys scripta elegans adult (b) and nest (c), chamaeleo chameleon (d). 269sightings and reproduction of allochthonous reptiles in calabria discussion our research showed the presence of three species of allochthonous reptiles in calabria. t. marginata and c. chamaeleon showed a localized distribution: our sightings have been made in widely different habitat types, often near human settlements, suggesting recent introduction. t. marginata is a non-native species for italy, although its introduction to sardinia and etruria dates back to ancient time (sindaco et al., 2006). data presented in this paper add to few undated records for peninsular italy: tuscany (sindaco et al., 2006) and latium (carpaneto, 2000). it is interesting to observe that calabrian population is located at an altitudinal range higher than the optimal one (0-400 m a.s.l.) showed by the sardinian records. the presence of the mediterranean chameleon in calabria represents a new datum: the species, in fact, has been reported for sicily (gasc et al., 1997) and for apulia (basso and calasso, 1991; fattizzo, 1996; fattizzo and marzano, 2002), where since 1980 observations lasted about ten years could indeed imply a small reproductive population, especially considering the limited longevity of the species. calabrian records cover years from 1994 to 2007: so, the naturalization and successful reproduction of the species could be possible. among allochthonous reptiles species founded in calabria, the most problematic one is the red-eared slider turtle (t. scripta elegans), introduced in the 1980s (tripepi and aceto, 2000). widespread as a pet, it was frequently abandoned in natural habitats and in urban areas. our research showed that this turtle has a widespread distribution in the study area, as also observed in other regions of italy: piedmont (andreone and sindaco, 1999), lombardy (ferri and di cerbo, 1995; bernini et al., 2004), trentino alto adige (caldonazzi et al., 2002), veneto (fracasso et al., 2000), friuli venezia giulia (lapini et al.,1999), liguria (doria and salvidio, 1994), tuscany (scoccianti and cigna, 1999), emilia-romagna (lanza and corti, 1993), latium (marangoni, 2000), molise (lanza and corti, 1993), apulia (scillitani et al.,1996) and sicily (turrisi and vaccaro, 1998). however, in some regions the presence of the species has occurred only in single localities or refers only to single individuals. not much is known about reproduction of t. scripta in natural habitats in italy. it seems that the species is certainly able to reproduce, not in the wild but only in urban or suburban pools, as in friuli venezia-giulia (lapini et al., 1999) and latium (marangoni, 2000). ferri and di cerbo (1998) reported for piedmont and lombardy only egg-depositions, but not hatchlings. ficetola et al. (2003) reported the case of the reproduction of this species for the po delta. this paper presents the first confirmed reproductive site in the wild for t. scripta in southern italy: its reproductive success was also confirmed in europe only for france (cadi et al., 2004) and spain (de roa and roig, 1997; martinez-silvestre, 1997; bertolero and canicio, 2000; capalleras and carretero, 2000). the high number of nests founded around the angitola lake disproves the hypothesis outlined by luiselli et al. (1997) that suggested that the danger of naturalization of the species is less serious than is thought. it is believed that this species can compete with emys orbicularis, the only native european pond turtle inhabiting calabria, currently in sharp decline due to alterations to water surfaces (cadi and joly, 2003, 2004). it is due to the fact that the chelonian communities in north-american freshwaters differ from the european ones: in fact, whereas most european freshwater habitats are commonly occupied by only one species, north-american habitats support 270 e. sperone et alii up to six species (gibbons, 1990). in such assemblages, species exhibit high competitive abilities that ought to favour their establishment in european waters, at the expense of the niche breadth of species such as emys orbicularis. in particular, the expected advantage of allochthonous species relies on lower age at maturity, higher fecundity and larger adult body size (rollinat, 1934; cagle, 1946; lebboroni and chelazzi, 1991; arvy and servan, 1998; cadi and joly, 2003, 2004). on the basis of these assumptions, and due to the fact that there are no sightings of individuals of e. orbicularis from the angitola lake since 1992 (tripepi and aceto, 2000; g. paolillo pers. comm.), it is possible to affirm that the presence of t. scripta in the study area could affect the fitness and survivorship rate of e. orbicularis. on the basis of the collected data, from a conservationistic point of view, we can affirm that: t. marginata and c. chamaeleon are alien, but not invasive species according to the iucn (2000) classification, and should thus be subjected to biodiversity conservation measures; t. scripta potentially threats natural populations of e. orbicularis and it can be considered as a serious invasive alien species and several european countries have stopped the importation of the red-eared slider from north-america. our results demonstrate a need for: estimating the size of t. scripta populations in calabria; evaluating their reproductive success; planning the removing of the exotic turtles from natural habitats, where possible. such actions are urgent and remain a challenge for the managements of natural environments. references andreone, f., sindaco, r. 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(1997): foreword. in: strangers in paradise: impact and management of nonindigenous species in florida, p. 9-10. simberloff, d., schmitz, d.c., brown, t.c. eds, washington dc, island press. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 7(1): 105-110, 2012 long term observations on the alimentation of wild eastern greek tortoises testudo graeca ibera (reptilia: testudines: testudinidae) in dobrogea, romania alexandru iftime1, oana iftime2 1 “grigore antipa” national museum of natural history, kiseleff bvd. no. 1, bucharest, romania. corresponding author. e-mail: alexandru_iftime@yahoo.com 2 university of bucharest, faculty of biology, department of genetics, aleea portocalelor 1-3, s6, bucharest, romania. submitted on: 2011, 25th september; revised on 15th december; accepted on 2012, 19th january. abstract. the wild diet of testudo graeca ibera in dobrogea, romania is investigated by direct observation. a clear predominance (over 95%) of plant matter is noticed, with 25 plant species consumed. moreover the ingestion of animal matter (carrion) as well as calcareous earth was observed. keywords. testudo graeca, alimentation, long term observations the spur-thighed tortoise, testudo graeca, including its eastern subspecies, t. g. ibera, is well known as a terrarium companion and also as a protected species. thus, numerous recommendation for the captive diet of t. graeca are available, and also data showing it as a generalist vegetarian that also takes occasional small quantities of animal food, i.e. invertebrates and carrion (buskirk et al., 2001). there are, however, few data about the feeding habits of the wild t. graeca ibera populations in the balkans and romania (beyond general data such as those of, e.g., fuhn and vancea, 1961). there is greater knowledge on the populations in spain (e.g. cobo and andreu, 1988; andreu et al., 2000; díaz-paniagua and andreu, 2009), the caucasus (bannikov et al., 1977) or north africa (el mouden et al., 2006; rouag et al., 2008). the similar species t. hermanni is also better known, especially as regards the western populations (e.g. nougarède, 1998; soler et al., 2007; mazzotti et al., 2007; muñoz et al., 2009; budó et al., 2009). greater knowledge on the type of food consumed is an addittive value for the conservation policies concerning this vulnerable species. this knowledge allows to adjust the care of captive bred populations to the natural condition of the species (cf. willemsen et al., 2002) and to manage wild populations. the natural diet of testudo spp. can be investigated into by two methods: direct observation (e.g. lagarde et al., 2003) and/or the analysis of faecal matter (e.g. cobo and andreu, 1988; el mouden et al., 2006, díaz-paniagua and andreu, 2009; munoz et 106 a. iftime and o. iftime al., 2009). we have chosen the first method. more than 500 specimens of t. graeca were observed in the wild, from march to october, in dobrogea (romania), over a period of 10 years (2000-2010). most of these were observed in a forest-steppe biotope, composed of open, mainly oak and hornbeam forest, with an understory of dogwood (cornus), hawthorn (crataegus) dog rose (rosa) etc., and steppe patches dominated by stipa. when the tortoises were observed feeding, notes and pictures what they consumed were taken, allowing, in most cases, identification of the food articles. the alimentation appears to be predominantly vegetarian; 96,5% of the cases in which t. greaca was observed feeding involved vegetal matter. the 25 plant species observed to be consumed are presented in table 1. we noticed a predilection for the ingestion of young leaves, especially those of ficaria, taraxacum, lotus, trifolium, medicago, fragaria, polygonum, poaceae, sonchus, teucrium, table 1. plants consumed by t. graeca, with note of the ingested part. class order family species consumed part frequency of consumption magnoliopsida ranunculales ranunculaceae ranunculus ficaria leaves, flowers low (1-5 %) asterales asteraceae taraxacum officinale leaves, flowers moderate (5-10%) sonchus sp. leaves low (1-5 %) crepis sp. leaves low (1-5 %) artemisia sp. (a. pontica?) leaves low (1-5 %) fabales fabaceae lotus corniculatus leaves high (>10%) trifolium sp. leaves, flowers high (>10%) medicago sp. leaves moderate (5-10%) vitales vitaceae vitis vinifera leaves low (1-5 %) rosales rosaceae fragaria sp. leaves moderate (5-10%) rosa sp. leaves low (1-5 %) pyrus sp. fruit low (1-5 %) prunus sp. fruit low (1-5 %) lamiales lamiaceae teucrium polium leaves moderate (5-10%) gentianales rubiaceae galium sp. leaves low (1-5 %) caryophyllales amaranthaceae atriplex sp. leaves low (1-5 %) amaranthus sp. leaves low (1-5 %) polygonaceae polygonum sp. leaves low (1-5 %) cornales cornaceae cornus mas fruit low (1-5 %) liliopsida poales poaceae festuca arundinacea, f. sp. leaves low (1-5 %) poa sp. leaves moderate (5-10%) dactylis glomerata leaves low (1-5 %) dichanthium sp. leaves low (1-5 %) . leaves moderate (5-10%) 107diet of testudo graeca in romania and less commonly those of artemisia, rosa, rubiaceae, chenopodiaceae; very young plants of lamiaceae and apiaceae were also consumed. fruit consumption is sporadic: cornus mas, pyrus sp. (fig. 1), prunus sp. flowers/inflorescences (taraxacum, fabaceae, ranunculaceae) are generally ingested together with leaves of the same plant. as compared to the diet observed for t. graeca in other regions, we can notice that plants pertaining to the families asteraceae, fabaceae and poaceae are frequently consumed, which is in accord with the data of bannikov et al. (1977), cobo and andreu (1988), el mouden et al. (2006) and díaz-paniagua and andreu (2009). even at the genus level, we can notice that lotus and medicago, staples in the alimentation of spanish t. graeca (díaz-paniagua and andreu, 2009) are also frequently consumed by romanian specimens. the recurrence of these plant families as the favourite food of t. graeca – despite the quite different environment in which the observations took place, especially from the iberian and north african data – supports the idea that this species is selective as regards the plant species it consumes (cf. el mouden et al., 2006). lamiaceae and rosaceae are also consumed with moderate frequency, whereas plants pertaining to other families are less commonly taken. t. graeca and other chelonians may ingest toxic plants species as anti-helminthics, (see the discussion in el mouden et al., 2006). on the basis of our observations, among the plants ingested, only artemisia cf. pontica (the roman wormwood, containing thujone fig. 1. t. graeca consuming pyrus fruit (wild pear). greci, tulcea county, romania. photo by oana iftime. 108 a. iftime and o. iftime fig. 2. t. graeca trying to eat from dead wild cat. cheia, constanţa county, romania. photo by alexandru iftime. fig. 3. t. graeca having consumed friable limestone, with limestone dust around snout, urluia, constanţa county, romania. photo by alexandru iftime. 109diet of testudo graeca in romania and sometimes used by herbalists as a vermifuge – hence its name – despite its toxicity to man in large doses) could fit into the cathegory of anti-helminthics (table 1). occasional intake of animal food, i.e. carrion, was also noticed. specimens were observed feeding on a dead wild cat (fig. 2), as well as on remains of dead birds, bovines and ovines, leftovers from the prey of jackals and/or feral dogs. overall, the carrion (and generally animal food) intake seems far less marked than in other studies (e.g. andreu et al., 2000), but we cannot exclude that this could be due to a method bias since through direct observation it is more difficult to find animals feeding upon carrion. geophagy in t. graeca is mentioned by fuhn (1969); török (2001) records the ingestion of kaolin (china clay). we have also noticed the ingestion of earth, especially clays containing limestone, or even degraded, friable limestone rock (fig. 3). this behaviour was mostly noticed in juveniles and sub-adults and should probably be conected to their special mineral needs (mostly calcium) – cf. liesegang et al. (2007). ingestion of non-calcareous pebbles (cf. gagno and alotto, 2010) was not observed by us. coprophagy, which is mentioned by fuhn and vancea (1961), was not observed by us. our data allows the conclusion that t. graeca is, as previously known, mainly a herbivore, quite selective in its choice of plant food, but it supplements its vegetal diet with animal protein and mineral intake, in minimal but probably necessary quantities. acknowledgements to dr. g. negrean (the botanical garden of bucharest) and to dr. m. petrescu (icem tulcea), for their kind support in the identification of plants. references andreu, a. c., díaz-paniagua, c., keller, c. (2000): la tortuga mora (testudo graeca l.) en doñana., monografías de herpetología, vol. 5, asociacion herpetologica española barcelona. bannikov, a. g., darevskyi, i. s., ishchenko, v. g., rustamov, a. k., shcherbak, n. n., (1977): opredelitel’ zemnovodnykh i presmykeyushchikhsya fauny sssr. prosveshcheniye, moskva. budó, j., capalleras, x., fèlix, j., font, j. (2009): aportacions sobre l’estudi de l’alimentació de la tortuga mediterrània (testudo hermanni hermanni) a la serra de l’albera (catalunya). butlletí de la societat catalana d’herpetologia, 18: 109-115. buskirk, j. r., keller, c., andreu, a. c. (2001): testudo graeca linnaeus, 1758 – maurische landschildkröten. in: fritz, u. (ed.): schildkröten (testudines) i, pp. 126-178. handbuch der reptilien und amphibien europas. aula verlag, wiebelsheim. cobo, m., andreu, a. c. (1988): seed consumption and dispersal by the spur-thigh tortoise testudo graeca. oikos, 51:267-273. díaz-paniagua, c., andreu, a. c. (2009): tortuga mora – testudo graeca. en: enciclopedia virtual de los vertebrados españoles. carrascal, l. m., salvador, a. (eds.). museo nacional de ciencias naturales, madrid. versión 21-07-2009. 110 a. iftime and o. iftime el mouden, e. h., slimani, t., ben kaddour, k., lagarde, f., ouhammou, a., bonnet, x. (2006): testudo graeca graeca feeding ecology in an arid and overgrazed zone in morocco. j. arid. environ. 64: 422-435. fuhn, i.e. (1969): broaşte, şerpi, şopîrle, pp. 233-236. ed. natura şi omul, bucureşti [in romanian] fuhn, i.e., vancea, şt. (1961): reptilia (ţestoase, şopîrle, şerpi). in: fauna rpr, 14 (2): 1-338. edit. academiei rpr, bucureşti. [in romanian] gagno, s., alotto, c. (2010): géophagie chez la tortue d’hermann, testudo hermanni gmelin, 1789 (chelonii, testudinidae), dans la région des maures (var, france). bull. soc. herp. fr. 135-136: 23-32 lagarde, f., bonnet, x., corbin, j., henen, b., nagy, k., mardonov, b., naulleau, g. (2003): foraging behaviour and diet of an ectothermic herbivore: testudo horsfieldi. ecography 26: 236-242. liesegang, a., hatt, j. m., wanner, m. (2007): influence of different dietary calcium levels on the digestibility of ca, mg and p in hermann’s tortoises (testudo hermanni). j. anim. physiol. an. n. 91: 459-464. mazzotti, s., bertolucci, c., fasola, m., lisi, i., pisapia, a., gennari, r., mantovani, s., vallini, c. (2007): la popolazione della testugine di herrmann (testudo herrmanni) del bosco della mesola. quad. staz. ecol., civ. mus. st. nat., ferrara 17: 91-104. muñoz, a., soler, j., martínez-silvestre, a. (2009): aportaciones al studio de la alimentación de testudo hermanni hermanni en el parque natural de la sierra de montsant. bol. asoc. herpetol. esp. 20: 54-58. nougarède, j.p. (1998): principaux traits d’histoire naturelle d’une population de tortue d’hermann (testudo hermanni) dans le sud de la corse. ephe thesis, université de montpellier, france. rouag, r. ferrah, c., luiselli, l., tiar, g., benyacoub, s., ziane, n., el mouden, e. (2008): food choice of an algerian population of the spur-thighed tortoise, testudo graeca. afr. j. herpetol. 57(2): 103-113 soler, j., martínez-silvestre, a., saez, a., peris, m. (2007): dieta de les tortugues mediterrànies testudo hermanni hermanni reintroduïdes al parc natural de la serra del montsant, 2006-2007. iii jornades del parc natural de la serra de montsant. falset. tarragona. török, zs. (2001): notă privind geofagia la testudo graeca ibera pallas 1814 (reptilia, testudines, testudinidae). satu mare – studii şi comunicări, ştiinţele naturii, ii-iii: 188-190 [in romanian] willemsen, r. e., hailey, a., longepierre, s., grenot, c. (2002): body mass condition and management of captive european tortoises. herpetol. j. 12: 115-121. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 8(2): 171-176, 2013 discovery of alien water frogs (gen. pelophylax) in umbria, with first report of p. shqipericus for italy dario domeneghetti1,*, giacomo bruni2, mauro fasola3, adriana bellati3 1 department of biology, university of rome “tor vergata”, via della ricerca scientifica, 1 00133 rome, italy. *corresponding author. e-mail: dario.eco.domeneghetti@gmail.com 2 department of biology, university of florence, via romana, 17 50125 florence, italy 3 department of earth and environmental science, university of pavia, via ferrata, 9 27100 pavia, italy submitted on 2013, 16th september; revised on 2013, 13th november; accepted on 2013, 19th november. abstract. allochthonous water frogs (gen. pelophylax) have been repeatedly introduced in several european countries, causing dramatic consequences for the conservation of indigenous taxa. in italy, invasive populations are known for northern regions, where they were introduced mainly for edible and scientific purposes. here, we report the first detection of an alien population of water frogs in central italy, along the resina valley (umbria). genetic analysis of the mitochondrial nd3 gene polymorphism assigned some specimens to two different pelophylax ridibundus clades widespread in central and eastern europe. by contrast, two samples matched the mitochondrial dna (mtdna) sequence of pelophylax lessonae bergeri, an autochthonous taxon widespread in central italy, suggesting possible hybridization between alien and indigenous frogs. finally, the specific haplotype of pelophylax shqipericus, the albanian pool frog, was also identified according to mtdna polymorphism. this record, firstly reported for italy, poses concerns for the conservation of this cryptic taxon, suggesting that international water frog trade may involve also particularly endangered species. keywords. pelophylax ridibundus, pelophylax shqipericus, european water frogs, alien species, conservation, mitochondrial dna, central italy. water frogs of the genus pelophylax (fitzinger, 1843) comprise several species widespread in a multitude of freshwater habitats across western palearctic. within the genus, several complexes of genetically similar taxa can naturally hybridize in the wild, posing questions for species determination and conservation (e.g., akin et al., 2010). for instance, in central and eastern europe, the pool frog pelophylax lessonae (camerano, 1882) can mate with the marsh frog pelophylax ridibundus (pallas, 1771), to produce the hybrid taxon pelophylax esculentus (linnaeus, 1758) according to a fascinating and unusual mode of reproduction (i.e., hybridogenesis). during hybridogenetic reproduction, p. esculentus discards the lessonae genome prior to meiosis in order to produce gametes containing only the ridibundus genome, which is clonally transmitted in each generation (tunner and heppich, 1981; pagano et al., 1997). the absence of strong reproductive barriers coupled with the weak morphological variation of different taxa pose serious issues for conservation, because invasive lineages may rapidly pollute the gene pool of indigenous populations spreading over a large geographic range without being detected (e.g., pagano et al., 2003; holsbeek and jooris, 2010; holsbeek et al., 2010). in recent years, molecular techniques proved to be extremely useful for species determination within the pelophylax genus, because distinct taxa can be identified according to genetic variation, despite the weak morphological and/or bioacustic differentiation of several species (lode and pagano, 2000; plötner and ohst, 2001; plötner et al., 2001, 2008, 2009; akin et al., 2010). 172 dario domeneghetti et al. despite exotic lineages of european water frogs have been introduced repeatedly due to uncontrolled commercial trade (e.g., belgium: holsbeek et al., 2010; france: schmeller et al., 2007; germany: mayer et al., 2013; great britain: zeisset and beebee, 2003; spain: arano et al., 1995), this group is currently classified in italy as less vulnerable among amphibians (andreone and luiselli, 2000). from a taxonomic point of view, the situation of italian populations is rather complicated by the presence of at least five different species and hemiclonally-reproducing hybrid lineages, living in mixed hybridogenetic populations. in northern italy, hybridogenetic populations of p. lessonae and p. esculentus occur, while southwards populations are assigned to two different taxa, pelophylax lessonae bergeri (günther, 1986) and the hybridogenetic pelophylax hispanicus (bonaparte, 1839). in sicily, a different and not yet described subspecific taxon exists (canestrelli and nascetti, 2008), while in sardinia, water frog populations have been introduced several times during the past two centuries from various regions of the italian peninsula as well as from other countries (e.g., eastern turkey, bellati et al., 2012). autochthonous populations of p. ridibundus occur only in the carso triestino (friulivenezia giulia, north-eastern italy, dolce, 1976; mezzena and dolce, 1978; lanza, 1983), but allochthonous populations have been repeatedly introduced in many italian regions from other european countries for edible and scientific purposes (e.g., andreone and sindaco, 1999; bressi, 2006b; razzetti et al., 2006). the most important geographic source of alien populations has been identified in the eastern mediterranean regions, such as turkey, greece and the balkans (see ficetola and scali, 2010 for a history of introductions). to date, documented introduction of alien taxa of european water frogs in italy are the balkan frog pelophylax kurtmuelleri (gayda, 1940) in liguria and piedmont regions (lanza, 1962), p. ridibundus in friuli venezia giulia and trentino alto adige (lapini and zanghellini, 1993; see also bressi, 2006 a) and probably pelophylax bedriagae (camerano, 1882) in emilia romagna. noteworthy, despite the detection of several alien populations of water frogs in northern italy, records from central regions (south of emilia-romagna) are scarce or even completely absent. in umbria (central italy) no allochthonous taxa of water frogs have been reported during last decades (ragni et al., 2006). during summer 2012, we found large-sized frogs in a pond located near the resina river, in the province of perugia (43.24679°n, 12.48821°e wgs84). in late september 2012, during field work, we observed adult and juvenile water frogs of the pelophylax complex. however, no correspondence of these specimens with any italian taxon was possible according to morphological characters. these specimens were characterized by large body size, wrinkled skin, small metatarsal tubercles, and males had black-coloured vocal sacs (table 1, fig. 1b). according to morphology, these frogs were classified as p. cf. ridibundus and clearly distinguished from the autochthonous central italian taxa observed at the same site (p. l. bergeri and p. hispanicus), which showed bright green and yellow colours and big metatarsal tubercles (lanza, 1983; günther, 1990; lanza et al., 2007, 2009). the unidentified water frogs were found together, or in immediate proximity, of a rich batrachofauna including lissotriton vulgaris, triturus carnifex, bufo bufo, hyla intermedia, rana italica and rana dalmatina. morphometric measurements were recorded by photographing them on a metric board and the images were then analyzed using quantum gis and arcview gis software. this allowed to take the standard morphometric measures suggested as diagnostic by previous authors (plötner et al., 1994): tibia length (tl); femur length (fl); inner metatarsal tubercles plus first toe length (mtftl); snout to vent length (svl); head width (hw); nostril to eye length (ne) and tympatable 1. morphometric measures (in mm) of the 17 specimens found in the surveyed pond within the resina river catchment and morphologically classified as pelophylax cf. ridibundus. specific assignments of individuals analyzed with molecular techniques have been reported on the basis of mitochondrial nd3 sequence polymorphism (rid, pelophylax ridibundus; ber, pelophylax lessonae bergeri; shq, pelophylax shqipericus). for the explanation of different measures’ acronyms, please refer to the text. juv, specimens metamorphosed in the year of sampling; ♀, female; ♂, male. idsex mtdna tl fl mtftl svl hw ne ty 1♂ ber 26.3 23.8 9.9 48.5 17.7 3.7 3.8 2♀ 26.2 26.8 9.5 49.6 17.3 3.4 4.1 3♀ rid 24.3 23.0 7.7 46.3 15.9 3.2 3.4 4♂ rid 23.1 22.9 7.7 44.7 15.2 2.8 3.4 5♂ 24.9 23.9 8.9 47.2 16.4 3.0 3.7 6juv. ber 13.5 14.9 5.0 30.6 11.0 1.9 2.7 7 rid 40.3 33.3 12.8 79.8 28.2 5.9 5.1 8♂ 27.4 19.1 10.5 53.4 19.1 3.6 4.2 9♂ 38.3 52.2 15.0 71.9 27.9 5.8 5.5 10♂ 24.6 24.2 8.1 47.4 16.3 3.3 3.8 11♂ 20.1 13.9 8.1 38.7 14.0 2.9 3.1 12♂ 31.0 38.3 11.0 58.8 21.6 4.0 4.7 13♀ rid 25.7 21.0 9.3 49.9 17.3 3.5 3.9 14♂ 35.8 43.3 13.2 68.7 23.8 4.6 5.6 15♂ 29.4 30.9 10.1 56.6 19.9 3.8 4.5 16juv. 11.9 13.8 2.5 26.0 9.9 1.7 2.1 17♀ shq 34.9 36.8 12.4 77.2 25.8 4.9 5.4 173first report of alien frogs (gen. pelophylax) in umbria num diameter (ty) (table 1). overall, we measured 17 of these questionable specimens. a small piece of tissue was also collected from seven specimens by toe-clipping and stored in 96% ethanol for genetic analysis. in the laboratory, genomic dna was purified with a standard salt-extraction procedure using the commercial kit “archivepure dna cell /tissue” kit (5 prime, hamburg, germany) and following the manufacturer’s instructions. a portion (340 base pair long) of the mitochondrial nd3 gene encoding for the subunit 3 of nadh dehydrogenase was amplified with already published primer pair (plötner et al., 2008) modified from meyer (1993). this marker appears to be diagnostic for pelophylax species (akin et al., 2010), allowing also the recognition of distinct geographic clades of p. ridibundus (plötner et al., 2008; akin et al., 2010). pcr products were purified with “gelelute extraction” kit (5 prime, hamburg, germany) and sequenced on an abi 3730xl using the forward primer (macrogen europe, amsterdam, the netherlands). sequences were compared with all the available homologous sequences available from genbank (www.ncbi. nlm.nih.gov/genbank/) using the basic local alignment search tool (blast, http://blast.ncbi.nlm.nih.gov/blast. fig. 1. pictures of (a) pelophylax ridibundus specimen number 9, (b) p. ridibundus specimen number 9, detail of the vocal sac black-coloured, (c) p. ridibundus specimen number 13, showing a short and flat metatarsal tubercle and (d) p. shqipericus specimen number 17. 174 dario domeneghetti et al. cgi) and setting the nucleotide blast (nblast) algorithm with default parameters. genetic analysis assigned four individuals (number 3, 4, 7 and 13) to european clades of p. ridibundus widespread in central and eastern europe, for which several geographic mitochondrial haplogroups have been described (plötner et al., 2008). in particular, sample 3, 7 and 13 (accession number: hg763871, hg763872 and hg763873 respectively) yielded a 100% match with the previously identified haplotype r17 (distributed in france, bulgaria, serbia, slovakia, romania, latvia, poland and russia), which is the most widespread haplotype in central european p. ridibundus, and with r18 and r19 (occurring in bulgaria and greece, respectively), while sample 4 (accession number: hg763874) was identical with haplotypes r13 (distributed in germany, lithuania, macedonia, romania, ukraine and latvia) and to r14 from greece, which is widely distributed in eastern europe (plötner et al., 2008). the genetic analysis of individuals 1 (accession number: hg763869) and 6 (accession number: hg763870, a young individual metamorphosed in the same year) assigned these specimens to p. l. bergeri with 100% probability, matching one published sequence from italy (akin et al., 2010). interestingly, these individuals, possessing bergeri-specific mtdna, but morphologically looking very similar to frogs genetically identified as p. ridibundus (tonality of brown colour, no yellow pigment on the hind legs, little metatarsal tubercles), may indicate hybridization between alien p. ridibundus and autochthonous p. l. bergeri. recent molecular analyses of serum albumin intron-1 polymorphism according to the technique by hauswaldt et al. (2012) for the detection of hybrids within the p. esculentus complex seem to support this conclusion (bellati a., unpubl. data). finally, individual 17 (accession number: hg763875, an adult female) was identical with the nd3 sequence of pelophylax shqipericus (hotz, uzzell, günther, tunner & heppich, 1987), the albanian pool frog, from lake skutari, montenegro (akin et al., 2010). this species inhabits a restricted geographic area between greece and southern montenegro, its populations are currently endangered due to rapid wetlands loss and harvesting for commercial purpose, particularly food consumption, and it is therefore under consideration for cites listing (gratwicke et al., 2010). the discovery of a frog carrying p. shqipericus mtdna was unexpected, but at the same time it posed major concerns about consequences of uncontrolled water frog trade. indeed, according to our results, translocations of individuals outside their native range could negatively affect not only indigenous populations, but also the persistence of cryptic and endangered taxa with very localized spatial distribution. further nuclear and eventually morphological analyses are needed to clarify this aspect. noteworthy, unpublished molecular investigations of serum albumin intron-1 polymorphism seem to corroborate mitochondrial evidence (bellati a., unpubl. data), since this individual showed a unique nuclear profile which also differs from all the species analyzed so far (hauswaldt et al., 2012), including p. ridibundus, p. kurtmuelleri, p. bedriagae, p. lessonae and p. l. bergeri. as suggested by several studies, the main risk for the genetic integrity of autochthonous populations results from the release of allochthonous pelophylax species and their hybridization with native water frogs (pagano et al., 2003; schmeller et al., 2007; holsbeek et al., 2008, 2010; holsbeek and jooris, 2010; luquet et al., 2011). accordingly, italian hybridogenetic populations could be threatened by the introduction of allochthonous european p. ridibundus lineages, which may provoke a rapid genetic loss of p. lessonae and p. l. bergeri due to hemiclonal exclusion of the lessonae genome in the hybrids (plötner et al., 2008). finally, we are aware that the syntopic presence of native italian taxa, p. ridibundus and p. shqipericus in the same small studied pond (approximately 20 square meters) could eventually lead to the production of hybrids whose genealogy would be difficult to assess. natural hybrids between p. ridibundus and balkan species such as p. shqipericus have been occasionally reported (hotz and uzzell, 1982). these individuals are obviously hybrids in constitution, do not exclude any parental genome, encounter meiotic disturbances and decrease in fitness. similarly, they are unable to reproduce hybridogenetically (ragghianti et al., 1999). inter-specific crosses between p. shqipericus and other european taxa (like p. lessonae) may also generate high hatching rate (plötner et al., 2010). therefore, further investigations with co-dominant nuclear markers (such as hypervariable microsatellites) are strongly needed to assess the real hybridization rate in central italian ponds of the resina river, in order to undertake efficient measures to control their possible spread. in conclusion, our work allowed for the first time the detection of non-native water frog taxa in central italy, whose invasive potential will be assessed in future by more extensive surveys and greater sampling effort. moreover, we confirmed the importance of molecular analyses for water frog identification, which appeared not possible just according to morphology and biometric features. acknowledgements we are grateful to silvio marta for useful discussions, suggestions and for the protocol to analyzing images in gis 175first report of alien frogs (gen. pelophylax) in umbria environmental, to walter cocca who helped in the laboratory analysis and to giulia severi and giulia ricciardi for the pictures and for their help in the field. references akin, ç., can bilgin, c., beerli, p., westaway, r., ohst, t., litvinchuk, s.n., uzzell, t., bilgin, m., hotz, h., guex, g.d., plötner, j. 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(2003): population genetics of a successful invaders: the marsh frog rana ridibunda in britain. mol. ecol. 12: 639-646. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 8(1): 53-57, 2013 first evidence of a paedomorphic population of the smooth newt (lissotriton vulgaris) in the czech republic václav gvoždík1,2, veronika javůrková3,4, oldřich kopecký5,* 1 department of zoology, national museum, cirkusová 1740, 193 00 prague, czech republic 2 laboratory of molecular ecology, institute of animal physiology and genetics, academy of sciences of the czech republic, 277 21 liběchov, czech republic 3 department of zoology, faculty of science, charles university in prague, viničná 7, 128 44, prague, czech republic 4 institute of vertebrate biology v. v. i., academy of sciences of the czech republic, květná 8, 603 65 brno, czech republic 5 department of zoology and fisheries, faculty of agrobiology, food and natural resources, czech university of life sciences, kamýcká 957, 165 21 prague, czech republic. * corresponding author. e-mail: kopeckyo@af.czu.cz submitted on 2012, 20th december; revised on 2013, 13th march; accepted on 2013, 27th march. abstract. facultative paedomorphosis is an environmentally induced polymorphism that is well known for many caudate species including newts. although facultative paedomorphosis has been documented in some smooth-newt populations, records of entirely paedomorphic populations outside the balkans are limited. here we present the first evidence of a paedomorphic population of the smooth newt in the czech republic with discussion of potential causes that need to be further tested. keywords. facultative paedomorphosis, urodeles, “triturus”, central europe. facultative paedomorphosis, the case of phenotypic heterochrony when some sexually mature individuals from a population (or some populations of a species) retain some states of larval morphology, has been widely documented for many caudates (review in denoël et al., 2005; laudet, 2011). in europe, facultative paedomorphosis regularly occurs in newts (former genus “triturus”) with high frequency in the alpine newt (ichthyosaura alpestris), especially in some areas of the balkans and alps (denoël et al., 2001). beside the alpine newt paedomorphosis has also been recorded in other newt species (summarized by wells, 2007): lissotriton helveticus, l. italicus, l.  vulgaris, triturus cristatus, t.  macedonicus, and ommatotriton ophryticus (başkale et al., 2011). in the smooth newt (l.  vulgaris), this phenomenon is also relatively common, although usually only a small part of a population or single individuals are paedomorphic. cases of peadomorphosis in the smooth newt have been reported from various places within almost the whole distribution range (review in schmidtler and franzen, 2004), but entirely paedomorphic populations of l. vulgaris are rarely reported (dely, 1967; dolmen, 1981; denoël et al., 2009). one such population from northern hungary was described like a different subspecies, triturus vulgaris tataiensis (dely, 1967), but validity of this subspecies was rejected (raxworthy, 1990). here, we report a presumably almost completely paedomorphic population of l. vulgaris from the czech republic. this is the first formally reported recent record of urodelan paedomorphosis for the country. paedomorphic smooth newts (fig. 1) were discovered (by vg and vj) on 28 april 2012 in a water reservoir in stará lysá (50°13’31.6”n, 14°47’57.1”e, ~190 m a.s.l.). the finding place is situated in “polabí”, which is a lowland area along the labe (elbe) river in the central bohemian region. this part of the czech republic is one of the warmest, with dry and mild winters and average annual temperature between 8-9 °c (quitt, 1971). the local landscape 54 václav gvoždík et al.  fig. 1. smooth newts observed in the stará lysá water reservoir, czech republic: a)  paedomorphic male in nuptial colouration; b) paedomorphic female in nuptial colouration; c) close-up view on another paedomorphic male showing the structure of gills; d) aquatic immature specimen with open gill slits (a-d photographed on 7 may 2012); e) paedomorphic male, and f) female later in the season with already reduced gills; g) different pair (adult male and female) demonstrating variation in the level of persistence of larval traits; h) immature paedomorphic specimen with large gills; i) metamorph-like aquatic immature specimen with open gill slits (e-i photographed on 23 august 2012); j) stará lysá water reservoir. 55paedomorphic smooth newts in the czech republic has been historically used for agriculture and so the locality is from three quarters surrounded by crop fields and from one quarter by a relatively dry cultivated pine forest (cf. covaciu-marcov et al., 2011). newts inhabit a water reservoir (originally used for fire protection) sized 25 × 15 m with maximal water depth of 1.5 m made from concrete blocks (fig. 1j). shorter sides of the reservoir are formed by vertical walls while longer sides slope in approximate 45°. the reservoir is supplied from a deep borehole (~60 m), and water is changed approximately once per three years and occasionally refilled (last change in 2010; v. vaněk, pers. comm.). no fish were observed. the reservoir is without vegetation, only filamentous algae grow later in the season over submerged objects and newts use them for wrapping eggs. apart from newts, several adult individuals of marsh frogs (pelophylax ridibundus) were observed and their numerous offspring later in the season. the water is rich on planktonic micro-crustaceans. generally, the reservoir meets conditions for occurrence of paedomorphic newts: does not freeze to the bottom in winter, do not dry in summer, few predators (no fish), abundant food resources (litvinchuk et al., 1996; denoël and poncin, 2001). the locality has been visited in several occasions (april, may, july, august, september, october 2012), and opportunistic visual surveys and dip-netting have been done. paedomorphic individuals were observed during all visits, with a visually assessed abundance in the spring. during the assumed peak of reproductive season (7 may 2012), we observed 70 adult smooth newt individuals when counting specimens visible along the reservoir walls during one walk around the tank. except of two seemingly metamorphic females (but without success to catch them and inspect for morphology) and one almost metamorphic juvenile (but with open gill slits; fig. 1d), all individuals were clearly paedomorphic (fig. 1a-c) and only three of them were males. sex ratio bias toward the females is common in paedomorphic individuals of this species (e.g., kalezić and džukić, 1986; litvinchuk et al., 1996; çiçek and ayaz, 2011), however the observed ratio is unusual. also situations when paedomorphic smooth newts outnumber metamorphic individuals have been only rarely reported (e.g., kalezić and džukić, 1986; rotnikcević et al., 2000) compared to the more common metamorph-biased population ratio in this species (e.g., banks, 1985; kalezić and džukić, 1985; çiçek and ayaz, 2011). however, it is important to mention that we cannot make clear conclusion about the ratio of peadomorphs vs. metamorphs in our smooth-newt population as we did not make a quantitative survey. potential bias toward paedomorphs might be also caused by differences in behaviour of the morphs with respect to the relatively high water depth. during the spring visits, many paedomorphic newts were resting in the water column along the walls to 50 cm below water surface and were easily counted. to observe reproductive behaviour, three paedomorphic individuals (one male, two females) were taken into captivity (7  may 2012). water temperature in the aquarium ranged 16.7-19. 4 °c and newts were kept under natural light conditions. the newts were housed together in an aquarium (50 × 25 × 25 cm) filled by settled tap water and with several waterweed plants (egeria densa). during the following days we had observed the male’s courtship behaviour (fan display), which is in newts similar for both morphs (denoël, 2002), and deposition of eggs by both females. as we expected (cf. banks, 1985; litvinchuk et al., 1996; litvinchuk, 2001), the male metamorphosed after 19 days, followed by the females which metamorphosed after 42 and 45 days in captivity, respectively (including closed gill slits). later in the season (23 august 2012), all captured adults (table 1) were already almost without crests and fin appendices, and with small, sometimes rather rudimental gills, nonetheless, still in aquatic phase and with open gill slits (fig. 1e-g). immature individuals, according to the body size and shape and level of development of the cloaca, were of two types: some had large gills and fins (fig. 1h), while some others resembled the adults, but being substantially smaller, by having rudimentary gills or only open gill slits (fig. 1i). beside these paedomorphic specimens (open gill slits as a common character), some typical larvae were also observed. it seems that adult individuals reduce their larval traits (gills) toward the winter (cf. denoël, 2003). however, it is currently unknown if the same individuals grow the larval traits again in the next season. a study of the demographic structure is currently ongoing using the capture-markrecapture method and shall shed more light onto this and other questions in near future. despite relatively strong evidence for the beneficial role of facultative paedomorphosis as an alternative table 1. morphometric characteristics in cm (svl snout-vent length, lt length of tail, dbl distance between front and hind limbs) of adult peadomorphic smooth newts (females n = 7; males n = 3) from stará lysá, czech republic.     mean min. max. sd females svl 3.45 3.13 3.82 0.27 lt 3.10 2.80 3.47 0.22 dbl 1.69 1.41 1.93 0.18 males svl 3.68 3.34 4.01 0.34 lt 3.50 3.19 3.82 0.32   dbl 1.67 1.57 1.77 0.10 56 václav gvoždík et al. ontogenetic pathway in many tailed amphibians, importance and influence of particular factors lying behind its evolution is not fully resolved. whilst paedomorphosis can be a highly heritable trait (e.g., harris et al., 1990; voss et al., 2003), the occurrence of facultative paedomorphosis in individuals and its maintenance in a population is most probably a result of interplay between genotype and heterogeneity of the environment (reviewed by denoël et al., 2005). among many factors, streambed or pond-bank structures (bennett and chippindale, 2006), slow desiccation rate (semlitsch et al., 1990), and high food availability in water (ryan and semlitsch, 2003) have been observed to induce occurrence of paedomorphs and the maintenance of resulted polyphenism in a population. all the above-listed factors, particularly high food availability and resource partitioning between different phenotypes seem to be feasible as possible factors responsible for the maintenance of paedomorphosis in our smooth-newt population. retention of larval feeding apparatus in paedomorphs may allow more efficient foraging on plankton and aquatic prey (whiteman et al., 1996; denoël, 2004). as a result, both microhabitat segregation and utilization of different food has been observed in particular morphs, with paedomorphs mainly foraging on plankton and distributed in both shallow and deep parts of water column compared to foremost shallower distributed metamorphs (denoël and joly, 2001; denoël and schabetsberger, 2003). as the water reservoir is rich in planktonic micro-crustaceans and areas of deep water column, we suppose the predominance of paedomorphs in our population to be inherent in these factors. it is however worth noting that predominance of paedomorphs in our smooth newt population could also be a result of an environmental-hormonal interplay, as both stress and steroid hormones were observed to significantly affect metamorphosis of larvae and paedomorphs to the adult stage (reviewed by laudet, 2011). detailed endocrinological studies are needed to shed light on these proximate mechanisms. acknowledgements the study was carried out in accordance to the permit sz-092744/2012kusk/3 issued by the regional office of the central bohemian region of the czech republic and with a support by vladimír vaněk (the mayor of stará lysá). we also would like to thank jiří moravec (national museum in prague) and an anonymous reviewer for valuable discussion and comments, respectively. the research was supported by the ministry of culture of the czech republic (dkrvo 2013/13, national museum, 00023272). vj is also grateful to the institutional research support of the charles university in prague and project svv-2012-265-206. references banks, b. 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(2004): triturus vulgaris linnaeus, 1758 – teichmolch. in: handbuch der reptilien und amphibien europas, bd. 4/2b, schwanzlurche (urodela) iib, pp. 847-967. grossenbacher, k., thiesmeier b., eds, aula verlag, wiesbaden. semlitsch, r.d., harris, r.n., wilbur, h.m. (1990): paedomorphosis in ambystoma talpoideum: maintenance of population variation and alternative life-history pathways. evolution 44: 1604-1613. voss, s.r., prudic, k.l., oliver, j.c., shaffer, h.b. (2003): candidate gene analysis of metamorphic timing in ambystomatid salamanders. mol. ecol. 12: 1217–1223. wells, k.d. (2007): the ecology and behavior of amphibians. the university of chicago press, chicago and london. whiteman, h.h., wissinger, s.a., brown, w.s. (1996): growth and foraging consequence of facultative paedomorphosis in the tiger salamander ambystoma tigrinum nebulosum. evol. ecol. 10: 433-446. acta herpetologica vol. 8, n. 1 june 2013 firenze university press the oogenic cycle of the caspian bent-toed gecko, cyrtopodion caspium (squamata: gekkonidae) in iran vida hojati1*, kazem parivar2, eskandar rastegar-pouyani3, abdolhossein shiravi1 altitudinal effects on life history parameters in populations of liolaemus pictus argentinus (sauria: liolaemidae) joel antú gutiérrez1,*, carla piantoni2, nora r. ibargüengoytía1,3 recent cryptic extinction of squamate reptiles on yoronjima island of the ryukyu archipelago, japan, inferred from garbage dump remains yasuyuki nakamura1,*, akio takahashi2, hidetoshi ota3 trophic niche and feeding biology of the italian wall lizard, podarcis siculus campestris (de betta, 1857) along western mediterranean coast marco a.l. zuffi*, chiara giannelli novel, non-invasive method for distinguishing the individuals of the fire salamander (salamandra salamandra) in capture-mark-recapture studies goran šukalo1,*, sonja đorđević2, dragojla golub1, dejan dmitrović1, ljiljana tomović2,3 going out tonight? when insular hierophis viridiflavus breaks the whip snakes rules delaugerre michel-jean first evidence of a paedomorphic population of the smooth newt (lissotriton vulgaris) in the czech republic václav gvoždík1,2, veronika javůrková4, oldřich kopecký5,* no detection of chytrid in first systematic screening of bombina variegata pachypus (anura: bombinatoridae) in liguria, northern italy stefano canessa1,*, an martel2, frank pasmans2 status of the european pond turtle, emys orbicularis (reptilia: testudines: emydidae) in vorarlberg, austria andreas kleewein1, günther wöss2 comparative cytogenetics of two species of ground skinks: scincella assata and s. cherriei (squamata: scincidae: lygosominae) from chiapas, mexico riccardo castiglia1,*, alexandra m.r. bezerra2, oscar flores-villela3, flavia annesi1, antonio muñoz4, ekaterina gornung1 polydactyly in the tyrrhenian wall lizard (podarcis tiliguerta) antigoni kaliontzopoulou1,*, daniele salvi1, verónica gomes1, joão p. m. c. maia1,2,3, panagiotis kaliontzopoulos4 book review: roberto sindaco, alberto venchi, cristina grieco. the reptiles of the western palearctic. 2. annotated checklist and distributional atlas of the snakes of europe, north africa, middle east and central asia, with an update to the vol. 1. edoardo razzetti acta herpetologica journal of the societas herpetologica italica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 8(2): 99-104, 2013 a skeletochronological study of parthenogenetic lizards of genus darevskia from turkey marine arakelyan1,*, ruzanna petrosyan1, çetin ilgaz2, yusuf kumlutaş2, salih hakan durmuş3, yahya tayhan4, felix danielyan1 1 yerevan state university, alek manoogian, 1, yerevan 0025, armenia, *corresponding author. e-mail: arakelyanmarine@yahoo.com 2 dokuz eylül university, faculty of sciences, department of biology, 35160, buca-i̇zmir, turkey 3 dokuz eylül university, faculty of education, department of biology, 35160, buca-i̇zmir, turkey 4 hakkari university, healty vocational college, hakkari, turkey submitted on 2012, 20th february; revised on 2013, 13th may; accepted on 2013, 10th december. abstract. the skeletochronological method has been used to assess age distribution and age-related differences in body size among populations of the parthenogenetic lizards darevskia sapphirina, d. uzzelli, d. armeniaca and d. unisexualis from turkey and armenia. the age distribution between d. armeniaca and d. unisexualis did not significantly differ and ranged from 1 to 8 years. maximum age for both d. uzzelli and d. sapphirina was 6 years, and 8 years for both d. unisexualis and d. armeniaca. in all the studied species, individuals reached sexual maturity after third hibernation. according to patterns of growth marks resorption, d. sapphirina is distinguished from all other rock lizards of genus darevskia by a narrowest periosteal bone as result of high rate of endosteal resorption resulting in complete destruction of hatchling line and line of the first hibernation. keywords. darevskia, parthenogenesis, age, svl, skeletochronology. introduction the genus darevskia includes seven parthenogenetic species of caucasian rock lizards widely distributed throughout the inner part of the armenian plateau on territories of central, northwestern and northern armenia, adjacent region of southern georgia, western azerbaijan and northeastern turkey. among parthenogenetic species, d. armeniaca, d. dahli, d. unisexualis and partly d. rostombekowi have comparatively wide areas of distribution, while d. uzzelli, d. sapphirina and d. bendimahiensis are endemics of eastern and northeastern turkey with limited distribution ranges. the habitats of the cited species are similar and occur primarily in steppe zone, where they prefer rocks and conglomerates of stones at elevation above 1500 m with drastic seasonal climatic variations (darevsky, 1967; darevsky and danielyan, 1977; schmidtler et al., 1994; ananjeva et al., 2006; ilgaz, 2006; arakelyan et al., 2011). previous studies of parthenogenetic darevskia species revealed low genetic variability in comparison with their bisexual parental species, in which genome diversity may arise as a result of mutation or multiple hybridization events (fu et al., 2000, moritz et al., 1992, martirosyan et al, 2002). according to results obtained with mitochondrial and allozyme markers (fu et al., 2000; murphy et al., 2000), parthenogenetic species d. unisexualis, d. uzzelli, d. sapphirina and d. bendimahiensis originated from hybridization events of the same biparental species d. raddei and d. valentini during reticulate evolution. thus, comparative studies about age and growth of parthenogenetic lizards may provide additional information about the origin of parthenogenetic species and possibility to study the influence of enviroment on phenotypic diversity in the clonal populations of lizards. however, the data on growth, longevity and age composition of populations of darevskia parthenogenetic species 100 marine arakelyan et al. are scarce. skeletochronological data for d. armeniaca, d. dahli, d. unisexualis and d. rostombekowi from armenia show that parthenogenesis does not influence growth rate, longevity and maturity and are correlated with body size rather than reproduction mode (arakelyan and danielyan, 2000; arakelyan, 2001; arakelyan, 2002). despite their conservation importance due to restricted ranges (akarsu et al., 2009, kaska et al., 2009), no data are currently available on the life history of endemic parthenogenetic species of genus darevskia from turkey. the purpose of this study is to determine and compare the age and body size of parthenogenetic species of genus darevskia from turkey and analyze differences in body size for each age group among populations of unisexual lizards. materials and methods a total of 255 individuals of partenogenetic species d. armeniaca (mehely, 1909), d. sapphirina (schmidtler et al, 1994), d. unisexualis (darevsky, 1966) and d. uzzelli (darevsky and danielyan, 1977) were collected from different localities from northeastern and eastern part of turkey and compared with populations of d. armeniaca and d. unisexualis from armenia (table 1 and figure 1). we used 70% ethanol preserved specimens that had already been collected and incorporated into the collections of zdeu (zoology department ege university, turkey) and yerevan state university, armenia. lizard snout-vent length (svl) was measured from the tip of snout to anal cleft by using a dial caliper with an accuracy of 0.01 mm. the age of individuals was determined by skeletochronological method (smirina, 1974; castanet and smirina, 1990) which is based on counting of numbers of arrested growth lines (lag) formed in the bones of reptiles as a result of seasonal growth processes. cross-sections of femur bones were used for counting of concentric arrested growth lines. the bones were decalcified in 5% nitric acid solution to one hour and rinsed in tap water for over 12 hours. the cross sections (10 mm thickness) of diaphysis of femur were prepared using a freezing microtome and then were stained with erlich’s haematoxylin for 20 min. the sections were mounted on the slide with a drop of glycerin and a cover slip was placed over the slide. the cross sections were examined under a light microscope and recorded with a digital canon a100 camera. lags were counted and verified independently by two authors. the cross-sections were processed by image analysis. for each individual at least three selected cross-sections from the middle of diaphysis were used for measurement. measurements were made on images of bone sections using the morphometric software tpsdig (rohlf, 2004). the square roots of mean values of longest and shortest axes of diameters of bone sections and diameters of marrow cavity were calculated. the periosteal bone width was expressed as difference between periosteal diameter (bone width) and the marrow diameters of bones. the hatchling line (neonatal line) and the first hibernation resting line suggests that the first table 1. data on the specimens of parthenogenetic rock lizard used for skeletochronological analysis (n – number of samples) species n locality date altitude coordinates d. armeniaca 76 ardahan, turkey artavazd vil., kotayk province, armenia 5 km of gyulagarak vil., lori province, armenia 09.07.2003 20.07.1999 30.06.1999 1750 1870 1400 41°04’n, 42°50’e 40°37’n, 44°33’e 40.59’n, 44°77’e d. uzzelli 52 28 km se of horasan, erzurum, turkey 7 km w of selim, kars, turkey 03.09.2002 29.06.2001 2000 1950 39°08’n, 43°06’e 39°08’n, 43°06’e d. unisexualis 112 28 km se of horasan, erzurum, turkey 7 km w of selim, kars, turkey 5 km of noratuz vil., gegharkunik province, armenia 2 km of tcovinar vil., gegharkunik province, armenia artavazd vil., kotayk province, armenia 03.09.2002 29.06.2001 07.06.2004 08.07.2000 20.07.1999 2000 1950 1930 1920 1870 39°08’n, 43°06’e 39°08’n, 43°06’e 40°23’n, 45°12’e 40°09’n, 45°27’e 40°37’n, 44°33’e d. sapphirina 15 26 km nw of erciş, van, turkey 19.06.2002 1950 39°08’n, 43°06’e fig. 1. sampling localities of parthenogenetic species (square: darevskia sapphirina, triangle: darevskia uzzelli, circle: darevskia unisexualis, star: darevskia armeniaca). 101a skeletochronological study of parthenogenetic lizards of genus darevskia from turkey lags deposited in juveniles are retained into later life time of lizards (smirina, 1974; castanet, 1994). both of these closely spaced lines were completely or partly present in all samples of d. armeniaca, d. unisexualis and d. uzzelli, which allowed a correct assessment of the number of their lags (fig. 2). in order to account for the number of resorbed lags in process of endosteal resorption in some samples of d. sapphirina, “back calculation” was performed by comparing the diameter of the marrow cavity of the adult lizards with that of the first lag and outer edge of a bone of the subadult lizards (smirina, 1974; castanet and smirina, 1990). all data were tested for normality (shapiro-wilk) and for homogeneity of variances (levene test). we used one-way analysis of variance (anova) and scheffé’s post hoc test to test for differences in snout-vent length (svl) or bone width within each age class in species or populations. significance was defined as p < 0.05. statistica software version 7.0 was used for data treatment (statsoft, 2004). results age structure of all studied species is characterized by a maximum age of 6-8 year and predominance of 4-5 year-olds among adult individuals (fig. 3). the maximum age estimated by skeletochronological method for d. armeniaca and d. unisexualis was 8 years, while the old a b c d fig. 2. femur bone cross–sections of four-year old parthenogenetic lizards of four species (a d. armeniaca, b d. unisexualis, c d. uzzelli, and d d. sapphirina). arrows indicate lags, mc marrow cavity, eb endosteal bone. 102 marine arakelyan et al. est lizards of d. uzzelli, d. sapphirina in our samples were 6 years. according to the presence of vitellogenic eggs, individuals reach sexual maturity after third hibernation in all studied species. the average age of adults in our samples was similar among species (mean ± se d. armeniaca: 4.46 ± 0.11 n = 73; d. unisexualis: 4.44 ± 0.11 n = 95; d. sapphirina: 4.44 ± 0.33 n = 10; d. uzzelli: 4.06 ± 0.25 n = 17) and not differed significantly (f3, 195= 1.02, p = 0.39) from one another. significant differences in svl between four parthenogenetic species in each age class (table 2) were found only between 4 years-old d. uzzelli and d. unisexualis (p < 0.05) and between 4 years-old d. uzzelli and d. armeniaca (p < 0.01) according to scheffé’s post-hoc test for one-way anova. the maximum svl of 71 mm was recorded for a 6 years-old d. armeniaca, 70 mm in a 6 years-old d. unisexualis, 67 mm in a 6 years-old d. uzzelli, 59 mm in a 5 years-old d. sapphirina. no differences in body size (svl) for each age class of d. unisexualis parthenogenetic lizards were detected among seven localities while among three populations of d. armeniaca significant differences were found only between populations from stepanavan in armenia and ardahan in turkey (p < 0.05) in 4-years old lizards (fig. 4). d. unisex ualis horasan, turkey noratuz, armenia hankavan, armenia t covinar, armenia 3 4 5 6 age 40 45 50 55 60 65 70 75 80 s v l d. arm eniaca ardahan, turkey stepanavan, armenia hankavan, armenia 3 4 5 6 age 45 50 55 60 65 70 75 sv l fig. 4. differences in body size (svl) between populations of d. unisexualis and d. armeniaca within each age group (yr.). vertical bars show0 .95 confidence intervals.   age n um be r o f l iz ar ds d.armeniaca d.unisexualis d.sapphirina d.uzzelli 3 4 5 6 7 8 0% 3% 5% 8% 10% 13% 15% 18% 21% fig. 3. distribution of adult age groups of parthenogenetic species. table 2. snout-vent length of adults of parthenogenetic species in each age group (n: number of samples, se: standard error of the mean, sd: standard deviation) species age group 3 4 5 6 n mean ± se sd n mean ± se sd n mean ± se sd n mean ± se sd d. armeniaca 9 55.25 ± 1.24 3.45 31 56.60 ± 0.67 3.42 26 61.84 ± 0.73 3.33 5 64.54 ± 1.67 4.13 d. unisexualis 16 53.35 ± 0.93 4.54 38 57.92 ± 0.61 3.85 29 61.27 ± 0.69 2.73 8 63.91 ± 1.32 2.86 d. uzzelli 6 58.66 ± 1.52 4.98 6 59.24 ± 1.52 7.01 3 60.41 ± 2.15 1.78 2 63.73 ± 2.64 3.90 d. sapphirina 2 48.64 ± 2.64 0.22 4 54.62 ± 2.64 2.67 4 57.85 ± 1.87 1.20 1 59.47 103a skeletochronological study of parthenogenetic lizards of genus darevskia from turkey we found differences between patterns of growth marks resorption among studied species. examination of stained cross sections of femur bones of d. unisexualis and d. uzzelli revealed the low endosteal resorption which did not destroy any part of first resting lines in 53% and 34% of cases respectively, whereas in others cases it locally destroyed up to 3/4 of the innermost rings. d. armeniaca had intermediate rate of resorption where the sections with intact first lines comprised 8%, and there were no sections with completely destroyed hatchling and first hibernation lines. d. sapphirina had considerably high resorption with 98% of cases completely destroyed hatching and line of first hibernation (fig. 2, 5). consequently, among compared four species, d. sapphirina had a significantly narrower periosteal bone (f3, 115 = 41.94, p < 0.0001) in comparison with d. unisexualis (scheffé’s post-hoc test p < 0.001), d. uzzelli (p < 0.001), and d. armeniaca (p < 0.001) (fig. 5). discussion although skeletochronology has been applied to four parthenogenetic darevskia species in armenia (arakelyan and danielyan, 2000), no studies have been conducted in d. sapphirina and d. uzzelli in turkey. the range of ages of d. uzzelli and d. sapphirina in our samples did not differ from other species of darevskia, where maximum age was 6 years, similar to d. rostombekowi and d. dahli (arakelyan and danielyan, 2000) and lower than in d. armeniaca and d. unisexualis. however, the oldest lizards of d. armeniaca (8 years) identified by skeletochronological methods in different populations of armenia and turkey did not represent the oldest individuals documented under natural conditions estimated by mark-andrecapture method. the long term observation of d. armeniaca in armenia showed that the maximum longevity is up to 10 year (galoyan, 2011). thus, the actual longevity of parthenogenetic lizards may exceed the maximum recorded by skeletochronological estimated age, especially for larger species, probably due to outer lags distances in older individuals that are very narrow or missing. there were no significant differences in the snoutvent length (svl) of parthenogenetic species among age groups, however d. rostombekowi had the smallest svl in each age group while d. uzzelli, d. unisexualis and d. armeniaca had larger sizes. among studied species d. unisexualis, d. uzzelli and d. sapphirina apparently originated from hybridization of the same parent species: d. raddei and d. valentini (murphy et al., 2000). the comparison of their svl indicated that the species d. unisexualis and d. uzzelli are close to each other, whereas d. sapphirina has a smaller size. it is possible that body size between parthenogenetic species originating from same parents does not change. the comparisons of body size of the same unisexual species will allow studies of influence of the environment on clonal organisms. we did not find significant differences in body size between populations of parthenogenetic species, with the exception of 4 yearsold d. armeniaca. however, we found some populationspecific variation in body size in each age group of d. armeniaca and d. unisexualis from armenia and turkey, where body size of lizards from turkey was larger than that of armenian lizards. the climatic and habitat characteristics of sampling localities in armenia and turkey were mostly similar. observed differences among populations could result from the temperature and the length of active periods at lower and higher elevations. previous studies showed that body length, growth rates, age of maturity, and longevity can vary considerably among populations of the same species, where lizards inhabiting high-elevation sites and northern latitudes live longer than those from low-elevation sites and southern latitudes in the northern hemisphere (arakelyan, 2000; wapstra and swain, 2001; roitberg and smirina, 2006). surprisingly, the periosteal bones of d. sapphirina were notably thinner than those of other species apparently as the result of intensive endosteal resorption. the rate of resorption in darevskia rock lizards is quite slow (arakelyan and danielyan 2000; arakelyan, 2002) and the first growth arrested lines are always locally present with with the exception of d. sapphirina. the apparently high rate of resorption may result from either a specific mutation or new allele combination following a hybridization event among bisexual species which gave rise to the parthenogenetic species.   d. sapphirina d. unisex ualis d. uzzelli d. arm eniaca3 4 5 6 age 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 b on e w id th fig. 5. the periosteal bone width (in µm) of four parthenogenetic species within each age group (yr.). vertical bars show 0.95 confidence intervals. 104 marine arakelyan et al. acknowledgments the authors wish to express their gratitude to dr. marco a.l. zuffi, museo di storia naturale e del territorio, università di pisa, and anonymous reviewers for helpful comments on earlier drafts of the manuscript. we thank project no. 2009. kb.fen.003 scientific research project coordination unit, dokuz eylül university, turkey for their support. references akarsu, f., tuniyev, b., ananjeva, n., agasyan, a., orlov, n., tuniyev, s. 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(2002): the study of age, growth, and longevity in triploid hybrids of rock lizards of genus darevskia in armenia. russian j. herpetol. 9: 63-68. arakelyan, m., danielyan, f., corti, c., sindaco, r., leviton, a. (2011): herpetofauna of armenia and nagorno-karabakh. ssar, salt lake city. castanet, j., smirina, e. (1990): introduction to the skeletochronological method in amphibians and reptiles. ann. sci. nat. zool. 11: 191-196. darevsky, i.s. (1967): rock lizards of the caucasus: systematics, ecology and phylogenesis of the polymorphic groups of caucasian rock lizards of the subgenus archeolacerta. nauka, leningrad (in russian). darevsky, i.s., danielyan, f.d. (1977): lacerta uzzeli sp. nov. (sauria, lacertidae) a new parthenogenetic species of rock lizard from eastern turkey. trudy zool. inst. 76: 55-59 (in russian). fu, j., murphy, r.w., darevsky, i.s. (2000): divergence of the cytochrome b gene in the lacerta raddei complex and its parthenogenetic daughter species: evidence for recent multiple origins. copeia 2: 432-440. galoyan, e.a. (2011). the role of social relations in the formation of space structure of the settlements of bisexual and parthenogenetic species of rock lizards. unpublished phd thesis. moscow state university, moscow (in russian). ilgaz, ç. (2006): on specimens of darevskia armeniaca (sauria: lacertidae: darevskia) collected from ardahan. turk. j. zool. 30: 47-54. kaska, y., kumlutaş, y., avcı, a., üzüm, n., yeniyurt, c., akarsu, f. (2009): d. sapphirina. in: iucn 2011. iucn red list of threatened species. version 2013.2. <www.iucnredlist.org>. downloaded on 22 may 2013. martirosyan, i.a., ryskov, a.p., petrosian, v.g., arakelian, m.s., aslanian, a.v., danielian, f.d., darevskiĭ, i.s., tokarskaia, o.n. (2002): variation of miniand microsatellite dna markers in populations of parthenogenetic rock lizard darevskia rostombekovi. rus. j. gen. 38: 691-698. moritz, c., uzzel, t., spolsky, c., hotz, h., darevsky, i., kupriyanova, l., danielyan, f. (1992): the maternal ancestry and approximate age of parthenogenetic species of caucasian rock lizards (lacerta: lacertidae). genetica 87: 53-62. murphy, r.w, fu, j., macculloch, r.d., darevsky, i.s., kupriyanova, l.a. (2000): a five line between sex and unisexuality: the phylogenetic constraints on parthenogenesis in lacertid lizards. zool. j. linn. soc. 130: 527-549. rohlf, f.j., 2004. tpsdig. version 1.40. suny, stony brook. roitberg, e.s., smirina, e.m. (2006): adult body length and sexual size dimorphism in lacerta agilis boemica (reptilia, lacertidae): between-year and interlocality variation. in: mainland and insular lizards: a mediterranean perspective, pp. 175-187. corti, c., lo cascio, p., biaggini, m., florence university press, florence: 175-187. schmidtler, j.f., eiselt, j., darevsky, i.s. (1994): untersuchungen an feldeidechsen (lacerta-saxicola-gruppe) in der östlichen türkei: 3. zwei neue parthogenetische arten. salamandra 30: 55-70. smirina, e.m. (1974): prospects of age determination by bone layers in reptilia. zool. zhur. 53: 111-117 (in russian). wapstra, e., swain, r. (2001): geographic and annual variation in life history traits in a temperate zone australian skink. j. herpetol. 35: 194-203. acta herpetologica vol. 8, n. 1 june 2013 firenze university press journal of the societas herpetologica italica acta herpetologica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 9(1): 25-31, 2014 doi: 10.13128/acta_herpetol-13117 high levels of prevalence related to age and body condition: host-parasite interactions in a water frog pelophylax kl. hispanicus mar comas1, alexis ribas2,*, concetta milazzo3, emilio sperone3, sandro tripepi3 1 departamento de zoología, facultad de ciencias, universidad de granada, granada, 18071, spain 2 biodiversity research group, faculty of science, udon thani rajabhat university, udon thani, 41000, thailand. *corresponding author. e-mail: alexisribas@hotmail.com 3 dibest, department of biology, ecology and earth science, university of calabria, via p. bucci, i-87036, rende (cs), italy submitted on 2013, 29th july; revised on 2014, 17th february; accepted on 2014, 20th february abstract. host traits can significantly influence patterns of infection and disease. here, we studied the helminths parasitizing the italian edible frog pelophylax kl. hispanicus, giving special attention to the relationship between parasites and host traits such as sex, snout vent length, weight and body condition. the helminth community was composed of seven species: three trematode species (diplodiscus subclavatus, gorgodera cygnoides, pleurogenes claviger), three nematode species (icosiella neglecta, oswaldocruzia filiformis, rhabdias sp.) and one acanthocephalan species (pomphorhychus laevis). we found that prevalence was positively correlated with snout-vent length and weight, but did not differ with body condition or sex. we found that prevalence and mean species richness increased with age. our results show that abundance of icosiella neglecta was positively correlated with higher values for host body condition. in fact, we found that high prevalence and mean species richness do not necessarily imply poorer body condition in the parasitized host. in conclusion, our results show that the helminth community in this taxon has great diversity, and this host-parasite system seems to be evolved to low levels of virulence, helminths maintaining a commensal relationship with this frog. keywords. acantocephala, body condition, italian edible frog, nematoda, trematoda. introduction wild amphibian populations are declining worldwide (whiles et al., 2006; wake, 2007) and factors such as global change and pollution may magnify the effect of pathogens and disease in this animal class (harvell et al., 2002; taylor et al., 2005; paull and johnson, 2011; macnab and barber, 2011; tinsley et al., 2011). for example, higher water temperatures lead to a rise in the incidence of parasitic diseases due to increased pathogen development transmission and host susceptibility (karvonen et al., 2010). parasites can have a significant influence on the population dynamics of the host species (longshaw et al., 2010) and understanding how host-parasite interactions will change in relation to host traits is one of the cornerstones of parasitology (mcalpine, 1997; dobson, 2009; lafferty, 2009). the relationship between prevalence and factors such as age or sex is not clear. in some studies an age-related increase in prevalence in amphibians has been reported (mcalpine, 1997; campiao et al., 2009). nevertheless, some studies describe a decrease in prevalence (ibrahim, 2008; raffel et al., 2009; tinsley et al., 2012), while others report no age-related patterns (garvin et al., 2003; hasselquist et al., 2007). age-related changes in prevalence may be due to several causes. an increase in prevalence with age may be caused by prolonged exposure to parasite accumulation (sanchis et al., 2000), while a decrease in prevalence with age may be linked to differential survival rates between individuals with and without parasites 26 m. comas et alii (parasitized individuals may be more likely to die and so over time there will be more non-infected individuals). a decrease in prevalence with age may also be influenced by changes in the immune response capacity of infected individuals. levels of disease risk in hosts of different ages will vary due to differences in their susceptibility to infection and in their age-acquired immunity (raffel et al., 2009; tinsley et al., 2012). nonetheless, prevalence may also decrease with time post-exposure due to parasite mortality, especially in the case of parasites that do not reproduce within a host (telfer et al., 2008; holland, 2009). relationships between sex and helminth prevalence show no clear patterns in amphibians. some authors (gonzález and hamann, 2012) found significant differences in the intensity of infection between sexes, with females having higher mean intensities than males. other authors found that levels of parasitism by sex depended on the studied species. for example, in wood frogs rana sylvatica (leconte, 1825) the parasite load in males was higher than in females (dare and forbes 2008), while in lithobates frogs the parasite load was higher in females (dare and forbes 2009a). in the northern leopard frogs rana pipiens (schreber, 1782), prevalence and mean abundance were higher in breeding female frogs than in breeding males, even though no sex differences were observed in non-breeding adults (dare and forbes 2009b). host traits can significantly influence patterns of infection and disease (mcalpine, 1997; dare and forbes 2008; raffel et al., 2009; tinsley et al., 2012). in the present study, we analysed relationships between hosts and their parasites in the italian edible frog pelophylax kl. hispanicus and give special attention to host-parasite relationships and host traits such as host age, sex, snout-vent length, weight and body condition. we also examined the possible seasonality of the parasites. the italian edible frog is a hybridogenic species. these frogs are kleptospecies derived from p. bergeri and p. ridibundus or from p. kl. esculentus, which is itself of hybrid origin (holsbeek and jooris, 2009; vorburger and reyer, 2003). it is endemic to italy, occurring on the italian mainland south from genoa to rimini and sicily. it is associated with a wide range of aquatic environments such as rivers, swamps and freshwater lakes and marshes, with either intermittent or permanent flow regimes. it is usually found in mixed populations with pelophylax bergeri (andreone et al., 2009) and normally occurs in sympatry with other amphibian species (talarico et al., 2004; sperone et al., 2009). as far as we know, there are no studies of the diet of the italian edible frog, although the parental specie p. ridibundus (pallas, 1771) has been reported to consume coleoptera, diptera, hymenoptera and, less frequently, vertebrates such as amphibians (mainly tadpoles), reptiles and even mammals (mollow et al., 2010). it has been shown that in other close related species of water frogs such as p. kl. esculentus and p. lessonae (camerano, 1882) no significant differences in trophic niche exist between the hybrid and the parental species, which share the same habitats (sas et al., 2007). herein, we addressed the following questions: 1) what is the helminth community of the italian edible frogs? 2) what are prevalence, diversity, abundance and intensity of infection of helminths found in this host? 3) is there a relationship between these parasitological parameters and host traits? 4) does the prevalence of parasites vary seasonally? material and methods study area and sampling our sampling site is located in the lower basin of a stream (beltrame) in calabria (southern italy; 38°44’ n, 16°32’ e) at 10 m a.s.l.. this stream’s flow regime is characterized by sudden floods, alternating with long periods of drought, particularly in summer (sabato and tropeano, 2004). the climate is mediterranean, with annual precipitation values of around 800–1000 mm (talarico et al., 2004). average temperatures range from 31 ºc in the warmest to 13 ºc in the coldest months. the vegetation is characterised by evergreen sclerophyllous formations (among which, quercus ilex and q. suber) and mediterranean scrub and shrubs with species such as tamarix sp. and nerium oleander. this study was carried out in autumn 2008 (81 individuals), in spring (17 individuals) and summer (19 individuals) 2009. in all, 117 frogs (54 males, 60 females and 3 unsexed) were netted along a 1-km transect along the lower course of the stream. snout vent-length (svl, nearest 0.1 mm) and body weight (nearest 0.1 g) were recorded for each individual. we measured the snout-vent length (svl) from the tip of the snout to the rear border of the vent. weight was measured using a precision balance. body condition was calculated from estimated residuals of the relationship body weight-body length (log-transformed; green, 2001). hosts were grouped into three age classes on the basis of their svl (juveniles: 35–60 mm; subadults: 61–70 mm; adults 71–110 mm) following lanza (1983). after collecting the biometric data, specimens were anaesthetized and killed with tricaine methane sulphonate, ms 222 (sigma-aldrich chemical co., st. louis, mo, usa). all procedures were carried out following the recommendations of the ethical committee of the university 27host-parasite interactions in a water frog of calabria and under the supervision of authorized investigators. then, gastrointestinal tract (oesophagus, stomach and small and large intestines), as well as lungs, urinary bladder, liver, heart and kidneys, were dissected and placed separately in petri dishes containing 0.9% saline solution. the muscle (dewlap tissue) was also examined. organs, muscle and gastrointestinal tract were then examined separately for helminths under a stereomicroscope. recovered helminths were placed in vials in 70% ethanol for subsequent examination. nematodes and acantocephalans were examined on a temporary mounting in amann lactophenol. trematodes were stained in acetic carmine, with hcl at 5% to remove excess dye, dehydrated in series alcohol, cleared in xylene and mounted in canada balsam for identification. helminth were identified according to the literature (e.g., anderson and bain 1982; brown 1987; skryabin 1964; slimane et al., 1993;  starzyska 1958). a subset of helminths recovered was deposited in the museu de zoologia de barcelona (mzb), catalonia, spain, with accession numbers mzb 2013–3683mzb 2013–3689. data analysis we calculated prevalence (the percentage of hosts that are infected), mean abundance (total number of individuals of a particular parasite species found in the sample, divided by the total number of hosts examined) and mean intensity (average abundance of a particular parasite considering only the infected members of the host species) of helminth species following bush et al. (1997). mean species richness of helminths was calculated as the sum of helminth species per individual divided by the total sample size of host individuals (bolek and coggins, 2001). the brillouin index (hb) of diversity was calculated following the equation: hb = (ln n! –∑ ln ni!) / n, where n is the total number of individuals and ni is the number of individuals in the ith species (magurran, 2004). numeric dominance was determined using the berger-parker dominance index, following the equation d = nmax/n, where nmax is the number of individuals of the most abundant species and n is the total number of individuals in the sample. to test the effect of sex, age and season on parasite prevalence a maximum likelihood chi-square test was used. the effect of host sex on parasite abundance, taking into account body size, was tested using an ancova. the effect of host sex on intensity was tested using a mann-whitney u test. differences in snout-vent length (svl) between males and females were tested with a t-student test. the difference in mean species richness among age classes was analysed using the kruskal-wallis test. we performed logistic regression analyses to test the relationships between snout-vent length (svl), weight and body condition of the frogs with the presence/ absence of parasites (i.e., prevalence). a one-way anova was performed to test whether helminth mean species richness (introduced as categorical factor with values between 0 and 4 species) was related to (a) body size, (b) weight and (c) body condition. in order to test how prevalence for each species of parasite affects body condition, an anova was performed. a multiple regression analysis was performed for each species of parasite in order to test how body condition changes with abundance. the assumptions of normality and homoscedasticity were checked with the shapiro-wilk’s test and levene’s tests, respectively. all statistical analyses were performed using statistica 10.0. results the helminth community consisted of seven species: three trematodes, three nematodes and one acanthocephalan (table 1). icosiella neglecta had the highest prevalence (61.54%) and was the dominant species (d = 0.51), while pleurogenes claviger was the rarest (0.85%). three of the seven species were rare, with low prevalence (< 6%) and low mean abundances (< 0.16 parasites/host). only 18 frogs were uninfected, while the remaining harboured 1–4 helminth species and 1–19 parasites. we found sexual dimorphism (t114 = 4.56, p < 0.0001), females (75.13 ± 14.8 mm; mean ± sd) being larger than males (62.3 ± 13.6 mm). our results showed a trend for higher prevalence in females than in males (77.7 % in males and 90 % in females; χ2 = 3.23, df = 1, p = 0.07). we also found that prevalence significantly increased depending on snout-vent length (χ2 = 8.476, df = 1, p = 0.0036) and weight (χ2 = 10.176, df = 1, p = 0.001), but not on body condition (χ2 = 1.457, df = 1, p = 0.227). our results show that prevalence increased with age (table 2). we did not find significant differences in the seasonal prevalence (χ2 = 0.74, df = 2, p = 0.69). prevalence in autumn 2008 was 82.72%, in spring 2009 was 88.24% and in summer 2009 was 89.47%. mean species richness did not differ between sexes (being 1.27 ± 0.9 in males and 1.55 ± 0.8 species/host in females; u = 1327, p = 0.097) and likewise, did not depend on either body size, weight or body condition (body size, f4,117 = 0.8, p = 0.53; weight, f4,117 = 1.6, p = 0.39; body condition, f4,117 = 0.74, p = 0.57). nevertheless, mean species richness increased with age (table 2). the brillouin index of diversity was 1.29. for both d. subclavatus and i. neglecta, abundance significantly covaried with host body size but not with host 28 m. comas et alii sex (table 3) and there are not sex link patterns in intensity for any species of parasite (table 4). body condition did not covaried with prevalence in any helminth species (table 5), although body condition was positively correlated with abundance of icosiella neglecta (t108 = 2.28, p = 0.024, β = 0.22) and was almost significant for pomphorhychus laevis (t108 = 1.96, p = 0.053, β = 0.18) (table 6). discussion our results show a significant increase in helminth prevalence related to snout vent length and weight in the italian edible frog. consequently, given that body size increases with age, older individuals are more parasitized. these imply that prolonged exposure to parasite accumulation is the most important factor explaining the high level of prevalence found in the studied population. as the time of exposure to parasites and vectors increase, parasite prevalence does too. this result is consistent with other studies that have found that prevalence increased with age (mcalpine, 1997; sanchis et al., 2000; abumadi et al., 2001; treml et al., 2012; haas et al., 2012). in other species of water frogs such as the levant water frog p. bedriagae, females live longer than males (çiçek et al., 2011) and so, because we found higher levels of prevalence with age, we expected females to harbour higher parasite loads than males. nevertheless, even though females were larger and older than males, our results showed no significant sex-linked patterns of prevalence (despite the fact that females tended to have higher parasite loads). moreover, we found no significant relationship between host body condition and prevalence or between host body condition and mean species richness, which may suggest that this population has a certain tolerance to these parasites. in fact, we found a positive correlation between host body condition and the abundance of two species of parasites, which was significant in icosiella neglecta and almost significant in pomphorhychus laevis. hosts with the greatest abundances of these two parasites species were in better body condition. when the table 1. number of parasitized hosts and prevalence (%), mean intensity (mi ± sd), mean abundance (ma ± sd), number of helminth recovered and occupied microhabitat in the host pelophylax kl. hispanicus from a stream (beltrame) in calabria, italy (n = 117). helminth species number of individuals parasitized (prevalence, %) mi ± sd (range) ma ± sd number recovered microhabitat trematoda diplodiscus subclavatus (pallas, 1760) 52 (44.4) 3.08 ± 2.85 1.37 ± 2.85 160 rectum (1-16) gorgodera cygnoides (zeder, 1800) 5 (4.27) 3.80 ± 3.35 0.16 ± 3.35 19 urinary bladder (1-9) pleurogenes claviger (rudolphi, 1819) 1 (0.85) 7.0 0.06 7 small intestine (7-7) nematoda rhabdias sp. 16 (13.68) 2.75 ± 2.74 0.38 ± 2.74 44 lungs (1-11) icosiella neglecta (diesing, 1851) 72 (61.54) 4.25 ± 3.19 2.61 ± 3.19 306 subcutaneous and intermuscular tissue (1-14) oswaldocruzia filiformis (goeze, 1782) 13 (11.11) 4.15 ± 4.18 0.46 ± 4.18 54 small intestine (1-13) acanthocephala pomphorhychus laevis (müller, 1776) 6 (5.13) 1.17 ± 0.41 0.06 ± 0.41 7 external wall intestine/stomach (1-2) total 99 (84.62) 6.03 ± 5.08 5.10 ± 5.08 597 29host-parasite interactions in a water frog host has evolved tolerance instead of resistance as a way of mitigating the harm caused by a parasite, a relationship may change from parasitic to commensal (miller et al. 2006; leung & poulin, 2008). however, when the abundance of a parasite becomes too great for the host, host survival may fall. consequently, we probably found high parasite loads in hosts that were able to tolerate this level of parasitism, that is, in those hosts that show higher body condition. the prevalence (84.6%) and mean species richness (1.4) found in the present study are high, especially in comparison with frogs from temperate regions, where prevalence values that range 7.2 – 92% (galeano et al., 1990; yoder and coggins, 2007; comas and ribas, 2014) and mean species richness of 0.8 (aho, 1990; muzzall et al., 2001) or even mean species richness as low as 0.072 (comas and ribas, 2014). therefore, our findings show that the italian edible frog in our study population harbours a community with high prevalence and mean helminth species richness. in our study area, the italian edible frog shares habitat with other amphibian species and also with a rich invertebrate community. a diverse community increases the likelihood of having more intermediate hosts and vectors available (aho, 1990) and so living in a speciesrich community may imply greater parasite loads. moreover, during dry summer periods, host density dramatically increases and leads to greater parasitism (arneberg et al., 1998; krasnov et al., 2002; harvell et al., 2002; marina et al., 2005; lindsey et al., 2009). however, we did not find any differences in prevalence among seasons, as also it occurs in other parasite studies where no seasonal patterns of occurrence were found (sanchis et al., 2000). in conclusion, we found that the italian edible frog harbours seven different helminth species and infestation by these species increases with snout vent length, weight table 2. prevalence (%), mean species richness and snout-vent length (svl in mm) for each age class (n = sample size). age group n prevalence (%) mean species richness ±sd (range) svl ± sd (range) juveniles 32 71.87% 1.06 ±0.94 (0-4) 52.06±6.86 (35-60) subadults 38 84.21% 1.29±0.8 (0-3) 65.84±2.69 (61-70) adults 47 93.61% 1.74±0.82 (0-3) 83.17 ± 10.21 (71-110) table 3. abundance according to sex for each species of parasite (ancova controlling for snout vent length (svl)). helminth species svl f(1, 111) svl p sex f(1, 111) sex p diplodiscus subclavatus 10.44 0.001 0.064 0.799 gorgodera cygnoides 1.887 0.172 0.000 0.976 pleurogenes claviger 0.098 0.754 1.184 0.278 rhabdias sp. 1.331 0.250 0.312 0.577 icosiella neglecta 35.76 0.000 1.200 0.275 oswaldocruzia filiformis 0.456 0.500 0.102 0.749 pomphorhychus laevis 0.150 0.698 0.334 0.564 table 4. intensity according to sex for each species of parasite (results of mann-whitney u test). helminth species u z p diplodiscus subclavatus 251.500 0.570 0.568 gorgodera cygnoides 2.500 0.288 0.772 pleurogenes claviger 0.000 0.000 1.000 rhabdias sp. 28.500 -0.317 0.750 icosiella neglecta 465.500 1.738 0.082 oswaldocruzia filiformis 15.000 0.731 0.464 pomphorhychus laevis 3.000 -0.654 0.512 table 5. results of the anova performed for each species of parasite in order to test how body condition changes with prevalence. helminth species f(1, 108) p diplodiscus subclavatus 0.363 0.547 gorgodera cygnoides 0.165 0.685 pleurogenes claviger 1.386 0.241 rhabdias sp. 0.688 0.408 icosiella neglecta 3.113 0.080 oswaldocruzia filiformis 0.004 0.944 pomphorhychus laevis 2.730 0.101 table 6. results of the multiple regression analysis performed for each species of parasite to test how body condition changes with abundance. helminth species beta t108 p diplodiscus subclavatus 0.085 0.895 0.372 gorgodera cygnoides -0.034 -0.365 0.715 pleurogenes claviger -0.103 -1.144 0.255 rhabdias sp. 0.149 1.576 0.117 icosiella neglecta 0.220 2.279 0.024 oswaldocruzia filiformis 0.023 0.236 0.813 pomphorhychus laevis 0.178 1.955 0.053 30 m. comas et alii and consequently, age. furthermore, we found that higher levels of infestation and parasite diversity do not seem to have any severe impact on host body condition. acknowledgements special thanks to gregorio moreno-rueda and the two anonymous referees who made helpful improvements in this manuscript. this research was carried out with the approval of the “ministero dell’ambiente e della tutela del territorio (direzione per la protezione della natura)”, permit number 2004/30911. references abu-madi, m.a., lewis, j.w., mikhail, m., el-nagger, m.e., behnke, j.m. 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(2007): helminth communities in five species of sympatric amphibians from three adjacent ephemeral ponds in southeastern wisconsin. j. parasitol. 93: 755–760. acta herpetologica vol. 8, n. 2 december 2013 firenze university press journal of the societas herpetologica italica acta herpetologica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah editorial the “peer” in “peer review” gad perry1, jaime bertoluci2, bruce bury3, robert w. hansen4, robert jehle5, john measey6, brad r. moon7, erin muths8, marco a.l. zuffi9,* 1 texas tech university, lubbock, tx, usa; journal of herpetology 2 universidade de são paulo, brazil; phyllomedusa 3 usgs, corvallis, or, usa; herpetological conservation and biology 4 clovis, ca, usa; herpetological review 5 university of salford, greater manchester, uk; herpetological journal 6 university of the western cape, south africa; african journal of herpetology 7 university of louisiana at lafayette, la, usa; herpetologica 8 usgs, fort collins, co, usa; journal of herpetology 9 museum natural history, university of pisa, italy; acta herpetologica peer review is the best available mechanism for assessing and improving the quality of scientific work. as herpetology broadens its disciplinary and geographic boundaries, highquality external review is ever more essential. we are writing this editorial jointly because the review process has become increasingly difficult. the resulting delays slow publication times, negatively affect performance reviews, tenure, promotions, and grant proposal success. it harms authors, agencies, and institutions (ware, 2011) in our review process, editors assign each new submission to a knowledgeable associate editor, who seeks sufficient expert reviewers to evaluate the manuscript. in recent years, editors have commented on the increasing difficulty of finding willing reviewers, and have speculated on its causes, often citing selfishness (ghazoul, 2011; hochberg, 2010; mcpeek et al., 2009; navarro et al., 2010; sheppard, 2000; thompson, 2010). there are certainly people who regularly decline requests to review, but our experience agrees with ware (2011) that they are the exception. why, then, is the problem getting worse? most reviewers reside at academic institutions and government agencies facing budget cuts, unfunded “accountability” measures, and increasing privatization and commercialization (perry et al., 2007). writing an informed and constructive review takes considerable work. as professional responsibilities increase each year, the time and recognition for such unpaid work diminish. moreover, potential reviewers are sometimes instructed to minimize their service activities (garrison, 2005). yet competent peer review is as vital a part of the scientific process as any experiment. * with the exception of the first author, authors are arranged in alphabetical order g. perry et al. our goal is to provide a publication process that is objective, efficient, timely, and pleasant. we are exploring various options for addressing the problem, and we need your help. please help us by taking the following steps: if you are asked to review a manuscript, please respond quickly. this will shorten the process. delays in review often begin with a tardy response to the request for a review. please do your best to say “yes.” if you do, please make sure to meet the deadline or explicitly request an extension to a specific date. we are increasingly facing delays because of chasing colleagues who missed the deadline, sometimes by many weeks. if you are not able to do the review, do not have the time to provide an in-depth review, or do not think you can meet the deadline, then please say “no” right away and suggest one or more alternate potential reviewers. involve your advanced graduate students in the review process while maintaining the confidentiality of the process. explain that this is an important part of their professional duties. advocate to administrators the value of reviewer service at every opportunity, explain that it is a performance-related part of your job that helps keep you up-to-date, and ask for it to be part of your annual evaluation. these are simple steps, but they will greatly help reduce delay and frustration. many thanks in advance, your editors references garrison, c. (2005): exploring new faculty orientation: the good, the bad, and making it better. essays in education 13: 1-9. ghazoul, j. (2011): reviewing peer review. biotropica 43: 1-2. hochberg, m.e. (2010): youth and the tragedy of the reviewer commons. ideas ecol. evol. 3: 8-10. mcpeek, m.a., deangelis, d.l., shaw, r.g., moore, a.j., rausher, strong, d.r., ellison, a.m., barrett, l., rieseberg, l., breed, m.d., sullivan, j., osenberg, c.w., holyoak, m., elgar, m.a. (2009): the golden rule of reviewing. am. natur. 173: e155-e158. navarro, l., sánchez, j.m., cortina, j., jiménez-eguizábal, l. (2010): en manos de la buena voluntad de los colegas… en nuestras manos. ecosistemas 19: 1-7. perry, g., mackessey, s., sites, j., ford, n. (2007): the ongoing privatization of public universities. iguana 14: 257-258. sheppard, c.( 2000): the poor referee. marine poll. bull. 40: 1-2. thompson, f.r., iii. (2010): the golden rule of reviewing. j. wildlife managem. 74: 191192. ware, m. (2011): peer review: recent experience and future directions. new rev. inform. network. 16: 23-53. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 7(1): 139-153, 2012 genetic variability of mesalina watsonana (reptilia: lacertidae) on the iranian plateau and its phylogenetic and biogeographic affinities as inferred from mtdna sequences jiří šmíd1,2,*, daniel frynta1 1 department of zoology, faculty of science, charles university in prague, viničná 7, 128 44, prague 2, czech republic. *corresponding author. e-mail: jirismd@gmail.com 2 department of zoology, national museum, cirkusová 1740, 193 00 prague, czech republic. submitted on: 2011, 15th november; revised on: 2012, 13th march; accepted on: 2012, 23rd april abstract. the lacertid lizard mesalina watsonana is widely distributed on the iranian plateau where it is one of the most common lizards. however, the intraspecific variability and the phylogenetic position of this species within the genus still remain unknown. we sequenced a 715bp long fragment of the mtdna cytochrome b gene from lizards sampled in 10 localities covering the iranian distribution range of the species. we identified four distinct and geographically isolated clades with an average genetic divergence between them ranging from 9.8 to 13.1% (p-distance) which is comparable to the values of genetic distance commonly reported between lacertidae species. analyses combining data from recently published phylogeny of the genus mesalina with our dataset confirmed the monophyly of m. watsonana. the isolation of this species from the rest of the genus points out the important role of the zagros mountains uplift during the miocene. it is possible that this geological event participated on the isolation of the ancestor of m. watsonana from the rest of the mesalina lizards and together with the upheaval of the whole iranian plateau provided suitable environmental conditions for rapid diversification of this species. keywords. mesalina watsonana, lacertidae, iran, zagros, mtdna, phylogeny. introduction the upland area of the iranian plateau represents a unique biogeographical element in the middle east, isolated from the neighboring territories (anderson, 1968, 1999; fisher, 1968; coad, 1998). it encompasses most of the territory of iran, reaching afghanistan and pakistan in the east. the geographical delimitation of the iranian plateau is determined by high mountain ranges of the zagros in the west, elborz and kopet dagh in the north, lofty peaks of hindu kush in afghanistan in the east and makran and sulaiman moun140 j. šmíd and d. frynta tain ranges in the south and east of pakistan (fisher, 1968; khan, 1980). this area has been extensively studied from the geological point of view due to persisting geological activity; thus, the orographical history and dating of major events are well-known (tchalenko and braud 1974; dersourt et al., 1986; mouthereau, 2011). the uplift of the zagros mountains was initiated by the collision of the arabian lithospheric plate moving in the north-east direction and the eurasian landmass, which took place from the oligocene to the miocene 35 – 20 million years ago (mya) (mouthereau, 2011). this event has led to a formation of unique environmental and climatic conditions on the uplifted plateau and thus separated iranian highland from mesopotamian lowland populations and resulted into (sub)specific differentiation of many species of amphibians and reptiles (wischuf and fritz, 1996; feldman and parham, 2004 mauremys; hrbek et al., 2006 – aphanius; rastegar-pouyani et al., 2006 asaccus). for a long time, the lizards of the genus mesalina gray, 1838 were considered a part of the genus eremias fitzinger, 1834, until szczerbak (1974) resurrected gray´s generic name and separated these small lizards from the more robustly built genus eremias distributed mostly in central asia. mesalina lizards inhabit dry regions of africa along the northern coast from senegal to somalia; some species penetrate through the sahara desert into the sahel. their distribution spans eastwards throughout the arabian peninsula and iranian plateau to northwestern india. the genus mesalina currently encompasses 14 described species. whereas the majority of the species occur west of the zagros mountains, there are only two species inhabiting the eastern part of the genus range, with m. watsonana being the only one living right on the iranian plateau (anderson, 1999; sindaco and jeremčenko, 2008). the first attempt to resolve phylogenetic relationships inside the genus mesalina using molecular data was done by joger and mayer (2002). their results brought justification for an elevation of the socotran m. kuri to a species level, but did not provide any further phylogenetic information concerning the relationships among other studied taxa. recently, kapli et al. (2008) used partial cyt b and 16s rrna sequences (mtdna) to infer phylogeny and phylogeography of the north african members of the genus mesalina. their study revealed high genetic differences between eastern and western populations of the widespread m. guttulata and across the distribution of m. brevirostris. with the application of molecular clock calibration, they showed these intraspecific evolutionary splits took place between 7.1 to 9 mya and may have been correlated with climate change in north africa and southwest asia during the miocene (kapli et al., 2008). however, neither joger and mayer (2002) nor kapli et al. (2008) included m. watsonana in their analyses, so the phylogenetic position of this species still remains unclear. according to its morphology, it is considered a part of a monophyletic group with m. guttulata (arnold, 1986). in spite of the fact that m. watsonana is considered abundand and extremely common (smith, 1935), we are not aware of any publication aimed directly on this species. in the present study, we focused on the genetic differentiation among the populations of m. watsonana from the iranian plateau and the phylogenetic position of this species within the genus using mitochondrial cyt b gene sequences. this marker is traditionally used for assessing both intraspecific variability within lacertid lizard species and relationships among different genera (poulakakis et al., 2003, 2005; carranza et al., 2004; podnar et al., 2004, 2005; pavlicev and mayer, 2009). an assessment of the iranian populations may 141genetic diversity of mesalina watsonana in iran help us not only uncover biogeographic history of the genus mesalina, but also provide a reliable model revealing evolutionary history of this region. materials and methods sampling a total of 16 individuals of m. watsonana from ten distinct geographical locations were used. the sampling sites were chosen to cover all the iranian range of the species´ distribution (fig. 1). sequences of other lacertids, m. guttulata and ophisops elegans, were used as outgroup species in the phylogenetic analyses. all tissue samples used in this study came from the material deposited in the collections of the faculty of science, charles university in prague and from the national museum in prague. to infer overall phylogenetic position of m. watsonana within the genus, sequences of other species already published elsewhere (whiting et al., 2003; kapli et al., 2008; rastegar-pouyani et al., 2010) were used. the complete list of the new material as well as genbank accession numbers of elsewhere published sequences used in this study are given in table 1. dna extraction, pcr and sequencing total genomic dna was extracted from tissue pieces (tail tips, thigh muscles) using qiagen dnaeasy® tissue kit (qiagen) according to the manufacturer’s protocol. we amplified a fragment of fig. 1. map of distribution of m. watsonana and the sample sites used for this study. numbers correspond to the locality numbers in tab. 1. dashed lines delimit individual clades recovered from the genetic analyses. 142 j. šmíd and d. frynta table 1. list of samples of m. watsonana including geographical origin and genbank accesion numbers, including sequences of mesalina species taken from genbank used for the taxon-wide phylogeny. species sample locality; number in fig 1 gps genbank acc no. source m. watsonana gah 09 gahkom, iran; 10 28°10’54.01”n, 55°49’20.13”e jn828632 this study m. watsonana ard 14 ardestan, iran; 3 33°18’19.64”n, 52°24’2.39”e jn828633 this study m. watsonana bard 05 bardeskan, iran; 7 35°14’57.92”n, 57°58’47.05”e jn828634 this study m. watsonana izad 03 izadkhast, iran; 4 31°31’58.45”n, 52°7’32.09”e jn828635 this study m. watsonana izad 04 izadkhast, iran; 4 31°31’58.45”n, 52°7’32.09”e jn828636 this study m. watsonana izad 08 izadkhast, iran; 4 31°31’58.45”n, 52°7’32.09”e jn828637 this study m. watsonana may 06 mayamey, iran; 6 36°24’10.17”n, 55°41’13.30”e jn828638 this study m. watsonana salaf 07 salafchegan, iran; 2 34°29’20.17”n, 50°28’12.90”e jn828639 this study m. watsonana tab 11 tabas, iran; 8 33°35’57.37”n, 56°54’44.12”e jn828640 this study m. watsonana vaz 01 vazireh, iran; 9 28°59’50.53”n, 54°46’52.49”e jn828641 this study m. watsonana vaz 10 vazireh, iran; 9 28°59’50.53”n, 54°46’52.49”e jn828642 this study m. watsonana kush 001 kushk-e nosrat, iran; 1 35°6’39.11”n, 50°53’51.71”e jn828643 this study m. watsonana kush 02 kushk-e nosrat, iran; 1 35°6’39.11”n, 50°53’51.71”e jn828644 this study m. watsonana kush 12 kushk-e nosrat, iran; 1 35°6’39.11”n, 50°53’51.71”e jn828645 this study m. watsonana kush 13 kushk-e nosrat, iran; 1 35°6’39.11”n, 50°53’51.71”e jn828646 this study m. watsonana anj anjireh, yazd, iran; 5 32°13’60.00”n, 54°22’60.00”e jn828647 this study m. guttulata jem 109 ghayl ba wazir, yemen 14°54´36”n, 49°02´14”e jn828648 this study m. guttulata jordan 31°15’11.16”n, 35°36’48.6”e ef555250 kapli et al., 2008 m. guttulata libya 30°27’57.24”n, 24°32’11.76”e ef555254 kapli et al., 2008 m. guttulata morocco 32°02’49.92”n, 4°24’31.68”w ef555255 kapli et al., 2008 m. guttulata morocco 31°24’06.48”n, 5°43’39.36”w ef555256 kapli et al., 2008 m. guttulata tunisia 33°31’21”n, 9°59’33”e ef555268 kapli et al., 2008 m. guttulata tunisia 33°09’00.72”n, 10°17’23.64”e ef555270 kapli et al., 2008 m. guttulata jordan 29°34’13.44”n, 35°24’45.68”e ef555279 kapli et al., 2008 m. guttulata egypt 28°25’58.79”n, 29° 4’58.21”e ay217815 whiting et al., 2003 m. brevirostris iran fj416173 rastegar-pouyani et al., 2010 m. brevirostris syria 35°25’02.64”n, 40°19’11.28”e ef555264 kapli et al., 2008 m. brevirostris syria 35°25’36.48”n, 40°01’40.08”e ef555266 kapli et al., 2008 m. brevirostris syria 34°17’35.16”n, 36°45’55.8”e ef555267 kapli et al., 2008 m. rubropunctata egypt 24°24’n, 33°01’01.2”e ef555274 kapli et al., 2008 m. bahaeldini egypt 28°32’26.88”n, 33°58’51.6”e ef555243 kapli et al., 2008 m. olivieri ef555247 kapli et al., 2008 m. olivieri ef555249 kapli et al., 2008 m. olivieri tunisia 32°07’43.32”n, 10°33’49.68”e ef555272 kapli et al., 2008 m. olivieri ef555273 kapli et al., 2008 m. simoni morocco 31°54’43.2”n, 7°30’18”w ef555259 kapli et al., 2008 ophisops elegans fam35 famenin, iran 35°08’45.68”n, 48°52’33.40”e jn828649 this study 143genetic diversity of mesalina watsonana in iran about 700bp of the mtdna cytochrome b gene with primers l14841 (kocher et al., 1989) and cb3h (palumbi, 1996). amplification involved an initial cycle of denaturation at 93 °c for 2 min, 41 subsequent cycles of 93 °c for 1 min, 46 °c for 1 min and 72 °c for 1 min, and final step of extension at 72 °c for 10 min. pcr products were subsequently purified using qiaquick® pcr purification kit (qiagen) following manual therein. sequencing was conducted on abi prism 3100 avant genetic analyzer at the laboratory of dna sequencing, faculty of science, charles university in prague. phylogenetic analyses alignment of concatenated sequences was performed with clustal w (thompson et al., 1994) as implemented in bioedit 7.0 (hall, 1999) and checked manually. prior to analyses, all sequences were translated into amino acids using vertebrate mitochondrial translation code. this did not reveal any stop codons or gaps suggesting that all protein coding sequences were functional and no pseudogenes were amplified. the alignment for the dataset including m. watsonana and selected outgroups (referred to as watsonana dataset hereupon) was 715 bp long. the second dataset including m. watsonana sequences and additional sequences of mesalina species retrieved from genbank (referred to as taxon-wide dataset hereupon) resulted in an alignment with a limited overlap of 321 bp of our own, and the genbank data. average genetic uncorrected distances (p-distance) between individuals and mitochondrial clades were calculated in mega 2.1 (kumar et al., 2001).three different phylogenetic analyses were performed: maximum parsimony (mp), maximum likelihood (ml) and bayesian inference (bi). mp analysis was performed in paup* 4.0b10 (swofford, 2003) under heuristic search criterion with 100 random stepwise addition and tree-bisection-reconnection (tbr) branch swapping. ml analyses were conducted using phyml 3.0 (guindon and gascuel, 2003) with the nearest neighbor interchange (nni) tree improvement, and adopting the hky+i+g model as the best fitting model of substitution for both datasets. the most appropriate model of sequence evolution under both akaike information criterion (aic) and bayesian information criterion (bic) was selected by jmodeltest 0.1.1 (posada, 2008). nodal support for mp and ml trees was assessed by 1000 bootstrap pseudoreplications (felsenstein, 1985). bayesian analyses were performed with mrbayes 3.1.2 (huelsenbeck and ronquist, 2001) implementing the hky+i+g model, with two runs and four chains for each run for 5x106 generations with the sampling frequency of every 100th generation that produced 50 000 sampled trees. after each analysis and after the assurance that the log-likelihood scores achieved stationarity (as plotted against generation time), the first 10% of the trees (5000) was discarded as a burn-in. a 50% majority rule consensus tree was then produced from the posterior distribution of the trees, and posterior probabilities calculated as the percentage of a sampled tree recovering any particular clade (huelsenbeck and ronquist, 2001). molecular clock calibration to estimate divergence dates among individual lineages, we used a bayesian coalescent approach as implemented in the software beast v1.6.1 (drummond and rambaut, 2007). because our major interest was to estimate the outbranching date of m. watsonana from the rest of the species, we performed the calibration on the dataset consisting of all available mesalina species from genbank. we used only one representative animal for each haplotype for this analysis. kapli et al. (2008) used the dating of the split between gallotia stehlini and g. simonyi calculated by maca-meyer et al. (2003) as a calibration point. however, the dating of diversification of gallotia lizards was recently reassessed by cox et al. (2010) and all the major diversification events were moved farther to the geological history. therefore, we based our calibration on the data of cox et al. (2003) and employed the splits between psammodromus lizards and gallotia stehlini and between g. stehlini and 144 j. šmíd and d. frynta g. simonyi as calibration points (17 – 20 mya, 11 – 13 mya respectively). as a next calibration point we used the diversification of timon lepidus from dalmatolacerta oxycephala 5.3 mya as used by hispley et al. (2009), which was based on estes´s (1983) paleontological findings. the following sequences used for molecular clock calibration were also used as outgroups in the taxon-wide dataset in other phylogenetic computations: p. algirus (eu116517), g. simonyi (af101219), g. stehlini (ay154899), t. lepidus (gq142119), d. oxycephala (gq142129). we employed the yule tree prior as suggested for the species-level phylogenies, with randomly generated starting tree, an uncorrelated lognormal relaxed molecular clock model and under the hky+i+g model. the priors for the most recent common ancestors (tmrca) for the calibration points were specified as having normal distribution. the analysis was run twice with 5x107 generations; achieved stationarity and convergence of both runs were checked in tracer 1.5 (rambaut and drummond, 2007) and a burn-in of 20% performed in treeannotator 1.6.1 (drumond and rambaut, 2007). results the watsonana dataset alignment consisted of 715bp out of which 263 positions were variable and 170 parimony informative. all phylogenetic methods resulted in nearly identical tree. mp resulted in 10 equally parsimonious trees, ml analysis resulted in a topology with lnl= -2975.12 which was comparable to the one recovered by mrbayes (mean lnl = -2992.16). the final parameter estimates (obtained from phyml) for this dataset were: gamma shape parameter = 0.188, ti/tv ratio = 10.544, base frequencies f(a)= 0.26, f(c)= 0.30, f(g)= 0.14, f(t)= 0.30. sixteen analyzed samples were represented by 12 different haplotypes. all m. watsonana samples formed a clade with high bootstrap and posterior probabilities support (mp/ml/bi: 100/93/1). four main and well-defined clades of this species were recovered from all analyses: 1) the southern lineage including three animals from localities gahkom and vazireh (100/89/-; average p-distance within this group equal to 0.7%); 2) the central lineage consisting of only one sample from anjireh, 3) the northern lineage including animals from bardeskan, tabas and mayamey (100/92/1; average within-group p-distance equal to 1.8%), with tabas and bardeskan samples clustering together with high support (93/98/1), and 4) the western clade, including samples from izadkhast, ardestan, salafchegan and kushk-e nosrat (90/93/0.99; average within-group p-distance equal to 4.5%). inside the last group, two sister lineages were recognized, one formed by three animals from izadkhast (100/99/1, two of them sharing the same haplotype), and another one from the six remaining samples (99/96/1, two haplotypes present twice). although the four major evolutionary branches were very well supported, mutual relationships among them remain unresolved (fig. 2). genetic distances between them were surprisingly high (uncorrected p-distance ranging from 9.8 to 13.1%). average between-group uncorrected genetic distances are summarized in tab. 2. mp analysis of the taxon-wide dataset produced 16 most parsimonious trees. the 50% majority-rule consensus tree was nearly identical to the results of other computational approaches except of low bootstrap values for higher clades. ml analysis resulted in a topology with lnl= -5494.08009, gamma shape parameter with 4 discrete categories = 0.219, ti/tv ratio = 8.798, nucleotide frequencies: f(a)= 0.27, f(c)= 0.29, f(g)= 0.14, f(t)= 0.30. mean log-likelihood values obtained from bayesian analysis was equal to -5461.980505. all computational methods provided the same topology for the subtree of 145genetic diversity of mesalina watsonana in iran m. watsonana as described above. in this taxon-wide phylogeny, all samples of mesalina form a monophyletic clade (82/79/0.92) consisting of two main branches – one represented by all of the samples of m. watsonana clustering together with one genbank sample of m. brevirostris (fj416173) (99/96/1) and the other lineage formed by all the remaining species, although this clade has only moderate support (-/79/0.94). mutual relationships among individual watsonana clades remain as described above. for comments on the genbank sample nested within watsonana, see discussion. within the second group, m. olivieri and m. simoni formed a separate clade (-/83/0.96) sister to a cluster of brevirostris, guttulata, bahaeldini and rubropunctata (-/74/0.94). m. brevirostris stands apart fig. 2. bayesian inference phylogram of m. watsonana based on 715bp fragment of cyt b. mp and ml bootstrap support values above 70% and bi posterior probabilities above 0.9 are indicated above branches. table 2. average uncorrected genetic distances (p-distance) between individual clades of m. watsonana from the iranian plateau based on 715 bp fragment of cyt b. within-group distances are reported in corresponding fields on the diagonal. s c n w south 0.007 central 0.100 north 0.099 0.098 0.018 west 0.131 0.120 0.108 0.045 146 j. šmíd and d. frynta from the other three species, however, with a very week support (-/76/-). the cluster of guttulata, bahaeldini and rubropunctata (-/-/0.95) forms an unresolved polytomy rendering guttulata paraphyletic. topology of this group is in a general agreement with the tree presented by kapli et al. (2008), except of paraphyly of m. guttulata with respect to rubropunctata and bahaeldnini and lower resolution of mutual relationships. we ascribe all this to shorter sequences of mtdna used in the taxon-wide analysis. the new sample of guttulata from yemen (jem109) clusters with its conspecific sequences. the bi tree of the taxon-wide dataset is depicted in fig. 3. average between-species uncorrected p-distances are reported in tab. 3. the molecular clock analysis brought the following results: separation of iranian m. watsonana from the other species occurred 15.9 mya, the 95% highest posterior density (hpd) interval ranged between 7.8 – 25.6 mya. within m. watsonana, the basal radiation resulting in the four independent clades took place 7.0 mya (hpd: 3.1 – 12.2). within the western clade, the specimens from the locality izadkhast branched out from fig. 3. taxon-wide phylogeny of the genus mesalina. the tree is a 50% majority rule consensus tree of the bayesian analysis. mp and ml bootstrap support values above 70% and bi posterior probabilities above 0.9 are indicated above branches. age estimates are indicated below nodes in bold and include the mean and the hpd 95% confidence interval. 147genetic diversity of mesalina watsonana in iran the rest 3.2 mya (hpd: 1.1 – 6.0). in the northern clade, the sample from mayamey (may06) separated from the two others (bard05, tab11) 1.0 mya (hpd 0.2 – 2.0). diversification dates of the remaining species predate the results of kapli et al. (2008) because of different calibration settings. in our analyses, the basal split between olivieri + simoni clade and the cluster of brevirostris, rubropunctata, guttulata and bahaeldini took place 11.9 mya (hpd: 5.8 – 19.6), brevirostris branched off 9.5 mya (hpd: 4.6 – 15.5) and the radiation of gutulata, rubropunctata and bahaeldini occured 7.0 mya (hpd: 3.3 – 11.5). discussion intraspecific variability of m. watsonana in our work, we used samples of m. watsonana from ten localities throughout the iranian species´ distribution range. phylogenetic analyses based on the 715bp fragment of cyt b indicate existence of four highly supported main clades of m. watsonana occurring in separate geographic regions across the iranian plateau: the southern clade from the southernmost tip of the zagros mountains, the central clade from the plains in the middle of iran, the western clade from the foothills of the zagros, and the northern clade. the western clade can be further divided into two separate lineages, one from the locality izadkhast, another from the localities salafchegan, ardestan and kushk-e nosrat. obvious geographic isolation of the m. watsonana clades is, however, remarkable. whereas the separation of the northern clade can be explained by the presence of vast desert areas of dasht-e lut and dasht-e kavir in central iran that were repeatedly flooded with saltwater (anderson, 1999), the divergence between western, central and southern clades can not be clarified by presence of any apparent natural barrier. moreover, high activity and low biotope preferences of mesalina lizards (smith, 1935; anderson, 1968; baker et al., 2004) suggesting high dispersal ability are in contrast with the presence of such diversified clades. on the other hand, high diversity of reptilian species or species groups was already reported from this particular territory for bunopus tuberculatus (červenka et al., 2008) and eremias persica (rastegar-pouyani et al., 2010). table 3. average uncorrected genetic distances (p-distances) between species or species groups of mesalina used in this study, based on 321 bp of cyt b. species with unresolved phylogeny are merged into one group. mo – mesalina olivieri, ms – m. simoni, mbr – m. brevirostris, mba – m. bahaeldini, mg – m. guttulata, mr – m. rubropunctata, mw – m. watsonana, mbr* probably misidentified m. watsonana determined as m. brevirostris by rastegar-pouyani et al. (2010). mo + ms mbr mba + mg + mr mbr* fj416173 mo + ms mbr 0.150 mba + mg + mr 0.148 0.151 mbr* fj416173 0.194 0.181 0.186 mw 0.170 0.179 0.188 0.121 148 j. šmíd and d. frynta all four watsonana clades are mutually separated by high genetic distances ranging from 9.8 – 13.1% (see tab.2). these distances are in concordance with those of other mesalina species found by kapli et al. (2008). according to their findings, genetically remotest populations of m. brevirostris are separated by 11.5%, intraspecific variability of m. gututlata reaching up to 15.6%. taking into account harris´s (2002) average genetic distance between congeneric species of reptiles being 13.6%, the real diversity of the genus mesalina is still underestimated and should be revised. our inability to resolve phylogenetic relationships among watsonana clades can be either caused by fast evolution and thus high saturation rate of the cyt b or by rapid radiation of mesalina in iran. fast radiation of the whole lacertidae was already suggested by mayer and pavlicev (2007) and pavlicev and mayer (2009) and could have taken place in mesalina as well. we did not, however, cover the entire distribution of m. watsonana. samples from eastern populations from afghanistan and pakistan might affect the tree topology and might disclose closer relationships among the clades. moreover, additional genetic data (including nuclear) and detailed morphological study encompassing material from the type locality (between karachi and sukkur, sind, pakistan) are essential for any taxonomic revisions. thus, we suggest to keep considering m. watsonana as a single species, although it has high intraspecific genetic variability. comments on the phylogeny of mesalina by combination of our data with the genbank sequences, we provide a taxon-wide phylogeny of the genus mesalina. according to the analyses of all mesalina sequences available, m. watsonana always forms a separate clade clearly distinct from all the other species. the lineage of genbank species has basically the same topology as published by kapli et al. (2008) with olivieri and simoni forming a clade sister to a lineage of brevirostris + guttulata + bahaeldini + rubropunctata. m. brevirostris forms a sister lineage to the three remaining species, albeit with low to moderate support. the clade consisting of guttulata + bahaeldini + rubropunctata remains polytomic. the position of rubropunctata differs from the results of kapli et al. (2008) based on 16s rrna, where it formed a clade close to brevirostris, although without a reliable bootstrap support. its placement within the guttulata clade in our analyses could result from the usage of a short fragment of mtdna for the analyses. the nesting of bahaeldini within guttulata remains consistent with kapli et al. (2008). in accordance with its geographic isolation, m. watsonana forms a monophyletic clade deeply separated from the rest of the genus. this species was long considered a subspecies of m. guttulata until arnold (1986) elevated this form to a species rank on the basis of detailed hemipenial morphology. taking this into account, one would suppose closer relationship of these two forms. not only they are separated by a high genetic distance, moreover there are also several other evolutionary lineages (=species) closer to m. guttulata. similar morphology leading former authors to consider m. watsonana a subspecies of m. guttulata is in this case nothing but phenotypic conservativeness of mesalina lizards. this may be caused by the adaptation to xeric environmental conditions and similar habitat types spanning across mesalina distribution range without any obvious selective pressure affecting their morphology. 149genetic diversity of mesalina watsonana in iran the new guttulata sample from yemen clusters well to the rest of m. guttulata specimens. genetic distance separating yemeni and egyptian m. guttulata (14.1%, data not shown) suggests a long-time independent evolution. yet another noteworthy sequence was a sample of m. brevirostris (fj416173) nested within the m. watsonana clade. this was most probably caused by misidentification of the animal belonging to the latter species. this animal was collected in a region where the distribution of both species overlap (anderson, 1999). however, if this specimen really fits the morphological characteristics of m. brevirostris, then we might be dealing with hybridizaiton and gene introgression from one species to another. in any case, detailed study of the zone of sympatry should be carried out in order to clarify the phylogenetic position of iranian populations of m. brevirostris with respect to the levantine populations of this species and of the sympatric m. watsonana. biogeography m. watsonana is the only member of the genus inhabiting the iranian plateau. although its distribution partly overlaps with m. brevirostris in the mesopotamian plain and in the southeastern pakistan, m. watsonana is the only species exceeding beyond the zagros and the sulaiman ranges (khan, 1980; anderson, 1999). this striking pattern can be, however, explained by the geological history of this region. the collision of the arabian tectonic plate to the eurasian landmass took place 35 20 mya (dercourt et al., 1986 mouthereau, 2011), its drifting movement in the northeast direction resulted in the continuous uplift of the zagros mountains which culminated 12.4 – 10 mya (sborshchikov et al., 1981; mouthereau, 2011). thus, the outbranching time of m. watsonana which is dated back to the middle miocene (15.9 mya) can be correlated with this geological event. after its formation, zagros established as the major biogeographic barrier in the middle east and hampered mesalina the east-west dispersion. our findings regarding the evolutionary history of mesalina lizards predate the results of mayer and pavlicev (2007) and of kapli et al. (2008) and they are in concordance with those of hipsley et al. (2009), who suggest earlier diversification of lacertids with the split of ethiopian and saharo-eurasian clades (sensu mayer and pavlicev, 2007) dated back to the middle eocene (43.2 ± 5.6 myr) and the radiation of saharo-eurasian xerophilous forms (acanthodactylus, omanosaura, ophisops, eremias and mesalina) following soon after (40.2 ± 5.1 myr). hipsley et al. (2009) date the divergence between m. guttulata and m. brevirostris to the late miocene (10.3 ± 2.9 myr). according to available data, the ancestors of the genus mesalina penetrated from the east africa into asia, spread over the southwest asia and re-entered north africa again (kapli et al., 2008). m. watsonana might have separated even before the uplift of the zagros mountains took place, but the ongoing raise of the massif isolated this species from the rest of the genus and thus prevented gene flow. subsequently, the remaining mesalina species then diversified in north africa in several steps ~ 11.9 mya (separation of olivieri + simoni), ~ 9.5 mya (separation of brevirostris) and ~ 7.0 mya (guttulata clade radiation). thus, as we can see, both dispersal and vicariance processes may have played an important role in the evolution of the mesalina group. within m. watsonana, the split into the four discovered lineages took place in the late miocene (5.6 mya). since there are no apparent geographical barriers 150 j. šmíd and d. frynta limiting the dispersal of these lizards on the iranian plateau, it remains disputable whether the four lineages became isolated via vicariant in situ diversification or by dispersion of already separated populations. in our study, we brought a new view on the phylogeny of the genus mesalina. the results presented confirm the monophyly of m. watsonana and its isolated position within the genus, which is supported by historical biogeographic events. nevertheless, broader sampling accross the entire distribution and more genes (including nuclear) involved might help bring closer insight into the phylogeny and phylogeography of these lizards. acknowledgements we thank pavel hulva and václav gvoždík for their kind help with computations, jan červenka, vojto baláž and pavel munclinger for laboratory support, lukáš kratochvíl for unpublished data. the research was supported by the grant agency of the czech academy of sciences (project no. 206/05/2334), by charles university in prague (project no. 9873) and by ministry of culture of the czech republic (dkrvo 00023272). we are indebted to monika malečová, silva lišková and two anonymous reviewers for correcting and significantly improving the english of the manuscript. references anderson, s.c. 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(1996): eine neue unterart der bachschildkröte (mauremys caspica ventrimaculata subsp. nov.) aus dem iranischen hochland. salamandra 32: 113122. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 6(1): 105-118, 2011 climate change and peripheral populations: predictions for a relict mediterranean viper josé c. brito1, soumia fahd2, fernando martínez-freiría1, pedro tarroso1, said larbes3, juan m. pleguezuelos4, xavier santos5 1 cibio, centro de investigação em biodiversidade e recursos genéticos da universidade do porto, campus agrário de vairão, r. padre armando quintas, 4485-661 vairão, portugal. corresponding author. e-mail: fmartinez_freiria@yahoo.es 2 département de biologie, faculté des sciences, université abdelmalek essaâdi, tétouan, morocco. 3 faculté des sciences biologiques et agronomiques, université m. mammeri. tizi-ouzou, algeria. 4 departamento de biología animal, facultad de ciencias, universidad de granada, e-18071 granada, spain. 5 departament de biologia animal, universitat de barcelona, av. diagonal 645, e-08028 barcelona, spain. submitted on: 2010, 26th december; revised on 2011, 10th may; accepted on 2011, 26h may. abstract. ecological niche-based models were developed in peripheral populations of vipera latastei in north africa to: 1) identify environmental factors related to species occurrence; 2) identify present suitable areas; 3) estimate future areas according to forecasted scenarios of climate change; and 4) quantify habitat suitability changes between present and future climatic scenarios. field observations were combined with environmental factors to derive an ensemble of predictions of species occurrence. the resulting models were projected to the future north african environmental scenarios. species occurrence was most related to precipitation variation. present suitable habitats were fragmented and ranged from coastal to mountain habitats, and the overall fragmented range suggests a relict distribution from wider past ranges. future projections suggest a progressive decrease in suitable areas. the relationship with precipitation supports the current unsuitability of most north africa for the species and predicts future increased extinction risk. monitoring of population trends and full protection of mountain forests are key-targets for long-term conservation of african populations of this viper. predicted trends may give indications about other peripheral populations of palearctic vertebrates in north africa which should be assessed in detail. keywords. climate change, conservation, mediterranean, biogeography, range regression, vipera latastei. mailto:fmartinez_freiria@yahoo.es 106 j.c. brito, s. fahd, f. martínez-freiría, p. tarroso, s. larbes, j.m. pleguezuelos and x. santos introduction the complex geographic shifts around the strait of gibraltar over the past 14 million years (de jong, 1998) resulted in high percentages of european and african species found in morocco and iberian peninsula, respectively (schleich et al., 1996; sindaco and jeremcenko, 2008). the pleistocene climatic oscillations have also produced shifts in species ranges (hewitt, 2000): during cold periods, european species in north africa expanded their range, but in warm periods, they have experienced severe reductions in the southern part of their range, with populations remaining isolated in mountainous areas (santos et al., 2009). currently, north-western africa has the highest diversity and number of europeanoriginated relicts of terrestrial reptiles in the mediterranean basin (bons and geniez, 1996; schleich et al., 1996; pleguezuelos et al., 2010). during the last 10,000 years, the region has been subjected to enormous landscape changes for both natural reasons (climate warming during the holocene) and human activities (charco, 1999). presently, it is estimated that only 4.7% of the original mediterranean forests remain (cuttelod et al., 2008). thus, many european taxa in north africa are presently restricted to isolated mountains where suitable habitats endure (bons and geniez, 1996; schleich et al., 1996). range reductions and shifts to higher elevations are expected in mountain specialists (peterson, 2003) and highlands may act as refuges against climatic constraints (nogués-bravo et al., 2007). climate change scenarios for north africa predict a decrease in rainfall of 10-200 mm by 2025 (paeth and thamm, 2007), which may increase the vulnerability of these populations to extinction. the lataste’s viper, vipera latastei, is an appropriate taxon to analyse potential effects of climate change in the extinction vulnerability of european-originated relicts in northwestern africa because: 1) it is a species of european origin that colonised north-western africa prior to the formation of the strait of gibraltar (saint-girons, 1980; authors, unpub. data); 2) the global distribution is well known (brito et al., 2008); 3) several lifehistory traits, such as low growth rates, frequency of reproduction and dispersion capacity, and feeding specialisation, make it prone to local extinction (brito and rebelo, 2003; pleguezuelos et al., 2007; santos et al., 2007a); 4) the rare reported occurrences in northwestern african, even in areas relatively well sampled (bons and geniez, 1996; real et al., 1997; fahd and pleguezuelos, 2001), suggest low population densities; and 5) its distribution is highly related to an environmental variable, the annual precipitation, at both regional and local scales (brito et al., 2008; martínez-freiría et al., 2008). populations of v. latastei in north africa were ascribed to the subspecies v. l. gaditana (saint-girons, 1977). they have been shown to constitute morphologically differentiated groups (brito et al., 2008) and genetical substructuring has been identified for morocco and algeria, probably related to the opening of the gibraltar strait (authors, unpub. data). african populations occur within a concise area, from the rif and middle atlas mountains of morocco to western tunisia, isolated by the mediterranean from the remaining european populations. previous biogeographical studies suggested that local environmental pressures are related with the african range of the species (brito et al., 2008). these factors, combined with the near-threatened status in morocco (pleguezuelos et al., 2010) and the rareness and fragmented character of the species in africa, stress the need for the development of regional evaluations of species vulnerability to climate change. analyses within geographical limits are useful (czech and krausman, 1997) 107climate change and relict viper populations because most decisions and budgets on the management of a species of conservation concern are planned independently by the different countries within the range of a species (rodrigues and gaston, 2002; samways, 2003). this study uses ecological niche-based models in african relict populations of v. latastei to: 1) identify suitable areas for species occurrence in present time; 2) estimate future suitable areas according to forecasted scenarios of climate change; and 3) quantify habitat suitability changes between present and future climatic scenarios. we intend to provide insights on the vulnerability to extinction of european-originated relict taxa in north africa to predicted climate change impacts. material and methods data the study area comprises northern regions of morocco, algeria and tunisia (fig. 1). a total of 33 viper localities (table 1) were collected from bibliographic records (boettger, 1883; dolfus and beaurieux, 1928; chpakowsky and chnéour, 1953; bons, 1958, 1963; saint-girons, 1977; mediani et al., 2009), museum collections (mnhn paris, mncn madrid, univ. salamanca), unpublished observations given to authors, and fieldwork conducted between 1989 and 2009 (fahd and pleguezuelos, 2001; fahd et al., 2005, 2007; authors, unpub. data). fieldwork observations were georeferenced to the gps precision (wgs84 datum). given the restricted range of microhabitats occupied by the species (santos et al., 2006; brito et al., 2008; martínez-freiría et al., 2008), bibliographic and museum localities were georeferenced with a precision of 1 × 1 km. environmental factors, or ecogeographical variables (hereafter egv), were selected according to their importance to the distribution of v. latastei (santos et al., 2006; brito et al., 2008; martínezfreiría et al., 2008): annual average temperature (ante), annual temperature range (tanr), maximum temperature of warmest month (tmax), annual precipitation (anpr) (hijmans et al., 2005), and one topographical grid (usgs, 2006) from which slope (slop) was derived with the geographical information system (gis) arcgis 9.2 (table 2). future climate data from three global circulation models (gcm: cccma, hadcm3 and csiro) and two ippc 3rd assessment emission scenarios (a2a and b2a) for three time periods (2020-2050, 2050-2080 and 2080-2100) (ipcc-tgica, 2007) fig. 1. location of the study area within the mediterranean context, distribution of observations of vipera latastei in north-western africa and major toponomies in the study area. 108 j.c. brito, s. fahd, f. martínez-freiría, p. tarroso, s. larbes, j.m. pleguezuelos and x. santos were obtained from worldclim (hijmans et al., 2005). the resolution of egvs was standardised to a grid cell size of 0.0110 degrees (about 1 × 1 km) for matching the resolution of observations. analyses were developed using a geographic coordinate system given the limited latitudinal extent of the study area. correlations between egvs were relatively negligible (r < 0.599 in all cases). the presence sample size available for developing ecological models was very small which is mostly related to the rareness and localised character of the species in africa (bons and geniez, 1996; schleich et al., 1996) and sampling restrictions in politically unstable areas. these constraints table 1. location of observations of vipera latastei used to develop the ecological models. the year and the origin of the observation are also included. locality, country year reference oued bou regieg, morocco 1959 bons, 1963 mamora forest, morocco 2008 unpub. data given to authors tanger, morocco 1982 boettger, 1883 jbel haouch ben lake’aa, morocco 2009 mediani et al., 2009 astouf, morocco 1990 collection dba granada jbel el alem, morocco 2000 authors, unpub. data oued laou, morocco 1986 unpub. data given to authors kelti, morocco 2005 authors, unpub. data ain rami, morocco 1992 fahd and pleguezuelos, 2001 chaouen, morocco 1992 fahd and pleguezuelos, 2001 fifi, morocco 2008 unpub. data given to authors azrou, morocco 1928 dolfus and beaurieux, 1928 talassemtane, morocco 2001 authors, unpub. data bou slimane, morocco 1992 fahd and pleguezuelos, 2001 béni m’hamed, morocco 2005 authors, unpub. data sidi ali aguelmane, morocco 2006 fahd et al., 2007 khandak lanasser, morocco 1992 fahd and pleguezuelos, 2001 jbel tidghine, morocco 2005 fahd et al., 2006 ain zora, morocco 1992 fahd and pleguezuelos, 2001 ras el ma, morocco 1908 collection mncn madrid moulouya river mouth, morocco 1988 collection univ. salamanca saïdia, morocco 1958 bons, 1958 aïn benian, algeria 1891 collection mnhn paris djebel heidzer, algeria 2005 authors, unpub. data tikjda, algeria 2004 authors, unpub. data darna, algeria 2004 authors, unpub. data darna, algeria 2005 authors, unpub. data ait ouabane, algeria 2005 authors, unpub. data akfadou, algeria 2006 authors, unpub. data mountain edough, algeria 1977 saint-girons, 1977 annaba, algeria 1901 collection mnhn paris ain soltane, tunisia 1953 chpakowsky and chnéour, 1953 ain draham, tunisia 1953 chpakowsky and chnéour, 1953 109climate change and relict viper populations forced using four localities (table 1) where vipers were observed outside the temporal range of present environmental data, 1950 to 2000 (hijmans et al., 2005). removing such localities would imply an even smaller sample size for calibrating models, which would probably increase uncertainties in model predictions. ecological niche-based models models were developed with maximum entropy approach, using maxent 3.3.0f (phillips et al., 2006). this modelling technique requires only presence data as input, but consistently performed well in comparison to other methods, especially at low samples sizes and in assessments of climate change effects (elith et al., 2006; hernandez et al., 2006; hijmans and graham, 2006; wisz et al., 2008). a total of 25 replicates were run with random seed, which allows a different random 20% test / 80% train data partition in each run. presence data for each replicate were chosen by bootstrap allowing sampling with replacement. models were run with auto-features (phillips et al., 2006), and the area under the curve (auc) of the receiver-operating characteristics (roc) plot was taken as a measure of individual model fit (liu et al., 2005). the importance of an egv for explaining the species distribution was determined by its average percent contribution to the model. the relationship between viper occurrence and egvs was determined by examination of response curves profiles from univariate models (martínez-freiría et al., 2008; brito et al., 2008, 2009). the individual model replicates (n = 25) were added to generate a mean forecast of probability of species presence under present climatic conditions (araújo and new, 2007; marmion et al., 2009). standard deviation between individual model probabilities of occurrence was used as an indication of prediction uncertainty (buisson et al., 2010; carvalho et al., 2010). the individual model replicates were projected for each gcm and emission scenario, resulting in 150 simulations for each year. models were averaged by year to generate a future probability of presence. the maximum standard deviation between replicate uncertainties across combinations of gcms and emission scenarios were taken as a measure of final prediction uncertainty. the consensus predictions of mean models were reclassified into three categories of habitat suitability: core habitats with more than 0.5 mean probability of occurrence, marginal habitats (between 0.25 and 0.5) and unsuitable habitats (less than 0.25). the area of each category was quantified and percentage change of each category from the present to the future predicted models were calculated (carvalho et al., 2010). total presence data (n = 33) were overlaid with present and future mean models to calculate percentages of presences in each habitat suitability category. table 2. environmental variation (minimum maximum) in the study area in the present time and predicted for 2020, 2050, 2080 from the ensemble of two emission scenarios (a2a and b2a) and three global circulation models (cccma, csiro and hadcm3). variable units current 2020 2050 2080 anpr mm 168 1430 156 1423 152 1370 131 1276 tanr ºc 18.4 36.9 18.3 38.6 17.8 39.7 18.2 40.9 tmax ºc 25.7 37.2 26.4 40.7 27.6 40.8 28.8 43.7 ante ºc 4.2 20.1 5.3 21.4 6.6 22.3 7.3 24.6 slop % 0 69 110 j.c. brito, s. fahd, f. martínez-freiría, p. tarroso, s. larbes, j.m. pleguezuelos and x. santos results the roc plots for the training and testing datasets exhibited high average aucs (above 0.934 and 0.886, respectively) with low standard deviations (table 3). all observations in the present model were identified as occurring in core and marginal habitat suitability areas. annual average precipitation was the egv most related to occurrence (average contribution above 58%), but slope (above 13%), annual temperature range (above 9%), and maxitable 3. sample sizes, average (and standard deviation) training and test auc, and average percent (and standard deviation) contribution of each variable for the 25 maximum entropy models projected to four climatic scenarios (present, 2020, 2050 and 2080), and number (and percentage) of observations of vipera latastei in north-western africa in each habitat suitability category in each climatic scenario. present 2020 2050 2080 n training samples 26 per model 26 per model 26 per model 26 per model n test samples 7 per model 7 per model 7 per model 7 per model training auc (sd) 0.935 (0.013) 0.934 (0.018) 0.934 (0.019) 0.935 (0.018) test auc (sd) 0.895 (0.067) 0.886 (0.093) 0.887 (0.087) 0.892 (0.087) anpr (sd) 57.7 (14.1) 64.7 (15.6) 63.7 (15.2) 63.0 (17.2) tanr (sd) 9.0 (5.4) 11.0 (9.4) 9.3 (6.6) 9.8 (8.3) tmax (sd) 9.1 (9.4) 7.9 (8.8) 9.4 (11.9) 8.2 (11.7) ante (sd) 6.4 (6.7) 3.6 (5.8) 4.0 (5.1) 3.6 (4.7) slop (sd) 17.7 (11.2) 12.8 (9.3) 13.6 (13.4) 15.5 (11.7) suitability category unsuitable (%) 0 (0) 3 (9.1) 6 (18.2) 8 (24.2) marginal (%) 16 (48.5) 14 (42.4) 17 (51.5) 22 (66.7) core (%) 17 (51.5) 16 (48.5) 10 (30.3) 3 (9.1) fig. 2. response curves for the most related environmental factors to the distribution of vipera latastei in north-western africa. curves depict probability of occurrence along the environmental gradients. 111climate change and relict viper populations fig. 3. mean probability of occurrence of vipera latastei in north-western africa, at a 1x1km scale, for the present and projected models (2020, 2050 and 2080), based on three global circulation models (gcm) and two emission scenarios (150 bootstrap models for each year). maximum standard deviation of predictions across gcms and scenarios are represented in smaller insets. 112 j.c. brito, s. fahd, f. martínez-freiría, p. tarroso, s. larbes, j.m. pleguezuelos and x. santos mum temperature of warmest month (above 8%) were also related (table 3). the profiles of the response curves suggest that the species is restricted to precipitation and slopes roughly above 900 mm and 15%, respectively, and maximum temperatures below 30 ºc (fig. 2). core habitat-suitability areas according to the present model (fig. 3) were fragmented and restricted to the rif and few cells in central middle atlas and coastal tangier in morocco, eastern tellian atlas in algeria, and el feidja in tunisia. marginal suitability habitats surrounded core areas and included also the middle atlas and coastal regions of salé, melilla, saidia and oran. the areas of prediction uncertainty were common to the habitats identified as marginal and core. future projection of models predicted a progressive decrease in the availability of suitable areas (table 4). compared with the model for the present time, a decrease of 89% and 57% in the availability of core and marginal habitats, respectively, is predicted by 2080. core habitat areas will be extremely fragmented and restricted to the rif and tellian mountains (fig. 3) and would include only 9% of present-day viper localities in core areas (table 3). the distribution of future predicted suitability areas suggests that most currentviper localities (67%) will be located in marginal habitats (table 4). areas of prediction uncertainty were restricted to few squares, especially for 2080 (fig. 3). discussion the low number of observations available, the large dimensions of the study area and the projection to future climates stressed the importance of incorporating distinct sources of uncertainty in model projections for future climatic conditions. first, average predictions from different gcms and emission scenarios were analysed, which allowed recovering patterns emerging from the noise associated with distinct model outputs (buisson et al., 2010; carvalho et al., 2010). secondly, average predictions from model replicates using distinct presence data sets were analysed, which partially accounted for the effects of low sample size (pearson et al., 2007). the maximum entropy algorithm was employed due to its good performance under climate change scenarios (hijmans and graham, 2006) and ability to deal with low sample sized data sets (elith et al., 2006; hernandez et al., 2006; wisz et al., 2008). however, uncertainties associated to modelling techniques have been emphasised (thuiller, 2004; wiens et al., 2009; buisson et al., 2010) and should be table 4. forecasted evolution of habitat suitability for vipera latastei in north-western africa. number of 1x1 km squares classified by the present model and 2020, 2050 and 2080 scenarios in each habitat suitability category (unsuitable, marginal and core habitat). percentage of gain (+) or loss (-) in number of squares relatively to the present model are given in brackets. unsuitable marginal core present 103958 47905 7609 2020 120751 (+16.2) 32493 (-32.2) 6228 (-18.1) 2050 131074 (+26.1) 25120 (-47.6) 3278 (-56.9) 2080 142193 (+36.8) 16440 (-65.7) 839 (-89.0) 113climate change and relict viper populations addressed in future studies. nevertheless, models were apparently robust and all presence data were identified in core and marginal areas of present model predictions. uncertainties in projections of models related to low sample size were mostly located in cells identified with core and marginal suitability, but not in cells of unsuitable habitat, suggesting that potential areas for the occurrence of the viper may actually be smaller, on average, than predicted. present models were calibrated with restricted-range of environmental conditions in comparison to the predicted environmental range for the future (table 2) which may produce biases in model projections for the future (barbet-massin et al., 2010). however, the lower precipitation and higher temperatures predicted for the future that fall outside the present variation, mostly located in lower altitude areas between the rif and middle atlas of morocco and south-eastern valleys of el feidja in tunisia (data not shown), correspond already to present-day unsuitable areas (fig. 3). the uncertainties arriving from these biases are thus negligible because these areas are very unlikely to become suitable habitats in the future. about 65% of the study area was quantified as unsuitable in the present model, which agrees with the biogeographical pattern of the peripheral limit of a species distribution. the proximity of the sahara desert as a true ecological barrier for v. latastei further supports the observed relationship between high annual precipitation and low maximum temperature with species presence. presently, v. latastei is less common in flatter areas, which correspond essentially to coastal and agricultural regions, and slope is probably acting as surrogate for habitat loss in plain areas where human activities tend to be more intense (charco, 1999; ramdani et al., 2001; cuttelod et al., 2008). in fact, local extinction in morocco was suggested for coastal regions, where recent intensive sampling effort (fahd and pleguezuelos, 2001; fahd et al., 2005, 2007; harris et al., 2008; authors, unpub. data) failed to confirm previous observations. these localities corresponded to coastal cells located in the salé beach, moulouya mouth and tangiers peninsula, and have been most affected recently by tourism urbanisation (ramdani et al., 2001; fahd et al., 2005) that probably induced severe habitat loss for the viper. local extinction in the coastal belt is a pattern also reported for the snake community of mediterranean coastal spain, deriving from intense tourism and agricultural activities (santos et al., 2006, 2007b). the modelling approach used in this study considered all observations available because the secretive behaviour of the viper hampers the accurate determination of local extinction. however, if the suggested disappearance from certain areas of coastal morocco is confirmed, then the current predictions of suitable habitats may be overestimated, as well as future range predictions. present suitable areas for v. latastei in north-western africa are fragmented and mostly restricted to mountain areas of low habitat change. most recent observations come from protected areas holding forests of high environmental and economic value, where grazing is relatively restricted to favour natural seedling. alarmingly, pine plantations (e.g., in northwestern tunisia, brito et al., 2008), cannabis culture (in the rif, fahd et al., 2005), and extensive agriculture and overexploitation of livestock outside protected areas, continue to threaten habitats (charco, 1999). the models further suggested that the species may be present in currently undetected mountains, such as in jbel bou naceur (morocco). likewise, large core habitat areas in algeria were predicted for eastern tellian atlas, but field work is needed to confirm viper presence in these politically unstable areas. 114 j.c. brito, s. fahd, f. martínez-freiría, p. tarroso, s. larbes, j.m. pleguezuelos and x. santos future projections are not optimistic for viper persistence, given the predicted declines in suitable habitats with no new suitable areas identified. the significant increase of temperature in north africa during the mid-piacenzian warm interval (ca 3 myr ago) of the late pliocene (jost et al., 2009) probably induced refugia in suitable mountain valleys during warming stages and triggered for the current fragmented range of v. latastei. thus, the projected decrease in precipitation (paeth and thamm, 2007) should imply even smaller suitable areas in the future. dispersal was suggested as a possible mechanism for decreasing the impacts of climate change (e.g., araújo et al., 2006), but severe population declines are expected in species with low dispersal capacities surrounded already by unsuitable habitats (foden et al., 2007). in the case of v. latastei, colonisation of new cells is highly unlikely during the time-period of the study, given its climatic specialisation in north africa and the systematically occurrence in preserved habitats (real et al., 1997; fahd and pleguezuelos, 2001). this pattern is consistent with the iberian peninsula, where this viper is present in various habitat types (from coastal dunes to highland shrublands) but preferably in localities where habitats are well preserved (santos et al., 2006). additionally, several biological traits of this viper make it a slow coloniser (brito and rebelo, 2003; pleguezuelos et al., 2007; santos et al., 2007b). according to future predictions, mountains will become climatic refugia, as predicted also for cedrus atlantica forests in morocco (cheddadi et al., 2009). therefore, the currently existing mountain parks are priority areas for the conservation of this species. although it is unknown if species will be able to adapt to future climate conditions and persist in the current range, monitoring of population trends and full protection of mountain forests are key-targets for long-term conservation of v. latastei in north africa. however, current predictions should also be evaluated for the first projected scenario (year 2020), with viper sampling in current suitable habitats and/or confirmation of forecasted decrease in precipitation. field sampling should take into account detectability biases related to rareness and cryptic behaviour (mazerolle et al., 2007). if predicted reductions in habitat suitability to 2020 are correct, conservation actions must be accomplished, including the strict protection of all areas with viper populations, the exclusion of grazing from these areas, and potentially population translocation. under the scenario of habitat suitability decrease in 2020, the delay of these management actions would be catastrophic for long-term conservation of this viper. trends in the availability of suitable habitats observed in this study may give indications about other european-originated vertebrates with relict populations in north africa, including fishes (barbus sp.), amphibians (salamandra algira), reptiles (coronella girondica), birds (cinclus cinclus), and mammals (mustela putorius), which should evidence similar environmental responses and extinction risks as the studied viper. the conservation of these species would 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revised on 2014, 2nd april; accepted on 2014, 6th may abstract. amphibian diseases are acknowledged as significant contributors to the decline and extinction of amphibian species. the main culprits currently considered are chytridiomycosis and ranavirus. in central america, highly endemic and geographical restricted terrestrial species may be at risk from these diseases. we collected 49 agalychnis callidryas larvae, one lithobates forrei and five unidentified larvae on the nicaraguan island ometepe, all deceased, and skin samples were taken. the presence of ranavirus was determined using pcr. ranavirus was found involved in 41 of 55 tadpoles. forty-one agalychnis callidryas, one lithobates forrei and another five unidentified anuran tadpoles. keywords. anurans, mortality, ranavirus, nicaragua, ometepe. amphibians are declining in all continents where they occur due to several causes like habitat destruction, habitat degradation, pollutants, climate change and diseases (stuart et al., 2008). besides chytridiomycosis caused by the fungal agents batrachochytrium dendrobatidis (voyles et al., 2009) and b. salamandrivorans (martel et al., 2013), ranaviruses globally contribute to amphibian declines (miller et al., 2011; robert and chinchar, 2012; brenes, 2013). ranaviruses belong to the family iridoviridae and have been recorded from asia, central-, southand north america and europe (whitfield et al., 2007; xu et al., 2010). although all amphibian life stages can be affected, larvae appear to be most susceptible to the disease and mortality rates are often high (green et al., 2002; greer et al., 2005; kik et al., 2011; miller et al., 2011). central america is one of the regions considered most struck by disease-driven amphibian declines, notably chytridiomycosis affecting mostly endemic high-elevation species with a restricted range (duellman, 1999; savage, 2002; lips et al., 2008). the volcanic islands in lake nicaragua contain complex high-elevation amphibian assemblages in a region where ranavirus has not been recorded before. in this study we found ranavirus to be involved in a mortality event comprising several anuran species in the crater lake of volcano maderas on ometepe island. ometepe (11°30’n, 85°35’w) is situated in lake nicaragua (8,264 km2) and is comprised out of two volcanos (mckaye, 1995; nemitz, 2008). our survey was conducted on the maderas volcano, the smallest one, with cloud forests on its upper slopes. this survey was carried out in october 2011, coinciding with the rainy period. surveys had a strong focus on locating and sampling anurans and their breeding waters. since breeding animals and vocalizing males were found to be most active during the hours between sunset and midnight, surveys were carried out between these. 126 tariq stark et alii on 21 october 2011 55 tadpoles of two anuran species (agalychnis callidryas and lithobates forrei), were found dead in the cloud forest of maderas at an elevation of 1,203 meters (nicaragua, 11°26’n, 85°30’e). from 54 of these tadpoles, skin swabs were collected and processed for the detection of bd using qpcr (boyle et al., 2004) and for the presence of ranavirus dna using the pcr described by mao et al. (1997). we tested for these two pathogens because these two are known to cause amphibian declines in central america. thirty-five out of 49 dead larvae of agalychnis callidryas (in various stages of development), one dead lithobates forrei tadpole and five dead unidentified tadpoles (due to post mortem decay), tested positive for ranavirus. none of these larvae tested positive for bd. ranavirus was associated in this case with a mortality event in the larvae of two anuran species on ometepe. however, only pcr was performed because it was not possible to collect dead animals to perform a histological examination. the latter is needed to confirm an etiological diagnosis (greer et al., 2005). during an outbreak, ranavirus has been known to kill almost 100% of larvae and post metamorphs (green et al., 2002). ranavirus may thus have a big impact on geographically restricted and small populations of amphibians on maderas like the micro-endemic bolitoglossa insularis (status vulnerable iucn, 2013), craugastor laevissimus (status endangered iucn, 2013). as the current study only encompassed a limited time frame, we cannot exclude an effect on other species fig. 1. ometepe island. the study area on volcano maderas, one of the two volcanoes that comprises the island, is marked with a. 127ranavirus associated mortality in assemblage of cloud forest amphibians in nicaragua within the entire community of amphibians in this cloud forest. if we consider the most likely scenario that the outbreak is due to a recent introduction of ranavirus on ometepe, the route of viral entry is hypothetical. few tourists venture up maderas due to the very poor accessibility of the volcano (personal observation). the infrastructure of both the lowland on ometepe and the accessibility of maderas are, however, being improved since 2011 (personal observation). with these changes it might be easier to reach the higher ranges of the volcano, which can lead to more people in the cloud forest acting as vectors for ranavirus. continuous monitoring of the amphibian assemblages at this site is highly recommended. acknowledgements specimens were collected under permit number 011102010/dgpn issued by the ministry of the environment and natural resources nicaragua (marena). we also thanks the universidad nacional autónoma de nicaragua-leon, león, nicaragua, for helping obtaining collecting permits through javier sunyer. we are very grateful to fundacion colibolca and josé m zolotoff-pallais (momabacho) for their guidance, assistance and use of the field station. we are also grateful for rachael antwis for supplying us with various field equipment. ignas dummer we wish to thank for creating the map of the research area depicted in this paper. jordi janssen is thanked for proof reading this paper. references boyle, d.g., boyle, d.b., olsen, v., morgan, j.a.t., hyatt, a.d. 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(2005): five amphibian mortality events associated with ranavirus infection in south central ontario canada, dis aquat organ. 67: 9-14. iucn (2013). iucn red list of threatened species. version 2013.2. <www.iucnredlist.org>. downloaded on 20 may 2014. kik, m., martel, a., spitzen-van der sluijs, a., pasmans, f., wohlseind, p., grönea, a., rijksa, j.m. (2012): ranavirus-associated mass mortality in wild amphibians, the netherlands, 2010: a first report. vet. j. 190: 284-286. lips, k.r., diffendorfer, j. mendelson, j.r., sears, m.w. (2008): riding the wave: reconciling the roles of disease and climate change in amphibian declines. plos biol. 6: 72. martel, a., spitzen-van der sluijs, a., blooi, m., bert, w., ducatelle, r., fisher, m.c., woeltjes, a., bosman, w., chiers, k., bossuyt, f., pasmans, f. (2013): batrachochytrium salamandrivorans sp. nov. causes lethal chytridiomycosis in amphibians. pnas 110: 1532515329. miller, d., gray, m., storfer, a. (2011): ecopathology of ranaviruses infecting amphibians. viruses 3: 23512373. mckaye, k.r., ryan, j.d., stauffer, j.r., perez, l.j.l., vega, g.i., van den berghe, e.p. (1995): african tilapia in lake nicaragua. ecosystem in transition. 45: 406-411. nemitz, d. (2008): an assessment of sampling detectability for global biodiversity monitoring: results from sampling grids in different climatic regions, unpublished master thesis. faculty of biology, georgaugust-universität göttingen. robert, j., chinchar, v.g. (2012): ranaviruses: an emerging threat to ectothermic vertebrates. report of the first international symposium on ranaviruses, minneapolis mn july 8, 2011. dev. comp. immunol. 2: 259-261. savage, j.m. (2002): the amphibians and reptiles of costa rica: a herpetofauna between two continents, between two seas. university of chicago press, chicago. stuart, s.n., hoffmann, m., chanson, j.s., cox, n.a., berridge, r.j., ramani, p., young, b.e. (eds.) (2008): threatened amphibians of the world. lynx edicions, barcelona, spain. voyles, j., young, s., berger, l., campbell, c., voyles, w.f., dinudom, a., cook, d., webb, r., alford, r.a., skerrat, l.f., speare, r. (2009): pathogenesis of chytridiomycosis, a cause of catastrophic amphibian declines. science 326: 582-585. whitfield, s.m., geerdes, e., chacon, i., ballesteros, e., jimenez, r., donnelly, m.a., kerby, j.l. (2013): infection and co-infection by the amphibian chytrid fungus and ranavirus in wild costa rican frogs. dis. aquat. organ. 104: 173-178. xu, k., zhu, d., wei, y., schloegel, l.m., chen, x., wang, x. (2010): broad distribution of ranavirus in free ranging rana dybowskii in heilongjiang, china. ecohealth 7: 18-23. acta herpetologica vol. 8, n. 2 december 2013 firenze university press journal of the societas herpetologica italica acta herpetologica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 7(2): 221-237, 2012 the significance of using satellite imagery data only in ecological niche modelling of iberian herps neftalí sillero1, josé c. brito2, santiago martín-alfageme3, eduardo garcíameléndez4, a.g. toxopeus5, andrew skidmore5 1 centro de investigação em ciências geo-espaciais (cicge), universidade do porto, faculdade de ciências, rua do campo alegre, 687, 4169-007 porto, portugal. corresponding author. e-mail: neftali. pablos@fc.up.pt 2 cibio, centro de investigação em biodiversidade e recursos genéticos da universidade do porto, campus agrário de vairão, rua padre armando quintas, 4485-661 vairão, portugal 3 servicio transfronterizo de información geografica, universidad de salamanca, c.m. san bartolome, pz. fray luis de león 1-8, 37008 salamanca, spain 4 área de geodinámica externa, facultad de ciencias biológicas y ambientales, universidad de león, campus de vegazana s/n, 24071, león, spain 5 itc, faculty of geo-information science and earth observation, university of twente, hengelosestraat 99, 7514 ae enschede, the netherlands submitted on: 2011, 24th october; revised on: 2012, 20th march; accepted on: 2012, 11th april. abstract. the environmental data used to calculate ecological niche models (enm) are obtained mainly from ground-based maps (e.g., climatic interpolated surfaces). these data are often not available for less developed areas, or may be at an inappropriate scale, and thus to obtain this information requires fieldwork. an alternative source of eco-geographical data comes from satellite imagery. three sets of enm were calculated exclusively with variables obtained (1) from optical and radar images only and (2) from climatic and altitude maps obtained by ground-based methods. these models were compared to evaluate whether satellite imagery can accurately generate enm. these comparisons must be made in areas with well-known species distribution and with available satellite imagery and ground-based data. thus, the study area was the south-western part of salamanca (spain), using amphibian and reptiles as species models. models’ discrimination capacity was measured with roc plots. models’ covariation was measured with a spatial spearman correlation. four modelling techniques were used (bioclim, mahalanobis distance, garp and maxent). the results of this comparison showed that there were no significant differences between models generated using remotely sensed imagery or ground-based data. however, the models built with satellite imagery data exhibited a larger diversity of values, probably related to the higher spatial resolution of the satellite imagery. satellite imagery can produce accurate enm, independently of the modelling technique or the dataset used. therefore, biogeographical analysis of species distribution in remote areas can be accurately developed only with variables from satellite imagery. keywords. remote sensing, geographical information system, ecological niche modelling (enm), reptiles, amphibians, spain. 222 neftalí sillero et al. introduction predictive modelling of species distribution is an important tool for conservation biology and biogeography. for instance, it is used to calculate the potential distribution of species (bombi et al., 2009; brito et al., 2009), evaluate the effects of climatic warming on species distribution (araújo et al., 2006), and the suitability of protected areas (garcía, 2006; doko et al., 2011). ecological niche models (hereafter referred to as enm) are produced with environmental variables such as climatic, topographical and habitat data. information about these environmental factors is obtained from a large variety of maps, produced by ground-based methods (climatic data from meteorological stations, altitude from geodesic methods, vegetation maps from analogical aerial photographies) and published in paper or in digital format. we defined ground-based methods as those making field observations, taking in situ measurements and performing land surveying (kerle et al., 2004). these data are frequently available for developed areas of the world (martínez vega, 1996), but may not be available for less developed areas, where particular types of data may not exist. currently, climate surfaces at 1 km2 resolution have been produced for almost all the earth by interpolation of data measured at meteorological stations (hijmans et al., 2005). the variables obtained in that study were precipitation, as well as minimum and maximum temperature. however, data quality across climate surfaces was not consistent because the number of meteorological stations in some areas is scarce (e.g., in the sahara and in siberia, see fig. 1 in hijmans et al., 2005). maximum data uncertainty occurred in mountains, in isolated islands and in poorly sampled areas (hijmans et al., 2005). furthermore, using climatic factors alone might not be sufficient for modelling species distributions. for instance, it has been shown that vegetation data clearly improves the explanatory power of bioclimatic models (thuiller et al., 2004; seoane et al., 2004). therefore, in less developed areas, it may be preferable to carry out fieldwork to collect environmental data. this might not be feasible, however, when fieldwork costs are high, the study area is very large, or there is no time for data collection (muldavin et al., 2001). these constraints hamper the development of enm. an alternative source of eco-geographical data comes from remote sensing platforms. variables obtained from satellite imagery that are traditionally used in predictive modelling include vegetation indexes (such as the normalized difference vegetation index, ndvi), land cover (kerr and ostrovsky, 2003), and land surface temperature (rogers et al., 2002). other variables, such as ground humidity (lavers et al., 1996), are used less frequently. land-cover maps are obtained through a classification process, meanwhile vegetation indexes are obtained through mathematical calculations (chuvieco, 2000). in comparison to data from ground-based maps, satellite imagery has several advantages, notably: 1) a worldwide coverage, allowing investigators to carry local, regional, national or continental studies; 2) several spatial resolutions, ranging from 1 km2 in the advanced very high resolution radiometer (avhrr) to 1 m2 in the ikonos; 3) several temporal resolutions, ranging from 16 days in the case of landsat 5 tm to every day in the case of avhrr; 4) a higher accuracy than ground-based methods, because satellite imagery data are not extrapolated as most climate data are; and 5) a lower cost in comparison with studies where it is necessary to obtain data from fieldwork. for example, many variables, such as altitude data from the shuttle radar topography mission (srtm: www2.jpl.nasa. 223using remote sensing data only for enms gov/srtm/); ndvi data from the avhrr sensor (http://edcsns17.cr.usgs.gov/earthexplorer/); or landsat imagery (www.glovis.usgs.org), are now publicly available at no cost (sillero and tarroso, 2010). for these reasons, an important application for satellite imagery is the biogeographical analysis of species occurring in remote areas, where environmental data do not exist or are not publicly available, especially at high resolutions (sader et al., 1991; martínez vega, 1996). remote areas often correspond to biodiversity hotspots where rare and endemic species are frequently the target of conservation planning (myers et al., 2000). most of the 25 regions recognized as the most important hotspots of biodiversity in the world (see list in myers et al., 2000) are located in less developed countries. nevertheless, there is a general lack of knowledge concerning the accuracy of enm calculated with satellite imagery in comparison with other data sources (thuiller et al., 2004). it is important to evaluate the different environmental data sets available to modellers as well as to advice how to choose them from the available data, but to date this has generally not been well explored in the literature. it has been shown that climatic satellite imagery data can improve enm accuracy when combined with ground-based maps (suarez-seoane et al., 2004). however, there are not consistent results about enm reliability when using satellite imagery only. for instance, similar enm were obtained with land-cover data (venier et al., 2004; using bird species) and with climatic and ndvi data (zimmermann et al., 2007; using tree species), but not with ndvi data only (parra et al., 2004; using bird species also). therefore, other remote sensing data, such as topographical data, should be evaluated and applied to other animal groups (e.g., amphibians, reptiles), where relationships between ground-based maps and satellite imagery variables might be different. moreover, it is very important to study the suitability and accuracy of satellite imagery as a data source in enm because in remote areas satellite imagery is frequently the only data source that is available. the objective of this paper was to analyse how ecological niche models are affected by the type of environmental variables used. can environmental variables obtained from different methods represent in the same way the ecological requirements of the species? therefore, will enms using different sets of variables identify those relationships between the species and the environment? specifically, we want to evaluate if enm developed separately with satellite imagery and ground-based map data can produce similar results. to achieve this, such a comparison must be made in areas where the species distribution is well known and where both satellite imagery and ground-based data are available. the province of salamanca, in central spain, was selected because robust chorological (sillero et al., 2005) and environmental data (león llamazares, 1991; hijmans et al., 2005) are available. amphibians and reptiles were selected as study species because they have a high correlation with environmental factors (soares and brito, 2007; sillero et al., 2009). also, to test the robustness of the statistical approaches and imagery used for predicting species models, the comparisons were performed with two ground-based maps and one satellite imagery dataset and four modelling methods. specifically, the objective was to compare the predictive power and accuracy of four sets of models calculated using satellite imagery data and ground-based map data only, and next to evaluate if satellite imagery variables could be a useful data source to develop reliable enm. 224 neftalí sillero et al. material and methods study area the study area corresponded to the range of a scene of the landsat 5 thematic mapper image, row 203, path 32, of june 23, 1999, on the salamanca province (fig. 1). this part of the province of salamanca, located in central western spain (fig. 1) was selected because it is an area with good information about the distribution of species as well as the eco-geographical characteristics of the region. salamanca is a 12500 km2 plain (12884 raster cells) with an average altitude of 700 to 900 m. to the south, this plain is interrupted by the mountains of the sistema central, ranging from 900 m to 2425 m, and to the north-west by the duero river canyon, ranging from 750 m to 112 m. the climate is mediterranean, with low precipitation (average of 400 mm throughout the year), and summer drought, and with cool temperatures in winter. these general climate characteristics are modified perceptibly by the relief to the south and north-west. the precipitation in the mountain ranges increases with altitude and the average exceeds 700 mm per year. species amphibians and reptiles were selected because they are taxonomic groups that can be modelled more accurately than other animal groups, as they exhibit low mobility and high correlation fig. 1. location of the landsat 5 thematic mapper image in salamanca. location of the salamanca province in the iberian peninsula (map 1) and range of the scene of the landsat 5 thematic mapper image, row 203, path 32, of june 23, 1999, on the salamanca province (map 2). the background is the hillshade model of the srtm dem with a spatial resolution of 100 m.   225using remote sensing data only for enms with environmental factors, such as temperature or precipitation (soares and brito, 2007; sillero et al., 2009). species distribution data were collected from the most recent herpetological atlas of salamanca (sillero et al, 2005), where observations were presented at the level of a 1 km x 1 km utm grid. for modelling purposes, a subset of species was selected according to two criteria: 1) the distribution area was accurately delimited, following opinions from specialists published in herpetological atlases (pleguezuelos et al., 2002; sillero et al., 2005); and 2) the extension of their distributions, because the predictive and the explanatory power of enm might depend on range size. species with restricted ranges may be modelled with a higher accuracy than widespread species, as it is easier to define their ecological niche (stockwell and peterson, 2002; seoane et al., 2005; marmion et al., 2009). using species of different biogeographical affinities (see sillero et al., 2009), the effect of species types in enms’ results is reduced. as in other statistical methods, in order to obtain reliable conclusion on method performances, it is necessary to include all the variation in the independent variables. therefore, four widespread species and four restricted-range species were selected (sillero et al, 2005). the widespread species were (fig. 2): three amphibians, pleurodeles waltl (n = 71 locations), salamandra salamandra (n = 87), bufo calamita (n = 129), and one reptile, natrix maura (n = 83). the restricted species were (fig. 2): one amphibian, rana iberica (n = 41), and three reptiles, acanthodactylus erythrurus (n = 37), lacerta schreiberi (n = 35), podarcis carbonelli (n = 46). in summary, four amphibians and four reptiles were used in the analysis. fig. 2. amphibian and reptile records for species distribution models in salamanca province. amphibians: a, pleurodeles waltl; b, salamandra salamandra; c, bufo calamita; d, rana iberica. reptiles: e, acanthodactylus erythrurus; f, lacerta schreiberi; g, podarcis carbonelli; h, natrix maura. each black square represents a record in a 1x1 km utm square grid.   226 neftalí sillero et al. environmental data sources three data sources were used to calculate ground-based map models: (1) a bioclimatic atlas of salamanca (león llamazares, 1991); (2) the worldclim series (hijmans et al., 2005; http://www. worldclim.org/); and (3) a digital elevation model (dem) built with the contour lines of 1:50000 topographic maps by the junta de castilla y león (spain). vegetation data were not included in ground-based variables as all maps available for the study area were produced by remote sensing techniques (in fact, all vegetation maps are currently performed using satellite imagery). these three data sources were combined in two datasets: grb-l, with variables from león llamazares (1991) and the dem; and grb-w, with variables from worldclim series and the dem. only variables with a correlation lower than ± 0.8 were included in the ground-based map models: grb-l dataset included 12 variables, and grb-w dataset included 7 variables. in the appendix 1 is described the methodology for obtaining and preparing the ground-based map variables (see table a1). satellite imagery was selected from the landsat 5 thematic mapper (tm) and the second version of the shuttle radar topography mission (srtm) adapted to the official spanish reference system (european datum 1950 for spain and portugal; http://topografia.montes.upm.es/informacion/ sig/mde/index.html). these two sensors have a high spatial resolution (30 m and 100 m, respectively) and a high spectral resolution (7 channels: three in the visible spectrum, four in the infrared; and 16 m of vertical precision, respectively) (chuvieco, 2000; rabus et al., 2003). landsat 5 tm classifies vegetation more accurately than other sensors (e.g., systeme pour l’observation de la terre, spot), because it has more channels specifically distributed to map vegetation types (gao, 1999). a total of 14 variables with a correlation lower than ± 0.8 were included in the satellite imagery (si) models (see table a2). the methodology for obtaining and preparing the satellite imagery variables is described in the appendix 1. in order to analyse the similarity among the variables of the three datasets, a spatial principal component analysis (spca) was performed for each dataset. then, the first component of each spca was used to calculate the correlation among the three datasets. therefore, a spatial spearman correlation (ssc) was used to measure this covariation among datasets with arcinfo software 9.2 (band collection statistics tool). it was not possible to calculate the ssc directly with the datasets’ variables due to the different number and types of variables. ecological niche models the activity patterns of species complicate the accurate determination of reliable absence data in a given locality. consequently, ground-based map and satellite imagery models were calculated using four modelling techniques that use presence-only occurrence data, thus calculating the ecological realized niche (sensu sillero, 2011): bioclim (nix, 1986), mahalanobis distance (sokal and rohlf, 1995), garp (stockwell and noble, 1992), and maxent (phillips et al., 2004, 2006). modelling techniques are explained in the appendix 1. in other words, four sets of models were executed for each dataset (grb-l, grb-w and satellite imagery) and for each of the eight species, resulting in a total of 96 models. comparisons of model predictive ability the models were compared in two ways: (1) all models were tested with receiver operated characteristics (roc) plots. the area under the curve (auc) of the roc plot was taken as a measure of the discrimination capacity of the models (liu et al., 2005; elith et al., 2006; vanderwal et al., 2009). comparisons were performed using an anova test for repeated measures when variables or 227using remote sensing data only for enms their logarithmic transformations were normal and homoscedastic; if not, the nonparametric wilcoxon test was used (sokal and rohlf, 1995). (2) a spatial spearman correlation (ssc) was used to measure the covariation among models by pairs of datasets with arcinfo software 9.2 (band collection statistics tool). models from several datasets can obtain high auc values (thus models performed well), but they can be spatially dissimilar: species are then predicted in different places. for this reason, it is necessary to measure how spatially similar they are. the auc values of both species groups (widespread and restricted) were compared using the mann-withney u test. statistical analyses were performed with openstat software (http://www.statpages.org/miller/openstat/). results similarity among datasets the first pair of each spca explained 73.44, 74.46, and 47.12% of the variance of the grb-l, grb-w, and si datasets, respectively. the most similar pair of datasets was grbw/si (ssc = 0.58), followed by the pair grb-l/si (ssc = -0.37). therefore, the less similar pair was grb-w/grb-l (ssc = -0.33). area under the curve examples of enm for two species are presented in fig. 3. auc values were higher than 0.5 in all the sets of models, i.e. results were not achieved by chance alone (fig. 4). only in three cases, auc value was lower than 0.8 (bioclim grb-l and si; and garp grb-l). in the garp and maxent models, natrix maura was always the species with the lowest auc value meanwhile rana iberica and lacerta schreiberi were the species with the highest values, in four and two datasets, respectively (fig. 4). in the bioclim and mahalanobis models, this pattern is not evident. considering each modelling method independently, there were highly significant differences in auc values among the three datasets (table 1 and fig. 4), in the case of bioclim (wilcoxon test; t = 2.366; p < 0.01; n = 24; df = 24) and garp (wilcoxon test; t = 1.96; p = 0.025; n = 24; df = 7). auc values from satellite imagery (si) dataset (bioclim, si mean = 0.836 ± 0.080; garp, si mean = 0.869 ± 0.084) were higher than the other two datasets (bioclim, grb-l mean = 0.669 ± 0.173; grb-w mean = 0.785 ± 0.072; garp, grb-l mean = 0.796 ± 0.114; grb-w mean = 0.801 ± 0.057). the differences in auc values were not significant when comparing the other two modelling methods: mahalanobis distance (anova for repeated measures: n = 24; df = 20; f = 1.50; p = 0.262); and maxent (wilcoxon test: n = 24; df = 7; t = 1.153; p = 0.124). there was not a clear pattern when comparing both groups of species (widespread vs. restricted); only in four cases (bioclim grb-l and grb-w; mahalanobis grb-w; and maxent si), auc values were higher in the restricted species (table 2 and fig. 4). spatial spearman correlations spatial spearman correlations values were in generally low; only few pairs were higher than 0.5, namely for the bioclim, garp and maxent models (fig. 5). here again, 228 neftalí sillero et al. fig. 3. examples of species distribution models. the species are rana iberica (first column of maps) and acanthodactylus erythrurus (second column of maps). the first row corresponds to bioclim models, the second to mahalanobis models, the third to garp models and the fourth to maxent models. inside each row, maps correspond to grb-l, grb-w and satellite imagery dataset, from left to right, respectively. grey scale stands for habitat suitability values with darker tones corresponding to maximum values.   229using remote sensing data only for enms table 1. mean and standard deviation (sd) of auc (area under the roc curve) values for the four modelling methods. variables from two datasets of ground-based maps (grb-l and grb-w) and one of satellite imagery (si) were used to calculate the species distribution models. see the text and appendix 1 for details. *: there was no solution in the mahalanobis model of l. schreiberi for the dataset grb-l. therefore, the auc mean did not include this model. method dataset mean sd bioclim grb-l 0.669 0.173 grb-w 0.785 0.072 si 0.836 0.080 mahalanobis grb-l* 0.986 0.010 grb-w 0.995 0.008 si 0.985 0.021 garp grb-l 0.796 0.114 grb-w 0.801 0.057 si 0.860 0.084 maxent grb-l 0.864 0.073 grb-w 0.830 0.074 si 0.874 0.058 fig. 4. auc values of species distribution models. models were calculated with two datasets of groundbased maps (grb-l and grb-w) and one of satellite imagery (si). ae: acanthodactylus erythrurus; bc, bufo calamita; ls: lacerta schreiberi; nm, natrix maura; pc, podarcis carbonelli; pw: pleurodeles waltl; rib, rana iberica; ss: salamandra salamandra. there was no solution in the mahalanobis model of l. schreiberi for the dataset grb-l.   230 neftalí sillero et al. table 2. results from mann-whitney u test (the first line corresponded to z statistics and the second line to the p value). auc (area under the roc curve) and spatial spearman correlations (ssc) values were compared between two species groups (widespread and restricted) for the four modelling methods. the widespread species were: pleurodeles waltl, salamandra salamandra, bufo calamita, natrix maura; the restricted species were: rana iberica, acanthodactylus erythrurus, lacerta schreiberi, podarcis carbonelli. variables from two datasets of ground-based maps (grb-l and grb-w) and one of satellite imagery (si) were used to calculate the species distribution models. see the text and appendix 1 for details. there was no solution in the mahalanobis model of l. schreiberi for the dataset grb-l. therefore, auc and ssc value was not included this model. method auc ssc grb-l grb-w si grb-l/si grb-l/grb-w grb-w/si bioclim 2.31 0.01 (**) 2.02 0.02 (**) 1.44 0.07 (ns) 1.15 0.12 (ns) 0.72 0.24 (ns) 0.29 0.39 (ns) mahalanobis 0.35 0.36 (ns) 1.73 0.04 (*) 0.29 0.39 (ns) 1.3 0.1 (ns) 2.12 0.02 (ns) 1.24 0.11 (ns) garp 1.44 0.07 (ns) 1.44 0.07 (ns) 1.59 0.06 (ns) 1.44 0.07 (ns) 1.15 0.12 (ns) 1.01 0.16 (ns) maxent 1.44 0.07 (ns) 1.44 0.07 (ns) 1.73 0.04 (*) 0.87 0.19 (ns) 1.15 0.12 (ns) 0.58 0.28 (ns) **: highly significant; *: significant; ns: non significant. table 3: mean and standard deviation (sd) of spatial spearman correlations (ssc) for the four modelling methods. variables from two datasets of ground-based maps (grb-l and grb-w) and one of satellite imagery (si) were used to calculate the species distribution models. see the text and appendix 1 for details. *: there was no solution in the mahalanobis model of l. schreiberi for the dataset grb-l. therefore, ssc were not calculated for the pair combinations with this model. method dataset mean sd bioclim grb-l/si 0.29 0.17 grb-w/grb-l 0.32 0.19 si/grb-w 0.20 0.10 mahalanobis grb-l/si* 0.08 0.10 grb-w/grb-l* 0.34 0.10 si/grb-w 0.43 0.10 garp grb-l/si 0.33 0.24 grb-w/grb-l 0.23 0.29 si/grb-w 0.29 0.12 maxent grb-l/si 0.49 0.20 grb-w/grb-l 0.60 0.11 si/grb-w 0.47 0.16 **: highly significant; *: significant; ns: non significant. 231using remote sensing data only for enms rana iberica and lacerta schreiberi were the species with the highest values when using the garp and maxent models; however, these two species had the lowest values using the bioclim model. in the mahalanobis models, natrix maura had the lowest values in two comparisons. there was no solution in the mahalanobis model of l. schreiberi for the dataset grb-l; therefore, this model was not included in the subsequent analysis. there were significant differences among dataset pairs of comparisons using the mahalanobis (wilcoxon test: n = 24; df = 7; t = 2.366; p = 0.009) and maxent models (wilcoxon test: n = 24; df = 7; t = 1.820; p = 0.034; table 3 and figure 5). ssc were higher when comparing the pair of datasets grb-l and grb-w than the other two pairs, in mahalanobis models, and when comparing the pair of datasets grb-w and si, in maxent models. for the other two sets of models, there were no significant differences among pairs of comparisons: bioclim (wilcoxon test: n = 24; df = 7; t = 0.700; p = 0.242; table 3); garp (wilcoxon test: n = 24; df = 7; t = 0.700; p = 0.242; table 3). also, there was no significant difference among ssc values of widespread and restricted species groups (table 2 and fig. 5). discussion the high accuracies obtained in almost all models based on satellite imagery and ground-based datasets indicated that, in general, all models performed reliably and are robust. the predictive power of the four sets of modelling methods was similar, with the fig. 5. spatial spearman correlation (ssc) values of species distribution models. ssc were calculated comparing pairs of different dataset models. ae: acanthodactylus erythrurus; bc, bufo calamita; ls: lacerta schreiberi; nm, natrix maura; pc, podarcis carbonelli; pw: pleurodeles waltl; rib, rana iberica; ss: salamandra salamandra. there was no solution in the mahalanobis model of l. schreiberi for the dataset grb-l.   232 neftalí sillero et al. auc values and spatial spearman correlations (ssc) not being significantly different in almost all cases. when there were significant differences, (bioclim and garp models), it was the satellite imagery dataset which obtained higher auc values. probably, this result is not due only to the dataset itself, but also may be produced by the modelling technique employed: bioclim and garp have a lower accuracy compared with other models (elith et al., 2006); and the dataset grb-l is probably the less accurate as it results from data interpolation. therefore, both methods together with the dataset may produce worst results. the lower accuracy of grb-l is confirmed by the ssc of spca results: si dataset is more similar to the other two datasets, than those datasets (grb-l and grb-w) between them. for the mahalanobis and maxent models, the ssc was higher when comparing the pair of datasets grb-l/grb-w and grb-w/si. as confirmed by the results of the spca ssc, dataset si is a good surrogate of grb-w. however, the high ssc between the models of the datasets grb-l and grb-w is not concordant: the value should be lower as they are the most dissimilar datasets. however, the p-value for maxent ssc comparisons was very low. in some models (bioclim, mahalanobis, and maxent; see table 2), species with a restricted range had high auc values, while widely dispersed species had lower auc values, suggesting that species with a restricted range could be more accurately modelled than widely dispersed species. on the other hand, in presence only models, the maximum achievable auc is lower in widespread species (phillips et al., 2006). this might further affect the auc differences between widespread and restricted range species. nevertheless, ssc values were similar for both groups of species. although this result had been confirmed clearly in earlier studies (e.g., stockwell and peterson, 2002; seoane et al., 2005; vanderwal et al., 2009), our results do not allow to assess the same conclusion. restricted species are modelled with a larger accuracy because their ecological niches have a smaller geographical extension, and therefore they are more easily identified (see vanderwal et al., 2009). hence, higher ssc values among restricted species should be expected in comparison with widespread species. in other studies, enm were improved when climate data from satellite imagery were combined with habitat variables (suarez-seoane et al., 2004). also, the incorporation of land cover data improved the explanatory power of enm calculated only with groundbased variables, although it did not improve the predictive power (thuiller et al., 2004). these results confirmed that satellite imagery data improve model results when added to ground-based data. however, enm calculated separately or in combinations with regional climate surfaces and from land cover data also had similar accuracy (venier et al., 2004). model performance was also similar when ndvi variables were combined with climatic and topographical datasets using ground-based data. zimmermann et al. (2007) found that ground-based variables outperform the satellite imagery predictors, although models calculated with only satellite imagery data provided high model accuracies, due to the correlation between climate and satellite imagery variables. our findings support results by vernier et al. (2004) and zimmermann et al. (2007), although these studies used only two datasets and one modelling technique. in another study (parra et al., 2004), models performed relatively well with two ground-based datasets (one with only climatic variables and other with only topographical data) and poorly with ndvi variables (used as unique data source, for deriving several variables). probably, these partial datasets were not able to define completely the species’ ecological niche. therefore, results could depend on the 233using remote sensing data only for enms technique or dataset implemented. in fact, bioclim was considered as the main factor for general low model performance (parra et al., 2004). as here, the aim of parra et al. (2004) was not to test among modelling methods but to test among environmental datasets. our results showed that satellite imagery data can produce enm of acceptable accuracy, independently of the modelling technique or the dataset used. this is very important when the study area is in a remote region, or when spatial resolution of the groundbased layers is coarse, as satellite imagery may be the only feasible source of data (martínez vega, 1996; sillero and tarroso, 2010). in fact, remote sensing data have been applied for many years in epidemiological studies (hay et al., 1996), particularly in tropical areas, where it is very difficult to measure environmental variables that require continuous monitoring (rogers et al., 2002). for example, accurate data on altitude (lower than 1 km2) is produced currently by radar images (e.g., srtm and aster gdem; see sillero and tarroso, 2010). moreover, for some countries (e.g., mauritania) digital topographical maps are not available. a source for altitude data is radar images. climatic data come from interpolation of weather stations, but in areas without such stations like the sahara desert, the interpolated results can be very inaccurate (see hijmans et al., 2005). using again the example of mauritania, all vegetation maps have been produced by satellite imagery data. however, works by parra et al. (2004), vernier et al. (2004) and zimmermann et al. (2007) did not compare the satellite imagery models with models calculated exclusively with ground-based data, to measure which type of variables produced more accurate enm. according to the present study, biogeographical analysis of species distribution in remote areas can be accurately calculated using variables from satellite imagery only. small differences may be found when modelling other species, namely in groups with low environmental dependency (e.g., birds, mammals). everything will depend on the capacity of remote sensing data in explaining the habitat species’ requirements. as satellite imagery (e.g., ndvi) observe the actual spatial variation (han et al., 2004), satellite imagery can describe the environment more accurately than ground-based data sources. satellite imagery is produced with higher spatial and spectral resolutions than conventional field methods used to produce ground-based data (kerr and ostrovsky, 2003; turner et al., 2003). this supposes a larger diversity of values in the variables obtained from the satellite imagery than those derived from the ground-based data. this is proved by the low proportion of explained variance presented by the first component of the pca analysis of the si dataset. whereas a climatic map obtained by interpolation shows only few values inside a particular area, satellite imagery may capture subtle differences in either the soil or the vegetation characteristics. for instance, an artificial grass field is not distinguished from a natural one by conventional aerial photography, but the infra-red image is able to detect the chlorophylical activity of the natural grass field (chuvieco, 2000). when using satellite imagery, two adjacent grass fields, one artificial and the other natural, therefore would be identified as two different structures. in an enm, the habitat suitability index of the two fields probably would be the same if satellite imagery was not used. this is also applicable to adjacent micro-habitats. furthermore, climate variables alone cannot distinguish possible reflectance differences between early and late plant species. therefore, in some situations, areas with different habitat suitability indexes can be detected more accurately with satellite imagery, resulting in more accurate enm (suarez-seoane et al., 2004). an example of this could be an enm of deciduous broad234 neftalí sillero et al. leaved trees, as multi-temporal imagery can distinct phenology compared to evergreen needle-leaf trees (zimmermann et al., 2007). satellite imagery has also disadvantages or short comings. the temporal record of satellite imagery is recent and short (chuvieco, 2000). thus, our capacity to perform historical studies with satellite imagery is lower than with interpolated-climate data (hijmans et al., 2005). moreover, one of the most important programmes of acquiring satellite imagery (landsat) is not completely functional any more (both landsat 5 and 7 are not working properly). other disadvantage of satellite imagery is the relatively low number of environmental variables that sensors can provide (sillero and tarroso, 2010). manual photo-interpretation can obtain more exact products than automatic image classification algorithms (although there are examples proving the opposite, such as the first edition of corine project for spain; pers. obs.). despite these disadvantages, an important advantage of satellite imagery data over ground-based data is its capacity to produce temporal series of information from the same area (hay et al., 1998; frança and setzer, 1998; azzali and menenti, 2000; lucas et al., 2000a; lucas et al., 2000b; sobrino, 2000; green and hay, 2002). therefore, it is possible to study the same ecological processes over time, such as habitat monitoring (kobayashi and dye, 2005), land cover mapping (stow et al., 2006) and climate change detection (roerink et al., 2003). future work should include more complete temporal series of climatological data, land cover and vegetation productivity. acknowledgements we would like to prof. j. martins for checking the english. ns has a post-doctoral grant (sfrh/bpd/26666/2006) from fundação para a ciência e tecnologia (fct, portugal) and jcb has a contract (programme ciência 2007) from fct. supplementary material supplementary material associated with this article can be found at: < http://www.unipv.it/ webshi/appendix > manuscript number 9891: appendix 1. references araujo, m.b., thuiller, w., pearson, r.g. 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(2004): climate and satellite-derived land cover for predicting breeding bird distribution in the great lakes basin. j. biogeogr. 31: 315-331. zimmermann, n.e., edwards, t.c., moisen, g.g., frescino, t.s., blackard, j.a. (2007): remote sensing-based predictors improve distribution models of rare, early successional and broadleaf tree species in utah. j. appl. ecol. 44: 1057-1067. acta herpetologica vol. 7, n. 2 december 2012 firenze university press advertisement call of species of the genus frostius cannatella 1986 (anura: bufonidae) flora a. juncá1, david l. röhr2, ricardo lourenço-de-moraes3, flávio j. m. santos1, airan s. protázio1, ednei a. mercês1, mirco solé4 amphibians in southern apennine: distribution, ecology and conservation notes in the “appennino lucano, val d’agri e lagonegrese” national park (southern italy). antonio romano1,*, remo bartolomei1, antonio luca conte1, egidio fulco2 the significance of using satellite imagery data only in ecological niche modelling of iberian herps neftalí sillero1, josé c. brito2, santiago martín-alfageme3, eduardo garcía-meléndez4, a.g. toxopeus5, andrew skidmore5 reproductive strategy of male and female eastern spiny lizards sceloporus spinosus (squamata: phrynosomatidae) from a region of the chihuahuan desert, méxico aurelio ramírez-bautista1,*, barry p. stephenson2, xóchitl hernández-ibarra1, uriel hernández-salinas1, raciel cruz-elizalde1, abraham lozano1, and geoffrey r. smith3 morphological variability of the hermann’s tortoise (testudo hermanni) in the central balkans katarina ljubisavljević1, georg džukić1, tanja d. vukov1, miloš l. kalezić1,2 the usefulness of mesocosms for ecotoxicity testing with lacertid lizards maria josé amaral1,2,*, rita c. bicho1, miguel a. carretero2, juan c. sanchez-hernandez3, augusto m. r. faustino4, amadeu m. v. m. soares1, reinier m. mann1,5 does acclimation at higher temperatures affect the locomotor performance of one of the southernmost reptiles in the world? jimena b. fernández*, nora r. ibargüengoytía advertisement call of scinax littoralis and s. angrensis (amphibia: anura: hylidae), with notes on the reproductive activity of s. littoralis michel v. garey1, thais r. n. costa2, andré m. x. de lima2, luís f. toledo3, marília t. hartmann4 book review: marine s. arakelyan, felix d. danielyan, claudia corti, roberto sindaco, alan e. leviton 2011. herpetofauna of armenia and nagorno-karabakh. society for the study of amphibians and reptiles stefano scali book review: rafaqat masroor 2012. a contribution to the herpetofauna of northern pakistan stefano scali evidence of high longevity in an island lacertid, teira dugesii (milne-edwards, 1829). first data on wild specimens. j. jesus first assessment of the endoparasitic nematode fauna of four psammophilous species of tropiduridae (squamata: iguania) endemic to north-eastern brazil markus lambertz1,*, tiana kohlsdorf2, steven f. perry1, robson waldemar ávila3, reinaldo josé da silva4 rediscovery and redescription of the holotype of lygosoma vittigerum (= lipinia vittigera) boulenger, 1894 yannick bucklitsch1, peter geissler1, timo hartmann1, giuliano doria2 , andré koch1,* reproductive phenology of the tomato frog, dyscophus antongili, in an urban pond of madagascar’s east coast ori segev1,*, franco andreone2, roberta pala2, giulia tessa2, miguel vences1 range extension of the critically endangered true poison-dart frog, phyllobates terribilis (anura: dendrobatidae), in western colombia roberto márquez1,*, germán corredor2, carlos galvis3 daniel góez2, & adolfo amézquita1 differences in habitat use of two sympatric species of ameiva in east costa rica esther sebastián-gonzález1, ramón gómez2 acta herpetologica journal of the societas herpetologica italica © firenze university press www.fupress.com/ah acta herpetologica 5(2): 245-253, 2010 incomplete albinism in discoglossus pictus (otth, 1837) filippo spadola1, gianni insacco2 1 laboratorio di metodologie veterinarie applicate alla fauna esotica e selvatica, dipartimento di scienze sperimentali e biotecnologie applicate, facoltà di medicina veterinaria di messina, polo universitario ss. annunziata i-98128, messina, italy. corresponding author. e-mail: fspadola@unime.it 2 centro regionale recupero fauna selvatica e tartarughe marine fondo siciliano per la natura e museo civico di storia naturale, via generale girlando 2, i-97013 comiso (rg), italy. e-mail: gianniinsacco@virgilio.it submitted on: 2009, 31th december; revised on: 2010, 26th august; accepted on: 2010, 20th september. abstract. the authors present an incomplete albinism case in a discoglossus pictus subject found in sicily. this is the first note for italian territory, the second for the species and the third for discoglossus genus. keywords. discoglossus pictus, albinism, alitidae, discoglossidae, amphibians. discoglossus pictus belongs to the alitidae family, previously called discoglossidae (lanza et al., 2007, 2009). two genera belong to alitidae: alytes (present in morocco, iberian peninsula, france, transalpine switzerland, southern belgium, southern netherlands and western germany) and discoglossus (distributed in maghreb africa, southern europe and israel) (table 1). discoglossus pictus is a western mediterranean species, distributed in sicily, malta and gozo island, tunis, including galita island, northern algeria. it is also a naturalized species in northern spain and southern france (lanza et al., 2007, 2009). discoglossus pictus presents three different dorsal colourations: a “spotted” phenotype with brown, brown-greenish, brown-yellowish or greenish stains on brown-reddish or brown-yellowish background, a “striped” phenotype characterized by a median band and two lateral bright bands, coupled with numerous spots similar to the previous phenotype, and a third “concolor” phenotype, brown, brown-reddish or reddish normally uniform or with small and shed dark sketches (lanza et al., 2007, 2009). as a premise, it should be remembered that albinism is due to a blockade of melanophores metabolic pathway leading to melanine synthesis (lanza et al., 2007, 2009). if metabolic alteration impacts skin and eye melanophores, complete albinism occurs; incomplete albinism occurs when metabolic alteration impacts only skin or eye melanophores (lanza et al., 2007, 2009). partial albinism, instead, consists of the presence of achromic areas in more or less wide and more or less numerous portions of the skin (lanza et al., 2007, 2009). 246 f. spadola and g. insacco in amphibians there are a number of reports of complete and incomplete albinism. thus it has been mandatory to concentrate bibliographic research on albinism cases in alitidae family (table 1). for discoglossus genus, albinism records are extremely rare. for instance, in 1879, lataste claims a lack of albino discoglossus pictus to complete its researches on tadpoles pigmentation (lataste, 1879). in the month of june 2009, in ragusa province (italy), precisely in marina di ragusa, few meters over the sea level and approximately 500 meters from the sea, an albino discoglossus pictus individual was found. the animal was in a small lake rich in nymphaea alba, in a big garden of a private owner. in the same area many subjects of the same species were present, all belonging to the spotted phenotype (fig. 2 c). studied subject jumped among other presenting a uniform white skin coloration with black eyes (fig. 1; fig. 2 a, b). it was a sub-adult (5 cm body length), incomplete albino individual, extremetable 1. summary of species of the family of alitidae and reports of albinism. fig. 1. discoglossus pictus with albinism incomplete. left view. 247incomplete albinism in discoglossus pictus ly vital and in apparently good health status (fig. 1; fig. 2 a, b). dimension and actual lack of secondary sexual characters did not allow us to determine gender, but we expect it was a female. the animal is still alive and it is the first case of discoglossus pictus incomplete albino on the italian territory. acknowledgements we wish to thank: pasquale spadola, salvatore viola, edoardo razzetti, annalisa zaccaroni, rafael marquez and iñigo martinez-solano for their cooperation. references benavides, j., viedma, a., clivilles, j., ortiz, a., gutiérrez, j.m. (2000): albinismo en alytes dickhilleni y salamandra salamandra en la sierra de castril (granada). bol. asoc. herpetol. esp. 11: 83. fig. 2. (a) right view; (b) dorsal view; (c) other discoglossus pictus “spotted” phenotype with the albino (photo by viola s.). 248 f. spadola and g. insacco boulenger, g.a. (1897): the tailless batrachians of europe. part i. ray society, london. capanna, e. (1968): osservazioni sul semialbinismo degli anfibi. boll. zool. 34: 100-101. capanna, e. (1969): albinismo parziale in una popolazione insulare di discoglossus sardus tschudi. boll. zool. 36: 135-141. diego-rasilla, f.j., luengo, r.m. (2007): varios casos de albinismo en alytes obstetricans (laurenti, 1768). bol. asoc. herpetol. esp. 18: 92. garcía-parís, m., montori, a., herrero, p. (2004): amphibia. lissamphibia. en: fauna iberica. vol. 24. ramos sánchez, m.a., et al. eds, museo nacional de ciencias naturales, madrid. gilboa, i., dowling, h.g. (1974): a bibliography on albinism in amphibians and reptiles, 1849-1972. a bibliographic service by herpetological information search systems, american museum of natural history, new york, publ. herpetol. 6: 3-11. héron-royer, b. (1878): recherches sur la fécondité des batraciens anoures alytes obstetricans, hyla arborea, et sur la fécondation des oeufs du bufo bufo dans l’obscurité. bull. soc. zool. france 3: 278-285. héron-royer, b. (1886): sur la reproduction de l’albinisme par voie héréditaire chez l’alyte accoucheur et sur l’accouplement de ce batracien. bull. soc. zool. france 11: 671679. lanza, b., andreone, f., bologna m.a., corti, c., razzetti, e. (2007): fauna d’italia. vol. xlii, amphibia, calderini, bologna. lanza, b., nistri, a., vanni, s., (2009): anfibi d’italia. ministero dell’ambiente e della tutela del territorio e del mare, ispra grandi & grandi editori, savignano sul panaro (mo). lataste, f. (1879): étude sur le discoglossus pictus otth. actes soc. l. bord. 4: 275-341. márquez, r. (2009): sapo partero ibérico – alytes cisternasii. en: enciclopedia virtual de los vertebrados españoles. salvador, a., ed, museo nacional de ciencias naturales, madrid. http://www.vertebradosibericos.org/. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 8(2): 177-178, 2013 book review: bo beolens, michael watkins, michael grayson. the eponym dictionary of amphibians edoardo razzetti museo di storia naturale, università degli studi di pavia, piazza botta 9/10, i-27100 pavia. e-mail: razzetti@unipv.it carolus linnaeus, the scientist who laid the foundations for the modern biological naming scheme passed away over 200 years ago but still new species are discovered every day and even amphibians, a rather small group of animals, had over 160 new species described last year (2012). describing new species is funny because it is one of the few instances in science where you can leave a permanent record and, at the same time, express yourself without too many constraints. zoologists, explorers, collectors and sometimes relatives, lovers, and friends of the taxonomist often had the honour to have their name latinised and used to name a previously unknown species. this is considered a great privilege as the scientific names can perpetuate the memory of a person for hundreds of years. the book “the eponym dictionary of amphibians” deals exactly with this subject: it provides information about scientific and common names that contain a person’s name. so if you consider an amphibian species as hydromantes strinatii (aellen, 1958) you will find a biography of the swiss biospeleologist pierre strinati to whom the species is dedicated (but not of villy aellen who described it because the describer is not considered an eponym). for each entry there is a list of amphibians named after the person, a short biography that ranges from a few words for persons that contributed only marginally (or not at all!) to science and culture in general, to over 25 lines for famous zoologists. since this book is organized as a dictionary it is not illustrated except for the nice front cover; the references are limited to a general list of books and journals, this latter could not be a problem in the internet age as a quick web search with carefully selected word usually delivers a wealth of information. names are arranged alphabetically and so, when needed, it is easy to check for a surname and obtain the information you are looking for. i tried to look for the societas herpetologica italica members and i found few people listed: franco andreone, emilio balletto, benedetto lanza for whom there are complete and correct data. i then tried to check if all the eponyms of italian amphibians were included and again i was amazed by completeness and the accuracy of the biography presented. i was a little surprised by the absence of paolo savi that is related to the italian common name of salamandrina perspicillata but then i realized that the most used english common name for this species is northern spectacled salamander, thus it does not include the word “savi”. this poses some questions about the choice of common names that, especially for amphibians and reptiles, are often controversial so that for some species a plethora of names is available. is this book really complete and up to date? i had the opportunity to keep it at hand on my desk for nearly three months, i checked it often and i am quite sure that the book is the result of a long and careful work; with 2668 eponyms listed to honour 1609 names i am fairly sure that is almost complete. even species recently described as lyciasalamandra yehudahi 2012 dedicated to the father of middle east herpetology yehudah werner were included. this book will unfortunately not going to be a bestseller as there are not so many people that are eager to know that uraeotyphlus oommeni is dedicated to the indian zoologist oommen v. oommen, but anyway i found it a very useful tool, especially when the eponym you are looking for is not so famous or when the name honoured is a living zoologist. 178 book review as a final note, i would like to add that the tireless authors of this book already published few other eponym dictionaries dedicated to reptiles and mammals that you’ll find listed among the references (while the ones dedicated to shark and rays, and to birds are in press). there are still many species on the planet that are waiting to be described and named, this is the reason why i sincerely hope that all these books will be updated on a regular basis, maybe taking in account also fossil species so that eobarbourula delfinoi the eponym of the president of our societas herpetologica italica could be listed. the book can be purchased at the cost of €43.99 from the publisher’s website (http://www.pelagicpublishing.com, isbn 978-1-907807-41-1). references beolens, b., watkins, m., grayson, m. (2009): the eponym dictionary of mammals. johns hopkins university press, baltimore. beolens, b., watkins, m., grayson, m. (2011): the eponym dictionary of reptiles. johns hopkins university press, baltimore. beolens, b., watkins, m., grayson, m. (2013): the eponym dictionary of amphibians. pelagic publishing, exeter. acta herpetologica vol. 8, n. 1 june 2013 firenze university press journal of the societas herpetologica italica acta herpetologica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 8(2): 159-162, 2013 a revised geographical range for liolaemus elongatus koslowsky, 1896 (squamata: liolaemini) in argentina: review of reported and new-data based distribution with new localities ignacio minoli1, cintia d. medina1, nicolás frutos2, mariana morando1, luciano j. avila1,* 1 grupo de herpetología patagónica, cenpat-conicet, boul. almt. g. brown 2915, u9120acd, puerto madryn, chubut, argentina. * corresponding author. e-mail: avila@cenpat.edu.ar 2cátedra fundamentos básicos de cartografía e introducción a sig – facultad de filosofía y humanidades – universidad nacional de córdoba. ciudad universitaria. córdoba, argentina submitted on 2013, 25th april; revised on 2013, 6th june; accepted on 2013, 3rd october. abstract. estimating the effective geographical ranges of species is central to species-oriented conservation and management. in this paper, we review the geographical distribution of liolaemus elongatus koslowsky, 1896 with three new records for northern chubut and southern río negro provinces, argentina. based on detailed locality records pooled from multiple data sources, including new records obtained for this study, we revise the range of l. elongatus sensu stricto and provide geographical distribution maps comparing the previously recognized range to that proposed herein. our results show that l. elongatus possesses a much more limited geographic distribution than previously thought, being restricted to areas south of 38°s latitude; the newly proposed range is merely half the species formerly recognized geographical distribution. keywords. lizards, geographical range size, liolaemus, biogeography, patagonia, argentina. the accurate estimation of the geographical range of a species is central to species-oriented conservation policy. nevertheless, species occurrence data are often biased, due to their opportunistic origin (newbold, 2010): in fact, they tend to be pooled around specific areas depending on the nature of collecting campaigns and/ or the accessibility of sampled areas (katzner et al., 2011; feeley and silman, 2011). in this general context, when widespread and diverse taxa are involved (e.g., liolaemidae; pincheira-donoso et al., 2008), the combination of a large sampling area with the uncertainty of taxonomic assignments may lead to possible overestimation or underestimation of the real distribution of the species under investigation, with obvious drawbacks, not only from the conservation point of view. the genus liolaemus may be a good example of such a problem. liolaemus is a south american genus of lizards with more than 230 described species (breitman et al., 2011; abdala et al., 2012), some of which are widely distributed in argentina. whereas recent work has resulted in changes in the systematic status of several liolaemus species (e.g., abdala et al., 2012; avila et al., 2012), herpetologists have not adequately updated species geographical distributions. one such lizard is liolaemus elongatus koslowsky (1896), a viviparous, insectivorous, medium-sized lizard (85 mm maximum snout-vent length, svl) which typically inhabits rocky outcrops of western patagonia steppe environments (cei, 1986). liolaemus elongatus was originally described from rocky outcrops of western chubut (“…el territorio del chubut, cerca de las cordilleras, donde vive en las grietas de las rocas…”; koslowsky, 1896). since the 1970s, this species has been considered widely distributed over patagonia, reaching northwestern areas of argentina including altoandina 160 ignacio minoli et al. environments (cei, 1974, 1986; avila and lobo, 1999; avila et al., 2000). phylogeographical analyses showed that southernmost populations from the agrio river basin (neuquén province) are genetically clustered and spatially separated from northern populations (morando et al., 2003). based on mitochondrial data, some northern l. elongatus populations appear more closely related to the liolaemus petrophilus complex (morando et al., 2003), while others, although being closely related to l. elongatus, maintain some degree of difference (medina pers. comm.). several of the northernmost populations considered by morando et al. (2003) to represent new candidate species were later described as new species (e.g., l. parvus and l. tulkas; quinteros, et al., 2008; l. smaug and l. choique; abdala et al., 2012; l. burmeisteri; avila et al., 2012). however, the knowledge of the geographical distribution of l. elongatus and related taxa remains fragmented and a detailed review of the geographical range of these species is lacking. this, along with other unknown aspects of the natural history of liolaemus elongatus, led avila et al. (2000) to consider its conservation status as “insufficiently known”. here, we evaluate the reported distribution of l. elongatus by reviewing all populations previously allocated to this species and comparing them with new geographical records of populations allocated to l. elongatus sensu stricto (morando et al., 2003; medina pers. comm.) in the río negro, chubut and southern neuquén provinces. between february 1998 and march 2010, we collected specimens of liolaemus elongatus by hand, snooze or forked stick, following visual survey from a vehicle along unpaved roads or walking transects. we recorded latitude, longitude and elevation for each locality, as determined by a garmin gps 12™ global position device. taxonomic identity was established for each collected specimen based on morphological analyses with a classical morphological approach (using scale counts, morphometry, and color pattern), taking into consideration the original species description of liolaemus elongatus koslowsky 1896 and comparison with syntypes deposited in museo de la plata collection (ferraro and williams, 2006). additionally, results of phylogeographic studies based on three mitochondrial genes (morando et al., 2003) and two mitochondrial and four nuclear genes (medina pers. comm.) were also considered for taxa identification. after capture, lizards were euthanized by a pericardial injection of sodium thiopenthotal pentovet®, fixed in 10-20% formalin and later transferred to 70% ethanol (simmons, 2002). voucher specimens from these collections were deposited in the herpetological collection (ljamm-cnp; http://www.cenpat.edu.ar/colecciones03.html) located in centro nacional patagónico (cenpat-conicet), puerto madryn, argentina. we followed animal handling procedures suggested by simmons (2002) and in agreement with regulations detailed in the argentinean national law #14346. each provincial fauna authority regulates collection permits, which are included in acknowledgments. we made updated geographical range maps based on ljamm-cnp collections records and a review of museum and literature data associated with vouchered specimens, in order to compare our new records with the distribution previously reported for this species. using the results of phylogeographic and phylogenetic studies (morando et al., 2003; avila et al., 2004; medina pers. comm.), morphological comparisons of sampled specimens with syntypes, and results of ongoing morphological studies to establish species limits (medina, pers. comm.), we considered as liolaemus elongatus sensu stricto all records located south of 38°s latitude. we georeferenced all records for l. elongatus obtained from published studies without geographical coordinates, but with an accurate locality description. all records in the resulting database were mapped using the program gvsig® version 1.11. from our review of published literature and museum data, we obtained 202 records distributed in 70 localities of liolaemus elongatus (fig. 1a; suppl. mat. table t1 and appendix a1). this set of data points supported a wide geographical range for this species, spanning from southern catamarca province southward along a narrow strip limited to the andes mountains of neuquén province, through río negro, and continuing south of chubut province (cei, 1974, 1986; avila and lobo, 1999; scolaro, 2005). the database obtained from our collection campaigns, consisted on 294 records from 75 localities (fig. 1b; suppl. mat. table t1 and appendix a1). three localities included in the 25 de mayo department (río negro province) and telsen department (chubut province) represent new geographic records; in detail: five specimens (ljamm-cnp 6227-6228, 6235-6237) from provincial route 5, 22 km nw el cain (41°35’48.9” s, 68°22’11.3” w; 1165 m a.s.l.), 25 de mayo department; three individuals (ljamm-cnp 10974-6) from provincial route 67, 11.2 km s río negro-chubut border (42°04’34.45” s, 68°09’43.11” w; 1407 m a.s.l.); one specimen captured (ljamm-cnp 7514) from provincial route 67, on the road to talagapa, 53.1 km n of gan gan city (42º13’50.8” s, 68º14’23.8” w; 1402 m a.s.l.), telsen department. by reviewing distributional data available for liolaemus elongatus from previous systematic studies, and adding localities from recent phylogeography studies as well as new records from recent field collections, our results 161liolaemus elongatus geographical range allows redefining the geographical distribution of l. elongatus in argentina. the geographical range assigned by previous publications for this species covers a much broader area than that supported by our data. since the 1970s, l. elongatus was thought to range from catamarca province (avila and lobo, 1999; avila et al., 2000) to southern chubut province (cei 1974, 1986). in contrast, our results show that the current distribution of l. elongatus sensu stricto ranges from south of agrio river basin (neuquén) to southern chubut province and includes several new sites providing a more detailed description of the distribution of the species within neuquén, río negro and chubut (fig. 1b). the updated range is only one-half of the species formerly recognized geographical distribution. this discrepancy is partly due to the taxonomic review of the species assignments, and partly due to the discovery of new occurrence sites. indeed, records from new localities represent the easternmost geographical points for this species, respectively located (straight line distance) at about 79 km, 93 km and 92 km from the closest known populations of sierra añueque (cei, 1986; cei and avila, 1998; fig. 1b). these records are particularly significant, because they represent the first ones about this species from the somuncurá plateau and related volcanic outcrops. precise geographical distribution studies are important (feeley and silman, 2011) as they provide basic information for systematic (debandi et al., 2011), biogeographic (corbalán and debandi, 2008; vera-escalona et al., 2010) and conservation (corbalán et al., 2011; katzner et al., 2011) studies. recently, analyses of museum-based collections data demonstrated numerous cases of lizard population extinctions worldwide (sinervo et al., 2010). the information presented in the present study will make a useful contribution to similar broad-scale analyses in the future and should facilitate more rapid development of conservation plans for l. elongatus, if necessary. acknowledgments we thank c. h. f. pérez, m. f. breitman, m. kozykariski, n. feltrin for field support. we also thank j. c. bagley and m. f. breitman for reviewing this manuscript. we thank funding for field work were provided by nsf-pire (oise 0530267, issued to j. johnson), and several grants from conicet and foncyt (issued to m. morando and l. j. avila). we thank the authorities from neuquén (160/07; 1028/09; 0154/10; 0155/11), río negro (74071-df-2005) and chubut (06530/11; 02304/12) provinces for collection permits. supplementary material associated with this article can be found at http://www.unipv.it/webshi/appendix manuscript number 12711. references abdala, c.s., díaz gómez, j.m., juárez heredia, v.i. (2012): from the far reaches of patagonia: new phylogenetic analyses and description of two new species of the liolaemus fitzingerii clade (iguania: liolaemidae). zootaxa 3301: 43-60. avila, l.j., lobo, f. (1999): new lizard records for la rioja and catamarca provinces, northwestern argentina. herpetol. rev. 30: 115-116. avila, l.j., montero, r., morando, m. (2000): categorización de las lagartijas y anfisbenas de argentina. in: categorización de los anfibios y reptiles de la república argentina, pp. 51-74. asociación herpetológica argentina, ed, san miguel de tucumán, argentina. avila, l.j., morando, m., pérez, c.h.f., sites jr., j.w. (2004): phylogenetic relationships of lizards of the liolaemus petrophilus group (squamata, liolaemidae), with description of two new species from western argentina. herpetologica 60: 187-203. fig. 1. a) distributional range in argentina of liolaemus elongatus (sensu lato) from literature and museum review. black dots: literature and museum records; white stars: río negro and chubut provinces new records; grey light shaded area: range from scolaro (2005); grey strong shaded area: range from cei (1986). b) new proposal for current geographic distribution in argentina of liolaemus elongatus. black triangles: ljamm-cnp database collection records; white stars: río negro and chubut provinces new records; white circles: closest known population of sierra añueque; dashed black and white line: agrio river. 162 ignacio minoli et al. avila, l.j., pérez, c.h.f., medina, c.d., sites jr., j.w., morando, m. (2012): a new species of lizard of the liolaemus elongatus clade (reptilia: iguania: liolaemini) from curi leuvu river valley, northern patagonia, neuquén, argentina. zootaxa 3325: 37-52. breitman, m.f., pérez, c.h.f., parra, m., morando, m., sites jr., j.w., avila, l.j. (2011): new species of lizard from the magellanicus clade of the liolaemus lineomaculatus section (squamata: iguania: liolaemidae) from southern patagonia. zootaxa 3123: 32-48. cei, j.m. (1974): revision of patagonian iguanids of the liolaemus elongatus complex. j. herpetol. 8: 219-229. cei, j.m. (1986): reptiles del centro, centro-oeste y sur de la argentina. herpetofauna de las zonas áridas y semiáridas. monografie iv. museo regionale di scienze naturali torino, italy. cei, j.m., avila, l.j. (1998): reconocimiento de la categoría de especie para liolaemus petrophilus (squamata, tropiduridae, liolaeminae). facena 14: 75-80. corbalán, v., debandi, g. (2008): la lacertofauna de mendoza: lista actualizada, distribución y riqueza. cuad. herpetol. 22: 5-24. corbalán, v., tognelli, m.f., scolaro, j.a., roig juñent, s.a. (2011): lizards as conservation targets in argentinean patagonia. j. nat. conserv. 19: 60-67. debandi, g., corbalán, v., scolaro, j.a., roig juñent, s.a. (2011): predicting the environmental niche of the genus phymaturus: are palluma and patagonicus groups ecologically differentiated? austral. ecol. 36: 1-9. feeley, k.j., silman, m.r. (2011): keep collecting: accurate species distribution modelling requires more collections than previously thought. divers. distrib. 17: 1132-1140. ferraro, d.p., williams, j.d. (2006): material tipo de la colección de herpetología del museo de la plata, buenos aires, argentina. cuad. herpetol. 19: 19-36. katzner, t.e., ivy, j.a.r., bragin, e.a., milner gulland, e.j., dewoody, j.a. (2011): conservation implications of inaccurate estimation of cryptic population size. anim. conserv. 14: 328-332. koslowsky, j. (1896): sobre algunos reptiles de patagonia y otras regiones argentinas. revista mus. la plata 7: 447-457. morando, m., avila l.j., sites jr., j.w. (2003): sampling strategies for delimiting species: genes, individuals, and populations in the liolaemus elongatus-kriegi complex (squamata: liolaemidae) in andean-patagonian south america. syst. biol. 52: 159-185. newbold, t. (2010): applications and limitations of museum data for conservation and ecology, with particular attention to species distribution models. prog. phys. geog. 34: 3-22. pincheira-donoso, d., scolaro, j.a., sura, p. (2008): a monographic catalogue on the systematics and phylogeny of the south american iguanian lizard family liolaemidae (squamata, iguania). zootaxa 1800: 85. quinteros, a.s, abdala, c.s., gómez, j.m.d., scrocchi, g.j. (2008): two new species of liolaemus (iguania: liolaemidae) of central west argentina. s. am. j. herpetol. 3: 101-111. scolaro, j.a. (2005): reptiles patagónicos: sur. guía de campo. universidad nacional de la patagonia, trelew, argentina. simmons, j. e. (2002): herpetological collecting and collections managements. herpetol. circ. 31: 1-154. sinervo, b., mendez de la cruz, f., miles, d.b., heulin, b., bastiaans, e., villagran santa cruz, m., lara resendiz, r., martínez méndez, n., calderón espinosa, m.l., meza lázaro, r.n., gadsden, h., avila, l.j., morando, m., de la riva, i.j., victoriano sepulveda, p., duarte rocha, c.f., ibargüengoytía, n., aguilar puntriano, c., massot, m., lepetz, v., oksanen, t. a., chapple, d.g., bauer, a.m., branch, w.r., clobert, j., sites jr., j.w. (2010): erosion of lizard diversity by climate change and altered thermal niches. science 328: 894-899. vera-escalona, i.m., coronado, t., muñoz-mendoza, c., victoriano, p.f. (2010): historical and current distribution of the lizard liolaemus pictus (dumeril & bibron 1837) (liolaemidae) and new continental southern limit of distribution. gayana 74: 139-146. supplementary material table t1. summary of georeferenced locality records for liolaemus elongatus. appendix a1. detailed localities of all the records obtained for liolaemus elongatus. © firenze university press www.fupress.com/ah acta herpetologica 5(2): 199-205, 2010 evidence of tail autotomy in the european plethodontid hydromantes (atylodes) genei (temmick and schlegel, 1838) (amphibia: urodela: plethodontidae) antonio romano1,*, felix amat2, xavier rivera3, giuseppe sotgiu4, salvador carranza5 1 dipartimento di biologia, università di roma “tor vergata”, via della ricerca scientifica, i-00133 rome, italy. corresponding author. e-mail: antonioromano71@gmail.com 2 àrea d’herpetologia, museu de granollers – ciències naturals, francesc macià 51, e-08403, granollers, catalonia, spain. e-mail: amatfelix@yahoo.co.uk 3 societat catalana d’herpetologia museu de zoologia, passeig picasso s/n, e-08003, barcelona, spain. e-mail: xavirivera@yahoo.es 4 no profit naturalistic association zirichiltaggi sardinia wildlife conservation, strada vicinale filigheddu 62/c, i-07100, sassari, italy. e-mail: sotgiuseppe@hotmail.com 5 institut de biologia evolutiva (csic-upf), passeig marítim de la barceloneta, 37-49, e-08003, barcelona, spain. e-mail: salvador.carranza@ibe.upf-csic.es submitted on: 2010, 5th may; revised on: 2010, 18th october; accepted on: 2010, 27th october. abstract. caudal autotomy is a defensive mechanism widely adopted by lungless salamanders (plethodontidae) from the new world. in contrast, in europe, this mechanism was not described until very recently for just one sardinian species, hydromantes (speleomantes) sarrabusensis. we report on tail autotomy observed in another species from the same island, hydromantes (atylodes) genei. in europe, self-amputation of the tail seems to be restricted to some plethodontids inhabiting sardinia, while continental species do not exhibit analogous antipredator strategies. keywords. autotomy, hydromantes, plethodontid. autotomy is the ability to cast off, spontaneously or by reflex, a part of the body and, in herpetology, it usually refers to the breakage and loss of the tail. autotomy is a widespread defence against predators among invertebrates and vertebrates (see references in cooper et al., 2004). salamander antipredator strategies include autotomy among the suites of behavioural and morphological traits employed against predators and competitors (brodie, 1983; stebbins and cohen, 1995). such anatomical adaptation evolved in some plethodontids and salamandrids from an initial strategy based on redirecting the attack by predators to the body and/or the head to the tail by means of adopting a defensive posture (brodie, 1977). 200 a. romano et alii morphological diversification in skeletal, tongue and digital characteristics have all played an important role as diversification drivers in the large adaptive radiation of the family plethodontidae, the most remarkable among salamanders, embracing from troglodytic species to entirely arboreal ones (wake and larson, 1987). there is some evidence that supports a complex pattern of independent origins of coevolved traits (wake, 1991). one of the most surprising traits, the ability to autotomize and regenerate the tail as defensive behaviour, has received little attention in evolutionary studies (mueller et al., 2004). among plethodontids, which are distributed predominantly in the americas (wake, 1966; amphibiaweb, 2010), all members of the tropical radiation, including the neotropical genus bolitoglossa, and a few species of other american genera (ensatina, hemidactylium and batrachoseps) have specialized region at the base of the tail that is related to autotomy but many plethodontids that do not have specialized autotomy zones nevertheless are capable of autotomy (for a detailed account see wake and dresner, 1967). the genus hydromantes occurs in western north america (california; see usgs national amphibian atlas, 2009) and in central mediterranean europe. in particular, european plethodontids occur on the mediterranean island of sardinia (five species) and in peninsular italy and a small southern-eastern portion of france (three species; sindaco et al., 2006; carranza et al., 2008). the north american hydromantes belong to the subgenus hydromantes, the mainland europe species and four of the five sardinian species are ascribed to the subgenus speleomantes and a species inhabiting the southwest of sardinia to the subgenus atylodes. all three subgenera have sometimes been considered as full genera as a result of the large geographical disjunction that exists between western north america and italy, but according to both the morphological differences and especially the low level of genetic differentiation that exists within the genus hydromantes it is considered here that it is more appropriate to treat them as subgenera (see et al., 2005 for the taxonomic debate; lanza et al., 2006; wake; crochet, 2007 for the alternative use of both terms; carranza et al., 2008 and van der meijden et al., 2009 for genetic differentiation within hydromantes). autotomy has been reported for european species only recently (favelli et al., 2007) in the sardinian hydromantes (s.) sarrabusensis (lanza, leo, forti, cimmaruta, caputo and nascetti, 2001). during fieldwork carried out in sardinia in 2006, we studied a population of hydromantes (a.) genei (temminck and schlegel, 1838), inhabiting a mine gallery near s. giovanni’s cave (domusnovas, carbonia-iglesias). each animal was measured and sexed and immediately released at the sampling site. during the handling of the specimens, tail autotomy was recorded in a subadult salamander (fig. 1). the salamander, after twisting movements of the tail, lost a small portion of its distal part without losing blood. in the same population we observed other salamanders with regenerated tails. since the plethodontid tail has locomotor, respiratory, behavioural and food storage functions (wake and dresner, 1967), its autotomy can be highly costly and therefore it plays an important role in the survival of the salamanders. tail autotomy represents a loss of fat and protein (energy), both that are stored in the tail and represent the main source of energy in the tail regrown process, however making possible constraints, for instance, to the reproductive input (maynard smith and parker, 1976; bernardo and agosta, 2005) and may impose other social costs, such as reduction in fighting ability (wise and jaeger, 1998). indeed, even in species which are highly specialised for caudal autotomy these very 201evidence of tail autotomy in the european plethodontid figure 1. specimen of hydromantes (a.) genei, showing tail autotomy: a. the salamander before autotomy; b. the same salamander after caudal autotomy; c. close-up of the autotomised tail; note that just the tail tip tail was shed. 202 a. romano et alii rarely drop their tails unless the situation is life threatening (beneski, 1989). an autotomic plethodontid observed by favelli et al. (2007) used the fingers of the person handling the animal as a fulcrum to break its tail. to the contrary, the salamander we observed was free on the palm when autotomised its tail. furthermore, as may be noted in fig.1, the shed portion of the tail was very small. the loss of small portion of distal part of the tail reduces the disadvantage previously mentioned. the modality of caudal autotomy that was observed could be considered a case of “economy of autotomy”, which has been known for many years also in some reptiles (e.g., woodland, 1920; bustard, 1968; cooper et al., 2009). the degree of specialisation for defensive tail loss varies among different plethodontids lineages. some of them have highly specialised tail morphologies with basal constrained tails (ensatina and hemidactylium) associated with adult terrestrial life. in those species, the breakage includes almost the entire tail to ensure a large portion of food, discouraging predators to attack the salamander (wake and dresner, 1967). other lineages do not posses a localised breakage plane in their tail but have just developed wound healing capabilities. finally, other lineages have no specialisation for caudal loss and their tails simply break mechanically as found in semi aquatic desmognathines and non-neotenic spelerpines (wake and dresner, 1967). in the genus hydromantes (i.e., european cave salamanders and the three californian species) tail autotomy is rare. in all american plethodontids exhibiting autotomy, rupture occurs between, rather than through, vertebrae as in lizards (see stebbins and choens, 1995). intervertebral breakage was confirmed also for the sardinian species h. (s.) sarrabusenis (favelli et al., 2007) and, likely, intervertebral breakage occurs also in h. (a.) genei. despite the fact that tail regeneration after accidental breakage has been reported for some european mainland populations of hydromantes (salvidio, 1997), tail autotomy is only known to occur in the sardinian species h. (s.) sarrabusensis (favelli et al., 2007) and h. (a.) genei (this note). this difference among continental and island plethodontids cannot be tested due to absence in research effort on h. ambrosii, h. italicus, and h. strinatii because these mainland species are more studied than their island congeners (cf. lanza et al., 2006) and specific tests conducted to assess the occurrence of tail autotomy in continental plethodontids gave always negative results (lanza et al., 2006). the tail plays an important role for climbing as it has been demonstrated for arboreal and saxicolous american salamanders of medium sizes (wake, 1966) but not for small sized species (alberch, 1981). with respect to the body dimension, in hydromantes (a.) genei and h. (s.) sarrabusensis the largest sex presents an adult size of up to 124 mm, which is smaller than other sardinian plethodontids (up to 127 mm for the smaller sex and up to 150 for the largest one) and comparable to the size of mainland species (up to 128 mm) (see lanza et al., 2007). however the actual differences in size among hydromantes species (less than 20%) are not comparable to size differences that exist among medium size and miniaturised salamanders (more than 100%) of the supergenus bolitoglossa (alberch, 1981). european hydromantes are cave-adapted species [with the exception of h. (s.) sarrabusensis] characterised by possessing large and webbed hands and feet that most probably are an adaptation to manoeuvre on the wet, smooth walls of caves (lanza, 1991), as has been reported for other plethodontids species adapted to similar environmental condition (e.g., chiropterotriton magnipes, jaekel and wake, 2007). furthermore, the positive allom203evidence of tail autotomy in the european plethodontid etric lengthening of the tail (meaning that larger individuals have proportionately longer tail), has a similar adaptive significance because cave salamanders use their prehensile and sensitive tail largely during climbing (lanza, 1991). the loss of the tail can thus likely affect their climbing performance. it would be of particular interest to investigate if the two sardinian taxa exhibiting caudal autotomy [h. (a.) genei and h. (s.) sarrabusensis] are more terrestrial than climbers or if they are too small to be affected by the loss of the tail on their climbing abilities. however little ecological information is available to allow any preliminary conclusions. hydromantes (s.) sarrabusensis is a terrestrial salamander living on the ground in a granitic area without caves of south-eastern sardinia (lanza et al., 2006) and, consequently, it can be hypothesised that it climbs less than the other european cave-adapted species. thus, in this species, the tail loss would affect its movement capacity less than in a cavernicolous species. on the contrary, hydromantes (a.) genei, although it also inhabits humid forested areas in the vicinity of streams, it is mainly a cavernicolous species, but no data are available to establish if this salamander spends more time on the ground than other european plethodontids. hydromantes (a.) genei is the sister and basal clade to all the other european cave salamanders and its ancestor colonised sardinia in the late miocene (9 million of year ago, ma), while h. (s.) sarrabusenis belongs to a different lineage of eastern sardinian plethodontids and originated approximately 5 ma (carranza et al., 2008; van der meijden et al., 2009). studies on the evolution of the caudal autotomy, could elucidate if this defensive trait was acquired independently by these two species and therefore can be considered a convergence. in hydromantes (a.) genei two distinct lineages (a and b), showing a deep genetic divergence, have been recognised (lanza et al., 1995; carranza et al., 2008; van der meijden et al., 2009). we only observed tail autotomy in members of lineage a. further studies need to verify the existence of tail autotomy in the other lineage and in other sardinian plethodontid species, whose ecological traits are substantially unknown. indeed, consideration should be given to conspecific populations, or populations of closely related species, which are exposed to diverse predator pressures and may exhibit different level of tail autotomy as defensive behaviour (cooper et al., 2004). acknowledgements specimens were handling under permission of from the ‘ministero dell’ambiente e della tutela del territorio e del mare’ (permit reference: dpn/2d/2006/7546). this study was supported by grant cgl2009-11663 from the minsterio de ciencia e innovación and grant 2009 sgr 1462 from the generalita de catalunya to sc. we would like to thank david wake and one anonymous referee for useful suggestions during the revision of the manuscript. references alberch, p. 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(1920): some observations on caudal autotomy and regeneration in the gecko (hemidactylus flaviviridis, ruppel). quart. j. micr. sci. 65: 63-100. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 8(1): 79-80, 2013 book review: roberto sindaco, alberto venchi, cristina grieco. the reptiles of the western palearctic. 2. annotated checklist and distributional atlas of the snakes of europe, north africa, middle east and central asia, with an update to the vol. 1. edoardo razzetti museo di storia naturale, università degli studi di pavia, piazza botta 9/10, i-27100 pavia. e-mail: razzetti@unipv.it the reptiles of the western palearctic is the second volume of an ambitious project: to create a checklist and an accurate atlas for all the reptile species of this complex biogeographic region, a huge area of nearly 27 millions of square kilometers that includes about 690 reptile species. gathering together all these data is a task that would deter anyone except roberto sindaco and colleagues. the realization of this project involved nearly twenty years spent traveling in remote areas for fieldwork (from the volcanoes of canary islands to the steppes of kazakhstan) and thousands of hours entering taxon/locality records from obscure literature into a database, georeferencing them, and plotting data on maps. the outcome of all these efforts is between my hands: a thick 17 × 24 cm hardcover volume printed on nice coated paper with the logos of societas herpetologica italica and societas europaea herpetologica on it. from the perspective of snakes, the western palearctic includes regions that enthralled the zoologists since the age of enlightenment and other that were largely overlooked. actually, by an herpetological point of view, europe is probably the most investigated region of the world, as the earliest accounts go back to aristotle, and comprehensive attempts of summarizing the knowledge of its ophidiofauna were repeatedly published more or less in every decade of the past century. conversely, little is known about the snakes of large areas of north africa (algeria, libya) and asia (e.g., syria, lebanon, georgia, afghanistan, yemen, oman, iran) where the relevant data are scattered among hundreds of scientific papers or were drawn from individual field notes and collections housed in several museums. the reptiles of the western palearctic begins with six general sections that represent a slightly modified and updated version of the same chapters in the first volume (sindaco and jeremčenko, 2008):. the first and the second detail the geographical extent and habitat types with the help of few (16) nice photographs of different landscapes; i wished a deeper coverage of these sections but this would be probably beyond the scope of this volume. next are “material and methods” with a list of the information provided in each species account and how the 52.000 records were selected and mapped. the status of the herpetological knowledge in the area is presented in the subsequent chapter with a list of reference text for each country and, sometimes, useful comments on outdated and inaccurate sources. next are the chapters about the biodiversity and biogeography which probably represent the strongest point of this book since the distributional records here presented are an invaluable tool for this kind of analyses and the authors master these topics particularly well. conservation issues are the subject of the next chapter; these are dealt with heavy emphasis on shrinking and fragmentation of natural habitat, introduction of alien species, iucn red list categories and legislation that leaves few room for “emotional” issues like pet trade or collection for scientific purposes. the next part is the annotated checklist which represents the core of the book, a bulk of 350 pages considering the associated distribution maps (184 figures) and the colour plates. from the start, it is clear that the authors’ objective is to gather together all published information about 80 book review taxonomy, biogeography and distribution for each snake species. references are cited in the text, which greatly improves the book’s usefulness and there is also a further list of maps data sources for each species. every account starts with reference to the original description and type locality. this could be not particularly interesting for most of the readers but it must be stressed that it’s quite unusual as most of the books usually just report author, year of description and the “restricted” type locality (that is often an unjustifiable emendation like for many species dealt by robert mertens). there is a distribution map based on a one by one degree grid for each genus (inserted in the text) and a map for each species (these are lumped together in a separate chapter); all the maps report the global distribution of taxa even if extralimital distribution include few dots and are often delimited just by a generic line. i would like to spend few more words about the 342 images of snakes which cover most of the species treated in the book, even some rare taxa that i wasn’t able to see depicted before; the quality of the images is outstanding with just two exceptions: gloydus himalayanus and montivipera latifi. my opinion is that reptiles of the western palearctic by roberto sindaco, alberto venchi and cristina grieco is an invaluable reference for professional and amateur herpetologists, and i feel that it will be considered a reference text for the next decades; anyway readers should keep in mind that this book do not follow the general trend of most recent herpetological monographs so you will never find any identification keys or any data about the ecology or biology as the emphasis is just on taxonomy and distribution. as final remark have to admit that i tried to find general mistakes and misspelled scientific names but to my shame the only ones i could find were included in the foreword that i personally wrote. the book can be purchased at the cost of € 62.00 (or € 52.00 for s.h.i. members) at the publisher’s website (http://www.edizionibelvedere.it/). references sindaco, r., jeremčenko, v.k. (2008): the reptiles of the western paleartic. 1. annotated checklist and distributional atlas of the turtles, crocodiles, amphisbaenians and lizards of europe, north africa, middle east and central asia. edizioni belvedere, latina. acta herpetologica vol. 8, n. 1 june 2013 firenze university press the oogenic cycle of the caspian bent-toed gecko, cyrtopodion caspium (squamata: gekkonidae) in iran vida hojati1*, kazem parivar2, eskandar rastegar-pouyani3, abdolhossein shiravi1 altitudinal effects on life history parameters in populations of liolaemus pictus argentinus (sauria: liolaemidae) joel antú gutiérrez1,*, carla piantoni2, nora r. ibargüengoytía1,3 recent cryptic extinction of squamate reptiles on yoronjima island of the ryukyu archipelago, japan, inferred from garbage dump remains yasuyuki nakamura1,*, akio takahashi2, hidetoshi ota3 trophic niche and feeding biology of the italian wall lizard, podarcis siculus campestris (de betta, 1857) along western mediterranean coast marco a.l. zuffi*, chiara giannelli novel, non-invasive method for distinguishing the individuals of the fire salamander (salamandra salamandra) in capture-mark-recapture studies goran šukalo1,*, sonja đorđević2, dragojla golub1, dejan dmitrović1, ljiljana tomović2,3 going out tonight? when insular hierophis viridiflavus breaks the whip snakes rules delaugerre michel-jean first evidence of a paedomorphic population of the smooth newt (lissotriton vulgaris) in the czech republic václav gvoždík1,2, veronika javůrková4, oldřich kopecký5,* no detection of chytrid in first systematic screening of bombina variegata pachypus (anura: bombinatoridae) in liguria, northern italy stefano canessa1,*, an martel2, frank pasmans2 status of the european pond turtle, emys orbicularis (reptilia: testudines: emydidae) in vorarlberg, austria andreas kleewein1, günther wöss2 comparative cytogenetics of two species of ground skinks: scincella assata and s. cherriei (squamata: scincidae: lygosominae) from chiapas, mexico riccardo castiglia1,*, alexandra m.r. bezerra2, oscar flores-villela3, flavia annesi1, antonio muñoz4, ekaterina gornung1 polydactyly in the tyrrhenian wall lizard (podarcis tiliguerta) antigoni kaliontzopoulou1,*, daniele salvi1, verónica gomes1, joão p. m. c. maia1,2,3, panagiotis kaliontzopoulos4 book review: roberto sindaco, alberto venchi, cristina grieco. the reptiles of the western palearctic. 2. annotated checklist and distributional atlas of the snakes of europe, north africa, middle east and central asia, with an update to the vol. 1. edoardo razzetti acta herpetologica journal of the societas herpetologica italica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 6(2): 137-147, 2011 changes in the blood composition of some anurans çiğdem gül*, murat tosunoğlu, didem erdoğan, dilşah özdamar çanakkale onsekiz mart university, faculty of sciences and arts, department of biology, zoology section, tr-17100 çanakkale-turkey. * correspondig author. e-mail: gulcigdem17@hotmail.com submitted on: 2010, 23th december; revised on: 2011, 28th april; accepted on: 2011, 10th may. abstract. we examined some hematological parameters (red blood cell count, white blood cell count, haemoglobin concentration, hematocrit value, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration and plasma total protein) on five anuran species of terrestrial (pseudepidalea viridis, pelobates syriacus and hyla arborea), semi-aquatic (rana dalmatina) and aquatic (pelophylax ridibundus) nature from çanakkale, turkey. differences between males and females in terms of haemoglobin, hematocrit and mean cell volume in p. viridis were statistically significant. the rbc count was higher in terrestrial and aquatic species than in semi-aquatic species. haemoglobin concentration, hematocrit value, mcv and mhc were higher in terrestrial species than in semi-aquatic and aquatic species. the mchc values were all similar to each other. the plasma total protein was higher in terrestrial species than in aquatic species. to sum up, variations were detected in the some hematological parameters under examination among the anuran species. keywords. anura, some hematological parameters, terrestrial, semi-aquatic, aquatic. introduction amphibia and reptilia species were affected by negative conditions of pollution in the environmental condition and destruction of biotopes. blood parameters of amphibia species were particularly affected by the negative environmental conditions. amphibians are potentially reliable and efficient bioindicators (welsh and ollivier, 1998; garg and hippargi, 2007). they are very sensitive to even the slightest fluctuations in the environmental settings (cunnigham and saigo, 1999). amphibians are a heterogeneous group of vertebrates with regard to their blood cell count and size. however, the blood cell counts in amphibia reported a wide individual variation and considerable interspecies differences (hutchison and szarski, 1965; szarski and czopek, 1966; rouf, 1969; sinha, 1983; atatür et al., 1999; cabagna et al., 2005) as 138 ç. gül et. alii well as in relation to body weight, age and sex (arvy, 1947; schermer, 1954; goniaakowska, 1973; sinha, 1983; banerjee, 1988; wojtaszek and adamowicz, 2003), season (zhukova and kubantsev, 1979; sinha, 1983; wojtaszek et al., 1997; arserim and mermer, 2008) and altitudinal distribution (arıkan, 1989; ruiz et al., 1989). most studies on hematology in various species of anura have dealt with blood cell counts (alder and huber, 1923; arvy, 1947; kaplan, 1952; stephan, 1954; schermer, 1954; hutchison and szarski, 1965; arıkan, 1989) and cell sizes (wintrobe, 1933; foxon, 1964; hartman and lessler, 1964; szarski and czopek, 1966; kuramoto, 1981; stöck and grobe, 1997; atatür et al., 1999; arıkan et al., 2003; cabagna et al., 2005; gül and tok, 2009). nevertheless, some hematological parameters of the anuran species, such as hematocrit value (pcv), haemoglobin concentration (hb), mean cell volume (mcv), mean cell haemoglobin (mch), mean cell haemoglobin concentration (mchc) and plasma total protein concentration, are very scarce and individual studies (prosser and weistein, 1950; haris, 1972; carmena-suero et al., 1980; sinha, 1983; ostejic et al., 2000; wojtaszek and adamowicz, 2003; coppo et al., 2005; arserim and mermer, 2008; dönmez, 2009). so, the possibilities of using quantitative and qualitative parameters of the blood in amphibians undoubtedly hold great interest (zhelev et al., 2006). in the clinical investigation, blood samples are of great diagnostic value and can easily be obtained (frye, 1991). the normal reference ranges of hematological parameters are also important in assessing the status of the species population, and deviation from the expected values can assess the impact of stress on the species (dickinson et al., 2002). therefore, the objective of this study was to determine some hematological parameters [red blood cell count (rbc), white blood cell count (wbc), haemoglobin concentration (hb), hematocrit value (pcv), mean cell volume (mcv), mean cell haemoglobin (mch), mean cell haemoglobin concentration (mchc) and plasma total protein value] in some anuran species (pelophylax ridibundus, rana dalmatina, pseudepidalea viridis, hyla arborea and pelobates syriacus). in addition, this study aims to investigate differences in the some hematological parameters of anuran species of terrestrial (p. viridis, p. syriacus and h. arborea), semi-aquatic (r. dalmatina) and aquatic (p. ridibundus) nature. material and methods specimens of the different anuran species used in this study (p. viridis: n =10, h. arborea: n = 15, p. syriacus: n = 13, r. dalmatina: n = 15 and p. ridibundus: n = 10) were collected from their natural habitat and various localities of canakkale, turkey. studies were carried out in the reproductive period from february to april between 2008 and 2010. blood samples of the live specimens were obtained in the laboratory within one day of their capture. the blood samples were taken from the etherized frogs by means of ventriculus punctures, via heparinized hematocrit capillaries (arıkan et al., 2003). the quantity of blood taken out from each specimens were 0.15 ml. the blood cell counts were performed utilizing a neubauer hemocytometer, and the standard hayem’s solution was used as diluting solutions for erythrocytes by thoma pipettes, while for the leukocytes, the method of jerrett and mays (1973) (which is a slight modification of blain’s methodsturkie, 1954) was utilized; i.e. a 1:1 mix of neutral red diluted to 1/5000 with 0.07% physiological saline and 12% formaline prepared with 0.07% physiological saline was used. 139haematology of turkish anurans blood from each frog or toad was placed into heparinised hematocrit capillaries and used to determine the hematological parameters. hematocrit value (pcv) was determined by the microhematocrit method. the tubes were then spun in a micro-hematocrit centrifuge for 5 min at 12000 rpm, and the hematocrit value (pcv) was calculated with a hematocrit reader. hemoglobin concentration (hb) was measured with a sahli’s hemometer. the mean cell volume (mcv), mean cell hemoglobin (mch) and mean cell hemoglobin concentration (mchc) were calculated mathematically from the above results. mcv was calculated by dividing hematocrit per liter of blood by total rbc count (tanyer, 1985). the blood samples were centrifuged at 3000 rpm for 10 min and it was ensured that the plasma section be isolated from the blood cells. the plasma total protein quantity in the plasma was measured using a refractometer. non-parametric tests and descriptive statistics were calculated using spss (v10.0). mann whitney u test was applied between males and females. results hematological parameters investigated in the present study are represented in table 1. there were some significant differences in some parameters when comparisons were made between male and female data for one species. differences between males and females in terms of hemoglobin, hematocrit and mean cell volume in p. viridis were statistically significant (p < 0.05). when the hematological values of anuran species were examined, the highest rbc count, wbc count, hemoglobin concentration and hematocrit value were found in pseudepidalea viridis, whereas the lowest rbc count, wbc count, hemoglobin concentration and hematocrit value were found in r. dalmatina. the mean cell volume was detected to be the highest in h. arborea and the lowest in r. dalmatina. the mean cell hemoglobin was found to be the highest in h. arborea and the lowest in p. ridibundus. the mean cell hemoglobin concentration was found to be the highest in pseudepidalea viridis and the lowest in p. ridibundus. the plasma total protein was found to be the highest in p. viridis and the lowest in p. ridibundus. the hematological values of anuran species are given in detail in table 1. discussion several authors, who worked on different species of anura, mentioned individual variations concerning both the rbc and wbc counts (alder and huber, 1923; klieneberger, 1927; schermer, 1954; hutchison and szarski, 1965; cabagna et al., 2005; chiesa et al. 2006). in the present study, significant differences were found between males and females only in p. viridis specimens in terms of hemoglobin, hematocrit and mean cell volume. the rbc count was higher in males than in females of species p. ridibundus, r. dalmatina, h. arborea and p. syriacus. similar observations were found by arvy (1947) in r. temporaria, where males (450000) showed a higher rbc count than females (330000). even sinha (1983) stated a higher count in males (320000) than in females (250000) in r. esculenta. wojtaszek and adamowicz (2003) have reported a higher number of rbc in males (340000) than in females (290000) of bombina bombina. however, the rbc count of p. 140 ç. gül et. alii ta bl e 1. r ed b lo od c el l co un ts ( r b c ) (n /µ l) , w hi te b lo od c el l co un t (w b c ) (µ l) , h ae m og lo bi n co nc en tr at io n (h b) ( g/ dl ), h em at oc ri t va lu e (p c v ) (% ), m ea n ce ll vo lu m e (m c v ) (f l) , m ea n ce ll ha em og lo bi n (m c h ) (p g/ ce ll) , m ea n ce ll ha em og lo bi n co nc en tr at io n (m c h c ) (% ) an d pl as m a to ta l p ro te in ( pt p) (g /l ). f: fe m al e, m : m al e, t : t ot al ; n : n um be r of s pe ci m en s, s d : s ta nd ar d de vi at io n. se x te rr es tr ia l ps eu de pi da le a vi ri di s pe lo ba te s sy ri ac us h yl a ar bo re a n m ea n sd r an ge n m ea n sd r an ge n m ea n sd r an ge r b c f 6 93 76 66 21 29 43 72 00 00 -1 30 00 00 11 76 59 09 19 18 18 56 00 00 -1 20 00 00 11 73 36 36 26 19 65 34 00 00 -1 29 00 00 m 4 97 50 00 94 33 9. 81 84 00 00 -1 04 00 00 2 65 25 00 31 81 9. 80 63 00 00 -6 75 00 0 4 64 75 00 21 28 18 40 00 00 -9 20 00 0 t 10 95 26 00 16 89 08 72 00 00 -1 30 00 00 13 74 84 61 18 04 44 56 00 00 -1 20 00 00 15 71 06 66 24 55 16 34 00 00 -1 29 00 00 w b c f 6 59 00 13 91 .4 0 45 00 -8 00 0 11 26 00 17 98 .3 3 12 00 -7 40 0 7 36 02 65 2. 83 20 00 -4 90 0 m 4 67 75 11 17 .6 6 57 00 82 00 2 32 50 35 3. 55 30 00 -3 50 0 2 28 00 11 31 .3 7 20 00 -3 60 0 t 10 62 50 13 02 .3 4 45 00 -8 20 0 13 27 00 16 62 .8 2 12 00 -7 40 0 9 36 02 60 4. 40 31 00 -4 90 0 h b f 6 15 .7 6 0. 79 14 .6 017 .0 0 11 10 .3 8 2. 46 6. 00 -1 2. 80 11 12 .2 1 1. 42 10 .0 013 .8 0 m 4 12 .9 5 0. 91 12 .0 014 .2 0 2 9. 30 3. 53 6. 80 -1 1. 80 4 11 .8 2 1. 22 10 .4 013 .4 0 t 10 14 .6 4 1. 65 12 .0 017 .0 0 13 10 .2 1 2. 50 6. 00 -1 2. 80 15 12 .1 1 1. 34 10 .0 013 .8 0 pc v m 6 58 .5 0 6. 94 52 .0 070 .0 0 5 40 .0 0 0. 08 27 .0 050 .0 0 11 50 .1 8 8. 64 40 .0 067 .0 0 f 4 43 .7 5 3. 30 40 .0 047 .0 0 2 38 .0 0 0. 11 30 .0 046 .0 0 3 45 .0 0 10 .0 0 35 .0 055 .0 0 t 10 52 .6 0 9. 40 40 .0 070 .0 0 7 40 .0 0 0. 08 27 .0 050 .0 0 14 49 .0 7 8. 81 35 .0 067 .0 0 m c v m 6 63 8. 63 96 .0 5 53 8. 46 -7 93 .0 6 5 46 8. 20 83 .0 7 36 6. 67 -5 95 .2 4 11 75 8. 10 28 5. 86 45 6. 59 -1 41 1. 76 f 4 45 0. 18 34 .1 0 40 3. 85 -4 76 .1 9 2 57 7. 35 14 3. 06 47 6. 19 -6 78 .5 2 3 87 8. 72 43 7. 86 54 6. 88 -1 37 5. 00 t 10 56 3. 25 12 2. 40 40 3. 85 -7 93 .0 6 7 49 9. 39 10 4. 15 36 6. 67 -6 78 .5 2 14 78 3. 94 30 8. 21 45 6. 59 -1 41 1. 76 m c h m 6 17 4. 18 33 .9 4 13 0. 77 -2 13 .8 9 11 13 8. 59 35 .3 4 10 0. 00 -2 28 .5 7 11 18 6. 89 77 .0 6 10 6. 98 -3 82 .3 5 f 4 13 3. 93 17 .5 3 11 5. 38 -1 52 .3 8 2 14 1. 37 47 .2 8 10 7. 94 -1 74 .8 1 4 19 4. 08 48 .4 8 14 4. 57 -2 60 .0 0 t 10 15 8. 08 34 .2 7 11 5. 38 -2 13 .8 9 13 13 9. 02 35 .0 4 10 0. 00 -2 28 .5 7 15 18 8. 81 68 .9 7 10 6. 98 -3 82 .3 5 m c h c m 6 27 .2 4 3. 10 23 .0 330 .7 7 5 28 .8 6 2. 73 25 .6 031 .9 0 11 24 .6 8 2. 91 20 .2 431 .1 6 f 4 29 .6 7 1. 94 27 .6 532 .0 0 2 24 .2 1 2. 19 22 .6 625 .7 6 3 26 .1 9 6. 87 18 .9 132 .5 7 t 10 28 .2 1 2. 86 23 .0 332 .0 0 7 27 .5 3 3. 30 22 .6 631 .9 0 14 25 .0 0 3. 77 18 .9 132 .5 7 pt p m 5 8. 16 1. 36 6. 00 -9 .5 0 4 7. 12 1. 65 5. 00 -9 .0 0 11 7. 47 1. 56 6. 00 -1 0. 00 f 4 7. 50 0. 57 7. 00 -8 .0 0 2 6. 50 0. 70 6. 00 -7 .0 0 2 7. 25 0. 35 7. 00 -7 .5 0 t 9 8. 00 1. 14 6. 00 -9 .5 0 6 6. 91 1. 35 5. 00 -9 .0 0 13 7. 44 1. 43 6. 00 -1 0. 00 141haematology of turkish anurans ta bl e 1. c on tin ue d. se m ia qu at ic a qu at ic r an a da lm at in a pe lo ph yl ax r id ib un du s r b c se x n m ea n sd r an ge n m ea n sd r an ge f 9 71 66 60 15 09 97 55 00 00 -1 02 00 00 5 88 60 00 82 94 5. 76 78 00 00 -9 80 00 0 m 6 64 83 30 84 24 1. 71 49 00 00 -7 20 00 0 5 76 20 00 20 65 67 65 00 00 -1 13 00 00 t 15 68 93 30 12 94 75 49 00 00 -1 02 00 00 10 82 40 00 16 21 52 65 00 00 -1 13 00 00 w b c f 2 27 00 98 9. 94 20 00 -3 40 0 3 31 06 14 62 .2 0 16 00 -4 52 0 m 4 26 75 96 0. 46 20 00 -4 10 0 2 25 50 14 84 .9 2 15 00 -3 60 0 t 6 26 83 86 5. 83 20 00 -4 10 0 5 28 84 13 08 .9 2 15 00 -4 52 0 h b f 9 8. 51 1. 29 6. 80 -1 0. 60 5 9. 52 1. 47 8. 20 -1 2. 00 m 6 8. 20 1. 21 6. 60 -1 0. 10 5 8. 66 1. 94 7. 40 -1 2. 10 t 15 8. 38 1. 23 6. 60 -1 0. 60 10 9. 09 1. 68 7. 40 -1 2. 10 pc v m 9 34 .9 1 6. 49 28 .5 747 .0 5 5 43 .1 9 4. 12 39 .4 548 .7 8 f 6 30 .2 6 2. 64 26 .8 033 .3 0 5 38 .1 9 13 .4 8 23 .5 259 .2 5 t 15 33 .0 5 5. 67 26 .8 047 .0 5 10 40 .6 9 9. 76 23 .5 259 .2 5 m c v m 9 49 5. 14 80 .9 9 39 9. 41 -6 03 .2 0 5 48 8. 83 38 .7 4 42 8. 80 -5 24 .5 2 f 6 47 2. 77 67 .8 3 40 2. 39 -5 91 .8 3 5 49 9. 71 10 5. 91 33 6. 00 -6 21 .7 9 t 15 48 6. 19 74 .3 0 39 9. 41 -6 03 .2 0 10 49 4. 27 75 .4 0 33 6. 00 -6 21 .7 9 m c h m 9 12 0. 58 15 .2 7 10 3. 92 -1 49 .1 5 5 10 7. 50 13 .2 8 95 .6 512 9. 03 f 6 12 9. 09 30 .1 8 92 .9 518 1. 63 5 11 4. 69 8. 07 10 5. 71 -1 24 .2 4 t 15 12 3. 98 21 .8 4 92 .9 518 1. 63 10 11 1. 09 11 .0 3 95 .6 512 9. 03 m c h c m 9 24 .6 1 2. 36 19 .9 727 .0 0 5 21 .9 5 1. 66 20 .3 924 .6 0 f 6 27 .1 6 3. 76 22 .2 030 .6 8 5 23 .7 8 5. 11 18 .7 231 .4 6 t 15 25 .6 3 3. 14 19 .9 730 .6 8 10 22 .8 7 3. 71 18 .7 231 .4 6 pt p m 4 5. 75 5. 75 5. 00 -6 .0 0 3 5. 50 0. 50 5. 00 -6 .0 0 f 2 5. 50 0. 70 5. 00 -6 .0 0 3 5. 83 0. 76 5. 00 -6 .5 0 t 6 5. 66 0. 51 5. 00 -6 .0 0 6 5. 66 0. 60 5. 00 -6 .5 0 142 ç. gül et. alii viridis was higher in females than in males. mahapatra et al. (2010) have reported a higher rbc count in females (530000) than in males (480000) of the polypedates maculatus. a similar observation was found by kaplan (1951, 1952) in rana pipiens and by arserim and mermer (2008) in r. macrocnemis. meanwhile, no sexual dimorphism was reported for the rbc count of r. pipiens (rouf, 1969), r. catesbeiana and r. calamitans (hutchison and szarski 1965) (table 2). the wbc counts vary depending on anuran species, season, sex, nutritional conditions and some physiological conditions, such as diseases and breeding (rouf, 1969; arıkan, 1989; wojtaszek and adamowicz, 2003). in the present study, significant differences were not found between males and females in all species in terms of wbc count. the wbc counts were higher in males than in females of species p. ridibundus, r. dalmatina and h. arborea. a similar observation was found by kaplan (1951, 1952) in r. pipiens (16,134 in males; 14,134 in females) and by wojtaszek and adamowicz (2003) in b. bombina (9,734 in males; 7030 in females). however, they were higher in females than in males of other species (p. syriacus and p. viridis). arserim and mermer (2008) put forth that the wbc count in r. macrocnemis is higher in females (3613) than in males (3445, table 2). the haemoglobin concentration (hb) and hematocrit values (pcv) were found to significantly differ between males and females in only p. viridis. similar observations were found by sinha (1983) in r. esculenta, by wojtaszek and adamowicz (2003) in b. bombina, by arserim and mermer (2008) in r. macrocnemis and by dönmez et al. (2009) in bufo bufo. kaplan (1954) found statistically significant sexual differences in the hematocrit values of r. pipiens. the hemoglobin concentration of p. maculatus was higher in females (7.80 g/100 ml) than in males (6.56 g/100 ml) (mahapatra et al., 2010). arserim and mermer (2008) put forth that the hematocrit values in rana macrocnemis are higher in females (35.00) than in males (32.00). also, a similar observation was found by haris (1972) (tables 1 and 2). the values of mcv were found to significantly differ between males and females in only p. viridis. in other species, there were no significant differences between males and females. generally, the mcv values were higher in females than in males of h. arborea, p. syriacus and p. ridibundus. similar mcv observations were found by dönmez et al. (2009) in b. bufo. sinha (1983) and arserim and mermer (2008) reported that the mcv value in r. esculenta and r. macrocnemis is higher in females than in males. mch values were higher in females than in males of p. syriacus, h. arborea, r. dalmatina and p. ridibundus. sinha (1983) reported that the mch value in r. esculenta is higher in males than in females. mchc values were higher in females than in males of p. viridis, h. arborea, r. dalmatina and r. ridibunda. mahapatra et al. (2010) has also reported a higher mchc value in females than in males of p. maculatus. however, dönmez et al. (2009) stated a higher mchc value in males than in females of b. bufo (tables 1 and 2). plasma total protein values were higher in males than in females of p. viridis, p. syriacus and h. arborea (table 1). atatür et al. (1999) determined differences in the size of erythrocytes from several species of anura in turkey and looked for the reasons in the different environmental conditions of the biotopes. therefore, several researchers (atatür et al., 1999; zhelev et al., 2006) have found that aquatic anurans have larger erythrocytes than semi-aquatic and terrestrial species. 143haematology of turkish anurans ta bl e 2. th e so m e he m at ol og ic al p ar am et er s in d iff er en t a nu ra s pe ci es r ef er ed b y va ri ou s au th or s. se x r b c w b c h b pc v a ld er a nd h ub er ( 19 23 ) h yl a ar bo re a 67 40 00 29 00 0 a rv y (1 94 7) r an a te m po ra ri a m 45 00 00 f 33 00 00 r ou f ( 19 69 ) r an a pi pi en s 31 94 00 6. 75 24 .6 5 h ar ri s (1 97 2) r an a pi pi en s m 16 00 00 -5 40 00 0 96 00 -3 80 00 2. 00 -9 .5 0 8. 00 -4 1. 50 f 11 00 00 -4 50 00 0 78 50 -2 51 50 2. 90 -1 2. 70 17 .0 047 .5 0 c ar m en asu er o et a l. (1 98 0) h yl a se pt en tr io na lis 6. 20 22 .4 0 r an a ca te sb ei an a 9. 50 40 .4 0 si nh a (1 98 3) r an a es cu le nt a m 32 00 00 7. 20 21 .8 0 f 25 00 00 5. 80 19 .8 0 a rı ka n et a l. (1 98 9) r an a ri di bu nd a 32 66 20 31 42 o st oj ic e t a l. (2 00 0) bu fo s pi nu lo su s lim en si s 63 40 00 39 .5 3 a rı ka n et a l. (2 00 3) pe lo dy te s ca uc as ic us 77 60 00 25 60 w oj ta sz ek a nd a da m ow ic z (2 00 3) bo m bi na b om bi na m 34 00 00 ( 19 00 00 -4 65 00 0) 97 34 7. 44 ( 4. 99 -1 2. 20 ) 13 .7 026 .2 0 f 29 00 00 ( 24 00 00 -3 55 00 0) 70 30 6. 78 ( 3. 38 -8 .3 1) 12 .0 023 .3 0 c op po e t a l. (2 00 5) r an a ca te sb ei an a 30 .1 0 (2 5. 00 -3 9. 00 ) a rs er im a nd m er m er ( 20 08 ) r an a m ac ro cn em is m 50 62 50 ( 28 00 00 -9 40 00 0) 34 45 ( 26 00 -4 20 0) 8. 12 ( 5. 60 -1 2. 10 ) 32 .0 0 (1 9. 00 -4 2. 00 ) f 52 40 00 ( 32 00 00 -9 00 00 0) 36 13 ( 28 00 -5 20 0) 8. 07 ( 6. 20 -1 1. 00 ) 35 .0 0 (1 6. 00 -4 6. 00 ) t 51 48 39 ( 28 00 00 -9 40 00 0) 35 27 ( 26 00 -5 20 0) 8. 10 ( 5. 60 -1 2. 10 ) 34 .0 0 (1 6. 00 -4 6. 00 ) d ön m ez e t a l. (2 00 9) bu fo b uf o m 90 00 00 ( 88 00 00 -9 20 00 0) 11 .8 0 (1 1. 20 -1 2. 40 ) 42 .3 6 (4 1. 53 -4 3. 20 ) f 87 00 00 ( 84 00 00 -9 00 00 0) 10 .1 0 (9 .4 010 .8 0) 33 .0 8 (2 8. 57 -3 7. 60 ) g ül a nd t ok ( 20 09 ) r an a ri di bu nd a 32 05 00 ( 20 00 00 -6 50 00 0) 25 36 ( 80 045 20 ) r an a da lm at in a 41 57 50 ( 31 00 00 -5 50 00 0) 26 83 ( 20 00 -4 10 0) bu fo v ir id is 72 17 50 ( 45 00 00 -9 00 00 0) 16 46 ( 90 025 00 ) bu fo b uf o 53 47 22 ( 45 30 00 -7 03 00 0) 23 25 ( 11 00 -3 80 0) h yl a ar bo re a 57 95 83 ( 40 50 00 -7 03 00 0) 29 80 ( 46 049 00 ) pe lo ba te s sy ri ac us 65 71 00 ( 54 30 00 -7 93 00 0) 12 16 ( 60 025 00 ) m ah ap at ra e t a l. (2 01 0) po ly pe da te s m ac ul at us m 48 00 00 ( 37 00 00 -5 80 00 0) 14 62 8 (1 24 00 -1 62 00 ) 6. 56 ( 5. 20 -8 .4 0) 28 .6 5 (1 8. 51 -3 7. 60 ) f 57 00 00 ( 40 00 00 -7 10 00 0) 16 64 2 (1 40 00 -2 00 00 ) 7. 80 ( 6. 40 -9 .0 0) 23 .8 0 (1 6. 00 -3 2. 07 ) 144 ç. gül et. alii ta bl e 2. c on tin ue d. se x m c v m c h m c h c pt p a ld er a nd h ub er ( 19 23 ) h yl a ar bo re a a rv y (1 94 7) r an a te m po ra ri a m f r ou f ( 19 69 ) r an a pi pi en s h ar ri s (1 97 2) r an a pi pi en s m f c ar m en asu er o et a l. (1 98 0) h yl a se pt en tr io na lis 27 .7 0 r an a ca te sb ei an a 23 .5 0 si nh a (1 98 3) r an a es cu le nt a m 70 7. 00 24 6. 00 33 .0 0 f 84 0. 00 25 0. 50 29 .5 0 a rı ka n et a l. (1 98 9) r an a ri di bu nd a o st oj ic e t a l. (2 00 0) bu fo s pi nu lo su s lim en si s 62 1. 90 17 2. 65 28 .0 6 a rı ka n et a l. (2 00 3) pe lo dy te s ca uc as ic us w oj ta sz ek a nd a da m ow ic z (2 00 3) bo m bi na b om bi na m 41 1. 70 -7 57 .7 0 15 8. 30 -2 68 .1 0 29 0. 60 -5 54 .0 0 f 36 3. 30 -9 16 .6 0 14 5. 30 32 0. 30 18 9. 00 -6 04 .1 0 c op po e t a l. (2 00 5) r an a ca te sb ei an a 70 9. 00 ( 50 5. 00 -7 88 .0 0) 15 7. 00 ( 12 1. 00 -1 97 .0 0) 23 .3 0 (2 0. 20 -3 1. 40 ) 4. 34 ( 3. 05 -5 .6 5) a rs er im a nd m er m er ( 20 08 ) r an a m ac ro cn em is m 67 4. 07 ( 42 5. 53 -1 00 0) 17 0. 48 ( 91 .4 922 9. 41 ) f 71 6. 39 ( 42 0. 45 -1 10 5. 26 ) 16 5. 10 ( 87 .7 825 7. 89 ) t 69 4. 54 ( 42 0. 45 -1 10 5. 26 ) 16 7. 88 ( 87 .7 825 7. 89 ) d ön m ez e t a l. (2 00 9) bu fo b uf o m 47 0. 74 ( 46 9. 56 -4 71 .9 3) 13 1. 02 ( 12 7. 27 -1 34 .7 8) 27 .8 3 (2 6. 97 -2 8. 70 ) f 37 8. 90 ( 34 0. 12 -4 17 .7 0) 11 5. 95 ( 11 1. 90 -1 20 .0 0) 30 .8 1 (2 8. 72 -3 2. 90 ) g ül a nd t ok ( 20 09 ) r an a ri di bu nd a r an a da lm at in a bu fo v ir id is bu fo b uf o h yl a ar bo re a pe lo ba te s sy ri ac us m ah ap at ra e t a l. (2 01 0) po ly pe da te s m ac ul at us m 58 2. 01 ( 47 4. 57 -7 00 .0 0) 13 7. 51 ( 11 9. 29 -1 46 .6 6) 26 .0 5 (2 0. 57 -3 2. 34 ) f 41 9. 56 ( 36 0. 57 -4 62 .1 6) 13 5. 82 ( 12 5. 00 -1 60 .0 0) 33 .4 9 (2 8. 06 -4 0. 00 ) 145haematology of turkish anurans in this study, the rbc counts were higher in terrestrial (p. viridis, p. syriacus and h. arborea) than in semi-aquatic (r. dalmatina) species. there were variations in wbc count of all species. hemoglobin concentration, hematocrit value, mcv and mhc were higher in terrestrial (p. viridis, h. arborea and p. syriacus) than in semi-aquatic (r. dalmatina) and aquatic (p. ridibundus) species. the mchc values were all similar to each other. the plasma total protein was higher in terrestrial species than in semi-aquatic species. in summary, it was reported that blood cell counts and sizes in anuran species displayed considerable individual variations and interspecies differences. furthermore, in this study, individual and interspecies variations were observed in the some hematological parameters examined on the anuran species. acknowledgements this study was supported by the scientific research foundation of çanakkale onsekiz mart university under project no: 2010/95. we would like to extend our gratitude to the foundation. references alder, a., huber, e. 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(2003): haematology of the fire bellied toad bombina bombina l., comp. clin. pathol. 12: 129-134. zhelev, z.m., angelov, m.v., mallov, i.a. (2006): a study of some metric parameters of the erythrocytes in rana ridibunda (amphibia: anura) derived from an area of highly developed chemical industry. acta. zool. bulp. 58: 235-244. zhukova, t.i., kubantsev, b.s. (1979): changes in the blood composition of salientians during hibernation. sov. j. ecol. 9: 379-382. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 7(2): 331-340, 2012 reproductive phenology of the tomato frog, dyscophus antongili, in an urban pond of madagascar’s east coast ori segev1,*, franco andreone2, roberta pala2, giulia tessa2, miguel vences1 1 zoological institute, technical university of braunschweig, mendelssohnstr. 4, 38106 braunschweig, germany. * corresponding author. e-mail: orisgv@gmail.com 2 museo regionale di scienze naturali, via g. giolitti 36, 10123 torino, italy submitted on: 2012, 29th june; revised on: 2012, 8th november; accepted on: 2012, 14th november. abstract. based on daily monitoring around an urban pond in the coastal town of maroantsetra, from 2003-2011, we provide an analysis of the yearly reproductive activity of the tomato frog (dyscophus antongilii), a large-sized and prominent red-coloured microhylid frog from north-eastern madagascar. frogs were observed all year round but despite the limited climatic seasonality in the region it was possible to identify a high activity period between january-may and a lower activity period between junedecember. freshly laid eggs were found in all months except november, and with highest incidence between january and may, while calling was heard in all months. we found a positive correlation between daily adult counts and minimum air temperature. on the contrary rainfall did not significantly predict activity, although boosts of calling and egg-laying especially in the austral winter were observed after heavy rainfall events. we define d. antongilii in maroantsetra as a sporadic wet season breeder that reproduces at irregular intervals following heavy rain events. keywords. amphibia, anura, microhylidae, maroantsetra, sporadic wet season breeder. anurans are characterized by a high variability in breeding seasonality among species, populations, and years. one end of the breeding phenology continuum constitutes of explosive breeders and the other of prolonged breeders (wells, 1977). environmental conditions or abiotic factors have been suggested as the determinants for anuran temporal breeding patterns (saenz et al., 2006; wells, 2007). duration and timing of breeding may be limited by rainfall (donnelly and guyer, 1994; bevier, 1997; gottsberger and gruber, 2004), temperature (bertoluci and rodrigues, 2002; saenz et al., 2006), or the availability of suitable sites for breeding (sullivan, 1982). temporally short breeding bouts were also suggested as an adaptation to reduce predation pressures (woodward and mitchell, 1990; lucas et al., 1996), cannibalism (petranka and thomas, 1995), and the energetic costs of reproduction (mccauley et al., 2000). in temperate and subtropical regions that are char332 ori segev et al. acterized by distinct dry and wet seasons, the reproduction of pond-breeding anurans occurs typically during the wet season while in those tropical and subtropical regions with little climatic variation throughout the year, more species demonstrate largely aseasonal breeding phenologies (crump, 1974). duellman and trueb (1994) divided an amazonian anuran community that reproduces all year round into continuous breeders that potentially breed every night, opportunistic breeders that breed regularly after heavy rains, and sporadic wet and dry breeders that breed at irregular intervals after heavy rains or throughout dry spells, respectively. they suggest that the availability of breeding sites dictates interspecies variation in breeding phenology. the true tomato frog or northern tomato frog, dyscophus antongilii is a representative of the madagascar-endemic microhylid subfamily dyscophinae that is related to asian microhylids (van bocxlaer et al., 2006; van der meijden et al., 2007). dyscophus is the sole genus in the subfamily, and it contains three species: d. insularis occurring in dry areas of madagascar’s west coast, d. antongilii, living in a small area of the north-east and east coast, and d. guineti, the false tomato frog, living in eastern mid-altitude rainforest areas (glaw and vences, 2007). dyscophus insularis is a medium sized (40-50 mm snoutvent length) and cryptically coloured species that breeds explosively in temporary as well as permanent ponds and has small tadpoles that complete their metamorphosis very fast (glos, 2003; grosjean et al., 2007; j. glos, pers. comm.), the two closely related eastern species d. antongilii and d. guineti are larger (60-101 mm svl), bright red-orange coloured, breed in temporary as well as larger permanent ponds and have large-sized tadpoles that, as far as known, require a longer time to complete metamorphosis (pintak, 1987; glaw and vences, 1994, 2007). while d. guineti is regularly exported from madagascar for the exotic pat-trade and assessed within the least concern threat category, d. antongilii is considered to be near threatened and is listed in the appendix i of the convention on the international trade in endangered species (cites) (raxworthy and nussbaum, 2000) which largely inhibits legal exports for the pet trade taking place from madagascar (andreone et al., 2005, 2008). due to the prominence of d. antongilii which in the area of the town of maroantsetra and the nearby masoala national park is considered as an important flagship species for conservation, several studies have addressed aspects of its genetic structure (chiari et al., 2006) and ecology (tessa et al., 2007, 2011). basic information on its activity, activity cycles, reproduction, and habitat choice is however still missing. here we contribute to closing this gap in knowledge and provide an analysis on the yearly activity of a population of d. antongilii located in an urban area within maroantsetra. this study was carried out in a pond within the urban area of maroantsetra, a small coastal town located in north-eastern madagascar that is located within an area of very high yearly rainfall. the town consists mainly of small buildings of one or two storeys built on sandy ground with a network of open ditches running throughout the town to drain rainwater and partly sewage water. most houses have gardens and numerous semiabandoned parcels are scattered throughout the town. d. antongilii occurs at many parts within the town, breeding both in ditches and in small and large ponds. the study pond (located at 15°25’47”s, 49°44’23”e) had a diameter of 6-7 m and was surrounded by fences and garden areas, very close to several huts and houses, partial333phenology of the tomato frog ly surrounded by a fence, and by some vegetation at the edges and in the water (fig. 1), and populated by domestic ducks. this anthropogenic habitat is in the property of a local nature guide in maroantsetra living in an adjacent house. the dyscophus population found in and around the study pond was relatively large. for instance, from 12-15 february 2003 we performed intensive searches and collected 78 individuals, and on 24 february 2004 we found six males (snout-vent length 62-67 mm, hereafter: svl) and 10 females (84-101 mm svl) of which 3-5 were toe-clipped recaptures from the previous year. during a total of nine years (2003-2011) the local nature guide in whose property the dyscophus study pond was located regularly undertook a daily monitoring of the tomato frog population in and around the pond, noting (1) the number of adult frogs fig. 1. photographs of the tomato frog, dyscophus antongilii and its habitat. (a) study pond with partial view of the garden. (b) adult male. (c) adult female. (d) study pond and surrounding vegetation. 334 ori segev et al. observed active on the ground and in the water, (2) presence or absence of egg clutches which float on the water surface, and (3) emission of calls by the frogs. because dyscophus males often call from concealed positions and the design of our study was so to obtain daily data by untrained observers, calling intensity was estimated in four relatively rough categories: 0 = no calls heard in the respective afternoon/evening, 1 = few isolated calls heard, 2 = regular calling, 3 = large choruses. a distinction between calls of different individuals was not possible, but the calling in large choruses certainly referred to multiple individuals calling simultaneously. presence of freshly laid eggs was scored as 0 = no eggs, 1 = few eggs, and 2 = large quantity of eggs corresponding to multiple clutches. when egg-laying took place on consecutive days, a distinction of newly laid eggs from those laid previously probably was not always possible for the untrained observers, but because the eggs hatch after 36 hours (pintak, 1987) this possible error is limited to a maximum period of two days. due to the limitations concerning the call and egg data, most of our statistical analyses are based on the counts of adults. although call, egg and adult count data is available for every year from 2003 to 2011, due to logistic reasons in several years there are gaps of several months in the dataset (e.g., no complete data for january-february 2003). for the period january 2003 to january 2004, data on daily minimum and maximum temperature (°c) as well as daily accumulated precipitation (mm) and daily mean relative humidity (%) were obtained from the meteorological station of maroantsetra, at a distance of less than 1 km from the study site; unfortunately, these meteorological data were not available for subsequent years. we used a non-parametric test, kruskal-wallis, to test for difference in adult numbers between years and between months. for the correlation analysis of 2003 climatic data with the phenological data we used pearson pairwise correlation for the adult counts and spearman rank correlation for the categorical eggs and calls. statistical analysis was carried out using the statistical software jmp in version 5.0 by sas institute, inc. and sigmaplot version 10.0 by systat software, inc. for the exponential curve-fitting of the non-linear regression analysis. a table with all original data has been deposited in the dryad data repository (http://dx.doi.org/10.5061/dryad.5h52h). our long term survey based on the data from 2003-2011 provides evidence that d. antongilii in maroantsetra breed almost all year round as reflected in the temporal distribution of adults, egg clutches, and calls (figs 2-3). the mean rank of the number of adults observed differed between months (kruskal-wallis; h = 20.87; df = 11; p > 0.05) and between years (kruskal-wallis; h = 16.26; df = 8; p > 0.05), with counts being particularly high through 2003 and particularly low through 2007 (fig. 4). despite the lack of apparent climatic seasonality in the region the breeding of d. antongilii can be roughly divided according to its activity level into a high activity period between january-may and a lower activity period between june-december (fig. 2). freshly laid eggs were found in all months except november, and with highest incidence between april and may, while calling was heard in all months (fig. 3) during both mornings and evenings. the number of adults observed was relatively stable over the years, with no trend of declining values (fig. 4). the maximum number of adult individuals observed per day during the transect walks around the pond were 12 in 2004, 20 in 2005, 17 in 2006, 20 in 2007, 35 in 2008, 27 in 2009, 30 in 2010, and 35 in 2011. 335phenology of the tomato frog we examined how d. antongilii breeding phenology co-varies with climatic variables recorded at maroantsetra meteorological station during 2003. interestingly, in 2003, weekly rainfall in maroantsetra was highest during some weeks of the austral winter, in june fig. 2. daily number of adults per month during 2003-2011. box boundaries indicate 25th-75th percentiles, the black line in the box indicates the median, the grey line indicates the mean, and the error bars indicate 10th-90th percentiles. fig. 3. mean number of days per month (± se) when eggs and calls were recorded, data from 2003-2011. note that bars are not cumulative but the number of days on which calls were heard also starts from zero. 336 ori segev et al. and july. this period of intense rainfall was then followed by breeding activity, with calls and eggs recorded. however, the more continuous breeding activity at constant high levels was observed in the austral summer, between march and may. consequently, we found a positive correlation between mean adult counts, and minimum air temperature (fig. 5). following this correlation analysis we performed a regression of daily mean adult counts versus daily minimum air temperature during 2003. adult activity increased nonlinearfig. 4. inter-year (2003-2011) variation in mean daily number (± se) of adults per month. numbers in bars indicate months per year included in the calculation. table 1. pearson pairwise correlation of daily mean adult counts, and spearman’s rank correlation of egg clutches, and calls recorded during 2003 with air temperature (daily maximum and minimum), daily rainfall, and daily mean relative humidity recorded at maroantsetra during 2003 (n = 10 months with frog data available, i.e., march-december). mean sd correlation coefficient p maximum air temperature (°c) 28.92 2.51 adults 0.44 0.204 eggs 0.265 0.459 calls -0.182 0.614 minimum air temperature (°c) 21.7 2.13 adults 0.657 0.039 eggs 0.524 0.120 calls 0.024 0.947 rainfall (mm) 17.14 10.91 adults -0.016 0.966 eggs 0.265 0.459 calls 0.194 0.590 humidity (%) 82.1 2.29 adults 0.059 0.870 eggs 0.226 0.530 calls 0.085 0.815 337phenology of the tomato frog ly with minimum temperature, and above 23°c there was an exponential increase in the mean daily number of adults (fig. 5). northern madagascar has two seasons, a hot, wet summer that lasts from november to april and a cooler, dry winter that lasts from may to october. the island climate is dominated by the south-eastern trade winds originating from the indian ocean. the eastern coast and particularly the bay of antongil area are exposed to almost constant blowing of these winds which carry hot humid air and heavy rains. despite the high research intensity recently targeting madagascar’s amphibian fauna the phenology of these animals has been little studied, with most of the breeding information collected during the wet season. malagasy reptiles reproduce typically during the wet season (glaw and vences, 1996) but breeding can also take place during the dry season, both along the west coast and on higher elevation mountains (vences et al., 2004). in areas of higher elevations, e.g. around 900 m above sea level where the highest species diversity of frogs occurs, low temperatures in the austral summer may be an important limiting factor for breeding activity. at low-elevation coastal areas, temperature is high year-round, but we nevertheless found that adult counts in maroantsetra co-varied with minimum air temperature, and breeding activity was not limited only to the wet season as evidenced by records of freshly laid eggs and calling individuals in all seasons (fig. 3). the bay of antongil area is featured by tropical wet and warm winter with no real dry season, mean annual temperatures of 24 °c and annual rainfall above 3000 mm. the combination of high temperatures and high rainfall during the entire year probably enables year-round reproduction for d. antongilii. adopting the reproduction mode division of duellman and trueb (1994) we thus define d. antongilii in maroantsetra as a sporadic wet season breeder that breeds at irregular intervals. fig. 5. relationship between daily mean number of adults per month (n = 11) versus minimum daily air temperature (°c) recorded at maroantsetra during 2003. 338 ori segev et al. a better knowledge of the phenology of dyscophus antongilii may have conservation implications beyond the species biology. understanding the interactions between climatic conditions, biological parameters (such as calling, mating, egg-laying), and their respective seasonality can serve to fine tune conditions for captive breeding, in case the species needs to be kept in captivity. one suggested factor driving the global decline of amphibian populations is the chytrid fungus (batrachochytrium dendrobatidis) (lips et al., 2006). global warming and changes in the thermoregulatory regimes of species might facilitate the global spread of the disease, with possible severe consequences for madagascar’s amphibians. in fact, the tomato frog has been shown to be susceptible to chytrid infection in captivity (von oevermann et al., 2005), and we can thus foresee that it might be one of the malagasy species affected by the pathogen in case of chytrid introduction to madagascar (andreone et al., 2012). human disturbance, as in our highly anthropogenic study site, does in principle not have negative consequences for d. antongilii conservation as indicated by the absence of a distinct decline in individual counts over the years (fig. 4). in maroantsetra, just close to the current study site, a small urban protected area has been purchased and enriched with artificial ponds (andreone, 2008) specifically for tomato frog conservation. our data suggest that it will probably be rather easy to ensure continuous reproduction of tomato frogs at such artificially created breeding sites. a crucial subject of future studies will be to obtain information how the development and survival of eggs, tadpoles and juveniles might be influenced by the disturbances prevailing in these urban environments (such as presence of ducks, sewage waters, or usage of fertilizers), in order to be able to boost the reproduction of this species under controlled conditions. acknowledgements we are grateful to numerous colleagues for their help during our madagascar work, in particular f. glaw, a. sarovy, and l. derussé. special thanks go to felix and to his family for carrying out the dyscophus monitoring thoroughly over all these years. the work in madagascar was made possible by cooperation agreements of the author’s institutions with the université d’antananarivo, département de biologie animale, and the parc botanique et zoologique de tsimbazaza. we are grateful to the malagasy authorities for research permits. the conservation and research on the tomato frog in maroantsetra was supported by grants of eaza, biopat, antongil conservation, and the zoo de doué de la fontaine. mv furthermore was supported by the volkswagen foundation and os by a fellowship of the minerva foundation. 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(2007): the ecology and behavior of amphibians. the university of chicago press, chicago and london. acta herpetologica vol. 7, n. 2 december 2012 firenze university press advertisement call of species of the genus frostius cannatella 1986 (anura: bufonidae) flora a. juncá1, david l. röhr2, ricardo lourenço-de-moraes3, flávio j. m. santos1, airan s. protázio1, ednei a. mercês1, mirco solé4 amphibians in southern apennine: distribution, ecology and conservation notes in the “appennino lucano, val d’agri e lagonegrese” national park (southern italy). antonio romano1,*, remo bartolomei1, antonio luca conte1, egidio fulco2 the significance of using satellite imagery data only in ecological niche modelling of iberian herps neftalí sillero1, josé c. brito2, santiago martín-alfageme3, eduardo garcía-meléndez4, a.g. toxopeus5, andrew skidmore5 reproductive strategy of male and female eastern spiny lizards sceloporus spinosus (squamata: phrynosomatidae) from a region of the chihuahuan desert, méxico aurelio ramírez-bautista1,*, barry p. stephenson2, xóchitl hernández-ibarra1, uriel hernández-salinas1, raciel cruz-elizalde1, abraham lozano1, and geoffrey r. smith3 morphological variability of the hermann’s tortoise (testudo hermanni) in the central balkans katarina ljubisavljević1, georg džukić1, tanja d. vukov1, miloš l. kalezić1,2 the usefulness of mesocosms for ecotoxicity testing with lacertid lizards maria josé amaral1,2,*, rita c. bicho1, miguel a. carretero2, juan c. sanchez-hernandez3, augusto m. r. faustino4, amadeu m. v. m. soares1, reinier m. mann1,5 does acclimation at higher temperatures affect the locomotor performance of one of the southernmost reptiles in the world? jimena b. fernández*, nora r. ibargüengoytía advertisement call of scinax littoralis and s. angrensis (amphibia: anura: hylidae), with notes on the reproductive activity of s. littoralis michel v. garey1, thais r. n. costa2, andré m. x. de lima2, luís f. toledo3, marília t. hartmann4 book review: marine s. arakelyan, felix d. danielyan, claudia corti, roberto sindaco, alan e. leviton 2011. herpetofauna of armenia and nagorno-karabakh. society for the study of amphibians and reptiles stefano scali book review: rafaqat masroor 2012. a contribution to the herpetofauna of northern pakistan stefano scali evidence of high longevity in an island lacertid, teira dugesii (milne-edwards, 1829). first data on wild specimens. j. jesus first assessment of the endoparasitic nematode fauna of four psammophilous species of tropiduridae (squamata: iguania) endemic to north-eastern brazil markus lambertz1,*, tiana kohlsdorf2, steven f. perry1, robson waldemar ávila3, reinaldo josé da silva4 rediscovery and redescription of the holotype of lygosoma vittigerum (= lipinia vittigera) boulenger, 1894 yannick bucklitsch1, peter geissler1, timo hartmann1, giuliano doria2 , andré koch1,* reproductive phenology of the tomato frog, dyscophus antongili, in an urban pond of madagascar’s east coast ori segev1,*, franco andreone2, roberta pala2, giulia tessa2, miguel vences1 range extension of the critically endangered true poison-dart frog, phyllobates terribilis (anura: dendrobatidae), in western colombia roberto márquez1,*, germán corredor2, carlos galvis3 daniel góez2, & adolfo amézquita1 differences in habitat use of two sympatric species of ameiva in east costa rica esther sebastián-gonzález1, ramón gómez2 acta herpetologica journal of the societas herpetologica italica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 9(1): 103-107, 2014 doi: 10.13128/acta_herpetol-12914 a preliminary report of amphibian mortality patterns on railways karolina a. budzik1, krystian m. budzik2 1 department of comparative anatomy, institute of zoology, jagiellonian university gronostajowa 9, 30-387 kraków, poland. corresponding author. e-mail: coffee8b@gmail.com 2 institute of botany, jagiellonian university kopernika 27, 31-501 kraków, poland on 2013, 6th november; revised on 2014, 20th february; accepted on 2014, 4th april 2014 abstract. in contrast to road mortality, little is known about amphibian railroad mortality. the aim of this study was to quantify amphibian mortality along a railway line as well as to investigate the relationship between the availability of breeding sites in the surrounding habitats and the monthly variation of amphibian railway mortality. the study was conducted from april to july 2011 along 45 km of the railway line kraków tarnów (poland, małopolska province). three species were affected by railway mortality: bufo bufo, rana temporaria and pelophylax kl. esculentus. most dead individuals (77%) were adult common toads. the largest number (14) of amphibian breeding sites was located in the most heterogeneous habitats (woodland and rural areas), which coincides with the sectors of highest amphibian mortality (42% of all accidents). as in the case of roads, spring migration is the period of highest amphibian mortality (87% of all accidents) on railroads. our findings suggest that railroad mortality depends on the agility of the species, associated primarily with the ability to overcome the rails. keywords. habitat effect, seasonality effect, common toad, poland. one of the main consequences of urbanization is the construction of new communication networks, e.g. linear infrastructures such as roads and railways. roads are physical barriers to animal migration, which may have negative consequences both in terms of animal mortality and habitat fragmentation (andrews and gibbons, 2005) and, in turn, may lead to isolation of populations through reduced movement and gene flow (gibbs, 1998; st. clair, 2003). among vertebrates, amphibians are the most affected by these threats (stuart et al., 2004). their requirement of aquatic habitats and reproduction-dependent seasonal migrations make them particularly vulnerable to the negative impact of road traffic (hels and buchwald, 2001; hamer and mcdonnell, 2008). apart from roads, railways may also act as migratory barriers and thus negatively affect amphibian populations (berthoud and antoniazza, 1998; ray et al., 2002). to date, the impact of railways on amphibians has not been established and, in contrast to the issue of amphibian road mortality (carr and fahrig, 2001; mazerolle, 2004; sirello, 2008; sutherland et al., 2010), data on amphibian mortality due to the presence of railways are very scarce (berthoud and antoniazza, 1998; vos et al., 2001; reshetylo and briggs, 2010). the aim of this study was to quantify amphibian mortality along a railway line and to investigate the effect of the surrounding habitat and the seasonal variation of railway mortality of amphibians. the study was conducted along 45 km of the line podłęże biadoliny (direction kraków tarnów, southern poland) (fig. 1). the railroad is constituted by two rail lines that split into several others where large stations occur. the track spacing is 1.435 m wide, and the height of the rail profile is 0.172 m. the substrate of the tracks is made of stones. the average daily number of trains running on this route in both directions is about 60. the trains run between 3:00 am and 23:00 pm. the average frequency of trains is 2-3 trains / h, increasing up to 3-4 trains / h from 14:00 to 20:00 (due to a lack of data, freight trains were not included). the study site included highly urbanized and agricultural 104 k.a. budzik, k.m. budzik areas, grasslands and forests. numerous ditches, oxbows and wetlands, as well as some larger water bodies, such as fish ponds, occur near the railway line and constitute potential breeding habitat for amphibians. the stretch was divided into 30 transects associated with different types of habitat. five types of transects were established: ‘woodland’ transects with woodland on both sides of the railroad (six transects; total length: 11.35 km), ‘woodland and rural areas’ transects with woodland on one side of the railroad and rural areas on the other side (four transects; total length: 6.16 km), ‘rural areas’ transects with rural areas on both sides of the railroad (10 transects; total length: 14.74 km), ‘open areas’ transects with open, natural areas (eight transects; total length: 9.8 km) and ‘urban areas’ transects (two transects; total length: 3.25 km). the study was conducted from april to july 2011. in april and may each transect was monitored twice a month, while in june and july, once a month. all transects were surveyed on foot. the duration of each survey was 1 to 3 hours. the surveys were conducted from the morning until the evening (often three or four transects a day), usually in sunny and dry weather. all findings of dead amphibians were georeferenced, photographed, and information on amphibian species and age (juvenile or adult) were taken. this detailed information ensured that we avoided recounting of dead individuals, even though we did not remove dead amphibians from the rails. additionally, the presence of dead reptiles was registered. a buffer zone of about 150 m on both sides of the railway was monitored for the presence of amphibians and potential reproductive sites at the same time as the railway mortality surveys. the inspections consisted of searching through all ditches, pools, puddles and water bodies, their edges and vicinities. the water reservoirs were also dipnetted. all individuals were released after identification in the field. the determination of amphibian presence was based on direct observations of adults and juveniles, as well as on observations of spawn, larvae and male mating calls. all observed green frogs were classified as pelophylax kl. esculentus. chi square tests were used to assess differences in railroad mortality depending on habitat type and month. additionally, differences in number of breeding sites in different habitat types were assessed. the analysis included only breeding sites of species affected by railroad mortality. then, differences between pairs of habitat types in respect to railroad mortality and breeding site abundance were tested. spearman’s correlation was used to measure the association between the number of dead specimens found on the railroad for each species with the number of reproductive sites found in the buffer zone. within the study area we found the following species (the number of breeding sites is given in parentheses): the agile frog rana dalmatina (23 sites), the common frog r. temporaria (7 sites), the moor frog r. arvalis (1 site), the green frogs pelophylax kl. esculentus (43 sites), the european tree frog hyla arborea (1 site), the fire-bellied toad bombina bombina (10 sites), the common toad bufo bufo (5 sites), the great crested newt triturus cristatus (4 sites), and the smooth newt lissotriton vulgaris (1 site). a total of 62 dead individuals of three species (b. bufo, r. temporaria and p. kl esculentus) were found within the area of the railway tracks. seven frog specimens were not identified. most dead amphibians were adult common toads (77%), and a large proportion of dead frogs (73%) were juveniles. the transect differed in terms of amphibian mortality (χ2 = 54.4, df = 4, p-value < 0.001): the majority of the amphibian mortality occurred in woodland and rural areas ( fig. 2, table 1). the buffer zone areas (habitat types) varied in terms of amphibian breeding site abundance (χ2 = 10.8, df = 4, p-value < 0.05). most of the breeding sites (of amphibians affected by railroad mortality) were located in the ‘woodland and rural areas’ type (table 2). the number of dead specifig. 1. location of the surveyed transect in poland. legend: a surveyed transect of railway line, b further railway line, c rivers, d forests. 105amphibian mortality on the railways mens and the number of reproductive sites occurring in the habitat types was not significantly correlated for any of the species. however, this association is present if all dead frogs (rana temporaria, pelophylax kl.esculentus and unspecified rana/pelophylax) are taken together (r = 0.436, p-value < 0.05). there is also a significant relationship between dead b. bufo and breeding sites abundance (r = 0.458, p-value < 0.001), if three breeding sites situated outside the buffer zone (up to 1.3 km in a straight line from the tracks) (budzik k. m., pers. inf.) are taken into account. the majority of dead amphibians were found at the beginning of the reproductive season (χ2 = 128.2, df = 3, p-value < 0.001; fig. 3). most dead amphibians found in april were spatially clustered, while in the following months the specimens were scattered. many of the toads (58%) were found within the railroad tracks and their remains were fragmented. the remaining individuals, as well as other dead amphibians, were not mechanically damaged. all dead frogs were found outside of the railroad track. additionally, in may we found one roadkilled fire-bellied toad under one of the rail viaducts. we found six dead grass snakes (natrix natrix), one of which was found near a dead common toad (fig. 4). to our knowledge, this study despite being largely exploratory reports the first empirical data on amphibian railway mortality. our results show that railway mortality is a real threat for amphibians, an issue that requires deeper evaluation for conservation planning. the amphibians found in the study area are common in this region of poland (głowaciński and rafiński, 2003). furthermore, two of the three species affected by railroad mortality (b. bufo, r. temporaria), are among the most common european amphibians, for which there is evidence of great road-mortality (orłowski, 2007; bonardi et al., 2011; matos et al., 2012). the high number of amphibians killed along woodland and rural areas is likely associated with the abundance of breeding sites in these types of habitats. however, the results predominantly relate to the common toad, therefore they are highly conditioned by this species, which typically inhabits heterogeneous habitats (pavignano et al., 1990). fig. 2. number of dead individuals (including their age) found in different types of habitat: woodland (a), woodland and rural areas (b), rural areas (c) and open areas (d). table 1. chi-square test comparing the railroad mortality between each pair of habitat types. “-” refers to low expected frequencies, test is not applicable. woodland woodland and rural rural open woodland and rural χ2 = 5.4, df=1, p < 0.05 rural χ2 = 11.5, df=1, p < 0.001 χ2 = 31.7, df=1, p < 0.001 open χ2 = 11.5, df=1, p < 0.001 χ2 = 27.7, df=1, p < 0.001 urban χ2 = 6.9, df=1, p < 0.01 χ2 = 13.2, df=1, p < 0.001 table 2. chi-square test comparing the number of b. bufo, r. temporaria, and p. kl esculentus breeding sites between each pair of habitat types. “-” refers to low expected frequencies, test is not applicable. ns: non significant p-value. woodland woodland and rural rural open woodland and rural χ2 = 1.7, df=1, ns rural χ2 = 2.7, df=1, ns χ2 = 8.4, df=1, p < 0.01 open χ2 = 1.1, df=1, ns χ2 = 4.8, df=1, p < 0.05 urban χ2 = 1.3, df=1, ns χ2 = 3.4, df=1, ns 106 k.a. budzik, k.m. budzik most dead amphibians were spatially clustered in april but this result appears conditioned by the large number of common toads and their mass migrations to breeding sites. toads were scattered after the breeding season, suggesting seasonal migrations towards feeding grounds. additionally, as in the case of roads (hels and buchwald, 2001; hamer and mcdonnell, 2008), spring migration seems to be the period of highest amphibian mortality on railroad tracks. undetermined frogs were probably representatives of the common frog or moor frog, which awakened from hibernation in april. the peak of green frog mortality in june may indicate dispersal in search of new habitats because of the gradual drying of habitat in ditches alongside the railroad tracks. the fragmentation of the remains of common toads clearly suggests that the direct cause of death was collision with a train. the short limbs of these animals reduce their ability of overcoming barriers such as rails. in addition, numerous studies have shown that amphibians are likely to remain immobile if faced with an approaching light (cornell and hailman, 1984; mazerolle et al., 2005). thus, it is possible that common toad activity can be disturbed under train light, increasing the risk of mortality. the toads that were not damaged, but trapped inside the track, probably died of dehydration. because dead frog individuals were not mechanically damaged, we suppose that they were probably hit by a train while trying to overcome the rails. the majority of dead frogs were juveniles. we suggest that most adults, able to hop farther and faster than the juveniles, may migrate more successfully. we did not find any dead individuals of the agile frog, the european tree frog, the fire-bellied toad or newts. as regards the latter, small-sized species may avoid the tracks because they are unable to cross them. to successfully migrate, their only option may be to avoid the rails and rather move along the viaducts: this suggestion is worthy of further investigation. however, this result may also be due to a sampling issue: on the one hand, small-sized amphibians dry up faster; on the other hand they may be crushed by a train; either way, this would make them very difficult to detect (dodd et al., 2004; mazerolle et al., 2005). there is also the possibility that small-sized amphibians may migrate through a gap under the railway. the agile frog and probably the other frogs seem to successfully cross the rails, probably thanks to their jumping ability. railroad mortality seems to depend on physical features (such as body size, limb length) and may be associated with the agility of the species. in the case of roads, agility was related mainly to velocity of the individual (schlupp and podloucky, 1994; hels and buchwald, 2001), while in the case of railroad tracks, agility relates primarily to the ability to overcome obstacles. due to its physical features, the common toad was more likely to become stranded at the rail, indicating that this species is more vulnerable to railway mortality. however, other species that do not cross the track because of their small body size may also be affected by the railroad, but at the level of gene flow (reh, 1989; vos et al., 2001) which represents a conservation issue that is worthy of further study. further investigations examining in detail the effect of individual physical features on amphibian railroad mortality, railway-related migration behavior of amphibians, as well as gene flow among amphibian populations isolated by railway line, are warranted. acknowledgement we would like to thank luca corlatti and a second anonymous reviewer for providing comments that greatly improved previous versions of this manuscript. fig. 3. number of dead individuals found in each month of the survey. fig. 4. dead common toad (bufo bufo, arrow) on the tracks with dead grass snake (natrix natrix). photo by k.m. budzik. 107amphibian mortality on the railways references andrews, k.m., gibbons, j.w. 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(2004): status and trends of amphibian declines and extinctions worldwide. science 306: 1783-1786. sutherland, r.w., dunning, p.r., baker, w.m. (2010): amphibian encounter rates on roads with different amounts of traffic and urbanization. conserv. biol. 24: 1626-1635. vos, c.c., antonisse-de jong, a.g., goedhart, p.w., smulders, m.j.m. (2001): genetic similarity as a measure for connectivity between fragmented populations of the moor frog (rana arvalis). heredity 86: 598-608. acta herpetologica vol. 8, n. 2 december 2013 firenze university press journal of the societas herpetologica italica acta herpetologica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 6(1): 59-80, 2011 the balearic herpetofauna: a species update and a review on the evidence samuel pinya1, miguel a. carretero2 1 espais de natura balear. conselleria de medi ambient del govern de les illes balears. c/ pintor josep coll bardolet, s/n. 07170 valldemossa, illes balears, spain. 2 cibio, centro de investigação em biodiversidade e recursos genéticos. campus agrário de vairão, 4485-661 vairão, portugal. corresponding author. e-mail: carretero@mail.icav.up.pt submitted on: 2011, 11th january; revised on 2011, 10th may; accepted on 2011, 10th may. abstract. here, we update the current list of amphibian and reptile fauna present in the balearic islands, probably the most outstanding case in the mediterranean and of the most in the world where massive species introduction is in conflict with the survivorship of highly restricted endemic taxa. resulting of a long term evolution in insularity, endemic herpetofauna was already decimated during the pleistocene but, after the human colonisation of the archipelago, the introduction of alien species, passive or deliberate, has been provoking new extinctions and range retractions in the native herpetofauna. such process is not interrupted but has even intensified during the last years. the current species list is composed by five amphibians (one native) and 21 reptiles (2 native). a critical review of the evidence on extinctions and introductions is provided together with the conservation implications. compared to the last review (mayol, 1985) six new reptile species are now naturalised or are in process of naturalization, colubrid snakes constituting the most conflicting element due to their predator role. keywords. balearic islands, amphibians, reptiles, alien species, conservation. introduction in a globalised world, the introduction of species out of their original ranges is becoming one of the most serious threats to biodiversity (iucn, 2000; nentwig, 2007). amphibians and reptiles are among the groups which are most widely introduced and simultaneously most threatened by introductions, the recent large monographs on this topic (lever, 2003; kraus, 2009) reflecting the acuteness of the problem. distinctions are to be made between alien (those transported and released outside their native ranges by human activities, intentionally or not), naturalised (those aliens maintaining reproductive populations) and invasive (those, aliens or not, that menace biodiversity) species (iucn, 60 s. pinya and m.a. carretero 2000; kraus, 2009). not all introduced species become naturalised although many do, and not all are invasive but some can be (kraus, 2009). of all the habitats and regions undergoing species introductions, islands are the most exposed since many alien species become invasive being harmful for local biota. native species evolving in insular conditions often loose ability for competing, eluding predators and facing disease, becoming vulnerable to alien competitors, predators or parasites (whittaker and fernández-palacios, 2007). as a result, many insular faunas deviate from equilibrium theory on the basis of geographical features (macarthur and wilson, 1967; whittaker and fernández-palacios, 2007) because of the repeated species invasions and substantial loss or decrease in their native species (iucn, 2000). amphibian and reptiles are not an exception to this rule and the cases of boiga irregularis in guam (savidge, 1987; engbring and fritts, 1988), anolis carolinensis in bonin islands (japan) (hasegawa et al., 1988) and anolis sagrei in cayman islands (losos et al., 1993) are especially paradigmatic. being one of the world biodiversity hotspots (myers et al., 2000), the mediterranean basin is unique in having so ancient interactions between biota and human activities (blondel and aronson, 1999). because of this, distinguishing autochthonous from allocthonous species and natural from modified habitats, may be difficult and often speculative. in fact, some authors have arbitrarily proposed neolithic for distinguishing between native and alien species within a region (manchester and bullock, 2000), although preneolitic interactions are also feasible. molecular tools and paleontological record are available for assessing both extinctions and introductions but these evidences are not free of uncertainty. actually, key remains may be lacking from fossil record and the best molecular calibrations do not unambiguously distinguish human introductions from recent natural colonisations. here, we update the current list of amphibian and reptile fauna present in the balearic islands, probably the most outstanding case in the mediterranean and of the most in the world where massive species introduction is in conflict with the survivorship of highly restricted endemic taxa. a critical review of the evidence on extinctions and introductions is provided together with the conservation implications. the (paleo)geographical framework and the human arrival the balearic islands are located in the western mediterranean, occupying an area of 4800 km2 (llorenç et al, 2007) which harbours a huge number of endemic species of fauna (pons and palmer, 1996) and flora (alomar et al., 1997). this high level of endemicity can be associated to the ancient isolation of the archipelago, which has not been connected to mainland since the end of the messinian, about 5.33 my bp (cavazza and wezel, 2003). the abrupt refilling of the mediterranean after this period (garcía-castellanos et al., 2009) was responsible for not only the definitive separation from the continent but also for the fragmentation between the pityusic (ibiza, formentera) and the gimnesic (mallorca, menorca) subarchipelagos. because both are separated by deep sea channels, they were never reconnected during the subsequent sea level fluctuations during the pliopleistocene. in contrast, shallow channels within subarchipelagos allowed repeated connections, especially during the glaciations (emig and geistdoerfer, 2004). http://en.wikipedia.org/wiki/robert_macarthur http://en.wikipedia.org/wiki/edward_o._wilson 61update of the balearic herpetofauna the first human settlements date from the third millennium bce (bover et al., 2008). paleontological mammal studies suggest coincidence with the disappearing of autochthonous species (myotragus balearicus bate 1909, hypnomys morpheus bate 1919, hypnomys mahonensis bate, 1919) and the first appearing of new species (atelerix algirus lereboullet, 1842, mustela nivalis linnaeus, 1766) in the fossil record (alcover, 1979a; bover et al., 2008). such coincidence of human arrival with extinctions, range retractions and introductions applies to herpetofauna and is common to other mediterranean islands (corti et al., 1999). nevertheless, human activities have intensified during the last century, completely modifying the appearance of the islands, particularly mallorca, the most damaged by the tourism industry. currently, pet keeping is also becoming extended, and fauna recovery centres are receiving a large list of different alien species. most of them have not succeeded in the naturalisation process, but those achieving it constituted an additional factor threatening native species. a recent analysis based on regression models establishes a link between anthropogenic factors and present-day extinctions and colonisations, at least for reptiles (ficetola and padoa-schioppa, 2009). however, some amphibian and reptile extinctions clearly predate the arrival of humans to the archipelago (bover et al., 2008). for menorca, plio-pleistocene reconnection to mallorca has been invoked as causative factor but reasons for the late pleistocene extinctions (i.e., in ibiza) remain unclear (bover et al., 2008), although they may be linked to temperature fluctuations. an updated species list the current list of amphibians and reptiles in the balearic islands is constituted by five amphibians (table 1) and 21 reptiles (table 2), including those reported here for the first time. mallorca is the island that harbours the highest species richness (22). the status of each species is briefly commented below. amphibians pelophylax perezi (seoane, 1885) the iberian water frog is considered introduced in unknown date probably for insect control. it is present in all the main islands but menorca (pleguezuelos, 2004; pérez-mellado, 2005) and it does not appear in the fossil record. however, water frogs are morphologically conservative and phylogenetic support for its allocthonous status and putative origin is still lacking (sumida et al., 2000; plötner and ohst, 2001). competitive interaction with the native alytes muletensis have been suggested (román, 2004) and predation on this species has been demonstrated (alcover et al., 1984). bufo balearicus boettger, 1880 among the green toads (bufo viridis complex), those inhabiting southern italy, corsica, sardinia and the balearics are now considered a distinct species (stöck et al., 2006). the presence of the species in the balearics is considered the result of an ancient, human62 s. pinya and m.a. carretero mediated introduction (mayol, 1985). lack of old fossils in the balearics and subfossils associated to neolithic settlements in the balearics, together with the similarities in mating calls and plasma proteins point to a tyrrhenian origin (hemmer et al., 1981). nevertable 1. list of amphibians of the balearic islands (ar: alien species without evidence of recent reproduction; na: naturalized alien species; nv: native species; -: absent). mallorca menorca ibiza formentera cabrera anura pelophylax perezi na ar na na bufo balearicus na na na alytes muletensis nv ex alytes obstetricans ar hyla meridionalis na table 2. list of terrestrial reptiles of the balearic islands (aa: acclimatised alien species without evidence of reproduction; ex: extinct; na: naturalised alien species; nr: native species without evidence of recent reproduction; nv: native species; -: absent). mallorca menorca ibiza formentera cabrera chelonia testudo hermanni na na testudo graeca na ex na emys orbicularis na na trachemys scripta na aa aa chrysemys picta na mauremys leprosa na aa chelydra serpentina aa squamata chamaeleo chamaeleon aa tarentola mauritanica na na na na na hemidactylus turcicus na na na na na podarcis lilfordi nv nv nv podarcis pytiusensis na nv nv podarcis sicula na na na? psammodromus algirus na scelarcis perspicillata na timon lepidus aa macroprotodon mauritanicus na na natrix maura na na malpolon monspessulanus aa na aa hemorrhois hippocrepis na na rhinechis scalaris aa na na aa 63update of the balearic herpetofauna theless, because transmarine colonisations have been demonstrated for this group (stöck et al., 2006) and the few samples analysed (stöck et al., 2006), more work is still needed to provide complete support for the polarity of the colonisation. the species is widespread in the gymnesics but populations in ibiza are decreasing (palerm, 1997). no interactions with local herpetofauna have been described (pleguezuelos, 2004). alytes muletensis (sanchiz and adrover, 1977) the autochthonous status of the balearic midwife toad is well supported by molecular, (fromhage et al., 2004, martínez-solano et al., 2004), morphological (martínez-solano et al., 2004) and paleontological evidence (alcover and mayol, 1981; bover et al., 2008). results from phylogenetic studies reasonably fit with a scenario of messinian vicariance with the betic-rifean alytes species (fromhage et al., 2004; martínez-solano et al., 2004). early pleistocene to holocene fossils (bover et al., 2008) suggest a widespread distribution in mallorca, the species being currently restricted to the serra de tramuntana mountain chain (román, 2004) where it was rediscovered alive only 30 years ago (mayol et al., 1981; mayol and alcover, 1981). holocene fossils from menorca, currently ascribed to a. muletensis (bover et al., 2008), also indicate recent extinction in this island. catastrophic predation by the viperine snake, natrix maura and, eventually competition with the pelophylax perezi, are considered responsible for this range retraction/extinction and continue menacing the surviving populations (román, 2004). experimental work, nevertheless, indicates that tadpoles may have developed anti-predatory response after the introduction of n. maura (griffiths et al., 1998). the accidental spread of the fungus batrachochytrium dendrobatidis, causative agent of the chytridiomycosis, to wild populations from captive breeding within a reintroduction program, constituted another serious threat for this species (walker et al., 2008). alytes obstetricans (laurenti, 1768) the common midwife toad is found in western europe, including great britain where it has been introduced (beebee and griffiths, 2000). the species was first reported for the balearics in 2007 in two localities of maó (menorca), where several individuals were observed, some of them males carrying egg masses (carrera and pons, 2010). its presence has been considered a consequence of trade of exotic live plants for ornamental purposes (carrera and pons, 2010) but phylogenetic studies are necessary to determine its origin considering the complexity of this group (martínez-solano et al., 2004). menorca is, then, the first island where its introduction has been documented (kraus, 2009). no interactions with native herpetofauna have been observed. hyla meridionalis (boettger, 1874) the stripeless tree fog is found in menorca where is considered introduced (esteban et al., 1994). no fossils exist but no menorcan specimens have been incorporated in the recent phylogenetic studies on this group (recuero et al., 2007; stöck et al., 2008). nevertheless, such studies coincide in indicating that even the iberian populations are introduced from north africa. further molecular studies should determine the origin of the menorcan populations. no inference with the extant native species is indicated (pleguezuelos, 2004). 64 s. pinya and m.a. carretero extinct amphibians painted frogs, discoglossus sp., have been described for the pleistocene of mallorca and plio-pleistocene of menorca (bover et al., 2008). the ancient origin of this genus (fromhage et al., 2004) suggests at least a messinian colonisation of the archipelago. the possible extinction of such forms during the middle pleistocene predates the arrival of humans and could be related to glaciations. latonia sp. (discoglossidae) is also known from the early pliocene of menorca but disappeared during the faunal turnover of the late pliocene, probably due to the contact with the mallorcan fauna (bover et al., 2008). reptiles testudo hermanni gmelin, 1789 the hermann’s tortoise ranges throughout the northern mediterranean basin and is found in many islands including mallorca and menorca, where is widespread. balearic subfossil remains from archaeological sites indicate ancient introduction (3000 year bp) probably as food source (mayol, 1985). molecular results corroborate the non-native status of this species in the balearics and a western mediterranean origin (fritz et al., 2006). no interactions with other herpetological species have been described but its role regarding herbivory and seed dispersal has not been investigated in the archipelago. testudo graeca linnaeus, 1758 the spur-thighed tortoise is native to north africa and the middle east. in the balearics it is restricted to w mallorca (calvià, e andratx and s puigpunyent) and a possibly extinct population in formentera. no fossil or subfossil remains are available, which advocates for a recent introduction, dated from 19th century (pleguezuelos, 2004). phylogenetic analysis support this hypothesis since no mtdna variation is found between mallorca and nw african coast (fritz et al., 2009). even if the species is not native to the balearics and other mediterranean inlands, these often healthy populations may be of conservation value eventually serving as back-ups if mainland populations were declining as considered by some authors (vamberger et al., 2011). similarly to the previous species, no information on interactions is available either with herpetofauna or with native flora. extinct terrestrial chelonians native land tortoises different from testudo are well represented in the post-messinian fossil record from the archipelago (bover et al., 2008). cheirogaster gimnesica (bate, 1914) is known from the pliocene of menorca and cheirogaster sp. from the plio-pleistocene of ibiza. both had disappeared during the climatic fluctuations in the middle pleistocene before the arrival of humans (bover et al., 2008). emys orbicularis (linnaeus, 1758) widespread in the western palaearctic, the european pond terrapin is widespread through the lagoons and the ponds of menorca and much localised in ne mallorca (albufera de alcudia). according to mayol (1985), the species would have been introduced as pet during the roman times or even earlier. this is supported by the lack of fossils (bover et al., 2008) and historical records (mayol, 1985). phylogenetic analysis (velo65update of the balearic herpetofauna antón et al., 2008) ascribes the menorcan specimens to the italian lineage, also reaching ne iberia, corsica and sardinia. no present interference with native species is detected and it is even menaced by the desiccation of wetlands (pleguezuelos, 2004). nevertheless, it has been hypothesised that may have contributed to the decline of alytes muletensis in both islands (pleguezuelos, 2004) at lowlands. trachemys scripta schoepff 1792 having its original range in the mississippi basin, the red-eared terrapin has now successfully established abundant breeding populations in central, south america, africa, asia and europe (lever, 2003). the arrival and breeding of this species to islands has also been confirmed (i.e., british virgin islands, owen et al., 2005; canary islands, rodríguezluengo, 2001; cayman islands, lever, 2003). the occurrence of this species in the balearics has previously been reported for mallorca (rivera and arrivas, 1993; avellà, 1998; pleguezuelos, 2004; pinya et al., 2007) and menorca (rivera and arrivas, 1993; avellà, 1998; filella, 1999; pleguezuelos, 2004; pérez-mellado, 2005), and its reproduction in mallorca (mas and perelló, 2001; pérez-mellado, 2005; pinya et al., 2007). now, its presence is extended to ibiza and formentera where its reproduction is still not confirmed. the origin in all cases is pet trade. in the natural park of s’albufera de mallorca, several dozens of animals are captured with funnel traps every year. recent studies in se france indicate that t. scripta excludes emys orbicularis from basking sites (crucitti et al., 1990; cadi and joly, 2003) decreasing its body condition and its increasing mortality (cadi and joly, 2004). potential effects for native herpetofauna as amphibian predator and pathogen transmitter have been suggested (martínez-silvestre et al., 2011). chrysemys picta (schneider, 1783) the painted turtle is native to united states and southern canada and has been imported as a pet to florida, philippine islands (uetz et al., 2010) and malta (despott, 1913). its presence has been reported for the s’albufera de mallorca natural park but its reproduction is still not confirmed (pinya et al., 2007). no interactions with native herpetofauna are known. mauremys leprosa (schweiger, 1812) the spanish terrapin has previously been reported for menorca (lortet, 1887; alcover, 1979b; alcover and mayol, 1981; rivera and arrivas, 1993), its arrival being related to pet trade (kraus, 2009). in mallorca, it is now successfully breeding in an abandoned lignite mine in the centre of the island (pinya et al., 2007). moreover, several males and females of adult sizes have recently been collected in the s’albufera de mallorca natural park (pinya et al., 2008). the same potential threatens mentioned for other terrapins apply to this species. chelydra serpentina (linnaeus, 1758) the common snapping turtle is native from north, central and northern south america. its introduction due to pet trade has been reported in other islands like the canary islands (pleguezuelos, 2004) or guam (leberer, 2003). the species was first reported for the balearics in 2004, when an almost 15 kg female was captured in the 66 s. pinya and m.a. carretero s’albufera de mallorca natural park (pinya et al., 2007). since then, two more adults have been captured when crossing roads. it is suspected that a small population may occur in this humid area but breeding is still not confirmed. in a second locality near palma de mallorca city, it has also been observed (pinya et al., 2007) without any evidence of reproduction. no interactions with the native or introduced herpetofauna have been observed although it exists a potential threaten to local aquatic fauna as birds, amphibians and reptiles, like e. orbicularis or p. perezi. chamaeleo chamaeleon (linnaeus, 1758) the common chameleon is a southern mediterranean species. it is widely distributed in most of the countries of north africa, middle orient, arabic peninsula, turkey, iberian peninsula, apulia (italy) and several islands from the aegean sea and malta (klaver, 1981, razzetti and sindaco, 2006). this range is at least partially resulting of introductions, and in some areas where the species was reported (i.e., sicily, crete) there is no evidence of naturalised populations. populations in southern iberia derive from two independent colonisations from morocco (paulo et al., 2002) whereas another species, chamaeleo africanus laurenti, 1768 was introduced from the nile delta, egypt to the peloponnese (dimaki et al., 2008). the common chameleon was never reported before for the balearic islands (mayol, 1985, bover et al., 2008). since 2004 some isolated individuals have been collected by fauna recovery centres across mallorca, but especially close to the most populated villages and cities. its presence appears to result from isolated individuals, as a consequence of pet trade. phylogenetic analysis of such specimens may reveal their putative relationships with the conspecific populations already analysed around the mediterranean basin (dimaki et al., 2008) although disentangle iberian from north-western african origin may be difficult. it is also noticed that transmarine colonisations have been demonstrated for other chameleon species (raxworthy et al., 2002). although some pregnant females have been collected in the wild, currently, the naturalization of the species in mallorca cannot be assumed. tarentola mauritanica (linnaeus, 1758) the moorish gecko ranges the iberian peninsula, nw africa and many other continental and insular areas throughout the mediterranean basin. phylogenetic evidence (harris et al., 2004a, 2004b; perera and harris, 2008; rato et al., 2010) suggests that the species has recently colonised the italian peninsula, the balkans and many mediterranean islands, probably by passive transport. selective forces have, however, been identified by comparing mtdna and ndna profiles (rato et al., 2010). samples from menorca, included in such studies, belong to the most widespread lineage found across the mediterranean clearly support its alien status, but the origin of the colonisation remains uncertain. on the other hand, unidentified gecko remains are also known from the pliocene of menorca (bover et al., 2008) which may suggest successive gecko colonisations and extinctions. although predation on lacertid juveniles has been reported, it shares habitat with podarcis lilfordi and p. pityusensis in many islets without any obvious negative effects on them (pleguezuelos, 2004). hemidactylus turcicus (linnaeus, 1758) the mediterranean gecko ranges the middle east, the mediterranean basin and also multiple sites around the gulf of mexico. analysis of mtdna (carranza and arnold, 67update of the balearic herpetofauna 2006) indicates a recent westwards expansion human-mediated from the middle east across the whole mediterranean region and then across the atlantic ocean. this includes the populations found in the balearic islands, the ascription of the menorcan pliocene gecko fossils to this species being unlikely. however, more recent analysis on ndna also suggests that selection may be an important force shaping the genetic diversity of this species (rato et al., 2011). no negative impact on native herpetofauna has been described (pleguezuelos, 2004). podarcis lilfordi (günther, 1874) the lilford’s wall lizard is endemic to the small islets around mallorca, menorca and cabrera. pliocenic menorcan fossils of small lacertids were identified as podarcis sp. whereas the abundant remain in both main islands from the early pleistocene to the holocene are ascribed to p. lilfordi (bover et al., 2008). molecular studies (brown et al., 2008; terrassa et al., 2009) are compatible with the paleogeological scenario, including the messinian colonisation, the vicariance between menorca and mallorca at the end of the pliocene and the pleistocenic separation of cabrera. in all cases, genetic distances between islets belonging to the same area are minute. interestingly, a population has survived in a tiny island inside a menorcan lagoon (triay, 2000). all this evidence, hence, corroborates both native status of current islet populations and its recent extinction in the main islands. such extinction is attributed to the introduction of alien predators, especially mammals and snakes (mayol, 1985), although the pliocenic ancestors of this species were in contact with snakes at least in menorca (bover et al., 2008). nevertheless, experimental studies have demonstrated relaxed mechanisms of tail autotomy in this species when compared to continental ones (pérez-mellado et al., 1997), which even decrease when rats are absent from the islets (pafilis et al., 2008). moreover, competitive exclusion by p. sicula has already taken place at least in one menorcan islet (pérez-mellado, 2002b). podarcis pityusensis (boscá, 1883) the ibiza wall lizard is endemic to ibiza, formentera and nearby islands. in contrast to p. lilfordi, it is present and abundant in the main islands and introduced populations are known for mallorca and the iberian peninsula (carretero et al., 1991; garcía-porta et al., 2001; pérez-mellado, 2002a). fossils attributed to podarcis sp. and to p. pityusensis are known for the pliocene and late pleistocene of ibiza, respectively. in the same way, phylogenetic studies (brown et al., 2008) support a messinian origin and the autochthonous status in the pityusic islands. the absence of macroprotodon mauritanicus from this subarchipelago is commonly invoked to explain the survival of the species in the main islands but experiments have also demonstrated that tail autotomy is easier in this species than in p. lilfordi (pérez-mellado et al., 1997). the single stable populations introduced in mallorca is urban and do not contact with p. lilfordi, which is restricted to the islets. podarcis sicula (rafinesque, 1810) the italian wall lizard mainly ranges the italian peninsula, corsica, sardinia and sicily, but some populations are also found in turkey, cyprus, israel, tunisia, france, iberian peninsula and united states. it is widespread in menorca where does not overlap with p. lilfordi and lacks fossil record. more recently, a colony has been in sant jordi, ses salines 68 s. pinya and m.a. carretero (mallorca). its phylogenetic affinities are with sicilian populations rather than with corsican or sardinian ones which suggest long distance introduction (podnar et al., 2005). together with this colonising capacity, this species is able either to displace native podarcis (nevo et al., 1972; downes and bauwens, 2002) or to hybridise with them (capula, 1993, 2002; capula et al., 2002). thus, it constitutes a serious threat for the balearic species, especially for p. lilfordi whose small and fragmented range makes it extremely vulnerable (pérez-mellado et al., 2008). in fact, if the information of an introduction from the mallorcan colony to cabrera would be confirmed, the important populations of native p. lilfordi will risk extinction. psammodromus algirus (linnaeus, 1758) widely distributed across se france, iberian peninsula and nw africa, the large psammodromus has recently been reported for several localities from se mallorca (masius, 1999; vicens, 2005). current observations in other parts of the island suggest that it is starting to spread out to s mallorca (llucmajor). further analysis of genetic markers will determine whether the source population is located in iberia or in north africa (carranza et al., 2006, carretero et al., 2009). it must be remarked that the high reproductive output (carretero and llorente, 1997), the favourable habitat (carretero et al., 2002) and the lack of other lacertid species in non urban areas of mallorca, may favour a rapid progression of the species in the island with unknown consequences. it is, for instance, expectable that if abundant it could serve as prey and keeping high numbers of predators as macroprotodon mauritanicus, or the newly introduced colubrids (see below), then preventing any eventual reintroduction of native lizards. scelarcis perspicillata (duméril and bribon, 1839) the moroccan rock lizard ranges the atlas and other mountain massifs of morocco, nw algeria as well as menorca where it has an irregular distribution associated bare rocks, walls and cliffs. it was considered introduced based on the disjoint distribution with the african main range, the association of some populations with human buildings and the lack of fossils (perera, 2002). however, the menorcan populations are, in fact, morphologically and genetically distinct from the moroccan populations studied to date (harris et al., 2003; perera et al., 2007). only a comparative study in the whole species range, including algerian populations, will allow demonstrating its alien status in menorca. the species does not contact with the native p. lilfordi and displays habitat segregation with the introduced p. sicula. timon lepidus (daudin, 1820) the ocellated lizard is a typical reptile from the western european regions under mediterranean climate (mateo, 2002) including nw italy, sw france and most of the iberian peninsula (mateo and cheylan, 1997). in the last decade some individuals have been observed in the se of palma de mallorca city (mallorca), in an old sandstone quarry. there is no evidence of reproduction in this site since neither juveniles nor egg were found. although overseas dispersal has been postulated for the genus (paulo et al., 2008), a single introduction of isolated individuals is more likely. in nature, the species predates upon small lacertids (castilla et al., 1991) but no native species are present in the mallorca main island. 69update of the balearic herpetofauna macroprotodon mauritanicus guichenot, 1850 after the recent taxonomic revision (wade, 2001; carranza et al., 2004) the false smooth snakes, macroprotodon sp., have been split into several taxa. one of them, m. mauritanicus, occupies n tunisia, nw algeria, mallorca and menorca. on the basis of the separate range, almost null genetic variation between mallorca and africa (carranza et al., 2004) and lack of fossil record (bover et al., 2008), the species is considered introduced in the balearics, probably from roman times (pleguezuelos, 2004). it has been considered responsible for the extinction of p. lilfordi in mallorca and menorca, due to its saurophagous diet and the temporal coincidence between its arrival to the balearics and the extinction of this species in the main islands (mayol, 1985). natrix maura (linnaeus, 1758) the viperine snake is distributed on both continental sides of western mediterranean, being considered introduced in sardinia, mallorca and menorca. in the gymnesics, where no fossils are available, the species would have been imported from ancient times (alcover and mayol, 1981). molecular studies corroborate its alien status in mallorca and point to a european origin (guicking et al., 2006). in serra de tramuntana (mallorca), the presence of this species is negatively correlated with tadpole density of a. muletensis (pleguezuelos, 2004) which suggests that its predation pressure constitute an important threat for the species (román, 2004). the species have been supposed to be responsible for the extinction of a. muletensis and discoglossus sp. in menorca (pleguezuelos, 2004). the first seems realistic while the second is unlikely since the painted frogs had disappeared earlier in the fossil record (bover et al., 2008). malpolon monspessulanus (hermann, 1804) the montpellier snake occupies a circum-mediterranean range although the western and eastern populations are strongly separated in the phylogeny and may represent different species (carranza et al., 2006). it has recently been reported for the balearics. namely, its arrival is associated with the trade of old olive trees used for ornamentation which snakes were found hidden inside the roots (oliver and álvarez, 2010; álvarez et al., 2010), the same way of p. sicula in hyères island, france (bruekers, 2003) and in rioja, iberian peninsula (valdeón et al., 2010). currently, there are several observations and captures of adult individuals of both sexes in mallorca, ibiza and formentera (álvarez et al., 2010), suggesting that breeding may be a matter of time. ensuring the origin of the introductions may be difficult if the samples carry the most widespread haplotypes that are found both in iberia and north africa (carranza et al., 2006). currently, the species had been considered introduced only in lampedusa, between sicily and tunisia (corti and lo cascio, 2002). in the future, this euryphagous snake may represent a serious threat for native lacertid species, especially in ibiza, and, if reached, in the small islets, which is likely considering its swimming abilities. hemorrhois hippocrepis (linnaeus, 1758) the horseshoe snake is found in the iberian peninsula and nw africa, as well as in some mediterranean islands (zembra, pantelleria, sardinia) were is said to be introduced (bruno and hotz, 1976; pleguezuelos and feriche, 2004). original observations indicate 70 s. pinya and m.a. carretero that this species arrived by the same way and almost at the same time than m. monspessulanus. its reproduction is confirmed by the observation of juveniles and pregnant females in capdepera, nw mallorca, although recently several individuals have been observed in the rest of the island. currently, there is a dense population in mallorca and it also has arrived to ibiza where it started to reproduce (oliver and álvarez, 2010; álvarez et al., 2010). uncertainty about the origin would also affect this species when molecular markers could be analysed due to the same reason than in the previous species (carranza et al., 2006). threatens for native herpetofauna could also be similar. rhinechis scalaris (schinz, 1822) the ladder snake is endemic to iberian peninsula and adjacent se france. it was previously reported as a member of the balearic herpetofauna (alcover and mayol, 1981; kotsakis, 1981; mayol, 1985) although its presence was restricted to menorca (esteban et al., 1994), where no fossils are known. the preliminary phylogeographic analysis of this species (nulchis et al., 2008), not including menorcan samples, found no variation in the mtdna. no new haplotypes are, hence, expected for menorca but if they would appear would pose doubts on its introduced status. recent observations, however, indicate its presence also in mallorca, ibiza and formentera, where it has arrived by the same way as m. monspessulanus and h. hippocrepis (oliver and álvarez, 2010¸ álvarez et al., 2010). nevertheless, its biological impact on herpetofauna is expected to be less serious in comparison to those species because it almost exclusively consumes birds and mammals (pleguezuelos et al., 2007). extinct snakes and amphisbaenians as previously commented, fossil snakes, namely, viperids (vipera natiensis bailon, garcia-porta and quintana-cardona, 2002 and vipera sp.) and colubrids (coluber sp.) are know for the early and middle pliocene of menorca (bailon et al., 2002; bover et al., 2008). an amphisbaenian (blanus sp.) is also found in the same assemblages (bover et al., 2008) in agreement other plio-pleistocenic remains from sardinia, sicily and the italian peninsula (delfino, 2003). both snakes and worm lizards disappeared from the balearic fossil record in the pleistocene (bover et al., 2008). to summarise, prior to the humans arrival, the balearic herpetofauna was the result of a long term evolution in insularity producing pliocenic amphibian and reptile lineages endemic to the three main island groups (figure 1). such endemic taxa were decimated during the pleistocene sea level and climate fluctuations. after the human colonisation of the archipelago, the introduction of alien species, passive or deliberate, caused new extinctions and range retractions in the native herpetofauna. such process is not interrupted but has even intensified during the last years (fig. 1). of the current herpetofauna of the balearic islands, just one amphibian (a. muletensis), and two reptiles (p. lilfordi and p. pityusensis) are considered native. thus, since humans arrived it has become an increasing of amphibian richness of five times and about 10.5 times in case of reptiles (fig. 2), being the squamata the order which included a highest number of new species. the last updated checklist of amphibians and reptiles (mayol, 1985) considered a total of four amphibians and 15 reptiles with a percentage of introduced 71update of the balearic herpetofauna species in amphibians of 75% and of 60% in reptiles. since then a total of six new species of reptiles have been naturalised or are in process of naturalization in the balearic islands. the considerable biogeographic differences between the alien species are to be taken into account. some of them come, in fact, from historical introductions which may suppose either that they are now integrated in the balearic ecosystems or that their removal would be even more traumatic. this may apply to the geckos, the european pond terrapin and the land tortoises. beyond their origin, their conservation actions require cautiousness, particularly when they are endangered in their original ranges (for a discussion on this topic, see böhme, 2002). in such cases (i.e., emys orbicularis) preventing the impact from new aliens as in the case of would be advisable. on the other hand, evidence is still insufficient fig. 1. ancient and recent changes in the balearic terrestrial herpetofauna. 72 s. pinya and m.a. carretero to determine the status of some species and a principle of caution suggest further research before taking management decisions (i.e., b. balearicus, h. meridionalis, s. perspicillata). on the other hand, colubrid snakes constitute the most conflicting aliens due to its predator role. in mallorca, they are expected to predate upon the geckos and the recently introduced psammodromus algirus, as well as local mammals and birds. however, in ibiza and formentera, the occurrence of snakes in the wild was never documented before. impact on p. pityusensis could be similar to the case of p. lilfordi after the introduction of m. mauritanicus and other vertebrates some centuries ago to mallorca and menorca which caused the extinction of its populations in the main islands. effects could be even more acute since the new alien snakes have a higher predator potential than the african false smooth snake. in consequence, it can be considered that the threat to the endemic herpetofauna in the balearic archipelago is increasing. although time passes, the list of amphibians and reptiles continues growing as it is showed since the eighties (mayol, 1985) and probably will not stop if legal effective measures are not taken seriously to control the entrance of alien species to these islands. acknowledgements the work of mac was supported by the projects ptdc/bia-bde/67678/2006 and ptdc/ bia-bec/101256/2008 funded by fundação para a ciência e a tecnologia (portugal). references alcover, j.a. 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(2007): island biogeography. oxford, oxford university. bbib67 ole_link1 ole_link2 bbib28 ole_link5 ole_link6 ole_link7 ole_link8 _goback ole_link1 ole_link2 ole_link3 ole_link4 ole_link1 ole_link2 ole_link19 ole_link20 ole_link21 ole_link29 ole_link3 ole_link4 ole_link5 ole_link31 ole_link14 ole_link15 ole_link12 ole_link13 ole_link16 ole_link17 ole_link22 ole_link23 ole_link24 ole_link8 ole_link9 ole_link10 ole_link11 ole_link18 ole_link27 ole_link28 ole_link25 ole_link26 ole_link6 ole_link7 ole_link34 ole_link37 ole_link38 acta herpetologica vol. 6, n. 1 june 2011 firenze university press widespread bacterial infection affecting rana temporaria tadpoles in mountain areas rocco tiberti extreme feeding behaviours in the italian wall lizard, podarcis siculus massimo capula1, gaetano aloise2 lissotriton vulgaris paedomorphs in south-western romania: a consequence of a human modified habitat? severus d. covaciu-marcov*, istvan sas, alfred ş. cicort-lucaciu, horia v. bogdan body size and reproductive characteristics of paedomorphic and metamorphic individuals of the northern banded newt (ommatotriton ophryticus) eyup başkale1, ferah sayım2 , uğur kaya2 genetic characterization of over hundred years old caretta caretta specimens from italian and maltese museums luisa garofalo1, john j. borg2, rossella carlini3, luca mizzan4, nicola novarini4, giovanni scillitani5, andrea novelletto1 the phylogenetic position of lygodactylus angularis and the utility of using the 16s rdna gene for delimiting species in lygodactylus (squamata, gekkonidae) riccardo castiglia*, flavia annesi localization of glucagon and insulin cells and its variation with respect to physiological events in eutropis carinata vidya. r. chandavar1, prakash. r. naik2* the balearic herpetofauna: a species update and a review on the evidence samuel pinya1, miguel a. carretero2 effects of mosquitofish (gambusia affinis) cues on wood frog (lithobates sylvaticus) tadpole activity katherine f. buttermore, paige n. litkenhaus, danielle c. torpey, geoffrey r. smith*, jessica e. rettig food composition of uludağ frog, rana macrocnemis boulenger, 1885 in uludağ (bursa, turkey) kerim çiçek preliminary results on tail energetics in the moorish gecko, tarentola mauritanica tommaso cencetti1,2, piera poli3, marcello mele3, marco a.l. zuffi1 climate change and peripheral populations: predictions for a relict mediterranean viper josé c. brito1, soumia fahd 2, fernando martínez-freiría1, pedro tarroso1, said larbes3, juan m. pleguezuelos4, xavier santos5 assessing the status of amphibian breeding sites in italy: a national survey societas herpetologica italica* osservatorio erpetologico italiano acta herpetologica journal of the societas herpetologica italica acta herpetologica rivista della societas herpetologica italica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 7(1): 69-80, 2012 is the exploratory behavior of liolaemus nitidus modulated by sex? jaime troncoso-palacios1, antonieta labra1,2,* 1 programa de fisiología y biofísica, facultad de medicina, universidad de chile, casilla 70005, correo 7, santiago, chile 2 university of oslo, department of biology, centre for ecological and evolutionary synthesis, p.o. box 1066 blindern, n-0316 oslo, norway. *corresponding author. e-mail: a.l.lillo@bio.uio.no submitted on: 2012, 10th january; revised on: 2012, 13th february; accepted on: 2012, 11th march. abstract. chemoreception is an important sensory modality used by lizards to assess their environments and to communicate. however, despite growing information regarding chemoreception in this taxon, its modulation by sex has been little explored, except in researches directly focused on reproductive aspects. in this study, we compared the responses of females and males of the iguanid lizard liolaemus nitidus to scents from conspecifics of the same sex, themselves (own), a predator, and a control. the only stimulus that induced different responses between sexes was the scent of conspecifics; males reacted sooner than females to these scents in agreement with their lower tolerance of potential sexual competitors. the similar ecology of the sexes may explain the similarities in their responses to the other scents we tested. however, independent of the scents, we found major behavioral differences between the sexes (e.g. males always tail waved for longer), pointing intrinsic sexual variation in behaviors associated to exploration. keywords. chemical recognition, sexual variation, ecology, predation risk, tail waving introduction lizards are highly dependent on the chemical modality to communicate as well as to explore and assess their environments, depending heavily for this on the vomeronasal organ (filoramo and schwenk, 2009; mason and parker, 2010). the tongue collects molecules from the environment and brings them to the vomeronasal organ, and the rate of tongue-flicking is usually used as a bioassay for chemorecognition (mason and parker, 2010). using this assay it has been shown that lizards discriminate between scents of different conspecifics (moreira et al., 2008), and between conspecifics and congeneric spe70 j. troncoso-palacios and a. labra cies (labra, 2011). more specifically, males can assess the competitive abilities of potential opponents (labra, 2006), as well as the reproductive condition of females (head et al., 2005), while females can assess the quality of males (martín and lópez, 2006) and discriminate their own offspring (main and bull, 1996). in non-social contexts, lizards are able to detect areas scented by prey (labra, 2007; besson et al., 2009) or predators (lloyd et al., 2009). results obtained by tongue-flicks have also been confirmed or reinforced by including other behavioral traits, such as headbobs (labra, 2006), tail displays (alonso et al., 2010), bites (khannoon et al., 2010), slow motion (labra and niemeyer, 2004) and time in motion (labra, 2007). despite the increasing volume of information on lizard chemoreception (mason and parker, 2010), almost no effort has been put into exploring the role of sex in this reception (see sampedro et al., 2008), except in studies directly focused on reproductive behavior: sexual selection (martín and lópez, 2008) and species recognition (barbosa et al., 2006; gabirot et al., 2010). these, as well as studies in taxa such as mammals (dorries et al., 1995; baum and keverne, 2002), have showed that sexual variation in response to scents is related to reproduction, which has been correlated with sexual dimorphism in brain areas and neural circuits involved in chemoreception (lizards: sampedro et al., 2008; mammals: suárez and mpodozis, 2009; stowers and logan, 2010). however, this sexual dimorphism does not necessarily imply sexual variation in the chemorecognition of non-reproductive scents. food, for example, might trigger the same behavior in both sexes (stowers and logan, 2010). to shed some light on whether sex modulates lizards’ chemorecognition, we compared the responses of males and females of the iguanid lizard liolaemus nitidus to nonreproductive scents normally found in the environment: from themselves, a snake predator, and a control (free of scents from l. nitidus or other species). we also compared the sexes’ response to conspecific of the same sex, which can be important in intersexual competition. materials and methods animals and their maintenance liolaemus nitidus is a common and widespread species from central chile (mella, 2005). during the austral spring of 2009 (november december) we collected lizards (10 males and 10 females) by noose in “el tabo” (33° 29’s77° 38’w), central chile. the mean snout-vent length of males and females were 82.71 ± 3.67 (se) and 74.24 ± 2.5 mm, respectively. four individuals (1 male and 3 females; snout-vent length: 60.9 ± 3.17 cm) of the snake philodryas chamissonis were collected from different localities in central chile, far away (~ 120 – 150 km) from “el tabo”. this snake species is one of two species that inhabit central chile, with the most saurophagus habits of these species (jaksic et al., 1982), and it preys on l. nitidus (lobos et al., 2009). in the laboratory, individuals of both species were sexed, weighed, and their snout-vent lengths were measured. lizards were placed in an indoor vivarium equipped with halogen lamps that kept the room with conditions similar to a typical local summer day: temperature between 12 and 32°c and a photoperiod of 13l:11d. animals were housed individually in plastic enclosures (42 x 29 x 24 cm) with a frontal window (10 x 14 cm) covered with plastic mesh. the lids of the enclosures were partially replaced by a plastic mesh (16 x 29 cm), which allowed more light and ventilation, and added extra surface to climb. enclosures had a substrate of sand (3 cm), a wooden stick used by lizards to climb and 71is the exploratory behavior of liolaemus nitidus modulated by sex? bask, and two bowls, one for water and the other, inverted, to provide a shelter and a basking place. water was supplied ad libitum and food (mealworms) was supplied every other day (always dusted with vitamins). snakes were maintained in a separate room in identical conditions to those used for lizards (i.e. terraria, photoperiod and temperature). they were fed once per week with a mouse of approximately 21% of snake weight (similar to the weight of an adult l. nitidus), as was approved by the bioethical committee. experiment design animals remained in their enclosures for one week without disturbance to allow them to acclimate to the experimental conditions and to release scents, since enclosures were used for substrate-borne scents. for the experiments, the focal individual was removed from its enclosure and held in a cloth bag for 10 min to reduce handling-associated stress (labra, 2011). thereafter, the bag was carefully opened allowing the animal to move freely into the experimental enclosure. lizards were tested individually and randomly in enclosures used by: (1) a conspecific of similar size and same sex as the tested lizard, (2) the tested lizard (own), (3) an individual of p. chamissonis (snake) and (4) an unused enclosure (control) without scents from a heterospecifc or conspecific, which contained new sand of the same type as the maintenance enclosures. for the experiments, the inhabitant of the experimental enclosure was removed just before the trial, together with the bowls and the stick. between trials, control enclosures were rinsed with abundant water containing a bit of detergent to eliminate residual chemical traces from the tested lizard; the sand was replaced. potential biases in behavior due to variations in body temperature were avoided by recording the cloacal temperature of the tested lizard at the end of the trial. if values were not close (± 3°c) to the mean selected body temperature of the species (35°c; labra, 1998), the trial was disregarded and repeated later. lizards were subjected to one trial per day, and at the end of the experiments, they were returned to their own enclosures in which they were kept undisturbed for at least three days. after the focal lizard was placed in the experimental enclosure, we recorded the latency to the first tongue-flick, i.e. time elapsed between the introduction of the lizard into the enclosure and the initiation of the first tongue-flick (see below). once the lizard licked, we filmed its behavior for 10 min with an 8-mm digital video camera placed 50 cm above the terrarium. from the videos, and based on previous studies (e.g., labra and niemeyer, 2004; labra, 2006, 2007), we recorded the following variables: (1) motion time: the total time that lizards made adjustments of the body posture, displacements, or head movements (scanning), excluding any motion arising from the behaviors listed below. (2) latency to the first movement of the tail: time elapsed between the first tongue-flick and the first tail movement, and (3) tail waving: the total time that the entire tail, or its posterior portion, was moved rapidly from side to side. these three variables were recorded directly with a stopwatch. (4) tongue-flicks: the number of protrusions and rapid retractions of the tongue, regardless of whether it touched the substrate or was waved in the air. other behaviors (gaping, digging, slow motion, headbobs, marking behaviors, and self-licking) were observed, but their low frequency precluded any further test, although one of this is discussed later. animals were maintained in good condition during the entire period of experimentation and were returned to their capture sites at the end of the study. statistical analysis to achieve normality of residuals, latency to the first tongue-flick and number of tongueflicks were log10 and square-root transformed, respectively. for these two variables, as well as for motion time, the effects of sex, treatment, and their interaction were determined by two-way 72 j. troncoso-palacios and a. labra anovas with repeated measurements for treatments. thereafter, lsd post-hoc tests were applied. because the residuals of the two variables related to the tail did not achieve normality even after transformations, they were analyzed using the non-parametric friedman’s anovas followed by conover post-hoc tests. sexual differences in these two variables were investigated using a mann– whitney test. data are shown as untransformed mean ± se. statistical significance was set at p< 0.05 and all tests were two-tailed. analyses were made with brightstat (stricker, 2008). results latency to the first tongue-flick was affected by sex and by its interaction with the treatment (table 1); males had shorter latency than females (lsd test: p = 0.0389). the interaction revealed that in conspecific enclosures, males tongue-flicked earlier than females (fig. 1; p = 0.0267). motion time was only affected by the treatment (table 1); lizards were more active in the enclosure of conspecifics than in their own or snake enclosures (fig. 2; p = 0.0012 and p = 0.0102, respectively), as well as in the control enclosure, although this was not statistically significant (p = 0.062). the treatment also affected the number of tongue-flicks (table 1); lizards did it less in their own enclosures than in those of conspecifics or snakes (fig. 3a; p = 0.0024 and p = 0.0275, respectively), and also less than in the control enclosure, although this was not statistically significant (p = 0.061). the interaction between sex and treatment was also significant (fig. 3b; table 1); females tongue-flicked less in their own enclosures than in the enclosures of conspecifics or controls (p = 0.0002 and p = 0.0279, respectively), and made fewer tongue-flicks in the snake enclosure than in conspecific enclosures (p = 0.0143). the latency to the first tail wave differed marginally among treatments (friedman anova x2 = 7.78, p = 0.0507); lizards waved the tail sooner in the snake and control enclosures than in their own enclosures (conover test: p < 0.05, in both cases). however, there was a clear sexual difference in this latency; males waved the tail before females (fig. 4a). the total time that lizards waved their tails differed among treatments (x2 = 10.9, p = 0.0120); this was shorter in their own than in the snake and control enclosures (conover test: p < 0.01). finally, males waved the tail for longer than females (fig. 4b). table 1. repeated measures analysis of variance, testing for the effects of sex, treatment (conspecific of the same sex, own, snake, and control enclosure), and their interaction on three response variables, recorded in liolaemus nitidus with statistically significant values in bold. df latency to the first tongue-flick motion time number of tongue-flicks f p f p f p sex 1,54 4.962 0.0389 1.620 0.2193 0.727 0.4050 treatment 3,54 0.236 0.8709 4.305 0.0086 3.600 0.0191 sex x treatment 3,54 2.981 0.0393 1.846 0.1498 3.012 0.0379 73is the exploratory behavior of liolaemus nitidus modulated by sex? discussion females and males of l. nitidus shared many similarities in their behavioral responses to the different chemical environments, which could be a consequence of their similar ecologies (see below). interestingly, however, independent of the actual scents they fig. 1. mean latency to the first tongue-flick (+se; seconds) exhibited by males and females in four experimental conditions. fig. 2. mean total motion time (+ se; seconds) exhibited by l. nitidus in four experimental conditions. 74 j. troncoso-palacios and a. labra encounter, the sexes showed major differences in the way they behave in the different environments. below, we discuss the responses related and unrelated to the encountered chemical stimuli separately. fig. 3. mean total number of tongue-flicks (+ se) displayed by l. nitidus in four experimental conditions. a. total average. b. data separated by sex. 75is the exploratory behavior of liolaemus nitidus modulated by sex? ichemorecognition of different scents conspecific. the only stimulus that caused different responses between sexes was the scent of conspecifics, which triggered faster chemical exploration in males (i.e. shorter latency to the first tongue-flick). this sexual difference can be consequence of males experiencing a higher selective pressure to recognize and react against conspecifics of the same fig. 4. mean a. latency to the first tail wave (+se; minutes) and b. total time waving the tail (+ se; seconds) exhibited by males and females of l. nitidus. the results of the mann–whitney tests are shown. 76 j. troncoso-palacios and a. labra sex, considering that male home ranges do not overlap, while those of females overlap by approximately 12% (fox and shipman, 2003). in this regard, a faster reaction of an invader male (the tested lizard) to scents found in the “territory” of another male speeds up the collection of information, which may allow the invader to assess the fighting abilities of the territory owner and react accordingly (labra, 2006). interestingly, cooper et al. (1996) found that both sexes of cordylus cordylus tongue-flicked more to conspecifics of the same sex, which was attributed to the high territoriality of both sexes in this species. thus, we can hypothesize that territoriality may be a modulator of response to scents of conspecific of the same sex, i.e. higher territoriality, stronger response to these scents. what explains the different reactions of the sexes of l. nitidus to conspecific scents before the first investigation with the tongue? scents are a blend of compounds with different degrees of volatility (weldon et al., 2008), and it has been proposed that the latency to the first tongue-flick can be modulated by olfaction through the nose of the volatile fraction of the blend (cowles and phelan, 1958). on this background, our hypothesis is that sexes may differ in the production of volatile compounds that activate olfaction, with males producing a different or more concentrated blend of certain volatile compounds, which then prompts a faster recognition, and initiation of the exploration. for example, in acanthodactylus boskianus males and females differ in the chemical composition of the femoral glands, which have pheromonal activity (khannoon et al., 2011). we know that there are interspecific and intraspecific differences in the chemical composition of the scents from precloacal secretions in several species of liolaemus, and these secretions are only present in males of the analyzed species (escobar et al., 2001). but we do not know about sexual differences in other sources of scents (labra et al., 2002). another non-exclusive hypothesis for this difference in latency to the first tongue-flick is that sexes differ in their sensibility to the volatile compounds produced by conspecifics of the same sex, with males being more sensitive than females to compounds coming from individuals of the same sex. results from iberolacerta cyreni partially support this hypothesis; sexes differ in their responses to different chemical compounds present in males’ femoral glands (martín and lópez, 2008). which of these hypotheses are correct will need further investigations based on chemical isolation and behavioral testing of pheromonal compounds from males and females. finally, the observation that both sexes moved more in enclosures of conspecifics may be interpreted as attempting to escape of the “territory” of an unfamiliar individual. self-recognition. a lower number of tongue-flicks toward the own vs. other scents is clear evidence of self-recognition (labra, 2008). the absence of a sex effect in self-recognition suggests that sexes may have the same capability to recognize familiar (own) scents. however, some caution is required in interpreting this result, considering that we found an interaction between sex and treatment, indicating that self-recognition based on tongue-flicks was strongly dependent on female behavior, opening up the possibility that sexes may differ in how they process their own scents. this requires further investigation with larger sample sizes. predator. males and females behaved similarly when confronted with snake scents, possibly a consequence of similar predation risk (jaksic and fuentes, 1980). two sets of 77is the exploratory behavior of liolaemus nitidus modulated by sex? evidence showed that lizards recognized the snake scent. first, lizards waved their tails sooner and for longer in the snake and control enclosures, as compared to their own enclosures. these two conditions, snake and control, would be the most threatening, because lizards may face known (snake) or unknown (control condition) risk, and tail waving can help to deflect attacks toward the tail instead of toward the body (telemeco et al., 2011), enabling lizards to escape by autotomizing the tail (bateman and fleming, 2009). secondly, the snake scent was the only stimulus that triggered slow motion (in three individuals), which may help to reduce detectability to a predator (labra and niemeyer, 2004). control condition. sexes behaved similarly in the control condition, probably a consequence of resemblance in their general exploratory behavior. it is known that explorations of environments made by lizards (i.e. total time that animals move) is positively correlated with the size of their home ranges (verwaijen and van damme, 2008), and both sexes of l. nitidus have similar home range size (fox and shipman, 2003). based on this, we expected that both sexes of l. nitidus behave and explore similarly in novel environment. interestingly, cooper et al. (2001) found no sexual differences in total movement in the field in five lizard species, although they gave no comments on their territorial behavior. iiexploratory behavior of the environment: sexual variation independent of the chemical scents found in the environment, females and males of l. nitidus showed three behavioral differences: latency to the first tongue-flick, latency to first tail wave, and total time waving the tail. males started chemical exploration (tongueflicks) sooner than females, which may suggest that they are more “eager” to explore any environment, but potentially increase their risk of being detected and attacked sooner by predators or conspecifics. increased tail waving may then help to deflect potential attacks to the tail, allowing the individual to escape, as we discussed earlier (bateman and fleming, 2009; telemeco et al., 2011). alonso et al. (2010) found that males of the gecko gonatodes albogularis tail waved more than females, and ascribed this to sexual variation in predation rate. this explanation does not make sense for of l. nitidus, as both sexes experience similar predation rates (jaksic and fuentes, 1980), but reinforces the relevance of the ecology of the group to understand their behavior. the fact that sexual differences were consistent across the treatments may be an indication of sexual variation in behavioral syndrome (sensu sih et al., 2004) of l. nitidus (schuett et al., 2010), as in other species. in the pygmy bluetongue lizard, tiliqua adelaidensis, males directed more tongue-flicks than females to a novel burrow, but there were no difference in the time that they inspected or remain in the burrow (fenner and bull, 2011). we summarize our results in l nitidus by concluding that the ecology of sexes seems to be a modulator of their responses to different scents; sexes behave similarly towards scents with similar ecological meaning (non-reproductive: predator, own, and control) and differ when confronted to scents that represent something different for them (reproductive: conspecifics of the same sex). we also found behavioral differences unrelated to the perceived scents that may be evidence of different behavioral syndromes in the sexes. 78 j. troncoso-palacios and a. labra acknowledgements the study was developed with the authorization of the bioethics committee, from universidad de chile, and the chilean governmental agency that regulated animal capture and maintenance (servicio agricola y ganadero). we thank j. constanzo for her invaluable help in collecting the animals, zoológico nacional de chile for providing two snakes, a. martínez and n. velásquez for their laboratory assistance, to t. f. hansen for his extremely important contribution to an early version of this manuscript, and to r. børsjø for improvements to the language. study funded by fondecyt 1090251 to al. references alonso, m.l.b., cotrina, j.m., pardo, d.a., font, e., molina-borja, m. 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(2008): natural products from the integument of nonavian reptiles. nat. prod. rep. 25: 1-39. © firenze university press www.fupress.com/ah acta herpetologica 5(2): 151-160, 2010 distribution of tadpoles of ollotis occidentalis (amphibia: anura: bufonidae) along the río salado, puebla, méxico guillermo a. woolrich-piña1, geoffrey r. smith1,2, luis oliver-lópez1, monica barbosa morales1, julio a. lemos-espinal1 1laboratorio de ecología, unidad de biología, tecnología y prototipos (fes-iztacala, unam), av. de los barrios s/n, 54090 los reyes iztacala, tlalnepantla, estado de méxico, mexico. corresponding author. e.mail: smithg@denison.edu 2department of biology, denison university, 43023 granville, ohio, usa. submitted on: 2010, 23th january; revised on 2010, 22th september; accepted on 2010, 6th october. abstract. we examined monthly variation in the characteristics of river sections along the río salado (puebla, mexico) and how these factors were associated with the distribution of the tadpoles of the toad ollotis occidentalis. tadpoles were observed in the river in march 2007, and from november 2007 through february 2008 and were only found in the main river channel. sections of the río salado with tadpoles were deeper, wider, and longer than sections without tadpoles. dissolved oxygen levels were higher and salinity was lower in river sections with tadpoles compared to the sections without tadpoles. there was no difference in temperature between sections with and without tadpoles. tadpoles were found in river sections that contained more vegetation than river section without tadpoles. our results suggest that the distribution of tadpoles of o. occidentalis is related to the permanence of water, the chemical nature of the water, and the presence of vegetation. keywords. dissolved oxygen, ollotis occidentalis, pool size, salinity, tadpoles. introduction one of the major goals of ecology is to understand what determines species distributions. for pond-, pool-, or stream-breeding amphibians the relevant question is often “why are tadpoles found in some locations but not others?” in some cases, the distribution of amphibians appears driven by the chemical or physical characteristics of the pool, pond, or stream (e.g., eason and fauth, 2001; richards, 2002; rocha et al., 2002; halverson et al., 2003; welch and macmahon, 2005; girish and krishnamurthy, 2009). in other cases, it appears that the distribution can be explained by the biotic community (e.g., blaustein and margalit, 1995; eason and fauth, 2001). in addition, the interaction of the 152 g.a. woolrich-piña et alii chemical or physical environment with the biotic environment can potentially determine the success of amphibian larvae, and thus could affect their distribution (e.g., sadinski and dunson, 1992; warner et al., 1993; skelly, 1996; smith et al., 2006). we investigated the distribution of the tadpoles of ollotis [bufo] occidentalis along the río salado (puebla, mexico) which, as its name suggests, can have relatively high salinity. amphibians are typically not found in saline conditions, and are usually thought to be unable to withstand elevated salinities (ultsch et al., 1999). however, several species of amphibians (exerodonta xera, hyla arenicolor, lithobates spectabilis, and o. occidentalis) are known to occur in the río salado. we examined monthly variation in water chemistry (salinity, dissolved oxygen), water temperature, physical characteristics (size, distance from main channel), and vegetative cover of river sections and we evaluated how these factors differed between sections of the río salado with and without tadpoles of o. occidentalis. ollotis occidentalis occurs from south-central to northern mexico. in southern puebla, o. occidentalis occurs in pine forest and fry tropical deciduous forests (oliver-lópez et al., 2000). in the valle de zapotitlán salinas, o. occidentalis breeds during the rainy season in slow moving water or in pools on the edges of rivers (oliver-lópez et al., 2000; smith and lemos-espinal, 2010). very little is known about the ecology and natural history of this toad beyond reports on its reproduction (duellman, 1961; oliver-lópez et al., 2000; oliver-lópez and ramírez-bautista, 2002; smith and lemos-espinal, 2010). materials and methods study area the río salado (see fig. 1) runs through el valle de zapotitlán salinas in southeastern puebla, mexico (see woolrich piña et al. 2005 for additional details). pools along the río salado are seasonal, forming during the dry season as the water level in the río salado falls. el valle de zapotitlán is part of the valle de tehuacan-cuicatlán that lies in central mexico, which is considered to be an ecologically important region due to its high levels of biodiversity and endemism (dávila-aranda et al., 1993). surveys of the herpetofauna have reported between 22 and 32 species of amphibians and reptiles (martín del campo and sánchez-herrera, 1979; canseco-márquez and gutiérrez-mayén, 1996). more recently, there have been studies examining the effects of habitat deterioration on amphibians and reptiles (mata-silva, 2000) and the reproduction of the anurans in the valle de zapotitlán salinas (oliver-lópez et al., 2000; oliver-lópez and ramírez-bautista, 2002). woolrich piña et al. (2005) provide a summary of the amphibians and reptiles of el valle de zapotitlán salinas. methods we conducted surveys along a 2 km segment of the río salado (fig. 1) monthly from february 2007 to june 2008 to determine the distribution of tadpoles of o. occidentalis and to characterize conditions along the río salado. the tadpole of this species could be actually differentiated from those of other co-occuring species in the field. we surveyed the first 10 – 19 sections of river or isolated pools we encountered on each of our surveys. we generally began the surveys in the same location and proceeding in the same direction along the river and so many of the river sections were similar among visits; however, on some visits the starting location and direction moved varied 153distribution of ollotis occidentalis tadpoles (e.g., when diff erent researchers conducted a monthly survey). in addition, because isolated pools dried up during some parts of the year, the selected isolated pools may have varied. our impression is that while selection of river sections and isolated pools may have caused some of the variation observed among months, we do not believe this to be a major factor in explaining monthly variation since there appeared to be much more homogeneity among sites within monthly samples than among monthly samples. fig. 1. photographs of the study site along the río salado, puebla, mexico. note that the close-up photograph was taken during one of the wetter months of the year. 154 g.a. woolrich-piña et alii surveys took place in the morning hours (e.g., 0800 to 1200 h). each river section (identified as a single continuous length of river of similar water flow, either a pool with still water or a section of river with greater flow between two pools) or isolated pool (a pool without direct connection to the main channel of the río salado) was visually searched for the presence of larval amphibians (the river sections were small enough, and the water clear enough and shallow enough to allow for the easy detection of any tadpoles). all river sections were contiguous with, or in the case of isolated pools, parallel to, the main river channel of the river (i.e., we did not skip sections of the river during our surveys). we measured the distance between each isolated pool and the main channel of the río salado. the main channel consisted of the primary path of the río salado and which contained water throughout its length and throughout the year. for surveyed section in the main channel, this distance was recorded as 0 m. we also measured the physical dimensions (length and width) of each river section. for each sampled river section or isolated pool we measured maximum depth, salinity, temperature, dissolved oxygen, and ph. we measured water chemistry and temperature approximately 20 – 30 cm from the river’s edge and at a depth of 10 – 20 cm. salinity, dissolved oxygen, and temperature were measured using a ysi model 85 handheld do/conductivity meter. we used df universal indicator paper ph 1-14 to measure ph. in addition, we visually estimated the percent cover of aquatic vegetation in each pool. to examine the differences in the characteristics of river sections where we found tadpoles and sections where we did not find tadpoles, we used a nested manova with month and tadpole presence (nested within month) as the independent variables and the physical and chemical parameters as the dependent variables. we limited this analysis to the november 2007 to february 2008 surveys (the months when tadpoles were present; see below). a significant manova was followed by univariate nested anovas to examine each dependent variable in greater detail and to determine which of the measured variables differed over time and between sections with and without tadpoles. due to the dynamic nature of the río salado (drying of pools and changes in water flow), we were not able to follow specific river sections from month to month, especially pools isolated from the main river channel (see above). we, therefore, could not use repeated measures anovas. results monthly variation depth of the river sections varied among months, with the lowest values found in august 2007 and an increase from this low through june 2008, with a temporary decrease in march 2008 (fig. 2a). width of the river sections also differed among months (fig. 2b), as did the length of river sections (fig. 2c). width and length were both higher early in the study period (e.g., march to august 2007) and lower from september 2007 through june 2008. the distance of a river section or isolated pool to the main river channel varied among months, with peaks in july and august 2007 and december 2007 (fig. 2d). dissolved oxygen was fairly constant but showed distinctly low values in june 2007, and february to june 2008 (fig. 2e). salinity showed a significant trend to higher values throughout the study period (fig. 2f). water temperature varied significantly among months (fig. 2g). the amount of vegetative cover in sections was low for most of the study period with distinct periods of high vegetative cover in march 2007 and from november 2007 to february 2008 (fig. 2h). 155distribution of ollotis occidentalis tadpoles fig. 2. variation in characteristics of surveyed river sections throughout the study period (february 2007 to june 2008) along the río salado. means are given ± 1 se. 156 g.a. woolrich-piña et alii comparison of sections with and without tadpoles tadpoles were observed in the river in march 2007 and from november 2007 through february 2008 (fig. 3). to further investigate the factors that might influence the distribution of tadpoles in the río salado, we limited our analyses to the november 2007 to february 2008 surveys (i.e., a single contiguous breeding season). the nested manova found significant differences in the river sections where we found tadpoles and sections where we did not find tadpoles (wilks’ l = 0.005, f32,186 = 18.7, p < 0.0001). there was also a significant month effect (wilks’ l = 0.009, f24, 146 = 24.84, p < 0.0001). water depth varied among months (fig. 4a; f3,57 = 3.96, p = 0.012), and tadpoles were found in deeper river sections (fig. 4a; f4,57 = 24.3, p < 0.0001). width of the river sections did not vary significantly from month to month (fig. 4b; f3,57 = 1.41, p = 0.25). sections of the river where tadpoles were found were wider than those lacking tadpoles (fig. 4b; f4,57 = 5.98, p = 0.0004). river section length did not change from month to month (fig. 4c; f3,57 = 1.17, p = 0.33). sections with tadpoles were longer than sections without tadpoles (fig. 4c; f4,57 = 5.88, p = 0.0005). distance to the main channel varied from month to month (fig. 4d; f3,57 = 6.39, p = 0.0008). sections of the river that contained tadpoles were closer to the main river channel than sections that did not contain tadpoles (fig. 4d; f4,57 = 17.0, p < 0.0001). in fact, no tadpoles were observed in sections that were not along the main river channel. dissolved oxygen levels remained relatively constant among months (fig. 4e; f3,57 = 0.4, p = 0.75). dissolved oxygen levels were higher in river sections where we found tadpoles compared to the sections without tadpoles (fig. 4e; f4,57 = 42.8, p < 0.0001). salinity varied among months (fig. 4f; f3,57 = 7.85, p = 0.0002). tadpoles were found in river sections that had significantly lower salinity than river sections where no tadpoles were found (fig. 4f; f4,57 = 360.2, p < 0.0001). the difference in salinity between sections with and without tadpoles was highest in november 2007 and then stabilized from december 2007 to february 2008 (fig. 4f). temperature showed a clear variation among months with a low in january 2008 (fig. 4g; f3,57 = 942.3, p < 0.0001). the temperature of sections in which we found tadpoles fig. 3. proportion of surveyed river sections with tadpoles during monthly surveys of the río salado from february 2007 to june 2008. number of surveyed river sections is provided for each survey. 157distribution of ollotis occidentalis tadpoles differed from the temperature of sections in which we found no tadpoles, but the difference was quite small (fig. 4g; f4,57 = 3.52, p = 0.01). the amount of vegetative cover did not vary from month to month (fig. 4h; f3,57 = 1.56, p = 0.21). tadpoles were found in river sections that contained more vegetation than river section without tadpoles (fig. 4h; f4,57 = 178.0, p < 0.0001). discussion the río salado and the pools along it are clearly a dynamic system, both in terms of the size and depth of the river sections and the physical and chemical aspects of the water. such a dynamic aquatic system is likely to influence the amphibians and other organisms that live in the río salado. indeed, there are clear changes in the presence of tadpoles of o. occidentalis throughout the year (see fig. 3). the dynamics of the appearance of o. occidentalis tadpoles in the río salado is similar to that described for a population in the nearby río zapotitlán (canseco-márquez et al., 2003). our results also make it clear that the distribution of tadpoles of o. occidentalis is influenced by the characteristics of the river. in particular it appears that factors related to the permanence of water, the chemical nature of the water, and the presence of vegetation, are important. the importance of water-permanence is suggested by the significant difference in river section length, width, and depth, with river sections with tadpoles having more water (i.e., wider, longer, deeper) than sections without tadpoles. thus, tadpoles are located in sections of the river where water will likely persist for longer periods. in addition, the fact that tadpoles were never found in pools located outside the main river channel suggests the importance of water permanence. in a community of stream-breeding frogs in sulawesi, indonesia, gillespie et al. (2004) found that the tadpoles of most species were found only in pools or still water along the main channel of the stream, and only one species was found to use pools not directly connected to the main channel. however, the preference for deeper sections of streams can vary depending on the species of tadpole, the stream surveyed, and on other characteristics of the stream (e.g., water flow, amount of vegetative cover) (eterovick and barata, 2006). in part this variation may result from variation in the overall permanence of the stream. in the case of the río salado, the permanence of any section of the river is variable, and thus tadpoles may prefer to be in sections of the river that might be more predictable in their duration. we also found that tadpoles of o. occidentalis were located in sections of the river with lower salinity. indeed, sections without tadpoles were nearly twice as saline as sections with tadpoles. thus, tadpoles use areas based in part on their salinity. other species of anurans are also known to be less prevalent in areas with higher salinity (e.g., davenport and huat, 1997; smith et al., 2007) or to avoid oviposition in water with higher salinity (haramura, 2008). however, salinity did not affect the distribution of buergeria japonica tadpoles fig. 4. mean characteristics of river sections of the río salado with (closed circles) and without (open circles) tadpoles. means are given ± 1 se. 158 g.a. woolrich-piña et alii (haramura, 2007). in contrast, the abundance of rhinella marina increased with salinity in introduced populations on puerto rico (ríos-lópez, 2008). it appears that at least some species of bufonids show local adaptation to salinity, and are able to tolerate higher levels of salinity as tadpoles (e.g., gómez-mestre and tejedo, 2003, but see beebee, 1985). we also found dissolved oxygen levels were higher in areas of the river with tadpoles than in areas of the river without tadpoles. this is consistent with previous results suggesting that anaxyrus terrestris tadpoles use microhabitats with higher levels of dissolved oxygen (noland and ultsch, 1981). dissolved oxygen also appears to be important in determining species richness of tadpoles in ponds along the middle paraná river in argentina (peltzer and lajmanovich, 2004). tadpoles also used sections of the river with higher levels of vegetative cover. for the most part, the vegetation consisted of algae, and thus this relationship of tadpole presence and vegetative cover could reflect greater food availability for the tadpoles. alternatively, the presence of more vegetative cover could simply reflect better quality water (i.e., lower salinity, higher dissolved oxygen content, greater permanence). given the apparent importance of water permanence and water chemistry in determining the distribution of tadpoles of o. occidentalis in the río salado, it is worth examining the potential conservation implications of the salt factories (“salineras”) that are located along the río salado, including just upstream of our study area. these salineras divert water from the río salado to harvest the salt by evaporation. thus these salineras have the potential to greatly alter both the amount of water and the chemistry of the water of the río salado. such alterations may have impacts on the amphibians of the río salado, either directly through changes in water levels or water chemistry, or indirectly by sublethal effects of changes in water chemistry (e.g., squires et al., 2008). it is not clear whether or not there is any current impact of the salineras on the río salado; however, any future increases in the number of extent of salineras should be carefully considered in light of the potential impacts on the native fauna, including the amphibians, living in the río salado. acknowledgments this research was supported by funds from dirección general de asuntos de personal académico through the project papiit-in221707 “factores que determinan la distribución de los anfibios en las pozas asociadas al río salado, puebla, méxico”; and for facultad de estudios superiores iztacala through the programa de apoyo a los profesores de carrera (papca) 2007-2008 with the project “caracterización de las pozas asociadas al río salado (puebla) y su influencia en la distribución de los anfibios: aspectos ecológicos y geográficos” and 2009-2010 “efecto de la salinidad sobre las larvas de anuros que habitan el río salado (puebla)”. the research was approved by the denison university institutional animal care and use committee (protocol 07-004). we thank two anonymous reviewers for comments that improved the manuscript. references beebee, t.j.c. 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(2005): anfibios y reptiles del valle de zapotitlán salinas, puebla. universidad nacional autónoma de méxico conabio. méxico. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 6(2): 289-295, 2011 cross-amplification of microsatellite loci reveals multiple paternity in halys pit viper (gloydius halys) evgeniy simonov1, 2, michael wink2 1 institute of systematics and ecology of animals, siberian branch of russian academy of sciences, frunze 11, 630091 novosibirsk, russia. corresponding author. e-mail: ev.simonov@gmail.com 2 institute of pharmacy and molecular biotechnology, heidelberg university, inf 364, d-69120 heidelberg, germany. submitted on: 2011, 26th july; revised on 2011, 29th august; accepted on 2011, 8th september. abstract. for halys pit viper (gloydius halys) species-specific microsatellite primers are not available. we tested a set of twenty primer pairs, originally developed for various crotalinae species, for cross-amplification with gloydius halys. the level of allelic polymorphism was assessed for eight successfully amplified loci via genotyping of a population sample. between three to 24 alleles per locus were recorded. we examined a female and seven of its embryos for multiple paternity using seven microsatellite loci. more than two paternal alleles were detected in two loci indicating that two or more fathers were involved. this is the first report of multiple paternity in the wild population of crotalinae. the life history characteristics of halys pit-viper that can be associated with multiple paternity are discussed. keywords. microsatellites, multiple paternity, gloydius halys, pit viper, crotalinae. during the last two decades the introduction of molecular techniques has greatly facilitated kinship analysis in wild populations of animals. multiple paternity has been documented for various taxonomic groups of vertebrates (laurila and seppa, 1998; wink and dyrcz, 1999; avise et al., 2002; westneat and stewart, 2003; gottelli et al. 2007; uller and olsson, 2008). in scaled reptiles (squamata) multiple paternity seems to be a very common phenomenon and reaches the highest levels known in vertebrates (uller and olsson, 2008). however, cases of multiple fecundation have only been reported for 36 out of more than 8900 squamate species and they are limited to nine out of 52 squamate families (uetz et al., 2007; uller and olsson, 2008; voris et al., 2008). recently, several authors have recognized the need to consider this phenomenon in scaled reptiles in the broader phylogenetic context (voris et al., 2008; wusterbarth et al., 2010) because our knowledge on the occurrence of multiple paternity in diverse squamate taxa is still insufficient. microsatellites are very useful for paternity studies due to their co-dominance, biparental inheritance and high variability (webster and reichart, 2005). the development of these 290 evgeniy simonov and michael wink markers is still relatively expensive and a time consuming task, while recent achievements in application of 454 pyrosequencing for microsatellite development have greatly facilitated this process (e.g. castoe et al., 2010; malausa et al., 2011). given that the flanking regions of microsatellite loci may be quite conserved in related taxa (moore et al., 1991; primmer et al., 1996), cross-species amplification is a widely used way to avoid the process of the microsatellite development for each newly studied species (bushar et al., 2001; anderson, 2006). because specific microsatellite primers were not available for halys pit viper (gloydius halys) a set of microsatellite primers from other snakes were tested for cross-species amplification. the primer set found was subsequently utilized in a paternity analysis of a female with seven embryos that had been found dead on a roadside. we performed cross-species microsatellite amplification on 156 individuals of adult g. halys that were sampled during 2008 2010 in the novosibirsk region (west siberia, russia). buccal swabs and scale clippings were used to obtain the tissue samples which were stored in 95% ethanol at 4 °c. after sampling and examination all snakes were released at the site of capture. in attempt to detect multiple paternity in g. halys we used a roadkilled gravid female, collected in august 2009 at the same study area. the dissection of the female revealed seven well-developed embryos that were measured and sexed. intercostal muscle tissue of the female and tail tips of the embryos were used for dna extraction. total genomic dna was isolated using standard proteinase k and phenol-chloroform protocols (sambrook et al., 1989). a panel of 20 microsatellite loci which had been developed for crotalinae species (genera crotalus and sistrurus) was tested for cross-species amplification with g. halys (table 1). initially we performed pcr with each primer pair in a set of six samples using the following conditions – initial denaturation at 94 °c for 5 min followed by 45 cycles of 60 s at 94 °c, annealing at 53 °c for 60 s, 60 s extension at 72 °c, and a final extension of 5 min at 72 °c. at a second step all primer pairs which showed positive results were employed in gradient pcr with tgradient termocycler (biometra). for this step we used three samples out of previous set and the same pcr conditions, but the annealing temperature ranged from 50 to 65 °c. after that we rejected all primers which had yielded ambiguous patterns (i.e. a lot of non-specific amplifications) at all thermal regimes. finally, we selected the optimal annealing temperature for each locus (table 1) and performed pcr with the six samples. the loci which exhibited more than two alleles were applied for further genotyping of the whole data set. the female and its embryos were genotyped twice to be sure that an observed pattern is not a result of an amplification/electrophoresis error. all pcrs were performed in 25 µl reaction mix containing 15-60 ng dna, 0.1 mm each of dgtp, dctp and dttp, 0.045 mm datp, 0.1 µl 33p-α-datp (amersham biosciences), 1.5 u of top-taq dna polymerase (bioron), 2.5 µl of 10 × amplification buffer (10 mm tris-hcl ph 8.5, 50 mm kcl and 2.5 mm mgcl2), and 10 pmol of forward and reverse primers. amplification products of all pcrs were separated by high-resolution electrophoresis in 6% denaturing polyacrylamide gels at 65 w for 1.5 h using a base acer sequencer (stratagene). after the gels were vacuum dried they were exposed for 24-96 h to x-ray film (biomax mr film, kodak). pcr products were sized with reference to a known sequencing reaction of the pbluescript sk+ plasmid. we checked for possible scoring errors due to the production of stutter bands, allele dropout and the presence of null alleles using micro-checker v. 2.2.3 software (oosterhout et al., 2004). test for link291cross-amplification of microsatellite loci reveals multiple paternity in halys pit viper age disequilibrium was performed in genepop web version 4.0.10 (rousset, 2008) using exact probability test with a markov chain approximation. the critical p-values were corrected for multiple tests by the benjamini and yekutieli (b–y) method (benjamini and yekutieli, 2001). table 1. microsatellite loci and results of their amplification with gloydius halys. reference and used species: amunguia-vega et al. (2009), crotalus tigris; bgoldberg et al. (2003), c. tigris; cvillarreal et al. (1996), c. horridus; dholycross et al. (2002), c. willardi; eoyler-mccance et al. (2005), c. viridis; fgibbs et al. (1998), sistrurus catenatus. na, number of observed alleles; ta, annealing temperature. *allele sizes are based on the size of the clone sequenced for each locus (gibbs et al. 1998). locus repeat motif allelic size, bp na (n) amplification with g. halys yes/no/ ambiguous allelic size, bp na (n) ta (°c) crti14a (gt)19 274-314 14 (25) no crti19a (ca)18 212-235 6 (25) ambiguous crti12aa (ca)22 219-240 6 (25) yes 206-220 7 (164) 60 crti37a (gt)12(ga)26 274-312 14 (25) yes 264-292 7 (164) 55 crti95a (ca)22 174-211 10 (25) yes 172-202 16 (164) 56 crti10b (gaa)48 219-300 22 (149) yes 240-312 23 (164) 55 crti12b (ca)14 217-225 5 (149) yes 216-232 3 (164) 57 ch5ac (ca)17 164-142 8 (29) yes 144-150 2 (6) 56 ch7-150c (ca)13 146-144 2 (32) yes 122-130 2 (6) 56 ch 5-183c (ca)11 136-124 7 (26) no ch 7-144c (ca)16(ga)12 116-100 5 (18) no ch 7-87c (ca)12 159-145 3 (16) yes 138-180 12 (164) 55 ch 3-155c (ca)13 146-122 4 (22) no cwa14d (ac)24 147-175 7 (54) yes 144-174 8 (164) 57 cwa29d (ac)13 160-190 5 (54) no cwb6d (ga)19 122-130 5 (54) ambiguous mfrd5e (tg)23 172-194 9 (192) yes 160-186 10 (157) 56 scu01f (ag)24 149* 12 (73) ambiguous scu16f (ac)17 167* 4 (74) no scu26f (ac)24 173* 5 (74) ambiguous results of cross-amplification testing are summarized in table 1. unambiguous results were obtained for ten out of twenty tested microsatellite loci initially amplified with six samples of g. halys. most of them (50%) were originally developed for crotalus tigris, 30% for c. horridus and 10% for c. willardi and c. viridis. no str locus of sistrurus catenatus could be successfully amplified with g. halys. eight of cross-amplified loci were used to screen with the whole data set (n = 164). polymorphism varied between three (crti12) to 24 (crti10) alleles per locus. number of alleles per locus and range of allelic sizes in halys pit viper was generally similar to that of the original species (table 1). there was no evi292 evgeniy simonov and michael wink dence for scoring errors resulting from stuttering or large allele dropout. however, microchecker detected the signs of presence of a null allele for locus crti12a with an estimated frequency of 0.08. no linkage disequilibrium tests were significant after b-y correction. we examined the female and its embryos for multiple paternity using seven microsatellite loci. more than two paternal alleles were detected for the locus crti95 (three paternal alleles) and crti10 (four paternal alleles). this is good evidence for multiple fertilization of the litter by two or more males (table 2). table 2. microsatellite dna genotypes of gloydius halys female and its embryos. locus maternal genotype offspring genotypes inferred paternal alleles1 (f ) 2 (f ) 3 (f ) 4 (f ) 5 (m) 6 (m) 7 (m) crti12a 212/206 212/206 212/212 212/212 212/208 212/212 212/206 212/206 212, 208 crti37 288/264 292/288 292/288 292/288 288/264 292/288 292/264 292/264 292, 288 or 264 crti95 172/196 194/196 198/196 172/196 198/196 172/196 172/196 172/196 194,198, 196 crti10 279/258 309/258 306/258 258/258 279/279 258/258 309/258 309/258 258, 279, 306, 309 crti12 230/216 230/216 232/216 216/216 232/216 232/230 216/216 232/216 216, 232 ch 7-87 156/156 156/156 156/156 156/156 156/156 156/152 156/152 156/156 152, 156 cwa14 174/172 172/144 174/174 172/144 174/172 172/144 172/144 172/144 144, 174 successful cross-species amplification of microsatellites in snakes is known for a number of loci and was used both in population genetics and paternity testing studies (e.g., clark et al., 2008; wusterbarth et al., 2010). the three loci tested in the present work (scu01, scu16 and scu26) have been previously cross-amplified with other pit vipers, as well as with representatives of colubridae (gibbs et al., 1998; anderson, 2006). surprisingly, we could not achieve satisfactory results with them in our analysis. however, pcr products belonging to microsatellites were produced for loci scu01 and scu26, but their interpretation was severely complicated by the numerous non-specific bands. further improvements of pcr conditions via selection of various pcr buffers may solve this problem, as it was shown in anderson (2006). in general, the 50% rate of successful amplifications reached in our study confirms that cross-species utilization of microsatellites may be considered as preferred convenient way to avoid the development of the specific loci for each newly studied species. to our best knowledge, only two documented cases of multiple paternity in vipers (viperidae) have been reported. occurrence of multiple paternity in the common adder (vipera berus) is a well established fact (e.g., stille et al., 1986; ursenbacher et al., 2009). in the pit viper (crotalinae) subfamily there is one report of multiple paternity in captive copperhead (agkistrodon contortrix) caused by sperm storage and revealed by the phenotypes of the offspring (schuett and gillingham, 1986). it is notable that attempts to 293cross-amplification of microsatellite loci reveals multiple paternity in halys pit viper detect multiple paternity in other pit vipers had failed. villarreal et al. (1996) had tested two litters of timber rattlesnake (crotalus horridus) using six loci, whereas gibbs et al. (1998) analysed two litters of massasauga (sistrurus catenatus). hence, we documented here the first case of multiple paternity in a free-living crotalinae, and g. halys is the second viperidae species for which this phenomenon has been documented in the wild. we encourage more intense sampling and paternity testing both for old world and new world pit vipers, considering that the prevalence of genetic monandry (instead of multiple paternity) may be a likely phenomenon in some snake lineages, as has been recently been shown by lukoschek and avice (2011) for true sea snakes of genus hydrophis. several life history traits of the halys pit viper suggest that multiple paternity may occur in this species. first, although there are no known recordings of multiple mating in this species, g. halys occurs at high population densities, at least at our study site (267 individual/hectare; simonov, 2007), with apparent breeding aggregations. importantly, it has been hypothesised that population density and male-biased operational sex ratio are linked to the occurrence (and frequency) of multiple paternity in snakes and other squamates (uller and olsson, 2008; voris et al., 2008). however, some recent studies fail to find evidence of such a link (blouin-demers et al., 2005; ursenbacher et al., 2009). next, mating has been observed in the middle and end of the activity season in g. halys (paraskiv, 1956; yakovleva, 1964; yakovlev, 1985), a behaviour known to be associated with sperm storage in snakes (e.g., halpert et al., 1982). thus, under the assumption that females multiple mate and store sperm, two common features in snake species (olsson and madsen, 1998), it is likely that multiple paternity is promoted in this species. in this study, we show that multiple paternity does occur, and now we recommend further studies to be undertaken in order to understand the extent and evolutionary consequences of multiple paternity in this species. acknowledgements this work was partly supported by the daad (fellowship for e.s.). we thank dr. j. gonzález, h. staudter, h. sauer-gürth and p. kremer for advice and technical assistance and also dr. v. zinchenko and i. dolgov for assistance during fieldwork. references anderson, c.d. 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[in russian] issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 8(1): 1-8, 2013 the oogenic cycle of the caspian bent-toed gecko, cyrtopodion caspium (squamata: gekkonidae) in iran vida hojati1*, kazem parivar2, eskandar rastegar-pouyani3, abdolhossein shiravi1 1 phd in developmental biology, department of biology, damghan branch, islamic azad university, damghan, iran. *corresponding author. e-mail: vida.hojati@gmail.com 2 phd in developmental biology, department of biology, science and research branch, islamic azad university, tehran, iran 3 phd in molecular evolution, department of biology, faculty of science, hakim sabzevari university, sabzevar, iran submitted on 2012, 14th june; revised on 2013, 12th january; accepted on 2013, 11th february. abstract. the caspian bent-toed gecko, cyrtopodion caspium, is one of the commonest lizards in northern iran. it is nocturnal, anthropophile and oviparous. in this study, the reproductive cycle of this species was studied by focusing on oogenesis, from april 5 to october 20, 2011. in total, 70 adult females were obtained from mazandaran, one of the northern provinces of iran where the climate is temperate. ovaries were removed and processed for histological and morphometric studies. the results show that oocyte growth starts in late april and ends in august. mating starts in spring, especially at the beginning of may, with oviposition occurring from late may to mid august. females lay 1-2 eggs per clutch with the possibility of producing a secondary clutch later in the season. maximum reproductive activity occurs in may and peaks in june. there was no significant difference between right and left side of reproductive system. with oogenesis occurring from april through july, c. caspium follows an oogenic cycle typical for temperate species. keywords. lizard, gecko, cyrtopodion caspium, oogenesis, ovary, reproduction. introduction lizards exhibit three general types of reproductive cycles: associated, dissociated and constant (pough et al., 2001). associated and dissociated reproductive cycles are characterized by the presence of a discontinuous mating season. in an associated reproductive cycle, gonadal activity increases immediately prior to the mating period in both males and females simultaneously. in this reproductive cycle, females have no need to store sperm due to it’s availability during the reproductive season. this cycle is common in species that live in predictable environments such as in the temperate zones (diaz et al., 1994), the subtropics (huang, 1997), and in the seasonal tropics (censky, 1995a, b). in a dissociated reproductive cycle, gonadal activity is low during the mating period and peaks during the non-mating period. in this type of reproductive cycle, male gonadal activity is shorter than that of females and sperm is stored by the female genital system for some months with fertilization occurring later (torki, 2006). a dissociated cycle is typically observed in species that live in temperate zones and have a brief mating season (van wyk, 1995). a constant reproductive cycle is exhibited by species in which gonadal activity is maintained at nearly maximum level almost year-round. several species of tropical lizards exhibit a constant reproductive cycle (jenssen and nunez, 1994). these relationships between reproductive cycles and climate suggest that reproduction in lizards is affected by environmental variables such as temperature (marion, 1982), precipitation (guillette and casas-andrew, 1987), and photoperiod (licht, 1967). phylogenetic constraints may also play a major role in shaping the reproductive characteristics of lizards (dunham and miles, 1985). if so, it may be beneficial to study the reproductive cycles of species within a single diverse and wide-ranging lineage. 2 vida hojati et al. the members of the family gekkonidae display different reproductive patterns, as evidenced by the presence of viviparous and oviparous species (nogueira et al., 2011). breeding season is determined by cycles of photoperiod, temperature, rainfall and availability of food (vitt and pianka, 1994). the caspian bent-toed gecko, cyrtopodion caspium (eichwald, 1831), is a nocturnal oviparous lizard of the family gekkonidae (anderson, 1999) and comprises two subspecies in the caspian sea region (szczerbak and golubev, 1996; anderson, 1999). cyrtopodion caspium caspium is widely distributed in the eastern part of the caucasus, middle asia including turkmenistan, uzbekistan, southern tajikistan, southwestern kazakhstan, northern afghanistan and iran (szczerbak, 2003). the northern border of its range is a line from komsomolets bay on the northeastern shore of the caspian sea to the northern coast of the aral sea and syr darya. in iran it is known to occur in the mazandaran and gorgan regions, northern and eastern khorasan, extending south to sistan from sea level up to 1700 m (anderson, 1999). cyrtopodion caspium insularis (akhmedov and szczerbak, 1978) occurs on the island of vulf in the caspian sea and is known only from the type locality. it differs from the nominate subspecies in having the first pair of postmental shields usually separated from each other by gular scales, though there may be a tiny dot-like contact, while in the nominate subspecies they are broadly in contact (akhmedov and szczerbak, 1978). cyrtopodion caspium is one of the most common lizards of iran especially in its northern range. because no data is available on the reproduction of this species, this project was conducted to characterize its reproductive cycle by focusing on oogenic cycle in northern iran. materials and methods study area the study locality was sari county (36°32’n, 54°7’e), in the mazandaran province in northern iran, located on the southern coast of the caspian sea. sari is situated inland from the caspian sea in the semi-tropical coastal plain to the north of the alborz mountains. the rainy season lasts about seven months, with an annual precipitation of more than 1,110 mm, giving the countryside a green and lush appearance. the climate of this area is wet and temperate (during this study, the mean temperatures of the coldest and warmest seasons were 1.6 °c and 22.5 °c, respectively), with the most dominant plants being grassy species belonging to the families asteraceae and poaceae (assadi et al., 2005). sampling sampling took place periodically every 15 days during the activity period of this species from april 5 to october 20, 2011. all specimens were collected by hand, with the aid of a torch, at night time. most of the specimens were collected from walls of old buildings and gardens. in total, 70 adult and mature females were captured (five specimens per sampling period) and sexed by the presence of preanal and femoral pores in males and their absence in females. our observations show that females reached sexual maturity at a body length (svl) about 42 mm, but we tried to capture specimens which had attained a larger svl to be sure they were sexually mature adults. some specimens were kept in terrarium in order to study the frequency of clutch deposition. methods the specimens were transferred alive to the zoology laboratory of islamic azad university, damghan branch and svl (south-vent length), tl (tail length), hl (head length) were measured. then they were anaesthetized by chloroform and anatomized. we observed oviductal eggs and vitellogenic follicles simultaneously in most specimens from may to june. rod (diameter of right ovary), lod (diameter of left ovary), row (weight of right ovary), low (weight of left ovary), rov (volume of right ovary), lov (volume of left ovary), rfn (number of right follicles), lfn (number of left follicles), maxrf (maximum diameter of right follicles), maxlf (maximum diameter of left follicles), minrf (minimum diameter of right follicles), minlf (minimum diameter of left follicles), mrf (mean diameter of right follicles), mlf (mean diameter of left follicles), roel (length of right oviductal eggs), loel (length of left oviductal eggs), roew (width of right oviductal eggs), loew (width of left oviductal eggs), roewe (weight of right oviductal eggs), loewe (weight of left oviductal eggs), roev (volume of right oviductal eggs), loev (volume of left oviductal eggs), mdfl (mean diameter of follicular layer), mdn (mean diameter of nucleus), mnn (mean number of nucleus) and mdn (mean diameter of nucleolus) were measured. length, width and diameter were measured by dial caliper with an accuracy of 0.02 mm. weight was measured by a scale with an accuracy of 0.001 g. gonads, once removed, were inspected for metric and meristic characters. the number, weight, diameter (length and width) and volume of immature, growing and mature follicles and oviductal eggs were inspected in right and left ovaries separately. after fixing the ovaries in 10% formalin, tissues were dehydrated, cleared in xylene, infiltrated and embedded with paraffin. sections were made at 5-7 microns, deparaffinized, re-hydrated, stained (hematoxylin and eosin) and mounted. the sections were studied by light microscopy at 100× and 400× magnification. photographs were prepared by digital camera. data were analyzed by spss 17 software and one-way anova to compare biometric data among monthly samples. 3the oogenic cycle of cyrtopodion caspium results in this study, the maximum svl and tl of females were 68.03 and 91.16 mm, respectively. the maximum hl was 17.56 mm. body length (svl) of the smallest mature female was 42.64 mm. the gecko c. caspium in the study area hibernates from late october to early april. geckos emerge in early april and begin oogenesis and vitellogenesis by mid april, while mating is observed in early may. oviposition occurs from from late may to early august. in the terrarium, the first egg was observed may 18, and hatching occurred after about 45-50 days post oviposition in july and august, while oogenesis and vitellogenesis stopped by mid august. according to our observations in the field and analyzing the captured animals, oviposition may occur twice a year and clutch size ranges from 1–2 eggs per clutch. the ovaries are paired and vesicular, consisting of 3-8 follicles (figure 1a). three types of follicles were observed: immature, growing and mature. in april, ovaries are white and small with an irregular shape. the growing follicles were observed in late april (figure 1b). ovary size increased immediately in may and mature follicles were observed in early may. ovaries are large in july and august with mature follicles present. ovary size decreases in september and october until the following april. the follicular layer is multilayered and polymorphic, composed of three cell types: small, intermediate and the large pyriform cells (figure 1c). the diameter of follicular layer varies between 30-70 μm in mature and immature follicles, respectively. the mean diameter of follicular layer is 50 μm. the diameter of the nucleus varies between 35 to 260 μm in immature and mature follicles, respectively. the nucleoli are very large and distinguishable from april to july and their numbers vary from 2 to 30 in immature and mature follicles, respectively (figure 1d). the diameter of nucleoli varies between 25 and 70 μm. the activity of nucleoli decreases after july and they disappear in the nucleus. mature oocytes enter the oviducts, where fertilization occurs. we observed follicullar atresia in some ovaries especially in the postbreeding period. the descriptive statistics of ovarian characters were described in table 1 and 2. analysis of variance of shows that weight, svl and tl of mature females were not significantly different among groups; but ovary weight and volume and mean diameter of follicles were significantly different between groups (p < 0.05, table 3 and 4). there are no significant differences in the ovarian characters between the left and right side of body (paired t-test, p > 0.05 in all cases). there is no significant difference between body length (svl) of mature females and gonadal weight in different months. oviductal eggs were observed between may 5 and august 5. the largest egg was observed in the left oviduct of a female with svl = 60.62 mm in early may. its size, weight and volume were 13.46 × 12.45 mm, 0.819 g and 0.20 mm3, respectively. the mean size and weight of oviductal eggs were 10.27 × 8.46 mm and 0.462 g, respectively. there was at least one large oviductal egg in right or left ovary in the adult females, but they usually had two eggs, one in each oviduct (figure 2a). there were 4 oviductal eggs in a specimen (10.32 × 12.45, 10.70 × 12.48, 5.15 × 5.40 and 5.20 × 5.80 mm) on may 20. eggs are white and oval shaped and were deposited from late may to early august and they were laid usually in moist and warm holes of the walls of old buildings and gardens as a natural incubator. the egg shells are soft but harden after 8-12 hours after deposition. in this study, the first egg (12.08 × 11.01 mm and 0.755 g) was laid on may 20 and we also observed two additional oviductal eggs (5.83 × 5.42 mm and 12.45 × 12.48 mm) in this female, after dissection. in this study, the number of laid eggs varied from 1-2 per clutch. we observed the first hatchling in 10 july. these results confirm that the incubation period ranges between 45 and 50 days, as we observed the first oviposition on 20 may and the first hatchling appeared on 10 july. one of the embryos (about 35 days old and with total length = 18.10 mm) was studied in early july (figure 2b). the body length (svl) and tail length (tl) of the smallest hatchling were 19.11 and 28.15 mm, respecfig. 1. cyrtopodion caspium: a) ovary with growing follicles; b) growing oocyte; c) follicular layer and d) nucleus with nucleoli. ic = interstitial cell; lc = large pyriform cell; sc = small cell; zp = zona pellucida. photos by v. hojati. 4 vida hojati et al. table 1. descriptive statistics for the examined characters in c. caspium. for abbreviations see methods. character n range minimum maximum mean se sd variance w (g) 70 4.915 2.718 7.633 4.681 0.129 1.084 1.175 svl (mm) 70 25.390 42.640 68.030 58.500 0.474 3.964 15.716 tl (mm) 70 26.670 64.490 91.160 80.298 0.645 5.399 29.149 hl (mm) 70 9.360 8.200 17.560 11.535 0.239 2.000 4.000 rod (mm) 70 5.990 2.130 8.120 4.32800 0.158 1.321 1.746 lod (mm) 70 5.440 2.430 7.870 4.16186 0.131 1.098 1.205 row (g) 70 7.335 0.005 7.340 0.16587 0.106 0.882 0.780 low (g) 70 0.859 0.006 0.865 0.07200 0.020 0.172 0.030 rov (mm3) 70 0.249 0.011 0.260 0.05516 0.005 0.042 0.002 lov (mm3) 70 0.210 0.030 0.240 0.05286 0.005 0.040 0.002 rfn 70 5.000 3.000 8.000 5.20000 0.157 1.314 1.728 lfn 70 5.000 3.000 8.000 5.17143 0.132 1.103 1.217 maxrf (mm) 70 3.800 1.190 4.990 2.407 0.112 0.935 0.875 maxlf (mm) 70 3.640 1.090 4.730 2.16729 0.097 0.813 0.661 minrf (mm) 70 1.580 0.340 1.920 0.870 0.0343 0.287 0.083 minlf (mm) 70 1.220 0.320 1.540 0.890 0.030 0.249 0.062 mrf (mm) 70 2.110 0.830 2.940 1.50857 0.054 0.455 0.207 mlf (mm) 70 1.630 0.720 2.350 1.40586 0.040 0.338 0.114 roel (mm) 21 8.170 5.420 13.590 10.674 0.460 2.110 4.448 loel (mm) 15 7.630 5.830 13.460 10.4960 0.585 2.267 5.140 roew (mm) 21 6.320 5.180 11.500 8.598 0.366 1.677 2.813 loew (mm) 15 7.260 5.190 12.450 8.76467 0.559 2.165 4.689 roewe (g) 21 0.749 0.069 0.818 0.503 0.054 0.248 0.062 loewe (g) 15 0.742 0.077 0.819 0.486 0.067 0.261 0.068 roev (mm3) 21 0.130 0.070 0.200 0.133 0.008 0.038 0.001 loev (mm3) 15 0.140 0.080 0.220 0.136 0.012 0.0475 0.002 mdfl (μm) 70 40.00 30.00 70.00 49.823 0.7861 6.570 43.169 mdn (μm) 70 94.69 35.81 260.50 121.680 2.791 23.352 545.302 mnn 45 28.00 2.000 30.000 14.6647 0.294 1.972 3.888 mdn ( μm) 45 45.00 25.00 70.00 32.7498 1.407 9.441 89.125 table 2. temporal measurements for egg and oviductal characters in c. caspium. characters april n = 10 may n = 10 june n = 10 july n = 10 august n = 10 september n = 10 october n = 10 mean diameter of ovary 3.67 4.78 4.42 4.91 3.78 3.84 3.83 mean diameter of nucleus 152.45 170.46 178.34 118.68 110.52 103.66 101.71 nucleolus diameter 25-70 25-60 30-60 40-50 – – – follicles numbers 4-8 4-8 3-7 3-7 4-6 3-6 4-6 mean diameter of follicles 1.02 1.50 1.74 1.85 1.41 1.59 1.43 mean size of oviductal eggs – 12.93×10.83 9.08×7.42 9.71×7.64 9.37×7.97 – – mean weight of oviductal eggs – 0.801 0.331 0.454 0.262 – – 5the oogenic cycle of cyrtopodion caspium table 3. anova of ovarian macroscopic characters in c. caspium. for abbreviations see methods. character variance sum of squares df mean square f p w between groups 20.945 13 1.611 1.500 0.147 within groups 60.161 56 1.074 total 81.106 69 svl between groups 149.274 13 11.483 0.688 0.767 within groups 935.105 56 16.698 total 1084.379 69 tl between groups 306.479 13 23.575 0.774 0.683 within groups 1704.824 56 30.443 total 2011.303 69 hl between groups 12.612 13 0.970 0.930 0.529 within groups 58.415 56 1.043 total 71.028 69 rod between groups 30.161 13 2.320 1.439 0.001 within groups 90.296 56 1.612 total 120.458 69 lod between groups 18.645 13 1.434 1.245 0.004 within groups 64.522 56 1.152 total 83.167 69 row between groups 14.790 13 1.138 1.634 0.003 within groups 38.997 56 0.696 total 53.787 69 low between groups 1.092 13 0.084 4.929 0.000* within groups 0.954 56 0.017 total 2.047 69 rov between groups 0.070 13 0.005 5.891 < 0.001 within groups 0.051 56 0.001 total 0.120 69 lov between groups 0.065 13 0.005 6.300 < 0.001 within groups 0.045 56 0.001 total 0.110 69 fig. 2. cyrtopodion caspium: a) oviductal eggs and mature follicles and b) 35 days old embryo. photos by v. hojati. 6 vida hojati et al. tively. the juveniles were commonly observed in july and august. the mean svl and tl of hatchlings were 22.38 and 30.20 mm, respectively. the maximum activity of oogenesis occurs in may. discussion since oogenesis occurred from april through august, the pattern followed an associated reproductive cycle typical of species from temperate areas. the results of our study on the oogenic cycle of c. caspium are reported for the first time from iran and west southern asia. the largest female body length in this study was 68.03 mm, whereas in previous studies, it has been reported to be 59.80 mm, in northern afghanistan (anderson, 1999). a female collected from northern afghanistan in mid-april had an oviductal egg about 5 mm long, whereas in the present study we observed oviductal eggs only in early may. this difference is probably due to the different climates of the two regions. all oocytes grow but only some of them enter vitellogenesis and reach the maximum size to later become shelled eggs. the weight, diameter and volume of the ovaries and oocytes increase between may and july and the females are heavier in mid-andlate spring due to the presence of eggs. clutch size was similar to other gekkonid lizards, such as cyrtopodion scabrum (anderson, 1999). the smallest juvenile collected in this present study had a svl = 19.11 mm in early august, whereas anderson (1999) reported the smallest juvenile with a svl = 20.7 mm in late august. although oocyte growth was accompanied by changes in the granulosa layer, zona radiata and thecal layers, the zona pellucida remained unchanged throughout. based on work on the gecko hemidactylus mabouia, the small cells of the granulosa layer are differentiated into three distinct cell types: small, intermediate and the large pyriform cells (moodley and van wyk, 2007). similar to most other squamates, the intermediate and pyriform cells at the onset of vitellogenesis regressed to a single cuboidal epithelium. following ovulation, the granulosa layer hypertrophied forming laical tissue (moodley and van wyk, 2007). the theca layer differentiated into two layers and septal invasion of the corpus luteum took table 4. anova of ovarian microscopic characters in c. caspium. for abbreviations see methods. character variance sum of squares df mean square f p rfn between groups 43.200 13 3.323 2.449 0.010 within groups 76.000 56 1.357 total 119.200 69 lfn between groups 29.543 13 2.273 2.339 0.014 within groups 54.400 56 0.971 total 83.943 69 mrf between groups 4.582 13 0.352 2.033 < 0.001 within groups 9.709 56 0.173 total 14.291 69 mlf between groups 4.299 13 0.331 5.164 < 0.001 within groups 3.587 56 0.064 total 7.886 69 mdfl between groups 1702.264 13 130.943 5.745 < 0.001 within groups 1276.426 56 22.793 total 2978.690 69 mdn between groups 25266.383 13 1943.568 8.806 < 0.001 within groups 12359.438 56 220.704 total 37625.821 69 mnn between groups 146.298 8 18.287 26.575 < 0.001 within groups 24.773 36 0.688 total 171.071 44 mdn between groups 2984.285 8 373.036 14.329 < 0.001 within groups 937.228 36 26.034 total 3921.513 44 7the oogenic cycle of cyrtopodion caspium place. at the time of oviposition, corpora lutea regressed to form ovarian scars (corpora albicantia). follicular atresia occurred in previtellogenic follicles (hydration stage) and seldom in vitellogenic follicles. the highest incidence of atresia occurred in the post-breeding period (moodley and von wyk, 2007) while atretic follicles were replaced by new growing follicles recruited into the follicular size hierarchy (jones et al., 1978). the gross morphology, oogenesis and folliculogenesis of the ovaries of c. caspium correspond to the general squamate pattern described for oviparous reptiles (moodley and van wyk, 2007). the ovarian stroma was characterized by follicles in different stages of development, and may contain corpora lutea, and corpora atretica. the follicular epithelium of the lizard oocytes undergoes structural and morphological modifications throughout oocyte growth. during this process the number of follicle cells increases and the epithelium acquires a multilayered and polymorphic organization which is characterized by the appearance of large follicle cells including intermediate and pyriform cells. the number of large cells also increases during oocyte growth and this increase parallels that of small cells. however, another study indicates that large follicle cells arise from the differentiation of small cells (filosa et al., 1979). in c. caspium, there was a germinal bed (gb) in each ovary. the germinal bed containing oogonia, oocytes, and primordial follicles, was most active during the vitellogenic period. in this species, the number of laid eggs varied from 1-2 per clutch. another advantage of geckos for comparative analysis of egg shape is that female geckos of all species lay invariant clutches of one or two eggs (shine and greer, 1991). consequently, female geckos have no more than one developing egg in a single ovary at a time. the egg shells harden soon after deposition, thus they undoubtedly offer a better protection against invertebrate predators and desiccating environmental conditions than a soft shell. in squamate reptiles, the generally flexible and poorly mineralized parchment-like egg shell is clearly plesiomorphic, with hard shells occurring only in the gecko family gekkonidae (andrews, 2004). four gekkotan families (carphodactylidae, diplodactylidae, eublepharidae and pygopodidae) lay soft-shelled eggs, while their close relatives (gekkonidae) lay hard-shelled eggs (doughty, 1997). this highly mineralized egg shell is much more costly to produce, as calcium is a limiting element for most terrestrial organisms. however, within gekkonids, small species lay more elongated eggs than larger species (kratochvil and frynta, 2005). in this study, we report more than one egg clutch per year. our results show that the reproductive cycle of c. caspium is of the associated type and occurrs during the well defined period in which oocytes were not found in the ovaries all year round. acknowledgement collecting permits and legal requirements were prepared by department of environment of mazandaran 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(2003): guide to the reptiles of the western palearctic. krieger, malabar, fl. torki, f. (2006): spermatogenesis of the agama trapelus lessonae in the central zagros mountains, iran. zool. middle east 38: 21-28. van wyk, j.h. (1995): the male reproductive cycle of the lizard, cordylus giganteus (sauria: cordylidae). j. herpetol. 29: 522-535. vitt, l.j., pianka, e.r. (1994): lizard ecology: historical and experimental perspectives. princeton university press, princeton. acta herpetologica vol. 8, n. 1 june 2013 firenze university press the oogenic cycle of the caspian bent-toed gecko, cyrtopodion caspium (squamata: gekkonidae) in iran vida hojati1*, kazem parivar2, eskandar rastegar-pouyani3, abdolhossein shiravi1 altitudinal effects on life history parameters in populations of liolaemus pictus argentinus (sauria: liolaemidae) joel antú gutiérrez1,*, carla piantoni2, nora r. ibargüengoytía1,3 recent cryptic extinction of squamate reptiles on yoronjima island of the ryukyu archipelago, japan, inferred from garbage dump remains yasuyuki nakamura1,*, akio takahashi2, hidetoshi ota3 trophic niche and feeding biology of the italian wall lizard, podarcis siculus campestris (de betta, 1857) along western mediterranean coast marco a.l. zuffi*, chiara giannelli novel, non-invasive method for distinguishing the individuals of the fire salamander (salamandra salamandra) in capture-mark-recapture studies goran šukalo1,*, sonja đorđević2, dragojla golub1, dejan dmitrović1, ljiljana tomović2,3 going out tonight? when insular hierophis viridiflavus breaks the whip snakes rules delaugerre michel-jean first evidence of a paedomorphic population of the smooth newt (lissotriton vulgaris) in the czech republic václav gvoždík1,2, veronika javůrková4, oldřich kopecký5,* no detection of chytrid in first systematic screening of bombina variegata pachypus (anura: bombinatoridae) in liguria, northern italy stefano canessa1,*, an martel2, frank pasmans2 status of the european pond turtle, emys orbicularis (reptilia: testudines: emydidae) in vorarlberg, austria andreas kleewein1, günther wöss2 comparative cytogenetics of two species of ground skinks: scincella assata and s. cherriei (squamata: scincidae: lygosominae) from chiapas, mexico riccardo castiglia1,*, alexandra m.r. bezerra2, oscar flores-villela3, flavia annesi1, antonio muñoz4, ekaterina gornung1 polydactyly in the tyrrhenian wall lizard (podarcis tiliguerta) antigoni kaliontzopoulou1,*, daniele salvi1, verónica gomes1, joão p. m. c. maia1,2,3, panagiotis kaliontzopoulos4 book review: roberto sindaco, alberto venchi, cristina grieco. the reptiles of the western palearctic. 2. annotated checklist and distributional atlas of the snakes of europe, north africa, middle east and central asia, with an update to the vol. 1. edoardo razzetti acta herpetologica journal of the societas herpetologica italica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 9(1): 129, 2014 doi: 10.13128/acta_herpetol-14038 book review: federico chacón, richard dennis johnson. amphibians and reptiles of costa rica. federico chacón, richard dennis johnson. amphibians and reptiles of costa rica. anfibios y reptiles de costa rica. sebastiano salvidio dipartimento di scienze della terra, dell’ambiente e della vita (distav), università di genova, i 16132 genova italy. e-mail: salvidio@ dipteris.unige.it this field guide is available in two different editions, one only in english consisting of 172 pages and one in both english and spanish of 215 pages. the format of both is 12 x 17 cm, and make them handy and valuable pocket friends. in this review i will comment only the english edition, but i verified that the bilingual bock covers exactly the same topics and number of species. the english guide begins with a four pages “introduction” in which the biological richness and the national nature conservation policy are very briefly described. few lines are dedicated also to the recent amphibian decline described for this region. then a tree pages section describes the symbols used in the species description and in the species range maps. the pocket guide then illustrates the 191 amphibians and the 227 reptiles found within costa rica boundaries, comprising extinct species [e.g. the endemic golden toad incilus (bufo) periglenes] and also introduced species (e.g., the cuba treefrog osteopilus septentrionalis). all species’ are described by a very short text and a small (2 x 2 cm) but clear map illustrates the species’ distribution. almost all species are depicted by small (usually 3.5 x 6 cm) but good quality and bright colour photos. however, 22 of the 42 reported salamanders are not illustrated. dangerous snakes for humans are marked by a skull symbol. the guide ends with a useful glossary, a systematic index and with a short cv of the authors. the variety of amphibian and reptile species and their extreme diversification is striking and gives to the reader interested in these animals a strong motivation to visit the country. if the principal aim of this field guide was to allow tourists visiting costa rica to appreciate, get interested and try to recognize the hundreds of herps present in the different ecosystems, it was surely achieved. i also suggest that all parents visiting costa rica buy a copy this guide for their child, because this booklet will surely constitute an unforgettable souvenir for all future naturalists and herpetologists. i personally missed only the absence of further suggested readings concerning one of the most remarkable wildlife regions of the world. the english pocket guide can be purchased at the cost of $14.95 (about €11), the english/spanish edition for $19.95 (about €15), from the publisher’s website (http://www.cornellpress.cornell.edu/ book/?gcoi=80140100952080, isbn 978-0-8014-7869-7). acta herpetologica vol. 8, n. 2 december 2013 firenze university press journal of the societas herpetologica italica acta herpetologica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 6(2): 247-259, 2011 escape by the balearic lizard (podarcis lilfordi) is affected by elevation of an approaching predator, but not by some other potential predation risk factors william e. cooper, jr.1, valentín pérez-mellado2 1department of biology, indiana university purdue university fort wayne, fort wayne, in 46805, usa. corresponding author. e-mail: cooperw@ipfw.edu 2departamento de biologia animal, universidad de salamanca, 37071 salamanca, spain. submitted on: 2011, 6th april; revised on: 2nd september; accepted on: 5th september. abstract. many predation risk factors to affect escape behavior by lizards, but effects of some potential risk factors are unknown or are variable among species. we studied effects of several risk factors on escape responses by the balearic lizard (podarcis lilfordi, lacertidae) on escape responses. escape was elicited by an approaching experimenter who recorded flight initiation distance (predator-prey distance when escape begins) and distance fled. when an experimenter approached from above (upslope), flight initiation distance and distance fled were longer than when the experimenter approached from below. this novel effect suggests that lizards exposed to aerial predation might have been naturally selected to respond rapidly to predators approaching from above or that effects of path inclination of escape ability may differ between predators and prey in a manner requiring a larger margin of safety during approaches from above than below. although sex differences in aspects of escape occur in some lizards, including lacertids, no sex difference was observed in p. lilfordi. because vigilance and some other aspects of antipredatory behavior exhibit cortical lateralization, we tested effects of approach from the left and right sides of lizards. as predicted by optimal escape theory, side of approach did not affect flight initiation distance. because many lizards have color vision and respond to pigmentation of conspecifics in social settings, researchers have often worn only drably colored clothing when simulating predators. this precaution may be unnecessary because flight initiation distance did not differ among investigator shirt colors (red, orange, olive). keywords. escape behavior, flight initiation distance, distance fled, laterality, predation risk, squamata. introduction optimal escape theory successfully predicts effects of many risk factors on escape decisions (stankowich and blumstein, 2005; cooper and frederick, 2007, 2010; cooper 248 w.e. cooper, jr. and v. pérez-mellado 2010a). although optimal escape theory was developed to predict flight initiation distance, the distance separating prey from an approaching predator when the prey begins to flee, its principles regarding predation risk, cost of escaping, and prey fitness are often applicable to other escape variables, including distance fled and probability of entering refuge (martín and lópez, 2003; lópez et al., 2005). numerous predation risk factors are known to affect escape decisions (lima and dill, 1990; stankowich and blumstein, 2005; cooper, 2010a). flight initiation distance increases with risk for diverse risk factors, and mounting evidence indicates that distance fled and probability of entering refuge also increase with predation risk in many circumstances (stankowich and blumstein, 2005; cooper, 2010a). despite extensive study in the past 15 years, effects of some potential risk factors on escape decisions remain unstudied. one such factor is the elevation of an approaching terrestrial predator with respect to the prey. many prey are subject to both aerial and terrestrial attack. because aerial predators often approach more rapidly than terrestrial predators and may be more maneuverable during chases, risk may often be high when prey are approached from above. even for terrestrial predators, greater maximal approach speed may be possible while moving downhill. predators approaching on the ground from below a prey’s position may have slower top running speed or acceleration than when on level ground (irschick and jayne, 1999; vanhooydonck and van damme 2001; warner and shine, 2000). in these circumstances, it may be predicted that flight initiation distance is longer when prey are approached from above than from below. for prey that do not enter refuges, distance fled is also predicted to be longer for approaches from above than below. predictions regarding refuge entry are less clear, but if risk is great enough to elicit hiding in refuge by a high proportion of individuals, a greater proportion of individuals are predicted to enter refuge when approached from above than from below. sex differences in escape behavior may occur for several reasons, including differences in body size, detectability, and escape ability. over a wide range of taxa, about one third of prey species exhibit sex difference in flight initiation distance (stankowich and blumstein, 2005). among 30 lizard species, flight initiation distance is longer in males than females in at least one population in five species, longer in females than males in only one species; it does not differ between sexes in the remaining 24 (80%) species (johnson, 1970; shallenberger, 1970; snell et al., 1988; burger and gochfeld, 1990; braña, 1993; bulova, 1994; stone et al., 1994; smith, 1996; whiting, 2002; cooper, 2003a, 2006, 2009, in press; lailvaux et al., 2003; capizzi et al., 2007; cooper and wilson, 2007a; vanhooydonck et al., 2007; brecko et al., 2008; cooper and avalos, 2010). a sex difference in distance fled has been detected in only one of 21 lizard species studied (snell et al., 1988; stone et al., 1994; smith, 1996; whiting, 2002; cooper, 2003a, 2006, 2009a, in press; lailvaux et al., 2003; cooper and wilson, 2007a; vanhooydonck et al., 2007; brecko et al., 2008; cooper and avalos, 2010); males fleeing a greater distance in that species (cooper, 2006). a sex difference in probability of entering refuge occurs in 2 of 5 lizard species, conspicuous males being more likely than less conspicuous females to enter refuge in both cases (lailvaux et al., 2003; cooper, in press). another possible risk factor is the side from which a predator approaches a prey. because sidedness affects many activities, prey having different escape latencies when approached from right or left (lippolis et al., 2002) or prey that exhibit left-right pref249escape by the balearic lizard erences for maintaining vigilance (martín et al., 2010 ) might also exhibit differences in flight initiation distance between approaches by predators from the left and right. if such differences occur, flight initiation distance is predicted to be longer for approaches from the side for which escape latency is shorter. if differential vigilance between sides is important, flight initiation distance might be longer during approaches from the side where the prey is more vigilant. on the other hand, because natural selection should favor prey able to escape from attacks from any angle, the side of approach may not affect flight initiation distance if the prey has detected the predator at a distance greater than the optimal flight initiation distance (cooper and frederick, 2007). many lizards have excellent color vision, and many species have bright, socially significant coloration (cooper, 1992). therefore, it has been common practice for researchers who simulate predators approaching lizards to wear drab coloration to avoid possible effects of color on escape behavior (e.g., martín and lópez, 1999; cooper 2003b). however, it has never been established whether the color of clothing worn by researchers affects escape parameters. here we examine possible influences on escape decisions by balearic lizards (podarcis lilfordi) of two predation risk factors not previously studied in any prey species, the elevation of an approaching predator with respect to the prey and the coloration of clothing used by investigators. in addition, we investigated the poorly known effect of the side from which the predator approaches and the possibility that the sexes differ in flight initiation distance and distance fled. methods animals and study sites podarcis lilfordi is a small, omnivorous lacertid lizard that occurs at high population densities on many islets off the coasts of the large islands mallorca and menorca (pérez-mellado and corti, 1993; pérez-mellado et al., 2008), balearic islands, spain. we conducted field experiments on effects of experimenter elevation during approach, lizard sex, and shirt color on escape on dragonera island adjacent to mallorca and of side from which approach occurred on aire adjacent to menorca. all data were collected in late june 2010 in sunny conditions when lizards were active. males are slightly larger than females on aire (67.8 mm vs. 59.3 mm snout-vent length) and some other, but not all, islets (pérez-mellado et al., 2000). only large adults were included in observations. on dragonera data were collected during 09:00-14:25 h at air temperatures of 27-29 °c in the study of elevation, 28-29 °c in the study of side of approach, and 23-25 °c in the study of shirt color. lizards were observed on and adjacent to unpaved pedestrian paths. lizards there escaped off the paths and could seek refuge beneath vegetation or in rock crevices, including those in stone fences in several locations. human beings are continually present on dragonera, a natural park where boats daily bring tourists to the islet who walk along the paths occupied by lizards. many known and potential predators are present. these include raptors, especially kestrels (falco tinnunculus) and booted eagles (aquila pennata), seagulls that capture lizards occasionally (yellow-legged gull, larus michahellis; audoin’s gull, l. audouinii; cramp and simmons, 1983)), and ship rats (rattus rattus), which are potential predators. on aire data were collected during 11:15-18:00 h at temperatures 22-26 °c. lizards were observed on open ground amidst patches of sparse vegetation. available refuges were low bushes, rock crevices, and holes at the bottom of some stone fences, as well as burrows from introduced rab250 w.e. cooper, jr. and v. pérez-mellado bits (oryctolagus cunniculus). aire has lacked permanent residents since the 1960s, but biologists, lighthouse maintenance personnel, and boaters often visit it during spring and summer. potentially predatory birds on aire include kestrels (falco tinnunculus), booted eagles (aquila pennata), seagulls of both species noted for dragonera, and shrikes. kestrels eat lizards in southern europe (cramp and simmons, 1980). kestrels are major predators on p. lilfordi populations on some menorcan islets, especially on islets where they breed. they often visit aire, but have not bred there for several years. the seagull larus michahellis is abundant on aire, but this species is not known to eat p. lilfordi in cabrera (araújo et al., 1977) or p. atrata in the columbretes islands (gómez, 1991; catalá et al., 1990). shrikes (lanius spp.) are major predators of lizards and occur on menorca and some of its islets, particularly during spring migration. however, they were only occasionally observed on aire. mammalian and ophidian predators are absent from aire (pérez-mellado, 1989). simulation of approach by a predator an investigator walked directly toward a lizard to elicit escape behavior. people are not natural predators of p. lilfordi. however, biologists and amateur collectors have removed many of these lizards from natural populations. even if such human predation has been unimportant, prey subject to attack by diverse predators are likely to be naturally selected to flee when approached directly by any potential predators. consequently, use of researchers as simulated predators is effective for study of escape responses in diverse prey (stankowich and blumstein, 2005), including lizards (e.g., cooper, 1997a, 2000a; martín and lópez, 1999b; martín et al., 2003a,b; cooper and wilson, 2007a,b). studies of escape by lizards using human simulated predators have consistently validated predictions of optimal escape theory about flight initiation distance for numerous predation risk factors and for factors that affect cost of fleeing (e.g., heatwole, 1968; burger and gochfeld, 1990; martín and lópez 1996; cooper 1997a-c, 1999, 2000; cooper et al. 2003a; cooper et al. 2006; cooper and whiting, 2007). simulation of a predator by a researcher has some important advantages. this method is efficient because humans can traverse rough terrain much better than inanimate predator models can. it also precludes actual predation that might occur if natural predators were used and facilitates control of differences among experimental conditions. several potential problems with this method have proven to be unimportant. one possible drawback is that apparent escape responses to people might be motivated by avoidance of trampling rather than avoidance of a predator. another is that some prey, such as chameleons, exhibit different responses to different types of predators (stuart-fox et al., 2006). in sceloporus virgatus, a lizard similar to p. lilfordi in being primarily terrestrial, no qualitative differences in escape occurred in response to approaching people and models of snakes and birds (cooper, 2008). predator-specific responses are unknown in p. lilfordi, which escapes by fleeing and often enters refuges (cooper et al., 2009a, b, 2010). to avoid experimenter bias, another potential drawback, we approached lizards in a standardized fashion: the same gait was used in all approaches and approach speeds were practiced to ensure consistency across trials. approach speeds were 0.8 ± 0.0 (se) m/s in the experiment on the effect of side from which the lizard was approached and 1.4 ± 0.1 (se) m/s in the other experiments. sample sizes for the speeds were 10 each. for each experiment the treatment order was selected prior to data collection to eliminate any unconscious choice of treatments to favor predicted outcomes. all approaches were made by wec. experimental design and analysis all experiments were conducted independently of each other. in the study of the effects of elevation of the approaching predator with respect to the prey, lizards were approached by an inves251escape by the balearic lizard tigator walking along a path either from below (downslope) or above (upslope). data were collected where slopes were moderate (ca. 15-30 °, corresponding to a difference of 30-60° between approaches from above and below). the experimenter walked upslope during several successive trials, and then downslope for the next series of trials in a different location. the overall number of trials was the same for each elevation (n = 26), and no sequential biases were possible due to differences in time of day, temperature, or other variables. in this and the other studies, the experimenter moved very slowly to a position 6-8 m from a lizard located on the ground, stopped for a few seconds, approached directly, and then stopped approaching as soon as the lizard began to flee. data recorded were sex of lizard, flight initiation distance and distance fled to the nearest 0.1 m, and whether or not the lizard entered refuge. analysis of variance for a single factor experiment was used to test for sex differences in flight initiation distance and distance fled. an analysis of covariance with flight initiation distance as covariate was used to further examine the effect of elevation of approacher on distance fled. in the absence of a sex difference, similar analyses were used to assess the significance of the difference between approaches from above and below in the two distance variables. a fisher exact probability test was used to examine the difference in proportion of individuals that entered refuge during approaches from above and below. to study the effects of side from which a lizard was approached, an investigator approached a the lizard’s left or right side from angles in an 60 ° arc centered perpendicular to the lizard’s longitudinal axis. eight-eight lizards were tested in counterbalanced sequence (n = 44 each). to study effects of shirt color the investigator wore a red, orange or olive t-shirt during approaches. fifteen lizards were tested using orange and olive shirt color and 20 were tested using red shirt color. five trials were conducted in sequence for a given shirt color before the investigator changed shirts to another color. in the studies of effects of approach side and shirt color, only flight initiation distance was measured. anova for single-factor experiments was used in each experiment to for differences in treatments. prior to analyses of variance, the assumptions of normality and homogeneity of variance were evaluated using kolmogorov-smirnov and levene’s tests, respectively. effect sizes are reported as η2, for which values < 0.30 are considered small (cohen, 1992). assumptions were met in all cases. significance tests were two-tailed with α = 0.05. results predator elevation and lizard sex flight initiation distance was significantly greater (f1, 50 = 10.19, p = 0.0024) when the predator approached from above than from below lizards on an inclined path (fig. 1). in an analysis of variance, distance fled was significantly longer (f1, 50 = 6.51, p = 0.0145) for lizards approached from above (1.0 ± 0.1 m) than from below (0.7 ± 0.1 m). distance fled increased as flight initiation distance increased for all lizards (r = 0.28; f1,50 = 4.39, p = 0.041). this relationship was significant for approaches from above (r = 0.42; f1,24 = 5.04, p = 0.034), but not from below (r = 0.05; f1,24 = 0.06, p = 0.81). slopes of distance fled on flight initiation distance differed substantially, but not significantly (f1,48 = 2.48, p = 0.12). in an ancova using flight initiation distance as covariate, the distance fled remained significantly greater during approaches from above than from below (f1,49 = 3.40, p = 0.036). the effect sizes were small: η2 = 0.20 for flight initiation distance and η2 = 0.12 (anova) or 0.06 (ancova) for distance fled. 252 w.e. cooper, jr. and v. pérez-mellado few lizards entered refuges: 3 of 26 when approached from above and 4 of 26 when approached from below. the approaching predator’s elevation did not affect the probability of entering refuge (fisher p = 0.71). flight initiation distance did not differ significantly between sexes (2.4 ± 0.7 m for males, n = 29; 2.6 ± 0.8 m for females, n = 23; f1, 50 = 0.57, p = 0.45). neither did distance fled for the same individuals (0.9 ± 0.4 m for males, 0.8 ± 0.5 m for females; f1, 50 = 0.06, p = 0.81). approach side and shirt color flight initiation distance did not differ significantly (f1,86 = 0.05, p = 0.82) between approaches from the lizard’s left (2.3 ± 0.1 m) and right (2.3 ± 0.2 m) sides (n = 44 each). the investigator’s shirt color (table 1) did not significantly affect flight initiation distance (f2,47 = 0.21, p = 0.81), distance fled (f2,47 = 1.53, p = 0.23), or probability of entering refuge (fisher p = 1.00 for olive versus red, 0.68 for olive versus orange, and 0.71 for red versus orange). fig. 1. flight initiation distances of podarcis lilfordi approached from above and below. error bars represent 1.0 se. table 1. flight initiation distance (m) and distance fled (m) by podarcis lilfordi when approached by an investigator wearing a shirt having one of the listed colors. shirt color flight initiation distance distance fled entered refuge n mean se mean se yes no olive 3.1 0.2 0.6 0.1 3 12 15 orange 3.0 0.3 0.7 0.1 5 10 15 red 2.8 0.2 0.8 0.1 5 15 20 253escape by the balearic lizard discussion lizards permitted closer approach before fleeing and fled for shorter distances when the experimental predator approached along a path from below them than from above. these effects, although novel, were small. in several species of lizards approached on level ground when perched on trees, flight initiation distance increases as perch height decreases (cooper, 1997a, 2006, 2009c). in effect, individuals higher on tree trunks are closer to refuge, i.e., to being out of reach above the predator. for lizards on the ground, flight initiation distance increases as distance to refuge increases because a longer time is required to flee into a more distant refuge (bulova, 1994; cooper, 1997a, 2000). in the studies of lizards on trees, flight initiation distance is longer when the predator is higher with respect to the lizard, but for a different reason in p. lilfordi. in the present study, approach from above positioned the experimenter higher with respect to the lizard than approach from below, but the distance to the nearest refuge would have been the same regardless of the elevation of approach. one hypothesis to explain the longer flight initiation distance and distance fled by lizards approached from above than below is that natural selection has favored long flight initiation distance and distance fled in response to aerial predators that approach more rapidly than terrestrial predators. this hypothesis assumes that lizards interpret standing human beings as aerial predators, which they might be more likely to do when approached from above than from below. because the predator approached at the same speed in all trials, the hypothesis also requires that natural selection has favored longer flight initiation distance at a given approach speed when the approach is from above than below. a second hypothesis is that flight initiation distance and distance fled are longer during approaches from above than below because terrestrial predators can run and/or accelerate fasterly when moving downslope than upslope (irschick and jayne, 1999; jayne and irschick, 2000). on steep slopes, flight initiation distance by the phrynosomatid lizard sceloporus virgatus did not differ from that in other terrestrial microhabitats, but distance fled was longer on the slopes (cooper and wilson, 2007b). the effect of slope on distance fled is opposite that of predator elevation in p. lilfordi. by fleeing upslope, s. virgatus could escape without incurring costs of entering refuge because the predator’s ability to pursue was diminished on the slope. no information is available about approach from above on escape by s. virgatus, but the different effects on distance fled in the two lizard species appear to reflect different aspects of escape strategies. future studies of effects of predator elevation during terrestrial approach on escape decisions should be conducted to verify the new findings, determine how widespread the effects are among lizards, and to ascertain the importance of the steepness of the approach slope. the infrequency of refuge use in this experiment despite the presence of abundant refuges might be a consequence of habituation to human presence. in areas where other lizard species frequently encounter human beings, flight initiation distance is shorter (burger and gochfeld, 1990; labra and leonard, 1999; eifler, 2001; cooper, 2006, 2009c, 2010b; cooper and whiting, 2007b; cooper and avalos, 2010). in columbian black-tailed deer, distance fled increases as flight initiation decreases, and both variables are shortened by habituation (stankowich and coss, 2007). in species that use refuges, shorter distance fled by habituated individuals presumably is accompanied by a lower probability of entering refuge when the effect of initial distance from refuge is taken into account. 254 w.e. cooper, jr. and v. pérez-mellado among diverse lizard taxa, sex differences in flight initiation distance occur in only a small proportion of species (cooper, in press). in lacertidae, flight initiation distance is longer in males than females in two of five species studied, iberolacerta horvathi and zootoca vivipara (braña, 1993; capizzi et al., 2007; brecko et al., 2008). however, no difference in flight initiation distance between sexes has been detected in podarcis (p. lilfordi: this study; p. melisellensis: brecko et al., 2008; p. muralis: braña, 1993). although the proportion of lacertid species having sex differences in flight initiation distance is more than twice that in all other lizards combined, the sample size for lacertids is too small to assess this difference with adequate statistical power. distance fled rarely differs between sexes in lizards, having been detected only in one species (anolis gundlachi: cooper, 2006) of 22 species tested, including p. lilfordi and others reviewed by cooper (in press). the side from which a predator approached a lizard did not affect flight initiation distance. brain function of many vertebrates is lateralized, the right eye and left hemisphere feeding being dominant in foraging and the left eye and right hemisphere in antipredatory contexts (martín et al., 2010). in the common wall lizard (podarcis muralis), the left eye is preferentially used to monitor predators once a lizard has entered refuge (martín et al., 2010). in horses, distance moved was greater when a person on the horse’s left than right side opened an umbrella (austin and rogers, 2007), suggesting a relationship between side bias and escape behavior. the absence of any laterality in flight initiation distance suggests that neural lateralization may not affect flight initiation distance in lizards. the decision to begin fleeing is based on degree of predation risk, cost of fleeing, and the prey’s initial fitness (cooper and frederick, 2007, 2010). only risk is likely to be affected by the side from which a predator approaches. laterality of flight initiation distance may be expected to occur only if escape ability differs between approaches from the left and right, which might occur due to interhemispheric differences in control of fleeing or to morphological asymmetry. lateralization is believed to be advantageous in allowing more rapid responses (mandel et al., 2008). however, in the scenario of optimal escape theory, a prey monitors the approach of a predator prior to fleeing. given time for monitoring and risk assessment, lateralization seems unlikely to affect flight initiation distance. nevertheless, lateralization might affect rapidity of response when a predator is closer than the optimal flight initiation distance. this can occur if the prey does not detect the predator until immediate flight is required (blumstein, 2003; stankowich and coss, 2006; cooper and frederick, 2007). in such circumstances, more rapid detection of a predator on one side could favor shorter latency to escape and, therefore, slightly longer flight initiation distance. the color of an investigator’s shirt did not affect flight initiation distance by p. lilfordi. although experimenters have been cautious about possible effects of clothing color on lizard escape behavior (martín and lópez, 1999; cooper, 2003b), wearing drab clothing of similar coloration throughout an experiment does not appear to be necessary in studies of flight initiation distance. it remains possible that colors known to affect lizard social behavior (cooper and crews, 1987; cooper, 1992; martín and forsman, 1999; weiss, 2006) might affect flight initiation distance, this seems very unlikely. given the huge differences in body size and shape between lizards and human beings, lizards are unlikely to misidentify researchers as conspecifics no matter what the colors of clothing worn by the researchers. 255escape by the balearic lizard acknowledgements the research was funded by the research project: cgl200912926c0202 from the spanish ministry of science and innovation. we thank martí mayol for permits allowing our research and providing accommodations in the natural park of dragonera. we are grateful to mario garrido and ana pérezcembranos for logistical help in aire island. we are also thankful for the scientific capture permits issued by the conselleria de medi ambient of balearic government. references araújo, j., muñoz-cobo, j., purroy, f.j. 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(2001): evolutionary traeoffs in locomotor capacities in lacertid lizards: are splendid sprinters clumsy climbers? j. evol. biol. 14: 46-54. warner, d.a., shine, r. (2000): morphological variation does not influence locomotor performance within a cohort of hatchling lizards (amphibolurus muricatus, agamidae). oikos 114: 126-134. weiss, s.l. (2006): female-specific color is a signal of quality in the striped plateau lizard (sceloporus virgatus). behav. ecol. 17: 726-732. whiting, m.j. (2002): field experiments on intersexual differences in predation risk in the lizard platysaurus broadleyi. amphibia-reptilia 23: 119-124. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 7(1): 111-118, 2012 testing the ability to store sperm: an experimental manipulation of mating opportunities in the common wall lizard, podarcis muralis daniele pellitteri-rosa1, roberto sacchi1, fabio pupin2, adriana bellati1, walter cocca1, augusto gentilli1, paolo galeotti1, mauro fasola1 1dipartimento di scienze della terra e dell’ambiente, laboratorio di eco-etologia, università degli studi di pavia, via ferrata 9, 27100 pavia, italy. corresponding author. e-mail: masterfauna@unipv.it 2museo tridentino di scienze naturali, via calepina 14, 38122 trento, italy submitted on: 2011, 24th october; revised on 2011, 23rd march; accepted on 2012, 29th march. abstract. female common wall lizards (podarcis muralis) typically reproduce annually and lay more than one clutch per season. in this study, we tested whether females store sperm between clutches and between years by manipulating mating opportunities of females through appropriate experiments. our results revealed that females are definitely unable to store sperm for medium or long-term, suggesting they necessary need to repeatedly mate with males to fertilize their eggs. finally, by comparing our results to other similar multi-clutched species, we conclude that sperm storage probably does not constitute a selective advantage for species with a promiscuous reproductive system based on multiple mating in populations with high densities. keywords. multiple mating, sperm storage, sexual selection, unfertilized eggs, common wall lizard. introduction in reptiles, multiple mating and sperm storage frequently occur (sever and hamlett, 2002), as documented for chelonians (johnston et al., 2006), ophidians (schuett, 1992) and saurians (villaverde and zucker, 1998; olsson et al., 2007), even though the organs devoted to sperm storage are relatively undifferentiated. moreover, sperm storage in reptiles shows a considerable variation in time duration, ranging from hours to months or even years (schuett and gillingham, 1986; pearse et al., 2001; olsson et al., 2007). the evolutionary causes of sperm storage are debated. in a review of multiple paternity in reptiles, uller and olsson (2008) have suggested that the main cause for sperm retention is selection for sperm longevity resulting from sperm competition when the turnover of 112 d. pellitteri-rosa et al. female reproductive cycles is quick. alternatively, sperm storage may also contribute to cryptic female choice, enabling female control of paternity and retention of optimal sperm across reproductive cycles (olsson and madsen, 1998; uller and olsson, 2008). noteworthy, these reasons are not mutually exclusive and can occur simultaneously within a species. multiple mating and female sperm storage could be promoted by sexual selection as they increase offspring diversity (genetic bet-hedging hp, watson, 1991; see also yasui, 1998 for a review) and elicit cryptic female choice or sperm competition, thereby incrementing the probability that eggs are fertilized by sperm from high-quality males (olsson and shine, 1997; yasui, 1997). opportunity for cryptic female choice could have driven the evolution of sperm storage especially when females are able to control paternity through the retention of sperm from high quality males over breeding cycles (olsson and madsen, 1998). apart from sexual selection, long-term sperm storage can evolve to delay the time between copulation and fertilization, when deferred emergence increases offspring survival (birkhead and møller, 1998; villaverde and zucker, 1998). the advantages of long time sperm storing are also particularly evident in species having few mating opportunities, due to the sexual segregation of both sexes in time and space (ruckstuhl and neuhaus, 2005), male sperm limitation (preston et al., 2003), or asynchrony among spermatogenesis, mating, and ovulation (schuett, 1992). for example, females of several snake species evolved long time sperm storage as a response to the lack of synchrony among spermatogenesis, mating, and ovulation (fox, 1952; saint girons, 1982; schuett, 1992). distinguishing between the different hypotheses explaining multiple mating and sperm storage represents one of the most interesting topic of mating systems, but it is also a hard question to be solved. this is because observational data collected during field researches frequently agree with different hypothesis. a first approach to address these issues is to determine whether a species is actually able to store sperm or not and this can be achieved only through experiments that manipulate mating opportunities for females (by reducing or increasing the number of copulations) as well as the timing of sperm storage (by shortening or lengthening the time between copulations), thus recording the effects on the production of fertilized eggs. due to their biological and reproductive traits, lizards represent an ideal model to investigate these subjects since, for example, most species are promiscuous, and lack post-hatching parental care (madsen et al., 1992; olsson et al., 1996, 2007; uller and olsson, 2008). the common wall lizard (podarcis muralis, laurenti 1768) is a small (snout-vent length, svl, 45-75 mm) lizard occurring in southern and central europe (arnold and burton, 1985). both males and females mate multiply during the same breeding season, and females produce on average two clutches per year (barbault and mou, 1986; brana and ji, 2007). the breeding season lasts from mid-march to late july (corti and lo cascio, 1999), although significant differences in the onset of mating can arise as a consequence of climate variation not only at macro-geographic scale, but even among different sites at local scale. recently, oppliger et al. (2007) showed that multiple paternity occurred in at least 87% of the clutches, thus suggesting possible shortterm effects of sperm storage (uller and olsson, 2008). however, to date the ability of this species to store sperm has not been demonstrated nor for medium-term nor for 113testing sperm storage in podarcis muralis long-term periods. therefore, it would be really interesting to verify if females are actually able to store sperm, and above all whether sperm is stored to fertilize two successive clutches laid by the same female in the same breeding season, or whether sperm is stored over successive breeding seasons. in this study we manipulated the mating opportunities of females in order to investigate if they are able to a) use sperm stored before the first clutch to fertilize the eggs of the second clutch without new copulations (hereafter medium-term sperm storage), and b) use sperm stored before last clutch in a breeding season to fertilize eggs in the first clutch of the next season without new copulations (hereafter long-term sperm storage). material and methods medium-term sperm storage experiment in spring 2007 (april to may), we captured by noosing 83 pregnant females in 6 sites in the surroundings of pavia (lombardy, northern italy). pregnancy was assessed by the presence of mating scars, inflicted by the male on the female belly at copulation (strijbosch et al., 1980). females were transferred to our laboratory (department of animal biology, university of pavia) within 2 hours from capture, and housed individually in outdoor plastic jars (60 × 50 × 50 cm) containing soil mixed with small stones as substrate and a single brick as shelter. we provided female with 3 mealworms, tenebrio molitor larvae, each day, and water ad libitum. each cage was also supplied a small tank (10 × 10 × 5 cm) filled by wet sand and partially covered by a tile. every morning tanks were carefully checked for egg presence and sprayed with water to keep sand wet. all lizards were maintained in captivity until egg laying, which occurred for 68 females within 15 days on average from collection. mean clutch size was 4.6 (range: 2-8). we randomly selected 20 lizards that had laid their first fertilized clutch to investigate if they were able to lay a second fertile clutch without new copulations. the other 63 females were returned to their capture sites. long-term sperm storage experiment during february and march 2008, we noosed 20 sexually mature virgin females in 5 sites located in the town of pavia and in its surroundings. virgin condition was inferred by the lack of male ventral scars, meaning that females had not mated yet in the current season. after captured, females were housed in our laboratory within 2 hours from capture. contrary to previous experiment, females were held indoor under a natural light–dark cycle in individual transparent plastic jars (20 × 30 × 20 cm) provided with a newspaper sheet as substratum, a water tank and a shelter, and fed with 3 mealworms each day. water was provided ad libitum. plastic jars were placed under an uv-b lamp (18 w) in order to provide the daily uv requirements for calcium and vitamin d fixation, and an incandescent lamp (25 w) for heating. uv lamps were switched on for 3 h a day (from 10.00 am to 13.00 pm), while incandescent lamps were set alight for 6 h a day (from 11.00 am to 17.00 pm). each cage was provided with a small tank (10 × 10 × 5 cm) filled by wet sand for egg laying. the aim of this second experiment was to check if females were able to fertilize eggs using the sperm eventually preserved by the copulations obtained in the previous breeding season. finally, females that had laid an infertile clutch were paired with a male in order to check if they were actually able to lay fertile eggs. the other females were returned to their capture sites. 114 d. pellitteri-rosa et al. results medium-term sperm storage experiment nine females did not lay eggs at all, while eleven produced clutches including only not fertilized eggs, i.e. eggs with flabby consistency, greyish colour and irregular shape (table 1). by contrast, fertilized and viable eggs appear white coloured with a tight and soft shell. as commonly occurring in most species of lizards, only fertilized eggs showed a clearly visible germinal disk that can be distinguished by the colour (orange to purple) inside the still moist egg shell. in addition, sterile eggs are usually smaller and not as firm as fertile ones (köhler, 2004; see fig. 1). mean clutch size was 4.4 (range: 3-6), while the mean time elapsed between first and second clutch was 31 ± 4 days (range: 16-53). all lizards were released in their capture site at the end of the experiment. long-term sperm storage experiment on the whole, 17 out 20 females laid a clutch, while the other three did not lay any egg at all (table 1). mean clutch size was 3.6 (range: 2-5), but none involved fertilized eggs. the mean time elapsed between capture and laying was 49 ± 2 days (range: 36-59). however, after having been paired with a male, 18 out 20 females laid clutches including fertilized eggs (table 1). table 1. number of the first and second clutches laid by common wall lizard females during mating manipulation experiments. clutches are distinguished according to the viability of eggs laid. female treatment 1st clutch female treatment 2nd clutch fertile infertile not laid fertile infertile not laid medium-term experiment mated in wild 20 0 0 not mated 0 11 9 long-term experiment not mated 0 17 3 mated in captivity 18 0 2 discussion our experiments clearly showed that common wall lizard females do not store sperm for medium or long-term. indeed, we have demonstrated that females who had laid a fertile first clutch are unable to produce a second one without mating again. this result clearly indicates that females are not able to keep the sperm collected during the period preceding the first deposition for enough time to fertilize the eggs of the second laying. this conclusion is reinforced by the fact that 11 females have anyway laid eggs, although unfertilized. these findings suggest that females of common wall lizard could produce eggs that 115testing sperm storage in podarcis muralis must be laid at the end of the ovarian cycle, irrespective of male fertilization. we conclude that this reproductive strategy depends on the high density this species is found in natural populations, which makes unlikely that females do not find males for mating. in this scenario, females are sure to fertilize their eggs by at least one male and the existence of a sperm storage system in the medium-term would not provide them any substantial selective advantage. the second experiment demonstrated the complete inability of females to store sperm between two consecutive breeding seasons. indeed, none of the 20 virgin females captured at the beginning of the breeding season was able to lay fertile eggs. nevertheless, as we found in the previous experiment, most females (17 out 20) laid an infertile clutch, reinforcing our hypothesis that females must necessarily lay the eggs after a limited time period, apart from their fertilization by a reproductive male. we can presume that in this species female have not evolved any particular sperm storage organs. we can exclude that the experimental conditions had affected eggs deposition by females, since in 2007 most of the 83 collected females were able to lay a good clutch after a period of captivity, and during 2008 experiment 90% of females (18 out of 20) that have laid a bad first clutch, were able to lay a good clutch after having been paired with a male. therefore, the observed deposition of bad clutches could be actually due to the lack of a previous eggs fertilization. comparing our results with other similar multi-clutched squamate species, we detected the existence of medium and/or long-term sperm storage mechanisms in many snakes and some lizards. for example, several female adders store sperm for weeks or even months, as an obligatory component of their reproductive cycle, with maintenance of fertilization capacity (schuett, 1992; isogawa and kato, 1995; almeida-santos and salomão, 1997). in lizards, the ability to store sperm has been proved in some species (for example, hemiergis peronii: smyth and smith, 1968; psammophilus dorsalis: srinivas et al., 1995; urosaurus ornatus: villaverde and zucker, 1998; niveoscincus ocellatus: wapstra et al., 1999; uta stansburiana: zamudio and sinervo, 2000; calotes versicolor: shanbhag, 2003; anolis sagrei: calsbeek et al., 2007; ctenophorus pictus: olsson et al., 2007), interpreting it as necessary reproductive choice when the mating season does not match the time of egg production, as occur in species with autumn mating and spring vitellogenesis and ovufig. 1. difference between a fertile (left) and a sterile egg (right) of podarcis muralis. 116 d. pellitteri-rosa et al. lation (uller et al., 2010); oviductal sperm storage strategy has been evolved in tropical lizards that lay eggs in multiple clutches possibly to eliminate repeated mating and reduce risk of predation (shanbhag, 2003). in all these cases the evolution of a sperm storage system seems to have been driven by natural selection, although we cannot exclude the possibility of a selective advantage involving the facilitation of female choice and sperm competition at least for some of those species (olsson and madsen, 1998). by contrast, our results clearly show that, at least for that concerning northern italian lowland populations, female common wall lizards do not store sperm between clutches nor for the same breeding season nor for two successive breeding seasons. it’s presumable that sperm storage does not constitute a selective advantage for such species characterized by promiscuous reproductive systems based on multiple mating in populations characterized by high densities, since females are certain to fertilize their eggs during each subsequent ovarian cycle, thus making sperm storage unnecessary. interesting enough, a previous study revealed the presence of multiple paternity within the same clutch in a highland population of common wall lizard (oppliger et al., 2007). in the light of our findings, we can reasonably conclude that multiple paternity in p. muralis must be better explained by multiple matings occurring during each ovarian cycle, rather than by sperm storage across subsequent clutches. acknowledgments we are very grateful to drs. mattia melpignano and marina teofilo pignati for their help with field and laboratory work. research was supported by phd grants (doctorate in ecology and geobotany) from university of pavia to d.p.r. and f.p. the study was carried out in conformity with the italian current laws for lizard collection and detention (aut. prot. dpn-2007-0035506 and dpn2008-0003606). references almeida-santos, s.m., salomão, m.g. 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(1997): a ‘‘good-sperm’’ model can explain the evolution of costly multiple mating by females. am. nat. 149: 573-584. yasui, y. (1998): the ‘genetic benefits’ of multiple mating reconsidered. trends ecol. evol. 13: 246-250. zamudio, k., sinervo, b. (2000): polygyny, mate-guarding, and post-humous fertilizations as alternative mating strategies. proc. natl. acad. sci. usa 97: 14427-14432. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 7(2): 281-296, 2012 does acclimation at higher temperatures affect the locomotor performance of one of the southernmost reptiles in the world? jimena b. fernández*, nora r. ibargüengoytía inibioma–conicet. universidad nacional del comahue, centro regional universitario bariloche, departamento de zoología, laboratorio de ecofisiología e historia de vida de reptiles. centro regional universitario bariloche, quintral 1250, barrio jardín botánico, san carlos de bariloche (8400), río negro, argentina. tel.: +54 294 4428505 int. 103; fax: +54 294 4428505. *corresponding author. e-mail: jimenafernandez@comahue-conicet.gob.ar; noraibarg@gmail.com submitted on: 2012, 28th march; revised on: 2012, 10th july; accepted on: 2012, 6th august. abstract. when an animal in the laboratory experiences a change in temperature, physiological processes are affected but they stabilize under the new temperature condition over a few weeks by a process of phenotypic plasticity called acclimation, but whether an organism can acclimate or not depends on the trait and the taxon. liolaemus sarmientoi is one of the southernmost reptiles in the world, inhabiting the extreme and arid environment of patagonia, argentina, characterised by great seasonal climatic variation and cold air temperatures throughout the year (mean air temperature of 8 °c; ranging from 1.2 to 14.1 °c). however, these lizards prefer body temperatures in the laboratory ranging from 26.3 to 37.8 °c (mean tpref = 34.4 ± 0.28 °c), temperatures that they rarely achieve in nature. herein, we explore the effects of thermal acclimation on performance of l. sarmientoi at a temperature higher than their mean natural environmental temperature during their activity period (austral spring-summer). we analysed the speed in sprint and long runs at medium and high temperatures in the field and again after a period of acclimation of 20 days at 21 °c. acclimation to higher and constant temperature resulted in a decrease in running speed in both  long and  sprint runs, suggesting potentially negative effects for natural populations if environmental temperature increases. keywords. lizard, liolaemus sarmientoi, acclimation, locomotor performance, high latitude. introduction ectotherms from temperate environments are subject to considerable daily fluctuations in environmental and body temperatures (bennett and dawson, 1976), but behavioural thermoregulation, particularly by reptiles, can modify the daily amplitude of body 282 j.b. fernández and n.r. ibargüengoytía temperature (cowles and bogert, 1944; hertz et al., 1993). moreover, the tolerance of organisms to low or high temperature extremes is not fixed, but is generally preconditioned by the thermal experience in their environment (schmidt-nielsen, 1997). when an animal is exposed to a change in temperature, physiological processes resulting in a form of phenotypic plasticity known as acclimation take place (bennett, 1990; schmidt-nielsen, 1997). for example, an acclimation period of exposure to either hot or cold temperatures, results in a gradually greater physiological tolerance to the induced temperature (angilletta, 2009). thermal acclimation in ectotherms can affect running or swimming performance (elphick and shine, 1998; kingsolver and huey, 1998; glanville and seebacher, 2006; wilson et al., 2007), lethal temperatures (kaufmann and bennett, 1989), and metabolism (jacobson and whitford, 1970; rogowitz, 1996), among others. in this regard, lizards are good biological models for testing effects of possible long-term climate variation on animals (sinervo et al., 2010). physiological responses to temperature in lizards have typically been measured by quantifying running velocity on a treadmill or race track (bennett, 1980; hertz et al., 1983; van damme et al., 1992; bauwens et al., 1995; angilletta et al., 2002; ibargüengoytía et al., 2007; aguilar and cruz, 2010; fernández et al., 2011; kubisch et al., 2011). lizards of the genus liolaemus from the cold temperate climate of patagonia show reduced locomotor performance at the lower temperatures, typical of their environment. yet, in most cases they have a thermal optimum for performance that is lower than their preferred temperature in laboratory (bonino et al., 2011; fernández et al., 2011; kubisch et al., 2011). in this study we assess the effects of thermal acclimation in liolaemus sarmientoi, one of the southernmost reptiles in the world. we test running speed in long and sprint runs, two ecologically relevant parameters of the behavioural responses, used to capture prey, escape from predators, and potentially other social activities, such as territorial competition and mating (bennett, 1980; christian and tracy, 1981; van berkum, 1988; huey et al,. 2009). liolaemus sarmientoi occurs between 49º and 53ºs latitude in isolated outcrops of ancient volcanic craters in an extreme and arid area of the patagonian steppe (santa cruz province, argentina; roig, 1989; oliva et al., 2001). experiencing a mean annual environmental temperature of 12 °c, l. sarmientoi achieves a body temperature of about 26 °c by behavioural thermoregulation, but when placed in a laboratory thermal gradient they prefer higher temperatures (mean tpref: 34.4 ± 0.28 °c; ibargüengoytía et al., 2010), which they rarely attain in their natural habitat. however, l. sarmientoi exhibits high running speeds in long and sprint runs in a wide range of temperatures, a range that exceeds temperatures they can achieve by thermoregulation in nature (fernández et al., 2011). the origin of liolaemus in warm and humid climate conditions of the early miocene of patagonia (gasparini et al., 1986; schulte et al., 2000; albino, 1994, 2011) could explain the consistent preference for high temperatures in the genus (medina et al., 2012) exceeding field body temperatures up to 6 °c (labra, 1998; medina et al., 2009 , 2012; rodriguez-serrano et al., 2009). the differences between field active and preferred body temperatures in the southernmost lizards, liolaemus magellanicus and l. sarmientoi (ibargüengoytía et al., 2010) and their greater running performance at high temperatures (fernández et al., 2011), together with recent glacial recessions occurring between 7600 and 2500 bp from areas now occupied by l. sarmientoi (schobinger, 1973; coronato et 283acclimation effects on performance of liolaemus sarmientoi al., 2007), suggests that while the miocene ancestors of l. sarmientoi lived at warmer and lower latitudes, their descendants have recently colonized southern and colder latitudes (cei, 1986; etheridge, 1995). therefore, we hypothesize that although southern populations evolved adaptations to colder temperatures and shorter activity seasons (fernández et al., 2010), they would retain some of their warm-climate adaptations, or lose them more slowly, resulting in greater phenotypic plasticity in thermal physiology compared to ancestral or northern populations. if selection against warm-climate traits was weak, then such plasticity would be broad in colonizing populations but would slowly narrow. herein, we test the prediction that individuals from l. sarmientoi will exhibit greater locomotor performance when acclimated to temperatures well above their normal field temperatures. materials and methods study sites and captures field work was carried out in santa cruz province, argentina (50°s, 72°w and 51ºs, 70ºw; 133 m a.s.l). the climate that prevails in this region is cold temperate, semiarid (soto and vázquez, 2001), dominated by sub-polar cold and humid air masses with winds increasing toward the south which contribute to aridity, a distinctive feature of the patagonian climate (camilloni, 2007). the mean annual air temperature is 8.04 °c (ranging from 1.2 to 14.1 °c), but the mean air temperature during the lizard’s activity period from october to march is 12.1 °c (according to the closest meteorological station in río gallegos, santa cruz; fig. 1). we captured 36 individuals of liolaemus sarmientoi by hand or noose during february 2009, when lizards were at the end of the reproductive season (fernández, pers. comm.). capture permission was obtained from the wildlife delegation of santa cruz province, according to disposition 09/09. experiments lizards were prompted to run in nature at low, high, and medium temperatures without acclimation (published in fernández et al., 2011). they were then brought to the laboratory where we measured their preferred body temperature (see ibargüengoytía et al., 2010). subsequently, the same individuals were acclimated for the present study for 20 days at a mean temperature of 21 ± 0.02 °c (from 18.3 to 23.6 °c). the 21 °c was selected because it is the mean micro-environmental air temperature recorded 1 cm above the ground of each lizard capture site during the day when they were showing activity outside the burrows (ibargüengoytía and cussac, 2002; ibargüengoytía et al., 2010), and it is expected to mimic a more constant and higher temperature than they would experience naturally. during the acclimation period, lizards were placed in an open-top terrarium (15 × 20 × 20 cm) with a sand floor, and were provided with water, mealworm larvae (tenebrio molitor), and house crickets (achetus domestica) ad libitum. considering that loss of body condition is extremely relevant, because lizards in losing reserves will tend to select for lower preferred temperatures (brown and griffin, 2003), we omitted data generated by seven of the original 36 animals captured because they had lost more than 20% of their body mass during captivity; this caution follows the protocol of hertz et al. (1983), huey et al. (1989), and kaufmann and bennett (1989). in addition, we analysed the relationship between the proportions of body mass lost and running speed to indicate whether 284 j.b. fernández and n.r. ibargüengoytía whole-animal physical condition was correlated with performance (following kaufmann and bennett, 1989). for that reason, we worked with 29 individuals (nine males, 11 females, and nine juveniles) of l. sarmientoi. the sex and reproductive condition were achieved by presence of pre-cloacal pores in males and morphology, respectively. there were not pregnant females in the sample. lizards were prompted to run along a plastic track (0.1 m wide and 1.5 m long), waiting 24 h after captures, at two body temperatures: medium (21 °c) and high (30 °c). these temperatures were controlled outdoors in appropriate microenvironments, where lizards achieved a body temperature approximately equal to 21 or 30 °c. the body temperature of each lizard (tb) was measured before each run using a thermocouple inserted 1 cm inside the cloaca (catheter probe tes tp-k01, 1.62 mm diameter), and connected to a tes 1302 digital thermometer (tes electrical electronic corp., taipei, taiwan, ± 0.01 °c). we used two types of runs following the methods of ibargüengoytía et al. (2007), fernández et al. (2011), and kubisch et al. (2011): 1) long runs (lr) of 1 m, each animal running three consecutive times while being stimulated by gently touching their tail or the back of their legs and taking care not to interfere with the running speed, and 2) sprint runs (sr) of 0.2 m, each animal running five consecutive times with a single initial stimulus. the sr represents the first burst or escape response from a predator, and the 0.2 m length is appropriate because the top velocity is reached usually in the first milliseconds of the response (bonino et al., 2011). after long runs, lizards were left in the shade for a minimum rest period of 4 h before undertaking the sprint runs. the runs were performed outdoors after 24 h of capture (fernández et al., 2011) and after the acclimation period. the runs were filmed using a sony dcr-sr 45 video camera recorded in ntsc fig. 1. air temperatures according to the meteorological station of río gallegos, santa cruz: maximum and minimum extremes (black dots and squares), maximum and minimum means (white dots and triangles), and mean (black triangle) temperatures throughout the year. there are also indicated the lizards’ activity season (dotted lines), mean field body temperature (short dashed line), mean preferred body temperature (long dashed line) and mean air temperature of microhabitats (solid line; which corresponds to acclimation temperature) from ibargüengoytía et al. (2010).   285acclimation effects on performance of liolaemus sarmientoi with error  of  ± 0.03  frame per second. videos were processed using microsoft  windows movie maker version 2.1.4026.0 (error ± 0.06 s) in order to register the time and determine the running speed (total error ± 0.0045 s). in our analysis of running speeds, only the speed of the fastest non-stop lr and sr was used for each lizard during each of the two temperature trials. we calculated the maximum speed achieved by each lizard (vmax i); the maximum vmax i which correspond to the speed of the fastest lizard for both lr (vmax lr) and sr (vmax sr); the thermal optimum (to; as the tb at vmax lr and vmax sr); and the performance breadth (b80; calculated as the range of tb at which performance is greater than or equal to 80% of the vmax for all individuals), following the methods of huey and stevenson (1979) and angilletta et al. (2002). the body temperature recorded for each individual before trials was used to calculate the b80 and analyse it in relation to the percentage of maximal performance in long and sprint runs. because we compared the same animals before and after acclimation, data from fernández et al. (2011; vmax lr, vmax sr, b80, and to) and ibargüengoytía et al. (2010; the set-point range as the central 50% of the preferred body temperatures, tpref) from unacclimated lizards were recalculated including only the individuals used in the acclimation experiment for the present study. statistical analyses for statistical analyses, speeds were standardized as the residuals of the regressions between the maximum speed of each lizard (vmax i) and their body temperature (tb), with the purpose to isolate the effect of the acclimation period on performance when running at medium and high temperatures trials. by using residuals we removed the differences we found in the individual’s body temperatures before and after acclimation at medium temperatures (wilcoxon signed rank test, long runs: w = -304.00, n = 27; sprint runs: w = -435.00, n = 29, p < 0.001) and high temperatures (paired t-test, sprint runs: t28 = -2.99, p = 0.01) and in long and sprint runs at medium temperatures (paired t-test, unacclimated: t27 = -6.78, p < 0.001) and high temperatures (paired t-test, acclimated: t26 = -2.24, p = 0.03). analyses were conducted using the statistical programs sigma stat 3.5®, spss 15.0®, and sigma plot 10.0®. the dependence between variables was analysed by linear regression. paired t-tests were used to test differences between two related samples and two-way repeated measures analysis of variance (two-way rmanova) for repeated samples, the holm-sidak test was used as a posteriori test. the assumptions of normality and homogeneity of variance for parametric procedures were checked using the kolmogorov-smirnov and levene’s tests, respectively. when either of these assumptions was not met, we used an equivalent nonparametric test such as wilcoxon signed rank for the comparison of medians of two related samples. the significance level used for all statistical tests was p < 0.05 (sokal and rohlf, 1969; norusis, 1986). best-fit regressions were chosen according the highest r-squared value. results relationship between running speed and body mass lost lizards showed a mean mass loss of 13.13% during acclimation, except in one individual that gained 4.17%. there was no relationship between percentage of mass lost during acclimation and the maximum speed achieved by each lizard (vmax i) in either lrs (linear regression, f1,27 = 2.08; r2 = 0.074; slope = -0.0151; p = 0.16) or srs (linear regression, f1,28 = 3.56; r2 = 0.117; slope = 0.0096; p = 0.07). 286 j.b. fernández and n.r. ibargüengoytía maximum running speed (vmax) for unacclimated and acclimated lizards in long and sprint runs no lizard was able to run as fast as before acclimation. in long runs, the maximum speed achieved by each lizard (vmax i; table 1) was higher in unacclimated than in acclimated individuals (paired t-test, t27 = 2.62, p = 0.014; fig. 2) and also in sprint runs (wilcoxon, w = -184.00, n = 29, p = 0.037; fig. 2). the vmax (maximum vmax i, which is the speed of the fastest lizard) for unacclimated lizards in long runs was 1.56 m/s (vmax lr) at thermal optimum (to) = 31 °c and in sprint runs was 1.54 m/s (vmax sr) at to = 27 °c. the relationship between unacclimated and acclimated lizards in long runs did not show a linear (r2 = 0.10; f1,27 = 2.93; p = 0.10) or quadratic regression (r2 = 0.10; f1,27 = 1.43; p = 0.24), but in sprint runs the best fit corresponded to quadratic regression (r2 = 0.63; f2,28 = 22.50; p < 0.01; fig. 2). comparisons between the speed of long and sprint runs in unacclimated and acclimated individuals. in unacclimated lizards the temperature trial (21 or 30 °c) did not affect their speed (two-way rmanova, f 1, 27 < 0.001; p = 1.000; fig. 3), although speed was affected by the type of run only at 30 °c (two-way rmanova, f 1, 27 = 10.41; p = 0.003; holmsidak, t27 = 3.44, p = 0.001; fig. 3, white dots). there was no interaction between the temperature trial and the type of run (two-way rmanova, f1, 27 = 2.33, p = 0.138). in acclimated lizards the speed also was not affected by the temperature trial (21 or 30 °c; two-way rmanova, f 1, 26 < 0.001; p = 1.000; fig. 3), although the speed was affected by the type of run (two-way rmanova, f 1, 26 = 3.57; p = 0.070; fig. 3). there also was no interaction between the temperature trial and the type of run (two-way rmanova, f1, 26 = 0.52, p = 0.821). table 1. running speeds (m/s) by temperature trial (ºc) for each unacclimated and acclimated lizards running long (lr) and sprint (sr) runs. it is shown the number of individuals (n), minimum, maximum, mean, standard error (± se), and variance for the maximum speed achieved by each lizard in each trial (vmax i). variable temperature trial (ºc) n minimum (m/s) maximum (m/s) mean (m/s) (±) se variance (m/s) unacclimated lr vmax i 21 28 0.313 1.149 0.766 0.039 0.044 30 28 0.238 1.563 1.063 0.078 0.170 sr vmax i 21 29 0.426 1.000 0.735 0.030 0.026 30 29 0.083 1.538 0.884 0.060 0.104 acclimated lr vmax i 21 27 0.314 0.935 0.630 0.037 0.037 30 27 0.258 1.449 0.858 0.056 0.084 sr vmax i 21 29 0.278 1.053 0.688 0.034 0.034 30 29 0.230 1.000 0.902 0.033 0.032 287acclimation effects on performance of liolaemus sarmientoi in unacclimated lizards the relationship between long and sprint runs did not show a linear regression at 21 °c (r2 = 0.13; f1,27 = 4.00; p = 0.06), but it did at 30 °c (r2 = 0.66; f1,27 = 50.02; p < 0.01; fig. 3), this also happen in acclimated lizards at 21 °c (r2 = 0.19; f1,26 = 5.77; p = 0.02; fig. 3) and at 30 °c (r2 = 0.20; f1,26 = 6.31; p = 0.02; fig. 3). fig. 2. regressions (black solid lines) and 95% confidence intervals (dashed lines) of standardized maximum speed (vmax i) for each unacclimated versus acclimated lizard in long runs (y = -0.10 + 0.24x) and sprint runs (y = -0.01 + 0.69x 1.16x2) of liolaemus sarmientoi. line of equality intersects the origin.   288 j.b. fernández and n.r. ibargüengoytía comparisons between the speed of unacclimated and acclimated lizards in long and sprint run in long runs the speed of lizards was not affected by the temperature trial (two-way rmanova, f1, 26 < 0.001, p = 1.000; fig. 4), however speed was affected by the acclimation only when lizards ran at 30 °c (two-way rmanova, f 1, 26 = 8.22; p = 0.008; holm-sidak, t26 = 4.22, p < 0.001; fig. 4, white dots). there was interaction between the temperature trial and acclimation (two-way rmanova, f1, 26 = 9.74, p = 0.004). fig. 3. linear regressions (black solid lines) of standardized speed for long versus sprint runs at medium (21 ºc, black dots) and high temperatures (30 ºc, white dots) for unacclimated and acclimated liolaemus sarmientoi. line of equality intersects the origin.   289acclimation effects on performance of liolaemus sarmientoi in sprint runs the speed of lizards also was not affected by the temperature trial (twoway rmanova, f 1, 28 < 0.001; p = 1.000; fig. 4), although the speed was affected by the acclimation (two-way rmanova, f 1, 28 < 0.001; p = 0.957; fig. 4). there was no interaction between the temperature trial and acclimation (two-way rmanova, f1, 28 = 0.14, p = 0.716). in long runs the relationship between unacclimated and acclimated lizards showed a linear regression at 21 °c (r2 = 0.36; f1,26 = 14.13; p < 0.01; fig. 4) and at 30 °c (r2 = 0.40; f1,26 = 16.73; p < 0.01; fig. 4), this also happen in sprint runs at 21 °c (r2 = 0.17; f1,28 = 5.73; p = 0.02; fig. 4) and at 30 °c (r2 = 0.30; f1,28 = 11.76; p < 0.01; fig. 4). fig. 4. linear regressions (black solid lines) of standardized speed for unacclimated versus acclimated individuals of liolaemus sarmientoi at medium (21 ºc, black dots) and high temperatures (30 ºc, white dots) trials for long and sprint runs. line of equality intersects the origin.   290 j.b. fernández and n.r. ibargüengoytía percentage of maximal performance in long and sprint runs and the set-point range of preferred body temperatures (tpref) in long runs, 65% of adults before acclimation and 10 % of them after acclimation ran at the same speed or faster than 80% of the adult vmax lr (1.56 m/s, n = 20) in a range of tb from 25 to 34 °c and 28 to 30 °c (b80), respectively (fig. 5, upper panels). in sprint runs before acclimation, 25 % of the lizards ran at the same speed or faster than 80% of the vmax sr in a range of tb from 27 to 31 °c, and after acclimation no lizard ran at an equal or greater speed than 80% of the vmax sr (fig. 5, lower panels). the range from 60 to 68% of the maximal speed for sprint runs was achieved by 55% of unacclimated lizards at temperatures ranging from 25 to 35 °c, and after acclimation it was achieved by 85% of them at temperatures ranging from 20 to 35 °c (fig. 5, lower panels). the set-point (the central 50%) of the preferred body temperatures (tpref) for adults analysed in the present study ranged between 27.3 and 39.7 °c (fig. 5). fig. 5. relationship between relative performance (% vmax i) with body temperature (ºc) of adults of liolaemus sarmientoi in unacclimated and acclimated long (upper panels) and sprint runs (lower panels). horizontal solid line indicates the 80 % of performance breadth (b80), and vertical dashed lines indicate the central 50% of the preferred body temperature (tpref) of adults in laboratory   291acclimation effects on performance of liolaemus sarmientoi discussion liolaemus sarmientoi, in one of the southernmost environment of the world, achieves the highest speed in long runs when their body temperature ranges from 25 to 35 °c (fernández et al., 2011). in contrast, the sympatric liolaemus magellanicus shows no difference in speed between long and sprint runs, and reaches its maximal performance only over a narrow range of high temperatures (from 30 to 34 °c), which are included within the set-point range of tpref, unlikely to be found in nature (fernández et al., 2011). liolaemus sarmientoi performance is consistent with an evolutionary trend toward the ability to run fast over a broader range of temperatures, especially in long runs, including those low temperatures they often experience in their current environment (fernández et al., 2011). in addition, contrary to our expectance, most of the lizards were not able to run as fast as before acclimation, that is, acclimation at higher temperatures in l. sarmientoi, rather than enhancing performance, resulted in a detrimental effect on running speed, more conspicuous in long runs. moreover, even the differences observed prior to acclimation, higher speeds in long than in sprint runs, disappeared following acclimation. thermal sensitivity of locomotor performance can vary among ectotherms, and thermal acclimation enhances some physiological performance in some species depending on the temperatures at which are acclimated (huey et al., 1999; angilletta et al., 2009). in amphibians after metamorphosis, the general pattern shows no beneficial acclimation in their locomotor performance to different temperatures (feder, 1986; knowles and weigl, 1990; marvin, 2003). thus, wilson and franklin (2000) predict that the locomotor performance of animals in terrestrial habitats should be more thermally insensitive than their performance in aquatic habitats. therefore, amphibians that shift from a thermally buffered aquatic environment to a variable terrestrial environment lose their ability to acclimate their locomotor physiology. in this sense, ectotherms like l. sarmientoi that live in a thermally variable environment exposed to a daily and yearly high fluctuations of body temperature, characteristic of high latitudes, could evolve locomotor capabilities independent of temperature fluctuations, with a corresponding loss of the ability to acclimate, such as happens in amphibians. the same pattern was observed in the desert night lizard (xantusia vigilis) that was incapable of acclimating locomotor performance to either 20 or 30 °c for 50 days (kaufmann and bennett, 1989). present results show that l. sarmientoi, in a manner similar to that of high-altitude temperate-zone organisms (see ghalambor et al., 2006), displays a broad thermal tolerance to cope with the large seasonal changes in temperature (fig. 1), but also shows poor locomotor abilities after acclimation at higher temperatures. the detrimental effect of high temperatures is also noticeable if we consider that speed was reduced in long runs only in lizards running at the high temperature trial but not in those running at medium temperatures (fig. 4). the absence of a correlation between weight lost in captivity and running performance, provides support for the rejection of the hypothesis that a decrement in speed after acclimation results from a deterioration of physical condition during captivity, although we could consider other unnoticed factors, aside from mass lost (such as the lack of exercise or dehydration, among others) that may have been artefacts of captivity. even so, the reduced locomotor performance found in l. sarmientoi at high temperatures may be affected by a lower aerobic capacity 292 j.b. fernández and n.r. ibargüengoytía during a long run at that temperature (bennett and licht, 1972), rather than an alteration in the contraction of muscles, which usually is not affected by high temperatures (bennett, 1985; else and bennett, 1987; kaufmann and bennett, 1989). in addition, in sprint runs after a period of acclimation, l. sarmientoi showed a broader range of temperatures (from 20 to 35 °c) than before acclimation when its performance is between 60 to 68% of its maximal speed. the ability to achieve high speed even at low temperatures suggests that lizards under constant and higher environmental temperatures, such as during a “benign” austral summer, could switch their actual preferences from long to sprint runs. these results also suggest that differences in fitness within a population, relative to thermoregulatory behaviour, could result in a differential use of microhabitats (switch between open to vegetated areas) and locomotor preferences. based on our results we propose that under a continued increase in environmental temperature this southern species of lizard, l. sarmientoi, assuming no adaptive or plastic behavioural compensation driven by rapid environmental change (sinervo et al., 2010), may suffer a decrement in running speed that could directly affect their ability to escape from predators, capture prey, and perform several social activities. climate-change scenarios predict increases in environmental temperature over a scale of decades (sinervo et al., 2010), which would likely result in increased aridity, changes in community dynamics, such as increased competition and a southward shift in the geographical ranges of predators from northern latitudes, resulting in declines of prey populations (lavilla, 2001). under this scenario, we consider that a deeper knowledge of species reaction norms (sensu stearns, 1989 and via et al., 1995) of ecologically pertinent traits, like locomotor performance, would help in predicting the fitness consequences on reptiles caused by rising global temperature. acknowledgements we thank alejandro scolaro, marlin medina, joel gutiérrez, and fabián tappari for their help in the collection of animals for this study, dante d’ arielli and rebeca fernández helped cared for lizards during captivity, and manuela martinez and joseph smith assisted during the trials. we also thank amanda manero (unpa) and guillermo clifton (inta) for their logistic support, and the fauna direction of the government of santa cruz for the permits to perform our field work. we also want to express our gratitude to john d. krenz for their constructive and insightful comments on the manuscript. this study was supported by the consejo nacional de investigaciones científicas y técnicas (conicet, pip 100271), argentina and by the agencia de investigación científica (foncyt, pict 1086). references aguilar, r., cruz, f.b. 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(2000): inability of adult limnodynastes peronii (amphibia: anura) to thermally acclimate locomotor performance. comp. biochem. physiol. a 127: 21-28. acta herpetologica vol. 7, n. 2 december 2012 firenze university press advertisement call of species of the genus frostius cannatella 1986 (anura: bufonidae) flora a. juncá1, david l. röhr2, ricardo lourenço-de-moraes3, flávio j. m. santos1, airan s. protázio1, ednei a. mercês1, mirco solé4 amphibians in southern apennine: distribution, ecology and conservation notes in the “appennino lucano, val d’agri e lagonegrese” national park (southern italy). antonio romano1,*, remo bartolomei1, antonio luca conte1, egidio fulco2 the significance of using satellite imagery data only in ecological niche modelling of iberian herps neftalí sillero1, josé c. brito2, santiago martín-alfageme3, eduardo garcía-meléndez4, a.g. toxopeus5, andrew skidmore5 reproductive strategy of male and female eastern spiny lizards sceloporus spinosus (squamata: phrynosomatidae) from a region of the chihuahuan desert, méxico aurelio ramírez-bautista1,*, barry p. stephenson2, xóchitl hernández-ibarra1, uriel hernández-salinas1, raciel cruz-elizalde1, abraham lozano1, and geoffrey r. smith3 morphological variability of the hermann’s tortoise (testudo hermanni) in the central balkans katarina ljubisavljević1, georg džukić1, tanja d. vukov1, miloš l. kalezić1,2 the usefulness of mesocosms for ecotoxicity testing with lacertid lizards maria josé amaral1,2,*, rita c. bicho1, miguel a. carretero2, juan c. sanchez-hernandez3, augusto m. r. faustino4, amadeu m. v. m. soares1, reinier m. mann1,5 does acclimation at higher temperatures affect the locomotor performance of one of the southernmost reptiles in the world? jimena b. fernández*, nora r. ibargüengoytía advertisement call of scinax littoralis and s. angrensis (amphibia: anura: hylidae), with notes on the reproductive activity of s. littoralis michel v. garey1, thais r. n. costa2, andré m. x. de lima2, luís f. toledo3, marília t. hartmann4 book review: marine s. arakelyan, felix d. danielyan, claudia corti, roberto sindaco, alan e. leviton 2011. herpetofauna of armenia and nagorno-karabakh. society for the study of amphibians and reptiles stefano scali book review: rafaqat masroor 2012. a contribution to the herpetofauna of northern pakistan stefano scali evidence of high longevity in an island lacertid, teira dugesii (milne-edwards, 1829). first data on wild specimens. j. jesus first assessment of the endoparasitic nematode fauna of four psammophilous species of tropiduridae (squamata: iguania) endemic to north-eastern brazil markus lambertz1,*, tiana kohlsdorf2, steven f. perry1, robson waldemar ávila3, reinaldo josé da silva4 rediscovery and redescription of the holotype of lygosoma vittigerum (= lipinia vittigera) boulenger, 1894 yannick bucklitsch1, peter geissler1, timo hartmann1, giuliano doria2 , andré koch1,* reproductive phenology of the tomato frog, dyscophus antongili, in an urban pond of madagascar’s east coast ori segev1,*, franco andreone2, roberta pala2, giulia tessa2, miguel vences1 range extension of the critically endangered true poison-dart frog, phyllobates terribilis (anura: dendrobatidae), in western colombia roberto márquez1,*, germán corredor2, carlos galvis3 daniel góez2, & adolfo amézquita1 differences in habitat use of two sympatric species of ameiva in east costa rica esther sebastián-gonzález1, ramón gómez2 acta herpetologica journal of the societas herpetologica italica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 7(2): 189-201, 2012 advertisement call of species of the genus frostius cannatella 1986 (anura: bufonidae) flora a. juncá1, david l. röhr2, ricardo lourenço-de-moraes3, flávio j.m. santos1, airan s. protázio1, ednei a. mercês1, mirco solé4 1 programa de pós-graduação em zoologia, universidade estadual de feira de santana, av. universitária, novo horizonte, cep 44036-900, feira de santana bahia, brazil 2 programa de pós-graduação em ecologia, universidade federal do rio grande do norte 3 programa de pós-graduação em ecologia e conservação da biodiversidade, universidade estadual de santa cruz (uesc), rodovia jorge amado, km 16, ilhéus, bahia, brazil, cep 45662-900 4 departamento de ciências biológicas, universidade estadual de santa cruz, rodovia jorge amado, km 16, ilhéus, bahia, brasil, cep 45662-900. corresponding author. e-mail: mksole@uesc.br submitted on: 2011, 25th october; revised on: 2012, 26th april; accepted on: 2012, 2nd may. abstract. frostius pernambucensis and f. erythrophthalmus are cryptic bufonid species recognized mainly by the iris color: yellow in f. permambucensis and red in f. erythrophthalmus. however, field studies showed that the iris color of f. erythrophthalmus could vary between yellow and red. to improve the recognition of these species we described the advertisement call of frostius pernambucensis and frostius erythrophthalmus and we tested if call characteristics are influenced by temperature, male size and perch height. we also report on a physical interaction between two males of f. pernambucensis and the associated vocalization, suggesting that f. pernambucensis has not a territorial call or encounter call. comparing the advertisement calls, the call of f. pernambucensis was lower, shorter and with a smaller number of notes than the call of f. erythrophthalmus. dominant frequency and fundamental frequency variation of the f. pernambucensis advertisement call were related to the male’s size, while the call emission rate was related to air temperature. however, we could not find relationship among the acoustic characteristic of f. erythrophthalmus and male size or temperature. keywords. vocalization, frostius pernambucensis, frostius erythrophthalmus, atlantic forest, specific recognition. introduction the genus frostius cannatella 1986 is endemic to the atlantic rainforest and is found in the brazilian states of paraíba (pimenta and caramaschi, 2007), pernambu190 flora a. juncá et al. co (bokermann, 1962), alagoas (peixoto and freire, 1998) and bahia (juncá and freitas, 2001; pimenta and caramaschi, 2007). frostius was suggested to be phylogenetically related to atelopus dumeril and bibron, 1841, dendrophryniscus jiménez de la espada, 1870, melanophryniscus gallardo, 1961, oreophrynella boulenger, 1895 and osornophryne ruiz-carranza and hernandez-camacho, 1976 (cannatella, 1986). the morphological characteristics and the cladistic analyses performed were indicative of a closer sister group relationship among the genus frostius + (atelopus + osornophryne) or frostius + atelopus (cannatella, 1986). the phylogenetical proximity with the genus atelopus is expected considering the morphological similarities already pointed out by bokermann (1962). based on dna characteristics, recent studies have indicated that osornophryne (frost et al., 2006), atelopus, melanophryniscus and dendrophryniscus (frost et al., 2006, pramuk et al., 2007) are basal bufonid genera, however, these authors did not include material from frostius in their analyses. very little is known about the biology of frostius species. males vocalize perched on tree trunks and bushes of various heights (juncá, pers. obs.). we know that f. pernambuscensis (bokermman, 1962) reproduces in bromeliad phytothelms, where the endothrophic tadpoles complete their growth (cruz and peixoto, 1982; juncá and borges, 2002), whereas nothing is known about the reproductive biology of f. erythrophthalmus pimenta and caramaschi, 2007, which is suspected to be similar to that of f. pernambuscensis (pimenta and caramaschi, 2007). the only two species known from this genus are very similar. they are distinguished by shape and color, the red iris of frostius erythrophthalmus being the most conspicuous characteristic (pimenta and caramaschi, 2007). however, individuals of f. erythrophthalmus have been found presenting yellow iris color which is a feature assigned to f. pernambucensis (solé, pers. obs.). the known distribution of f. erythrophthalmus is restricted to the south of bahia state, while f. pernambucensis is known from the states of paraíba, pernambuco, alagoas and northeastern bahia. apparently there is no overlap between the respective geographic distributions of these species. the morphological similarity between the two species of the genus calls for efforts to find other diagnostic characteristics, for example, the recognition of their respective advertisement calls. since the main function of this type of vocalization is to attract conspecific females (wells, 2007), some degree of divergence between the advertisement calls of the two species is expected. although the use of the advertisement call characteristics have been widely used in taxonomy (e.g., castroviejo-fisher et al., 2009, vieites et al., 2009, funk et al. 2012) , only a few are recognized as statics characteristics, with low individual variation (e.g., dominant frequency and pulse emission rate, gerhardt, 1991). in many species these statics characteristics can be influenced by the male’s size (e.g., asquith et al., 1988), temperature (e.g., castellano and giacoma, 2000) geographic variation (catellano et al., 2002, smith et al., 2003) and habitat and micro-habitat (ryan et al., 1990; ryan and wilczynsky, 1991; preininger et al., 2007). to contribute to the recognition of the two frostius species, we compared the advertisement calls of f. pernambucensis and f. erythrophthalmus and tested if the size of males, air temperature and perch height influence the analyzed acoustic characteristics. we also describe a physical interaction between two males of f. pernambucensis and the associated vocalization. 191advertisement call of species of frostius material and methods males of the two frostius species vocalized more during the night, but also during daytime, at different times. we recorded specimens of frostius at night, from 1900 to 2300 h. we studied a frostius pernambucensis population at serra da jibóia, municipality of santa terezinha, bahia (12°51’s, 39°28’w). the hills extend for about 6 km with a maximum altitude of 850 m, with caatinga (typical xeric vegetation of brazil) in the valleys, humid atlantic rainforest on the slopes and rocky fields on the hilltops. they belong to a range of disconnected hills which extend from the coast of bahia state towards the northwest and north of baia de todos os santos region (juncá, 2006). we recorded the advertisement call of frostius erythrophthalmus at three sites in the southern region of bahia state: rppn (private natural heritage reserve) capitão, itacaré municipality (five males), rppn boa união, ilhéus municipality (two males) and michelin ecological reserve, igrapiuna municipality (one male). the michelin reserve has fragments of dense atlantic rainforest (13°50’s, 39°10’w, altitude 90 to 383 m) surrounded by banana, cocoa and rubber tree plantations. the annual precipitation reaches 2000 mm (camurugi et al., 2010). the rppn boa união and capitão are located in the south of the state of bahia and are classified as tropical lowland (oliveirafilho and fontes, 2000). according to köppen (1936) the climate is classified as af being hot and humid without a dry season; the average temperature is around 24 °c degrees and the annual rainfall can reach 2200 mm (sá et al., 1982; landau et al., 2003). the forest of this region consists of a mosaic in different successional stages, with a large concentration of epiphytes, bromeliads, great lianas, and a dense sub-canopy (thomas et al., 1998; jardim 2003). in october 2008, we measured the advertisement call’s sound pressure level (spl) and snoutvent length (svl) of 30 males of frostius pernambucensis together with the air temperature. we used a decibel meter minipa® msl – 1351c to measure the spl of three consecutive calls of each male at a distance of fifty centimeters from the individual. the arithmetic average of the three measurements was considered the sound intensity for each specimen. in april 2009, we recorded the advertisement call of twenty two males of frostius pernambucensis using a sony® wm-d6 digital audio tape recorder, a directional microphone yoga® ht-32oa, keeping the microphone 50 cm from the vocalizing male. during the two year study, we measured the height of the vocalization site and the air temperature with a digital minipa® mv 363 thermometer. after recording and measuring the call intensity, we measured the svl of each male, putting light pressure on each individual’s body using a wooden board, obtaining the measurement with a caliper (0.05 mm precision, 2008) and a plastic ruler (0.10 mm precision, 2009). voucher specimens were deposited in the zoology museum of uefs (mzuefs 3708-3709). in 2010, we recorded the advertisement call of frostius erythrophthalmus with a marantz® pmd 660 digital recorder and a yoga® em 9600 unidirectional microphone. after each recording, we collected the males and took the air temperature with a digital type k thermometer with an accuracy of 0.1 ºc (pce group-222). all the males recorded were photographed with a digital camera, whilst still alive to register the iris coloration. males of frostius erythrophthalmus were deposited at the zoological collection of the santa cruz state university, zoological museum of uefs (mzuefs 3687) and zoology museum ‘professor adão josé cardoso’, state university of campinas (zuec 16631-33). the archives of the vocalization sound of frostius pernambucensis and frostius erythrophthalmus are deposited in the sound library sonoteca uefs (frostius pernambuscensis suefs 30.01-30.22; f. erythrophthalmus suefs100.09-100.12, 100.34, 100.35, 100.43 and 100.44) call analyses we digitalized and analyzed the calls using the canary 1.2.4 program. the following characteristics were measured: call duration (ms), interval between calls (s), call rate (call/s), pulse number, pulse rate (pulse/s), dominant frequency (khz) and fundamental frequency (khz). we calcu192 flora a. juncá et al. lated the mean and standard deviation for each characteristic analyzed of the males which had more than one call recorded. we verified if call characteristics are influenced by temperature, svl and perch height (the last variable was only tested for f. pernambucensis) using multiple linear regression with bioestat 5.0 software (ayres et al., 2007). we used past 2.06 software (hammer et al., 2001) to perform a principal component analysis (pca) to verify if the acoustic characteristics of the advertisement call of these two species could separate the individuals into two groups, through the use of a correlations matrix, considering that the variables used were in different units. the characteristics used in this analysis were dominant frequency, fundamental frequency, number of pulses, pulse rate and call duration. the characteristics that were influenced by svl or temperature had their values corrected by the partial coefficient regression, which is obtained in partial linear multiple regression. we used broken the stick method to identify the number of principal components to be retained (hammer et al., 2001). to check the stability of the analysis we used bootstrap with 1000 randomizations. to determine if the call characteristics of f. pernambucensis are significantly different from those of f. erythrophthalmus, we used unequal variance t-test. the accepted probability was 0.05. results frostius pernambucensis in 2008, the males vocalized while perched on the vegetation with height variation from 0.20 to 200.00 cm (x = 114.62, sd = 52.5, n = 30). the vocalization spl varied from 72.50 to 95.20 db (x = 84.39 sd = 5.75, n = 30). the average svl was 27.80 mm (sd = 0.22; n = 30) and the temperature varied from 17.60 to 22.10 oc (x = 19.67, sd = 0.98, n = 30). the multiple linear regression was nearly significant for spl (f3,26 = 2.648, p = 0.07) and showed a significant relationship between spl and temperature (partial coefficient regression b = 2.309, t1,26 = 2.447, p = 0.02), while the relationships between spl and svl (partial coefficient regression b = -7.2438, t1,26 = -1.7587, p = 0.09) or perch height (partial coefficient regression b = 0.0226, t1,26 = -1.3892, p = 0.18) were not significant. that year, we observed an interaction between two males, which lasted about 5 minutes. only the advertisement call was emitted and by only one of the males. the vocalization was alternated with visual displays. in these displays there was an extension of the hind limbs (fig. 1). physical interaction between these two males was common: one of the males passed beneath the other (fig. 1a, 1b and 1e). during the observation the silent male tried to remove the resident male that was vocalizing and vice-versa. the sex of the silent individual was confirmed by a dilated gular region. in 2009 we recorded three calls for each male of frostius pernambucensis which vocalized perched in the vegetation with height variation from 0.30 to 2.53 m (x = 1.29, sd = 0.59, n = 21 males). the advertisement call of f. pernambucensis is long, lasting approximately 8 s, composed of a series of around 54 pulses and an emission rate of 7 pulses/s (table 1, fig. 2). the call emission rate was around 2 call/min. the dominant frequency was around 2.36 khz (table 1). a discreet frequency modulation is only observed in the first pulse and also in some calls in the second pulse (fig. 2b). the svl average for the males was 36.79 mm (sd= 0.21). the air temperature varied from 20 to 23 oc (x = 20.88, sd = 0.83, n = 21). the multiple linear regression was significant for dominant frequency (f3,17 = 4.122, p = 0.02), fundamental frequency (f3,17 = 3.166, p = 0.05) and call emission rate (f3,17 193advertisement call of species of frostius = 3.190, p = 0.05).the svl was important for the dominant frequency variation (partial coefficient regression b = -0.2401, t = 2.917, p = 0.01) and fundamental frequency (partial coefficient regression b = -0.3435, t = 2.447, p = 0.02), whereas the temperature had a significant influence on the call emission rate (partial coefficient regression b = 0.2208, t = 2.650, p = 0.02). the perch height was not significantly related with any acoustic variable. frostius erythrophthalmus the number of calls recorded for each male of frostius erythrophthalmus varied from 1 to 3. the males vocalized at an approximate height of 1 m (x = 1.07, sd = 0.45, n = 8). fig. 1. interactions between two males of frostius pernambucensis, from municipality of santa terezinha, bahia state, brazil. the photos are in temporal sequence.   194 flora a. juncá et al. table 1. analyzed acoustic characteristics of the advertisement call of frostius species. f. pernambuscensis (n = 96 calls) f. erythrophthalmus (n = 21 calls) serra da jibóia ilhéus, itacaré e igrapiuna call duration (s) 7.54 ±1.53 (1.94-9.84) 10.59 ±3.10 (4.84-15.30) pulse number 52.64 ±9.93 (15-75) 69.23 ±22.48 (41-121) pulse rate (pulse/s) 7.02 ±0.44 (6.01-8.20) 6.77 ±1.30 (4.49-8.47) call rate (call/min) 1.68 ±0.38 (1.00-2.33) 1.94 ±0.70 (1.18-2.86) interval between calls (s) 28.49 ±9.56 (11.70-72.03) 29.42 ±11.73 (19.82-42.50) dominant frequency (khz) 2.37 ±0.10 (2.11-2.54) 3.07 ±0.19 (2.76-3.47) fundamental frequency (khz) 1.76 ±0.21 (0.75-2.04) 2.54 ± 0.32 (1.83-2.95) fig. 2. advertisement call of frostius pernambucensis, from municipality of santa terezinha, bahia state, brazil. a) power spectrum, b) audiosectogram and c) oscillogram.   195advertisement call of species of frostius the svl varied from 20.00 to 23.24 mm (x = 21.30, sd = 1.03, n = 8) and the air temperature from 21.1 to 31.5 °c. the advertisement call of f. erythrophthalmus is similar to the call of f. pernambucensis in its general structure (fig. 3) and they differed primarily in measured dominant frequency and fundamental frequencies, all of them higher in f. erythrophthalmus (table 1). the multiple linear regression did not detect any relationship between the characteristics of the advertisement call of f. erythrophthalmus, svl and temperature. using the broken stick method only the principal components 1 and 2 were retained and together they explained 84.9% of the variance (table 2). the dispersion graph of the scores of the principal components 1 and 2 separated the two species (fig. 4). all the varifig. 3. advertisement call of frostius erythrophthalmus, from municipality of itacaré, bahia state, brazil. a) power spectrum, b) audiosectogram and c) oscillogram.   196 flora a. juncá et al. ables showed loadings above 0.70 in pc1, except the note rate, that was the most important characteristic in the pc2 (table 2). the t-test showed a significant difference among population averages for the dominant frequency (t = 2.590, p = 0.033, df = 7.71) and fundamental frequency (t = 4.644, p < 0.002, df = 7.77), number of pulses (t = -2.608, p = 0.032, df = 7.79) and call duration (t = -2.850, p = 0.022, df = 7.94), but didn’t indicate a difference in the pulse rate (t = 0.180, p = 0.861, df = 7.67). during field work in the studied populations of frostius erythrophthalmus we observed males with red and also yellow (itacaré and ilhéus) or only yellow irises (igrapiuna). table 2. variable loadings, eigenvalue and percentage of the variance for each principal component. pc1 pc2 pc3 pc4 pc5 call duration 0.7647 -0.6279 0.1448 -0.00333 0.001066 pulse number 0.8255 0.4905 -0.2257 -0.1646 6.35e-07 pulse rate 0.8392 0.3821 -0.3542 0.1558 -1.05e-06 dominant frequency 0.8497 -0.2423 0.4682 0.01039 -0.00093 fundamental frequency -0.04673 0.844 0.5337 0.02561 0.000526 eigenvalue 0.7647 -0.6279 0.1448 -0.00333 0.001066 % variance 53.89 31.036 14.03 1.0431 0.00005 fig. 4. value dispersion of pc1 and pc2. full circles frostius pernambucensis and empty circles frostius erythrophthalmus.   -3 -2 -1 0 1 2 3 component 1 -3 -2 -1 0 1 2 3 c o m p o n en t 2 197advertisement call of species of frostius discussion the two species of frostius have similar advertisement calls, which are composed of a series of pulses emitted at regular intervals, a pattern of advertisement call found in many other bufonid species (martin, 1972). comparing the two species, the call of f. pernambucensis was lower, shorter and with a smaller number of notes than the call of f. erythrophthalmus. the differences of frequency, duration of the call and pulse emission rate are common among bufonid species which are phylogenetically close and show similar advertisement calls (gergus et al., 1997; garda et al., 2010). although the pulse rate averages didn’t show any significant differences between the two species of frostius, it was an important variable which explained the variation in axis 2 of the pca. various atelopus species have the advertisement call registered (cocroft et al., 1990) and at the very least only one call has ever been registered of osornophryne guacamaio by a single male in the presence of a female (gluesenkamp, 2001). these two genera are phylogenetically close to frostius (cannatella, 1986), and both genera have calls also formed by a series of pulses. just as in the species of frostius, atelopus species also show advertisement calls with the same basic structure, but pulse rate, dominant frequency, and call length differ among species (cocroft et al., 1990). many species of the genera atelopus (lötters et al., 1999) and at least osornophryne guacamayo (gluesenkamp, 2001) also have shiny coloration on the vent, including palmar and plantar surfaces and osornophryne spawning has eggs with a lot of vitellus (mcdiarmid and gorzula, 1989; glusenkamp, 2001), similar to frostius pernambucensis. however, unlike frostius species, the majority of species of both genera don’t have tympanic membranes and the acoustic communication was considered less important than tactile or visual communication (cocroft et al., 1990; gluesenkamp, 2001). this way, interaction between two males of frostius pernambucensis indicates that visual and tactile communication could also be important. the observed behavior during interaction indicates a dispute of vocalization site, suggesting the specie doesn’t have a territorial or agonistic call, as reported for atelopus chiriquiensis in aggressive encounters (jaslow, 1979). usually, temperature has major influence on temporal properties of anuran acoustic signals (navas and bevier 2001, lingnau and bastos 2007), while the svl has an influence on the spectral properties (e.g., castellano et al., 2002, giasson and haddad, 2006). this relationship was also registered in frostius pernambucensis, where the dominant frequency variation and the fundamental frequency of the advertisement call were related to the male’s size, while the call emission rate was related to air temperature. the same analysis did not show any influence of the svl or temperature on the characteristics of the advertisement call of f. erythrophthalmus. however, small sample size needs to be taken into account when interpreting this result. the sound pressure level measured to the advertisement call of f. pernambucensis is in the range of other anuran species, that vary from 72 to 116 db (wells, 2007). as f. pernambucensis, at least to some species the call intensity is not related to svl (e.g., passmore, 1981), although in other species large males tend to produce louder calls (given, 1987; arak, 1988). a possible influence of temperature on call spl still remains an unexplored subject, although it was not found in at least one anuran species (tárano, 2001). although the perch height could be important to sound propagation (e.g., kime et al., 2000), it did not influence any acoustic variable of f. pernambucensis. 198 flora a. juncá et al. pimenta and caramaschi (2007) did not report any variation in iris coloration in the populations they studied, however, the populations of f. erythrophthalmus in this study showed yellow (igrapiuna) or yellow and red (itacaré and ilhéus) iris coloration. the identification of f. erythrophthalmus in these populations was made using other diagnostic characteristics reported by the authors of the specie (e.g., dorsal and ventral coloration, size and shape of tympanum, shape of fingers and toes). so, this study contributes to the diagnosis of two species in the field through the differences in the advertisement call, considering that the iris coloration can vary in frostius erythrophthalmus. acknowledgements we are grateful to f. camurugi, l. neves, b. tanure, s. alencar, t. nilo, r. moreira and e. menezes for help during field work, to coordenação de aperfeiçoamento de pessoal de nível superior (capes) to a fellowship to second, third, fourth and fifth authors, to conselho nacional de desenvolvimento científico e tecnológico for a fellowship to the first (cnpq. process 305457/20098) and last (cnpq. process 309217/2009-1) authors, to instituto brasileiro do meio ambiente e dos recursos naturais renováveis (ibama/sisbio), for the license to carry out the work (process 02010.000026/2007-49, sisbio n° 14299-1) and to the center for biodiversity studies of the michelin ecological reserve, iesb instituto de estudos socioambientais do sul da bahia and milton a. de castro (rppn boa união) for logistic support. this study was supported by universidade estadual de feira de santana, universidade estadual de santa cruz and cnpq. 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(2009): vast underestimation of madagascar’s biodiversity evidenced by an integrative amphibian inventory. proc. natl. acad. sci. u. s. a. 106: 8267-8272. acta herpetologica vol. 7, n. 2 december 2012 firenze university press advertisement call of species of the genus frostius cannatella 1986 (anura: bufonidae) flora a. juncá1, david l. röhr2, ricardo lourenço-de-moraes3, flávio j. m. santos1, airan s. protázio1, ednei a. mercês1, mirco solé4 amphibians in southern apennine: distribution, ecology and conservation notes in the “appennino lucano, val d’agri e lagonegrese” national park (southern italy). antonio romano1,*, remo bartolomei1, antonio luca conte1, egidio fulco2 the significance of using satellite imagery data only in ecological niche modelling of iberian herps neftalí sillero1, josé c. brito2, santiago martín-alfageme3, eduardo garcía-meléndez4, a.g. toxopeus5, andrew skidmore5 reproductive strategy of male and female eastern spiny lizards sceloporus spinosus (squamata: phrynosomatidae) from a region of the chihuahuan desert, méxico aurelio ramírez-bautista1,*, barry p. stephenson2, xóchitl hernández-ibarra1, uriel hernández-salinas1, raciel cruz-elizalde1, abraham lozano1, and geoffrey r. smith3 morphological variability of the hermann’s tortoise (testudo hermanni) in the central balkans katarina ljubisavljević1, georg džukić1, tanja d. vukov1, miloš l. kalezić1,2 the usefulness of mesocosms for ecotoxicity testing with lacertid lizards maria josé amaral1,2,*, rita c. bicho1, miguel a. carretero2, juan c. sanchez-hernandez3, augusto m. r. faustino4, amadeu m. v. m. soares1, reinier m. mann1,5 does acclimation at higher temperatures affect the locomotor performance of one of the southernmost reptiles in the world? jimena b. fernández*, nora r. ibargüengoytía advertisement call of scinax littoralis and s. angrensis (amphibia: anura: hylidae), with notes on the reproductive activity of s. littoralis michel v. garey1, thais r. n. costa2, andré m. x. de lima2, luís f. toledo3, marília t. hartmann4 book review: marine s. arakelyan, felix d. danielyan, claudia corti, roberto sindaco, alan e. leviton 2011. herpetofauna of armenia and nagorno-karabakh. society for the study of amphibians and reptiles stefano scali book review: rafaqat masroor 2012. a contribution to the herpetofauna of northern pakistan stefano scali evidence of high longevity in an island lacertid, teira dugesii (milne-edwards, 1829). first data on wild specimens. j. jesus first assessment of the endoparasitic nematode fauna of four psammophilous species of tropiduridae (squamata: iguania) endemic to north-eastern brazil markus lambertz1,*, tiana kohlsdorf2, steven f. perry1, robson waldemar ávila3, reinaldo josé da silva4 rediscovery and redescription of the holotype of lygosoma vittigerum (= lipinia vittigera) boulenger, 1894 yannick bucklitsch1, peter geissler1, timo hartmann1, giuliano doria2 , andré koch1,* reproductive phenology of the tomato frog, dyscophus antongili, in an urban pond of madagascar’s east coast ori segev1,*, franco andreone2, roberta pala2, giulia tessa2, miguel vences1 range extension of the critically endangered true poison-dart frog, phyllobates terribilis (anura: dendrobatidae), in western colombia roberto márquez1,*, germán corredor2, carlos galvis3 daniel góez2, & adolfo amézquita1 differences in habitat use of two sympatric species of ameiva in east costa rica esther sebastián-gonzález1, ramón gómez2 acta herpetologica journal of the societas herpetologica italica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 7(1): 29-39, 2012 quantifying the intersexual and interspecific morphometric variation in two resembling sympatric lacertids: iberolacerta horvathi and podarcis muralis anamarija žagar1,2,*, nadja osojnik3, miguel a. carretero2, al vrezec4 1 university of ljubljana, biotechnical faculty, department of biology, večna pot 111, si-1000 ljubljana, slovenia. *corresponding author. e-mail: anamarija.zagar@gmail.com 2 cibio, centro de investigação em biodiversidade e recursos genéticos, campus agrário de vairão, 4485-661 vairão, portugal 3 cesta iii/4, si-3320 velenje, slovenia 4 national institute of biology, večna pot 111, si-1000 ljubljana, slovenia submitted on: 2011, july 10th; revised on 2011, october 20th; accepted on 2011, november 26th. abstract. podarcis muralis and iberolacerta horvathi are sympatric, frequently syntopic, lacertids through the entire range of i. horvathi and very similar in their general body size and shape, as well as in most ecological traits. we morphologically compared adults from the area of sympatry using biometric measurements and performed analyses to investigate their sexual size and shape dimorphism. a total of 34 males and 24 females of i. horvathi, and 25 males and 23 females of p. muralis, all adult individuals, were measured. both species showed sexual size dimorphism with females being longer (snout-vent length, svl) than males. after svl correction (ancova), head width, length and height and mass showed to be sexually dimorphic in both species. males carry relatively wider, longer and higher heads and were heavier than conspecific females. i. horvathi heads were more flattened than those of p. muralis and p. muralis were heavier than i. horvathi. both species displayed the same pattern of sexual dimorphism regarding body size, head size and shape not only in direction but also in magnitude. all results confirm that both species are very similar in studied biometric characters and, together with their ecological similarities, these suggest in absence of other factors they are likely to interact when living together. keywords. biometric characters, sexual dimorphism, southern slovenia, podarcis muralis, iberolacerta horvathi, lacertidae introduction the common wall lizard (podarcis muralis) and horvath’s rock lizard (iberolacerta horvathi) are sympatric and frequently syntopic lacertids through the entire range of i. 30 a. žagar et al. horvathi (tiedemann, 1997). the range of latter as known today extends from pre-alpine and alpine part of north eastern italy, to north western slovenia (lapini et al., 2004; rassati, 2010) and southern austria (cabela et al., 2007), and dinaric alps of central and southern slovenia and velebit in croatia (de luca, 1989; krofel et al., 2009). allotopic i. horvathi populations are only known from high elevations where p. muralis is absent; in the north of its distributional range above 900 m a.s.l. (e.g., lapini et al., 1993; cabela et al., 2007). both species are said to be very similar in their general body size and shape, the average snout-vent length of adults being between 55-65 mm (e.g. de luca, 1989; aleksić and ljubisavljević, 2001). reproductively, i. horvathi is monoestrous while p. muralis may lay a second clutch when conditions are favourable (lapini et al., 1993; capula et al., 1993). the dietary niches of both species are highly overlapping (de luca, 1992; richard and lapini, 1993). in the area of sympatry both species start their activity period in march/april and go to hibernation in october/november, exhibiting the diurnal activity pattern typical of lacertids in temperate zone (de luca, 1992; lapini et al., 1993). the overall habitat use of both species is also highly similar (cabela et al., 2007). they mostly occupy rocky habitats with sparse vegetation or open rocky screes in forests. on the microhabitat scale, some differences in slope and vegetation cover selection between species have been detected, i. horvathi tending to use more rocks and vertical surfaces than p. muralis (arnold, 1987; lapini et al., 1993; cabela et al., 2007). moreover, similarity in the overall appearance between both species has frequently led to misidentification in the past (e.g. brelih, 1954; de luca, 1989). to determine the species it is usually necessary to catch or make a close-up photograph of the individual to see the position of rostral and frontonasal scales that are frequently in contact in i. horvathi and separated in p. muralis (e.g. arnold et al., 2007). because of that, recent intensive studies discovered unknown populations of i. horvathi in many parts of its range where the species had previously been misidentified as p. muralis or not recorded yet (lapini et al., 2004; žagar, 2008 a, b; krofel et al., 2009; cafuta, 2010; rassati, 2010). sexual dimorphism in size and shape occurs in most species of lacertids. size (snoutvent length and body mass) can be either maleor female-biased (e.g., kaliontzopoulou et al., 2007; kratochvíl et al., 2003) and males in most species have bigger and wider heads and longer limbs (e.g. herrel et al., 1996). such sexual differentiation in lacertids is hypothesised to be derived from different selection pressures: males having bigger head proportions to have a firm grasp of a female’s trunk during copulation and females having longer trunks to hold the eggs (kaliontzopoulou et al., 2007, 2008 a, b). in the case of p. muralis, instances of both male-biased (strijbosch et al., 1980; barbault and mou, 1988; braña and ji, 2000; allan et al., 2006; bruner and constantini, 2007; gracceva et al., 2008; aleksić et al., 2009) and female-biased (gracceva et al., 2008; kaliontzopoulou and carretero, unpublished) sexual dimorphism have been described. in contrast, for i. horvathi a single comparative study of sexual dimorphism published so far showed a female-biased sexual size dimorphism (de luca, 1989) as described for some other species of this genus (e.g. for i. galani; arribas et. al., 2006). the main aim of our study is to reveal morphometric (dis)similarity in two very resembling species of lacertids, p. muralis and i. horvathi living in the area of sympatry. in the past, most comparisons of size and shape of both species only referred to descriptive, often qualitative data and were not targeted to biometric characters in order to detect 31morphometric variation in two sympatric lacertids possible subtle interspecific differences. we aimed to compare differences at intra(intersexual) and interspecific level in order to evaluate morphological divergences of considered sympatric lacertids. for the first time, we quantitatively investigate biometric characters of these two species from a sympatric area in southern slovenia. intersexual differences in size and shape in lacertids are a result of sexual selection rather than resulting from niche segregations (carretero, 2004) and therefore we expect it exists in both species. material and methods study area and individuals studied lizards were captured at 19 localities in the sympatric area of p. muralis and i. horvathi in kočevsko region, southern slovenia (45˚28’37’’n, 14˚48’34’’e) in spring and summer periods between 2007 and 2010 (fig. 1). the maximal distance between locations was 30 km that ensured similar environmental conditions. climate was moderate continental and mountainous influenced by mediterranean, inland and atlantic ocean (kordiš, 1993) with total annual precipitation of 1600 to 1800 mm and mean temperature in july 17.9 °c and in january -2.8 °c (puncer, 1980). typical habitats of both lacertid species in the region are natural or artificial rock cliffs with no or sparse vegetation and rocky outcrops on the forest edge (žagar, 2008 a). sampling sites were chosen to have similar microhabitat characteristics. all individuals caught were measured and photographed, and released afterwards on the same location. sex was verified by inspection of colouration, cloacal region and femoral pores. biometric characters to quantify intraand interspecific differences we measured six biometric characters: snout vent length (svl), head length (hl) from the tip of the snout to the posterior border of the collar, pileus length (pl), head width (hw), head height (hh), and mass (m) (only for specimens caught in 2010, measuring scheme after kaliontzopoulou et al., 2007). all linear measurements were taken to the closest 0.1 mm, using digital callipers and mass was measured to the closest 0.1 g, using scale with a hook type pesola max. 30 g. statistical analysis variables were logarithmically transformed to meet the assumptions of normality and homogeneity of variances. considering that sexual dimorphism either in size (ssd) or in shape (sshd) is common in lacertids (see introduction) sex was taken into account in all analyses. differences in svl were tested through a two-way anova with sex and species as factors. differences for the other measurements were relativized to svl by using a twoway ancova design with sex and species as factors and svl as covariate for size correction. all statistical analyses have been done using statistica 10 (statsoft, 2011). 32 a. žagar et al. results a total of 34 males and 24 females of i. horvathi, and 25 males and 23 females of p. muralis, all adult individuals, were measured (tab. 1). both species showed sexual size dimorphism with females being longer (svl) than males, but no interspecific differences either in svl or in sexual dimorphism were found (tab. 1 and 2). after size correction (ancova), only pl did not show to be dependent on sex, but hw, hl, hh and m proved to be sexually dimorphic in both species (tab. 2). in fact, males of both species had relatively wider, longer and higher heads and were heavier than the conspecific females (tab. 1). moreover, for both sexes, i. horvathi heads were more flattened than those of p. muralis and p. muralis were heavier than i. horvathi but no interspecific differences were found for the remaining variables (tab. 2). discussion our results comparing body size, head size and head shape of adult p. muralis and i. horvathi form the area of sympatry, indicated both sexual dimorphism and (limited) interspecific variation which were independent one from another. we have also confirmed that species were similar to each other in their external appearance. considering each sex   fig. 1. locations of the sampling sites (a) of common wall lizards (podarcis muralis, numbers 1, 2, 7-9, 16 and 19) and horvath’s rock lizards (iberolacerta horvathi, numbers 3-6, 10-15, 17 and 18) and the location of the study area in slovenia (b) with distributions of both species (p. muralis in grey and i. horvathi in black stripes). 33morphometric variation in two sympatric lacertids separately, there were no significant differences in body size and some head dimensions between the species, except for the head height (see tab. 2). however, i. horvathi’s heads tended to be more flattened, whereas the heads of p. muralis have a higher arc. flatness of i. horvathi heads has been reported already before and is a trait that is often used in general description of this species (e.g. radovanović, 1951; mršić, 1997; arnold and ovetable 1. descriptive statistics of biometric characters for two species of lacertids, podarcis muralis and iberolacerta horvathi, from southern slovenia. for each variable next values are given: mean±sd, minmax and n (numbers of individuals). legend: svl – snout-vent length, hh – head height, pl – pileus length, hl – head length, hw – head width and m – mass. variable iberolacerta horvathi podarcis muralis males females males females svl 53.46±3.90 43.7-60.0 34 58.30±4.27 48.2-65.9 24 54.71±4.62 41.9-62.8 25 58.85±3.36 50.1-64.6 23 hh 6.37±0.38 5.7-7.0 19 6.19±0.38 5.5-6.9 23 6.96±0.88 4.5-8.5 25 6.87±0.66 5.3-8.2 23 pl 13.29±1.73 10.7-21.4 33 12.57±0.80 10.5-13.8 24 14.14±1.00 11.9-15.6 25 13.24±0.80 11.5-14.8 23 hl 19.72±1.55 16.7-23.0 34 18.83±1.32 16.1-21.4 24 20.10±1.48 17.1-22.5 25 18.82±1.58 13.3-21.3 23 hw 8.96±0.91 7.0-10.5 33 8.71±0.71 6.9-9.8 24 9.14±0.81 7.0-11.0 25 8.43±0.39 7.6-9.2 23 m 4.25±0.58 2.8-5.3 19 4.01±0.83 2.5-5.5 23 4.26±1.11 1.5-6.3 25 4.55±0.90 2.5-5.5 23 table 2. results of an(c)ova comparisons for species and sex (variables are svl corrected, except svl) for two species of lacertids, podarcis muralis and iberolacerta horvathi, from southern slovenia. legend: svl – snout-vent length, hh – head height, pl – pileus length, hl – head length, hw – head width and m – mass. variable denominator species sex species*sex df f df p f df p f df p svl 105 1.20 1 0.2730 30.0 1 <0.0001* 0.20 1 0.6939 hh 89 35.00 1 <0.0001* 15.83 1 0.0001* 0.01 1 0.9173 pl 104 0.0006 1 0.9813 2.29 1 0.1337 1.23 1 0.2705 hl 105 0.04 1 0.8390 64.06 1 < 0.0001* 0.31 1 0.5759 hw 104 1.73 1 0.1911 67.33 1 <0.0001* 3.30 1 0.0723 m 89 5.45 1 0.0220* 25.79 1 <0.0001* 2.43 1 0.1225 34 a. žagar et al. den, 2002, cabela et al., 2007; lapini et al., 1993, 2004). flat head was also described as a general trait for all species of genera iberolacerta (arnold et al., 2007). most of the species from genera podarcis and iberolacerta use crevices as escape sites and their body and heads are moderately to very depressed (arnold and oveden, 2002). both species also displayed the same pattern of sexual dimorphism regarding body size, head size and shape not only in direction but also in magnitude. females had longer snout-vent lengths whereas males carried relatively bigger heads in all dimensions but length. in the family lacertidae, considerable lability in the direction of sexual size dimorphism have been found both between (reviewed in cox et al., 2007) and within species where either males or females are bigger depending on the population (roitberg and smirina, 2006 a, b; roitberg, 2007) in response to geographic variations in the relative contributions of sexual, fecundity and natural selection forces (kaliontzopoulou et al., 2010 a, b). across populations of lacertids, size (snout-vent length and body mass) was described to be either male-biased with males having larger body sizes (vogrin, 2005; kaliontzopoulou et al., 2007; brecko et al., 2008; kaliontzopoulou et al., 2008 a; aleksić et al., 2009), or female-biased with females having larger size dimensions on account of a longer trunk (kratochvíl et al., 2003; liu et al., 2008). more specifically in the case of p. muralis, the direction of the sexual size dimorphism observed in our study (females longer than males) was not always found. namely, in some populations no significant difference in snout-vent length between adults of the two sexes was observed (barbault and mou, 1988; strijbosch et al., 1980; allan et al., 2006) whereas in others even there was the opposite trend, males being longer than females (gracceva et al., 2008), than showed here. the results from this study corroborate that interpopulation variability in sexual size dimorphism is very high in p. muralis. on the other hand, comparing data for i. horvathi with the only extensive morphometric study previously carried out for this species, our study show similar trends: females were significantly larger in snout-vent length than males (de luca, 1989). however, in the study of de luca (1989), data of females and males from two populations were pooled together which posed doubts on the reliability of the results since inter-population variation in lacertids is known to be high and snout-vent length tends to be sexually dimorphic in lacertid lizards (i.e. kaliontzopoulou et al, 2010 a, b). hence, it is crucial to size-adjust other morphological traits before comparing them (kratochvíl et al., 2003). more populations of i. horvathi, as well as of p. muralis, should be studied in the future to recognize if different patterns of sexual size and shape dimorphisms are exhibited in different populations. regarding the sexual shape dimorphism, there were head shape differences in both species with males having relatively wider and higher (but not longer) heads. for i. horvathi, bischoff (1984) and de luca (1989) already suggested a similar pattern. likewise, mostly all morphometric studies of p. muralis populations also found the same trend, e.g. males having larger heads adjusted to snout-vent length than females (gracceva et al., 2008) or males having larger jaw sizes and larger heads than females (aleksić et al., 2009). the direction of sexual shape dimorphism in the head robustness is common to whole family lacertidae in which males have wider and bigger relative head measurements and also longer limbs (herrel et al., 1996; kratochvíl et al., 2003; bruner et al., 2005; vogrin, 2005; kaliontzopoulou et al., 2007; kaliontzopoulou et al., 2008 a; ljubisavljević et al., 2008; aleksić et al., 2009). although the direction in shape sex dimorphism is common, its magnitude is highly variable between species (verwaijen et al., 2002; kaliontzopoulou 35morphometric variation in two sympatric lacertids et al., 2007; ljubisavljević et al., 2008), but even though, in our case almost no differences in the amount of sexual shape dimorphism were detected between both species living in the area of sympatry. actually, the analysis revealed that sexual dimorphism was in general higher (significant differences in all but one biometric characters) than species variation (significant differences only in head height and relative body mass, tab. 2). summarizing, the lacertids p. muralis and i. horvathi show remarkable similarities not only in general size and shape (and in colouration; de luca, 1989; arnold and oveden, 2002), but also in their sexual dimorphism patterns. our study comparison of interand intraspecific sexual dimorphism patterns form localities in the sympatric area confirmed that p. muralis and i. horvathi are very similar. together with their ecological similarities these facts suggest that, in absence of other factors, they are likely to be in interaction when living together. with our analysis we could not yet identify what type of interspecific interaction is present between both species, but most probably acts as interference which is commonly found in predator guilds (e.g. carothers and jaksić, 1984; robinson and terborgh, 1995; linnell and strand, 2000), and even in lacertids (downes and bauwens, 2002, 2004). in this perspective, results suggest potential for competition between species since interspecific effects in sympatric populations were not expressed in morphological, i.e. coevolved divergences. perhaps such effects are expressed in other phenotypic or ecological adaptations, e.g. spatial segregation or resource partitioning (tokeshi, 1999) that need to be investigated in the future. thus, biometric comparisons could provide a first evaluation of competitive potential between lacertids of similar size belonging to the same predator guild (valverde, 1967). if interspecific size differences are smaller than intraspecific, i.e. sexual dimorphism, this might suggest species to be potential competitors. on the other hand, equal or larger interspecific size differences could indicate already coevolved niche differentiation and consequently lower competition potential, i.e. “ghost of competition” pattern (begon et al., 2006). multipopulation comparison of sexual dimorphism and morphological divergence between both species in allopatry and sympatry should be carried out to test the hypotheses of character displacement and relaxation of interspecific competition (butler et al., 2007). acknowledgements collecting permits were provided by agency for environment (ministry for environment and space, slovenia). we would like to thank lara kastelic, martin kavšček, bia rakar, teo delić, tea romih, polona raspor, darja osojnik and alja kastelic, who helped during sampling campaigns. the work of mac was 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(2008a): pomen presvetlitev za plazilce (reptilia) v gozdni krajini. univerza v ljubljani, biotehniška fakulteta, oddelek za biologijo, ljubljana. žagar, a. (2008b): the lowest altitudinal record of horvath’s rock lizard (iberolacerta horvathi) in slovenia. nat. slov. 2008/2: 59-62. žagar, a., planinc, g., krofel, m. (2008): records of horvath’s rock lizard (iberolacerta horvathi) from notranjsko podolje region (central slovenia). nat. slov. 9/2: 43-44. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 9(1): 61-73, 2014 doi: 10.13128/acta_herpetol-13269 a new, recently extinct subspecies of the kuroiwa’s leopard gecko, goniurosaurus kuroiwae (squamata: eublepharidae), from yoronjima island of the ryukyu archipelago, japan yasuyuki nakamura1, akio takahashi2, hidetoshi ota3 1 tropical biosphere research center, university of the ryukyus, senbaru 1, nishihara, okinawa 903-0213, japan. corresponding author. e-mail: ynaka.riukiaria@gmail.com 2 department of zoology, faculty of science, okayama university of science, ridai-cho 1-1, kita-ku, okayama, okayama 700-0005, japan 3 institute of natural and environmental sciences, university of hyogo, and the museum of nature and human activities, yayoi-gaoka 6, sanda, hyogo 669-1546, japan submitted on: 2013, 28th august; revised on: 2014, 19th january; accepted on: 2014, 7th march abstract. a new subspecies of the eublepharid gecko, goniurosaurus kuroiwae, is described on the basis of fragmentary bones recovered from a midden on yoronjima island, ryukyu archipelago, japan. it differs from the other conspecific subspecies in having a unique combination of osteological character states: in particular, the maxilla contains a posteriorly extended maxillary shelf and a scarcely inclined lateral wall above the posterior tooth row, and the frontal contains a widened anterior section and a laterally overhanging anterior part of lateral prefrontal facet, both of which differentiate this new subspecies from the morphologically most similar g. k. kuroiwae. the new subspecies, endemic to yoronjima island, may have gone extinct, together with several other amphibians and reptiles on the island — most likely due to human-related deforestation and increased predation pressure from introduced weasels. keywords. biodiversity, extinction, human impacts, lizard, subfossil. introduction kuroiwa’s leopard gecko, goniurosaurus kuroiwae (namiye, 1912), sensu grismer et al. (1994), is a eublepharid gecko endemic to the central part of the ryukyu archipelago, japan, in the subtropical northwestern pacific. it is isolated from the rest of its congeners occurring in southeastern continental china, northern vietnam, and adjacent coastal islands (grismer et al., 2002; ziegler et al., 2008; orlov et al., 2008; wang et al., 2010, 2013), and the species represents the easternmost old world member of the family (grismer, 1988; grismer et al., 1994). because this species (or species complex, see below) consists of a number of allopatric and apparently diagnosably divergent insular populations, its classification has been the subject of much controversy. in the latest revision (grismer et al., 1994), its populations were combined into a single species comprising five extant subspecies: g. k. kuroiwae (namiye, 1912); g. k. orientalis (maki, 1930); g. k. splendens (nakamura and uéno, 1959); g. k. toyamai grismer et al., 1994; and g. k. yamashinae (okada, 1936). later, grismer et al. (1999, 2002), adopting the evolutionary species concept, referred to each of these taxa as full species, and this taxonomic treatment has been widely accepted in the context of the recent trend of disuse of the subspecies rank by herpetologists. however, we believe that such changes in taxonomic rank are premature, because diagnostic characters originally proposed to define each of these taxa are few in number and, moreover, include those with highly variable character states. for example, yellow-brown to gold iris color was considered as one of the character states that 62 y. nakamura, a. takahashi, h. ota discriminates g. k. yamashinae from the other subspecies whose irises are blood-red in color (grismer et al., 1994), but g. k. kuroiwae populations from okinawajima and adjacent islets also include individuals with more or less yellow irises (h.ota, unpublished observations). we therefore prefer to take a conservative stance with respect to the taxonomic treatment of g. kuroiwae sensu lato by maintaining the framework of grismer et al. (1994). the species has been recorded from 10 islands of the okinawa island group and one island of the amami island group (grismer et al., 1994; kurita and kawamura, 2011). its patchy insular distribution has baffled biogeographers. this is best exemplified in the species’ absence from islands between tokunoshima island in the amami island group, where g. k. splendens occurs, and islands of the okinawa island group, where the other four subspecies occur (grismer et al., 1994). this distributional gap is now partially filled by our discovery of bone fragments from a presumed cultural deposit on yoronjima island, an islet representing the southwesternmost extremity of the amami group (nakamura et al., 2013; fig. 1). the available skeletal elements of the yoronjima g. kuroiwae are rather few in number and fragmentary, but they deserve careful morphological investigation as these are the only available evidence for the population on yoronjima that has recently become extinct (see below). our detailed morphological investigations using comparative specimens, including representatives of all known conspecific subspecies, revealed that the yoronjima g. kuroiwae has a unique suite of character states that differentiates it from the others, warranting recognition as a new subspecies as described below. given the presumed recency of the bones, the present finding most likely represents a human-induced extinction of an insular squamate taxon, which has never before been documented in japan (including the ryukyus). material and methods yoronjima is a small (20.5 km2), flat (97 m elevation) limestone island lying 40 km southwest of okinoerabujima island in the amami island group, and 30 km northeast of okinawajima island of the okinawa island group (fig. 1). brief descriptions of yoronjima and the sampling site are presented in nakamura et al. (2013). the skeletal remains of goniurosaurus kuroiwae subspecies that are described here were retrieved from deposits under a limestone rock shelter located approximately 150 m east of the takachiho shrine, asato (27°02’05”n, 128°26’02”e; fig. 1). the site appeared to be a midden, as the deposits were mixed with soil, numerous marine fish bones, and modern artifacts (such as fragments of tableware and glass bottles). among the artifacts, the fragmentary glass bottles were estimated to be from no earlier than the 19th century. based on the species composition and preservation of terrestrial vertebrate elements (four species of frogs, ten species of squamate reptiles [including g. kuroiwae], a shrew, and some non-native rodents such as the black rat [rattus rattus], all of which were mostly preserved in fine condition), we believe that this accumulation of skeletal remains is derived from animals that were attracted by invertebrates associated with garbage (nakamura et al., 2013). the site yielded at least 19 bone fragments of g. kuroiwae, of which some were reported preliminarily in our preceding paper, along with associated remains of several other squamates (nakamura et al., 2013). the yoronjima g. kuroiwae material was compared with partially skeletonized specimens of known g. kuroiwae subspecies (see appendix). one of the difficulties in this study arose from the scarcity of available g. kuroiwae specimens for osteological comparisons. most of the subspecies are poorly represented by museum specimens because their populations are currently endangered due to loss of habitat, predation by exotic mammals, and frequent illegal collections for pet trade; thus, they are under the strict protection of the prefectural laws of okinawa and kagoshima (tanaka, 2005a, b, c, d; ota, 2010). unfortunately, we were unable to examine the maxilla of g. k. toyamai, and the axis and the frontal of g. k. toyamai and g. k. yamashinae. likewise, disarticulated skeletal elements, such as frontals, available for comparison were very limited (see table 2). moreover, with regard to some specimens of g. k. kuroiwae (derived from roadkill), some of the elements were not observable due to fracture. body size (snout–vent length, svl) of the yoronjima material was estimated based on measurements taken from certain elements of extant g. kuroiwae skeletons with size data. the relevant regressions (where x = bone measurement and y = svl) were: length of the dentary tooth row (right), y = -9.8 + 9.04x, r2 = 0.79, p < 0.0001, n = 16; interorbital width of the fig. 1. map of the central ryukyus showing the location of yoronjima island and the distribution of goniurosaurus kuroiwae subspecies. star indicates the study site. 63new subspecies of goniurosaurus kuroiwae frontal, y = -20.8 + 45.5x, r2 = 0.73, p < 0.01, n = 8; length of the humerus, y = -29.5 + 8.54x, r2 = 0.96, p < 0.0001, n = 8 (five right and three left bones from eight individuals); and the length of the femur, y = -33.4 + 7.39x, r2 = 0.9, p < 0.01, n = 8 (the same side as the humerus). morphological comparisons were performed using a binocular microscope (nikon smz-10), and drawings were made using a camera lucida. measurements were taken with imagej (us national institutes of health, bethesda, md; http://imagej. nih.gov/ij/) or an ocular micrometer. numerical calculations were performed using the program r (r development core team, 2011). osteological terminology followed that of camp (1923), estes et al. (1988), and conrad (2004). all of the examined subfossil material was deposited in the fujukan, museum of the university of the ryukyus, nishihara, okinawa, japan (rumf). results systematics goniurosaurus kuroiwae yunnu nakamura, takahashi and ota, subsp. nov. (figs. 2-4; tables 1, 2) type material holotype: rumf-gf-4076, a well-preserved left maxilla, collected by akio takahashi, kentaro hagari, and motohiro okade on 19 november 2011. paratypes: one right maxilla (rumf-gf-4075), three right and three left dentaries (rumf-gf-4077 to 4082), six frontals table 1. summary of selected morphometric and meristic data for the subspecies of goniurosaurus kuroiwae. numerals in parentheses indicate sample sizes, and those in brackets indicate medians (for ratios) or means ± 1 standard error (for others). nc = not compared. r = right, l = left. all measurements are in mm. subspecies yunnu kuroiwae orientalis splendens toyamai yamashinae n 5 4 6 2 1 maxilla (left) tooth count 52 (1) 41-50 (4) [45.5 ± 1.85] 42-51 [47 ± 1.96] 44-50 [46.2 ± 0.83] nc 43 frontal width at the point of the base of the anterolateral processes (wbap) 3.22 (1) 2.74-3.27 (4) [3.12 ± 0.13] 2.8 (1) 2.98-3.25 (4) [3.09 ± 0.06] nc nc interorbital width (wi) 1.92, 1.94, 2.08, 2.13 (4) [2.02 ± 0.05] 2.04-2.47 (4) [2.3 ± 0.09] 2.08 (1) 2.0-2.16 (4) [2.09 ± 0.04] nc nc wbap/wi (%) 168 (1) 132-142 (4) [134.5] 135 (1) 144-152 (4) [148] nc nc dentary (right, unless noted) surface length of anterodorsal margin of lateral coronoid facet (lalcf ) 1.09, 0.96, 0.83 (2r, 1l) [0.96 ± 0.08] 0.65-0.96 (4r, 1l) [0.85 ± 0.06] 0.63-1.41 [1.06 ± 0.16] 0.68-0.9 [0.74 ± 0.03] 0.79, 0.9 0.79 length of ventral margin of lateral coronoid facet (lvlcf ) 2.18, 1.97, 1.95 (2r, 1l) [2.03 ± 0.07] 1.41-2.6 (4r, 1l) [1.99 ± 0.2] 1.06-1.76 [1.33 ± 0.15] 0.91-1.53 [1.23 ± 0.09] 1.3, 1.74 1.09 lvlcf/lalcf (%) 200, 205, 235 (2r, 1l) [205] 198-280 (4r, 1l) [227] 114-168 [119.5] 125-187 [173] 165, 193 138 tooth count 50, 53, 51, 52 (2r, 2l) [51.5 ± 0.65] 41-53 [47.6 ± 2.23] 47-51 [49.5 ± 0.96] 46-52 [49.8 ± 0.98] 52, 50* 48 tooth row length 9.8, 10.4, 10, 9.9 (2r, 2l) [10.1 ± 0.12] 8.5-11.4 [10.4 ± 0.51] 9.1-10.2 [9.7 ± 0.27] 8.9-9.8 [9.4 ± 0.15] 11, 10.7* 10.3 * denotes unreliable character observation due to breakage or abnormality. 64 y. nakamura, a. takahashi, h. ota (rumf-gf-4083 to 4088), two right posterior mandibles (rumf-gf-4089 and 4090), one axis (rumf-gf-4091), one right humerus (rumf-gf-4092), and one right femur (rumf-gf-4093). of these, two right (rumfgf-4077 and 4078) and one left (rumf-gf-4080) dentaries have the same data as the holotype; others were collected by akio takahashi and yasuyuki nakamura on 24-25 november 2007. etymology the subspecific name is derived from “yunnu,” an old vernacular name for yoronjima island. diagnosis the bone fragments recovered are referred to the genus goniurosaurus based on their association with maxillary and dentary teeth having the derived condition of an expanded and ridged occlusal margin that is unique to goniurosaurus among gekkotans (grismer, 1988), and to g. kuroiwae based on its exclusive occurrence in the ryukyus (see also accounts of certain skeletal elements presented below). goniurosaurus kuroiwae yunnu is a relatively small-sized (estimated adult svl 66.6-84.2 mm) subspecies. it differs from all known conspecific subspecies in having the following combination of morphological characteristics: posteriorly extended maxillary shelf one tooth’s width wide at level of posterior-most tooth; scarcely inclined lateral wall of posterior maxilla above posterior part of tooth row; ratio of width at the point of base of anterolateral processes of frontal to interorbital width high (1.68); laterally overhanging anterior parts of lateral prefrontal facets of frontal; ventral margin of lateral coronoid facet of dentary straight or slightly curved dorsally — more than twice as long as surface length of antertable 2. comparisons of morphological characteristics of the subspecies of goniurosaurus kuroiwae. numerals in parentheses indicate sample sizes. nc = not compared. subspecies yunnu kuroiwae orientalis splendens toyamai yamashinae n 5 4 6 2 1 maxilla maxillary shelf width at level of posterior-most tooth: one tooth’s width more (+) or less (-) than a half of one tooth’s width + (1) + nc lateral wall above posterior tooth row: nearly upright (+) or inclined considerably (-) + (1) + nc + frontal anterior part of lateral prefrontal facet: overhangs laterally (+) or not (-) + (2) (4) (1) nc nc nc dentary lateral coronoid facet: ventral margin straight (+) or bowed ventrally (-) + (3) + posterolateral margin below lateral coronoid facet convex (1) convex (4)/ straight (1) convex (3)/ straight (1) recessed straight (1)/ recessed (1) convex posterior mandible retroarticular process: longitudinal ridge present (+) or absent (-) + (2) + + (3)/(1)* + + prearticular-surangular suture below posterior mylohyoid foramen: position to the midpoint between the foramen and ventral edge dorsal (1)/ same (1) dorsal (4), (1)** same (1)/ ventral (3) ventral (3), (1)** ventral same** axis hypapophysis: ventral keel thick (+) or not (-) + (1) + (4) + (1) (3) nc nc * denotes unreliable character observation due to breakage or abnormality. ** denotes inferred state owing to the overlying angular. 65new subspecies of goniurosaurus kuroiwae odorsal margin of facet; convex posterolateral margin of dentary ventral to lateral coronoid facet, forming a broad and posteriorly-oriented process; prearticular-surangular suture below posterior mylohyoid foramen situated at, or dorsal to, midpoint between foramen and ventral edge of prearticular; longitudinal ridge on dorsal surface of retroarticular process; thick keel on axial hypapophysis. description of holotype nearly complete left maxilla lacking part of posterior margin of facial process and a number of teeth (figs. 2a-d). teeth pleurodont, peg-like, undifferentiated, set closely (ctenodont), forming continuous straight edge ventrally, 52 tooth loci and 36 functional teeth (including posteriormost one) (fig. 2b); tooth crowns exposed fig. 2. maxilla and frontals of goniurosaurus kuroiwae yunnu new subspecies. a-d, the holotype left maxilla (rumf-gf-4076) in lateral (a), medial (b), and ventral (c) views, and close-up of the posterior part in ventral view (d); e-h, frontal (rumf-gf-4083) in dorsal (e), ventral (f), anterior (g), and left lateral (h) views; i, frontal (rumf-gf-4084) in ventral view. abbreviations: alp, anterolateral process; amp, anteromesial process; fp, facial process; lpff, lateral prefrontal facet; ms, maxillary shelf; plp, posterolateral process; pmp, posterior maxillary process; sl, subsurface lamina; sy, symphysis. the arrow in d indicates the lateral wall above the posterior tooth row, which is scarcely visible from the ventral side. broken lines represent inferred original outlines. scale bars equal 1 mm. 66 y. nakamura, a. takahashi, h. ota below labial wall (fig. 2a), each expanded, more elongated mediolaterally than anteroposteriorly, and consisting of labiolingual series of (usually) four cusps, yielding flat occlusal surface in mediolateral views (figs. 2a, b); facial process steep and three times the anterior height of bone, triangular in shape, with thin and acute dorsal tip (figs. 2a, b); seven supralabial foramina, forming row at lateral surface, and additional row of two associated foramina dorsally (fig. 2a); anteromesial process prominent, medial edge thick and ridged ventrally (figs. 2b, c); maxillary shelf broadest at level of 30th tooth, extending posteriorly beyond tooth row by more than one tooth’s width medially to posteriormost tooth (figs. 2c, d); bases of some posteriormost teeth unexposed medially due to ventrally turned maxillary shelf (fig. 2b); a toothless deep depression, the length of two teeth, follows posteriorly to tooth row (fig. 2d); lateral wall above posterior part of tooth row scarcely inclined laterally, lateral surface hardly visible from ventral side (fig. 2d); posterior maxillary process disproportionally truncated, with length from posteriormost tooth to tip of process smaller than length of four posteriormost teeth, although we cannot confirm the process is intact (fig. 2d); tip of process curves dorsomedially (figs. 2a-c). measurements (mm): maximum length, 10.1; maximum width, 1.92; maximum height (without teeth), 3.49; tooth row length, 9.33; anteromesial process length, 1.1; maximum length of maxillary shelf from tooth row, 1.54; length from widest point of bone to posterior tip, 4.95. descriptions of paratypes the paratypical maxilla (right: rumf-gf-4075) is much more worn, so that it furnishes no morphological characteristics. like the holotype, it is identified as this species on the basis of the expanded occlusal margins of the tooth crowns (a synapomorphy of g. kuroiwae and some other goniurosaurus species: grismer et al., 1999, 2002; wang et al., 2010) usually with four cusps (a unique character state seen in g. kuroiwae: sumida and murphy, 1987; grismer, 1988; nikitina and ananjeva, 2009). of the six paratypical frontals recovered, none is complete. these unpaired frontals possess ventrally fused lateral descending processes (subolfactory processes), which is a shared derived character state of the extant gekkotans (kluge, 1967; estes et al., 1988; see also daza et al., 2013). the frontals also possess the posterolateral processes, which are anteroposteriorly elongated, unlike those of other japanese gekkotans examined (all gekkonids) whose posterolateral processes are thin anteroposteriorly. the most complete of the referred frontals (rumf-gf-4083: figs. 2e-h) retains the anterior section of the bone, which is anteriorly elongated and widened, while the anterior third of both anterolateral processes and the intervening subsurface lamina for the nasals are lost. the length from the subsurface lamina to the parietal facet is 8.0 mm, and the width at the point of the base of the anterolateral processes (3.22 mm) is 1.68 times wider than the narrowest width of the interorbit (1.92 mm) (figs. 2e, f; table 1). the smooth dorsal surface is concave, most prominently posterior to the postorbitofrontal facets. the anterolateral processes are tilted laterally (fig. 2g). in lateral view, the bone is deepest at the anterior end of the ventral contact of the lateral descending processes (fig. 2h). anteriorly, the paired lateral descending processes terminate at a much more posterior level of the nasal overlap, yielding a flat ventral surface of the more anterior part (fig. 2f). posterior to the ventral contact, the processes become a pair of swellings that extend posteriorly to the posterolateral edges. the ventral surface of the posterolateral margin is thin and flat. in this specimen and the second best preserved specimen of the referred frontals (rumf-gf-4084: fig. 2i), the lateral prefrontal facets are straight and overhang laterally throughout their length (figs. 2e-g, i). although the second best specimen lacks the anterior fourth or fifth of the lateral prefrontal facets (fig. 2i), the width at the anteriormost intact part (3.15 mm) is much greater, 1.48 times, than that at the orbital constriction (2.13 mm), this suggests that the high ratio between the width at the point of the base of the anterolateral processes and interorbital width in the most complete specimen (see above) is not a malformation. six dentaries (three right and three left, fig. 3) are referred to g. kuroiwae on the basis of tooth morphology (fig. 3f), which is identical to that of the maxillae (see above). the total tooth count is 50 and 53 in two right dentaries and 51 and 52 in two left dentaries (table 1). the tooth crowns are exposed above the labial wall. the meckelian groove is fused completely. the subdental shelf is well developed. the mental foramina are arranged in a longitudinal row at the middle of the lateral surface; there are a total of seven (n = 2) or eight (n = 3) foramina. these foramina are anteroposteriorly elongated and housed within depressions. the posterolateral margin is posterodorsally excavated for the lateral process of the coronoid (henceforth referred to as “the lateral coronoid facet”; fig. 3b). the ventral margin of the lateral coronoid facet is straight (figs. 3d, e) or weakly curved dorsally (figs. 3b, c), and it is approximately twice as long as the surface length of the anterodorsal margin (tables 1, 2). the best preserved right dentary (rumf-gf-4077: figs. 3a-c) retains an almost intact posterolateral mar67new subspecies of goniurosaurus kuroiwae gin, except for the tip of the angular process. ventral to the lateral coronoid facet, the margin is strongly convex, forming a posteriorly-oriented broad process. ventral to the process, it is weakly notched. despite the posterolateral parts being broken, at least two other dentaries (rumf-gf-4078, 4080: figs. 3d, e) confirm that the posterolateral dentary margins are not excavated anteriorly beyond the levels of the posterior ends of the lateral coronoid facets. two specimens of right postdentary bones of mandibles are preserved (rumf-gf-4089, 4090; figs. 4a-c), both of which lacks the dentary, coronoid, angular, and splenial (plus posterior half of the retroarticular process in rumf-gf-4090). they are referred to gekkotans on the basis of having an anteriorly constricted and spoon shaped retroarticular process with a medial offset and a lateral notch of the base (estes et al., 1988; daza et al., 2013; fig. 4a) and to g. kuroiwae on the basis of having an angulated dorsal edge of the surangular (which is round in other examined japanese gekkotans) (fig. 4a). in both specimen, a short, anteroposteriorly oriented ridge is present immediately lateral to the foramen chorda tympani of the dorsal surface of the retroarticular process (fig. 4a). both the posterior surangular foramen and the posterior mylohyoid foramen are distinct at the posterior part, with the latter foramen being located at almost the midpoint of the mandible’s height (figs. 4b, c). in rumf-gf-4090, the surangular-prearticular suture fig. 3. dentaries of goniurosaurus kuroiwae yunnu new subspecies. a-c, right dentary (rumf-gf-4077) in medial (a) and lateral (b) views, and close-up of the posterodorsal part in lateral view (c); d, right dentary (rumf-gf-4078) in lateral view; e and f, left dentary (rumf-gf-4080) in lateral view (e) and close-up of the posterior teeth in occlusal view (f). abbreviations: alcf, anterodorsal margin of the lateral coronoid facet; ap, angular process; lcf, lateral coronoid facet; mf, mental foramen; vlcf, ventral margin of the lateral coronoid facet. scale bars equal 1 mm. 68 y. nakamura, a. takahashi, h. ota traverses across the lateral surface as nearly parallel with the ventral edge of the prearticular, although its posterior extent is unclear (fig. 4c). at the position below the posterior mylohyoid foramen, the suture is situated at the midpoint (rumf-gf-4089), or dorsally to the midpoint (rumf-gf-4090), between the foramen and the ventral edge of the prearticular (figs. 4b, c). one nearly entire axis (rumf-gf-4091: figs. 4d-f) lacking the synapophysis and postzygapophysis on the left side is referred to the new subspecies. it is referred to gekkotans on the basis of having a squarish outline in the ventral view, distinct subcentral foramina, a round condyle, and posteriorly directed lateral projections at the lateral sides of the axial hypapophysis (camp, 1923; hoffstetter and gasc, 1969); and to g. kuroiwae on the basis of having a procoelous condyle and a fused notochordal canal, both of which indicate a eublepharid origin (camp, 1923; hoffstetter and gasc, 1969; kluge, 1987; grismer, 1988). it is 3.7 mm in length (without the dens, 3.2 mm), 2.3 mm in maximum width, and 3.7 mm in maximum height. the condyle is slightly inclined dorsoventrally. its transverse width of 0.85 mm is much smaller than that of the posterior end of the centrum at 1.27 mm. the hypapophysis bears a thick and well-developed keel. appendicular skeletons are represented by one almost complete right humerus (rumf-gf-4092, 12.0 mm long; fig. 4g) and one complete right femur (rumf-gf-4093, 14.4 mm long; fig. 4h). association of the humerus is based on its size, straight overall shape, slender brachial, relatively undeveloped entepicondyle, and ovoid radial condyle that elongates in parallel with fig. 4. posterior mandibles, axis, humerus, and femur of goniurosaurus kuroiwae yunnu new subspecies. a and b, right posterior mandible (rumf-gf-4089) in dorsal (a) and lateral (b) views; c, right posterior mandible (rumf-gf-4090) in lateral view; d-f, axis (rumfgf-4091) in right lateral (d), posterior (e), and ventral (f) views; g, right humerus (rumf-gf-4092) in ventral view; h, right femur (rumf-gf-4093) in medial view. abbreviations: con, condyle; ec, entepicondyle; fct, foramen chorda tympani; hy, hypapophysis; pa, prearticular; pmf, posterior mylohyoid foramen; psf, posterior surangular foramen; rap, retroarticular process; rc, radial condyle; sa, surangular; sf, subcentral foramen. scale bars equal 1 mm. 69new subspecies of goniurosaurus kuroiwae the bone’s axis. the femur is referred on the basis of its size and straight and slender shape. body size svl estimates of the yoronjima material are 78.884.2 mm based on the length of three dentary tooth rows (9.8-10.4 mm, two right and one left: rumf-gf-4077, 4078, and 4080), 66.6-76.1 mm based on the interorbital width of four frontals (1.92-2.13 mm, rumf-gf-4083 to 4086), and 73.0 mm based on the length of the humerus (rumf-gf-4092) and the femur (rumf-gf-4093). the skeletal elements used for the estimation are considered to be derived from skeletally mature individuals judging from their fully ossified state and, in the dentaries, the large number of teeth. comparisons the unique combination of osteological character states in those skeletal elements from the yoronjima differentiates goniurosaurus kuroiwae yunnu from all other currently recognized subspecies of g. kuroiwae (see tables 1, 2). most similar is the geographically closest g. k. kuroiwae. however, the yoronjima material differs from g. k. kuroiwae (and some other known conspecific subspecies) in having: a posteriorly extended maxillary shelf that is one tooth’s width wide at the level of the posteriormost tooth (the shelf is less posteriorly extended in g. k. kuroiwae [see also fig. 6 in grismer, 1988], g. k. splendens, and g. k. yamashinae; table 2); a very weak lateral inclination of the lateral wall of the posterior part, above the posterior tooth row, of the maxilla (this part is considerably inclined in g. k. kuroiwae and g. k. splendens, as in their posterior maxillary processes, and the lateral surfaces are clearly visible from the ventral sides; table 2); laterally overhanging anterior parts of lateral prefrontal facets of the frontal (vs. these parts vertical in g. k. kuroiwae and g. k. orientalis; table 2); and an interorbital constriction and a widened anterior section of the frontal, resulting in a high ratio (1.68) of the width at the point of the base of the anterolateral processes to interorbital width (the ratio is lower in g. k. kuroiwae, g. k. orientalis, and g. k. splendens; table 1). additionally, the number of left maxillary teeth of g. k. yunnu, at 52, is greater than that observed in g. k. kuroiwae and other subspecies (table 1). indeed, a range of 46-50 left maxfig. 5. line drawings of mandibles (right, except g. k. kuroiwae) of extant goniurosaurus kuroiwae subspecies in lateral views, showing variation in shapes of the posterolateral dentaries and the height of the prearticulars. a, g. k. kuroiwae (kuz r67952) reversed left; b, g. k. orientalis (rumf-zh-570); c, g. k. splendens (kuz r71533); d, g. k. toyamai (omnh r3974) posterior retroarticular process is omitted; e, g. k. yamashinae (rumf-zh-206). anterior dentaries and most teeth are omitted. in all specimens the posterior extent of the surangularprearticular suture is unclear. abbreviations: ang, angular; cor, coronoid; den, dentary; pa, prearticular; pmf, posterior mylohyoid foramen; rap, retroarticular process; sa, surangular. scale bars equal 1 mm. 70 y. nakamura, a. takahashi, h. ota illary tooth counts from eight “g. kuroiwae” (presumably g. k. kuroiwae) has been reported by nikitina and ananjeva (2009). moreover, the estimated adult svl of g. k. yunnu at 66.6-84.2 mm, though apparently close to the adult svl of most other subspecies (75-95 mm for g. k. orientalis, 65-81 mm for g. k. splendens, and 75-85 mm for g. k. toyamai and g. k. yamashinae: ota, 2003; tanaka, 2005a, b, c), is somewhat smaller, particularly at the upper limit, than that of g. k. kuroiwae (76-100 mm for the okinawajima population: tanaka and nishihira, 1989; 80-92 mm for the yagajijima population: kurita and kawamura, 2011). however, the body size of lizard populations can vary drastically in time and space (e.g., pregill, 1986 and literature cited therein; sumner et al., 1999), and, thus, caution should be used in using body size as a character. goniurosaurus kuroiwae yunnu also differs from extant conspecific subspecies other than g. k. kuroiwae in having: an elongated ventral margin of the lateral coronoid facet of the dentary that is twice as long as the surface length of the anterodorsal margin and that is straight or slightly curved dorsally (vs. a less-elongated and, as a whole, ventrally curved ventral margin in g. k. orientalis, g. k. splendens, g. k. toyamai, and g. k. yamashinae; tables 1, 2); a strongly convex posterolateral margin of the dentary ventral to the lateral coronoid facet (vs. a straight or recessed margin in g. k. splendens and g. k. toyamai; table 2); prearticular-surangular suture at the position below the posterior mylohyoid foramen situated at, or dorsal to, the midpoint between the foramen and the ventral edge of the prearticular (vs. a suture situated ventrally to the midpoint in g. k. splendens and g. k. toyamai; table 2); a longitudinal ridge on the dorsal surface of the retroarticular process (vs. its absence in g. k. splendens; table 2); and a thick keel of the axial hypapophysis (vs. an obscured or rudimentary keel in g. k. splendens; table 2). discussion the yoronjima goniurosaurus kuroiwae, described here as a new subspecies, g. k. yunnu, can be regarded as an endemic form, morphologically distinguishable from all known conspecific subspecies. further circumstantial support for this taxonomic conclusion is provided by the distributions of extant subspecies. four currently recognized subspecies of the okinawa island group are separated from each other by straits that are less than 300 m deep (see the bathymetry of oshima et al., 1988). separation from other landmasses by straits greater than 400 m deep suggests that yoronjima has been isolated from other landmasses for a relatively long period, and it is therefore unlikely that the yoronjima g. kuroiwae belongs to any known g. kuroiwae subspecies. in addition to its diagnosability, such putative geographical isolation warrants the recognition of g. k. yunnu as a distinct subspecies like all the others. our comparisons demonstrated the existence of a degree of morphological variation in certain skeletal elements of these g. kuroiwae subspecies, which in some cases could be comparable to that between full eublepharid species (kluge, 1962; grismer, 1988; and other papers) and there seems to be further inter-subspecies variation in some elements of their skulls (y.n. unpublished data). although our systematic interpretation of the osteological variation is inconclusive due to the lack of prepared skulls of some g. kuroiwae subspecies and the appropriate outgroup samples, the osteological information is seemingly promising and should be incorporated into future taxonomic studies of this group. the discovery of goniurosaurus kuroiwae yunnu should be marked as a highly likely instance of a taxon-level anthropogenic extinction in insular squamate reptiles. the excellent state of preservation of the material and its association with modern debris strongly suggest that this animal survived until recently, whereas the complete absence of sampling or observation records of live animals from the island, despite recent repeated herpetofaunal surveys (see literature cited in maenosono and toda, 2007), supports the idea of complete extinction of this gecko subspecies in recent times. there is little doubt that such recent extinction on an island at the postmodern human colonization stage was related to human activities (case et al., 1998). unlike the cases of largesized lizards (pregill and worthy, 2003; pregill and steadman, 2004), the possibility of human exploitation can be discounted by the relatively small body size of the animal, although the remains were associated with artifacts. the majority of historic extinctions of insular lizards are attributable to predation by introduced mammalian predators (case et al., 1998). this may possibly be true with respect to g. k. yunnu, as well as at least three other species of yoronjima’s indigenous herptiles that disappeared after the 1960s, presumably as a result of predation by the exotic weasel, mustela itatsi, which was introduced to the island in the middle of the 1950s (nakamura et al., 2009, 2013). nevertheless, the contribution of other factors to the extinction, such as deforestation, remains open to discussion. on the other hand, there is a faint hope that this gecko still survives on yoronjima, as it is usually difficult to confirm the extinction of relatively small, nocturnal, and secretive animals, even in such small, topographi71new subspecies of goniurosaurus kuroiwae cally-uncomplicated and hence thoroughly surveyable islands. it is, however, hardly conceivable that for several decades this obligate terrestrial, slow-moving gecko, which had long been isolated on the originally carnivora-free island of yoronjima, has been able to elude the established weasel, which is swift, predominantly nocturnal (masuda and watanabe, 2009), and notorious for predation on lizards when introduced to islands (e.g., hasegawa, 1999; sekiguchi et al., 2002). our findings illustrate the susceptibility of g. kuroiwae to population extinction, a notion which has been suggested previously (e.g., tanaka, 1996, 2005d; ota, 2010). together with “rather incomplete” insular distribution of the species (grismer et al., 1994), the present results may imply the existence of other recently eradicated (but nevertheless unnoticed) insular populations of this species. documenting the missing pieces, particularly those wiped out by unnatural causes, demands priority, as it is a necessary complement to explain the diversity of the species and its relationship with the geohistory of the ryukyus. acknowledgements we thank seiji moriyama and the late shin-ichiro moriyama (yoron town) for permission to sample on their land; kiyotaka hatooka (omnh), takeshi sasaki (rumf), and mamoru toda (university of the ryukyus) for allowing us to dissect specimens in their care; takaki kurita (university of the ryukyus) for advice on the gender of the extant goniurosaurus kuroiwae specimens examined; kentaro hagari and motohiro okade for field assistance at the site; and thomas ziegler (cologne zoo, germany) for providing literature. we are grateful to two anonymous reviewers for their helpful comments on the earlier version of the manuscript. this study was partially supported by a grant-in-aid to the 21st century coe program at the university of the ryukyus from the ministry of education, culture, sports, science and technology, japan (monbu-kagaku-sho). references camp, c.l. 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(2005d): goniurosaurus kuroiwae kuroiwae. in: threatened wildlife in okinawa, second edition, animals, red data okinawa, pp. 111-113. okinawa prefectural government, ed, okinawa prefectural government, naha. (in japanese) tanaka, s., nishihira, m. (1989): growth and reproduction of the gekkonid lizard eublepharis kuroiwae kuroiwae. in: current herpetology in east asia, pp. 349-357. matsui, m., hikida, t., goris, r.c., eds, herpetological society of japan, kyoto. wang, y.-y., yang, j.-h., cui, r.-f. (2010): a new species of goniurosaurus (squamata, eublepharidae) from 73new subspecies of goniurosaurus kuroiwae yingde, guangdong province of china. herpetologica 66: 229-240. wang, y.-y., yang, j.-h., grismer, l.l. (2013): a new species of goniurosaurus (squamata: eublepharidae) from libo, guizhou province, china. herpetologica 69: 214-226. ziegler, t., nguyen, q.t., schmitz, a., stenke, r., rösler, h. (2008): a new species of goniurosaurus from cat ba island, hai phong, northern vietnam (squamata: eublepharidae). zootaxa 1771: 16-30. appendix specimens examined dissected specimens of goniurosaurus kuroiwae examined. acronyms are kuz = kyoto university museum zoological specimens, omnh = osaka museum of natural history, and rumf = fujukan, university museum of the university of the ryukyus. goniurosaurus kuroiwae kuroiwae—okinawajima (kunigami vil.): kuz r52392 (male), r65827 (gender unknown), r67952 (female), r71631 (male), r71909 (male). goniurosaurus kuroiwae orientalis—tokashikijima: kuz r71908 (male), rumf-zh-569 (female), 570 (female), 572 (male). goniurosaurus kuroiwae splendens—tokunoshima: kuz r34580 (female), r71533 (female), r71895 (male), r71896 (female), r71897 (female), r71898 (female). goniurosaurus kuroiwae toyamai—iheyajima: omnh r3974 (male), r3975 (female). goniurosaurus kuroiwae yamashinae—kumejima: rumf-zh-206 (male). acta herpetologica vol. 8, n. 2 december 2013 firenze university press journal of the societas herpetologica italica acta herpetologica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 6(1): 1-10, 2011 widespread bacterial infection affecting rana temporaria tadpoles in mountain areas rocco tiberti department of animal biology, university of pavia, via ferrata 1, i-27100 pavia, italy. e-mail: rocco. tiberti@unipv.it submitted on: 2010, 29th january; revised on:2010, 15th november; accepted on:2010, 30th december. abstract. periodic mass die-offs of rana temporaria tadpole populations have occurred in the ponds of prealpine mountain areas of brescia (northern italy) since the early 2000s. the author reports some observational data and analytical results from three sites: tadpoles from mortality events had erythema, especially on the legs, suggestive of septicemia. bacterial culture of these tadpoles revealed aeromonas hydrophila and aeromonas sobria, two organisms often associated with red leg disease. egg mass counts from 29 pastureland ponds did not revealed breeding activity declines over five years in the monte guglielmo area. aeromonas hydrophila and aeromonas sobria usually behave as opportunistic bacteria that can become pathogenic after suppression of the immune system by endogenous or exogenous stressors. thus, a plurality of environmental factors may contribute to mortality events; some of them are discussed, including loss of high altitude breeding ponds resulting in overcrowding and poor water quality in remaining ponds and the presence of other pathogens. keywords. mass die-offs, red leg, aeromonas sp., rana temporaria, tadpoles. introduction since 1990, an increasing number of studies have reported declines in amphibian populations (wake, 1991), and several contributing factors have been proposed (gardner, 2001), including disease (berger et al. 1998; daszak et al., 2000; kiesecker et al., 2001; kiesecker et al., 2004). therefore a lot of conservation efforts have been created by several organizations attempting to understand and monitor the decline. the need of taking full account of each episode of abnormal mortality has been emphasized. in the prealpine mountain areas of brescia (southern italian alps), mass die-offs among rana temporaria tadpoles have been reported since the early 2000s. the purpose of this text is documenting the occurrence of these mortality events using retrospective data from bagolino and odeno and new data from monte guglielmo massif. therefore, 2 r. tiberti from 2005 to 2009, in order to highlight any effect of mass die-offs on populations, rana temporaria breeding activity has been monitored in the monte guglielmo area by counting egg masses in 29 ponds and collecting biological samples, including live and dead tadples, cloacal swambs on adult animals and water samples. material and methods study area. all the samples have been collected in high altitude breeding ponds. usually the ponds are artificially dug in high altitude pastures for watering cows; ponds persistence depends on human maintenance. the studied ponds are shallow (from a few centimeters to just over one meter) and subject to strong daily and seasonal variations of chemical and physical parameters; because of cattle presence the ponds are regularly subject to nutrient pollution in summer time. the studied areas have a typical mountain climate and the ponds freeze during the winter; in a few cases, they dry at the end of summer. samples come from three distinct prealpine areas of brescia (northern italy): bagolino, odeno and monte guglielmo (fig. 1). the sampling sites cover a wide longitudinal range in the prealpine area of brescia. bagolino (lat.: 45°49’35’’; long.: 10°27’38’’; alt.: 769 m a.s.l.) is a village in the valley of the river caffaro on the right side of sabbia valley; odeno (lat.: 45°44’50’’; long.: 10°20’12’’; alt.: 917 m a.s.l.) is a hamlet of pertica alta in the sabbia valley; monte guglielmo is a rather isolated mountain range that straddles the watershed between trompia valley and camonica valley. there are no further information on the exact location of sampling site from bagolino and odeno, while accurate data are available for monte guglielmo as shown in fig. 1 and table 2. sampling and analytical methods. in order to closely observe any sign of disease, several frogs and tadpoles were captured using a landing net or with bare hands. some of the caught animals underwent further treatments or were sampled: five adult frogs underwent cloacal swamb sampling for bacterial examination; fecal samples were collected gently rubbing the perianal area of adult frogs using sterilized swambs with culture medium; treated animals were immediatly released. moreover several infected tadpoles (dead or still alive) were sampled; dead tadpoles were stored in test tube, while still alive tadpoles in containers allowing their survival until the refrigeration. finally, five water samples from the infected sites were collected for bacteriological analysis. all samples were preserved at low temperature (4 °c) and examined within one day in the izs (istituto zooprofilattico sperimentale) in brescia. each sample underwent bacteriological examination; parasitological and viral examinations were performed for the tadpoles sampled in monte guglielmo area. details of the analytical methods are given in the following paragraphs. parasitological examination. microscopic examinations were performed to identify several parasites: 1-2 drops of sample were diluted in lugol solution to observe flagellate and ciliate protozoans; nematodes and cestodes eggs were separated by flotation in a sodium nitrate satured solution; trematodes were separated by sedimentation and centrifugation and gross specimens were observed. bacteriological examination. since rana temporaria is an ectothermic organism, the sample was obtained from internal tissues and organs and was directly cultured on gassner agar and blood agar plates and stored at variable temperature for 24-48 hours. further, sub cultures were transferred from agar plates to differential and selective media and the morphology of the colony was observed; bacterial colonies were identified at the specific level thanks to specific biochemical test. enterobacteria and non-enterobacteria identification was performed thank to identification systems as api-20e and api-20ne. viral examination. viral examination with transmission electron microscopy (tem) were performed directly on aqueous suspensions of internal organs and tissues to find mature virions. the samples were repeatedly frozen and thawed to facilitate the release of viral particles in the aquehttp://en.wikipedia.org/wiki/caffaro_(river) http://en.wikipedia.org/wiki/valle_sabbia 3bacterial infection affecting rana temporaria tadpoles ous suspension. the suspension was centrifuged twice at low speed (6000 and 10000 rpm, for 20 minutes) to remove coarse debris; a small amount of the second supernatant was added to microtubes containing formvar-coated copper grids for tem and underwent ultracentrifugation (80000 rpm). the grid was drawn and coloured with 2% sodium phosphotungstate (napt) for 1.5 minutes and finally examined by tem. data analysis. since 2005 to 2009, the author had been looking for the effects of mass dieoffs on the abundance of rana temporaria populations. thus rana temporaria breeding activity in the monte guglielmo massif was monitored by counting egg masses in 29 ponds. the presence of 4 metapopulations of rana temporaria (a-d) was assumed to test for population declines; each metap   fig. 1. study area; (1): position on the alpine range; (2): position in the provincial administration of brescia; (3): studied ponds and groups of ponds (a-d) in monte guglielmo area. 4 r. tiberti opulation was sustainded by a group of ponds less than one kilometer far away one from another (fig. 1). three different linear mixed-effects model (lme) fitted by restricted maximum likelihood had been used to explore the effects of time on the number of egg-masses layed in the ponds (eggs), as a dependent variable. a time variable (year) and a binary variable standing for triturus carnifex presence (tricar) (which looked likely to be important in determining rana temporaria breeding sites) had been added as fixed effects. the group of ponds (group) had been added as random effect and each pond (pond) as nested random effect. the models were compared each other using a log-likelihood ratio test and the accuracy of the last model was verified by testing the normality of the residuals. results early signs of disease were observed in the advanced stage of larval development and rarely before the appearance of hind legs. initial signs of disease included pettechia and ecchymosis within the caudoventral and lateroventral body and tail. in the later stages of the disease, larvae swam slowly (lethargy) even when pursued by for capture. in the terminal stage of the disease, moribund individuals hung listlessly at the surface or at the bottom of the pond. the disease was highly contagious, often involving the entire tadpole population in the infected pond and most of the ponds, even in large areas. nevertheless adult rana temporaria or other anuran (bufo bufo) and urodele (triturus carnifex) species, table 1. summary table of examination results. mg: samples from monte guglielmo area; o: samples from pertica alta – odeno area; b: samples from bagolino area; cfu: colony-forming units; en dash: examination has not been performed. sample date area bacterial examination viral examination parasitological examination 1 6 dying tadpoles 26 june 2006 mg negative negative negative 2 6 died and 16 dying tadpoles 26 june 2006 mg aeromonas hydrophila infection negative negative 3 1 dying tadpole 03 july 2006 mg negative negatine negative 4 2 dying tadpoles 03 july 2006 mg aeromonas sobria infection negative negative 5 5 anal swambs 03 july 2006 mg negative 6 water 03 july 2006 mg aeromonas sp. 52000 cfu 7 water 03 july 2006 mg negative 8 3 dying tadpoles 17 june 2003 o negative 9 2 dead tadpoles 17 june 2003 o negative 10 2 dead tadpoles 17 june 2003 o aeromonas hydrophila infection 11 10 dying tadpoles 31 may 2004 o aeromonas sp. infection 12 10 helthy tadpoles 31 may 2004 o negative 13 water 26 may 2003 o aeromonas hydrophila 160000 cfu 14 water 26 may 2003 o aeromonas hydrophila 31000 cfu 15 tadpoles 04 june 2003 b aeromonas sp. infection 16 water 04 june 2003 b aeromonas hydrophila 13000 cfu 17 water 04 june 2003 b aeromonas sobria 16000 cfu 5bacterial infection affecting rana temporaria tadpoles cohabitating the same ponds, did not display clinical evidence of disease. five anal tampons were collected as confirmatory test on apparently healthy adult frogs and no pathological bacteria were found. the examination of died or dying tadpoles, from all the study sites, always highlights a negative response for viral and parasitological examination, while the bacterial examinations often pointed out septicaemic infections caused by motile aeromonas group (aeromonas hydrophila and aeromonas sobria) (table 1). similarly, the same pathogen was table 2. geographical data of ponds, rana temporaria egg masses counting (eggs) and triturus carnifex presence (tricar) in m. guglielmo area, since 2005 to 2009. lat: latitude; long: longitude; alt: altitude; na: not available data; x: triturus carnifex presence. pond lat long alt (m) 2005 2006 2007 2008 2009 eggs tricar eggs tricar eggs tricar eggs tricar eggs tricar a1 45°47’26’’n 10°11’57’’e 1434 0 x 13 x 0 x 0 x 0 x a2 45°46’58’’n 10°11’14’’e 1389 3 40 7 6 0 x a3 45°47’16’’n 10°11’29’’e 1472 14 30 x 53 30 70 a4 45°47’14’’n 10°11’33’’e 1505 6 15 48 115 73 a5 45°47’33’’n 10°11’36’’e 1562 27 6 99 22 79 a6 45°47’33’’n 10°11’36’’e 1652 na na x 20 1 28 a7 45°47’03’’n 10°11’11’’e 1371 na na 37 30 35 b1 45°45’43’’n 10°11’59’’e 1313 60 162 45 12 10 b2 45°45’35’’n 10°11’56’’e 1309 9 22 26 14 50 b3 45°45’28’’n 10°11’36’’e 1373 136 70 15 17 13 b4 45°45’22’’n 10°11’39’’e 1408 45 na 4 na 28 b5 45°44’43’’n 10°11’30’’e 1535 0 5 na 2 5 b6 45°44’53’’n 10°11’21’’e 1569 0 7 2 0 15 b7 45°45’14’’n 10°11’02’’e 1681 na 10 4 na 35 b8 45°44’55’’n 10°11’38’’e 1674 0 4 0 0 0 b9 45°44’60’’n 10°11’47’’e 1672 5 16 34 24 57 b10 45°44’48’’n 10°11’52’’e 1664 10 16 3 199 53 c1 45°46’46’’n 10°07’11’’e 1166 0 x 0 x 0 x 0 x 0 x c2 45°46’57’’n 10°07’16’’e 1175 0 x 0 x 0 x 0 0 x c3 45°46’40’’n 10°07’55’’e 1361 0 x 0 x 0 x 0 0 x d1 45°44’48’’n 10°09’52’’e 1564 17 30 0 0 1 d2 45°44’47’’n 10°09’52’’e 1563 6 4 1 0 1 d3 45°44’49’n 10°09’44’’e 1577 na 0 0 1 2 d4 45°45’14’n 10°09’44’’e 1734 18 7 na 0 0 d5 45°45’16’n 10°09’43’’e 1739 50 26 18 2 0 d6 45°45’20’n 10°09’46’’e 1760 na na na 0 0 d7 45°45’30’n 10°09’21’’e 1749 3 na na 6 7 d8 45°45’40’n 10°09’30’’e 1853 0 na na 0 0 d9 45°45’42’n 10°09’29’’e 1863 0 13 9 0 7 tot. 409 496 425 481 569 6 r. tiberti found in pond water samples by counting the number of bacterial colonies on a culture medium (table 1). testing the hypothesis that the number of egg masses per pond would have decreased from 2005 to 2009 (table 2), no significant relationship between eggs and year was found (table 3). in mod1 we set year and tricar as fixed effects and group as random effect. in mod2, pond has been added as nested random effect to test the effect of every pond on eggs. mod1 was compared with mod2 with a log-likelihood ratio test (lrt = 18.06, p < 0.0001). since the presence of triturus carnifex influenced the variance structure of mod2 (f = 22.16, p < 0.0001), mod2 was weighted on the tricar binary variable. the updated model (mod3) was compared with mod2 (lrt = 4.830825 , p < 0.05). the accuracy of mod3 was verified by testing the normality of the residuals (w = 0.99, p = 0.25). based on the models, the population of rana temporaria has not significantly declined since 2005; otherwise, the importance of triturus carnifex as a factor in disturbing rana temporaria breeding activity is detectable in all the three models. discussion the only pathogens isolated in the samples were aeromonas hydrophila and aeromonas sobria. aeromonas sp. occurs widely in fresh and estuarine waters (cahill, 1990; hazen et al., 1978; massa et al., 2001) and is considered a primary and secondary pathogen of aquatic and terrestrial animals. these bacterial pathogens can infect anuran (hubbard, 1981; carey, 1993; bradford, 1991; marquez et al., 1995; razzetti and bonini, 2001; taylor et al., 1999; nyman, 1986; hird et al., 1981; sherman and morton, 1993; colt et al., 1984; hayes and jennings, 1986; glorioso et al., 1974; rigney et al., 1978), urodeles (kaplan and glaczenski, 1965; boyer et al, 1971), fishes, reptiles, birds, and mammals, including humans (panigrahy et al., 1981; cahill, 1990; gorden et al., 1979; davis et al., table 3. fixed effect results from mixed models testing the hypothesis that number of egg masses per pond will decrease since 2005 to 2009. the groups of pond and the ponds were included as a random effect and nested random effect in these models. ns: not significant. model source of variation beta df f p mod1 intercept 149.95 121 13.4 < 0.001 year -0.07 121 0.5 ns tricar -1.83 121 17.0 <0.001 mod2 intercept 116.35 96 11.5 <0.001 year -0.06 96 0.6 ns tricar -1.26 96 7.1 <0.01 mod3 intercept 147.74 96 11.7 <0.001 year -0.07 96 1.3 ns tricar -1.26 96 9.5 <0.01 7bacterial infection affecting rana temporaria tadpoles 1978). aeromonas hydrophila is one of the pathogenic agents of red leg disease. gross signs observed during mortality events within the prealpine study sites included ettechia and ecchymosis within the caudoventral and lateroventral body and tail, lethargy and coma and were consistent with those reported for red leg disease (nace, 1968; marquez et al., 1995; nyman, 1986; hird et al., 1981; carey, 1993). however, aeromonas sp. is not the only agent in this pathology, which is the result of a complex interaction between other gram negative bacteria including pseudomonas spp., proteus spp., flavobacterium indologenes and f. meningosepticum (hubbard, 1981; taylor et al., 1993; anver and pond, 1984). moreover, aeromonas sp. presence by itself does not necessarily give evidence of disease; these bacteria can live in free waters, on the skin and in the digestive tract of amphibians without causing any disease (hubbard, 1981; carey, 1993; hird et al., 1981). consequently, even if aeromonas sp. is also considered a primary pathogen, several stressors are often involved in outbreaks of disease and aeromonas sp. is considered to behave as an opportunistic pathogen, infecting immunodepressed hosts. for example carey (1993) reported that aeromonas hydrophila infection occurred in bufo boreas after the immune system of the amphibians was suppressed by environmental factors. natural and anthropogenic stressors, including pre-existing diseases, may be involved in the occurrence of red leg; as a matter of fact, aeromonas sp. can behave as a secondary pathogen and its irregular isolation in the samples (table 1) might suggest the presence of other primary pathogens. moreover, the occurrence of the same above mentioned dieoffs with gross signs of systemic hemorrhagic disease are considered to be indicators of viral infection (gray et al., 2009; converse and green, 2005; cunningham et al., 1996 ) and although aeromonas sp. can cause red leg disease, retrospective studies showed that ranaviruses are also often present and may be the primary pathogen. nevertheless, viral examination seems to rule out ranaviruses presence at least in the samples from monte guglielmo, even if no confirmatory diagnosis (pcr and immunohistochemistry methods) (hyatt et al., 2000; reddacliff and whittington, 1996) has been performed and the examined samples were few. additionally, gross lesions, similar to the observed ones, were pointed out in diseases caused by batrachochytrium dendrobatidis in adult amphibians (green and converse 2005). however chytridiomycosis is a fatal disease of post-metamorphic frogs infecting keratinized skin of adults and can be ruled out as primary pathogen of the observed mass die offs; since tadpoles have keratin only in mouthparts, chytrid infection on larval anurans commonly results in reduced developmental rate and foraging efficiency (venesky et al., 2010) without causing mass die-offs. lots of other factors may be implicated in determing the pathogenicity of aeromonas sp. for example, during the last decades farmers reduced the number of cows in the mountain areas and consequently the demand for water; mountain ponds were often abandoned and underwent a drying process. this may result in a more extreme habitat for tadpoles. still existing ponds are subject to wider depth fluctuations and the reduced basin volume is worsened by stronger seasonal and daily variations, resulting in increased total solids, eutrophication and tadpoles crowding; everyone of these factors can potentially suppress the tadpoles immune system and facilitate the disease. for instance, the abandonment of mountain ponds can affect the immune system of tadpoles in the wild and this process is potentially linked with diurnal oxygen supersaturation which can occur in eutrophic ponds: evaluating the importance of dissolved gas, colt et al. (1984) exposed rana catesbeiana tadpoles to supersaturated water causing signs of gas bubble disease followed by red leg disease. 8 r. tiberti finally, several limitations should be pointed out in the current study, since the obtained results show that it is not possible to state unequivocally whether aeromonas sp. acts as a primary pathogen or whether there are pre-existing environmental or infective stressors in the studied populations. several unexplored factors could be involved in the observed mass die-offs, weakening the immune system of tadpoles; even some short-term acute stressors can induce immunosuppression lasting for days (ellsaesser and clem, 1986; pickering et al., 1982; carey, 1993), making even more difficult to determine conclusively which environmental factor (or which combination of environmental factors) may have caused a sufficient degree of stress to induce disease in the prealpine populations. however, in monte guglielmo area, the number of egg masses has not significantly declined since 2005 and the consequences of the disease were not found in a reduced breeding activity of rana temporaria, suggesting that this species is able to withstand high larval mortality occurred in the last years. regardless, evidence of widespread mass die-offs in prealpine areas has been reported since the early 2000s and previous declines should not be ruled out. the measured abundance could be the result of a decline lasting for years and reducing the population to the current minimum. finally, the extent of the phenomenon and the amplitude of the affected area should arouse the interest for other conservation issues, such as the health and the consevation status of other amphibians living in the same areas (including bombina variegata and triturus carnifex, listed in annex ii of the habitat directive) and the preservation of important habitats such as high altitude ponds. acknowledgements i am particularly grateful to mr. giuseppe tomasini and dr. franco panunzi for their kind help in data collection. i want to thank prof. giuseppe bogliani for his support, dr. cristian pasquaretta for the precious help in data analysis and tito tiberti for his kind proofreading of the text. i thank the comunità montana of valle trompia and the provincial administration of brescia. sample examination would not have been possible without the contribution of the izs (istituto zooprofilattico sperimentale) of brescia. references anver, m.r., pond, c.l. 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(1991): declining amphibian populations. science 253: 860. http://www.ncbi.nlm.nih.gov/pubmed/20135192 http://www.ncbi.nlm.nih.gov/pubmed/20135192 bbib67 ole_link1 ole_link2 bbib28 ole_link5 ole_link6 ole_link7 ole_link8 _goback ole_link1 ole_link2 ole_link3 ole_link4 ole_link1 ole_link2 ole_link19 ole_link20 ole_link21 ole_link29 ole_link3 ole_link4 ole_link5 ole_link31 ole_link14 ole_link15 ole_link12 ole_link13 ole_link16 ole_link17 ole_link22 ole_link23 ole_link24 ole_link8 ole_link9 ole_link10 ole_link11 ole_link18 ole_link27 ole_link28 ole_link25 ole_link26 ole_link6 ole_link7 ole_link34 ole_link37 ole_link38 acta herpetologica vol. 6, n. 1 june 2011 firenze university press widespread bacterial infection affecting rana temporaria tadpoles in mountain areas rocco tiberti extreme feeding behaviours in the italian wall lizard, podarcis siculus massimo capula1, gaetano aloise2 lissotriton vulgaris paedomorphs in south-western romania: a consequence of a human modified habitat? severus d. covaciu-marcov*, istvan sas, alfred ş. cicort-lucaciu, horia v. bogdan body size and reproductive characteristics of paedomorphic and metamorphic individuals of the northern banded newt (ommatotriton ophryticus) eyup başkale1, ferah sayım2 , uğur kaya2 genetic characterization of over hundred years old caretta caretta specimens from italian and maltese museums luisa garofalo1, john j. borg2, rossella carlini3, luca mizzan4, nicola novarini4, giovanni scillitani5, andrea novelletto1 the phylogenetic position of lygodactylus angularis and the utility of using the 16s rdna gene for delimiting species in lygodactylus (squamata, gekkonidae) riccardo castiglia*, flavia annesi localization of glucagon and insulin cells and its variation with respect to physiological events in eutropis carinata vidya. r. chandavar1, prakash. r. naik2* the balearic herpetofauna: a species update and a review on the evidence samuel pinya1, miguel a. carretero2 effects of mosquitofish (gambusia affinis) cues on wood frog (lithobates sylvaticus) tadpole activity katherine f. buttermore, paige n. litkenhaus, danielle c. torpey, geoffrey r. smith*, jessica e. rettig food composition of uludağ frog, rana macrocnemis boulenger, 1885 in uludağ (bursa, turkey) kerim çiçek preliminary results on tail energetics in the moorish gecko, tarentola mauritanica tommaso cencetti1,2, piera poli3, marcello mele3, marco a.l. zuffi1 climate change and peripheral populations: predictions for a relict mediterranean viper josé c. brito1, soumia fahd 2, fernando martínez-freiría1, pedro tarroso1, said larbes3, juan m. pleguezuelos4, xavier santos5 assessing the status of amphibian breeding sites in italy: a national survey societas herpetologica italica* osservatorio erpetologico italiano acta herpetologica journal of the societas herpetologica italica acta herpetologica rivista della societas herpetologica italica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 8(1): 59-63, 2013 no detection of chytrid in first systematic screening of bombina variegata pachypus (anura: bombinatoridae) in liguria, northern italy stefano canessa1,*, an martel2, frank pasmans2 1 arc centre of excellence for environmental decisions, school of botany, university of melbourne, 3010 vic, australia. *corresponding author. e-mail: canessas@unimelb.edu.au 2 faculty of veterinary medicine, ghent university, merelbeke 9820, belgium submitted on: 2012, 7th decembre; revised on: 2013, 10th april; accepted on: 2013, 19th april. abstract. the apennine yellow-bellied toad bombina variegata pachypus, a small anuran endemic to peninsular italy, has been declining throughout its range over the last 30 years. although mortality by chytridiomycosis, caused by the fungus batrachochytrium dendrobatidis, was first reported for the species in 2004, its role in the decline has not yet been assessed. between 2011 and 2012 we sampled eight populations of b. v. pachypus in liguria, northern italy, swabbing 86 and 143 individuals respectively, corresponding to between 24 and 80% of the estimated individuals within each population. we did not detect chytrid in any the samples collected. for the three largest populations in the region, we can rule out infections of prevalence greater than 10% with at least 98% confidence. research at a larger scale is urgently needed to clarify the role of b. dendrobatidis in the decline of this and other amphibians in italy. keywords. bombina, chytrid, confidence, liguria, monitoring, population size, prevalence. the amphibian chytrid fungus batrachochytrium dendrobatidis is the main pathogen driver of the global amphibian decline (fisher et al., 2009). die-offs and extinctions have occurred in all continents (berger et al., 1998; conradie et al., 2011; swei et al., 2011) including several cases in europe (garner et al., 2005). in italy, infection by chytridiomycosis has been documented for several species in the last decade (reviewed in tessa et al., 2013): further information on the distribution of b. dendrobatidis in italy is needed to understand the causes of local and regional declines and inform conservation efforts. the apennine yellow-bellied toad bombina variegata pachypus (anura: bombinatoridae) was the first amphibian species in italy with confirmed mortality by chytridiomycosis, reported by stagni et al. (2004) on captive individuals caught from three populations in the northeastern apennines. recent evidence suggests chytrid is present in populations of b. v. pachypus along the italian peninsula, with infections dating back as early as the late 1970s (canestrelli et al., 2013). b. v. pachypus has declined steeply over the last 30 years, with widespread local disappearances: although habitat loss is believed to represent the main driver of the decline (mirabile et al., 2009; canessa et al., 2013), chytridiomycosis is accepted as a potential threat to the species (see for example its red list assessment in iucn, 2011). however, to our knowledge, no systematic assessment of the spread of chytrid among populations of b. v. pachypus in italy has been undertaken to date. in order to clarify this issue at least in part, we conducted surveys throughout the northern italian region of liguria in 2011 and 2012. a recent assessment of the status of b. v. pachypus in this area showed that over 50% of the known populations disappeared between 2001 and 2010: habitat loss relating to the abandonment of traditional farming practices has been suggested as the main cause of decline (canessa et al., 2013). a small-scale survey (five individuals tested) carried out in 2005 at a single site in the region failed to detect any presence of b. dendrobatidis (adams et al., 2008). 60 stefano canessa et al. in 2011, between 31 july and 4 august we visited all seven known breeding sites of b. v. pachypus in liguria, located within the catchments of the lavagna and vara rivers in the eastern part of the region, encompassing an area of approximately 800 km2. in 2012, we repeated our visits at five of these seven populations: the three sites believed to host the largest populations were sampled three times during the 2012 season in order to maximize the overall proportion of the population that could be tested (22 june, 24 july and 26 july). we also tested all the animals present at a small captive breeding centre within the study area: these individuals had been caught in the wild and kept in a semi-natural enclosure with no contact with other amphibians during the study period. we captured all detected individuals by hand and photographed them to record their unique ventral pattern: this allowed us to calculate the number of unique individuals tested in addition to the total number of swabs collected at sites that were visited multiple times. to minimize the chance of spreading pathogens, we followed recommendations by speare et al. (2004) when handling individuals, and sites within different catchments were never sampled during the same day. we collected samples as described by hyatt et al. (2007) using cotton-tip swabs and then released individuals at the point of capture. samples were stored at –4 °c until further analysis. we only tested post-metamorphic individuals: although pathogen prevalence can be higher in tadpoles, facilitating detection, we chose to avoid any impact to the few remaining populations of the species in the region that could result from applying lethal or nonlethal sampling to larvae. swabbing is less sensitive than other methods, and can still damage tadpole mouthparts and facilitate infections in addition to stress resulting from handling (retallick et al., 2006). b. dendrobatidis dna in the samples, real-time taqman pcr assays were conducted on a cfx96 real time system (biorad). amplification conditions, primer and probe concentrations were according to boyle et al. (2004). due to economic constraints, samples were run in duplicate. consistent results between the two runs were classified as correct: we considered a test result as positive where both qpcr runs gave results above 0.1 ge, with standard deviation smaller than the average of both repeats and suitable amplification curves. after obtaining the test results, we estimated the mean prevalence (with 95% credible intervals) of the pathogen in the population using a bayesian equal-tailed jeffrey prior (brown et al., 2001). to assess the sensitivity of our sampling program, we then calculated the probability of detecting 5, 10 and 30% infections with the number of individuals sampled at each site, by solving c = 1 – (1 – p)n where c is the probability of detecting at least one infected individual from n tested in a population with p prevalence of the disease (digiacomo and koepsell, 1986). additionally, for the three largest populations in the study we were able to use an existing multi-year markrecapture database to estimate population size at the time of sampling (canessa, unpubl. data). to account for open-population dynamics such as temporary emigration, often observed for b. variegata (hartel, 2008), we used an open-population jolly-seber model (wagner et al., 2011). we fit the model in a bayesian framework using dateand population-dependent recapture rates (code in kéry and schaub, 2011). we used jags (plummer, 2005) to obtain samples from three markov chains over 100,000 iterations, after discarding the first 10,000 as a burn-in. in 2011, we collected 81 swabs from 81 individuals in seven wild populations, corresponding to between 24 and 42% of the estimated sizes of respective populations (as estimated from 95% confidence intervals from the jollyseber model): at two sites we captured only one and four individuals respectively (table 1). in 2012, we captured and swabbed a total of 143 individuals at four of the wild locations on four separate occasions, corresponding to between 66 and 80% of the estimated sizes of the tested populations. several individuals were re-captured and swabbed in two or three occasions in 2012, yielding a total of 224 swabs for that season. all individuals at the captive breeding centre were also caught and tested (four in 2011 and three in 2012): we did not calculate confidence intervals or sampling power for this site. all samples tested negative for b. dendrobatidis. we captured more than 10 individuals only at four sites: in 2011, this resulted in upper 95% confidence intervals (ci) of prevalence between 0.09 and 0.13 (table 1). for three of these populations, in 2012 we were able to reduce the upper cis to 0.05 or less; at the fourth site, only seven individuals were captured in 2012, resulting in an upper ci of 0.23. for the three most intensively sampled populations, we can be at least 97% confident that chytrid would have been detected at these populations for a prevalence of at least 10%, and more than 83% confident for a prevalence of at least 5% (table 1). for the smaller populations sampled (less than 10 individuals), we could make no reliable inference regarding the prevalence of infection, although in sites with more than one capture, the probability of having missed large infections (> 30% prevalence) was still lower than 50%. although reliable estimates of abundance could not be obtained for the smaller populations in the study, we 61no detection of chytrid in ligurian b. variegata observed relatively high recapture rates (> 40%) for the larger populations, suggesting the small sample sizes at some sites might reflect actual abundances. nevertheless, the estimates we present for populations with less than 10 sampled individuals can only be considered as preliminary and do not allow reliable inference. for the largest populations in the region the large sample sizes, particularly for 2012, allows us to rule out with sufficient confidence infections of prevalence greater than 10%. additional information about the expected prevalence of chytrid infection in b. v. pachypus is needed to make more precise inference: for example. canestrelli et al. (2013) recently found that prevalence of b. dendrobatidis in present and historical specimens of b. v. pachypus across the italian peninsula we never below 12%. amongst populations of b. variegata in hungary, the infection rate has been estimated between 12% and 29% (vörös, pers. comm.). in the netherlands and flanders (belgium), spitzen-van der sluijs et al. (2010) reported a prevalence of infection of 5.9% for b. variegata out of 255 individuals sampled. holding this relatively high base rate as a guideline, the failure to detect chytrid in the ligurian populations may indicate lower susceptibility due to local conditions, bias related to the date of sampling (patterns of chytrid infection are highly seasonal: see kriger and hero, 2007), or possibly actual absence of the pathogen from the region at present. in addition, breeding sites of b. v. pachypus in liguria have been regularly monitored throughout the breeding season since 2008 and no obvious sign of chytrid infection (e.g. individual mortality or abnormal behaviour) has ever been reported (arillo et al., 2009; arillo et al., 2011). it is difficult to infer whether chytridiomycosis might have had a role in past local extinctions in the region. most disappearances in the region in recent years have occurred at sites in agricultural areas (canessa et al., 2013). however, the existing literature suggests such sites might be less likely to host permanent foci of chytrid infection, due to their low elevation and open vegetation types, resulting in higher water temperatures (> 30 °c in august) and regular desiccation, unsuitable for the pathogen (johnson et al., 2003). for example, hyne et al. table 1. results for all screened populations of b. v. pachypus in 2011 and 2012.     population fav lo1 lo2 lop pav per pin tev 2011 total swabs1 4 17 21 8 17 1 13 1 individuals swabbed2 4 17 21 8 17 1 13 1 population size3 68 ± 2 67 ± 2 35 ± 4 47 ± 3 % tested4 25.0 31.3 48.6 27.7 prevalence (95% ci)5 0.36 0.11 0.09 0.21 0.11 0.77 0.13 0.77 detection p = 0.056 0.19 0.58 0.66 0.34 0.58 0.05 0.49 0.05 p = 0.17 0.34 0.83 0.89 0.57 0.83 0.10 0.75 0.10 p = 0.38 0.76 1.00 1.00 0.94 1.00 0.30 0.99 0.30 2012 total swabs1 106 60 7 51 individuals swabbed2 55 43 7 35 population size3 69 ± 2 69 ± 2 35 ± 4 48 ± 2 % tested4 79.7 62.3 20.0 72.9 prevalence (95% ci)5 0.04 0.04 0.23 0.05 detection p = 0.056 0.94 0.89 0.30 0.83 p = 0.17 1.00 0.99 0.52 0.97 p = 0.38 1.00 1.00 0.92 1.00 1 total number of swabs collected 2 total numbers of unique individuals swabbed, where repeated visits were carried out (lo1, lo2 and pin in 2012) 3 population size at the time of sampling (± standard deviation), estimated using the jolly-seber open population model – a dash indicates no estimate could be made for that population/year 4 proportion of the estimated population size (mean) that was tested 5 upper 95% equal-tailed jeffrey prior confidence interval (lower interval is 0 in all cases). 6, 7, 8 probability of detecting at least one infected individual if the pathogen were present in the population with a prevalence of 0.05, 0.1 and 0.3 respectively. 62 stefano canessa et al. (2009) found lowland species persisting without noticeable population declines in the presence of chytrid in an agricultural landscape with relatively warm climate. conversely, particularly high impacts of chytrid worldwide have occurred in forest areas with permanent hydroperiods and cooler mesoclimates (berger et al., 1998): all remaining large populations of b. v. pachypus in liguria occur in such areas and therefore require special attention. at the moment, monitoring must continue across all known sites to ensure prompt detection of possible outbreaks. quantifying the efficiency of monitoring (e.g. capture rates of individuals and test sensitivities) can help inform screening programs to achieve the best results for the effort invested (lachish et al., 2012). although b. v. pachypus is recognized as an endangered species in italy, this is, to our knowledge, the first systematic screening of any of its populations for b. dendrobatidis. conservation plans for the species include habitat restoration and ex-situ initiatives (di cerbo and ferri, 2002; gentilli et al., 2002; arillo et al., 2011). additional information about the role of chytridiomycosis in the decline of b. v. pachypus is urgently needed, both to inform actions (for example, habitat restoration may not be sufficient where disease is the main threat) and to raise awareness of the need to implement safety protocols (phillott et al., 2010). in general, further research about the distribution and prevalence of chytrid amongst amphibian species in italy should be seen as a priority, particularly in areas of known and ongoing declines. surveys at the local and regional scale, whilst relatively inexpensive, can provide valuable information about the spread and impact of the pathogen and help optimize conservation strategies. acknowledgements we thank a. morán ordoñez, f. oneto, d. ottonello, p. piana and s. salvidio for help with surveys. comments from two anonymous referees helped improve an earlier version of the manuscript. capture and handling permits granted by the ministero dell’ambiente e della tutela del territorio (dpn2010-370, 12-01-297 2010). field work was supported by funding from the societas europaea herpetologica and the society for the study of amphibians and reptiles. manuscript preparation was supported by the university of melbourne and the arc centre of excellence for environmental decisions. references adams, m.j., galvan, s., scalera, r., grieco, c., sindaco, r. 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(2011): the superpopulation approach for estimating the population size of ’prolonged’breeding amphibians: examples from europe. amphibia-reptilia 32: 323-332. acta herpetologica vol. 8, n. 1 june 2013 firenze university press the oogenic cycle of the caspian bent-toed gecko, cyrtopodion caspium (squamata: gekkonidae) in iran vida hojati1*, kazem parivar2, eskandar rastegar-pouyani3, abdolhossein shiravi1 altitudinal effects on life history parameters in populations of liolaemus pictus argentinus (sauria: liolaemidae) joel antú gutiérrez1,*, carla piantoni2, nora r. ibargüengoytía1,3 recent cryptic extinction of squamate reptiles on yoronjima island of the ryukyu archipelago, japan, inferred from garbage dump remains yasuyuki nakamura1,*, akio takahashi2, hidetoshi ota3 trophic niche and feeding biology of the italian wall lizard, podarcis siculus campestris (de betta, 1857) along western mediterranean coast marco a.l. zuffi*, chiara giannelli novel, non-invasive method for distinguishing the individuals of the fire salamander (salamandra salamandra) in capture-mark-recapture studies goran šukalo1,*, sonja đorđević2, dragojla golub1, dejan dmitrović1, ljiljana tomović2,3 going out tonight? when insular hierophis viridiflavus breaks the whip snakes rules delaugerre michel-jean first evidence of a paedomorphic population of the smooth newt (lissotriton vulgaris) in the czech republic václav gvoždík1,2, veronika javůrková4, oldřich kopecký5,* no detection of chytrid in first systematic screening of bombina variegata pachypus (anura: bombinatoridae) in liguria, northern italy stefano canessa1,*, an martel2, frank pasmans2 status of the european pond turtle, emys orbicularis (reptilia: testudines: emydidae) in vorarlberg, austria andreas kleewein1, günther wöss2 comparative cytogenetics of two species of ground skinks: scincella assata and s. cherriei (squamata: scincidae: lygosominae) from chiapas, mexico riccardo castiglia1,*, alexandra m.r. bezerra2, oscar flores-villela3, flavia annesi1, antonio muñoz4, ekaterina gornung1 polydactyly in the tyrrhenian wall lizard (podarcis tiliguerta) antigoni kaliontzopoulou1,*, daniele salvi1, verónica gomes1, joão p. m. c. maia1,2,3, panagiotis kaliontzopoulos4 book review: roberto sindaco, alberto venchi, cristina grieco. the reptiles of the western palearctic. 2. annotated checklist and distributional atlas of the snakes of europe, north africa, middle east and central asia, with an update to the vol. 1. edoardo razzetti acta herpetologica journal of the societas herpetologica italica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 7(1): 155-162, 2012 loggerhead turtles (caretta caretta) foraging at drini bay in northern albania: genetic characterisation reveals new haplotypes can yilmaz1, oguz turkozan1,*, fevzi bardakci1, michael white2, esmeralda kararaj3 1 adnan menderes university, faculty of science and arts, department of biology, 09010 aydin, turkey. * corresponding author. e-mail: turkozan@adu.edu.tr 2 mediterranean association to save the sea turtles, 1c licavitou street, athens, greece. 3 school of biological sciences, tirana university, tirana, albania. submitted on: 2011, 24th november; revised on: 2012, 5th january; accepted on: 2012, 6th february. abstract. loggerhead turtles (caretta caretta) was studied over 3 summers in a nearshore habitat, the patoku area in the southern part of driniki bay, albania. tissue samples were collected from 40 loggerhead turtles incidentally captured in stavnike fish-traps (a type of pond-net). a fragment of 859 base-pair mt-dna d-loop region was amplified from these turtles and compared with previously described haplotypes. haplotype cc-a2.1 (93%) was the dominant haplotype in the region. two previously unknown haplotypes, cc-a6.1 and cc-a10.4, were described with this study. furthermore, haplotype cc-a.2.8 was also observed which was previously recorded from italy. haplotype and nucleotide diversity were 0.14615 and 0.00017, respectively. keywords. caretta caretta, foraging ground, mitochondrial dna, haplotype, albania introduction during their life history, loggerhead turtles (caretta caretta; linnaeus 1758) generally experience two different ecological stages, oceanic and neritic (bolten, 2003). the oceanic stage includes the time from when the hatchlings leave the nesting beach until they return to coastal benthic foraging habitats as juveniles (bolten & balazs, 1995). according to mcclellan and read (2007), the shift from oceanic to neritic waters is ‘’complex and reversible’’ and therefore is not a discrete ontogenetic shift. although these stages have long been studied across the distributions of loggerhead turtles, in recent decades, molecular tools have become very helpful for the detection of developmental migrations (bolten et al., 1998) and determination of stock compositions and in foraging and wintering areas. 156 c. yilmaz et al. drini bay in northern albania is one of the important foraging grounds for loggerhead turtles in the adriatic sea (white et al., 2011). in this study, we collected tissue samples from loggerhead turtles in a nearshore habitat in the southern part of drini bay (fig. 1) to generate preliminary information about the genetic composition of the foraging population. this is the first genetic characterization study in this area using a fragment of the d-loop mtdna. material and methods sea turtles were studied over three summers (2008-2010) by medasset (mediterranean association to save the sea turtles). dna samples were collected in 2009-2010 from 40 loggerhead turtles in a nearshore habitat, the patoku area in the southern part of drini bay (fig. 1). nearly all turtles (99%) were incidentally captured in ‘stavnike’ fish-traps (a type of pound-net), and subsequently measured and tagged. it was only possible to collect biopsies from a small number of captured turtles due to limited research funds. preference was given to larger turtles, as there may be a fig. 1. the map showing the study area 157new haplotypes in the loggerhead turtles foraging at drini bay better chance that their dna matches existing haplotypic information obtained from nesting beaches (encalada et al., 1998; laurent et al., 1998; carreras et al., 2007; garafalo et al., 2009; yılmaz et al., 2011). all haplotypic data on loggerhead and green turtles are housed in the archie carr centre for sea turtle research database (http://accstr.ufl.edu/). in this database there two forms of haplotypic data occurs, shorter (380bp) and longer (over 800bp). the longer haplotypes named according to their similarity to shorter haplotypes till 380 bp. if the longer form is identical to shorter haplotypes until 380 bp and differ after it these longer haplotypes are called as the variants of shorter haplotypes. bowen et al. (2004) stated that nesting populations contribute more to neighbouring mixed stocks than to more distant mixed stocks. the mediterranean region, therefore, is the possible source of turtles foraging in drini bay. we therefore compared our results with other shorter d-loop sequences available for this species for the mediterranean (encalada et al., 1998; laurent et al., 1998; carreras et al., 2007; garafalo et al., 2009; yılmaz et al., 2011). adult and juvenile males were also included in the sampled animals because they represent a significant portion of this foraging population (white et al., 2011). laparoscopy or hormonal analysis was unavailable, so sex was determined by using ccl (curved carapace length) measurements and tail morphology. adult males have a greatly extended tail enabling copulation to occur while juvenile males show signs of caudal development the tail widening proximally and extending distally (m. white pers. com. 2012). the minimum ccl for adults was selected as being 70.0 cm although two adult males were slightly smaller than this (white, unpublished data). short-tailed turtles with a ccl greater than 70.0 cm were assumed to be adult females. sex of the smaller short-tailed turtles could not be determined, as these could be juveniles of either sex. tissue samples were taken from the axillary region near the anterior limbs. the sampling site was wiped with antiseptic (betadine) prior to excision with a scalpel blade of a small section of skin (1 mm2). the sample was then placed into a tube with 96% ethanol, which was changed after 10 days. morphometric data (ccl and ccw, curved carapace length and width, respectively) were also collected from all captured turtles following bolten (1999). date of capture and the sex of sampled turtles were also recorded. lab work isolation of genomic dna was carried out using the phenol-chloroform method (hillis et al., 1996). a fragment of 859 base-pair (bp) of the mtdna d-loop region was amplified by polymerase chain reaction (pcr mastercycler personnel, eppendorf, germany) using the primer pair lcm15382 and h950 (abreu-grobois et al., 2006). the pcr protocol was carried out over 35 cycles at 94 oc for 30 seconds, 55 oc for 1 min and 72 oc for 1 min. pcr products were visualized in agarose gel and purified with the genelute pcr clean-up kit, (sigma, germany). purified pcr products were sequenced in both forward and reverse directions using a 3730xl capillary system automatic sequencer (macrogen inc., s. korea). sequences were aligned by eye using the programme bioedit ver 7.0.9 (hall, 1999) and compared with previously described haplotypes recorded in the archie carr centre for sea turtle research database (http://accstr.ufl.edu/). in addition, previous data from other sites in the mediterranean were also included for a general assessment of mediterranean populations. results the mean ccl of the sampled turtles was 68.8 cm (sd = ± 10.3 cm; range = 32.084.5 cm; n = 40). haplotype cc-a2.1 (93%) was the dominant haplotype obtained in the 158 c. yilmaz et al. table 1. data on the loggerhead turtles captured at patoku region of albania (novel haplotypes are in bold). date ccl ccw sex tag haplotype 7/22/09 72.0 67.0 adult male al0112 cc-a2.1 7/22/09 61.0 56.0 undetermined al0113 cc-a2.1 7/22/09 57.0 53.0 undetermined al0114 cc-a2.1 7/22/09 64.0 58.5 undetermined al0115 cc-a2.1 7/23/09 82.0 72.0 adult male al0116 cc-a2.1 7/24/09 58.0 53.5 juvenile male al0117 cc-a2.1 7/24/09 67.0 64.0 undetermined al0118 cc-a2.1 7/28/09 32.0 30.0 undetermined no tag cc-a2.1 7/29/09 55.5 51.0 undetermined al0119 cc-a2.1 7/29/09 65.0 64.0 juvenile male al0120 cc-a2.1 7/29/09 72.0 64.0 juvenile male al0121 cc-a2.1 7/29/09 64.5 58.0 undetermined al0122 cc-a2.1 7/30/09 59.0 59.0 undetermined al0123 cc-a2.1 7/31/09 70.0 68.0 adult female al0124 cc-a2.1 7/31/09 66.5 62.0 juvenile male al0125 cc-a2.1 7/31/09 72.0 66.0 adult female al0126 cc-a2.1 7/31/09 74.0 69.0 adult female al0127 cc-a2.1 7/31/09 74.5 71.0 juvenile male al0128 cc-a2.1 8/1/09 73.0 67.5 adult female al0129 cc-a2.1 8/1/09 68.0 61.5 juvenile male al0130 cc-a2.1 8/1/09 70.0 64.0 juvenile male al0131 cc-a2.1 8/2/09 47.5 43.5 undetermined al0132 cc-a2.1 8/2/09 65.5 59.0 undetermined al0133 cc-a6.1 8/2/09 74.5 70.5 adult male al0134 cc-a2.1 8/3/09 72.0 69.5 juvenile male al0136 cc-a10.4 8/4/09 76.5 72.0 adult female al0137 cc-a2.1 8/4/09 68.0 64.0 juvenile male al0138 cc-a2.1 8/5/09 73.0 64.5 adult male al0140 cc-a2.1 6/23/10 84.0 78.0 adult female al0204 cc-a2.1 6/23/10 84.5 77.0 adult male al0222 cc-a2.1 6/30/10 83.0 75.0 adult female al0228 cc-a2.8 6/28/10 73.0 70.0 juvenile male al0229 cc-a2.1 6/28/10 70.5 65.0 juvenile male al0230 cc-a2.1 6/28/10 66.0 61.0 juvenile male al0231 cc-a2.1 6/28/10 73.5 65.0 adult male al0233 cc-a2.1 7/14/10 76.0 68.0 adult female al0245 cc-a2.1 7/14/10 82.0 72.0 adult male al0246 cc-a2.1 7/17/10 55.5 52.0 undetermined al0254 cc-a2.1 7/30/10 70.0 65.0 adult female al0274 cc-a2.1 7/30/10 78.0 74.5 adult female al0278 cc-a2.1 159new haplotypes in the loggerhead turtles foraging at drini bay region sampled for this study (table 1). however, two previously unknown haplotypes (according to the archie carr database mentioned above) were also found (cc-a6.1 and cc-a10.4; genbank accession numbers jq350705 and jq350706, respectively). the last observed haplotype, cc-a.2.8 was first identified from a juvenile stranded in puglia (south adriatic sea, italy) in may 2008 (garafalo, 2010). haplotype and nucleotide diversity were 0.14615 and 0.00017, respectively. discussion the haplotype with the highest frequency at the study site was cc-a2.1, which is not surprising since it is a variant of the short haplotype cc-a2; the most common loggerhead haplotype in the mediterranean (table 2). the short forms of novel haplotypes cc-a6.1 and cc-a10.4 are variants of cc-a6 and cc-a10; which have only been recorded from greek islands and greece (table 2). thus, it is probable that the loggerheads in drini bay mainly originate from nesting populations in greece. the female tagged at patoku (tag # al0127) that nested at sekania (margaritoulis, pers. com.), zakynthos, in july 2011, supports this possibility. furthermore, lazar et al. (2004) reported loggerheads in croatian waters, in the north of albania, that had been tagged while nesting at zakynthos, greece. in another study, the foraging grounds off the coasts of western mediterranean have been shown to be inhabited mainly by turtles from the eastern mediterranean (carreras et al., 2006). moreover, that study revealed a difference in genetic structure between northern african and western mediterranean stocks that could be explained by prevailing ocean currents and water masses. in conclusion, the new haplotypes of this study are coming from an unsampled nesting site. however, the current findings suggest that the source population of loggerhead turtles foraging in drini bay is most likely from greece based on the information that nesting populations contribute more to neighbouring mixed stocks than to more distant mixed stocks (bowen et al., 2004), but the sample size must be increased in order to carry out mixed stock analysis and evaluate the contribution of multiple nesting colonies to this foraging ground. acknowledgements this study was carried out by permission from ministry of environment, forests and water administrations of republic of albania and tissues were imported to turkey with cites permission (tr180110/35/001). the analysis was funded by the university of adnan menderes-department of biology, turkey. the sampling was conducted within the framework of the project “monitoring and conservation of important sea turtle feeding grounds in the patok area of albania, 2008-2010”. the project was funded by medasset, the global environment facility’s small grant programme (gef/sgp), the regional activity centre for specially protected areas (rac/spa), the united nations environment programme mediterranean action plan (unep/map), the british chelonia group (bcg), the j.f. costopoulos foundation (greece), the spear charitable trust (uk) and the panton trust (uk). the authors would like to thank bryan wallace for his valuable comments and linguistic review of earlier version of the paper. 160 c. yilmaz et al. ta bl e 2. h ap lo ty pe fr eq ue nc ie s in th e m ed ite rr an ea n th at r ep re se nt p os si bl e so ur ce p op ul at io ns fo r th e lo gg er he ad s fo ra gi ng in d ri ni b ay , a lb an ia . sa m pl in g si te n h ap lo ty pe s (% ) so ur ce c c -a 2 c c -a 3 c c -a 6 c c -a 10 c c -a 13 c c -a 20 c c -a 29 c c -a 31 c c -a 32 c c -a 43 c c -a 52 c c -a 53 c c -a 3. 2 z ak yn th os 20 85 5 10 c ar re ra s et a l., 2 00 7 k yp ar is si a 21 90 10 en ca la da e t a l., 1 99 8 la ko ni ko s 19 95 5 c ar re ra s et a l., 2 00 7 g re ec e 10 90 10 la ur en t e t a l., 1 99 8 c re te 19 10 0 c ar re ra s et a l., 2 00 7 c yp ru s 10 10 0 c ar re ra s et a l., 2 00 7 c yp ru s 35 10 0 en ca la da e t a l., 1 99 8 le ba no n 9 10 0 c ar re ra s et a l., 2 00 7 is ra el 19 84 16 c ar re ra s et a l., 2 00 7 w es te rn t ur ke y 16 94 6 c ar re ra s et a l., 2 00 7 ea st er n tu rk ey 32 59 41 la ur en t e t a l., 1 99 8 c al ab ri a 47 59 .6 36 .2 4. 2 g ar af al o et a l. 20 09 d al ya n 40 62 .5 37 .5 yı lm az e t a l. 20 11 d al am an 20 25 75 yı lm az e t a l. 20 11 w t r 76 78 .9 5 21 .0 5 yı lm az e t a l. 20 11 m t r 48 95 .8 3 2. 08 3 2. 08 3 yı lm az e t a l. 20 11 et r 72 83 .3 3 11 .1 1 1. 39 1. 39 1. 39 1. 39 yı lm az e t a l. 20 11 161new haplotypes in the loggerhead turtles foraging at drini bay references abreu-grobois, f., horrocks, j., formia, a., leroux, r., velez-zuazo, x., dutton, p., soares, l., meylan, p., browne, d. 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(2003): active swimmers-passive drifters: the oceanic juvenile stage of loggerheads in the atlantic system. in: loggerhead sea turtles, pp. 63-78. bolten, a.b., witherington, b.e., eds, smithsonian books, washington d.c. bowen, b.w., bass, a.l., chow, s.-m., bostrom, m., bjorndal, k.a., bolten, a.b., okuyama, t., bolker, b.m., epperly, s., lacasella, e., shaver, d., dodd, m. hopkins-murphy, s.r., musick, j.a., swingle, m., rankin-baransky, k., teas, w., witzell, w., dutton, p.h. (2004): natal homing in juvenile loggerheads (caretta caretta). mol. ecol. 13: 3797-3808. carreras, c., pont, s., maffucci, f., pascual, m., barcelo, a., bentivegna, f., cardona, l., alegre, f., sanfelix, m., fernandez, g., aguilar, a. 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(2009): loggerhead turtle (caretta caretta) matrilines in the mediterranean: further evidence of genetic diversity and connectivity. mar. biol. 156: 2085-2095. garofalo, l. (2010): “the genetics of the loggerhead turtle (caretta caretta) in italy: from scientific data to public knowledge” phd thesis. università tor vergata, roma. hall, t.a. (1999): bioedit: a user friendly biological sequence aligment editor and analysis programme for windows 95/98/nt. nucleic acids symp. ser. 41: 95-98. 162 c. yilmaz et al. hillis, d.m., moritz, c., mable, b.k. (eds) (1996): molecular systematics 2nd edition sinauer associates, sunderland, ma. laurent, l., casale, p., bradai, m.n., godley, b.j., gerosa, g., broderick, a.c., schroth, w., schierwater, b., levy, a.m., freggi, d., abd el-mawla, e.m., hadoud, d.a., gomati, h.e., domingo, m., hadjichristophorou, m., kornaraky, l., demirayak, f., gautier, c. (1998): molecular resolution of marine turtle stock composition in fishery bycatch: a case study in the mediterranean. mol. ecol. 7: 1529-1542. lazar, b., margaritoulis, d., tvrtkovic n. (2004): tag recoveries of the loggerhead sea turtle caretta caretta in the eastern adriatic sea: implications for conservation. j. mar. biol. assoc. uk 84: 475-480. mcclellan, m.c., read, a.j. (2007): complexity and variation in loggerhead sea turtle life history. biol. lett. 3: 592-594. white, m., boura, l., venizelos, l. (2011): medasset’s three‐year project: monitoring an important sea turtle foraging ground at patok, albania. mtn in press yılmaz, c., türkozan, o., bardakcı, f. (2011): genetic structure of loggerhead turtle (caretta caretta) populations in turkey. biochem. syst. ecol. 39: 266-276. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 6(1): 119-126, 2011 assessing the status of amphibian breeding sites in italy: a national survey societas herpetologica italica* sede museo regionale di scienze naturali, torino. * corresponding author: sebastiano salvidio, dipteris – università di genova, corso europa 26, i-16132 genova, italy. e-mail: salvidio@dipteris.unige.it submitted on: 2011, 3rd february; revised on: 2011, 5th february; accepted on: 2011, 27th may. abstact. the ecological status of 203 amphibian aquatic breeding sites, selected from the national database of the societas herpetologica italica (shi), was surveyed in the period 2008-2009 to assess their ecological status. sites were randomly extracted, after stratification by the three biogeographical regions present in italy, besides sardinia and sicily. the field surveys, conducted by professionals, amateurs and volunteers, showed that since 1979 about 11% of the sites were destroyed or no more suitable for the reproduction of amphibians that bred in the same site in the past. the percentage of destroyed or altered sites was 8%, both in the mediterranean and alpine biogeographical regions, and 15% in the continental one. however, there were no statistical significant differences among the regions, suggesting that the rate of amphibian site loss was similar in different parts of italy. this nation-wide monitoring project demonstrated that in italy, during the last thirty years, a relevant proportion of amphibian breeding habitats has been destroyed or altered. the main cause of site alteration were land reclamation and water extraction. keywords. amphibian breeding sites, conservation, freshwater habitat loss, italian amphibian survey, scientific monitoring, volunteers. introduction amphibians are the vertebrate class more at risk of extinction, and nearly one-third of the living species are currently considered threatened (stuart et al., 2004). in fact, amphibian populations are declining in different parts of the planet, especially in mountain ecosystems in central and south america, at high elevations in western unites states, and in south-eastern australia (e.g. alford and richards, 1999; houlahan et al., 2000; lannoo, 2005). the causes of these declines are still under investigation and natural and anthropogenic factors such as habitat modifications, infectious diseases, introduction of exotic spe120 societas herpetologica italica cies, toxic chemicals, increase in uv-b irradiation, and global climatic change have been considered (e.g., beebee and griffiths, 2005; lannoo, 2005). to date there are no quantitative nation-wide data on the status of amphibian species in italy, although there have been general evaluations based on their overall distribution and natural history (andreone and luiselli, 2000, 2001; sindaco, 2000, 2006). therefore in 2008 the societas herpetologica italica (shi), promoted a national monitoring project to assess the status of aquatic breeding sites of italian amphibians. this project was based on the shi database, containing more than 15,000 amphibian records that were collected by hundreds of professional herpetologists and volunteers, in addition to historical and museum data. these records were used to produce the atlas of italian amphibians and reptiles (sindaco et al., 2006), and the national data base ckmap (s.h.i. in ruffo and stock, 2005). each record of the shi database is referred to a 10 × 10 km utm grid square, administrative region, province, municipality, precise locality (if available) and type of record (bibliographic, museum specimen or unpublished observation). the shi monitoring project aimed to produce a national assessment based on field surveys carried out over a short time period, as already conducted in switzerland for both amphibians (schmidt and zumbach, 2005) and reptiles (monney and meyer, 2005). in this pilot project however, only aquatic amphibian species were taken into consideration, since freshwater breeding sites are usually easy to recognize and to locate in the field, in particular by amateur herpetologists and volunteers. material and methods survey design this study was planned by a national coordination committee that acted on behalf of the shi. thus, site selection was centralized to reduce the possibility of “choosing” well-known or easy-to-find amphibian sites. the random selected sites were listed by administrative regions and assigned to a regional coordinator committee, formed by one or more shi associates. the regional coordinator(s) performed the surveys personally or in collaboration with local herpetologists or volunteers. thus, the management and the structure of project was similar to the one of the italian herpetological atlas (sindaco et al., 2006). all the sites stored in the shi database were sorted by date and only those dating after 1979 were retained for monitoring. the sites were selected by the past occurrence of selected species. then, the sites were stratified by the three biogeographical regions represented in italy: alpine, continental and mediterranean (condé et al., 2002). a total of 340 sites were randomly extracted from these strata in relation to their surface (see table 1), assuring however that at least 20 sites each were from the two main islands (sardinia and sicily), characterized by peculiar amphibian faunas. thereafter the monitoring project was divided in two phases. in the first, amphibian breeding sites had to be visited to assess their current ecological status, and in particular if they were still suitable for amphibian reproduction. in the second, those sites found suitable should have been sampled intensively for two consecutive breeding seasons (i.e., years), to obtain presence/absence data of amphibian species with three surveys per breeding season (schmidt and zumbach, 2005). 121assessing the status of amphibian breeding sites in italy: a national survey field sampling protocol all participants to the monitoring project had to compile a field data sheet. in the first part of the sheet the operator had to clearly state if the exact location of the amphibian breeding site was found. if not, she or he had to stop the survey and ask the national coordinators for a substitute site. in this case, a new site with the same species located within the same biogeographic region was randomly extracted the stratified database. if the original site was localised with a relative good confidence, then the compiler had to give precise utm wgs 84 coordinates, habitat description, and a subjective but convincing judgement on the ecological status of the site in particular about the suitability for the reproduction of amphibian species previously reported. moreover, she or he had to describe the reasons that caused the observed alteration or destruction of the site, choosing from a list of threats ranging from natural vegetation succession, introduction of alien species up to the complete destruction caused by man. results during the period 2008-2009, only 203 (60%) out of the selected 340 sites were surveyed. localisation of the sites surveyed during this study shows that their distribution covered the entire italian peninsula, comprising the alps, sardinia and sicily (fig. 1). according to local coordinators, missing sites could not be surveyed mainly at cause of lack of funds to cover travel expenses and shortage of trained volunteers. these logistic problems hindered the survey of many high-altitude and poorly accessible sites in the alps and in the apennines (fig. 1, table 1). due to the difficulties to complete the first phase of the project, the second one, which demanded a much more intensive field effort by the operators, was stopped by the national coordination committee in accordance with the regional coordinators. despite the anticipated end of the program, the data collected underline a negative picture of the present conservation status of amphibians in italy. indeed, the percentage of destroyed sites was 8% both in the alpine and mediterranean biogeographic region, while it was 15% in the continental one. however, there were no significant differences in the distribution of destroyed sites among the regions according to a chi-square analysis (χ2 = 2.802; df = 2, p = 0.246). the overall percentage of destroyed amphibian breeding sites in italy in the last 30 years was 11%. the main causes of the destruction or alteration of the breeding sites are resumed in table 2. it is clear that land reclamation, construction and drainage were the main causes of the loss of amphibian breeding sites. discussion the present study represents the first scientific assessment of the ecological status of amphibian breeding sites in italy. one of the main conclusions concerns the efficiency of an italian national survey exclusively based on volunteers and not sustained by any kind of public funding. the herpetological association shi has not been able to ensure any kind 122 societas herpetologica italica of reimbursement to its associates or to volunteers involved in the monitoring programme. this caused the premature interruption of the programme, as only 60% of the selected sites were assessed. therefore, the second phase of the programme, which requested the   fig. 1. distribution of the 203 amphibian breeding sites assessed for their ecological status in 2008-2009, in italy. table 1. number of randomly selected sites per biogeographical region, number of surveyed and destroyed/unsuitable amphibian sites. biogeographical region sites extracted sites surveyed (% of extracted) site destroyed (% of surveyed) alpine 40 12 (30%) 1 (8 %) continental 90 82 (91%) 13 (15%) mediterranean 210 109 (52%) 9 (8%) total 340 203 (60%) 23 (11%) 123assessing the status of amphibian breeding sites in italy: a national survey monitoring of amphibian populations on a species-based protocol, never began. in other countries, where some kind of public coordination or funding is assured, as is the case of switzerland (schmidt and zumbach, 2005) and the netherlands (van der meij et al., 2009), amphibian monitoring is successfully maintained over relatively long-periods by volunteers coordinated by scientific staff. indeed, these kind of mixed programmes provide important insights on how to assure persistency and efficiency of monitoring environmental projects, providing scientific data concerning amphibian populations trends. in any case, the present partial results confirm the general claim that amphibian aquatic habitats are disappearing and that amphibian populations are declining. indeed for the first time, quantitative data on the rate and causes of alteration or modification of amphibian breeding sites in italy have been provided. the main cause of aquatic habitat destruction or alteration was by far the direct modification of their ecological status by man (i.e., drainage for land reclamation), while other indirect causes, such as introduction of invasive species, were also relevant. in addition to this latter phenomenon, already relatively well studied in italy (lillo et al., 2011; gherardi et al., 1999), we have to consider also the possible spreading of infectious diseases, such as the chitrid fungus batrachochitridium dendrobatis (weldon et al., 2004), that still have not yet been evaluated at the italian-wide level. moreover, we have to consider the great alteration and the fragmentation of terrestrial habitats (berdini, 2009) that may also cause amphibian population widespread long-term declines. unfortunately, the results of this study, limited to the assessment of aquatic habitats, does not allow any evaluation of these threats. moreover, this study was not conceived to assess or census newly-formed habitats suitable for amphibian reproduction. in fact, it is possible that in the last 30 years some new breeding sites were generated or restored by natural hydrological processes, such as river floods, or by the creation of artificial wetlands. however, in developed countries the dynamic processes of streams and rivers are usually highly limited by humans in order to provide new land for agriculture, urban development or infrastructures (brierley, fryirs, 2005). conversely, some recent amphibian conservation projects have been active (e.g., bressi, 1996; bressi et al., 2000; lapini, 2007). these conservation projects surely increased the number of pond habitats for biodiversity and in particular for amphibians, at the local level. therefore, the mean rate of disappearance of breeding sites estimated by the present study (11% in the last 30 year) is probably slightly overestimated. table 2. causes of destruction/alteration of amphibian breeding sites in italy. cause of destruction/alteration number of sites (%) natural succession of vegetation 1 (4.3) land reclamation/water drainage or extraction 14 (60.9) water pollution 1 (4.3) site management (unsuitable for reproduction) 1 (4.3) site abandonment (artificial sites) 1 (4.3) introduction of alien species (crayfish, fish etc.) 2 (8.7) unknown 3 (13.1) total 23 (100.0) 124 societas herpetologica italica finally, the assessment of the conservation status of all the amphibian species listed in the european directive “habitat and species” 92/43/cee is requested by article 17, and should be reported to the european commission in 2012. however, to date there are no available nation-wide monitoring data on the trends of amphibian populations, and the present study clearly shows that in italy a wide-scale monitoring is not feasible only through the excusive use of volunteers, without an official engagement of regional and national environmental authorities. acknowledgements all persons who contributed to this project are listed in alphabetical order: andreone franco, angelini jacopo, associazione natura cascina bellezza onlus, associazione zirichiltaggi, bassu lara, bennati r., bernini franco, bettiol katia, biancardi, biggi emanuele, bionda radames, bocca massimo, bonato lucio, buzzetti filippo maria, carafa marco, carletti silvia, cassol michele, casu viviana, cavalieri c., cornetti luca, corti claudia, dal lago antonio, damico maurizio, delisio lorenzo, delmastro giovanni battista, di cerbo anna rita, di francesco n., di tizio luciano, donelli o., doria giuliano, emiliani davide, eusebio bergò paolo, evangelista massimo, faraone francesco paolo, fattizzo tiziano, ferri vincenzo, ferro m., fiacchini david, ficetola francesco, foglia gessica, fulco egidio, giacalone g., giberti p., gola, grieco cristina, guarino fabio maria, iannelli a., iantorno a., incao, jiménez gonzales pilar, lamagni luca, lillo francesco, lo valvo mario, manca j., marchesi m., mazzotti stefano, menegon michele, mezzasalma marcello, miserocchi danio, montioni francesca, mosini andrea, nistri anna maria, nitti nicola, novaga r., odierna gaetano, oneto fabrizio, ottonello dario, parolin enrico, pedrini paolo, pellegrini mario, pellitteri rosa daniele, penazzi rocco, petruzzi f., piazzini sandro, picariello orfeo, poggiani luciano, pollo r., pupin fabio, razzetti edoardo, richard jacopo, riservato elisa, romanazzi enrico, romano antonio, romano antonio vito, rossini marco, sacchi roberto, sala luigi, salvidio sebastiano, sassi a., scali stefano, scirocco t., seglie daniele, semenzato massimo, silvano fabrizio, sindaco roberto, sommacal monica, spada arianna, sperone emilio, spilinga cristiano, svanella abbondio, tormen fausto, tormen giuseppe, tripepi sandro, vaccaro angelo, vanni stefano, ventrella pasquale, nulchis valentina, zampogno elena, zuffi marco a.l. a special thanks are due to sebastiano salvidio and roberto sindaco for having supervised the project and present contribution. references alford, r.a., 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(ed.) atti i congresso nazionale societas herpetologica italica, torino, museo regionale di scienze naturali torino, 821 pp. sindaco, r. (2006): erpetofauna italiana: dai dati corologici alla conservazione. pp. 679 695. in: sindaco, r., doria, g., razzetti, e., bernini, f. (eds): atlante degli anfibi e rettili d’italia – atlas of amphibians and reptiles in italy, shi, edizioni polistampa firenze, 792 pp. 126 societas herpetologica italica sindaco, r., doria, g., razzetti, e., bernini, f. (2006): atlante degli anfibi e rettili d’italia – atlas of amphibians and reptiles in italy, shi, edizioni polistampa firenze, 792 pp. stuart, s.n., chanson, j.s., cox, n.a., young, b.e., rodriguez, a.s., fischman, d.l., waller, r.w. (2004): status and trends of amphibian declines and extinctions worldwide. science 306: 1783−1786. van der meij, t., van strien, a., smit, g., goverse e. 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(2004): origin of the amphibian chytrid fungus. emerging infectious diseases, 10 (12): 2100-2105. bbib67 ole_link1 ole_link2 bbib28 ole_link5 ole_link6 ole_link7 ole_link8 _goback ole_link1 ole_link2 ole_link3 ole_link4 ole_link1 ole_link2 ole_link19 ole_link20 ole_link21 ole_link29 ole_link3 ole_link4 ole_link5 ole_link31 ole_link14 ole_link15 ole_link12 ole_link13 ole_link16 ole_link17 ole_link22 ole_link23 ole_link24 ole_link8 ole_link9 ole_link10 ole_link11 ole_link18 ole_link27 ole_link28 ole_link25 ole_link26 ole_link6 ole_link7 ole_link34 ole_link37 ole_link38 acta herpetologica vol. 6, n. 1 june 2011 firenze university press widespread bacterial infection affecting rana temporaria tadpoles in mountain areas rocco tiberti extreme feeding behaviours in the italian wall lizard, podarcis siculus massimo capula1, gaetano aloise2 lissotriton vulgaris paedomorphs in south-western romania: a consequence of a human modified habitat? severus d. covaciu-marcov*, istvan sas, alfred ş. cicort-lucaciu, horia v. bogdan body size and reproductive characteristics of paedomorphic and metamorphic individuals of the northern banded newt (ommatotriton ophryticus) eyup başkale1, ferah sayım2 , uğur kaya2 genetic characterization of over hundred years old caretta caretta specimens from italian and maltese museums luisa garofalo1, john j. borg2, rossella carlini3, luca mizzan4, nicola novarini4, giovanni scillitani5, andrea novelletto1 the phylogenetic position of lygodactylus angularis and the utility of using the 16s rdna gene for delimiting species in lygodactylus (squamata, gekkonidae) riccardo castiglia*, flavia annesi localization of glucagon and insulin cells and its variation with respect to physiological events in eutropis carinata vidya. r. chandavar1, prakash. r. naik2* the balearic herpetofauna: a species update and a review on the evidence samuel pinya1, miguel a. carretero2 effects of mosquitofish (gambusia affinis) cues on wood frog (lithobates sylvaticus) tadpole activity katherine f. buttermore, paige n. litkenhaus, danielle c. torpey, geoffrey r. smith*, jessica e. rettig food composition of uludağ frog, rana macrocnemis boulenger, 1885 in uludağ (bursa, turkey) kerim çiçek preliminary results on tail energetics in the moorish gecko, tarentola mauritanica tommaso cencetti1,2, piera poli3, marcello mele3, marco a.l. zuffi1 climate change and peripheral populations: predictions for a relict mediterranean viper josé c. brito1, soumia fahd 2, fernando martínez-freiría1, pedro tarroso1, said larbes3, juan m. pleguezuelos4, xavier santos5 assessing the status of amphibian breeding sites in italy: a national survey societas herpetologica italica* osservatorio erpetologico italiano acta herpetologica journal of the societas herpetologica italica acta herpetologica rivista della societas herpetologica italica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 8(2): 163-166, 2013 datadotdna: an alternative marking system for tortoises of genus testudo luca brugnola1,2,*, carlo biancardi3, nicoletta di francesco2, luciano di tizio2, adrian gheorghiu4 1 corpo forestale dello stato, servizio cites territoriale, viale della riviera n° 301, 65123 pescara, italy. *corresponding author. e-mail: l.brugnola@corpoforestale.it 2 s.h.i. – sezione abruzzo-molise “a. bellini”, via salomone 112, 66100 chieti, italy 3centro studi faunistica dei vertebrati della sisn, milano, italy 4 datadot italia s.r.l., via giuseppe lazzati, 185, 00166 roma, italy submitted on 2013, 14th october; revised on 2013, 18th november; accepted on 2013, 14th december. abstract. it was analyzed the effectiveness of one method of individual and unique marking, alternative to the application of microchip, on 17 specimens of testudo hermanni through datadotdna technology. this technology has proven to be an effective system of marking of testudo spp., answering the need for unambiguous identification of individuals. further advantages are the easy application and reading, the long-term resistance as well as the difficulties of possible fraudulent tampering. keywords. testudo hermanni, testudo graeca, testudo marginata, datadotdna, marking. the species testudo hermanni gmelin, 1789, testudo graeca linnaeus, 1758 and testudo marginata schoepff, 1792 are enclosed in annex a to council regulation (ec) no 338/97 and subsequent amendments and revisions. council regulation (ec) no 338/97 deals with the protection of species of wild fauna and flora by regulating the trade therein, harmonising the implementation of the “washington convention on international trade in endangered species of wild fauna and flora” at european level. the legal obligation to mark all the living individuals of species included in annex a has been introduced to control the pet trade, in accordance with the law 7 february 1992 no 150 and the mentioned council regulation. the animals should be univocally identified by microchip iso 11784:1996 (e) and 11785:1996 (e) compliant. due to the commonly used microchip dimensions (mm 12 x 2.12), and in order to guarantee the animals welfare, the cites management authority (in italy the ministry for environment and territory and sea) provided, for individuals under the age of five years, the possibility to use a photographic identification archive, instead. the recent commercial availability of smaller microchip (mm 7 x 1.25) allowed the marking procedure of individuals within their first year of age, namely when their carapace reached the length of 5 cm. however, microchip inoculation is an invasive action that entails intrinsic risks, which are common to all the surgical procedures, but more hazardous when performed on small and very young animals. hence, in order to minimise all the risk factors, it has been tested the possible implementation of the datadotdna technology for marking testudo hermanni, which growth rate in the first years is lower than those of the other two species t. graeca and t. marginata. microdot identification technology has been applied for several years in australia, united kingdom, italy and many other countries, to the prevention of car theft or to guarantee the authenticity of art and collector objects. more recently, it has been employed both to oppose the oyster theft (honan, 2007; sydney morning herald, 2007), and in entomological researches, to investigate the orchid pollination by wasps (whitehead and peakall, 2013). this technology is based on polymer discs (diameter one millimetre or less) assembled with a unique laser 164 luca brugnola et al. impressed alphanumeric code. discs are secured on the involved surface using different kinds of adhesive. the objectives of our research were: i) prove that datadotdna identification technology is an efficient alternative, or complementary marking method for testudo tortoises; ii) test its resistance to the removal by means of natural agents (rubbing or other), or by illicit management (removal and reuse of disks); iii) verify that all the procedures were in compliance with the animal welfare requirements, and in particular that they do not affect the individual growth rate. seventeen individuals of unspecified gender of testudo hermanni, between 1 and 3 years old, were divided into two homogeneous groups (a: experimental; b: control), constituted by 9 and 8 individuals, respectively. during the trial period, the two groups were placed in large monitored enclosures, in semi-natural conditions, ambient temperature and same food supply. an identification form was drawn up for each subject. the main biometric measures (carapace length, width and height; plastron length; body mass) were recorded and filled in the form at the beginning of the trial. the shell of each subject of group a was cleaned and dried before placing the datadotdna technology id system. the identification system was composed by a 9 characters alphanumeric code, impressed in specular alternating rows on discs of 0.5 mm of diameter (fig. 1). discs were secured with three different adhesives based on: i) aqueous solution of dipropylene-glycol-monomethylether (dgm); ii) methyl cyanoacrylate (cm); iii) n-butyl acetate (nb). the dgm based adhesive has uv fluorescence characteristics, which allow for easier detection of discs glued on the shell. if necessary, the fluorescent pigment (ciba uvitex nfw optical brightener) could be easily included in the composition of the other two kinds of glue, following the manufacturing instruction for component ratios. for each subject, three polyester discs were separately suspended in each of the adhesive solutions and placed, with the aid of a small pad, on one vertebral and two coastal scutes. after the drying time, which ranged from 5 to 20 minutes (quicker for cm, longer for nb), the readability of the discs was tested with both a portable optical microscope (100×), and a portable digital microscope (27-108×) (fig. 2). at the end of these operations, the animals were brought back to their enclosures. biometric measurements were taken monthly during the trial period, according to the hibernation periods, for a total of 10 records for 13 individuals, and 11 records for the remaining 4. during these surveys, the conditions and readability of each disc were checked, as well as the presence of localised or generic growth aberrations of the shell, or on-going pathological conditions. since the trial period started before for 4 tortoises born between 2008 and 2009, and equally distributed in experimental and control groups and later for the others (born in 2010 fig. 1. microdot disc of 0.5 mm in diameter. fig. 2. experimental phases: placement (upper) and reading test (lower) of microdot discs. 165marking testudo with datadotdna and 2011), an interval of 15 months, included one winter latency period, has been taken into consideration for each animal. the differences between the last and the first measurement, over such interval of time, were recorded for each biometric parameter (table 1, 2). anova for repeated measures cannot be applied to analyse two groups of paired data (huck and mclean, 1975). feasible alternatives are: i) t-test for independent groups on the differences between last and first measurements (hopkins, 1997), or ii) analysis of covariance (ancova) with the experimental and control groups as independent variable, the last measurement as dependent variable and the first one as covariate. in these cases, the latter is considered the most powerful test (huck and mclean, 1975). covariate permits to remove the influence of differences of the starting measurement within and between groups. neither abnormal development of carapace or plastron, nor local or systemic pathologies were found, in any subject, during the experimental period. the biometric data recorded were congruent with the age of any single tortoise, and the growth rates were in line with literature data (cheylan, 1981). all the alphanumeric code reading tests had positive result, for all three adhesives employed, but some discs were lost by three tortoises. reading was reasonably easy with the portable optic microscope at 100×, while with the digital one at 108× it was pretty hard, due to difficulties to focus a bended surface, like the carapace. the only relevant reading problem was caused by discs applied on carapace black spots, because they were transparent with the code impressed in black. ancova gave not significant differences, at α = 0.05, between the mean value of all biometric measures (table 3). therefore, we can confidently conclude that datadotdna marking technology did not affect the growth of subjects included in group a. datadotdna technology proved to be an efficient marking system for tortoises of testudo genus, alternative, or complementary, to other currently adopted techniques (stubbs et al., 1984; guyot and clobert, 1997), at least for tortoises raised in controlled conditions. this methodoltable 1. experimental group (a). first measurements and, in brackets, differences between last and first measurements. start date: date of marking and first measurement; lc: carapace length; wc: carapace width; hc: carapace height; lp: plastron length; mb: body mass. datadot birth year start date (dd-mm-yyyy) lc (mm) wc (mm) hc (mm) lp (mm) mb (g) cfs 001018 2011 27-03-2012 37.5 (7.5) 33 (4) 18 (4) 32 (3) 10 (8) cfs 001009 2011 27-03-2012 45 (11) 39 (6) 24 (7) 39 (8) 16 (20) cfs 001004 2011 27-03-2012 39 (12) 34 (8) 21 (7) 33 (9) 14.5 (14.5) cfs 001000 2011 27-03-2012 42 (13) 37 (9) 21 (7) 37 (9) 15.9 (16.1) cfs 001007 2011 27-03-2012 40 (12) 37 (7) 22 (6) 35 (9) 14 (15) cfs 001012 2011 27-03-2012 40 (9) 36 (3) 20 (5) 34 (6) 13.9 (8.1) cfs 001019 2011 27-03-2012 37 (8) 33 (4) 18 (5) 31 (6) 9 (9) cfs 001002 2009 9-07-2011 45 (28) 37 (21) 23 (16.5) 38 (22) 15 (63) cfs 001020 2008 9-07-2011 51 (26) 45 (19) 28 (19) 42 (20) 36 (68) table 2. control group (b). first measurements and, in brackets, differences between last and first measurements. start date: date of first measurement; lc: carapace length; wc: carapace width; hc: carapace height; lp: plastron length; mb: body mass. id birth year start date (dd-mm-yyyy) lc (mm) wc (mm) hc (mm) lp (mm) mb (g) citespe-270312-18 2011 27-03-2012 42 (9) 36 (5) 20 (6.5) 34 (7) 13 (13) citespe-270312-19 2010 27-03-2012 47 (12.5) 39 (10) 23 (8) 40 (10) 19.3 (19.7) citespe-270312-20 2011 27-03-2012 37 (12) 34 (8) 21 (8) 32 (6) 11.7 (13.3) citespe-270312-24 2011 27-03-2012 39 (14) 35 (10.5) 20 (9) 34 (9) 11.9 (16.1) citespe-270312-25 2011 27-03-2012 42 (5) 36 (5) 21 (4.5) 36 (4) 13 (7) citespe-270312-35 2011 27-03-2012 39 (11) 37 (11) 20 (8) 34 (7) 12 (15) pllmra-090711-6 2008 9-07-2011 57 (21) 48 (15) 31 (17) 47 (17.5) 36 (58) pllmra-090711-7 2008 9-07-2011 52 (20.5) 41 (16) 28 (14.5) 42 (18) 25 (49) 166 luca brugnola et al. ogy meets the current regulation requirements in terms of unambiguous identification of individuals, durability on the marked animal, ease placement and reading and difficulty of possible illicit tampering of the mark. long-term durability tests of microdot discs on testudo individuals are currently in progress, as one of the objectives of our further research. however, the following tests on durability were carried out by an independent laboratory of melbourne (australia): accelerated aging treatments (high and low air temperature, high humidity) and removal tests (high pressure cold water/detergent cleaner test, high pressure hot water cleaner test). those experiments gave permanence rates, in the range of 90-100% of the marking, for over 20 years (johnson, 2010). the current (2013) price, excluding vat, for a kit of 500 microdots (0.5 mm) is about 15 euros. the only remarkable limit of this method is the impossibility to read discs placed on black surfaces. however, this problem could be solved by laser micro engraving the code on different substrata, like ceramic. this new technique 0.2 mm micro-tags, up to 15 alphanumeric characters, better readability has been recently developed by datadot technologies ltd. (australia), and should be tested in the field, as well. moreover, the fraudulent removal of discs is possible, as is of micro-chips, but their reuse appear to be very hard. in fact, the removal process damages the discs, which get included into the adhesive layer used to fix them. readability of discs was negatively affected by the thickness of the adhesive layer. the deterrent factors against illicit removal of marks are supported by: i) the dimensions of the polyester discs: 0.3 mm with 7 characters/row code, 0.5 mm with 11 characters/row code or 1.0 mm with 21 characters/row code. the smaller the disc, the more difficult will be their visual detection; ii) the quantity of discs, with the same code, that could be placed on each animal: the more the discs placed, the harder will be their complete removal. hence, the rational could be using several small discs (0.3 mm), fixed on different parts of the shell. this should be a strong deterrent against illicit marking removal. acknowledgements the experimental animals belong to a group of testudo hermanni that have been entrusted in judicial custody in the structures of the state forestry corps. we thanks datadot italia s.r.l. for their kind willingness to share useful ideas during the design of the experiments, and for granting free-of-charge all the equipment, including adhesives and reading devices, used during the trials. we thanks two anonymous reviewers for their suggestions. references cheylan, m. (1981): biologie et écologie de la torte d’hermann testudo hermanni, gmelin 1789. mémoires et travaux de l’institut de montpellier (e.p.h.e.), 13, montpellier. guyot, g., clobert, j. (1997): conservation measures for a population of hermann’s tortoise testudo hermanni in southern france bisected by a major highway. biol. cons. 79: 251-256. honan, k. (2007): oyster farmers excited by dots. abc rural. retrieved from: http://www.abc.net.au/rural/ content/2007/ s1950390.htm hopkins, w.g. (1997): a new view of statistics. will g. hopkins. retrieved from: http://www.sportsci.org/ resource/stats/index.html huck, s. w., mclean, r. a. (1975): using a repeated measures anova to analyze the data from a pretestposttest design: a potentially confusing task. psychol. bull. 82: 511-518. johnson, c. (2010): test report emanar consultans environmental testing data dots. vipac reference: 30v-10-0248-tpr-586446-0, 23 sep. 2010, vipac engineers & scientists ltd. melbourne, australia. stubbs, d., hailey, a., pulford, e., tyler, w. (1984): population ecology of european tortoises: review of field techniques. amphibia-reptilia, 5: 57-68. sydney morning herald (2007): dotty oysters thwart thieves. general news 04.04.2007. whitehead, m. r., peakall, r. (2013): short-term but not long-term patch avoidance in a orchid-pollinating solitary wasp. behav. ecol. 24: 162-168. table 3. means and standard deviations of the biometric measures within the experimental (a) and control (b) groups. lc: carapace length; wc: carapace width; hc: carapace height; lp: plastron length; mb: body mass. group lc wc hc lp mb a average 14.05 9.00 8.50 10.22 24.63 standard dev. 7.59 6.56 5.38 6.44 23.55 b average 13.12 10.06 9.44 9.81 23.89 standard dev. 5.43 4.07 4.18 5.22 18.77 ancova fdf, p f1,14 = 1.59, p = 0.23 f1,14 = 0.01, p = 0.93 f1,14 = 0.15, p = 0.70 f1,14 = 1.28, p = 0.28 f1,14 = 0.50, p = 0.49 acta herpetologica vol. 8, n. 1 june 2013 firenze university press journal of the societas herpetologica italica acta herpetologica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 7(2): 341-345, 2012 range extension of the critically endangered true poison-dart frog, phyllobates terribilis (anura: dendrobatidae), in western colombia roberto márquez1,*, germán corredor2, carlos galvis3 daniel góez2, adolfo amézquita1 1 department of biological sciences, universidad de los andes, aa 4976. bogotá d.c., colombia. *corresponding author. e-mail: roberto.marquez33@gmail.com 2 biology department, universidad del valle, aa 25360, cali, colombia 3 cali zoological foundation, carrera 2 oeste calle 14 esquina, barrio santa teresita. cali, colombia submitted on: 2012, 26th september; revised on: 2012, 22nd october; accepted on: 2012, 24th october. abstract. the poison-dart frog phyllobates terribilis is currently classified as endangered or critically endangered due to its extremely restricted geographic distribution and intensive smuggling by pet traffickers. based on molecular data, we here report two new localities representing a 60 km northward extension of its previously recognized range. the identity of other phyllobates populations in western colombia is discussed, as well as the current morphological criteria used to distinguish p. terribilis and the similar p. bicolor. keywords. phyllobates terribilis, geographic distribution, dna barcoding, conservation status. the golden poison-dart frog, phyllobates terribilis, is one of the three species that was used by the emberá people of western colombia to poison darts, and is by far the most toxic among them. the species was described by myers et al. (1978), based on specimens collected at quebrada guangüí (type locality) and la brea, about 8 km west of the type locality, in the departamento del cauca, colombia, which remained the only known localities for almost 20 years. while topotypic specimens are yellow to orange, a mintcolored morph was later reported from la sirpa near the mouth of the saijá river, about 15 km west of quebrada guangüí (lötters et al., 1997). in the same paper, two series of phyllobates frogs collected about 50 km south of buenaventura (departamento del valle del cauca), and 70 km north of the type locality, were reported to exist at the herpetological collection of the universidad del valle, cali (uvc 7208-7210, 7135-7137). these were tentatively diagnosed as phyllobates bicolor, based on their snout-vent length (svl) and their blackish distal limb and throat coloration. moreover, it was suggested that p. terribilis 342 roberto márquez et al. could represent an extreme of clinal variation in p. bicolor (lötters et al., 1997). recent molecular analyses have, however, unambiguously supported p. bicolor and p. terribilis as two separate and well differentiated species (grant et al., 2006). because it was unclear whether specimens of phyllobates occurring south of buenaventura, belonged to either p. bicolor, or p. terribilis, we used dna barcoding to diagnose frogs from two localities in southern departamento del valle del cauca: (1) río naya (3°17’n, 77°24’w) and (2) boca yurumanguí (3°23’n, 77°18’w), in the naya and yurumanguí river drainages, respectively (fig. 1). we refrain from providing more detailed coordinates due to the intensive smuggling suffered by this frog species and its very restricted distribution range, which led castro (2004) to declare p. terribilis as one of the few critically endangered amphibian species in colombia. we captured four individuals at río naya and two at boca yurumanguí. we measured their svl and collected tissue samples by mouth-swabbing. two frogs from each locality were maintained as vouchers (field numbers gecoh 355, gecoh 434, gecoh 643, and gecoh 1163; fig. 2) and the other ones released. specimens from río naya ranged 38-41 mm (mean 39.75, standard deviation 1.22) and those from yurumanguí were 37 and 40 mm in svl. dna was extracted from the collected epithelial tissue using dneasy animal blood and tissue kits (qiagen), and the 5’ fragment of the cytochrome c oxidase i (coi) mitofig. 1. reported localities of phyllobates terribilis, including those reported in this paper. 343distribution of phyllobates terribilis chondrial gene was sequenced by using the dglco1490 and dghco2198 primers (meyer et al., 2005). the obtained sequences were deposited in genbank under accession numbers jx887784-jx887787. we then used the barcode of life database identification system (bold ids; http://www.boldsystems.org/) to identify the sequences that best matched the query sequences from our specimens. to further corroborate our results, we inferred bayesian and maximum likelihood gene trees using mrbayes (ronquist and huelsenbeck, 2003) and raxml (stamatakis, 2006), respectively, from our sequences and all co1 sequences from phyllobates species available in genbank (fig. 3; grant et al., 2006). the two sequences from río naya were 99.8% similar among them, and those from boca yurumanguí were identical. the bold ids yielded a 0.992 probability of assigning the two boca yurumanguí sequences to p. terribilis, and 0.987–0.988 probabilities of assigning the río naya individuals to the same species, which was the best match for the four sequences. assignment probabilities to p. bicolor (0.940, 0.935–0.936, respectively) were lower in all cases. regarding the gene tree, the four query sequences were nested within a very well supported clade that included all the other p. terribilis sequences (fig. 3). hence, both the barcoding and phylogenetic analyses strongly support the frogs from boca yurumanguí and río naya as part of p. terribilis. given the short geographic distance (fig. 1) between the localities reported here and the other two localities referred to by lötters et al. (1997) and the similar body size (all the collected adults range from 38.2–41.2 mm) among all these frogs, as well as their fig. 2. ventral and dorsal views of p. terribilis from a-b. río naya and c-d. boca yurumanguí. 344 roberto márquez et al. blackish distal limb and throat coloration, it is most likely that frogs referred by them (including those tentatively assigned to p. bicolor) are indeed p. terribilis. myers et al. (1978) suggested that the only useful morphological characters at distinguishing bicolor and terribilis were color pattern and body size. our data suggest otherwise, posing a potential conflict between genetic and morphologic (traditionally based on body size and color) diagnosis of both species. further taxonomic and phylogenetic studies with much wider sampling are urgently needed to clarify this issue. our results expand the distribution of p. terribilis about 60 km north of its previously known range, with the proposed distribution now including localities in two departments of western colombia (cauca and valle del cauca). since one of the motives leading to the classification of p. terribilis as critically endangered (rueda-almonacid et al., 2004) or endangered (bolivar and lötters, 2004) was its very restricted extension of range, our findings could improve the conservation status of the species. however, further field censuses and studies are necessary to conduct a thorough reevaluation of the conservation status of p. terribilis. fig. 3. bayesian gene tree inferred from co1 sequences of phyllobates spp. available in genbank and those of frogs collected in two new localities (in bold). the numbers on internodes represent bayesian posterior probabilities followed by ml bootstrap values (1000 pseudoreplicates). 345distribution of phyllobates terribilis acknowledgements the authors would like to thank the epeara siapidara indigenous community for their support and hospitality, as well as the fundación zoológica de cali and the faculty of sciences at universidad de los andes for providing financial support. rm and aa were supported by a colombia biodiversa scolarship from the alejandro ángel escobar foundation. we would also like to thank an anonymous reviewer and ylenia chiari whose comments improved the quality of the manuscript. references bolívar, w., lötters, s. (2004): phyllobates terribilis. in: iucn red list of threatened species. version 2012.1. http://www.iucnredlist.org/details/biblio/55264/0. sept. 15 2012. castro, f. (2004): rana venenosa dorada phyllobates terribilis. in: libro rojo de los anfibios de colombia, pp. 178–182. rueda-almonacid, j. v., lynch, j. d., amézquita, a., eds, conservación internacional colombia, instituto de ciencias naturales, universidad nacional de colombia, ministerio del medio ambiente. bogotá, colombia. grant, t., frost, d.r., caldwell, j.p., gagliardo, r., haddad, c.f.b., kok, p.j.r., means, d.b., noonan, b.p., schargel, w.e., wheeler, w.c. (2006): phylogenetic systematics of dart-poison frogs and their relatives (amphibia: athesphatanura: dendrobatidae). bull. amer. mus. nat. hist. 299: 1-262. lötters, s., castro, f., köhler, j., richter, r. (1997): notes on the distribution and color variation of poison frogs of the genus phyllobates from western colombia (anura: dendrobatidae). rev. fr. aquariol. 24: 55-58. meyer, c.p., geller, j.b., paulay, g. (2005): fine scale endemism on coral reefs: archipelagic differentiation in turbinid gastropods. evolution 59: 113-125. myers, c.w., daly, j., malkin, b. (1978): a dangerously toxic new frog (phyllobates) used by emberá indians of western colombia, with discussion of blowgun fabrication and dart poisoning. bull. amer. mus. nat. hist. 161: 311-366. ronquist, f., huelsenbeck, j.p. (2003): mrbayes 3: bayesian phylogenetic inference under mixed models. bioinformatics 19: 1572-1574. stamatakis, a. (2006): raxml-vi-hpc: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. bioinformatics 22: 2688-2690. acta herpetologica vol. 7, n. 2 december 2012 firenze university press advertisement call of species of the genus frostius cannatella 1986 (anura: bufonidae) flora a. juncá1, david l. röhr2, ricardo lourenço-de-moraes3, flávio j. m. santos1, airan s. protázio1, ednei a. mercês1, mirco solé4 amphibians in southern apennine: distribution, ecology and conservation notes in the “appennino lucano, val d’agri e lagonegrese” national park (southern italy). antonio romano1,*, remo bartolomei1, antonio luca conte1, egidio fulco2 the significance of using satellite imagery data only in ecological niche modelling of iberian herps neftalí sillero1, josé c. brito2, santiago martín-alfageme3, eduardo garcía-meléndez4, a.g. toxopeus5, andrew skidmore5 reproductive strategy of male and female eastern spiny lizards sceloporus spinosus (squamata: phrynosomatidae) from a region of the chihuahuan desert, méxico aurelio ramírez-bautista1,*, barry p. stephenson2, xóchitl hernández-ibarra1, uriel hernández-salinas1, raciel cruz-elizalde1, abraham lozano1, and geoffrey r. smith3 morphological variability of the hermann’s tortoise (testudo hermanni) in the central balkans katarina ljubisavljević1, georg džukić1, tanja d. vukov1, miloš l. kalezić1,2 the usefulness of mesocosms for ecotoxicity testing with lacertid lizards maria josé amaral1,2,*, rita c. bicho1, miguel a. carretero2, juan c. sanchez-hernandez3, augusto m. r. faustino4, amadeu m. v. m. soares1, reinier m. mann1,5 does acclimation at higher temperatures affect the locomotor performance of one of the southernmost reptiles in the world? jimena b. fernández*, nora r. ibargüengoytía advertisement call of scinax littoralis and s. angrensis (amphibia: anura: hylidae), with notes on the reproductive activity of s. littoralis michel v. garey1, thais r. n. costa2, andré m. x. de lima2, luís f. toledo3, marília t. hartmann4 book review: marine s. arakelyan, felix d. danielyan, claudia corti, roberto sindaco, alan e. leviton 2011. herpetofauna of armenia and nagorno-karabakh. society for the study of amphibians and reptiles stefano scali book review: rafaqat masroor 2012. a contribution to the herpetofauna of northern pakistan stefano scali evidence of high longevity in an island lacertid, teira dugesii (milne-edwards, 1829). first data on wild specimens. j. jesus first assessment of the endoparasitic nematode fauna of four psammophilous species of tropiduridae (squamata: iguania) endemic to north-eastern brazil markus lambertz1,*, tiana kohlsdorf2, steven f. perry1, robson waldemar ávila3, reinaldo josé da silva4 rediscovery and redescription of the holotype of lygosoma vittigerum (= lipinia vittigera) boulenger, 1894 yannick bucklitsch1, peter geissler1, timo hartmann1, giuliano doria2 , andré koch1,* reproductive phenology of the tomato frog, dyscophus antongili, in an urban pond of madagascar’s east coast ori segev1,*, franco andreone2, roberta pala2, giulia tessa2, miguel vences1 range extension of the critically endangered true poison-dart frog, phyllobates terribilis (anura: dendrobatidae), in western colombia roberto márquez1,*, germán corredor2, carlos galvis3 daniel góez2, & adolfo amézquita1 differences in habitat use of two sympatric species of ameiva in east costa rica esther sebastián-gonzález1, ramón gómez2 acta herpetologica journal of the societas herpetologica italica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 9(1): 33-41, 2014 doi: 10.13128/acta_herpetol-13406 microclimatic variation in multiple salamandra algira populations along an altitudinal gradient: phenology and reproductive strategies daniel escoriza ¹, jihene ben hassine² 1institute of aquatic ecology, university of girona, campus montilivi, 17071 girona, spain. corresponding author. e-mail: daniel_ escoriza@hotmail.com 2 faculty of sciences of tunis, department of biology, university tunis-el manar 2092 tunis, tunisia submitted on 2013, 10th october; revised on 2013, 15th november; accepted on 2014, 3rd april abstract. salamandra algira is one of the southernmost species of the genus, and most of its ecology remains poorly known. we studied the microhabitat conditions of the sites occupied by several populations of s. algira along an altitudinal gradient, and the use of water bodies for reproduction. the microclimate conditions were analysed at six sites in northern morocco: one site in beni snassen massif (s. algira spelaea), two in the middle atlas and central rif mountains (s. algira splendens), and three in the western rif mountains and peninsula tingitana (s. algira tingitana), where a viviparous population also occurs. the microclimate was characterized using temperature and relative humidity data loggers for a period of two years. we also measured the surface area and depth of the water bodies where we found s. algira larvae. our results showed an autumn-winter reproductive period for all ovoviviparous populations studied. in most of the aquatic habitats examined, larvae appeared between november and march, although this period could extend to may at higher altitudes. larval abundance and their size variability did not correlate with water body size or microclimate conditions. the decrease in the number of larvae per water body coincided with the existence of suitable conditions for post-metamorphic dispersal. salamandra algira occurred in regions with moist conditions (annual average relative humidity greater than 64 %) and with mean annual temperatures of 13.6-18.6 °c, but populations were largely segregated along a gradient of humidity, with some showing higher and more constant values than others. the viviparous population occurs in a region with maritime influence and greater microclimate stability than the other sites studied. keywords. viviparity, fire salamander, microhabitat, north africa. introduction salamandra is a genus that occurs along the western palaearctic, appearing especially diversified in the mediterranean region (steinfartz et al., 2000). the southern boundaries of the geographic range of salamandra species occur in northern morocco and northern israel, where they reach latitudes of 34°n–32°n (warburg, 1986; salvador, 1996). at these southern boundaries, salamandra populations are associated with evergreen forests at low-medium altitudes (below 2000 m a.s.l.), occurring in isolated areas, in some cases at low densities (escoriza and comas, 2007; warburg, 2007). the distribution of salamandra algira bedriaga, 1883 extends from northwestern of morocco to the north–eastern tip of algeria (stuart et al., 2008), but is discontinuous, and some populations are separated from one another by major geographical barriers, such as the semiarid moulouya valley (escoriza et al., 2006a). little is known about the phenology and habitat preferences of s. algira, except for scattered observations of its activity and presence (doumergue, 1901; pasteur and bons, 1959; schleich et al., 1996; martínez-medina, 2001; beukema et al., 2013; escoriza et al., 2013). numerous studies have examined the ecological niche of the amphibians on the basis of bioclimatic mod34 d. escoriza, j. ben hassine els, although this type of analysis can produce a false estimation of the species’ fundamental niche (araújo et al., 2013). these models at coarse resolution may overestimate the exposure to climate variation that a species experiences, owing to the buffering effect of the microhabitat on temperature (scheffers et al., 2014). this fact is especially relevant in terrestrial salamanders, whose occurrence is strongly dependent on microclimatic gradients that show rapid variation at fine spatial resolution (peterman and semlitsch, 2013). in the case of the genus salamandra, microhabitat conditions have a significant effect on its occurrence, because salamandra species show strong site fidelity and move in relatively small territories, covering distances up to 1300 m (bonato and fracasso, 2003; bar-david et al., 2007; schulte et al., 2007). the activity of terrestrial salamanders is strongly controlled by two environmental parameters, relative humidity (rh) and air temperature (spotila, 1972). salamanders show increased surface activity at relatively low temperatures (although not below 0° c) and high air humidity (welsh and lind, 1995; helfer et al., 2012). this activity is critical for their survival because it is related to foraging, reproductive migration and post-metamorphic dispersal (baldauf, 1952; feder and londos, 1984; liebgold and jaeger, 2007). the surface activity of the salamander species (such as salamandra infraimmaculata and lycisalamandra helverseni) that inhabit southern mediterranean regions occurs mainly between autumn and early spring (degani and warburg, 1978; polymeni, 1994). it is possible that the reproductive activity of s. algira also occurs during late autumn, winter, and early spring, because most observations of adults and larvae reported in the literature have been recorded during this period (doumergue, 1901; pasteur and bons, 1959; martínez-medina et al., 1997; escoriza et al., 2006a). given that s. algira occupies elevations of 100–2010 m a.s.l. in morocco (bons and geniez, 1996), we anticipated that local climatic conditions would cause differences in the onset of reproductive activity and larval occurrence. we analysed the effects of hydroperiod on larval development and abundance, because this factor has a significant impact on the larval stages of other species of salamander (semlitsch and wilbur, 1988; rowe and dunson, 1995). this analysis of environmental conditions should also provide new data on the conditions in which the viviparous population of s. algira inhabits. in other ectothermic vertebrates, this parity mode has evolved independently in several species that appear in cold regions (pyron and burbrink, 2014). in the case of the genus salamandra, viviparity is a reproductive strategy that presumably also evolves under harsh environmental conditions, such as extreme temperatures and the absence of surface water (veith et al., 1998; veloantón et al., 2007). however, there is not necessarily a relationship between these conditions and the development of viviparous reproductive strategies (dopazo and korenblum, 2000; garcía-parís et al., 2003). in northern morocco, the only known viviparous population of s. algira occurs in a mountain range near the coast, on a limestone substrate that lacks surface water bodies (martínez-medina et al., 1997; martínez-medina, 2001). in this study, we characterized the microclimate at six sites along the distribution of s. algira in northern morocco, analysing the relationships between environmental variables, reproductive mode (viviparous vs. ovoviviparous), and larvae presence. first we determine whether a microclimatic gradient exist among these populations of s. algira. then we assessed whether larvae presence and their development correlate with environmental conditions on a small spatial scale. materials and methods the study was conducted in northern morocco (fig. 1). climatic data were obtained in areas inhabited by six isolated populations of salamandra algira. population 1 (northern tip of tingitana peninsula, fig. 1), represents a population of s. algira tingitana, which is viviparous. populations 2 and 3 (north-western rif ) represent a different lineage from population 1. these two populations (2 and 3) are located at different altitudes (table 1). populations 4 (central rif ) and 5 (middle atlas) correspond to two lineages of s. algira splendens, separated geographically by a river valley. population 6 represents s. algira spelaea, a population genetically closer to the algerian populations than to the   fig. 1. study area and sites (white circles). 1) northern tip of tingitana peninsula, viviparous population of s. algira tingitana, 2) north western rif, low altitude population of s. algira tingitana, 3) north western rif, high altitude population of s. algira tingitana 4) central rif, s. algira splendens, 5) middle atlas, s. algira splendens, 6) beni snassen, s. algira spelaea. 35microclimate conditions in salamandra algira other populations occurring in morocco (escoriza and comas, 2007). we installed a hobo® pro v2 data logger temp / rh at each of these localities, near one or more water bodies where we had previously found larvae, during the two-year period (march 2010 to march 2012). these data loggers were programmed to record temperature and rh with a two-hour periodicity, with an accuracy of about ± 0.21 °c and ± 2.5 %, respectively. the data loggers were placed at ground level, in areas sheltered from the sun, oriented north, and approximately 100 m from the nearest water body. environmental parameters were measured at the local scale because the presence and density of amphibian species in forest habitats can show significant variations with very fine spatial resolution (stoddard and hayes, 2005; olson et al., 2007). we found no water body containing the larvae of population 1 (viviparous), so in this case the data logger was placed where we observed adults. each site was sampled in september, november, january, march, and may, because we expected to find greater reproductive activity in autumn, winter and springtime, based on previous surveys and the literature (see above). the water bodies surveyed were temporary ponds and springs. at two localities (western rif, high altitude site, and middle atlas) we included two water bodies, because they were found nearby but show important variation in their surface area. this makes a total of seven surveyed aquatic habitats, a sufficient number to draw ecological conclusions in descriptive and time-sequenced studies of aquatic communities (ruhí et al., 2013). we measured the surface area of the water body and its average depth, as a proxy for hydroperiod (brooks and hayashi, 2002). the surface area of each water body was estimated on the basis of the maximum length of the longitudinal and transverse axes, assuming an elliptical shape. in water bodies larger than 100 m², the surface area was obtained using a garmin dakota 100 gps unit. average depth was defined as the mean value of five successive measurements from the shore to the centre. the number of larvae was determined from a minimum of 10 dip nets in ponds with a surface area of less than 50 m² and from up to 60 dip nets in ponds over 1,000 m² in an attempt to include all possible mesohabitats (from shallow water to deep zones). we also measured the length of the larvae, since the variability in their size (standard deviation of larval length), could indicate the existence of sequential episodes of larval depositions, i.e., greater variability in size may be associated with the existence of several larval cohorts in the same pond. the total length of larvae was measured to the nearest 0.1 mm using a digital calliper and larvae were released afterwards. the positions of the studied sites according to the temperature and rh gradients were examined with a canonical correspondence analysis (cca) conducted with the vegan package (oksanen et al., 2012) of the software r (r development core team, 2011). the existence of significant differences between the climatic conditions, when one site was compared with the rest, was established with permutational multivariate analysis of variance (permanova; anderson, 2005). we also assessed the variability in the microclimate conditions per site using a permutational analysis of multivariate dispersions (permdisp; anderson, 2004). permdisp provides an estimate of the dispersion of the data around a centroid, based on a statistic of average dissimilarities (ad). this statistic can be interpreted as a proxy for microclimate stability per site (i.e., greater ad indicates greater instability in the microclimatic conditions). because of computational limits, the statistics in these analyses were calculated for a random sample of 400 measures per site. the presence of larvae in the water bodies and the standard deviations of larval size were correlated with environmental variables, specifically with the water body size (surface area of the water body x average depth), the temperature at 12:00 h (local time), and the number of days per month with optimal conditions for activity (optimal days, od). the size of the water body was studied to determine the possible effects of this variable on larval abundance and development. air temperature at 12:00 h is related to water temperature and therefore to the rate of variation in larval size (petranka, 1984). the value of air temperature included in the analysis corresponds to that measured on the 15th day of the month. od is the number of days per month on which air temperature was within the range of 4.0-14.9 °c and rh was above 80 % for at least two hours. these parameters correspond to the surface activity intervals described for other southern mediterranean salamandra species (degani, 1996; garcía-parís et al., 2004; rebelo, 2008). therefore, this analysis examined the possible correlation between environmental conditions and larval abundance, which could be result of variations in adult surface activity. this correlation was assessed with the relate routine based on euclidean distance table 1. geographic and altitudinal location of the studied sites, relative position in the microclimatic gradient and intra-annual microclimate variation. cca scores: scores obtained in the first axis of the canonical correspondence analysis, based on the variables of annual temperature / relative humidity (rh). perm: p values obtained by permanova comparing the values of annual temperature / rh of one site against the rest. ad: average dissimilarities, dispersion of the values of annual temperature / rh per site. lat / long altitude (m a.s.l.) cca scores permutation p ad north peninsula tingitana 35.8º n, 5.4º w 328 0.80 0.001 0.72 western rif (low) 35.4º n, 5.4º w 113 0.30 0.002 1.03 western rif (high) 35.2º n, 5.4º w 1006 -0.18 0.86 1.24 central rif 35.0º n, 5.0º w 1488 -0.87 0.001 1.30 middle atlas 34.0º n, 4.1º w 1470 -0.32 0.001 1.63 beni snassen 34º n, 2º w 860 0.40 0.001 0.95 36 d. escoriza, j. ben hassine matrices (clarke et al., 2008), after applying bonferroni’s correction. these analyses were performed with the primer v6.1.11 package (primer-e ltd., plymouth). results the studied populations occurred within a mean annual temperature range of 13.6-18.6 °c, at a mean annual rh exceeding 64 % (fig. 2). the cca provided a high eigenvalue for the first axis (eigenvalue = 0.40, explained variance = 88 %), indicating that this axis represents a strong environmental gradient, whereas the second axis described a weaker gradient (eigenvalue = 0.05, explained variance = 12 %). on the first axis, rh contributed more (biplot score = 0.66) than temperature (biplot score = 0.14). the cca scores for each site on the first axis are shown in table 1. the site of viviparous s. algira tingitana showed the highest cca score (i.e., highest rh values), whereas the site in the central rif had the lowest score. the permanova analysis revealed that most of the studied populations occupied a distinct position on this gradient (table 1). the sites occupied by these populations showed variable microclimatic stability: the site of the viviparous population had the lowest ad values (i.e., greatest microclimatic stability), whereas both populations of s. algira splendens (located at higher altitudes) had the highest ad values (i.e., lowest microclimatic stability; table 1). our data reveal the existence of suitable conditions for surface activity between november and april at all sites, although these conditions may extend from october to may at higher altitudes (fig. 3). this period coincided with the appearance of larvae in all surveyed water bodies during november, although at one site larvae were detected from september (table 2). the presence of larvae in the water bodies extended until the month of march, and in may, larvae were only observed in one water body. the standard deviation of larval size (i.e., size variability) was greatest in january and the maximum mean larval size was observed in march (table 2). in all the cases, larval abundance and size variability did not correlate significantly with the size of the water body, od, or air temperature (table 3). discussion our results indicated an autumn-winter reproductive period for all the ovoviviparous populations of salamandra algira examined. at the beginning of this period, there was an increase in the suitable conditions for activity in this species, which possibly correlate with the onset of reproductive activity. winter breeding activity is usually found in circum-mediterranean populations of the genus salamandra (degani and warburg, 1995; rebelo and leclair, 2003), possibly in response to higher autumn-winter precipitation and the occurrence of mild temperatures. the first larval cohort was found in november, and the larvae were small and uniformly sized. the increase in size heterogeneity observed in january may be attributable to the co-occurrence of several larval cohorts (produced by several episodes of larval deposition) within a single pond. in most of the water bodies examined, there was a reduction in the number of larvae and their size variability in march, which indicated the end of new larval recruitment, the metamorphosis of the first cohorts, and the possible exist     fig. 2. plot of mean temperatures (a) and relative humidity (b). northern tip of tingitana peninsula (n tingitana), middle atlas, central rif, western (w) rif high, western (w) rif low and beni-snassen. 37microclimate conditions in salamandra algira ence of an increasing cannibalistic pressure exerted by the larger larvae (escoriza et al., 2006b). this decline in larval abundance was not correlated with water body size or microclimatic conditions. as in other salamander species, there is a minimum time period required for metamorphosis, which may be related to water temperature and available trophic resources rather than to the hydroperiod (maret and collins, 1994; díaz-paniagua et al., 2005; ryan, 2007). this result also suggested that, in some populations, although there were suitable conditions for adult activity, individuals did not breed in late winter / early spring. however, at one site, large larvae were detected in mid-september, possibly caused by an episode of larval deposition during late spring and the persistence of the larvae in the water body during the summer (escoriza et al., 2006b). metamorphosis occurred at larval lengths of 62-69 mm. at all sites, the decline in the number of larvae per water body (probably due to the beginning of larval metamorphosis) coincided with the existence of suitable air temperature / rh conditions for post-metamorphic dispersal (temperatures of 4-14 °c and 80 %-100 % rh). at sites located at higher altitudes, activity may cease for a few days in the month of january, when temperatures drop below 2 °c. at lower altitudes, conditions were possibly suitable for activity throughout the winter.   fig. 3. days with optimal conditions per site, number of days per month in which environmental conditions are suitable for surface activity (air temperature between 4-14.9 ºc and relative humidity > 80 %). northern tip of tingitana peninsula, black (n tingitana); middle atlas, aquamarine; central rif, green; western (w) rif high, blue; western (w) rif low, red; beni-snassen amethyst. table 2. data measured on five sampling occasions during one year period at seven aquatic habitats that correspond to different populations of salamandra algira. w: western. at two sites (w rif high and middle atlas) two water bodies were sampled. w rif (low) w rif (high) w rif (high) central rif middle atlas middle atlas beni snassen number of s. algira larvae september november january march may 0 4 3 0 0 0 5 5 1 0 0 14 13 1 0 0 9 13 0 0 0 6 7 5 0 2 19 19 12 3 0 7 4 1 0 average total length of larvae (mm) september november january march may 0 43.4 52.3 0 0 0 38.8 55.5 62.9 0 0 40.9 47.3 69.2 0 0 40.9 48.9 0 0 0 42.3 43.1 56.2 0 56.5 35.0 40.1 51.1 53.9 0 34.6 41.9 64.9 0 standard deviation of total length of larvae september november january march may 0 4.2 5.2 0 0 0 3.5 2.9 0 0 0 3.6 3.9 0 0 0 3.8 8.9 0 0 0 3.4 11.5 5.0 0 0.7 7.4 8.0 2.8 1.9 0 3.2 5.3 0 0 surface area × average depth september november january march may 0 24 69 60 32 0 28 28 28 21 0 9 4 4 3 2 2 2 2 2 0 10 13 3 0.6 0.7 0.7 1 0.7 0.7 0 0.1 0.1 0.1 0.1 air temperature (ºc) at 1200 h september november january march may 25.6 15.9 11.0 18.6 26.8 28.3 14.4 7.7 18.5 25.8 28.3 14.4 7.7 18.5 25.8 19.8 7.0 3.5 16.5 17.8 27.5 12.9 7.6 17.9 20.8 27.5 12.9 7.6 17.9 20.8 21.1 9.7 7.4 15.4 18.1 38 d. escoriza, j. ben hassine our results indicated that the examined populations were largely segregated along a gradient of humidity, with some populations showing higher and more constant values of rh than others. this suggests a variation in the suitability of environmental conditions across sites, which may be related to variations in adult density, as described for other species (degani and warburg, 1978). however, in the beni snassen massif, where s. algira is extremely localized (escoriza and comas, 2007), local microclimate conditions were very suitable for this species. this could be an effect of the spatial resolution at which the data were obtained. the discontinuous vegetation cover in the beni snassen massif (tobi et al., 2000) could cause rapid spatial changes in environmental parameters when the buffering effects of vegetation are lost (rykken et al., 2007). this fact would explain the confinement of this population to a few favourable areas. the viviparous population occupied a climatic niche distinct from that occupied by the other studied populations of s. algira, with more stable microclimatic conditions throughout the year. similarly, based on macroclimatic niche models, beukema et al. (2010) also found that these localities showed a lower annual thermal range when compared with those occupied by ovoviviparous populations. this microclimate stability arise from the particular conditions of the gibraltar strait region, with its constantly high air humidity (mejías et al., 2007). this population inhabits a calcareous massif with poor vegetation cover (martínez-medina et al., 1997). a dense vegetation cover reduces the oscillation of air temperature and soil moisture (rittenhouse et al., 2008), and for this reason the occurrence of terrestrial salamanders is usually related to the structure of vegetation (herbeck and larsen, 1999). however, as in other coastal populations of salamanders (diller and wallace, 1994), the constant air humidity may reduce the salamanders’ dependence on the buffering effects of vegetation cover. our data offered new insights into the ecology of a little-known salamander species that is distributed in relict populations in northern morocco. these data suggest a link between the presence of s. algira and high moisture levels. environmental moisture is most stable in mature forests with a closed canopy and dense shrub cover, but may change dramatically with perturbation of the plant cover. in recent decades, there has been a significant loss of forest areas in northwest africa (zaimeche and sutton, 1997) with the spread of agriculture, and this loss has reached 42 % of the surface area in the rif mountains (taïqui, 1997). proper protection of these forests, with consequent preservation of their unique microclimatic environments, will be essential for the survival of most populations of s. algira. acknowledgments fieldwork in morocco was authorised by various scientific permits provided by the haut commissariat aux eaux et forêts et à la lutte contre la désertification (hceflcd / dlcdpn / cff), rabat, morocco. this work was supported by a grant from the mohamed bin zayed species conservation fund (project 13057819). the authors are also grateful to two anonymous reviewers whose comments have helped in improving the manuscript. references anderson, m.j. 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(1997): the future of the environment in algeria, morocco and tunisia. j. north afr. stud. 2: 40-57. acta herpetologica vol. 8, n. 2 december 2013 firenze university press journal of the societas herpetologica italica acta herpetologica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 8(2): 105-113, 2013 easy life of males? indirect evidence that growth is easier than egg production in mangrove-dwelling monitor lizards (varanus indicus) petra frýdlová1, jan hnízdo2, petr velenský3, olga šimková1, veronika cikánová1, lenka chylíková2, daniel frynta1,* 1department of zoology, faculty of science, charles university in prague, viničná 7, cz-128 44 praha 2, czech republic. *corresponding author. e-mail: frynta@centrum.cz 2animal clinic, bílá hora, čistovická 44/413, cz-163 00 praha 6, czech republic 3prague zoo, u trojského zámku 3, cz-171 00 praha 7, czech republic submitted on 2013, 4th march; revised on 2013, 17th july; accepted on 2013, 18th july. abstract. in male-larger species of animals, males typically continue to grow after the age of female sexual maturation has been reached. consequently, a switch of energy allocation occurs as the female investment from growth is shifted into egg production. we focus on the transitional period when both sexes heavily invest into anabolic processes; males invest in the development of body tissues while females predominantly invest in egg production. in captive mangrovedwelling monitor lizards, we found that relative food intakes as well as quantitative estimates of anabolic processes (relative growth and egg production rates) are fairly comparable between the sexes. in spite of this biochemical clinical values and body condition indices revealed sex differences suggesting costs of reproduction in females. these results clearly illustrate that growth and egg production still substantially differ in associated physiological costs. this may be attributed to qualitative requirements (nutrients, minerals, etc.) of these processes. our results correspond well with the higher susceptibility and mortality rates of females than males in many lizard species in captivity. keywords. bimaturism, biochemical clinical values, growth rate, reproductive investment, sexual size dimorphism, varanidae introduction in most animals, fundamental differences between the biological roles of males and females arise as an inherent consequence of sexuality (trivers, 1972, 2002). ova are considerably larger gametes than sperms and consequently, the value of minimal expenditure per offspring is much higher in females than in conspecific males (trivers, 1972, 2002). this potential inequality in reproductive investment between the sexes responsible for sexual selection is especially highlighted in animal taxa producing large offspring, e.g., terrestrial vertebrates (clutton-brock, 1988). in these animals, females are imprisoned to pay heavy metabolic costs to produce eggshells and yolk (or equivalent nutrition for the embryos in the case of viviparous taxa). in contrast, male investment (except in species with paternal care) is more variable and a considerable part of it is allocated to activities associated with intrasexual competition, e.g., territory defence, male combat, and mate guarding (darwin, 1871; andersson, 1994). in reptiles, the females’ cost of reproduction was demonstrated repeatedly (itonaga et al., 2012; vitousek et al., 2010; for a review see schwarzkopf, 1994). fitness of an individual depends, besides other factors (e. g., behavioural traits), on the decisions determining the allocation of available resources to particular life-history traits. consecutive hierarchical allocation of female resources was clearly demonstrated in paroedura picta, a small male-larger gecko with an invariant clutch size. its females’ investment is first allocated to growth, second to reproduction and finally to fat storage (kubička 106 petra frýdlová et al. and kratochvíl, 2009). also, in other male-larger lizards, females usually slow their growth after maturation and allocate further investments preferably to reproduction. in contrast, males of the same age continue to grow intensively as larger male size is usually attained by prolongation of the growth period, resulting in sexual bimaturism (stamps and krishnan, 1997; frynta et al., 2010). supposing that other energetic expenses (e.g., basal metabolism, locomotion, etc.) are comparable between the sexes and that animals allocate all remaining energy to anabolic processes, these should also be comparable between males and females during this transitive period. nevertheless, males spend the entire energy budget into the building of body tissues, while females allocate the resources saved in the expense of growth into the production of eggs. investments into the production of eggs are constrained by the availability of minerals as calcium (de buffrénil and francillon-vieillot, 2001; kratochvíl and frynta, 2006). thus, we may expect qualitative differences between the production of eggs and body tissues. we hypothesise that it is not the energetic expenses, but rather the qualitative differences that may determine the relative costs of male and female types of investment. the period of ontogeny when both sexes are pressed to maximize anabolic processes, but differ in their preferred allocation, provides a promising model. males allocate the energy into the growth of their bodies, while females into the egg production. we can compare their investments in terms of produced biomass, required energy (both scaled to body mass and/or metabolism), and associated health costs. the mangrove-dwelling monitor, varanus indicus (daudin, 1802) belongs to the subgenus euprepiosaurus (cf. ast, 2001; böhme, 2003). this group consists mainly of closely related forms that were recently split from v. indicus sensu stricto (ziegler et al., 2007; koch et al., 2009). varanus indicus is capable of indeterminate (de buffrénil et al., 2004) and rapid growth (cf. cortical vascularization of long bones; de buffrénil et al., 2008). the mangrove-dwelling monitor lizard is among the most sexually dimorphic monitor lizards of the family varanidae (frýdlová and frynta, 2010). this sexual dimorphism especially concerns body size, while shape differences between the sexes are rather small (frýdlová et al., 2011). under natural conditions, males are about 2.66 times heavier (mean body mass = 1287 g) than females (484 g; wikramanayake and dryden, 1988). the degree of sexual size dimorphism (ssd) computed from asymptotic body weights estimated in captivity was as high as 2.81 (4594 g in males versus 1635g in females; frynta et al., 2010). similarly to other species of monitor lizards (auffenberg, 1981; lemm et al., 2004), v.indicus is not only large, but also a surprisingly fast-growing animal. the sexes differ both in their asymptotic body sizes and the timing of growth (corresponding to somewhat delayed inflection points of the logistic growth curves of males compared to that of females). in contrast, maximum growth rate (in the exponential phase of the growth curves) are almost the same in males and females. nevertheless, the absolute growth rates, and partially also relative growth rates in the next phase of growth (at the age of 500-1000 days), continue to be high in males, while females gradually stop to grow and switch the energy investment into egg production (frynta et al., 2010). in this paper, we analysed data concerning males and females of v.indicus during the phase when males still continue to grow intensively while females retard their growth in favour of reproduction. we estimated growth rates, egg production and food intake. the data concerning the investment of the sexes into anabolic processes were compared with chemical clinical values collected at the same period to characterize the health and nutritional status of the examined animals. this allowed us to test the hypotheses that (1) the investment into anabolic processes is comparable in both sexes; (2) female investment in egg production is more costly (in terms of health parameters) than corresponding male investment into their own body tissues. material and methods the experimental animals were 34 individuals (21 males and 13 females) of mangrove-dwelling monitors, varanus indicus, reared at the prague zoo. they were siblings belonging to four subsequent clutches hatched in november 2006, january, february and june 2007. the animals were housed individually in terrariums gradually changed for larger ones to satisfy the needs of the growing animals (ending with terrariums 145 x 100 x 130 cm large). the ambient temperature in the breeding room varied from 28 to 30 ºc with hotter basking sites. chicken livers or necks were provided weekly (ad libitum), supplemented with vitamins and minerals (nutri mix, trouw nutrition biofaktory, ltd.). ad3 and e vitamins (pharmagal s.r.o.) were provided monthly. in order to provide unambiguous sex determination (cf. schildger et al., 1999; horn, 2004) and to assess the reproductive status, we employed ultrasonographic imaging. on 15 february 2009, we performed ultrasonographic examination of abdominal cavities using a linear electronic 7.5 – 14 mhz probe (sonix op, kanada) with the animal lying in dorsal recumbence. all abdominal ultrasound scans were performed systematically in a standardized fashion from cranial to caudal direction in both sections (longitudinal and sagital) and were performed by one examiner (j.h.). in females, follicle size was measured to assess the stage of ovarian cycle that may affect their physiological state. ultrasonic imaging revealed that matured follicles or ova 107growth vs. reproduction in monitor lizards were already present at the age of 506 days (frynta et al., 2010). the first clutch of unfertilized eggs was recorded at the age of 578 days (frýdlová, pers. comm.). ultrasonography revealed sexual maturation in experimental animals of both sexes and this conclusion was further confirmed by observation of mating during the experimental encounters. since the age of five months, we allowed short-time encounters between the sexes to test signs of sexual behaviour and recorded the first mating attempt of a male at the age of 522 days. the experimental animals were regularly weighed every two weeks (before feeding) and snout-vent length was measured every three months. increments of body weight (body weight as assessed at a given moment minus that at a previous weighting) were calculated for each experimental animal and each two-week period within the range of 500-1000 days. these were divided by the exact length of the period (in days) and scaled to body weight at the beginning of the period. the observed increment represents the mass of produced tissues that should be roughly proportional to the allocation of energy to anabolic processes. in females, the partial increments were affected by ovarian cycles involving gradual increase of total body weight during the growth of ova followed by an abrupt body weight loss at egg deposition. however, these temporal effects affecting the variation of the increments had no effect on mean values, because increases were fully compensated by weight losses when averaging weight increments assessed during the time frame exceeding the length of the ovarian cycle (two months). the amount of available body tissue strongly determines the quantity of metabolic processes. thus, we can reasonably expect that the body weight at the beginning of the period roughly reflects the metabolic (both anabolic and catabolic) capacity of the individual and thus, it is a reliable variable for the standardization of body weight increments. because the exponent of the allometric relationship between metabolic rate and body weight is usually (kleiber´s law; schmidt-nielsen, 1984) closer to 0.75 than to 1 (corresponding to isometry), we alternatively scaled the body weight increments to the initial body weight^0.75. the egg production of each female was monitored throughout the study and each egg was weighed prior to its placement into the incubator. because the intervals between clutches were fairly regular, lasting about two months, we were well informed about the approximate timing of egg laying. during this period, the behaviour (e.g., digging) of the females was observed to prevent egg cannibalization and/or damage. food intake was measured as the amount of food that disappeared from the feeder (plastic box with surplus food corresponding to about 30% of the body weight) during 24 hours. it was assessed once on 19-20 january 2009 (liver diet; further referred to as period 1) and repeatedly 4-5 times from 2 march to 6 july 2009 (chicken neck consumption; further referred to as period 2). the age of the experimental animals at the time of food intake assessment varied within the range of 590-810 and 632-978 for periods 1 and 2, respectively. food conversion rate was calculated as food consumption (in grams) divided by body weight increment assessed in a given two-week period (in grams). the health condition of the experimental animals was monitored regularly. on the 14 december 2008, 15 february 2009 and 25 april 2009, we performed complex veterinary surveys including the collection of blood samples for the assessment of biochemical clinical values. blood samples were taken from the ventral caudal tail vein. the blood was sampled by direct puncture with a 20g needle and a 2 ml heparinized syringe. about 0.8 to 1.5 ml of whole blood was aspirated and directly sent for biochemical analysis, blood smear and blood count. for biochemical testing whole blood was analysed using the dry chemistry system spotchem ez (medista, cz). we measured 8 biochemistry values: concentration of albumin (alb), transferins = other protein (trans; calculated as total protein alb), total bilirubin (t bil), glucose (glu), calcium (ca), inorganic phosphorus (ip), uric acid (ua), alkaline phosphatase (alp). the concentrations of proteins (alb, trans) are usually treated as markers of nutritional status and body reserves; glucose (glu) levels reflect the actually available energy; relationship between the concentrations of main minerals (ca, ip) reflects the calcium balance; and the elevated concentrations of excretion products of metabolism (t-bil, ua) and alp are treated as markers of uncompensated metabolic load (campbell, 2006; diethelm and stein, 2006). scaled mass index (smi) reflecting body condition was calculated after peig and green (2009, 2010): smi = mi * (lo/ li)bsma (mi = body mass; li = body length, i.e., svl in our dataset; lo = arbitrary value of li, e.g., close to arithmetic mean values of body length in study population; bsma is a slope of reduced major axis of body mass versus body length allometric relationship). empirical rma slope calculated from our data was 3.3225, lo was set to 400 mm). the rma slope was calculated from entire data points (pseudoreplicates) available for the range of 500-1000 days. prior to this analysis, we confirmed that the slope of the allometric relationship of body weight and svl did not differ between the sexes. for this purpose, we run a marginal gls model (see below) accounting for repeated measures that revealed no effect of svl*sex interaction (f = 0.03 and p = 0.8557). the values of smi were highly correlated (r = 0.957) with those of fulton’s index of condition which is calculated as k = m/l3. thus, both these indices of body condition were mutually interchangeable and, following peig and green (2010), we selected smi for further analyses of body condition at the time of blood sampling. we used statistica analysis system (version 6.0) for most calculations. the marginal models including accounting for repeated measures was performed in r version 2.10.0 (r development core team 2009) using gls function as implemented in the nlme package. symmetric correlation structure was selected for these analyses. the experiments were performed in accordance with the czech law implementing all corresponding european union regulations and were approved by the institutional animal care and use committee (5530/2008-30, 8388/2011-30). results investment to body growth and egg production median values for body size of males and females during the study period were: 432.4 mm (n = 21, range 108 petra frýdlová et al. 333.0 – 550.7 mm) and 369.6 mm (n = 13, range 308.3 – 433.2 mm), respectively. weight increments per day scaled to body weight^0.75 were examined using marginal models accounting for repeated measuring of the same individual (pseudoreplications). first, we included sex, log-transformed age and their interaction as factors. the effect of the interaction was not significant (f(1,985) = 0.34, p = 0.5606) which suggested homogeneity of slopes and allowed us to remove the interaction from the model (this decision was confirmed by the log-likelihood ratio test at α = 0.05). the reduced model revealed that weight increments per day scaled to body weight^0.75 were higher in males than in females (f(1,986) = 103.47, p < 0.0001, coefficient for males = 0.00888, se = 0.000873) and that they decreased sharply with log-transformed age (f(1,986) = 47.90, p < 0.0001, coefficient = 0.02004, se = 0.00275; fig. 1). thus, male investments into the production of new body tissues is higher than that of females of the same initial body mass and this difference may be interpreted as the anabolic capacity saved by the female in expense of growth. in order to estimate the amount of the anabolic capacity saved during a single ovarian cycle by a typical female, we performed the following calculations. per two month period (= the typical inter-clutch interval) and initial female body weight^0.75, a female typically saves the amount of reserves needed to build 0.533 grams of its body (= 0.00888[grams per day]*60[days]). considering that at the age of 500-1000 days, the mean female body mass was 1071 g (n = 368 pseudoreplicates), which corresponds to 187.7 in terms of weight^0.75, a female saved 99.8 g of its body weight (= 0.533*187.7) that may be invested elsewhere (e.g., into egg production). because mean weight of eggs produced by these females was 25.21 g, this represents a rough equivalent of four eggs (99.8/25.21 = 3.96 eggs). we alternatively performed this calculation based on the relationship between body weight increment and untransformed body weight of the female. the difference between the intercepts of this relationship in males and females (i.e., the coefficient for the male sex) was 0.066145. thus, per two month period and per gram of its initial body weight, a female typically saves the amount of reserves needed to build 0.066145 grams of her body (0.066145 = 0.001102409 [grams per day]*60[days]). a female of mean body weight of 1071 g thus saved the investment of 70.8413 g (= 0.066145*1071), which corresponds to 2.81 eggs. during the study period, we recorded 28 clutches (n females = 10) consisting of 1-10 eggs. the mean clutch size and mean egg weight were 3.96 eggs and 25.21 g, respectively. food intake, its scaling and rates of food conversion into body mass we found no sex differences in food intake per body weight (period 1: means = 0.599 and 0.611 in males and females, respectively; n (males) = 21, n (females) = 13; t = -1.09, p = 0.2841; period 2: 0.405 and 0.426; t = -0.79, p = 0.4351). when food intake was scaled to body weight^0.75, these values appeared somewhat higher in males than in females. nevertheless, this difference was statistically significant in period 2 only (period 1: 2.690 and 2.405; t = 1.694, p = 0.1000; period 2: 1.812 and 1.436; t = 3.40, p = 0.0018). in males, allometric relationships between food intake and body weight were clear (r = 0.802 and 0.554; ps<0.0001), the intercepts (a) and slopes (b) of these relationships were a = 1.3271, b = 0.702 (seb = 0.120, n = 21) and a = 1.2377, b = 0.658 (seb = 0.109, n (pseudoreplicates) = 84), in the first and second period, respectively. the confidence intervals of the slopes overlap the theoretical values 0.75. in females, this allometric relationship was significant in the first period (a = 2.1266, b = 0.568, seb = 0.212, n = 13, r = 0.631, p = 0.0209), but not in the second period (a = 8.0803, b = -0.340, seb = 0.299, n (pseudoreplicates) = 53, r = -0.157, p = 0.2608). the homogeneity of slopes test revealed sexual differences in allometric slopes fig. 1. plot of the relationship between relative weight increments (rwi, grams per day per weight^0.75) and log-transformed age (ln age) in captive mangrove-dwelling monitor lizards at the age of 500-1000 days. lines indicate ordinary least-square (ols) regressions computed separately for males and females. the equations of these regressions are as follows: females: y = 0.128960606 0.0180518898*x (r = -0.1788, p = 0.0006); males: y = 0.160058468 0.0214532419*x (r = -0.2630, p < 0.0001). triangles are males, circles are females. 109growth vs. reproduction in monitor lizards during the second (f(1,133) = 12.53, p = 0.0006; f(1, 119.95) = 16.04, p<0.001), but not the first period (f(1,30) =0.27, p = 0.6096). in the first period, the mean food conversion rate was 18.2% in males and 13.8% in females (mann-whitney test, n(males) = 21, n(females) = 13, z = 0.62, p = 0.5351). in the second period, the food conversion rate was 16.8% in males and 1.3% in females, and this difference (computed from mean values for each individual) was significant (mann-whitney test, n(males) = 21, n(females) = 13, z = 3.07, p = 0.0022). biochemical values and body condition index means and other descriptive statistics of non-transformed biochemical values are given in table 1. we performed pca in order to examine the correlation structure among the studied biochemical values to reduce the number of examined traits. pc1 and pc2 accounted for 34.1% and 21.3% of variation in the original log-transformed variables, respectively (see fig. 2 for loadings). pc1 is correlated positively with bilirubin, phosphor and uric acid (i.e. metabolic residues) and negatively with albumin, glucose and calcium (i.e., the actually available sources of amino acids, energy and minerals). thus, it may be interpreted as a measure of body reserves deficit. pc2 scores increase with values of albumin, calcium, transferins and uric acid. pc scores were further examined to assess the effects of sex; pc1 scores were smaller in males than in females and this difference was significant (marginal gls: f(1,88) = 12.98, p = 0.0005, intercept = 0.6327297, se = 0.2059295, coefficient for males = 0.8899365, se = 0.2470415). no significant sex differences were found in pc2 scores (marginal gls: f(1,88) = 1.96, p = 0.1648). in females, pcs were apparently not associated with follicle size (pc1: n = 10; r = 0.133, p = 0.7139; pc2: n = 10; r = -0.209, p = 0.5624), nor the presence/absence of egg laying during the week preceding blood sampling (n (pseudoreplicates): absent = 19, present = 8; pc1: t = 1.20, p = 0.2426; pc2: t = 1.14, p = 0.2664). the scaled mass index (smi) was slightly, but significantly higher (f(1,83.54) = 6.24, p = 0.014) in males (mean = 1603; se = 34.99) than in females (mean = 1498; se = 23.07). nevertheless, the relationship between this body condition index and pc1 of biochemical values was not tight and interacted with sex (f(1,61.5) = 6.84, p = 0.011; fig. 3). table 1. means, standard deviations (sd), medians, percentiles (5 and 95%) of the clinical chemical values in the males and females of the mangrove-dwelling monitor lizards (varanus indicus). the results of the testing of the sex differences by repeated measures glmms (f value, degrees of freedom and p value) are provided in the right column. abbreviations: m – male, f – female, n – number of pseudoreplicates (blood samples), alb – albumin, trans transferins (= other protein calculated as total protein alb), t-bil total bilirubin, glu glucose, ca calcium, ip inorganic phosphorus, ca/ip – ratio of calcium and inorganic phosphorus, ua uric acid, alp alkaline phosphatase. significant comparisons are indicated by asterisks. sex n mean sd median 5% 95% f (df ) p alb m 63 20.59 3.87 21.0 13.0 27.0 1.77 (1, 74.4) (g/l) f 36 19.28 4.37 19.0 12.0 28.0 0.188 trans m 63 47.19 7.03 46.0 38.0 58.0 0.24 (1, 60.1) (g/l) f 36 48.19 12.16 46.0 31.0 76.0 0.624 t-bil m 63 8.70 4.39 7.0 5.0 17.0 4.64 (1, 80.0) (µmol/l) f 36 12.75 6.04 12.0 4.0 23.0 0.034* glu m 63 6.55 1.09 6.5 5.2 8.4 12.30 (1, 69.3) (mmol/l) f 36 6.13 1.50 6.0 4.2 8.9 0.001* ca m 63 2.84 0.32 2.9 2.2 3.3 1.20 (1, 77.7) (mmol/l) f 36 2.81 0.46 2.8 2.1 3.7 0.277 ip m 63 1.67 0.34 1.6 1.2 2.3 7.27 (1, 77.9) (mmol/l) f 36 2.02 0.80 1.9 1.2 4.5 0.009* ca/ip m 63 1.78 0.42 1.7 1.1 2.4 (mmol/l) f 36 1.55 0.52 1.5 0.7 2.4 ua m 63 194.75 59.49 188.0 112.0 322.0 50.15 (1, 64.49) (µmol/l) f 36 309.50 97.11 302.5 180.0 528.0 <0.001* alp m 63 6.41 1.20 6.5 4.2 8.4 <0.01 (1, 86.8) (µkat/l) f 36 6.27 1.33 6.2 4.1 8.2 0.961 110 petra frýdlová et al. discussion investment to body growth and egg production authors studying maternal investment in lizards adopted the concept distinguishing between income and capital breeders (schwarzkopf, 1994 and references herein; for concept see drent and daan, 1980). income breeders produce eggs exclusively in the expense of actual food intake. in contrast, capital breeders are able to accumulate and store fat reserves for a long time period and invest them all at once. nevertheless, these strategies represent a continual scale rather than a strict dichotomy. it was demonstrated in the scink eulamprus tympanum that litter size depends on fat reserves while new-borns’ body size depends on the actual energetic income (doughty and shine, 1998). recently, warner et al. (2008) measured isotope contents in australian jacky dragons (amphibolurus muricatus) and demonstrated that the energy allocation strategy used for reproduction differs sharply even among the egg components. egg lipids follow the capital breeding strategy, while egg proteins follow both the income and capital ones. this may be explained by easier storing of fat compared to proteins. as many other tropical lizards, mangrove-dwelling monitor lizards are predominantly income breeders. this assumption was supported by their growth patterns (frynta et al., 2010; this study). in order to quantify sex differences in the quantity of anabolic processes during the examined period (age 500-1000 days), we measured the gains of body weight as well as the weight of produced eggs. in v. indicus, mature females produce clutches in regular intervals of approximately two months. our females produced about 100 g of eggs per single ovarian cycle. this value fits well in our estimates of the mean difference between the body weight gains of males and females per two month period (rescaled to mean female body size in the study period). it equals to 99.8 g or 70.8 g, for allometric models with slopes 0.75 and 1, respectively. thus, the egg production is realized mainly in the expense of saved metabolic reserves that are devoted to growth in males. nevertheless, the energetic costs associated with the production of the body tissue may be somewhat lower than those associated with the production of the same amount of egg mass. although the energetic value of eggs expressed in calories per dry weight is only slightly higher to that of body tissues (congdon et al., 1978), we are aware that the differences in water content between these substances may affect this comparison considerably. the temporal coincidence and quantitative correspondence between growth reduction and beginning of egg laying provides no conclusive information about the direction of the putative causal relationship between these two events. to solve this, experimental manipulafig. 2. variation structure of the clinical chemical values in captive mangrove-dwelling monitor lizards visualized by loadings of the principal components (pc1 and pc2). abbreviations: alb – albumin, trans transferins (= other protein calculated as total protein alb), t-bil total bilirubin, glu glucose, ca calcium, ip inorganic phosphorus, ca/ip – ratio of calcium and inorganic phosphorus, ua uric acid, alp alkaline phosphatase. fig. 3. the relationship between the principal component 1 of the clinical chemical values (pc1) and the scaled mass index (smi) of the body condition at the time of blood sample collection in the captive mangrove-dwelling monitor lizards (varanus indicus). triangles are males, circles are females. 111growth vs. reproduction in monitor lizards tions with egg size and/or number (landwer, 1994; sinervo, 1994), castration (cox et al., 2010) and food intake (kubička and kratochvíl, 2009) are required. food intake, its scaling, and rate of food conversion into body mass it should be considered that our experimental study uses captive-bred animals. ad libitum feeding, the absence of predation pressure, stability of environmental condition and the lack of intraspecific agonistic interactions would affect the experimental animals considerably. compared to wild animals, the captive ones are capable to invest more energy to the body growth and reproduction (mendyk, 2011). retes and bennett (2001) notify extreme cases of multiclutching (14 consecutive clutches in a 14-month period) in females of v. glauerti when treated under ad libitum conditions. in our study, the relative amount of food consumed by males was roughly equal or even somewhat higher than that consumed by conspecific females. as food was provided ad libitum, the female metabolic loads did not substantially exceed those of the males during the examined period. although males were larger than females, this conclusion remained substantially unchanged irrespective of scaling the food intake alternatively to body weight or predicted baseline metabolism (estimated as body weight^0.75). there are theoretical arguments supporting each of these scaling strategies (sibly et al., 2012) and our empirical data suggest that the exponents of the allometric relationship between food intake and body weight support the exponent 0.75 when ols regression (supposing that the error in body weight is negligible) is considered. however, the rma regression model (considering errors in both axes) would result in somewhat higher slopes close to 1. moreover, during period 2, there was no clear association between food intake and body weight in females, probably as a result of the great variation in both the food consumption and the actual body weight in egg-laying females. we found that the rates of food conversion into body mass were initially (period 1) almost the same in males and females. nevertheless, in period 2, these rates dramatically decreased due to the reduction of female investment into growth and the beginning of egg production. biochemical values and body condition index the clinical chemical values reported by previous authors in other species of monitor lizards (v. varius: jessop et al., 2011; v. komodoensis: gillespie et al., 2000; lemm et al., 2004; v. dumerilii: bertelsen et al., 2007) are generally comparable to those of this paper. the differences are small enough to be attributed to differences in the methods of the assessment and small sample sizes. we found that during the examined ontogenetic period, males of v.indicus exhibited higher values of glucose and lower values of urea, bilirubin, and phosphor, than females. we interpret these differences as indirect evidence that the body reserves and health condition of females were more limited than those of males. this corresponds well with the sex differences in the scaled mass index suggesting better body condition of males. because the relative food intake, as well as the rates of the produced body/egg mass were at least comparable between the sexes (see above), the impaired body reserves and/or health condition of females cannot be explained by the increased loads of metabolic and/or anabolic processes in this sex. thus, qualitative rather than quantitative differences between the loads associated with the production of eggs and body mass should be considered. this may lead to the conclusion that the cost of female duties is heavier than that of male ones. although fairly intuitive, such evidence was not previously reported, at least not in large carnivorous lizards. however, temporal instability of calcium, phosphor and magnesium levels was clearly demonstrated in the reproducing females of komodo dragon (v. komodoensis; lemm et al., 2004). recently, laver et al. (2012) revealed substantially higher mortality rates in females than in males of wild v. komodoensis and attributed this finding to the heavy costs of reproduction in females. this further biases the operational sex ratio in favour of males and intensifies the strength of the sexual selection. according to life-history models (charnov, 2011; charnov and warne, 2011), increased mortality should further reduce the investment into growth, health and longevity in females. in conclusion, the empirical experience of breeders that females are a more susceptible and gentle sex was supported even in the model lizard with quantitatively comparable metabolic and anabolic rates in the sexes. this may be interpreted as a demonstration of a load associated with female life-history. obviously, this load results from qualitative rather than quantitative differences between the metabolic duties of the sexes. this study was performed to stimulate further attention to these phenomena. nevertheless, as any observational study, it is limited to correlative results and their interpretations. thus, there is an urgent call for manipulative experiments enabling to uncover the causative relationships and providing independent controls (e.g., for sex differences in the absence of growth and/or reproduction). 112 petra frýdlová et al. acknowledgements we thank ivan rehák for encouragement and valuable comments and mr. v. straka, medista, who kindly provided equipment (spotchem ez analyzer) and material for biochemical analysis of the blood samples. we are grateful to fritz rickhoff and silvie lišková who kindly improved the english. the project was supported by the grant agency of the charles university (no. 62910/2010). references andersson, m. 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(2002): natural selection and social theory. oxford university press, u.s.a. vitousek, m.n., mitchell, m.a., romero, l.m., awerman, j., wikelski, m. (2010): to breed or not to breed: physiological correlates of reproductive status in a facultatively biennial iguanid. horm. behav. 57: 140146. warner, d.a., bonnet, x., hobson, k.a., shine, r. (2008): lizards combine stored energy and recently acquired nutrients flexibly to fuel reproduction. j. anim. ecol. 77: 1242-1249. wikramanayake, e.d., dryden, g.l. (1988): the reproductive ecology of varanus indicus on guam. herpetologica 44: 338-344. ziegler, t., schmitz, a., koch, a., böhme, w. (2007): a review of the subgenus euprepriosaurus of varanus (squamata: varanidae): morphological and molecular phylogeny, distribution and zoogeography, with an identification key for the members of the v. indicus and the v. prasinus species groups. zootaxa 1472: 1-28. acta herpetologica vol. 8, n. 1 june 2013 firenze university press journal of the societas herpetologica italica acta herpetologica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 8(2): 129-140, 2013 road ecology and neotropical amphibians: contributions for future studies paula eveline ribeiro d’anunciação1,*, priscila silva lucas2, vinícius xavier silva1, alex bager2 1laboratório de ecologia de fragmentos florestais (ecofrag), instituto de ciências da natureza (icn), universidade federal de alfenas, r. gabriel monteiro da silva, 700, alfenas, mg, cep: 37130-000, brazil. * corresponding author. e-mail: paulaevel@yahoo.com.br 2laboratório de manejo e conservação ambiental, setor de ecologia, departamento de biologia, universidade federal de lavras, campus universitário, cep 3037, lavras, minas gerais, cep 37200-000, brazil submitted on 2012, 16th february; revised on 2013, 25th july; accepted on 2013, 27th september. abstract. many species of amphibians have suffered serious population declines. several factors contribute separately or jointly to these declines. however, the reduction of an available habitat due to human expansion is still the main cause, and roads are a major mean for this expansion. both the construction phase and the subsequent use of roads have negative consequences for amphibians. we reviewed the literature on the subject within the neotropical context. to this end, the paper begins with a summary of recent reviews and proceeds through an analysis of sampling methods used in roadkill studies, mitigation measures and the neotropical scenario and concludes with several suggestions to guide future studies. more attention will be given to roadkills, which is one of the primary impacts on wildlife that is caused by roads. even in the neotropical zone most studies are foot-based, the richness and abundance of amphibians affected are higher in regions outside the neotropics. one possible explanation is that in the other regions, the proportion of studies exclusively on amphibians is bigger. regarding mitigation measures, most studies only indicates what should be used, but do not implement or evaluate their effectiveness. keywords. neotropical amphibians, roads, roadkill, mitigation, review. introduction currently, amphibians have attracted the attention of researchers and conservationists due to population declines that have been recorded worldwide (43% of species), with 168 species being considered extinct (global amphibian assessment, 2012), the highest values than any other group of vertebrates (stuart et al., 2004). several factors appear to contribute separately or jointly to these declines, such as fragmentation, loss or destruction of habitat, diseases such as chytridiomycosis, overexploitation, reduction of the ozone layer and acidic rain (weygoldt, 1989; young et al., 2001; eterovick et al., 2005). the primary cause, however, appears to be the reduction of suitable habitats due to an expansion of human activities on natural areas, which is largely enhanced by roads (glista et al., 2008). there are several recent reviews on the environmental impacts of roads (e.g. fahrig and rytwinski, 2009; rytwinski and fahrig, 2012) and how they affect populations and communities of amphibians (e.g. colino-rabanal and lizana, 2012; beebee, 2013). however, there is no study with emphasis on neotropical proposing the standardization of methods that facilitate future comparisons between different studies. the neotropical region includes countries with the largest absolute richness of amphibians and with the largest number of endangered species in the world (global amphibian assessment, 2012). many of the measures that would help reducing the impact of roads on amphibians can be used for other animal groups and geographic regions. both the construction phase and the subsequent use of the roads have negative consequences for amphib130 paula eveline ribeiro d’anunciação et al. ians. several indirect effects can be mentioned: chemical pollution, for example, the application of salt to remove ice (denoël et al., 2010), sound pollution with the traffic noise interfering with male vocalization near roads (hoskin and goosem, 2010), light pollution that is known to mainly promote instant immobilization, which makes amphibians more susceptible to vehicle collisions when crossing roads (mazerolle et al., 2005) and genetic effetcs, with roads reducing gene flow and decreasing genetic diversity in amphibians (lesbarrères et al., 2003). however, death caused by vehicle-wildlife collisions is considered to be the greatest cause of non-natural deaths of vertebrates (forman and alexander, 1998), and amphibians are the most frequently killed vertebrates on roads (puky, 2004). even though vehicle-animals collisions are considered one of the greatest impacts, the road mortality rates still is underestimated, since most studies do not account the carcass removal and detection rate. properly defining road mortality rates is important to identify road stretches where concentrating mitigation measures and to determine the effectiveness of these measures (teixeira et al., 2013). in the neotropics there are no studies on the implementation of mitigation measures, or assessing their effectiveness to amphibians. in this context, the present paper aims to: i) compare the methods that have been used to evaluate roadkills impacts on neotropical amphibians with studies from other regions, and ii) assess what mitigation measures are the most frequently advocated by researchers. material and methods we conducted a literature review using eight major databases (isi web of knowledge, scopus, elsevier, jstor, sciencedirect, springerlink, wiley inter science and scielo). we also consulted the references identified in the articles resulting from the database search. for the search, we used the following keywords in various combinations: road, amphibians, vertebrates, roadkill, road effect, road impact, road mortality and mitigation measures. for the comparison of methods were selected only amphibian roadkill studies, regardless of criteria. possible problems in the methodology were identified and discussed. for the analysis of mitigation measures we selected studies with some mitigation proposals or studies that tested the measures for amphibians. the number of species of the neotropical zone and other regions was compared by mann-whitney test, since sample normality was rejected by shapiro-wilk test (p <0.05), as well as the traffic intensity in both regions. the relationship between the number of vehicles per day on the roads and the number of individuals roadkilled was tested by simple linear regression (only studies that have provided this information). the proportion of studies that has provided the information or used given method was compared between neotropics and other regions by chi-2 test. the parameters used in this test were number of studies exclusively about amphibians, survey methods, paving, sampling period, landscape description, sampling time and roadkill rates. for all tests the level of significance was 0.05. results methodological analysis in the neotropics we found 19 studies, being 12 in brazil (63.2%), three in mexico (15.8%), two in argentina (10.5%) and two in colombia (10.5%). for the studies conducted outside of neotropical zone (n = 26), 13 (50%) were performed in the united states, nine (34.6%) in europe, two (7.7%) in australia, two (7.7%) in canada, one (2.6%) in china, and one (2.6%) in turkey. the parameters studied are described in table 1. the number of amphibian species affected was higher in studies conducted in other regions than in the neotropics (u = 115.5, z(u) = 2.25, p = 0.023). the average number of individuals roadkilled was about 10 times greater in studies conducted outside of neotropical zone. there was no relationship between the number of individuals roadkilled and the number of vehicles per day on the roads, both for studies of neotropical zone (f1,6 = 0.32, r²= 0.05 p = 0.595) as for the other regions (f1,6= 3.19, r² = 0.34, p = 0.122) and for the regions pooled together (f1,14 = 0.0003, r² = 0 p = 0.987). there was no difference in traffic volume between neotropics and the other regions either (u = 18, z(u) = 1.91, p = 0.056). the proportion of studies that exclusively surveyed amphibians in the neotropics (21%) was lower than other regions (55.2%, χ2= 15.35, df = 1, p <0.001). the species most affected by roadkills in the neotropics are represented by the genera leptodactylus and rhinella. the usual survey methods used in the studies inside and outside of neotropical zone differ. the proportion of studies using patrolling by car was different (χ2= 11.87, df= 1, p < 0.001); outside neotropical zone approximately 42% were conducted by car, while in the neotropics were 15.8%. foot-based counts showed similar proportions (χ2= 0.016, df= 1, p =0.991), 36.8% for the neotropics e 37.9% for the other regions. paved roads were the major target of these studies (χ2= 0.223, df= 1, p = 0.691), both in the neotropics (92%) and outside (85.7%). the sampling period usually encompassed more than one season in both regions (χ2 = 1.10, df = 1, p = 0.335) and the proportion of studies was 61.5% in the neotropical zone and 73.7% in the other regions. furthermore, the percentage of neotropical studies (36.8%) that performed a detailed description of the studied area was 131road ecology and neotropical amphibians ta bl e 1. in fo rm at io n av ai la bl e in s tu di es o f r oa d ec ol og y co nd uc te d in si de a nd o ut si de o f n eo tr op ic s, in cl ud in g am ph ib ia ns , b ut n ot e xc lu si ve ly . r ef er en ce c ou nt ry to ta l d is ta nc e sa m pl ed ( k m ) pa vi ng tr affi c in te nsi ty ( nu m be r of v eh ic le s pe r da y) sa m pl in g m et ho ds sa m pl in g du ra tio n (d ay s) a m ph ibi an ri ch ne ss a m ph ib ia n ab un da nc e sa m pl in g tim e pe ri od (d iff er en t se as on s= df , p oi nt sa m pl in g= ps *) la nd sca pe de sc ri ptio n st ud ie s ex cl us iv ely a bo ut am ph ib ia ns r oa dki ll ra te (n um be r of r oa dk ill am ph ib ia ns pe r km ) outside neotropicsa re sc o, 2 00 5 u sa 0. 7 pa ve d 21 50 0 on fo ot 13 15 12 84 0 df 0. 87 c ar va lh o an d m ir a, 2 01 1 po rt ug al 26 pa ve d 49 57 .5 ca r 52 14 10 93 df ye s 0. 80 c le ve ng er e t a l., 2 00 3 c an ad a 11 7 pa ve d 19 11 5 ca r 2 48 m or ni ng df 7. 35 5 e4 c ol em an e t a l., 2 00 8 u sa 14 .8 un pa ve d ca r 10 4 5 58 m or ni ng an d ev en in g df 0. 04 d od d jr e t a l., 2 00 4 u sa 3. 2 pa ve d 11 00 0 on fo ot 72 8 16 47 al l d ay df ye s 7. 14 es te sz um pf e t a l., 2 01 0 u sa 8 on fo ot 4 20 df ye s fa hr ig e t a l., 1 99 5 c an ad a 84 .3 pa ve d 85 00 on fo ot 6 18 56 ev en in g ps ye s ye s 3. 67 g ib bs a nd s hr iv er , 2 00 5 u sa 1 on fo ot 6 1 66 ps ye s 11 g lis ta e t a l., 2 00 8 u sa 12 ca r 12 4 9 98 09 df ye s ye s 6. 59 g ry z an d k ra uz e, 2 00 8 po la nd 2. 51 pa ve d 35 on fo ot 51 5 10 46 m or ni ng df 81 .7 g ol di ng ay a nd t ay lo r 20 06 a us tr al ia 0. 2 on fo ot 13 8 10 29 m or ni ng ps ye s 39 5. 8 g u et a l., 2 01 1 c hi na 11 on fo ot 60 3 49 4 m or ni ng ps ye s ye s 21 .9 5 h ar te l e t a l., 2 00 9 r om en ia 83 .3 ca r 26 2 16 31 ev en in g ps ye s 0. 75 h os ki n an d g oo se m , 2 01 0 a us tr al ia 0. 4 pa ve d 69 50 on fo ot 7 ev en in g an d m or ni ng df ye s ye s k ob yl ar z, 2 00 1 u sa 4 pa ve d + un pa ve d on fo ot 10 10 18 5 ev en in g ps ye s 4. 62 la ng en e t a l., 2 00 7 u sa 35 5 pa ve d 72 09 on fo ot + c ar 96 10 m or ni ng an d ev en in g ps 19 .3 3 la ng en e t a l., 2 00 9 u sa 43 8. 4 on fo ot 10 10 39 4 m or ni ng ps ye s 0. 9 m az er ol le , 2 00 4 c an ad a 20 pa ve d 18 ca r 37 10 36 57 df ye s 4. 9 m ee k, 2 01 2 fr an ça 10 pa ve d bi cy cl e 36 0 7 53 9 aft er no on df ye s o rl ow sk i, 20 07 po la nd 48 .8 ca r 17 2 1 95 7 aft er no on df ye s ye s o rl ow sk i e t a l., 2 00 8 po la nd 52 .3 55 45 on fo ot + c ar 21 6 10 37 42 m or ni ng an d ev en in g df ye s ye s 82 .6 9 sa nt os e t a l., 2 00 7 sp ai n 28 ca r 3 3 11 5 ps ye s ye s sh w iff e t a l., 2 00 7 u sa 10 pa ve d ca r 10 95 3 39 m or ni ng df 0. 00 3 si lle ro e t a l., 2 00 8 sp ai n ca r 17 14 31 2 ev en in g ps ye s 0. 23 sm ith a nd d od d jr , 2 00 3 u sa 3. 2 pa ve d on fo ot 15 6 7 83 3 df 1. 67 to k et a l., 2 01 1 tu rk ey 14 4 ca r 8 82 df 0. 00 4 m ea n ± st an da rd d ev ia tio n   59 .1 2± 10 9. 4   84 82 ±7 12 4. 9   18 1. 9± 34 3. 9 7± 4. 2 12 76 ±2 03 6. 1         30 .7 ±8 7. 05 (c on tin ue d) 132 paula eveline ribeiro d’anunciação et al. r ef er en ce c ou nt ry to ta l d is ta nc e sa m pl ed ( k m ) pa vi ng tr affi c in te nsi ty ( nu m be r of v eh ic le s pe r da y) sa m pl in g m et ho ds sa m pl in g du ra tio n (d ay s) a m ph ibi an ri ch ne ss a m ph ib ia n ab un da nc e sa m pl in g tim e pe ri od (d iff er en t se as on s= df , p oi nt sa m pl in g= ps *) la nd sca pe de sc ri ptio n st ud ie s ex cl us iv ely a bo ut am ph ib ia ns r oa dki ll ra te (n um be r of r oa dk ill am ph ib ia ns pe r km ) neotropicsa nd ra de a nd m ou ra , 2 01 1 br az il 4. 5 pa ve d + un pa ve d ca r 6 20 df ye s a tt ad em o et a l., 2 01 1 a rg en tin a 6 pa ve d 60 25 on fo ot 30 7 35 2 m or ni ng df 1. 63 c ai ro a nd z al ba , 2 00 7 a rg en tin a 2 pa ve d 12 00 on fo ot 1 25 aft er no on df ye s ye s c oe lh o et a l., 2 01 2 br az il 4. 41 pa ve d 21 32 on fo ot 16 13 14 33 m or ni ng df ye s ye s 20 .3 g on za le zg al lin a et a l., 2 01 3 m éx ic o 11 2 pa ve d 64 on fo ot + c ar 34 1 1 aft er no on df ye s 0 g ro ss el et e t a l., 2 00 8 m éx ic o 1. 2 bi cy cl e 36 2 10 0 m or ni ng an d aft er no on ps 2. 31 h en ge m üh le a nd c ad em ar to ri , 2 00 8 br az il 12 pa ve d on fo ot 21 24 df 0. 00 6 m el o an d sa nt os -f ilh o, 2 00 7 br az il 63 pa ve d ca r 25 4 11 m or ni ng df ye s 0. 00 6 m ill i a nd p as sa m an i, 20 06 br az il 28 pa ve d m ot or cy cl e 70 2 4 m or ni ng ps 0. 00 2 m or al es -m áv il et a l., 1 99 7 m éx ic o 8 pa ve d on fo ot 51 5 9 13 9 df ye s 0. 03 pr ad o et a l., 2 00 6 br az il 19 .2 ca r 12 11 m or ni ng df 0. 04 q ui nt ér oá ng el e t a l., 2 01 2 c ol ôm bi a 6. 4 pa ve d 19 68 on fo ot 15 3 2 7 ev en in g df ye s 0. 00 7 sa nt an a, 2 01 2 br az il 40 0 m ot or cy cl e 12 5 39 m or ni ng df ye s 0. 04 sa nt os e t a l., 2 01 2 br az il 13 25 0 bi cy cl e 36 3 3 m or ni ng df 0. 00 6 si lv a et a l., 2 00 7 br az il 28 60 on fo ot + c ar + bi cy cl e 12 0 9 51 m or ni ng an d ev en in g df 0. 00 3 si lv a et a l., 2 01 1 br az il 35 pa ve d m ot or cy cl e 32 10 3 ps ye s ye s 0. 09 so uz a et a l., 2 01 0 br az il 4. 45 pa ve d 36 50 21 90 1 1 m or ni ng df tu rc i a nd b er na rd e, 2 00 9 br az il 11 0 m ot or cy cl e 30 2 68 ps 0. 02 v ar ga ssa lin as e t a l., 2 01 1 c ol om bi a 96 on fo ot 40 1 5 ps 0. 05 m ea n ± st an da rd d ev ia tio n   50 .2 ±9 2. 4   19 18 .5 ±2 07 3. 7   19 8. 3± 52 7. 1 4. 3± 3. 6 12 6± 32 7. 1         1. 53 ±5 .1 4 * s am pl in g pe rf or m ed in a g iv en p er io d, n ot in cl ud in g m or e th an o ne s ea so n. = in fo rm at io n w as n ot a va ila bl e in th e st ud y ci te d. ta bl e 1. c on tin ue d. 133road ecology and neotropical amphibians similar to studies from outside (about 42.3%, χ2 = 0.38, df = 1, p = 0.613). in most studies sampling was performed during morning (χ2 = 2.38, df = 1, p = 0.145), both for the neotropics (77%) and for outside (59%). the calculation of roadkill rates of amphibians in the neotropics was uncommon and was only performed by four studies (21%), as well as in the other regions (nine studies; 34.5%, χ2 = 3.28, df = 1, p = 0.093). mitigation measures out of the 22 studies analyzed, only three evaluated the effectiveness of proposed mitigation measures and two tested the preferences for specific attributes of crossing structures. among these studies, none is located in the neotropics. all studies that tested the efficiency, considered that the measure is effective to reduce roadkill. a large majority of studies performed in the neotropics (77%) only indicated measures without testing them. wildlife crossings (amphibian tunnels and culverts) associated with drift fences or barrier walls are among the most adopted and frequently advocated mitigation measures (table 2). discussion methodological analysis the greatest richness of amphibians in the world, which is found in the neotropics, mainly brazil (segalla et al., 2012), is not represented in the studies on the neotropical species killed by traffic. the expectation that this low number of species and individuals could be the table 2. studies that have proposed or analyzed the effectiveness of mitigation measures. reference country mitigation measure(s) monitoring duration was effectiveness tested? are measures effective? was the mitigation only proposed? o th er r eg io ns aresco, 2005 usa temporary drift fence + drainage culvert 2.5 years yes yes no clevenger et al., 2003 canada culverts no yes dodd jr et al., 2004 usa barrier wall + culvert 2 years yes yes no fahrig et al., 1995 canada barrier wall + underpasses no yes gibbs and shriver., 2005 usa barrier wall, tunnels, transporting individuals no yes gu et al., 2011 china underpasses and traffic control measures no yes hoskin and goosem, 2010 australia bridges, concrete barriers no yes kobylarz, 2001 usa barriers, underpasses, traffic signs no yes lesbarrères et al., 2004 frança amphibian tunnels 1 month * no lesbarrères et al., 2010 frança habitat replacement 4 years yes yes no mata et al., 2005 espanha culverts, underpasses and overpasses 2 months no no patrick et al., 2010 usa drift fence + culvert 3 months no no woltz et al., 2004 usa several road crossing structures 2 months * no n eo tr op ic al z on e andrade and moura, 2011 brazil drift fence + culvert no yes attademo et al., 2011 argentina underpasses or culverts + barrier wall no yes cairo and zalba, 2007 argentina underpasses no yes coelho et al., 2012 brazil passages + drift and barrier fences no yes gonzález-gallina et al., 2013 méxico drainage culverts and overpasses no yes hengemühle and cademartori, 2008 brazil traffic signs, speed bumps, passages, barrier wall no yes melo and santos-filho, 2007 brazil traffic signs, speed bumps, fences and tunnels no yes santana, 2012 brazil unspecified no yes souza et al., 2010 brazil traffic signs, speed bumps, passages, barrier wall no yes * studies that tested the preferences for specific attributes of crossing structures. = information was not available in the study cited. 134 paula eveline ribeiro d’anunciação et al. result of a lower number of vehicles traveling per day on roads in the neotropics was not confirmed, because the traffic volume in the neotropics and other regions was equal. however, one of the major characteristics of roads that have an influence on the mortality of amphibians and also of other animals is the volume of traffic. a higher number of vehicles traveling on the roads per day increases the chance of small animals such as amphibians to be roadkill (fahrig et al., 1995; hels and buchwald, 2001). the low number of roadkilled amphibians in the neotropics may also be due to samplings that included wild vertebrates in general, that is, studies not specific for quantifying roadkilled amphibians. in these studies amphibians are considered the most underestimated group (milli and passamani, 2006; coelho et al., 2008; hengemühle and cademartori, 2008; souza et al., 2010; attademo et al., 2011; santos et al., 2012; gonzález-gallina et al., 2013). the inclusion of more than one group of vertebrates in the survey objective is not often accompanied by people trained to identify all animal groups, which skews the results. although most neotropics studies have been conducted on foot, the amphibian richness affected is higher outside neotropical zone. monitoring performed at lower speeds, such as on foot or by bicycle, facilitates the finding of small specimens (silva et al., 2007; hengemühle and cademartori, 2008). at higher speed, the visualization is limited. for example, langen et al. (2007) showed that the number of animals detected by on foot surveys was approximately 50 times higher than a survey made by car. moreover, the carcasses of small animals do not remain on the road for long, either due to predators or because they deteriorate faster (slater, 2002; taylor and goldingay, 2004; antworth et al., 2005). certain behaviors of some amphibian species also affect the roadkill rates. these animals are mostly active during rainy periods and/or after rainfalls, while the ground is still wet (cairo and zalba, 2007). however, activity peak may be different among species. for example, in a study conducted in denmark, rana temporaria and r. arvalis were active soon after sunset, while bufo bufo was active between 10 and 11 p.m. (hels and buchwald, 2001). the speed and daytime movement patterns of species are important characteristics of vulnerability, as species that are slow and diurnal are more likely to be roadkilled are those (cairo and zalba, 2007). nevertheless, the migration of amphibians occurs mainly at night, when traffic volume is reduced (approximately 80% of traffic volume occurs during the day; festin, 1996). in the neotropics only one study was made at night, this may have reduced detection rate. spatio-temporal factors can influence amphibian roadkills, coelho et al. (2012) demonstrated a significant concentration of amphibian roadkills in summer and showed that variables such as average daily temperature, precipitation and photoperiod (variables strictly related to amphibian activities) were the most important factors related to the temporal distribution of roadkills of amphibians in general and of particular species, such as l. latrans, r. icterica and hypsiboas faber. the spatial distribution of roadkills was not random. amphibian mortality was related to types of land cover, distance from water bodies, artificial light and roadside ditches, which indicated the importance of local characteristics for the occurrence of mortality hotspots (coelho et al., 2012). with the increase of the impact of the roadkills and the need to understand the factors that influence them, more complex studies began to be developed in the neotropics, which considered the influence of the sampling effort in estimating wildlife roadkills (bager and rosa, 2011) and the spatial-temporal approaches linked to these traffic-induced mortalities (rosa and bager, 2012; coelho et al., 2012). mitigation measures there is consensus that a system including construction of tunnels and barriers is better to anurans, as it would allow all seasonal migrations, including those of juveniles (jochimsen et al., 2004; puky and vogel, 2004; puky, 2006; andrews et al., 2008; schmidt and zumbach, 2008; glista et al., 2009; lesbarrères and fahrig, 2012). the quantification of the animals that use these structures, not only by the simple counting of individuals but by methods that include capture-mark-recapture, can assist in the estimation of the real proportion of amphibian populations that benefit from this measure (schmidt and zumback, 2008). hylids and other arboreal amphibians, however, rarely use these passages. specific tests for these groups are rare and necessary, as well as alternative methods of passing as rope bridges used by primates and other arboreal mammals (weston et al., 2011). although many of the problems can be related to inappropriate design or the lack of planning for these structures (puky and vogel, 2004; andrews et al., 2008; lesbarrères and fahrig, 2012), perhaps the lack of scientific rigor and in the efficiency assessment is one of the major causes (lesbarrères and fahrig, 2012). additional issues are related to wildlife crossings, such as studies without replications, lack of information between pre and post-construction and relatively short monitoring periods. studies testing the effectiveness of given mitigation methods is essential for planning strategies for the most affected taxa. some of these studies can also increase the 135road ecology and neotropical amphibians ability to anticipate and prevent large number of roadkills, such as those using collision models applied them for mitigation projects (gunson et al., 2011). nevertheless, the implementation of wildlife crossings does not always benefit the amphibian populations. when the implementation of mitigation measure is in roadkill hotspots, must be directed to safe crossing sites by extensive barrier fencing. however, tunnels may focus predation on high local concentrations of individuals. occasional mass mortality by flooding or oil seepage is also possible. tunnel and fence systems also require continuous maintenance to work well, and this is often not sustainable (allaback and laabs, 2003, beebee, 2013). another point to be considered in the determination of mitigation measures is that amphibians have some preference regarding the design features of the tunnels. for instance rana esculenta, r. dalmatina, r. pipiens and r. clamitans prefer tunnels with specific type of substrate, for example, soil (lesbarrères et al., 2004; woltz et al., 2008), that are brighter and have larger diameter openings (woltz et al., 2008). as for the tunnel length, r. pipiens and r. clamitans prefer tunnels that are 6.1-9.1 m in length (woltz et al., 2008). alternative measures that have worked well for amphibians include the temporary closure of roads, usually at night. although amphibian death on the roads could occur before the daily closure time, this method appears to be efficient because it protects different age structures and life stages within the population (schmidt and zumback, 2008), and can be reasonably applied, for example, to roads that cut through protected areas. however, in many situations, roads are not closed to traffic because people are not willing to use alternative routes (jochimsen et al., 2004; schmidt and zumback, 2008). the road ahead regardless of a longor short-term sampling, studies demonstrate the high amphibian mortality among vertebrates (fahrig et al., 1995; hels and buchwald, 2001; smith and dodd jr, 2003; semlitsch et al., 2007). however, as many contradictory studies are published due to differences in the sampling protocols, the definition of the methodology of amphibian roadkill studies must be supported by several points, certain of which are controllable, and others of which are not. actual roadkills depend on variables that are not often considered in the studies, such as: features of the surrounding landscape, species traits (type and breeding season, population density, seasonal activity and movement speed), characteristics of the road (traffic intensity and number of lanes) (balkenhol and waits, 2009; garcia-gonzales et al., 2012; santana, 2012) and even the intention of drivers to kill wild animals or certain species that are considered somehow less charismatic, such as the cane toad (rhinella marina) (beckmann and shine, 2012). however, variables that influence road mortality can be controlled to optimize the results. among those variable are the following: type of sampling (on foot or by car), period of the day during which the data are collected and number of daily collections, survey of species in the vicinity of the road, sampling effort, training of the team (experience of the observers), detection and removal rates. that is, the result of the sampling will depend on the techniques used, on the experience and skills of those conducting the job of detecting and identifying the organisms, the time spent and the faunal composition in each location (silveira et al., 2010). therefore, defining a practical and accurate methodology to enable comparisons between different studies and, consequently, conclusions that can be more generalizable has a paramount importance. the use of appropriate methodology will enable an even more accurate location of priority areas for mitigation or areas that should be avoided in the planning of new roads (lesbarrères and fahrig, 2012). characteristics of the surroundings of the road can influence the roadkill rates, such as the occurrence of specific habitats and potential breeding areas (malo et al., 2004). the composition, configuration and quality of water bodies (findlay et al., 2001; mazerolle and desrochers, 2005; orlowski, 2007) and also the presence of forested areas near the roads (carvalho and mira, 2011) are indicative of areas with high mortality rates for amphibians. in a study conducted in china, the mortality was higher for the stretches of road with a greater proportion of wet grasslands within a 1-km radius (gu et al., 2011). the comparison of amphibian roadkills is difficult because of the different methods used and conditions of the local population (elzanowski et al., 2009). in general, samplings conducted on foot allow the detection of a greater number of live or dead species and also of small animals and juveniles compared to samplings performed by car (taylor and goldingay, 2004, langen et al., 2007). the monitoring of the wildlife of the surroundings is essential because the effects of roads on different species are related to their biological, ecological and behavioral characteristics (eigenbrod et al., 2008). in addition, there are many local factors (population density and landscape, among others) that can affect the richness and abundance of roadkilled species (coffin, 2007). therefore, a survey performed exclusively on the road should not be considered to be representative of the total area, precisely because many species avoid the roads. moreover, there are several studies criticizing the use of roads as a method of species survey (case, 1978; enge and wood, 2002; 136 paula eveline ribeiro d’anunciação et al. steen and smith, 2006). however, this criticism does not imply that data collected on roads cannot be used to extrapolate the potential distribution of species recorded in the area (mccarthy et al., 2012). the sampling effort of the study may also interfere in the quantification of roadkilled animals. as amphibians experience a high temporal variation in population, long-term studies are ideal to identify these fluctuations. however, studies of this magnitude are not always feasible, and there is also the need for a quick response. an alternative is to assess many areas with different degrees of impact (fahrig et al., 1995). amphibians also show seasonal variation of activity; the periods of greatest activity are the wettest months when reproduction peaks (smith and dodd jr, 2003; glista et al., 2008; attademo et al., 2011). clearly, roads near or crossing water bodies will exhibit a greater incidence of mortality (langen et al., 2009). the ideal sampling effort should vary according to the size and heterogeneity of the area, and one of the methods to assess the effort is through rarefaction curves (bager and rosa, 2011). a comparison of mortalities between roads is impractical if only raw data are considered due to differences in the characteristics of roads (e.g., extent) and sampling design (rosa et al., 2012). an alternative that facilitates the comparison is to analyze roadkill rates (ratio between roadkill number and time or space unit) (gummier-costa and speber, 2009; turci and bernarde, 2009). moreover, the addition of factors determining observation error into population models increases the accuracy of results: the incorporation of sampling effort into analyses may improve the reliability of estimates (bonardi et al., 2011). human resources directly affect the detection of roadkilled animals. the team must be properly trained, qualified and competent for carcass detection and species identification. the effectiveness of sampling will depend on the skill and experience of the team because trained personnel can find more species and individuals (silveira et al., 2010). another factor that should be taken into account is the position of the observer on the road. samplings in which the team walks in all directions and lanes during all collections ensure a greater accuracy of the results. if the detection and persistence rates are not considered, the number of individuals is underestimated. carcass persistence on road can be affected by scavengers activity, weather (slater, 2002) and traffic flow, whereas detectability can be influenced by carcass size (morrison, 2002), amount of roadside vegetation, number of surveys, methodology used and skills and experience of the team involved in the census (colino-rabanal and lizana, 2012). based on the abovementioned information, we could focus on the following points for reflection by taking into account the goal of obtaining a roadkill rate closer to the reality of the road effect and the development of a sampling protocol. the aim of this protocol is to improve the measurement of roadkill rate, allowing comparison between landscapes. we suggest the following: i. sampling performed on foot. if the area is too large, we suggest a combination of two sampling methods (on foot and by car) to produce a more accurate measure of the roadkill rate, as well as a greater representativeness of the species (silveira et al., 2010). the selection of stretches of road where the sampling will be performed on foot can be made through draws, selection of points located at regular intervals, random points distributed throughout the road and points purposely selected due to their features (e.g., areas that cut through water bodies). consider all direction and all lanes that compose the road during the sampling is important too. ii. conducting a pilot study to define the sampling method according to the species, such as because the best time of the day and months for sampling. if a pilot study is not feasible, the sampling should be performed during multiple periods of the day to include species with different behaviors and to prevent the loss of carcasses. iii. determining specific characteristics of the surrounding landscape to verify correlation with stretches of road near water bodies, forested areas or certain land uses. iv. quantify the traffic volume (fahrig et al., 1995; hels and buchwald, 2001; mazerolle, 2004). v. evaluate the persistence rates of carcasses and the detection rate (teixeira et al., 2013). vi. whenever possible, the research team should collect individuals or tissue samples and include them in zoological collections (balkenhol and waits, 2009). vii. concomitantly with the surveys, the team should also perform an inventory of the fauna in the area surrounding the studied road (rosa et al., 2012). viii. prioritize, whenever possible, long-term (andrews et al., 2008) and comparative studies with controls replication. when there is a need for quick response, the minimum sampling effort for surveys of more than one year should be weekly or even more frequent collection. in areas exhibiting high environmental seasonality, we suggest twice a week collection for at least one year. however, to include the most affected species, a bi-weekly monitoring is sufficient (bager and rosa, 2011). ix. use time series analyses to determine which mitigation measure is more appropriate. x. to monitor the mitigation measures, we recommend comparisons of roadkills before and after the methods are applied. 137road ecology and neotropical amphibians xi. use the geographic information system gis tools and visit the site before implementing new roads (andrews et al., 2008). xiii. form multidisciplinary teams, involving different social actors (e.g., government members, engineers, conservationists and local residents). multidisciplinary teams may better evaluate / limit issues before the construction of the road, and implement monitoring and mitication after road constructions (andrews et al., 2008; 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(2001): population declines and priorities for amphibian conservation in latin america. conserv. biol. 15: 1213-1223. acta herpetologica vol. 8, n. 1 june 2013 firenze university press journal of the societas herpetologica italica acta herpetologica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 8(1): 19-34, 2013 recent cryptic extinction of squamate reptiles on yoronjima island of the ryukyu archipelago, japan, inferred from garbage dump remains yasuyuki nakamura1,*, akio takahashi2, hidetoshi ota3 1 tropical biosphere research center, university of the ryukyus, senbaru 1, nishihara, okinawa 903-0213, japan. *corresponding author. e-mail: ynaka.riukiaria@gmail.com 2 department of zoology, faulty of science, okayama university of science, ridai-cho 1-1, kita-ku, okayama, okayama 700-0005, japan 3 graduate school of human science and environment, university of hyogo, yayoi-gaoka 6, sanda, hyogo 669-1546, japan submitted on 2012, 28th december; revised on: 2013, 24th february; accepted 2013, on 24th april. abstract. we report recent skeletal remains of squamate reptiles screened from an old garbage dump deposit found on yoronjima island, a small island of the ryukyu archipelago, in the subtropical northwestern pacific. identified remains include at least three species of terrestrial snakes, one sea snake, and seven species of lizards. among these, the ryukyu short-legged skink, ateuchosaurus pellopleurus; kuroiwa’s leopard gecko, goniurosaurus kuroiwae; and one unidentified gekko species constitute entirely new findings. for a further two snake species and one lizard species, the evidence presented here is the first specimen-based documentation that these species existed on this island, where they are now evidently absent. the present absence of these species on yoronjima is mainly attributable to predation by the japanese weasel, mustela itatsi, introduced in the mid-1950s. our results demonstrate a prominent case of recent, sizable deterioration of insular herpetofaunal diversity, which has never been documented with certainty in the ryukyu archipelago, and suggest that human influence should be taken into account in biodiversity research in this area. keywords. extinction, ryukyu archipelago, squamata, weasel, yoronjima. introduction our knowledge of the natural diversity of many insular terrestrial vertebrate faunas in subtropical and tropical regions remains comparatively limited. currently, these faunas are often depauperate in indigenous elements compared to their original states, due mainly to past activities of humans and their associated animals (e.g., henderson, 1992; case et al., 1998). even where documentation of past faunal states is regarded as comprehensive, caution should be exercised not to underestimate an island’s natural biodiversity, as this renders unreliable conclusions from biogeographical and ecological studies that consider only the historically known fauna (steadman, 2006; whittaker and fernández-palacios, 2007). in a number of islands in the lower latitudes, reptilian bone fragments associated with buried human contexts have offered unique opportunities to reveal the existence of several currently missing elements as well as to investigate the history of insular terrestrial reptile communities under anthropogenic influence (e.g., pregill, 1998; pregill and worthy, 2003; pregill and steadman, 2004). however, there have not been many previous studies focusing on reptile remains (which tend to be small and easily overlooked), suggesting there remains a large gap in our understanding of the diversity of insular reptiles. the ryukyu archipelago, forming the most southwesterly part of japan, is a cluster of more than one hundred continental fragments in the subtropical northwestern pacific. the islands have been explored zoologically since the mid-19th century (stejneger, 1907), so the extant herpetofauna is considered to be well documented 20 yasuyuki nakamura et al. (ota, 1998; maenosono and toda, 2007). habitat degradation by humans has been the most serious threat to the biological diversity of indigenous amphibians and reptiles (ota, 2000; ministry of the environment, 2010), which are predominant elements of the terrestrial vertebrate faunas. as is the case with many islands in other regions, there has recently been a growing concern for the negative effects of exotic mammalian predators such as domestic dogs, cats, mongooses, and weasels (e.g., watari et al., 2008; yamada et al., 2009; ministry of the environment, 2010). until recently, the herpetofaunas of this archipelago have nevertheless been regarded as healthy because neither extinction nor serious, irreparable population extirpation (such as on a whole-island level) of japanese indigenous amphibians and reptiles has been known (environment agency, 2000; ministry of the environment, 2010); this concerns the period at least since the mid-19th century, which marked the start of the westernization and modernization of japan. this view is however no longer warranted, stemming merely from a past scarcity of knowledge. indeed, hikida et al. (1992) and ota (2003b) have suggested that documented and possibly unique plestiodon skink populations may have disappeared from tairajima and akusekijima islands in the northern part of this archipelago, due to predation by the introduced japanese weasel, mustela itatsi. as shown below, it seems appropriate that such instances not be regarded as isolated cases. on yoronjima island, a small island in the central ryukyus, we confirmed the recent disappearance of a rhacophorid tree frog (rhacophorus viridis) population by examining some very recent skeletal remains collected from an old garbage dump (nakamura et al., 2009). this is the first confirmed case of an island-level extirpation in modern terrestrial herptiles in japan. vertebrate remains collected at the same time include several species of snakes and lizards, some of which seem to have no previous record or only old unverified records for this island. in this study, we report the results of a detailed identification of these recent reptilian remains and demonstrate the first specimen-based evidence for recent and sizable losses of components of the squamate faunas in this archipelago. material and methods brief description of yoronjima and its terrestrial vertebrate fauna yoronjima is a relatively small (area 20.5 km2) and flat (elevation 97 m) island, lying 30 km northeast of okinawajima island and 40 km southwest of okinoerabujima island in the central ryukyus (fig. 1). it consists mainly of terraced limestone beds deposited in the middle pleistocene (odawara and iryu, 1999). yoronjima has a relatively long history of human settlement. archaeological records indicate that this island was first colonized more than two thousand years ago (the late jomon period; takamiya and chinen, 1984). at a minimum, this island is likely to have been occupied continuously since the early 15th century, at which time a medieval fort was constructed (motoyama, 1988). the known terrestrial non-volant vertebrate fauna on yoronjima is poor in species when compared to other islands in the central ryukyus. all the recorded species are common on islands of the central ryukyus, and no endemic taxa have been found. the current terrestrial herpetofauna on yoronjima consists of three species of frogs (hallowell’s tree frog, hyla hallowellii; ryukyu kajika frog, buergeria japonica; okinawa narrow-mouth toad, microhyla okinavensis), four species of lizards (hokou gecko, gekko hokouensis; house gecko, hemidactylus frenatus; ryukyu five-lined skink, plestiodon marginatus; okinawa green grass lizard, takydromus smaragdinus), and two species of snakes (okinawa green snake, cyclophiops semicarinatus, and brahminy blind snake, ramphotyphlops braminus) (ota, 1986; maenosono and toda, 2007). of these, hemidactylus frenatus has presumably been unintentionally introduced to yoronjima by humans (lever, 2003; ota et al., 2004), which may also be the case for ramphotyphlops braminus (e.g., ota et al., 2004). the occurrence of four other reptile species (okinawa keelback snake, amphiesma pryeri; ryukyu odd-tooth snake, dinodon semicarinatum; okinawan tree lizard, japalura polygonata polygonata; common four-clawed gecko, gehyra mutilata) has also been reported (koba, 1956; nakamura and uéno, 1959; takara, 1962; morita, 1988; samejima, 1991), but it has been pointed out that these insufficient and mostly verbal records need further confirmation (ota, 2003a; maenosono and toda, 2007). terrestrial mammals are poorly represented and consist of the ryukyu flying fox, pterofig. 1. map of the northern and central parts of the ryukyu archipelago, showing the location of yoronjima island. star indicates the study site. 21recent extinction of squamates pus dasymallus; watase’s shrew, crocidura watasei; musk shrew, suncus murinus; norway rat, rattus norvegicus; black rat, r. rattus; and house mouse, mus musculus (see morita, 1988; funakoshi et al., 2006; motokawa, 2007). in addition, the oriental free-tailed bat, tadarida insignis, was recorded (morita 1988). all rodents and the musk shrew are believed to have been introduced (e.g., motokawa, 2007), as has the japanese weasel, mustela itatsi (see morita, 1988). study site skeletal remains of terrestrial vertebrates examined here were collected from an old garbage dump, which was found by a. takahashi in 2007, deposited under the sheltered overhang of a limestone rock wall located in asato (27°02’05”n, 128°26’02”e; fig. 1). precise deposition dates are unknown but probably no earlier than the late 19th century, as they were found in mixture with fragments of various modern artifacts such as tableware and glass bottles. apart from the remains of livestock and marine fishes, there were remains of small terrestrial vertebrates. these were collected by careful fine-scale (1 mm) sieving of sandy soil deposits. four frog species identified from the remains have already been reported (nakamura et al., 2009). mammalian remains identified included crocidura watasei, rattus norvegicus, r. rattus, and mus musculus. all these species live on yoronjima today (see above). identification collected reptile skeletal elements were identified with the help of comparisons to skeletal specimens of extant squamata species occurring in the central ryukyus and adjacent regions (appendix). owing to the virtual absence of useful information on the systematic identification of most fragmentary squamate reptile bone elements, our comparisons were based on overall similarity. however, for major taxonomic assignment of lizard bones we mostly followed available apomorphy-based systematic hypotheses. our identifications at species level were primarily based on the current distributions of certain taxa. in particular, we regarded a present occurrence on yoronjima or nearby islands as important information for species-level identification, unless contradictory evidence was present. this approach appears justified because of the very recent status of the bone remains examined here. we present brief descriptions of identified remains (for snakes in particular) to provide basic information for the systematic identification of their isolated skeletal elements. all observations were made under stereoscopic microscopes (nikon smz-10 or smz-1000). measurements were taken with an embedded micrometer. osteological terminology follows laduke (1991) and holman (2000) for snakes and rieppel (1984) and estes et al. (1988) for lizards. division of the regions of snake vertebral columns followed hoffstetter and gasc (1969), and for subregions of the trunk vertebrae, laduke (1991). the described skeletal remains were deposited in the collection of the museum of the university of the ryukyus (fujukan), nishihara, okinawa, japan. results systematic accounts suborder serpentes family colubridae subfamily colubrinae cyclophiops semicarinatus (hallowell, 1861) (fig. 2a) referred specimens—24 middle and posterior trunk vertebrae (rumf-gf-4060). mni = at least two individuals, on the basis of size. the referred vertebrae are as long as wide and nearly square across the zygapophyseal articular facets. the neural spine is square anteriorly and angularly undercut posteriorly. it is of moderate height at the anterior edge, roughly twice as long as high, and below the height of the neural canal. viewed anteriorly, the zygosphene is dorsally convex; viewed from above, its anterior edge is weakly convex. the lateral inclinations of the zygosphenal articular facets are moderate, ~40° from the sagittal plane. the neural arch is slightly depressed. the posterior neural arch swells dorsally, so that the height of the posterior part of the neural spine is less than half the height of the anterior part. posteriorly, the neural arch laminae bear barely developed epizygapophyseal spines, which extend posterolaterally beyond the margins of the postzygapophyseal articular facets. the shapes of the prezygapophyseal articular facets are ovoid, while those of the postzygapophyseal articular facets are rounded. the prezygapophyseal accessory processes are blunt and directed laterally in dorsal view. viewed from above, the anterior edges are usually bowed posteriorly. the cotyle and the condyle are rounded, except for those of smallsized vertebrae where they are usually depressed. they are equal in size to the neural canal, but large-sized vertebrae tend to have relatively large cotyles and condyles. in lateral view, the condyle is moderately oblique; viewed ventrally, the transverse width of the condyle almost equals the width of the precondylar part of the centrum. the subcentral ridges are weakly defined, and are straight or somewhat bowed dorsally in lateral view. the hemal keel is deep and narrow, and laterally deeply excavated by the deep subcentral grooves. the presence of the distinct neural spines, well-developed prezygapophyseal accessory processes, well separated diapophyses and parapophyses, and the lack of the hypapophysis indicate that the referred vertebrae are middle or posterior trunk vertebrae of colubrine snakes (rage, 1984; but see also malnate, 1972; ikeda, 2007). they would thus expect to be derived from either cyclophiops semicarinatus or dinodon semicarinatum, representatives of colubrine snakes in the central ryukyus. the trunk vertebrae 22 yasuyuki nakamura et al. have been identified as those of c. semicarinatus, on the basis of their relatively low neural spines lower than the height of the neural canals, square anterior margins of the neural spines, rounded cotyles and condyles (except for small-sized vertebrae), posteriorly bowed anterior edges of the prezygapophyseal accessory processes, the presence of epizygapophyseal spines that extend beyond the margins of the postzygapophyseal articular facets, rounded postzygapophyseal articular facets, and very weak precondylar constrictions of the centra. dinodon semicarinatum (cope, 1860) (fig. 2b) referred specimens—21 middle and posterior trunk vertebrae (rumf-gf-4061). mni = at least two individuals (one juvenile and one adult), on the basis of size. referred vertebrae are short and broad, with relatively large cotyles and condyles. the neural spine is thin, short, and high, and about as high as long. its height is greater than that of the neural canal in anterior view. it overhangs fig. 2. trunk vertebrae of snakes. (a) referred vertebra of cyclophiops semicarinatus (one of 24 registered as rumf-gf-4060), (b) referred vertebra of dinodon semicarinatum (one of 21 registered as rumf-gf-4061), (c) referred vertebra of amphiesma pryeri (one of 39 registered as rumf-gf-4062), and (d) referred vertebra of hydrophiinae sp. (unidentified sea snake) (rumf-gf-4063). left to right: anterior, lateral (left), posterior, dorsal, and ventral views. abbreviations are: con, condyle; cot, cotyle; di, diapophysis; ezs, epizygapophyseal spine; hk, hemal keel; hy, hypapophysis; na, neural arch; nc, neural canal; ns, neural spine; pa, parapophysis; pap, parapophyseal process; prz, prezygapophyseal accessory process; przf, prezygapophyseal articular facet; pozf, postzygapophyseal articular facet; sr, subcentral ridge; zy, zygosphene; zyf, zygosphenal articular facet; zygaf, zygantral articular facet. scale bars equal 1 mm. 23recent extinction of squamates anteriorly and posteriorly, and the anterodorsal corner is rounded. the dorsal roof of the zygosphene is convex; its anterior margin is convex anteriorly. the lateral inclinations of the zygosphenal articular facets are moderate, ~40° from the sagittal plane. viewed posteriorly, the neural arch is more or less depressed. viewed laterally, its posterodorsal part swells, so that the depth of the posterior neural spine is half the depth of the anterior edge. the neural arch laminae above the zygantra extend posteriorly beyond the posterior ends of the facets of the zygantra (hereafter referred to as zygantral articular facets). the epizygapophyseal spines are usually absent, or rudimentary if present. the blunt and gently tapered prezygapophyseal accessory processes are directed laterally in dorsal view, with rounded or weakly pointed tips. the shapes of the prezygapophyseal articular facets are ovoid; those of the postzygapophyseal articular facets are rectangular, more extensive mediolaterally than anteroposteriorly. the cotyle and the condyle are oval in outline, broader transversely than sagittally, with moderate inclinations. viewed ventrally, the centrum tapers posteriorly to the condyle, and the condyle is much wider than the precondylar part of the centrum. the subcentral ridges are weakly to moderately defined and bowed dorsally in lateral view. the hemal keel is narrow but widens at the posterior part. it is shallow in middle trunk vertebrae (fig. 2b) and deep in posterior trunk vertebrae. the state of the subcentral grooves ranges from shallow in middle trunk vertebrae (fig. 2b) to deep in posterior trunk vertebrae. most of the middle and posterior trunk vertebrae of dinodon semicarinatum are separated from those of cyclophiops semicarinatus by their short and wide general shapes, high neural spines that are usually higher than the neural canals, anteriorly overhanging anterior edges of the neural spines, dorsoventrally depressed cotyles and condyles (except for small-sized vertebrae), anteriorly bowed anterior edges of the prezygapophyseal accessory processes, much wider transverse width of the condyles than the precondylar parts of the centra, laterally elongated postzygapophyseal articular facets, posteriorly elongated neural arch laminae that hide the zygantral articular facets from above, and the lack of the epizygapophyseal spine. subfamily natricinae amphiesma pryeri (boulenger, 1887) (fig. 2c) referred specimens—39 precaudal trunk vertebrae (rumf-gf-4062). mni = 1. referred precaudal vertebrae have generally slender and elongate overall shapes. the neural spine is thin, and is much thinner laterally than the hypapophysis. it is moderately high and long. the length is more than twice the height at the anterior edge, and the height is about equal to that of the neural canal. the neural spine overhangs anteriorly and posteriorly. the zygosphene is very slightly dorsally convex; its anterior margin is convex. the lateral inclinations of the zygosphenal articular facets are slight, ~25° from the sagittal plane. viewed posteriorly, the neural arch laminae are bent at the midpoints. the dorsal elevation of the posterior neural arch is slight; it is confined to almost the same level as the zygosphene. the epizygapophyseal spines are barely developed, with tips extending posteriorly to the margins of the postzygapophysial articular facets. the distinct parapophyseal processes protrude anteriorly. the shapes of the prezygapophyseal articular facets are sub-round or ovoid, while those of the postzygapophysial articular facets are rounded. the cotyle and the condyle are rounded and smaller than the neural canal. the condyle is slightly wider than the precondylar part of the centrum in ventral view, and the dorsoventral inclination is moderate. the prezygapophyseal accessory processes are thin or blunt and directed anterolaterally. the hypapophysis is broad, deep, pointed posteriorly, and squared anteroventrally. its distal part extends posteriorly beyond the condyle tip. viewed laterally, the subcentral ridges are well defined and slightly bowed dorsally, and viewed ventrally, the shallow subcentral grooves run throughout the ventral side of the centrum. precaudal vertebrae of amphiesma pryeri differ from the middle and posterior trunk vertebrae of cyclophiops semicarinatus and dinodon semicarinatum in being narrow and elongate and having well developed hypapophyses, distinct and anteriorly projected parapophyseal processes, anterolaterally directed prezygapophyseal accessory processes (e.g., ikeda, 2007), less inclined laterally facing zygosphenal articular facets, a posterior neural arch that extends dorsally very slightly, posterior neural arch laminae bent at the midpoints, and small condyles and cotyles smaller than the neural canals. it differs further from middle and posterior trunk vertebrae of c. semicarinatus in having overhanging anterior edges of the neural spines and anteriorly bowed anterior edges of the prezygapophyseal accessory processes; and from d. semicarinatum in having rounded condyles and cotyles, rounded postzygapophysial articular facets, and weaker precondylar constrictions. although anterior trunk vertebrae of these colubrine snakes also possess hypapophyses, these vertebrae are easily identified on the basis of their dorsoventrally elongate and anteroposteriorly short general shapes (laduke, 1991). these precaudal vertebrae highly resemble those of amphiesma pryeri, while there are some other non-colubrine cenophidian snakes in the central ryukyus that display hypapophyses throughout their precaudal vertebrae 24 yasuyuki nakamura et al. (e.g., ikeda, 2007; see suppl. fig. s1-3). precaudal vertebrae of the amami odd-scaled snake, achalinus werneri, may be distinguished from those of am. pryeri by e.g., less elongated centra, less developed hypapophyses, thick neural spines that are flattened on the dorsal edges, and flat neural arch laminae. likewise, those of elapid snakes (the coral snakes sinomicrurus japonicus japonicus and s. j. boettgeri, and the sea kraits laticauda colubrina, l. laticaudata, and l. semifasciata) differ in being heavily-built and having short centra, lower and thicker neural spines (of the same thickness as the hypapophyses), depressed posterior neural arches (which are even laterally expanded in laticauda), and mostly spine-like hypapophyses (see also ikeda, 2007). precaudal vertebrae of the black-headed sea snake, hydrophis melanocephalus, and the turtleheaded sea snake, emydocephalus ijimae, resemble those of am. pryeri in being elongate, lightly-built, and having thin neural spines, but they are readily distinguished by high neural spines that are confined to the posterior of the zygosphenes and by less developed hypapophyses. viperid vertebrae can easily be excluded, as they are generally anteroposteriorly short and have both short accessory processes and thick hypapophyses that are dorsoventrally long and anteroposteriorly short (szyndlar, 1991; ikeda, 2007). unfortunately, no information is available on vertebral characteristics of the kikuzato’s stream snake, opisthotropis kikuzatoi, an extremely rare snake endemic to kumejima island in the central ryukyus. this species is substantially smaller-bodied than am. pryeri and is strictly confined to a small number of mountain stream systems of an island far from yoronjima (okinawa prefectural board of education, 1993). therefore, here, we tentatively reject the possibility that o. kikuzatoi is represented. family elapidae hydrophiinae sp. (unidentified sea snake) (fig. 2d) referred specimen—one trunk vertebra (rumfgf-4063). mni = 1. one trunk vertebra is referred to a sea snake. this specimen displays a characteristic elongated neural arch that extends posteriorly beyond the condyle tip. viewed posteriorly, the neural arch is broad and vaulted, with the posterior margin around the midline rising dorsally to near the posterodorsal corner of the neural spine. the neural spine lost the anterodorsal corner, but the anterior edge is clearly inclined posteriorly and the base is located considerably behind the zygosphene. the dorsal edge slopes up posteriorly, and the posterior corner overhangs. the height of the neural spine is less than its length. the dorsal roof of the zygosphene is weakly convex dorsally, and the rounded anterior margin strongly protrudes when viewed from above. lateral inclinations of the small zygosphenal articular facets are moderate (38°). the prezygapophyseal accessory processes are thin, long, pointed, and directed anterolaterally. they are positioned distinctly ventrally of the prezygapophyseal articular facets, which are rounded and small. the parapophyseal processes barely protrude anteriorly. the diapophyses are anterolaterally directed, reaching the level of the cotyle rim, and are almost invisible from above. weakly developed epizygapophyseal spines are present. viewed laterally, the condyle is truncated with a slight inclination, and viewed ventrally, it is slightly wider than the precondylar part of the centrum. the hypapophysis is shallow, and its tip directs ventrally and extends posteriorly only to the level of the condyle base. in lateral view, the weakly defined subcentral ridge is bowed dorsally. the subcentral grooves are weak and confined to the regions along with the anterior part of the hypapophysis. the referred material clearly differs from vertebrae of the three species discussed above. among cenophidian snakes occurring in and around the central ryukyus, some character states possessed by this vertebra are confined to sea snakes, without being universal. these are the neural spine, which rises out considerably behind the zygosphene; the neural arch, which extends posteriorly beyond the condyle; and anterolaterally directed diapophyses, which are almost invisible from above (suppl. fig. s2, 3). however, this vertebra specimen differs from examined sea snakes (three laticauda species, emydocephalus ijimae, and hydrophis melanocephalus) in the sloping dorsal edge of the neural spine, the posterior neural arch that flares out dorsally at the midline, and the pronouncedly anteriorly protruding zygosphene. although we were unable to examine respective skeletal specimens, we consider this unique vertebra to be derived from one of three other hydrophiine sea snake species known from the waters surrounding the central ryukyus: hydrophis cyanocinctus, h. ornatus maresinensis, and pelamis platura (see toriba, 1996). suborder lacertilia family agamidae japalura polygonata polygonata (hallowell, 1861) (fig. 3a, b) referred specimens—six right and four left maxillae (rumf-gf-4064), three right and three left dentaries (rumf-gf-4065), a proximal part of the right humerus, and a proximal part of the left femur. mni = 6. 25recent extinction of squamates the referred dentaries are labiolingually flattened, with anterior tips that are curved dorsally (fig. 3b). the meckelian groove is open along the entire length. four specimens preserve complete tooth rows, which are composed of three canine-like anterior teeth and 12 (right: n = 1) or 13 (left: n = 3) tricuspid posterior teeth. the anterior teeth are conical and curved, and attach to the inner aspect of the labial wall (i.e., pleurodont), while the posterior teeth are firmly united with the dorsal portion of the labial wall (acrodont). posterior teeth are compressed laterally, with a pair of small additional cusps on each anterior and posterior side. dentition of the maxillae is identical to that of the dentaries (fig. 3a), consisting of two or three canine-like pleurodont anterior teeth and 12 (right: n = 2), 13 (right: n = 2), or 14 (left: n = 1) acrodont posterior teeth. dentition of the referred maxillae and dentaries exhibits a combination of character states exclusive to agamid lizards: canine-like pleurodont anterior teeth and deltoid, tricuspid, and labiolingually flattened acrodont posterior teeth, as well as a lack of indentation for the coronoid in the posterolateral dentaries (estes et al., 1988; see also smith et al., 2011). these specimens were positively identified as japalura polygonata polygonata, the only representative of agamid lizards in the central ryukyus. additionally, fragments of a humerus and a femur were also identified. all of these referred bones are essentially identical to the same elements in living j. p. polygonata. family scincidae ateuchosaurus pellopleurus (hallowell, 1861) (fig. 3c) referred specimen—one frontal (rumf-gf-4066). mni = 1. the referred material is an intact frontal. it is undivided and fused completely, thin and slightly convex dorsally, and the lateral descending processes are shallow downturned lobes that have no ventral contact. posteriorly, it bears a prominent notch for the parietal foramen at the midline and a pair of posterolateral projections at the lateral sides. such a single, unpaired frontal bone with shallow lateral descending processes is unique among japanese lizards to so-called lygosomine skinks. among them, or even among scincid species, the only known species to possess a single frontal with a parietal foramen are members of the genus ateuchosaurus (see greer, fig. 3. lizard remains referred to japalura polygonata polygonata (a, b), ateuchosaurus pellopleurus (c), plestiodon marginatus (d), and takydromus smaragdinus (e). (a) right maxilla (one of 10 registered as rumf-gf-4064) in medial view, (b) left dentary (one of 6 registered as rumf-gf-4065) in medial view, (c) frontal (rumf-gf-4066) in dorsal view, (d) right dentary (rumf-gf-4067) in medial view, and (e) left dentary (rumf-gf-4068) in medial view. abbreviations are: mg, meckelian groove; pf, parietal foramen. scale bars equal 1 mm. 26 yasuyuki nakamura et al. 1970). this material was definitely identified as a. pellopleurus, as ateuchosaurus is exclusively represented by this species in the ryukyu archipelago. plestiodon marginatus hallowell, 1861 (fig. 3d) referred specimens—one right dentar y (rumfgf-4067), two right and one left posterior mandibles, five right and one left humeri, one right femur, and one right and one left pelvic girdle. mni = 5. the referred dentary fragment is deep and truncated. the meckelian groove is fully open to the symphysis. dentition is pleurodont; there are 17 teeth and seven vacant tooth positions. teeth are blunt and tapering cylinders whose crowns are slightly thinner than the rest of the teeth. each of the intact teeth has a concave and striated lingual surface at the crown and a pair of cusps that are labiolingually doubled and lingually inclined. this dentary specimen is referred to scincids on the basis of the cylindrical teeth and striated and lingually concave tooth crowns (e.g., estes, 1983). of the three currently recognized scincid species in the central ryukyus, ateuchosaurus pellopleurus and the barbour’s blue-tailed skink, plestiodon barbouri, may be excluded because the dentary is of too great a size and depth (see suppl. fig. s4). although the use of the body size for identification of lizard remains requires special caution (e.g., pregill, 1986), the material could be referable to p. marginatus, and the present occurrence of the species on yoronjima (see above) supports this conclusion. certain elements of scincid remains were also referred to this species on the basis of size and general morphological similarities. although two subspecies of p. marginatus have been recognized, we here have omitted subspecific status as the distinction of these subspecies has been cast into doubt by recent research (e.g., honda et al., 2008). family lacertidae takydromus smaragdinus (boulenger, 1887) (fig. 3e) referred specimens—one left dentary (rumf-gf-4068), and one right and one left pelvic girdle. mni = 1. the referred left dentary is small, thin, and shallow. the meckelian groove is open and ventrally located in the anterior part. there are 16 slender and cylindrical pleurodont teeth and 10 or 11 vacant alveoli. dentition is heterodont; some posterior intact teeth show tricuspid tooth crowns while anterior teeth are clearly unicuspid. these character states (especially of the teeth) are typical in takydromus lizards (arnold, 1997; arnold et al., 2007). this specimen was assigned to t. smaragdinus, as this is the only representative of the genus in the central ryukyus. one right and one left pelvic girdle were also referred to this species on the basis of distinct tubercles on proximal dorsal edges of the ilia. family gekkonidae gekko hokouensis pope, 1928 (fig. 4a-d) referred specimens—three right and three left maxillae (rumf-gf-4069), two right and three left dentaries (rumf-gf-4070), five frontals (rumf-gf-4071). mni = 5 dentary the completely fused meckelian grooves and pleurodont tooth rows of these shallow dentaries display the unique character states of geckos (kluge, 1967; estes et al., 1988), and the peg-like simple and slender teeth indicate a gekkonid origin (fig. 4b). among seven described and one undescribed species of gekkonid occurring in the central ryukyus (toda, 2008; toda et al., 2008), the moderately sized material from the garbage pile does not seem to be referable to any of the three relatively largesized species (the takara gecko, gekko shibatai, the amami gecko, g. vertebralis, and the undescribed species of the okinawa island group) nor to the small-sized species, the mourning gecko, lepidodactylus lugubris (see ota, 1996; toda, 2008; toda et al., 2008). among the remaining four equivalent-sized gekkonid species (gehyra mutilata; gekko hokouensis; the bowring’s gecko, hemidactylus bowringii; and h. frenatus), gehyra mutilata apparently differs from the referred specimens in having thin teeth and elongated tooth crowns (suppl. fig. s5a). for the rest of the species, however, we were unable to find any useful morphological difference between these and the referred gekkonid dentaries. we have tentatively referred these dentary fragments as gekko hokouensis because other certain elements of gekkonid remains from this site were also assigned to gekko species, whereas none have been clearly identified as hemidactylus. maxilla an intact specimen of the referred maxillae displays a pronounced process at the anterior part of the medial shelf, the anteromesial process (fig. 4a), which is one of the synapomorphies of geckos (rieppel, 1984; 27recent extinction of squamates estes et al., 1988). the posterior maxillary process bears a short ascending wall (hereafter referred to as the posterior maxillary lamina) at the lateral side of the medial shelf. this character state was found in examined species of gekko, which possess jugals that are laterally covered by the posterior maxillary laminae (suppl. fig. s6b-e), but is lacking in examined hemidactylus species and in gehyra mutilata, whose jugals wrap almost throughout these parts (suppl. fig. s6a, f, g). these maxillae can thus be identified as those of gekko hokouensis, the only extant representative of gekko species on yoronjima. frontal these referred frontals are lightly built and unpaired (fig. 4c, d). they were assigned to geckos because they form depressed cylinders due to the lateral descending processes that meet and fuse ventrally (kluge, 1967; estes et al., 1988). their anteroposteriorly truncated general shapes and rounded ventral sides indicate that these frontals are of gekkonid origin and do not belong to the eublepharid goniurosaurus. the dorsal surfaces of the referred frontals are concave, the lateral edges of the orbital margins are acute, and the prefrontal sutures laterfig. 4. lizard remains referred to gekko hokouensis (a-d), gekko sp. (a large-sized gekko species) (e-h), and goniurosaurus kuroiwae (i, j). (a) right maxilla (one of 6 registered as rumf-gf-4069) in medial view, (b) right dentary (one of 5 registered as rumf-gf-4070) in medial view, (c, d) frontal (one of 5 registered as rumf-gf-4071) in dorsal (c) and ventral (d) views, (e) right maxilla (one of 3 registered as rumf-gf-4072) in medial view, (f) right dentary (one of 3 registered as rumf-gf-4073) in medial view, (g, h) frontal (one of 3 registered as rumf-gf-4074) in dorsal (g) and ventral (h) views, (i) left maxilla (rumf-gf-4076) in medial view, and (j) right dentary (rumf-gf-4077) in medial view. abbreviations are: amp, anteromesial process; pml, posterior maxillary lamina; pmp, posterior maxillary process. arrows in c and g indicate the posterior extents of the prefrontal sutures. scale bars equal 1 mm. 28 yasuyuki nakamura et al. ally extend posteriorly beyond the narrowest parts of the bones (fig. 4c). among the four equivalent-sized gekkonid species of the central ryukyus (see above), the frontal of gehyra mutilata is distinguished from the referred frontals by the heavily-built body, the posterior dorsal surface without a concavity, and rolled orbital margins (suppl. fig. s7a). likewise, frontals of hemidactylus differ in that the prefrontal sutures do not extend posteriorly beyond the narrowest parts of the bones (suppl. fig. s7f, g). gekko hokouensis is the only species that has comparable size and morphological details to those of the yoronjima specimens. gekko sp. (a large-sized gekko species) (fig. 4e-h) referred specimens—three right maxillae (rumfgf-4072), two right and one left dentaries (rumfgf-4073), three frontals (rumf-gf-4074), and three left pterygoids. mni = 3. certain elements of the referred material appear identical to bones of gekko hokouensis, but the specimens are too large to belong to this species. some of the material’s character states, especially the presence of posterior maxillary laminae of the maxillae (fig. 4e) and the prefrontal sutures of the frontals which extend to the narrowest parts of the bones (fig. 4g), readily exclude all japanese lizards examined other than species of the genus gekko (see above). however, the widths at the narrowest parts of the two frontals (1.8 and 1.9 mm) and the length of the maxillary tooth row (8.7 mm) show that these elements were derived from animals with ~56, 59, and 65 mm snout-vent lengths (svls), respectively (estimates were obtained from measurements of 14 gecko specimens from yoronjima and okinawajima islands). by contrast, the svl range of the extant yoronjima population of g. hokouensis, the only known gekko species from yoronjima (see above), is merely up to 51.1 mm (male: 43-47.9 mm, n = 2; female: 48-51.1 mm, n = 4; see also toda, 2008 for svl of g. hokouensis from four islands in the okinawa island group [~56.4 mm, n = 170]). although lizard fossils have frequently exhibited aberrant body size when compared with those of modern counterparts (arnold, 1976; pregill, 1986), in this case certain elements referred to g. hokouensis at a size comparable to those of the extant conspecific population were recovered. the referred elements compare favorably with largebodied gekko species, particularly those widely distributed in the central ryukyus: g. vertebralis of the amami island group and the undescribed gekko species known from the okinawa island group (toda, 2008; toda et al., 2008). skeletons of these species and g. hokouensis resemble each other, and since in sum these yoronjima specimens do not preserve enough traits to permit specific identification, here we simply refer to them as a largesized gekko. family eublepharidae goniurosaurus kuroiwae (namiye, 1912) [sense grismer et al., 1994] (fig. 4i, j) referred specimens—one right and one left maxillae (rumf-gf-4075 to 4076) and four right and three left dentaries (rumf-gf-4077 to 4083). mni = 4. the occlusal margins of tooth crowns in these maxillae and dentaries are expanded. the unique dental character is a derived feature diagnosing some mostly insular species of goniurosaurus including g. kuroiwae (e.g., grismer et al., 1999, 2002). no other goniurosaurus occurs in and around the ryukyu archipelago (grismer et al., 1994). we therefore refer these bones to g. kuroiwae. five subspecies of goniurosaurus kuroiwae are currently recognized (grismer et al., 1994; but see grismer et al., 1999), but none of these are known to originate from yoronjima. a detailed morphological comparison of the yoronjima material and known g. kuroiwae subspecies will be presented in a separate paper. discussion in total, three species of terrestrial snakes, one sea snake, and seven species of lizards were recovered from the old garbage dump deposits, in addition to four frog species previously reported by us (nakamura et al., 2009). among these, the discovery of skeletal elements of the ryukyu short-legged skink, ateuchosaurus pellopleurus; one large-sized gekko species; and kuroiwa’s leopard gecko, goniurosaurus kuroiwae, are of particular note, as none of these species have ever been recorded from yoronjima. the results not only increase the total number of indigenous terrestrial squamate species on yoronjima to 10, but also provide the first specimen-based documentation that some of these species once existed on this island. with regard to amphiesma pryeri, dinodon semicarinatum, and japalura polygonata polygonata, their previous records from yoronjima were not supported by extant voucher specimens or results of recent surveys. hence, their occurrence on yoronjima has been questioned (ota, 2003a; maenosono and toda, 2007). the occurrence of takydromus smaragdinus on yoronjima was 29recent extinction of squamates for the first time reported by toda and takahashi (2002), with some doubt regarding its native status. the present results confirm the native occurrence of these species on yoronjima. by contrast, the occurrence of a sea snake bone from these inland deposits is seemingly unnatural; it is likely to have been deposited as a result of consumption by humans (as like laticauda species are consumed locally) or by a bird. although the site appeared to be an old garbage dump, the possibility that these animals had been brought from nearby islands by humans is very low, because this subfossil assemblage is comprised mainly of small non-attractive species, which possess no economic value. additionally, the small and easily overlooked bones (size of the vertebrae are usually ~1 mm) of ramphotyphlops braminus apart, it may also be significant that the absence of hemidactylus frenatus in the subfossil assemblage. conceivably the environment around the site was unsuitable for this synanthropic species, which prefers buildings and artificially lighting places, or the remains concerned predate the invasion of the gecko (before 1958: nakamura and uéno, 1959). it is evident that the natural species diversity of the terrestrial squamate fauna on yoronjima has been seriously underestimated. three out of 10 indigenous species that historically existed on the island were missing from previous accounts, together with another three species whose occurrence in the fauna has been doubted (see above). there is a parallel case in yoronjima amphibians: until we recovered the species’ bones from garbage dump deposits, the native status of the okinawa green tree frog, rhacophorus viridis, had been questioned because there was no plausible record from this island other than a few old museum specimens (nakamura et al., 2009). it is notable that these newly documented species of herptiles have never been recorded in faunal surveys conducted in the past five decades (maenosono and toda, 2007; nakamura et al., 2009), although based on literature records and museum specimens (see below), three of them (japalura polygonata polygonata, dinodon semicarinatum, and rhacophorus viridis) are considered to have occurred there at least in the 1950s. despite the infrequency of surveys, the faunal impoverishment shown by recent investigations is considered to be an accurate reflection of the current state of the herpetofauna due to yoronjima’s small scale, flat profile and resulting thoroughly surveyable nature. it seems safe to state that most of the remains examined here are no older than the late 19th century (although the possibility of intrusions of some heterochronic elements cannot be entirely ruled out; see below). together, these conclusions suggest that the present results illustrate a prominent case of recent mass disappearance of indigenous herptile populations from an island, something that has never been documented in the ryukyu archipelago. the markedly recent character of the event suggests human involvement in the present case. due to the relatively impoverished state of the current yoronjima vegetation (e.g., ohno, 1991), it is likely that deforestation played a major role in the demise of these mostly woodland-dwelling species. current vegetation cover (including forest, shrub, and grassland) is estimated at 4.94 km2 (national institute for japanese islands, 1994), less than a quarter of the total surface area, and much of the forest has been fragmented. however, the current species diversity of indigenous terrestrial squamates on yoronjima (four species) is far lower than would be expected from the carrying capacity of the remnant forest. according to available data, at least 11 out of 21 major islands (> 1 km2) in the central ryukyus hold comparable or smaller vegetated areas than yoronjima (national institute for japanese islands, 1994). all but one of these islands are known to possess richer indigenous terrestrial squamate faunas, based on records of existent specimens (maenosono and toda, 2007; kojima et al., 2012). the exception, kudakajima island (area: 1.38 km2, vegetated area: 0.65 km2), has only four known species and mirrors the disproportionally poor nature of yoronjima’s current squamate fauna. therefore, deforestation does not satisfactorily account for the disappearance of the discussed reptile and frog species from yoronjima. some other mechanism must have been at work. a more plausible explanation for the disappearance of these herptiles assumes an exotic mammalian predator, the japanese weasel, mustela itatsi, which was introduced in the mid-1950s (see below), as the causal agent. this agile and mostly nocturnal mustelid (masuda and watanabe, 2009) had been introduced to several islands of the ryukyu archipelago to control introduced rodents (e.g., uchida, 1969; shiraishi, 1982). in this archipelago where most islands lack native carnivorous mammals, these weasels have in multiple instances (at least 14 islands) been implicated in the declines of certain indigenous reptiles (e.g., shibata, 1964; toyama, 1983; hikida et al., 1992; ota and masunaga, 2004; toyama, 2005 and cited therein). a good deal of notoriety has attended the weasel because its introduction resulted in near extinction of an indigenous skink (plestiodon latiscutatus, as eumeces okadae) on miyakejima island in the izu islands of japan (hasegawa, 1999). yoronjima is a small and isolated island, and as a consequence the population size of native herptiles would have been limited. there is little doubt that predation by weasels was sufficient to rid the island of these squamate reptiles and of the frog rhacophorus viridis. although j. p. polygonata and r. viridis are arboreal species, their frequent use of ground habitats may have left them vulnerable to the weasels’ predation. 30 yasuyuki nakamura et al. chronological correlation also corroborates the idea that predation by weasels might have been responsible for the herptile extinctions on yoronjima. the introduction of weasels to yoronjima occurred in 1953, when 13 individuals were imported, and in 1955 or 1956, 60 more individuals (possibly more than 200: iha, 1966) followed (yotsumoto, 1959; morita, 1988). the last records for japalura polygonata polygonata and dinodon semicarinatum are from 1955, those for rhacophorus viridis from 1959 (koba, 1956; nakamura et al, 2009). in both cases, cessation of sightings seems to have followed closely on the introduction of the weasel. causes and timing of the disappearance of the other species concerned (amphiesma pryeri, ateuchosaurus pellopleurus, a large-sized gekko species, and goniurosaurus kuroiwae) are unknown; it is however likely that they shared the same fate. the extent to which recent or earlier human activities have affected the composition of local herpetofaunas in the ryukyu archipelago remains to be clarified by expanding records of late quaternary fossils and subfossils of terrestrial vertebrates. documenting anthropogenic losses of terrestrial vertebrates would allow us to predict the relative ability of species or populations to persist under human impacts, as well as informing conservation management of currently endangered species/populations. the present study shows that some populations of indigenous squamates vanished from yoronjima. although their conspecifics (or members of the same subspecies) still exist on other islands, their disappearance from yoronjima may be a matter of serious concern because there is no guarantee that the currently accepted taxa provide appropriate units for conservational considerations (ota, 2000). moreover, these populations have disappeared before their taxonomic status could have been verified by modern taxonomic techniques. taxonomic investigation of amphibians and reptiles of the ryukyu archipelago is far from completion, as illustrated by a series of recent findings (e.g., toda et al., 2008; matsui, 2011). it is now evident that in terms of population genetics and morphology, there are several levels of intraspecific diversity in certain island populations of amphibians and reptiles in this archipelago (e.g., shibaike et al., 2009). these circumstances raise the question about whether the losses on yoronjima can be treated as just the demise of local populations. a concluding question to be addressed here is how the rich accumulation of terrestrial vertebrate remains in the investigated garbage dump was developed. this site appears to have no structural peculiarity that would turn it into a natural trap for these animals, but it has to some extent acted as a sink for surface waste. owl activity as the most frequently attributed factor for the accumulation of small terrestrial vertebrate remains is not in evidence here, as the components of this bone assemblage are a mixture of diurnal (japalura and skinks) and nocturnal species (others) with a considerable range of body sizes, ranging from tiny ateuchosaurus to rattus norvegicus. the only known resident owl on yoronjima is the brown hawk-owl, ninox scutulata (amami ornithologists’ club, 2009); it is a relatively small-sized bird (~30 cm in total length) and therefore its exclusive involvement in the accumulation of these vertebrate remains seems to be improbable. with the exception of the sea snake, dietary use of these vertebrates by humans is also unlikely; many of the identified species of squamates and frogs seem to be too small to use as food resources for humans, and none of the material from the site showed any sign of cooking or having been worked. we suppose that some of the finds may be incidental remains from animals died in the vicinity, but mostly the animals found here may have foraged in the dump for arthropods and small vertebrates associated with garbage and leftovers from the inhabitants, and have died or been killed there. co-occurrence of their remains suggests that rodents and shrews were likely to be involved, although none of the examined reptilian and amphibian remains showed clear evidence of marks attributable to these scavengers. if this conjecture holds true, remains of old garbage dumps, very common facilities anywhere in the world, would offer effective data sources to reveal recent changes in the biodiversity of terrestrial vertebrate faunas, particularly those of limestone islands in lower latitudes. acknowledgements we thank seiji moriyama and the late shinichiro moriyama for permission to carry out sampling on his land; jumpei ichikawa, shun`ichi matsumura, and yoshitaka tahara for providing us with comparative material; mamoru toda (university of the ryukyus) for permission of use of specimens in his care; and kentaro hagari and motohiro okade for help of part of fieldwork. we also thank julien claude (ise-m, france) and krister t. smith (senckenberg research institute and natural history museum, germany) for valuable comments to the manuscript. this study was partially supported by a grant-inaid to the 21st century coe program at the university of the ryukyus from the ministry of education, culture, sports, science and technology, japan (monbu-kagaku-sho). supplementary material associated with this article can be found at http:// www.unipv.it/webshi/appendix manuscript number 11924. references amami ornithologists’ club (2009): birds of amami. bun-ichi sogo shuppan, tokyo. 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(2007): island biogeography: ecology, evolution and conservation (second edition). oxford university press, oxford. appendix skeletal specimens examined listed those of snakes and lizards occurring in the central ryukyus, extralimital taxa were omitted. acronyms are kuzr = kyoto university museum zoological specimens and npn = y. nakamura private collection. snakes—achalinus werneri: okinawajima (npn 1091); amphiesma pryeri: amamioshima (npn 774), okinawajima (npn 650, 651, 774, 775, 776, 1067, 1090); cyclophiops semicarinatus: okinawajima (npn 511, 512, 648, 649, 858); dinodon semicarinatum: okinawajima (npn 009, 513, 653, 777, 778, 779, 780, 859); emydoceph34 yasuyuki nakamura et al. alus ijimae: okinawajima (npn 1085, 1087); hydrophis melanocephalus: iriomotejima (npn 1080), okinawajima (npn 1086); laticauda colubrina: iriomotejima (npn 857); laticauda laticaudata: iriomotejima (npn 726); laticauda semifasciata: miyakojima (npn 1074); protobothrops flavoviridis: okinawajima (npn 1093); ovophis okinavensis: okinawajima (npn 1094); ramphotyphlops braminus: okinawajima (npn 1102); sinomicrurus japonicus japonicus: amamioshima (npn 1088); sinomicrurus japonicus boettgeri: okinawajima (npn 656). lizards—ateuchosaurus pellopleurus: amamioshima (npn 510), okinawajima (npn 1051, 1096); gehyra mutilata: ukejima (npn 504); gekko hokouensis: okinawajima (kuzr 62307, 62308, 62317, 62319, 62424, npn 1070, 1071), ukejima (npn 509), yoronjima (kuzr 62240, 62264, 62265, npn 644, 645); gekko shibatai: takarajima (kuzr 62272, 62274, 62275, 62279, 62345, 62364); gekko vertebralis: kotakarajima (kuzr 62266, 62267, 62268, 62269, 62280, 62388, 62389); gekko sp.: okinawajima (kuzr 62354, 62390, 62392, 62397, 62410, 62421, 62422, 62423, 62435, npn 1095); goniurosaurus kuroiwae kuroiwae: okinawajima (kuzr 52392, 65827, 67952, 71631, 71909); goniurosaurus kuroiwae orientalis: tokashikijima (kuzr 71908); hemidactylus bowringii: ukejima (npn 506); hemidactylus frenatus: okinawajima (npn 1068, 1069, 1097, 1098), yoronjima (npn 646, 647); japalura polygonata polygonata: amamioshima (npn 767), okinawajima (npn 1042, 1051), tokashikijima (npn 632, 633, 634, 635, 636); lepidodactylus lugubris: okinawajima (npn 1046, 1047); plestiodon barbouri: okinawajima (kuzr 62980, 62981); plestiodon marginatus: okinawajima (npn 382, 654, 655); takydromus smaragdinus: okinawajima (npn 285, 643, 1099, 1100). supplementary material photographs of selected skeletal elements of snakes and lizards occurring in the central ryukyus fig. s1. middle trunk vertebrae of typhlopid (a) and colubrid (b-e) snakes. (a) ramphotyphlops braminus (npn 1102), (b) achalinus werneri (npn 1091), (c) cyclophiops semicarinatus (npn 512), (d) dinodon semicarinatum (npn 513), and (e) amphiesma pryeri (npn 774). left to right: anterior, lateral (left), posterior, dorsal, and ventral views. scale bars equal 1 mm. fig. s2. middle trunk vertebrae of viperid (a, b) and elapid (c, d) snakes. (a) ovophis okinavensis (npn 1094), (b) protobothrops flavoviridis (npn 1093), (c) emydocephalus ijimae (npn 1087), and (d) hydrophis melanocephalus (npn 1080). left to right: anterior, lateral (left), posterior, dorsal, and ventral views. scale bars equal 1 mm. fig. s3. middle trunk vertebrae of elapid snakes (continued). (a) laticauda colubrina (npn 857), (b) l. laticaudata (npn 726), (c) l. semifasciata (npn 1074), and (d) sinomicrurus japonicus boettgeri (npn 656). left to right: anterior, lateral (left), posterior, dorsal, and ventral views. scale bars equal 1 mm. fig. s4. maxilla and mandibles of agamid (a, b), scincid (c-e), and lacertid (f) lizards (in medial views). (a) right maxilla of japalura polygonata polygonata (npn 1051), (b) right mandible of j. p. polygonata (npn 1051), (c) right mandible of ateuchosaurus pellopleurus (npn 1096), (d) right mandible of plestiodon barbouri (kuz r62980), (e) right mandible of p. marginatus (npn 665), and (f) right mandible of takydromus smaragdinus (npn 643). scale bars equal 1 mm. fig. s5. mandibles of gekkotan lizards (in medial views). (a) right mandible of gehyra mutilata (npn 504), (b) right mandible of gekko hokouensis (npn 644), (c) right mandible of g. shibatai (kuzr 62274), (d) right mandible of g. vertebralis (kuzr 62267), (e) right mandible of gekko sp. (kuzr 62392), (f) right mandible of hemidactylus bowringii (npn 506), (g) right mandible of h. frenatus (npn 1098), (h) right mandible of lepidodactylus lugubris (npn 1046), and (i) left mandible of goniurosaurus kuroiwae kuroiwae (kuzr 71631). scale bars equal 1 mm. fig. s6. maxillae of gekkotan lizards (in medial views). (a) left maxilla of gehyra mutilata (npn 504), (b) right maxilla of gekko hokouensis (npn 644), (c) right maxilla of g. shibatai (kuzr 62274), (d) right maxilla of g. vertebralis (kuzr 62267), (e) right maxilla of gekko sp. (kuzr 62392), (f) right maxilla of hemidactylus bowringii (npn 506), (g) right maxilla of h. frenatus (npn 1098), (h) right maxilla of lepidodactylus lugubris (npn 1046), and (i) right maxilla (dorsal part broken) of goniurosaurus kuroiwae kuroiwae (kuzr 71631). scale bar equals 1 mm. fig. s7. frontals of gekkotan lizards (in dorsal [right] and ventral [left] views). (a) gehyra mutilata (npn 504), (b) gekko hokouensis (npn 644), (c) g. shibatai (kuzr 62274), (d) g. vertebralis (kuzr 62267), (e) gekko sp. (kuzr 62392), (f) hemidactylus bowringii (npn 506), (g) h. frenatus (npn 1098), (h) lepidodactylus lugubris (npn 1046), and (i) goniurosaurus kuroiwae orientalis (kuzr 71908) with anterior part of the skull. scale bars equal 1 mm. acta herpetologica vol. 8, n. 1 june 2013 firenze university press the oogenic cycle of the caspian bent-toed gecko, cyrtopodion caspium (squamata: gekkonidae) in iran vida hojati1*, kazem parivar2, eskandar rastegar-pouyani3, abdolhossein shiravi1 altitudinal effects on life history parameters in populations of liolaemus pictus argentinus (sauria: liolaemidae) joel antú gutiérrez1,*, carla piantoni2, nora r. ibargüengoytía1,3 recent cryptic extinction of squamate reptiles on yoronjima island of the ryukyu archipelago, japan, inferred from garbage dump remains yasuyuki nakamura1,*, akio takahashi2, hidetoshi ota3 trophic niche and feeding biology of the italian wall lizard, podarcis siculus campestris (de betta, 1857) along western mediterranean coast marco a.l. zuffi*, chiara giannelli novel, non-invasive method for distinguishing the individuals of the fire salamander (salamandra salamandra) in capture-mark-recapture studies goran šukalo1,*, sonja đorđević2, dragojla golub1, dejan dmitrović1, ljiljana tomović2,3 going out tonight? when insular hierophis viridiflavus breaks the whip snakes rules delaugerre michel-jean first evidence of a paedomorphic population of the smooth newt (lissotriton vulgaris) in the czech republic václav gvoždík1,2, veronika javůrková4, oldřich kopecký5,* no detection of chytrid in first systematic screening of bombina variegata pachypus (anura: bombinatoridae) in liguria, northern italy stefano canessa1,*, an martel2, frank pasmans2 status of the european pond turtle, emys orbicularis (reptilia: testudines: emydidae) in vorarlberg, austria andreas kleewein1, günther wöss2 comparative cytogenetics of two species of ground skinks: scincella assata and s. cherriei (squamata: scincidae: lygosominae) from chiapas, mexico riccardo castiglia1,*, alexandra m.r. bezerra2, oscar flores-villela3, flavia annesi1, antonio muñoz4, ekaterina gornung1 polydactyly in the tyrrhenian wall lizard (podarcis tiliguerta) antigoni kaliontzopoulou1,*, daniele salvi1, verónica gomes1, joão p. m. c. maia1,2,3, panagiotis kaliontzopoulos4 book review: roberto sindaco, alberto venchi, cristina grieco. the reptiles of the western palearctic. 2. annotated checklist and distributional atlas of the snakes of europe, north africa, middle east and central asia, with an update to the vol. 1. edoardo razzetti acta herpetologica journal of the societas herpetologica italica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 9(1): 109-113, 2014 doi: 10.13128/acta_herpetol-12658 meiotic behavior of two polyploid species of genus pleurodema (anura: leiuperidae) from central argentina nancy e. salas1, julián a. valetti2, pablo r. grenat1,3,*, manuel a. otero1, adolfo l. martino1 1ecología-educación ambiental, departamento de ciencias naturales, facultad de ciencias exactas, físico-químicas y naturales, universidad nacional de río cuarto, ruta nacional n° 8 km 601, (x5804bya) río cuarto, argentina. *corresponding author. email: pgrenat@exa.unrc.edu.ar 2genética de poblaciones, departamento de ciencias naturales, facultad de ciencias exactas, físico-químicas y naturales, universidad nacional de río cuarto, ruta nacional n° 8 km 601, (x5804bya) río cuarto, argentina 3conicet fellowships submitted on: 2013, 22nd february; revised on: 2013, 19th december; accepted on: 2014, 11th january abstract. polyploidy is an important evolutionary force but rare in vertebrates. however, in anurans, the genus pleurodema has polyploid species, two of them tetraploid and one octoploid. the manner in which the chromosomes join in diakinesis can vary among species and, crucially, if they differ in their ploidy levels. in this work, we describe the meiotic configurations in two cryptic species from central argentina, with different ploidy levels, pleurodema kriegi (tetraploid) and p. cordobae (octoploid). a total of 306 diakineses from 19 individuals were analyzed. in meiosis, p. kriegi form 22 bivalents, whereas p. cordobae exhibits variation in meiotic figures. we discuss the possible alloand autopolyploid origin of these species, and we consider that the autopolyploid origin of p. cordobae from p. kriegi might be the most feasible. keywords. pleurodema cordobae, polyploidy, cytogenetics, diakinesis. polyploidy is an important evolutionary force in some plant groups, (white, 1973; lacadena, 1996; otto and whitton, 2000), but this mechanism is rarer in vertebrates (orr, 1990; holloway et al., 2006). however, polyploidy in anuran amphibians has been documented in numerous families (barrio and rinaldi de chieri, 1970; bogart, 1980; kawamura, 1984; kuramoto, 1990; beçak and beçak, 1998; otto and whitton, 2000; martino and sinsch, 2002; stöck et al., 2002; rosset et al., 2006; mable et al., 2011; bogart and bi, 2013). the frog genus pleurodema tschudi, 1838 is distributed from panama throughout south america to southern chile and argentina and currently is represented by 15 species, of which 10 have been recorded in argentina (valetti et al., 2009; kolenc et al., 2009; maciel and nunes, 2010; faivovich et al., 2012). this genus is exceptional in including species with three levels of ploidy. according to cytogenetic studies conducted so far, pleurodema includes three polyploid species, p. kriegi (müller 1926) and p. bibroni tschudi 1838 which are 2n = 4x = 44, and p. cordobae valetti, salas and martino 2009 which is 2n = 8x = 88, while the remaining species are diploid (2n = 2x = 22) (brum-zorrilla and sáez, 1968; barrio and rinaldi de chieri, 1970; veloso et al., 1972; duellman and veloso, 1977; schmid et al., 1993; lourenço et al., 2006; valetti et al., 2009). during meiosis, chromosome pairing and crossing over of homologous chromosomes occurs in prophase i, and is in diakinesis as can be seen by the more condensed chromosomes and the formation of chiasmata (macgregor, 1993). the way in which chromosomes are joined forming chiasmata may vary between species, particularly if they are of different ploidy levels, resulting in different meiotic configurations (salas and martino, 2009). 110 n.e. salas et alii in this paper, we analyze the meiotic figures of pleurodema kriegi (tetraploid) and pleurodema cordobae (octoploid). pleurodema kriegi and p. cordobae are two polyploid cryptic species endemic to the sierra grande of cordoba, argentina. despite having different ploidy levels, they maintain a high degree of morphological and acoustical similarity (valetti et al., 2009). the size of adult individuals in both species varies between 28 and 41 mm, p. cordobae is slightly larger than p. kriegi and, in both cases, females are larger than males (valetti, 2012). these species breed from november to march (austral spring-summer) and are crepuscular and nocturnal (valetti et al., 2009). samples of p. kriegi from two localities of sierra de achala (31º36’46’’ s, 64º52’29’’ w and 31º36’23’’ s, 64º52’6’’ w) and p. cordobae from two sites of sierra de comechingones (32º23’58’’ s, 64º55’35’’ w and 32°22’13” s, 64°55’55” w) were collected during spring and summer months between 2006 and 2010. the sierra de córdoba constitutes the eastern group of the sierras pampeanas system embracing the sierra grande, sierra norte and sierra chica. sierra grande includes the sierra de achala to the north and the sierra de comechingones to the south (miró, 1999; ramos, 1999). this region is characterized by a humid temperate climate with snow in winter. the annual rainfall is 800– 900 mm, distributed mainly in the spring-summer period (capitanelli, 1979). the environment both in the pampa de achala and the sierra de comechingones is very similar. the water bodies in which the species were sampled were semi-permanent ponds in highland pastures. after treatment in vivo with 0.3% colchicine, individuals were anesthetized and sacrificed by immersion in a 1% solution of ms-222 (pukhta and blazhek, 2004). meiotic chromosomes, prepared from testis, were studied in 64 spermatocytes in diakinesis of four p. kriegi individuals and 242 spermatocytes in diakinesis from 15 p. cordobae individuals. all techniques, hypotonic treatment, fixation of cells and staining were performed according to schmid, (1978a, b); schmid et al., (1979) and schmid et al., (1990). chromosomal preparations, when dried and stained, were examined using a zeiss axiophot d-7082 fluorescence microscope with a zeiss axiocam hrc digital camera. images were captured using the software axiovision version 4.8. the diakineses were examined using the image analysis program adobe ® photoshop ™ cs2. the analysis of spermatocytes in diakinesis revealed that all individuals of the tetraploid species p. kriegi formed 22 bivalents rings (fig. 1a, 2a). in contrast, the analysis of diakinesis in individuals of p. cordobae revealed different meiotic configurations (table 1). the most common configuration (68%) comprised an octavalent (viii), 15 tetravalents (iv) and 10 bivalents (ii) (fig. 1b, 2b). however, other figures were observed. in 23% of the cases two types of configurations were observed: 1) an octavalent, 14 tetravalent and 12 bivalents; and 2) an octavalent, 16 tetravalents and eight bivalents. a small number (9%) of cells analyzed correspond to other types of configurations (table 1). fig. 2. karyotype of the diakinesis of (a) pleurodema kriegi, la posta, pampa de achala, ((2n = 4x = 44) and (b) pleurodema cordobae, los tabaquillos, sierra de comechingones, (2n = 8x = 88). fig. 1. spermatocytes in diakinesis. (a) pleurodema kriegi, la posta, pampa de achala, (2n = 4x = 44). (b) pleurodema cordobae, los tabaquillos, sierra de comechingones, (2n = 8x = 88). table 1. patterns of meiotic figures in pleurodema cordobae. meiotic configurations total observed diakineses 1 viii 15 iv 10 ii 165 1 viii 14 iv 12 ii 30 1 viii 16 iv 8 ii 25 15 iv 14 ii 6 13 iv 18 ii 6 1 viii 13 iv 14 ii 5 1 viii 14 iv 12 ii 5 111meiotic behavior of two polyploid pleurodema diakinetic analysis demonstrated that the chromosomes of tetraploid p. kriegi join forming bivalent instead of tetravalent figures, leading to 22 rings in all cases analyzed. barrio and rinaldi de chieri (1970) interpreted these same results as reflecting an allopolyploid origin. barrio (1977) suggested that the ancestral species of p. kriegi and p. bibroni (both tetraploid) would have originated by allopolyploidy from p. thaul and related species (possibly extinct) in semiarid western argentina, and subsequently spread eastward from the plains to the atlantic coast. finally, in the pleistocene, the ancestral species would have undergone cladogenesis, giving rise to the extant species p. kriegi in the central area of argentina and p. bibroni in uruguay. in the analysis of p. cordobae diakinesis, chromosomes showed no consistent pattern. in contrast, we observed different numbers of rings in their cells, including multivalent meiotic configurations. multivalent configurations have been described in several polyploid anuran species (beçak et al., 1966, 1967, 1970; schmid et al., 1985; salas and martino, 2009). although multivalent formations can occur in an allopolyploid if the hybridizing species are closely related (chenuil et al., 1999), the multivalent formations observed in p. cordobae could be evidence of autopolyploid origin. moreover, recently faivovich et al., (2012) performed a phylogenetic analysis of the genus pleurodema based on sequences of nuclear and mitochondrial genes. these authors obtained a clade comprising the three polyploid species, where p. cordobae is the sister of p. kriegi and these two species together form the sister taxon of p. bibroni. as the authors demonstrate, these results also would be consistent with an autopolyploid origin of p. cordobae from p. kriegi, although they make clear that cytogenetic studies and a population-level approach will be required to confirm this. therefore, under the hypothesis of a recent autopolyploid origin of p. cordobae from p. kriegi, both geographically close, it could be expected that the chromosomes are paired forming tetrads, in diakinesis. this is because in the potential ancestral species the chromosomes are paired forming dyads. however, as noted above, the behavior of the chromosomes was not as expected. bogart and bi (2013) conclude that polyploid species generally have higher levels of genetic diversity than related diploid species and that these polyploids accumulate genes from such diploid associations by having additional linkage groups. therefore, meiotic aberrations are necessary for the initial evolution of polyploids and may persist to maintain the genomic integrity (bogart and bi, 2013; stenberg and saura, 2013). consequently, the origin of p cordobae by polyploidy could be relatively recent and the meiotic behavior to be responding to the same fact. although in p. cordobae we did not observe a consistent pattern, the presence of an octavalent ring in all diakinesis tested is notable. however, in accordance with expected pattern and the studies realized to date, we cannot interpret such meiotic behavior. cytogenetic studies by gish technique in cells of p. cordobae hybridized with probe of total genomic dna of p kriegi, would determine the number of chromosome complements shared by the two species. this method would enable evaluate the hypothesis of an autopolyploid origin of p. cordobae from p. kriegi. the polyploid individuals produce viable gametes if these are balanced in chromosome number; otherwise the gametes would be infertile. macgregor (1993) stated that this problem could be solved by mechanisms to prevent unequal multivalent formation, or by strengthening the effective meiosis diploidization yielding only bivalents. the results obtained from our analysis of meiotic configurations of p. cordobae may indicate that the gametes formed in this species have low viability. however, populations of p. cordobae are quite numerous and biological data on the species indicate that these populations are reproducing without major problems (valetti et al., 2011). future studies analyzing chromosome behavior in diakinesis in greater detail and evaluating fertility in p. cordobae will be critical to the understanding of this particular chromosome pairing mode at the stage prior to the generation of gametes. moreover, the octoploid condition of p. cordobae and the features shown in this study make this species an interesting model for genetic research. acknowledgements the secretary of research and technology of the national university of río cuarto (secyt-unrc) provided funds under grant ppi 18/c350. the second and third authors thank conicet – argentina (consejo nacional de investigaciones científicas y tecnológicas) for fellowships granted. our study was authorized by the córdoba environmental agency (a01/2013). references barrio, a. 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(1973): animal cytology and evolution. cambridge university press, london. acta herpetologica vol. 8, n. 2 december 2013 firenze university press journal of the societas herpetologica italica acta herpetologica acta herpetologica 9(2): 147-158, 2014 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-13473 comparative morphology of liolaemus lizards precloacal glands soledad valdecantos1,*, virginia martínez1, antonieta labra2,3 1 ibigeo (instituto de biología y geología del noa), universidad nacional de salta-conicet, avenida bolivia 5150, 4400, salta, argentina. *corresponding author. e-mail: solevaldecantosp@gmail.com 2 programa de fisiología y biofísica, facultad de medicina, universidad de chile, casilla 70005, correo 7, santiago, chile 3 university of oslo, department of biosciences, centre for ecological and evolutionary synthesis, p.o. box 1066 blindern, n-0316 oslo, norway submitted on 2013, 25th october; revised on 2014, 5th may; accepted on 2014, 4th june editor: giovanni scillitani abstract. liolaemid lizards and amphisbaenids have precloacal pores in the anterior border of the cloaca, where epidermal glands drain and expel pheromonal secretions. precloacal glands occur usually only in males, but in those few species where both sexes have precloacal glands, these are larger in males. only the morphology and/or histology of precloacal glands of amphisbaenids have been described, and it is unknown whether in lizards these glands differ across ages, sexes and/or species, and if the lack of pores is associated with a lack of glands. we investigated for the first time the morphology and histology of lizard precloacal glands, by studying three liolaemus species that differ in the presence of pores in their cloaca: l. irregularis, in which adults and juveniles of both sexes have pores; l. poecilochromus, in which only adult males have pores, and l. neuquensis, in which the adults of both sexes lack pores. results show that the number of pores varies among species and sexes, but not between ages of a species. adults, but not juveniles, of l. irregularis have sexual dimorphism in pore sizes; these are larger in males than in females. in addition, pores are larger in adult males of l. irregularis than in l. poecilochromus. glands are tubuloalveolar with holocrine secretion, having similar structure across individuals, although adult males have larger glands than females and juveniles. finally, the structure of liolaemus precloacal glands is very similar to those of the amphisbaenid precloacal glands and the femoral glands of other lizard species. keywords. epidermal gland, histology, histochemistry, sexual dimorphism, ontogenetic variation. introduction many lizard species have epidermal glands with secretions, which are a complex mix of lipids and proteins, involved in intraspecific chemical communication (mason, 1992; louw et al., 2007; weldon et al., 2008; martín and lópez, 2011; louw et al., 2011). externally, glands are recognized by patches of modified scales that allow the release of the secretions. one type, the generational glands, lacks a collecting portion and the associated scales have a glandular appearance through which secretions are expelled directly, without a pore (van wyk and mouton, 1992). the other type of glands, the precloacal/femoral glands, have a unique and clear pore where the collecting portion of the gland drains secretions to be expelled (cole, 1966a; maderson and chiu, 1970; baig and böhme, 1991; van wyk and mouton, 1992; jared et al., 1999). the precloacal pores are situated on the anterior border of the cloaca, while the femoral ones are located on the ventral surface of the thighs (alberts et al., 1992; imparato et al., 2007; labra, 2008). pores are usually present only in adult males (chauhan, 1986; dujsebayeva et al., 2009; lobo et al., 2012). in species where both sexes have pores, these are larger in males (cole, 1966b, athavale et al., 1977, ferreira, 2007), and if juveniles have pores, these are larger in adults than in juveniles (cole, 1966b; alberts et al., 1992). in a few species, both sexes have secondarily lost these pores (darevsky and szczerbak, 1997; lobo, 2005). 148 s. valdecantos et alii the morphology of the femoral glands has received significant attention (e.g. athavale et al., 1977; chauhan, 1986; ferreira, 2007; imparato at al., 2007; chamut et al., 2009), and they can be lobed (antoniazzi et al., 1993; imparato et al., 2007) or tubular, simple (chiu and maderson, 1975), branched (chiu and maderson, 1975; ferreira, 2007) or acinar (khannoon et al., 2013). in contrast, the knowledge about the precloacal glands morphology is confused, because a variety of glands located in the cloacal area had been called precloacal glands, such as some generation glands and callose scales of several cordylid (e.g. van wyk and mouton, 1992) and agamid lizards (e.g. dujsebayeva et al., 2007). the other group of glands, those traditionally called precloacal glands (etheridge, 1995; pinna et al., 2010), is located on the border of the cloaca, which have a recognizable secretory and collecting portion, and are found in amphisbaenids and liolaemid lizards. focusing on these later precloacal glands, we can say that the knowledge on their morphology is limited to some studies on amphisbaenas (e.g. antoniazzi et al., 1993). however, because the structure of the glands of liolaemid lizards is unknown, the understanding of the extent of morphological precloacal gland variation as compared to the femoral gland, it is not possible. the lack of information regarding the morphology of precloacal glands and pores in liolaemus lizards, the most diverse liolaemidae genus (lobo et al., 2010a), with more than 240 species (uetz and hošek, 2014), contrasts with the extensive use of precloacal pores as a taxonomic character, as well as for sex discrimination (lobo, 2005). gland secretions are involved in social recognition (labra, 2008), and their lipid compositions differ among species and populations (escobar et al., 2001; 2003). the glands apparently share a function across liolaemus, but the variation in morphology among species, sexes or age classes (i.e., juveniles vs. adults) is largely unknown, and it is unclear if the lack of external pores is associated with a complete absence of these epidermal glands. to shed some light on these questions, we studied the external, anterior ventral border of the cloaca in three liolaemus species that differ in the occurrence of precloacal pores: 1l. irregularis: adults and juveniles of males and females have pores (valdecantos and lobo, 2007); 2 l. poecilochromus: only adult males have pores (laurent, 1986); and, 3l. neuquensis: adults of both sexes lack pores (scolaro et al., 2007). materials and methods animals we processed individuals of the herpetological collection of the museo de ciencias naturales of universidad nacional de salta (argentina), in order to have an adequate sample size without affecting negatively the natural populations. specimens were fixed in 10% buffered formalin solution and preserved in 70% alcohol, and were collected in consecutive summers (december-february of 2007, 2008, 2009), not in their mating season (ramírez-pinilla, 1991). we used 83 individuals of l. irregularis, 38 of l. poecilochromus and two of l. neuquensis. the description of the color of the secretions, however, was based on field observations of wild lizards. histology we removed the external, anterior ventral border of the cloaca of 31 l. irregularis (♂: 7 juveniles, 8 adults; ♀: 9 juveniles, 7 adults), 22 l. poecilochromus (♂: 5 juveniles, 8 adults; ♀: 2 juveniles, 7 adults) and two l. neuquensis adults (1♂, 1♀), and dehydrated in graded series of ethanol solution, cleared in xylene and embedded in paraffin. thereafter, paraffin sagittal serial sections (5-7 µm) were obtained with a rotary microtome (reichter, germany) and were stained with hematoxylin-eosin (h & e). based on previous studies on sexual maturity (valdecantos and lobo, 2007), we considered juveniles of l. irregularis and l. poecilochromus individuals which snout-vent lengths ranged from 32.76 to 48.63 mm, without including newborns since they were not available in the collection. adults of l. irregularis had sizes from 70.4 to 99.1 mm, and l. poecilochromus, from 62.14 to 75.03 mm. no age classes were studied in l. neuquensis, and the adult male measured 60.44 mm, while the adult female measured 59.25 mm. gland descriptions and their measurements were done on the three best central mid-sagittal sections (i.e., not on their borders) of the best-sectioned gland from each specimen. descriptions follow the nomenclature of cole (1966b), geneser (2000), and gartner and hiatt (1994). histochemistry to identify neutral and acid mucosubstances, paraffin sections were treated separately with periodic acid schiff ’s reagent (pas) and alcian blue (ab) ph 2.5 (martoja and martojapierson, 1970). separately, we stained new slides with bromophenol blue to identify proteins (imparato et al., 2007). we did not search for lipids, because they solubilize in alcohol, which was used to preserve the lizards. slides were observed under an olympus bx40 microscope (tokyo, japan) and photographed with a digital camera olympus dp25 (tokyo, japan). scanning electron microscopy we processed the external, anterior ventral cloacal border of four l. irregularis, one of each sex and age (juvenile, adult), and one adult male of l. poecilochromus. the tissues were dehydrated by critical point, dried with liquid carbon dioxide, sputtered with gold, and examined under a scanning electron microscopy (jeol jsm-6480, tokyo, japan). 149precloacal glands of liolaemus lizards morphometry of precloacal pores and glands we determined the number and diameter (mm) of the precloacal pores of l. irregularis and l. poecilochromus (see table 1 for sample size). to obtain the pore diameters, we measured the length of the minor and major axes of each pore, using a binocular arcano magnifying glass (hg 272931, shangai, china) with a graduated ocular lens. each individual was characterized by the average value of the diameters of all its pores. using a microscope olympus magnifying glass (tokyo, japan) with a graduated ocular lens, and following cole (1966b), we measured the maximum length of the glands from the anterior end to the posterior end of the basal layer of germinative epithelial cells. the maximum width was measured in the transverse axis of the precloacal gland (see table 2 for sample size and fig. 5 for indications of how these measurements were taken). to remove the effect of body size in the statistical analyses of pores and glands sizes, we measured the snout-vent length of these lizards (see below). statistics the numbers of precloacal pores were not normally distributed, and comparisons were made with non-parametric mann-whitney tests. the log10 transformed pore diameters and the log10 length and width of the precloacal glands were normally distributed, and were compared using analysis of covariance (ancova) with snout-vent length as covariate, followed by a posteriori tukey tests (zar, 1999). results gross morphology and morphometry juveniles and adults of both sexes of l. irregularis (figs. 1a, b, c, d), and all males of l. poecilochromus (fig. 1e), had a line of precloacal pores, and each pore was located in a single modified scale. two adult males of l. poecilochromus had, immediately posterior to the line of precloacal pores, a row of “secondary pores” (fig. 1f), consisting of three to four pores, smaller than the primary ones. because these “secondary pores” were uncommon, they were not considered in the final analyses of pore morphology. three out of the seven adult females of l. poecilochromus had one or two precloacal pores (fig. 1g), and one of these females had a gland that opened inside the cloaca, rather than with a pore in a scale. the remaining females did not have any trace of pores (fig. 2f). finally, three out of five juvenile males of l. poecilochromus had in the scales of the cloacal border, deep invaginations without openings, i.e. clefts (fig. 1h). the inspection of the two l. neuquensis individuals did not reveal pores or clefts. the secretions of living animals had an orange color (fig. 1a). in adult males of l. irregularis and l. poecilochromus secretions were always visible as a solid and compact pack of dead squamous cells and secretory cells (fig. 2a), which in most adult males emerged through the pores as long and rigid cylinders, resembling wax crayons (figs. 1a, e). in contrast, in females and juveniles little of the glandular secretions emerged out of the pore (figs. 1b, c, d). the morphometry of precloacal pores is in table 1. males (adults and juveniles) of l. irregularis had more precloacal pores than females (adults: w = 338.50, p = 0.0001; juveniles: w = 25.00, p = 0.004). in addition, adult males had larger pores than adult and juvenile females (figs. 2a, c), but there were no differences between juveniles of both sexes (figs. 2b, d). differences in pore size between ages were observed in females, but not in males (f = 26.51, p<0.0001). finally, l. irregularis had more (w = 122.00, p<0.0001, figs, 1a, e, f) and larger precloacal pores (f = 9.14, p = 0.0054, figs. 2a, e) than l. poecilochromus. histology each pore was associated to a tubuloalveolar gland, compound in adult (figs. 3a, e), simple in juveniles (figs. 4a, c). glands always had secretion in the lumen, independent of the age and sex of the individuals. the morphology, but not the size of these glands was similar between sexes and ages (table 2; length: f = 9.16, p = 0.0004; width: f = 21.06, p<0.0001). adult males of l. irregularis had longer glands than adult females and juveniles, but there were no differences between juveniles of both sexes. the width of the glands was larger in adult males than in females, but no differences were found between age classes by sex. finally, adult males of l. irregularis had longer (f = 5.93, p = 0.0331), but not wider (f = 0.0001, p = 0.9910), precloacal glands than l. poecilochromus. the size of the glands increased with the body size in adult males of l. irregularis (figs. 5a, b), which are large enough to touch each other (fig. 3a). the following description corresponds to the glands of adult males of l. irregularis (fig. 3a) and l. poecilochromus (fig. 3e). the gland is surrounded by an envelope of a thin layer of connective tissue (figs. 3b, e), which extends into the partitions that divide the gland into several elongated lobules (figs. 3b, c, e). each lobule has several tubules and alveoli that converge in a single and short central duct that collects the secretions, which finally reach the pore (figs. 3a, e). tubules and alveoli are internally coated by a layer of basal cells, the germinative epithelium (figs. 3d, f). this layer consists of small, flat to cubic cells that have oval to spherical nuclei, with conspicuous nucleoli, which occupy the larg150 s. valdecantos et alii fig. 1. ventral view of adult and juvenile of liolaemus lizards showing the cloacal area. liolaemus irregularis: (a) adult male, (b) adult female, (c) juvenile male, (d) juvenile female. l. poecilochromus: (e) and (f) adult males, (g) adult female, (h) juvenile male. the white arrowhead from (a) to (g) shows the precloacal pores with secretions and in (h), the clefts. the white circle in (f) indicates a “secondary pore”. figures (a), (b) and (e) were taken in wild lizards in the field, showing the normal condition of these secretions. the other pictures were taken from specimens from the herpetological collection. 151precloacal glands of liolaemus lizards est part of the cells (figs. 3e, f). mitoses are observed in the germinative stratum (fig. 3f), indicating a continuous proliferation of cells that replenish the dead ones. as cells proliferate, they move toward the periphery, and during this migration, they enlarge, increasing their cytoplasmic content with granules (figs. 3d, g), which show a weak pas positive reaction (i.e., pink tones; fig. 6a). towards the center of the tubules and alveoli, most free/ luminal cells are degenerated as evidenced by the gradual pyknosis of their nuclei or because they are enucleated, with a disaggregated cytoplasm (figs. 3d, g). inside the central duct, the structural integrity of the cells is not recognizable any more (fig. 3g). the final secretion appears as a mixture of secretory granules and cell debris, corresponding to a holocrine secretion (figs. 3g, 4b, d). three out of seven adult females of l. irregularis had precloacal glands with similar structure as those of adult males. the glands of the remaining adult females (n=4), those of the adult females of l. poecilochromus (n=3) with precloacal pores, and those of juveniles of both sexes of l. irregularis had fewer or non-existent partitions and alveoli, although they had secretions (figs. 4a, b, c, d). fig. 2. scanning electron microscopy of the precloacal pores of liolaemus irregularis (a to d) and l. poecilochromus (e and f). (a) adult male, (b) juvenile male, (c) adult female, (d) juvenile female, (e) adult male, (f) adult female. : pack of dead squamous cells and secretory cells. 152 s. valdecantos et alii table 1. descriptive statistics (mean ± sd) of snout-vent length (svl, mm) of the individuals of l. irregularis and l. poecilochromus measured to characterize the precloacal pores, considering separately both sexes and age classes. %ip: percentage of individuals with precloacal pores from the total number on individuals examined. the characteristics of the precloacal pores of these two liolaemus species are: n°p: number and pd: diameter (mm) of pores. in parentheses, the minimum and maximum values. n: sample size. l. irregularis l. poecilochromus juveniles adults adults females n = 9 males n = 8 females n = 16 males n = 15 males n = 15 svl 36.6 ± 3.4 (31.7–43.9) 38.9 ± 2.6 (36.7–45.0) 73.5 ± 6.1 (62.0–85.0) 82.0 ± 11.25 (66.3–97.6) 68.2 ± 5.3 (58.3–76.1) % ip 67 100 100 100 100 n°p 4.1 ± 3.2 (0–8) 8.4 ± 1.5 (7–11) 5.9 ± 1.9 (1–8) 8.4 ± 1.1 (7–10) 4.0 ± 1.2 (3–7) pd 0.08 ± 0.06 (0.09–0.15) 0.15 ± 0.04 (0.12–0.23) 0.22 ± 0.05 (0.16–0.33) 0.54 ± 0.19 (0.25–0.81) 0.49 ± 0.14 (0.18–0.75) fig. 3. histological sections of the anterior cloacal borders of an adult male of liolaemus irregularis (a to d and g) and an adult male of l. poecilochromus (e and f). (a) general view of the precloacal glands draining, each one, in a single scale. (b) magnification of the box in (a), showing details of the envelope surrounding the gland, a thin layer of connective tissue, and the division of glands in lobules. (c) magnification of the box in (b), showing details of the partitions that divide the gland into several elongated lobules and the germinative epithelium that coated tubules and alveoli, which consists of small, flat to cubic cells, with oval to spherical nuclei. (d) magnification of the box in (c), with details of the different cell types. (e) general view of precloacal glands. (f) details with immersion of the box shown in (e), showing germinative cells with spherical nuclei and conspicuous nucleoli and mitoses in the germinative stratum. (g) details with immersion of a basal portion of alveoli of precloacal gland where the different cell types and degenerated cells can be appreciated. degenerated cells are enucleated with a disaggregated cytoplasm where cytoplasmatic membranes cannot be recognized anymore. abbreviations: cd: central duct, cgrc: cell with granular cytoplasm, cpyn: cell with pyknotic nucleus, e: epidermis, ec: enucleated cells, en: envelope, gc: germinative cells, l: lobule, m: mitosis, n: nucleus, nu: nucleolus, p: pore, pa: partition, pg: precloacal gland, prg: proctodeal gland, s: secretion, sc: scale, spta: secretory portion tubule alveolar, t: tubule, ua: unstained area; : enucleated cells with a disaggregated cytoplasm and degenerated cells. stain: hemalum-eosin. 153precloacal glands of liolaemus lizards the clefts of the juvenile males of l. poecilochromus are invaginations of the epidermis on the cloacal border (fig. 4e), without secretions, which are located in the same position where the large pores of adults are observed. the “secondary pores” found in two adult males of l. poecilochromus were morphologically different from the precloacal glands, as they are a sac filled with a material whose nature probably is secretion (fig. 4f). no traces of precloacal pores or clefts were observed in either sex of l. neuquensis. fig. 4. histological section of the cloacal border of l. irregularis (a to d) and l. poecilochromus (e and f). (a) precloacal gland of a juvenile female showing the precloacal gland. (b) magnification of the box in (a), with details of the different cell types. (c) precloacal gland of a juvenile male with a single partition. (d) magnification of the box in (c), showing details of partition, germinative cells and secretion. (e) cleft of a juvenile male. (f): “secondary pore” of an adult male. abbreviations: c: cleft, cpyn: cell with pyknotic nucleus, e: epidermis, ec: enucleated cells, gc: germinative cells, pa: partition, pg: precloacal gland, s: secretion, sp: secondary pore. stain: hemalum-eosin. table 2. descriptive statistics (mean ± sd) of the length (lg; mm) and width (wg; mm) of precloacal glands of l. irregularis and l. poecilochromus, considering separately both sexes and age classes. n: sample size. l. irregularis l. poecilochromus juveniles adults adults females n = 9 males n = 5 females n = 7 males n = 7 females n = 3 males n = 7 lg 0.13 ± 0.04 0.21 ± 0.04 0.26 ± 0.06 0.90 ± 0.41 0.16 ± 0.05 0.43 ± 0.19 wg 0.12 ± 0.03 0.19 ± 0.05 0.23 ± 0.06 0.83 ± 0.22 0.20 ± 0.08 0.58 ± 0.20 154 s. valdecantos et alii histochemical observations the secretion in the glandular lumen showed positive reaction to pas (i.e. pink tones, fig. 6a) and ab (i.e., light blue tones, fig. 6b). intracytoplasmatic granules and secretions were positive to bromophenol blue (fig. 6c). the cytoplasms of many cells showed unstained areas, independent of the stain used (figs. 3d, g). discussion each precloacal pore in liolaemus is located in a unique scale, without modification, such as those observed in teiids, where the scales resemble a rosette (e.g. imparato et al., 2007). each pore has an associated epidermal gland, and if glands are present, they are always functional, i.e. they have secretions in the lumen, independent of the sex and age of the individual. in l. irregularis, there is sexual dimorphism in the size and number of these pores, biased towards males. in the case of l. poecilochromus, we found “secondary pores” in some adult males, and juvenile males had clefts, which would represent the origin of the adult precloacal pores, as what was reported for the femoral pores of crotaphytus (cole, 1966b). to our knowledge, we are describing for the first time in liolaemus these clefts, likely because they are not easily detected by the naked eye. the “secondary pores” had been described in some other liolaemus species, which are used as a taxonomic character (e.g. lobo et al., 2010b), fig. 5. length (a) and width (b) of precloacal glands in relation to the snoutvent length of l. irregularis and l. poecilochromus. each point represents an individual. the regression values were obtained including all the individuals measured in this study, independent of the species. each graph has an insert showing how glands were measured. fig. 6. histological section of precloacal glands of adult males with different stains. (a) details of a partition and cells with granular cytoplasm of l. poecilochromus, stained with pas. the pink cytoplasms indicate a weak positive reaction. (b) details of the secretion and glands of l. irregularis stained with ab. the light blue color indicates a weak positive reaction. (c) details of a partition and cells with granular cytoplasm and secretion of l. poecilochromus, stained with bromophenol blue. the intense blue tone indicates a strong positive reaction. abbreviations: cgrc: cell with granular cytoplasm, l: lobule, pa: partition, pg: precloacal gland, s: secretion. 155precloacal glands of liolaemus lizards although their function is unknown, and so, if their product might be involved in chemical communication. individuals of l. irregularis had functional precloacal glands from early in the ontogeny (i.e. juveniles), which experience a marked ontogenetic change in males; the number of tubules, alveoli and the size of glands increase from juveniles to adults. even though we did not include newborns of l. irregularis (individuals with ~ 27 mm of snout-vent length), the fact that all juveniles have functional secreting glands, suggests that individuals areborn with precloacal glands. the observed ontogenetic variation of the l. irregularis glands is similar to what was reported for the femoral glands; in crotaphytus collaris there is an increase in the size and complexity of glands after birth in males, but not in females (cole, 1966b). in cordylus polyzonus polyzonus, the variation of the males’ gland size is correlated with spermatogenic activity (van wyk, 1990), and in iguana iguana, the activity of the glands varies across the reproductive cycle, and they are always larger in males than in females (ferreira, 2007). unlike l. irregularis, the precloacal glands of l. poecilochromus are absent in juveniles, which only have invaginations of the epidermis, in the same position where pores are located in adults, which suggests that glands start to develop when males are juveniles, reaching maturity in adults. all together, observations suggest that gland development would be associated with the individual’s sexual maturity, as was reported for other liolaemus species (e.g. valdecantos and lobo, 2007). the liolaemus precloacal secretions contain acid mucosubstance, which contrast to the neutral mucosubstances found in other species of lizards and amphisbaenids (antoniazzi et al., 1993; imparato et al., 2007). unfortunately, there are no studies exploring the role played by these mucosubstances. the precloacal secretions also contain proteins, as has been reported for the femoral/precloacal gland secretions of other species (antoniazzi et al., 1993; imparato et al., 2007). it was proposed that proteins of the femoral secretions of iguana iguana might act as a barrier to reduce lipid evaporation (alberts, 1991). in addition, the observed intra and interspecific variability in the protein profiles may provide significant information about the identity of the owner (alberts et al., 1993). on the other hand, it is well known that femoral/precloacal secretions contain lipids (imparato et al., 2007; martín and lópez, 2011), also described in the precloacal secretions of liolaemus species (escobar et al., 2001; 2003). in this study, we could not explore the presence of lipids because specimens were preserved in alcohol, but the unstained areas observed in the glands suggest that the content would be of lipidic nature. the secretions are expelled as rigid cylinders, in opposition to the flexible condition of femoral secretions (e.g. “paste-like secretions”) reported by chauhan (1986). probably precloacal secretions are delivered passively in thin layers while lizards perform their daily activities. in fact, we never observed pieces of these cylinders on lizard perches in the field, nor individuals with broken cylinders of secretion. a passive delivery is facilitated by the position of these pores, as has been suggested for amphisbaena alba (jared et al., 1999). moreover, secretions may be adhered to faeces during defecation, contributing to their pheromonal properties (labra, 2008). this does not exclude, however, the possibility that secretions may be more actively delivered by dragging the cloaca in the substrate (labra et al., 2002). the precloacal glands of these liolaemus species have a general histological structure similar to the precloacal glands of amphisbaenas (antoniazzi et al., 1993; jared et al., 1999), and to the femoral glands of lizards from different families (cole, 1966b; imparato et al., 2007). thus, it may be possible to consider that the femoral and precloacal glands are homologous; both are holocrine with pheromonal secretions (martín and lópez, 2011) and have the same embryological origin (ectoderm). it is intriguing however, what determines variation in the area where glands appear (cloaca vs. thighs). in liolaemus, there is a significant intra and interspecific variation in the number of precloacal pores, and moreover, some few species have secondarily lost them (etheridge, 1995; lobo, 2005). in most species (> 90%) pores are only present in males, and it is unclear which factors determine the loss (e.g. in some species) or gain (e.g. in females) of these glands, or their occurrence early in the ontogeny (e.g. l. irregularis). in cordylidae, the presence of generational glands correlated with the environment temperature; females, and in few cases males, of species that inhabit at high altitude lack these glands (cordes et al., 1995). in contrast, escobar et al. (2001) found that liolaemus species that live at higher altitude have more pores than those from lower altitude, which was explained as a need to produce more secretions due to a faster degradation of these at high altitude. as for the species studied here, altitude/environment temperature cannot explain the difference in the number of precloacal pores, as both species with pores, l. irregularis and l. poecilochromus, inhabit high elevations and the presence of precloacal pores in females differed between these two species. moreover, l. neuquensis is one of the few species of the subgenus liolaemus (lobo et al., 2010a) that lacks precloacal pores although it lives at low elevations (< 500 masl), while other species in this liolaemus subgenus that inhabit at high elevations, have precloacal pores (e.g. 156 s. valdecantos et alii martínez oliver and lobo, 2002). potentially, phylogenetic constraints may explain the interspecific difference between l. irregularis and l. poecilochromus that belong to distinct subclades of eulaemus (schulte et al., 2000; pincheira-donoso et al., 2008; lobo et al., 2010a). further than phylogenetic constraints upon the occurrence of the precloacal pores, their evolution may also be tightly related to selective forces upon chemical communication, considering that precloacal secretions are involved in social communication (labra, 2008). if so, the lack of precloacal glands (i.e. l. neuquensis) may be related to a reduction in the use of chemical communication, unless other pheromonal sources fulfill the function of these glands. in contrast, l. irregularis, the species in which pores are present in all individuals, may use significantly more chemical communication, and precloacal secretions may encode important information for different aspects of their social communication, while secretions in l. poecilochromus would play only a key role in the reproductive success of adult males. it is unclear, however, why some females of l. poecilochromus develop these glands. it seems intuitive that this species is experiencing selective forces to use more the precloacal secretions in chemical communication, which also may explain the occurrence of “secondary pores” in some males, and precloacal glands in some females. in summary, this first morphological study on lizard precloacal glands shows that in liolaemus their presence varies across sexes and species, although, they are always functional, producing secretions. future studies will unravel the selective forces that determine gains and losses of these glands across species, sexes, and ages. acknowledgements authors thank fernando lobo, donald griffin and two anonymous reviewers for their significant contribution to an early version of this manuscript and especially to hervé seligmann, who also fixed the language. we are indebted with oscar leone for his invaluable help with the laboratory work and morphological descriptions. sv thanks conicet for a visitor fellowship that contributed to develop part of this study. funds come from ciunsa nº 1918 (universidad nacional de salta) to sv and vm, and from fondecyt 1120181 to al. references alberts, a.c. 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(1999): biostatistical analysis. 4th ed. prentice hall, new jersey, usa.figures issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 6(2): 149-160, 2011 kitobo forest of kenya, a unique hotspot of herpetofaunal diversity patrick k. malonza*, beryl a. bwong, vincent muchai section of herpetology, national museums of kenya, p. o. box 40658-00100, nairobi, kenya. *corresponding author. e-mail: kmalonza@museums.or.ke submitted on: 2011, 4th january; revised on: 2011, 5th september; accepted on 2011, 8th november. abstract. herpetologically, the remoteness of kitobo forest in south-eastern kenya has partly contributed to it remaining virtually un-explored until 2007. three surveys were conducted in december 2007, december 2009 and april 2010 aimed at generating a comprehensive list of the forest amphibians and reptiles. using largely timedspecies count method, 13 species of amphibians representing eight families and 32 reptiles belonging to 11 families were recorded. overall species diversity was highest during the 2007 sampling. the richness and abundance of amphibians was highest during the april 2010 sampling period when the amount of rainfall was also highest. the results of species accumulation curves of the three sampling periods did not plateau demonstrating that more species occur in this forest. pressure on this forest fragment from the adjacent local people is high which in addition to the annual floods threatens its long-term survival. for example the distribution and abundance of some forest associated species such as the tree frogs leptopelis flavomaculatus and hyperolius puncticulatus appear to fluctuate with flood events and may decline in future. considering the forest associated herpetofanua recorded, kitobo forest is zoogeographically assignable to the east african coastal forest biodiversity hotspot. the documentation of high species richness and diversity in this small forest fragment strongly highlight its biodiversity importance and place it among the most important sites for the conservation of reptiles and amphibians in kenya. keywords. herpetofaunal diversity, lowland forest, rainfall, floods, zoogeography. introduction in the last decade in kenya there has been increased interest in exploration of reptiles and amphibians in forests. this has resulted to production of species lists e.g. lower tana river forests (malonza et al., 2006), kakamega forest (schick et al., 2005, lötters et al., 2007; wagner and böhme, 2007; wagner et al., 2008) and the taita hills (malonza et al., 2010). what is evident from these recent studies is that they are all from the well 150 p.k. malonza, b.a. bwong and v. muchai known world biodiversity hotspots namely the guineo-congolian rainforest, eastern arc and coastal forests of east africa (mittermeier et al., 2004). kitobo forest is one of the little known arid land forest which possibly due to its remoteness has escaped the attention of biodiversity surveyors. no past biodiversity work has ever been done in this forest despite being a government gazetted community trust forest. we here present the first ever baseline data on its reptiles and amphibians diversity across three sampling wet seasons of varying rainfall intensity. from the results we try to assess the biogeographical affinity of the kitobo forest using herpetofauna as indicator species. while highlighting its herpetofaunal diversity we briefly discuss the observed threats and their implications for sustainable conservation of the forest. study area the kitobo forest is a ground water forest located about 10 km south-east of taveta town in the taita-taveta county, south-eastern kenya (fig. 1). it is approximately 250 km inland from the coast and on the extreme lowland north-east of the tanzanian eastern arc mountain block of north pare mountains (newmark, 2002), near the kenya-tanzania border (3o25.777’s; 37o36.660’e). it covers an area of ca. 160 ha at an altitude of about 730 m above sea level. it is a gazetted community trust forest. it is largely an evergreen indigenous forest surrounded by arid lands of acacia bushes. it owes its existence to the eruption on its edge of a large njoro spring and other small ones inside the forest originating from the volcanic mt. kilimanjaro. the springs then develop into a permanent river that flow through the forest dividing it into two major blocks. floods of varying intensity from rains in mt. kilimanjaro highlands in the north seasonally covers parts of the forest with some water stagnating throughout in some parts of the forest that has resulted in drying of trees. otherwise rainfall in the area is low, unreliable and erratic. the estimated annual mean rainfall and temperature is 530 mm and 22 oc respectively (jätzold and schmidt 1983). majority of the local multiethnic people depend on agriculture for their livelihood. through irrigation they grow a wide range of crops such as bananas, onions, kales, cabbages, tomatoes, cucumbers, maize, citrus, pepper, mangoes, and coconuts mostly for outside market. materials and methods surveys were conducted on three occasions (december 2007, december 2009 and april 2010 each covering about two weeks. all these three periods fall within the ordinary wet season for the area but the weather conditions varied from very low rains to high, from the first to the third sampling occasion respectively. similarly flooding events and intensity also increased from the first to the last sampling period. counting of amphibians and reptiles was done by using a timed species count method similar to those described by karns (1986); heyer et al. (1994); sutherland (1996). this entails quietly walking and intensively searching within all possible herpetofaunal microhabitats such as under leaves, debris, decomposing tree stumps and logs , on tree, shrubs, bushes, wetlands including digging for burrowing species. this was done for one person hour both day and night by two observers. opportunistic day and night visual and acoustic encounter surveys were made (rödel and ernst, 2004; veith et al., 2004). trapping using x-shaped drift fence with pitfall traps, a modification of that used by corn (1994) with segments of 5 m length upright plastic sheet (drift fence) stretching between 151kitobo forest of kenya, a unique hotspot of herpetofaunal diversity the buckets was used. the pitfall traps consisted of 10 litre plastic buckets flush with the ground; in total every trap array had five buckets. two trap sets were established in the forest interior for five days in the first occasion, seven days in the second and third sampling. traps were used for detection of small primarily nocturnal crawling herpetofauna not easily detected through other methods. the use of a combination of methods was to ensure detection of as many species as possible. quantitative data analysis used data generated from timed species count only as the others either yielded less data like the traps or were un-standardized such as opportunistic visual encounter surveys (ves). voucher specimens collected except amphibian larvae were fixed in 10% formalin (after euthanasia). tissues of selected specimens were preserved in absolute alcohol for the possibility of later molecular analyses. tadpoles were fixed in 95% ethanol. colour photos of selected species and their habitats were taken. frog calls where possible were recorded by means of an analogue marantz pmd-222 audio cassette recorder and a sennheiser k6-me66 directional microphone or a sony d20 digital camera. gps data were determined using a 12 channel garmin receiver. all these data are deposited in the national museums of kenya (nmk), nairobi. species richness and diversity analysis in the context of this study species richness and diversity refer to the number of different species and abundance in the number of individuals of each species observed (magurran, 1988). the observed species richness was estimated using the estimates 8.2 program (colwell, 2009). herpetofaunal species diversity was measured with shannon index (h’). a number of species richness estimators were used: – chao 1, ace, and jacknife 1. species accumulation curves fig. 1. map showing the location of kitobo forest. inset: map of kenya showing the location of the study area. 152 p.k. malonza, b.a. bwong and v. muchai were calculated and generated using the software programme estimates using 1000 randomizations. these were compared to the observed species. the taxonomy for amphibians followed frost et al. (2006) and frost (2007) while that of reptiles follows spawls et al. (2002) except for scincid lizards that follows brandley et al. (2005). selected individuals of underrepresented species were kept as voucher specimens and deposited in national museums of kenya (nmk). statistical analyses the variation in mean species diversity over the three sampling occasions (year or season) was quantified using the non-parametric kruskal-wallis h test. data was analyzed with statistica 6.0 software (statsoft, 2001) at 5% significance level. results species richness, diversity and composition a total of 13 amphibian species representing eight families and 32 reptile species representing 11 families were recorded excluding one snake species (atractaspis bibronii a. smith, 1849) recorded only during a one day reconnaissance survey in 2006 (appendix 1). majority of the species were lizards with terrestrial/arboreal trachylepis maculilabris (gray, 1845) and the burrowing melanoseps loveridgei brygoo & roux-estève, 1981 being the most abundant especially during the dry periods (appendix). more species were recorded on the forest edge than the interior. some species such as leptopelis flavomaculatus (günther, 1864) and hyperolius puncticulatus (pfeffer, 1893) were restricted to the forest interior. hemisus marmoratus (peters, 1854) and amietophrynus gutturalis (power, 1927) were the two most abundant species caught in traps. using data from timed species counts (tsc), the total number of species recorded during the sampling periods was almost the same with amphibians increasing with increasing rainfall intensity from 2007 through 2009 to 2010. however, the mean species diversity of hereptofauna per sampling was highly significant and different (kruskal-wallis h = 24.55, df = 2, n = 117, p < 0.001) and highest in 2007 (table 1). the non-parametric species richness accumulation curves did not reach an asymptote (figure 2). the mean number of species plus or minus the standard deviation (sd) per sampling by the other estimators was always higher than the observed species (sobs) (table 1). natural history notes during our surveys we observed biological information of some species previously unknown. while the advertisement call of leptopelis flavomaculatus is known, the species breeding and tadpoles were largely unknown. we managed to collect three tadpoles within water spring puddles inside the forest and they had a labial tooth row formula (ltrf) 153kitobo forest of kenya, a unique hotspot of herpetofaunal diversity fig. 2. species accumulation curves of the timed species count samples showing species observed and other species richness estimators during the three sampling periods. december 2007 april 2010 0 5 10 15 20 25 30 35 40 45 0 5 10 15 20 25 number of samples n um be r of s pe ci es species observed ace chao 1 jacknife 1 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40 45 50 number of samples n um be r of s pe ci es species observed ace chao 1 jacknife 1 0 5 10 15 20 25 30 35 40 45 0 10 20 30 40 50 60 number of samples n um be r of s pe ci es species observed ace chao 1 jacknife 1 december 2009 154 p.k. malonza, b.a. bwong and v. muchai of 4(2-4)/3. the other biological notes were of the species of the limbless burrowing skink, melanoseps loveridgei which like the other melanoseps species was assumed to lay eggs. during our sampling a gravid female was dug-out within a decomposing log and while being handled gave birth into a young one of about 60 mm total length. species range extensions this study extended the distribution records for a number of species in kenya. these are leptopelis flavomaculatus, hyperolius puncticulatus (pfeffer, 1893), hyperolius tuberilinguis smith, 1849, xenopus muelleri (peters, 1844), hyperolius glandicolor (peters, 1879), cnemaspis africana (werner, 1895), lygodactylus luteopicturatus pasteur, 1964, melanoseps loveridgei, trachylepis maculilabris, thelotornis mossambicanus (bocage, 1895), dasypeltis medici (bianconi, 1859), and dendroaspis angusticeps (a. smith, 1849) see appendix. zoogeographical affinity the findings of this study demonstrate that kitobo forest consists of a combination of species found in the east african coastal forests, eastern arc mountains and widespread african savanna species. however, considering forest associated species most of them are affiliated to the lowland coastal forest. examples are leptopelis flavomaculatus, hyperolius puncticulatus, hyperolius tuberilinguis, xenopus muelleri, lygodactylus luteopicturatus, dendroaspis angusticeps among others (see appendix). discussion the results presented here show that kitobo forest has a very rich and diverse herpetofauna. the results on local amphibian and reptile species richness in the forest show that there are more species than observed in this lowland forest as evident from the species accumulation curves that did not plateau. this is particularly due to the influx of species from the surrounding arid lands that use the evergreen forest as a refuge. in particular we expect more reptiles especially snakes than recorded because these are normally very cryptic and detect the presence of an observer and then disappear before being spottable 1. the mean species richness, diversity and richness estimators±1sd for the three sampling periods species richness index december 2007 december 2009 april 2010 species observed (sobs) 31 ± 8.07 29 ± 6.95 32 ± 7.30 abundance based cover estimate (ace) 39.28 ± 8.41 33.54 ± 7.77 39.68 ± 8.93 chao 1 36.79 ± 9.21 33.50 ± 8.30 34.57 ± 7.61 jacknife 1 39.61 ± 10.31 37.80 ± 8.73 41.80 ± 8.95 shannon 3.0 2.65 2.75 155kitobo forest of kenya, a unique hotspot of herpetofaunal diversity ted. the higher species diversity in 2007 than 2009 and 2010 was due to the increase in abundance of open water breeding amphibians with increasing amount of rainfall. the presence of high species richness and diversity in kitobo forest concurs with other studies elsewhere. these have found that high species richness in lowland ecosystems is associated with high energy and productivity (see hawkins et al., 2003; willig et al., 2003). energy in form of temperature and water are known indirect measures of net primary productivity which in turn results to high species richness (e.g. van rensburg et al., 2002; sanders et al., 2003). in kitobo forest there is substantial ground water from springs much of the year and optimum temperatures (jätzold and schmidt, 1983) favourable for high habitat productivity and in turn species diversity. the high species richness along the forest edge was due the presence of basking sites for reptiles and open water breeding sites for amphibians (duellman and trueb, 1994; zug et al., 2001). this high species richness and diversity follows the phenomenon of edge effects that provide more habitat niches for species co-existence (fagan, 1999). zoogeographically, the presence of leptopelis flavomaculatus, hyperolius puncticulatus, xenopus muelleri, lygodactylus luteopicturatus and dendroaspis angusticeps which are mostly associated with east african coastal forests (see howell, 1993; schiøtz, 1999; spawls et al., 2002; channing and howell, 2006; burgess et al., 2007) clearly shows the close affinity of kitobo forest to this biodiversity hotspot. the key threat to kitobo forest herpetofauna is floods resulting from rainfall in the highlands of mt. kilimanjaro. floods are one of the results of global climate change. elsewhere studies have found varying direct and indirect effects of floods on amphibian and reptile communities (borczyk, 2001; maltchick et al., 2007; moreira et al., 2008; furlani et al., 2009). the direct effects include affecting the species breeding phenology while indirectly they destroy the breeding and foraging sites. these effects are species specific resulting in either population decline, fluctuation, increase or no change. less affected are the arboreal ones because of their ability to climb on trees (borczyk, 2001). in kitobo forest floods have resulted in destruction of the habitat with part of the forest where stagnant water remains resulting to drying of plants. this was observed to result’s in shifts in the abundance of certain forest associated species. for example with increasing flood events from 2007 through 2009 to 2010 the abundance of the tree frog leptopelis flavomaculatus fluctuated by avoiding recently flooded sites and establishing new populations in un-flooded sites of the forest. apart from floods there high human population adjacent to the forest due to the intensive agriculture (irrigation schemes) relies on the forest for fire wood, palm tree material and plant poles that continue to open-up the forest. kitobo forest is an island in a ‘sea’ of intensive agricultural development, and concern is raised here for its continued protection so as to remain a species refuge. more work on herpetofauna including other forms of biodiversity is needed covering different times of the year to come up with a comprehensive species list of this forest refuge and get a clear picture of its biogeographical assignment. acknowledgements many thanks go to our nmk colleague, joash nyamache who helped in field data collection. we are very grateful johana mwawasi nusu, the kenya forest service guard of kitobo communi156 p.k. malonza, b.a. bwong and v. muchai ty forest for his assistance and good knowledge of the forest. thanks to the kenya forest service administration for permitting us to work in the forest. this study was funded by the mohamed bin zayed (mbz) species conservation fund, project number 0925565 and partly the national museums of kenya, nairobi. references borczyk, b. 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(2007): dynamics of the terrestrial amphibian assemblage in a flooded riparian forest fragment in a neotropical region in the south of brazil. braz. j. biol. 68: 763-769. mittermeier, r.a., robles-gil, p., hoffmann m., pilgrim, j.d., brooks, t.m., mittermeier, c.g., lamoreux, j.l., fonseca, g. (2004): hotspots revisited: earth’s biologically richest and most endangered terrestrial ecoregions. cemex, mexico city. moreira, l.f.b., machado, i.f., lace, a.r.g.m., maltchik, l. (2008): anuran amphibians dynamics in an intermittent pond in southern brazil. acta limnol. brasil 20: 205-212. newmark, w.d. (2002): conserving biodiversity in east african forests: a study of the eastern arc mountains. springer-verlag, berlin, heidelberg. rödel, m.o., ernst, r. (2004): measuring and monitoring amphibian diversity in tropical forests. i. an evaluation of methods with recommendations for standardization. ecotropica 10: 1-14. sanders, n.j., moss, j., wagner, d. 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(2001): herpetology. an introductory biology of amphibians and reptiles. academic press. appendix. the distribution and abundance of the 32 reptile and 13 amphibian species in kitobo forest during the three sampling occasions using timed species count method species 2007 2009 2010 habitat habits amphibians pipidae xenopus muelleri (peters, 1844) 0 0 9 interior/edge aquatic bufonidae amietophrynus gutturalis (power, 1927) 16 4 22 interior/edge terrestrial hemisotidae hemisus marmoratus (peters, 1854) 2 9 10 interior/edge fossorial arthroleptidae leptopelis flavomaculatus (günther, 1864) 3 18 53 interior arboreal hyeproliidae hyperolius glandicolor (peters, 1879) 16 42 66 interior/edge arboreal hyperolius puncticulatus (pfeffer, 1893) 5 18 10 interior arboreal hyperolius tuberilinguis smith, 1849 10 2 34 interior/edge kassina senegalensis (duméril & bibron, 1841) 0 0 1 edge terrestrial 159kitobo forest of kenya, a unique hotspot of herpetofaunal diversity species 2007 2009 2010 habitat habits ptychadinidae ptychadena anchietae (bocage, 1867) 6 4 25 interior/edge fossorial ptychadena mascareniensis (duméril & bibron, 1841) 19 33 94 interior/edge terrestrial phrynobatrachidae phrynobatrachus acridoides (cope,1867) 14 51 30 interior/edge arboreal phrynobatrachus natalensis (smith, 1849) 0 0 3 edge arboreal pyxicephalidae tomopterna cryptotis (boulenger, 1907) 0 0 2 edge terrestrial sub-total number of species 9 9 13 reptiles gekkonidae lygodactylus cf scheffleri sternfeld, 1912 0 1 1 edge arboreal lygodactylus luteopicturatus pasteur, 1964 2 15 15 interior/edge arboreal hemidactylus platycephalus peters, 1854 7 7 11 interior/edge arboreal hemidactylus mabouia (moreau de jonnés, 1818) 4 16 23 interior/edge arboreal hemidactylus squamulatus tornier, 1896 1 0 0 edge terrestrial cnemaspis africana (werner, 1895) 2 0 0 interior arboreal chamaeleonidae chamaeleo dilepis leach, 1819 7 9 1 edge arboreal scincidae melanoseps loveridgei brygoo & roux-estève, 1981 14 2 1 interior fossorial lygosoma sundevalli (a. smith, 1849) 5 2 2 interior/edge fossorial panaspis wahlbergii (a. smith, 1849) 1 0 0 edge terrestrial trachylepis maculilabris (gray, 1845) 8 74 15 interior/edge arboreal trachylepis striata (peters, 1854) 2 14 29 interior/edge arboreal trachylepis planifrons (peters, 1878) 0 1 2 edge arboreal trachylepis brevicollis (weigmann, 1837) 0 5 3 edge terrestrial lacertidae latastia longicaudata (reuss, 1834) 1 4 2 edge terrestrial heliobolus spekii günther, 1872 0 0 2 interior/edge terrestrial agamidae agama lionotus boulenger, 1896 1 2 19 edge arboreal gerrhosauridae gerrhosaurus major duméril, 1851 1 1 3 edge terrestrial gerrhosaurus flavigularis wiegmann, 1828 0 4 0 edge terrestrial varanidae varanus niloticus (linnaeus, 1766) 2 4 0 interior/edge terrestrial varanus albigularis (daudin, 1802) 0 1 0 edge terrestrial leptotyphlopidae leptotyphlops scutifrons merkeri (werner, 1909) 2 0 0 interior fossorial pythonidae python natalensis a. smith, 1840 0 0 2 interior terrestrial colubridae lycophidion capense (a. smith, 1831) 1 0 0 interior fossorial philothamnus battersbyi loveridge, 1951 6 3 2 edge arboreal 160 p.k. malonza, b.a. bwong and v. muchai species 2007 2009 2010 habitat habits philothamnus punctatus peters, 1866 0 1 1 interior arboreal thelotornis mossambicanus (bocage, 1895) 1 0 0 edge arboreal dasypeltis medici medici (bianconi, 1859) 2 0 0 interior terrestrial prosymna stuhlmanni (pfeffer, 1893) 0 1 0 edge terrestrial elapidae naja melanoleuca hallowell, 1857 0 0 1 interior terrestrial dendroaspis angusticeps (a. smith, 1849) 1 interior arboreal crocodylidae crocodylus niloticus laurenti, 1768 nile 1 0 0 interior aquatic sub-total number of species 22 20 19 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 6(1): 81-85, 2011 effects of mosquitofish (gambusia affinis) cues on wood frog (lithobates sylvaticus) tadpole activity katherine f. buttermore, paige n. litkenhaus, danielle c. torpey, geoffrey r. smith*, jessica e. rettig department of biology, denison university, granville, oh, 43023, usa. *corresponding author. e-mail: smithg@denison.edu submitted on: 2011, 11th january; revised on 2011, 13th april; accepted on 2011, 11th may. abstract. we examined the changes in activity of wood frog (lithobates sylvaticus) tadpoles exposed to combinations of visual, chemical, and mechanical cues of the invasive mosquitofish (gambusia affinis). we also examined whether the responses of the tadpoles to the predator cues were influenced by the short-term accumulation of chemical cues in the experimental container. in our experiment, the activity of wood frog (l. sylvaticus) tadpoles was not affected by the presence of various cues from mosquitofish. our experiment demonstrated that the repeated use of trial water can influence the activity level of tadpoles, regardless of the predator cue treatment used. tadpoles in the first trial tended to be less active than tadpoles in subsequent trials. this effect does not appear to be mediated by the accumulation of predator cues since there was no significant interaction term. our results suggest that short-term accumulation of predator chemical cues do not affect the behavior of wood frog tadpoles: however, our results suggest that the repeated use of the same water in consecutive trials may affect tadpole behavior, perhaps through the accumulation of conspecific chemical cues. keywords. activity, behavior, conspecific cues, gambusia affinis, lithobates sylvaticus, predator cues. introduction non-native species are among the many threats to amphibian populations around the world, and in particular, the introduction of non-native fish frequently results in declines of native amphibians (see kiesecker, 2003). such non-native predators frequently have greater effects on native prey than native predators do because the prey are naïve to the predator and thus may not respond to them as a predator (banks and dickman, 2007; salo et al., 2007). 82 k.f. buttermore, p.n. litkenhaus, d.c. torpey, g.r. smith and j.e. rettig one of the most widespread introduced fish are the mosquitofish (gambusia affinis and gambusia holbrooki, biology and ecology reviewed in pyke, 2005, 2008). mosquitofish are known predators of amphibians, both within their native range (baber and babbitt, 2003; stanback, 2010) and outside their native range (e.g., komak and crossland, 2000; gregoire and gunzburger, 2008; segev et al., 2009). previous studies have shown that some species of tadpoles show anti-predator behaviors, such as reducing their activity or changing their space use, in response to the presence of mosquitofish or mosquitofish cues (e.g., lawler et al. 1999; burgett et al. 2007; smith et al. 2009, 2010, 2011), but other species do not (e.g., hamer et al. 2002; smith et al. 2008, 2009). we examined the changes in activity of wood frog (lithobates sylvaticus) tadpoles exposed to combinations of visual, chemical, and mechanical cues of mosquitofish to determine which cues, if any, elicited a change in behavior. we predicted that (1) tadpoles would have reduced activity in the presence of a mosquitofish (based on a previous study of wood frogs by burgett et al., 2007), and that (2) tadpoles would reduce their activity in the presence of an uncaged predator compared to with a caged predator (i.e., greater reduction of activity with greater risk – a threat-sensitive response; helfman, 1989; stankowich and blumstein, 2005). in addition, we examined whether the responses of the tadpoles to the predator cues were influenced by the short-term accumulation of chemical cues, both from predators and conspecifics, in the experimental container. we predicted that responses would be greater in later trials than in earlier trials in the same container. material and methods we collected l. sylvaticus egg masses (n = 8) from a pond on the denison university biological reserve, granville, licking co., ohio on 13 march 2010. this pond occasionally has mosquitofish in years in which it retains water year-round, but not during 2010. eggs were allowed to hatch in the laboratory. tadpoles from multiple egg masses (3-4) were maintained in mixed groups in large plastic containers for two weeks and fed ad libitum until used in the experiment. mosquitofish used in this experiment were collected from another pond on the denison university biological reserve. we began the experiment when tadpoles had reached gosner stage 26 (gosner, 1960; mean mass = 0.023 g). we used female mosquitofish (total length = 4.5-5 cm) that had not been fed for at least 24 h. we established three treatments: a control treatment with a tadpole and an empty cage (i.e., no predator cues), a caged predator treatment with a tadpole and one caged mosquitofish (i.e., visual and chemical predator cues), and an uncaged predator treatment with a tadpole, an uncaged mosquitofish, and an empty cage (i.e., visual, chemical, and physical predator cues). cages were 17 cm length × 12 cm width × 13.5 cm height and made of fine mesh (1 mm) netting (measured light transmittance ≈ 100%). after allowing the tadpole to acclimate for five minutes, we recorded its activity as either swimming or non-swimming every 60 seconds for 15 minutes. at the end of each trial, we immediately replaced the tadpole and repeated the procedure. after three trials, we replaced the water with aged tap-water and the mosquitofish. we did not use any tadpole more than once. each treatment was replicated 11 times. we used a two-way anova to assess treatment effects on the number of times the tadpoles were observed swimming, and the effect of trial number (i.e., did the accumulation of cues affect activity?). preliminary analyses indicated no statistical difference in the 2nd or 3rd trials (p > 0.05), so we pooled these trials for the analyses. 83effects of predation on tadpole activity results predator cue treatment had no effect on swimming activity of the wood frog tadpoles (table 1; f2,27 = 0.15, p = 0.86). tadpoles in the first trial were less active than tadpoles in the second or third trial (table 1; f1,27 = 4.47, p = 0.044). the interaction between predator cue treatment and trial number was not signficant (table 1; f2,27 = 0.34, p = 0.71). discussion in our experiment, the activity of wood frog (l. sylvaticus) tadpoles was not affected by the presence of cues from mosquitofish, whether those cues came from caged or uncaged fish. this result is not consistent with another study on the effects of mosquitofish on the activity of wood frog tadpoles. burgett et al. (2007) found that activity in wood frog tadpoles was lower in the presence of chemical cues from mosquitofish. however, the amount of chemical cue used in burgett et al. (2007) was probably much higher than the cues used in our experiment. burgett et al. (2007) used chemical cues from several mosquitofish accumulated over a much longer period of time in their experiment than we used in our experiment. however, their experiment did not include visual or physical cues. an additional possible explanation might be that there is genetic variation in the ability to respond to mosquitofish cues in this wood frog population and the variation in response ability may reflect such genetic variability. indeed, smith et al. (2010) found variation in behavioral responses to mosquitofish cues among sibships of green frog tadpoles (l. clamitans) with some sibships showing changes in activity levels and others not. the lack of a response to mosquitofish cues by the wood frog tadpoles is not unique among ranids, a group in which responses to mosquitofish are variable among species and even within species. for example, burgett et al. (2007) found that l. sylvaticus tadpoles decrease activity when exposed to cues from mosquitofish, as did l. clamitans tadpoles (smith et al., 2010, in press), whereas tadpoles of l. catesbeianus did not respond to the presence of mosquitofish (smith et al. 2008). lawler et al. (1999) found that younger tadpoles (gosner stage 26) of rana draytonii reduced activity levels in the presence of mosquitofish, but older tadpoles (gosner stage 33-36) showed no reduction in activity. variation in antipredator responses among species is not entirely unexpected since mosquitofish are not native and thus responses may depend on the ability of each species to generalize their anti-predator response between native and non-native predator species. table 1. effect of predator treatment and experimental trial on the proportion of observations during which wood frog (lithobates sylvaticus) tadpoles were swimming. means are given ± 1 s.e. trial number predator treatment control caged predator uncaged predator first 0.24 ± 0.12 0.23 ± 0.19 0.17 ± 0.08 other 0.42 ± 0.13 0.37 ± 0.11 0.52 ± 0.14 84 k.f. buttermore, p.n. litkenhaus, d.c. torpey, g.r. smith and j.e. rettig our experiment demonstrated that the repeated use of trial water can influence the activity level of tadpoles, regardless of the predator cue treatment used. tadpoles in the first trial tended to be less active than tadpoles in subsequent trials. this effect does not appear to be mediated by the accumulation of predator cues since there was no significant interaction term. it may be that the accumulation of conspecific cues, in the absence of consumption cues (no tadpoles were consumed by the mosquitofish in the trials), may induce greater activity. indeed, previous studies have shown higher levels of activity in the presence of higher densities of conspecifics in tadpoles (e.g., golden et al., 2001; relyea, 2002; awan and smith, 2007b; smith and awan, 2009). perhaps reflecting an increase in foraging activity in the presence of competitors or due to a perceived reduction of predation risk in the presence of conspecifics. however, awan and smith (2007a) found no change in activity level in wood frog tadpoles with changes in tadpole density (see also golden et al., 2000 for xenopus laevis). thus it may be the increased activity of the wood frog tadpoles in our experiment is the result of accumulated conspecific cues, but additional work is needed to more fully explain the results. acknowledgments we thank w. and l. smith for help collecting egg masses. this experiment was approved by the denison university iacuc (protocol 10-003). references awan, a.r., smith, g.r. 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(2002): competitor-induced plasticity in tadpoles: consequences, cues, and connections to predator-induced plasticity. ecol. monogr. 72: 523-540. salo, p., korpimaki, e., banks, p.b., nordstrom, m., dickman, c.r. (2007): alien predators are more dangerous than native predators to prey populations. proc. r. soc. 274b: 1237-1243. segev, o., mangel, m., blaustein, l. (2009): deleterious effects by mosquitofish (gambusia affinis) on the endangered fire salamander (salamandra infraimmaculata). anim. conserv. 12: 29-37. smith, g.r., awan, a.r. (2009): the roles of predator identity and group size in the antipredator responses of american toad (bufo americanus) and bullfrog (rana catesbeiana) tadpoles. behaviour 146: 225-243. smith, g.r., boyd, a., dayer, c.b., ogle, m.e., terlecky, a.j. (2009): responses of grey treefrog and american toad tadpoles to the presence of cues from multiple predators. herpetol. j. 19: 79-83. smith, g.r., boyd, a., dayer, c.b., ogle, m.e., terlecky, a.j., dibble, c.j. (2010): effects of sibship and the presence of multiple predators on the behavior of green frog (rana clamitans) tadpoles. ethology 116: 217-225. smith, g.r., boyd, a., dayer, c.b., winter, k.e. (2008): behavioral responses of american toad and bullfrog tadpoles to the presence of cues from the invasive fish, gambusia affinis. biol. invas. 10: 743-748. smith, g.r., terlecky. a.j., dayer, c.b., boyd, a., ogle, m.e., dibble, c.j. (2011): effects of mosquitofish and ammonium nitrate on activity of green frog (lithobates clamitans) tadpoles: a mesocosm experiment. j. freshwater ecol. 26: 59-63. stanback, m. (2010): gambusia holbrooki predation on pseudacris feriarum tadpoles. herpetol. conserv. biol. 5: 486-489. stankowich, t., blumstein, d.t. (2005): fear in animals: a meta-analysis and review of risk assessment. proc. r. soc. 272b: 2627-2634. bbib67 ole_link1 ole_link2 bbib28 ole_link5 ole_link6 ole_link7 ole_link8 _goback ole_link1 ole_link2 ole_link3 ole_link4 ole_link1 ole_link2 ole_link19 ole_link20 ole_link21 ole_link29 ole_link3 ole_link4 ole_link5 ole_link31 ole_link14 ole_link15 ole_link12 ole_link13 ole_link16 ole_link17 ole_link22 ole_link23 ole_link24 ole_link8 ole_link9 ole_link10 ole_link11 ole_link18 ole_link27 ole_link28 ole_link25 ole_link26 ole_link6 ole_link7 ole_link34 ole_link37 ole_link38 acta herpetologica vol. 6, n. 1 june 2011 firenze university press widespread bacterial infection affecting rana temporaria tadpoles in mountain areas rocco tiberti extreme feeding behaviours in the italian wall lizard, podarcis siculus massimo capula1, gaetano aloise2 lissotriton vulgaris paedomorphs in south-western romania: a consequence of a human modified habitat? severus d. covaciu-marcov*, istvan sas, alfred ş. cicort-lucaciu, horia v. bogdan body size and reproductive characteristics of paedomorphic and metamorphic individuals of the northern banded newt (ommatotriton ophryticus) eyup başkale1, ferah sayım2 , uğur kaya2 genetic characterization of over hundred years old caretta caretta specimens from italian and maltese museums luisa garofalo1, john j. borg2, rossella carlini3, luca mizzan4, nicola novarini4, giovanni scillitani5, andrea novelletto1 the phylogenetic position of lygodactylus angularis and the utility of using the 16s rdna gene for delimiting species in lygodactylus (squamata, gekkonidae) riccardo castiglia*, flavia annesi localization of glucagon and insulin cells and its variation with respect to physiological events in eutropis carinata vidya. r. chandavar1, prakash. r. naik2* the balearic herpetofauna: a species update and a review on the evidence samuel pinya1, miguel a. carretero2 effects of mosquitofish (gambusia affinis) cues on wood frog (lithobates sylvaticus) tadpole activity katherine f. buttermore, paige n. litkenhaus, danielle c. torpey, geoffrey r. smith*, jessica e. rettig food composition of uludağ frog, rana macrocnemis boulenger, 1885 in uludağ (bursa, turkey) kerim çiçek preliminary results on tail energetics in the moorish gecko, tarentola mauritanica tommaso cencetti1,2, piera poli3, marcello mele3, marco a.l. zuffi1 climate change and peripheral populations: predictions for a relict mediterranean viper josé c. brito1, soumia fahd 2, fernando martínez-freiría1, pedro tarroso1, said larbes3, juan m. pleguezuelos4, xavier santos5 assessing the status of amphibian breeding sites in italy: a national survey societas herpetologica italica* osservatorio erpetologico italiano acta herpetologica journal of the societas herpetologica italica acta herpetologica rivista della societas herpetologica italica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 7(2): 239-252, 2012 reproductive strategy of male and female eastern spiny lizards sceloporus spinosus (squamata: phrynosomatidae) from a region of the chihuahuan desert, méxico aurelio ramírez-bautista1,*, barry p. stephenson2, xóchitl hernández-ibarra1, uriel hernández-salinas1, raciel cruz-elizalde1, abraham lozano1, geoffrey r. smith3 1centro de investigaciones biológicas, universidad autónoma del estado de hidalgo, méxico, a.p. 1-69 plaza juárez, c. p. 42001, pachuca, hidalgo, méxico. *corresponding author. e-mail: ramibautistaa@ gmail.com 2department of biology, mercer university, 1400 coleman ave., macon, ga 31207, usa 3department of biology, denison university, granville, oh 43023, usa submitted on 2012, 3rd january; revised on 2012, 27th september; accepted on 2012, 30th september. abstract. we examined the reproductive strategy of male (n = 84) and female (n = 62) s. spinosus from a single population in san luis potosí, méxico. the male reproductive cycle peaked in march and april and declined from may to september, and was not correlated with fat body mass, but was positively correlated with liver mass. the female reproductive cycle peaked in april and may and declined from june through november, and was not correlated with fat body mass, but was correlated with liver mass. mean clutch size based on oviductal eggs was 17.5 ± 1.9 (n = 12), and was not correlated with female snout-vent length. our results for s. spinosus are generally similar to those of other populations of the s. spinosus species group. however, there are differences in some traits (e.g., timing of the initiation of the female reproductive cycle; clutch size), suggesting that the s. spinosus group could serve as another model group within sceloporus to explore ecological and evolutionary causes of among population life history variation. keywords. clutch size, lizard, méxico, reproductive cycle, sceloporus spinosus. introduction studies of lizards in the genus sceloporus have contributed greatly to our understanding of the factors influencing life history variation among populations, especially reproductive characteristics (ballinger, 1977; dunham, 1978; hernández-salinas et al., 2010). widespread species such as s. undulatus (lemos-espinal et al., 2003; niewiarowski et al., 240 aurelio ramírez-bautista et al. 2004), s. grammicus (hernández-salinas et al., 2010), and s. variabilis (benabib, 1994) have been used in numerous studies to examine reproductive variation among populations. many of these same species have also been well studied at the level of a single population in the context of reproductive cycles (e.g., s. grammicus: guillette and bearce, 1986; jiménez-cruz et al., 2005) or among multiple populations in the context of lifehistory variation (s. grammicus: hernández-salinas et al., 2010; ramírez-bautista et al., 2011; s. jarrovii: ballinger, 1973, 1979; ballinger et al., 1996; ramírez-bautista et al., 2002; gadsden et al., 2008; s. variabilis: benabib, 1994). collectively this research has provided considerable insights into the factors shaping the evolution of life-history traits in lizards (benabib, 1994; hernández-salinas et al., 2010). however, these well-studied species represent only a small sampling of the more than 90 species that comprise the genus sceloporus [see wiens and reeder (1997) and wiens et al. (2010) for a detailed phylogeny of sceloporus with associated species groups]. all of these studies have documented variations in reproductive timing between females and males within a single population or among populations of the same species, as well as variation in reproductive traits (clutch size, egg size, offspring size at birth, etc.). in addition, studies in sceloporus have shown that fat body and liver mass influence sperm production and vitellogenesis (s. variabilis: benabib, 1994; ramírez-bautista et al., 2006; s. jarrovii: ramírez-bautista et al., 2002; s. pyrocephalus: ramírez-bautista and olvera-becerril, 2004; s. grammicus: jiménez-cruz et al., 2005; hernández-salinas et al., 2010; ramírez-bautista et al., 2012; s. formosus: ramírez-bautista and pavón, 2009), as well as reproductive characteristics such as snoutvent length (svl) at sexual maturity, clutch size, and offspring svl (ballinger, 1977; benabib, 1994), but this may not always be the case (e.g., male s. mucronatus, méndez de la cruz et al., 1988; male s. utiformis, ramírez-bautista and gutiérrez-mayén, 2003) or there may be plasticity in the relationship (e.g., warne et al., 2012). however, we need additional information about these reproductive characteristics with studies on a single population or multiple populations of the same species to understand better the life history variation among lizard populations (ramírez-bautista and olvera-becerril, 2004; ramírez-bautista et al., 2011). thus, conclusions drawn about variation in reproductive cycles and life history traits in sceloporus based exclusively on the relatively few well-studied species could be biased by aspects of species-specific ecology or phylogenetic history, obscuring more general patterns characteristic of the genus overall (dunham and miles, 1985). very little has been published to date on the reproductive characteristics of sceloporus spinosus (fitch, 1978, 1985; valdéz-gonzález and ramírez-bautista, 2002; calderón-espinosa et al., 2006), a relatively large oviparous spiny lizard common in saxicolous habitats in central méxico. this lizard has a relatively large range, occurring across the states of durango and western tamaulipas in the north to northern jalisco, michoacán, hidalgo, and puebla on the mexican plateau in the south (wiens and reeder, 1997; wiens et al., 2010). sceloporus spinosus is part of the s. spinosus species group containing three species, all of which are endemic to méxico (wiens and reeder, 1997; wiens et al., 2010). here we report the first direct observations on reproduction in males and females of s. spinosus in a population from the chihuahuan desert, san luis potosí, méxico. we were specifically interested in determining the extent to which this population of s. spinosus conforms to the reproductive phenology described for other populations of s. spinosus, as well as that of another member of the s. spinosus species group, s. horridus. for example, previous 241reproduction in sceloporus spinosus research indicates that peak male reproductive activity (inferred from testes size) occurs in april and may for s. spinosus from puebla, méxico and in s. horridus; a secondary peak in the fall also occurs for the puebla population of s. spinosus (valdéz-gonzález and ramírez-bautista, 2002). female reproductive activity in these previously studied populations also exhibits strong seasonality for a variety of traits, including onset and duration of vitellogenesis, clutch size, and the timing and duration of egg deposition (valdézgonzález and ramírez-bautista, 2002). in general, vitellogenesis is initiated in the winter or spring, and ovulation occurs in the spring and summer. in s. spinosus from puebla, at least some females were capable of depositing two clutches in a given summer (valdézgonzález and ramírez-bautista, 2002). if phylogeny plays a major role in determining the reproductive strategies and life history characteristics of s. spinosus, we expected our study population to exhibit similar patterns to those of other populations in the s. spinosus species group. alternatively, if local ecological or evolutionary conditions are more important, we expected to find evidence of different reproductive strategies and other life history traits in our population of s. spinosus relative to those observed in other populations of the s. spinosus complex. we were also interested in determining the extent to which indices of energy stores (fat body and liver mass) co-varied with male and female reproductive cycles. two main strategies for energetic balance during the breeding season have been previously identified in sceloporus. in one strategy, lizards accumulate fat stores only prior to the onset of reproduction, and these reserves are consumed over the course of the primary breeding season due to energetic demands and time constraints imposed by reproductive behaviors and activities (valdéz-gonzález and ramírez-bautista, 2002; jiménez-cruz et al., 2005). in the alternative strategy, lizards continue to feed and thus accumulate fat bodies and liver mass until the end of the reproductive period, perhaps by reducing their reproductive activities into a smaller portion of their daily energy and activity budgets (ramírezbautista, 1995; ramírez-bautista and vitt, 1997). fat body and liver mass influence sperm production in males and vitellogenesis in females in some species of sceloporus (s. variabilis: benabib, 1994; ramírez-bautista et al., 2006; s. jarrovii: ballinger, 1977; ramírezbautista et al., 2002; s. pyrocephalus: ramírez-bautista and olvera-becerril, 2004; s. grammicus: jiménez-cruz et al., 2005; hernández-salinas et al., 2010; ramírez-bautista et al., 2012; s. formosus: ramírez-bautista and pavón, 2009), suggesting an important role for these organs in supporting aspects of reproduction in these species. materials and methods study area this study was conducted using specimens collected from june 1999 to may 2000 near the community of las lagunas (22º30´n, 100º23´w, datum: wgs84; elevation 2230 m) in the municipality of guadalcázar, san luis potosí, méxico. this region consists of characteristic high-elevation chihuahuan desert, and common vegetation communities of the study site include mesquite scrubgrassland, oak-juniper woodland, and cactus forest dominated by oaks (quercus polymorpha, q. laeta), mesquite (prosopis juliflora) and juniper (juniperus flaccida) (rzedowski, 1978). mean annu242 aurelio ramírez-bautista et al. al temperature and precipitation based on monthly means was 25.8 ºc and 600 mm, respectively (garcía, 1981). collection and euthanization techniques a total of 84 adult male and 62 adult female s. spinosus was used in this study. lizards were captured by hand or noose using standard field techniques (casas-andreu et al., 1991). in the laboratory, lizards were anesthetized by freezing and sacrificed by nuchal injection of 10% formalin (ramírez-bautista et al., 2012). specimens were fixed in 10% formalin (ramírez-bautista et al., 2008) and deposited in the laboratory of the centro de investigaciones biologicas at universidad autónoma del estado de hidalgo, pachuca, hidalgo, for use in subsequent morphological analyses. male reproduction we removed the testes and weighed them to the nearest 0.0001 g to obtain testes mass (tm). livers and fat bodies were removed and weighed (to the nearest 0.0001 g) so that seasonal cycles in the mass of organs potentially related to reproduction could be described. the mass or size of the testes is frequently correlated with spermatogenic or breeding activity in lizards (e.g., mckinney and marion, 1985; van sluys, 1993; ochotorena et al., 2005). we considered males sexually mature if they had enlarged testes and convoluted epididymides consistent with sperm production (goldberg and lowe, 1966). to analyze monthly variation in gonad, fat body, and liver mass, we used the residuals of the regression of each variable (log10 transformed) on log10-transformed svl in anovas with month as the independent variable. we regressed the residuals of fat body and liver mass on the residuals of gonad mass to examine the relationship between energy storage and testicular size (ramírez-bautista and vitt, 1997). means are given ± 1 se unless otherwise indicated. female reproduction non-vitellogenic follicles (nvf), vitellogenic follicles (vf) in the ovary (= follicular mass [nvf plus vf] = fm), and oviductal eggs were removed and weighed to the nearest 0.0001 g. in reproductive females, the largest egg (of the oviductal eggs, or ovarian vf or nvf) on each side of the body was weighed to the nearest 0.0001 g and multiplied by either the number of eggs (egg mass), or vf or nvf (gonad mass) on the same side of the body as appropriate, in order to estimate total gonad mass, indicated here as either gonad mass (gm) or egg mass (em). clutch size was determined by counting the number of eggs in the oviducts (oe) of adult females during the reproductive season (i.e., we only counted shelled eggs for clutch size, not vf or nvf). livers and fat bodies were removed and weighed (to the nearest 0.0001 g) so that seasonal cycles in the mass of organs potentially related to reproduction could be described. we used the svl of the smallest female with enlarged vitellogenic follicles or eggs to estimate minimum size at sexual maturity (ramírez-bautista and vitt, 1997, 1998). there were significant relationships between svl and gonad mass, fat body mass, and liver mass (all log10 transformed). thus, we performed anovas on the residuals of gonad mass, liver, and fat body mass (log10 transformed) with month as the independent variable to describe the organ cycles (ramírez-bautista and vitt, 1997, 1998). we calculated a pearson product-moment correlation coefficient to test for a relationship between clutch size and female svl. means are reported ± se, unless otherwise indicated. 243reproduction in sceloporus spinosus results male reproduction all of the 84 males we sampled were sexually mature and their testes ranged in mass from 0.006 – 0.870 g. there was a significant relationship between log10-svl and log10gonad mass (r2 = 0.42, f1,83 = 58.6, p < 0.0001), log10-fat body mass (r2 = 0.52, f1,83 = 87.5, p < 0.0001), and log10-liver mass (r2 = 0.07, f1,83 = 6.14, p = 0.015). anovas on the residuals of these regressions revealed significant month effects for testes mass (f8,75 = 5.25, p < 0.0001) and fat body mass (f8,75 = 2.34, p = 0.026), but not liver mass (f8,75 = 1.84, p = 0.084). residual testes mass in males varied significantly among months, with a peak from march to april, followed by a generally steady decline from may to september. this was followed by a sharp increase in october (fig. 1a), the latest month in the calendar year for which data were available. residual fat body mass varied significantly among months, with a steady decline from february through may, followed by a steady increase from may through october (fig. 1b). residual liver mass showed no significant monthly variation; however, maximum mass was observed during september and october (fig. 1c). residual testicular mass was positively related to residual liver mass (n = 84, r2 = 0.06, p = 0.021), but not to residual fat body mass (n = 84, r2 = 0.008, p = 0.41). female reproduction sixty–two adult females were used to study the reproductive cycle, and these showed a significant relationship between log10-svl and log10-gonad mass (r2 = 0.16, f1,61 = 11.4, p = 0.001), log10-fat body mass (r2 = 0.20, f1,61 = 14.6, p = 0.0003), and log10-liver mass (r2 = 0.46, f1,61 = 51.2, p < 0.0001). as with males, we removed the effects of female size by using the residuals from these regressions to describe gonad mass, fat body mass, and liver mass cycles (fig. 2a-c). there was significant monthly variation in residual gonad mass (f1,53 = 5.17, p < 0.0001), but not in residual fat body mass (f8,53 = 1.75, p = 0.108) or residual liver mass (f8,53 = 1.65, p = 0.1315). residual gonad mass peaked in april and may and declined from june through november (fig. 2a; f8,53 = 5.13, p < 0.0001, n = 62). the visual pattern for residual fat body mass shows a small peak in april followed by a decline in may, and then a gradual increase from june through october (fig. 2b). although there was no statistically significant variation across months in residual liver mass, inspection of fig. 2c suggests that liver mass increased sharply from september to november, as compared with the relatively flat pattern observed from march to august. females showed variation in gonad development across months (table 1). no individual female showed both vitellogenic follicles (in the ovary) and oviductal eggs simultaneously, as would be expected if females can lay multiple clutches during a single reproductive season. females started to produce oviductal eggs on 15 april, and the first day that hatchlings were observed (n = 1) was on 5 july, indicating an incubation period of about 82 days. residual gonad mass in females was correlated significantly with residual liver mass (r = 0.28, p = 0.027, n = 62), but not residual fat body mass (r = 0.061, p = 0.64, n = 62). mean clutch size estimated from oviductal eggs was 17.5 ± 1.9 (range 7-28, n = 12). clutch size showed no significant correlation with female svl (r = 0.54, p = 0.07, n = 12) or female 244 aurelio ramírez-bautista et al. mass (both log-transformed, r = 0.08, p = 0.79, n = 12). total egg mass was also not significantly correlated with female svl (both log-transformed, r = 0.33, p = 0.290, n = 12). mean egg mass was 12.1 ± 1.5 g (range 5.8-24.1 g, n = 12) and was not significantly related to clutch size (n = 10, r2 = 0.18, p = 0.23). mean hatchling svl was 34.6 ± 0.8 mm (range 25.7-40.9 mm, n = 33). hatchlings were observed in the field from july to december. fig. 1. annual cycles of residual a) testes mass, b) fat body mass, and c) liver mass for male sceloporus spinosus from las lagunas, municipality of guadalcázar, san luis potosí, méxico. means are given ± 1 se. 245reproduction in sceloporus spinosus discussion male reproduction male s. spinosus in las lagunas, s.l.p. appear to have a bimodal seasonal reproductive cycle. a peak in testicular mass in april was followed by a gradual decline throughout fig. 2. annual cycles of residual a) gonad mass, b) fat body mass, and c) liver mass for female sceloporus spinosus from las lagunas, municipality of guadalcázar, san luis potosí, méxico. means are given ± 1 se. 246 aurelio ramírez-bautista et al. the spring, summer, and early fall, which in turn was followed by a secondary peak in october. the presence of enlarged testes in october suggests that s. spinosus may exhibit a second bout of fall breeding activity, although a lack of samples from november to january prevents more definitive conclusions. although cytological studies are needed to confirm the patterns of seasonality we observed in this study, it is well-established that testicular mass in male lizards is generally greater during the breeding season than before or after (ramírez-bautista and vitt, 1998; hernández-salinas et al., 2010). such a bimodal seasonal pattern of male reproductive activity may be common in s. spinosus specifically, and perhaps more generally among members of the spinosus group. a previous study of s. spinosus from puebla, méxico found an initial peak in male testicular size in april and may and a secondary peak in the fall (valdéz-gonzález and ramírez-bautista, 2002). another member of the spinosus group, s. horridus, showed an initial peak in testicular size in april and may (valdéz-gonzález and ramírez-bautista, 2002). the onset of the long (march-november) seasonal reproductive cycle of males may be related to one or several environmental factors, such as photoperiod, temperature, or precipitation as has been observed in other lizards (see marion, 1982). in populations that exhibit two or more breeding seasons within a single year, the onset of each period of reproductive activity may be triggered by different environmental stimuli such as photoperiod or temperature, with the second peak influenced by precipitation, as occurs in many lizards (marion, 1982; ramírez-bautista and vitt, 1997, 1998). indeed, a similar pattern showed by males in the population of s. spinosus from puebla, méxico is related to precipitation (valdézgonzález and ramírez-bautista, 2002). variation in the timing and number of reproductive peaks may indicate the existence of a plastic response to common environmental factors in oviparous species of sceloporus more generally, or it may be that species of the spinosus group respond in a similar way to environmental factors as a function of shared evolutionary history (miles and dunham, 1992; wiens and reeder, 1997). in male s. spinosus from las lagunas, testes mass was positively correlated with liver mass, but not with fat body mass. inspection of fig. 2b reveals that fat body mass decreased sharply during may, immediately following the peak of the primary reproductive season. depletion of male fat reserves across the breeding season has been observed in other lizard species, and is consistent with the idea that reproductive activities have a high energetic cost (ramírez-bautista and vitt, 1997; ramírez-bautista et al., 2002; hernández-salinas et al., 2010). thus, male s. spinosus are apparently unable to maintain a net positive (or neutral) energy balance during the spring reproductive season, despite the relative availability of food resources during this period (ramírez-bautista, 1995; ramírezbautista and vitt, 1997). this pattern has been observed in several other species of scetable 1. number of female sceloporus spinosus with non-vitellogenic follicles (nvf), vitellogenic follicles (vf), and oviductal eggs (oe) in each month of the study. march april may june july august september october november nvf 4 (66.7%) 2 (40%) 1 (12.5%) 8 (50%) 7 (70%) 3 (60%) 7 (100%) 3 (100%) 3 (100%) vf 2 (33.3%) 0 4 (50%) 6 (37.5%) 0 1 (20%) 0 0 0 oe 0 3 (60%) 3 (37.5%) 2 (12.5%) 3 (30%) 1 (20%) 0 0 0 247reproduction in sceloporus spinosus loporus from méxico, including s. formosus (guillette and sullivan, 1985), s. grammicus (guillette and casas-andreu, 1981; guillette and bearce, 1986; ramírez-bautista et al., 2009), s. mucronatus (méndez-de la cruz et al., 1994), s. pyrocephalus (ramírez-bautista and olvera-becerril, 2004), s. utiformis (ramírez-bautista and gutierrez-mayén, 2003), and s. variabilis (benabib, 1994). the alternative strategy, where males maintain stable fat body and liver volumes during the reproductive season, has also been documented in mexican spiny lizards (e.g., s. dugesii: ramírez-bautista and dávila-ulloa, 2009; s. gadoviae: ramírez-bautista et al., 2005; s. jalapae: ramírez-bautista et al., 2005; s. grammicus: hernández-salinas et al., 2010). female reproduction female s. spinosus also have a seasonal reproductive period, extending from march to august (table 1). gonad mass increased sharply in april and may, a finding congruent with an increase in the number of vitellogenic follicles and oviductal eggs during this timeframe (fig. 2a, table 1). the onset of the female reproductive period in this population of s. spinosus is delayed compared to a population from puebla, méxico; at that site, vitellogenesis was initiated in late january, with ovulation occurring in the spring and early summer (valdéz-gonzález and ramírez-bautista, 2002). the female reproductive cycle of s. horridus (another member of the spinosus group) showed a somewhat similar extension in reproductive activity (valdéz-gonzález and ramírez-bautista, 2002). male and female s. spinosus exhibit synchronous reproduction; thus, as in males, females begin producing vitellogenic follicles in march coincident with increased temperature and photoperiod, and the end of reproduction in august coincides with increased precipitation (see garcía, 1981). this pattern is similar to that observed in other lizard species (marion, 1982; ramírez-bautista and olvera-becerril, 2004), indicating that these three environmental factors can often play an important role in determining the onset and duration of reproductive activity in lizards (marion, 1982; hernández-salinas et al., 2010). at las lagunas, female s. spinosus appear to produce only a single clutch per year (table 1). at puebla, females can produce up to two clutches per year, each apparently slightly smaller (12.6%) than the single clutch of females from las lagunas (valdézgonzález and ramírez-bautista, 2002; this study). these results could be explained by variation in predation pressure in the environments that these populations inhabit (ramírez-bautista, 1995). all species of the spinosus group have a larger clutch size than other species of sceloporus (valdéz-gonzález and ramírez-bautista, 2002; see ramírez-bautista and olverabecerril, 2004), which could reflect population or species-specific differences in predation pressure (ballinger, 1979; benabib, 1994; ramírez-bautista, 1995). the extended reproductive cycle of the puebla population permits females to have two clutches, perhaps because the wet season at puebla is longer and results in greater total precipitation than at las lagunas (valdéz-gonzález and ramírez-bautista, 2002). however, this interpretation should be considered with caution, since the population studied by valdéz-gonzález and ramírezbautista (2002) was conducted using individuals from a wide geographic area in méxico. female gonad mass was positively correlated with liver mass, but not fat body mass. as liver mass was relatively high and fat body mass relatively low during the period of peak reproductive activity in females, the energy invested in vitellogenic follicles and eggs 248 aurelio ramírez-bautista et al. probably comes from fat body masses exclusively (or nearly so). these data provide more evidence that reproduction incurs a high energetic cost in female lizards (ramírez-bautista and vitt, 1997, 1998; ramírez-bautista and olvera-becerril, 2004; hernández-salinas et al., 2010), in addition to other costs of reproduction (ballinger, 1977; ramírez-bautista, 1995). our results suggest that this population of s. spinosus could contribute to our understanding of the role of fat body and liver energy reserves in affecting juvenile and adult growth rate, and the onset of sexual maturity in lizards (see benabib, 1994). mean clutch size in our population of s. spinosus was 17.5 ± 1.9 eggs, similar to that reported for the puebla population (15.5 ± 1.2 eggs: valdéz-gonzález and ramírez-bautista, 2002). clutch size for females from las lagunas is greater than that for the populations of the spinosus species group reported in calderón-espinosa et al. (2006; range of means = 9.2-16.6 eggs), as well as that of a population of s. spinosus from oaxaca (mean = 12.66; fitch, 1978, 1985). it is also larger than that reported for two populations of another member of the spinosus group, s. horridus (mean = 14.0, valdéz-gonzález and ramírezbautista, 2002; mean = 9.3, ramírez-bautista, 2003). thus, there is clearly substantial variation in clutch size among populations of the spinosus species group. further work is needed to examine potential causes of this variation. obtaining information on additional populations of s. spinosus or other members of the spinosus species group, as well as data from populations of species groups closely related to the spinosus group, will help in elucidating the relative contributions of environmental factors and phylogeny (ballinger, 1979; miles and dunham, 1992; benabib, 1994). in lizards, body size usually positively influences clutch size [see fitch (1985) and dunham et al. (1988) for reviews]. interestingly, we did not find a significant relationship between female body size and clutch size, which is an adaptive strategy found in populations of lizards influenced by variation in climate, predation intensity, female size (vitt and price, 1982), or variation in rainfall and drought directly affecting food availability and consequently clutch size and offspring size (abell, 1999). however, at las lagunas the trend for larger females (n = 7, females > 100 mm svl had clutch sizes ranging from 15 – 28 eggs, with the exception of a single female of 108 mm svl which had 7 eggs) to produce larger clutches was nearly significant (p = 0.071). there was also no such relationship in the previously-studied population of s. spinosus from puebla or the population of s. horridus (valdéz-gonzález and ramírez-bautista, 2002), suggesting that the relationship between female size and clutch size is non-existent or very weak in the s. spinosus species group. this correlation pattern has been documented in both oviparous (benabib, 1994; ramírez-bautista and olvera-becerril, 2004; ramírez-bautista et al., 2005) and viviparous species (ramírez-bautista et al., 2008). conclusions our results for male and female reproduction in s. spinosus suggest that they are generally similar in some reproductive characteristics (see above) to other members of the s. spinosus species group. however, we found some evidence of variation in reproductive traits (e.g., timing of the initiation of the female reproductive cycle; clutch size) relative to other populations of s. spinosus and other species within the spinosus group. these results suggest that the s. spinosus group could serve as another model group within sceloporus 249reproduction in sceloporus spinosus to explore ecological and evolutionary causes of interpopulational variation in life history traits. future efforts should target s. edwardtaylori, the only member of the spinosus group lacking baseline reproductive data. however, studies of additional populations of s. spinosus and s. horridus will also be crucial in determining both the extent of geographic variation in reproductive traits, as well as assessment of the relative importance of environmental factors and evolutionary history on life history characteristics. acknowledgements we thank r. torres-cervantes, a. leyte-manrique, and o. ramos-flores for their assistance in this research. we also thank semarnat for approving this research (permit #hssx1304811). this work was supported by the projects conabio r045, conacyt-27618-n, s52552 and fomix-conacyt-43761, 95828. references abell, a.j. 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(2010): phylogenetic relationships of phrynosomatid lizards based on nuclear and mitochondrial data, and a revised phylogeny for sceloporus. mol. phylogenet. evol. 54: 150-161. acta herpetologica vol. 7, n. 2 december 2012 firenze university press advertisement call of species of the genus frostius cannatella 1986 (anura: bufonidae) flora a. juncá1, david l. röhr2, ricardo lourenço-de-moraes3, flávio j. m. santos1, airan s. protázio1, ednei a. mercês1, mirco solé4 amphibians in southern apennine: distribution, ecology and conservation notes in the “appennino lucano, val d’agri e lagonegrese” national park (southern italy). antonio romano1,*, remo bartolomei1, antonio luca conte1, egidio fulco2 the significance of using satellite imagery data only in ecological niche modelling of iberian herps neftalí sillero1, josé c. brito2, santiago martín-alfageme3, eduardo garcía-meléndez4, a.g. toxopeus5, andrew skidmore5 reproductive strategy of male and female eastern spiny lizards sceloporus spinosus (squamata: phrynosomatidae) from a region of the chihuahuan desert, méxico aurelio ramírez-bautista1,*, barry p. stephenson2, xóchitl hernández-ibarra1, uriel hernández-salinas1, raciel cruz-elizalde1, abraham lozano1, and geoffrey r. smith3 morphological variability of the hermann’s tortoise (testudo hermanni) in the central balkans katarina ljubisavljević1, georg džukić1, tanja d. vukov1, miloš l. kalezić1,2 the usefulness of mesocosms for ecotoxicity testing with lacertid lizards maria josé amaral1,2,*, rita c. bicho1, miguel a. carretero2, juan c. sanchez-hernandez3, augusto m. r. faustino4, amadeu m. v. m. soares1, reinier m. mann1,5 does acclimation at higher temperatures affect the locomotor performance of one of the southernmost reptiles in the world? jimena b. fernández*, nora r. ibargüengoytía advertisement call of scinax littoralis and s. angrensis (amphibia: anura: hylidae), with notes on the reproductive activity of s. littoralis michel v. garey1, thais r. n. costa2, andré m. x. de lima2, luís f. toledo3, marília t. hartmann4 book review: marine s. arakelyan, felix d. danielyan, claudia corti, roberto sindaco, alan e. leviton 2011. herpetofauna of armenia and nagorno-karabakh. society for the study of amphibians and reptiles stefano scali book review: rafaqat masroor 2012. a contribution to the herpetofauna of northern pakistan stefano scali evidence of high longevity in an island lacertid, teira dugesii (milne-edwards, 1829). first data on wild specimens. j. jesus first assessment of the endoparasitic nematode fauna of four psammophilous species of tropiduridae (squamata: iguania) endemic to north-eastern brazil markus lambertz1,*, tiana kohlsdorf2, steven f. perry1, robson waldemar ávila3, reinaldo josé da silva4 rediscovery and redescription of the holotype of lygosoma vittigerum (= lipinia vittigera) boulenger, 1894 yannick bucklitsch1, peter geissler1, timo hartmann1, giuliano doria2 , andré koch1,* reproductive phenology of the tomato frog, dyscophus antongili, in an urban pond of madagascar’s east coast ori segev1,*, franco andreone2, roberta pala2, giulia tessa2, miguel vences1 range extension of the critically endangered true poison-dart frog, phyllobates terribilis (anura: dendrobatidae), in western colombia roberto márquez1,*, germán corredor2, carlos galvis3 daniel góez2, & adolfo amézquita1 differences in habitat use of two sympatric species of ameiva in east costa rica esther sebastián-gonzález1, ramón gómez2 acta herpetologica journal of the societas herpetologica italica acta herpetologica 5(2): 249-x, 2010 © firenze university press www.fupress.com/ah a new approach for surveying the alpine salamander (salamandra atra) in austria ursula reinthaler-lottermoser1, magdalena meikl1, ana gimeno1, elisabeth weinke2, robert schwarzenbacher1,* 1 molecular biology universität salzburg, billrothstraße 11, a-5020, salzburg, austria.*corresponding author. e-mail: roberts@sbg.ac.at 2 geoinformatik universität salzburg, hellbrunnerstraße 34, a-5020, salzburg, austria. submitted on: 2010, 6th june; revised on: 2010, 26th august; accepted on: 2010, 8th october. abstract. the alpine salamander is a small pitch black amphibian which is endemic to the european alps and the dinarides. it is strictly protected according to the european ffh guidelines. despite its central role in the alpine ecosystem our actual published record in austria is small. in order to resolve this shortcoming our project explores its distribution in austria. it uses a participatory and community based approach to gather data. everybody can enter and look at alpine salamander observations on our website www.alpensalamander.eu. this approach also allows us to establish an “oral history” of salamander observations in the past 50 years by conducting interviews in the local community. since july 2009 the website and salamander report database are online. from the actual data (more than 5600 records) we already obtained an overview about the present distribution and data quality. the data are an excellent basis for detailed scientific studies on these remarkable amphibians. with this new and highly interactive approach science and education are combined to initiate protection measures with the public. keywords. alpine salamander, salamandra atra, amphibian, austria, alps. the alpine salamander is on the red list of endangered animals in austria (klewen, 1991; nöllert and nöllert, 1992; greven, 1998; guex and grossenbacher, 2003; kyek and maletzky, 2006) and strictly protected according to the european habitats directive on the conservation of natural habitats and of wild fauna and flora (european union law, 2010). with less than 800 observations, the actual amount of published records in austria is very small. in fact, its present distribution tendency in the austrian alps, its ecology and its habitat are still unknown. in order to resolve this shortcoming, our project explores the distribution of the alpine salamander focusing on austria. in the next years this effort will be extended to the other alpine countries, where the alpine salamander occurs. the main goal is to map occurrence, population size and development as well as the genetic structure of the alpine salamander. 250 u. reinthaler-lottermoser et alii we have established the website www.alpensalamander.eu, where everybody can report alpine and fire salamander observations. this community based approach enables the combination of research, education and dissemination by interactive participation. some technical details of the alpensalamander.eu portal database: www.alpensalamander.eu is a standalone wordpress blog that contains information about the salamanders, the research project and a video-blog. it is the central resource for users and hosts the salamander report interface. the salamander report interface is an embedded google map running an application programming interface (api) that allows for navigation, location and entry of salamander observations, which are subsequently stored in the mysql database. the data base stores the entry identification number, date and time, the name and the email address of the user, the type and number of salamanders (alpine or fire salamander), the latitude, longitude and the altitude of the location of the salamander observation, a remark (text entry), and a photograph. the weather conditions, such as sun/rain, temperature and humidity are collected from the zamg (zentralanstalt für meteorologie und geodynamik, austria) according to coordinates and date. currently all entries are recorded and manually inspected by the research team for their credibility. questionable entries such as putative misidentifications between alpine salamander and alpine newt, are investigated by contacting the user. questionable data are deleted or flagged, and can be excluded for analysis at will. the dataset is freely available to academia. this web 2.0 approach is a new step to involve the population actively in research and protection of these vulnerable amphibians and their habitats. a second part of our project is to record “the oral history” of the alpine salamander observations in the past 50 years, to see whether the populations are stable or decreasing. therefore, we conducted interviews in the local community with alpinists, farmers, hunters, national park staff and mineral collectors to preserve their well-versed local knowledge of the alpine salamander. there were also several articles in national and local newspapers to inform the population and ask for participation. in addition we collaborate with several national parks (hohe tauern, berchtesgaden) and museums to establish the project in schools, wildlife and mountaineering organizations. website and salamander report database are online since july 2009. as of august 2010, more than 5681 alpine salamander reports in austria were gathered. until now there are 1058 different users, 338 are regular users and the community is still growing. the 5681 records are displayed as 578 clusters (fig. 1). the criteria for clustering the salamander records are more than 4 individuals per hectare. although the project is still in the initial stages, the data already picture a preview of the alpine salamander distribution in austria and allow for a preliminary analysis including the assessment of data quality. as mentioned before, all records were evaluated in person. obviously questionable reports, like coordinates in a lake, were checked by contacting the user and corrected or deleted. this aggregated data set is the basis for our preliminary analysis where we consolidated the data with different maps. the results are shown on the following pages. the datum in percent is related to the clusters as described before. compared to the geology (fig. 1a), the main distribution of the alpine salamander is in the northern limestone alps (47.2%) and the central alps (42.7%). we observed 6.6% in the grauwackenzone and 2.6% in the flysch zone. only a few findings were in the 251a new approach for surveying the alpine salamander in austria figure 1. distribution of salamandra atra in austria. dataset collected from june 2009 to august 2010. a) the distribution of salamandra atra in comparison with different geology. pink: central alps; dark blue: southern limestone alps; light blue: northern limestone alps; yellow: vienna basin; light green: south-eastern foothills of the alps; orange: eastern foothills of the alps; violet: grauwacken zone; lavender: basin of klagenfurt; brown: graniteand gneiss upland; dark green: flysch zone. b) the distribution of salamandra atra across land covered areas. brown: agricultural areas; rose: artificial surfaces; green: forest and seminatural areas; dark blue: water bodies; light blue: wet lands. c) the distribution of salamandra atra in the different austrian climate types: dark blue: alpine climate; rose: middle-europe transitional climate; light blue: pannonian climate; orange: illyric climate. d) the distribution of salamandra atra per altitude (m a.s.l.). dark green: 115 – 500; light green: 500 – 1000; light orange: 1000 – 1500; orange: 1500 -2000; pink: 2000 – 2500; lavender: 2500 – 3000; white: 3000 3798. 252 u. reinthaler-lottermoser et alii southern limestone alps (0.15%) as well as in the foothills of the alps (0.15%) and the granite and gneiss upland (0.3%).there are no records for the vienna basin, the southeastern and north-eastern foothills of the alps. the second map (fig. 1b) shows the distribution of salamandra atra across land covered areas in accordance to the corine landcover classification from 2000. in this map it is shown that over 91.5% of the alpine salamanders were found in forest or in semi-natural areas. only 5% was found in agricultural areas and 3.1 % was found in artificial surfaces. on the third map (fig. 1c) the distribution of salamandra atra is compared with the austrian climate types. 95.8% of the recorded salamanders can be found at an alpine climate. this climate predominates mostly in the alpine regions of the austrian federal states vorarlberg, tyrol, salzburg as well as parts of styria, upperand lower austria. the other 4.2% were recorded on middle european transitional climate. these observations took place in lower austria, some spots in upper austria and in the west of vorarlberg. fig. 1d shows the distribution of salamandra atra per altitude. most of the alpine salamanders (42.6%) were found on altitudes between 1500 and 2000 meter a.s.l., 33.8% was found on lower sides at 1000 – 1500 m. only 0.3% was observed at high altitudes between 2500–3000 m. in lower regions, ranging from 115–500 m, five salamanders (0.9%) were recorded. the records correlate with increasing altitude, the best altitude for alpine salamander observations seems to be between 1500 and 2000 meters. taken together, our new approach that involves the community in a scientific project proofs very successful for monitoring the alpine salamander in austria. since july 2009 we were able to record 5681 alpine salamander reports. a broad community was built in short time with the help of 1058 different users and this community is still growing. the records on our website increased in correlation to our degree of popularity. to get even more attention, we started publishing reports in different national and local newspapers. in these articles we invited the population to report salamanders when they find some. the feedback was actually positive. a second positive effect is that the population is getting more attentive on the small black salamanders and so it gets easier to protect those animals. in comparison, the biodiversity database from the natural history museum in vienna contains 754 recordings for the alpine salamander (cabela et al., 2001). our community based approach generates much more data and immediately publishes them to the public. on our website anybody is able to report findings of salamanders. in comparison with other amphibians, it is very difficult to find the alpine salamander; they only come to the surface when the weather is adequate (i.e., in the early morning hours or while/after a summer rain). with the vast amount of records it is possible to plan further monitoring activities much more efficiently. some positive effects of these data are: • a salamander distribution map was generated with almost no cost for austria. • the map is updated continuously and allows monitoring salamander populations. • dense salamander occurrences and peculiar observations, like high altitude populations, can be used for research studies like a genetic monitoring to analyze the relationships between the individuals. with the different geological maps (see results), it becomes obvious that these animals appear basically in the alpine climate on altitudes between 1500 and 2000 m. our data 253a new approach for surveying the alpine salamander in austria are in line with published findings of (cabela et al., 2001, as well as guex and grossenbacher, 2003). this indicates that the data from the public are of high quality and that the abuse of the database is negligible. the main distribution area of the alpine salamander is in austria. so it is necessary to sensitize the population and especially the children, to spark their interest for these amazing animals. we are aimed at collaborating with national parks and museums throughout europe to effectively disseminate the project and its results in wildlife and mountaineering organizations but also at schools and universities. the web portal www.alpensalamander.eu allows collection of salamander observations by the public which is serving two main purposes as i) data collection for new scientific project initiatives and ii) education of the public through direct and interactive dissemination of salamander observations. we believe that protection of amphibians and their habitats is only possible by actively involving the population, as participatory research has shown. the alpine salamander is one of the heraldic animals of europe and it is our obligation to put every effort into research on it, in order to preserve the significant moment of observing these amazing animals for future generations. acknowledgments we thank prof. günter fachbach for his time and valuable suggestions. two anonymous reviewers and the ah editorial staff helped in improving previous drafts of the ms. references cabela, a., grillitsch, h., tiedemann, f. (2001): atlas zur verbreitung und ökologie der amphibien und reptilien in österreich. umweltbundesamt, wien. greven, h. (1998): survey of the oviduct of salamandrids with special reference to the viviparous species. j exp zool. 282: 507 – 525 guex, g.d., grossenbacher, k. (2003): salamandra atra laurenti, 1768 alpensalamander. in: handbuch der reptilien und amphibien europas, bd.4/ii b, schwanzlurche (urodela) ii b, p. 975-1028. grossenbacher, k., thiesmeier, b., eds, aula-verlag, wiesbaden. klewen, r. (1991): die landsalamander europas, teil 1. die neue brem-bücherei, wittenberg lutherstadt, ziemsen. kyek, m., maletzky, a. (2006): atlas und rote liste der amphibien und reptilien salzburgs, amt der salzburger landesregierung, naturschutzabteilung. nöllert, a., nöllert, c. (1992): die amphibien europas. bestimmung – gefährdung – schutz. franckh-kosmos verlag, stuttgart. © firenze university press www.fupress.com/ah acta herpetologica 5(1): 63-85, 2010 free gis for herpetologists: free data sources on internet and comparison analysis of proprietary and free/open source software neftalí sillero1, pedro tarroso2 1 centro de investigação em ciências geo-espaciais (cicge) da universidade do porto. r. campo alegre, 687, 4169-007 porto, portugal. corresponding author. e-mail: neftali.pablos@fc.up.pt 2 cibio, centro de investigação em biodiversidade e recursos genéticos da universidade do porto, campus agrário de vairão, r. padre armando quintas, 4485-661 vairão, portugal. e-mail: pedro. tarroso@gmail.com submitted on: 2009, 5th november; revised on 2010, 26th april; accepted on 2010, 3rd may. abstract. geographical information systems (gis) have been used widely in zoology and ecology, particularly in herpetology. the use of spatially explicit analysis has increased during the last decade, with the consequent expansion of gis application in ecology. during the last years, geo-information technology has been developed within the free/open source software (foss) community, resulting in new open source formats and several gis packages. however, proprietary packages seem still to be the first choice for herpetologists, thus involving non negligible costs for gis technology adoption. additional costs arise from environmental data, which are usually expensive, worsening in the case of large study areas. an alternative solution is to use freely available data, despite a possible decrease of resolution. in this review, we aim to show the feasibility of spatial analysis within foss gis packages, rank these packages using the number of available tools and list several data sources freely available on the internet. we listed several websites providing the most important free data for spatial analysis, i.e. altitude and derived data; past, current and future climatic series data; and satellite derived data. we provide also a list of the most commonly used functions in gis analysis and their availability in the six software compared in this study (arcgis; gvsig; ilwis; quantum gis; grass; and diva-gis). the software gvsig is the one with more functions (106) followed by quantum gis with 94 and grass with 84. keywords. geographical information system, free/open source software, internet data sources, herpetology. introduction a geographical information system (gis) is a specific type of information system, i.e., a set of processes, executed on raw data through observation, measurement, description, 64 n. sillero and p. tarroso explanation and forecasting, to produce new information useful in decision-making. some authors extend the scope of the gis definition from a simple geographical information system to a broader geographical information science (e.g., longley et al., 1991). gis differs from other information systems because it uses geographically referenced data (maguire, 1991; sillero et al., 2002). commonly viewed as a simple software package, gis is in fact an integrated collection of software, hardware and people (sillero et al., 2002). although other computer programs can use spatial data (e.g., computer-aided design or cad and some statistics packages), gis can perform spatial operations. in fact, any cad is able to perform geometrical operations in a cartesian space, using the same entity-attribute model as in gis vector data, but the user is not compelled to operate in a geographical datum and coordinate reference system context. moreover, a “non-geographical” spatial information system is completely unable to deal with angular positioning systems (i.e., latitude and longitude data in degrees). the dissemination of gis occurred after the 70s with small programs that could handle and operate with spatial data and during the 80s with packages like arcinfo or grass, the seeds of the most widely used software nowadays (longley et al., 1991). the application of gis covers a great diversity of fields, such as geography, agriculture, economics, photogrammetry and surveying. furthermore, gis has been used widely in zoology and ecology, particularly in herpetology. for example, gis is an essential tool for producing herptile chorological atlases (sillero et al., 2002, 2005; loureiro and sillero, 2008). in fact, the last herpetological atlases published in europe used gis to store, analyse and map species distribution data: e.g., spain (pleguezuelos et al., 2002), italy (sindaco et al., 2006), western palearctic (sindaco and jeremcenko, 2008), or portugal (loureiro et al., 2008). gasc et al. (1997) were pioneers on the application of gis to map species distributions, creating the first herpetological atlas of europe. the ecological modelling of amphibians and reptiles relies also on gis to calculate and process potential distribution models (guisan and hofer, 2003; segurado and araújo, 2004; anadon et al., 2006; arntzen, 2006) and habitat suitability models (raxworthy et al., 2003; kearney and porter, 2004; dayton and fitzgerald, 2006; kaliontzopoulou et al., 2008), as well as in the analysis and forecast of species richness distribution (soares and brito, 2007; ribeiro et al., 2009), climate change effects on species (meynecke, 2004; thomas et al., 2004; araújo and pearson, 2005; parra-olea et al., 2005; araújo et al., 2006; araújo et al., 2008) and expansion process of autochtonous species (sillero, 2009, 2010) or invasive species (ficetola et al., 2007, 2009; real et al., 2008). in conservation and management of reptiles, gis is used in nature reserve design (ferrier et al., 2002), conservation studies (root et al., 2003; santos et al., 2006, 2009; carretero et al., 2008), forecasting the impacts of road networks (benayas et al., 2006) and amphibian road-kills (clevenger et al., 2003; glista et al., 2007; sillero, 2008). gis is widely used in herpetological biogeography, namely in descriptive biogeographical analysis and identification of biogeographical areas (teixeira et al., 2001, 2002; wiens et al., 2006; sillero et al., 2009), phylogeography analysis (arntzen and alexandrino, 2004; real et al., 2005), detection of sympatry areas and hibridization processes (brito and crespo, 2002; costa et al., 2008; martínez-freiría et al., 2008), and speciation mechanisms (kozak and wiens, 2007). inside a gis framework is possible to analyse species dispersion processes through permeability matrix (ray et al., 2002), detect migration paths (joly et al., 2003), identify animal movements (brito, 2003) and assess home range areas (osterwalder et al., 2004; knapp and owens, 2005; kerr and bull, 2006; waldron 65free gis for herpetologists et al., 2006; greenberg and mcclintock, 2008; imansyah et al., 2008). gis has also been applied to taxonomical studies in order to identify different taxa by modelling their ecological niches (sá-sousa, 2000; brito et al., 2008). these works confirm the increasing demand of spatial explicit analysis and the expansion of gis in ecology during the last decade. although ecology is a marginal field in need of geospatial tools, it is not surprising that geo-information technology is also being developed within the free/open source software (foss) community. the concept of “free software” was first defined by richard m. stallman in the form of the so-called four freedoms (mitasova and neteler, 2004; tufto and cavallini, 2005): 1) the freedom to run a program, for any purpose; 2) the freedom to study how the program works, and adapt it to your needs; 3) the freedom to redistribute copies; and 4) the freedom to improve the program, and release your improvements to the public. points 2 and 4 need obligatorily the access to the program source code. stallman conceived the gnu-project in 1983 to support the free software concept, and a year later he created the free software foundation. here, the term free refers not only to the price of acquisition of the software but to a complex of freedoms that allows the user to copy, analyse the source code and even to modify it. the gnu general public license (gpl) is a software licensing form developed by the free software foundation that grants and protects the four freedoms describes above. under the gpl, the software code and its modifications must remain “open”. gpl is the most widely used license for foss nowadays. over the past few years several foss gis projects have been implemented. most of them can be found at “freegis project” web site (www.freegis.org). these projects play an important role in adaptation of gis technology by stimulating new experimental approaches and by providing access to gis for a wide spectrum of users. this foss philosophy has been extended in some way to data sources availability. spatial data is in fact essential to perform gis analysis. everyday there are more institutions offering their spatial data for free (kozak et al., 2008). in this review, we aim to show the feasibility of spatial analysis within foss gis packages, rank these packages using the number of available tools and list several data sources freely available on the internet. this review is organized in three sections: 1) the review of free sources of geographical data; 2) the comparative analysis of proprietary and free gis packages and 3) the final remarks. free spatial data sources on internet spatial analysis performed by ecologists relate the biology of the species with environmental factors, such as climatic, topographical and habitat data (e.g., sillero et al., 2009). there are several sources for obtaining these variables, namely topographical, thematic maps and satellite imagery. in some cases, it may be necessary to collect environmental data by fieldwork, which might not be feasible when the costs are high, the study area is very large, or there is no time for data collection (muldavin et al., 2001). the solution is to acquire data from specialized spatial data providers. these products are usually expensive, worsening in the case of large study areas. this constraint hampers the development of ecological spatial analysis. an alternative solution is to use freely available data, despite a possible decrease of resolution. 66 n. sillero and p. tarroso there are a large amount of environmental data freely available on internet. these data are dispersed over several dedicated sites and researchers usually find difficult to retrieve free data from the internet. an authoritative source of free gis data is the osgeo public geospatial data project (www.osgeo.org/geodata), as well as its geonetwork opensource metadata harvesting tool (www.geonetwork-opensource.org). these sites are a valid starting point to search for freely available, authoritative data. kozak et al. (2008) presented a table with links to a few websites, often considered the most important with free environmental data. available data can be grouped in several different types of spatial information: administrative (country limits, road networks), geographical (country populations), climatic (temperature, precipitation), topographical (altitude, slope), satellite imagery and aerial photography. spatial data are stored at different resolutions, either spatial (e.g., resolutions of 200 m, 10 km) or temporal (e.g., daily data, annual data). the area covered by the data is also variable: from small areas (municipalities) to the whole world. the antarctic continent is not included in most spatial data sources (e.g., worldclim, shuttle radar topography mission). we distributed free spatial data sources in five groups: digital elevation models (dem), satellite imagery, climatic data, geographical data and environmental data. we listed websites (table  1) that we commonly use and searched the internet for others storing these type of data or referencing it (e.g., “freegis project”: www.freegis.org). other authors provided several sources (e.g., kozak et al., 2008) that we also add. four of the listed websites should be mentioned here: the shuttle radar topography mission (srtm), worldclim, glovis, and spot vegetation. these provide the most important data for spatial analysis in herpetology, i.e., altitude and derived data; past, current and future climatic series data; and satellite derived data. in fact, these are the websites most accessed by researchers. these data are enough for performing complex biogeographical studies of amphibians and reptiles (e.g., sillero et al., 2008, 2009). the shuttle radar topography mission (srtm) site offers altitude data at three different spatial resolutions: global dataset is available at 3 arc second and 30 arc second resolutions (approximately 90 m and 1 km, respectively); the current strm3 version 2.1 global dem is at 90 m, whereas the so-called “unfinished” 1 arc second (~ 30 m) covers only the conterminous usa. the srtm was a project of nasa, the national geospatialintelligence agency, and the german and italian space agencies, performed in february 2000 during 11 days on board shuttle endeavour. it used dual radar antennas to acquire interferometric radar data, processing digital topographic data at 1 arc sec resolution (farr et al., 2007). recently, other global dem has became available: the aster dem, with a spatial resolution of 30 m. the srtm is in the third version, with most errors corrected. aster dem is still in its first version. both dem are currently the most complete nearglobal high-resolution database of earth’s topography. worldclim (hijmans et al., 2005) offers current series of climatic data: precipitation, mean temperature, maximum temperature and minimum temperature, together with a set of 19 climatic derived variables. there are several spatial resolutions available: 30 arc seconds, 2.5, 5 and 10 arc minutes. also, climatic variables are available for several future scenarios (e.g., a2a and b2a hadcm3) and years (2020, 2050 and 2080), and for two past periods, the last interglacial period and the last glacial maximum. the global visualization viewer (glovis) offers satellite imagery from all the landsat series (1, 3, 4 and 5 mss; 4 and 5 tm; and 7 etm+). these landsat series cover a 67free gis for herpetologists ta bl e 1. d at a so ur ce s fr ee ly a va ila bl e on t he i nt er ne t. th e w eb si te s ha ve b ee n or ga ni ze d in f ou r gr ou ps : d ig ita l e le va tio n m od el s, s at el lit e im ag er y, c lim at ic da ta , g eo gr ap hi ca l d at a, a nd e nv ir on m en ta l d at a. n am e d es cr ip tio n li nk o w ne r d ig ita l e le va tio n m od el s g t o po 30 u sg s d em 3 0 se g ht tp :// ed c. us gs .g ov /p ro du ct s/ el ev at io n/ gt op o3 0/ gt op o3 0. ht m l u sg s g lo ba l l an d o ne -k m b as e el ev at io n pr oj ec t n at io na l o ce an og ra ph ic d at a c en te r d em ht tp :// w w w .n gd c. no aa .g ov /m gg /t op o/ gl ob e. ht m l n o a a sr t m sh ut tle r ad ar t op og ra ph y m is si on ht tp :// sr tm .c si .c gi ar .o rg / n a sa sa te lli te g eo de sy sr t m 30 _p lu s b at hy m et ry ht tp :// to pe x. uc sd .e du /w w w _h tm l/s rt m 30 _p lu s. ht m l u ni ve rs ity o f c al ifo rn ia sa n d ie go g lo ba l a st er d em a st er g lo ba l d ig ita l e le va tio n m od el 3 0m w w w .g de m .a st er .e rs da c. or .jp n a sa /m et i h y d r o 1k h y d r o 1k e le va tio n d er iv at iv e d at ab as e ht tp :// ed c. us gs .g ov /p ro du ct s/ el ev at io n/ gt op o3 0/ hy dr o/ u sg s sa te lli te im ag er y fr ee v eg et a t io n d is tr ib ut io n si te sp o t v eg et at io n se ns or -f re e pr od uc ts ht tp :// fr ee .v gt .v ito .b e/ sp o t g lc f g lo ba l l an d c ov er f ac ili ty ht tp :// gl cf .u m ia cs .u m d. ed u/ in de x. sh tm l n o a a g lo ba l l an d 1k m a v h r r pr oj ec t a dv an ce d v er y h ig h r es ol ut io n r ad io m et er da ta ht tp :// ed c2 .u sg s. go v/ 1k m / n o a a ge on et w or k fi nd i nt er ac tiv e m ap s, g is d at as et s, s at el lit e im ag er y an d r el at ed a pp lic at io ns ht tp :// w w w .fa o. or g/ ge on et w or k/ sr v/ en /m ai n. ho m e fa o se a m le ss se am le ss d at a d is tr ib ut io n sy st em , e ar th r es ou rc es o bs er va tio n an d sc ie nc e ht tp :// se am le ss .u sg s. go v/ w eb si te /s ea m le ss / u sg s la nd sa t.o rg la nd sa t p ro gr am m e ho m e pa ge ht tp :// w w w .la nd sa t.o rg / n a sa la a d s le ve l 1 a nd a tm os ph er e a rc hi ve a nd d is tr ib ut io n sy st em ht tp :// la ds w eb .n as co m .n as a. go v/ n a sa (c on tin ue d) 68 n. sillero and p. tarroso n am e d es cr ip tio n li nk o w ne r lp d a a c a st er a nd m o d is l an d d at a pr od uc ts a nd se rv ic es ht tp s: // lp da ac .u sg s. go v/ u sg s w is t w ar eh ou se i nv en to ry s ea rc h to ol ht tp s: // w is t.e ch o. na sa .g ov / n a sa g lo ba l v is ua liz at io n v ie w er sa te lli te im ag er y ht tp :// gl ov is .u sg s. go v/ u sg s ea rt he xp lo re r sa te lli te im ag er y ht tp :// ed cs ns 17 .c r.u sg s. go v/ ea rt he xp lo re r/ u sg s c lim at ic d at a w o r ld c li m c lim at ic d at a fr om h ijs m an e t a l 2 00 5 ht tp :// w w w .w or ld cl im .o rg / h ijs m an e t a l bi o c li m bi oc lim at ic p ar am et er s fo r m od el lin g so ftw ar e bi oc lim ht tp :// cr es .a nu .e du .a u/ ou tp ut s/ an uc lim /d oc /b io cl im . ht m l a us tr al ia n n at io na l u ni ve rs ity g lo ba lpe t a nd g lo ba la ri di ty da ta se ts g lo ba l p ot en tia l e va po -t ra ns pi ra tio n an d a ri di ty i nd ex ht tp :// cs i.c gi ar .o rg /a ri di ty /i nd ex .a sp c g ia rc on so rt iu m fo r sp at ia l i nf or m at io n g c m d at a po rt al fu tu re c lim at ic s ce na ri os ht tp :// gi sw eb .c ia t.c gi ar .o rg /g c m pa ge / ip c c g eo gr ap hi c da ta a tla s of th e bi os ph er e h um an im pa ct s, la nd u se , e co sy st em s, w at er re so ur ce s ht tp :// w w w .s ag e. w is c. ed u/ at la s/ sa g e d ig ita l c ha rt o f t he w or ld a dm in is tr at iv e da ta ht tp :// w w w .m ap ro om .p su .e du /d cw / pe nn s ta te u ni ve rs ity g eo d at a po rt al en vi ro nm en ta l a nd g eo gr ap hi ca l d at a ab ou t w or ld ht tp :// ge od at a. gr id .u ne p. ch / u ni te d n at io ns en vi ro nm en t pr og ra m m e gd at a fr ee g eo gr ap hi c (g is ) da ta fo r an y co un tr y in th e w or ld ht tp :// bi og eo .b er ke le y. ed u/ bg m /g da ta .p hp b er ke le y u ni ve rs ity ge on et w or k fi nd i nt er ac tiv e m ap s, g is d at as et s, s at el lit e im ag er y an d r el at ed a pp lic at io ns ht tp :// w w w .fa o. or g/ ge on et w or k/ sr v/ en /m ai n. ho m e fa o c ou nt ry f ile s (g n s) c om pl et e fi le s of g eo gr ap hi c n am es fo r g eo po lit ic al a re as ht tp :// ea rt hin fo .n ga .m il/ gn s/ ht m l/c nt ry _fi le s. ht m l g n s (c on tin ue d) ta bl e 1. ( co nt in ue d) . 69free gis for herpetologists n am e d es cr ip tio n li nk o w ne r g is d at af in de r a dm in is tr at iv e da ta ht tp :// w w w .li b. un c. ed u/ re fe re nc e/ gi s/ da ta fin de r/ in de x. ht m l d av is l ib ra ry r ef er en ce d ep ar tm en t ea rt ht re nd s li nk s to r es ou rc es ht tp :// ea rt ht re nd s. w ri .o rg /i nd ex .p hp w or ld r es ou rc es in st itu te en vi ro nm en ta l d at a g eo d at a po rt al en vi ro nm en ta l a nd g eo gr ap hi ca l d at a ab ou t w or ld ht tp :// ge od at a. gr id .u ne p. ch / u ni te d n at io ns en vi ro nm en t pr og ra m m e eu ro pe an e nv ir on m en t a ge nc y en vi ro nm en ta l d at a ab ou t e ur op e ht tp :// w w w .e ea .e ur op a. eu / eu ro pe an u ni on eu ro pe an d is tr ib ut ed i ns tit ut e of t ax on om y g lo ba l a nd e ur op ea n g is la ye rs ht tp :// ed it. cs ic .e s/ g is do w nl oa ds .h tm l eu ro pe an u ni on g ia m g lo ba l m ap o f i rr ig at ed a re a ht tp :// w w w .iw m ig ia m .o rg /i nf o/ m ai n/ in de x. as p iw m i h y d e h is to ry d at ab as e of th e g lo ba l e nv ir on m en t ht tp :// w w w .m np .n l/e n/ th em as ite s/ hy de /i nd ex .h tm l n et he rl an ds en vi ro nm en ta l a ss es sm en t a ge nc y n o d c n o a a n at io na l o ce an og ra ph ic d at a c en te r ht tp :// w w w .n od c. no aa .g ov / n o a a ta bl e 1. ( co nt in ue d) . 70 n. sillero and p. tarroso large temporal interval: 1 and 3 mss, 1973-1976; 4 and 5 mss, 1984-1987; 4 and 5 tm, 1984-2003; 7 etm+, 1999-2003. the landsat 5 tm and 7 etm+ are currently operative, but with some important problems hampering to record images in a correct way. the vegetation site offers global data on 10 days-temporal series of normalized difference vegetation index (ndvi), with a spatial resolution of 1 km. vegetation is a sensor of spot satellite, recording daily ndvi data. there are other sites providing data from satellite sensors. for instance, the largest temporal series of ndvi (from 1989 to present) comes from the advanced very high resolution radiometer (avhrr), located at earthexplorer website (together with other satellite data). modis also offers data on vegetation indexes, surface temperature and land cover. the global land cover facility focus on research using satellite data to assess land cover change for local to global systems. proprietary and free/opern source gis softwares currently, proprietary packages (e.g., arcgis 9.3 by esri www.esri.com and idrisi version taiga by clark labs www.clarklabs.org) seem to be the first choice for herpetologists (and biologists, in general). in fact, arcgis family products is one of the most used gis softwares. arcgis is an integrated collection of gis software products for building and deploying a complete gis (i.e., with vector and raster functions). the variety of arcgis applications can run on a great range of hardware, from simple desktops to servers and handhelds, providing gis tools wherever needed. in the last years, many government agencies have adopted esri’s standard vectorial format (shapefile) as the common format for their geographical products. there is also an open source format: the geography markup language (gml), a grammar defined by the open geospatial consortium (ogc) to express geographical features, including not only conventional vector data, but also coverages and raster data. nowadays there is a large availability of free gis softwares, most of them open source and released under the terms of the gpl, running on a variety of platforms (windows, linux, mac os). one of the oldest gis software is grass (geographic resources analysis support system: grass.osgeo.org; current version is 6.4.0 rc5) originally developed by the us army construction engineering research laboratories as a tool for land management and environmental planning, and currently maintained as foss. grass is oriented for raster analysis, including tools for spatial modelling, visualization, management and processing of satellite imagery. the analysis of vectorial data improved in the newer versions. grass has been under continuous development since 1982, involving a large number of federal us agencies, universities, and private companies. other foss is quantum gis (qgis: www.qgis.org; version 1.4.0 enceladus), which supports vector and raster formats. qgis includes some spatial analysis tools for vectorial data and a grass plug-in, allowing the use of a full grass installation within qgis. this software can easily connect to a postgis/postgresql database server to retrieve geographical information. openmodeller (openmodeller.sourceforge.net) is a qgis based project specifically oriented at species distribution modelling. in fact, it is a heavily modified qgis, with several modelling tools added using the native qgis plugin mechanism. 71free gis for herpetologists gvsig (www.gvsig.gva.es; version 1.9) is also a vectorial gis capable of using most common vector and raster formats. grass, qgis and ilwis are also able to read (and partially write) almost all known gis vector and raster formats, since they rely on frank warmerdam gdal/ogr interoperability library, which gvsig uses in part. sextante (www.sextantegis.com; version 0.55) provides gvsig with both raster and vector geographical analysis capabilities with over 150 extensions for geo-statistics and hydrological analysis, creation of vegetation indexes and profiles. it is also provided a lighter version of gvgis for mobile devices. both qgis and gvsig work in several operating systems. grass, qgis and gvsig can read geographical data from remote sources through wms1, wcs2 or wfs3 standard protocols. the integrated land and water information system (ilwis; www.itc.nl/ilwis/default. asp; version 3.6) is a windows-based raster gis and remote sensing software, developed commercially by the itc (faculty for geo-information science and earth observation of the university of twente, the netherlands) up to its last release (version 3.4) and since 2007 is maintained as foss. ilwis comprises a complete package of image processing, spatial analysis and digital mapping. diva-gis (version 7.1.7) is focused on vector analysis and developed particularly for mapping and analysing biodiversity data. it provides tools to predict species distributions using the bioclim or domain models. there are other softwares mainly for visualizing vector and raster format such as kosmo by saig (sistemas abiertos de información geográfica; www.saig.es), gaia (www.thecarbonproject.com/gaia.php), openjump (www.openjump.org) and arcreader (www.esri.com). we listed in the appendix 1 the most commonly used functions in gis and their availability in the six software compared in this study (arcgis 9.3, gvsig 1.9, ilwis 3.6, quantum gis 1.4.0, grass 6.4.0 rc5, and diva-gis 7.1.7). these functions are explained in appendix 2 and have been separated in three groups: vector (with 59 functions), raster (with 58) and typographic and layout production (with 12). as explained above, most of gis programs are initially designed to work exclusively in either the vector or raster domain, with some capability to operate in the other. therefore, a particular software could have a high number of functions for a specific format, but none or a few in the other. for example, the former versions of gvsig had several vector functions and few raster functions. our intention is to rank the six gis software by the number of available functions in each groups. the highest ranked software will be the software with more functions available. arcgis 9.3 occupies the first position (table 2), thus it is the software that presents most of the tools considered here. arcgis has a modular structure, i.e., the functions are included in several modules. to use all the functions, the user need to buy all modules. for example, raster functions are included in the module spatial analyst and without this module, arcgis is mainly a vector-oriented gis: it can read raster files just as images and 1 the open geospatial consortium (ogc) defines a web map service (wms) as a standard protocol for serving georeferenced map images over the internet that are generated by a map server using data from a gis database. 2 the opengis® web coverage service interface standard (wcs) is a standard interface and operations that enables interoperable access to geospatial coverages, as defined by the ogc. 3 the open geospatial consortium web feature service interface standard (wfs) provides an interface allowing requests for geographical features across the web, using platform-independent calls, as defined by the ogc. 72 n. sillero and p. tarroso not as true rasters, without any processing capability except for colour palette adjustments and false-colour band assignment, and even the geoprocessing of vectorial data is limited in the most basic license with native tools. however, this modular scheme is a advantage for proprietary software: the user can buy only the packages that cover his needs and does not have to buy other specific tools. nevertheless, arcgis is capable of use external tools written in several programming languages (i.e., visual basic for applications, python) enhancing its range of application. a high number of these scripts are free and available at esri’s website (http://arcscripts.esri.com). therefore, some functions not available in arcgis out of the box can be performed through an external script. gvsig 1.9 ranks second, with 106 functions in total. it has also a modular structure, with freely available modules. one of the major advantages is the ability of reading directly several formats (without importing them previously), both vector and raster, and process them. it has good graphical production capabilities. despite being mainly focused on territory management, gvsig has enough functions for ecological analysis. qgis 1.4.0 is a good software for visualizing data and is capable of reading a large number of file formats. it has many functions for spatial analysis with the grass plug-in, although it only works directly with grass formats (without importing data previously through gdal/ogr library). it has good graphical production capabilities, including tools to build scales, legends and it works with several maps. this last version includes many vectorial tools, for analysis, management, research and geoprocessing. it also has a tool to import scripts to add new functions from several repositories. these features, and especially the user-friendly interface, extends the potential use of this software. grass 6.4.0 rc5 and ilwis 3.6 have many functions, particularly oriented towards rasters and imagery processing. grass and ilwis can read many formats through the gdal/ogr library. however, grass has a well defined project that often results in duplication of data. while the grass project may present several advantages for advanced users, the inexperienced user will find difficult to apprehend the grass paradigm. for users with large databases of spatial data on other formats, this limitation can hamper the use of these foss gis. grass works with projects that have a previously user defined extent (before entering data) and where external files should be imported. the grass location/mapset paradigm avoids tampering with projections and datums, and it is the only physical representation explicitly designed to allow true concurrent, multi-user access. a common mistake in esri products is not to define the raster analysis environment; the user can mix rasters with different cell sizes and awkward coordinate table 2. global results of available functions on gis softwares. see in appendix 1, table 3 for vector functions, table 4 for raster functions and table 5 for layout functions. see appendix 2 for function definitions. software versions: arcgis 9.3; gvsig 1.9; ilwis 3.6; qgis 1.4.0; grass 6.4.0 rc5; diva 7.1.7. arcgis gvsig ilwis qgis grass diva vector 56 50 28 50 38 15 raster 46 46 38 37 40 23 layout 12 10 9 7 60 6 total 114 106 75 94 84 44 73free gis for herpetologists reference systems, which could cause severe errors not only in map algebra but also in geostatistical applications. grass data structures instead completely eliminate the problem. on the other hand, ilwis is not capable of working with very large files. overall, these packages are very useful for ecological analysis. diva-gis 7.1.7 is a software oriented specifically to biogeography and diversity studies. despite a small number of functions (15 vector, 23 raster and six layout functions), those are extremely useful in biogeography studies. furthermore, some functions are exclusive to diva-gis (e.g. similarity indexes), constituting a good complement to other software. briefly, gvsig is the foss with more functions (106) followed by qgis with 94 and grass with 84. ilwis has 75 functions. we can considered gvsig the most complete foss specially for its capacity of working directly with several data formats out of the box, both vector and raster files. qgis has more functions than grass thanks to the new vector tool plug-in included in the last version (1.4.0 enceladus). final remarks events like the elimination of selective availability in gps devices in 2000 with the increase of signal quality and precision, the increased performance of altitude data with the shuttle radar topography mission in 2000, and the public availability of all archives of landsat satellites in 2009 were very important to support the foss philosophy, firstly proposed by richard m. stallman, as explained above. science is the most important motor providing progress to the humanity. to achieve this objective, gathered data should be made available publicly and freely. in the scope of this review, we should emphasize some of the advantages of working with free/open source software (foss): most of the packages presented here interact with each other and with other foss (e.g., server databases, statistical packages) and communicate with open formats, common to a huge range of software (e.g., graphical production software). this constitutes a vast collection of functions that cover virtually all potential applications to ecology. moreover, most of the foss is being continuously updated and upgraded with new functionalities. this increases the difficulty of ranking gis software, due to the fact that the evaluation of the presence of a particular feature is not straightforward: some software do not present an easy access to a feature, and it does not means the feature is not present trough a command line, for instance. therefore, the rank presented in this review reflects the user-friendliness of the gis packages. this review does not cover functionalities neither software for more advanced users. one example of a function of this type is the use of a command line for batch processing. this function is common between arcgis, gvsig, grass and ilwis, for instance, and is extremely useful for processing/production of large datasets. the use of the command line and scripts can overcome the apparent limitations of a software. for a paradigmatic example, the r statistical computing environment (r development core team, 2009) is a free programming language widely used in science and has modules for spatial and geostatistical analysis. one great advantages of this language is the advanced control of the processing, wide range of available modules and, as an example, the interaction with grass. 74 n. sillero and p. tarroso acknowledgements ns is supported by a postdoctoral grant (sfrh/bpd/26666/2006) and pt by a doctoral grant (sfrh/bd/42480/2007), all from fundação para a ciência e tecnologia (fct, portugal). a previous version of the manuscript was improved considerably by damiano g. preatoni and stefano scali. references anadon, j.d., gimenez, a., martinez, m., martinez, j., perez, i., esteve, m.a. 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(2006): evolutionary and ecological causes of the latitudinal diversity gradient in hylid frogs: treefrog trees unearth the roots of high tropical diversity. am. nat. 168: 579-596. appendix 1 table 3. list of vectorial functions, grouped by similar characteristics. six softwares were analysed: one proprietary (arcgis 9.3); and five foss (gvsig 1.9, ilwis 3.6, quantum gis 1.4.0, grass 6.4.0 rc5 and diva-gis 7.1.7). we counted the number of functions available per software. the most complete software will be the software with more available functions. see in this appendix, table 4 for raster functions and table 5 for layout functions. see table 2 on manuscript for global results. see appendix 2 for function definitions. vector arcgis gvsig ilwis qgis grass diva basic operations shapefile reading yes yes yes yes import/export shapefiles yes yes yes yes yes yes vector to raster yes yes yes yes yes yes vector projection yes yes yes yes yes yes attributes reading yes yes yes yes yes yes modeller yes yes yes total 6 6 5 5 4 5 map digitalization vector digitalization yes yes yes yes yes vector edition yes yes yes yes yes image georeferencing yes yes yes yes yes yes total 3 3 3 3 3 1 selection selection by attributes yes yes yes yes yes yes selection by location yes yes yes yes total 2 2 1 2 2 1 (continued) 79free gis for herpetologists vector arcgis gvsig ilwis qgis grass diva tables operations table edition yes yes yes yes yes table calculator yes yes yes yes yes table statistics yes yes yes yes yes yes table join yes yes yes yes yes table link yes yes yes summarize tables yes graphs yes yes yes total 7 6 6 4 4 1 point operations points to lines yes points to polygons yes yes add data points yes yes yes yes yes yes calculate coordinates yes yes yes vectorial buffers yes yes yes yes merge yes yes yes yes yes dissolve yes yes yes yes clip yes yes yes yes break features yes yes yes convex hull yes yes yes yes yes voronoi tessellation yes yes yes yes delaunay tessellation yes yes yes yes select at random yes yes yes autocorrelation yes yes yes yes total 14 10 4 11 8 5 polylines operations lines to polygons yes yes yes yes yes lines to points yes yes yes yes yes vectorial buffers yes yes yes yes merge yes yes yes yes yes dissolve yes yes yes yes clip yes yes yes yes vectorial grids yes break features yes yes yes yes generalization yes yes yes yes total 8 8 4 9 7 0 polygon operations polygons to points yes yes polygons to lines yes yes yes yes yes (continued) 80 n. sillero and p. tarroso vector arcgis gvsig ilwis qgis grass diva intersection yes yes yes yes union yes yes yes yes difference yes yes yes yes vectorial buffers yes yes yes yes merge yes yes yes yes dissolve yes yes yes yes clip yes yes yes yes spatial join yes yes yes yes vectorial grids yes yes yes centroids calculation yes yes yes yes count points in polygons yes yes calculate area yes yes break features yes yes yes total 13 12 4 14 9 1 network analysis euclidean distance yes yes yes yes cost distance yes yes nearest neighbour yes yes yes yes yes total 3 3 1 2 1 1 total 56 50 28 50 38 15 table 4. list of raster functions, grouped by similar characteristics. see table 3 for more information. see in this appendix, table 3 for vector functions and table 5 for layout functions. see table 2 on manuscript for global results. see appendix 2 for function definitions. software versions: arcgis 9.3; gvsig 1.9; ilwis 3.6; qgis 1.4.0; grass 6.4.0 rc5; diva 7.1.7. raster arcgis gvsig ilwis qgis grass diva basic operations import/export ascii yes yes yes yes yes yes import/export raw data yes yes yes yes yes yes raster to vector yes yes yes yes yes raster projection yes yes yes yes pixel information yes yes yes yes yes yes raster stacks yes yes yes fusion bands yes yes yes histogram yes yes yes yes colour table yes yes yes yes yes radiometric enhanced yes yes yes total 9 9 8 7 7 5 (continued) 81free gis for herpetologists raster arcgis gvsig ilwis qgis grass diva spatial basic analysis raster algebra yes yes yes yes yes yes buffer rasters yes yes yes yes raster descriptive statistics yes yes yes yes yes yes random value rasters yes yes yes yes clip rasters yes yes yes yes raster spatial join yes yes values composites yes yes yes neighbourhood statistics yes yes yes yes yes zonal statistics yes yes aggregate yes yes yes yes yes yes resample yes yes yes yes mosaic yes yes yes yes yes total 12 9 7 8 8 7 raster statistics raster correlation yes yes yes yes yes yes raster regression yes yes yes yes yes raster multiple regression yes yes yes raster pca yes yes yes yes raster factor analysis yes yes cluster analysis yes yes yes fit distribution analysis yes total 4 6 4 2 4 4 raster comparisons cross-tabulation yes yes yes yes kappa yes yes yes yes similarity analysis yes roc analysis yes yes total 0 1 2 2 2 4 image analysis reclassification yes yes yes yes yes yes masks yes yes yes yes yes filters yes yes yes yes yes raster classification yes yes yes yes accuracy assessment vegetation indexes yes yes image transformations yes yes yes density slicing yes total 4 6 6 4 5 1 (continued) 82 n. sillero and p. tarroso raster arcgis gvsig ilwis qgis grass diva interpolation semivariogram yes yes yes idw yes yes yes yes yes spline yes yes yes kriging yes yes yes trend surface yes yes yes yes yes tin yes yes yes total 6 5 4 4 3 0 digital elevation models slope yes yes yes yes yes yes aspect yes yes yes yes yes hillshading yes yes yes yes visibility analysis yes yes yes yes contour analysis yes yes yes yes 3-d visualization yes yes yes yes total 6 5 2 6 6 2 hydrology flow analysis yes yes yes yes yes direction analysis yes yes yes yes yes watershed analysis yes yes yes yes yes fill analysis yes yes yes yes yes stream calculation yes yes yes yes total 5 5 5 4 5 0 total 46 46 38 37 40 23 table 5. list of layout functions, grouped by similar characteristics. see table 3 for more information. see in this appendix, table 3 for vector functions and table 4 for raster functions. see table 2 on manuscript for global results. see appendix 2 for function definitions. software versions: arcgis 9.3; gvsig 1.9; ilwis 3.6; qgis 1.4.0; grass 6.4.0 rc5; diva 7.1.7. layout arcgis gvsig ilwis qgis grass diva basic operations export a map yes yes yes yes yes yes palette colours yes yes yes yes yes yes scale yes yes yes yes yes yes north arrow yes yes yes yes yes yes legend yes yes yes yes yes text yes yes yes yes yes yes (continued) 83free gis for herpetologists layout arcgis gvsig ilwis qgis grass diva figure yes yes yes yes total 7 7 7 7 5 6 advanced operations insert several maps yes yes yes advanced text yes yes freehand text yes insert grid yes yes insert geogrid yes yes yes total 5 3 2 0 1 0 total 12 10 9 7 6 6 appendix 2 vector functions shapefile reading: reads directly files in shapefile format, without importing them previously. import/export shapefiles: imports and/or exports shapefiles. vector to raster: transforms vector files to raster files. vector projection: transforms a vector file from its original projection to another one. attributes reading: reads the attributes stored in a table. modeller: functions can be concatenated and stored for future uses. vector digitalization: transforms data on a physical map or an image to a vector file. vector edition: modifies the topology of a vector file. image geo-referencing: correctly places an image inside a given coordinate reference system. selection by attributes: selects entities and attributes using boolean operators on an attribute table. selection by location: selects entities and attributes using topological criteria. table edition: modifies table values. table calculator: performs mathematical calculations among table fields. table statistics: descriptive statistics of table fields. table join: joins two tables through a common field. table link: the values of a table are read in a second table. summarize tables: applies an aggregation function such as sum, mean, count, to a field by grouping its values based on unique values from another field. graphs: table attributes can be represented in a graph. points to lines, points to polygons; lines to points, lines to polygons; etc: transforms a specific type of vector file to another type. add data points: imports a list of coordinates to a point vector file. calculate coordinates: adds coordinates to the attribute table of a point vector file. vectorial buffers: calculates polygons of constant distance around points, lines or polygons. merge: joins several vector files of the same type (point, line or polygon). dissolve: joins item of a vector file by the same attribute. clip: extracts a part of a vector file with a polygon. break features: exports each item of a point, line or polygon vector file to a different and independent file. 84 n. sillero and p. tarroso convex hull: calculates the minimum convex polygon enclosing a group of points. voroni/delaunay: two particular types of vectorial interpolations. select at random: selects a sample of points from a file. autocorrelation: calculates the correlation among the points of the same file. vectorial grids: builds rectangular polyline or polygon cartographical grids at constant distances. generalization: reduces nodes in a line or polygon vector, preserving its attributes but yielding a coarser geometry. intervals are generally not regular, since generalisation algorithms must leave as much nodes as possible to preserve the original curvature of an entity. intersection: joins the spatially common parts of two vector files in only one file. union: joins two vector files in only one file, either spatially common or non-common parts. difference: joins the not common parts of two vector files in only one file. spatial join: transfers attributes between two vectors (of any type) based on coincident locations. centroids calculation: calculates the central point of a polygon. count points in polygons: counts the number of points inside the limits of a polygon. calculate area: adds the perimeter/area values to the items of a polygon table file. euclidean distance: calculates the euclidean distance between two points. cost distance: calculates the cost of going from a place to another place along a specific path depending on the value of an attribute. raster functions import/export ascii: imports/exports raster files in ascii format. import/export raw data: imports/exports raster files to other formats. raster to vector: transforms raster files to vector files. raster projection: transforms a raster file from its original projection to another one. pixel information: reads pixel values. raster stacks: includes several raster files or bands inside an unique file. raster algebra: performs mathematical operations among rasters. raster buffers: calculates a buffer of constant distance. raster descriptive statistics: descriptive statistics of pixel values. random value rasters: creates a raster with random values. clip rasters: extracts a part of a vector file with a polygon or another raster. raster spatial join: transfers the attributes of a raster file to a point file. values composites: calculates a new raster file from a list of raster using a mathematical function. neighbourhood statistics: quantifies and visualizes spatial variation in pixel. zonal statistics: calculates statistics on values of a raster within the zones of another dataset. aggregate: increases the size of pixels (and reduces the spatial resolution) using a mathematical operator. resample: changes the pixel size in the image. raster correlation: calculates the correlation among a group of rasters. raster regression: calculates a regression between two rasters. raster multiple regression: calculates a multiple regression between several rasters. raster pca: calculates a principal component analysis between two rasters. raster factor analysis: calculates a factorial analysis between two rasters. cluster analysis: calculates a cluster analysis between two rasters. fit distribution analysis: fits the values of a rasters to a specific distribution curve. data sampling: extracts randomly pixel values. autocorrelation: measures the spatial autocorrelation inside a raster. cross-tabulation: compares a group of raster by their common values (normally for categorical data). 85free gis for herpetologists kappa: a statistical measure of similarity between two raster. similarity analysis: index for measuring the similarity among values of two raster. reclassification: modifies pixel values using rules. masks: classifies a part of a raster as background. filters: modifies pixel values using mathematical operators in a square with a specific size. raster classification: statistical method for splitting a image in significant groups. accuracy assessment: method for validating a raster classification. vegetation indexes: indexes for detecting vegetation in satellite imagery. image transformations: transformations of satellite imagery, namely for classification purposes. density slicing: classifies a image by groups with the same number of pixels. semivariogram: function expressing the influence of the value of a spatial variable measured in a given point on its neighbours at increasing distances and at different directions. idw, spline, etc: different interpolation methods. slope: calculates the slope inside each pixel of a dem. aspect: calculates the cardinal orientation inside each pixel of a dem. hillshading: calculates a shade model from a dem. visibility analysis: calculates visible areas from a point. contour analysis: extracts lines connecting points at equal values (isopleths) from continuous raster data such as elevation or rainfall. 3-d visualization: visualizes a raster in a 3-d environment. flow analysis: calculates the amount of water flow through each pixel of a dem. direction analysis: calculates the direction of water through each pixel of a dem. watershed analysis: calculates the watersheds in a dem. fill analysis: eliminates depressions in a dem. stream calculation: calculates stream lines in a dem. layout functions export a map: produces a map which is exported in a particular image format or in pdf format. palette colours: the features inside the map can be represented with different colours and classify methods. scale: inserts a graphical scale in the map. north arrow: inserts a north arrow in the map. legend: inserts the legend of the features represented in the map. text: inserts text in the map. figure: inserts figures in the map. insert several maps: inserts several maps in the same sheet. advanced text: the text can be rotated. freehand text: the text can be placed over a curved segment. insert grid: inserts a non geo-referenced grid. insert geogrid: inserts a geo-referenced grid. acta herpetologica 9(2): 231-235, 2014 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-14209 maximum body size and age distribution in the italian stream frog, rana italica dubois 1987 (amphibia: anura) vincenzo buono1, fabio maria guarino2, leonardo vignoli1,3,* 1 dipartimento di scienze, università “roma tre”, viale g. marconi 446, 00146 rome, italy. *corresponding author. e-mail: leonardo. vignoli@uniroma3.it 2 dipartimento di biologia, università degli studi di napoli federico ii, via cinthia 21, 80126 naples, italy 3 center for evolutionary ecology, largo san leonardo murialdo, 1, 00146 rome, italy submitted on 2014, 21st march; revised on 2014, 5th may; accepted on 2014, 30th may editor: marco mangiacotti abstract. data on mean longevity and age structure of rana italica are very scanty. skeletochronological data comparisons with other european brown frogs showed that life span of r. italica is similar to those of other congeneric species. in this study we report the age structure of a population of r. italica from central italy with a new record of species maximum size. the study population inhabits a perennial stream in a lowland oak wood. all the individuals were marked by means of toe clipping. skeletochronological analyses showed that adult age ranged from 3 to 5 years in males and from 2 to 6 years in females with no intersexual difference. in our study, the oldest individual (estimated age was 6 years) was also the biggest (68 mm), representing the new body size record for the species. previous skeletochronological studies on r. italica have shown that the mean longevity for this species is four years but the maximum age recorded is 5 and 8 years in low and high elevation populations, respectively. the ages recorded for the study population inhabiting a hilly area showed higher average and maximum values than those found in lowland populations. keywords. rana italica, maximum size, age, skeletochronology. rana italica (dubois 1987) is an anuran amphibian belonging to the brown frogs group, widely distributed within the italian peninsula, at elevations ranging between 30 and 1400 m a.s.l. (picariello et al., 2006). despite its abundance and distribution, the knowledge about the ecology of this species is scanty and is mostly the result of general observation rather than that of specific studies. the italian stream frog inhabits small streams within the wet, broad-leaf forests of the apennines from central liguria to calabria (picariello et al., 2006). it usually prefers fast flowing streams with a rocky substrate but it also occurs in small ponds, mountain peat bogs and in some man-made habitats such as water troughs. adult males are smaller than adult females, with a maximum reported snout-to-vent length of 60 mm for males and of 65 mm for females (lanza et al., 2009). in addition, males are brownish-grey while females are reddish-brown. both males and females remain near stream pools for most of the year, using rocks on the bottom of deeper pools with permanent water as refuges against predators, as well as for mating and egg laying (lanza, 1983; picariello et al., 2006). breeding occurs from late january to early may and oviposition peaks in february-march depending on the altitude (guarino et al., 1993). the only data on mean longevity and age structure of rana italica derive from skeletochronological studies by guarino et al. (1995a, b) performed on two populations of southern italy. comparisons with skeletochronological data obtained from other european brown frogs show that life span of r. italica (8 yr) is similar to that of rana dalmatina (7 yr, guarino et al., 1995a; sarasola-puente et al., 2011), of rana iberica (9 yr; esteban and sanchiz, 2000) but much lower than that of rana temporaria (up 232 v. buono et alii to 17 yr; ishchenko, 1996; miaud et al., 1999; guarino et al., 2008) and of rana arvalis (11 yr; ishchenko, 1996). however, it should be taken into consideration that often inter-specific and inter-populational differences in longevity and age at maturity occur depending on latitude and/or altitude (guarino et al., 2003; 2008). in this study we report a new maximum body size record for r. italica and data on frog age from a population living at the neighbouring of rome (central italy) by using skeletochronological technique. we collected 101 individuals (40 females, 25 males, and 36 juveniles) of rana italica by dip netting at cineto romano (latium, central italy: 42°02′52.39′′n 12°56′39.35′′e, about 400 m a.s.l.) during the breeding season (march-april) of 2011. sex was determined on the basis of external morphology and the smallest frogs that did not show unequivocal secondary sexual characters were classified as juveniles. the study population inhabits an oak wood surrounding a small river in the lucretili mountains, immediately outside the lucretili regional park boundaries. all the frogs collected were measured from snout-to-vent (svl) and marked by means of toeclipping, this technique apparently not affecting frog survival (guarino et al., 2003). in order to assess the age of the frogs, the phalanxes from the third toe of the left hind leg from a representative sample of collected animals (18 males, 23 females, 11 juveniles) were fixed in 70% ethanol and analysed by means of the standard skeletochronological technique (guarino et al., 1995a; 1998). phalanxes were decalcified in 5% nitric acid for about 1h 15 min, rinsed with tap water and cross-sectioned at diaphyseal level using a cryostat. phalangeal sections, 12 μm thick, were stained with mayer’s acid hemalum for about 25 min and mounted in synthetic resin mounting media (hi-mo, bio-optica). in order to count the lines of arrested growth (lags) in the periosteal bone, for each phalanx, about twenty sections were observed under light microscope (motic ba 340) equipped with digital camera (nikon coolpix 5000). we assumed that in the phalanx of this species possible lags were deposited annually according to what was observed for other temperate frogs (paton et al., 1991; guarino et al., 2003). all the slides were examined and the age was estimated independently by two researchers (vb and fmg). in case of contrasted estimation made by the two observers, the individual was discarded from the analyses. all the collected animals were released at the place of capture just after measuring and toe-clipping. lags were identifiable in all examined phalanges. although the distinctiveness of lags was different from one animal to the other and often within a single bone section, age estimation by lags counting was possible in all individuals examined. age was assessed with an error rate of one year in 17% of the males (n = 3) and 26% of the females (n = 6) due to the presence of weakly stained lines interpreted by us as a lag but that alternatively could be interpreted as supplementary line (sensu castanet et al., 1993). in about 28% of males (n = 5) and 30% of females (n = 7), we encountered difficulties in distinguishing the most peripheral lags because too close to each other. we used non-parametric mann-whitney u-test to compare body size among adult frogs (performed on both all sampled individuals and those investigated by means of skeletochronology), and the spearman’s rank correlation to test the relationship between age and svl. all the statistical analyses were carried out by using spss software (version 17) with alpha set at 0.05. with regards to svl, sexes did not show significant differences (mean ± standard deviation, females: 52.1 ± 0.46 mm; males: 51.5 ± 0.31 mm; mann-whitney u-test, u = 442.5, n = 65, p = 0.438; fig. 1a). mean svl of juvenile was 29.5 ± 4.1 mm. concerning the skeletochronological analyses, all the juveniles resulted to be one-year old. adult age ranged from 2 to 6 years in females and from 3 to 5 years in males (fig. 1b) and did not significantly differ between the sexes (u = 149, p = 0.131). in both sexes, svl was highly correlated with age (spearman’s rank correlation, males: rs = 0.67, p = 0.004; females, rs = 0.79, p < 0.001) but a wide overlapping between the sizes of individuals from different age classes was found (fig. 2). among the largest adults, three exceeded 60 mm, and were all females. two of them measured 64.5 mm (4 and 5 years old, respectively) and the third 68.0 mm (6 years old; fig. 3). most anurans show sexual size dimorphism (in which males are usually smaller than females) due to sexual selection and different life-history traits between genders (monnet and cherry, 2002). in our sample males were slightly older than females and this could explain the lack of size dimorphism found between sexes. the maximum body length of rana italica so far reported was 65 mm in females and 60 mm in males (lanza et al., 2009). therefore, the svl of 68 mm measured on a female in this study represents a new record of maximum length for this species. it is also noteworthy that we observed several other big-sized individuals (two females with svl of 64 mm and three males with svl close to 60 mm). a possible explanation of our findings is that the study population is very long-lived for reasons yet to be ascertained. it is known that amphibians have indeterminate growth but their body size is not always a reliable indicator of age because a widespread extensive overlap in body size among age classes (halliday and ver233maximum size in rana italica rel, 1988; guarino and erismis, 2008). on the contrary, skeletochronology has proven to be one of the most reliable techniques to assess individual age and growth in amphibians (castanet et al., 1993) and it is well applicable also to r. italica, as confirmed in this as well as in previous studies (guarino et al., 1995a, b). previous skeletochronological studies on r. italica have shown that the mean longevity for this species was four years and the maximum age recorded was 5 and 8 years in low and high elevation populations, respectively (guarino et al., 1995b). in our study, the oldest individual was also the biggest and its estimated age was 6 years. we did not recorded adult males younger than three years, this lack likely being due to research defect. the ages recorded for the study population inhabiting a hilly area, on average, are higher than those found for the species in lowland populations (guarino et al., 1995b). for comparison, we analysed the body size reported for another amphibian species, salamandrina perspicillata, well studied in its whole range from a biometric point of view (romano and ficetola, 2010), living syntopically with r. italica at the study site. the population of s. perspicillata from the study site differed in body size with respect to other 10 salamander populations from central italy sited at comparable elevation (range 240500 m a.s.l.) and reported in romano and ficetola (2010) and vignoli et al. (2011, 2012) (cineto population: mean fig. 1. body size (a) and age (b) assessed by lags counting of rana italica from the study site. data on svl refer to 52 individuals selected for skeletochronological analysis (juveniles: n = 11; males: n = 18; females: n = 23). data on age refer to adult individuals only. fig. 2. correlation between age classes and body size (svl) in juvenile (plus), female (circles), and male (crosses) individuals of rana italica from the studied population. fig. 3. phalangeal cross-section of female of rana ialica, 68.3 mm svl. arrows show lines of arrested of growth; d: double lines, mc: marrow cavity. bar scale: 110 µm. 234 v. buono et alii svl + sd = 38.6 ± 2.6 mm; other populations: mean svl range = 30.9-41.7 mm). interestingly, the population from cineto showed a statistically significant larger size in eight comparisons (80%), smaller size in one comparison, and non-significant difference in one comparison, with no altitudinal trend (rs = 0.261; n = 11; p = 0.465). the observed body size pattern (i.e., larger body size in comparison to other population at comparable elevation at the same latitude) shared by two amphibians would deserve further in depth analyses. as a general rule, among anurans from temperate areas, intraspecific large variations in life history traits such as body size, age at maturity and mean longevity occurs depending on latitudes or altitudes, which cause different climatic influences (hemelaar, 1988; sagor et al., 1998; miaud et al., 1999). however, large difference in body size, growth rates and age at maturity may also occurs between neighbouring populations living at similar altitudes and experiencing similar climatic conditions, as shown for r. temporaria (augert and joly, 1993). in this case, it was hypothesized that the inter-populational differences are related to a difference in the quality of the environment surrounding the water basins. unfortunately, demographic information about this taxon is scanty and we do not know if other populations with large individuals occur in its areal. furthermore, because few data are available for young frogs (i.e., no males were younger than 3 years), maybe due to a sampling bias, the mean age values obtained in the studied population could be biased towards higher life expectancy when compared with populations of r. italica of similar altitude and latitude. further studies deepening the potential causal relationships between demographic patterns and ecological parameters are in progress to clarify this point. acknowledgements we would like to thank dr. alessandra bissattini for her invaluable help in the field, and three anonymous referees for their useful suggestions. the italian ministry of the environment gave us authorization for the temporary capture of amphibians (authorization dpn-20090005106). no animals were damaged or killed during this project, and all were handled according to the standards of the italian ministry for scientific research and technology. l. vignoli is gratefully inspired by roger federer, whose very effective choice to change the wilson tool for doing the job is really appreciated, and dedicated this paper to his friends aldo the apache for his determination in fighting alien species in the 40’s and dr. king schultz for his honesty and his passion for siegfried. references augert, d., joly, p. (1993): plasticity of age at maturity between two neighbouring populations of the common frog (rana temporaria l.). can. j. zool. 71: 26-33. castanet, j., francillon-vieillot, h., meunier, f.j., ricqlès, a.de (1993): bone and individual aging. in: bone growth, pp. 245-283. hall, b.k., ed., crc press, boca raton. della rocca, f., vignoli, l., bologna, m.a. (2005): the reproductive biology of salamandrina terdigitata (caudata, salamandridae). herpetol. j. 15: 273-278. denton, j.s., beebee, t.j.c. (1993): reproductive strategies in a female-biased population of natterjack toads, bufo calamita. anim. behav. 46: 1169-1175. esteban, m., sanchiz, b. (2000): differential growth and longevity in low and high altitude rana iberica (anura, ranidae). herpetol. j. 101: 19-26. guarino, f.m., di fiore, m.m., caputo, v., iela, l., angelini, f., rastogi, r.k. 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(2003): a skeletochronological study of growth, longevity and age at sexual maturity in a population of rana latastei (amphibia, anura). j. biosci. 28: 775-782. guarino, f.m., erismis, u. (2008): age determination and growth by skeletochronology of rana holtzi, an endemic frog from turkey. ital. j. zool. 75: 237-242. guarino, f.m., di già, i., sindaco, r. (2008): age structure by skeletochronology in a declining population of rana temporaria from northern italy. acta zool. acad. sci. hung. 54: 99-112. halliday, t.r., verrell, p.a. (1988): body size and age in amphibians and reptiles. j. herpetol. 22: 253-265. hemelaar, a. (1988): age, growth and other population characteristics of bufo bufo from different latitudes and altitudes. j. herpetol. 22: 369-388. 235maximum size in rana italica ishchenko, v.g. (1996): demography and declining populations of some euroasiatic brown frogs. russian j. herpetol. 3: 143-151. lanza, b. (1983): anfibi, rettili: (amphibia, reptilia). guide per il riconoscimento delle specie animali delle acque interne italiane, 27. centro nazionale delle ricerche, verona. lanza, b., nistri, a., vanni, s. (2009): anfibi d’italia. quaderni di conservazione della natura. vol. 29. ministero dell’ambiente e della tutela del territorio e del mare, i.s.p.r.a., grandi e grandi ed., modena. miaud, c., guyetant r., elmberg, j. (1999): variations in life history traits in the common frog rana temporaria (amphibian: anura): a literature review and new data from the french alps. j. zool. 246: 61-73. monnet, j.m., cherry, m.i. (2002): sexual size dimorphism in anurans. proc. r. soc. lond. b 269: 23012307. paton, d., juarranz, a., sequerosm, e., perez-campo, r., lopez-torres, m., barja de quiroga, g. (1991): seasonal age and sex structure of rana perezi assessed by skeletochronology. j. herpetol. 25: 389-394. picariello, o., guarino, f.m., bernini, f. (2006): rana italica dubois, 1987. in: atlante degli anfibi e rettili d’italia/atlas of italian amphibians and reptiles, pp. 358-361. sindaco, r., doria, g., razzetti, r., bernini, f., eds, societas herpetologica italica, edizioni polistampa, firenze. romano, a., ficetola, f.g. (2010): ecogeographic variation of body size in the spectacled salamanders (salamandrina): influence of genetic structure and local factors. j. biogeogr. 37: 2358-2370. sagor, e.s., quellet, m., barten, e., green, d.m. (1999): skeletochronology and geographic variation in age structure in the wood frog, rana sylvatica. j. herpetol. 32: 469-474. sarasola-puente, v., gosa, a., oromi, n., madeira, m.j., lizana, m. (2011): growth, size and age at maturity of the agile frog (rana dalmatina) in an iberian peninsula population. zoology 114: 150-154. vignoli, l., silici, r., brizzi, r., bologna, m.a. (2010): in vivo sexual discrimination in salamandrina perspicillata: a cross-check analysis of annual changes in external cloacal morphology and spermic urine release. herpetol. j. 20: 17-24. vignoli, l., silici, r., bissattini, a.m., bologna, m.a. (2012): aspects of olfactory mediated orientation and communication in salamandrina perspicillata (amphibia, caudata): an experimental approach. ethol. ecol. evol. 24: 165-173. © firenze university press www.fupress.com/ah acta herpetologica 5(2): 207-215, 2010 electrophoretic comparison of blood-serum proteins of apathya cappadocica (sauria, lacertidae) subspecies from anatolia çetin ilgaz1, hüseyin arıkan2, yusuf kumlutaş3, aziz avcı4 ¹ dokuz eylül university, fauna and flora research and application center, 35160, buca-i̇zmir, turkey. correspondig author. e-mail: cetin.ilgaz@deu.edu.tr ² ege university, faculty of science, department of biology, section of zoology, 35100, bornova-i̇zmir, turkey e-mail: huseyin.arikan@ege.edu.tr 3 dokuz eylül university, faculty of education, department of biology, 35160, buca-i̇zmir, turkey. e-mail: yusuf.kumlutas@deu.edu.tr 4 adnan menderes university, science and art faculty, department of biology, 09010, aydın-turkey. e-mail: aavci@adu.edu.tr submitted on: 2010, 26th july; revised on: 2010, 26th august; accepted on: 2010, 8th october. abstract. blood-serum proteins of the known subspecies of apathya cappadocica (werner, 1902) were studied comparatively by polyacrylamide disc gel electrophoresis. in order to obtain useful biochemical data for classification, differences between the electrophoreograms of the samples included in the morphologically different subspecies were distinguished qualitatively and quantitatively. these comparisons indicated that electrophoretic results supported morphological discrimination of the known subspecies of a. cappadocica. keywords. apathya cappadocica, blood-serum proteins, lacertidae, anatolia. introduction apathya is a small genus of lacertid lizard including 2 species [apathya cappadocica (werner 1902) and apathya yassujica (nilson, rastegar-pouyani, rastegar-pouyani and andrén 2003)] found in central, east, south and southeastern anatolia, northern iraq, and west iran (eiselt, 1979; baran and atatür, 1998; nilson et al., 2003; arnold et al., 2007). a. cappadocica was first described as lacerta cappadocica from erciyes mountain in kayseri, turkey (werner, 1902). a. cappodocica is a polytypic species and includes five subspecies [a. c. cappadocica (werner, 1902) – type locality: erciyes mountain, turkey; a. c. urmiana (lantz-suchow, 1934) – type locality: 20 km sw of rezaiyeh, iran; a. c. wolteri (bird, 1936) – type locality: 16 km w of gaziantep, turkey; a. c. muhtari (eiselt, 1979) – type locality: 26 km sw of bitlis, turkey and a. c. schmidtlerorum (eiselt, 1979) – type 208 ç. ilgaz et alii locality 10 km s of diyarbakır, turkey] (lantz and suchow, 1934; bird, 1936; eiselt, 1979; baran and atatür, 1998; sindaco et al., 2000). after a detailed study by eiselt (1979), regional studies on morphology and ecology of a. cappadocica in its distribution sites were conducted (bischoff and schmidtler, 1994; schmidtler and bischoff, 1995; schmidtler, 1997; anderson, 1999; kumlutaş and olgun, 1999). schmidtler and bischoff (1995) examined specimens separated into four groups collected in gaziantep, hatay, adana and mersin in terms of pholidolial characteristics. they found considerable differences between the specimens dependent on climatic conditions in terms of examined pholidolial characteristics. they finally stated that colorpattern features of the specimens belonging to three known subspecies a. c. wolteri, a. c. muhtari and a. c. schmidtlerorum show variability dependent on their habitats. the taxonomical status of known subspecies of a. cappadocica is doubtful. there was no information on blood serum electrophoretic pattern in the comparison between the known subspecies of a. cappadocica. in order to confirm the present taxonomical status of known subspecies of a. cappadocica, it is necessary to obtain further information outside of morphological studies and the purpose of the present study is to identify the similarities and differences of the patterns of blood serum proteins in known subspecies of a. cappadocica. materials and methods adult male and female specimens of a. cappadocica were collected from known distribution sites of each subspecies in the southern and southeastern part of turkey between 10 june to 15 june 2005 by y. kumlutaş, ç. ilgaz and a. avcı. the specimens were transferred to the laboratory to obtain blood samples for electrophoretic analysis. after obtaining blood samples they were fixed with 5% formaldehyde in 70% ethanol and then preserved in 70% ethanol according to the method described by başoğlu and baran (1977). collection numbers (zdeu, zoology department of ege university) were given to the specimens kept in the lab. of the department of biology at buca education faculty of dokuz eylül university. data on specimens used for electrophoretic analysis is given in table 1. all blood-serum protein study specimens were of similar length, i.e. they were of similar age. table 1. data on the specimens of a. cappadocica used for electrophoretic analysis (n: number of specimens) subspecies n locality collection date altitude (m) coordinates a. c. cappadocica 2 (1♂ 1♀) pozantı, adana, turkey 10.06.2005 1210 n 3733292 – e 3458453 a. c. wolteri 2 (1♂ 1♀) between kilis and hassa 32. km, kilis, turkey 11.06.2005 540 n 3650280 – e 3707517 a. c. urmiana 2 (1♂ 1♀) hasankeyf, batman, turkey 15.06.2005 479 n 3742742 – e 4124547 a. c. muhtari 2 (1♂ 1♀) küçükalanlı village, şanlıurfa, turkey 12.06.2005 799 n 3710524 – e 3838335 a. c. schmidtlerorum 2 (1♂ 1♀) between diyarbakır and siverek, diyarbakır, turkey 14.06.2006 1058 n 3750897 – e 3942794 209comparison of blood-serum proteins of apathya cappadocica for electrophoretic analysis, blood samples were obtained from two specimens (one male and one female) for each subspecies of a. cappadocica. blood samples were obtained from the postorbital sinuses of living specimens via heparinized hematocrit capillary tubes according to the method described by maclean et al. (1973). samples were centrifuged for 5 minutes at 600 g and were stored in equal amounts (4 μl) at -20°c for each separation until analysis. blood-serum samples were separated using polyacrylamide-disc electrophoresis according to davis (1964), slightly modified by özeti and atatur (1979). electrophoretic separations were carried out at room temperature (20-25°c) with a canalco model 1200 electrophoresis apparatus. separation gels were first stained with 0.5% amido black, and then de-stained passively with repeated 7% acetic acid baths. gels were qualitatively evaluated directly from the electrophoretograms and densitometric tracing curves of the separations were obtained using a gelman acd-15 model 39430 densitometer scanning at 500 nm. results all specimens examined were sexually mature and no obvious difference was recorded in serum protein phenograms (in densitometric curves) between sexes in all subspecies. consequently, the specimens were pooled by sex for further evaluation. we also pooled the same aliquots of serum together and so this situation is important for the densitometric quantitative analysis. significant differences were established among the subspecies from the viewpoints of fraction numbers, electrophoretic mobilities and densities of the blood proteins which suggests that all subspecies of a. cappadocica are clearly distinct at the subspecific level (fig. 1). gel electrophoretograms of the blood protein samples of the five subspecies are shown in fig. 1. the gel electrophoretograms of blood-serum proteins of a specimen from each subspecies, together with their densitometric tracing curves, are shown in figs 2, 3, 4, 5 and respectively. in a. c. cappadocica, a. c. wolteri and a. c. urmiana the blood protein fractions were divided into 14 fractions or fraction groups (1 albumin-like fraction and 1 postalbuminlike fraction at albumin region and 12 globulin-like fractions at globulin region) while the total number of fractions or fraction groups were found to be 12 (2 albumin-like fractions and 1 postalbumin-like fraction at albumin region and 9 globulin-like fractions at globulin region) in a. c. schmidtlerorum. finally the total blood protein fractions were divided into 11 fractions or fraction groups (1 albumin-like fraction and 1 postalbumin-like fraction at albumin region and 9 globulin-like fractions at globulin region) (figure 2-6). although all subspecies except a. c. schmidtlerorum show similarity in terms of albumin fractions, the fractions matched to each other at the globulin region show both qualitative and quantitative differences among all subspecies. discussion many researchers have highlighted the taxonomic importance of the number of fractions, and the mobility and density of blood-serum proteins obtained from electrophoretic separation (dessauer and fox, 1956; chen, 1967; ferguson, 1980; arıkan, 1990; arıkan et 210 ç. ilgaz et alii al., 1998, 1999; kumlutaş et al., 2007). ferguson (1980) stated that while the quantitative difference of fractions could reflect gender, age, environmental and physiological factors, the qualitative differences of fractions could be caused by genetic variations. so, qualitative differences are important for taxonomic evaluations. fig. 1. blood protein samples electrophoretograms of the five subspecies of apathya cappadocica. a: a. c. cappadocica, b: a. c. wolteri, c: a. c. urmiana, d: a. c. schmidtlerorum, e: a. c. muhtari. (s: start, junction between the stacking and separation gels). fig. 2. gel photograph showing the electrophoretic separation of the blood protein sample obtained from a. c. cappadocica, together with its densitometric tracing curve. od: optical density, s: start (junction between the stacking and separation gels), g1-g11: globulins zone, pa: postalbumine zone, a: albumine zone. 211comparison of blood-serum proteins of apathya cappadocica fig. 3. gel photograph showing the electrophoretic separation of the blood protein sample obtained from a. c. wolteri, together with its densitometric tracing curve. for further explanation, see legend to figure 2. fig. 4. gel photograph showing the electrophoretic separation of the blood protein sample obtained from a. c. urmiana, together with its densitometric tracing curve. od: optical density, s: start (junction between the stacking and separation gels). 212 ç. ilgaz et alii fig. 5. gel photograph showing the electrophoretic separation of the blood protein sample obtained from a. c. schmidtlerorum, together with its densitometric tracing curve. od: optical density, s: start (junction between the stacking and separation gels). fig. 6. gel photograph showing the electrophoretic separation of the blood protein sample obtained from a. c. muhtari, together with its densitometric tracing curve. od: optical density, s: start (junction between the stacking and separation gels). 213comparison of blood-serum proteins of apathya cappadocica there is no information concerning its serological characterization on known a. cappadocica subspecies. the results obtained in the blood-serum electrophoretic separation indicate significant differences among known subspecies of a. cappadocica in terms of the number of fractions, and the mobility and density of blood-serum fractions. while a. c. cappadocica, a. c. wolteri and a. c. urmiana show significant differences quantitatively, there is a considerable difference between a. c. schmidtlerorum and a. c. muhtari qualitatively. finally, it should be stated that each subspecies has special electrophorenograms. the taxonomical status of this species, which has formed the subject of the study since 1902, the date when it was first identified, up to this day, has been continuously controversial. the species which was identified as lacerta cappadocica by werner in 1902 was included in a new genus under the name of apathya, taking these features into consideration: subdigital lamellae being obviously crytopodion, being black in color and having a semi-opal palpebral aperture that is generally composed of 6-8 scales in the lower eyelid; the existence of tiny postnasal plates in the lower part of the nostrils; the first supraocular being multi partial; the existence of many small scales that form a transversal line on the posterior of anal; the occipital being wider than interparietal; and the existence of pterygoid teeth (mehely, 1907). mehely’s apathya genus was mentioned as the synonym of latastia (bedriaga, 1884) by boulenger (1907). later, this taxon was accepted in the world of science in the following century (mertens, 1924). the taxon, which was evaluated under the species of apathya until the mid 20th century, was included again in the species of lacerta in the following decades (mertens, 1952; clark and clark, 1973; böhme, 1971). one genus which is evaluated in the lacertidae family, but whose taxonomical condition has not been clearly highlighted, is apathya (mayer and arribas, 2001). in the study which observed the morphological and molecular evaluation of 19 taxa in total, which are included in the lacertini group in palearctic – oriental area; cappadocica form was regarded in apathya (arnold et al., 2007). in the study, as a result of both the evaluation of 64 different morphological characters and the observation of the mitochondrial dna of taxa that formed the subject of the research with molecular techniques, the result was that apathya has a close relationship with hellonolacerta. in another study carried out recently (pavlicev and mayer, 2009), the result was contrary to the results obtained by arnold et al. (2007); a clear interpretation cannot be achieved regarding the taxonomical status of a. cappadocica and close relative forms of it (e.g., timon lepidus and lacerta agilis). as can be understood from the explanations given above, more studies need to be carried out at the molecular level together with morphological data in order to clarify the current taxonomical situation and relative relationship of apathya more clearly. acknowlegements this work forms part of a project (project no. tbag-104t017) supported by tübi̇tak (the scientific and technical research council of turkey). 214 ç. ilgaz et alii references anderson, s.c. (1999) the lizard of iran. soc. for the study of amphibians and reptiles, oxford (oh). arıkan, h. (1990): rana ridibunda (anura, ranidae) populasyonları üzerinde morfolojik ve serolojik araştırmalar. turk. j. zool. 14: 40-83. arıkan, h., atatür, m.k., mermer, a. 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(1952): amphibien und reptilien aus der türkei. i̇stanbul üniversitesi fen fakültesi mecmuası 18: 41-75. nilson, g., rastegar-pouyani, n., rastegar-pouyani, e., andrén, c. (2003): lacertas of south and central zagros mountains, iran, with descriptions of two new taxa. russ. j. herpetol. 10: 11-24. özeti, n., atatür, m.k. (1979): a preliminary survey of the serum proteins of a population of mertensiella luschani finikensis başoglu and atatür from finike in southwestern anatolia. i̇stanbul üniversitesi fen fakültesi mecmuası 44: 23-79. pavlicev, m., mayer, w. (2009): fast radiation of the subfamily lacertinae (reptilia: lacertidae): history or methodical artefact? mol. phylog. evol. 52: 727-734. schmidtler, j.f., bischoff, w. (1995): beziehungen zwischen lebensraum und morphologie bei lacerta cappadocica werner, 1902 in der türkei. die eidechse 6: 13-21. schmidtler, j.f. (1997): anmerkungen zur lacertiden-fauna des südlichen zentral anatolien. die eidechse 8: 1-9. sindaco, r., venchi, a., carpaneto, g.m., bologna, m. (2000): the reptiles of anatolia: a checklist and zoogeographical analysis. biogeographia 21: 441-554. werner, f. (1902): die reptilien und amphibienfauna von kleinasien. sb. kaiserl. akad. wiss. wien, mathem.-naturw. 111: 1057-1121. acta herpetologica 9(2): 253-258, 2014 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-14968 molecular assessment of podarcis sicula populations in britain, greece and turkey reinforces a multiple-origin invasion pattern in this species iolanda silva-rocha1,*, daniele salvi1, d. james harris1, susana freitas1, chris davis2, jim foster2, guntram deichsel3, chloe adamopoulou4, miguel a. carretero1 1 cibio-inbio-up, research centre in biodiversity and genetic resources, universidade do porto, campus agrário de vairão, rua padre armando quintas 7, 4485-661 vairão, vila do conde, portugal. *corresponding author. e-mail: irocha@cibio.up.pt 2 amphibian & reptile conservation, the witley centre, witley, godalming, surrey gu8 5qa, united kingdom 3 friedrich-ebert-str. 62, biberach an der riss, germany de-88400 4 zoological museum, department of biology, university of athens, panepistimioupolis, gr-15784, greece submitted on 2014, 2nd september, revised on 2014, 17th october, accepted on 2014, 21st october editor: gentile francesco ficetola abstract. biological invasions are a challenge to conservation and constitute a threat to biodiversity worldwide. the italian wall lizard podarcis sicula has been widely introduced, and seems capable of adapting to most of the regions where it is established and to impact on native biota. here we construct a phylogenetic framework to assess the origin of the introduced populations in the united kingdom, greece and turkey comparing cytochrome-b gene sequences of lizards from five locations to published sequences from the native range and other non-native locations. the results support an origin from central italy for the united kingdom population, from the adriatic region for the greek population and from calabria for the population from turkey. these results emphasise the multiple-source pattern of introduction of this species identified in previous studies. the improvement in the knowledge of the origin and pathways by which invaders arrive in new areas, as well as the monitoring of their populations, are crucial for successful strategies to deal with exotic species. keywords. biological invasions, italian wall lizard, cytochrome b, human-mediated introductions. biological invasions are a major concern to biodiversity conservation due to the threat to native biota (simberloff et al., 2013). the italian wall lizard, podarcis sicula, is one such reptile species that has been widely introduced (kraus, 2009). from its native distribution in the italian peninsula, sicily and the north adriatic coast, this species is considered to have been introduced in several other places, such as the tyrrhenian islands, corsica and sardinia, menorca in the balearics and in islands and coastal areas of the eastern adriatic sea (corti, 2006). besides these regions, scattered introduced populations are also known from the iberian peninsula, in cantabria (meijide, 1981), almería (mertens and wermuth, 1960), lisbon (gonzález de la vega et al., 2001), la rioja (valdeón et al., 2010) and near barcelona (rivera et al., 2011); in southern france, in toulon and château d’if island (morgue, 1924; orsini, 1984); in switzerland (schulte and gebhart, 2011); in turkey, in istanbul surroundings and the marmara islands (mollov, 2012; ilgaz et al., 2013); in north africa, in tunisia and tripoli (arnold and ovenden, 2002); and in the united states, in philadelphia, kansas, new york and california (deichsel et al., 2010; kolbe et al., 2013). very recently, two additional introductions have been reported in the united kingdom (hodgkins et al., 2012) and in greece (adamopoulou, 2014). this mediterranean lizard is very eclectic regarding habitat choice, being found both in natural areas, agricultural environments and in urban areas (capula, 1994; corti 2006). exotic populations of p. sicula have become 254 i. silva-rocha et alii established and undergone expansion in different regions encompassing a wide spectrum of environmental conditions, namely in north america (burke et al., 2002; burke and ner, 2005). therefore, this lizard appears to be an effective and successful coloniser. the ecological and behavioural traits of this species also contribute to the success in the new area, causing a great impact on native lizards with which p. sicula is able to compete. behavioural interference with native podarcis species have been reported, namely with p. melisellensis in the adriatic coast (downes and bauwens, 2002) which can result in the extinction of the latter when the introduction of p. sicula takes place in small islets (nevo et al., 1972). moreover, hybridisation with other podarcis species is also documented, namely with the endemic p. tiliguerta in sardinia (capula, 2002), with p. raffonei in the aeolian islands (capula et al., 2002) and with p. wagleriana in sicily (capula, 1994). these negative effects on native biota, hence, qualify this species as a problematic invader (kraus, 2009). given the potential adverse effects of p. sicula out of its native range, it becomes crucial to develop effective management strategies. understanding the colonisation patterns of this successful invader provides the basis for delineating more effective preventive measures (dorcas et al., 2010). a single source of introduction might facilitate the introduction of control measures on target populations, regions and invasion pathways, while multiple sources would indicate a general invasive character of the species requiring more global measures at a species level. molecular evidence supporting the origin of p. sicula is still lacking for many introduced populations. recently, taking advantage of the available phylogeographic information for the native range of the species podnar et al. (2005), silva-rocha et al. (2012) and kolbe et al. (2013) revealed multiple sources for the populations of the iberian peninsula and menorca, and of the united states. both studies concluded that the pathways by which the species is being introduced are well distinct between cases, ranging from the pet trade, to cargo and the nursery trade of olive trees (valdeón et al., 2010; rivera et al. 2011) as well as escaping from captivity and deliberate release (deichsel et al., 2010). in this study, we use phylogenetic analyses to assess the putative origin of three other recently introduced populations, occurring in the united kingdom, greece and turkey. the uk population was introduced in 2010 and has already been eradicated (hodgkins et al., 2012). in greece, the population was described as a very recent introduction and was detected in 2014 (adamopoulou, 2014). on the other hand, the population from turkey was introduced historically and is in apparent expansion (mollov, 2012; ilgaz et al., 2013). the identification of the origin of these three additional introduced populations is expected to improve the picture of the colonization pattern revealed by the previous studies. one sample of p. sicula was collected from mudanya (locality reported by mollov 2009; 40º22’5.844”n, 28º54’7.56”e), one sample from güzelyah (40º21’ 52.416”n, 28º54’7.56”e) and one sample from iznik (40º25’43.6434”n, 29º 43’ 45.7674”e) in turkey, three samples from palaio faliro (athens; 37º55’9.38”n, 23º42’ 0.50”e) in greece and one sample from buckinghamshire (52º1’47.604”n, 1º1’10.308” w) in the united kingdom. total genomic dna was extracted from tail tissue and a fragment of 687 base pairs of the mitochondrial gene cytochrome b (cyt-b) was amplified by pcr using the same primers and procedures described in silva-rocha et al. (2012). the sequences generated in the present study (genbank accession numbers: kp036396-kp036402) were aligned with 116 sequences downloaded from genbank: 39 sequences from individuals of p. sicula from the native range (podnar et al., 2005), accession numbers: ay185095, ay185094, ay770869–ay77090; 16 sequences from the iberia peninsula and 7 from menorca introduced populations (silva-rocha et al., 2012), accession numbers: jx072938-jx072960; one sequence from switzerland (schulte and gebhart, 2011); 1 sequence from california (deichsel et al., 2010), accession number: hq154646; and 52 sequences from kolbe et al. (2013), from both the introduced populations of the united states (nine sequences) and from the native populations (43 sequences), accession numbers: jx186516-jx186568. three sequences from podarcis muralis and p. melisellensis were also downloaded from genbank and used as outgroup (accession numbers ay185096, ay185029 and ay185057), following podnar et al. (2005). we performed a maximum likelihood (ml) phylogenetic analysis to infer the relationships between the cyt-b haplotypes using the software mega 6 (tamura et al., 2013). the model hky + gamma was selected as the best model of sequence evolution under the bayesian information criterion (bic), chosen using mega 6. tree searches were performed using the heuristic search mode. node support was calculated over 1000 bootstrap replicates. in addition to the tree-building approach, we analysed the genealogical relationships among the native and non-native haplotypes clustered in the ‘sicula’ clade (podnar et al., 2005) by means of a statistical parsimony network using the software tcs 1.21 (clement et al., 2000), in order to get a better resolution on relationships between closely related haplotypes. the final alignment includes 127 sequences of 687 base pairs. four new haplotypes were identified from the five introduced locations here studied, and 78 haplotypes 255origin of introduced p. sicula fig. 1. ml phylogenetic estimate of relationships between cytochrome b (cyt-b) haplotypes from native podarcis sicula populations (podnar et al. 2005, kolbe et al. 2013) and those from introduced populations generated in this study (turkey and united kingdom) and by silvarocha et al. 2012 and kolbe et al. 2013 (almeria, cantabria, la rioja, lisbon, menorca, new jersey, california, new york and kansas). p. muralis and p. meliselensis were used as outgroups (not shown). sequences downloaded from genbank are named according to their accession number. main p. sicula haploclades are indicated by grey boxes and subclades are named according to the geographic origin of haplotypes (native samples in italic). samples from united kingdom, greece and turkey are highlighted in grey and underlined. bootstrap support values are indicated above the nodes of interest. 256 i. silva-rocha et alii were identified from the sequences generated by the previous studies (podnar et al., 2005; deischsel et al., 2010; schulte and gebhart, 2011; silva-rocha et al., 2012; kolbe et al. 2013). in particular, one different haplotype was found in the british and greek locations each, while two different haplotypes occurred in the turkish populations. the estimate of relationships based on ml indicates that lizards from the united kingdom belong to the “rome” clade found by kolbe et al. (2013) (fig. 1). this result supports the hypothesis of hodgkins et al. (2012) that the origin of the lizards introduced in the united kingdom was from a locality close to rome. these authors stated that the lizards were found in june 2010 on a consignment of tufa (a type of soft, porous limestone) imported from italy in march 2010 for a restoration project of an 18th century landscape garden in stowe, buckinghamshire. this hypothesis was also supported by j. foster (pers. comm.) who indicated tivoli (ca. 30 km east of rome) as the origin of the building materials. lizards from greece are included in the “campestrissicula” clade, sharing the haplotype with lizards sampled in the adriatic region by podnar et al. (2005) and with lizards sampled in new jersey by kolbe et al. (2013). therefore, the most probable origin for the greek population is the adriatic region, which is geographically close to greece. the population was found in a narrow zone of sand (dimensions approx. 90 × 15 m) between an overcrowded beach and the tram station on the main avenue. the occupied area is a small artificial “park” of various trees planted on bare sand. the park officer noticed the presence of the lizards since he started to plant the trees, so this could be a probable vector by which the animals arrived. unfortunately, it is not possible to know the origin of the trees since they were collected from the garbage. furthermore, we cannot exclude other potential introduction pathways, since there is a big yacht marina less than 500 meters away from the colony and a large port, port of piraeus, approximately 8km away (adamopoulou, pers. comm.). fig. 2. map of the introduced populations of podarcis sicula analysed. (a) populations in iberian peninsula, united kingdom and switzerland, (b) populations in united states, (c) populations in greece and turkey. 257origin of introduced p. sicula regarding the turkish population, their cyt-b haplotype clusters in the “sicula” clade of podnar et al. (2005) (fig. 1). the haplotype from turkey seems to correspond to the calabrian stock, since is closely related to the ones found in the calabrian region. accidental historical introductions by people or merchant vessels are possible pathways through which this population arrived in turkey from southern italy (mollov, 2009). our ml results are in accordance with previous studies (silva-rocha et al., 2012; kolbe et al., 2013), as can be seen both in the ml tree (fig. 1) and in the summary map of fig. 2. based on the additional sequences generated by kolbe et al. (2013) from lizards sampled in the native range, it is possible to refine the inferred origin for the populations of lisbon and cantabria (northern spain). indeed, our ml tree supports an origin from northwestern tuscany for the cantabria population in and from central italy around rome for the lisbon population which clustered within the “rome” clade. the results of this study reinforce the multiple source and pathways pattern suggested by silva-rocha et al. (2012) and kolbe et al. (2013) and confirm the invasive potential of this species as a whole (fig. 2). this reveals once more the tendency of p. sicula to use man-made objects as refuges and the role of these as an effective vector for the introductions of this lizard. regarding the british population, the early detection and fast collection of the individuals was the key to preventing the expansion of this species from the formal garden where it was first observed (see details of the eradication in hodgkins et al. 2012). the greek population is established and already has at least 50-60 individuals (adamopoulou, 2014). early eradication of this population is recommended. on the other hand, the turkish populations seem to be of more longstanding origin and are currently in expansion towards the south of the marmara sea (mollov, 2009; tok et al., 2014). a specific monitoring program would be needed to assess in detail the extent and progress of this expansion. overall, results obtained here accumulated to the previous evidence demonstrating that the italian wall lizard p. sicula can be an effective invader. its successful acclimatization to environmental conditions different for those prevailing in its original mediterranean range such as those in switzerland or central usa increases conservation concern, since the probability to become invasive is boosted by the adaptability of the species. certainly, documenting the origin and pathways of introduced populations and monitoring the expansion of the populations are needed to define effective management strategies (kraus, 2009; simberloff et al., 2013). acknowledgments ds is supported by the fct post-doctoral grant sfrh/bpd/66592/2009 and is-r by the fct phd grant sfrh/bd/95745/2013 under the programa operacional potencial humano – quadro de referência estratégico nacional funds from the european social fund and portuguese ministério da educação e ciência. the molecular work of this study was funded by fcomp-01-0124-feder-007062 fct project ptdc/bia-bec/102179/2008, ptdc/bia-bec/101256/2008, and partially financed by the project “biodiversity, ecology and global change” co-financed by north portugal regional operational programme 2007/2013 (on.2 – o novo norte), under the national strategic reference framework (nsrf), through the european regional development fund (erdf) and by the project “biodiversity conservation in a changing world” financed by the portuguese integrated program of ic&dt call nº 1/saesctn/alent-07-0224-feder-001755. references adamopoulou, c. 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(2013): the desire for variety: italian wall lizard (podarcis siculus) populations introduced to the united states via the pet trade are derived from multiple native-range sources. biol. invasions. 15: 775-783. kraus, f (2009) alien reptiles and amphibians: a scientific compendium and analysis. springer, new york. meijide, m. (1981): una nueva población de lacerta sicula rafinesque para el norte de españa. acta vertebrata 8: 304-305. mertens, r. and wermuth, h. (1960): die amphibien und reptilien europas. verlag waldemar kramer, frankfurt am main. mollov, i. (2009): a new locality of the italian wall lizard podarcis siculus (rafinesque-schmaltz, 1810) from turkey. zoonotes 6: 1-3. morgue, m. (1924): note succinte sur les espèces de lacerta muralis des îles du golfe de marseille. bull. soc. linn. lyon 3: 55. nevo, e., gorman, g. c., soulé, m., yang, e. j., clover, r., jovanovic, v. (1972): competitive exclusion between insular lacerta species (sauria, lacertidae). oecologia 10: 183-190. orsini, j.p. (1984): a propos du lézar sicilien podarcis sicula en provence. bull. c.r.o.p 6: 8. podnar, m., mayer, w., tvrtković, n. (2005): phylogeography of the italian wall lizard, podarcis sicula, as revealed by mitochondrial dna sequences. mol. ecol. 14: 575-588. rivera, x., arribas, o., carranza, s., maluquer-margalef, j. (2011): an introduction of podarcis sícula in catalonia (ne iberian peninsula) on imported olive trees. bull. soc. catalana herpetol. 19: 79-85. schulte, u. gebhart, j. (2011): geographic origin of a population of the italian wall lizard podarcis siculus (rafinesque-schmaltz,1810), introduced north of the alps. herpetozoa 24: 96-97. silva-rocha, i., salvi, d., carretero, m.a. (2012): genetic data reveal a multiple origin for the populations of the italian wall lizard podarcis sicula (squamata: lacertidae) introduced in the iberian peninsula and balearic islands. ital. j. zool. 79: 502-510. simberloff, d., martin, j.l., genovesi, p., maris, v., wardle, d., aronson, j., courchamp, f., galil, b., garciaberthou, e., pascal, m., pysek, p., sousa, r., tabacchi, e., vila, m. (2013): impacts of biological invasions: what’s what and the way forward. trends ecol. evol. 28: 58-66. tamura, k., stecher, g., peterson, d., filipski, a., kumar, s. (2013). mega6: molecular evolutionary genetics analysis version 6.0. mol. biol. evol. 30: 2725-2729. tok, c.v., çiçek, k., hayretdag s., tayhan, y., yakin, b.y. (2014). range extension and morphology of the italian wall lizard, podarcis siculus (rafinesque-schmaltz, 1810) (squamata: lacertidae), from turkey. turk. j. zool. 38: 1-7. valdeón, a., perera, a., costa, s., sampaio, f., carretero, m.a. (2010): evidencia de una introducción de podarcis sicula desde italia a españa asociada a una importación de olivos (olea europaea). bol. asoc. herpetol. esp. 21: 122-126. acta herpetologica 10(2): 161-162, 2015 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-17181 book review: pietro giovacchini, valentina falchi, sergio vignali, giacomo radi, luca passalacqua, fausto corsi, marco porciani, fabrizio farsi. atlante degli anfibi della provincia di grosseto (20032013) edoardo razzetti museo di storia naturale, università di pavia, piazza botta 9, i-27100 pavia, italy. e-mail: razzetti@unipv.it in the past twenty years there has been a great interest in the geographic distribution of amphibians and reptiles and considerable efforts have been spent to collect, analyze and publish these records. a careful evaluation of distribution data is of fundamental importance to establish the conservation priorities (e.g., rondinini et al., 2013) and to develop correct conservation programs. for the italian territory, herpetological atlases are available for most of the regions and a complete national atlas was published few years ago with a synthesis of records collected over two decades (sindaco et al., 2006). tuscany has not been left behind and was the subject of specific herpetological researches that led to the publication of a regional atlas (vanni and nistri, 2006) and two provincial ones: prato (fancelli et al., 2005) and siena (piazzini et al., 2005, 2010). tuscany is generally considered well-studied since there are several local researchers, but few gaps in the distribution maps still exist as it is proved by the publication of this atlas of amphibians of the province of grosseto. the book has been realized by a group of zoologists coordinated by pietro giovacchini of the provincial office for protected areas and biodiversity with the cooperation of a local ornithological group (gruppo ornitologico maremmano studi naturalistici “a. ademollo”). the colophon sports the patronage of societas herpetologica italica. the atlas is completely written in italian, consists of 113 pages and is a nice paperback edition in octavo (24×17 cm). the volume is hosted in the “quaderni delle aree protette” series which includes books that deal about monumental trees, protected areas and archeology. it opens up with general chapters about geology, hydrography, climate and vegetation which are covered in good detail. next is the section about material and methods which describe how data have been collected and analyzed. the grid used for the maps in this study is the widely adopted utm 10×10 km. with such large grid map there is the risk of overestimating the presence of the species also in a large province like grosseto (4504 km2): a 5×5 km grid would probably be better even if research efforts required to ensure adequate coverage would be much higher (sindaco et al., 2015). it is interesting the approach used to collect data that is quite unusual (but is rather common on the ornithological ones), the maps have been based on a completely independent dataset (collected between 2003 and 2013) this is the reason why the distribution maps presented for some species are slightly different (e.g., rana italica) or, sometimes, striking different (e.g., bombina pachypus) from the atlas of tuscany (vanni and nistri, 2006). i feel that this makes the book even more valuable and original as there is no risk to confirm old doubtful data that cannot be verified. at the same time, this approach has a few potential drawbacks. for example i am wondering why the common salamander (salamandra salamandra) was not discussed at all in the book even if three bibliographic data are available for the province (cf. vanni and nistri, 2006). maybe those data are questionable, maybe they are just very old, i have no idea why and i will have to discover it by my own. 162 edoardo razzetti each species account (12 species are listed) opens with a “general description” particularly useful for field identification which includes also few taxonomic remarks. next is “distribution in italy” and “ecological and biological notes” both well-written and supported by the essential citations. the “distribution in the province of grosseto” is the core subject of this book and is a comprehensive evaluation of all the record collected by the authors together with altitudinal distribution and habitat types (for which maps and histograms are detailed in the opposite page). all in all the coverage of the data is excellent and apparently no areas have been overlooked. sections about the “status and conservation measures” and “museum’s specimens” close each account. the final chapters are dedicated to the “conclusive insights” including wide area analyses, conservation status, laws protecting local amphibians and the action plans. since in italy the provinces have legislative powers on the protection of biodiversity and the protection of the territory, these chapters have been written with care and competence. finally one special mention goes to the pictures of the book that, despite the fact that are quite small, are some of the best i have ever seen. they usually include a full picture, larval stages and a portrait for most of the species. giacomo radi, that realized them, not only is able to capture the essence of amphibians in their habitat, but also to create pleasing images that show the most important aspects of their biology. the printed version of the book can be requested at the province of grosseto, whereas the e-book is available at no cost from several websites including the one of societas herpetologica italica. references fancelli e., nistri a., vanni s. (2005): anfibi e rettili, biodiversità in provincia di prato, vol. 1. amministrazione provinciale di prato, arcidosso (gr). piazzini s., favilli l., manganelli g. (2005): atlante degli anfibi della provincia di siena (1999-2004). quaderni naturalistici 1, amministrazione provinciale di siena, siena. piazzini s., favilli l., manganelli g. (2010): atlante dei rettili della provincia di siena (2000-2009). quaderni naturalistici 2, amministrazione provinciale di siena, siena. sindaco r., doria g., razzetti e., bernini f. (2006): atlante degli anfibi e dei rettili d’italia / atlas of italian amphibians and reptiles. societas herpetologica italica, edizioni polistampa, firenze. sindaco r., razzetti e., liuzzi c. (2015): il nuovo progetto atlante della societas herpetologica italica. in: atti x congresso nazionale della societas herpetologica italica (genova, 15-18 ottobre 2014), pp. 21-30. doria g., poggi r., salvidio s., tavano m., eds, ianieri edizioni, pescara. rondinini, c., battistoni, a., peronace, v., teofili, c. (2013): lista rossa iucn dei vertebrati italiani. comitato italiano iucn e ministero dell’ambiente e della tutela del territorio e del mare, roma. vanni, s., nistri a. (2006): atlante degli anfibi e dei rettili della toscana. università degli studi di firenze, museo di storia naturale, sezione zoologica “la specola”, regione toscana, firenze. acta herpetologica vol. 10, n. 1 june 2015 firenze university press acta herpetologica journal of the societas herpetologica italica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 8(1): 65-67, 2013 status of the european pond turtle, emys orbicularis (reptilia: testudines: emydidae) in vorarlberg, austria andreas kleewein1, günther wöss2 1 university of vienna, department of evolutionary biology, althanstraße 14, a-1090 vienna, austria. corresponding author. e-mail: andreas.kleewein@gmx.net 2 natural history museum of vienna, herpetological collection, burgring 7, a-1010 vienna, austria submitted on: 2013, 21st march; revised on 2013, 12th may; accepted on: 2013, 15th may. abstract. prehistoric and historic records of emys orbicularis (linnaeus, 1758) for the western austrian province of vorarlberg and adjacent regions are reviewed. two recently captured pond turtles allowed the first analyses of mitochondrial cytochrome b haplotypes for the province. both turtles represent lineage iv haplotypes, whereas lineage ii is expected to be native. we conclude that native e. orbicularis are extinct in vorarlberg. keywords. emys orbicularis, austria, vorarlberg, mtdna, distribution the possible occurrence of the european pond turtle, emys orbicularis (linnaeus, 1758), in the western austrian province of vorarlberg has long been debated (gemel, 2001). it is well known that these animals were formerly massively traded as lenten food and as pets (fritz, 2001; fritz et al., 2004), so that any records could also refer to escaped or released non-native turtles. however, since prehistoric pond turtle records are known for vorarlberg, the occurrence of native turtles cannot be completely excluded. nevertheless, e. orbicularis is no longer included as a native species in the newest red list for the herpetofauna of vorarlberg (aschauer et al., 2008). for the entirety of austria, the species is classified as ‘critically endangered’ (gollmann, 2007), and in older red lists for austria it was concluded that the occurrence of e. orbicularis in vorarlberg could only be secured by constant reintroduction (häupl and tiedemann, 1983). the aim of this study is to review the present status of e. orbicularis in vorarlberg and to examine two newly discovered pond turtles genetically to clarify whether these individuals could be native. theoretically, native turtles may still occur in regions with historic or prehistoric records, as is the case with vorarlberg (compare kinzelbach, 1988; sommer et al., 2007). it should be highlighted that the oldest prehistoric e. orbicularis in austria were discovered in the rhine valley near koblach, vorarlberg. these remains originate from the late mesolithic, approximately 8,000 years ago (kunst and gemel, 2000). moreover, european pond turtles probably served as food for prehistoric humans in vorarlberg, as indicated by turtle bones in prehistoric settlements (vonbank, 1965). according to historic records, the european pond turtle was once widespread around lake constance, and turtles were ordered from local fishermen by the dukes of baden (bernauer and jacoby, 1994; gemel, 2001). it should therefore be assumed that e. orbicularis once was also abundant in the vorarlberg portion of lake constance and in the neighbouring rhine valley. however, budde (1996) considers the species as extinct in the lake constance area and in upper swabia, while gemel (2001) believes that e. orbicularis disappeared in the austrian lake constance region only towards the end of the 20th century. grillitsch and cabela (2001) consider that e. orbicularis is ‘localized’ and ‘released’ in vorarlberg and the lake constance region. whether any european pond turtle caught in vorarlberg is native or not can be, at least in part, elucidated by genetic analyses. using the mitochondrial cytochrome b 66 a. kleewein, g. wöss gene as a marker (fritz et al., 2007, 2009), it is often possible to differentiate between native and introduced turtles (fritz et al., 2004). recent research has shown that ten deeply divergent mitochondrial lineages with many haplotypes exist, which correspond to a pronounced phylogeographic structure (lenk et al., 1999; fritz et al., 2007, 2009). according to the general distribution pattern of extant and extinct pond turtle populations (cf. sommer et al., 2009), the expected mitochondrial lineage in vorarlberg is lineage ii. consequently, any pond turtle bearing haplotypes of other lineages cannot be native. during a project on allochthonous turtles in vorarlberg, supported by the ‘inatura – erlebnis naturschau gmbh’, the authors studied in 2011 a wild-caught couple of e. orbicularis. the male was discovered in the same year in the municipality of mäder, feldkirch district, on a parking lot beneath a car (47°20’57’’n, 9°36’56’’e; 415 m a.s.l.) and had, when examined, a carapace length of 15.7 cm and a body mass of 552 g. the female was discovered in 2010 in the market municipality of lustenau, dornbirn district (47°24’27’’n, 9°39’19’’e; 406 m a.s.l.). in 2011, its carapace measured 18.2 cm and its body mass was 911 g. the turtles had been taken into care and were therefore available for study. buccal swabs of both turtles were sent to a laboratory for determining the haplotype of the mitochondrial cytochrome b gene. haplotype classification followed lenk et al. (1999) and fritz et al. (2007, 2009). the obtained sequences were compared to genbank data. the male turtle represents a lineage iv haplotype, which could not be more closely determined due to short sequence length, and the female bears haplotype ivb. the latter haplotype has so far only been found on the island of cephalonia, off the west coast of greece (fritz et al., 2007), indicating that this turtle is not native. however, turtles from cephalonia are known to be very small, with carapacial lengths below 12 cm (richter and mayer, 1990; fritz, 2001). hence, the large size of the vorarlberg female suggests that it was raised in captivity and possibly even presents a captive-bred mixture of different lineages. west of the adriatic, the range of lineage iv stretches from the coastal po valley down to southern italy, while it reaches from istria and dalmatia to the peloponnese and boeotia on the adriatic east coast (lenk et al., 1999; fritz et al., 2007, 2009). thus, both examined specimens were either released or escaped from garden ponds. the situation in vorarlberg seems therefore to be similar to what fritz (2001) described for southern germany, switzerland, the rest of austria and the bohemian depression. most pond turtle populations have disappeared there very early, due to the human impact. however, the situation is further complicated by the historic trade with european pond turtles. since e. orbicularis was not considered meat in historic times, it could be eaten during lent and there was a demand, leading to the massive import of turtles from elsewhere (fritz, 2001). in addition, until approximately 1980, huge numbers of european pond turtles were sold as pets, including turtles originating from regions where lineage ii haplotypes occur (fritz et al., 2004). therefore, we can assume that turtles bearing different haplotypes were also imported to vorarlberg, and some of them may have been released or escaped, like in other austrian provinces (kunst and gemel, 2000). thus, even any future record of lineage ii turtles in vorarlberg would be no unambiguous evidence for the native occurrence of pond turtles. taking all available evidence together, we conclude that native e. orbicularis should be considered extinct in the whole of vorarlberg. acknowledgements our sincere thanks go to ‘inatura dornbirn’ for approving a project on allochthonous turtles in vorarlberg, during which we had the opportunity to extract genetic material from e. orbicularis. references aschauer, m., grabher, m., huber, d., loacker, i., tschisner, c., amann, g. (2008): rote liste gefährdeter amphibien und reptilien vorarlbergs. inatura, dornbirn. bernauer, a., jacoby, h. (1994): bodensee, naturreichtum am alpenrand. naturerbe verlag j. resch, überlingen. budde, m. (1996): kartierung der europäischen sumpfschildkröte (emys orbicularis linnaeus, 1758) in naturschutzgebieten oberschwabens und des angrenzenden bodenseegebietes unter dem aspekt der autochthonie. diploma thesis. university of ulm, ulm. fritz, u. (2001): emys orbicularis (linnaeus, 1758) europäische sumpfschildkröte. in: handbuch der reptilien und amphibien europas. schildkröten i, pp. 343-515. fritz, u., ed, aula-verlag, wiebelsheim. fritz, u., guicking, d., lenk, p., joger, u., wink, m. (2004): when turtle distribution tells european history: mtdna haplotypes of emys orbicularis reflect in germany former division by the iron curtain. biologia 59 (suppl. 14): 19-25. fritz, u., guicking, d., kami, h., arakelyan, m., auer, m., ayaz, d., ayres fernández, c., bakiev, a.g., celani, a., džukić, g., fahd, s., havaš, p., joger, u., khabibullin, v.f., mazanaeva, l.f., široký, p., tripepi, s., 67status of emys orbicularis in vorarlberg, austria valdeón vélez, a., velo antón, g., wink, m. (2007): mitochondrial phylogeography of european pond turtles (emys orbicularis, emys trinacris) – an update. amphibia-reptilia 28: 418-426. fritz, u., ayaz, d., hundsdörfer, a.k., kotenko, t., guicking, d., wink, m., tok, c.v., çiçek, k., buschbom, j. (2009): mitochondrial diversity of european pond turtles (emys orbicularis) in anatolia and the ponto-caspian region: multiple old refuges, hotspot of extant diversification and critically endangered endemics. org. divers. evol. 9: 100-114. gemel, r. (2001): zum vorkommen der europäischen sumpfschildkröte. in: atlas zur verbreitung und ökologie der amphibien und reptilien in österreich: auswertung der herpetofaunistischen datenbank der herpetologischen sammlung des naturhistorischen museums in wien, pp. 716-736. cabela a., grillitsch, h., tiedemann, f., eds, umweltbundesamt, wien. gollmann, g. (2007): rote liste der in österreich gefährdeten lurche (amphibia) und kriechtiere (reptilia). in: rote listen gefährdeter tiere österreichs, pp. 37-60. zulka, k.p., ed, böhlau, wien. grillitsch, h., cabela, a. (2001): reptilien. emys orbicularis linnaeus, 1758. in: atlas zur verbreitung und ökologie der amphibien und reptilien in österreich: auswertung der herpetofaunistischen datenbank der herpetologischen sammlung des naturhistorischen museums in wien, pp. 442-455. cabela a., grillitsch, h., tiedemann, f., eds, umweltbundesamt, wien. häupl, m., tiedemann, f. (1983): rote liste der in österreich gefährdeten kriechtiere (reptilia) und lurche (amphibia). in: rote listen gefährdeter tiere österreichs, pp. 63-66. gepp, j., ed, bundesministerium für gesundheit und umweltschutz, wien. kinzelbach, r. (1988): die europäische sumpfschildkröte (emys orbicularis) im einzugsgebiet des rheins. zeitschr. angew. zool. 75: 385-420. kunst, g.k., gemel, r. (2000): zur kulturgeschichte der schildkröten unter besonderer berücksichtigung der bedeutung der europäischen sumpfschildkröte, emys orbicularis (l.) in österreich. in: die europäische sumpfschildkröte, pp. 21-62. hödl, w., rössler, m., eds, linz (stapfia 69). lenk, p., fritz, u., joger, u., wink, m. (1999): mitochondrial phylogeography of the european pond turtle, emys orbicularis (linnaeus 1758). mol. ecol. 8: 19111922. richter, k., mayer, w. (1990): einige bemerkenswerte herpetologische beobachtungen in griechenland. herpetozoa 2: 159-161. sommer, r.s., persson, a., wieseke, n., fritz, u. (2007): holocene recolonization and extinction of the pond turtle, emys orbicularis (l., 1758), in europe. quaternary sci. rev. 26: 3099-3107. sommer, r.s., lindqvist, c., persson, a., bringsøe, h., rhodin, a.g.j., schneeweiss, n., široký, p., bachmann, l., fritz, u. (2009): unexpected early extinction of the european pond turtle (emys orbicularis) in sweden and climatic impact on its holocene range. mol. ecol. 18: 1252-1262. vonbank, e. (1965): vorarlberg. in: lexikon urund frühgeschichtlicher fundstätten österreichs, pp. 181184. franz, l., neumann, a.r., eds, hollinek-habelt, wien. acta herpetologica vol. 8, n. 1 june 2013 firenze university press the oogenic cycle of the caspian bent-toed gecko, cyrtopodion caspium (squamata: gekkonidae) in iran vida hojati1*, kazem parivar2, eskandar rastegar-pouyani3, abdolhossein shiravi1 altitudinal effects on life history parameters in populations of liolaemus pictus argentinus (sauria: liolaemidae) joel antú gutiérrez1,*, carla piantoni2, nora r. ibargüengoytía1,3 recent cryptic extinction of squamate reptiles on yoronjima island of the ryukyu archipelago, japan, inferred from garbage dump remains yasuyuki nakamura1,*, akio takahashi2, hidetoshi ota3 trophic niche and feeding biology of the italian wall lizard, podarcis siculus campestris (de betta, 1857) along western mediterranean coast marco a.l. zuffi*, chiara giannelli novel, non-invasive method for distinguishing the individuals of the fire salamander (salamandra salamandra) in capture-mark-recapture studies goran šukalo1,*, sonja đorđević2, dragojla golub1, dejan dmitrović1, ljiljana tomović2,3 going out tonight? when insular hierophis viridiflavus breaks the whip snakes rules delaugerre michel-jean first evidence of a paedomorphic population of the smooth newt (lissotriton vulgaris) in the czech republic václav gvoždík1,2, veronika javůrková4, oldřich kopecký5,* no detection of chytrid in first systematic screening of bombina variegata pachypus (anura: bombinatoridae) in liguria, northern italy stefano canessa1,*, an martel2, frank pasmans2 status of the european pond turtle, emys orbicularis (reptilia: testudines: emydidae) in vorarlberg, austria andreas kleewein1, günther wöss2 comparative cytogenetics of two species of ground skinks: scincella assata and s. cherriei (squamata: scincidae: lygosominae) from chiapas, mexico riccardo castiglia1,*, alexandra m.r. bezerra2, oscar flores-villela3, flavia annesi1, antonio muñoz4, ekaterina gornung1 polydactyly in the tyrrhenian wall lizard (podarcis tiliguerta) antigoni kaliontzopoulou1,*, daniele salvi1, verónica gomes1, joão p. m. c. maia1,2,3, panagiotis kaliontzopoulos4 book review: roberto sindaco, alberto venchi, cristina grieco. the reptiles of the western palearctic. 2. annotated checklist and distributional atlas of the snakes of europe, north africa, middle east and central asia, with an update to the vol. 1. edoardo razzetti acta herpetologica journal of the societas herpetologica italica acta herpetologica 10(2): 121-124, 2015 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-15421 notes on the reproductive ecology of the oaxaca mud turtle (kinosternon oaxacae) in the vicinity of mazunte, mexico alma g. vázquez gómez1, martha harfush2, rodrigo macip-ríos3,* 1 escuela de biología. benemérita universidad autónoma de puebla. blvd. valsequillo y av. san claudio, edificio 112-a, ciudad universitaria, puebla, puebla, c.p. 72570 2 centro mexicano de la tortuga. domicilio conocido, santa maría tonameca, mazunte, oaxaca, c.p. 70902 3 escuela nacional de estudios superiores, unidad morelia universidad nacional autonoma de mexico. antigua carretera a pátzcuaro no.8701 col. ex hacienda de sán josé de la huerta, morelia, michoacan c.p. 58190. *corresponding author. e-mail: rmacip@enesmorelia.unam.mx submitted on 2015, 1st january; revised on 2015, 12th may; accepted on 2015, 29th may. editor: paolo casale abstract. kinosternon oaxacae is one of the least known species of mexican turtles. we conducted a preliminary survey from august to november 2013 in three localities on the coast of oaxaca with the objective to increase and complement the knowledge of the reproductive ecology of kinosternon oaxacae, including field data on nesting season, clutch size, egg size, and relative clutch mass. we found 21 females with eggs in their oviducts. with this sample we determined that the reproductive season extends from early may to early november. clutch size averaged two eggs (± 1), and egg size averaged 30.61 (± 1.89) mm in length, 17.11 (± 0.69) mm in width, and 7.64 (± 0.57) g in mass. relative clutch mass (clutch mass/gravid body mass) was 0.046 (± 0.023), and the smallest gravid female was 118.8 mm carapace length. no correlations of reproductive traits were found with body size, and no variation in reproductive traits or body size was detected among localities. comparisons with related (scorpioides group) species were made. keywords. egg size, clutch size, nesting season, oaxaca, relative clutch mass. mexico is the second richest country in turtle species in the world (legler and vogt, 2013; van dijk et al., 2014). from the 61 known taxa of mexican nonmarine turtles (legler and vogt, 2013; flores-villela and garcia-vazquez, 2014; van dijk et al., 2014), 57% are endemic. unfortunately the general knowledge of the mexican turtle fauna is relative meager, and according to legler and vogt (2013), kinosternon oaxacae “is one of the least known species in mexico”. there are only a few published data available, including its description (berry and iverson, 1980) and some preliminary natural history data (iverson, 1986). this species is known from 44 specimens from only 15 localities from the ometepec river basin near guerrero to the western limits of isthmus of tehuantepec (iverson, 1986; carr, 1993; legler and vogt, 2013). reproductive data of k. oaxacae came from a sample of 21 individuals examined by iverson (1986). the available information can be summarized as follows: vitelogenesis extends from april to july and mating and ovulation occur (“probably”) in june or/and july. data from females for a natural reproductive event (oviductal eggs or corpora lutea) are absent of the literature, and nesting season ‘probably’ begins in mid july, but it is not known when it ends. estimated clutch size could be between two to five eggs, and no data about egg size is known whatsoever (iverson, 1986). for species with limited distributions as such k. oaxacae (21120 km2 aprox.), quality data are necessary to develop proper conservation strategies (klemens, 2000; primack, 2012). the coast of oaxaca is an economically poor region with high environmental impact due to tour122 alma g. vázquez gómez et alii ism (supposedly environmental friendly), meteorological phenomena (hurricanes, tropical storms, etc.), and non-sustainable land management policies. the accelerated development of other activities, such cattle ranching, agriculture, and unorganized urban growth could impact the future of this scarcely known endemic species. the aim of this study was to increase/complement the knowledge of the reproductive ecology of kinosternon oaxacae, including field data on nesting season, clutch size, egg size, and relative clutch mass. for that we conducted a preliminary survey from august to november 2013 in the vicinity of mazunte, oaxaca, at the localities of el aguacate (15°43’18’’n, 96°23’15’’w; 13 surveys), escobilla (15°43’55.77’’n, 96°43’59.55’’w; 14 surveys), and san roque (15°46’43’’n, 96°27’33’’w; 12 surveys), nad 27 datum. we consider a survey as one day of fieldwork from 9:00 to 17:00 h. we used fyke net traps and hand captures to gather females with a minimum carapace length (cl) of 90 mm as described as the minimum size of reproduction in other similar and related species (iverson, 1986; iverson, 2008; macip-rios et al., 2009; iverson, 2010; iverson et al., 2013). hand captures were made by an average of five persons per collect event, averaging a 520 hours/person. traps sum 112 hours/trap for escobilla and 96 hours/trap for san roque. females were brought to the lab at the centro mexicano de la tortuga (cmt) were they were measured, weighed, and marked with an individual code by shell notching (cagle, 1939). no radiograph was used but all females were injected with a dose of 1.5 ml/kg of oxytocin to induce oviposition (gibbons and greene, 1979). injected turtles were placed in individual enclosures with a sand substrate for oviposition. deposited eggs were measured (length and width) with a dial caliper (0.02 mm) and weighed with a triple beam ohaus junior tj611 balance (0.1 g). reproductive effort was estimated by the relative clutch mass (rcm) formula modified by cuellar (1984): clutch mass/gravid females body mass. we compared clutch size, egg size and relative clutch mass among localities by a non-parametric (rank based) kruskalwallis test (zar, 1999). we also used a non-parametric spearman ρ (rho) to identify correlations between body size (carapace length) and reproductive parameters. all statistical tests were ran with jmp ver. 5.0.1 (sas institute, 2002) with an α = 0.05. we also searched in the archives of the cmt for field journals and data sheets with information and data gathered by the cmt personnel from discontinuous surveys from the years 2009 and 2010. we used these data to support our direct observations in 2013. turtles were collected under the permit sgpa/dgvs/04572/13 issued by the ministry of natural resources of the mexican federal government (semarnat). no ethics permits are required in mexico when organisms are used for scientific research, however no turtle were killed or harm during the study, and all individuals were handled and managed under international ethics on animal issues. a total of 57 females were injected with oxycitocin, but only 21 females oviposited in the lab; 11 females were from san roque, eight from escobilla, and two from el aguacate. females with oviductal eggs were detected from late september to early november. the smallest female with eggs in the three localities was 118.8 mm cl. clutch size (cs) ranged 1-4 eggs, with a mean of 1.95 (± 1.0) (mean ± sd) eggs; egg length (el) averaged 30.61 (± 1.89) mm, egg width (ew) 17.11 (± 0.69) mm, and egg mass 7.64 (± 0.57) g. relative clutch mass was 0.046 (± 0.023). the comparison of reproductive traits among localities did not show significant variation in clutch size (h2 = 0.29, p = 0.86); either el (h2 = 3.81, p = 0.14), ew (h2 = 0.87, p = 0.64), em (h2 = 3.73 p = 0.15), and rcm (h2 = 0.94, p = 0.62). means and variation by localities are presented in table 1. body size (cl) was not correlated with table 2. non parametric correlations of reproductive traits. ρ is spearman rho; cl is carapace length; rcm is relative clutch mass. x y ρ p body size (cl) egg length 0.28 0.21 body size (cl) egg width -0.14 0.52 body size (cl) egg mass 0.35 0.11 body size (cl) clutch size 0.12 0.58 clutch size rcm 0.92 <0.0001 table 1. variation in mean reproductive traits in kinosternon oaxacae across the three localities in the vicinity of mazunte, méxico. rcm = relative clutch mass (= clutch mass/gravid females body mass). n = sample size. numbers in parenthesis are standard deviations. locallity (n) clutch size egg length (mm) egg width (mm) egg mass (g) rcm san roque (11) 1.8 (0.87) 30.07 (1.72) 17.20 (0.82) 7.62 (0.46) 0.048 (0.02) escobilla (8) 2.0 (1.06) 31.56 (2.07) 17.06 (0.62) 7.73 (0.74) 0.040 (0.02) el aguacate (2) 2.5 (2.12) 29.75 (0.07) 16.85 (0.10) 7.11 (0.01) 0.060 (0.04) 123reproductive ecology of oaxaca mud turtle (kinosternon oaxacae) any reproductive trait. only rcm was correlated with clutch size (table 2). archive data showed that eggs were laid in captivity from early may to early september, with the following average measurements for 21 captive eggs: el 27.29 (± 2.81) mm, range 22-30 mm; ew 17.15 (± 1.79) mm, range 15-22 mm; em 4.28(± 1.48) g, range 2.6-7.1g. based on our combined data (field survey and archive data), nesting season probably extends from early may to early november, significantly longer than iverson’s (1986) hypothesis of mid july to an unknown date. actually, reproductive season of k. oaxacae is quite similar to that of the closely related kinosternon integrum, for which reproductive season extends from may to early november (iverson, 1999; macip-ríos et al., 2009), and k. scorpioides, which reproduce from may through november across its range (iverson, 2010). the oaxaca mud turtle shows a similar reproductive season to those turtle species in the seasonal (dry) tropics of the pacific coast of méxico, concentrating all of their reproductive activity in the rainy season (macip-ríos, 2010), which in the study area extends from may to october (trejo, 2004), even though in the study area water is available all year round in rivers, streams, ponds, and small lagoons. the smallest female with oviductal eggs (118.8 mm of cl), a proxy of minimum body size at first reproduction, was also quite similar to that of k. integrum (maciprios et al., 2009; 2011; 2013) and k. scorpioides (iverson, 2008; iverson, 2010). apparently turtles of the scorpioides group mature around 114-120 mm cl, which is large compared with others kinosternids from higher latitudes (macip-ríos, 2010). clutch size was possibly underestimated because we did not use x-ray photographs. instead we estimated clutch size by the number of eggs oviposited after oxytocine was injected, a proceeding repeated for three days if no egg was oviposited, assuming all eggs were laid. this method produced clutches of one egg to four eggs. iverson (1986) suggested a clutch size might range from two to five eggs for the oaxaca mud turtle. our estimate was similar, but we suspect that a higher clutch size could be attained, because congeners with similar body size and morphology (k. scorpioides and k. integrum) are capable of producing clutches from 2 to 10 eggs (iverson, 1999; macip-ríos et al., 2009; iverson, 2010). macip-ríos (2010) collected two gravid k. oaxacae (141 and 136 mm cl) in july 2008 from chacalapa, oaxaca (a locality 5.8 km northwest from san roque), and found clutches (estimated by x-ray photographs) of three eggs in both individuals. clutch size may actually be reduced in k. oaxacae compared with its relatives of the same genus (macipríos et al., 2013). k. oaxacae showed smaller variation in egg width (coefficient of variation = 4.07) compared with egg length (coefficient of variation, cv = 6.18), and egg mass (cv = 7.52). egg size was similar to that other related kinosternids of about the same size, which depends in part on female body size and its relation with clutch size. rcm was similar to the average reported within related species (iverson et al., 1991); however, it was lower than the calculated value for the two chacalapa females (0.065 and 0.064) reported by macip-ríos (2010). comparisons between localities and correlations between reproductive traits did not show any pattern. the strong correlation between clutch size and rcm may reflect an underestimation of clutch size. a direct correlation between clutch size and rcm represents an isometric investment of body mass by each egg of the clutch, a previously unknown pattern which may be an artifact of the sample size, since it is absent in other species of mud turtles (lovich et al., 2012; macip-ríos et al., 2013). finally, this is the first study that documents a minimum reproductive size, egg size (length, width, and mass), and reproductive output in kinosternon oaxacae. this paper also extends the known nesting season from early may to early november. it also demonstrates that the reproductive phenology and major traits are quite similar to those of k. integrum, but more data are needed to confirm the reproductive traits of this locally endemic species in oaxaca. acknowledgements we thank the mexican turtle center (cmtconanp) for led us use their facilities during this study. founding for was provided by the grant program issued by the vice rectory of research and graduate studies (viep in spanish) of the meritorious university of puebla, méxico. we also want to tank richard c. vogt for their assistance in fieldwork. turtles were collected under the permit sgpa/dgvs/04572/13 issued by the ministry of natural resources of the mexican federal government (semarnat). john b. iverson made useful comments on early drafts of this manuscript. references berry, j., iverson, j.b. 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(cords.). instituto de biología (unam)fondo oaxaqueño para la concervación de la naturaleza-wwf, méxico. van dijk p.p., iverson, j.b., rhodin, a.g.j., shaffer, h.b., bour. r. (turtle taxonomy working group). (2014): turtles of the world, 7th edition: annotated checklist of taxonomy, synonymy, distribution, and conservation status. in: conservation biology of freshwater turtles and tortoises: a compilation project of the iucn (ssc tortoise and freshwater turtles specialist group, pp. 329-479. rhodin, a.g.j., pritchard, p.c.h., van dijk, p.p., saumure, r.a., buhlmann, k.a., iverson, j.b., mittermeier, r.a. eds chelonian research monographs 5: 329-479. zar, j. (1999): biostatiscal analysis. 4th edition, prentice hall, new jersey. acta herpetologica vol. 10, n. 1 june 2015 firenze university press acta herpetologica journal of the societas herpetologica italica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 9(1): 1-14, 2014 doi: 10.13128/acta_herpetol-12988 new cranial characters in the tribe hydropsini (serpentes: dipsadidae: xenodontinae) diego o. di pietro1,*, leandro alcalde1,2, jorge d. williams1 1 sección herpetología, división zoología vertebrados, facultad de ciencias naturales y museo (unlp). paseo del bosque s/n (cp1900), la plata, buenos aires, argentina. * corresponding author. email: dipietro@fcnym.unlp.edu.ar 2 sección herpetología, área sistemática, instituto de limnología dr. r. a. ringuelet (conicet). boulevard 120 y 62 (cp1900), la plata, buenos aires, argentina submitted on: 2013, 28th june; revised on: 2013, 13th november; accepted on: 2013, 10th december abstract. we here describe the skull in four species of the three genera of the tribe hydropsini (serpentes: dipsadidae: xenodontinae): helicops infrataeniatus, h. leopardinus, hydrops caesurus and pseudoeryx plicatilis. we compare them with several genera of dipsadidae. we found that the unpaired foramen on the parabasisphenoid with anterior position is the only skull feature shared by all hydropsini genera. this feature also occurs in semi-aquatic (erythrolamphrus semiaureus) and fully-aquatic (farancia abacura) dipsadids. all species of hydrops with available skull descriptions and pseudoeryx plicatilis share four features: (1) the anterior border of the angular is higher than the posterior border of the splenial, (2) the vomerine processes of the premaxilla are long, (3) the ascending process of the premaxilla overlaps the horizontal lamina of the nasals, and (4) an anterior projection of the prefrontal is present. all species of helicops with available skull descriptions and pseudoeryx plicatilis share three features: (1) a vertical lamina of the nasal with a notch, (2) a single foramen rotundum, and (3) the presence of a ventral projection of the transverse crista of the basioccipital. finally, we found small, paired parietal foramina in most of the dipsadids studied here, which are filled with a sudan-black-positive tissue of possible nervous origin. keywords. cranial osteology, serpentes, hydropsini introduction roze (1957 a, b) was the first to propose sister relationships of the genera helicops, hydrops, and pseudoeryx on the basis of the presence of a single internasal scale and the dorsally-positioned external nares. until a series of recent molecular phylogenies were published (see below), relationships among these genera were rejected (e.g., neill, 1964; cadle, 1984; ferraresi, 1994). zaher (1999) studied the hemipenes of xenodontinae finding no useful characters to group the three genera of hydropsini. despite this, he considered that helicops, hydrops, and pseudoeryx shared two features: (1) the wide origin of the muscle adductor mandibularis externus superficialis, and (2) the viviparous reproductive mode. other studies additionally reported oviparity in hydropsini (scrocchi et al., 2005), including different species of the same genus (e.g., helicops: see cunha and nascimento, 1981; rossman, 1973, 1984). albuquerque (2002) agrees with zaher (1999) in placing helicops, hydrops, and pseudoeryx in tribe hydropsini. in all recently published molecular phylogenies, the south american xenodontinae form a clade that comprises several monophyletic units (tribes), one of which consists of the three genera of the tribe hydropsini (helicops, hydrops and pseudoeryx; vidal et al., 2000; lawson et al., 2004; zaher et al., 2009; vidal et al., 2010; pyron et al., 2011; grazziotin et al., 2012; pyron et al., 2013). complete information on the bony skull is available for nearly a third of the dipsadidae genera (i.e., 29 gen2 d.o. di pietro, l. alcalde, j.d. williams era) whereas fragmentary descriptions were published for additional 29 genera (cundall and irish, 2008). information on the bony skull of hydropsini is more complete, since descriptions are available for helicops carinicaudus, h. infrataeniatus (yuki and lema, 2005), hydrops marti, h. triangularis (albuquerque, 2002), and pseudoeryx plicatilis (cundall and rossman, 1984). a comparison of the cranial osteology among the three hydropsini genera may prove useful characters to discuss the systematic value of previously studied features and to identify new ones that could unravel relationships among species within the tribe. thus, the main goal of this work is to describe the bony skull variation of helicops infrataeniatus, h. leopardinus, hydrops caesurus, and pseudoeryx plicatilis and compare it to that of dipsadidae species (see appendix). this comprehensive comparison includes terrestrial, semi-fossorial, fossorial, semi-aquatic, fully-aquatic, and arboreal forms of dipsadidae. fig. 1. skull dorsal views. helicops infrataeniatus (a), h. leopardinus (b), pseudoeryx plicatilis (c), and hydrops caesurus (d). abbreviations: ap, ascending process of the premaxilla; f, frontal; n, nasal; ex, exoccipital; pa, parietal; paf, parietal foramina; pf, prefrontal; po, postorbital; pr, prootic; px, premaxilla; pxf, premaxillary foramen; q, quadrate; so, supraoccipital; st, supratemporal; sx, septomaxilla; tp, transverse process of the premaxilla; ?, unnamed foramina. 3cranial characters in the tribe hydropsini material and methods we studied double stained and cleared skulls of the following hydropsini species (i.e., our ingroup taxa): helicops infrataeniatus (n=5), h. leopardinus (n=5), hydrops caesurus (n=2) and pseudoeryx plicatilis (n=1). for skull comparisons we chose representatives of dipsadidae (i.e., our outgroup taxa) which appear most closely related to hydropsini in the most recent molecular phylogenies of colubroidea (zaher et al., 2009; vidal et al., 2010; pyron et al., 2011; grazziotin et al., 2012; pyron et al., 2013): hydrodynastes gigas (n = 3), erythrolamprus semiaureus (n=3), leptodeira annulata pulchriceps (n=1), oxyrhopus rhombifer (n= 2), phalotris bilineatus (n=1), philodryas patagoniensis (n=3), psomophis obtusus (n=1), sibynomorphus turgidus (n=1), thamnodynastes chaquensis (n=1), t. hypoconia (n=2), and farancia abacura (n=1). specimens are from the reptilian collection of the museo de la plata (mlp.r. and mlp. jw.), buenos aires province, argentina. the body of each voucher specimen and the corresponding skull are deposited in its respective collection, using separate numbers. detailed voucher information is listed in the appendix. we prepared the skulls using the technique of taylor and van dyke (1985). in addition to the double-stained and cleared skulls, we also studied selected species using dried skulls that were partially disarticulated (see appendix). furthermore, to identify the nervous tissue associated with particular cranial openings (e.g., parietal foramina), we dissected one specimen of helicops leopardinus and one of h. infrataeniatus and stained them with sudan black following the technique of song and parenti (1995). the terminology used throughout this study follows cundall and irish (2008) for cranial bones and grazziotin et al. (2012) for taxonomic arrangements. results snout and bones of the nasal region premaxillae. the triangular premaxilla has three processes: the paired and laterally projecting transverse process, the paired and posteriorly-directed vomerine process, and the unpaired dorsally-projecting ascending process (fig. 1a, 2a, 3a, b). the premaxilla is pierced by a pair of premaxillary foramina (fig. 1a, 2c). in hydropsini, the ascending process of the premaxilla and the anterior end of the horizontal lamina of each nasal relate to one another in two ways. in pseudoeryx plicatilis and hydrops caesurus the ascending process of the premaxilla overlaps the anterior end of the horizontal lamina of the nasals, whereas in helicops infrataeniatus and h. leopardinus these bones lack contact (fig. 3b, d). this second way, also occurs in the outgroup species erytrolamprus semiaureus, thamnodynastes chaquensis, and t. hypoconia, whereas the first way also occurs in hydrodynastes gigas and philodryas patagoniensis. in contrast to hydropsini, in some of the outgroup species (farancia abacura, leptodeira annulata, oxyrhopus rhombifer, phalotris bilineatus, psomophis obtusus, and sibynomorphus turgidus) there is contact but no overlap between the ascending process of the premaxilla and the anterior end of the horizontal nasal lamina. the shape and length of the vomerine processes are another source of variation. the two studied helicops have short processes (fig. 2a, b), as in the outgroup species hydrodynastes gigas, leptodeira annulata, psomophis obtusus, thamnodynastes chaquensis, and t. hypoconia. the other hydropsini, hydrops caesurus and pseudoeryx plicatilis, have a long vomerine processes of the premaxilla (fig. 2c, d), as in the outgroup species eryhtrolamprus semiaureus, farancia abacura, oxyrhopus rhombifer, phalotris bilineatus, and philodryas patagoniensis. sibynomorphus turgidus is the only studied species with an azygous median process (probably by fusion of left and right separate vomerine process of the other species). the shape of each vomerine process of the premaxilla can be rounded, as in helicops leopardinus and pseudoeryx plicatilis (fig. 2b, c) or more acute and pointed, as in h. infrataeniatus and hydrops caesurus (fig. 2a, d). finally, the vomerine processes of the premaxilla and the anterior processes of the septomaxilla are overlapped in all studied species. nasals. each nasal is formed by two bony laminae, the horizontal and the vertical lamina (fig. 1, 3, 4). in general, the vertical lamina has not been described except in the work of cundall and shardo (1995). the vertical lamina of each nasal bone functions as a bony septum between the left and the right nasal capsules. each horizontal lamina have anterior and posterior processes that extend from the anterior and posterior margins. the shape of the horizontal lamina, the extension and shape of the vertical lamina, and the length of the anterior nasal process vary in hydropsini (fig. 1, 3, 4). the horizontal lamina is clearly triangular (only in helicops infrataeniatus and h. leopardinus; fig. 1a, b; 4a, c) or almost square-shaped (hydrops caesurus; fig. 1d, 4g), with an intermediate rounded condition (in pseudoeryx plicatilis; fig. 1c, 4e). the lateral end of the horizontal lamina is rounded also in philodryas patagoniensis and thamnodynastes chaquensis, whereas it is square-shaped in the remaining outgroup species. both vertical lamina are emarginated in pseudoeryx plicatilis, helicops infrataeniatus and h. leopardinus (fig. 3a-c; 4b, d, f), but not in hydrops caesurus (fig. 3d, 4h). the outgroup species that have such emargination are erythrolamprus semiaureus, farancia abacura, sibynomorphus turgidus, thamnodynastes chaquensis, and t. hypoconia. vomers. these complex bones and the septomaxillae are deeply integrated on each side. each vomer has two 4 d.o. di pietro, l. alcalde, j.d. williams parts (fig. 2, 3, 5a-d): (1) a triangular-shaped vertical plate at each side of the midline of each vomer, and (2) a cup-shaped or capsular expansion that limits the vomeronasal organ and grows laterally from the vertical plate. the vertical plate bears two foramina at its caudoventral corner (fig. 5a, b). the smallest foramen is only present in h. leopardinus and the largest one occurs in all hydropsini and outgroup species. the posterior margin of the vertical plate of the vomer is straight in all hydropsini and most outgroup species (fig. 5b), whereas it is emarginated in some outgroup species (oxyrhopus rhombifer, phalotris bilineatus, and sibynomorphus turgidus). the capsular expansion has multiple dorsal foramina with the branches of the vomeronasal nerve running through it (fig. 5c). septomaxillae. the septomaxilla contacts the vertical plate of the vomer. each septomaxilla has a rounded body enclosing anteriorly the vomeronasal organ. the body of the bone projects three processes: the anterior, the lateral, and the posterior (fig. 2, 3, 5e-g). the anterior process varies from slightly bifid in helicops infrataeniatus and h. leopardinus to rounded in hydrops caesurus and pseudoeryx plicatilis. the anterior septomaxillae process is bifid also in farancia abacura, phalotris bilineatus, psomophis obtusus, sibynomorphus turgidus, both thamnodynastes fig. 2. skull ventral views. helicops infrataeniatus (a), h. leopardinus (b), pseudoeryx plicatilis (c), and hydrops caesurus (d). abbreviations: avc, anterior vidian opening; bo, basioccipital; col, columella; lf, lacrimal foramen; pb, parabasisphenoid; pvc, posterior vidian opening; v, vomer; (vl), ventral lamina of the parabasisphenoid; v2 and v3, ramus of the trigeminal nerve. other references in figure 1. 5cranial characters in the tribe hydropsini species, and leptodeira annulata, whereas it is rounded in the remaining outgroup species. the lateral process grows dorsally from the body of the septomaxilla, forming the lateral bony parts of the nasal capsule (fig. 3). the posterior end of the posterior septomaxillary process articulates with the frontal whereas the lateral part of this process curves ventro-anteriorly. the features of the septomaxillae did not vary in any ingroup or outgroup species. braincase parabasisphenoid (sphenoid bone). the irregular, almost octagonal-shaped parabasisphenoid forms the skull floor across the otic and orbital regions and functions as part of the palate by completing the space between pterygoids and palatines (fig. 2). interestingly, the parabasisphenoid consists of a double-floored horizontal bone lamina (dorsal and ventral) connected through a low midline vertical lamina (fig. 3a-c). the ventral horizontal lamina constitutes the exposed face of the bone and forms the anterior end of the parabasisphenoid (fig. 2). the dorsal horizontal bone lamina is visible in lateral view only (fig. 3a-c). this lamina fills the space between the two trabecula crani. the very low interorbital septum projects dorsally from the dorsal lamina of the parabasisphenoid. in hydrops caesurus, fig. 3. skull lateral views. helicops infrataeniatus (a), h. leopardinus (b), pseudoeryx plicatilis (c), and hydrops caesurus (d). abbreviations: dl, dorsal lamina of the parabasisphenoid; fr, foramen rotundum; ls, laterosphenoid; of, optic foramen; ov, oval window; uf, unnamed foramen; vf, vagal foramen; vl, ventral lamina of the parabasisphenoid; vof, vomerine foramen; vp, vomerine process; vw, vertical wall of the parabasisphenoid. other references in figures 1 and 2. 6 d.o. di pietro, l. alcalde, j.d. williams the vertical lamina and the short septum are completely obscured by the well-developed descending flanges of the frontals (fig. 3d). in the other hydropsini, the descending flanges of the frontals are less developed. consequently, the short interorbital septum is less visible in hydrops caesurus than in helicops and pseudoeryx plicatilis (see the description of the frontals). the anterior projection of the parabasisphenoid varies. it can be wide with three points and double notched, as in h. leopardinus and hydrops caesurus (fig. 2b, d), or wide with a single point and no notches, as in h. infrataeniatus (fig. 2a). in contrast, pseudoeryx plicatilis has a narrow parabasisphenoid anterior projection with a single point (fig. 2c). in the outgroups, a narrow anterior end of the parabasisphenoid with a single point occurs in leptodeira annulata, oxyrhopus rhombifer, phalotris bilineatus, and sibynomorphus turgidus, a wide anterior end with three points occurs in erythrolamprus semiaureus, philodryas patagoniensis, and both studied thamnodynastes, and, finally, a rounded anterior end occurs in hydrodynastes gigas and psomophis obtusus. all studied hydropsini display a short axial ridge along the mid-ventral face of the parabasisphenoid. there are five paired foramina along each side of the median ridge. the identity of such foramina is uncertain and undescribed in the literature, except for the anterior-most and posterior-most largest pairs of foramina representing the anterior and the posterior vidian openings, respectively (fig. 2a). the anterior vidian openings can vary in their position. they open by the suture between the parietal and parabasisphenoid in hydrops caesurus, pseudoeryx plicatilis (fig. 2c, d), in one specimen of h. infrataeniatus (mlp.r.5624), and also in the outgroup species oxyrhopus rhombifer. these openings are entirely embedded in the parabasisphenoid in helicops leopardinus and the remaining specimens of h. infrataeniatus (fig. 2a, b), as well as in most outgroup species. all studied hydropsini have an unpaired midlineplaced foramen that is positioned anteriorly with respect to the anterior vidian openings (fig. 2a-d). such an unpaired foramen occurs also in the outgroups erythrolamprus semiaureus and farancia abacura. in philodryas patagoniensis, the unpaired parabasisphenoid foramen is found between the anterior and posterior vidian openings. the remaining outgroup taxa lack this foramen. finally, the parabasisphenoid varies in presence/ absence of lateral projections at its postorbital level. these lateral projections are present in helicops infrataeniatus and h. leopardinus (fig. 2a, b), but are absent in hydrops caesurus and pseudoeryx plicatilis (fig. 2c, d). among outgroup species, such lateral projections are found in fig. 4. nasal dorsal views. helicops infrataeniatus (a), h. leopardinus (c), pseudoeryx plicatilis (e), and hydrops caesurus (g). lateral views of the right nasals of helicops infrataeniatus (b), h. leopardinus (d), pseudoeryx plicatilis (f), and hydrops caesurus (h). abbreviations: ap, anterior process of the nasal; hl, horizontal lamina of the nasal; nn, nasal notch; pp, posterior process of the nasal; vl, vertical lamina of the nasal. figure is not scaled. the arrows point toward anterior. 7cranial characters in the tribe hydropsini erythrolamprus semiaureus, hydrodynastes gigas, philodryas patagoniensis, thamnodynastes chaquensis, and t. hypoconia. prefrontals. in hydrops and pseudoeryx species, the dorsal margin of the prefrontal projects forming a rounded anterior process (fig. 1c, d). this process is absent in both studied helicops species (fig. 1a, b). in the outgroup species, three conditions of the prefrontal anterior process exist: (1) it is absent in leptodeira annulata and phalotris bilineatus; (2) it is short (i.e., as long as or smaller than its base) in most outgroup species; and (3) it is long (i.e., clearly longer than its base) in oxyrhopus rhombifer. the prefrontal-frontal articulation is strongly curved (v-shaped) in h. leopardinus (fig. 1b), whereas it is almost straight in all other examined species. finally, the anterior wall of the prefrontal is pierced by one large lacrimal foramen, two small foramina (sometimes absent), and one dorsal unnamed foramen (fig. 1, 2, 3). frontals. the anterior margin of each frontal is irregular and the lateral margin is expanded, forming part of the orbit roof, between the prefrontal and the postorbital bones. the descending flange of the frontal closes the orbit medially and also forms the anterior and dorsal margin of the optic foramen (fig. 3). these flanges reach the parabasisphenoid in hydrops caesurus (fig. 3d) where they are larger than in other hydropsini. large descending flanges of the frontals also occur in the outgroup species farancia abacura, leptodeira annulata, oxyrhopus rhombifer, phalotris bilineatus, psomophis obtusus, and sibynomorphus turgidus. shorter descending flanges that do not reaches the level of the parabasisphenoid occur in the remaining outgroup species, and in the two helicops species and pseudoeryx plicatilis (fig. 3a-c). the anterior end of each ventral flange of the frontal forms a cotyle to articulate with the condylus of the posterior process of the septomaxilla. the frontal-parietal suture is straight in h. leopardinus (fig. 1b), whereas it is oblique and irregular in the other hydropsini (fig. 1a, c, d). the naso-frontal articulation is absent in all hydropsini we studied. within the present work, presence of such articulation was verified only for semi-fossorial and fossorial (oxyrhopus rhombifer, and phalotris bilineatus respectively) species, and also for the aquatic farancia abacura as was first mentioned by haines (1967). postorbitals. each small and arched postorbital surrounds the posterodorsal end of the orbit. a full contact between postorbital and parietal is only found in hydrops caesurus, but not in the other hydropsini (fig. 1d, 3) or the outgroup species. the postorbital extends to the cranial floor at the level of the orbit (i.e. without overpassing the floor) in hydropsini species (fig. 3) and most of the outgroup species, whereas it overpasses the cranial floor in erythrolamprus semiaureus, hydrodynastes gigas, leptodeira annulata, and sibynomorphus turgidus. fig. 5. left vomer and left septomaxilla of helicops leopardinus. lateral (a), medial (b), dorsal (c), and ventral (d) views of the left vomer. dorsal (e), ventral (f), and lateral (g) views of the left septomaxilla. the arrows point toward anterior. abbreviations: aps, anterior process of the septomaxilla; ce, capsular expansion of the vomer; lps, lateral process of the septomaxilla; lvs, large vomer foramen; pps, posterior process of the septomaxilla; sb, septomaxillary body; svf, small vomer foramen; vp, vertical plate of the vomer; vnf, vomeronasal nerve foramina. figure is not scaled. 8 d.o. di pietro, l. alcalde, j.d. williams parietal. we found three variable traits in the parietal of hydropsini: (1) a contact between the parietal and the postorbital (see above the description of the postorbitals); (2) a parietal participation in the closure of the orbital margins; and (3) presence or absence of traces of the suture between the left and the right parietals. the orbital projections of the parietal are present in all hydropsini, but they reach the orbit only in helicops infrataeniatus and hydrops caesurus (fig. 1a, d), whereas helicops leopardinus and pseudoeryx plicatilis lack the parietal contribution to the orbits (fig. 1b, c). the parietal participation to the orbits occurs in all outgroup species, except farancia abacura, oxyrhopus rhombifer, and sibynomorphus turgidus. interestingly, some specimens of h. leopardinus have unknown independent ossifications instead of the orbital projections of the parietal. helicops infrataeniatus, h. leopardinus and pseudoeryx plicatilis have an incomplete fusion between the left and the right parietal, as indicated by an anterior median notch that bisects the frontals at the level of the parietal (fig. 1a-c). in contrast, hydrops caesurus has no such suture (fig. 1d). phalotris bilineatus, philodryas patagoniensis, psomophis obtusus, and thamnodynastes chaquensis are the only outgroup species possessing the anterior notch of the parietal. interestingly, one pair of small foramina is sometimes found at each side of the midline of the parietal and near the posterior end of the bone in helicops infrataeniatus, h. leopardinus, and pseudoeryx plicatilis (fig. 1b, c). although presence of such parietal foramina varies within these species with only one foramen opening in some cases, they were completely absent in hydrops caesurus. the parietal foramina represent very small areas where the cranial cavity connects to the exterior. blood vessels are absent from this site. no nerves were found to pass through the foramina that were filled with a sudan-blackpositive tissue. some specimens have one or two additional foramina irregularly disposed near the parietal foramina. parietal foramina occur in all outgroup species, except farancia abacura, hydrodynastes gigas, phalotris bilineatus, psomophis obtusus, and sibynomorphus turgidus. finally, all studied taxa have a sagittal crest of similar height that bifurcates anteriorly, forming an y-shaped pattern (fig. 1). prootics. the oval window and some additional important foramina open into the prootics. two of them are always present and constitutes the openings for the maxillary (v3) and mandibular (v2) branches of the trigeminal nerve. these openings are separated by the lamellar laterosphenoid, a bone that is incorporated to the prootic (fig. 3). the v2 foramen opens either on the prootic (all hydropsini and most of the outgroup species; fig. 3) or on the prootic-parietal suture (leptodeira annulata, oxyrhopus rhombifer, and sibynomorphus turgidus). other openings vary bilaterally (two to four small foramina ventral to v2, v3 and the oval window; fig. 3). the dorsal face of the bone is opened by a small unnamed foramen that is consistently present in all species. this foramen is tapered by the supratemporal in most hydropsini and outgroups species, whereas it is exposed by the short supratemporals in hydrops caesurus, leptodeira annulata, phalotris bilineatus, and sibynomorphus turgidus. supraoccipital. the supraoccipital bone has a low dorsal crest that is continuous with the sagittal crest of the parietal (fig. 1). the supraoccipital is excluded from the margins of the foramen magnum by the exoccipitals (fig. 1, 6). many small unnamed foramina are observed on the dorsum of the supraoccipital. two axially arranged pairs are almost always present. in addition, a medial pair and one unpaired medial foramen can occur in helicops leopardinus and hydrops caesurus. in the latter species the last foramen is slightly displaced towards the left. basioccipital. the hexagonal-shaped basioccipital forms the most posterior part of the skull floor and integrates the main body of the occipital condyle (fig. 2, 3, 6). the suture between the parabasisphenoid and the basioccipital is straight with no projections in hydrops caesurus (fig. 2d) and the outgroup species farancia abacura and oxyrhopus rhombifer, whereas it bears a mid-posterior projection in the remaining hydropsini (fig. 2a-c) and outgroups. the basioccipital has a characteristic transverse crista that projects ventrally from the body of the bone. this crista is present in helicops infrataeniatus, h. leopardinus and pseudoeryx plicatilis (figs. 2a-c, 6a-c) and in some of the outgroup species (farancia abacura, hydrodynastes gigas, philodryas patagoniensis, and psomophis obtusus), whereas it is absent in hydrops caesurus (figs. 2d, 6d) and the remaining outgroup species. exoccipitals. these bones close the foramen magnum dorsolaterally, forming the dorsolateral parts of the occipital condyle, and close the oval window posteriorly (fig. 2, 3, 6). the foramen rotundum opens ventrally to the oval window, whereas the hypoglossal foramina and the vagal foramen open between the rotundum and the magnum foramen (fig. 3b, 6). the foramen rotundum can be simple or double. it is simple in helicops infrataeniatus, h. leopardinus, and pseudoeryx plicatilis and the outgroup species farancia abacura, hydrodynastes gigas, philodryas patagoniensis, and both studied thamnodynastes species. it is double in hydrops caesurus and the outgroup species erythrolamprus semiaureus, leptodeira annulata, oxyrhopus rhombifer, phalotris bilineatus, psomophis obtusus, and sibynomorphus turgidus. 9cranial characters in the tribe hydropsini palatomaxillary arch maxillae. each maxilla bears a pair of posterior processes, the medial and the lateral, at the site where the maxilla articulates with the ectopterygoid. the lateral process is always rounded, whereas the medial process is either rounded (pseudoeryx plicatilis and hydrops caesurus; fig. 7f, h) or square-shaped (helicops infrataeniatus and h. leopardinus; fig. 7b, d). the medial process of the outgroup taxa is either rounded (farancia abacura, phalotris bilineatus, sibynomorphus turgidus, thamnodynastes hypoconia) or squared-shaped (erythrolamprus semiaureus, hydrodynastes gigas, oxyrhopus rhombifer, philodryas patagoniensis, psomophis obtusus, and thamnodynastes chaquensis). leptodeira annulata lacks the medial process of the maxilla. the palatine process of the maxilla is always triangular with a rounded to slightly pointed tip (fig. 7b). palatines. the posterior borders of the palatines, that articulate with the pterygoids, are v-shaped (fig. 7a, c, e, g). within the ingroup, no important differences have been noted in relation to the square-shaped choanal processes (fig. 7a). the maxillary process, however, varies from long and triangular-shaped (in hydrops caesurus, helicops infrataeniatus and h. leopardinus; fig. 7a, c, g) to short and square-shaped (in pseudoeryx plicatilis; fig. 7e). the maxillary process of the palatine is square-shaped also in hydrodynastes gigas, leptodeira annulata, phalotris bilineatus, psomophis obtusus, and sibynomorphus turgidus, whereas it is longer than wide in erythrolamprus semiaureus, farancia abacura, oxyrhopus rhombifer, philodryas patagoniensis, thamnodynastes chaquensis, and t. hypoconia. in all taxa, the maxillary processes are placed slightly anterior to the choanal processes (fig. 7). ectopterygoids. we found only one ectopterygoid feature that varies within the ingroup: the length of the anterior processes by which the ectopterygoid embraces the maxilla. these processes may be of subequal length (helicops infrataeniatus, h. leopardinus and pseudoeryx plicatilis; fig. 7a, c, e) or the medial process may be longer than the lateral process (hydrops caesurus; fig. 7g). this latter state also occurs in most of the outgroup species: erythrolamprus semiaureus, hydrodynastes gigas, oxyrhopus rhombifer, phalotris bilineatus, philodryas patagoniensis, psomophis obtusus, thamnodynastes chaquensis, and t. hypoconia. finally, the lateral process is longer than the medial process in the outgroup species farancia abacura, leptodeira annulata, and sibynomorphus turgidus (but this feature was not observed in hydropsini). the shape of the lateral process of the ectopterygoid is also variable. this process is square-shaped in all studied hydropsini and in most of the outgroup species (fig. 7a, c, e, g), and is rounded only in leptodeira annulata and sibynomorphus turgidus. pterygoids. the teeth-bearing pterygoid is the largest and most posterior element of the palatomaxillary arch (fig. 7). we observed no variable features concerning this bone. fig. 6. skull posterior view. helicops infrataeniatus (a), h. leopardinus (b), pseudoeryx plicatilis (c), and hydrops caesurus (d). abbreviations: exc, extracolumella; fm, foramen magnum; oc, occipital condyle; sfp, stapedial footplate. other references as in figures 1-3. 10 d.o. di pietro, l. alcalde, j.d. williams bones of the suspensorium supratemporals. the width and length of the supratemporal bones vary within hydropsini. supratemporals are of similar width along their entire length in helicops infrataeniatus and h. leopardinus, as well as in erythrolamprus semiaureus, hydrodynastes gigas, oxyrhopus rhombifer, and psomophis obtusus. the anterior half of the supratemporals is wider than the posterior half in hydrops caesurus and pseudoeryx plicatilis (fig. 1), and the remaining outgroup species. we found three conditions of the relative length of the supratemporal bone, two of which occur in hydropsini: (1) helicops infrataeniatus, h. leopardinus, and pseudoeryx plicatilis have long supratemporals in which the anterior margin of the bone reaches or exceeds the parietal-prootic suture, and the posterior margin of the bone runs behind the posterior margin of exoccipitals (fig. 1a-c, 3a-c); and (2) hydrops caesurus has short supratemporals that never extend posterior than exoccipitals and do not reach the parietal-prootic suture (fig. 1d, 3d). the second condition also occurs in phalotris bilineatus that has dorsalized supratemporals (and, therefore, the length of the supratemporals should be assessed in a dorsal view of the skull). the third condition was found in leptodeira annulata and sibynomorphus turgidus, which have very short supratemporals not reaching the middle of the prootic. the remaining outgroup taxa have long supratemporals. quadrates. each quadrate has an ovoid-shaped bony process to receive a pair of ligaments, one projecting from the distal end of the stapes, and the other projecting from the prootic. such bony processes are surrounded by alcian-blue-positive capsules (fig. 2, 3c). the quadrate displays only very little variability in all studied ingroup and outgroup species. columellae. the rod-shaped, proximally-expanded, and partially ossified columella runs between the oval window and the quadrate. the columella contacts the quadrate through a ligament (fig. 2, 3). the proximal expansion of the stapes forms the ovoid-shaped stapedial footplate that almost completely covers the oval window. the cartilaginous distal portion of the columella (extracolumella) is alcian-blue-positive and is feather-shaped (fig. 2, 6). the bony part of the columella thus represents half (one specimen of h. leopardinus mlp.r.5627 and hydrops caesurus) or two-thirds (remaining hydropsini and outgroup taxa) of the total length of the structure (columella and extracolumella). mandibles compound bones. three distinctive regions can be recognized: the articular region, the retroarticular process, and the adductor fossa (fig. 8a, e, f). in all cases, the articular region represents the articular facet, is dorsally concave, runs perpendicular to the long axis of the mandible, and is shorter than the blunt retroarticular process. the studied species of hydropsini vary in the height of the medial margin of the adductor fossa (or mandibular fossa). although all studied species have the surangular crest taller than the prearticular crest, the grade of development of the prearticular crest may be expressed in three character conditions. the prearticular crest of the adductor fossa is clearly lower than the surangular crest in helicops infrataeniatus and h. leopardinus (fig. 8a, b), whereas hydrops caesurus is the only species having both crests approximately of the same height (the prearticular crest is slightly lower than the surangular crest) (fig. 8d). the prearticular crest in pseudoeryx fig. 7. palate left side. dorsal (a, c, e, g) and ventral (b, d, f, h) views. helicops infrataeniatus (a, b), h. leopardinus (c, d), pseudoeryx plicatilis (e, f), and hydrops caesurus (g, h). abbreviations: chp, choanal process; ec, ectopterygoid; mp, maxillary process; mx, maxilla; p, pterygoid; pp, palatine process; pt, palatine. 11cranial characters in the tribe hydropsini plicatilis shows an intermediate condition (not as low as in helicops spp. nor as high as in hydrops caesurus) (fig. 8c). in some dipsadids (erythrolamprus semiaureus, hydrodynastes gigas, psomophis obtusus, thamnodynastes chaquensis, and t. hypoconia), we found the prearticular crest lower than the surangular crest, as in helicops. in the other studied dipsadids (farancia abacura, leptodeira annulata, oxyrhopus rhombifer, phalotris bilineatus, and sibynomorphus turgidus), the prearticular crest shows an intermediate condition, as in pseudoeryx plicatilis. angulars. the triangular-shaped angulars do not contact the dentaries and the meckel’s grooves are very well developed (fig. 8e). the angular houses the posterior mylohyoid foramen that, only in h. infrataeniatus, is clearly bigger than the anterior mylohyoid foramen (fig. 8e). in the remainder hydropsini (fig. 8f, g, h) and all outgroup species, the foramina are almost equal in size. splenials. the triangular-shaped splenials are slightly smaller than the angulars. they contact the dentaries and angulars and bear the anterior mylohyoid foramen (fig. 8e). the extent of the contact area between splenial and angular can vary. both bones may have the same height at the contact point, as in helicops infrataeniatus and h. leopardinus (fig. 8e, f), and also erythrolamprus semiaureus, farancia abacura, hydrodynastes gigas, philodryas patagoniensis, psomophis obtusus, and thamnodynastes chaquensis. in contrast, the anterior border of the angular is higher than the posterior border of splenial in hydrops caesurus and pseudoeryx plicatilis (fig. 8g, h), and also phalotris bilineatus, leptodeira annulata, oxyrhopus rhombifer, sibynomorphus turgidus and thamnodynastes hypoconia. dentaries. these v-shaped bones have two processes, the posterodorsal (dentigerous) and the posteroventral one (fig. 8). in all studied species, the mental foramen and the foramen of the mandibular branch of the trigeminal nerve are of equal size (fig. 8a). discussion although hydropsini is monophyletic in all recently published molecular phylogenies, relationships within the tribe remain controversial, as hydrops appears to form a clade with helicops (zaher et al., 2009; pyron et al., 2010; grazziotin et al., 2012) or with pseudoeryx (vidal et al., 2000, 2010; pyron et al., 2013). in relation to the monophyly of hydropsini, we found only one feature of the bony skull shared by the three genera of the tribe: the unpaired foramen on the parabasisphenoid placed anterior to the anterior vidian openings. this feature occurs also in the semi-aquatic erythrolamprus semiaureus, and the highly aquatic farancia abacura. with respect to the internal relationships within the tribe, our observations support the relationship of hydrops with pseudoeryx. hydrops caesurus and other species of the genus (hydrops martii, and h. triangularis) with published skull descriptions (albuquerque, 2002) share four feafig. 8. right mandible. lateral (a-d) and medial (e-h) views. helicops infrataeniatus (a, e), h. leopardinus (b, f), pseudoeryx plicatilis (c, g), and hydrops caesurus (d, h). abbreviations: af, adductor fossa; an, angular; amf, anterior mylohyoid foramen; arr, articular region; cb, compound bone; dn, dentary; ftn, foramen trigeminal nerve; mf, mental foramen; mg, meckel´s groove; pda, posterodorsal arm of the dentary; pmf, posterior mylohyoid foramen; pva, posteroventral arm of the dentary; rp, retroarticular process; sp, splenial. 12 d.o. di pietro, l. alcalde, j.d. williams tures with pseudoeryx plicatilis, and none with the helicops species studied here (helicops infrataeniatus and h. leopardinus) or for which published skull descriptions exist (h. carinicaudus: yuki and lema, 2005). however, helicops species and pseudoeryx plicatilis share only three features. helicops species are characterized by four features (putative synapomorphies): (1) a pointed lateral margin of the horizontal lamina of the nasal (unambiguous), (2) no anterior projection of the prefrontal, (3) short vomerine processes of the premaxilla, and (4) a supratemporal of similar width across its length. the lateral projections of the parabasisphenoid present in helicops also occur in the two species of hydrops described by albuquerque (2002) and, therefore, they are not a synapomorphy of the species of helicops. in contrast to the helicops species studied here, helicops carinicaudus studied by yuki and lema (2005) displays a contact between the ascending process of the premaxilla and the anterior end of the horizontal lamina of the nasal. the studied species of hydrops share three features: (1) short supratemporals, (2) both lateral and medial walls of the adductor fossa of same height (unambiguous), and (3) a full bone contact between the parietal and the postorbital (unambiguous). other features (e.g., vertical lamina of the nasals not emarginated, dorsal foramen of the prootic not tapered by the supratemporal, and straight suture between parabasisphenoid and the basioccipital) are not shared by the hydrops species described by albuquerque (2002). two characters are unique to pseudoeryx plicatilis: (1) an intermediate development of the medial margin of the adductor fossa, and (2) a rounded lateral end of the horizontal lamina of the nasal. the narrow parabasisphenoid anterior projection with a single point found in pseudoeryx plicatilis is also found in hydrops triangularis (albuquerque, 2002). hydrops species and pseudoeryx plicatilis share four features: (1) an angular that is higher than the splenial at the level of the common suture of these bones, (2) long vomerine processes of the premaxilla, (3) an ascending process of the premaxilla that overlaps the anterior end of the horizontal lamina of the nasal, and (4) the presence of a anterior projection of the prefrontal. the medial articular process of the maxilla is rounded in the hydrops and the pseudoeryx species studied here, but not in the hydrops species studied by albuquerque (2002). when considering the species studied here together with those for which are available skull descriptions, helicops and hydrops share no features uniquely. within the sample studied by us, we found that they share a long and triangular-shaped maxillary process of the palatine, but this was not found in the species of helicops and hydrops studied by yuki and lema (2005) and albuquerque (2002), respectively. helicops species and pseudoeryx plicatilis share three features: (1) a transverse crista of the basioccipital projected ventrally, (2) a vertical lamina of the nasal with a notch (absent in hydrops caesurus and not described in the literature for the other species of helicops and hydrops), and (3) a single foramen rotundum (double in hydrops caesurus, and not described in the literature for helicops carinicaudus, hydrops marti, and hydrops triangularis). other features shared by helicops and pseudoeryx in our study (e.g., medial and lateral processes of the ectopterygoid of same length, anterior notch of the parietal present, and parabasisphenoid-basioccipital suture projected mid-posteriorly) are not shared by other species of these genera (albuquerque, 2002; yuki and lema, 2005). finally, we found parietal foramina in the dipsadids erythrolamprus semiaureus, helicops infrataeniatus, h. leopardinus, leptodeira annulata pulchriceps, philodryas patagoniensis, pseudoeryx plicatilis, thamnodynastes chaquensis, t. hypoconia, and oxyrhopus rhombifer. the unpaired parietal foramen of other tetrapods is usually filled by the sensory anterior (third eye) and the secretory posterior (pineal gland or epiphysis) organs (quay, 1979). the parietal foramen is a midline skull roof opening that communicates with the cranial cavity independent of the presence of a pineal eye (crumly, 1982). crumly (1982) noted that, in contrast to previous evidence (e.g., gaffney, 1975), a parietal foramen is present in many testudinids. although the loss of the parietal foramen is considered one of the ophidian synapomorphies (e.g., gauthier et al., 1988; lee and scanlon, 2002), we observed small paired foramina in our taxa where the parietal foramen usually opens in other lepidosaurians. apparently, neither nerves nor blood vessels pass through these foramina, although the foramina were filled by a sudan black-positive tissue, probably of nervous origin. these foramina are variably present within some species (e.g., h. infrataeniatus), whereas in other species they are either consistently present (e.g., h. leopardinus) or consistently absent (e.g., hydrops). these features should be evaluated with caution given the small sample sizes studied. parietal foramina have also been reported in pituophis catenifer (bullock and tanner, 1966), phalotris matogrossensis (lema et al., 2005), helicops carinicaudus (yuki and lema, 2005), notechis scutatus, and atractus erythromelas (cundall and irish, 2008). these cranial-roof openings need further study in order to clarify their function and taxonomic value. interestingly, the pineal organ of snakes is composed solely of pinealocytes, which presumably have no sensory function (ekström and meissl, 2003). 13cranial characters in the tribe hydropsini acknowledgements d. di pietro thanks cic (buenos aires) and conicet for the doctoral scholarship. l. alcalde thanks conicet for support of his research. the authors thank the organismo provincial para el desarrollo sostenible de la provincia de buenos aires (opds, disposición n°003/2011) for collecting permits. to the reviewers and editors which have improved the manuscrit during the revision process. references albuquerque, n.r. 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(1985): revised procedures for staining and clearing small fishes and other vertebrates for bone and cartilage study. cybium 9: 107-109. vidal, n., kindl, s.g., wong, a., hedges, s.b. (2000): phylogenetic relationships of xenodontine snakes inferred from 12s and 16s ribosomal rna sequences. mol. phylogenet. evol. 14: 389-402. vidal, n., dewinter, m., gower, d.j. (2010): dissecting the major american snake radiation: a molecular phylogeny of the dipsadidae bonaparte (serpentes: caenophidia). c. r. biologies 333: 48-55. yuki, r.n., lema, t. (2005): análise comparativa entre as cobras d’água meridionais (helicops carinicaudus (wied, 1825) e h. infrataeniatus jan, 1865) com a descrição do crânio e hemipênis (serpentes: colubridae: xenodontinae). comun. mus. ciênc. tecnol. pucrs, sér. zool. 18: 85-128. zaher, h. (1999): hemipenial morphology of the south american xenodontine snakes, with a proposal for a phylogenetic xenodontinae and a reappraisal of colubroid hemipenes. bull. am. mus. nat. hist. 240: 1-168. zaher, h., grazziotin, f.g., cadle, j.e., murphy, r.w., moura-leite, j.c., bonatto, s.l. (2009): molecular phylogeny of advanced snakes (serpentes: caenophidia) with an emphasis on south america xenodontinaes: a revised classification and descriptions of new taxa. pap. av. zool. 49: 115-153. appendix studied specimens and voucher information. references: ds&c (double stained and cleared skull), d (dried skull). erythrolamprus semiaureus (dipsadidae: xenodontinae: xenodontini: semi-aquatic) from punta lara (buenos aires province, argentina): 1 male (tl: 642 mm, voucher mlp. jw.1549, ds&c skull mlp.r.5629); 2 females (tl: 1067mm, voucher mlp.jw.1548, ds&c skull mlp.r.5630; tl: 1150 mm, voucher mlp.jw.1831, ds&c skull mlp.r.5655); farancia abacura (dipsadidae incertae sedis: fully-aquatic) from alaucha county (florida, usa): 1 male (tl: 772 mm, voucher mlp. jw.345, ds&c skull mlp.r.5651); helicops infrataeniatus (dipsadidae: xenodontinae: hydropsini: fully-aquatic) from isla del ibicuy (entre ríos province, argentina): 1 male (tl: 645 mm, voucher mlp.r.5017, ds&c skull mlp.r.5625); 2 females (tl: 543 mm, voucher mlp.r.5191, ds&c skull mlp.r.5624; tl: 487 mm, voucher mlp.r.5192, ds&c skull mlp.r.5645); helicops infrataeniatus from punta lara (buenos aires province, argentina): 1 female (tl: 335 mm, voucher mlp.jw.1528, ds&c skull mlp.r.5689); helicops infrataeniatus from unknown locality: d skull (mlp.r.5957); helicops leopardinus (dipsadidae: xenodontinae: hydropsini: fully-aquatic) from bella vista (corrientes province, argentina): 4 females (tl: 444 mm, voucher mlp.jw.633, ds&c skull mlp.r.5626; tl: 529 mm, voucher mlp.jw.632, ds&c skull mlp.r.5627; tl: 364 mm, voucher mlp.r.5643, ds&c skull mlp.r.5644; tl: 407 mm, voucher mlp.jw.631, ds&c skull mlp.r. 5654); 1 male (tl: 680 mm, voucher mlp. jw.668, ds&c skull mlp.r.5690); hydrodynastes gigas (dipsadidae: xenodontinae: hydrodynastini: semi-aquatic) 2 specimens from from unknown locality: d skull (mlp.r.5638), d skull (mlp.r.5954) with no voucher specimen; hydrodynastes gigas from ituzaingó (corrientes province, argentina): 1 male (tl: 980 mm, voucher mlp.r.5649, ds&c skull mlp.r.5650); hydrops caesurus (dipsadidae: xenodontinae: hydropsini: fully-aquatic) from yaciretá (corrientes province, argentina): 1 female (tl: 436 mm, voucher mlp.jw.1944, ds&c skull mlp.r.5628); hydrops caesurus from isla yaciretá (río paraná, paraguay): 1 female (tl: 610 mm, voucher mlp.jw.1693, ds&c skull mlp.r.5688); leptodeira annulata pulchriceps (dipsadidae: dipsadinae: imantodini: arboreal) from unknown locality: 1 male (tl: 502mm, voucher mlp.r.5642, ds&c skull mlp.r.5648); oxyrhopus rhombifer (dipsadidae: xenodontinae: pseudoboini: semi-fossorial) from medanos (buenos aires province, argentina): 1 female (tl: 895 mm, voucher mlp.jw.932, ds&c skull mlp.r.5631); oxyrhopus rhombifer from balcarce (buenos aires province, argentina): 1 female (tl: 763 mm, voucher mlp.jw.1626, ds&c skull mlp.r.5646); phalotris bilineatus (dipsadidae: xenodontinae: elapomorphini: fossorial) from sierra de la ventana (buenos aires province, argentina): 1 female (tl: 324 mm, voucher mlp.r.5640, ds&c skull mlp.r.5641); philodryas patagoniensis (dipsadidae: xenodontinae: philodryadini: terrestrial) from tandil (buenos aires province, argentina): two males (tl: 753 mm, voucher mlp.jw.092, ds&c skull mlp.r.5632; tl: 732 mm, voucher mlp.jw.093, ds&c skull mlp.r.5633); philodryas patagoniensis from sierra de la ventana (buenos aires province, argentina): 1 male (tl: 659 mm, voucher mlp.r.5955, ds&c skull mlp.r.5956); pseudoeryx plicatilis (dipsadidae: xenodontinae: hydropsini: fully-aquatic) from paraguay: 1 female (tl: 658 mm, voucher mlp.r.5572, ds&c skull mlp.r.5634); psomophis obtusus (dipsadidae: xenodontinae: psomophiini: terrestrial) from esteros del iberá (corrientes province, argentina): 1 female (tl: 372 mm, voucher mlp.r.5455, ds&c skull mlp.r.5639); sibynomorphus turgidus (dipsadidae: dipsadinae: dipsadini: terrestrial) from ibera (corrientes province, argentina): 1 female (tl: 492 mm, voucher mlp.r.5441, ds&c skull mlp.r.5647); thamnodynastes chaquensis (dipsadidae: xenodontinae: tachymenini: terrestrial) from alto paraná (argentina): 1 female (tl: 609 mm, voucher mlp.jw.1888, ds&c skull mlp.r.5635); thamnodynastes hypoconia (dipsadidae: xenodontinae: tachymenini: terrestrial) from alto paraná (argentina): 1 female (tl: 567 mm, voucher mlp.jw.1887, ds&c skull mlp.r.5636); thamnodynastes hypoconia from azára, apóstoles (misiones province, argentina): 1 male (tl: 610mm, voucher mlp.r.5652, ds&c skull mlp.r.5653). acta herpetologica vol. 8, n. 2 december 2013 firenze university press journal of the societas herpetologica italica acta herpetologica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 7(1): 163-169, 2012 evidence of phoresy by leeches (hirudinoidea) on rhinella abei (anura: bufonidae) in the atlantic rainforest in the state of santa catarina, southern brazil thiago maia-carneiro1,*, thiago arnt dorigo1, milena wachlevski2, carlos frederico duarte rocha1 1 departamento de ecologia, universidade do estado do rio de janeiro, rua são francisco xavier 524, 20550-019, rio de janeiro, rj, brazil *corresponding author. e-mail: thiagomaianc@gmail.com 2 pós-graduação em ecologia, universidade federal de santa catarina, campus universitário reitor joão davi ferreira lima trindade, 88040-970, florianopolis, sc, brasil submitted on: 2012, 26th march; revised on: 2012, 17th april; accepted on: 2012, 29th may. abstract. in the atlantic rainforest of the parque estadual da serra do tabuleiro, state of santa catarina, southern brazil, we found rhinella abei (bufonidae) infested by leeches (hirudinoidea). we captured 27 toads on the margin of a lagoon both inside and outside water in a survey carried out during one night, and 13 of which had a total of 30 leeches (mean intensity of infestation = 2.3 ± 1.3). we did not observe wounds, scars and/or hemorrhages caused by the leeches on the toads examined, no hemorrhaging after the removal of the leeches, and the leeches removed from the toads were empty of blood. this evidence led us to theorize that the leeches were not parasitizing the toads but had a phoretic relationship. the leeches were found on both dorsal and ventral surfaces (13 on each) of the toads and were predominantly on the toads’ axils followed by back and thighs. the average snout-vent length of the toads was 69.2 ± 5.3 mm and their average body mass was 27.1 ± 6.9 g. the number of leeches found on a toad was not related to its snout-vent length or body mass. we are unaware of any previous records of leeches using anurans as dispersal agents, as suggested in the present study. keywords. leeches, phoresy, rhinella, brazil. in recent decades records of phoretic associations among aquatic organisms have become more common (calisto and goulart, 2000). phoresy occurs when an organism uses another as a temporary “host” in order to disperse (see davies et al., 1982; khan and frick, 1997; calisto and goulart, 2000; bajerlein and bloszyk, 2004; eng et al., 2005; santos et al., 2005; zawal, 2006; beaulieu et al., 2008). the phenomenon has been frequently reported for invertebrates such as diptera (calisto and goulart, 2000), acari (bajerlein 164 t. maia-carneiro et al. and bloszyk, 2004; zawal, 2006; beaulieu et al., 2008), nematoda (eng et al., 2005), and pseudoscorpionida (santos et al., 2005) using animals for transport. it may also be widespread in hematophagous species of leeches (hirudinoidea) to disperse to other water bodies (davies et al., 1982; khan and frick, 1997). for example khan and frick (1997) found the leech erpobdella punctata attached to spotted salamanders (ambystoma maculatum) in a pond in south carolina, usa and concluded that the leech was using the salamander as a phoretic agent. to our knowledge, however, there are no reports of leeches using anurans for transport. most species of leeches are considered ectoparasites that feed through sucking the blood and other body fluids from their hosts, and most of the species included in this group are parasites of small invertebrates, such as gastropods, crustaceans, and annelids, although they can also parasite on vertebrates such as reptiles (readel et al., 2008) and amphibians (briggler et al., 2001; toledo, 2005; loebmann et al., 2008; romano and cerbo, 2007; stead and pope, 2010; tiberti and gentilli, 2010). generally, leeches obtain food through structures of the foregut as a protractile pharyngeal proboscis or a cutting structure similar to slicing jaws or blades that cut the host tegument and anchor by pressing the mouth against it, and they may eat several times their own weight (brusca and brusca, 2003). also, some leeches produce anticoagulants which allows the maintenance of the hemorrhage even after the removal of the leeches (brusca and brusca, 2003; tiberti and gentilli, 2010). at least two species of leeches have been reported to be associated with amphibians in southern brazil, feeding on tadpoles, on juveniles and on adults of anurans of different families (loebmann et al., 2008). rhinella abei (baldissera jr., caramaschi and haddad, 2004) (bufonidae), inhabits remnants of the brazilian atlantic rainforest throughout the eastern of the states of paraná and santa catarina and north to the state of rio grande do sul, with its geographic distribution limited by the serra do mar to the north and by the serra geral to the west (baldissera jr. et al., 2004). this species may be encountered in both forested habitats and clearings in natural or anthropogenically modified areas. studies of r. abei are scarce, and are mainly on its distribution and habitat use (garey, 2007; lucas, 2008; armstrong and conte, 2010; cunha et al., 2010; wachlevski and rocha, 2010), and one study concerning its phylogeography (thomé et al., 2010). none of these studies mentioned an association of leeches with r. abei. in the atlantic rainforest of the parque estadual da serra do tabuleiro, state of santa catarina, brazil, we found individuals of r. abei externally infested by leeches. this led us to question whether the leeches were feeding on the toads or were in a phoretic relationship with them. the study was carried out in a remnant of dense ombrophilous forest in the parque estadual da serra do tabuleiro (pest) (27°44’s, 48°48’w), santo amaro da imperatriz municipality, state of santa catarina, southern brazil. the pest is one of the most relevant conservation units of integral protection in santa catarina because it shelters five of the vegetal physiognomies occurring in the state. moreover, it represents an important fitogeographic divisor, presenting high local biodiversity (klein, 1981), including several anuran species (wachlevski and rocha, 2010). the climate in the area has a mean annual rainfall of 1200 mm and a mean annual temperature of approximately 20.5 °c (cecca, 1997). the individuals of rhinella abei were collected in july 2009 during one night of survey using visual encounter survey methods (hereafter ves) (crump and scott, 1994). we 165phoresy by leeches on rhinella searched for anurans along the shoreline of a permanent lagoon that the toads used as reproductive site. the gender of the toads was identified by their vocalizations. in this species, males vocalize a release call when they are laterally compressed on both sides of the body in the region between abdomen and thorax, while females do not (personal observation). we carefully examined all body surfaces of each toad captured for leeches or evidence of parasitism (hemorrhages, wounds and/or scars) caused by the leeches. we recorded the region of the body (arm, axils, back, eye, foot, forearm, groin, and thigh) where leeches or scars were found and how many leeches were found attached to each region. the frequency (f, in %) of use of the body regions was represented by the percentage of leeches found attached to each region. we also measured the snout-vent length (svl) of the toads using a caliper (precision of 0.1 mm) and the body mass using a dynamometer pesola© with 100 g of capacity (precision of 0.1 g). the prevalence of infestation on r. abei by the leeches was represented by the percentage of anurans found infested among all toads sampled and examined, and the intensity of infestation was estimated by the mean number of leeches encountered attached on the anurans infested. to evaluate whether there were relationships between the number of leeches and toads’ svl and between the number of leeches and toads’ mass we performed simple regression analysis (zar, 1999), using only infested individuals. we captured 27 individuals of rhinella abei during the ves, of which 26 were males and one was a female. the identified female did not vocalize and was the largest (both svl and body mass) toad sampled in this study, matching the expected sexual dimorphism in this species with females larger than males (see baldissera jr. et al., 2004). we found a total of 30 leeches on 13 r. abei (range: 0 – 4, n = 27); the female had one leech attached on the ventral surface of the right thigh. as we sampled only one female of r. abei during this study we cannot say if a particular sex may be more or less related to leech phoresys as found by khan and frick (1997). in this study, all infested toads did not present evidence of being parasitized by leeches; we never observed the presence of wounds, hemorrhages or scars in their teguments caused by action of leeches as would be expected (linnaeus, 1758; tiberti and gentilli, 2010). also, none of the leeches were turgid, indicating that they were not currently consuming the blood of the toads. furthermore, the leeches were removed by hand from the body surface of the toads without any evidence of causing injuries on the toads’ tegument. these observations suggest that the relationship between r. abei and the leeches was not parasitism but phoresy. the average intensity of infestation was 2.3 ± 1.3 leeches per individual toad. the only other study we could find that reported an intensity of infestation by phoretic leeches on amphibians found 1.04 leeches per salamander (khan and frick, 1997). the leeches mainly occupied the toads’ axils (n = 7, f = 23.3%), followed by the back and the thighs (both n = 6, f = 20.0%) (fig. 1). in a phoretic association between salamanders ambystoma maculatum (shaw, 1802) (ambystomatidae) and leeches erpobdella punctata (leidy, 1870) (erpobdellidae) the phoronts attached mainly to the regions near the forelimbs of the salamanders (khan and frick, 1997). briggler et al. (2001) reported the parasitism of four species of anurans and two of salamanders by the leech desserobdella picta (verrill, 1872) (glossiphoniidae) and observed a preference of bufo americanus by the leeches, which occupied predominantly the axillaries regions of the front limbs. in another study, 166 t. maia-carneiro et al. leeches were observed attached to the legs or to the skin at the base of their vocal sac of anurans of different families (bufonidae, leptodactylidae, cycloramphidae and hylidae) (loebmann et al., 2008). the study of loebmann et al. (2008) reported the presence of leeches on the animals’ bodies but did not report evidence of wounds, scars or hemorrhages caused by leeches, and therefore may have been incorrectly reported as parasitism instead of phoresy. however, under laboratorial conditions, one toad rhinella arenarum (hensel, 1867) exposed to 15 leeches of the same species found attached on rhinella dorbignyi (duméril and bibron, 1841) died quickly (less than 20 minutes; loebmann et al., 2008). in general, based on a review of available studies and our own, it seems that the leeches are predominantly found on the limbs and on the axillaries regions of anurans (khan and frick, 1997; briggler et al., 2001; tiberti and gentilli, 2010; loebmann et al., 2008), probably because the leeches attached there are less vulnerable to being removed by contact to the substrate and/or by action of the anurans. we found leeches on almost half (48.1%) of the r. abei sampled in serra do tabuleiro. similarly khan and frick (1997) found phoront leeches on 49.1% of the salamanders they sampled. another study that found a parasitic relationship between frogs (rana temporaria) and leeches found leeches on 7 of 15 (46.7%) frogs, although four of the remaining eight frogs had dark globular clusters on their skin (tiberti and gentilli, 2010). in four (9.5%) of the 42 adults anurans of different species were found at least one leech (loebmann et al., 2008). the average svl of the toads was 69.2 ± 5.3 mm (range: 60.8 – 86.0 mm) and their average body mass was 27.1 ± 6.9 g (range: 19 – 55). we predicted that larger toads would carry more leeches due to their greater available surface area, but neither svl nor body mass were related with the number of leeches found on the toads’ bodies (f1,11 = 0.194, p = 0.668and f1,11 = 0.196, p = 0.666, respectively). we also expected that more leeches would be attached to dorsal surfaces of the toad’s body compared to ventral surfaces, because on dorsal surfaces the leeches would be less vulnerable to direct contact with the substrate, decreasing the possibility of being mechanically removed from 23.3% 20.0% 20.0% 13.3% 10.0% 6.7% 3.3% 3.3% 0 2 4 6 8 axils back thigh groin foot arm eye forearm n um be r of le ec he s body regions fig. 1. number of leeches (n = 30) recorded attached on each of the body regions (n = 8) of rhinella abei (n = 13) in the atlantic rainforest of the parque estadual da serra do tabuleiro, in the state of santa catarina, southern brazil. values above the bars indicate the frequency (%) of leeches on each body region. 167phoresy by leeches on rhinella the tegument. however, we found no difference in the rate of occupation on the dorsal and ventral surfaces with 13 leeches on each. however, in this study the back of the toads was the second most occupied body region following axils. there are only a small number of studies reporting phoresy with vertebrates as “hosts” (davies et al., 1982; khan and frick, 1997), and no reports of anurans as the dispersal agents. while our study involved a fairly small number of individuals collected at one site in one month, it still provides evidence of a possible phoretic relationship between anurans r. abei and at least one unidentified species of leech. additional research is needed to verify this relationship both spatially and temporally and to determine the species of leeches involved. acknowledgements this study was supported by research grants from the conselho nacional de desenvolvimento científico e tecnológico (cnpq) (processes 304791/20010-5 and 470265/2010-8) to c. f. d. rocha. t. maia-carneiro and t.a. dorigo received msc grants from the coordenação de aperfeiçoamento de pessoal de nível superior (capes). m. wachlevski received a ph.d. grant from capes. thanks to the resort plaza caldas da imperatriz logistical support in the field. specimens were collected under collecting permit number 1448 sisbio/instituto chico mendes and fatma (fundação do meio ambiente de santa catarina). references armstrong, c.g., conte, c.e. 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(2005): predation of juvenile and adult anurans by invertebrates: current knowledge and perspectives. herpetol. rev. 36: 395-400. wachlevski, m., rocha, c.f.d. (2010): amphibia, anura, restinga of baixada do maciambu, municipality of palhoça, state of santa catarina, southern brazil. check list 6: 602-604. zar, j.h. (1999): biostatistical analysis. prentice-hall, englewood cliffs, nj. zawal, a. (2006): phoresy and parasitism: water mite larvae of the genus arrenurus (acari: hydrachnidia) on odonata from lake binowskie (nw poland). biol. lett. 43: 257-276. acta herpetologica 10(1): 7-15, 2015 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-15149 haplotype variation in founders of the mauremys annamensis population kept in european zoos barbora somerová1, ivan rehák2,*, petr velenský2, klára palupčíková1, tomáš protiva1, daniel frynta1 1 department of zoology, faculty of science, charles university in prague, viničná 7, cz-12844, prague 2, czech republic 2 prague zoo, u trojského zámku 3, cz-171 00 prague 7, czech republic. * corresponding author. e-mail: ophis@tiscali.cz submitted on 2014, 15th august; revised on 2014, 11th november; accepted on 2014, 12th november editor: uwe fritz abstract. the critically endangered annam leaf turtle mauremys annamensis faces extinction in nature. because of that, the conservation value of the population kept in european zoos becomes substantial for reintroduction programmes. we sampled 39 specimens of m. annamensis from european zoos and other collections (mainly founders, imports and putatively unrelated individuals), and also four specimens of mauremys mutica for comparison. in each animal, we sequenced 817 bp of the mitochondrial nd4 gene and 940 bp of the nuclear r35 intron that were used as phylogenetic markers for mauremys mutica-annamensis group by previous authors. the sequences of the r35 intron, which are characteristic for m. annamensis and which clearly differ from those characteristic for m. mutica and/or other mauremys species, were mutually shared by all of the examined m. annamensis. they also possessed mitochondrial haplotypes belonging to the annamensis subclades i and ii, distinctness of which was clearly confirmed by phylogenetic analyses. thus, both nuclear and mitochondrial markers agreed in the unequivocal assignment of the examined individuals to m. annamensis. although no obvious hybrids were detected within the founders of the captive population, further careful genetic evaluation using genom-wide markers is required to unequivocally confirm this result. keywords. mauremys, geoemydidae, conservation, mt gene nd4, nuclear intron r35, vietnam, hybridization. introduction asian turtles face an extinction crisis due to habitat destruction and high demands from the chinese markets (van dijk et al., 2000; le et al., 2004; cheung and dudgeon, 2006; turtle conservation coalition, 2011). one of the heavily exploited species is mauremys annamensis, the annam leaf turtle. (siebenrock, 1903). this species of the family geoemydidae has a very limited and fragmented distribution and is restricted only to central vietnam (le et al., 2004; parham et al. 2006). mauremys annamensis is almost extinct in the wild, with limited numbers in ex-situ populations in vietnam, europe and the usa. it is listed in the appendix ii of cites and is globally redlisted as critically endangered by the iucn (2013). captive breeding seems to be one of the long-term solutions for the survival of asian turtles (hudson and buhlmann, 2002; turtle conservation coalition, 2011). mauremys annamensis has been repeatedly bred in some european zoos, including prague zoo (velenský, 2006; raffel and meier, 2013). currently, these zoos have started co-ordinated ex situ conservation breeding of the species associated with a repatriation project. among the programmes’ top priorities at present is the repatriation of the best captive-bred specimens. the situation of conservation breeding is complicated by hybridization among distinct species and even genera of the geoemydids (galgon and fritz, 2002; fritz and mendau, 2002; fritz et al., 2004; schilde et al., 2004; spinks et al., 2004; buskirk et al., 2005; stuart and par8 b. somerová et alii ham, 2006; shi et al., 2008). hybridization among mauremys annamensis, m. mutica, m. sinensis, m. nigricans, cuora amboinensis and c. trifasciata was reported both in captivity and in the wild (parham et al., 2001; shi and parham, 2001; fong and chen, 2010). the current events of natural hybridization between m. mutica and m. sinensis on taiwan island (fong and chen, 2010) represent an especially interesting case. the phylogenetically closest species of m. annamensis is m. mutica (barth et al., 2004; feldman and parham, 2004; spinks et al., 2004) and these species may interbreed (fong et al., 2007). this represents a serious problem for the efforts to build sustainable ex-situ breeding programs enabling the reintroduction and establishment of sustainable populations of m. annamensis in the wild. hybridization events in the annamensis-mutica complex were demonstrated by striking incongruence among phylogenies of the individual genes, i.e., the mitochondrial and nuclear markers. some of these incongruences may result from recent translocation and consequent hybridization; however, hybridization events that took place in the past are even more likely. fong et al. (2007) clearly demonstrated such incongruence in the hainan population of m. mutica, which differs from the “true mutica” of the eastern continental china by the presence of mitochondrial haplotypes forming a clade branching within those belonging to the m. annamensis. in contrast, sequences of r35 intron of hainan m. mutica are even less related to the corresponding sequences of the m. annamensis than those of the “true mutica”. moreover, it was clearly demonstrated that mitochondrial haplotypes of the m. annamensis was split into two deeply divergent haplogroups, which are referred to as the annamensis subclade i and ii (fong et al., 2007; fong, 2008). the phylogeographic pattern of these subclades is, however, unclear due to the extinction of most of the original populations in the nature. to organize proper ex-situ captive breeding and to remove potential hybrids from the rescue population, it is necessary to examine the genetic variation of the founders of the m. annamensis population. in this study, we focused on the founders, imported and putatively unrelated individuals of the m. annamensis kept in european zoos and other collections. we sequenced mitochondrial (nd4 gene) and nuclear (r35 intron) parts of dna to (1) verify the species determination of the founders, (2) assess sequence variation of the captive population, (3) assign captive specimens into the main haplogroups (subclades i and ii) and to (4) exclude the discovered interspecific species hybrids from the breeding pool. for comparison, we also included a few specimens of m. mutica into the analyses. material and methods in this paper, we examined 39 specimens of mauremys annamensis from european zoos and other collections (founders, imported and putatively unrelated individuals, i.e., captive born specimens having no shared maternal ancestors in their pedigree), and also four specimens of m. mutica were included for comparison (table  1). for all individuals, we sequenced a combination of mitochondrial (mtdna) and nuclear dna (nudna). for sampling of individuals, we used a non-invasive method: we took the tip of the claw from each sampled animal and stored in eppendorf tubes with 96% ethanol at -20°c prior dna extraction. we isolated the total genomic dna with dnaeasy tissue kit (qiagen, hilden, germany), following the manufacturer’s recommendations. using standard conditions and the primers l-nd4 and h-leu, we amplified an 892 bp fragment of mtdna containing the nadh dehydrogenase subunit 4 (nd4) gene and parts of trna (stuart and parham, 2004). following the conditions in fujita et al. (2004), and using the primers r35ex1 and r35ex2, we amplified the fragment of nudna containing 1133 bp of the rna fingerprint protein 35 (r35) gene intron 1. patterns from the sequencing chromatograms indicated that at the r35 locus, some individuals were heterozygous for a length polymorphism, which usually corrupts the sequence reads downstream of the indel site (see bhangale et al., 2005, fig. 1b). for sequencing the r35 intron, we used internal forward and reverse primers (spinks and shaffer, 2007) in combination with external primers (fujita et al., 2004) for the putative length-polymorphic individuals (spinks and shaffer, 2007). sequences of both mtdna and nudna fragments were aligned and manually checked using chromas lite 2.01 (technelysium pty ltd), bioedit (hall, 1999) and clustal x 1.81 (thompson et al., 1997). analyses of the estimates of evolutionary divergence between the sequences of nd4 gene and r35 intron were conducted using the maximum composite likelihood model (tamura et al., 2004). the included codon positions were 1st+2nd+3rd+noncoding. all positions containing gaps and missing data were eliminated. evolutionary analyses were conducted in mega6 (tamura et al., 2013). bayesian analysis (ba) was conducted with mrbayes 3.1 (huelsenbeck and ronquist, 2001; ronquist and huelsenbeck, 2003). the best-fit model (hky+g) was selected by hlrt in modeltest 3.7 (posada and crandall, 1998). two independent runs of bayesian analyses were conducted with a random starting tree and run for 30x106 generations, with trees sampled every 100 generations. the burn-in command was used to discard the first 10% of trees (3,000,000 generations), which were generated before the chain reached equilibrium in the distribution of trees. for these phylogenetic analyses, we also included some mtdna and nudna sequence data used in intrageneric studies about the mauremys mutica-annamensis complex (fong et al., 2007; fong and chen, 2010) and some species from the family geoemydidae, which were used as outgroups (genbank numbers are listed in appendix 1). 9haplotype variation in mauremys annamensis results in an alignment of the mitochondrial nd4 gene (817 bp), we detected 16 haplotypes, 25 variable sites and 17 parsimony-informative sites. all individuals of m. annamensis examined in this study possessed the mitochondrial nd4 gene (p-distaces ranging from 0.127% to 1.826%) typical for this species (fong et al., 2007). phylogenetic analyses containing our sequences in the context of those available in the genbank confirmed haplogroups and the general topology of previously published trees (fong et al., 2007). the ba tree (fig. 1) suggests a principal split between the “true mutica clade” (ba posterior probability  =  1.00) and a clade (ba  =  1.00) containing both the m. annamensis and hainan m. mutica. the latter clade further splits into three distinct clades (all ba probabilities  =  1.00). these are an “annamensis subclade i”, “annamensis subclade ii” and the “hainan mutica clade”. average uncorrected p-distance between the “annamensis subclade i” and “annamensis subclade ii was 1.968%”. the sister relationship between the “annamensis subclade i” and the “hainan mutica clade” is moderately supported (ba = 0.82). nd4 sequences of our m. annamensis samples belong to the haplogroups previously described as the “annamensis subclade i” and “annamensis subclade ii” (13 and 26 cases, respectively). out of four examined samples of the putative m. mutica, nd4 sequences branch within the “true mutica clade” and one within the “annamensis subclade i”. p-distances among these four clades computed from all available sequences (including genbank sources) suggest low mutual divergence among both the “annamensis” and “hainan mutica” clades (table 1). in an alignment of nuclear r35 intron (918 bp), we detected 25 haplotypes, 20 variable sites and 7 parsimony-informative sites. all 39 specimens putatively belonging to the m. annamensis shared mutually similar sequences of r35 intron (p-distaces from 0.132% to 0.932%). the r35 sequences in three of four m. mutica samples clearly differed from those of the m. annamensis. phylogenetic analysis of these sequences and those available in the genbank (alignment of 940 bp, see fig. 2) confirmed the presence of the three previously described clades (fong et al., 2007) within the annamensis-mutica complex: the “hainan mutica clade” (ba posterior probability  =  0.98) is the sister group of the true mutica-annamensis clade (ba  =  1.00), which contains a group of mutica sequences corresponding to the “true mutica clade” (ba  =  0.53) and a well-supported “annamensis clade” (ba = 1.00). in ba tree, the “true mutica” is paraphyletic with respect to the “annamensis clade”, however, most of the sequences of this group form a single branch with low support (ba = 0.53). the ba analysis placed all 39 examined sequences of the m. annamensis into the “annamensis clade”. out of four of the m. mutica sequences, one belongs to the “hainan mutica clade”, one into the “annamensis clade” and the remaining two into the “true mutica” (table 2). discussion we have no evidence suggesting the presence of the interspecific hybrids among the examined founders of the m. annamensis kept in european collections. of course, without an application of expensive genome-wide markers (like snps, extensive number of microsatellites), it is impossible to entirely rule out partial introgression of the genomes of other related geoemydids into some founders of the european population of the m. annamensis (i.e., presence of hybrids of a higher order f2 and higher generations and backcrosses). also, without cloning, we are unable to evaluate the affinity of potential heterozygots of the r35 intron to individual mitochondrial subclades. nevertheless, when considering other supportive evidence (age, origin), the presence of hybrids seems to be fairly unlikely. the original geographic distribution of the mauremys annamensis is unknown, only few records document it. that is why it is hard to understand the significance of the two distinct mitochondrial clades, which we, as well as previous authors, detected in the m. annamensis. it is unclear whether these clades occur or occurred in the wild in syntopy or allopatrically. the sequence divergence table 1. average values of estimates of evolutionary divergence between sequences (the p-values are expressed in per cents).   annamensis subclade ii annamensis subclade i hainan mutica clade true mutica clade annamensis subclade ii 0.083-0.167     annamensis subclade i 0.844-1.193 0.167-0.420   hainan mutica clade 1.020-1.281 0.589-0.934 0.083-0.167 true mutica clade 3.952-4.895 3.549-4.782 3.952-4.782 0.083-1.554 10 b. somerová et alii between the two clades is only about 0.84-1.19 %. thus, we cannot reject the possibility that retention of ancestral polymorphism is a cause of a simultaneous occurrence of these related, but still distinct clades, in the sampled population of the m. annamensis. ancestral polymorphism may be irrelevant to an original population structure of the species prior to its recent decline leading to near extinction in the wild. the distinction between the mitochondrial haplotype groups i and ii has been recognized only recently and thus, the species has been treated as a single conservation unit in most zoos and collections. however, it is possible to keep the animals of the two groups apart. this would be recommended especially in the case of animals producing offspring suitable for repatriation projects. nevertheless, such a precaution cannot subfig. 1. bayesian tree of mitochondrial dna (nd4) of the genus mauremys. numbers at branches are support values, only values >  0.95 are shown. samples sequenced in this study, which correspond to table 1, in bold, remaining samples were sequenced by previous authors (fong et al., 2007; fong and chen, 2010), mm  =  mauremys mutica, ma  =  mauremys annamensis. countries of the origins of samples are shown at individuals with reliable locality. 11haplotype variation in mauremys annamensis table 2. list of samples used in this study containing information about species, breeder, nuclear and mitochondrial haplotype subclades. nr.   species breeder nd4 r35 mauremys annamensis mtdna subclade ii 1 mauremys annamensis h. becker annamensis subclade ii annamensis clade 2 mauremys annamensis h. becker annamensis subclade ii annamensis clade 3 mauremys annamensis h. becker annamensis subclade ii annamensis clade 3d mauremys annamensis d. frynta annamensis subclade ii annamensis clade 5d mauremys annamensis d. frynta annamensis subclade ii annamensis clade 6 704774 mauremys annamensis rotterdam annamensis subclade ii annamensis clade 8 704525 mauremys annamensis rotterdam annamensis subclade ii annamensis clade 10 704524 mauremys annamensis rotterdam annamensis subclade ii annamensis clade 11 705067 mauremys annamensis rotterdam annamensis subclade ii annamensis clade 12 3 mauremys annamensis münster annamensis subclade ii annamensis clade 13 4 mauremys annamensis münster annamensis subclade ii annamensis clade 15 9 mauremys annamensis münster annamensis subclade ii annamensis clade 16 1 mauremys annamensis münster annamensis subclade ii annamensis clade 17 2 mauremys annamensis münster annamensis subclade ii annamensis clade 19 6 mauremys annamensis münster annamensis subclade ii annamensis clade 20 mauremys annamensis m. schilde annamensis subclade ii annamensis clade 23 m53 mauremys annamensis praha annamensis subclade ii annamensis clade 25 f21 mauremys annamensis praha annamensis subclade ii annamensis clade 26 f9 mauremys annamensis praha annamensis subclade ii annamensis clade 32 roo718 mauremys annamensis leipzig annamensis subclade ii annamensis clade 35 32 mauremys annamensis panuška annamensis subclade ii annamensis clade 36 33 mauremys annamensis panuška annamensis subclade ii annamensis clade 37 34 mauremys annamensis panuška annamensis subclade ii annamensis clade 126 cos679 mauremys annamensis chester annamensis subclade ii annamensis clade 127 cos678 mauremys annamensis chester annamensis subclade ii annamensis clade 130 cos349 mauremys annamensis chester annamensis subclade ii annamensis clade mauremys annamensis mtdna subclade i 1d mauremys annamensis d. frynta annamensis subclade i annamensis clade 2d mauremys annamensis d. frynta annamensis subclade i annamensis clade 4d mauremys annamensis d. frynta annamensis subclade i annamensis clade 7 704212 mauremys annamensis rotterdam annamensis subclade i annamensis clade 9 704523 mauremys annamensis rotterdam annamensis subclade i annamensis clade 18 8 mauremys annamensis münster annamensis subclade i annamensis clade 21 mauremys annamensis m. schilde annamensis subclade i annamensis clade 22 mauremys annamensis m. schilde annamensis subclade i annamensis clade 24 m7 mauremys annamensis praha annamensis subclade i annamensis clade 33 roo720 mauremys annamensis leipzig annamensis subclade i annamensis clade 34 roo719 mauremys annamensis leipzig annamensis subclade i annamensis clade 128 cz/921 mauremys annamensis chester annamensis subclade i annamensis clade 129 cz/922 mauremys annamensis chester annamensis subclade i annamensis clade mauremys mutica 4   mauremys mutica h. becker true mutica clade true mutica clade 5 mauremys mutica h. becker true mutica clade true mutica clade animals of hybrid origin 28 3 mauremys mutica praha true mutica clade annamensis clade 27 2 mauremys mutica praha annamensis subclade i hainan mutica clade 12 b. somerová et alii stantiate the elimination of the descendants of parents belonging to different clades from the studbook population. there is an urge call for further research of the genetic variation in the m. annamensis using multiple nuclear markers and/or advanced genomic methods, especially to enable a better understanding of the divergence of the two distinct subclades. 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(2013): mega6: molecular evolutionary genetics analysis version 6.0. mol. biol. evol. 30: 2725-2729. thompson, j. d., gibson, t. j., plewniak, f., jeanmougin, f., higgins, d. g. (1997): the clustal_x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. nucl. acids res. 25: 4876-4882. turtle conservation coalition (2011): turtles in trouble: the world’s top 25+ most endangered tortoises and freshwater turtles 2011. available from: http://www. iucn-tftsg.org/top-25-2011/ (may 11, 2014). van dijk, p.p., stuart, b.l., rhodin, a.g.j. (2000): asian turtle trade: proceedings of a workshop on conservation and trade of freshwater turtles and tortoises in asia, chelonian research monographs, chelonian research foundation, lunenburg, massachusetts. velenský, p. (2006): chovatelství – plazi. výroční zpráva 2006, zoo praha. available from: http://www.zoopraha.cz/vse-o-zoo/vyrocni-zpravy/7572-vyrocni-zprava-2006 (may 31, 2014). appendix 1 list of sequences containing their genbank numbers used in this study. name of sequence species nd4 r35 1ma m. annamensis ef034098 ef587934 10ma m. annamensis af348280 dq386668 11mm.hainan m. annamensis ef034096 ef87929 12ma m. annamensis ef034105 — 13ma.hainan m. annamensis ef034104 ef587915 14mm.hainan m. mutica ef034097 ef587925 15.mm.hainan m. mutica ef034101 ef587917 16.mm.hainan m. mutica ef034095 ef587930 17mm.vietnam m. mutica af348278 dq386664 18ma m. annamensis ef034104 ef587923 19ma m. annamensis ef034106 ef587928 20ma m. annamensis ef034107 ef587924 21ma m. annamensis ef034112 dq386656 22ma m. annamensis ef587914 ef587921 23ma m. annamensis ef034099 ef587919 24ma m. annamensis ef034100 — 25ma m. annamensis ef034108 — 26mm m. mutica ef034092 — 27mm m. mutica ef034093 ef587931 28mm m. mutica ef034089 ef587932 29mm m. mutica af348278 dq386666 2ma m. annamensis ay337338 ef587933 30mm m. mutica ef034090 — 31mm m. mutica ef034092 ef587916 32mm m. mutica ef034094 ef587927 3ma m. annamensis ef034103 — 4ma m. annamensis ef034105 ef587922 15haplotype variation in mauremys annamensis name of sequence species nd4 r35 6ma m. annamensis ef034102 ef587929 7ma m. annamensis ef034109 ef587926 8ma m. annamensis ef034113 dq386655 9mm.vietnam m. mutica af348279 –––– cuora amboinensis cuora amboinensis ef011357 hq442382 cuora galbinifrons cuora galbinifrons ay364617 –––– cuora pani cuora pani — ef011442 cuora trifasciata cuora trifasciata — jq596437 cyclemys dentata cyclemys dentata — am931697 leucocephalon yuwonoi leucocephalon yuwonoi — am931708 m. nigricans m. nigricans ef034111 –––– m. reevesii m. reevesii ef034110 –––– m. reevesii.taiwan4 m. reevesii gq259438 gq259459 m. reevesii.taiwan7 m. reevesii gq259441 gq259464 m. sinensis.hainan13 m. sinensis ay337345 dq386678 m. sinensis.taiwan9 m. sinensis gq259443 gq259465 m. caspica m. caspica ay337340 –––– mauremys japonica mauremys japonica — hq442386 mm.taiwan19 m. mutica gq259452 gq259471 mm.taiwan20 m. mutica gq259453 gq259472 mm.taiwan21 m. mutica gq259454 gq259473 mm.taiwan25 m. mutica gq259457 gq259474 mm.taiwan26 m. mutica gq259458 gq259475 ocadia glyphistoma ocadia glyphistoma — dq386663 sacalia quadriocellata sacalia quadriocellata — hq442384 siebenrockiella siebenrockiella leytensis — am931708 siebenrockiella crassicollis siebenrockiella crassicollis — ay954913 acta herpetologica vol. 10, n. 1 june 2015 firenze university press obituary: valery k. eremchenko (1949-2014) leo j. borkin1, tatjana dujsebayeva2, roberto sindaco3, matthias stöck4 haplotype variation in founders of the mauremys annamensis population kept in european zoos barbora somerová1, ivan rehák2,*, petr velenský2, klára palupčíková1, tomáš protiva1, daniel frynta1 reproductive ecology of sichuan digging frogs (microhylidae: kaloula rugifera) wei chen1,*, lina ren2, dujuan he2, ying wang2, david pike3 toxic effects of carbaryl on the histology of testes of bufotes variabilis (anura: bufonidae) özlem çakici basal frequency of micronuclei and hematological parameters in the side-necked turtle, phrynops hilarii (duméril & bibron, 1835) maría a. latorre 1,2,*,#; evelyn c. lópez gonzález1,2, #; pablo a. siroski1,2,3; gisela l. poletta1,2,4 into a box interiors: clutch size variation and resource allocation in the european pond turtle marco. a.l. zuffi1,*, simonetta citi2, elena foschi1, francesca marsiglia1, eva martelli1 where to “rock”? choice of retreat sites by a gecko in a semi-arid habitat andreia penado1,2, ricardo rocha3,4,*, marta sampaio3, vanessa gil3, bruno m. carreira3, rui rebelo3 age structure, growth and longevity in the common toad, rhinella arenarum, from argentina clarisa de l. bionda1,2,*, silvia kost 4, nancy e. salas1, rafael c. lajmanovich3, ulrich sinsch4, adolfo l. martino1 on a putative type specimen of pleurodema bibroni tschudi, 1838 from chile (anura: leptodactylidae) daiana paola ferraro re-description of the external morphology of phyllomedusa iheringii boulenger, 1885 larvae (anura: hylidae), with comments on the external morphology of tadpoles of the p. burmeisteri group samanta iop¹, victor mendes lipinski¹, bruno madalozzo¹, franciele pereira maragno¹, sonia zanini cechin¹, tiago gomes dos santos² book review: harold heatwole, john w. wilkinson (eds). amphibians biology. volume 11 status of conservation and decline of amphibians. eastern hemisphere. part 4 . southern europe and turkey sebastiano salvidio book review: antonio romano. atlante degli anfibi del parco nazionale del cilento vallo di diano e alburni distribuzione, biologia, ecologia e conservazione sebastiano salvidio acta herpetologica journal of the societas herpetologica italica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 8(2): 167-170, 2013 food composition of ocellated skink, chalcides ocellatus (forskal, 1775) (squamata: scincidae), from the cyprus island kerim çiçek*, bayram göçmen ege university, faculty of science, biology department, zoology section, tr-35100, bornova-izmir, turkey. *corresponding authr: e-mail: kerim.cicek@hotmail.com or kerim.cicek@ege.edu.tr submitted on 2013, 10th october; revised on 2013, 2nd december; accepted on 2013, 6th december. abstract. we examined the food composition of the museum specimens of chalcides ocellatus (forskal, 1775) collected from morphou (= güzelyurt) and gönyeli (nicosia district, northern cyprus). the stomach contents of 41 (23 males, 11 females, and 7 juveniles) individuals were analyzed, and totally 86 prey items were detected. the species was found to feed mainly on a variety of insects (94.3%) and particularly on coleopterans (62.1%). no statistically significant sexor age-dependent difference was observed in the feeding regime. in conclusion, the diet of c.ocellatus was based mainly on insects and other arthropods. keywords. chalcides ocellatus, ocellated skink, food analysis, cyprus the ocellated skink, chalcides ocellatus (forskal, 1775), is a medium-sized semi-fossorial lizard which is mainly distributed from north africa, the middle east, and the most part of the mediterranean basin (anderson, 1999; kornilios et al., 2010; uetz and hošek, 2013). the species occupies a wide range of habitats such as archaeological sites, cultivated fields, hedges, gardens, open forest, mediterranean scrub, and patches of vegetation on coastal sands (schleich et al., 1996; anderson, 1999; kalboussi and nouira, 2004; budak and göçmen, 2005; baha eldin, 2006; taylor et al., 2012). chalcides ocellatus is a predominantly insectivorous lizard which feeds on various terrestrial insects. data on the food composition of c. ocellatus were investigated in turkey (mermer, 1996), tunisia (kalboussi and nouira, 2004), egypt (attum et al., 2004; taylor et al., 2012), and italy (capula and luiselli, 1994; rugiero, 1997; lo cascio et al., 2008; carretero et al., 2010). only anecdotal data on the food composition of the species from northern cyprus are available. the present study aims to determine the food composition of the northern cypriot population and to contribute to the limited knowledge of its biology. we examined 41 (23 males, 11 females, and 7 juveniles) preserved specimens of c. ocellatus deposited at the zmhru (the zoology museum of harran university, şanlıurfa, turkey). they were used to determine the northern cypriot herpetofauna as a continuation of the previous study (göçmen et al., 2008). lizards were collected from morphou (= güzelyurt, lat.: 35.209389°, long.: 32.954021°, sea level) and gönyeli (lat.: 35.238192°, long.: 33.299273°, 178m asl.), nicosia district, northern cyprus on july 2 and 3, 2008. for all individuals, we measured the snout-vent length (hereinafter ‘the svl’) with a dial caliper to the nearest 0.01 mm. sex was determined by direct observation of the gonads during dissection. according to the gonad development, we considered those with a svl less than 55 mm juveniles. the stomachs were dissected, and prey items were identified under a stereomicroscope. we identified the stomach contents to the lowest possible taxa. the food contents were assessed in terms of the numeric proportion (the number of a particular prey item in all prey items, n%) and the frequency of occurrence (the frequency of lizard stomachs containing a particular prey type, f%). the trophic niche overlap was 168 kerim çiçek, bayram göçmen measured using pianka’s index (o, 1973). this index ranges from 0 (no similarity) to 1 (totally similar). the food-niche breadth was determined using shannon’s index (h, shannon, 1948). the values of this index typically range from 1.5 (narrow niche breadth) to 3.5 (wide niche breadth) (macdonald, 2003). all niche calculations were done by making use of the “ecosim version 7.72” program (gotelli and entsminger, 2012). normality of the svl and tl distributions for both males and females was tested with kolmogorov-smirnov d test, and since they were normally distributed (p ≥ 0.05), the parametric t-test was used for comparison. the age classes (juveniles, males, and females) were compared using the non-parametric kendall’s rank correlation and kruskal-wallis tests due to the data which were not normally distributed (the kolmogorov–smirnov d test, p ≤ 0.05). the alpha level was set at 0.05. in the results section, the mean values are provided with their standard deviations. the average svl of the 41 (23 males, 11 females, and 7 juveniles) individuals of c. ocellatus from northern cyprus under examination was 48.0 (sd = 3.75, range = 42.8–52.2) mm for juveniles, 71.9 (6.86, 55.0–83.2) mm for males, and 72.10 (5.46, 64.5–81.3) mm for females. there are no statistical differences in size between the sexes (t-test, t = 0.107, p = 0.916). in the stomach contents of 41 individuals, 86 prey items (9 in juveniles, 49 in males, and 28 in females) were detected, with their sizes varying between 3 and 20 mm, and the median number of prey items was 1 (range = 1–3) in juveniles, 2 (1–6) in males, and 1 (1–6) in females. a rather weak correlation was observed between the svl and the number of prey items (kendall τ = 0.31, p = 0.02). no statistical difference in the number of prey items in the stomach contents was present among males, females, and juveniles (kruskal-wallis test, χ2 = 4.678, p = 0.096). a total of 86 prey items in the stomach contents of c. ocellatus were found to belong to 3 classes and 5 orders (table 1). sand and gravel materials were found in the stomach contents, and it is most likely that they were ingested accidentally during foraging. the prey taxa included classes arachnida (araneae), chilopoda (geophilomorpha), and insecta (orthoptera, hymenoptera, coleoptera, and lepidoptera). when compared with other classes, insecta contains the highest number of prey groups (94.3%). among these prey items, the largest groups by numeric proportion (n%) found in the stomach contents were coleoptera (62.1%), orthoptera (13.8%), and formicidae (8.1%), respectively. the largest rate by frequency of occurrence (f%) also belonged to these groups: coleoptera (70.7%), orthoptera (26.8%), and formicidae (7.3%). however, only 2 (2.4%) larval prey items were consumed by lizards. as it is seen, order coleoptera is the favorite prey taxon in the sexes. according to the pianka’s niche overlap index (o), the food composition of males, females and juveniles is mostly similar (ojuveniles, males = 0.87, ojuveniles, females = 0.91, and omales, ofemales = 0.93), and the food contents considerably overlapped. this means that adults and juveniles use the same microhabitat for foraging. additionally, the close food-niche breadth (shannon’s index, h) was observed in the age classes (hjuveniles = 1.53, hmales = 1.40, and hfemales = 1.79). according to the index values, the cypriot population has a narrow food-niche breadth. although the food composition of c. ocellatus quite varies according to the region it inhabits, it generally feeds mainly on various arthropods and predominantly on the prey items included in class insecta. the most preferred prey items in the food composition of the tunisian population are coleoptera (n% = 56.1%, f% = 84.3% for the northern population; 7.1%, 22.9% for the southern population), isopoda (38.6%, 40% for the southern population) and insect larvae (12.4%, 45.7% for the northern population; 19.3%, 34.3% for the southern population) (kalboussi and nouira, 2004). capula and luiselli (1994) stated that isopoda (n% = 16.7%), gastropoda (16.7%), oligochaeta (14.6%), and araneae (11.4%) were the most frequently consumed prey items for the central sardinian (italy) population. an insular (lampione, italy) population was examined, and its food composition was observed to consist substantially of coleoptera (n% = 18.3%, f% = 66.7%) and insect larvae (8.5%, 30.3%) (carretero et al., 2010). these authors also stated that the species partially consumed plant material (n% = 60.8%, f% = 75.8%) as well. rugiero (1997) detected (n%) 42.4% araneae, 15.1% isopoda, and 15.1% coleopteran larvae in the food composition of the central italian population. lo cascio et al. (2008) stated that lampedusa and conigli (the pelagie islands, italy) populations of c. ocellatus primarily fed on arthropods and that the main prey items for the individuals were heteroptera (n% = 32.1%), coleoptera (21.1%), gastropoda (11.9%), and formicidae (10.0%) for lampedusa and coleoptera (38.0%), formicidae (12.0%), and insect larvae (16.0%) for conigli. attum et al. (2004) reported that coleoptera and hymenoptera were the most observed prey items in the food composition of the species from the sinai peninsula (egypt). the favorite prey item groups in the southern egyptian population are insect larvae (n% = 37%, f% = 40%), and it was detected to feed on orthoptera (20%, 23%), coleoptera (20%, 13%), and plant material (14%, 13%) (taylor et al., 2012). the food composition of c. parallelus generally consisted of coleoptera (n% = 54.8%), blattodea (12.3%), araneae (6.8%), and gastropoda (5.5%) from the chafarinas islands in north 169diet of the ocellated skink, chalcides ocellatus, in cyprus africa (civantos et al., 2013). mermer (1996) stated that coleoptera (n% = 83.3%), formicidae (40%) and gastropoda (33.3%) were the most encountered groups in the food composition of the turkish population. coleoptera, orthoptera and formicidae were substantially observed in the food composition of the northern cypriot population, respectively. we observed a few seeds in the stomach contents of a female individual. in the ocellated skink, plant material was observed in the food composition of insular populations (lo cacsio et al., 2008; carretero et al., 2010), whereas no or a low proportion of plant remains were found in the food of continental populations (e.g. kalboussi and nouira, 2004; civantos et al., 2013). carretero et al. (2010) claimed that there was a trend for increasing the degree of plant consumption with isolation and for decreasing it with the island area. when compared to other conspecific populations, partly herbivory feeding of c.ocellatus could be related to limited food sources in island populations. skink lizards are positive energy balance (huey et al., 2001) and generally have food in their stomachs. we detected that the individuals of cypriot population had at least one prey item in their stomach. capula and luiselli (1994) observed a positive correlation between the svl and the number of prey items. there was a quite weak relationship in the cypriot population, and the food composition did not fully overlap between the sexes in the southern egyptian population of the species (o = 0.61), and it was indicated that its reason might be related to the small number of specimens (taylor et al., 2012). the niche overlap we observed among the age groups in the cypriot population reinforces this probability. in conclusion, the ocellated skinks (c.ocellatus) mainly fed on arthropods and especially on terrestrial insects. there were no differences in diet between the sexes and among the age groups. the most frequently consumed prey items with respect to numeric proportion were coleoptera, orthoptera, and formicidae. other prey groups were chilopoda and araneae. the food composition of c.ocellatus was based mainly on insects and other arthropods and carabids constituted nearly half of its food. acknowledgements the collection of specimens was permitted by the environmental protection agency in the ministry of environment and natural resources of the turkish republic of northern cyprus (protocol no. ç.k.0.00.23.89.7.07.1202 of november 13, 2007). thanks are due to two anonymous reviewers for their constructive contribution. table 1. food composition of chalcides ocellatus (7 juveniles, 23 males, and 11 females) from northern cyprus. n (%): prey numbers and their proportion in the lizard stomach, f (%): frequency of occurrence of the prey item in the stomach of all individuals. prey taxa juveniles n (%) males n (%) females n (%) total n (%) f (%) chilopoda geophilomorpha 1 (2%) 1 (3.6%)) 2 (2.3%) 1 (2.4%) arachnida araneae 1 (11.1%) 1 (3.6%) 2 (2.3%) 2 (4.9%) insecta undetermined insects 2 (22.2%) 5 (10.0%) 7 (8.1%) 3 (7.3%) orthoptera 2 (22.2%) 8 (16.0%) 2 (7.1%) 12 (13.8%) 11 (26.8%) hymenoptera non-formicidae 1 (3.6%) 1 (1.1%) 1 (2.4%) formicidae 2 (22.2%) 5 (17.9%) 7 (8.1%) 3 (7.3%) coleoptera  undetermined coleopterans 2 (7.1%) 2 (2.3%) 1 (2.4%) coleoprean larvae 2 (7.1%) 2 (2.3%) 1 (2.4%) carabidae 2 (22.2%) 32 (64%) 7 (25%) 41 (47.1%) 22 (53.7%) cerambycidae 1 (3.6%) 1 (1.1%) 1 (2.4%) coccinellidae 1 (2.0%) 1 (3.6%) 2 (2.3%) 1 (2.4%) tenebrionidae 1 (2.0%) 5 (17.9%) 6 (6.9%) 4 (9.8%) lepidoptera 1 (2.0%) 1 (1.1%) 1 (2.4%) number of prey items 9 49 28 86 h 1.53 1.40 1.79 170 kerim çiçek, bayram göçmen references anderson, s.c. (1999): the lizards of iran. society for the study of amphibians and reptiles, ithaca, ny. attum, o., covell, c., eason, p. (2004): the comparative diet of three saharan sand dune skinks. afr. j. herpetol. 53: 91-94. baha eldin, s.m. (2006): a guide to the reptiles and amphibians of egypt. american university in cairo press, cairo. budak, a., göçmen, b. (2005): herpetology. ege üniversitesi fen fakültesi kitaplar serisi, no. 194, ege üniversitesi basimevi, bornova-izmir. [in turkish] capula, m., luiselli, l. (1994): resource partitioning in a mediterranean lizard community. boll. zool. 61: 173177. carretero, m.a., cascio, p.l., corti, c., pasta, s. (2010): sharing resources in a tiny mediterranean island? comparative diets of chalcides ocellatus and podarcis filfolensis in lampione. bonn zool. bull. 57: 111-118. civantos, e., ortega, j., lópez, p., pérez-cembranos, a., pérez-mellado, v., martín, j. 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(2010): phylogeography of the ocellated skink chalcides ocellatus (squamata: scincidae), with the use of mtdna sequences: a hitch-hiker’s guide to the mediterranean. mol. phylogenet. evol. 54: 445456. lo cascio, p., corti, c., carretero, m.a., pasta, s. (2008): dati preliminari sulla dieta di due popolazioni insulari di chalcides ocellatus. herpetologia sardiniae 8: 314-317. macdonald, g.m. (2003): biogeography: space, time, and life. john wiley & sons inc., new york, usa. mermer, a. (1996): biological and taxonomical investigations on chalcides ocellatus (sauria: scincidae) in anatolia. turk. j. zool. 20: 77-93. pianka, e.r. (1973) the structure of lizard communities. annu. rev. ecol. evol. syst. 4: 53-74. rugiero, l. (1997): on the ecology and phenology of chalcides chalcides (linnaeus 1758) in central italy (squamata; sauria; scincidae). herpetozoa 10: 81-84. schleich, h.-h., kästle, w., kabisch, k. (1996): amphibians and reptiles of north africa. koeltz scientific publishers, koenigstein, germany. shannon, c.e. (1948): a mathematical theory of communication. bell syst. tech. j. 27: 379-423 and 623-656. taylor, d.j., titus-mcquillan, j., bauer, a.m. (2012): diet of chalcides ocellatus (squamata: scincidae) from southern egypt. bull. peabody mus. nat. hist. 53: 383-88. uetz, p., hošek, j. (2013): the reptile database http:// www.reptile-database.org, accessed: aug 1, 2013. acta herpetologica vol. 8, n. 1 june 2013 firenze university press journal of the societas herpetologica italica acta herpetologica acta herpetologica 10(2): 143-148, 2015 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-16427 correlation between development and increase of number of labial tooth rows in ghost frog tadpoles (anura: heleophrynidae) werner conradie1,2,*, christa conradie3 1 department of herpetology, bayworld, p.o. box 13147, humewood, port elizabeth 6013, south africa. *corresponding author. e-mail: werner@bayworld.co.za 2 south african institute for aquatic biodiversity, p/bag 1015, grahamstown, 6140, south africa 3 23 st. tropez, verdun road, lorraine, port elizabeth 6070, south africa submitted on 2015, 14th july; revised on 2015, 14th october; accepted on 2015, 15th october editor: rocco tiberti abstract. the family heleophrynidae is restricted to southern africa and comprises two genera with seven species. tadpoles are well adapted, with huge sucker mouths, to live in the fast flowing headwaters of mountain streams. the unique sucker-like mouth has numerous transverse rows of labial teeth, which are used to attach themselves to the rock surface and to scrape algae from the rocks. in this paper data on the ontogenetic increase of labial tooth rows in tadpoles of four species of ghost frog are presented. notes on the development of tadpoles and mouthparts are also presented. keywords. feeding, algae, labial tooth rows, correlation, size. ghost frogs belong to the family heleophrynidae, which consists of two genera: hadromophryne van dijk 2008 and heleophryne sclater 1898, containing one and six species respectively (du preez and carruthers, 2009). this anuran family is endemic to south africa, lesotho, and swaziland (boycott, 1999). this is an evolutionarily distinct family, their common ancestor and the remainder of all the neobatrachians having split from archaeobatrachians around the jurassic period (± 160 mya: biju and bossuyt, 2003) to form a sister group to all the other neobatrachians (san mauro et al., 2005; frost et al., 2006). both adults and tadpoles are well adapted to live in the headwaters of mountain streams, tadpoles in particular possessing a huge sucker-like mouth and a dorso-laterally flattened body to overcome the fast flowing water (fig. 1). this sucker-like mouth possesses numerous transverse rows of labial teeth, which are used to anchor the tadpole to the rock surface and for feeding. tadpoles feed on algae growing on the surface of submerged rocks by scraping it off with the aid of numerous labial tooth rows (see fig. 2; rose, 1926; boycott, 1972; boycott and de villiers, 1986). the cooler temperatures in these streams are responsible for an extended larval life stage, which can last up to 18-24 months (boycott, 1988; passmore and carruthers, 1995; channing, 2001; du preez and carruthers, 2009). these highly specialised species have been trapped within the upper limits of specific catchments, thus leading to high vulnerability due to loss and degradation of natural habitat resulted from deforestation, fires, floods, erosion, damming and introduction of predatory fish. this has led to two species of heleophrynidae having been classified as endangered (boycott, 2004; iucn, 2010). the situation is likely to be worsened by the effect of global warming on reduction in stream flow. the table mountain ghost frog (heleophryne rosei) is considered critically endangered and hewitt’s ghost frog (heleophryne hewitti) is rated endangered, while the remaining species are currently considered least concern (iucn, 2010). anuran tadpoles have very complex mouthparts. especially the number, shape and size of teeth and their 144 werner conradie, christa conradie arrangement in rows varies both interspecifically and ontogenetically within species (altig and mcdiarmid, 1999), which indicates an ecomorphological adaptation towards the pressures of the environmental conditions of the specific species, as well as to exploit different parts of the available food sources within an ecosystem (khan and mufti, 1994; venesky et al., 2011). labial teeth play a facultative role in feeding, functioning to anchor the oral disc to the substrate and, in conjunction with the jaw sheaths, to rake material off substrate (venesky et al., 2010a,b, 2011). for example, many rows of labial teeth may be advantageous for taxa inhabiting fastflowing streams where more teeth allow better anchorage to substrates to avoid being swept downstream (eg., hadromophryne spp., heleophryne spp., trichobatrachus robustus, conraua spp., petropedetes spp.; channing et al., 2013). alternatively, fewer labial tooth rows may be advantageous for taxa living in lentic (standing water) habitats where they are not under selective pressures to anchor themselves firmly to a substrate (venesky et al., 2010a,b). despite the high morphological diversity found in tadpoles, they are often overlooked and understudied relative to other consumer groups in freshwater systems. there is also a lack of information on interspecific and ontogenetic changes in oral morphology, and how differences in tadpole mouthpart arrangements across species correlate with selective feeding (altig et al., 2007). all species of ghost frog tadpoles are characterised by a high number of keratinised labial tooth rows. the labial tooth row formula (ltrf) ranges from 4/14 to 4/17 (upper jaw/lower jaw) in each species, with the first row of posterior labial tooth rows usually divided (hewitt, 1913; rose, 1950; wager, 1965; van dijk, 1966; du preez and carruthers, 2009; channing et al., 2012). we follow the use of ltrf laid out in altig and mcdiarmid (1999). hadromophryne natalensis tadpoles differ from heleophryne tadpoles by possessing a lower keratinised jaw sheath (van dijk, 2008; du preez and carruthers, 2009; channing et al., 2012). visser (1971, 1985) described the development of individual fang-like teeth, which are replaced by rows of smaller teeth at 20-22 days in heleophryne tadpoles. boycott (1988) made a personal observation: “the number of tooth-rows depends on the age of the tadpoles”. in this study, tadpoles of four species of ghost frogs were examined to investigate and document the pattern of ontogenetic increase in labial tooth rows in heleophrynidae in relationship to body size. two hundred and thirty one tadpoles of four species of ghost frogs were examined: heleophryne purcelli (n = 36), heleophryne regis (n = 77), heleophryne hewitti (n = 75) and hadromophryne natalensis (n = 43). new material was collected by lifting rocks in streams, with a standard aquarium net suspended below to catch dislodged tadpoles. tadpoles were euthanized with tricaine methanesulfonate (ms222) solution and preserved in 10% buffered formalin. voucher specimens are all hosted in the port elizabeth museum (pem; appendix a), apart from a few tadpoles of h. hewitti that were measured, photographed and examined in the field. tadpoles identifications are based on known distributions of respective species (minter et al., 2004). body length (tip of snout to the anterior part of the vent) represented overall size, and was measured using a mitutoyo caliper (accuracy of 0.05 mm) and rounded off to the nearest 0.1 mm. as the number fig. 1. dorso-lateral photo of heleophryne hewitti tadpole in situ (witte river, baviaanskloof, eastern cape, south africa) fig. 2. close-up view of heleophryne hewitti tadpole showing the big sucker-like mouth and numerous transverse labial tooth rows (grid represents 1cm x 1cm) 145increased number of labial tooth rows in ghost frogs of anterior labial tooth rows is constant for all four species, only the posterior labial tooth rows with visible teeth were counted with the aid of a nikon smz1270 dissecting microscope, or a hand lens in field observations. due to environmental conditions or the effect of high amphibian chytrid infections in heleophrynidae (tarrant et al., 2013) some labial teeth get damaged. tadpoles belonging to heleophryne and hadromophryne cannot be accurately staged (gosner, 1960), as the hind limb buds, used in standard tadpole staging, develop in a small pouch. data analysis was carried out with program r (r development core team, 2014) and summarised in table 1. spearman rank-order correlation analysis was run with only body length and the number of posterior labial tooth rows. the number of posterior labial tooth rows varied from 5-17. it is also noted that the posterior labial tooth rows can be divided into two groups: primary posterior labial tooth rows and secondary posterior labial tooth rows (fig. 2). primary posterior labial tooth rows are characterised by being well developed, evenly spaced, continuous and darkly pigmented; they ranged between 4-6 (mainly 5) rows. secondary posterior labial tooth rows varied from 1-10: small, broken, narrowly spaced rows that fade away posteriorly. a strong positive correlation existed between body length and the number of posterior labial tooth rows in all four species and is statistical significant (table 1 and fig. 3). correlation data indicates that the body length explains 34-64% of the variation in number of posterior labial tooth rows. the strongest correlation existed within had. natalenis (r = 0.64; p = 0) and the lowest for hel. purcelli (r = 0.34; p = 0.04). in general, a strong support exists to indicate that number of posterior labial tooth rows increase with body size. remaining variations are due to environmental factors, such as feeding behaviour, food availability, flooding, and damage by the amphibian chytrid fungus (smith et al., 2007). visser (1985) recorded a beak with fang-like teeth that precedes the tooth rows during the first month of development, though he only recorded the total length of tadpoles. body length is more reliable than total length, as the tail could have been damaged and adds an additional unnecessary variable. his measurements thus had to be extrapolated to be able to include his data into this paper, achieved by multiplying visser (1985) total length measurements with a factor of 0.38342. this factor was derived by dividing body length with the total length of the tadpoles studied in this paper, and applied to visser’s data (table 2). the shaded area in fig. 3 represented the period of fang-liked teeth described by visser (1985). the lack of data for specimens smaller than 7 mm (body length) in museum collections indicates that these tadpoles make use of an alternative microhabitat during early developmental stages and thus elude capture by researchers. two years of bimonthly monitoring of tadpole numbers in the elandsberg mountains failed in detecting tadpoles smaller than 7 mm in main currents (w. conradie pers. comm.). it was also reported by visser (1985) that early developmental tadpoles are found between aggregations of small rocks where they are protected from the stronger currents. visser (1985) found no support that the early beak and fang-like teeth are used for grasping and indications are that they are solely used for feeding, however the sucker develops before these structures and thus fulfils the role of attachment from hatching to avoid getting swept away. the true function of this fang-like teeth needs further investigation and could fill the same function as for leptodactylon spp. tadpoles, in acting as a sieve to pick up detritus or preventing larger gravel/sand through (mapouyat et al., 2014). visser (1985) further hypothesised that the loss of the beak and fang-like teeth enables the tadpoles to switch between particular food sources, supported by the description and figure included in his paper, showing a tadpole adapted for feeding on detritus. these food sources are expected to be mostly available in the gullies or slow flowing sections of mountain streams close to where eggs would have been deposited, thus restricting the developing tadpoles to these habitats for the first few table 1. summary table indicating body length, number of posterior labial tooth rows, correlation between the two characters of southern african ghost frog tadpoles. body length (mm) number of posterior labial tooth rows spearman rank-order correlation (r) two-tailed value (p) heleophryne h. purcelli (n = 36) 15.6±4.07 7 to 17 0.34194 0.04437 h. regis (n = 77) 13.2±3.57 6 to 16 0.5429 0 h. hewitti (n = 75) 13.5±3.57 5 to 14 0.48837 1e-05 hadromophryne h. natalensis (n = 43 ) 18.8±3.26 13 to 16 0.64413 0 146 werner conradie, christa conradie months. it can’t be disregarded that a smaller suctorial mouth and weaker suction power will further limit smaller tadpoles to this microhabitat. as the tadpoles develop additional rows of teeth, their feeding behaviour moves to scraping algae from rock surfaces, thus subsequently moving into faster flowing water as the suction ability of the suctorial mouth increases. with development, the number of labial tooth rows increases to allow for a maximum surface area for this feeding mechanism. in order to expand and enhance our knowledge of heleophrynid tadpoles, their ecology, especially feeding behaviour, still needs to be studied further. acknowledgements the authors wish to thank the following people and organisations: klaas basson (mto: longmore plantafig. 3. graph showing the relationship between the number of posterior labial tooth rows and body length (mm) of four species of ghost frog tadpoles. the lack of data in the shaded area are explained by visser (1985) description of fang-like teeth development (see main text) table 2. early development of tadpole labial teeth according to visser (1985): * body length was extrapolated by multiplying by a factor of 0.38342 and inserted into the table as additional data. day average total length (mm) average body length* (mm) description of labial teeth development 1 11.27 4.32 hatchling, no teeth 8 to 10 12.5 4.79 first fang-like teeth develop above 11 to 13 13.7 5.25 single row of fang-like teeth with 15-19 above and 2-6 below 14 13.8 5.29 20-26 fang-like teeth above; 6-8 fang-like teeth on each aspect below 20 to 22 15.95 6.12 1st and 2nd continuous labial tooth rows develop below fang-like teeth; fang-like teeth start to drop out <30 16.54 6.34 4 labial tooth rows above and below; fang-like teeth all gone 147increased number of labial tooth rows in ghost frogs tion manager) and karen kirkman (mto: environmental consultant) who gave permission to carry out fieldwork on private land at the time of the study; zoological society of london edge fellowship program for funding; bayworld for fieldwork vehicle use and access to collections. research and collection permit: cro 64/10cr and cro 65/10cr. we thank the two reviewers for their insightful comments, which improved the quality of this note. references altig, r.a., mcdiarmid, r.w. 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(2006): the amphibian tree of life. bull. am. mus. nat. hist. 297. new york, pp. 370. gosner, k.l. (1960): a simplified table to stage anuran embryos and larvae with notes on identification. herpetologica 6: 183-190. hewitt, j. (1913): description of heleophryne natalensis, a new batrachian from natal; and notes on several south african batrachiens and reptiles. ann. s. afri. mus. 2: 475-484. iucn (2010): the iucn red list of threatened species. version 2015.2 <www.iucnredlist.org>. downloaded on 6 july 2015. khan, m.s., mufti, s.a. (1994): oral disc morphology of amphibian tadpole and its functional correlates. pak. j. zoo. 26: 25-30. mapouyat, l., hirschfeld, m., rödel, m.-o., liedtke, h.c., loader, s.p., gonwouo, l.n., dahmen, m., doherty-bone t., barej m.f. (2014): the tadpoles of nine cameroonian leptodactylodon species (amphibia, anura, arthroleptidae). zootaxa 3765: 29-53. minter, l.r., burger, m., harrison, j.a., braack, h.h., bishop, p.j. & kloepfer, d., ed. (2004): atlas and red data book of the frogs of south africa, lesotho and swaziland. first edition. washington, smithsonian. passmore, n.i., carruthers, v.c. (1995): south african frogs, 2ed. southern book publishers and witwatersrand university press, johannesburg. r development core team. (2013): r: a language and environment for statistical computing. r foundation for statistical computing vienna, austria. available from: <http://www.r-project.org>. rose, w. (1926): some field notes on the batrachia of the cape peninsula. ann. s. afr. mus. 20: 433-450. rose, w. (1950): the reptiles and amphibians of southern africa. maskew miller, cape town. san mauro, d., vences, m., alcobendas, m., zardoya, r., meyer, a. (2005): initial diversification of living amphibians predated the breakup of pangaea. am. nat. 165: 590-599. smith, k.g., weldon, c., conradie, w., du preez, l.h. (2007): relationships among size, development, and batrachochytrium dendrobatidis infection in african tadpoles. dis. aquat. org. 74: 159-164. tarrant, j., cilliers, d., du preez, l.h., weldon, c. (2013): spatial assessment of amphibian chytrid fungus (batrachochytrium dendrobatidis) in south africa confirms endemic and widespread infection. plos one 8(7): e69591. doi:10.1371/journal.pone.0069591. van dijk, d.e. (1966): systematic and field key to the families, genera and described species of southern african anuran tadpoles. ann. natal mus. 18: 231286. van dijk, d.e. (2008): clades in heleophrynid salientia. afr. j. herp. 57: 43-48. 148 werner conradie, christa conradie venesky, m.d., wassersug, r.j., jorgensen, r.m., parris, m.j. (2011): comparative feeding kinematics of temperate pond-dwelling tadpoles (anura, amphibia). zoomorphology 130: 31-38. venesky, m.d., wassersug, r.j., parris, m.j. (2010a): how does a change in labial tooth row number affect feeding kinematics and foraging performance of a ranid tadpole (lithobates sphenocephala)?  biol. bull. 218:160-168. venesky, m.d., wassersug, r.j., parris. m.j. (2010b): the impact of variation in labial teeth on the feeding kinematics of southern leopard frog (lithobates sphenocephala) tadpoles. copeia 2010: 481-486. visser, j.d. (1971): hunting the eggs of the ghost frog (heleophryne purcelli orientalis fitzsimons). afr. wildl. 25: 22-24. visser, j.d. (1985): the fang-like teeth of the early larvae of some heleophryne (anura: leptodactylidae). s. afr. j. sci. 81: 200-202. wager, v.a. (1965): the frogs of south africa. purnell, cape town. appendix a list of material examined, ‘pem t’ refers to the tadpole collection of the port elizabeth museum and the numbers in brackets refer to the number of tadpoles in each lot: heleophryne purcelli: pem t275 (4), pem t277 (11), pem t278 (19), pem t285 (2). heleophryne regis: pem t30 (8), pem t34 (20), pem t36 (4), pem t284 (9), pem t286 (11), pem t287 (2), pem t288 (18), pem t420 (5). heleophryne hewitti: pem t327 (2), pem t408 (7), pem t373 (2), pem t419 (5), pem t694 (4), pem t694 (6), 49 field observations. hadromophryne natalensis: pem t82 (24), pem t214 (16), pem t274 (1), pem t301 (2). acta herpetologica vol. 10, n. 1 june 2015 firenze university press acta herpetologica journal of the societas herpetologica italica acta herpetologica 9(2): 131-138, 2014 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-13188 in vitro temperature dependent activation of t-lymphocytes in common wall lizards (podarcis muralis) in response to pha stimulation roberto sacchi1,*, enrica capelli1, stefano scali2, daniele pellitteri-rosa1, michele ghitti1, emanuele acerbi1, elena pingitore1 1 dipartimento di scienze della terra e dell’ambiente, università degli studi di pavia, via ferrata 9, i-27100 pavia, italy. *corresponding author. e-mail: roberto.sacchi@unipv.it 2 museo civico di storia naturale, c.so venezia 55, i-20121 milano, italy submitted on 2013, 28th october; revised on 2014, 23rd june; accepted on 2014, 24th june editor: uwe fritz abstract. ecological immunology attempts to explain the variability of immune response among individuals by invoking costs and trade-offs, which may optimize the immune defence against pathogens. in ectotherms body temperature is correlated to that of the surrounding environment, so that their entire physiology, including immune functions, is influenced by the environmental temperature. we used in vitro phytohaemoagglutinin (pha) stimulation in order to assess the effects of temperature on cell mediated adaptive response in male and female common wall lizards (podarcis muralis). cell cultures were prepared from blood samples, inoculated with pha and incubated at 22°c, 25°c, 32°c, and 38°c for three days. pha stimulation caused proliferation of t-lymphocytes, but the effect depended on the incubation temperature. lymphocyte proliferation was significantly impaired at both 22°c and 38°c compared to 32°c, which represented the highest levels of activation. furthermore, lymphocyte activation was more variable in males while females were less immune suppressed than males at low temperatures. differences between sexes suggest a possible influence of steroid hormones. keywords. ecological immunology, adaptive immune response, temperature dependent effects, phytohaemoagglutinin pha, t-lymphocyte proliferation, ectotherms. introduction ecological immunology investigates the physiological or molecular basis of immune responses, by placing them in the context of ecology and adaptation (sheldon and verhulst, 1996; norris and evans, 1999; sadd and schmid-hempel, 2009). there is extensive evidence that immune defence is costly, requiring investment of energy, nutrients, and time, during the development, maintenance, and use of the immune system (klasing and leshchinsky, 1999; lochmiller and deerenberg, 2000). when resources are limited, allocation of energy to immune defences may be modulated by the need to spend energy on other functions such as growth, reproduction, and maintenance (nelson and demas, 1996). thus, multiple trade-offs occurring between immune functions and other compartments and life-processes cause severe constraints on the evolution of immune response and the other fitness related traits. costs of development, maintenance and use of immune defence have been widely investigated on homeotherm vertebrates, and especially birds (norris and evans, 1999; martin et al., 2008). by contrast, ecthotermic vertebrate have been less extensively studied (see zimmerman et al., 2010), even if a large amount of researches had been done on fish (e.g., bly and clem, 1992; collazos et al., 1996; köllner and kotterba, 2002; ndong et al., 2007; prophete et al., 2009). ectotherms are particularly interesting under an eco-immunological perspective, as body temperature is correlated to that of 132 r. sacchi the surrounding environment, so that their entire physiology, including immune functions, is influenced by the environmental temperature (zimmermann et al., 2010). this offers an interesting opportunity to investigate the costs of the immune response, as the trade-offs mediated by the immune function are likely to have different settings at different environmental temperatures. both endotherms and ectotherms show seasonal variation in immune-response, but ectotherms display higher variability due to their dependence on environmental temperatures as heat source (zimmermann et al., 2010). the immune system of ectotherms has been shown to respond across a wide range of temperatures, but strongest responses occur at species-specific temperature ranges, with impaired responses at temperatures above and below the optimal threshold (le morvan et al., 1998; mondal and rai, 2001; merchant et al., 2003; merchant and britton, 2006; raffel et al., 2006). for example, experiments carried out on carp (cyprinus carpio) immunized against bovine serum albumin, revealed that the primary antibody response is suppressed at low temperatures (avtalion, 1969), particularly that mediated by t-helper cells (le morvan et al., 1996). similarly, mondal and rai (2001) reported that the highest levels of phagocytosis and cytotoxicity of splenic macrophages from hemidactylus flaviviridis occurred at 25°c, with impaired macrophage function at both higher and lower temperatures. in reptiles, the studies on temperature effects on immune system chiefly focused on the seasonal variation of immune response in both innate (mondal and rai, 2001; merchan et al., 2003, 2004) and adaptive immunity (muñoz and de la fuente, 2001; but see zimmermann et al., 2010 for a review). surprisingly, no one has specifically investigated the response of the immune system to short time variation of temperature such that occurring daily or with weather instability. short time effects of temperature are particularly interesting under an eco-immunological perspective, since pathogen ability to infect ectotherms can be heavily affected by fast temperature-dependent fluctuations of host immune system (wright and cooper, 1981), with direct consequences on the behavioural, ecological and physiological strategies that individuals adopt to optimize fitness. in the present study we measured the t-cell mediated immune response of common wall lizards podarcis muralis under different temperatures, covering the typical thermal-activity range of the species. to do this, we set up cell cultures from blood samples that were activated with phytohaemagglutinin (pha) and incubated at four different temperatures. pha is a lectin found in plants, especially legumes, which causes local swelling and oedema, driven by mitogenesis and infiltration of t-lymphocytes into the injected tissue (goto et al., 1978). the common wall lizard is a small lizard (snout-vent length, svl, 45-75 mm) widespread in southern and central europe, which mates multiply and produces two clutches per year on average (sacchi et al., 2012) during its life (max lifetime 5 years, barbault and mou, 1988; pers. obs.). breeding season starts from late february and ends in july (sacchi et al., 2012), and body temperature during activity is near 33°c, being slightly higher (33-36°c) in warmer regions (e.g. central italy) and lower (32°c) in mountain areas (avery, 1978; braña, 1991; tosini and avery, 1994). the immune-system has been previously investigated in relation to polymorphic ventral colouration (sacchi et al., 2007; galeotti et al., 2010) or immunocompetence handicap hypothesis (oppliger et al., 2004), but earlier study have not examined the influences of the environmental factors on the immune system response. materials and methods individual collection from 6 july to 4 august 2012, we collected by noosing 24 (12 males and 12 females) adult common wall lizards (snoutvent length, svl > 50 mm, sacchi et al., 2012) in a farm in the surrounding of pavia (northern italy, 45°11’31”n, 9°9’11”e). we carried out five sampling sessions in which at least one male and one female were collected. after capture, lizards were measured by a digital calliper (accuracy ± 0.1 mm) for svl and transferred in cloth bags to the laboratory at the university of pavia. blood sampling and cell cultures blood samples (15-20 μl) were collected in heparinized capillary tubes from the postorbital sinus (maclean et al., 1973) and inoculated in 15 ml of rpmi 1640 medium supplemented with 10% bovine serum. cell suspension was then subdivided into two 7 ml sub-cultures, one of which was inoculated with 1% pha solution (pha-p sigma l-8754, 50 mg in 10 ml phosphate buffered saline (oppliger et al., 2004; sacchi et al., 2007). the remaining solution (1 ml) was used to assess starting cell concentration (scc) using a neubauer chamber, and only live lymphocytes were counted. each sub-culture was then distributed in four 1.5 ml culture tubes, and incubated at 22°c, 25°c, 32°c, and 38°c for 3 days. afterwards, cell were collected, re-suspended and newly counted. this second count involved only proliferating lymphocytes. stimulation of t-cell after incubation was evaluated by determining the colony-forming units per ml (cfu) and the ratio between the total cell recovery to scc (growth index, gi). unfortunately, 30 samples incubated at 32°c were lost because the stove broke up during experiment. so our final sample included 48 cultures for 22°c, 25°c and 38°c, but only 18 for 32°c. 133temperature effects on t-cells in p. muralis statistical analyses preliminarily, we ran a t-test for matched pairs to check for differences in lymphocyte stimulation between the phatreated and the control cultures at each of the four incubation temperatures. the cfu in the full sample showed a poissonlike distribution and thus its relationship with sex, treatment (pha vs control) and temperature (analysed as a four levels factor) was investigated by a glmm with poisson error distribution and log link function. the number of colonies counted after the three day incubation was the dependent variable, while the individual identity entered the model as random factor. all main effects and two-way interactions sex × temperature, sex × treatment and temperature × treatment were included in the models as fixed effects. we also included the svl as covariate in order to account for possible effects of individual size on immune-response. the growth index gi was slightly skewed, and achieved normality after four root transformation. the relationship between gi and sex, treatment and temperature was thus analysed using a linear mixed model including individual identity as random factor, and the same fixed factors, covariate and their interactions used in the analysis of cfu. both models were then optimized by removing all non-significant terms (using likelihood-ratio tests) until only significant terms were retained (zuur et al., 2009). all tests were performed using r 2.13.1 statistical package (r development core team, 2010), and unless otherwise stated, values reported are means ± se. results peripheral blood lymphocytes of both sexes were actually stimulated by pha at all incubation temperatures, as both cfu and gi were significantly far higher in activated cultures than in controls. however, the difference between the gi values of females in pha-activated and control recorded at 32°c was marginally not significant, probably because of the small sample (see table 1 for statistics). pha-activated lymphocytes showed the characteristic activated morphology with enlarged size (2-3 times) and spikes and cell aggregates (clones) containing from few to several dozen cells (fig. 1). the glmm confirmed the capacity of pha to activate lymphocytes proliferation, but the intensity of the response was highly affected by both incubation temperatures and sex (fig. 2a). indeed, the cfu significantly increased in pha treated cultures with respect to controls, but strictly depending on the incubation temperature (temperature × treatment: lrt-χ² = 29.34, df = 3, p < 0.0001, fig. 2a). the highest activation was recorded at 32°c (8056 ± 1197 colonies/ml), while the minimum occurred at both 25°c (2760 ± 489 colonies/ml) and 38°c (2617 ± 531 colonies/ml). moreover, males showed on average a significant higher activation than females, irrespective of the incubation temperatures (males: 3583 ± 523 colonies/ml, females: 3482 ± 452 colonies/ml; sex × treatment: lrt-χ² = 13.74, df = 1, p = 0.00021), even though pha-activation was higher in females than in males when cultures were incubated at 22°c (sex × temperature: lrt-χ² = 40.97, df = 3, p < 0.0001, fig. 2a, table 1). finally, the size of individuals did not significantly affect the activation of lymphocytes (p-value at removal > 0.66). the final model for gi included only the interaction temperature × treatment (lrt-χ² = 13.24, df = 3, p = 0.0041), confirming that cell proliferation was higher at 32°c (gi = 13.27 ± 2.26) than in all other temperatures (gi22°c = 8.56 ± 1.93, gi25°c = 7.69 ±1.65, gi38°c = 5.13 ± 0.88, fig. 2b). the gi was higher in males than in females at 32°c (fig. 2b), but the effect of sex was not significant (p > 0.81), and was removed from the final model. as for cfu, the size of individuals had no significant effects on immune-response. finally, the intensity of the lymphocyte activation was highly variable among individuals, as both models revealed a highly significant effect of the random factor table 1. in vitro effects of pha administration on t-lymphocyte proliferation assessed by colony-forming units (cfu) and growth index (gi) in male and female common wall lizards at four different temperatures. phaactivated controls n t p cfu males 22°c 2643 ± 694 26 ± 26 12 3.774 0.0031 25°c 2890 ± 692 0 12 4.176 0.0015 32°c 8489 ± 1767 1484 ± 1484 6 3.000 0.030 38°c 2760 ± 787 0 12 3.505 0.0049 females 22°c 4414 ± 791 0 12 5.581 0.00016 25°c 2630 ± 721 26 ± 26 12 3.570 0.0044 32°c 7187 ± 1005 0 3 7.155 0.019 38°c 2473 ± 746 195 ± 195 12 2.970 0.013 gi males 22°c 5.85 ± 1.16 1.09 ± 0.22 12 3.906 0.0024 25°c 6.17 ± 1.24 1.25 ± 0.45 12 6.578 0.00039 32°c 16.7 ± 2.18 1.28 ± 0.67 6 3.416 0.019 38°c 4.95 ± 0.93 0.91 ± 0.29 12 4.958 0.00043 females 22°c 11.26 ± 3.58 1.58 ± 0.67 12 5.745 0.00012 25°c 9.20 ± 3.07 1.56 ± 0.57 12 5.437 0.00020 32°c 6.32 ± 1.05 0.82 ± 0.81 3 3.237 0.084 38°c 5.30 ± 1.53 1.47 ± 0.54 12 3.487 0.0051 134 r. sacchi (cfu: lrt-χ² = 406.38, df = 1, p < 0.0001, gi: lrt-χ² = 22.01, df = 1, p < 0.0001). discussion in this paper we analysed the effect of short-time variation of temperature on the lymphocyte activation in male and female common wall lizards, assessed by pha injection in blood cultures. despite the fact that temperature is expected to affect the immune response more extensively in ectotherms than in endotherms, the susceptibility to sudden changes of environmental temperature by immune response of the same individual, to our knowledge, had never been performed in a terrestrial reptile. previous immunological studies in reptiles used pha injection to examine the negative effect of steroids on cell-mediated immunity (belliure et al., 2004; oppliger et al., 2004; berger et al., 2005; huyghe et al., 2009), morph-specific differences in immune response (sacchi et al., 2007; huyghe et al., 2009), or female preference for males with better immune response (lópez and martín, 2005). several components of the defence mechanisms of reptiles including phagocyte bactericidal activity, leukocyte mobilization and humoral mediators of inflammation have been reported to be temperature-dependent (farag and el ridi, 1984; muñoz and de la fuente, 2001; merchant et al., 2003, 2004, keller et al., 2005). however, most studies did not analysed the response of the same individual to different temperatures, rather compared the responses of different groups of individuals to different temperatures (farag and el ridi, 1984; muñoz and de la fuente, 2001). so, these findings cannot actually be considered a demonstration of a direct effect of temperature on the immune system, since several other factors may affect immune-response (e.g., hormones mondal and rai, 2002; belliure et al., 2004; huyghe et al., 2009). the in vitro activation of lymphocytes enabled us to set up an experimental design with repeated measures within individual, in which every lizard has been exposed to all the incubation temperatures. this design allowed us to actually evaluate the change in the immune function of a given individual in response to the variation of thermal condition. thus any significant change in lymphocytes proliferation can be attributed to the direct effect of environmental temperature. in this study we found that the lymphocytes proliferation following pha stimulation was significantly impaired in cultures kept at both 22°c and 38°c compared to that kept at 32°c, which represented the highest levels of activation in both sexes. since the ability of lymphocytes to proliferate following a mitogenic stimulus in vitro is an indication of immune response in vivo fig. 1. morphology of lymphocytes of common wall lizards in blood cultures: a) a single lymphocyte (black arrow) among erythrocytes (white arrow); b) a lymphocyte with spikes and a small (c) and a large (d) cell aggregates. fig. 2. the in vitro effect of temperature on colony-forming unit (cfu, a) and growth index (gi, b) of t-lymphocytes of male (white) and female (gray) common wall lizards incubated with (pha-activated) or without (controls) phytohaemoagglutinin. 135temperature effects on t-cells in p. muralis (clem et al., 1984), our data suggest that common wall lizards keep a temperature range in which immune response is optimal, while temperatures above and below this range exert a suppressive effect. our data agree with past researches on thermal activity, which indicated that common wall lizards reach the maximum efficiency near to 33°c (avery, 1978; braña, 1991; tosini and avery, 1994), and suggest that physiological processes in this species are optimized near this temperature. this conclusion agrees also with previous findings in other species of reptiles: the highest levels of phagocytosis and cytotoxicity of splenic macrophages of hemidactylus flaviviridis occurred at 25°c, with impaired macrophage function at both higher (37°c) and lower temperatures (7°c and 15°c, mondal and rai, 2001). in alligator mississippiensis antimicrobial and amoebacidal activity occurred between 5°c and 40°c, and was significantly reduced at temperatures below 15°c and above 30°c (merchant et al., 2003, 2004). accordingly, most physiological processes are optimized near 30-31°c (merchant et al., 2003, 2004). earlier reports on the effects of temperature on the immune system were aimed to sustain the hypothesis that reptiles might be immunosuppressed during the winter months, when body temperatures are far below the optimum. in our study, however, we found that immune suppression may already occur during the breeding season, in coincidence with cold periods, or even during night time. indeed, body temperatures of lizards remain remarkably constant during the day, but decrease substantially both in the early morning and in the late evening (braña, 1991). these findings have relevant consequences in the perspective of ecological-immunology, since short-time fluctuations of temperature might severely affect the trade-offs between immune function and other compartments and life-processes, with remarkable effects on behavioural and physiological fitness-related traits. for example, temperature-dependent suppression of immune function might increase the susceptibility to parasite infections during the breeding season and consequently reduce the reproductive efforts of less resistant individuals. furthermore, the immunocompetence handicap hypothesis (folstad and karter, 1992) states that androgens have a dual effect in stimulating the expression of the secondary sexual characters while suppressing the immune functions. the reduction in the immune response following unpredictable fluctuations of temperature below or above the optimum, might amplify the suppressive action of androgens, with negative effects on the expression of secondary sexual traits of males. however, these additional “thermal” costs might be paid differentially by the different individuals; for example lizards keeping less quality territories including low quality basking sites might suffer the highest levels of temperature-dependent immune suppression. actually, both individuals settled in marginal territories and subordinate individuals might spend much more time to reach optimal body temperature than dominant individuals occupying the best basking sites, and therefore might be exposed for longer time to the consequences of temperature-dependent immune suppression. the effects of temperature on the immune functions in ectotherms should, ultimately, increase the variance of male quality, and consequently amplify the difference in the expression of the secondary sexual traits among them. in this scenario, endotherms are not expected to pay additional costs in immunocompetence due to fluctuations of environmental temperatures, since they use metabolic heat to maintain body temperature within the optimal physiological range. so, under high variable temperature regimes (e.g. in temperate climates) endotherms might reveal a lower variability in male secondary sexual traits than ectotherms, and this difference should be reduced in more stable climatic conditions (e.g. in tropical regions). a second relevant result of this study was the difference in temperature-dependent immune suppression between males and females. earlier studies in mammals have well established sex differences in cell-mediated immune responses, with females generally having enhanced immune-reactivity than males (ansar ahmed et al. 1985; grossman, 1989; cannon and st. pierre, 1997; klein, 2004). among reptiles, sex differences in immune response have been reported in the striped sand snake psammophis sibilans and in the yellow-bellied house gecko h. flaviviridis, in which females have higher response than males (saad, 1989; mondal and rai, 1999, 2002). in both cases, sex-associated immune differences have been related to sex steroids, even if experimental studies have shown that immunosuppressive effects of sex hormones act mainly on innate immunity (mondal and rai, 1999, 2002). indeed, gonadectomy of both males and females causes a considerable increase in percentage phagocytosis, while in vitro administration of dihydrotestosterone (dht) and 17β-estradiol (e2) suppresses phagocytosis, cytotoxic activity of splenic macrophages and il-1 secretion (mondal and rai, 1999, 2002). on the other hand, con a and pha stimulations did not cause significant differences between sexes in the t-lymphocytes proliferation in both caspian pond turtle and loggerhead sea turtles (muñoz and de la fuente, 2001; keller et al., 2005), suggesting that adaptive response is similar in males and females. contrary to these latter, we showed that pha stimulation in common wall lizards causes dependent activation of t-lymphocytes, suggesting that adaptive immune response might be actually 136 r. sacchi affected by sex steroids. however, this difference between sexes varied at different temperatures, and immunosuppression was more severe on males at temperatures below the optimum and more severe on females at temperatures above the optimal. this different response by sexes might be explained by different and opposite effects of male and female hormones on the immune function, but future researches are needed to highlight the specific effect of hormones on lymphocyte activation in this species. in conclusion, in vitro activation of immunoresponse is a powerful and effective tool to investigate the effect of biotic and abiotic factors on the immune function in natural populations of reptiles. in particular, the possibility to analyse immune-response in vitro might allow researchers to repeatedly test the same individual, improving the opportunities to focus on the currencies that mediate the cost of immune responses in life history trade-offs. acknowledgements we thank walter cocca, davide tronconi and alan coladonato for their help with field and laboratory work. the study was carried out in conformity with the italian and european current laws for lizard collection and detention (aut. prot. pnm-2012-0009344). references ansar ahmed, s., penhale, w.j., talal, n. 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(2009): mixed effects models and extension in ecology with r. springer, new york. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 8(2): 81-91, 2013 life history of the marbled whiptail lizard aspidoscelis marmorata from the central chihuahuan desert, mexico héctor gadsden1, gamaliel castañeda2 1 instituto de ecología, a. c.-centro regional chihuahua, cubículo 29c, miguel de cervantes no. 120, complejo industrial chihuahua, c. p. 31109, chihuahua, chihuahua, méxico. corresponding author. e-mail: hector.gadsden@inecol.edu.mx 2 facultad de ciencias biológicas. universidad juárez del estado de durango. avenida universidad s/n. fraccionamiento filadelfia. gómez palacio, 35070, durango, méxico submitted on 2012, 5th december; revised on 2013, 10th august; accepted on 2013, 3rd september. abstract. the life history of a population of marbled whiptail lizard, aspidoscelis marmorata, was examined from 1989 to 1994 in the sand dunes of the biosphere reserve of mapimí, in northern méxico. lizards were studied using markrecapture techniques. reproduction in females occurred between may and august, with birth hatchlings matching the wet season in august. reproductive activity was highest in the early wet season (july). males and females reached adult size class at an average age of 1.7 years and 1.8 years, respectively. body size of males attained an asymptote around 90 mm snout-vent length and females around 82 mm snout-vent length, at an age of approximately 3.6 years and 3.0 years, respectively. the density varied from 7 to 85 individuals / 1.0 ha. the mexican population had late maturity, relatively long life expectancy, and fewer offspring. overall, the observed data for a. marmorata and the expectations of life history theory for a late maturing species (k-rate selection) are in agreement. keywords. aspidoscelis marmorata, lizard, mexico, life history, reproductive cycle, chihuahuan desert. introduction four life history traits (age of maturity, clutch size, reproductive effort, and longevity) are used regularly to segregate populations into one of two states generally recognizing but ignoring that a continuum exists between these two. the contrasting states are: short-lived populations characterized by individuals with short lives, early sexual maturity, large broods, and high annual reproductive effort; and long-lived populations characterized by individuals with long lives, delayed sexual maturity, small broods, and low annual reproduction effort. life history theory provides two opposing models for the evolution of these two population types. the deterministic model postulates that high levels of density-independent mortality result in fluctuating population density, which favors a high intrinsic growth rate (r) and produces short-lived populations. in the stochastic model (optimization), high density-independent mortality produces variable, and usually low, juvenile survivorship, which favors a stable and long-lived adult population. the conflict arises from the generality of the models and their attempt to explain life-histories evolution of diverse organisms (zug, 1993). life history studies within discrete taxonomic groups (e.g., tinkle et al., 1970; ballinger, 1973; dunham, 1980, 1981; james, 1991; chapple, 2003; sears and angilletta jr., 2004, mata-silva et al., 2008, 2010) and among conspecific populations (e.g., pianka, 1970; tinkle and ballinger, 1972; parker and pianka, 1975; ballinger, 1979; dunham, 1982; van devender, 1982; niewiarowski and roosenburg, 1993; lemos-espinal and ballinger, 1995; stearns, 2000; niewiarowsky et al., 2004; du et al., 2005; rojasgonzález et al., 2008) have been somewhat more successful in revealing the relationships among life-history traits and different environmental regimes. even in these cases, difficulties are encountered that make conclusions less 82 héctor gadsden, gamaliel castañeda robust than desirable. a major difficulty is the discrimination between a trait´s variation arising from selection pressures for individuals to adapt to their present habitat / location and variation resulting from a proximate response to current disturbance in local conditions. awareness of the difficulties in the data and interpretation promotes caution but does not deny the evolution of life-history patterns (zug, 1993). life history of a species can be summarized by demographic parameters such as rates of birth and death, migratory movement, and structure of populations through time, as they deal with the interaction between longevity, reproductive age, growth, and age-specific mortality versus the environment (zug, 1993; stearns, 2000). interactions among these parameters through life make demographics of populations subject to constant variation through time. the demography of lizard populations can be influenced by various environmental factors, such as temperature (adolph and porter, 1993; parker, 1994), precipitation (andrews, 1988; bull, 1994), food availability (ballinger, 1977; howland, 1992; smith, 1996), as well as morphological and phylogenetic constraints (ballinger, 1983; stearns, 1984; dunham and miles, 1985). some life history patterns can be singled out relating the dynamics and structure of populations (tinkle, 1969a, stearns, 2000). for example, lizards with delayed sexual maturity tend to have longer life cycles, along with major energetic investments in maintenance instead of reproduction (tinkle, 1969a; tinkle et al., 1970), and lower adult mortality (hasegawa, 1990; bull, 1995). lizards with early sexual maturity have shorter life cycles and higher mortality rates for the young and adults (ballinger and congdon, 1981; andrews and nichols, 1990), resulting in a higher rate a of population turnover (ferguson et al., 1980; tinkle et al., 1993). this dichotomy represents the extremes of a life-history gradient, but many intermediate combinations exist (dunham et al., 1994; stearns, 2000). the population structure can also be influenced by the mating system. populations with higher incidence of polygyny tend to have higher male death rates than populations where polygyny is reduced (schoener and schoener, 1980; stamps, 1983), generating a female biased sex-ratio. life history theory was developed mainly from data of temperate lizard species in the united states (pianka, 1970; ballinger, 1983), nevertheless in the vast desert regions of northern mexico, which has a rich diversity of lizards, long-term studies of life history, population structure and dynamics are limited (e.g., gadsden et al., 1995; gadsden et al., 2001b; gadsden and estrada-rodriguez, 2007). here we study the life-history of a population of aspidoscelis marmorata from the central chihuahuan desert in northern mexico (hendricks and dixon, 1986; dixon, 2009). although there is extensive knowledge of several aspects of the ecology and life history of a. tigris from its northern arid distribution (turner et al., 1969; pianka, 1970; parker, 1972; asplund, 1974; vitt and ohmart, 1977; mitchell, 1979; hendricks and dixon, 1984; anderson, 1994; sullivan, 2009), little is known about demography and life history of a. marmorata from the desert zones of northern méxico (gadsden et al., 1995; díaz-gómez, 2009; dixon, 2009; mata-silva et al., 2008, 2010). herein we identify seasonal patterns of variation in population size, age structure, sex ratio, growth, reproduction, survivorship, and life history attributes of the lizard a. marmorata in the central chihuahuan desert, and make comparisons with other populations of the same species and a. tigris, to evaluate predictions of life history theory. materials and methods study area we conducted field work in a 1 ha study plot (26°52’n, 103°32’w) within the mapimian subprovince of the chihuahuan desert in the mapimí biosphere reserve, chihuahua, mexico (1250 m elev.) (barbault and halffter, 1981). the study plot was gridded with wooden stakes placed 20 m apart over an area of about 100 × 100 m. data from this study plot were used in the evaluation of demographic characteristics of this population. the habitat was sandy dune, and the vegetation is dominated by desert thornscrub of acacia constricta, acacia gregii, larrea tridentata, yucca elata, and lycium berlandieri (breimer, 1985). the climate of this region is seasonal and receives monsoon summer rains, with the highest temperature and rainfall occurring in june through september. temperatures range from an average winter low of 3.9 ºc to an average summer high of 36.1 ºc. mean annual precipitation is 230 mm, but variation between years is high (cornet, 1988). reproductive analysis during 1990 we collected monthly samples of a. marmorata from other sand dunes (within several kilometers to the marked population) for analysis of reproductive condition. these were brought into the laboratory and necropsied within 24 h. in total we collected 48 female specimens by noosing or shot with bb rifles, euthanized with nembutal, preserved in 10% formalin (gadsden and palacios-orona, 1997; gadsden et al., 2001a), and deposited the samples in the collection of instituto de ecología, a. c. (voucher specimens-inecol-ct-1-48). we used the smallest females that showed vitellogenic follicles or oviductal eggs to estimate the minimum snout-vent length (svl) at sexual maturity (ramírez-bautista and vitt, 1997). we recorded number of non-vitellogenic follicles, vitellogenic follicles and oviductal eggs 83life history of the marbled whiptail lizard from the central chihuahuan desert, mexico for females. we determined litter size by counting oviductal eggs of adult females during reproductive season. population structure and dynamics in the staked study plot we caught individual lizards of a. marmorata using pitfall cans (100 traps of two gallons volume each one) and funnel traps in spring (4-8 may 1989, 6-12 may 1990, 20-27 apr. 1991, 13-21 may 1992, 8-11 may 1993, and 20-24 apr. 1994), summer (11-17 sep. 1989, 7-16 jul. 1990, 15-23 jul. 1991, 21-25 aug. 1992, 18-22 jul. 1993, and 21-24 sep. 1994), and fall (4-14 nov. 1989, 27-30 oct. and 1-3 nov. 1990, 3-11 nov. 1991, 13-17 nov. 1992, 10-16 nov. 1993, and 1-5 dec. 1994). traps were checked every day in the morning during different periods of study and were removed after each sampling period. each individual was permanently marked by toeclipping, and a number was painted on the back for quick identification (tinkle, 1967). for each capture, the following data were recorded: date, time of day, sex, and snout-vent length (svl), measured to the nearest 1 mm with a ruler. in addition, abdomens of females were carefully palpated to determine whether oviductal eggs were present. once data was acquired, lizards were released immediately at the exact point of capture (james, 1991). in order to increase recapture data with individuals previously marked on the back, the site searches followed a standardized procedure. on each sampling date, each person (3 people) chose one row of quadrants (100 × 20 m) and walked slowly across the entire area remaining at all times between two rows of stakes (tinkle, 1967; howland, 1992). each bush and other hiding places were disturbed when necessary to locate and capture each lizard. field work was conducted between 9:00 and 14:00 h every day. we recorded svl, weight and sex for marked and recaptured lizards and estimated the growth curves of females and males. the changes in length (dsvl) and time intervals (dt) were used to estimate body growth rates (gr = dsvl/dt). data were analyzed considering the following recapture time interval > 60 and < 460 days. additionally, averages of growth and snout-vent length of lizards recaptured more than once were used to estimate rates of growth representative of the population. average length during the interval for each lizard (mean snout-vent length) was the average of the first and last snout-vent length observed for each. we used the von bertalanffy model to evaluate growth rate of a. marmorata (von bertalanffy, 1951, 1957; fabens, 1965; lemosespinal et al., 2005). this model predicts that growth rate will be maximal for small body size (juveniles) and will decrease as the size increases (lemos-espinal and ballinger, 1995) following the linear function: gr = a – bmeansvl (1) where a is the initial growth rate and b is the decrease coefficient. meansvl is used instead of original size because growth is measured over a limited period and may overestimate the gr for initial svl (van devender, 1978). asy mptot ic size is pre dic te d as z = -a/b equation (1) can be expressed as follows: a bmeansvl = a [1-svl/z] which is the derivation of fabens (1965) of differential equation model of von bertalanffy growth. knowing the size of lizards at birth (svlo), and using the z and b values obtained from gr = a bmeansvl, the growth curve can be obtained from: svl = z (1 ke-bt) where svl is length reached by a lizard after a time t (from birth), k is a constant that can be calculated if svl0 is known, and t is the number of days elapsed (age of lizard). the estimate of k is performed as follows: k = 1 svlo/z we used the following fabens (1965) equation to estimate svl of a lizard at time t + d (svl2) in terms of the svl at time t (svl1): svl2 = z (z svl1) e-bd where d is the time interval for growth. to test how well this model fits the real growth of a. marmorata, lizards of known age were compared with sizes predicted by the model. linear regressions were used to determine the relationship between gr and svl. the regressions were calculated separately for each sex and were compared using analysis of covariance (ancova), using svl as a covariate. for all statistical analysis we used only one observation per individual, which was selected at random. all values are given as means ±1 se. densities were estimated from lizards caught in pitfall cans and from visual sightings. we estimated seasonal density (1989-1991) using jolly-seber method (jolly, 1965; seber, 1982) for open populations; therefore we did not need to assume the absence of recruitment and mortality. the estimation of population size was obtained from the simple relationship: 84 héctor gadsden, gamaliel castañeda population size = size of marked population / proportion of lizards marked. random sampling becomes the crucial assumption, and we assume that: 1) every individual has the same probability (αt) of being caught in the tth sample, where it is marked or unmarked. the critical assumption of equal catchability was tested for leslie, chitty, and chitty test of equal catchability (leslie et al., 1953). 2) every marked individual has the same probability of survivorship (øt) from the tth to the (t + 1)st sample. 3) individuals do not lose their marks, and marks are not overlooked at capture. 4) sampling time is negligible in relation to intervals between samples. density estimates using jolly-seber method did not include data for period 1992-1994. the population abundance of this species declined dramatically during this last period (likely due to a prolonged regional drought) and the data were not suitable for this method. results reproductive cycle body size (svl) of adult females averaged 78.2 mm (se = 0.62, range 70-90 mm, n = 48). the mean svl of gravid females was 80.0 mm (se = 0.84, n = 9). gravid females represented 28% of all sexually mature females caught during the reproductive season (n = 32). reproductive activity in females (fig. 1) began in may and declined in middle aug., with oviductal eggs present from may to august. we observed hatchlings from aug. to sep., when most of the annual precipitation occurred and food was abundant. in jul. and aug., 50% and 38% of females respectively had eggs in utero. the embryonic developmental period was estimated from the date at which the first female had freshly ovulated eggs in utero (early-may) to when the first hatchling was found (lateaug.). these data suggest a gestation of ca. 75 days and incubation was ca. 45 days. morphometric data field mean svl in adult males was 80.95 mm (se = 0.73, range 70-92 mm, n = 70) and in adult females was 76.80 mm (se = 0.70, range 70-90 mm, n = 36). average body mass of males was 14.67 g (se = 0.42, range, 8.0–26.5 g, n = 69) and in females was 12.00 g (se = 0.53, range 8.0-22.00 g, n = 36). based on comparisons of males and females, males attained a significantly greater svl and body mass than females (f1, 105 = 13.29, p < 0.0001 and f1, 104 = 14.49, p < 0.0001, respectively). population structure and dynamics growth was significantly higher (anova, f4, 82 = 19.6, p < 0.0001) in juveniles and subadults (x = 0.12 ± 0.01 mm × day-1, n = 13 and x = 0.10 ± 0.01 mm × day-1, n = 22, respectively) than hatchlings (x = 0.06 ± 0.01 mm × day-1, n = 5), adults (x = 0.02 ± 0.003 mm × day-1, n = 36), and old adults (x = 0.005 ± 0.003 mm × day-1, n = 7). females grew faster than males (0.046 ± 0.008 mm/ day and 0.040 ± 0.006 mm/day, respectively), however this difference is not significant (ancova f1, 52 = 0.012, p = 0.91). the growth rate decreased significantly with respect to svl (f1, 52 = 45.09, p < 0.0001), suggesting a growth curve of von bertalanffy type. growth rates varied inversely with the average svl for males and females (fig. 2). the values used to estimate constants of the growth curves were the same for females and males, corresponding to an svl of 37.9 mm, with an estimated age of 30.0 days. using these constants in the equation of fabens (1965), provides longevity for females of approximately 3.0 yr and 3.6 yr for males, at an svl of 82 mm and 90 mm, respectively (fig. 3). longevity for males larger than 90 mm and females larger than 82 mm was very high and may not be representing reality. the age of an individual can be estimated from data on the earliest date of hatchling emergence, the mean growth rate of individuals in different size classes, and the date of capture. based on the growth curves of both sexfig. 1. percentage of aspidoscelis marmorata in various reproductive stages in each month of the year. sample sizes appear above bars. 85life history of the marbled whiptail lizard from the central chihuahuan desert, mexico es, males reached the adult size class around 76 mm svl and females around 75 mm svl, at an average age of 1.7 yr and 1.8 yr, respectively. comparing this estimate with the data obtained from the reproduction data (i.e., the data obtained from 1990), the smallest sexually mature female with oviductal eggs was 75 mm svl, whereas the smallest male measured with enlarged testes was 74 mm svl. body size of males attained an asymptote around of 90 mm svl and females around of 82 mm svl, at an age of approximately 3.6 yr and 3.0 yr, respectively (fig. 3). the population structure of a. marmorata (fig. 4) in spring and fall were similar but not in summer (χ2 = 50.44, df = 8, p < 0.005). a notable feature of summer is the presence of hatchlings and juveniles. the relative number of adult females and males (svl ≥ 70 mm) did not differ significantly among the six years (χ2 = 1.19, df = 5, p > 0.25). lizards in the 70-82 mm svl size class were the most numerous in spring and summer. the seasonal sex ratio (1989-1994 pooled) was heavily biased toward males in spring, summer and fall (1.7, 1.6 and 4.0, respectively). in fact, the overall sex ratio (3 seasons pooled) was 1.75 (63 males and 36 females), which is significantly different from 1:1 (χ2 = 7.3, df = 1, p = 0.0068). the mean density in summer, fall, and spring (19891991) of a. marmorata (table 1) was 32.0 animals/1.0 ha (se = 7.5, range 24.5-39.6, n = 2), 40.6 animals/1.0 ha (se = 19.3, range 21.3-60.0, n = 2), and 46.0 animals/1.0 ha (se = 39.1, range 6.9-85.1, n = 2), respectively. the mean total density (summer, fall, and spring combined) was 39.5 animals/1.0 ha (se = 11.7, range 6.9-85.1, n = 6). relationship existed between precipitation and density (r = 0.82, p < 0.04, n = 6). seasonal density estimates did not include data for the period 1992-1994. the abundance of this population declined dramatically during this last period probably caused by a prolonged regional drought. estimates of probability of survival (ø) from sample time t to sample time t +1 (table 1) indicated higher survival in 1989 than in other years. we estimated the total fig. 2. growth in males and females of the lizard aspidoscelis marmorata in mapimí, durango. each point represents the body growth rate per day which has a given average svl. fig. 3. growth curves of von bertalanffy model for females and males of the lizard aspidoscelis marmorata in mapimi, durango. abscissa shows age (days); ordinate shows svl (mm). estimated from the equation of fabens (1965), with an initial svl = 37.9 mm vs. 30 days old. the values used were a = 0.0022 and b = 0.190 for females, and a = 0.0016 and b = 0.156 for males. open rectangles and open diamonds represent means. fig. 4. number of individuals of aspidoscelis marmorata in different size classes in spring, summer, and fall. 34-40 mm = hatchlings, 41-49 mm = juveniles, 50-69 mm = subadults, 70-82 mm = adults, and 83-94 mm = old adults. open bar represents adult males; diagonal-lined bar represents adult females; and horizontal lines represent females and males of hatchlings, juveniles, and subadults. 86 héctor gadsden, gamaliel castañeda number of new additions to the marked population that were made at sampling t (called z´t). thus, over this period (1989-1991) we observed a total of 47 new individuals entering the marked population and we predicted (z´t values) a total of nine new individuals, and a 19% underestimate. this result is probably biased, and it suggests unequal catchability for the animals within the marked population. this sort of bias could arise if socially dominant individuals are easier to trap. discussion reproductive cycle the reproductive season for a. marmorata in sand dunes of the mapimí biosphere reserve, began in may (spring) and declined in aug. (middle summer) during the dry season in may.-jun. and wet season in jul.aug. most of the annual rainfall occurs during summer (particularly aug. and sep.), and we suggest that hatchling emergence (between aug. and sep.) corresponds to the wet season when food abundance is high in order to promote juvenile growth and survivorship, as occurs in populations of a. tigris in arizona (parker, 1972), and nevada (tanner and jorgensen 1963), and a. marmorata in texas (milstead, 1957). gadsden and palacios-orona (2000) found in the same dune area in mexico that a. marmorata eats insects in abundance during the fall season, mainly isopters, lepidopters, and coleopters. this could increase the reproductive potential of this species of lizard in the spring to emerge from its winter dormancy, due to an increase of fat stored in their fat bodies. the reproductive cycle peaks in summer and is different to that previously reported for a. marmorata in spring (mata-silva et al. 2010) and similar to that registered for a. tigris and a. marmorata (mccoy and hoddenbach, 1966; goldberg and lowe, 1966; goldberg, 1976; vitt and ohmart, 1977), see table 2. nevertheless the known differences in reproductive traits among populations of a. marmorata and a. tigris may be manifesting proximate environmental effects. the smallest females of a. marmorata probably reach early sexual maturity at approximately 20 mo of age. likewise, data available for other female a. tigris from low elevations (530-1176 m, see burkholder and walker, 1973), females reach sexual maturity after emerging from their second hibernation in 22-24 mo at 70 mm svl. burkholder and walker (1973) estimated size at sexual maturity of a. tigris females to be 67-70 mm svl. in the northeast of the sonoran desert in arizona, some individuals of a. tigris reached mature size in their first spring at ages of about 8-11 mo, but many others did not do so until their second spring at ages of 20-23 mo. this is similar to ages at first breeding reported for other areas (turner et al., 1969; tinkle, 1969a). population structure and dynamics in this study, a. marmorata was observed in the dunes from early spring (apr.) to early fall (oct.), as recorded by hendricks and dixon (1984) for a. marmorata (cited as cnemidophorus tigris) in texas. according to these authors, juveniles and subadults represent the majority of the population by early fall, probably because the older adults have already begun torpor. young a. marmorata may emerge during warm weather throughout the winter, as evidence by several specimens observed in early winter. growth in reptiles is influenced by both phylogenetic and environmental factors (andrews, 1982). aspidoscelis marmorata exhibits differences in growth rates from a. tigris populations possibly related to proximate environmental influences, such as temperature, photoperiod, rainfall and food availability (tinkle, 1967, andrews, 1982). cuellar (1993) studied one high-latitude a. tigris population in utah, which had lower growth rates (0.07 mm × d-1 subadults and 0.008 mm × d-1 adults) than this study. however parker (1972) studied a daily growth rate of a. tigris populations in arizona, which had higher growth rates for adults (0.04 mm × d-1) than this study. in mapimí biosphere reserve the mean growth rates for a. marmorata was 0.1 mm × d-1 subadults and 0.01 mm × d-1 adults. the discrepancy may represent the inclusion of many immature individuals in the constructed size classes (cuellar, 1993), whereas all adult a. marmorata table 1. population estimates by used of jolly-seber model of population estimation. seasonal samples (1989-1991) of aspidoscelis marmorata from mapimí biosphere reserve, chihuahua, mexico. sp=spring, su=summer, fa=fall. population sample proportion marked (α) total number (n) probability of survival (ø) se n se ø sp-1989 0.00 0.0 0.38 0.09 su-1989 0.57 24.5 0.98 5.4 0.52 fa-1989 0.53 60.0 0.90 24.7 0.62 sp-1990 0.47 85.1 0.27 48.0 0.19 su-1990 0.36 39.6 0.28 21.2 0.28 fa-1990 0.40 21.3 0.47 20.5 0.47 sp-1991 0.80 6.9 4.8 su-1991 0.33 87life history of the marbled whiptail lizard from the central chihuahuan desert, mexico mentioned above were reproductive. however, reciprocal transplant experiments could be useful for examining the relative importance of genetic vs. proximate environmental effects as sources of variation among a. marmorata and a. tigris rates to identify whether differences in rates are adaptatives. furthermore, according to tinkle (1967) and lemos-espinal et al. (2003), like other lizards, growth rates of this species possibly fluctuate with the proximate environment on both seasonal and annual timescale. numerous population density data exist for a. tigris, pianka (1970) found lower densities for a. tigris located in high-latitudes (4-5 individuals per hectare) than in our low-latitudes (mean 39 individuals per hectare), see table 1 and table 2. nevertheless, these density values are within the ranges for other areas summarized by turner et al. (1969) and are also similar to census estimations by pianka (1970). the density of adult individuals a. marmorata in mapimí biosphere reserve was similar to that found in a population of a. tigris in nevada, fluctuating between 1 to 49 individuals per hectare (turner et al., 1969); suggesting that, if any relationship between density and individual growth does exist, it is not inversely density dependent as is usually assumed and observed (scott, 1990). instead lizard density is influenced by a complex variety of factors, including availability of food resources and thermal environment (pianka, 1970; rose, 1982; christian and tracy, 1985; sinervo, 1990). the density of a. marmorata was higher in spring 1990 when compared to the others six seasons (table 1). this large increase may be due to a larger adult recruitment in spring 1990 in response to previous above normal precipitation. pianka (1970) and whitford and creusere (1977) suggested that the density of most lizard species varies directly with changes in productivity and relative abundance of arthropods. likewise the availability of insect prey change according to the distribution and amount of rain throughout the year (maury, 1995). although the availability of arthropods was not evaluated in this study, we found a relationship between rainfall and density of lizards. mean probability of survival (ø = 0.55) obtained in this study (table 1) was similar to rates registered in a high-latitude population of a. tigris in southern nevada (ø = 0.57), see turner et al. (1969). during summer 1990 in mapimí biosphere reserve (i.e., aug. through sep.) high precipitation increased the availability of insect prey. in sand dunes of mapimí, lizards showed an abrupt prey shift in fall, eating many more isoptera and hemiptera and fewer other insects (gadsden-esparza and palacios-orona, 1997). increase in food abundance has been shown to affect food utilization in a. tigris (anderson, 1994), and apparently appear to affect reproductive output (whitford and creusere, 1977; dunham, 1981). therefore, the autoecological and population responses of a. marmorata to environmental variation may be regarded as normal. life-history characteristics of populations in a. marmorata (table 2) did not deviate from estimates of fitch (1985), and burkholder and walker (1973). in fact, burkholder and walker (1973) suggest that southern populations of a. tigris produce a minimum of two clutches per season. according to these authors the difference in table 2. comparison of life history data between aspidoscelis marmorata (this study, texas1, and texas 2) and aspidoscelis tigris (arizona, california, colorado and idaho). m-s = mata-silva et al. (2010), mh = mccoy and hoddenbach (1966), s = schall (1978), t = taylor et al. (2001), pi = pianka (1970), p = parker (1972), m = mitchell (1979), g = goldberg (1976), vo = vitt and ohmarth (1977), bw = burkholder and walker (1973). chihuahua texas1 texas2 new mexico arizona california colorado idaho elevation (m) 1129 1215 500-1845 1059 450-500 1585-1830 432 530-1176 length of growing season (days) 240 210 130 165 length of reproductive season (days) 120 100 130 75 100 90 density (individuals/ha) 7-85 1-36 1-12 4-5 hatchling size (mm svl) 34-37 48 37-41 32-35 size range mature males (mm) 70-92 68-94 71-96 63-97 size range mature females (mm) 70-90 75-87 60-93 70-80 71-93 69-102 average size adult males (mm) 80.2 83.7 83.5 81.5 average size adult females (mm) 76.8 80.9 76.7 81.2 clutch size 2.6 3.3 2.0-2.2 2.2 2.0-2.2 2.3-4.1 2.9-3.4 2.6 clutch frequency 2 2 2 2 2 1 1 date hatchling appear august june june 14-june mid-august july mid-august source this study m-s mh, s t pi, p, m pi, g vo bw, pi 88 héctor gadsden, gamaliel castañeda clutch size of northern and southern populations and number of clutches laid per year pose some unique problem to be considered. the first factor worth considering is the length of activity period. mccoy and hoddenbach (1966) suggested that in northern populations the activity period is too short to allow the production of two clutches. therefore, northern populations partially make up for this by producing one large clutch in each season. the second possibility is that northern populations are larger in average body size and greater longevity, which allows them to produce a larger clutch than those in southern areas. this would again be related to latitude and elevation, and possibly to other factors such as food abundance. however the clutch size varies erratically and geographic trends are not consistent. in most instances, at least, clutch size is probably proportional to body size (fitch, 1985). in other lizard species, tanner (1972) suggest that with increasing elevation uta populations live live longer, attain larger size, and have more eggs per clutch. alternatively, nussbaum and diller (1967) predicted that high latitude and/or high altitude uta populations show shorter growing season, shorter reproductive season, reduced per season fecundity (clutch frequency is reduced) and greater survivorship than low altitude and/or low latitude populations. these two life history patterns may be significant in a. marmorata, and would be another way of adapting to a short or longer activity season. pianka (1970) propose that reduced predation, reduced time exposed to predation (shorter active season) in northern populations of a. tigris could explain in part the higher average clutch size and lower clutch frequency in these populations. tinkle (1969b) has reached similar results in his studies on uta stansburiana. the studied population of a. marmorata had a late maturity, a long life expectancy, and few offspring. overall, the observed data and the expectations of current life history theory for a late maturing species (ballinger, 1983; dunham et al., 1994; stearns, 2000) are in agreement with k-rate selection. further studies on other a. marmorata populations in mexico are needed to define latitudinal and elevational tendencies. intraspecific variations of a. marmorata from dissimilar geographic and climatic locations would amplify the understanding of differing environmental conditions and their influence on the life history of the marbled whiptail lizard. these studies will need to gather data on body size, longevity, temperature, moisture, predator pressure, parasites, and availability of food, which potentially affect life history parameters of lizards. acknowledgments this study was supported by a conacyt grant (1367n9206). we thank the herrera family for field assistance. permit semarnap-sgpa/dgvs/88173 and it is also a contribution to the unesco-mab program. references adolph, s.c., porter, w.p. 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(1993): herpetology – an introductory biology of amphibians and reptiles. san diego. academic press. acta herpetologica vol. 8, n. 1 june 2013 firenze university press journal of the societas herpetologica italica acta herpetologica acta herpetologica 10(1): 39-45, 2015 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-15003 into a box interiors: clutch size variation and resource allocation in the european pond turtle marco a.l. zuffi1,*, simonetta citi2, elena foschi1, francesca marsiglia1, eva martelli1 1 museum of natural history, university of pisa, via roma 79, i-56011 calci (pisa), italy. corresponding author. e-mail: marco.zuffi@ unipi.it 2 department of veterinary clinic. university of pisa, viale delle piagge, 2 i-56124 pisa, italy submitted on 2014, 26th september; revised on 2015, 25th january; accepted on 2015, 21st february editor: sandra hochscheid abstract. in order to highlight the temporal dynamics of different stages of follicula distribution on reproductive output, we analysed the female size and the reproductive frequency of the european pond turtle, emys orbicularis, using 191 ultrasonographic and 67 x-ray images (collected from 2000 to 2011 in two different localities of a coastal plain area of northwestern italy). to compare digital image results we also used anatomical topography of autopsied females. although reproductive females were significantly longer in one locality and larger in the second one, clutch size did not differ between localities. two main clutches were produced during a year, with occasionally a third one. shelled eggs were frequent in may, june and july, while follicula were present till august, with a decrease in follicular size especially in july and august. despite the presence of a number of follicles in late summer and in autumn, a third expected clutch was only an exceptional event, differently from what happens in other sites of the species’ distribution range. the permanence of follicles after the last deposition is interpreted as a possible extra energy source during the very hot and dry summers of coastal central italy and for the hibernating phase. key words. european pond turtle, ultrasonography, x-rays; reproductive investment. introduction life history strategies represent the ways in which animals acquire and expend resources. the amount of energy gained with food and stored in fat bodies, liver and muscles indicates the potentiality of maximum reproductive capacity of each individual. the ecological consequences of energy storage have attracted much attention quite recently (hom, 1988; bonnet et al., 1998; santos and llorente, 2004). the amount of stored energy and its seasonal duration may play an important role in the reproduction of every organism. furthermore, the patterns of the subsequent use of the amount of energy may represent important aspects of interspecific and intra-specific variation in life histories. in fact, differences between the endothermic and the ectothermic model of energy storage and maintenance are marked, such as in mammals versus reptiles (else and hulbert, 1981). specifically, oviparous reptiles capitalize on their food income in the same season of reproduction and utilize only endogenous energy stores (congdon and tinkle, 1982; bonnet et al., 1998; santos and llorente, 2004). pathways from energy income to fat storage may be resumed as food, digestion and assimilation in body reserves. income breeders do not store lipids to any great extent. the process of the production of youngsters, involves a complex series of biochemical steps that cannot be avoided. the synthesis of vitellogenin macro-molecules involves a large amount of energy as well as their incorporation and deposition in the oocytes. yolk deposition is a 40 marco a.l. zuffi et alii continuous process which requires stored substances, even from short-term storage, in order to moderate the discontinuous effects of the activities of feeding and digesting. thus, the physiological pathways leading from resource acquisition to yolk deposition are similar among taxa, the differences depending on the magnitude and schedule of body reserve storage before reproduction (allen, 1976; congdon and tinkle, 1982; bonnet et al., 1998). the energy budget is primarily stored in the corpora lutea, subsequently used for follicular formation, ovulation process and egg formation. from a series of multiple follicles, females ovulate to produce shelled eggs. in chelonians multiple clutches represent the rule: it is the case in all marine turtles, in most terrestrial tortoises and freshwater turtles. radiography to assess reproductive status, clutch size and clutch frequency has been used for many years in chelonians (gibbons and greene, 1979; andreu and villamor, 1986; zuffi et al., 1999; servan and roy, 2004; bertolero and marín, 2005). the ultrasonographic technique instead has been applied only recently (table 1). on the contrary, the post-reproductive period of chelonians has received virtually no attention. this period could offer further information on the evolution of remnant follicular eggs of adult turtle and tortoise species, from immediately after the last egg laying to the end of the active season, prior to wintering. this contribution is aimed at describing the occurrence and the seasonal variability of developed and undeveloped follicula, their size and their possible function, during and especially after the reproductive period, considering also the influence of body structure (e.g. shell size and shape). as model species we selected the european pond turtle (emys orbicularis) as it is abundant in europe (podloucky, 1997; sillero et al., 2014), and in many italian areas (zuffi, 2000; zuffi et al., 2011), it lays multiple clutches per year (e.g. zuffi and odetti, 1998; fritz, 2003, and data revision herein), and its reproductive variation strongly correlates to localities, habitat features and female size (mitrus table 1. comparative source data (in chronological order) of ultrasonographic and radiographic techniques to assess reproductive status in chelonians species ultrasound n x-ray n kinosternon subrubrum x 3 gibbons and greene, 1979 deirochelys reticularia x 2 gibbons and greene, 1979 sternotherus odoratus x 2 gibbons and greene, 1979 chrysemys picta x 129 congdon and tinkle, 1982 pseudemys scripta x 65 congdon and gibbons, 1983 emydoidea blandingi x 90 congdon et al., 1983 chelydra serpentina, chrysemys picta dorsalis deirochelys reticularia pseudemys concinna pseudemys floridana pseudemys scripta terrapene carolina clemmys marmorata gopherus polyphemus kinosternum subrubrum sternotherus odoratus trionyx ferox x x x x x x x x x x x x 4 1 13 1 11 88 8 2 1 19 9 1 congdon and gibbons, 1985 congdon and gibbons, 1985 congdon and gibbons, 1985 congdon and gibbons, 1985 congdon and gibbons, 1985 congdon and gibbons, 1985 congdon and gibbons, 1985 congdon and gibbons, 1985 congdon and gibbons, 1985 congdon and gibbons, 1985 congdon and gibbons, 1985 congdon and gibbons, 1985 testudo graeca x 38 andreu and villamor, 1986 mauremys leprosa x 27 andreu and villamor, 1986 chrysemys picta x 17 christens and bider, 1987 chelydra serpentina x 68 congdon et al., 1987 emydoidea blandingi x 280 congdon and van loben sels, 1991 emys orbicularis x 15 zuffi et al., 1999 emys orbicularis x 30 servan and roy, 2004 cuora amboinensis x 13 haies and kalb, 2010 cuora flavomarginata x 24 x 24 ting-yu et al., 2011 emys orbicularis x 191 x 66 present research 41follicles and eggs of the fresh water turtle and zemanek, 1998; zuffi et al., 1999, 2007; kotenko, 2000; zinenko, 2004). despite it being among the species listed in the eu habitat directive and in several european red lists, it is much less studied than expected and should certainly be the object of further research. materials and methods sampled individuals were from san rossore (average coordinate: 9 m asl, 43°42’51”n, 10°18’30”e) and from the us army camp darby areas (10 m asl, average coordinate: 43°38’03”n, 10°20’11”e), both in the natural regional park of “migliarino san rossore massaciuccoli” (pisa, tuscany, central italy) (fig. 1). the two areas are separated by the arno river, a natural barrier that has been intensively used by humans since the time of the second world war, including the construction of houses, a touristic harbour and anchoring hundreds of boats along the river. we are confident that no exchange of individuals has occurred since then. the two areas are characterised by a mixed wood of pines and oaks, artificial canals, ponds and marshy areas, fully described elsewhere (zuffi and rovina, 2006; zuffi et al., 2007). no data nor information is however available on animal movements in this area. thus we considered two populations as independent. wild individuals were captured by hand and with fishing nets, placed along borders of artificial canals and ponds, from 2000 to 2011. adult females were marked individually with notches on the marginal scutes, as described in stubbs et al. (1984) and zuffi et al. (1999), in order to recognize them when recaptured and avoid any pseudoreplication in analyses. we examined 191 e. orbicularis galloitalica adult females (secondary sexual characters described in zuffi et al., 1999), to assess adulthood features and body size variation. carapace and plastron measurements, body mass recording, individual marking or recognition took on average 10 minutes. we processed each individual in the field and turtles were released immediately afterwards. during ovulation and the egg-laying period, that is from early-mid may to late july (see zuffi et al., 2007, and references herein), we re-captured marked females and we assessed their reproductive condition using manual inguinal palpation and ultrasonography (respectively to detect follicles or eggs, and quantify oviductal and shelled eggs), and x-ray techniques to measure clutch size before laying (gibbons and greene, 1979; zuffi et al., 1999, 2005). data were also collected from several females during late july and august, a period when females were reported to reproduce rarely or not reproduce at all (e.g. mitrus and zemanek, 1998; zuffi and odetti, 1998; fritz, 2003). ovulating and or checked adult females were brought to the vet lab and kept 24 hours at maximum in captivity. after ultrasonographic and x-ray analysis, the individuals were released. clutch size was determined by x-ray techniques only. as a consequence, sample size of females bearing follicular eggs differed from sample size of females bearing shelled eggs. two autopsied preserved females (corsica, 2 may 1998, msnt-1127 and 2 june 1998, msnt-1128, natural history museum herpetological collection), belonging to the same subspecies (fritz, 2003), were used to take direct images of the various statuses of developing follicula and of shelled eggs (fig. 2). follicular and oviductal egg estimation of each female was obtained by summing (from ultrasonographic images) the visible eggs on the right side plus those visible on the left side, then dividing the results by two. even if just one single ultrasonographic image could represent most of the internal environment, we used both side images to build the average estimation, in order to minimize the risk of underestimating the whole follicular set. the reproductive phenology was considered as a whole, merging data of the two populations. we measured turtles with standard methods, following zuffi et al. (1999): variables considered were carapace length and width, plastron length and width, carapace height, tail length (from cloaca distal opening to tail tip), and body mass. measurements were  ±  1 mm and mass  ±  1 g accuracy. a caliper and an electronic dynamometer were used for measurements. biometric data of two sampling localities were analyzed for normality, then processed with the student t-test. ultrasound and x-ray data were analyzed between localities with the mann-whitney u test. egg data were tested for normalfig. 1. sampling areas for emys orbicularis in north-western coastal tuscany. empty ovals indicate female sampling areas. 42 marco a.l. zuffi et alii ity, then processed with the non-parametric kolmogorovsmirnov ranking test using month as factor; they were also analyzed for skewness and kurtosis (http://mvpprograms.com/help/mvpstats/distributions/skewnesscriticalvalues) to detect any asymmetry of the distribution patterns. spearman correlation test was then applied in analyzing number of developing follicular eggs and month. significance was selected at 0.05. analyses were carried out with ibm spss 21.0.0 release. results considering body size of the sampled reproductive females and sampling areas, san rossore females were larger than camp darby females in terms of carapace length and smaller than camp darby females as regards carapace width (sr carapace length: 136.9  ±  13.7cm, cd carapace length: 132.4  ±  11.3 cm; unequal variances, student’s t88,224, 2.174, p  =  0.032; sr carapace width: 95.3  ±  9.4 cm, cd carapace width: 98.8  ±  7.5 cm; unequal variances, student’s t86,188, -2.447, p  =  0.016) with no differences in the other variables. number of x-ray eggs did not differ between localities (san rossore, 6.2  ±  1.1, n  =  48; camp darby, 5.9  ±  1.2, n  =  18; mann-whitney u test  =  1.017, p  =  0.309). ultrasound eggs were significantly different between localities (san rossore, 5.3  ±  2.6, n  =  152; camp darby, 3.3  ±  1.5, n  =  39; mann-whitney u test  =  4.017, p  <  0.0001), while no difference was found in ultrasound egg size between localities (san rossore, 11.7  ±  5.9, n  =  151; camp darby, 12.7  ±  5.5, n  =  39; mann-whitney u test = 1.096, p = 0.273). we found reproductive females with shelled eggs in may, june and july, with an average clutch size (x-ray) of 6.1  ±  1.1 eggs (n  =  66, range 2-9). adult females with calcified eggs were found in may (n  =  9, 27%), in june (n  =  25, 67.6%) and in july (n  =  2, 5.4%). on the whole, developing follicular eggs were 4.9  ±  2.5 (n  =  191), with an average size of 11.8  ±  5.9 mm. developing follicular eggs were 3.2  ±  1.9 in may (range 1-8, n  =  19, 9.5%), 4.2  ±  1.9 in june (range 1-9, n  =  64, 33.7%), 5.4  ±  2.5 in july (range 0-16, n  =  77, 40.5%) and 6.1  ±  3.2 in august (range 1-11, n  =  31, 16.3%) (table 2). the skewness was significant in may and july (p  <  0.05 and p  <  0.01, respectively). difference of ultrasound eggs distribution among months was significant (rank kolmogorovsmirnov test  =  24.708, 3 df, p  <  0.0001; fig.  3). developing follicular eggs were 19.5  ±  9.1 mm in may (range 8-35.8 mm, n  =  19), 14.1  ±  6.1 mm in june (range 6.2-35 mm, n  =  64; fig.  4), 9.6  ±  2.3 mm in july (range 4.5-13.8 mm, n  =  76) and 7.9  ±  2.3 mm in august (range 5.3-10.9 mm, n  =  31). the skewness was significant only in june (p  <  0.01). egg size differences among months was significant (rank kolmogorov-smirnov test  =  63.983, 3 df, p < 0.0001; fig. 5). fig. 2. autopsied female emys orbicularis, showing eggs (se = shelled eggs) and follicula (fe = follicular eggs) distribution. table 2. clutch laying temporal distribution versus follicle distribution. values are expressed in %. distribution may june july august clutch 27.0 67.7 5.4 0.0 follicles 9.5 33.7 40.5 16.3 fig. 3. ultrasonographic distribution of developing follicula per month. 43follicles and eggs of the fresh water turtle the number of developing follicular eggs were positively correlated with month, while size of developing follicular eggs were negatively correlated with month (spearman ρnumber, 0.345, p  <  0.0001, n  =  191; spearman ρsize, -0.581, p < 0.0001, n = 190). discussion external shell morphology and size differences of reproductive females between localities are limited to carapace length and width. our results agree only in part with previously published papers on the same populations, where differences were much more marked and significant for almost all variables (zuffi et al., 2004, 2007). sample size of x-rayed females is however a limited fraction of the much larger adult female sample we have been monitoring for other purposes for more than 25 years. it is evident that our sampled females are more similar in size and in reproductive output (they actually produced the same number of shelled-eggs; see results) as expected from previous results (see zuffi et al., 2004). in all the turtle species studied to date, the reproduction period lasts some months (ernst et al., 1994). the constitutive elements of the clutch (e.g. follicles, oviductal eggs, corpora lutea) are present at the same time in the female body and characterize a reproductive feature of all chelonians. the follicles show two distinct stages,  <  10 mm diameter and  >  10 mm diameter (congdon and gibbons, 1985). as observed (congdon and tinkle, 1982), the presence of discrete classes of enlarged follicles shows evidence of multiple clutches. in addition, we were also able to assess the presence of discrete classes of enlarged follicles. in our study area adult females monitored in late summer bear a large number of follicles, especially in july. follicle diameters range on average from 8 to 10 mm, as is the case in preovulatory females. in july we recorded only two reproducing females (with shelled eggs), while in no other cases (using manual palpation, ultrasonography, or radiography) did we have any evidence of a third clutch. a third reproduction is therefore particularly rare, if not exceptional, in this area. in fact, from previous ecological research, a maximum of two clutches was found in emys orbicularis (zuffi and odetti, 1998; zuffi et al., 1999), and a third deposition was only hypothesized. however, three or even more depositions have been recorded in other parts of the species’ range (see revision in fritz, 2003). interestingly, even if not surprisingly, the number of undeveloping follicles decreases after the last reproductive period (i.e. july, august). reasonable questions are i) do late summer follicles have a function, and if so, which is it? and ii) what does this extra amount of energy reserve represent? do small follicles simply represent the extra amount of undeveloped eggs? in all probability, during the post reproductive phase, follicula that have not been used in reproduction may indicate the start of an absorbing process, and prior to estivation during hot summers in dry canals and ponds (naulleau, 1991) they may represent an energy reserve to compensate for metabolic demand (hutton et al., 1960; huey, 1982). alternatively, the unused follicles fig. 4. ultrasonographic image of a june female with different stages of follicula (on the left: developing follicles; on the right: transverse image of a shelled egg). fig. 5. distribution of size of developing follicula per month determined through ultrasonography. 44 marco a.l. zuffi et alii may remain inactive (not enlarging, not being absorbed) for the last part of the season (and of the year) and will contribute to the next year’s clutch (congdon and tinkle, 1982). in the painted turtle, chrysemys picta, it has been found that each female shows three groups of follicles, with different developing stages, suggesting a three year cycle to complete the potential maximum reproductive output (christiansen and moll, 1973; callard et al., 1978). as a member of the emydidae family, emys orbicularis could reasonably be expected to follow the same pattern recorded in north-american species. on the contrary, our ultrasonographic (and x-ray) data which are, as far as we are aware, the only available dataset for the species, seem to contradict the patterns found elsewhere, suggesting that the reproductive cycle may likely be completed within one year or at maximum in two years (many follicles of a smaller size in august, see figs. 4 and 5). however, it remains unclear which pattern follicles do follow in autumn (september-november), prior to wintering. keller et al. (1998) found that in south western spain, the european pond turtle (emys orbicularis occidentalis) lays multiple clutches regularly, especially during wet summers (up to five clutches in one year), suggesting how abundant rains enable turtles to reproduce frequently. however, in other parts of the species’ distribution range, where multiple clutches were also recorded (fritz, 2003), climatic data and turtle reproductive patterns were not studied or available data are still scarce or anecdotal (see rogner, 2009). thus, it is not simple to shed light on the biological and ecological reproductive model of the european pond turtle, emys orbicularis, which appears to be highly variable, likely depending on site climatic conditions, food and water abundance and on female size. forthcoming research should cover analysis of any adult preserved museum specimens captured in autumn to verify the presence and the developmental stage of follicula and in vivo study of the gonadal status of adult post reproductive females. acknowledgements we thank collaborators and students that helped in the field: f. di benedetto, m.e. fabbri, m. giusti, m. marconcini, f. odetti, l. rovina, a. teti. ministero dell’ambiente e della tutela del territorio provided permissions to m.a.l.z. (dpn/iid/2005/28177 7/11/2005; dpn-2007-0009336, 3/04/2007) to capture, handle and measure turtles; permissions to enter natural park migliarino san rossore massaciuccoli was given to m.a.l.z. (2232/9-5.1 on 26/02/2001; 2770/7-2.1 on 23/03/2005; 8057/7-2.1 on 04/08/2005; 3739/7-2.1 on 7/04/2006). chris powell (international, pisa) revised the english version. no one of the authors has any conflict of interest to declare. finally, many thanks to sandra hochscheid and martin bona for their precious suggestions for revision and comments to the submitted ms. references allen, w.v. (1976): biochemical aspects of lipid storage and utilization in animals. am. zool. 16: 631-648. andreu, a.c., villamor, m.c. (1986): reproduction of testudo graeca in doñana, sw spain. in: studies in herpetology, pp. 589-592. rocek, z., ed, prague. bertolero, a., marín, a. 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(2006): habitat characteristics of nesting areas and of predated nests in a mediterranean population of the european pond turtle, emys orbicularis galloitalica. acta herpetol. 1: 37-51. acta herpetologica vol. 10, n. 1 june 2015 firenze university press obituary: valery k. eremchenko (1949-2014) leo j. borkin1, tatjana dujsebayeva2, roberto sindaco3, matthias stöck4 haplotype variation in founders of the mauremys annamensis population kept in european zoos barbora somerová1, ivan rehák2,*, petr velenský2, klára palupčíková1, tomáš protiva1, daniel frynta1 reproductive ecology of sichuan digging frogs (microhylidae: kaloula rugifera) wei chen1,*, lina ren2, dujuan he2, ying wang2, david pike3 toxic effects of carbaryl on the histology of testes of bufotes variabilis (anura: bufonidae) özlem çakici basal frequency of micronuclei and hematological parameters in the side-necked turtle, phrynops hilarii (duméril & bibron, 1835) maría a. latorre 1,2,*,#; evelyn c. lópez gonzález1,2, #; pablo a. siroski1,2,3; gisela l. poletta1,2,4 into a box interiors: clutch size variation and resource allocation in the european pond turtle marco. a.l. zuffi1,*, simonetta citi2, elena foschi1, francesca marsiglia1, eva martelli1 where to “rock”? choice of retreat sites by a gecko in a semi-arid habitat andreia penado1,2, ricardo rocha3,4,*, marta sampaio3, vanessa gil3, bruno m. carreira3, rui rebelo3 age structure, growth and longevity in the common toad, rhinella arenarum, from argentina clarisa de l. bionda1,2,*, silvia kost 4, nancy e. salas1, rafael c. lajmanovich3, ulrich sinsch4, adolfo l. martino1 on a putative type specimen of pleurodema bibroni tschudi, 1838 from chile (anura: leptodactylidae) daiana paola ferraro re-description of the external morphology of phyllomedusa iheringii boulenger, 1885 larvae (anura: hylidae), with comments on the external morphology of tadpoles of the p. burmeisteri group samanta iop¹, victor mendes lipinski¹, bruno madalozzo¹, franciele pereira maragno¹, sonia zanini cechin¹, tiago gomes dos santos² book review: harold heatwole, john w. wilkinson (eds). amphibians biology. volume 11 status of conservation and decline of amphibians. eastern hemisphere. part 4 . southern europe and turkey sebastiano salvidio book review: antonio romano. atlante degli anfibi del parco nazionale del cilento vallo di diano e alburni distribuzione, biologia, ecologia e conservazione sebastiano salvidio acta herpetologica journal of the societas herpetologica italica acta herpetologica 10(1): 67-72, 2015 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-15547 re-description of the external morphology of phyllomedusa iheringii boulenger, 1885 larvae (anura: hylidae), with comments on the external morphology of tadpoles of the p. burmeisteri group samanta iop¹,*, victor mendes lipinski¹, bruno madalozzo¹, franciele pereira maragno¹, sonia zanini cechin¹, tiago gomes dos santos² ¹ universidade federal de santa maria.  programa de pós graduação em biodiversidade animal – centro de ciências naturais e exatas. prédio 17, sala 1140 d, cidade universitária camobi, km 29 cep: 97105-900 santa maria, rs – brasil. *corresponding author. e-mail: samantaiop@yahoo.com ² universidade federal do pampa, campus são gabriel. av. antônio trilha, 1847 cep: 97300-000 – são gabriel, rs – brasil submitted on 2015, 12th february; revised on 2015, 28th april; accepted on 2015, 30th april editor: sebastiano salvidio abstract. phyllomedusa iheringii is a leaf frog endemic to the uruguayan savanna, which reproduces in water bodies in open areas. here, based on the lack of some informative characteristics observed on the first description of this species, we re-describe the larval external morphology of p. iheringii from localities in brazil and uruguay, and compared them with other species from the p. burmeisteri group. the tadpoles of p. iheringii belong to the suspensionrasper guild. the body length corresponds to approximately one-third of the total length. the body is pyriform in dorsal view and laterally triangular. the snout is rounded in a dorsolateral view. the spiracle is single and almost ventral, not forming a free tube, and sinistral. the opening of vent tube is dextral. the oral disc is anteroventral and anteriorly directed, with single ventral emargination. marginal papillae uniseriate, interrupted in a wide dorsal gap, and with pointed tips. the labial tooth row formula is 2(2)/3(1). the third lower row is four times shorter than the others. furthermore, the tadpoles of p. iheringii showed morphological differences in relation to other species of the p. burmeisteri group, this species seems to be smaller in average and have fewer marginal papillae in the oral discs. keywords. tadpole morphology, phyllomedusinae, uruguayan savanna ecoregion. the genus phyllomedusa currently has 30 species and is widely distributed in the neotropics, occurring from panama, the pacific slopes of colombia, south america east of the andes, and southwards to northern argentina and uruguay (frost, 2014). species of this genus are distinguished from other neotropical hylids by their contrasting colour patterns, vertical pupils, and an arboreal reproductive mode (caramaschi, 2006). in brazil, 23 species of phyllomedusa are recorded and all have external larval morphology known (segalla et al., 2012). nevertheless, larval internal oral features and chondrocranium are poorly known for most species. similarly, the larval morphology still requires to be described or re-described, because some previous descriptions raise doubts concerning the identification of some structures, as is the case of p. iheringii. the group of phyllomedusa burmeisteri comprises five species (p. bahiana, p. burmeisteri, p. distincta, p. iheringii, and p. tetraploidea) that share similarities in external morphology, vocalization, and cytogenetic features, and possess a complex pattern of strips on the thighs as a diagnostic characteristic (pombal jr. and haddad, 1992; silva-filho and juncá, 2006; faivovich et al., 2010). species of the p. burmeisteri group show a parapatric or allopatric distribution throughout the atlantic rainforest and replace each other throughout the geographic distri68 samanta iop et alii bution of the group (brunes et al., 2010). phyllomedusa iheringii is the only species of the group that occurs in open areas and is endemic to the uruguayan savanna, occurring in uruguay and southern of the brazilian state of rio grande do sul (olson et al., 2001; brunes et al., 2010; frost, 2014). phyllomedusa iheringii reproduces in both lentic or lotic water bodies (but in backwater zones), and uses marginal vegetation for both vocalization and spawning (maneyro and carreira, 2012). males call whilst perched on vegetation and the eggs are laid in leaf nests. tadpoles are exotrophic and drop into lentic water to complete their development (mode 24; haddad and prado, 2005). the first description of the p. iheringii tadpole was provided by de sá and gerhau (1983) was based on a single individual hatched from on single egg mass, from one single locality. this first effort to describe the species, lack of information concerning the sizes, lenght, and positions of some body structures treated as diagnostic characteristics. the dataset provided may not represent with precision the morphological intraspecific variation of the species within its distributional range and may have limited the understanding of intraspecific variation as well as inter-specific comparisons with other species of the group. here, we re-describe the external morphology of the tadpole of p. iheringii, accounting for intraspecific variation and providing comparisons with other species of the p. burmeisteri group. in addition, we present data on natural history. the tadpoles of phyllomedusa iheringii were collected at four sites in the uruguayan savanna: encruzilhada do sul (30°33’50.7’’ s, 52°34’13.1’’ w, 414 m a.s.l.; about 100 km from the type locality; zufsm 8166), santa maria (29°49’25.3” s, 53°37’19.3” w, 229 m a.s.l.; zufsm8831), and são sepé (30°15’27.5’’ s, 53°34’52.4’’ w, 346 m a.s.l.; zufsm 8832-3) in rio grande do sul, brazil; and in cerro chapeu, rivera, uruguay (30°57’11’’ s,  55°28’22’’ w, 257 m a.s.l.; rml 2448). the larvae were collected and immediately fixed in a formalin 10% solution in field. the re-description was based on eight specimens of  stage  37 (gosner, 1960) (seven individuals from são sepé and one from rivera),  from which 21 morphological measurements were taken which according to  mcdiarmid and altig (1999), and altig (2007) and lavilla and scrocchi (1986). to allow  comparison  within  p. iheringii and with other species from p. burmeisteri  group,  we also took the same measures of other 32 individuals (stages 26-31, 34-36; gosner, 1960) (table 1). tadpole measurements were made using a stereomicroscope (0.01 mm precision), except for tl, which was measured with a digital caliper (0.01 mm precision) (table 2). external morphology the tadpoles of phyllomedusa iheringii belong to the suspension-rasper guild (mcdiarmid and altig 1999; both et al., 2011), and have an elongated body (bh/bw: 0.92; tab. 1). the body length corresponds to approximately one-third of the total length (bl/tl: 0.34). the body is piriform in dorsal view and triangular in lateral view (fig. 1a, b). the snout is rounded in a dorsolateral view. the nostrils are rounded and dorsolateral (nd: 0.25  ±  0.04). the internostril distance is larger than the distance between the nostrils and the snout (ind: 3.60  ±  0.22; nsd: 1.44  ±  0.27). eyes are lateral (ed: 2.15  ±  0.19). the interorbital distance is greater than the distance between the eyes and the snout (iod: 7.75  ±  0.41; esd: 6.16  ±  0.35). the spiracle is sinistral, single and almost ventral, not forming a free tube. the opening of vent tube is dextral. the tail is pointed, with flagellum, and longer and taller than the body (tal/ tl: 0.66; mth/bh: 1.33). the tail musculature is higher than wide (tmh/tmw: 1.16). the dorsal fin originates anterior of dorsal tail-body junction, the ventral fin is higher than the dorsal fin (vmh: 5.93  ±  1.01; dmh: 1.74  ±  0.24). the oral disc is anteroventral and anteriorly directed, not emarginate. marginal papillae uniseriate, interrupted in a wide dorsal gap, and with pointed tips. submarginal papillae laterally aggregate in the oral disc, not forming rows. the upper jaw sheath is curved, but flat in the middle, not serrated and its width is approximately seven times greater than high (ujsw/ujsh: 7.86). the lower jaw has a ‘v’ shape, not serrated and approximately six times longer than high (llj/hlj: 6.06). the labial tooth row formula is 2(2)/3(1) and the third lower row is four times shorter than the others (fig. 1c). coloration usually, the dorsum of the body and tail varies from yellowish to greenish. the lateral portion of the body is grey-violet, with brown pigmented areas in the middle of fin. the fins are translucent and have small, uniformly distributed melanophores. the intestine region is bluish in lateral and ventral views. there are two darkened blotches on the head, between the eyes and the nostrils. when preserved in formalin (10%), the tadpoles are uniformly yellowish or greyish. variation the tadpoles of phyllomedusa iheringii do not have ontogenetic variation, though geographic variation 69the tadpole of phyllomedusa iheringii occurs. such variation may be the result of different environmental conditions (temperature, ph), food availability or presence of predators among water bodies. although tadpoles from the three localities are morphologically similar, some of them have small variations (table 1). indeed, some individuals in different developmental stagtable 1. morphological measurements of 40 tadpoles of phyllomedusa iheringii from different localities of the uruguayan savana. mean, standard deviation and range (mm) are shown. the stage and sample size are in parentheses. bh – body height, bl – body length, bw – body width, dmh – dorsal fin height, ed – eye diameter, esd – eye–snout distance, hlj –lower jaw sheath height, ind – inter-narial distance, iod – interorbital distance, llj –lower jaw sheath length, mth – maximum tail height, nd – nostril diameter, nsd – nostril–snout distance, od – oral disc diameter, tal – tail length, tl – total length, tmh – tail musculature height, tmw – tail musculature width, ujsh – upper jaw sheath height, ujsw – upper jaw sheath width, vmh – ventral fin height. encruzilhada do sul (stage 27, n = 1) santa maria (stage 27, n = 3) são sepé (stage 26-31, n = 18) são sepé (stage 34-36, n = 7) rivera (stage 36, n = 3) são sepé and rivera (stage 37, n = 8) bh 5.60 4.80 ± 0.12 (4.67 – 5.60) 6.45 ± 0.71 (4.74 – 7.41) 7.07 ± 0.94 (8.52 6.04) 9.77 ± 0.93 (9.16 – 10.84) 7.74 ± 0.64 (8.35 – 6.41) bl 8.60 6.85 ± 0.14 (6.72 – 8.60) 11.48 ± 1.54 (8.85 – 13.86) 14.78 ± 1.26 (16.79 – 13.43) 16.05 ± 1.24 (14.77 – 17.25) 15.02 ± 1.86 (16.51 – 10.95) bw 5.08 4.45 ± 0.10 (4.39 – 5.08) 6.87 ± 0.69 (5.30 – 7.83) 8.25 ± 0.81 (9.23 – 6.94) 8.69 ± 0.33 (8.31 – 8.89) 8.42 ± 0.76 (9.39 – 7.32) dmh 1.15 0.95 ± 0.07 (0.87 – 1.15) 1.27 ± 0.14 (1.00 – 1.28) 1.59 ± 0.14 (1.76 – 1.34) 1.66 ± 0.12 (1.56 – 1.80) 1.74 ± 0.24 (2.06 – 1.4) ed 1.60 1.37 ± 0.03 (1.29 – 1.60) 2.03 ± 0.35 (1.29 – 2.42) 2.55 ± 0.27 (2.74 2.09) 2.10 ± 0.14 (2.02 – 2.26) 2.50 ± 0.19 (2.77 – 2.18) esd 3.47 3.00 ± 0.16 (2.87 – 3.47) 4.73 ± 0.65 (3.54 – 5.85) 5.97 ± 0.51 (6.58 – 5.25) 5.52 ± 0.37 (5.26 – 5.94) 6.16 ± 0.35 (6.9 – 5.75) hlj 0.13 0.10 ± 0.01 (0.09 – 0.13) 0.14 ± 0.05 (0.10 – 0.29) 0.16 ± 0.06 (0.3 – 0.11) 0.15 ± 0.03 (0.13 – 0.19) 0.15 ± 0.04 (0.19 – 0.1) ind 2.00 1.80 ± 0.02 (1.78 – 2.00) 2.95 ± 0.29 (2.32 – 3.44) 3.40 ± 0.22 (3.08 3.18) 3.61 ± 0.11 (3.52 – 3.73) 3.60 ± 0.22 (3.84 – 3.22) iod 3.88 3.69 ± 0.03 (3.65 – 3.88) 5.73 ± 0.7 (4.42 – 6.63) 7.36 ± 0.79 (8.61  –  6.02) 7.09 ± 0.53 (6.48  –  7.46) 7.75 ± 0.41 (8.32  –  7.2) llj 0.58 0.58 ± 0.08 (0.54 – 0.67) 0.73 ± 0.18 (0.54 – 1.28) 0.90 ± 0.14 (1.2  –  0.81) 1.21 ± 0.25 (0.93  –  1.42) 0.91 ± 0.09 (1.05  –  0.77) mth 6.92 5.50 ± 0.13 (3.72 – 6.92) 7.78 ± 1.29 (3.72 – 9.15) 9.88 ± 0.70 (10.48  –  8.6) 9.06 ± 1.30 (8.06  –  10.53) 10.33 ± 1.26 (12.22  –  8.38) nd 0.13 0.14 ± 0.03 (0.11 – 0.16) 0.20 ± 0.06 (0.13 – 0.33) 0.23 ± 0.04 (0.23  –  0.18) 0.21 ± 0.02 (0.20  –  0.23) 0.25 ± 0.04 (0.31  –  0.21) nsd 0.92 0.56 ± 0.24 (0.40 – 0.92) 1.09 ± 0.25 (0.75 – 1.54) 1.49 ± 0.24 (1.74  –  1.5) 1.47 ± 0.07 (1.41  –  1.55) 1.44 ± 0.27 (1.85  –  1.1) od 1.28 1.49 ± 0.21 (1.25  –  1.62) 2.58 ± 0.50 (1.77 – 4.15) 3.21 ± 0.62 (4.57  –  2.79) 3.41 ± 0.68 (2.89  –  4.18) 3.12 ± 0.18 (3.32  –  2.82) tal 15.70 15.58 ± 1.02 (14.41 – 16.26) 23 ± 3.12 (17.02 – 29.73) 28.21 ± 3.35 (32.78  –  23.64) 23.53 ± 0.86 (22.72  –  24.44) 29.61 ± 2.06 (32.28  –  26.98) tl 24.30 22.43 ± 1.13 (21.13 – 24.30) 34.48 ± 4.01 (26.11 – 40.2) 42.99 ± 4.41 (48.93  –  37.07) 39.58 ± 2.10 (37.49  –  41.69) 44.63 ± 2.55 (47.73  –  40.88) tmh 2.85 2.21 ± 0.18 (2.09 – 2.85) 3.63 ± 0.73 (2.32 – 4.5) 4.70 ± 0.56 (5.02  –  3.74) 3.11 ± 0.14 (3.00  –  3.27) 4.52 ± 0.50 (4.86  –  3.3) tmw 1.99 1.52 ± 0.24 (1.30 – 1.99) 3.02 ± 0.56 (2.02 – 3.82) 3.99 ± 0.58 (4.5  –  3.03) 2.43 ± 0.24 (2.20  –  2.67) 3.88 ± 0.59 (4.79  –  2.85) ujsh 0.16 0.12 ± 0.03 (0.10 – 0.16) 0.23 ± 0.07 (0.14 – 0.46) 0.44 ± 0.53 (0.36  –  0.210) 0.31 ± 0.06 (0.24  –  0.36) 0.22 ± 0.04 (0.29  –  0.18) ujsw 0.99 0.84 ± 0.01 (0.22 – 0.99) 1.39 ± 0.31 (0.97 – 2.38) 1.54 ± 0.68 (2.53  –  1.58) 1.95 ± 0.36 (1.68  –  2.36) 1.73 ± 0.07 (1.82  –  1.63) vmh 3.33 2.63 ± 0.14 (2.30 – 3.33) 4.02 ± 0.56 (2.80 – 4.88) 5.05 ± 0.42 (5.26  –  4.92) 4.87 ± 1.17 (3.75  –  6.09) 5.93 ± 1.01 (7.55  –  4.08) 70 samanta iop et alii es (25, 26, 31, 36 and 37) have an oral formula of 2(2)/3, instead of the usual formula of 2(2)/3(1), and the third lower row in some cases, not linked to a specific developmental stage, is much smaller in comparison with the other ones. the marginal papillae can be light white or have several melanophores. most individuals have a few blotches on the body and tail, but many others had fewer of these markings, which were less dark. some individuals from são sepé had melanophores on the ventral fins and blueish intestines. however, specimens from encruzilhada do sul, santa maria, and cerro chapeu had no melanophores on the ventral fins, and the intestine ranged from brown to black. natural history tadpoles of phyllomedusa iheringii occurred throughout the spring and summer of the austral region (from september to march), and inhabit deeper and non-vegetated areas of ponds, pools of streamlets, and small dams used in cattle raising. at least, larvae of other 13 anuran species occurred syntopically with larvae of phyllomedusa iheringii at the studied waterbodies: dendropsophus minutus, elachistocleis bicolor, hypsiboas pulchellus, leptodactylus gracilis, l. latrans, odontophrynus americanus, physalaemus cuvieri, p. gracilis, p. henselii, pseudopaludicola falcipes, scinax fuscovarius, s. granulatus, and s. uruguayus. larvae usually forage near the water surface (head up, tilted upwards at 45º), but dive quickly when disturbed (without forming schools). on two occasions, we observed predation on tadpoles of p. iheringii in a temporary pond at são sepé: in the first incident (december 2003), a tadpole was devoured by a beetle larva (dytiscidae) early in the night, near the water surtable 2. comparison of external morphological characteristics between tadpoles from the phyllomedusa burmeisteri species group. *the original description uses the stage viii of rugh (1951). stages were assessed according to gosner (1960). species stage oral formula ventral gap marginal papillae reference phyllomedusa bahiana lutz, 1925 34–36 2(2)/3(1) present mostly uniserial; biserial at emargination silva – filho and juncá, 2006 phyllomedusa burmeisteri boulenger, 1882 35 2(2)/3(1) absent two series, with four or more series on the labial emargination cruz, 1982 phyllomedusa distincta a. lutz in b. lutz, 1950 37 2(2)/3(1) absent two series, with a few scattered papillae on the labial emargination cruz, 1982 phyllomedusa iheringii boulenger, 1885 28* 2(2)/3 absent uniserial de sá and gerdau, 1983 phyllomedusa iheringii boulenger, 1885 37 2(2)/3(1) 2(2)/3 absent uniserial, with submarginal papillae laterally aggregate in the oral disc, not forming rows present study phyllomedusa tetraploidea pombal jr. and haddad, 1992 37 2(2)/3(1) absent uniserial, with numerous series on the emargination pombal jr. and haddad, 1992 *the original description uses the stage viii of rugh (1951). stages were assessed according to gosner (1960). fig. 1. tadpole of phyllomedusa iheringii (museum catalog zufsm 10015) at gosner stage 36: (a) dorsal view (scale = 10 mm); (b) lateral view (scale  =  10 mm); (c) oral disc (scale  =  1 mm). drawnigs by b.m. and t.g.s 71the tadpole of phyllomedusa iheringii face; the second occasion (november 2011) occurred in the afternoon, when a great kiskadee bird (pitangus sulfuratus, tyrannidae) repeatedly plunged into the water to catch and eat several tadpoles. the morphological characteristics presented here for phyllomedusa iheringii are similar to those reported by de sá and gerhau (1983). however, we found variation in the labial tooth row formula (table 2), the color of papillae, body, tail, fins, and the intestine (which ranges from black to blue). the variation in coloration is probably related to the structural characteristics of the pond and to avoid predation and competitors (thibaudeau and altig, 2012). indeed, experiments among tadpoles of several species in the presence of predators exhibit differences in coloration and morphology (mccollum and leimberger, 1997; van buskirk and mccollum, 2000; touchon and warkentin, 2008). tadpoles assessed here were collected in ponds with distinct structural variables and subject to different predation pressures, and thus we suggest that future studies could test the adaptive value of tadpole coloring on different pressures of predators and competitors. several characteristics of the phyllomedusa iheringii tadpole are shared with other species of the p. burmeisteri group, such as the triangular shape of the body in dorsal view, ventral fin higher than the dorsal fin, and the oral formula 2(2)/3(1), with a reduced third row of lower teeth and dorsal gap (cruz, 1982; de sá and gerhau, 1983; pombal jr. and haddad, 1992; silva-filho and juncá, 2006). however, the tadpole of p. iheringii is distinguished from other species of the p. burmeisteri group by fewer marginal papillae in the oral disc (only one series), whereas p. burmeisteri has two rows of papillae in the anterior and posterior portion of the oral disc, and four series on the labial emargination (cruz, 1982); p. bahiana has one row in the anterior and posterior region and a double row on the labial emargination (silva-filho and juncá, 2006); p. distincta has double series (cruz, 1982); p. tetraploidea has one row, but with numerous rows on the labial emargination (pombal jr. and haddad, 1992), and p. bahiana is the only species in the group that has a ventral gap of marginal papillae (silva-filho and juncá, 2006). in summary, the tadpole of p. iheringii is distinct from other species of the p. burmeisteri group by the oral disc, mainly by the number of marginal papillae and the labial emargination. the species belonging to the genus phyllomedusa possess complex polypeptides on the skin that can be toxic and cause irritation, which might potentially protect the species against predation (duellman and trueb, 1986; caramaschi and cruz, 2002). however, several species of vertebrates and invertebrates prey on eggs, larvae, and adults of phyllomedusa (castanho, 1996; feltrim and cechin, 2000; toledo et al., 2005; dias et al., 2012). here, we add two predators of larvae of this genus (a great kiskadee bird and a beetle larva). until now, odonate naiads, belostomatid bugs, spiders and fishes were identified as predators of tadpoles of phyllomedusa (azevedoramos et al., 1992; gascon, 1992; schmidt and amézquita, 2001; santos-silva et al., 2013) tadpoles of phyllomedusa iheringii are similar to those of other species of p. burmeisteri group, but smaller in average and with fewer marginal papillae in the oral disc (not emarginated). variation (between populations and individuals) on larvae of p. iheringii was primarily related to size, the oral disc formula (and the size of the third lower row) and the color pattern of the intestines and fins. in this sense, we believe our results add important information about the external morphology and natural history of p. iheringii, an endemic species of the uruguayan savanna ecoregion. we suggest future studies to focus on frog species that are relatively common in the neotropics, such as p. iheringii, but that lack basic information as aspects of behavior, reproductive biology, and morphological descriptions of tadpoles. acknowledgements we are grateful to raúl maneyro landó for providing specimens from uruguay. s.i, v.m.l, b.m and f.p.m. thank capes, for the doctoral fellowships. t.g.s and s.z.c. are grateful to cnpq for research fellowships (proc. 307352/2013-7 and 304929/2012-3, respectively). collecting permits (#29509 and 29508) were provided by the instituto chico mendes de conservação da biodiversidade (icmbio). we thank to the anonymous reviewers for their comments and suggestions on the manuscript. references altig, r. 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(2000). functional mechanisms of an inducible defence in tadpoles: morphology and behaviour influence mortality risk from predation. j. evol. biol. 13: 336-347. acta herpetologica vol. 10, n. 1 june 2015 firenze university press obituary: valery k. eremchenko (1949-2014) leo j. borkin1, tatjana dujsebayeva2, roberto sindaco3, matthias stöck4 haplotype variation in founders of the mauremys annamensis population kept in european zoos barbora somerová1, ivan rehák2,*, petr velenský2, klára palupčíková1, tomáš protiva1, daniel frynta1 reproductive ecology of sichuan digging frogs (microhylidae: kaloula rugifera) wei chen1,*, lina ren2, dujuan he2, ying wang2, david pike3 toxic effects of carbaryl on the histology of testes of bufotes variabilis (anura: bufonidae) özlem çakici basal frequency of micronuclei and hematological parameters in the side-necked turtle, phrynops hilarii (duméril & bibron, 1835) maría a. latorre 1,2,*,#; evelyn c. lópez gonzález1,2, #; pablo a. siroski1,2,3; gisela l. poletta1,2,4 into a box interiors: clutch size variation and resource allocation in the european pond turtle marco. a.l. zuffi1,*, simonetta citi2, elena foschi1, francesca marsiglia1, eva martelli1 where to “rock”? choice of retreat sites by a gecko in a semi-arid habitat andreia penado1,2, ricardo rocha3,4,*, marta sampaio3, vanessa gil3, bruno m. carreira3, rui rebelo3 age structure, growth and longevity in the common toad, rhinella arenarum, from argentina clarisa de l. bionda1,2,*, silvia kost 4, nancy e. salas1, rafael c. lajmanovich3, ulrich sinsch4, adolfo l. martino1 on a putative type specimen of pleurodema bibroni tschudi, 1838 from chile (anura: leptodactylidae) daiana paola ferraro re-description of the external morphology of phyllomedusa iheringii boulenger, 1885 larvae (anura: hylidae), with comments on the external morphology of tadpoles of the p. burmeisteri group samanta iop¹, victor mendes lipinski¹, bruno madalozzo¹, franciele pereira maragno¹, sonia zanini cechin¹, tiago gomes dos santos² book review: harold heatwole, john w. wilkinson (eds). amphibians biology. volume 11 status of conservation and decline of amphibians. eastern hemisphere. part 4 . southern europe and turkey sebastiano salvidio book review: antonio romano. atlante degli anfibi del parco nazionale del cilento vallo di diano e alburni distribuzione, biologia, ecologia e conservazione sebastiano salvidio acta herpetologica journal of the societas herpetologica italica acta herpetologica 10(2): 85-91, 2015 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-16297 variation of growth rate and survival in embryos and larvae of rana temporaria populations from the pyrenees neus oromi1,2,*, sebastià camarasa1, isabel sanuy1, delfi sanuy1 1 escola tècnica superior d’enginyeria agrària, departament de producció animal (fauna silvestre), university of lleida, av. rovira roure 191, 25198 lleida, spain. *corresponding author. e-mail: noromi@ulg.ac.be 2 laboratory of fish and amphibian ethology, behavioural biology unit, department of biology, ecology and evolution, university of liege, 22 quai van beneden, 4020 liege, belgium submitted on 2015, 4th june; revised on 2015, 2nd october 2015; accepted on 2015, 5th october editor: sebastiano salvidio abstract. variations on embryonic and larval life history traits of ectotherm organisms are strongly affected by temperature conditions. however, these effects can vary between species and populations depending on the mechanisms that act in a determinate local habitat. in the present study, we analysed the effects of temperature on several embryonic and larval traits (survival, development and growth rate until the metamorphosis) of rana temporaria in two populations living at different altitude (1540 and 2100 m) in the pyrenees. five spawns from each population were distributed in a common garden experiment at different temperature treatments according to the normal temperature range that tadpoles might experience in the field and also considering a high treatment (24 °c) to test a possible effect of global warming. like in other studies of the same species in a latitudinal gradient, the temperature effects depended on the analysed trait. our results support the general rule that the rate of development is faster at higher temperatures, although survival was significantly affected by the highest temperature in the highland population. size varied at embryonic and larval stages and was largest at metamorphosis in the highland population. in concordance, the growth rate was higher in the highland population suggesting a countergradient variation in response to the short growing season. however, this possible adaptation can be altered in a global warming scenario with an increase of mortality and limited growth. keywords. rana temporaria, development, growth, altitudinal variation. introduction variation in climatic conditions determines intraspecific differences in life histories especially in ectotherm organisms. in general, low temperature reduces their physiological rates, decreasing the development and growth rates at higher latitudes and altitudes. however, many studies have found increased growth and development rates at higher latitudes and altitudes (e.g. conover and schultz, 1995; arendt, 1997). the effects of temperature in anuran tadpoles can influence some traits that are basic to population survival in one specific habitat. in addition, the warmer waters can compromise survival because of decreased oxygen availability, oxidative stress due to higher metabolic rate and the build-up of microbes from decomposing food or nitrogenous waste products (e.g., courtney jones et al., 2015). the increased temperature can reduce the metamorphic size in some species (e.g., álvarez and nicieza, 2002: discoglossus galganoi; orizaola and laurila, 2009: rana lessonae) whereas in other species growth is accelerated by high temperatures (e.g., arendt and hoang, 2005: spea hammondi; sanuy et al., 2008: bufo calamita). the mechanisms that determine the variation of these larval life history traits can be understood in a local habitat scale. therefore, intraspecific studies of geographic variation have pro86 neus oromi et alii vided evidences of the occurrence of microevolution as a response to local ecological conditions (e.g., conover and schultz, 1995; huey et al., 2000; laugen et al., 2005). several evolutionary models can explain the occurrence of the different mechanisms that determine the variation of larval life history across a latitudinal or altitudinal gradient (yamahira and conover, 2002). environmental influences and microevolutionary pressures can act synergistically on the phenotypic variation of life history traits (cogradient selection) when both factors tend to either reduce or enhance the trait in question occurrence of microevolution as a response to local ecological conditions (e.g., conover and schultz, 1995). considering this hypothesis, organisms are adapted to the temperature that they most commonly experience in the natural environment (levinton and monahan, 1983; lonsdale and levinton, 1985). therefore, in an altitudinal gradient, populations living at low altitudes are expected to grow faster at high temperatures, whereas high altitude populations can show a higher growth rate at a low temperature. however, if the phenotypic response does not reflect the microevolutionary process responsible for the population genetic differentiation (arendt and wilson, 1999; ghalambor et al., 2007; crispo, 2008), the environmental factor can be antagonistic against the effect imposed on the phenotypic variation by natural selection (conover and schultz, 1995). from this perspective, considering the breeding period along an altitudinal gradient, as the length of the growth season declines across higher altitudes, the tadpoles living at high altitudes have less time to grow and develop than those in low altitudes (conover and schultz, 1995). consequently, highland populations are expected to have a higher capacity for growth and development in all temperatures, in order to be able to compensate for the shorter growth season. the essential prediction of this hypothesis is that there should be an inverse relationship between the length of the growth season and the capacity for growth and development (conover and schultz, 1995; yamahira and conover, 2002). this phenomenon is known as “countergradient variation” (conover and schultz, 1995; levins, 1969). although larval growth and development rates have been considered important in the determination of performance during later life stages (mousseau and fox, 1998; lindström, 1999), very little is known about life history variation in embryonic and larval traits along altitudinal gradients. in rana temporaria, muir et al. (2014a) in scotland reported a phenotypic plasticity variation in an altitudinal gradient. in our study we also analysed a phenotypic variation depending on temperature treatments, in the case of high temperatures than those recorded in the field. global average surface temperature is predicted to further rise by 2 to 6 °c at the end of the 21st century (ipcc, 2007). global warming can significantly change the behaviour, growth rate, and reproductive phenology of a species, specially affecting the ectotherms organisms (brattstrom, 1963). there is a notable lack of knowledge about the impact of global warming on the survival and life history traits of the populations from cold habitats. as high latitude ectotherms can be highly vulnerable to climate warming in temperate zones (gerick et al., 2014) we expect a similar effect at high altitudes. in this study we want to investigate the effects of temperature in a large-wide distributed european species, the common frog rana temporaria, across an altitudinal gradient in the pyrenees. previous studies on this anuran species have focused in embryonic and larval life history traits at latitudinal scales. several works have found that larval growth and development rates increase with latitude (merilä et al., 2000; stählberg et al., 2001; laugen et al., 2002; laurila et al., 2001, 2002). however, other studies show that the variation in the embryonic development and growth in r. temporaria cannot be explained in terms of a latitudinal gradient in season length whereas adaptation to a latitudinal variation in temperature might contribute to explain the variations observed (laugen et al., 2003b). lindgren and laurila (2005, 2010) found increased growth rate at higher latitudes, evidencing that the populations from higher latitudes had higher growth efficiency. in our study, we analyse the effects of temperature on embryonic and larval life history traits of r. temporaria across an altitudinal gradient in the pyrenees. the length of breeding season declines, as well as the ambient temperature, from the low to the highland populations. we analyse if seasonal time constraints are mainly responsible for the observed variations or if the adaptation to the prevailing temperature is the important factor. in addition, we aim to assess the survival effect at high temperatures in larvae of r. temporaria. at high altitude, the tadpoles of r. temporaria appear to have the potential to adapt physically to surviving at low temperatures (muir et al., 2014a) but what occurs when the temperature exceeds their normal exposure range is unknown. material and methods study zone the study zone was situated in a pasture area of the vall d’aneu (42°37’n, 1°03’e) in the catalonian pyrenees (iberian peninsula). the lowland population was located near a stream (riera del tinter) at 1540 m. the highland population was situated in the same valley in a breeding site near a small river (les cabanyes) at 2100 m. both populations reproduce 87growth rate and survival in larvae of rana temporaria in temporary ponds formed as a consequence of the thawing of snow and ice in spring. the climate is typical of temperate mountain regions, with short bursts of heavy rainfall in april and may, and stable periods of high pressure between september and november. the temperature conditions of the zone have been extrapolated from the data of the national park of aigüestortes in the pyrenees (from 2005 to 2008) at altitudes of 1600 and 2000 m. the temperatures during spring and summer varied from 3.8 to 22 °c at 1600 m and 1.1 to 19.5 °c at 2100 m (from may to september; minimum and maximum air temperature respectively). we considered the length of the growth season as continuous period, from spring (may) to winter (october) where mean air temperature exceeds +5 °c. it was approx. of 120 days for the lowland population and 90 days for the highland population. we considered that breeding did not occur below 5 °c, as reported muier et al. (2014) on populations of r. temporaria at high altitude in scotland. the onset of the breeding season differs between populations (the highland population reproduced 1 month after the lowland population). experimental design rana temporaria eggs were collected on april 5th and may 5th 2012, from the lowland and highland population respectively, at embryonic stage 0-3 of gosner (1960). the eggs were taken to the laboratory of the university of lleida (spain) and immediately used in the experiment. the temperature treatments (16, 20 and 24 °c) were selected according to the normal temperature range that tadpoles might experience in the field and also considering a high treatment (24 °c) to test a possible effect of global warming in the growth and development of r. temporaria in the pyrenees. the experiment was formed by 3 controlled temperature systems: 16, 20 and 24 °c. each temperature system was formed by 4 plastic boxes (57 × 77 × 20 cm) of 40 litres connected between them to avoid temperature fluctuation. eggs from each population (represented by 5 spawns per population) were randomly distributed in the different plastic boxes and individualised (225 larvae per population) in floating plastic glasses (0.5 l) in each temperature condition. the remained eggs were released in the same site where were collected. the temperature in each plastic box (12 plastic boxes, 4 per temperature) was measured every day (sd = ± 0.3 °c). water of each plastic glasses was changed every three days. when tadpoles arrived at stage 25, they were fed with fish food ad libitum and excess food was removed every day. larval mortality was monitored every day. the body length (without the tail (length) of each larva in the different treatments was also regularly measured, using a digital camera and a millimetre scale. we obtained the measures of length with the corel draw 7.0 program using the “dimensioning lines” tool to measure the distance between two points. the distance was corrected using the millimetre scale available in each digital photo. the response variables measured from each tadpole were: 1) age at hatching (defined as the number of days between gosner stages 0-3 and 25), 2) age at metamorphosis (defined as the number of days between gosner stages 0-3 and 42), 3) size at hatching (measured by body length at stage 25), 4) size at metamorphosis (measured by body length at stage 42) and 5) larval growth rate (between gosner stages 25 and 42 divided by the days between these two stages). at the end of the experiment, all the survival animals were released to the field. statistical analyses the laboratory experiment was a factorial design with population (lowland and highland) and temperature (16, 20 and 24 °c) as factors. all the data of the response variables were first tested for normality and variance heterogeneity before analyses. age at hatching and metamorphosis, and growth rate were logtransformed to archive normality. survival was performed on arcsin-transformed proportions, which effectively normalized the error variances. the effects of temperature and population, as well as their interaction, on survival and size at hatching and metamorphosis were analysed in a 2-factorial anova. to test the differences in the egg size between populations in the beginning of the experiment, we used a 1-factorial anova analysis. all the analyses were based on the procedures of the statistical package jmp 9.0.1 with a significance level of alpha = 0.05. results survival the survival at hatching was affected by population and temperature treatment without significant interaction (lowland population: 99, 98.9 and 96% at 16, 20 and 24 °c; highland population 95.6, 95.4 and 82% respectively; table 1). at the end of the study, the survival at metamorphosis was lower in the highland population (92, 93.3 and 84% at 16, 20 and 24 °c respectively) than in the lowland population (98.6, 98.6 and 94.6%). although, the highest temperature reduced the survival in both populations, there were not statistical differences between temperature treatments (table 1). development time age at hatching and metamorphosis varied significantly between temperature treatments without population effects at early time of development (table 1, fig.1a, b). the populations had higher age at hatching and metamorphosis as temperature treatment increased. the significant interaction between temperature and population factors at age at metamorphosis shows that temperature treatment did not affect in the same level the two populations. in fact, in contrast to the results in the other temperatures, the highland population developed slower than the lowland population at 24 °c. 88 neus oromi et alii size and growth rate at the beginning of the experiment, the eggs of the highland population, 2.48 ± 0.39 (sd) mm, had similar size than those in the lowland population, 2.39 ± 0.11 (sd) mm (1-factorial anova; f = 2.76, p = 0.09). the population effect was significant on size at hatching and metamorphosis. the temperature was significant on size at metamorphosis but not at hatching (table 1, fig. 2a,b). size at hatching and metamorphosis varied depending on population origin and temperature treatment (table 1, fig. 2a,b). however, the growth rate, which allows to measure the exploitation of resources, was significantly higher in the highland population (2 factorial anova, f1,188 = 101.66, p < 0.00001) without temperature treatment effects (f2,188 = 1.41, p = 0.247) and interaction (“temperature*population”: f2,188 = 2.93, p = 0.056, fig. 3). discussion the present work shows a relevant effect of the temperature on the development and growth of rana temporaria that depends on the altitude. mortality increased weakly at highest temperature in all populations, especially notable at highest temperature in the highland population. this is probably due to the fact that the highland population is unlikely exposed in the nature to temperatures above 22 °c. as it was found in other amphibian species (gerick et al., 2014) in a global warming scenario, table 1. results of anova for the effects of temperature (16, 20 and 24 °c) and population (lowland and highland) on survival, age and size at hatching and metamorphosis of rana temporaria tadpoles. (ss= sum of squares; df= degrees of freedom). response source ss df f p survival at hatching population temperature temperature x population 7414.27 2276.57 1073.53 1, 835 2, 835 2, 835 30.54 4.68 2.21 < 0.001 0.009 0.110 age at hatching population temperature temperature x population 0.02289 1.41241 0.02936 1,1499 2,1499 2,1499 1.81 57.57 1.20 0.174 < 0.0001 0.305 hatching size population temperature temperature x population 415.789 24.7488 49.0953 1, 330 2, 330 2, 330 68.04 1.88 3.72 < 0.0001 0.154 0.025 survival at metamorphosis population temperature temperature x population 362.049 244.136 244.136 1, 690 2, 690 2, 690 3.99 1.35 1.35 0.045 0.260 0.260 age at metamorphosis population temperature temperature x population 5.89946 13.5599 1.43157 1, 694 2, 694 2, 694 28.14 32.34 3.41 < 0.0001 < 0.0001 0.033 metamorphosis size population temperature temperature x population 42.4891 10.4598 42.4891 1, 188 2, 188 2, 188 5.02 0.62 2.50 0.026 0.541 0.085 fig. 1. mean days (± sd) of development time of two rana temporaria populations at three temperature treatments. a) age at hatching and b) age at metamorphosis. 89growth rate and survival in larvae of rana temporaria some populations will experience maximum summer temperatures above their maximum thermal limit that can promote the disappearance of these populations. however, others studies about temperature tolerance (duarte et al., 2012) at different stages and altitudes are needed to evaluate the possible effects of global warming in the perpetuation of r. temporaria in its present distribution. we found different temperature effects and variations that depend on the analysed trait, as e.g., lindgren and laurila (2005) showed in a latitudinal gradient in scandinavian r. temporaria populations. for example, while age at metamorphosis increased at low temperatures and in the highland population, size was similar between temperature treatments at hatching and higher in the lowland population. in contrast, the size at metamorphosis was not affected by the temperature, being larger in the highland population. the temperature affected the earliest stages of development but the population effects or altitudinal effect, began to be notable after embryonic stages, past the stage 25. it is possible that the time constraint imposed on embryonic stages is not as severe as in the larval development, because the larvae may compensate the delays in hatching (räsänen et al., 2002). in addition, a high development rate can limit the hatchling size, that is a more important fitness factor than age at hatching (laugen et al., 2003a). a larger hatching size is often beneficial because it increases competitive ability and in many systems brings a survival advantage or other improvements in later fitness (kaplan, 1998; pechenik et al., 1998; lindström, 1999). in our study, the differences on age at hatching between populations are not much notable and the highest hatching size in the lowland population seems to be an altitudinal effect. others studies on r. temporaria have shown that embryos from low altitude develop slower than those from higher altitudes (angelier and angelier, 1968; martin and miaud, 1999). however, pahkala et al. (2002) and laugen et al. (2003a) did not find a clear pattern of embryonic development across a latitudinal cline (scandinavia). in our study, we found clearly that development rates increase with altitude only at low temperature (16 °c), similarly to the results showed with latitude in r. temporaria of scandinavia (martin and miaud, 1999; merilä et al., 2000; laurila et al., 2001, 2002) and in an altitudinal range in scotland (muir et al., 2014a). the growth rate showed a clear variation, suggesting an increase of this trait along the altitudinal gradient across the pyrenees. other authors found similar results in the same species across a latitudinal gradient in scandinavia, but in those cases, the development rates of r. temporaria increased in the northern populations (e.g., merilä et al., 2000; laugen et al., 2003b; lindgren and laurila, 2005). tadpoles in the highland populations can exhibit higher activity of the enzymatic levels deriving in a larger size (latitudinal gradient: laurila et al., 2008; altitudinal gradient: muir et al., 2014a) that increases the competitive ability of high altitude tadpoles as compared to low altitude conspecifics. in addition, the higher fig. 2. average size (± sd) at different temperature treatments of two populations of rana temporaria. a) hatching size and b) metamorphosis size. fig. 3. growth rate (gr) (± sd) of two populations of rana temporaria at different temperatures. 90 neus oromi et alii growth rate was maintained in all temperature treatments, suggesting an adaptation to season length rather than to prevailing temperature (conover and schultz, 1995; laugen et al., 2003b; palo et al., 2003). survival at high altitude is probably facilitated by mechanisms that permit faster growth rate in r. temporaria (muir et al., 2014b). overall, in the line of a large number of ectotherms studies, our results support the general rule that development rate is faster at higher temperatures. however, hatching size is not affected by temperature treatments, which is the opposite to the pattern commonly found in ectotherms (atkinson and sibly, 1997). probably the energetic costs of development at low temperature limit a larger size that can be derivate of a long embryonic development. as muir et al. (2014b) reported in the altitudinal gradient of scotland, we found a higher larval growth rate in the highland population that suggests an adaptation that has occurred to maximize growth during the short growing season. however, this possible adaptation can be altered in a global warming scenario with an increase of mortality and limited growth. acknowledgments thanks to pepe guillen for the field work and to lluis camarero for providing the meteorological data. we also thank to marc rivas, jordi burguet and bernat nadal for their help in the experimental part. the project has the approval of the care committee at university of lleida (spain). the study zone is not a protected area and the present work did not involve endangered or protected species. the permission for eggs collection was approved by the generalitat de catalunya (direcció general del medi natural i biodiversitat; refernce number: sf/281). the project was financed by the ministerio de economía y competitividad, gobierno de españa ref. cgl200912767-c02-01. thanks are also due to two anonymous reviewers. references álvarez, d., nicieza, a.g. 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(2014a). behavioural and physiological adaptations to low-temperature environments in the common frog, rana temporaria. evol. biol. 14: 110. muir, a.p., biek ,r., thomas, r., mable, b.k. (2014b). local adaptation with high gene flow: temperatura parameters drive adaptation to altitude in the common frog (rana temporaria). mol. ecol. 23: 561-574. orizaola, g., laurila, a. (2009): microgeographic variation in temperature-induced plasticity in an isolated amphibian population. evol. ecol. 23: 979-991. pahkala, m., laurila, a., merilä, j. (2002): effects of ultraviolet-b radiation on common frog rana temporaria embryos from along a latitudinal gradient. oecologia 13: 458-465 palo, j.u., o’hara, r.b., laugen, a.t., laurila a., primmer, c.r., merilä, j. 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(2002): intravs. interspecific latitudinal variation in growth: adaptation to temperature or seasonality? ecology 83: 1252-1262. acta herpetologica vol. 10, n. 1 june 2015 firenze university press acta herpetologica journal of the societas herpetologica italica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 9(1): 115-117, 2014 doi: 10.13128/acta_herpetol-13105 clutch size in wild populations of alytes muletensis samuel pinya1,*, valentín pérez-mellado2 1 herpetological research and conservation centre, associació per a l’estudi de la natura. balearic islands, spain. * corresponding author. e-mail: sampinya22@gmail.com 2 department of animal biology, universidad de salamanca, spain, e-mail: valentin@usal.es submitted on 2013, 17th july; revised on 2013, 21st november; accepted on 2014, 16th april abstract. clutch size of genus alytes presents strong interand intra-specific variability. the mallorcan midwife toad (a. muletensis) is a threatened endemism of the island of mallorca (spain), and has the smallest clutch size of the genus. most of the previously published clutch size information was obtained from animals breeding in captivity. in this study we analysed a large number of clutches from wild populations, and we compared their size (number of eggs) with previously published data from both captive and wild populations. our results showed that clutch sizes of wild males carrying a single clutch were similar to those observed in captivity. however, multiple clutches were more common in the wild than in captivity. keywords. alytes muletensis, clutch size, wild populations, endemic species, balearic islands in alytes species it is males that care for eggs. the female expels a strand of eggs which are fertilised by the male before he wraps them around his legs to protect them from predators. when the eggs are ready to hatch, the male wades into shallow ponds or streams to allow tadpoles to go into the water (stebbins and cohen, 1997). five separate species of midwife toads are found across western europe, northern africa, and the island of mallorca: alytes obstetricans, alytes cisternasii, alytes dickhilleni, alytes maurus and alytes muletensis (martinez-solano et al., 2004). the clutch size of the genus alytes is highly variable both, interand intra-specifically (table 1). the mallorcan midwife toad, a. muletensis, is a threatened endemism of the island of mallorca (balearic islands, spain; serra et al., 2009), with the smallest clutch size of the genus (alcover et al., 1984, fig. 1). as in other alytes species, the clutch size of a. muletensis is fairly variable (table 2). during studies in captivity bush (1993, 1996) observed the presence of double clutches for the first time in a. muletensis, that is, one male carrying the eggs released by two females. he also found that single clutches (less than 15 eggs) had an average of 10.00 eggs (range from 4 to 14 eggs, bush, 1993, 1996). taking into account double clutches, the average increased to 11.36 eggs (range from 4 to 27; bush, 1996). additionally, and also in captivity, the maximum clutch size observed by román and mayol (1997) had 34 eggs, while the average clutch size was 11 eggs. in order to establish demographic parameters of wild a. muletensis individuals, between 2007-2010, we counted the number of eggs carried by 92 males from 11 natural, plus 7 artificial, breeding sites in the wild. an artificial breeding site for the purpose of this study is a man-made structure such as drinking ponds for cattle or old water cisterns/tanks being used by wild a. muletensis for breeding. these sites were established as a conservation measure for the release of captive-bred animals during the 1990s. the current sample represents the descendants of these introduced toads. on the other hand, streams and isolated natural pools are considered natural breeding sites. the clutch size was determined by counting the number of eggs of empty strings and egg capsules after the tadpoles had emerged, and within a short time after the males had come out of the water. 116 s. pinya, v. pérez-mellado to show clutch size variability in wild populations of a. muletensis, descriptive statistics are provided (table 2). we used one way anova to compare (1) clutch size data from alcover et al. (1984) with that from the present study, (2) clutch size data from bush (1996; captive populations) with that from the present study, and (3) clutch size data for artificial and natural breeding sites from the present study. a chi-square test was used to compare frequencies of multiple clutches in captive (from bush, 1996) and wild (from the present study) populations of a. muletensis. all statistical analyses were carried out with statistica (ver. 6.0). our data revealed an average clutch size of 12.04 eggs in the wild (table 2). we found no significant difference (f1,99 = 1.40, p = 0.24) when our data was compared to previously published averages for wild individuals (alcover et al., 1984), maybe because of the small sample size in the 1984 study. there was also no significant difference between clutch size data in our study (f1,319 = 0.83, p = 0.36) and that published by bush (1996) for animals in captivity. additionally, there was no significant difference between natural (n=41) and artificial (n=51) breeding sites in the present study (f1,92 = 1.31, p = 0.26). the observed range of clutch size found in this study is the largest known for both wild and captive populations, and coincides with the minimum and maximum values published previously (alcover et al., 1984; bush, 1996; román and mayol, 1997). multiple clutches are common in natural populations of continental midwife toads (bush, 1996). between 56-61 % of a.obstetricans and 80 % of a. cisternasii eggcarrying males carry two or more clutches simultaneously (reading and clarke, 1988; márquez 1990). in a. muletensis, double clutches are rare (12-14 % according to bush, 1996). on the other hand, triple clutches, those with more than 28 eggs, were first reported by román and mayol, (1997) with a record of 34 eggs, but their frequency was not provided. our data shows that, in wild populations 23.91 % of clutches should be considered multiple clutches, with 3.26 % of them consisting of triple clutches. when comparing the occurrence of multiple clutches, between wild individuals in this study and data for captive individuals (bush, 1996), we found a significantly higher frequency of multiple clutches in the wild population (χ2 = 5.30, p < 0.02). the low incidence of multiple clutches in captivity may be due to the short time availtable 1. clutch sizes of continental alytes species. average eggs per clutch, minimum and maximum eggs per clutch, and source of information. species average range reference a. obstetricans 63 35-95 crespo, 1979 104 46-143 lópez-jurado et al., 1979 51 24-142 buchholz, 1989 38 5-120 galán et al., 1990 27 21-33 galán et al., 1990 55 24-77 galán et al., 1990 34 6-53 galán et al., 1990 77 32-171 reading, clarke, 1998 a. cisternasii 73 42-119 crespo, 1979 104 46-143 lópez-jurado et al., 1979 73 48-116 malkmus, 1983 87 20-180 marquez, 1989 a. dickhilleni 74 28-149 gonzález-miras, garcía-cardenete & tejedo, 2012 a. maurus 60-70 donaire-barroso, bogaerts, 2003 table 2. clutch size in a. muletensis: summary of literature and original (present study) data. population type, average clutch size, sample size (when available), minimum and maximum eggs per clutch, and source of information. population type average n range variance reference natural 12.04 92 4-34 32.53 present study natural 9.78 9 7-12 alcover et. al, 1984 captive 10-24 martínez-rica et al., 1984. captive 11.43 9-15 tonge & bloxam, 1989 captive 11.36 220 4-27 14.05 bush, 1996 captive and natural 11 -34 román & mayol, 1997 fig. 1. male a. muletensis carrying eggs (island of mallorca, spain). 117clutch size in alytes muletensis able for a male to obtain the second clutch from a female (bush, 1996). males in wild populations may have longer time intervals during which they can accept additional clutches. in addition, the operational sex-ratio during the breeding season also determines the potential for multiple clutches. if more females are available males will be able to mate with more than one female in a short period of time and hence brood more than one clutch (bush, 1996). it is also possible that wild individuals breed at higher densities than captive individuals, and wild receptive males may be able to obtain a second clutch more frequently simply because they are more likely to encounter more than one gravid female. aknowledgements we would like to thank joan mayol, joan a. oliver, mª antònia vanrell and joan c. salom of the balearic environmental agency, who gave us administrative support and provided the capture permits (19/2007, 44/2008, 07/2009, 13/2010) for our study with the mallorcan midwife toad. we are also grateful to all the people who provided support during the field-work, specially xavier manzano and jaume bonnin. montserrat carbonell made an excellent revision of english language and gave us very useful suggestions. references alcover, j.a., mayol, j., jaume, d., alomar, g., jurado j. (1984): biologia i ecologia de les poblacions de baleaphryne muletensis a la muntanya mallorquina. in: història biològica del ferreret. monografies científiques 3, pp. 129-152. hemmer, h., alcover, j.a., eds, editorial moll, palma de mallorca. buchholz, s. (1989): untersuchungen zur fortpflanzungsbiologie und populationsdynamik einer freilandpopulation von alytes obstetricans (amphibia, anura, discoglossidae). diplomarbeit, universität würzburg, 81 s. bush, s.l. (1993): courtship and male parental care in the majorcan midwife toad, alytes muletensis. unpublished doctoral dissertation. university of east anglia, norwich. bush, s. l. (1996): why is double clutching rare in the majorcan midwife toad?. anim. behav. 52: 913-922. crespo, e.g. (1979): contribuçao para o conhecimento da biología dos alytes ibéricos, alytes obstetricians boscai lataste 1879 e a. cisternasii boscá 1879: a problamática da especiaçao de a. cisternasii. unpublished doctoral dissertation. university of lisboa. donaire-barroso, d., bogaerts, s. (2003): datos sobre taxonomía, ecología y biología de alytes maurus (pasteur & bons, 1962) (anura; discoglossidae). bull. soc. cat. herp. 16: 25-41. galán, p., vences, m., glaw, f., fernádenz-arias, b., garcía-parís, m. (1990): beobachtungen zur biologie von alytes obstetricans in nordwestiberien. herpetofauna 12: 17-24. gonzález-miras, e., garcía-cardenete, l., tejedo m. (2012): historia natural. in: seguimiento de alytes dickhilleni: informe final. monografías sare 02, pp. 17-22. bosch, j., gonzález-miras, e., eds. asociación herpetológica española, ministerio de agricultura, alimentación y medio ambiente, madrid. malkmus, r. (1983): zur fortpflanzungsbiologie von a. cisternasii. weinstadt 5: 30-34. márquez, r. (1990): male parental care, sexual selection, and the mating systems of the midwife toads alytes obstetricans and alytes cisternasii. unpublished doctoral dissertation. university of chicago. martínez-rica j.p., pardo, m.p., cervantes, j. (1984): la reproducción y la conducta en cautividad del sapillo balear, baleaphryne muletensis. in: història biològica del ferreret. monografies científiques 3, pp. 175-191. hemmer, h., alcover, j.a., eds, editorial moll, palma de mallorca. martínez-solano, i., gonçalves, h.a., arntzen, w., garcía-parís, m. (2004): phylogenetic relationships and biogeography of midwife toads (discoglossidae: alytes). j. biogeogr. 31: 603-618 reading, c.j., clarke, t. (1988): multiple clutches, egg mortality and mate choice in the midwife toad, alytes obstetricans. amphibia-reptilia 9: 357-364 román, a., mayol, j. (1997): la recuperación del ferreret, alytes muletensis. documents tècnics de conservació 1. conselleria de medi ambient, ordenació del territori i litoral, palma de mallorca. serra, j.m., griffiths, r., bosch, j., beebee, t., schmidt, b., tejedo, m, lizana, m., martínez-solano, i., salvador, a., garcía-parís, m., gil, e.l., arntzen, j.w. (2009). alytes muletensis. in: iucn 2013. iucn red list of threatened species. version 2013.2. <www.iucnredlist. org>. downloaded on 24 march 2014. stebbins, r.c., cohen n.w. (1995): a natural history of amphibians. princeton university press. chichester, west sussex. tonge, s., bloxam, q. (1989): breeding the mallorcan midwife toad alytes muletensis in captivity. int. zoo yb 28: 45-53. acta herpetologica vol. 8, n. 2 december 2013 firenze university press journal of the societas herpetologica italica acta herpetologica © firenze university press www.fupress.com/ah acta herpetologica 5(1): 87-89, 2010 unusual colour form of siberian pit viper (gloydius h. halys) from the northern edge of its area evgeniy simonov institute of systematics and ecology of animals, siberian branch of russian academy of science, frunze 11, 630091 novosibirsk, russia. e-mail: ev.simonov@gmail.com submitted on: 2009, 28th august. revised on: 2010, 21st april. accepted on 2010, 30th april. abstract. in this report, i describe the unusual colour form of siberian pit viper (gloydius h. halys) from the marginal population in novosibirsk region (west siberia, russia). this morph was named “bronze” and occurs of 16% of individuals. to the best of my knowledge, such colour pattern does not known from other parts of g. halys area. keywords. pit viper, gloydius halys, “bronze” pattern. diverse colour patterns of squamates perform different important functions: thermoregulation, predators avoidance, mimicry, crypsis, protection of vital organs, and some other (jackson et al., 1976; sweet, 1985; brodie, 1993; brodie and janzen, 1995; tanaka, 2005). within one species various colour forms (morphs) may reflect local adaptations to a given stimulus. for instance an increase of dark (melanistic) individuals may be affected by non-optimal thermal conditions as dark coloured individuals have some advantages for thermoregulation (e.g., tanaka, 2005). on the other hand, proportion of different morphs may be influenced by random genetic drift (e.g., bittner and king, 2003). the halys pit viper [gloydius halys (pallas, 1776)] is the most widespread species of the subfamily crotalinae with 6 to 9 recognised subspecies (orlov and barabanov, 1999; uetz and hallermann, 2009). since 2006, i have been carrying out on ecological and morphological research on this species in the southeastern part of novosibirsk region (west siberia, russia) in the valley of river berd’. a detailed description of study area is provided in simonov (2009). this locality represents the isolated northwesternmost point of halys pit vipers’ distribution. nearest neighbour populations are between 190 and 250 km distance (simonov, 2008). during four years, 106 individuals of g. h. halys (except neonates) were captured and examined. to avoid repeated measurements, i used individual recognition of head coloration patterns combined with clipping of ventral scales. most of pit vipers in this population have a typical colour pattern, but 16% (n = 17) of snakes have a reduced band pattern. these individuals have a uniform color of brown88 e. simonov ish tints covering dorsal and lateral parts of body, whereas head and ventral side display common pattern (fig. 1). some of them have a light or dark narrow vertebral strip and/or dotted lateral strips (table 1). in addition, all abnormal coloured snakes have a light marks on the nape of the neck. this morph was named “bronze”, as nilson et al. (1995) named very similar colour form encountered among vipera dinniki and vipera lotievi. within g. h. halys the “bronze” morph occurs in both sexes, but it was also met in one juvenile snake. although the halys pit viper is a very polymorphic species, individuals without typical banded pattern are very rare. occurrences of completely black and red individuals of g. halys are known from kirghizia and south kazakhstan (yakovleva, 1964; kolbincev, 2006). pestov (2003), who first discovered the considered population of g. halys, shortly mentioned the encounter of two “monotonous brown” specimens. besides it, no additionfig. 1. bronze colour form (a) and normal colour pattern (b) of gloydius halys halys from novosibirsk region. table 1. frequencies of siberian pit viper (g. h. halys) colour forms in examined population from novosibirsk region. colour form frequency, n (%) normal 89 (84%) bronze (uniform) 10 (9.4%) bronze (striped) 7 (6.6%) bronze (total) 17 (16%) 89unusual colour form of siberian pit viper al records about bronze, not banded, individual snakes, apart those presently described, are available to date. references bittner, t.d., king, r.b. (2003): gene flow and melanism in garter snakes revisited: a comparison of molecular markers and island vs. coalescent models. biol. j. linn. soc. 79: 389-399. brodie, e.d. iii (1993): differential avoidance of coral snake banded patterns by freeranging avian predators in costa rica. evolution 47: 227-235. brodie, e.d. iii, janzen, f.j. (1995): experimental studies of coral snake mimicry: generalized avoidance of ringed snake patterns by free-ranging predators. funct. ecol. 9: 186-190. jackson, f.j., ingram, w. iii, campbell, h.w. (1976): the dorsal pigmentation pattern of snakes as an antipredator strategy: a multivariate approach. am. nat. 110: 10291053. kolbincev, v.g. (2006): amphibians and reptiles of aksu-djabagly reserve and features of its ecology. selevinia 2006: 160-172. [in russian] nilson, g., tuniyev, b.s., orlov, n., höggren, m., andrén, c. (1995): systematics of the vipers of the caucasus: polymorphism or sibling species? asiatic herpetol. res. 6: 1-26. orlov, n.l., barabanov, a.v. (1999): classification of the agkistrodon halys-intermedius complex: a critical review. rus. j. herpetol. 6: 167-192. pestov, m.v. (2003): halys pit viper – the new species of novosibirsk region’s fauna. in: amphibians and reptiles in west siberia, p. 35-38. “revi-k” press, novosibirsk. [in russian] simonov, e.p. (2008): revision of north distribution’s board of halys pit viper (gloydius (agkistrodon) halys) and its biotopical location on west siberia territory. bull. mordovia univ., biol. sci. 2: 65-70. [in russian] simonov, e.p. (2009): differences in habitat use, daily activity patterns and preferred ambient temperatures of adult and neonate gloydius halys halys from an isolated population in southwest siberia: preliminary data. herpetol. notes 2: 1-7. sweet, s.s. (1985): geographic variation, convergent crypsis and mimicry in gopher snakes (pituophis melanoleucus) and western rattlesnakes (crotalus viridis). j. herpetol. 19: 55-67. tanaka, k. (2005): thermal aspects of melanistic and striped morphs of the snake elaphe quadrivirgata. zool. sci. 22: 1173-1179. uetz, p., hallermann, j.: the reptile database, http://www.reptile-database.org, accessed 23 august 2009. yakovleva, i.d. (1964): reptiles of kirghizia. frunze. [in russian] acta herpetologica 11(1): 91-94, 2016 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-16038 correlation between endoscopic sex determination and gonad histology in pond sliders, trachemys scripta (reptilia: testudines: emydidae) david perpiñán1, albert martínez-silvestre2,3, ferran bargalló3, marco di giuseppe4, jorge orós5, taiana costa6,* 1 hospital for small animals, royal (dick) school of veterinary studies, university of edinburgh. easter bush campus, midlothian eh25 9rg, scotland, united kingdom 2 crarc, centre de recuperació d’amfibis i rèptils de catalunya, masquefa 08783, barcelona, spain 3 zoològic badalona veterinària, conquesta 74, badalona 08912, barcelona, spain 4 centro veterinario per animali esotici. viale regione siciliana sud-est 422-426, 90129, palermo, italy. 5 department of morphology, school of veterinary science, universidad de las palmas de gran canaria. arucas 35413, las palmas de gran canaria, spain. 6 division of infection and immunity, the roslin institute, university of edinburgh. easter bush campus, midlothian eh25 9rg, scotland, united kingdom. *corresponding author. e-mail: taiana.costa@roslin.ed.ac.uk submitted on 2015, 18th march; reviewed on 2015, 12th november; accepted on 2015, 4th december editor: sandra hochscheid abstract. coelioscopy has been proven to be a valuable technique to determine the sex of juvenile chelonians. however, there is a disagreement regarding the proper way to identify testes and ovaries, which is a direct consequence of the lack of studies correlating the results of endoscopic examination with the histology of the gonad. in this blinded study we assessed two methods of sex determination in juvenile pond sliders (trachemys scripta) using endoscopy: via coelioscopy, with visualization of the gonads, and via cloacoscopy, with visualization of phallus/clitoris; we then compared the results of these procedures with the histology of the gonad. the results of gonad histology correlated 100% with the results from coelioscopic examination, but only 57.77% of the results obtained by cloacoscopy were accurate. using cloacoscopy, 83.33% of males and 38.46% of females were misdiagnosed. sex determination of juvenile pond sliders was considered accurate when coelioscopy was used, but inaccurate when cloacoscopy with identification of phallus/clitoris was attempted. keywords. reptiles, turtles, chelonians, anatomy, diagnostic imaging, endoscopy, sex determination. most chelonians are long-lived animals where external/secondary sexual characteristics are only evident after several years of age (mushinsky et al., 1994). for this reason, coelioscopy has been proven to be a valuable technique to determine the sex of juvenile chelonians before they show secondary sexual characteristics (kuchling, 2006; hernandez-divers et al., 2009; kuchling and griffiths, 2012). however, there has been a disagreement about the proper identification of testes and ovaries in juvenile chelonians using coelioscopy (hernandez-divers et al., 2009; kuchling, 2009; innis 2012). this controversy arose from the lack of studies correlating the results of sex determination using endoscopic images with the histology of the gonad. we therefore designed a study to correlate the results of endoscopic sex determination in juvenile pond sliders (trachemys scripta) via coelioscopy (with visual inspection of the gonad) and cloacoscopy (with visual inspection of the phallus/clitoris) with the result of postmortem histological examination of the gonads. 92 david perpiñán et alii fifteen juvenile pond sliders, weighing 93-164 g (mean = 124.5 g) and measuring 7.5-9.9 cm (mean = 8.74 cm) of straight carapace length, were captured near the city of barcelona (ne spain) under the pest control project (permit number 002363 from the departament de medi ambient of the generalitat de catalunya). animals were maintained at 22 °c and fastened for 48 hours prior to the procedures. each animal was anaesthetised with alfaxalone (alfaxan, vetoquinol, spain) at 10 mg/ kg iv (subcarapacial vein) and injected with lidocaine sc (0.1 ml of a 1 mg/ml dilution) in the left prefemoral area. coelioscopies and cloacoscopies were performed in each animal using a 2.7 mm, 30o viewing rigid endoscope with a protective sheath and a videocamera (karl storz endoscopia ibérica sa, madrid, spain). briefly, for coelioscopic examination a 3-mm skin incision was performed in the centre of the prefemoral fossa and access to the coelomic cavity was performed separating the associated muscles; the endoscope was then inserted into the coelom and 10-20 ml of sterile saline (nacl 0.9%) was used to insufflate the coelomic cavity; the endoscope was directed dorsocaudally in order to inspect the reproductive system (divers et al., 2009). for cloacoscopic examination, the endoscope was inserted through the cloaca and directed cranially; 10-15 ml of air was used to insufflate the cloaca to visualize phallus/clitoris located in the ventral aspect of the proctodeum (spadola and insacco, 2009; selleri et al., 2013). following the procedures, turtles were not allowed to recover and were euthanased with an overdose of intravenous sodium thiopental (2 ml of tiobarbital braun, braun, spain). necropsies were performed immediately after death and the gonads were collected, fixed in 10% neutral buffered formalin, processed routinely for histology, stained with haematoxylin and eosin and assessed under light microscopy. this was a blinded study, where the veterinarian performing coelioscopies (dp) and the veterinarian performing cloacoscopies (am) did not know the results generated by each other; in addition, the pathologist (tc) did not know the results from coelioscopies and cloacoscopies. furthermore, videos of the endoscopic procedures (both coelioscopies and cloacoscopies) were recorded and submitted to two independent veterinarians with experience in chelonian anatomy (jo and mg) for additional blinded evaluations. the fleiss’ kappa statistical measure was used for assessing the reliability of agreement between observers (geertzen, 2012). histology was considered the gold standard for gonad identification. results are shown in table 1. gonad histology revealed that 13 turtles were female and two turtles were male. results from coelioscopies correlated 100% with the results from gonad histology, and the statistical analysis showed a perfect agreement (ϰ = 1) between observers. however, only 57.77% of the sexing results made via cloacoscopy were accurate, and the statistical analysis showed a poor agreement (ϰ = -0.324) between observers. using cloacoscopy, males were misdiagnosed as females in 83.33% of cases and females were misdiagnosed as males in 38.46% of cases. table 1. comparison of sex determination of 15 pond sliders (trachemys scripta) using coelioscopy, cloacoscopy and gonad histology. animal gonad histology celioscopy cloacoscopy observer 1 observer 2 observer 3 observer 1 observer 2 observer 3 1 ♀ ♀ ♀ ♀ ♀ ♀ ♂ 2 ♀ ♀ ♀ ♀ ♂ ♀ ♀ 3 ♀ ♀ ♀ ♀ ♀ ♀ ♂ 4 ♂ ♂ ♂ ♂ ♀ ♀ ♂ 5 ♀ ♀ ♀ ♀ ♂ ♀ ♂ 6 ♀ ♀ ♀ ♀ ♂ ♀ ♀ 7 ♀ ♀ ♀ ♀ ♂ ♀ ♀ 8 ♀ ♀ ♀ ♀ ♀ ♀ ♂ 9 ♂ ♂ ♂ ♂ ♀ ♀ ♀ 10 ♀ ♀ ♀ ♀ ♀ ♀ ♂ 11 ♀ ♀ ♀ ♀ ♂ ♀ ♀ 12 ♀ ♀ ♀ ♀ ♀ ♂ ♂ 13 ♀ ♀ ♀ ♀ ♀ ♂ ♂ 14 ♀ ♀ ♀ ♀ ♀ ♀ ♀ 15 ♀ ♀ ♀ ♀ ♀ ♂ ♀ 93endoscopy in turtles on coelioscopic examination, ovaries were long, irregular and translucent organs. they were loosely attached to the dorsal coelomic wall by long and loose ovarian ligaments on both poles of the organ (fig. 1a). ovarian previtellogenic follicles varied in density, size and appearance, from a few small, white to yellow round follicles embedded in the organ, to multiple prominent large yellow round follicles distributed through most of the parenchyma (fig. 1b). the different features of the ovarian follicles were not associated with body weight or carapace length. dark pigment and small vessels were appreciated in some ovaries (fig. 1c). adrenal glands were not always easily identified. the oviduct was a white, straight and tubular structure with small blood vessels on its surface. it ran parallel to and extended further cranially and caudally than the ovary, associated with the ovarian ligaments. it was observed either dorsally or ventrally to the ovary; occasionally, it was dorsal in the cranial part of the ovary and ventral in the caudal part. the diameter of the oviduct was variable, and sometimes it had a similar diameter as the ovary. testes were slightly oval, white to yellow, and had a net of blood vessels on the surface of the organ, irradiating from the area of the mesorchium (fig. 1d). the testicle was closely attached to the coelomic wall and only tight and short ligaments were visible on both poles of the organ (fig. 1e). a black and relatively diffuse structure (believed to be the epididymis) was closely associated with the mesenteric surface of the testicle. melanic spots were not found on the surface of testes. vas deferens was only seen in one animal (50% of males) as a thin and white straight tubular structure, only visible arising from the caudal aspect of the testicle. on cloacoscopic examination, phallus and clitoris had an identical appearance; they were heart-shaped structures (with the tip of the heart pointing caudally) situated on the ventral aspect of the proctodeum. both proctodeum and phallus/clitoris were pigmented (fig. 1f). appropriate description of gonads and associated structures are provided in the present article. it should be noted that interspecific differences in gonad morfig. 1. endoscopic images of ovary (a, b, c), testicle (d, e) and clitoris (f) in pond sliders (trachemys scripta). ovary (ov), oviduct (du), intestines (in), testicle (te), epididymis (ep), vas deferens (vd), kidney (ki), proctodeum (pr), clitoris (cl), and accessory vesicles (av). 94 david perpiñán et alii phology may exist; for example, images provided by hernandez-divers et al. (2009) showed melanic testes in chinese box turtles (cuora flavomarginata); testes with pink and orange colours have also been described in chelonians (kuchling, 2006); testes from aldabra tortoises (aldabrachelys gigantea) in a study by kuchling and griffiths (2012) were much more elongated that the ones observed in the present study with pond sliders. we observed variation in density, size, colour and shape of ovarian follicles in pond turtles of similar size and body weight. it has been described that follicles become more yellow, larger and more abundant as the animal ages (kuchling, 2006). in our study, sex identification of juvenile pond sliders by visualization of phallus/clitoris via cloacoscopy was unreliable and therefore this method is not recommended for sex identification. however, visualization of gonads via coelioscopy was 100% accurate at determining the sex of juvenile pond sliders and, therefore, it is recommended for sexing chelonians whose secondary sexual characteristics are not evident. the small number of males included in our study should be considered a potential source of bias. a different endoscopic technique through cloacoscopy and access to the urinary bladder or accessory urinary vesicles has been described for sex determination in adult european freshwater turtles (emys orbicularis) (spadola and insacco, 2009) and juvenile mediterranean tortoises (testudo hermanni) (selleri et al., 2013). this technique offers the advantage of being less invasive than coelioscopy, and further studies will need to assess efficacy and safety in juvenile pond sliders or other chelonian species. acknowledgments we would like to thank jordi grífols from zoològic badalona veterinària for his support to this study. references divers, s.j., stahl, s.j., camus, a. (2010): evaluation of diagnostic coelioscopy including liver and kidney biopsy in freshwater turtles (trachemys scripta). j. zoo. wild. med. 41: 677-687. geertzen, j. (2012): inter-rater agreement with multiple raters and variables. retrieved november 7, 2015, from https://nlp-ml.io/jg/software/ira/ hernandez-divers, s.j., stahl, s.j., farrell, r. (2009): an endoscopic method for identifying sex of hatchling chinese box turtles and comparison of general versus local anesthesia for coelioscopy. j. am. vet. med. assoc. 234: 800-804. innis, c.j. (2012): testes morphology in cuora flavomarginata: a response to kuchling and griffiths (2012; chelonian conservation and biology 11[1]). chelonian conserv. biol. 11: 273-274. kuchling, g. (2006): endoscopic sex determination in juvenile freshwater turtles, erymnochelys madagascariensis: morphology of gonads and accessory ducts. chelonian conserv. biol 5: 67-73. kuchling, g. (2009): differences among studies on sex identification on hatchling turtles. j. am. vet. med. assoc 235: 932-933 (letter answered by hernandezdivers and stahl). kuchling, g., griffiths, o. (2012): endoscopic imaging of gonads, sex ratios, and occurrence of intersexes in juvenile captive-bred aldabra tortoises. chelonian conserv. biol. 11: 91-96. mushinsky, h.r., wilson, d.s., mccoy, e.d. (1994): growth and sexual dimorphism of gopherus polyphemus in central florida. herpetologica 50: 119-128. selleri, p., di girolamo, n., melidone, r. (2013): cystoscopic sex identification of posthatchling chelonians. j. am. vet. med. assoc. 242: 1744-1750. spadola, f., insacco, g. (2009): endoscopy of cloaca in 51 emys trinacris (fritz et al., 2005): morphological and diagnostic study. acta herpetol. 4: 73-81. acta herpetologica vol. 11, n. 1 june 2016 firenze university press feeding habits of mesoclemmys vanderhaegei (testudines: chelidae) elizângela silva brito1,*, franco leandro souza2, christine strüssmann3 morphology, ecology, and behaviour of hylarana intermedia, a western ghats frog ambika kamath1, rachakonda sreekar2,3 a new account for the endangered cerrado rocket frog allobates goianus (bokermann, 1975) (anura: aromobatidae), with comments on taxonomy and conservation thiago ribeiro de carvalho1,2,*, lucas borges martins1,2, ariovaldo antonio giaretta1 molecular phylogenetics of the pristimantis lacrimosus species group (anura: craugastoridae) with the description of a new species from colombia mauricio rivera-correa, juan m. daza* population structure and activity pattern of one species of adenomera steindachner, 1867 (anura: leptodactylidae) in northeastern brazil maria juliana borges-leite1,*, joão fabrício mota rodrigues2, patrícia de menezes gondim1, diva maria borges-nojosa1 vocal repertoire of scinax v-signatus (lutz 1968) (anura, hylidae) and comments on bioacoustical synapomorphies for scinax perpusillus species group marco antônio peixoto1,*, carla silva guimarães1, joão victor a. lacerda2, fernando leal2, pedro c. rocha2, renato neves feio1 amendment of the type locality of the endemic sicilian pond turtle emys trinacris fritz et al. 2005, with some notes on the highest altitude reached by the species (testudines, emydidae) federico marrone*, francesco sacco, vincenzo arizza, marco arculeo photo-identification in amphibian studies: a test of i3s pattern marco sannolo1,*, francesca gatti2, marco mangiacotti3,4, stefano scali3, roberto sacchi4 no evidence for the ‘expensive-tissue hypothesis’ in the dark-spotted frog, pelophylax nigromaculatus li zhao, min mao, wen bo liao* no short term effect of clinostomum complanatum (trematoda: digenea: clinostomatidae) on survival of triturus carnifex (amphibia: urodela: salamandridae) giacomo bruni1,*, claudio angelini2 yeasts in amphibians are common: isolation and the first molecular characterization from thailand srisupaph poonlaphdecha, alexis ribas* introduction of eleutherodactylus planirostris (amphibia, anura, eleutherodactylidae) to hong kong wing ho lee1, michael wai-neng lau2, anthony lau3, ding-qi rao4, yik-hei sung5,* correlation between endoscopic sex determination and gonad histology in pond sliders, trachemys scripta (reptilia: testudines: emydidae) david perpiñán1, albert martínez-silvestre2,3, ferran bargalló3, marco di giuseppe4, jorge orós5, taiana costa6,* book review: john w. wilkinson. amphibian survey and monitoring handbook. pelagic publishing franco andreone book review: susan newman. frogs amphibians and their threatened environment. discovery and expression through art k-3. frogs are green franco andreone issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 7(2): 297-308, 2012 advertisement call of scinax littoralis and s. angrensis (amphibia: anura: hylidae), with notes on the reproductive activity of s. littoralis michel v. garey1, thais r.n. costa2, andré m.x. de lima2, luís f. toledo3, marília t. hartmann4 1 departamento de zoologia e botânica, instituto de biociências, letras e ciências exatas, universidade estadual paulista, brazil. corresponding author. e-mail: michelgarey@gmail.com 2 programa de pós-graduação em ecologia e conservação, universidade federal do paraná, brazil 3 museu de zoologia “prof. dr. adão josé cardoso”, universidade estadual de campinas, brazil 4 universidade federal da fronteira sul, campus de erechim, brazil submitted on: 2012, 15th may; revised on: 2012, 13th october; accepted on: 2012, 13th october. abstract. scinax littoralis and s. angrensis are poorly known atlantic forest endemic species from the scinax catharinae group, which is known from southern and southeastern brazil. herein, we describe the advertisement calls of these two species and compare them to other species within the s. catharinae group. additionally, we provide information on breeding sites, and calling activity in s. littoralis. advertisement calls of s. angrensis and s. littoralis are composed of multi-pulsed notes with multiple frequency bands and frequency modulation. scinax littoralis has a higher minimum and maximum frequency, and lower dominant frequency than s. angrensis, while scinax littoralis has a longer call. scinax littoralis appears to be a habitat generalist, occurring from secondary to mature forests, and breeding both in temporary and in permanent ponds. male s. littoralis were usually observed calling perched on shrub stems, and displayed satellite behavior when the male density was high. keywords. bioacoustics, breeding activity, satellite behavior, vocalization, treefrog. introduction vocalization is the most widespread means of communication for frogs, especially for mate attraction (ryan, 2001). call types are usually categorized into mating or advertisement, territorial, release, distress, and warning calls (bogert, 1960), but for several species, one signal type may communicate many things to conspecifics (wells, 2007). the advertisement call is mainly a reproductive signal, but may also provide information about spe298 michel v. garey et al. cies recognition or territory defense (ryan, 2001; wells, 2007). because frogs have species-specific calls, the study of acoustic signals can be useful for researchers: field research dependent on auditory surveys (e.g., line transects, zimmerman, 1993), or research focusing on behavior (bee et al., 1999), natural history (lima et al., 2010) or taxonomic goals (e.g., cocroft and ryan, 1995; pombal et al., 1995; toledo et al., 2007) can all capitalize on differences in calls among species to gain information relevant to a specific study. both scinax littoralis and s. angrensis belong to s. catharinae group, which is within the s. catharinae clade (faivovich, 2002). both species are endemic to the brazilian atlantic forest and are morphologically similar (cryptic species) and apparently allopatric. however, their life history and ecology remain poorly known. the distribution of s. littoralis ranges from southern portion of the state of são paulo (pombal and gordo, 1991) to the state of paraná (conte et al., 2009) and s. angrensis may be found from the state of rio de janeiro (carvalho-e-silva et al., 2008) to the northeastern portion of the state of são paulo (araújo et al., 2009). the reproductive season of s. littoralis is prolonged, with breeding occurring throughout the year (narvaes et al., 2009). males of this species usually call from high perches over permanent ponds (narvaes et al., 2009). females display two distinct reproductive modes: eggs may be deposited in ponds or in bromeliad leaf axils, and females have been observed depositing eggs in temporary ponds (toledo et al., 2012). scinax angrensis males have been recorded calling around temporary and permanent ponds and around stream zones (carvalho-e-silva et al., 2008). one reproductive mode was observed: females lay eggs in ponds (hartmann et al., 2010). herein, we describe the advertisement calls of both s. littoralis and s. angrensis and provide novel data on the reproductive and calling behavior of s. littoralis. these calls are compared to other closely related species, which may provide insight into relationships within the group. material and methods call description the advertisement call of s. littoralis was recorded in candonga village (25⁰35’26”s, 48⁰48’47”w, 230 m a.s.l.), in the municipality of morretes (fig. 1), state of paraná, in november 2010, at 2230h – 0100h (air temperature = 23 °c). the advertisement calls of this species were recorded with a tascam dr-08 digital recorder with external microphone seinnheser me66. the advertisement call of s. angrensis was recorded in the serra do mar state park (23°21’53”s, 44°49’27”w, 50 m a.s.l.), municipality of ubatuba (fig. 1), state of são paulo, in january 1994, at 2000h-2130h (air temperature = 23.5°c). the advertisement calls of this species were recorded with a nagra-e tape recorder with an external microphone seinnheser me66. calls were digitalized and analyzed in raven pro 1.3 (cornell bioacoustics research program) at 16 bits, 44 khz, a fft frame length of 512 samples, and hann window type. the brightness was adjusted to 68%, the contrast was adjusted to 80%, and overlap was adjusted to 50%. the terminology used in these descriptions follows toledo and haddad (2005a). for s. littoralis, 21 advertisement calls of three males were analyzed (n = 75 notes), and for s. angrensis 17 calls of two individuals were analyzed (n = 43 notes). we analyzed the number of notes per call, number of frequency bands, call duration, note duration, number of pulses per note, pulse rate, minimum, maxi299advertisement call of scinax mum and dominant frequency, inter-note interval and call rate. to analyze the difference in internote intervals between s. littoralis advertisement calls, and to determine if there were differences in call parameters among s. littoralis and s. angrensis we used a student’s t-test. all variables were also tested for normality and variance homogeneity statistical analysis. data are presented as mean ± standard deviation. the advertisement calls of scinax littoralis (fnjv 12855-12862) and s. angrensis (fnjv 12863) were deposited in fonoteca neotropical “jacques vielliard”, instituto de biologia, universidade estadual de campinas, campinas, state of são paulo, brazil. voucher specimens of s. littoralis were deposited in the coleção herpetológica of the museu de zoologia universidade de são paulo, são paulo, brazil (mzusp 137947-48). we collected individuals from the same population of s. angrensis, however, we did not collect the specimens from which the advertisement calls were recorded. the specimens of the population of s. angrensis from serra do mar state park, nucleus picinguaba, were deposited in the amphibian collection of célio f. b. haddad, departamento de zoologia, instituto de biociência, são paulo state university, rio claro, brazil (cfbh 7912-7916). fig. 1. map of southern brazil showing the municipalities of species type-localities and study sites. white circle: scinax angrensis type-locality; black circle: s. angrensis study-site; black square: scinax littoralis type-locality; white and gray square: s. littoralis study-sites. 300 michel v. garey et al. reproductive activity of s. littoralis the reproductive behavior of s. littoralis was studied at reserva natural salto morato (rnsm; 25º09’s; 48º16’-48º20’w), in the municipality of guaraqueçaba (fig. 1), on the north coast of the state of paraná, southern brazil. rnsm is composed of a mosaic of landscapes due to its land use history. the protected area cover comprises ~2.500 ha of atlantic forest, with altitude ranging from 25 to 930 m a.s.l. in this region, the climate according to the köppen-geiger classification (peel et al., 2007) is of the type cfa: temperate climate with no dry season, and an average annual temperatures of around 21 °c. annual rainfall is elevated; with more than 2,000 mm y-1 and mean relative humidity is 85% (maack, 2002). we sampled nine lentic water bodies, one per night, using the visual and acoustic survey at breeding sites technique (scott and woodward, 1994). data were collected between september 2006 and april 2007. surveys generally began at 1900 h and were finished at midnight. we searched for frogs at swamps, temporary and permanent ponds in early secondary forest, late secondary forest and mature forest (see gatti, 2000), three water bodies in each successional forest stage. the water bodies were located between 20 to 200 m a.s.l. for each male found calling, we measured the perch height and the horizontal distance to the margin of pond. in each survey area, the number of calling males was estimated by counting all individuals in each surveyed area by walking around the perimeter of each breeding site (vasconcelos and rossa-feres, 2005). males were observed for approximately one hour per night using animal focal sampling (altmann, 1974). differences in perch height and distance to ponds margin among the forest succession stages were analysed using anova. differences among treatments were determined using the tukeykramer hsd test. results call description the advertisement call of s. angrensis is composed by 1-7 multi-pulsed notes that present multiple frequency bands and an irregular frequency modulation (table 1). the mean duration of the advertisement call is 0.42 ± 0.1 s, the mean note duration is 0.025 ± 0.012 s (first note = 0.025 ± 0.009 s, second note = 0.034 ± 0.011 s, third note = 0.015 ± 0.011 s, fourth note = 0.023 ± 0.024 s, fifth note = 0.034 s, sixth note = 0.005 s, and seventh note = 0.009 s), with 17.88 ± 5.18 pulses per note (first note = 12.6 ± 0.009, second note = 14.4 ± 5.6, third note = 8.7 ± 8.9, fourth note = 10.5 ± 12, fifth note = 15, sixth note = 4, and seventh note = 6 pulses per note). the inter-note interval was 50.13 ± 15.74 ms (23-76, n = 23). notes have 2-4 frequency bands (fig. 2). the minimum frequency of the fundamental band is 1.67 ± 0.23 khz, the maximum frequency of the highest band is 4.51 ± 0.69 khz, while the dominant frequency is 3.13 ± 0.37 khz, which is the second band (first note = 3.29 ± 0.33 khz, second note = 3.39 ± 0.23 khz, third note = 3.17 ± 0.45 khz, fourth note = 3.01 ± 0.24 khz, fifth note = 2.76 khz, sixth note = 2.41 khz, and seventh note = 3.49 khz). the pulse rate is 575 ± 7.5 pulses/s, and pulse rate was higher in the middle of the notes. the call rate was 16 calls/min, and note rate was 0.072 note/s. the last note tends to be shorter than the others when the call is composed of three or more notes. the advertisement call of s. littoralis consists of 3-4 multi-pulsed notes, with 3-10 simultaneously frequency bands (table 1). the mean duration of the advertisement call is 301advertisement call of scinax ta bl e 1. c al l p ar am et er s of t he s ci na x ca th ar in ae s pe ci es g ro up . n n: n um be r of n ot es /c al l; d ur at io n: d ur at io n of t he a dv er tis em en t ca ll; p ul se s/ no te : n um be r of p ul se s pe r no te ; p ul se s/ s: r at e of p ul se s pe r se co nd ; f m ax : m ax im um fr eq ue nc y; f m in : m in im um fr eq ue nc y; f do m : d om in an t f re qu en cy ; s tr uc : s tr uc tu re o f ca ll, p re se nc e of p ul se s = p an d/ or p re se nc e of h ar m on ic s = h ; n fb = n um be r of fr eq ue nc y ba nd s. d at a ar e sh ow n as : m ea n ± st an da rd d ev ia tio n (r an ge , n ). n n d ur at io n (s ) pu ls es /n ot e pu ls es /s fm ax ( kh z) fm in ( kh z) fd om ( kh z) st ru c n fb r ef er en ce sc in ax a ng re ns is (1 -7 ) 0. 42 ± 0 .1 (0 .1 70. 7, n = 43 ) 17 .8 8 ± 5. 18 (7 -2 8, n = 3 3) 43 4. 65 ± 6 3. 28 (2 89 .4 754 2. 86 , n = 33 ) 4. 51 ± 0 .6 9 (3 .1 25. 75 ; n = 75 ) 1. 67 ± 0 .2 3 (1 .1 12. 13 ; n = 75 ) 3. 13 ± 0 .3 7 (2 .1 53. 7, n = 42 ) p/ h 28 pr es en t s tu dy s. li tt or al is (3 -4 ) 0. 55 ± 0 .1 2 (0 .2 10. 79 ; n = 75 ) 23 .6 8 ± 2. 73 (8 -3 0, n = 3 7) 46 2. 49 ± 12 9. 43 ( 31 4. 29 10 00 , n = 3 7) 4. 65 ± 0 .7 9 (2 .8 75. 57 , n = 42 ) 2. 2 ± 0. 31 (1 .4 72. 79 , n = 42 ) 2. 65 ± 0 .2 9 (1 .8 93. 53 ; n = 75 ) p/ h 310 pr es en t s tu dy s. a gi lis ( no te “ a” ) 2 0. 39 ± 0 .7 6 (0 .3 80. 40 , n = 12 ) 85 .6 7 ± 5. 07 (7 496 , n = 1 2) 7. 66 ± 0 .1 8 (7 .4 57. 92 , n = 12 ) p 0 n un es e t a l., 2 00 7 s. a gi lis ( no te “ b” ) 2 2. 5 ± 5. 3 (1 .0 63. 2, n = 10 4) 7. 15 ± 1 .2 2 (5 -1 1, n = 1 04 ) 7. 02 ± 0 .4 8 (5 .6 -7 .8 8, n = 10 4) p 0 s. a lb ic an s 1 0. 7 4. 2 2. 8 (3 .3 -4 .1 ) p/ h po m ba l e t a l., 1 99 5* s. a rg yr eo rn at us (5 -2 80 ) (0 .8 -2 5) (2 -2 5) 9. 0 3. 6 (5 .0 -6 .3 ) p 0 po m ba l e t a l., 1 99 5 s. c an as tr en si s (6 -7 ) 5. 6 1. 4 p 0 c ar do so a nd h ad da d, 19 82 s. c at ha ri na e > 2 3. 1 2. 2 p/ h po m ba l e t a l., 1 99 5* s. c en tr al is 2 0. 37 ± 0 .0 6 (n = 4 ) 6. 2 2. 8 (3 .2 -4 .6 ) p 0 po m ba l a nd b as to s, 19 96 s. h ie m al is 2 7. 0 2. 0 (2 .2 -4 .3 ) p/ h (2 -4 ) h ad da d an d po m ba l, 19 87 s. m ac ha do i (6 -7 ) 0. 05 5. 2 0. 15 3. 5 h b ok er m an n an d sa zi m a, 1 97 3 s. r an ki (4 -5 ) 5. 5 1. 5 p 0 a nd ra de a nd c ar do so , 19 87 s. r iz ib ili s (7 -2 3) (1 .2 92. 74 ) (1 572 ) 5. 5 2. 0 (2 .8 -4 .0 ) p 0 po m ba l e t a l., 1 99 5 s. s ka io s (4 273 ) (4 .4 27. 9) 9. 2 ± 3. 33 (5 -1 6, n =1 0) (2 .2 12. 24 ) p 0 po m ba l e t a l., 2 01 0 * d at a of o th er a ut ho rs p re se nt ed b y po m ba l e t a l. 19 95 . 302 michel v. garey et al. 0.55 ± 0.12 s, the mean note duration is 0.050 ± 0.013 s (first note = 0.054 ± 0.011 s, second note = 0.053 ± 0.001 s, third note = 0.047 ± 0.016 s, and fourth note = 0.023 ± 0.001 s), with 23.68 ± 2.73 pulses per note (first note = 21.9 ± 4.17, second note = 22.43 ± 2.11, third note = 18.53 ± 6.81, fourth note = 7 ± 1 pulses per note). the minimum frequency of the fundamental band is 2.20 ± 0.31 khz, the maximum frequency of the highest frequency band is 4.65 ± 0.79 khz, whereas the dominant frequency is 2.65 ± 0.29 khz, which is the third band (first note = 2.58 ± 0.26 khz, second note = 2.72 ± 0.20 khz, third note = 2.67 ± 0.20 khz, and fourth note = 2.47 ± 0.19 khz). the first two notes were simifig. 2. waveform (above) and spectrogram (below) of the advertisement calls of s. angrensis (air temperature = 23.5 °c), and scinax littoralis (air temperature = 23 °c). 303advertisement call of scinax lar in irregular frequency modulation. the third note varied: in some cases, it was modulated like the first two notes, while in others it was of relatively constant frequency. the pulse amplitude increased across the note. the interval between the first and second notes was 41.69 ± 9.43 ms (24-66; n = 51), and the interval between the third and fourth note was 43.22 ± 8.33 ms (27-63; n = 23). the call rate was 6.37 ± 2.73 call/min (4.8-9.52; n = 3), and note rate was 0.082 note/s. the advertisement call of s. angrensis and s. littoralis present similarities in the structure but some parameters differed. scinax littoralis has a greater call length (t = 5.97; p = 0.0001, df = 120), note length (t = 9.49; p = 0.0001; df = 120), and number pulses per note (t = 7.20; p = 0.0001; df = 120), than s. angrensis. however, s. angrensis has a higher dominant frequency (t= 4.88; p = 0.0001; df = 120), and lower minimum frequency (t = -10.86; p = 0.0001; df = 120), maximum frequency (t = 2.32; p = 0.022; df = 120), in relation to s. littoralis. these species did not differ in relation to notes per call (t = -0.06; p = 0.949; df = 120), and number of frequency bands (t = 1.38; p = 0.069; df = 120). fig. 3. horizontal distance from the water of the perches used for calling by males scinax littoralis in three vegetation succession stage sites in the reserva natural salto do morato, municipality of guaraqueçaba, state of paraná, brazil. positive values indicate that a calling site was located outside of the breeding pool; negative values indicate that a calling site was located within a pool; zero value is the margin of the pool. dots indicate the means and bars indicate standard deviation. early = early secondary forest; late = late secondary forest; old-growth = old growth forest. 304 michel v. garey et al. reproductive activity of s. littoralis scinax littoralis were found calling in every forest type sampled and bred in both temporary and permanent ponds. males were found calling perched on shrubs ranging from 23-220 cm above the ground (69.73 ± 33.35 cm, n = 49) located 0-308 cm horizontally from pond edges (88.37 ± 95.83 cm, n = 49). the height of the perch used by s. littoralis was the same in all forest stages (f(2, 48) = 2.37, p = 0.106). however, perches used in the mature forest were farther from the water in comparison to the others (fig. 3; f(2, 48) = 6.50, p = 0.003). scinax littoralis calls during the night and may exhibits a satellite-male strategy (n = 5). satellite behavior was observed at nights with density of calling males > 20 males per pond, in the late secondary forest and mature forest. satellite males had a distance of approximately 19 ± 3 cm (n = 3) from the calling male. satellite males were standing still near the calling male, not in low posture, however, we did not record any satellite male attempting to intercept gravid females. discussion multi-pulse structure, common to both s. littoralis and s. angrensis, is widespread within the genus scinax (e.g., haddad and pombal, 1987; pombal et al., 1995; present study). other call features common in s. catharinae group are present in the calls of these two species, such as short note lengths and harmonic structure (pombal et al., 1995). the presence of harmonics in species of the scinax catharinae group may be questionable (see vielliard, 1993). in our results it is possible to identify different frequency bands, however, only two of it would be integer multiples of the fundamental frequency, while the other seems as additional frequency modulation. therefore, some of these frequency bands may be sidebands (vielliard, 1993). despite of its origin, multiple frequency band structure in the advertisement call is common among the congeneric species (e.g., haddad and pombal, 1987). anyway, despite morphological and ecological similarities between s. littoralis and s. angrensis, the differences among the call features of those species are evident, especially regarding the dominant frequency level and call rate. few species in the s. catharinae group have higher dominant frequencies than scinax angrensis, such as s. agilis (nunes et al., 2007), and s. argyreornatus (pombal et al., 1995). these species can be distinguished from s. angrensis by the absence of multiple frequency bands. the advertisement call of s. angrensis has more notes per call than s. albicans (pombal et al., 1995), s. centralis (pombal and bastos, 1996), and s. hiemalis (haddad and pombal, 1987), although s. angrensis has lower number of notes per call than s. argyreornatus (pombal et al., 1995), s. canastrensis (cardoso and haddad, 1982), s. machadoi (bokermann and sazima, 1973), scinax rizibilis (pombal et al., 1995), and s. skaios (pombal et al., 2010). scinax angrensis has a higher minimum frequency than s. machadoi as well, s. machadoi has the shortest call duration documented in the group (pombal et al., 1995). scinax angrensis has a shorter call than do s. skaios (pombal et al., 2010), s. catharinae, s. rizibilis, and s. argyreornatus (pombal et al., 1995). scinax angresis differs from s. ranki by harmonic structure of advertisement call. for some species in this group 305advertisement call of scinax (e.g., s. catharinae) no full description of advertisement calls exists, thus we are unable to compare their calls to others in the s. catharinae group (table 1). some species in the s. catharinae group have higher dominant frequencies than scinax littoralis, such as s. agilis (nunes et al., 2007), s. albicans (pombal et al., 1995), and s. argyreornatus (pombal et al., 1995). most species have a similar range of dominant frequency (table 1). scinax littoralis can be distinguished from s. argyreornatus, s. rizibilis (pombal et al., 1995), and s. skaios (pombal et al., 2010) by the presence of multiple frequency bands, lower number of notes per call, and call length. scinax littoralis has higher call duration than s. centralis (pombal and bastos, 1996), s. catharinae, and s. machadoi (pombal et al., 1995), although s. littoralis has lower call duration than s. albicans (pombal et al., 1995). advertisement call of s. ranki has no multiple frequency bands (andrade and cardoso, 1987). scinax hiemalis has higher maximum frequency than s. littoralis (haddad and pombal, 1987). scinax littoralis also has fewer notes per call than s. canastrensis (cardoso and haddad, 1982), and s. machadoi (bokermann and sazima, 1973). as already know for the genus (e.g., toledo and haddad, 2005b; abrunhosa et al., 2006), scinax littoralis either exhibits a prolonged breeding season (sensu wells, 1977a). the prolonged breeding season is common among tropical amphibians (wells, 2007), and, despite our study site is at a subtropical zone, it is still likely the pattern already observed for s. littoralis in the type locality (estação ecológica juréia-itatins, narvaes et al., 2009). males of this species may adopt satellite strategy. satellite males are common within the most neotropical amphibian families (e.g., haddad, 1991; barreto and andrade, 1995; pombal and haddad, 2005). four hypotheses, not mutually exclusive, explain satellite behavior in amphibians: 1) satellite males are sexual parasites that try to mate with females attracted by their calling neighbor (wells, 1977b); 2) satellite males are waiting for a change in the physical condition of the dominant male, due to the high energetic cost of calling (bevier, 1997; pombal and haddad, 2005); 3) satellite males are inferior or subordinate males waiting for a calling site to be vacated by the dominant male when he enters amplexus (wells, 1977b); 4) the satellite strategy can decrease predation risk by acoustically oriented predators (e.g., tuttle et al., 1981). we observed satellite behavior only in high densities, as suggested by gerhardt and klump (1988). since we did not observe any female interception attempt by satellite males (hypothesis 1), we suggest that the other three hypotheses are therefore more likely to happen in this species. although s. littoralis appears to be constrained to the forest habitats, breeding activity seems to be independent of the successional stage of the forest and of pond stability. perch heights did not vary between areas, but males vocalized farther from the margin of the water in mature forest. these differences in calling site may be related to the availability of bromeliads around the margin of the spawning site (see toledo et al., 2012): and bromeliads were most abundant in mature forest, including around the margins of breeding sites. we found low abundance of bromeliads in late secondary forest, and none in early secondary forest. we present here data on the natural history of two endemic species of atlantic forest of brazil, which is an endangered biome with lacks on the life history knowledge of most amphibian species. the calling comparisons presented here aid in easy distinction among some species of the s. catharinae group in further surveys or to study the ecology and the evolution of the of advertisement call for the reproduction of scinax clade. 306 michel v. garey et al. acknowledgements we are indebted to kristine kaiser for helpful suggestions on the manuscript and for reviewing the english. m.v. garey thanks the fundação grupo o boticário de proteção à natureza (agreement fbpn-ufpr 623) for financial support and access to the study area. we thank ibama for assistance and for our collection permit (no. 107/06-ran), and programa de pós graduação em ecologia e conservação of the universidade federal do paraná for general support. m.t. hartmann thanks capes for financial support through the programa de apoio a projetos institucionais com a participação de recém-doutores (prodoc). during the preparation of this paper m.v. garey was supported by fapesp fellowship (processes: 2008/50575-1) and unesp/prope (edital 05/2012). l.f. toledo thanks fapesp for a grant and scholarship. references abrunhosa, p.a., wogel, h., pombal jr., j.p. 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(1993): audio strip transects. in: measuring and monitoring biological diversity. standard methods for amphibians, pp. 92-97. heyer, w.r., donnelly, m.a., mcdiarmid, r.w., hayek, l.a.c., foster, m.s., eds, smithsonian institution, washington. acta herpetologica vol. 7, n. 2 december 2012 firenze university press advertisement call of species of the genus frostius cannatella 1986 (anura: bufonidae) flora a. juncá1, david l. röhr2, ricardo lourenço-de-moraes3, flávio j. m. santos1, airan s. protázio1, ednei a. mercês1, mirco solé4 amphibians in southern apennine: distribution, ecology and conservation notes in the “appennino lucano, val d’agri e lagonegrese” national park (southern italy). antonio romano1,*, remo bartolomei1, antonio luca conte1, egidio fulco2 the significance of using satellite imagery data only in ecological niche modelling of iberian herps neftalí sillero1, josé c. brito2, santiago martín-alfageme3, eduardo garcía-meléndez4, a.g. toxopeus5, andrew skidmore5 reproductive strategy of male and female eastern spiny lizards sceloporus spinosus (squamata: phrynosomatidae) from a region of the chihuahuan desert, méxico aurelio ramírez-bautista1,*, barry p. stephenson2, xóchitl hernández-ibarra1, uriel hernández-salinas1, raciel cruz-elizalde1, abraham lozano1, and geoffrey r. smith3 morphological variability of the hermann’s tortoise (testudo hermanni) in the central balkans katarina ljubisavljević1, georg džukić1, tanja d. vukov1, miloš l. kalezić1,2 the usefulness of mesocosms for ecotoxicity testing with lacertid lizards maria josé amaral1,2,*, rita c. bicho1, miguel a. carretero2, juan c. sanchez-hernandez3, augusto m. r. faustino4, amadeu m. v. m. soares1, reinier m. mann1,5 does acclimation at higher temperatures affect the locomotor performance of one of the southernmost reptiles in the world? jimena b. fernández*, nora r. ibargüengoytía advertisement call of scinax littoralis and s. angrensis (amphibia: anura: hylidae), with notes on the reproductive activity of s. littoralis michel v. garey1, thais r. n. costa2, andré m. x. de lima2, luís f. toledo3, marília t. hartmann4 book review: marine s. arakelyan, felix d. danielyan, claudia corti, roberto sindaco, alan e. leviton 2011. herpetofauna of armenia and nagorno-karabakh. society for the study of amphibians and reptiles stefano scali book review: rafaqat masroor 2012. a contribution to the herpetofauna of northern pakistan stefano scali evidence of high longevity in an island lacertid, teira dugesii (milne-edwards, 1829). first data on wild specimens. j. jesus first assessment of the endoparasitic nematode fauna of four psammophilous species of tropiduridae (squamata: iguania) endemic to north-eastern brazil markus lambertz1,*, tiana kohlsdorf2, steven f. perry1, robson waldemar ávila3, reinaldo josé da silva4 rediscovery and redescription of the holotype of lygosoma vittigerum (= lipinia vittigera) boulenger, 1894 yannick bucklitsch1, peter geissler1, timo hartmann1, giuliano doria2 , andré koch1,* reproductive phenology of the tomato frog, dyscophus antongili, in an urban pond of madagascar’s east coast ori segev1,*, franco andreone2, roberta pala2, giulia tessa2, miguel vences1 range extension of the critically endangered true poison-dart frog, phyllobates terribilis (anura: dendrobatidae), in western colombia roberto márquez1,*, germán corredor2, carlos galvis3 daniel góez2, & adolfo amézquita1 differences in habitat use of two sympatric species of ameiva in east costa rica esther sebastián-gonzález1, ramón gómez2 acta herpetologica journal of the societas herpetologica italica acta herpetologica 11(1): 69-73, 2016 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-15529 no evidence for the ‘expensive-tissue hypothesis’ in the dark-spotted frog, pelophylax nigromaculatus li zhao, min mao, wen bo liao* key laboratory of southwest china wildlife resources conservation (ministry of education), china west normal university, nanchong 637009, p. r. china. *corresponding author. e-mail: liaobo_0_0@126.com submitted on 2015, 10th february; revised on 2015, 9th november; accepted on 2016, 15th january editor: giovanni scillitani abstract. increased brain size significantly contributes to the performance and fitness of organisms. the expensive-tissue hypothesis (eth) based on studies of the correlation between brain size and size of the other energetically costly organs in mammals predicts that energy investment increased in one energetically costly tissue necessitates a decrease of investments in other costly tissues. here, we test this hypothesis in an ectothermic species, the dark-spotted frog, pelophylax nigromaculatus. we found that relative brain size was not correlated with relative sizes of testes, heart, liver, spleen, kidneys or limb muscles within each sex. moreover, we also failed to find significantly negative correlation among the expensive organs (i.e., testes, heart, liver, spleen, kidneys or limb muscles) in this frog. however, we observed a significantly positive correlation between liver residuals and kidney residuals. our finding suggests that energetic costs of one expensive tissue do not direct necessarily affect the investment in another expensive tissue, but rather may scatter its effect on all other expensive tissues. keywords. pelophylax nigromaculatus, brain size, expensive-tissue hypothesis. brain size is an important characteristic that affects the performance and fitness of organisms (allman, 2000; liao et al., 2015a). brain size is often used as an indicator of the brain’s evolutionary development state in response to cognitive benefits (see review, striedter, 2005). previous studies have indicated that variations in brain size can be explained by the social brain hypothesis, the expensive-tissue hypothesis (eth) and the sexual selection hypothesis (aiello and wheeler, 1995; pitnick et al., j 2006; dunbar and shultz, 2007; barton and capellini, 2011). however, the brain needs more energy per unit weight than the other somatic tissues, making it metabolically expensive (mink et al., 1981). according to the eth, investment in one metabolically costly tissue requires a decrease of investments in other tissues (aiello and wheeler, 1995). in the past two decades, the eth has received mixed support in vertebrates (aiello and wheeler, 1995; jones and maclarnon, 2004; isler and van schaik, 2006; pitnick et al., 2006; barrickman and lin, 2010; navarrete et al., 2011). some studies find that investment in a metabolically expensive tissue is negative significantly correlated with the other metabolically expensive tissues, thus supporting the eth (aiello and wheeler, 1995; kaufman et al., 2003; isler and van schaik, 2006; barrickman and lin, 2010; jin et al., 2015; tsuboi et al., 2015). however, other studies found that there were either non-significant or even positive correlations between metabolically costly tissues (isler and van schaik, 2006; lemaître et al., 2009; barrickman and lin, 2010; navarrete et al., 2011; liu et al., 2014). most studies of the eth have been performed among species, though some intraspecific studies have been conducted (fish, warren and iglesias, 2012; liu et al., 2014; frogs, jin et al., 2015). however, no consistent results have confirmed the patterns of intraspecific variation in brain size, and the eth in anurans need further study. in this 70 li zhao et alii study, we tested the eth in the dark-spotted frog pelophylax nigromaculatus, a species widely distributed in plains, hilly paddy fields, ponds, lakes, rivers and mountains at an altitude below 2200m (fei and ye, 2001). previous studies of the eth have recognized brain, heart, kidneys, liver, testes and gut tissues as being metabolically costly (aiello and wheeler, 1995; pitnick et al., 2006; isler and van schaik, 2006; barrickman and lin, 2010; navarrete et al., 2011; warren and iglesias, 2012). our aim was to explore whether brain size was negatively correlated with any other organs (i.e., heart, liver, spleen, kidneys, testes, and limb muscles mass) in p. nigromaculatus. we captured specimens by hand in ponds in yingxi town of nanchong city (30°50’n, 106°07’e, 338 m a.s.l.) in sichuan, china (mao et al., 2014). all individuals were caught at night during the breeding season from april 11 to 19 in 2010. we collected a total of 84 individuals (45 males and 39 females), and then brought them to the laboratory. the sex was determined by observing the differences in secondary sex characteristics. before processing, the frogs were kept in rectangular tanks (1.0*0.5*0.4 m; l*w*h) with a water depth of 5 cm at room temperature. animals were sacrificed by using double-pithing. we measured body size (snout to vent length, svl) to the nearest 0.01 mm by a caliper, and body mass to the nearest 0.1 mg by an electronic balance. frogs were dissected and all tissues (i.e., brains, livers, hearts, spleen, kidneys, testes, and limb muscles) were removed and weighed. we analyzed the male and female data separately. all data were log-transformed prior to analysis. in order to control the effect of body mass on energetically expensive tissues, we used relative size of expensive tissues to analyze correlations between brain size and the other organs. the relative size of the expensive tissue was the residual of observed expensive tissues size to that predicted on the basis of the regressions of brain, heart, spleen, kidneys, liver, testes mass and limb muscles on body mass. we estimated body condition using the residuals from a regression of body mass on svl. some tissues were affected by body condition. if the effect of body condition on each of the expensive tissues was significant, we then used a partial analysis and body condition as a covariate to test relationships between brain mass and each of the expensive tissues. statistical tests were parametric, using type iii sums of squares tests with the spss (22.0) statistical package. in the male dark-spotted frog, p. nigromaculatus, the results showed that brain residuals did not negatively correlate with residuals of testes, heart, liver, spleen, kidneys or limb muscles (table 1). for other tissues (testes, table 1. regressions of brain mass residuals and body condition on other organ size residuals in male pelophylax nigromaculatus. coefficient estimates from regressions are given with 95% ci in brackets, and β and p-values associated with each regression are also provided. organ size brain mass body condition estimates [±95% ci] β p-value estimates [±95% ci] β p-value testes 0.010[-0.205,0.224] 0.015 0.927 0.017[-0.082,0.115] 0.056 0.731 heart -0.051[-0.388,0.286] -0.047 0.761 0.111[-0.265, 0.043] -0.217 0.152 liver 0.086[-0.223,0.394] 0.085 0.578 0.104[-0.038,0.055] 0.220 0.147 spleen -0.027[-0.144,0.090] -0.071 0.644 -0.009[-0.064,0.046] -0.052 0.733 kidneys 0.158[-0.158,0.473] 0.152 0.320 -0.003[-0.153,0.146] -0.007 0.965 limb muscles 0.428[-0.324,1.179] 0.172 0.257 0.048[-0.309,0.405] 0.041 0.788 table 2. regressions of brain mass residuals and body condition on other organ size residuals in female pelophylax nigromaculatus. coefficient estimates from regressions are given with 95% ci in brackets, and β, p-values associated with each regression are also provided. organ size brain mass body condition estimates [±95% ci] β p-value estimates [±95% ci] β p-value heart -0.080[-0.393,0.239] -0.085 0.605 -0.292[-0.308,-0.277] -0.252 0.121 liver 0.050[-0.193,0.293] 0.069 0.678 -0.083[-0.238,0.073] -0.175 0.287 spleen 0.012[-0.111,0.135] 0.032 0.847 -0.063[-0.140,0.014] -0.264 0.104 kidneys 0.215[-0.058,0.489] 0.254 0.119 -0.023[-0.206,0.161] -0.041 0.804 limb muscles 0.063 [-0.591,0.716] 0.032 0.847 -0.370[-0.775,0.036] -0.290 0.073 71the expensive-tissue hypothesis lacking in a frog liver, spleen, kidneys, limb muscles), we found only one significant positive correlation between the liver residuals and the kidney residuals (fig. 1a). we found the same relationships between metabolically expensive tissues in females (table 2, fig. 1b). the results of our study did not find a significant correlation between body condition and other organs either in males or females (table 1, table 2). however, a significantly positive correlation between liver residuals and kidney residuals still existed when controlling for the effect of body condition (male: r = 0.442, p = 0.005; female: r = 0.527, p < 0.001). the expensive tissue hypothesis states that  organisms can reduce the size of other expensive tissues in their body to maintain a relatively larger brain size (aiello and wheeler, 1995). previous most convincing studies in favor of the expensive tissue hypothesis result from ectothermic animals such as the elephant nose fish gnathonemus petersii (kaufman et al., 2003), or the guppy poecilia reticulata (kotrschal et al., 2013), 73 species of lake tanganyika cichlids (tsuboi et al., 2015), the omei wood frog rana omeimontis (jin et al., 2015). in contrast to the eth, we did not find clear evidence to support this hypothesis in p. nigromaculatus. there is a similar study investigated eth in a fish which did not find support for eth (liu et al., 2014). the energy trade-off hypothesis predicts that organisms, in order to maintain relatively larger brain, will reduce investment in reproduction (isler and van schaik, 2006). pitnick et al. (2006) found significantly negative correlations between investment in testes and brains in bats, supporting this theory. however, we did not find a negative correlation between the size of the brain and testes in the dark-spotted frog. there are two possible evolutionary paths to fuel energetic requirements for brain enlargement in animals: (i) increase overall energy budget and (ii) re-allocate energy budget. the metabolic constraints hypothesis concerns the first possibility, and eth and the energy trade-off hypothesis concern the second possibility. the lack of support for the eth found in this study suggest that energetic constraints operates in frog brain size evolution, despite copious evidence that brain tissue is metabolically costly to develop and maintain in another frog (jin et al., 2015). this difference in the eth between the two frogs’ species might relate to difference in their habitat use (fei and ye, 2001). muscle tissue can consume a considerable proportion of the organism’s energy at rest and it must therefore be included in the eth (aiello and wheeler, 1995). for amphibians, individuals with high energy costs of locomotive capability have strong ability to search for mates and to avoid predation (duellman and trueb, 1986; liao et al., 2012; jin et al., 2015; liao et al., 2015b). as a result, individuals with greater reproductive fitness selected larger brains are associated with an elevated cognitive ability (i.e., the ability to process information; striedter, 2005). however, we did not find an increase brain mass increasing with the mass of limb muscles, as proxy for the costs of locomotion. the kidneys are the primary organs for excreting metabolic waste and maintaining ph balance in organisms (moore, 1995; ganong, 2005). the liver is one of the most important energy storage organs in animals (ji et al., 2002; yang and wu, 2006). the eth predicts a significant negative correlation between the size of the brain and both the liver and the kidneys. nonetheless, our findings demonstrated that there were no significant relationships among them in p. nigromaculatus. the critically important organs (testes, liver, spleen, kidneys, and limb muscles) can change in size and metabolic activity in different life-cycle periods (piersma, fig. 1. correlations between residuals liver mass and residuals of kidneys mass in pelophylax nigromaculatus, controlling for body condition (a: male; b: female). 72 li zhao et alii 2002). in this study, we did not find significant relationships between brain mass and organ mass of any of the major metabolically expensive organs. this lack of correlation supports the hypothesis that energetic costs of one expensive tissue do not necessarily directly affect the investment in another expensive tissue, but rather may scatter its effect on all other expensive tissues (lemaître et al., 2009). moreover, we did not find evidence that there were negative correlations among testes, spleen, limb muscles, liver or kidneys, suggesting that there was no trade-offs between metabolically expensive tissues due to differences in activity level or growth among individuals. however, we found a positive correlation between liver and kidneys in both sexes. acknowledgements we thank the national sciences foundation of china (31471996), sichuan province outstanding youth academic technology leaders program (2013jq0016) and sichuan province department of education innovation team project (14td0015; 15td0019) for providing the financial support. the reported experiments comply with the current laws of china concerning animal experimentation, and permission to collect frogs was received from the ethical committee for animal experiments in china west normal university. references aiello, l.c., wheeler, p. 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(2014): sexual dimorphism in the limb muscles of the dark-spotted frog, pelophylax nigromaculata. herpetol. j. 24: 147-153. mink, j. w., blumenschine, r. j., adams, d.b. (1981): ratio of central nervous system to body metabolism in vertebrates—its constancy and functional basis. am. j. physio. 241: r203-r212. moore, k. (1995): essential clinical anatomy. lippincott williams and wilkins, philadelphia. 73the expensive-tissue hypothesis lacking in a frog navarrete, a., van schaik, c.p., isler, k. (2011): energetics and the evolution of human brain size. nature 480: 91-93. piersma, t. (2002): energetic bottlenecks and other design constraints in avian annual cycles. integr. comp. biol. 42: 51-67. pitnick, s., jones, k.e., wilkinson, g.s. (2006): mating system and brain size in bats. proc. roy. soc. b 273: 719-724. striedter, g.f. (2005): principles of brain evolution. sunderland: sinauer associates. warren, d.l., iglesias, t.l. (2012): no evidence for the ‘expensive-tissue hypothesis’ from an intraspecific study in a highly variable species. j. evol. biol. 25: 1226-1231. xu, x. f., wu, y.l., ou, y.y., (2002): water and energy content variation of the major energy reserves in adult grass lizards, takydromus septentrionalis. zool. res. 23: 44-48. yang, j.q., wu, b.w. (2006): textbook of physiology. science press, beijing. tsuboi, m., husby, a., kotrschal, a., hayward, a., buechel, s.d., zidar, j., lovlie, h., kolm, n. (2015): comparative support for the expensive tissue hypothesis: big brains are correlated with smaller gut and greater parental investment in lake tanganyika cichlids. evolution 69: 190-200. acta herpetologica vol. 11, n. 1 june 2016 firenze university press feeding habits of mesoclemmys vanderhaegei (testudines: chelidae) elizângela silva brito1,*, franco leandro souza2, christine strüssmann3 morphology, ecology, and behaviour of hylarana intermedia, a western ghats frog ambika kamath1, rachakonda sreekar2,3 a new account for the endangered cerrado rocket frog allobates goianus (bokermann, 1975) (anura: aromobatidae), with comments on taxonomy and conservation thiago ribeiro de carvalho1,2,*, lucas borges martins1,2, ariovaldo antonio giaretta1 molecular phylogenetics of the pristimantis lacrimosus species group (anura: craugastoridae) with the description of a new species from colombia mauricio rivera-correa, juan m. daza* population structure and activity pattern of one species of adenomera steindachner, 1867 (anura: leptodactylidae) in northeastern brazil maria juliana borges-leite1,*, joão fabrício mota rodrigues2, patrícia de menezes gondim1, diva maria borges-nojosa1 vocal repertoire of scinax v-signatus (lutz 1968) (anura, hylidae) and comments on bioacoustical synapomorphies for scinax perpusillus species group marco antônio peixoto1,*, carla silva guimarães1, joão victor a. lacerda2, fernando leal2, pedro c. rocha2, renato neves feio1 amendment of the type locality of the endemic sicilian pond turtle emys trinacris fritz et al. 2005, with some notes on the highest altitude reached by the species (testudines, emydidae) federico marrone*, francesco sacco, vincenzo arizza, marco arculeo photo-identification in amphibian studies: a test of i3s pattern marco sannolo1,*, francesca gatti2, marco mangiacotti3,4, stefano scali3, roberto sacchi4 no evidence for the ‘expensive-tissue hypothesis’ in the dark-spotted frog, pelophylax nigromaculatus li zhao, min mao, wen bo liao* no short term effect of clinostomum complanatum (trematoda: digenea: clinostomatidae) on survival of triturus carnifex (amphibia: urodela: salamandridae) giacomo bruni1,*, claudio angelini2 yeasts in amphibians are common: isolation and the first molecular characterization from thailand srisupaph poonlaphdecha, alexis ribas* introduction of eleutherodactylus planirostris (amphibia, anura, eleutherodactylidae) to hong kong wing ho lee1, michael wai-neng lau2, anthony lau3, ding-qi rao4, yik-hei sung5,* correlation between endoscopic sex determination and gonad histology in pond sliders, trachemys scripta (reptilia: testudines: emydidae) david perpiñán1, albert martínez-silvestre2,3, ferran bargalló3, marco di giuseppe4, jorge orós5, taiana costa6,* book review: john w. wilkinson. amphibian survey and monitoring handbook. pelagic publishing franco andreone book review: susan newman. frogs amphibians and their threatened environment. discovery and expression through art k-3. frogs are green franco andreone issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 8(2): 141-146, 2013 diet of tadpoles of physalaemus biligonigerus (leiuperidae) from agricultural ponds in the central region of argentina clarisa de l. bionda1,4,*, elisa luque2, noemi gari2, nancy e. salas1, rafael c. lajmanovich3, adolfo l. martino1 1 ecología-educación ambiental, departamento de ciencias naturales, facultad de ciencias exactas, físico-químicas y naturales, unrc, ruta nacional nº 36-km 601, río cuarto, córdoba, argentina. *corresponding author. email: cbionda@exa.unrc.edu.ar 2 botánica sistemática, departamento de ciencias naturales, facultad de ciencias exactas, físico-químicas y naturales, unrc, argentina 3 ecotoxicología, escuela superior de sanidad “dr. ramón carrillo”, facultad de bioquímica y ciencias biológicas, paraje “el pozo” s/n, santa fe, argentina 4 consejo nacional de investigaciones científicas y técnicas (conicet), argentina submitted on 2012, 3th october; revised on 2013, 11th june; accepted on 2013, 24th june. abstract. the intensification of agriculture has led an important loss of natural habitats, with significant consequences for biodiversity. in this sense, the studies on anuran amphibian tadpoles inhabiting these environments are relevant, because the larval stage is a phase of population regulation. the aim of this study was to analyze the diet in physalaemus biligonigerus tadpoles, an anuran species widely distributed in south america and that inhabit agroecosystems. three sites were sampled; two agroecosystems with different alteration degrees (ag1 and ag2) and an uncultured (sm) third place. the captured tadpoles were anesthetized, fixed and preserved in formaldehyde (10%). subsequently, the complete intestine was removed and analyzed for food items under a binocular microscope. the diet in p. biligonigerus tadpoles has a dominance of algae bacillariophyceae, mainly in agroecosystems, due to the presence of the genera navicula, nitzschia and gomphonema. there was a considerable abundance of the gomphonema genus in the ag2 site. in addition, in the ag1 site several non-diatom algae were particularly abundant in the diet, such as the genera euglena, oedogonium and chaetophora. in the sm site, the non-diatom genus oscillatoria was well represented in the diet. tadpoles inhabiting the site with abundant crop and livestock (ag1) ingested a significantly smaller amount of food. the presence of certain algae associated with eutrophic environments could indicate some pollution in agroecosystems (ag1 and ag2). larval diet is suggested as a potential bioindicator of environmental health for these areas. keywords. agriculture, algae, bioindicators, gut content. amphibian populations are experiencing worldwide declines (collins and crump, 2009), which are caused by multiple factors including trematode parasites, ultraviolet (uv-b) radiation and chemical pollutants (e.g. blaustein et al., 2003; henle et al., 2008; johnson et al., 2008; peltzer et al., 2011). the conversion of forest to agricultural land is occurring at rapid rates in many neotropical areas (pengue, 2005). the central region of argentina has been greatly affected by agricultural development, mainly to soybean cropland (glycine max merril) (schnepf et al., 2001). thus, agriculture has become the main cause of reduction and fragmentation of natural habitats. due to environmental degradation, agriculture is also considered an important factor in the decline in amphibians recorded the last decades (collins and crump, 2009). principally, in agriculturally dominated landscapes the amphibians depend on patches of natural vegetation and ephemeral ponds for their survival and reproduction (peltzer et al., 2003). the decrease in quality and hydroperiod of aquatic environments may influence both time and size at meta142 clarisa de l. bionda et al. morphosis of tadpoles, and may cause increasing mortality or predation pressure (carey and bryant, 1995; altig et al., 2007). in this sense, the adult population would vary in relation to changes in larval success, hence the larval stage is considered a phase of population regulation (heyer, 1974; wilbur, 1980). recent evidence has revealed that the deterioration of agricultural ponds might modify the trophic availability, affecting the development and growth to amphibian tadpoles that are sustained in such environments (bionda et al., 2012). in addition, the diet of anuran tadpoles is usually diverse, can be composed of plant fragments, protozoa, rotifers, anuran eggs, and even other tadpoles, but most tadpoles are primarily herbivorous consuming a wide variety of algal taxa (kupferberg, 1997; altig et al., 2007). bioindicators include biological processes, species, or communities and are used to assess the quality of the environment and how it changes over time (holt and miller, 2011). in this way the diet of these organisms may constitute a bioindicator for characterization and monitoring of environmental quality (blanco et al., 2004; bionda et al., 2012). the aim this study was analized the diet of physalaemus biligonigerus (cope, 1861) tadpoles in temporary ponds from agroecosystems for the central region of argentina. the study region belongs to the pampa plains of central argentina. moderately undulating plains and a temperate climate characterize the area, with an annual mean temperature of 23 ºc in january and 6 ºc in july and with mean annual temperature of 18 ºc. the region is characterized by rainy and dry seasons, with rains typically starting in october and continuing through the warm months until march (mean annual rainfall of 800-1 000 mm) (gatica et al., 2012). we sampled three sites in rio cuarto (33º08’s, 64º24’w; 434 m a.s.l.; córdoba province, argentina). two of these sites were agricultural lands with different alteration degrees (ag1, 9 ha: 33°05’s 64°26’w; ag2, 12 ha: 33°05’s 64°25’w). these study areas are sparsely vegetated with low growing plants (heights <0.5 m) and permanent and temporary ponds. in ag1, the cropland is around ponds (within approx. 10 m), in ag2 is farther (about 200 m). during the study period, both agroecosystems were devoted to soybean crop and were used for cattle grazing. the third site (sm) was a suburban pond located on the campus of national university of rio cuarto (33º06’s, 64º25’w; 465 m a.s.l.). this is an area with permanent and temporary ponds, and a small strip of forest occurred all around the edge of the ponds. physalaemus biligonigerus is a species of anuran widely distributed in south america (kwet et al., 2012), is a representative species of amphibian communities in the region and their populations are abundant in the central region of argentina (bionda et al., 2011; 2012). the p. biligonigerus tadpoles were captured in december 2007, along transect from the margin of the water body to 2 m therein. the captured tadpoles were anesthetized with chloroethane, fixed and preserved with 10% buffered formalin. subsequently, for a total of 5-6 tadpoles per site, between stages 36-39 of gosner (1960), the complete intestine was removed and their contents analyzed (only first third). the food items were determined under a zeiss optical microscope at 400 x, according to the technique described by hill et al. (2000). items were identified to the genus level. the algal density estimate was based on quantitative samples, following villafañe and reid (1995). the percentage frequency of occurrence (fo%, percentage of individuals that consumed a given category of prey) and numerical frequencies (n%) for each food item were calculated. the values of food items consumed for each site were compared within the pairs using mann-whitney u tests followed by bonferroni correction. to describe the dietary diversity among different populations, we calculated the shannon and weaver (1949) diversity index (h). furthermore, we measured the evenness (e) in for each population diet following magurran (1987). the diet of p. biligonigerus consists primarily of microalgae, mainly diatoms in the sites ag2 and sm. table 1 shows the results for the more abundant algal genera recorded in the gut contents of the tadpoles for the three sites. in particular, the classes chlorophyceae (nine genera in total) due mainly to the presence of the genera oedogonium and chaetophora, and euglenophyceae (four genera) with the genus euglena, were important in the tadpoles diet in ag1. the class bacillarophyceae (13 genera) mainly represented by the genera gomphonema and nitzschia, was predominant in the diet of ag2. finally, the class bacillarophyceae (11 genera) due mainly to the presence of the genus navicula, and cyanophyceae (five genera) with the genus oscillatoria, were important in the diet of sm. there were significant differences in the total items consumed by tadpole between ag1 and the other sites (mann-whitney, p< 0.017). we detected a significantly lower amoung ot food in tadpoles inhabiting the site most modified by crop and livestock (ag1). the amount of food consumed by p. biligonigerus in ag1 site was 451 548 ± 238 351 average items by gut, in ag2 was 4 057 099 ± 364 666 and in sm was 3 151 682 ± 2 324 009. thus, tadpoles of ag1 site consume only 11.1% and 14.3% of food amount consumed by the tadpoles of ag2 and sm, respectively. however, the ag1 site had the diet with higher diversity (ag1: h= 2.32; ag2: h= 1.55 and sm: h= 1.61) and evenness (ag1: e1= 0.69; ag2: e1= 0.43 and sm: e1= 0.50). 143diet of tadpoles of physalaemus biligonigerus (leiuperidae) from agricultural ponds in the central region of argentina table 1. percent n (%) and occurrence frequencies fo (%) of the more abundant algal genera recorded in the gut contents of physalaemus biligonigerus tadpoles of two agroecosystems sites ag1 (n= 5), ag2 (n= 5) and the uncultured place sm (n= 6) in central argentina. values followed by different letters differ significantly (mann-whitney, p <0.017). ag1 ag2 sm n (%) fo (%) n (%) fo (%) n (%) fo (%) bacillariophyceae craticula 0.12 40 0.02 80 cyclotella 0.30 100 0.15 100 0.01 20 cymbella 0.03 40 0.01 20 achnanthes 0.33 100 0.01 20 amphora 0.06 40 0.01 40 fragilaria 0.10 40 surirella 0.01 20 fallacia 0.03 40 0.12 100 9.21 100 gomphonema 0.04 20 83.12 100 0.02 40 hantzschia 0.07 40 0.13 100 0.50 100 navicula 0.82 100 2.95 100 41.60 100 nitzschia 11.64 100 11.75 100 8.23 100 pinnularia 4.01 100 0.01 40 5.84 100 sellaphora 0.05 40 neidium 0.02 20 chlorophyceae chlorococcal 0.05 40 monoraphidium 0.02 20 ankistrodesmus 0.01 20 chloroficeae 0.04 20 oedogonium 14.29 100 0.09 17 microspora 6.04 80 chaetophora 14.92 100 pediastrum 0.02 20 scenedesmus 0.38 100 0.91 100 staurastrum 0.02 20 tetraedron 0.04 20 0.01 20 ulothrix 0.01 20 cladynomonas 0.01 20 cyanophyceae aphanocapsa 0.41 80 chrooccoccus 0.02 20 cyanophyceae 4.56 80 0.06 20 0.40 20 gomphosphaeria 0.01 20 0.01 20 merismopedia 0.01 20 lyngbia 0.34 20 oscillatoria 9.70 100 29.29 100 anabaena 0.11 40 spirulina 0.82 70 euglenophyceae phacus 6.35 100 0.01 20 0.55 100 euglena 18.60 100 0.02 40 1.57 100 trachelomonas 0.23 80 0.01 20 0.91 100 strombomonas 0.57 100 lepocinclis 6.84 100 0.03 50 mean amount (± sd) of food items consumed by tadpoles 451 548 ± 238 351a 4 057 099 ± 364 666b 3 151 682 ± 2 324 009b 144 clarisa de l. bionda et al. average cattle density at wetlands during our study was 11.3 head (sd = 3.05) per wetland ha per month in ag1, and 26.3 head (sd = 3.51) per wetland ha per month in ag2. there were fewer cattle in ag1, but they stayed closer to wetlands than the cattle in ag2. amphibian larvae may be at greater risk from the effects of contaminants that adults, since water bodies receive runoff from agricultural lands, and also receive direct aerial fumigation. in anuran larvae, diet is usually indicative of the type and abundance of food resources in their environments (heyer, 1974; lajmanovich, 2000). in this sense, our results indicated that the tadpoles of the most altered cropland site (ag1), showed a significantly lower amount of food in their intestine. the surveyed ponds are surrounded by an agricultural matrix and are also used by livestock, these activities may affect food availability in ponds to tadpoles of p. biligonigerus, considering that agriculture causes eutrophication of surface waters (sharpley et al., 2003; withers and haygarth, 2007) and the eutrophic environments have lower food quality (smol, 2002). the ag1 diet showed the highest diversity and evenness, however, the tadpoles consume little food there. similar results were found by bionda et al. (2012) for a study performed with tadpoles of the bufonid rhinella arenarum (hensel, 1867) for these same sites. rhinella arenarum tadpoles also consumed less food items in the ag1 site, despite their diet was most diverse and equitable. in adittion, the r. arenarum tadpoles for ag1 reported a lower mass and poorer body condition. pollution may interfere with the food acquisition and digestion, affecting the amount of food consumed by tadpoles and thus expecting a smaller mass of tadpoles (carey and bryant 1995). in this sense, studies have shown that nitrogen fertilizers reduce the feeding activity of tadpoles of some species (marco et al., 1999; hatch and blaustein, 2000). however, further studies are needed to elucidate this question. diatoms are useful bioindicators of water conditions (tison et al., 2008). several species of diatoms are associated with the presence of certain nutrients such as nitrogen or phosphorus (blanco et al., 2004); many of the agrochemicals used by farmers are among its principal components to nitrogen and phosphorus. among diatom algae, the genera navicula, nitzschia and gomphonema were important in the diet of the tadpoles in agroecosystems (ag1 and ag2). these same algae were most abundant in the diet of r. arenarum (bionda et al., 2012). the genera navicula, nitzschia and gomphonema are considered the main eutrophic indicators among diatoms, and are especially resistant to environments with high electrical conductivity (palmer, 1977; kelly et al., 2005). moreover, several non-diatom algae such as euglena, oedogonium and chaetophora were abundant in ag1 site. the predominance of the genera euglena and oedogonium could indicate elevated content of organic matter in these ponds (fabrizi, 2012). furthermore, also the diatom nistzchia, considered among the most widespread algae in sewage ponds (palmer, 1977), was frequent in the diet of ag1 site. significant abundance of these genera in the diet of tadpoles of ag1 could correspond to the presence of livestock and its staying close to wetlands. the amount of food in the ag2 site was similar to sm site, although is important to note that the 83.1% of the ag2 diet corresponds to the genus gomphonema. gomphonema is a dominant diatom genus in environments with elevated levels of phosphorus (nather khan, 1990; kelly et al., 2005). in the study that was performed with the toad r. arenarum, gomphonema was abundant in the diet of ag1 site; this would raise serious questions about environmental health in these agroecosystems (bionda et al., 2012). the navicula algae were predominant in the diet of the tadpoles of sm site. as we mentioned before, the presence of navicula is associated with some pollution, but it can occur at high densities in both unpolluted and polluted sites (nather khan, 1990). oscillatoria was another genus of non-diatom algae that was well represented into the diet of sm site. the predominance of oscillatoria in the sm tadpoles might be associated with the presence of higher amount of organic matter in the ponds of this site (fabrizi, 2012). while crops are absent in the surroundings of sm site and, therefore, it was considered as a reference site to be compared with agroecosystems, this environment shows some degree of disturbance, as it surrounded by residences, which may determine an increase in the amount of organic material and therefore the predominance of this genus. in summary, the presence of certain algae registered on the tadpoles diet from different agricultural sites could correspond to their levels of alteration. is possible thus consider these algae as bioindicators, as they are directly associated with eutrophic environments. as similar results were recorded in a previous study realized to these same sites but using the species r. arenarum, then the larval diet is suggested that as a potential bioindicator of environmental health for these areas. acknowledgements we thank the argentinean national research council for science and technology (conicet). the investigation was conducted according to the guidelines for use of live amphibians and reptiles in field research compiled by asih, hl & ssar (2001) and the state law “protection and conser145diet of tadpoles of physalaemus biligonigerus (leiuperidae) from agricultural ponds in the central region of argentina vation of wild fauna” (argentina national law nº 22.421). our study was authorized by cordoba environmental agency (a.c.a.s.e.). references altig, r., whiles, m.r., taylor, c.l. 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(2007): agriculture, phosphorus and eutrophication: a european perspective. soil use manage. 23: 1-4. acta herpetologica vol. 8, n. 1 june 2013 firenze university press journal of the societas herpetologica italica acta herpetologica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 6(2): 175-208, 2011 ecological associations and genetic divergence in black-bellied salamanders (desmognathus quadramaculatus) of the southern appalachian mountains jessica a. wooten1, leslie j. rissler2 1department of biology, the university of findlay, findlay, oh 45840, usa. 2 department of biological sciences, the university of alabama, tuscaloosa, al 35487, usa. corresponding author. e-mail: rissler@as.ua.edu submitted on: 2011, 7th march; revised on: 2011, 31th july; accepted on: 2011, 09th october. abstract. the discovery and subsequent description of cryptic biodiversity is often challenging, especially for groups that have undergone rapid lineage accumulation in the relatively recent past. even without formal descriptions, understanding genetic diversity patterns as they relate to underlying ecological or historical processes can be important for conservation. the dusky salamanders of the genus desmognathus, with 20 described species, comprise the second largest genus of plethodontid salamanders in the eastern united states. however, due to the presence of high genetic diversity and relatively few morphological synapomorphies, the number of species is likely to increase. for the three nominal species within the d. quadramaculatus species complex, including d. quadramaculatus, d. folkertsi, and d. marmoratus, we used a portion of the mitochondrial genome and nuclear markers in the form of amplified fragment length polymorphisms (aflp) to uncover spatial patterns of genetic diversity. within d. quadramaculatus and d. marmoratus, we uncovered four well-supported lineages with the mitochondrial sequences; phylogeographic patterns were not congruent with the aflp data. both sets of markers identified a clear isolation by stream distance. using multiple regressions, we found that historical river drainages and terrestrial ecoregions explained the phylogeographic patterning we observed for d. quadramaculatus. keywords. salamanders, desmognathus quadramaculatus, desmognathus marmoratus, aflp, streams, desmognathus folkertsi, mtdna, ecoregions. introduction in light of the constant threat of anthropogenic habitat alteration (davis et al., 2008) and the ubiquity of cryptic biodiversity (bickford et al., 2006), the discovery and subsequent descriptions of unique evolutionary lineages are central to understanding the full 176 j.a. wooten and l.j. rissler extent of biodiversity on earth. however, the discovery and subsequent description of cryptic species is challenging, especially for organismal groups that have experienced rapid lineage accumulation in the recent past (kozak et al., 2006; wiens et al., 2006) and that lack obvious morphological differentiation. this is certainly true in lungless salamanders (plethodontidae) where many species boundaries and evolutionary relationships have been concealed by the absence of obvious morphological synapomorphies (wake, 1966; larson et al., 1981; wake et al., 1983; carr, 1996; adams and rohlf, 2000; highton and peabody, 2000; chippindale et al., 2004; mueller et al., 2004; wiens et al., 2006; tilley et al., 2008). salamanders are a challenging group for which to delimit species boundaries and uncover cryptic biodiversity. many salamander taxa exhibit extensive intraspecific variation in traits, such as color and pattern. at the same time, they exhibit extreme interspecific morphological similarity, which is usually coupled with high levels of genetic diversity (wake, 1966, 1991; wake et al., 1983; highton and peabody, 2000; wake and jockusch, 2000; gao and shubin, 2003; mueller et al., 2004; kozak et al., 2006; wiens et al., 2006; beamer and lamb, 2008; tilley et al., 2008). the plethodontidae is the most species-rich family of salamanders containing approximately 68% of all known species (amphibiaweb.org), with new species being described regularly because of extreme morphological similarities and widespread homoplasy (e.g., tilley and mahoney, 1996; garcía-parís and wake, 2000; jockusch et al., 2001; camp et al., 2002; mccranie et al., 2005; hanken et al., 2007). the dusky salamanders (desmognathus) comprise one of the most species-rich genera within plethodontidae (vieites et al., 2007), with the hotspot of biodiversity in the appalachian mountains (kozak et al., 2006; wiens et al., 2006; vieites et al., 2007). most desmognathan species are restricted to, or are associated with, swift-flowing mountain streams in the appalachian mountains of eastern north america. many recent studies agree that the 20 currently recognized species may be a significant underestimation of the full extent of the biodiversity within the genus (kozak et al., 2005; beamer and lamb, 2008), and the number of recognized species will likely increase due to the presence of extensive morphological stasis, rampant homoplasy, and high levels of genetic diversity among and within groups. to complicate modern-day taxonomy, studies have revealed a more complex and intertwined evolutionary history for many species within the genus desmognathus (titus and larson, 1996; rissler and taylor, 2003; kozak et al., 2005; jones et al., 2006; beamer and lamb, 2008; tilley et al., 2008), especially for d. marmoratus and d. quadramaculatus (titus and larson, 1996; rissler and taylor, 2003; jackson, 2005; jones et al., 2006). interestingly, these studies have suggested that some populations of d. quadramaculatus are more closely related to populations of d. marmoratus, a wholly aquatic and relatively rare salamander species (titus and larson, 1996; rissler and taylor, 2003; jackson, 2005; jones et al., 2006) with a limited geographic distribution. however, to date, there have been no published studies focusing exclusively on the phylogeography of d. quadramaculatus across its range. the use of nuclear markers in plethodontid salamander species, with the exception of allozymes, is uncommon for studies of fine-scaled population structure, although sequencing nuclear genes is becoming more common. that said, amplified fragment length polymorphism (aflp; vos et al., 1995) is a pcr-based, anonymous dominant nuclear-marker technique that has been extensively used to investigate intraspecific phylogeographic patterns, uncover new species, and delimit species boundaries in many groups (shaw, 2002; seman et al., 2003; wang et al., 2003; creer et al., 2004; finn et al., 2006; mendelson and simons, 2006; garoia et al., 2007; kinkead et al., 2007; nicolè et al., 2007), especially in 177ecology and genetic of black-bellied salamanders plants, (e.g., hoarau et al., 2001; garcia et al., 2004; albach et al., 2006; agrimonti et al., 2007; andrade et al., 2007; assefa et al., 2007; nicolè et al., 2007), microbes and fungi (e.g., vos et al., 1995; blears et al., 1998; bensch and åkesson, 2005), and invertebrates (wilding et al., 2001; shaw, 2002; carisio et al., 2004; mock et al., 2004; pizzo et al., 2006). studies that use aflp in vertebrate taxa are limited (bensch and åkesson, 2005), but this number has increased over the past few years (ogden and thorpe, 2002; wang et al., 2003; makowsky et al., 2008). at present, there are only a few known studies that involve the use of aflp in salamander taxa (voss and shaffer, 1997; curtis and taylor, 2003; riberon et al., 2004; lowe et al., 2006; whitlock et al., 2006; jehle et al., 2007; kinkead et al., 2007; milá et al., 2010), even though aflp is a useful marker that can be used to investigate population structure and genetic variability at the species level (mueller and wolfenbarger, 1999). the goals of our study were to use both mitochondrial and aflp data to: 1) address the phylogeographic and genetic patterns across populations of d. quadramaculatus, and 2) assess the spatial patterns of divergence, specifically testing the role of drainages (i.e., current versus historical river drainages) as barriers of gene exchange. materials and methods the study taxon desmognathus quadramaculatus is a species complex comprised of at least three nominal species, including, d. quadramaculatus (black-bellied salamander), d. folkertsi (dwarf black-bellied salamander), and d. marmoratus (shovel-nosed salamander). desmognathus quadramaculatus exhibits the largest geographic range of the three and is a semi-aquatic species that can be found in swift-flowing, montane streams extending from southern west virginia southward through the great smoky and unicoi mountains, and into the blue ridge escarpment of western north carolina. this species reaches its southern-most geographic limit in the upper piedmont of northern georgia (jensen et al., 2008). desmognathus folkertsi and d. marmoratus both exhibit relatively small geographic ranges and are often syntopic with that of d. quadramaculatus. desmognathus folkertsi is a recently described, semi-aquatic species (camp et al. 2002) known from northern georgia and abutting areas of the carolinas; whereas, d. marmoratus is an aquatic species that has a larger geographic distribution extending from southern virginia, south along the blue ridge mountains to northern georgia. desmognathus folkertsi appears to represent a cohesive, monophyletic taxon (jackson, 2005; wooten 2007; wooten et al., 2010). however, d. marmoratus, as it is currently recognized, consists of several lineages and may be a paraphyletic taxon (jackson, 2005; jones et al., 2006; kozak et al., 2006). jones et al. (2006) suggested the recognition of southernmost populations of d. marmoratus as a separate species (d. aureatus). sampling locations we examined 281 individuals from 56 populations d. quadramaculatus from the southern appalachian mountains in the eastern united states, as well as closely related species, including d. monticola (n = 15), d. folkertsi (n = 23), and d. marmoratus (n = 8; fig. 1). detailed information on locality, museum voucher number, genbank accession number, and river drainage are presented in appendix 1. voucher specimens were euthanized in using ethyl 3-aminobenzoate methanesulfonate 178 j.a. wooten and l.j. rissler (ms 222; sigma-aldrich) at a concentration of 2 g / l. an approximately 1 cm piece of tail tissue was removed for genetic analysis and frozen (-80oc) in the permanent tissue archive at the university of alabama herpetology collection (uahc). specimens were then fixed in 10% formalin and preserved in 70% ethanol. all specimens were deposited in the uahc stored at the university of alabama, tuscaloosa or in the appalachian state herpetology collection (appsu), boone, north carolina. mitochondrial dna extraction and sequencing whole genomic dna was extracted from approximately 5 mm of tail tissue using the dneasy tissue-kit protocol for animal tissues (qiagen, valencia, ca). amplification of the 12s rrna and a portion of the valine transfer trna regions of the mitochondrial genome was completed on all individuals by using primers b and g from titus and larson (1996) and following a modified methodology of rissler and taylor (2003). this portion of the mitochondrial genome has been shown to be a highly informative marker for discriminating intraand interspecific relationships in desmognathus (titus and larson, 1996; crespi et al., 2003; rissler and taylor, 2003; rissler et al., 2004). exosapit (u.s. biochemicals corp., cleveland, oh) at 37 oc for 45 min and 80 oc for 15 min was used to purify the pcr products. cycle-sequencing was completed in forward and reverse directions on the purified pcr products using bigdye terminator (applied biosystems, foster city, ca), followed by purification with sephadex g-50 (sigma aldrich corp., st. louis, mo), and visualized on an abi 3100 automated dna sequencer. additional mtdna sequences were retrieved from genbank for d. wrighti (outgroup), d. quadramaculatus, d. marmoratus, d. aeneus, and d. monticola, and these accession numbers are listed in appendix 2. collapse 1.2 (http://darwin.uvigo.es/software/collapse.html) was used to remove any identical haplotypes before phylogenetic analyses. amplified fragment length polymorphism our aflp procedures were modified from the protocol by voss et al. (1995) and blears et al. (1998). digestion reactions in 20 µl volumes were performed on whole genomic dna with a concentration of 25 ng / µl using the following cocktail per reaction: 2 µl ecori 10x restriction buffer, 1.0 µl ecori, 0.2 µl mse, and 12 µl h2o. following a 5 h incubation period at 37 oc, 20 µl of ligation mixture consisting of 12 µl h2o, 4 µl 10x t4 dna ligase buffer, 1.5 µl of each double-stranded adapter pair (75 pmol), and 1.0 µl of t4 ligase was added to the digestion solution. the double-stranded adaptor pairs were constructed from the following complementary single-stranded oligonucleotides: ecori adaptor: 5’-ctc gta gac tgc gta cc-3’ and 5’-aat tgg tac gca tac-3’. ligation was performed for 10 h at 16 oc. following ligation, each solution was diluted with 160 µl h2o. two increasingly selective pcr amplifications of the ligated dna were performed. for preselective amplification, 10µl of the diluted restriction-ligation product was used as a template and added to 40 µl preselective amplification solution consisting of: 5 µl 10x pcr buffer, 1.3 µl of each preselective primer (15 pmol), 4.0 µl dntp (10 mm), 1.0 µl formamide, 2.5 µl mgcl2 (25 mm), 24.75 µl h2o, and 0.5 µl taq dna polymerase. four preselective primer combinations were used including: ecori + ac / mse + ca; ecori + ca / mse + ca; ecori + ca / mse + cg; and ecori + ca / mse + gc. the pcr cycling conditions were a preliminary 72 oc extension for 60 sec followed by 20 cycles of 94 oc for 50 sec, 56 oc for 60 sec, and 72 oc for 120 sec. preselective solutions were diluted with 125 µl of h2o following pcr cycling. for selective pcr, 5 µl of diluted preselective solution was added to 20 µl of selective pcr solution which consisted of 0.5 µl formamide, 10 µl h2o, 2.5 µl 10x pcr buffer, 3 µl mgcl2 (25 mm), 3 µl dntp (10 mm), 1.5 µl (0.5 pmol) of the flourophore (6-fam)-labeled ecori primer, 1.5 µl (25 pmol) of the unlabeled mse selective primer, and 0.5 µl taq dna polymerase. the pcr 179ecology and genetic of black-bellied salamanders cycling conditions were: a touchdown cycle of 94 oc for 50 sec, 57 oc for 60 sec, and 72 oc for 120 sec, followed by 20 cycles of 94 oc for 50 sec, 56 oc for 60 sec, and 72oc for 120 sec. the 20 cycles were followed by a final extension at 72 oc for 10 min. three combinations of selective primer combinations were chosen including: ecori + caa / mse + ca; ecori + caa / mse + cg; and ecori + caa / mse + gc from a primer screening procedure in another study (wooten and tolley-jordan, 2009). when combined, these combinations produced between 450 and 555 well-defined bands per sample, distributed widely across the 60-350 scoring window. the selective amplification products were purified using fine sephadex g-50 (sigma-aldrich corp., st. louis, mo). we loaded 1.5 µl of purified product per sample along with 0.5 µl genescan-500 rox ladder (perkin-elmer) into 96-well plates and samples were analyzed using an automated abi 3100 with genescan software. electropherograms were imported into genemarker v1.6 for further analyses. phylogenetic reconstruction – mtdna and aflp approximately 600 bp of the 12s rrna and valine transfer trna regions were sequenced. forward and reverse sequences were assembled, and any ambiguous sites were verified using sequencher 4.6 (gene codes, ann arbor, mi). clustal w procedure in bioedit v7.0.9 (http://www. mbio.ncsu.edu/bioedit/bioedit.html) was used to align all sequences. the alignment was manually corrected. the sequences were sufficiently similar to allow manual correction following alignment without restrictions based upon the gap positions in the hypothesized loop regions of the 12s rrna sequences. we used d. wrighti as the outgroup in all phylogenetic reconstructions; d. wrighti is currently accepted as the basal member of the genus desmognathus (titus and larson, 1996; rissler and taylor, 2003; jones et al., 2006; tilley et al., 2008). phylogenetic relationships among haplotypes using mtdna sequences were estimated using maximum likelihood and bayesian procedures. for the bayesian procedure, the model of sequence evolution that best fit the mtdna sequence data was determined using mrmodeltest v2.2 (http:// www.abc.se/~nylander). mrbayes v3.1 was then used to conduct a bayesian phylogenetic analysis (huelsenbeck and ronquist, 2001) using the trn + γ model of evolution. six heated markov chains were run for 10×106 generations, sampling every 1000 generations for a total of 10,000 samples. three replicate searches were conducted to verify that the analyses were optimal by producing similar topologies and ln-likelihood scores (huelsenbeck and bollback, 2001). the burn-in procedure was used to eliminate topologies generated before the ln-likelihood was stabilized. posterior probabilities were estimated as the proportion of trees sampled after the burn-in procedure containing each of the observed bipartitions (larget and simon, 1999). for the maximum-likelihood procedure, modeltest v3.7 (posada and crandall, 1998) was used to estimate the model of sequence evolution. garli v0.951 (zwickl, 2006) was used to reconstruct phylogenetic relationships among haplotypes using the maximum-likelihood procedure with the trn + γ model of evolution. statistical support for each branch was assessed using bootstrap analysis with 1000 replications in garli v0.951 (zwickl, 2006). for aflp analyses, each primer combination was used to create a unique template to normalize the data across the different runs, and this step standardized the calling of peaks across the four different primer combinations using genemarker v1.6 (softgenetics, state college, pennsylvania). peaks were called between 60-350bps, and these data were entered into a binary matrix as discrete variables (1 for presence and 0 for absence). the binary matrix from each primer combination was combined into one data set and analyzed as phenotypes. a jaccard dissimilarity matrix was generated in r package v4.0 (casgrain and legendre, 2001) from the combined binary matrix. a phylogram was generated in paup* (swofford, 2002) using the minimum evolutionary procedure. statistical support for each branch was assessed using both neighbor-joining and parsimony analyses with 1000 replications in paup* (swofford, 2002). for analyses, hardy-weinberg equilibrium was assumed for the d. quad180 j.a. wooten and l.j. rissler ramaculatus, d. folkertsi, and d. marmoratus populations, and we assumed that each peak (i.e., fragment) represented a unique sequence (zhivotovsky, 1999; kinkead et al., 2007; measey et al., 2007). genetic diversity indices patterns of genetic diversity within and across lineages and river drainage basins were investigated using dnasp v4.0 (rozas and rozas, 1999; rozas et al., 2003) for populations of d. quadramaculatus. for the three nominal species in the d. quadramaculatus complex, haplotype diversity (nei, 1987), sequence diversity (k; tajima, 1983), nucleotide diversity (p; nei, 1987), the number of mutations per site (θ; nei, 1987), and the total number of mutations (h; nei, 1987) were calculated to measure sequence diversity. in order to test predictions made by the neutral theory of molecular evolution (kimura, 1983) and for evidence of population-expansion events, we calculated tajima’s d (tajima, 1989), fu and li’s d* test statistic (fu and li, 1993), fu and li’s f* test statistic (fu and li, 1993) and fu’s fs statistic (fu, 1997). for aflp data, we used structure v2.2 (pritchard et al., 2000; falush et al., 2003; falush et al., 2007), which is a clustering method that assigns individuals to populations using multilocus genotype data (pritchard et al., 2000; falush et al., 2003), to discern between lineages of the d. quadramaculatus species complex. we assigned individuals to five groups based upon the bayesian phylogram to test for congruence between the phylogeny and the group placement using the aflp fragments, a multiloci, dominant, nuclear markers, using structure v2.2 (pritchard et al., 2000; falush et al., 2003; falush et al., 2007). in structure v2.2 (pritchard et al., 2000; falush et al., 2003; falush et al., 2007), we ran 10,000 burn-in with 100,000 iterations for two – 10 (k) groups with the admixture ancestry model and the option of correlated allele frequencies between populations (falush et al., 2003). we allowed structure v2.2 to infer the degree of admixture alpha from the aflp data. when alpha is close to zero, most individuals are from one population; whereas, when alpha is greater than one, individuals are admixed (evanno et al., 2005). we set lambda, which was a parameter of the distribution of allelic frequencies, to one (pritchard et al., 2000; evanno et al., 2005). we completed a pilot run using the aflp data, and we found that a burn-in of 1000 and mcmc of 10,000, with 10 replications for each value of k, was not sufficient. because of this, we increased the burn-in to 10,000 and the mcmc to 100,000, with 10 replications for each value of k; we found that the likelihood values and the amount of variation for the likelihood of each k stabilized across replications. finally, we chose the option of ancestdist, which calculates the 90% probability interval that a particular individual would be assigned to a distinct group. for aflp data, we used aflp-surv v1.0 (vekemans, 2002; vekemans et al., 2002) to estimate genetic diversity with dominant alleles using the lynch and milligan (1994) method. statistical analysis of aflp fragments was based on the assumption that the fragments act as diploid, dominant markers either being present (scored as 1) or absent (scored as 0). nei’s genetic distance, which is the average, expected heterozygosity of the marker loci was used to estimate genetic diversity from the aflp fragments. we assumed hardy-weinberg equilibrium to examine total gene diversity (hj), mean gene diversity within populations (hw), average gene diversity among populations (hb), and fst across the landscape (vekemans, 2002; vekemans et al., 2002). because discrete genotypes are not generated from aflp fragments, each fragment was treated as a single locus with two alleles (blears et al., 1998). in aflp-surv, we chose the bayesian non-uniform distribution with 1000 permutations for estimates of gene diversity. in addition, we constructed a multidimensional scaling (mds) plot based on jaccard’s dissimilarity measure (1-similarity; gower 1971) using statistica v.6 (statsoft) and r package v.4.0 (casgrain and legendre, 2001) to see if distinct clusters formed based on dissimilarity among populations or between d. folkertsi and d. quadramaculatus. in arlequin v.3.11 (excoffier et al., 2005), we calculated an amova using the aflp binary matrix for unique haplotypes and used the same scheme for assigning individuals to populations and groups as in the mtdna analysis. finally, we calculated nei’s (1978) unbiased genetic distance (d) between lineages 181ecology and genetic of black-bellied salamanders of d. folkertsi and again between d. folkertsi and d. quadramaculatus using the aflp binary matrix in genealex 6.1 (peakall and smouse, 2006). spatial partitioning of genetic variation desmognathus quadramaculatus, like d. folkertsi, is a semi-aquatic salamander, and gene flow can occur via aquatic or terrestrial routes (wooten et al., 2010). therefore, we evaluated isolation by distance (wright, 1943; slatkin, 1993) using both land and aquatic routes of dispersal. we calculated distances among streams using network analyst in arcgis v9.0. an arbitrary value of 5,000,000 km was assigned to represent streams that were in different river drainages. straight-line distances (km) via land were calculated for each individual using latitude and longitude between each geographic locality using the r package v4.0. we used mantel tests in the r package v4.0 (casgrain and legendre, 2001) to test these associations. analysis of molecular variance (amova; excoffier et al., 1992) was used to test the partitioning of genetic variation within and across streams and river drainage basins using arlequin v3.1 (excoffier et al., 2005). the amova φ statistics are equivalent to f statistics (wright, 1931; wright, 1965) and are determined to be significant based on at least 1000 nonparametric permutations of individuals, populations, or groups of populations. in our analyses, individuals were assigned to a stream and then grouped based on river drainage basin. we used multiple regression to examine whether patterns of genetic diversity were explained by the following phylogeographic breaks: 1) current taxonomic classification (i.e., d. folkertsi, d. quadramaculatus, and d. marmoratus), 2) current drainage patterns, 3) historical drainage patterns from the pliocene (kozak et al., 2006), 4) freshwater ecoregions (categorized according to sampling location and information available in abell et al., 1999), and 5) terrestrial ecoregions (categorized according to sampling location and the classification of temperate broad leaf and mixed forests ecoregions in ricketts et al., 1999). to test these hypotheses, we generated uncorrected genetic distances for each mitochondrial haplotype calculated from paup* (swofford, 2002). these values were considered the dependent variable. for each of the five independent variables, characters were assigned based on geographic locality or taxonomic group, depending on the phylogeographic break, and a matrix of distances between all pairs of haplotypes was generated using r package v4.0 (casgrain and legendre, 2001). these matrices were imported into permute! v3.4 (casgrain, 2001); multiple-regressions were computed and 1000 permutations were completed (legendre et al., 1994) to assess the probability that each break explains the patterns of genetic diversity observed. backwards regression was used to determine the importance of the independent variables. results overall genetic variation for the nominal species within the d. quadramaculatus complex, 327 sequences representing 800 aligned bases were reported. for d. quadramaculatus, 201 unique haplotypes were identified from a total of 281 individuals and 56 sampling localities (appendix 1). the trn + γ model of evolution was chosen from mrmodeltest v2.2 with nucleotide frequencies of a = 0.2828, c = 0.2201, g = 0.2025, and t = 0.2946; substitution model rate matrix: r(a) [a-c] = 1.0000, r(b) [a-g] = 2.5812, r(c) [a-t] = 1.0000, r(d) [c-g] = 1.0000, r(e) [c-t] = 2.1188, and r(f ) [g-t] = 1.0000; γ distribution shape parameter equaled 0.4069. we reported bootstrap values of ≥ 70% (hillis and bull, 1993) and pos182 j.a. wooten and l.j. rissler terior probabilities ≥95% (wilcox et al., 2002) to represent well-supported nodes for the phylogenetic hypothesis. for d. quadramaculatus, the mean number of aflp fragments generated by each aflp primer combination was 187.67, and this varied from 167 fragments for primer combination ecori + caa / msei + gc to 212 fragments for primer combination ecori + caa / msei + cg. when all aflp fragments were considered, a total of 563 fragments were generated from three primer combinations. of these, 536 were polymorphic for d. quadramaculatus from 38 populations. we tested for aflp fragment-size homoplasy and found that there was no significant correlation between polymorphic aflp fragment size and frequency of fragment occurrence (r = -0.089, p = 0.348; vekemans et al., 2002). phylogenetic analysis – mtdna the bayesian analysis and the maximum-likelihood phylograms yielded similar topologies for d. quadramaculatus species complex and the outgroup. because of this, we represented the phylogenetic hypothesis of the d. quadramaculatus species complex with the bayesian phylogram with the posterior probabilities and maximum-likelihood bootstrap values for common branches. we generated a 50% majority-rule consensus phylogram from the bayesian analysis with a mean ln-likelihood = -15898.7 following a burn-in procedure to remove 2600 topologies that were generated before stability of the ln-likelihood was established. haplotypes within the d. quadramaculatus complex formed a well-supported monophyletic group when all three nominal species were included in the analysis (figs. 2a and 2b). there were two major lineages recovered (lineage 1 and 2) with strong statistical support; lineage 1 was comprised of d. folkertsi, which formed a monophyletic group, and lineage 2 was composed of d. quadramaculatus and d. marmoratus (fig. 2a). within lineage 2, several well-supported lineages were formed; two lineages were found south of the french broad river (fig. 2a; lineage 2a and 2b) and two lineages were found north of the french broad river (fig. 2b; lineages 2c and 2d). lineage 2a was comprised of both nominal species d. quadramaculatus and d. marmoratus; whereas; lineage 2b consisted only of d. quadramaculatus. both lineages 2a and 2b consisted of individuals from georgia, south carolina, and southwestern north carolina. the northern lineage (lineage 2c and 2d) consisted of d. quadramaculatus and d. marmoratus individuals from west virginia, virginia, and northern tennessee. the population from northern tennessee (fig. 1, map locality 5) formed a monophyletic, well-supported clade; this population is undergoing further research. lineage 2d consisted of a single population in north carolina (fig. 1, map locality 13); this population formed a well-supported and basal lineage to lineage 2c. however, because lineage 2d consisted of a single population in an isolated geographic area, surveys for d. quadramaculatus populations are underway in surrounding streams. phylogenetic analysis – aflp desmognathus folkertsi and d. quadramaculatus formed well-supported, distinct lineages when the aflp data were used, but within the species, no clear geographic patterns were found (data not shown). desmognathus marmoratus individuals were nested within 183ecology and genetic of black-bellied salamanders d. quadramaculatus for the aflp, but these nodes were not well-supported. furthermore, the mds plot did not reveal any distinct groups for d. quadramaculatus, d. folkertsi, or d. marmoratus. fig. 1. study localities in the southern appalachian mountians. sampling localities spanned parts of west virginia, virginia, north carolina, tennessee, south carolina, and georgia. 184 j.a. wooten and l.j. rissler statistical analyses of genetic data haplotypes within the d. quadramaculatus species complex exhibited typical to high levels of genetic diversity. desmognathus folkertsi exhibited the lowest level of genetic diversity among the three nominal species with a k = 1.16%; whereas, d. quadramaculatus and d. marmoratus exhibited high levels of diversity with a k = 12.02% and k = 20.08%, respectively (table 1). both d. quadramaculatus and d. marmoratus exhibited tajima’ d, fu and li’s d*, fu and li’s f*, and fu’s f statistics were not significant; however, these same measures for d. folkertsi were significantly negative (table 1). this suggests that populations of d. quadramaculatus and d. marmoratus have been established for a long period of time and have not experienced recent range expansions. however, for d. folkertsi, the phylogeny had shallower branches than expected under the neutral model of evolution, which is consistent with a recent range expansion. when the data were partitioned by river drainage for d. quadramaculatus, the catawba river drainage exhibited the highest levels of diversity k = 184.164; whereas, the fig. 2. (part 2a) bayesian phylogram using the 12s rrna portion of the mitochondrial genome with the maximum-likelihood model of evolution trn + g for unique haplotypes of d. quadramaculatus, d. folkertsi, and d. marmoratus. lineage 1 is d. folkertsi. lineage 2a is basically d. quadramaculatus and d. marmoratus that occur in southern areas including northern georgia, north carolina, and southern tennessee. lineage 2b contains d. quadramaculatus from populations of georgia, south carolina, and north carolina. (part 2b) northern populations of d. quadramaculatus. lineage 2c contains populations from virginia, west virginia, north carolina, and tennessee. lineage 2c-3 is a clade in tennessee (see text). only support values on major nodes are given (bootstrap values from 1000 replicates). see appendix 1 and 2 for more detail on specific localities and samples. 185ecology and genetic of black-bellied salamanders tennessee, savannah, new, chattahoochee, and coosa/tallapoosa river drainages exhibited moderate levels of genetic diversity, k = 140.599, k =163.422, k = 112.131, k = 70.099, and k = 156.990, respectively, for mtdna (table 2). only 4.47% of the genetic diversity occurred among drainages but 90.37% of the genetic diversity was partitioned within drainages (table 3). this pattern is indicative of gene flow across the landscape, either by natural or human-mediated means. the expected heterozygosity values (hj) calculated using the aflp data for the two main mitochondrial lineages within the d. quadramaculatus species complex, lineage 1 (d. folkertsi) and lineage 2 (d. quadramaculatus plus d. marmoratus) were equal to 0.241 and 0.229, respectively (table 4). to calculate the expected heterozygosity values (hj) in only d. quadramaculatus, individuals were divided into two groups using lineages derived from the mitochondrial topology (i.e., lineages 2a and 2b and lineages 2c and 2d). group 1 included d. quadramaculatus individuals from lineages 2a and 2b, and group 2 included d. quadramaculatus individuals from lineages 2c and 2d. group 1 and group 2 table 1. comparative summary statistics of the mitochondrial sequence variation in desmognathus quadramaculatus, d. folkertsi, and d. marmoratus. π: nucleotide diversity per site with jukes and cantor correction; k: mean number of nucleotide differences; η: total number of mutations; θ: the amount of variation expected at each nucleotide site if there is neutral evolution. d. quadramaculatus d. folkertsi d. marmoratus haplotype diversity ± sd 0.950 ± 0.008 0.822 ± 0.083 0.978 ± 0.054 π ± sd 0.230 ± 0.0154 0.012 ± 0.004 0.333 ± 0.039 k 96.184 6.866 160.667 η 439 61 335 θ per site 0.177 0.028 0.245 tajima’s d 0.967 (p > 0.10) -2.305 (p < 0.01) 1.785 (0.10 > p > 0.05) fu and li’s d* -1.551 (p > 0.10) -2.956 (p < 0.05) 1.166 (p > 0.10) fu and li’s f* -0.342 (p > 0.10) -3.227 (p < 0.02) 1.494 (0.05 < p < 0.10) fu’s fs statistic 18.63 -2.401 3.569 table 2. comparative summary statistics of the 12s rrna mitochondrial sequence variation in desmognathus quadramaculatus partitioned by river drainage. symbols as in table 1. tennessee (n = 64) coosa/ tallapoosa (n = 21) chattahoochee (n = 14) savannah (n = 10) new river (n = 10) catawba (n = 11) haplotype diversity ± sd 0.977 ± 0.010 0.948 ± 0.031 1.000 ± 0.027 1.000 ± 0.045 0.878 ± 0.025 1.000 ± 0.039 π ± sd 0.227 ± 0.022 0.284 ± 0.035 0.128 ± 0.006 0.283 ± 0.069 0.2013 ± 0.031 0.307 ± 0.056 k 140.599 156.990 74.099 163.422 112.131 184.164 η 538 369 407 388 375 401 θ per site 0.224 0.186 0.220 0.238 0.141 0.228 186 j.a. wooten and l.j. rissler exhibited expected heterozygosity (hj) values equal to 0.222 and 0.237, respectively. there was little genetic differentiation among study sites of d. quadramaculatus with a total gene diversity (ht) equal to 0.236 and an fst equal to 0.028 (table 5). analysis of molecular variance (amova) for the aflp data using three populations (lineage 1, group 1, and group 2; see description above) and two groups derived from the maximum-likelihood topology (fig. 2; lineage 1 and lineage 2) supported the mtdna conclusions that most of the variation (88.44%) is found within the populations (φct). table 3. partition of genetic variation for mitochondrial sequences in desmognathus quadramaculatus determined by amova. d.f. sum of squares variance component percentage of variation among groups (lineages*) 1 640.945 3.893 5.160 among population (drainages**) within groups 3 440.933 3.376 4.470 within populations 188 12821.412 68.199 90.370 total 192 13903.290 75.468 100.000 * lineages (fig. 2). ** five major river drainage basins including new river, catawba, tennessee, chattahoochee, coosa/tallapoosa, savannah were used. table 4. genetic diversity derived from aflp data partitioned by lineage inferred from the mitochondrial genome for desmognathus quadramaculatus, d. folkertsi, and d. marmoratus. n number of loci % polymorphic loci hj* ± se lineage 1 (d. folkertsi) 10 563 71.4 0.241 ± 0.008 lineage 2a + 2b (d. quadramaculatus and d. marmoratus)** 27 563 68.4 0.222 ± 0.007 lineage 2c + 2d (d. quadramaculatus and d. marmoratus)*** 31 563 71.9 0.237 ± 0.007 *hj: nei’s gene diversity; expected heterozygosity under hardy-weinberg genotypic proportions. ** southern populations. *** northern populations table 5. genetic diversity derived from aflp data computed over all populations for desmognathus quadramaculatus. hw: mean nei’s gene diversity; mean within-population expected heterozygosity under hardy-weinberg genotypic proportions; ht: total gene diversity; hb: nei’s dst; average gene diversity among populations in excess of that observed within populations; fst: wright’s fixation index; proportion of total gene diversity that occurs among as opposed to within populations. n hw ± se ht ± se hb ± se fst ± se 58 0.2293 ± 0.0074 0.2358 ± 0.0013 0.0066 ± 0.0001 0.0279 ± 0.0312 187ecology and genetic of black-bellied salamanders the proportion of the variation explained by among groups (φsc) and among populations within groups (φst) was small, comprising only 5.94% and 5.62%, respectively (table 6). nei’s d value between the lineage 1 and lineage 2 (fig. 2) in the d. quadramaculatus species complex was 0.028, and that between d. quadramaculatus and d. folkersi was 0.0542. the optimal number of populations (k) computed in structure v2.2 (pritchard et al., 2000; falush et al., 2003; falush et al., 2007) using the aflp fragment data was three; however, those three groups were not congruent with the three studied taxa. the estimated probability of three populations (ln p(d)) was equal to –16557.7, with a variance of ln p(d) equal to 1048.5, α = 0.2552, and fst = 0.3317. desmognathus folkertsi was placed into the first inferred cluster 43.3% of the time, more often than in either the second or third inferred cluster. desmognathus quadramaculatus and d. marmoratus were grouped into the first inferred cluster 64.0% of the time, and 10.9% and 25.0% for the second and third inferred clusters, respectively. testing phylogeographic breaks we examined the impact of river drainages and ecoregions on phylogeographic patterns in d. quadramaculatus. we found individuals of d. quadramaculatus to be isolated table 6. partition of genetic variation in desmognathus quadramaculatus determined by amova using aflp fragment data. d.f. sum of squares variance component percentage of variation among groups (species*) 1 231.951 5.264 5.940 among populations (lineages**) within groups 1 222.043 4.978 5.620 within populations 65 5094.329 78.374 88.440 total 67 5548.323 88.616 100.000 * d. folkertsi and d. quadramaculatus only table 7. multiple regression tests of genetic distance, historical and modern river drainage connections, freshwater and terrestrial ecoregions assignment based on geographic locality, and species group based upon uncorrected genetic distance and current taxonomic assignment. matrix b p species-level group* 0.101 0.003 current drainage basin -0.049 0.085 historical drainage basin -0.108 0.037 freshwater ecoregions 0.002 0.474 terrestrial ecoregions 0.116 0.043 * d. quadramaculatus, d. folkertsi, or d. marmoratus 188 j.a. wooten and l.j. rissler by stream (r = 0.335, p < 0.0001) and straight-line (r = 259.526, p = 0.001) distance using the mitochondrial sequences. however, when using the aflp fragment data, d. quadramaculatus populations were only isolated by stream distance (r = 0.135, p = 0.024). we found that current taxonomic species, pliocene river drainage basin, and terrestrial ecoregions explained the observed phylogeographic pattern in d. quadramaculatus (r2 = 0.017; p = 0.013; table 7). neither current river drainage basin nor freshwater ecoregions helped to explain the phylogenetic patterns in d. quadramaculatus (table 7). discussion lineages and population structure – mitochondrial sequences the phylogenetic analysis within the d. quadramaculatus species complex generated from the mitochondrial sequences revealed two major lineages, lineage 1 and lineage 2 (fig. 2). lineage 1 consisted of only d. folkertsi; lineage 2 consisted of both northern and southern populations of d. quadramaculatus and d. marmoratus. the lineages of d. marmoratus formed well-supported branches within d. quadramaculatus. within lineage 2, which contained both d. quadramaculatus and d. marmoratus, four well-supported lineages were uncovered (fig. 2). overall, lineage 2 was separated geographically, with distinct northern (lineage 2c and 2d) and southern (lineage 2a and 2b) lineages. one lineage of particular interest is a single population of d. quadramaculatus found in north carolina (lineage 2d) which formed a well-supported and basal relationship to d. quadramaculatus in lineage 2c (fig. 2). because this basal relationship was formed using individuals only from an isolated, single population located north of the french broad river (fig. 1, map locality 13), future research is underway to investigate surrounding geographic areas to reveal the phylogenetic patterning in this area. overall, lineage 2c appears to be a polytomy with few well-supported branches, which may be indicative of a recent population expansion. within lineage 2c, a single population from tennessee (fig. 1, map locality 5) appears to be a unique evolutionary lineage that is well nested within lineage 2c and is of future research interest (fig. 2). although our sample size was limited for d. marmoratus due to drought conditions during sampling years, the inclusion of these samples in our analyses allowed us to make some comparisons with d. quadramaculatus. our mitochondrial sequence data suggests that southern d. marmoratus populations are nested within southern d. quadramaculatus lineages (see lineage 2a) with strong statistical support (fig. 2). however, due to a small sample size for southern d. marmoratus and the fact that we only used one portion of the mitochondrial genome to discern these fine-scale genetic patterns, our results for the relationship between southern d. quadramaculatus and southern d. marmoratus do not corroborate the published phylogenetic relationships that suggest a sister relationship between southern populations of d. marmoratus with d. folkertsi (jackson, 2005; jones et al., 2006). for northern populations of d. marmoratus (lineage 2c), we uncovered a complex, intertwined relationship between d. marmoratus and d. quadramaculatus, where popu189ecology and genetic of black-bellied salamanders lations of d. marmoratus are more closely related to populations of d. quadramaculatus, rather than to populations of itself. these results corroborate the findings of other authors, and indicate that d. marmoratus, especially northern populations, may not be an exclusive species in relation to d. quadramaculatus (titus and larson, 1996; rissler and taylor, 2003; jackson, 2005; jones et al., 2006). past research using mitochondrial sequence evidence revealed that d. marmoratus and d. quadramaculatus are not monophyletic (titus and larson, 1996; rissler and taylor, 2003; jones et al., 2006); our data revealed similar phylogenetic patterns. voss et al. (1995) found that of 16 allozyme loci, eight were fixed between populations of d. marmoratus north and south of the eastern continental divide. in an unpublished master’s thesis, jackson (2005) revealed that d. quadramaculatus and d. marmoratus from populations in the northern part of the geographic range were nested together when phylogenetic analysis was completed using three portions of the mitochondrial genome (cytochrome b, nd4, and 12s) and a nuclear gene (gapdh). more research that includes additional and strategic population sampling is needed to tease apart these complex evolutionary relationships among d. folkertsi, d. quadramaculatus, and d. marmoratus. lineages and population structure – aflp fragments our study is one of only a few that used aflps to investigate population genetics in plethodontid salamanders (lowe et al., 2006; wooten, 2007; wooten et al., 2010). our aflp fragment data were sufficient to differentiate between d. folkertsi and d. quadramaculatus, but were not able to distinguish between d. marmoratus and d. quadramaculatus. the results from the aflp data and the mitochondrial data were not congruent, indicating that these markers may not be suitable for fine-scale phylogenetic analysis in desmognathan taxa. regardless, future taxonomic revisions will likely be necessary, because nether d. marmoratus or d. quadramaculatus do not appear to be monophyletic lineages. genetic diversity among populations patterns of genetic diversity can often be explained by historical drainage connections for many freshwater species, including fishes and amphibians (mayden, 1988; burridge et al., 2006; jones et al., 2006; kozak et al., 2006). for d. quadramaculatus populations, we found that there was as much genetic variance explained within a single stream as there was between streams or drainages. semi-aquatic salamander species, including d. folkertsi, d. quadramaculatus, and d. monticola, are typically used as fish bait, particularly for fishing for black bass (genus micropterus), and, therefore, bait-bucket release has been used to explain phylogenetic and disjunct geographic range patterns in some taxa (martof, 1953; jensen and waters, 1999; bonett et al., 2007). bait-bucket release may explain some of the genetic partitioning among populations of these and other salamander species (wooten et al., 2010); however, fine-scale population studies and phylogenetic analysis may reveal that some of these populations are disjunct, relict populations that are isolated and not associated with bait-bucket release (camp and wooten, ms under review). 190 j.a. wooten and l.j. rissler phylogeographic breaks and phylogeography current river drainages do not significantly explain the phylogenetic patterns that are observed in the d. quadramaculatus species complex. in fact, our data suggest that historical drainages have played a more important role in shaping these patterns. this is not surprising considering that modern drainages are composites of historical drainages and there is a strong association between historic drainage patterns and phylogenetic relationships (kozak et al., 2006; jones et al., 2006). in our analyses, individuals from the upper savannah river drainage form sister relationships with individuals from the lower tennessee river drainage (fig. 2; quadramaculatus 78-81). in addition, individuals from the catawba river drainage form relationships with individuals from the new river drainage; it has been hypothesized that the catawba river was once part of the new river drainage system (map localities 8-10; jones et al., 2006) and this would explain the observed phylogenetic patterning. it is also possible that the observed phylogeographic patterns are a result of movement among and between populations, across both aquatic and terrestrial routes. however, it is unknown how far individuals of the d. quadramaculatus species complex move across either route; it is difficult to estimate dispersal rates and distances (milá et al., 2010) and these data are absent for most species (grant et al., 2010). that said, we do know, however, it is not unlikely that during times of flooding that these salamanders can use terrestrial routes for dispersal, augmenting gene flow among populations (camp and wooten, ms under review). we also found that the phylogenetic patterns can be explained by breaks in terrestrial, but not freshwater ecoregions. this may help support the idea that salamanders in the d. quadramaculatus complex exhibit phylogenetic patterns due to limited dispersal abilities across terrestrial terrain without the aid of flooding, but not freshwater ecoregions, where the salamanders can move along the waterways for dispersal, which may help to stabilize their populations (grant et al., 2010). in d. quadramaculatus, the patterns of genetic diversity across the landscape have been shaped by historical drainage patterns that have occurred throughout the history of the appalachian mountains. because of the complex topology, including the presence of large rivers and valleys and steep topological relief, many barriers to gene flow exist; phylogeographic patterns of d. quadramaculatus reflect this isolation. we found that paleodrainage patterns influence the genetic diversity of d. quadramaculatus more than current drainage patterns. these patterns are similar to those reported in eurycea bislineata (kozak et al. 2006), and support the idea that paleodrainages directly influence phylogenetic patterns in many fish and salamander that are restricted or may rely on water as a means of dispersal. results presented here further corroborate and extend previous mitochondrial studies that suggest that historical river drainage patterns influence the phylogenetic patterns of desmognathan species (voss et al., 1995; rissler and taylor, 2003; jackson, 2005; jones et al., 2006). conclusions undescribed species, parallel evolution, and morphological conservatism are factors that contribute to the complexity of desmognathan taxonomy (jackson, 2005). desmog191ecology and genetic of black-bellied salamanders nathus quadramaculatus and d. marmoratus exhibited high levels of genetic diversity for desmognathan salamanders; however, this may be due to small sample size for d. marmoratus and the presence of undescribed species in d. quadramaculatus. the genetic diversity of d. quadramaculatus was not partitioned by current river drainage, but the phylogeographic patterns may be explained, at least in part, by historical river drainage connections. in addition, terrestrial ecoregions, rather than freshwater ecoregions, explained the genetic distances between individuals. this provides additional evidence that d. quadramaculatus likely disperses via both terrestrial and aquatic pathways, with gene flow being enhanced through terrestrial routes, but constrained within drainages. thus, future conservation programs that may be developed to protect these salamanders should consider the terrestrial environment in addition to stream quality. in summary, more research is needed to tease out the complexly interwoven nature of the evolutionary history shared by these three salamander species. acknowledgements this manuscript is a result of a chapter from the ph.d. dissertation of jessica wooten under the direction of l. rissler. h. smith-somerville, p. harris, j. lopez-bautista, and c. camp provided comments on earlier versions of the manuscript. many people helped in the field or obtained tissue for us, including s. eagle, w. van devender, a. van devender, c. camp, d. beamer, m. chadwick, c. cox, s. parker, j. hodgson, d. merritt, j. humphries, j. waldron, z. felix, b. sutton, r. makowsky, c. makowsky, s. fields, and w. smith. w. holznagel, l. tolley-jordan, and e. toorens provided assistance with the laboratory work and aflp fragment analysis. p. bradford extracted the stream distances for each locality. all salamander research was approved by the institutional animal care and use committee (iacuc) protocol number 05-242-3 to leslie rissler at the university of alabama. this research was funded by: a nsf deb 0414033 awarded to leslie rissler, american museum of natural history grant awarded to jessica wooten, and the university of alabama. references abell, r., olson, d., dinerstein, e., hurley, p., diggs, j., eichbaum, w., walters, s., wettengel, w., allnutt, t., loucks, c., hedao, p. 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84 .3 90 eu 55 23 42 m on tic ol a 38 c oo sa /t al la po os a g ilm er g a bi g tu rn ip to w n c re ek 34 .7 17 84 .3 90 eu 55 22 73 m on tic ol a 39 c oo sa /t al la po os a g ilm er g a o w lto w n c re ek 34 .6 63 84 .4 41 eu 55 22 45 m on tic ol a 40 c oo sa /t al la po os a lu m pk in g a n im be lw ill c re ek 34 .5 82 84 .1 84 eu 55 22 47 m on tic ol a 54 te nn es se e h ab er sh am g a d em er es t 34 .5 60 83 .5 39 eu 55 22 51 m on tic ol a 49 te nn es se e u ni on g a h el to n c re ek 34 .7 49 83 .9 26 eu 55 22 50 m on tic ol a 40 c oo sa /t al la po os a lu m pk in g a n im be lw ill c re ek 34 .5 82 84 .1 84 eu 55 22 48 m on tic ol a 51 te nn es se e to w ns g a h ia w as se e r iv er 34 .8 24 83 .7 33 eu 55 22 74 m on tic ol a 32 te nn es se e c la y n c m us kr at b ra nc h 35 .0 47 83 .6 09 eu 14 42 07 m on tic ol a 32 te nn es se e c la y n c m us kr at b ra nc h 35 .0 47 83 .6 09 eu 14 42 08 m on tic ol a 53 c ha tt ah oo ch ee w hi te g a sm ith ga ll w oo ds 34 .6 90 83 .7 70 eu 55 22 80 m on tic ol a 39 c oo sa /t al la po os a g ilm er g a h ol ly c re ek 34 .6 63 84 .4 41 eu 14 41 75 2a 1 qu ad ra m ac ul at us 35 c oo sa /t al la po os a m ur ra y g a la ur el c re ek 34 .7 95 84 .6 03 eu 55 22 30 2a 1 qu ad ra m ac ul at us 39 c oo sa /t al la po os a m ur ra y g a o w lto w n c re ek 34 .6 63 84 .4 41 eu 55 22 32 2a 1 qu ad ra m ac ul at us 35 c oo sa /t al la po os a m ur ra y g a la ur el c re ek 34 .7 95 84 .6 03 eu 55 22 31 2a 1 qu ad ra m ac ul at us 40 c oo sa /t al la po os a lu m pk in g a n im be lw ill c re ek 34 .5 82 84 .1 84 eu 55 22 34 2a 1 qu ad ra m ac ul at us 38 c oo sa /t al la po os a g ilm er g a bi g tu rn ip to w n c re ek 34 .7 17 84 .3 90 eu 55 22 33 2a 1 qu ad ra m ac ul at us 10 c at aw ba c al dw el l n c d ix on c re ek 36 .1 07 81 .7 82 eu 55 23 16 2a 2 qu ad ra m ac ul at us 56 sa va nn ah c la y n c tr ib ut ar y of h ia w as se e r iv er 34 .7 60 84 .0 04 eu 14 41 47 2a 2 qu ad ra m ac ul at us 7 n ew r iv er w at au ga n c h ow ar d c re ek 36 .2 81 81 .7 21 eu 14 42 15 2a 2 qu ad ra m ac ul at us 7 n ew r iv er w at au ga n c h ow ar d c re ek 36 .2 81 81 .7 21 eu 55 23 13 201ecology and genetic of black-bellied salamanders li ne ag e sp ec ie s m ap lo ca lit y r iv er d ra in ag e c ou nt y st at e lo ca lit y la tit ud e lo ng itu de a cc es si on n um be r 2a 2 qu ad ra m ac ul at us 7 n ew r iv er w at au ga n c h ow ar d c re ek 36 .2 81 81 .7 21 eu 55 23 11 2a 2 qu ad ra m ac ul at us 12 n ew r iv er bu rk e n c st ee le ’s c re ek 35 .9 06 81 .8 29 eu 55 23 27 2a 2 qu ad ra m ac ul at us 7 n ew r iv er w at au ga n c h ow ar d c re ek 36 .2 81 81 .7 21 eu 55 23 09 2a 2 qu ad ra m ac ul at us 7 n ew r iv er w at au ga n c h ow ar d c re ek 36 .2 81 81 .7 21 eu 55 23 12 2a 2 qu ad ra m ac ul at us 7 n ew r iv er w at au ga n c h ow ar d c re ek 36 .2 81 81 .7 21 eu 55 23 14 2a 2 qu ad ra m ac ul at us 12 n ew r iv er bu rk e n c st ee le ’s c re ek 35 .9 06 81 .8 29 eu 55 23 31 2a 2 qu ad ra m ac ul at us 11 n ew r iv er a ve ry n c n or th h ar pe r c re ek 36 .0 07 81 .8 55 eu 55 23 37 2a 2 qu ad ra m ac ul at us 11 n ew r iv er a ve ry n c n or th h ar pe r c re ek 36 .0 07 81 .8 55 eu 55 23 33 2a 2 qu ad ra m ac ul at us 10 c at aw ba c al dw el l n c g re en m ou nt ai n c re ek 36 .1 07 81 .7 82 eu 55 23 23 2a 2 qu ad ra m ac ul at us 11 n ew r iv er a ve ry n c n or th h ar pe r c re ek 36 .0 07 81 .8 55 eu 55 23 35 2a 2 qu ad ra m ac ul at us 11 n ew r iv er a ve ry n c n or th h ar pe r c re ek 36 .0 07 81 .8 55 eu 55 23 36 2a 2 qu ad ra m ac ul at us 12 n ew r iv er bu rk e n c st ee le ’s c re ek 35 .9 06 81 .8 29 eu 55 23 28 2a 2 qu ad ra m ac ul at us 12 n ew r iv er bu rk e n c st ee le ’s c re ek 35 .9 06 81 .8 29 eu 55 23 29 2a 2 qu ad ra m ac ul at us 11 n ew r iv er a ve ry n c n or th h ar pe r c re ek 36 .0 07 81 .8 55 eu 55 23 34 2a 2 qu ad ra m ac ul at us 27 te nn es se e g ra ha m n c ta po co 35 .3 81 83 .9 01 eu 55 23 00 2a 3 qu ad ra m ac ul at us 21 sa va nn ah pi ck en s sc w ild ca t c re ek 35 .0 37 82 .7 08 eu 55 22 37 2a 3 qu ad ra m ac ul at us 23 te nn es se e ja ck so n n c pu m pk in to w n c re ek 35 .2 51 83 .2 89 eu 55 22 95 2a 3 qu ad ra m ac ul at us 22 te nn es se e m ac on n c r ic km an c re ek 35 .2 73 83 .4 01 eu 55 23 03 2a 3 qu ad ra m ac ul at us 22 te nn es se e m ac on n c r ic km an c re ek 35 .2 73 83 .4 01 eu 55 23 02 2a 3 qu ad ra m ac ul at us 26 sa va nn ah o co ne e sc tr ib ut ar y of c ha tt og a r iv er 34 .8 08 83 .1 21 eu 55 23 55 2a 3 qu ad ra m ac ul at us 26 sa va nn ah o co ne e sc tr ib ut ar y of c ha tt og a r iv er 34 .8 08 83 .1 21 eu 55 23 56 2a 3 qu ad ra m ac ul at us 23 te nn es se e ja ck so n sc pu m pk in to w n c re ek 35 .2 51 83 .2 89 eu 55 22 96 2a 4 qu ad ra m ac ul at us 36 c oo sa /t al la po os a g ilm er n c r oc k c re ek 34 .7 81 84 .3 28 eu 55 22 60 2a 4 qu ad ra m ac ul at us 36 c oo sa /t al la po os a g ilm er g a r oc k c re ek 34 .7 81 84 .3 28 eu 55 22 61 2a 4 qu ad ra m ac ul at us 37 c ha tt ah oo ch ee g ilm er g a st an le y c re ek 34 .7 83 84 .3 03 eu 55 22 63 2a 4 qu ad ra m ac ul at us 37 c ha tt ah oo ch ee g ilm er g a r oc k c re ek 34 .7 83 84 .3 03 eu 55 22 62 2a 4 qu ad ra m ac ul at us 35 c oo sa /t al la po os a g ilm er g a h ol ly c re ek 34 .7 95 84 .6 03 eu 14 41 76 2a 4 qu ad ra m ac ul at us 51 te nn es se e to w ns g a h ia w as se e r iv er 34 .8 24 83 .7 33 eu 55 22 75 202 j.a. wooten and l.j. rissler li ne ag e sp ec ie s m ap lo ca lit y r iv er d ra in ag e c ou nt y st at e lo ca lit y la tit ud e lo ng itu de a cc es si on n um be r 2a 4 qu ad ra m ac ul at us 47 te nn es se e u ni on g a h el to n c re ek 34 .7 48 83 .9 09 eu 14 41 93 2a 4 qu ad ra m ac ul at us 47 te nn es se e u ni on g a h el to n c re ek 34 .7 48 83 .9 09 eu 14 41 92 2a 4 qu ad ra m ac ul at us 47 te nn es se e u ni on g a h el to n c re ek 34 .7 48 83 .9 09 eu 14 41 89 2a 4 qu ad ra m ac ul at us 43 te nn es se e u ni on g a c oo pe r’s c re ek 34 .7 90 84 .0 31 eu 14 42 05 2a 4 qu ad ra m ac ul at us 42 te nn es se e u ni on g a fl at c re ek 34 .7 48 84 .0 26 eu 14 41 97 2a 4 qu ad ra m ac ul at us 43 te nn es se e u ni on g a c oo pe r’s c re ek 34 .7 90 84 .0 31 eu 14 42 02 2a 4 qu ad ra m ac ul at us 44 te nn es se e u ni on g a w ol f c re ek 34 .7 68 83 .9 46 eu 55 22 81 2a 4 qu ad ra m ac ul at us 28 te nn es se e m on ro e t n tu rk ey c re ek 35 .3 47 84 .1 93 eu 55 23 72 2a 4 qu ad ra m ac ul at us 28 te nn es se e m on ro e t n tu rk ey c re ek 35 .3 47 84 .1 93 eu 55 23 71 qu ad ra m ac ul at us 51 te nn es se e to w ns g a h ia w as se e r iv er 34 .8 24 83 .7 33 eu 55 22 77 qu ad ra m ac ul at us 33 te nn es se e po lk t n bi g lo st c re ek 35 .1 60 84 .4 69 eu 55 23 69 qu ad ra m ac ul at us 51 te nn es se e to w ns g a h ia w as se e r iv er 34 .8 24 83 .7 33 eu 14 41 87 qu ad ra m ac ul at us 27 te nn es se e g ra ha m n c ta po co 35 .3 81 83 .9 01 eu 55 23 01 qu ad ra m ac ul at us 29 te nn es se e m on ro e t n tr ib ut ar y of t el lic o r iv er 35 .2 58 84 .0 90 eu 55 23 74 qu ad ra m ac ul at us 29 te nn es se e m on ro e t n tr ib ut ar y of t el lic o r iv er 35 .2 58 84 .0 90 eu 55 23 75 qu ad ra m ac ul at us 51 te nn es se e to w ns g a h ia w as se e r iv er 34 .8 24 83 .7 33 eu 55 22 76 qu ad ra m ac ul at us 22 te nn es se e m ac on n c r ic km an c re ek 35 .2 73 83 .4 01 eu 55 23 03 qu ad ra m ac ul at us 16 te nn es se e sw ai n n c n ol an d c re ek 36 .2 81 81 .7 21 eu 55 23 05 qu ad ra m ac ul at us 16 te nn es se e sw ai n n c n ol an d c re ek 36 .2 81 81 .7 21 eu 55 23 06 qu ad ra m ac ul at us 15 te nn es se e bl ou nt t n a br am ’s c re ek 35 .6 08 83 .9 36 eu 55 23 61 qu ad ra m ac ul at us 14 te nn es se e se vi er t n la ur el f al ls 35 .6 67 83 .5 97 eu 55 23 66 qu ad ra m ac ul at us 14 te nn es se e se vi er t n la ur el f al ls 35 .6 67 83 .5 97 eu 55 23 67 2b 1 qu ad ra m ac ul at us 55 c ha tt ah oo ch ee r ab un g a n an cy to w n c re ek 34 .5 04 83 .4 81 eu 55 22 35 2b 1 qu ad ra m ac ul at us 55 c ha tt ah oo ch ee r ab un g a n an cy to w n c re ek 34 .5 04 83 .4 81 eu 55 22 56 2b 1 qu ad ra m ac ul at us 55 c ha tt ah oo ch ee r ab un g a n an cy to w n c re ek 34 .5 04 83 .4 81 eu 55 22 57 2b 1 qu ad ra m ac ul at us 55 c ha tt ah oo ch ee r ab un g a n an cy to w n c re ek 34 .5 04 83 .4 81 eu 55 22 58 2b 1 qu ad ra m ac ul at us 54 c ha tt ah oo ch ee h ab er sh am g a d em er es t 34 .5 60 83 .5 39 eu 55 22 52 2b 1 qu ad ra m ac ul at us 54 c ha tt ah oo ch ee h ab er sh am g a d em er es t 34 .5 60 83 .5 39 eu 55 22 54 203ecology and genetic of black-bellied salamanders li ne ag e sp ec ie s m ap lo ca lit y r iv er d ra in ag e c ou nt y st at e lo ca lit y la tit ud e lo ng itu de a cc es si on n um be r 2b 1 qu ad ra m ac ul at us 54 c ha tt ah oo ch ee h ab er sh am g a d em er es t 34 .5 60 83 .5 39 eu 55 22 53 2b 1 qu ad ra m ac ul at us 24 te nn es se e m ac on n c g le nn f al ls 35 .0 35 83 .2 37 eu 55 22 86 2b 1 qu ad ra m ac ul at us 24 te nn es se e m ac on n c g le nn f al ls 35 .0 35 83 .2 37 eu 55 22 87 2b 1 qu ad ra m ac ul at us 22 te nn es se e m ac on n c r ic km an c re ek 35 .2 73 83 .4 01 eu 55 23 04 2b 1 qu ad ra m ac ul at us 25 sa va nn ah o co ne e sc tr ib ut ar y of c ha tt og a r iv er 34 .9 74 83 .1 10 eu 55 23 51 2b 1 qu ad ra m ac ul at us 25 sa va nn ah o co ne e sc tr ib ut ar y of c ha tt og a r iv er 34 .9 74 83 .1 10 eu 55 23 52 2b 1 qu ad ra m ac ul at us 23 te nn es se e ja ck so n n c pu m pk in to w n c re ek 35 .2 51 83 .2 89 eu 55 22 94 qu ad ra m ac ul at us 26 sa va nn ah o co ne e sc tr ib ut ar y of c ha tt og a r iv er 34 .8 08 83 .1 21 eu 55 23 54 2b 1 qu ad ra m ac ul at us 19 te nn es se e tr an sy lv an ia n c lo ok in g g la ss f al ls 35 .2 89 82 .7 61 eu 55 22 88 2b 1 qu ad ra m ac ul at us 23 te nn es se e ja ck so n n c pu m pk in to w n c re ek 35 .2 51 83 .2 89 eu 55 22 97 2b 1 qu ad ra m ac ul at us 21 sa va nn ah pi ck en s sc w ild ca t c re ek 35 .0 37 82 .7 08 eu 55 22 36 2b 1 qu ad ra m ac ul at us 18 te nn es se e h ay w oo d n c h un gr y c re ek 35 .3 68 82 .8 17 eu 55 22 89 2b 1 qu ad ra m ac ul at us 18 te nn es se e h ay w oo d n c h un gr y c re ek 35 .3 68 82 .8 17 eu 55 22 90 2b 1 qu ad ra m ac ul at us 21 sa va nn ah pi ck en s sc w ild ca t c re ek 35 .0 35 83 .2 37 eu 55 23 53 2b 1 qu ad ra m ac ul at us 20 sa va nn ah pi ck en s sc ea st at oe c re ek 35 .0 51 82 .8 19 eu 55 23 50 2b 2 qu ad ra m ac ul at us 40 c oo sa /t al la po os a lu m pk in g a n im be lw ill c re ek 34 .5 82 84 .1 84 eu 55 22 46 2b 2 qu ad ra m ac ul at us 37 c ha tt ah oo ch ee g ilm er g a st an le y c re ek 34 .7 83 84 .3 03 eu 55 22 64 2b 2 qu ad ra m ac ul at us 41 te nn es se e u ni on g a su ch es c re ek 34 .7 01 84 .0 40 eu 14 42 06 2b 2 qu ad ra m ac ul at us 51 te nn es se e to w ns g a h ia w as se e r iv er 34 .8 24 83 .7 33 eu 14 41 88 2b 2 qu ad ra m ac ul at us 51 te nn es se e to w ns g a h ia w as se e r iv er 34 .8 24 83 .7 33 eu 55 22 78 2b 2 qu ad ra m ac ul at us 52 c ha tt ah oo ch ee h ab er sh am g a sp oi lc an e c re ek 34 .7 60 83 .7 52 eu 55 22 82 2b 2 qu ad ra m ac ul at us 32 te nn es se e c la y n c m us kr at b ra nc h 35 .0 47 83 .6 09 eu 14 42 09 2b 3 qu ad ra m ac ul at us 40 c oo sa /t al la po os a lu m pk in g a n im be lw ill c re ek 34 .5 82 84 .1 84 eu 55 22 49 2b 3 qu ad ra m ac ul at us 44 te nn es se e u ni on g a w es t f or k of w ol f c re ek 34 .7 68 83 .9 46 eu 14 41 71 2b 3 qu ad ra m ac ul at us 54 c ha tt ah oo ch ee h ab er sh am g a d em er es t 34 .5 60 83 .5 39 eu 55 22 55 2b 3 qu ad ra m ac ul at us 34 c oo sa /t al la po os a fa nn in g a st ar c re ek 34 .8 71 84 .2 03 eu 55 22 70 2b 3 qu ad ra m ac ul at us 43 te nn es se e u ni on g a c oo pe r’s c re ek 34 .7 90 84 .0 31 eu 14 42 00 2b 3 qu ad ra m ac ul at us 43 te nn es se e u ni on g a c oo pe r’s c re ek 34 .7 90 84 .0 31 eu 14 41 99 204 j.a. wooten and l.j. rissler li ne ag e sp ec ie s m ap lo ca lit y r iv er d ra in ag e c ou nt y st at e lo ca lit y la tit ud e lo ng itu de a cc es si on n um be r 2b 3 qu ad ra m ac ul at us 34 c oo sa /t al la po os a fa nn in g a st ar c re ek 34 .8 71 84 .2 03 eu 55 22 69 2b 3 qu ad ra m ac ul at us 34 c oo sa /t al la po os a fa nn in g a st ar c re ek 34 .8 71 84 .2 03 eu 55 22 71 2b 3 qu ad ra m ac ul at us 44 te nn es se e u ni on g a w es t f or k of w ol f c re ek 34 .7 68 83 .9 46 eu 14 41 67 2b 3 qu ad ra m ac ul at us 44 te nn es se e u ni on g a w es t f or k of w ol f c re ek 34 .7 68 83 .9 46 eu 14 41 68 2b 3 qu ad ra m ac ul at us 44 te nn es se e u ni on g a w es t f or k of w ol f c re ek 34 .7 68 83 .9 46 eu 14 41 70 2b 3 qu ad ra m ac ul at us 47 te nn es se e u ni on g a h el to n c re ek 34 .7 48 83 .9 09 eu 14 41 94 2b 3 qu ad ra m ac ul at us 42 te nn es se e u ni on g a fl at c re ek 34 .7 48 84 .0 26 eu 14 41 96 2b 3 qu ad ra m ac ul at us 44 te nn es se e u ni on g a w es t f or k of w ol f c re ek 34 .7 68 83 .9 46 eu 14 41 69 2b 3 qu ad ra m ac ul at us 43 te nn es se e u ni on g a c oo pe r’s c re ek 34 .7 90 84 .0 31 eu 14 41 98 2b 3 qu ad ra m ac ul at us 36 c oo sa /t al la po os a g ilm er g a r oc k c re ek 34 .7 81 84 .3 28 eu 55 22 59 2b 3 qu ad ra m ac ul at us 38 c oo sa /t al la po os a g ilm er g a bi g tu rn ip to w n c re ek 34 .7 17 84 .3 90 eu 55 23 48 2b 3 qu ad ra m ac ul at us 38 c oo sa /t al la po os a g ilm er g a bi g tu rn ip to w n c re ek 34 .7 17 84 .3 90 eu 55 23 44 qu ad ra m ac ul at us 38 c oo sa /t al la po os a g ilm er g a bi g tu rn ip to w n c re ek 34 .7 17 84 .3 90 eu 55 23 49 qu ad ra m ac ul at us 38 c oo sa /t al la po os a g ilm er g a bi g tu rn ip to w n c re ek 34 .7 17 84 .3 90 eu 55 23 43 qu ad ra m ac ul at us 38 c oo sa /t al la po os a g ilm er g a bi g tu rn ip to w n c re ek 34 .7 17 84 .3 90 eu 55 23 45 qu ad ra m ac ul at us 38 c oo sa /t al la po os a g ilm er g a bi g tu rn ip to w n c re ek 34 .7 17 84 .3 90 eu 55 23 47 qu ad ra m ac ul at us 38 c oo sa /t al la po os a g ilm er g a bi g tu rn ip to w n c re ek 34 .7 17 84 .3 90 eu 55 23 46 2b 3 qu ad ra m ac ul at us 37 c ha tt ah oo ch ee g ilm er g a st an le y c re ek 34 .7 83 84 .3 03 eu 55 22 65 2b 3 qu ad ra m ac ul at us 37 c ha tt ah oo ch ee g ilm er g a st an le y c re ek 34 .7 83 84 .3 03 eu 55 22 66 2b 3 qu ad ra m ac ul at us 37 c ha tt ah oo ch ee g ilm er g a st an le y c re ek 34 .7 83 84 .3 03 eu 55 22 67 qu ad ra m ac ul at us 2 n ew r iv er n ic ho la s w v c ol lis on c re ek 38 .1 13 81 .1 44 eu 14 41 42 qu ad ra m ac ul at us 1 n ew r iv er fa ye tt e w v g la de c re ek 38 .1 91 80 .8 97 eu 55 23 96 2c 1 qu ad ra m ac ul at us 46 te nn es se e u ni on g a u nn am ed tr ib ut ar y of n ot te ly r iv er 34 .7 50 83 .8 50 eu 55 22 68 2c 1 qu ad ra m ac ul at us 6 n ew r iv er h en de rs on n c w es t f or k of f re nc h br oa d r iv er 36 .2 27 81 .4 35 eu 55 23 07 2c 1 qu ad ra m ac ul at us 9 c at aw ba w at au ga n c g re en m ou nt ai n c re ek 36 .1 14 81 .7 78 eu 55 23 18 2c 1 qu ad ra m ac ul at us 9 c at aw ba w at au ga n c m id dl e fo rk a t p ay ne ’s br an ch 36 .1 14 81 .7 78 eu 55 23 24 205ecology and genetic of black-bellied salamanders li ne ag e sp ec ie s m ap lo ca lit y r iv er d ra in ag e c ou nt y st at e lo ca lit y la tit ud e lo ng itu de a cc es si on n um be r qu ad ra m ac ul at us 6 n ew r iv er h en de rs on n c w es t f or k of f re nc h br oa d r iv er 36 .2 27 81 .4 35 eu 55 22 83 2c 2 qu ad ra m ac ul at us 2 n ew r iv er n ic ho la s w v c ol lis on c re ek 38 .1 13 81 .1 44 eu 14 41 44 2c 2 qu ad ra m ac ul at us 4 n ew r iv er g ile s va la ur el b ra nc h 37 .3 92 80 .6 42 eu 55 23 82 2c 2 qu ad ra m ac ul at us 2 n ew r iv er n ic ho la s w v c ol lis on c re ek 38 .1 13 81 .1 44 eu 55 22 41 2c 2 qu ad ra m ac ul at us 6 n ew r iv er h en de rs on n c w es t f or k of f re nc h br oa d r iv er 36 .2 27 81 .4 35 eu 55 23 15 2c 2 qu ad ra m ac ul at us 6 n ew r iv er h en de rs on n c w es t f or k of f re nc h br oa d r iv er 36 .2 27 81 .4 35 eu 55 23 32 2c 2 qu ad ra m ac ul at us 3 n ew r iv er m er ce r w v u nn am ed tr ib ut ar y of b lu est on e r iv er 37 .3 77 81 .2 24 eu 55 24 06 2c 2 qu ad ra m ac ul at us 4 n ew r iv er g ile s va la ur el b ra nc h 37 .3 92 80 .6 42 eu 55 23 81 2c 2 qu ad ra m ac ul at us 4 n ew r iv er g ile s va la ur el b ra nc h 37 .3 92 80 .6 42 eu 55 23 79 2c 2 qu ad ra m ac ul at us 6 n ew r iv er h en de rs on n c w es t f or k of f re nc h br oa d r iv er 36 .2 27 81 .4 35 eu 55 22 84 2c 2 qu ad ra m ac ul at us 1 n ew r iv er fa ye tt e w v g la de c re ek 38 .1 91 80 .8 97 eu 55 24 02 2c 2 qu ad ra m ac ul at us 3 n ew r iv er m er ce r w v u nn am ed tr ib ut ar y of b lu est on e r iv er 37 .3 77 81 .2 24 eu 55 24 09 2c 2 qu ad ra m ac ul at us 2 n ew r iv er n ic ho la s w v c ol lis on c re ek 38 .1 13 81 .1 44 eu 55 22 38 2c 2 qu ad ra m ac ul at us 4 n ew r iv er g ile s va la ur el b ra nc h 37 .3 92 80 .6 42 eu 55 23 83 2c 2 qu ad ra m ac ul at us 1 n ew r iv er fa ye tt e w v g la de c re ek 38 .1 91 80 .8 97 eu 55 24 03 2c 2 qu ad ra m ac ul at us 6 n ew r iv er h en de rs on n c w es t f or k of f re nc h br oa d r iv er 36 .2 27 81 .4 35 eu 55 22 98 2c 2 qu ad ra m ac ul at us 4 n ew r iv er g ile s va la ur el b ra nc h 37 .3 92 80 .6 42 eu 55 23 80 2c 2 qu ad ra m ac ul at us 6 n ew r iv er h en de rs on n c w es t f or k of f re nc h br oa d r iv er 36 .2 27 81 .4 35 eu 55 23 08 2c 2 qu ad ra m ac ul at us 3 n ew r iv er m er ce r w v u nn am ed tr ib ut ar y of b lu est on e r iv er 37 .3 77 81 .2 24 eu 55 24 13 2c 3 qu ad ra m ac ul at us 5 te nn es se e su lli va n t n h ar pe r’s c re ek 36 .4 75 82 .0 90 eu 55 23 57 206 j.a. wooten and l.j. rissler li ne ag e sp ec ie s m ap lo ca lit y r iv er d ra in ag e c ou nt y st at e lo ca lit y la tit ud e lo ng itu de a cc es si on n um be r 2c 3 qu ad ra m ac ul at us 5 te nn es se e su lli va n t n h ar pe r’s c re ek 36 .4 75 82 .0 90 eu 55 23 60 2c 3 qu ad ra m ac ul at us 5 te nn es se e su lli va n t n h ar pe r’s c re ek 36 .4 75 82 .0 90 eu 55 23 68 2c 3 qu ad ra m ac ul at us 5 te nn es se e su lli va n t n h ar pe r’s c re ek 36 .4 75 82 .0 90 eu 55 23 65 2c 3 qu ad ra m ac ul at us 5 te nn es se e su lli va n t n h ar pe r’s c re ek 36 .4 75 82 .0 90 eu 55 23 73 2c 3 qu ad ra m ac ul at us 5 te nn es se e su lli va n t n h ar pe r’s c re ek 36 .4 75 82 .0 90 eu 55 23 64 2c 3 qu ad ra m ac ul at us 5 te nn es se e su lli va n t n h ar pe r’s c re ek 36 .4 75 82 .0 90 eu 55 23 58 2c 3 qu ad ra m ac ul at us 5 te nn es se e su lli va n t n h ar pe r’s c re ek 36 .4 75 82 .0 90 eu 55 23 59 2c 3 qu ad ra m ac ul at us 5 te nn es se e su lli va n t n h ar pe r’s c re ek 36 .4 75 82 .0 90 eu 55 23 63 2c 3 qu ad ra m ac ul at us 5 te nn es se e su lli va n t n h ar pe r’s c re ek 36 .4 75 82 .0 90 eu 55 23 70 qu ad ra m ac ul at us 1 n ew r iv er fa ye tt e w v g la de c re ek 38 .1 91 80 .8 97 eu 55 23 94 qu ad ra m ac ul at us 1 n ew r iv er fa ye tt e w v g la de c re ek 38 .1 91 80 .8 97 eu 55 24 01 qu ad ra m ac ul at us 3 n ew r iv er m er ce r w v u nn am ed tr ib ut ar y of b lu est on e r iv er 37 .3 77 81 .2 24 eu 55 24 10 qu ad ra m ac ul at us 1 n ew r iv er fa ye tt e w v g la de c re ek 38 .1 91 80 .8 97 eu 55 24 00 qu ad ra m ac ul at us 1 n ew r iv er fa ye tt e w v g la de c re ek 38 .1 91 80 .8 97 eu 55 24 04 qu ad ra m ac ul at us 2 n ew r iv er n ic ho la s w v c ol lis on c re ek 38 .1 13 81 .1 14 eu 55 22 40 qu ad ra m ac ul at us 1 n ew r iv er fa ye tt e w v g la de c re ek 38 .1 91 80 .8 97 eu 55 23 98 qu ad ra m ac ul at us 4 n ew r iv er g ile s va la ur el b ra nc h 37 .3 92 80 .6 42 eu 55 23 77 qu ad ra m ac ul at us 3 n ew r iv er m er ce r w v u nn am ed tr ib ut ar y of b lu est on e r iv er 37 .3 77 81 .2 24 eu 55 24 12 qu ad ra m ac ul at us 6 n ew r iv er h en de rs on n c w es t f or k of f re nc h br oa d r iv er 36 .2 27 81 .4 35 eu 55 22 85 qu ad ra m ac ul at us 4 n ew r iv er g ile s va la ur el b ra nc h 37 .3 92 80 .6 42 eu 55 23 84 qu ad ra m ac ul at us 6 n ew r iv er h en de rs on n c w es t f or k of f re nc h br oa d r iv er 36 .2 27 81 .4 35 eu 55 23 38 qu ad ra m ac ul at us 1 n ew r iv er fa ye tt e w v g la de c re ek 38 .1 91 80 .8 97 eu 55 23 99 qu ad ra m ac ul at us 3 n ew r iv er m er ce r w v u nn am ed tr ib ut ar y of b lu est on e r iv er 37 .3 77 81 .2 24 eu 55 24 11 207ecology and genetic of black-bellied salamanders li ne ag e sp ec ie s m ap lo ca lit y r iv er d ra in ag e c ou nt y st at e lo ca lit y la tit ud e lo ng itu de a cc es si on n um be r qu ad ra m ac ul at us 6 n ew r iv er h en de rs on n c w es t f or k of f re nc h br oa d r iv er 36 .2 27 81 .4 35 eu 55 23 39 qu ad ra m ac ul at us 3 n ew r iv er m er ce r w v u nn am ed tr ib ut ar y of b lu est on e r iv er 37 .3 77 81 .2 24 eu 55 24 06 qu ad ra m ac ul at us 1 n ew r iv er fa ye tt e w v g la de c re ek 38 .1 91 80 .8 97 eu 55 23 95 qu ad ra m ac ul at us 2 n ew r iv er n ic ho la s w v c ol lis on c re ek 38 .1 13 81 .1 44 eu 55 22 43 qu ad ra m ac ul at us 4 n ew r iv er g ile s va la ur el b ra nc h 37 .3 92 80 .6 42 eu 55 23 76 qu ad ra m ac ul at us 1 n ew r iv er fa ye tt e w v g la de c re ek 38 .1 91 80 .8 97 eu 55 23 97 qu ad ra m ac ul at us 2 n ew r iv er n ic ho la s w v c ol lis on c re ek 38 .1 13 81 .1 44 eu 55 22 39 qu ad ra m ac ul at us 3 n ew r iv er m er ce r w v u nn am ed tr ib ut ar y of b lu est on e r iv er 37 .3 77 81 .2 24 eu 55 24 14 qu ad ra m ac ul at us 3 n ew r iv er m er ce r w v u nn am ed tr ib ut ar y of b lu est on e r iv er 37 .3 77 81 .2 24 eu 55 24 14 qu ad ra m ac ul at us 9 c at aw ba w at au ga n c g re en m ou nt ai n c re ek 36 .1 14 81 .7 78 eu 55 23 19 qu ad ra m ac ul at us 7 n ew r iv er w at au ga n c h ow ar d c re ek 36 .2 81 81 .7 21 eu 14 42 14 qu ad ra m ac ul at us 2 n ew r iv er n ic ho la s w v c ol lis on c re ek 38 .1 13 81 .1 44 eu 55 22 42 qu ad ra m ac ul at us 7 n ew r iv er w at au ga n c h ow ar d c re ek 36 .2 81 81 .7 21 eu 14 42 13 qu ad ra m ac ul at us 9 c at aw ba w at au ga n c g re en m ou nt ai n c re ek 36 .1 14 81 .7 78 eu 55 23 17 qu ad ra m ac ul at us 8 c at aw ba w at au ga n c m id dl e fo rk a t p ay ne ’s br an ch 36 .1 85 81 .6 54 eu 55 23 25 qu ad ra m ac ul at us 8 c at aw ba w at au ga n c m id dl e fo rk a t p ay ne ’s br an ch 36 .1 85 81 .6 54 eu 55 23 26 qu ad ra m ac ul at us 12 n ew r iv er bu rk e n c st ee le ’s c re ek 35 .9 06 81 .8 29 eu 55 23 30 2d qu ad ra m ac ul at us 13 te nn es se e m ad is on n c tr ib ut ar y of b ig l au ra l c re ek 35 .9 83 82 .6 62 eu 55 22 91 2d qu ad ra m ac ul at us 13 te nn es se e m ad is on n c tr ib ut ar y of b ig l au ra l c re ek 35 .9 83 82 .6 62 eu 55 29 2 2d qu ad ra m ac ul at us 13 te nn es se e m ad is on n c tr ib ut ar y of b ig l au ra l c re ek 35 .9 83 82 .6 62 eu 55 29 3 *i nd iv id ua ls fr om lo ca lit ie s 17 , 3 0, 3 1, a nd 4 8 w er e de le te d w he n id en tic al h ap lo ty pe s w er e re m ov ed . v ou ch er n um be rs : 1 u a h c 1 58 24 , 2 u a h c 1 58 25 , 3 u a h c 1 58 27 , 4 u a h c 1 58 26 , 5 u a h c 1 58 29 , 6 u a h c 1 58 30 , 7 u a h c 1 58 32 , 8 u a h c 1 58 31 , 9 u a h c 15 81 9, 10 u a h c 1 58 21 , 1 1 u a h c 1 58 34 , 1 2 u a h c 1 58 45 , 1 3 a pp su 2 54 02 , 1 4 a pp su 2 54 03 , 1 5 u a h c 1 58 38 , 1 6 u a h c 1 58 33 , 1 7 u a h c 1 58 42 , 1 8 u a h c 1 58 44 , 19 a pp su 2 45 04 , 2 0 u a h c 1 58 43 , 2 1 u a h c 1 56 52 , 2 2 u a h c 1 56 53 , 2 3 a pp su 2 45 09 , 2 4 a pp su 2 45 07 , 2 5 a pp su 2 45 10 , 2 6 a pp su 2 45 05 , 2 7 a pp su 2 45 06 208 j.a. wooten and l.j. rissler appendix 2. individuals from genbank used in the genetic analyses. lineage species accession number county state citation monticola af437369 giles va rissler and taylor 2003 monticola af437362 craig va rissler and taylor 2003 monticola af437353 giles va rissler and taylor 2003 monticola ay549660 giles va rissler et. al. 2004 monticola ay549650 craig va rissler et. al. 2004 monticola af437351 giles va rissler and taylor 2003 monticola ay549666 giles va rissler et. al. 2004 monticola ay549644 craig va rissler et. al. 2004 monticola ay549657 giles va rissler et. al. 2004 monticola ay549679 unknown nc rissler et. al. 2004 monticola ay549673 montgomery va rissler et. al. 2004 monticola af437369 giles va rissler and taylor 2003 2-c1 marmoratus af437329 caldwell nc rissler and taylor 2003 2-c3 marmoratus af437336 smyth va rissler and taylor 2003 2-b1 quadramaculatus af437409 henderson nc rissler and taylor 2003 2-c1 quadramaculatus af437331 giles va rissler and taylor 2003 2-c1 quadramaculatus af437332 smyth va rissler and taylor 2003 2-c1 quadramaculatus af437320 giles va rissler and taylor 2003 2-c1 quadramaculatus af437325 giles va rissler and taylor 2003 2-c1 quadramaculatus af437321 giles va rissler and taylor 2003 2-c1 quadramaculatus af437322 giles va rissler and taylor 2003 2-c1 quadramaculatus af437326 giles va rissler and taylor 2003 2-c1 quadramaculatus af437324 giles va rissler and taylor 2003 2-c1 quadramaculatus af437333 giles va rissler and taylor 2003 quadramaculatus af437323 giles va rissler and taylor 2003 quadramaculatus af437319 giles va rissler and taylor 2003 quadramaculatus af437330 giles va rissler and taylor 2003 quadramaculatus af437335 giles va rissler and taylor 2003 quadramaculatus af437328 giles va rissler and taylor 2003 quadramaculatus af437327 giles va rissler and taylor 2003 quadramaculatus af437334 giles va rissler and taylor 2003 2-d quadramaculatus af437337 giles va rissler and taylor 2003 aeneus af437410 graham nc rissler and taylor 2003 fuscus af437405 giles va rissler and taylor 2003 fuscus af437406 giles va rissler and taylor 2003 imitator af437408 unknown tn rissler and taylor 2003 wrighti af437317 macon nc rissler and taylor 2003 wrighti ay135559 sevier tn crespi et al. 2003 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 8(2): 115-122, 2013 the advertisement and release calls of melanophryniscus pachyrhynus (miranda-ribeiro, 1920) from the central region of rio grande do sul, southern brazil vinícius matheus caldart¹,*, tiago gomes dos santos², raúl maneyro³ ¹ universidade federal de santa maria, programa de pós-graduação em biodiversidade animal. av. roraima, n° 1000. cep 97.105-900. santa maria, rs, brazil. *corresponding author. email: viniciuscaldart@yahoo.com.br ² universidade federal do pampa (unipampa). av. antônio trilha, nº 1847. cep 97.300-000. são gabriel, rs, brazil ³ laboratorio de sistemática e historia natural de vertebrados, facultad de ciencias, udelar. igua, 4225. cp 11400. montevideo, uruguay submitted on 2013, 17th april; revised on 2013, 11th june; accepted on 2013, 22nd august. abstract. the redbelly toad melanophryniscus pachyrhynus (miranda-ribeiro, 1920) is a poorly known toad belonging to the m. tumifrons group, which inhabits rocky open areas on top of hilly landscapes of the uruguayan savanna ecoregion in uruguay and southern brazil. in this study we describe the advertisement and release calls from a redbelly toad population from the central rio grande do sul, southern brazil, based on recordings obtained during the winter season of 2011. the advertisement call consists of a complex call with two segments, the first segment comprising a series of single, unpulsed notes, and the second comprising a multi-pulsed note (a trill) highly variable in duration. the mean dominant frequency of the call is 2668 ± 154 hz (2261–2932 hz) and mean call duration is 37.07 ± 23.97 s (6.64–75.20 s). this feature, however, is highly variable depending on the duration of the second segment, which may be notably longer compared to calls of other species of the genus melanophryniscus. the release call was recorded during amplexus of up to four individuals. it consists of a short trill with a dominant frequency of approximately 2245 hz, emitted as a series of five to six pulsed notes, each comprising five or six pulses. the advertisement call of melanophryniscus pachyrhynus is compared with those of four other melanophryniscus species, and the similarities in habitat requirements and in the temporal pattern of reproduction for species of the m. tumifrons group are discussed. keywords. call description, reproduction, redbelly toad, bufonidae, grasslands, uruguayan savanna ecoregion. introduction the bufonid genus melanophryniscus, currently composed of 26 species of small neotropical toads, occurs from the interandean valleys of southern bolivia to paraguay, northern argentina, southern and southeastern brazil, and uruguay (frost, 2013). according to morphological features, these toads are arranged into three phenetic species groups: the m. moreirae, m. stelzneri and m. tumifrons groups (cruz and caramaschi, 2003). the m. tumifrons group currently includes eight species that share features such as highly developed warts with an apical corneous spine on the dorsal surfaces and flanks, a dorsal color pattern without contrasting dots, and the presence of a frontal skin gland between the eyes (cruz and caramaschi, 2003; naya et al., 2004). melanophryniscus pachyrhynus (miranda-ribeiro, 1920) is a poorly known toad belonging to the m. tumifrons group, but which is relatively abundant in southern brazil (vaz-silva et al., 2008). recently, based on a comparison of external morphology, osteological and molecular characters, baldo et al. (2012) placed m. orejasmirandai as a junior synonym of m. pachyrhynus and showed that the distribution of m. pachyrhynus currently com116 vinícius matheus caldart, tiago gomes dos santos, raúl maneyro prises localities from central and eastern rio grande do sul, in brazil, and from southern and northeast uruguay. its distribution seems to be associated with upland environments in the uruguayan savanna ecoregion, where it inhabits rocky open areas on top of hilly landscapes (vazsilva et al., 2008; baldo et al., 2012). this toad calls from herbaceous vegetation on the ground and breeds in small temporary streams (santos et al., 2011). anuran advertisement calls are a useful taxonomic character because they are species-specific and serve as a mechanism of reproductive isolation (wells, 1977). in spite of this, no information on the vocalizations of m. pachyrhynus is available, nor for the populations previously referred to as m. orejasmirandai. a recent bioacoustic analysis of the genus melanophryniscus considered the calls of 13 species from the three phenetic groups, but m. pachyrhynus calls were not included (baldo, 2011). in fact, there is a lack of call descriptions for species from the m. tumifrons and m. moreirae groups, in contrast with the call descriptions available for the m. stelzneri group (e.g., baldo and basso, 2004; kwet et al., 2005; ferrari and vaira, 2008; kurth et al., 2013). in this study we describe the advertisement and release calls of m. pachyrhynus for a population from são sepé, a municipality located in the central region of rio grande do sul state, southern brazil, comparing its advertisement call with those of four other species of melanophryniscus. additionally, based on data available in the literature, we discuss the similarities in habitat requirements and in the temporal pattern of reproduction for species of the m. tumifrons group. material and methods calls were recorded in the municipality of são sepé (30°14’56.2”s, 53°35’22.4”w, 162 m a.s.l.), state of rio grande do sul, brazil, on 18th and 19th june 2011. this area belongs to the planalto sul-rio-grandense (or serra do sudeste), a pampean region characterized by rock crystalline shield outcrops covered by a natural mosaic of grassland (campos) and seasonal forests (ibge, 2004). historically, land use in the region was based on cattle raised on natural vegetation, but this economic activity is now being replaced by soybean (summer season) and wheat (winter season) cultivation. the local climate is classified as subtropical wet (cfa in the köppen classification), with rainfall evenly distributed throughout the year (1200–1600 mm), i.e., with no dry season (overbeck et al., 2007). summer temperatures are high (maximum 40 ºc), while winter temperatures are low, with median values less than 15 ºc during the three months period when frosts are common. thus, climatic seasonality is mostly determined by variation in temperature and photoperiod (both et al., 2008). calls were recorded with an olympus vn-6200pc digital recorder with an internal microphone, at a distance of approximately 50 cm from individuals, between 16:00 h and 21:00 h during two rainy days in the winter season. air temperature at the calling sites ranged from 16 ºc to 17 ºc, and water temperature ranged from 16 ºc to 18 ºc. the advertisement call of m. pachyrhynus was described based on a total of 12 calls recorded from six individuals, and was compared to the advertisement calls described for four congeners, whose descriptions included similar call parameters: m. krauczuki (baldo and basso, 2004), m. atroluteus (baldo and basso, 2004), m. montevidensis (kwet et al., 2005) and m. dorsalis (kwet et al., 2005). the release call of m. pachyrhynus was described based on five calls recorded from one individual. we could not analyze the release calls of more individuals because the emission of these calls was sporadic and most calls were masked by the advertisement calls of other males. three specimens were collected and deposited in the herpetological collection of the universidade federal de santa maria (voucher specimens: zufsm 5193, 5195, 5243). calls were analyzed with soundruler software v. 0.9.6.0 (http://soundruler.sourceforge.net; bee, 2004; gridi-papp, 2004) at a sampling frequency of 44100 hz and a resolution of 16 bits. oscillograms and audiospectrograms were also produced in soundruler software, with the following parameters: fast fourier transformation (fft) with 256 points, 100% frame, hanning window type and 90% overlap. the following call parameters were analyzed: dominant frequency (hz), call duration (s), 1st segment duration (s), number of notes of 1st segment, duration of notes of 1st segment (s), internote intervals of 1st segment (s), notes per s of 1st segment, 2nd segment duration (s), number of pulses of 2nd segment, duration of pulses of 2nd segment (s), interpulse intervals of 2nd segment (s), and pulses per s of 2nd segment. values of call parameters are presented in the text as mean ± sd and range. terminology used for call parameters follows heyer et al. (1990) and duellman and trueb (1994), and the definition of the term “note” follows the criteria of mclister et al. (1995), in which a note refers to a unit of sound consisting of one or more pulses, produced during a single airflow cycle. while recording the vocalizations of m. pachyrhynus, it was evident that each individual note of the first segment of the call corresponded to an airflow movement of the vocal sac, whereas pulses of the second segment (the trill) were all the result of a single airflow movement of the vocal sac, i.e., in the trills the vocal sac remained inflated all the time, with no airflow movements of the vocal sac as observed for the production of notes of the first segment. it should be noted that the definition of mclister et al. (1995) has been adopted in recent studies because it establishes a physiological basis for note emission (orrico et al., 2009). moreover, such definition could facilitate interpretations of the parameters of compound calls, such as those known for melanophryniscus species (e.g., kwet et al., 2005). results the advertisement call of melanophryniscus pachyrhynus consists of a compound call with two segments (fig. 1 and suppl. a1). the first segment comprises a series of 117the advertisement and release calls of melanophryniscus pachyrhynus fig. 1. advertisement call of melanophryniscus pachyrhynus recorded in the municipality of são sepé, rio grande do sul, brazil, on 18 june 2011, ~20:00 h, air temperature 16.5 ºc, water temperature 16 ºc. a) oscillogram (top) and audiospectrogram (bottom) of the entire call, b) oscillogram (top) and audiospectrogram (bottom) of a six second section of the call (1st and 2nd segments), c) oscillogram (top) and audiospectrogram (bottom) of one second section of the 2nd segment (the trill) of the call. 118 vinícius matheus caldart, tiago gomes dos santos, raúl maneyro single, unpulsed notes (17 ± 7 notes; range 8–28 notes) with a mean duration of 0.017 ± 0.004 s (0.010–0.026) and a mean time interval of 0.234 ± 0.034 s (0.082–0.322 s) between them, emitted at a mean rate of 3.9 ± 0.5 notes per second (2.9–4.3 notes/s). the first segment of the call has a mean duration of 4.44 ± 1.88 s (1.85–7.67 s). the second segment of the call comprises a multipulsed note (a trill), highly variable in duration (25.27 ± 15.80 s; 4.79–45.75 s), with a mean of 818 ± 490 pulses (164–1382 pulses). the mean pulse duration is 0.012 ± 0.002 s (0.007–0.023 s) and the mean time interval between pulses is 0.014 ± 0.003 s (0.007–0.030 s). pulses are emitted at a mean rate of 32.8 ± 1.5 pulses per second (30.2–34.3 pulses/s). the mean dominant frequency of the call is 2668 ± 154 hz (2261–2932 hz), and the mean call duration is 37.07 ± 23.97 s (6.64–75.20 s), but it is highly variable due to the duration of the second segment, which may be notably longer in comparison to calls of other species of the genus melanophryniscus (table 1). the advertisement call of m. pachyrhynus may be emitted in two different ways, i.e., the second segment (the trill) alone or both segments in sequence. no males were observed emitting only the first segment, and we did not observe any apparent behavioral differences related to call emission. males of m. pachyrhynus called along a temporary stream (50 cm wide) flowing through cultivated land (wheat), in shallow water among herbaceous vegetation based on the ground. the highest male abundance of m. pachyrhynus in calling activity occurred at night (~21:00 h), totaling about 23 individuals. during this period we observed that males alternated calling activity with short dislocations (up to ~40 cm) down and/or upstream searching by females. at least three times we observed moving males being clasped by other males, resulting in an amplexus of up to four individuals. in these cases, clasped males tried to escape and displace the other male by moving along the substrate or by emitting a release call. the release call consists of a short trill, emitted as a series of five to six pulsed notes composed of five or six pulses each, with a dominant frequency of approximately 2245 hz (fig. 2 and suppl. a2). the mean duration of the table 1. comparison of temporal and spectral parameters of the advertisement call of melanophryniscus pachyrhynus (12 calls from six males; air temperature: 16–17 ºc; water temperature: 16 °c) and four other melanophryniscus species. values are presented as mean, followed by range in parenthesis. call parameters m. pachyrhynus 1 m. krauczuki 2 m. atroluteus 2 m. montevidensis 3 m. dorsalis 4 dominant frequency (hz) 2668 (2261–2932) 3300 – 3000 – – (2100–2800) – (2300–3200) call duration (s) 37.07 (6.64–75.20) 32.699 (25.013–36.646) 7.523 (5.09–10.35) – – 1st segment duration (s) 4.44 (1.85–7.67) 2.031 (1.128–3.160) 4.143 (2.575–5.36) 1.98 (1.0–4.5) 1.89 (1.0–2.3) number of notes of 1st segment 17 (8–28) 8.6 (6–12) 20.6 (15–25) 17 (7–28) 13 (6–18) duration of notes of 1st segment (s) 0.017 (0.010–0.026) 0.009 (0.005–0.023) 0.102 (0.006–0.174) 0.0313 (0.021–0.039) 0.0424 (0.02–0.05) internote intervals of 1st segment (s) 0.234 (0.082–0.322) 0.217 (0.147–0.837) 0.091 (0.006–0.229) 0.1035 (0.078–0.13) 0.1402 (0.08–0.17) notes per s of 1st segment 3.9 (2.9–4.3) – – – (8–10) – (4–7) 2nd segment duration (s) 25.27 (4.79–45.75) 30.4548 (23.784–33.408) 3.012 (1.832–4.303) 1.58 (1.2–2.0) 1.50 (0.6–2.2) number of pulses of 2nd segment 818 (164–1382) 1298.5 (1018–1502) 222.38 (139–321) 147 (100–192) 112 (54–162) duration of pulses of 2nd segment (s) 0.012 (0.007–0.023) – – 0.0051 (0.0045–0.006) 0.0071 (0.007–0.009) interpulse intervals of 2nd segment (s) 0.014 (0.007–0.030) – – 0.0052 (0.005–0.006) 0.0069 (0.0065–0.0075) pulses per s of 2nd segment 32.8 (30.2–34.3) 43.67 (42.35–44.95) 75.44 (74.31–76.8) – (85–95) – (74–78) populations from: 1 são sepé, central rio grande do sul, brazil (present study); 2 misiones, argentina (baldo and basso, 2004); 3 la paloma, uruguay (kwet et al., 2005); 4 torres, rio grande do sul, brazil (kwet et al., 2005). 119the advertisement and release calls of melanophryniscus pachyrhynus trills is 0.131 ± 0.009 s (0.119–0.141) and the mean interval between them is 0.699 ± 0.157 s (0.533–0.854). additionally, we observed two male–female pairs in axillary amplexus (suppl. fig. s1) during the night period between 20:00–20:40 h, but we did not observe the egg deposition. we returned to the breeding site after one week and observed m. pachyrhynus tadpoles in the pools remaining in the dry stream bed. discussion the advertisement call of m. pachyrhynus differs from those of m. atroluteus (kwet and miranda, 2001; baldo and basso, 2004), m. dorsalis and m. montevidensis (kwet et al., 2005) in having shorter notes in the first segment and because it has a noticeably longer second segment, consequently presenting a greater number of pulses. on the other hand, the advertisement call of m. pachyrhynus is similar to the call of m. krauczuki (baldo and basso, 2004), at least in the variable duration of the call and the greater number of pulses in the second segment, although the call of m. pachyrhynus can be longer. the mean dominant frequency of the advertisement call of m. pachyrhynus has an intermediate value in comparison with the calls of these species. no other formal call descriptions from the m. tumifrons group are available, but maneyro and kwet (2008) report that the advertisement call of m. devincenzii (m. tumifrons group) from northern uruguay is similar to that of other species in the m. stelzneri group, although also presenting a longer second segment. advertisement calls composed of long trills are known for species of rhinella (guerra et al., 2011; sãopedro et al., 2011), peltophryne (hernández et al., 2010), frostius (juncá et al., 2012), and also for species of melanophryniscus (baldo and basso, 2004), among other bufonids. although some species with trilled calls are known to increase their call duration in response to other males (wells, 2007), the highly variable duration of the advertisement call of m. pachyrhynus is remarkable within the genus melanophryniscus, given that the shortest call recorded has 6.64 s and the longest has 75.20 s. evidence gathered for anurans that call in dense choruses and at high rates revealed that this behavior implies a high energetic cost (wells, 2001), although males can produce long calls at a slow rate in dense choruses, or produce short calls at a high rate in sparse choruses (wells and taigen, 1986). as explosive breeders (sensu wells, 1977), males of m. pachyrhynus probably experience a dense concentration of males over the course of a few days at the breeding sites. thus, it is possible that the variation in call duration may be related to sexual selection dependent on the abundance of conspecific competitors at the breeding sites, since male-male competition for females is expected to be more significant than female choice in explosive breeders (wells, 1977). nevertheless, although the adverfig. 2. release call of a male of melanophryniscus pachyrhynus emitted while being clasped by another male. details with the arrows indicate the pulses of the release call, and the single note that appears around the third pulse onwards represents the beginning of an advertisement call of another nearby male. call recorded in the municipality of são sepé, rio grande do sul, brazil, on 18 june 2011, ~20:30 h, air temperature 16.5 ºc, water temperature 16 ºc. from top to bottom: oscillogram and audiospectrogram. 120 vinícius matheus caldart, tiago gomes dos santos, raúl maneyro tisement call of m. pachyrhynus constitutes a remarkable sexual signal, the functional significance of this exaggerated acoustic signal remains unclear and deserves further studies. release calls usually consist of a series of repeated broad-spectrum notes emitted by males when clasped by other males, indicating to a clasping male that he has grabbed an inappropriate mate (wells, 2007). thus, release calls constitute a signal used for sex recognition (marco et al., 1998; liao and lu, 2009). unlike advertisement calls, release calls are much less studied, although these calls are known for several bufonid species with explosive breeding (garda et al., 2010; guerra et al., 2011; sanabria and quiroga, 2012). release calls of the genus melanoprhyniscus were described for the first time recently for four species, consisting of a short trill which may or may not be frequency-modulated (baldo, 2011). the release call of m. pachyrhynus described here has this same structure and was recorded during dense aggregations of males, in which we observed amplexus of up to four individuals. reproductive aggregations in some melanophryniscus species are known to be male-biased, such as in the case of melanophryniscus rubriventris and melanophryniscus aff. montevidensis from argentina, whose males outnumbered females at all breeding events studied (vaira, 2005; cairo et al., 2013). moreover, males of melanophryniscus rubriventris were found actively searching in order to obtain mates (vaira, 2005). these results suggest that release calls may be common in species of melanophryniscus. most species of the m. tumifrons group breed in temporary or permanent small streams, usually after heavy rainfall (maneyro and kwet, 2008; santos et al., 2011). melanoprhyniscus pachyrhynus was found breeding in small temporary and permanent streams during spring and autumn in uruguay (prigioni and langone, 1990; lavilla and langone, 2004; maneyro and carreira, 2012), and during early summer (santos et al., 2011) and winter (present study) in ephemeral streams in southern brazil. these results indicate that m. pachyrhynus may breed at any season throughout the year, since there is enough rainfall to create ephemeral streams. similar observations were made for melanophryniscus cambaraensis in the campos de cima da serra region, southern brazil (santos et al., 2010), and for melanophryniscus devincenzii in northern uruguay (maneyro and kwet, 2008). based on these findings, we suggest that species of the m. tumifrons group probably share similar habitat requirements, as well as a similar temporal pattern of reproduction, i.e., without a marked climatic seasonality and dependent on rainfall patterns. additional studies are still necessary in order to test this hypothesis for the m. tumifrons species group, although at least for one species of the m. stelzneri group the breeding activity showed annual variation due to variation in rainfall patterns (vaira, 2005). the genus melanophryniscus currently has 26 species of small neotropical toads, but for many of them basic aspects of their natural history and ecology are still poorly known. this scenario is evident for the case of m. pachyrhynus, which for many years was known only for its type locality, and is now considered relatively common in rio grande do sul (vaz-silva et al., 2008). with regard to acoustic communication, call descriptions for species of the m. tumifrons and m. moreirae groups are necessary for a better comparative approach within the species groups, and attention should also be given to the occurrence of release calls within the genus. furthermore, basic investigations including descriptions of breeding habitats, breeding behavior and calling activity, as well as reproductive events, could provide valuable information for this taxon. acknowledgements the authors are grateful to axel kwet and an anonymous reviewer for their valuable suggestions on the manuscript. the first author is grateful to coordenação de aperfeiçoamento de pessoal de nível superior (capes) by the doctorate fellowship granted. authorization for capture of specimens was issued by the instituto chico mendes de conservação da biodiversidade (mma/icmbio) under the license nº 29509-1/2011. supplementary material associated with this article can be found at http://www.unipv.it/webshi/appendix manuscript number 12680. references baldo, d., basso, n.g. 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(2011): breeding habitat and natural history notes of the toad melanophryniscus pachyrhynus (mirandaribeiro, 1920) (anura: bufonidae) in southern brazil. herpetol. bull. 116: 15-18. são-pedro, v.a., medeiros, p.h., garda, a.a. (2011): the advertisement call of rhinella granulosa (anura, bufonidae). zootaxa 3092: 60-62. vaira, m. (2005): annual variation of breeding patterns of the toad, melanophryniscus rubriventris (vellard, 1947). amphibia-reptilia 26: 193-199. vaz-silva, w., balestrin, r.l., di-bernardo, m. (2008): rediscover y of melanophryniscus pachyrhynus (miranda-ribeiro, 1920) (amphibia: anura: bufonidae) in southern brazil, with addenda to species redescription. s. am. j. herpetol. 3: 36-42. wells, k.d. (1977): the social behaviour of anuran amphibians. anim. behav. 25: 666-693. wells, k.d. (2001): the energetic of calling in frogs. in: anuran communication, pp. 45-60. ryan, m.j., ed, smithsonian institution press, washington dc. wells, k.d. (2007): the ecology and behavior of amphibians. the university of chicago press, chicago and london. wells, k.d., taigen, t.l. (1986): the effect of social interactions on calling energetic in the gray treefrog (hyla versicolor). behav. ecol. sociobiol. 19: 9-18. supplementary material fig. s1. amplectant pair of melanophryniscus pachyrhynus observed in the municipality of são sepé (30°14’56.2”s, 53°35’22.4”w, 162 m a.s.l.), rio grande do sul, brazil, on 18 june 2011, ~20:30 h, air temperature 16.5 ºc, water temperature 16 ºc. audio file a1. advertisement call of melanophryniscus pachyrhynus recorded in the municipality of são sepé (30°14’56.2”s, 53°35’22.4”w, 162 m a.s.l.), rio grande do sul, brazil, on 18 june 2011, ~20:30 h, air temperature 16.5 ºc, water temperature 16 ºc. audio file a2. release call of a male of melanophryniscus pachyrhynus emitted while being clasped by another male, recorded in the municipality of são sepé (30°14’56.2”s, 53°35’22.4”w, 162 m a.s.l.), rio grande do sul, brazil, on 18 june 2011, ~20:30 h, air temperature 16.5 ºc, water temperature 16 ºc. the single notes that appear around the third pulse onwards represent the beginning of an advertisement call of another nearby male. acta herpetologica vol. 8, n. 1 june 2013 firenze university press journal of the societas herpetologica italica acta herpetologica acta herpetologica 9(2): 187-202, 2014 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-14384 geographic variation in the morphology of macrovipera lebetina (linnaeus, 1758) (ophidia: viperidae) in iran naeim moradi1,2,*, nasrullah rastegar-pouyani1,2, eskandar rastegar-pouyani1,3 1 iranian plateau herpetology research group (iphrg), faculty of sciences, razi university, 6714967346 kermanshah, iran. *corresponding author. e-mail: naeim.moradi@yahoo.com 2 department of biology, faculty of sciences, razi university, 6714967346 kermanshah, iran 3 department of biology, faculty of sciences, hakim sabzevari university, sabzevar, iran submitted on 2014, 14th april; revised on 2014, 10th september; accepted on 2014, 10th september editor: marco a.l. zuffi abstract. the levantine viper, macrovipera lebetina, has an extensive geographical range being distributed in central asia and the middle east. the species exhibits high levels of polymorphism, especially in colouration and pattern. recent studies revealed significant morphological differences between the two subspecies from northeastern and western portions of iran. however, considering limited geographic samplings, taxonomic status of iranian macrovipera are controversial. in this study, uniand multivariate statistical techniques were used to analyze geographic variation in 31 morphological characters measured in 117 specimens of macrovipera lebetina covering its entire range in iran. sexual dimorphism was obvious in number of scales across the head and subcaudals. univariate analyses detected substantial geographic variation in several meristic characters. pholidosis exhibits a general north-south pattern of variation and most scale counts averaged higher in southern regions. colouration displayed a pattern of strong clinal variation among three broad areas consisting of the combined western and northwestern, northeastern, and southern highland regions. also, morphometric characters exhibited a general north-south pattern of geographic variation and some characters averaged lower in southern regions. populations from the southern regions remained clearly distinct in principal component, cluster and discriminant analyses. in the light of these differences, it is concluded that the southern iranian populations should not be identified as belonging to macrovipera lebetina obtusa (dwigubsky, 1832), which occurs in northwestern and western regions of the iranian plateau. keywords. viperidae, macrovipera lebetina, geographic variation, morphology, statistical analyses, iranian plateau. introduction the genus macrovipera has been described by reuss in 1927, and then resurrected by herrmann et al. (1992). according to lenk et al. (2001), just two species are known: macrovipera schweizeri (werner, 1935), which is endemic to a few islands in the western cyclades (nilson and andrén, 1988) and macrovipera lebetina (linnaeus, 1758). the levantine viper, macrovipera lebetina is distributed from central asia to the middle east (nilson and andrén, 1988; nilson et al., 1988; böhme and wiedl, 1994; göçmen et al., 1996; david et al., 1999; atatür and göçmen, 2001; ananjeva et al., 2006; göçmen et al., 2006). traditionally, up to six distinct subspecies were recognized of m. lebetina: m. l. chernovi (chikin and szczerbak, 1992), m. l. lebetina (linnaeus, 1758), m. l. obtusa (dwigubsky, 1832), m. l. peilei (murray, 1892), m. l. transmediterranea (nilson and andrén, 1988) and m. l. turanica (terentiev and chernov, 1940). recently, stümpel and joger (2009) and stümpel (2012) in their molecular analysis showed that haplotypes of macrovipera lebetina are subdivided into five major lineages which support the validity of the allopatric subspecies lebetina, euphratica, obtusa, turanica and chernovi 188 n. moradi et alii with no samples available from peilei (southern afghanistan and pakistan) and transmediterranea (north africa). one more lebetina-like taxon from south-central iran (jiroft/baft mts.) is genetically very distinct from all other macrovipera lebetina taxa (stümpel, 2012). macrovipera lebetina obtusa (dwigubsky, 1832) has a southernmost range from turkey south to n jordan, eastwards through the caucasus to kashmir and n india (sindaco et al., 2013). macrovipera lebetina chernovi (chikin and szczerbak, 1992) occurs in north-eastern iran, south turkmenistan, and parts of northern afghanistan, tadjikistan, and northern pakistan (phelps, 2010; stümpel, 2012) and is replaced by macrovipera lebetina turanica in central asia. the body colour of blunt-nosed vipers can be virtually any shade of brown, even pinkish, dark or light grey. specimens from cyprus tend to be paler. the bluntnosed viper can possess faint or bold markings and many seem uniform in colour. markings, however distinct, take the form of cross bands, dorsal blotches, and vertical lateral bars of a darker hue than the ground colour. some handsome specimens have a more contrasting colour pattern (mermer et al., 2012). the ventral surface is usually a paler version of the ground colour with or without darker stippling (phelps, 2010). in recent years, iranian researchers such as afroosheh and kazemi (2011), rajabizadeh et al. (2011) and oraie et al. (2012) have done valuable surveys on the taxonomic status of macrovipera lebetina in iran. all of the previous researches have focused on the western and northeastern populations of m. lebetina, whereas southern populations were not investigated. in order to clarify their taxonomic status, in this paper geographic variation in morphology of all populations of macrovipera lebetina across the whole distribution range in iran is discussed. materials and methods specimens and sampling in this study, 117 specimens of m. lebetina were examined across the entire range of species distribution in iran (see appendix). of these, 77 mature and intact individuals (29 males and 41 females) were selected for analysis of metric and meristic characters used in morphological studies of vipers, and 99 specimens were investigated in non-parametric analysis of colour pattern. because of the immaturity or lack of revealed colour pattern, 65 specimens among them were selected for parametric analysis. thirty specimens were captured from june 2012 to late october 2013; more than twenty were released subsequent to measuring morphological characters in the field, whereas the remainders according to pisani (1973) were preserved in ethanol 96% and deposited at the razi university zoological museum (ruzm), museum of shahid bahonar university of kerman (zmsbuk), and sabzevar university herpetological collection (suhc). additional specimens subject of this study come from the herpetological collections (table 1). also, six pictures were selected for analyzing the colour pattern. characters characters used in this research are listed in table 2. all of the specimens were examined for eight metric, seventeen meristic, and six colour pattern characters (table 2). metric characters were evaluated with a digital caliper to the nearest 0.01 mm. because expressing body measurements as a percentage of snout-vent length (svl) does neutralize allometry, we calculated the residual values of each body measurements as a function of svl (babocsay, 2003). all mensural characters as compared to svl were analyzed. table 1. number of specimens subject of present study come from the herpetological collections and used for meristic, mensural, and color pattern analysis. type of analysis herpetological collections zcri zmsbuk suhc ruzm suzm fdoi meristic 43 4 6 4 2 4 mensural 43 4 6 4 2 4 colour pattern 55 7 6 6 3 4 total 61 8 9 8 3 4 zcri: zoological collection of the department of venomous animals and antiserum production, razi vaccine and serum research institute, hesarak, karaj zmsbuk: zoological museum of shahid bahonar university of kerman suhc: sabzevar university herpetological collection ruzm: razi university zoological museum suzm: shiraz university zoological museum fdoi: species diversity museum of department of environment, fars 189geographic variation in the iranian macrovipera lebetina statistical analyses the principal components analysis (pca) based on a correlation matrix of meristic data was used to determine whether distinct geographic units exist within macrovipera lebetina. independent multivariate analyses were conducted on combined sexes. in addition, the cluster observation method was used to explore the population’s groupings, based on significant characters which had r2 > 50%. also discriminant analysis was used for meristic characters, in order to evaluate the actual degree of discrimination among the a priori groups as well as to predict their group membership. moreover, generalized squared distances between groups were computed to show the distance between each pair of groups. to describe dispersal pattern among morphological characters of different localities, descriptive statistical parameters including minimum, maximum, mean and standard deviatable 2. morphological characters used in this study. abbreviation characters meristic characters prev number of preventrals + small gular scales along the midline of the ventral side of the head ven number of ventral scales scd number of subcaudal scales dors1 number of dorsal scale rows, counting across the forepart of the body [one head length behind the posterior end of head]1 dors2 number of dorsals, counted across mid-body (at mid-svl length) dors3 number of dorsals, counted across the hind part of the body, one head length before the anal plate1 ap number of apical scales blspl number of scales between last supralabial scales across the head can number of canthal scales interocular number of scales across head, between the dorsal parts of two eyes spl* number of supralabials scales ifl* number of sublabial (infralabial) scales incir* number of inner circumocular scales outcir* number of outer circumocular scales (around eye and supraocular scales) lor* number of loreal scales supraoc number of supraocular scales bespl number of scales between eye and supralabials mensural characters (mm) svl snout-vent length tal tail length hl head length (from anterior tip of snout to the angle of jaws) hw head width hh head height dbn distance between nostrils iod inter ocular distance sl snout length (distance between rostrum to the anterior edge of eye) colour pattern characters scn the area of the black or dark pigmentation in and around the dorsal blotch, in number of scales. a scale was counted half if it was pigmented over < 50% and one if > 50% of its area (mean of three blotches, halfway between the rostrum and the anus)2 l/wdbr length to width ratio for a dorsal blotch at mid-svl. l/ddbr length of dorsal blotch to distance between two blotches at mid-svl l/dlbr length of lateral blotch to distance between two blotches at mid-svl dbc dorsal base colour: g) gray; gb) grayish brown; b) brown; r) reddish brown ventp ventral pattern: d) scattered dots on the entire ventral surface; dd) ventral surface densely dotted; s) scattered dots on the entire ventral surface with a trapezoid splotch * meristic characters were recorded from both sides and analyzed separately. 1 campbell et al., 2004. 2 babocsay, 2003. 190 n. moradi et alii tion were employed separately for each group. data normality was checked by drawing residual plots: residual versus fits and main effects plot for each character to carrying out the analyses. the existence of sexual dimorphism was checked in all mature specimens using independent sample t-test. because some data was missing, the general linear model anova was used to explore the significant differences by gender, but also sexes combined. statistical analyses for all meristic and colour pattern characters were performed using one-way anova followed by fisher’s lsd method comparison post hoc test to explore the patterns of morphological variation among combined sexes. also, considering that dbc and ventp are qualitative characters, non-parametric analyses for these characters were performed using kruskal-wallis test. the significance level for all the statistical tests was set at p < 0.05. statistical analyses were performed using the minitab® (ver. 16) for the windows. results descriptive statistics ranges and mean ± sd for studied characters in each group are provided in table 3. the specimens were classified according to the different colour patterns and recent studies (oraie et al., 2012), and the distribution of each pattern in iran was mapped (fig. 1). specimens were grouped in four geographic samples based on geographic units: i. khorasan province (kopet dagh mts.); ii. golestan province (area between kopet dagh mts. and alborz mts.); iii. alborz mts. along with the northern part of zagros mts.; iv. southern part of zagros mts. (latitude 33°n) extended south to the persian gulf coast and continuing southeast to sistan mts. (longitude 58°e). results of anova analyses for meristic characters analysis of meristic data indicated that nine characters in males [ven, ap, blspl, spl(l), spl(r), ifl(l), ifl(r), outcir(l) and supraoc], ten characters in females [ven, scd, spl(l), ifl(l), ifl(r), incir(l), incir(r), outcir(l), lor(r) and supraoc] and thirteen characters when sexes were combined [prev, ven, scd, can, interocular, spl(l), spl(r), ifl(l), ifl(r), incir(l), outcir(l), lor(l) and lor(r)] showed statistically significant differences (p < 0.05). results of anova for meristic characters in combined sexes show that prev in group iii was significantly lower (p < 0.05) compared to other groups. the number of ven in group iv are significantly (p < 0.0001) higher than in other groups, whereas scd in group ii are significantly lower compared to other groups. characters of ifl(l) and ifl(r) in group iv show significant higher values (p < 0.0001) in comparison with groups ii and iii, whereas respective values form group iv overlap with those of group i. lor(l) in group ii were significantly higher in comparison with group iv. scale counts of lor(r) and spl(l) in groups i and iv are significantly increased compared to groups ii and iii and separated from them, whereas, scale counts of supraoc in these groups (i and iv) was significantly decreased with respect to groups ii and iii. also, scale counts of spl(r) in group iv shows a significant increase (p < 0.05) in comparison with groups ii and iii and separated from them, whereas this group overlaps with group i. results of anova analyses for metric characters results of anova analyses showed that iod in males, tal in females, iod and dbn in combined sexes showed significant differences (p < 0.05). results for mensural characters in combined sexes indicated that vipers from eastern populations have broader heads than those from western and northwestern populations. results of anova and non-parametric analyses for colour patterns analysis of colouration indicates that three characters (scn, l/wdbr and l/ddbr) in combined sexes show statistically significant differences (p < 0.05) among groups. in four groups, results of anova for colour pattern show that scn in group iv has significantly lower number than in group iii, but it overlaps with values from groups i and ii. character of l/wdbr in groups i and iv were significantly decreased (p < 0.05) with respect to groups ii and iii. character of l/ddbr in groups i and iv were significantly lower (p < 0.05) with respect to group iii. analysis of two non-parametric characters among 99 individuals indicated that both dorsal base colour (dbc) and ventral pattern (ventp) in combined sexes showed powerful significant differences (p < 0.0001) among groups. the median of dbc among four groups was 74.07% r for group i; 50% b for group ii; 72% gb for group iii and 75.67% g for group iv. also, the median of ventp among groups was 100% dd for group i; 70% b for group ii; 56% d for group iii and 86.48% s for group iv. these results indicated that each geographical group have a particular pattern, however, some individuals were difficult to classify with an ambiguous colour patterns such as uniform ground colour without recognizable pattern. according to these results and our observations, three different colour patterns existed among the iranian populations (fig. 2). 191geographic variation in the iranian macrovipera lebetina table 3. descriptive statistics of meristic, mensural, and colour pattern characters in four groups of macrovipera lebetina. all mensural characters as compared to svl were analyzed. character sex i mean ± sd (n) (range) ii mean ± sd (n) (range) iii mean ± sd (n) (range) iv mean ± sd (n) (range) prev m 4.60 ± 0.54 (5) 4-5 4.50 ± 0.57 (4) 4-5 4.00 ± 0.00 (4) 4-4 4.70 ± 0.58 (18) 3-5 f 4.75 ± 0.50 (4) 4-5 4.71 ± 0.48 (7) 4-5 4.18 ± 0.60 (11) 3-5 4.57 ± 0.60 (19) 3-5 ven m 170.20 ± 1.79 (5) 168-172 167.25 ± 5.12 (4) 162-173 170.50 ± 2.52 (4) 168-174 173.35 ± 2.00 (18) 171-178 f 168.75 ± 2.63 (4) 165-171 167.29 ± 2.69 (7) 164-172 169.09 ± 2.59 (11) 165-173 174.00 ± 2.98 (19) 169-179 scd m 46.80 ± 3.11 (5) 44-52 44.5 ± 3.11 (4) 42-49 46.00 ± 2.45 (4) 43-49 47.25 ± 3.99 (18) 35-53 f 44.25 ± 4.03 (4) 40-49 39.86 ± 5.46 (7) 29-46 45.60 ± 3.75 (11) 37-49 45.78 ± 3.06 (19) 42-52 dors 1 m 24.20 ± 1.09 (5) 23-25 25.00 ± 0.00 (4) 25-25 24.50 ± 1.00 (4) 23-25 24.11 ± 1.23 (18) 21-25 f 25.00 ± 0.00 (4) 25-25 24.14 ± 1.06 (7) 23-25 24.45 ± 0.93 (11) 23-25 23.94 ± 1.54 (19) 21-27 dors 2 m 25.40 ± 0.89 (5) 25-27 25.00 ± 0.00 (4) 25-25 25.00 ± 0.00 (4) 25-25 25.22 ± 0.64 (18) 25-27 f 25.00 ± 0.00 (4) 25-25 25.00 ± 0.00 (7) 25-25 25.18 ± 0.60 (11) 25-27 25.21 ± 0.63 (19) 25-27 dors 3 m 19.00 ± 0.00 (5) 19-19 18.50 ± 1.00 (4) 17-19 19.00 ± 0.00 (4) 19-19 18.77 ± 0.64 (18) 17-19 f 19.00 ± 0.00 (4) 19-19 19.00 ± 0.00 (7) 19-19 19.18 ±0.60 (11) 19-21 19.00 ± 0.00 (19) 19-19 ap m 6.80 ± 0.44 (5) 6-7 7.00 ± 0.00 (4) 7-7 6.00 ± 0.00 (4) 6-6 6.38 ± 0.50 (18) 6-7 f 7.00 ± 0.81 (4) 6-8 6.28 ± 0.48 (7) 6-7 6.81 ± 0.87 (11) 6-9 6.31 ± 0.58 (19) 6-8 blspl m 23.80 ±0.83 (5) 23-25 25.25 ± 0.50 (4) 25-26 26.00 ± 0.81 (4) 25-27 25.20 ± 1.20 (18) 23-28 f 25.25 ± 1.70 (4) 23-27 25.71 ± 0.75 (7) 25-27 25.36 ± 1.28 (11) 23-27 26.12 ± 1.02 (19) 24-28 can m 2.20 ± 0.44 (5) 2-3 2.00 ± 0.00 (4) 2-2 2.50 ± 0.57 (4) 2-3 2.18 ± 0.40 (18) 2-3 f 2.25 ± 0.50 (4) 2-3 2.00 ± 0.00 (7) 2-2 2.50 ± 0.52 (11) 2-3 2.15 ± 0.37 (19) 2-3 interocular m 12.00 ± 0.70 (5) 11-13 10.50 ± 0.57 (4) 10-11 11.75 ± 1.25 (4) 10-13 11.11 ± 0.67 (18) 10-12 f 11.75 ± 0.95 (4) 11-13 11.28 ± 0.75 (7) 10-12 12.09 ± 1.22 (11) 10-14 11.21 ± 0.97 (19) 9-13 spl(l) m 10.40 ± 0.54 (5) 10-11 10.00 ± 0.81 (4) 9-11 9.75 ± 0.50 (4) 9-10 10.72 ±0.57 (18) 10-12 f 10.50 ± 0.57 (4) 10-11 9.85 ± 0.37 (7) 9-10 10.27 ± 0.64 (11) 9-11 10.73 ± 0.65 (19) 10-12 spl(r) m 10.60 ± 0.54 (5) 10-11 9.75 ± 0.50 (4) 9-10 9.50 ± 0.57 (4) 9-10 10.83 ± 0.61 (18) 10-12 f 10.50 ± 0.57 (4) 10-11 10.28 ± 0.75 (7) 9-11 10.45 ± 0.52 (11) 10-11 10.52 ± 0.61 (19) 10-12 192 n. moradi et alii character sex i mean ± sd (n) (range) ii mean ± sd (n) (range) iii mean ± sd (n) (range) iv mean ± sd (n) (range) ifl(l) m 13.80 ± 0.44 (5) 13-14 12.25 ± 0.50 (4) 12-13 13.00 ± 0.81 (4) 12-14 14.27 ± 0.89 (18) 13-16 f 13.75 ± 0.50 (4) 13-14 13.28 ± 0.75 (7) 13-15 13.36 ± 0.67 (11) 13-15 14.42 ± 1.01 (19) 13-17 ifl(r) m 13.80 ± 0.83 (5) 13-15 12.50 ± 0.57 (4) 12-13 13.00 ± 0.00 (4) 13-13 14.16 ± 0.85 (18) 13-16 f 13.50 ± 0.57 (4) 13-14 13.57 ± 0.78 (7) 12-14 13.45 ± 0.68 (11) 13-15 13.42 ±0.83 (19) 13-16 incir(l) m 15.60 ± 1.51 (5) 14-17 14.50 ± 1.00 (4) 13-15 16.00 ± 1.55 (4) 15-17 15.11 ± 1.07 (18) 14-17 f 14.50 ± 1.29 (4) 13-16 16.14 ± 0.69 (7) 15-17 15.90 ± 1.04 (11) 14-17 14.78 ± 1.08 (19) 13-17 incir(r) m 15.60 ± 2.70 (5) 13-19 14.25 ± 0.95 (4) 13-15 15.50 ± 1.29 (4) 14-17 15.61 ± 1.09 (18) 14-18 f 14.75 ± 1.25 (4) 13-16 16.71 ± 0.75 (7) 16-18 16.18 ± 1.66 (11) 13-19 14.94 ± 1.12 (19) 13-17 outcir(l) m 23.20 ± 0.83 (5) 22-24 22.25 ± 1.50 (4) 21-24 20.75 ± 1.70 (4) 19-23 22.88 ± 1.27 (18) 21-25 f 20.75 ± 1.70 (4) 19-23 21.71 ± 0.95 (7) 20-23 21.36 ± 1.69 (11) 20-26 22.89 ± 1.15 (19) 21-25 outcir(r) m 22.80 ± 1.30 (5) 21-24 22.00 ± 2.16 (4) 20-25 22.75 ± 2.87 (4) 19-26 22.88 ± 1.67 (18) 20-25 f 21.50 ± 2.38 (4) 19-24 22.14 ± 1.77 (7) 20-25 22.72 ± 2.19 (11) 19-27 22.94 ± 1.90 (19) 18-25 lor(l) m 16.40 ± 1.51 (5) 14-18 13.00 ± 1.41 (4) 12-14 14.50 ± 0.57 (4) 14-15 15.46 ± 2.29 (18) 12-20 f 14.25 ± 3.20 (4) 11-17 13.14 ± 1.67 (7) 11-16 14.00 ± 2.79 (11) 9-19 15.62 ± 1.74 (19) 12-18 lor(r) m 16.80 ± 2.16 (5) 14-20 14.00 ± 1.41 (4) 13-15 14.75 ± 1.89 (4) 12-16 14.92 ± 1.97 (18) 12-19 f 15.25 ± 2.75 (4) 12-18 13.42 ± 1.90 (7) 11-16 13.81 ± 2.52 (11) 9-17 15.75 ± 1.43 (19) 13-18 supraoc m 1.40 ± 0.89 (5) 1-3 2.00 ± 0.81 (4) 1-3 2.75 ± 0.50 (4) 2-3 1.33 ± 0.68 (18) 1-3 f 1.50 ± 1.00 (4) 1-3 2.71 ± 0.48 (7) 2-3 2.81 ± 0.40 (11) 2-3 1.15 ± 0.50 (19) 1-3 bespl m 3.00 ± 0.00 (5) 3-3 3.00 ± 0.00 (4) 3-3 3.00 ± 0.00 (4) 3-3 2.94 ± 0.23 (18) 2-3 f 3.00 ± 0.00 (4) 3-3 3.00 ± 0.00 (7) 3-3 3.00 ± 0.00 (11) 3-3 2.89 ± 0.31 (19) 2-3 tal/svl m 0.13 ± 0.00 (5) 0.13 ± 0.01 (4) 0.13 ± 0.00 (4) 0.13 ± 0.01 (17) f 0.13 ± 0.01 (4) 0.12 ± 0.00 (7) 0.13 ± 0.01 (11) 0.13 ± 0.01 (19) hl/svl m 0.04 ± 0.00 (5) 0.04 ± 0.00 (4) 0.04 ± 0.00 (4) 0.04 ± 0.00 (15) f 0.04 ± 0.00 (4) 0.04 ± 0.00 (7) 0.04 ± 0.00 (11) 0.04 ± 0.00 (16) hw/svl m 0.02 ± 0.00 (5) 0.02 ± 0.00 (4) 0.0 ± 0.00 (4) 0.02 ± 0.00 (17) f 0.02 ± 0.00 (4) 0.02 ± 0.00 (7) 0.02 ± 0.00 (11) 0.02 ± 0.00 (19) hh/svl m 0.01 ± 0.00 (5) 0.01 ± 0.00 (4) 0.01 ± 0.00 (4) 0.01 ± 0.00 (18) f 0.01 ± 0.00 (4) 0.01 ± 0.00 (7) 0.01 ± 0.00 (11) 0.01 ± 0.00 (19) dbn/svl m 0.01 ± 0.00 (5) 0.00 ± 0.00 (4) 0.00 ± 0.00 (4) 0.00 ± 0.00 (15) f 0.01 ± 0.00 (4) 0.00 ± 0.00 (7) 0.00 ± 0.00 (11) 0.00 ± 0.00 (16) table 3. (continued). 193geographic variation in the iranian macrovipera lebetina character sex i mean ± sd (n) (range) ii mean ± sd (n) (range) iii mean ± sd (n) (range) iv mean ± sd (n) (range) iod/svl m 0.01 ± 0.00 (5) 0.01 ± 0.00 (4) 0.01 ± 0.00 (4) 0.01 ± 0.00 (15) f 0.01 ± 0.00 (4) 0.01 ± 0.00 (7) 0.01 ± 0.00 (11) 0.01 ± 0.00 (16) sl/svl m 0.01 ± 0.00 (5) 0.01 ± 0.00 (4) 0.01 ± 0.00 (4) 0.01 ± 0.00 (15) f 0.01 ± 0.00 (4) 0.01 ± 0.00 (7) 0.01 ± 0.00 (11) 0.01 ± 0.00 (16) scn 16.66 ± 7.35 (7) 6.33-27.33 17.83 ± 3.62 (4) 12.66-21.00 19.82 ± 4.80 (11) 12.00-25.66 13.47 ± 4.95 (25) 7.00-27.00 l/wdbr 0.37 ± 0.12 (7) 0.21-0.52 0.69 ± 0.11 (4) 0.53-0.75 0.68 ± 0.22 (11) 0.34-1.07 0.37 ± 0.12 (25) 0.10-0.61 l/ddbr 0.86 ± 0.38 (7) 0.36-1.25 1.44 ± 0.22 (4) 1.14-1.67 1.61 ± 0.70 (11) 0.65-3.10 0.78 ± 0.41 (25) 0.32-1.97 l/dlbr 0.82 ± 0.32 (7) 0.48-1.38 0.95 ± 0.31 (4) 0.63-1.24 0.93 ± 0.32 (12) 0.52-1.78 0.64 ± 0.37 (26) 0.31-2.07 table 3. (continued). fig. 1. distribution map of macrovipera lebetina in iran, each coloured region represents a studied group. each circle on the map represents a location from which at least one snake was examined. 194 n. moradi et alii principal component analysis multivariate analysis of the meristic characters was carried out to determine whether distinct geographic units exist within macrovipera lebetina populations. five principal components (pcs) explained 55.8%, and the first eight principal components revealed 73.5% of the total variation. of this total, 18.9% explained by pc1 in which supraoc, ven, and ifl were mainly responsible for this variation; 31.8% explained by pc2 which is mainly attributed to interocular and incir; 41.6% explained by pc3 mainly attributed to outcir; and 49.1% explained by pc4 in which scd and dors3 were more important. these results showed that the supraoc, ven and interocular are most important characters in separating populations (table 4). the magnitude and sign of the loadings on pc1 and pc2 showed significant separation among groups and the high degree of separation of southern populations (group iv) from other groups. also, group i tends to separate from two residual groups (fig. 3). cluster analysis a pearson distance dendrogram based on two meristic characters (ven and supraoc) with p < 0.05 and r2 > 50% clusters all four geographic groupings used in this study. males and females are treated together because there is no sexual significant difference between them in these two characters. in figure 4, the first major dichotomy separates the southern populations (cluster iv) with a considerable distance from all other clusters. the second major dichotomy separates the northeastern populations (cluster i) with a distance from clusters ii and iii, which, in turn, constitute two major branches; one branch encompasses gorgan populations (cluster ii) from northern iran, whereas the other branch is composed of all the other localities in northwest, west and western parts of alborz mts., approaching cluster iv in the south. discriminant analysis the reclassifications were compared to the original cluster designations to determine how well the original model was supported by the discriminant analysis. due to existence of missing values, twelve cases were removed from the multivariate analyses. discriminant analysis was performed on the 58 remaining snakes with sexes combined. classification results and predicted group membership showed that 98.3% of the original allocations were correctly classified. though vipers from the northeastern population (group i) were correctly classified to 88.9%, this value is 100% for other three groups. however, classification results with cross-validation indicated that a total of 72.4% of the original groupings were correctly classified. proportions correctly classified are 66.7% for group i, 77.8% for group ii, 46.2% for group iii, and 85.2% for group iv. the generalized squared distances among groups were computed to show the distance between each pair of groups as follow: 17.37 between group ii and iii; 22.63 between group i and ii; 19.69 between group i and iv; 31.32 between group ii and iv; 19.49 between group i and iii; and 28.03 between group iii and iv (table 5). fig. 2. typical pattern of macrovipera lebetina from northeastern populations (left); northern, western and northwestern populations (middle); and southern populations (right) (sketch by: naeim moradi). 195geographic variation in the iranian macrovipera lebetina table 4. factor loadings on the first eight principal components extracted from a correlation matrix of 22 meristic characters in macrovipera lebetina from iran. characters pc1 pc2 pc3 pc4 pc5 pc6 pc7 pc8 prev 0.214 -0.009 0.048 -0.298 0.361 0.068 -0.008 0.013 ven 0.323 -0.127 0.192 0.169 -0.307 0.182 0.156 0.059 scd 0.172 -0.104 -0.018 0.426 -0.076 -0.216 0.042 -0.263 dors 1 -0.024 0.196 0.065 0.150 0.085 -0.213 0.101 0.613 dors 2 0.106 0.269 0.265 0.104 -0.021 -0.368 0.139 -0.300 dors 3 -0.062 0.292 0.155 0.302 -0.096 0.034 0.097 -0.360 ap 0.032 0.188 0.095 0.207 0.563 -0.178 0.011 -0.057 blspl -0.010 0.167 -0.109 0.264 -0.259 0.118 -0.550 0.092 can -0.093 0.244 0.003 0.322 0.218 0.399 -0.248 -0.066 interocular 0.028 0.425 -0.033 0.032 0.167 -0.080 0.281 0.154 spl(l) 0.267 0.056 0.135 0.051 0.096 -0.085 -0.498 0.032 spl(r) 0.230 0.139 0.023 -0.354 0.138 -0.080 -0.330 -0.049 ifl(l) 0.304 0.172 0.345 -0.079 -0.189 0.193 0.085 0.155 ifl(r) 0.304 0.230 0.236 -0.176 -0.146 -0.025 -0.135 0.024 incir(l) -0.124 0.389 -0.234 -0.209 -0.199 0.015 -0.005 -0.023 incir(r) -0.112 0.349 -0.083 -0.204 -0.372 -0.115 0.076 -0.074 outcir(l) 0.302 0.022 -0.368 -0.020 -0.018 -0.287 0.030 0.011 outcir(r) 0.175 0.017 -0.494 -0.023 -0.003 -0.194 -0.106 -0.248 lor(l) 0.340 0.139 -0.253 0.030 0.061 0.277 0.237 0.027 lor(r) 0.289 0.074 -0.330 0.163 0.065 0.383 0.163 0.048 supraoc -0.368 0.263 -0.105 -0.030 0.062 0.131 -0.048 0.058 bespl -0.035 0.044 0.147 -0.283 0.135 0.312 0.080 -0.435 eigenvalue 4.1650 2.8393 2.1580 1.6449 1.4744 1.4230 1.3280 1.1291 proportion 0.189 0.129 0.098 0.075 0.067 0.065 0.060 0.051 cumulative 0.189 0.318 0.416 0.491 0.558 0.623 0.683 0.735 fig. 3. scatterplot of individuals on the first two principle components. 196 n. moradi et alii fig. 4. pearson distance dendrogram resulting from the cluster analysis based on two meristic characters (ventral and supraocular scales) of macrovipera lebetina in iran. table 5. linear discriminant function for groups. characters i ii iii iv prev 119 125 120 121 ven 88 88 88 90 scd -22 -23 -22 -23 dors 1 48 49 48 49 dors 2 61 62 58 59 dors 3 242 246 243 244 ap -15 -16 -16 -19 blspl 78 81 79 80 can -24 -29 -20 -17 interocular -41 -43 -40 -43 spl(l) 72 70 73 72 spl(r) 64 62 61 66 ifl(l) -162 -164 -159 -164 ifl(r) -56 -58 -59 -55 incir(l) 39 40 41 39 incir(r) -0 0 -1 -1 outcir(l) 131 133 130 134 outcir(r) -56 -56 -53 -56 lor(l) -11 -12 -11 -10 lor(r) -32 -33 -35 -34 supraoc 39 45 44 38 bespl 371 384 376 368 constant -12071 -12320 -12210 -12495 table 6. standardized coefficients for determining differences among four groups of macrovipera lebetina. characters function 1 2 3 prev 0.070 -0.421 0.447 ven -0.234 0.755 -0.001 scd -0.045 0.151 -0.204 dors 1 -0.001 -0.097 0.175 dors 2 0.142 -0.261 -0.022 dors 3 0.135 -0.067 0.001 ap 0.135 -0.422 -0.490 can -0.345 0.546 0.049 interocular 0.121 0.651 -0.567 spl(l) -0.219 0.061 -0.347 spl(r) -0.228 0.108 -0.131 ifl(l) -0.208 0.012 0.077 ifl(r) -0.369 0.198 0.444 incir(l) 0.115 0.131 -0.141 incir(r) 0.098 -0.472 0.188 outcir(l) -0.059 0.179 0.391 outcir(r) 0.163 0.249 0.282 lor(l) 2.340 7.021 2.551 lor(r) -2.137 -6.654 -2.474 supraoc 0.874 0.267 0.195 bespl 0.418 -0.082 -.295 197geographic variation in the iranian macrovipera lebetina based on additional discriminant function analysis (dfa) of meristic characters using spss® ver. 20, the first three functions yielded 100% of total information in which, the first function explained 73% of the total variance with characters supraoc, ifl, spl and incir having the highest values. the loadings of each character on a particular function are shown in table 6. function 1 is heavily weighted by numbers of supraocular scales (supraoc) and the number of loreal scales (lor). function 2 is weighted by ventral (ven), canthal (can), loreal (lor) and interocular scales, while function 3 is weighted by loreal scales count (lor). totally, loreal scale count was the most powerful character to separate four groups. based on this analysis, with the ordination of four groups along the first two functions (function 1 against function 2), the centroids of each geographic group is well separated from any other group (fig. 5). results of sexual dimorphism analyses firstly, two sample t-tests using all meristic data showed significant sexual dimorphism (p < 0.05) in scd and blspl. this indicates that males have more subcaudal scales (scd) and females have more scales across the head. a principal component analysis (pca) for sexes was performed; the magnitude and sign of the loadings on pc1 and pc2 showed apparent sexual dimorphism pattern between males and females. although, the scatterplot did not show a clear separation between the sexes, but a weak pattern of sexual dimorphism is apparent (fig. 6). fig. 5. patterns of variation expressed by the two discriminant functions (function 1 against function 2) for four groups of macrovipera lebetina. 210-1-2 3 2 1 0 -1 -2 p2 p 1 f m sex scatterplot of p1 vs p2 fig. 6. scatterplot of principal component analysis for males and females of macrovipera lebetina. 198 n. moradi et alii discussion latifi in 1983 assigned the iranian macrovipera to the subspecies of macrovipera lebetina obtusa (dwigubsky, 1832). however, chikin and szczerbak in 1992 described a new subspecies from khorasan province. until now, the iranian macrovipera consisted of two subspecies: macrovipera lebetina obtusa (dwigubsky, 1832) (fig. 7) distributed in west, northwest, and north of iran, and macrovipera lebetina chernovi (chikin and szczerbak, 1992) (fig. 8) occurring in northeastern iran. our results indicated that the separation of the northeastern populations of m. lebetina from the northwestern and western populations could be corroborated by morphological characters, as previously suggested by chikin and szczerbak (1992), ananjeva et al. (2006), rajabizadeh et al. (2011), and oraie et al. (2012). however, the character expression in southern populations likely amounts to another taxonomic status in the iranian macrovipera taxa. m. lebetina populations from southern region of the iranian plateau showed significant differences to all northern populations regarding several morphological characters. for example, pholidosis showed a general north-south pattern of geographic variation for most meristic characters. ventral, infralabial, outer circumocular and supralabial scales averaged higher, and interocular, inner circumocular and supraoculars averaged lower in the southern regions. many ophidians display latitudinal pattern of geographic variation in pholidosis. christman (1980) examined 15 species of snakes in florida and founded a latitudinal pattern of geographic variation in two characters, ventral and subcaudal scales, both of which increased in number from north to south in most species. higher scales count in the southern regions could also be the result of selection for larger body sizes, which in turn may reflect longer annual growing periods in southern regions. substantial geographic variation in colouration was observed among the iranian macrovipera as well. vipers from the southern populations have the narrowest crossbars on dorsum with high distances between them. they also entail lower scale counts compared to northwestern and northeastern populations. the general pattern of body crossbars and blotches in macrovipera lebetina appeared to be influenced by latitudinal pattern and elevation. the alborz mts. of the northern and northwestern regions combine high latitude and high elevation, and consequently annual temperatures there are among the coldest in the species’ range. high number of dark pattern elements would be expected to increase the ability of a snake to thermoregulate at high elevations. integumentary colour and reflectance are known to exert a strong influence on the thermal biology of snakes (peterson et al., 1993). also, according to non-parametric analysis, there was a clinal difference between northern and southern populations, so that with toward the southern latitudinal, dorsal body colour become brighter and ventral dots pattern become denser and dots form a trapezoid-shaped spots and belly will have a mottled appearance. geographic variation in pattern may be strongly related to habitat. the differences between the northeastern and western regions may be due to the lower elevations and warmer summers of the northeastern regions and different landscape features in kopet dagh mts. in addition, the southern populations remained clearly distinct in all multivariate analyses and the cluster analyses showed that the southern populations were distinguishable with a considerable distance from all the other clusters. multivariate statistical analyses are the fig. 7. an adult specimen of macrovipera lebetina obtusa from takab, western azerbaijan province, iran (photograph by omid mozaffari). fig. 8. an adult specimen of macrovipera lebetina chernovi from khorasan province, iran (photograph by hasan moghimi). 199geographic variation in the iranian macrovipera lebetina most comprehensive when applied to morphological data, inasmuch as the rely upon multiple aspects of the phenotype. the formal recognition of distinct geographic units within macrovipera lebetina in supported by this study. gorgan populations (cluster ii) in the multivariate analyses virtually separated from other northeastern and northern-western populations, however, existence of overlapping in pholidosis and colour pattern in some characters can be a sign of a hybrid zone between two major northeastern and northern-western populations, this confirms the existence of two subspecies of m. l. obtusa and m. l. chernovi. in spite of that cannot be recognized a distinct border between ranges of two northern subspecies, individuals of gorgan populations have more similar to the northern-western subspecies. eventually, individual characters often showed clear patterns of variation. in all of multivariate analyses, characters of ventral and supraocular scales were identified as important and significant variables and were used for mapping geographic variation. differences in dorsal and ventral scale counts are often diagnostic for different species of macrovipera (nilson and andrén, 1988; joger, 1984; göçmen et al., 1999; phelps, 2010) and chikin and szczerbak, 1992 early reliance on supraoculars to delineate subspecies of iranian macrovipera lebetina. most recently, stümpel (2012) in molecular analyses on mtdna sequences mentioned that the specimen from southeastern iran were completely distinct from other specimens of m. lebetina. however, studies of reptiles have demonstrated the early classification based on a few specimens or single morphological characters often do not agree with more comprehensive molecular and morphological analyses. nevertheless, we accept, provisionally, considering the some morphological evidences, the hypothesis of the specific status of the southern iranian populations as macrovipera sp. (fig. 9). however, complementing phylogenetic investigation is required to gain a deeper understanding of the evolution of this species complex. new combinations of morphological characters are used to device a key for identification of iranian macrovipera, partly based on diagnostic characteristics gathered from chikin and szczerbak (1992), david et al. (1999), joger (1984) and oraie et al. (2012). iranian macrovipera identification key 1aventral scales 156-188 (less than 171 in 81.08% of specimens) ............................................................................... 2 1bventral scales 169-179 (more than 172 in 83.78% of specimens), supraocular entire, and infralabials 13-17, dorsum brownish gray with narrow crossbars with distinguishable distance between them, each crossbar includes 13.47 ± 0.99 pigmented scales at mid snout-vent length. belly covered with dark blotch (3-5 trapezoid spots in posterior part of each ventral scales). some southeastern specimens’ uniform glazy black ................. macrovipera sp. 2asupraocular entire or divided and larger than circumoculars, snout dark (one third of anterior part of the head). dorsum reddish brown or dark with large unfilled blotch which sometimes connected together, each dorsum blotch includes 16.66 ± 2.78 pigmented scales at mid snout-vent length. the ratio of interocular distance to head length is 0.39 ± 0.02. belly often light and splotched by dark dots ....... macrovipera lebetina chernovi (chikin and szczerbak, 1992) 2bthree supraoculars present and same as the other circumoculars, head uniform, with narrow strips between the corner of the mouth and the posterior part of the eyes, and also between the eye and fourth supralabial scale. dorsum brown or dark brown with rectangular dark marking and arrange in zigzag pattern, each dorsum blotch includes 19.29 ± 1.16 pigmented scales at mid snout-vent length. the ratio of interocular distance to head length is 0.36 ± 0.01. belly often light and splotched with dark dots .............. ......................macrovipera lebetina obtusa (dwigubsky, 1832) acknowledgements this study was performed based on official permission number 91/49936 issued by department of the environment of iran. also, this project was supported by the iranian plateau herpetology research group (iphrg) of fig. 9. an adult male, macrovipera sp. from masjed soleyman, khuzestan province, iran (photograph by naeim moradi). 200 n. moradi et alii the razi university of kermanshah. we would also like to thank the a. zare mirakabadi, curators of zoological collections in the department of venomous animals and antisera production, razi vaccine and serum research institute, hesarak, karaj, and s. shafiei and h. r. esmaeili for borrowing preserved specimens. also thank to k. mebert for helpful comments. our special thanks dedicated for f. todehdehghan, m. e. sehati-sabet, k. rabiei, b. fathinia, a. sharifi, o. mozaffari, h. oraie and f. masjedi for their helps and cooperation during this study. references afroosheh, m., kazemi, s.m. 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(in press): taxonomic status of the iranian macrovipera lebetina (linnaeus, 1758) (ophidia: viperidae) based on morphological and venom electrophoresis comparisons. turk. j. zool. peterson, c.r., gibson, a.r., dorcas, m.e. (1993): snake thermal ecology: the causes and consequences of body-temperature variation. in: snakes: ecology and behavior, pp. 241-314. seigel r.a., collins j.t., eds, 201geographic variation in the iranian macrovipera lebetina mcgraw-hill, new york. phelps, t. (2010): old world vipers, a natural history of the azemiopinae and viperinae. edition chimaira, frankfurt. pisani, g.r. (1973): a guide to preservation techniques for amphibians and reptiles. (herpetological circulars). society for the study of amphibians and reptiles, salt lake city. rajabizadeh, m., joger, u. nilson, g. (2011): review of new findings in taxonomy and distribution of vipera (s.l.) of iran. seh european congress of herpetology and dght deutscher herpetologentag: luxembourg and trier. reuss, t. (1927): sechs europšische giftschlangengattungen. zool. anzeiger leipzig 72: 124-129. sindaco, r., venchi, a., grieco, c. (2013): the reptiles of the western palearctic, volume 2: annotated checklist and distributional atlas of the snakes of europe, north africa. middle east and central asia, with an update to vol. 1. edizioni belvedere, italy. stümpel, n. (2012): phylogenie und phylogeographie eurasischer viperinae unter besonderer berücksichtigung der orientalischen vipern der gattungen montivipera und macrovipera. unpublished dissertation. university of braunschweig, germany. stümpel, n., joger, u. (2009): recent advances in phylogeny and taxonomy of near and middle eastern vipers–an update. zookeys 31: 179-191. terentjev, p.v., chernov, s.a. (1940): a field guide to amphibians and reptiles of the ussr. second, revised, and enlarged edition, uchpedgiz, leningrad (in russian). appendix. herpetological collections, locality, and collection numbers (no.) of the specimens of macrovipera lebetina used in present study. specimens locality no. zcri kalat, near mashhad, khorasan razavi prov. 2349 kalat, near mashhad, khorasan razavi prov. 2350 kalat, near mashhad, khorasan razavi prov. 2363 kalat, near mashhad, khorasan razavi prov. 2351 kalat, near mashhad, khorasan razavi prov. 2346 kalat, near mashhad, khorasan razavi prov. 2342 kalat, near mashhad, khorasan razavi prov. 2344 gorgan, golestan prov. 1313 gorgan, golestan prov. 2154 gonbad e kavoos, golestan prov. 2158 tang-e-rah (gonbad e kavoos), golestan prov. 2260 tang-e-rah (gonbad e kavoos), golestan prov. 2261 gonbad e kavoos, golestan prov. 2256 gonbad e kavoos, golestan prov. 2259 tang-e-rah (gonbad e kavoos), golestan prov. 2262 gorgan, golestan prov. 2151 rahmat abad (rasht), gilan prov. 2143 rudbar, gilan prov. 2150 arak, markazi prov. 2267 alajigh, between tehran & saveh 2292 ashk island, orumie lake 129382 mahallat, markazi prov. 1314 orumie lake island 129381 rezaeie, west azarbaijan prov. 129380 alajigh, between tehran & saveh 2180 arezu island, orumie lake 129386 bijar, kordistan prov. 1249 ashk island, orumie lake 129383 specimens locality no. kermanshah, kermanshah prov. 972 semirom, esfahan prov. 2274 khansar, esfahan prov. 2237 khansar, esfahan prov. 2238 meymahe, esfahan prov. 2280 semirom, esfahan prov. 2159 eghlid (aso pas), fars prov. 2144 albaji, ahvaz, khuzistan prov. 2309 tang-e-karun, eghlid, fars prov. 2210 albaji, ahvaz, khuzistan prov. 2142 tang-e-karun, eghlid, fars prov. 2212 shushtar, khuzistan prov. 2248 abadeh, fars prov. 2298 albaji, ahvaz, khuzistan prov. 2234 masjed soleyman, khuzistan prov. 2299 albaji, ahvaz, khuzistan prov. 2195 aligudarz, lorestan prov. 2208 tang-e-karun, eghlid, fars prov. 2211 masjed-soleyman, khuzistan prov. 2170 mamasani, fars prov. 119204 masjed-soleyman, khuzistan prov. 2168 ramhormoz, khuzistan prov. 121817 kalat, near mashhad, khorasan razavi prov. (picture) 426 kalat, near mashhad, khorasan razavi prov. (picture) 979 kalat, near mashhad, khorasan razavi prov. 969 mashhad, khorasan razavi prov. 1385 mashhad, khorasan razavi prov. 1272 mashhad, khorasan razavi prov. 1273 dare gaz, khorasan razavi prov. 608 202 n. moradi et alii specimens locality no. dare gaz, khorasan razavi prov. 2228 dare gaz, khorasan razavi prov. 1304 masjed-soleyman, khuzistan prov. 2171 masjed-soleyman, khuzistan prov. 2166 suhc khorasan razavi prov. erp 986 khorasan razavi prov. erp 987 babmaran, kerman prov. erp 143 bahrame gur protected area, fars province erp 1531 bakhtegan national park, fars prov. erp 1518 pariz, kerman prov. erp 3732 balvard, sirjan, kerman prov. erp 1941 sabzevar, khorasan razavi prov. erp 666 shimbar protected area, khuzistan prov. erp 1741 ruzm kermanshah, kermanshah prov. vv.31.3 kermanshah, kermanshah prov. vv.31.1 kermanshah, kermanshah prov. vv.31.2 kermanshah, kermanshah prov. vv.31.5 asmari mts, masjed soleyman, khuzistan prov vv.31.6 asmari mts, masjed soleyman, khuzistan prov vv.31.7 abdanan, ilam prov. unlabeled abdanan, ilam prov. unlabeled zmsbuk khabr national park, kerman prov. a3 khabr national park, kerman prov. a18 khabr national park, kerman prov. a12 sirjan, kerman prov. a46 sirjan, kerman prov. a57 khabr national park, kerman prov. a58 pariz, kerman prov. a110 baft, kerman prov. a32 suzm shiraz, fars prov. unlabeled specimens locality no. bamoo national park, fars prov. unlabeled bamoo national park, fars prov. unlabeled fdoi sarvestan, fars prov. unlabeled seyf abad, fars prov. unlabeled seyf abad, fars prov. unlabeled sarvestan, fars prov. unlabeled picture abdanan, ilam prov. -khojir protected area, tahran prov. -bazman, siatan prov. -tangestan, bushehr prov. -capture & release golestan national park, golestan prov. -golestan national park, golestan prov. -golestan national park, golestan prov. -damghan, semnan prov. -shimbar protected area, khuzistan prov. -shimbar protected area, khuzistan prov. -golgir, masjid-soleyman, khuzistan prov. -balvard, sirjan, kerman prov. -balvard, sirjan, kerman prov. -balvard, sirjan, kerman prov. -pariz, kerman prov. -pariz, kerman prov. -ghatruye national park, fars prov. -bamoo national park, fars prov. -bamoo national park, fars prov. -gorm protected area, jahrom, fars prov. -turan national park, semnan prov. -shahrud, semnan prov. -shahrud, semnan prov. -rudbarak protected area, semnan prov. -zcri: zoological collection of the department of venomous animals and antiserum production, razi vaccine and serum research institute, hesarak, karaj zmsbuk: zoological museum of shahid bahonar university of kerman suhc: sabzevar university herpetological collection ruzm: razi university zoological museum suzm: shiraz university zoological museum fdoi: species diversity museum of department of environment, fars issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 8(1): 35-39, 2013 trophic niche and feeding biology of the italian wall lizard, podarcis siculus campestris (de betta, 1857) along western mediterranean coast marco a.l. zuffi*, chiara giannelli museum natural history and territory, university of pisa, via roma 79-i-56011 calci (pisa), italy. *corresponding author. e-mail: marcoz@museo.unipi.it submitted on 2012, 30th october; revised on 2012, 30th december; accepted on 2013, 2nd january. abstract. trophic niche of the italian wall lizard was studied at three different sites in tuscany (central northern italy), two along the mediterranean sea, one inland. fecal pellet analysis was carried out on 71 pellets (37 of male and 34 of female adult lizards), accounting on the whole for 184 prey items. coleoptera, hymenoptera (ants), araneae and gastropoda were the most represented taxa (numerical abundance of ca 22, 7, 7, 6% respectively). we found brillouin diversity index similar in females and in males, with a marked overlap between sexes, but differences in niche overlapping among localities. diet spectrum was quite different with that found in other central italy localities, in the tuscan archipelago, or in areas where p. siculus has recently introduced. our study confirms the opportunistic pattern and adaptability of this lizard species, and increases the range of sampled localities within the species’ range. keywords. italian wall lizard, podarcis siculus, diet, feeding ecology, western tuscany, italy. the italian wall lizard, podarcis siculus, is very spread in italy and dalmatia (corti et al., 2011), assessed by iucn as near threatened (crnobrnja-isailovic et al., 2008), and protected by european, regional and local laws. despite well known for distributive and faunistical data set (henle and klaver, 1986; corti, 2006; vanni and nistri, 2006; corti et al., 2011) it is a relatively less studied species (herrel et al., 2008; biaggini et al., 2009). feeding ecology is relatively much more studied. diet ranges from insects, spiders to small terrestrial crustaceans (see henle and klaver, 1986 and literature therein). on average, variability in feeding ecology has been found as habitat dependent on geographical position and ecological conditions (e.g., continental vs insular; pérez-mellado and corti, 1993) or according to season and to local prey fauna composition (rugiero, 1994). when arthropod preys are scarce, p. siculus may integrate its diet with small molluscs, plant matters (plants, seeds, fruit; pérezmellado and corti, 1993). exceptionally, large individuals may prey upon conspecifics, mainly hatchlings (rugiero, 1994; grano et al., 2011), but also to other lizard species or small mammals (capula and aloise, 2011). variability of feeding ecology of podarcis siculus has been tested on some localities (ouboter, 1981; sorci, 1990; capula et al., 1993; perez-mellado and corti, 1993; rugiero, 1994; bombi and bologna, 2002; salvador, 2006; herrel et al., 2008). recently, herrel et al. (2008) found that an introduced population in a small island of the dalmatian coast, with different habitat structure and scarcity of prey taxa, rapidly evolved, modifying the alimentary tract with the formation of intestinal caeca (as in typically herbivory reptiles) and adapting body structure (fore and hind limbs ratio on snout to vent length) to the new habitat. due to the species’ wide distribution, it is worth-studying diet variability in many different contexts (see perez-mellado and corti, 1993). in this paper we are aimed at testing i) variability in trophic niche of p. siculus along multiple sites and ii) comparing differences in diet, if any, with the other known studied italian populations. sampling localities were in western and central northern tuscany. we sampled lizards in marchjune 2009 in lucca (viareggio-lecciona, 43°50’47”n, 10°14’52”e), and in pisa (san rossore, within the region36 m.a.l. zuffi, c. giannelli al natural “parco di migliarino san rossore massaciuccoli”, 43°43’11”n, 10°16’41”e) province. we sampled in march-june 2008 and 2009 in pistoia (lamporecchio and casalbosco, 43°56’29”n, 10°59’52”e) province. sampling areas in lucca and pisa provinces represented coastal samples, while the pistoia area represented a continental area. in lucca and pisa sites, p. siculus was the unique lizard present; in pistoia sites, p. muralis and l. viridis were also present. fecal pellets were collected at lizard capture. after measurement (snout to vent length, body mass), the animal was then released at the capture point. we collected 71 pellets (37 of male and 34 of female adult lizards): 14 from lucca, 32 from pisa and 25 from pistoia. each sample was analyzed under stereomicroscope, determining prey composition at the lowest taxonomical rank as possible (order), with assistance of a specialist of main taxa (see acknowledgments). data obtained from fecal pellet analysis were used to describe the trophic niche width (diversity niche index and niche overlap). as shannon-wiener h’ index assumes random sampling, which is quite doubtful since diet selection has been reported for different lacertids (see carretero, 2004 and references therein), we preferred to measure the species diversity using brillouin index, suitable for non-random samples, and simpson diversity indices. brillouin index considers the total number of species and the frequency with which individuals are distributed within a species. high brillouin index values underline an increase of specific diversity in the considered community. simpson’s high index values (as reciprocal) indicate an higher width in resources use (e.g., many resources are used with high frequency). both indices were used to compare our data with other data set and references. trophic niche overlapping of males and females, and among areas (sampled sub-groups of the coastal vs continental areas) was calculated with morisita’s overlap indices. we tested the overlapping degree or the exclusion degree in prey types by males and females and overlapping or exclusion between sexes of the considered areas. to calculate width and overlapping values, we used the free source software bio-dap (thomas, 2000). biodap calculates diversity indices that correspond to those shown by magurran (1988). analyses were not performed on the unidentified groups (see results). parametric and non parametric analyses were performed with spss 13.0, two tailed with α at 0.05. on average, in podarcis siculus pellets, we found 17 different groups, accounting for 277 items: coleoptera, orthoptera, hymenoptera, hemiptera, blattoidea, dermaptera, diptera, aracnida (aranaea), anellida, gastropoda, reptilia, insect wings (thereafter insects), furs, plant matters, sand, unidentified arthropoda, unidentified matters (table 1). within coleoptera, the identified families are aphodiidae (brindalus porcicollis), curculionidae, carabidae, chrysomelidae, tenebrionidae, cerambycidae, staphylinidae (carabidae and curculionidae more abundant). among hymenoptera, only the family formicidae. brillouin diversity index was 1.80 and 1.95 respectively for males and females, and niche overlap was 0.952 (cmh, similarity morisita-horn, bio-dap). these indices underlined a strong similarity and a marked overlap, respectively, in the trophic spectrum between sexes (table 2). more in depth, the trophic niche as resulting from simpson’s index on 15 suitable categories was 0.181 for males and 0.138 for females, and reciprocal was 5.513 and 7.225 respectively. considering sampled matters divided per sex, we did not find any significant difference (nine prey categories with n > 5, and sex, with a χ2 = 8.141, 8 df, ns), suggesting the absence of any sexual preference in diet in these populations of the italian wall lizards. for the observed similarity of diet of the two sexes, we merged all sampled data, dividing them among the three main sites (table 3): lucca, pisa and pistoia (n = 47, 120 and 88 respectively). we firstly tested data normality in sampling effort (normality test, z = 1.11, p = 0.17, ns), and any difference in prey and items distribution, finding slight differences only for lucca-pisa (anova, f = 2.845, p = 0.078, 2 df, lsd post-hoc test p = table 1. frequency of predated categories on the total sample of podarcis siculus. category n % coleoptera 61 22.022 orthoptera 11 3.972 hymenoptera 22 7.942 hemiptera 5 1.805 blaptoidea 1 0.361 dermaptera 1 0.361 diptera 1 0.361 insects 21 7.581 araneae 19 6.859 anellida 1 0.361 gastropoda 17 6.138 reptilia 3 1.083 hairs 11 3.971 vegetals 1 0.361 sand 9 3.249 unidentified arthropoda 36 12.996 unidentified material 57 20.577 total 277 100 37trophic niche in p. siculus 0.026). we then excluded from the analysis also the “unidentified material” and “unidentified arthopods”: at this stage, after removal unsuitable matters, anova analysis on distribution of consumed taxa was not significant (f = 2.13, p = 0.144, 2 df, with not significant post-hoc tests). being no significant differences in sample size among localities, we considered the examined sample well balanced on the whole. the trophic niche of the whole sample (simpson index on 15 categories), was 0.163, and reciprocal (1/ simpson’s index), was 6.151, showing a marked diversity niche, indicating euriphagy. simpson’s index, on 15 usable categories among the three localities was 0.150 for lucca, 0.187 for pisa and 0.140 for pistoia; reciprocal (1/ simpson index), was 6.679, 5.359 and 7.141 for lucca, pisa and pistoia respectively. brillouin index was 1.66, 1.74 and 1.91 for lucca, pisa and pistoia, respectively, indicating a strong similarity among samples. diet of podarcis siculus campestris is characterized by a wide range of invertebrates, mainly insects and arthropods. it is worth noting that a great part of the examined sample is composed by parts and small pieces of arthopodes sensu lato, even if arachnida and gastropoda are furthermore relevant in term of sample size. the reptile items were lizard tail scales and mammal item were small micro-mammal hairs (insectivore or rodent, items too digested to make firm determination). our data set shows the actually variable diet spectrum of the italian wall lizard. comparatively with previously published papers, there are slight, but worth mentioning different results: in central italy (latium, in a coastal, sandy habitat not far from rome) isopoda represented fairly 50% of the detected preys (rugiero, 1994). lepidoptera, coleoptera, araneida and gasteropoda are other well represented taxa. also a juvenile was preyed upon a large male. thus, arthropods represent about 90% of the total preys taxa. this is not surprising at all. arthropods are the larger group of invertebrates living in almost of terrestrial habitats. opportunistic cannibalism and predation upon other reptiles and vertebrates was furthermore recorded in this and in other small lizards (rugiero, 1994; capula and aloise, 2011; grano et al., 2011). bombi and bologna (2002), in another locality in latium, showed that podarcis siculus diet differed quite markedly with respect to the sympatric p. muralis: p. siculus population seems more euriphagous than p. muralis, that shows a diet basically based on formicidae. in the wider and more comprehensive paper of perèz-mellado and corti (1993; cumulative data set from the six islands of the tuscan archipelago), p. siculus mainly fed on diptera, and with minor occurrence on coleoptera and hymenoptera (formicidae). from the same study, it is worth mentioning that p. lilfordi showed a seasonal shift in prey consumption, with coleopterans in spring and isopoda-formicidae in summer. in menorca island, p. siculus fed mainly on spiders (23.8%), coleopterans (21.42%), and isopoda (11.9%), and with lower frequency on other invertebrate taxa (salvador, 2006). while variation in diet composition was not significant in p. siculus between menorca and the tuscan archipelago, in this archipelago, interspecific differences were on the contrary significant (pérez-mellado and corti, 1993). the podarcis lizards that consumed a high proportion of plant matters in their diet, also showed table 2. diversity and similarity indices in p. siculus samples (roman numbers denote a diversity rank; i = higher). males females lucca pisa pistoia brillouin hb 1.80 1.95 1.66 1.74 1.91 ii i iii ii i simpson’s 0.181 0.138 0.150 0.187 0.140 1/simpson’s 5.513 7.225 6.679 5.359 7.141 ii i ii iii i morisita-horn 0.952 lucca vs pisa ii 0.922 pisa vs pistoia i 0.942 lucca vs pistoia iii 0.903 table 3. frequency of predated categories by podarcis siculus in three tuscany sites. category lucca % pisa % pistoia % coleoptera 11 32.354 29 37.665 21 28.767 othoptera 2 5.882 4 5.196 5 6.849 hymenoptera 1 2.941 9 11.688 12 16.438 hemiptera 2 5.882 0 0.0 3 4.109 blaptoidea 0 0 0 0.0 1 1.369 dermaptera 0 0 1 1.298 0 0.0 diptera 0 0 0 0.0 1 1.369 insects 6 17.647 6 7.785 9 12.328 araneae 3 8.824 8 10.390 8 10.958 anellida 0 0 0 0.0 1 1.369 gastropoda 4 11.764 9 11.690 4 5.479 reptilia 1 2.941 2 2.598 0 0.0 hairs 1 2.941 6 7.794 4 5.479 vegetals 0 0 0 0.0 1 1.369 sand 3 8.824 3 3.896 3 4.109 total 34 100 77 100 73 100 38 m.a.l. zuffi, c. giannelli high frequency of aggregated preys, mainly ants. in the balearic islands, p. lilfordi and p. pityusensis, were strictly stenophagous (homoptera, some coleopteran families, but especially ants), independently of their phylogenetic relationships (as discussed in carretero, 2004). it has then supposed that myrmecophagy, widely shared among insular populations, is a very good strategy in arid environments or during prolonged drought. several studies suggest that podarcis species have an active role in pollen load and transport (pérez-mellado et al., 2000a); in addition, herbivory in podarcis is more adaptive than random and that coevolution between lizards and plants is a matter of fact (see castilla, 1999; pérez-mellado et al., 2000a, b; riera et al., 2002; espinoza et al., 2004; rodríguezpérez et al., 2005), despite this phenomenon is not strictly the case for p. siculus (but see herrel et al., 2008; vervust et al., 2010). in the tuscan archipelago drought during mid-end summer is less pronounced than on the main land and, therefore, we can find more flowering plants, associated insects and arthropods taxa than on the continent (pérez-mellado and corti, 1993). in non sympatric greek podarcis populations (podarcis milensis, p. gaigeae and p. erhardii) of the aegean archipelago, it has been noted that the preys aggregated to plant matters were usually related to a different food source (nectar) (adamopoulou et al., 1999). interestingly, comparing the niche indices of continental, insular, native and not native populations [hierarchical cluster analysis to 1/h simpson’s index, recalculated from the percent expressed raw data in peréz-mellado and corti (1993), rugiero (1994), bombi and bologna (2002), and burke and mercurio (2002)], we wish to underline that our podarcis siculus campestris had a trophic niche i) quite similar to that found by peréz-mellado and corti (1993), ii) relatively distant from that found by rugiero (1994) and by bombi and bologna (2002), and iii) much different from results obtained in the long island population (burke and mercurio, 2002) (fig. 1). specifically it is also worth mentioning that the predation on mammals, or the scavenging on (see capula and aloise, 2011) is not so uncommon, likely being a pattern with a low occurrence. furthermore, cannibalism (in our sample, remains of caudal scales, nails and fingers make predation an evident matter) has been usually reported as male peculiarity (grano et al., 2011), while in our sample the pattern was recorded in females only, and limited to the coastal area. any conclusion on directional cannibalism is however preliminary, due to the very small sample size. euriphagy and marked opportunistic behaviour is widely reported in all cases studied to date in podarcis siculus spp. we therefore underline that future research will consider more comprehensive data set on multiple sites, at least during two to three consecutive years (on average a population life span, zuffi et al., unpublished), and with analysis on prey energetics/lizard metabolic demands. acknowledgements particular thanks to marco dellacasa (museum natural history, university of pisa) for taxonomic determination of some material. handling permission during 2007-2008 were released by italian minister of environment (permission dpn/2007/0009336, on 3/04/2007 to malz), capture and handling permissions in 2009 were directly provided by regional park san rossore-migliarino-massaciuccoli, in accordance to the regional law (lr 56/2000, regione toscana). i specially thank paolo casale and an anonymous referee that commented and highly improved the first submitted version. references adamopoulou, c., valakos, e.d., pafilis, p. 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(2000): bio-dap. clearinghouse for ecology softwares. illinois natural history survey-prairie research institute. http://nhsbig.inhs.uiuc.edu/wes/ populations.html vanni, s., nistri, a. (2006): atlante degli anfibi e dei rettili della toscana. regione toscana, giunta regionale e museo storia naturale dell’università di firenze, sezione di zoologia “la specola”. vervust, b., pafilis, p., valakos, e.d., van damme, r. (2010): anatomical and physiological changes associated with a recent dietary shift in the lizard podarcis sicula. physiol. biochem. zool. 83: 632-642. acta herpetologica vol. 8, n. 1 june 2013 firenze university press the oogenic cycle of the caspian bent-toed gecko, cyrtopodion caspium (squamata: gekkonidae) in iran vida hojati1*, kazem parivar2, eskandar rastegar-pouyani3, abdolhossein shiravi1 altitudinal effects on life history parameters in populations of liolaemus pictus argentinus (sauria: liolaemidae) joel antú gutiérrez1,*, carla piantoni2, nora r. ibargüengoytía1,3 recent cryptic extinction of squamate reptiles on yoronjima island of the ryukyu archipelago, japan, inferred from garbage dump remains yasuyuki nakamura1,*, akio takahashi2, hidetoshi ota3 trophic niche and feeding biology of the italian wall lizard, podarcis siculus campestris (de betta, 1857) along western mediterranean coast marco a.l. zuffi*, chiara giannelli novel, non-invasive method for distinguishing the individuals of the fire salamander (salamandra salamandra) in capture-mark-recapture studies goran šukalo1,*, sonja đorđević2, dragojla golub1, dejan dmitrović1, ljiljana tomović2,3 going out tonight? when insular hierophis viridiflavus breaks the whip snakes rules delaugerre michel-jean first evidence of a paedomorphic population of the smooth newt (lissotriton vulgaris) in the czech republic václav gvoždík1,2, veronika javůrková4, oldřich kopecký5,* no detection of chytrid in first systematic screening of bombina variegata pachypus (anura: bombinatoridae) in liguria, northern italy stefano canessa1,*, an martel2, frank pasmans2 status of the european pond turtle, emys orbicularis (reptilia: testudines: emydidae) in vorarlberg, austria andreas kleewein1, günther wöss2 comparative cytogenetics of two species of ground skinks: scincella assata and s. cherriei (squamata: scincidae: lygosominae) from chiapas, mexico riccardo castiglia1,*, alexandra m.r. bezerra2, oscar flores-villela3, flavia annesi1, antonio muñoz4, ekaterina gornung1 polydactyly in the tyrrhenian wall lizard (podarcis tiliguerta) antigoni kaliontzopoulou1,*, daniele salvi1, verónica gomes1, joão p. m. c. maia1,2,3, panagiotis kaliontzopoulos4 book review: roberto sindaco, alberto venchi, cristina grieco. the reptiles of the western palearctic. 2. annotated checklist and distributional atlas of the snakes of europe, north africa, middle east and central asia, with an update to the vol. 1. edoardo razzetti acta herpetologica journal of the societas herpetologica italica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 9(1): 43-49, 2014 doi: 10.13128/acta_herpetol-13541 fire salamander (salamandra salamandra) in larzac plateau: low occurrence, pond-breeding and cohabitation of larvae with paedomorphic palmate newts (lissotriton helveticus) mathieu denoël*, laurane winandy laboratory of fish and amphibian ethology, behavioural biology unit, department of biology, ecology and evolution, university of liege, 22 quai van beneden, 4020 liege, belgium. * corresponding author. e-mail: mathieu.denoel@ulg.ac.be submitted on 2013, 6th november; revised on 2014, 18th january; accepted on 2014, 31st january abstract. alternative reproductive strategies are widespread in caudate amphibians. among them, fire salamanders (salamandra salamandra) usually rely on streams to give birth to aquatic larvae but also use ponds, whereas palmate newt larvae (lissotriton helveticus) typically metamorphose into terrestrial juveniles, but can also reproduce in retaining their gills, a process known as paedomorphosis. here we report repeated observations of an unusual case of coexistence of these two alternative traits in the same pond (larzac, france). the prevalence of fire salamanders in southern larzac was very low (pond occupancy: 0.36%). the observed abundance of fire salamander larvae and paedomorphic newts was also low in the studied pond. on one hand, the rarity of this coexistence pattern may suggest that habitat characteristics may not be optimal or that competition or predation processes might be operating. however, these hypotheses remain to be tested. on the other hand, as this is the only known case of breeding in southern larzac, it could be considered to be at a high risk of extirpation. keywords. pond-breeding, facultative paedomorphosis, syntopy, coexistence, conservation, fire salamander, palmate newt. introduction fire salamanders (salamandra salamandra) typically give birth to gilled larvae in epigeous running waters such as streams throughout their widespread distribution range in europe (thiesmeier, 1994; steinfartz et al., 2000; manenti et al., 2009b; ficetola et al., 2012). some subspecies and populations have evolved differently, skipping the aquatic developmental stage (buckley et al., 2007). between these two extremes, fire salamanders have also specialized in using alternative aquatic habitats: epigeous stagnant waters such as ponds (egea-serrano et al., 2006; schulte, 2008; caspers et al., 2009) and hypogeous (i.e., subterranean) springs or pools (manenti et al., 2009a; 2013). fire salamander larvae can coexist with other caudate species. this occurs frequently with newts in ponds and similar lentic water bodies (wiestermann, 2004; segev and blaustein, 2007; thiesmeier and dalbeck, 2011). however, in running waters, they are more often the only caudate species present (thiesmeier and dalbeck, 2011). exceptions remain, such as in the pyrenees, where fire salamander larvae can be seen in the same streams as pyrenean brook newts (calotriton asper). however, even in this case, there is often spatial partitioning between the two species (guillaume, 2006). alternative developmental strategies also occur in newts. the most widespread pattern implies the metamorphosis of aquatic gilled larvae into juveniles that mature on land (wells, 2007). however, in some populations, larvae skip metamorphosis and retain gills at the adult stage; a process known as paedomorphosis (whiteman, 1994). in the palmate newt (lissotriton helveticus), 44 m. denoël, l. winandy this occurs with the highest incidence on the larzac plateau in southern france (gabrion et al., 1977; denoël, 2007). although found in the springs and streams of peripheral areas of southern larzac (geniez and cheylan, 2012), cases of reproduction of fire salamanders have not yet been described on the plateau itself and, to our knowledge, coexistence patterns with paedomorphic newts have not been reported in the literature. in this context, the aim of this study was to determine the occurrence of fire salamander larvae on the larzac plateau, to present the characteristics of a pond-breeding population in cohabitation with both newt morphs, and to propose hypotheses on this unusual pattern. material and methods a total of 277 ponds were surveyed for the presence of amphibians, with a special focus on palmate newts as part of a long-term survey on the southern larzac plateau (2001-2013) in the hérault and gard departments (languedoc-roussillon region). this encompassed all the ponds, potentially adequate for amphibians which were found using varied sources: literature searches, topographical maps, ortho-image analyses and local contacts. running waters are extremely rare on the plateau and consist in small springs that water directly some ponds, and springs of larger streams on the limits of the plateau, i.e. almost not running in the plateau itself. these habitats were all investigated during the present survey. the region is a limestone area with traditional agriculture, particularly at its southern limits (durand-tullou, 1959). the blandas plateau was also included in this study, since it is globally part of larzac. most sampling occurred in spring months during the peak of the newt breeding season (670 censuses, i.e., an average ± se of 2.4 ± 0.1 visits per pond), but also in summer and autumn in some years. the present study focused on the pond where fire salamander larvae were found (see results). at this pond, the sampling of amphibians was done during day-time on 7 june 2011, 27 march 2013, and 13 july 2013. sampling in the study pond was done as exhaustively as possible by dip-netting, i.e., stopping netting when no more adult newts or salamander larvae where found after several trials in all the micro-habitats of the pond. the same technique was used in the other larzac ponds, but seining was also used for the deepest ones (see denoël and ficetola, 2014). given the high capture effort in each habitat and the rather high fecundity of fire salamanders, it is very unlikely that breeding events were missed in ponds. although the study was aimed first at studying palmate newts, larvae of fire salamanders would not have been overlooked as a special focus was done in all ponds for the larvae of palmate newts, which are much different from those of fire salamanders. however, the abundances determined are likely underestimated as some individuals, particularly in the larval cohorts can hide more easily than adults in ponds. in the population inhabited by fire salamander larvae, the census included both adult newt morphs and fire salamander larvae. paedomorphic palmate newts were distinguished from metamorphic ones by the presence of gill slits and external gills. adulthood was assessed by the presence of a developed cloaca (denoël et al., 2001). snout-vent length (from the tip of the snout to the end of cloaca) was measured in the field. because of the very low number of paedomorphs, values were pooled across sexes, so focusing only at the level of species and phenotypes. since the mesh size of the dip net was 4 mm, it is likely that the smallest palmate newt larvae were not caught. consequently, the results on larvae are indicative of the size of individuals from the largest cohort only. the non-parametric mann-whitney test was used because of the non-normality of data even after transformation. some individuals were likely the same in the analyses done at different dates so the data sets are not fully independent. for this reason, comparisons were done only within each sampling date. all tests were computed in statistica 10 (http://www.statsoft.com). some habitat variables were recorded in the field: maximum water depth, pond size, presence of aquatic vegetation, oxygen concentration, conductivity, ph and turbidity. forest cover was obtained from analysis of ortho-images and elevations above sea level from a digital terrain model (institut geographic national, france) in arcgis 10.2 (http://www.esri.com). results fire salamander larvae (salamandra salamandra terrestris) were found in only one out of the 277 sampled ponds (0.36% occurrence; fig. 1). this population is located on the plateau de courcol at one of the southern margins of larzac at an elevation of 711 m a.s.l. this area is part of the forêt domaniale notre-dame de parlatges and of the saint-pierre de la fage municipality (hérault department). the pond is on the plateau but close to the rupture of slopes making the transition with the foothills and lowlands (fig. 1). it is a man-made pond built in concrete for game management. a small ditch drives rain water to the pond. on average, the pond’s surface area covered 19 m², the maximum depth was 55 cm, conductivity 212 µs/ cm, and oxygen concentration 6.2 mg/l. the ph of the pond in june 2011 was 8.1. no vegetation was present in the pond, except on the border, and a layer of green algae covered parts of the sediment layer in july 2013. the water was muddy in 2011 and transparent in 2013. forests covered 51% of the 20-m radius and 77% of the 100-m radius around the focal pond. only the south-eastern side of the pond was directly bordered by tree plantations, mainly comprising austrian pines (pinus nigra). fire salamander larvae were present at each of the three visits (n = 19, 5 and 9, respectively, for each sampling session; fig. 2), always in presence of adult palmate newts (n = 8, 62 and 64, respectively) and their larvae 45coexistence of fire salamander larvae and paedomorphic newts (even more than 60, march 2013). paedomorphic newts were found only at the two 2013 sampling sessions: four in march and three in july. some parsley frogs (pelodytes punctatus) but no marbled newts (triturus marmoratus) were found in the pond. the mean ± se snout-vent length of fire salamander larvae was 26.6 ± 3.2 mm in march 2013 (n = 5) and 29 ± 0.6 mm in july 2013 (n = 9), whereas for paedomorphs it was 36.3 ± 1.6 mm (n = 4) and 36.3 ± 1.5 mm (n = 3) and for metamorphs it was 41.7 ± 0.6 mm (n = 47) and 42.5 ± 0.7 mm (n = 40) for the two dates, respectively. in march 2013, the snout-vent length of the largest cohort of palmate newt larvae was 20.4 ± 0.2 mm (n = 15). the size of fire salamander larvae was significantly smaller than that of metamorphs (mann-whitney u test: z= −3.632, p < 0.001 and z = −2.404, p < 0.05 in march and july 2013, respectively) and paedomorphs in july (z = −4.634, p < 0.001), but not in march (z= −1.152, p = 0.08), although the small sample size needs to be taken into account for the interpretation of the p-value. the size of fire salamander larvae was larger than that of larval palmate newts in july 2013 (z = 4.067, p < 0.001). discussion the occupancy of an alternative reproductive habitat (i.e., a pond) led to an unusual coexistence between fig. 1. localization of the studied pond with fire salamander larvae (large star) and the other inventoried ponds without fire salamanders (full circles: some appear superposed) on the southern larzac plateau, france. the background of the map represents the relief (the lowest elevations are darker). the continuous and interrupted traits represent, respectively, the a75 highway and the boundaries between administrative departments (hérault, gard and aveyron). see text for discussions on external records in valleys and foothills outside the study area. 46 m. denoël, l. winandy aquatic larvae of a terrestrial species, the fire salamander, and an alternative paedomorphic phenotype of a newt species. such situations are valuable for understanding the success of strategies in presence of potential competitors or predators (rheinhardt et al. 2013; werner et al., 2013). in the case studied herein, the low prevalence of both alternative strategies (i.e., pond use by the fire salamander and paedomorphosis) may suggest that the habitat is not optimal, either because of the lack of adequate environmental features for both alternatives or because the cohabitation reduced individual payoffs as a consequence of competition or predation pressures (segev and blaustein, 2007; wissinger et al., 2010). long-term or experimental studies including foraging and comparative analyses are needed to test these hypotheses. the preliminary results presented here suggest that habitat characteristics are not fully optimal for paedomorphosis, which may explain its low rate (maximum 6%) in the population. a multivariate analysis of the ecological correlates of paedomorphosis in larzac ponds showed the importance of deep water for the occurrence and abundance of paedomorphic palmate newts (denoël and ficetola, 2014). the advantage of deep ponds for paedomorphs is in that they are less likely to dry up and they can provide habitat heterogeneity favourable to the persistence of the dimorphism (denoël, 2003; 2005; 2006). here the shallow depth and the size of the studied pond (55 cm, 19 m²) make it a homogeneous habitat that might occasionally dry up in summer. fire salamanders are among the top foragers in pond environments that drive the composition of aquatic communities (blaustein et al., 1996; reinhardt et al., 2013). although their larvae can coexist with other amphibian species, they are often the only reported amphibian in the habitats where they are found (thiesmeier and dalbeck, 2011). fire salamander larvae reduce the abundance of newt larvae through both competition for invertebrates and predation of small newt larvae (segev and blaustein, 2007) and avoidance–attraction mechanisms (guillaume, 2006). in the studied larzac pond, fire salamander larvae accounted for a small part of the amphibian community in both 2013 sampling sessions but not in june 2011. it is therefore too premature to conclude on the direction of possible competitive or predatory interactions. however, it can be expected that larval and paedomorphic newts may share some feeding similarities with fire salamander larvae given that they also rely on the aquatic suction mechanisms of aquatic invertebrates (reilly, 1996; denoël and joly, 2001; denoël, 2004; denoël et al., 2004; reinhardt et al., 2013). it is unlikely that fire salamanders were more abundant in larzac in the past few centuries than during the present study (2001-2013). many larzac ponds are recent and larzac used to be less forested (at least since agriculture was established in the area), i.e., factors that are usually not favourable for the fire salamander (manenti et al., 2009b). specifically, the austrian pine plantations around the pond date from the 1930s. the atlas of languedoc-roussillon amphibians highlights a gap in the distribution map, corresponding to the larzac and blandas plateaux (geniez and cheylan, 2012). when fire salamanders were present at nearby localities outside the plateau, they were associated with springs and running waters but, at a broader scale in languedoc-roussillon, they were also found in ponds (geniez and cheylan, 2012). the only published record of the presence of the species strictly on the plateau was made in an area known as lac des rives – a temporary lake – but it refereed to an adult individual (observation of m.f. lapeyrie in 1995: geniez and cheylan, 2012). no larvae were seen in this area when the lake and surrounding ponds were flooded in 2004 (m. denoël, pers. obs.). in contrast, the fire salamander was reported more frequently in the hills north-east of the blandas plateau and in the cevennes (geniez and cheylan, 2012). the species is also present in the wooded slopes at the southern limits of larzac, for instance in cirque de labeil and cirque du bout du monde (p. arnaud, pers. comm.) as well as at varied localities of the bottom of vis canyon, such as mas du pont, madières and grenouillet (g. hanula, m. salze, r. servaye, pers. comm.). west of southern larzac, terrestrial fire salamander individuals were also found in the southern foothills of the plateau de guilhaumard (a satellite plateau of larzac) in the upper orb valley, close to a running water network (midi-pyrénées database: pottier, 2008; lpo aveyron database). the near absence of springs and running waters on the larzac plateau itself has probably limited the presence of this species. moreover, the studied area fig. 2. fire salamander larva (plateau de courcol, larzac, france). 47coexistence of fire salamander larvae and paedomorphic newts and its southern foothills are at the margin of its distribution range, with fire salamanders absent from the lowlands along the mediterranean in languedoc-roussillon (geniez and cheylan, 2012; riberon and miaud, 2012). more inventories should be done in springs and small streams in the foothills at the edges of larzac to determine the exact distribution and ecology of the species and to better understand its rarity on the plateau. as the studied pond was only recently created (in 1995), colonization may be interpreted as recent, which may have been facilitated by the movement capabilities of this species (schmidt et al., 2007; schulte et al., 2007) and by the tree plantations around the pond. the closest observations of the fire salamander were made around 0.8 km north-west (parlatges), 2 km south-west (champ du lac) and 2.4 km south-east (salces) of the study area, all referring to larvae in streams and/or springs and to adults on land (p. arnaud and g. hanula, pers. comm.). the linear distance, and particularly the large differences in altitude (up to 400 m), and the steep slopes separating these places from the pond studied herein, suggest that they may have been connected through dispersal of some individuals rather than by regular exchanges. this hypothesis is supported by the fact that reported values of home ranges in the literature (e.g., an average of 1295 m²; schulte et al., 2007) are lower than the distances to the nearby locations. the easiest connection pathway may have been from the south-west as several dozens of terrestrial fire salamanders were seen there, with some individuals up to the slope rupture (p. arnaud, pers. comm.) and because there are no major breaks in elevation from this observation site to the studied pond. this case constitutes the highest number of adult salamanders reported in the area. the number of adult individuals that used the present pond is not known because only few larvae were observed. the larvae could thus result from either a single or very few parturition acts. however, the repeated observations in 2011 and 2013 suggest that this is a local specialization or a breeding opportunism. more research (e.g., microsatellite analyses) to find the closest related populations could help to determine possible differentiations. one more potentially favourable pond, intermediate between the records of fire salamanders under the slope rupture and the studied pond also remains to be sampled. finally the rarity of the fire salamander in southern larzac (0.36% of water bodies inhabited, all included) makes this site highly vulnerable. the observed accumulations of sediments in the pond may be detrimental for both fire salamander larvae and newts, particularly paedomorphs, because it reduces water availability (denoël and ficetola, 2014). local interventions to remove excess sediments helped in this perspective. no external signs of dermocystid parasites were seen in the fire salamander larvae and palmate newt individuals of this population, although these pathogens were found on newts at other larzac ponds (gonzález-hernández et al., 2010). both introduced fish and crayfish were seen nearby on the plateau, whereas crayfish were also found in streams peripheral to larzac (m. denoël, pers. obs.; g. hanula, pers. comm.). palmate newt populations from larzac are declining due to these fish introductions (crochet et al., 2004; denoël et al., 2005), whereas fire salamanders from other areas suffered from both fish and crayfish introductions (martinez-solano et al., 2003; cruz et al., 2006; ficetola et al., 2011). studies at other edges of the fire salamander distribution range also showed high risks of local extirpations (spitzen-van der sluijs et al., 2013). consequently, much attention needs to be paid to the fire salamander populations from larzac and its foothills. acknowledgements we are grateful to b. thiesmeier and two anonymous reviewers for their comments on this manuscript, g. pottier and l. weber (midi-pyrénées “batz” database), p. geniez (languedoc-roussillon “malpolon” database), r. liozon (lpo aveyron database), p. arnaud (office national de la chasse et de la faune sauvage), g. hanula, m. salze and r. servaye for providing locality data on fire salamanders, b. fernandez and g. rieffe (office national des forêts) for sharing information on the studied area, and to pond owners, municipalities and the o.n.f. for allowing access to their ponds. the capture permit was provided by dreal languedoc-roussillon. all animal handling followed ethical standards. the scientific and ethical aspects of the protocol were approved by the conseil national de la conservation de la nature (france). this research received a fonds de la recherche scientifique f.r.s.-fnrs grant j.0008.13, and a fonds spéciaux pour la recherche grant c11/23 (university of liège). this is a publication of the applied and fundamental fish research center (affish-rc). m. denoël and l. winandy are a research associate and a research fellow, respectively, at f.r.s. – fnrs. references blaustein, l., friedman, j., fahima, t. 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(2004): feuersalamander salamandra salamandra (linnaeus, 1758). in: die lurche und kriechtiere sachsen-anhalts. verbreitung, ökologie, gefährdung und scutz, pp. 50-56. meyer, f., buschendorf, j., zuppke, u., braumann, f., schädler, m., grosse, w.-r., eds., laurenti verlag, bielefeld. wissinger, s.a., whiteman, h.h., denoël, m., mumford, m.l., aubee, c.b. (2010): consumptive and nonconsumptive effects of cannibalism in fluctuating agedstructured populations. ecology 91: 549-559. acta herpetologica vol. 8, n. 2 december 2013 firenze university press journal of the societas herpetologica italica acta herpetologica acta herpetologica 9(2): 159-166, 2014 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-14013 new insights into the taxonomy of the skittering frog euphlyctis cyanophlyctis complex (schneider, 1799) (amphibia: dicroglossidae) based on mitochondrial 16s rrna gene sequences in southern asia asghar khajeh1,5, zeinolabedin mohammadi2,5,*, fatemeh ghorbani2, javad meshkani3, eskandar rastegar pouyani3, adam torkamanzehi4 1 department of crop productions technology, faculty of agriculture and natural resource,, university of sistan & baluchestan, zahedan, iran 2 department of biology, faculty of sciences, ferdowsi university of mashhad, mashhad, iran. * corresponding author. e-mail: mohammadi.zeinal@gmail.com 3 department of biology, faculty of sciences, hakim sabzevari university, sabzevar, iran 4 department of biology, faculty of sciences, university of sistan & baluchestan, zahedan, iran 5 both authors contributed equally to this manuscript submitted on 2014, 2nd february; revised on 2014, 8th august; accepted on 2014, 12thseptember editor: uwe fritz abstract. the skittering frog (euphlyctis cyanophlyctis) is considered to be a species complex distributed in southern and southeastern asia. genetic diversity and taxonomic status of populations across their ranges is unclear and existence of several cryptic species is expected. in this study we used sequence variation in the mitochondrial 16s ribosomal rna gene to elucidate the taxonomic status of iranian populations of e. cyanophlyctis and compare their genetic diversity and divergence to populations from the indian subcontinent. phylogenetic analysis indicated that the populations of e. cyanophlyctis from iran, bangladesh-assam (northeastern india), southern india, and sri lanka are partitioned into different clades. at least four different haplogroups were detected which are here proposed to be considered as allopatric cryptic species. the sedimentation of the helmand river into the sistan depression during the neogene and subsequent formation of dry land barriers are proposed to have caused the iranian populations of skittering frogs to be disconnected from those of the indian subcontinent, resulting in differentiation of these lineages. in addition, some populations from southern india and those from sri lanka that were previously recognized as e. cyanophlyctis belong to e. mudigere. a preliminary investigation on the genetic diversity of the populations from southeastern iran highlights the low genetic diversity among these populations. keywords. euphlyctis cyanophlyctis, phylogeny, 16s rrna gene, differentaition. introduction the genus euphlyctis (family dicroglossidae) consists of six species of which euphlyctis cyanophlyctis (schneider, 1799) is one of the most widespread in southern asia. this species has been assigned to various genera over time, including rana (rana cyanophlyctis boulenger, 1920), bufo (bufo cyanophlyctis latreille, 1801), and dicroglossus (dicroglossus cyanophlyctis deckert, 1938), and it has been revised in its current name by dubois (1975). the type locality of this species is probably tranquebar in southeastern india (bauer, 1998) and the species is believed to be distributed throughout southern asia from southeastern iran to vietnam and sri lanka (frost, 2013). the distribution and taxonomy of iranian populations of e. cyanophlyctis have been debated by several authors. boulenger (1920) mentioned that blanford 160 a. khajeh et alii recorded it from makran, persian baluchestan. earlier, nikolskii (1899) described rana cyanophlyctis seistanica from sistan, iran based on its smaller tympanum and longer snout. this subspecies has only been reported from the type locality and along the border of iran, pakistan, afghanistan (khan, 2004). anderson (1963) later recorded rana cyanophlyctis from iran. baluch and kami (1995) reported rana cyanophlyctis cyanophlyctis from southern and southeastern iran and proposed rana cyanophlyctis seistanica to be a synonym. additionally, khan (1997) described a new subspecies, euphlyctis cyanophlyctis microspinulata from khuzdar, baluchestan, pakistan and recognized three subspecies in his checklist of the amphibians of pakistan (khan, 2004). the previous phylogenetic analysis by kosuch et al. (2001) based on a fragment of the mitochondrial 16s ribosomal rna gene suggested a close relationship between e. cyanophlyctis and e. ehrenbergii. a subsequent analysis suggested a sister relationship between e. cyanophlyctis and e. hexadactylus (kurabayashi et al., 2005). alam et al. (2008) studied the genetic divergence of two species in this genus, e. cyanophlyctis and e. hexadactylus, and recognized at least two cryptic species among the populations of e. cyanophlyctis from india, sri lanka and bangladesh. in addition, a new species, euphlyctis mudigere, was described recently from southwestern india (joshy et al., 2009). during the late paleocene and early eocene, the indian plate, which had broken away from gondwana in the early cretaceous, collided into the eurasian landmass and formed the sistan depression along its western part on the iranian microcontinent. later, during the neogene, the sistan depression was filled as a result of continuous sedimentation of helmand river (molnar and tapponnier, 1975; klootwijk and pierce, 1979). the region is today a transition between the oriental and palaearctic faunas and contains a mixture of elements of both (misonne, 1959; borkin and litvinchuk, 2012). however, the helmand basin serves as a low altitude barrier (macey et al., 1998; rastegar pouyani et al., 2010) between iranian plateaue and indian subcontinent. this area in southeastern iran comprises dry lands irrigated by the helmand river and includes hamoon lake (whitney, 2006). the region also comprises a part of the lut desert, one of the most arid deserts in the world extending to the dashti margo (desert of death) in afghanistan. low precipitation and a large excursion between day and nighttime temperatures leads to a low diversity of amphibian in this region except for infrequent aquatic habitats associated with the helmand river that provide suitable habitats for populations of e. cyanophlyctis. in this study, we address the taxonomic status of different populations of e. cyanophlyctis from sistan, southeastern iran. we analyzed intraspecific variation within different populations of e. cyanophlyctis of southern asia and also investigated the phylogenetic relationships among populations of five species of the genus euphlyctis in southern asia to assess the taxonomic status of the species complex. materials and methods specimens a total of 41 individuals of e. cyanophlyctis were collected using hand nets in 2010 to 2011 from nine localities in sistan and baluchestan province in southeastern iran (fig. 1). identification of specimens was based on baluch and kami (1995). all specimens are deposited in the zoological laboratory of faculty of agriculture and natural resource, department of crop productions technology,, saravan, iran. tissue samples were available from 32 specimens of the 41 specimens of e. cyanophlyctis as well as two pseudepidalea viridis (family bufonidae) from iran that were used as outgroups (table 1 and fig. 1). all tissue samples were taken from the musculature of the hind limbs and preserved in 80% ethanol. laboratory procedures total genomic dna was extracted from ethanol-preserved muscle using a standard phenol/chloroform protocol (sambrook et al., 2001). standard polymerase chain reaction (pcr) amplifications were performed to amplify 492 bp of the of the mitochondrial 16s ribosomal rna gene fragment (hereafter 16s). pcr amplification was performed using the universal forward primer 16sl (5´-cgcctgtttatcaaaaacat-3´) and the reverse primer 16sh (5´-ccggtctgaactcagatcacg-3´) following the protocol of palumbi (1991). the amplified dna fragments were sequenced commercially using the automated sequencer abi prism 3700 at macrogen inc. (south korea). newly obtained sequences were deposited in genbank (kf992800 to kf992833) (table 1). in addition to specimens from iran, 21 additional 16s sequences of the genus euphlyctis and one of the dicroglossid hoplobatrachus tigerinus were obtained from genbank (table 1). phylogenetic analyses nucleotide sequences were aligned using clustalw (thompson et al., 1994) as implemented in the bioedit sequence alignment editor, v.7.0.9 (hall, 1999). gaps in the alignment were treated as missing data. genetic distances were calculated with mega v.4.0 (tamura et al., 2007) with the p-distances model using the complete deletion option (kimura, 1980). phylogenetic reconstructions were carried out under the maximum 161on the taxonomy of the euphlyctis cyanophlyctis parsimony (mp) and the maximum likelihood (ml) methods in paup* v.4.0b10 (swofford, 2000). appropriate substitution models were determined using the akaike information criterion, as implemented in modeltest (posada and crandall, 1998). maximum likelihood analyses were carried out using a heuristic tree search with 10 random addition sequence replicates and tree-bisection-reconnection (tbr) branch swapping. maximum parsimony analyses were performed using heuristic searches with tbr branch swapping and random addition sequence with 1000 replicates. support for internal nodes was estimated via nonparametric bootstrapping (500 and 5000 replicates for ml and mp, respectively) (felsenstein, 1985) with a single random addition sequence replicate per bootstrap replicate. bayesian inference (bi) reconstructions were performed using mrbayes v.3.2.2 (huelsenbeck et al., 2011). four simultaneous markov chain monte carlo (mcmc) chains with incremental heating temperature 0.2 were run 6000,000 generations and sampled every 100 generations. the burn-in size was determined by checking the convergence of −log likelihood (−inl) values, and the first 10% of the mcmc chain was discarded. the bayesian posterior probability (pp) was evaluated from the remaining trees. haplotype and nucleotide diversity indexes for evaluation of dna polymorphism were estimated using dnasp v.5 (librado and rozas, 2009). the parsimony network of genealogical relationships within e. cyanophlyctis was obtained tcs v.1.21 (clement et al., 2000). results phylogenetic analyses were conducted on a final alignment of 492 bp of 16s for 53 individuals of euphlyctis, as well as two p. viridis and one h. tigerinus that served as outgroups. the alignment contained 81 variable sites, of which 48 were parsimony informative. gaps were coded as missing and there were no ambiguous sites. the best-fit model obtained from modeltest was a general time-reversible substitution model (gtr+i+g) (-lnl=1782.6420), with a proportion of invariable sites i= 0.3651, variable sites g=0.4488, empirical base frequencies (a: 0.3035; c: 0.2413; g: 0.2079; t: 0.2473), and fig. 1. map showing the sampling localities of the euphlyctis cyanophlyctis used for this study. 162 a. khajeh et alii substitution rates (rate[a–c] 8.0429, rate[a–g] 15.1827, rate[a–t] 6.5556, rate[c–g] 0.9254, rate[c–t] 33.0808, rate [g-t]1.0000) estimated from the data set. these parameters were used for ml analysis. the tree topologies are well resolved and identical among different analyses (fig. 2). two strongly supported primary clades (a/b and c) are recognized in all analyses. clade (a) includes e. cyanophlyctis from iran (a1) and e. cyanophlyctis from bangladesh-assam (a2). clade (b) consists of e. cyanophlyctis from the southern india (b1), e. ehrenbergii (b2) beside e. mudigere and specimens from india and sri lanka that were previously identified as e. cyanophlyctis (b3) (bossuyt and milinkovitch, 2000; alam et al., 2008). the third clade (c) comprises sequences of e. aloysii and e. hexadactylus (fig. 2). the sister relationship between the clade (a1) and (a2) was well supported across all optimality criteria (99% parsimony bootstrap value, 70% likelihood bootstrap and 100% bayesian posterior probability). in addition, our analyses reveal low genetic distances within each clade, including low inter-population genetic divergence between iran and bangladesh-assam specimens (1.7%) and high divergence between bangladesh-assam and india (4.7%) (table 2). specimens of e. cyanophlyctis from iran and the southern india had the lowest intrapopulation genetic distance (0.1%) while specimens from bangladesh-assam showed the highest intra-population genetic distance (0.5%) (table 2). in total, 12 unique 16s haplotypes were identified in 49 individual skittering frogs. low haplotype diversity was found in iran populations (hd= 0.353) in comparison to higher diversity found in the bangladesh-assam populations (hd= 0.857). additionally, the populations of skittering frogs from iran demonstrate the lowest nucleotide diversity (π = 0.00072) as compared to the populations from all the other regions. bangladesh-assam table 1. tissue samples and genbank accession numbers as well as locality information for the euphlyctis cyanophlyctis specimens used in this study. number of specimens (n), number of haplotype (h), haplotype diversity (hd ±sd) and nucleotide diversity (π±sd). sequences not generated in the framework of this study were obtained from genbank from following publications: a: bossuyt and milinkovitch (2000); b: kosuch et al. (2001); c: kurabayashi et al. (2005); d: alam et al. (2008); e: joshy et al. (2009); f: kotaki et al. (2010); g: hasan et al. (2012). sequences with accession numbers starting with kf were generated for the present study. species collecting site n locality number h hd π accession number country locality e. cyanophlyctis iran karvandar 3 1 2 0.353± 0.084 0.00072 ± 0.00017 kf992818-kf992820 iran irendeghan 3 2 kf992821-kf992823   iran jalgh 3 3 kf992809-kf992811 iran apatan 6 4 kf992800-kf992805 iran zaboli 3 5 kf992829-kf992831 iran ashar 2 6 kf992827 and kf992828 iran sarbaz 3 7 kf992824-kf992826 iran domaki 3 8 kf992806-kf992808 iran tiran 6 9 kf992812-kf992817 india padil 2 10 2 0.567± 0.129 0.00262± 0.00066 ab272603-ab272604d india mangalore 1 10 ab488901f india konaje 1 11 jf832392 india madikeri 1 11 ab167938c india cochin 1 13 ay014366b india assam 1 18 5 0.857± 0.108 0.00484± 0.00075 ab290420d bangladesh mymensingh 3 17 ab272602d, ab530495g, ab272601d bangladesh cox’s bazar 4 16 ab530494g, ab530496-ab530498g e. mudigere sri lanka 2 15 3 0.833 ± 0.222 0.00378± 0.00378 ab290418-ab290419d india mudigere 2 12 ab377109e, af249053a e. aloysii india adyar 1 14 ab272606e e. ehrenbergii yemen 1 ay014367 b e. hexadactylus india mangalore 1 10 ab167941 c h. tigerinus india padil 1 10 ab272594 d p. viridis iran zabol 2 kf992832-kf992833 163on the taxonomy of the euphlyctis cyanophlyctis fig. 2. maximum likelihood phylogeny for euphlyctis cyanophlyctis (using a 492 bp portion of the mitochondrial 16s ribosomal rna gene). numbers represent mp and ml bootstrap values (mp/ml), respectively (500/5000 replicates). the posterior probability values from the bayesian analysis are indicated at the > 99 % (**) and > 95 % (*) significance levels. 164 a. khajeh et alii populations have the highest nucleotide diversity (π = 0.00484). iran populations exhibit the lowest number of 16s rrna haplotypes (h=2) whereas the highest number of 16s rrna haplotypes was found in bangladesh-assam (h=5). these 12 haplotypes were grouped in four separate haplogroups that correspond to the geographic distribution of these samples (table 1 and fig. 3). the first haplogroup includes two haplotypes from iran and the second haplogroup was identified as related to nominal e. cyanophlyctis from the southern india. the third haplogroup consists of five 16s rrna haplotypes from bangladesh-assam specimens and the fourth haplogroup has three haplotypes that included specimens from sri lanka, southern india, and one specimen of e. mudigere (fig. 3). discussion the phylogenetic analyses, genetic distances, and haplotype network analyses strongly suggest that e. cyanophlyctis populations are split into four main genetic lineages separated by a high level of nucleotide divergence (between 1.7% and 4.7% k2p distance). these lineages correspond to the eastern clade (a1) from iran, the northern clade (a2) specimens bangladesh-assam, the southern clade (b1) that we consider to be nominal e. cyanophlyctis because of its proximity to the type locality, and the clade from southern india and sri lanka that includes e. mudigere. morphometric, morphological and molecular similarities (low haplotype and low nucleotide diversity) between populations of skittering frogs from southeastern iran casts strong doubts on the validity of e. cyanophlyctis seistanica. in contrast, there is substantial divergence between populations of e. cyanophlyctis from iran and the indian subcontinent. formation and subsequent sedimentation of the sistan depression during the neogene (whitney, 2006) might have played a role as the main barrier, causing geographic isolation and divergence between populations of skittering frogs on the indian subcontinent and in the iranian helmand basin. genetic divergence is a major consequence of vicariance and both dispersal events and isolation by distance can drive speciation (lomolino et al., 2005). the formation of the helmand barrier was proposed to have driven vicariance of different clades within the genus laudakia and the eremias persica complex (macey et al., 1998; rastegar pouyani et al., 2010). the close relationship of specimens from iran and bangladesh is difficult to explain in terms of geological and dispersal events. this could be the result of molecular homoplasy or ancestral state polymorphism (avise, 1994) caused by recent fragmentation rather than recent dispersion. on the other hand, the specimens from sri lanka and india constitute a monophyletic group with e. mudigere suggesting that all of these specimens are referable to that species. the occurrence of e. mudigere in sri lanka and india was not unexpected since sri lanka is a part of the deccan plateau and was connected to the indian subcontinent until the late miocene (deraniyagala, 1992). although specific values of genetic divergence should not serve as the only basis for delimitation of species, a threshold of 3% genetic divergence could be used for recognition of a new species (based on fouquet et al. 2007 for 16s). we recovered genetic divergences more than 3% between bangladesh-assam and southern indian (4.7%) as well as between iran and indian populations (4.1%). these results are concordant with the study by alam et al. (2008) that identified several cryptic species including at least four cryptic species currently identified as e. cyanophlyctis. these cryptic lineages are probably the result of allopatric speciation and a detailed taxonomic revision is strongly recommended. table 2. p-distances of the 16s gene of euphlyctis species. withingroup divergences in bold. species 1 2 3 4 5 6 7 1. e. cyanophlyctis (iran) 0.1 2. e. cyanophlyctis (bangladesh-assam) 1.7 0.5 3. e. cyanophlyctis ( india ) 4.1 4.7 0.1 4. e. mudigere 3.8 4.5 3.4 0.4 5. e. ehrenbergii 6.7 7.4 5.4 5.1 6. e. hexadactylus 9.9 10.3 9.8 9.9 11.5 7. e. aloysii 10.8 10.7 11.1 10.2 11.5 7.7 fig. 3. haplotype networks of euphlyctis cyanophlyctis (49 individuals) based on a 492 bp portion of the mitochondrial 16s ribosomal rna gene. networks were not joined if haplotypes were separated by more than 9 mutations. (h1, h2): iran; (h3, h4): india; (h5, h6, h7, h8, h9): bangladeshassam; (h10, h11, h12): mudigere (ab377109), india and sri lanka. 165on the taxonomy of the euphlyctis cyanophlyctis acknowledgments this research was supported by grants from the research department of the university of sistan & baluchestan, zahedan, iran under project no. 89013. permission to collect was authorized by the iran department of environment (permission number: 912-n/13/1; 2009; 15th august). we are grateful to professor muhammad sharif khan for his kind cooperation in identification of the specimens. our special thanks go to david c. blackburn and professor kraig adler for editing the manuscript and their valuable comments on the manuscript. the first author is deeply indebted to seyed saeed hoseinian for his kind help. references alam, m.s., igawa, t., khan, m.m.r., islam, m.m., kuramoto, m., matsui, m., kurabayashi, a., sumida, m. 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(2006): geology, water, and wind in the lower helmand basin, southern afghanistan. us geological survey scientific investigation report. acta herpetologica 10(1): 73-74, 2015 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-15321 book review: harold heatwole, john w. wilkinson (eds). amphibians biology. volume 11 status of conservation and decline of amphibians. eastern hemisphere. part 4 . southern europe and turkey sebastiano salvidio dipartimento di scienze della terra, dell’ambiente e della vita (distav), università di genova, i 16132 genova italy. e-mail: salvidio@dipteris.unige.it this book consists of 158 pages, a soft cover and is available from pelagic publishing, exeter united kingdom. it is part of a series of similar volumes recently published on the conservation status of amphibians in western europe (heatwole and wilkinson, 2013), asia (heatwole and das, 2014) and north africa (busach and heatwole, 2014). the book covers, with a specific chapter each, the following countries (in alphabetical order): albania, bosnia and herzegovina, bulgaria, croatia, cyprus, greece, hungary, italy, macedonia, malta, montenegro, republic of serbia, romania, slovenia and turkey. the reader may notice the absence of portugal and spain, that in fact were included in a previous volume (heatwole and wilkinson, 2013). the list of contributors includes 25 authors, all of them well known herpetologists being, without any doubt, among the major experts of the region they are called to illustrate within this book. each chapter is organised in short sections, generally similar but not identical for all countries. generally, an introductory section presents and discuss the diversity of the batrachofauna, with usually at least one table listing the species found in each territory, their international and national level of protection and their local conservation status. these sections are very useful and informative at the same time, because they provide immediate information about the current situation of the local amphibian diversity found in each country. a second section is devoted to species of special concerns (e.g. rare, highly isolated or declining) or to urgent conservation problems, such as the diffusion of chytrids, the introduction of invasive species that threaten amphibians or specific cases of habitat degradation or destruction. the third section is specifically dedicated to “conservation measures and monitoring programmes” and, in my personal opinion, provides the most interesting and original information of the entire book. unfortunately, the quantity and quality of the information provided for the different chapters is highly heterogeneous, going from the three full pages of slovenia, to only five lines of macedonia, without speaking of bosnia and herzegovina where this section is completely missing. each chapter ends with a list of references. this book is informative as it provides updated information about the status of amphibian populations in the many countries treated. reading it, i found many useful data concerning different places, species or populations and i think the book will be of interest to professional herpetologists and to conservationists as well. however, i regret that these positive result could have been even more successful with some additional effort to standardise the different contributions that are often too unbalanced, at least concerning the quantity of the information provided. finally, readers would have surely appreciated a better quality of the printed colour illustrations. references busack, s.d., heatwole, h. (2014). amphibian biology. volume 11. status of conservation and decline of amphibians: eastern hemisphere part 2: north africa. basic appl. herpetol. 27: 1-117. 74 s. salvidio heatwole, h., das, i. (2013). amphibian biology. volume 11. status of conservation and decline of amphibians: eastern hemisphere part 1: asia. natural history publications, kota kinabalu, malaysia. heatwole, h., wilkinson, j.w. (2013). amphibian biology. volume 11 part 3: status of conservation and decline of amphibians: eastern hemisphere: western europe. pelagic publishing, exeter uk. acta herpetologica vol. 10, n. 1 june 2015 firenze university press obituary: valery k. eremchenko (1949-2014) leo j. borkin1, tatjana dujsebayeva2, roberto sindaco3, matthias stöck4 haplotype variation in founders of the mauremys annamensis population kept in european zoos barbora somerová1, ivan rehák2,*, petr velenský2, klára palupčíková1, tomáš protiva1, daniel frynta1 reproductive ecology of sichuan digging frogs (microhylidae: kaloula rugifera) wei chen1,*, lina ren2, dujuan he2, ying wang2, david pike3 toxic effects of carbaryl on the histology of testes of bufotes variabilis (anura: bufonidae) özlem çakici basal frequency of micronuclei and hematological parameters in the side-necked turtle, phrynops hilarii (duméril & bibron, 1835) maría a. latorre 1,2,*,#; evelyn c. lópez gonzález1,2, #; pablo a. siroski1,2,3; gisela l. poletta1,2,4 into a box interiors: clutch size variation and resource allocation in the european pond turtle marco. a.l. zuffi1,*, simonetta citi2, elena foschi1, francesca marsiglia1, eva martelli1 where to “rock”? choice of retreat sites by a gecko in a semi-arid habitat andreia penado1,2, ricardo rocha3,4,*, marta sampaio3, vanessa gil3, bruno m. carreira3, rui rebelo3 age structure, growth and longevity in the common toad, rhinella arenarum, from argentina clarisa de l. bionda1,2,*, silvia kost 4, nancy e. salas1, rafael c. lajmanovich3, ulrich sinsch4, adolfo l. martino1 on a putative type specimen of pleurodema bibroni tschudi, 1838 from chile (anura: leptodactylidae) daiana paola ferraro re-description of the external morphology of phyllomedusa iheringii boulenger, 1885 larvae (anura: hylidae), with comments on the external morphology of tadpoles of the p. burmeisteri group samanta iop¹, victor mendes lipinski¹, bruno madalozzo¹, franciele pereira maragno¹, sonia zanini cechin¹, tiago gomes dos santos² book review: harold heatwole, john w. wilkinson (eds). amphibians biology. volume 11 status of conservation and decline of amphibians. eastern hemisphere. part 4 . southern europe and turkey sebastiano salvidio book review: antonio romano. atlante degli anfibi del parco nazionale del cilento vallo di diano e alburni distribuzione, biologia, ecologia e conservazione sebastiano salvidio acta herpetologica journal of the societas herpetologica italica acta herpetologica 9(2): 237-242, 2014 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-14040 age structure of a population of barbarophryne brongersmai (hoogmoed 1972) (anura, bufonidae) inhabiting an arid environment in the central jbilets (west-morocco) abderrazzak fattah1, tahar slimani1, el hassan el mouden1, odile grolet2, pierre joly2,* 1cadi ayyad university, faculty of sciences semlalia, biodiversity and ecosystems dynamic laboratory, po box 2390, marrakech 40 000, morocco 2 lyon1 university, umr 5023 ecology of natural and anthropized hydrosystems, entpe, cnrs, bât. darwin c, 69622 villeurbanne, france. *corresponding author. e-mail: pierre.joly@univ-lyon1.fr submitted on 2014, 7th february; revised on 2014, 14th may; accepted on 2014, 18th august editor: giovanni scillitani abstract. we estimated age structure and some life-history traits in a population of barbarophryne brongersmai at the northern border of the distribution area of this species by using skeletochronology to establish individual age and age at sexual maturity. maturity was reached at 2-4 years in males and 3-5 years in females, which was late relatively to small body size. longevity was estimated to be 12 and 8 years for males and females, respectively, whereas mean body size is 45.6 mm in both sexes. however, this estimate suffers from small sample size in females. we did not detect a positive relationship between age and size, suggesting that adult individuals cease growing after maturity. conversely, body size varied within an age class, suggesting great variability of growth rate among individuals during the juvenile stage. collectively, these traits characterize a life history strategy that could be an evolutionary response to habitat unpredictability in arid regions. keywords. endemic species, skeletochronology, life history traits, arid environments, morocco in arid zones, the reproduction of the amphibians is exposed to high environmental stochasticity because both the amount of rainfall and the hydroperiod of the breeding sites are usually variable and unpredictable. we assume that increased probability of reproduction failure due to pond drying is compensated by higher longevity as suggested by life history theory (roff, 1992) and already shown in amphibians (joly and morand, 1994; church et al., 2007; cayuela et al., 2014). in the perspective of increasing the data set about this issue, we investigated the age structure of a small-bodied toad, barbarophryne brongersmai, at the northern edge of the sahara desert to compare it with those established in other bufonidae breeding in less unpredictable environments. described and separated from bufotes viridis by hoogmoed (1972), barbarophryne brongersmai is a smallbodied toad endemic to south-western morocco (in the present paper, we consider recent taxonomic revisions of the bufo genus by pyron and wiens, 2011, and beukema et al., 2013). the studied population inhabits an arid grassland with sparsely scattered jujube trees (ziziphus lotus), located 25 km north of marrakech in the central jbilets (31°37’n, 8°02’w), a range of low hills near the northern border of the distribution area of the species (guillon et al., 2004). in this region, annual precipitation rarely reaches 300 mm (le houérou, 2001) and rare rain events of short duration and high intensity provoke sudden water discharges in the wadis (temporary rivers), usually occurring from the end of february to mid-april. the studied area is heavily grazed by large herds of sheep and goats in permanent movement that keep the grass very short, even during the rainy season. our popula238 a. fattah et alii tion breeds at a temporary pond (15 m in diameter and 80 cm depth) that appears in the bed of a temporary river (defla wadi), the hydroperiod of which did not exceed 18 days from 2009 to 2012 because of strong evaporation and intensive use for livestock watering. because the toads come to the pond immediately after it fills with water (guillon et al., 2004; fattah, 2008; slimani et al., 2012), sampling occurred from february to april, at night (from 20 to 00h gmt) using hand nets. we measured the toads from snout to cloaca by means of a calliper and weighed them using a field balance (accuracy 0.1g). we determined gender by the presence of nuptial callosities and calling in males. after measurements, we collected the third toe of the hind leg and we immediately released the toad at the place of its capture. we sectioned phalanges in order to quantify numbers of lines of arrested growth (lags), following the method by castanet and smirina (1990) based on the detection of line of arrested growth generated by the cold and/or dry season (each lag is interpreted as 1 year of age). the studies by barbault et al. (1979) on “bufo”pentoni, esteban et al. (1999) on pelophylax saharicus, and doglio et al. (2008) on b. brongersmai have confirmed that long bones reliably record yearly growth arrest in arid regions bordering sahara desert. however, this method is suspected to regularly underestimate age because of bone remodelling and rapprochement of peripheral lines, especially in the oldest individuals (wagner et al., 2011). we made sections in the medium part (diaphysis) of the third phalanx because the two distal phalanxes are known to exhibit strong medullary resorption (francillon-vieillot, 1987; fretey and le garff, 1992). all the sections where age estimation was doubtful (because of medullary resorption or other cause) were discarded from the results. three observers (a.f., o.g. and p.j.) independently counted the lags of each individual and re-examined the specimen and discussed the results when their estimations diverged. when the result remained uncertain, the specimen was not taken into account. age at maturity was estimated by the sudden and permanent rapprochement of the lags that reflects the slowing of growth rate after sexual maturity is reached. data normality was first assessed (kolmogorovsmirnov test) before testing for difference between means with mann-whitney u test, while correlation between age and body size was assessed by spearman test. means are reported with standard deviation and tests were considered significant at α = 0.05. during the 4-years survey, we sampled 80 individuals (71 males and 9 females, sex ratio = 0.89), but we succeeded in establishing age in only 37 males and 6 females. the bone sections show clear lines of arrested growth (lags) that are almost circular and continuous in most individuals (68.6%, fig. 1). we considered the perimeter of the bone as corresponding to the last winter since the toads were sampled in february, march and fig. 1. transverse sections within the diaphysal region of phalanx bones in the studied population. (a) male with 6 visible lags (cm: medullary cavity; lm: line of metamorphosis). (b) male with 9 visible lags. (c) female with 8 visible lags. 239age structure of a population of barbarophryne brongersmai april. we clearly detected the decrease in the distance that separates the lags suggesting that sexual maturity is reached at 2-4 years in males, and 3-5 years in females (fig. 1b, c), although their low number prevents any firm conclusion. at the breeding site, we sampled some males younger than 4 years, although 4-year individuals were the most frequently observed (fig. 2). highest estimated age is 12 years in males and 8 years in females. body size varied from 35.5 to 52.2 mm in males (45.4 ± 2.9 mm, n = 37) and from 40.0 to 50.5 mm in females (45.6 ± 4.2 mm, n = 6), with a mass of 6.5 ± 1.2 g in males and 7.4 ± 1.9 g in females and the observed pattern of sexual dimorphism in body size was not significant (mann–whitney u-test: u = 110.5, z=0.017, p = 0.10). because we did not detect a significant relationship between age and body size in males (r = -0.073, p = 0.667, n = 37), or in females (r = 0.529, p = 0.279, n = 6) (fig. 3), the oldest individuals were not always the largest ones. our study provides data about population structure that brings new light on the adaptive strategy of this species, despite the low confidence of our conclusions about growth and longevity in females, because of the low numbers of sampled individuals. this apparent bias in sex ratio could be due to (i) higher detectability of males because of conspicuous breeding behaviour, (ii) longer stay at breeding site, (iii) visiting the breeding location too early after rainfalls for the females to be present, and (iv) the possibility that the reproductive cycle of females is biennial when that of males is annual. sexual maturity is reached after about 3-4 years in most males and 5 years in females. however age structure in males is skewed since we would expect the 2 and 3 years-old toads to be more numerous than those aged 4 as a result of mortality. a first hypothesis for explaining this gap in young males is that not all the males reach sexual maturity before reaching 4 years, whereas a second one is that some males reach sexual maturity when two years old. we do not have enough evidence to conclusively reject either of these hypotheses. the one-year difference in age at maturity between males and females is commonly observed in the anurans, and is usually attributed to the higher investment of females in gametes (miaud et al., 1999; bastien and leclair, 1992; kyriakopoulou-sklavounou and grumiro, 2002; eaton et al., 2005). however, males and females can reach sexual maturity at the same age in some toads and other anurans (lykens and forester, 1987; friedl and klump, 1997; kyriakopoulou-sklavounou et al., 2008), probably when resources are abundant. the late age at maturity in our population suggests a low growth rate in juveniles, strongly constrained by the shortness of the activity period and perhaps the scarcity of food. as a consequence, the difference in age at maturity between genders is not surprising in such an environment. however, the low number of female sampled prevents any firm conclusion about this supposed lack of difference between genders. the absence of a relationship between age and body size is surprising because anurans often exhibit continuous growth, particularly females because of the strong relationship between fecundity and body size (joly, 1991; prado and haddad, 2005; lengagne et al., 2007). increasing fecundity at the expense of reducing somatic growth is theoretically possible, leading thus females keeping a fig. 3. relationship between estimated age (x-axis in year) and body size (y-axis; snout-vent length in mm) in the studied population. dark dots: females; open dots: males. fig. 2. frequency of barbarophryne brongersmai at defla wadi in 2009-2012 according to estimated age in the studied population. filled bars: females; open bars: males. 240 a. fattah et alii constant body size while increasing reproductive investment when ageing (castellano et al., 2004). however, such a theoretical option of resource allocation dynamics is not common in the amphibians that usually increase fecundity along life through indeterminate growth. furthermore, the absence of sexual dimorphism in body size also suggests a selection for low annual fecundity that could be compensated by a longer lifespan. in males, the observed longevity exceeds by 4 years that estimated in jbel saghro (anti atlas, morocco) by doglio et al. (2008). this greater longevity in the central jbilets could be due to greater sampling effort in our population. by visually extrapolating the decreased number of individuals as age increases, we predict that life expectancy could reach 13-14 years in the studied population. longevity (potential lifetime under optimal life conditions) should be longer first because skeletochronology usually underestimates current age due to bone remodelling and rapprochement of peripheral lines (wagner et al., 2011) and second because natural populations are exposed to external causes of mortality (predation, disease, drought, decreased food availability) that reduce life expectancy. in other anurans, longevity estimated in the field is around threefold lower than that measured under optimal rearing conditions. in bombina variegata for example, longevity reaches 29 years in artificially reared individuals when it does not exceed 12-13 years in the field (plytycz and bigaj, 1993; unpubl. res.). we thus suspect b. brongersmai to have a potential longevity much higher than expected on the basis of its body size. this hypothesis is supported by the fact that most toad species with body size larger than that of b. brongersmai exhibit shorter longevity and smaller age at maturity even in species with higher body size (table 1). these comparisons suggest that small-bodied b. brongersmai is characterized by slow growth during the juvenile stage. the interplay of high longevity, slow growth during the juvenile stage, absence of continued growth at the adult stage, and small body size constitutes a specific demographic strategy. we assume that this life history style constitutes a response to the unpredictability of breeding habitats and the shortness of activity period by increasing the number of lifetime breeding occasions (seger and brockmann, 1987; roff, 1992). the test of this hypothesis needs to establish age structure in other populations, including large numbers of females, and to investigate physiological mechanisms that support the great longevity of this species. acknowledgements. this work has received the support of hassan ii academy of sciences and technics (icgvsa project). it was authorized by the high commissariat for water and forest (morocco), scientific permits 98/2009, 15/2010 and 234/2012 hceflcd/dlcdp/ references barbault, r., castanet, j., francillon, h., ricqles, a.d. (1979): détermination de l’âge chez un anoure déserticole bufo pentoni anderson, 1893. rev. écol. (terre vie) 33: 129-141. bastien, h., leclair, r. (1992): aging wood frogs (rana sylvatica) by skeletochronology. j. herpetol. 26: 222-225. beukema, w., de pous, p., donaire-barroso, d., bogaerts, s., garcia-porta, j., escoriza, d., arribas, o.j., el mouden, e.h., carranza, s. (2013): review of the systematics, distribution, biogeography and natural history of moroccan amphibians. zootaxa 3661: 1-60. castanet, j., smirina, e.m. (1990): introduction to the skeletochronological method in amphibians and reptiles. ann. sci. nat, zool. biol.anim. 11: 191-196. table 1. comparison of longevity and age at maturity in several bufonids. species longevity (year) age at maturity (year) body size (average mass of the species/ average mass of barbarophryne brongersmai) remarks data source bufo bufo 8 2-4 3 to 4 higher at elevation >1800m hemelaar, 1988; frazer, 1966; fretey, 1995; gittins et al., 1980; gittins et al., 1982 epidalea calamita 9 2-3 2 higher at elevation > 2200 m stevens et al., 2003; leskovar et al., 2006; oromi et al., 2012 bufotes viridis 9 2-5 2 sinsch et al., 2004 anaxyrus hemiophrys 9 1-2 eaton et al., 2005 241age structure of a population of barbarophryne brongersmai castellano, s., cucco, m., giacoma, c. (2004): reproductive investment of female green toads (bufo viridis). copeia 2004: 659-664. cayuela, h., besnard, a., bonnaire, e., perret, h., rivoalen, j., miaud, c., joly, p., 2014. to breed or not to breed: past reproductive status and environmental cues drive current breeding decisions in a long-lived amphibian. oecologia 176: 107-116 church, d.r., bailey, l.l., wilbur, h.m., kendall, w.l., hines, j.e. (2007): iteroparity in the variable environment of the salamander ambystoma tigrinum. ecology 88: 891-903. doglio, s., seglie, d., kabiri, l. (2008): first skeletochronology results of bufo (epidalea) brongersmai from the moroccan anti atlas (anura, bufonidae). in: herpetologia sardiniae, pp. 226-230. corti, c., ed., societas herpetologica italica, edizioni belvedere, latina. eaton, b.r., paszkowski, c.a., kristensen, k., hiltz, m. (2005): life-history variation among populations of canadian toads in alberta, canada. can. j. zool. 83: 1421-1430. esteban, m., garcia-paris, m., buckley, d., castanet, j. (1999): bone growth and age in rana saharica, a water frog living in a desert environment. ann. zool. fenn. 36: 53-62. fattah, a. (2008): contribution à la connaissance de l’ecologie de bufo brongersmai (amphibia, anura, bufonidae) dans les jbilets centrales (maroc occidental). implication en termes de conservation. mémoire de master, faculté des sciences semlalia, marrakech. francillon-vieillot, h. (1987): la croissance des os longs chez les amphibiens et son utilisation comme critère d’âge. phd thesis, université paris vii, france. frazer, j.f.d. (1966): a breeding colony of toads (bufo bufo l.) in kent. brit. j. herpetol. 3: 236-252. fretey, t., le garff, b. (1992): apports de la squelettochronologie dans la démographie du crapaud commun, bufo bufo (l.) (anura, bufonidae) dans l’ouest de la france. in: tissus durs et âge individuel des vertébrés, pp. 409-419. baglinière, j.l., castanet, j., conand, f., meunier, f.j., eds., orstom-inra, paris, france. fretey, t. (1995): biologie d’une population reproductrice de crapaud commun, bufo bufo (linné, 1758) en forêt de rennes (ille-et-vilaine, france). phd thesis, université de rennes, france. friedl, t.w.p., klump, g.m. (1997): some aspects of population biology in the european treefrog hyla arborea. herpetologica 53: 321-330. gittins, s.p., parker, a.g., slater, f.m. (1980): population characteristics of the common toad (bufo bufo) visiting a breeding site in mid-wales. j. anim. ecol. 49: 161-173. gittins, s.p., steeds, j.e., williams, r. (1982): population age-structure of the common toad (bufo bufo) at a lake in mid-wales determined from annual growth rings in the phalanges. brit. j. herpetol. 6: 249-252. guillon, m., le liard, g., slimani, t. (2004): nouvelles données sur la répartition et l’écologie de reproduction de bufo brongersmai, bufo mauritanicus et bufo viridis (anoura, bufonidae) dans les jbilets centrales, maroc occidental. bull. soc. herp. fr. 111-112: 37-48. hemelaar, a.s.m. (1988): age, growth and other population characteristics of bufo bufo from different latitudes and altitudes. j. herpetol. 22: 369-388. hoogmoed, m.s. (1972): on a new species of toad from southern morocco. zool. meded. 47: 49-67. joly, p. (1991): interdemic variations of size and fecundity in the common frog, rana temporaria. alytes 9: 79-88. joly, p., morand, a. (1994): theoretical habitat templets, species traits, and species richness: amphibian in the upper rhône and its floodplain. freshwater biol. 31: 455-468. kyriakopoulou-sklavounou, p., grumiro, i. (2002): body size and age assessment among breeding populations of the tree frog hyla arborea in northern greece. amphibia-reptilia 23: 219-224. kyriakopoulou-sklavounou, p., stylianou, p., tsiora, a. (2008): a skeletochronological study of age, growth and longevity in a population of the frog rana ridibunda from southern europe. zoology 111: 30-36. le houérou, h. n. (2001). biogeography of the arid steppeland north of the sahara. j. arid environ., 48: 103-128 lengagne, t., arthaud, f., cormier, m., joly, p. (2007): cost of sexually embracing a large female offset by the number of eggs fertilised for small male bufo bufo l. biol. j. linn. soc. 92: 755-762. leskovar, c., oromi, n., sanuy, d., sinsch, u. (2006): demographic life history traits of reproductive natterjack toads (bufo calamita) vary between northern and southern latitudes. amphibia-reptilia 27: 365-375. lykens, d.v., forester, d.c. (1987): age structure in the spring peeper: do males advertise longevity? herpetologica 43: 216-223. miaud, c., guyétant, r., elmberg, j. (1999): variations in life history traits in the common frog rana temporaria (amphibia, anura): a literature review and new data from the french alps. j. zool., london 249: 61-73. oromi, n., sanuy, d., sinsch, u. (2012): altitudinal variation of demographic life-history traits does not mimic latitudinal variation in natterjack toads (bufo calamita). zoology 115: 30-37. 242 a. fattah et alii plytycz, b., bigaj, j. (1993): studies on the growth and longevity of the yellow-bellied toad, bombina variegata, in natural environments. amphibia-reptilia 14: 35-44. prado, c.p.a., haddad, c.f.b. (2005): size-fecundity relationships and reproductive investment in female frogs in the pantanal, south-western braz. herp. j. 15: 181-189. pyron, r.a., wiens, j.j. (2011): a large-scale phylogeny of amphibia including over 2800 species, and a revised classification of extant frogs, salamanders, and caecilians. mol. phylogenet. evol. 61: 543-583. roff, d.a. (1992): the evolution of life histories: theory and analysis. chapman and hall, london. seger, j., brockmann h. j. (1987): what is bet-hedging? in: oxford surveys in evolutionary biology, p. 182– 211. harvey, p. h., partridge, l., eds.,oxford university press, oxford, uk. sinsch, u., leskovar, c., drobig, a., könig, a., grosse, w.r. (2004): life-history traits in green toad (bufo viridis) populations: indicators of habitat quality. can. j. zool. 85: 665-673. slimani, t., fattah, a., el mouden, e.h., radi, m., doglio, s. (2012): ecology and population parameters of bufo (pseudepidalea) brongersmai (hoogmoed, 1972) living in small temporary ponds in the arid area of jbilets (western morocco). summary in seventh world congress of herpetology [wch 7], p. 666-667. vancouver, canada, august 8-14. stevens, v., wesselingh, r., baguette, m. (2003): demographic processes in a small, isolated population of natterjack toads (bufo calamita) in southern belgium. j. herpetol. 12: 59-67. wagner, a., schabetsberger, r., sztatecsny, m., kaiser, r. (2011): skeletochronology of phalanges underestimates the true age of long-lived alpine newts (ichthyosaura alpestris). herpetol. j. 21: 145-148. © firenze university press www.fupress.com/ah acta herpetologica 5(2): 161-177, 2010 reptiles of sardinia: updating the knowledge on their distribution daniele salvi1, pierluigi bombi2 1 cibio, centro de investigação em biodiversidade e recursos genéticos, campus agrário de vairão, 4485-661 vairão, portugal. 2 via maria giudice 23, i-00135 rome, italy. corresponding author. e-mail: pierluigi.bombi@gmail. com submitted on: 2009, 22th october; revised on: 2010, 3rd march; accepted on: 2010, 29th july. abstract. sardinia shows a clear lack of herpetological data due to an evident paucity of herpetological surveys. this gap of knowledge is worthy of attention, since sardinia hosts a rich herpetofauna with a large proportion of endemic species, and distribution data are crucial for targeting conservation efforts. in this paper we provide new distribution data for sardinian reptiles, with the aim of updating our knowledge on their specific distribution. data were opportunistically recorded during ten years of field research in sardinia, carried out in more than twenty campaigns from april 1999 to june 2009. all the eighteen reptile species belonging to the sardinian fauna were recorded. a total amount of 293 faunistic data were collected from 178 different localities covering the entire study area. within this dataset, 137 faunistic data fall outside known species’ ranges as reported in the atlas of italian amphibians and reptiles. in conclusion, data presented here produced a remarkable increase of knowledge on sardinian reptiles’ distribution. nevertheless, it should be evidenced that, notwithstanding the present updating, the knowledge of sardinian reptiles’ distribution is probably still far from being exhaustive. thus, further investigations are strongly required for obtaining a complete picture and identifying conservation priorities in terms of isolated species/populations and areas of high diversity and endemicity. keywords. herpetofauna, reptiles, faunistics, italy, sardinia. introduction the knowledge of species’ distribution is crucial for their conservation. thus, many studies focused on amphibian and reptile distribution in italy. most of these researches consisted in atlas projects carried out at different geographic scale, from single protected areas (e.g., gentilli and barbieri, 2002; lapini, 2006), to several level of administrative units such as cities (e.g., bologna et al., 2003), municipalities (e.g., lapini, 1997), provinces (e.g., 162 d. salvi and p. bombi fiacchini, 2003; piazzini et al., 2005; bologna et al., 2007), and regions (e.g., doria and salvidio, 1994; mazzotti et al., 1999; bologna et al., 2000; vanni and nistri, 2006). local atlas projects has represented a first step towards the synthesis of data coming from a plethora of different sources, from bibliographic data and museum collections, to field observations by volunteers and researchers. the importance of these atlases appears evident in their role of sound and reliable basis for conservation initiatives and prioritization proposals (e.g., brotons et al., 2004; araújo et al., 2004; rodríguez et al., 2006). a national-scale project started in 1994 aimed to gather a preliminary picture on the distribution of amphibians and reptiles found in italy (societas herpetologica italica, 1996). in recent times this large amount of data has been further updated and increased leading to a cartographic synthesis in the ckmap project (stoch, 2000-2005) and in the atlas of the italian amphibians and reptiles (sindaco et al., 2006). this book represents the state of the art of the knowledge on the distribution of amphibians and reptiles in italy and is based on more than 70,000 records. the overall coverage of these data is on the whole relatively good, except for two regions, as evidenced by the same authors. basilicata and sardinia regions show a clear lack of herpetological data (sindaco et al., 2006: p. 132). concerning sardinia, but this is probably true also for basilicata, this pattern come from an evident paucity of herpetological surveys rather than a low level of herpetological diversity as evidenced by preliminary data reported in bombi and salvi (2008). this gap of knowledge is worthy of attention, since sardinia hosts a rich and distinctive herpetofauna with a large proportion of endemic species, and distribution data are crucial for targeting conservation efforts. concerning reptiles, sardinia is inhabited by 18 recognised species, representing 35% of the reptiles diversity found in italy (sindaco et al., 2006). three species are endemic to sardinia and corsica, algyroides fitzingeri, podarcis tiliguerta, archaeolacerta bedriagae (lacertidae), and another, euleptes europaea (sphaerodactylidae), is sub-endemic to these two islands being also present in some coastal localities of tuscany and liguria (italy). podarcis sicula (lacertidae) and natrix natrix (colubridae) occur in sardinia with distinctive populations respect to those spread on the continent, and are considered as sardinian endemic subspecies (p. s. cetti) or even incipient species (n. n. cetti) (thorpe, 1980; aprea et al., 2000). most of sardinian reptiles have a high intrinsic conservation value and are listed as species with conservation priorities in the annexes ii and iv of the habitats directive 92/43/eec (euleptes europaea, emys orbicularis, testudo hermanni, testudo graeca, and testudo marginata) or considered threatened in the iucn red list (e.g., natrix natrix cetti as critically endangered) (iucn, 2010). to date no one paper reviewed the knowledge about the entire sardinian herpetological diversity at a regional scale, while several studies are available for specific areas (e.g. cesaraccio and lanza, 1984; poggesi et al., 1996) or single species (lecis and norris, 2003; bombi and vignoli, 2004). the aim of this paper is providing new distribution data for sardinian reptiles, from an investigation carried out throughout sardinia, and updating our knowledge of sardinian reptiles distribution. materials and methods we considered the entire territory of the sardinian region, amounting to more than 24,000 km2. sardinia is the second-largest island of italy and in general of the mediterranean sea, placed south to 163update of reptile distribution in sardinia the island of corsica, west from the italian peninsula, north from tunisia, and east from the balearic islands. it has a mediterranean climate, with rainfall concentrated in autumn and winter and hot/dry summers, which supports characteristic mediterranean forests, woodlands, and scrub vegetation. contextually to a ten-years long field-based research in sardinia, focused on ecology, distribution and phylogeography of some endemic reptile species (e.g. bombi et al., 2009a, 2009b; salvi et al., 2010), faunistic observations were opportunistically recorded. fieldwork was carried out in more than twenty campaigns from april 1999 to june 2009, with a field effort of approximatively 50 weeks of activity. we surveyed all the main sardinian domains, including mountain, mid-elevation, coastal, and micro-insular sites, and considering different reptile habitats. surveys were carried out mainly during day between 0800 h and 1900 h from april to october. only original data from direct observations has been included in the present study. we focused our attention to terrestrial and freshwater reptiles, thus excluding marine turtle species. we also excluded amphibian species because they need searching methodologies conflicting with our main field activity. most of the individuals were captured by hands, nosed, or by means of rubber bands. each captured animal was sexed, measured, photographed, and then released in the exact capture point. a low fraction of data came from road-killed specimens or from visual encounter surveys. tracks of animal were not considered as indicator of species’ presence since in most of the case they do not allow an unambiguous species identification. for each locality geographic coordinates were recorded by using a gps device, and toponyms were assigned by identifying the closest locality reported in maps from the “istituto geografico militare”. for conservation reasons, in the light of the threatened status and vulnerability of many sardinian species, we provided in appendix 2 only low-resolution coordinates. more details shall be available from the authors upon specific request. for each species, our data were compared with the currently known species distribution as reported in sindaco et al. (2006). for each species we produced updated maps of distribution by overlapping our data with those from sindaco et al. (2006) at 10×10 utm grid scale. from these data we calculated the species richness per cell for identifying the hotspot of reptile diversity in sardinia. we utilised taxonomic names as in sindaco et al. (2006) and we did not considered subspecies since for most of sardinian reptiles the intraspecific taxonomy is under debate [e.g. for archaeolacerta bedriagae (salvi et al., 2009a, 2009b, 2010); for podarcis sicula (podnar et al, 2005); for podarcis tiliguerta (capula, 1996; harris et al., 2005)]. results all the 18 reptile species belonging to the sardinian fauna were recorded. these species include: two emydidae, three testudinidae, three gekkota, four lacertidae, two scincidae, and four colubridae. the inventory of the sampled localities is provided in appendix 2, and their location reported in figure 1. a total amount of 293 faunistic data were collected from 178 different localities covering the entire study area. within this dataset, 137 faunistic data fall outside utm squares where species presence was documented before by sindaco et al. (2006). these localities of occurrence produce an extension of the documented distribution ranges for of all but two species (i.e., chalcides chalcides and hemorrhois hippocrepis). in particular, emys orbicularis has been observed in two localitites outside its known range, trachemys scripta in one, testudo hermanni in two, testudo graeca in one, testudo marginata in one, euleptes europaea in three, hemidactylus turcicus in five, tarentola mauritanica in nine, algyroides fitzingeri in 12, archaeolacerta bedriagae in one, podarcis tiliguerta in 36, podarcis sicula in 26, chalcides ocellatus in 10, hierophis viridiflavus in 24, natrix natrix in one, and natrix maura in three. the updating of atlas 164 d. salvi and p. bombi fig. 1. sampling localities. for details on geographic coordinates and toponyms see appendix 2. 165update of reptile distribution in sardinia fig. 2. distribution maps for nine reptiles of sardinia. grey circles: utm squares of presence reported in the atlas of italian amphibians and reptiles (sindaco et al., 2006); black circles: new utm squares of presence from this study. 166 d. salvi and p. bombi fig. 3. distribution maps for nine reptiles of sardinia. grey circles: utm squares of presence reported in the atlas of italian amphibians and reptiles (sindaco et al., 2006); black circles: new utm squares of presence from this study. 167update of reptile distribution in sardinia maps from sindaco et al. (2006) are provided in figure 2 and 3. in table 1 and in appendix 1 are reported for each species the localities and utm squares relative to their observation. the geographic pattern of reptile species richness is shown in figure 4. discussion data presented here produced a remarkable increase of knowledge on sardinian reptiles distribution compare to that reported in the last available syntheses of the atlas of italian amphibians and reptiles (sindaco et al., 2006). among almost 300 data, 47% concerns localities placed outside the squares of documented presence as reported in above mentioned studies. on the basis of the updated knowledge, it is possible to highlight that five species are widespread across sardinia (algyroides fitzingeri, podarcis tiliguerta, podarcis sicula, chaltable 1. number of utm squares and number of localities for each species. in parentheses are evidenced the number of utm squares where species’ presence was not documented before and the number of localities falling in these cells. species number of utm squares number of localities emys orbicularis (linnaeus, 1758) 2 (2) 2 (2) trachemys scripta (schoepff, 1792) 1 (1) 1 (1) testudo hermanni gmelin, 1789 3 (2) 3 (2) testudo graeca linnaeus, 1758 1 (1) 1 (1) testudo marginata (schoepff, 1792) 2 (1) 2 (1) euleptes europaea (gené, 1839) 5 (3) 5 (3) hemidactylus turcicus (linnaeus, 1758) 9 (5) 9 (5) tarentola mauritanica (linnaeus, 1758) 11 (6) 17(9) algyroides fitzingeri (wiegmann, 1834) 16 (9) 20 (12) archaeolacerta bedriagae (camerano, 1885) 14 (1) 23 (1) podarcis tiliguerta (gmelin, 1789) 56 (26) 99 (36) podarcis sicula rafinesque-schmaltz, 1810 38 (21) 45 (26) chalcides chalcides (linnaeus, 1758) 1 (0) 1 (0) chalcides ocellatus (forskål, 1775) 9 (7) 12 (10) hemorrhois hippocrepis (linnaeus, 1758) 1 (0) 1 (0) hierophis viridiflavus (lacépède, 1789) 30 (19) 43 (24) natrix natrix (linnaeus, 1758) 3 (1) 3 (1) natrix maura (linnaeus, 1758) 4 (3) 6 (3) 168 d. salvi and p. bombi cides ocellatus, hierophis viridifl avus). on the contrary, the other species are restricted to specifi c areas. th e distribution range has been signifi cantly increased for two out of three endemic species (a. fi tzingeri and p. tiliguerta). concerning the endemic species a. bedriagae one data (locality 148) would extend the range of this species in the northern part of the sette fratelli mountains district. th is area represents the southernmost portion of the species’ range and it is of particular importance in the light of the isolation and distinctiveness of these southern populations (guillaume, 1987; bombi et al., 2009a, 2009b; salvi et al., 2009a, 2009b). th e record of trachemys scripta represents a serious threat for insular freshwater ecosystems. th is threat is made still more real by the presence in the same area of emys orbicularis. in fact, the interaction between these two species, even if its eff ect is controverfig. 4. distribution maps for reptile diversity in sardinia. the number of species recorded in each cell is proportional to the relative gray tone (white = 0 species; black = 12 species). 169update of reptile distribution in sardinia sial, could lead to the extinction of the european pond terrapin. recently, some other data have been reported for other areas of sardinia (bassu et al., 2008), suggesting a spreading of the red-eared terrapin across the island. thus, further surveys and deeper investigations should be planned for evaluating the current status of this species in the area and setting management action to avoid its further spread. the finding of a new area where testudo hermanni occurs, in the extreme north-east of the island, is interesting for the conservation of this species. the hermann’s tortoise is known to be present in sardinia only in localized and deeply isolated populations (mazzotti, 2006; bassu et al., 2008). in addition, it should be taken into account that this area is not protected, differently from other presence sites (i.e., asinara island and sinis peninsula). therefore, this new area could represent a new opportunity for hermann’s tortoise conservation and specific measures should be focused on this area. the horseshoe whip snake hemorrhois hippocrepis is likely more common in southern sardinia than believed and it could be hypothesized that its mainly nocturnal pattern of activity account for such underestimation (l. luiselli, pers. comm.). thus, we probably encountered this species just once because our fieldwork was concentrated during the day. it should be underlined that h. hippocrepis is one of the most endangered reptiles in italy (filippi and luiselli, 2000). therefore, the improvement of our understanding of the biology and distribution of the horseshoe whip snake assumes a crucial importance for guaranteeing the long-term conservation of this species. our surveys did not afford any data about zamenis longissimus / z. lineatus. currently, the presence of this species in sardinia is considered highly controversial. two data from museum specimens have been referred to z. longissimus or to z. lineatus by different authors (see razzetti and zanghellini, 2006). other two data, coming from field observations, were reported in razzetti and zanghellini (2006) and in bassu et al. (2008), but in both cases not a single live specimen has been collected in sardinia. taking into account the large numbers of data collected for other snakes (e.g., 43 for hierophis viridiflavus), we consider more likely z. longissimus / z. lineatus to be not present in sardinia as autochtonous species, but at least present with localized acclimatized allochthonous individuals, as proposed by puddu et al. (1998), despite further specific research should be carried out on these taxa. the main hotspots of reptile diversity in sardinia correspond to the iglesiente area (south-western sardinia), to the main mountain massifs of eastern sardinia, and to some coastal areas of northern sardinia. most of this diversity hotspots represent also areas of endemicity. indeed, the iglesiente district together with the main mountainous areas of eastern sardinia (sette fratelli mount, gennargentu massif, and limbara mount) host many of the endemic herpetofauna (e.g. archaeolacerta bedriagae, natrix natrix cetti, euproctus platycephalus, speleomantes species). this congruence has a double interest. on the one hand, it gives preliminary indications for selecting areas with high conservation priority. on the other hand, it would provide a intriguing scenario for testing biogeographic and evolutionary hypotheses, underlying the origin of the observed pattern of diversity and endemism. in conclusion, data presented here give a significant contribution for clarifying the distribution of sardinian reptiles and compiling the inventory of the reptiles diversity occurring in different areas of the island. nevertheless, it should be evidenced that, not170 d. salvi and p. bombi withstanding the present updating, the knowledge of sardinian reptiles’ distribution is probably still far from being exhaustive. thus, further investigations are strongly required for obtaining a complete picture and identifying conservation priorities in terms of isolated species/populations and areas of high diversity and endemicity. aknowledgments we wish to thank several people, who have contributed to samplings or supported this research: c. argiolas, d. blam, m. a. bologna, v. bordoni, m. a. carretero, r. corbeddu, m. d’amen, n. cabitta, a. devaddis, c. fassò, d. j. harris, a. loddo, l. luiselli, p. mesina, i. pasquale, d. pinna, d. spagnesi, p. tolu, r. xavier, and l. vignoli. we would also thank for logistic support oasi wwf monte arcosu and ente foreste demaniali sardegna. references aprea, g., odierna, g., capriglione, t., caputo, v., guarino, f.m. 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(2006): atlante degli anfibi e dei rettili della toscana. regione toscana, università degli studi di firenze, museo di storia naturale, sezione zoologica “la specola”, firenze. 173update of reptile distribution in sardinia appendix 1 localities of observation for each species. in bold are evidenced localities falling outside the utm squares where species presence is currently known. family species localities emydidae emys orbicularis (linnaeus, 1758) 35, 47 trachemys scripta (schoepff, 1792) 47 testudinidae testudo hermanni gmelin, 1789 21, 23, 170 testudo graeca linnaeus, 1758 38 testudo marginata (schoepff, 1792) 9, 31 gekkonidae euleptes europaea (gené, 1839) 21, 31, 73, 137, 142 hemidactylus turcicus (linnaeus, 1758) 15, 61, 73, 87, 115, 124, 156, 167, 175 tarentola mauritanica (linnaeus, 1758) 7, 10, 15, 19, 20, 40, 137, 142, 146, 150, 152, 156, 158, 160, 162, 163, 177 lacertidae algyroides fitzingeri (wiegmann, 1834) 1, 2, 6, 10, 22, 25, 26, 31, 46, 49, 50, 74, 77, 88, 91, 102, 103, 118, 152, 158 archaeolacerta bedriagae (camerano, 1885) 1, 5, 10, 19, 20, 25, 31, 44, 46, 49, 51, 54, 77, 91, 109, 137, 148, 149, 150, 151, 154, 155, 157 podarcis tiliguerta (gmelin, 1789) 1, 2, 3, 4, 8, 10, 12, 14, 18, 19, 20, 25, 30, 31, 32, 33, 37, 43, 44, 47, 49, 50, 51, 52, 53, 54, 55, 57, 59, 60, 62, 63, 64, 65, 67, 68, 69, 70, 71, 73, 74, 75, 77, 79, 81, 83, 86, 88, 89, 90, 91, 94, 96, 99, 101, 105, 106, 107, 108, 110, 112, 113, 117, 118, 119, 120, 125, 126, 127, 129, 132, 133, 135, 136, 137, 139, 141, 144, 145, 147, 148, 149, 151, 152, 153, 154, 155, 157, 159, 160, 162, 163, 166, 168, 169, 170, 171, 177, 178 podarcis sicula rafinesque-schmaltz, 1810 15, 23, 25, 41, 47, 48, 56, 57, 68, 72, 76, 83, 85, 92, 110, 113, 114, 115, 116, 117, 119, 120, 121, 122, 124, 125, 130, 131, 132, 133, 134, 135, 136, 137, 139, 141, 143, 144, 145, 146, 156, 160, 163, 165, 176 scincidae chalcides chalcides (linnaeus, 1758) 174 chalcides ocellatus (forskål, 1775) 25, 29, 30, 61, 72, 73, 78, 123, 124, 128, 156, 158 colubridae hemorrhois hippocrepis (linnaeus, 1758) 164 hierophis viridiflavus (lacépède, 1789) 1, 10, 11, 13, 16, 17, 20, 24, 25, 27, 28, 31, 33, 34, 36, 37, 39, 42, 45, 58, 66, 68, 77, 80, 82, 84, 89, 97, 100, 102, 104, 108, 111, 124, 132, 138, 140, 141, 160, 161, 166, 173, 174 natrix natrix (linnaeus, 1758) 95, 98, 152 natrix maura (linnaeus, 1758) 23, 55, 93, 152, 166, 172 174 d. salvi and p. bombi appendix 2 sampling localities. coordinates are provided in decimal degree format. locality ids correspond to those reported in the map in figure 1. locality id igm toponym municipal district latitude longitude 1 punta falcone santa teresa di gallura 41,26 9,23 2 faro di punta falcone santa teresa di gallura 41,25 9,23 3 p. acuta santa teresa di gallura 41,24 9,16 4 la ficaccia 1 santa teresa di gallura 41,24 9,22 5 capo testa santa teresa di gallura 41,24 9,14 6 la ficaccia 2 santa teresa di gallura 41,24 9,22 7 s. teresa gallura santa teresa di gallura 41,24 9,19 8 porto di longone santa teresa di gallura 41,24 9,19 9 la torraccia santa teresa di gallura 41,24 9,19 10 p.ta dei colmi la maddalena 41,23 9,39 11 c. guardioli la maddalena 41,23 9,42 12 madonnetta la maddalena 41,23 9,37 13 becco di vela la maddalena 41,23 9,47 14 poggio stefano la maddalena 41,23 9,48 15 c. susini la maddalena 41,23 9,43 16 guardia vecchia la maddalena 41,22 9,40 17 p. nido d’aquila la maddalena 41,22 9,38 18 ferracciolo la maddalena 41,22 9,47 19 teialone la maddalena 41,21 9,47 20 pietraiaccio la maddalena 41,21 9,46 21 l’isuledda palau 41,20 9,32 22 m. iacheddu palau 41,19 9,34 23 la sciumara santa teresa di gallura 41,19 9,31 24 bonifica di barrabisa palau 41,19 9,32 25 l’orso palau 41,17 9,41 26 st.zi pittorra santa teresa di gallura 41,17 9,31 27 m. piseddu arzachena 41,15 9,43 28 st.zo silvaredda arzachena 41,14 9,43 29 rio mannu arzachena 41,13 9,44 30 p. arzachena arzachena 41,13 9,44 31 m. moro arzachena 41,11 9,52 32 nibbaragghia arzachena 41,10 9,50 33 p.ta rumazzino arzachena 41,09 9,56 34 c. nuova arzachena 41,08 9,46 35 r. di petralonga arzachena 41,08 9,47 36 r. di l’umpitrato arzachena 41,07 9,46 37 cugnana olbia 41,01 9,50 38 m. lu naracu sant’antonio di gallura 41,00 9,28 39 m.giu santu calangianus 40,98 9,25 40 li iunchi badesi 40,97 8,86 41 l’approdo stintino 40,97 8,23 42 m. attunisi luras 40,96 9,21 175update of reptile distribution in sardinia locality id igm toponym municipal district latitude longitude 43 cala di vacca stintino 40,95 8,23 44 m. pino telti 40,95 9,37 45 catala calangianus 40,94 9,26 46 m. della croce aggius 40,93 9,06 47 r. le prugne aggius 40,93 9,06 48 st.zo l’agnulu calangianus 40,91 9,17 49 li reni tempio pausania 40,86 9,18 50 campianatu tempio pausania 40,86 9,13 51 p.ta balistreri tempio pausania 40,85 9,18 52 mad.na della neve tempio pausania 40,85 9,17 53 giugantinu tempio pausania 40,85 9,17 54 sa berritta tempio pausania 40,85 9,17 55 rio contra manna berchidda 40,84 9,14 56 lampianu sassari 40,81 8,20 57 p.ta turrita monti 40,80 9,32 58 font.na di nicolau ala’ dei sardi 40,71 9,37 59 cant.ra di s. anna ianna de sant’anna 40,58 9,63 60 funt.na patronu bitti 40,58 9,47 61 s. lucia siniscola 40,58 9,77 62 pinn.ta tres montes ozieri 40,58 8,97 63 n.ghe loelle budduso’ 40,57 9,32 64 font.na sa mela siniscola 40,57 9,64 65 f. tirso budduso’ 40,56 9,33 66 sa janna renosa bitti 40,55 9,34 67 m. mesanu bultei 40,52 9,07 68 m. fgheras villanova monteleone 40,50 8,37 69 m. romasino villanova monteleone 40,49 8,37 70 monte albo lula 40,47 9,53 71 pizzonchi lula 40,46 9,49 72 foresta burgos burgos 40,42 8,94 73 sa minda orosei 40,42 9,75 74 funt.na coloras padria 40,39 8,63 75 tres funtanas bolotana 40,35 8,89 76 c. scala e attos bosa 40,35 8,48 77 n.ghe ortachis bolotana 40,35 8,91 78 p.ta palai bolotana 40,34 8,92 79 c. tentizzos bosa 40,32 8,45 80 m. muradu bosa 40,32 8,51 81 s. n. de su monte nuoro 40,32 9,37 82 p.te di olos oliena 40,30 9,48 83 sorg.te de su cologone oliena 40,29 9,49 84 cant.ra abbadtzu suni 40,29 8,61 85 portolato bortigali 40,28 8,85 86 calagonone dorgali 40,28 9,64 87 c.le s. giorgio bosa 40,28 8,49 176 d. salvi and p. bombi locality id igm toponym municipal district latitude longitude 88 sollapiolos dorgali 40,27 9,60 89 m. bonacoa dorgali 40,27 9,61 90 nuragheddu dorgali 40,26 9,62 91 giulia oliena 40,25 9,43 92 maidreu dorgali 40,23 9,52 93 gianna ferru mamoiada 40,18 9,30 94 codula de luna urzulei 40,17 9,56 95 donurtei fonni 40,07 9,26 96 m. spada fonni 40,07 9,29 97 cu.le qualbu fonni 40,07 9,27 98 riu su trocu fonni 40,06 9,26 99 funt.na masiai fonni 40,06 9,28 100 qu.le sos beraniles fonni 40,06 9,25 101 qu.le is rulas desulo 40,04 9,26 102 funt.na curadore desulo 40,03 9,23 103 tala sazias tonara 40,03 9,18 104 zerroi san vero milis 40,02 8,46 105 sa code desulo 40,02 9,28 106 br.cu spina villagrande strisaili 40,02 9,30 107 arcu gennargentu villagrande strisaili 40,01 9,32 108 funt.na ‘e piraona desulo 40,00 9,30 109 p.ta la marmora arzana 39,99 9,32 110 st.gno pauli e sali cabras 39,95 8,51 111 parduierru cabras 39,92 8,53 112 can.le brabbau oristano 39,90 8,53 113 c.lo ‘e s’accu ‘e su suergiu laconi 39,88 9,09 114 idrov. a sassu arborea 39,82 8,58 115 su varongu pau 39,80 8,77 116 scala seremida sini 39,76 8,91 117 pauli maiori tuili 39,74 8,96 118 pauli ‘e palla cammisa gesturi 39,74 9,00 119 funt.na ortu gesturi 39,74 9,00 120 arridroxiu tuili 39,73 8,97 121 s. luisa tuili 39,73 8,97 122 su nuraxi barumini 39,71 8,99 123 planu senis suelli 39,61 9,14 124 perda quaddu guspini 39,56 8,61 125 montevecchio guspini 39,55 8,57 126 s.s. del gerre ballao 39,54 9,34 127 piscinas arbus 39,54 8,45 128 p.ta cugui arbus 39,52 8,58 129 r. casciera arbus 39,52 8,50 130 f. flumendosa armungia 39,51 9,41 131 sa mandara villacidro 39,48 8,73 132 coddu de su medaueddu villacidro 39,47 8,72 133 r. coxinas villacidro 39,46 8,71 177update of reptile distribution in sardinia locality id igm toponym municipal district latitude longitude 134 villacidro villacidro 39,46 8,73 135 lavera fluminimaggiore 39,43 8,50 136 sa domo de sani villasalto 39,42 9,31 137 br.cu berritta villasalto 39,42 9,31 138 costa pireddu muravera 39,41 9,60 139 qu.le sa gotti sa perda burcei 39,40 9,39 140 is cucureddus dolianova 39,40 9,20 141 cuili piccinnu sinnai 39,39 9,29 142 su piroi burcei 39,38 9,36 143 p.ta cucurnia iglesias 39,37 8,52 144 serpeddieddu sinnai 39,36 9,29 145 s. giovanni domusnovas 39,34 8,63 146 cuili becciu muravera 39,34 9,57 147 campu omo sinnai 39,31 9,39 148 mitza poni fogu sinnai 39,30 9,43 149 punta sa ceraxa sinnai 39,29 9,44 150 riu maidopis 1 sinnai 39,29 9,41 151 riu maidopis 2 sinnai 39,29 9,41 152 riu maidopis 3 sinnai 39,29 9,41 153 riu maidopis 4 sinnai 39,29 9,41 154 perd ‘asub’ ‘e pari sinnai 39,28 9,42 155 aqueddas 1 sinnai 39,28 9,43 156 rio samasa gonnesa 39,28 8,43 157 aqueddas 2 sinnai 39,28 9,43 158 c. cappai maracalagonis 39,27 9,31 159 arcu su crabiolu sinnai 39,27 9,43 160 cast.o d’acquafredda siliqua 39,26 8,82 161 c. deas siliqua 39,26 8,82 162 n.ghe sa fraigada sinnai 39,26 9,40 163 dispensa vecchia sinnai 39,25 9,43 164 su rulloni cagliari 39,22 9,16 165 s. lucia uta 39,20 8,94 166 c. camboni siliqua 39,20 8,82 167 can.le sperrimeddas uta 39,20 8,91 168 sa canna uta 39,20 8,91 169 sera cannisoni siliqua 39,19 8,77 170 riu su strumpu castiadas 39,17 9,56 171 c. perdu melis 1 uta 39,16 8,87 172 c. perdu melis 2 uta 39,16 8,87 173 molentis villasimius 39,14 9,56 174 manunzas villasimius 39,14 9,54 175 sa tanca san giovanni suergiu 39,13 8,52 176 is pes san giovanni suergiu 39,12 8,52 177 sa corona arrubia calasetta 39,05 8,38 178 p.te s’accorradroxu domus de maria 38,94 8,83 acta herpetologica 10(2): 125-127, 2015 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-15394 on the taxonomic status of liolaemus filiorum pincheira-donoso & ramírez, 2005 (iguania: liolaemidae): a response to pincheira-donoso jaime troncoso-palacios programa de fisiología y biofísica, instituto de ciencias biomédicas (icbm), facultad de medicina, universidad de chile, independencia 1027, santiago, chile. e-mail: jtroncosopalacios@gmail.com submitted on: 2015, 27th january; revised on: 2015, 15th august; accepted on: 2015, 27th august editor: aaron m. bauer abstract. i discuss the arguments put forth recently by pincheira-donoso, in which the author attempts to revalidate liolaemus filiorum pincheira-donoso & ramírez, 2005, a species which i had previously considered a junior synonym of l. puritamensis. the author of this revalidation omitted important information including: 1) the description was published without peer review, 2) one of the two types was deposited in a personal collection, 3) the diagnosis is weak and unclear, 4) the holotype was not explicitly described or illustrated. additionally, the author did not discuss key aspects of my paper, most particularly, the incorrect designation of the holotype of l. filiorum. keywords. holotype, species description, synonymy, liolaemus, iczn. the lizard liolaemus filiorum pincheira-donoso & ramírez, 2005 was described in a book entitled “fauna del altiplano y desierto de atacama” edited by the authors themselves (pincheira-donoso and ramírez, 2005). the species description was not subjected to peer review, contains several errors and omissions and has created taxonomic instability in liolaemus. the practice of publishing without peer review is generally strongly discouraged by the systematic community and may sometimes lead to taxonomic instability (iverson et al., 2001; kaiser et al., 2013). the description of l. filiorum is based on two specimens collected in the antofagasta region of chile, at two different localities: the holotype was listed using the specimen number mnhn-3829 and was said to be from cerro las papas and deposited in the museo nacional de historia natural de chile (mnhn) in santiago. the paratype, from taira, was listed as chdpd-01069, indicating that it had been deposited in the private collection of the book’s senior author, daniel pincheira-donoso. deposit of specimens in private collections is also discouraged and has been criticized (iczn, 1999; kaiser et al., 2013), and some journals, e.g., acta herpetologica, reject this practice. in the species description, no photograph, specific scalation data or snout-vent length of the holotype is provided. in 2013, eight years after the description of l. filiorum, together with mr. herman núñez, collection manager and curator at the mnhn, i attempted to locate the holotype of l. filiorum. we thoroughly searched for the specimen (mnhn-3829) on both the type specimen shelf and in the general collection. we found only one specimen with the label number 3829, a paratype of liolaemus puritamensis. at no time did pincheiradonoso and ramírez (2005) indicate that a paratype of l. puritamensis had also been designated as the holotype of l. filiorum. mr. núñez indicated to me that there is no other mnhn-3829 and that no other specimen referable to the “holotype” of l. filiorum is present in the mnhn collection (pers. comm.). although it is of course possible to designate a paratype of one species as the holotype of another, this implies that an inadvertent error was made in the assignment of the paratype, and this is certainly an important fact for the taxonomy of the older species. i also reviewed the entire database of the mnhn herpetological collection available at that time (gbif, 126 jaime troncoso-palacios 2013). although i found five specimens from the cerro las papas (mnhn 4087-89, 4218-19) none of them is indicated as a specimen examined by pincheira-donoso and ramírez (2005) and also only one mnhn 3829 was found (indicated as l. puritamensis). in a recent peer-reviewed publication (troncosopalacios, 2014), i therefore proposed that liolaemus filiorum is a junior synonym of l. puritamensis núñez & fox, 1989 because the putative holotype of the former nominal taxon (mnhn-3829) is part of the type series of the latter and indistinguishable from it (troncosopalacios, 2014). this was a taxonomic decision facilitated by the fact that in the original description of l. filiorum, the holotype is identified only by a specimen number; no photographs or specimen specific data are provided. pincheira-donoso and ramírez (2005) rather described variation within the species. for example, pincheira-donoso and ramírez (2005: 354) indicated that l. filiorum may exceed 80 mm in svl and on the same page they stated that it may exceed 85 mm, with no indication of the actual svl of the holotype. the diagnosis is confusing, indicating that size comparisons between l. filiorum (n = 2) and l. hajeki núñez, pincheira-donoso & garín 2004 (sample size not indicated) were performed with a student’s t-test after confirming normality using a shapiro-wilk test (pincheira-donoso and ramírez, 2005:353). without indication of sample size, such a diagnosis is extremely weak. shortly after my publication appeared, pincheiradonoso (2014) replied to my synonymisation, arguing that my work was an example of “negligent observations” and proposed to revalidate liolaemus filiorum. in this paper, pincheira-donoso (2014) claimed that “this specimen was recently transferred to the museo nacional de historia natural de chile collection from another collection” (without specifying which collection, although this may refer to the collection of the departamento de biología celular y genética of the universidad de chile, dbcuch, where several paratypes of l. puritamensis were previously located) and claimed that the mnhn-3829 number was duplicated. although, in fact, mnhn-3829 also has an older dbcuch label, the duplication of “mnhn-3829” is highly unlikely for several reasons. first, our search for mnhn-3829 specimen in 2013 ascertained that only one specimen with that number was present in the entire collection and even in the online database there was only one mnhn-3829 (gbif, 2013). furthermore, the timeline for the transfer of dbcuch specimens to the mnhn negates the argument that an error was committed by anyone other than pincheira-donoso and ramírez (2005). whereas ortiz and díaz-páez (2006) only stated that the transfers occurred “recently”, the dates become clearer when examining additional specimen accessions. for example, pincheira-donoso and núñez (2005:261) studied mnhn-3810 a specimen of l. ubaghsi esquerré, troncoso-palacios, núñez & garín 2013 (esquerré et al., 2014). they (2005:190) also studied mnhn 3833–3837, specimens of l. cf. moradoensis, currently l. bellii (esquerré et al., 2014). thus, specimens bracketing the registration number of the holotype of l. filorum were unambiguously present in 2005. thus, when pincheira-donoso and ramírez (2005) described l. filiorum, mnhn-3829 must have already been deposited in the national collection. to counter the argument that the holotype should be illustrated in the species description “when it is possible” (iczn, 1999: recommendation 16f), pincheira-donoso (2014) provided a photograph of another specimen (not the mnhn-3829 that i had examined) that he stated was the holotype, claiming that the collection number of the figured specimen was mnhn-3829. unfortunately, it is not possible to independently verify that this specimen is indeed the holotype of l. filiorum, because no photograph or specific data for the holotype were provided in the original description (pincheira-donoso and ramírez, 2005). several of pincheira-donoso’s taxonomic works have been noted as at least “controversial” by some authors, because they presented cases of inappropriate descriptions of species (lobo et al., 2010, 2012). examples include the assignment of paratype specimens of l. puna lobo & espinoza, 2004 as paratypes of l. barbarae pincheira-donoso and núñez, 2005 without an appropriate diagnosis (quinteros and lobo, 2009) and listing a frog specimen as a paratype of the lizard l. hermannunezi pincheira-donoso, scolaro & schulte, 2007 (troncoso-palacios, 2014). even if the specimen illustrated by pincheira-donoso (2014) is the true holotype, it is unclear why it was not deposited in the collection indicated in the original description for more than eight years post-publication and why it was not possible illustrate it in the original description. pincheira-donoso (2014) has criticized the peer review process of the journal cuadernos de herpetología, where i published my work (troncoso-palacios, 2014), claiming it did not meet an adequate scientific standard, even though the description of l. filiorum (pincheiradonoso and ramírez, 2005) was not subjected to peerreview in the first place. i strongly disagree with this criticism, given that cuadernos de herpetología is one of the most prestigious south american herpetology journals, where the most prominent specialists in the taxonomy of liolaemidae have published (e.g., avila, 1995; laurent, 1995; etheridge, 1998; lobo, 2000; abdala and quinteros, 2014; breitman et al., 2014); the journal has highly quali127on the taxonomic status of liolaemus filiorum fied editors and reviewers. it is also especially remarkable that pincheira-donoso (2014), whose publication was narrowly focused on criticizing my work (troncoso-palacios, 2014), misspelled my name throughout the entire manuscript (“troncoso-palacio”) and indicated incorrect pagination: “1-7” (see references in this paper for the correct citation and pages). in conclusion, as attested to by the positive reviews of my earlier paper and the narrative regarding specimen numbers and timelines presented herein, my original conclusion to consider liolaemus filiorum as a junior synonym of l. puritamensis due to inappropriate description and designation of the holotype should be considered correct. acknowledgements i thank the colleagues who encouraged me to write this response and the two anonymous reviewers who helped me to improve the early draft. h. kaiser and s. thomson for their invaluable comments. finally, thanks to the reviewer and editor of acta herpetoplogica. references abdala, c.s., quinteros, a.s. 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(2001): la ornamentación de los hemipenes en liolaemus (iguania: tropiduridae) cuad. herpetol. 14: 145-151. lobo, f., cruz, f.b., abdala, c.s. (2012): multiple lines of evidence show that phymaturus agilis scolaro, ibargüengoytía & pincheira-donoso, 2008 is a junior synonym of phymaturus spectabilis lobo & quinteros, 2005. cuad. herpetol. 26: 21-27. lobo, f., espinoza, r.e., quinteros, s. (2010): a critical review and systematic discussion of recent classification proposals for liolaemid lizards. zootaxa 2549: 1-30. ortiz, j.c., díaz-páez, h. (2006): estado de conocimiento de los anfibios de chile. gayana 70: 114-121. pincheira-donoso, d. (2014): no evidence for conspecificity between two high andes liolaemus lizards (squamata: liolaemidae). acta herpetol. 9: 249-252. pincheira-donoso, d., núñez, h. (2005): las especies chilenas del género liolaemus. taxonomía, sistemática y evolución. publ. ocas. mus. nac. hist. nat. chile 59: 1-487. pincheira-donoso, d., ramírez, g.m. (2005): desplazamiento de caracteres como evidencias de un posible caso de especiación simpátrica entre dos liolaemus del grupo jamesi en la provincia de el loa, con la descripción de una nueva especie. in: fauna del altiplano y desierto de atacama. vertebrados de la provincia de el loa, pp. 350-365. ramírez, g.m., pincheira-donoso, d., eds, phrynosaura ediciones, calama, chile. quinteros, a.s., lobo, f. (2009): the iguanian lizard liolaemus barbarae pincheira-donoso and núñez is a junior synonym of liolaemus puna lobo and espinoza. j. herpetol. 43: 336-339. troncoso-palacios, j. (2014): revisión del estatus taxonómico de liolaemus filiorum pincheira-donoso y ramírez, 2005 (iguania: liolaemidae). cuad. herpetol. 28: 111-117. acta herpetologica vol. 10, n. 1 june 2015 firenze university press acta herpetologica journal of the societas herpetologica italica acta herpetologica 9(2): 259-263, 2014 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-14736 sexing freshwater turtles: penile eversion in phrynops tuberosus (testudines: chelidae) joão f. m. rodrigues1,2,3,*, diego de o. soares2, josé r. f. silva3 1 programa de pós-graduação em ecologia e evolução, universidade federal de goiás, departamento de ecologia, goiânia, go, brazil. *corresponding author. e-mail: fabriciorodrigues303@gmail.com 2 núcleo regional de ofiologia da universidade federal do ceará (nurof-ufc), fortaleza, ce, brazil 3 programa de pós-graduação em ecologia e recursos naturais, universidade federal do ceará, centro de ciências, fortaleza, ce, brazil submitted on 2014, 25th july; revised on 2014, 15th november; accepted on 2014, 17th november editor: paolo casale abstract. here, we described a noninvasive method for sexing freshwater turtles by stimulating penile eversion. we immobilized the neck and limbs of animals using fingers and, after some seconds, turtles everted their penis. this method was tested in 33 male phrynops tuberosus, and 28 everted the penis. the efficiency of the method was not dependent of animal size, which reinforces its applicability. our method allows sexing turtles in the field, avoiding killing the animal or causing major injuries in order to assess the sex. keywords. defensive behaviour, penis, pleurodira, sexing method. determining the gender of individuals is useful in most population studies. however, sexing might be difficult for many species, especially freshwater turtles, because some species lack clear morphological differences between males and females (rueda-almonacid et al., 2007). examples of general noninvasive sexing methods include hormone profiles of blood and amniotic fluid (wibbels et al., 1987; gross et al., 1995; xia et al., 2011), chromosome analysis in species having genetic sex determination (gsd) (ferreira júnior, 2009), searching for mature eggs through palpation on the inguinal region of females (rueda-almonacid et al., 2007), sexual dichromatism in head spots (moll et al., 1981; bulté et al., 2013) and morphometry (gibbons and lovich, 1990; valenzuela et al., 2004; casale et al., 2005; rueda-almonacid et al., 2007; readel et al., 2008). this last method is the most used in field conditions. tail size and shape, cloaca position, claws length, and plastron morphology are examples of secondary sexual characteristics, being tail analysis applicable for all turtles (rueda-almonacid et al., 2007; reed and tucker, 2012). however, not experienced researchers might have difficulty to evaluate if a tail is large or small. penises of turtles are found in the cloaca’s ventral surface and are inflatable copulatory organs (zug, 1966; carvalho et al., 2010; cabral et al., 2011). a method to sex turtles is to insert the finger into the cloaca in order to find the penis (rueda-almonacid et al., 2007). however, this method is invasive and useless in small animals. manual hemipenile eversion (copulatory structure found in squamata) is a common method used to sex snakes, lizards, and amphisbaenians (reed and tucker, 2012). however, in chelonians, this method has been explored only recently (de solla et al., 2001; dustman, 2013; lefevbre et al., 2013). de solla et al. (1998) used this technique to sex individuals of chelydra serpentina, but they did not explain how it was performed. recently, lefevbre et al. (2013) described a method to stimulate penial erection 260 j.f.m. rodrigues et alii and ejaculation by applying vibrations to the animal shell, while de solla et al. (2001) and dustman (2013) stimulated penis eversion in chelydra serpentina by gently shaking the turtle horizontally and vertically, respectively. in july 2012, we observed that when captured male turtles were much stressed (trying to bite, moving their limbs, head, and tail very much), they everted the penis during handling after a few seconds [a behaviour already observed in chelydra serpentina (de solla et al., 2001) and hydromedusa maximiliani (famelli et al., 2011), which may be considered a defensive display]. in august 2012, when we were attempting to measure a stressed male of phrynops tuberosus, we held its neck and limbs against its body in order to safely and accurately measure it. when the specimen was held in this manner, the male rapidly everted the penis. these observations were the starting point for the development of the method described herein. this study aims to describe a method for sexing freshwater turtles, based in penile eversion, which do not require any additional material and may be easily performed in field conditions. the sexing method was tested in 33 males of phrynops tuberosus (peters, 1870) (phrynops geoffroanus sensu latu, populations of ceará referred by rueda-almonacid et al. (2007) as phrynops tuberosus), captured at the banabuiú river, village of laranjeiras (05º17’s, 38º31’w, datum wgs84), banabuiú, ceará, northern brazil, between august and november 2012. phrynops tuberosus is a chelid freshwater turtle distributed in northeastern south america (rueda-almonacid et al., 2007). turtles were hand-captured using snorkels, measured (carapace length (cm) – cl), sexed through tail size (males having a larger precloacal length than females) following rueda-almonacid et al. (2007) and marked following cagle (1939). males had their limbs and head immobilized (fig. 1). in some occasions, gently applying a little pressure on hind limbs and head was necessary to accelerate penile eversion in males that were calm (they did not try to bite or move their limbs, head and tail; their only action was to retract the neck and limbs when handled). the method was tested at a maximum twice in each male, the second trial being performed only if the first one had a negative result, in order to avoid excessive stress. eversion occurred after a few seconds, which was dependent on animal stressing level (most stressed individuals completely everted the penis in 9 s, while calmer turtles took approximately 30 s to only partially evert it). all animals retracted the penis after release and had no alteration in their normal behaviour. we also observed a small cloacal wall eversion in some females (see dustman, 2013). however, it was not employed an evaluation for females as systematic as it was performed for males. captures were authorized by the responsible environmental institution (see license number in acknowledgments). no animal suffered injuries or mistreatment in capture and handling processes, and no invasive method was performed. we used a logistic regression to evaluate the relationship between the efficiency of our method and male size. a chi-square test was used to compare the number of successes and failures using the method. the sexing method reliability was assessed as a proportion of males that exposed their penises. statistical tests were performed in r ver. 3.0.1 (r development core team, 2013). descriptive statistics are reported as mean ± standard deviation. we found that 28 males everted the penis when we used the method reported herein. the reliability of the method was 84.85% (28/33) and the vast majority of males were correctly sexed (χ2 = 16.03, df = 1, p < 0.001). males ranged in cl from 7.63 to 26.91 cm and averaged 20.96 ± 4.02 cm. the efficiency of the method was independent of animal size (logistic regression: z = -1.39, p = 0.16) (fig. 2). all stressed animals everted the penis. the five individuals that did not evert were calm. probably there was no error in sex identification, since these males had evident male characteristics, such as a large precloacal distance. if they were females, we would have observed a small cloacal wall evertion, but their tail did not show any response to the method. the development of non-lethal techniques to sex turtles is essential in conservation and demographic studies (rueda-almonacid et al., 2007; lefevbre et al., 2013). the method proposed here was able to properly sex the   fig. 1. illustration of a male phrynops tuberosus captured in banabuiú river, being sexed following the method explained in this study (rodrigues et al.). a) male p. tuberosus with head and limbs hold; b) male p. tuberosus everting the penis after a little pressure in the hind limbs and neck. note the arrows indicating the pressure points. arrows and letters were included in the original drawing using adobe photoshop™. drawings: diego de oliveira soares. 261freshwater turtles sexing method vast majority of captured males. our study is the first to describe a technique of penis eversion tested in pleurodira turtles. previous methods were successfully tested in chelydra serpentina (de solla et al., 2001; dustman, 2013) and emydoidea blandingii (lefevbre et al., 2013). the efficiency of most methods using penile eversion was tested in only one species (including ours). however, the potential contribution of these methods to field studies cannot be underestimated due to this limitation, and future studies in other turtle populations may provide more data about their success. penis is an inflatable copulatory organ, which also depends on blood influx to become everted and erected (zug, 1966; carvalho et al., 2010; cabral et al., 2011). stressing conditions, such as handling, may increase heart-beating rate in freshwater turtles (cabanac and bernieri, 2000), which could raise blood flow, easing penile eversion. a high efficiency of a penis eversion method in stressed turtles was also found in lefebvre et al. (2013). there were reports of other freshwater turtles everting penis after being handled (de solla et al., 2001; famelli et al., 2011), which reinforce the potential application of our method in other species. although our method was not efficient enough to sex all males, it had a high reliability (84.85%). manual hemipenile eversion in squamata also has some problems and is difficult to use in large bodied species, with muscular tails or in fragile species (reed and tucker, 2012). penile eversion could be used in parallel to traditional methods based on tail length size, in order to have an improved result. the lack of relationship between method efficiency and animal size is advantageous, because it reinforces its general applicability. a male with carapace length of 7.63 cm, for example, was successful sexed (see fig. 2 for more details about the lack of relationship between efficiency and animal size). most sexing methods of chelonians are size-dependent and could be performed only in large individuals, such as secondary sexual characteristics identification (for example, plastron concavity and claws size) and penis identification inside the cloaca (ruedaalmonacid et al., 2007; reed and tucker, 2012). unfortunately, we cannot make a prediction about the applicability of our method to immature individuals based on our data, which could be an interesting topic for future studies regarding this method. penis display may be considered a defensive behaviour in freshwater turtles (de solla et al., 2001; famelli et al., 2011). it may be an explanation for the eversion as a consequence of immobilization, because males everted the penis when they had been completely immobilized, with all their limbs and head held. hence, this behaviour would be a final strategy to try to scary the predator (the researchers, herein) and escape. our method allows sexing turtles in the field, avoiding killing the animal or causing major injuries in order to assess the sex. studies reporting sexing methods based in penile eversion are increasing, reinforcing the impor   fig. 2. relationship between the probability of success of the method and carapace length of phrynops tuberosus in centimeters. 262 j.f.m. rodrigues et alii tance of invest in such methods, which are already very common for squamata. finally, this manuscript enriches the list of studies of noninvasive and reliable sexing methods for turtles, and its employment may be useful in future population studies with these animals. acknowledgments we thank all the friends that helped in field survey. flávio de barros molina gave valuable suggestions on the first version of the manuscript and provided literature. diogo borges provete and roger bour gave valuable suggestions in the last version of the manuscript. cnpq and programa de pós-graduação em ecologia e recursos naturais da universidade federal do ceará provided a master fellowship to jfmr. instituto chico mendes de conservação da biodiversidade (icmbio) granted a measuring, marking, and capture license (sisbio/icmbio 32548-1). references bulté, g., germain, r.r., o’connor, c.m., blouin-demers, g. (2013): sexual dichromatism in the northern map turtle, graptemys geographica. chelonian conserv. biol. 12: 187-192. cabanac, m., bernieri, c. (2000): behavioural rise in body temperature and tachycardia by handling of a turtle (clemmys insculpta). behav. process. 49: 61-68. cabral, s.r.p., santos, r.l.s., franco-belussi, l., zieri, r., zago, c.e.s., oliveira, c. (2011): anatomy of the male reproductive system of phrynops geoffroanus (testudines: chelidae). acta sci. biol. sci. 33: 487-492. cagle, f.r. (1939): a system of marking turtles for future identification. copeia 1939: 170-173. carvalho, r.f., oliveira, s.c.r., bombonato, p.p., oliveira, a.s., sousa, a.l. (2010): morfologia dos órgãos genitais masculinos do jurará kinosternon scorpioides (chelonia: kinosternidae). pesq. vet. bras. 30: 289-294. casale, p., freggi, d., basso, r., argano, r. (2005): size at male maturity, sexing methods and adult sex ration in loggerhead turtles (caretta caretta) from italy waters investigated through tail measurements. herpetol. j. 15: 145-148. de solla, s.r., bishop, c.a., van der kraak, g., brooks, r.j. (1998): impact of organochlorine contamination on levels of sex hormones and external morphology of common snapping turtles (chelydra serpentina serpentina) in ontario, canada. environ. health persp. 106: 253-260. de solla, s.r., portelli, m., spiro, h., brooks, r.j. (2001): penis displays of snapping turtles (chelydra serpetina) in response to handling: defensive or displacement behavior. chelonian conserv. biol. 4: 187-189. dustman, e.a. (2013): sex identification in the common snapping turtle (chelydra serpentina): a new technique and evaluation of previous methods. herpetol. rev. 44: 235-238. famelli, s., bertoluci, j., molina, f.b., matarazzo-neuberger, w.m. (2011): structure of a population of hydromedusa maximiliani (testudines, chelidae) from parque estadual da serra do mar, an atlantic rainforest preserve in southeastern brazil. chelonian conserv. biol. 10: 132-137. ferreira júnior, p.d. (2009): aspectos ecológicos da determinação sexual em tartarugas. acta amaz. 39: 139-154. gibbons, j.w., lovich, j.e. (1990): sexual dimorphism in turtles with emphasis on the slider turtle (trachemys scripta). herpetol. monogr. 4: 1-29. gross, t.s., crain, d.a., bjorndal, k.a., bolten, a.b., carthy, r.r. (1995): identification of sex in hatchling loggerhead turtles (caretta caretta) by analysis of steroid concentrations in chorioallantoic/amniotic fluids. gen. comp. endocr. 99: 204-210. lefevbre, j., carter, s., mockford, s.w. (2013): new experimental method for semen extraction in freshwater turtles. herpetol. rev. 44: 595-600. moll, e.o., matson, k.e., krehibel, e.b. (1981): sexual and seasonal dichromatism in the asian river turtle callagur borneoensis. herpetologica 37: 181-194. r development core team (2013): r: a language and environment for statistical computing. r foundation for statistical computing, viena. readel, a.m., dreslik, m.j., warner, j.k., banning, w.j., phillips, c.a. (2008): a quantitative method for sex identification in emydid turtles using secondary sexual characters. copeia 2008: 643-647. reed, r.n., tucker, a.d. (2012): determining age, sex, and reproductive condition. in: reptile biodiversity: standard methods for inventory and monitoring, pp. 151-163. mcdiarmid, r.w., foster, m.s., guyer, c., gibbons, j.w., chernoff, n., eds, university of california press, berkeley. rueda-almonacid, j.v., carr, j.l., mittermeier, r.a., rodriguez-marecha, j.v., mast, r.b., vogt, r.c., rhodin, a.g., ossa-velásquez, j., rueda, j.n., mittermeier, c.g. (2007): las tortugas e los cocodrilianos de los países andinos del trópico. serie de guias tropicales de campo, nº 6. conservación internacional editorial panamericana, formas e impresos, bogotá. valenzuela, n., adams, d.c., bowden, r.m., gauger, a.c. (2004): geometric morphometric sex estimation for 263freshwater turtles sexing method hatchlings turtles: a powerful alternative for detecting subtle sexual shape dimorphism. copeia 2004: 735-742. wibbels, t., owens, d.w., morris, y.a., amoss, m.s. (1987): sexing techniques and sex ratios for immature loggerhead sea turtles captured along the atlantic coast of the united states. in: ecology of east florida sea turtles, pp. 65-74. witzell, w.n., ed, national oceanic and atmospheric administration, national marine fisheries service, miami, florida. xia, z., li, p., gu, h., fong, j.j., zhao, e. (2011): evaluating noninvasive methods of sex identification in green sea turtles (chelonia mydas) hatchlings. chelonian conserv. biol. 10: 117-123. zug, g.r. (1966): the penial morphology and the relationships of cryptodiran turtles. occ. papers. 647: 1-24. acta herpetologica 10(2): 149-153, 2015 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-17170 eryx jaculus (linnaeus, 1758): a new species for the italian herpetofauna (squamata: erycidae) gianni insacco1, filippo spadola2, salvatore russotto3, dino scaravelli4 1 museo civico di storia naturale, via degli studi 9, 97013 comiso (rg), italy. corresponding author. e-mail: g.insacco@comune. comiso.rg.it 2 museo della fauna del dipartimento di scienze veterinarie dell’università degli studi di messina, polo universitario ss. annunziata. i-98100 messina, italy 3 contrada grassura mollaka faia, s.n. 92027 licata (ag), italy 4 dipartimento di scienze mediche veterinarie, università di bologna, via tolara di sopra 50, ozzano emilia (bo) e st.e.r.n.a., museo ornitologico “f. foschi” forlì, via pedriali 12, 47121 forlì, italy submitted on 2015, 9th october; revised on 2015, 16th october; accepted on 2015, 23rd october editor: sebastiano salvidio abstract. the presence of a population of the javelin sand boa eryx jaculus in sicilia is here reported for the first time. observations of live snakes and road-killed individuals have been obtained from the region of licata (province of agrigento), in the southern part of sicily. data on the distribution and pholidosis from four specimens are presented. the large area of occurrence of the snake in sicily, that bears also a local vernacular name suggests an ancient origin of the colonization. the presence of this species, increases the numbers of snakes living in italy. keywords. eryx jaculus, sicily, italy, new record. eryx jaculus (linnaeus, 1758), commonly known as the javelin sand boa, has been recently assigned to the erycidae family (pyron et al., 2014). javelin sand boa can be found in southern balkans, on many islands in the aegean sea, transcaucasia, eastern ciscaucasia, north africa and middle east (tokar and obst, 1993; gasc et al.,1997; sindaco et al., 2013). it is a medium-sized species that usually grows up to 30-60 cm of total length (schleich et al., 1996 ; fig. 1a). the javelin sand boa prefers sandy and loose soils where it can easily burrow holes for breeding and sheltering. it is a predominantly crepuscular species (boulenger, 1913). it feeds on lizards, mice and snails. it is an ovoviviparous species; female specimens generate 6-12 eggs which hatch directly in the oviduct, and deliver live offspring in july (fuhn and vancea, 1961; fuhn, 1969). on 19 june 2006 a local tv station “tv alfa licata”, reported the first sighting of an adult individual, in licata, agrigento. subsequently, a series of field research and interviews to locals allowed to collect data on the presence of this species in the licata territory, along the salso river valley where 6 specimens were located. for the classification was followed arnold and ovenden (2002). before the present study, only anecdotal information were available (cf. tokar and obst, 1993), and the only museum specimen (natural history museum of florence) was considered mislabeled (razzetti and sindaco, 2006). corine land cover layers, provided by the regional environmental office of the sicily region, were elaborated using arcgis (9.2 esri) to categorize habitat types in the investigated area. a buffer area of 2000 m around each collection point was established and the third hierarchical level in corine land cover classes (bossard et al., 2000) was used to evaluate the percentage of each category. during our study two specimens were found dead on the road, one found drowned in a shaft and three were captured alive. these were kept for a few days in captivity, in order to take measurements and pictures and 150 insacco et alii then released in the same location where they have been captured. two dead specimens were preserved in the collection of the comiso natural history museum (msnc inventory 4501: 1, 2). the location data for each specimen are listed below. 1. adult, 19/06/2009; lat. 37°6’46.44”n; long. 13°56’20.35”e; via londra, licata (ag); rif.: youtube.com – “tv alfa licata, la città e i suoi problemi, la vipera”. 2. tl 422 mm; adult male; gr. 76; 09/06/2015; lat. 37°9’18.23”n; long. 13°53’32.53”e; contrada conca, licata (ag); captured and released, fig. 1a, b, c); fig. 2a, fig. 3a. 3. tl 445 mm; adult male; gr. 78; 13/06/2015; 37°8’7.08”n; 13°51’57.31”e; contrada s. francesco di paola, licata (ag); captured and released, fig. 2b; fig. 3b. 4. tl 190 mm; juvenile female; 13/06/2015; 37°8’7.05”n, 13°51’57.19”e; contrada s. francesco di paola, licata (ag); found drowned in a shaft, largely decomposed. fig. 3c. 5. tl 215 mm; juvenile female; gr. 9, 03/07/2015; 37°8’7.21”n, 13°51’57.13”e; contrada s. francesco di paola, licata (ag); captured and released; fig. 2c; fig. 3d. 6. tl 510 mm; adult female; 07/07/2015; lat. 37°8’26.51”n; long. 13°54’54.57”e; contrada calannino, licata (ag); rif. coll. msnc 4501-1; run over by a car, mummified, preserved in the museum in comiso; fig. 3e. 7. tl 585 mm; adult female; gr. 173; 21/07/2015; lat. 37°7’47.29”n; long. 13°52’54.49”e; poggio cuterizzio – ss 115; licata (ag); rif. coll. msnc 4501-2; run over by a car, preserved in the museum in comiso; fig. 2d; fig. 3f. the area where eryx jaculus was observed extends for about 40 km2 along the south coastal area of sicily, in the gela gulf. it is an alluvial plain called “la piana” created by the action of the river salso and it is mostly flat with small hills. this geological substrate is mainly constituted of montmorillonite clays that greatly dries out during the xeric season and cracks creating characteristic deep fractures in the arid soil. the local climate is typically mediterranean with mild and rainy winters and hot dry summers, with a constant marine breeze. the average temperature in licata is 17.7 °c with annual precipitation up to 405 mm (climate-date.org). from the analysis of the corine land cover data, in the buffer area the sighting points were mostly located in not irrigated arable lands (77,93%), followed by natural pastures and dry meadows (9,59%), agricultural mosaics (3,65%), and agricultural area with natural elements (3,11%). meristical analysis conducted on four specimens are listed in fig. 2, following eskandarzadeh et al. (2013), wconfirms they are indeed eryx jaculus the specimen b, fig. 1. eryx jaculus adulte, male, from southern sicily. a) view front body; b) details of the ventral body with claw-like spurs; c) ventral view of the mental groove. specimen pin ben be a 2 3 7 b 3 3 7 c 2 3 7 d 2 3 7 fig. 2. meristic scale counts of the head in specimens of eryx jaculus from sicily. number of scales posterior to internasal (pin); number of scales between eye and nasal (ben); number of scales between eyes (be). 151eryx jaculus in sicily which has 3 pin scales, is considered within the intraspecific variability (boulenger, 1913; tokar and obst, 1993). considering the data collected and occasional observations of e. jaculus by local people (table 1), there are information since 1930 in this area. the high frequency of sightings, as well as the presence of local vernacular names for the species (apita, aspit surdu, spitu) strongly suggests that this species was not recently introduced. given its nocturnal habits and very elusive nature, it is likely that this species simply went unnoticed for a long time. ancient introductions of snakes for religious cults or for war rituals were also proposed by masseti and zuffi (2011), with a particular reference to the greeks who inhabited the area for a long time and fought two important battles on those lands in 405 a.c. and in 310 a.c. (di blasi, 1844). the presence of a consolidate population in this sicilian valley, composed by adults and juveniles is underlining the biogeographic importance and the priority of conservation of this species. also it is necessary to highlight that being the e. jaculus a protected repfig. 3. eryx jaculus founds in licata (ag). a) male, alive in c.da conca; b) male, alive in c.da s. f. di paola; c) juvenile, female, drowned inside of well in c.da s. f. di paola; d) juvenile female, alive in c.da s. francesco di paola; e) mummified individual in c.da calannino; f) female, in poggio cuterizzio. 152 insacco et alii tile listed in the appendix iv of the 92/43/cee directive, in appendix ii of cites, and appendix iii of the bern convention, its presence in sicily calls for a prompt implementation of the relevant regulatory framework and safeguards activities as provided for under the law for the protected wild fauna in italy. the range of the species in nearby areas in sicily is also under study by the authors. a genetic study will be realized to verify subspecies attribution and identify the possible origin of this population. acknowledgment thanks to all the locals living in licata who helped us by reporting precious sightings and putting forward suggestions; in particular luigi caci, epifanio bonelli, rosa e calogero porrello, giuseppe casa. also thanks to morena priori for linguistic assistance and bruno zava who provided useful suggestions. since the species was not known in italy, research permits were not issued. finally we thank two anonymous reviewers that improved a first draft of the paper. references arnold, n., ovenden, d. (2002): collins field guide to reptiles and amphibians of britain and europe. collins, london. bossard, m., feranec, j., otahel, j. (2000): corine land cover technical guide. addendum 2000. technical report n° 40, european environment agency. boulenger, g.a. (1913): the snakes of europe. methusen and co. ltd, london. di blasi, g.e. (1844): storia del regno di sicilia, vol i. edizioni dafni catania, distribuzione tringale editore, stamperia oretea palermo. eskandarzadeh, n., darvish, j., rastegar-pouyani, e., ghassemzadeh, f. (2013): reevaluation of the taxonomic status of sand boas of the genus eryx (daudin, 1803) (serpentes: boidae) in northeastern iran. turk. j. zool. 37: 348-356. fuhn i.e. (1969): broaste, serpi, sopârle. ed. ştiinţifică, bucureşti. fuhn, i.e., vancea, şt. (1961): fauna r.p.r. reptilia (ţestoase, şopârle, şerpi). 9(2). editura academiei r.p.r., bucureşti. gasc, j.p., cabela, a., crnobrnja-isailovic, dolmen, d., grossenbacher, k., haffner, p., lescure, j., martens, h., marinez rica, j.p., maurin, h., oliveira, m.e., sofianidou, t.s., veith, m., zuiderwijk, a. (1997): atlas of amphibians and reptiles in europe. societas europaea herpetologica et muséum national d’histoire naturelle paris. masseti, m, zuffi, m.a.l. (2011): hypotheses on the origin of the population of asp viper, vipera aspis hugyi schinz, 1833, of the island of montecristo, in the northern tyrrhenian sea (tuscan archipelago, italy). table 1. sightings localities of eryx jaculus by local people, since 1930 to 2013. date locality and position number of specimens age/sex name of observer 2006-2013 contrada san francesco di paola, licata; 37°8’7.15°n, 13°51’57.27°e 10, alive adults salvatore russotto 1999-2013 contrada grasciura, licata; 37°7’48.32°n 13°52’31.17°e 8, alive adults salvatore russotto 2004 torre s. nicola, licata; 37°6’46.70°n 13°52’8.01°e 1, dead adult female salvatore russotto 1986-1991 contrada pozzillo, licata; 37°6’47.70°n 13°52’59.47°e 2, alive adult females luigi caci 1982-1987 contrada renella, licata; 37°8’25.16°n 13°55’59.98°e 4, alive adults epifanio bonelli 1987 contrada poggio cuterizzio, licata; 37°7’46.84°n 13°52’52.83°e 1, alive adult epifanio bonelli 1984 contrada san francesco di paola, licata; 37°8’7.15°n, 13°51’57.27°e 1, alive adults salvatore russotto 1980 contrada pisciotto, licata; 37°7’44.17°n 13°50’57.94°e 1, alive adult epifanio bonelli 1930-1970 contrada renella, licata; 37°8’37.72°n 13°55’44.77°e numerous, alive adults giuseppe casa 1930-1960 contrada culazzo, licata; 37°8’59.71°n 13°55’36.86°e numerous, alive adults giuseppe casa 153eryx jaculus in sicily br. herpetol. bull. 117: 1-9. pyron, r.a., reynolds, r.g., burbrink, f.t. (2014): a taxonomic revision of boas (serpentes, boidae). zootaxa 3846: 249-260. razzetti, e., sindaco, r. (2006): taxa non confermati o meritevoli di conferma. in: atlante degli anfibi e rettili d’italia atlas of italian amphibians and reptiles, pp. 645-652. sindaco, r., doria, g., razzetti, e., bernini, f. eds, edizioni polistampa, firenze. schleich, h.h., kastle, w., kabish, k. (1996): amphibians and reptiles of north africa, biology, systematics, field guide. koeltz scientific books, koenigstein. sindaco, r., venchi, a., grieco, c. (2013): the reptiles of the western palearctic. 2. annotated checklist and distributional atlas of the snakes of europe, north africa. middle east and central asia, with an update to the, vol 1, edizioni belvedere, latina. tokar, a., obst, f.j. (1993): eryx jaculus westliche sandboa. in: handbuch der reptilien und amphibien europas, band 3/i., schlangen (serpentes) i, pp. 35-54. böhme,w. ed., aula-verlag wiesbaden. acta herpetologica vol. 10, n. 1 june 2015 firenze university press acta herpetologica journal of the societas herpetologica italica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 7(2): 253-262, 2012 morphological variability of the hermann’s tortoise (testudo hermanni) in the central balkans katarina ljubisavljević1, georg džukić1, tanja d. vukov1, miloš l. kalezić1,2 1 department of evolutionary biology, institute for biological research “siniša stanković”, university of belgrade, bulevar despota stefana 142, 11060 belgrade, serbia. corresponding author. e-mail: katarina.ljubisavljevic@ibiss.bg.ac.rs 2 institute of zoology, faculty of biology, university of belgrade, studentski trg 16, 11000 belgrade, serbia submitted on: 2012, 9th may; revised on: 2012, 4th june; accepted on: 2012, 10th july. abstract. variation in a number of morphological characters were analysed in five populations of testudo hermanni from serbia and montenegro. tortoises from serbia appeared significantly larger than those from montenegro. an insular population of montenegro was distinct due to its extremely small body size and mass and dark plastral pigmentation. although the majority of tortoises had the supracaudal scute divided, many tortoises had the scute undivided. effects of possible genetic divergence and/or adaptation to different environmental conditions on observed morphological variation were discussed. keywords. land tortoises, balkan peninsula, morphological characteristics, plastral pigmentation, supracaudal scute. introduction in the last decade, there has been a great deal of interest in the phylogeny, morphology and taxonomy of the land tortoises (testudinidae) of the western palaearctic (van der kuyl et al., 2002; perälä, 2002a; fritz et al., 2005, 2006, 2007, 2009; de lapparent de broin et al., 2006a, b; parham et al., 2006; fritz and bininda-edmonds, 2007). one testudo species, the hermann’s tortoise (t. hermanni gmelin, 1789) with a patchy distribution in the northern mediterranean (bour, 1997; cheylan, 2001), is a particularly intriguing example for the complicated situation. it was placed in another genus by some authors (de lapparent de broin et al., 2006a, b) while others elevated its two subspecies to full species (t. hermanni, t. boettgeri) and recognized dalmatian populations as a third species (t. hercegovinensis; perälä, 2002b, 2004; bour, 2004). although these changes were not widely accepted (fritz et al., 2006; parham et al., 2006; fritz and bininda-edmonds, 2007), the balkan peninsula yet appeared as a particularly important and intriguing area for the complex pattern of diversi254 katarina ljubisavljević et al. fication within t. hermanni. thus, the higher genetic diversity within the balkan subspecies t. h. boettgeri compared with that in the western mediterranean t. h. hermanni was found to be probably related to existence of several refugia in the balkan peninsula during the pleistocene glaciations (van der kuyl et al., 2002; fritz et al., 2006). up to now, the studies on populations of t. hermanni in the region of the central part of the balkan peninsula (serbia and montenegro) included aspects related to their ecology (meek and inskeep, 1981; meek, 1985, 1989), sexual dimorphism (đorđević et al., 2011) and commercial export and the impact of overharvesting (ljubisavljević et al., 2011). however, some basic analyses, such as main morphological characteristics and their geographic variability, are still lacking. considering these facts, the main aim of this paper is to provide basic data about morphological characteristics of the hermann’s tortoise in the central balkans and to depict their variation pattern. materials and methods morphological parameters were analysed using five samples from serbia and montenegro (fig. 1): 1. limljani in montenegro (42°12’n, 19°06’e, 156 m a.s.l.); 2. starčevo island in lake skadar in montenegro (42°11’n, 19°13’e, 15 m a.s.l.); 3. eastern serbia (localities: stara brza, 44°28’n, 22°27’e, 67 m a.s.l.; petrovo selo, 44°37’n, 22°27’e, 435 m a.s.l. and vratna monastery, 44°23’n, fig. 1. map showing the distribution range of t. hermanni in the former yugoslavia (shaded area) with the numbers referring to sampling areas for the examination of morphological variation (according to ljubisavljević et al., 2011). 1. limljani; 2. starčevo island; 3. eastern serbia; 4. central serbia; 5. southern serbia. 255morphological variability of testudo hermanni 22°21’e, 165 m a.s.l.); 4. central serbia (localities: kolare, 43°54’n, 21°14’e, 160 m a.s.l. and ivković monastery 43°52’n, 21°13’e, 218 m a.s.l.); 5. southern serbia (locality: starac mt., 42°20’n, 21°52’e, 810 m a.s.l.). samples from several populations at distinct localities in the eastern and central parts of serbia were grouped together to obtain sufficient data for analysis. tortoises were spotted by walking through the habitat during daylight hours (9-18 h). they were measured and weighed in the field as described by stubbs et al. (1984), photographed, and then released immediately afterwards at the point of capture. sex was determined by plastral concavity and by the presence of larger tails in males in specimens larger than 10 cm carapace length. all animals smaller than 10 cm carapace length were considered to be juveniles (stubbs et al., 1984) and were excluded from analysis because of possible ontogenetic variation in morphology (guyot and devaux, 1997). since the size at maturity in t. hermanni varies substantially among localities (willemsen and hailey, 1999a), individuals were classified as male or female rather than as subadult, adult male or adult female (see willemsen and hailey, 2002). the following quantitative parameters were recorded: m – body mass; scl – midline straight carapace length, from the front of the nuchal scute, to the rear of the carapace; cw – carapace width (at widest point); ch – carapace height, the maximum vertical height from carapace to plastron. mechanical calipers (0.1 mm precision) was used for measuring scl, while other linear measurements were taken to the nearest mm using specially constructed at-bed calipers, a “tortometer” (stubbs et al., 1984). m was taken with an electronic balance (1 g accuracy). we considered as qualitative characteristics the supracaudal scute (undivided, divided) and the extent of plastral black pigmentation in eleven states distinguished by the degree and fragmentation of pigmentation on plastral plates (fig. 2). this was done according to modified scheme of guyot and devaux (1997). descriptive statistics (mean, standard error, range) for quantitative traits, and percentages of states for each qualitative trait were calculated. for subsequent analyses, quantitative characters were log-transformed to ensure data normality and to generate homogeneous variances (sokal and rohlf, 1981). after assessing the normality of the distribution (kolmogorov-smirnov test) and homogeneity of variances (bartlett test) the existence of significant geographic (among-localities) variation and sexual dimorphism was tested by analysis of variance (anova) for scl and by analysis of covariance (ancova, with scl as covariate) for other quantitative traits. williams’ corrected g test on actual counts was performed to examine differences in qualitative characters between sexes and among localities. for quantitative traits, the tukey hsd test for unequal sample sizes was used for average comparisons between samples. preliminary analyses revealed no significant sex related variations in the frequencies of qualitative traits (williams’ corrected g test, p > 0.05, for all comparisons), and therefore, data from both sexes of the same sample were pooled for further analyses. all statistical analyses were performed using the computer package statistica® (statistica for windows. statsoft, inc., tulsa, ok). results body size variation descriptive statistics of body size measurements of adult females and males from population samples are presented in table 1. sexual dimorphism in samples from montenegro appeared to be weak and was limited to a significant intersexual difference in the ch and cw in limljani population. on the other hand, samples from serbia consistently showed significant sexual differences for scl and cw. overall, females had for all characters significantly higher values than males. 256 katarina ljubisavljević et al. in females, statistically significant differences among samples were found for scl, and m with scl held constant (anova, scl: f4,61 = 17.61, p << 0.001; ancova, m: f4,60 = 7.18, p < 0.001). paired testing between the samples revealed significant differences in all comparisons (tukey hsd test, p < 0.001) for m, while scl significantly differed between samples from montenegro and serbia (tukey hsd test, p < 0.01 in all comparisons). for this character, samples from serbia showed significantly larger values compared with the samples from montenegro. fig. 2. range of black plastral pigmentation (modified scheme of guyot and devaux, 1997): (1) continuous black pigmentation; (2) black spots on humeral scutes are isolated; (3) black spots on anal scutes are isolated; (4) black spots on anal and humeral scutes are isolated; (5) black spots on anal and femoral scutes are isolated; (6) black spots on anal, humeral and femoral scutes are isolated (7) black spots are isolated on each scute and reduced in size; (8) black spots are lacking on anal scutes, while on humeral and femoral scutes are isolated; (9) black spots are lacking on anal scutes, while on other scutes are isolated and reduced in size; (10) black spots are lacking on anal and humeral scutes, while on other scutes are isolated and reduced in size; (11) black pigmentation is lacking. 257morphological variability of testudo hermanni table 1. descriptive statistics of quantitative characters of male and female t. hermanni from serbia and montenegro. mean values of mass (m) are given in grams; all other measures are expressed in millimeters. it is also reported the statistical significance of differences between sexes (p) tested by anova (for scl) and ancova with scl as covariate (for other characters): ns, non significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001. abbreviations of characters are given in “materials and methods”. character site males mean ± se (n; range) females mean ± se (n; range) p m limljani 800.00 ± 115.77 (8; 320.00 – 1350.00) 1016.48 ± 51.30 (21; 316.00 – 1420.00) ns starčevo island 525.57 ± 80.84 (7; 288.00 – 805.00) 747.78 ± 120.22 (9; 258.00 – 1250.00) ns eastern serbia 1146.00 ± 67.24 (15; 425.00 – 1550.00) 1577.94 ± 93.44 (18; 298.00 – 2035.00) ns central serbia 1114.09 ± 71.72 (11; 650.00 – 1435.00) 1808.33 ± 87.64 (9; 1375.00 – 2160.00) ns southern serbia 1237.50 ± 100.11 (10; 755.00 – 1685.00) 2025.56 ± 177.02 (9; 1080.00 – 2790.00) * cw limljani 119.50 ± 5.63 (8; 90.00 – 139.00) 125.14 ± 2.67 (21; 83.00 – 145.00) * starčevo island 107.00 ± 8.09 (7; 79.00 – 138.00) 113.22 ± 7.50 (9; 82.00 – 143.00) ns eastern serbia 149.25 ± 3.04 (16; 114.00 – 162.00) 155.56 ± 4.96 (18; 89.00 – 175.00) ** central serbia 151.00 ± 4.74 (11; 116.00 – 170.00) 165.11 ± 2.68 (9; 149.00 – 175.00) *** southern serbia 153.50 ± 4.57 (10; 130.00 – 173.00) 166.22 ± 5.17 (9; 137.00 – 185.00) *** scl limljani 151.00 ± 8.48 (8; 108.00 – 182.00) 164.62 ± 3.74 (21; 106.00 – 188.00) ns starčevo island 132.57 ± 8.42 (7; 104.00 – 164.00) 145.67 ± 10.21 (9; 101.00 – 188.00) ns eastern serbia 177.13 ± 4.35 (16; 126.00 – 206.00) 203.89 ± 6.11 (18; 108.00 – 229.00) ** central serbia 173.73 ± 4.11 (11; 143.00 – 193.00) 211.56 ± 3.82 (9; 195.00 – 229.00) *** southern serbia 182.80 ± 6.38 (10; 148.00 – 208.00) 218.11 ± 8.52 (9; 170.00 – 247.00) ** ch limljani 77.38 ± 3.54 (8; 59.00 – 91.00) 86.67 ± 1.68 (21; 63.00 – 97.00) ** starčevo island 67.86 ± 3.28 (7; 57.00 – 79.00) 74.89 ± 4.23 (9; 56.00 – 89.00) ns eastern serbia 89.44 ± 2.06 (16; 64.00 – 97.00) 86.67 ± 1.68 (18; 57.00 – 120.00) ns central serbia 88.73 ± 2.31 (11; 75.00 – 98.00) 103.11 ± 2.23 (9; 93.00 – 113.00) ns southern serbia 96.90 ± 3.48 (10; 77.00 – 111.00) 108.56 ± 4.18 (9; 88.00 – 126.00) ns 258 katarina ljubisavljević et al. in males, there were also significant differences in scl between the samples (anova, scl: f4,57 = 10.34, p < 0.001), with the males from starčevo island (montenegro) showing significantly lower values than serbian samples (tukey hsd test, p < 0.01). in addition, cw and ch significantly differed between the samples when scl held constant (ancova, cw: f4,45 = 7.18, p < 0.001; ch: f4,45 = 3.09, p = 0.02). paired testing between the samples revealed significant differences in all comparisons for ch (tukey hsd test, p < 0.01), while cw significantly differed between samples from montenegro and serbia (tukey hsd test, p < 0.001 in all comparisons). for this variable, samples from serbia showed significantly larger values than samples from montenegro. qualitative characteristics there was no statistically significant variance among samples with respect to the condition of the supracaudal scute (g = 2.99, df = 4, p > 0.05). in all samples, a divided scute prevailed, although an undivided scute also occurred in a considerable frequency (mean: 23.6%; se: 7.5%; range: 14.3-33.3%). the extent of plastral black pigmentation did not significantly vary among the samples (g = 38.87, df = 40, p > 0.05). pairwise comparisons revealed a significant difference fig. 3. relative frequency (%) of black plastral pigmentation patterns in the five samples of t. hermanni. shading corresponds to plastral pigmentation codes given in fig. 2. 259morphological variability of testudo hermanni between the samples from starčevo island and southern serbia (g = 16.54, df = 8, p = 0.03) in that tortoises from the island had more dark pigmentation, and tortoises from southern serbia had lower and distinctly fragmented pigmentation. states 1, 2 and 3 were evident in 25% of specimens from starčevo island, but not in the sample from southern serbia; there were no states 9, 10 and 11 in the sample from the island, while in the sample from southern serbia they were present in approximately 32% of specimens. the highest heterogeneity was found in the sample from eastern serbia, where nine out of 11 states were present, while the lowest was in samples from starčevo island and southern serbia, with six states in each. the fragmentation and reduced pigmentation were most evident in the sample from southern serbia (fig. 3). discussion population-specific variation and phenotypic plasticity in testudo species are common (guyot and devaux, 1997; široký and fritz, 2007; fritz et al., 2009). accordingly, our results that the condition of the supracaudal scute previously considered as reliable diagnostic character for distinguishing t. hermanni (divided scute) and t. graeca (undivided scute) is variable, match this pattern. other authors also indicated the presence of undivided scute in t. hermanni populations (radovanović, 1941; meek and inskeep, 1981; cheylan, 2001), thus limiting its reliability for taxonomic purposes. however, body size distinction between serbian and montenegrin populations of t. hermanni boettgeri may reflect molecular differences within this taxon, corresponding with the parapatric ranges of two mitochondrial haplotypes (b13 for adriatic coast and b1 for inland balkan populations; fritz et al., 2006). the initial differentiation within t. h. boettgeri yet suggests limited gene flow, although the distribution gap corresponds to the zone of high mountains between serbia and montenegro, which represents major barriers in spreading of t. hermanni (fritz et al., 2006). an alternative but  not mutually exclusive hypothesis is that the variation in adult size of t. hermanni is adaptive, owing to variation in adult survival rates (e.g., larger the size and higher the survival) as suggested by willemsen and hailey (2001). in addition, the larger body size in serbian vs. montenegrin samples matches bergmann’s rule, as suggested previously for this species (willemsen and hailey, 1999a; sacchi et al., 2007) and other chelonians (ashton and feldman, 2003). however, although the size and mass of tortoises from serbia fit the latitudinal pattern presented in willemsen and hailey (1999a), the southern serbian population appeared to be larger than the more northern ones. although our survey was partly limited, due to a few samples of a modest size, our data could be in agreement with willemsen and hailey’s (1999a) suggestion that body dimensions may reach a maximum north of greece and then the energetic and thermoregulation may limit body size further north. the insular montenegrin population is characterized by small body size and mass and very dark plastral pigmentation. it appeared noticeably smaller than other mainland populations of the same subspecies (willemsen and hailey, 1999a; cheylan, 2001), but within the range of some adriatic populations (meek and inskeep, 1981; meek, 1985, 1989). the presence of small-sized tortoises along the adriatic coast could be explained by larger adult mortality correlated with higher environmental temperature causing the more 260 katarina ljubisavljević et al. frequent fires than in inland areas (willemsen and hailey, 1999a). besides, the starčevo population could also be the example of an “island dwarfism” prevalent generally in larger vertebrates (lomolino, 2005) as well as in turtles (georges, 1985; aponte et al., 2003; lomolino, 2005). this phenomenon is caused by limited resources and predatory release and is especially pronounced on smaller islands (lomolino, 2005). although the small islands of the skadar lake are of relatively recent, postglacial origin (stanković, 1976) it is known that genetic bottlenecks or environmental pressure can lead to substantial morphological changes in island tortoises over a short period of time (georges, 1985; aponte et al., 2003). on the other hand, there are many examples that such size differences in chelonians are not limited to island populations (e.g., fritz et al., 2010, 2012). considerable size differences may exist in different geographic regions within the same clade and could be the result of general phenotypic plasticity that appeared to play the major role in shaping external morphology of tortoises (fritz et al., 2005, 2007, 2010, 2012). great interand intrapopulation variability in plastral pigmentation has already been observed among populations of t. h. boettgeri (guyot and devaux, 1997; willemsen and hailey, 1999b). generally, isolation and/or climate may influence the origin and maintenance of high levels of melanism (bittner and king, 2003). increased dark plastral pigmentation in the southern populations of the hermann’s tortoise has been explained by thermal advantage in increasing heat loss to the substrate by infrared radiation during activity (willemsen and hailey, 1999b). in addition, darker pigmentation could be the result of genetic drift in small insular tortoise populations (georges, 1982). it seems that both factors can play a role in the small-bodied southern insular population in montenegro. acknowledgements we would like to acknowledge the constructive improvements suggested by two anonymous reviewers and marco mangiacotti. we thank miroslav marković for help in the field and ljiljana tomović for valuable suggestions. this study was supported financially by the serbian ministry of education and science, grant no 173043. references aponte, c., barreto, g., terborgh, j. 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(2002): body mass condition in greek tortoises: regional and interspecific variation. herpetol. j. 12: 105-114. acta herpetologica vol. 7, n. 2 december 2012 firenze university press advertisement call of species of the genus frostius cannatella 1986 (anura: bufonidae) flora a. juncá1, david l. röhr2, ricardo lourenço-de-moraes3, flávio j. m. santos1, airan s. protázio1, ednei a. mercês1, mirco solé4 amphibians in southern apennine: distribution, ecology and conservation notes in the “appennino lucano, val d’agri e lagonegrese” national park (southern italy). antonio romano1,*, remo bartolomei1, antonio luca conte1, egidio fulco2 the significance of using satellite imagery data only in ecological niche modelling of iberian herps neftalí sillero1, josé c. brito2, santiago martín-alfageme3, eduardo garcía-meléndez4, a.g. toxopeus5, andrew skidmore5 reproductive strategy of male and female eastern spiny lizards sceloporus spinosus (squamata: phrynosomatidae) from a region of the chihuahuan desert, méxico aurelio ramírez-bautista1,*, barry p. stephenson2, xóchitl hernández-ibarra1, uriel hernández-salinas1, raciel cruz-elizalde1, abraham lozano1, and geoffrey r. smith3 morphological variability of the hermann’s tortoise (testudo hermanni) in the central balkans katarina ljubisavljević1, georg džukić1, tanja d. vukov1, miloš l. kalezić1,2 the usefulness of mesocosms for ecotoxicity testing with lacertid lizards maria josé amaral1,2,*, rita c. bicho1, miguel a. carretero2, juan c. sanchez-hernandez3, augusto m. r. faustino4, amadeu m. v. m. soares1, reinier m. mann1,5 does acclimation at higher temperatures affect the locomotor performance of one of the southernmost reptiles in the world? jimena b. fernández*, nora r. ibargüengoytía advertisement call of scinax littoralis and s. angrensis (amphibia: anura: hylidae), with notes on the reproductive activity of s. littoralis michel v. garey1, thais r. n. costa2, andré m. x. de lima2, luís f. toledo3, marília t. hartmann4 book review: marine s. arakelyan, felix d. danielyan, claudia corti, roberto sindaco, alan e. leviton 2011. herpetofauna of armenia and nagorno-karabakh. society for the study of amphibians and reptiles stefano scali book review: rafaqat masroor 2012. a contribution to the herpetofauna of northern pakistan stefano scali evidence of high longevity in an island lacertid, teira dugesii (milne-edwards, 1829). first data on wild specimens. j. jesus first assessment of the endoparasitic nematode fauna of four psammophilous species of tropiduridae (squamata: iguania) endemic to north-eastern brazil markus lambertz1,*, tiana kohlsdorf2, steven f. perry1, robson waldemar ávila3, reinaldo josé da silva4 rediscovery and redescription of the holotype of lygosoma vittigerum (= lipinia vittigera) boulenger, 1894 yannick bucklitsch1, peter geissler1, timo hartmann1, giuliano doria2 , andré koch1,* reproductive phenology of the tomato frog, dyscophus antongili, in an urban pond of madagascar’s east coast ori segev1,*, franco andreone2, roberta pala2, giulia tessa2, miguel vences1 range extension of the critically endangered true poison-dart frog, phyllobates terribilis (anura: dendrobatidae), in western colombia roberto márquez1,*, germán corredor2, carlos galvis3 daniel góez2, & adolfo amézquita1 differences in habitat use of two sympatric species of ameiva in east costa rica esther sebastián-gonzález1, ramón gómez2 acta herpetologica journal of the societas herpetologica italica acta herpetologica 11(1): 63-68, 2016 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-17198 photo-identification in amphibian studies: a test of i3s pattern marco sannolo1,*, francesca gatti2, marco mangiacotti3,4, stefano scali3, roberto sacchi4 1 cibio, research centre in biodiversity and genetic resources, inbio, universidade do porto, campus agrário de vairão, vila do conde, portugal. *corresponding author. e-mail: marco.sannolo@gmail.com 2 via garibaldi, 20090 vimodrone, milano, italy 3 museo civico di storia naturale di milano, corso venezia, 55, 20121 milano, italy 4 dipartimento di scienze della terra e dell’ambiente, università di pavia, via taramelli 24, 27100, pavia, italy submitted on 2015, 21st october; revised on 2016, 8th march; accepted on 2016, 9th march editor: rocco tiberti abstract. photo-identification is used for individual recognition of several animal species. it gives the possibility to take photos of large species from a distance or to avoid invasive marking techniques in small animals. for amphibians, the use of non-invasive marking methods is even more relevant in the light of their global decline. here we use the photo-identification data from a population of triturus carnifex to validate the photo-identification software i3s pattern. this recently developed utility has never been applied to amphibians. the software proved to be efficient and accurate for individual recognition for this species. contrarily to the previous releases of the i3s family, i3s pattern is particularly suitable for amphibians characterized by a complex individual pattern of large blotches or irregular spots, which are not readily identified by eye. keywords. triturus carnifex, computer vision, capture-mark-recapture, photo-identification, surf algorithm, nonparametric manova. introduction individual recognition is important in ecological studies such as capture-mark-recapture designs (cmr). this kind of research is of primary importance because it provides data on variables of interest like abundance, density, habitat use and dispersion, among others (seber, 1973; pradel, 1996; beirinckx et al., 2006; mazerolle et al., 2007). cmr studies require the individual marking of captured animals, and rely on the assumption that the marking method unequivocally identifies each individual during the study period (williams et al., 2002; mazerolle et al., 2007). photo-identification (hereafter photo-id) is a wellestablished technique for individual recognition in cmr studies: it has been successfully applied to invertebrates (frisch and hobbs, 2007; caci et al., 2013), fish (speed et al., 2007; van tienhoven et al., 2007), amphibians (gamble et al., 2008; ribeiro and rebelo, 2011; zaffaroni caorsi et al., 2012; bendik et al., 2013; moya et al., 2015), reptiles (perera and perez-mellado, 2004; sacchi et al., 2010) and mammals (hammond et al., 1990; kelly, 2001). this approach is based on the identification of individually distinct and permanent features of a certain body region (e.g., patterns of colouration, scars), which are little variable over time or at least during the study period. photoid has long been performed through by-eye comparison of photos. in the case of large photo-id catalogues, this approach can be time-consuming and prone to recognition errors (arntzen et al., 2004) the steady development of digital cameras and computers allowed overcoming these issues, and in the past thirty years various software and algorithms had been developed and are now available (e.g., i3s, van tienhoven 64 marco sannolo et alii et al., 2007; wild-id, bolger et al., 2012; mantha matcher, town et al., 2013; mydas, carter et al., 2014; aphis, moya et al., 2015). nevertheless, these utilities were often designed ad hoc for one or few species to maximize their efficiency, therefore their application to other species requires preliminary validation (sacchi et al., 2016). using photo-id as a non-invasive marking technique is particularly recommended for amphibians since they are facing a worldwide decline (blaustein and wake, 1990; pounds et al., 2006) and many other marking techniques could be harmful (bloch and irschick, 2004; heemeyer et al., 2007; waddle et al., 2008) and ethically debated (may, 2004). however, many amphibian species possess complex skin patterns, often characterized by large blotches and irregular spots. on the one hand such skin properties allow the unique identification of each individual. on the other hand, if the study species is characterized by extremely complex pattern, error rate might increase sensibly. therefore, there is the need of software specifically developed to perform such task. in particular, we focused our attention on i3s pattern (http://www.reijns.com/i3s/), the latest release of the i3s family (van tienhoven et al., 2007) because of five main reasons: i) it has been specially designed for those species characterized by big and irregular spots or blotches, such as many amphibians species; ii) it is apparently robust to non-standard photographic settings (as in the case of many field works); iii) it requires little image pre-processing procedure (saving operator time); iv) being freeware, it could be addressed to a broad audience among herpetologists; v) it has not yet been validated on amphibians. i3s pattern employs the algorithm surf (speededup robust features) developed in the field of computer vision (bay et al., 2007). this free utility allows a fast comparison of the image of the individual to be identified with all the previously stored images and ranks the images from the best to the worst match. to do this, it requires the user to process each image only once by a two steps protocol where: i) setting three homologous “reference points” that allow translating all images in the same two-dimensional reference system (see also sacchi et al., 2010 for further details); ii) defyining a rectangular area from which the software will automatically search and extract the “interest points” on which a unique profile of the image is built (bay et al., 2007). the chosen area should obviously refer to the same body region, but it has not necessarily be perfectly replicated in each image. the two steps require only few seconds, and a “.fgp” file containing the pattern (i.e., position of reference points and interests points) is created and stored. when a new image is processed and its pattern extracted, the software compares it with the database and computes the dissimilarity score between the new and the stored patterns (see software manual for computational details; http://www.reijns.com/i3s/download/i3s pattern. pdf ). at the end of the comparison process, the software shows side by side the new image and all possible matches ranked by increased dissimilarity score. all proposed matches can be visually inspected to assess if the match is correct. we used the photo-id data from an ongoing longterm study on the italian crested newt triturus carnifex to test the performance of i3s pattern. t. carnifex is a good model species to test the software because adult newts show a ventral pattern of irregular spots and large blotches that is unique for each individual and stable over time (arntzen and wallis, 1999). therefore, the aims of this research are to assess i) if i3s pattern successfully recognizes triturus carnifex individuals, and ii) its potential application to cmr herpetological studies. material and methods study species and sampling sessions we sampled 324 t. carnifex in the groane regional park (lombardy, northern italy, 45°38’n 9°6’e), in a natural pond located in a typical moor area (gatti and sannolo, 2014). newts were caught using funnel traps during nine sampling sessions every week between march and june 2014. we measured the snout-to-vent-length (svl) and the tail length (tl) of each newt and we noted their sex (table 1). we took photos of the ventral pattern of each newt in standard conditions: each newt was put on graph paper at 20 cm from the lens (using a nikon d-90 with an 18-55 mm lens) while keeping its body as straight as possible; each newt was kept wet, and we conducted the whole operation with wet hands and as fast as possible, in order to avoid the injuries and minimize any stress. to simulate a recapture, after one hour we took a second photo of each individual using the same procedure. between the two photo sessions, newts were kept in individual 1l plastic box filled with water. following measurements and photo identification, newts were released at the exact point of capture. two authors (ms and fg) identified by eye every individual captured during all field sessions, checking for recaptures and providing a doublechecked reference against which the software performances were evaluated (arntzen et al., 2004; bendik et al., 2013). all images were then processed using i3s pattern. statistical validation of the software we first evaluated the effect of the two primary sources of error in which the operator could incur, following sacchi et al. (2010): the error in selecting reference points and research area (drep1), and the prospective error due to non-perfect alignment and positioning of the newt under the camera (drep2). 65photo-identification in amphibian studies drep1 is, therefore, the dissimilarity score between the same image processed two times, while drep2 is the dissimilarity between two images of the same individual taken in one session of sampling (i.e., a simulated recapture). we also calculated a third measure, dpop, that is the mean dissimilarity score of each newt with all other individuals. if the software is able to discriminate consistently among individuals, drep2 should be lower than dpop. we also expected drep1 being always greater than zero and drep2 always greater than drep1 since it combines the errors of digitalization with the variability due to both newt positioning under the camera and photo quality (e.g., light conditions). the software allows adjusting nine additional parameters for a fine tuning of the algorithm. changing the number of interest points sampled in each photo might be the single parameter that affect more heavily the calculated distances among individuals. thus, we focused on the effect of the number of interest points used to calculate the dissimilarity score. the default number is 35 and has been optimized on sea turtles. therefore, we resampled the database also at 25, 30, 40, 45 interest points. to assess if the number of interest points might affect the dissimilarity score, we applied a non-parametric distance-based manova (anderson, 2001; mcardle and anderson, 2001) using the three scores (drep1, drep2 and dpop) as dependent variables and the number of interest points, the sex, and their interaction as the independents. for this test, we used 30 adult newts, 15 males, and 15 females. the np-manova was performed using the function “adonis” of the “vegan” r package (oksanen et al., 2013) in r ver. 3.1.0 (r core team, 2014). the p-values were assessed by setting the number of permutations to 999, and we tested the assumption of homogeneity of dispersions among distance (anderson, 2001; warton et al., 2012) using the functions “betadisper” and “permutest” (anderson, 2006; borcard et al., 2011) of the “vegan” r package. field validation of the software the ability of the software to identify correctly each individual in the context of a cmr study was then assessed using the newts’ images obtained during the nine session of field capture-mark-recapture. we processed 852 photos relative to 324 adults of t. carnifex sampled in the field between march and may 2014 (table 2). among them, 55 individuals were captured at least twice (34 males and 21 females). for these individuals, we randomly selected one photo and searched in the whole database for the matching. we noted if the software correctly matched the individual, the assigned rank, and the dissimilarity score between the sample image and the first correct match in the database. this score is equivalent to drep2 calculated in the previous test. while the false positive rate (far) is usually very low and easy to control using a countercheck method (byeye comparison), the false rejection rate (frr) is a major issue with photo-identification (bolger et al., 2012). frr has therefore been calculated as the ratio between the number of correct matches on the number of total attempts. we accepted a match as correct if it was at least one of the first 10 listed images. results descriptive statistics for the calculated distances are reported in table 1. the np-manova detected a significant main effect of both sex (p < 0.001) and interest points on distance (p < 0.001), but not of the interaction term (p = 0.64). sex has a significant effect on drep2 (kruskal-wallis test, kw = 13.5, p < 0.001, df = 1) and on dpop (kw = 4.4, p < 0.001, df = 1); female dissimilarity scores were always greater than male ones (table 1). the effect of interest points was significant only at dpop level (kw = 122, p < 0.001, df = 4). increasing the number of interest points, the difference between drep2 and dpop clearly decreased reaching its minimum at 45 interest points, but it was always highly significant (ks test at 45 interest points = 1, p < 0.001, one-tailed, fig. 1). the three scores were clearly separated (fig. 1). in particular, dpop was always greater than drep2, which in turn is greater than drep1. this result means that the table 1. mean dissimilarity scores ± standard deviation in relation to sex and number of interest points. note that females’ scores are always larger than males’ ones. no. of points 25 30 35 40 45 drep1 males 0.85 ± 0.44 0.78 ± 0.46 0.72 ± 0.52 0.72 ± 0.46 0.79 ± 0.41 females 0.95 ± 0.76 0.94 ± 0.70 0.77 ± 0.44 0.85 ± 0.52 0.83 ± 0.43 drep2 males 9.73 ± 5.51 8.24 ± 3.50 7.83 ± 3.85 7.99 ± 4.19 7.39 ± 3.54 females 13.4 ± 5.47 11.8 ± 4.97 10.7 ± 4.85 9.88 ± 3.86 8.91 ± 3.17 dpop males 48.8 ± 5.97 36.4 ± 3.74 30.3 ± 3.27 25.3 ± 3.06 22.2 ± 2.62 females 57.6 ± 9.65 42.1 ± 6.34 35.2 ± 5.61 27.9 ± 3.73 23.9 ± 2.56 table 2. mean ± standard deviation snout-to-vent-length (svl) and tail length of triturus carnifex divided by sex and age. sex n svl tl males 195 6.56 ± 0.38 4.69 ± 0.28 females 129 6.89 ± 0.39 5.51 ± 0.37 juveniles 12 4.64 ± 0.36 3.73 ± 0.35 66 marco sannolo et alii software can correctly distinguish among different newts. both drep1 and drep2 decreased across groups (table 1), but the difference was not significant. dpop showed an exponential decrease for the mean values. the sex had a significant effect in the model, while the interaction term sex×no. of points was not significant, which means either that the sex effect was constant across groups or that the test lacked enough statistical power. in table 1 it is shown that female means and variances were always larger than male ones. when the i3s was applied in the context of a cmr study, it was able to correctly match all 55 recaptured newts out of 324 individuals during the nine sampling sessions. in 42 cases the software proposed the correct match as the first one in the list, while for the other 13 cases, the correct match was between the second and the fifth position in the list. the frr of the i3s was 0.24, but it rapidly dropped to 0 after the first five matches were visually inspected. the mean distance between the tested image and the first correct match (equivalent to drep2), was 9.55 ± 3.17 sd. discussion according to our results, i3s pattern performs reliable images classification and can be applied to photo-id of t. carnifex. as expected from a good classifier, dpop was greater than drep2, and this latter greater than drep1. this outcome means that a greater dissimilarity was calculated when comparing the images of two different individuals with respect to images representing the same newt (table 1, fig. 1). the ability of the software to assign a smaller score to the pair of images representing the same newt should translate into a correctly proposed match as output. indeed, we found that i3s pattern always suggested the correct match as one of the first five candidates, and most often as the first one. moreover, this software proved to be robust against false negatives. in fact, one of the central issues using a new software or algorithm is that the rate of misidentification is unknown, especially the false negative rate (sacchi et al., 2010, 2016), which could systematically bias the estimate of demographic parameters (davis and ovaska, 2001; mccarthy et al., 2009). our estimation of frr, instead, varied from 0 to 0.24 depending on how restrictive was the selection of the false negative. these values are similar to those estimated by other studies (bolger et al., 2012). a second outcome is that the number of interest points had a significant effect on the dissimilarity score: increasing the number of interest points from the default setting of 35 to 45 did not lead to an increase in performance. therefore, at least for this species, 35 interest points covered the vast majority of inter-individual variation, managing to tell apart different newts. apparently, increasing the number of interest points led to sample newt belly in uninformative areas, making more similar two images that represented different individuals. for example, fig. 2 (on the right newt) summarizes as reflected light may be recognized as a coloration pattern by the software and sampled as interest points. on the contrary, reducing the interest points led to increasing the distance between drep2 and dpop. so, for this species, and maybe for other similar ones (sharing the same pattern), it is possible that reducing the number of interest points could improve the performance of i3s pattern, leading to the sampling of highly informative areas only. sex had a significant effect in our analysis, and both the mean and the variance were greater for females than for males (tab. 1). this result might be due to phenotypic differences in male and female bellies. indeed, in our population the female pattern is less contrasted, and the blotches are often less sharp in comparison with male ones. if this difference is consistent among the sexes, as we believe, it is possible that i3s pattern repeatedly calculated a greater dissimilarity scores for female. in the light of the results of the present study, we are confident that i3s pattern and its underlined algorithm surf could be successfully applied not only to species characterized by a regular and clearly distinctive pattern, but also to species keeping highly complex and irregular patterns. this is the case for many species of bufonidae, fig. 1. dissimilarity scores compared across groups (no. of interest points). note that drep1 and drep2 are stable across groups while both mean and variance of dpop tend to decrease with increasing number of interest points. 67photo-identification in amphibian studies ranidae, salamandridae and ambystomatidae, which exhibit various cryptic or aposematic patterns that are not easily matched by eye (vitt and caldwell, 2013). acknowledgements the present study received no financial support from any funding agency in the public, commercial or not-forprofit sectors. the research was conducted with respect to the current italian laws in relation to amphibians capture, marking and detention (aut.min.conc. prot. 0008880/ pnm). the authors would thank gariboldi l., wayne, b., and the “elba emperor”, zuffi m.a.l., for their help and advice during the research period. two anonymous reviewers provided valuable comments that improve an earlier version of the manuscript. references anderson, m.j. 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(2012): clip or snap? an evaluation of toe-clipping and photo-identification methods for identifying individual southern red-bellied toads, melanophryniscus cambaraensis. sajh 7: 79-84. acta herpetologica vol. 11, n. 1 june 2016 firenze university press feeding habits of mesoclemmys vanderhaegei (testudines: chelidae) elizângela silva brito1,*, franco leandro souza2, christine strüssmann3 morphology, ecology, and behaviour of hylarana intermedia, a western ghats frog ambika kamath1, rachakonda sreekar2,3 a new account for the endangered cerrado rocket frog allobates goianus (bokermann, 1975) (anura: aromobatidae), with comments on taxonomy and conservation thiago ribeiro de carvalho1,2,*, lucas borges martins1,2, ariovaldo antonio giaretta1 molecular phylogenetics of the pristimantis lacrimosus species group (anura: craugastoridae) with the description of a new species from colombia mauricio rivera-correa, juan m. daza* population structure and activity pattern of one species of adenomera steindachner, 1867 (anura: leptodactylidae) in northeastern brazil maria juliana borges-leite1,*, joão fabrício mota rodrigues2, patrícia de menezes gondim1, diva maria borges-nojosa1 vocal repertoire of scinax v-signatus (lutz 1968) (anura, hylidae) and comments on bioacoustical synapomorphies for scinax perpusillus species group marco antônio peixoto1,*, carla silva guimarães1, joão victor a. lacerda2, fernando leal2, pedro c. rocha2, renato neves feio1 amendment of the type locality of the endemic sicilian pond turtle emys trinacris fritz et al. 2005, with some notes on the highest altitude reached by the species (testudines, emydidae) federico marrone*, francesco sacco, vincenzo arizza, marco arculeo photo-identification in amphibian studies: a test of i3s pattern marco sannolo1,*, francesca gatti2, marco mangiacotti3,4, stefano scali3, roberto sacchi4 no evidence for the ‘expensive-tissue hypothesis’ in the dark-spotted frog, pelophylax nigromaculatus li zhao, min mao, wen bo liao* no short term effect of clinostomum complanatum (trematoda: digenea: clinostomatidae) on survival of triturus carnifex (amphibia: urodela: salamandridae) giacomo bruni1,*, claudio angelini2 yeasts in amphibians are common: isolation and the first molecular characterization from thailand srisupaph poonlaphdecha, alexis ribas* introduction of eleutherodactylus planirostris (amphibia, anura, eleutherodactylidae) to hong kong wing ho lee1, michael wai-neng lau2, anthony lau3, ding-qi rao4, yik-hei sung5,* correlation between endoscopic sex determination and gonad histology in pond sliders, trachemys scripta (reptilia: testudines: emydidae) david perpiñán1, albert martínez-silvestre2,3, ferran bargalló3, marco di giuseppe4, jorge orós5, taiana costa6,* book review: john w. wilkinson. amphibian survey and monitoring handbook. pelagic publishing franco andreone book review: susan newman. frogs amphibians and their threatened environment. discovery and expression through art k-3. frogs are green franco andreone acta herpetologica 10(1): 17-21, 2015 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-14594 reproductive ecology of sichuan digging frogs (microhylidae: kaloula rugifera) wei chen1,*, lina ren2, dujuan he2, ying wang2, david pike3 1 ecological security and protection key laboratory of sichuan province, mianyang normal university, mianyang, 621000, china. *corresponding author. e-mail: wchen1949@163.com 2 college of life science and biotechnology, mianyang normal university, mianyang, 621000, china 3 school of marine and tropical biology and centre for tropical environmental and sustainability science, james cook university, townsville, queensland, 4811, australia submitted on 2014, 3rd june; revised on 2014, 24th december; accepted on 2014, 27th december editor: rocco tiberti abstract. we investigated the reproductive ecology of sichuan digging frog (microhylidae: kaloula rugifera) in mianyang, china during the wet season (from may to septemper) of 2013. male k. rugifera first appeared at temporary ponds following the first heavy rain of the wet season and initiated calling. male frogs formed choruses throughout the wet season during the evenings and nights after rainstorms. females arrived at ponds shortly after males start calling and leaved the pond once they lay egg masses. amplexus lasted up to three hours. females were larger than males in terms of body size, but we found no evidence of size-assortative mating. clutch size varied from 920 to 2200 eggs, with egg diameter ranging from 1.33 to 1.93  mm. larger females laid more eggs, and there was no correlation between egg number and egg size. embryos hatched from eggs within 18-20 hours of oviposition, and grew for 25-40 days before complete metamorphosis occurred. growth was fastest immediately after hatching, and declined asymptotically with increasing tadpole body size. overall, k. rugifera have a breeding biology characterized by strong malemale competition with prolonged breeding coinciding with the annual wet season. keywords. breeding ecology, kaloula rugifera, life history, mating system. microhylidae günther, 1858 (fei et al., 2009; frost, 2013) is a geographically widespread family of frogs, that includes 426 species (fei et al., 2009), five (mo et al., 2013) of which occur in china (kaloula borealis; k. nonggangensis; k. pulchra; k. rugifera; and k. verrucosa). the reproductive ecology of chinese microhylidae is largely unknown and only the reproductive ecology of k. verrucosa has been reported (fei et al., 2009). in the present study, some aspects of the breeding ecology of sichuan digging frogs (microhylidae: k. rugifera) are described, providing basic information about the breeding season, reproductive behavior, duration of the larval period, and tradeoffs between egg number/size and female size. kaloula rugifera is distributed in the northeastern sichuan and southernmost gansu provinces in southwestern china (fei et al., 2009; fei and xie, 2004). this small species (adults snout to vent length range from 35-55 mm; fei et al., 2009) is often found in hilly areas near villages, and has been recorded sheltering in treeholes. this species breeds in rain-filled, temporary pools and small ponds (fei and ye, 1983; fei and xie, 2004). fieldwork was conducted at a temporary pond (104°46˝e and 31°29˝n; 459 m a.s.l. elevation) in mianyang, sichuan, china from may to september 2013. this area is characterized by annual average temperature of 16.3°c, and total annual precipitation averages of 865.5 mm (fig.  1). during the field season, the max-min range of temperature was 21.4-25.7°c. the temporary pond was located in dense woodland situated on mianyang normal university campus. the pond had an area of 7 m2, a max18 w. chen et alii imum seasonal depth of 10 cm and some wilted leaves in the water. the study site was visited after sudden rainstorm during all the breeding season. during the breeding season (may-september), we hand-collected 36 frogs (14 pairs found in amplexus, and four males and four females not in amplexus). we measured the snout-vent length (svl, to the nearest 0.1 cm) of all the collected frogs (fig.  1) and we transported each pairs to our laboratory (50 m away) using buckets containing pond water. after laying eggs, we determined the number of eggs per clutch, and photographed the eggs of eight clutches using a canon camera with macro lens (eos 550) with a scale ruler in order to measure egg size (chen et al., 2013a). we quantified egg size by measuring 10-30 eggs per clutch from digital photos, and averaged the minimum and maximum diameter of each egg (to the nearest 0.01 mm) to obtain a measure of egg size (chen et al. 2013b). we haphazardly selected two egg clutches from females with different size to study time to reach hatching and tadpole growth rates, and released all other egg clutches and frogs back into the study pond after data collection. the first was collected in late may, while the second in late june. tadpoles were reared in groups of 20-30 individuals in plastic buckets (10 liter/capacity) containing a 2 cm substrate of mud and 10 cm of water, both collected from our study pond. the buckets were maintained at room temperature in our laboratory. we fed tadpoles with 2-2.5 g of crushed goldfish feed daily (shangdong dongpinghu feed co., ltd, china: 32% crude protein, 5% crude fat, 3% crude fibre, 10% moisture, and 12% crude ash). a single investigator measured the tadpoles’ body size (svl) at 9:00-10:00 am of every day, until metamorphosis occurred. body size measures were used to estimate the tadpole growth rates using von bertalanffy’s (1957) equation. the equation takes the form st = smax (1 – e–kt+b), where st is body size at age t (time, in days), smax is the estimated asymptotic maximum body size that tadpoles can reach before metamorphosis occurred, k is a growth coefficient, and b is a constant. then, growth rate can be calculated as r  =  ds/ dt = k (smax – st). due to the small sample size, we used non parametric spearman’s correlation to explore the relationship between female body size and reproductive output (egg number and size), as well as between clutch size and egg size. we used spss software (version 16.0. chicago, spss inc) for statistical analysis, all statistics are two-tailed and summary statistics are presented as means  ±  standard deviation (sd). k. rugifera initiated breeding just after the first sudden rainstorm at the outset of the annual rainy season (25 may 2013; the rainy season lasted from late may-early september; fig.  1), when the rain filled the study pond. breeding continued throughout most of the rainy season due to sudden rainstorm, but became less frequent in september (we found 13 pairs in may-august, but just one pair in september), when temperatures became cooler and rainfall less frequent (fig. 1). these changes in weather are associated with gradual drying of temporary ponds (5-8 cm in depth) and, thus, with the end of the reproductive season of k. rugifera. in the evening following the first major rainstorm of the season, male frogs arrived at the temporary pool and began to chorus strongly. we observed females arriving at the pool two to three hours after males began advertising, then females engaged in amplexus within the first minutes of arriving at ponds (5.6 ± 2.3 min, n = 8; range: 3-9  min, fig.  2b). females generally laid eggs within three hours of initiating amplexus (116  ±  42 mins, n  =  6, range: 60-180 mins). after fertilization occurred, the pairs separated and females left the breeding pond after mating. males remained at the pond and called unless there were no females at the breeding site. adult males were significantly smaller than adult females in terms of body size (z = -5.004, p < 0.001;       te m pe ra tu re  ( °c )   r ai nf al l  ( m m )   month   fig. 1. monthly mean air temperatures and mean rainfall during the reproductive season of sichuan digging frogs (kaloula rugifera) at a seasonal pool at mianyang normal university campus (mianyang, sichuan, china). 19reproductive ecology of sichuan digging frogs males svl = 3.77  ±  0.23 cm; range: 3.3-4.3 cm, n = 19; females svl= 4.57  ±  0.30 cm; range: 4.1-5.0 cm, n  =  18; fig.  2a). despite the wide range of body sizes of both males and females, the body lengths of males and females found in amplexus were not significantly correlated (rs  =  0.157, p = 0.592, n = 14; fig.  3). thus, no evidence of size-assortative mating (a significant positive relationship between male and female body lengths of pairs in amplexus) was detected, as both large and small males successfully amplexed and mated with both large and small females. the number of eggs per egg clutch ranged from 9202200 eggs (1430  ±  456 eggs, n = 18 clutches), and egg diameter ranged from 1.33-1.93 mm (1.62  ±  0.12 mm, n = 355 eggs from eight clutches). we found significant positive correlations between female body size (svl) and clutch size (rs = 0.772, p < 0.001, n = 18; fig. 4a), however, we did not find a significant correlation between female body size and egg size (rs = 0.429, p = 0.289, n  =  8; fig.  4b) as well as between the number of eggs per clutch and egg size (rs = 0.5, p = 0.207, n = 8; fig 4c). eggs developed and hatched within 18-20 hours, tadpoles grew for 25-40 days before complete metamorphosis occurred. the initial body length of tadpoles ranged from 2-3 mm (fig. 5a). the individual growth rate of tadpoles was fastest immediately after hatching, and declined asymptotically with increasing body size (figs.  5a, b).the tadpoles hatching from eggs laid during late june had a larger initial body size and grew faster than tadpoles hatching on late may (kmay = 0.035; kjune = 0.046, fig. 5). k. rugifera breed along the entire rainy season, which lasts approximately four months, making this species a prolonged breeder (wells, 1977). breeding was initiated in late may, after the first heavy rainfall of the season, suggesting that the species’ reproductive phenology is driven by precipitation and ambient temperature (fei and ye, 1983), similar to con-generic species k. verrucosa (verrucous digging frog he et al., 2006). breeding aggregations at a single pond are relatively small in both species (18-20 males; he et al. 2006) similarly to k. verrucosa, also k. rugifera lay free-floating eggs, probably as a reproductive strategy to speed embryo development by exposing the entire egg surface to the high water temperatures (warkentin et al., 2005). larger and older female amphibians with larger abdominal cavities are often able to allocate more energy to reproduction (wells, 2007) by laying more eggs than smaller and younger conspecifics (dziminski and alford, 2005; chen et al., 2011). many amphibian females invest a lot of energy into body growth, enabling the incubation of a larger number of eggs and thus increasing fecundity (czarnoleski and kozlowski, 1998). in our study, larger  fig. 2. sichuan digging frogs (kaloula rugifera): a male (a) and a couple in amplexus (b) being approached by another male (top). photographs by wei chen.     m al es  ( cm )   females   (cm)   fig. 3. body size relationship of amplexing males and females (svl measurements) of sichuan digging frog (kaloula rugifera; n = 14). 20 w. chen et alii female k. rugiferalaid eggs with identical size in comparison with the little ones. two possible explanations for this pattern are that: 1) larger females can invest more energy into egg production, which results into larger clutch size (duellman and trueb, 1986; roff, 1992); and/ or 2) the females in this population are below maximum physical constraint and egg size is mainly constrained by proximate factors prior to breeding, such as energy availability during vitellogenesis (kaplan and salthe, 1979). many amphibians have tradeoff between clutch size and egg size (wells. 2007), but k. rugifera shows no correlations between egg number and size. this can be explained by the fact that limited physical clutch holding capacity cannot increase simultaneously the clutch size and egg size (jorgensen, 1981), which leading to the fact that females do not trade off clutch size with egg size. in our study, tadpoles finished metamorphosis within 25-40 days, much slower than tadpoles from chengdu, which matured with 20-23 days (fei and ye, 1983). these differences on developmental time may be due to the dependence of tadpole growth on temperature or density (chen et al. 2013c). von bertalanffy’s (1957) equation provided a realistic simulation of tadpole growth during development and showed that tadpoles raised during late june show faster growth rates than those individuals raised during late may. in late june, the faster growth strategy of tadpoles may be adaptive to the fast dry-offs of the ponds. this still need further study to understand the cause of differentiated development rates. acknowledgements financial support was provided by the scientific research foundation of mianyang normal university fig. 5. growth curve (a) and growth rate (b) of sichuan digging frog (kaloula rugifera) tadpoles hatching in late may (lm, solid lines) and late june (lj, dotted lines).         cl ut ch  s iz e     eg g   si ze  ( m m )   eg g   si ze  ( m m )   body  size   (cm)   body  size   (cm)   clutch  size   a   b   c   fig. 4. relationship between (a) female body size and clutch size (n = 18,); (b) female body size and egg size (n = 8), and (c) reproductive parameters such as clutch size (egg number) and egg size (n = 8) in sichuan digging frogs (kaloula rugifera).       b od y   le ng th  ( m m )   g ro w th  r at e   days   a   b   lj   lm   lm   lj   21reproductive ecology of sichuan digging frogs (no. qd2012a13) and the foundation of sichuan provincial department of education (no.  11zb138; 15za0298). all field and laboratory work was performed under licenses from the wildlife protection law of china and capture permits was granted by sichuan forestry bureau. references berven, k.a. (1988): factors affecting variation in reproductive traits within a population of wood frogs (rana sylvatica). copeia 1988: 605-615. chen, w., tang, z.h., fan, x.g., wang, y., pike, d.a. (2013a): maternal investment increases with altitude in a frog on the tibetan plateau. j. evol. biol. 12: 2710-2715. chen, w., zhao, l., wang, y., li, h., he, d.j., ren, l.n., tang, z.h., lu, x. (2013b): reproductive output of the brown frog rana kukunoris at high altitude of the tibetan plateau. acta herpetol. 8: 153-157. chen, w., you, z.q., liu, h., tang, z.h., guan, t.p., du, s.z. (2013c): summary of researches on life history of amphibians. j. mianyang norm. univ. 32: 44-52. chen, w., yu, t.l., lu, x. (2011): age and body size of rana kukunoris, a high-elevation frog native to the tibetan plateau. herpetol. j. 21: 149-151. czarnoleski, m., kozlowski, j. (1998): do bertalanffy’s growth curves result from optimal resource allocation? ecol. lett. 1: 5-7. duellman, w.e., trueb, l. (1986): biology of amphibians. mcgraw-hill inc, new york. dziminski, m.a., alford, r.a. (2005): patterns and fitness consequences of intraclutch variation in egg provisioning in tropical australian frogs. oecologia 146: 98-109. fei, l., hu, s.q., ye, c.y., huang, y.z. (2009): fauna sinica. amphibia. volume 2. anura. science press, beijing. fei, l., xie, f. (2004): kaloula rugifera. in: iucn 2013. iucn red list of threatened species. fei, l., ye, c. (1983): taxonomic study on hynobiidae including a new genus pseudohynobius (amphibia: caudata). acta herpetol. sinica 2: 31-37. frost, d.r. (2013): amphibian species of the world: an online reference. version 5.6 (9 january 2013). american museum of natural history, new york. jørgensen, c.b. (1981): ovarian cycle in a temperate zone frog, rana temporaria, with special reference to factors determining number and size of eggs. j. zool. 195: 449-458. kaplan, r.h., salthe, s.n. (1979): the allometry of reproduction: an empirical view in salamanders. am. nat. 113: 671-689. mo, y.m., zhang, w., zhou, s.c., chen, t.b., tang, h.x., meng, y.j., chen, w.c. (2013): a new species of kaloula (amphibia: anura: microhylidae) from southern guangxi, china. zootaxa 3710: 165-178. roff, d.a. (1992): the evolution of life histories. chapman and hall, new york. von bertalanffy, l. (1957): quantification laws in metabolism and growth. q. rev. biol. 32: 217-231. warkentin, k.m., gomes-mestre, i., mcdaniel, j.g. (2005): development, surface exposure, and embryo behavior affect oxygen levels in eggs of the red-eyed tree frog, agalychnis callidryas. physiol. biochem. zool. 78: 956-966. wells, k.d. (1977): the social behaviour of anuran amphibians. anim. behav. 25: 666-693. wells, k.d. (2007): the ecology and behavior of amphibians. the university of chicago press, chicago and london. acta herpetologica vol. 10, n. 1 june 2015 firenze university press obituary: valery k. eremchenko (1949-2014) leo j. borkin1, tatjana dujsebayeva2, roberto sindaco3, matthias stöck4 haplotype variation in founders of the mauremys annamensis population kept in european zoos barbora somerová1, ivan rehák2,*, petr velenský2, klára palupčíková1, tomáš protiva1, daniel frynta1 reproductive ecology of sichuan digging frogs (microhylidae: kaloula rugifera) wei chen1,*, lina ren2, dujuan he2, ying wang2, david pike3 toxic effects of carbaryl on the histology of testes of bufotes variabilis (anura: bufonidae) özlem çakici basal frequency of micronuclei and hematological parameters in the side-necked turtle, phrynops hilarii (duméril & bibron, 1835) maría a. latorre 1,2,*,#; evelyn c. lópez gonzález1,2, #; pablo a. siroski1,2,3; gisela l. poletta1,2,4 into a box interiors: clutch size variation and resource allocation in the european pond turtle marco. a.l. zuffi1,*, simonetta citi2, elena foschi1, francesca marsiglia1, eva martelli1 where to “rock”? choice of retreat sites by a gecko in a semi-arid habitat andreia penado1,2, ricardo rocha3,4,*, marta sampaio3, vanessa gil3, bruno m. carreira3, rui rebelo3 age structure, growth and longevity in the common toad, rhinella arenarum, from argentina clarisa de l. bionda1,2,*, silvia kost 4, nancy e. salas1, rafael c. lajmanovich3, ulrich sinsch4, adolfo l. martino1 on a putative type specimen of pleurodema bibroni tschudi, 1838 from chile (anura: leptodactylidae) daiana paola ferraro re-description of the external morphology of phyllomedusa iheringii boulenger, 1885 larvae (anura: hylidae), with comments on the external morphology of tadpoles of the p. burmeisteri group samanta iop¹, victor mendes lipinski¹, bruno madalozzo¹, franciele pereira maragno¹, sonia zanini cechin¹, tiago gomes dos santos² book review: harold heatwole, john w. wilkinson (eds). amphibians biology. volume 11 status of conservation and decline of amphibians. eastern hemisphere. part 4 . southern europe and turkey sebastiano salvidio book review: antonio romano. atlante degli anfibi del parco nazionale del cilento vallo di diano e alburni distribuzione, biologia, ecologia e conservazione sebastiano salvidio acta herpetologica journal of the societas herpetologica italica © firenze university press www.fupress.com/ah acta herpetologica 5(2): 217-222, 2010 impact of otter (lutra lutra) predation on amphibians in temporary ponds in southern spain dan cogălniceanu1, rafael márquez2, juan francisco beltrán3 1 university ovidius constanţa, faculty of natural sciences, aleea universităţii 1, corp b, r-900470, constanţa, romania. corresponding author. e-mail: dcogalniceanu@univ-ovidius.ro; dan_cogalniceanu @yahoo.com 2 fonoteca zoológica, dept. de biodiversidad y biología evolutiva. museo nacional de ciencias naturales (csic). josé gutierrez abascal 2, e-28006, madrid, spain. 3 departamento de fisiología y zoología, facultad de biología, universidad de sevilla. avda. reina mercedes 6, e-41012, sevilla, spain. submitted on: 2009, 21th october; revised on: 2010, 27th, august; accepted on: 2010, 22th october. abstract. we report the observation of an event of mortality of ribbed newts (pleurodeles waltl) and iberian spadefoot toads (pelobates cultripes) due to predation by a pair of otters (lutra lutra) in a temporary pond complex in southern spain. the peculiar predation mode on ribbed newts, with extraction of soft organs through an incision in the upper part of the thorax, may result in an underestimation of the importance of this species in the diet of otters. the high number of dead amphibians killed by two otters in only several hours suggests that the presence of these predators may pose a serious threat to amphibian populations. the risk is especially high in arid areas, with few ponds, synchronous reproductive migration, and high density of animals. we consider that measures promoting the conservation and population and range increase of otters may have a negative impact on amphibians. keywords. amphibians, pleurodeles waltl, pelobates cultripes, otter, predation, conflicting conservation goals. the goal of conservation is the persistence of biodiversity (williams and araujo, 2000), so current methods of planning include multiple species and landscape features. most conservation priorities and initiatives are focused on endangered and threatened species. these often result in specific action plans aimed at maintaining and increasing the effectives, density or area covered by the target species. negative secondary effects occur when the target species is a major predator or competitor of other endangered or threatened species. this is a major challenge for conservation, posing conflicting goals and requiring serious prioritization. until now, most reports were focused on the conflicts between humans and wildlife (drechsler, 2004; maikhuri et al., 2001; marrs et al., 2007). 218 d. cogălniceanu, r. márquez and j.f. beltrán we report an event of massive predation of ribbed newts (pleurodeles waltl) and iberian spadefoot toads (pelobates cultripes) by a pair of otters (lutra lutra) in a temporary pond complex in southern spain. the ponds are located in the south of spain, in the parque natural de la sierra norte de sevilla, navas del berrocal, almadén de la plata, sevilla (n 37º47’29.0” w 06º04’30.0”, elevation 439 m a.s.l.). the study area is of restricted access, and part of the west sierra morena environment, with hot, dry summers (fernández-alés, 1979). for the interval 1991-2001, mean annual temperature was 16.8 °c, and mean annual rainfall 768.4 mm (junta de andalucía, 2002). the landscape has a gentle slope, surrounded by scattered oaks (quercus ilex) and cork-oaks (quercus suber), and interspersed with shrubs of cistus, lavandula, rosmarinus, retama and pistacia lentiscus. the major pond is approximately 20 m in diameter and is part of a group of four ponds of similar size; two of them reaching depths of 40-60 cm and the other two being shallower 20-30 cm. these ponds typically fill with water with the fall rains and dry up along the spring, although in rainy years water can be present throughout the year. in addition to p. waltl and p. cultripes, the ponds are used for reproduction by pelodytes ibericus and bufo calamita and less than 100 m away is a temporary stream inhabited by a breeding population of alytes cisternasii which has been studied over several years (penna et al., 2006; márquez et al., 2007, 2008; tejedo et al., 2008). the autumn rains started in 2006 on october 18, after an extremely dry summer. this triggered a massive reproductive migration of amphibians towards the ponds. on the night of october 19, 2006, we found partially consumed bodies of 24 ribbed newts (pleurodeles waltl) and rests of more than five adult iberian spadefoot toads (pelobates cultripes). two additional bodies of ribbed newts were collected during daytime the day after. the predators, two otters (lutra lutra) were identified initially by one fresh scat found on land, one meter away from the edge of the water and later the two adult otters were directly observed in the vicinity of a pond. the remains of the ribbed newts were nearly complete bodies showing a characteristic wound in the upper thoracic area (fig. 1) which appeared open and emptied, as described by alarcos et al. (2006). in all cases, the heart, lungs and liver were consumed. some of the bodies were still moving, indicating that predation was very recent. newts were sexed based on the presence of male nuptial pads, totaling 11 males and 14 females and one individual that could not be sexed. collected bodies were measured in the laboratory to the nearest 0.5 mm with a digital caliper (table 1). only snout-vent length was significantly larger in females than in males (t test, p < 0.05). the remains of iberian spadefoot toads found consisted in shreds of skin and part of the digestive tract (stomach) and egg-masses in females. all the remains found were fresh, most likely from the same night and all but two ribbed newts were found on land, less than 50 cm away from the edge of the pond. the two ribbed newts found in the water were floating less than one meter away from the shore. although previous occurrences of massive predation events of toads have been reported for otters (lizana and pérez-mellado, 1990; lizana et al., 1996; bartralot and bonet-arbolí, 2000), and there is even a recent report of presumed otter predation on ribbed newts (alarcos et al., 2006), this is the first instance with visual confirmation of the nature of the predators, and the event with the highest mortality reported for a single night. this is particularly true since the otters were observed around midnight by the 219impact of otter predation on amphibians in temporary ponds researchers (only three hours after sunset) and they could have continued their predation if not interrupted. ten additional living ribbed newts were observed. the peculiar predation mode on ribbed newts, with the selective consumption of a small amount of soft organs, may cause the underestimation of this type of predation in diet studies of otters based on scat analyses (e.g., clavero et al., 2005). the ponds where this occurred were visited yearly in fall by researchers since 2001 and no similar predation events were ever observed (tejedo et al., 2008). the fact that in previous years iberian parsley frogs (pelodytes ibericus) were never heard in this pond fig. 1. close-up of ventral view of some predated pleurodeles waltl (© r. márquez). table 1. mean and standard deviation of body size measurements of male and female pleurodeles waltl killed by otters. n = number of individuals; svl = snout-vent length; tl = tail length; hw = head width. measurements are in mm following fontanet and horta (1989). significant differences between sexes (p < 0.05; t-test) are marked with *. n svl tl hw males 11 79.8 ± 4.5 * 89.2 ± 5.4 15.0 ± 0.5 females 14 85.8 ± 6.6 * 82.9 ± 9.9 15.6 ± 1.3 220 d. cogălniceanu, r. márquez and j.f. beltrán may suggest that the otters, who are typically found near the shore of a dam which is more than one km away downstream, may have located the ponds through the calls of the parsley frogs. otters typically have home ranges that can be of more than 10 km2 (erlinge, 1967). a similar survey in 2007, at the start of the fall rains found only six adult ribbed newts. while locally important, the otter predation reported did not eliminate the prey populations and its medium-term impact has still to be assessed. the scarcity of breeding sites for pond-dwelling amphibians in our study area results in high concentrations of amphibians in these temporary ponds, making them extremely vulnerable to the threat posed by opportunistic predators. both p. waltl and p. cultripes are considered near threatened in the spanish red book (montori et al., 2002, tejedo and reques, 2002) and in the iucn global and european red lists (iucn 2007; temple and cox, 2009), but given the general climatic trends in their home ranges, their threat status is likely to increase. repeated massive killing events combined with dry years may thus pose a severe threat to the survival of amphibian communities in the area. thus, during a severe winter, otters fed on european pond turtles (emys orbicularis) killing almost 200 individuals within a protected area (lanszki et al., 2006). reintroduction and management plans focused on increasing otter populations should consider mitigating the impact on prey populations, especially endangered ones. the persistence of amphibians is important for otters, since they represent an important trophic resource, so management measures that would allow their persistence are required (e.g., creation of artificial ponds). these conflicting conservation goals involving protected species require solid priority settings and trade-offs in decision-making. overall, present species-based conservation approaches appear biased and unable to foresee and prevent this type of conflicts. a more complex, integrated management approach is thus required. acknowledgements synthesys es-taf-1964 provided travel funds for dc. fieldwork was funded by ministerio de educacion y ciencia (spain) projects tempura, cgl2005-00092/bos and acura, cgl200804814-co2/bos, and cgl2006-27892-e/bos. permits to work in the natural park and scientific collection permits were granted by consejería de medio ambiente, junta de andalucía. we thank dr. miguel lizana for providing bibliography and two anonymous reviewers for helpful comments. references alarcos, g., ortiz, m., fernández, m.j., lizana m. (2006): depredación del gallipato (pleurodeles waltl) por nutria en los arribes de duero, salamanca. bol. asoc. herpetol. esp. 17: 85-88. bartralot, e., bonet-arbolí, v. (2000): depredación de mustélidos sobre sapo común (bufo bufo). bol. asoc. herpetol. esp. 11: 32-33. clavero, m., prenda, j., delibes, m. (2005): amphibian and reptile consumption by otters (lutra lutra) in a coastal area in southern iberian peninsula. herpetol. j. 15: 125-131. 221impact of otter predation on amphibians in temporary ponds drechsler, m. (2004): model-based conservation decision aiding in the presence of goal conflicts and uncertainty. biodiv. conserv. 13: 141-164. erlinge, s. (1967): home range of the otter lutra lutra. oikos 19: 81-98. fernández-alés, r. (1979): características ecológicas de la sierra norte de sevilla. jornadas sobre la gestión del parque natural de la sierra norte de sevilla. c.e.p.a. fontanet, x., horta, n. (1989): biometría y dimorfismo sexual en pleurodeles waltl michahelles, 1830 amphibia, salamandridae de una población del ne de la península ibérica. misc. zool. 13: 202-206. iucn (2007): iucn red list of threatened species. www.iucnredlist.org. junta de andalucía (2002): la información ambiental de andalucía. publicación anual en cd. consejería de medio ambiente. junta de andalucía. sevilla. lanszki, j., molnár, m., molnár, t. (2006): factors affecting the predation of otter (lutra lutra) on european pond turtle (emys orbicularis). j. zool., lond. 270: 219-226. lizana, m., pérez-mellado, v. (1990): depredación por la nutria (lutra lutra) del sapo de la sierra de gredos (bufo bufo gredosicola). doñana, acta vertebrata 17: 109-112. lizana, m., martín-sánchez, r., antín, j., lópez, j., morales, j.j., gutiérrez j., del arco, g. (1996): nuevos datos sobre la depredación de anfibios por nutrias (lutra lutra) en zonas altas de la sierra de gredos. actas de gredos 1993. boletín universitario uned 13: 9-16. maikhuri, r.k., nautiyal, s., rao, k.s., saxena, k.g. (2001): conservation policy–people conflicts: a case study from nanda devi biosphere reserve (a world heritage site), india. forest policy econ. 2: 355-365. márquez, r., bosch, j., penna, m. (2007): sound pressure level of advertisement calls of alytes cisternasii and alytes obstetricans (anura, discoglossidae). bioacoustics 16: 27-37. márquez, r., bosch, j., eekhout, x. (2008): intensity of female preference quantified through playback set points: call frequency versus call rate in midwife toads. anim. behav. 75: 159-166. marrs, r.h., galtress, k., tong, c., cox, e.s., blackbird, s.j., heyes, t.j., pakeman, r.j., le duc, m.g. (2007): competing conservation goals, biodiversity or ecosystem services: element losses and species recruitment in a managed moorland-bracken model system. j. environ. manage. 85: 1034-1047. montori, a., llorente, g.a., santos, x., carretero, m.a. (2002): pleurodeles waltl. in: atlas y libro rojo de los anfibios y reptiles de españa, p. 51-53. pleguezuelos, j.m., márquez, r., lizana, m., eds. dirección general de conservación de la naturaleza-asociación herpetológica española, madrid. penna, m., márquez, r., bosch, j., crespo, e.g. (2006): non-optimal propagation of advertisement calls of midwife toads in iberian habitats. j. acoust. soc. am. 119: 1227-1237. tejedo, m., reques, r. (2002): pelobates cultripes. in: atlas y libro rojo de los anfibios y reptiles de españa, p. 94-96. pleguezuelos, j.m., márquez, r., lizana, m., eds. dirección general de conservación de la naturaleza-asociación herpetológica española, madrid. tejedo, m., márquez, r., beltrán, j.f., marangoni, f., llusia, d. cambron, m., gómezmestre, i., meier, a., eekhout, x.,bowker, r.g., benítez, m., moreira, c.n., crespo, 222 d. cogălniceanu, r. márquez and j.f. beltrán e.g., sousa do amaral, j.p., penna, m.c. (2008): las investigaciones sobre anfibios en el parque natural de la sierra norte. in: investigación científica y conservación en el parque natural sierra norte de sevilla, p. 3-16. menor, a., cuenca, i. eds., junta de andalucía, consejería de medio ambiente, sevilla. temple, h.j., cox, n.a. (2009): european red list of amphibians. office for official publications of the european communities, luxembourg. williams, p.h., araujo, m.b. (2000): using probability of persistence to identify important areas for biodiversity conservation. proc. r. soc. lond. b 267: 1959-1966. acta herpetologica 11(2): 227-231, 2016 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-17922 heavy traffic, low mortality tram tracks as terrestrial habitat of newts mikołaj kaczmarski*, jan m. kaczmarek university of life sciences, institute of zoology, wojska polskiego 71c, 60-625 poznań, poland. * corresponding author. e-mail: traszka. com@gmail.com submitted on 2016, 3nd february; revised on 2016, 29th april; accepted on 2016, 10th june editor: adriana bellati abstract. amphibian mortality caused by rail traffic has not attracted much attention in comparison to road mortality. density of railways in landscape, as well as traffic intensity, is usually much lower than in case of roads. as a consequence, their overall effect on amphibian populations is tacitly assumed to be less negative. to test whether very intensive rail traffic can cause substantial mortality in population of a small amphibian, we investigated a smooth newt lissotriton vulgaris population located in the city of poznań, w poland, where tram tracks border isolated breeding ponds. we performed controls during the peak of autumn migratory activity along the tracks. less than 1% of all individuals found during the survey were killed by rail traffic. observed mortality was very low despite large number of individuals present on the track and intensive tram traffic. as negative effects of traffic are low, rail or tram embankments can provide an important terrestrial habitat for small european newts. keywords. amphibian, lissotriton vulgaris, mortality, railway, smooth newt, traffic. amphibian mortality caused by rail transport has not attracted much attention in comparison to road mortality, which is investigated by a growing number of papers (coffin, 2007; elzanowski et al., 2009; glista et al., 2009; cayuela et al., 2015; franch et al., 2015). density of railways in landscape, as well as traffic intensity, is usually much lower than in case of roads. as a consequence, their overall effect on amphibian populations is tacitly assumed to be less negative. despite that, amphibians crossing the railway line suffer from mortality by train collisions (budzik and budzik, 2013) or turbulence effect (barandun, 1991). railway infrastructure can block or hinder migration, especially in case of large newts or toads (etienne et al., 2003). however, the structure of a typical railway track could enable small, crawling amphibians, like european lissotriton newts, to move under the rails, therefore minimizing the risk of collision. on the other hand, in case of small species, dead individuals are more likely to disappear quickly after death, because of scavenging (beckmann and shine, 2014), drying (budzik and budzik, 2013) or physical destruction by traffic (hels and buchwald, 2001). as a consequence, rail traffic mortality rates for species like lissotriton newts are likely to be underestimated, just as they are for road traffic (elzanowski et al., 2009; cayuela et al., 2015). to verify whether very intensive rail traffic can cause substantial mortality in populations of a small amphibian, we investigated a smooth newt lissotriton vulgaris linnaeus, 1758 population located in the poznań city, poland (52°23’31.1”n, 16°58’33.5”e), where the breeding site is located near double tram tracks with very heavy traffic load. smooth newt is considered the most widespread newt of the old world (arntzen et al., 2009; sparreboom, 2014). although l. vulgaris is a common species, detailed analyses show that it undergoes a broad decline in western europe, analogous to that of other newt species (denoël, 2012; denoël et al., 2013). it breeds in a vast diversity of water bodies and is generally associated with different kinds of wetlands (bell, 1977). the breeding sites are two small ponds (about 700 m2 and 900 m2, 228 mikołaj kaczmarski, jan m. kaczmarek respectively) completely overgrown by phragmites australis (cav.)  trin.  ex  steud,  1841 within an intensively managed urban park (fig. 1). until 1960s, the studied area was the part of suburban farmland with a small river valley; urban development in 1970-1980s isolated l. vulgaris from surrounding populations. the park borders with housing estates and a forested buffer of an industrial area, containing a fish-inhabited pond that is the breeding site for common toads bufo bufo linnaeus, 1758. the double tram track is located along the southern border of the park. daytime traffic load is ca. 20 trams/hour, peaking at late evening and early morning hours (over 40 trams/hour). the structure of the tram track is analogous to a typical railway track: wooden sleepers (fig. 2c) on crushed stone aggregate (fig. 2a, b). we performed 11 controls between 6th and 19th october 2014 (peak of autumn migratory activity). we searched for newts between 20:00 and 24:00 along the 300-m transect at the edge of the tram tracks in their section closest to the breeding ponds. we also searched for newts crossing pedestrian trails within the park and roads along park boundaries. in total, 303 individuals were noted along tram tracks in the whole survey period. we found individuals of both sexes, as well as juveniles (table 1). captured individuals were measured (svl, body mass) using digital caliper and electronic scale. however, we did not mark individuals, so numbers are given separately for each day, with the highest number of animals observed on october 15th (68 individuals; fig. 3). only 3 individuals killed by tram were found during the survey period. dead newts were found only at pedestrian crossings. newts were observed at the edge of the track as well as between the rails (fig. 3). no individuals were found elsewhere (forested area, park trails or roads along the park boundaries). it has already been suggested that railway tracks do not necessarily block the migration efforts of newts. no significant genetic effects were found in populations of the alpine newt ichtyosaura alpestris laurenti, 1768 at both sides of the 30-year old railway, which could have been a consequence of newt migration across the track (prunier et al., 2014). in contrast, in case of north american salamander ambystoma opacum gravenhorst, 1807 a 100-years old railway led to total genetic isolation of two subpopulations divided by tracks (bartoszek and greenwald, 2009). such difference could be ascribed to the time of isolation or the lesser mobility of ambystoma opacum compared to european newts. also, the populafig. 1. situational map showing the area of the research. 229tram tracks as terrestrial habitat of newts tion researched by bartoszek and greenwald (2009) was already isolated and had relatively low number of individuals, while prunier et al. (2014) performed analysis on landscape scale with large number of interconnected populations. another evidence for low level of isolation provided by railway lines is the work of budzik and budzik (2013), who found no dead newts along the 45-km transect along the railway line in se poland. budzik and budzik (2013) suggested that either newts disappear shortly after death due to their small body size, or they are not subject to mortality caused by rail traffic. our results provide evidence for the latter hypothesis, as observed mortality was low compared to high number of live specimens and great intensity of tram traffic, much exceeding the train traffic intensity in budzik and budzik (2013). dead newts were found only at pedestrian crossings, where newt movement under the tracks is impossible, and animals are forced to move over instead of under the rails. we confirmed that, in late autumn, l. vulgaris from the urban population use the tram track as their terrestrial habitat, and are not subject to extensive mortality. the timing of the controls enabled us to search for dead individuals just after the evening peak of tram traffic (and, presumably, the peak of newt activity and mortality). temperature and humidity during all controls were likely to prevent the dead individuals from quick drying. as dead individuals were collected during the night, the risk of scavenging by birds was minimal. we could not exclude the possibility of scavenging by mammals (e.g., hedgehogs, feral cats, foxes, rats), but we treat it unlikely as the controls were performed early in the night when tram and human traffic was still relatively intensive, restricting the mammalian activity. thus, we feel that the detection probability of dead newts was higher than usually assumed for estimating road mortality (hels and buchwald, 2001; elzanowski et al., 2009). although the length of the transect was short compared to some other studies (e.g., budzik and budzik, 2013), we stress that it was enough to cover most of the terrestrial habitat of the investigated population, as the surrounding areas are either build-up or do not provide enough shelter for the newts (kaczmarek et al., 2014.). therefore, we suggest that the rail aggregate consisting of sleepers and stones is a crucial winter habitat for the investigated population (fig. 2). a large number of shelters within rail aggregate, providing humid and prey-rich terrestrial habitat, enable the persistence of a large newt population, despite isolation and deteriorating quality of the breeding pond. additionally, driving trams directly scare away large fig. 2. (a) male smooth newt l. vulgaris during the searching for prey among crushed stone aggregate; (b) crushed stone aggregate as newts habitat with the rail in the background; (c) female smooth newt found feeding on earthworm on the surface of a wooden sleeper (october, 2014). table 1. morphometric values of l. vulgaris from 14th october. x min max sd n svl [mm] mm 38.78 31.03 45.07 3.17 25 ff 38.82 29.67 46.11 4.36 28 juv 22.92 19.86 27.45 2.93 7 body mass [g] mm 1.79 1.18 2.93 0.40 25 ff 1.91 0.72 3.12 0.58 28 juv 0.32 0.12 0.59 0.16 7 fig. 3. number of individuals during each day of our survey with mortality (15th october, one female; 18th october, one male and one juvenile). 230 mikołaj kaczmarski, jan m. kaczmarek predators, thus reducing predation risk. easily warming up stone aggregate could also affect newt activity pattern, as active individuals were observed until the end of december (pers. comm.). presence of juvenile individuals (up to 38% in daysamples over 20 individuals) is interesting (fig. 3). malmgren (2002) showed that out of the pond movement of juvenile newts was more random than of the adult individuals, which is in accordance with theoretical framework presented by pittman et al. (2014). however, the majority of juveniles in malmgren (2002) chose similar direction as adults, i.e., towards the forest. abundance of juvenile individuals on the track suggests that their movements are nonrandom. if juvenile newts used ‘normal’ cues, signaling the proximity of optimal terrestrial habitat (e.g., humidity gradient, shadow, slope axis), they should move along the old riverbed and remain within the park. therefore, we suppose that at least some juvenile individuals follow the adults using olfactory cues (hayward et al., 2000). in contrast to low rail traffic mortality, european newts are vulnerable to road traffic mortality (denoël, 2012). for slow-moving newts, the probability of being killed is very high regardless of traffic intensity (hels and buchwald, 2001). in a setup similar to this study (500m transect along a local road, breeding newt population migrating from pond located 300 m from the transect), the road  kills-to-spawners ratio for l. vulgaris was higher than for any other amphibian, with more than half of the population killed each year (elzanowski et al., 2009). in such a context, rail traffic seems much less detrimental for newt populations, despite potential mortality caused by herbicide spraying (brühl et al., 2013). it seems that in the studied population, where the managed urban greenery does not provide a sufficient number of high-quality hibernation sites, cavities within the rail aggregate act as the key wintering habitat. it requires further investigation whether the rail/tram aggregate and sleepers are a suitable all-year habitat for juvenile newts. whereas it is indisputable that roads and railways negatively affect animals, some positive effects on birds and reptiles were also reported (morelli et al., 2014). we argue that for small, crawling amphibians like l. vulgaris the benefits from new habitats created by railway infrastructure can outweigh the potential costs of traffic-induced mortality. acknowledgements animal collection was performed according to rdos permit no. wpn-ii.6401.245.2014.ag. we are very thankful to piotr tryjanowski, anna m. kubicka, and katarzyna pędziwiatr for encouragement and discussion during preparation of the manuscript. the authors thank m. piasecka, m. machura and other students for field assistance. we are also grateful to three anonymous reviewers whose comments greatly improved this paper. references arntzen, j.w., kuzmin, s., beebee, t., papenfuss, t., sparreboom, m., ugurtas, i.h., anderson, s., anthony, b., andreone, f., tarkhnishvili, d., ishchenko, v., ananjeva, n., orlov, n., tuniyev, b. (2009): lissotriton vulgaris. the iucn red list of threatened species. version 2014.3. <www.iucnredlist.org> (accessed: 6 january 2015). barandun, j. (1991): amphibienschutz an bahnlinien. natur und landschaft 66: 305. bartoszek, j., greenwald, k.r. (2009): a population divided: railroad tracks as barriers to gene flow in an isolated population of marbled salamanders (ambystoma opacum). herpetol. conserv. biol. 4: 191-197. bell, g. (1977): the life of the smooth newt (triturus vulgaris) after metamorphosis. ecol. monograph. 47: 279-299. beckmann c., shine, r. (2014): do the numbers and locations of road-killed anuran carcasses accurately reflect impacts of vehicular traffic? j. wildlife manage. 79: 92-101. brühl, c.a., schmidt, t., pieper, s., alscher, a. (2013): terrestrial pesticide exposure of amphibians: an underestimated cause of global decline? sci. rep. 3: 1135. budzik, k.a., budzik, k.m. (2013): a preliminary report of amphibian mortality patterns on railways. acta herpetol. 9: 103-107. cayuela, h., quay, l., dumet, a., léna, j.p., miaud, c., rivière, v. (2015): intensive vehicle traffic impacts morphology and endocrine stress response in a threatened amphibian. oryx 2015: 1-7. coffin, a.w. (2007): from roadkill to road ecology: a review of the ecological effects of roads. j. transp. geogr. 15: 396-406. denoël, m. (2012): newt decline in western europe: highlights from relative distribution changes within guilds. biodivers. conserv. 21: 2887-2898. denoël, m., perez, a., cornet, y., ficetola, g.f. (2013): similar local and landscape processes affect both a common and a rare newt species. plos one 8: e62727. elzanowski, a., ciesiołkiewicz, j., kaczor, m., radwańska, j., urban, r. (2009): amphibian road mortality in 231tram tracks as terrestrial habitat of newts europe: a meta-analysis with new data from poland. eur. j. wildlife res. 55: 33-43. etienne, r.s., vos, c.c., jansen, m.j. (2003): ecological impact assessment in data-poor systems: a case study on metapopulation persistence. environ. manage. 32: 760-777. franch, m., silva, c., lopes, g., ribeiro, f., trigueiros, p., seco, l., sillero, n. (2015): where to look when identifying roadkilled amphibians? acta herpetol. 10: 103-110. glista, d.j., devault, t.l., dewoody, j.a. (2009): a review of mitigation measures for reducing wildlife mortality on roadways. landsc. urban plan. 91: 1-7. hayward, r., oldham, r.s., watt, p.j., head, s.m. (2000): dispersion patterns of young great crested newts (triturus cristatus). herpetol. j. 10: 129-136. hels, t., buchwald, e. (2001): the effect of road kills on amphibian populations. biol. conserv. 99: 331-340. kaczmarek, j., kaczmarski, m., pędziwiatr, k. (2014): changes in the batrachofauna in the city of poznań over 20 years. in: urban fauna. animal, man, and the city – interactions and relationships, pp. 169-178. indykiewicz, p., böhner, j., eds, artstudio, bydgoszcz. malmgren, j.c. (2002): how does a newt find its way from a pond? migration patterns after breeding and metamorphosis in great crested newts (triturus cristatus) and smooth newts (t. vulgaris). herpetol. j. 12: 29-35. morelli, f., beim, m., jerzak, l., jones, d., tryjanowski, p. (2014): can roads, railways and related structures have positive effects on birds? – a review. transport. res. d-tr. e. 30: 21-31. pittman, s.e., osbourn, m.s., semlitsch, r.d. (2014): movement ecology of amphibians: a missing component for understanding population declines. biol. conserv. 169: 44-53. prunier, j.g., kaufmann, b., léna, j.p., fenet, s., pompanon, f., joly, p. (2014): a 40-year-old divided highway does not prevent gene flow in the alpine newt ichthyosaura alpestris. conserv. genet. 15: 453-468. sparreboom, m. (2014): salamanders of the old world. 1st edition. knnv publishing, zeist. acta herpetologica vol. 11, n. 2 december 2016 firenze university press predator-prey interactions between a recent invader, the chinese sleeper (perccottus glenii) and the european pond turtle (emys orbicularis): a case study from lithuania vytautas rakauskas1,*, rūta masiulytė1, alma pikūnienė2 effective thermoregulation in a newly established population of podarcis siculus in greece: a possible advantage for a successful invader grigoris kapsalas1, ioanna gavriilidi1, chloe adamopoulou2, johannes foufopoulos3, panayiotis pafilis1,* the unexpectedly dull tadpole of madagascar’s largest frog, mantidactylus guttulatus arne schulze1,*, roger-daniel randrianiaina2,3, bina perl3, frank glaw4, miguel vences3 thermal ecology of podarcis siculus (rafinesque-schmalz, 1810) in menorca (balearic islands, spain) zaida ortega*, abraham mencía, valentín pérez-mellado growth, longevity and age at maturity in the european whip snakes, hierophis viridiflavus and h. carbonarius sara fornasiero1, xavier bonnet2, federica dendi1, marco a.l. zuffi1,* redescription of cyrtodactylus fumosus (müller, 1895) (reptilia: squamata: gekkonidae), with a revised identification key to the bent-toed geckos of sulawesi sven mecke1,*,§, lukas hartmann1,2,§, felix mader3, max kieckbusch1, hinrich kaiser4 the castaway: characteristic islet features affect the ecology of the most isolated european lizard petros lymberakis1, efstratios d. valakos2, kostas sagonas2, panayiotis pafilis3,* sources of calcium for the agamid lizard psammophilus blanfordanus during embryonic development jyoti jee1, birendra kumar mohapatra2, sushil kumar dutta1, gunanidhi sahoo1,3,* mediodactylus kotschyi in the peloponnese peninsula, greece: distribution and habitat rachel schwarz1,*, ioanna-aikaterini gavriilidi2, yuval itescu1, simon jamison1, kostas sagonas3, shai meiri1, panayiotis pafilis2 swimming performance and thermal resistance of juvenile and adult newts acclimated to different temperatures hong-liang lu, qiong wu, jun geng, wei dang* olim palus, where once upon a time the marsh: distribution, demography, ecology and threats of amphibians in the circeo national park (central italy) antonio romano1,*, riccardo novaga2, andrea costa1 on the feeding ecology of pelophylax saharicus (boulenger 1913) from morocco zaida ortega1,*, valentín pérez-mellado1, pilar navarro2, javier lluch2 notes on the reproductive ecology of the rough-footed mud turtle (kinosternon hirtipes) in texas, usa steven g. platt1, dennis j. miller2, thomas r. rainwater3,*, jennifer l. smith4 heavy traffic, low mortality tram tracks as terrestrial habitat of newts mikołaj kaczmarski*, jan m. kaczmarek book review: sutherland, w.j., dicks, l.v., ockendon, n., smith, r.k. (eds). what works in conservation. open book publishers, cambridge, uk. http://dx.doi.org/10.11647/obp.0060 sebastiano salvidio acta herpetologica 11(1): 95-97, 2016 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-17820 book review: john w. wilkinson. amphibian survey and monitoring handbook. pelagic publishing franco andreone museo regionale di scienze naturali, via g. giolitti, 36, i-10123 torino, italy. e-mail: franco.andreone@regione.piemonte.it people in need to conduct survey and ecological studies on amphibians (for pure research or for applied works) will certainly welcome the booklet authored by john w. wilkinson, a passionate conservationist and amphibian lover. the publisher pelagic publishing already produced in the last years several books and monographs dedicated to the natural history and conservation of habitats and species. the reviewed book is realised in octavo, soft bounded, 120 pages long, with colour photographs and black and white drawings. it belongs to the series entitled “conservation handbooks”, which also includes two other handbooks dedicated to the barn owl conservation and to monitoring of marine mammals. just to make a naturalistic “outing”: amphibians’ most typical representatives, such as frogs, toads, newts, and salamanders (not excluding the “odd” caecilians) have wonderful and peculiar life habits and for sure are among the most ideal subjects for a series of activities and studies (harvey pough et al., 2001). this is also due to the fact that many species from temperate areas usually aggregate around water pools where they breed. there, they can be found with rather good facility, having two life phases, one with aquatic larvae (known as tadpoles in anurans), and the other with terrestrial or sub-aerial adults. so far, they can be observed and collected with facility, just using hand nets, or a simple array of terrestrial and aquatic traps. when i started studying newts for my master thesis, it was nice to observe and collect them in a great number in temporary ponds using pit-falls and other simple devices. in fact, during the breeding phase (usually in springtime) many of them come back to water, and they may conduct quite a long aquatic phase. terrestrial urodeles, such as alpine salamanders (i.e., salamandra atra and s. lanzai) and cave salamanders (speleomantes spp.), are also abundant elements of the local biomasses and amphibians are also abundant animals in other biomes, such as tropical rainforests, where are also quite easy to observe. their activity is usually almost extended (going in winter periods as well) and represent good subjects to establish conservation priorities and biodiversity hotspots (veith et al., 2004). so far, herpetologists and biology students have the pleasure and necessity to know which are the best practises to collect and identify species in the field, as well to collect ecological data. evidently, wilkinson wished to fill this gap and presented all these topics in five numbered and three unnumbered chapters. the first chapter of the booklet is entitled “introducing amphibians” and it presents the amphibian diversity, a “must” when you deal with these vertebrates. with around than 7500 described species (estimate at january 2016 according to amphibiaweb, 2016), one third at least of which in danger of extinction (stuart et al., 2006), amphibians are often among the most favourite targets of ecologists and taxonomists, as new species are discovered all the times. this chapter also introduces the typical amphibians with the aid of nice colour photographs. to be sincere, i would have appreciated if this chapter was a little bit more extended, with more photographs. in a general context, more information would have been useful, showing, for example, the diffusion of amphibians around the world, and some more colourful or weird species. just for the sake of delighting eyes and for attracting people. 96 franco andreone chapter 2 is much more extended and is entitled “before you start surveying”. it includes important recommendations that a herpetologist should always take into consideration when she/he goes in the field. these span from the practical ways to collect data to instructions for preparation of handbooks and field procedures. an important sub-chapter deals with the “survey permissions and licenses”. although it is quite focussed on uk reality, it stresses the need to request and get all the documentations and authorisation before doing field work and handling live animals. i must confess that this kind of activities and procedures were much less important and respected in the past, but nowadays with the augmented level of legal protection of species and habitats it is really compulsory to follow strictly all the protocols and respect current legislation. it is also important to submit proposals of research and taking with us the research authorisations. two other subchapters are particularly worth of interest and must be read with great attention. one informs on how to handle amphibians (when this is strictly necessary) and provides useful information (in many cases amphibians can suffer for thermal shock and/or dehydratated). the other subchapter provides information about hygienic protocols. indeed, these arguments are closely correlated: it is extremely important to know that handling can be stressing for these small and delicate vertebrates, and also that this can be a way to diffuse dangerous pathogens. the deleterious effects of chytrid fungi (bratrachochytrium dendrobatidis and b. salmandrivorans) around the world are well-known and pose serious conservation problems for amphibians (weldon et al., 2004: martel et al., 2013). all people involved in amphibian surveys need to know how to disinfect hand nets and personal boots, since they can be vectors for these and other pathogens (dejean et al., 2007). it seems something trivial today, but it is important to recall, anyhow. the safety concept also includes attention for the human component, something that is often under-considered when going in the field. indeed, conducting research is not a simple and easy matter, and it might be really dangerous. the use of portable telephones a pleasure (and damnation!) in contemporary life is obviously important when going searching for amphibians, especially during nocturnal hours, when amphibians are more active, although it might be a little problem when conducting research in the tropics, often in areas far from electricity. chapter 3 (during your survey: amphibian survey methods) enters more in depth about the methods of survey, carefully listing the material necessary for the data collecting and providing information about the most relevant survey methods, among which: visual survey, egg counting, torching/night survey, funnel trapping, mesh funnel, ortmann-type traps. i also read with great interest and pleasure the information on how to build an artificial egg mop. in my early carrier i also tried this, and i remember that it was really amazing to see eggs promptly laid on it. the rest of the chapter deals with descriptions of survey methods to be used for terrestrial amphibians, as well as the application of the well-known pit-fall and drift fences methods. finally, for anurans wilkinson also provided indication about call recognition systems as well as an overview on hsis (habitat suitability indices). in chapter 4, “after your study”, wilkinson provides some synthetic indications on the statistical analyses that could be carried out, as well as indication on how to arrange and present the results, especially in a paper way. some last sub-chapters deal with further kinds of field studies, including population research, radio-tracking, taxonomy and gis studies. the last chapter, “resources to help you”, gives information on some format sheets that can be used as well as a list of field guides, textbooks, and equipment suppliers. the last sub-chapters presents a list of herpetological societies. amazingly, seen that this review is done on the official journal of societas herpetologica italica, it is quite a pity that this was not been listed, like the societé herpétologique de france. moreover, being a book exclusively dedicated to amphibians, i did not find any reference to the international society for the study and conservation of amphibians as well as to the iucn ssc amphibian specialist group and to the amphibian survival alliance. these should be corrected in the future, if other editions will follow. three last, unnumbered chapters, present a bibliographic list as well as a glossary, and an alphabetical index. to conclude this review i must say that i liked the book by wilkinson and i would recommend it. it is a nice compendium putting together an array of information and in such an extent it is surely useful for people at the beginning of their activity. notwithstanding, i would have appreciated a wider breadth of treated topics. for example, all the discussions and recommendations seem to me too much anglo-saxon centred. amphibians are so important at a global level that more examples should have been given for ongoing researches and methods in tropical countries, with maybe some more indications on problems and methods to be used there. although the book is clearly focussed on ecological and quite traditional methods, i would have read with interest about the application of new methodologies, such as the application of barcoding for species determination in the field, to edna and to automatic acoustic detection. probably, 97book review some more information on statistical analyses would have been welcome and useful as well. anyhow, all these points could be easily addressed in a forthcoming edition. the current one is already worth of been purchased and will represent a good deal for passionate batrachologists. references amphibiaweb (2016): amphibiaweb: information on amphibian biology and conservation. berkeley. collins, j.p., crump, m. (2009): extinction in our times. global amphibian decline. oxford university press, new york. dejean, t., miaud, c., ouellet, m. (2007): proposition d’un protocole d’hygiène pour réduire les risques de dissémination d’agents infectieux et parasitaires chez les amphibiens lors d’intervention sur le terrain. bull. soc. herp. fr. 122 : 40-48 griffiths, r.a. (1996): newts and salamanders of europe. poyser natural history, london. harvey pough, f., andrews, r.m., cadle, j.e., crump, m.l., savitzky, a-h, wells, k.d. (2001): herpetology. second edition. prentice hall, upper saddle river. martel, a., ppitzen-van der slulis, a., blooi, m., bert, w., ducatelle, r., fisher, m.c., woeltjes, a., bosman, w., chiers, k., bossuyt, f., pasmans, f. (2013): batrachochytrium salamandrivorans sp. nov. causes chytridiomicosis in amphibians. proc. nat. acad. sci. usa 110: 15325-15329. stuart, s., chanson, j. s., cox, n. a., young, b. e., rodrigues, a. s. l., fishman, d. l., waller, r. w. (2004): status and trends of amphibian declines and extinctions worldwide. science 306: 1783-1786. veith, m., lötters, s., andreone, f., rödel, m. o. (2004): measuring and monitoring amphibian diversity in tropical forests. ii. estimating species richness from standardized transect censing. ecotropica 10: 85-99. weldon, c., du preez l.h., hyatt, a.d., muller, r., speare, r. (2004): origin of the amphibian chytrid fungus. emerg. infect. dis. 10: 2100-2105. acta herpetologica vol. 11, n. 1 june 2016 firenze university press feeding habits of mesoclemmys vanderhaegei (testudines: chelidae) elizângela silva brito1,*, franco leandro souza2, christine strüssmann3 morphology, ecology, and behaviour of hylarana intermedia, a western ghats frog ambika kamath1, rachakonda sreekar2,3 a new account for the endangered cerrado rocket frog allobates goianus (bokermann, 1975) (anura: aromobatidae), with comments on taxonomy and conservation thiago ribeiro de carvalho1,2,*, lucas borges martins1,2, ariovaldo antonio giaretta1 molecular phylogenetics of the pristimantis lacrimosus species group (anura: craugastoridae) with the description of a new species from colombia mauricio rivera-correa, juan m. daza* population structure and activity pattern of one species of adenomera steindachner, 1867 (anura: leptodactylidae) in northeastern brazil maria juliana borges-leite1,*, joão fabrício mota rodrigues2, patrícia de menezes gondim1, diva maria borges-nojosa1 vocal repertoire of scinax v-signatus (lutz 1968) (anura, hylidae) and comments on bioacoustical synapomorphies for scinax perpusillus species group marco antônio peixoto1,*, carla silva guimarães1, joão victor a. lacerda2, fernando leal2, pedro c. rocha2, renato neves feio1 amendment of the type locality of the endemic sicilian pond turtle emys trinacris fritz et al. 2005, with some notes on the highest altitude reached by the species (testudines, emydidae) federico marrone*, francesco sacco, vincenzo arizza, marco arculeo photo-identification in amphibian studies: a test of i3s pattern marco sannolo1,*, francesca gatti2, marco mangiacotti3,4, stefano scali3, roberto sacchi4 no evidence for the ‘expensive-tissue hypothesis’ in the dark-spotted frog, pelophylax nigromaculatus li zhao, min mao, wen bo liao* no short term effect of clinostomum complanatum (trematoda: digenea: clinostomatidae) on survival of triturus carnifex (amphibia: urodela: salamandridae) giacomo bruni1,*, claudio angelini2 yeasts in amphibians are common: isolation and the first molecular characterization from thailand srisupaph poonlaphdecha, alexis ribas* introduction of eleutherodactylus planirostris (amphibia, anura, eleutherodactylidae) to hong kong wing ho lee1, michael wai-neng lau2, anthony lau3, ding-qi rao4, yik-hei sung5,* correlation between endoscopic sex determination and gonad histology in pond sliders, trachemys scripta (reptilia: testudines: emydidae) david perpiñán1, albert martínez-silvestre2,3, ferran bargalló3, marco di giuseppe4, jorge orós5, taiana costa6,* book review: john w. wilkinson. amphibian survey and monitoring handbook. pelagic publishing franco andreone book review: susan newman. frogs amphibians and their threatened environment. discovery and expression through art k-3. frogs are green franco andreone issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 9(1): 75-88, 2014 doi: 10.13128/acta_herpetol-13759 first evidence of the effects of agricultural activities on gonadal form and function in rhinella fernandezae and dendropsophus sanborni (amphibia: anura) from entre ríos province, argentina laura c. sanchez1,*, rafael c. lajmanovich1, paola m. peltzer1, adriana s. manzano2, celina m. junges1, andrés m. attademo1 1 national council for scientific and technical research (conicet) – faculty of biochemistry and biological sciences fbcb-unl, paraje el pozo s/n (3000), santa fe, argentina. corresponding author. e-mail: lauracecilias@gmail.com 2 centre of scientific investigation and transference of technology to the production (cicyttp-conicet), materi and españa s/n (3105), diamante, entre ríos, argentina – uader (autonomic university of entre ríos) submitted on 2013, 22nd december; revised on 2014, 20th april; accepted on 2014, 23rd april abstract. the relationship between male gonadal abnormalities and habitats with different degrees of agricultural activities was quantified in two anuran species, rhinella fernandezae and dendropsophus sanborni. the study sites were selected along a gradient of increasing agricultural land use in south-western entre ríos province (argentina): an agroecosystem, a natural wetland (a non-agricultural site adjacent to monoculture zones), and a natural forest (not associated with agriculture). rhinella fernandezae and d. sanborni were manually captured from each environment during field surveys. a scaled mass index (mi) was evaluated for each animal. specimens of r. fernandezae from the agroecosystem and the natural wetland site presented poorly developed seminiferous tubules, lower testicular volume, and a lower number of seminiferous tubules, primary spermatogonia, and spermatids than specimens from the natural forest site. additionally, we observed fewer primary spermatocytes in the agroecosystem group than in the natural forest group. individuals of d. sanborni from the agroecosystem and the natural wetland site presented poorly developed tubules, higher proportions of irregularly shaped testes, and a reduced number of primary and secondary spermatogonia compared with specimens from natural forest sites. consequently, the affected anurans are likely to have reduced reproductive success. we suggest that agrochemical use may be associated with decreased testicular development and function in both r. fernandezae and d. sanborni occurring in agroecosystems and nearby environments. buffer zones are needed to prevent contamination, preserve wildlife, and enhance the conservation value of pristine natural forests. keywords. agriculture, anomalies, testicular histology, germ cells, rhinella fernandezae, dendropsophus sanborni. introduction intensification of agricultural activities has become a major threat to biodiversity in different parts of the world. agriculture can alter natural systems in different ways, such as habitat loss and the creation of isolated fragments by conversion of natural habitats to arable land (e.g., joly et al., 2001; grau et al., 2005; morton et al., 2006), as well as through the possible deleterious impacts of chemicals on native flora and fauna (e.g., smith et al., 2000; khan and law, 2005; peltzer et al., 2011; amaral et al., 2012). exposure of amphibians to endocrine-disrupting chemicals affects the functioning of endocrine systems involved in reproduction, development, and metamorphosis (crump, 2001; mcdaniel et al., 2008; trachantong et al., 2013). as a result of abnormal development of reproductive organs or structures, recruitment can 76 l.c. sanchez et alii decrease, which has been as noted a proximate (direct) cause of amphibian declines (hayes et al., 2010). estrogenic chemicals, such as pesticides, are extensively used in natural environments, being distributed through sewage effluents, agricultural runoff, aerial drift, and possibly rainfall (colborn et al., 1993; storrs-méndez and semlitsch, 2010). there is increasing evidence of endocrine disruption and reproductive abnormalities in amphibian populations inhabiting agricultural areas (e.g., hayes et al., 2003; mccoy et al., 2008; mcdaniel et al., 2008; orton and routledge, 2011). in latin america, the use of genetically modified soybean crops has been largely increased, with the associated increase in pesticide application. soybean crop expansion has been driven by: (1) crop prices; (2) government and agro-industrial support; and (3) increasing demand from countries such as china (pengue, 2005). in argentina, a great expansion of soybean planting has occurred since 2005 (altieri and pengue, 2006). almost 100% of this increase involves the use of genetically modified ‘‘roundup ready’’ soybeans, resistant to glyphosatebased herbicides; therefore, the use of these chemicals has also increased (lajmanovich et al., 2010). several studies have suggested that glyphosate-based herbicides affect development (paganelli et al., 2010) and reproduction (oliveira et al., 2007; soso et al., 2007; romano et al., 2010) in wild fauna. in addition, glyphosate herbicides are rarely applied alone, but in combination with other biocides (herbicides, insecticides, and fungicides) that may have their own effects and interact with glyphosate in various ways (jergentz et al., 2005). according to laboratory experiments, the most widely used insecticides in argentina (chlorpyrifos, cypermethrin and endosulfan) also have negative effects on the reproductive system (usha and harikrishnan, 2005; jeong et al., 2006; singh and singh, 2008; rey et al., 2009). however, the possible associations between agricultural practices and changes in histoarchitecture or gonadal function of wild animals have been poorly explored in argentina, with no records of the effect on amphibians being reported despite the high susceptibility of this group. indeed, amphibians have biphasic life cycle, which makes them especially vulnerable to perturbations of both aquatic and terrestrial environments; highly permeable skin at all life history stages; restricted home range and limited dispersal abilities (jansen and healey, 2003). accordingly, these kinds of field studies are necessary because of the rapid agricultural expansion and intensification and would be very important for amphibian conservation and evaluation of the ecological risks associated with agriculture. in the present study we quantified the relationship between male anuran gonadal abnormalities and habitats with different degrees of agricultural activity. specifically, we hypothesized that gonadal form and function would be altered by agricultural activity in soybean fields. materials and methods site selection we selected three sites (an agroecosystem, a continental natural wetland, and a natural forest of island) located in southwestern entre ríos province (fig. 1). the agroecosystem site (ag) was a soybean field (glycine max (l.) merril) situated in diamante department (32º 06´12.3´´s; 60º 37´17.5´´w; 23 ha). it was cultivated by direct seeding (soybean in spring – summer, and wheat in autumn – winter). specifically, soybean is usually sown in november/december and harvested in march/ april. a natural water body flows across the field, forming a small wetland. several agrochemicals were applied at different doses during the study period. before sowing, two herbicides were used to remove weeds (glyphosate 66% 2 l/ha and 2-4 d 1 l/ha). after soybean emergence and at the beginning of bloom, glyphosate 48% 3.5 to 4 l/ha was applied twice. moreover, cypermethrin 0.1 l/ha and endosulfan 1l/ha were applied once or twice to eliminate caterpillars and bugs (cooperativa agrícola de diamante, pers. comm.). the natural wetland site (nw) is located between the agricultural and natural forest zones; this site is directly exposed to pluvial runoff from soybean fields due to the slope. the vegetation in the nw site was dominated by wooded and shrubby species, such as phytolacca dioica, rapanea laetevirens, fagara hyemalis, and celtis spinosa. the vegetation in water bodies and flooding areas was characterized by eichhornia azurea, lemna gibba, ludwigia peploides, panicum elephantipes, and polygonum punctatum. this site is located in the continental zone of predelta national park (pdnp) (32º 07´17.6´´s; 60º 38´02.2´´w). pdnp is a wetland reserve (2458 ha) located 2 km away from the agroecosystem, in the paraná river floodplain close to the starting point of paraná delta, which includes a continental zone as well as several islands (apn, 2003; aceñolaza et al., 2004). this reserve presents the typical vegetation of fluvial gallery forest (bó, 2006). the natural forest (nf) was located in the island region of the pdnp (32º 07´30.7´´s; 60º 38´11.6´´w). this is a pristine sector preserved from human impact and not exposed to direct runoff carrying agricultural chemicals. the dominant woody vegetation in nf site was salix humboldtiana, tessaria integrifolia, and albizia inundata. herbaceous vegetation was mainly composed by panicum prionitis. vegetation in the lower zones is the typical of areas prone to flooding, such as aspilia silphiodes, eichhornia azurea, enydra anagallis, eringium pandanifolium, and pontederia rotundifolia. field surveys according to sanchez et al. (2013) anuran species richness in ag site was s = 16, whereas in nw and nf sites it was s = 21 and s = 20, respectively. the most abundant species in 77effects of agriculture on amphibian gonads ag were hypsiboas pulchellus (hylidae), leptodactylus gracilis, l. latinasus, l. mystacinus (leptodactylidae) and odontophrynus americanus (odontophrynidae). the dominant species both in nw and nf were r. fernandezae (bufonidae), dendropsophus nanus, d. sanborni, h. pulchellus (hylidae), and l. latrans (leptodactylidae). rhinella fernandezae (gallardo, 1957) and dendropsophus sanborni (schmidt, 1944) were selected based on their high representation in amphibian communities of the study area. estimated total abundance was 251 individuals in r. fernandezae (nag = 45, nnw = 130, nnf = 76) and 226 individuals in d. sanborni (nag = 66, nnw = 95, nnf = 65). these values included adults, juveniles and tadpoles (sanchez et al., 2013). we used these species to test our hypothesis, as these wild populations were less likely to be adversely affected by the removal of a few individuals for the study. both species are found in natural and anthropogenic ecosystems and are widely distributed in northeastern argentina, southern paraguay, uruguay and southern brazil (iucn, 2011). we conducted field surveys during the anuran reproductive period in this region (peltzer and lajmanovich, 2007) from december 2007 to april 2008. this period coincides with the sowing and harvest of soybean crops. in each environment, we recorded amphibians using nocturnal searches. nocturnal searches combine visual encounter surveys (crump and scott, 1994) and audio strip transects (zimmerman, 1994). we conducted four searches per month, inspecting all sites during the same night and spending at least 90 minutes per person per site. we captured individuals of the selected species by hand. only individuals with evident secondary sexual characters (following cei, 1980) were captured (r. fernadezae: external vocal sac and dark thumb pads in the first and second fingers; d. sanborni: external vocal sac). fig. 1. location of study sites in diamante, south-western entre ríos province, central-eastern argentina, southern south america. (ag) agroecosystem, (nw) natural wetland, and (nf) natural forest. pictures: (ag) agroecosystem (back) and wetland formed from the natural water body (at the front); (nw) pond in the natural wetland where the agroecosystem can be observed on the hill (at the bottom); (nf) island pond in the natural forest site with surrounding vegetation and, at the bottom, levees with remnants of riparian forest. 78 l.c. sanchez et alii general measurements and dissection of reproductive organs individuals of r. fernandezae (rf) and d. sanborni (ds) were euthanized following the nrc (1996) guide, and according to the requirements of the ethical committee of our institution. specifically, a noninhaled agent (ethanol 20%) was used (avma, 2013). we weighed each individual using a digital balance (means ± se in g: rf = 13.179 ± 1.323; ds = 0.345 ± 0.008) and measured their snout-vent length (svl) with a digital caliper (means ± se in mm: rf = 48.896 ± 1.327; ds = 17.622 ± 0.141). to compare the nutritional condition of individuals among populations, we calculated the scaled mass index (mi) for each animal (peig and green, 2009, 2010), which was computed as follows: mi = mi (l0/li) bsma where mi and li are body weight (g) and length (mm), respectively, of individual i; bsma is the scaling exponent estimated by the sma regression of m on l; l0 is the arithmetic mean value of body length for the whole dataset; and mi is the predicted body weight for individual i when the linear body measurement is standardized to l0. we removed the testes and immediately measured their length (means ± se in mm: rf = 5.304 ± 0.203; ds = 1.193 ± 0.031) and width (means ± se in mm: rf = 1.818 ± 0.059; ds = 0.755 ± 0.016) with a digital caliper, under binocular stereo microscope. we estimated testicular volume using the formula for the volume of a prolate spheroid (dunham, 1981). additionally, the presence of vitellogenic oocytes in bidder´s organs in r. fernandezae was explored, following mccoy et al. (2008). although both testes are of the same size, we analyzed the right testis in order to standardize the procedure. the right testis of each species was fixed in 4% formaldehyde solution for 1 to 8 hours, depending upon size (which varies widely between species) because 4% formaldehyde penetrates at an approximated rate of 1 mm per hour (fox et al., 1985). afterwards, testes were transferred to 70% alcohol for histological processing. the specimens were deposited in the herpetological collection of centre of scientific investigation and transference of technology to the production (cicyttpconicet), diamante, entre ríos, argentina. histological evaluation we evaluated testis histology using light microscopy. after dehydration in increasing concentrations of ethanol, and clearing with xylene, testes were embedded in paraffin (bancroft and gamble, 2002). transverse sections (7 μm) were cut and stained with hematoxylin–eosin (bancroft and gamble, 2002). for each male, we examined 10 seminiferous tubules randomly selected from five different sections in the central part of each testis (díaz-páez and ortiz, 2001; ferreira et al., 2008). in turn, the five sections analyzed were separated by at least five sections of distance, to avoid evaluating the same tubules in different sections. for each section, testicular area was measured, the total number of seminiferous tubules was determined, and the shape of the testis was classified as round or irregular, according to gyllenhammar et al. (2009). in addition, we measured the total area of each of the 10 seminiferous tubules selected per individual. we classified all cysts, depending on the maturation stage of the germ cells, into one of the six categories: primary spermatogonia (i sg), secondary spermatogonia (ii sg), primary spermatocytes (i sc), secondary spermatocytes (ii sc), spermatids (sp), and spermatozoa (sz), following tsai et al. (2005) and gyllenhammar et al. (2009). the number of spermatozoa in the lumen of the seminiferous tubules was estimated and ranked from zero to three, with zero corresponding to the seminiferous tubules with no spermatozoa and three, to those with the highest number of spermatozoa (gyllenhammar et al., 2009). in addition, we analyzed sections to detect possible testicular anomalies (hecker et al., 2006), such as lack of development of the seminiferous tubules (e.g., sower et al., 2000; hayes et al., 2003), numerous pigment-containing cells (e.g., patiño et al., 2006; kloas et al., 2009), testicular oocytes (e.g., coady et al., 2005; mcdaniel et al., 2008), and lack of elongated spermatids (e.g., murata et al., 2002; edwards et al., 2006). all histological evaluations were made by one person to prevent observer bias. prior to evaluation, we randomized slides so that the observer was unaware (blind) of the origin site of each specimen. we used an arcano l 1200b htg microscope equipped with a sony dsc-w55 digital camera for taking photographs. histomorphometric measurements were made using image j software, version 1.32j (imagej, national institutes of health, bethesda, usa). data analysis we performed all statistical analyses with statistica software, version 6.0 (statistica for windows, statsoft inc., tulsa, usa). for each parametric analysis, we assessed normality of data distribution (kolmogorov–smirnov test) and homogeneity of variances (barlett test). we performed natural logarithmic transformations when it was necessary to meet the assumptions of the parametric tests. to compare testicular volume of individuals from different sites, the effect of svl had to be removed because, in general, larger individuals have greater testicular volume (e.g., ortega et al., 2005; sanabria et al., 2007). thus, we ran an ancova with site group as the fixed effect, testicular volume as the dependent factor, and svl as the covariate (mccoy et al., 2008). to test for significant differences between study sites, post-hoc comparisons within the ancova were made using the tukey’s test. before running the test, we checked the assumption of parallelism (homogeneity of slopes). mi, testicular area, total number of seminiferous tubules, and seminiferous tubular area were compared among study sites using a one-way analysis of variance (anova) combined with a tukey’s test. number of i sg cysts, ii sg cysts, i sc cysts, ii sc cysts, sp cysts, sz cysts per seminiferous tubule, and spermatozoa rank in the lumen are not independent of one another (since a cell type gives rise to another) and this can make interpretation difficult. therefore, these data were analyzed using a multivariate analysis of variance (manova) to assess if there were group differences in all the response variables considered simultaneously. after performing the manova, we used uni79effects of agriculture on amphibian gonads variate anova on each response variable to assess which variables were responsible for significant main effects. this was followed by post hoc comparisons (tukey’s tests) to test for significant differences among study sites (quinn and keough, 2002). in addition, we compared frequencies of irregularly shaped testes and testes with anomalies among sites using the test of difference between proportions (martínez-gonzález et al., 2006). results general measurements the manual captures of adult males resulted in n = 28 individuals of r. fernandezae and n = 29 individuals of d. sanborni. there were no differences in scaled mass index (mi) for both species among study sites (rf: f2,25 = 0.245, p = 0.784; ds: f2,26 = 2.567, p = 0.096). nevertheless, in r. fernandezae, the testicular volume was significantly different among site groups (f2,24 = 10.706, p = 0.0005). ag and nw groups had significantly reduced testicular volume than the nf group (tukey’s post-hoc test: p = 0.025 and p = 0.0005, respectively). dendropsophus sanborni showed no variation in testicular volume among sites (f2,25 = 0.116, p = 0.891). histological evaluation in r. fernandezae the seminiferous tubules/testis was significantly different among sites (f2,25 = 3.476, p = 0.046). there were significantly fewer seminiferous tubules/testis in the ag and nw groups than in nf group (tukey’s post-hoc test: p = 0.043 and p = 0.047 respectively). testicular area and loge (seminiferous tubular area) were not different among sites (f2,25 = 1.696, p = 0.204 and f2,25 = 2.476, p = 0.104, respectively; table 1). dendropsophus sanborni showed no significant differences among sites in these variables (testicular area: f2,26 = 0.066, p = 0.936; seminiferous tubules/testis f2,26 = 0.828, p = 0.448; and seminiferous tubular area f2,26 = 1.085, p = 0.353; table 1). numbers of germ cell cysts were significantly different among sites in both species (table 2). in r. fernandezae, primary spermatogonia, primary spermatocytes, and spermatid cysts per seminiferous tubule were different among sites (fig. 2a, table 2). tukey’s posthoc test showed that there were significantly fewer i sg and sp cysts per seminiferous tubule in the ag and nw groups than in nf group (i sg: p = 0.027 and p = 0.002 respectively; sp: p = 0.02 in both cases) and that there were significantly fewer i sc cysts per seminiferous tubule in the ag group than in the nf group (tukey’s post-hoc test: p = 0.008). number of secondary spermatogonias cysts, secondary spermatocytes cysts, spermatozoa cysts, and spermatozoa rank in the lumen were not different among sites (fig. 2a, table 2). in d. sanborni the primary and secondary spermatogonia cysts per seminiferous tubule were different among sites (fig. 2b, table 2). there were significantly fewer i sg cysts per seminiferous tubule in the ag and nw groups than in nf group (tukey’s post-hoc test: p = 0.044 and p = 0.01 respectively), and there were significantly fewer ii sg cysts per seminiferous tubule in the nw group than in the nf group (tukey’s post-hoc test: p = 0.049). cyst number of primary spermatocytes, secondary spermatocytes, spermatids and spermatozoa, and the spermatozoa rank in the lumen were not different among sites (fig. 2b, table 2). in r. fernandezae, the incidence of irregularly shaped testes did not differ among groups (nf = 33.3%, nw = 63.6%, ag = 62.5%). however, in d. sanborni, the incidence of irregularly shaped testes increased with agricultural intensity (nf = 30.0%, nw = 50.0%, ag = 72.7%), table 1. histological evaluation of testes of adult males of rhinella fernandezae and dendropsophus sanborni in agroecosystem (ag), natural wetland (nw), and natural forest (nf) sites in south-western entre ríos province, argentina. data are expressed as means ± se. means followed by different superscript letters (a or b) are significantly different from each other (p < 0.05, anova test combined with tukey’s test). site testicular area mm2 number of seminiferous tubules/testis * seminiferous tubular area mm2 ag n = 8 0.946 ± 0.254 52.775 a ± 4.455 0.015 ± 0.003 r. fernandezae nw n = 11 1.035 ± 0.172 53.545 a ± 4.045 0.020 ± 0.003 nf n = 9 1.431 ± 0.162 65.756 b ± 2.762 0.023 ± 0.002 ag n = 11 0.308 ± 0.027 22.564 ± 1.880 0.016 ± 0.002 d. sanborni nw n = 8 0.304 ± 0.025 26.975 ± 1.919 0.012 ± 0.001 nf n = 10 0.319 ± 0.034 24.460 ± 3.018 0.015 ± 0.002 * log transformed variable in rhinella fernandezae before performing the parametric significance test. however, for clarity, nontransformed data are presented. 80 l.c. sanchez et alii with statistically significant differences being found only between ag and nf (table 3). the analysis of testicular anomalies revealed cases of poorly developed seminiferous tubules, pigmented cells, testicular oocytes, and lack of elongated spermatids in both species. in r. fernandezae the incidence of poorly developed tubules (fig. 3a, b) increased with agricultural intensity (nf = 0.0%, nw = 27.3%, ag = 50.0%). this anomaly was significantly increased in ag and nw groups compared to the nf group, but no difference was found between ag and nw groups. the presence of pigmented cells distributed in the testicular interstitium (fig. 3c) was greater in ag (50%) and nw groups (63.6%) than in nf group (22.2%), and the incidence of this anomaly was significantly increased in nw compared to nf. only one individual with testicular oocytes was found; it belonged to the nw group (fig. 3d). additionally, only one individual with vitellogenic bidder´s organ was recorded. the specimen was collected in the nw site. in both cases, because of the small number of records, no statistical analyses were performed. the presence of specimens with lack of elongated spermatids increased with agricultural intensity (nf = 0.0%, nw = 18.2%, ag = 62.5%). ag group had a significantly higher number of individuals with no elongated spermatids than nw and nf groups; however, no differences were found between nw and nf groups (table 3). on the other hand, in d. sanborni the incidence of poorly developed tubules (fig. 3e, f) increased with agricultural intensity (nf = 0.0%, nw = 25.0%, ag = 27.3%). this anomaly was significantly increased in ag group compared to the nf group; however, no difference was found either between ag and nw groups or between nw and nf groups. the presence of pigmented cells distributed in the testicular interstitium (fig. 3g) was similar table 2. results of manova for overall effects of habitat types on numbers of cysts and anovas for each response variable. (i sg) primary spermatogonia, (ii sg) secondary spermatogonia, (i sc) primary spermatocytes, (ii sc) secondary spermatocytes, (sp) spermatids, (sz) spermatozoa, (sz in lumen) spermatozoa rank in the lumen. f d.f. p r. fernandezae manova 2.105 14,38 0.035 anova s i sg 8.372 2,25 0.002 ii sg 1.098 2,25 0.349 i sc 5.722 2,25 0.009 ii sc 1.465 2,25 0.250 sp 5.516 2,25 0.010 sz 0.835 2,25 0.446 sz in lumen 0.485 2,25 0.622 d. sanborni manova 2.025 14,40 0.041 anova s i sg 5.754 2,26 0.009 ii sg 3.713 2,26 0.038 i sc 2.371 2,26 0.113 ii sc 1.825 2,26 0.181 sp 0.197 2,26 0.823 sz 0.464 2,26 0.634 sz in lumen 3.223 2,26 0.056 fig. 2. histological evaluation of germ cell cysts in the testes of adult males of rhinella fernandezae (a) and dendropsophus sanborni (b) collected from the agroecosystem (dark gray), natural wetland (light gray), and natural forest (white) sites, in south-western entre ríos province, argentina. (i sg) primary spermatogonia, (ii sg) secondary spermatogonia, (i sc) primary spermatocytes, (ii sc) secondary spermatocytes, (sp) spermatids, (sz) spermatozoa. box-plot diagram shows the quartiles, median (band inside the box), mean (square inside the box), minimum and maximum (ends of the whiskers), and presence of extreme values (black dots). 81effects of agriculture on amphibian gonads in the three study sites (nf = 40.0%, nw = 37.5%, ag = 36.4%). three individuals with testicular oocytes were recorded, one in each environment (nf = 10.0%, nw = 12.5%, ag = 9.1%; fig. 3h). as a result, the observed proportions were not different among sites. we found only one individual with lack of elongated spermatids, which belonged to the nw group. because we recorded a small number of cases, we did not perform a statistical analysis on this testicular anomaly (table 3). discussion this is the first work exploring the association of abnormal gonadal form and function with agricultural activities in wild r. fernandezae and d. sanborni. specifically, testicular volume and shape, development of seminiferous tubules, and presence of several spermatogenic stages were affected with increasing agricultural intensity. table 3. summary of proportion tests for variables analyzed by means its frequency in rhinella fernandezae and dendropsophus sanborni. (ag) agroecosystem, (nw) natural wetland, and (nf) natural forest. variable comparison r. fernandezae d. sanborni z p z p irregularly shaped testes nf – nw 1.417 0.078 0.875 0.192 nf – ag 1.255 0.104 2.163 0.015 nw ag 0.051 0.480 1.024 0.154 poorly developed tubules nf – nw 2.031 0.021 1.633 0.052 nf – ag 2.828 0.002 2.031 0.021 nw ag 1.024 0.154 0.112 0.456 pigmented cells nf – nw 2.064 0.020 0.108 0.456 nf – ag 1.237 0.108 0.171 0.433 nw ag 0.596 0.274 0.051 0.480 testicular oocytes nf – nw _ _ 0.166 0.433 nf – ag _ _ 0.071 0.472 nw ag _ _ 0.234 0.409 lack of elongated spermatids nf – nw 1.564 0.059 _ _ nf – ag 3.652 0.0002 _ _ nw ag 2.142 0.016 _ _ fig. 3. histological sections of anuran testes. rhinella fernandezae: (a) testis with normal seminiferous tubules, (b) testis with poorly developed tubules, (c) testis showing pigmented cells, (d) testis with a single oocyte. dendropsophus sanborni: (e) testis with normal seminiferous tubules, (f) testis with poorly developed tubules, (g) testis showing pigmented cells, (h) testis with multiple oocytes. (st, solid line) normal seminiferous tubules, (p-st, dotted line) poorly developed seminiferous tubules, (pc) pigmented cells, (o) oocytes. in all images, individuals from the natural wetland are shown. bar represents 100 μm. 82 l.c. sanchez et alii gonadal abnormalities, such as those reported here, are likely to reduce the reproductive success of affected individuals and could help explain the declines in amphibian populations reported in environments exposed to pesticides (davidson et al., 2002; davidson, 2004; mccoy et al., 2008). general measurements the scaled mass index (mi) for adult males of r. fernandezae and d. sanborni did not differ significantly among the environments studied, indicating that the three populations of each species would not present differences in their nutritional state or energy capital accumulated in the body as a result of feeding (peig and green, 2009). on the other hand, we observed a decrease in testis volume of r. fernandezae in environments with greater exposure to agricultural activities (ag and nw). these observations might be influenced by the amplitude of the time window in which the r. fernandezae specimens were collected (december 2007 to april 2008). the time window might have influenced the testis volume measured if individuals had been captured from each site at different times within that period. however, the species were collected simultaneously from the three sites, and we did not expect differences in the mean values of any reproductive parameter among sites. additionally, martori et al. (2005) investigated possible changes in testicular volume of r. fernandezae between september 1999 and april 2000 in a field in córdoba province, argentina. as a result, the authors found no significant differences in that variable between those months, and they interpreted that testes are potentially active and are ready for reproduction throughout the spring-summer period. an alternative explanation is provided by taveramendoza et al. (2002). they reported that exposure to 21 μg/l atrazine for 48 h during development resulted in decreased testicular volume (57%) and decreased numbers of primary germ cells among male xenopus laevis (amphibia, pipidae) tadpoles (70%). accordingly, in the present study, individuals from ag may have been exposed to various pesticides at some time during their development, since the agroecosystem was sprayed with glyphosate, 2-4 d, cypermethrin and endosulfan, and the main implementation period coincides with the breeding season of amphibians (lorenzatti et al., 2004; peltzer and lajmanovich, 2007). during the time that these chemicals are used, rainfall can cause intense runoff, carrying agrochemicals to other nearby water bodies (peltzer et al., 2008), such as that located in nw; this fact might explain the observation of decreased testicular volume both in ag and nw anurans. on the other hand, d. sanborni showed no variation in testicular volume among sites. one explanation for the apparently greater vulnerability of r. fernandezae would be associated with terrestrial habitat use (sanchez and busch, 2008; sanchez et al., 2010). both species breed in ponds, flooded areas, temporary swamps, and the periphery of permanent lakes, where tadpoles live (sanchez et al., 2009, 2010), but newly metamorphosed r. fernandezae individuals build caves in the moist soil of the periphery of the water body (gallardo, 1969). consequently, despite having left the aquatic environment, they are potentially exposed to chemicals that may be in the water, because the sediment that forms the cave is embedded in water. in addition, unlike the specimens of d. sanborni, which can be frequently observed in the vegetation at a height of up to 1.5 m (toledo et al., 2003; conte and machado, 2005), r. fernandezae would be more directly exposed to pesticide sprays and the related surface runoff due to its burrowing-terrestrial habits. histological evaluation testes have many different vital structures (ogielska and bartmańska, 2009) involved with their main function: spermatozoa production. seminiferous tubules are of primary importance because they hold and release the sperm that is necessary to fertilize eggs (dutta and meijer, 2003). there are no data available on the gametogenic cycles of r. fernandezae and d. sanborni; however, studies in related species of the bufonidae and hylidae families in the region (e.g., bufonidae: rhinella arenarum, r. major; hylidae: dendropsophus minutus, hypsiboas riojanus, lysapsus limellum) indicate the presence of potentially uninterrupted gonadal activity and continuous annual spermatogenesis (cei, 1949; santos and oliveira, 2007; ferreira et al., 2008). these species are characterized by keeping constant values of various morphometric parameters of the testis (e.g., testicular weight, area and diameter of the seminiferous tubules) and almost constant presence of all spermatogenic stages throughout the year (santos and oliveira, 2007; ferreira et al., 2008). nevertheless, basso (1990) suggests that females of d. sanborni have a seasonal cycle, with breeding taking place only in spring and summer. in our study, in the anuran testes from the nf site, the overall structure showed a regular pattern of seminiferous tubules surrounded by a layer of interstitial tissue. by contrast, the overall structure of the ag (r. fernandezae = 50.0%; d. sanborni = 27.3%) and nw testes (r. fernandezae = 27.3%; d. sanborni = 25.0%) was disrupted and the shape of the tubules was not well defined. the potential exposure of anuran individuals to agrochemi83effects of agriculture on amphibian gonads cals could induce damage to the layers of interstitial tissue surrounding the seminiferous tubules, making them less visible (dutta and meijer, 2003). this could explain the lower number of seminiferous tubules recorded in both ag and nw r. fernandezae groups. the structure of the seminiferous tubules might also be severely disrupted. similar results were found by hayes et al. (2003) in anurans exposed to atrazine (field and laboratory experiments), by dutta and meijer (2003) in bluegills exposed to diazinon (laboratory experiences), and by aljahdali and bin bisher (2007) in rats treated with sumithion insecticide (laboratory experiments). in addition, the number of cysts of germ cells inside the seminiferous tubules was generally decreased in r. fernandezae and d. sanborni collected from environments with some level of influence of agricultural activities (ag and nw). accordingly, sower et al. (2000) reported a lack of development in the seminiferous tubules and reduced numbers of spermatogonias in individuals of lithobates clamitans and l. catesbeianus (amphibia, ranidae), and associated these results with endocrine disrupting chemicals. those results reinforce our observations that agricultural pesticides might be driving the abnormalities observed in gonadal form and function. in addition, other testicular anomalies were recorded, such as pigmented cells and testicular oocytes. pigmented cells increased in ag and nw r. fernandezae toads, whereas they were recorded in similar proportions in d. sanborni in the three sites. many studies suggest that the general function of these cells is focalization of destruction, detoxification, or recycling of endogenous and exogenous materials (ellis, 1980; herraez and zapata, 1986). there are several examples of the increase of pigmented cells in gonads, kidney or spleen in relation to pollutants, and they have been promoted as biomarkers of environmental exposure to pollutant chemicals both in frogs and fishes (wolke et al., 1995; couillard and hodson, 1996; patiño et al., 2006; kloas et al., 2009). in addition, a recent study shows that actin filaments in pigmented cells of x. laevis are affected by roundup formulations (glyphosate-based herbicide), and that, consequently, intracellular transport of pigment and aggregation of melanin to the cell centre are inhibited, rendering the cells a dark color (hedberg and wallin, 2010). accordingly, pigmented cells may be present in higher numbers in environments with greater agricultural exposure, or may not be more numerous but may have become more conspicuous because of damage in the transport mechanism of melanin. on the other hand, only one r. fernandezae with testicular oocytes was found and it was in nw group, whereas similar proportions of this anomaly were recorded for d. sanborni in the three environments. there has been some still unresolved debate in the literature regarding the relevance of testicular oocytes in amphibians and whether there may be a background rate resulting from natural processes in development, at least in juvenile individuals (e.g., jooste et al., 2005; storrs-méndez and semlitsch, 2010), or may be induced by exposure to pesticides or other estrogenic endocrine disrupting compounds (e.g., hayes et al., 2003; mcdaniel et al., 2008; tompsett et al., 2012, 2013; trachantong et al., 2013). with regard to vitellogenic bidder’s organ, only one individual of r. fernandezae with this condition was recorded and it was in nw site. vitellogenic oocytes within bidder’s organs in r. marina (amphibia, bufonidae) have been recently associated with agricultural sites in florida, united states (mccoy et al., 2008). these authors suggest that the testes of these toads may not function normally to suppress bidder’s organ oogenesis (mccoy et al., 2008). in general, pathological conditions in the testes, such as those reported here, may be associated with nutritional or environmental factors that contribute to these abnormalities (siddiqui et al., 2005). however, in this case, the nutritional status of the amphibian populations (analyzed by means of the scaled mass index) of each studied species did not differ among sites (peig and green, 2009). however, the frequency of testicular anomalies was found to increase, in general, with agricultural activity and was very low or absent in the site with zero agriculture, suggesting that these anomalies would not occur at a high frequency in areas not affected by agriculture (mccoy et al., 2008). evidence suggests an increased vulnerability of r. fernandezae compared with d. sanborni (based on greater evidence of affected morphology and gonadal histoarchitecture). this agrees with the results reported by lajmanovich et al. (2010), who indicate this bufonid as one of the species at highest ecological risk due to conversion of native environments to soybean crops. conclusions the reduced presence of germ cells and the high numbers of testicular anomalies in r. fernandezae and d. sanborni from environments with great agricultural exposure indicate an overall negative effect of agricultural land use upon testes (both abnormal testicular development and function) in these wild anurans from the southwestern entre ríos province, argentina. these animals are exposed to pesticides in soybean crops (jergentz et al., 2005). previous works have detected residues of organochlorine pesticides (e.g., chlordane, heptachlor, and endosulfan) in tissues of native anurans from the region (laj84 l.c. sanchez et alii manovich et al., 2002), and the concentrations recorded were strongly influenced by the use of intensively cropped lands (lajmanovich et al., 2005). therefore, the establishment of buffer zones around pristine natural forests could help reduce runoff from surrounding agricultural fields, increasing the conservation value of such areas. several factors should be taken into account in the design of buffer zones, such as type of vegetation in the buffer, the slope, infiltration rate and soil texture (hawes and smith, 2005). structurally diverse vegetation (combined presence of trees, shrubs and grasses) is much more effective at capturing a wide range of pollutants than a buffer with only trees or grass. in this sense, native plants are able to acquit better benefit than alien ones (essien, 2012). in addition, the speed of water flow increases with increasing slope. therefore, the steeper the land within the buffer, the wider it needs to be to have time to slow the flow of water and absorb the pollutants within it (wenger, 1999). on the other hand, it is important to investigate the roles of agricultural activities and the effectiveness of regulations on agrochemicals, particularly in latin american countries, in causing ecological risk for amphibians as native ecosystems are converted to soybean cultivations (lajmanovich et al., 2010). although the sample size is low, the extensive global use of pesticides and conversion of land use into agriculture, affecting amphibians, stresses the importance of the present findings. the data obtained in this study can help assess the ecological risk of land use for agriculture to argentine anurans. acknowledgements we thank dr. krista a. mccoy and dr. cecilia berg for their critical reviews and helpful comments on the earlier versions of the manuscript. jorgelina brasca revised the english text. we thank dr. dante a. paz for assistance, and dr. pablo aceñolaza for establishing contact with the cooperativa agrícola de diamante. we thank andrés sarquis and marcos galeano for their help in the field, and the landowners who allowed us to have access to their property. permits for access and field work in pre-delta national park were provided by delegación técnica de parques nacionales regional noreste. we thank park rangers for their support during the study. we are thankful to the conicet (consejo nacional de investigaciones científicas y técnicas) for providing the financial support as fellowships during the first author’s doctoral thesis. this work was also supported by grants am conicet pip112-200801-00225 and rl pict 1148. references aceñolaza, p.g., povedano, h.e., manzano, a.s., muñoz, j., areta, j.i., ronchi virgolini, a.l. 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(2013): effects of atrazine herbicide on metamorphosis and gonadal development of hoplobatrachus rugulosus. maejo int. j. sci. technol. 7: 60-71. tsai, p.s., kessler, a.e., jones, j.t., wahr, k.b. (2005): alteration of the hypothalamic-pituitary-gonadal axis in estrogenand androgen-treated adult male leopard frog, rana pipiens. reprod. biol. endocrinol. 3: 2. usha, s., harikrishnan, v.r. (2005): endosulfan: fact sheet and answers to common questions. interna88 l.c. sanchez et alii tional pops elimination network (ipen) pesticide working group, thanal, trivandrum, kerala. wenger, s. (1999): a review of the scientific literature of riparian buffer width, extent and vegetation. institute of ecology, university of georgia, athens, georgia. wolke, r.e., george, c.j., blazer, v.s. (1995): pigmented macrophage accumulations (mmc; pmb): possible monitors of fish health. in: parasitology and pathology of the world oceans, pp. 27-33. hargis, w.j., ed, national marine fishery service, washington d.c. zimmerman, b.l. (1994): audio strip transects. in: measuring and monitoring biological diversity standard methods for amphibians, pp. 92-97. heyer, w.r., donelly, m.a., mcdiarmid, r.w., hayek, l.c., foster, m.s., eds, smithsonian institution press, washington and london. acta herpetologica vol. 8, n. 2 december 2013 firenze university press journal of the societas herpetologica italica acta herpetologica acta herpetologica 10(1): 75-76, 2015 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-16084 book review: antonio romano. atlante degli anfibi del parco nazionale del cilento vallo di diano e alburni distribuzione, biologia, ecologia e conservazione sebastiano salvidio dipartimento di scienze della terra, dell’ambiente e della vita (distav), università di genova, i 16132 genova italy. e-mail: salvidio@dipteris.unige.it the “parco nazionale del cilento vallo di diano e alburni” (pncvda) is the largest and one of the most diverse national protected areas in italy. it is located in the southern part of the country, in the region of campania, not far from the boundary with basilicata and calabria. the park is really huge, covering more than 180.000 ha, and ranges from the mediterranean sea coast to the inner apennine mountains. from a naturalistic point of view, the pncvda is particularly well known for its plant and bird diversities, while the entire region offers to its visitors one of the most genuine and tasty examples of the true “mediterranean diet”. in recent years, there have been several herpetological publications in campania (e.g. romano et al., 2008, 2010a, b; guarino et al., 2012), suggesting an increasing interest on these zoological groups inhabiting the southern apennine environment. therefore the publication of this atlas appears as an obvious synthesis of these previous works and updates both the recent regional atlas of guarino et al. (2012) and the italian atlas that has been published less than 10 years ago (sindaco et al., 2006). the “atlante degli anfibi del parco nazionale del cilento vallo di diano e alburni” is completely written in italian, counts 164 pages and is forwarded by two presentations, the first by the park’s president and the second one by the president of the societas herpetologica italica, one of the many organisations that sponsored the volume. this book is organised in six main chapters and a reference list. the first two chapters treat the general park features (geology, flora, wildlife and aquatic habitats) and give some biological generalities on amphibians. an identification key for the species inhabiting the park, both textual and iconographic, is also provided. the third chapter is dedicated to the historical overview on the previous herpetological studies, while the fourth describes the methodology used to collect field data. the fifth chapter describes the distribution, ecology and conservation status of the eleven morphospecies found within the study territory: salamandra salamandra, salamandrina terdigitata, lissotriton italicus, triturus carnifex, bombina pachypus, bufo balearicus, bufo bufo, hyla intermedia, pelophylax kl. esculentus & p. lessonae (water frogs are treated together), rana dalmatina and rana italica. each species has its own colour map, an histogram visualising the altitudinal distribution, a pie chart informing about the different kinds of breeding habitats (e.g., running water sites, lakes and marshes, artificial tanks and so on). moreover, each species is illustrated by many photos describing phenotypic variation of adults, larvae and eggs, together with some examples of the species typical breeding habitats. in general, the species accounts are very informative about the ecology and current species conservation at the global and local level, and it is clear that all this information is truly first hand. the final chapter is devoted to the analysis of the amphibian community found in the pncvda. different issues are here discussed and analysed: biogeography, altitudinal distribution, ecological niches, diffusion, rarity and the general conservation status of amphibian populations. 76 s. salvidio the reference list is extensive, as it counts more than 150 entries. this should be a good start for anyone interested in southern apennine batrachofauna. in conclusion, the “atlante degli anfibi del parco nazionale del cilento vallo di diano e alburni” is a well written, informative and should be useful for all herpetologists working in or visiting the italian apennines and looking for amphibians in natural or artificial habitats. the book layout is agreeable and illustrations are printed in high quality. the only minor criticism to this concise but informative volume, is that the typesetting is relatively small, maybe to spare space (but there are large and under-used page margins), while the symbols that are used to describe the species conservation status are so tiny that are difficult to read, for example the “bern convention” symbol is so small that becomes almost un-recognisable and the letters under the logo are difficult to read even with a magnifying 20x lens. this volume may be requested writing to “parco nazionale del cilento e del vallo di diano p.zza s. caterina, 8, i-84078 vallo della lucania (sa), italy or by email: l.deriso@cilentoediano.it references guarino, f.m., aprea, g., caputo, v., maio, n., odierna, g., picariello, o. (2012): atlante degli anfibi e dei rettili della campania. massa editore, napoli. sindaco r., doria g., razzetti e., bernini f (2006): atlante degli anfibi e rettili d’italia – atlas of amphibians and reptiles in italy. edizioni polistampa, firenze. romano, a., spilinga, c., ventre, n., de riso, l. (2008): gli anfibi del parco nazionale del cilento e vallo di diano (campania) dati preliminari. in: herpetologia sardiniae, pp. 425-238. corti, c. ed, edizioni belvedere, latina. romano a., ventre n., de riso l., pignataro c., spilinga c., (2010a): amphibians of the “cilento e vallo di diano” national park (campania, southern italy): update check list, distribution and conservation notes. acta herpetol. 5: 233-244. romano a., de riso l., pignataro c., ventre n., spilinga c., (2010b): gli anfibi del massiccio degli alburni nel parco nazionale del cilento e vallo di diano (campania). in: atti viii congresso nazionale societas herpetologica italica, pp: 117-120. di tizio, l., di cerbo, a.r., di francesco, n., cameli, a. eds, ianieri edizioni, pescara. acta herpetologica vol. 10, n. 1 june 2015 firenze university press obituary: valery k. eremchenko (1949-2014) leo j. borkin1, tatjana dujsebayeva2, roberto sindaco3, matthias stöck4 haplotype variation in founders of the mauremys annamensis population kept in european zoos barbora somerová1, ivan rehák2,*, petr velenský2, klára palupčíková1, tomáš protiva1, daniel frynta1 reproductive ecology of sichuan digging frogs (microhylidae: kaloula rugifera) wei chen1,*, lina ren2, dujuan he2, ying wang2, david pike3 toxic effects of carbaryl on the histology of testes of bufotes variabilis (anura: bufonidae) özlem çakici basal frequency of micronuclei and hematological parameters in the side-necked turtle, phrynops hilarii (duméril & bibron, 1835) maría a. latorre 1,2,*,#; evelyn c. lópez gonzález1,2, #; pablo a. siroski1,2,3; gisela l. poletta1,2,4 into a box interiors: clutch size variation and resource allocation in the european pond turtle marco. a.l. zuffi1,*, simonetta citi2, elena foschi1, francesca marsiglia1, eva martelli1 where to “rock”? choice of retreat sites by a gecko in a semi-arid habitat andreia penado1,2, ricardo rocha3,4,*, marta sampaio3, vanessa gil3, bruno m. carreira3, rui rebelo3 age structure, growth and longevity in the common toad, rhinella arenarum, from argentina clarisa de l. bionda1,2,*, silvia kost 4, nancy e. salas1, rafael c. lajmanovich3, ulrich sinsch4, adolfo l. martino1 on a putative type specimen of pleurodema bibroni tschudi, 1838 from chile (anura: leptodactylidae) daiana paola ferraro re-description of the external morphology of phyllomedusa iheringii boulenger, 1885 larvae (anura: hylidae), with comments on the external morphology of tadpoles of the p. burmeisteri group samanta iop¹, victor mendes lipinski¹, bruno madalozzo¹, franciele pereira maragno¹, sonia zanini cechin¹, tiago gomes dos santos² book review: harold heatwole, john w. wilkinson (eds). amphibians biology. volume 11 status of conservation and decline of amphibians. eastern hemisphere. part 4 . southern europe and turkey sebastiano salvidio book review: antonio romano. atlante degli anfibi del parco nazionale del cilento vallo di diano e alburni distribuzione, biologia, ecologia e conservazione sebastiano salvidio acta herpetologica journal of the societas herpetologica italica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 7(2): 309-313, 2012 evidence of high longevity in an island lacertid, teira dugesii (milne-edwards, 1829). first data on wild specimens j. jesus centro de competências de ciências da vida, universidade da madeira, campus universitário da penteada, 9020-105 funchal, portugal. e-mail: jesus@uma.pt submitted on: 2012, 13th march; revised on: 2012, 23rd april; accepted on: 2012, 18th april. abstract. using the technique of capture, mark and recapture, here is reported a case of high longevity in the madeiran lizard, teira dugesii. it is one of the highest reported values for lacertid lizards (16 years or more) in a wild population. keywords. longevity, teira dugesii, madeira island. teira dugesii is a lacertid lizard endemic to the madeiran archipelago, and was introduced to some islands of the azores in the xix century (malkmus, 1985, 2004) and to the harbour area of lisbon (sá sousa, 1995). it is an omnivorous and oportunistic lacertid that inhabits a wide range of habitats, from sea level to the highest altitude of madeira archipelago (1871 m – pico ruivo, madeira island). it can be found in almost every island/islet of the archipelago (jesus et al., 2009). since the earliest 1990’s we have being studying t. dugesii in their native environments. most of the studies dealt with phylogeny and population genetics (e.g. brehm et al. 2001, 2002, 2003; jesus et al., 2005a). other studies have also been performed in this species, for example on diet (sadek, 1981), predation (jesus et al., 2005b), morphological variability (cook, 1979; báez and brown, 1997; jesus et al., 2006), and colour pattern variability (crisp et al., 1979; báez, 1990). curiously, no data exists on longevity in wild populations in the native area. the ecological aspects and life histories traits of wild populations are practically unknown, with a few studies performed on captive lizards (e.g., galan and vicente, 2003). in 1996 we started capture-mark recapture sessions on madeiran lizards to study growth, and some other aspects such as time of courtship, pregnancy, egg laying, hatchling and male maturation cycles, using toe-clipping. sessions lasted two years and several captures/recaptures were performed essentially in four localities of madeira island, using pitfall traps. the first was ponta de são lourenço located in the eastern part of the island, at approximately 70 m of altitude, a notably arid location. the second locality was pico 310 j. jesus do arieiro, with 1850 m of altitude, in the central mountain ridge, the third was curral das freiras, with 950m of altitude, in a inland position in the island, and the fourth was ribeira do alecrim, paul da serra, a plateau in the western part of the island, in the central mountain ridge at about 1300 m. in 2006, a unique event occurred in paul da serra. two (individuals 105 and 108) of the 7 lizards that were caught, were recaptured. according to svl values, in 1997, the individuals 105 and 108 were probably around or less than one year old (table 1). forty-six mm is the typical size of juveniles. although variation exists, according to cook (1979), animals of 45 mm or less are probably less than one year old. juveniles and young adults between 46 and around 60 are one or two years old, while larger ones are older mature adults. in 2011, another session of capture was performed. eighteen lizards were captured, three of which were recaptures. individual 10 was probably one year old in july 1996. regarding individual 204, it was difficult to estimate the age although probably, in september 2006, it was at least 2 years old (table 2). the most unexpected value belongs to individual 500. in 1997 it was already an adult female. the svl at that time suggests that the female was older than 2 years, and probably was born in 1995 or earlier, making her at least 16 years old in 2011. in this population males with svl longer than 75 (and even 80) mm are frequent, and it is even possible to find females with a similar size, almost reaching a svl of 80 mm. thus, although variability exists, there is a high probability to find females that are even older than this one. table 1. date of captures and svl of individuals 105 and 108 from paúl da serra individual 105 (female) individual 108 (male) date svl (mm) date svl (mm) 02 jun 1997 59 23 jun 1997 46 15 apr 1999 65 06 sep 2006 66 06 sep 2006 70 in 2006: 9 years * in 2006: 9 years* * estimated age table 2. date of captures and svl of individuals 105 and 108 of paúl da serra individual 10 (female) individual 204 (male) individual 500 (female) date svl date svl date svl 10 jul 1996 54 02 may 1997 67 15 apr 1999 66 06 sep 2006 68 19 jul 2011 73 19 jul 2011 78 19 jul 2011 75 at least 15 years * at least 6 years* at least 16 years* * estimated age 311high longevity in teira dugesii in 2011, one session of capture was performed in pico do arieiro, another high-altitude site, separated from paul da serra by deep valleys. nine individuals were captured, of which one (individual 75) was a recapture (table 3). probably in 1997, this individual was at least two years old, and was born in 1995 or earlier. in 2011, two attempts of capture in ponta de são lourenço were performed. fiftyseven lizards were captured and none were marked. these longevities are high when compared with other lacertid species, although most of the published data are from terrarium animals. slavens and slavens (1992, 1993) reported high longevities for captive reptiles including lacertids. for example they refer the following maximum ages: timon lepidus, 14 years and 5 months; lacerta trilineata, one individual 7 years and 9 months old and another 4 years and 4 months old; gallotia galloti, 5 years; podarcis pityusensis, 4 years and 7 months (still living at that time). a specimen of t. lepidus living in museum koenig has an estimated age of at least 28 years (philipp wagner, pers comm). bannert (1998) refers higher values for captive lacertid lizards, with 12 years for gallotia atlantica, gallotia caesaris and t. dugesii, 13 years for gallotia stehlini, lacerta bilineata and podarcis hispanica, 15 years for gallotia galloti eisentrauti and podarcis tiliguerta and 18 years for p. pityusensis. the value for t. dugesii is lower than our estimated age for wild specimens of this species. according to avery (1975) the mean expectation of life for zootoca vivipara was about 5 years. saint girons et al. (1989) obtained different longevities in two populations of lacerta viridis (5 and 8 or more). for podarcis muralis, castanet and roche (1981) obtained 6 years, bourliére (1946) 4 to 6 years, and flower (1937) 4 to 10 years. in lacerta agilis, in an alpine area of italy guarino et al. (2010) found that longevity was 4 years for males and 3 for females. in the same species, living in daghestan (russia), roitberg and smirina (2006) found longevities of 6 years for males and 5 years for females in the population from lowland and submontane regions (until 600 m. a.s.l.), but 7 years for males and 6 years for females in the population from highlands (starting from 960 m. a.s.l), emphasizing the variability in longevity. in the western coast of sweden olsson and shine (1996) also observed in l. agilis that maximum longevity was 11 years for males and 12 for females. although high, this value is still lower than that observed for t. dugesii. although the data is limited, an interesting aspect is that the longevity seems to be higher in high altitude populations. however, further captures are needed at different altitudes to confirm this. normally, these high longevities are common on islands forms, such as gallotia simonyi (20 years) (castanet and báez, 1991) and the extinct scincid macroscincus coctei table 3. date of captures and svl of individuals 105 and 108 of paúl da serra individual 75 (male) date svl (mm) 06 mar 1997 69 07 aug 2011 74 at least 15 years * * estimated age 312 j. jesus (16 years) (andreone and guarino, 2003). t. dugesii is usually much smaller than these two species, so it is probably one of the smallest lacertid lizard with such a high longevity. finally, this finding could be in accordance to the island theory, which states that species tend to have higher longevity than their mainland relatives (blondel, 1986). in fact, despite the lack of published data, there is evidence that scelarcis perspicillata, regarded as the mainland sister taxon to t. dugesii (harris et al., 1998), has lower longevity, about 4-5 years (ana perera, pers comm). acknowledgements i would like to acknowledge my friend james harris for helpful suggestions and assistance. i am also grateful to philipp wagner and dan cogalniceanu and sergé bogaerts for the precious bibliography and comments on reptile longevity. references andreone, f., guarino, f. (2003): giant and long-lived? age structure in macroscincus coctei, an extinct skink from cape verde. amphibia-reptilia 24: 459-470. avery, r. (1975): age-structure and longevity of common lizard (lacerta vivipara) populations. j. zool. 176: 555-558. báez, m. (1990): observaciones sobre colorido y diseño de podarcis dugesii en la isla de madeira. vieraea 18: 197-203. báez, m., brown, r. (1997): testing multivariate patterns within-island differentiation in podarcis dugesii from madeira. j. evol. biol. 10: 575-587. bannert, b. (1998): zur lebenserwartung lacertiden im terrarium. die eidechse 9: 59-66. blondel, j. (1986): biogéographie évolutive. masson, paris. bourliére, f. (1946): longevité moyene et logevité maximum chez les vertébrés. année boil. 22: 249-270. brehm, a., khadem, m., jesus, j., andrade, p., vicente, l. (2001): lack of congruence between morphometric evolution and genetic differentiation suggests a recent dispersal and loal habitate adaptation of the madeiran lizard lacerta dugesii. genet. sel. evol. 33: 671-685. brehm, a., harris, d.j., alves, c., jesus, j., thomarat, f., vicente, l. (2002): structure and evolution of the mitochondrial dna complete control region in the lizard lacerta dugesii (lacertidae, sauria). j. mol. evol. 55: 1-8. brehm, a., jesus, j., spínola, h., alves, c., vicente, l., harris, d. j. (2003): phylogeography of the madeiran endemic lizard lacerta dugesii inferred from mtdna sequences. mol. phylogenet. evol. 26: 222-230. castanet, j., báez, m. (1991): adaptation and evolution in gallotia lizards from the canary islands: age, growth, maturity and longevity. amphibia-reptilia 12: 81-102. castanet, j., roche, e. (1981): détermination de l’âge chez le lézard des murailles, lacerta muralis (laurenti, 1768) au moyen de la squelletochronologie. rev. suisse zool. 88: 215-226. 313high longevity in teira dugesii cook, l. (1979): variation in the madeiran lizard lacerta dugesii. j. zool. 187: 327-340. crisp, m., cook, l., hereward, f. (1979): color and heat balance in the lizard lacerta dugesii. copeia 1979: 250-258. flower, s. (1937): further notes on the duration of life in animals. iiireptiles. proc. zool. soc. lond. 107:1-39. galán, p., vicente, l. (2003). reproductive characteristics of the insular lacertid teira dugesii. herpetol. j. 13: 149-153. guarino, f., già, i., sindaco, r. (2010): age and growth of the sand lizards (lacerta agilis) from a high alpine population of north-western italy. acta herpetol. 5: 23-29. harris, d.j., arnold, e.n., thomas, r.h. (1998): relationships of lacertid lizards (reptilia: lacertidae) estimated from mitochondrial dna sequences and morphology. proc. r. soc. lond. b 265: 1939–1948. jesus, j., brehm, a., harris, d.j. (2005a): is c-mos phylogenetically informative at lower taxonomic levels in reptiles ? an assessment of variation within lacerta (teira) dugesii milne-edwards, 1829 (squamata: sauria: lacertidae). herpetozoa 18: 55-59. jesus, j., sampaio, l., silva, l. (2005b): incidence of lacerta dugesii milne-edwards, 1829 (sauria, lacertidae) in the diet of kestrels (falco tinnunculus canarinsis koenig, 1889; aves, falconidae) in a semiarid zone of madeira. herpetol. bull. 93: 14-16. jesus, j., sampaio, l., crespo, e. (2006): high frequency of lack of occipital scale in madeiran lizard lacerta dugesii milne-edwards, 1829 (sauria, lacertidae), on a very small island, selvagem pequena (selvagens, portugal). bol. asoc. herpetol. esp. 17: 88-93 jesus, j., teixeira, s., teixeira, d., freitas, t., russo, d. (2009): vertebrados terrestres autóctones dos arquipélagos da madeira e selvagens. direcção regional de ambiente. funchal. malkmus, r. (1985): zur verbreitung von rana perezi und lacerta dugesii auf den azoren. nachrichten naturw. mus. aschaffenburg 92: 37-69. malkmus, r. (2004): amphibians and reptiles of portugal, madeira and the azores-archipelago. a.r.g. gantner verlag k.g., ruggell, germany. olsson, m., shine, r. (1996): does reproductive success increase with age or with size in species with indeterminate growth? a case study using sand lizards (lacerta agilis). oecologia 105: 175-178. roitberg, e., smirina, e. (2006): age, body size and growth of lacerta agilis boemica and l. agilis strigata: a comparative study of two closely related lizard species based on skeletochronology. herpetol. j. 16: 133-148. sá sousa, p. (1995): the introduced madeiran lizard, lacerta (teira) dugesii. amphibiareptilia 16: 211-214. sadek, r. (1981): the diet of the lizard lacerta dugesii. biol. j. linn. soc. 73: 313-341. saint girons, h., castanet, j., bradshaw, s., baron, j. (1989). démographie comparée de deux populations françaises de lacerta viridis (laurenti, 1768). rev. ecol. (terre vie) 44: 361-386. slavens, f., slavens, k. (1992): reptiles and amphibians in captivity. breeding-longevity and inventory. slaveware, seattle, washington. slavens, f., slavens, k. (1993). reptiles and amphibians in captivity. breeding-longevity and inventory. slaveware, seattle, washington. acta herpetologica vol. 7, n. 2 december 2012 firenze university press advertisement call of species of the genus frostius cannatella 1986 (anura: bufonidae) flora a. juncá1, david l. röhr2, ricardo lourenço-de-moraes3, flávio j. m. santos1, airan s. protázio1, ednei a. mercês1, mirco solé4 amphibians in southern apennine: distribution, ecology and conservation notes in the “appennino lucano, val d’agri e lagonegrese” national park (southern italy). antonio romano1,*, remo bartolomei1, antonio luca conte1, egidio fulco2 the significance of using satellite imagery data only in ecological niche modelling of iberian herps neftalí sillero1, josé c. brito2, santiago martín-alfageme3, eduardo garcía-meléndez4, a.g. toxopeus5, andrew skidmore5 reproductive strategy of male and female eastern spiny lizards sceloporus spinosus (squamata: phrynosomatidae) from a region of the chihuahuan desert, méxico aurelio ramírez-bautista1,*, barry p. stephenson2, xóchitl hernández-ibarra1, uriel hernández-salinas1, raciel cruz-elizalde1, abraham lozano1, and geoffrey r. smith3 morphological variability of the hermann’s tortoise (testudo hermanni) in the central balkans katarina ljubisavljević1, georg džukić1, tanja d. vukov1, miloš l. kalezić1,2 the usefulness of mesocosms for ecotoxicity testing with lacertid lizards maria josé amaral1,2,*, rita c. bicho1, miguel a. carretero2, juan c. sanchez-hernandez3, augusto m. r. faustino4, amadeu m. v. m. soares1, reinier m. mann1,5 does acclimation at higher temperatures affect the locomotor performance of one of the southernmost reptiles in the world? jimena b. fernández*, nora r. ibargüengoytía advertisement call of scinax littoralis and s. angrensis (amphibia: anura: hylidae), with notes on the reproductive activity of s. littoralis michel v. garey1, thais r. n. costa2, andré m. x. de lima2, luís f. toledo3, marília t. hartmann4 book review: marine s. arakelyan, felix d. danielyan, claudia corti, roberto sindaco, alan e. leviton 2011. herpetofauna of armenia and nagorno-karabakh. society for the study of amphibians and reptiles stefano scali book review: rafaqat masroor 2012. a contribution to the herpetofauna of northern pakistan stefano scali evidence of high longevity in an island lacertid, teira dugesii (milne-edwards, 1829). first data on wild specimens. j. jesus first assessment of the endoparasitic nematode fauna of four psammophilous species of tropiduridae (squamata: iguania) endemic to north-eastern brazil markus lambertz1,*, tiana kohlsdorf2, steven f. perry1, robson waldemar ávila3, reinaldo josé da silva4 rediscovery and redescription of the holotype of lygosoma vittigerum (= lipinia vittigera) boulenger, 1894 yannick bucklitsch1, peter geissler1, timo hartmann1, giuliano doria2 , andré koch1,* reproductive phenology of the tomato frog, dyscophus antongili, in an urban pond of madagascar’s east coast ori segev1,*, franco andreone2, roberta pala2, giulia tessa2, miguel vences1 range extension of the critically endangered true poison-dart frog, phyllobates terribilis (anura: dendrobatidae), in western colombia roberto márquez1,*, germán corredor2, carlos galvis3 daniel góez2, & adolfo amézquita1 differences in habitat use of two sympatric species of ameiva in east costa rica esther sebastián-gonzález1, ramón gómez2 acta herpetologica journal of the societas herpetologica italica acta herpetologica 9(2): 265-266, 2014 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-15002 book review: andrea ambrogio, sergio mezzadri. girini d’italia tadpoles of italy edoardo razzetti museo di storia naturale, università degli studi di pavia, piazza botta 9/10, 27100 pavia, italy. e-mail: razzetti@unipv.it writing a review of this book is quite difficult as it is unusual in some aspects, i would define it “a field guide to the tadpole of italy” but since its dimensions are 21 × 30 cm i am afraid that it would not fit in the category of pocket size books. the text is completely bilingual and divided in two columns with italian on the left and english on the right, this solution increases the total number of (coated) pages (102) and thus the weight of the book, but makes it more useful and interesting for many foreign batrachologists. the frontispiece shows the logo of societas herpetologica italica, which patronized the publication, and the handwritten notation of which copy out the set of 800 copies the particular book is (this is a limited edition of numbered copies). another unusual solution is the cloth hardcover binding with just the title engraved in golden letters and no dust jacket, which again is atypical for a book published in the xxi century. the book starts with a nice foreword by giuliano doria followed by an acknowledgment list. there is a long introduction that deals not only with general aspects of anuran larvae and their biology but also gives useful suggestions on how to breed them and take good pictures, which can be useful to obtain a reference set of images to compare when the identification is apparently not reliable. it is important to tell that the book was written as a tool for tadpole field identification through the observation of external morphology, colour pattern and behavioural features (!); while the “standard” study of cheratodonts was not reported because it is necessary to kill or anesthetize the tadpoles to adopt it. next is a proposal of a dichotomous keys for the identification of live tadpoles (34 to 39 gosner stages); in general i do not rely too much on identification keys because they are based on a limited number of morphological characters that can easily bring to mistakes (cf. dubois, 1998), but i cannot deny the fact that these keys can be very useful to focus on the most useful characters or when experience is lacking. i would expect some data on young tadpoles (some species like bufo bufo are easy to distinguish in the field) or egg masses identification but this monograph deals only with well-developed larval stages. the core of the book consists of the species accounts and the plates that represent about the 90% of the book. the 13 full colour plates that depict the tadpoles are brilliant pieces of art. they are by far the best drawings of tadpoles i’ve ever seen, and i feel that andrea ambrogio succeeded well in the difficult task of giving the tadpoles a “living” appearance. one of the aspects of the plates that i appreciate most is that they do not depict the “standard” tadpole but they represent (when needed) different individuals to show variation in shape and colours which can be crucial to obtain a reliable identification. each species account starts with a detailed description similar to the ones proposed by bühler et al. (2007). most of the characters proposed are new and allow to distinguish even difficult species like rana dalmatina, rana latastei, rana italica and rana temporaria; this is surprising as in the past i realized how can be tough to identify a brown frog’s tadpole from the northern apennines or the po plain. it is difficult to know if these characters are really reliable, only time and experience will tell, but i am quite confident that, given the passion and experience of the authors, these have been carefully pondered. the rest of the species accounts deals in great detail with the biology of the larvae including geographic and altitudinal distribution, egg-laying habitat and period, water temperature at deposition, incubation and development time, diet, overwintering, predators and syntopy with other amphibians. the reference lists (each one located at the end of the species accounts) are accurate and complete. 266 edoardo razzetti is this book really useful? to me the answer is absolutely yes: even if there are several guides that offer identification keys and descriptions for tadpoles (the “classic” ones: lanza, 1983; nöllert and nöllert, 1992; arnold, 2002; lanza et al., 2007) most of them show drawings that are just modified version of previous authors works (sometimes of the xix century). ambrogi and mezzadri correct some mistakes that have been repeated several times, including the corrugated superior part of the body of the american bullfrog lithobates catesbeianus (which is probably an artefact due to alcohol preservation) and point out some obvious identification characters previously omitted (in european literature) like the black spots on the body (again in l. catesbeianus). as final argument i would like to add that the identification of anuran larvae can be an invaluable tool during amphibian surveys: just think that one of the most important (maybe the most important) italian population of pelobates fuscus has been detected, over 20 years ago, on the basis of few tadpoles only (gentilli et al., 1994). tadpoles of italy can be purchased from the publisher’s website (http://www.dranae.it/). references ambrogio, a., mezzadri, s. (2014): girini d’italia -tadpoles of italy. gavia edizioni, piacenza. arnold, e.n. (2002): a field guide to the reptiles and amphibians of britain and europe (collins field guides.). harper collins publ. ltd., london. bühler, c., cigler, h., lippuner, m. (2007): larve degli anfibi della svizzera: chiave di determinazione, fauna helvetica vol.19. centre suisse de cartographie de la faune. dubois, a. (1998). mapping european amphibians and reptiles: collective inquiry and scientific methodology. alytes, paris 15: 176-204. gentilli, a., scali, s., zuffi, m. (1996): confirmation of the presence of pelobates fuscus insubricus in province of varese (amphibia, anura, pelobatidae). natura bresciana, ann. mus. civ. sc. nat., brescia 30: 259-262. lanza, b. (1983): guide per il riconoscimento delle specie animali delle acque interne italiane. 27. anfibi, rettili (amphibia, reptilia). [collana del progetto finalizzato «promozione della qualità dell’ambiente» aq/1/205]. consiglio nazionale delle ricerche, roma. lanza, b., andreone, f., bologna, m.a., corti, c., razzetti, e., (eds) (2007): fauna d’italia, vol. xlii, amphibia, calderini, bologna. nöllert, a., nöllert, c. (1992): die amphibien europas. bestimmung, gefährdung, schutz. franckh-kosmos verlags-gmbh & co., stuttgart. issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 9(1): 15-23, 2014 doi: 10.13128/acta_herpetol-12931 the tadpole of odontophrynus barrioi cei, ruiz, and beçak, 1982 (anura: odontophrynidae): a comparison with the other tadpoles of the genus exequiel gonzález1, guillermina galvani1, eduardo sanabria2,*, diego a. barrasso3, leandro alcalde4, lorena quiroga1 1 departamento de biología, facultad de ciencias exactas físicas y naturales, universidad nacional de san juan. av. josé ignacio de la rosa y meglioli, cp5400 san juan, argentina 2 conicet. instituto de ciencias básicas, facultad de filosofía humanidades y artes, universidad nacional de san juan. av. josé ignacio de la roza 230 (o). capital. san juan. cp: 5400. * corresponding author. e-mail, sanabria.eduardoa@gmail.com 3 centro nacional patagónico (cenpat-conicet). blvd. brown 2915 (u9120acd), puerto madryn, chubut, argentina 4 área sistemática, sección herpetología, instituto de limnología ‘‘dr. raúl a. ringuelet’’ (ilpla-conicet), 120 y 62 (cp1900), la plata, buenos aires, argentina submitted on 2013, 2nd september; revised on 2014, 7th january; accepted on 2014, 20th february abstract. we describe the tadpole of odontophrynus barrioi including external larval features, chondrocranial and cranial musculature, and compare it with the other species of the genus. tadpoles of o. barrioi at stages 31–37 are about 52.46 mm long. the body is slightly depressed in lateral view and ovoid in dorsal view. oral disc is emarginate laterally, anteroventral and small with a single row of marginal papillae, a single large dorsal gap, and labial tooth row formula 2(2)/3(1). despite small interspecific variations, like different labial tooth row formulae, the general aspect of species is quite similar. although poorly-known yet, the chondrocranium and cranial musculature display some variations within the genus. keywords. chondrocranium, cranial muscles, external morphology, odontophrynidae, odontophrynus barrioi, tadpole. introduction the genus odontophrynus reinhardt and lütken (1862) is distributed in southern and eastern south america. it is composed of 11 species (frost, 2014), that are divided into three species groups (savage & cei, 1965; cei, 1987; caramaschi, 1996; amaro et al., 2009). the o. occidentalis group includes odontophrynus barrioi cei, ruiz, and beçak, 1982, o. occidentalis (berg, 1896), and o. achalensis di tada, barla, martori, and cei, 1984. the species under study is associated with arid and semiarid environments of central and western argentina in the chaco seco eco-region (torrella and adamolí, 2005). the o. occidentalis group is characterized by possessing enlarged postorbital, temporal, and parotoid glands, and other glands on the tibia (savage and cei, 1965). the adults of o. barrioi distinguish from the other species of the group by the following combination of characters: presence of irregularly arranged rounded dorsal glands; lack of a light vertebral stripe; presence of a great number of closely arranged post-orbital, temporal, and parotoid glands; absence of keratinous spines; presence of a well-developed gland between the eye and the maxilla; light brown background with dark brown spots; among others (for more details see rosset et al., 2007). the tadpole of o. barrioi was described only synthetically by cei et al. (1982) and compared in some morphometrical measurements with the tadpole of o. occidentalis by cei and crespo (1982). the aim of this work is to provide a detailed description of the external larval features, chondrocranial and 16 e. gonzález et alii cranial musculature of the tadpole of odontophrynus barrioi and to compare these characteristics with the available information for other species of the genus. materials and methods all specimens studied for the present work are housed at the amphibian collection of the museo de la plata (buenos aires, argentina). odontophrynus barrioi tadpoles were collected on 27 january 2011 in las tumanas stream (30º86’083”s, 67º32’628”w; 920 m a.s.l. – mlp 5690), la mesada stream (30º99’132”s, 67º29’694”w; 784 m a.s.l. – mlp 5691), astica stream (30º93’822”s, 67º34’055”w; 916 m a.s.l. – mlp 5692), and agua de las totoras (30º08’18”s, 67º 53’17.7”w; 1311 m a.s.l. – mlp 5693), valle fértil department, san juan, argentina. the valle fértil department is located in the chaco seco eco-region (torrella and adamolí, 2005). the vegetation of the area is diverse; predominantly tree species such as prosopis spp. (algarrobos), aspidosperma quebracho-blanco (quebracho blanco), ziziphus mistol (mistol) and shrub of larrea spp. (jarillas) are present, among others. the other anurans inhabiting in the area were tadpoles and adults of rhinella arenarum (bufonidae), leptodactylus latrans, l. bufonius (leptodactylidae), and phyllomedusa sauvagii (hylidae) (sanabria and quiroga, 2008). the average annual temperature is 17 ºc, annual mean minimum is 10.2 ºc and annual mean maximum 25.2 ºc, with rainfall concentrated in summer, with an annual average of 225 mm (cabrera, 1976). the tadpoles were anesthetized (2.5 ml of 2% xylocaine and 2% lidocaine hcl, astrazeneca labs, buenos aires, argentina) after capture and immediately fixed in 10% buffered formalin. seven tadpoles of odontophrynus barrioi (mlp 5694) were employed for the external morphology descriptions at stages 31–37 (gosner, 1960), also attaching morphological measurements at stages 26–30 and 38–41. to clearly visualize oral disc papillae, it was stained with 1% of giemsa solution, for 10 min. three stage 37–38 tadpoles of o. barrioi (mlp 5695) were double stained following the technique of taylor and van dyke (1985) to study the chondrocranium and associated muscles. muscles were observed before the clearing of specimens, after which the chondrocranium was studied. measurements were taken under a lancet stereomicroscope (10–40x) with an essex digital caliper (accuracy 0.01 mm). terminology follows haas (1995), and d’heursel and de sá (1999) for chondrocranium; alcalde and rosset (2003) for chondrocranial measurements; haas (2001) for mandibular muscles; haas and richards (1998) and haas (2003) for hyobranchial muscles; schlosser and roth (1995) for innervations; altig and mcdiarmid (1999), lannoo (1999), and grenat et al. (2009) for external morphology; altig and mcdiarmid (1999) for oral disc morphology; and altig and johnston (1989) for tadpole ecomorphological types. we measured 17 morphological variables: total length (tl), body length (bl), tail length (tal), body height (bh), maximum tail height (mth), tail muscle height (tmh), dorsal fin height (dfh), ventral fin height (vfh), maximum body width (mbw), body width at nares (bwn), tail muscle width (tmw), eye diameter (ed), interorbital distance (iod), internarial distance (ind), eye-narial distance (end), snout-narial distance (snd), oral disc width (odw). measurements follow lavilla and scrocchi (1986), altig and mcdiarmid (1999), and grenat et al. (2009). the morphological measurements are in millimeters (mm) and are reported as mean ± standard error. the identification of the tadpole was based in the fact that odontophrynus barrioi is the only species of this genus inhabiting at the valle fértil department (san juan, argentina: see rosset et al., 2007). results external morphology description: tadpoles at stages 31–37 are 52.46 ± 6.96 mm of tl. body is slightly depressed in lateral view and ovoid in dorsal view (bh/mbw = 0.86 ± 0.05), shorter than half of total length (bl/tl = 0.38 ± 0.004). the snout is rounded in dorsal view, and abrupt and blunt in lateral view. the nostrils are dorsal, directed anterodorsally, scarcely visible laterally, and positioned closer to the eyes than the tip of the snout (snd/end = 1.49 ± 0.13). the nostrils are elliptical, kidney-shaped, with a rounded and thin rim all around. rounded eyes, small and in dorsal position, directed dorsolaterally, vistable 1. morphometric measurements (mm, mean ± se) for tadpoles of odontophrynus barrioi. 40 larvae at stage: 26–30, 7 larvae at stage 31–37 and 10 larvae at stage 38–41 from valle fértil, san juan, argentina. character mean ± se 26–30 31–37 38–41 total lenght tl 32.21 ± 4.17 52.46 ± 6.96 76.76 ± 7.64 body lenght bl 12.65 ± 1.61 20.15 ± 2.95 28.85 ± 2.80 tail lenght tal 20.40 ± 3.23 34.12 ± 3.92 48.10 ± 5.35 body height bh 5.69 ± 1.03 9.12 ± 2.32 13.52 ± 2.58 maximum tail height mth 6.39 ± 0.93 9.49 ± 0.89 9.97 ± 3.58 tail muscle height tmh 3.16 ± 0.72 5.30 ± 0.80 8.22 ± 1.27 dorsal fin height dfh 2.90 ± 0.50 4.52 ± 0.58 8.08 ± 0.67 ventral fin height vfh 2.65 ± 0.58 4.36 ± 0.52 6.50 ± 1.44 maximum body width mbw 6.77 ± 1.04 11.24 ± 1.72 17.48 ± 4.89 body width at nares bwn 4.92 ± 0.88 7.35 ± 1.84 11.55 ± 2.62 tail muscule width tmw 1.87 ± 0.47 4.19 ± 1.07 7.15 ± 0.93 eye diameter ed 1.28 ± 0.23 2.03 ± 0.31 2.60 ± 0.32 interorbital distance iod 2.92 ± 0.43 4.32 ± 1.28 7.20 ± 0.68 internarial distance ind 1.54 ± 0.23 2.15 ± 0.51 2.47 ± 0.38 eye-nares distance end 1.87 ± 0.36 2.98 ± 0.61 4.40 ± 0.39 snout-narial distance snd 2.73 ± 0.47 4.14 ± 0.70 5.27 ± 0.52 oral disc width odw 2.45 ± 0.46 3.5 ± 1.15 4.61 ± 1.01 17the tadpole of odontophrynus barrioi ible in dorsal and lateral views. the spiracle is single, short, sinistral, lateroventrally positioned, spiracle opening oval, posterodorsally directed and with the inner wall of the opercular tube absent, visible in lateral, dorsal and ventral views. neuromasts of posterior supraorbital, supraorbital, infraorbital angular and posterior infraorbital are noticeable. the tail is large (tal/tl = 0.65 ± 0.01), with its maximum tail height similar to the body height (mth/bh = 1.19 ± 0.13) and its end portion rounded. myomeres are more evident in the anterior portion of the tail, with the musculature almost reaching the tip of the tail. the dorsal fin height is slightly higher than ventral fin height (dfh/vfh = 1.16 ± 0.09) and originates at body-tail junction. the vent tube is medial, attached to ventral fin, opening dextrally. oral disc: the oral disc is anteroventral, small (od/ mbw = 0.37 ± 0.03), laterally emarginated with absent papillae in the emarginated edges (fig. 1 d). it has a single row of marginal papillae, conical, with pointed tip, of variable sizes. a single large dorsal gap is present (approximately 85% of odw), without ventral gap. single or paired submarginal papillae are present in the supra and infraangular zone, more common and numerous in the later. labial teeth single, with one cusp, and posterior ridges curved. labial tooth row formula 2(2)/3(1). jaw sheaths keratinized, with upper jaw totally pigmented, and lower jaw pigmented on its upper half; serrated edges in both jaws; upper jaw sheath smooth arch-shaped with lateral processes and lower jaw sheath v-shaped. color in preservative: the body is black pale in the abdominal region and light brown in the anterior body region, with perinarial region darker. the ventral region is pale and transparent allowing the visualization of the visceral system. tail musculature is yellow pale, slightly darker at the junction of the dorsal fin with the body, and it shows grouped chromatophores forming a dense reticulate. fins are opalescent, in some cases finely reticulated and with irregular dark blotches. chondrocranium and cranial muscles neurocranium oval (width/length = 0.87) and depressed (height/width = 0.46), with greatest width at level of the arcus subocularis. the tetrapartite cartilago suprarostralis has the pars corpora medially joined by ligament (fig. 2 d). a proximal bridge connects the pars corpora with the respective pars alaris at each side. the par alaris bears processus anterior dorsalis and processus posterior dorsalis. spherical and small adrostral cartilages present. the cornu trabeculae diverges anterolaterally from the planum ethmoidale and comprises 27% of chondrocranial length. they are uniformly wide and bear a well-developed processus lateralis trabeculae. both the lamina orbitonasalis and the septum nasi are present at the studied stages. the fenestra frontoparietalis is limited posteriorly by the tectum synoticum, laterally by the taenia tecti marginalis, and anteriorly by the taenia tecti ethmoidalis. the taenia tecti transversalis divides the fenestra in two parts, the frontal and parietal ones, being the later subdivided in left and right parietal fenestrae by the taenia tecti medialis. the cartilaginous lateral walls formed by the cartilagines orbitalis are open at level of the optic, metoptic, and trochlear foramina, and fisura prootica. at the studied stages, the basi cranii is completely closed. the capsula auditiva comprises 30% of the chondrocranial length and its anterior copula is slightly overlapped with the dorsum of the processus ascendens. the larval crista parotica with processus anterolateralis and posterolateralis well developed. the medial wall of the capsula auditiva is alcian-blue negative and its openings could not be observed. the superior perilymphatic foramen opens in the posterior wall of the capsule. the operculum is present. the palatoquadrate bears processus articularis quadrati, processus muscularis quadrati, commissura quadrato-cranialis anterior, commissura quadrato-orbitalis, long processus pseudopterygoideus, and processus ascendens. the processus ascendens meets the pila antotica just posterior to the oculomotor foramen (intermediate union) forming a straight angle with the cranial floor. the lower jaw is composed by cartilagines infrarostrales and cartilago meckeli. the later with processus retroarticularis, processus dorsomedialis and processus ventromedialis well developed. the commissura intramandibularis is alcian-blue negative (fig. 2d, e). fig. 1. external morphology of the tadpole of odontophrynus barrioi at stage 38 (mlp.a-5695). (a) dorsal, (b) lateral, and (c) ventral views (scale bar = 10 mm); (d) oral disc (scale bar = 1 mm). drawer: gonzalez e. 18 e. gonzález et alii the hyobranchial apparatus lacks copula i. all ceratohyale processes (anterohyal lateral, anterohyal, posterohyal and articular) are well developed. the ceratohyalia is medially joined by the pars reuniens. the copula ii bears a short processus urobranchialis. the ceratobranchiales i, ii, and iv are continuous to the planum hypobranchiale; the third joins the planum by a smooth connection. the commissura proximalis is absent in all ceratobranchiales and all them are distally joined by a double distal commissure. the processus branchialis is closed and the fourth spiculae are well developed. the exoccipital, frontoparietal, and parasphenoid are the only ossification centres present at the studied stages (not drawings in fig. 2). respect to the cranial muscles, the ramus mandibularis of the nervus trigeminus runs laterally to all mm. levatorae mandibulae. in the table 2 are detailed the origin and insertion of each muscle. discussion external morphology the comparison of the larval external morphology of odontophrynus barrioi with the available larval descriptions for other species of the genus allow us to propose the following tadpole characterization of odontophrynus with small variations characteristic of each species: (1) small to medium sized tadpoles (tl = 30–75 mm, stages 31–39), (2) globose/ovoid in dorsal view and somewhat depressed or flattened below in lateral view, (3) rounded snout in dorsal view, (4) single and sinistral spiracle, directed dorsolaterally, (5) medial vent tube, (6) dorsal nostrils, (7) medium-sized tail, being always larger than 1/2 tl with robust musculature, (9) anteroventral oral disc, laterally emarginated (except in o. cultripes and o. salvatori), with a large dorsal gap and a single row of marginal papillae. submarginal papillae occur in the species o. barrioi, o. lavillai, o. cordobae, and o. maisuma only, (10) the most common ltrf for the genus is 2(2)/3(1) (o. barrioi, o. occidentalis, o. americanus, o. lavillai, o. cordobae, o. maisuma, o. cultripes, and o. salvatori), but 2/3(1) (o. maisuma and o. carvalhoi), 2/3 (o. maisuma), and 2(2)/3 (o. cultripes) are also present (see table 3), and (11) exotrophic, lentic and benthic tadpoles. regarding the odontophrynus occidentalis group to which o. barrioi belongs alongside o. occidentalis and o. achalensis, they are externally similar; the only difference that can be highlighted is that o. barrioi has the tip of the tail rounded contrary to o. occidentalis and o. achalensis in which the tip of the tail is acute. selected external morphological characters of odontophrynus tadpole fig. 2. chondrocranium of odontophrynus barrioi at stage 38. (a) dorsal view, (b) ventral view of hyobranchial apparatus (scale bar = 3 mm), (c) frontal view of cartilago suprarostralis (scale bar = 1 mm). references: ac: adrostral cartilage, as: arcus subocularis, ca: capsula auditiva, cb: ceratobranchiales, ch: ceratohyale, cot: commissura terminalis, cp: copula posterior and processus urobranchialis, cqo: commissura quadrato-orbitalis, ct: cornua trabeculae, h: hypobranchiale, pa: pars alaris, pab: processus anterior branchialis, pad: processus anterior dorsalis, pah: processus anterior hyalis, pal: processus anterolateralis hyalis, par: processus articularis, pas: processus ascendens, pc: pars corporis, plh: processus lateralis hyalis, pmq: processus muscularis quadrati, pp: processus pseudopterygoideus, ppd: processus posterior dorsalis, pph: processus posterior hyalis, pq: processus quadrato-ethmoidalis, pr: pars reuniens, pra: processus anterolateralis, s: spiculae i–ii, sn: septum nasi, tm: taenia tecti marginalis, ts: tectum synoticum, ttm:taenia tecti medialis, ttt: taenia tecti transversalis. 19the tadpole of odontophrynus barrioi table 2. origin and insertion of the cranial muscles on tadpoles of odontophrynus barrioi at stages 37–38. muscle origin insertion nervus trigeminus (cranial nerve v), mandibular musculature levator mandibulae internus processus ascendens cartilago meckeli levator mandibulae longus superficialis arcus subocularis cartilago meckeli levator mandibulae longus profundus arcus subocularis both muscles insert together in the pars alaris by a common tendon levator mandibulae externus profundus processus muscularis levator mandibulae externus superficialis processus muscularis pars alaris levator mandibulae articularis processus muscularis cartilago meckeli levator mandibulae lateralis processus retroarticularis pars alaris submentalis cartilago infrarostrale median raphe intermandibularis cartilago meckeli median raphe mandibulolabialis inferior cartilago meckeli oral disc mandibulolabialis superior absent nervus facialis, (cranial nerve vii), hyoid musculature suspensoriohyoideus processus muscularis and arcus subocularis ceratohyale suspensorioangularis processus muscularis processus retroarticularis orbitohyodeus processus muscularis ceratohyale quadratoangularis anteroventral on palatoquadrate processus retroarticularis hyoangularis lateralis ceratohyale cartilago meckeli, retroarticular hyoangularis medialis absent interhyoideus ceratohyale median raphe interhyoideus posterior ventral wall on branchial chamber diaphragmatopraecordialis median endof m. interhyoideus posterior septum transversus nervus glossopharyngeus (cranial nerve ix), branchial musculature levator arcuum branchialium i arcus subocularis commissura terminalis i subarcualis rectus i dorsal head on ceratobranchiale i. ventral heads on ceratobranchiales ii and iii ceratohyale constrictor branchialis i absent nervus vagus (cranial nerve x), branchial musculature constrictor branchialis ii ceratobranchiale ii commissura terminalis i constrictor branchialis iii ceratobranchiale ii commissura terminalis ii constrictor branchialis iv ceratobranchiale iii commissura terminalis iii diaphragmatobranchialis peritoneal wall commissura terminalis iii levator arcuum branchialium ii anterolateral on capsula auditiva and posterior arcus subocularis commissura terminalis ii levator arcuum branchialium iii posterolateral on capsula auditiva commissura terminalis iii levator arcuum branchialium iv posteroventral on capsula auditiva ceratobranchiale iv subarcualis obliquus ii ceratobranchiales ii–iii processus urobranchialis subarcualis rectus ii–iv ceratobranchiale iv ceratobranchiale i tympanopharyngeus m. levator arcuum branchialium iv pericardium dilatator laryngis posteroventral on capsula auditiva constrictor laryngis constrictor laryngis forms an annulus rounding the larynx transversus ventralis iv absent nervus hypoglossus (spinal nerve ii), hypobranchial musculature geniohyoideus hypobranchial at level of ceratobranchiale iii cartilago infarostrale rectus cervicis peritoneal wall ceratobranchiale iii 20 e. gonzález et alii table 3. main morphological features of the tadpoles of odontophrynus. abbreviations: dg (dorsal gap), dv (dorsal view), ltrf (labial tooth row formula), lv (lateral view), s (sinistral), tot (tip of tail). with asterisks (*) are marked the characters inferred from illustrations. (**)o. salvatori was transferred to proceratophrys, should be remarked that possible this species also pertain to this genus, but provisionally is considered as a species of odontophrynus not assigned to anygroup. the tadpole of odontophrynus monachus was not included because it remains unknown (caramaschi and napoli, 2012). species reference body shape oral disc ltrf spiracle vent tube nostrils tail eyes occidentalis group o. barrioi present study ovoid in dv, depressed in lv laterally emarginated, dg present 2(2)/3(1) s, opening posterodorsally directed medial, dextral opening dorsal, elliptical large, tot rounded small, dorsal, directed dorsolaterally o. barrioi cei et al. 1982; cei and crespo 1982; cei 1987 — — — s, opening dorsolaterally directed — — tot rounded — o. occidentalis savage and cei 1965; cei 1980; cei 1987 depressed in lv laterally emarginated, dg present 2(2)/3(1) s medial, dextral opening dorsal large, tot rounded dorsal, directed upwards o. achalensis di tada et al. 1984; cei 1987 short laterally emarginated, dg present* 2(2)/3(1)* s — dorsal large, tot acute dorsolaterally americanus group o. americanus savage and cei 1965; cei 1980 ovoid in dv laterally emarginated, dg present 2(2)/3(1) s medial dorsal large, tot acute dorsal, directed laterally o. lavillai lavilla and scrocchi 1991; fabrezi and vera 1997 ovoid in dv, depressed in lv laterally emarginated, dg present 2(2)/3(1) s medial, dextral opening dorsolaterally, elliptical large, tot acute large, lateral o. cordobae grenat et al. 2009 ovoid in dv, depressed in lv laterally emarginated, dg present 2(2)/3(1) s medial, dextral opening dorsal, elliptical large, tot acute small, directed dorsolaterally o. maisuma borteiro et al. 2010 ovoid in dv, depressed in lv laterally emarginated, dg present 2(2)/3(1), 2/3(1) and 2/3 sometimes s, opening posterodorsally directed medial, dextral opening dorsal, rounded large, tot rounded large, dorsal, directed laterally cultripes group o. cultripes savage and cei 1965; cei 1980 ovoid in dv, depressed in lv not laterally emarginated, dg present 2(2)/3, 2(2)/3(1) s, opening posterodorsally directed* medial, dextral opening dorsal large, tot acute dorsal, directed laterally o. carvalhoi caramaschi 1979 ovoid in dv, triangular elongated in lv laterally emarginated, dg present 2/3(1) s medial, dextral opening dorsal tot acute large, dorsal, directed laterally no group o. salvatori** brandão and batista 2000 ovoid in dv, depressed in lv not laterally emarginated, dg present 2(2)/3(1) s, opening posterodorsally directed medial, dextral opening dorsal, rounded tot rounded large, dorsal, directed laterally 21the tadpole of odontophrynus barrioi known to date are summarized in table 3 complementing the table done by do nascimento et al. (2013). chondrocranium and cranial muscles the cranial morphology of the odontophrynus occidentalis group is known only for o. achalensis (haas, 1997; 2003). the chondrocranium of this species was illustrated and its cranial muscles and chondrocranial characters were described in the context of a broad phylogenetic analysis of anurans. in addition, only chondrocranial features (lacking muscle descriptions) are available for three species of the o. americanus group (o. americanus, o. lavillai, o. maisuma; fabrezi and vera, 1997; do nascimento et al., 2013) and two species of the o. cultripes group (o. carvalhoi, o. cultripes; do nascimento et al., 2013). the chondrocranial features were described for five species of proceratophrys (dos santos dias et al., 2013) but not for the other genus that composed odontophrynidae (macrogenioglottus). do nascimento et al. (2013) provided a chondrocranial characterization of odontophrynus based on the following combination of characters: (1) tetrapartite suprarostral cartilage, (2) processus lateralis trabeculae present, (3) crista parotica distinct, (4) processus pseudopterygoideus present, (5) low attachment of the processus ascendens, (6) commissural quadratoorbitalis present, (7) processus lateralis hyalis present, (8) tectum parietale present, (9) open processus branchialis, and (10) fourth spicule modified into a small plate. there is a notable variation in the character (1) of this characterization within odontophrynus and proceratophrys: although always tetrapartite, both corpora may be either free or distally joined by cartilage, the corpus – ala union may be absent, proximally present, or both proximally and distally present. the same condition of the characters 3, 4, 6, and 9 are present in species of proceratophrys with available chondrocranial descriptions (see dos santos dias et al., 2013), whereas the characters 2, 7, and 10 were not clearly described by dos santos dias et al. (2013) for the proceratophrys they studied. the characters 5 and 8 are variable within odontophrynus since they have a different condition in o. barrioi (intermediate union of the processus ascendens and absence of tectum parietale at advanced stages of development). furthermore, we have verified both characters in a sequence of larval stages of a tetraploid population of o. americanus (from buenos aires province, argentina) housed at the herpetological collection of the museo de la plata (mlp. 5111). these specimens have an intermediate union of the processus ascendens and lack the tectum parietale (at least at stage 42) as we found here for o. barrioi. the fenestra frontoparietalis may be posteriorly closed by the formation of a tectum parietal (o. achalensis at least at stage 40); other condition is the presence of two fenestrae parietales limited by the tectum synoticum and the taenia tecti transversalis et medialis (o. barrioi at stage 38); and the fenestra frontoparietalis remains widely opened by the existence of a tectum synoticum only (o. americanus and o. lavillai at stages between 31–37). however, these features are clearly due to the heterochronic development observed at different developmental stages across the species. finally, we wish to make a brief comment about the presence/absence of adrostral cartilages in the species of odontophrynus. odontophrynus barrioi is the unique species of the genus in which the adrostrals seem invariable present. in other species they may be either present or absent as in o. americanus (absent in the specimens studied by do nascimento et al., 2013; but present in those studied by fabrezi and vera, 1997) and o. achalensis (see haas, 2003); or merely present as adrostral tissue mass (not chondrified, o carvalhoi, o. maisuma). the comparison of the cranial muscles of o. achalensis (haas, 1997, 2003), with the muscles we described for o. barrioi shows a single variable feature between both species: the insertion site of the m. rectus cervicis (ceratobranchiale iii in o. barrioi, ceratobranchiale iii and iv in o. achalensis). in summary, the external morphology of the tadpole of odontophrynus barrioi is similar to the other species of the genus with small variations between them, and the internal larval features are controversial (e.g. morphology of the adrostral cartilages) to diagnose the species groups of odontophrynus. acknowledgments we thank e. espejo for providing housing and t. gonzalez and r. aguilar for helping with the english. authorities of san juan fauna office provide permits (sa y ds nº 1300-47362011). we special thank to p. pastor, a. navas, m. herrera, d. moreno, a. cataldo for the field work assistance and two anonymous reviewers that greatly contributed to ameliorate the manuscript. this work was partially supported by a postdoctoral fellowship from conicet, awarded to eas. references alcalde, l., rosset, s.d. 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(2005): situación ambiental en la ecoregión del chaco seco. in: la situación ambiental de argentina 2005, pp. 74-100. brown, a., ortiz, u., acerbi, m., corchera, j. eds., fundación vida silvestre, buenos aires, argentina. acta herpetologica vol. 8, n. 2 december 2013 firenze university press journal of the societas herpetologica italica acta herpetologica issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 9(1): 119-123, 2014 doi: 10.13128/acta_herpetol-13740 consequences of haemogregarine infection on the escape distance in the lacertid lizard, podarcis vaucheri isabel damas-moreira1,2,*, d. james harris1, daniela rosado1,2, isabel tavares1,2, joão p. maia1,2,3, daniele salvi1, ana perera1 1 cibio research centre in biodiversity and genetic resources, inbio, universidade do porto, campus agrário de vairão, rua padre armando quintas, nº 7, 4485-661 vairão, vila do conde, portugal. *corresponding author. e-mail: isabeldamas.m@gmail.com 2 departamento de biologia, faculdade de ciências, universidade do porto, rua do campo alegre fc4 4169-007 porto, portugal 3 institut de biologia evolutiva (csic universitat pompeu fabra). passeig marítim de la barceloneta, 37-49. 08003 barcelona, spain submitted on 2013, 18th december; revised on 2014, 5th april; accepted on 2014, 23rd april abstract. nowadays it is widely accepted that parasites play a significant role in the community structures in which they occur, and ultimately upon ecosystems. furthermore, infection by parasites might be associated with considerable deterioration of individual host fitness. while the apicomplexan parasites belonging to the genus hepatozoon can provoke severe deleterious effects in some mammals, impact on other hosts, such as reptiles, is still unclear. we assessed the effect of hepatozoon parasites on podarcis vaucheri flight-initiation distance from a simulated predator, a behaviour that is determinant for a successful escape and is therefore likely to have major implications on a lizard’s survival. we found that flight-initiation distance was not dependent on the time of the day or tail condition. subadults exhibited worse body condition than adults and females had worse body condition than males. regarding intensity of parasitism, subadults showed higher parasitemia levels. escape distance was not associated with parasitic load or any of the other studied features, which is indicative of limited impact of the parasite. this negligible effect might explain the remarkably high prevalence (more than 96%) of this parasitic group within this p. vaucheri population. keywords. flight-initiation distance, hepatozoon, lacertid. parasitism is a major selective force driving organisms’ life history traits, and is usually associated with a deterioration of host body condition (oppliger et al., 1996). the apicomplexan haemogregarines (apicomplexa: adeleorina) are the most common blood parasites in reptiles, and among them, the genus hepatozoon miller, 1908 is one of the most frequent (telford, 2009). although the deleterious effects of some hepatozoon species in domestic animals are well-known (baneth et al., 1998; 2003), the impact for wild reptile hosts is still uncertain and vague. hepatozoon gamonts infect erythrocytes, possibly causing an oxygen deficit as a result of the destruction of red blood cells (telford, 2009). therefore, hepatozoon infection consequences might be relevant in situations requiring high oxygen transportation, such as escaping from predators. concerning this, haemogregarine load was found to be associated with lower antipredatory performance both in terms of burst speed (oppliger et al., 1996; garrido and pérez mellado, 2014), and tail regeneration rate (oppliger and clobert, 1997). the distance a lizard allows potential predators to approach before fleeing (the flight-initiation distance), is determinant for a successful escape (cooper, 2006). the optimal escape theory (ydenberg and dill, 1986) is based on the principal that a prey will only escape from its predator when the risk of predation equals the cost of escaping. these costs are not only energetic but also include abdicating foraging (cooper and pérez-mellado, 2004), missing social opportunities (ydenberg and dill, 1986) and reducing basking time (martín and lópez, 1999). 120 i. damas-moreira et alii in this study we analyse the influence of the haemogregarine hepatozoon on the flight-initiation distance in the lizard podarcis vaucheri boulenger, 1905. following the optimal escape theory and previous studies, if parasites negatively affect host fitness or locomotor performance, we hypothesise that this can be reflected in lizards’ flight-initiation distance. thus, we expect that lizards with higher parasite load will have a faster response and run to the shelter sooner than non-parasitized lizards, as hepatozoon sp. may decrease their locomotor speed which can be compensated by running away before the predator gets as close. the tests were performed in may 2013 in oukaimeden (morocco, 31º12’n, 7º51’w) on a total of 55 individuals of podarcis vaucheri. of these, 24 were males (including three subadults), and 31 were females (including seven subadults). individuals above 45 mm snoutvent length were considered adults, and below, subadults (schleich et al., 1996). the study area consists mainly of rock outcrops with fissures or large rocks among grass. at this location, podarcis vaucheri presents high densities and hepatozoon parasites have been previously reported and characterized genetically (maia et al., 2011). escape distance trials consisted of standardized approaches to lizards simulating a predator attack. given that speed and direction of the approach might influence a lizard response (martín and lópez, 1999; cooper, 2006) approaches were always performed in the same way by the same researcher with the same outfit. the researcher simulating the predator (dr) slowly walked towards the lizard, with arms next to the body and making no abrupt movements until the lizard fled. the distance between the researcher and the place where the lizard was at the moment it escaped was recorded using a laser distance measurer (±2 mm over 30 m). the distance from the lizards to the nearest refuge at the time of fleeing was not recorded, as all lizards were no more than 30 centimetres away from a shelter (idm, pers. obs.). only one trial was performed per lizard and only individuals that were basking at the initiation of the trial were included. after each trial, the lizard was captured and tail condition (intact, regenerated or recently lost) and time of capture were recorded. all trials were conducted between 10 and 16 hours. individuals were measured for snoutvent length (svl), weighed and a piece of tail tip was taken and stored in 96% ethanol for genetic analyses. blood resultant from the tail was smeared across a glass slide and air-dried for posterior treatment in the laboratory. all animals were released immediately after at the sample site. blood smears were fixed with absolute methanol for 2 min, stained with giemsa (1:9 distilled water) for 45 min and air-dried. using an olympus cx41 microscope, prevalence was estimated as the percentage of infected individuals within the sampled population and the individual parasitemia load was considered as the percentage of infected red blood cells out of 2500 erythrocytes. for these counts, random pictures were taken at x400 using cell^b 3.4 olympus® software, and counted manually using the imagej 1.46® program. molecular methods were used to confirm the identity of the detected parasites. dna was extracted from the blood of five randomly selected infected lizards using the high salt method (sambrook et al., 1989). a fragment of the 18s rrna gene was amplified using the primers hepf300 and hepr900 (ujvari et al., 2004), as described in harris et al. (2011). positive pcr products were purified and sequenced by a commercial facility (macrogen, the netherlands). sequences are available at genbank with the accession numbers kj659858 to kj659862. all parasite sequences analysed were part of the same lineage of hepatozoon infecting lizard hosts (lineage 2 identified in maia et al., 2012). prevalence was 96.4% (53/55) and intensity of infection varied from 0 to 11.7%, with a total mean intensity (± sd) of 1.86% (± 2.44). six individuals had more than 5% infected erythrocytes and two of these lizards, both females, more than 10% erythrocytes infected. prior to statistical analysis all continuous variables were log transformed to meet normality assumptions. in order to infer if the time of capture or tail condition were interfering with the flight-initiation distance, analysis of variance (anova, lm function) were performed for each variable independently. in order to assess the need to include svl and body condition (bc, considered as the ratio of svl/weight) as covariates in the analysis, spearman correlations were determined with all the remaining continuous variables (cor function). differences between sexes and ages in body size and body condition were assessed using an anova (lm function). differences in parasite prevalence between sexes and ages were tested using a generalized linear model (glm) with a logistic regression (mass package, venables and ripley, 2002), while differences in parasite intensity (considering only positive samples) were tested with an analysis of covariance (ancovas, with lm function) including bc as covariate. to evaluate if bc, svl or intensity of parasitism were related to flight-initiation distance, non-parametric spearman correlations (cor function) were determined. differences in flight escape distance were assessed using ancovas, where we tested the effect of host sex, age and presence of the parasite, including bc as covariate. finally, ancovas were performed only on infected 121haemogregarine impacts on podarcis vaucheri escape distance individuals, to assess for any effect of sex and age, after correcting by bc and intensity as covariates. all statistical tests were conducted using r v. 2.15.2 (r development core team, 2012). flight-initiation distance was not dependent on the time of the day (f = 0.973, df = 1, p = 0.328), or tail condition (f = 0.241, df = 2, p = 0.787), and thus these were not considered in further analyses. males and females did not differ significantly in body size (f = 2.232, df = 1, p = 0.141). body condition, however, differed between sexes (f = 38.510, df = 1, p < 0.001) and ages (f = 37.918, df = 1, p < 0.001), with females and subadults exhibiting worse bc. all individuals analysed were infected, with the exception of two lizards. given the low sample size of non-infected individuals, differences in prevalence between sexes or ages could not be tested statistically. considering only infected individuals and correcting for body condition, intensity of parasitism was not associated with sex (f = 0.346, df = 1, p = 0.56), but it differed between age classes (f = 4.136, df = 1, p = 0.048), with younger lizards having higher parasite load (table 1). flight-initiation distance was not correlated either to body condition (spearman correlation, r = -0.102, p = 0.458) or to parasite intensity (r = 0.024, p = 0.861). moreover, escape distance did not differ between infected and uninfected lizards (ancova, f = 0.007, df = 1, p = 0.933), sexes (f = 0.714, df = 1, p = 0.402) or ages (f = 1.175, df = 1, p = 0.284) after correcting for bc. finally, considering only infected individuals, we did not find differences in escape distance between sexes and ages (ancova, using bc as covariate, in all cases, p > 0.05). our results show that flight initiation distance was independent of parasite intensity. one possible explanation for this lack of differences might be that heavily infected individuals suffer higher mortality, and consequently our sampling was performed on individuals that were not exposed to higher infection levels. however, this is unlikely to be the case, since several of the individuals exhibited moderately high parasitemia levels, when compared to other studies with podarcis (0.1% in amo et al., 2005 b; 0.9% in garrido and pérez-mellado, 2013). hepatozoon prevalence in lizards are highly variable: while some studies reported about 20% infected individuals in populations of sphenodon, calotes and hemidactylus (herbert et al., 2010; godfrey et al., 2011; gupta et al., 2013), others report higher levels in podarcis from the iberian peninsula (amo et al., 2005 b; martín et al., 2008; roca and galdón, 2010). nevertheless, our prevalence values are extremely high, with only two of the 55 sampled individuals not infected with hepatozoon, which precludes any conclusion regarding the effect of parasite presence on the flight escape distance. in fact, as far as we know, this is the highest prevalence recorded in a continental population of podarcis. this estimate is similar to the values found in the insular podarcis lilfordi showing 95% haemogregarine prevalence of infection, albeit with an average of only 1% parasitemia levels (garrido and pérez-mellado, 2013). these differences may be the result of different availability of competent vectors, or to a seasonal variation of the parasite itself or their vectors, although this still needs to be verified. our study revealed that after correcting for body condition, younger individuals were more heavily infected, contradicting the trend found in other studies with reptilian haemogregarines (amo et al., 2004; molnár et al., 2013) and also with hepatozoon (salkeld and schwartable 1. number of individuals analyzed (n), mean flight-initiation distance (m), mean intensity of infection (%), and prevalence among genders and ages. males females adults n = 21 subadults n = 3 adults n = 24 subadults n = 7 distance          mean distance 1.8700 1.4237 1.9588 1.5364 ± sd 0.8562 0.2224 0.7736 0.4337 overall gender mean ± sd 1.814 ± 0.815 1.863 ± 0.727 parasites          mean intensity 1.72 1.52 1.51 3.11 ± sd 1.90 1.61 2.46 3.76 prevalence (%) 100 66.66 95.83 100 overall gender mean intensity ± sd 1.77 ± 1.85 1.93 ± 2.84 overall gender prevalence (%) 95.83 96.77 122 i. damas-moreira et alii zkopf, 2005). however, this result is also concordant with other works conducted in hepatozoon spp. in reptiles, in which parasite intensity declined with increased host size or age (madsen et al., 2005; brown et al., 2006; godfrey et al., 2011). this may be explained by natural selection removing susceptible reptiles and only those displaying moderate parasite load are able to survive until older ages (madsen et al., 2005; brown et al., 2006), or simply occur because younger individuals have had less time to acquire immunity, which might lead to higher infection intensity (hudson and dobson, 1997) and plausibly lower body condition, as observed in our study. these results seem to indicate that hepatozoon infection has no strong impact on the flight-initiation distance of p. vaucheri from oukaimeden. this suggests a solid and stable evolutionary interaction between host and parasite (combes, 2001; amo et al., 2005 b), where lizards from this population evolved to tolerate the infection rather than to fight it (sheldon and verhulst, 1996). both intensity and prevalence levels can thus indicate how well established this parasite is in this population, in which the mean intensity (near 2%) might be the result of immune system counterbalancing the high prevalence values (near 96.4%) in the population. moreover, flight-initiation distance might not be affected by the virulence of this hepatozoon strain, but can be influenced by other variables, allied or not with the occurrence of parasites. future research should integrate the study of other endo or ectoparasites, as studies in lizards revealed that their presence can also interfere in host fitness, including nematodes (amo et al., 2005 a), ticks (main and bull, 2000) or mites (sorci et al., 1995). despite the lack of evidence of parasite interference in the flight-initiation behaviour obtained in this study, these are complex parasite systems and clearly warrants further investigation. acknowledgements special thanks to beatriz tomé for her help in the laboratory. fieldwork was carried out under the permit of the haut commisariat aux eaux and forets of morocco (hceflcd/ dlcdpn/dprn/dff n°14/2010) and was supported by the percy sladen memorial fund (to djh), by the british herpetological society (to idm), and by the chicago herpetological society (to jpm). jpm, ds, and ap are supported by grants and contract by fundação para a ciência e tecnologia (fct, portugal) under the programa operacional potencial humano – quadro de referência estratégico nacional funds from the european social fund and portuguese ministério da educação e ciência (jpm: phd grant sfrh/bd/74305/2010; 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(1986): the economics of fleeing from predators. adv. stud. behav. 16: 229-249. acta herpetologica vol. 8, n. 2 december 2013 firenze university press journal of the societas herpetologica italica acta herpetologica acta herpetologica 10(1): 47-54, 2015 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-15304 where to “rock”? choice of retreat sites by a gecko in a semi-arid habitat andreia penado1,2, ricardo rocha3,4,*, marta sampaio3, vanessa gil3, bruno m. carreira3, rui rebelo3 1 school of life sciences, university of sussex, falmer, brighton, east sussex, bn1 9rh, united kingdom 2 cibio/up, jardim botânico tropical/iict, tv. conde da ribeira, 9, 1300-142 lisboa, portugal 3 centre for ecology, evolution and environmental changes, faculdade de ciências da universidade de lisboa. 1749-016 lisboa, portugal. *corresponding author. e-mail: ricardo.nature@gmail.com 4 metapopulation research centre, faculty of biosciences, university of helsinki, po box 65 (viikinkaari 1), fi-00014 helsinki, finland submitted on 2015, 6th january; revised on 2015, 14th march; accepted on 2015, 19th march editor: marco mangiacotti abstract. the selvagens gecko (tarentola boettgeri bischoffi joger, 1984) is a medium sized gecko endemic to the selvagens archipelago, madeira, portugal. the biology of this gecko is poorly known and in this study we present the first evidence regarding its habitat use. in 2010 (spring and autumn) and 2011 (spring), we collected data on the characteristics of the habitat surrounding 168 rocks used by these geckos as retreat sites, as well as on 75 randomly selected rocks. we also recorded body measurements of the individuals caught under each rock. in both seasons retreat site occupancy was found to be related to rock area, with geckos being found mainly under large rocks. interestingly, we found that in spring heavier males, in better body condition, occupied the largest rocks and larger geckos occupied rocks closer to creek beds. our results shed some light upon the behavioural ecology of this nocturnally active ectotherm, that spends the day under a retreat site: i) intraspecific competition may be an ecological factor prevalent in this species, since larger individuals occupy larger rocks, located in a presumably high quality micro-habitat; ii) the possibility of spring territoriality in males, that compete for good quality shelters. keywords. selvagens, habitat use, retreat sites, territoriality, tarentola boettgeri bischoffi. introduction geckos (gekkota) show a high diversity among reptiles and play a remarkable ecological role, particularly in warm and arid environments (vitt and caldwell, 2009). however, knowledge of gecko’s ecology lags behind that of other vertebrate taxa (e.g., downes and shine, 1998; ikeuchi et al., 2005), and only recently attracted increasing attention (hibbits et al., 2012; lisicic et. al., 2012; vasconcelos et al., 2012a; ibrahim, 2013). we suspect that the slow progress in knowledge about this group has been mainly due to their specific features (e.g., largely nocturnal, mostly living in inaccessible areas and difficult to capture). understanding the use of a species’ microhabitat is key for a good knowledge of its ecology and for guiding conservation actions (huey, 1991; kacoliris et al., 2009). due to their dependence on environmental temperature, the selection of suitable retreat sites is of particular importance to ectotherm lizards such as geckos, particularly in temperate zones. the habitat surrounding retreat sites may influence physiological performance and individual behaviour (huey, 1991; hibbits et al., 2012) and, consequently, impact upon population and community dynamics (pulliam and danielson, 1991; rosenzweig, 1991). the choice of suitable retreat sites may also promote social interactions, because favourable sites may be used by more than one individual (kearney et al., 2001), and this may lead to the development of site-defence behaviour (preten and case, 1998). territorial behaviour is common among lizards (e.g., baird et al., 1996), and has been gradually shown in 48 a. penado et alii geckos. adults of the australian gecko heteronotia binoei defend retreat sites (bustard, 1968), whereas most males of the barking gecko ptenopus garrulus garrulus establish their territory before the females’ emergence from winter dormancy (hibbits et al., 2012). studies in captivity also showed gecko territorial behaviour: paired male leopard geckos eublepharis macularius displayed more territorial marking than single males when exposed to a neutral test arena (sakata et al., 2002), and encounters between male desert geckos coleonyx reticulatus triggered aggressive behaviour (dial, 1978). however, we have no knowledge of any kind of territoriality in other species (frankenberg, 1992; johnston and bouskila, 2007). the selvagens gecko (tarentola boettgeri bischoffi joger, 1984) is a medium sized crepuscular and nocturnal gecko endemic to the selvagens archipelago, madeira, portugal. this sub-species can be found in three isolated populations in the three main islands: selvagem grande, selvagem pequena and ilhéu de fora, occurring entirely within a protected area (rebelo, 2008). in the island of selvagem grande, it is distributed from the sea level to the arid central plateau, where it reaches the highest abundance (oliveira et al., 2010). the simple landscape of the central plateau provides an ideal site for the study of gecko ecology, as every single retreat site (loose stones) can be verified over large areas. in this study we aimed to assess the habitat use by t. b. bischoffi, comparing the features of confirmed retreat sites, in the spring and the autumn seasons, with the features of potential retreat sites where the geckos were absent. we hypothesized that a key feature of the retreat site – its size – should be positively selected, as well as features associated with the resources that the immediate vicinity of the retreat site may provide, such as distance to shrubs or to humid sites. furthermore, we also tested if there was a relationship between the quality of the retreat site and the individual traits (sex, body size and body condition) of the geckos that used them, looking for indirect evidence of retreat site defense. materials and methods study area this study was carried out in the volcanic island of selvagem grande (30°8΄45˝n, 15°51΄51˝w), located in the eastern atlantic, 300 km south of madeira and 150 km north of the canaries archipelago. the island’s surface is roughly 270 ha and the main topographical feature is a 150 ha stony desert plateau (at 200 m a.s.l.) that supports an arid ecosystem (oliveira et al., 2010) (fig.  1). the climate is sub-tropical and semi-arid, with a rainy season in the boreal winter (campos and granafig. 1. central plateau of selvagem grande. a dry creek bed can be seen in the foreground, and virtually all the regularly-spaced shrubs are suaeda vera 49retreat sites of selvagens gecko deiro, 1999). annual precipitation is low, but torrential, producing flash floods that created a network of approximately 0.5 m deep dry creek beds criss-crossing the plateau. there are no major rocky formations over the plateau, and practically all loose rocks can be lifted. plant cover is increasing, after the successful eradication of rabbits and mice in 2003 (oliveira et al., 2010). herbaceous annuals now cover large parts of the plateau after the rains, but from may to october most of the plateau is devoid of herbs. during these months plant cover is restricted to regularly spaced shrubs, mainly of shrubby sea-blite suaeda vera with some individuals of the macaronesian endemic schizogyne sericea. due to wind action, all shrubs are pyramid shaped and rarely grow over 1 m high. the distance among shrubs is remarkably similar (0.82 ± 0.35 m; r. rebelo, pers. obs.) over most of the plateau, reflecting low water availability during long periods. selvagem grande is an important breeding station for several species of seabirds and, other than t. b. bischoffi, has two other resident terrestrial vertebrates, namely an endemic sub-species of madeira wall lizard teira dugesii selvagensis and the berthelot’s pipit anthus berthelotii berthelotii (oliveira et al., 2010). data collection we sampled three 1-ha plots on three occasions, grouped in two distinct seasons: spring (april 2010 and may 2011) and autumn (september 2010). in each visit, we lifted all loose rocks in each site, checking for the presence of geckos. this procedure was repeated twice in each sampling season. we collected data on habitat characteristics surrounding 168 rocks used as a retreat site by the geckos (spring n = 129, autumn n = 39), as well as on 75 randomly selected rocks (spring n = 45, autumn n = 30). we measured the following habitat characteristics: rock area (cm2; width x length, square root transformed for the statistical analyses), distances to the nearest rock and to the nearest shrub (cm), the maximum height of the nearest shrub (cm), canopy diameter (cm; estimated by the square root of the product of the longest canopy axis by its perpendicular) and registered the presence or absence of creek beds in the immediate vicinity (<  5 cm) of each rock. abbreviations used are as follows: rock area (ra), distances to the nearest rock (dr), distances to the nearest shrub (ds), maximum height of the nearest shrub (smh), canopy diameter (cd) and creek beds (cb). whenever possible, we captured geckos by hand and recorded their snout-vent length (svl) with callipers, to the nearest 0.1 mm, and their body mass with spring scales to the nearest 0.05 g. we assigned one of the following reproductive categories to each gecko: adult female (pregnant and non-pregnant), adult male – identified by the two post-cloacal prominences corresponding to the hemipenes – or juvenile. other studies of geckos found no differences in size at maturation between both sexes (leclair and leclair, 2011; kubistch et al., 2012), and therefore individuals with svl ≥ 4 cm were considered adults, based on the smallest size of gravid females, as the eggs are easily seen through the body wall (rebelo, pers. obs.). all captured individuals had both eyes photographed and were individually identified by their iris pattern using the matching software interactive individual identification system (rocha et al., 2013). the iris pattern of t. b. bischoffi has been observed to remain stable during periods up to four years and consequently the method can be deemed suitable for long-term mark-recapture studies (rocha and rebelo, 2014). to avoid pseudoreplication, recaptured geckos were excluded from the analysis. after the eyes were photographed and the body measurements were taken, all geckos were released at their initial point of capture. statistical analyses to compare the rocks used as retreat sites and the habitat surrounding them with the random selection of rocks in each season, we performed generalized linear models (glms) with a binomial error distribution and a logistic link function. as no correlation was found between the variables, we fitted all possible simple models relating gecko presence/absence with the habitat characteristics using the dredge function from the mumin r package and then we ranked them following the akaike information criterion with the correction for small sample sizes (aicc). we then employed a model averaging approach on models with δaicc ≤ 2 to obtain parameter estimates across the range of best final models and computed the odds ratios (or) of the model-averaged selected variables for each season. values of or > 1 indicate a positive association between the variable and the presence of geckos, while or < 1 indicate a negative association. to assess the condition of the geckos occupying the larger rocks, we first performed linear regressions of the cubic root of body mass against svl for each sex/age class (females, males and juveniles), and used the residuals as an estimate of body condition of all the individuals captured in spring (the relatively low sample size did not allow us to address this question for the autumn). the data met all the assumptions of linear regression. to further explore the question of which selvagens geckos occupy larger rocks during spring, we used glms relating rock area with svl, mass (cubic root transformed) and body condition for each sex/age. statistical analyses were conducted with the r software, version 2.15.3 (r development core team 2013). results the number of geckos found and their body parameters are depicted in table 1. although ~95% of the individuals were found alone, in several occasions there were two geckos sharing the same rock. interestingly, in almost every case the two individuals were of different sexes, suggesting that males (the larger sex) may be more tolerant of females than of other males. however, the sample size was too low to proceed to further analyses. data dredging originated four models that better explained the retreat site use by geckos in spring and seven models in autumn (table  2). in both seasons, rock 50 a. penado et alii area was present in all the models. in autumn, distance to the nearest shrub was included in three models, height of the nearest shrub and creek beds in two, and shrub canopy diameter in only one (table 2). according to the averaged-models from both seasons, for a one-unit increase in rock area we expect a probability slightly higher than 50% of finding a gecko under a rock (spring or = 1.10; autumn or = 1.09; table 3). considering the entire spring sample, the larger rocks were occupied by individuals with longer svl (p < 0.05; table 4), heavier mass (p < 0.05; table 4) and in better body condition (p < 0.05; table 4). considering each sex separately, a similar pattern was found only for adult males – heavy individuals in better body condition occupied large rocks (p < 0.05; table 4). furthermore, individuals found in the rocks close to the creek beds were larger (p < 0.05; table 5) than individuals found distant to the creek beds. when considering sex/ age classes separately, no differences were found (table 5). we did not analyse the relations between gecko body dimensions and habitat variables in the autumn due to the relatively small sample size, referring to data collected only in one year. discussion this study showed that the distribution of the selvagens gecko in this island’s plateau may be explained by the size of available retreat sites (rock area). furthermore, we also showed that heavy males and in good condition tended to occupy the larger retreat sites, and larger individuals tended to occupy retreat sites close to the dry creek beds. the size of retreat sites has already been demonstrated to be a good predictor of its use by other species of rock-dwelling geckos (shah et al., 2004; croak et al., 2008; vasconcelos et al. 2012a). the area of a retreat site table 2. models originated by the data dredging procedure for both spring and autumn seasons. cb = creek beds, cd = canopy diameter, dr = distance to nearest rock, ds = distances to the nearest shrub, ra = rock area and smh = maximum height of the nearest shrub; df stands for degrees of freedom. rank model estimate ± se of slope coefficient aicc aicc weight df spring null model y ~ 1 1.02 ± 0.19 172.09 146 1 y ~ ra -1.22 ± 0.59 150.00 0.38 2 2 y ~ ra + cb -1.26 ± 0.60 150.80 0.25 3 3 y ~ ra + ds -1.50 ± 0.69 151.38 0.19 3 4 y ~ ra + cd -1.41 ± 0.75 151.43 0.18 3 autumn null model y ~ 1 0.19 ± 0.26 87.37 61 1 y ~ ra -1.99 ± 0.84 80.17 0.23 2 2 y ~ ra + ds -2.66 ± 0.99 80.33 0.22 3 3 y ~ ra + smh -2.46 ± 1.19 81.57 0.12 3 4 y ~ ra + cb -1.90 ± 0.85 81.58 0.11 3 5 y ~ ra + ds + smh -3.22 ± 1.30 81.63 0.11 4 6 y ~ ra + cd -2.61 ± 1.05 81.73 0.11 3 7 y ~ ra + ds + cb -2.55 ± 0.99 81.78 0.10 4 table 3. parameter estimates, p-values (p) and odds ratio for the averaged models of each season. results are shown only for significant variables: *, p < 0.05; **, p < 0.01; ***, p < 0.001. mean and standard error of occupied rocks (ocrs) and control rocks (ctrs) are reported per season. variables estimate p odds ratio mean ± se ocrs mean ± se ctrs spring slope coefficient -1.64 ± 0.70 rock area 0.10 ± 0.03 *** 1.10 31.95 ± 1.19 23.26 ± 1.06 autumn slope coefficient -2.68 ± 1.18 rock area 0.09 ± 0.03 ** 1.09 33.21 ± 1.93 25.16 ± 1.63 table 1. number of geckos, mean and standard error for svl (mm) and mass (g) during spring and autumn. individuals independent variable spring autumn mean ± se n mean ± se n all individuals svl 56.45 ± 0.76 123 51.27 ± 1.59 39 males 61.50 ± 0.51 54 58.10 ± 0.90 15 females 57.28 ± 0.63 47 54.45 ± 0.98 12 juveniles 42.25 ± 1.67 22 39.56 ± 2.70 12 all individuals mass 7.48 ± 0.25 122 5.86 ± 0.43 39 males 8.77 ± 0.16 54 8.44 ± 0.78 15 females (all) 8.04 ± 0.34 47 5.92 ± 0.33 12 females (pregnant) 8.80 ± 0.42 28 females (nonpregnant) 6.93 ± 0.47 19 juveniles 2.88 ± 0.33 21 2.57 ± 0.37 12 51retreat sites of selvagens gecko table 4. results of glms relating gecko morphological features with occupancy of larger rocks during spring. *, p < 0.05; **, p < 0.01; ***, p < 0.001; df, degrees of freedom. individuals independent variable estimate ± se df t p all individuals svl 0.34 ± 0.13 126 2.678 ** males 0.25 ± 0.45 51 0.556 0.581 females 0.40 ± 0.45 46 0.898 0.374 juveniles -0.19 ± 0.22 21 -0.842 0.410 all individuals mass 12.02 ± 3.47 115 3.463 *** males 24.79 ± 12.27 44 2.020 * females (all) 17.97 ± 10.05 43 1.788 0.081 females (pregnant) 0.91 ± 15.98 24 0.057 0.955 females (nonpregnant) 29.10 ± 14.84 18 1.960 0.067 juveniles -6.80 ± 6.31 20 -1.078 0.294 all individuals body condition 16.22 ± 7.91 114 2.052 * males 55.48 ± 21.57 45 2.573 * females (all) 19.42 ± 11.82 43 1.643 0.108 females (pregnant) 3.81 ± 17.76 24 0.214 0.832 females (nonpregnant) 28.56 ± 19.57 18 1.459 0.163 juveniles 9.12 ± 7.50 19 -1.217 0.239 table 5. glms results relating geckos morphological features with the proximity to creek beds during spring.*, p < 0.05; **, p < 0.01; ***, p < 0.001; df, degrees of freedom. individuals independent variable estimate ± se df t p all individuals svl 0.08±0.03 125 2.375 * males 0.02±0.08 52 0.190 0.849 females 0.11 ± 0.08 46 1.282 0.200 juveniles 0.17 ± 0.07 21 0.235 0.814 all individuals mass 2.44±0.94 115 2.585 ** males 2.86±2.31 45 1.239 0.215 females (all) 4.07 ± 2.16 43 1.881 0.060 females (pregnant) 6.52 ± 3.82 24 1.704 0.088 females (nonpregnant) 1.32 ± 2.89 18 0.457 0.648 juveniles 0.39 ± 2.33 20 0.166 0.868 all individuals body condition 0.21±1.43 115 0.146 0.884 males 5.33±3.32 44 1.608 0.108 females (all) 3.28± 2.20 43 1.491 0.136 females (pregnant) 7.40 ± 4.06 24 1.823 0.068 females (nonpregnant) -1.95 ± 3.87 18 0.503 0.614 juveniles -3.60 ± 3.51 20 -1.026 0.305 52 a. penado et alii might not be necessarily the environmental cue used by the selvagens gecko in order to choose a rock. it might simply correlate with other equally or more important attributes, such as size and three-dimensional structure of the space used as a retreat site (shah et al., 2004; croak et al., 2008); or with the thickness of the rock (croak et al., 2008; huey et al., 1989), that may enhance thermoregulation, provide effective protection from predators, and/or a site for encountering potential mates (schlesinger and shine, 1994; downes and shine, 1998; shah et al., 2004). for a nocturnally active ectotherm that spends the day under a retreat site, the quality and availability of this resource could thus be an important determinant of habitat selection with potential consequences for its fitness. in fact, patterns of habitat use during the mating and reproductive seasons in our study site (spring) were mainly influenced by the size of the available retreat sites and, for larger geckos, by the presence of a scarce resource, i.e., water. water availability has an obvious importance in organisms inhabiting harsh and seasonal environments (beck and jennings, 2003). even if only available in soil moisture, water may contribute to an increase in food (insect) availability in the immediate vicinity of the retreat sites. these two aspects can, in turn, shape social interactions, particularly retreat site defence by males. in contrast to more complex environments, the landscape in the selvagem grande central plateau is strongly dominated by only one species of shrub, s. vera, and the bushes are very similar in size and shape, making the vegetation highly homogeneous. other studies carried out in arid habitats reported a weak or even the lack of association between saxicolous reptiles, including geckos, and canopy cover (croak et al., 2012). in this study, habitat variables were selected in all the models, but there wasn’t a variable consistently selected in both seasons. in this landscape, shrubs constitute the only effective wind barrier; their dead leaves, as well as other vegetable material, accumulate more easily beneath shrubs than in the open areas, and presumably insect abundance may also be higher below shrubs. t. b. bischoffi co-occurs in the selvagens archipelago with madeira wall lizards t. d. selvagensis and both species’ selection of retreat sites can be influenced by their ecological interactions. however, while t. d. selvagensis are very abundant along the sea bird colonies on the cliffs, they are relatively rare on the central plateau. our results allow us to infer some interesting aspects of the selvagens gecko behavioural ecology that merit further studies. first, intraspecific competition may be an ecological factor prevalent in this species whereby larger individuals (especially males) occupy larger rocks. second, as the breeding season corresponds to the spring months, our results suggest the occurrence of spring territoriality in males that compete for good quality shelters. published literature shows that smaller subordinate male geckos are forced by larger dominant males to occupy low quality shelters (downes and shine, 1998), or that juvenile geckos occupy smaller rocks than adults (vasconcelos, et al., 2012a). moreover, male selvagens geckos were not observed sharing retreat-sites with other males (rebelo, pers. obs.), similarly to what has been found in other arid habitats, (schlesinger and shine, 1994; downes and shine, 1998; vasconcelos et al., 2012a), reinforcing our suspicions about the occurrence of spring territorial behaviour in the selvagens gecko. to the extent of our knowledge, this is the first study on habitat use patterns of tarentola boettgeri bischoffi. this study aimed to shed some light in the basic ecological aspects of a threatened subspecies, living on an island whose vegetation is recovering from centuries of occupation from rabbits and mice. this species belongs to the most species-rich genus of geckos of nw africa and the eastern atlantic islands (vasconcelos et al., 2012b). given the conservative morphology of all the species of this genus, we expect that our findings will trigger future research on habitat selection patterns and territoriality in this group. acknowledgments the madeira natural park issued the permits to visit this area with restricted access, as well as to capture and handle the animals. we are grateful to the pnm staff, and particularly to paulo oliveira, dília menezes, carolina santos, and the selvagens natural reserve’s wardens for logistical support. we would also thank sasha vasconcelos, dave goulson and two anonymous reviewers who provided useful comments. funding was provided by fct (portugal) through its pluriannual funding program to r. rebelo. references baird, t.a., acree, m.a., sloan, c.l. 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(2009): herpetology – an introductory biology of amphibians and reptiles. third edition. academic press, london. acta herpetologica vol. 10, n. 1 june 2015 firenze university press obituary: valery k. eremchenko (1949-2014) leo j. borkin1, tatjana dujsebayeva2, roberto sindaco3, matthias stöck4 haplotype variation in founders of the mauremys annamensis population kept in european zoos barbora somerová1, ivan rehák2,*, petr velenský2, klára palupčíková1, tomáš protiva1, daniel frynta1 reproductive ecology of sichuan digging frogs (microhylidae: kaloula rugifera) wei chen1,*, lina ren2, dujuan he2, ying wang2, david pike3 toxic effects of carbaryl on the histology of testes of bufotes variabilis (anura: bufonidae) özlem çakici basal frequency of micronuclei and hematological parameters in the side-necked turtle, phrynops hilarii (duméril & bibron, 1835) maría a. latorre 1,2,*,#; evelyn c. lópez gonzález1,2, #; pablo a. siroski1,2,3; gisela l. poletta1,2,4 into a box interiors: clutch size variation and resource allocation in the european pond turtle marco. a.l. zuffi1,*, simonetta citi2, elena foschi1, francesca marsiglia1, eva martelli1 where to “rock”? choice of retreat sites by a gecko in a semi-arid habitat andreia penado1,2, ricardo rocha3,4,*, marta sampaio3, vanessa gil3, bruno m. carreira3, rui rebelo3 age structure, growth and longevity in the common toad, rhinella arenarum, from argentina clarisa de l. bionda1,2,*, silvia kost 4, nancy e. salas1, rafael c. lajmanovich3, ulrich sinsch4, adolfo l. martino1 on a putative type specimen of pleurodema bibroni tschudi, 1838 from chile (anura: leptodactylidae) daiana paola ferraro re-description of the external morphology of phyllomedusa iheringii boulenger, 1885 larvae (anura: hylidae), with comments on the external morphology of tadpoles of the p. burmeisteri group samanta iop¹, victor mendes lipinski¹, bruno madalozzo¹, franciele pereira maragno¹, sonia zanini cechin¹, tiago gomes dos santos² book review: harold heatwole, john w. wilkinson (eds). amphibians biology. volume 11 status of conservation and decline of amphibians. eastern hemisphere. part 4 . southern europe and turkey sebastiano salvidio book review: antonio romano. atlante degli anfibi del parco nazionale del cilento vallo di diano e alburni distribuzione, biologia, ecologia e conservazione sebastiano salvidio acta herpetologica journal of the societas herpetologica italica acta herpetologica 11(2): 189-195, 2016 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-18616 swimming performance and thermal resistance of juvenile and adult newts acclimated to different temperatures hong-liang lu, qiong wu, jun geng, wei dang* hangzhou key laboratory for animal adaptation and evolution, school of life and environmental sciences, hangzhou normal university, hangzhou 310036, zhejiang, china. *corresponding author. e-mail: dang.wei@hotmail.com submitted on 2016, 11th july; revised on 2016, 20th october; accepted on 2016, 22nd october editor: rocco tiberti abstract. thermal acclimatory adjustments of locomotor performance and thermal tolerance occur commonly in ectothermic animals. however, few studies have investigated ontogenetic differences in these acclimatory responses, and thus, their causes remain unclear. in this study, juvenile and adult chinese fire-bellied newts (cynops orientalis) were acclimated to one of two temperatures (16 or 24 °c) for 4 weeks to examine ontogenetic differences in acclimation effect on burst swimming speed, and critical thermal minimum (ctmin) and maximum (ctmax). swimming performance was thermally acclimated in both juvenile and adult c. orientalis. adult newts had greater absolute swimming speeds than juveniles, which may simply result from their larger sizes. cold acclimation enhanced low-temperature resistance, and warm acclimation enhanced high-temperature resistance in both juveniles and adults. despite no ontogenetic difference in ctmin, adult newts had greater ctmax and acclimation response ratio than juveniles, indicating their greater abilities to withstand extreme high temperatures and manage rapid temperature shifts. ontogenetic change in the thermal acclimatory responses of newts may be related to changes in the thermal environment they experience. keywords. salamandridae, ontogeny, thermal acclimatory response, swimming performance, thermal tolerance. introduction acclimation is the process that modulates the physiological and behavioural performance of organisms, allowing them to adjust to fluctuating environmental factors such as temperature, humidity, and salinity (lagerspetz, 2006). thermal acclimation of physiological and behavioural traits has been widely investigated in various organisms and has been shown to vary considerably among different species (angilletta et al., 2002; lagerspetz and vainio, 2006). due to the potential impact on determining resilience to future climate change, the thermal acclimatory ability of ectothermic species has attracted increasing attention in recent years (gvoždík, 2012; sandblom et al., 2014; seebacher et al., 2015). locomotor performance and thermal tolerance are fitness-related traits, and may determine the survival of animals that are exposed to high predation pressures or extreme environmental temperatures (arnold, 1983; leroi et al., 1994; willett, 2010). consequently, the locomotor performance and thermal tolerance of animals acclimated to various environmental conditions have frequently been assessed (wilson et al., 2000; gvoždík et al., 2007; měráková and gvoždík, 2009; grigaltchik et al., 2012; xu et al., 2015). such acclimation effects also vary at different ontogenetic stages (brooks and sassaman, 1965; menke and claussen, 1982; wilson and franklin, 2000; wilson et al., 2000). previous studies on anuran species have showed that thermal acclimatory ability on locomotor performance could easily be observed before metamorphosis, but was lost after metamorphosis, which was explained by an ontogenetic shift in the living environment (wilson and franklin, 2000; wilson et al., 2000). acclimatory ability on locomotor performance 190 hong-liang lu et alii should be reduced when metamorphosed frogs migrate from thermally stable aquatic habitats to terrestrial habitats with large daily temperature fluctuations (wilson and franklin, 2000; wilson et al., 2000). in most amphibian species, the critical thermal minimum (ctmin) and maximum (ctmax) generally increase with increasing acclimation temperatures (floyd, 1983; gvoždík et al., 2007; shi et al., 2012), but sometimes warm-acclimated metamorphosing tadpoles do not necessarily have higher ctmax than those that are cold-acclimated (cupp, 1980; menke and claussen, 1982). this might also be partly due to a shift in the thermal regime of a species (cupp, 1980; sherman, 1980; menke and claussen, 1982). however, studies on ontogenetic differences in the thermal acclimation of locomotor performance and thermal tolerance are still limited in amphibian species. since the mechanisms underlying the ontogenetic change in thermal acclimatory response are still not completely understood, it is necessary to collect more extensive data. the chinese fire-bellied newt, cynops orientalis, is a small-sized (up to 80 mm snout-vent length, svl) primarily aquatic newt that is widely distributed in central and eastern china, and can be commonly found in permanent ponds, rice terraces, and ditches (fei et al., 2006). c. orientalis individuals are predominantly aquatic, but occasionally migrate short distances across land to other water bodies. mating and oviposition occur between march and july when water temperature is between 15 and 23 °c (yang and shen, 1993). although the histology, sexual behaviour, and breeding ecology of this species have been studied during the past decades (yang and shen, 1993; sparreboom and mouta faria, 1997; xie et al., 2012; jin et al., 2016), none of these studies has focused on thermal physiological performance. here, we acclimated juvenile and adult c. orientalis to two temperatures for 4 weeks to examine ontogenetic differences in thermal acclimatory performances. on the basis of results from previous studies on the thermal acclimatory responses of amphibian species, we predict the following: (1) the ability to thermally acclimate locomotor performance should not disappear, (2) and ability to withstand extreme temperatures should be enhanced from juvenile to adult stages in predominantly aquatic newts. materials and methods all newts (16 metamorphosed juveniles and 24 adults) used in the present study were collected from fuyang mountainous area (hangzhou, zhejiang, eastern china) in july 2015 and transferred to our laboratory at hangzhou normal university. prior to thermal acclimation, animals were maintained in six 60 (l) × 50 (w) × 40 (h) cm3 aquaria (6−7 individuals per aquarium) with a water depth of 15 cm at 20 °c and on an l:d 12:12 photoperiod for 2 weeks. each aquarium was provided with pieces of tiles and some aquatic plants that served as refuges. the newts were then randomly divided into two groups (8 juveniles and 12 adults in each group), each of which was assigned to one temperature treatment: 16 or 24 °c. these temperatures were chosen because they may approximate the range of optimal temperatures for newt activity in the field (yang and shen, 1993). each group of animals was housed in five identical aquaria (4 juveniles or 4 adults per aquarium) in one of two temperature-controlled rooms held at the experimental temperatures. aquaria (photoperiod l:d 12:12) were placed on the same shelf to minimize water temperature difference among aquaria. water temperature of each aquarium was confirmed multiple times using a ut-325 electronic thermometer (uni-trend group, shanghai, china), and it varied less than 1 °c. newts were maintained at the designated temperatures for 4 weeks. throughout the experiment, newts were fed with tubifex worms or fish meat. all newts were measured for burst swimming performances at test temperatures of 16 and 24 °c, and allowed to rest for 48 h between trials. during the resting period, newts were maintained in their aquaria at corresponding acclimation temperature. to avoid possible test sequence effects, newts were randomly assigned to different test orders (different acclimation and test temperatures). the test temperatures of newts were achieved by placing them into an incubator at the corresponding temperatures for approximately 1 h prior to each trial. newts were placed into a racetrack (120 × 10 × 20 cm3) filled with water to a depth of 10 cm at the test temperature and then encouraged to swim by tapping the tails with a paintbrush. a panasonic hdc-hs900 digital video camera (panasonic co., japan) was positioned laterally to record the swimming performance of each newt. each newt was tested twice with a minimum of 30 min rest between the trials. to minimise the possible diel and photophasic effects, measurements on any given day started at 13:00 and ended within 3 h. all video-clips were examined using mgi videowave iii software (mgi software co., canada) for maximal speed over 25 cm. in the following text, speed was expressed as two metrics: absolute speed (cm/s) and relative speed (the ratio of absolute speed to svl for each individual, svl/s). we used the dynamic method for determining the ctmin and ctmax of the newts (kour and hutchison, 1970; lutterschmidt and hutchison, 1997). trials were conducted in water baths between 10:00 and 15:00. the newts were cooled or heated from their acclimation temperatures at a rate of 0.3 °c min-1 until individuals lost righting response, and their body temperatures were measured by inserting the probe of an electronic thermometer into the cloaca (lutterschmidt and hutchison, 1997; xu et al., 2015). we ran tests at 1-week intervals to minimise possible interactions between ctmin and ctmax. the newts were maintained in their aquaria during the intervals between trials. the thermal resistance range (trr) was calculated as the difference between ctmax and ctmin (van berkum, 1988), and the acclimation response ratio (arr) was calculated by dividing the tolerance change by the change in acclimation temperature (claussen, 1977). 191thermal acclimation in a chinese fire-bellied newt we used statistica 6.0 (statsoft, tulsa, usa) to analyse the data. data were tested for normality using kolmogorovsmirnov tests, and for homogeneity of variances using bartlett’s test. the primary analyses indicated that aquarium had no visible effects on swimming performance (mixed model anovas with aquarium as the random factor, all p > 0.532), so repeatedmeasure anovas were used to determine whether ontogeny, acclimation temperature and test temperature affected swimming performance. two-way anovas were used to determine whether ontogeny and acclimation temperature affected ctmin and ctmax. results there were no differences between groups in the body sizes of juveniles (svl: 39.9 ± 1.1 mm vs 41.5 ± 0.8 mm, t = 1.21, df = 14, p = 0.246; mass: 1.89 ± 0.09 g vs 1.84 ± 0.11 g, t = 0.35, df = 14, p = 0.731) or adults (svl: 64.2 ± 1.4 mm vs 65.4 ± 0.7 mm, t = 0.73, df = 22, p = 0.470; mass: 6.48 ± 0.49 g vs 7.00 ± 0.35 g, t = 0.86, df = 22, p = 0.400) prior to the beginning of the experiment. the absolute swimming speed of c. orientalis was significantly affected by acclimation, test temperature, and ontogeny (table 1, fig. 1a, b). overall, newts that acclimated and tested at high temperature swam faster than those acclimated and tested at low temperature. moreover, adults swam faster than juveniles (fig. 1a, b). the absolute speeds of newts were positively related to their svls (linear regression analysis, all p < 0.05). with regard to relative speed, the differences between acclimation temperatures and between test temperatures were still evident, but not between adult and juvenile individuals (table 1, fig. 1c, d). the interaction between test temperature and acclimation temperature, and between ontogeny and acclimation temperature had no significant effects on relative speed of newts (table 1). both mean ctmin and ctmax of juvenile and adult newts significantly increased as acclimation temperature increased (table 2, fig. 2a, b). overall, the mean ctmax of adults was significantly higher than that of juveniles (fig. 2b), but there was no significant difference in ctmin between adults and juveniles (table 2, fig. 2a). the effect of thermal acclimation on ctmax differed significantly between adults and juveniles, but this effect on ctmin did not (table 2). there was a significant increase in the ctmax of adults as acclimation temperature increased (t = 7.78, df = 22, p < 0.0001), but not in that of juveniles (t = 1.56, df = 14, p = 0.141) (fig. 2b). similarly, acclimation temperature significantly affected the trr of newts (table 2, fig. 2c). the acclimation temperature effect differed between adult and juvenile individuals. the trr of adults increased as acclimation table 1. results of repeated-measures anovas on swimming performance variables (absolute and relative speed) measured for juvenile and adult cynops orientalis acclimated to two different temperatures. swimming performance absolute speed relative speed acclimation temperature f1, 36 = 4.73, p = 0.036 f1, 36 = 6.27, p = 0.017 test temperature f1, 36 = 7.96, p = 0.008 f1, 36 = 10.31, p = 0.003 ontogeny f1, 36 = 13.43, p < 0.001 f1, 36 = 0.12, p = 0.726 acclimation × test temperature interaction f1, 36 = 0.12, p = 0.730 f1, 36 = 0.13, p = 0.716 acclimation temperature × ontogeny interaction f1, 36 = 0.22, p = 0.641 f1, 36 = 0.13, p = 0.720 test temperature × ontogeny interaction f1, 36 = 0.61, p = 0.439 f1, 36 = 0.03, p = 0.853 acclimation × test temperature × ontogeny interaction f1, 36 = 0.01, p = 0.905 f1, 36 = 0.02, p = 0.900 juvenile test temperature (oc) 16 24 r el at iv e sp ee d (s v l/ s) 0 1 2 3 4 5 6 16 24 a bs ol ut e sp ee d (c m /s ) 0 8 16 24 32 40 acclimated to 16oc acclimated to 24oc adult a b c d 1 2 fig. 1. mean values (+se) for swimming performance (absolute and relative swimming speed) of juvenile and adult cynops orientalis acclimated to different temperatures. 192 hong-liang lu et alii temperature increased, but slightly decreased in juveniles (table 2, fig. 2c). the arr values of ctmin and ctmax at acclimation temperatures between 16 and 24 °c were 0.08 and 0.07 for juveniles, and 0.12 and 0.26 for adults, respectively. discussion our results showed that thermal acclimation significantly affected the locomotor performance of c. orientalis. warm-acclimated newts appeared to have better locomotor performance than those that were cold-acclimated, which is not consistent with the beneficial acclimation hypothesis that predicts acclimation to a particular temperature should enhance an animal’s performance or fitness at that temperature (leroi et al., 1994). the effect of thermal acclimation on locomotor performance has been shown to vary among different amphibian species. for example, constant temperature acclimation failed to affect aquatic and terrestrial locomotor performance in adult ambystoma tigrinum nebulosum and ichthyosaura alpestris (else and bennett, 1987; šamajová and gvoždík, 2010), or only had acclimatory capacity in terrestrial locomotion to warm temperatures in triturus dobrogicus (gvoždík et al., 2007), or in aquatic locomotion to cold temperatures in eurycea guttolineata and pseudotriton ruber (marvin, 2003a, b). the fire-bellied newts living in permanent aquatic habitats in mountainous areas may experience limited temperature fluctuations at both juvenile and adult stages (fei et al., 2006). therefore, unlike those newts and salamanders that spend more than one-half of the year on land, such as a. tigrinum nebulosum and i. alpestris (else and bennett, 1987; šamajová and gvoždík, 2010), c. orientalis individuals do not lose the ability to acclimate their aquatic locomotor performance when they reach sexual maturity. this is consistent with our aforementioned prediction. in fact, the explanation proposed by wilson and franklin (2000) for the reduced acclimatory ability was based on the absence of thermal acclimatory responses of terrestrial locomotor performance rather than aquatic locomotor performance. aquatic locomotor performance can still be thermally acclimated in adults of fully aquattable 2. results of two-way anovas on critical thermal minimum, critical thermal maximum, and thermal resistance range of juvenile and adult cynops orientalis acclimated to two different temperatures. critical thermal minimum critical thermal maximum thermal resistance range acclimation temperature f1, 36 = 88.77, p < 0.0001 f1, 36 = 36.60, p < 0.0001 f1, 36 = 5.15, p = 0.029 ontogeny f1, 36 = 0.27, p = 0.606 f1, 36 = 9.76, p = 0.004 f1, 36 = 11.64, p = 0.001 acclimation temperature × ontogeny interaction f1, 36 = 3.07, p = 0.088 f1, 36 = 12.51, p = 0.002 f1, 36 = 8.35, p = 0.006 c ri tic al th er m al m ax im um (o c ) 35 36 37 38 39 th er m al r es is ta nc e ra ng e (o c ) 34 35 36 37 38 juvenile adult c ri tic al th er m al m in im um (o c ) 0.0 0.5 1.0 1.5 2.0 acclimated to 16oc acclimated to 24oc a b c 1 2 fig. 2. mean values (+se) for critical thermal minimum, critical thermal maximum, and thermal resistance range of juvenile and adult cynops orientalis acclimated to different temperatures. 193thermal acclimation in a chinese fire-bellied newt ic or semi-aquatic species (wilson et al., 2000; marvin, 2003a, b; gvoždík et al., 2007; wu et al., 2013; mineo and schaeffer, 2014; xu et al., 2015). inconsistent with the results of previous studies on one species of triturus newt and two species of ambystoma salamander (shaffer et al., 1991; wilson, 2005; landberg and azizi, 2010), adults swam faster than juveniles in c. orientalis. this might simply result from larger body size at adulthood because there was no significant ontogenetic difference in relative speed. the reduced swimming performance in adult urodeles amphibians is interpreted as a consequence of changes in tail shape, rather than a negative size effect on performance (landberg and azizi, 2010). the effect of ontogenetic change in tail shape on the swimming performance of c. orientalis should be investigated in future studies. the ability to withstand extreme temperatures may determine the survival of animals. the ctmin value for c. orientalis (0.5–1.5 °c) falls within the values reported for other fully aquatic or semi-aquatic urodeles (-1.9–3.9 °c for four desmognathus, one plethodon, and one eurycea salamanders, layne and claussen, 1982a, b, 1987), whereas the ctmax for c. orientalis (36.2–38.3 °c) is similar to the values reported for most other urodeles, and is higher than those for some high-latitude or high-altitude species (hutchison, 1961; brooks and sassaman, 1965; sealander and west, 1969; berkhouse and fries, 1995; gvoždík et al., 2007). compared with anuran species, for c. orientalis, the ctmin (tadpoles: 7.4−8.9 °c for fejervarya limnocharis, 8.7−11.7 °c for microhyla ornata, shi et al., 2012; but 0–1.6 °c for rana catesbeiana, menke and claussen, 1982; adults: 2.1−5.1 °c for three hyla treefrogs, layne and romano, 1985; 4.1−4.9 °c for rhinella arenarum and odontophrynus occidentalis, sanabria et al., 2012, 2013) and ctmax (tadpoles: 37–43 °c, cupp, 1980; sherman, 1980; navas et al., 2010; shi et al., 2012; simon et al. 2015; adults: 41.5–43.7 °c for two hyla treefrogs, blem et al., 1986; but 35.0–37.8 °c and 34.1–36.1 °c for r. arenarum and o. occidentalis, sanabria et al., 2012, 2013) were lower than those for most frog and toad species. therefore, thermal tolerance varies among amphibian species, and is believed to be correlated with habitat and geographic distribution (hutchison, 1961). moreover, adult c. orientalis had a greater ctmax than did juveniles, which was also found in other urodeles and anurans, such as e. nana, notophthalmus viridescens, bufo woodhousii fowleri, and hoplobatrachus chinensis (hutchison, 1961; sherman, 1980; berkhouse and fries, 1995; fan et al., 2012). as reported for other amphibian species (e.g., brooks and sassaman, 1965; sealander and west, 1969; menke and claussen, 1982; gvoždík et al., 2007; shi et al., 2012), low-temperature resistance can be enhanced by cold acclimation, whereas high-temperature resistance can be enhanced by warm acclimation in c. orientalis. warmacclimated adult newts had a relatively wider trr than those that were cold-acclimated, but this pattern was not observed in juveniles. contrarily, the trr of tadpoles decreased with increasing acclimation temperature (20, 25 and 30 °c) in two other anuran species, f. limnocharis and m. ornata (shi et al., 2012). although partially reflecting a difference in temperature treatment, the differential results from these studies may also reflect different optimal temperatures that enable animals to exhibit a high thermal resistance. those thermal conditions resembling environmental temperatures in animals’ natural habitats may be propitious for enhancing their thermal resistance (xu et al., 2015). the magnitude of the resistance response to thermal acclimation may reflect the ability to manage temperature shifts. it has been assumed that the species living in environments with large daily temperature variations should have a greater ability to withstand rapid temperature shifts than those living in thermally stable environments (sandblom et al., 2014). surprisingly, the arr of ctmax for adult c. orientalis is greater than that of other semi-aquatic urodeles (0.02–0.17, hutchison, 1961; sealander and west, 1969; gvoždík et al., 2007). despite no significant ontogenetic difference in acclimation effect on ctmin, the arrs of critical thermal limits in adult c. orientalis appeared to be greater than those of juveniles. combined with the greater ctmax and trr, our results indicate that adult c. orientalis have greater abilities to withstand extreme high temperatures and manage rapid temperature shifts than juveniles do. this is consistent with our second prediction. such ontogenetic shifts in thermal resistance may be related to  changes  in the  thermal environments experienced by active newts. animals living in warmer and more thermally variable environments are believed to have greater resistance abilities than those living in cooler, less variable environments (brooks and sassaman, 1965; berkhouse and fries, 1995). adult c. orientalis can be active over a wider area, and occasionally migrate from aquatic environments to humid-land environments. consequently, adult individuals are likely to be exposed to higher and more variable temperatures than are juveniles. acknowledgments our experimental procedures complied with the current laws on animal welfare and research in china, and were specifically approved by the animal welfare and ethics committee of hangzhou normal university (hznu194 hong-liang lu et alii 201506-005). this work was supported by grants from the natural science foundation of china (31670399) and zhejiang province (lq12c03003, ly15c030006), and the china scholarship council. references angilletta, m.j., niewiarowski, p.h., navas, c.a. 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(1993): studies on breeding ecology of cynops orientalis. zool. res. 14: 215-220. acta herpetologica vol. 11, n. 2 december 2016 firenze university press predator-prey interactions between a recent invader, the chinese sleeper (perccottus glenii) and the european pond turtle (emys orbicularis): a case study from lithuania vytautas rakauskas1,*, rūta masiulytė1, alma pikūnienė2 effective thermoregulation in a newly established population of podarcis siculus in greece: a possible advantage for a successful invader grigoris kapsalas1, ioanna gavriilidi1, chloe adamopoulou2, johannes foufopoulos3, panayiotis pafilis1,* the unexpectedly dull tadpole of madagascar’s largest frog, mantidactylus guttulatus arne schulze1,*, roger-daniel randrianiaina2,3, bina perl3, frank glaw4, miguel vences3 thermal ecology of podarcis siculus (rafinesque-schmalz, 1810) in menorca (balearic islands, spain) zaida ortega*, abraham mencía, valentín pérez-mellado growth, longevity and age at maturity in the european whip snakes, hierophis viridiflavus and h. carbonarius sara fornasiero1, xavier bonnet2, federica dendi1, marco a.l. zuffi1,* redescription of cyrtodactylus fumosus (müller, 1895) (reptilia: squamata: gekkonidae), with a revised identification key to the bent-toed geckos of sulawesi sven mecke1,*,§, lukas hartmann1,2,§, felix mader3, max kieckbusch1, hinrich kaiser4 the castaway: characteristic islet features affect the ecology of the most isolated european lizard petros lymberakis1, efstratios d. valakos2, kostas sagonas2, panayiotis pafilis3,* sources of calcium for the agamid lizard psammophilus blanfordanus during embryonic development jyoti jee1, birendra kumar mohapatra2, sushil kumar dutta1, gunanidhi sahoo1,3,* mediodactylus kotschyi in the peloponnese peninsula, greece: distribution and habitat rachel schwarz1,*, ioanna-aikaterini gavriilidi2, yuval itescu1, simon jamison1, kostas sagonas3, shai meiri1, panayiotis pafilis2 swimming performance and thermal resistance of juvenile and adult newts acclimated to different temperatures hong-liang lu, qiong wu, jun geng, wei dang* olim palus, where once upon a time the marsh: distribution, demography, ecology and threats of amphibians in the circeo national park (central italy) antonio romano1,*, riccardo novaga2, andrea costa1 on the feeding ecology of pelophylax saharicus (boulenger 1913) from morocco zaida ortega1,*, valentín pérez-mellado1, pilar navarro2, javier lluch2 notes on the reproductive ecology of the rough-footed mud turtle (kinosternon hirtipes) in texas, usa steven g. platt1, dennis j. miller2, thomas r. rainwater3,*, jennifer l. smith4 heavy traffic, low mortality tram tracks as terrestrial habitat of newts mikołaj kaczmarski*, jan m. kaczmarek book review: sutherland, w.j., dicks, l.v., ockendon, n., smith, r.k. (eds). what works in conservation. open book publishers, cambridge, uk. http://dx.doi.org/10.11647/obp.0060 sebastiano salvidio issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 8(1): 41-45, 2013 novel, non-invasive method for distinguishing the individuals of the fire salamander (salamandra salamandra) in capture-mark-recapture studies goran šukalo1,*, sonja đorđević2, dragojla golub1, dejan dmitrović1, ljiljana tomović2,3 1faculty of science, university of banja luka. mladena stojanovića 2, 78000 banja luka, republic of srpska, bosnia and herzegovina. *corresponding author. e-mail: sukalogoran@yahoo.com 2university of belgrade, faculty of biology, institute of zoology. studentski trg 16, 11000 belgrade, serbia 3institute for biological research “siniša stanković”, university of belgrade. bulevar despota stefana 142, 11000 belgrade, serbia submitted on: 2013, 16th january; revised on: 2013, 3rd march; accepted on: 2013, 12th march. abstract. recently we started implementing a highly efficient, non-invasive method of direct individual marking (i.e., typifying) in a population study of the fire salamander, salamandra salamandra. our technique is based on the unique alphanumeric code for every individual, generated upon the numbers of openings of repellent/toxic skin glands in the yellow areas of the selected regions of the body. this code was proved reliable in the sample of 159 individuals from two separate populations and enabled easy and quick recognition of recaptured animals. the proposed method is inexpensive, easily applicable in the field, involves minimum stress for the animals and does not affect their behaviour and the possibility of repeated captures of “marked” (i.e., coded) individuals. it is particularly suitable for dense populations. keywords. salamandra salamandra, individual recognition, non-invasive, natural markings. the capability to accurately identify individual animals within a population is fundamental for capture– mark–recapture (cmr) studies, which provide basic information regarding population ecology. individual identification is indispensable for gaining information on individual growth rates, attainment of sexual maturity, rates of reproduction, survival and other life-history parameters and for the studies of various aspects of behaviour (davis and ovaska, 2001). the accurate assessment of population size and density is vital for efficient management and protection of species (bradfield, 2004). numerous techniques are used for individual marking of amphibians: toe clipping (donnelly et al., 1994; mccarthy and parris, 2004; liner et al., 2007), pattern mapping (donnelly et al., 1994; arntzen and teunis, 1993; gamble et al., 2008), branding, polymers and pigments (donnely et al., 1994), photographic identification (kurashina et al., 2003; plăiaşu et al., 2005; gamble et al., 2008; clemas et al., 2009), passive integrated transported (pit) tags (jehle and hödl, 1998), visible implant elastomer (vie) tags (campbell et al., 2009), and visible implant alphanumeric (via) tags (clemas et al., 2009; osbourn et al., 2011). being cheap and simple, the most frequently used invasive marking method is toe clipping (donnelly et al., 1994). however, this method involves stress and tissue damaging, which can increase, for example, the risk of infection (mccarthy and parris, 2004). also, the toes can regenerate; therefore these markings cannot be considered permanent (donnelly et al. 1994; davis and ovaska, 2001; ursprung et al., 2011). in addition, recent studies demonstrated decreased rates of recapture following the marking by toe clipping (davis and ovaska, 2001; mccarthy and parris, 2004; mccarthy et al., 2009). 42 goran šukalo et al. currently, photo-identification is the most frequently used non-invasive method in population studies of amphibians. photo-identification was previously used in population studies of several amphibian species; however, it was often proved reliable only in short-time studies because the patterns of spots, i.e. markings on the skin change over time (arntzen and teunis, 1993; kurushina et al., 2003; plăiaşu et al., 2005). another frequently stated drawback of individual identification based on colouration pattern was the time-consuming analyses of the photographs (plăiaşu et al., 2005). previous “marking” schemes considered solely the colouration pattern; we propose the inclusion of glands openings, supposing they vary less than the colour pattern. this modification considerably reduces the time necessary to analyse the photographs. to simplify the recognition of recaptured individuals in large populations, the addition of toe clipping to non-invasive approaches was suggested (plăiaşu et al., 2005). however, it should be kept in mind that amphibians’ toes (or complete limbs) can be deformed and/or lost due to various naturally causes, e.g. sublethal predation or infection (gray et al., 2002; stopper et al., 2002; bowerman et al., 2010; sessions and ballengée, 2010); also, amphibians can regenerate clipped toes (ursprung et al., 2011). besides, davis and ovaska (2001) showed that the number of recaptured animals with clipped toes was only 40%, compared to 60% of animals with fluorescent markings; that result suggested higher mortality or altered behaviour among the animals with clipped toes. here we propose a highly efficient, non-invasive method for recognizing individual animals, particularly suited for the fire salamander (salamandra salamandra). the method is based on the combination of the numbers of openings of the excretory ducts of skin glands in certain body regions. it causes minimum stress and there is no need to firmly restrain or anesthetize the animals. to our knowledge, our study is the first capture-mark-recapture assessment of salamandra salamandra populations ever performed in the balkan peninsula. the present study is based on the sample of 159 fire salamander individuals (157 adults, 2 juveniles), which were captured and photographed in the field. the study was conducted from march to november 2012, in two localities near the city of banja luka (republic of srpska, bosnia and herzegovina). the samples from the two populations consist of 91 and 68 individuals, respectively. we made three photographs of every individual: its left parotoid gland, right parotoid gland, and the entire dorsal side of the body. we used the digital camera sony dsc-s980. also, we clipped the fourth toe from the right hind limb. immediately after processing the individuals were released at the exact places of capture. the analysis of the number of toxic glands openings (black dots) on yellow surfaces in the selected regions of the body was conducted in the laboratory, based on the obtained photographs. a database of photographs was formed, necessary for subsequent identification of potential recaptures. we assigned an identification code to every individual, which consisted of three combinations of numbers and letters. the first combination depicts the number of gland openings on the yellow surface on the left parotoid gland, the second shows the number of gland openings on the yellow surface on the right parotoid gland, and the third set presents the number of gland openings on the yellow surfaces on the mid-dorsal side of the body, along the vertebral column (see fig. 1). the codes were entered into the excel database, along with individual descriptions, which enabled easy and quick search. the identification code for the individual shown in fig. 1 is: 14l/17r/14v, and it represents the following: 14 glands openings on the yellow surface covering the left parotoid gland (a), 17 openings on the yellow surface on the right parotoid (b), and 14 glands openings on the yellow surfaces along the trunk and tail in the two parallel rows following the vertebral column (c). note that we considered only the fully circular glands openings: five incomplete ones on the right side of the head were not counted. fig. 1. fire salamander’s body regions with the skin glands openings important for generating the unique identification code 43novel identification method for salamandra salamandra the number of the glands openings on the yellow surface on the left parotoid gland ranged from 0 to 26, and on the right parotoid it was between 1 and 29; on the yellow areas following the vertebral column along trunk and tail there were 0 to 36 glands openings (pooled sample, 159 individuals; fig. 2). this range of variation allows huge numbers of possible combinations within populations, i.e. there is only a minute probability of finding several individuals with the identical code and pattern. we assumed that the numbers of glands openings (i.e., yellow surfaces they are encircled by) do not considerably change over time, at least not in adult animals. however, in this initial phase of the capture-mark-recapture study, we performed the standard toe-clipping procedure in order to have an independent validation of photo-identification. among the 159 processed animals, we recaptured eleven individuals; all were recognized by the means of individual codes and spot patterns. time lag between the initial capture and subsequent recapture ranged from one to six months. using only the part of the alphanumeric code representing the numbers of poison glands openings on the yellow areas covering the parotoid glands, we successfully distinguished 72.5% and 89.7% of the samples from the two populations. with the inclusion of an additional trait, i.e. the number of glands openings on the yellow patches along the midline of the trunk and tail (two parallel rows of openings directly above the vertebral column, see fig. 1), the confidence of identification reached 100%, i.e. we precisely distinguished all the individuals from both populations. our alphanumeric code was proved unique in almost all individuals from the two sampled populations. comparison of the codes of all 159 individuals (pooled sample, both localities) showed that only two individuals (1.26%) had the identical codes, i.e. the matching combination of glands openings on the head and body (8l/12r/0v). however, checking the photographs in the database enabled quick and successful distinguishing of fig. 2. histogram of glands opening numbers in the selected body regions. 44 goran šukalo et al. these two animals, based on the differences in the pattern of yellow patches on the dorsal side of the body. the technique we propose satisfies the demands for the ideal marker (beausoleil et al., 2004; gibbons and andrews, 2004): an animal is subjected to no pain and to minimum stress, it is identifiable, the technique is easily applicable in the field (no software is necessary), it’s economical, does not lead to increased mortality and/ or reduced growth or reproduction, it has no effect on the behaviour of marked animals (or of the other animals towards the marked ones), and does not affect the probability of future capturing of marked compared to unmarked animals. if the cameras with high resolution and zooming performances are available, the animals can even be photographed directly in their natural environment, without the need of disturbance and capturing (compare, for example, lambert et al., 2012). considering all the above mentioned, the technique we propose is highly suitable for salamandra salamandra populations, such as the two sampled in this study. toe clipping, which we also applied in this preliminary phase of the cmr study, facilitated the recognition of recaptured individuals. this invasive procedure shall be abandoned if we prove satisfying accuracy of our newly proposed methodology. although the colour patches pattern is liable to changes during ontogeny (e.g. beukema, 2011), we can assume that in adults it does not change dramatically. on the other hand, the arrangement of salamanders’ poisonous glands does not seem to change during lifetime (mcmanus, 1935). in the years to come it is necessary that we continue monitoring this trait in our study populations, in order to check the possible pattern changes in various life stages. acknowledgements in the period when this research was conducted, a list of protected amphibian species did not exist in bosnia and herzegovina; 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(2011): toe regeneration in the neotropical frog allobates femoralis. herpetol. j. 21: 83-86. acta herpetologica vol. 8, n. 1 june 2013 firenze university press the oogenic cycle of the caspian bent-toed gecko, cyrtopodion caspium (squamata: gekkonidae) in iran vida hojati1*, kazem parivar2, eskandar rastegar-pouyani3, abdolhossein shiravi1 altitudinal effects on life history parameters in populations of liolaemus pictus argentinus (sauria: liolaemidae) joel antú gutiérrez1,*, carla piantoni2, nora r. ibargüengoytía1,3 recent cryptic extinction of squamate reptiles on yoronjima island of the ryukyu archipelago, japan, inferred from garbage dump remains yasuyuki nakamura1,*, akio takahashi2, hidetoshi ota3 trophic niche and feeding biology of the italian wall lizard, podarcis siculus campestris (de betta, 1857) along western mediterranean coast marco a.l. zuffi*, chiara giannelli novel, non-invasive method for distinguishing the individuals of the fire salamander (salamandra salamandra) in capture-mark-recapture studies goran šukalo1,*, sonja đorđević2, dragojla golub1, dejan dmitrović1, ljiljana tomović2,3 going out tonight? when insular hierophis viridiflavus breaks the whip snakes rules delaugerre michel-jean first evidence of a paedomorphic population of the smooth newt (lissotriton vulgaris) in the czech republic václav gvoždík1,2, veronika javůrková4, oldřich kopecký5,* no detection of chytrid in first systematic screening of bombina variegata pachypus (anura: bombinatoridae) in liguria, northern italy stefano canessa1,*, an martel2, frank pasmans2 status of the european pond turtle, emys orbicularis (reptilia: testudines: emydidae) in vorarlberg, austria andreas kleewein1, günther wöss2 comparative cytogenetics of two species of ground skinks: scincella assata and s. cherriei (squamata: scincidae: lygosominae) from chiapas, mexico riccardo castiglia1,*, alexandra m.r. bezerra2, oscar flores-villela3, flavia annesi1, antonio muñoz4, ekaterina gornung1 polydactyly in the tyrrhenian wall lizard (podarcis tiliguerta) antigoni kaliontzopoulou1,*, daniele salvi1, verónica gomes1, joão p. m. c. maia1,2,3, panagiotis kaliontzopoulos4 book review: roberto sindaco, alberto venchi, cristina grieco. the reptiles of the western palearctic. 2. annotated checklist and distributional atlas of the snakes of europe, north africa, middle east and central asia, with an update to the vol. 1. edoardo razzetti acta herpetologica journal of the societas herpetologica italica acta herpetologica 9(2): 139-146, 2014 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-13237 presence of chitinase enzymes in crocodilians pablo a. siroski1,2,*, gisela l. poletta1,3, maría v. parachú marcó1,4, hugo h. ortega2, mark e. merchant5 1proyecto yacaré laboratorio de zoología aplicada: anexo vertebrados (fhuc unl / maspyma), a. del valle 8700, santa fe, argentina. *corresponding autor. e-mail: psiroski@fcv.unl.edu.ar 2 instituto de ciencias veterinarias del litoral (icivet conicet/unl), r. p. kreder 2805. s3080hof — esperanza, santa fe, argentina 3cat. toxicología, farmacología y bioquímica legal, fbcb, unl, santa fe, argentina/iegeba, fceyn (conicet-uba), buenos aires, argentina 4cic y ttp conicet. dr. materi y españa, (3105) diamante, entre ríos. argentina 5 department of chemistry, mcneese state university, p.o. box 90455, lake charles, louisiana 70609, usa submitted on 2013, 15th august; revised on 2014, 16th june; accepted on 2014, 4th september editor: aaron m. bauer abstract. chitin is an abundant bio-polymer present as a structural component of many organisms such as arthropods, nematodes, mollusks, insects, and fungi, among others.  chitinolytic enzymes are synthesized for organisms to defend themselves against chitin-containing pathogens.  chitotriosidase (cht) is a chitinase enzyme and one of the main proteins secreted by activated macrophages. it plays an important role in mechanisms of immunity by hydrolyzing chitin, thus protecting against chitin-containing pathogens. in this study, cht was detected in  caiman latirostris  plasma, and characterized under laboratory controlled conditions of temperature, reaction time, plasma concentration, ph and salinity. the results complement other immunological studies performed in caimans and demonstrate that they possess an efficient and well-developed immune system that resists the attack of some pathogens.  based on the current knowledge of the properties and homologies of cht, it would be highly valuable to evaluate its possible therapeutic application in the veterinary clinical setting. keywords. chitinases, crocodilians, innate immunity, immunology. introduction chitin, a repeating β (1→4)-linked homopolymer of n-acetyl-β-d-glucosamine (glcnac), is the second most abundant biopolymer on earth next to cellulose (daimon et al., 2003). it can be found as a structural component in coatings of many species, including the integument of arthropods (neville et al., 1976), the microfilarial sheath of parasitic nematodes (fuhrman and piessens, 1985; araujo et al., 1993), mollusks (badariotti et al., 2007), the gut linings of insects (shahabuddin and kaslow, 1994), the cell walls of fungi (kuranda and robbins, 1991) and some algae (badariotti et al., 2007). production of chitin depends on chitin synthase, while degradation or remodeling of chitin-containing structures requires chitinases. these enzymes are essential for maintaining normal life cycle functions such as morphogenesis of arthropods (merzendorfer and zimoch, 2003) or cell division and sporulation of yeast and other fungi (kuranda and robbins, 1991). chitinolytic enzymes have also been found in organisms that do not contain chitin polymers themselves, such as viruses, bacteria, plants and animals (badariottia et al., 2007). indeed, chitinases also have an important role in parasitic invasion of chitinous hosts. several studies have demonstrated that chitinases are also produced constitutively, or inducibly, as pathogen resistant proteins; thus vertebrates synthesize chitinases to defend themselves against chitin-containing pathogens such as protozoa, fungi, insects and nematodes (renkema et al., 1995; gooday, 1999). mammals express two active chitinases, chitotriosidase (cht, ec number: 3.2.1.14) and acidic mammalian 140 p.a. siroski et alii chitinase (amcase). only amcase has been detected in the gastro-intestinal tract, probably due to its acidic ph (bussink et al., 2008). chitotriosidase enzyme glycosyl hydrolase, is one of the main proteins secreted by activated macrophages and has been characterized at the protein and gene level (hollack et al., 1994). the enzyme is synthesized in massive amounts and is predominantly secreted as a 50 kda protein by specifically-activated macrophages. about one third of the enzymes synthesized are proteolytically processed to a 39 kda form that accumulates in lysosomes. in the blood stream, the secretory 50-kda cht occurs predominantly, whereas in tissues, the 39-kda form is most abundant (renkema et al., 1997). the 39 kda enzyme is c-terminally truncated and lacks a chitin-binding domain. chitotriosidase is located in specific granules of polymorphonuclear cells and secreted following stimulation with granulocyte macrophage colony-stimulating factor (gm-csf). in addition, gm-csf induces expression of cht in macrophages, not expressed in monocytes that constitutively secrete the enzyme and partially accumulate it in their lysosomes in a number of pathological settings period. it is also produced in vitro after prolonged cultivation (renkema et al., 1998). chitotriosidase activity had been known to cleave both colloidal chitin and the cell wall chitin of candida albicans, and inhibits cryptococcus neoformans, and mucor rouxii proliferation, suggesting a role in defense against chitinous human pathogens (boot et al., 2001; van eijk et al., 2005). elevated chitinase activity has also been detected in the blood and tissues of guinea pigs with systemic aspergillus fumigatus infection (overdijk et al., 1996; 1999). plasma chitotriosidase activity is increased in several lysosomal (brinkman et al., 2005; ries et al., 2006; vedder et al., 2006) and nonlysosomal diseases (michelakakis et al., 2004). serum and plasma cht is used as diagnostic marker of gaucher disease. elevated cht reflects a gradual intralysosomal accumulation in the liver, spleen, or bone marrow, which is indicative of gaucher cell (lipid-loaded macrophages). macrophages overloaded with the enzyme accumulated in lysosomal material (lipids) were shown to secrete cht. in contrast, low levels of cht have been associated with susceptibility to infection by chitin containing parasites (bussink et al., 2006). in fact, many studies have been performed to detect cht activity towards chitin-containing pathogens both in vitro and in vivo (van eijk et al., 2005). crocodiles exhibit a versatile and efficient nonspecific immune system adapted to the environments in which they commonly live. generally, these animals live in environments (both natural and captive) that contain high concentrations of pathogenic microorganisms. for different reasons, serious injuries occur and sometimes animals lose entire limbs as a consequence of social disputes (webb and manolis, 1983). however, crocodilians normally tolerate these injuries and generally do not show signs of illness (siroski et al., 2009). crocodilians have been shown to have immune components with an apparently higher activity than others animals, including humans (siroski, 2011), which make them interesting models for the elucidation of those mechanisms. these immunological effector mechanisms necessary for the efficient control of infectious agents are dependent on the distinct defense strategies. some components involved in these routes of the defense system have been identified and characterized. these include serum complement cascades (merchant et al., 2003; siroski et al., 2009), dipeptidilpeptidase (merchant et al., 2010; siroski et al., 2011), and phospholiphases (merchant et al., 2009a; 2011; siroski et al., 2013). all these features may converge in the antimicrobial properties detected in different crocodilian tissues (shaharabany et al., 1999; merchant et al., 2003; siroski et al., 2009). to our knowledge, cht activity in the plasma of crocodilians has not been studied to date. due to the important role of cht enzymes in the generation of the innate immune system, we aimed to analyze the presence of this enzyme in caiman latirostris plasma under different biochemical conditions (plasma concentration, time, temperature, ph, and salinity-dependence) to characterize its activity. material and methods for this study, wild caiman latirostris (n=13; 7 females and 6 males) were captured from different pristine natural areas in the province of santa fe, argentina. due to the influence of temperature on the physiology of these animals, all samples were collected during the summer. animals were bled and measured (range 1.51 to 2.31 m of total length), and then returned to their environment within an hour of capture. the blood samples were collected immediately after capture to avoid stress in the animals. blood samples were obtained from spinal vein (zippel et al., 2003) using heparin as an anticoagulant. whole blood was centrifuged immediately at 1000 g for 20 min, at room temperature (approximately 24°c). the plasma was frozen at -20°c and cht enzyme assays were conducted within seven days of the collection of samples. plasma cht enzyme activity was determined as described in hollack et al. (1994) using the artificial substrate 4-methylumbelliferyl-β-d-n,n´,n´´-triacetylchitotrioside (4 mu-chitotrioside; sigma chemical co., st. louis, mo). the enzyme assay mixture contained 15 µl of plasma and 200 µl (0.022 mm) of the substrate dissolved in citrate-phosphate buffer, ph 5.2, in a total volume of 215 µl. the reaction was stopped with 2 ml of glycine-sodium hydroxide (stop buffer), 141chitinase enzymes in crocodilians ph 10.6. this mixture was incubated under different conditions depending on the type of assay, and the fluorescence of each sample was determined with a spectrofluorimeter (excitation and emission wavelength of 365 and 450 nm, respectively). the cht activity was determined by plotting a standard curve of the 4-methylumbelliferone product against the relative fluorescence units. caiman cht activity temperature dependence. to evaluate the effect of temperature on enzyme activity, cht assays were performed at different temperatures (from 5°c to 40°c, with intervals of 5°c). prior to the onset of the reaction, aliquots of plasma and substrate dissolved in buffer were incubated separately for 15 min to reach the corresponding temperature of the study. components of the reaction were mixed and incubated for 30 min at the same temperatures. caiman cht activity plasma concentration dependence. to determine the effect of plasma concentration on cht activity, different amounts of caiman plasma (0, 1, 2, 5, 10, 20, 50 and 100 µl) were used. the reaction was stopped after 30 min of incubation. caiman cht activity time dependence. to evaluate the kinetics of the cht activity, the substrate was incubated with the caiman plasma for varying amounts of time (0, 5, 10, 15, 20, 30, 60, and 90 min). aliquots of plasma and substrate dissolved in citrate-phosphate buffer were mixed by quadruplicate. the reaction was stopped with 2 ml of glycine-sodium hydroxide buffer after the different time points. caiman cht activity ph dependence. the effects of the ph on the cht activity were evaluated using several buffers (mcilvain buffer containing dihydrogen phosphate in different concentrations to regulate the ph) varying from ph 4 to 10. after 30 min incubations, stop buffer was used to terminate the reaction. caiman cht activity salinity dependence. to determine the effects of salinity on cht activity, different volumes of 1 m nacl solution (from 0 to 100 µl) were added to the enzyme assay mix (plasma and substrate) and the total volume was balanced with distilled water and incubated for 30 min. statistical analyses. all assays were performed in quadruplicate and the results are expressed as µm ± standard error (se). statistical analyses were performed using the software spss 14.0 for windows (spss for windows, 2005). nonlinear regression analyses were used to describe the effects of each variable on cht activity, and a p value ≤ 0.05 was considered statistically significant. results the cht activity of c. latirostris was dependent on the incubation temperature during the reaction, showing a positive relationship up to 35°c (r=0.8907) (fig. 1). chitotriosidase activity was compromised at low temperatures (5, 10 and 15°c; p <0.05), and at 40°c. in the assays performed to determine the influence of plasma concentration on cht of c. latirostris, the activity exhibited an increase when volumes of plasma increased (r2= 0.8956, fig. 2) showing a volume-dependent activity. low volumes of plasma produced a significant increase in cht activity. the 50% of the maximum activity was reached with approximately 15 µl of caiman plasma and the higher cht activity was obtained with the addition of 30 µl of plasma. time of incubation was found to have a positive relationship with the substrate (r2=0.6425). caiman plasma cht exhibited high activity immediately after the addition of substrate, gradually increased with time. activity was elevated from 5 to 40 min of incubation with fluorescent substrate, the moment at which a plateau was reached by the curve until the last point of time studied fig. 1. chitotriosidase activities in caiman plasma increased with incubation temperature (r=0.8907). samples were analyzed in quadruplicates, and are presented as means ± standard error. fig. 2. the increment of plasma concentration was positively correlated with the product formed as consequence of chitotriosidase activity (r= 0.8956; p <0.05). samples were treated as independent in quadruplicates, and presented as means ± standard error. 142 p.a. siroski et alii (fig. 3). figure 4 displays the assay performed to evaluate the optimum functioning ph for cht activity in c. latirostris plasma (r2=0.7497). incubation of caiman plasma at different ph showed that the optimum ph for enzyme activity is 7. activity decreased to 50% of the maximum at ph 6 and 8, and practically no activity was detected at both ph extremes tested. the cht activity of c. latirostris plasma was assayed under different salinity conditions and increased with salinity (r2=0.5697) at low concentrations (fig. 5), reaching the highest value with the addition of different volumes of 1 m nacl. the addition of 80 µl (or more) of saline solution resulted in a reduction of cht activity at levels even lower than base line. discussion chitinases are ubiquitous enzymes that hydrolyze chitin, and have been identified in various organisms; they are involved in several biological processes including defense against chitin containing pathogens, such as fungi (sahai and manocha, 1993). presently, these enzymes are receiving considerable attention due to their importance in very diverse processes such as the life cycles of chitin-containing organisms, food processing, defense against pathogens, and innate immunity. particularly, some of these processes have been proposed to be very effective in crocodilians (siroski et al., 2011, 2013). based on the results obtained from the reaction between caiman plasma and 4 mu-(chitotrioside), the specific substrate of cht allowed us to identify the first known crocodilian chitinase, c. latirostris cht. this cht is structurally homologous to chitinases from various species (barone et al., 2007). they are secreted by activated macrophages and probably play a role in the defending against chitin-containing pathogens, in tissue remodeling and cell migration. in this study, we report similar values in animals apparently healthy or without any signs of illnesses. there are few studies of chitinase activity in other reptiles. several genera of old world lizards including, anguis, uromastyx, chamaeleo and lacerta (jeuniaux, 1961, 1962, 1963); and two new world lizards, anolis carolinensis (jeuniaux, 1962, 1963) and sceloporus undulatus garmani (marsh et al., 2001) have been reported fig. 5. the effects of the several salinities conditions reached with differents quantities of nacl on the chitotriosidase activity in samples of caiman latirostris plasma in quatriplicate and presented as means ± standard error. fig. 3. chitotriosidase activity in caiman latirostris plasma reached a very high level over a short period. similar behavior was detected in others enzymes activities studied involved in the caiman immune system. fig. 4. the ph functioning for chitotriosidase activity in caiman latirostris plasma showed the highest enzyme activity around 7 and a wide tolerance to work under and above this. 143chitinase enzymes in crocodilians to secrete chitinase. these studies have shown that some vertebrates produce chitinase in the whole digestive tube in order to metabolize chitin as a food source (karasov, 1989). thus, the production of chitinase would allow them to utilize the chemical potential energy in their food more efficiently. polymorphonuclear leukocytes, but not lymphocytes and monocytes, are a major source of cht in blood. chitotriosidase hydrolyzes chitin substrates similarly to chitinases that are found in a variety of species (boot et al., 1998), but this study is the first report of the presence and characterization of cht in crocodilian plasma, or for that matter in any reptile species. in our study, we learned that cht exhibits optimal activity in a narrow range of biochemical conditions. we chose several factors that are known to affect the rate at which the enzymes work, including temperature, salinity, ph, plasma concentration, and kinetic of incubation. because any of these parameters might affect the rate of cht enzyme reaction, each must be carefully controlled if we attempt to study the effects of changes in the enzyme itself. in all assays carried out the cht enzymes of c. latirostris demonstrated high levels of activity. ectothermic vertebrates are considered appropriate models to assess the influence of temperature on a variety of physiological functions (pxytycz and zkowicz, 1994). plasma cht activity demonstrated a positive relationship with temperature (fig. 1). when the incubation temperature is 15°c, cht thermal activity was found to be very low, but increased at higher temperatures. crocodilian physiology is considered practically “null” at temperatures below 15°c, but c. latirostris plasma cht activity detected at these temperatures could be attributed to the greater climatic tolerance of c. latirostris compared with other related species (siroski, 2004). maximum cht activity was detected between 30 and 35°c. this is reasonable because crocodilians have been demonstrated to maintain body temperature within a range of 28-33°c by using natural thermal gradients, and this range is close to the preferred temperature of c. latirostris for carrying out normal physiological processes (bassetti, 2002). to our knowledge, there are no published studies concerning the temperature dependence of cht activity in other species, and we have found that caiman cht requires a specific environment and conditions within which to function. this is to be expected in ectotherms in which biochemistry and physiology are highly dependent on temperature (siroski et al., 2012; siroski et al., 2013). the effect of increased volumes of caiman plasma with constant amounts of substrate solution was associated with increased cht activity up to a peak at which activity remains high, independent of the plasma volume added. with this extreme sensitivity reproducible results with minimal variance can be obtained with small volumes of plasma, which can be extremely useful for studies in which it is not possible to obtain high volumes of blood. similarly low volumes of plasma were found to be sufficient to produce measurable activities with high precision in this and related crocodilian species in the determination of crocodilian plasma phospholipase, pla2 (merchant et al., 2010; siroski et al., 2013) and dipeptidilpeptidase, dppiv enzymes (merchant et al., 2009b; nevalainen et al., 2009; siroski et al., 2012), as well as with respect to serum complement activities (merchant et al., 2005; merchant and britton, 2006; siroski et al., 2010). our findings demonstrated that caiman cht exhibited time-dependent activity. after only five minutes of incubation of substrate with caiman plasma, cht activity rapidly increased. high concentrations of the reaction products were detectable at 2 min, and steadily increased up to a maximum at 30 min, at which time, the cht activity showed a plateau until the last time point evaluated. these results support those observed in similar studies performed in dppiv and pla2 in the same species (siroski et al., 2011). observations such as these highlight immunological properties of caiman serum to to respond to challenges from microbial infection. the ph of a solution is important for the structure and function of enzymes. outside the range of the optimum ph of an enzyme, its structure can be compromised, and thus the activity can decrease dramatically. high temperatures and changes in ph can denature the enzyme, or result in conformation changes. the phactivity relationship observed in this species differs from that in other species and other types of chitinases. at intermediate ph values (6-8), plasma cht activities were elevated. it is expected that the ph optimum for each chitinase isoform is close to the ph of the environment in which it is active in vivo, and may differ depending on tissue origin. chitinolytic  activity  was present in extracts of stomach, small intestine and pancreas, with the stomach and pancreas having the highest specific  activities (jeuniaux et al., 1982). for reptiles, optimum ph for intestinal and pancreatic chitinase is 4.5, with no activity at ph 1.0, whereas stomach chitinase has an optimum at ph 3.0, with strong activity at ph 1.0 (micha et al., 1973; jeuniaux et al., 1982). in this context, it seems desirable to investigate why the cht activity in our samples occurred over such a broad ph range, but this feature could be another adaptive advantage that is part of an efficient defense mechanism. it is expected that cht isoforms are close to the osmolality of environmental conditions and may be seasonally-related. under these conditions, maximum activi144 p.a. siroski et alii ties correspond to each species and may differ depending on tissue origin, or in response to tissue-specific metabolic demands. in this study, the effect of increasing concentrations of salinity  affected cht caiman plasma activity. such ionic strength dependence is characteristic of a number of enzymes whose activities are inhibited or diminished at low or high salt concentrations; one would expect that changes in salt concentration cause conformational changes in these enzymes. to our knowledge, there are no studies concerning the salinity tolerance of proteins in crocodilians. however, those few crocodilian species that inhabit water of high or low salinity, or estuarine waters with fluctuating salinities probably exhibit enzymes that can act across a broader range of salinities than those of caimans. such species have the ability to actively increase their body fluids in low or high salinity environments. hyperregulation is accompanied by permanent exchange of ions from the internal and external milieu, and thus, mechanisms to counteract dilution or concentrated of body fluids had to be developed. changes in salinity can alter the functions of enzymes and hormones with serious consequences, resulting in a variety of metabolic changes identical to those caused by water stress, as detected by lance et al. (2010), and are probably mediated by hormonal signals (elsey, 2005). out of this tolerance range, one would expect cht activity to fall under the severity of the imposed salinity stress. we found the highest enzyme activity values reasonably close to the physiological ph and salinity conditions of caiman plasma. despite this, caiman plasma cht activities also were detected under extreme ph conditions, but at lower activities. the fact that this enzyme exhibits activity outside of normal physiological conditions (at which the enzymes of only a few species would be active) could possibly be another reason for the effectiveness of the caiman’s immune system. additional evidence for a role of cht during immunological responses is the observation that the enzyme is rapidly and acutely up-regulated with pro-lactin, ifn-γ, tumour necrosis factor α, and lps, but not with il-10 (malaguarnera et al., 2004; di rosa et al., 2005). involvement of cht in caiman immune defenses was not investigated but it would be very interesting to measure its plasma and tissue expression level after both bacterial and lps challenges. the high activity of cht detected in caiman plasma suggests that future investigations may reveal interesting information regarding the properties and homologies of this enzyme in crocodilians. based on the importance and function of cht enzyme, it could be used to study molecular immune mechanisms in a phylogenetic context or could have a possible clinical application as a biomarker of individual health status. references araujo, a.c., souto-padrón, t., de souza w. 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(1983): crocodylus johnstoni in the mackinlay river, n.t. v. abnormalities and injuries. aust. wildl. res. 10: 407-409. zippel, k.c., lillywhite, h.b., mladnich, c.r. (2003): anatomy of the crocodilian spinal vein. j. morphol. 258: 327-335. acta herpetologica 11(2): 111-118, 2016 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-18075 effective thermoregulation in a newly established population of podarcis siculus in greece: a possible advantage for a successful invader grigoris kapsalas1, ioanna gavriilidi1, chloe adamopoulou2, johannes foufopoulos3, panayiotis pafilis1,* 1 section of zoology and marine biology, department of biology, national and kapodistrian university of athens, panepistimioupolis, gr-15784, greece. *corresponding author. e-mail: ppafil@biol.uoa.gr 2 zoological museum, department of biology, national and kapodistrian university of athens, panepistimioupolis, gr-15784, greece 3 school of natural resources and environment, university of michigan, ann arbor, mi 48109, usa submitted on 2016, 3rd march; revised on 2016, 15th june; accepted on 2016, 21th june editor: daniele pellitteri-rosa abstract. temperature affects all aspects of reptilian biology. in order to colonize new habitats and support viable populations lizards have to successfully deal with their thermal environment. podarcis siculus is a notorious example of a successful colonizer that has invaded numerous habitats outside its natural distribution range. though certain features of its thermal biology have been assessed so far, the thermoregulatory abilities of the species remain poorly described. here we investigated a recently discovered population in greece and evaluated the effectiveness of thermoregulation measuring three main thermal parameters: set-point range, operative and field body temperatures. the greek p. siculus appear to be accurate, precise and effective thermoregulators achieving e = 0.96. this effective thermoregulation may be used to explain, among other special characteristics, its spreading success. keywords. temperature, thermoregulation, invasive species, italian wall lizard, greece. introduction thermoregulation is crucial in ectotherms, shaping all features of their overall biology (bartholomew, 1982). in small reptiles like lizards, thermoregulation is most times achieved behaviorally through appropriate movements between warmer and cooler microhabitat sites, shade and sun (avery, 1982; stevenson, 1985). systematic research of reptilian thermal biology dates back to the mid-1940s (cowles and bogert, 1944). in 1976, huey and slatkin introduced a concise and detailed model to evaluate thermoregulation in ectotherms. this paradigm remained in use for the next two decades until hertz and his partners (1993) fundamentally changed the way thermoregulation was perceived and proposed a thorough research protocol to answer a question of paramount importance: how effectively do lizards thermoregulate? in order to answer this question, hertz et al. (1993) took into account three main parameters: body temperatures (tb, the temperature that animals achieve in the field), operative temperatures (te, the temperature that a non-regulating animal would achieve in the field, measured with the use of models), and the species’ setpoint range (tset, the temperature that animals select in a laboratory setting in the absence of any ecological constraints). therefore tb can be viewed as the result of the species’ mean tset, which is considered a thermal utopia under ideal conditions (sagonas et al., 2013a) and its interactions with a biotope’s te. by considering these variables together, we are able to assess the effectiveness, accuracy and precision of thermoregulation of any given species (hertz et al., 1993). the italian wall lizard podarcis siculus (rafinesqueschmaltz, 1810) (sauria, lacertidae) is definitely not any 112 grigoris kapsalas et alii given species. it is a small-bodied (snout-vent length, svl, up to 90 mm; corti and lo cascio, 2002), diurnal, heliothermic lacertid that feeds mainly on terrestrial invertebrates (corti, 2006), although its diet may include unusual food resources such as rodents, geckos and even conspecifics (capula and aloise, 2011; grano et al., 2011), revealing a flexible and opportunistic feeder (zuffi and giannelli, 2013), as most lacertids are (scali et al., 2015). a native lizard of the italian peninsula and north adriatic coasts, p. siculus has recently expanded its distribution in many other mediterranean countries by establishing numerous new populations (crnobrnja isailovic et al., 2009). its excellent dispersal abilities (vignoli et al., 2012) are underscored by the fact that it is the only podarcis lizard that can be found in four continents: europe, asia, north america and africa (arnold and ovenden, 2002; kolbe et al., 2013; tok et al., 2015). as such, p. siculus has been widely used as a model organism in numerous ecological, physiological, behavioral and phylogenetic studies (fulgione et al., 2004; podnar et al., 2005; bonacci et al., 2008; biaggini et al., 2009; vervust et al., 2010; raia et al., 2010). several aspects of its thermal biology have been also studied (avery, 1978; ouboter, 1981; van damme et al., 1990; tosini et al., 1992, 1996). however, its thermoregulation effectiveness (sensu hertz et al., 1993) remains undetermined. in this study, we worked with a new, recently established population in greece (adamopoulou, 2015). we focused on the thermoregulatory capacity of p. siculus by measuring the three main thermal features, that is tb, te, and mean tset. furthermore, we compared the published data on set-point range temperatures from different sites within the species range. we hypothesized that since p. siculus is a highly successful colonizer capable of adapting easily to new habitats, it should achieve effective thermoregulation. materials and methods the study site (palaio faliro) is located along the back side of a sandy beach in the athens, greece (37o55’9.38”n, 23o42’0.50”e). it is a heavily modified habitat planted with oleanders, tamarisks and yuccas delimited by a crowded beach on the front and a highway avenue on the back. the only other reptile recorded on site was the ocellated skink (chalcides ocellatus). the site represents the only known location of p. siculus in greece (adamopoulou, 2015), which probably originates from the adriatic region (silva-rocha et al., 2014). in may 2015 we measured the operative temperatures of the site using 31 hollow, electroformed copper models that mimic the size, shape and reflectance of the species and were validated against live animals in the field (bakken, 1992; dzialowski, 2005). to simulate the heat capacity of the lizards, we added 2.5-3 ml of water into each model, both ends of which were sealed with plasticine, except for a narrow opening where the probe of a data-logger (hobo u12 4-channel external data logger u12-008) was inserted (diaz, 1997; grbac and bauwens, 2001). the models were placed on various spots on site so as to cover all types of microhabitats available to lizards (huey, 1991). we recorded te every 15 minutes for two consecutive days (09:00-19:00). to ensure that the temperature responses between the copper models and the study animals were similar, we tested their cooling and heating rate (lutterschmidt and reinert, 2012). an adult male lizard and a copper model were placed side by side near a heating source (two 140 w lamps) for 45 minutes. subsequently, the heating source was turned off and the subjects were left to cool down for 45 minutes, resulting in a total period of 90 minutes. during this period we recorded the temperature of the model and the lizard every five minutes with a weber quick reading cloacal thermometer. a linear regression of tb on te suggested that there was a good fit between the model and the animal responses (regression statistics ± se; slope = 1.305 ± 0.118, intercept = -7.290 ± 3.557, r2 = 0.928, p < 0.001). the body temperatures (tb) of 30 individuals were measured on site during the same dates that tes were sampled. lizards were caught by hand or noose from all occupied microhabitats and their temperature was measured within 20 seconds using a quick-reading cloacal thermometer (t-4000, miller & weber, inc., queens, ny) to the nearest 0.1 °c. for every lizard caught we also measured svl with digital calipers (silverline 380244, accurate to 0.01 mm) while sex was determined by inspection of the femoral pores. finally, 11 adult males (since sampling took place within the reproductive period we tried to avoid the impact of sex on set-point range temperatures; carneiro et al., 2015) were transferred to the laboratory facilities of the department of biology at the university of athens to measure tset. the lizards were placed in a specially designed terrarium (100 x 25 x 25 cm) where a thermal gradient (10-60 °c) was achieved with the use of two incandescent heating lamps (100 w and 60 w) at one end and two ice bags on the opposite (van damme et al., 1991). subsequently their body temperature was measured every 30 min for four consecutive hours (castilla and bauwens, 1991) using a quick-reading cloacal thermometer (t-4000, miller & weber, inc., queens, ny). we used the inter-quartile range of the body temperatures selected by lizards in the thermal gradient (hertz et al., 1993). to evaluate the effectiveness of thermoregulation we used the formula: ε = 1 (db − / de − ), where db − represents the mean deviation of field tbs from mean tset and de − provides a measure of thermoregulation accuracy and the mean deviation of tes from mean tset, (hertz et al., 1993). mean db provides an index of the accuracy of thermoregulation whereas de − sketches out the thermal quality of the habitat. e may range from zero (perfect thermoconformers) to one (perfect thermoregulators). complementary to the classical evaluation of the thermoregulatory effectiveness (hertz et al., 1993), we also employed an alternative approach that quantifies the extent of departure from perfect thermoconformity (blouin-demers and weatherhead, 2001). in the latter, positive values of the differ113thermoregulatory effectiveness of podarcis siculus ence between de − and db − describe thermoregulation, zero represents animals demonstrating perfect thermoconformity, and negative values describe animals avoiding habitats of high thermal quality. the magnitude of the difference (de − db −) provides an index of the thermoregulatory effectiveness (blouin-demers and weatherhead, 2001). results our results (male svl = 74.3 ± 3.5 mm, n = 9; female svl = 65.3 ± 7.0 mm, n = 20; t-test, t = -3.650, df = 27, p = 0.001) corroborated the typical pattern of sexual body size differences according to which male p. siculus are larger than females (henle and klaver, 1986). males did not differ from females in their tb (t-test, t = 0.758, df = 28, p = 0.455) and achieved similar body temperatures in the field (mean tb for males = 33.2, mean tb for females = 32.6). the mean value for the pooled tb data was 32.8°c (table 1). the majority of tb fell within the spectrum of mean tset while the diel variation of body temperatures was limited (fig. 1). operative temperatures ranged from 19.5 °c at 09:00 h (minimum) to 53.4 °c at 14:15 (maximum) and mean te was 31.7 °c (table 1, fig. 1). tset values ranged from 30.0°c to 37.0 °c (table 1). lizards achieved a mean set-point temperature of 33.8 °c (table 1). the mean deviation of tb from mean tset was 0.1 °c while the mean deviation of te from mean tset was 3.2 °c (table 1). these values were used to estimate the effectiveness of thermoregulation (sensu hertz et al., 1993), that was e = 0.96. this value indicates that p. siculus is an active thermoregulator, as expected from the closeness of body temperatures to mean tset. the complementary approach we used (blouin-demers and weatherhead, 2001), also revealed high thermoregulatory effectiveness (de − db − = 3.1) discussion this study provides a comprehensive analysis of the thermoregulation effectiveness of the italian wall lizard. in line with our first hypothesis, p. siculus proved to be an effective thermoregulator and able to achieve body temperatures within mean tset. body temperatures that lizards achieve in the field vary considerably among different biotopes (avery, 1978; van damme et al., 1990; tosini et al., 1992), in accordance with our second prediction. operative temperatures at the study site revealed – at least during the study period – a benign habitat lacking extreme temperatures (table 1). nonetheless, the site offers the lizards the required thermal heterogeneity for behavioral thermoregulation. mean de received a low value (3.2), indicating the high thermal quality of the habitat. in other words, lizards can easily achieve body temperatures within mean tset. set-point range temperatures are critically important in reptiles as they determine the optimal overall performance of the animal (clusella-trullas et al., 2007). many factors, such as season, sex, age, reproductive status, body size and habitat may affect tset (andrews et al., 1999; cartable 1. the thermal variables used in this study: operative temperatures (te), body temperatures in the field (tb), set-point range (tset), deviation of te from tset (de) and deviation of tb from tset (db). variable n mean (°c) range (°c) sd se te 31 31.7 19.5 – 53.4 7.10 0.22 de 31 3.2 0 – 16.4 3.30 0.10 tb 30 32.8 26.8 – 35.8 1.94 0.35 db 30 0.1 0 – 3.2 0.59 0.11 tset 11 33.8 30.0 – 37.0 2.25 0.30 fig. 1. distribution of the mean body temperature in the field (tb) and the mean operative temperature (te). the shaded area indicates the set-point range (tset) measured in the laboratory. 114 grigoris kapsalas et alii retero et al., 2005; carretero, 2008; veríssimo and carretero, 2009; sagonas et al., 2013 a, b). the mean tset for p. siculus was 1°c lower than the only other value reported in literature (avery 1978) and falls within the upper percentile of the thermal range of other podarcis species (table 2). to the best of our knowledge there are three published studies that present data on the set-point range and body temperatures of p. siculus (avery, 1978; van damme et al., 1990; tosini et al., 1992). the greek population achieved higher tbs in the field than lizards in pisa (32.04 °c, tosini et al., 1992) but lower compared to the populations from tuscany (35.16 °c, avery, 1978) and corsica (33.89 °c, van damme et al., 1990). in regard to set-point range temperatures the mean tset in the italian population is approximately 1 °c higher (34.79 °c, avery, 1978) than the one estimated in the present study. an interesting finding was that tbs did not vary considerably during the day, contrary to other podarcis lizards (e.g., adamopoulou and valakos, 2005; sagonas et al., 2013a), a feature that was also reported by previous studies (van damme et al., 1990; tosini et al., 1992). the limited range of tbs is an indication of high precision in thermoregulation (hertz et al., 1993). as predicted by the low de − that was mentioned above, an impressive 93% of all tbs fell within the measured range of mean tset. this corresponded to a very low mean db (0.1), which suggests high thermoregulation accuracy (hertz et al., 1993). thus, p. siculus appears to be not only a precise thermoregulator (van damme et al., 1990; tosini et al., 1992) but also an accurate one. the index of thermoregulation effectiveness took a high value (e = 0.96), which is among the highest that have been reported so far, not only for podarcis lizards (table 2) but among all lacertids. when animals are unable to thermoregulate, e will approach zero, whereas when they can successfully regulate their body temperature, e will be close to one (herzt et al., 1993). according to our results, p. siculus, at least in the study ecosystem, is actively thermoregulating with great success. this is directly related to the small deviation of tbs from mean tset. at this point we have to stress out that the evaluation of e was based on mean tset that was calculated from exclusively male individuals and also that a limited number of tbs were obtained in a short period of time. in order to invade new ecosystems and establish viable populations, animals have to fullfill certain requirements (kolar and lodge, 2001). ectotherms need to furthermore meet additional thermal demands: they have to adapt swiftly to the environmental temperatures of the new biotope while, at the same time, perform close to optimal levels (kraus, 2009). thermal specialists, which achieve optimal performance in a narrow range of temperatures, are less apt to invade new habitats than thermal generalists (angilletta et al., 2003; angilletta, 2009). the italian wall lizard is a successful colonizer thanks to the quick acclimatization and adaptability to the environmental conditions of the new “home” and to the numerous ways of dispersal (deichsel et al., 2010; valdeón et al., 2010; silva-rocha et al., 2012, 2014). our findings suggest that p. siculus is able to effectively, accurately and precisely regulate its body temperature. thanks to this table 2. studies on mean set-point range (tset) and effectiveness of thermoregulation (e) in podarcis lizards. species location e tset reference p. siculus athens, greece 0.96 33.8 this study p. liolepis columbretes island, spain 0.95 34.2 bauwens et al., 1996 p. milensis milos island, greece 0.95 33.4 adamopoulou and valakos, 2005 p. lilfordi menorca island, spain 0.88 35 ortega et al., 2014 p. gaigeae skyros island, greece 0.87 33.7 sagonas et al., 2013a p. levendis pori island, greece 0.84 33.9 lymberakis et al., 2015 p. muralis cres, croatia 0.81 31.9 grbac and bauwens, 2001 p. peloponnesiacus peloponnese, greece 0.76 34 pafilis, 2003 p. erhardii andros island, greece 0.66 35.1 pafilis, 2003 p. melisellensis cres, croatia 0.63 33.5 grbac and bauwens, 2001 p. tiliguerta corsica, france 35.47 van damme et al., 1990 p. bocagei spain 35.15 bauwens et al., 1995 p. tauricus peloponnese, greece 33.8 pafilis, 2003 p. vaucheri ketama, morocco 33.43 verissimo and carretero, 2009 p. hispanicus bellaterra, spain 33.07 carretero et al., 2006 p. hispanicus galera, spain 31.65 carretero, 2012 115thermoregulatory effectiveness of podarcis siculus effective thermoregulation, it may overcome the thermal challenges of new environments. as p. siculus settles new populations, there is strong evidence that it competes with native lacertids. downes and bauwens (2002) found that p. siculus is more aggressive and dominant than podarcis melisellensis and occupies better thermal microhabitats. in laboratory experiments, p. siculus was also more aggressive and eventually suppressed activity levels in podarcis tiliguerta (vanhooydonck et al., 2000). furthermore, p. siculus hybridizes with other endemic podarcis lizards (capula, 1993; capula, 2002; capula et al., 2002). the above considerations highlight the risk that p. siculus posses for autochthonous species, above all for greece, which is home to seven endemic lacertids (valakos et al., 2008). since p. siculus has become established in athens, it is only matter of time before it invades places that host endemic lizards (e.g., peloponnese, milos, skyros, crete). to eliminate the danger of further dispersal and the concomitant negative effects, the investigated athens population of p. siculus needs to be exterminated. the hellenic herpetological society inaugurated an eradication project in spring 2015. more than 150 individuals have been captured so far indicating that the initially estimated 50-60 animals (adamopoulou, 2015) have multiplied to a much larger actual population in only a couple of years. this study, in spite of sampling flaws in field and lab temperature measurements, paves the way for delineating the particular features of ectotherms, which, like p. siculus, rapidly expand their distribution. further research that will assess the thermoregulation pattern of p. siculus in diverse habitats throughout its range is badly required. effectiveness of thermoregulation and mean tset should be the focal points. thereby it will be clarified whether the successful dispersal of the species is due to a standardized, conservative thermal pattern (the “static” view, bogert, 1949) or just a response to environmental factors (the “labile” view, huey, 1982). if e will receive equally high values and mean tset will be similar to the studied population, then p. siculus will be indeed an effective thermoregulator with a conservative thermal physiology. our results are the first step to this end. acknowledgements we are grateful to natalia gourgouliani, ana pereira, kostantina mitsi and stratos kafentzis for their valuable help with fieldwork. all work was conducted with permission from the hellenic ministry for environment and energy (permit code 61σμ465φθη-ες6).  references adamopoulou, c. 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(2013): trophic niche and feeding biology of the italian wall lizard, podarcis siculus campestris (de betta, 1857) along western mediterranean coast. acta herpetol. 8: 35-39. acta herpetologica vol. 11, n. 2 december 2016 firenze university press predator-prey interactions between a recent invader, the chinese sleeper (perccottus glenii) and the european pond turtle (emys orbicularis): a case study from lithuania vytautas rakauskas1,*, rūta masiulytė1, alma pikūnienė2 effective thermoregulation in a newly established population of podarcis siculus in greece: a possible advantage for a successful invader grigoris kapsalas1, ioanna gavriilidi1, chloe adamopoulou2, johannes foufopoulos3, panayiotis pafilis1,* the unexpectedly dull tadpole of madagascar’s largest frog, mantidactylus guttulatus arne schulze1,*, roger-daniel randrianiaina2,3, bina perl3, frank glaw4, miguel vences3 thermal ecology of podarcis siculus (rafinesque-schmalz, 1810) in menorca (balearic islands, spain) zaida ortega*, abraham mencía, valentín pérez-mellado growth, longevity and age at maturity in the european whip snakes, hierophis viridiflavus and h. carbonarius sara fornasiero1, xavier bonnet2, federica dendi1, marco a.l. zuffi1,* redescription of cyrtodactylus fumosus (müller, 1895) (reptilia: squamata: gekkonidae), with a revised identification key to the bent-toed geckos of sulawesi sven mecke1,*,§, lukas hartmann1,2,§, felix mader3, max kieckbusch1, hinrich kaiser4 the castaway: characteristic islet features affect the ecology of the most isolated european lizard petros lymberakis1, efstratios d. valakos2, kostas sagonas2, panayiotis pafilis3,* sources of calcium for the agamid lizard psammophilus blanfordanus during embryonic development jyoti jee1, birendra kumar mohapatra2, sushil kumar dutta1, gunanidhi sahoo1,3,* mediodactylus kotschyi in the peloponnese peninsula, greece: distribution and habitat rachel schwarz1,*, ioanna-aikaterini gavriilidi2, yuval itescu1, simon jamison1, kostas sagonas3, shai meiri1, panayiotis pafilis2 swimming performance and thermal resistance of juvenile and adult newts acclimated to different temperatures hong-liang lu, qiong wu, jun geng, wei dang* olim palus, where once upon a time the marsh: distribution, demography, ecology and threats of amphibians in the circeo national park (central italy) antonio romano1,*, riccardo novaga2, andrea costa1 on the feeding ecology of pelophylax saharicus (boulenger 1913) from morocco zaida ortega1,*, valentín pérez-mellado1, pilar navarro2, javier lluch2 notes on the reproductive ecology of the rough-footed mud turtle (kinosternon hirtipes) in texas, usa steven g. platt1, dennis j. miller2, thomas r. rainwater3,*, jennifer l. smith4 heavy traffic, low mortality tram tracks as terrestrial habitat of newts mikołaj kaczmarski*, jan m. kaczmarek book review: sutherland, w.j., dicks, l.v., ockendon, n., smith, r.k. (eds). what works in conservation. open book publishers, cambridge, uk. http://dx.doi.org/10.11647/obp.0060 sebastiano salvidio acta herpetologica 12(1): 109-112, 2017 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-19155 where is my place? quick chorus structure assembly in the european tree frog michal berec faculty of agriculture, university of south bohemia, studentská 13, 370 05 české budějovice, czech republic. e-mail: michal.berec@ seznam.cz submitted on 2016, 4th october; revised on 2016, 24th november; accepted on 2017, 3rd april editor: giovanni scillitani abstract. lek mating systems are characteristic of anurans that use oviposition sites that cannot be easily monopolized by individual males. the dynamics of the chorus structure in leks is not well known. here, we examine the relationship between the movement activity of individual males during the breeding season and their size. according to our observations, the site fidelity of males is not completely random, with the larger males moving significantly shorter distances than smaller males. however, this difference applies only to the distance between the first and second captures. whether higher site fidelity contributes to higher mating success needs further investigation. keywords. vocalization, chorus, movement, size, tree frog. individuals of the majority of amphibian species live their own solitary lives throughout the year except during the reproductive period, when they gather in defined spaces to find appropriate sexual partners. males of some of these species do not occupy territories during the reproductive period but rely on physical characteristics during short and intensive bouts (scramble competition) to overpower mating competitors (duellman and trueb, 1994). males of the other species divide the space into defended segments and lure individual females by acoustic or visual stimuli (wells, 2007). in lek mating systems, males typically occupy and defend small areas in which they vocalize (höglund and alatalo, 1995). whenever intruders of the same sex approach too closely, they fight against them. females choose their mates by moving both within and between several aggregations of males (höglund and alatalo, 1995). surprisingly, there are only a handful of studies describing lek structure and dynamics in anurans (emlen, 1976, tárano, 2009), although many species of frogs and toads form typical lek aggregations (wells, 2007). this lack of research is surprising, as the positions of individual males within the lek can strongly influence their mating success (arita and kaneshiro, 1985; kokko et al. 1998; howard et al. 2011; but see sæther et al. 2005 for the opposite results). at the same time, the size of individual males is the most important determinant of attractiveness in many anurans (wells, 2007 and references therein). here, we report the results of our survey of movement dynamics within a lek system of european tree frog (hyla arborea). we specifically focused on the relationship between the movement of males during the reproductive season and their size (as a proxy for age). our goal was to examine whether leks in this tree frog species show spatiotemporal structure. despite its possible relevance to complex frog reproductive behaviour, this subject has not previously been analysed in detail. we investigated a population of european tree frog (hyla arborea) in velký vávrovský pond (1.5 ha, 390 m a.s.l., average depth 40 cm, maximum depth 120 cm, 48°59’25.18”n, 14°25’55.24”e) at the outskirts of české 110 m. berec budějovice in south bohemia, czech republic. this pond is used for carp production and is also a breeding site used by other anurans (bombina bombina, bufo bufo, rana dalmatina, pelophylax kl. esculentus). this pond is nearly square in shape. three of its four sides are overgrown by willow shrubs (salix sp.), and the fourth side is paved. the littoral vegetation consists of sparse clumps (less than 3% of water area) of carex sp., typha angustifolia, and alisma plantago-aquatica. the frogs were therefore easily observed and caught. tree frog males were collected during the breeding season, beginning at the onset of calling activity every day from april 13 to june 17, 2005, excluding the days with unsuitable conditions (pouring rain, storms). each night, all males in the chorus were captured and measured. as all males called within four metres from the shore, we walked slowly around the pond and entered the water only to catch males to reduce disturbance to the chorus. all males were caught by hand and individually marked by toe-clipping, and the snout-urostyle length (sul) was measured to the nearest millimetre using a vernier calliper. only one digit per limb was toeclipped, with a maximum of three digits per individual. for toe-clipping, we used scissors disinfected in alcohol. no analgesic was used. we did not observe any signs of pain or distress in the frogs when handled. additionally, we did not observe any negative effect on survival (we have used this method repeatedly in previous studies). some males resumed calling a few minutes after toe clipping (see funk et al., 2005 for comments on the toeclipping method). the position of each male was determined using coloured tags with numerical codes positioned along the breeding-site shoreline at 2 m intervals. when an individual frog was recaptured on a subsequent night or nights, we measured its position to the nearest centimetre from the previous position, using a measuring tape in cases of distances within a radius of five metres. for greater distances, we measured the distance covered by using the marks on the map and rounded the value to the nearest ten centimetres. we did not use gps or similar system because the measurement error with such devices is at least as large as that with our approach. all statistical tests were performed using the software package statistica 10.0 (statsoft, 2012). all distances were log-transformed before analysis. in total, 188 males were captured and individually marked. during the season, 44% of males were captured only once, 22% were captured twice, 15% were captured three times, 13% were captured four times, 3% were captured five times, and 3% were captured six times. the number of captures of the same individual was not dependent on the sul of males (kruskal-wallis anova by ranks: h (5, n = 188) = 0.581, p = 0.988). meanwhile, the sul of males captured did not depend on the day of the season (r = 0.082; r2 = 0.007; f1,186 = 1.266; p = 0.262). in contrast, the sul of tree frog males partially determined the movement pattern during the breeding season. our analysis revealed significant relationships between sul and distances between subsequent captures (r = -0.382; r2 = 0.146; f1,222 = 37.848; p < 0.001; fig. 1). however, only the distance between the 1st and 2nd capture was clearly associated with the above mentioned relationship. here, smaller males covered longer distances between the first and the second capture than did fig. 2. relationship between the sul of males and the distance between the first and second captures. fig. 1. relationship between the sul of males and the average distance between all pairs of subsequent captures. 111movement dynamics of tree frogs larger males (fig. 2). none of the subsequent movements showed a significant association with sul (table 1). although the distance covered by males steadily decreased between subsequent captures (table i), this effect was not statistically significant (kruskal-wallis anova by ranks: h (4 n = 171) = 0.127, p = 0.998). in general, larger males usually spend more nights in a lek, which is related to their mating success in lekbreeding anurans (pröhl, 2003; friedl and klump, 2005; castellano et al. 2009; botto and castellano, 2016). contrary to this, our data did not support this pattern, as the number of captures (i.e., nights when the male attended the lek) was not dependent on the sul of males. here, the data suggest another scenario, in which males of different sizes spend similar amounts of time (numbers of nights) in a lek. however, the size of males determined their movement behaviour. male size is generally the most important determinant of mating success in explosive competitors (holliday and tejedo, 1995), whereas the situation is much more complex in lek-breeding anurans (höglund and alatalo, 1995). studies of lekking non-anuran vertebrates suggest that individual position in the lek can be very important (kokko et al., 1998; howard et al., 2011). surprisingly, despite the large number of lek-breeding anurans, hardly any studies linking mating success of males and their individual positions within a lek have been published. as many lek-breeding anurans establish nonrandom spacing patterns during reproduction events (whitney and krebs, 1975; brenowitz et al., 1984; tárano, 2009), different outcomes from different positions in the lek could be predicted. the lek is re-established every night in lek-breeding anurans, including european tree frog (grosse, 2009). according to our data, the site fidelity of males is not completely random and is determined by their size during part of the breeding season. in fact, the larger males (i.e., older sensu kyriakopoulousklavounou and grumiro, 2002) moved significantly shorter distances than smaller (younger) males. however, this difference applies only to the distance between the first and second captures. after that, no differences were observed in movement activity between subsequent captures. emlen (1976) f ound a different pattern of chorus formation in bullfrogs (lithobates catesbeianus). he described leks in bullfrogs as “both spatially and temporally ephemeral” but subsequently found that larger (older) males occupied more centrally located territories. on the other hand, the locations of individual males changed rapidly, and they moved from one aggregation to another during the breeding season. the fact that larger males occupied much “stable” area implies question of the advantage of this behaviour. this can be the result of their previous experience as larger males are presumed to be older ones (friedl and klump, 1997). thus, larger males have better knowledge about the locality from previous reproductive seasons and should improve their position according to their previous mating success. simultaneously, experienced males can also recognize better mating positions (e.g., determined by abundance and structure of vegetation cover, distance from the shore, or oviposition site, etc.) in shorter time than smaller ones. moreover, physical superiority of larger males enables the territories to be hold for longer period. more intensive movement activity of smaller males can therefore be only the consequence of their effort to find better mating opportunities. site fidelity has been documented in several anuran species. various authors explain this behaviour as advantageous for male-male competition (davis, 1987; brenowitz and rose, 1994; marshall et al., 2003), search strategies of females (fellers, 1979; murphy and gerhardt, 2002), female attraction or choice (bradbury and gibson, 1983; grafe, 1997), female paths (grafe, 1997), or sound transmission (narins and hurley, 1982; wells and schwartz, 1982; marshall et al., 2003). using dynamic programming, the theoretical model by switzer (1993) of sequential settlement decisions predicted site fidelity to be positively correlated to age. unfortunately, previous studies did not relate the observed behaviour directly to the size of males. the reason why larger tree frog males chose and returned to the same positions more precisely than smaller males is not obvious at this time and deserves further investigation. table 1. mean distances ± standard deviation between two subsequent captures, as well as results of regression analyses between the sul of males and distances between subsequent captures. capture mean distance ± sd (cm) range (cm) r r2 f d.f. p 1st -2nd 801 ± 870 0-4660 -0.533 0.284 45.311 1,104 <0.001 2nd -3rd 614 ± 494 0-2450 -0.182 0.033 2.163 1,63 0.143 3rd -4th 541 ± 498 0-2020 -0.237 0.056 2.016 1,34 0.165 4th -5th 414 ± 294 52-840 -0.311 0.097 0.966 1,9 0.351 112 m. berec acknowledgements permission for conducting the study was issued by the ministry of environment of the czech republic (permission number 1211/32/sop/e/05/456/cho). i would like to thank irena šetlíková, luděk berec, and two anonymous reviewers for valuable comments on the earlier versions of the manuscript. research was financially supported by the gaju 081/2016/z. references botto, v., castellano, s. (2016): attendance, but not performance, predicts good genes in a lek-breeding treefrog. behav. ecol. 27: 1141-1148. bradbury, j.w., gibson, r.m. (1983): leks and mate choice. in: mate choice, pp. 109-138. bateson, p., ed, cambridge university press, cambridge. brenowitz, e.a., rose, g.j. (1994): behavioural plasticity mediates aggression in choruses of the pacific treefrog. anim. behav. 47: 633-641. brenowitz, e.a., wilczynski, w., zakon, h.h. (1984): acoustic communication in spring peepers. j. comp. physiol. a 155: 585-592. castellano, s., zanollo, v., marconi, v. berto, g. (2009): the mechanisms of sexual selection in a lek-breeding anuran, hyla intermedia. anim. behav. 77: 213–224. davis, m.s. (1987): acoustically mediated neighbor recognition in the north american bullfrog, rana catesbeiana. behav. ecol. sociobiol. 21: 185-190. duellman, w.e., trueb, l. (1994): biology of amphibians. john hopkins university press. emlen, s.t. 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(2003): plasticity of aggressive signalling and its evolution in male spring peepers, pseudacris crucifer. anim. behav. 65: 1223-1234. murphy, c.g., gerhardt, h.c. (2002): mate sampling by female barking treefrogs (hyla gratiosa). behav. ecol. 13: 472-480. narins, p.m., hurley, d.d. (1982): the relationship between call intensity and function in the puerto rican coqui (anura: leptodactylidae). herpetologica 38: 287-295. pröhl, h. (2003): variation in male calling behaviour and relation to male mating success in the strawberry poison frog (dendrobates pumilio). ethology 109: 273-290. sæther, s.a., baglo, r., fiske, p., ekblom, r., höglund, j., kålås, j.a. (2005): direct and indirect mate choice on leks. am. nat. 166: 145-157. statsoft inc. (2012): statistica (data analysis software system), version 9.1 (version 9.1). available from www.statistica.com. switzer, p.v. (1993): site fidelity in predictable and unpredictable habitats. evol. ecol. 7: 533-555. tárano, z. (2009): structure of transient vocal assemblages of physalaemus fischeri (anura, leiuperidae): calling site fidelity and spatial distribution of males. s. am. j. herp. 4: 43-50. wells, k.d. (2007): the ecology and behavior of amphibians. the university of chicago press, chicago. wells, k.d., schwartz j.j. (1982): the effect of vegetation on the propagation of calls in the neotropical frog centrolenella fleischmanni. herpetologica 38: 449-455. whitney, c.l., krebs, j.r. (1975): spacing and calling in pacific tree frogs, hyla regilla. can. j. zool. 53: 15191527. acta herpetologica vol. 12, n. 1 june 2017 firenze university press morphological variation and sexual dimorphism in liolaemus wiegmannii (duméril & bibron, 1837) (squamata: liolaemidae) from uruguay joaquín villamil1,*, arley camargo2, raúl maneyro1 sex does not affect tail autotomy in lacertid lizards panayiotis pafilis1,*, kostas sagonas2, grigoris kapsalas1, johannes foufopoulos3, efstratios d. valakos4 fire salamander (salamandra salamandra) males’ activity during breeding season: effects of microhabitat features and body size raoul manenti*, andrea conti, roberta pennati call variation and vocalizations of the stealthy litter frog ischnocnema abdita (anura: brachycephalidae) pedro carvalho rocha1,2,*, joão victor a. lacerda2,3, rafael félix de magalhães2,3, clarissa canedo4, bruno v. s. pimenta5, rodrigo carrara heitor6, paulo christiano de anchieta garcia2,3 feeding ecology of two sympatric geckos in an urban area of northeastern brazil josé guilherme g. sousa1, adonias a. martins teixeira2,*, diêgo alves teles2, joão antônio araújo-filho2 and robson waldemar ávila3 a pattern-based tool for long-term, large-sample capture-mark-recapture studies of fire salamanders salamandra species (amphibia: urodela: salamandridae) jeroen speybroeck1,*, koen steenhoudt2,$ skeletal variation within the darwinii group of liolaemus (iguania: liolaemidae): new characters, identification of polymorphisms and new synapomorphies for subclades linda díaz-fernández*, andrés s. quinteros, fernando lobo marking techniques in the marbled newt (triturus marmoratus): pit-tag and tracking device implant protocols hugo le chevalier1, olivier calvez2, albert martínez-silvestre3, damien picard4, sandra guérin4,5, francis isselin-nondedeu6, alexandre ribéron1, audrey trochet1,2,* evidence for directional testes asymmetry in hyla gongshanensis jindongensis qing gui wu1, wen bo liao2,* the advertisement call of pristimantis subsigillatus (anura, craugastoridae) florina stănescu1,4, rafael márquez2, paul székely3,4,*, dan cogălniceanu1,4,5 nematodes infecting anotosaura vanzolinia (squamata: gymnophthalmidae) from caatinga, northeastern brazil bruno halluan s. oliveira¹, adonias a. martins teixeira¹,*, romilda narciza m. queiroz¹, joão a. araujo-filho¹, diêgo a. teles¹, samuel v. brito2, daniel o. mesquita¹ where is my place? quick chorus structure assembly in the european tree frog michal berec a possible mutualistic interaction between vertebrates: frogs use water buffaloes as a foraging place piotr zduniak1,*, kiraz erciyas-yavuz2, piotr tryjanowski3 good vibrations: a novel method for sexing turtles donald t. mcknight1,2,*, hunter j. howell3, ethan c. hollender1, and day b. ligon1 errata to mecke, s., hartmann, l., mader, f., kieckbusch, m., kaiser, h. (2016): redescription of cyrtodactylus fumosus (müller, 1895) (reptilia: squamata: gekkonidae), with a revised identification key to the bent-toed geckos of sulawesi. acta herpetologica 11(2): issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 8(2): 147-152, 2013 water-soluble proteinase activity of gastric fluids in the european whip snake, hierophis viridiflavus: an experimental preliminary survey antonio felicioli1, simona sagona1, michele corazza1, carlo pretti1,*, marco a.l. zuffi2 1 dipartimento di scienze veterinarie, università di pisa, viale delle piagge 2, i-56124 pisa, italy. *corresponding author. e-mail: c.pretti@vet.unipi.it 2 museo di storia naturale, università di pisa, via roma 79, i-56011, italy submitted on 1st march 2013; revised on 7th october 2013; accepted on 6th november 2013. abstract. understanding the mechanism of metabolic digestion and of food assimilation in wild snake species is of fundamental importance to compare trophic niches, their adaptive significance and evolutionary patterns of diet selection for different snake species. from two adult hierophis viridiflavus males in fasting condition, we sampled gastric fluids using fiberoptic endoscopy. we analyzed the proteolytic activities (optimal ph and temperature, response to inhibitors) of snake samples by zymography techniques. the two major protease activity bands were always detected at every tested ph. activity bands were also detectable over 37 °c; in particular both bands showed the highest activity ranging from 55 °c to 60 °c. ranging from 65 °c to 85 °c, one band remained visible while the other band disappeared over 60 °c. the pattern of proteolytic enzymes activity of sampled gastric fluids highlighted a scenario composed of few active proteases, reflecting the feeding status (e.g., fasting) of studied snakes. detection of proteolytic activity until 85 °c, as in prokaryotic organisms, supported the hypothesis about the presence of proteases of exogenous source. keywords. european whip snake, hierophis viridiflavus, water-soluble proteinase, stomach. food (e.g., prey type, prey abundance and feeding frequency) acts as a primary influencing factor in all biological processes of an organism life. energy metabolism, food consumption (i.e., energy gain), and digestive performance have been measured quite rarely in reptiles in the wild (congdon et al., 1982; wikelsky et al., 1993; henen et al., 1998), while most effort has been done on wild snakes under laboratory conditions (e.g., secor and nagy, 1994; secor et al., 1994; hopkins et al., 2004; zuffi et al., 2010). nevertheless, recent research indicates that vicariance may cause differences of feeding patterns in snakes (zuffi, 2001; luiselli et al., 2005), or influences the digestive performance in lizards (pafilis et al., 2007). the relative patterns of growth that are dependent on the quality of food (i.e., different prey types, different frequencies) have been analyzed more in depth (bonnet et al., 2001; cox and secor, 2007; secor et al., 2007; zuffi, 2007). it has been emphasized how newborn and juvenile snakes can grow to different sizes and show different phenotypes according to frequency and quality of food supply (bonnet et al., 2001). evidence of such a pattern is widely documented in the literature (see shine, 1993 for a comprehensive review): snakes species may show deep inter-population body size differences due to area effect [i.e., insular gigantism or dwarfism (boback, 2003)], to different food resource allocation (shine, 1993; rugiero et al., 1995; pleguezuelos et al., 2007) or to both effects (fornasiero et al., 2007). analysis of biochemical and physiological patterns of digestion in reptiles and in snakes could allow further comparisons of metabolic demands among species with different food regimes (bradshaw et al., 1987; luiselli, 2006), from highly specialized (houston and shine, 1994; luiselli et al., 2001, 2007), to broadly generalist (rugiero and luiselli, 1995; pleguezuelos and fahd, 2004; filippi et al., 2005) snake species. in snakes, different diet regimes 148 antonio felicioli et al. and food preferences could lead to a different pattern of digestion (e.g., efficiency; zuffi et al., 2010) or to different enzymatic activity processes (secor, 2008). as model species we used the european whip snake, hierophis viridiflavus. this oviparous colubrid snake has a very wide prey spectrum (heimes, 1993; capula et al., 1997; zuffi, 2001, 2007), and also displays an ontogenetic shift in diet composition (rugiero and luiselli, 1995; zuffi, 2007), as typically observed in several other snake species (luiselli et al., 1996, 2007; shine et al., 2002). it can reach up to 200 cm total length, with an average body length of around 130 to 140 cm, while insular populations are significantly smaller than continental ones (fornasiero et al., 2007; zuffi, 2007). nevertheless, most of the available knowledge on food preference or food ecology of the european whip snake refer to some localities only (i.e., capula et al., 1997), thus limiting comparison of feeding rate and food preference patterns among populations. therefore, analyzing and understanding the mechanism of metabolic digestion (i.e. duration, sexual variation) and of food assimilation in wild species may be of fundamental importance to understand and compare i) trophic niches in different snake species and or between sexes, ii) the adaptive significance of selecting a different diet and evolutionary patterns of diet selection for snake species, characterized by different food regimes. endoscopic examination. all experiments were performed with no lethal methods and the snakes survived at the end of the experiment; they were released immediately after the experiment. two adult males (970 and 1010 mm snout to vent length, tombolo di pisa, pisa, central tuscany, northern italy; examined the day after capture; maintained at 27°c two hours before examination, 12:12 light-darkness cycle) were examined with flexible fiberscope gif q10 olympus 100 cm, ø 10mm and a xenon light source cle 10 olympus, without anesthesia, restraining them manually in a dorsal position on a thermal pad at 38°c. the endoscope tip was gently introduced in the mouth and in both cases it was spontaneously swallowed. gastrointestinal fluids were collected by a catheter injecting before a 100 mm tris-hcl buffer (ph 8.0, 38 °c) that was aspirated after 30 seconds. handling and experimentation were conducted under the approval of the university of pisa (italy) animal care and use committee and “ministero dell’ambiente e della tutela del territorio e del mare. direzione per la protezione della natura” (permissions # n dpn/iidiv/2006/7167 to mal zuffi). zymography. protein concentration was measured according to bradford’s method (bradford, 1976). sdspage was performed according to laemmli (laemmli, 1970). proteins from the gastric fluid sample underwent proteolytic activity-staining electrophoresis (heussen and dowdle, 1980), using 0.5% gelatine as precast protein substrate in a discontinuous 12% t laemmli’s gel in mini vertical unit se260 electrophoresis system (amersham bioscience) under semi-denaturing conditions (before loading, samples were 1:2 diluted with 2% sds without boiling). no β-mercaptoethanol was added. each clarified extracts (~5 μg of proteins) were loaded in each lane. electrophoresis run at 20ma at 15°c for 3 hours (felicioli et al., 2004; piccolomini et al., 2006). after electrophoresis, gels were shaken gently at room temperature for 30 min in 100 ml 2% triton x-100 in water to remove sds and restore full enzyme activity. gels were then transferred to a bath containing 100 mm tris-hcl buffer, ph 8.0 and kept under mild shaking, at 37 °c for 2 hours, then stained with 0.1% blue r250 coomassie and de-stained with 40% methanol and 10% acetic acid. for the ph activity assay (jousson et al., 2007), electrophoretic slabs were incubated in 2% triton x-100 for 30 min, then at 37 °c for 2 h in a 0.1m buffer. the buffer was tris-acetate at ph 3, 4, 5 and 6; tris–hcl at ph 7-8 and; and borate at ph 9-10. for the thermal stability assay, 10 lanes of gel corresponding to 10 aliquots of the crude extract of the sample after electrophoretic run were separately incubated for 2 hours in 100 mm trishcl buffer, ph 8.0 at 4 °c, 15 °c, 22 °c, 37 °c, 55 °c, 60 °c, 65 °c, 70 °c, 75 °c, 80 °c and 85 °c. all the replicates have been scanned with an epson expression 1680 pro scanner and analyzed using quantity one 4.2.3 version bio-rad laboratories (milano, italy). phenylmethylsulfonylfluoride (pmsf) and pepstatin a (sigma-aldrich) were used as inhibitors according to barrett (1994). two major activity bands were chosen for optimal ph activity, thermal stability, molecular mass estimation, and inhibition experiments. proteolytic pattern variations. the gastric area (fig. 1) was correctly identified by endoscopic examination and gastric fluids were sampled.. gastric fluid samples (crude extracts, ph 7.2) showed 0.66 mg/ml of total proteins. figure 2 shows the zymography on a porcine gelatine precast gel of crude extracts from the gastric fluid of fasting h. viridiflavus. four major activity bands of different intensities were found (lane 2), showing estimated molecular weights of 93 kda (a), 64 kda (b), 43 kda (c) and 38 kda (d), respectively. within the major activity bands, the 43 kda and 38 kda bands showed the highest intensity. no activity bands were detected at 23.4 kda [lane 1, porcine pancreatic trypsin standard (t)], revealing the absence of trypsin-like protease. for subsequent experi149gastric protease in hierophis ments, we focused attention only on c and d bands. figure 3 shows the ph activity of crude extracts from the gastric fluid of fasting h. viridiflavus. the two major bands (c and d) were always detected at every tested ph. densitometric results showed very low activity levels of band d until ph 7, while band c exhibited higher activity levels than band c (3-4-fold at ph 7, ph 8 and ph 9). figure 4 shows the thermal activity of crude extracts from the gastric fluid of fasting h. viridiflavus. activity bands were detectable over 37 °c; in particular both bands showed the highest activity ranging from 55 °c to fig. 1. endoscopic image of gastric area fig. 2. gelatine-precast zymography page of crude extract of h. viridiflavus gastric fluid. lane 1: porcine pancreas trypsin; lane 2: h. viridiflavus gastric fluid. molecular weights are shown on the left side. under zymography figure, densitometric results of main bands (expressed as rm, relative mobility) are also shown. fig. 3. gelatine-precast ph-activity zymography page of crude extract of h. viridiflavus gastric fluid. each lane corresponds to a specific ph incubation. molecular weights are shown on the left side. under ph activity figure, densitometric results of main bands (expressed as rm, relative mobility) are also shown (represented values are expressed as mean of two independent experiments). fig. 4. gelatine-precast thermal activity zymography page of crude extract of h. viridiflavus gastric fluid. each lane corresponds to a specific temperature (°c) incubation. molecular weights are shown on the left side. under thermal activity figure, densitometric results of main bands (expressed as rm, relative mobility) are also shown (represented values are expressed as mean of two independent experiments). 150 antonio felicioli et al. 60 °c. ranging from 65 °c to 85 °c the c band remained visible while the d band disappeared over 60 °c. as presented in this research, it is evident how very large meals requiring compensatory adjustments in blood flow, acid secretion, and regulation of acid-base homeostasis (barboza et al., 2010) are processed by carnivores, such as snakes (intermittently feeders). consequently, the digestive system appears to be a suitable model, because of its phenotypic plasticity and its integration with other physiological systems, including reproduction, circulation or thermoregulation (barboza et al., 2010). heat is the main energy that snakes (and all the other reptiles) use to perform metabolic activities. the maximum recorded temperature of voluntary activity is the temperature at which most of the physiological, metabolic, and biological processes occur in several reptile species (bradshaw, 1986). lelièvre et al., 2010 reported that the european whip snake showed maximum thermal optimum at about 31-32 °c, with measured temperature of 28.7 ± 0.9 °c during digestion and 27.9 ± 1°c during post-prandial condition. digestion processes of large meal feeders could be driven by the quantitative and qualitative composition of gastric fluids. since few information are available about in vivo gastric secretion in reptiles, including partial or complete characterization of gastric protease, actual knowledge of in vivo gastric enzymatic patterns could contribute to the understanding of selected feeding ecology issues. in the present study, the pattern of proteolytic enzymes activity from the extracted gastric fluids reported a scenario composed of few active proteases, reflecting the feeding status (fasting) of studied snakes. zhalka and bdolah (1987) reported low levels of gastric protease activity in natrix tessellata. the observed ph value (7.2) of gastric fluid samples was consistent to those of other snakes (cox and secor, 2010; bessler and secor, 2012) in fasting condition. in addition, the absence of trypsinlike activity bands in our samples supports the hypothesis that endoscopy really sampled the gastric area, since trypsin (used as 23.4 kda proteolytic standard) is a pancreatic protease secreted into the gut. zymogram analysis revealed two main protease activity bands on which our attention was focused (band c and d). regarding ph-activity, band c revealed proteolytic activity all over the tested ph, with an optimal activity at basic ph, while band d appeared to be active only at basic ph. these observations, together with the inhibition experiments, supported the hypothesis that the observed proteases may belong to a class of alkaline proteases; in fact pepstatin a (90 μm), a typical acidic protease inhibitor, did not inhibited both the bands while pmsf (1.3 mm), a typical serine protease inhibitor, strongly inhibited both the bands (data not shown). regarding the thermo-activity, contrasting information are available about high thermo-stable protease in ectothermic vertebrates. de souza bezerra et al. (1999) observed in a tropical fish (colossoma macropum) 35 °c as optimum temperature for stomach acid protease activity, whereas 65 °c was found as the optimum value for the pyloric caeca (gut) alkaline protease activity. moreover, band c surprisingly revealed high activity until 85 °c supporting the hypothesis about the presence of an exogenous origin protease. in fact some prokaryotic organisms possess such thermostable protease: kuzu et al. (2012) described the production of a thermostable chitinase by bacillus thuringiensis subsp. kurstaki with high levels of activity at 110 °c into an alkaline ph range. regarding reptile chitinases, a putative one was also identified in the stomach of a phrynosomatid lizard (marsh et al., 2001), but in an acidic ph range. no information about temperature range are available to date. in conclusion, we can summarise that: a) fiberoptic endoscopy could be a suitable non-lethal method for gastrointestinal sampling in snakes; b) the absence of trypsin-like protease in sampled fluids confirmed the correct gastric location of the endoscopic probe; c) four major proteolytic activity bands were detected in sampled fluids; d) fasting condition of snakes resulted in a not so various pattern of proteolytic enzymes; e) the two major proteolytic activity bands (c and d) seemed to belong to a class of alkaline proteases; f ) the high temperature stability at alkaline ph of band c activity supported the hypothesis of a protease of exogenous source (e.g., bacteria); g) band d, with a thermal activity until 65 °c in an alkaline range, seemed to be a putative protease. while several laboratory analyses were carried out on pet snakes (e.g., python species; ott and secor, 2007; cox and secor, 2008; secor, 2008), wild species were considered quite rarely (mccue, 2007; chia-wei et al., 2009; secor et al., 2012), preventing in depth comparative analyses. furthermore, because wild snake feeding behavioural physiology is virtually unknown, this study may lead to forthcoming research such as experiments on different prey regimes. references barboza, p.s., bennet, a., lignot, j.h., mackie, r.i., mcwhorter, t.j., secor, s.m., skovgaard, n., sundset, m.a., wang, t. 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(2010): adaptive significance of food income in european snakes: body size is related to prey energetics. biol. j. linn. soc. 100: 307-317. acta herpetologica vol. 8, n. 1 june 2013 firenze university press journal of the societas herpetologica italica acta herpetologica acta herpetologica 11(1): 1-13, 2016 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-17147 feeding habits of mesoclemmys vanderhaegei (testudines: chelidae) elizângela silva brito1,*, franco leandro souza2, christine strüssmann3 1 programa de pós-graduação em biologia de água doce e pesca interior, instituto nacional de pesquisas da amazônia, av. andré araújo 2936, 69080–971 manaus, amazonas, brazil. *corresponding author. e-mail: eliz.chelidae@gmail.com 2 centro de ciências biológicas e da saúde, universidade federal de mato grosso do sul, av. costa e silva s/n, 79070–900 campo grande, mato grosso do sul, brazil 3 departamento de ciências básicas e produção animal, faculdade de agronomia, medicina veterinária e zootecnia, universidade federal de mato grosso, av. fernando correa da costa 2367, 78060–900 cuiabá, mato grosso, brazil submitted on 2015, 10th october; revised on 2015, 10th december; accepted on 2016, 27th january editor: sebastiano salvidio abstract. we studied the feeding habits of mesoclemmys vanderhaegei in small water bodies in the central region of the brazilian cerrado. we captured 80 individuals, 79 of which [41 (52%) females, 27 males (34%), 11 (14%) juveniles] had stomach contents for analysis. we identified 64 food item categories. the turtles were eating more animal than plant material, aquatic invertebrates being the most conspicuous diet item found. plant material was more abundant in females and rare in the diet of juveniles. mesoclemmys vanderhaegei proved to be an omnivorous and opportunistic chelonian, feeding on a wide range of food items. although there are differences in the consumption of food items among sexes and age categories, the most consumed food categories were common in all water bodies sampled. keywords. cerrado ecoregion, diet, food habits, foraging ecology, freshwater turtle, prey items, stomach contents, stomach flushing. introduction studies of freshwater turtle trophic ecology have shown that feeding habits in populations of the same species vary with the availability of food, life stage, sex, presence of other species of turtles (moll, 1976; vogt, 1980; fachín-terán et al., 1994; fachín-terán et al., 1995), habitat type and seasonality, among other factors (moll and moll, 2000). diet is one of the most often-discussed aspects of turtle natural history (souza, 2004). freshwater turtles in the family chelidae are generally considered omnivorous (souza, 2004), and ontogenetic dietary shifts are known in some species (e.g., bouchard and bjorndal, 2006). variations in the diet of turtles in relation to size and sex appear to be important to avoid niche overlap and intraspecific competition (fachín-terán et al., 1995; souza and abe, 1998). although some aquatic turtles possess a general morphology and behavior patterns adapted for carnivory, a variety of plant items are also regularly consumed, including stems, flowers, leaves and fruit (moll and moll, 2004). entirely or primarily carnivorous species can consume items ranging from zooplankton and a large number of aquatic and non-aquatic invertebrates to vertebrates (moll and moll, 2004). studies on feeding habits of turtles may involve direct observation (focal animal method, see sabino, 1999), analysis of stomach contents (legler, 1977) and/or fecal material (caputo and vogt, 2008) from live or preserved specimens. knowledge of the composition and variation in diet and feeding habits across different species can underpin broad ecological and evolutionary studies, as well as aid conservation and management planning (souza, 2004). human interference (e.g., trash, offering of arti2 elizângela silva brito et alii ficial food, impoundments) may cause important changes in the diet of free-living aquatic organisms, as reported by sabino et al. (2005), who found “obese” fish populations in areas subjected to intense public visitation in southwestern brazil. changes in the availability of food resources due to water pollution can also reduce the amplitude of the trophic niche and enhance competition amongst sympatric freshwater turtles (luiselli et al., 2004). the vanderhaege’s toad-headed turtle, mesoclemmys vanderhaegei (bour, 1973), is a central south american chelid most often recorded in water bodies within ecosystems with naturally open vegetation, such as the cerrado and chaco (brandão et al., 2002; souza, 2005; rueda-almonacid et al., 2007; vinke et al., 2013; marques et al., 2014). this species can occur in mountains and on high plateaus, in oligotrophic streams with clear water (brandão et al., 2002; brito et al., 2009; brito et al., 2012). however, individuals have also been found in swampy lowland environments with heavy aquatic vegetation cover, as well as in medium-sized rivers (vinke et al., 2013), dams and ponds (marques et al., 2013, 2014). the species also occurs in urban environments, though less frequently (brito et al., 2012). although generally considered carnivorous (ruedaalmonacid et al., 2007; vinke et al., 2013; marques et al., 2014), no detailed studies of the feeding habits of mesoclemmys vanderhaegei have been published. this study aims to identify and quantify the composition of the diet of m. vanderhaegei in streams in the cerrado ecoregion of central brazil. we investigate the effects of sex, age category, and site on dietary habits of this species and whether individuals are diet specialists or generalists. materials and methods study site the study was conducted in the chapada dos guimarães national park (15°25’s; 55°20’w; datum wgs 84) and its immediate surroundings, in the chapada dos guimarães fig. 1. map illustrating study site in the chapada dos guimarães national park and surrounding areas, mato grosso, brazil. black dots represent sampled streams. 3diet of mesoclemmys vanderhaegei municipality, mato grosso state, central brazil (fig. 1). the area is located in the southwestern portion of the cerrado ecoregion, a savanna-like vegetation that originally covered nearly two thousand square kilometers, mostly across central brazil. seven water bodies were sampled (table 1), at altitudes between 600 and 800 m a.s.l. obtention of specimens and stomach contents captures were made between 05 of january and 30 of march 2007. individuals were hand captured during visual searches or with the aid of baited funnel traps. for details of size, type and arrangement of funnel traps see brito et al. (2009). at each collecting site, seven traps operated 24 hours a day, for six consecutive days (a total sampling effort of 1008 trap hours per site). traps were visited three times a day (06:00, 14:00, and 21:00 h). visual searches by two observers were conducted during trap visits, totaling 36 hours/observer at each collection site. individuals were marked following the methods of cagle (1939), measured (to the nearest 0.05 mm, with a 300 mm vernier caliper: straight-line carapace length – cl, carapace width – cw; straight-line plastron length – pl; plastron width – pw), weighed (body mass, in g), and sexed, after which they were released at the capture site. sex was determined by external examination of secondary sexual characteristics: chelid males usually possess a longer and thicker tail than females (brito et al., 2009). individuals in which sex could not be determined (n = 11; all with cl < 100 mm) were treated as juveniles and grouped together in this category. to sample the diet of m. vanderhaegei we collected stomach contents using the stomach flushing technique developed by legler (1977). we considered the stomach empty when, after two consecutive flushing attempts, totaling 60 ml of water, individual turtles regurgitated nothing but water. samples of stomach contents were stored in containers with 70% alcohol (plant samples and invertebrates) or 10% formalin (vertebrates). diet analyses stomach contents were examined under a stereo-microscope and the food items were quantified and identified to the lowest possible taxonomic level. plant fragments that could not be identified because they were in an advanced state of decomposition were categorized as “unidentified plant material”. parts of exoskeleton, legs, wings and antennae of invertebrates that could not be identified were grouped in the category “unidentified arthropoda”. aquatic invertebrates were identified by using dichotomous keys (pérez, 1988; costa et al., 2006) and by consulting experts (see acknowledgements). total length (mm) of entire prey was measured to the nearest 0.01 mm using a digital caliper (bts®). data on stomach contents were analysed by sex (females and males), age categories (adults and juveniles), and water bodies. for each food category we determined the frequency of occurrence, the numerical frequency, and percentage volume (hyslop, 1980; marrero, 1994), measured by the displacement of a water column in a 1 ml syringe (graduated to 0.01 ml increments). when food item classes had a volume less than 0.01 ml, we regrouped such items into broader categories for volumetric analysis. the final analytical categories for volume were: aquatic invertebrates, non-aquatic invertebrates, fish, adult amphibians, larval amphibians, vertebrate bones (except fish), mammalian teeth, reptile scales, unidentified animal material, stems, leaves, fruits, seeds, aquatic plants (submerged vegetation), unitable 1. characteristics of seven capture sites of mesoclemmys vanderhaegei in chapada dos guimarães municipality (mato grosso, brazil) and number of turtles captured on each site. abbreviations: water body type: le lentic; lo lotic; le/lo mixed. natural/human impacted: n natural; hi human impacted. presence of aquatic vegetation (submerged and floating): ++ abundant; + present; absent. bankside vegetation: tr trees; sr shrubs; gr grasses. substrate: mu mud; sa sand; ro rock. water body captures coordinates water body type natural/ human impacted submerged vegetation floating vegetation bankside vegetation substrate monjolinho 18 15º24’s 55º48’w le/lo hi ++ ++ tr mu/sa dam 1 7 15º26’s 55º45’w le hi ++ + gr sa dam 2 5 15º26’s 55º45’w le hi + gr sa aldeia velha 5 15º26’s 55º45’w lo n + + tr/sr/gr sa/ro quineira 24 15º27’s 55º44’w le/lo hi ++ + tr mu/sa independência 8 15º24’s 55º50’w lo n + tr/sr sa/ro congonhas 13 15º23’s 55º50’w lo hi tr/sr/gr sa/ro 4 elizângela silva brito et alii dentified plant material, sediment (sand/stone), material from human activities (food scraps and religious rituals), periphyton, and unidentified material. statistical analyses we used analysis of variance (anova) to test if differences existed in the proportions of animal and plant items consumed by different sexes and ages of m. vanderhaegei. for this analysis, food items of animal origin were grouped in four categories: aquatic invertebrates, non-aquatic invertebrates, fish, and amphibians. we excluded the contributions of unidentified invertebrates and small bone fragments, scales and teeth of vertebrates, scarcely represented in our sample. similarly, plant items were grouped into four categories: aquatic plants, fruits, leaves, and plant material not identified; seeds were excluded due to poor representativity. an anova was also performed to investigate the differences between the consumption of animal and plant items as a whole. for both analyses, data on the proportions of each of the categories considered were transformed into arc sine square root, a transformation recommended when using frequency-based data that is not normally distributed (zar, 1996). the paired-samples t test (zar, 1996) was used to compare frequency of occurrence of animal and plant material consumed by mesoclemmys vanderhaegei in the seven water bodies sampled. to test the relationship between the total volume of ingested plant matter and the carapace length (cl) of captured individuals, and between the size of the entire ingested prey and cl, we employed a simple linear regression. for statistical analyses we used the application systat (wilkinson, 1990). the level of significance in statistical tests was set at ≤ 0.05. based on abundance (quantitative) and presence/absence (qualitative) data for the 12 food categories with higher values of frequency of occurrence, we performed an ordination analysis (non-metric multidimensional scaling – nmds), using the application pcord (version 4.0) to reduce the dimensionality of diet composition. we did this for male and female m. vanderhaegei (sites combined) and for each studied water body (sexes combined). to give the same weight to each sample (individual), we used the bray-curtis index as a measure of dissimilarity between matrices of abundance and presence/absence. results we captured 80 individuals of mesoclemmys vanderhaegei of which 79 had stomach contents. of these, 41 (52%) were females, 27 were males (34%), and 11 (14%) were juveniles. we identified 64 food item categories. items of animal origin were found in 97% (n = 77) of individuals examined, and items of plant origin in 63% (n = 65). among the animal items, invertebrates of the class insecta were the most diverse (with material from 25 families in 11 orders; table 2). macro-invertebrate species at aquatic larval stage were the most frequently consumed insects, mainly from the orders odonata, diptera and hemiptera. non-aquatic invertebrates (hymenoptera, lepidoptera, araneae, diplopoda) were also present, though generally with a frequency of less than 5%. among vertebrates, fish of the genus astyanax (characidae: characiformes) were most commonly consumed. amphibians occurred both at low frequency and small numbers when compared to other animal categories, and were mostly tadpoles. for plant material, the most often consumed items were aquatic plants, fruits (mainly from ficus sp., moraceae, and from mauritia flexuosa, arecaceae) and leaves, plus periphyton and material from the unidentified category. females of mesoclemmys vanderhaegei consumed more plant material than either males or juveniles (fig. 2), but recorded differences between the three categories were not significant (f3,27 = 0.26, p = 0.41). juveniles consumed more animal items than adult males and females (f3,106 = 0.26, p = 0.02). males consumed significantly more aquatic invertebrates than females (f3,76 = 0.33, p < 0.01). however, individuals in the two sexes did not differ in the consumption of non-aquatic invertebrates (f3,19 = 0.53, p = 0.06), fish (f3,6 = 0.67, p = 0.73) or amphibians (f3,12 = 0.76, p = 0.26). there was no significant relationship between turtle cl (n = 66) and the abundance of aquatic invertebrates consumed (r2 = 0.01, p = 0.38). fig. 2. frequency of occurrence of animal and plant material consumed by 79 individuals of mesoclemmys vanderhaegei captured in the chapada dos guimarães region (mato grosso, brazil) between january and march 2007. abbreviations: f = females (n = 41), m = males (n = 27) and j = juveniles (individuals ≤ 100 mm; n = 11). 5diet of mesoclemmys vanderhaegei table 2. composition of stomach contents of 79 individuals of mesoclemmys vanderhaegei from chapada dos guimarães, mato grosso, brazil, sampled between january and march 2007. frequency of occurrence and numerical frequency of each prey type by sex and age category: m adult males (n = 27); f adult females (n = 41); j juveniles (individuals ≤ 100 mm; n = 11). abbreviations: n.i. not identified due to digestion state. food items’ life stage: a adult (invertebrates and vertebrates; i imago (invertebrates only); l larva (vertebrates only); p pupa. food items frequency of occurrence numerical frequency m f j m f j invertebrates 322.09 268.05 418.14 74.47 57.71 75.16 odonata anisoptera (i) 14.81 (4) 9.75 (4) 9.09 (1) 1.11 (4) 1.31 (5) 1.07 (1) libellulidae (i) 25.92 (7) 19.51 (8) 9.09 (1) 15.59 (56) 12.36 (47) 3.22 (3) gomphidae (i) 11.11 (3) 4.87 (2) 2.50 (9) 1.31 (5) aeshnidae (i) 14.81 (4) 17.07 (7) 2.78 (10) 2.89 (11) odonata zygoptera (i) 2.43 (1) 0.26 (1) coenagrionidae (i) 2.43 (1) 0.26 (1) lestidae (i) 14.81 (4) 9.75 (4) 20.89 (75) 7.63 (29) blattaria (a) 2.43 (1) 0.26 (1) orthoptera (a) 14.81 (4) 12.19 (5) 18.18 (2) 1.11 (4) 1.31 (5) 2.14 (2) neuroptera (i) 4.87 (2) 9.09 (1) 0.52 (2) 1.07 (1) corydalidae (i) 3.70 (1) 0.27 (1) diptera (i) 3.70 (1) 4.87 (2) 9.09 (1) 0.27 (1) 0.78 (3) 4.30 (4) diptera culicomorpha (a) 3.70 (1) 14.63 (6) 9.09 (1) 0.27 (1) 1.57 (6) 1.07 (1) chironomidae (p) 29.62 (8) 17.07 (7) 18.18 (2) 3.34 (12) 2.89 (11) 2.15 (2) culicidae (p) 3.70 (1) 4.87 (2) 3.34 (12) 1.31 (5) simuliidae (p) 3.70 (1) 9.09 (1) 1.11 (4) 2.15 (2) diptera brachycera (a) sarcophagidae (a) 2.43 (1) 0.26 (1) hemiptera (a) 14.81 (4) 12.19 (5) 1.11 (4) 1.31 (5) hemiptera auchenorrhyncha (a) 14.81 (4) 2.43 (1) 27.27 (3) 1.39 (5) 0.26 (1) 8.60 (8) hemiptera heteroptera belostomatidae (a) 3.70 (1) 9.09 (1) 0.27 (1) 1.07 (1) corixidae (a) 2.43 (1) 9.09 (1) 0.26 (1) 1.07 (1) naucoridae (a) 9.09 (1) 0.26 (1) 1.07 (1) pleidae (a) 3.70 (1) 18.18 (2) 0.27 (1) 3.22 (3) trichoptera annulipalpia (i) 11.11 (3) 7.31 (3) 9.09 (1) 1.11 (4) 1.05 (4) 1.07 (1) hydropsychidae (i) 7.31 (3) 18.18 (2) 1.31 (5) 2.15 (2) macronema sp. (i) 7.40 (2) 2.43 (1) 18.18 (2) 1.11 (4) 0.52 (2) 5.37 (5) polycentropodidae (i) 2.43 (1) 9.09 (1) 0.52 (2) 4.30 (4) trichoptera integripalpia leptoceridae (i) 3.70 (1) 9.09 (1) 0.27 (1) 1.07 (1) coleoptera (a) 3.70 (1) 12.19 (5) 18.18 (2) 1.31 (5) 2.15 (2) coleoptera (i) 2.43 (1) 9.09 (1) 0.26 (1) 1.07 (1) coleoptera adephaga (a) dytiscidae (a) 3.70 (1) 7.31 (3) 9.09 (1) 1.11 (4) 1.05 (4) 1.07 (1) coleoptera elateroidea (a) elateridae (a) 3.70 (1) 0.27 (1) ephemeroptera (i) 14.81 (4) 2.43 (1) 9.09 (1) 1.94 (7) 0.52 (2) 1.07 (1) ephemeroptera pisciforma (i) baetidae (i) 14.81 (4) 4.87 (2) 9.09 (1) 5.57 (20) 0.78 (3) 5.37 (5) hymenoptera (a) 9.75 (4) 18.18 (2) 1.31 (5) 2.15 (2) formicidae (a) 3.70 (1) 9.75 (4) 18.18 (2) 0.27 (1) 1.57 (6) 2.15 (2) (continued) 6 elizângela silva brito et alii among the four categories of commonly eaten plant material, there was a significant difference between sexes and age categories for the consumption of leaves (f3,11 = 1.00, p < 0.01), aquatic plants (f3,21 = 0.96, p < 0.01), and plant material not identified (f3,19 = 0.86, p < 0.01), but not for fruit (f3,14 = 0.17, p = 0.35). females were the largest consumers of plants. the two axes of the nmds captured much of the variation of m. vanderhaegei diet (62% for qualitative data – presence/absence, and 59% for quantitative data – abundance), but it was not possible to separate the diets of males and females (fig. 3). items of animal origin represented the largest proportion of stomach contents volume for females (46%), males (53%), and juveniles (75%; table 3). for all these three categories, sediment constituted less than 1% of total stomach contents volume. for animal prey the commonest food items by volume were fish and aquatic invertebrates, for plants it was fruits. periphyton also occupied large volumes. although periphyton was the bulkiest item, only four individuals (one juvenile, one female and two males, 5% of stomachs analyzed) contained this type of food item. in 4% of females and juveniles and 7% of males, the stomachs contained material from human activities: e.g. beans, eggshell and popcorn. this type of material represented 15% of the volume of the diet of individuals caught in congonhas stream, the food items frequency of occurrence numerical frequency m f j m f j lepidoptera (i) 2.43 (1) 0.26 (1) cossidae (i) 3.70 (1) 0.27 (1) notodontidae (i) 3.70 (1) 9.09 (1) 0.27 (1) 1.07 (1) araneae 3.70 (1) 2.43 (1) 0.27 (1) 0.26 (1) diplopoda (a) 2.43 (1) 0.26 (1) oligochaeta 3.70 (1) 0.55 (2) unidentified arthropoda (i) 3.70 (1) 4.87 (2) 18.18 (2) 0.27 (1) 0.78 (3) 2.15 (2) unidentified arthropoda (a) 55.55 (15) 41.46 (17) 72.72 (8) 5.57 (20) 8.94 (34) 10.75 (10) amphibians 18.05 7.30 9.09 2.49 1.04 1.07 leptodactylidae (l) 3.70 (1) 0.27 (1) hylidae dendropsophus minutus (a+l) 4.87 (2) 0.78 (3) amphibians (n.i.) (a) 7.40 (2) 0.55 (2) amphibians (n.i.) (l) 7.40 (2) 2.43 (1) 9.09 (1) 1.67 (6) 0.26 (1) 1.07 (1) fish 48.13 58.51 27.27 5.54 12.09 3.22 astyanax sp. 37.03 (10) 53.65 (22) 27.27 (3) 4.45 (16) 11.57 (44) 3.22 (3) rivulus sp. 3.70 (1) 0.27 (1) vertebrate bones (n.i.) 3.70 (1) 0.55 (2) reptile scales 3.70 (1) 2.43 (1) 0.27 (1) 0.26 (1) mammalian teeth (n.i) 2.43 (1) 0.26 (1) plant material 96.26 136.55 72.72 7.75 22.06 8.59 aquatic plants (submerged vegetation) 22.22 (6) 34.14 (14) 18.18 (2) 2.22 (8) 4.73 (18) 2.15 (2) stems 7.40 (2) 14.63 (6) 9.09 (1) 0.55 (2) 1.57 (6) 1.07 (1) leaves 18.51 (5) 21.95 (9) 18.18 (2) 1.11 (4) 2.36 (9) 2.15 (2) seeds 3.70 (1) 2.43 (1) 0.27 (1) 0.26 (1) fruit of ficus sp. (fig) 11.11 (3) 17.07 (7) 0.83 (3) 8.15 (31) fruit of mauritia flexuosa (buriti) 7.40 (2) 17.07 (7) 18.18 (2) 0.55 (2) 1.84 (7) 2.15 (2) unidentified plant material 25.92 (7) 29.26 (12) 9.09 (1) 2.22 (8) 3.15 (12) 1.07 (1) algae periphyton 7.40 (2) 3.70 (1) 3.70 (1) 0.27 (1) 0.27 (1) 0.27 (1) material from human activities (religious food-offerings) 29.62 (8) 7.30 (3) 9.09 (1) 6.12 (22) 4.99 (19) 5.37 (5) sediment (sand/gravel) 29.62 (8) 14.62 (6) 54.54 (6) 3.06 (11) 1.57 (6) 6.44 (6) table 2. (continued). 7diet of mesoclemmys vanderhaegei only locality in which such items were recorded from m. vanderhaegei stomachs. in all water bodies sampled, diet items of animal origin were more commonly consumed than those of plant origin (t = 2.77; df = 6; p = 0.03; fig. 4). water bodies where there was greatest consumption of plant items were mostly dammed streams (monjolinho, quineira, dam 1, and dam 2), all anthropogenic environments. fruit consumption was recorded only in monjolinho and quineira dammed streams. the volume of plant (r2 < 0.03, p = 0.13) and animal material (r2 < 0.01, p = 0.72) in the stomachs was not significantly related to the cl of individuals caught. similarly, there was no significant relationship between prey size and the cl of captured individuals (r2 = 0.01, p = 0.13, n = 45 entire prey). qualitative data on sampling sites captured much of the variation in the diet between water bodies (63%), but the resulting ordination did not allow us to group water bodies according to the consumed food items (fig. 5). discussion in the area, and during the period of study, mesoclemmys vanderhaegei was found to be omnivorous with a general tendency to carnivory, as briefly pointed in a recent species’s account by marques et al. (2014). in many communities of vertebrates, omnivorous species represent the greater part of the consumers (polis, 1991; polis and strong, 1996), and species identified as primarily carnivorous may include plant material in their diet (mctigue and zimmerman, 1991). omnivory has been reported frequently among members of the family chelidae (e.g., medem, 1960; mittermeier et al., 1978; souza, 2004; caputo and vogt, 2008; martins et al., 2010). however, some species of this family, such as chelus fimbriatus, hydromedusa tectifera, phrynops hilarii, and platemys platycephala are exclusively carnivorous (vogt and villarreal, 1993; alcalde et al., 2010). mesoclemmys vanderhaegei is one of the largest aquatic vertebrates in the water bodies studied, along with the cuvier’s dwarf caiman (paleosuchus palpebrosus) and the green anaconda (eunectes murinus), and like these two, appears to occupy the higher levels of the local trophic chain (e. brito, pers. comm. 2007). items of animal origin were those most frequently used by all age and sex categories of m. vanderhaegei, with aquatic invertebrates being the most important, both in terms of proportional contribution to the diet and as number of individuals. high consumption of aquatic invertebrates (larvae and pupae) is frequently recorded for freshwater turtles (e.g., lagler, 1943; moll, 1990; souza and abe, 1998, 2000; moll and moll, 2004; caputo and vogt, 2008; bonino et al., 2009; alcalde et al., 2010; martins et al., 2010; brasil et al., 2011). however, consumed non-aquatic invertebrates fig. 3. non-metric multidimensional scaling (nmds) plots of food items consumed by mesoclemmys vanderhaegei from chapada dos guimarães, mato grosso, brazil, according to (a) presence/absence and (b) abundance. only turtles that were sexed were considered (n = 69; f females; m males). 8 elizângela silva brito et alii include adults of orthoptera, coleoptera and formicidae, indicating that m. vanderhaegei is an opportunistic predator, a feature common among aquatic turtles (moll, 1976; chessman, 1984; souza and abe, 1998, 2000; luiselli et al., 2004; alcalde et al., 2010; brasil et al. 2011). fish, particularly those of the genus astyanax (characiformes), are among the three most common animal items consumed by m. vanderhaegei. they are also the commonest prey item by volume recorded during the study. although they are known to be important in the diet of carnivorous and omnivorous freshwater turtles belonging to suborder cryptodira, fish are generally considered to be more easily captured by individuals belonging to those turtle species with longer necks (moll and moll, 2004) – the pleurodira – for which hydromedusa tectifera is a good example (alcalde et al., 2010). on several occasions during the field study, individuals of m. vanderhaegei were observed to chase fish of the genus astyanax. however, none of these attempts resulted in a successful capture. it appeared that individuals of m. vanderhaegei soon gave up chasing individuals when they were not caught immediately and would go in pursuit of other fish when there were schools on site (e. brito, pers. comm. 2005). at least five species of astyanax occur in the chapada dos guimarães national park, all of them considered regular and abundant members of the local fish community (icmbio, 2009). additionally, species in the genus astyanax can form large schools (e.g., suzuki and orsi, 2008). some of the individuals from these schools occasionally enter in the funnel traps (e. brito, pers. comm. 2007), where they could be more easily captured by imprisoned turtles and result in an overestimation of fish as a prey for m. vanderhaegei. although both sexes have higher consumption of animal items than of plants, the consumption is proportionately greater in females than in males. similar studies in other species of chelidae give conflicting results for this aspect: for the neotropical species phrynops table 3. composition of stomach contents of 79 individuals of mesoclemmys vanderhaegei from chapada dos guimarães, mato grosso, brazil, sampled between january and march 2007. volume (v) in ml and in percentual of each food item category, by sex and age category: m adult males (n = 27); f adult females (n = 41); j juveniles (individuals ≤ 100 mm; n = 11). food items’ life stage: a adult; l larva. food item category age category m f j v (ml) v (%) v (ml) v (%) v (ml) v (%) invertebrates 10.98 16.96 15.91 17.11 2.27 17.53 aquatic invertebrates 8.41 12.99 13.39 14.40 1.06 8.19 non-aquatic invertebrates 2.57 3.97 2.52 2.71 1.21 9.34 vertebrates 10.58 16.32 26.05 28.01 7.46 57.55 amphibians (a) 0.12 0.19 0.00 0.00 0.00 0.00 amphibians (l) 1.92 2.96 0.06 0.06 0.02 0.15 fish 7.86 12.13 23.24 24.99 7.44 57.40 vertebrate bones (except fish) 0.10 0.15 0.00 0.00 0.00 0.00 mammalian teeth 0.00 0.00 0.05 0.05 0.00 0.00 reptilian scales 0.08 0.12 0.60 0.65 0.00 0.00 unidentified vertebrates 0.50 0.77 2.10 2.26 0.00 0.00 plant material 1.76 3.64 15.05 16.16 0.77 5.94 stems 0.19 0.29 1.18 1.27 0.02 0.15 leaves 0.13 0.20 0.05 0.05 0.04 0.31 fruits 1.34 2.07 12.04 12.95 0.50 3.86 seeds 0.10 0.15 0.02 0.02 0.00 0.00 aquatic plant (submerged vegetation) 0.16 0.25 0.87 0.94 0.11 0.85 unidentified plant material 0.44 0.68 0.89 0.96 0.10 0.77 sediment (sand/gravel) 0.07 0.11 0.06 0.06 0.04 0.31 material from human activities (religious food-offerings) 2.61 4.03 7.05 7.58 0.50 3.86 algae periphyton 26.60 41.26 20.89 27.17 1.30 9.87 unidentified material 11.30 17.67 8.00 3.88 0.62 4.93 9diet of mesoclemmys vanderhaegei geoffroanus, no significant differences were found in diet composition of males and females (fachín-terán et al., 1994), while in emydura krefftii from australia (georges, 1982), and acanthochelys spixii, in the brazilian cerrado (brasil et al., 2011), females consumed more plant material, while males ate more animal items. considering the different reproductive strategies of the sexes, with females having to increase their weight and maintain nutritional reserves during egg development and laying (schoener, 1971), such differences in the diets of males and females are not unexpected. the fact that our study found no differences in the composition and abundance of food items consumed by male and female m. vanderhaegei suggests a possible food niche overlap between the two sexes. this could be related to the opportunistic consumption of the most abundant items in the environment by both sexes. although another chelid – phrynops geoffroanus – is frequently recorded in lower sections of the streams studied, no other freshwater turtle but m. vanderhaegei occurs at the study site. therefore, it is also possible that in the absence of competition, individuals in this species have such a broad niche that there is no need for betweensex partitioning. the occurrence of dietary changes in the presence of potential competitors is an interesting hypothesis that could be tested for m. vanderhaegei, in the drainages mentioned here. on the contrary, the greater frequency of occurrence of plant materials in the stomachs of individuals of mesoclemmys vanderhaegei may be simply the result of accidental ingestion during predation of animal items. in fact, when analyzing the diet of omnivorous turtles, it is often not possible to determine whether the plant material was ingested intentionally or accidentally (parmenter and avery, 1990; lindeman, 1996; rowe and parsons, 2000). another aspect potentially indicating accidental ingestion of plant items was the fact that only small fragments of such material were found in the stomach contents. on the other hand, the large volume of periphyton found in four stomachs of m. vanderhaegei during our study is suggestive of deliberate intake (tables 2 and 3). the same can be inferred for bulkier plant items – fruits of ficus sp. and exocarp and mesocarp of mauritia flexuosa were found almost whole in the stomachs, suggesting that they had been purposely ingested. in general, chelid turtles appear to supplement their diets with fruits or seeds, particularly when these items are readily available or when aquatic prey becomes more dispersed, as in the rainy season (fachín-terán et al., 1995; lima et al., 1997; caputo and vogt, 2008). our results reinforce the suggestion by caputo and vogt (2008) that turtle diets ideally should be investigated by using both stomach flushing and fecal analyses. in a study of the diet of rhinemys rufipes in brazilian amazonia, those authors found that bulky stomach contents (e.g., large fruit seeds), generally not regurgitated by turtles by using the stomach flushing fig. 4. frequency of occurrence of animal and plant material consumed by mesoclemmys vanderhaegei in the seven water bodies sampled in the chapada dos guimarães region, mato grosso, brazil. subtitle: av aldeia velha stream; co congonhas stream; in independência stream; mo monjolinho stream; qui quineira stream; d1 dam 1; d2 dam 2. fig. 5. non-metric multidimensional scaling (nmds) plots of food items consumed by mesoclemmys vanderhaegei in the seven sites sampled in chapada dos guimarães municipality, mato grosso, brazil. a = independência stream; c = congonhas stream; v = aldeia velha stream; l = dam 1; d = dam 2; m = monjolinho stream; q = quineira stream. 10 elizângela silva brito et alii technique, do however appear in analyses of fecal contents (caputo and vogt, 2008). the consumption of animal items in greater numerical frequency and percentage volume by juveniles than by adult males and females corroborates what is reported elsewhere. the record of ontogenetic diet changes in chelonian species is common and usually juvenile freshwater turtles are more carnivorous, while adult freshwater turtles are more herbivorous (clark and gibbons, 1969; moll, 1976; parmenter and avery, 1990; kennett and tory, 1996; spencer et al., 1998) or omnivorous (bouchard and bjorndal, 2006). this change may be related to protein requirements for growth (schoener, 1971), considering that the juvenile turtles are more susceptible to predation and need to grow fast. change in diet upon reaching reproductive maturity has also been reported in emydura krefftii (chelidae), a species where juveniles are carnivorous and adults omnivorous (georges, 1982), and in hydromedusa maximiliani (chelidae) (souza and abe, 1998), where small crabs accounted for 75% of all food items consumed by juveniles but only 5% of the food items consumed by adults. a curious result of this study is the similarity of m. vanderhaegei diet in water bodies with apparently very different environmental characteristics. in congonhas stream, for example, there are no fish (j. penha, pers. comm. 2007) but a sporadic supply of biological material from human activities does occur (picnics and religious food-offerings). this material comprised a large variety of fruits (e.g., kiwi, grapevines, pear, peach, apple, banana, watermelon), vegetables (okra), cooked grains (rice, popcorn), beans, and eggs, arranged in plastic or wood trays, at the margins of the stream. the broad trophic plasticity recorded for m. vanderhaegei indicates that individuals of this species have very generalist diets. the identification of unusual food items, for example, scales of reptiles (snakes and lizards) and mammalian teeth, as well as material from human activities (see table 2), may also reflect a common tendency for freshwater turtles to be scavengers (souza, 2004). the categories of food eaten by individuals of m. vanderhaegei indicate that they may forage in several different strata of the water column. benthic foraging was indicated by the consumption of such aquatic invertebrates as trichoptera (which live mostly under rocks, logs and plant materials, in running water; pérez, 1988) and odonata (whose larvae have strong associations with lentic and aquatic vegetation, but may also occur in lotic environments, where they live buried in the sand; pérez, 1988). meanwhile, the consumption of large amounts of fish (astyanax sp.), of fruits (e.g., ficus sp.), and of nonaquatic invertebrates active on understory vegetation (e.g. various ant species) indicate that individuals of m. vanderhaegei also forage both nektonically and directly from the surface. as with most omnivorous vertebrates, in which feeding is related to a variety of foraging opportunities in their habitat, female, male and juvenile m. vanderhaegei all showed a mixed diet of plants and animals items. while a mixed diet implies less efficient digestion, since it requires a diverse gut microflora, it also involves less loss of time and energy in the search for more nutritious foods (bjorndal, 1991). in addition, a mixed plant-animal diet provides a broader spectrum of nutrients than does an item-restricted diet, which may explain why most organisms are to some extent omnivorous and hence have an enhanced ability to optimize their nutritional balance (bjorndal, 1991). the present study provides detailed information on the diet of mesoclemmys vanderhaegei in natural and anthropogenic disturbed environments in headwater streams in cerrado. much of the original area occupied by this ecoregion is presently modified by anthropogenic changes derived from agriculture. such modifications have strongly impacted aquatic ecosystems, severely altering species composition, functioning and structure of freshwater habitats throughout cerrado (agostinho et al., 2005), in such an extent that this ecoregion is considered a global biodiversity hotspot (klink and machado, 2005). a close relative of m. vanderhaegei – mesoclemmys hogei, native to another brazilian hotspot, the atlantic forest – was recently recognized by the governmental environmental agency as critically endangered, being the first and the only continental brazilian chelonian to be considered as a threatened species (icmbio, 2015). additional studies on actual and potential effects of anthropogenic and natural disturbances on the feeding habits of m. vanderhaegei, among other biological aspects, might help to prevent extinction risks to this species. acknowledgments the authors are grateful to environmental analysts and experts from the chapada do guimarães national park for their support during field data collection. we also thank the centro nacional de pesquisa e conservação de répteis e anfíbios (ran) for licenses to capture m. vanderhaegei (permit # ran/ibama 031/2005). thanks are also due to pastor henrique, murcio, and the authorities from chapada dos guimarães for allowing field study at properties under their care, respectively, the buriti evangelical school, the aldeia velha farm, and parque municipal do quineira. we particularly thank 11diet of mesoclemmys vanderhaegei all the people who assisted in the collection of field data and capes for a scholarship grant to esb and a prodoc (programa de apoio a projetos institucionais com a participação de recém-doutores) grant to cs that led to the completion of the field work. wesley oliveira, from leta (laboratório de ecologia e taxonomia de artrópodes), helped to identify aquatic invertebrates. cs and fls thank cnpq for research productivity grants (processes # 309541/2012-3 and 303006/2014-5, respectively). rafael valadão helped with the map. adrian barnett and richard carl vogt helped with the english and gave suggestions that improved the text. we also thank two anonymous reviewers. references agostinho, a.a., thomaz, s.m., gomes, l.c. 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(1996): biostatistical analisys. prentice hall, new jersey. acta herpetologica vol. 11, n. 1 june 2016 firenze university press feeding habits of mesoclemmys vanderhaegei (testudines: chelidae) elizângela silva brito1,*, franco leandro souza2, christine strüssmann3 morphology, ecology, and behaviour of hylarana intermedia, a western ghats frog ambika kamath1, rachakonda sreekar2,3 a new account for the endangered cerrado rocket frog allobates goianus (bokermann, 1975) (anura: aromobatidae), with comments on taxonomy and conservation thiago ribeiro de carvalho1,2,*, lucas borges martins1,2, ariovaldo antonio giaretta1 molecular phylogenetics of the pristimantis lacrimosus species group (anura: craugastoridae) with the description of a new species from colombia mauricio rivera-correa, juan m. daza* population structure and activity pattern of one species of adenomera steindachner, 1867 (anura: leptodactylidae) in northeastern brazil maria juliana borges-leite1,*, joão fabrício mota rodrigues2, patrícia de menezes gondim1, diva maria borges-nojosa1 vocal repertoire of scinax v-signatus (lutz 1968) (anura, hylidae) and comments on bioacoustical synapomorphies for scinax perpusillus species group marco antônio peixoto1,*, carla silva guimarães1, joão victor a. lacerda2, fernando leal2, pedro c. rocha2, renato neves feio1 amendment of the type locality of the endemic sicilian pond turtle emys trinacris fritz et al. 2005, with some notes on the highest altitude reached by the species (testudines, emydidae) federico marrone*, francesco sacco, vincenzo arizza, marco arculeo photo-identification in amphibian studies: a test of i3s pattern marco sannolo1,*, francesca gatti2, marco mangiacotti3,4, stefano scali3, roberto sacchi4 no evidence for the ‘expensive-tissue hypothesis’ in the dark-spotted frog, pelophylax nigromaculatus li zhao, min mao, wen bo liao* no short term effect of clinostomum complanatum (trematoda: digenea: clinostomatidae) on survival of triturus carnifex (amphibia: urodela: salamandridae) giacomo bruni1,*, claudio angelini2 yeasts in amphibians are common: isolation and the first molecular characterization from thailand srisupaph poonlaphdecha, alexis ribas* introduction of eleutherodactylus planirostris (amphibia, anura, eleutherodactylidae) to hong kong wing ho lee1, michael wai-neng lau2, anthony lau3, ding-qi rao4, yik-hei sung5,* correlation between endoscopic sex determination and gonad histology in pond sliders, trachemys scripta (reptilia: testudines: emydidae) david perpiñán1, albert martínez-silvestre2,3, ferran bargalló3, marco di giuseppe4, jorge orós5, taiana costa6,* book review: john w. wilkinson. amphibian survey and monitoring handbook. pelagic publishing franco andreone book review: susan newman. frogs amphibians and their threatened environment. discovery and expression through art k-3. frogs are green franco andreone acta herpetologica 9(2): 243-247, 2014 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-14184 reproduction of endangered big-headed turtle, platysternon megacephalum (reptilia: testudines: platysternidae) yik-hei sung1,2,*, billy c.h. hau1, nancy e. karraker1,3 1school of biological sciences, the university of hong kong, hong kong sar, china 2 present address: department of biology, hong kong baptist university, hong kong sar, china. *corresponding author. e-mail: heisyh@gmail.com 3 present address: department of natural resources science, university of rhode island, kingston, ri, usa submitted on 2014, 13th march; revised on 2014, 20th october; accepted on 2014, 24th october editor: sandra hochscheid abstract.; the big-headed turtle (platysternon megacephalum) is heavily harvested to support tremendous demands from food and pet markets, and thus its ecology remains poorly understood. the presence of self-sustaining populations in hong kong (22°09’-22°37’n, 113°50’-114°30’e) provides important opportunities to advance our understanding of this species. we employed mark-recapture surveying, radio-tracking of two gravid females, and directed streamside searches to document the reproductive ecology of the species between september 2009 and june 2011 in hong kong. we found seven gravid females between 20-27 june 2010 and 2011, and which subsequently oviposited on average three eggs (range 2-8), with mean length and width of 36 mm and 21 mm, in early july. there was positive correlation between the size of females and clutch sizes. we found one clutch in leaf litter 1.6 m away from the stream, which hatched between 14 to 18 october. the incubation period was estimated to be between 103 and 110 days. the results of this study provide important information to formulate conservation plan for this endangered species. keywords. asian turtle crisis, incubation, oviposition, turtle conservation. big-headed turtles (platysternon megacephalum) is currently listed as endangered in the iucn red list (iucn, 2013) and was up-listed to appendix i of cites (cites, 2011). however, turtles are still captured and commonly sold in food and pet markets (cheung and dudgeon, 2006; gong et al., 2009). it is evident that populations of p. megacephalum have been heavily depleted and are in drastic decline in china (hendrie, 2000; tana et al., 2000; gong et al., 2006). the iucn red list status of this species was recently proposed to be changed from endangered to critically endangered to highlight the immediate need to conserve this species (horne et al., 2012). given the acute nature of declines of p. megacephalum throughout its range (horne et al., 2012), the presence of self-sustaining populations in hong kong provides important opportunities to advance our understanding of this species (sung et al., 2013). to our knowledge, the reproductive biology of wild p. megacephalum such as clutch size and timing of reproduction has not been documented. in this study, we aimed to investigate the reproductive biology of p. megacephalum to answer the following questions: 1) when is the reproductive season, including when do they lay eggs and their eggs hatch? 2) what are the clutch sizes and egg sizes? 3) where do they lay eggs? 4) is the number of eggs by clutch related to the size of females? our overarching goal in delineating the reproductive ecology of the species was to generate information that is useful to design conservation plans for this species. we conducted this study within the hong kong special administrative region, china (22°09’-22°37’n, 113°50’-114°30’e) from 1 september 2009 to 27 june 2011. study sites included four streams located within national parks and a stream in kadoorie farm and 244 y.-h. sung et alii botanic garden (kfbg), a private reserve in which access by the public is controlled. the elevation of the study sites are between 250 to 800 m above sea level. in order to investigate the reproductive ecology of p. megacephalum we employed three methods: radio-tracking of gravid females, mark-recapture surveys, and daytime searches for oviposition sites. we conducted a mark-recapture study, regularly surveying for turtles using baited hoop traps and visual encounter surveys in all five study sites following methods in sung et al. (2013). we made 261 captures of 138 individuals, of these, 26, 30 and 82 were adult males, females and juveniles, respectively. female turtles achieve maturity when they reach 100 mm in carapace length (sung et al., in press), thus we regarded all female turtles over 100 mm in carapace length as adults. for every adult female p. megacephalum captured, we assessed the presence of eggs by palpation (kuchling, 1999), and potentially gravid females were transported to the wild animal rescue centre of kadoorie farm and botanic garden for x-ray radiography to confirm the presence of oviductal eggs and document clutch sizes. we conducted linear correlation analysis between the clutch size and the carapace length of females using software r (r core team, 2014). the sizes of eggs were measured from radiograph images using imagej software (national institutes of health; http://rsb.info.nih.gov/ij/). a total of 25 eggs were measured. we used the straightline carapace length as reference for measurement. we compared the carapace length and plastron length between gravid females and nongravid females by t-test using software r. data were log-transformed to meet the assumption of normality. in june 2010, we attached radio-transmitters (lf1-2477-rs-t, l.l. electronics, usa) on the rear side of the carapace of two gravid females using epoxy putty glue and released them back to their original locations within 24 hours of capture. we used a communication receiver (ic-r10, icom inc., usa) and a flexible 3-element yagi antenna (biotrack ltd., uk) to relocate the radio-tracked females every evening after release in an attempt to locate egg laying sites. we palpated the turtles to determine whether turtles had laid eggs. we conducted opportunistic and systematic daytime searches for eggs in potential nesting sites, including in leaf litter and soil within 0.5 to 20 m from streams during the wet season (i.e. between 27 april 2011 to 2 september 2011). searching effort for nests totaled 15 person-hours over the study period. the sizes of eggs discovered in the field were carefully measured to the nearest 1 mm by caliper (mitutoya corporation, japan) and weighed to the nearest 1 g with a spring balance (pesola, baar, switzerland). we visited identified nests every three to four days to determine the time of hatching. during our mark-recapture study, which captured a considerable number of females from march-october, we only detected gravid females by palpation in june (fig. 1). among 105 captures of females all year round, we found a total of seven different gravid females between 20 june to 27 june in both 2010 and 2011. the average straightline carapace length and plastron length of gravid females were 125.1 mm (sd = 14.0, range 110.5-152.9 mm, n = 7) and 103.7 mm (sd = 11.6, range 90.3-127.2, n = 7), respectively, and the average weight was 393.3 g (sd = 121.9, range 276-624, n = 7). the average straight-line carapace length and plastron length of nongravid females were 119.7 mm (sd = 12.0, range 105.6-153.2 mm, n = 23) and 100.8 mm (sd = 10.1, range 88.8-127.1, n = 23), respectively, and the average weight was 326.3 g (sd = 105.3, range 180-595, n = 23). there were no significant differences between females that we found gravid and nongravid females in carapace length (t = -1.50, p = 0.18) and plastron length (t = -0.89, p = 0.42). average clutch size was 3.6 (sd = 2.0, range 2–8, n = 7). the clutch size was positively correlated with the carapace length of females (r2 = 0.70, p = 0.02). analysis of x-ray radiographs revealed that the mean length of oviductal eggs was 35.8 mm (sd = 0.8, range 27.4-43.7, n = 25) and the average width of eggs found in nests was 20.5 mm (sd = 1.1, range 18.0-21.9, n = 25). we radio-tracked two gravid females in june and july 2010, however, they were relocated to the same place where we captured them. they were gravid until we found that they laid their eggs on 1 or 2 july and on 3 or 4 july. despite extensive searching during radio-tracking, we were unable to find these nests. during daytime searches for nest sites, we were only able to find one clutch containing two eggs in leaf litter at 5 cm soil depth, 1.6 m from the stream edge, and at figure 1. number of captures of all females and gravid females platysternon megacephalum in hong kong between 2009 and 2011. 245reproduction of platysternon megacephalum 689 m elevation on 26 july 2011. unfortunately, one of the eggs was damaged during searching and thus measurements were only taken for one egg. the egg measured 38.5 × 21.6 mm in size and weighed 11.0 g. the egg hatched between 14 october and 18 october 2011. based on our observations in the field, at least some female p. megacephalum deposit eggs in early july (1-4 july in this study) and the embryonic developmental period may be between 103 and 110 days. this is the first study to document a nest of wild p. megacephalum. some freshwater turtles travel large distances on land to seek out suitable nesting sites (congdon et al., 1987). however, it appears that some p. megacephalum nest within 2 m of streams. a radio-tracking study found that females remained near (i.e. less than 5 m) streams during reproductive seasons (y.h. sung, unpublished data), and another radio-tracking study of relocated adults showed that the maximum distance from the streams was 5.8 m (shen et al., 2010). these provided further evidence that females likely lay eggs near streams. females apparently dig a shallow nest and lay eggs in leaf litter. our observations indicated that much of the riparian area along the study streams appears to be suitable for nesting. there may be other suitable nesting sites that are more obscure and difficult to find, such as rock crevices and deeper within the soil. platysternon megacephalum in hong kong appears to have consistent and synchronized breeding seasons during the wet season, as we only detected gravid females in late june. in addition, we observed males and females inhabiting the same small pool on multiple occasions (over five times) in may from a mark-recapture study (y.h. sung, unpublished data). such observations were rare in other times of the year, and suggest that pairing and copulation may occur in may. as with many other turtle species, the onset of reproduction, and the period between mating and egg laying, of p. megacephalum is concordant with the wet season, when food is abundant for gravid females (wang et al., 2011). however, it is not known if females in this species store sperm and if fertilization of eggs can be accomplished from previous years’ mating. females of many species of turtles are capable of sperm storage (pearse and avise, 2001), and this is a possibility for p. megacephalum. the breeding season among individual p. megacephalum is relatively consistent because we were only able to detect gravid females in late june and early july, despite considerable number of females captured throughout the wet season (fig. 1). females of other asian turtle species are gravid for a longer period of time, for example, between late april to june for cuora mouhotti (wang et al., 2011) in hainan and mid-april to mid-july for cuora flavomarginata in taiwan (chen and lue, 1999). we did not x-ray females regularly to detect oviductal eggs when they could not be detected by palpation. regular x-raying of females in the wet season may help us to better understand the reproductive season of p. megacephalum. the single clutch of eggs located in the field in this study hatched in mid-october, which was concordant with observations of newly-hatched neonates in october and november in hong kong (kendrick et al., 2011). however, there was an exceptional capture of newlyhatched neonates in april (kendrick et al., 2011). there are two possible explanations for this observation. one explanation is that in some cases embryos may develop over the winter period or eggs may hatch and hatchlings may remain in nests until spring. an alternative explanation is that p. megacephalum lays eggs during the winter. the latter is unlikely as we have documented through radio-telemetry that most p. megacephalum are inactive during winter (y.h. sung et al., unpublished data). in captive situations, female p. megacephalum lay one to three eggs as a single clutch per year (bonin et al., 2006; zhou and wang, 2009), and we confirmed that the clutch sizes of most females were three. notably, one female at a protected site contained eight eggs, and it was the largest female captured in this study (plastron length = 127.5 mm). the largest female caught among the four other sites (plastron length = 105.5 mm), where illegal harvesting has occurred, were at least 15% smaller (sung et al., 2013). the absence of large females at unprotected sites was likely caused by past illegal harvesting of turtles (sung et al., 2013). we found that clutch size was positively correlated to the size of females, removal of large females severely reduces the resilience of populations to over-exploitation (paitz et al., 2007). this may explain why populations of p. megacephalum decline quickly in places where harvesting occurs. the size of eggs may be underestimated as the eggs may be tilted inside the turtles during x-ray, however, this was still a valuable method because this was the only non-invasive way to provide information of egg size of this species. for many imperiled turtle species under extremely high pressure from harvesting, captive breeding is believed to be valuable to help their conservation (horne et al., 2012). populations of p. megacephalum in many places have been heavily depleted because of unflagging demand in the food and pet markets, as enforcement has been inadequate to stop illegal harvesting. captive breeding has been suggested as a means to conserve p. megacephalum, yet it has rarely been successful (shi et al., 2007). one major reason is the lack of knowledge about their natural history, including reproductive biology, diet, 246 y.-h. sung et alii and behavior. we hope the information yielded from this study in hong kong, where relatively healthy populations remain, may improve methods for captive breeding of p. megacephalum. given the currently unresolved taxonomy of platysternon across its broad distribution and severe harvesting pressure throughout much of its range, we recommend that increased effort be made for in-situ conservation and research. a major constraint to our ability to conserve asian freshwater turtle species has been that populations are so severely diminished by over-collection that it becomes impossible to conduct ecological research that would provide a basis for conservation actions, as has been the case with many cuora species and has nearly been the case with p. megacephalum. while fairly robust populations of some of these threatened turtles remain, it is imperative that we begin collecting baseline data to inform conservation programs as we work to reduce the threat that harvesting presents to freshwater turtle populations. acknowledgements we thank a. brown for permission to conduct this study within kadoorie farm and botanic garden the private reserve. we are grateful to m. lau, g. ades, p. crow and a. grioni for project support. we thank s.p. gong for helpful discussion. we are grateful to the agricultural fisheries and conservation department of the hong kong government for issuing a research permit. we thank many students from the university of hong kong and other volunteers for their generous help in the field. this study was approved by the committee on the use of live animals in teaching and research, the university of hong kong (#culatr 2249-10) and department of health, the government of hong kong special administrative region (# (10-5) dh/ha&p/8/2/3 pt.17). references bonin, f., devaux, b., dupre, a. (2006): turtles of the world. johns hopkins university press, baltimore. chen, t.h., lue, k.y. (1999): population characteristics and egg production of the yellow-margined box turtle, cuora flavomarginata flavomarginata, in northern taiwan. herpetologica 55: 487-498. cheung, s.m., dudgeon, d. (2006): quantifying the asian turtle crisis: market surveys in southern china, 2000-2003. aquat. conserv. 16: 751-770. cites (2011): proposals for amendment of appendices i and ii. twelfth meeting of the conference of the parties, proposal 20. congdon, j.d., breitenbach, g.l., van loben sels, r.c., tinkle, d.w. (1987): reproduction and nesting ecology of snapping turtles (chelydra serpentina) in southeastern michigan. herpetologica 43: 39-54. gong, s., shi, h., xu, r. (2006): a survey of freshwater turtles in jianfengling nature reserve, hainan province, china. chin. j. zool. 41: 80-83. gong, s.p., chow, a.t., fong, j.j., shi, h.t. (2009): the chelonian trade in the largest pet market in china: scale, scope and impact on turtle conservation. oryx 43: 213-216. hendrie, d. (2000): status and conservation of tortoises and freshwater turtles in vietnam. in: asian turtle trade: proceedings of a workshop on conservation and trade of freshwater turtles and tortoises in asia. chelonian research monographs, pp. 63-73. van dijk, p.p., stuart, b., rhodin, a.g.j., eds, mtc printing, lunenburg. horne, b., poole, c., walde, a. (2012): conservation of asian tortoises and freshwater turtles: setting priorities for the next ten years. recommendations and conclusions from the workshop in singapore, february 21-24, 2011. wildlife conservation society, singapore. iucn (2013): iucn red list of threatened species. version 2013.2. available from < http://www.iucnredlist. org>. kendrick, r.c., lau, m.w.n., crow, p.a., ades, g.w.j. (2011): notes on wild population of big-headed turtle in hong kong. kadoorie farm and botanic garden publication series no. 9. kadoorie farm and botanic garden, hong kong. kuchling, g. (1999): the reproductive biology of the chelonia. springer press, berlin. paitz, r.t., harms, h.k., bowden, r.m., janzen, f.j. (2007): experience pays: offspring survival increases with female age. biol. lett. 3: 44-46. pearse, d.e., avise, j.c. (2001): turtle mating systems: behavior, sperm storage, and genetic paternity. j. hered. 92: 206-211. r core team (2014): r: a language and environment for statistical computing. r foundation for statistical computing, vienna, http://www.r-project.org. shen, j.w., pike, d.a., du, w.g. (2010): movements and microhabitat use of translocated big-headed turtles (platysternon megacephalum) in southern china. chelonian conserv. biol. 9: 154-161. shi, h., parham, j., lau, m., chen, t. (2007): farming endangered turtles to extinction in china. conserv. biol. 21: 5-6. sung, y.h., hau, b.c.h., lau, m.w.n., crow, p.a., kendrick, r.c., buhlmann, k.a., ades, g.w.j., karraker, 247reproduction of platysternon megacephalum n.e. (in press): growth rate and an evaluation of age estimation for the endangered big-headed turtle (platysternon megacephalum) in china. j. herptol. sung, y.h., karraker, n.e., hau, b.c.h. (2013): demographic evidence of illegal harvesting of an endangered asian turtle. conserv. biol. 27: 1421-1428. tana, t., hour, p., thach, c., sopha, l., sophat, c., piseth, h., kimchay, h. (2000): overview of turtle trade in cambodia. in: asian turtle trade: proceedings of a workshop on conservation and trade of freshwater turtles and tortoises in asia. chelonian research monographs, pp. 55-57. van dijk, p.p., stuart, b., rhodin, a.g.j., eds, mtc printing, lunenburg. wang, j., gong, s., shi, h., liu, y., zhao, e. (2011): reproduction and nesting of the endangered keeled box turtle (cuora mouhotii) on hainan island, china. chelonian conserv. biol. 10: 159-164. zhou, t., wang, w. (2009): altas of turtles farmed in china. china agriculture press, beijing. acta herpetologica 11(1): 75-79, 2016 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-17774 no short term effect of clinostomum complanatum (trematoda: digenea: clinostomatidae) on survival of triturus carnifex (amphibia: urodela: salamandridae) giacomo bruni1,*, claudio angelini2 1 viale palmiro togliatti, 101, 50019 sesto fiorentino, italy. *corresponding author. e-mail: giacomo.b90@gmail.com 2 via guglielmo marconi, 30, 04018 sezze, italy submitted on 2015, 30th december; reviewed on 2016, 3rd march; accepted on 2016, 15th march editor: daniele pellitteri-rosa abstract. in this paper we evaluated the effects of the metacercariae of c. complanatum on body condition and survival probability of the italian crested newt. we studied two populations of t. carnifex, with one of the two infected by the trematode, in central italy, for a year. we found that: i) paedomorphic newts were more likely to be infected; ii) cysts were mostly located on the head and into the mouth; iii) the infection did not affect the body size of newts; iv) survival probabilities of infected and not infected newts from the same population did not differ, and it was similar to the survival of newts from the not-infected population. we conclude that, at least in the short term, the metacercariae of c. complanatum are not damaging for t. carnifex. keywords. short term survival, triturus carnifex, clinostomum complanatum, host-pathogen interaction, capturemark-recapture the global amphibian decline rekindled the interest in amphibian diseases and the pathogens that cause them. in particular, the fungi batrachochytrium dendrobatidis and b. salamandrivorans, iridoviruses and bacterial infections have received much attention, since they caused mass mortalities worldwide (daszak et al., 1999; green et al., 2002; wake and vredenburg, 2008; martel et al., 2014). infections by other parasites got less attention, but it is known that a variety of organisms, such as protozoans and nematoda (schmidt and roberts, 2009), maxillopoda (marquardt et al., 2000) and trematoda, can be amphibians’ pathogens. a remarkable number of species of trematoda infecting amphibians is reported (e.g., http://www.colorado. edu/eeb/facultysites/pieter/parasite_database/trematodes.html). trematoda cause harmful infections in fish (kim and nagasawa, 1996; lane and morris, 2000) and reduced growth and poor body condition, limb malformation and even death in amphibians (hsu et al., 2004; johnson and mckenzie, 2008). we have studied the infection of the digenetic trematode clinostomum complanatum rudolphi, 1814 at the expense of a population of triturus carnifex (laurenti, 1768). clinostomum infect snails as first intermediate hosts and fish or amphibians as second intermediate ones. in amphibians the infection usually consists of dermal cysts (sutherland, 2005) which contain metacercariae (yellow grub). the final host is represented by fisheating birds where metacercariae mature in and eggs are released via bird feces or from their beak. clinostomum species have been found to have infected several species of amphibians (reviewed in caffara et al., 2014) resulting in pathological changes (miller et al., 2004). reports of amphibians infected by clinostomum were from north america only, until caffara et al. (2014) found a site in central italy where lissotriton vul76 giacomo bruni, claudio angelini garis and t. carnifex were infected by c. complanatum. the present epidemiological study mainly focuses on the prevalence of the yellow grub infection and its effects on body condition and survival in this italian crested newt population. we studied two sites in tuscany, central italy. the site where newts are infected by yellow grubs (already reported in caffara et al., 2014; from this point forward it will be coded as sf) is an artificial pond built in 2008, 35 m a.s.l., surface 256 m2 and maximum depth 2 m, inside the sci-spa it51140011, in sesto fiorentino municipality. as a control site, we studied a natural pool (coded as an) in a xerophytic wood, 655 m a.s.l., surface 53 m2 and maximum depth 1 m, in anghiari municipality. both sites host l. vulgaris and t. carnifex; no potential predators like fish or crayfish have been found inside; water level remains almost constant all year round in both sites. the italian crested newts were sampled by dip-netting from march 2012 to march 2013 at sf (16 capture occasions, one every 26.7 days on average), and from july 2012 to may 2013 at an (seven capture occasions, one every 53 days on average); capture sessions lasted about two hours. at sf two funnel traps were also placed in the pond for the whole night before the dip-netting session. newts were marked by photographing their ventral spot pattern, they were sexed, and most of them were measured (snout-vent length, svl henceforth; weight was not recorded since we supposed it would have varied during the study period); the life stage (paedomorphic or metamorphosed adult) has also been recorded. paedomorphic individuals were discriminated from larvae on the basis of cloaca shape and by the presence of secondary sexual characteristics. the metacercariae of clinostomum sp. show a predilection for the skin (sutherland, 2005), causing evident cysts (gray et al., 2007), thus they are easy to be found by visual inspection. cysts similar to clinostomum ones may be caused by mesomycetozoan organisms, such as amphibiocystidium, but they can be distinguished by a trained observer (see also raffel et al., 2008). we also inspected the inner mouth by using a kimura spatula. the number and location of cysts of each single newt have been recorded. newts were immediately released after this in-field procedure. the data we collected at sf has been used: i) to evaluate the association between infection (infected/ not-infected newts), life stage (paedomorphic/metamorphosed) and sex (log-linear analysis, delta = 0.5, 100 maximum iterations, convergence criterion = 0.01); ii) to assess if the cysts were randomly distributed on the whole body or if they were clustered (chi-square test); iii) to evaluate if the infection per se affected the body size (for the ancova the covariate was the interaction term between sex and life stage) or if the number of metacercariae did (spearman correlation). parametric statistics were applied when the data met normality assumption and when appropriate; the software statistica 7 (statsoft inc.) has been used for statistical analyses. capture-mark-recapture (cmr) data were used to evaluate if the infection affected the individual survival. estimates of survival probability were obtained via the program mark (white and burnham, 1999) under the cormack-jolly-seber (cjs) model. for sf we considered 334 newts for an amount of 567 captures. basing on this dataset, we built 24 models, considering the following parametrizations for survival: constant [phi(.)], time dependent [phi(t)], dependency on life stage [phi(.*l)], interaction between time and life stage [phi(t*l)], interaction between life stage and sex [phi(.*l*s)], interaction between time, life stage and sex [phi(t*l*s)]; and the following parametrizations for capture probability: constant [p(.)], time dependent [p(t)], dependency on life stage [p(.*l)], interaction between time and life stage [p(t*l)]; further, the same 24 models were run including the number of parasites as individual covariate for survival, thus obtaining an amount of 48 models at sf. for the sake of comparison, a cmr study was also carried out at an (141 newts were captured, 230 captures). no newts in this site showed visible lesions consistent with c. complanatum, and only 3 paedomorphic newts were captured (they have been removed from further analyses). consequently, we ran eight cmr models under cjs model, considering survival as phi(.), phi(t), phi(.*s) or phi(t*s), and capture probability as p(.) or p(t). before the analyses, a closure test confirmed that the populations were open [stanley and burnham (1999) closure test: sf: chi-square = 420.04, d.f. = 25, p < 0.001; an: chi-square = 205.57, d.f. = 13, p < 0.001]. program u-care (choquet et al., 2005) was used to evaluate the goodness of fit of the cjs model to both data-sets. cjs model fits the data collected at sf (chi-square = 115.43, d.f. = 134, p = 0.87), but not at an (chi-square = 27.84, d.f. = 16, p < 0.05) due to trap-happiness of females. however, since the resulting c-hat was quite low (1.73), we only corrected for overdispersion at an by adjusting the c-hat in the program mark. consequently, the evaluation of the best model is based on aicc for sf, and on qaicc for an. out of 340 newts captured at sf, 98 were infected (28.8%; no individual out of 141 from an was infected), with one to eight yellow grubs. log-linear analysis returned as the model that best fits the data the association sex×life stage and life stage×infected/not-infected (chi-square = 0.68, d.f. = 2, p = 0.71). a significantly 77no short term effect of clinostomum complanatum on survival of triturus carnifex larger proportion of paedomorphic newts were infected (29.5%) in comparison with metamorphosed newts (23.5%) (chi-square = 7, d.f. = 1, p < 0.01). forty-one infected newts were captured more than once. the number of parasites changed in 22 newts without any apparent time pattern. two individuals that became infected and four that lost their only parasite were removed from further analyses. cysts were found in the head (86 cysts in 57 newts), into the mouth (51 in 41 newts), in the trunk (7 in 7 newts), limbs (5 in 3 newts) and tail (10 in 8 newts) (chisquare = 163.46, d.f. = 4, p < 0.001). the average number of parasites per individuals (1.74 ± 0.12) was not correlated with svl (rs = -0.11, p = 0.3, n = 82). body size of infected newts did not differ from the size of not-infected newts (f1,297 = 0.59, p = 0.44), even when taking into account life stage and sex (ancova, f1,294 = 1.92, p = 0.16; a two-way anova revealed significant differences between life stages, sexes and their interaction). model fitting for survival analysis reported phi(t) p(.*l) as the best model for sf, with an aicc weight of 0.998. consequently, none of the models including the number of parasites as covariate got support. survival estimates from the best model are reported in fig. 1. the best model for an, phi(t)p(.), did not have a strong support, with a model weight equal to 0.59; the sixth ranked model had a δqaicc equal to 5.95 and model weight 0.03, the seventh model had weight < 0.001 and δqaicc equals to 13.52, thus estimates of survival at an in fig. 1 are based on model averaging (survival differences among sexes got a global weight of 0.13, meaning numerical difference at the third decimal number; for convenience, the figure reports only female survival, which was the lower one). our findings show that clinostomum infection neither caused higher mortality rate nor affected body size of t. carnifex. in the infected sf population survival did not differ between infected and not-infected newts, and it was very similar to survival of the non-infected an population (during the time period the two studies overlapped). usually, infectious diseases are studied at individual level, while few studies evaluated the effects of an infection on population demography (woodhams et al., 2011). we showed that during a short time period yellow grubs infection did not affect adult newts survival. sf population had been continuously monitored since 2008, and the first record of c. complanatum dates back to october 2010. clinostomum metacercariae can survive in the host for about 4 years (elliot and russert, 1949), consequently any possible effect on survival could have been evident at the time of the study (march 2012-march 2013). therefore, although the data were collected in a short time, our findings may be considered substantial. the abundant presence of metacercariae on the head and into the mouth may be linked to the exposure of fig. 1. survival probabilities at sf (squares) and an (circles) sites, with 95% c.i. 78 giacomo bruni, claudio angelini these regions while feeding. in fact the diet of t. carnifex includes freshwater molluscs (fasola and canova, 1992), that are intermediate host of clinostomum. the cephalic region could also be selected by the parasite as the location with the main organs of the newts, where the parasite can cause damage and inhibit some vital functions (miller et al., 2004), likely making it easier for the birds to prey on newts. the success of the infestation by trematodes is strongly influenced by environmental and ecological characteristics, including abundance of intermediate hosts (e.g., johnson and chase, 2004) and the presence of seasonal definitive hosts. sf pond is surrounded by wetlands where bird species, like ardea cinerea, a. purpurea, a. alba, ardeola ralloides, nycticorax nycticorax and egretta garzetta, which can host adults of c. complanatum (al-salim and ali, 2010; shamsi et al., 2013) have been frequently observed. in the pond there are also various species of freshwater molluscs. thus, all the hosts required for the life cycle of c. complanatum to be completed are present. on the contrary, an is a small, isolated basin in a wooded area, where we did not observe any waterfowls. however, it is also important to take into account that occurrence of helmint infections in amphibians may indicate an environmental stress or a disequilibrium in the trophic web (johnson and lunde, 2005; green et al., 2009). environmental modifications, such as water pollution by pesticides and introduction of allochthonous species might foster the parasitosis emergence (daszak et al., 2000) and modify the natural dynamics between host and parasite (rohr et al., 2008; poulin, 2010). indeed, the area surrounding sf are heavily influenced by agriculture and infested by allochthonous species like procambarus clarkii, gambusia holbrooki, pseudorasbora parva, trachemys scripta and myocastor coypus. on the contrary, the wooded area surrounding an could preserve it from environmental modifications. a larger proportion of paedomorphic newts were infected compared to metamorphic ones. this suggests that the probability of infection is related to the time spent in water by the newts. metamorphic newts spent part of the year on land (vanni et al., 2007), therefore they are less exposed to the parasite. on the contrary, the aquatic larval and paedomorphic stages are prone to be infected. during our surveys we did not search deliberately for larvae, and the few we captured apparently were not infected. even if further monitoring is required to analyse the long term survival, basing on our findings adult survival does not seem lowered by yellow grubs. however, it is important to assess if clinostomum can represent a threat for amphibians by acting on larvae, something that can be achieved by including the recruitment rate in the study of populations’ dynamic. acknowledgements we are grateful to elia serafini for let us know the site of anghiari and to monica caffara and giulia ricciardi for the critical reading of the paper. italian ministry of environment, land and sea permit no 0019536 10/09/2012 pnm-ii and 0029422 23/10/2012 – pnm-ii. references al-salim, n.k., ali, a.h. 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(2011): mitigating amphibian disease: strategies to maintain wild populations and control chytridiomycosis. front. zool. 8: 8. acta herpetologica vol. 11, n. 1 june 2016 firenze university press feeding habits of mesoclemmys vanderhaegei (testudines: chelidae) elizângela silva brito1,*, franco leandro souza2, christine strüssmann3 morphology, ecology, and behaviour of hylarana intermedia, a western ghats frog ambika kamath1, rachakonda sreekar2,3 a new account for the endangered cerrado rocket frog allobates goianus (bokermann, 1975) (anura: aromobatidae), with comments on taxonomy and conservation thiago ribeiro de carvalho1,2,*, lucas borges martins1,2, ariovaldo antonio giaretta1 molecular phylogenetics of the pristimantis lacrimosus species group (anura: craugastoridae) with the description of a new species from colombia mauricio rivera-correa, juan m. daza* population structure and activity pattern of one species of adenomera steindachner, 1867 (anura: leptodactylidae) in northeastern brazil maria juliana borges-leite1,*, joão fabrício mota rodrigues2, patrícia de menezes gondim1, diva maria borges-nojosa1 vocal repertoire of scinax v-signatus (lutz 1968) (anura, hylidae) and comments on bioacoustical synapomorphies for scinax perpusillus species group marco antônio peixoto1,*, carla silva guimarães1, joão victor a. lacerda2, fernando leal2, pedro c. rocha2, renato neves feio1 amendment of the type locality of the endemic sicilian pond turtle emys trinacris fritz et al. 2005, with some notes on the highest altitude reached by the species (testudines, emydidae) federico marrone*, francesco sacco, vincenzo arizza, marco arculeo photo-identification in amphibian studies: a test of i3s pattern marco sannolo1,*, francesca gatti2, marco mangiacotti3,4, stefano scali3, roberto sacchi4 no evidence for the ‘expensive-tissue hypothesis’ in the dark-spotted frog, pelophylax nigromaculatus li zhao, min mao, wen bo liao* no short term effect of clinostomum complanatum (trematoda: digenea: clinostomatidae) on survival of triturus carnifex (amphibia: urodela: salamandridae) giacomo bruni1,*, claudio angelini2 yeasts in amphibians are common: isolation and the first molecular characterization from thailand srisupaph poonlaphdecha, alexis ribas* introduction of eleutherodactylus planirostris (amphibia, anura, eleutherodactylidae) to hong kong wing ho lee1, michael wai-neng lau2, anthony lau3, ding-qi rao4, yik-hei sung5,* correlation between endoscopic sex determination and gonad histology in pond sliders, trachemys scripta (reptilia: testudines: emydidae) david perpiñán1, albert martínez-silvestre2,3, ferran bargalló3, marco di giuseppe4, jorge orós5, taiana costa6,* book review: john w. wilkinson. amphibian survey and monitoring handbook. pelagic publishing franco andreone book review: susan newman. frogs amphibians and their threatened environment. discovery and expression through art k-3. frogs are green franco andreone acta herpetologica 10(2): 93-101, 2015 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-16150 helminth parasites in the toad rhinella major (bufonidae) from chaco region, argentina monika i. hamann*, cynthya e. gonzález centro de ecología aplicada del litoral (cecoal), consejo nacional de investigaciones científicas y técnicas (conicet), ruta 5, km 2.5, w 3400 amd corrientes, argentina. *corresponding author. e-mail: monika_hamann@yahoo.com submitted on 2015, 27th april; revised on 2015, 12th september; accepted on 2015, 9th october editor: rocco tiberti abstract. the present study describes the diversity of helminth parasites of rhinella major (anura: bufonidae) in relation to their body size in 4 subhumid vs. semiarid sampling sites from the argentine chaco region. helminths were found in 81% of the specimens examined (n = 85). fifteen species (13 in subhumid and 7 in semiarid areas) of helminth parasites were found, and most of them were nematodes. parasites were found in all the examined organs, with highest prevalence and intensity in the digestive tract. parasite transmission to the toad host occurs by skin penetration or oral ingestion. maximum helminth richness ranged between 2 and 4 species per infected toad. the most abundant species was aplectana hylambatis. body size of the host was the main factor in determining parasite richness. the helminth parasite fauna was rather different in hosts from subhumid vs. semiarid sites, but the dominant (aplectana hylambatis) and codominant species (cylindrotaenia sp. and rhabdias elegans) were the same. mean species richness and mean species diversity of helminths were significantly different between the zones. these results suggest that the amount of rainfall and associated humidity affects the distribution and development of the parasite fauna of this toad. keywords. parasitic assembly, helminth community, habitat use, rhinella major, chaco region, argentina. introduction the small toad rhinella major (müller and hellmich, 1936) is a member of the toad family bufonidae, which can be found in south america. its distribution in argentina includes the provinces of chaco, formosa, salta, jujuy, santiago del estero, santa fe, and tucumán (frost, 2014), where it is able to withstand extremely dry conditions that predominate most of the year. r. major is a nocturnal species, living and feeding almost exclusively in terrestrial habitats. its diet is neither generalised nor specialised, with predominance of formicids (i.e. insectivore) (duré et al., 2009). only the systematics of its helminth parasites have been reported (see campião et al., 2014), whereas the respective host-parasite interactions are still poorly understood. the diversity of helminth parasite species in amphibian hosts is often correlated with the habitat inhabited by the latter, e.g., terrestrial amphibian tend to host more nematode parasites with direct life cycles (luque et al., 2005; yoder and coggins, 2007; ibrahim, 2008; santos and amato, 2010, 2013; hamann et al., 2006a, 2012, 2013a), while aquatic/semiaquatic amphibian are especially infected by trematode parasites, many of which have complex life cycles that require several hosts and may take place entirely within the aquatic habitat (wetzel and esch, 1997; mcalpine and burt, 1998; zelmer et al., 1999; muzzall et al., 2001; hamann et al., 2006b, 2013b; campião et al., 2012). such ecological factors may affect the likelihood of colonization by larval parasites in the intermediate hosts and among the vertebrate definitive hosts (thieltges et al., 2008). the present study of the toad r. major was performed in two areas from the argentine chaco region. one zone is subhumid with longer humid periods, and characterized by dense forests interspersed with open 94 monika i. hamann, cynthya e. gonzález floodplain grassland, while the second zone is semiarid, with a shorter and relatively unstable humid period, and dominated by sparser, lower and less diverse forests combined with spiny shrubs. in this context, our aim is to describe the helminth fauna infecting r. major in habitats contrasting for their climatic features (semiarid vs. subhumid habitats), and compare the level of parasitic infection of differently sized toads. material and methods study sites toad populations were captured in four widely separated locations of the chaco region of argentina between october, 2011 and november, 2012. these locations span two different zones: subhumid zone (two localities: concepción del bermejo: 26°36’s, 60°57’w and las lomitas: 24°42’s, 60°35’w) and semiarid zone (two localities: taco pozo: 25°36’s, 63°15’w and ingeniero juárez: 23°54’s, 61°51’w) (fig. 1). compared to the semiarid sampling sites, the subhumid zone is characterized by a more dense vegetation with hardwood trees (e.g., schinopsis balansae and tabebuia avellanedae), secondary woody vegetation (e.g., zizyphus mistol, acacia caven, bumelia obtusifolia and acanthosyris falcata), and herbaceous species comprising patches of grasses (e.g., elionurus muticus) and terrestrial bromeliads (e.g., aechmea distichantha and bromelia serra). the climate of this zone is characterized by high temperatures and dry north winds in the months of august to november, whereas rainfall is often higher from december to march (table 1). during the rainy season rivers overflow favours the development of numerous marshes, swamps and lagoons that occupy former riverbeds and meanders. in the semiarid zone, the forest is adapted to dry conditions (xerophytic deciduous forest), with predominance of smallleaved deciduous and thorny species adapted to fluctuations in water availability, as well as to seasonal thermal variations. woody vegetation (e.g., bulnesia sarmientoi, prosopis ruscifoli, stetsonia coryne and trithinax biflabellata) is sparse, and the herbaceous species are predominantly grasses (trichloris sp., gouinia sp. and setaria argentina). the landscape is flat, gently sloping toward the east. the climate is characterized by low rainfall (mean anual about <700 mm), and high temperatures, at times exceeding 47 ºc since this area comprises part of the south american heat pole (naumann, 2006). table 1 presents the values of precipitation and temperature at the study localities. collection and examination of toads and parasites samples of r. major were collected in subhumid (n = 61) and semiarid (n = 24) zones (table 1). toads were transported alive to the laboratory and then euthanized in chloroform (chcl3) solution. their snout-vent length (svl) was recorded. at necropsy, hosts were sexed and the esophagus, stomach, gut, lungs, liver, urinary bladder, kidneys, body cavity, musculature, skin, and brain examined for parasites. helminths were observed in vivo, counted, and killed in hot distilled water and preserved in 70% ethanol. for identification, the digeneans, and cestodes were stained with hydrochloric carmine, cleared in creosote, and mounted in canada balsam. nematodes were cleared in glycerine or lactophenol, and examined as temporary mounts. helminths were deposited in the helminthological collection of centro de ecología aplicada del litoral (cecoal) consejo nacional de investigaciones científicas y técnicas (conicet), corrientes city, corrientes, argentina [accession numbers cecoal, 11101902, haematoloechus longiplexus; 12110819, travtrema sp.; 11100008, strigeidae gen. sp. 1; 12110803, strigeidae gen. sp. 6; 12110820, cylindrotaenia sp.; 12110817, mesocestoides sp.; 12110816, cosmocerca podicipinus; 12110823, aplectana hylambatis; 11101906, oswaldocruzia mazzai; 12110825, schulzia travassosi; 11101902, rhabdias elegans; 12110813, r. pseudosphaerocephala; 12110814, porrocaecum sp.; 11100003, physaloptera sp.; 12110827, ascarididae gen. sp.]. a sample of toad was deposited in the herpetology collection of cecoal-conicet, with accession number lhc, 5081. statistical analysis helminth fauna description. prevalence, intensity, and abundance were calculated according to bush et al. (1997). helminth communities have been classified at the infracommunity level (all helminth populations within a single r. major), component community level (all helminth infracommunities within a population of the r. major) and parasite fauna (all the parasite species that exploit a given host across its geographic range) fig. 1. map showing the location of the study area and sampling sites in the subhumid (las lomitas and concepción del bermejo) and semiarid (ingeniero juárez and taco pozo) zones. 95helminth parasites in rhinella major (bush et al., 1997; poulin, 1997). the measures of community richness and diversity employed included: the total number of helminth species (richness), mean richness (the sum of helminth species, per individual toads, divided by the total sample size), shannon index (h´) (shannon and weaver, 1949), evenness (j’) as h´/h´ max (pielou, 1966; zar, 2010). the bergerparker index of dominance (d) was used to determine the most abundant species (magurran, 2004). helminth fauna in semiarid vs. subhumid habitats. sorensen similarity qualitative index was used for comparing the degree of similarity in helminth communities between habitats. the statistic z was used to compare 2 proportions (prevalence) of helminth infection. a t-test was used to test for significant difference in the log10 helminth diversity (hutcheson, 1970) between zones. the importance of each parasite in the community was examined according to the methodology outlined by thul et al. (1985), in which helminth species are classified into four groups (dominant, codominant, subordinate and unsuccessful pioneer species) by taking into account their prevalence, intensity, and maturity factor (equal to 1.0 if at least 1 mature specimen of species j is found and equal to 0 otherwise), which is related to the degree of host specificity. since the samples sizes were different in semiarid vs. subhumid habitats, we used rarefaction methods (implemented in the software ecosim 7.7; gotelli and entsminger, 2004) for comparing the mean diversity and the mean richness of the helminth species. rarefaction uses probability theory to derive expressions for the expectation and variance of species diversity and richness for a sample of a given size. this method “rarefies” its samples down to a common abundance level and then compares species diversity and richness. the process was repeated 1,000 times to generate a mean and a variance of species diversity and richness. wilcoxon’s v-test was used to compare differences in species richness between semiarid and subhumid habitats. helminth fauna and r. major size. spearman’s rank test (rs) and pearson’s coefficient correlation (r) were used to indicate the relationship between host body size and helminth species richness. all statistical analyses were performed using xlstat 7.5 (addinsof, 2004) and mvsp (kovach, 1999). results general patterns fifteen helminth species were found in the 85 r. major individuals collected in the chaco region. the predominant groups of parasites were nematodes (9 species), followed by trematodes (4 species) and cestodes (2 species). parasites were found in all major organs, with highest prevalence and intensity in the small intestine (anterior and middle portion of intestine), large intestine and stomach (table 2). however, no parasites were found in the muscles. eight species were found at the adult stage, and for these r. major is the definitive host; 7 species were at a larval stage and infect r. major as intermediate or paratenic hosts. parasite transmission to the toad host occurs by skin penetration (60% of the species) or oral ingestion (40% of the species) (table 2). comparison of the parasite fauna in semiarid vs. subhumid habitats the relative importance of species within the parasite community was as follows: a. hylambatis was strongly characteristic of the community in both habitat types (table 3). seven codominant species contributed to a table 1. variables recorded in rhinella major and climatic data of two zones from the chaco region, argentina. subhumid zone semiarid zone concepción del bermejo summer 2012 las lomitas spring 2012 taco pozo spring 2011 ingeniero juárez spring 2011 collected toads number of toads 31 30 10 14 males/females 15/16 26/4 8/2 7/7 mean length (mm) 44.68 57.23 51.60 39.48 min-max length (mm) 37-61 48-68 42-60 32-47 precipitations (mm) 1 mean anual 937 919 620 689 total annual 649 737 456 *805 total season 169 76 223 45 temperature (°c) 2 mean annual 20 to 22 22 to 24 20 to 22 22 to 24 1data from rohrmann h. (pers. comm.) and data from gómez et al. (2013). 2data from naumann (2006).*exceptional situation. 96 monika i. hamann, cynthya e. gonzález lesser degree in the helminth community, and two of them (cylindrotaenia sp. and r. elegans) were common to the semiarid and subhumid zones. two species were uncommon or rare in the community (e.g., h. longiplexus). seven that were able to enter the host but not to reach maturity contributed little to the community and are characteristic of different hosts (e.g., physaloptera sp.). in both zones, helminths exhibited a low diversity with a less equitable distribution (tabla 4). the most abundant species (d) was a. hylambatis. there were 5256 individuals of diverse helminth species in the subhumid zone, while 646 individuals were found in the semiarid zone. the value of sorensen qualitative similarity index was low (40%). infection prevalence showed no significant differences between zones (z = -0.94; df = 1; p > 0.05). using a rarefaction method, the number of individuals was combined and the values of mean richness and mean diversity for each of the zones were obtained (table 5). mean species richness of helminth parasites differed between the two habitats, i.e., it was greater in the subhumid zone (wilcoxon v-test = 85; p = 0.003; n1 = 13; n2 = 13). mean species diversity of helminth parasites also differed between the two habitats, being higher in the semiarid zone (wilcoxon v-test = 0; p = 0.0002; n1 = 13; n2 = 13). in both habitats, the helminth parasites were similar in a set of 4 species (a. hylambatis, physaloptera sp., r. elegans and strigeidae gen. sp. 6). diversity of these helminth parasites showed significant difference between zones (t-test = -1.983; v = 732.19; p < 0.05); the subhumid zone (h’ = 0.030) showed greater inequality of species abundance compared to the semiarid zone (h’ = 0.054). helminth species richness related to host body size of the 85 toads examined from the two zones, infection prevalence was 81%, and significant positive correlation was found between toad body length and helminth species richness (r = 0.43; n = 69; p < 0.0001). toads from the subhumid zone showed high infection prevalence (79%), and parasite species richness was significantly correlated with body length of the host (rs = 0.46; n = 48; p = 0.001). toads from the semiarid zone presented even higher infection prevalence (88%), and also significant correlation between parasite species richness and body length of the host (rs = 0.59; n = 21; p < 0.01). table 2. helminths recorded in rhinella major from the chaco region, argentina. site of infection: si-small intestine, li-large intestine, pzpharyngeal zone, gb-gall bladder, m-mesentery, gm-gastric mucosa, lu-lung; stage (s): adult (a) and larval (l); mode of transmission of parasites (t): oral ingestion (i), skin penetration (p); prevalence: %; mean intensity: mi ± sd; total number: #; prevalence in chaco region: cr. helminths and site of infection s t subhumid zone semiarid zone cr las lomitas concepción bermejo ingeniero juárez taco pozo % mi ± sd # % mi ± sd # % mi ± sd # % mi ± sd # % trematoda haematoloechus longiplexus lu a i 10 1.0 1 1 travtrema sp. m l p 3 1.0 ± 0.0 1 1 strigeidae gen. sp.1 m l p 3 3.0 ± 0.0 3 1 strigeidae gen. sp. 6 m l p 3 9.0 ± 0.0 9 7 1.0 ± 0.0 1 2 cestoda cylindrotaenia sp. si a i 17 5.2 ± 1.5 26 70 2.1 ± 1.4 15 14 mesocestoides sp. m l i 10 1.3 ± 0.5 4 4 nematoda cosmocerca podicipinus li/ si a p 23 6.0 ± 5.5 42 8 aplectana hylambatis li/si a i/p 97 142.7 ± 77.0 4139 55 54.9 ± 55.5 934 86 17.0 ± 21.2 204 90 45.6 ± 38.2 410 79 oswaldocruzia mazzai si a p 10 2.0 ± 0.0 2 1 schulzia travassosi si/ gb a p 10 6.0 ± 4.1 18 3 23.0 ± 0.0 23 5 rhabdias elegans lu a p 7 3.5 ± 0.5 7 30 3.0 ± 2.2 9 6 r. pseudosphaerocephala lu a p 17 1.6 ± 0.5 8 6 porrocaecum sp. pz l i 3 1.0 ± 0.0 1 1 physaloptera sp. gm l i 30 4.3 ± 3.3 39 3 1.0 ± 0.0 1 21 1.30 ± 0.5 4 15 ascarididae gen. sp. si l i 3 1.0 ± 0.0 1 1 97helminth parasites in rhinella major discussion in general, the results of the present study indicate that the helminth fauna (both adults and larvae) of r. major is relatively rich, with a composition similar to those previously described for other bufonid toads (rhinella fernandezae and melanophryniscus klappenbachi) from the chaco region of argentina (hamann et table 3. classification of helminth species in rhinella major from the chaco region, argentina. dominant species (i ≥ 1.0), codominant species (0.01 ≤ i < 1.0), subordinate species (0 < i < 0.01), unsuccessful pioneer species (i = 0). combined data (c). classification of helminth* subhumid zone semiarid zone las lomitas conc. del bermejo c ingeniero juárez taco pozo c dominant species aplectana hylambatis 99.262 99.742 99.544 99.472 96.471 98.865 cylindrotaenia sp. 2.745 codominant species cylindrotaenia sp. 0.108 0.055 0.805 h.longiplexus 0.026 cosmocerca podicipinus 0.243 0.125 schulzia travassosi 0.045 0.144 0.070 oswaldocruzia mazzai 0.052 0.015 rhabdias elegans 0.088 0.706 0.207 r. pseudosphaerocephala 0.033 0.017 subordinate species rhabdias elegans 0.006 h. longiplexus 0.008 unssucesfull pioneer species porrocaecum sp. 0.000 0.000 physaloptera sp. 0.000 0.000 0.000 0.000 0.000 ascarididae gen. sp. 0.000 0.000 travtrema sp. 0.000 0.000 strigeidae gen. sp. 1 0.000 0.000 strigeidae gen. sp. 6 0.000 0.000 0.000 0.000 mesocestoides sp. 0.000 0.000 *according to the methodology from thul et al. (1985) table 4. summary of ecological indices of helminth communities in rhinella major from chaco region, argentina. combined data (c). variables subhumid zone semiarid zone las lomitas conc. del bermejo c ingeniero juárez taco pozo c component community richness 11 5 13 3 5 7 diversity (h’) 0.09 0.08 0.10 0.05 0.13 0.12 evenness (j) 0.09 0.11 0.09 0.11 0.18 0.14 dominance (d) 0.96 0.96 0.95 0.99 0.94 0.97 identification of (d) aplectana hylambatis aplectana hylambatis infracommunity range richness 1 4 0 2 0 4 0 2 0 4 0 4 mean richness 2.17 ± 0.93 1.22 ± 0.42 1.81 ± 0.91 1.33 ± 0.47 2.33 ± 0.94 1.76 ± 0.89 mean diversity (hb) 0.06 ± 0.08 0.02 ± 0.07 0.05 ± 0.08 0.06 ± 0.10 0.13 ± 0.11 0.08 ± 0.10 mean evenness (e) 0.17 ± 0.24 0.08 ± 0.22 0.13 ± 0.24 0.20 ± 0.33 0.39 ± 0.37 0.28 ± 0.37 98 monika i. hamann, cynthya e. gonzález al., 2013a, 2014). the helminthological fauna of r. major is dominated by nematode parasites. this pattern is also observed in other bufonid toads from temperate region (bolek and coggins, 2003; yoder and coggins, 2007) and tropical region (bursey et al., 2001; goldberg and bursey, 2003; luque et al., 2005; ibrahim, 2008; santos and amato, 2010, 2013; hamann et al., 2013a), in which the helminth community consisted mainly of nematodes. an exception is the study by hamann et al. (2014) who found that nematode species show low infection levels in the toad m. klappenbachi; these results were related essentially to the thicker integument of this particular host, as well as by the presence of alkaloids in the skin glands that could avoid the parasitic attack. the species a. hylambatis was the dominant parasite in r. major. this had also been observed by hamann et al. (2012) for leptodactylus bufonius (leptodactylidae) from corrientes province in argentina, a frog with a more terrestrial habit, whose microhabitat preference significantly contributed to the high infection by a. hylambatis. likewise, in other reports for rhinella fernandezae (bufonidae) from brazil (santos and amato, 2010), and for the toad amietophrynus regularis (bufonidae) from egypt (ibrahim, 2008) the parasitic community was dominated by the genus aplectana (cosmocercidae). in particular, the fact that a. hylambatis -a nematode with direct life cyclepredominates in r. major could indicate a relationship with the mainly terrestrial habits of this amphibian. infection by aquatic metacercariae (travtrema sp., strigeidae gen. sp. 1 and strigeidae gen. sp. 6) showed low richness, reflecting the fact that r. major spends only little time in the water. thus, the present study confirms that the parasite communities of r. major exhibit the general characteristics of a terrestrial amphibian, coinciding that terrestrial amphibians contain greater proportion of nematode parasites (bursey et al., 2001; goldberg and bursey, 2003; bolek and coggins, 2003; luque et al., 2005; yoder and coggins, 2007i; ibrahim, 2008; santos and amato, 2010, 2013; hamann et al., 2013a). the infections recorded in r. major show that this anuran species represents the definitive host for more than 50% of the species found parasitizing them; apart from this, this toad had a minor role as paratenic and/ or intermediate host, reflected by the presence of larvae at different infection sites, e.g. infection by larvae of physaloptera sp., which persist in the gastric mucosa for varying periods of time without reaching the adult stage (see anderson, 2000), or by larvae of mesocestoides sp. which remain encapsulated in the mesenteries (see prudhoe and bray, 1982). these infections occur when toads eat invertebrates (e.g., insects and oribatid mites), and subsequently the ingested larval forms of these parasites culminate their biological life cycles in some birds and mammals (e.g., by predation of r. major by fox; see schalk and morales, 2012). many of the helminth species recorded in this toad were generalists, given that its presence has been reported in amphibian species belonging to other families in our region (see campião et al., 2014; hamann et al., 2014). proportionately, each zone had a similar number of dominant and codominant helminth species in the community, and the three species (a. hylambatis, cylindrotaenia sp. and r. elegans) shared by toads from both table 5. summary of main results of the rarefaction method. the number of individuals (n) was unified to obtain values of mean diversity (md) and mean richness (mr) 1 ± sd for subhumid and semiarid zones. n subhumid zone semiarid zone md ± sd mr ± sd md ± sd mr ± sd 50 0.16 ± 0.12 2.47 ± 1.03 0.22 ± 0.12 2.78 ± 0.95 100 0.18 ± 0.09 3.70 ± 1.23 0.24 ± 0.08 3.76 ± 0.95 150 0.19 ± 0.08 4.51 ± 1.26 0.24 ± 0.07 4.39 ± 0.96 200 0.20 ± 0.06 5.27 ± 1.28 0.25 ± 0.05 4.81 ± 0.92 250 0.20 ± 0.05 5.68 ± 1.27 0.26 ± 0.04 5.28 ± 0.92 300 0.20 ± 0.05 6.21 ± 1.25 0.26 ± 0.04 5.56 ± 0.87 350 0.20 ± 0.05 6.55 ± 1.27 0.26 ± 0.03 5.81 ± 0.84 400 0.20 ± 0.04 6.90 ± 1.25 0.26 ± 0.03 6.05 ± 0.76 450 0.20 ± 0.04 7.11 ± 1.21 0.26 ± 0.03 6.28 ± 0.71 500 0.21 ± 0.04 7.39 ± 1.18 0.26 ± 0.02 6.50 ± 0.61 550 0.21 ± 0.04 7.59 ± 1.22 0.26 ± 0.02 6.68 ± 0.52 600 0.21 ± 0.03 7.83 ± 1.22 0.26 ± 0.01 6.84 ± 0.39 650 0.21 ± 0.03 8.00 ± 1.22 0.26 ± 0.00 7.00 ± 0.00 99helminth parasites in rhinella major zones were categorized the same way (table 3); i.e., the relative importance of these species was similar in both habitats. nevertheless, more larval species were present in toads from the subhumid zone. this difference may be influenced in part by low water availability in the semiarid zone given by shorter humid periods. thus, because of this factor some invertebrates that act as intermediate hosts are not active, and therefore the development and survival of infective stages does not progress to allow completion of the parasite life cycle in this zone. in the same way, soil with high contents of sand combined with scarce humidity can also affect the survivorship and transmission of infective nematode larvae (see pizzatto et al., 2013). on the other hand, the dominant species (a. hylambatis) confers a relatively high level of predictability regarding the parasite communities of this toad, such that any sample of r. major from a single locality is likely to include this common helminth. this cosmocercid species that reaches high local prevalence also tends to have a wide geographical range (see poulin, 2007). finally, the helminthological composition showed little similarity between the sampling sites, and this result clearly highlights the important role of local biotic and abiotic factors in determining the proportion of infected toads in a population and the number of helminth species infecting them. available information suggests that parasite species richness increases with host size. for instance, several authors in north america (bolek and coggins, 2003; yoder and coggings, 2007) have found a positive relationship between species richness and size of anaxyrus americanus (bufonidae), and ibrahim (2008) in egypt has shown that host age of a. regularis played a significant role in determining community species richness. the results of the present study also indicate that host size plays an important role in parasitic infections, in agreement with the results of previous analyses of argentine amphibians (hamann et al., 2010, 2013a, 2014); these results suggest that larger (i.e., older) toads have been exposed to parasites for a longer time and also expose a larger surface area for parasitic attack. in the present study, this is supported by the fact that in the subhumid zone the amphibians present larger body sizes with higher infection level. in conclusion, this study revealed that the size and habitat of r. major influence its helminth infections. in particular, our results suggest that climate (e.g. amount of rainfall and soil moisture) is an important factor affecting the distribution and development of the parasite fauna of r. major. acknowledgments we thank ing. rohrmann, h. of office of the provincial water administration (apa) of the chaco province, argentina who provided information and documents necessary to our work. we also thank dr. cuevas, m.a. of the office of the secretary of natural resources, department of fauna and protected areas of the production ministry of chaco province, argentina, and dr. bray, h.d of the secretary of natural resources and environmental quality of the production and environment ministry of formosa province, argentina to authorize the capture of the toads for this research. financial support was provided by the consejo nacional de investigaciones científicas y técnicas (conicet) of argentina, through grant pip 11220090100345. references addinsoft. 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(1999): the role of habitat in structuring halipegus occidualis metapopulations in the green frog. j. parasitol. 85: 19-24. acta herpetologica vol. 10, n. 1 june 2015 firenze university press acta herpetologica journal of the societas herpetologica italica acta herpetologica 10(2): 155-158, 2015 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-16457 testes asymmetry of bufo gargarizans in relation to body condition and age tong lei yu1, yan shu guo2 1 college of life science, xinyang normal university, sd 464000, henan, china. corresponding author. e-mail: yutonglei_00000@163. com 2 college of life sciences, china west normal university, sd 637000, sichuan, china. submitted on 2015, 29th july; revised on 2015, 24th october; accepted on 2015, 28th october. editor: gentile francesco ficetola abstract. testes asymmetry theory predicts that males in good condition or mature age may be able to develop larger degrees of directional asymmetry than males in poor condition or younger age. in this study, we tested the testes asymmetry theory in the bufo gargarizans. we found that b. gargarizans has a significant directional asymmetry in testes mass, with left testis being significantly larger than right testis. male body condition was correlated with relative testes mass and absolute testes asymmetry, but not correlated with relative testes asymmetry. therefore, we suggested that males in good body condition have the higher ability of sperm competition than males in poor body condition. additionally, male age was correlated with absolute testes asymmetry, but not correlated with relative testes mass and testes asymmetry, thus not supporting the hypothesis that males with a higher degree of directional asymmetry survive better. keywords. testes asymmetry, body condition, age, bufo gargarizans. testes asymmetry theory predicts that directional asymmetry should be the norm because in many cases only one of the two testes is functional, with the other test having a compensatory role (schärer and vizoso, 2007). in this case, males in good condition or relatively older age may be able to develop larger degrees of directional asymmetry than males in poor condition or small age (møller, 1994; birkhead et al., 1997; graves, 2004). directional testes asymmetry has been previously observed in birds (birkhead et al., 1997) and mammals (yu, 1998). interestingly, in recent years similar results have been observed in some amphibians (hettyey et al., 2005; zhou et al., 2011; liu et al., 2011; liu et al., 2012). the asiatic toad (bufo gargarizans) is widely distributed in east asia, including china and portions of the russian far east, but is relatively rare on the korean peninsula. these toads breed during late-winter in large ponds or paddy fields (yu and guo, 2013), while overwinter in the sand or ponds. this species avoids dense forests, and is associated with open habitats such as grasslands, open forests, and cultivated areas. they prefer humid areas, and seldom live at altitudes of more than 800 meters (yu and guo, 2013). however, we know little about testes asymmetry of b. gargarizans in relation to body condition and age. in this study, we examined whether the directional testes asymmetry occurs in b. gargarizans. moreover, we tested whether positive correlations existed between degree of directional testes asymmetry, body condition and age. data were collected in vegetable fields of nanchong city (30°35’n, 104°45’e; 300 m above sea level) in southern sichuan, china. a total of 56 male toads were collected by hand at night in early-may, early-june, and early-september 2006, because the testis masses does not significantly change during the post-breeding season (i.e., after may) in the site (reviewed by liu et al., 2011). then, we took them to the laboratory and put those toads individually in a circular bucket (15l) with fresh water of 15 cm deep at room temperature for one night. 156 tong lei yu, yan shu guo snout–vent length (svl) of each captured toad was measured using a plastic ruler to the nearest 1 mm, and its body mass was measured using an electronic balance to the nearest 0.01 g. then, 56 male toads were killed by double-pithing. we dissected the toads and took out their two testes, weighing them to the nearest 0.1 mg. following møller and swaddle (1997) and liu et al. (2011), we used two measures of testes to confirm degree of directional testes asymmetry: 1) absolute testes asymmetry, mass of the left testis minus the mass of the right (liu et al., 2011); 2) relative testes asymmetry, the difference in testis mass / 0.5 (left-testis + right-testis mass) (liu et al., 2011; liu et al., 2012). we used paraffin section and ehrlich’s haematoxylin stain to produce histological sections of the phalanges. age was determined by counting the number of lines of arrested growth (lags) in the sections. this technique has been shown to be a reliable tool for determining age for a variety of amphibian species (smirina, 1994), including rana chensinensis (lu et al., 2006), bufo andrewsi (liao and lu, 2012). schulte-hostedde et al. (2001) suggested that the measurement of live animals’ condition is typically done by regressing body mass on body size and the residuals of this regression are used as an index of body condition. body condition was calculated using linear regression by entering body mass as a dependent variable and svl as independent variable, and the residual mass was used as an index. similarly, relative testes mass was defined by entering testes mass as a dependent variable and svl as independent variable using linear regression, and residual mass was used as an index. we used paired t-test to evaluate difference in masses of the left and right testis. we performed pearson correlations to analyse the relationship between male age and body size or body mass. the relationships between male condition (dependent variable) and absolute testes asymmetry or relative testes asymmetry or relative testes mass (independent variable) were analyzed by linear regression analysis. also, we used linear regression to test the relationships between male age (dependent variable) and absolute testes asymmetry or relative testes asymmetry or relative testes mass (independent variables). statistical tests were performed by the software spss 14.0 for windows (statistical product and service solutions company, chicago). all values are reported as mean ± se and all statistical tests were two-tailed. average svl and body mass of males were 8.09 ± 0.17 cm and 79.78 ± 4.64 g, respectively. males had mean age of 2.32 ± 0.11 years (range: 1 to 4 years). age was correlated significantly with male svl (pearson’s correlation coefficient: r = 0.83, n = 56, p < 0.001) and body mass (r = 0.94, n = 56, p < 0.001). testes mass of b. gargarizans did not vary significantly during the post-breeding season (one-way anova: f2, 53 = 2.48, p = 0.09). the testes mass of males showed directional asymmetry (paired t-test: t = 3.17, df = 55, p = 0.002), with the left testes being significantly larger than the right ones in 71.43% of all the samples. the degree of absolute testes asymmetry was significantly correlated left testis mass with (r = 0.51, n = 56, p < 0.001). we regressed body mass on male svl, showing a highly significant regression (f1, 54 = 398.13, r2 = 0.881, p < 0.001, body mass = 25.78×svl (cm) – 128.796). male condition was significantly correlated with the degree of absolute testes asymmetry (body condition = 0.21×absolute testes asymmetry – 1.522, f1, 54 = 4.94, p = 0.03; r2 = 0.084, fig. 1a), but not related to relative testes asymmetry (f1, 54 = 0.99, p = 0.32; r2 = 0.018, fig. 1c). relative testes mass significantly increased in males with better body condition (body condition = 0.044×relative testes mass (mg) 0.051, f1, 54 = 5.77, p = 0.02; r2 = 0.097, fig. 1e). male age was significantly correlated with the degree of absolute testes asymmetry (age = 0.026×absolute testes asymmetry + 2.14, f1, 54 = 19.28, p < 0.001; r2 = 0.263, fig. 1b), but not related with relative testes asymmetry (f1, 54 = 1.44, p = 0.23; r2 = 0.026, fig. 1d). there was also no significant correlation between age and relative testes mass (f1, 54 = 0.02, p = 0.88; r2 = 0.00, fig. 1f). our study revealed that the left testis mass was significantly larger than the right in the b. gargarizans. this result was similar to the pattern of testes asymmetry in other anuran species (rana nigromaculata, zhou et al., 2011; hylarana guentheri, liu et al., 2011; rana omeimontis, liu et al., 2012), while male frogs from all populations in rana temporaria have larger right testes (hettyey et al., 2005). additionally, we found a significant positive relationship between the mass of the left testis and the degree of absolute testes asymmetry. this result was consistent with the hypothesis that left testes only are functional, and the right testis would increase in size if the left testis became nonfunctional (møller, 1994; birkhead et al., 1997). testes asymmetry might occur because of multiple reasons. for instance, maintaining two large, functional testes may be costly (møller, 1994), and one testis may have higher efficiency in sperm production due to constraints during embryonic development (kempenaers et al., 2002). previous studies have suggested that testis asymmetry may not be a good measure of male body condition (birkhead et al., 1997). our results showed that male 157testes asymmetry of bufo gargarizans body condition was correlated with the degree of absolute testes asymmetry in b. gargarizans, but not correlated with the degree of relative testis asymmetry, suggesting that large males in good body condition had not a larger degree of testis asymmetry than small males in poor condition. this result was not consistent with hettyey et al. (2005) and møller (1994). interestingly, male body condition was positively correlated with relative testis mass (simmons and kotiaho, 2002), suggesting that males in good body condition have the higher ability of sperm competition than males in poor body condition. game theory models predict that ejaculate investment should increase with the risk and intensity of sperm competition (parker, 1998). the testes should thus be relatively large when the likelihood of sperm competition is high (møller and briskie, 1995). consequently, males in good fig. 1. relationships between (a) body condition and absolute testes asymmetry, (b) age and absolute testes asymmetry, (c) body condition and relative testes asymmetry, (d) age and relative testes asymmetry, (e) body condition and relative testes mass, (f) age and relative testes mass. displayed values are untransformed data for easier interpretation. 158 tong lei yu, yan shu guo conditions are expected to have larger testes than males in poor condition with increased risk of sperm competition (olsson et al., 1997). previous studies have shown that testes asymmetry is age-related (e.g., birkhead et al., 1997; graves, 2004; liu et al., 2012), suggesting that males with a higher degree of directional asymmetry survive better. however, we did not found age was positively correlated with the degree of relative testes asymmetry and relative testes mass. liu et al. (2011) and zhou et al. (2011) observed similar results in hylarana guentheri and rana nigromaculata, suggesting that older males did exhibit a higher degree of testis asymmetry than younger males. a possible explanation was males have already attained full reproductive maturity during the first breeding year (reviewed by liu et al., 2011). acknowledgments we would also like to thank w.h. qi, y.l. han, and j.z. ning for their assistance in the field. the collection of all the toads used in the study was permitted by forestry bureau of nanchong, following all the applicable institutions of the animal care guidelines in china. the study was funded by key  project  of  he’nan educational committee (grant no. 12b180033). references birkhead, t.r., buchanan, k.l., devoogd, t.j., pellatt, e.j., szèkely, t., catchpole, c.k. (1997): song, sperm quality and testes asymmetry in the sedge warbler. anim. behav. 53: 965-971. graves, g.r. (2004): testicular volume and asymmetry are age dependent in black-throated blue warblers (dendroica caerulescens). auk 121: 473-485 hettyey, a., laurila, a., herczeg, g., jönsson, k.i., kovács, t., merilä, j. (2005): does testis weight decline towards the subarctic? a case study on the common frog, rana temporaria. naturwissenschaften 92: 188-192. kempenaers, b., peer, k., vermeirssen, e.l.m., robertson, r.j. (2002): testes size and asymmetry in the tree swallow tachycineta bicolor: a test of the compensation hypothesis. avian sci. 3: 115-122. liao, w.b., lu, x. (2012): adult body size = f (initial size + growth rate× age): explaining the proximate cause of bergman’s cline in a toad along altitudinal gradients. evol. ecol. 26: 352-364. liu, w.c., huang, y., liao, y.m., li, c. (2012): testes asymmetry of chinese endemic frog (rana omeimontis) in relation to body condition and age. northwest. j. zool. 8: 390-393. liu, y.h., liao, w.b., zhou, c.q., mi, z.p., mao, m. (2011): asymmetry of testes in guenther’s frog, hylarana guentheri (anura: ranidae). asian herpetol. res. 2: 234-239. lu, x., li, b., liang, j.j. (2006): comparative demography of a temperate anuran, rana chensinensis, along a relatively fine elevational gradient. can. j. zool. 84: 1789-1795. møller, a.p. (1994): directional selection on directional asymmetry: testes size and secondary sexual characters in birds. proc. roy. soc. lond b 258: 147-151. møller, a.p., briskie, j.v. (1995): extra-pair paternity, sperm competition and the evolution of testis size in birds. behav. ecol. sociobiol. 36: 357-365. møller, a.p., swaddle, j.p. (1997): asymmetry, developmental stability, and evolution. oxford: oxford university press. olsson, m., madsen, t., shine, r. (1997): is sperm really so cheap? costs of reproduction in male adders, vipera berus. proc. royn. soc. lond. b 264: 455-459. parker, g.a. (1998): sperm competition and the evolution of ejaculates: towards a theory base. in: birkhead t.r., møller a.p. (eds.), sperm competition and sexual selection. san diego, ca: acad. press, 3-54. schärer, l., vizoso, d.b. (2007): phenotypic plasticity in sperm production rate: there’s more to it than testis size. evol. ecol. 21: 295-306. schulte-hostedde, a.i., millar, j.s., hickling, g.j. (2001): evaluating body condition in small mammals. can. j. zool. 79: 1021-1029. simmons, l.w., kotiaho, j.s. (2002): evolution of ejaculates: patterns of phenotypic and genotypic variation and condition dependence in sperm competition traits. evolution 56: 1622-1631. smirina, e.m. (1994): age determination and longevity in amphibians. gerontology 40: 133-146. yu, t.l., guo, y.s. (2013): differential response to abiotic conditions, predation risk, and competition determine breeding site selection by two anuran species. anim. cells syst. 17: 127-132. yu, z.h. (1998): asymmetrical testicular weights in mammals, birds, reptiles and amphibia. int. j. androl. 21: 53-55. zhou, c.q., mao, m., liao, w.b., mi, z.p., liu, y.h. (2011): testis asymmetry in the dark-spotted frog rana nigromaculata. herpetol. j. 21: 181-185. acta herpetologica vol. 10, n. 1 june 2015 firenze university press acta herpetologica journal of the societas herpetologica italica acta herpetologica 11(2): 151-160, 2016 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-17874 redescription of cyrtodactylus fumosus (müller, 1895) (reptilia: squamata: gekkonidae), with a revised identification key to the benttoed geckos of sulawesi sven mecke1,*,§, lukas hartmann1,2,§, felix mader3, max kieckbusch1, hinrich kaiser4 1  department of animal evolution and systematics and zoological collection marburg, faculty of biology, philipps-universität marburg, karl-von-frisch-straße 8, 35032 marburg, germany. *corresponding author. e-mail: meckes@staff.uni-marburg.de 2  current address: department of ecology and evolution, johann wolfgang goethe-universität – biologicum, max-von-laue-straße 13, 60438 frankfurt am main, germany 3 janusstraße 5, 93051 regensburg, germany 4  department of biology, victor valley college, 18422 bear valley road, victorville, california 92395, usa; and department of vertebrate zoology, national museum of natural history, smithsonian institution, washington, dc 20013, usa § co-first authors submitted on 2016, 27th january; revised on 2016, 16th august; accepted on 2016, 30th august editor: aaron m. bauer abstract. the binominal cyrtodactylus fumosus has frequently been used for populations of bent-toed geckos occurring on some indonesian islands, including java, bali, sulawesi, and halmahera. unfortunately, incorrect usage of this name for different geographic lineages has resulted in confusion about the true identity of c. fumosus. examination of the type specimen and additional specimens from rurukan and mount masarang, north sulawesi province, indonesia, revealed that this population is distinct from other forms heretofore called ‘fumosus’ by a combination of unique morphological characters. in order to stabilize the taxonomy of c.  fumosus sensu stricto, and to prevent further confusion, we provide a comprehensive redescription of this species, whose distribution we herein restrict to north sulawesi. cyrtodactylus fumosus is one of the most distinctive species among the six bent-toed geckos recorded from sulawesi, and it differs from sulawesi congeners by the presence of (1) precloacofemoral scales, including three porebearing scales on each thigh, separated from 10 or 11 pore-bearing scales in the precloacal region by 9-11 interscales in males, (2) a precloacal groove in adult males, (3) flat dorsal tubercles in 4-7 irregularly arranged longitudinal rows at midbody, and (4) a distinct lateral fold lacking tubercles. we also provide a revised identification key to the benttoed gecko species of sulawesi. keywords. cyrtodactylus fumosus, c.  marmoratus, lacertilia, bent-toed geckos, reptiles, north sulawesi, indonesia, morphology. introduction the bent-toed gecko fauna of sulawesi consists of six species, including cyrtodactylus batik iskandar et al., 2011; c.  fumosus (müller, 1895); c.  hitchi riyanto et al., 2016; c.  jellesmae (boulenger, 1897); c.  spinosus linkem et al., 2008; and c.  wallacei hayden et al., 2008. two of these, c.  fumosus and c.  jellesmae have been reported from north sulawesi province, indonesia (e.g., boulenger, 1897; koch et al., 2009; iskandar et al., 2011; koch, 2012). cyrtodactylus fumosus was described by müller (1895a) based on a single specimen (nmb-rept 2662; adult female), collected by paul benedict sarasin (1856-1929) and karl friedrich (“fritz”) sarasin (1859-1942) in the 152 sven mecke et alii “boelawa mountains” (= huidu matabulawa) of northern sulawesi. following its original description, c.  fumosus was also reported from localities outside of sulawesi (e.g., de rooij, 1915; mertens, 1929, 1934; manthey and grossmann, 1997; mckay, 2006; oliver et al., 2009; das, 2010; koch, 2012; de lisle et al., 2013; riyanto et al., 2013, 2015), leading to the perception of a wide distribution and a rather inconsistent or even erroneous definition of the taxon, since the name became applied to bent-toed gecko species not representing c.  fumosus sensu stricto (see hartmann et al., 2016). boulenger (1897) was the only author who provided a detailed, though not entirely correct (see hartmann et al., 2016: footnote 1), species account for c.  fumosus sensu stricto, based on specimens from north sulawesi. the recent identification of new species from the sunda islands masquerading under the name c.  fumosus (riyanto et al., 2015; hartmann et al., 2016) and re-examination of c.  fumosus specimens from north sulawesi, however, show that the taxonomy of c. fumosus is in disarray, and this makes it necessary to properly redescribe this conspicuous taxon based on a multitude of eidonomic characters, some of which have never been provided in the literature. whereas hartmann et al. (2016) already published remarks on the taxonomy of c.  fumosus and provided selected comparative morphological data for this species, a comprehensive redescription of c.  fumosus is necessary to stabilize the taxonomy of a species that has experienced prominent use in the literature, but whose identity has regularly been misconstrued. this redescription, featured below, can serve as solid basis for the delineation and description of additional new species of bent-toed geckos currently recognized as c.  fumosus, and allows the correction of comparative literature data. materials and methods our redescription of cyrtodactylus fumosus is based on the examination of four specimens of that taxon, including the holotype (nmb-rept 2662) and three additional specimens (nmb-rept 2663; bmnh 1895.2.27.7, 1896.12.9.3). for each specimen used in the redescription, we recorded data for 31 eidonomic characters (see table 1 for definitions and abbreviations). of these, 14 were metric and 16 meristic. we also calculated the following ratios: axiall/svl, arml/svl, legl/svl, headl/svl, headw/headl, snoutl/headl, snoutl/orbd, and mentalh/mentalw. all measurements were taken to the nearest 0.1 mm using digital calipers. scale counts and observations of external morphology were made using a dissection microscope. characters occurring bilaterally were measured or counted on the right side of specimens, unless stated otherwise; for femoral pores, interscales, and labial scales, we provide counts for both sides of the body (the prefixes “r” and “l” are used to distinguish characters counted on the right or left side, respectively). in our diagnosis, ranges are followed by means ± standard deviations. for descriptions of pattern and coloration we apply the terminology of köhler (2012). numbers in parentheses behind the respective capitalized color name refer to the coding therein. the terminology used to distinguish between different depressed precloacal areas follows mecke et al. (2016). drawings are based on photographs of ethanol-preserved specimens and were prepared using the program coreldraw graphics suite x3. museum abbreviations follow sabaj pérez (2014). results cyrtodactylus fumosus (müller, 1895) (figs 1; 2) gymnodactylus fumosus müller, 1895a: 833 (holotype nmb-rept 2662; type locality: “boelawa gebirge,” sulawesi, elevation 1200 m) gymnodactylus fumosus—müller, 1895b: 865 gymnodactylus fumosus—boulenger, 1897: 204 gymnodactylus fumosus (part.)—de rooij, 1915: 16 gymnodactylus fumosus—brongersma, 1934: 168 gymnodactylus fumosus—brongersma, 1953: 172 gymnodactylus fumosus—kramer, 1979: 160 cyrtodactylus fumosus (part.)—manthey and grossmann, 1997: 222 cyrtodactylus fumosus (part.)—grismer, 2005: 429 cyrtodactylus fumosus (part.)—grismer and leong, 2005: 588 cyrtodactylus fumosus (part.)—biswas, 2007: 19 cyrtodactylus fumosus (part.)—hayden et al., 2008: 109 cyrtodactylus fumosus (part.)—rösler and glaw, 2008: 14 cyrtodactylus fumosus (part.)—chan and norhayati, 2010: 50 cyrtodactylus fumosus (part.)—das, 2010: 209 cyrtodactylus fumosus (part.)—iskandar et al., 2011: 65 cyrtodactylus fumosus (part.)—koch, 2012: 161 cyrtodactylus fumosus—hartmann et al., 2016: 556 cyrtodactylus fumosus (part)—riyanto et al., 2016: 69 cyrtodactylus fumosus—mecke et al., 2016: 356 holotype: nmb-rept 2662 (fig. 1a and table 2; hartmann et al. 2016: fig.  5): adult female (svl = 77.8 mm) collected by paul and fritz sarasin in 1894; terra typica: “boelawa gebirge” (= huidu matabulawa), corrected to “bone mountains” (= pegunungan bone, north sulawesi province, indonesia) by boulenger (1897). referred specimens: nmb 2663 (fig. 1b): mount masarang; bmnh 1895.2.27.7 (fig. 1c; same specimen figured in boulenger, 1897: plate vii, fig. 2), 1896.12.9.3 (fig. 1d): rurukan. definition: cyrtodactylus fumosus is a moderatelysized bent-toed gecko species (maximum svl = 78 mm) 153redescription of cyrtodactylus fumosus that can be readily distinguished from all other congeners occurring in the greater and lesser sunda islands, sulawesi, and the maluku islands by the following combination of characters: (1) a single series of precloacofemoral scales, including three pore-bearing scales on each thigh, separated from 10 or 11 pore-bearing scales in the precloacal region by 9-11 interscales in males (fig. 2a), (2) a precloacal groove in adult males (fig. 2a), (3) posterior precloacal scales (fig. 2a), (4) flat and smooth (unkeeled) dorsal tubercles in 4-7 irregularly arranged longitudinal rows at midbody (fig. 2b), (5) a distinct lateral fold lacking tubercles, (6) 37-50 longitudinal rows of ventral scales at midbody, (7) 17-23 scales under 4th toe, and (8) a horizontal slit-like ear opening. comparisons: characters distinguishing cyrtodactylus fumosus from all other species of cyrtodactylus occurring on the sunda islands and sulawesi were provided by mecke et al. (2016: table 2). here, our comparisons are limited to sulawesi taxa, with characters of c.  fumosus provided in parentheses. cyrtodactylus batik can be table 1. metric and meristic characters with abbreviations used in this study. character abbreviation definition snout-vent length svl from tip of snout to cloaca axial length axiall from axilla to groin tail length taill from cloaca to tip of tail arm length arml from insertion of brachium into body wall to claw of longest finger leg length legl from insertion of thigh into body wall to claw of longest toe head length headl from tip of snout to articulation of quadrate bone with lower jaw head width headw measured at level of ear openings head height headh measured at level of ear openings snout length snoutl from tip of snout to anterior margin of orbit orbit-ear distance orbeard from posterior margin of orbit to anterior margin of ear opening orbital diameter orbd from anterior to posterior margin of orbit ear length earl from anterior to posterior margin of ear opening mental length mentall maximum length of mental shield mental width mentalw maximum width of mental shield dorsal tubercle rows dtr number of longitudinal tubercle rows on dorsum at midbody, counted in one row between lateral folds paravertebral tubercles pvt number of tubercles counted in a longitudinal row between posterior insertion of forelimb and anterior insertion of hindlimb ventral scales vs number of ventral scales at midbody, counted in one row between lateral folds precloacofemoral scales pfs number of enlarged precloacofemoral scales, counted along lowest, pore-bearing series femoral pores fp number of femoral pores on enlarged scales on thigh interscalesa inters number of enlarged poreless scales between series of pore-bearing precloacal scales and series of pore-bearing femoral scales on thigh precloacal pores pp number of precloacal pores situated in precloacal groove postcloacal tubercles pct number of postcloacal tubercles subdigital lamellae under 4th toe lt4 number of subdigital scales under 4th toe, counted from first enlarged scale (lamellae) on lower side of toe to scale proximal to apical scale supralabial scales 1 supralab1 number of labial scales of upper jaw, beginning with first enlarged scale bordering rostral shield, ending with last enlarged scale bordering labial angle supralabial scales 2 supralab2 number of labial scales of upper jaw, beginning with first enlarged scale bordering rostral shield, ending with enlarged scale below anterior margin of eye infralabial scales infralab number of labial scales of lower jaw, beginning with first scale bordering mental shield, ending with last enlarged scale bordering labial angle internasal scales internas number of scales between rostronasals, bordering rostral supraciliar scales sc number of enlarged scales extending from anterior-ventral to posterior-dorsal edge of orbit interorbital scales ios number of scales counted in a row between the medial edges of orbits across head gular scales guls number of gular scales bordering pair of first postmentals a rösler et al. (2007); hartmann et al. (2016); and mecke et al. (2016) referred to interscales as “infrascales.” 154 sven mecke et alii distinguished from c.  fumosus by a larger size of adults with a maximum svl of 115 mm (78 mm); the absence of pfs (pfs present); the absence of pp and fp in both sexes (pp and fp present in males); the absence of a precloacal depression in both sexes (precloacal groove present in males); 23-26 slightly keeled dtr (4-7 unkeeled dtr); the presence of tubercles on the lateral skin fold (tubercles on lateral skin fold absent); 24-27 lt4 (17-23 lt4); and the presence of transversely enlarged subcaudal scales in a single row (enlarged, paired median subcaudals) (iskandar et al., 2011; riyanto et al., 2016). cyrtodactylus hitchi can be distinguished from c.  fumosus by the absence of pfs (pfs present); the absence of pp and fp in both sexes (pp and fp present in males); the absence of a precloacal depression in both sexes (precloacal groove present in males); the presence of 18-20 keeled dtr (4-7 unkeeled dtr); the presence of tubercles on the lateral skin fold (tubercles on lateral skin fold absent); and the presence of transversely enlarged subcaudal scales in a single row (enlarged paired median subcaudals) (riyanto et al., 2016). cyrtodactylus jellesmae can be distinguished from c.  fumosus by the absence of pfs (pfs present); the absence of pp and fp in both sexes (pp and fp present in males); the absence of a precloacal depression in both sexes (precloacal groove present in males); the presence of 13-22 raised dtr (4-7 flat dtr); the presence of tubercles on the lateral skin fold (tubercles on lateral skin fold absent); and the absence of enlarged subcaudal scales (enlarged paired median subcaudals present) (boulenger, 1897; mecke et al., 2016, pers. obs.). cyrtodactylus spinosus can be distinguished from c.  fumosus by the absence of a continuous series of enlarged precloacal and femoral scales (pfs present); by widely spaced femoral scales (femoral scales juxtaposed); the presence of a shallow precloacal pit in males (deep precloacal groove in males); the presence of lateral and caudal spines (spines absent); and the presence of a prehensile tail (tail not prehensile) (linkem et al., 2008; harvey et al., 2016). cyrtodactylus wallacei can be distinguished from c.  fumosus by a larger size of adults, reaching a maximum svl of 114 mm (78 mm); the absence of pfs (pfs present); the absence of pp and fp in both sexes (pp and fp present in males); the absence of a prefig. 1. dorsal views of the known specimens of cyrtodactylus fumosus: (a) nmb-rept 2662 (holotype, adult female); (b) nmbrept 2663 (subadult male); (c) bmnh 1895.2.27.7 (adult female); (d) bmnh 1896.12.9.3 (adult male). photographs by sven mecke. bmnh 1895.2.27.7 is also figured (in dorsal view) in boulenger (1897: plate vii, fig. 2). fig. 2. diagnostic characters of cyrtodactylus fumosus. (a) precloacofemoral region (with pore-bearing precloacal scales and groove shaded in grey) of a male specimen (bmnh 1896.12.9.3), showing precloacal and femoral pores. scale bar equals 2 mm (b) dorsum, showing tubercles (holotype nmb-rept 2662). scale bar equals 2 mm. (c) ventral side of anterior part of head (holotype nmb-rept 2662). scale bar equals 1 mm. drawings prepared by felix mader based on photographs by sven mecke. 155redescription of cyrtodactylus fumosus cloacal depression in both sexes (precloacal groove present in males); and the presence of 23-25 slightly keeled, trihedral dtr (4-7 unkeeled and flat dtr) (hayden et al., 2008). description of the holotype. general habitus, metrics, and ratios: adult female; svl = 77.8 mm; axiall = 35.2 mm; taill (broken, only tail stump present) = 8.7 mm; arml = 35.7 mm; legl = 43.9 mm; headl = 21.3 mm; headw = 14.2 mm; headh = 9.2 mm; snoutl = 8.8 mm; orbeard = 6.6 mm; orbd = 5.2 mm; earl = 1.2 mm; head rather short (headl/svl = 0.27) and wide (headw/headl = 0.67), clearly depressed between eyes, distinct from neck; snout rather elongate (snoutl/headl = 0.41), longer than orbd (snoutl/orbd = 1.69), canthus rostralis distinct; foreand hindlimbs of moderate size (arml/svl = 0.46; legl/svl = 0.56), without webbing between digits; relative length of fingers = iv > iii > v > ii > i; relative length of toes = iv > iii > v > ii > i; lateral skin fold distinct, lacking tubercles. scalation: dorsal scales granulate, interspersed with slightly enlarged, flat, roundish and irregularly arranged dorsal tubercles (fig. 2b), 5 dtr; 13 pvt; tubercles on occiput, neck, and hindlimbs similar in shape to those on dorsum (no tubercles present on the forelimbs). thirty-eight vs, distinctly larger than those on dorsum, juxtaposed; a single series of 46 poreless pfs; enlarged posterior precloacal scales present, arranged in a chevron-like shape consisting of five series of scales (from anterior to posterior: 10/ 8/ 7/ 6/ 2 scales); 2 flat pct; number of lamellae under fingers: i 12, ii 16, iii 16, iv 18, v 16; number of lamellae under toes: i 13, ii 15, iii 17, iv 17, v 16. rostral shield rectangular, 2.2 times as wide as high, partly divided by a median, vertical furrow, in contact with 1st supralab, 2 rostro-nasals and a single internas; naris surrounded by rostral, 1st supralab, a single rostronasal, and three post-nasals; r12 l12 supralab1, r6 l5 supralab2, separated from orbit by three rows of small granular scales; r9 l11 infralab; cephalic scales small, rounded, granulate and juxtaposed; tubercles on occiput and neck flat and unkeeled; 40 sc; 46 ios; mental triangular, wider than long (mentalw/mentall = 1.7); one pair of enlarged 1st postmentals, enlarged 2nd postmentals absent (fig. 2c); pair of 1st postmentals bordered by mental, 1st infralab, and 9 guls (fig. 2c); scales on throat minute and rounded. coloration: natural color and pattern altered due to preservation. ground color of dorsum cinnamondrab (50); head darker than dorsum, burnt umber (48) in color, with indistinct warm sepia (40) stripe running from posterior border of orbits along neck, forming a collar at level of posterior margin of forelimbs; labial scales buff (5), stippled with darker color, with stipples most concentrated at edges of some scales; dorsum with irregular, faint dark drab (45) blotches, not arranged in distinct pairs, most visible on vertebral region between forelimbs and on mid-dorsum; ground color of dorsal surface of limbs similar to ground color of dorsum; limbs with diffuse dark drab (45) markings; venter, throat and lower surface of limbs uniformly smoke grey (266), heavily dotted; color of dorsal and ventral surfaces of tail stump similar to dorsal and ventral ground color, respectively. table 2. metric (in mm) and meristic data for the known specimens of cyrtodactylus fumosus. abbreviations are defined in table 1. characters occurring bilaterally were measured or counted on the right side of specimens, unless stated otherwise; for femoral pores, interscales, and labial scales we provide counts for both sides of the body (the prefixes “r” and “l” are used to distinguish characters counted on the right and left side, respectively). n/a = not applicable. our metric data of bmnh 1895.2.27.7, the only known specimen with an original tail (taill = 67.1), well agree with the measurements listed by boulenger (1897), who also provided a drawing of a specimen (plate vii, fig. 2) identifiable as bmnh 1895.2.27.7. nmb-rept 2662 (holotype) nmb-rept 2663 bmnh 1895.2.27.7 bmnh 1896.12.9.3 sex female male female male svl 77.8 56.6 60.7 77.5 axiall 35.2 22.2 28.3 31.4 arml 35.7 22.1 24.9 32.9 legl 43.9 29.6 32.9 42.0 headl 21.3 15.7 16.8 20.4 headw 14.2 10.6 11.9 14.5 headh 9.2 7.0 6.7 9.5 snoutl 8.8 6.9 7.7 9.4 orbeard 6.6 4.1 4.3 6.3 orbd 5.2 3.6 4.0 4.1 earl 1.2 1.2 2.0 2.3 dtr 5 7 4 6 pvt 13 16 14 18 vs 38 37 47 50 pfs 46 45 46 39 fp 0 r3 l3 0 r3 l3 inters n/a r10 l9 n/a r10 l11 pp 0 11 0 10 lt4 (proximal) 7 8 10 9 (l) lt4 (distal) 10 11 13 12 (l) lt4 17 19 23 21 (l) supralab1 r12 l12 r13 l13 r11 l12 r11 l12 supralab2 r6 l5 r6 l6 r6 l6 r6 l6 infralab r9 l11 r10 l10 r11 l10 r8 l8 guls 9 8 7 8 156 sven mecke et alii intraspecific variation: our assessment of the variation is based on the holotype and three additional specimens from north sulawesi (one adult and one subadult male, one adult female) unless stated otherwise. measurements (in mm) are listed as range followed by mean ± standard deviation provided in parentheses: svl = 56.677.8 (68.2 ± 11.1); axiall = 22.2-35.2 (29.3 ± 5.5); taill (original tail) = 67.1 (n = 1); arml = 22.1-35.7 (28.9 ± 6.4); legl = 29.6-43.9 (37.1 ± 6.9); headl = 15.7-21.3 (18.6 ± 2.7); headw = 10.6-14.5 (12.8 ± 1.9); headh = 6.7-9.5 (8.1 ± 1.5); snoutl = 6.9-9.4 (8.2 ± 1.1); orbeard = 4.1-6.6 (5.3 ± 1.3); orbd = 3.6-5.2 (4.2 ± 0.7); earl = 1.2-2.3 (1.7 ± 0.6). ratios: axiall/svl = 0.39-0.47 (0.43 ± 0.03); arml/svl = 0.39-0.46 (0.42 ± 0.03); legl/svl = 0.52-0.56 (0.54 ± 0.02); headl/svl = 0.27-0.28 (0.27 ± 0.01); headw/headl = 0.67-0.71 (0.69 ± 0.02); snoutl/ headl = 0.41-0.46 (0.44 ± 0.02); snoutl/orbd = 1.692.29 (1.96 ± 0.25); rostralw/rostralh = 1.53-2.18 (1.91 ± 0.28); mentalw/mentall = 1.29-1.83 (1.64 ± 0.24). scale counts are listed as range followed by mean ± standard deviation provided in parentheses: dtr = 4-7 (5.75 ± 1.3); pvt = 13-18 (15.25 ± 2.2); vs = 37-50 (43 ± 6.5); pfs = 39-46 (44 ± 3.4), only a single series present; enlarged posterior precloacal scales consisting of 5 or 6 series; pct = 2-3, flat in shape; lt4 = 17-23 (19 ± 2.8); supralab1 = 11-13 on right side of head and 12-13 on left side of head; infralab = 8-11 on right side of head and 8-11 on left side of head; sc = 32-40 (33.5 ± 4.4); ios = 45-49 (47.3 ± 2.1); guls = 7-9. furthermore, all specimens possess a distinct lateral skin fold lacking tubercles and a horizontal, slit-like ear opening. a distinctive row of 5 or 6 tubercles on the dorsal surface of the upper leg is present in three specimens (absent in the holotype). specimens with unregenerated tails possess two strongly enlarged median subcaudal rows. unlike female specimens, male specimens of cyrtodactylus fumosus (n = 2) possess three pore-bearing scales on each thigh, separated from 10 or 11 pore-bearing precloacal scales by 9-11 inters. a distinct precloacal groove is fully developed in adult males (n = 1) only. data of measurements and scale counts for the main characters of the holotype and additional specimens used for the diagnosis are provided in table 2. ground color of dorsal surface of body, head, and tail varies considerably between the specimens available to us and appears to depend on the respective preservation method. hence, ground color of dorsal surface varies from cinnamon (255) over cinnamon-drab (50) to drab (19), with the specimens housed in nmb being darker than the ones housed in bmnh; dorsum with 4-7, sometimes indistinct, warm sepia (40) blotches; original tail (n = 1) with six warm sepia (40) blotches; regenerated tail of one specimen (bmnh 1896.12.9.3) possesses three indistinct, partially interrupted, warm sepia (40) lines, running from base to tip of tail; dorsal surface of limbs and head with diffuse warm sepia (40) or dark drab (45) markings; venter, lower surface of limbs, and throat uniformly pale buff (1) or smoke grey (266 and 267). see fig. 1 for coloration and pattern of preserved specimens. distribution and natural history: although the name cyrtodactylus fumosus has frequently been applied to bent-toed gecko populations from java, bali, halmahera, and the entire island of sulawesi (e.g., de rooij, 1915; grismer, 2005; das, 2010; de lisle et al., 2013; riyanto and mumpini, 2013; riyanto et al., 2015), c.  fumosus sensu stricto is only known from the four specimens featured herein, all of which were collected in north sulawesi (müller, 1895a, b; boulenger, 1897; see fig. 3). the occurrence of c.  fumosus on lembeh island, off the coast of northern sulawesi (grismer, 2005: appendix 1, grimser and leong, 2005: appendix  1), appears to be based on misidentified specimens, since the data (including key characters for diagnosis) provided by grismser (2005: table 2) and grismer and leong (2005: table 2) do not match those of c.  fumosus sensu stricto as reported herein. moreover, the data provided by grismer (2005) and grismer and leong (2005: table 2) seem to be partly based on the erroneous description of c.  fumosus provided by de rooij (1915) (see hartmann et al., 2016). according to the data provided by müller (1895a, b), specimens of cyrtodactylus fumosus sensu stricto were collected at elevations 1200-1260 m, in a terrain that is, based on satellite images (google earth, viewed on 24 january 2016), covered with montane rainforest. although there are only limited data available on the natural history of c.  fumosus, we believe the species to be restricted to montane rainforest habitats in north sulawesi. the distribution of c.  fumosus, as currently known, overlaps with the range of c.  jellesmae, the only other species of cyrtodactylus known from north sulawesi. figure 3 shows the distribution of the six benttoed geckos currently known from sulawesi. remarks on the identity of cyrtodactylus fumosus from java: hartmann et al. (2016) discussed the status of cyrtodactylus fumosus populations outside of sulawesi and came to the conclusion that these records were based on erroneous data provided in the literature (e.g., de rooij, 1915) and/or misidentified specimens. recently, riyanto et al. (2015) applied the name c.  fumosus to populations of bent-toed geckos from java, which are unequivocally identifiable as belonging to the c.  marmoratus (gray, 1831) complex. these authors largely based their 157redescription of cyrtodactylus fumosus assumption on de rooij (1915), who mainly distinguished between c.  fumosus and c.  marmoratus by a continuous or discontinuous pore series, respectively. however, de rooij (1915) largely based her definition of c.  fumosus on boulenger (1897), who erroneously reported this species to have a continuous pore series, and her personal examination of specimens housed in the collections of bmnh and smf, which are conspecific with c.  halmahericus (mertens, 1929) (see hartmann et al., 2016: footnote 1). cyrtodactylus halmahericus, unlike c. fumosus, possesses a continuous pore series in males (a redescription of c.  halmahericus is currently underway). whereas the lectotype of c.  marmoratus (rmnh. rena 2710a.1; adult male), all other adult male paralectotypes housed in rmnh (rmnh.rena 2710a.2-a.5, 2710.1-2), and several other adult male specimens we have examined personally, possess a continuous series of pores (precloacofemoral pores), this character may vary ontogenetically (brongersma, 1953, pers. obs.), between sexes (rösler et al.; 2007, mecke et al., 2016), and between c.  marmoratus sensu stricto and morphologically similar species masquerading under this name. cyrtodactylus fumosus can be easily distinguished from c.  marmoratus as currently defined by the following characters: (1) a discontinuous series of precloacal (10 or 11) and femoral pores (three on each thigh) in males, (2) the absence of pores in females, (3) the presence of posterior precloacal scales, (4) the presence of widely scattered, roundish, flat, and smooth dorsal tubercles in 4-7 rows at midbody (11-19 in the type series of c.  marmoratus at rmnh), (5) 14-18 paravertebral tubercles (22-29 in in the type series of c.  marmoratus at rmnh), and enlarged paired median subcaudals (enlarged subcaudals absent in c. marmoratus). it is obvious that the male specimen (mzb.lace 12903) identified as cyrtodactylus fumosus by riyanto et al. (2015) and depicted in their fig. 4b is not conspecific with c.  fumosus, because it possesses a continuous pore series and lacks posterior precloacal scales. the precloacofemoral region of that specimen rather matches that of c.  marmoratus sensu stricto (see hartmann et al., 2016: fig. 3h, mecke et al., 2016: fig. 1a). since riyanto et al. (2015) failed to properly identify c.  fumosus and c.  marmoratus, their comparative table 3 should not be used for the identification of these taxa. the example well demonstrates the importance of examining relevant type specimens before taxonomic decisions are made. discussion the phylogenetic affinities of cyrtodactylus fumosus remain unclear. the presence of pores, a precloacal depression in males, and posterior precloacal scales are shared with other species of cyrtodactylus from the region, e.g., c.  halmahericus (halmahera) and c.  klakahensis hartmann et al., 2016 (eastern java), with which it may be closely allied1. by contrast, c. fumosus might represent an offshoot of an exclusive clade containing sulawesi bent-toed geckos only. results of studies on sulawesi amphibians and reptiles suggest that this island is herpetogeographically complex, supporting taxa of both sundaic and australopapuan affinities (koch, 2011, 2012), including endemics (e.g., how and kitchener, 1997; whitten et al., 2001; koch, 2011, 2012). the restriction of cyrtodactylus fumosus to sulawesi underscores that this island holds a significant amount 1 cyrtodactylus petani riyanto et al., 2015 also shares with c.  fumosus the presence of pores and posterior precloacal scales. riyanto et al. (2015) provided inconsistent data on whether a precloacal groove is present in male specimens of c.  petani. however, male c.  petani lack a precloacal groove or pit (awal riyanto, in litt.; mecke et al., 2016). fig. 3. map of sulawesi showing the distribution of the six species of cyrtodactylus currently recognized from this island: cyrtodactylus batik (inverted black triangle), c.  fumosus (black star), c.  hitchi (black circle), c. jellesmae (white circle), c. spinosus (black triangle), and c.  wallacei (black diamond). records are based on specimens listed in the appendices and data provided in hayden et al. (2008), linkem et al. (2008), iskandar et al. (2011), wanger et al., (2011), koch (2012), riyanto et al., (2016). a white circle with a black dot represents a photo-voucher for c. jellesmae available to us. base map modified from wikipedia © sadalmelik / wikimedia commons / cc-by-sa-3.0 by max kieckbusch. 158 sven mecke et alii of endemism. the species is apparently only found in the mountains of north sulawesi province, and such a limited range exemplifies that isolated geographic features in this region (e.g., mountain ranges) may be the key locales for such endemism. according to koch (2012: table 11) more than 20 amphibians and reptiles (including candidate species) are endemic to northern sulawesi. most of these appear to be endemic to offshore islands, but we hypothesize that the north sulawesi mountain ranges may harbor a higher number of endemic herpetofaunal taxa than generally assumed as well. we disagree with iskandar et al. (2011), who considered that the sulawesi herpetofauna is impoverished compared to other regions in southeast asia, largely due to natural factors alone. the high rate at which new amphibian and reptile species are being discovered on sulawesi contradicts this hypothesis, and the relatively low diversity may simply reflect the limited amount of fieldwork conducted there to date. since 2000, 16 reptile species have been described from sulawesi (e.g., tropidophorus baconi hikida et al., 2003; calamaria butonensis howard and gillespie, 2007; rabdion grovesi amarasinghe et al., 2015), a number that equals ~15% of the reptiles known from this island. the number of described species of cyrtodactylus in sulawesi alone increased by 200% during the last decade. preliminary examination of preserved bent-toed geckos from sulawesi in museum collections suggests that at least one undescribed species of bent-toed gecko is present on the island. photographic images of specimens in life available to us indicate that a further three species of cyrtodactylus from sulawesi are yet to be described. therefore we agree with e.g., linkem et al. (2008), and koch (2011, 2012), who considered the herpetological diversity of sulawesi to be underestimated. key to the species of the genus cyrtodactylus of sulawesi this key is applicable to identify adult bent-toed geckos based on non-sexually dimorphic characteristics, although characters present in males only may accompany a choice. 1a long spines on lateral fold and lateral portion of tail present; tail prehensile c. spinosus 1b long spines on lateral fold and lateral portion of tail absent; tail not prehensile 2 2a enlarged precloacofemoral scales present in both sexes, bearing a total number of 16 or 17 pores in males, 10 or 11 of which are precloacal pores and 3 of which are femoral pores; pore-bearing scales separated by 9-11 enlarged interscales; precloacal groove present in males; no tubercles on lateral fold c. fumosus 2b enlarged precloacofemoral scales; pores; precloacal groove; and tubercles on lateral fold absent 3 3a enlarged median subcaudals absent c. jellesmae 3b enlarged median subcaudals present 4 4a enlarged subcaudals in multiple rows c. wallacei 4b enlarged subcaudals in a single row for most of the tail’s length 5 5a 24-27 lamellae under 4th toe; svl in adults 103-113 mm c. batik 5b 18-21 lamellae under 4th toe; svl in adults 62-79 mm c. hitchi acknowledgements the authors thank denis vallan and urs wüest (nmb), patrick campbell (bmnh), esther dondorp (rmnh), raffael ernst and markus auer (mtkd), christopher j. raxworthy, david a. kizirian, david a. dickey, and lauren vonnahme (amnh), joseph martinez and josé rosado (mcz), and gunther köhler and linda acker (smf), for allowing examination of material in their care. we also thank ka schuster (philippsuniversität marburg, germany) for reading and commenting on a draft of this publication, and olivier s.g. pauwels (rbins) and lee l. grismer (lsuhc) for their helpful reviews, which greatly improved this publication. this study was supported by an amnh collection study grant to sm. references amarasinghe, a.a.t., vogel, g., mcguire, j.a., sidik, i., supriatna, j., ineich, i. 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(1997): biogeography of indonesian snakes. j. biogeogr. 24: 725-735. howard, s.d., gillespie, g.r. (2007): two new calamaria (serpentes) species from sulawesi, indonesia. j. herpetol. 41: 237-242. iskandar, d.t., rachmansah, a., umilaela (2011): a new bent-toed gecko of the genus cyrtodactylus gray, 1827 (reptilia, gekkonidae) from mount tompotika, eastern peninsula of sulawesi, indonesia. zootaxa 2838: 65-78. kluge, a.g. (1985): notes on gekko nomenclature (sauria: gekkonidae). zool. meded. 59: 95-100. koch, a. (2011): the amphibians and reptiles of sulawesi: underestimated diversity in a dynamic environment. in: biodiversity hotspots, pp. 383-404. zachos, f.e. & habel, j.c., eds, springer-verlag, heidelberg. koch, a. (2012): discovery, diversity and distribution of the amphibians and reptiles of sulawesi and its offshore islands. chimaira, frankfurt am main. koch, a., arida, e., riyanto, a., böhme, w. (2009): islands between the realms: a revised checklist of the herpetofauna of the talaud archipelago, indonesia, with a discussion about its biogeographic affinities. bonn. zool. beitr. 56: 107-129. köhler, g. (2012): color catalogue for field biologists. herpeton, offenbach. kramer, e. (1979): typenkatalog der echsen im naturhistorischen museum basel (bm). stand 1978. rev. suisse zool. 86: 159-166. linkem, c.w., mcguire, j.a., hayden, c.j., setiadi, i.m., bickford, d.p., brown, r.m. (2008): a new species of bent-toed gecko (gekkonidae: cyrtodactylus) from sulawesi island, eastern indonesia. herpetologica 64: 224-234. lisle, h.f. de, nazarov, r.a., raw, l.r.g., grathwohl, j. (2013): gekkota. catalog of recent species. private printing. manthey, u., grossmann, w. (1997): amphibien & reptilien südostasiens. natur und tier-verlag, münster. mckay, j.l. (2006): a field guide to the amphibians and reptiles of bali. krieger publishing company, malabar. mecke, s., kieckbusch, m., hartmann, l., kaiser, h. (2016): historical considerations and comments on the type series of cyrtodactylus marmoratus gray, 1831, with an updated comparative table of the bent-toed geckos of the sunda islands and sulawesi. zootaxa. mertens, r. (1929): zwei neue haftzeher aus dem indoaustralischen archipel (rept.). senckenbergiana 11: 237-241. mertens, r. (1934): die amphibien und reptilien der deutschen limnologischen sunda-expedition. suppl.bd. 12: tropische binnengewässer, bd. 4: arch. hydrobiol. 12: 677-701. müller, f. (1895a): reptilien und amphibien aus celebes, (i. bericht). verh. naturforsch. ges. basel 10: 825843. müller, f. (1895b): reptilien und amphibien aus celebes, (ii. bericht). verh. naturforsch. ges. basel 10: 862869. oliver, p., edgar, p., mumpuni, iskandar, d.t., lilley, r. (2009): a new species of bent-toed gecko (cyrtodactylus: gekkonidae) from seram island, indonesia. zootaxa 2115: 47-55. 160 sven mecke et alii riyanto, a., mumpini (2013): herpetofauna di taman nasional bali barat. prosiding seminar nasional biologi-ipa (conference paper), surabaya, indonesia: 1-17. riyanto, a., grismer, l.l., wood, jr., p.l. (2015): the fourth bent-toed gecko of the genus cyrtodactylus (squamata: gekkonidae) from java, indonesia. zootaxa 4059: 351-363. riyanto, s., kurniati, h., engilis, jr., a. (2016): a new bent-toed gecko (squamata: gekkonidae) from the mekkonga mountains, south east sulawesi, indonesia. zootaxa 4109: 59-72. rooij, n., de (1915): the reptiles of the indo-australian archipelago. vol. i.: lacertilia, chelonia, emydosauria. e. j. brill, leiden. rösler, h., richards, s.j., günther, r. (2007): remarks on morphology and taxonomy of geckos of the genus cyrtodactylus gray, 1827, occurring east of wallacea, with descriptions of two new species (reptilia: sauria: gekkonidae). salamandra 43: 193-230. rösler, h., glaw, f. (2008): a new species of cyrtodactylus gray, 1827 (squamata: gekkonidae) from malaysia, including a literature survey of mensural and meristic data in the genus. zootaxa 1729: 8-22. sabaj pérez, m.h. (ed.) (2014): standard symbolic codes for institutional resource collections in herpetology and ichthyology: an online reference. version 5.0 (22 september 2014). american society of ichthyologists and herpetologists, washington, d.c., usa. available from http://www.asih.org/ (accessed: 14 january 2016). wanger, t.c., motzke, i., saleh, s., iskandar, d.t. (2011): the amphibians and reptiles of the lore lindu nationa park area. central sulawesi, indonesia. salamandra 47: 17-29. whitten, t., henderson, g.s., mustafa, m. (2001): ecology of sulawesi. periplus editions, singapore. appendix specimens examined for diagnosis and comparison cyrtodactylus fumosus.—indonesia: north sulawesi province: bone mountains (pegunungan bone, 1200 m a.s.l.): nmb 2662 (holotype); mount masarang: nmb 2663; rurukan: bmnh 1895.2.27.7, 1896.12.9.3. cyrtodactylus halmahericus.—indonesia: north maluku province: north halmahera: mcz herp r-19279, smf 8230 (paratype); central halmahera: oba (payahe): smf 8232 (paratype); soah konorah (soakonora): smf 8233 (holotype). cyrtodactylus jellesmae.—indonesia: central sulawesi province: malakosa, kuala navusu: amnh r142969-73; tolai, sungai river: amnh r142974; north sulawesi province: kema: nmb-rept 2659 (paralectotype); buol: nmb-rept 2660 (lectotype); mount masarang: nmb-rept 2661 (paralectotype); pulau biaro: mcz 171466; south sulawesi province: lowah (muara loa): mcz 25337. cyrtodactylus klakahensis.—indonesia: jawa timur province: lumajang, klakah: smf 22476 (holotype); smf 22477-79 (paratypes). cyrtodactylus marmoratus.—indonesia: java: rmnh.rena 2710.1-8 (paralectotypes), rmnh.rena 2710a.1 (lectotype), rmnh.rena 2710a.2-6 (paralectotypes), mtkd 8903-05, smf 8218; west java: rmnh.rena 9847, zma.rena 15387 (three specimens); jawa barat province: garoet (garut regency): rmnh.rena 9846 (three specimens), rmnh.rena 10114 (two specimens), kamodjang (kawah kamojang): rmnh.rena 9849; jawa tengah province: “goewa djatidjadjar jdjoe bagelen” (= gua jatijajar, kebumen); karangpucung: smf 92361; jawa timur province: malang: rmnh.rena 9848 (two specimens). cyrtodactylus petani.—indonesia: jawa timur province: toelong agoeng (tulungagung regency): zma.rena 11353. acta herpetologica vol. 11, n. 2 december 2016 firenze university press predator-prey interactions between a recent invader, the chinese sleeper (perccottus glenii) and the european pond turtle (emys orbicularis): a case study from lithuania vytautas rakauskas1,*, rūta masiulytė1, alma pikūnienė2 effective thermoregulation in a newly established population of podarcis siculus in greece: a possible advantage for a successful invader grigoris kapsalas1, ioanna gavriilidi1, chloe adamopoulou2, johannes foufopoulos3, panayiotis pafilis1,* the unexpectedly dull tadpole of madagascar’s largest frog, mantidactylus guttulatus arne schulze1,*, roger-daniel randrianiaina2,3, bina perl3, frank glaw4, miguel vences3 thermal ecology of podarcis siculus (rafinesque-schmalz, 1810) in menorca (balearic islands, spain) zaida ortega*, abraham mencía, valentín pérez-mellado growth, longevity and age at maturity in the european whip snakes, hierophis viridiflavus and h. carbonarius sara fornasiero1, xavier bonnet2, federica dendi1, marco a.l. zuffi1,* redescription of cyrtodactylus fumosus (müller, 1895) (reptilia: squamata: gekkonidae), with a revised identification key to the bent-toed geckos of sulawesi sven mecke1,*,§, lukas hartmann1,2,§, felix mader3, max kieckbusch1, hinrich kaiser4 the castaway: characteristic islet features affect the ecology of the most isolated european lizard petros lymberakis1, efstratios d. valakos2, kostas sagonas2, panayiotis pafilis3,* sources of calcium for the agamid lizard psammophilus blanfordanus during embryonic development jyoti jee1, birendra kumar mohapatra2, sushil kumar dutta1, gunanidhi sahoo1,3,* mediodactylus kotschyi in the peloponnese peninsula, greece: distribution and habitat rachel schwarz1,*, ioanna-aikaterini gavriilidi2, yuval itescu1, simon jamison1, kostas sagonas3, shai meiri1, panayiotis pafilis2 swimming performance and thermal resistance of juvenile and adult newts acclimated to different temperatures hong-liang lu, qiong wu, jun geng, wei dang* olim palus, where once upon a time the marsh: distribution, demography, ecology and threats of amphibians in the circeo national park (central italy) antonio romano1,*, riccardo novaga2, andrea costa1 on the feeding ecology of pelophylax saharicus (boulenger 1913) from morocco zaida ortega1,*, valentín pérez-mellado1, pilar navarro2, javier lluch2 notes on the reproductive ecology of the rough-footed mud turtle (kinosternon hirtipes) in texas, usa steven g. platt1, dennis j. miller2, thomas r. rainwater3,*, jennifer l. smith4 heavy traffic, low mortality tram tracks as terrestrial habitat of newts mikołaj kaczmarski*, jan m. kaczmarek book review: sutherland, w.j., dicks, l.v., ockendon, n., smith, r.k. (eds). what works in conservation. open book publishers, cambridge, uk. http://dx.doi.org/10.11647/obp.0060 sebastiano salvidio acta herpetologica 11(2): 233-234, 2016 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-18614 book review: sutherland, w.j., dicks, l.v., ockendon, n., smith, r.k. (eds). what works in conservation. open book publishers, cambridge, uk. http://dx.doi.org/10.11647/obp.0060 sebastiano salvidio dipartimento di scienze della terra, dell’ambiente e della vita (distav), università di genova, i 16132 genova italy. e-mail: salvidio@dipteris.unige.it the book “what works in conservation” is the product of the “conservation evidence” project (www.conservationevidence.com) which consists of four parts: i) a searchable database, ii) synopses of the evidence reported in the database for different species, habitats and conservation interventions, iii) the book itself and iv) the journal “conservation evidence”. in this open access journal, evidences of management actions and their post hoc monitoring are always reported on, usually by the comparison with a control or a previous situation. by the way, it is worth noticing that a recent special issue of the journal dedicated to amphibians has been recently published (meredith et al., 2016). the volume “what works in conservation” consists of an short “introduction” (pages 1-7) and seven chapters dedicated to different animal taxa, habitats or conservation interventions: 1) amphibian conservation (pages 9-65); 2) bat conservation (pages 67-93); 3) bird conservation (pages 95-244); 4) farmland conservation (pages 245-284); 5) some aspects of control of freshwater invasive species (pages 285-292); 6) some aspects of enhancing natural pest control (pages293-315) and finally 7) enhancing soil fertility (pages 317-338). the interventions are listed according to iucn categories, while worldwide conservation evidences were obtained by reviewing the available scientific literature in english and, when needed, also in other languages. two criteria are requested to be included in the assessment: first the intervention was fully implemented in the field and second the effects of intervention were monitored scientifically, to allow some kind of statistical inference about the results. therefore, this approach excludes predictive species modelling and also correlative studies that are sometimes used to plan or realize conservation projects. the book is not descriptive or based on illustrated case studies, as is the case of conventional books on conservation (e.g., sutherland, 2000), but is a synthetic guide intended to provide a rapid overview of the scientific evidence as obtained from specialized literature. effectiveness and harmful effects of conservation actions or management interventions are assessed by a panel of experts cited in the first page of each chapter. experts were asked to classify interventions in six categories from “beneficial” to “likely to be ineffective or harmful” (table 1). the experts were asked to judge anonymously the evidence and the certainty for each intervention and to review their own judgment after seeing the overall scores and comments from the entire panel. revision rounds were stopped after a large consensus among experts was achieved. this method, based on published data judged by experts, is a modified delphi technique, which is now becoming a relevant decision tool in ecology and biodiversity conservation (mukherjee et al., 2015). references to the reviewed literature are not reported within the book, but the link to the online literature database is always given and, therefore, the reader is bound to a web connection to retrieve citations and deepen each conservation outcome. in this review i will comment only on the first chapter regarding “amphibian conservation”. the expert panel for amphibians was composed by 28 scientists and man234 sebastiano salvidio agers and at a first glance it is clearly european union biased (14/28 = 50% of experts) with a large prevalence of uk experts (10 out of 14). experts from usa constituted the 19% (5/28), africans the 15% (4/28) and asians only the 7% (2/28). in this panel, the scarcity of south american amphibian conservationists, represented by only one member, is also noticeable. many different threats were assessed in the chapter “amphibian conservation”: agriculture, urban development, transportation, collecting, logging and habitat modification. for each threat, a table with the final judgment of the experts on the conservation action is given, following the classification given in the “introduction” (see also table 1). then, a short text explaining the scientific bases on how the consensus was reached and in particular the number of studies, countries in which the actions were implemented and their main results is shortly given. in addition, the experts scored “effectiveness”, “certainty” and “harms” related to the intervention, expressed as percentages. going through the many different conservations actions assessed to reduce amphibian threats, some well-known interventions are confirmed to have large beneficial effects, with little or no harm at all, such as “remove or control fish by drying out ponds”, “deepen ponds to prevent desiccation” or “create ponds”. on the other hand, there are some interesting responses to some long-debated conservation actions, such as “commercially breed amphibians for the pet trade” or “use amphibians sustainably”, for which no scientific evidence was found. another example is the response about interventions to reduce population declines of amphibians crossing paved roads. thus, the common practice to “use humans to assist migrating amphibians across roads” (i.e., the use of volunteers to rescue toads on roads), was evaluated by the panel of experts as “unlikely to be beneficial”. in this specific case, the best alternative conservation action should be “close roads during seasonal amphibian migration” or “modify gully pots or kerns”. in short, the volume “what works in conservation” is an original, useful and practical tool for conservationists, managers, activists of non-governmental organizations and also for amphibian ecologists, all of them will obtain relevant information about conservation actions to be realized or eventually to be avoided, this latter information almost never discussed in classic conservation textbooks. the book should always be consulted before (and i stress the word “before”) planning any kind of conservation intervention to correctly evaluate, not only possible positive outcomes but, also non-desired and collateral harmful effects. it should also be used by local and national authorities that are charged to judge and fund biodiversity conservation actions. these actions are sometimes based not on scientific evidence but only on some self-assessed evaluation. the fact that “what works in conservation” is online and downloadable free of charges should facilitate its wide consultation by private and public entities working on the long-term conservation of amphibian populations. references meredith, h.m.h., van buren, c., antwis, r.e. (2016): making amphibian conservation more effective. conservation evidence 13: 1-6. mukherjee, n., hugé, j., sutherland, w.j., mcneill, j., van opstal, m., dahdouh-guebas, f., koedam, n. (2015). the delphi technique in ecology and biological conservation: applications and guidelines. methods ecol. evol. 6: 1097-1109 sutherland, w.j. (2000):the conservation handbook. research, management and policy. blackwell science, oxford, uk. table 1. synthesis of the categories for judging conservation interventions used in “what works in conservation”. intervention result short definition beneficial evidence for high effectiveness and no harm likely to be beneficial evidence for medium effectiveness and low harm trade-off between benefit and harms both effectiveness and harm present; to be evaluated according to local circumstances unknown effectiveness insufficient or inadequate quality of data unlikely to be beneficial lack of effectiveness not clearly demonstrated by data; no agreement among experts likely to be ineffective or harmful ineffectiveness or harm clearly demonstrated by data acta herpetologica vol. 11, n. 2 december 2016 firenze university press predator-prey interactions between a recent invader, the chinese sleeper (perccottus glenii) and the european pond turtle (emys orbicularis): a case study from lithuania vytautas rakauskas1,*, rūta masiulytė1, alma pikūnienė2 effective thermoregulation in a newly established population of podarcis siculus in greece: a possible advantage for a successful invader grigoris kapsalas1, ioanna gavriilidi1, chloe adamopoulou2, johannes foufopoulos3, panayiotis pafilis1,* the unexpectedly dull tadpole of madagascar’s largest frog, mantidactylus guttulatus arne schulze1,*, roger-daniel randrianiaina2,3, bina perl3, frank glaw4, miguel vences3 thermal ecology of podarcis siculus (rafinesque-schmalz, 1810) in menorca (balearic islands, spain) zaida ortega*, abraham mencía, valentín pérez-mellado growth, longevity and age at maturity in the european whip snakes, hierophis viridiflavus and h. carbonarius sara fornasiero1, xavier bonnet2, federica dendi1, marco a.l. zuffi1,* redescription of cyrtodactylus fumosus (müller, 1895) (reptilia: squamata: gekkonidae), with a revised identification key to the bent-toed geckos of sulawesi sven mecke1,*,§, lukas hartmann1,2,§, felix mader3, max kieckbusch1, hinrich kaiser4 the castaway: characteristic islet features affect the ecology of the most isolated european lizard petros lymberakis1, efstratios d. valakos2, kostas sagonas2, panayiotis pafilis3,* sources of calcium for the agamid lizard psammophilus blanfordanus during embryonic development jyoti jee1, birendra kumar mohapatra2, sushil kumar dutta1, gunanidhi sahoo1,3,* mediodactylus kotschyi in the peloponnese peninsula, greece: distribution and habitat rachel schwarz1,*, ioanna-aikaterini gavriilidi2, yuval itescu1, simon jamison1, kostas sagonas3, shai meiri1, panayiotis pafilis2 swimming performance and thermal resistance of juvenile and adult newts acclimated to different temperatures hong-liang lu, qiong wu, jun geng, wei dang* olim palus, where once upon a time the marsh: distribution, demography, ecology and threats of amphibians in the circeo national park (central italy) antonio romano1,*, riccardo novaga2, andrea costa1 on the feeding ecology of pelophylax saharicus (boulenger 1913) from morocco zaida ortega1,*, valentín pérez-mellado1, pilar navarro2, javier lluch2 notes on the reproductive ecology of the rough-footed mud turtle (kinosternon hirtipes) in texas, usa steven g. platt1, dennis j. miller2, thomas r. rainwater3,*, jennifer l. smith4 heavy traffic, low mortality tram tracks as terrestrial habitat of newts mikołaj kaczmarski*, jan m. kaczmarek book review: sutherland, w.j., dicks, l.v., ockendon, n., smith, r.k. (eds). what works in conservation. open book publishers, cambridge, uk. http://dx.doi.org/10.11647/obp.0060 sebastiano salvidio acta herpetologica 10(2): 129-134, 2015 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-16512 body size, reproduction and feeding ecology of pleurodema diplolister (amphibia: anura: leiuperidae) from caatinga, pernambuco state, northeastern brazil josé guilherme g. sousa¹,*, robson waldemar ávila² ¹ programa de pós-graduação em bioprospecção molecular, departamento de química biológica, universidade regional do cariri – urca, brazil. *corresponding author. e-mail: sousajgg@gmail.com ² departamento de ciências biológicas, universidade regional do cariri urca, brazil submitted on 2015, 29th august; revised on 2015, 25th september; accepted on 2015, 28th september editor: sebastiano salvidio abstract. we present data about body size, sexual dimorphism, reproductive traits and diet ecology of pleurodoma diplolister. this species is sexually dimorphic with females larger than males, corroborating others leiuperidae species. the number of eggs varied from 62 to 1241 and we found a positive relationship between svl of females and number of mature ovarian eggs but there is no relationship between svl and volume of eggs. the diet of p. diplolister was composed by 11 categories of which formicidae, coleoptera and orthopterans were the most important items and showed generalist and oportunistic predator habits. data presented here should be considered in the development of future conservation strategies of anurans from caatinga biome and other semiarid/arid environments. keywords. ecology, body size, reproduction, sexual dimorphism, diet, pleurodema diplolister. frogs of the genus pleurodema inhabit mainly dry forests and open areas (andrade and vaz-silva, 2012). they are medium sized anurans that frequently show adaptations to arid environments, such as morphology, explosive reproduction strategies associated to rainfall, fossorial habits and fast larval development (gould and vrba, 1982; hodl, 1992; cardoso and arzabe, 1993). pleurodema diplolister (peters, 1870) is the only known species of the genus inhabiting the caatinga, which is an arid and exclusive biome of brazil (cardoso and arzabe, 1993), but p. diplolister could also be found in brazilian cerrado and transition zones between caatinga-cerrado (vitt et al., 2005; valdujo et al., 2011; andrade and vazsilva, 2012; roberto et al., 2013). data about ecology of p. diplilister are scarce and based on reproduction behavior (hodl, 1992) and feeding ecology (santos et al., 2003) of two diferent localities from caatinga biome. herein, we present data on body size, sexual dimorphism, reproduction and feeding ecology of the frog p. diplolister at a gallery forest, from caatinga, pernambuco state, northeastern brazil. the study was conducted on february 2012 at the angico farm (08°07’s; 40°05’w), located at the rural zone of the municipality of ouricuri, pernambuco state, brazil. the vegetation is characterized mainly by deciduous forest and hypoxerophytic caatinga (velloso et al., 2001). local climate is hot and semiarid, with the rainy period ranging from october to april and the mean annual precipitation ranging from 500-800 mm (velloso et al., 2001). we collected p. diplolister specimens by pitfall traps with drift fences and by hand, in a gallery forest near a temporary river during and after one of the few rains occurred in that year. four sets of pitfalls traps were placed, each set is composed by 4 buckets (20 l) organized in a “y” form (cechin and martins, 2000) with the central bucket connected to three buckets at extremities by a drift fence about 8 m in lenght and 50 cm in height. 130 josé guilherme g. de sousa, robson waldemar ávila the specimens were euthanatized with a lethal injection of lidocaine chlorohydrate 2%, fixed in 10% formalin and then preserved in 70% ethanol. voucher specimens were deposited at coleção herpetológica da universidade regional do cariri (urca-h: 1068, 2852-2915, 29252926). for each adult individual we measured with a digital caliper the following variables (± 0.01 mm): snout-vent length (svl), head length (hl), head width (hw), interorbital distance (iod), internarinal distance (ind), eyenostril distance (end), eye diameter (eyd), tympanum diameter (td), femur length (fml), tibia length (tl), tarsal length (tal) and foot length (fol). sex was assigned by dissection and direct examination of the gonads. to examine morphological variation due to sex, we made a multivariate discriminant function analysis with residuals of morphological variables to test if significant sexual differences exist in the body shape (using all morphometric variables) and among individual morphometric variables. the residuals from a linear regression between each variable and svl were used to remove the effect of body size in the analysis of sexual dimorphism. clutch size was determined by counting a sample and then estimating the number of mature ovarian eggs. we recorded length and width (± 0.01 mm) of five eggs in females and testicles in males and estimated the volume for each using the ellipsoid formula: v = 4 3 π w 2 ⎛ ⎝⎜ ⎞ ⎠⎟ 2 l 2 ⎛ ⎝⎜ ⎞ ⎠⎟ where w and l represent width and length, respectively. we consider males analyzed as reproductively mature (adult) because they all showed convoluted epididymides. we considered females as reproductive actively when they possessed mature ovarian eggs. we use residuals of morphometric variables to remove the effect of size and test for sexual dimorphism when showed ovarian eggs or convoluted oviducts. we tested for relationships between body size and either egg number (females) or testicle volume (males) using the non-parametric spearman correlation test. to verify if svl influenced the volume of eggs in the ovarian, we use a simple linear regression. stomachs were removed from preserved specimens and examined under a stereomicroscope to identify prey items to the most inclusive taxonomic level possible (e.g., order). prey items that were too fragmented to allow a reliable estimate of volume were excluded, and items that could not be identified due to advanced stage of digestion were included in the “other arthropods” category. we recorded length and width (0.01 mm) of intact items with digital calipers and estimated prey volume (v) using the ellipsoid formula detailed above. diet composition was determined based on number (n), volume (v) and frequency (f) of each prey type in the stomachs for individual frog and for pooled stomachs. to determine the relative contribution of each prey category, we calculated the relative importance index (i) using the formula (powell et al., 1990): i = f%+ n%+v% 3 where f%, n% and v% are the percentages of frequency, number and volume of preys, respectively. dietary niche breadth (b) was calculating using the inverse of simpson’s diversity index (simpson, 1949): b = 1 / i=1 n pi 2 where p is the numeric proportion of prey category i, and n is the number of categories. values of b vary from one (usage of a single category = specialized diet) to n (equal usage of all categories = generalist diet). to evaluate the food niche overlap between the males and females, we use pianka’s overlap (pianka, 1973) index in ecosim 7.0 (gotelli and entsminger, 2004): øjk = i=1 n pij pik i=1 n pij 2( ) pik 2( ) where i is the category of the resource, p is the proportion of resource category i, j and k represents compared sexes and n is the total number of categories. the overlap ranges from 0 (no overlap) to 1 (total overlap). to evaluate the relationship between prey volume and svl, hl and hw we performed spearman correlations. unless otherwise stated, we performed all statistical analysis using statistica software version 10.0 (statsoft, inc 2011), presented means with ± standard deviation and a significance level of α = 0.05. we collected 67 specimens of p. diplolister (28 adult males, 37 adult females and two juveniles females). females were significantly larger than males in the body shape (wilks λ = 0.6617; f12,51 = 2.1726; p = 0.027) and individuals variables showed significantly difference in tl (wilks λ= 0.72; f1,51= 0.91; p = 0.033) and tal (wilks λ= 0.76; f1,51 = 7.90; p = 0.007) (table 1). 131ecology of pleurodema diplolister thirty-three females presented ovarian egg, ranging from 62 to 1241 (mean = 573.18 ± 289.06). mature ovarian eggs (n = 18915) averaged 1.08 ± 0.13 mm in length, 0.93 ± 0.11 mm in width and 0.68 ± 0.23 mm3 in volume. the smallest reproductive female was 25.33 mm svl. we found no relation between female svl and volume of eggs (r² = 0.7853; f1,31 = 2.6421; p = 0.11), however, there was positive significant correlation between female svl and number of eggs (rs = 0.595; p < 0.05). the smallest reproductive male was 23.65 mm svl and there was no correlation between males svl and testicles volume (rs = 0.395; p > 0.05). only twenty-two specimens (32.8% of the total, being 21 adult individuals, 11 males, 10 adult females, and one juvenile female) had a total of 59 prey items in their stomachs. mean diversity of preys by stomach was 1.75 ± 1.22, with females having more diversified diet (2.22 ± 1.39), than males (1.18 ± 0.6). females juvenile ingested four prey types. eleven categories of prey items were identified in diet of p. diplolister: hymenopetarns of the family formicidae (45.76%) and the order coleoptera (20.34%) were more important numerically in diet composition, whereas coleoptera (29.56%), orthoptera (29.02%) and formicidae (19.19%) were more important volumetrically. regarding the relative importance of each prey item, formicidae (31.56%), coleoptera (22.94%), orthoptera (14.07%) were the main contributors in the diet composition (table 2). niche breadth of p. diplolister was 3.74 in number and 4.45 in volume. food niche overlap between the males and females was 0.7071. there was no correlation between prey volume and: svl (rs = 0.1968; p > 0.05), hl (0.1111; p >0.05), or hw (0.2415; p > 0.05). sexual differences in body size are common among anurans, with approximately 90% of the species having females larger than males, but males could be equal size or larger than females in those species which males engage physical combat (shine, 1979). that pattern was also found in p. diplolister, where females tend to be larger and more robust than males. many leiuperids follow this trend at many places of south american (table 3). additionaly, different populations of pleurodema thaul (lesson, 1827) could (or not) present sexual dimorphism, as was demonstrated by iturra-cid et al. (2010) from chile at different latitudes/longitudes. in the present study, the average egg volume remains constant and independent of female svl. however, larger females produce more eggs, resulting in the large difference in the amount of eggs found in ovaries. this pattern can most likely increase the reproductive success rate, once larger females producing more eggs will result in a greater amount of individuals that will hatch and reach maturity. the number of ovarian mature eggs found here was similar to that reported by hodl (1992) in a population of p. diplolister from paraiba state, with 528-742 eggs reported in foam nests. apparently, body size has an influence on egg number of leiuperid species, since larger species presented table 1. morphological variables of pleurodema diplolister from a caatinga area, pernambuco state, northeasthern brazil: discriminant function analyses results (dfur). agmv= all grouped morphometric variables, end = eye-nostril distance, eyd = eye diameter, fol= foot length, hl= head length, hw = head width, ind= internarial distance, iod = interorbital distance, td = tympanum diameter, fml= femur length, svl= snout-vent length, tl= tibia length, tal= tarsal length. all values are given in mm. females (n= 37) mean (range) males (n = 28) mean (range) dfur wilks λ (p-value) svl 33.8 (25.3–38.0) 31.4 (23.6–36.4) 0.807 (0.002) hl 14.3 (12.7–15.7) 13.6 (10.1–15.8) 0.662 (0.960) hw 13.3 (11.5–15.0) 12.6 (9.1–14.1) 0.679 (0.254) iod 5.9 (5.2–6.8) 5.7 (4.7–6.6) 0.665 (0.638) end 3.1 (2.3–3.5) 2.9 (1.2–3.6) 0.662 (0.913) ind 3.2 (2.9–4.1) 3.2 (2.4–3.7) 0.665 (0.622) eyd 4.5 (3.6–5.4) 4.5 (3.2–5.1) 0.665 (0.617) td 1.9 (1.2–2.4) 1.9 (1.9–2.6) 0.693 (0.128) fml 13.5 (11.7–15.3) 12.9 (9.6–14.7) 0.670 (0.438) tl 13.6 (12.0–14.7) 13.2 (10.0–15.0) 0.724 (0.033) tal 8.5 (7.0–10.3) 7.9 (5.9–9.5) 0.764 (0.007) fol 14.8 (13.4–16.4) 14.2 (11.2–16.5) 0.664 (0.707) agmv 0.6617 (0.027) table 2. diet composition of pleurodema diplolister from a caatinga area, pernambuco state, northeasthern brazil. f = frequency; n = abundance; v = volume; i = relative importance index. prey item f f % n n% v v% i araneae 4 10.81 4 6.78 33.79 6.61 8.07 coleoptera 7 18.92 12 20.34 151.07 29.56 22.94 diptera 1 2.70 1 1.70 1.22 0.24 1.55 hemiptera 1 2.70 1 1.70 1.12 0.22 1.54 hymenoptera formicidae 11 29.73 27 45.76 98.09 19.19 31.56 others 2 5.41 3 5.09 53.32 10.43 6.97 lepidoptera 2 5.41 2 3.39 0.82 0.16 2.99 neuroptera 1 2.70 1 1.70 0.98 0.19 1.53 orthoptera 3 8.11 3 5.09 148.31 29.02 14.07 others arthropods 4 10.81 4 6.78 10.95 2.14 6.58 seeds 1 2.70 1 1.70 11.4 2.23 2.21 total 37 59 511.07 niche breadth   3.74 4.45   132 josé guilherme g. de sousa, robson waldemar ávila more eggs both in ovarian and in foam nests; on the other hand, breeding activity patterns (explosive or continuous) does not affect egg number in this anuran family (see table 3). additional studies with another populations of p. diplolister are necessary to confirm this patterns. amphibians of arid environments usually are active only two-three months in a year (during rainy season), and then start aestivation process (carvalho et al., 2010). in caatinga, p. diplolister stay on aestivate ten or eleven months, emerging from the burrow at first rains, and start to reproduce explosively in temporary water bodies (carvalho et al., 2010). feeding in p. diplolister starts just after reproduction (carvalho et al., 2010), which may explain the large number of empty stomachs observed in this study. we found no correlation between svl and prey size. based on the numeric and volumetric niche breadth, we consider p. diplolister as a generalist and opportunistic predator. the food niche overlap between males and females was relatively high, which was already expected since males and females use similar microhabitats to forage and reproduce in the sampled area (sousa pers. obs.). the diet of pleurodema species has been reported from some countries of south america: in brazil, santos et al. (2003) found chilopoda, isoptera and coleoptera as the most important prey item for p. diplolister, whereas formicidae, coleoptera and orthoptera were the most important prey categories found in the present study; in chile, ants and insect larvae were the most important item for pleurodema bufoninum  bell, 1843 (pincheiradonoso, 2002) and diptera and arachnids for pleurodema thaul (schneider, 1799) (díaz-páez and ortiz, 2003); in argentina, dipterans (stratiomyidae), spiders (araneidae) and beetles (coleoptera) to pleurodema cinereum cope, 1878 (hulse, 1979) and hemiptera and hymenoptera to pleurodema nebulosum (burmeister, 1861) (sanabria et al., 2007). considering that p. diplolister is a generalist and opportunist predator, these differences in diet composition are probably due to variation in abundance and prey diversity of each studied area. the population of p. diplolister studied showed generalist and opportunistic predator habits, and males and females showed high food niche overlap, which is probably due the similarity in microhabitat use. pleurodema diplolister also presented sexual dimorphism (females larger than males), whereas probably as result of sexual selection, larger females (and probably older) had greater amount of mature ovarian eggs than smaller females. other studies with leiuperines are required to confirm the reproductive pattern here discussed. data presented here should be considered in the development of future conservation strategies of anurans from caatinga biome and other semiarid/arid environments. acknowledgements we would like to thanks to pró-reitoria de pós graduação e pesquisa da universidade regional do cariri for financial support (chamada pública prpgpurca 03/2013). to fundação cearense de apoio ao desenvolvimento científico e tecnológico – funcap for the research grant awarded to rwa (bpi-006700006.01.00/12) and fellowship to jggs. to samuel brito for provided helpful comments on previous version of the manuscript. the frogs were collected by permit of instituto chico mendes de conservação da biodiversidade icmbio (permit 29613-1). table 3. data of eleven leiuperines from south america, with snout vent─length of males and females (svl m and f), average number of mature ovarian eggs (eggs), egg diameter (ed), sexual size dimorphism (ssd), breeding activity patterns (bap) and source. specie svl eggs ed ssd bap reference m f physalaemus albonotatus 29.6 30.2 1474 0.98 absent continuous rodrigues et al. (2004) physalaemus centralis 36.3 34.5 1842 1.3 absent explosive brasileiro and martins (2006) physalaemus cuvieri ─ ─ 473.5 1.3 present ─ barreto and andrade (1995) physalaemus fernandezae 23.2 26.6 ─ ─ present ─ marangoni et al. (2011) physalaemus kroyeri 31.1 30.2 1332 absent continuous gally and zina (2013) physalaemus maculiventris 23.3 20.5 190 ─ ─ ─ heyer et al. (1990) physalaemus nattereri 47.3 51.3 3765 1.2 present explosive rodrigues et al. (2004) physalaemus signiffer 25 27.4 273.14 ─ present continuous wogel et al. (2002) pleurodema diplolister 31.44 33.79 573.18 1.08 present explosive present study pleurodema diplolister 30.7 34.3 678 ─ ─ explosive hodl (1992) pleurodema guayapae ─ 41.7 1137 1.44 ─ explosive valetti et al. (2013) 133ecology of pleurodema diplolister references andrade, s.p., vaz-silva, w. 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(2001): ecorregiões propostas para o bioma caatinga. associação plantas do nordeste; instituto de conservação ambiental. the nature conservancy do brasil, recife. 134 josé guilherme g. de sousa, robson waldemar ávila vitt, l., caldwell, j., colli, g., garda, a., mesquita, d., frança, f., shepard, d., costa, g., vasconcellos, m., silva, v. (2005): uma atualização do guia fotográfico dos répteis e anfíbios da região do jalapão no cerrado brasileiro. special publications in herpetology, sam noble oklahoma museum of natural history 2: 1-24. wogel, h., abrunhosa, p.a., pombal jr, j.p. (2002): atividade reprodutiva de physalaemus signifer (anura, leptodactylidae) em ambiente temporário. iheringia, sér. zool. 92: 57-70. acta herpetologica vol. 10, n. 1 june 2015 firenze university press acta herpetologica journal of the societas herpetologica italica acta herpetologica 11(1): 47-52, 2016 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-16940 population structure and activity pattern of one species of adenomera steindachner, 1867 (anura: leptodactylidae) in northeastern brazil maria juliana borges-leite1,*, joão fabrício mota rodrigues2, patrícia de menezes gondim1, diva maria borges-nojosa1 ¹ programa de pós-graduação em ecologia e recursos naturais, departamento de biologia, universidade federal do ceará – ufc, campus do pici, cep 60455-760, fortaleza, ce, brazil. *corresponding author. e-mail: jborgesleite@gmail.com 2 programa de pós-graduação em ecologia e evolução, departamento de ecologia, centro de biociências, universidade federal de goiás – ufg, campus samambaia, cep 74001-970, goiânia, go, brazil submitted on 2015, 23rd september; revised on 2015, 8th november; accepted on 2015, 16th november editor: fabio maria guarino abstract. we analyzed the population structure and sexual size dimorphism of an adenomera species occurring in the municipality of são gonçalo do amarante, from september 2010 to august 2011, using pitfall traps and active searches. we captured 116 individuals; 36 males, 23 females and 57 juveniles. sexual size dimorphism was not observed. the smallest individuals were found in the middle of the rainy season, and the higher abundance of juveniles during this period may be related to recruitment. females were captured in pitfall traps more often than males, while males were captured during active searches more often than females due to their calling behavior. we provide basic information regarding this adenomera population, located in a region of severe environmental degradation, which may serve as a source of information for future studies of the area aiming to evaluate how the construction of industrial complexes affects anuran populations. keywords. amphibian, ceará, leptodactylus marmoratus group, sexual size dimorphism. constant environmental degradation, mainly caused by human action, has brought about serious changes in global diversity, with habitat loss being the greatest threat to biodiversity (vitousek et al., 1997). elimination or modification of specific anuran microhabitats has been considered the main factor responsible for population declines of several species of this taxonomic group (young et al., 2001; becker et al., 2007). basic information studies detailing the population structure of anurans have increased over the years, but they are concentrated in biomes such as the cerrado (giaretta and menin, 2004; kokubum and giaretta, 2005; giaretta et al., 2008), neotropical rainforest (sá et al., 2014) and amazon forest (menin et al., 2007; kokubum and sousa, 2008; waldez et al., 2011). some studies report data on the abundance, size classes and population recruitment (which mainly occurs in the wet period) of amphibian species, and sexual dimorphism, with females typically larger than males (e.g., giaretta and menin, 2004; kokubum and giaretta, 2005; kokubum and sousa, 2008; waldez et al., 2011). in addition, adult and juvenile size, and sex-ratio data are important to better understand the population structure, expanding on knowledge that generally focuses on reproduction and diet. members of the genus adenomera are recognized by their small size (snout vent length average is 18-25mm), have terrestrial habits and some species lay their eggs in underground chambers with tadpoles completing development in foam nests (angulo et al., 2003; kokubum and giaretta, 2005; kok et al., 2007; kokubum and sousa, 2008), or in pools originated by flooding of the nest (de la riva, 1995; almeida and angulo, 2002; carvalho and 48 maria juliana borges-leite et alii giaretta, 2013) or in holes in fallen tree trunks (menin and rodrigues, 2013). in this paper, we aim to analyze the population structure, the activity pattern, and sexual size dimorphism of one species of adenomera in an area that suffers strong disturbance due to the installation of an industrial complex. furthermore, we aim to expand the knowledge of the adenomera group, and provide data for future studies that seek to resolve its current taxonomic confusion. we performed the fieldwork in the municipality of são gonçalo do amarante, ceará state, brazil (fazenda maceió da taíba: 03°30’54”s, 38°55’07”w) from september 2010 to august 2011. the climate is defined by two periods: dry (july-december) and rainy (january-june). mean annual rainfall is 1,026 mm and mean annual temperature is 27°c (funceme–fundação cearense de meteorologia e recursos hídricos). the study area is a forest fragment mostly composed of coastal, inter-dune, semi-deciduous forest (mata de tabuleiro), where approximately 382 plant species have been recorded (castro et al., 2012), largely consisting of small trees. the nearest water source is an inter-dune pond located approximately 500 to 1000 meters from the sampling point. we conducted samplings once a month lasting three days per field trip to observe a population of adenomera sp. this undescribed species has been recorded until now only from são gonçalo do amarante (borges-leite et al., 2014), and has been analyzed and confirmed by experts as a new species (kokubum, pers. comm.). this species has a seasonal reproductive pattern with breeding occurring only in wetter months, and tadpole development has no aquatic stage (borges-leite et al., 2015). active searches, unlimited by time, were performed by two people mainly in areas of forest without a water source, from 3 p.m to 12 a.m. pitfall traps were also disposed in a forest fragment of semi-deciduous forest (mata de tabuleiro), but in a different location to those used for active searches. pitfall traps were composed of a five-station line; each station (trap) consisting of four plastic buckets of 60 l arranged in a “y” (heyer et al., 2001), totaling five traps per day of effort. we sexed all captured individuals based on external morphology (shovel-shaped snout on males) and measured snout-vent length (svl) with a digital caliper (accurate to 0.01 mm) to ascertain the presence of sexual size dimorphism. to classify individuals as juvenile or adult, we used the method of waldez et al. (2011), in which the size of the smallest calling male is used to classify juvenile individuals (juveniles had svl < 18.8 mm in our study). individuals larger than 18.8 mm were classified in males or females according to the external morphology described above. we used welch’s t test to analyze sexual dimorphism in snout-vent length, since this variable had a normal distribution. we used the kruskal-wallis (k-w) test to evaluate variation in svl of juveniles, males, females, and the total population between the months in which they were sampled. we used chi-squared tests to evaluate the number of males and females captured in each sampling method used in this study (active searches and pitfall traps). all statistical analyses were performed using stats packages available in r, version 2.13.2 (r development core team, 2014). the alpha value was set to 5% for all statistical tests. descriptive statistics are presented in this paper as mean ± standard deviation. after 12 months of sampling, the applied effort was 180 traps × days (five pitfall traps × three days × 12 months) and 720 hours of active searches (10 hours × three days × 12 months × two persons). we captured 116 individuals: 36 males, 23 females and 57 juveniles (fig. 1-2 and table 1). adult females were captured in all samplings during the rainy season (january-june) and adult males were captured between january and april. we captured juveniles from february to june, with a peak in number in may. juveniles were sampled throughout the rainy season, except at its onset, in january, and their size varied during the sampling period (k-w test, h = 37.68, df = 4, p < 0.001, fig. 3a), being small at the beginning of the rainy season and larger close to the dry season. males had svl ranging between 18.8-22.6 mm (mean = 20.77 ± 0.87 mm, n = 36), while females ranged between 18.8-25.5 mm (mean = 21.46 ± 1.94 mm, n = 23). the size of males did not change during the study (k-w test, h= 2.38, df = 3, p = 0.50, fig. 3b), but svl of females (k-w test, h = 11.49, df = 4, p = 0.04, fig. 3c) and the total population (k-w test, h = 43.81, df = 4, p < 0.001, fig. 3d) were larger at the beginning of the rainy season. there was no evidence of sexual dimorphism in size (t = 1.62, df = 27.66, p = 0.11). morphological differences between males and females were observed, with males having a more shovel-shaped snout. the proportion of males and females captured in pitfall traps was significantly different with a higher number of females than males captured (χ2 = 17.19, df = 1, p < 0.001; table 1). the captures were also sex-biased in active searches, with a higher number of males than females (χ2 = 26.95, df = 1, p < 0.001; table 1). significant differences in sex ratio with a male-biased sample may result from behavioral differences between the sexes (giaretta and menin, 2004) or even the choice of sampling method (fogarty and vilella, 2002). active search, for example, may bias the result of the sex ratio towards males (fogarty and vilella, 2002; waldez et al., 2011). the male-biased capture in active search may be 49population structure and activity pattern of one species of adenomera fig. 1. snout-vent length (svl) class-size distribution in adenomera sp. females, males and juveniles. fig. 2. number of adenomera sp. males, females and juveniles sampled each month from january – june 2011 at são gonçalo do amarante, ceará, brazil. 50 maria juliana borges-leite et alii due to researchers being guided by the calling behavior of males, while females go unnoticed. this bias was also found in our results for active searches (table 1). in most anurans, there is no difference in activity pattern between males and females (lemckert, 2004). however, we found that females were captured in pitfall traps more frequently than males, suggesting higher mobility for this sex because active animals are more captured by this trap than inactive ones. females usually use larger areas than males due to increased energetic demands and nutritional requirements (muths, 2003). this difference in movement pattern was not observed by kokubum and giaretta (2005), who found a non-significant difference in the number of individuals of each sex captured in pitfall traps in uberlândia, minas gerais. amphibians that have a long reproductive period can recruit juveniles during most of the year (lópez et al., 2011). some studies of species with terrestrial reproduction showed that a peak in reproductive activity and juvenile growth occurs in the rainy season (moreira and lima, 1991; ovaska, 1991; kokubum and giaretta, 2005; borges-leite et al., 2015). once the number of juveniles is increasing in the population, a decrease in the average body size of the population is expected, as previously observed in other genera, suggesting the occurrence of recruitment events (menin et al., 2007; waldez et al., 2011). in our study the rainy season was marked by a variation in the size and number of captured juveniles, and by the occurrence of a greater abundance of juveniles than adults in the last months of the season. these characteristics suggest the existence of recruitment events in this population in the rainy period, beginning in february and peaking in may. this recruitment can also be observed by the decrease in svl of the total population during the study (fig. 3d). it can also be seen that in may, three months after recruitment started, the size of females is smaller than in previous months, indicating that some of the individuals previously classified as juveniles have already become adult. this observation in females supports the results of sá et al. (2014), with regard to adult body size variation in anurans with prolonged reproduction, although we did not find the same variation in males. although there are some studtable i. number and sex of adenomera sp. individuals captured using pitfall traps and active search methods. categories method of capture pitfall traps active searches male 1 35 female 20 3 fig. 3. variation in snout-vent length of adenomera sp. from january – june 2011. a) juveniles; b) males; c) females; and d) total population (1 = january, 2 = february, 3 = march, 4 = april, 5 = may and 6 = june). 51population structure and activity pattern of one species of adenomera ies in brazilian coastal regions focusing on reproductive patterns and sexual dimorphism of anurans (e.g. giasson and haddad, 2007), to our knowledge, our study is the first to explore the population structure of a coastal anuran. our study showed that in this population of adenomera there is no sexual size dimorphism, contrary to 90% of frog species in which females are larger than males (shine, 1979) and also to other species of the genus adenomera (e.g. kokubum and giaretta, 2005; kok et al., 2007). this sexual difference in anurans may arise, for example, because females delay their reproduction and invest more time in growth than males, which grow up fast and start reproductive activity early (zhang and lu, 2013). however, in an environment where the rainy season is reduced, such as in our study area, it may be advantageous for females not to delay reproduction. we conclude that juvenile recruitment in this population of adenomera occurs in the rainy season, and there was no sexual size dimorphism. hence, we add new important data to the problematic adenomera group. the taxonomic problems of this group, which include great intraand inter-population morphological variation and cryptic species (de la riva, 1996; angulo et al., 2003), need more population studies in order to help understand its variation. regarding the population from são gonçalo do amarante, this study may be a starting point for future studies aiming to evaluate the impact of the port and industrial complex over a year-long interval in an anuran population. at the current moment, the adenomera population has a high recruitment, favoring its maintenance, but only future detailed studies will be able to evaluate its ability to co-occur alongside strong human disturbance. acknowledgements we thank mr. and mrs. borges for accommodation and permission to use their property for research; the instituto chico mendes de conservação da biodiversidade (icmbio) for collection permits (authorization # 25935-1 sisbio); and daniel cassiano, rafael carvalho and james harris for reviewing the manuscript and providing interesting suggestions. m.j. borges-leite and j.f.m. rodrigues thank capes for a graduate fellowship. references almeida, a.p., angulo, a. (2002): adenomera aff. marmorata (ncn). reproduction. herpetol. rev. 33: 197198. angulo, a., cocroft, r.b., reichle, s. (2003): species identity in the genus adenomera (anura: leptodactylidae) in southeastern peru. herpetologica 59: 490504. becker, c., fonseca, c.r., haddad, c.f.b., batista, r.b., prado, p.i. (2007): habitat split and the global decline of amphibians. science 318: 1775-1777. borges-leite, m.j., rodrigues, j.f.m., borges-nojosa, d.m. (2014): herpetofauna of a coastal region of northeastern brazil. herpetol. notes 7: 405-413. borges-leite, m.j., rodrigues, j.f.m., gondim, p.m., borges-nojosa, d.m. (2015): reproductive activity of adenomera aff. hylaedactyla (anura: leptodactylidae) in a coastal area of ceará state, brazil. anim. biol. 65: 101-111. castro, a.s.f., moro, m.f., menezes, m.o.t. (2012): o complexo vegetacional da zona litorânea no ceará: pecém, são gonçalo do amarante. acta bot. bras. 26: 108-124. carvalho, t.r., giaretta, a.a. (2013): taxonomic circumscription of adenomera martinezi (bokermann, 1956) (anura: leptodactylidae: leptodactylinae) with the recognition of a new cryptic taxon through a bioacoustic approach. zootaxa 3701: 207–237. de la riva, i. (1995): a new reproductive mode for the genus adenomera (amphibia: anura: leptodactylidae): taxonomic implications for certain bolivian and paraguayan populations. stud. neotrop. fauna environ. 30: 15-29. de la riva, i. (1996): the specific name of adenomera (anura: leptodactylidae) in the paraguay river basin. j. herpetol. 30: 556-558. fogarty, j.h., vilella, f.j. (2002): population dynamics of eleutherodactylus coqui in cordillera forest reserves of puerto rico. j. herpetol. 36: 193-201. giaretta, a.a., menin, m. (2004): reproduction, phenology and mortality sources of a species of physalaemus (anura: leptodactylidae). j. nat. hist. 38: 1711-1722. giaretta, a.a., menin, m., facure, k.g., kokubum, m.n.c., oliveira filho, j.c. (2008): species richness, relative abundance, and habitat of reproduction of terrestrial frogs in the triângulo mineiro region, cerrado biome, southeastern brazil. iheringia, sér. zool. 98: 181-188. giasson, l.o.m, haddad, c.f.b. (2007): mate choice and reproductive biology of hypsiboas albomarginatus (anura: hylidae) in the atlantic forest, southeastern brazil. south am. j. herpetol. 2: 157-164. heyer, r.w., donnelly, m.a., hayek, l.c., foster, m.s. (2001): medición y monitoreo de la diversidad biológica: métodos estandarizados para anfíbios. editorial universitária de la patagônia, chubut. 52 maria juliana borges-leite et alii kok, p.j.r., kokubum, m.n.c., macculloch, r.d., lathrop, a. (2007): morphological variation in leptodactylus lutzi (anura, leptodactylidae) with description of its advertisement call and notes on its courtship behavior. phyllomedusa 6: 45-60. kokubum, m.n.c., giaretta, a.a. (2005): reproductive ecology and behavior of a species of adenomera (anura, leptodactylidae) with endotrophic tadpoles: systematic implications. j. nat. hist. 39: 1745-1758. kokubum, m.n.c., sousa, m.b. (2008): reproductive ecology of leptodactylus aff. hyleadactylus (anura, leptodactylidae) from an open area in northern brazil. s. am. j. herpetol. 3: 15-21. lemckert, f.l. (2004): variations in anuran movements and habitat use: implications for conservation. appl. herpetol. 1: 165-181. lópez, j.a., scarabotti, p.a., ghirardi, r. (2011): seasonal patterns of abundance and recruitment in an amphibian assemblage from the paraná river floodplain. interciência 36: 538-544. menin, m., rodrigues, d.j. (2013): the terrestrial tadpole of leptodactylus andreae (anura, leptodactylidae), a member of the lepdotactylus marmoratus species group. zootaxa 3683: 92-94. menin, m., lima, a.p., magnusson, w.e., waldez, f. (2007): topographic and edaphic effects on the distribution of terrestrially reproducing anurans in central amazonia: mesoscale spatial patterns. j. trop. ecol. 23: 539-547. moreira, g., lima, a.p. (1991): seasonal patterns of juvenile recruitment and reproduction in four species of leaf litterfrogs in central amazonia. herpetologica 47: 295-300. muths, e. (2003): home range and movements of boreal toads in undisturbed habitat. copeia 2003: 160-165. ovaska, k. (1991): reproductive phenology, population structure, and habitat use of the frog eleutherodactylus johnstonei in barbados, west indies. j. herpetol. 25: 424-430. r development core team (2014): r: a language and environment for statistical computing (on-line), vienna, r foundation for statistical computing. accessed january 20, 2014 at http://www.r-project.org/. sá, f.p., zina, j., haddad, c.f.b. (2014): reproductive dynamics of the neotropical treefrog hypsiboas albopunctatus (anura, hylidae). j. herpetol. 48: 181185. shine, r. (1979): sexual selection and sexual dimorphism in the amphibia. copeia 1979: 297-306. vitousek, p.m., mooney, h.a., lubchenco, j., melillo, j.m. (1997): human domination of earth’s ecosystems. science 277: 494-499. waldez, f., menin, m., rojas-ahumada, d.p., lima, a.p. (2011): population structure and reproductive pattern of pristimantis aff. fenestratus (anura: strabomantidae) in two non-flooded forests of central amazonia, brazil. s. am. j. herpetol. 6: 119-126. young, b.e., lip, k.r., reaser, j.k., ibañes, r., salas, a.w., cedeño, j.r., coloma, l.a., ron, s., marca, e., meyer, j.r., muñoz, a., bolamos, f., chaves, g., romo, d. (2001): population declines and priorities for amphibian conservation in latin america. conserv. biol. 15: 1213-1223. zhang, l., lu, x. (2013): sexual size dimorphism in anurans: ontogenetic determination revealed by an across-species comparison. evol. biol. 40: 84-91. acta herpetologica vol. 11, n. 1 june 2016 firenze university press feeding habits of mesoclemmys vanderhaegei (testudines: chelidae) elizângela silva brito1,*, franco leandro souza2, christine strüssmann3 morphology, ecology, and behaviour of hylarana intermedia, a western ghats frog ambika kamath1, rachakonda sreekar2,3 a new account for the endangered cerrado rocket frog allobates goianus (bokermann, 1975) (anura: aromobatidae), with comments on taxonomy and conservation thiago ribeiro de carvalho1,2,*, lucas borges martins1,2, ariovaldo antonio giaretta1 molecular phylogenetics of the pristimantis lacrimosus species group (anura: craugastoridae) with the description of a new species from colombia mauricio rivera-correa, juan m. daza* population structure and activity pattern of one species of adenomera steindachner, 1867 (anura: leptodactylidae) in northeastern brazil maria juliana borges-leite1,*, joão fabrício mota rodrigues2, patrícia de menezes gondim1, diva maria borges-nojosa1 vocal repertoire of scinax v-signatus (lutz 1968) (anura, hylidae) and comments on bioacoustical synapomorphies for scinax perpusillus species group marco antônio peixoto1,*, carla silva guimarães1, joão victor a. lacerda2, fernando leal2, pedro c. rocha2, renato neves feio1 amendment of the type locality of the endemic sicilian pond turtle emys trinacris fritz et al. 2005, with some notes on the highest altitude reached by the species (testudines, emydidae) federico marrone*, francesco sacco, vincenzo arizza, marco arculeo photo-identification in amphibian studies: a test of i3s pattern marco sannolo1,*, francesca gatti2, marco mangiacotti3,4, stefano scali3, roberto sacchi4 no evidence for the ‘expensive-tissue hypothesis’ in the dark-spotted frog, pelophylax nigromaculatus li zhao, min mao, wen bo liao* no short term effect of clinostomum complanatum (trematoda: digenea: clinostomatidae) on survival of triturus carnifex (amphibia: urodela: salamandridae) giacomo bruni1,*, claudio angelini2 yeasts in amphibians are common: isolation and the first molecular characterization from thailand srisupaph poonlaphdecha, alexis ribas* introduction of eleutherodactylus planirostris (amphibia, anura, eleutherodactylidae) to hong kong wing ho lee1, michael wai-neng lau2, anthony lau3, ding-qi rao4, yik-hei sung5,* correlation between endoscopic sex determination and gonad histology in pond sliders, trachemys scripta (reptilia: testudines: emydidae) david perpiñán1, albert martínez-silvestre2,3, ferran bargalló3, marco di giuseppe4, jorge orós5, taiana costa6,* book review: john w. wilkinson. amphibian survey and monitoring handbook. pelagic publishing franco andreone book review: susan newman. frogs amphibians and their threatened environment. discovery and expression through art k-3. frogs are green franco andreone acta herpetologica 11(1): 99-100, 2016 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-18119 book review: susan newman. frogs amphibians and their threatened environment. discovery and expression through art k-3. frogs are green franco andreone museo regionale di scienze naturali, via g. giolitti, 36, i-10123 torino, italy. e-mail: franco.andreone@regione.piemonte.it it is my strong conviction that science and research are useless without a parallel diffusion and dissemination to people. indeed, well-known evaluation methods, such as impact factor (if) and hirsch (h) indices, are rather good methods established by measuring the quotation of a paper by other (scientific) people, usually scientists in scientific journals. in other words, citation indices still remain in the same “environment”, with a scientific community basically quoting itself. on the opposite, i believe that one of the best and real indicators of a paper (or of man’s career) is the capacity to pass through walls and reach common people, in order to transmit information and provide useful education and instruction. at the end, whether this is done in early or adult age, it does not really matter. personally, i believe that this can be done in an easiest and most efficient way by specifically addressing to children. as we all know, kids are eager to learn and to understand. everything. in such a sense, talking to them about amphibians maybe a weird idea, but, at the end, a good proposal indeed, and a good way to address their interests and sensitivities in the future, specially seen the need for new generations to conserve amphibians and their environments (amphibiaweb, 2016). in such a sense the book “built” (not only and not simply written) by susan newman is for me a real jewel. in many ways. this book is mainly destined to educators and provides a true roadmap to teach young readers and explorers the love for amphibians. susan has coped her strong passion for frogs (she is the founder and the promoter of “frogs are green”, a non profit organisation created in 2009 to bring awareness to what’s happening on our planet, especially to the fact that frogs everywhere are disappearing: http://frogsaregreen.org/) with art. in such a way the aim of the organisation is to give kids the opportunity to learn about the environment, frogs and other amphibians, and to express themselves through art. in this way susan’s love for science has been put together with the great and somehow innate interest that children (in this case up to the age of 3 years) have for frogs and drawings. the book chapters (in reality, much more than a simple book, susan’s work is a sort of handbook: easy to be followed) has been designed directly by susan (who is a graphic by profession) to give educators a roadmap to teach kids about amphibians, animals that, at the end, are not easy to find in nature and even to spot in zoological parks, although frogs and toads are true characters which often inhabit childhood tales. the pages of the book are full of interesting and correct information, accompanied by a wealth of photographs as well (many of which represents frogs from my beloved madagascar), with explicit and clear indications about youtube links where looking at videos. photos represents animals and environments. although, of course, frogs are dominant all through the text, many other animal species are represented. this is because the final aim of the book is to talk about environments with the excuse of dealing with amphibians. frogs depicted in the photos are species with peculiar and fantastic colourations and forms, but also more common species and, in several cases, dead animals, as in the case of chytrid effects. i believe that is really important to show that animals and nature are not only and not always beautiful and attractive, but in many cases suffer of pol100 franco andreone lution and other kinds of threats. in this ways, kids learn and understand what should be done. as already said, one of the ways children are driven to understand (maybe love) amphibians pass through art. all through the text kids are asked to draw what they heard and learned. this is really important, since drawing activity becomes a true way to approach the inner feelings and transform the “simple” and sometimes trivial science teaching in a real sentiment for conserving fragile creatures and environments. the book is structured in six chapters. in the first chapter susan presents general aspects about frogs and amphibians. this is done with the use of photographs and solicit the small readers to draw using different colours and shapes. chapter two (the food chain) introduces the variety of ecological adaptations in amphibians using slideshows, videos and discussions. chapter three (frog friendly habitats) presents rainforests and other habitats in the world hosting a great variety of amphibians. this argument is much more given in details in chapter four (biodiversity and the rainforest). in this teachers invite kids to paint themselves as frogs. in chapter five (reproduction & care of young) the variety of breeding modes and development is illustrated in detail, soliciting the surprise of young people using videos and links. finally, in chapter six (threats to survival), amphibians are presented as fragile organisms and major threats are discussed using photos, videos and direct explanations. at the end, susan’s book turns out to be an interesting experiment where passion for animals goes together with education. this is something that researchers and academics should always take in mind when doing science: conservation, in a changing world, is possibly the first target. and passes always through comprehension and understanding. references amphibiaweb (2016): amphibiaweb: information on amphibian biology and conservation. berkeley. acta herpetologica vol. 11, n. 1 june 2016 firenze university press feeding habits of mesoclemmys vanderhaegei (testudines: chelidae) elizângela silva brito1,*, franco leandro souza2, christine strüssmann3 morphology, ecology, and behaviour of hylarana intermedia, a western ghats frog ambika kamath1, rachakonda sreekar2,3 a new account for the endangered cerrado rocket frog allobates goianus (bokermann, 1975) (anura: aromobatidae), with comments on taxonomy and conservation thiago ribeiro de carvalho1,2,*, lucas borges martins1,2, ariovaldo antonio giaretta1 molecular phylogenetics of the pristimantis lacrimosus species group (anura: craugastoridae) with the description of a new species from colombia mauricio rivera-correa, juan m. daza* population structure and activity pattern of one species of adenomera steindachner, 1867 (anura: leptodactylidae) in northeastern brazil maria juliana borges-leite1,*, joão fabrício mota rodrigues2, patrícia de menezes gondim1, diva maria borges-nojosa1 vocal repertoire of scinax v-signatus (lutz 1968) (anura, hylidae) and comments on bioacoustical synapomorphies for scinax perpusillus species group marco antônio peixoto1,*, carla silva guimarães1, joão victor a. lacerda2, fernando leal2, pedro c. rocha2, renato neves feio1 amendment of the type locality of the endemic sicilian pond turtle emys trinacris fritz et al. 2005, with some notes on the highest altitude reached by the species (testudines, emydidae) federico marrone*, francesco sacco, vincenzo arizza, marco arculeo photo-identification in amphibian studies: a test of i3s pattern marco sannolo1,*, francesca gatti2, marco mangiacotti3,4, stefano scali3, roberto sacchi4 no evidence for the ‘expensive-tissue hypothesis’ in the dark-spotted frog, pelophylax nigromaculatus li zhao, min mao, wen bo liao* no short term effect of clinostomum complanatum (trematoda: digenea: clinostomatidae) on survival of triturus carnifex (amphibia: urodela: salamandridae) giacomo bruni1,*, claudio angelini2 yeasts in amphibians are common: isolation and the first molecular characterization from thailand srisupaph poonlaphdecha, alexis ribas* introduction of eleutherodactylus planirostris (amphibia, anura, eleutherodactylidae) to hong kong wing ho lee1, michael wai-neng lau2, anthony lau3, ding-qi rao4, yik-hei sung5,* correlation between endoscopic sex determination and gonad histology in pond sliders, trachemys scripta (reptilia: testudines: emydidae) david perpiñán1, albert martínez-silvestre2,3, ferran bargalló3, marco di giuseppe4, jorge orós5, taiana costa6,* book review: john w. wilkinson. amphibian survey and monitoring handbook. pelagic publishing franco andreone book review: susan newman. frogs amphibians and their threatened environment. discovery and expression through art k-3. frogs are green franco andreone issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 6(2): 209-221, 2011 preliminary results on biological aspects of the grass snake, natrix natrix in the southern coastal area of the caspian sea faraham ahmadzadeh1,7, miguel a. carretero2, konrad mebert3, afshin faghiri4, saeedeh ataei5, samaneh hamidi6, wolfgang böhme7 1department of biodiversity and ecosystem management, environmental sciences research institute, shahid beheshti university g.c., iran. corresponding author. e-mail: fahmadza@uni-bonn.de 2cibio, centro de investigação em biodiversidade e recursos genéticos, universidade do porto, campus agrário de vairão, 4485-661 vairão , portugal. 3siebeneichenstrasse 31, 5634 merenschwand, switzerland . 4department of biology, damghan branch, islamic azad university, damghan, iran. 5department of molecular genetics, national institute for genetic engineering and biotechnology, tehran, iran. 6department of zoology, faculty of biological sciences, shahid beheshti university g.c., iran. 7zoologisches forschungsmuseum alexander koenig (zfmk), adenauerallee 160. d-53113 bonn, germany. submitted on: 2011 15th january; revised on 2011 1st november; accepted on 2011 9th november. abstract. natrix natrix is, together with n. tessellata, the only representative of water snakes found in iran. the lack of ecological studies on this species in iran stimulated the current preliminary research on some basic biological traits. from april to july 2008, a total of fifty five snakes were collected from two stations in the southern part of the caspian sea coast, iran: gomishan wetland and sari. basic morphometrics were compared between sexes and stations and gonadal development was compared between stations and seasons. at the two stations, adult sex ratio was not different from 1:1 and juveniles composed a predominant portion of the sample. both sexes had unimodal length class size with a peak at 40-45 cm snout-vent length (svl). body sizes were unusually small for the species and no svl sexual dimorphism was detected although males were relatively heavier than females for the same svl. snakes of both sexes attained larger size with better body condition at sari than in gomishan. males and females carried more mature gonads in summer. however, in sari males developed relatively larger testes earlier in the season and both sexes displayed less synchronic reproduction at this station. these results are best explained by local variations in habitat, trophic availability and degree of environmental disturbance. keywords. natrix natrix, sexual dimorphism, reproduction, iran. 210 f. ahmadzadeh et alii introduction in iran, the water snakes in the genus natrix are represented by two species: the dice snake natrix tessellata (laurenti, 1768) and the grass snake natrix natrix (linnaeus, 1758). the former is a medium-sized (up to 1.2 m total length), semi-aquatic snake that prefers relatively open, and often rocky shore habitat of any aquatic system where it hunts mostly fish (mebert, 2011a). it is widely distributed across the north, northwest, west, southwest and south of the country, whereas the second has a very limited distribution range (firouz, 2000; latifi, 2000; bagherian and kami, 2009). the second semi-aquatic snake, the grass snake, attains a slightly larger total length (occasionally > 1.5 m), and is typically associated with lentic water habitats, such as ponds and lakes, but inhabits also streams, rivers and grasslands where is forages predominantly on anurans (beebee and griffiths, 2000; latifi, 2000; szczerbak, 2003). the grass snake has a huge distribution, ranging from britain, iberian peninsula and north-western africa in the west to lake baikal and northwest china in the east (thorpe, 1981; orlov and tuniyev, 1987; szczerbak, 2003). in iran, it is found in large numbers in the northern provinces, including gilan, mazandaran and golestan (latifi, 2000; bagherian and kami, 2009). currently, the grass snake is regarded as a serious pest in some fish farming areas and causes harm to the industry by feeding on juvenile fish, especially in open and uncontrolled farms like lakes (patimar, pers. comm.). despite their abundance, the ecology of snakes is less well known compared to other reptiles (greene, 2001; pough et al., 200l). nevertheless, the natricine snakes are among the better studied (gregory, 2004), and n. natrix is no exception (e.g., thorpe, 1975, 1984; madsen and shine, 1993a, 1993b; mertens, 1995; filippi et al., 1996; blosat, 1998; borczyk, 2004, 2007; consul et al., 2009). however, biological studies providing quantitative data for this species in iran are still lacking. therefore, the aim of this preliminary study is to stimulate the research on associated topics, by providing more detailed biological information, namely on basic morphometrics and reproduction, for the iranian populations of this species. we focused on interpopulational, intersexual and seasonal variation, as well as on habitat features and conservation status. materials and methods study areas the study was carried our near the south-eastern coast of the caspian sea, in northern iran (fig. 1). initially, sampling was conducted at three stations: gomishan wetland (37°09’07.3”n and 53°54’32”e), ghaz harbour (36°47’41.0”n and 53°56’43.0”e), and sari (36°32’42.9”n and 53°06’41.9”e). ghaz harbour was subsequently removed from the analysis due to low sample size. the gomishan wetland is a protected brackish water body at the south-eastern corner of the caspian sea coast with an area of approximately 20,000 hectares. the main wetland area contains salt marsh vegetation and seasonally-inundated flats of salicornia mixed with halostachys and halocnemum grasses. the sari station is located in the vicinity of fish farms near the city of sari, the capital of mazandaran province, at about 130 km from gomishan wetland. at this station, the rainy season lasts about seven months with an annual precipitation of more than 1,110 mm, enabling green 211preliminary results on biological aspects of iranian natrix natrix and lush vegetation typical for a hyrcanian forest habitat. all specimens at this station were collected along streams in the woodland and the area around a large fish pond (scott, 1995; kiabi et al., 1999). following physicochemical measurements were recorded at both stations once, using a hach portable device: temperature (°c), ph, salinity (‰) and total dissolved solids tds (g/l) during the first week of may. field sampling field methods were the same for both stations. sampling was carried out from april to july 2008, the first and fourth week of each month, in the morning from 08:00-10:00 hrs. we recorded diet through anecdotal observation of prey capture or occasional regurgitation (but no methodical investigation), as well as types of primary microhabitat and behaviour. all specimens were collected by hand and transferred to the zoology laboratory of environmental sciences research institute, shahid beheshti university, sacrificed humanely with the permission by the institutional authority and fixed in 10% formalin. individuals were considered adults when longer than 30 cm svl according to mertens (1995). adult specimens were weighted to the nearest gram (body mass, bm). the following measurements were recorded: svl (snout-vent length) and lct (length of complete tail from vent to tip of tail). in addition, rtl and ltl (right and left testes lengths) in males and rol and lol (right and left ovarian lengths) in females were measured to assess their reproductive status. all linear measurements were recorded to the nearest centimetre. no other variables, like the number of follicles or their sizes could be recorded due to methodical constraints. fig. 1. study sites in the south-eastern coast of the caspian sea, iran. 212 f. ahmadzadeh et alii statistical analysis the juveniles were excluded from the statistical analyses. for adults, the overall sex ratio was assessed using chi-square (χ2) test (zar, 1999). measurements were log-transformed and normality and homoscedasticity were ensured prior to the analyses. a two-way anova was performed to compare adult svl differences between stations and sexes. for the other length measurements like tail length (lct) and lengths of testes (tls) and ovaries (ols), two-way ancovas with station and sex as factor and svl as covariate were run. in the same way, gonad measurements were compared through two-way ancovas with station and season as factors and svl and bm as covariates. no pholidotic variables could be recorded. all statistical analyses were performed in statistica 10 (statsoft, 2011) and significance level was set at 0.05. results in early may, gomishan wetland exhibits higher values of water physical features than equivalent measurments show from sari, including water temperatures, ph, salinity (%) and tds (g/l) (table 1). a broad range of potential prey types for n. natrix was observed at all sites. the scattered observations indicated the consumption of amphibians, such as pelophylax ridibundus and rana macrocnemis, one legless lizard, anguis fragilis, and some cultured fish (barbus sp., cyprinidae). the number of specimens collected by station, season and class is shown in table 2. at the two study sites, the grass snake was relatively common. in total, 55 grass snakes including 18 males, 21 females and 16 juveniles were collected from both sites during two field seasons. the overall sex ratio was not significantly different from 1:1 (χ2 = 0.23, df = 1, p > 0.05). juveniles (< 30 cm svl) constituted a large percentage of the collected specimens, 29% and 28% in spring and summer, respectively. the total length of adult males table 1. physical traits of water, temperature (t), ph, salinity and total dissolved solids (tds) in the two sampling stations from the south-eastern caspian sea, iran. station t(oc) ph salinity (‰) tds (g/l) gomishan wetland 21.9 8.97 28.7 22.2 sari 16.9 8.2 1.3 20.4 table 2. the number of iranian natrix natrix collected by station, season and class. gomishan wetland sari males females juveniles males females juveniles spring 5 5 1 4 7 8 summer 4 4 4 5 5 3 213preliminary results on biological aspects of iranian natrix natrix ranged between 46.0 and 85.0 cm and adult females between 46.0 and 76.7 cm. the snoutvent length frequency distribution of snakes from the two stations indicated that the dominant length class was 40-45 cm for both sexes, representing 33% of males and 38% of females for both stations lumped (fig. 2). descriptive statistics of the morphometric variables and ancova comparisons are shown in table 3. snakes attained longer svl in sari than in gomishan but tests failed to detect significant differences either between sexes or interactions between sex and station (table 3). moreover, relatively to their svl, males were heavier than females and individuals from sari were heavier than those from gomishan, although there was no variation of sexual dimorphism between stations (table 3, fig. 3). finally, no differences in tail length relative to svl were detected either between sexes, between stations or their interaction (table 3). the smallest adult male with thickened muscular tail root was 35 cm svl. relative to svl, both ovaries were longer in summer than in spring. whereas right ovaries showed no differences between the stations, thus following the same seasonal pattern (ancova station f1,15 = 1.07, p = 0.32; season f1,15 = 49.74, p < 0.0001; station*season f1,15 = 0.01, p = 0.92), left ovaries were slightly longer in grass snakes from the gomishan site when compared to those from sari (ancova station f1,16 = 7.34, p = 0.02; season f1,16 = 63.44, p = 0.0000001; station*season f1,16 = 1.46, p = 0.24). the same patterns persisted when both svl and bm were used as covariates to account for body condition either for the right testis (ancova station f1,14 = 0.000001, p = 0.99; season f1,14 = 54.15, p < 0.0001; station*season f1,15 = 0.16, p = 0.69) or for the left testis (ancova station f1,14 = 9.96, p = 0.007; season f1,14 = 70.09, p < 0.000001; station*season f1,15 = 0.63, p = 0.44). the smallest male with thickened efferent ducts, indicating sexual maturity, was 32 cm svl. relative (to svl) length of the testes was significant higher in sari than in gomishan, but seasonal variation, spring being lower than summer, was slight and only detected in the left testis (ancova right testis station f1,13 = 34.27, p < 0.0001; season f1,13 = 6.18, p = 0.03; station*season f1,13 = 3.18, p = 0.10; left testis station f1,13 = 10.73, p = 0.006; season f1,13 = 0.42, p = 0.53; station*season f1,13 = 0.48, p = 0.50). fig. 2. length frequency distribution for grass snake natrix natrix from the south-eastern caspian sea, iran. 214 f. ahmadzadeh et alii discussion this study presents the first ecological picture of the grass snake of iran, and thus, augments our knowledge on the biology of the widespread palaearctic water snake, natrix table 3. descriptive statistics of the morphometric variables for the two analysed populations of adult n. natrix from iran and ancova comparisons for site and sex; all but the first using svl as covariate. bm: body mass; svl: snout-vent length; lct length of complete tail. numbers indicate mean ± se, range and sample size. variable gomishan sari ancova (station, sex, station*sex) males females males females f df p svl 40.32 ± 1.60 40.33 ± 1.14 54.20 ± 2.41 48.67 ± 2.04 1.73 1,35 0.20 32-47 47-35 45-67 38-62 35.12 1,35 < 0.0001 9 9 9 12 1.98 1,35 0.17 bm 59.00 ± 3.26 49.78 ± 2.57 82.87 ± 3.78 74.25 ± 4.67 7.17 1,34 0.01 47-72 60-36 67-98 51-112 4.51 1,34 0.04 9 9 9 12 1.83 1,34 0.18 lct 12.49 ± 0.65 11.44 ± 0.47 12.86 ± 1.07 12.68 ± 0.37 0.96 1,34 0.33 10-15 14-9 9-18 11-15 0.03 1,34 0.87 9 9 9 12 2.27 1,34 0.14 fig. 3. variation of the relative body mass (relativized to svl) between sexes and stations in adult natrix natrix from the south-eastern caspian sea, iran. vertical bars denote 95% confidence intervals. 215preliminary results on biological aspects of iranian natrix natrix natrix. the results and conclusions have still a preliminary character, yet they warrant their relevance due to virtually non-existing information of that species from the south-eastern corner of it global distribution. the incidental observations on diet are in accordance with the generally recorded wide food niche based on amphibians, fish, and rarely lizards, as was reported from other areas (e.g., reading and davies, 1996; luiselli et al., 1997; kabisch, 1999). this distinguishes the grass snake from the syntopic and more piscivorous dice snake, n. tessellata, which at the same sites consumed only fish and has never been found as far from water as n. natrix (ahmadzadeh et al., 2011). similar food niche differences have been detected in other syntopic populations, as in italy (capula et al., 2011), greece (ioannidis and mebert, 2011), and croatia (janev hutinec and mebert, 2011). the balanced sex ratio at the two populations show a normal pattern as generally described for snakes in parker and plummer (1987). luiselli et al. (2011) calculated a near equal sex ratio in n. tessellata from italy, whereas sex ratio for another natricine snake, nerodia sipedon, was slightly elevated with 1.5:1 and 1.3:1, respectively (feaver, 1977; king, 1986), and locally much higher with 2.7 for the closely related n. maura (hailey and davies, 1987). the mature to immature ratio found here (2.5:1) was clearly lower than in a population in central germany (1.1:1, mertens (1995). although this could be the result of a high recruitment, the effect of a low sample size can also be hypothesised. concerning adult snakes, mertens (1995) found the 60–70 cm svl size class the most numerous in males in central germany. similarly in other german populations, waitzfig. 4. variation of the gonad length between spring (april) and summer (july) in adult male and female natrix natrix from the south-eastern caspian sea, iran. vertical bars denote 95% confidence intervals. 216 f. ahmadzadeh et alii mann (1991) found the size class 70–79 cm total length for adult males to be dominant (incl. 20–25% tail length), whereas kühnel’s (1993) sample contained mostly smaller adult males in the class 50–60 cm total length (tail included). on the other hand, females displayed bimodal size distribution with one peak in medium sizes and another in the very large specimens (> 70 cm). the pattern for females in the studies by waitzmann (1991) and kühnel (1993) was similar to those of males, but in 10 cm higher size class, i.e., with 80–89 cm and 60–70 cm being the most frequent snakes. this reveals a strong geographic variation on body size, possibly resulting from local environmental effects on growth. unlike mertens’s finding, the size distribution of iranian grass snakes is unimodal in both sexes with small modal values (40–45 cm). two results are remarkable in reference to the adult sizes and their frequencies at both iranian sites: first, no sexual size dimorphism was detected in tail length relative to svl; and, second, males yielding slightly larger svl than females. both results contrast not only with other studies on the same species in other parts of its range (thorpe, 1984; and refs. in kabisch, 1999, in blanke et al., 2008; and in pleguezuelos, 2010), but also with other natrix species (schätti, 1982; gruschwitz et al., 1999; santos et al., 2000; mebert, 2011b), as well as with other natricines (king, 1989; shine, 1994; mebert, 2010). regarding sexual dimorphism, in most snakes, females attain a larger body size (svl) than males, whereas males have longer and thickened tail roots (shine, 1994; pough, 2001). however, in this study, statistical analyses were not able to detect differences between sexes either for svl or for relative tail length. significantly, specimens from both study sites exhibited a rather small size compared to other populations in germany (e.g., wisniewski, 1958; waitzman, 1991), but were similar to those sampled from a sub-population of kühnel’s (1993) study sites in berlin, northern germany. the interpretation of the last author suggested that a visibly sharp decline in green frogs, the principal prey, had resulted in the temporary survival or local persistence of smaller individuals only. such conditions may apply temporarily to the iranian sites as well. certainly, it could be hypothesised that both results (lack of sexual dimorphism and small size) from the iranian grass snakes are linked, that is, that the overall reduction in body size has minimised the sexual size dimorphism to the degree of becoming statistically undetected. however, kühnel’s study (1993) across a greater area in berlin found a significant sexual dimorphism with approximately the same number of grass snakes and a similar average size. in addition, males tended to be more robust than females for the same size in both populations studied. in contrast, capula et al. (1994) found female n. natrix on sardinia exhibiting higher body conditions than males. at this stage of the study, we can only speculate about the causes for the larger males or the lack of large females in the samples, including a) an artefact of low sample sizes (~ 5 adult snakes per season and sex), b) that the sampled males are older than females in the area, c) show a more rapid growth rate at the same age as females due to earlier emergence and feeding after hibernation, or c) occurrence of a negative selection against females, such as: 1) suffering a higher incidence of predation due to their larger size, 2) dying before reaching a large size/age due to increasing toxic load from the copious amounts of herbicides and pesticides applied in the agriculture surrounding the sites, 3) migrating outside the sites for oviposition. grass snakes from sari attained larger sizes with better body condition than those from gomishan, which could be the result of a stable food source as provided by the fish 217preliminary results on biological aspects of iranian natrix natrix farms. overall, the various studies on n. natrix populations indicate that body length in both sexes can vary greatly among sites and possibly even fluctuate within a site depending on periodically varying food availability. finally, the number of adult snakes in this study is too small for any further conclusion on the high frequency of small females and lack of sexual dimorphism of tail length. hence, only increasing the sample size will likely produce a conclusive answer in this context. regarding the reproductive organs, across its wide range, grass snakes are active from the beginning of march (in the south) or may (in the north) until september or november, respectively (kabisch, 1999; szczerbak, 2003). reproductive activity lasts from may to mid-july. in may, after a quiescent period of 7-8 months, females start vitellogenesis followed by an increase of the length of both ovaries. all females collected showed some reproductive activity with enlarged ovaries in the summer, when likely maximum levels are reached, as oviposition is normally from june to august (kabisch, 1999; meyer et al., 2009). in contrast, males showed equal gonadal activity over a longer period, the increase of testis length being nearly equal in spring and in summer. this is likely due to their postnuptial spermatogenesis, i.e. spermatogenesis and growth of testes occurs in summer and continues into the fall, a period that was not measured in this study. such a spermatogenetic pattern was confirmed very recently for grass snake populations from the same area in iran (faghiri et al., 2011). this is also known for the other natrix species (for n. tessellata: see rutishauser, 1996; and for n. maura: see santos et al., 2000; santos et al., 2001). since climate is very similar between both field stations, the difference of testicular growth between populations derive from other local ecological factors. the larger testes in grass snakes from sari may be the result of a larger energy input early in the season due to a higher food availability at the fish farms, hence, being less seasonal than in gomishan. the growth pattern of the ovaries seems less clear, but female grass snakes in gomishan may start earlier and be less synchronic than those of sari which will be all peaking in summer. this kind of local environmental constraints have also been reported for n. maura in a river delta from spain (santos and llorente, 2004; santos et al., 2005). generally, n. natrix inhabits in a wide range of aquatic habitats: riverbanks, swamps and mountain streams up to 2400 m a.s.l. (madsen, 1984; mertens, 1995; beebee and griffith, 2000; szczerbak, 2003). comparing the two stations in this study, the gomishan wetland is a more open habitat type, situated in close proximity to the caspian sea, with sparse submerged vegetation, and higher values of physicochemical characters, a biotope and habitat structure typical for piscivorous n. tessellata (see refs. in mebert 2011a). this contrasts to the wooded site at sari, which represents a typical hyrcanian forest habitat. if the conclusions of other studies on n. natrix, summarized in kabisch (1999) and blanke et al. (2008), can be extended to iran, the forests of sari with an abundance of anuran prey are expected to provide more suitable habitat structures and ecological resources for n. natrix than the gomishan wetland. wooded areas combined with the fish farms at sari, yielding an additional, easy accessible food source, could be the factors producing markedly higher population densities of n. natrix, composed by larger individuals in better condition, which are able to start reproduction earlier. these results are in correspondence to those published for n. natrix (e.g., mertens, 1995; beebee and griffith, 2000; szczerbak, 2003) and similar for n. maura (pleguezuelos and feriche, 2003; santos et al., 2005, 2011). 218 f. ahmadzadeh et alii acknowledgements this research of fa was supported by shahid beheshti university of tehran (grant no. 665020). therefore, financial support from the home university is gratefully acknowledged. the work of mac was supported by the projects ptdc/bia-bde/67678/2006 and ptdc/biabec/101256/2008 funded by fundação para a ciência e a tecnologia (portugal). references ahmadzadeh, f., mebert, k., ataei, s., rezazadeh, e., goli, l.a., böhme, w. 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(1999): biostatistical analysis, 4th ed. prentice hall, upper saddler river, new jersey. acta herpetologica 12(1): 29-36, 2017 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-18115 fire salamander (salamandra salamandra) males’ activity during breeding season: effects of microhabitat features and body size raoul manenti*, andrea conti, roberta pennati dipartimento di bioscienze, università degli studi di milano, via celoria, 26 – 20133 milano (italy).*corresponding author. e-mail: raoulmanenti@gmail.com submitted on 2016, 15th march; revised on 2016, 17th november; accepted on 2016, 19th november editor: rocco tiberti abstract. after metamorphosis, fire salamander is considered fully terrestrial, usually inhabiting wooded areas around aquatic habitats. it is often reported that only females go back to water for laying the larvae. the aim of this study is to assess if sites where males are active during the breeding seasons have specific features among microhabitat determinants and distance from the breeding sites. in the autumns of 2013 and 2014, we surveyed 26 transects and 72 plots around six isolated breeding sites in north-western italy. during rainy nights, we recorded males position and distance from breeding pools, while during daytime we characterized the environmental features of the plots. males detection probability was relatively high (mean ± se: 81.0 ± 4.3%). several males (15% of the observations) were encountered inside breeding pools where females were laying larvae. males occurrence was positively related to plots closer to breeding pools and higher leaf litter depth. larger males were found closer to the breeding pools. this case study shows that the distribution of fire salamander males during the breeding season depends on the breeding sites. keywords. breeding pond, ecology, mating behaviour. introduction the assessment of ecological relationships between amphibians and their terrestrial habitat are very important to understand proper management actions and enhance conser vation policies. among european amphibians with extensive terrestrial habits there is the fire salamander, salamandra salamandra. this is a widespread species in europe, that has been extensively studied in its use of different breeding sites, while studies on terrestrial habitat requirements were less frequent (denoël, 1996; carafa and biondi, 2004; catenazzi, 2016). for breeding, s. salamandra is generally linked to small lotic environments (manenti et al., 2009), but it can also use a wide variety of habitats like ponds (egea-serrano et al., 2006; denoël and winandy, 2014; caspers et al., 2015) and hypogeous (i.e., subterranean) springs or pools (ianc et al., 2012; manenti et al., 2015). moreover, some populations evolved complete viviparity, skipping the aquatic larval stage (velo-anton et al., 2007). adults are strictly terrestrial and generally live in broadleaved forests (joly, 1968). even if it is generally reported that only females migrate to wetlands to give birth to the aquatic larvae while males do not go back to water after metamorphosis (joly, 1968; nöllert and nöllert, 1992; denoël, 1996; lanza et al., 2009; ficetola, 2012), it is known that the species lives around water bodies without strong displacements from them (ficetola, 2012). in general the species seems to prefer deciduous mixed woodlands, especially those mostly dominated by the common beech fagus sylvatica (lanza et al., 2009; balogova and uhrin, 2014). studies performed in the alps show that the fire salamander prefers areas which are characterized by a mean annual precipitation > 600 30 r. manenti, a. conti, r. pennati mm (schauer et al., 2012). salamanders tend to be active at temperature as low as 1 °c, with a thermal optimum in terms of trade-off between thermoregulation and metabolic costs at 8 °c (catenazzi, 2016). estivation occurs at temperatures above 16 °c (catenazzi, 2016). s. salamandra seems to avoid dry forests (especially spruce monocultures) and its ecology in floodplains and coniferous forests needs further studies (lanza et al., 2009). recent studies provided evidences of the importance that a complex ensemble of determinants, linked to both terrestrial and aquatic habitats, plays in allowing the occurrence of stable populations (ficetola et al., 2011). however few studies are available in terms of microhabitat use in the forest environment. these data could allow to gain a better knowledge on proper forests habitat management for conservation purposes. adults are considered to be territorial even if some superimpositions of home ranges may occur (lanza et al., 2009) and during winter individuals may aggregate in shelters (baumgart, 1981; balogova and uhrin, 2014). the adult activity usually shows two peaks during the year, coinciding with high rainfall periods: one during spring, and one in autumn (joly, 1968; lanza et al., 2009). these are also the two seasons in which both mating and larvae laying occur, especially south to alps (romeo et al., 2015). females usually lay larvae during night and may stay around their breeding sites for several nights, as females repeatedly give birth to only some of the larvae they retain (joly, 1986); moreover in these occasions they likely mate for the following year’s brood (steinfartz et al., 2006). mating happens on land habitats, most often in spring and in autumn months, and usually at night (lanza et al., 2009). the reproductive males are reported to show a frequent posture during the courtship, in particular they rise on their anterior limbs and show a quick bucco-pharyngeal respiration. this posture can be frequently observed in the males of s. salamandra (bruno, 1973; catenazzi, 1998) and also in males of other congeneric species as s. lanzai and s. atra, also in absence of females and in more or less plane and open areas (manenti and pennati pers. obs.). in s. salamandra the display of this posture has been interpreted both as a territorial and competitive behaviour among males (bruno, 1973), and as a strategy allowing a more effective perception or attraction of the females (lanza et al., 2009). the advantage of an affective female detection could also favour a non-random choice by the males of the microhabitat where being active during the mating seasons. however, specific ethological and ecological studies are missing and, at present, it is impossible to say if males spatial patterns are linked to specific reproductive behaviours. the general hypothesis of the present study is that, to increase their chances of mating, males should stay close to the females as soon as they are ready to mate, i.e., after they laid their larvae. our specific hypotheses are that getting closer to the breeding ponds should be the most effective spatial strategy for males and that they should compete for the territory patches closer to the breeding sites. we therefore tested if the sites where males are active during the breeding seasons have specific features among some microhabitat determinants and distance from the breeding site. moreover, we want to assess if male size (as a proxy of their reproductive quality) have a role in determining the location of activity sites. these information could increase our understanding of the spatial and reproductive ecology of s. salamandra and confirm that males compete for the occupancy of some sites/ microhabitats and could be useful to perform further ethological studies on their reproductive and territorial behaviour. material and methods during the months of october and november 2013 and 2014, we performed surveys around 6 distinct breeding sites of the fire salamander located in the districts of lecco and monza e brianza, lombardy, north-western italy (fig. 1). the study area is characterized by hilly reliefs with a good cover of broadleaved woodlands dominated by castanea sativa and quercus robur. it is crossed by a dense hydrographic network characterized by different typologies of water bodies such as creeks, streams, brooks, resurgences and rivers. breeding sites were characterised by isolated pools, fed by small springs, with an area ranging from 5 to 15 m2. each isolated pool corresponded to a different locality. the maximum distance between the 6 sites studied was 14 km (average 6.9 km). linear transect surveys we looked for fire salamander males during night surveys in rainy nights (between 9:00 pm and 12:00 pm), during which we also check for larvae depositions by females. these surveys were performed along 26 linear straight transects (4 linear transect around 4 pools and 5 linear transect around other two pools) beginning at distances comprised between 110 and 75 m (average ± se: 97.2 ± 5.3 m) from the breeding sites and ending in them. the starting positions of the transects were randomly chosen during the daytime of the first field survey, and distinctive marks were placed to distinguish them to each other. then we walked in the direction of the breeding site measuring the covered distance with a measuring strip and tracking and mapping the transect with a gps garmin eterx 10 (precision 3 m). the width of the surveyed area around the transect was 5 meters (2.5 m on each side). for each survey season (octobernovember 2013 and 2014), we performed at least two surveys 31fire salamander males activity around breeding sites for transect (range 2-4 surveys per transect, average ± se: 2.76 ± 0.11 times). surveys for each transect lasted from 30 to 45 minutes. during night surveys, we caught all the fire salamanders that we encountered. we placed the individuals in small holed bags in the same point of observation following protocols used for other salamanders (lunghi et al., 2015) in order to do the measures successively, without affecting the position of the individuals occurring in the successive parts of the transect. the point of encounter was registered with the same gps garmin etrex 10. at each survey we re-recorded the starting point of the transects and the pool position, tracking all the transects length. after the capture of all the observed individuals we took a picture of each individual and registered their position. each individual was photographed on a plastic millimetre paper to allow successive accurate measures of the total length, and weighed with a precision dynamometer pesola (precision ± 0.1 g). the total lengths (precision ± 1 mm) of the males was obtained through the software “imagej”. in 2014 we also took a picture of the dorsal patterns to allow the individual identification of the salamanders. for each salamander, we used gps data to record the distance from the pool, and we measured the maximum slope inclination of the capture point with a digital inclinometer digital angle bevel box 360 (precision ± 0.2°) placed over the middle of a flat wooden board of 10 × 10 cm. sex determination was performed on the basis of their cloaca features. as males develop a swollen cloaca when they are sexually mature (raffaëlli, 2007), the individuals shorter than the smallest recognizable male were considered juveniles. sample areas/plots surveys along each transect, we chose a total of 72 circular plots with a 2.5 m ray, separated each other by at least 5 meters, and placed within 30 m from the breeding pools (average number ± se of plots per transect was 2.7 ± 0.2, range: 2-5). the number of plots was chosen to specififig. 1. localization of the breeding sites (triangles) of fire salamander around which we positioned linear transects and plots .numbers indicate the site id; some of the sites are superimposed due to geographic proximity. 32 r. manenti, a. conti, r. pennati cally cover the part of the transects placed closer to the breeding sites, and their position was randomly chosen within a buffer of 30 m around each breeding site to gather detailed information on the microhabitats. in these plots, we recorded five environmental variables during daytime surveys: litter leaf depth, maximum slope inclination, number of trees with a diameter > 50 cm, distance from the breeding pool, bush cover. leaf litter depth may be linked to shelter availability, humidity level of the soil and prey availability: it was measured placing a rigid ruler in the point of the plot with the maximum litter depth and measuring its height from the soil. maximum slope may be important to explain salamander distribution (manenti et al., 2011; werner et al., 2014): inclination was measured placing digital inclinometer over a rigid wooden board (20 × 20 cm) in the point of the plot with the maximum inclination. the trees diameter was assessed using a measuring strip: all the trees with a diameter > 50 cm occurring in the plots were counted, in order to detect the possible effect of the occurrence of large and old trees that are often shelters for salamanders (apodaca and godwin, 2015). the distance of the plot from the breeding sites was assessed using measuring strips with a precision of 0.01 m. the bush cover was estimated following the point-intercept method described by dodd (2010), using a 2 m long pole placed vertically in the plot center and numbering the times it intercepts any piece of vegetation. the position of each plot was recorded through gps and each plot was marked with small, temporary but visible during night marks over trees or rocks. statistical analyses linear transect surveys we built a linear mixed model (lmm) to assess factors influencing the distance from the breeding sites at which males were found during night samplings. we limited this analysis to the males observations of 2014 (n = 80), because the identification of each individual (based on their dorsal pattern) was performed only in the second year. we added the males square-root transformed total length as dependent variable, and log-transformed distance from the breeding site, and log-transformed slope inclination as independent variables. as we found recaptures, we added the individuals identity as random effect. variables transformation was performed to increase normality (tested through a shapiro test). since there is a strong exponential linear dependence between weight (w) and total length (l) of males (w = 0.02 l2.67; r2 = 0.82), we considered only total lengths to analyse the effects of male size on their distribution. the variables significance was assessed through a wald f test (bolker et al., 2008). sample areas/plots surveys we used presence 5.5 to assess in each plot the probability of detection per visit of males and females (hines, 2006). accounting for the probability of detection it is important because a non-detection record (equivalent to a recorded absence) represents a lack of evidence that the species occurs in a specific site (gomez-rodriguez et al., 2012). we used multiple season models (mackenzie et al., 2003) to understand the predictors (survey and environmental variables) that satisfactorily described the collected records while accounting for imperfect detection (mackenzie et al., 2002). meaning that we considered the story of detection and non-detection per visit per plot considering both autumn 2013 and autumn 2014. on the basis of this history, presence returns a probability to have correctly assessed the presence/ absence of the species in the plots, such as a probability of occurrence (mackenzie et al., 2003). to disentangle the role of the predictors it is necessary to build models comprising all their possible combinations and to select the best reliable model. we used the akaike’s information criterion (aic values) to select the best model explaining males probability of occurrence in the plots (rolls, 2011; lele et al., 2013). the model with the lowest aic and the highest weight was considered the best model describing species detectability (burnham and anderson, 2002). since a model with no covariates for neither occupancy nor detectability (model ψ(.) p(.)) showed that the detection probability of males was relatively high (mean ± se: 81.0 ± 4.3%), when building the other occupancy models, we only varied the predictor variables for occupancy and always used a model with constant detection probability (i.e., model ψ(predictor variables) p(.)). thanks to the relative small number of variables considered, we were able to test all the possible combinations among them. in order to reduce the retention of overly complex models we excluded from the candidate set those models that were more complicated versions of any model with a lower qaic value (richards et al., 2011). this approach reduces model uncertainty, and improves the performance of model selection (richards et al., 2011). we present all the candidate models with some supporting information, e.g. delta-aic < 4, qaic weight > 0.1. to assess the relative role of the variables composing the best model we used a wald f test (bolker et al., 2008) with “car” package in r environment. to asses which environmental factors affect males sites choice, we preferred to use the estimates of males probability of occupancy at a given plot (calculated with 33fire salamander males activity around breeding sites presence) rather than the observed “naïve” absence/ presence. we considered this value as the dependent variable of a generalized linear mixed model (glmm) with a quasi-binomial error distribution using the log-transformed microhabitat features recorded in the plots as independent variables. we included pool locality as random factor, to account for the fact that several plots can surround the same pool. all the analyses were performed by r software using the packages “lmertest”, “lme4” and “car” (r development core team, 2013). results linear transects surveys during the two sampling seasons, we caught 213 salamanders 147 of which were males. males total length was 17.1 cm on average (maximum 21.6 cm, minimum 8 cm, sd = 2.39), while weight was 32.5 g on average (maximum 45 g, minimum 4 g, sd = 10.0). females were 17.5 cm long on average (maximum 21.2 cm, minimum 8.5 cm, sd = 3.3) and weighed 43.4 g on average (maximum 78 g, minimum 6 g, sd = 18.7). in 67% of night surveys, we observed ponding females near the pools borders or on floating elements in the pools. the 15 % of the observed males were encountered inside breeding pools where females were laying larvae. average males distance from the pools was 21.2 m (maximum 106 m, minimum 0 m, se = 3.44). we detected a significant effect of males size on their distribution. in particular, on average, longer males were closer to the breeding pools (table 1). sample areas/plots surveys detection probability on the surveyed plots was higher for males (p = 0.81 ± 4.3% se) than for females (p = 0.54 ± 6.2% se). the plots in which males were encountered showed a higher mean litter depth (1,5 cm ± 0.2 se vs. 0.9 cm . ± 0.1 se), slower slope inclination (5.48° ± 1.2 se vs. 9.25° ± 1.7 se) and lower number of trees (1.46 ± 0.2 se vs. 2.63 ± 0.3 se). the best occupancy model was that including distance to breeding pools, slope inclination, number of trees and leaf litter depth (table 2). the occupancy probability of males was higher in plots closer to breeding pools (f = 22.29; p < 0,001) and with higher leaf litter depth (f = 10.11; p < 0,01). there was a non-significant negative tendency to both slope inclination and trees density. discussion our results provide novel data on the fire salamander males spatial activity around breeding sites. in particular, our study provides evidence that during the breeding season males use microhabitats with specific features like deeper leaf litter and closer to breeding sites. generally the use of terrestrial habitats by the salamanders of the genus salamandra has been investigated to study their site fidelity to a restricted area and their relatively small home ranges (schulte et al., 2007; ficetola, 2012). most of the information about the use of the terrestrial environment is at the landscape scale and underlines the importance of suitable woodland habitats in riparian, mountainous and agricultural areas (manenti et al., 2009; tanadini et al., 2012; manenti et al., 2013); while studies on terrestrial microhabitat preference in the genus salamandra are lacking. our study was carried out when females reach the breeding sites to lay the larvae and focuses on adult males, to understand their microhabitat choice when they likely look actively for mating. in the fire salamander, copulation may occur at almost any period of the year except during the winter (francis, 2002); it haptable 1. results of lmms analysis showing the relationship between the size of the encountered males considered as dependent variable and, slope inclination and distance from the breeding pools of the encountering point considered together as independent variables (numdf = degrees of freedom in the numerator; dendf = degrees of freedom in the denominator). variable estimate numdf dendf f p distance from the breeding site -0.29 1 67 11.2 0.001 slope inclination 0.03 1 67 0.17 0.68 table 2. model selection of the best fitted site-occupancy models (only models with δqaic ≤ 4 are shown). the symbol (.) indicates a constant parameter with no covariate and k is the number of parameters in the model. δaic is the difference between the qaic score of the model and the best ranked model and w is the akaike model weight. model k qaic δqaic w ψ(distance + leaf litter depth + slope + n. of trees) p(.) 5 137.41 0.00 0.416 ψ(distance + n. of trees) p(.) 4 138.06 0.65 0.300 ψ(distance + leaf litter depth) p(.) 4 139.42 2.01 0.152 ψ(distance) p(.) 6 140.89 3.48 0.073 34 r. manenti, a. conti, r. pennati pens generally during night, and both in spring and autumn it is quite frequent (rivera et al., 1999; lanza et al., 2009). the higher probability of finding males was found in the plots where the trees were less numerous and slope inclination lower, even if the tendency was not significant. a similar result has been already reported by denoël (1996) who observed a high encounter rate for footpaths and open areas. it is likely that this choice may be linked to the possibility to enhance mating opportunities through a more effective females detection. some previous studies showed the existence of a role played by the slope inclination since the species generally visits more frequently sites with inclinations lower than 35° (manenti et al., 2011; werner et al., 2014) and, especially in mountainous landscapes, salamanders are active on paths and microhabitats with lower inclinations (pantuso et al., 2015). we found males in plots with a lower mean slope inclination, and this variable was included in the best occupancy model. no information is available on the role played by leaf litter; even if its depth may be easily linked to prey occurrence, shelters availability and optimal humidity. the sex ratio observed during our study (69% males) is similar to those found by klewen (1985) and schulte et al. (2007) in autumn. the observed differences between sexes are correlated with general differences in activity patterns (schulte et al., 2007), with females that likely are less active outside shelters than males which probably remain active to maximize their mating opportunities. our results clearly show that males are more active in microhabitats close to the breeding sites. this fact may be incidental to the use of the breeding sites by females, with males exploiting the pools and their surroundings to enhance the probabilities to mate. although other explanations may exist, as males could prefer the surroundings of the water bodies because humidity or prey availability could be higher. this is the first report demonstrating that the activity of adult fire salamanders of both sex depends to the vicinity of the water, while it was generally believed that after metamorphosis only females go back to the water bodies (lanza, 1983; joly, 1986; nöllert and nöllert, 1992; ficetola, 2012). moreover, our results reveal that there are differences among males in their distribution around the breeding sites. in particular, larger males exploit the microhabitats closest to the pools thus increasing their probability to meet the females. thus, a competition and a territoriality for the sites is likely to occur. the existence of possible territoriality in s. salamandra at least during the breeding seasons has been reported (catenazzi, 1998). moreover in the close relative s. algira the existence of fights between males has been reported (bogaerts and donairebarroso, 2005). males that are likely to be looking for mating display a typical posture rising on their anterior limbs; this position has also been reported as a fighting posture (bruno, 1973) but may be interpreted as an olfactory strategy allowing a more effective perception of the females presence, or as a behavioural display to attract females, related to male quality (wells, 2007; lanza et al., 2009). we did not precisely quantified the males exhibiting this posture because we took any care to avoid disturb and fear responses but this aspect may be of particular ethological interest and further researches should be conducted to relate males behaviour and microhabitats choice; not only in the fire salamander, but also in its fully terrestrial congeneric species like s. atra and s. lanzai in which the same behaviour is very common (andreone, 1992) and the knowledge of the ecological and ethological factors favouring active mate searching by males may be of importance for proper habitat managements. in general our study underlines that factors facilitating females perception like vicinity to the breeding sites drive the male distribution patterns. the extensive exploitation of terrestrial habitats surrounding the breeding pools also has implications for planning proper conservation actions for s. salamandra, as the maintenance of favourable microhabitats allowing females perception may enhance mating possibilities for males. the terrestrial environment surrounding water bodies appears to play a fundamental role in the ecology and ethology of the fire salamander. aknowledgments we are grateful to rocco tiberti and two anonymous reviewers for precious suggestions to previous version of the manuscript. we thank chiara galliani for the professional english revision of the text. the research was approved by the lombardy region authority and authorized complying with the regional law 10/2008, p. n. f1.2013.0002091, started on january 30 2013, ended on december 31 2015. adults were manipulated as less as possible, and released in the collecting point according to the permission prescriptions. references andreone, f. 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(2014): analysis of habitat determinants in contact zones of parapatric european salamanders. j. zool. 292: 31-38. acta herpetologica vol. 12, n. 1 june 2017 firenze university press morphological variation and sexual dimorphism in liolaemus wiegmannii (duméril & bibron, 1837) (squamata: liolaemidae) from uruguay joaquín villamil1,*, arley camargo2, raúl maneyro1 sex does not affect tail autotomy in lacertid lizards panayiotis pafilis1,*, kostas sagonas2, grigoris kapsalas1, johannes foufopoulos3, efstratios d. valakos4 fire salamander (salamandra salamandra) males’ activity during breeding season: effects of microhabitat features and body size raoul manenti*, andrea conti, roberta pennati call variation and vocalizations of the stealthy litter frog ischnocnema abdita (anura: brachycephalidae) pedro carvalho rocha1,2,*, joão victor a. lacerda2,3, rafael félix de magalhães2,3, clarissa canedo4, bruno v. s. pimenta5, rodrigo carrara heitor6, paulo christiano de anchieta garcia2,3 feeding ecology of two sympatric geckos in an urban area of northeastern brazil josé guilherme g. sousa1, adonias a. martins teixeira2,*, diêgo alves teles2, joão antônio araújo-filho2 and robson waldemar ávila3 a pattern-based tool for long-term, large-sample capture-mark-recapture studies of fire salamanders salamandra species (amphibia: urodela: salamandridae) jeroen speybroeck1,*, koen steenhoudt2,$ skeletal variation within the darwinii group of liolaemus (iguania: liolaemidae): new characters, identification of polymorphisms and new synapomorphies for subclades linda díaz-fernández*, andrés s. quinteros, fernando lobo marking techniques in the marbled newt (triturus marmoratus): pit-tag and tracking device implant protocols hugo le chevalier1, olivier calvez2, albert martínez-silvestre3, damien picard4, sandra guérin4,5, francis isselin-nondedeu6, alexandre ribéron1, audrey trochet1,2,* evidence for directional testes asymmetry in hyla gongshanensis jindongensis qing gui wu1, wen bo liao2,* the advertisement call of pristimantis subsigillatus (anura, craugastoridae) florina stănescu1,4, rafael márquez2, paul székely3,4,*, dan cogălniceanu1,4,5 nematodes infecting anotosaura vanzolinia (squamata: gymnophthalmidae) from caatinga, northeastern brazil bruno halluan s. oliveira¹, adonias a. martins teixeira¹,*, romilda narciza m. queiroz¹, joão a. araujo-filho¹, diêgo a. teles¹, samuel v. brito2, daniel o. mesquita¹ where is my place? quick chorus structure assembly in the european tree frog michal berec a possible mutualistic interaction between vertebrates: frogs use water buffaloes as a foraging place piotr zduniak1,*, kiraz erciyas-yavuz2, piotr tryjanowski3 good vibrations: a novel method for sexing turtles donald t. mcknight1,2,*, hunter j. howell3, ethan c. hollender1, and day b. ligon1 errata to mecke, s., hartmann, l., mader, f., kieckbusch, m., kaiser, h. (2016): redescription of cyrtodactylus fumosus (müller, 1895) (reptilia: squamata: gekkonidae), with a revised identification key to the bent-toed geckos of sulawesi. acta herpetologica 11(2): issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 9(1): 51-59, 2014 doi: 10.13128/acta_herpetol-12667 habitat selection in the fossorial toad pelobates fuscus insubricus (amphibia: pelobatidae): does the soil affect species occurrence? loredana carisio1, roberto sacchi2, daniele seglie3, roberto sindaco4,* 1 regione piemonte, osservatorio regionale sulla fauna selvatica, corso stati uniti 21, 10100 torino, italy 2 dipartimento di scienze della terra e dell’ambiente, university of pavia, via taramelli 24, 27100, pavia, italy 3 department of life sciences and systems biology, university of torino, via accademia albertina, 13, 10123 torino, italy 4 istituto per le piante da legno e l’ambiente, corso casale, 476, 10132 torino, italy. *corresponding author. e-mail: rsindaco@gmail.com submitted on 2013, 13th april; revised on 2013, 21st november; accepted on 2014, 5th march abstract. for their rapid and alarming decline, the italian populations of the italian spadefoot toad (pelobates fuscus insubricus) are of high conservation importance. in this study we examined habitat use by the spadefoot toads in north-west italy, where one of the largest remaining populations lives. we used compositional analysis and logistic regression models to elaborate land-use and soil composition in order to determine the habitat preferences of the spadefoot toads. we found that the two main variables predicting the occurrence of spadefoot toads in the study area were the percentages of sandy-loam soil texture and the presence of entisols. our results showed that spadefoot toads preferred soils that keep soft and malleable structure: entisols with sand texture, where sand represents the major component, and mature soils, with a high degree of pedogenesis and a relatively high natural fertility and humidity. conversely, pelobates fuscus avoids inceptisols, probably too hard to be dug by the species. this study demonstrates that, at least in the italian range, the choice of areas in which to reintroduce p. fuscus, or where re-create favourable habitats for it, must take into account the soil types, which, in intensely cultivated areas, seems to be decisive for the possibility of survival of the species in the medium and long time. keywords. spadefoot toad, terrestrial habitat, soil, compositional analysis, conservation. introduction a central goal of conservation is to identify the main factors affecting the presence and abundance of species at large scales over long periods of time. only when favourable and unfavourable factors are known, it is possible to take appropriate conservation measures, in order to reduce the negative impacts and improve the environmental conditions to the advantage of the species. the spadefoot toad (pelobates fuscus) is a fossorial amphibian widely distributed in europe. in last century populations declined dramatically in the northern and western parts of its range, where several local extinctions were documented (france: lescure, 1984; parent, 1985; dubois, 1998; belgium: rappè, 1982; perczy, 1994; netherlands: pelt and van bree, 1965; denmark: fog et al., 1997; gislen and kauri, 1959; sweden: berglund, 1998). in italy, where the species was probably widespread in the whole po plain in the xix century, it currently occurs in a few isolated populations (andreone, 2006). despite new breeding sites have been recently discovered (mazzotti et al., 2002; andreone et al., 2004; mercurio and li vigni, 2007), the total number of known breeding sites has dramatically reduced since the end of the xx century (andreone et al., 2004), and the species is considered one of the most threatened amphibians in italy (rondinini et al., 2013). for example, in piedmont, out of 21 reproductive sites known in 1985, only 7 still exist despite several conservation projects (d. seglie, pers. obs.). 52 l. carisio, r. sacchi, d. seglie, r. sindaco the decline of spadefoot toad in italy is due to several concurrent causes, including the drastic reduction and alteration of suitable habitats, the increase of intensive agriculture, the change of agricultural practices in the ricefields, the worsening of water-quality, and the progressive fragmentation of the residual natural habitats. additional threats are the introduction of fishes and allochthonous crayfishes (e.g., procambarus clarkii), and possibly the introduction of allochthonous amphibians such as lithobathes catesbeianus (andreone, 2006; andreone et al., 2007), which may convey amphibian pathogens like batrachochytrium dendrobatidis. however, this fungus has not yet been found on pelobates fuscus (federici et al., 2008) in the areas of the piedmont where it affects other amphibians (adams et al., 2008). the conservation of the italian populations of the spadefoot toad is of particular interest, since they are considered as belonging to an endemic subspecies, p. f. insubricus, which is listed in the annexes ii and iv of the habitat directive 92/43/cee as priority species. from the taxonomic point of view, this subspecies is separated from the other european populations on the base of genetic distinctiveness and some morphological and acoustic features (héron-royer, 1888; andreone and piazza, 1990; andreone et al., 1993). however, while several authors recognize the italian populations as a distinct subspecies (nöllert and nöllert, 1992; andreone et al. 1993), its validity is still debated (andreone et al., 2007; crottini et al., 2007). the genetic study of crottini et al. (2007) confirmed that po plain populations keep a very high genetic variability within the “western” lineage, and a high number of haplotypes not found elsewhere in europe. this high genetic variability indicates that the po basin was one of the most important glacial refugia for the species during the pleistocene. as far as its ecology, in northern europe the occurrence of p. fuscus is determined by the interaction between pond features and hydroperiod with the occurrence of large predators such as fishes and crayfishes. breeding sites are large permanent ponds with high spring temperatures, high concentrations of phosphorus and oxygen, and a shoreline with a high proportion of steep banks (strijbosch, 1979; nyström et al., 2002). by contrast, composition of the terrestrial habitat close to the ponds and traffic has no or low effect on p. fuscus occurrence (nyström et al., 2002). in italy p. fuscus occurs in lowlands up to 400 m a.s.l. (andreone, 2006) and does not appear to be very selective for habitats with the exception of areas with substrates soft enough to allow burrowing, such as sandy soils (lanza, 1983; andreone et al., 1993; gentilli and scali, 2001). indeed, it has been found in woods, meadows, poplar plantations, cereal crops and ricefields (lanza, 1983; andreone et al., 1993; fortina and andreone, 1999; mazzotti et al., 2002; scali and gentilli, 2003). during the breeding season the species has been reported to use different types of wetlands, from ponds and marshes to ditches, almost all temporary sites, usually in open areas (lanza, 1983; scali and gentilli, 2003; andreone et al., 2007). however, no quantitative analyses on habitat preference have been carried out so far in italy, probably because of species rarity, the low consistence of residual populations and the difficulty to locate the spadefoot toads in the field (andreone et al., 2004). similarly, few studies have been carried out on habitat use outside the breeding season (eggert, 2002; bosman and van den munckhof, 2006). in this paper, we analysed the habitat preference of spadefoot toads in the last population settled south the city of turin; the main objective was to detect some of the habitat features surrounding breeding areas, with particular emphasis on soil, which could explain the presence/absence of the species in order to supply detailed information useful to develop management and conservation programs. material and methods study area and collecting data the study area (fig. 1) includes a portion of the alluvial plain south the city of turin and is delimited southwards by the towns of carmagnola (44°50’n, 7°53’e), poirino (44°55’n, 7°50’e) and santena (44°56’n, 7°46’e). this area hosts one of the last populations of spadefoot toads of piedmont, which breeds in several small and medium-large ponds that are included within the site of community importance (hereafter sci) “stagni di poirino-favari” (it111035), extended over an area of about 1,840 hectares (sindaco et al., 2009). since the discovery of the species (by g. boano in 1989), the area was thoroughly monitored by many researchers (see acknowledgments), mostly during night transects conducted through low-speed driving on paved and gravel roads. the data were collected between 1988 and 1991, during march-may (corresponding to the activity period of the species in this area), and account for 70 individuals. the research effort has been constant over the entire area, as all the roads of the study area were covered with the same intensity. the position of each toad was accurately recorded on the topographic maps of the italian military geographical institute (i.g.m.) at the scale 1:25.000, and then mapped using a gis. the localities of p. fuscus in the turin area (fig. 2) are derived from the available literature and the database of the regional herpetological atlas (andreone and sindaco, 1999). 53habitat selection in pelobates fuscus insubricus habitat variables we investigated habitat selection by spadefoot toads by assessing the evolution and texture of soil and the land use in the 70 spots were p. fuscus was found (fig. 1), and in 150 spots randomly selected within a 50 m buffer along the transects. the habitat variables accounted for soil type, soil texture and landcover, and were measured using digital maps of the regione piemonte (surveyed at the scale scale 1 : 10.000, published at the scale 1:50.000) (http://www.regione.piemonte.it/ agri/area_tecnico_scientifica/suoli/suoli1_50/carta_suoli.htm), reporting the degree of pedogenesis and soil texture according to the usda classifications (http://soils.usda.gov/technical/classification/taxonomy/). our study area involved six main soil types, i.e., alfisols with or without hydromorphisms (classes a1 and a3 respectively), inceptisols with or without hydromorphisms (classes b1 and b2 respectively), and entisols with or without hydromorphisms (classes c1 and c2 respectively). since classes a1, b2, and c2 covered less than 5% of the study area, they were grouped in the same class. therefore, in our analyses we used only four classes of soil evolution: alfisols (a3), inceptisols (b1), entisols (c1), and other soils (a1, b2, c2 combined). only five out of the twelve possible soil textures are present in the study area, with different percentages of sand and silt, from sandy soils (sand and loamy-sand classes, with more than 90% and 70% of sand respectively, in weight), intermediate soils (sandy-loam and loam classes, with 40-70% of sand) and silty soils (silt-loam class, with less than 40% of sand). in all these five classes clay occurs in no more than 20-25%. although the soil data go back over about 15 years ago, these data are still valid, because in agricultural landscapes the pedogenesis will stop in the topsoil, or at least slow down significantly, while in depth it continues. however, the soil composition does not change in a few decades (i. boni, pers. comm.). finally, habitat types were grouped in five main categories: urban areas (urban areas, parks and gardens within towns), woods (oak-hornbeam and locust woods), cultivated fields (maize, sunflower, and alfalfa), and poplar plantations. remnant habitat types were combined in a single group, which cover less than 5% of the study area. to compare the incidence of the aforementioned variables in places where p. fuscus individuals were detected, we considered 220 plots with a 50-m radius, centred on the point of observation of p. fuscus (70 plots), and on 150 random points (see above). moreover, we calculated the distance (in meters) from each plot to the border of the nearest urbanized area and to the nearest pond. fig. 1. findings of pelobates fuscus insubricus in the sci it1110035 “stagni di poirino favari” between 1988 and 1991. 54 l. carisio, r. sacchi, d. seglie, r. sindaco statistical analyses we used a t-test to check for differences between species and random plots in mean distances from the nearest urban area and the nearest pond. since soil variables were highly intercorrelated (suppl. mat. table t1), we used the compositional analysis (aebischer et al., 1993) to rank the soil evolution, soil texture and land-use variables according to their importance in promoting (or disfavouring) spadefoot toad occurrence. habitat availability was estimated using the random plots, while the wilk’s lambda, determined by randomization tests, was used to assess the significance of the ranking matrix fig. 2. historical and current distribution of pelobates fuscus insubricus in the plain south of turin. grey (intermediate gray): urbanized areas; red (dark gray): high suitable soils; yellow (light gray): suitable soils; white: unsuitable soils. populations: 1) piobesi t.se env., 2) right bank of po river between carignano and carmagnola; 3) carmagnola – san michele; 4) carmagnola – cervirola and tetti grandi; 5) carmagnola – casanova and env.; 6) moncalieri – testona, 7) rivoli, 8) torino – vanchiglia, 9) the study area. the species has been confirmed in recent times only in loc. 2 and 9. colour legend between brackets refers to the printed version of the image (black and white). 55habitat selection in pelobates fuscus insubricus (aebischer et al., 1993). in order to assess the potential effects of soil and land use variables on the occurrence of spadefoot toads, logistic regression models were developed to identify the most important variables likely affecting toad presence. since variables were intercorrelated, we developed all possible threepredictor models using one variable for soil evolution, one for soil texture and one for habitats respectively, in a way that every variable within each environmental group entered the same number of models. following this procedure we built 100 models (4 texture soils × 5 soil evolution classes × 5 land-use variables). inference from models was made according to the information-theoretic approach (anderson et al., 2000; anderson and burnham, 2002; mazerolle, 2006): for each model, we computed the differences with the minimum aic (δaic) and akaike weights (waic), and we ranked models according to this last index. the relative importance of predictor variables was measured by the sum of the models akaike weights (σw) where each variable appeared (anderson and burnham, 2002). to quantify the effects of the predictor variables the β (partial regression coefficients) were weighted and averaged on the models obtained β. the unconditional sampling variance (var β) and 95% confidence intervals were computed for all predictors in order to assess their statistical significance (anderson and burnham, 2002). all analyses were performed using the software r (r development core team, 2010), and data reported are means and standard errors, unless otherwise stated. to meet the assumptions of the analyses, data in proportions were arcsine transformed prior to analysis. results the p. fuscus presence plots were significantly closer to the nearest pond (presence plots: 557.3 ± 43.7 m, random plots: 1014.2 ± 82.7 m; welch two samples t-test: t = 7.581, df = 180.8, p < 0.001) and significantly apart from the nearest urban area (presence plots: 213.7 ± 26.9 m, random plots: 147.7 ± 14.0 m; welch two samples t-test: t = 2.172, df = 107.7, p = 0.032) than randomly selected ones. the compositional analysis for the variables concerning the evolution of soil generated a highly significant ranking matrix (λ = 0.752, p = 0.002), confirming that alfisols and entisols were preferred by the spadefoot toads, whereas inceptisols were significantly avoided (table 1). the ranking matrix was highly significant also as far as soil textures (λ = 0.475, p = 0.002), and indicated that sandy-loam soils were clearly preferred by toads, followed by sand and silt-loam ones, whereas loamy-sand soils were overall avoided (table 2). the ranking of habitat variables by compositional analysis was less clearly defined with respect to that obtained for the soil variables. indeed, even if the ranking matrix was highly significant (λ = 0.339, p = 0.002), it did not show a striking preference for a specific habitat but only a small preference for poplar plantations (table 3). as expected, urbanized areas were avoided by the species. the multi-model inference showed that p. fuscus occurrence was mainly influenced by sandy-loam soil texture, entisols, poplar plantations, and urban areas (table 4). the relatively major importance of the sandyloam soil texture and entisols was empirically supported by the high values of the sum of akaike weights for the models where the variables appeared (sandyloam soils: σw = 0.98; entisols: σw = 0.96). their effects table 1. ranking matrix of soil evolution variables comparing proportional soil use within plots of species occurrence with proportions of total available soil types evaluated in random plots. each mean element in the matrix is represented by its sign. when the sign occurs three times it represents a significant deviation from random at p < 0.05. alfisols entisols other soils inceptisols alfisols + + + + + + + entisols − + + + + + + other soils − − − − − − + + + inceptisols − − − − − − − − − table 2. ranking matrix of soil texture variables comparing proportional texture use within plots of species occurrence with proportions of total available texture types evaluated in random plots. symbols as in table 1. sandyloam sand silt-loam loam loamysand sandy-loam + + + + + + + + + + + + sand − − − + + + + + + + silt-loam − − − − loam − − − − − − − − − + + + loamy-sand − − − − − − − − − − − − table 3. ranking matrix of habitat variables comparing proportional habitat use within plots of species occurrence with proportions of total available habitat types evaluated in random plots. symbols as in table 1. poplar plantations woods cultivated fields other habitats urban areas poplar plantations + + + + + + + + woods − + + + + + + + cultivated fields − − other habitats − − − − − − − − − + + + urban areas − − − − − − − − − − − − 56 l. carisio, r. sacchi, d. seglie, r. sindaco on the occurrence of p. fuscus were largely positive as expressed by estimating the regression coefficient using model averaging (sandy-loam soils: β = 3.59; entisols: β = 2.21), even when models uncertainty was accounted for (β 95% confidence interval ranged from 2.02 to 5.15 for sandy-loam soils and from 1.35 to 3.06 for entisols). poplar plantations (σw = 0.51) and urban areas (σw = 0.36) were the third and the fourth predictors in order of importance. percentage of poplar plantations had a positive effect on spadefoot toad occurrence (β = 1.46; 95% confidence interval ranged from 1.19 to 1.73), whereas the effect of urban areas was negative (β = -0.76; 95% confidence interval ranged from -0.94 to -0.58). all other variables had σw less than 0.10, having a minor effect on p. fuscus settlement. discussion habitat selection in this paper we showed that the spadefoot toad has specific habitat requirements in our study area; its occurrence was primarily associated with the conditions of soils, and the two most important variables predicting its distribution were the percentage of sandy-loam soil texture and the presence of entisols. nonetheless, these two variable were not highly intercorrelated (pearson’s rp = -0.18), but sandy-loam texture was mainly associated to alfisols (rp = 0.45), whereas entisols related principally with sand soil texture (rp = 0.79). therefore, spadefoot toads occurs in two opposite soil conditions: on one hand the soils showing any profile development other than a small a horizon, basically unaltered from their parent material, where sand represents the major component (entisols with sand texture); on the other hand the mature soils, with an high degree of pedogenesis, a well evident three-horizons profile, and a relatively high native fertility and humidity. in this last case, the percentage of sand remained high, but a relevant portion of silt and clay was also present. despite their different composition, both types of soils keep a soft and malleable structure that can easily be dug. spadefoot toads in our study area avoided the inceptisols, which are of relatively recent origin, having only a weak appearance of the horizons produced by pedogenesis. the humus does not accumulate in layers, but is mixed with the mineral matrix in a more thin texture (loam and silk-loam; suppl. mat. table t1) with respect to the two other dominant soils. consequently, the soil is denser and uneasy to be dug by the toads. a second relevant result of this study was the negative impact of urban areas, which confirms that the disturbance by human presence and activities is among the main causes of the decline of the species. the negative effect of urban areas can be explained by several concomitant conditions: the progressive erosion of suitable habitats due to the expansion of the metropolitan area of turin (inhabited by about 1,700,000 people), the increase of barriers (mainly roads) among breeding sites that segregates the population in smaller and more isolated subpopulations, and the direct killing due to traffic load. table 4. multi-model inference on models parameters and relative importance of the 14 soil and land-use variables used for analysing habitat selection by p. fuscus: σw, akaike weights; β, averaged weighted partial regression coefficient; se(β), standard error of β; lower ci95% and higher ci95%, confidence interval at 95%. variabile σw β se(β) lower ci95% higher ci95% sandy-loam 0.9810 3.5857 0.7998 2.0180 5.1534 entisols 0.9619 2.2067 0.4346 1.3549 3.0585 poplar plantations 0.5085 1.4634 0.1389 1.1912 1.7356 urban areas 0.3583 -0.7623 0.0912 -0.9410 -0.5836 woods 0.0820 0.2689 0.0619 0.1475 0.3902 other habitats 0.0264 -0.0187 0.0128 -0.0437 0.0063 cultivated fields 0.0248 0.0006 0.0038 -0.0068 0.0081 loamy-sand 0.0190 -0.0601 0.0132 -0.0859 -0.0343 inceptisols 0.0155 -0.0185 0.0039 -0.0261 -0.0109 other soils 0.0126 -0.9625 11.5472 -23.5950 21.6700 alfisols 0.0099 0.0143 0.0031 0.0082 0.0203 sand 4.6×10-08 1.1×10-07 2.63×10-08 6.2×10-08 1.6×10-07 silt-loam 5.7×10-10 -1.3×10-09 3.6×10-10 -2.0×10-09 -6.0×10-10 loam 3.7×10-10 -3.3×10-10 9.1×10-11 -5.0×10-10 -1.5×10-10 57habitat selection in pelobates fuscus insubricus despite traffic load had not been found to be a relevant threat in northern europe populations (hels and buchwald, 2001; nyström et al. 2002), the proportion of toads killed might be relevant in our study area, since many of the observed specimens were found dead on road. these results agree with the past and current presence of the spadefoot toad in the area south of turin (fig. 2) where it is evident the presence of populations in areas with suitable soil and the extinction of some populations due to urbanization. finally, we also found that poplar plantations were among the preferred habitats of the spadefoot toads, as previously reported for other italian populations (andreone et al., 2007). the preference for this type of habitat is only marginally dependent on the quality of soils where poplars are planted: even though poplars are cultivated preferentially in sandy-loam soils (rp = 0.18), the value of the correlation coefficient is too low to fully explain the positive selection of poplar plantations by toads as a simple correlation with soils texture. two possible reasons leading spadefoot toads to prefer poplar plantations might be: 1) the fact that the large part of intensively cultivated areas of piedmont, and in particular the corn-fields, are too disturbed by deep plowing and show a fauna of invertebrates very depleted compared to poplar plantations (casale et al., 1993); 2) the milling, which farmers regularly do in order to keep the ground under the canopy free from weeds; this practice turns over and fragments the soil, which might become softer and suitable for spadefoot toads irrespective of its original structure and texture. conservation due to its rarity, several conservation actions have been carried out in italy during the last decades to protect the spadefoot toad (andreone, 1984, 2001; andreone et al., 1993; fortina et al., 2000; scali et al. 2001; ferri, 2002; but see andreone et al., 2004). the initial projects had mainly dissemination and education purposes (andreone et al., 1993; andreone, 2001), whereas the more recent ones involved also habitat management actions (sindaco et al., 2013). the latter gave priority to identification and conservation of the main breeding sites and the surrounding areas (andreone et al., 2004), restoring of degraded ponds and habitats previously used for breeding (scali et al., 2001), looking for new populations (mazzotti et al., 2002, mercurio and li vigni, 2007), digging of new ponds and tadpoles translocation (scali et al., 2001). the reintroduction attempts failed because some essential parameters were not considered, particularly the type of breeding sites and soil characteristics (e.g., in vanzago lombardy, and in bellinzago novarese and agrate conturbia piedmont). despite based on a dataset dating back more than 20 years, the present paper provides soil parameters useful to identify areas suitable for the species on wide areas of the po valley, where the totality of the italian populations of the spadefoot toad occurs. as well as many areas of the po plain, the landscape of the plain south to turin has been rapidly transformed from an area dominated by small scale farming and numerous fish-ponds, to an area with large scale agriculture, increasing urbanization and industry. several original wetlands suitable for spadefoot toads went lost, and the long term survival of the species in most of its italian range is strictly dependent from the conservation of the residual artificial ponds within a habitat matrix with features fitting its habitat requirements. the information obtained in this study might help researchers to better identify and protect the areas characterized by the most suitable habitats for the species, to design actions of habitat restoring to improve the quality of the existing habitats, and to detect more efficiently where the digging of new ponds might provide the best results for the breeding performance of the species. lastly, combining the digital maps of soils now available with detailed land-use maps, large-scale models of habitat suitability for the species could be developed. these maps could help researchers to detect the areas where to concentrate the efforts to locate other yet unknown populations of spadefoot toads. acknowledgements we wish to thank the people who provided many of the records of pelobates used for this article: riccardo fortina, roberto marocco, giovanni boano, giovanni battista delmastro, franco andreone. paolo martalò (i.p.l.a., istituto per le piante da legno e l’ambiente, torino) provided information on soil 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(1979): habitat selection of amphibians during their aquatic phase. oikos 33: 363-372. supplementary material table t1. pearson correlation matrix (rp) among soil and land-use variables measured by the gis in the 220 plots (70 with spadefoot toad and 150 randomly selected). acta herpetologica vol. 9, n. 1 june 2014 firenze university press new cranial characters in the tribe hydropsini (serpentes: dipsadidae: xenodontinae) diego o. di pietro1,*, leandro alcalde1,2, jorge d. williams1 the tadpole of odontophrynus barrioi cei, ruiz, and beçak, 1982 (anura: odontophrynidae): a comparison with the other tadpoles of the genus exequiel gonzález1, guillermina galvani1, eduardo sanabria2,*, diego a. barrasso3, leandro alcalde4, lorena quiroga1 high levels of prevalence related to age and body condition: host-parasite interactions in a water frog pelophylax kl. hispanicus mar comas1, alexis ribas2,*, concetta milazzo3, emilio sperone3, sandro tripepi3 microclimatic variation in multiple salamandra algira populations along an altitudinal gradient: phenology and reproductive strategies daniel escoriza ¹, jihene ben hassine² fire salamander (salamandra salamandra) in larzac plateau: low occurrence, pond-breeding and cohabitation of larvae with paedomorphic palmate newts (lissotriton helveticus) mathieu denoël*, laurane winandy habitat selection in a fossorial toad pelobates fuscus insubricus (amphibia: pelobatidae): does the soil affect species occurrence? loredana carisio1, roberto sacchi2, daniele seglie3, roberto sindaco4,* a new, recently extinct subspecies of the kuroiwa’s leopard gecko, goniurosaurus kuroiwae (squamata: eublepharidae), from yoronjima island of the ryukyu archipelago, japan yasuyuki nakamura1, akio takahashi2, hidetoshi ota3 first evidence of the effects of agricultural activities on gonadal form and function in rhinella fernandezae and dendropsophus sanborni (amphibia: anura) from entre ríos province, argentina laura c. sanchez1,*, rafael c. lajmanovich1, paola m. peltzer1, adriana s. manzano2, celina m. junges1, andrés m. attademo1 a review of the genus gonionotophis in north-eastern africa (squamata: lamprophiidae) benedetto lanza1, donald g. broadley2 observations on the use of tarantula burrows by the anurans leptodactylus bufonius (leptodactylidae) and rhinella major (bufonidae) in the dry chaco ecoregion of bolivia christopher m. schalk1, marco sezano2 a preliminary report of amphibian mortality patterns on railways karolina a. budzik1, krystian m. budzik2 meiotic behavior of two polyploid species of genus pleurodema (anura: leiuperidae) from central argentina nancy e. salas1, julián a. valetti2, pablo r. grenat1,3,*, manuel a. otero1, adolfo l. martino1 clutch size in wild populations of alytes muletensis samuel pinya1,*, valentín pérez-mellado2 consequences of haemogregarine infection on the escape distance in the lacertid lizard, podarcis vaucheri isabel damas-moreira1,2,*, d. james harris1, daniela rosado1,2, isabel tavares1,2, joão p. maia1,2,3, daniele salvi1, ana perera1 death in the clouds: ranavirus associated mortality in assemblage of cloud forest amphibians in nicaragua tariq stark1,*, carlijn laurijssens¹, martijn weterings¹,², annemarieke spitzen-van der sluijs³,4, an martel4, frank pasmans4 book review: federico chacón, richard dennis johnson. amphibians and reptiles of costa rica. federico chacón, richard dennis johnson. amphibians and reptiles of costa rica. anfibios y reptiles de costa rica. sebastiano salvidio acta herpetologica journal of the societas herpetologica italica acta herpetologica 12(2): 125-132, 2017 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-20740 meristic and morphometric characters of leptopelis natalensis tadpoles (amphibia: anura: arthroleptidae) from entumeni forest reveal variation and inconsistencies with previous descriptions susan schweiger1, james harvey2, theresa s. otremba1, janina weber1, hendrik müller1,* 1 institut für spezielle zoologie und evolutionsbiologie mit phyletischem museum, friedrich schiller universität jena, erbertstrasse 1, 07743 jena, germany. *corresponding author. e-mail: hendrik.mueller@uni-jena.de 2 41 devonshire avenue, howick, 3290, south africa submitted on: 2017, 5th june; revised on: 2017, 31st august; accepted on: 2017, 3rd october editor: rocco tiberti abstract. the tadpole of leptopelis natalensis is described based on a series of 32 specimens from entumeni forest, kwazulu-natal, south africa. previous descriptions are brief, lack morphometric data, or are based on specimens of imprecise origin. the tadpole resembles other leptopelis tadpoles and is generally in agreement with existing accounts, although some differences exist. some of these differences seem to fall within the range of natural variation. others, such as the presence of a fifth anterior row of keratodonts, might be indicative of variation at the population level and should be considered in future taxonomic revisions. leptopelis natalensis tadpoles seem to be most readily distinguished by their more narrowly keratinized beaks from the geographically overlapping or adjacent l. mossambicus and l. xenodactylus. keywords. south africa, kwazulu-natal, eastern cape, taxonomy, buccal morphology, ontogenetic variation. introduction the genus leptopelis currently comprises 53 described species (frost, 2017) of medium to large tree frogs that are distributed throughout most of sub-saharan africa (schiøtz, 1999). the most southerly distributed species of the genus is l. natalensis, which is found in a variety of habitats along the eastern region of the south african provinces of kwazulu-natal and part of eastern cape (schiøtz, 1999; bishop, 2004; venter and conradie, 2015). although some, mostly brief, descriptions and illustrations of l. natalensis tadpoles have been published (wager, 1930, 1965; van dijk, 1966; lambiris, 1988; channing, 2001; du preez and carruthers, 2009; channing et al., 2012), channing (2001) considered none of the south african leptopelis tadpoles to have been adequately described. the various descriptions furthermore differ in a number of diagnostic characters, such as labial keratodont formula. these differences in the existing descriptions could be the result of variation or population-specific differences that might be significant in future taxonomic revisions (penske et al., 2015). to differentiate between these options, it is important to provide detailed locality information, which is lacking in some works providing information on tadpole morphology of l. natalensis (e.g., du preez and carruthers, 2009). while detailed, or at least limited, locality data are provided by wager (1930, 1965, 1986), pickersgill (2007) and channing et al. (2012), these accounts provide mostly meristic data but few or no morphometric data that would help in assessing subtle differences that might exist between populations. we here provide a description and measurements of an ontogenetic series of tadpoles of l. natalensis based on a series collected at entumeni forest, 126 susan schweiger et alii kwazulu-natal, assess variability and ontogenetic changes, and highlight differences between these and previous descriptions. materials and methods a total of 32 tadpoles were collected at entumeni forest, kwazulu-natal, south africa on 3 december 2014. the tadpoles were collected in a small forest stream (28.888334°s, 31.365928°e). some tadpoles were preserved on the day of collection, while others were raised on a diet of commercial aquarium fish food and sampled in regular intervals to obtain different stages of development. identification of the tadpoles was confirmed by raising some through metamorphosis. specimens were euthanized in an aqueous solution of tricaine methanesulfonate (ms222; fluka), fixed in 4% neutral buffered formalin, and transferred to 70% ethanol. voucher specimens have been deposited in the herpetological collection of the museum für naturkunde berlin, germany (zmb85717). staging followed gosner (1960). standard measurements and labial tooth row formula followed altig and mcdiarmid (1999) and description of buccopharyngeal morphology wassersug (1976). drawings were prepared with the aid of a camera lucida attached to a zeiss sv12 stereomicroscope. for inspection of the buccopharyngeal morphology, one tadpole of stage 36 was dissected, postfixed with 1% osmium tetroxide, dehydrated and critical point dried in an emitech k850 critical point dryer, sputter coated with gold-palladium using an emitech k500 and investigated using a phillips xl30 esem scanning electron microscope with a digital image capture system. description tadpole. the description is based on 32 tadpoles from gosner (1960) stages 25 to 42 (see table 1 for measurements and detailed information). the tadpole is slender overall, with a moderately elongated, slightly dorsoventrally flattened body (wider than deep) and a relatively long tail with low dorsal and ventral fins. the widest point of the body is just behind the eyes (fig. 1). no nares are visible until stage 34. from stage 35 to 37, the nares are indicated as light coloured spots, but do not seem to be open until stage 38. when fully formed, the nares are positioned dorsolaterally, about twice as far from the eye than the tip of the snout in lateral view. the eyes are positioned dorsally. a small anlage of the developing eyelid is first visible at stage 40, anterior of the eye. the spiracle is sinistral, about as far from the snout as from the body-tail junction. the spiracular opening is an upright oval, slightly slanted forward; its largest diameter almost as large as the diameter of the eye lens. the spiracular tube is angled upwards at about 45°; the posterior end, including its inner wall, is free from the body. the tail is about 2.5 times as long as the body (see table 1 for measurements) and very muscular. the myomeres are visible in the posterior half of the tail, but are otherwise indistinct or obscured by the dense pigmentation. the tailfins are very low, with the dorsal fin marginally deeper than the ventral fin. the dorsal fin has a low origin on the base of the tail, just behind the tail-body junction, and gradually rises towards the middle of the tail, where it reaches its maximum height. the ventral tailfin is very even, with the margin of the fin running more or less parallel to the ventral edge of the muscular tail. the overall deepest point of the tail is at about half its length. tip of tail is pointed, with the muscular tail terminating some distance before the tail tip (fig. 1b). the vent tube is attached to the right side of the ventral tailfin, with a very large opening forming a pointed arch. the coiled gut is well visible through the ventral body wall. oral disc. the oral disc is positioned subterminally and is not visible in dorsal view. the oral disc is lightly emarginated and has a broad rostral gap. one row of globular marginal papillae is present anterolaterally and laterally; posteriorly, two rows of papillae are present. papillae are largest anterolaterally and laterally, and smaller posteriorly. a few submarginal papillae are present laterally. keratodont formula is 4(2-4)/3(1) in the majority of the examined specimens (see below for variation). moving inwards, supralabial keratodont rows are progressively smaller (fig. 1a), infralabial rows are of nearly equal length, with p3 being slightly shorter than p1 and p2. interruption of p1, if present, is very narrow in some specimens (fig. 1a) but more pronounced in others. keratodonts are about equally sized in most rows, except in p3 where they get progressively smaller laterally. the individual keratodonts are spoon-shaped and have eight or nine cusps along their margins, with the apical cusps being larger than the more laterally positioned ones (see inset in fig. 2a). the jaw sheaths are serrated but only narrowly keratinised (indicated by the dark pigmentation). by stage 42, the lower jaw sheath and all keratodonts are absent and the papillation is much reduced in extent. buccopharyngeal morphology. the prenarial area of the buccal roof (fig. 2a) is somewhat elongated and contains a few scattered pustules. in addition, a pair of short ridges is present and somewhat slanted laterally. the orientation of the choana is oblique to the midline (about 45 degrees) and both choanae form a forward-pointing angle of approximately 90 degrees. the jagged narial valves project deep into the buccal cavity and obscure the choanal openings. anterolateral of each choana is a broad, flaplike papilla with a pustulate edge. three to four thick, broad-based papillae are present posterolaterally to each 127tadpole of leptopelis natalensis table 1. measurements of leptopelis natalensis. all measurements in millimeters (arithmetic mean ± sd). * indicates a damaged tail in one of the specimens of the series, which was omitted from the calculations. gosner stage 25 (n=3) 31 (n=1) 34 (n=1) 35 (n=2) 36 (n=14) 37 (n=2) 38 (n=2) 39 (n=1) 40 (n=1) 41 (n=1) 42 (n=4) total length 32.1 ± 2.3* 29.7 34.9 34.1 ± 1.2 36.5 ± 2.7* 35.3* 37.9 ± 0.8 41.7 39.1 37.2 36.2 ± 2.3 body length 7.1 ± 0.6 8.2 9.5 9.2 ± 0.8 10.2 ± 0.8 10.7 ± 0.0 10.7 ± 0.8 11.8 11.2 11.0 10.9 ± 0.3 body width 3.8 ± 0.2 4.0 4.9 4.5 ± 0.3 5.2 ± 0.6 5.9 ± 0.1 5.9 ± 0.1 6.0 6.1 5.5 4.3 ± 0.6 body height 2.9 ± 0.1 3.6 4.2 3.4 ± 0.4 4.3 ± 0.4 4.8 ± 0.1 4.9 ± 0.1 5.3 4.4 5.0 4.2 ± 0.2 tail length 16.7 ± 1.6* 21.2 25.2 24.9 ± 0.1 26.4 ± 2.3* 24.5* 27.1 ± 0.4 30.0 27.6 27.0 26.1 ± 1.8 tail height 3.3 ± 0.3 3.6 4.2 4.1 ± 0.4 4.6 ± 0.3 4.4 ± 0.6 5.1 ± 0.4 5.0 4.9 5.0 4.1 ± 0.5 tail muscle height 1.9 ± 0.3 2.3 3.0 2.8 ± 0.2 3.2 ± 0.4 3.2 ± 0.1 3.1 ± 0.1 3.5 3.0 2.9 2.8 ± 0.3 width of oral disc 1.2 ± 0.2 1.4 1.6 1.6 ± 0.1 1.9 ± 0.2 1.9 ± 0.1 2.0 ± 0.1 2.4 2.2 2.0 1.6 ± 0.2 interorbital distance 2.7 ± 0.3 3.3 3.9 3.5 ± 0.8 4.1 ± 0.3 4.4 ± 0.1 4.3 ± 0.1 4.6 4.3 4.7 4.7 ± 0.4 internarial distance 1.9 ± 0.9 2.0 1.9 1.7 1.4 ± 0.2 snout-naris distance 1.6 ± 0.1 1.6 1.7 1.6 1.2 ± 0.3 snout-eye distance 2.5 ± 2.6 2.9 3.3 3.4 ± 0.1 3.3 ± 0.2 3.5 ± 0.2 3.4 ± 0.1 3.9 4.0 3.8 3.1 ± 0.1 snout-spiracle distance 4.9 ± 0.5 5.4 6.1 6.3 ± 0.4 6.7 ± 0.4 27.1 ± 0.3 7.1 ± 0.2 8.1 7.4 7.2 naris-eye distance 1.9 ± 0.1 2.1 2.0 2.1 2.2 ± 0.1 eye diameter 0.6 ± 0.1 0.8 1.1 1.0 ± 0.0 1.2 ± 0.1 1.2 ± 0.1 1.2 ± 0.1 1.5 1.5 1.5 1.4 ± 0.1 fig. 1. oral disc (a), lateral (b) and dorsal (c) view of a gosner stage 36 tadpole of leptopelis natalensis from entumeni forest, kwazulu natal, south africa. scale bar equals 0.5 mm in (a) and 5 mm in (b) and (c). 128 susan schweiger et alii fig. 2. scanning electron microscope images of the (a) buccal roof and (b) buccal floor of a gosner stage 36 tadpole of leptopelis natalensis. inset in (a) shows a close-up of a keratodont. scale bars in (a) and (b) equal 1 mm, and 5 µm in the inset. 129tadpole of leptopelis natalensis choana, and about six small pustules are present in the area between them. the median ridge separating postnarial arena and buccal roof arena is very prominent and forms an almost semi-circular flap. the buccal roof arena is fringed by eight pairs of medium to large papillae of similar sizes as in the buccal floor arena. an additional three to four pairs of smaller papillae are present in second row towards the posterior part of the buccal roof arena. there is furthermore a group of three to four short lateral roof papillae at each side of the arena. within the buccal roof arena and posterior to it are ca. 100 pustules. a well-defined glandular zone with numerous secretory pits is present; it is broader laterally and has a relatively narrow medial gap. the dorsal velum has a broad medial gap and number of smaller papillae and pustules along its edge and sides. additional pustules are present posterior to the dorsal velum and within its median gap. in the buccal floor (fig. 2b), a pair of large, flaplike infralabial papillae, with smaller pustules along their margins, is present immediately inside the oral cavity on each side. an additional large, flap-like infralabial papilla is present medially just behind the lower jaw sheath. four large lingual papillae are present on the tongue anlage. the area immediately behind the lingual papillae is marked by a transverse groove. to the left and right of this grove is a fairly large, bulbous structure. the buccal floor arena is fringed by nine to ten pairs of medium to large buccal floor papillae, all curved towards the buccal floor arena, except for the posteriormost pair of papillae, which point backwards (possibly an artefact of preservation). around 60 pustules cover the posterior two thirds table 2. ontogenetic variation in labial keratodont formula. number in brackets indicates number of specimens exhibiting the labial keratodont formula; asterisk (*) indicates asymmetry in the last (innermost) anterior keratodont row, with keratodonts present on one side only; keratodont data were only taken for 13 of the 14 investigated specimens of stage 36. stage labial keratodont formula 25 4(2-4)/3(1) [2]; 4(2-4*)/3 [1] 31 5(2-5)/3(1) [1] 34 5(2-5*)/3(1) [1] 35 4(2-4)/3(1) [2] 36 4(2-4)/3(1) [6]; 4(2-4)/3 [1]; 5(2-5*)/3(1) [1]; 5(2-5)/3(1) [5] 37 5(2-5)/3(1) [2] 38 4(2-4)/3(1) [2] 39 5(2-5)/3(1) [1] 40 5(2-5*)/3(1) [1] 41 5(2-5*)/3(1) [1] table 3. summary of published information on leptopelis natalensis tadpoles. ec – eastern cape province, kzn – kwazulu-natal province, g – gosner (1960) stage. reference locality keratodont formula oral disc characters maximum length /tail length as multiple of body length this study entumeni forest (kzn) 4(2-4)/3(1) or 5(2-5)/3(1), rarely 4(2-4)/3 double row of marginal papillae posteriorly, single row of slightly larger papillae laterally; jaw sheaths delicate and narrowly keratinized; disc emarginated 42mm (g39)/2.5x wager (1930) port st. johns (ec) 4(2-4)/3 double row of marginal papillae posteriorly, single row laterally; disc emarginated 51mm/2.5x wager (1965) port st. johns (ec) 4(2-4)/3 double row of marginal papillae posteriorly, single row laterally; disc emarginated 49mm/2.75x durban (kzn) 4(2-4)/3 35mm/2.5x nkandla (kzn) 5(2-5)/3 50mm/2.3x lambiris (1988) 4(2-4)/3 double row of marginal papillae posteriorly, single row laterally 50mm (g38)/pickersgill (2007) hillcrest (kzn) 4(2-4)/3, sometimes 4(24)/3(1) double row of marginal papillae posteriorly, single row laterally; jaw sheaths narrowly keratinized; disc not emarginated 49mm (g37?)/2.5x du preez and carruthers (2009) 4(2-4)/3, sometimes 4(24)/3(1) double row of marginal papillae posteriorly, single row laterally; jaw sheaths delicate 50mm/2.6x (figured tadpole) channing et al. (2012) kzn 4(2-4)/3 double row of marginal papillae posteriorly, single row of slightly larger papillae laterally; jaw sheaths delicate and keratinized along margins; disc emarginated 35mm/2.2x 130 susan schweiger et alii of the buccal floor arena, as well as the area immediately posterior to it. in addition, ca. 15 pustules are present anterolaterally of the buccal floor arena, just in front of the buccal pockets. the buccal pockets are simple, narrow but deep, curved slits, with no associated papillae or pustules. it is unclear whether the buccal pockets are perforated to provide a bypass to the atrial chamber (wassersug, 1976) or whether these end blind. the ventral velum is wide, with four marginal projections on each side, and a deep medial notch that exposes the glottis. the ventral velum contains secretory pits along its margin. coloration in life. a nearly uniform dark olive, with a scatter of iridiophores across the entire dorsal and lateral sides of body and tail. ventral side more sparsely pigmented anteriorly, but skin above the abdominal cavity completely unpigmented and translucent in younger stages but becoming somewhat more opaque in older specimens. coloration in preservative. dorsal body densely pigmented and overall homogenously dark brown in colour. lateral line system very well visible as pigment-free spots in clearly defined lines along the body. ventral body sparsely pigmented anteriorly but pigment-free above the abdominal cavity, with the coiled gut clearly visible. pigmentation becomes somewhat less dense on tail and individual melanophores more easily discernible. pigmentation on the dorsal tail-fin similar to the muscular tail, but distalmost edge pigment-free. ventral fin free of pigment and translucent, with only some scattered melanophores present along the basal edge and towards the posterior end. variation. overall, little variation is present within the examined material. specimens differ slightly in the distribution of melanophores on the tail-fins, with some specimens having a ventral tailfin that is almost entirely free of pigmentation except for the very tip of the tail, whereas others show scattered pigment to a various extent within the posterior half of the fin. pigmentation of the dorsal fin also slightly less or more dense in some specimens. the most variation is seen in the oral disc, in particular the number and arrangement of keratodont rows. slightly more than half of the specimens (14 of 27; see table 2) have four anterior rows of keratodonts, with the first always undivided, and the remainder divided. the rest of the specimens have an additional, innermost fifth keratodont row (a5). in all specimens, the last anterior row is usually rather short and in a number of specimens present on one side only (table 2). the first posterior row is usually divided by a small gap of variable width, but undivided in two of the 27 specimens examined that have an oral apparatus. discussion overall, the tadpole of l. natalensis resembles other leptopelis tadpoles in general shape and appearance (see channing et al., 2012, and barej et al., 2015, for most recent and comprehensive treatments of the group). in south africa, the range of l. natalensis is close to or overlaps with the ranges of l. xenodactylus and l. mossambicus (channing, 2001; minter et al., 2004). based on the available information, the tadpole of l. mossambicus appears to be somewhat larger and proportionally shorter-tailed than that of l. natalensis, and overall darker in colouration, being more brown than olive (du preez and carruthers, 2009). leptopelis mossambicus further appears to differ from l. natalensis by having slightly higher tailfins, a very broad rostral gap in the papillation of the oral disc that is almost as broad as the disc itself, and somewhat more robust jaw sheaths that are keratinized for about half their width (du preez and carruthers, 2009; channing et al., 2012). the tadpole of l. xenodactylus is very similar to l. natalensis and these species appear to be indistinguishable by overall shape and colouration alone. however, in contrast to l. natalensis, l. xenodactylus tadpoles do seem to have a more robustly keratinized jaw (channing, 2008; du preez and carruthers, 2009; channing et al., 2012), which should help facilitate a correct identification. in addition there are differences in papillation, with more submarginal papillae being present in l. xenodactyloides and the posterior papillae differing in size (inner row of shorter, more globular papillae and outer row of relatively long papillae; channing 2008). the two species have so far not been found in sympatry, although they occur in close proximity to each other in central kwazulu-natal. a number of descriptions of the tadpole of l. natalensis have been provided before (summarized in table 3), but most of them are brief, lack measurements, or do not provide precise locality information. all previous accounts and our observations agree on the overall shape and colouration of the tadpoles, but some differences especially in total length, oral disc morphology, and keratodont formula exist. while most accounts provide a maximum length of around 50 mm, wager (1965) and channing et al. (2012) reported 35 mm as total length, at least for some populations, but did not indicate what stage the examined specimens were at. furthermore, there is some variation regarding the length of the tail, but all of this seems to be within the limit of normal variation, given that maximum length is largely dependent on condition. while most previous investigators reported or figured a slightly emarginated oral disc, which matches our own observations, pickersgill (2007) illustrated a disc 131tadpole of leptopelis natalensis that is not emarginated. assuming all observations to be correct, this would indicate a more substantial difference between that population and others. both van dijk (1966) and du preez and carruthers (2009) reported the presence of an elygium in the eye of l. natalensis tadpoles, although van dijk (1966) indicated that an ocular elygium might not generally be present and is not easily detected. in our specimens, an ocular elygium is not present, but dorsally the pigmented skin seems to somewhat extend onto the eyeball, which may represent an epidermal elygium (see kruger et al., 2013). most previous descriptions gave the keratodont formula of l. natalensis tadpoles as 4(2-4)/3, indicating an undivided p1 (wager, 1930, 1986; lambiris, 1988; pickersgill, 2007; du preez and carruthers, 2009; channing et al., 2012), but pickersgill (2007) and du preez and carruthers (2009) also stated that p1 can sometimes be divided. in our series, only two specimens had an undivided p1, the rest all had a divided p1 although the gap was very slight in some specimens. while this may be an indication of interpopulational variation, it seems possible that previous reports might have simply overlooked a narrow gap in p1. similar variations in the presence of a divided vs. undivided p1 have been reported by penske et al. (2015) for leptopelis cf. grandiceps. furthermore, only slightly more than half of the specimens (14, see table 2) of our ontogenetic series of l. natalensis tadpoles had four anterior rows of keratodonts. almost as many (13 specimens) had an additional, divided a5 and a resulting keratodont formula of 5(2-5)/3(1). although also present in some younger specimens, the presence of an a5 seemed to be more pronounced in older tadpoles (table 2). variation in the number of keratodont rows has been reported for a number of species, including l. calcaratus, l. gramineus, l. vannutelli and l. yaldeni (see channing et al., 2012). an ontogenetic increase has further been reported for l. aubryoides (barej et al., 2015), l. calcaratus (lamotte and perret, 1961) and l. viridis (rödel 2000), and specimens with an additional a5 have been reported for l. modestus, l. spiritusnoctis and l. rufus (barej et al., 2015). in many anuran tadpoles, anterior keratodont rows are added during ontogeny and the presence of an a5 in some leptopelis might be related to overall tadpole size. it is therefore possible that previous investigations did not examine specimens of a sufficient age for an a5 to be expressed. at the same time, the maximum length of 50 mm reported by several authors for l. natalensis (e.g., wager, 1930; lambiris, 1988; see table 2 for full list), which substantially exceeds the 42 mm of the largest specimen in our sample, would argue against this. only wager (1965) reported tadpoles with an a5 from nkandla forest, kwazulu-natal. entumeni forest, the origin of the specimens examined here and only other reported population of l. natalensis tadpoles with a fifth anterior row of keratodonts, is less than 30 km away from nkandla forest. given the current state of knowledge, these two populations seem diagnosably different from other l. natalensis populations at the level of tadpole morphology. future studies of phylogeography of l. natalensis should include these populations and investigate their degree of differentiation compared to others. acknowledgements we would like to thank ezemvelo kzn for issuing the necessary permits (op4976/2013, op635/2014) to undertake our research and in particular adrian armstrong and sharon louw for their generous advice and help facilitating our work. for help in the field we are grateful to lars möckel and katrin friedemann. funding was provided through a german science foundation (dfg) grant to hm (mu2914/2-1). references altig, r., mc diarmid, r.w. (1999): body plan – development and morphology. in: tadpoles – the biology of anuran larvae, pp. 24-51. mc diarmid, r.w., altig, r., eds, chicago university press, chicago. barej, m.f., pfalzgraff, t., hirschfeld, m., liedtke, h.c., gonwouo, n.l., penner, j., dahmen, m., dohertybone, t., schmitz, a., rödel, m.o. (2015): the tadpoles of eight west and central african leptopelis species (amphibia: anura: arthroleptidae). amphib. rept. cons. 9: 56-84. bishop, p.j. (2004): leptopelis natalensis (smith, 1849). in: atlas and red data book of the frogs of south africa, lesotho and swasiland, pp. 160-162. minter, l.r., burger, m., harrison, j.a., braack, h.h., bishop, p.j., kloepfer, d., eds, smithsonian institution, washington, d.c. channing, a. (2008): the mud dwelling tadpole of the long-toed tree frog, leptopelis xenodactylus (arthroleptidae). herp. rev. 39: 288-290. channing, a. (2001): amphibians of central and southern africa. comstock publishing associates, cornell university press, ithaca. channing, a., rödel, m.o., channing, j. (2012): tadpoles of africa. edition chimaira, frankfurt-am-main. du preez, l., carruthers, v. (2009): a complete guide to the frogs of southern africa. struik nature, cape town. 132 susan schweiger et alii frost, d.r. (2017): amphibian species of the world: an online reference. version 6.0 (accessed 23 may 2017). electronic database accessible at http:// research.amnh.org/herpetology/amphibia/index.html. american museum of natural history, new york. gosner, k.l. (1960): a simplified table for staging anuran embryos and larvae with notes on identification. herpetologica 16: 183-190. kruger, d.j.d., weldon, c., minter, l.r., du preez, l.h. (2013): morphology of the elygium and developing umbraculum in the eye of amietia vertebralis tadpoles. j. morphol. 274: 551-556. lambiris, a.j.l. (1988): a review of the amphibians of natal. lammergeyer 39: 1-212. lamotte, m., perret, j.l. (1961): contribution à l’étude des batraciens de l’ouest africain xiii. les formes larvaires de quelques espèces de leptopelis: l. aubryi, l. viridis, l. anchietae, l. ocellatus et l. calcaratus. bulletin de l’institut fondamental d’afrique noire, serie a 3: 855-885. minter, l.r., burger, m., harrison, j.a., braack, h.h., bishop, p.j., kloepfer, d., eds. (2004): atlas and red data book of the frogs of south africa, lesotho and swasiland. si/mab series 9. smithsonian institution, washington, d.c. penske, s., gvoždík, v., menegon, m., loader, s.p., müller, h. (2015): description of the tadpole of leptopelis cf. grandiceps (amphibia: anura: arthroleptidae) from the uluguru mountains, tanzania. herpetol. j. 25: 61-64. pickersgill, m. (2007): frog search: results of expeditions to southern and eastern africa. edition chimaira, frankfurt-am-main. rödel, m.o. (2000): herpetofauna of west africa. volume i: amphibians of the west african savanna. edition chimaira, frankfurt-am-main. schiøtz, a. (1999): treefrogs of africa. edition chimaira, frankfurt-am-main. van dijk, d.e. (1966): systematic and field keys to the families, genera and described species of south african anuran tadpoles. ann. natal. mus. 18: 231-286. venter, a.j., conradie, w. (2015): a checklist of the reptiles and amphibians found in the protected areas along the south african wild coast, with notes on conservation implications. koedoe 57: 1-25 wager, v.a. (1930): the breeding habits and life-histories of two rare south african amphibia. i. hylambates natalensis a. smith. ii. natalobatrachus bonebergi hewitt and methuen (plates v-x). trans. roy. soc. s. afr. 19: 79-91. wager, v.a. (1965): the frogs of south africa. purnell and sons, cape town. wager, v.a. (1986): frogs of south africa: their fascinating life stories. delta books, craighall. wassersug, r.j. (1976): oral morphology of anuran larvae: terminology and general description. occ. pub. mus. nat. hist. univ. kansas 48: 1-23. acta herpetologica vol. 12, n. 2 december 2017 firenze university press meristic and morphometric characters of leptopelis natalensis tadpoles (amphibia: anura: arthroleptidae) from entumeni forest reveal variation and inconsistencies with previous descriptions susan schweiger1, james harvey2, theresa s. otremba1, janina weber1, hendrik müller1,* brown anole (anolis sagrei) adhesive forces remain unaffected by partial claw clipping austin m. garner*, stephanie m. lopez, peter h. niewiarowski species and sex comparisons of karyotype and genome size in two kurixalus tree frogs (anura, rhacophoridae) shun-ping chang1,2, gwo-chin ma2,3,4, ming chen2,5,6,7,8,*, sheng-hai wu1,* non-native turtles in a peri-urban park in northern milan (lombardy, italy): species diversity and population structure claudio foglini1, roberta salvi2,* species composition and richness of anurans in cerrado urban forests from central brazil cláudia márcia marily ferreira1,*, augusto cesar de aquino ribas2, franco leandro de souza3 the life-history traits in a breeding population of darevskia valentini from turkey muammer kurnaz, alı i̇hsan eroğlu, ufuk bülbül*, halıme koç, bılal kutrup influence of desiccation threat on the metamorphic traits of the asian common toad, duttaphrynus melanostictus (anura) santosh mogali*, srinivas saidapur, bhagyashri shanbhag predation of common wall lizards: experiences from a study using scentless plasticine lizards jenő j. purger*, zsófia lanszki, dávid szép, renáta bocz reproductive timing and fecundity in the neotropical lizard enyalius perditus (squamata: leiosauridae) serena najara migliore1,2,*, henrique bartolomeu braz2,3, andré felipe barreto-lima4, selma maria almeida-santos1,2 observations on the intraspecific variation in tadpole morphology in natural ponds eudald pujol-buxó1,2,*, albert montori1, roser campeny3 and gustavo a. llorente1,2 reliable proxies for glandular secretion production in lacertid lizards simon baeckens diet of juveniles of the venomous frog aparasphenodon brunoi (amphibia: hylidae) in southeastern brazil rogério l. teixeira1, ricardo lourenço-de-moraes2, débora c. medeiros3, charles duca3, rogério c. britto4, luiz c. p. bissoli5, rodrigo b. ferreira3,* who are you? the genetic identity of some insular populations of hierophis viridiflavus s.l. from the tyrrhenian sea ignazio avella, riccardo castiglia, gabriele senczuk* acta herpetologica 12(2): 187-191, 2017 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-19981 reproductive timing and fecundity in the neotropical lizard enyalius perditus (squamata: leiosauridae) serena najara migliore1,2,*, henrique bartolomeu braz2,3, andré felipe barreto-lima4, selma maria almeida-santos1,2 1 setor de anatomia, departamento de cirurgia, faculdade de medicina veterinária e zootecnia, universidade de são paulo, av. orlando marques de paiva 87, cidade universitária, são paulo, sp, brazil. 05508-270 2 laboratório de ecologia e evolução, instituto butantan av. dr. vital brazil, 1500 butantã, são paulo sp, brazil. 05503-900 3 school of life and environmental sciences, heydon-laurence building, a08, university of sydney, nsw, 2006, australia 4 laboratório de herpetologia, departamento de zoologia, instituto de ciências biológicas, campus darci ribeiro, universidade de brasília df, brazil. 70910-900 *corresponding author. e-mail: serena_891@hotmail.com submitted on: 2016, 16th december; revised on: 2017, 24th february; accepted on: 2017, 29th july editor: giovanni scillitani abstract. enyalius perditus is a semi-arboreal lizard species whose reproduction is poorly known. here, we combine information obtained from preserved and live specimens to describe the reproductive timing (vitellogenesis, gravidity, and egg-laying) and fecundity (clutch size, egg size, and relative clutch mass) in females of e. perditus. female reproduction is remarkably seasonal and occurs in the warmer and wetter periods of the year. secondary vitellogenesis occurs from mid to late spring, whereas gravidity and egg-laying occur in early summer. mating appears to be synchronized with secondary vitellogenesis, indicating an associated reproductive cycle. we suggest that e. perditus females produce only a single clutch per reproductive season. clutch size ranged from three to 11 eggs and was positively correlated with female body size. finally, the relative clutch mass was high, a recurrent feature to “sit-and-wait” foragers. keywords. clutch size, reproductive biology, reproductive cycle, seasonal reproduction. detailed information on the reproductive biology of a number of species is critical for elaborating and testing ecological-evolutionary hypotheses and providing informed decisions on conservation strategies (shine and bonnet, 2009; vitt, 2013). despite the recent increase in the number of studies on the reproduction of neotropical lizards (e.g., balestrin et al., 2010; ferreira et al., 2009; vieira et al., 2001), knowledge about the reproductive biology of several species is still scarce. enyalius is composed of insectivorous, diurnal, and semi-arboreal lizards (jackson, 1978; rautenberg and laps, 2010; sturaro and silva, 2010; barreto-lima and sousa, 2011; barreto-lima et al., 2013). enyalius is distributed mostly in atlantic forest areas but some species may also occur in the amazon forest, forest galleries in ‘cerrado’, and isolated forested areas in ‘caatinga’ (barreto-lima, 2012). enyalius perditus is commonly found in atlantic forest areas in southeastern brazil, where it may occur in sympatry with e. brasiliensis, e. iheringii, and e. bilineatus (barreto-lima, 2012). many studies have addressed several aspects of the natural history of the species, including feeding ecology, activity patterns, microhabitat use, sexual dimorphism, and behavior (barreto-lima and sousa, 2006, 2011, sturaro and silva, 2010; barreto-lima et al., 2013; migliore et al., 2014). recently, migliore et al. (2014) summarized the reproductive information available for e. perditus and e. iheringii and found that published data is 188 serena najara migliore et alii limited to punctual observations on clutch size, courtship and mating behavior, and timing of mating and gravidity. this limited information impairs both an overview on the reproduction of the species and broad comparisons across other species. to fill this gap, we combine information obtained from both museum and live specimens to describe the reproductive timing (vitellogenesis, gravidity, and egg-laying) and fecundity (clutch size, egg size, and relative clutch mass) in females of e. perditus. we analyzed 35 sexually mature females of e. perditus housed in six scientific collections from brazil (appendix 1). the specimens were mostly collected throughout the atlantic forest domain. the climate in the area is seasonal and characterized by a distinct hot, rainy season from spring to summer (october-march) and a dry season from autumn to winter (april-september) associated with lower temperatures (mendonça and danni-oliveira, 2007). females were considered sexually mature if they contained vitellogenic follicles, oviductal eggs, or folded oviducts. for each individual, we recorded the: (1) snout-vent length (svl) to the nearest 1 mm, (2) number of ovarian follicles or eggs, (3) diameter of the largest ovarian follicle, and (4) length and width of oviductal eggs. additional observations on body sizes, egg-laying, egg size, and clutch size were obtained from two gravid females collected in the biological reserve of boracéia, são paulo state, on 8th december 2015. these females were kept in terrarium containing branches and leaf litter and allowed to oviposit naturally. procedures for egg measurements and incubation were used according to migliore et al. (2014). we determined the timing of secondary vitellogenesis using a scatterplot of the diameter of the largest ovarian follicle (almeida-santos et al., 2014). relative clutch mass (rcm) was calculated by dividing the total clutch mass by maternal body mass after oviposition + total clutch mass (vitt and price, 1982). we used simple linear regression to determine the relationship between maternal svl and clutch size (both log-transformed; king, 2000) and significance was assumed at p ≤ 0.05. mean values are always followed by ± standard deviation. body sizes of e. perditus females ranged from 58 to 91 mm (mean = 74.7 ± 7.8 mm; n = 36). females with follicles in primary vitellogenesis were found throughout the year (fig. 1). substantial increases in follicular size (and thus secondary vitellogenesis) were observed from mid to late spring (november-december; fig. 1). gravid females (n = 11) were observed in early-summer (january; fig. 1). no female contained follicles in secondary vitellogenesis simultaneously with oviductal eggs. egglaying was recorded in early summer (table 1). the two wild-caught females laid eight and six eggs each on 26th december 2015 and 1st january 2016, respectively (fig. 1). all eggs spoiled over incubation due to fungal contamination. clutch size (including preserved and captive specimens) averaged 7.1 ± 2.4 eggs (range: 3-11 eggs; n = 13 clutches). clutch size was positively correlated with maternal svl (r = 0.70; n = 13; p = 0.008; fig. 2). egg length in all gravid females averaged 15.45 ± 1.36 mm (range: 12.45-17.80 mm; n = 92 eggs from 13 females) and egg width averaged 8.15 ± 0.56 mm (range: 7.38fig. 1. reproductive timing in females of enyalius perditus. the graph shows the seasonal variation in the diameter of the largest ovarian follicle or oviductal egg and the timing of egg-laying. closed circle: ovarian follicles; open circle: oviductal eggs; arrows: egg-laying. table 1. morphometrics of two clutches of enyalius perditus from biological reserve of boracéia, são paulo state, brazil. rcm: relative clutch mass.  individual date laid clutch size female mass1 (g) total clutch mass (g) rcm2 egg length (mm) egg width (mm) egg mass (g) female 1 26 dec 2015 8 12.3 6.91 0.36 16.71 ± 0.28 9.58 ± 0.12 0.86 ± 0.02 female 2 1 jan 2016 6 11.4 4.28 0.27 16.09 ± 0.43 8.66 ± 0.26 0.71 ± 0.01 1post parition mass. 2rcm was calculated by dividing the total clutch mass by maternal body mass after oviposition plus total clutch mass (vitt and price, 1982). 189reproduction in enyalius perditus 9.58 mm; n = 92 eggs from 13 females). rcm for the two wild-caught females that laid eggs in captivity was 0.27 and 0.36. reproductive timing in e. perditus females is remarkably seasonal, with secondary vitellogenesis, gravidity, and egg-laying occurring within three months, from november to january (see sturaro and silva, 2010; barreto-lima and sousa, 2011 for additional records of vitellogenesis and gravidity). therefore, reproductive timing in e. perditus females is associated with the warmer and wetter periods of the year. reproductive information for other enyalius species is rather limited. however, the reproductive timing in e. perditus appears to be concentrated within a shorter period of time than other enyalius from the atlantic forest (marques and sazima, 2004; teixeira et al. 2005; rautenberg and laps, 2011; migliore et al., 2014). this short reproductive season in females of e. perditus suggests that females produce only a single clutch per reproductive season. indeed, this is corroborated by the absence of females containing follicles in secondary vitellogenesis simultaneously with oviductal eggs (almeida-santos et al., 2014). associated reproductive cycles are common in lizards and consist of reproductive events in males and females (i.e., sperm production, mating, and ovulation) occurring at the same period (crews and gans, 1992; méndez-de la cruz et al., 2014). courtship and mating in e. perditus have been reported in spring (november-december: barreto-lima and sousa, 2006; migliore et al., 2014) and thus are synchronized with the timing of secondary vitellogenesis and ovulation. this suggests that e. perditus exhibits associated reproductive cycles. however, histological investigations of the reproductive cycle of e. perditus males are required to confirm if the species exhibits associated reproductive cycles. mean clutch size in e. perditus is low relative to at least three other congenerics (14.0 eggs in e. iheringii: rautenberg and laps, 2010; migliore et al., 2014; 12.3 eggs in e. leechi: vitt et al., 1996; and 11.5 eggs in e. brasiliensis: teixeira et al., 2005) but high relative to another congeneric (4.4 eggs in e. bilineatus: teixeira et al., 2005). these differences may be explained by interspecific differences in mean body size since all enyalius species that showed higher clutch size than e. perditus also exhibited larger body sizes (see rand, 1982; vitt et al., 1996; teixeira et al., 2005; rautenberg and laps, 2010). this idea is corroborated by our finding that the clutch size in e. perditus increased with maternal svl, as observed in other enyalius (teixeira et al., 2005; vitt et al., 1996) and in many lizard species with variable clutch size (fitch, 1970; tinkle et al., 1970). the rcm for e. perditus (0.27-0.36) is similar to that reported for a congeneric (0.38 in e. iheringii; migliore et al., 2014). in lizards, rcm tends to be relatively low in “wide forager” species and relatively high in “sit-andwait” foragers (vitt and price, 1982). females of enyalius species appear to be “sit-and-wait” foragers (sousa and cruz, 2008; borges et al., 2013) and move shorter distances than males (barreto-lima et al., 2013). the rcm for enyalius is higher than the upper limit reported for other oviparous lizards that forage widely (~ 0.21) and consistent with values reported for “sit-and-wait” foragers (vitt and price, 1982), thus in agreement with the association between rcm and foraging tactics. acknowledgements we thank g. puorto, p. r. manzani, j. c. mouraleite, m. r. s. pires, r. n. feio, and v. silva for allowing access to specimens under their care, m. t. rodrigues for providing some specimens, m. teixeira-junior for assistance in field work. we also thank p. monteiro, n. torello-vieira and k. banci for criticism on an earlier draft. we thank capes (coordenação de aperfeiçoamento de pessoal de nível superior) for providing financial support (master’s scholarship to s. n. migliore), sisbio (sistema de autorização e informação em biodiversidade; permit number sisbio 47011-4), and butantan institute animal ethics committee (approval number 3491021015) for the permission to collect and maintain the lizard specimens at the ecology and evolution lab–butantan institute. fig. 2. relationship between maternal snout-vent length and clutch size (both log-transformed) in enyalius perditus. closed circles: data from preserved specimens; open circles: data from freshly laid clutches. 190 serena najara migliore et alii references almeida-santos, s.m., braz, h.b., santos, l.c., sueiro, l.r., barros, v.a., rojas, c.a., kasperoviczus, k.n. 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(1970): evolutionary strategies in lizard reproduction. evolution 24: 55-74. vieira, g.h.c., wiederhecker, h.c., colli, g.r. báo, s. n. (2001): spermiogenesis and testicular cycle of the lizard tropidurus torquatus (squamata, tropiduridae) in the cerrado of central brazil. amphibia-reptilia 22: 217-233. vitt, l.j. (2013): walking the natural-history trail. herpetologica 69: 105-117. 191reproduction in enyalius perditus vitt, l.j., ávila-pires, t.c.s., zani, p.a. (1996): observations on the ecology of the rare amazonian lizard, enyalius leechii (polychrotidae). herpetol. nat. hist. 4: 77-82. vitt, l.j., price, h.j. (1982): ecological and evolutionary determinants of relative clutch mass in lizards. herpetologica 38: 237-255. appendix appendix 1. list of museums and voucher specimens of enyalius perditus examined. collection (abbreviation) voucher number instituto butantan, são paulo, coleção de referência (ibspcr) são paulo: são josé do barreiro (ibspcr 407). museu de zoologia, universidade estadual de campinas (zuec) são paulo: ilhabela (zuec 2934, 2935, 2936, 2938, 2942), ubatuba (zuec 1887). museu de zoologia joão moojen (mzufv) minas gerais: lambari (mzufv 633). coleção herpetológica alfred russel wallace (charw) minas gerais: lambari (charw 94, 155, 158, 160, 161, 194, 289, 293, 296, 321), alfenas (charw 322), boa esperança (charw 317). coleção herpetológica da universidade federal de ouro preto (ufop) minas gerais: itatiaia, serra de ouro branco (ufop 939s, 968s, 976s, 978s, 993s, 995s, 996s, 997s, 1042s, 1062s, 1077s, 1087s). museu de história natural capão da imbuia (mhnci) paraná: telêmaco borba (mhnci 3128, 12956, 12966). acta herpetologica vol. 12, n. 2 december 2017 firenze university press meristic and morphometric characters of leptopelis natalensis tadpoles (amphibia: anura: arthroleptidae) from entumeni forest reveal variation and inconsistencies with previous descriptions susan schweiger1, james harvey2, theresa s. otremba1, janina weber1, hendrik müller1,* brown anole (anolis sagrei) adhesive forces remain unaffected by partial claw clipping austin m. garner*, stephanie m. lopez, peter h. niewiarowski species and sex comparisons of karyotype and genome size in two kurixalus tree frogs (anura, rhacophoridae) shun-ping chang1,2, gwo-chin ma2,3,4, ming chen2,5,6,7,8,*, sheng-hai wu1,* non-native turtles in a peri-urban park in northern milan (lombardy, italy): species diversity and population structure claudio foglini1, roberta salvi2,* species composition and richness of anurans in cerrado urban forests from central brazil cláudia márcia marily ferreira1,*, augusto cesar de aquino ribas2, franco leandro de souza3 the life-history traits in a breeding population of darevskia valentini from turkey muammer kurnaz, alı i̇hsan eroğlu, ufuk bülbül*, halıme koç, bılal kutrup influence of desiccation threat on the metamorphic traits of the asian common toad, duttaphrynus melanostictus (anura) santosh mogali*, srinivas saidapur, bhagyashri shanbhag predation of common wall lizards: experiences from a study using scentless plasticine lizards jenő j. purger*, zsófia lanszki, dávid szép, renáta bocz reproductive timing and fecundity in the neotropical lizard enyalius perditus (squamata: leiosauridae) serena najara migliore1,2,*, henrique bartolomeu braz2,3, andré felipe barreto-lima4, selma maria almeida-santos1,2 observations on the intraspecific variation in tadpole morphology in natural ponds eudald pujol-buxó1,2,*, albert montori1, roser campeny3 and gustavo a. llorente1,2 reliable proxies for glandular secretion production in lacertid lizards simon baeckens diet of juveniles of the venomous frog aparasphenodon brunoi (amphibia: hylidae) in southeastern brazil rogério l. teixeira1, ricardo lourenço-de-moraes2, débora c. medeiros3, charles duca3, rogério c. britto4, luiz c. p. bissoli5, rodrigo b. ferreira3,* who are you? the genetic identity of some insular populations of hierophis viridiflavus s.l. from the tyrrhenian sea ignazio avella, riccardo castiglia, gabriele senczuk* acta herpetologica 10(1): 23-29, 2015 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-14780 toxic effects of carbaryl on the histology of testes of bufotes variabilis (anura: bufonidae) özlem çakici ege university, science faculty, biology department, zoology section 35100 bornova-i̇zmir, turkey. e-mail: ozlem.cakici@ege.edu.tr submitted on: 2014, 13th august; revised on: 2014, 22th december; accepted on: 2014, 29th december editor: sebastiano salvidio abstract. this study was designed to investigate histopathologic effects of carbaryl on the testes of adult toad, bufotes variabilis. to that end, animals were exposed to carbaryl once by oral gavage (low dose: 50 µg/g, medium dose: 100 µg/g and high dose: 200 µg/g). after 96 h, toads were euthanized. in low-dose group, some seminiferous tubules lost their regular shape. also, the enlargement of interstitial spaces among tubules and germ cell necrosis were determined. a weak hemorrhage was observed among some tubules. in medium-dose group, germ cell necrosis was detected in many seminiferous tubules. this time, a weak hemorrhage was detected within tubules. in the high dose group, an increase in the number of disorganized tubules were observed. vacuolization and necrosis in germ cells of seminiferous tubules were frequently seen. according to these findings, carbaryl caused dose-related histopathological damage in testis of b. variabilis. based on these findings, this study clearly shows that carbaryl affects male fertility in b. variabilis. keywords. amphibia, anura, bufonidae, bufotes variabilis, toad, carbaryl, testes, histopathology introduction wide range use of pesticides may cause serious problems in ecosystem. in fact, rani et al. (2013) stated that even though pesticides are present in the environment in small quantities when compared to other contaminants such as industrial wastes and fertilizers, they are responsible for public and scientific concern because of their high biological activity, and many species are badly affected by this pesticide use. for instance, one of the biggest problem in conservation biology is the decline of many amphibian species (e.g. alford et al., 2001; stuart et al., 2004) and pesticides play an important role in these declines (davidson, 2004; punzo, 2005; mann et al., 2009). according to davidson (2004), cholin-esterase inhibiting pesticides such as organophosphates and carbamates have more effect in amphibian declines than other different pesticide classes. carbaryl (1-naphthyln-methylcarbamate) is included in carbamates and is used as a contact and systemic insecticide on agricultural products to control various kind of pests including moths, beetles, cockroaches, ants, ticks and mosquitoes (tomlin, 2000; murty et al., 2012). garber et al. (2007) also reported that adverse effects of carbaryl on endangered anuran populations is relevant. to that end, a number of studies were conducted to determine the effects of carbaryl in the early developmental stages of amphibians. punzo (2005) examined the time spent in activity (tail movement) and swimming speed of tadpoles of the rio de grande leopard frog, rana berlandieri. author stated that time spent in activity and the swimming speed decreased after carbaryl exposure and this may influence an increased rate of predations. in addition, because of the slower growth rates, tadpoles needed more time to complete metamorphosis. carbaryl had also teratogenic effects on the embryos of the clawed frog, xenopus laevis and caused abnormal tail flexure (bacchetta et al., 2008). similarly, kang et al. (2010) reported that carbaryl was harmful in the oriental fire-belleed toad, bombina orientalis embryos and exposure to carbaryl caused some 24 ö. çakici external deformities. boone et al. (2013) stated that carbaryl could have long-lasting effects on the brain development when exposure occured at sensitive developmental stages. as a result of aquatic exposure to the carbaryl, both reduced survival and increased time to metamorphosis were observed in the aquatic phase of the american toad, bufo americanus (distel and boone, 2009). the effects of a 24 hours exposure to carbaryl increased fatigue in juvenile spotted salamander, ambystoma maculatum (mitchkash et al., 2014). cholinesterase and carboxylesterases were strongly inhibited by carbaryl in the larvae of the toad rhinella arenarum (ferrari et al., 2011). based on above-mentioned studies, it can be concluded that carbaryl have severe adverse effects on early life stages of amphibians. naturally, early life development stages are usually considered to be the most sensitive to the adverse effects of toxic compounds. however, it is also important to analyse the effects of pesticides in adult amphibians because they may have a potential to irreversibly damage the organs. for this reason, studies are needed to determine the effects of carbaryl on the gonadal structure of adult amphibians. considering all, histopathologic effects of carbaryl on the testis of levantine frog, pelophylax bedriagae were examined by çakıcı (2013). several histopathologic findings indicated that carbaryl had the potential to affect germ cell development and possibly to have negative effects on spermatogenesis. accordingly the present study aimed at the evaluation, on the basis of tissue histopathology, of the negative effects of carbaryl in the variable toad, bufotes variabilis a species widely distributed throughout turkey (başoğlu et al., 1994). material and methods animal groups and experimental design adult individuals of b. variabilis were caught around bornova, i̇zmir province in turkey. before experiments, toads were acclimatized to the laboratoty conditions at a 12 h dark/ light cycle, at 22 ± 3° c temperature and 45 ± 5 % relative humidity for 15 days. then, the toads were randomly assigned to either the carbaryl-treated groups (low, medium and high dose groups) or to the control group, each group consisting of 8 mature male toads. as formerly mentioned by durant et al. (2007), contaminated insects are an important way of exposure to pesticides for insectivorous vertebrates inhabiting areas that receive pesticide application. as adult toads fed on insects, they may exposed to carbaryl by eating prey that’s covered in carbaryl. however only one study made by fair et al. (1995) directly quantified carbaryl residues of terrestrial invertebrates and determined that grasshoppers had mean residues of 17 µg/g two days following rangeland application of 0.5 kg active ingredient/ha. by means of this insect residue data and carbaryl application rates, which can vary from 1.12 to 22.42 kg active ingredient/ha (epa 2004), durant et al. (2007) stated that a 10 g lizard consuming 1 g prey could ingest close concentrations ranging between 3.9-78.5 µg/g 2 days following carbaryl application and factoring the short-half life into their estimates, scientists selected three doses that encompassed the entire range of concentrations that they believed lizards could encounter in the environment. therefore, these results were used to calculate the concentrations of carbaryl used in this study and the author is confident that these concentrations of carbaryl are the ones which toads could encounter in their environment. in addition, durant et al. (2007) stated that studies related to the acute exposure of carbaryl are ecologically relevant because carbaryl have a short half-life. experimental groups contained low (50 µg/g), medium (100 µg/g) and high doses (200 µg/g) of carbaryl insecticide (purity 98 %) supplied by agrobest grup (i̇zmir, turkey). carbaryl was dissolved in acetone and prior to experiments, acetone was used as control and no important histologic effects were observed in testicular tissue of the toads. carbaryl was administrated once by oral gavage to experimental groups. histological analyses following 96 h the exposure to carbaryl, toads were euthanized and their testes were quickly removed. after fixation in bouin for 24 h, testis samples were dehydrated in ethanol, cleared in xylol and embedded in paraffin. tissues were serially sectioned at 5 μm, then stained with mayer’s haematoxylin and eosin and photographed with olympus cx31 (tokyo/japan). results during this study, there was no mortality in the experimental bufotes variabilis after exposure to carbaryl for 96 h. according to light microscopic examinations, spermatogenesis appeared normal in the control group animals (fig. 1a), with germ cells in cystic organizations of a seminiferous tubule were easily observed (fig. 1b). this investigation showed that carbaryl treatment caused dose-related histopathologic changes in b.variabilis. in the low-dose group, some seminiferous tubules lost their regular shape and enlargement of interstitial spaces among tubules was clearly observed (fig. 2a). in addition, germ cell necrosis within seminiferous tubules was also detected (fig. 2b), with a weak hemorrhage that was present among some tubules (fig. 2c). in the medium-dose group, germ cell necrosis was clearly present in many seminiferous tubules (figs 3a and b), again with a weak hemorrhage present within tubules (fig. 3c). finally, in the high-dose group, an increase in the number of disorganized tubules was observed (fig. 4a), 25effect of carbaryl on bufotes variabilis while vacuolization in germ cells was also detected in many seminiferous tubules (fig. 4b) and evident germ cell necrosis was conspicuous (fig. 4c). discussion it is a common characteristic of male amphibians that germ cells in the same stage of differentiation constitute clusters in arranged in cystic structures with a synchronism development (lofts, 1974; rastogi, 1976; jorgensen et al., 1986; 1988; rastogi et al., 1988; oliveira et al., 2002). for example carezzano et al (2013) stated that seminiferous locules distinguished in cysts with spermatogenic cells at different stages of development  1    2    3    1    2    3    4   fig. 1. histologic section of testis in control group. a) general view of testis. normal appearance of seminiferous tubules (asterisk); b) normal spermatogenesis in seminiferous tubules (asterisk) in the control group. note the germ cells in a cystic organization (arrowhead). fig. 2. histologic section of testis in low dose group. a) general view of testis. the enlargement of interstitial spaces (asterisk); b) germ cell necrosis (arrow); c) note the weak hemorrhage (arrow) between two disorganized seminiferous tubules. 26 ö. çakici  1    2    3    4    1    2   3    4  fig. 3. histologic section of testis in medium dose group. a) general view of testis. note the disorganized tubules (asterisk); b) prominency in germ cell necrosis (arrow); c) a weak hemorrhage (arrow) within seminiferous tubules. fig. 4. histologic section of testis in high dose group. a) general view of testis: an increase in the number of disorganized tubules (asterisk); b) vacuolization (v) in germ cells; c) prominent germ cell necrosis (arrow) in a seminiferous tubule. 27effect of carbaryl on bufotes variabilis were present in the testes of the argeentine horned frog, ceratophrys ornata. ferreira et al. (2008) described the testis structure of the harlequin frog pseudis limellum. these authors stated that germ cells were organized as cysts and spermatogenesis is similar to other anurans, but with some peculiarities related to organization of the germ cells (ferreira et al., 2008). santos and de oliveira (2008) reported that in the male tree frog dendropsophus minitus, spermatogenesis occured in seminiferous tubule where germ cells were arranged in spermatogenic cysyts, and each cyst included cells in the same stage of differentiation. in other words, sperm maturation followed a similar pattern in many amphibian species and, in genereal terms, bufotes variabilis’ spermatogenesis appeared similar to the above mentioned species. due to the limited research about the effects of carbaryl on gonadal structure of fish, amphibians or reptiles, the present research can only be compared with few other studies. çakıcı (2013) studied the histopathologic effect of carbaryl on the testes of levantine frog, pelophylax bedriagae. in that study, three doses of carbaryl, corresponding to 50, 100 and 200 µg/g, respectively, were used and carbaryl caused dose-related histopathologic changes in the testes of the experimental animals were observed (çakıcı (2013). similarly, doserelated histopathologic findings were determined in the toad bufotes variabilis, as reported in the present study. in particular, shrinkage of seminiferous tubules and disintegration in germ cells were determined in the low dose group when compared to the control one. cakici and akat (2012) also studied the toxic effect of carbaryl on testes of the snake-eyed lizard ophisops elegans. these authors used low (2.5 µg/g), medium (25 µg/g) and high (250 µg/g) carbaryl doses, finding no important histopathologic defects on seminiferous tubules in the low dose group of animals. cakici and akat (2012) observed the tubular asymmetry and blood cell infiltration in interstitial space of tubules in the testes of o. elegans, while çakıcı (2013) stated that most tubule lost their normal appearance in high dose carbaryl treated group of p. bedriagae. similarly, germ cell necrosis was conspicuous in b. variabilis. cakici and akat (2012) determined the vacuolization in germ cells in the testis of o. elegans but this effect was much more commonly seen in b. variabilis. a study made by sanchez et al. (2014) investigated the connection between male gonadal abnormalities and the effects of agricultural activities in two anuran species, the toad rhinella fernandezae and the frog dendropsophus sanborni. these authors determined some abnormalities such as poorly developed seminiferous tubules and reduction in number of germ cells in both species. in view of their findings, they concluded that agrochemical use may have negative effects on the reproductive success on these two species. saxena and mani (1987) examined the effects of some pesticides (fenitrothion and carbofuran) on the testicular recrudescence in the freswater murrel, channa punctatus. according to this study carbofuran, a carbamate insecticide, delayed the formation of spermatids and sperms. in addition, necrosis of spermatogonia and spermatocytes were also observed. harilal and sahai (1990) determined that carbaryl accumulated in the gonadal tissues even after 4 days exposure in the catfish heteropneustes fossilis. jyothi and narahan (1999) examined the toxic effects of carbaryl on gonadal structure of a freshwater fish, clarias batrachus and according to this study, exposure to carbaryl caused some histopathologic changes, such as vacuolation and necrosis in gonads, while basement membrane thickness was also observed in the testis. similarly, vacuolization and necrosis were determined in the testis of bufotes variabilis. lauan and ocampo (2013) determined that exposure to carbaryl caused some histopathologic alterations in nile tilapia, oreochromis niloticus such as hemorrhagic necrosis detected in the peripheral and interlobular interstitium of the seminiferous lobules. in addition, they stated that as the treatment doses increased, disorganization of the testes structure also appeared to become more serious (lauan and ocampo, 2013). mensah et al (2012) reported that sublethal doses of carbaryl in the environment may have an adverse effect on the reproductive success of zebrafish. according to toppari et al. (1996), to affect spermatogenesis, pesticides acts by hormonal or genotoxic pathways by passing through the blood testis barrier, while fattahi et al. (2012) asserted that pesticides could also have direct effects on testicular tissue. to better understand this relevant issue, more comprehensive studies should be made to clarify the physiologic mechanisms of insecticides on the male reproductive system in particularly in amphibians. in summary, all the above studies showed that the pesticide carbaryl may have severe negative impacts on the germ cell development in the testes of various terrestrial and freshwater animals. similarly, many histopathologic findings presented in this study indicated that carbaryl affected the development of germ cells and had negative effects on the spermatogenesis in b. variabilis. as it is known, decline of many amphibian species is a global problem and the negative effects of wide range use of pesticides on the viability and reproduction of amphibians should be better investigated, in particular on adult individuals. 28 ö. çakici acknowledgements this study was approved by ministry of forestry and water affairs (date: 22 september 2011, number: 11875) and by the animal ethical committee of ege university, faculty of medicine (2011-166). references alford, r.a., dixon, p.m., pechman, j.h.k. 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(1996): male reproductive health and environmental xenoestrogens. environ. health perspect. 104: 741-803. acta herpetologica vol. 10, n. 1 june 2015 firenze university press obituary: valery k. eremchenko (1949-2014) leo j. borkin1, tatjana dujsebayeva2, roberto sindaco3, matthias stöck4 haplotype variation in founders of the mauremys annamensis population kept in european zoos barbora somerová1, ivan rehák2,*, petr velenský2, klára palupčíková1, tomáš protiva1, daniel frynta1 reproductive ecology of sichuan digging frogs (microhylidae: kaloula rugifera) wei chen1,*, lina ren2, dujuan he2, ying wang2, david pike3 toxic effects of carbaryl on the histology of testes of bufotes variabilis (anura: bufonidae) özlem çakici basal frequency of micronuclei and hematological parameters in the side-necked turtle, phrynops hilarii (duméril & bibron, 1835) maría a. latorre 1,2,*,#; evelyn c. lópez gonzález1,2, #; pablo a. siroski1,2,3; gisela l. poletta1,2,4 into a box interiors: clutch size variation and resource allocation in the european pond turtle marco. a.l. zuffi1,*, simonetta citi2, elena foschi1, francesca marsiglia1, eva martelli1 where to “rock”? choice of retreat sites by a gecko in a semi-arid habitat andreia penado1,2, ricardo rocha3,4,*, marta sampaio3, vanessa gil3, bruno m. carreira3, rui rebelo3 age structure, growth and longevity in the common toad, rhinella arenarum, from argentina clarisa de l. bionda1,2,*, silvia kost 4, nancy e. salas1, rafael c. lajmanovich3, ulrich sinsch4, adolfo l. martino1 on a putative type specimen of pleurodema bibroni tschudi, 1838 from chile (anura: leptodactylidae) daiana paola ferraro re-description of the external morphology of phyllomedusa iheringii boulenger, 1885 larvae (anura: hylidae), with comments on the external morphology of tadpoles of the p. burmeisteri group samanta iop¹, victor mendes lipinski¹, bruno madalozzo¹, franciele pereira maragno¹, sonia zanini cechin¹, tiago gomes dos santos² book review: harold heatwole, john w. wilkinson (eds). amphibians biology. volume 11 status of conservation and decline of amphibians. eastern hemisphere. part 4 . southern europe and turkey sebastiano salvidio book review: antonio romano. atlante degli anfibi del parco nazionale del cilento vallo di diano e alburni distribuzione, biologia, ecologia e conservazione sebastiano salvidio acta herpetologica journal of the societas herpetologica italica acta herpetologica 9(2): 203-217, 2014 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-12875 factors determining gekkotan (reptilia, sauria) distribution in tunisia (north africa) wided tlili1,*, aymen nefla1, michel delaugerre2, ridha ouni3, saïd nouira1 1 unité de recherche “biodiversité et biologie des populations”, 05/ur/09-10. département de biologie, faculté des sciences mathématique, physique et biologique de tunis (fst), campus universitaire 2092, el manar tunis, tunisia. *corresponding author. e-mail: wideddada@gmail.com 2 conservatoire du littoral, résidence saint marc, rue du juge falcone, f20200 bastia, france 3 tunisian wildlife conservation society. faculté des sciences mathématique, physique et biologiques de tunis (fst), campus universitaire 2092, el manar tunis, tunisia submitted on 2013, 5th may; revised on 2014,2nd june; accepted on 2014, 3rd october editor: ernesto filippi abstract. tunisian geckos count nine species (1 is insular relict, 1 is endemic, 2 are ubiquitous and 5 are enfeoffed). we aim to determine factors influencing their distributions. surveys were founded on environmental divisions. presence/absence data for 113 grids were analyzed using multivariate tools. 18 environmental variables were revealed and clustered into five factors to model species distributions. established models were further projected on non-explored areas within tunisian territory. the distribution of continental geckos follows an indirect bidirectional gradient; the south-northward one is physiologically stressful and the north-southward one is biologically stressful. five biogeographic regions were established showing concordance with climatic and vegetation regionalization. the distribution of non-anthropophilic species is positively correlated to thermal amplitudes gradient. the distribution of anthropophilic taxa is positively correlated to agricultural land-use. oasis, sebkhas and chotts are particular landscapes that disturb both distributions. predicted areas follow the yielded distribution patterns despite some discrepancy for s. sthenodactylus. the niche characterizing shows that land use and altitude increase the probability of occurrence of h. turcicus and t. mauritanica. alternatively, they decrease the probability of the presence of t. deserti, t. neglecta, t. tripolitanus and s. petrii. models could also show that the absence of s. sthenodactylus in northern regions is attributed to high altitudes and cereal land-use. as to t. fascicularis, the displacement of the northern limits of its range is mostly attributed to an improvement of field investigations. established model of its distribution shows a restricted area of probable occurrence in central tunisia confirming its endemism. keywords. gekkota, tunisia, biogeography, clustering, upgma, ecological factors, principal component analysis, ecological niche modelling. introduction recent overviews of the distribution of some geckos occurring in tunisia have been established (nouira, 1997; joger, 2003; rufray et al., 2003; delaugerre et al., 2011; tlili et al., 2012 a, b). reported data have marginally extended the known ranges of the two phyllodactylidae tarentola fascicularis and tarentola deserti (tlili et al., 2012 a). most importantly, they have reported new record of stenodactylus sthenodactylus in sub-humid regions (tlili et al., 2012 b). these expansions may occur either because of variations in ecological factors or because of traits of the natural history of the species (macarthur, 1972; holt, 2003). alternatively, it may be due to anthropogenic impact such as involuntary introduction accompanying human transport. guisan et al. (1999) and guisan and zimmermann (2000) demonstrated that environmental variables usually replace a combination of dif204 w. tlili et alii ferent resources and do not have direct physiological consequence for a species performance. to shed light on the factors that shape species’ ranges, scientists need to classify regional faunas into discrete groups (guisan and zimermann, 2000). in the case of tunisia, this task is complicated by its location in the mediterranean basin. besides, it is a transition area between the palearctic and the afrotropical ecozones and thereby should be treated differently than the center of these two biogeographical regions (kreft and jetz, 2010). moreover, located astride the atlas domain and the old african continent, tunisia might harbor species of many origins (nouira, 1996). in such cases, several authors suggested to start by selecting appropriate environmental variables that might explain the observed distribution of species (e.g., franklin et al., 2000; guisan and zimermann, 2000; hirzel et al., 2002; holt, 2003). such studies have been already undertaken for several groups of reptiles in tunisia including lacertidae (nouira, 1996) and scincidae (kalboussi, 2006). parallel work for gekkotan fauna is lacking (tlili et al., 2012 a, b) and questions relative to which factor influence the species distribution and how species will be distributed in time and space remain unanswerable (heikinheimo et al., 2007). for instance, why did s. sthenodactylus (an arid species) extend its distribution only to a particular sub-humid region (cape bon peninsula) among other similar regions in the extreme north of tunisia (tlili et al., 2012b)? besides, why did some juxtaposed areas show variation concerning their biodiversity despite their proximal locations and similar abiotic characteristics (tlili et al., 2012a)? given the above, this work has several aims: 1) to elaborate a map of biogeographical regions based on gekkotan species richness; 2) to extract the environmental variables constraining their distributions in tunisia, 3) to identify areas of probable occurrence and 4) to assess their ecological niche (habitat). material and methods study area located in the southern shore of the mediterranean sea, tunisia is separated from europe by the channel of sicily (140 km). tunisian landscape is naturally regionalized into units with characteristic climate, landforms, soil and vegetation (fakhfakh and laclavère, 1979). the landscape is marked by a general low relief crossed by the dorsal mountain chain (fig. 1a). within the cap bon and medjerda lowland regions all the original woodlands and forests have been cleared for agriculture. khroumirie and mogod regions are areas in the north-west comprising mountainous mediterranean forest and maquis. the central steppe region marks the transition zone between the dorsal mountain and the desert and harbors many salt lakes including ‘chott el jrid’. jeffara and dhahara regions are areas of subdesert, desert and sahara landscapes where the stony ergs and the large sand-dunes of the great eastern erg occur. the climate of tunisia is mainly mediterranean divided into 5 bioclimatic stages: humid, sub-humid, semi-arid, arid and saharan (emberger, 1950; tlili et al., 2012b). sampling and mapping preliminary sampling took place in spring and summer seasons from 1996 to 2012 as described in tlili et al. (2012 b). however, concentration of occurrence data was showed around locations of anthropophilic geckos’ populations and differences in sampling efforts have been revealed from north (highly inventoried) to south (less inventoried). thus, on the basis of distributions maps (tlili et al., 2012 a, b), 0.3°×0.3° equal-area grids were chosen as a sampling unit to re-sample an equal number of replicates per environmental combination (fig. 1b). teams of work followed climatic gradient where each bioclimatic stage was surveyed by two observers in spring and summer seasons of 2011 and 2012 (graham and hijmans, 2006; kreft and jetz, 2010). observers alternated between day and night attempting to seek for one specimen of each species for each grid (guisan and zimmermann, 2000). maps were composed using quantum-gis software (sillero and tarroso, 2010). species distributions and regionalization absence/presence data were converted into a species per site incidence matrix in order to calculate species richness (s.r.) and grids similarities. a matrix of similarity coefficients between grids was established using jaccard’s index (jaccard, 1908) suitable for binary data (real and vargas, 1996; kreft and jetz, 2010): ȷ = c/n (c = number of attributes present in both operational taxonomic units otus, n = total number of attributes). the statistical significance of obtained pairs of otus was tested using the statistical table of probability values (real, 1999). herein, the probabilities associated with jaccard’s index depend only on the total number of attributes (n) present in either of the two otus being compared (n = p + q c). the 113 grids were clustered according to their species richness. a matrix of dissimilarities was established from jaccard’s distance (d (j, k) = 1 ȷ). obtained distances range from 0 to 1 (0 when both units have the same attributes, 1 when they share no attributes). the unweighted pair-group average (upgma (sneath and sokal, 1973)) was used as a linkage rule. the retained number of clusters was based on the number of bioclimatic stages (guidi, et al., 2009). biogeographical patterns environmental variables were automatically selected by following environmental gradients detected from the clusters’ structure (guisan and zimmerman, 2000; hirzel et al., 2002). 205gekkotan distribution in tunisia the 113 cells were characterized with variables relating to: 1) relief (the mean value), 2) climate (emberger coefficient, thermal amplitudes (°c) and precipitation levels (mm)), 3) lithology (dominant type), 4) soil (dominant type), 5) water plan (presence of sebkhas, chotts or lakes), 6) vegetation cover (woodland, degraded forest, steppe, subdesert, and desert), 7) agriculture (cereal, olive, palm) and 7) urbanism (habitations, industrial, touristic and road densities). spatial data were provided by the “office tunisien de la cartographie” as printed maps and/ or numerical data. taking into account the strong correlations between most of these variables (appendix 1), a linear transformation of correlated variables into uncorrelated variables was made via a principal component analysis pca (kaplunovsky, 2005; lawley et al., 2011). the extraction of principal components amounts to a variance maximizing (varimax) rotation of the original variable space (harrell et al., 1996; guisan and zimmermann, 2000; clark et al., 2003; kaplunovsky, 2005; brito et al., 2011; lawley et al., 2011). to confirm the orthogonal character of obtained factors, clusters of items and rotated axes were identified; then, correlations between those (oblique) factors were computed, and that correlation matrix of oblique factors is further factor-analyzed to yield a set of five orthogonal factors that divide the variability in the items into that due to common variance (secondary factors), and unique variance due to the clusters of similar items in the analysis (primary factors) (appendix 2). species distribution modeling modeling of the distribution of the eight gekkotan species in tunisia was assessed by means of maxent 3.3.3 (phillips et al., 2006, philips and dudik, 2008). the model for each of the eight gekkotan species expresses the suitability of each grid cell as a function of its environmental variables by mean of maximum entropy method. we considered variables extracted by factor analysis to produce “features” which constrain the probability distribution. for every cell occurring within tunisian borders, maxent produced a value of probability for the presence of a gecko species, ranging from 0 to 1. if p(x) is the raw output for environmental conditions x, the corresponding logistic value is c p(x) / (1 + c p(x)) for a particular value of c (namely, the exponential of the entropy of the raw distribution). for easier interpretation, cumulative probability values were transformed into three main distribution classes: 1) the ‘out of range’ area, 2) the ‘suboptimal’ area, and 3) the optimal area (anadón et al., 2012). the importance of each environmental factor in explaining the observed distribution of geckos was evaluated by the fig. 1. map of tunisia. geographic location and natural regions (a); sampling units numbers (b) 206 w. tlili et alii percent of contribution (pc) and permutation importance. response curves obtained from maxent were also used to discuss the niche of tunisian gekkota. results taxonomic status and species richness tunisian geckos belong to three gekkotan families: gekkonidae, phyllodactylidae and sphaerodactylidae (table 1). figure 2 illustrates the spatial variation of the species richness to latitudinal, climatic and geomorphological gradients. beyond the latitude 36°, only two anthropophilic species t. mauritanica and h. turcicus occur (22.2% of the total gecko-diversity). between latitude 35° and 36°, s. sthenodactylus occurs as well as the two anthropophilic species; it occupies 56.6% of the 113 grid cells. between latitude 34° and 35°, five gekkotan species were found: t. mauritanica, t. fascicularis, h. turcicus, t. tripolitanus and s. sthenodactylus. southward the latitude 34°, only species highly adapted to drastic saharan conditions occurred: s. sthenodactylus, s. petrii, t. deserti, t. neglecta and t. tripolitanus. considering bioclimatic stages (fig. 2a), species richness ranges from two to five species. each stage contains only two gekkotan families. only euleptes europaea (relict of northern islands) occupies humid regions. given its insular statute, it was not included in the following parts of analysis. the two anthropophilic species are widely distributed and occupy all bioclimatic stages. six of the nine gekkotan species occur in arid environments. according to lithology and vegetation (fig. 2b), we note that the presence of geckos is heavily dependent on natural shelters availability such as rocky crevices, stones, sandy borrow or tree barks. most importantly, we noticed that the aggregation of the non-anthropophilic geckos (in central and southern regions) is related to local variations of vegetal landscape. t. fascicularis is usually found in open steppe type in which s. sthenodactylus occupies either the bark of trees or small shrubs. t. tripolitanus is associated with burrows under rocks. in ergs, s. sthenodactylus and s. petrii are found exclusively at the base of the rare and dispersed plants. table 1. taxonomic status of tunisian gekkota. s.i. species incidences. family genus species global distribution s.i. statutes sphaerodactylidae euleptes europaea (gené, 1839) mainly restricted to western mediterranean island. some small continental isolated populations have been reported in south of france and west of italy (salvidio and delaugerre, 2003; renet et al., 2008; delaugerre et al., 2011; salvidio et al., 2010). --insular relict phyllodactylidae tarentola mauritanica (linnaeus, 1758) it ranges from the iberian peninsula to italy in the north of mediterranean sea; and from morocco to the nile delta in the south (kluge, 2001) 59.3% ubiquitous neglecta strauch, 1895 it is known with certainty in algeria north-western libya, southern tunisia and chad (schleich et al., 1996). 11.5% desert habitat deserti boulenger, 1891 it is present in morocco, algeria and tunisia (schleich et al., 1996, harris et al., 2006). 44.25% desert habitat fascicularis (daudin, 1802) this species was recently elevated to the specific rank by joger and bshaenia (2010). 13.3% endemic to central tunisia gekkonidae hemidactylus turcicus (linnaeus, 1758) a recent phylogeny assigned populations of north africa to the arid clade. this taxon has a large circummediterranean distribution. 56.6% ubiquistous stenodactylus petrii anderson, 1896 it is found in africa, the middle east and the southwest of asia 23% desert habitat sthenodactylus (lichtenstein, 1823) it has been reported in africa, the middle east and saudi arabia (schleich et al., 1996; padial, 2006) 56.6% sandy areas tropiocolotes tripolitanus peters, 1880 it ranges in north africa, middle east and parts of asia. populations of the middle east have been transferred to the species t. somalicus. those of asia have been moved to asiocolotes genera. individuals from north africa are now part of the species t. tripolitanus which is located in tunisia, libya and egypt (loveridge, 1974; schleich et al., 1996) 41.6% desert habitat (hamada) 207gekkotan distribution in tunisia fig. 2. distribution of gekkota species richness according to: bioclimatic stages (a), geomorphology (b), lithology (c) and vegetal cover. maps were provided by the office of the ministry of agriculture (forestry management department) and further processed with quantum-gis software (sillero and tarroso, 2010). 208 w. tlili et alii grids clustering and regionalization species richness varies from two to five species per grid cell. calculated values of ȷ indices between the grids were all significant at the probability level 0.05. the dendrogram of fig. 3a split at ȷ value of 0.6 to form two groups. the first group (yellow shades) assemblages grids occurring on calci-magnesian and isohumic soils, under precipitation levels higher than 150 mm and with thermal amplitude lower than 20°c. this sub-branch contains two clusters: 1) northern regions with the sahel region (grids 49-56-57-61); and 2) central region with golf de gabes region (grids 68-73-78-61). the second group (brown shades) gathers together the grids occurring on hydromorphic and poorly developed soils, under precipitation levels lower than 150 mm and with thermal amplitude higher than 20°c. it contains four clusters: 1) chott el jrid region (grids 65-66-70-73-74); 2) tyaret region (grids 109-110-111-112-113); 3) jbil region (grids 83-84-85-89-91-92-93-102-103-107-108) and 4) a cluster with no defined geographical area (grids 65, 66, 71 112, 113). a combination of corresponding colors was used to delineate regions according to cluster memberships (fig. 3b). fig. 3. dendrogram (a) and map (b) resulting from upgma hierarchical clustering of grid cell assemblages of gekkota based on jaccard dissimilarity matrix at the species level. the six major biogeographical divisions are highlighted in the dendrogram with large colored rectangles. 209gekkotan distribution in tunisia environmental factors 18 environmental variables were identified based on the hierarchical tree (fig. 3a) and shared distribution patterns (fig. 3). most of these variables are highly correlated producing great redundancy. a linear transformation of correlated variables into uncorrelated variables via pca retained five factors with eigenvalues ≥ 1.0 (according to the kaiser criterion). the total variance explained by all variables was 71.33% (appendix 2). the orthogonal character of obtained factors was confirmed and clusters of items and rotated axes were identified (table 2). five orthogonal factors were yielded containing 11 environmental layers to develop gekkotan distribution models (table 3). distribution model the 11 environmental layers produced “features”, which constrain the distribution of computed probabilities. in our case, the set of features depend on the number of presence records for the species. the receiver operating characteristic (roc) curves show that models for all phyllodactylidae (appendix 3) and most of gekkonidae (appendix 4) will well perform in predicting occurrences compared to a random selection of points. in fact, auc values are all higher than 0.7 except for s. sthenodactylus which is 0.578 (table 4). also, table 4 shows threshold models that permitted to identify suitable cells for each species. the analysis of variables’ contributions table 2. factor loadings with varimax as a rotation method. underlined loadings are >0.6; bold values are negative correlations. s.r. species richness. factor 1 factor 2 factor 3 factor 4 factor 5 rain-thermal -0.895183 0.006325 -0.048915 0.078812 0.127187 precipitations 0.746916 0.350094 0.080001 0.105304 0.116798 lithology 0.108549 -0.353286 0.17636 -0.65443 0.16875 soil 0.745688 -0.035934 -0.069964 -0.020273 -0.053747 accident 0.447775 -0.419113 0.322996 -0.214502 0.421146 vegetation 0.874349 0.07004 0.136885 -0.045448 -0.140143 cereal 0.884682 0.019462 0.048258 0.036604 -0.107117 olive 0.26776 0.424258 -0.215968 0.250303 0.130972 almond 0.27634 -0.052531 -0.188399 0.430077 -0.303976 palm -0.248537 0.132684 -0.045195 0.10083 0.839285 water plan 0.045374 -0.040519 -0.03302 0.893364 0.145629 coast 0.140236 0.887385 0.028427 -0.052517 0.032802 altitude 0.581316 -0.466223 0.147382 -0.33244 0.023178 urban 0.48289 0.551476 0.204528 0.190292 -0.02548 tourism 0.131498 0.753782 0.056591 0.080994 0.147267 species richness 0.049573 0.009893 -0.961705 0.101292 0.012076 anthropophlic s.r. 0.858163 0.215614 -0.200664 0.148998 0.019184 natural s.r. -0.665359 -0.184663 -0.650443 -0.027808 -0.009454 explained variance 5.67628 2.589464 1.711235 1.74057 1.129378 proportion .total 0.315349 0.143859 0.095069 0.096698 0.062743 table 3. extended factor loading matrix: correlations of variable clusters (oblique factors) with primary factors. cluster 1 cluster 2 cluster 3 cluster 4 cluster 5 secondary 1 0.242825 0.552793 -0.336319 0.545912 0.059906 secondary 2 0.590997 0.220702 0.375875 -0.132976 -0.353716 primary 1 0.769258 0.000000 0.000000 0.000000 0.000000 primary 2 0.000000 0.803561 0.000000 0.000000 0.000000 primary 3 0.000000 0.000000 0.863486 0.000000 0.000000 primary 4 0.000000 0.000000 0.000000 0.827223 0.000000 primary 5 0.000000 0.000000 0.000000 0.000000 0.933432 210 w. tlili et alii to each model (table 5) shows that three to four variables are involved in controlling distributions of ubiquitous species (h. turcicus, s. sthenodactylus, t. mauritanica, t. deserti and t. tripolitanus). only one variable contribute with more than 70% in modeling distributions of scarce and rare species (t. neglecta and s. petrii). response curves (fig. 4) show how the logistic prediction changes as each environmental variable is varied, keeping all other environmental variables at their average sample value. for instance, s. sthenodactylus does not show specific environmental requirement; however the probability of it occurrence decreases with increased cereal land use. the probability of occurrence of t. neglecta increases with thermal amplitudes. in fact, areas with thermal amplitudes higher than 19 are particularly favorable for t. fascicularis, t. deserti, t. tripolitanus and s. petrii. t. fascicularis showed very narrow environmental requirements; it occurs in area with 200 mm of precipitation, between 20 and 22 of thermal amplitudes, 200 to 400 m asl, less than 100 of population density and 200 quintal of cereal yielded per hectare. variable jackknife showed that the environmental variable that decreases the gain the most when table 4. auc (area under curves), cumulative threshold (c.t.), logistic threshold (l.t.) and corresponding fractional predicted area (f.p.a.) for the mtp (minimum training presences), mtss (maximum training sensitivity plus specificity) and eed (equaled entropy of thresholded and original distributions). t.m. tarentola mauritanica, t.n. t. neglecta, t.d. t. deserti, t.f t. fascicularis, h.t. hemidactylus turcicus, s.p. stenodactylus petrii, s.s. s. sthenodactylus, t.t. tropiocolotes tripolitanus. h.t. s.p. s.s. t.d. t.f. t.m. t.n. t.t. a.u.c. 0.743 0.853 0.578 0.768 0.831 0.788 0.896 0.723 mtp c.t. 3.197 5.835 1.902 4.437 9.081 3.108 3.161 4.648 l.t. 0.166 0.277 0.280 0.223 0.215 0.226 0.338 0.247 f.p.a. 0.623 0.362 0.931 0.499 0.442 0.524 0.290 0.643 mtss c.t. 6.55 13.186 12.861 4.434 17.655 8.489 17.017 12.569 l.t. 0.28 0.394 0.452 0.223 0.366 0.72 0.390 0.411 f.p.a. 0.587 0.303 0.779 0.499 0.326 0.463 0.216 0.512 eed c.t. 3.197 2.595 1.121 2.331 50.819 2.429 3.161 3.185 l.t. 0.166 0.235 0.236 0.152 0.141 0.221 0.338 0.208 f.p.a. 0.653 0.401 0.954 0.548 0.524 0.537 0.290 0.679 table 5. analysis of variable contributions t.m. tarentola mauritanica, t.n. t. neglecta, t.d. t. deserti, t.f. t. fascicularis, h.t. hemidactylus turcicus, s.p. stenodactylus petrii, s.s. s. sthenodactylus, t.t. tropiocolotes tripolitanus. variable percent contribution permutation importance h.t. s.p. s.s. t.d. t.f. t.m. t.n. t.t. h.t. s.p. s.s. t.d. t.f. t.m. t.n. t.t. thermal amplitudes 43.6 0 10.4 0.2 0.1 13.3 3.8 1.2 70.4 0 14.3 0.3 4.3 75.4 8.1 0 cereal 20.9 75.1 37.4 71.9 0.4 55.9 2.5 0.1 12.3 41.8 17.5 32.8 25.5 15.4 0 4.3 precipitations 13.6 1.3 1.8 0 0 0.6 9.2 0 0.8 14.3 2.4 0 0 2 76.2 0 urbanism 10.1 0 0.3 4.7 32.1 23.1 0 39.5 13.8 0 11.8 9.9 2.5 5.6 0 39.6 emberger coefficient 5 0.3 17 0.6 20.1 0.3 78.2 0.2 0 1.8 9 23.3 0 0 0 6.6 vegetation 3.8 0.3 12.9 17.6 22.4 6.7 0 49.5 0 0 9 15.9 35 0 0 41.3 soil 1.7 4.4 8.6 3.1 12.5 0 1 2.6 0 3.9 16.7 8.5 18.1 0 0.6 1.6 coast 0.6 0 0 0 1.7 0 0 1.4 1.7 0 0 0 13.5 0 0 4.1 palm 0.5 0 0.1 0 1.4 0.1 0 0.1 0.9 0 0 0 0.2 0.9 0 0 lithology 0.1 1.1 3.2 0.6 1.3 0 5.03 1.6 0 4.4 4.5 0 1 0.7 14.4 0 waterplan 0 0 0.2 0 0 0 0 3.8 0 0 0 0 0 0 0 1.1 tourism 0 0 2.6 0.8 1.9 0 0 0 0 0 9.6 8.1 0 0 0 1.2 olive 0 0.4 0 0 0 0 0 0 0 1.6 0 0.1 0 0 0 0 altitude 0 1.7 1.6 0 2.6 0.1 0 0 0 3.9 1.7 0 0 0 0 0 accident 0 3.2 0 0 0 0 0 0 0 3.9 0 0 0 0 0 0 almond 0 0 3.8 0.4 0.5 0 0 0 0   3.5 1.2 0 0 0 0 211gekkotan distribution in tunisia fig. 4. response curves explaining gekkotan niches. climatic items (a), relief (b) and land-use-items (c). 212 w. tlili et alii fig. 5. predicted probabilities of presence of gekkonidae within tunisian territory and habitat quality classes. h. turcicus (a), s. sthenodactylus (b), s. petrii (c) and t. tripolitanus (d). 213gekkotan distribution in tunisia fig. 6. predicted probabilities of presence of phyllodactylidae within tunisian territory and habitat quality classes. t. mauritanica (a), t. fascicularis (b), t. neglecta (c) and t. deserti (d). 214 w. tlili et alii it is omitted is thermal amplitudes for h. turcicus, cereal for stenodactylus genus, soil for t. fascicularis, thermal amplitudes for t. mauritanica, precipitations for t. deserti, and vegetation for t. tripolitanus. figures 5 and 6 show the projection of the model to unexplored grids covering tunisian territory. discussion tunisian geckos count nine species showing expansions of their ranges (tlili et al., 2012 a, b). they also presented distinct biogeographical affinities mostly concordant with the different habitat selection patterns (fig. 3). this variation does not concern climatic affinities because 77.8% of tunisian geckos are adapted to arid climate (unlike tunisian lacertidae) (nouira, 1996; nouira and blanc, 2004) and scincidae (kalboussi, 2006). however, within this climatic frame, species of the same genus are latitudinally replaced to form several guilds according to vegetal and soil characteristics. the so far recognized distribution of gekkotan fauna shows that species distribution follows a bidirectional gradient. the south-north direction is physically stressful considering the increasing humidity and development of vegetal cover; these two parameters are constraining the species occurrences and only ubiquitous ones are present. the north-south direction is biologically stressful taking into account the increasing species richness (macarthur, 1972; real et al., 1997; guisan and zimmermann, 2000). assumptions yielded from field observations were confirmed and shared distribution patterns were determined (mclaughlin, 1992; holt and keitt, 2005; heikinheimo et al., 2007; escalante, 2009; kreft and jetz, 2010). thereby, a biogeographical regionalization of tunisia according to gekkotan species richness could be established yielding five regions (fig. 3). region i harbors anthropophilic geckos widely distributed generally occupying habitats proximal to the sea (coastal line) or to an important water body (lagoon, lac …). the greatest parts of these lands are widely exploited for agricultural or industrial purposes. the south-western part of this region harbors mediterranean taxa specific to north africa; it represents the connecting point between tunisia and southern neighboring countries and contains a large traffic network. region ii represents some culminant points in central tunisia (dorsal mountain) and harbors endemic geckos in addition to anthropophilic ones. region iii harbors saharo-sindian geckos which occupy area of arid steppe lands characterized by the continental influence and drastic conditions of life. as to region iv, it harbors saharan geckos that occupy the grand erg oriental. finally region v harbors desert species that prefers rocky lands and small shrubs habitats. the gecko-based regionalization observed in southern tunisia coincides with that based on lacertidae fauna (nouira, 1996; 2004). for instance, saharan and saharo-sindian lacertidae were reported in southern tunisia; their differentiation was attributed to edaphic gradient and climatic conditions (nouira and blanc, 2003; 2004). while acathodactylus scutellatus inhabits bordering regions of the sahara, a. dumerilli occupies sandy habitats of barchans regions and a. longipes inhabits sandy saharan biotopes of the grand erg oriental. the gecko-based regionalization is also concordant with our first assumptions yielded from field work. nevertheless, we note the presence of a sixth cluster grouping spatially disjointed grids representing western and southern regions. also, faunas of northern regions are composed of two widespread cosmopolitan species which provide little useful biogeographical information. the question was then which of the environmental variables evidenced by clustering analysis is a more accurate representation of the distribution of geckos (lawley et al., 2011; strand, 2011). it is known that for ectotherms, including lizards, climate has been proposed as a key factor of their distribution (e.g. adolph and porter, 1993; doughty and shine, 1995; arad et al., 1997; kaspari and valone, 2002; carretero, 2008; salvidio and oneto, 2008). temperature and rainfall intervene indirectly by co-limiting primary production and physiologically limiting access to that production (kaspari et al., 2000). edaphic and relief characteristics take part in burrows selection (zaady and bouskila, 2002). results provided herein suggest retaining five oblique factors to explain gekkotan distributions: 1) environmental items, 2) coastal items, 3) biotic items, 4) sebkhas and chotts landscape items and 5) oasis landscape items. it also shows that gekkotan species richness is positively correlated to environmental items (climate and natural land-use). it is negatively correlated to coastal items (urban and touristic land-use) and geomorphology (altitudes and geological accidents). a strong relationship has been revealed between reptiles’ occurrences and the orographic structures in tunisia (nouira, 1996). for instance, the dorsal mountain constitutes a barrier to the expansion of saharan species toward the palearctic domain and vice versa. so far, this is in accordance with our observations concerning the distribution of the species s. sthenodactylus being limited to the south-eastern limit of the dorsal. the absence of the species in northern regions remains not explained by climate requirements alone; it could be related to geographical barriers and to their history of colonization and settlement. in 215gekkotan distribution in tunisia fact, s. sthenodactylus, considered as saharan, becomes related to widely mediterranean species and expand its ecological niche to sub-humid regions. a better understanding of the niche of the species is shown in figure 4 where the probability of occurrence of the species decreases with high precipitations, low thermal amplitudes, altitudes higher than 250m, but most importantly, cereal land-use. maps of the fig. 2 show that these are the same characteristics of the extreme north of tunisia which explains the absence of the species within this area. map b of fig. 5 shows that the suboptimal area of occurrence of s. sthenodactylus lays beyond the dorsal mountain and in the cap bon peninsula. however, area of very low probabilities of occurrence did appear in the map despite the certain presence of the species within this area. for the other gekkotan species, predicted areas follow the general distribution patterns previously yielded. besides, established models defined suitable habitats for each species and confirmed the latitudinal gradient of distribution. land use and altitude increase the probability of occurrence of h. turcicus and t. mauritanica. alternatively, they decrease the probability of the presence of t. deserti, t. neglecta, t. tripolitanus and s. petrii. h. turcicus occurs within area with high cereal land use; this is in accordance with field observations that revealed that the species inhabits preferentially hay fields. as to t. fascicularis, it showed a little displacement of the northern limit of its range towards semiarid regions. being a newly described species this displacement must be the result of better field explorations, however, modeling of its niche predicted areas of probable occurrence within the central arid steppe. high probabilities of occurrence of the species are restricted to domains characterized by less than 200 mm of precipitations, 22 to 21 thermal amplitudes, 200 m of altitudes and very low land use (fig. 4). acknowledgments we thank the office of the ministry of agriculture (forestry management department) who provided necessary authorization for animal collecting (n° 902 and 1294). we also thank the officials from the meteorological stations and the tunisia wildlife conservation society twcs who provided information and documents necessary to our work. special thanks are addressed to marc cheylan, claudia corti and josé d. anadón for their helpful advices. our gratitude is addressed to doghri moadha, mrabet kais, doghri ali, khemiri rajeh, and doghri aymen for material help. supplementary material supplementary material associated with this article can be found at: <http://www.unipv.it/webshi/appendix > manuscript number 12875. appendix 1, appendix 2, appendix 3 a, appendix 3 b. references adolph, s.c., porter, w.p. 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(2002): lizards burrows association with successional stages of biological soil crusts in an arid sandy region. j. arid environ. 50: 235-246. acta herpetologica 9(2): 167-177, 2014 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-14194 head shape variation in eastern and western montpellier snakes marco mangiacotti1,*, laura limongi1, marco sannolo1, roberto sacchi2, marco a. l. zuffi3, stefano scali1 1 museo civico di storia naturale, corso venezia 55, i-20121 milano, italy. *corresponding author. e-mail: marco.mangiacotti@gmail. com 2 dipartimento di scienze della terra e dell’ambiente, università di pavia, via taramelli 24, i-27100, pavia, italy 3 museo di storia naturale, università di pisa, via roma 79, i-56011 calci (pisa), italy submitted on 2014, 19th march; revised on 2014, 27th august; accepted on 2014, 1st september editor: sebastiano salvidio abstract. the montpellier snake malpolon monspessulanus is a wide-ranging species that inhabits western and eastern europe, north africa and middle east. four clades have been recognised as two species, m. insignitus and m. monspessulanus, each with two subspecies. clades have been substantially identified on the basis of molecular data, pholidosis and colouration, while morphometric traits have been ignored. we compared head shape of 54 specimens belonging to three out of the four clades (m. insignitus insignitus, m. i. fuscus, and m. monspessulanus monspessulanus) by means of geometric morphometrics. we found a significant differentiation: the supraocular and frontal area showed the largest amount of variation, being respectively much thinner in m. i. insignitus, a bit less thin in m. i. fuscus and definitely wider in m. m. monspessulanus. our findings are fully in agreement with the genetic studies and phylogeny explains more than 20% of the observed variation, supporting the taxonomic distinction inside the genus malpolon. the functional and/or adaptive meaning of the observed differences is not clear, but it seems unlikely that it may be related to diet. combining morphological data with phylogeography and environmental features, we formulated an explanatory hypothesis that allowed a precise and testable prediction. keywords. taxonomy, phylogeography, psammophiinae, malpolon, mediterranean, geometric morphometrics, nonparametric manova, variation partitioning. introduction the genus malpolon fitzinger 1826 belongs to the subfamily psammophiinae (family lamprophiidae; pyron et al., 2013), a taxonomic group of snakes which includes 49 species (uetz and hošek, 2013) with an afro-asiatic distribution (sindaco et al., 2013; uetz and hošek, 2013). the genus currently counts two mediterranean species (böhme and de pury, 2011), which show allopatric geographic distributions (fig. 1): malpolon monspessulanus hermann 1804 (western montpellier snake), which ranges from liguria (w italy) through france, iberian peninsula, morocco, and western sahara (fahd and pleguezuelos, 2001; geniez et al., 2006; sindaco et al., 2013); m. insignitus geoffroy sant-hilaire 1827 (eastern montpellier snake), which spreads from eastern morocco eastward around the mediterranean sea, reaching anatolia and balkans, middle east, iraq and iran, and also caucasia and transcaucasia (sindaco et al., 2013). two subspecies are currently recognized within each species (fig. 1, see also sindaco et al., 2013; uetz and hošek, 2013): m. insignitus insignitus geoffroy sant-hilaire 1827, which occurs in the eastern coast of mediterranean africa (from eastern morocco to egypt) and middle east (de haan, 1999; carranza et al., 2006), and m. i. fuscus fleischmann 1831, which occupies the remnant part of the areal (balkans, anatolia, caucasia and transcaucasia, iraq and iran; fig. 1; de haan, 1999); m. monspessulanus monspessulanus hermann 1804, which ranges from eastern morocco to south-western europe, and m. m. saharatlanticus geniez, 168 m. mangiacotti et alii cluchier and de haan 2006, a vicariant of the nominal subspecies, in western morocco and western sahara (geniez et al., 2006). specific and subspecific distinctions are based on molecular data and phenotypic traits. the phylogenetic reconstruction using mitochondrial dna (cyt b and 12s rrna; carranza et al., 2006) revealed that i) the genetic distance between the eastern (m. insignitus) and western (m. monspessulanus) clades justifies their raising to the status of distinct species (carranza et al., 2006), ii) the occurrence in the eastern clade of a greater genetic diversity, which suggests maintaining the subspecies m. i. fuscus, and iii) the genetic variability of the western clade is far lower, even comparing moroccan populations to european ones, supporting the hypothesis that current european populations are descendant of a recent recolonization from morocco through gibraltar (80,000– 170,000 years ago; carranza et al., 2006). since molecular investigations did not cover the westernmost part of the m. monspessulanus range in africa, they failed to detect the subspecies m. m. saharatlanticus, whose description is then based only on morphology (geniez et al., 2006). nevertheless, as noticed by geniez et al. (2006), a partial molecular support to its validity comes from the sole specimen from the northern limit of m. m. saharatlanticus (from essaouria, sw morocco; fig. 1; carranza et al., 2006), which has been included in the genetic analysis, in fact this specimen partly diverges from m. m. monspessulanus, suggesting a possible genetic differentiation also within the western clade. in addition to the genetic differences, a set of phenotypic traits allows discrimination among the species and subspecies of the genus (table 1; szyndlar, 1988; de haan, 1999; geniez et al., 2006). these traits concern pholidosis (i.e., scales arrangement), colour pattern and skull morphology, but, interestingly, none of them involves morphometric features. actually, as far as we know, the only attempt to differentiate members of the genus malpolon through morphometric measures was carried out by geniez et al. (2006). their study considers three out of the four malpolon clades (m. i. fuscus was excluded) and, besides colouration and pholidosis, it is focused on the head shape, represented by four linear measures (see table 1 in geniez et al., 2006). even though their analysis has been able to discriminate the three clades, the contribution of the morphometric characters appears to be negligible, with little differences in head shape among clades (at least for the measured traits). despite geniez et al. (2006) did not address explicitly this issue, the analysis of the data presented in their article supports this deduction because: i) all the linear measures are highly correlated to each other and to snout-to-vent length (see the loadings of the first principal component in table 5 of geniez et al., 2006); ii) pc1 fig. 1. left: map showing the distribution of the genus malpolon taking into account its species and subspecies; the general distribution map was based on sindaco et al. (2013), while the approximate limits of each subspecies were obtained from de haan (1999) and geniez et al. (2006). since it was not possible to obtain a precise localization of each analysed specimen, two types of localities are shown: unmistakably identified localities (dots), and localities arbitrarily chosen inside the state where specimens were collected (stars). right: phylogenetic tree inferred from cytochrome b and 12s rrna with recognizable clades (modified from carranza et al., 2006). names of the tips of the tree are the original ones by carranza et al., 2006 and correspond to the accepted scientific names at the time of their publication. clades are named with the current nomenclature. 169head shape variation in eastern and western montpellier snakes axis is a surrogate of the specimen’s size, thus clades differentiation along pc1 represents the linear size difference among subsamples (directly appreciable from table 2 in geniez et al., 2006); iii) pc2 axis is weakly correlated to morphometric measures; iv) coherently with the points above, the authors do not use morphometrics to explain the observed differentiation (see table 6 and discussion in geniez et al., 2006). in other words, the analysis, by merging size with shape, does not allow detecting the actual occurrence of morphometric differentiation, therefore a study explicitly addressing this issue and comparing malpolon clades from a morphometric perspective is still lacking. in order to start filling the gap, we decided to focus on the head for at least two reasons. firstly, the snake head is thought to be subject to strong ecological and evolutionary constraints (shine, 1991; bonnet et al., 2001; aubret et al., 2004; manier, 2004; vincent et al., 2006; gentilli et al., 2009; hampton, 2011; llorente et al., 2012; henderson et al., 2013), so the different evolutionary histories undergone by each clade may have left traces on the head shape (shine, 1991; grudzien et al., 1992; forsman and shine, 1997; hibbitts and fitzgerald, 2005; vincent et al., 2006a; herrel et al., 2008). secondly, the genus malpolon is characterised by a peculiar head shape: the combination of large eyes and oblique supraocular scales gives this snake a typical “grim facial expression” (kreiner, 2007). this trait is shared with other species of psammophiinae (even if not so accentuated; coborn, 1991), suggesting its possible genetic base and thus a potential systematic value. in this scenario, our study aims to answer three main questions: i) does head shape vary among the malpolon clades? ii) is the morphometric variation in agreement with the phylogenetic relationship among clades? iii) is there any relationship between geographic pattern of head shape variation and environmental features? materials and methods samples and geometric morphometrics we analysed 54 specimens (36 males and 18 females) obtained from the collections preserved in the natural history museums of milan (msnm), florence (“la specola”, mzuf) and genoa (“giacomo doria”, msng; table 2; table s1). only adult males and females were considered (age was assessed following feriche et al., 2008) and specimens from lampedusa and cyprus were excluded in order to avoid a possible “island” effect on the size and shape of the head (bobak, 2006). unfortunately, all collections lacked specimens of the subspecies m. m. saharatlanticus, which, consequently, was not included in our comparison. each specimen was assigned to one of the remnant three subspecies on a geographic base (fig. 1; de haan, 1999; carranza et al., 2006; geniez et al., 2006; sindaco et al., 2013). the species identification was confirmed by pholidosis and colouration, whenever possible. for each specimen we recorded sex and snout-to-vent length (svl) and we took a picture of the dorsal view of the head, using a nikon d50 professional camera at 6.1 megapixel resolution equipped with a nikkor 60 mm af-s micro lens, at a fixed distance of 18 cm. photos were subsequently used to perform the analysis of the shape by means of geometric morphometrics. geometric morphometrics is a well-established technique (claude, 2008; kaliotzopoulou, 2011; adams et al., 2012) that effectively allows removing non-shape information from geometric structures (bookstein, 1997; dryden and mardia, 1998) and has been proved to be more powerful than traditional morphometrics (rohlf, 2000). fourteen two-dimensional landmarks and eight semi-landmarks (fig. 2; table s2) were digitized for each specimen using tpsdig2 (rohlf, 2010; available at: http://life.bio.sunysb.edu/morph/), which resulted in 22 pairs of coordinates that together described head shape. these configurations were superimposed using a general procrustes analysis (gpa; rohlf and slice, 1990) in order to remove information concerning size and orientation and to preserve only information related to shape (dryden and mardia, 1998; claude, 2008). since head is a bilaterally symmetric structure and we were not interested in the quantification of the deviation from this condition, we followed the procedure suggested by klingenberg et al. (2002) to remove left-right asymmetry from table 1. main phenotypic traits used to distinguish malpolon species and subspecies from literature (szlyndar, 1988; de haan, 1999; geniez et al., 2006). “saddle-complex” refers to the dark drawing of the dorsal region of male body, which may assume a characteristic saddle-like shape. “no. of basioccipital processes” refers to the shape of the posterior part of the basioccipital bone, which may have two posterior processes (even reduced to callosities) or a single well-developed spur. for a more exhaustive description of colour patterns (also in females), see table 6 in geniez et al. (2006) and de haan (1999). species subspecies saddle-complex no. of scales at mid-body basioccipital processes m. monspessulanus monspessulanus present, limited to the first third of the trunk 19 1 m. monspessulanus saharatlanticus present, extended on the major part of the body 19 1 m. insignitus insignitus absent 19 2 m. insignitus fuscus absent 17 2 170 m. mangiacotti et alii the global shape. the procedure involves three steps: 1) generating a reflected copy of each configuration; 2) superimposing the combined collection of original and mirrored configurations; 3) averaging the coordinates of corresponding landmarks in the original and mirrored configuration of each specimen in order to obtain a perfectly symmetric configuration. we specifically implemented the first and third step in r (version 3.0.1; r core team, 2013), while general procrustes superimposition (second step) was performed by the “gpagen” function of the “geomorph” r-package (adams and otarola-castillo, 2013). head shape variation among clades differences in head shapes were analysed by means of a non-parametric distance-based manova (hereafter npmanova, anderson, 2001; mcardle and anderson, 2001). this method substitutes the usual raw data matrix of the dependent variables of manova with a distance matrix computed between each pair of observations; hence it decomposes the sum of the square pairwise distances in an anova-like way (see anderson, 2001 for details). the technique allows covariates and interactions and the p-values of the model terms are assessed via permutation of the observations (anderson, 2001). we applied the np-manova to the matrix of the pairwise procrustes distances (claude, 2008) computed on the superimposed configurations, using “clade” (three levels factor: m. m. monspessulanus, m. i. insignitus, m. i. fuscus), “svl” (covariate) and their interaction as independent variable. svl was included to take into account a possible allometric effect and it was log-transformed according to the non-linearity of the general allometric function (gayon, 2000; claude, 2008). the variable “sex” was excluded from the model because of the low and unbalanced number of females in our sample (we had only two adult females for m. i. fuscus; table 2). since “sex” could affect head shape, we tried to ensure the reliability of the results by assessing the coherence of the outcomes of a model that considers only males (male) with those of a model that considers both sexes (both). both models were based on the same set of independent variables, i.e., clade, svl and their interaction. male tests if head shape of males varies among the clades, taking into account allometry; both should allow a generalization of male results under the a priori assumption that no sexual dimorphism occurs in head shape. to give some support to this assumption, we tested the occurrence of intersexual difference in head shape in the m. m. monspessulanus clade, where males and females were balanced and numerous enough (table 2). np-manova was performed with the function “adonis” of the “vegan” r package (oksanen et al., 2013), and we tested the assumption of homogeneity of dispersions among clades fig. 2. configuration of the landmarks (black dots) and semi-landmarks (white dots) taken on each image. for a detailed definition of each landmark see table s2. 171head shape variation in eastern and western montpellier snakes (anderson, 2001, 2006; warton et al., 2012) with the functions “betadisper” and “permutest” (anderson, 2006; borcard et al., 2011) of the “vegan” r package obtaining always a non-significant difference among the three groups (p > 0.05, no. of permutations = 4999). relationship between head shape and phylogeny the relationship between phylogeny and morphology was assessed by the combination of two analyses. firstly, we used the partitioning of the determination coefficient (the so-called “variation partitioning”; peres-neto et al., 2006) of male and both models to disentangle the pure contribution of the variables “clade” and “svl” from their shared effects. secondly, we compared the topology of the phylogenetic tree linking the analysed clades with that of an analogous tree built on the morphometric distances among the mean shapes predicted by the models for each clade. indeed, if head shape had a partial genetic base, we would expect that the predicted forms for the two subspecies of m. insignitus should resemble each other more than with the predicted shape for m. m. monspessulanus. since we had only three clades, we lacked the power to test statistically our expectation (only three topologies are possible with three groups), so we just assessed the coherence of the trees. partition variation was performed with the function “varpart” of the vegan r package (oksanen et al., 2013), while the morphometric tree was generated with the base function “hclust” in r (r core team, 2013). relationship between head shape and environment to characterize the areas inhabited by each clade we used the map by sindaco et al. (2013) and extracted from it the set of one-degree cells where malpolon has been reported to occur. each cell was assigned to a clade on the basis of its geographic position (de haan, 1999; geniez et al., 2006; carranza et al., 2006). then, for each cell, we computed the mean value of two climatic variables, mean temperature of the coldest month (mtcm) and annual precipitation (ap), both obtained from www.worldclim.org (hijmans et al., 2005) at 10 arc-minutes resolution. we chose these variables to take into account the thermophily of montpellier snakes (ottonello et al., 2006; morenorueda et al., 2009) and to capture climate-habitat relationship at this spatial resolution via correlation (mcgill, 2010; mangiacotti et al., 2013). the two variables were weakly correlated (spearman correlation coefficient: rs = 0.22), indicating their ability to load different kind of environmental information. the environmental difference among the areas inhabited by the four malpolon clades was assessed by a np-manova: the pairwise euclidean distances of the scaled (standardized) values of the climatic variables of each occurrence cell (legendre and legendre, 1998) were used as the dependent matrix and “clades” (four levels-factor) as the independent variable. given the low number of clades, the analysis of the relationship between morphometry and environment was just qualitative. results head shape variation among clades the difference in head shape among the three malpolon clades was highly significant for both male and both models (table 3). no sexual dimorphism in head shape was detected in m. m. monspessulanus (“sex”: f1,20 = 0.90, p > 0.05, no. of permutations = 9999). the visual comparison of the predicted forms for each group allowed recognizing two main patterns of variation, loaded by different regions of the head (fig. 3a; fig. s1). the most evident one regarded the supraocular area and the external margin of the parietal scales, which showed a clear differentiation between the two species (m. i. insignitus and m. i. fuscus on one side and m. m. monspessulanus on the other side; fig. 3b, 3c). in particular the two subspecies of m. insignitus had: i) a less flared frontal scale; ii) thinner and longer supraocular scales, which determine a more acute silhouette of the anterior part of the head; iii) a more arched margin between the frontal and supraocular scales; iv) a less curved posterolateral margin of the parietal scales. the difference between the subspecies of m. insignitus were less evident and were loaded by the prefrontal and paritable 2. characteristics of the samples used in the present study. only adults were considered. svl stand for “snout-to-vent” length and is expressed in millimeters. n svl (mean ± sd) m. m. monspessulanus ♂♂ 12 918 ± 249 ♀♀ 12 812 ± 110 m. i. insignitus ♂♂ 11 838 ± 251 ♀♀ 4 800 ± 34 m. i. fuscus ♂♂ 13 780 ± 85 ♀♀ 2 769 ± 16 table 3. significance of the terms included in the models tested via np-manova with 9999 permutations. male refers to the model based only on males, both to the model based on both males and females. f is the f-statistic of the model, p the associated p-value obtained by randomization; df = degrees of freedom; svl is logtransformed; clade x svl represents the interaction term. term df male both f p f p clade 2 6.78 0.0001 7.25 0.0001 svl 1 2.57 0.0170 2.37 0.0310 clade x svl 2 1.44 0.1314 1.32 0.1893 172 m. mangiacotti et alii etal regions: the intersection point between prefrontal and frontal scales as well as the intersection between the posterior margin of the parietals were shifted to the front in m. i. fuscus with respect to m. i. insignitus (fig. 3d). the np-manova also detected a significant allometric effect (table 3), which was shared by all clades (the interaction term was indeed not significant). allometry seemed to act mainly on the prefrontal scales, leading to a relative elongation of the scales with increasing size (fig. 4). correspondingly, the anterior margin of the frontal scale and the posterior margin between the parietals became more acute. relationship between head shape and phylogeny the partition of the determination coefficient assigned a remarkable part of variation to the factor “clade” (more than 20% in both models; fig. 5a), while the effect of size appeared negligible (no more than 1%). the overlap between the two variables was null in both models (fig. 5a), indicating that the pure effect of the phylogenetic factor weighted considerably more than allometry. the morphometric tree obtained from the mean predicted shapes showed that the two subspecies of m. insignitus were more similar to each other than to m. m. monspessulanus (fig. 5b), i.e., the morphometric relationship among clades predicted by the factor “clade” matched the phylogenetic one. relationship between head shape and environment the environmental features of the occurrence cells are significantly different among clades (f3,349 = 60.28, p ≤ 0.0001, no. of permutations = 9999; fig. 6a). in parfig. 3. a) head configurations predicted by the model male for each clade at its mean svl. b) deformation grid highlighting the most stressed zones when mean predicted shape of m. m. monspessulanus is converted to the mean predicted shape of m. i. insignitus. c), d) same as b) but with the other two clades comparisons. in panels b, c and d, shape differences are amplified 0.5 times. no amplification is used in panel a. 173head shape variation in eastern and western montpellier snakes ticular, m. m. monspessulanus and m. i. fuscus inhabit relatively colder and rainier areas than the couple m. m. saharatlanticus – m. i. insignitus, which, conversely, share a drier and warmer environment (fig. 6a). the analysis of the climatic distance confirmed this crossed resemblance between the least related clades (fig. 6b). discussion the species and subspecies of the genus malpolon have been substantially identified on the basis of molecular data and, to a less extent, on pholidosis and colouration pattern (de haan, 1999; carranza et al., 2006; geniez et al., 2006). on the other hand, morphometric traits have been practically ignored, even though such information can be very helpful, not only for taxonomic purposes, but also in order to shed light on the evolutionary mechanisms that had driven the differentiation of malpolon lineages. with this in mind, we looked for the occurrence of a morphometric diversification in three out of the four recognised clades of the montpellier snakes, by focusing on head shape because of its potential genetic, evolutionary and adaptive meaning (shine, 1991; forsman and shine, 1997; bonnet et al., 2001; manier, 2004; vincent et al., 2006a; herrel et al., 2008; gentilli et al., 2009; hampton, 2011; llorente et al., 2012; henderson et al., 2013). we found three important results, i.e., i) head shape varies significantly among the three clades; ii) the differences are fully in agreement with the previous phylogenetic analyses and phylogeny explains a relevant part of the observed shape variation; iii) the tree obtained from climatic data does not match that from phylogeny. the largest difference in head shape occurs between the western and eastern clades and is loaded by the frontal and supraocular regions: the frontal scale is more flared in m. insignitus than in m. monspessulanus and, contemporarily, the supraocular scales are larger and with a continuous joint with the parietals. the two subspecies of the eastern species are less differentiated between each other, with m. i. fuscus showing a little enlargement of the pre-parietal zone and an anterior shift along the mid-line of the intersection point between the frontal and prefrontal scales as well as the posterior intersection between the parietal scales. all these differences cannot be ascribed neither to a combination of allometric effect nor to an inhomogeneous distribution of size classes in our sample (table 2) because svl is not statistically different among clades (f2,33 = 1.42; p > 0.05), allometry has an overall low effect (fig. 5a), and it does not involve the same regions of clade (allometry involved mainly the prefrontal region, whereas clades were loaded by the frontal, supraocular and partly parietal one; compare fig. 3 and fig. 4). even when some overlapping effect of clade and size occurs (eastern clades, where specimens have smaller svl; table 2), shape changes due to clade are opposite to what could be expected on the basis of size differences between samples (fig. 3d and fig. 4). our results suggest that head shape patterns of montpellier snakes match the phylogenetic reconstruction based on genetic data. a similar result has been already obtained for other snakes (e.g., vipera aspis, gentilli et al., 2009; coronella austriaca, llorente et al., 2012), but we found that this effect seems more pronounced in the genus malpolon. indeed, the differences in head shapes showed in fig. 4. allometric effect: head configurations predicted by the model male for each clade when svl is set to the minimum and maximum observed value. 174 m. mangiacotti et alii fig. 3a were not amplified, contrary to what was done by both gentilli et al. (2009) and llorente et al. (2012), supporting the intensity of the “clade” effect. therefore, we can reliably state that our results corroborate the distinction of the two species (m. monspessulanus and m. insignitus) and also the recognition of two subspecies inside the insignitus clade, as suggested by both molecular analysis (carranza et al., 2006) and pholidosis (de haan, 1999). the functional and/or adaptive meaning of the observed shape differences is not clear. herpetologists have usually related head shape modification in snakes to diet and prey size (shine, 1991; grudzien et al., 1992; bonfig. 5. a) variation partitioning plot: percentage contribution of each variable in the model male (bold) and both (italic). the intersection of the circles shows the variation arbitrarily assignable to clade or size. b) the tree obtained from the matrix of pairwise procrustes distances among the mean predicted configurations. figure 6. a) climatic comparison among geographic areas occupied by the four clades of the genus malpolon: black symbols represent clade centroid; vertical and horizontal lines centred on each symbol represent the standard deviation of the climatic variable on the y and x axis. mmm = m. m. monspessulanus; mms = m. m. saharatlanticus; mii = m. i. insignitus; mif = m. i. fuscus. b) the tree obtained from the matrix of pairwise climatic distances among the clades. before computing the euclidean distances, each climatic variable was standardized (scaled). 175head shape variation in eastern and western montpellier snakes net et al., 2001; king, 2002; aubret et al., 2004; boback, 2006; vincent et al., 2006a, b; hampton, 2011), but this idea does not seem to fit the malpolon case because of two main reasons: firstly, the head features associated with prey size are mainly related to head width and snout length/ width (shine, 1991; bonnet et al., 2001; hampton, 2011); these traits are expected to mould the shape of the parietal scales, while our results focused mainly on the pre-parietal area. secondly, the (scanty) data about malpolon diet do not highlight any sort of difference between eastern and western populations and describe montpellier snake as an opportunistic predator of items of various sizes (de haan, 1999; ottonello et al., 2006; plezueguelos et al., 2009). the occurrence of a systematic difference in the size of available prey is a prerequisite for a diet-shape relationship (aubret et al., 2004). since it does not seem so, we do not think feeding is the major key to understand the observed pattern of head shape variation in montpellier snakes, even though further studies are needed to exclude the diet divergence hypothesis. an alternative hypothesis to explain the observed pattern of head shape variation may come from the combination of the morphological data with the phylogeography of the genus and with the environmental features of the geographical areas occupied by each clade of malpolon. each clade inhabits climatically and topographically different areas, suggesting that these environmental factors could be among the causal factors for the observed morphological variation. more interestingly, the comparison identifies two main climatic groups: m. i. fuscus and m. m. monspessulanus on the one hand, m. i. insignitus and m. m. saharatlanticus on the other hand. if we compare the mean head shape of the three analysed clades (fig. 3), we notice that the enlargement of frontal and supraocular scales of m. i. fuscus with respect to m. i. insignitus seems to shift the insignitus head to the monspessulanus form (fig. 5b). in the light of the phylogeographic reconstruction by carranza et al. (2006), the two above considerations allow formulating a testable hypothesis: after the speciation event that led to the distinction of the eastern and western montpellier snake, m. insignitus dispersed eastward along the mediterranean coast of north africa, penetrated into middle-east and, through turkey into balkans and caucasia. here it encountered different environmental conditions, which have induced the modification of the head shape (this is the hypothetical assumption: the environment-shape causal relationship). since the conditions of this region partially match those occurring in the western part of the malpolon range, m. m. monspessulanus and m. i. fuscus may have shown a certain degree of evolutionary convergence in head shape. even if this interpretation is quite speculative, it has the advantage of giving a symmetric prediction for the other two clades: under the same assumption head shape of m. m. saharatlanticus should slightly differ from that of the nominal subspecies in the direction that tends towards m. i. insignitus. a further and complementary consideration is that the region of the head which shows the greatest variability involves the typical trait of the genus, i.e., the “grim facial expression” (kreiner, 2007): this suggests that the key to the interpretation of the observed head shape variation may lie in the functional meaning of this trait. even if no information is available about it (as far as we know), some indications may come from the hunting style of the montpellier snakes: preys are spotted by sight and the first third of the trunk is often uplifted in order to have a better view of the surroundings (arnold and burton, 1985; de haan, 1999; kreiner, 2007). such a tactic justifies the great development of the eyes and, consequently, the expansion of the supraoculars and the parallel reduction of the frontal. it is not improbable that also the degree of binocular vision could have taken advantage from this particular configuration, improving hunting performance (hibbitts and fitzgerald, 2005). so, different environmental characteristics may have required slightly different eyes-supraoculars-frontal packages and the observed among-clades differences may be the result of this adaptation. in conclusion our study gives strong support to the taxonomic and phylogenetic relationships among three out of the four currently recognized clades inside the genus malpolon; it highlights the potential importance of head shape in the reconstruction of the evolutionary history of the montpellier snakes; it shows how geometric morphometrics can be a powerful instrument to validate molecular phylogeny and to investigate the underlying causal mechanisms (rohlf, 2002; kaliontzopoulou, 2011). acknowledgements we would like to thank giuliano doria, and the technicians of the natural history museum of genoa, and annamaria nistri for the opportunity of photographing and measuring specimens from the collections of msng and mzuf, respectively. we would also thank manuela springolo for the unforgettable sweets which greatly helped doing the analyses and all the other occasional collaborators of the mipipa herp-group. we thank also two anonymous reviewers for improving the ms with their constructive comments. supplementary materials associated with this article can be found at http://www-3. unipv.it/webshi/appendix/welcome.htm. 176 m. mangiacotti et alii references adams, d.c., otarola-castillo, e. 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(1991): why do large snakes eat larger prey items? funct. ecol. 5: 493-502. szyndlar, z. (1988): two new extinct species of the genera malpolon and vipera (reptilia, serpentes) from the pliocene of layna (spain). acta zool. cracov. 31: 687-706. uetz, p., hošek, j. (2013): the reptile database, http:// www.reptile-database.org [accessed on january, 3rd 2014]. vincent, s.e., dang, p.d., herrel, a., kley, n.j. (2006a): morphological integration and adaptation in the snake feeding system: a comparative phylogenetic study. j. evol. biol. 19: 1545-1554. vincent, s.e., moon, b.r., shine, r., herrel, a. (2006b): the functional meaning of “prey size” in water snakes (nerodia fasciata, colubridae). oecologia 147: 204-211. warton, d.i., wright, t.w., wang, y. (2012): distancebased multivariate analyses confound location and dispersion effects. met. ecol. evol. 3: 89-101. acta herpetologica 10(2): 159-160, 2015 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-16436 book review: florian doré, marc cheylan, pierre grillet. le lézard ocellé un géant sur le continent européen sebastiano salvidio dipartimento di scienze della terra, dell’ambiente e della vita (distav), università di genova, i 16132 genova italy. e-mail: salvidio@dipteris.unige.it the ocellated lizard, timon lepidus (daudin, 1802) is found in portugal, spain, in southern and western france and in italy, but only in the north-western part of liguria, where the populations are in direct continuity with those of provence (sindaco and jeremćenko, 2008; salvidio et al., 2009; mateo, 2011). in the last years, this large and attractive lizard has been object of two monographic books, the first published by cheylan and grillet (2004) and the second one making the object of this review. “le lézard ocellé un géant sur le continent européen” (the ocellate lizard a giant on the european continent) is divided in four main sections. the first “origine et caractéristiques du genre timon” (origin and characteristics of the genus timon) synthetizes the systematics, taxonomy, evolution and morphology of this large mediterranean lizards. the approach is a practical naturalistic one, providing information on species variability and also the means to detect the lizard’s presence by illustrating its traces and in particular burrows, footprints, feces and shredded skin. the second part “un hôte des garrigues et maquis méditerranéen” (an inhabitant of mediterranean garrigue and maquis) gives the detailed and updated situation of the ocellated lizard populations distribution in different countries, with particular emphasis on french populations, that have been surveyed by the authors since many years. the connection between the species and its habitat and microhabitat characteristics is well explained and described by many illustrations of lizards in their natural environment. in this part, the authors stress the importance of burrows and shelters for the persistence of populations, suggesting a strict connection with other mediterranean digging species, such as the rabbit oryctolagus cuniculus and the european bee-eater merops apiaster. the third section “histoire naturelle du lézard ocellé” (natural history of the ocellated lizard) gives detailed information on the trophic niche, seasonal activity pattern, reproductive behavior, individual growth, population structure, displacement and predators. the last part of this book “une espèce en déclin” (a declining species) is based on the many scientific reports and publications written in recent years by the authors on the long-term survey and monitoring of several populations in different regions of france. the examples reported show some striking cases of local decline, sometimes attributable to natural vegetation succession and urbanization, others more enigmatic as is the case of the one from the crau plain, in which the ocellated lizard was very abundant about twenty years ago. it has to be said that since many years, doré, cheylan and grillet proposed and implemented specific survey protocols to assess the temporal trend of ocellated lizard populations (e.g., doré et al., 2011). these methods, that account for the species detectability or observability (mackenzie et al., 2002, 2003), are robust and give sound results, thus permitting to evaluate in an objective way the population trend and, therefore, should be taken as examples when establishing monitoring protocols for other lizards species. finally in this part of the book, the authors stress again the importance of artificial burrows and underground man-made shelters for the conservation of ocellated lizard populations living in open habitats, in which natural rocky shelters are missing or in which tunnel-digging animals are rare or absent. indeed, 160 sebastiano salvidio providing artificial shelters could represent a low-cost strategy to locally ameliorate the species conservation status or at least to increase its observability, thus permitting a better assessment of its presence and abundance. this hard cover book of 192 pages is robustly bound and is illustrated by dozen of really fantastic photos of lizards and their allies in their natural habitat. it is not only extremely informative but is also graphically appealing and full, really full of first-hand informations about the natural history of this fascinating animal, that should be considered as an emblem of non-forested mediterranean habitats in sw europe. since many years, the authors are giving original contributions on the conservation and ecology of many reptile species in southern europe and the present book should be of interest not only for herpetologists but also for ecologists and habitat managers dealing with conservation of mediterranean biodiversity. this book is available from biotope éditions (http://www.biotope-editions.com/) and is priced € 28.00. references cheylan, m., grillet, p. (2004): le lézard ocellé. editions belin, paris. doré, f., grillet, p., thirion, j-m., besnard, a., cheylan, m., (2011): implementation of a long-term monitoring program of the ocellated lizard (timon lepidus) population on oleron island. amphibia-reptilia 32: 159-166. mackenzie, d.i., nichols, j.d., lachman, g.b., droege, s., royle, j.a., langtimm, c.a. (2002): estimating site occupancy rates when detection probabilities are less than one. ecology 83: 2248-2255. mackenzie, d.i., nichols, j.d., hines, j.e., knutson, m.g., franklin, a.b. (2003): estimating site occupancy, colonization, and local extinction when a species is detected imperfectly. ecology 84: 2200-2207. mateo, j. a. (2011). lagarto ocelado timon lepidus. in: enciclopedia virtual de los vertebrados españoles, pp: 1-52. salvador, a., marco, a. eds. museo nacional de ciencias naturales, madrid. http://www.vertebradosibericos.org/ salvidio s., bologna m.a., cheylan m. (2011). timon lepidus (daudin, 1802), pp. 441-449. in: corti c., capula m., luiselli l., razzetti e., sindaco r. eds. fauna d’italia reptiliia. xlv. calderini, bologna. sindaco, r., jeremčenko, v.k. (2008): the reptiles of the western palearctic. 1. annotated checklist and distributional atlas of the turtles, crocodiles, amphisbaenians and lizards of europe, north africa, middle east and central asia. monografie della societas herpetologica italica i edizioni belvedere, latina (italy). acta herpetologica vol. 10, n. 1 june 2015 firenze university press acta herpetologica journal of the societas herpetologica italica acta herpetologica 12(1): 79-88, 2017 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-19158 marking techniques in the marbled newt (triturus marmoratus): pit-tag and tracking device implant protocols hugo le chevalier1, olivier calvez2, albert martínez-silvestre3, damien picard4, sandra guérin4,5, francis isselin-nondedeu6, alexandre ribéron1, audrey trochet1,2,* 1 cnrs, enfa, umr5174 edb (laboratoire evolution et diversité biologique), université paul sabatier, 118 route de narbonne, toulouse f-31062, france. *corresponding author. e-mail: trochet.audrey@wanadoo.fr 2 station d’ecologie théorique et expérimentale, umr 5321, moulis f-09200, france 3 crarc avinguda maresme s/n 08783 masquefa, barcelona, spain 4 gecco groupe ecologie et conservation des vertébrés, université d’angers, 2 boulevard lavoisier, f-49045 angers, france 5 centre d’ecologie fonctionnelle et evolutive; umr 5175; 1919 route de mende, f-34293 montpellier cedex 5, france 6 departement aménagement et environnement ecole polytechnique de l’université françois rabelais de tours, cnrs  ; umr 7324 citeres équipe ipape; 33-35 allée ferdinand de lesseps, 37200 f-tours, france submitted on 2016, 10th october; revised on 2016, 31th october; accepted on 2016, 5th december editor: marco sannolo abstract. individual marking has become essential for studying population dynamics and ecological requirements. however, marking small-bodied species such as amphibians is becoming a challenge in the last decades. amphibian surveys may require to mark manually individuals, using toe clipping, polymers and pigments, or passive integrated transponders (pit-tags). even if ethics committees have recently recommend avoiding toe clipping in amphibians, the use of pit-tags led to controversial results because low tag retention reported in some studies. here, we describe a protocol of potentially life-long pit-tag marking in a protected species, the marbled newt triturus marmoratus. in addition, we also detailed a second procedure of surgery for the implantation of transmitters needed in radio-tracking surveys. during both procedures, we found that the newt phase (either aquatic or terrestrial) strongly affected the anesthesia duration. indeed, newts in aquatic phase were more quickly anesthetized than newts under terrestrial phase. we then recommend to pay attention of this physiological particularity when performing this kind of procedure. improving our knowledge on ecological requirements and population dynamics of this species is crucial for management and conservation plans, and could be extended to other large newts. keywords. anesthesia, transmitter implantation, triturus marmoratus, pit-tagging, newts, skin permeability introduction individual marking has become an essential method for studying ecological requirements, population dynamics or colonization rates (mccarthy and parris, 2004). two different methods are widely used for monitoring species: capture-marking-recapture (cmr) and radiotracking surveys. the cmr method is a powerful method for estimating population parameters such as abundance (thompson et al., 1998), survival, recruitment, and population growth rate (lettink and armstrong, 2003). for cmr studies, marking of individuals can be performed using several techniques (ferner, 2007), such as color pattern (non-invasive method often used in some salamanders and anurans species; see delarze et al., 2000; lama et al., 2011; ribeiro and rebelo, 2011; waye, 2013; elgue et al., 2014), the use of passive integrated transponders (pit-tags; jehle and hödl, 1998), toe clipping, or the use 80 h. le chevalier et alii of polymers and pigments (brannelly et al., 2014). toe clipping is a technique commonly applied to amphibians, but may be useful for a limited time due to their regeneration ability (andreone, 1986). indeed, time intervals for regeneration range between less than one year to several years or not observable regenerations, depending on the species (ferner, 2007). this technique could also be a source of stress and involves tissue damage and a risk of infection in many species and may also interfere with behaviour and movement pattern of individuals (parris and mccarthy, 2001; mccarthy and parris, 2004; funk et al., 2005). photo matching is a relatively low-costly and non-invasive powerful technique allowing individuals to be recognize using photographic identification of external body markings (arntzen et al., 2004). this technique had been used in a variety of species, including newts (drechsler et al., 2015; mettouris et al., 2016; see also diego-rasilla and luengo, 2002) but photo-identification might be difficult to apply in some cases (for instance in the marbled newt during the aquatic phase, females are very dark colours with very low contrast). observers can correctly match 100% of photo pairs “by eye” (morrison et al., 2016), but this process is highly time-consuming, and was considered as relatively not appropriate for studies on large populations (arntzen et al., 2004). since several years, many automatic recognition softwares have been developed (wild-id, bolger et al., 2012; aphis, moya et al., 2015; hotspotter, crall et al., 2013; amphident, matthé et al., 2008; i3s pattern, van tienhoven et al., 2007) to reduce the time of individual identification. many studies demonstrated that, using automated processes for photographic re-identification, pattern mapping is a successful approach for the identification of individuals, even in large populations (drechsler et al., 2015; mettouris et al., 2016). however in some cases, these tools failed to match many image pairs (morrison et al., 2016), and might induce significant bias in the cmr analysis. pit-tagging is a relatively new marking technique (christy, 1996) providing rapid recognition of individuals during recapture sessions and limiting recognition errors (using recorder scanning device for instance). also, some scanning devices can read the pit-tags at several centimeters of the marked individual, which reduce the stress induced during the manipulation. along with the demonstration of drawbacks of this method in amphibians (tracy et al., 2011; brannelly et al., 2014), such as low tag retention among marked individuals and the expense, ethics committees have recently recommend avoiding toe clipping in amphibians. indeed, other recent studies showed pertinent results using pit-tagging for amphibian monitoring (connette and semlitsch, 2011; heard et al., 2012) with no significant effects of marking on survival rate or body condition (perret and joly, 2002). miniaturization process of pit-tags and similar marking techniques has increased these last years, being less invasive, and the size should continue to decrease in the future (gibbons and andrews, 2004; cooke et al., 2013). pit-tagging (relatively costly and rapid recognition) and photo identification (low-costly and non-invasive) are often compared for studying the benefits and disadvantages of each technique (arntzen et al. 2004), and to identify the more appropriate method to apply on amphibian populations. the choice of technique depends on financial and human costs, but also on the study species, study duration and sampling proportion (arntzen et al. 2004). the use of photo-identification technique is well documented in the literature (drechsler et al., 2015; mettouris et al., 2016; sannolo et al., 2016) but clear and detailed protocols about the use of pit-tagging still remain rare, in particular in newts. moreover, it is noteworthy that individuals tested here were captured for locomotion and mobility experimental tests under controlled conditions (see below) and that each individual from host captive wildlife within french establishments authorized must be marked with pit-tagging since august 2004 (art. r. 413-30; 10 august 2004). radio-tracking surveys are also commonly used in many species, even in small-bodied urodeles (jehle and arntzen, 2000; ribéron and miaud, 2000; rittenhouse and semlitsch, 2006). for radio-tracking studies, an active transmitter is either attached to the different parts of the animal body, depending on the taxon (stuck on carapace, neck or wings) or surgically implanted into the coelomic cavity. then, tracked individuals can then be located using a receiver. contrary to cmr surveys, radiotracking allows precise and remote detection of individuals allowing determination of habitat use and daily movements. here, we describe the protocol of a successful pittag marking in the marbled newt. we also detail a surgical procedure for the implantation of transmitters in this species. details on individual morphology, anesthesia duration, and the effect of individual phase (terrestrial or aquatic phase) are explored. studies on ecological requirements and population dynamics of this species are crucial for management and conservation plans. technological progress over the last few years in transmitter size allows the use of small transmitters without an external part. developing an efficient protocol for both pit-tagging and implantation of transmitters could strongly be useful and applicable in other large newt species. 81marking techniques in the marbled newt materials and methods study species the marbled newt (triturus marmoratus) is a protected species listed on both the bern convention and the european habitat directive (annex iii and annex iv respectively; least concern on the iucn redlist), with declining populations (arntzen et al., 2009). during the breeding season (from march to may), this species lives in a variety of temporary or permanent water sources, such as well-vegetated ponds, pools, ditches and streams (nöllert and nöllert, 2003). newts have a permanent permeable skin. but during the breeding season their skin is more permeable, allowing for respiration, but also resulting in significant water loss. a nearby water source therefore constitutes critical habitat for this species during reproduction (wells, 2007). after breeding, the skin of newts undergoes a seasonal change in osmotic permeability and becomes less permeable (wells, 2007). newts become more terrestrial and move out of water to winter and feeding habitats, notably wet habitats such as forests or under stones, to hibernate. within this terrestrial phase, the mobility of the marbled newt is estimated to be less than 1 kilometer (jehle, 2000; jehle and arntzen, 2000). newt sampling we captured a total of 46 marbled newts (22 females and 24 males) from two sites to minimize the impact on newt populations and also to test if radio-tracking survey data were similar between individuals from different landscapes (see trochet et al., 2017). we sampled marbled newts in southern france, in the department of ariège (n = 30; 12 females and 18 males; 43.076347°n, 1.351639°e) between the 1st and the 29th april 2015, and in the department of gers (n = 16; 10 females and 6 males; 43.671781°n, 0.504308°e) between the 24th april and the 6th of may 2015. individuals were caught using a landing net and transported to the station d’ecologie théorique et expérimentale cnrs (42.958285°n, 1.086455°e; moulis, france), an ecological research station of the national center for scientific research situated in the foothills of the pyrenees. animals were housed in groups of 6 to 8 individuals in aquaria of 60×30×30 cm and kept at a temperature of 20°c, and were fed with mealworms. for each individual, we measured the snoutvent length (svl) using a caliper and the body mass (bm) with an electronic scale (precision 0.01 g). pit-tagging was performed between the 2nd april and the 11th may 2015, during the aquatic phase of the breeding season when newts had large crest in males and highly permeable skin (table 1). during this aquatic phase, the skin is smoother and less coloured. transmitter implantations were then performed between the 28th may and the 19th june 2015 during the postbreeding migration when newts were in the terrestrial phase, recognizable by the small crest in males and low permeable skin (individuals more coloured and grainy skin; table 1). all individuals were pit-tagged, with 24 individuals also implanted with transmitters. animals were released after 40 to 70 days at their site of capture after locomotion and mobility experimental tests (data not shown here). anesthesia procedure both procedures were performed in sterile conditions using diluted chlorexidine 0.75%. prior to each procedure, individuals were rinsed and placed in individual boxes. considering the average weight of newts (mean ± sd: 11.53 ± 3.32 g; min-max: 5.54 – 20.23 g; table 1) and the concentration of lidocaine/prilocaine in emla ointment (5% lidocaine 2.5% and prilocaine 2.5%; astrazeneca gmbh laboratories, germany, emla), the final dosage for anesthesia was 450 mg/kg, by percutaneous absorption of the ointment in a cutaneous squared surface of 1cm × 1 cm. we applied one spot of cutaneous anesthetic cream on their left flank, until the muscular system was relaxed and animals stopped moving. we considered animals to be surgically anesthetized (only cardiac impulse was present) when individuals lost the “withdrawal reflex” (i.e. no response when pinching digits and tail; fig. 1a) and “righting reflex” (i.e. unable to right themselves when put on their back; fig. 1b; mitchell, 2009). animals were rinsed before the procedures to remove excess cream. anesthesia and recovery durations were recorded for both protocols. pit-tagging protocol (n = 46) during anesthesia, the newt skin was firstly disinfected before surgery with diluted chlorexidine 0.75%. we then placed a pit-tag (rfid standards iso 11784 & 11785 type fdx-b, 1.4×8 mm, 134.2 khz from biolog-id, fr; fig. 1c) into the dorsal side using an injector, previously disinfected with diluted chlorexidine 0.75%. the mean mass of pit-tags was 0.03 g, representing approximately 0.26% (range min-max: 0.15-0.54 %) of the newt body mass. the needle was inserted on the left side, at the site of anesthetic application, from the bottom of the back and pushed up under the skin to install the pit-tag lateral to the hepatic area between the posterior and anterior limbs. the injection site was immediately disinfected after injection with chlorexidine 0.75%. pit-tags were not removed from individuals before releasing. transmitter implantation (n = 24) during anesthesia, a longitudinal incision of about 1 centimeter, matching the width of the transmitter, was made on the right flank using surgical scissors in two steps, for the skin and then muscle tissue. the transmitter (v1|10a ultimate lite implants: battery life around 54 days; biotrack, uk) was placed into the abdominal cavity and fitted between the internal organs (fig. 2a). the mean mass of transmitters was 1.77 ± 0.04 g, representing approximately 16% of the newt body mass (average 10.65 ± 2.23 g), a proportion considered unlikely to impact displacements (madison and farrand, 1998). the muscle tissue and the skin were then pulled over the transmitter and sutured. the muscle tissue was sutured with running subcuticular clo82 h. le chevalier et alii table 1. individual data on the 46 marbled newts used for both pit-tagging and transmitter implantations. id: individual number (corresponding to the seven last digits of pit-tag number); svl: snout-vent length (cm); bm: body mass (g). capture pit-tags transmitter implantations morphology release date site date id date transmitter frequency surgery date sex svl bm ariege 01/04/2015 3891004 02/04/2015 150.2431 01/06/2015 m 6.30 8.51 10/06/2015 ariege 01/04/2015 3891021 02/04/2015 150.3037 02/06/2015 f 6.75 9.41 10/06/2015 ariege 26/04/2015 3560269 26/04/2015 150.1231 02/06/2015 m 6.20 9.38 10/06/2015 ariege 26/04/2015 3797561 26/04/2015 150.2730 02/06/2015 f 7.60 12.67 10/06/2015 ariege 26/04/2015 3910400 26/04/2015 m 6.40 8.37 10/06/2015 ariege 26/04/2015 3910360 26/04/2015 150.0941 01/06/2015 m 6.10 7.51 dead ariege 28/04/2015 3560248 29/04/2015 150.0040 28/05/2015 f 7.80 14.69 08/06/2015 ariege 28/04/2015 3793003 29/04/2015 150.2141 28/05/2015 m 7.90 14.75 08/06/2015 ariege 28/04/2015 3563668 29/04/2015 150.0327 02/06/2015 f 7.05 10.73 08/06/2015 ariege 28/04/2015 3793285 29/04/2015 150.1530 02/06/2015 f 7.00 12.99 08/06/2015 ariege 28/04/2015 3910145 29/04/2015 150.1832 02/06/2015 m 7.00 9.20 08/06/2015 ariege 28/04/2015 3560302 29/04/2015 f 6.50 9.16 08/06/2015 ariege 28/04/2015 3910246 29/04/2015 m 7.30 12.00 08/06/2015 ariege 28/04/2015 3560049 29/04/2015 m 6.90 9.90 08/06/2015 ariege 28/04/2015 3793236 29/04/2015 m 7.75 14.57 08/06/2015 ariege 28/04/2015 3910116 29/04/2015 m 7.10 10.31 08/06/2015 ariege 28/04/2015 3560290 29/04/2015 150.3333 01/06/2015 f 7.45 11.95 10/06/2015 ariege 28/04/2015 3910488 29/04/2015 150.7132 01/06/2015 m 7.30 11.38 10/06/2015 ariege 28/04/2015 3560094 29/04/2015 150.0636 02/06/2015 f 7.70 13.69 10/06/2015 ariege 28/04/2015 3560259 29/04/2015 150.7424 01/06/2015 m 7.10 10.33 dead ariege 29/04/2015 3563674 30/04/2015 m 7.40 11.95 08/06/2015 ariege 29/04/2015 3909734 30/04/2015 m 8.05 16.89 08/06/2015 ariege 29/04/2015 3796489 30/04/2015 m 7.60 13.04 08/06/2015 ariege 29/04/2015 3796891 30/04/2015 m 7.40 11.57 08/06/2015 ariege 29/04/2015 3793419 30/04/2015 f 7.90 18.67 08/06/2015 ariege 29/04/2015 3890964 30/04/2015 f 7.25 16.27 08/06/2015 ariege 29/04/2015 3909958 30/04/2015 f 7.80 20.02 08/06/2015 ariege 29/04/2015 3563699 30/04/2015 f 7.90 20.23 08/06/2015 ariege 29/04/2015 3560116 30/04/2015 m 7.90 14.12 08/06/2015 ariege 29/04/2015 3560253 30/04/2015 m 7.70 13.97 08/06/2015 gers 26/04/2015 3560111 26/04/2015 150.772 18/06/2015 f 6.70 7.58 23/06/2015 gers 26/04/2015 3910195 26/04/2015 f 6.70 10.29 23/06/2015 gers 26/04/2015 3910288 26/04/2015 f 7.10 9.53 23/06/2015 gers 26/04/2015 3910201 26/04/2015 m 6.75 8.67 23/06/2015 gers 06/05/2015 3792718 11/05/2015 150.623 17/06/2015 m 6.90 9.61 23/06/2015 gers 06/05/2015 3796526 11/05/2015 150.922 17/06/2015 f 7.55 14.39 23/06/2015 gers 06/05/2015 3560285 11/05/2015 150.532 18/06/2015 m 6.60 9.47 23/06/2015 gers 06/05/2015 3560266 11/05/2015 150.562 18/06/2015 f 6.65 11.12 23/06/2015 gers 06/05/2015 3909919 11/05/2015 150.651 18/06/2015 m 6.40 8.93 23/06/2015 gers 06/05/2015 3909854 11/05/2015 150.802 18/06/2015 f 7.15 10.60 23/06/2015 gers 06/05/2015 3910535 11/05/2015 150.831 18/06/2015 m 6.10 10.09 23/06/2015 gers 06/05/2015 3910390 11/05/2015 150.502 19/06/2015 f 6.60 7.96 23/06/2015 gers 06/05/2015 3560126 11/05/2015 150.592 19/06/2015 f 7.20 8.63 23/06/2015 gers 06/05/2015 3910153 11/05/2015 m 5.80 5.54 23/06/2015 gers 06/05/2015 3563687 11/05/2015 f 7.10 12.23 23/06/2015 gers 06/05/2015 3560239 11/05/2015 f 6.50 7.35 23/06/2015 83marking techniques in the marbled newt sure (fig. 2b) using absorbable material (ethilon, diameter 3/0). the skin was sutured with simple interrupted suture (3, 4 or 5 surgeon’s knots depending on the length of the incision; fig. 2c, 2d) using absorbable material (ethilon, diameter 3/0). sutures were performed using an iris cutting needle (a c-shaped needle 7 mm long) and surgical silk. transmitters were not removed from individuals before releasing. recovery phase after each protocol, anesthetized individuals were then placed in a recovery room, into wet boxes, where the ventral portion of the body was placed into water to promote the expulsion of the anesthetic agent. we considered newts to be recovered with the return of the righting reflex. they were then placed into a well-vegetated terrarium, with water, soil substrate, shelter (mud) and food (tubifex worms) and kept under observation for 5 to 10 days. no visible impact on health and behaviour was observed after the pit-tagging procedure. however after transmitter implantation, triggering of a molt on 14 newts was observed two days after operation. the return of the feeding behaviour occurred in the four days after the operation. statistical analyses we performed a total of 70 fully-recorded procedures (46 pit-tag implants, 24 transmitter implants). in order to test if skin permeability (i.e., the date of both operations) could have an effect both on the anesthesia and the recovery durations (both not normally distributed), we used non-parametric spearman correlation tests. we also compared both the anesthesia and the recovery durations and their variability (i.e., using a coefficient of variation defined as standard variation/mean × 100) depending on the two procedures tested here using wilcoxon tests. we then calculated a condition index (hereafter ci) for each newt estimated by the residuals of the regression of log(bm) on log(svl) (jakob et al., 1996), a useful index in amphibian studies (denoël et al., 2002; bancila et al., 2010) to test if the morphology could be related to both anesthesia and recovery durations in the two different procedures using spearman correlations. finally, we tested if the sex had an influence on both anesthesia and recovery durations using wilcoxon tests. all statistical analyses were performed using r 3.0.1. (r development core team, 2014). results anesthesia duration our results showed that the anesthesia duration was shorter among individuals in aquatic phase (mean ± sd: 5.17 ± 3.09 minutes) than among newts in terrestrial phase (mean ± sd: 10.15 ± 4.49 minutes) and that anesthesia duration was variable as both during pit-tagging fig. 1. anesthesia procedure after application of emla cream: loss of pain reflex (a); loss of righting reflex (b); pit tag implantation (c). fig. 2. surgical implantation of transmitter: insertion of transmitter in the abdominal cavity (a); completed suture of muscle tissue using a simple continuous suture pattern (b); skin suture using the horizontal mattress suture technique (c); and completed procedure (d) 84 h. le chevalier et alii as in transmitter implantation procedures (wilcoxon test: w = 1, p = 1). the anesthesia duration was significantly different between both procedures (wilcoxon test: w = 125.5, p ≤ 0.01) and increased from the first procedures performed (i.e., pit-tagging) at the beginning of april 2015 (aquatic phase with high permeable skin) to the final procedures (i.e., transmitter implantation) performed at the end of june 2015 (terrestrial phase with less permeable skin; spearman correlation: rs = 0.39, p < 0.01; fig. 3). we found non-significant relationship between the anesthesia duration and newts ci (spearman correlations: rs = -0.08, p = 0.50). our results also showed no influence of sex on the anesthesia duration (wilcoxon tests: w = 560.5, p = 1). recovery duration our results did not found difference in recovery duration variability between both procedures (wilcoxon test: w = 0, p = 1). we found a strong difference in the recovery duration between the two procedures (wilcoxon tests: w = 43, p < 0.01). the recovery duration after pit-tagging (mean ± sd: 76.45 ± 27.51 minutes) was significantly shorter than the recovery duration after transmitter implantation (mean ± sd: 309.80 ± 87.93 minutes; spearman correlation: rs = 0.60, p = < 0.01; fig. 4). contrary to the anesthesia duration, newt ci influenced recovery duration (spearman correlations: rs = -0.27, p = 0.02) where the largest newts woke up faster. our results showed no influence of sex on the recovery duration (wilcoxon tests: w = 635, p = 0.36). pit-tags and transmitter surveys no pit-tags were reported lost, even several months after implantation. before releasing, we reported the number of days for which newts kept their pittags: 44.02 days in average; range min-max: 39-69 days. we also noted that none of the tags moved from the insertion site. newts with transmitters were then radiotracked up to 48 days after release (see trochet et al., 2017). only one newt (id number: 3910145) was found dead in the field during the survey (seven days after release). fig. 4. recovery duration (in minutes) for both procedures. fig. 3. anesthesia duration (in minutes) depending on procedure date related to the number of newts in terrestrial phase (less permeable skin). pit-tagging procedures are represented by black dots and transmitters implantations are represented by white dots. 85marking techniques in the marbled newt discussion marking amphibians is a challenge for studying population dynamics. indeed, their small size and their semiaquatic life cycle increase the difficulty to mark them. toe clipping is still in use in amphibian monitoring, even if this process is actually criticized (parris and mccarthy, 2001). as a non-invasive and successful method for individual identification, photo-matching is becoming a powerful technique largely used in amphibian species since many years. but this method can be time-consuming, even using automatic processes for photographic re-identification. moreover, pattern mapping may be not appropriate for studies on large populations (but see drechsler et al., 2015; mettouris et al., 2016). pit-tagging is a recent advance in animal marking technique and is now frequently used in a variety of species, including amphibians (ott and scott, 1999; cucherousset et al., 2008) but this method is relatively costly and had demonstrated contrasting results. the recent cost reduction and miniaturization of pit-tags (in this study: pit-tag length 1.4×8 mm, approximately $2.50 each) make this technique particularly interesting in amphibian monitoring surveys. while photo-identification has been explored in several amphibian species, a detailed and powerful method of pittagging still remains rare, especially in newts. here, we specified how perform pit-tagging in the marbled newt to limit pit-tag loss respecting the welfare of individuals. also, this kind of marking became mandatory in french establishments authorized to host captive wildlife, and for which a clear protocol is needed. anesthesia and recovery durations the use of a cutaneous anesthetic agent permits the performance of reliable procedures in a less invasive way. physiological state should be closely observed during the procedures and dosage recommendations followed. our findings also demonstrated that the recovery duration in newts after anesthesia was more influenced by the invasiveness of the procedure than by the phase of the individuals. we reported deaths after both marking procedures only for two newts operated on twice after the use of surgical adhesive (see below). hence, we recommend following these protocols to increase the likely success of surveys, even with the constraint of requiring sterile conditions. pit-tagging as a robust method for amphibian marking pit-tag marking has been used in many species, allowing assessment of movement patterns or growth and survival rates (jehle and hödl, 1998; ott and scott, 1999; perret and joly, 2002). this method is often preferred in long-term population surveys (perret and joly, 2002; arntzen et al., 2004) and in behavioral studies (winandy and denoël, 2011), even in relatively small-bodied salamanders (connette and semlitsch, 2011). indeed, no significant effect of pit-tagging on growth and survival has been demonstrated (ott and scott, 1999; perret and joly, 2002; connette and semlitsch, 2011). pit-tags are permanent and internal transponders, and this technique is generally considered as less invasive than other methods such as toe clipping which might be a stressful marking method for individuals (parris and mccarthy, 2001). most studies using pit-tagging reported pit-tag loss and failure, or mortality of individuals, but these results were likely caused by improper implantation (gibbons and andrews, 2004). tag loss can also occur just after pittag injection if the tag exits through the opening caused by the needle (12.1% of tag loss in feldheim et al., 2002). most of these studies, which inject pit-tags without anesthesia, tended to reject the use of these transponders because of low tag retention rate (33.3% tag loss in brannelly et al., 2014). here, no pit-tag expulsion was recorded. hence, in order to minimize pit-tag loss, we strongly recommend anesthetizing individuals before pit-tagging. this results in the immobilization of the individual for several minutes which reduces the chance of expulsion and stress to the individual thereby improving healing. we note that animals can be released just after recovery (i.e., 70.08 ± 20.23 minutes) and kept less than one day in captivity. using the procedure described here, all newts kept their pit-tags, even after 69 days. we also applied this protocol previously to bufo bufo (data not shown here) and individuals kept their pit-tags over 183 days, suggesting that pit-tags had not been lost by individuals for a long time, and that long-term survey studies should be successful using the protocol described here. safe protocol for transmitter implantation for radio-tracking surveys most radio-tracking studies in amphibians showed no significant effect on feeding, body mass or survival rate in implanted individuals (olders et al., 1985; madison and farrand, 1998). in amphibians, the use of internal implants is highly recommended, as external tags may hinder mobility and feeding, despite the use of transmitters with various modes of attachment (fukuyama et al., 1988; fiorito et al., 1994; golay, 1996; tramontano, 1997). distances and data obtained by radio-tracking are very useful and can be used both in population dynamics and conservation biology studies. to this day, radio86 h. le chevalier et alii tracking is the only technique that can be used to determine habitat use during seasons where cryptic behavior is displayed, as well as providing indispensable data for conservation management. using our protocol of implantable transmitters, we observed no infection after operating on newts. an important observation was made concerning the use of surgical adhesive (3m vetbond tissue adhesive, 3m animal care products, united states) for the 10 first operations, which was applied to the sutures at the end of these procedures. surgical adhesive may be placed over a suture line for added protection from dehiscence and to serve as a waterproof coating (wright and whitaker, 2001). it was observed that these newts had ruptured their sutures due to the solidified surgical adhesive. newts that had ruptured their sutures were then operated on a second time. two marbled newts died after this second operation (id numbers 3910360 and 3560259; table 1). we believe that these deaths would not have occurred if we not used the surgical adhesive and been obliged to perform a second procedure. as a consequence, the use of surgical adhesive for this particular protocol of transmitter implantation is ill-advised. to summarized, 87.5% animals survived up to 48 days in the wild for successful radio-tracking surveys (see trochet et al., 2017) post-surgery, until the battery life expired. transmitter implantation seemed to not impact behaviour of newts, as already observed in other amphibian species (olders et al., 1985; madison and farrand, 1998; johnson, 2006; marcec et al., 2016). radio-tracking studies however have their limitations: the precision of tracking depends on the number of location points and the presence of an observer may influence the behaviour of the studied animal, even if this last effect is reduced (brown et al. 2013). moreover, in small-bodied animals such as newts, the main limitation of radio-tracking studies using implantable transmitters is the transmitter size and battery duration, leading to a restricted signal range (gourret et al., 2011). despite the reduction of transmitter size and increased battery life, this restriction could become problematic with very mobile species, for example during migration (van gelder et al., 1986; sinsch, 1990). conclusion we demonstrated that, in disinfected conditions and following an anesthesia protocol before marking, pit-tagging can be a very useful marking method, without any tag loss or significant impact on individual health and behavior. surgical procedures, such as internal transmitter implantation could also be performed under the same anesthesia protocol. our findings also showed that anesthesia duration was strongly dependent on the phase of newts (aquatic or terrestrial), which is related to certain modifications of the skin, such as permeability. contrary to the anesthesia duration, the recovery duration was only related to the procedure used (pit-tagging vs. transmitter implantation). acknowledgments financial support was provided by the fondation de france and by the french agence nationale de la recherche through the anaee project (‘analysis and experimentation on ecosystems’; project no. 312690), and partly with the feder and aelb funds managed by the epl (etablissement public loire) and snb (french national biodiversity strategy). this work was supported by the french laboratory of excellence project “tulip” (anr-10-labx-41; anr-11-idex-0002-02) and benefited from an “investissement d’avenir” grant managed by agence nationale de la recherche (ceba, ref. anr10-labx-25-01). authors have complied with all applicable animal care guidelines, and all required french permits (capture permit and authorization for experimentation with animals) have been obtained. at and hlc collected newts (permit nos. 09-2014-14 and 32-2014-07). dp and sg developed the transmitter implantation protocol in the marbled newt. oc performed surgery (animal experimentation accreditation n°a09-1). hlc and at analyzed the data and wrote the manuscript, which was corrected by all authors. references andreone, f. 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(2001): amphibian medicine and captive husbandry, 1st edition. krieger publishing company, malabar. acta herpetologica vol. 12, n. 1 june 2017 firenze university press morphological variation and sexual dimorphism in liolaemus wiegmannii (duméril & bibron, 1837) (squamata: liolaemidae) from uruguay joaquín villamil1,*, arley camargo2, raúl maneyro1 sex does not affect tail autotomy in lacertid lizards panayiotis pafilis1,*, kostas sagonas2, grigoris kapsalas1, johannes foufopoulos3, efstratios d. valakos4 fire salamander (salamandra salamandra) males’ activity during breeding season: effects of microhabitat features and body size raoul manenti*, andrea conti, roberta pennati call variation and vocalizations of the stealthy litter frog ischnocnema abdita (anura: brachycephalidae) pedro carvalho rocha1,2,*, joão victor a. lacerda2,3, rafael félix de magalhães2,3, clarissa canedo4, bruno v. s. pimenta5, rodrigo carrara heitor6, paulo christiano de anchieta garcia2,3 feeding ecology of two sympatric geckos in an urban area of northeastern brazil josé guilherme g. sousa1, adonias a. martins teixeira2,*, diêgo alves teles2, joão antônio araújo-filho2 and robson waldemar ávila3 a pattern-based tool for long-term, large-sample capture-mark-recapture studies of fire salamanders salamandra species (amphibia: urodela: salamandridae) jeroen speybroeck1,*, koen steenhoudt2,$ skeletal variation within the darwinii group of liolaemus (iguania: liolaemidae): new characters, identification of polymorphisms and new synapomorphies for subclades linda díaz-fernández*, andrés s. quinteros, fernando lobo marking techniques in the marbled newt (triturus marmoratus): pit-tag and tracking device implant protocols hugo le chevalier1, olivier calvez2, albert martínez-silvestre3, damien picard4, sandra guérin4,5, francis isselin-nondedeu6, alexandre ribéron1, audrey trochet1,2,* evidence for directional testes asymmetry in hyla gongshanensis jindongensis qing gui wu1, wen bo liao2,* the advertisement call of pristimantis subsigillatus (anura, craugastoridae) florina stănescu1,4, rafael márquez2, paul székely3,4,*, dan cogălniceanu1,4,5 nematodes infecting anotosaura vanzolinia (squamata: gymnophthalmidae) from caatinga, northeastern brazil bruno halluan s. oliveira¹, adonias a. martins teixeira¹,*, romilda narciza m. queiroz¹, joão a. araujo-filho¹, diêgo a. teles¹, samuel v. brito2, daniel o. mesquita¹ where is my place? quick chorus structure assembly in the european tree frog michal berec a possible mutualistic interaction between vertebrates: frogs use water buffaloes as a foraging place piotr zduniak1,*, kiraz erciyas-yavuz2, piotr tryjanowski3 good vibrations: a novel method for sexing turtles donald t. mcknight1,2,*, hunter j. howell3, ethan c. hollender1, and day b. ligon1 errata to mecke, s., hartmann, l., mader, f., kieckbusch, m., kaiser, h. (2016): redescription of cyrtodactylus fumosus (müller, 1895) (reptilia: squamata: gekkonidae), with a revised identification key to the bent-toed geckos of sulawesi. acta herpetologica 11(2): acta herpetologica 12(1): 113-116, 2017 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-20574 a possible mutualistic interaction between vertebrates: frogs use water buffaloes as a foraging place piotr zduniak1,*, kiraz erciyas-yavuz2, piotr tryjanowski3 1 department of avian biology and ecology, faculty of biology, adam mickiewicz university in poznań, umultowska 89, 61-614 poznań, poland. *corresponding author. e-mail: kudlaty@amu.edu.pl 2 ornithology research center, ondokuz mayis university, 55139 kurupelit samsun, turkey 3 institute of zoology, poznań university of life sciences, wojska polskiego 71c, 60-625 poznań, poland submitted on 2017, 23rd march; revised on 2017, 19th april; accepted on 2017, 20th april editor: marco mangiacotti abstract. mutualisms shape biodiversity by influencing the ecology and the evolution of populations and communities. for example, among many others, birds commonly forage in association with large mammals, including livestock, but so far no similar relationship has been described for amphibians. in this note we describe the association between the marsh frog (pelophylax ridibundus) and the anatolian water buffalo (bubalus bubalis) in turkey and provide possible explanations for the existence of direct relations between these representatives of two vertebrate classes. we hope that our note stimulates future research on this subject. keywords. bubalus bubalis, interaction, pelophylax ridibundus. interspecific interactions such as mutualism are main processes that shape biodiversity by influencing the ecology and the evolution of populations and communities (bascompte, 2009; sazima et al., 2010). among many others, birds commonly forage in association with large mammals, including livestock, wild ungulates and pachyderms (dean and macdonald, 1981; sazima, 2011). in particular, associations with cattle and buffaloes are one of the best known and occur in many geographical regions (bradshaw and white, 2006; sazima, 2011). however, searching through different kinds of scientific information sources, we did not find any records on similar relationships between amphibians and large mammals. thus, the purpose of this note is to describe the association of the marsh frogs (pelophylax ridibundus) and the anatolian water buffaloes (bubalus bubalis) in turkey and to provide a possible explanation for the existence of direct relations between these representatives of two vertebrate classes. the observations were carried out in the kızılırmak delta (n turkey) that stretches along the black sea coast from 41°30'n and 41°45'n to 35°43'e and 36°08'e. the delta is one of the largest wetlands in the middle east and an important area for migratory birds (erciyas-yavuz et al., 2015). the delta is located at an altitude between 0 to 15 m above sea level and its total surface area is about 56,000 ha. while 70% of the delta is intensively used by people, the remainder is a natural habitat including open water, freshwater and semi-saline lakes, marsh vegetation, sand dunes, woodland, and farmland, including pastures for the local buffalo race (barış et al., 2005). anatolian water buffaloes play a major role in structuring the vegetation in the kızılırmak delta, including its permanent and ephemeral wetlands and adjacent coastal sand dune systems. they stay outside during april-november and the rest of the year they are kept in farms (sullivan et al., 2016). to confirm the rarity and novelty of our observations, we searched information on possible relationships 114 p. zduniak, k. erciyas-yavuz, p. tryjanowski between frogs and buffaloes, domestic cattle and similar large mammals using the internet-based search engines of thomson-reuters (web of science, zoological record) and scopus databases, google scholar and google books. additionally, we searched for amphibian and mammal (generally, and specifically frog and buffalo) records in previously undescribed sources including internet web searches for trip reports, images and videos carried by google, google images, flickr and youtube. we searched not only the english and latin but also included numerous other languages. browsing internet graphic sources and searching for the sitting frogs on buffaloes, four photographs were found. three were from south asia and one from hungary. in all cases these were single buffaloes with the head protruding out of the water, where frogs were present but no insects were observed. no data about the described phenomenon were found in other sources including scientific on-line databases. real data in the field were also collected. the random and independent 12 observations of anatolian water buffalo took place during october 3rd and 10th 2012. we recorded on 10 occasions the situation, when marsh frogs were present on buffaloes (tab. 1). frogs were recorded both on resting and standing mammals in different part of their body including the head. in most cases frogs hunted flies (fig. 1). further random observations carried out in the next year confirmed that the described phenomenon only occurs in autumn, when the density of frogs is much higher than during spring (pers. obs.). our observations indicate an association between frogs and buffaloes and may have a biological meaning. the observed behaviour was not incidental or loosely structured. frogs foraged on buffaloes in a similar manner as birds on large mammals (heatwole, 1965; dean and macdonald, 1981; yosef and yosef, 1991; sazima, 2011). the food habits of marsh frogs are generalist and the species may change its diet in response to local variation in frequency of available prey items, mainly insects, but sometimes also fish, amphibians, and small mammals (çiçek and mermer, 2007; mollov et al., 2010). because the diet of the species also includes fly species, and many of them are parasites or main disease vectors for large mammals, including buffaloes (bengis et al., 2002; altintas, 2004), the recorded association between frogs and buffaloes can be considered as a possible mutualistic interaction (bascompte, 2009; sazima et al., 2010). mutualistic interactions between large mammals and birds may originate and intensify rapidly under specific local conditions (bradshaw and white, 2006). such situations probably also occurred in the kızılırmak delta, where frogs were present on buffaloes only in autumn, when the density of amphibians is high compared to spring. however, because we did not study the diet of the frogs, we cannot prove that water buffaloes have tangible benefit from the presence of the frogs. hence, the observed relationship can be also interpreted as commensalism (dickman, 1992), where only frogs benefit from the buffaloes without affecting them. the reasons why the associations between frogs and free ranging water buffaloes were not previously reported are unclear. this could be due to frog and buffalo density in one specific area, but also other local factors like density of insects or habitat, like small water bodies constructed by buffaloes during body washing, grazing and excrements (jansen and healey, 2003; hartel and von wehrden, 2013; musitelli et al., 2016). photographs from south asia and europe with frogs resting (not foraging) on buffaloes suggest that the occurrence of frogs on buffaloes may be occasional and accidental and the probability of such observations may depend on frog and buffalo densities. however, our observations where in most cases frogs hunt flies present on buffaloes confirm the existtable 1. basic data about the following observations of frogs on buffaloes carried out in the kızılırmak delta in october 2012. no. observation date air temperature (c°) no. buffaloes no. buffaloes with frogs no. frogs on buffaloes mean no. frogs per buffalo 1 03.10 17 9 7 31 4.4 2 03.10 20 1 1 27 27.0 3 04.10 17 3 2 9 4.5 4 04.10 21 6 6 14 2.3 5 05.10 18 2 2 5 2.5 6 06.10 18 2 2 2 1.0 7 07.10 16 3 2 5 2.5 8 07.10 23 1 1 19 19.0 9 08.10 24 2 2 13 6.5 10 10.10 21 4 3 22 7.3 overall mean ± sd – 19.5 ± 2.7 3.3 ± 2.5 2.8 ± 2.0 14.7 ± 9.9 7.7 ± 8.5 115interaction between frogs and buffaloes ence of an interspecific interaction between amphibians and large mammals. an additional explanation for the observed phenomenon could be the use of buffaloes by frogs as an efficient heat source, which can be important for heterothermic amphibians especially at low ambient temperatures (sinsch, 1984). to the best of our knowledge, these are the first observations of an interaction between marsh frogs and water buffaloes, or even more generally between amphibians and large mammals. however, our data provide the first evidence of such associations. we hope that our note will stimulate further research on this subject. acknowledgments we thank m. kaczmarski, a.p. møller, kerim çiçek, cemal özsemir and nizamettin yavuz for their help in the field, comments and literature search. we also thank two reviewers for the detailed comments on the manuscript. more pictures documenting frogs behaviour, as well as results of internet search can be send on request. references altintas, n. 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(1991): tristram’s grackles groom nubian ibex. wilson bull. 103: 520-522. acta herpetologica vol. 12, n. 1 june 2017 firenze university press morphological variation and sexual dimorphism in liolaemus wiegmannii (duméril & bibron, 1837) (squamata: liolaemidae) from uruguay joaquín villamil1,*, arley camargo2, raúl maneyro1 sex does not affect tail autotomy in lacertid lizards panayiotis pafilis1,*, kostas sagonas2, grigoris kapsalas1, johannes foufopoulos3, efstratios d. valakos4 fire salamander (salamandra salamandra) males’ activity during breeding season: effects of microhabitat features and body size raoul manenti*, andrea conti, roberta pennati call variation and vocalizations of the stealthy litter frog ischnocnema abdita (anura: brachycephalidae) pedro carvalho rocha1,2,*, joão victor a. lacerda2,3, rafael félix de magalhães2,3, clarissa canedo4, bruno v. s. pimenta5, rodrigo carrara heitor6, paulo christiano de anchieta garcia2,3 feeding ecology of two sympatric geckos in an urban area of northeastern brazil josé guilherme g. sousa1, adonias a. martins teixeira2,*, diêgo alves teles2, joão antônio araújo-filho2 and robson waldemar ávila3 a pattern-based tool for long-term, large-sample capture-mark-recapture studies of fire salamanders salamandra species (amphibia: urodela: salamandridae) jeroen speybroeck1,*, koen steenhoudt2,$ skeletal variation within the darwinii group of liolaemus (iguania: liolaemidae): new characters, identification of polymorphisms and new synapomorphies for subclades linda díaz-fernández*, andrés s. quinteros, fernando lobo marking techniques in the marbled newt (triturus marmoratus): pit-tag and tracking device implant protocols hugo le chevalier1, olivier calvez2, albert martínez-silvestre3, damien picard4, sandra guérin4,5, francis isselin-nondedeu6, alexandre ribéron1, audrey trochet1,2,* evidence for directional testes asymmetry in hyla gongshanensis jindongensis qing gui wu1, wen bo liao2,* the advertisement call of pristimantis subsigillatus (anura, craugastoridae) florina stănescu1,4, rafael márquez2, paul székely3,4,*, dan cogălniceanu1,4,5 nematodes infecting anotosaura vanzolinia (squamata: gymnophthalmidae) from caatinga, northeastern brazil bruno halluan s. oliveira¹, adonias a. martins teixeira¹,*, romilda narciza m. queiroz¹, joão a. araujo-filho¹, diêgo a. teles¹, samuel v. brito2, daniel o. mesquita¹ where is my place? quick chorus structure assembly in the european tree frog michal berec a possible mutualistic interaction between vertebrates: frogs use water buffaloes as a foraging place piotr zduniak1,*, kiraz erciyas-yavuz2, piotr tryjanowski3 good vibrations: a novel method for sexing turtles donald t. mcknight1,2,*, hunter j. howell3, ethan c. hollender1, and day b. ligon1 errata to mecke, s., hartmann, l., mader, f., kieckbusch, m., kaiser, h. (2016): redescription of cyrtodactylus fumosus (müller, 1895) (reptilia: squamata: gekkonidae), with a revised identification key to the bent-toed geckos of sulawesi. acta herpetologica 11(2): acta herpetologica 10(1): 1-6, 2015 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-16160 obituary: valery k. eremchenko (1949-2014) leo j. borkin1, tatjana dujsebayeva2, roberto sindaco3, matthias stöck4 1 st. petersburg, russia 2 almaty, kazakhstan 3 turin, italy 4 berlin, germany on 14 april 2014 a leading herpetologist of central asia and a former member of the societas herpetologica italica, valery konstantinovich eremchenko (fig. 1), passed away in bishkek, kyrgyzstan, after several years of severe illness. valery is survived by his second wife anna zemtseva and two daughters: victoria (now in israel) and darya (a schoolgirl, in bishkek). we lost a remarkable expert of the herpetofauna of middle asia, a friend and collaborator. an obituary containing a brief biography was recently published on amphibia-reptilia (amre, borkin et al., 2015). therefore, below are only listed the main steps of the scientific career of v.k. eremchenko. for more details, please read the aforementioned obituary, which appeared in amre. 1949 dr. eremchenko was born on june, 27, in morozovsk, rostov province, u.s.s.r. (now russian federation). 1970 technical assistant at the institute of biology, academy of sciences of the kirghiz soviet socialist republic, frunze (now bishkek). 1977 valery attended the 4th soviet herpetological conference in leningrad, where he was introduced to the herpetological community of the former soviet union. 1977 graduated from the frunze state university with a diploma in biology, including the license to teach biology and chemistry in normal schools. 1979-1981 probationer of research (research stazhor) in the laboratory of vertebrates, the institute of biology, academy of sciences of the kirghiz ssr, frunze. 1980 eremchenko (together with valentina toropova, his first wife) published his first paper in herpetology devoted to reptilian fauna of the tokmak town environs (chu valley); formerly they published several papers in ornithology. 1981 senior laboratory technician, and after several months, junior research fellow, a first permanent research position in the former u.s.s.r. fig. 1. valery konstantinevich eremchenko in 2005 (photograph courtesy françois soulard, traversees). 2 l.j. borkin et alii 1981 postgraduate student at the institute of zoology, ukrainian academy of sciences in kiev, advised by n. n. szczerbak. 1984 eremchenko successfully defended his thesis at the institute of zoology, ukrainian academy of sciences in kiev and received the candidate of science degree, an equivalent to a ph.d. 1985 senior research fellow at the institute of biology, academy of sciences of the kirghiz s.s.r.. 1988 head of the zoological museum at the institute of biology, from then on the leading kirghiz s.s.r. scientific institution in herpetology. 1990 in october eremchenko and some colleagues, including leo borkin, were invited scientists to chengdu, china, by prof. zhao ermi and other chinese herpetologists. it was valery’s first visit outside the former soviet union. 1992 in july valery participated in the first asian herpetological meeting, held in huangshan, china. 2004 head of the exhibition department in the kyrgyzrussian (slavic) university. 2008 valery retired. after the collapse of soviet union (december 1991), unlike other scientists who left the previous soviet republics due to growing nationalistic pressure and economic disaster, valery decided to stay in the now independent kyrgyz republic. in a “first world” country, one can only roughly imagine what it means surviving and performing scientific research in a country ranging in the lowest third of world poverty. during this period, valery struggled for both, the continuation of his scientific work and surviving with his family in a rapidly transforming country that was shaken by several regime changes since. valery continued various herpetological studies in the kyrgyz republic and neighboring parts of kazakhstan, in cooperation with herpetologists from several countries: russia (leo j. borkin), switzerland (notker helfenberger, gaston guex), kazakhstan (tatiana dujsebaeva), italy (emilio balletto, cristina giacoma, roberto sindaco), and germany (matthias. stöck, at the time, berkeley and lausanne), sometimes supported by the russian foundation of basic research and earthwatch institution (usa). from 2005, valery also started working for regional offices of nabu, a german ngo, devoted to nature conservation in central asia, where he got employed under several contracts until 2014. he was a member of the soviet herpetological society (now a.m. nikolsky herpetological society, st.  petersburg), asiatic herpetological society (berkeley, chengdu) and italian herpetological society (turin). according to personal files, kept at the bishkek institute of biology and soil, eremchenko (co-) authored at least 55 full papers, mostly in russian; five of them were devoted to birds of kirghizia. his name eremchenko was transliterated also as jeremčenko (scientific transliteration), jeriomtschenko, and yeriomchenko. his herpetological publications spanned the period from 1977-2011, and primarily covered systematics, distribution, and faunistics of amphibians and reptiles of kyrgyzstan, and other republics of former soviet middle asia. valery described 17 new taxa of reptiles, most of these descriptions and names are still valid (table 1). in honor of valery, a  mountain skink from kyrgyzstan, asymblepharus eremchenkoi panfilov, 1999 has been described. this list of zoological papers was compiled to the best of our ability, but may be incomplete. in the last years, valery published in local magazines that have been virtually inaccessible to us. despite our efforts, it was not possible to obtain further information from kyrgyz institutions or from his former colleagues. 1. toropova, v.i., eremchenko, v.k. (1975): [on winter feeding of long-eared owl asio otus l. in condition of cultural landscape]. izvestiya academii nauk kirgizskoy s.s.r. [=  proc. nat. acad. sci. kyrgyz s.s.r.], frunze 5: 67-68. [in russian]. 2. toropova, v.i., eremchenko, v.k. (1977): [additions to the list of nesteing birds of chu valley]. in: yurlov, k.t. (ed.). [migrations of birds in asia]. nauka press, trudy biologicheskogo institut, sibirskogo otdeleniya akademii nauk sssr [trans. biol. inst., siberian branch, ussr acad. sci.], novosibirsk 33: 222. [in russian]. 3. eremchenko, v.k., szczerbak (“shcherbak”), n.n. (1980): [about generic allocation of ablepharid lizards (reptilia, sauria, scincidae) of fauna of the u.s.s.r.]. vestnik zoologii [bull. zool.], kiev 4: 10-15. [in russian, with english summary]. 4. eremchenko, v.k., szczerbak (“shcherbak”), n.n. (1980): [a new species of lidless lizard ablepharus lindbergi sp. n. (reptilia, sauria, scincidae) from afghanistan]. vestnik zoologii [bull. zool.], kiev 6: 84-86. [in russian, with english summary]. 5. toropova, v.i., eremchenko, v.k. (1980): [on the breeding of the common stonechat (saxicola torquata l.) in kirgiziya]. problemi bioecologii zhivotnikh i rasteniy i okhrana okruzhaushchei sredi [the problems of animal and plant biology and environment protection.]. sbornik dokladov molodikh uchenikh instituta biologii academii nauk kirgizskoi s.s.r. [= collected reports of young scientists inst. biol. acad. sci. kyrgyz s.s.r.] ilim press, frunze: 47-48. [in russian]. 6. toropova, v.i., eremchenko, v.k. (1980): [migrations of birds in kar-kara (kirghizia)]. in: abdusalyamov, 3obituary i.a. (ed.), [migrations of birds in asia]. dushanbe: donish press: 119-125. [in russian]. 7. toropova, v.i., eremchenko, v.k. (1980): [species composition and number of reptile fauna in the environs of tokmar town]. in: tokobaev, m.m., kozhevnikova, n.d. (eds.), [geography of plant and animal life of kirghizia]. izvestiya kirghizskogo geographicheskogo obshchestva [= proc. kyrgyz geogr. soc.] 14: 94-96. [in russian]. 8. eremchenko, v.k., shukurov e.d. (1981): the finding of the turkestan agama agama lehmanni (nik.) in kirghizia. izvestiya academii nauk kirgizskoy s.s.r. [= proc. nat. acad. sci. kyrgyz s.s.r.], frunze 2: 65. [in russian]. table 1. reptile taxa described by v.k. eremchenko original name authors journal present name / status family gekkonidae alsophylax (altiphylax) tokobajevi n. sp. jeriomtschenko and szczerbak, 1984 vestnik zoologii, kiev, 2: 47-50 altiphylax tokobajevi (eremchenko & szczerbak, 1984) altiphylax n. subgen. jeriomtschenko and szczerbak, 1984 vestnik zoologii, kiev, 2: 47 accepted genus cyrtopodion narynensis n. sp. eremchenko, tsarinenko and panfilov, 1999 izvestiya natsionalnoy academii nauk kyrgyzskoy respubliki, bishkek, 3-4: 48-55 mediodactylus narynensis (eremchenko, tsarinenko & panfilov, 1999) family lacertidae dimorphea n. subgen. eremchenko, 1999 izvestiya natsionalnoy academii nauk kyrgyzskoy respubliki, bishkek, 1: 73 accepted subgenus eremias kokshaaliensis n. sp. eremchenko and panfilov, 1999 nauka i novye tekhnologii, bishkek, 4: 118-120. accepted species eremias multiocellata szczerbaki n. ssp. eremchenko [“jeriomtschenko”] and panfilov, 1992 konspect issledovanii po tsitogenetike i sistematike nekotorik asiatskik vidov (scincidae i lacertidae), ilim press, bishkek: 69-71 eremias szczerbaki eremchenko and panfilov, 1992 eremias velox borkini n. subsp. eremchenko and panfilov, 1999 nauka i novye tekhnologii, (1): 122-123 accepted subspecies family scincidae ablepharus darvazi n. sp. jeriomtschenko et panfilov, 1990 izvestiya academii nauk kirghizskoi s.s.r, khimiko-tekhnologicheskie i biologicheskie nauki, 4: 56-63 accepted species asymblepharus n. gen. eremchenko et szczerbak (“shcherbak”), 1980 vestnik zoologii, kiev, 4: 10-15. accepted genus asymblepharus alaicus yakovlevae n. ssp. eremchenko, 1983 vestnik zoologii, kiev 1983 (2): 35-42. accepted subspecies asymblepharus ladacensis stimsoni n. ssp. eremchenko [“jeriomtschenko”], 1992 konspect issledovanii po tsitogenetike i sistematike nekotorik asiatskik vidov (scincidae i lacertidae), ilim press, bishkek: 58-65 accepted subspecies asymblepharus mahabharatus n. sp. jeremčenko, shah and panfilov, 1998 izvestiya natsionalnoy academii nauk kyrgyzskoy respubliki, bishkek, 4: 42-43. accepted species asymblepharus nepalensis n. sp. eremchenko and helfenberger, 1998 izvestiya natsionalnoy academii nauk kyrgyzskoy respubliki, bishkek, 4: 41-45 accepted species himalblepharus n. subgen. eremchenko [“jeriomtschenko”], 1987 izvestiya academii nauk kirgizskoy s.s.r., frunze, khimiko tekhnologicheskie nauki, 2: 54-57. accepted subgenus of asymblepharus kaestlea n. gen. eremchenko and das, 2004 hamadryad, 28 (1-2): 43-50. accepted genus livorimica n. gen. eremchenko, 2003 izvestiya vuzov, bishkek, 1-2: 20-28. currently considered a synonym of sphenomorphus (nguyeng et al. 2011) livorimica bacboensis n. sp. eremchenko, 2003 izvestiya vuzov, bishkek, 1-2: 20-28. sphenomorphus bacboensis (eremchenko, 2003) 4 l.j. borkin et alii 9. eremchenko, v.k. (1983): [distribution and geographical variability of asymblepharus alaicus (sauria, scincidae)]. vestnik zoologii [bull. zool.], kiev 2: 35-42. [in russian, with english summary]. 10. eremchenko, v.k., szczerbak n.n. (1984): [a new gecko species (reptilia, gekkonidae) from tien shan]. vestnik zoologii [bull. zool.], kiev 2: 46-50 [in russian, with english summary]. 11. eremchenko, v.k., shurukov, e.f., szczerbak, n.n. (1985): [kashgar racerunner, eremias buechneri a new species for the u.s.s.r. fauna.]. vestnik zoologii [bull. zool.], kiev 4: 79-80. [in russian]. 12. eremchenko, v.k., szczerbak n.n. (1985): [distribution and ecology of the gecko, alsophylax tokobajevi jeriomtschenko et szczerbak, 1984 (reptilia, gekkonidae)]. izvestiya academii nauk kirgizskoy s.s.r. [= proc. nat. acad. sci. kyrgyz s.s.r.], frunze 3: 60-63. [in russian]. 13. eremchenko, v.k., szczerbak n.n. (1986): [ablepharine lizards of the fauna of the u.s.s.r. and neighboring countries]. ilim press, frunze. [in russian]. 14. eremchenko, v.k. (1987): [systematics and relationships of indo-himalayan scincells (sauria: scincidae)]. izvestiya academii nauk kirgizskoy s.s.r., khimiko-tekhnologicheskie nauki [= proc. nat. acad. sci. kyrgyz s.s.r., chem.-tech. sciences], frunze, 2: 54-57. [in russian]. 15. szczerbak, n.n., eremchenko, v.k. (1987): [new findings of reptiles on the territory of the u.s.s.r.]. vestnik zoologii [bull. zool.], kiev 5: 85. [in russian]. 16. eremchenko, v.k. (1988): [amphibians and reptiles of kirghizia]. izvestiya academii nauk kirgizskoy s.s.r. khimiko-tekhnologicheskie [= proc. nat. acad. sci. kyrgyz s.s.r., chem.-tech. sciences], frunze, (1987) 4: 26-30. [in russian] 17. eremchenko, v.k. (1989): [scheme of treatment of collection specimens of reptiles. 1b. skinks of the genera ablepharus asymblepharus]. in: szczerbak n.n (ed.). rukovodstvo po izucheniyu zemnovodnykh i presmykayushchikhsya [handbook to the study of amphibians and reptiles]. kiev: i.i. schmalhausen institute of zoology, academy of sciences, ukrainian s.s.r.: 30-31. [in russian]. 18. eremchenko, v.k., panfilov, a.m. (1989): [karyologycal study of asymblepharus alaicus (elpat., 1901); sauria, scincidae]. izvestiya academii nauk kirgizskoy s.s.r., khimiko-tekhnologicheskie nauki [= proc. nat. acad. sci. kyrgyz s.s.r., chem.-tech. sciences] (1988) 4: 88-92. [in russian]. 19. tokobaev, m.[m.], toropova, v.[i.], eremchenko v.[k.] (1989): movement to the harmony. in: priroda i chelovek: 1989 god [nature and man: 1989]. kyrgyzstan press, frunze: 17-21. [in russian]. 20. eremchenko, v.k., panfilov, a.m. (1990): [ablepharus darvazi sp. nov. a new species of the lidless skink (sauria, scincidae) from tadjikistan.]. izvestiya academii nauk kirghizskoi s.s.r, khimiko-tekhnologicheskie i biologicheskie nauki 4: 56-63. [in russian]. 21. eremchenko, v.k. (1992): [on systematics of asymblepharus ladacensis (gunther, [sic!] 1864), (sauria: scincidae, lygosominae), pp. 58-65. in: eremchenko, v.k., panfilov, a.m, tsarinenko, e.i. (eds.), [research conspectus on cytogenetics and systematics of some asian species of the scincidae and lacertidae]. ilim press, bishkek. [in russian]. 22. eremchenko, v.k., panfilov, a.m., tsarinenko e.i. (1992): [eremias multiocellata-complex: solution of some disputable problems of systematics of multiocellated racerunners of kirghizia (sauria, lacertidae, eremias), pp.  65-80. in: eremchenko, v.k., panfilov, a.m, tsarinenko, e.i. (eds.), [research conspectus on cytogenetics and systematics of some asian species of the scincidae and lacertidae]. ilim press, bishkek. [in russian]. 23. eremchenko, v.k., panfilov, a.m., tsarinenko, e.i. (1992): [catalogue of the collection of amphibians and reptiles of the zoological museum of the institute of biology of the academy of sciences of the republics of kyrgyzstan], pp. 91-176. in: eremchenko, v.k., panfilov, a.m, tsarinenko, e.i. (eds.), [research conspectus on cytogenetics and systematics of some asian species of the scincidae and lacertidae]. ilim press, bishkek. [in russian]. 24. panfilov, a.m., eremchenko, v.k. (1992). [multiple nors in reptiles], pp. 5-57. in: eremchenko, v.k., panfilov, a.m, tsarinenko, e.i. (eds.), [research conspectus on cytogenetics and systematics of some asian species of the scincidae and lacertidae]. ilim press, bishkek. [in russian]. 25. tsarinenko, e.i., eremchenko, v.k. (1992): [variation in the kirghiz racerunner eremias nicolskii [sic!] bedriaga, 1905 (sauria, lacertidae), pp.  80-90. in: eremchenko, v.k., panfilov, a.m, tsarinenko, e.i. (eds.), [research conspectus on cytogenetics and systematics of some asian species of the scincidae and lacertidae]. ilim press, bishkek. [in russian]. 26. eremchenko, v.k., helfenberger, n., shah, k., panfilov, a.m. (1998): [two new species of skinks (scincidae: ligosominae sic!) from nepal]. izvestiya natsionalnoy academii nauk kyrgyzskoy respubliki [= proc. nat. acad. sci. kyrgyz republic], bishkek 4: 41-45. [in russian] 5obituary 27. eremchenko, v.k., panfilov, a.m., kasiev, ?, portnyagina, v.i. (1998): [what frightens experts of the union of scientists of kyrgyz.republic]. bulleten obshchestvennogo tsentra ekologicheskoi informatsii [bull. pub. cent. ecol. inform.], bishkek 8: 3-5. 28. eremchenko v.k. (1999): [nomenklatura aziatskikh jašcurok eremias wiegmann, 1834 v svjazi s proceduroj posledujušcego oboznacenija monotipii]. izvestija natsional’noj academii nauk kyrghyzskoj respubliki [= proc. nat. acad. sci. kyrgyz republic], bishkek 1: 72-73. 29. eremchenko, v.k. (1999): [nomenclature of asian racerunners eremias wiegmann, 1834 in connection with procedure of subsequent designation of monotypy (sauria: lacertidae)]. izvestiya natsionalnoy academii nauk kyrgyzskoy respubliki [= proc. nat. acad. sci. kyrgyz republic], bishkek 1: 72-73. [in russian]. 30. eremchenko. v.[k.], panfilov, a[m.] (1999): [taxonomic position and geographic relations of a lacertid lizard eremias velox from the issyk-kul lake depression, tien shan mountains, kyrgyzstan]. nauka i novye tekhnologii [science and new technologies], bishkek, special issue 1, pp. 119-125. 31. eremchenko, v.k., panfilov, a.m. (1999): [taxonomic position and biogeographic connections of alsophylax loricatus strauch, 1887 (reptilia: gekkonidae)]. nauka i novye tekhnologii [science and new technologies], bishkek 2: 182-183. [in russian, with very short english summary]. 32. eremchenko, v.k., panfilov, a.m. (1999): [some methodological problems of taxonomy and phylogeny of toad agamas on the example of phrynocephalus helioscopus (pallas, 1771) (sauria: agamidae)]. nauka i novye tekhnologii [science and new technologies], bishkek 3: 116-122 [in russian, with very short english summary]. 33. eremchenko, v.k., panfilov a.m. (1999): [taxonomic position of multiocellated racerunners of the eremias multiocellata complex of kyrghyzstan and neighbour china (sauria: lacertidae: eremias)]. nauka i novye tekhnologii [= science and new technologies], bishkek 4: 112-124. [in russian, with very short english summary]. 34. eremchenko, v.k., panfilov, a.m., borkin, l.j., helfenberger, n. (1999): [new data on the distribution of ablepharus deserti strauch, 1868 in kazakhstan and kyrghyzstan, with notes on ecology and cytogenetics]. izvestiya natsionalnoy academii nauk kyrgyzskoy respubliki [= proc. nat. acad. sci. kyrgyz republic], bishkek 4: 106-109. [in russian]. 35. eremchenko, v.k., tsarinenko, e.i., panfilov, a.m. (1999): [intraspecific relations in geckos cyrtopodion russowii (strauch) and taxonomic position of tien shan population]. izvestiya natsionalnoy academii nauk kyrgyzskoy respubliki [= proc. nat. acad. sci. kyrgyz republic], bishkek 3-4: 48-55. [in russian]. 36. panfilov, a.m., eremchenko, v.k. (1999): [new data on karyology of six species of skinks (sauria: scincidae) of eurasia]. science and new technologies, bishkek 2: 61-67. [in russian]. 37. panfilov, a.m., eremchenko, v.k. 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(2008): the reptiles of the western palearctic. 1. annotated checklist and distributional atlas of the turtles, crocodiles, amphisbaenians and lizards of europe, north africa, middle east and central asia. monografie della societas herpetologica italica i edizioni belvedere, latina (italy). 51. eremchenko, v.k. (2009): [the first record of autumn mating in the middle asian tortoise (testudo horsfieldii)]. selevinia (kazakhstansky zoologichesky zhurnal) [= the zool. j. of kazakhstan], almaty (2008): 253. [in russian]. 52. eremchenko, v.k., chirikova, m.a. (2009): intraspecies predation in reptiles and the first record of cannibalism in the steppe-runner eremias arguta in kirghizia. selevinia (kazakhstansky zoologichesky zhurnal) [= the zool. j. of kazakhstan], almaty (2008): 253-254. [in russian] 53. fritz, u., auer, m., chirikova, m.a., duysebayeva, t.n., eremchenko, v.k., kami, h.g., kashkarov, r.d., masroor, r., moodley, y., pindrani, a., široký, p., hundsdörfer, a. (2009): mitochondrial diversity of the widespread central asian steppe tortoise (testudo horsfieldii gray, 1844): implications for taxonomy and relocation of confiscated tortoises. amphibia-reptilia 30: 245-257. 54. eremchenko, v.k. (2010): [truth and lie about our lesser brothers. review of the “red book of kyrgyz republic”. second edition. bishkek, 2007]. selevinia (kazakhstansky zoologichesky zhurnal) [= the zool. j. of kazakhstan], almaty (2009): 287-295. [in russian]. 55. harder, t., kustareva. l., eremchenko, v., toropova, v., kulagin, s., sagymbaev, s. (2011): wildlife of kyrgyzstan (species checklist in four languages; also available at: http://www.wildlife.kg/indexe.html). published by nabu kyrgyzstan. 56. eremchenko, v.k. (in press): skinks of kashmir: the genus asymblepharus eremchenko & sczcerbak, 1980 (sauria: scincidae: lygosominae). bulletin kyrghyz-russian slavic university, bishkek. references borkin, l.j., dujsebayeva, t., sindaco, r., stöck, m. (2015): obituary: valery k. eremchenko (1949-2014). amphibia-reptilia 36: 1-4. nguyen, t.q., schmitz, a., nguyen, t.t., orlov, n.l., böhme, w., ziegler, t. (2011): review of the genus sphenomorphus fitzinger, 1843 (squamata: sauria: scincidae) in vietnam, with description of a new species from northern vietnam and southern china and the first record of sphenomorphus mimicus taylor, 1962 from vietnam. j. herpetol. 45: 145-154. acta herpetologica vol. 10, n. 1 june 2015 firenze university press obituary: valery k. eremchenko (1949-2014) leo j. borkin1, tatjana dujsebayeva2, roberto sindaco3, matthias stöck4 haplotype variation in founders of the mauremys annamensis population kept in european zoos barbora somerová1, ivan rehák2,*, petr velenský2, klára palupčíková1, tomáš protiva1, daniel frynta1 reproductive ecology of sichuan digging frogs (microhylidae: kaloula rugifera) wei chen1,*, lina ren2, dujuan he2, ying wang2, david pike3 toxic effects of carbaryl on the histology of testes of bufotes variabilis (anura: bufonidae) özlem çakici basal frequency of micronuclei and hematological parameters in the side-necked turtle, phrynops hilarii (duméril & bibron, 1835) maría a. latorre 1,2,*,#; evelyn c. lópez gonzález1,2, #; pablo a. siroski1,2,3; gisela l. poletta1,2,4 into a box interiors: clutch size variation and resource allocation in the european pond turtle marco. a.l. zuffi1,*, simonetta citi2, elena foschi1, francesca marsiglia1, eva martelli1 where to “rock”? choice of retreat sites by a gecko in a semi-arid habitat andreia penado1,2, ricardo rocha3,4,*, marta sampaio3, vanessa gil3, bruno m. carreira3, rui rebelo3 age structure, growth and longevity in the common toad, rhinella arenarum, from argentina clarisa de l. bionda1,2,*, silvia kost 4, nancy e. salas1, rafael c. lajmanovich3, ulrich sinsch4, adolfo l. martino1 on a putative type specimen of pleurodema bibroni tschudi, 1838 from chile (anura: leptodactylidae) daiana paola ferraro re-description of the external morphology of phyllomedusa iheringii boulenger, 1885 larvae (anura: hylidae), with comments on the external morphology of tadpoles of the p. burmeisteri group samanta iop¹, victor mendes lipinski¹, bruno madalozzo¹, franciele pereira maragno¹, sonia zanini cechin¹, tiago gomes dos santos² book review: harold heatwole, john w. wilkinson (eds). amphibians biology. volume 11 status of conservation and decline of amphibians. eastern hemisphere. part 4 . southern europe and turkey sebastiano salvidio book review: antonio romano. atlante degli anfibi del parco nazionale del cilento vallo di diano e alburni distribuzione, biologia, ecologia e conservazione sebastiano salvidio acta herpetologica journal of the societas herpetologica italica acta herpetologica 9(2): 249-252, 2014 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-14562 no evidence for conspecificity between two high andes liolaemus lizards (squamata: liolaemidae) daniel pincheira-donoso laboratory of evolutionary ecology of adaptations, school of life sciences, university of lincoln, brayford campus, lincoln, ln6 7dl, lincolnshire, united kingdom. e-mail: dpincheiradonoso@lincoln.ac.uk submitted on 2014, 26th may; revised on 2014, 10th september; accepted on 2014, 11th october editor: aaron m. bauer abstract. the remarkable taxonomic, ecological and geographic diversity achieved by the south american lizard genus liolaemus has inspired persistent debate about species boundaries and the reliability of phenotypic predictors/ indicators of reproductive isolation between species (i.e., signatures of speciation). many aspects of these debates remain unsettled and part of the diversity of the genus remains under controversy. factors such as small samples, or lack of molecular data to quantify genetic differences between species can be regarded as legitimate limitations on the ability to draw definite taxonomic conclusions. however, conclusions drawn from careless and negligent observations should be taken with high degree of caution. a recent paper offers a clear example of this latter scenario, in which it is suggested that liolaemus filiorum pincheira-donoso and ramirez, 2005 is a synonym of liolaemus puritamensis nuñez and fox, 1989 based on qualitative analyses of the “holotype” of the former which is, in fact, not the holotype of this taxon, but rather one of the paratypes of the latter species. editors and referees should play a central role in preventing publication of studies of this nature. keywords. liolaemus, speciation, synonymy, taxonomy, integrative taxonomy. the iguanian genus liolaemus is one of the most species-rich groups of living vertebrates (pincheira-donoso et al., 2013a). the prolific adaptive radiation undergone by this lineage has resulted in 240+ species adapted to a remarkable diversity of ecological conditions, encompassing all climatic extremes found in central and southern south america, including deserts, tropical environments, and some of the highest elevations/latitudes recorded among reptiles globally (morando et al., 2003; espinoza et al., 2004; schulte et al., 2004; pincheira-donoso et al., 2008, 2013b; labra et al., 2009; pincheira-donoso, 2011; pincheira-donoso and tregenza, 2011; meiri et al., 2013). not surprisingly, the causes and consequences, and the genetic and phenotypic expressions behind such diversity have been the focus of persistent debate and controversy (morando et al., 2003, 2004; pincheira-donoso and nuñez, 2005; avila et al., 2006; labra, 2011; pincheira-donoso, 2012). opinions remain divergent, and debates remain unsettled. in recent years, the development of the ‘integrative taxonomy’ approach has made explicit the need for taxonomic conclusions to be drawn from quantitative and multivariate analyses of the various components involved in the process of species formations, i.e., the search for signatures of speciation (padial et al., 2009, 2010). this approach adds scientific objectivity to taxonomic conclusions, and has in fact increasingly been adopted by liolaemid researchers (avila et al., 2006; morando et al., 2007; pincheira-donoso et al., 2007; breitman et al., 2011a, b; morando et al., 2013; scolaro et al., 2013). however, a recent study (troncoso-palacio, 2014) that proposed a conclusion with major implications (conspecificity between two species) not only employs a purely typological and qualitative analytical approach, but is based on a fundamental error. in this study, troncoso-palacio (2014) concludes that liolaemus puritamensis nuñez and fox, 1989 and l. filiorum pincheira-donoso and ramirez, 2005 are the same species (given the temporal priority of 250 daniel pincheira-donoso l. puritamensis, the latter taxon is treated as a subjective junior synonym of the former). this conclusion is based on comparisons between three specimens, two l. puritamensis and (as claimed by the author) the “holotype” of l. filiorum. although the lack of a quantitative analytical approach and the extremely limited sample sizes may be viewed as inappropriate to support the hypothesis of conspecificity between these taxa, the very basic flaw that invalidates the conclusions of troncoso-palacio (2014) is that this “holotype” is not the holotype of l. filiorum but, in fact, one of the paratypes of l. puritamensis itself (fig. 1). this specimen was recently transferred to the museo nacional de historia natural de chile collection from another collection and erroneously given the same number, mnhn-3829, as the l. filiorum holotype, although the data accompanying the specimen clearly identify it as a paratype of l. puritamensis. by focusing on the museum collection number of the specimen rather than on details of locality, date and collectors provided in the original description of l. filiorum, troncoso-palacio compared specimens in the same type series and not surprisingly concluded that these specimens were conspecific. thus, no evidence exists to conclude that these two liolaemus are the same species. here, i provide images of the real holofig. 1. liolaemus from the high andes of chile. liolaemus filiorum: dorsal views of the holotype (a), paratype (b), and two living adult male specimens (c, d) from the type locality, cerro las papas, chile. liolaemus puritamensis: a living specimen of from vilaco, chile (e). 251no conspecificity in liolaemus type (mnhn-3829) and paratype (chdpd-01069) of l. filiorum, and pictures of both species in life (fig. 1). taxonomy has seen a tendency to be treated as a marginal discipline, especially in high-impact scientific journals, and has been considered the ‘cinderella’ of biology (e.g., padial and de la riva, 2007). while this view disregards the overwhelmingly vital role of taxonomic research as the basis for understanding biodiversity, and thus for areas such as ecology and evolution (see isaac et al., 2004, for a discussion), it is essential that taxonomists conduct this science with due care and scientific rigor. taxonomy is a science and, as such, it depends on the use of scientific approaches to thoroughly and quantitatively test hypotheses (e.g., hypotheses of speciation events as the basis for recognizing different species, or evidence that the genomes of two species fail to show signals of speciation events, which can include phenotypic or genetic signals). in this context, it is crucial for editors, referees and colleagues to identify studies that do not employ such standards before they are approved for publication. acknowledgements the author thanks an anonymous referee for constructive comments on an earlier version of this manuscript. also, thanks to the financial support provided by a university of lincoln research investment fund grant (rif). references avila, l.j., morando, m., sites, j.w. (2006): congeneric phylogeography: hypothesizing species limits and evolutionary processes in patagonian lizards of the liolaemus boulengeri group (squamata : liolaemini). biol. j. linnean soc. 89: 241-275. breitman, m.f., avila, l.j., sites, j.w., morando, m. (2011a): lizards from the end of the world: phylogenetic relationships of the liolaemus lineomaculatus section (squamata: iguania: liolaemini). mol. phylogenet. evol. 59: 364-376. breitman, m.f., perez, c.h.f., parra, m., morando, m., sites, j.w., avila, l.j. (2011b): new species of lizard from the magellanicus clade of the liolaemus lineomaculatus section (squamata: iguania: liolaemidae) from southern patagonia. zootaxa 3123: 32-48. espinoza, r.e., wiens, j.j., tracy, c.r. (2004): recurrent evolution of herbivory in small, cold-climate lizards: breaking the ecophysiological rules of reptilian herbivory. proc. natl. acad. sci. usa 101: 16819-16824. isaac, n.j.b., mallet, j., mace, g.m. (2004): taxonomic inflation: its influence on macroecology and conservation. trends ecol. evol. 19: 464-469. labra, a. (2011): chemical stimuli and species recognition in liolaemus lizards. j. zool. 285: 215-221. labra, a., pienaar, j., hansen, t.f. (2009): evolution of thermal physiology in liolaemus lizards: adaptation, phylogenetic inertia, and niche tracking. am. nat. 174: 204-220. meiri, s., bauer, a.m., chirio, l., colli, g.r., das, i., doan, t.m., feldman, a., castro-herrera, f., novosolov, m., pafilis, p., pincheira-donoso, d., powney, g., torres-carvajal, o., uetz, p., van damme, r. (2013): are lizards feeling the heat? a tale of ecology and evolution under two temperatures. global ecol. biogeogr. 22: 834-845. morando, m., avila, l.j., sites, j.w. (2003): sampling strategies for delimiting species: genes, individuals, and populations in the liolaemus elongatus-kriegi complex (squamata: liolaemidae) in andean-patagonian south america. syst. biol. 52: 159-185. morando, m., avila, l.j., baker, j., sites, j.w. (2004): phylogeny and phylogeography of the liolaemus darwinii complex (squamata: liolaemidae): evidence for introgression and incomplete lineage sorting. evolution 58: 842-861. morando, m., avila, l.j., turner, c.r., sites, j.w. (2007): molecular evidence for a species complex in the patagonian lizard liolaemus bibronii and phylogeography of the closely related liolaemus gracilis (squamata: liolaemini). mol. phylogenet. evol. 43: 952-973. morando, m., avila, l.j., perez, c.h.f., hawkins, m.a., sites, j.w. (2013): a molecular phylogeny of the lizard genus phymaturus (squamata, liolaemini): implications for species diversity and historical biogeography of southern south america. mol. phylogenet. evol. 66: 694-714. nuñez, h., fox, s.f. (1989): liolaemus puritamensis, a new species of iguanid lizard previously confused with liolaemus multiformis (squamata: iguanidae). copeia 1989: 456-460. padial, j.m., de la riva, i. (2007): taxonomy, the cinderella of science, hidden by its evolutionary stepsister. zootaxa 1577: 1-2. padial, j.m., miralles, a., de la riva, i., vences, m. (2010): the integrative future of taxonomy. front. zool. 7: 16. padial, j.m., castroviejo-fisher, s., kohler, j., vila, c., chaparro, j.c., de la riva, i. (2009): deciphering the products of evolution at the species level: the need for an integrative taxonomy. zool. scripta 38: 431-447. pincheira-donoso, d. (2011): predictable variation of range-sizes across an extreme environmental gradient 252 daniel pincheira-donoso in a lizard adaptive radiation: evolutionary and ecological inferences. plos one 6: e28942. pincheira-donoso, d. (2012): cautionary comments on the influence of chemical-based interactions as potential drivers of sexual speciation in liolaemus lizards. j. zool. 288: 231-233. pincheira-donoso, d., nuñez, h. (2005): las especies chilenas del género liolaemus. taxonomía, sistemática y evolución. publ. ocas. mus. nac. hist. nat. chile, 59: 1-487. pincheira-donoso, d., ramirez, g.m. (2005): desplazamiento de caracteres como evidencias de un posible caso de especiación simpátrica entre dos liolaemus del grupo jamesi en la provincia de el loa, con la descripción de una nueva especie. in: fauna del altiplano y desierto de atacama. vertebrados de la provincia de el loa, pp. 350-365. ramírez, g.m., pincheira-donoso, d., eds, phrynosaura ediciones, calama, chile. pincheira-donoso, d., tregenza, t. (2011): fecundity selection and the evolution of reproductive output and sex-specific body size in the liolaemus lizard adaptive radiation. evol. biol. 38: 197-207. pincheira-donoso, d., scolaro, j.a., schulte, j.a. (2007): the limits of polymorphism in liolaemus rothi: molecular and phenotypic evidence for a new species of the liolaemus boulengeri clade (iguanidae, liolaemini) from boreal patagonia of chile. zootaxa 1452: 25-42. pincheira-donoso, d., hodgson, d.j., tregenza, t. (2008): the evolution of body size under environmental gradients in ectotherms: why should bergmann’s rule apply to lizards? bmc evol. biol. 8: 68. pincheira-donoso, d., bauer, a.m., meiri, s., uetz, p. (2013a): global taxonomic diversity of living reptiles. plos one 8: e59741. pincheira-donoso, d., tregenza, t., witt, m.j., hodgson, d.j. (2013b): the evolution of viviparity opens opportunities for lizard radiation but drives it into a climatic cul-de-sac. global ecol. biogeogr. 22: 857-867. schulte, j.a., losos, j.b., cruz, f.b., nuñez, h. (2004): the relationship between morphology, escape behaviour and microhabitat occupation in the lizard clade liolaemus (iguanidae: tropidurinae: liolaemini). j. evol. biol. 17: 408-420. scolaro, j.a., jara, m., pincheira-donoso, d. (2013): the sexual signals of speciation? a new sexually dimorphic phymaturus species of the patagonicus clade from patagonia argentina. zootaxa 3722: 317-332. troncoso-palacio, j. (2014): revisión del estatus taxonómico de liolaemus filiorum pincheira-donoso y ramírez, 2005 (iguania: liolaemidae). cuad. herpetol. 28: 1-7. acta herpetologica 12(2): 209-214, 2017 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-20283 who are you? the genetic identity of some insular populations of hierophis viridiflavus s.l. from the tyrrhenian sea ignazio avella, riccardo castiglia, gabriele senczuk* dipartimento di biologia e biotecnologie “charles darwin”, università di roma “la sapienza”, 00151, roma, italy. *corresponding author. e-mail: gabriele.senczuk@uniroma1.it submitted on: 2017, 3rd february; revised on: 2017, 13th april; accepted on: 2017, 6th june editor: adriana bellati abstract. this work investigates the genetic identity of hierophis viridiflavus s.l. specimens from insular populations, to determine which of the two previously identified species is present on each island. here, the authors hypothesise about times and modes of colonization and discuss the faunistic value of the obtained results. this follows the recent proposal to consider the two clades as two different species. specimens from the islands of favignana, lipari and vulcano belong to h. carbonarius and probably all belong to putative sicilian source populations. conversely, all individuals from the pontine islands (ponza, palmarola, ventotene) should be considered to belong to h. viridiflavus. even if genetically identical to the specimens from the tyrrhenian italian coast, these individuals show a darker colouration, very similar to the one usually shown by h. carbonarius specimens. considering that the pontine h. viridiflavus populations probably have a very recent origin, the dark livery of these individuals could be the result of a rapid morphological adaptation to insular environments. keywords. colour pattern, hierophis viridiflavus, islands, nd4, phylogeography. the western whip snake hierophis viridiflavus s.l. (lacépède, 1789) is a colubrid snake with a wide distribution range. it can be found in central europe from eastern spain to central france, luxemburg, switzerland, slovenia and croatia. its range also includes all of the italian peninsula, sicily, sardinia and most of the smaller italian islands, corsica and some croatian islands (vanni and nistri, 2006; zuffi, 2007). the species is found from sea level to 1500-1800 m a.s.l., although it is extremely rare above 1500 m in the alps (farinello and bonato, 2000). interestingly, individuals of this species show two main phenotypes, one named “viridiflavus” (usually brown/blackish with yellow stripes and spots) and the other named “carbonarius”, typically melanic (completely black with blackish/grey ventral colouration) or melanotic (almost completely black livery, but paler/ yellowish head scales and ventral surface). individuals from some of the islands of the tuscan archipelago, sardinia and corsica show a third phenotype, a middle ground between “carbonarius” and “viridiflavus”, called “abundistic” (zuffi, 2008). in the past, four h. viridiflavus s.l. subspecies have been described for italy, mainly due to the chromatic pattern of the analysed individuals: h. v. viridiflavus (rimpp, 1979), h. v. carbonarius (bonaparte, 1833), h. v. kratzeri (kramer, 1971) and h. v. antoniimanueli (capolongo, 1984). however, based on mitochondrial and nuclear dna evidences, they have all been recently rejected (vanni and zuffi, 2011). indeed, nagy et al. (2002) and rato et al. (2009) showed the presence of two mitochondrial distinct haplogroups: the first, roughly corresponding to the subspecies h. v. viridiflavus (clade w) includes individuals from spain, france, corsica, sardinia and central to north-western italy on the west side of the apennines; the second, in part matching with the subspecies h. v. carbonarius (clade e), occurs on the other side of the apennines, from north-eastern 210 ignazio avella et alii italy to southern italy (including sicily). more recently, a study based on morphometric, genetic and karyological data, proposed the elevation of the two genetic groups to species status (mezzasalma et al., 2015). in particular, the authors emphasized significant differences in sexual chromosomes, as while females from the eastern group have a submetacentric w sex chromosome, females from the western group have a telocentric w sex chromosome. thus, individuals from the eastern clade have been recognized as hierophis carbonarius, while individuals from the western clade have been recognized as hierophis viridiflavus. interestingly, the relationship between colour variation and genetic repartition does not match completely (zuffi, 2008). the brown/blackish colouration with yellow stripes and spots pattern, generally almost exclusive of the h. viridiflavus range, can be found also in h. carbonarius specimens (rato et al., 2009). although the distribution of the two species in the italian peninsula and on the largest mediterranean islands (sardinia, corsica and sicily) has already been studied (nagy et al., 2002; rato et al., 2009; mezzasalma et al., 2015), there is a lack of molecular data from smaller italian archipelagos. the aim of the present work is to determine the genetic identity of individuals collected in some tyrrhenian islands including: ponza, palmarola and ventotene from the pontine archipelago; vulcano and lipari from the aeolian islands; and favignana from the aegadian islands, in order to update the distribution of the two species of whip snakes. we sampled a total of seven individuals of h. viridiflavus s.l. from six different islands between march 2014 and july 2015 (geographic locations are reported in table 1 and showed in fig. 1). snakes were caught and handled following standard protocols (fowler, 1978), and some ventral scales were removed and preserved in pure ethanol. in one case (rs296 from lipari), the tissue was obtained from a shedded skin. genomic dna was extracted following the protocol described in aljanabi and martinez (1997). a fragment including the terminal portion of the nadh dehydrogenase subunit 4 (nd4) was amplified by standard pcr protocols using primers published by arèvalo et al. (1994). amplification conditions were the same as described by pinho et al. (2006). the pcr products were purified with a sure clean (bioline©) purification kit and the sequencing reactions were run under big-dye tm terminator cycling conditions by a commercial company, macrogen (www.macrogen.com). the electropherograms were checked using the software finchtv (http://www.geospiza.com/finchtv/) to ensure the absence of double peaks and ambiguous positions. the obtained sequences were deposited to genbank (accession numbers: ky923281ky923287) and joined with additional 91 nd4 sequences of hierophis viridiflavus s.l. retrieved from genbank (accession numbers: fj430621-fj430660, rato et al., 2009; ln552045-ln552095, mezzasalma et al., 2015). nucleotide sequences were translated into amino acids with mega 6.0 (tamura et al., 2013) using the vertebrate mitochondrial genetic code in order to assess the absence of pseudogenes. one nd4 sequence of hierophis gemonensis (laurenti, 1768) was downloaded from genbank (accession number: ay487044, nagy et al., 2004) and included in the analysis as outgroup, as it is considered the closest related species to h. viridiflavus (schätti, 1988). the software jmodeltest (posada, 2008) was used to determine the most appropriate model of sequence evolution for the nd4 dataset. according to the akaike information criterion (aic), the most supported evolutionary model was the trn + i, therefore applied in the subsequent analysis. to reconstruct phylogenetic relationships, we used a coalescent bayesian approach as implemented in mrbayes 3.2.6 (ronquist et al., 2012). we run 2 million generations, with 4 markov chains sampling every 1000 steps. after a burn-in of 10%, the remaining trees were used to compute a 50% majority rule consensus tree. in addition, a statistical parsimony network under 95% probability connection limits was constructed using tcs 1.21 (clement et al., 2000). number of haplotypes, nucleotide diversity (π) and haplotype diversity (h) were table 1. individuals analysed including sampling location, group of islands, colour pattern, haplotype number and haplogroup. sample code locality archipelago colour pattern haplotype species rl22 ponza pontine “abundistic” h1 h. viridiflavus rl66 ponza pontine “abundistic” h1 h. viridiflavus rl79 ventotene pontine “abundistic” h1 h. viridiflavus rl80 palmarola pontine “abundistic” h1 h. viridiflavus rs85 favignana aegadian “carbonarius” h10 h. carbonarius rs276 vulcano aeolian “carbonarius” h9 h. carbonarius rs296 lipari aeolian “carbonarius” h9 h. carbonarius 211genetic identity from tyrrhenian insular populations of hierophis viridiflavus s.l. also calculated for each group using dnasp 5.1 (librado and rozas, 2009). the final nd4 alignment (568 bp) of 99 sequences returned 67 polymorphic sites and 13 haplotypes. the phylogenetic analysis confirmed the presence of two well defined mitochondrial clades (fig. 1a), as already stated in previous works and corresponding to the two species h. viridiflavus and h. carbonarius (rato et al., 2009; a hv24-11 v39-15 hv6-29 v10-79 hv17-52 hv32-62 hv42-40 hv8-30 hv18-14 rl66-2 v4-31 hv30-33 rs276-6 hv26-74 v13-57 hv49-70 hv43-47 hv38-72 v19-55 v30-77 v20-51 v27-42 hv22-41 hv25-69 hv41-65 v26-45 v7-31 v1-46 hv35-37 rl80-3 hv45-67 v12-67 hv14-28 v32-75 v25-45 v21-53 hv13-18 v8-31 v42-43 hv28-54 hv5-57 hv16-21 hv40-36 hv29-73 hv46-13 hv4-34 rs85-4 hv9-10 v9-79 v14-57 v15-57 v38-26 hv37-78 hv33-56 hv2-23 v41-66 hv3-76 hv34-49 hv1-12 v11-79 hv19-35 v3-58 v40-22 hv48-68 v22-67 v16-57 v29-77 hv21-32 v33-34 hv12-9 hv36-27 hv31-38 rl79-1 hv20-8 hv51-24 rl22-2 v17-17 hv23-63 v31-77 v35-16 v6-31 v24-44 v2-48 hv47-7 hv11-20 hv15-61 v23-39 v28-64 hv27-59 v36-21 hierophis gemonensis hv10-28 v18-50 hv50-25 hv39-71 hv7-19 hv44-60 rs296-5 v37-7 0,99 0,99 0,8 0,98 1 c h1 hierophis viridiflavus hierophis carbonarius h11 h9 h5 h13 h12 h6 h7 h8 h10 h2 h3 h4 61 56 52 41 60 57 49 59 54 47 3236 40 62 3435 3337 38 67 29 28 30 27 25 23 19 20 21 24 14 12 10 9 13 18 11 7 8 69 74 73 78 72 71 6870 63 65 76 44 55 46 58 5051 45 48 53 43 3942 31 66 26 17 16 15 2264 79 77 75 ponza 2 favignana 4 vulcano 5 ventotene 3 palmarola 1 lipari 6 b h10 h6 h8 h1 h4 h3 h2 h5 h9 h12 h13 h7 h11 fig. 1. bayesian phylogenetic tree (a) based on nd4 sequences for 98 ingroup specimens of h. viridiflavus s.l. and one outgroup (h. gemonensis). the posterior probabilities are indicated at each node. each label indicates the specimen code, the locality number and the relative haplotype. insular individuals are shown in bold. geographic distribution (b) of the two mitochondrial lineages corresponding to h. viridiflavus (blue) and h. carbonarius (red). statistical parsimony network (c) connecting haplotypes. the circle size is proportional to the sequence frequencies and each filled rectangle represent one substitution. 212 ignazio avella et alii mezzasalma et al., 2015). nei’s standard genetic distance between the two species was 4.2%. specimens from favignana, vulcano and lipari, belong to the species h. carbonarius (fig. 1a). this clade showed the presence of nine haplotypes (fig. 1c, see table a1 in supplementary materials for all the haplotype references) with h = 0.623 ± 0.071, and π = 0.00389 ± 0.00057 (mean ± sd). the specimens from vulcano (rs276) and lipari (rs296) shared the same haplotype (h9) with individuals from localities seven and eight (fig. 1b), corresponding to iria and lago spartà (sicily). these results may suggest a recent colonization, either human-mediated or by oversea dispersal, from sicily to the aeolian islands. on the other hand, the specimen from the island of favignana (rs85) showed a new private haplotype (h10), separated by one mutational step from haplotype h9. in this case, the single fixed substitution may have occurred on the island through a vicariant mechanism. indeed, during the last glacial phase this island was connected to sicily and become separated following the last glacial maximum because of the sea level drop. a similar scenario has also been suggested to explain the genetic differentiation observed in other reptiles from favignana (mizan, 2015; senczuk et al., 2017). however, due to the small sample size from sicily, we cannot completely rule out that insular distinctiveness may have derived from a recent dispersal process of a haplotype not yet sampled in sicily. all the individuals from the pontine islands (rl22, rl66, rl79, rl80) shared one single haplotype (h1) and should therefore be recognized as belonging to h. viridiflavus (fig. 1a). this clade is genetically less differentiated than h. carbonarius clade (h = 0.128 ± 0.067; π = 0.00023 ± 0.00012; mean ± sd) and is composed by four haplotypes: a single highly represented haplotype (h1; with an allele frequency of 94%) and three derived and extremely localized haplotypes (h2, h3 and h4). this result may suggest an anthropic introduction in modern times or a recent colonization of the pontine islands from the tyrrhenian coast of the italian peninsula. interestingly, the four specimens from the pontine islands showed a colour pattern which resemble the “abundistic” morph, which is in the middle between the “carbonarius” (melanic/melanotic) and the “viridiflavus” (black and yellowish) colour patterns. the “abundistic” phenotype was previously reported only in sardinia, corsica and the tuscan archipelago. however, schätti and vanni (1986) reported similarities between specimens from the pontine islands and the dark coloured ones from emilia romagna now considered belonging to h. carbonarius. in particular, the individual sampled from ventotene (rl79, fig. 2) had a very dark dorsal with a yellowish ventral colouration, showing a phenotype which could easily be mistaken with the one observed in many populations of h. carbonarius. this observation confirms that colour pattern alone cannot help identifying the species to which a specimen belongs. the four h. viridiflavus specimens from the pontine islands are genetically indistinguishable from the usually brown-yellowish western whip snakes located on the tyrrhenian coast of italy, but show a darker phenotype. it has been reported in previous works that colour variation in reptiles can be associated to adaptive processes (norris and lowe, 1964; rosenblum et al., 2004). for example, darker or melanotic colouration may give a benefit in terms of thermoregulation (trullas et al., 2007; broennimann et al., 2014) and reproduction (capula and luiselli, 1994), and similar conclusion had been already drawn by rato et al. (2009) and zuffi (2007), as they consider the colour types in hierophis viridiflavus s.l. a by-product of different environmental conditions. therefore, the dark colouration of the snakes from the pontine islands could be the result of adaptive morphological evolution which occurred in a very short time, a phenomenon already observed in other insular reptile populations (losos et al., 1997; herrel et al., 2008). finally, despite changes in colour polymorphism might also be the outcome of nonadaptive processes (king, 1988; lorioux et al., 2008), the independent recurrence of the “abundistic” chromatism in all the northern tyrrhenian islands suggests a prominent role of adaptive forces acting in similar insular environmental conditions, which would deserve further studies. acknowledgements thanks to dario d’eustacchio, emanuela de simone, marco basile and mattia menchetti for providing samples, to laura gramolini for helping in laboratory and fig. 2. the specimen from ventotene island (rl79). the individual was found stuck in a mist net trap and later released. 213genetic identity from tyrrhenian insular populations of hierophis viridiflavus s.l. to brinna barlow for editorial assistance. we thanks sara riello for providing photos of the ventotene specimen. all the tissue samples used in this work were collected with permission of the italian environment ministry for the environment, land and sea to rc. 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(2008): colour pattern variation in populations of the european whip snake, hierophis viridiflavus: does geography explain everything? amphibiareptilia 29: 229-233. acta herpetologica vol. 12, n. 2 december 2017 firenze university press meristic and morphometric characters of leptopelis natalensis tadpoles (amphibia: anura: arthroleptidae) from entumeni forest reveal variation and inconsistencies with previous descriptions susan schweiger1, james harvey2, theresa s. otremba1, janina weber1, hendrik müller1,* brown anole (anolis sagrei) adhesive forces remain unaffected by partial claw clipping austin m. garner*, stephanie m. lopez, peter h. niewiarowski species and sex comparisons of karyotype and genome size in two kurixalus tree frogs (anura, rhacophoridae) shun-ping chang1,2, gwo-chin ma2,3,4, ming chen2,5,6,7,8,*, sheng-hai wu1,* non-native turtles in a peri-urban park in northern milan (lombardy, italy): species diversity and population structure claudio foglini1, roberta salvi2,* species composition and richness of anurans in cerrado urban forests from central brazil cláudia márcia marily ferreira1,*, augusto cesar de aquino ribas2, franco leandro de souza3 the life-history traits in a breeding population of darevskia valentini from turkey muammer kurnaz, alı i̇hsan eroğlu, ufuk bülbül*, halıme koç, bılal kutrup influence of desiccation threat on the metamorphic traits of the asian common toad, duttaphrynus melanostictus (anura) santosh mogali*, srinivas saidapur, bhagyashri shanbhag predation of common wall lizards: experiences from a study using scentless plasticine lizards jenő j. purger*, zsófia lanszki, dávid szép, renáta bocz reproductive timing and fecundity in the neotropical lizard enyalius perditus (squamata: leiosauridae) serena najara migliore1,2,*, henrique bartolomeu braz2,3, andré felipe barreto-lima4, selma maria almeida-santos1,2 observations on the intraspecific variation in tadpole morphology in natural ponds eudald pujol-buxó1,2,*, albert montori1, roser campeny3 and gustavo a. llorente1,2 reliable proxies for glandular secretion production in lacertid lizards simon baeckens diet of juveniles of the venomous frog aparasphenodon brunoi (amphibia: hylidae) in southeastern brazil rogério l. teixeira1, ricardo lourenço-de-moraes2, débora c. medeiros3, charles duca3, rogério c. britto4, luiz c. p. bissoli5, rodrigo b. ferreira3,* who are you? the genetic identity of some insular populations of hierophis viridiflavus s.l. from the tyrrhenian sea ignazio avella, riccardo castiglia, gabriele senczuk* acta herpetologica 11(1): 81-84, 2016 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-17406 yeasts in amphibians are common: isolation and the first molecular characterization from thailand srisupaph poonlaphdecha, alexis ribas* biodiversity research group, faculty of science, udon thani rajabhat university, udon thani 41000, thailand. *corresponding author. e-mail: alexisribas@hotmail.com submitted on: 2015, 6th november; revised on: 2016, 21th march; accepted on: 2016, 25th march editor: fabio maria guarino abstract. a survey of the presence of yeasts in frogs and toads in thailand was conducted using standard mycological examination techniques. the results, which were confirmed with molecular techniques, revealed the presence of five yeast species – cryptococcus liquefaciens, c. heveanensis, pseudozyma hubeiensis, rhodotorula mucilaginosa and r. minuta – in the bile of these amphibians. although previous works have isolated yeasts from amphibian gastrointestinal tracts and skin, it is questionable whether these yeasts were acquired by ingestion or were commensals on adult individuals. frog farms, an urban area and protected natural areas were surveyed and all tested positive for yeasts, which shows their ubiquity in both wild and farm-reared frogs. additionally, the finding of yeasts in five different species of frogs and toads shows that there is a wide spectrum of hosts in this vertebrate group. our results thus suggest that yeasts are likely to be widespread among amphibians in different habitat types and in a wide range of host species. keywords. amphibian, yeast, rhodotorula, pseudozyma, cryptococcus frogs are parasitized by several taxa, the most-studied of which include helminths, bacteria, viruses and the chytrid fungus batrachochytrium dendrobatidis. parasites cause high mortality rates in wild amphibian populations (densmore and green, 2007), while infectious diseases are a concern in amphibians kept in captivity as pets or reared on farms as a food source. although not strictly pathogenic, symbiont organisms have been found on frog skins, where they are reported to act as a barrier against chytridiomicosis, and have also been isolated in the guts of these vertebrates (walke et al., 2014). anecdotal reports of yeasts – opportunistic pathogens that are ubiquitous in nature – exist for tadpoles and adult amphibians. steinwascher (1979) found that candida humicola was abundant in the faeces of some large tadpoles of rana clamitans in australia, where it acts as a mutualistic symbiont. rhodotorula glutinis was isolated from the tadpoles of marsh frogs (limnodynastes peronii) in australia, where it plays a role in suppressing the growth of the mosquito larvae that inhabit the same pond (mokany and shine, 2003). in the study by sammon et al. (2010), yeast were isolated from the faeces and the skin of adult australian green tree frogs (litoria caeruela), although no subsequent molecular confirmation was undertaken. as all studies of yeast from the gastrointestinal tracts of amphibians (tadpoles and adults) to date have only focused on faeces, it is still unclear whether or not yeasts form part of the natural microflora of amphibians or whether they are acquired from the environment through the ingestion of water and thus appear in faeces. the yeast candida humicola can modify the behavior of tadpoles (kiesecker et al., 1999) and so should be considered pathogenic rather than symbiotic, as has been previously reported (steinwascher, 1979). consequently, the distribution of yeasts in both wild and farm-reared populations of amphibians is of interest. in the course of a parasitological survey of adult frogs and toads in thailand, the observation under the microscope of yeast-like cells 82 srisupaph poonlaphdecha, alexis ribas in bile led us to suspect that yeasts were present in these amphibians. we thus conducted culture and molecular characterization to confirm the validity of these microscopic observations. fieldwork was carried out in march–june 2014. frogs were collected by hand in three types of habitats: frog farms, urban areas and protected natural areas. on farm 1, where chicken and fish are also bred, frogs are raised in walled concrete enclosures at high densities. on farm 2, frogs are raised in shallow ponds which were covered by plastic on the floor and net as a wall, it also has chickens in the very close area. on farm 3, frogs are raised in ponds and coexist with fish and ducks, although they are physically separated by netting. all farms are located in udon thani province. the second habitat surveyed was an urban area on the udon thani rajabhat university campus, where there are many buildings and green spaces. the third environment surveyed were two protected forest areas, na yungnam som natural park (344 km2, udon thani province) and phu wua wildlife sanctuary (186.5 km2, bueng kan province), that both harbour great floral and faunal biodiversity. once collected, all samples were immediately transported alive to a laboratory at udon thani rajabhat university and identified following taylor (1962) and the nomenclature used in an online database of the world’s amphibians (frost, 2015). amphibians were anesthetized, killed using ms222 (ethyl-4-aminobenzoate) and then dissected. their gall bladders were removed and placed on sterile petri dishes. syringes were used to suck bile from the gall bladders, which was then transferred onto yeast malt agar (himedia laboratories) and cultured using a spread-plate technique. the ym culture plates were incubated at 25°c for 5–7 days. single colonies were selected, streaked onto ym agar plates and then incubated at 25°c for 5–7 days. the purified colonies were examined microscopically and those with yeast-like shapes were subjected to further molecular studies. isolation of dna was carried out by boiling of cells with lysis buffer according to the methods of maniatis et al., (1982) with slight modification. a loopful of yeast cells was transferred to 1.5 ml eppendorf tube. the 100 µl of lysis buffer was added. cell suspensions were boiled in water bath or metal block bath for 15 min. after boiling, 100 µl of 2.5 m potassium acetate (ph 7.5) was added and placed on ice for 1 hour, and centrifugated at 14,000 rpm for 5 min. supernatant was extracted twice with 100 µl of chloroform: isoamyl alcohol (24:1 v/v). dna was precipitated with isopropanol, placed at 20°c for 10 min and centrifugated at 15,000 rpm for 15 min. dna pellet was rinsed with 70% and 90% ethanol and then dried up (15-30 min at room temperature). the dried dna was dissolved in 30 µl milli q water. the amplification and sequencing of 13 isolates were performed at the national center for genetic engineering and biotechnology (biotec), bangkok (thailand). the divergent d1/d2 domain of 26s rdna was amplified with primers nl-1 (5’-gca tat caa taa gcg gag gaa aag-3’) and nl4 (5’-ggt ccg tgt ttc aag acg g-3’) (kurtzman and robnett, 1998). amplification was carried out in a 100 μl reaction mixture containing 100 ng of genomic dna, 2.5 u of taq polymerase (thermo scientific), 20 mm of each dntp, 40 mm of each primer, 10 mm tris-hcl and 1.5 mm mgcl2. the reaction was pre-denatured at 94°c for 5 min, then repeated for 30 pcr cycles with denaturation at 94°c for 1 min, annealing at 55°c for 1 min, extension at 72°c for 2.5 min and then final extension at 72°c for 10 min. the amplified dna was purified using a qiaquick pcr purification kit according to the manufacturer’s instructions. the nucleotide sequences of d1/d2 domain of 26s rdna were directly determined using pcr products following kurtzman and robnett (1998) with slight modifications. cyclic sequencing of the d1/d2 domain was conducted with an nl1 forward primer (59-gca tat caa taa gcg gag gaa aag-39) and an nl4 reverse primer (59-ggt ccg tgt ttc aag acg g-39) using an abi prismtm bigdyetm terminator cycle sequence ready reaction kit (applied biosystems, stafford, usa) following the manufacturer’s instructions. the thirteen obtained sequences (576 to 612 base pairs) of d1/d2 domain of 26s rdna were compared using a blast homology search (http://www.ncbi. nlm.nih.gov/blast): af070432, af181515, af189948, dq008953 and fj534905. our obtained sequences were deposited in genbank, accession numbers ku893128 to ku893140. growth of yeast colonies on ym agar (pink and white) was observed in samples from 12 amphibians from 3 of the 90 farms (3.33%), 2 of the 7 amphibians from the urban area (28.57%) and 7 of the 31 amphibians (21.87%) from the wild (total=129 specimens). one specimen of hoplobatrachus rugulosus from a farm harboured two gross colony morphologies (table 1). molecular characterization confirmed the presence of cryptococcus heveanensis, c. liquefaciens, pseudozyma hubeiensis, rhodotorula minuta and r. mucilaginosa (see table 1). the yeasts isolated from the amphibians are common in the environment and have been reported as opportunistic pathogens in vertebrates. cryptococcus liquefaciens is a common yeast that is able to survive in extreme habitats such as the deep sea (buzzini and margesin, 2013) and can be found in the microbiota of human 83yeasts in amphibians skin (zhang et al., 2011). it has also been reported as an opportunistic pathogen (takemura et al., 2015) and is able to cause fatalities in humans (conde-pereira et al., 2015), but has never before been reported in amphibians. cryptococcus cuniculus has previously been reported from rabbits (shin et al., 2006). pseudozyma hubeiensis, a basidiomycetous yeast, was recently isolated in china from the leaves of a number of plant species (wang et al., 2006), while liou et al. (2009) have described a new species of this genus in thailand from the region in which the current study was carried out. to our knowledge, there are no previous reports of this genus of yeast in vertebrates. the genus rhodotorula is a common yeast found in air, soil, lakes, ocean water, milk and fruit juice (wirth and goldani, 2012), and is able to survive in adverse conditions. rhodotorula spp. including the two species found in amphibians in the present study have been observed as opportunistic pathogens in humans (wirth and goldani, 2012). recent studies have reported the presence of rhodotorula yeasts in vertebrates (chitkomckown et al., 2013) including a female lamb (but with no associated pathology), in skin lesions in sea lions in an aquarium (alvarez-perez et al., 2010) and as a normal component of the microbiota of salmonids (raggi et al., 2014). our results suggest that the role of yeasts in wild and farmed amphibians as pathogens has to date been underestimated and it is likely that extrinsic and intrinsic factors induce their pathogenicity. our results show a high diversity of yeast species (5) in amphibians compared to the number of species found in vertebrates. however, only a few studies of yeasts in vertebrates have ever been conducted. it is possible that the aquatic habitat of adult amphibians (partially dependent on water and varying according to the species) may contribute to the life cycle of these yeasts. our examination of bile samples revealed that yeasts colonize the internal organs of frogs. although the presence of candida spp. in human gall bladders has previously been reported (diebel et al., 1996), we believe that this is the first report of yeast from the gall bladders of non-human vertebrates. the study by sammon et al. (2010) found a minimum of six species of yeasts (rhodotorula sp., trichosporon spp., candida sp., c. famata and c. glabrata) in faeces from five frogs and our findings confirm this high diversity. we isolated five species of yeasts, which suggests that the biodiversity of yeasts in frogs has been insufficiently studied. additionally, our study confirms the ubiquity of yeasts in these amphibians since they were found in two wild protected areas, in an urban area and on frog farms. our study also shows that yeasts are widespread in a number of species of amphibians, including both those associated with aquatic habitats and those with more terrestrial habitats (toads). acknowledgements this study was supported by a grant from the research and development institute, udon thani rajabhat university. we would like to thank renu sirimongkonthaworn and tiwarat thalernkietleela from udon thani inland fisheries research and development center, taweep kampaengmuang, director of phu wua wildlife sanctuary, and rungsun loupha, director of na yung-nam som protected area. the research in phu wua wildlife sanctuary and na yung-nam som protected area was approved by the department of national table 1. distribution of the yeast species confirmed by molecular analysis according to habitat and host. sampling point habitat anuran species species of yeasts % similarity farm 2 farm hoplobatrachus rugulosus rhodotorula mucilaginosa 100.00% farm 2 farm hoplobatrachus rugulosus rhodotorula mucilaginosa 100.00% udru urban area kaloula pulchra rhodotorula mucilaginosa 100.00% phu wua wildlife sanctuary wild sylvirana faber rhodotorula mucilaginosa 99.80% udru urban area duttaphrynus melanostictus rhodotorula mucilaginosa 99.50% phu wua wildlife sanctuary wild kaloula pulchra rhodotorula minuta 99.70% farm 2 farm hoplobatrachus rugulosus cryptococcus liquefaciens 100.00% phu wua wildlife sanctuary wild sylvirana faber cryptococcus liquefaciens 100.00% na yung-nam som protected area wild duttaphrynus melanostictus cryptococcus liquefaciens 100.00% na yung-nam som protected area wild kaloula pulchra cryptococcus liquefaciens 100.00% udru urban area duttaphrynus melanostictus cryptococcus heveanensis 99.80% na yung-nam som protected area wild fejervarya limnocharis psudozyma hubeiensis 99.80% na yung-nam som protected area wild duttaphrynus melanostictus psudozyma hubeiensis 99.80% 84 srisupaph poonlaphdecha, alexis ribas parks, wildlife and plant conservation and the national research council of thailand (number 0002/7387). this study would not have been possible without the enthusiastic work by udru students, above all thanyarat phorsi and amornrat leenat. references alvarez-perez, s., mateos, a., dominguez, l., martineznevado, e., blanco, j.l., garcia, m.e. 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(2012): epidemiology of rhodotorula: an emerging pathogen. interdiscip. perspect. infect. dis. 465717. zhang, e., tanaka, t., tajima, m., tsuboi, r., nishikawa, a., sugita, t. (2011): characterization of the skin fungal microbiota in patients with atopic dermatitis and in healthy subjects. microbiol. immunol. 55: 625-632. acta herpetologica vol. 11, n. 1 june 2016 firenze university press feeding habits of mesoclemmys vanderhaegei (testudines: chelidae) elizângela silva brito1,*, franco leandro souza2, christine strüssmann3 morphology, ecology, and behaviour of hylarana intermedia, a western ghats frog ambika kamath1, rachakonda sreekar2,3 a new account for the endangered cerrado rocket frog allobates goianus (bokermann, 1975) (anura: aromobatidae), with comments on taxonomy and conservation thiago ribeiro de carvalho1,2,*, lucas borges martins1,2, ariovaldo antonio giaretta1 molecular phylogenetics of the pristimantis lacrimosus species group (anura: craugastoridae) with the description of a new species from colombia mauricio rivera-correa, juan m. daza* population structure and activity pattern of one species of adenomera steindachner, 1867 (anura: leptodactylidae) in northeastern brazil maria juliana borges-leite1,*, joão fabrício mota rodrigues2, patrícia de menezes gondim1, diva maria borges-nojosa1 vocal repertoire of scinax v-signatus (lutz 1968) (anura, hylidae) and comments on bioacoustical synapomorphies for scinax perpusillus species group marco antônio peixoto1,*, carla silva guimarães1, joão victor a. lacerda2, fernando leal2, pedro c. rocha2, renato neves feio1 amendment of the type locality of the endemic sicilian pond turtle emys trinacris fritz et al. 2005, with some notes on the highest altitude reached by the species (testudines, emydidae) federico marrone*, francesco sacco, vincenzo arizza, marco arculeo photo-identification in amphibian studies: a test of i3s pattern marco sannolo1,*, francesca gatti2, marco mangiacotti3,4, stefano scali3, roberto sacchi4 no evidence for the ‘expensive-tissue hypothesis’ in the dark-spotted frog, pelophylax nigromaculatus li zhao, min mao, wen bo liao* no short term effect of clinostomum complanatum (trematoda: digenea: clinostomatidae) on survival of triturus carnifex (amphibia: urodela: salamandridae) giacomo bruni1,*, claudio angelini2 yeasts in amphibians are common: isolation and the first molecular characterization from thailand srisupaph poonlaphdecha, alexis ribas* introduction of eleutherodactylus planirostris (amphibia, anura, eleutherodactylidae) to hong kong wing ho lee1, michael wai-neng lau2, anthony lau3, ding-qi rao4, yik-hei sung5,* correlation between endoscopic sex determination and gonad histology in pond sliders, trachemys scripta (reptilia: testudines: emydidae) david perpiñán1, albert martínez-silvestre2,3, ferran bargalló3, marco di giuseppe4, jorge orós5, taiana costa6,* book review: john w. wilkinson. amphibian survey and monitoring handbook. pelagic publishing franco andreone book review: susan newman. frogs amphibians and their threatened environment. discovery and expression through art k-3. frogs are green franco andreone acta herpetologica 10(2): 77-84, 2015 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-14979 one scute ring per year in testudo graeca? a novel method to identify ring deposition patterns in tortoises roberto c. rodríguez-caro1,*, eva graciá1, renata morais dos santos1, josé d. anadón2, andrés gimenez1 1 area of ecology, dept. of applied biology, miguel hernández university, av. de la universidad, 03202, elche, spain. * corresponding author. e-mail: r.rodriguez@umh.es 2 dept. of biology. city university of new york. new york. usa submitted on 2014, 10th september; revised on 2015, 11th may; accepted on 2015, 26th may. editor: paolo casale abstract. a reliable estimation of individuals’ age is helpful to conduct demographic studies on wildlife populations. in tortoises, many studies have estimated individuals’ age by counting growth rings on their scutes, assuming one ring per year (1:1 ratio). however, the accuracy of this method is controversial. the ring deposition pattern can vary depending on species, or even populations, and should be studied comprehensively. we studied the ring deposition pattern of testudo graeca in southeastern iberian peninsula, using recaptures of 156 individuals between 2004 and 2010. we used a novel approach to explore the ring deposition pattern and to test possible differences between localities and individuals. our results revealed that most analysed individuals (57.4%) showed a 1:1 ratio, in which rings were deposited during months of activity (spring to autumn). however, we found a trend to count less rings than years, which underestimated 1 year every 3 or 4 years. no differences in the deposition patterns were found among sites, sizes or sexes because the halt in growth during hibernation equally affects all tortoises in all sites. our results support that the assumed 1:1 ratio in the assignment of individuals’ age is too simplistic. since ring deposition patterns are complex, the use of statistical approaches capable of handling deviations from the assumed deposition ratios can help to better depict population age structures. keywords. growth rings, age estimation, spur-thighed tortoise, ring deposition pattern. introduction an accurate estimation of individuals’ age is key for the study of life history and population dynamics of species (ricklefs, 1990). a population’s age distribution is also a basic parameter to assist with management and conservation strategies (slobodkin, 1963). in turtles and tortoises, there are three main approaches to estimate age: skelotochronology (zug et al., 1986), growth models (carr and goodman, 1970) and growth ring counts (agassiz, 1857; hereinafter grc). each one of the methods has its pros and cons (wilson et al., 2003; curtin et al., 2008; armstrong and brooks, 2014). firstly, although skelotochronology can provide reliable data about age it is an invasive method that just can be used with death animals and also shows some biases (curtin et al., 2008). on the other hand, growth data models allow to produce age estimates in big long-lived species (armstrong and brooks, 2014), but some papers exemplifies case studies where size is weakly related to age (e.g., carr and goodman, 1970; congdon et al., 2001). in the present work, we focus into the grc method, probably the most extensively used, but that it is not free of criticisms (wilson et al., 2003). grc was first described by agassiz (1857) and was used to estimate age in chrysemys picta. although several studies have supported it (graham, 1979; zug, 1991; germano and bury, 1998; bertolero et al., 2005), others have questioned its use (tracy and tracy, 1995; kennett, 1996; brooks et al., 1997). in particular, wilson et al. (2003) 78 roberto c. rodríguez-caro et alii reviewed 143 papers that provided age data by counting growth rings. most papers (67%) presented no data to support the use of grc, and several researchers cited sexton (1959) as a validation method. however, in that paper, the author built a theoretical model without testing the relationship between rings and age. in accordance with wilson et al. (2003), we identified four critical sources of mismatches that question the accuracy of grc as an age estimator. these sources can be divided into two groups. firstly, deposition of rings may not follow the usually assumed one ring per year (1:1 ratio) because: 1) some individuals may deposit two rings or more in the same season during periods or in habitats with good resources availability (tracy and tracy, 1995; aresco and guyer, 1998; berry, 2002); 2) some individuals may stop growing, and may not even deposit any ring for years (turner et al., 1987). secondly, even if ring deposition follows a 1:1 ratio, other factors may negatively affect the accuracy of the technique: 3) scutes worn-out from friction may make it impossible to discern individual rings, thus age may be underestimated (aresco and guyer, 1998; litzgus and brooks, 1998; berry, 2002); and 4) identification of rings requires an observer’s arbitrary decision, and repeated measures may yield different results. this inherent subjectivity of ring counts may be exacerbated by those rings produced in years after reaching maturity as they might be too small to be accurately counted (lambert, 1982; galbraith and brooks, 1987; zug, 1991; germano and bury, 1998). understanding the deposition pattern of rings in tortoises is a premise for using the grc technique in age inferences (germano and bury; 1998, wilson et al., 2003; bertolero et al., 2005). for age estimates from grc to be reliable, ring production has to be cyclical (i.e., regular) and at known deposition rates (germano, 1988; zug, 1991; germano and fritts, 1994; wilson et al. 2003). however, deposition rates can vary among species, or even populations, if they withstand different climatic conditions (e.g., moll and legler, 1971; berry, 2002). in the testudo genus, the results of previous studies show that the reliability of grc as an age estimator diminishes with the individual’s age (diaz-paniagua et al., 2001 for t. graeca) and some authors consider the grc technique is only reliable for juveniles (bertolero et al., 2005, attum et al., 2011; for t. hermanni and t. kleinmanni, respectively). these results suggest uncertainty in the accuracy of age-based demographic studies since species dynamics relies heavily on adult dynamics (diaz-paniagua et al., 2001). therefore, comprehensive studies to address the accuracy of the grc method are required. the purpose of this work is to study the growth  ring deposition pattern of the spur-thighed tortoise (t. graeca) population in southeastern iberian peninsula. in particular the specific goals of this study are twofold: 1) to test whether the deposition pattern follows a 1:1 ratio and understand the temporal context; and 2) to explore possible differences between sizes, sexes, stages and samples sites. in order to address these objectives, we start by analysing the mismatch between the number of deposited rings and the elapsed time between captures of individuals and develop a novel method to infer the deposition ring patterns of the species. our results will provide valuable information to further demographic studies of this endangered population. moreover, the undertaken approach can prove useful to study other chelonian populations as a first step to address the accuracy of the grc technique. materials and methods study system and data collection t. graeca is a long-lived species and individuals can live up to 30 years (diaz paniagua et al., 2001). in southeastern iberian peninsula the species displays strong seasonal activity patterns (perez et al., 2002). the main activity season occurs in spring (march to june), when the species’ principal breeding season (mating and laying of eggs) takes place, and a secondary activity season occurs in autumn (september to november) when most, but not all, individuals are active (perez et al., 2002). these two activity periods are separated by two inactivity periods, summer and winter, due to scarce food resources and extreme temperatures (perez et al., 2002). monitoring was carried out at 18 sampling sites over 6 years (2004-2010), which are representative of the species’ entire range in southeastern areas of the iberian peninsula (fig. 1), although one locality comprises more than half of the data (galera, n = 99, 63% of the individuals analysed). surveys were conducted during the active period walking though the habitat in line transects and capturing the tortoises in opportunistic encounters. individuals were marked with an individual code by notching the marginal scutes. from each individual, carapace length (cl) and scute ring count was recorded. rings were counted from the third costal scute to the right of the carapace (fig. 2) because, as in other tortoises, the rear part of the carapace receives less wear than the front part or the plastron (germano, 1988). ring width was measured to the nearest millimetre using a digital calliper. sex of individuals was identified by secondary characters (lopez-jurado et al., 1979). unsexable immature individuals were classified as subadults. tortoises were released in the same place that it was found. measurements were taken by 19 different specialists. to minimize errors and bias, these specialists followed the same measurement protocol and were trained for standardization under supervision of experienced researchers with captive animals before fieldwork. in total we captured 156 tortoises at least twice (74 females, 70 males and 12 juveniles). only one recapture per tortoises was used in order to maintain the independence of the data. 79growth ring patterns in testudo graeca mismatches due to errors in measurements in a first step, we excluded the individuals for whom ring counting was not feasible due to worn-out scutes. in order to ensure that the number of rings deposited between captures was not strongly affected by inaccurate observations, we first checked the reliability of our data. in particular, we attempted to identify the last rings of the previous capture during subsequent captures using ring width and relative ring position (fig. 2). those individuals where this identification was not possible were removed from further analyses. we compared the size distribution before and after remove the individuals using a chi-squared analysis to test whether the size frequencies are maintained after the individuals were discarded. we also compared the size of the discarded males and females with those non-discarded individuals by means of a t-statistic (α = 0.95). in order to test out the errors made by the specialists, we used a chi-squared analysis to test the frequencies of the accurate and inaccurate counts that they carried out. ring deposition pattern we analysed the temporal deposition patterns of rings, by means of comparing the increment of the number of rings (dr) and the increment of time between captures according to different hypotheses of deposition patterns (dtdp). the difference between these two values is the bias between time and rings, under the five different hypothesised deposition patterns (btrdp). btrdp = dr dtdp deposition pattern hypotheses (dp, table 1) were formulated considering that the detection of a new ring requires more fig. 1. samples sites inside the distribution area in the southeast of spain, between the provinces of murcia and almeria. the black points are the sampled sites and the black star is the site called “galera”, the place with the higher number of captured tortoises. the area in the all samples sites is 1 km2. 80 roberto c. rodríguez-caro et alii than half the time contemplated in the hypothesis, because we consider that we can detect the ring “in growth” before the complete ring is deposited. for example, under the first hypothesis of one ring per year (dty), we assumed that we would be able to identify a new ring after the sixth month. under the second and third hypotheses of one ring per spring (dts) and one ring per autumn (dta), we could be able to see the ring after two spring months, and after one and a half autumn month, respectively. under the fourth hypothesis of one ring per active season, including both spring and autumn (dtsa), we should detect a new ring after 3.5 months of spring or autumn. finally, our fifth hypothesis states that one ring is deposited in spring and another in autumn (dts+a), thus one ring should be detected in at least two spring months and another ring in, at least, one and a half autumn months (table 1). the fit between the hypothesized deposition patterns and the observed data was tested by a t-test. according to the hypotheses put forward, if the btrdp did not significantly differ from zero (α = 0.95), we assumed that the number of rings represents the time interval. differences between sexes, between stage classes (adults and juveniles), and among samples sites (comparing “galera” with the other sample sites) were tested by means of a wilcoxon test. we used linear regression to assess how the best fitted btrdp varied with the time that elapsed between capture and recapture (dtdp) and with a body measure (cl, fig. 2). results of the 156 recaptured individuals, 41 (26.3%) were ruled out from further analyses given the possible mismatches due to errors in measurements in their observafig. 2. tortoise carapace showing the third scute and the carapace length (cl) measure. a) the rings counted in the first capture and b) the rings counted in the recapture, where dr is the difference between a) and b). table 1. abbreviations for the bias between time and rings (btrdp) and time elapsed between captures at different hypotheses of deposition pattern (dtdp). deposition pattern (dp) hypothesis bias variables (btrdp) time variables (dtdp)** equation* 1 ring per year btry dty dty=no. months/12 1 ring per spring btrs dts dts=no. months in spring/4 1 ring per autumn btra dta dta=no. months in autumn/3 1 ring per active season, spring and autumn btrsa dtsa dtsa=no. months in spring or autumn/7 1 ring per spring and another ring per autumn btrs+a dts+a dts+a=no. months in spring/4 + no. months in autumn/3 *springs include four months (march, april, may and june) and autumn three (september, october and november). **we rounded off dtdp to create a discrete variable. 81growth ring patterns in testudo graeca tions. in seven tortoises, scutes were very worn-out (4.5% of the total sample) and, in 34 tortoises the pattern of ring measures between recaptures vastly differed and did not allow an accurate estimation of the number of rings deposited (21.8%). the size class distribution was no different before and after ruling them out (χ2 = 2.35, df = 10, p = 0.99; fig. 3). we found no differences in size between discarded and non-discarded female tortoises (147.7 ± 14.9 mm and 143.8 ± 12.9 mm, respectively; p > 0.05). nevertheless, discarded males (116.9 ± 8.9 mm) were significantly larger than non-discarded ones (112.5 ± 10.1 mm; p = 0.034). juveniles not were analysed because only one juvenile was discarded. we detected no differences among all the specialists when comparing their number of accurate and inaccurate observations (χ2 = 14.53, df = 13, p = 0.34), nor even between those 10 observers who, together, achieved more than 80% of observations (χ2 = 10.77, df = 9, p = 0.29). ring deposition pattern we analysed the deposition pattern in 115 individuals (57 females, 47 males and 11 juveniles). the mean interval between captures was 1.0 ± 1.2 years. in all the different hypothetical temporal deposition patterns tested, the mean of the time that had elapsed between captures was longer than the mean of the number of deposited rings 0 5 10 15 20 25 <8 8 – 8.9 9 – 9.9 10 – 10.9 11 – 11.9 12 – 12.9 13 – 13.9 14 – 14.9 15 – 15.9 16 – 16.9 >=17 n um be r of in di vi du al s size (cm) juveniles males females fig. 3. size distribution patterns in the population after some individuals were ruled out for error measures (n=115). 0 5 10 15 20 25 30 35 -3 -2 -1 0 1 2 3 4 5 % o f i nd iv id ua ls btrsa juveniles males females fig. 4. frequency of individuals with a bias according to btrsa (bias variable based on the hypothesis that one ring is deposited per active season – spring and autumn). when btrsa is 0, there is a 1:1 ratio (one ring per year) table 2. descriptive statistical data calculated for the bias variables (after some tortoises were ruled out for errors measures, n=115). bias variables mean sd t-test (x=0), df =114 p.value btry -0.27 1.119 -2.58 0.011 btrs -0.29 1.114 -2.76 0.007 btra -0.26 1.093 -2.56 0.012 btrsa -0.16 1.048 -1.60 0.112 btrs+a -1.36 2.053 -7.09 0.000 btr is the bias variable obtained with different temporal hypothesis (more information in table 1). 82 roberto c. rodríguez-caro et alii (table 2). the temporal deposition pattern btrsa (one ring jointly developed in spring and autumn) was the only pattern that statistically matched the observed data (table 2). based on this assumption, 57.4% of the analysed tortoises exactly followed a 1:1 deposition pattern (i.e., btrsa = 0, fig. 4). nonetheless, the btrsa variable correlated negatively with the time that had elapsed between captures (r2 = 0.0153; p < 0.001). namely, the trend was to count fewer rings than expected (fig. 5). finally, no differences were found in the temporal deposition patterns between males and females, between adults and juveniles or between the galera location and the remaining sites (w = 1287.5; w = 519; w = 1011; respectively, p > 0.05), and btrsa also had no relation with individual sizes (p > 0.05). discussion activity periods and rings deposition in testudo graeca it has been proposed that the number of rings per year in the scutes of tortoises depends on the number of inactive periods experienced by the individuals, during which growth diminishes or stops (wilson et al., 2003). the physiological process underlying ring deposition is based on the growth of the epidermis, when outer cells die and keratinise forms the scute layer. our results fit this biological process and indicate that the seasonal activity pattern of t. graeca in southeastern iberian peninsula strongly influences its ring deposition pattern. the majority of individuals deposited one ring when one spring and one autumn had elapsed. we can, thus, infer that growth occurs during these activity seasons, and even in summer, when individuals grow, but probably more slowly. we were unable to detect differences in the number of deposited rings among males, females and juveniles, indicating that the growth halt during hibernation affects the entire population. do deposition patterns follow a one–ring-per-year ratio? according to our results, the deposition of a complete ring requires both activity periods (spring and autumn) and a growth halt during the hibernation, what is the equivalent to 1 year. more than half of the analysed individuals followed a one-ring-per-year pattern, 57.4% of the analysed tortoises showed exactly a 1:1 deposition pattern (i.e., btrsa = 0), and 90.4% revealed biases less than or equal to 1 year (fig. 4). overall our results support the one–ring-per-year ratio reported for other species (germano, 1988; iverson, 1988; germano and bury, 1998). however, slight individual deviations in the assumed pattern can result in inadequate age estimations, something which is especially problematic since chelonians are long-lived species. we identified a significant trend to visualise fewer rings than years, according to which 1 year is underestimated every 3 or 4 years (fig. 5). the cumulative effect of this underestimation would specially affect accurate age assignments, whereas the consideration of departure in more complex statistical approaches could help better depict population age structures (i.e., a probabilistic assignation of individuals to age class intervals according to the inferred number of rings). may other factors negatively affect the accuracy of the technique? this work demonstrates that there is a relation between number of rings and tortoises’ age, but possible sources of error may lead to inaccurate estimates. it is worth mentioning that 26.3% of recaptured individuals were ruled out to avoid inaccurate estimations of the number of rings deposited between recaptures. although the majority of discards were due to inconsistencies in the data obtained by the specialists, no differences were found in accurateness between them, probably because they received the same training. previous works that have reporting significant differences among observers found that discrepancies were related mainly with their ability to distinguish when a ring starts and finishes (legler, 1960; galbraith and brooks, 1989; germano and bury, 1998). fig. 5. regression between btrsa (bias variable based on the hypothesis that one ring is deposited per active season – spring and autumn) and the time elapsed between recaptures (dtsa). bold line is the result of the regression. 83growth ring patterns in testudo graeca the technique can be improved repeating counts by different specialist and training them according to possible ambiguities. these recommendations could be essential to guarantee data soundness, especially in long-term studies. the other cause that led us to rule out some individuals was that tortoises showed worn-out or damaged scutes, especially the largest males of the population, in whom it was impossible to accurately infer the number of deposited rings among recaptures. we hypothesised that the higher rates of male movements lead to greater scute wear than in females, and rings are also more easily inferred in females due to their larger sizes (anadon et al., 2012; rodríguez-caro et al., 2013). for spur-thighed tortoises in the doñana national park in southwestern spain, díazpaniagua et al., (2001) pointed out that the grc method is reliable only for those individuals aged up to 20 years, older tortoises show too worn-out scutes to be counted conclusions we assessed that grc technique can be used to infer the approximate age of testudo graeca in southeastern iberian peninsula and we develop a novel method to infer the deposition ring patterns in tortoises. we conclude that this population generally follows a one-ringper-year ratio. thus the present work support, with some cautions, the use of grc to estimate age in further studies of this population, and also encourage the use of this approach to test the deposition patterns of other chelonians populations. despite the general trend (1:1 ratio), we also found notable variability associated with the individual’s heterogeneity in ring deposition, and also due to mismatches among observations. individual’s heterogeneity could be assumed using statistical approaches capable of handling the deviations observed from deposition ratios, such as probabilistic assignations of individuals to age intervals instead of using specific age estimations. regarding ring counting, we share the usual recommendation of training observers or increase the number of captured individuals, but also it would be convenient duplicate counts by different observers in order to avoid possible mistakes. in our study, we validated our data by at least two observations using recaptures at different times, but data could be easily improved using independent counts carried out by two specialist at the same time. acknowledgements we thank dr. f. botella for his support in this work. we also thank all the people who participated in the fieldwork. the spanish ministry of science funded this work through projects cgl2009-08251 and cgl201233536. permits for the field work and capture animals were provided by “la dirección general de gestión del medio natural de la junta de andalucía” (sgb/foa/ afr) and “la delegación general de medio natural de la comunidad autónoma de la región de murcia” (fa/aut/cap-06, aut/fa/und/186-08, aut/et/ und/48/2010). r. rodriguez-caro and e. graciá were 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(1986) age determination of loggerhead sea turtles, caretta caretta, by incremental growth marks in the skeleton. smithson contrib. zool. 427: 1-34. acta herpetologica vol. 10, n. 1 june 2015 firenze university press acta herpetologica journal of the societas herpetologica italica acta herpetologica 12(2): 157-165, 2017 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-18179 species composition and richness of anurans in cerrado urban forests from central brazil cláudia márcia marily ferreira1,*, augusto cesar de aquino ribas2, franco leandro de souza3 1 programa de pós-graduação em ecologia e conservação, universidade federal de mato grosso do sul, centro de ciências biológicas e da saúde, cidade universitária, caixa postal 549, cep 79070-900, campo grande, ms, brazil. *corresponding author. e-mail: claudiamarily@hotmail.com 2 universidade federal de mato grosso do sul, faculdade de computação, caixa postal 549, cep 79070-900, campo grande, ms, brazil 3 universidade federal de mato grosso do sul, centro de ciências biológicas e da saúde, cidade universitária, caixa postal 549, cep 79070-900, campo grande, ms, brazil submitted on: 2016, 21st april; revised on: 2017, 15th april; accepted on: 2017, 25th may editor: gentile francesco ficetola abstract. brazil harbors the greatest diversity of amphibians on the planet although there are few studies dealing with urban fauna. the objective of this study is to describe the species composition and richness of anurans in urban cerrado fragments from campo grande municipality, mato grosso do sul state, central brazil. the specimens were sampled in three stages through pitfall traps and visual/acoustic surveys. seventeen species were recorded (17.7% of anuran species registered in mato grosso do sul), with leptodactylidae and hylidae being the most represented families. the existence of a high number of green areas and water bodies in the urban area likely favors anuran species in the region. the anuran communities in urban areas of campo grande were dominated by species which use a broad range of habitats. in this study there was the record of a new species of anuran, proceratophrys dibernardoi, for the state of mato grosso do sul. the forest fragments that had the highest similarity for species composition were those with similar environmental conditions. the knowledge of the fauna that occurs in urban areas is important because natural habitats suffer severe fragmentation and degradation and species present in these areas may disappear in a shorter period of time. keywords. anuran, city, urbanization, cerrado, habitat fragmentation. introduction there are approximately 7,600 amphibian species in the world (frost, 2017), most of them occurring in tropical regions where natural landscapes have been altered by human activities (ellis and ramankutty, 2008). brazil has 1,080 amphibian species (segalla et al., 2016) and at least 209 are recorded for the cerrado and adjoining biomes (valdujo et al., 2012). the cerrado is a biodiversity hotspot being currently affected by landscape changes (soares-filho et al., 2014) and so conservation strategies must be viewed as a priority (overbeck et al., 2015). threats to the cerrado domain and associated biodiversity include land conversion for agriculture and pasture as well as urban expansion (klink and machado, 2005), which in turn can result in the isolation and reduction of the population size of several species and cause local extinctions. amphibians are amongst the most endangered vertebrate groups in the world (stuart et al., 2004; hamer and mcdonnell, 2008; verdade et al., 2010). some factors that affect amphibian populations are habitat destruction, introduced species, climate change, uv-b radiation, pollution and disease (young et al., 2001; verdade et al., 2010). 158 cláudia márcia marily ferreira et alii urbanization causes habitat fragmentation, loss, isolation and degradation (hamer and mcdonnell, 2008) and associated biotic and abiotic habitat changes (aronson et al., 2014), being a process that promotes profound changes in the environment. many amphibian species are threatened by the expansion of urban areas (hamer and mcdonnell, 2008). in urban habitats the major factors impacting amphibians are changes in natural vegetation and hydrological courses (hamer and mcdonnell, 2008), aquatic and terrestrial pollution (paul and meyer, 2001; croteau et al., 2008), predation by domestic animals (woods et al., 2003), roadkills (andrews et al., 2008), and diseases (croteau et al., 2008). urban development may change the species composition and decrease the richness and diversity of amphibians (rubbo and kiesecker, 2005; hamer and mcdonnell, 2008; mckinney, 2008; hamer and mcdonnell, 2010). many species recorded in the urban environment use multiple types of habitat, being habitat generalists (hamer and mcdonnell, 2008). however several species with specific habitat requirements, still poorly known to science or endangered are also recorded in this type of environment (grandinetti and jacobi, 2005; knispel and barros, 2009; ferreira et al., 2010; silva et al., 2011; pereira et al., 2013). at least 84% of the population within brazil resides within urban areas (ibge, 2015) and the state of mato grosso do sul (central brazil) has one of the highest urbanization rates in the country (almeida, 2009). in mato grosso do sul the predominant cerrado vegetation with its distinct physiognomies such as palm swamps (veredas), gallery forests, wet campos, and closed woodlands harbors 97 amphibian species (souza et al., in press). despite this considerable richness, there are few studies on urban species in this region (ávila and ferreira, 2004). the objectives of this study were to describe fig. 1. localization of the 11 urban cerrado fragments in campo grande municipality, mato grosso do sul state, central brazil. 1. itanhangá square (is); 2. sóter ecological park (sep); 3. coqueiral farm (cf); 4. anhanduí ecological park (aep); 5. imbirussu environmental education center (ieec); 6. mato grosso do sul federal university fish center (ufmsf); 7. dom bosco university (dbu); 8. mato grosso do sul federal university cerrado (ufmsc); 9. military college (mc); 10. prosa state park (psp); 11. matas do segredo state park (mssp). 159urban forest anurans in central brazil the species composition and richness of anurans in urban cerrado fragments from campo grande municipality, mato grosso do sul state, central brazil. material and methods study site campo grande municipality has 840,000 habitants living in an area of 8,100 km2. the altitude is 532 m a.s.l. and climate is characterized by a dry (april to september) and wet period (october to march). mean annual rainfall is 1,530 mm and mean annual temperature ranges between 18 and 28.8 oc. around 21% of the area exhibits native cerrado vegetation while the remainder is covered by pastures, agricultural fields and developed areas (planurb, 2015). the urban perimeter of campo grande has an area of 154.4 km2 and a population density of 104 inhabitants/km2. the city has 28 headsprings and 18 green areas (parks and protected areas) covering about 864 hectares (planurb, 2015). eleven urban forest fragments with size ranging from 1.8 to 170.2 ha were selected for this study (fig. 1): itanhangá square (is; 1.8 ha), sóter ecological park (sep; 3.4 ha), coqueiral farm (cf; 10 ha), anhanduí ecological park (aep; 10.8 ha), imbirussu environmental education center (ieec; 13.2 ha), mato grosso do sul federal university fish center (ufmsf; 15.4 ha), dom bosco university (dbu; 28.8 ha), mato grosso do sul federal university cerrado (ufmsc; 35.2 ha), military college (mc; 48.6 ha), prosa state park (psp; 128 ha), and matas do segredo state park (mssp; 170.2 ha). there are bodies of water in all forest fragments, except: dbu, ufmsc and mc. data collection anurans were sampled in three stages from november 2012 to march 2013, november 2013 to april 2014, and november 2014. sampling methodology included pitfall traps and visual/acoustic surveys (heyer et al., 1994). active sampling was made independent of the pitfall trap sampling. the pitfall traps were used in all periods of the study, while visual/ acoustic surveys were used during the second and third stage. each pitfall trap line consisted of four 30 l plastic buckets connected by 50 cm height drift-fences. the number of lines varied from one to two according to fragment size (areas larger than 20 ha had two lines). pitfall traps were kept opened during four consecutive days by month in the first stage of the study and seven consecutive days by month from the second and third stage (to increase the sampling effort), and checked every 48 hours. visual/acoustic surveys were realized at night in one 30 x 30 m quadrat in each fragment. in forest fragments with water bodies, the active sampling was conducted in the wet areas or in their vicinity. in each forest fragment five nights of sampling were conducted, once a month (totaling five months of sampling), except in ufmsc and dbu where we made three and four nights of sampling, once a month, respectively. visual/ acoustic surveys had an average duration of two hours, starting at dusk (18:30 h). voucher specimens were deposited at the coleção zoológica de referência of the universidade federal de mato grosso do sul (zufms, campo grande, mato grosso do sul state). nomenclature followed frost (2017). data analysis to evaluate the sampling effort, we used a species accumulation curve based on the samples, often known as mao tau estimate (chiarucci et al., 2008; colwell et al., 2012). these data were analysed with a presence-absence matrix, with taxa in rows and samples in columns; mao tau estimate is suitable when a number of samples are available (hammer et al., 2001). each forest fragment was considered a sampling unit and the sufficiency in sampling was considered when the slope of the curve approached zero. to estimate the similarity in anuran species composition among forest fragments sampled we used a cluster analysis (upgma) based on a similarity matrix constructed with the sørensen index for the community. for this analysis, only presence and absence data of species on each site were used to be able to concatenate presence of species from the different methods used. it was also used the k-means algorithm to create several partitions forming a cascade from 2 to 6 groups. calinski criteria (calinski-harabasz, 1974) was used to determine the optimum number of groups for the k-means. the groups assigned to each local were then used to determine the indicator value (dufrene and legendre, 1997) of each species to evaluate its importance in determining the local group. all analyses were performed with r (r core team, 2016), using vegan (oksanen et al., 2016) and indicspecies (de caceres and legendre, 2009) packages. results seventeen anuran species from four families were recorded (table 1). leptodactylidae was the richest family (11 species), followed by hylidae (four species) and bufonidae and odontophrynidae (one species each). dendropsophus nanus, hypsiboas punctatus, hypsiboas raniceps and leptodactylus syphax were sampled exclusively by visual/acoustic search while leptodactylus cf. elenae and physalaemus nattereri were sampled only by pitfall traps. richness among sites ranged from one to 10 (table 1). cumulative species curve reached the asymptote with eleven samples (fig. 2), with an expected total richness of 17. based on the calinski criteria two major groups for the sampled locals (fig. 3) were observed, with the group is and sep more similar (75%), followed by psp and mssp (67%) and aep, cf, ufmsf, palm swamps areas, with 57% or more of similarity (table 2). hypsiboas punctatus and leptodactylus podicipinus had the higher indi160 cláudia márcia marily ferreira et alii table 1. anuran species recorded at 11 cerrado urban fragments in campo grande municipality, mato grosso do sul state, central brazil. see text for site abbreviation. species/family sites is sep cf aep ieec ufmsf dbu ufmsc mc psp mssp bufonidae rhinella schneideri (werner, 1894) x x x x x x x x hylidae dendropsophus nanus (bounlenger, 1889) x x x x hypsiboas punctatus (schneider, 1799) x x x x x hypsiboas raniceps cope, 1862 x x scinax fuscovarius (lutz, 1925) x x x leptodactylidae adenomera diptyx (boettger, 1885) x x x x x x x x leptodactylus cf. elenae heyer, 1978 x leptodactylus fuscus (schneider, 1799) x x leptodactylus labyrinthicus (spix, 1824) x x x leptodactylus mystacinus (burmeister, 1861) x x x x leptodactylus podicipinus (cope, 1862) x x x x leptodactylus syphax bokermann, 1969 x x x physalaemus albonotatus (steindachner, 1864) x x x x physalaemus centralis bokermann, 1962 x x physalaemus cuvieri fitzinger, 1826 x x x physalaemus nattereri (steindachner, 1863) x x x odontophrynidae proceratophrys dibernardoi brandão, caramaschi, vaz-silva and campos, 2013 x x x richness 4 4 4 10 7 8 3 1 3 10 8 fig. 2. cumulative species curve (solid line) for 11 cerrado urban fragments in campo grande municipality, mato grosso do sul state, central brazil. gray space represents 95% confidence intervals. fig. 3. similarity cluster analysis (upgma) with the sørensen index for 11 cerrado urban fragments in campo grande municipality, mato grosso do sul state, central brazil. see text for abbreviations. 161urban forest anurans in central brazil cator values for group 1, while no species were distinctiveness for group 2 (table 3). discussion seventeen species of anurans were recorded in the urban area of campo grande, representing 17.7% of the species reported for mato grosso do sul state. the existence of a high number of green areas and water bodies in the urban environment can favor anuran species in the region. most species detected in campo grande are able to tolerate environments altered by anthropogenic factors. some of the species observed are endemic to the cerrado and a new species (p. dibernardoi) was recorded in the state. forest fragments with similar environmental conditions have similar anuran composition. the predominance of leptodactylidae and hylidae species is an expected result because of the large number of species of these families in the neotropics (duellman, 1988), also configuring the most specious families in brazil (segalla et al., 2016). on the other hand, the few species recorded for bufonidae and odontophrynidae in the present study can reflect the low diversity of these taxa in cerrado areas (valdujo et al., 2012). although none of the species recorded in this study table 2. similarity values of sørensen index between 11 cerrado urban fragments in campo grande municipality, mato grosso do sul state, central brazil. ufmsc cf mc ieec is aep ufmsf psp mssp sep cf 0.40 mc 0.00 0.00 ieec 0.00 0.36 0.20 is 0.40 0.50 0.00 0.18 aep 0.18 0.57 0.00 0.59 0.57 ufmsf 0.22 0.67 0.00 0.67 0.33 0.67 psp 0.18 0.43 0.46 0.47 0.29 0.50 0.67 mssp 0.22 0.50 0.36 0.40 0.33 0.56 0.62 0.67 sep 0.00 0.25 0.00 0.36 0.75 0.57 0.17 0.14 0.17 dbu 0.00 0.00 0.33 0.00 0.00 0.00 0.00 0.31 0.18 0.00 table 3. group to which each species was classified, followed by the indicator value for the group 1 (cf, ieec, aep, ufmsf, psp and mssp) and group 2 (ufmsc, mc, is, sep and dbu) and the respective significance for use as an indicator species.   group iv for group 1 iv for group 2 p adenomera diptyx 1 0.563 0.130 0.224 dendropsophus nanus 1 0.667 0.000 0.055 eupemphix nattereri 1 0.208 0.075 1.000 hypsiboas punctatus 1 0.833 0.000 0.016 hypsiboas raniceps    1 0.333 0.000 0.452 leptodactylus cf. elenae 2 0.000 0.200 0.467 leptodactylus fuscus    1 0.333 0.000 0.460 leptodactylus labyrinthicus 2 0.049 0.282 0.562 leptodactylus mystacinus  2 0.152 0.218 1.000 leptodactylus podicipinus 1 0.833 0.000 0.015 leptodactylus syphax  2 0.049 0.282 0.529 physalaemus albonotatus  1 0.667 0.000 0.054 physalaemus centralis 2 0.076 0.109 1.000 physalaemus cuvieri  1 0.208 0.075 1.000 proceratophrys dibernardoi 1 0.208 0.075 1.000 rhinella schneideri  1 0.714 0.114 0.047 scinax fuscovarius 1 0.500 0.000 0.175 162 cláudia márcia marily ferreira et alii was assigned to any threat status category (icmbio, 2014), some points must be considered. the recently described proceratophrys dibernardoi (brandão et al., 2013) represents a new species record for mato grosso do sul state and was found at least in three sampled urban areas. furthermore, physalaemus centralis, p. nattereri e p. dibernardoi are typical cerrado species (valdujo et al., 2012; brandão et al., 2013). as a biodiversity hotspot (myers et al., 2000), the cerrado is suffering from intensive landscape changes resulting from land use (ratter et al., 1997) which requires ongoing research on management, conservation and urban planning (soaresfilho et al., 2014; overbeck et al., 2015). most (70.6%) of the anuran urban species found in campo grande municipality utilize a broad range of habitats and are found in anthropogenic environments, such as dendropsophus nanus (reichle et al., 2004). anurans from urban areas are mainly represented by habitat generalist species (hamer and mcdonnell, 2008; ferreira et al., 2010; zocca et al., 2014) such as scinax fuscovarius (aquino et al., 2010), common species in the urban environment (ávila and ferreira, 2004; grandinetti and jacobi, 2005; rodrigues et al., 2008; torres, 2012; pereira et al., 2013). species that live in urban areas also have high fertility (hamer and mcdonnell, 2010), and reproduce in degraded aquatic environments (lane and burgin, 2008). rhinella schneideri, for example, a species present in urban areas of brazil (ávila and ferreira, 2004; shibatta et al., 2009; corrêa et al., 2014), uses a broad variety of habitats (aquino et al., 2004), females produce more than 5.000 eggs (uetanabaro et al., 2008) and individuals of this species were found in forest fragments in campo grande whose water bodies are contaminated by sewage and garbage. despite the predominance of habitat generalists in urban systems, species habitat specialists are also recorded (ferreira et al., 2010) and species still little known to science (santana et al., 2008). new anuran species are being discovered in the urban environment (santana et al., 2012; feinberg et al., 2014). this means that cities can harbor a considerable biological community that provides interesting appeal for management and environmental education programs (feinberg et al., 2014). the species physalaemus centralis and p. nattereri are more sensitive to anthropogenic habitat disturbances (aquino et al., 2004; colli et al., 2004) than others species recorded in this study; maybe for this reason there were low numbers recorded in the urban area (melo et al., 2007; present study). proceratophrys dibernardoi is a species with few biological data available because it was recently discovered (brandão et al., 2013). the forest fragments with high similarity among species were those that presented similar environmental conditions, such as types of vegetation and levels of anthropogenic impact. itanhangá square and sep have the highest similarity between species. they are smaller fragments and are much modified by human activities. the two green areas are surrounded by built-up areas and suffer great pressure of the surroundings. matas do segredo state park and psp are the largest fragments present in the urban area of campo grande and also the best preserved ones. the mssp and psp harbor headsprings and have similar cerrado physiognomies (imasul, 2009, 2011). the psp is surrounded by roads and other human constructions, whereas the mssp has part of its area facing rural area. the palm swamps of aep, cf and ufmsf have a small area and suffer great anthropogenic surrounding pressures. anhanduí ecological park is a palm swamp in regeneration and the entire fragment is surrounded by roadways and is affected by sporadic fires. coqueiral farm is a small privately owned area and harbor bandeira’s headspring. the site is completely surrounded by roads and is impacted with urban development in its vicinity. mato grosso do sul federal university fish center has a lot of garbage and the cabaça stream is polluted by sewage (semadur, 2015). the fragment is completely surrounded by human constructions. the knowledge of urban biodiversity is important since forest fragments in cities suffer high ecological impacts mainly associated to border effect with direct consequence for forest and litter dwelling species. longterm and multidisciplinary studies are required to understand the dynamic connections between environmental and socioeconomic processes in the cities (tanner et al., 2014), resulting in important tools for conservation and management programs for urban ecosystems. preserving green areas inside an urban matrix is important for biodiversity protection (cornelis and hermy, 2004). for anurans, urban forest fragments are extremely important sites since they represent areas for shelter, feeding, dispersion and reproduction (becker et al., 2008; hamer and macdonnell, 2008; sabbag and zina, 2011). in this way, some actions for conserving anurans in urban areas should include green areas restoration and management, including connecting corridors to facilitate the possibility of species dispersal. acknowledgments we thank instituto de meio ambiente de mato grosso do sul for authorization to carry out this study in state conservation units; secretaria municipal de meio ambiente e desenvolvimento urbano for authorizing this study in parks and other green areas of the 163urban forest anurans in central brazil city. we thank commanders of the military college (colonels gil de melo rolim and ricardo guilherme ribeiro de almeida), hemerson pistori and the owner of the coqueiral farm by authorizations for the collection respectively at the mc, the dbu and cf. we thank the diego santana, jose luiz massao, liliane piatti and vanda lucia ferreira for helping us with species identification; jeffrey himmelstein for reviewing the english. we thank cnpq for project financing, capes for the scholarship granted to author cláudia márcia marily ferreira and icmbio/ sisbio by the authorization (nº. 10379) to collect and transport the animals. franco leandro de souza receives grant from cnpq (303006/2014-5). references almeida ra. 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(2014): uso do espaço por anuros em ambiente urbano de santa teresa, espírito santo. bol. mus. de biol. mello leitão (n. sér.) 35: 105-117. acta herpetologica vol. 12, n. 2 december 2017 firenze university press meristic and morphometric characters of leptopelis natalensis tadpoles (amphibia: anura: arthroleptidae) from entumeni forest reveal variation and inconsistencies with previous descriptions susan schweiger1, james harvey2, theresa s. otremba1, janina weber1, hendrik müller1,* brown anole (anolis sagrei) adhesive forces remain unaffected by partial claw clipping austin m. garner*, stephanie m. lopez, peter h. niewiarowski species and sex comparisons of karyotype and genome size in two kurixalus tree frogs (anura, rhacophoridae) shun-ping chang1,2, gwo-chin ma2,3,4, ming chen2,5,6,7,8,*, sheng-hai wu1,* non-native turtles in a peri-urban park in northern milan (lombardy, italy): species diversity and population structure claudio foglini1, roberta salvi2,* species composition and richness of anurans in cerrado urban forests from central brazil cláudia márcia marily ferreira1,*, augusto cesar de aquino ribas2, franco leandro de souza3 the life-history traits in a breeding population of darevskia valentini from turkey muammer kurnaz, alı i̇hsan eroğlu, ufuk bülbül*, halıme koç, bılal kutrup influence of desiccation threat on the metamorphic traits of the asian common toad, duttaphrynus melanostictus (anura) santosh mogali*, srinivas saidapur, bhagyashri shanbhag predation of common wall lizards: experiences from a study using scentless plasticine lizards jenő j. purger*, zsófia lanszki, dávid szép, renáta bocz reproductive timing and fecundity in the neotropical lizard enyalius perditus (squamata: leiosauridae) serena najara migliore1,2,*, henrique bartolomeu braz2,3, andré felipe barreto-lima4, selma maria almeida-santos1,2 observations on the intraspecific variation in tadpole morphology in natural ponds eudald pujol-buxó1,2,*, albert montori1, roser campeny3 and gustavo a. llorente1,2 reliable proxies for glandular secretion production in lacertid lizards simon baeckens diet of juveniles of the venomous frog aparasphenodon brunoi (amphibia: hylidae) in southeastern brazil rogério l. teixeira1, ricardo lourenço-de-moraes2, débora c. medeiros3, charles duca3, rogério c. britto4, luiz c. p. bissoli5, rodrigo b. ferreira3,* who are you? the genetic identity of some insular populations of hierophis viridiflavus s.l. from the tyrrhenian sea ignazio avella, riccardo castiglia, gabriele senczuk* acta herpetologica 13(2): 141-146, 2018 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-20920 the relationship between brain size and digestive tract length do not support expensive-tissue hypothesis in hylarana guentheri ya ting liu, yi luo, jun gu, sha jiang, da yong li, wen bo liao* key laboratory of southwest china wildlife resources conservation (ministry of education), china west normal university, nanchong, 637009, china. *corresponding authors. e-mail: liaobo_0_0@126.com submitted on: 2017, 11th september; revised on: 2018, 15th march; accepted on: 2018, 6th may editor: giovanni scillitani abstract. the brain is among the most energetically costly organs in the vertebrate body. the expensive-tissue hypothesis (eth) predicts that increasing the size of another costly organ, such as the gut, should compensate for the cost of a small brain. to date, this hypothesis has mainly been tested in homoeothermic animals and in some ectothermic animals (e.g., fishes and anurans). here, we undertake a test of the eth by analyzing the relationship between brain size variation and length of the digestive tract in hylarana guentheri. after controlling for geographical situation and body size, we did not find a correlation between brain mass and the length of the digestive tract in h. guentheri. our findings suggest that the variation of brain size did not follow general patterns in this species and that the effect of diet quality cannot play a role in the variation of brain. keywords. brain size; energetic constraints; expensive-tissue hypothesis; hylarana guentheri. introduction as an important cognitive organ of animals, increased brains confirm to improve the cognitive ability (striedter, 2005; jiang et al. 2015; luo et al., 2017; wu et al., 2016; yu et al., 2018). however, brains are one of the most metabolically costly tissues in the vertebrate body (mink et al., 1981). the high amount of energy related to maintaining brain tissues should constrain on evolution of brain size (striedter, 2005; isler and van schaik, 2006), although the brains can confer cognitive ability (kotrschal et al., 2013; liao et al., 2015a; zeng et al., 2016; kotrschal et al., 2017; samuk et al., 2018). the expensive-tissue hypothesis (eth) predicts that the enlarged brains will inevitably decrease the sizes of other metabolically costly tissues, such as gut (aiello and wheeler, 1995). there are evidences that the evolution of brain size follows the prediction of the eth in animal kingdom (isler and van schaik, 2006; navarrete et al., 2011; jin et al., 2015; tsuboi et al., 2015; liao et al., 2016a; sukhum et al., 2016). since the eth was developed, primarily comparative studies have tested their predictions. while some studies have shown that negative associations between brain mass and gut size support the eth (kaufman et al., 2003; tsuboi et al., 2015; liao et al., 2016a), other studies do not find such patterns (lemaître et al., 2009; barrickman and lin 2010; navarrete et al., 2011). most studies on the cost of brain size evolution have mainly focused on birds and mammals (e.g., aiello and wheeler, 1995; jones and maclarnon, 2004; isler and van schaik, 2006; pitnick et al., 2006; navarrete et al., 2011). in recent years, the cost of brain size evolution in ectothermic vertebrates has been investigated at inter-specific species level (tsuboi et al., 2015; liao et al., 2016a; sukhum et al., 2016) and intra-specific species level (kotrschal et al., 2013; liu 142 ya ting liu et alii et al., 2014; jin et al., 2015; zhao et al., 2016; gu et al., 2017; yang et al., 2018). for instance, a negative correlation between brain mass and the length of the digestive tract have been found in rana omeimontis (jin et al., 2015) while zhao et al. (2016) do not find any correlation between them in the dark-spotted frog (pelophylax nigromaculata). here we provide the test of the relationship between the brain mass and length of the digestive tract among hylarana guentheri populations. based on the eth, we predict that a larger brain should accompany a smaller digestive tract in the frog. material and methods the guenther’s frog, hylarana guentheri is found in the subtropical forests in china at elevations ranging from 500 to 1100 m (fei and ye, 2001). the species exhibits a variable activity period. males actively look for females and used advertisement calls to attract them (liu et al., 2011). in this species, mating and egg-laying extends from march to may, as a prolonged breeder (wells, 1977). we collected a total of 135 frogs from six populations along a 420-km latitudinal and 341-m altitudinal transect across the hunan, hubei and sichuan provinces in china between june and august in 2016 (figure 1). all individuals were captured by hand in paddy fields and pools at night and confirmed at the adult stage by direct observations of secondary sexual characteristics (see details in liao and lu, 2010; jin et al., 2015; liao et al., 2015b). we recorded the altitude, latitude and longitude of each population because environmental factors will affect morphology and physiology of organism (chen et al., 2016; yang et al. 2017; liao et al., 2016b; tang et al., 2018; zhong et al., 2017; wang et al., 2017; liao et al., 2018; qin et al., 2018; wu et al., 2018). we took all individuals to the laboratory and kept individually in rectangular tanks (0.5 × 0.4 × 0.4 m) before being anesthetized with benzocaine. each individual was killed by single-pithing and preserve in 4% buffered formalin in a phosphate buffer (liao et al., 2016a; lüpold et al., 2017; mai et al. 2017a, b). we measured body size of all individuals (snout-vent length: svl) to the nearest 0.01 mm with a caliper, and body mass to the nearest 0.1 mg with an electronic balance. all frogs were dissected, and their brains were removed (see detail in liao et al., 2015a; jin et al., 2016). we then weighed and measured all brains to the nearest 0.1 mg with an electronic balance. we used a canon sx530 hs digital camera to take digital images of digestive tract with a caliper. we then measured length of digestive tract using a motic images 3.1 digital camera mounted on a moticam 2006 light microscope at a 400× magnification. all statistical analyses were performed using spss 22.0 (statistical product and service solutions company, chicago, usa). we ran a linear mixed models (lmms) with log10fig. 1. topographic original maps showing the sampling locations of the six populations in hylarana guentheri in china. 143no evidence for eth in a frog transformed brain mass as a dependent variable, population as a random factor, and log10-transformed digestive tract length, latitude, longitude, sex and altitude as fixed effects, log10-transformed body size as covariate to test the original eth. within each sex, we also used lmms to test the correlation between brain size and digestive tract length. results we at first analyzed the association between relative brain size and relative digestive tract length to evaluate the original eth. we found that relative brain size was not correlated with relative size of digestive tract when controlling for the effect of the body size (table 1). there was a non-significant variation in brain size among populations (z = 0.656, p = 0.512). moreover, sex, latitude, longitude and altitude did not affect the relative brain size (table 1). the lmms revealed that for males relative brain size was not correlated with relative length of digestive tract (fig. 2; f1, 80.413 = 0.573, p = 0.451) and the environmental factors (e.g., latitude: f1, 1.97 = 1.069, p = 0.411; longitude: f1, 2.117 = 0.01, p =0.93; altitude: f1, 2.1 = 0.674, p = 0.494). however, there was a significant correlation between brain size and body size (f1, 79.354 = 96.174; p < 0.001). for females, the relative brain size was not correlated with digestive tract length (fig. 3; f1, 49 = 2.034, p = 0.16) and the environmental factors (e.g., latitude: f1, 49 = 0.002, p = 0.969; longitude: f1, 49 = 0.050; p = 0.943). however, we also found that brain size was positively correlated with the body size (f1, 49 = 57.732; p < 0.001). discussion our results uncover a non-significant association between brain size and digestive tract size after controlling for several environmental factors and body size in h. guentheri, which do not support the original “brain versus gut” prediction arising from the eth. thus, our study does not support the existence of energetic constraints as important factors influencing patterns of brain size diversification among individuals in frogs. below, we discuss our results in more detail considering the main hypothesis addressed. although the data set on which the eth is based refers to primates (aiello and wheeler, 1995), most contable 1. regression models of brain size on digestive tract in relation to various predictor variables in species when controlling for body size. source d.f. predictor f p brain size 1, 133.961 digestive tract 1.182 0.279 1, 134.993 sex 1.104 0.295 1, 2.002 altitude 3.517 0.201 1, 1.999 longitude 2.152 0.280 1, 1.981 latitude 0.028 0.883 1, 110.113 body size 150.942 <0.001 fig. 2. plot of relative brain size and relative digestive tract length of the sampled male individuals of hylarana guentheri. fig. 3. plot of relative brain size and relative digestive tract length of the sampled female individuals of hylarana guentheri. 144 ya ting liu et alii vincing evidences favoring the eth come from ectothermic animals (e.g., gnathonemus petersii: kaufman et al., 2003; rana omeimontis: jin et al., 2015; poecilia reticulata: kotrschal et al., 2013; tanganyika cichlids: tsuboi et al., 2015; anurans: liao et al., 2016a). the negative relationship between brains and guts is not observed in homothermic animals (isler and van schaik, 2006, jones and maclarnon, 2004; barrickman and lin, 2010; navarrete et al., 2011). hence, the eth might explain cases of encephalization in specific lineages of homoeotherms but it is not valid in overall these groups (aiello et al., 2001). in contrast to the homoeotherms where brain mass corresponds to 1-2% of total body mass (striedter, 2005), frogs and toads have smaller brain mass that, on average, corresponds to 0.3% of body mass (liao et al., 2016a). the robust demonstrations of negative associations between brain and gut across species suggest that energetic constraints play an important role in vertebrate, especially ectotherm brain evolution. for h. guentheri, the energetic constraints cannot explain brain size variation because of the lack of correlation between brain size and digestive tract length. there is at first an inverse correlation between one very costly organ, the brain, and the very organ that procures energy, the gut. potentially this correlation may materialize if an increased brain enables a potentially cognitively driven shift towards a more nutritious diet (liao et al., 2016a). there is evidence that the smallestbrained species are primates, with the largest guts feed on plant matter, while the larger-brained species are more omnivorous and humans, with the largest brain and smallest gut, even outsourced part of its gut function to cooking pots (aiello and wheeler, 1995). previous studies have argued to find support for the eth in primates by using diet quality as a proxy of gut morphology (fish and lockwood, 2003). however, diet quality does not correlate with brain size in lemurs and lorises (gonzalez-voyer et al., 2009; allen and kay, 2011). within the anurans, because most are insectivorous as adults, the potential for dietary divergence may be limited. however, anurans may feed on insects of different nutritive value, which may demand varying levels of cognitive ability to catch (liao et al., 2016a). hence, diet quality might affect the brain size variation in anuran species. the energetic constraints play an important role in promoting brain size variation within single species because there are negative associations between brains and guts (kotrschal et al., 2013; jin et al., 2015). however, inconsistent with the eth, the relative brain size did not exhibit a negative correlation with digestive tract length in h. guentheri. similar pattern has been supported in the other two frogs (e.g., p. nigromaculata: zhao et al., 2016; fejervarya limnocharis: yang et al., 2017). the discrepancy among above studies also may result from the diet quality (isler and van schaik, 2006). there is evidence that a high diet quality combined with a large brain and a low diet quality combined with small brain has been reported in a frog r. omeimontis (jin et al., 2015), which suggests that more clever individuals (larger brains) make use of better food and therefore develop smaller guts. however, we did not find a correlation between brain and gut in h. guentheri, suggesting that diet quality is not combined with brain size variation. in conclusion, our findings suggest that the expensive-tissue hypothesis is not supported in h. guentheri because brain size and gut length are not inversely related. thus, the energetic constraints do not play an important role in promoting brain size variation for this species. acknowledgements financial support was provided by the national natural sciences foundation of china (31471996; 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(2017): altitudinal variation in organ size in polypedates megacephalus. herpetol. j. 27: 235-238. acta herpetologica vol. 13, n. 1 june 2018 firenze university press acta herpetologica 13(2): 101-108, 2018 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-22886 yellow patterns polymorphism of the fire salamander salamandra salamandra in poland anna najbar1,3,*, agnieszka konowalik1,3, bartłomiej najbar2, maria ogielska1 1 department of evolutionary biology and conservation of vertebrates, university of wrocław, sienkiewicza 21, 50-335 wrocław, poland. *corresponding author. e-mail: anna.najbar@uwr.edu.pl 2 faculty of biological sciences, university of zielona góra, prof. z. szafrana 1, 65-516 zielona góra, poland 3 a. najbar and a. konowalik contributed equally to this work submitted on: 2018, 13th march; revised on: 2018, 15th may; accepted on: 2018, 9th july editor: marco sannolo abstract. we analysed variation of dorsal yellow patterns in 2077 individuals that represented 23 populations from the northern parts of the sudetes and the carpathian mountains. we distinguished four types of yellow patterns: spotted (50.1%), spotted-and-striped (42.8%), zig-zag (5.8%), and striped (1.3%). spotted form dominated in the east (69.2%, the carpathians) and its frequency decreased to the west (46.4%, the sudetes), where spotted-and-striped forms became more common. the rarest in both mountain ranges (0.9% in the carpathians, 1.3% in the sudetes) was striped type. the exception was the westernmost population represented by 15.8% of the striped individuals. we did not find evidence of differences between yellow colouration variants and genotypes of 557 individuals defined by 10 microsatellite loci. no differences were found between females and males. we concluded that patterning does not constitute a unique feature at population and mountain ranges levels. keywords. amphibians, colouration, dorsal yellow patterns, poland, salamandra salamandra. introduction the fire salamander salamandra salamandra (linnaeus, 1758) is highly polytypic and widely distributed urodelan in europe (kuzmin et al., 2009). the species consists of over a dozen divergent geographical subspecies traditionally distinguished by various patterns of yellow dots and/or stripes on the black background of the dorsal side, with a great variety observed at regional, local, and individual levels (e.g., freytag, 1955; eiselt, 1958; veith, 1992; thiesmeier, 2004; velo-antón and buckley, 2015). for many years, yellow-black colouration had been explained as a cryptic colouration closely linked to climate and quality of the substrate or background of habitats (e.g., frish, 1920). nowadays, it is known that phenotype depends on heritable genetic variation, and functions as an aposematic colouration (thiesmeier, 2004; thiesmeier and grossenbacher, 2004). the differences among populations are explained by evolutionary processes, such as natural selection. among presumed factors, e.g., van alphen and arntzen (2016) mentioned predation intensity or thermal conditions. current classification of salamandra species and subspecies was supported by molecular studies, and some morphologically recognizable subspecies were also genetically differentiated (steinfartz et al., 2000; steinfartz and tautz, 2003). on the other hand, phenotypical similarities exist between closely related species e.g., s. salamandra and s. infraimmaculata, or subspecies e.g., s. s. salamandra and s. s. terrestris (degani, 1986; thiesmeier, 2004). colour polymorphism is mostly evident among the fire salamanders inhabiting the iberian peninsula (alcobendas et al., 1996; thiesmeier and grossenbacher, 2004; reis et al., 2011; veloantón and buckley, 2015), but no genetic or geographical 102 anna najbar et alii divergence was recently revealed between phenotypically differentiated subspecies s. s. bernardezi or s. s. alfredschmidti (beukema et al., 2016). therefore the authors suggested to revoke subspecies status of the latter taxon. poland is inhabited by the nominative subspecies s. s. salamandra, characterized by yellow, or rarely orange, irregular spots and stripes on the dorsal part of the body (juszczyk, 1987; zakrzewski, 2007). the classification of dorsal yellow patterns described by eiselt (1958) was further applied by juszczyk (1987) and zakrzewski (2007) for individuals collected in the carpathians. originally it contained four forms: spotted, striped, striped-spotted, and spotted-striped. furthermore paluch and profus (2004) proposed to distinguish the fifth, i.e., zig-zag form. according to available data, spotted and striped-spotted forms dominate, and striped one is the rarest among the fire salamanders in poland (juszczyk, 1987; paluch and profus 2004; zakrzewski, 2007). in addition, zig-zag form was described as extremely rare (zakrzewski, 2007). here, we present diversity and distribution of dorsal yellow patterns of the fire salamanders from the species entire range in poland. the aim of this study was to characterize differences at population and mountain range levels, and between females and males. we assume that a gradient of dorsal patterns exists with domination of spotted form in the east (the carpathians), and striped form in the west (the sudetes). we also tested the possible relationship between pattern types and genotypes based on 10 microsatellite loci. material and methods sampling and study area 2077 individuals were collected during active seasons (april-october) in the years 2004-2016 from 23 populations in poland. the study area represents the western, central and eastern sudetes mountains (16 populations), and the outer western and outer eastern carpathian mountains (seven populations). they constitute two main mountain ranges in the country, isolated by the moravian gate (depression between the eastern sudetes and the western carpathians). polish populations are located at the northern margin of the species range and inhabit mountainous areas including their foothills table 1. characteristic of sampling sites, years and the number of the collected individuals of the fire salamander salamandra salamandra from poland. population number mountain range locality altitude (m a.s.l.) years of samples collection (number of collected individuals) 1 the sudetes eastern upper lusatia 283 2015 (22), 2016 (16) 2 the sudetes izera mts. 470 2008 (51), 2009 (59) 3 the sudetes katzbach mts. 494 2008 (24), 2009 (71) 4 the sudetes kaczawskie piedmont 392 2008 (46), 2009 (57) 5 the sudetes kaczawskie piedmont 394 2005 (116), 2006 (20), 2008 (7), 2009 (53) 6 the sudetes wałbrzyskie mts. 509 2008 (26), 2009 (58) 7 the sudetes stołowe mts. 589 2008 (40), 2009 (64) 8 the sudetes ślęża massif 263 2004 (139), 2005 (28) 9 the sudetes bardzkie mts. 407 2008 (42), 2009 (60) 10 the sudetes bardzkie mts. 356 2006 (59), 2007 (38) 11 the sudetes bardzkie mts. 279 2008 (41), 2009 (52) 12 the sudetes bardzkie mts. 418 2008 (45), 2009 (67) 13 the sudetes bardzkie mts. 294 2008 (40), 2009 (53) 14 the sudetes śnieżnik massif 372 2007 (5), 2008 (42), 2009 (54) 15 the sudetes golden mts. 496 2007 (122), 2009 (57), 16 the sudetes opawskie mts. 401 2014 (44), 2015 (18) 17 the carpathians bielsko-biała city, silesian piedmont 394 2015 (35), 2016 (43) 18 the carpathians rożnów piedmont 351 2014 (4), 2016 (38) 19 the carpathians the ciężkowice piedmont 378 2014 (8), 2015 (9), 2016 (41) 20 the carpathians strzyżów piedmont 326 2014 (5), 2015 (2) 21 the carpathians lower beskids 417 2014 (2), 2016 (42) 22 the carpathians lower beskids 440 2014 (12), 2015 (40) 23 the carpathians bieszczady mts. 615 2016 (60) 103polymorphism of s. salamandra from poland (głowaciński and zakrzewski, 2003). dominant vegetation component in the sudetes sampling sites are mixed or deciduous forests (including dentario enneaphylli-fagetum) with predominance of the european beech fagus sylvatica and admixture of e.g. spruce picea sp., fir abies sp. or sycamore acer sp. (see ogrodowczyk et al., 2010). in the carpathians, salamanders inhabit large beech dentario glandulosae-fagetum or mixed forests with f. sylvatica as dominant species and highly shaded bottom with thick layer of litter. the exception is population no. 17 that inhabits small, highly isolated and contaminated heterogeneous forest, located within urbanized area of bielsko-biała city (for details see najbar et al., 2017). although the carpathians reach higher altitudes than the sudetes, populations from both ranges are located at an average altitude of 400 m above the sea level. altitudes of each study site were determined in the center of a sampled site using google earth software (google inc.). detailed description of the study area is presented in table 1 and fig. 1. straight line minimum distance was approx. 1.1 km from population no. 11 to population no. 13 in the sudetes, and maximum distance reached approx. 580.6 km between population no. 1 and 23 (fig. 1). distances between populations were designated in quantum gis 2.18.2 software (qgis development team 2013). in each population, transects were established by a breeding stream and salamanders were collected in the buffer of approx. 50 m up to 100 m on its both sides. statistical and molecular analysis statistical analyses were performed in statistica 12 software (statsoft, usa). pearson’s χ2 and post-hoc χ2 were used to find differences in compositions of dorsal yellow patterns of individuals: a) from the sudetes and the carpathians, b) from westernmost population no. 1, the sudetes and the carpathians, and c) between males and females. pearson’s χ2 was not used to test differences between populations due to small number or absence of striped and zig-zag forms, but frequencies of dorsal yellow patterns are presented in table 2 and supplementary table s1. to determine possible relationship between genetic variation and type of dorsal colour pattern we used polymorphism of 10 microsatellite loci: sal3, sal23, sal29, sale5, sale6, sale7, sale8, sal e11, sale12, sale14 (steinfartz et al., 2004), that were obtained in our previous study concerning population genetics of s. salamandra in poland (for protocol see najbar et al., 2015; konowalik et al., 2016). genotypes of 557 individuals representing 19 populations from the entire polish range were grouped according to five forms (see below preliminary results) and four forms in applied classification (table 3). values of fst between pairs of colour variants were calculated in arlequin 3.5 (excoffier and lisher, 2010). their significance was tested with 10 000 permutations. dorsal yellow patterns classification according to the criteria of eiselt (1958), then applied by juszczyk (1987), paluch and profus (2004), and zakrzewski (2007), the classification of s. salamandra dorsal yellow patterning includes five distinct forms: spotted, striped, zig-zag, striped-spotted and spotted-striped (fig. 2, supplementary table s1). however, the last two (striped-spotted and spottedfig. 1. salamandra salamandra distribution range (kuzmin et al., 2009) and the study sites in the sudetes (left, populations 1 – 16) and the carpathians (right, populations 17 – 23) in poland. mg – the moravian gate. map was created in coreldraw x3 software (corel corporation, canada). 104 anna najbar et alii striped) reveal difficulties in their unquestionable recognition because intermediate forms may occur. to avoid significant mistakes while comparing various studies, we considered whether we were allowed to pool striped-spotted and spottedstriped forms into one spotted-and-striped form. preliminary results supported simplification of the classification because 1) their frequencies were comparable in the entire studied area (supplementary table 2), and 2) no relationship between genotypes and patterning existed (table 3a). although we found significant differences in frequencies of striped-spotted and spotted-striped forms between the sudetes and the carpathians (post hoc χ2 = 13.13, p = 0.001), the above arguments supported this procedure. therefore, in this study we applied the simplified classification of yellow patterns including four forms: a) spotted, b) striped, c) zig-zag, and d) spotted-and-striped (fig. 2). all the collected salamanders were documented in the pictures and checked in order to avoid analysis of the same individual twice or more times. moreover, selected salamanders from the entire range were marked by toe clipping for further research what helped to recognize previously sampled individuals. results spotted form dominated in the entire s. salamandra range and constituted 50.1% of all the collected individuals. however, its frequency was higher in the carpathians (69.2%) than in the sudetes (46.4%). the frequency of the remaining forms in the range was: 42.8% of spotted-and-striped form, 5.8% of zig-zag, and only 1.3% of striped form. the exception was population no. 1 where 15.8% of individuals represented striped form and the table 2. frequency of dorsal yellow patterns of the fire salamander salamandra salamandra within populations, mountain ranges and sexes. the carpathian populations are presented in bold. location n frequency [%] spotted striped zig-zag spotted-and-striped the entire species range in poland 2077 50.1 1.3 5.8 42.8 the sudetes 1736 46.4 1.3 6.5 45.8 the carpathians 341 69.2 0.9 2.3 27.6 in each population: 1 38 13.2 15.8 13.2 57.9 2 110 60.0 0.0 4.5 35.5 3 95 46.3 1.1 4.2 48.4 4 103 65.0 0.0 2.9 32.0 5 196 27.0 0.0 1.0 71.9 6 84 57.1 1.2 10.7 31.0 7 104 47.1 1.9 1.0 50.0 8 167 47.3 0.6 4.8 47.3 9 102 43.1 2.0 3.9 51.0 10 97 50.5 0.0 14.4 35.1 11 93 52.7 1.1 8.6 37.6 12 112 51.8 1.8 13.4 33.0 13 93 50.5 1.1 9.7 38.7 14 101 43.6 1.0 12.9 42.6 15 179 46.4 0.0 6.7 46.9 16 62 32.3 8.1 1.6 58.1 17 78 55.1 2.6 1.3 41.0 18 42 73.8 0.0 0.0 26.2 19 58 77.6 0.0 8.6 13.8 20 7 42.9 0.0 0.0 57.1 21 44 63.6 0.0 0.0 36.4 22 52 76.9 1.9 1.9 19.2 23 60 76.7 0.0 1.7 21.7 females 936 47.9 1.0 5.9 45.3 males 884 46.5 1.9 5.4 46.2 juveniles 252 70.2 0.0 7.1 22.6 105polymorphism of s. salamandra from poland lowest frequency (13.2%) of spotted salamanders was found. in addition, population no. 16 also revealed higher number (8.2%) of striped forms. detailed predominance and frequency of dorsal colour patterns within populations are presented in table 2 and fig. 3a, and supplementary table s1. statistical analyses showed significant differences in the frequencies of dorsal yellow patterns between salamanders from the sudetes and the carpathians (χ2 = 60.72, p < 0.001), and while comparing population no. 1 with western (the sudetes) and eastern (the carpathians) groups of populations (χ2 = 138.48, p < 0.001). post-hoc table 3. dorsal yellow patterns pairwise fst (below diagonal) for: a) classification of eiselt (1958), applied by juszczyk (1987), paluch and profus (2004) and zakrzewski (2007) for polish populations, and b) simplified classification used in this study. above diagonal fst p values are presented. a n spotted striped zig-zag striped-spotted spotted-striped 297 spotted 0.758 0.887 0.412 0.273 8 striped -0.004 0.849 0.703 0.915 22 zig-zag -0.003 -0.008 0.368 0.934 84 striped-spotted 0.001 -0.005 -0.002 0.719 146 spotted-striped 0.001 -0.009 -0.004 -0.001 b n spotted striped zig-zag spotted-and-striped 297 spotted 0.763 0.886 0.158 8 striped -0.004 0.841 0.842 22 zig-zag -0.003 -0.008 0.870 230 spotted-and-striped 0.001 -0.007 -0.003 fig. 2. classification of colour patterns of the fire salamander salamandra s. salamandra. a – spotted form: irregular spots also on vertebral line; b – striped form: two rows of full or discontinuous stripes, vertebral line remains black without any spots; c – zig-zag form: continuous, rarely interrupted in one or two places, linear pattern that covers vertebral line; d – spotted-and-striped form: spots and striped, vertebral line remains black or may be disturbed by yellow spots; this intermediate form consists of striped-spotted and spotted-striped forms described by eiselt (1958), and applied for polish populations of the species by juszczyk (1987), paluch and profus (2004) and zakrzewski (2007). 106 anna najbar et alii χ2 results revealed differences between mountain ranges in the proportion of spotted (χ2 = 59.46, p < 0.001), zig-zag (χ2 = 9.00, p = 0.003), and spotted-and-striped individuals (χ2 = 38.69, p < 0.001). population no. 1 differed in the proportion of spotted (χ2 = 17.23, p < 0.001) and striped (χ2 = 62.18, p < 0.001) forms with salamanders from the sudetes, and in the proportion of all forms with individuals collected in the carpathians (for all p < 0.001). we did not find differences between males and females (χ2 = 3.36, p = 0.340). values of pairwise fst between forms of dorsal patterning varied from -0.008 to 0.001 (for all p > 0.05; table 3b). the results suggested no evidence of genetic differences between individuals that represent defined patterns at least according to variation of selected molecular markers used in this study. discussion it has been recognized that spotted forms of s. salamandra are characteristic of eastern europe, while striped individuals with two yellow bands are dominant in the west (e.g., eisely, 1958; thiesmeier, 2004). in poland, s. s. salamandra is mainly represented by individuals with irregular spots and/or stripes on the dorsal side of the body. in our study, spotted form was the most common, whereas striped form was the rarest (table 2). predomination of spotted and striped-spotted forms (a part of spotted-and-striped form according to classification applied for this study, fig. 2d) was previously reported by juszczyk (1987), paluch and profus (2004), and zakrzewski (2007). this result complies with distribution of s. salamandra subspecies recognized on the basis of variety in regionally observed colouration. in relation to other populations, substantial predominance of striped form, characteristic of s. s. terrestris (lacépede, 1788), was observed in site no. 1. this population is located at the polish-german border along the lusatian neisse (najbar and najbar, 2015), where ranges of these two subspecies contact (thiesmeier, 2004). frequency of striped form in this site was higher than in the remaining sites (table 2), while spotted and zig-zag represented the minority of observed individuals. significant dominance of striped patterns in the extremely western population no. 1 may suggest natural admixture with s. s. terrestris, as was also suggested by thiesmeier (2004). such results indicated an increase in the frequency of striped form towards the west. on the other hand, relatively large number of striped salamanders was observed also in population no. 16 located in the opawskie mountains (fig. 1). likewise, paluch and profus (2004) reported 4.8% of salamanders with the striped dorsal pattern in population from morzyszów, which is distanced of about 130 km to the east of population no. 1. although we observed striped and zig-zag salamanders in both the sudetes and the carpathians (table 2), these patterns were confirmed to be rare similarly to other studies from poland (juszczyk, 1987; paluch and profus, 2004; zakrzewski, 2007). phenotypes of several salamandra species and their subspecies tend to be misleading in their recognition (e.g., bickford et al., 2007), and the fire salamanders collected in this study resemble at least two subspecies, e.g. s. s. terrestris and s. s. beschkovi (obst, 1981). kowalski and młynarski (1965) reported similarities in yellow patterning between some of the collected individuals from poland and those representing western european subspecies s. s. terrestris, whereas juszczyk (1987) found similarities to s. s. algira, s. s. infraimmaculata or s. s. bejarae fig. 3. frequency of dorsal yellow patterns: a) in the carpathians and the sudetes, and b) the carpathians, the sudetes (excluding population no. 1), and the westernmost population no. 1. 107polymorphism of s. salamandra from poland according to subspecies classification of eiselt (1958). the first two do not exist in the current systematics anymore (joger and steinfartz, 1995; steinfartz et al., 2000) and were recognized as distinct species: s. algira (bedriaga, 1883) and s. infraimmaculata (mertens, 1948). on the other hand, the existence of several distinct phenotypes may be observed in spanish populations of s. s. bernardezi (beukema et al., 2016). sexual polymorphism has been frequently reported for amphibian species, including body size, colour dichromatism, pigmentation, quality or quantity of patterns (e.g., kupfer, 2007; davis and grayson, 2008; hoffman and blouin, 2008). in s. salamandra, sex-related morphological features, such as size and shape of the body, were usually observed (e.g., zakrzewski, 2007; labus et al., 2013; balogová and uhrin, 2015). our study did not reveal relationship between the occurrence of exact dorsal patterns and sexes, and correspond to previous conclusions of zakrzewski and wójcik (1982) that females and males did not differ in the topography of spots. however, a recent study of balogová and uhrin (2015) from the slovakian carpathians revealed the existence of sexual dichromatism in s. s. salamandra, and concluded that males were covered with larger yellow spots on dorsal side than females. genetic diversity in 10 microsatellite loci did not showed correlation with a variety of dorsal yellow patterns. however, the westernmost population no. 1 was not included to genetic analyses because the site was discovered later than when we conducted population genetics study (najbar et al., 2015; konowalik et al., 2016). veith (1992) excluded the possibility to recognize of s. s. salamandra and s. s. terrestris by color patterns in wide zone of hybrid populations from germany. therefore, further genetic analyses should be performed to confirm our hypothesis about possible occurrence of the second subspecies, s. s. terrestris, in poland. acknowledgements we thank editor and two anonymous reviewers for useful comments that helped to improved our manuscript. we also thank wiesław babik for useful suggestions, and tomasz januszkiewicz, ewa kunicka-najbar, monika malinowska, joanna górajewska, katarzyna serwa, and anna szwedor for help with samples collection. the project was approved by polish ministry of environment (permit no. dopog.-4201-02-74/05kl), general directorate for environmental protection (permit no. dzp-wg.6401.02.7.2014.jro), regional directorate for environmental protection in wrocław (permit no. wpn.6401.211.2015.mr.2), and the ii local ethics commission for animal experiments in wrocław (resolution no. 63/2008, 78/2014 and 68/2015). the study was financed by grants kbn nn 303 4147737, ds 1076/s/ ibś/2014, and 0420/1409/16. supplementary material supplementary material associated with this article can be found at <http://www.unipv.it/webshi/appendix> manuscript 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(2015): genetic structure and differentiation of the fire salamander salamandra salamandra at the northern margin of its range in the carpathians. amphibia-reptilia 36: 301-311. najbar, a., rusek, a., najbar, b. (2017): zagrożenia i propozycje ochronne salamandry plamistej salamandra salamandra w zurbanizowanym siedlisku w bielsku-białej. chrońmy przyrodę ojczystą 71: 300303. ogrodowczyk, a., ogielska, m., kierzkowski, p., maślak, r. (2010): występowanie salamandry plamistej salamandra s. salamandra linnaeus, 1758 na dolnym śląsku. przyroda sudetów 13: 179-192. paluch, a., profus, p. (2004): status i rozmieszczenie salamandry plamistej salamandra salamandra (linneus, 1758) w polsce ze szczególnym uwzględnieniem populacji gatunku w górach bardzkich (sudety środkowe). chrońmy przyrodę ojczystą 60: 49-77. reis, d.m., cunha, r.l., patrão, c., rebelo, r., castilho, r. (2011): salamandra salamandra (amphibia: caudata: salamandridae) in portugal: not all black and yellow. genetica 139: 1095-1105. steinfartz, s., veith, m., tautz, d. (2000): mitochondrial sequence analysis of salamandra taxa suggest old splits of major lineages and postglacial recolonizations of central europe from distinct source populations of salamandra salamandra. mol. ecol. 9: 397-410. steinfartz, s., tautz, d. (2003): the fire salamander: source for new species. german research. life sciences 25: 14-16. steinfartz, s., küesters, d., tautz, d. (2004): isolation and characterization of polymorphic tetranucleotide microsatellite loci in the fire salamandra salamandra salamandra (amphibia: caudata). mol. ecol. notes 4: 626-628. thiesmeier, b. (2004): der feuersalamander. laurentiverlag. bielefeld. thiesmeier, b., grossenbacher, k. (2004): salamandra salamandra (linnaeus, 1758) – feuersalamander. pp. 1059-1132 in: thiesmeier b., grossenbacher k. (eds.) handbuch der reptilien und amphibien europas. schwanzlurche iib. aula verlag, wiebelsheim. veith, m. (1992): the fire salamander, salamandra salamandra l., in central europe: subspecies distribution and intergradation. amphibia-reptilia 13: 297-313. velo-antón, g., buckley, d. (2015): salamandra común – salamandra salamandra (linnaeus, 1758). in: salvador, a., martínez-solano, i. (eds.): enciclopedia virtual de los vertebrados españoles. museo nacional de ciencias naturales, madrid. zakrzewski, m., wójcik, s. (1982): charakterystyka ubarwienia rasy nominalnej salamandry plamistej salamandra salamandra, (l.) pochodzącej z trzech różnych populacji. rocznik naukowo-dydydaktyczny. prace zoologiczne iv (82): 131-139. wsp kraków im. komisji edukacji narodowej w krakowie. zakrzewski, m. (2007): salamandra plamista. rozmieszczenie, biologia i zagrożenia. wydawnictwo naukowe ap, kraków. acta herpetologica vol. 13, n. 1 june 2018 firenze university press issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 9(1): 89-97, 2014 doi: 10.13128/acta_herpetol-11929 a review of the genus gonionotophis in north-eastern africa (squamata: lamprophiidae) benedetto lanza1, donald g. broadley2 1museo zoologico “la specola” (sezione del museo di storia naturale dell’università), via romana 17, 50125 firenze, italy 2curator emeritus, natural history museum of zimbabwe, p.o. box 240, bulawayo, zimbabwe. corresponding author submitted on 2013, 1st july; revised on 2013, 27th march; accepted on 2014, 14th may abstract. the status of the material of the genus gonionotophis from north-eastern africa (north of latitude 12’s and east of longitude 28’e) is reconsidered. the northernmost specimens of g. nyassae (günther, 1888) fall within the known range of variation for that species. the available specimens of the g. capensis (a. smith, 1847) complex from this region indicate that the number of postoculars varies from none to three, so that the ‘diagnostic’ lack of postoculars in m. fiechteri scortecci, 1929 is invalid. the somali specimens should be assigned to the north-eastern form g. chanleri (stejneger, 1893), of which simocephalus unicolor boulenger, 1910 is a synonym. data for g. chanleri is summarised from throughout its extensive range, and compared with data for adjacent populations of g. capensis and g. savorgnani (mocquard, 1877). keywords. reptilia, squamata, lamprophiidae, gonionotophis, north-eastern africa, taxonomy, zoogeography. introduction in the last revision of the genus mehelya csíki, 1903, loveridge (1939) recognized three subspecies of m. capensis (a. smith, 1847), the typical form from southern africa, m. c. fiechteri scortecci, 1929 (based on only two specimens from somalia), while other records from southern somalia (including the type of m. somaliensis lönnberg and andersson, 1913) were assigned to m. c. savorgnani (mocquard, 1877). parker (1949) accepted loveridge’s views, although sceptical of the alleged extensive range of the latter taxon. laurent (1956) reinstated m. c. unicolor (boulenger, 1910) on the basis of two specimens from rwanda. hoevers and johnson (1982) recorded m. nyassae (günther, 1888) from somalia for the first time and assigned three snakes to m. c. savorgnani, although noting that they had the dark venter of m. c. unicolor. lanza (1983, 1990) indicated the range of m. c. savorgnani in somalia. broadley (2005) pointed out that the lost type specimen of heterolepis capensis a. smith, 1847 appears to have been a female of the taxon now called gonionotophis savorgnani, which it matched in colour pattern (no white vertebral stripe) and a high ventral count of 241. accordingly bmnh 91.9.15.9 from delagoa bay, the only specimen available to boulenger when he prepared the species account for his catalogue of snakes in 1893, was nominated the neotype of h. capensis a. smith, 1847. kelly et al. (2011) published molecular data which showed that gonionotophis brussauxi (mocquard, 1889; type species) was nested within mehelya, and as the former generic name had priority, the latter had to be placed in synonymy. these data also suggested that the taxa of the g. capensis group (represented by capensis and unicolor) constituted a distinct clade, and also that g. unicolor was specifically distinct from g. capensis. unfortunately obtaining tissues samples of these rare snakes is very difficult, and further progress with a comprehensive molecular phylogeny for the genus will be slow. materials and methods this study was based on the material available in italian museums, the california academy of sciences, and various 90 b. lanza, d.g. broadley museums in africa, europe and the united states visited by dgb. additional data was incorporated from the publications of laurent (1956, 1960), vesey-fitzgerald (1958), de witte (1966) and derleyn (1978). institutional acronyms follow leviton et al. (1985), with the addition of kmh = field numbers of k.m. howell, university of dar-es-salaam, and msnssm = museo di storia naturale e della strumentazione scientifica dell’università di modena. gender was determined by checking for the presence of hemipenes by dissection. standard counts were taken of dorsal scale rows, ventrals, subcaudals (counted according to the method of dowling, 1951), upper labials (and those entering orbit), lower labials (and those in contact with the anterior sublinguals), preoculars, postoculars and temporals. the anal was entire in all specimens examined. there is great intraspecific variation in proportions and number of head shields, as previously indicated by loveridge (1939), so these characters were not analysed. snout-vent length and tail length were measured to the nearest mm with a white-face tape, or with fishing line if set in coils. there appears to be no change in colouration between live and preserved specimens of file snakes. results within the g. capensis complex, g. chanleri was readily diagnosed by its quadricarinate vertebral scale row, while g. savorgnani differed from g. capensis in lacking a white vertebral scale row and having higher ventral counts in both sexes (table 1). the much smaller g. nyassae differs in having juxtaposed dorsal scale rows and very low ventral counts (table 1). species accounts gonionotophis capensis (a. smith, 1847) southern file snake heterolepis capensis a. smith, 1847, ill. zool. s. africa, rept., pl. lv. type locality: ‘eastern parts of cape province, south africa’. type lost and type locality rejected, neotype designated by broadley (2005): bm 91.9.15.9 delagoa bay [= maputo, mozambique], donated by south african museum. heterolepis gueinzii peters, 1874, monatsber. dtsch. akad. wiss. berlin: 163, pl., fig. 2. type locality: port natal [= durban], south africa. heterolepis bicarinatus (not duméril, bibron and duméril) pfeffer, 1889: 9. simocephalus poensis (not a. smith) pfeffer, 1893: 86. simocephalus capensis boulenger 1893: 345 (part) and 1896: 617. mehelya capensis barbour and loveridge, 1928: 114; vesey-fitzgerald, 1958: 40; 1975: 12. mehelya capensis capensis loveridge, 1939: 142; 1951: 189; moreau and pakenham, 1942: 62; broadley and pitman, 1960: 439; pakenham, 1983: 25; laurent, 1956: 99; spawls et al., 2002. diagnosis. a large species with dorsal scales in 15 rows at midbody, vertebral scale row bicarinate, dorsal scale rows widely separated by interstitial skin, dorsals with a subserrate median keel with fragmented secondary keels; ventrals 193-224 without sexual dimorphism; anal entire; paired subcaudals 39-60 without sexual dimorphism. supralabials 7, third and fourth entering orbit. dark brown to black above, with vertebral scale row white and other dorsals white-tipped, venter white but outer ends of ventrals black. description. eye moderate, its diameter subequal to its distance from the lip; loreal little longer than deep; preocular usually single, but two in mcz 23201 and nmk 1225; postoculars 1-2; temporals usually 1+2, but 1+3 on one side of mcz 21011; supralabials 7, but 6 on one side of nmk 1227 and 8 on one side of nmk 1225; infralabials 8. dorsal scales in 15-17 rows on neck, 15 at midbody, 15 before the vent, vertebral row bicarinate; ventrals 215-221 in males, 213-224 in females; anal entire; subcaudals 51-60 in males, 40-52 in females. dentition.teeth firmly ankylosed, but all are replaced simultaneously, so that there may be duplicated rows of functional teeth throughout. table 1. comparison of ventral and subcaudal counts for four species of gonionotophis from northeastern africa. species ventrals subcaudals males females males females n range mean sd n range mean sd n range mean sd n range mean sd capensis 8 215-221 217.62 2.13 7 213-224 219.0 3.70 5 51-60 55.80 3.49 6 40-52 48.00 4.69 savorgnani 12 220-235 228.17 4.86 11 225-239 232.2 5.27 9 51-60 55.33 2.92 8 45-61 50.25 5.90 chanleri 21 215-233 223.28 4.38 20 218-243 225.4 5.67 15 44-62 56.07 4.77 15 44-57 48.73 4.53 nyassae 5 164-168 166.20 1.79 6 170-178 175.2 2.71 6 62-78 69.67 6.15 6 55-67 62.83 4.26 91a review of the genus gonionotophis in north-eastern africa (squamata: lamprophiidae) size. largest female from mbala, zambia 1245 + 155 = 1400 mm (vesey-fitzgerald, 1958). diet. a specimen from ruponda, tanzania, contained a gerrhosaurus nigrolineatus (loveridge, 1951). a zanzibar specimen, 1240 mm in total length, contained a boaedon 889 mm long, whose tail protruded from the file snake’s mouth (pakenham, 1983). habitat. savanna woodland. distribution. southeastern africa, north to eastern angola, zambia, south-eastern drc (katanga province), malawi and southeastern tanzania as far north as the eastern usambara mountains, where parapatric with g. chanleri. localities. democratic republic of congo (katanga). albertville [kalemie] mrac 4275, 9992, 16502 (laurent, 1956). zambia. abercorn [mbala] irsnb ---; vesey-fitzgerald, 1958. tanzania. amani mcz 21011, 23201-2, 23204, nmk 1225; dar es salaam nmk 1226; kwamkoro/kwamsambia forest reserve nmzb 16382; magrotto hill nmzb 14796; nanguru mission, lindi kmh 6002; ruponda, lindi mcz 50070; songea nmk 1227; zanzibar (pakenham, 1983). gonionotophis savorgnani (mocquard, 1877) congo file snake heterolepis savorgnani mocquard, 1887, bull. soc. philom. paris (7) 11: 27, pl. ii, fig. 4-4b. type locality: ogowe, gabon. simocephalus phyllopholis werner, 1901, zool anz. 24: 301, fig. 3-4. type locality: cameroon. simocephalus butleri boulenger, 1907, ann. mag. nat. hist. (7) 20: 489. type locality: between wau and chakchak, bahr el ghazel province, sudan; boulenger, 1911: 164 (bussu). mehelya (or simocephalus) lamani lönnberg, 1910, arkiv. zool. 8 (20): 2. type locality: mukimbungu, lower congo (drc). mehelya chanleri (not stejneger) loveridge, 1929: 21; pitman, 1936: 271, pl. iv, fig. 5 and pl. d, fig. 1. mehelya capensis savorgnani loveridge, 1939: 136 (part), fig. 1; laurent, 1956: 101, 372; de witte, 1966: 64; 1975: 72; pitman, 1974: 86, pl. iv, fig. 5 and pl. d, fig. 1; largen and rasmussen, 1993: 352; spawls et al., 2002; largen and spawls, 2010: 486 (gambela). mehelya poensis (not a. smith) de witte, 1941: 182. diagnosis. a large species with dorsal scales in 15 rows at midbody, vertebral scale row bicarinate, dorsal scale rows widely separated by interstitial skin, subserrate median keel with fragmented secondary keels; ventrals 211-241 without sexual dimorphism; anal entire; paired subcaudals 45-62 without sexual dimorphism. supralabials 7, third and fourth entering orbit. dark brown to black above, uniform, or each scale with a light apical spot, giving a speckled effect, venter white but outer ends of ventrals dark. description. eye moderate, its diameter one and a half times its distance from the lip; loreal slightly deeper than long; preocular usually single, but two in mrac 18078-9 and on one side of mrac 18077; postoculars 1-3; temporals usually 1+2, but 2+3 on mrac 18077 and on one side of mrac 18078; supralabials 7, but 6 on mrac 14272 and on one side of bm 1908.10.20.12; infralabials 8-9, but 7 in nmk 2890. dorsal scales in 15-21 rows on neck, 15 at midbody, 15 before the vent, vertebral row bicarinate; ventrals 220-235 in males, 225239 in females; anal entire; subcaudals 51-60 in males, 45-61 in females. dentition. teeth firmly ankylosed, but all are replaced simultaneously, so that there may be duplicated rows of functional teeth throughout. size. largest male (irsnb --bagbele, parc nat. garamba, drc) 1122 + 180 = 1302 mm (de witte, 1966); largest female (irsnb --parc nat. garamba, drc) 1405 + 200 = 1605 mm (de witte, 1966). diet. in uganda, pitman (1974) records snakes of the genera causus, boaedon and psammophis, lizards (gerrhosaurus), and frogs and toads. habitat. in eastern drc, commonly found in marshy areas (laurent, 1956). in uganda, recorded from larger forests and along the shores of lake victoria (pitman, 1974). the only specimen from ethiopia was found in marshy grassland near gambela at an altitude of ca. 500 m (largen and spawls, 2010). distribution.--cameroon, gabon, congo-brazzaville and northern angola, east through central african republic and d.r.c. (sympatric with g. chanleri at uvira) to southwestern sudan, south sudan, western ethiopia (gambela: largen and rasmussen, 1993), uganda and western kenya. localities.---sudan. boma national park nmk 2890; wau to chakchak bm 1946.1.14.44 (type of s. butleri). south sudan. magwe fmnh 62337; molongori fmnh 62335; torit fmnh 62336. democratic republic of congo. bagbele (de witte, 1966); butembo mrac 21600; garamba national park nmzb 16655 (de witte, 1966); lubile, pangi, maniema mrac 18082; mambasa mzuf 32391;manguretshipa, kivu mrac 17078-9; mutsora, ruwenzori mrac 14274; sake, lac kivu mrac 14272; teturi, mambasa mrac 18077; uvira lacm 49571; virunga national park – kabiro, katare, katuakamabi, rugetsi, rutshuru and rwindi (de witte, 1975). uganda. budongo forest bmnh 1935.10.11.7; bussu bm 1911.7.8.5-6; entebbe bm 1929.8.5.19; mabira forest bm 1908.10.20.12; ntan92 b. lanza, d.g. broadley di, bwamba forest lacm 46392; rhino camp, west nile fmnh 13129; saroti nmk 1418. kenya. chemilil, kisumu cas 150937; kakamega cas 111795, 150988; kisumu-nyalenda nmk 2882; kitale nmk 3151. gonionotophis chanleri (stejneger, 1839) unicolor file snake figs. 1, 2 simocephalus chanleri stejneger, 1893, proc. u.s. natl. mus. 16: 726. type: usnm 20126, wange [not manda island], kenya; boulenger, 1896: 617; 1915a: 621. simocephalus unicolor boulenger, 1910, ann. mag. nat. hist. (8) 5: 512. type: bmnh 1946.1.13.91, fort hall [= maranga], kenya; boulenger, 1915a: 621. simocephalus poensis (not a. smith) lepri, 1911: 323 (mogadiscio, somalia). mehelya (simocephalus) somaliensis lönnberg and andersson, 1913, arkiv. zool. (20) 8: 2. type: nhrm ---kismayu [= chisimaio], somalia. simocephalus butleri (not boulenger, 1907), boulenger, 1915b: 647. mehelia (sic) (simocephalus) fiechteri scortecci, 1929, atti. soc. ital. sci. nat. 68: 269. type: msnm 936, villaggio duca degli abruzzi [= giohar], somalia. mehelia (sic) poensis (not a. smith) testi, 1935: 507 (eastern eritrea). mehelya chanleri chanleri loveridge, 1936: 24 (rhino camp, west nile province, uganda). mehelya capensis savorgnani (not mocquard) loveridge, 1939: 137 (part), fig. 1; parker, 1949: 56; hoevers and johnson, 1982: 185; lanza, 1983: 225 and 1990: 438; largen and rasmussen, 1993: 352 (part, haud). mehelya capensis fiechteri loveridge, 1939: 141; parker, 1949: 57 (haud, 09’n-44’e). mehelya capensis unicolor laurent, 1956: 104; 1960: 24; bourgeois, 1968: 220; derleyn, 1978: 728-732; broadley, 1985: 60; broadley and howell, 1991: 26; hinkel, 1992: 439, pl. 338; spawls et al, 2002. mehelya capensis (not a. smith) vesey-fitzgerald, 1975: 12; largen, 1997: 90. mehelya unicolor largen and spawls, 2010: 486 (haud). description. eye moderate to large, its diameter subequal to, or larger than its distance from the lip; loreal longer than deep, in mzuf 27065 contacting the eye below the preocular on the left side; preocular usually single, but two on both sides of mzuf 1354 and on one side of msnm 977; postoculars 1-3, but absent on both sides of the type of m. fiechteri and bmnh 1949.2.1.86, and on right side of msnm 977, postoculars may be fused with the supraocular and/or the fourth supralabial; temporals usually 1+2, but 1+1 in mzuf 1355 and 2+3 in five specimens recorded by laurent (1956, 1960) from rwanda and kivu, and on one side of cas 122368 from voi; supralabials 7, but 6 on one side of mcz 55495 and mrac 20786, 8 in nmk 1341; infralabials 8, but 7 fig. 2. gonionotophis chanleri: scalation at midbody of mzuf 1354 from afgoi, somalia. fig. 1. gonionotophis chanleri: dorsal, lateral and ventral views of the head of mzuf 1354 from afgoi, somalia. 93a review of the genus gonionotophis in north-eastern africa (squamata: lamprophiidae) in nmk 2749 and 2996, and on one side of msnm 936, mzuf 1355 and mcz 55495. dorsal scales in 17-19 rows on neck, 15 at midbody, 15-17 before the vent, vertebral row often quadricarinate [unique in the genus]; ventrals 215-233 in males, 218-243 in females; anal entire; subcaudals 44-62 in males, 44-57 in females, paired, but the proximal ones sometimes, more or less irregularly, single, as follows: mogadiscio ? female, 6 single + 3 paired + 7 single + all paired (lepri, 1911) mzuf 1354 female, 2 paired + 1 single + all paired mzuf 2061 female, 3 single + all paired mzuf 27064 female, 9 single + 1 paired + 3 single + all paired (hoevers and johnson, 1982) mzuf 27065 male, 2 paired + 1 single + all paired mzuf 27643 female, 2 single + all paired dark brown above, lighter brown below, chin and throat and distal edges of ventrals white. however the type of s. savorgnani had a yellow venter, while nmk 1417 from voi is white and brown laterally with a brown stipple mesially, and cas 122368 from near voi has light patches mesially. dentition. in nmzb 3186 from arusha, tanzania, two rows of teeth are present on all dentigerous bones, the maxillary and dentary teeth are being replaced medial to the bones, while the palatine and pterygoid teeth are being replaced lateral to the bones. simultaneous replacement of teeth does not seem to have been recorded in any other genus. size. largest male (mrac 20786 kabuye, astrida, rwanda) 1311 + 174+ mm (tail truncated). largest female (kidwange, astrida, rwanda: laurent, 1960) 1249 + 166 = 1415 mm. diet. at fort hall, kenya, a specimen was killed while swallowing a 53 cm causus rhombeatus (loveridge, 1939). in burundi, captive specimens consumed all snakes offered to them, including the genera lycophidion, gonionotophis (accidental), boaedon, philothamnus, and juveniles of bitis arietans and b. gabonica, together with chameleons of the genera chamaeleo and trioceros (derleyn, 1978). habitat. in burundi, collected on the ruzizi plain and along the shores of lake tanganyika (derleyn, 1978). in kenya, usually in savanna woodland (spawls et al., 2002). in tanzania, montane forest in the arusha national park (nmzb 3186). distribution. eastern democratic republic of the congo (kivu province), rwanda, burundi, northern and western tanzania, kenya, somalia and eritrea (fig. 4). localities. eritrea. ‘ eastern slope’ (anonymous, 1933; testi, 1935) msnssm ----. somalia. afgoi mzuf 1354; alessandra island mzuf 2061; balad mzuf 1355; eggi mzuf 2298; gelib mzuf 27643; giohar [villaggio duca degli abruzzi] msnm 936 [type of s. fiechteri], 977; haud at 9’n, 44’e bmnh 1949.2.1.86; kismayu nmrm 2419 [type of m. somaliensis]; mareri cas 153341, mzuf 27064-5. kenya. kiboko range research station nmk 2996, 3029; kirwa, meru nmk 3155; makuweni, machakos nmk 1341; malindi nmk 2654, 2749; fort hall [muranga] bmnh 1946.1.13.91 [type of s. unicolor], nmk 1345; nyambeni mts nmk 1719; sotik nmk 1343; voi cas 122368, nmk 1417; wange usnm 20126 [type of s. chanleri]; watamu cas 184312. tanzania. iku, rukwa valley mcz 55495; kibondo nmzb 227; samaria nmzb 17104; tengeru, arusha nmzb 3186. rwanda. no precise locality mnhn 1996.7544; buhanga-ndara mrac 20785; kabuye mrac 20786-7; kidwange (laurent, 1960); nyakatare mrac 8305, 10988. burundi. no precise locality mnhn 1990.4626; bujumbura (derleyn a2-6), rnhm 26257; imbo sud [= rumonge](derleyn a1); ntita (derleyn a7). democratic republic of congo (kivu). remera (laurent, 1960); uvira mrac 21412. gonionotophis nyassae (günther, 1888) dwarf file snake fig. 3 simocephalus nyassae günther, 1888, ann. mag. nat. hist. (6) 1: 328. type: bmnh 1946.1.14.52, lake nyassa [= malawi]; boulenger, 1915a: 622. gononotophis degrijsi werner, 1906, zoöl. anz. 30: 53. type locality: usambara mountains, tanzania. mehelya nyassae loveridge, 1936: 243; 1939: 148; 1955: 183; 1956: 11; 1959: 38; parker et al., 1940; moreau and pakenham, 1941: 108; hoevers and johnson, 1982: 186; pakenham, 1983: 25; spawls et al., 2002; spawls et al., 2006: 102. diagnosis. a small species with 15 scale rows throughout the length of the body; dorsal scales imbricate, with a strong median keel and short apical keels laterally; ventrals 164-177 in males, 170-193 in females; subcaudals 62-78 in males, 54-74 in females; eye small to moderate in size; supralabials usually seven, the third and fourth entering the orbit. uniform dark brown to black above, venter brown, white or mottled. description. eye small, its diameter less than its distance from the lip; loreal nearly twice as long as deep; preocular 1; postocular 1 (rarely 2); temporals 1+2; supralabials 7, the third and fourth entering the orbit; infralabials 8, the first 5 (rarely 4) in contact with the anterior sublinguals. scales in 15 rows, dorsals with a simple median keel and two short lateral ones apically (but fully tricarinate in nmzb 11805, a 218 mm juvenile from zanzibar), vertebral scale row bicarinate; ventrals 94 b. lanza, d.g. broadley 164-168 in males, 170-178 in females; anal entire; subcaudals 62-78 in males, 55-67 in females. uniform dark brown to black above, lighter brown below, the ventrals with the distal margin bordered with whitish. dentition. teeth hinged (savitsky, 1981), which is presumably an advantage as the diet consists largely of well-armoured fossorial skinks (mochlus and afroablepharus), in addition, this does not appear to be a powerful constrictor. size. largest male (pakenham, 1983, from zanzibar) 485+118 mm, largest female (mzuf 27066 – mareri, somalia) 455+115 mm. diet. the stomach of a female from kilwa contained two skinks of the genera mochlus and afroablepharus (loveridge, 1956). two males from liwale each held an afroablepharus wahlbergii (loveridge, 1959). breeding. a gravid female from wema contained three eggs measuring 10 x 4 mm on june 13 (loveridge, 1936). habitat. in kenya, one taken in grass on the banks of the tana river (loveridge, 1939). at liwale, two males were taken among rotting vegetation bordering gardens following a night of fairly heavy rain (loveridge, 1959). low savanna, coastal thicket and woodland, from sea level to ca. 1 200 m altitude (spawls et al., 2006). distribution. from southern somalia south to kwazulu-natal, south africa, extending inland to rwanda, burundi, eastern democratic republic of congo, zambia, botswana and northern namibia. localities. somalia. mareri cas 153342; mzuf 27066. kenya. wema, ngatana mcz 40482. tanzania. no precise locality zmb 17412; kilwa mcz 5451920, nmk 973; liwale i 8311, 8320; mcz 52643; makonde scarp 2 kmh 26741; tamota mcz 54820; tunduru mcz 52644; usambara mts nmw --[type of g. degrijsi werner]; zanzibar (pakenham, 1983) nmzb 11805. rwanda. zmh --between lakes tanganyika and kivu. key to the north-east african species of gonionotophis based on external characters. 1a. dorsal scale rows widely separated by interstitial skin; secondary keels on body scales all strongly developed; ventrals 215-243; subcaudals 40-62; maximum total length over 1600 mm 2 1b. dorsal scale rows juxtaposed; secondary keels on dorsal scales reduced to two short ones apically; ventrals 164-178; subcaudals 55-78; maximum total length 645 mm nyassae 2a. vertebral scale row bicarinate; venter white, ends of each ventral dark 3 2b. vertebral scale row often quadricarinate; venter usually dark brown to black chanleri 3a. vertebral scale row white; ventrals 215-221 in males, 213-224 in females capensis 3b. vertebral scale row dark; ventrals 220-235 in males, 225-239 in females savorgnani discussion the three taxa previously considered subspecies of gonionotophis capensis are readily distinguished by their colour patterns. g. capensis has a well defined white stripe on the bicarinate vertebral scale row and a white venter. g. savorgnani has the dorsum uniformly dark, or with the scales white-tipped, the venter is white. the north-eastern form g. chanleri is uniformly dark above and below, or with the dorsal scales white-tipped, or the chin and throat white and white median patches on some ventrals (only uniformly pale in the type of s. chanleri). table 1 shows ventral and subcaudal counts for g. chanleri from throughout its range, compared with populations of g. capensis, g. savorgnani and g. nyassae (fig. 3) from north of latitude 12’s and east of longitude 28’e. fig. 3. gonionotophis nyassae: dorsal, ventral and lateral views of the head of mzuf 27066 from mareri, somalia. 95a review of the genus gonionotophis in north-eastern africa (squamata: lamprophiidae) while the former two taxa are clearly separated on the basis of ventral counts, those for g. chanleri completely overlap them both. when he reinstated m. unicolor as a subspecies of m. capensis, laurent (1956) also presented data showing that in the eastern democratic republic of congo there was little overlap in ventral counts between m. c. capensis (215-222) and m. c. savorgnani (221-236). l. chanleri differs from all other species in the genus in the strong development of secondary keels on the vertebral scale row, this is particularly well marked in the holotype, somali material, and the specimen from eritrea, where the vertebrals are distinctly quadricarinate. acknowledgements we thank barry hughes and dr goran nilson for sending us data for the haud and kismayu specimens respectively. manuela mascherini prepared the illustrations of the gonionotophis specimens (figs 1-3) under the direction of bl. references anonymous (? testi, f.) 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(1847): illustrations of the zoology of south africa, reptilia. smith, elder, and co., london. spawls, s., howell, k., drewes, r., ashe, j. (2002): a field guide to the reptiles of east africa. academic press, san diego. spawls, s., howell, k., drewes, r. (2006): pocket guide to the reptiles and amphibians of east africa. a&c black, london. stejneger, l. (1893): on some collections of reptiles and batrachians from east africa and the adjacent islands, recently received from dr w.l. abbott and mr william astor chanler, with descriptions of new species. proc. u.s. natl. mus. 16: 711-741. testi, f. (1935): sulla presenza di mehelia (sic) poensis in eritrea. arch. ital. sci. med. colon. 16: 507-509. vesey-fitzgerald, d.f. (1958): the snakes of northern rhodesia and the tanganyika borderlands. proc. trans. rhodesia sci. assoc. 46: 17-102. vesey-fitzgerald, d.f. (1975): a guide to the snakes of the tanzania and kenya borderlands. j. east afr. nat. hist. soc. natl. mus. 149: 1-26. werner, f. (1906): neue reptilien aus deutsch-ostafrika. zoöl. anz. 30: 53-55. acta herpetologica vol. 8, n. 2 december 2013 firenze university press journal of the societas herpetologica italica acta herpetologica acta herpetologica 10(1): 55-62, 2015 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-15142 age structure, growth and longevity in the common toad, rhinella arenarum, from argentina clarisa de l. bionda1,2,*, silvia kost4, nancy e. salas1, rafael c. lajmanovich3, ulrich sinsch4, adolfo l. martino1 1 ecología-educación ambiental, departamento de ciencias naturales, universidad nacional de río cuarto, ruta nacional n° 36-km 601, x5804bya, río cuarto, córdoba, argentina. *corresponding author. e-mail: cbionda@exa.unrc.edu.ar; claribionda@yahoo.com.ar 2 consejo nacional de investigaciones científicas y técnicas (conicet) 3 facultad de bioquímica y ciencias biológicas, universidad nacional del litoral, ciudad universitaria el pozo s/n (3000) santa fe, argentina 4 department biology, university of koblenz-landau, universitaetsstr. 1 d-56070 koblenz, germany submitted on 2014, 7th november; revised on 2014, 23th april, 2015; accepted on 2015, 25th april editor: rocco tiberti abstract. age structure, growth and longevity was determined in the common toad, rhinella arenarum, from a suburban pond located in the pampa plains, central argentina during two breeding seasons, in 2000 and 2008 by using skeletochronology, which relies on the analysis of the annual lines of arrested growth (lags) in bones. both females and males were captured in 2008, while only males were recorded in 2000. females were significantly larger than males. mean population age was 2.4  ±  0.9 years in 2000. in 2008, the difference in age was not significant between the sexes (males: 3.0  ±  0.7, n  =  21; females: 2.6  ±  0.9, n  =  12), neither between males in 2000 and 2008. the longevity in males of 2000 was 6 lags and exceeded that of males (5 lags) and females (4 lags) in 2008. von bertalanffy curves showed that the growth coefficient in the males of 2000 (k  =  2.97  ±  0.47) was almost double that of females (k  =  1.21  ±  0.10) and males (k  =  1.01  ±  0.14) of 2008. males and females rhinella arenarum show different morphological and life history traits and the year of sampling can significantly influence the estimation of the studied parameters such as age at maturity and growth rates. keywords. demographic traits, age, rhinella arenarum toad, skeletochronology, von bertalanffy model. introduction the common south american toad, rhinella [bufo] arenarum, belongs to the family bufonidae. this toad has a wide distribution in south america and it is found in argentina, bolivia, brazil, uruguay and paraguay. it has been assessed at least concern (lc) according to iucn red list of threatened species (iucn, 2015). adults normally congregate in large breeding groups at lentic water bodies. r. arenarum inhabits in a wide range of habitats, including coastal environments, subtropical or tropical forests, and rural or urban areas. the habitat diversity and large population size make this toad a particularly conspicuous species. although ecology, the feeding strategy and habitat preference of this anuran have been recently investigated (sanabria et al., 2007; quiroga et al., 2009; bionda et al., 2011a; 2012; 2013), the demographic life-history traits still remain largely unknown (but see echeverria and filipello, 1990). regarding amphibians and reptiles, skeletochronology is a widely used method for retrospective age estimation of individuals with unknown recapture history 56 clarisa de l. bionda et alii (hemelaari, 1988; smirina, 1994; sinsch, 2015), and an excellent tool to assess population age structure. skeletochronology relies on the analysis of annual growth zones alternating with dense lines of arrested growth (lags) in bones. lag formation is considered to be mainly due to a genetically controlled based circannual rhythm, which is synchronized with and reinforced by seasonal cycles such as hibernation and occasionally aestivation in temperate zone species (kleinenberg and smirina, 1969; castanet et al., 1993; smirina, 1994; sinsch et al., 2007). this study focusses on the dynamics of age structure and related life history parameters in a single population of r. arenarum breeding in a temporary pond in 2000 and 2008. members of this population have annual hibernation and short-term aestivation periods. aims of this study were (1) to report the size and age at sexual maturity, the longevity and the age structure in the common toad, rhinella arenarum, and (2) to test for the presence of sexual dimorphism with respect to ageand size-related life-history traits and their variability between the study years. materials and methods study area the study area belongs to the pampa plains of central argentina. the area is characterized by moderately undulating plains and a temperate climate with an annual mean temperature of 23ºc in january and 6ºc in july and with mean annual temperature of 18ºc. rainy seasons alternate with dry ones, with rains typically starting in october and continuing through the warm months until march with a mean annual rainfall of 800-1000 mm (gatica et al., 2012). the study pond was located in the campus of national university of rio cuarto (33º06’s, 64º25’w; 465 m a.s.l., pond size 966 m2). this is an area with permanent and temporary ponds which are surrounded by a small strip of forest. data collection a total of 159 r. arenarum were collected during two breeding seasons, 2000 and 2008 (105 males and two juveniles in 2000; 39 males and 15 females in 2008) from september to december, the period of major breeding activity of this species in this region (bionda et al., 2011a; 2012; 2013). the individuals were hand-captured during surveys at the pond shore, mostly after rainfall. we measured the snout-vent length (svl; mm) of each individual with a vernier caliber (0.01 mm precision). the sex was determined using external secondary sexual characters: presence of vocal sacs and nuptial pads, and coloration (males with brownish or greenish back; females’ greyish or light brown back, scattered with large dark or dark brown blotches). then a toe of the forelimb was clipped at the level of the penultimate phalanx and stored in 70% ethanol until being processed for skeletochronological analysis. the toe clip was used as a batch mark to prevent resampling (bionda et al., 2011b; 2013). after biometric measurement and toe-clipping, we released the animals at the sampling site. laboratory procedures followed the standard methods of skeletochronology (sinsch et al., 2001): (1) decalcification of bones (5-10% formic acid, 24 h), (2) fixation in bouin’s solution (for sample of 2000) or formol 4% (for sample of 2008) (at least 12 h), (3) historesintm (jung) (for sample of 2000) or paraffin embedding (for sample of 2008), (4) cross sectioning of the diaphysis at 8-10 μm using a jung rm2055 (for sample of 2000) and an arcano rotation (for sample of 2008) microtome, (5) staining with 0.05% cresylviolet (5-10 min, sample collected in 2000) or with ehrlich’s haematoxylin (2 min, sample collected in 2008), (6) light microscopic count of the number of lines of arrested growth (=  lag) using an olympus bx 50 (for sample of 2000) and a zeiss axiophot-axiolab (for sample of 2008), (7) documenting the most informative cross sections with a axiocamhrc zeiss digital camera using axio vision 4.3. the number of lines of arrested growth (lags) in each section was counted in the periosteal part of the bone by at least two authors (sk, us, cb, am). the criteria for incomplete, faint or double lines, as well as lags potentially lost due to endosteal resorption were applied according to tsiora and kyriakopoulousklavounou (2002), guarino et al. (2011). consequently, we define age as the number of lags counted. the adult sample used for skeletochronology was 105 males collected in 2000, and 21 males and 12 females collected in 2008. growth rate was estimated using the von bertalanffy’s (1938) model (e.g., üzüm and olgun, 2009; liao and lu, 2010; guarino et al., 2011). we used the following equation: svlt  =  svlmax – (svlmax – svlmet) e– k (t – tmet), where svlt  =  average svl at age t, svlmax  =  average maximum svl, svlmet  =  average svl at metamorphosis (fixed to 11.5 mm according to bionda, 2011), t  =  number of growing season experienced (age), tmet  =  proportion of the growing season until metamorphosis (age at metamorphosis), and k  =  growth coefficient (shape of the growth curve). the von bertalanffy growth model was fitted to the empiric age-size data using the least square procedure (e.g., cogălniceanu and miaud, 2002; cicek et al., 2011). estimates of svlmax and k are given with the corresponding 95% confidence interval. the following demographic variables were calculated according to leskovar et al. (2006): (1) age at maturity: the minimum number of lags counted in breeding individuals; (2) longevity: the maximum number of lags counted in reproductive individuals; (3) potential reproductive lifespan: the difference between longevity and age at maturity; (4) size at maturity: the average snout-vent length of all first breeders with the minimum number of lags; (5) modal lifespan: median of age distribution. data analysis the significance level used in all tests was p  <  0.05. descriptive statistics are given as mean  ±  standard deviation, 57age structure, growth and longevity in rhinella arenarum but as some subsets of age and svl data differed from a normal distribution, we used the non-parametric mann-whitney u-test to test for significant differences between the two sexes and between years. growth parameters were statistically compared based on the range of the ci95% interval. tests were performed using the statistical packages statistica 6.0 (statsoft inc., usa 2001) and stagraphics centurion xvi (statpoint, inc., 2014). results body length the average svl of males sampled was 99.4  ±  8.8 mm (range 76.1-117.7, n  =  105) in 2000 and 101.5  ±  7.1 mm (range 89.4-112.5, n  =  39) in 2008. in the females, the average svl was 108.6  ±  9.6 mm (range 82.6-123.3, n  =  15) in 2008. the svl of the individuals in 2008 differed significantly between sexes (mann-whitney, u  =  194.5; p  <  0.05), but there was no difference in svl between the males in the two sampled years (mannwhitney, u  =  10.4; p  =  0.51). the size distribution of males and females did not differ from a normal distribution (shapiro-wilk test: males w  =  0.977, p  =  0.37, females w = 0.882, p = 0.11; fig. 1). age structure all adults studied showed well-defined lines of arrested growth (lags) in the periosteal bone layer, which allowed assessing the individual age (fig. 2). endosteal resorption was present in 33% of the sample (e.g., fig. 2d), but the resorption did not hamper age determination because the first lag was never completely reabsorbed. in many cases the outer most lines were closely adjacent, but at the insertion site of the phalangeal ligament it was possible to discern the peripheral lags and to reliably count them (fig. 2c). double lags (two very closely adjacent growth marks) were rarely observed and were counted as a single lag (figs. 2a, b). the demographic life history traits are summarized in table 1. mean age was 2.4  ±  0.9 lags (= years) in the male sample of 2000, whereas that of the male and female sample of 2008 was 3.0  ±  0.7 and 2.6  ±  0.9 lags, respectively (fig. 3). the modal age did not significantly differ between the sexes in 2008 (u  =  128.5; p  =  0.93), and did neither between males in 2000 and 2008 (u  =  25.0; p  =  0.46). the modal age was 2 lags in males in 2000, and 3 lags in males and 2 lags in females in 2008 (table 1). the minimum number of lags counted in the reproductive individuals was 1 lag for males in 2000 (n  =  18 individuals). in 2008, the age at sexual maturity was 2 lags both in males (n  =  5) and females (n  =  2). the longevity and potential reproductive lifespan were higher in males in 2000 (table 1). size at sexual maturity of males was not significantly different between 2000 and 2008 (mann-whitney, u = 15.6; p  =  0.39), but reached during the first year of life in the individuals collected in 2000, whereas the individuals from 2008 needed two years to achieve the same size. females were significantly larger at maturity than males (mann-whitney, u = 9; p < 0.05). fig. 1. snout-vent length distribution (5 mm classes) of r. arenarum. males sampled in 2000 (grey bars, secondary vertical axis), males (filled bars) and females (empty bars) sampled in 2008. fig. 2. examples of cross sections of phalanges of adult r. arenarum. (a) female, 4 lags, svl  =  113.9 mm; (b) female, 4 lags, svl  =  113.9 mm; (c) female, 2 lags, svl  =  100.7 mm; (d) male, 5 lags, svl  =  96.8 mm. arrows indicate line of arrested growth (lag). eb: endosteal bone. mc: medullar cavity. rl: resorption line. 58 clarisa de l. bionda et alii sex-specific growth pattern growth curves of males and females sampled in 2008 using the von bertalanffy’s model (fig. 4) did not differ significantly with respect to the growth coefficient (kmales  =  1.01, ci95%: 0.73-1.30; kfemales  =  1.21, ci95%: 1.00-1.42; p > 0.05). a marked decrease of growth rate was observed from the 2nd to the 3rd lag which is after the presumed attainment of sexual maturity. the growth coefficient of males was significantly greater in 2000 than in 2008 (k  =  2.97, ci95%: 2.04-3.91; p < 0.05). the maximum asymptotic svl in 2008 differed significantly between males (svlmax  =  105.6 mm, ci95%: 101.0-110.2) and females (svlmax  =  114.0 mm, ci95%: 110.6-117.4; p < 0.05), i.e. svl was marginally female-biased. in contrast, modeled maximum svl of males did not vary significantly between 2000 and 2008 (svlmax  =  100.3 mm, ci95%: 98.5-102.0; p < 0.05). for all samples, the maximum asymptotic svl was slightly greater than measured average svl in this study (males 2000  =  99.4  ±  8.8; males 2008  =  101.5  ±  7.1 mm; females 2008  =  108.6  ±  9.6 mm). models were in good agreement with empirical values (r2: males2000  =  0.94, males2008 = 0.99, females2008 = 0.99). table 1. demographic life history traits of r. arenarum. sex n age mean (lags) mode (frequency) age at sexual maturity (lags) longevity (lags) potential reproductive lifespan (years) svl at sexual maturity (mm) males 2000 105 2.4 ± 0.9 (1-6) 2 (39.1%) 1 6 5 96.1 ± 8.3 males 2008 21 3.0 ± 0.7 (2-5) 3 (65%) 2 5 3 91.8 ± 2.2 females 2008 12 2.6 ± 0.9 (1-4) 2 (41.6%) 2 4 2 106.8 ± 3.7 fig. 3. age distribution (in lags) of r. arenarum. males sampled in 2000 (grey bars, secondary vertical axis), males (filled bars) and females (empty bars) sampled in2008. fig. 4. age-size relationship in males (a) and females (b) sampled in 2008, an in males sampled in 2000 (c). lines represent the von bertalanffy models of growth: (a) svl [mm]  =  105.63 mm (105.63 mm -114.5 mm)*e(-1.01387*lags); (b) svl [mm] =  114.0 mm (114.0 mm -114.5 mm)*e(-1.2106*lags); (c) svl [mm] = 100.28 mm (100.28 mm -114.5 mm)*e(-2.97484*lags). 59age structure, growth and longevity in rhinella arenarum discussion skeletochronological age assessment is an essential tool for investigations on demographic traits (e.g., sinsch et al., 2007; sinsch, 2015). moreover, it is a nonlethal destructive technique that can be performed on the phalanges, without sacrificing the animals (guarino et al., 2008). lag formation is considered to be mainly due to a genetically controlled based circannual rhythm (alcobendas and castanet, 2000; morrison et al., 2004, miaud et al., 2007; marangoni et al., 2009; 2012; sinsch, 2015). several studies confirmed the formation of one lag per year, equivalent to the number of hibernations of each individual, as the most common observed pattern of lag formation in palaearctic, tropical and subtropical amphibian species (smirina, 1994; guarino et al., 2008; marangoni et al., 2012), but precision decreases considerably in individuals older than 8 years (sinsch, 2015). moreover, the annual periodicity of lag formation is more pronounced when the climate of the sampling area is characterized by a marked seasonal variation (guarino et al., 2011), as is the case in r. arenarum. this toad species reproduces, when winter ends, therefore the last (outermost) lag and the perimeter of the phalange were very close together due to the absence of substantial bone growth. aestivation does not seem to have physiological effects on growth, as no typical growth marks (sinsch et al., 2007) for interrupted summer growth were found. we found no evidence that local individuals reproduce twice a year, a potential cause of double line formation (díaz paniagua and mateo, 1999; marangoni et al., 2012). the seasonal ovary cycle of r. arenarum leans support for a single reproductive period per year, the preovulatory oogenesis begins between july and august, the ovulatory period with mature oocytes extends from september to november, and a post reproductive period occurs between december and june (medina et al., 2004). consequently, the low incidence of double lines (5% in the total sample) has probably other causes, for example, an interruption of the hibernation (sinsch et al., 2007). sexual size dimorphism (ssd) sexual size dimorphism is observed in 90% of the 589 amphibian species surveyed by shine (1979). in our study as well in those of echeverria and filipello (1990) and bionda et al. (2011a), the body size in r. arenarum is female-biased. if adjusted for age (e.g., ma and lu, 2009; guarino et al., 2010; liao et al., 2015; ma et al., 2015), female-biased ssd still remains significant, but at a marginally significant level. in agreement with echeverria and filipello (1990) size at maturity of males is about 90-100 mm and of females about 100 110 mm, indicating that svl increment during the adult period is very low. since male and female growth rate are statistically indistinguishable, female-biased ssd may be the result of sex-specific differences in the threshold size for attaining sexual maturity. as in bufo bufo the age of maturity is variable, while a threshold size is mandatory for attaining maturity (hemelaar, 1988). the fact that the females are larger than males has been referenced to an increase of egg production, either to produce larger eggs, or to lay more eggs (cummins, 1986). in r. arenarum females the number of eggs was independent of body size, but female mass and mass loss after spawning were positively related indicating that condition rather than size determines egg production (bionda et al., 2011a). currently, ssd in anurans is thought to be largely a function of distinct life-history strategies of males and females (monnet and cherry, 2002). as juvenile growth is the main determinant of adult size (sinsch et al., 2010), females often delay maturity up to one year compared with the males and allocate the energy to somatic growth, thus reaching sexual maturity at larger sizes (marangoni et al., 2012). alternatively, females may show significantly increased preor post-maturity growth rates compared with males (ma and lu, 2009; guarino et al., 2008; 2011; liao and lu, 2012). in agreement with echeverria and filipello (1990), our study demonstrates that age at maturity and growth rate k did not differ significantly between the sexes, but the size threshold for attaining sexual maturity of females is greater (table 1). therefore, we assume that females continue to grow a few months longer than males at the same rate. since the temporal unit detectable with skeletochronology is one years and additional female’s growth period a few months, prolonged female growth cannot be detected by a difference in lag number. growth rate can vary considerably among years (males in 2000 vs males in 2008) indicating that k is probably influenced by local environmental factors. in contrast, maximum size is almost invariable (this study, echeverria and filipello, 1990). adult size variation in r. arenarum is about the same in all age classes, rendering an age assessment based on size classes unreliable as in most other anurans (e.g., guarino et al., 2010; li et al., 2013). demographic life history parameters early maturity often implies a short lifespan, while late maturity can be frequently observed in long-lived amphibians species (smirina, 1994; guarino et al., 2010; oromi et al., 2012). in our study population, minimum 60 clarisa de l. bionda et alii age at maturity of r. arenarum varied between 1 and 2 years, while longevity was male-biased (6 vs 4 years). echeverria and filipello (1990) found a minimum age at maturity of 2 years, and a greater longevity in females (7 years) than in males (5 years) in the buenos aires region. the different longevity observed in the present study is probably due to the small sample size. evidence for a possible trade-off between age at maturity and longevity requires a larger dataset from a larger number of populations. the age distribution (fig. 3) demonstrates that most individuals do not reproduce more than once or twice in a lifetime being age at maturity 1-2 lags and the percentage of individuals with more than 4 lags only 10.9%. in females, short longevity is possibly associated with breeding effort as shown in first-breeding rana temporaria females which show a higher annual mortality compared to males (guarino et al., 2008). it remains unclear whether the delay of maturity from one to two years in r. arenarum in 2008 reflects an adaptive response to variable environmental conditions, such as climate. for example, some variance in levels of average annual rainfall and temperature has been evidenced in the last decade in the study region (bionda, 2011) and, in general, climatic variability can affect the development and reproductive performance of amphibians (beebee, 1986; carey and bryant, 1995; blaustein and kiesecker, 2002; collins and crump, 2009; lópezalcaide and macip-ríos, 2011). the variation of temperature and precipitation are expected to affect activity patterns, microhabitat use, and thermoregulation and mediate modifications in individual growth rate, reproductive effort, and age structure. acknowledgements we thank the argentinean national research council for science and technology (conicet). the secretary of science and technology of national university of río cuarto (secyt-unrc) provided funds by grant ppi 18/c416. the investigation was conducted according to the state law “protection and conservation of wild fauna” (argentina national law nº 22.421). our study was authorized by cordoba environmental agency (a.c.a.s.e.), environmental secretary of córdoba government. references alcobendas, m., castanet, j. 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(2009): age and growth of the southern crested newt, triturus karelinii (strauch 1870), in a lowland population from northwest turkey. acta zool. acad. sci. hung. 55: 55-65. von bertalanffy, l. (1938): a quantitative theory of organic growth. hum. biol. 10: 181-213. acta herpetologica vol. 10, n. 1 june 2015 firenze university press obituary: valery k. eremchenko (1949-2014) leo j. borkin1, tatjana dujsebayeva2, roberto sindaco3, matthias stöck4 haplotype variation in founders of the mauremys annamensis population kept in european zoos barbora somerová1, ivan rehák2,*, petr velenský2, klára palupčíková1, tomáš protiva1, daniel frynta1 reproductive ecology of sichuan digging frogs (microhylidae: kaloula rugifera) wei chen1,*, lina ren2, dujuan he2, ying wang2, david pike3 toxic effects of carbaryl on the histology of testes of bufotes variabilis (anura: bufonidae) özlem çakici basal frequency of micronuclei and hematological parameters in the side-necked turtle, phrynops hilarii (duméril & bibron, 1835) maría a. latorre 1,2,*,#; evelyn c. lópez gonzález1,2, #; pablo a. siroski1,2,3; gisela l. poletta1,2,4 into a box interiors: clutch size variation and resource allocation in the european pond turtle marco. a.l. zuffi1,*, simonetta citi2, elena foschi1, francesca marsiglia1, eva martelli1 where to “rock”? choice of retreat sites by a gecko in a semi-arid habitat andreia penado1,2, ricardo rocha3,4,*, marta sampaio3, vanessa gil3, bruno m. carreira3, rui rebelo3 age structure, growth and longevity in the common toad, rhinella arenarum, from argentina clarisa de l. bionda1,2,*, silvia kost 4, nancy e. salas1, rafael c. lajmanovich3, ulrich sinsch4, adolfo l. martino1 on a putative type specimen of pleurodema bibroni tschudi, 1838 from chile (anura: leptodactylidae) daiana paola ferraro re-description of the external morphology of phyllomedusa iheringii boulenger, 1885 larvae (anura: hylidae), with comments on the external morphology of tadpoles of the p. burmeisteri group samanta iop¹, victor mendes lipinski¹, bruno madalozzo¹, franciele pereira maragno¹, sonia zanini cechin¹, tiago gomes dos santos² book review: harold heatwole, john w. wilkinson (eds). amphibians biology. volume 11 status of conservation and decline of amphibians. eastern hemisphere. part 4 . southern europe and turkey sebastiano salvidio book review: antonio romano. atlante degli anfibi del parco nazionale del cilento vallo di diano e alburni distribuzione, biologia, ecologia e conservazione sebastiano salvidio acta herpetologica journal of the societas herpetologica italica acta herpetologica 11(2): 119-125, 2016 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-18009 the unexpectedly dull tadpole of madagascar’s largest frog, mantidactylus guttulatus arne schulze1,*, roger-daniel randrianiaina2,3, bina perl3, frank glaw4, miguel vences3 1 hessisches landesmuseum darmstadt (hlmd), friedensplatz 1, 64283 darmstadt, germany. *corresponding author. e-mail: arne. schulze@hlmd.de 2 département de biologie animale, université d’antananarivo, bp 906, antananarivo 101, madagascar 3 division of evolutionary biology, zoological institute, technical university of braunschweig, mendelssohnstr. 4, 38106 braunschweig, germany 4 zoologische staatssammlung münchen (zsm-snsb), münchhausenstr. 21, 81247 münchen, germany submitted on 2016, 11th february; revised on 2016, 13th april; accepted on 2016, 9th july editor: marco sannolo abstract. the madagascar-endemic mantellid genus mantidactylus contains one subclade with two described frog species characterized by very large body sizes. this subclade is classified as the subgenus mantidactylus and is widespread in eastern and northern madagascar, but their reproductive biology and larval stages are still unknown. we here provide a detailed description of the larvae of one species in this subgenus, m. guttulatus, on the basis of genetic assignment (16s dna barcoding). the tadpoles were collected in the dry season from shallow waters near a stream in the mahajanga province in northwestern madagascar. their body and tail shape is remarkably generalized as typical for stream-adapted tadpoles, and the oral disc and labial keratodont row formula (4(2-4)/3(1)) are similar to those of other lotic mantellid frog larvae with generalized mouthparts like those in the subgenus brygoomantis. the well-separated positions of these subgenera in the mantellid phylogeny suggest extensive homoplasy in the evolution of larval mouthpart morphology within mantidactylus. keywords. amphibia, madagascar, mantellidae, mantidactylus, generalized oral disc, tadpole morphology. introduction among madagascar’s native frogs, the family mantellidae is the most diverse clade with 212 named species (amphibiaweb, 2016) and numerous undescribed species (vieites et al., 2009; perl et al., 2014). mantellids are endemic to madagascar and the comoros and include a fascinating diversity in ecomorphology and reproductive modes. the largest mantellids are classified in a wellsupported subclade of the genus mantidactylus (i.e., in the nominal subgenus mantidactylus): mantidactylus guttulatus, m. grandideri, and the candidate species m. sp. aff. grandideri “north”, although their alpha-taxonomy is in need of revision (see comments under materials and methods). with up to 120 mm snout-vent length m. guttulatus is the largest frog in madagascar and is common in rainforest streams of the northern and eastern part of the island (glaw and vences, 2007). despite their size and local abundance, information on the reproduction of this frog species is scarce and basically limited to one report of a calling specimen (vences et al., 2004). because for decades no tadpoles could be assigned to mantidactylus guttulatus or its close relatives, it was assumed that these species lack a larval phase or that the pre-metamorphic tadpoles develop in a nesting burrow (glaw and vences, 1994, 2007). altig and mcdiarmid (2006) described a tadpole with reduced oral structures from the ranomafana region and tentatively 120 arne schulze et alii assigned it as belonging to m. guttulatus. randrianiaina et al. (2011) provided molecular evidence for an assignment of these tadpoles to m. majori whose juveniles are morphologically similar to those of m. guttulatus. during a survey in northwestern madagascar, we obtained a small series of three mantidactylus tadpoles that we initially identified by morphology as belonging into the subgenus brygoomantis, despite being more elongated than other, syntopic brygoomantis larvae. molecular evidence demonstrated that these tadpoles instead belonged to mantidactylus guttulatus, and we provide a detailed description of their morphology. materials and methods three tadpoles (field numbers zcmv 13332, 13333 and 13334) were collected by r.d. randrianiaina, f.m. ratsoavina, a.s. rasamison, a. rakotoarison, d.r. vieites, and m. vences in the dry season on 29 june 2010. they were found in an opportunistic encounter survey near a large stream close to the analamisondrotra mobile phone pylon, between 56-57 km along the national road n°31 from bealanana to antsohihy (14.72602°s, 048.55497°e; 1175 m a.s.l.) in the mahajanga province. tadpoles were euthanized in a chlorobutanol solution shortly after collection. a tissue sample from the first third of the tail musculature of each tadpole was preserved in 99% ethanol. after tissue sampling, all specimens were preserved in 5% formalin and two of them were deposited in the zoologische staatssammlung münchen, germany (zsm; collection numbers zsm 704/2010, zsm 705/2010). tadpoles were identified by dna barcoding based on a fragment of the mitochondrial 16s rrna gene (thomas et al., 2005). the fragment of about 550 bp was amplified with primers 16sar-l and 16sbr-h from palumbi et al. (1991) and standard protocols resolved on automated sequencers were compared to a nearly complete database of sequences of adult malagasy frog species. dna sequences were deposited in genbank (accession numbers kx023902, kx023903, kx023904). for species names in the subgenus mantidactylus, we here follow the taxonomy suggested by glaw and vences (2007) who defined m. guttulatus as the species with a rather tubercular dorsum occurring mostly in northern madagascar, and m. grandidieri as the species with smooth skin widespread in the southern and central east of the island. this differs from the definition of altig and mcdiarmid (2006) who applied the name m. guttulatus to populations from the southern central east. however, it is obvious that this taxonomy is in need of revision and it is likely that the available names (rana guttulata boulenger, 1881; mantidactylus grandidieri mocquard, 1895; and rana pigra mocquard, 1900, currently a synonym of m. guttulatus) will have to be applied in a different way to the biological entities in the subgenus than in current practice. in fact, the tadpoles described herein might turn out to belong to a yet undescribed species. independent from this taxonomic conundrum, however, the subgenus mantidactylus is well defined and the molecular data leave no doubt that the tadpoles described herein belong into this clade. a canon dslr with 100 mm 2.8l and mp-e 65 mm lenses mounted on an electronic-driven macro rail was used to obtain the digital images of the preserved specimens. a stack of 10-15 images was taken and merged with helicon pro software to achieve images with a wide depth of focus. morphological descriptions and measurements were done on the basis of digital and scaled images of preserved tadpoles. terminology of morphological characters follows altig and mcdiarmid (1999). gosner’s (1960) classification was used to identify developmental stages. structures of the oral apparatus were described according to altig (1970), except for the term “keratodont,” which is used for the keratinized structures on the labia of the oral disc and presented as the labial keratodont row formula (lkrf). marginal papillae are considered separately for the region of the upper labium, the lateral region, and the region of the lower labium and the “marginal papillae row formula” (mprf) is provided according to schulze et al. (2015). all morphological landmarks and distances considered for the description are described and specified in table 1. comparing measurements, we consider them as “almost equal” if ratios of the measured values are 95-96% or 104-105%, “equal” if they are in the range 97-103%, as almost “in the middle” if they are in the range 45-46% or 54-55% and “in the middle” if they are in the range 47-53% (randrianiaina et al., 2011). results three tadpoles identified by 16s dna barcoding as mantidactylus guttulatus were collected within rainforest and close (ca. 20 m) to a large stream of 25 m width. the tadpoles were in a seepage area of a small, very shallow puddle (1-2 cm deep) with a slow steady flow of water. the 16s rdna sequences of these tadpoles were 99% identical to a reference sequence of an adult m. guttulatus from the tsaratanana massif (genbank accession no. fj559237). the following description refers to one of these tadpoles in gosner stage 26 (field number zcmv 13332 / zsm 704/2010, body length 9.5 mm, tail length 30.7 mm; figs. 1, 2; tab. 1). in dorsal view body elliptical, maximal body width attained almost at mid-body length, snout narrowly rounded. in lateral view, body depressed, maximal body height attained between the 3/5 and 4/5 of the body length, snout rounded. eyes moderately large, not visible from ventral view, positioned high laterally and directed anterolaterally, situated between the 2/10 and 3/10 of the body length. distance between eyes wide. nares rounded and small, marked with a marginal rim, positioned moderately high dorsolaterally and directed anterolaterally, situated closer to snout than to eye and lower than eye. distance between nares wide. spiracle sinistrally positioned and short, directed posteriorly, visible from ventral view, invisible from dorsal 121tadpole of mantidactylus guttulatus view and perceptible from lateral view; posterior third of inner wall free from body and formed that aperture is lateroposteriorly directed, its opening rounded, narrower than tube, situated between the 2/5 and 3/5 of the body length, located low on the body at the height of the hind limb insertion. long medial vent tube with dextral wall shorter than sinistral, causing a dextral directed opening, fully attached to ventral fin. glands absent. tail long, maximal tail height higher than body height, tail height at mid-tail higher than body height and as high as maxitable 1. measurements of landmarks (in mm) and their ratios (in %) of the preserved tadpole specimen of mantidactylus guttulatus (zcmv 13332 / zsm 704/2010) at gosner stage 26: a1 = first upper keratodont row; bh = maximal body height; bl = body length; bw = maximal body width; df = dorsal fin height at region of mid-tail; dg = size of the gap of marginal papillae in the region of the upper labium; dmth = distance of maximal tail height from the tail-body junction; ed = eye diameter; hab = height of the point where the axis of the tail myotomes contacts the body, measured from the lower curve of the belly; ind = inter-narial distance, measured from the centre of the eyes; iod = inter-orbital distance; jw = maximal width of keratinized upper jaw sheath; mth = maximal tail height; nh = naris height, measured from the lower curve of the belly to the centre of the naris; np = naris-pupil distance; odw = maximum width of opened oral disc; rn = rostro-narial distance, measured from the centre of the nares; sbh = distance between snout and the point of maximal body height; sbw = distance between snout and the point of maximal body width; se = snout-eye distance, measured to the centre of the pupil; sh = spiracle height; sl = spiracle length, measured from its visible edges; ss = snout-spiracle distance, measured from the centre of the spiracle opening; tal = tail length, measured from medium point of body-tail junction; th = tail height at the body-tail junction; thm = tail height at mid-tail; tl = total length; tmh = tail muscle height at the body-tail junction; tmhm = tail muscle height at mid-tail; tmw = tail muscle width at the body-tail junction; vf = ventral fin height at mid-tail. landmarks mm ratio % bh 4.1 sbw bl 57 bl 9.5 bw bh 117 bw 4.8 sbw bl 57 df 0.9 ed bl 11 dg 1.4 se bl 25 dmth 7.5 iod bw 71 ed 1.0 nd bl 3 eh 2.1 nh bh 46 hab 2.8 rn np 47 ind 2.3 ind iod 67 iod 3.4 sl bl 8 jw 1.2 ss bl 53 mth 4.8 sh bh 34 nh 1.9 sh hab 50 np 1.7 tal bl 213 odw 2.5 mth bh 117 rn 0.8 thm bh 117 sbh 6.5 thm mth 100 sbw 5.4 th bh 88 se 2.4 tmw bw 54 sh 1.4 tmh bh 63 sl 0.8 mth 54 ss 5.0 tmhm thm and mth 60 tal 20.3 df tmhm 31 th 3.6 vf tmhm 38 thm 4.8 df vf 82 tl 30.7 dmth tal 37 tmh 2.6 hab bh 68 tmhm 2.9 odw bw 52 tmw 2.6 dg odw 56 vf 1.1 a1 odw 83 jw odw 48 122 arne schulze et alii mal tail height, tail height at body-tail junction lower than body height. caudal musculature well developed. tail muscle reaches tail tip. tail fins very low, dorsal fin slightly lower than ventral fin at mid-tail, but slightly higher in posterior third. dorsal fin originates slightly behind dorsal body-tail junction, with shallow, gradually rising until the anterior 1/3 of the tail where it increases brusquely to attain its maximal height behind mid-tail and then continues gradually until the posterior 3/4 of the tail where it descends abruptly towards the tail tip. ventral fin originates at the ventral terminus of the body, rises meticulously until the anterior 1/4 of the tail, and then remains almost parallel to the ventral border of the tail muscle until close to the tail tip. maximal tail height located behind mid-tail, lateral line vein and myosepta imperceptible, point where the axis of the tail myotomes contacts the body located in the upper half of the body height, axis of the tail myotomes parallel with the axis of body length. tail tip narrowly rounded. moderately wide generalized oral disc, positioned almost ventrally and directed anteroventrally, clearly laterally emarginated. oral disc not visible from dorsal view, upper labium as a continuation of snout. marginal papillae uniseriate and interrupted by a wide gap on the upper labium, gap on the lower labium absent, total number of marginal papillae 48 (mprf: (1)/1/1). sixteen submarginal papillae present (8 on each side of the jaw sheaths folds). lkrf 4(2-4)/3(1), a1 keratodont row very long. density of keratodonts varies from 20/mm to 71/mm, a1 59/mm (total 118). gap in the a2 row narrow (>1% of a2 row) and distinctly wider in a3 and a4. gap in the p1 row less than the width of three keratodonts. alignment of anterior and posterior rows regular and nearly of same length. distal keratodonts of same length as those in the centre; prominent space between marginal papillae and keratodont rows. jaw sheaths partially keratinized, only the half section close to the edge coloured black; with finely pointed serrations. upper jaw sheath moderately wide and slightly arched, with a very shallow medial concavity. lower jaw sheath v-shaped, partially keratinized and partially hidden by the upper jaw sheath when closed. colouration in life uniformly dark brownish. dorsally, body covered by homogeneous dark brown melanophoric pigments. laterally, area below eyes, flank, and abdominal region densely reticulated. ventrally, oral disc and gular region reticulated, branchial regions reddish and spotted, beating heart visible; venter transparent, regularly spiralled intestinal coils visible. tail musculature yellowish coloured, and coarsely reticulated. fins patched with dark small spots with fringy edges. fig. 1. images of the living tadpole specimen of mantidactylus guttulatus (zcmv 13332 / zsm 704/2010) at gosner stage 26; a-c), in dorsal, lateral and ventral view (scale bar = 10 mm). 123tadpole of mantidactylus guttulatus colouration in preservative uniformly brownish coloured. brown melanic pigment in layers deeper than the skin covered the dorsum and flank, leaving laterally a slightly transparent area. some dark brown blotches scattered on the dorsum skin, condensed to form dark patches above the brain and the vertebral region. laterally, area below eyes and flank covered by dark brown reticulations, leaving out a perceptible transparent spiracle on the body wall. lower part of the flank spotted. tail musculature overlaid by dark brown spots which condensed in some area to form reticulations. fins covered by brown spots. ventrally, oral disc, gular and branchial regions reticulated; venter pale and spotted, intestinal coils visible with regular spiral shaped. in total, three tadpoles were captured, but due to a transportation problem, the second specimen (zcmv 13333) was destroyed. the external morphology of the third voucher specimen (zcmv 13334 / zsm 705/2010; gs 25) from the same locality shows the same characters and an identical oral disc configuration as the described above. discussion for decades, searches for the tadpole of mantidactylus guttulatus and its relatives in the subgenus mantidactylus have been unsuccessful, and scientists eventually hypothesized a nidicolous developmental mode for this species with a nest hidden very deep in the soil or even direct development (e.g., glaw and vences, 2007). during our tadpole surveys in many streams in ranomafana national park mainly during the rainy seasons between 2006 and 2009, no tadpole assignable to this subgenus was encountered (strauß et al., 2013), even at sites where many adults were present. one possible explanation for the absence might be a shifted onset of their reproductive season. contrary to many other species that start their reproductive efforts at the beginning of the warm-rainy season, the reproduction period of these frogs might peak at the end of each rainy season towards the beginning of the cool-dry season. the avoidance of reproductive competition with co-occurring species would be one benefit of this shift. an indication for this hypothesis is the early developmental stage of these tadpoles which suggests that they hatched in may. on the other hand, the single report of a calling individual from february (vences et al., 2004) indicates that some reproductive activity occurs during the peak of the warm-rainy season. the noticeable fact that the tadpoles were found in very shallow water and, moreover, in the seepage area of a small water body could be seen as an indication of fossorial habits. however, morphological adaptions for fossoriality like a prominent tubular spiracle or particularly small eyes present in other fossorial tadpoles e.g. otophryne robusta (wassersug and pyburn, 1987), leptobrachella mjobergi (haas et al., 2006) or micrixalus herrei (senevirathne et al., 2016) are absent in mantidactylus guttulatus. due to small sample size and the close vicinity of a large stream from which the tadpoles could have been washed away during a heavy rainfall this enigma requires further studies. also, because we did not hypothesize these tadpoles would belong to m. guttulatus when encountering them in the wild we undertook no special efforts to further investigate the seepage area in which they occurred. for instance, we cannot exclude that upstream the seepage would originate from some kind of cavity, more suitable for such a large frog to deposit its eggs. fig. 2. images of the preserved tadpole specimen of mantidactylus guttulatus (zcmv 13332 / zsm 704/2010) at gosner stage 26; a-c) in dorsal, lateral and ventral view (scale bar = 10 mm); d) wide open oral disc with anterior (a1-a4) and posterior (p1-p3) keratodont rows (white outline for better visibility, median gap in a1 row caused by preparation, scale bar = 1 mm); e) spiracle and f ) vent tube in closer view (white outline for better visibility, scale bar = 1 mm). 124 arne schulze et alii mantidactylus guttulatus tadpoles show the typical morphology of stream-adapted, orton (1953) type iv tadpoles with a large and muscular tail and low fins. according to altig and johnston (1989) they can be classified as lotic-benthic and thus assigned to the ecomorphological guild section i, guild 7. the tadpoles of m. guttulatus are similar to those of the subgenus brygoomantis (schmidt et al., 2009) which are considered as rather generalized lotic tadpoles. they share an oral disc with a large dorsal gap of marginal papillae, and a lkrf of 3-5 keratodont rows on the anterior labium with only the first being continuous and the others are interrupted by medial gaps, and three keratodont rows on the posterior labium of which the first usually has a very small medial gap. instead, the larvae of several other subgenera have highly specialized mouthparts, such as funnelshaped structures (chonomantis), poorly developed and reduced keratinized parts (ochthomantis, hylobatrachus) or a reduced number of keratodont rows in combination with unpigmented jaw sheaths (maitsomantis) (glaw and vences, 1994; vejarano et al., 2006; grosjean et al., 2011; randrianiaina et al., 2011). the well resolved phylogeny of wollenberg et al. (2011) suggests that the subgenera with generalized mouthparts (mantidactylus and brygoomantis) are not sister clades. while the subgenus mantidacylus branches off from the basal node of the mantidactylus clade, brygoomants is a sister clade to chonomantis (wollenberg et al., 2011). if these relationships are confirmed, it suggests extensive homoplasy in the evolution of tadpole mouthparts — either multiple independent evolution of specialized mouthparts, or reversal towards generalized mouthparts in the brygoomantis clade. it is surprising that a frog like mantidactylus guttulatus, whose reproductive mode has intrigued researchers for decades, has such a dull tadpole as described herein. the reproductive behaviour and the unusual microhabitat of the species still remains a mystery. where the species deposits its eggs and whether it displays any kind of pre-hatching parental care requires being elucidated by future studies. acknowledgements we are grateful to f. m. ratsoavina, a. s. rasamison, a. rakotoarison, and d. r. vieites for assisting during fieldwork for this study. this study was carried out in the framework of a cooperation accord between the département de biologie animale of the university of antananarivo, madagascar and the technical university of braunschweig. permission for collection was granted by the ministère des eaux et forêts, service de la gestion faune et flore; autorisation de recherche 064/070/10/ mef/sg/dgf/dcb.sap/slrse, delivered 25 march 2010;  including the export permit 135n-eao07/mg10, delivered 8 july 2010. financial support was granted by the volkswagen foundation to mv, rdr, and as, and by the deutscher akademischer austauschdienst to rdr. references altig, r. 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(2011): speciation in little: the role of range and body size in the diversification of malagasy mantellid frogs. bmc evol. biol. 11: 217. acta herpetologica vol. 11, n. 2 december 2016 firenze university press predator-prey interactions between a recent invader, the chinese sleeper (perccottus glenii) and the european pond turtle (emys orbicularis): a case study from lithuania vytautas rakauskas1,*, rūta masiulytė1, alma pikūnienė2 effective thermoregulation in a newly established population of podarcis siculus in greece: a possible advantage for a successful invader grigoris kapsalas1, ioanna gavriilidi1, chloe adamopoulou2, johannes foufopoulos3, panayiotis pafilis1,* the unexpectedly dull tadpole of madagascar’s largest frog, mantidactylus guttulatus arne schulze1,*, roger-daniel randrianiaina2,3, bina perl3, frank glaw4, miguel vences3 thermal ecology of podarcis siculus (rafinesque-schmalz, 1810) in menorca (balearic islands, spain) zaida ortega*, abraham mencía, valentín pérez-mellado growth, longevity and age at maturity in the european whip snakes, hierophis viridiflavus and h. carbonarius sara fornasiero1, xavier bonnet2, federica dendi1, marco a.l. zuffi1,* redescription of cyrtodactylus fumosus (müller, 1895) (reptilia: squamata: gekkonidae), with a revised identification key to the bent-toed geckos of sulawesi sven mecke1,*,§, lukas hartmann1,2,§, felix mader3, max kieckbusch1, hinrich kaiser4 the castaway: characteristic islet features affect the ecology of the most isolated european lizard petros lymberakis1, efstratios d. valakos2, kostas sagonas2, panayiotis pafilis3,* sources of calcium for the agamid lizard psammophilus blanfordanus during embryonic development jyoti jee1, birendra kumar mohapatra2, sushil kumar dutta1, gunanidhi sahoo1,3,* mediodactylus kotschyi in the peloponnese peninsula, greece: distribution and habitat rachel schwarz1,*, ioanna-aikaterini gavriilidi2, yuval itescu1, simon jamison1, kostas sagonas3, shai meiri1, panayiotis pafilis2 swimming performance and thermal resistance of juvenile and adult newts acclimated to different temperatures hong-liang lu, qiong wu, jun geng, wei dang* olim palus, where once upon a time the marsh: distribution, demography, ecology and threats of amphibians in the circeo national park (central italy) antonio romano1,*, riccardo novaga2, andrea costa1 on the feeding ecology of pelophylax saharicus (boulenger 1913) from morocco zaida ortega1,*, valentín pérez-mellado1, pilar navarro2, javier lluch2 notes on the reproductive ecology of the rough-footed mud turtle (kinosternon hirtipes) in texas, usa steven g. platt1, dennis j. miller2, thomas r. rainwater3,*, jennifer l. smith4 heavy traffic, low mortality tram tracks as terrestrial habitat of newts mikołaj kaczmarski*, jan m. kaczmarek book review: sutherland, w.j., dicks, l.v., ockendon, n., smith, r.k. (eds). what works in conservation. open book publishers, cambridge, uk. http://dx.doi.org/10.11647/obp.0060 sebastiano salvidio acta herpetologica 13(2): 171-175, 2018 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-23366 helminths infecting the black false boa pseudoboa nigra (squamata: dipsadidae) in northeastern brazil cicera silvilene l. matias1,*, cristiana ferreira-silva2, josé guilherme g. sousa3, robson w. ávila1,3 1 laboratório de herpetologia, departamento de química biológica, universidade regional do cariri, campus do pimenta, cep 63105000, crato, ce, brazil. *corresponding author. e-mail: silvilenematias@gmail.com 2 programa de pós-graduação em ciências biológicas (zoologia), departamento de parasitologia, instituto de biociências, universidade estadual paulista, cep 18080-970, botucatu, sp, brazil 3 programa de pós-graduação em ecologia e recursos naturais, departamento de ciências biológicas, universidade federal do ceará, campus universitário do pici, cep 60021970 fortaleza, ce, brazil submitted on: 2018, 8th june; revised on: 2018, 28th august; accepted on: 2018, 13th september editor: daniele pellitteri-rosa abstract. knowledge about endoparasites of snakes is essential to understand the ecology of both parasites and hosts. herein, we present information on helminths parasitizing the black false boa pseudoboa nigra in northeastern brazil. we examined 32 specimens from five brazilian states (ceará, piauí, pernambuco, maranhão and rio grande do norte). we found six helminths taxa: two acanthocephalans (acanthocephalus sp. and oligacanthorhychus sp.), three nematodes (hexametra boddaertii, physaloptera sp. and physalopteroides venancioi), and one cestode (ophiotaenia sp.). all parasites are reported for the first time infecting p. nigra, providing relevant information on infection patterns in this snake. keywords. acanthocephala, cestoda, nematoda, reptilia, snake. surveys of endoparasites associated with wild animals are key features to understand ecology, natural history, life cycles and evolution of parasites and their hosts (silva, 2008). parasites can influence their hosts in different ways, affecting their physiological and behavioural patterns (levri, 1999), which can cause changes in coloration, and decrease in reproductive capacity, resulting in reduced reproductive success (schall and dearing, 1987; dunlap and schall, 1995). studies focusing on endoparasites in reptiles has grown in the past few years, and the diversity of parasites tends to increase as new hosts are studied (ávila and silva, 2010). in brazilian snakes there is also an increase, especially in parasite species descriptions and new records of hosts (bursey and brooks, 2011; norval et al., 2012; pinto et al., 2012; araújo-filho et al., 2013; ávila et al., 2013; kuzmin et al., 2014, 2016; mati et al., 2015). however, little is known about infection patterns in snakes from brazil (almeida et al., 2008). the black false boa pseudoboa nigra (duméril, bibron and duméril, 1854) is widely distributed in brazil, bolivia, paraguay and argentina (etchepare et al., 2015). several aspects of its biology are known, such as diet and reproduction (orofino et al., 2010; gaiarsa et al., 2013; mesquita et al., 2013). however, data on parasitism in this snake is restricted to the description of protozoa trypanosoma serpentis (viola et al., 2009) and a report of the pentastomid raillietiella furcocerca (alcantara et al., 2014). herein, we describe both the helminths richness and infection patterns (prevalence and intensity) in p. nigra from northeastern brazil. we examined 32 specimens of p. nigra from five brazilian states in the northeastern region: ceará (n = 20), maranhão (n = 1), piauí (n = 4), pernambuco (n = 5) 172 cicera silvilene l. matias et alii and rio grande do norte (n = 2). we used specimens deposited at coleção herpetológica da universidade regional do cariri (urca-h 379, 507, 916, 2222, 2381, 3377, 3432, 3742, 3982, 4510, 4755, 4916, 5104, 5632, 5694, 5885, 5886, 7090, 8507, 9400, 9482, 9536, 10043, 10594, 10629, 11031, 11348, 11075, 11899, 11900, 12108, 12266), collected from 2011 to 2016. we checked carefully for helminths, under a stereomicroscope, the gastrointestinal tract (esophagus, stomach, small and large intestines), liver, kidneys, lungs, gallbladder and coelomic cavity. for helminths identification, we stained acanthocephalans and cestodes with alcoholic hydrochloric acid-carmine and cleared in eugenol, while nematodes were diaphanized in lactic acid (amato and amato, 2010). after identification, all helminths species were deposited at coleção parasitológica da universidade regional do cariri (urca-p 476–521). we calculated parasitological parameters (prevalence, mean abundance and mean intensity of infection) according to bush et al. (1997). of the black false boas analyzed, 23 specimens contained parasites, represented by six helminths species: two acanthocephalans (prevalence 59.3%) (acanthocephalus sp., and oligacanthorhychus sp.), three nematodes (43.75%) (hexametra boddaertii, physaloptera sp. and physalopteroides venancioi), and one cestode (3.1%) (ophiotaenia sp.). the overall prevalence was 71.9%, mean abundance 230.5 ± 126.5, mean intensity of infection 320.7 ± 173.4 (range: 1-3648) and richness 1.12 ± 0.15 in p. nigra (n = 32; svl 579.8 ± 166.2) (table 1). p. nigra presented high helminth overall prevalence (71.9%), similarly to other snakes, such as in bothrops moojeni (68%; barrella and silva, 2003), crotalus durissus terrificus (66%; dias et al., 2004), erythrolamprus miliaris (44.4 %, 72.7%; roldan and fiorillo, 2016 and mati et al., 2015, respectively). the richness and abundance of parasitic groups that contribute to high prevalence in these snake species vary according to the habits of their hosts. for example, nematoda richness tends to be higher in terrestrial snakes, such as p. nigra (3 spp.; present study), c. d. terrificus (7 spp.; dias et al., 2004) and crotalus mitchelli (5 spp.; goldberg et al., 2013), while trematoda richness is higher in aquatic species, such as e. miliaris (17 spp.; mati et al., 2015) and n. natrix (5 spp.; yildirimhan, 2007). thus, the richness, abundance and high prevalence of nematodes may be directly related to the host habitat (brouat et al., 2007). acanthocephalans present indirect life cycle and use snakes as paratenic host (e.g., travassos, 1917; pizzatto and madi, 2002; pizzatto and marques, 2006; smales, 2007). oligacanthorhynchus is represented actually by 34 species (amin, 2013), two of these known to infect south american snakes: oligacanthorhynchus spira infecting boa constrictor, boiga dendrophila, bothrops jararacussu, bothrops neuwiedi, clelia clelia, erythrolamprus aesculapii, erythrolamprus miliaris, erythrolamprus poecilogyrus, mastigodryas bifossatus, micrurus corallinus, philodryas olfersii, xenodon merremii and xenodon histricus (travassos, 1917; pizzatto and madi, 2002; pizzatto and marques, 2006); and oligacanthorhynchus taenioides infecting b. constrictor, b. dendrophila, b. jararacussu, b. neuwiedi, c. clelia, e. aesculapii, m. bifossatus, p. olfersii, x. merremii and x. histricus (travassos, 1917). cystacanths of oligacanthorhynchus sp. have also been reported in philodryas patagoniensis (smales, 2007). another acantocephalan found in the present study belongs to the genus acanthocephalus. from the 54 known species (amin, 2013; amin et al., 2014), only acanthocephalus lutzi has been reported infecting brazilian snakes (x. merremii and x. neuwiedii; smales, 2007). table 1. helminths parasites associated with pseudoboa nigra in five states from northeastern brazil. ce = ceará; pe = pernambuco; pi = piauí; rn = rio grande do norte, p = prevalence; ma = mean abundance (± standard error); mii = mean intensity of infection (± standard error); si= site of infection; (cc = coelomic cavity, s = stomach, li = large intestine, si = small intestine). helminths states p % ma ± se mii ± se si acanthocephala 62.5 377.74 ± 207.94 acanthocephalus sp. ce, pe 6.25 2.5 ± 1.74 40.0 ± 3.0 s oligacanthorhychus sp. ce, pe, pi 56.25 225.75 ± 126.5 401.33 ± 218.52 cc, si, li nematoda 43.75 14.43 ± 9.51 hexametra boddaerti ce, pe, pi 15.62 1.28 ± 0.69 8.20 ± 3.07 cc, s, si physaloptera sp. ce, pe, rn, 31.25 4.83 ± 4.55 49.66 ± 47.02 cc, s, li physalopteroides venancioi ce 3.13 0.03 ± 0.03 1.0 si cestoda 3.13 4.0 ophiotaenia sp. ce 3.13 0.12 ± 0.12 4.0 si 173helminths infecting pseudoboa nigra from brazil in the present study, only cystacanths were found, suggesting that p. nigra acts as paratenic host for acanthocephalus, with insects being the intermediate and birds the definitive hosts (travassos, 1917; schmidt and roberts, 1996), thus suggesting that acanthocephalus spp. may use p. nigra to reach its definitive hosts. identification of acanthocephalus sp. and oligacanthorhynchus sp. found in this study was not possible due to immature condition of specimens (cystacanths), making it difficult to analyze the proboscis hook formulas. nematodes were represented by hexametra boddaerti, physaloptera sp. and physalopteroides venancioi. h. boddaerti is commonly found in lizards (ávila and silva, 2010) and snakes (bursey and brooks, 2011). the genus hexametra contains 7 species (baker, 1987), of which h. boddaerti is known to infect the following snakes in south american: bothrops sp., crotalus durissus, oxyrhopus trigeminus, philodryas baroni, and philodryas patagoniensis (skrjabin, 1916; sprent, 1978; hartdegen and gamble, 2002; pinto et al., 2010). among the 105 physaloptera species known (pereira et al., 2014; luiz et al., 2015) p. monodens infects boa constrictor, p. obitusissima infects bothrops jararaca (skrjabin and sobolev, 1964) and physaloptera sp. infects micrurus surinamensis (ávila et al., 2013). physalopteroides venancioi is the only species with occurrence in the south america and is a generalist parasite of lizards (ávila and silva, 2010) and amphibians (campião et al., 2014). however, this is the first record of p. venancioi infecting snakes. we suggest that infection of p. nigra by p. venancioi is accidental, being the parasite acquired from the feeding of the snake, since its diet consists mainly of lizards (orofino et al., 2010). in brazil, there are five ophioteania species infecting snakes: ophiotaenia calmetti, o. elongata, o. flava, o. hyalina, and o. macrobothria. only a few brazilian snakes have been reported as host for ophiotaenia species, among them, bothrops jararaca, bothrops atrox, corallus caninus and micrurus corallinus (silva et al., 2006). in conclusion, we found a high prevalence (71.9%) of helminths in the black false boa, reporting it as a new host for all helminth species here recorded, contributing to the increase of diversity and knowledge on parasites. acknowledgements we thank the conselho nacional de desenvolvimento científico e tecnológico (cnpq) for providing a research fellowship to rwa (# 303622/2015-6), scientific initiation fellowship to cslm (121944/2017-4) and fellowship to cfs (140871/2017-9). jggs thanks to coordenação de aperfeiçoamento de pessoal de nível superior – capes for the fellowship. we are grateful to the cnpq/funcap/capes for  financial support (protax – processes 440511/2015-1; 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(2009): phylogenetic analyses based on small subunit rrna and glycosomal glyceraldehyde-3-phosphate dehydrogenase genes and ultrastructural characterization of two snake trypanosomes: trypanosoma serpentis n. sp. from pseudoboa nigra and trypanosoma cascavelli from crotalus durissus terrificus. j. eukaryot microbiol. 56: 594-602. yildirimhan, h.s., bursey, c.r., goldberg, s.r. (2007): helminth parasites of the grass snake, natrix natrix, and the dice snake, natrix tessellata (serpentes: colubridae), from turkey. comp. parasitol. 74: 343-354. acta herpetologica vol. 13, n. 1 june 2018 firenze university press acta herpetologica 10(2): 135-141, 2015 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-16194 malformations and body injuries in a hybrid zone of crested newts (caudata: salamandridae: triturus cristatus superspecies) zdeněk mačát1, lenka jeřábková2, antonín reiter3, martin rulík1, daniel jablonski4 1 department of ecology and environmental sciences, palacký university in olomouc, šlechtitelů 27, 78371, olomouc, czech republic. corresponding author. e-mail: zdenek.macat@gmail.com 2 nature conservation agency of the czech republic, kaplanova 1931/1, 14800, praha 11 chodov, czech republic 3 south moravian museum in znojmo, přemyslovců 129/8, 66902, znojmo, czech republic 4 department of zoology, comenius university in bratislava, mlynská dolina b-1, 84215, bratislava, slovakia submitted on 2015, 12th may; revised on 2015, 6th october; accepted on 2015, 9th october editor: sebastiano salvidio abstract. morphological abnormalities occur frequently in wild amphibian populations. we analysed malformation and injuries in the hybrid zone of three crested newt species, in the czech republic. in total, 274 individuals from 35 localities in south moravia (czech republic) were examined during the period 2010-2014. malformations were found in eight newts (2.9%) from seven localities. injuries were recorded on 59 newts (21.5%). proportions of tail crest injuries was significantly higher (p ˂ 0.1) in males than in females and the probability of being injured was significantly higher (p ˂ 0.01) for adult individuals. we discuss gene mutation, parasitism and predation as possible explanations for our observations. keywords. amphibia, triturus cristatus, morphology, abnormalities, polydactyly, limb damages, contact zone, czech republic preor post-natal malformations are regularly reported in different amphibian species (canestrelli et al., 2006; piha et al., 2006; sas and kovacs, 2006; machado et al., 2010; jarvis, 2011; henle, et al. 2012; gatti and sannolo, 2014). in general, amphibian abnormalities include malformation and injuries, and according to reeves et al. (2008) and hassine et al. (2011), the most common malformations in amphibian are: i) eye abnormalities such as anophtalmia or microphtalmia, ii) skeletal injuries such as brachydactyly (short digits), ectrodactyly (missing digits) or ectromelia (partial limb formation), iii) skeletal malformations such as amelia (missing limb), polydactyly (extra digits), syndactyly (digits fused), brachygnathia (short jaw), iv) surficial abnormalities such as incomplete tail resorption or skin trauma (e.g., scars, edema). along with colour abnormalities (e.g., leucism, albinism or axanthism) these malformations are recorded in the wild life of amphibians (jablonski et al., 2014). malformations could be a result of genetic mutation caused by environmental pollutants, parasites, diseases, prenatal stress, genetic predisposition or uv radiations (blaustein and johnson, 2003). on the other hand, injuries in wild animals can originate from diseases, intraspecific aggression, and most of all by predation (lima and dill, 1990). injuries are usually studied in animals that have the ability to autotomize a body part (cooper et al., 2004; maginnis, 2006) or to regenerate a missing part, which is after this process actually different from the original part (maginnis, 2006). frequency and types of injuries are probably connected with density and diversity of predators, with geographical differences in injury rates among population (placyk and burghardt, 2005) and sexes (sexual dimorphism) in the breeding season (kopecký, 2013). in this article, we present results of our study on morphological abnormalities found in crested newts from 136 zdeněk mačát et alii their hybrid zones in the czech republic. studied localities are southern parts of the czech republic (znojmo region), where the ranges of three crested newt species meet: triturus cristatus (laurenti, 1768), t. dobrogicus (kiritzescu, 1903), and t. carnifex (laurenti, 1768) (wielstra et al., 2014). their distribution is parapatric with mixed contact zone in south moravia, the only known among these species in the czech republic (mikulíček et al., 2012). despite malformations of amphibians are relatively well known, the following paper provides a new point of view on their causes. during the period 2010-2014 we recorded body abnormalities and injuries at triturus cristatus superspecies in znojmo region (south moravia, czech republic). during the breeding season, 274 individuals (136 males, 127 females and 11 subadults) in the water phase from 35 localities, were examined. newts were captured by funnel collapsible nylon baited traps (bock et al., 2009; madden and jehle, 2013), sexed, measured and photographed from dorsal and lateral side of the body. each locality was visited once, in case of sufficient number of caught newts (more than 10 individuals). otherwise these localities were repeatedly visited. individuals from all populations were recognized by belly pattern from photographs (according to hagström, 1973; jehle et al., 2011). differences between males and females and between bigger and smaller individuals were performed on tail injuries by the software statistica 12 (hill and lewicki, 2007), using a nonparametric mann-whitney u test. malformations were recorded at seven localities (table 1, fig. 1). each malformation was recorded in different locality, except two cases (locality podmolí 2). artificial pond was the most frequent type of habitat with morphological abnormalities (four cases); two were situated to the wood ponds and other cases represented different types of ponds. the distance between the two nearest localities was 0.2 km (podmolí 1 and podmolí 2) separated by road. there is no evidence about identical belly pattern between newts in these localities. the longest distance between two localities was 18.8 km (bojanovice and chvalatice). eight out of 274 individuals (2.9%) showed malformations of three different types: bidactyly, polydactyly and syndactyly (table 1, fig. 2). bidactyly was recorded five times (1.9% of all individuals) while polydactyly two times (0.7%) and syndactyly one a time (0.4%). overall, three male individuals (average svl: 71.9 mm) and five females (86.8 mm) were recorded with malformations. fifty-nine out of 274 newts (21.5%) were injured. male individuals were injured in 28 cases (47.5%) and females in 31 cases (52.5%). injuries could be divided into six types. the first type consisted of damage to the front limb (including arms and fingers; fig. 3e, g, h) and the second type consisted of a damage to the hind limb (fig. 3f). two types of injuries were recorded on tail: damage (ragged) on the tail crest and missing the tip of the tail (fig. 3a-d). there was some interesting differences between males and females: while males were injured on the crest of tail, females were injured on the tip of the tail. the last two injuries types were recorded on the body (trunk) and the head. the most common injury was damage to the tail (32 cases, 71.2%, fig. 4). the second most recorded was damage to the limb (15 cases, 25.4%). furthermore, two atypical injuries were found: one female had fresh scars on the trunk and tail (fig. 3i) and another female had a hole in the throat (fig. 3j). the proportion of tail crest injuries was significantly higher for males (14 cases) compared to females (p < 0.1). the probability of injuries was significantly higher for bigger newts (average 76.9 mm) compared to smaller ones (average 72.1 mm; p < 0.01). we observed three types of malformation in the t. cristatus superspecies (bidactyly, polydactyly and syndactyly). several cases of morphological malformation among the genus triturus are known from available literature: bidactyly in three individuals of t. cristatus from the united kingdom (jarvis, 2011), polydactyly in two t. carnifex from italy (gatti and sannolo, 2014), polymetable 1. malformation recorded in triturus cristatus superspecies during the study (svl snout-to-vent length; tl tail length). figure sex svl tl malformation locality gps altitude pond type date 1a f 78.4 52.9 bidactyly čížov 48.88n, 15.88e 400 m artificial 28 april 2010 1b f 89.9 69.8 polydactyly onšov 48.90n, 15.84e 460 m natural forest 22 april 2011 1c f 94 80.9 bidactyly podmolí 2 48.84n, 15.93e 406 m artificial 25 may 2011 1d m 75.4 56.1 bidactyly podmolí 2 48.84n, 15.93e 406 m artificial 25 may 2011 1e f 90 60.8 bidactyly podmolí 1 48.84n, 15.93e 412 m fish pond 27 april 2012 1f f 82 54 bidactyly lukov 48.86n, 15.89e 444 m forest wetland 29 april 2013 1g m 66.4 51.6 polydactyly bojanovice 48.94n, 16.00e 350 m artificial 10 may 2013 1h m 73.9 52.8 syndactyly chvalatice 48.93n, 15.74e 446 m artificial 2 april 2014 137malformations in triturus cristatus ly in t. marmoratus (recuero-gil and campos asenjo, 2002) and malformation of digits between hybrids of t. cristatus and t. marmoratus in western france (arntzen and wallis, 1991). malformation rates in amphibians do not exceed 5% in healthy populations (blaustein and johnson, 2003). in comparison with other authors studying crested newts (jarvis, 2011; gatti and sannolo, 2014; mester et al., 2015), the malformation rate found in this study is relatively high. on the other hand, malformation rate shows much higher fluctuations among other species of caudata: e.g., about 3.9% at calotriton arnoldi (martínez-silvestre et al., 2014) or about 40-90% at cryptobranchus alleganiensis (hiler et al., 2005). amphibians seem particularly prone to malformation (e.g., polydactyly). the extreme sensibility to environmental changes and habitat loss are well known in amphibians, including crested newts (beebee and griffiths, 2005). however, we have no data to suppose that pollution (some localities are situated into national park, where we can assume low levels of pollution) caused observed abnormalities. five main factors could possibly cause malformations: i) hyper-regeneration after predator attempts or accidents (eaton et al., 2004; ballengée and sessions, 2009), ii) exposition to high uv-b radiation (blaustein et al., 1997; pahkala et al., 2003), iii) chemical pollution from industry and agriculture (kiesecker, 2002; piha et al., 2006), iv) degradation of the environment (houlahan et al., 2000) and v) parasite infection (e.g., ribeiroia trematodes; kiesecker, 2002; johnson and chase, 2004). in addition, body malformations could also be caused by hybridization. different crested newt species cross in narrow hybrid zones (arntzen et al., 2014). arntzen and wallis (1991) discussed the relatively high proportion of malformation within triturus hybrid zone caused by collapsed genetic homeostasis. a much lower proportion of malformation is reported away from crestfig. 1. distribution map of triturus cristatus superspecies (t. cristatus pink, t. carnifex purple, t. dobrogicus orange; the colours in the overlap indicate putative contact or hybrid zones; sensu wielstra et al., 2014) in study area and locations of localities with records of malformations (1 chvalatice, 2 onšov, 3 lukov, 4 čížov, 5 podmolí 2, 6 podmolí 1, 7 bojanovice; see table 1.). 138 zdeněk mačát et alii ed newt hybrid zones (jarvis, 2011; gatti and sannolo, 2014, mester et al., 2015). therefore, hybridization could be the likely explanation for our results. crested newts are active swimmers, therefore, the injuries of tail or limbs could negatively affect their fitness. under natural conditions, injured individuals are easily liable to become a prey for predators (kopecký, 2013). the relatively high percentage of injured individuals in our study (compare with székely and nemes, 2003; kopecký, 2013) could be probably caused by predators, such as water birds, small mammals or invertebrates (e.g., dragonfly nymph, bowerman et al., 2010). moreover, some injuries could emerge from stocking fish for fishing, such as carassius gibelio (bloch, 1782), pseudorasbora parva (temminck and schlegel, 1846) and ameiurus nebulosus (lesueur, 1819) (lusk et al., 2010). taking into account the syntopic occurrence of the three crested newt species, competition within species is also possible. considerable sexual dimorphism during breeding phase is well known, in which males are more brightly coloured than females (e.g., griffiths, 1996; jehle et al., 2011). moreover, males are territorial and fights between them are known (griffiths, 1996). thus, we can expect that males are probably more frequently injured than females (cf. kopecký, 2013). our results concerning tail injuries partly support this hypothesis. as a simple explanation, we consider male tails to be more visually attractive for predators and they have different shape fig. 2. malformation recorded during the study of crested newt (for details see table 1). fig. 3. examples of injuries recorded during the study of crested newts (a-d tail damage, e-h missing toes or limb, i-j scars and tissues damage). 139malformations in triturus cristatus and dimension than female tails. kopecký (2013) did not find any intersexual difference, although injuries at tail tip were more frequent (but not significantly) in females. kopecký (2013) offers an explanation for mechanical damage to tail during underground movement in narrow spaces at terrestrial phase/hibernation. it is undisputed, that all types of injuries (loss of limbs, fingers or tail) can significantly impair walking, running, swimming, gliding, diving and could be a risk for disease infection (cooper et al., 2004; maginnis, 2006; marvin, 2010). our results suggest that larger (and presumably older) individuals are injured more often probably because body size at crested newts increases significantly with age (rehák, 1983; halliday and verrell, 1988). moreover, older animals will also accumulate injuries on their body. nevertheless, these accumulations do not need to be necessarily lethal for amphibians as was found by mott and steffen (2013) who reported the association between body size and nonlethal injuries in amphibians. acknowledgements we are very grateful to ben wielstra (sheffield) and two anonymous reviewers for their valuable comments, which improved previous versions of the manuscript. also, we would like to thank adam bednařík (olomouc) for technical support and people, which were helpful during the field survey: lenka reiterová (čížov), miroslav maštera (kostelní myslová), václav křivan (štěměchy) and podyjí national park administration for providing important facilities. field study was supported by iga přf upol: no. iga_prf_2015_008, norway grants: no. 76213/env/10, 5458/610/10 and monitoring program of nature conservation agency of the czech republic: no. ppkb 10/09/11. all handling of animals was carried out under permits number 32118/env/11-1156/620/11-pp18 and 27586/env/14-1544/630/14. references arntzen, j.w., wallis, g.p. 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(2005): geographic variation in the frequency of scarring and tail stubs in eastern gartersnakes (thamnophis s. sirtalis) from michigan, usa. amphibia-reptilia 26: 353-358. recuero-gil, e., campos asenjo, o. (2002): triturus marmoratus (marable newt): polymely. herpetol. bull. 82: 31-32. reeves, m.k., dolph, c.l., zimmer, h., tjeerdema, r.s., trust, k.a. (2008): road proximity increases risk of skeletal abnormalities in wood frogs from national wildlife refuges in alaska. environ. health persp. 116: 1009-1014. rehák, i. (1983): changes in body measures during the growth of the newts triturus vulgaris, t. alpestris and t. cristatus (amphibia: urodela). věst. čs. zool. spol. 47: 51-67. sas, i., kovacs, e.h. (2006): hexadactyly case at a rana kl. esculenta sample from the north-western part of 141malformations in triturus cristatus romania. an. u. o. fasc. biol. 13: 52-53. székely, p., nemes, s. (2003): the incidence of mutilations and malformations in population of pelobates fuscus. russ. j. herpetol. 10: 145-148. wielstra, b., sillero, n., vörös, j., arntzen, j.w. (2014): the distribution of the crested and marbled newt species (amphibia: salamandridae: triturus) an addition to the new atlas of amphibians and reptiles of europe. amphibia-reptilia 35: 376-381. acta herpetologica vol. 10, n. 1 june 2015 firenze university press acta herpetologica journal of the societas herpetologica italica acta herpetologica 11(1): 15-20, 2016 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-15172 morphology, ecology, and behaviour of hylarana intermedia, a western ghats frog ambika kamath1, rachakonda sreekar2,3 1 museum of comparative zoology and department of organismic and evolutionary biology, harvard university, 26 oxford street, cambridge, ma 02138, usa. corresponding author. e-mail: akamath@fas.harvard.edu 2 agumbe rainforest research station, agumbe, karnataka, india  3 current address: school of biological sciences, university of adelaide, sa 5000, australia submitted on 2015, 1st december; revised on 2016, 12th march; accepted on 2016, 18th march. editor: giovanni scillitani abstract. despite being common in the western ghats-sri lanka biodiversity hotspot, golden-backed frogs (hylarana, ranidae) remain poorly studied. in this paper, we present some preliminary data on the morphology, behaviour, and habitat use of hylarana intermedia, a member of the hylarana aurantiaca species group. we find evidence for female biased size dimorphism, as well as potential shape differences between the sexes in this species. additionally, we investigate the relationships between traits that may contribute to male conspicuousness (call rates, dorsal coloration, and body size) in two breeding habitats, a paddy field and a trench. our results suggest both size-dependent and environment-dependent variation in call rate and colour in this species. specifically, we find evidence against the adoption of sneaker mating strategies by small males in h. intermedia, and instead find variation both within and between populations in traits contributing to male conspicuousness. we conclude by proposing future directions for research on this common frog species. keywords. calling rate; colour; hylarana intermedia; microhabitat; size; western ghats. introduction golden-backed frogs (hylarana, ranidae) are widespread in the biodiversity hotspot of the western ghats and sri lanka, and are commonly encountered in a wide variety of habitats in this region (daniel, 2002; purushotham and tapley, 2011). a recent phylogenetic study has revealed extensive genetic variation among south asian golden-backed frogs, prompting the description of seven new species in the genus hylarana (biju et al., 2014). however, the ecology and behaviour of this diverse group of frogs are almost completely unknown. hylarana intermedia is a member of the hylarana aurantiaca species group, and is found north of the palghat gap in the western ghats of india (biju et al., 2014). a nocturnal frog, h. intermedia begins to breed with the arrival of the south-west monsoon, from june to august. large aggregations of calling males are found in pools of water and the surrounding vegetation (purushotham and tapley, 2011). in this paper, we present some preliminary data on the morphology, behaviour, and habitat use of h. intermedia, and propose future directions for research on this common frog species. sexual size dimorphism is widespread in frogs. females are larger than males in almost 90% of all anuran amphibians (shine, 1979). however, the presence of male-biased sexual size dimorphism in a frog species could reveal interesting biology, such as the occurrence of physical combat as part of male-male competition (shine, 1979) or an atypical age-structure of the breeding population (monnet and cherry, 2002). in a first step towards characterizing the morphology of this species, we inves16 ambika kamath, rachakonda sreekar tigate whether h. intermedia conforms to the pattern of female-biased sexual size dimorphism seen in most frogs. we also describe patterns of sexual shape dimorphism to identify potential morphological targets of sex-specific selection in this species. across animals, variation in body size is often related to variation in mating strategy (andersson, 1994). in frogs, a male’s mating success is primarily determined by his conspicuousness to females. phenotypically, male frogs can be conspicuous through both auditory and visual means. auditory conspicuousness is achieved through male calling behaviour, and abundant evidence indicates that a male frog’s vocalization is an important component of his conspicuousness and contributes to his mating success (e.g., arak, 1988; passmore et al., 1992; grafe, 1997; pröhl, 2003; reviewed in wells, 2007). visual conspicuousness in frogs can be influenced by size, coloration, and the performance of visual signals (wells, 1980; haddad and giaretta, 1999; grafe and wanger, 2007), even in nocturnal frogs that are active in conditions of low light (e.g., sheldon et al., 2003; vasquez and pfennig, 2007; gomez et al., 2009). a common mating strategy for smaller male frogs is to remain inconspicuous and behave as a satellite near a large, calling male, only to sneak matings with females that are attracted to the conspicuous male (emlen, 1976; blackwell and passmore, 1991). males of h. intermedia vary in body size, call rate, and dorsal coloration (fig. 1) and we test whether smaller male h. intermedia are in fact less conspicuous than larger males by examining the relationships of calling rates and dorsal coloration with body size. a male’s choice of microhabitat provides the environmental context in which his conspicuousness is assessed by both females and predators (e.g. leal and fleishmann, 2002; galeotti et al., 2003; amézquita and hödl, 2004). the strength and direction of selection and constraints acting on his phenotype thus depend on microhabitat, and different habitats provide different ranges of available microhabitats for males to choose among. the relationships between conspicuousness traits might therefore differ substantially between habitats (ohmer et al., 2009). in h. intermedia, breeding pools can be of different shapes and sizes, and within each pool, breeding males perch at and call from a variety of locations at different distances from the edge of the pool. we ask if h. intermedia differ phenotypically between two breeding sites that differ in their spatial structure: a large, disused paddy field and a narrow trench. we also examine if phenotypic variation in the larger, more varied habitat (the paddy field) is structured by an environmental variable (distance from dry land). we use our results to make predictions about how selection might act on the phenotypic traits determining conspicuousness in different breeding habitats of h. intermedia. materials and methods study site and data collection this study was conducted during the southwest monsoon period, from 5-19 july 2011, at the agumbe rainforest research station, agumbe, karnataka, india (13°50’n, 075°09’e; 560 m above sea level). we observed frogs in two locations: a large, disfig. 1. variation in the dorsal coloration of hylarana intermedia from light (left; colour score of one) to dark (right; colour score of five; see materials and methods for details of colour scoring). 17behaviour, ecology, and morphology of hylarana intermedia used paddy field and a narrow trench in an open forest clearing. we divided each site into non-overlapping sections, and only one section was sampled per night and not subsequently resampled, to minimize repeated observations of the same individuals. up to 11 individuals were observed and caught per section. male frogs, which can be differentiated from females by the presence of vocal sacs, were located either visually or by following their calls. male frogs were observed, without disturbing them, for 5 minutes, during which we counted the number of vocalizations produced. after the observation, a single observer (ak) scored the colour of the frog on a scale of 1 to 5 as follows: 1 = pale golden, 2 = light with intermediate spots, 3 = intermediate, 4 = intermediate with dark spots, 5 = dark (fig. 1). the same light source was used to illuminate all frogs to ensure a consistent scoring of colour. while dorsal colour can change within minutes (e.g., on being captured), changes in coloration were not noticed during observations. in the paddy field habitat, we also measured the shortest distance from the frog’s position to dry land. because the centres of ponds in the habitat had less vegetation than the edges (pers. obs.), the distance to dry land is an approximate indicator of habitat openness. we then caught the frogs to measure the following variables: snout-vent length (svl), mass, head length, head width, trunk length, femur length, tibia length, foot-to-toe length, humerus length, and radius-to-finger length. all limb measurements were made on only the left side of the frog. after all frogs were measured, they were released into the section of the site in which they were captured. morphological measurements were also made on additional male and female frogs captured at both sites as well as near trails between the two sites. statistical analyses a wilcoxon rank-sum test was conducted on snout-vent length (svl) to compare body size between males and females. to compare the morphology of male and female frogs, a principal components analysis was conducted on all morphological variables, and pc1 and pc2 were compared between males and females using wilcoxon rank-sum tests. to examine if male frogs in the paddy field and trench habitats differed in the traits contributing to conspicuousness, we compared both svl and calling rates between frogs from the two habitats using wilcoxon rank-sum tests. dorsal colour scores were compared between the two habitats using a chisquared test. analysing data separately for the paddy field and trench habitats, we tested whether male body size (svl) was correlated with calling rate using spearman rank correlations. we tested if frogs of different colours differed in size using jonckheereterpstra tests, which are similar to kruskal-wallis tests but apply to populations that are ordered by a factor (in this case, colour score). again, we considered data from the trench and paddy field habitats separately. finally, for the paddy field site, we used spearman rank correlations to test if the distance to dry land was correlated with svl or calling rate, and a jonckheere-terpstra test to examine if distance to dry land varied with colour. we conducted all analyses in r v 3.2.2 (r core team, 2015), and used the clinfun package (seshan, 2015) to perform the jonckheere-terpstra tests, using 5000 permutations to estimate p-values. results we observed and caught a total of 68 male frogs (38 individuals in the paddy field and 30 individuals in the trench). additionally, 43 males and seven females were caught for morphological measurements only. male and female h. intermedia differed significantly in svl: males were smaller in size than females (mean ± standard deviation in svl for males: 40.9 ± 2.3mm; females: 52.1 ± 2.4mm; w7, 111 = 777; p < 0.001). the first principal component axis (pc1) from the pca on morphological variables yielded a composite variable reflecting overall body size (i.e., positive loadings of similar magnitudes on all variables; table 1), and pc1 also differed significantly between the sexes (w7, 111 = 777; p < 0.001; fig. 2). the second principal component axis loaded positively on humerus length and trunk length, and negatively on head length and width. there was a trend towards higher pc2 in females than males (w7, 111 = 552; p = 0.06; fig. 2), suggesting a pattern of relatively longer trunks and humeri in females and relatively large heads in males. male frogs’ svl did not differ between the paddy field and trench habitats (mean ± standard deviation; paddy field: 41.2 ± 2.6 mm; trench: 41.4 ± 3.9 mm; w44, 71 = 1590.5, p = 0.87). calling rates also did not differ between male frogs from the two habitats (mean ± standard deviation; paddy field: 21.9 ± 20.7; trench: 20.2 ± 15.2; w38, 30 = 551, p = 0.82). however, frogs were lighter table 1. loadings on the first and second principal component axes (pc1 and pc2) from a principal components analysis on morphological variables from males and females. bold numbers indicate loadings with a magnitude greater than 0.25. pc1 pc2 % variance explained 76% 6% svl 0.34 -0.14 head length 0.39 -0.64 head width 0.32 -0.40 femur 0.32 0.18 tibia 0.34 0.12 foot 0.33 0.13 humerus 0.27 0.42 ulna+hand 0.33 0.06 trunk length 0.28 0.39 mass 0.34 -0.06 18 ambika kamath, rachakonda sreekar in colour in the paddy field than the trench (median colour score; paddy field = 3; trench: 4; χ4 = 17.1, p = 0.002). frogs of different colour did not differ in svl, within either the trench or paddy field habitat (paddy field: jt5, 38 = 262, p = 0.73; trench: jt5, 30 = 177.5, p = 0.31). calling rate did not vary with svl (spearman’s rho = 0.08; s38 = 8427, p = 0.64) in the paddy field. however, in the trench habitat, smaller frogs called more often than larger frogs (spearman’s rho = -0.47; s30 = 6587, p = 0.01; fig. 3). in the paddy field, frogs further from dry land called more often (spearman’s rho = 0.36; s38 = 5820, p = 0.025), and were darker in colour (jt5, 38 = 330.5, p = 0.03) than frogs closer to dry land. however, svl did not vary with distance to dry land (spearman’s rho = -0.08; s = 9861, p = 0.64). discussion to our knowledge, this paper is among the first on the behaviour and ecology of a south asian species in the genus hylarana. our conclusions lend insight into the phenotypic variation of these frogs and yield testable hypotheses that can be addressed by measuring the strength of sexual selection and viability selection on phenotypic traits of male h. intermedia in different habitats. the pattern of sexual size dimorphism in h. intermedia is similar to a majority of anuran amphibians: males are smaller than females (shine, 1979). a trend towards shape differences between the sexes could result from male and female reproductive success being limited by mate attraction and fecundity respectively. relatively larger heads in males might function to accommodate the vocal sac, which plays a crucial role in mate attraction. relatively longer trunks in females might be necessary for females to accommodate eggs. though little research been conducted on shape dimorphism in frogs, the patterns seen in h. intermedia seem to concur at least partially with other studies. for example, herrel et al. (2012) found that male xenopus tropicalis have relatively longer heads than females, and schauble (2004) found that male limnodynastes peronii have relatively wider heads than females. further comparisons between males and females, with a larger sample size for females, can confirm these patterns in h. intermedia. though a satellite mating strategy is often adopted by smaller frogs (emlen, 1976; blackwell and passmore, 1991), smaller h. intermedia males do not appear to fig. 2. plot of the first vs. the second principal component axes (pc1 vs. pc2) from a principal component analysis on the morphology of hylarana intermedia. blue and red circles represent male and female frogs respectively. squares represent means of pc1 and pc2 by sex, bars indicate standard error, and ellipses indicate 95% confidence intervals. fig. 3. correlations between call rate and snout-vent length (svl) of hylarana intermedia in trench (brown circles) and paddy field (green circles) respectively. lines represent linear regression lines, and are provided for visualization purposes only. 19behaviour, ecology, and morphology of hylarana intermedia adopt this tactic. smaller males neither call less frequently nor are darker in colour than larger male h. intermedia in either the paddy field or the trench habitat. in fact, smaller frogs called more frequently than larger frogs in the trench. we also found that frogs differed in colour between the two sampled breeding sites. interpopulation differences not only in traits contributing to conspicuousness but also in relationships between these traits indicate that male h. intermedia do not adopt consistent alternative reproductive strategies. in the paddy field, frogs far from dry land are both darker and call more frequently than frogs close to dry land. this result is consistent with at least two nonmutually-exclusive predictions based on sexual selection and viability selection, each of which is testable. first, the attention of females, who approach breeding ponds from land, may be attracted differently depending on their distance to the male frog. different types of signals attenuate differently with increasing distance between the signaller and receiver (endler, 1992; shine, 2005; bradbury and vehrencamp, 2011). we predict that, in h. intermedia, auditory conspicuousness is more effective in attracting females over long distances and visual conspicuousness more effective over short distances. this would lead to frogs in the centre of the paddy field, at large distances from the females found on dry land, to rely more on auditory conspicuousness and therefore call frequently. second, we predict that predation pressure from visual predators is higher in the more open centre of breeding ponds than at the more vegetated edges, leading frogs to adopt darker, less conspicuous, coloration in the centre. there is growing evidence that multimodal signals combining visual and auditory cues are important for male frogs attracting females, even in nocturnal species (e.g., sheldon et al., 2003; vasquez and pfennig, 2007; gomez et al., 2009). future studies on h. intermedia can begin by assessing whether dorsal colour is an indicator of some aspect of male condition or quality (sheldon et al., 2003; vasquez and pfennig, 2007), and whether females are preferentially attracted to lighter males relative to darker males, through either positive phototaxis or active choice (jaeger and hailman, 1971). however, signal traits do not evolve in isolation but coevolve in an environmental context that can bias signalling through sensory drive (endler, 1992). our results indicate that different phenotypic traits contributing to h. intermedia conspicuousness, as well as the correlations among them, can be spatially structured and habitat-dependent. we therefore caution future researchers to account for environmental variation when attempting to understand sexual selection and viability selection on the phenotypic traits contributing to conspicuousness in male h. intermedia. the environmental variation to be considered includes not only spatial components such as those investigated here but also temporally varying factors such as auditory interference from calls of sympatric frogs (e.g. zakerana sp.) and the monsoon rain. rapid habitat loss in the western ghats (anand et al., 2010), little previous research on even common taxa in the region, and the continued discovery of novel species imply that ecologists and natural historians are fighting a battle against time to document the biology of species in one of the world’s biodiversity hotspots (aravind et al., 2007). our aim in this paper is not to reach definite conclusions about the reproductive biology of h. intermedia, but instead to provide a framework and preliminary data on which future investigations can be based. we hope that this study stimulates further research on the behavioural ecology of h. intermedia, yielding interesting insights into how phenotypic variation is maintained both within and between populations of this common western ghats frog. acknowledgements since our study area did not fall within a protected area, and hylarana intermedia is not mentioned as a scheduled species in the wildlife (protection) act (1972) and subsequent amendments, we did not require government permits to conduct our study. our study did not entail collection of individuals and we made all efforts to minimize discomfort to the animal by ensuring careful handling and quick release of each captured individual back into the habitat they were first observed in. we thank neeti mahesh, gautam ramachandra, shyam rao, katya saini, naren srinivasan, quentin fournier, and francis crawley for help with data collection. siddharth rao helped to formulate the project and sampling methods. logistical support was provided by the agumbe rainforest research station, agumbe, karnataka. yoel stuart, jonathan losos, katie boronow, and two anonymous reviewers made suggestions that improved the quality of the manuscript. references amézquita, a., hödl, w. 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(2007): the ecology and behaviour of amphibians. the university of chicago press, chicago, il, usa. acta herpetologica vol. 11, n. 1 june 2016 firenze university press feeding habits of mesoclemmys vanderhaegei (testudines: chelidae) elizângela silva brito1,*, franco leandro souza2, christine strüssmann3 morphology, ecology, and behaviour of hylarana intermedia, a western ghats frog ambika kamath1, rachakonda sreekar2,3 a new account for the endangered cerrado rocket frog allobates goianus (bokermann, 1975) (anura: aromobatidae), with comments on taxonomy and conservation thiago ribeiro de carvalho1,2,*, lucas borges martins1,2, ariovaldo antonio giaretta1 molecular phylogenetics of the pristimantis lacrimosus species group (anura: craugastoridae) with the description of a new species from colombia mauricio rivera-correa, juan m. daza* population structure and activity pattern of one species of adenomera steindachner, 1867 (anura: leptodactylidae) in northeastern brazil maria juliana borges-leite1,*, joão fabrício mota rodrigues2, patrícia de menezes gondim1, diva maria borges-nojosa1 vocal repertoire of scinax v-signatus (lutz 1968) (anura, hylidae) and comments on bioacoustical synapomorphies for scinax perpusillus species group marco antônio peixoto1,*, carla silva guimarães1, joão victor a. lacerda2, fernando leal2, pedro c. rocha2, renato neves feio1 amendment of the type locality of the endemic sicilian pond turtle emys trinacris fritz et al. 2005, with some notes on the highest altitude reached by the species (testudines, emydidae) federico marrone*, francesco sacco, vincenzo arizza, marco arculeo photo-identification in amphibian studies: a test of i3s pattern marco sannolo1,*, francesca gatti2, marco mangiacotti3,4, stefano scali3, roberto sacchi4 no evidence for the ‘expensive-tissue hypothesis’ in the dark-spotted frog, pelophylax nigromaculatus li zhao, min mao, wen bo liao* no short term effect of clinostomum complanatum (trematoda: digenea: clinostomatidae) on survival of triturus carnifex (amphibia: urodela: salamandridae) giacomo bruni1,*, claudio angelini2 yeasts in amphibians are common: isolation and the first molecular characterization from thailand srisupaph poonlaphdecha, alexis ribas* introduction of eleutherodactylus planirostris (amphibia, anura, eleutherodactylidae) to hong kong wing ho lee1, michael wai-neng lau2, anthony lau3, ding-qi rao4, yik-hei sung5,* correlation between endoscopic sex determination and gonad histology in pond sliders, trachemys scripta (reptilia: testudines: emydidae) david perpiñán1, albert martínez-silvestre2,3, ferran bargalló3, marco di giuseppe4, jorge orós5, taiana costa6,* book review: john w. wilkinson. amphibian survey and monitoring handbook. pelagic publishing franco andreone book review: susan newman. frogs amphibians and their threatened environment. discovery and expression through art k-3. frogs are green franco andreone acta herpetologica 10(2): 103-110, 2015 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-16441 where to look when identifying roadkilled amphibians? marc franch1,2,*, cristiano silva3, gil lopes3, fernando ribeiro3, paulo trigueiros3, luis seco1,4, neftalí sillero1 1 cicge (geo-space sciences research centre), prof. manuel de barros astronomical observatory, university of porto, (portugal). * corresponding author. e-mail: apoarmatu@gmail.com 2 department of animal biology (vertebrates), university of barcelona, (spain) 3 school of engineering of the university of minho, algoritmi research center, guimarães (portugal) 4 ismai, university institute of maia, maia (portugal) submitted on 2015, 27th july; revised on 2015, 18th october; accepted on 2015, 18th october editor: marco sannolo abstract. roads have multiple effects on wildlife; amphibians are one of the groups more intensely affected by roadkills. monitoring roadkills is expensive and time consuming. automated mapping systems for detecting roadkills, based on robotic computer vision techniques, are largely necessary. amphibians can be recognised by a set of features as shape, size, colouration, habitat and location. this species identification by using multiple features at the same time is known as “jizz”. in a similar way to human vision, computer vision algorithms must incorporate a prioritisation process when analysing the objects in an image. our main goal here was to give a numerical priority sequence of particular characteristics of roadkilled amphibians to improve the computing and learning process of algorithms. we asked hundred and five amateur and professional herpetologists to answer a simple test of five sets with ten images each of roadkilled amphibians, in order to determine which body parts or characteristics (body form, colour, and other patterns) are used to identify correctly the species. anura was the group most easily identified when it was roadkilled and caudata was the most difficult. the lower the taxonomic level of amphibian, the higher the difficulty of identifying them, both in anura and caudata. roadkilled amphibians in general and anura group were mostly identified by the form, by the combination of form and colour, and finally by colour. caudata was identified mainly on form and colour and on colour. computer vision algorithms must incorporate these combinations of features, avoiding to work exclusively in one specific feature. keywords. roadkills, amphibians, jizz, computer vision, algorithms, survey methodology. introduction roads have multiple effects on wildlife, such as animal mortality, habitat and population fragmentation, as well as modification of animal behaviour (trombulak and frissell, 2000; jaeger and fahrig, 2004; jaeger et al., 2005; epps et al., 2007; lengagne, 2008; fahrig and rytwinski, 2009). amphibians in particular, due to their activity patterns, population structure, and preferred habitats, are strongly affected by traffic intensity and road density and die massively on roads (fahrig et al., 1995; glista et al., 2008; gryz and krauze, 2008; sillero 2008; garriga et al., 2012). monitoring roadkills is expensive and time consuming, and depends mainly on volunteers (grilo and ascensão, 2011; garriga et al., 2012; beebee, 2013). surveys can be performed by car (santos et al., 2007; sillero, 2008; garriga et al., 2012; matos et al., 2012), bike (ashley and robinson, 1996; collinson, 2013), or by foot (ashley and robinson, 1996; collinson, 2013; ruiz-capillas et al., 2015). cheap, easy to implement, and automatic methods for detecting roadkills over larger areas (broad monitoring) and along time (continuous monitoring) are necessary. in this sense, mapping systems provide the capacity to detect automatically the casualties of roadkills by intelligent algo104 marc franch et alii rithms (chambon and moliard, 2011; varadharajan et al., 2014), improving current monitoring systems. in the context of the research project roadkills (ptdc/bia-bic/4296/2012), funded by fundação para a ciência e a tecnologia (fct) of portugal, we developed a mobile mapping system installed on a trailer and composed of standalone power generation, computer imaging capturing, and recording under a meter gps receiver and one linear controlled and standardized lighting. to capture images, we adapted a line scan camera, commonly used in industry and in controlled environments. this camera model has an optical resolution between 250 µm/ pixel at 35 km/h, 500 µm/pixel at 70 km/h and 1000 µm/ pixel at 140 km/h. the camera acquire sequential images of 4096 pixels width and one pixel length (approx. 1.0 m × 0.25 mm). the construction of an image or a frame is obtained through the acquisition of several lines that the line scan camera captures. the obtained frame (as a result of the sum of several sequential lines) is the work unit of the algorithms with 4096 pixels width and a variable number of lines as its length. this new methodology improves passive sampling as car speed can be higher than currently used in visual surveys (20 km/h) (sillero, 2008; garriga et al., 2012; matos et al., 2012). the proposed mobile mapping system can be implemented on road maintenance vehicles and on surveillance or maintenance vehicles in protected areas without an expert for their correct use. analysis may be in real time from software resident in the mapping system itself or by transferring to a remote server. these systems are based on robotic computer vision techniques such as object recognition or structure from motion and are widely used in many computer vision applications. computer vision techniques usually involve three distinct steps: feature detector, description and matching (moreels and perona, 2007; derntl, 2014). most of the proposed features provide both a detector and a descriptor algorithms, which can be combined each other like in sift (scale-invariant feature transform), surf (speeded up robust features), brief (binary robust independent elementary features) or orb (oriented and rotated brief), all implemented in the opencv library (rublee et al., 2011). opencv (open source computer vision library) is an open source computer vision and machine learning software library. opencv was built to provide a common infrastructure for computer vision applications and to accelerate the use of machine perception in the commercial products (yu et al., 2004). bag of words or bag of features is one of the popular visual descriptors used for visual data classification. it is a sparse vector of occurrence counts of a vocabulary of local image features (gang, 2012). overall, the algorithms work looking for the detected patterns in the “problem” images in known patterns from images library. initially, known patterns of the library are provided by us. once designed the algorithm and running, it is self-sufficient in images that stock the library (with our supervision). computer vision algorithms try to emulate human vision when recognising an object or a part of it. in herpetology, amphibians are not recognised immediately by a unique feature, but by a set of particular and subtle characteristics, together with knowledge of the species (arnold and ovenden, 2003). professionals and amateurs cannot clearly define what these particular features are in order to identify an amphibian (ellis, 2011). the informal term among naturalists and scientists of this integrated identification form is “jizz” and is based on shape, size, colouration, habitat and location (macdonald, 2002; daston, 2008; ellis, 2011). currently there are no data about the particular features on amphibians’ “jizz” although this identification way is rather trivial. which criteria we follow, as experts, at the moment of identification? on which basis do we identify a roadkilled amphibian? species identification of the roadkilled amphibian may be very difficult due to the corpse state (ashley and robinson, 1996; glista et al., 2008). moreover, there is a gradient from intact corpses to completely unidentifiable carcasses depending on the violence of the impact with the vehicle, the time of permanence on the road and the weather or environmental conditions (glista et al., 2008). despite this, how “jizz” works in expert identification? are all used characters subtle or do some characteristics emphasise over others? is there a prioritisation process in the moment to generate an idea about the identification? thus, it is necessary to understand how we prioritise these features when deciding if a roadkilled amphibian is in fact a roadkilled amphibian (e.g. a roadkilled slag) or not. computer vision algorithms will work in a similar way to human vision. they will use our prioritisation process when analysing an image. in order to answer these questions, we asked to several amateur and professional herpetologists to answer a simple test composed of numerous pictures of roadkilled amphibians. specifically, we aimed to determine which parts or characteristics of the roadkilled amphibian body are used by experts to identify correctly the species, when a specific part of the body is essential to identify a species, or by the contrary, when experts use all the amphibian body to obtain a final conclusion. thus, we wanted to give a numerical priority sequence of particular characteristics to improve the computing and learning process of algorithms. our results will be essential to improve the ability of algorithms applied to images obtained for the mobile 105where to look when identifying roadkilled amphibians? mapping system developed for the automatic detection of amphibian roadkills on roads. when algorithms are developed, they will be tested under controlled conditions (roadkilled amphibians’ 3d plastic models) and on the field (real roadkilled amphibians) with and without the obtained identification parameters. material and methods structure of the test/survey the test consisted of five sets with ten images each and an associated questionnaire. the test was built with google sheets, which is able to provide reports and data tables. all images were collected in the iberian peninsula (mainly catalonia and portugal). besides amphibians, images also included some reptiles, invertebrates, birds, and mammals, in order to include false positives. thus, we included 50 images (table 1), 43 with roadkilled amphibians and seven with other groups (four reptiles: one lacerta bilineata; three podarcis sp.; two small mammals, talpa europaea and rattus sp.; and one invertebrate, an indeterminate caterpillar) in order to maintain the participant’s attention and avoid trends in their responses. we included pictures of 29 anura (15 bufo bufo) and 14 caudata (seven salamandra salamandra), proportion corresponding to relative species abundances (matos et al., 2012). two images of b. bufo were very difficult to identify. we obtained the test images from our own repository and the university of barcelona. we assigned a random order to the images to avoid possible trends of subjective grouping. test images were not possible to expand. although for some collaborators this was a problem, our idea was that the user tries to identify in the same conditions as the computer algorithm. we included a brief introductory letter on the front cover of the first questionnaire (with the presentation of the survey). at the beginning of each set of images, identification was requested, as anonymous or identified contributor. the questionnaire for each image was composed of six questions (suppl. mat. appendix a1-survey). the first question (q1): is it possible for you to identify a roadkilled amphibian in this image? was a binary question (‘yes’ or ‘no’) with the possibility of including comments. if the user answered ‘no’, the questionnaire for this image was finished. if the user answered ‘yes’, the test proceeded to the second question (q2): which is the taxonomic level do you get? taxonomical identification was possible to levels of order, genus and species. selecting an option in a higher taxonomic level did not conditioned options at lower taxonomic levels. the third question (q3): which features did you use for the identification? had multiple choices: form, colour, others patterns, form & colour, form & others patterns, colour & others patterns, or form & colour & others patterns. they could select one of three options or a combination of them. in the following three questions (q4: form: general body shape; head; forelimbs; hind-limbs; tail or other characters; q5: colour: general design pattern; specific colours; specific designs or other characters; and q6: others patterns: recognition of bones / carcasses; recognition of skins or other characters.), the user was able to select only one option. when selecting the option “other characters”, the user must specify the criteria in a text box, or any other comment. once each set was finished, the results were recorded and the link to the next set appeared. in the final set, we added a field where the user can introduce additional comments, views, or constructive reviews to the test. participants the diffusion of the survey was carried from mailing lists of known contacts, calling on researchgate (scientific and social networking service), on facebook (social networking service), from a post in road ecology group (642 members at july of 2014), and through the website, facebook and twitter (social networking service) accounts of the spanish herpetological society (ahe). potential participants were selected for their experience in the study of roadkills. we also offered the possibility to the potential participants of spreading the test to their contacts with experience in his research field. data processing the survey was online during six months (july 2014 to january 2015). we obtained the generated tables for each set after closing the site. we extracted proportions of each category/subcategory and applied chi square test in order to find differences in frequencies between groups and categories. table 1. list of species and number of test images for each species. order genus species images caudata salamandra salamandra 7 caudata triturus marmoratus 6 caudata pleurodeles waltl 1 anura bufo bufo 15 anura bufo calamita 4 anura hyla meridionalis 3 anura discoglossus galganoi 2 anura rana iberica 2 anura alytes obstetricans 1 anura discoglossus pictus 1 anura pelophylax perezi 1 reptilia podarcis hispanica 3 reptilia lacerta bilineata 1 mammalia rattus sp. 1 mammalia talpa europaea 1 lepidoptera (indeterminate caterpillar) 1 total: 50 106 marc franch et alii results hundred and five people answered the survey, of which 63 (60%) identified themselves and 42 (40%) were anonymous. participants’ contributions were different between sets. the answers to the first sets were higher than for the last sets: from 105 contributors in first set to 53 in the fifth one. although question 1 (q1: is it possible for you to identify a roadkilled amphibian in this image?) only supported an affirmative answer if contained a roadkilled amphibian, we admitted as affirmative answer in the group of “fool” pictures if the participants tried to identify the image by comments. for this question we obtained 3241 responses with about 60% correctly identified a roadkilled amphibian in the image. detailed results for taxonomical groups are found on table 2. we found highly significant differences in the correct identification among the three groups (χ2 = 54.12, df = 2, p < 0.001). anura was the group most easily identified as roadkilled of the three groups (between anura and the rest of groups; χ2 = 14.72, df = 1, p < 0.001). fools were clearly identified as a no roadkilled amphibian (between fools and the rest of groups; χ2 = 43.91, df = 1, p < 0.001). caudata was the most difficult group to identify as roadkilled amphibian (no differences were found between caudata and the rest of groups; χ2 = 0.47, df = 1, p = 0.492). about 52% of 2776 possible responses in q2 (in the case of identifying an amphibian, what is the taxonomic level do you get?) identified the order in the image, 28% the genus and 20% the species. detailed results are found on table 3. we found highly significant differences among taxonomic level (χ2 = 855.89, df = 2, p < 0.001) and also among the taxonomic levels of anura (χ2 = 984.18, df = 2, p < 0.001) and caudata (χ2 = 30.88, df = 2, p < 0.001), indicating an increase on the difficulty with the taxonomical level. there were significant differences between anura and caudata in assigning the order (χ2 = 187.43, df = 2, p < 0.001); assignment was easier for anura. there were no differences between anura and caudata for identifying the genus (χ2 = 0.13, df = 2, p = 0.715). species were identified with higher difficulty in anura than in caudata (χ2 = 20.03, df = 2, p < 0.001). in q3 (what have you based for identification? form, colour, others patterns, form & colour, form & others patterns, colour & others patterns or form & colour & others patterns), we have obtained 1755 responses, 1253 for anura and 412 for caudata (table 4). we found highly significant differences among identification elements (χ2 = 1784.60, df = 6, p < 0.001). in table 2. answers to q1 about the possibility of identification a roadkilled amphibian in the images. category roadkilled amphibian identification id. yes id. no total general responses: 1913 1328 3241 %%: 59.02 40.98 100.00 caudata responses: 473 393 866 %%: 54.62 45.38 100.00 anura responses: 1390 520 1910 %%: 72.77 27.23 100.00 fools responses: 415 50 465 %%: 89.25 10.75 100.00 table 3. correct answers to q2 about the taxonomic level identified for respondents in the images of the test. category taxonomy order genus species total general correct responses: 1660 897 641 3198 %%: 51.91 28.05 20.04 100.00 caudata correct responses: 354 284 246 884 %%: 40.05 32.13 27.83 100.00 anura correct responses: 1306 613 395 2314 %%: 56.44 26.49 17.07 100.00 table 4. results of q3 about identification criteria based on form (f), colour (c), other patterns (op) and their combination. category identifying criteria f c op f&c f&op c&op f&c&op total general responses: 677 259 69 522 76 34 118 1755 %%: 38.58 14.76 3.93 29.74 4.33 1.94 6.72 100.00 caudata responses: 102 130 3 151 9 5 12 412 %%: 24.76 31.55 0.73 36.65 2.18 1.21 2.91 100.00 anura responses: 527 128 60 349 60 29 100 1253 %%: 42.06 10.22 4.79 27.85 4.79 2.31 7.98 100.00 107where to look when identifying roadkilled amphibians? general, roadkilled amphibians were mostly identified by the form, in a lesser extent by the combination of form and colour, and finally by colour. the other elements and combinations had much lower frequencies. anura presented the same pattern (χ2 = 1366.55, df = 6, p < 0.001). for caudata, the identification was based mainly on form and colour and on colour in less proportion. the other elements and combinations had much lower frequencies. there were highly differences in frequency (χ2 = 517.65, df = 6, p < 0.001). in q4 (form: general body shape; head; forelimbs; hind-limbs; tail or other characters) we have obtained 1381 responses, 1025 for anura and 278 for caudata (suppl. mat. table t1). we found highly significant differences among identification form features (χ2 = 2995.32, df = 5, p < 0.001), mainly by the general body shape, and hind-limbs in lesser importance. the other features had much lower frequencies. the same on pattern for anura (χ2 = 2283.24, df = 5, p < 0.001) as well as for caudata (χ2 = 788.10, df = 5, p < 0.001). we obtained 883 responses in q5 (colour: general design pattern; specific colours; specific designs or other characters), 565 responses for images of anura and 291 responses for caudata (suppl. mat. table t2). we found highly significant differences among identification colour features (χ2 = 611.04, df = 3, p < 0.001) and for anura (χ2 = 581.33, df = 5, p < 0.001), based mainly on general design pattern. for caudata, we also found highly significant differences (χ2 = 206.13, df = 5, p < 0.001), but based mainly in specific colours. in q6 (others patterns: recognition of bones / carcasses; recognition of skins or other characters), we obtained 391 responses, 326 for anura and 42 for caudata (suppl. mat. table t3). we found significant differences among others patterns identification features (χ2 = 526.89, df = 2, p < 0.001), as well as for anura (χ2 = 431.35, df = 2, p < 0.001) and caudata (χ2 = 71.36, df = 2, p < 0.001). for all of them, participants identified roadkilled amphibians by other patterns based on recognition of skins. fig. 1. radar graphic of the “jizz” sequence for the different parameters of form, colour and other patterns together for general amphibians’ roadkills, for anurans and for caudata. 108 marc franch et alii we extracted the prioritisation sequence based on the percentage of all well identified images (figure 1). general sequence for amphibians’ roadkills is: jizz = 0.3858*f + 0.2974*f&c + 0.1476*c + 0.0672*f&c&op + 0.0433*f&op + 0.0393*op + 0.0194*c&op where f (form); c (colour) and op (other patterns) regarding the sequence for the different parameters of form, colour and other patterns together: jizz = 0.3686*gbs + 0.1669*gdp + 0.1390*hl + 0.0970*sc + 0.0725*rs + 0.0602*sd + 0.0247*h + 0.0205*fl + 0.0138*t + 0.0095*rb/c + 0.0266*oc where gbs (general body shape); gdp (general design pattern); hl (hind-limbs); sc (specific colours); rs (recognition of skins); sd (specific designs); h (head); fl (forelimbs); t (tail); rb/c (recognition of bones / carcasses) and oc (other characters) detailed sequences for anura and caudata and their specific characters can be found in the supplementary files (suppl. mat. appendix a2-jizz sequence). from this sequence we extracted, in general terms, the high importance of form (alone or in combination with colour) for a roadkilled amphibian identification. for specific patterns, the main one was gbs (general body shape: 36%) followed by gdp (general design pattern: 16.7%). discussion almost 60% of the q1 responses were correct. anura was the group most easily identified as roadkilled (73%), because the large hind-limbs of frogs and toads. results on body features confirmed this conclusion (see below). the correct identification of salamanders and newts was more difficult (55%) because they have similar fore and hind-limbs. in fact, the tail was the second more important feature for their recognition (see below). participants clearly identified fools as non roadkilled amphibians (almost 90%). therefore, participants were much more effective in identifying correctly what was not a roadkilled amphibian. this is a surprising result, because there are many features on roads that can be confounded with a roadkilled amphibian (e.g. small mammals, snails, tree sheets, rubbish). however, non-amphibian roadkills can be very difficult to identify correctly from inside a car but not from a close sight. participants identified correctly in 61% cases the order of the animals, but genus and species below 30% and 21%, respectively. this rank of taxonomical levels appeared also in anura and caudata. therefore, the lower the taxonomic level of amphibian, the higher the difficulty of identifying them, both in anura and caudata. however, the correct assignment of the order and species was easiest in anura than in caudata, confirming the easiest identification of anurans. the difficulty in assigning the genus was similar for both groups of amphibians. this lack of differences between both groups could be due to the lower number of genera in caudata. roadkilled amphibians in general and anura group were mostly identified by the form, in a lesser extent by the combination of form and colour, and finally by colour. the correct identification of caudata group was based mainly on form and colour and on colour in less proportion. these results confirmed that people in fact uses “jizz” to identify roadkilled amphibians. there was not one unique feature to get the correct identification, but a combination of features, although the most important was the form for anura but form and colour for caudata. this last result can be due to the easy identification of salamanders because of their clear pattern of black and yellow colours. colours alone were not sufficient as diagnostic feature as there are many species with similar or cryptic colours. in consequence, roadkills were correctly identified by general body shape and hind-limbs in anura and caudata groups, by general design pattern in anura and by specific colours in caudata, as well as by recognition of skins in both groups. again, these results confirmed the use of “jizz” to recognise herps. also, q5 answers were coherent with q4 ones. here, amphibian skin properties were useful to distinguish from fools. not all the people answered all the sets of the test due to limitations on survey design. we should not have included anonymous people as this reduces the sample size of our analysis about the effectiveness on answering depending on the profession and skills of the participants. our results may be better if we had included more images of other taxa (like insects, reptiles, birds and mammals), objects, vegetables, and other variables related to images for improving the test. the obtained results probably would be more robust but it was not the goal of the survey: we aimed to obtain a sequence of features and characteristics to identify a roadkilled amphibian. main conclusions “jizz” is the method used to identify herps. there is not a unique feature for the correct diagnoses of amphib109where to look when identifying roadkilled amphibians? ians but a combination of several features, depending mainly on the shape of the body and colours. more importantly, different combinations of shapes and colours are essential to identify one species from others. computer vision algorithms must incorporate these combinations of features to have success, avoiding to work exclusively on one specific feature. acknowledgments special thanks to all respondents (suppl. mat. appendix a3-contributors / respondents) and to all who have contributed to diffusion and promotion of the roadkills survey: road ecology group on facebook, asociación herpetológica granadina, asociación herpetológica española (ahe), herpnet and all individuals who have also believed appropriate the disclosure of the survey. thanks to an anonymous reviewer for english linguistic advising and for the comments and suggestions that greatly improved the last version of the manuscript. m. franch and c. silva are funded by a grant (uminho/ bi/175/2013, uminho/bi/172/2013) (from the foundation for science and technology portugal (fct) and cofinanced by feder through compete –pofc (“intelligent systems for mapping amphibian mortality on 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(2004): ch opencv for interactive open architecture computer vision. adv. eng. softw. 35: 527-536. acta herpetologica vol. 10, n. 1 june 2015 firenze university press acta herpetologica journal of the societas herpetologica italica acta herpetologica 11(1): 53-57, 2016 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-16490 vocal repertoire of scinax v-signatus (lutz 1968) (anura, hylidae) and comments on bioacoustical synapomorphies for scinax perpusillus species group marco antônio peixoto1,*, carla silva guimarães1, joão victor a. lacerda2, fernando leal2, pedro c. rocha2, renato neves feio1 ¹ museu de zoologia joão moojen, departamento de biologia animal, universidade federal de viçosa, vila gianetti, n° 32. cep 36570-000. viçosa, minas gerais, brazil. *corresponding author. e-mail: marco.peixotom@gmail.com ² laboratório de herpetologia, instituto de ciências biológicas, departamento de zoologia, universidade federal de minas gerais, cep 31270-901, belo horizonte, minas gerais, brazil submitted on 2015, 12th august; revised on 2015 4th december; accepted on 2015, 10th december editor: sebastiano salvidio abstract. herein we describe the vocal repertoire of scinax v-signatus, compare it to the other species belonging to the s. perpusillus species group and discuss the bioacoustical synapomorphies proposed for the group. we recorded an advertisement call and an aggressive call of s. v-signatus. the advertisement call is similar to the one described for others species of the scinax perpusillus group. similarly to scinax cosenzai, the first note in the advertisement call of s. v-signatus is the longest. the aggressive call was preceded by a series of advertisement calls. we suggest that bioacoustical parameters proposed as synapomorphies for the scinax perpusillus group are not valid, as they are also observed in species belonging to other groups within the genus. keywords. advertisement call, bromeligenous species, aggressive call, atlantic rain forest, taxonomy, anuran bioacustic, raven. the neotropical genus scinax wagler, 1830 include 113 recognized species (frost, 2015) distributed from southern mexico to the east coast of argentina (faivovich, 2002). it is composed of the s. ruber and s. catharinae clades with the later including the s. perpusillus and s. catharinae species groups (faivovich, 2002). scinax perpusillus species group is endemic to the brazilian atlantic forest and was first suggested based on its bromeligenous habitat and absent or reduced membrane between toes i-ii and ii-iii (peixoto, 1987). the group currently includes 13 described species and its monophyly has never been formally tested (alves-silva and silva, 2009). furthermore, pombal and bastos (2003) suggested bioacustical characters as possible synapomorphies. the specificity of anurans’ calling repertoire is an important taxonomic and phylogenetic tool (duellman and trueb, 1994; goicoechea et al., 2010; taucce et al., 2012). despite this importance, only eight species of the scinax perpusillus group had their call described: s. arduous (pombal and bastos, 2003), s. belloni (peres and simon, 2011), s. cosenzai (lacerda et al., 2012.), s. littoreus (pontes et al., 2013.), s. peixotoi (brasileiro et al., 2007) and s. perpusillus (pombal and bastos, 2003). moreover, s. arduous (pombal and bastos, 2003), s. littoreus (pontes et al., 2013) and s. perpusillus (pombal and bastos, 2003) had their aggressive/territorial calls described and s. insperatus (silva and alves-silva, 2011) and s. v-signatus (alves-silva and silva, 2009) had an antiphonic interaction described. besides those studies, 54 marco antônio peixoto et alii heyer et al. (1990) described the advertisement call of an unnamed species belonging to the scinax perpusillus group from estação biológica de boracéia, municipality of salesópolis, state of são paulo. scinax v-signatus (lutz, 1968) is endemic to the serra dos órgãos mountain range, rio de janeiro state, southeastern brazil (silva and alves-silva, 2013). alvessilva and silva (2009) described its vocalization based on males in antiphony and restricted their study to a behavioral/reproductive focus. herein we describe the vocal repertoire of s. v-signatus and compare its advertisement call with others species belonging to the s. perpusillus group. furthermore, we also compare it to the calls described for its sister group – s. catharinae group sensu faivovich 2002 – and discuss the use of bioacoustical data as a synapomorphy for the s. perpusillus group. we recorded 261 calls from six specimens of scinax v-signatus. recordings were made at different localities throughout the serra dos órgãos mountain range, rio de janeiro state, southeastern brazil. voucher specimens are housed at the coleção herpetológica of universidade federal de minas gerais (ufmg-amp) and coleção de anuros do museu de biodiversidade do cerrado, universidade federal de uberlândia (aag). two individuals (only one of which was collected: ufmg 14106) were recorded on 18 january 2013, at são pedro da serra district, municipality of nova friburgo, (22°18’47.03”s, 42°20’13.92”w; 25 °c; datum wgs84). in the same district, we recorded two other individuals on 19 january 2013. one individual was recorded on 23 january 2013 at macaé de cima district, also in the municipality of nova friburgo (22°28’40.08”s, 43°15’9.36”w; 25 °c; datum wgs84). one individual (aag-ufu 0742) was recorded on 14 december 2011, at maria mendonça district, municipality of trajano de moraes (22°11’32.60”s, 42°11’16.29”w; 20 °c; datum wgs84). whilst this individual was being kept in a plastic bag, it emitted a different type of call. recordings were made using digital recorders marantz pmd 660 coupled with a sennheiser me66 unidirectional microphone in all individuals, apart from those recorded on 19 january 2013, in which we used a sony icd p620 with internal microphone. the first featured sample rate of 44.1 khz, whereas the second 11.025 khz. both recorders had a 16 bit resolution and each recording was made at a distance of 20 cm to 1 m between specimen and microphone. oscillograms and spectrograms were produced and analyzed using software raven pro 1.4 (fft = 256, 89% overlap and hann window). terminology follows duellman and trueb (1994) and toledo et al. (2015).  parameters measured include call duration, interval between calls, note duration, interval between notes, number of notes per call, dominant frequency and peak frequency. temporal parameters were measured directly from the waveform. dominant frequency is given as range and is considered as the frequency band comprising 90% of energy within the call. it was acquired through the parameters “frequency 5%” and “frequency 95%” from raven pro 1.4 (charif et al., 2010). the peak frequency represents the peak of energy within the call and was acquired using the parameter “peak frequency” from raven pro 1.4. we compared our results to all data available in the literature. whenever values in tables and textual description presented incoherencies, we used the textual description. silva and alves-silva (2011) referred as  notes and pulses what is commonly treated as calls and notes (see discussion in lacerda et al., 2012). therefore, we treated it as calls and notes. alves-silva and silva (2009) did not stablish bioacoustics parameters for the call of scinax v-signatus, we did not compare it to our results. the advertisement call of scinax v-signatus (fig. 1a) consist of a series of 1-9 pulsed notes. it features duration fig. 1. oscillogram and spectrogram of advertisement and aggressive calls of scinax v-signatus: a) two distinct male in an interaction; b) mixed call of scinax v-signatus, i refers to the initial advertisement call and ii refers to the aggressive call. 55description of scinax v-signatus vocal repertoire between 77 and 1,076 ms (602 ± 184 ms; n = 240 calls) (table 1) and the interval between calls ranges between 142-2702 ms (1141 ± 0.483 ms; n = 183 intervals), with a tendency to shrink at the end of a series of calls. the first note is the longest with duration of 45-127 ms (73.0 ± 13.8 ms; n = 260 notes). the remaining notes have duration of 14-101 ms (52.0 ± 13.8 ms; n = 946 notes). interval between notes ranged from 26 to 242 ms (87.0 ± 25.0 ms; n = 784 intervals between notes). the notes had 1-23 pulses (16 ± 7 pulses). dominant frequency ranged between 2,282-5,250 hz. the peak frequency varied among populations, ranging between 2,497 and 4,500 hz (3,937 ± 0.407). whilst being kept on a plastic bag, one male emitted an aggressive call preceded by a series of 20 advertisement calls (fig. 1b). this call was emitted by one male, alone and inside a plastic bag. thus, we suggest that this type of call has an aggressive context (toledo et al., 2015). this call consisted of a series of 360 pulsed notes ranging from 20 to 350 ms (40.0 ± 39.0) at rate of 451.8 notes per minute. dominant frequency ranged from 2,625 to 5,625 hz and peak frequency of 2,812-4,875 hz (4,312 ± 695 hz). our results indicate that the advertisement calls of scinax v-signatus resemble the ones from s. arduous, s. belloni, s. cosenzai, s. littoreus, s. perpusillus, and s. peixotoi. those species share a similar pattern of call duration, interval between calls, number of notes, pulses per note, and dominant frequency. on the other hand, the call of s. peixotoi had higher interval between notes, and s. belloni has more pulses per note that s. v-signatus’ advertisement call. this high similarity is also observed in the aggressive call, where only the call duration and number of notes had a higher value than others. when analyzing the call of eleutherodactylus coqui, narins and capranica (1978) showed that the first note has a territorial importance. in their study, neighboring males only responded when the first note was played and the presence of the second note had no influence on male’s behavior. calls that carry multiple information (e.g. advertisement and territorial calls) are likely to reduce energy expenditure (wells, 1988). heterogeneous character was found on call of scinax v-signatus. the first notes of all calls are longer than others ones. these same characters are related to s. cosenzai, other species table 1. advertisement and agressive (*) calls of scinax perpusillus group species. species call duration (ms) call interval (ms) number of notes note duration (ms) note interval (ms) number of pulses/ note pulse duration (ms) dominant frequency (hz) reference s. arduous 198.0-328.0 234.0-283.0 4-6 14.0-45.0 23.0-51.0 5-13 2.0-4.0 3802-4682 pombal and bastos (2003) s. belloni 590.0-690.0 1410-3960 2 17.0-21.0 28.0-36.0 28-35 3078 peres and simon (2011) s. belloni 100.0-120.0 1410-3960 3 16.0-24.0 27.0-36.0 24-31 3078 peres and simon (2011) s. cosenzai 177.0-2066 1900-4320 2-14 1.8-111.0 10.0-516.0 1-33 177.02066 3375.9-4571.2 lacerda et al. (2012) s. littoreus 174.0-287.0 980.0-14197 2-5 118.0-348.0 14.0-38.0 2-9 4.0-24.0 4306.6-4651.2 pontes et al. (2013) s. littoreus 204.0-282.0 1009-7027 2-4 118.0-291.0 24.0-38.0 2-8 4306.6-4478.9 pontes et al. (2013) s. littoreus 174.0-287.0 980.0-2781 2-9 255.0-348.0 26.0-29.0 3-9 4512.7-4651.2 pontes et al. (2013) s. littoreus 201.0-223.0 2122-14197 3-4 230.0-371.0 25.0-27.0 2-4 4306.6 pontes et al. (2013) s. perpusillus 92.0-174.0 776.0-1067 3-6 7.0-18.0 9.0-59.0 3-5 2.0-5.0 4554-4856 pombal and bastos (2003) s. aff. perpusillus 250.0-400.0 4-5 30.0-70.0 4-10 3500-5900 heyer et al. (1990) s. peixotoi 146.0-232.0 438.0-2400 3-5 9.0-28.0 438.0-2400 4-9 1.0-3.0 3617-3963 brasileiro et al. (2007) s. v-signatus 77.0-1076 142.0-2702 1-9 14.0-127.0 26.0-242.0 1-23 2282-5250 this study s. arduous* 53.0-64.0 82.0-116.0 1 53.0-64.0 18-22 2.0-4.0 3760-4445 pombal and bastos (2003) s. insperatus* 25-50a 630.0-860.0 155-434b 4479-4665c silva and alves-silva (2011) s. insperatus* 23-47a 50.0-631.0 223-500b 4479-4665c silva and alves-silva (2011) s. littoreus* 267.0-322.0 981.0-1590 14-22 20.0-49.0 9.0-18.0d 2-4 1.0-4.0e 5304.6-5439 pontes et al. (2013) s. littoreus* 274.0-349.0 689.0-915.0 16-31 16.0-34.0 12.0-27.0d 2-4 1.0-4.0e 5356.2-5439 pontes et al. (2013) s. littoreus* 220.0-298.0 1228-1374 8-12 22.0-58.0 3.0-23.0d 2-4 1.0-4.0e 5292 pontes et al. (2013) s. perpusillus* 314.0-400.0 748.0-792.0 6-12 8.0-42.0 8.0-64.0 1-6 4.0-9.0 4902-4918 pombal and bastos (2003) s. perpusillus* 183.0-508.0 1 183.0-508.0 4770-4743 pombal and bastos (2003) s. v-signatus* 47820.0 360 2.0-350.0 12.0-183.0 2625-5625 this study 56 marco antônio peixoto et alii of scinax perpusillus species group (jvl pers. obs.). in the same way, the aggressive call of s. v-signatus was preceded by an advertisement call, composing a mixed call series. according to pombal and bastos (2003), the scinax perpusillus species group is distinguishable from the s. catharinae group and the s. ruber clade by having advertisement calls composed of three to six pulsed notes spaced by 23-59 ms. however, those parameters overlap in some species belonging to the s. catharinae group (e.g. s. angrensis and s. littoralis, garey et al., 2012). on other hand, some species signed to the s. perpusillus species group have advertisement calls with more than six notes per call and longer intervals between notes (i.e. s. littoreus, s. peixotoi and s. v-signatus). based on our results, we suggest that bioacoustical parameters should no longer be considered synapomorphies for the s. perpusillus species group. however, new studies focusing on relationships within scinax considering bioacoustical characters using modern phylogenetic approaches could elucidate this issue. the range of bioacoustical terminology in the literature reinforces the difficulty of establishing comparisons between calls of different species. within the genus scinax, species recognition and identification is not simple (pombal et al., 1995; lourenço et al., 2014). the high complexity of their vocal repertoires does not make it easier. in view of this discussion, we emphasize the importance of the careful use of bioacoustics in a genus known for its high diversity and difficult taxonomy. note added in proof the authors did not includes in this manuscript, the nomenclatural modifications, proposed to the genus scinax by "duellmann, w., marion, a., blair, h. (2016). phylogenetics, classification, and biogeography of the treefrogs (amphibia: anura: arboranae). monograph zootaxa 4104: 001-109" as either the results of the article "carvalho, t.r., martins, l.b., giaretta, a.a. (2015). the complex vocalization of the scinax cardosoi (anura: hylidae), with comments on adverrisement call in the s. ruber clade. phyllomedusa 14: 127-137)" because the manuscript was accepted before the publication of these articles. aknowledgements the authors are grateful to the coordenação de aperfeiçoamento pessoal de nível superior (capes) for financial support, and, to the conselho nacional de desenvolvimento científico e tecnológico (cnpq) for financial of the project “biogegrafia e conservação de anfíbios no complexo serrano da mantiqueira, sudeste do brasil” (068437-2014/06); lucas martins and thadeu santos for making records available; the instituto brasileiro do meio ambiente e dos recursos naturais renováveis (ibama) issued the collect permit 10504-1. financial support was given to rnf through a scientific productivity fellowship by cnpq. comments of anonymous reviewers helped to improve earlier version of this manuscript. references alves-silva, r., silva, h.r. 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(1988): the effect of social interactions on anuran vocal behavior. in: the evolution of the amphibian auditory system, pp. 433-454. fritzsch, b., ryan, m.j., hetherington, t.e., walkowiak, w. eds, wiley, new york. acta herpetologica vol. 11, n. 1 june 2016 firenze university press feeding habits of mesoclemmys vanderhaegei (testudines: chelidae) elizângela silva brito1,*, franco leandro souza2, christine strüssmann3 morphology, ecology, and behaviour of hylarana intermedia, a western ghats frog ambika kamath1, rachakonda sreekar2,3 a new account for the endangered cerrado rocket frog allobates goianus (bokermann, 1975) (anura: aromobatidae), with comments on taxonomy and conservation thiago ribeiro de carvalho1,2,*, lucas borges martins1,2, ariovaldo antonio giaretta1 molecular phylogenetics of the pristimantis lacrimosus species group (anura: craugastoridae) with the description of a new species from colombia mauricio rivera-correa, juan m. daza* population structure and activity pattern of one species of adenomera steindachner, 1867 (anura: leptodactylidae) in northeastern brazil maria juliana borges-leite1,*, joão fabrício mota rodrigues2, patrícia de menezes gondim1, diva maria borges-nojosa1 vocal repertoire of scinax v-signatus (lutz 1968) (anura, hylidae) and comments on bioacoustical synapomorphies for scinax perpusillus species group marco antônio peixoto1,*, carla silva guimarães1, joão victor a. lacerda2, fernando leal2, pedro c. rocha2, renato neves feio1 amendment of the type locality of the endemic sicilian pond turtle emys trinacris fritz et al. 2005, with some notes on the highest altitude reached by the species (testudines, emydidae) federico marrone*, francesco sacco, vincenzo arizza, marco arculeo photo-identification in amphibian studies: a test of i3s pattern marco sannolo1,*, francesca gatti2, marco mangiacotti3,4, stefano scali3, roberto sacchi4 no evidence for the ‘expensive-tissue hypothesis’ in the dark-spotted frog, pelophylax nigromaculatus li zhao, min mao, wen bo liao* no short term effect of clinostomum complanatum (trematoda: digenea: clinostomatidae) on survival of triturus carnifex (amphibia: urodela: salamandridae) giacomo bruni1,*, claudio angelini2 yeasts in amphibians are common: isolation and the first molecular characterization from thailand srisupaph poonlaphdecha, alexis ribas* introduction of eleutherodactylus planirostris (amphibia, anura, eleutherodactylidae) to hong kong wing ho lee1, michael wai-neng lau2, anthony lau3, ding-qi rao4, yik-hei sung5,* correlation between endoscopic sex determination and gonad histology in pond sliders, trachemys scripta (reptilia: testudines: emydidae) david perpiñán1, albert martínez-silvestre2,3, ferran bargalló3, marco di giuseppe4, jorge orós5, taiana costa6,* book review: john w. wilkinson. amphibian survey and monitoring handbook. pelagic publishing franco andreone book review: susan newman. frogs amphibians and their threatened environment. discovery and expression through art k-3. frogs are green franco andreone acta herpetologica 13(1): 33-42, 2018 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-20871 electric circuit theory applied to alien invasions: a connectivity model predicting the balkan frog expansion in northern italy mattia falaschi1,2,*, marco mangiacotti3, roberto sacchi3, stefano scali2, edoardo razzetti4 1 dipartimento di scienze e politiche ambientali, università degli studi di milano, via celoria 26, 20133 milano, italy 2 museo civico di storia naturale di milano, corso venezia 55, 20121 milano, italy 3 dipartimento di scienze della terra e dell’ambiente, università di pavia, via taramelli 24, 27100 pavia, italy 4 museo di storia naturale, università di pavia, piazza botta 9, 27100 pavia, italy *corresponding author. e-mail: matt_fala@hotmail.it submitted on 2017, 23rd june; revised on 2017, 15th november; accepted on 13th december 2017 guest editor: sebastiano salvidio abstract. invasive species are a major threat to biodiversity and alien herpetofauna already caused conservation problems in italy and in the world. pelophylax kurtmuelleri (gayda, 1940) is a water frog native to southern balkans, that was introduced in nw italy (liguria) in 1941, from where it has spread to piedmont, lombardy and recently to emilia romagna. surprisingly, during its expansion, the balkan frog seemed not to be able to cross the po river (with a single exception). so, we investigated the reasons for such directional limitation of range expansion, focusing on the potential role of the po river. combining ecological niche and connectivity models, we adopted a two-step process in order to assess: (i) if the habitat suitability differs between the two sides of the po plain; (ii) if the po river could act as a barrier in terms of dispersal. results showed that the northern side of the plain seems less suitable than the southern one, even if many suitable patches occur along the main left tributaries of the po river and in the hilly part of the plain. the po river behaves like a barrier for the balkan frog, but the only known point on the north bank of the river indicates that it may be able to only slow the dispersal and not to completely stop it. so, the most probable interpretation for the absence of the balkan frog from the northern po plain, is the combination of habitat suitability and connectivity issues: the former makes less probable that new viable populations can establish in the north, the latter decreases the northwards flux of the frogs. given these findings, the balkan frog seems able to spread in the whole northern italy and the colonization of the northern po plain could happen shortly. keywords. balkan frog, pelophylax kurtmuelleri, invasive alien species, connectivity models, italy. introduction invasive alien species (ias) are among the main threats to biodiversity at global scale (clavero and garcía-berthou, 2005), notably for freshwater ecosystems (gallardo et al., 2016). indeed, throughout a variety of interactions with the native biota (e.g., competition for space and resources, carmona-catot et al., 2013; predation on native fauna, roy et al., 2012; genetic loss due to hybridization, muhlfeld et al., 2014; spread of disease, gherardi, 2006), ias have been proved to have the potential to radically affect the invaded ecosystems. herpetological examples of ias are available (e.g., rhinella marina, shine, 2010; boiga irregularis, rodda and fritts, 1992; lever, 2003) and alien herpetofauna already caused conservation problems in italy (e.g., trachemys scripta; ficetola et al., 2009; xenopus laevis, lillo et al., 2011; lithobates catesbeianus, federici et al., 2008; ficetola and scali, 2010; pelophylax ridibundus/kurtmuelleri, ficetola and scali, 2010). as a consequence, the issue of ias has 34 mattia falaschi et alii started to be listed among the most important threats to the conservation of the italian herpetofauna (scali, 2008). pelophylax kurtmuelleri (gayda, 1940) is native to greece, albania, serbia, and macedonia. it was introduced in italy in western liguria region in 1941 for food purposes (lanza, 1962). in the subsequent years, it spread in liguria and piedmont (andreone, 1999). the species has been reported in several localities of southern lombardy between 2005 and 2010 (razzetti et al., 2010). in 2016, the first occurrences for emilia romagna were reported (http://www.ornitho.it/; accessed on 15/01/2017). this invasive species may potentially threat native water frogs (p. lessonae and p. kl. esculentus) through several negative interactions, as competition for habitat and trophic resources (ficetola and scali, 2010), hybridization (ellstrand and schierenbeck, 2000; pagano et al., 2003), or increasing the spread of pathogens. indeed, iass have been proved to work as vectors (garner et al., 2006) and the presence of the fungal pathogen batrachochytrium dendrobatidis has been assessed in p. kurtmuelleri (bellati et al., 2012). by combining these potential threats p. kurtmuelleri has the potential to negatively affect native amphibian populations and it calls for urgent and targeted studies on the real consequences of the introduction of this species in the italian freshwaters. beside this need, from a conservation perspective, the knowledge of possible expansion routes of this species could give a substantial benefit in planning future monitoring and control strategies. habitat connectivity has been recognized essential by both researchers and nature conservationists (rödder et al., 2016) and maintaining it has become a crucial conservation strategy because it determines numerous ecological phenomena (mcrae et al., 2016). for example, connectivity can mitigate the effects of climate change, by allowing species to shift habitat ranges in a rapidly changing climate (mcguire et al., 2016). moreover, connectivity is fundamental for population survival in a fragmented landscape: isolation makes populations more vulnerable to processes such as inbreeding, genetic drift as well as demographic stochasticity, resulting in higher local extinction probabilities (fahrig and merriam, 1985; taylor et al., 1993). however, the creation of ecological networks could be a double-edged sword, accelerating the spread and establishment of invasive species (cowley et al., 2015). in this scenario, the dispersal of the balkan frog in italy seems to be a suitable case study for analyzing the potential effect of connectivity on the spread of an alien species. on the one side, the invasive range of this frog is expanding and every year several new records from previously unoccupied areas are reported (http:// www.ornitho.it/); on the other side, looking at the current distribution and at the past expansion of this species, it seems that the po river is acting as a barrier, preventing the balkan frog to colonize the northern part of the po plain (fig. 1a). indeed, while being widespread south to the po river, the species is almost absent northwards (with the only exception of a single location in travacò siccomario (pv) at the confluence between po and ticino river, e. r. pers. obs.). nor its absence can be reasonably explained by a sampling bias, because i) the species can be easily identified by its call; ii) in this region many herpetologists and qualified naturalists usually work, (iii) the new technologies have made very easy to share data on animal occurrence (e.g., ornitho.it, i-naturalist.org), and (iv) targeted field surveys in some areas of piedmont and lombardy failed to find the balkan frog (e.r. and s.s., pers. obs.). classical biotic-abiotic-mobility theory for species distribution (soberón and peterson, 2005) predicts that a species occurs in a given place only if three main conditions are fulfilled: the area is appropriate in terms of the fundamental niche (habitat suitability), it meets the suite of biotic conditions necessary for the maintenance of viable populations (e.g., interspecific competition), and it has been accessible to the species over relevant time periods (connectivity). following this framework and assuming that no major discrepancies occur in the herpetological communities on both sides of the po plain (bernini et al., 2006), we might interpret the absence of the balkan frog as the consequence of (i) the lack of suitable areas to the north of the po river (abiotic factors hypothesis) or (ii) the barrier effect of the po river itself, which constitutes an almost impermeable obstacle to the northwards dispersal (movement factor hypothesis). so, the present study aims at (i) assessing if the habitat suitability differs between the two sides of the plain, supporting the abiotic factor hypothesis, and, if not, (ii) evaluating if the po river could be an almost impermeable barrier in terms of dispersal, according to the movement factor hypothesis. to answer these two questions we used a combination of both ecological niche models (enms; sensu sillero, 2011) and connectivity models (cms): the former allows us to compare the two sides of the plain in term of availability of suitable habitats; the latter let us testing which level of po river permeability may translate into an effective reduction in the availability of northwards dispersal routes. notably, cms were obtained applying the electrical circuit theory (mcrae, 2006; mcrae et al., 2008) to the landscape where a species occurs. in this framework, the landscape is treated as a conductance surface where each element (cell in a raster) is linked to 35predicting the balkan frog expansion its neighbors and has its own conductance (i.e., permeability to current flow). occurrences become the points in the network where current enters or exits the circuit (mcrae, 2006), and current intensity across the landscape measures the animal potential flux (mcrae et al., 2008). the outcomes are current maps representing the most probable connection pathways among suitable areas (mcrae, 2006; mcrae et al., 2008). since this theory was introduced in the ecological field, this approach has been used for a wide range of purposes, including the analysis of ias (cowley et al., 2015). material and methods occurrence data the occurrence data of p. kurtmuelleri from its invasive range in italy were obtained from different sources: historical published records, authors’ personal observations, and records by experienced herpetologists (see acknowledgments for a full list). all locations (fig. 1a) were georeferenced on a 1 × 1 km grid. the study area was delimited to northern and central italy (fig. 1a). since records of balkan frog from new localities are reported almost every week we decided to stop the data collection at the end of 2016. ecological niche model enm was fitted using maxent (phillips et al., 2004)(phillips et al., 2004), version 3.3.3k (maxent, 2017), which have proved to outperform other methods based on presence-only or presence/background data (elith et al., 2006). a set of nine raster maps at a 1 × 1 km resolution were arranged in order to account for ecologically meaningful factors: distance from streams, land cover (i.e., the relative coverage inside each 1 × 1 km cell for each land cover class) and elevation (table 1). all environmental variables were derived from maps freely available on the web (ispra, 2017): a vector layer of the italian hydrographic network, a raster layer of the land use (corine land cover) referred to 2012, at 100 × 100 m resolution, and a digital elevation model at 75 × 75 m resolution. to take into account multicollinearity among predictors (which can lead to model overfitting; warren and seifert, 2011), the variance inflation factor (vif) was obtained for each variable and used as diagnostic statistic: values around one indicate no substantial problems, while the more they depart from one, the more the multicollinearity is serious (e.g., a value of 10 represents an unacceptable value; faraway, 2004). since the variable “notirri” (not irrigated crops; table 1) showed a high vif (7.80), this predictor was removed. conversely, all the other predictors showed low multicollinearity (table 1) and had low bivariate correlation coefficients (supplementary material s1). the availability of environmental factors was determined on the basis of 10 000 background cells randomly drawn from the whole study area. hsm performance was assessed using a crossfold validation procedure considering the area under the receiver operating curve (auc) as performance score (merow et al., 2013): the occurrence dataset was randomly split into ten equal parts; each of them was used as the test dataset for the model built with the remnants nine. accordingly, all records were used nine times for training and one for testing the model, and all the observations are equally weighted in the training and in the testing of the model (merow et al., 2013). the final hsm was obtained by averaging the prediction of the ten enms from each replicate. in order to avoid overfitting, maxent was run allowing only “linear”, “product” and “quadratic” features (warren and seifert, 2011). data preparation and modeling were carried out in r environment (r core team, 2017), using the raster (hijmans, 2016) and dismo (hijmans et al., 2015) packages. fig. 1. (a) map showing the study area and the range expansion of pelophylax kurtmuelleri from the introduction to 2016. polygons represent the invasive range of this species at different points in time. dots represent occurrence points. pv: occurrence locality north of the po river; (b) habitat suitability map for pelophylax kurtmuelleri. 36 mattia falaschi et alii to compare the suitability of the northern and southern sides of the po plain, we computed a kernel density estimations of the distribution of suitability values: we firstly cut hsm with a 20 km buffer around the po river, and then we fitted and graphically compared two separate gaussian kernels with fixed bandwidth (0.03; obtained using the native r function “density”) for the north and south riversides. connectivity models cms were built using circuitscape (shah and mcrae, 2008), version 4.0.5 (circuitscape.org, 2017), a software designed to model habitat connectivity using the electrical circuit theory (mcrae, 2006; mcrae et al., 2008). conductance maps were derived from hsm by rescaling it between 0 (minimum suitability) and 1000 (maximum suitability), in order to reduce computational time due to floating points values. further, to better fit the known ecology of the species, which is never found above 800 meters a.s.l., in both its native and invasive range (sacchi et al., 2011), the cells at higher elevation were considered absolute barriers. for this purpose, these cells have been assigned an na value, completely excluding them from the cms computation. this modified suitability map was used as the conductance map for the reference model (cmref ), i.e., the model that considers the suitability as the only parameter affecting the potential spread. to evaluate the barrier effect of the po river, six more cms were built, by forcing the conductance of the cells intersecting the po river at increasing values: 0 (absolute barrier, cm0), 1 (minimum permeability, cm1), 10 (cm10), 50 (cm50), 100 (cm100), and 500 (cm500). circuitscape was run in advanced mode (mcrae et al., 2013) to allow for the use of arbitrary current sources and grounds: all the occurrence cells were set as current sources, while the cells at the perimeter of the study areas represented the ground. so, the model was able to predict the possible spread of this invasive species considering all possible directions in one fell swoop (cowley et al., 2015). when multiple sources are set simultaneously, a possible “repulsive” effect with no ecological basis can be observed. indeed, current flows from the sources (positive) to the ground (negative) and is prevented from moving between two sources (positive poles). to avoid this problem circuitscape was run separately for each source cell while leaving ground unchanged. then, the final model was created by cumulating all the current maps obtained in this way. for this purpose, a custom r function was developed (supplementary material s3). to test possible changes of the predicted spread under different po permeability scenarios, each out of the six connectivity maps was compared to the cmref (i.e., the map obtained without manipulating the po permeability). the comparison was made using map comparison kit software, version 3.2.3 (mck, 2017), a tool specifically developed for understanding the nature and the spatial distribution of the differences between map pairs (visser and de nijs, 2006). the software allows for a pixel-by-pixel comparison, whose outcome is, in turn, a map highlighting the location of the differences. further, it computes an overall similarity index between a map pair: zero stands for two completely different maps, one stands for an identical pair (visser and de nijs, 2006). given that cms are numerical, the “fuzzy numerical” algorithm was applied (visser and de nijs, 2006). results ecological niche model a total of 299 occurrence points were collected (corresponding to 160 occupied cells). the enm had a good performance (auc = 0.807 ± 0.012; mean ± standard error), with the highest variable contribution of altitude (43.6 ± 0.4%), followed by distance from the nearest stream (27.1 ± 0.6%). the response curves showed that suitability scores decrease with increasing altitude and table 1. list of the environmental variables considered in building hsm. “source” refers to the original map from which the data were derived or drawn. vif (variance inflation factor): the vif values for the whole set of variables (before) and for the subset obtained eliminating “notirri”, the most collinear variable (after). name description range source vif before after dem average altitude 1-4180 m digital elevation model 1.51 1.55 streamdist minimum distance from a stream 0-37000 m streams vector 1.13 1.11 irri percentage coverage of irrigated fields 0-100% land use 1.57 1.04 notirri percentage coverage of non-irrigated fields 0-100% land use 7.80 removed open percentage coverage of meadows 0-100% land use 1.17 1.01 shrubs percentage coverage of bushes 0-100% land use 2.19 1.44 urban percentage coverage of urban areas 0-100% land use 2.17 1.12 wetlands percentage coverage of wetlands 0-100% land use 1.30 1.02 woods percentage coverage of woodlands 0-100% land use 4.68 1.28 37predicting the balkan frog expansion distance from stream (supplementary material s2). suitability areas crossed the whole study area (fig. 1b), closely matching the hydrographic network: the suitability increases where the network is dense, the opposite where it rarefies. on average, the southern side of the po river appeared more suitable than the northern one, where many unsuitable areas can be found just next to the riverside (fig. 1b). indeed, the suitability distribution of the two sides of the po river showed a north-south asymmetry (fig. 2): the southern side has more high-scoring cells than the northern one, which, in turn, still maintains some highly suitable areas. the distribution of the observed occurrences agreed with the hsm to the south, but not to the north of the po river. connectivity models the reference connectivity model (cmref, fig. 3a) depicted a situation where the current was able to flow both southwards and northwards of the po. by manipulating the permeability, the obtained cms behaved in two sharply different ways (fig. 3): when the po river was set as an absolute barrier (cm0, fig. 3b), current mainly flowed southeast, and only a very small amount of current flowed in the northern side of the map; high current flow was visible along the entire coast of liguria up to northern tuscany and also starting from southern lombardy across all emilia romagna, reaching northern marche region. on the opposite, as soon as the po permeability was increased (cm1, cm10, cm50, cm100, cm500; respectively from fig. 3c to fig. 3g), the northern part of the map suddenly matched cmref, with current flowing along all the main po’s tributaries such as dora baltea, sesia, ticino, lambro, adda, serio, mincio, and reaching also other major rivers like the adige. this convergence could be already observed in cm1 (fig. 3c), i.e., the model corresponding to the lowest permeability scenario. differences and similarities were confirmed by the fuzzy numerical comparison performed in mck: similarity maps (fig. 4) showed nearly no differences between cmref and maps were po is not set as an absolute barrier (cm1 to cm500; from fig. 4b to fig. 4f), while the comparison between cmref to cm0 (fig. 4a) lead to significant differences in the northern part of the map. the rapid convergence towards cmref was noticeable considering the sudden growth of the overall similarity indices among cm models and cmref (fig. 5): when po permeability increased from 0 to 1 (i.e., the minimum allowable permeability value) the similarity index rose from 0.36 to 0.97. fig. 3. connectivity models showing the current flow at the variation of po permeability. (a) reference model without any modification to po permeability cmref. polygons represent the temporal range expansion (see fig. 1a); (b) po as an absolute barrier cm0; (c) permeability set at minimum value of one cm1; (d) permeability set at 10 cm10; (e) permeability set at 50 cm50; (f) permeability set at 100 cm100; (g) permeability set at 500 cm500. fig. 2. comparison of the available suitability of the northern and southern side of the po river. density estimation was restricted to a 20 km buffer (to the left and to the right of the river) and a fixed bandwidth (0.03) was used in both cases. 38 mattia falaschi et alii discussion the establishment of the balkan frog in italy started in 1941 from western liguria (lanza, 1962). since then, the species is still spreading over northern italy, even it has not been actually able to occupy the northern side of the po plain. hsm showed that the northern plain offers less suitable areas than the southern side, at least within the 20 km buffer zones along the riverbed (fig. 1b and 2). at the same time, cms pointed out that the river may act as a barrier itself, preventing the species to reach the northern corridors (i.e., the left-side tributaries of the po river, fig. 3). together, these findings suggest that the absence of the balkan frog from the north po plain may be due to the combination of these two factors, rather than to a prevalent effect of one of them. indeed, the abiotic factor hypothesis is only partially supported by hsm. according to the known ecology of the species, suitable areas include lowland and hilly sites, near to streams and rivers, regardless of the habitat types (sacchi et al., 2011). even though these patches are more common and dense south to the po, they undoubtedly occur also on the opposite riverside. similarly, the movement factor hypothesis is fully supported only when the po river is set as an absolute barrier (cm0, fig. 2b). even a minimum permeability would result in a widespread invafig. 5. growth of the similarity index among cm models and cmref over the increase of po permeability. fig. 4. similarity maps of the fuzzy numerical comparison performed in mck. comparison between (a) cmref and cm0, (b) cmref and cm1, (c) cmref and cm10, (d) cmref and cm50, (e) cmref and cm100, (f) cmref and cm500. 39predicting the balkan frog expansion sion of the northern italy through the main tributaries of the po river. further, the occurrence of a record north to the po river (pv in fig. 1a), makes the absolute barrier hypothesis unrealistic. assuming that the record was not related to anthropogenic transport, it would indicate that the balkan frog actually keeps the potential of crossing the po, even though very limited. consequently, the lack of records of this species in the north may be due to a co-occurrence of both connectivity and suitability constraints, i.e., the barrier effect of the po river and the spatial structure of the suitable habitat patches in the northern po plain. while river banks are often suitable habitat for many amphibians, the river itself could act as a biogeographical barrier for several reasons, such as large size of the riverbed (lampert et al., 2003; li et al., 2009; geissler et al., 2015); therefore, the po river might have played a role in mitigating the colonization of the north bank. furthermore, the spatial distribution of suitable habitat patches may also have played a significant role. as mentioned above, suitable patches are more common and denser south to the po, while northwards they concentrate along the major po tributaries, which head north-south (fig. 1b). in this context, colonization of new habitat patches can happen only if migration of individuals occurred in northern direction. combining these two phenomena, the result is that the gap between the northern and southern distribution of balkan frog may not be there in the future, and the colonization of the northern po plain is just a matter of time. in this scenario, once colonized the major corridors (i.e., alpine tributaries, such as dora baltea, sesia, ticino, lambro, adda, serio, and mincio, as well as other major rivers like the adige), the balkan frog could easily spread in the whole northern italy. in the last years, connectivity modeling became a widely used tool to study connectivity among populations, either for conservation purposes or to study species movement (chetkiewicz et al., 2006; mcrae et al., 2016). until now, electrical circuit theory has been applied to ecology mainly to study connectivity in a network of habitat patches already occupied by native species and only one study focused on modeling the spread of alien species (cowley et al., 2015). in our study, we showed how connectivity models could be a useful tool to deal with alien invasive species. we are aware that a number of points have to be improved. habitat selection applies both to residency and movements (chetkiewicz et al., 2006) and for this reason hsm has been recently used for deriving landscape connectivity (rödder et al., 2016). however, the use of habitat suitability as a proxy for landscape connectivity has been recently criticized, because animals might be able to move through less suitable areas, particularly during long-distance movements (keeley et al., 2017). furthermore, hybrid models that consider population dynamics should allow to better understand the processes driving biological invasions and make more reliable predictions (gallien et al., 2010). anyway, connectivity models and circuitscape software in particular, are still useful for conservation and management related to alien species, and they can return information on where to intervene to try to contain the phenomenon. about northern italy invasion by balkan frog, our conclusions suggest two possible scenarios: i) po river is an absolute barrier and is preventing the balkan frog from spreading in the northern italy; ii) po river is not an absolute barrier and is only mitigating the balkan frog’s breakthrough. so, where should conservation efforts be focused? if po is an absolute barrier, our suggestion is to understand which features play a role in this mechanism (e.g., permanent water, large riverbed; lampert et al., 2003), in order to take advantage of those characteristics by preserving or reinforcing those features, thus avoiding the northern italy invasion. if po river is not an absolute barrier, the situation would be much more critical and the only tool currently at our disposal might be the monitoring of the phenomenon, focusing the effort on the mayor corridors identified by this work (i.e., left po tributaries) and on the northern side of the plain. what research and nature conservation policy will be able to do in the future will be fundamental to preserve the environment from this invasion. despite several possible negative interactions between the balkan frog and italian indigenous water frogs (ficetola and scali, 2010), no specific study has ever been carried out to explore if and how far p. kurtmuelleri is threatening local biodiversity. hybridization is widespread in water frogs (holsbeek and jooris, 2010) and many alien amphibians serve as pathogen vector (garner et al., 2006; measey et al., 2016). carrying out specific studies about these last topics is an urgent need in our opinion, whereby, given the magnitude that this invasion seems to be able to reach, we hope that this manuscript will serve as an incentive for this purpose. acknowledgments the authors would like to thank roberto sindaco, claudio fiorini, danio miserocchi, armando gariboldi, cristina bruno, fabio cavagnini, franco bernini, elvire laurens, massimo gigante, luca artoni and fabio simonazzi, which made possible the creation of this work, by collecting and sharing with us the field data regarding the presence of the balkan frog in italy. we 40 mattia falaschi et alii also thank two anonymous reviewers which valuable contributed to improve the manuscript. supplementary material supplementary material associated with this article can be found at <http://www-3.unipv.it/webshi/appendix> manuscript number 20871. references andreone, f. 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(2011): ecological niche modeling in maxent: the importance of model complexity and the performance of model selection criteria. ecol. appl. 21: 335-342. acta herpetologica vol. 13, n. 1 june 2018 firenze university press species diversity and distribution of lizards in montenegro katarina ljubisavljević1,2,*, ljiljana tomović3, aleksandar urošević1, slađana gvozdenović2, vuk iković2, vernes zagora2, and nenad labus4 the first demographic data and body size of the southern banded newt, ommatotriton vittatus (caudata: salamandridae) abdullah altunişik diet and helminth parasites of freshwater turtles mesoclemmys tuberculata, phrynops geoffroanus (pleurodira: chelidae) and kinosternon scorpioides (criptodyra: kinosternidae) in a semiarid region, northeast of brazil antonio marcos alves pereira*, samuel vieira brito, joão antonio de araujo filho, adonias aphoena martins teixeira, diêgo alves teles, daniel oliveira santana, vandeberg ferreira lima, waltécio de oliveira almeida electric circuit theory applied to alien invasions: a connectivity model predicting the balkan frog expansion in northern italy mattia falaschi1,2,*, marco mangiacotti3, roberto sacchi3, stefano scali2, edoardo razzetti4 first report of bufo bufo (linnaeus, 1758) from sardinia (italy) ilaria maria cossu1, salvatore frau1, massimo delfino2,3, alice chiodi4, claudia corti1,5*, adriana bellati4 natural history and conservation of the nurse frog of the serranía del perijá allobates ignotus (dendrobatoidea: aromobatidae) in northeastern colombia hernán darío granda-rodríguez1,2, andrés camilo montes-correa3,*, juan david jiménez-bolaño3, marvin anganoy-criollo4,5 exploring body injuries in the horseshoe whip snake, hemorrhois hippocrepis juan m pleguezuelos*, esmeralda alaminos, mónica feriche how effectively do european skinks thermoregulate? evidence from chalcides ocellatus, a common but overlooked mediterranean lizard grigoris kapsalas, aris deimezis-tsikoutas, thanos georgakopoulos, ismini gkourtsouli-antoniadou, kallirroi papadaki, katerina vassaki, panayiotis pafilis thermal tolerance for two cohorts of a native and an invasive freshwater turtle species jun geng, wei dang, qiong wu, hong-liang lu* diet of a restocked population of the european pond turtle emys orbicularis in nw italy dario ottonello1, fabrizio oneto1,2, monica vignone2, anita rizzo2, sebastiano salvidio2,* helminths of the lizard colobosauroides cearensis (squamata, gymnophthalmidae) in an area of caatinga, northeastern brazil aldenir f. da silva neta*, robson w. ávila acta herpetologica 13(1): 75-82, 2018 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-21192 how effectively do european skinks thermoregulate? evidence from chalcides ocellatus, a common but overlooked mediterranean lizard grigoris kapsalas*, aris deimezis-tsikoutas, thanos georgakopoulos, ismini gkourtsouli-antoniadou, kallirroi papadaki, katerina vassaki, panayiotis pafilis department of zoology and marine biology, faculty of biology, national and kapodistrian university of athens, panepistimiopolis, ilissia, 15784, greece. *correspondence author. e-mail: gkapsalas@gmail.com submitted on: 2017, 7th september; revised on: 2018, 28th february; accepted on: 2018, 6th march editor: marco sannolo abstract. effective thermoregulation is of vital importance since body temperature affects virtually all physiological and biochemical processes. yet, our current knowledge in reptilian thermoregulation is largely based on a few, wellstudied taxonomic groups. this is especially true in europe, where our insights derive primarily from studies on the numerous lacertids of the continent. skinks on the other hand remain understudied despite being abundant around the mediterranean. in this paper we examine the thermoregulation effectiveness of the ocellated skink, a common lizard whose thermal biology has been overlooked, focusing on a population from a typical mediterranean habitat in mainland greece. we recorded body temperatures in the field and the lab and assessed the thermal quality of the habitat through operative temperatures. our findings suggest that chalcides ocellatus is a poor thermoregulator that stands very close to thermoconformity. the high thermal quality of the habitat allows the ocellated skink to regulate its temperature with less effort and lower accuracy. this indicates that c. ocellatus may have adopted a distinct thermoregulation strategy, most probably due to the particular life style of skinks. keywords. lizard, scincidae, temperature, habitat quality, thermal biology, greece. introduction the regulation of body temperature is arguably one of the most vital processes in reptilian life since most physiological and biochemical processes are temperature-dependent (angilletta et al., 2002; angilletta, 2009). unlike endotherms, reptiles do not rely on metabolism for thermoregulation but utilise their thermal environment instead: most species shuttle constantly within various microhabitats in search of favourable temperatures, while others move slowly between microhabitats and perform within a wide range of body temperatures (pough and gans, 1982; stevenson, 1985). the question of “how effectively does a lizard thermoregulate?” preoccupies herpetological research for decades (e.g., baldwin, 1925; sagonas et al., 2017). limitations in assessing reptilian thermoregulation were described in detail by hertz et al. (1993), who proposed an index of effectiveness using three variables: field body temperatures (tb), i.e., temperatures that lizards achieve in the field, operative temperatures (te), i.e., temperatures that non-regulating animals would achieve in the field, and preferred temperatures (tpref), i.e., the temperature (or range of temperatures, the set-point range tset) that animals select in the lab in an unconstrained thermal experimental setting. this approach, taken together with more recent modifications (e.g., blouin-demers and weatherhead, 2001) is widely accepted as the most accurate and exhaustive methodology and has been applied in numerous lizard species. in europe, lacertids comprise the largest lizard group (arnold and ovenden, 2002; uetz, 2017) and studies on 76 grigoris kapsalas et alii their thermoregulation have been on the front line for the past 30 years, yielding valuable insights. case studies that include viviparous (van damme et al., 1986; gvoždík and castilla, 2001) or oviparous species (díaz et al., 2005; sagonas et al., 2013a), gravid or non-gravid females (braña, 1993; veríssimo and carretero, 2009), populations that live on tiny islets (ortega et al., 2014; pafilis et al., 2016) or in alpine habitats (monasterio et al., 2009; ortega et al., 2016) describe in detail various alternative thermoregulatory patterns. however, in a striking contrast, all the other european lizard families remain largely understudied in terms of their thermal biology, and this applies to scincidae as well. the reason for this discrepancy should be sought primarily in the number of species: of the more than 1600 skinks currently described, only eight species occur in europe (uetz, 2017). additionally, the cryptic lifestyle of skinks and their low population densities limit thermoregulatory studies even more. as a result, only a handful of papers provide some, mostly descriptive, data on european skinks’ temperatures (al-sadoon and spellerberg, 1985; hailey et al., 1987; daut and andrews, 1993; herczeg et al., 2007) and, to the best of our knowledge, there are no works investigating their thermoregulation effectiveness sensu hertz et al. (1993). in this study we examine for the first time the thermoregulation of a european skink, the ocellated skink (chalcides ocellatus), the species with the widest range within the genus (schneider, 1997; lymberakis et al., 2009). taking into account that the species’ biology deviates in many ways from that of the lacertids (e.g., mainly fossorial life style with limited climbing abilities, viviparity) it would be presumable that its thermal physiology would also differ. our aim was on the one hand to provide the first systematically collected field and lab thermal data for a european skink, filling a knowledge gap on the ecophysiology of a common but understudied lizard group, and on the other hand to quantitatively assess the species’ thermoregulatory effectiveness (hertz et al., 1993; blouin-demers and weatherhead, 2001). material and methods study system chalcides ocellatus (forskål, 1775) (squamata, scincidae) is a common viviparous skink of the mediterranean. though its distribution is wide, including large parts of africa and southwestern asia, in europe it occurs mainly on mediterranean islands such as sardinia, sicily and malta and their surrounding islets (schneider, 1997). its distribution in greece comprises a few disconnected areas in the mainland and many islands (e.g., crete, rhodes, karpathos, chios) (valakos et al., 2008). interestingly, new records come to enhance the insular range of the species (belasen et al., 2012; itescu et al., 2016), most probably due to human transportation (kornilios et al., 2010). chalcides ocellatus is a fairly robust lizard, with a cylindrical body, a thick tail, short limbs, a relatively small head and a snout to vent length (svl) of up to 15 cm. it inhabits a variety of habitats including shrublands, sandy areas and farmlands and mainly feeds on coleoptera, formicidae, various larvae and snails (valakos et al., 2008; carretero et al., 2010). it is active during the day, but usually hides underneath rocks or buries itself in the sand (al-sadoon and spellerberg, 1985; valakos et al., 2008). on 23 april 2016, field work was conducted on the campus of the national and kapodistrian university of athens in greece (37°58’01”n, 23°47’20”e). located at the foothills of mount hymettus, the study site consists of an extensive area of maquis vegetation dominated by kermes oak (quercus coccifera), aleppo pine (pinus halepensis), gum tree (eucalyptus sp.) and green olive tree (phillyrea latifolia), as well as patches of open grassland and phrygana. the terrain is fairly rough and rocky, providing ideal hiding spots for the skinks. apart from c. ocellatus, two more lizards are present on the site, the balkan green lizard (lacerta trilineata) and the european snake-eyed skink (ablepharus kitaibelii). adult male lizards were captured by hand after overturning stones, rocks and tree trunks, and were subsequently taken to the facilities of the faculty of biology at the national and kapodistrian university of athens. female lizards were not used in the experiment to avoid possible effects of gravidity on thermoregulation (daut and andrews, 1993; corso et al., 2000; carretero et al., 2005). the captured lizards were housed for one week in individual glass terraria (80 × 30 × 40 cm) with a sand substrate before the experiment started. stones and bricks were placed on the substrate to serve as hiding spots. access to water was provided ad libitum and mealworms (tenebrio molitor) were fed to the lizards every other day. the lizards were held at 25°c under a controlled photoperiod (12l:12d). a 60 w incandescent lamp over each terrarium allowed the animals to thermoregulate for 8 hours per day. the animals were released back to the study site after the end of the experiment. field temperatures (tb and te) we measured the body temperature of 14 males in the field. tbs were measured to the nearest 0.1 °c, using a quickreading cloacal thermometer (t-4000, miller & weber inc., queens, ny) within 10 sec after capture (veríssimo and carretero, 2009; osojnik et al., 2013). air temperature (tair) at the capture point of each individual was also recorded. the relatively small sample size reflects the sparse population density of c. ocellatus on the site and its cryptic lifestyle. we recorded operative temperatures using 28 hollow copper models that approximated the size and reflectance of c. ocellatus (bakken, 1992; dzialowski, 2005). the models were closed at both ends and filled with 2.5-3 ml of water in order to replicate the heat storage capacity of the lizards (grbac and bauwens, 2001; lutterschmidt and reinert, 2012). at one end of the models a narrow slot was left open where the log77thermoregulation effectiveness of chalcides ocellatus ger probes (hobo u12-008 4-channel external data logger) were plugged in (díaz, 1997). the models were placed to cover as many of the microhabitat types available to lizards as possible (huey, 1991), which in this case were grouped in four different microhabitats: open ground, on relatively flat medium-sized rocks (vasconcelos et al., 2012), inside shrubs and underneath rocks. operative temperatures were recorded at 30 min intervals from 10:30 to 17:30 on the same day that field body temperatures (tb) were measured. to ensure the similarity of the temperature responses between models and lizards (hertz, 1992), we performed a laboratory experiment comparing their heating rates, before placing the models in the field (lutterschmidt and reinert, 2012). a lizard and a model were placed side-by-side under a 150 w lamp. we then measured their temperature every 5 min for a 45 min period. a linear regression of tb on te (slope = 0.84 ± 0.14, intercept = 4.68 ± 4.59, r2 = 0.83, n = 10) suggests that the copper model temperature is a significant predictor of the animal body temperature (f1,8 = 38.04, p = 0.0003). lab measurements (tpref and tset) we measured the preferred temperature and set-point range of 12 males in a specially designed terrarium (100 × 25 × 25 cm) that had two ice bags at one end and a 150 watt heating lamp at the other end, thus providing a thermal gradient ranging from 15 to 50 °c (van damme et al., 1986). prior to the experiment, the svl of each individual was recorded using a digital caliper (silverline 380244, accurate to 0.01 mm). each lizard was then allowed to acclimate inside the thermal gradient for 60 min prior to the actual measurements (sagonas et al., 2013a) and subsequently its body temperature was recorded every 30 min for an 180 min period (hertz et al., 1993). the measurements took place from 09:00 to 13:00, which coincides with the animal’s activity period based on our observations in the field. we calculated tpref for each individual as the mean of the body temperatures that the individual selected in the thermal gradient and then calculated the population tpref as the mean of individual tprefs. we estimated tset for each individual by using the lower and upper bounds of the central 50% of values (1st and 3rd quartile respectively) of the body temperatures that each individual selected in the thermal gradient. the tset for the population was subsequently calculated as the mean lower and mean upper bound of individual tsets (hertz et al., 1993; christian and weavers, 1996). effectiveness of thermoregulation the effectiveness of thermoregulation was assessed with the formula proposed by hertz et al. (1993), e = 1 – , where is the mean deviation of field tb from tset and denotes the accuracy of thermoregulation and is the mean deviation of te from tset and describes the thermal quality of the habitat. the values of e range from zero (perfect thermoconformers) to one (perfect thermoregulators) (hertz et al., 1993). e has some limitations though: being a ratio, the index is sensitive to extreme values and, additionally, different and combinations may yield equivalent estimates of thermoregulation effectiveness (christian and weavers, 1996; blouin-demers and weatherhead, 2001). thus, we implemented a complementary method proposed by blouin-demers and weatherhead (2001) to quantify the extent of departure from perfect thermoconformity . in this approach, a value of zero indicates a perfect thermoconformer, positive values describe a thermoregulator, whereas negative values denote animals that actively avoid habitats of high thermal quality. the magnitude of the difference between and provides an index of the effectiveness of thermoregulation (blouin-demers and weatherhead, 2001). statistics the shapiro-wilk test was used to test for normality. the non-parametric kruskal-wallis rank sum test was used to test te differences between microhabitats, since the assumption of normality was violated (p < 0.05). the relationship between tb and tair was assessed using linear regression. 95% confidence intervals for e were determined using bootstrap resampling: 1000 values of e were computed using random sampling with replacement from the observed tb and te distributions and subsequently the 2.5th and 97.5th percentiles were used as confidence interval limits (percentile method, hertz et al., 1993). the same procedure was also applied to the index (blouindemers and weatherhead, 2001). all tests were performed in r 3.3.2 (r core team, 2016). results the mean preferred temperature was 31.8°c, ranging from 27.9 to 34.6 °c (table 1). set-point range was estimated at 30.4 to 33.6 °c. the mean field body temperature was 27.2°c, ranging from 23.1 to 30.6 °c (table 1). and were 3.2 and 4.6 °c respectively (table 1). the placement of the lizard models in the field covered successfully the thermal diversity of the habitat, as revealed by the tes (table 2) recorded across different microhabitat types (kruskal-wallis rank sum test, χ2 = 39.229, df = 3, p < 10-6). the mean operative temperature was 30.1 °c, ranging from 18.2 °c at 11:00 (lowest te) to 57.0 °c at 15:30 (highest te) (table 1). field body temperature (tb) was positively correlated with air temperature (tair) at the point of capture (pearson’s r = 0.709) and the linear model that describes the relationship between the two variables had a strong fit (tb = 1.04 * tair + 2.6, r2 = 0.502, f(1,12) = 12.1, p = 0.0046, fig. 1). the effectiveness of thermoregulation sensu hertz et al. (1993) was estimated as e = 0.31 (bootstraped mean: 0.31, 95% confidence intervals: 0.03-0.56, n = 1000). thermoregulation effectiveness sensu blouin78 grigoris kapsalas et alii demers and weatherhead (2001) was evaluated at 1.44 (bootstraped mean: 1.44, 95% confidence intervals: 0.212.72, n = 1000). discussion in this study we assessed for the first time the thermoregulatory profile of a european skink. we recorded body temperatures in the field and the lab and, taking into account the thermal availability of the environment (te), we estimated the accuracy, precision and effectiveness of thermoregulation. only a few descriptive studies have dealt with aspects of the thermal biology of c. ocellatus: to the best of our knowledge this is the first time that thermoregulation effectiveness (hertz et al., 1993) was assessed for this species. because of the scarcity of thermal data for c. ocellatus from europe, we also compare our findings with conspecific populations outside europe and with other european chalcides as well. body temperatures in the field reached a mean value of 27.2 °c (table 1). lo cascio and corti (2008) reported similar tbs from an insular population in italy in the springtime (lampedusa island: 26.9 ± 1.7 °c), with tbs rising higher in the summer (lampedusa island: 32.0 ± 0.6 °c, isola dei conigli: 31.3 ± 1.2 °c). the congeneric chalcides bedriagai from spain achieved higher tbs and a wider range (mean tb = 30.5 °c, range = 25-35 °c; hailey et al., 1987). since there are no other tb measurements for c. ocellatus, it is hard to estimate the nature of these results. however, the diel variation of tbs was limited, a finding that suggests high precision in thermoregulation. table 1. variables used in this paper for the study of thermoregulation. tb: field body temperatures, te: operative temperatures, tpref: preferred temperatures, db: deviation of tb from tset, de: deviation of te from tset. variable n mean range sd se tb 14 27.23 23.10-30.60 2.41 0.64 te 28 30.05 18.22-56.98 7.24 0.35 tpref 12 31.83 27.93-34.58 2.03 0.59 db 14 3.19 0-7.30 2.39 0.64 de 28 4.62 0-23.41 4.21 0.21 table 2. operative temperatures (te) across microhabitat types within the study site. microhabitat n mean range sd se open ground 6 33.18 21.82-56.98 9.23 0.97 rock surface 9 31.18 19.39-44.38 6.27 0.54 inside shrubs 9 28.14 20.48-53 6.33 0.54 under rocks 4 27.06 18.22-36.47 5.29 0.68 fig. 2. relative frequency of field body temperatures (tb, grey shade), operative temperatures (te, solid line) and set-point range (tset, dotted lines) for the ocellated skink. fig. 1. linear regression of field body temperature (tb) on air temperature (tair). shaded area denotes 95% confidence intervals. 79thermoregulation effectiveness of chalcides ocellatus preferred temperatures were higher than tbs and achieved a mean value of 31.8 °c (table 1). interestingly, tpref had a wider range of variation compared to lacertids. nonetheless, the tprefs of the greek population were lower than the values reported from egyptian populations. al-sadoon (1986) measured a mean tpref of 34°c, similarly to pough and andrews (1985) who found a mean tpref of 34.4 °c. also, the range of tpref has been reported to vary from 28 to 37 °c (al-sadoon and spellerberg, 1985). these deviations in tpref between the european and african populations could be attributed to environmental conditions. preferred temperatures depend on the particular conditions of the habitat (scheers and van damme, 2002; pafilis et al., 2016). populations from warmer habitats are known to achieve higher tpref (sagonas et al., 2013b), though thermal optima have been also reported to show resilience despite environmental divergence in temperature (osojnik et al., 2013; clusella-trullas and chown, 2014). we believe that the lower tpref of the ocellated skink in greece is due to the temperate, cooler climate of the north mediterranean costs. nonetheless, we cannot rule out that methodological differences (e.g. sex, size class, time, season) with the abovementioned papers might be responsible for the observed deviations. operative temperatures ranged within a broad thermal window, varying from 18.2 to 57.0 °c (table 1). this wide range indicates high thermal heterogeneity, in other words a diverse thermal mosaic where lizards can shuttle between different microhabitats (table 2). the mean te was 30.1 °c, which lies within the values that have been reported from other greek habitats (adamopoulou and valakos, 2005; sagonas et al., 2013 a, b; kapsalas et al., 2016; pafilis et al. 2016; belasen et al., 2017; sagonas et al., 2017). based on these operative temperatures, the thermal quality index (sensu hertz et al., 1993) received a low value ( = 4.6), which is actually among the lowest ever reported in the north mediterranean (see table 1 at pafilis et al., 2016). the low indicates a habitat of high quality (hertz et al., 1993) that provides many thermal opportunities for successful thermoregulation. contrary to the low , received a value of 3.2, which is rather medial according to hertz et al., (1993). low implies high accuracy, since most of the observed field body temperatures fall within the animal’s preferred temperature range. in the case of the ocellated skink, tbs fell outside the set-point range in 93% of the cases (all of which were below tset) (fig. 2), resulting in the observed . this value suggests low accuracy in thermoregulation. however, we have to stress out the relativity of the latter notion. the lack of thermoregulatory studies on european skinks or within the genus chalcides, precludes us from understanding the magnitude of the observed value. for instance, a of 3.2 is rather high for lacertids (díaz et al., 2006; monasterio et al., 2009; ortega et al., 2016; sagonas et al., 2017) but not particularly high for certain anoles (hertz et al., 1993, woolrich-pina et al 2015), monitor lizards (christian and weavers, 1996) or geckos (rock et al., 2002, hitchcock and mcbrayer, 2006). the wide spectrum of operative temperatures and the low sketch out a rather benign thermal habitat. this fact is reflected in the low index of thermoregulation (e = 0.31). according to theory, e values close to zero stand for animals that do not thermoregulate actively whereas values close to one describe animals that thermoregulate carefully (hertz et al., 1993). chalcides ocellatus appears to be a poor thermoregulator, at least during this given period of the year. this finding is further supported by the alternative approach proposed by blouin-demers and weatherhead (2001). although their index does not take values within a specific range (contrary to e, hertz et al., 1993), the value reported here (1.44) points to a poor thermoregulator as well. low thermoregulatory effectiveness might be the norm for chalcides lizards. hailey et al. (1987) categorized the congeneric chalcides bedriagai as a thermoconformer, interpreting the high correlation between field body temperatures and substrate temperatures. this finding is in agreement with the observed thermoregulatory behaviour of the ocellated skink in our study, as seen both by the correlation of the ocellated skink’s tb with air temperature, as well as by the values of e and . according to our results, the focal population lives in a habitat of high thermal quality and heterogeneity. thermally challenging habitats dictate high thermoregulatory effectiveness to guarantee survival under extreme conditions (hertz et al., 1993; ortega et al., 2016; pafilis et al., 2016). this is not the case for c. ocellatus, which thanks to the aforementioned favourable habitat, can afford a less effective thermoregulation. its thermoregulation is also characterized by high precision and low accuracy. further studies that would include more species and populations and different seasons are required to thoroughly understand the thermal biology of european skinks. thermoregulation may vary with season (huey and pianka, 1977; hertz et al., 1993; díaz and cabezasdíaz, 2004), altitude (spencer and grimmond, 1994; zamora-camacho et al., 2016), insularity (sagonas et al., 2013b) and body size (sagonas et al., 2013a). it may also change according to organism needs depending on internal factors (e.g. pregnancy, body condition and water loss rate) (carretero et al., 2005). thus, the species’ thermoregulation strategy on mediterranean islands, in high80 grigoris kapsalas et alii er altitudes, in different latitudes or even at our study site during different seasons, may deviate from the present results. acknowledgements we are grateful to anna karelou, konstantina lentza, evdokia gigiza and nikos kargopoulos, for their valuable help during fieldwork. field sampling, animal handling and housing was conducted according to the provisions of greek legislation (presidential decree 67/1981 and law 3937/2011). references adamopoulou, c., valakos, e.d. 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(2016): thermoregulation in the lizard psammodromus algirus along a 2200-m elevational gradient in sierra nevada (spain). int. j. biometeorol. 60: 687697. acta herpetologica vol. 13, n. 1 june 2018 firenze university press species diversity and distribution of lizards in montenegro katarina ljubisavljević1,2,*, ljiljana tomović3, aleksandar urošević1, slađana gvozdenović2, vuk iković2, vernes zagora2, and nenad labus4 the first demographic data and body size of the southern banded newt, ommatotriton vittatus (caudata: salamandridae) abdullah altunişik diet and helminth parasites of freshwater turtles mesoclemmys tuberculata, phrynops geoffroanus (pleurodira: chelidae) and kinosternon scorpioides (criptodyra: kinosternidae) in a semiarid region, northeast of brazil antonio marcos alves pereira*, samuel vieira brito, joão antonio de araujo filho, adonias aphoena martins teixeira, diêgo alves teles, daniel oliveira santana, vandeberg ferreira lima, waltécio de oliveira almeida electric circuit theory applied to alien invasions: a connectivity model predicting the balkan frog expansion in northern italy mattia falaschi1,2,*, marco mangiacotti3, roberto sacchi3, stefano scali2, edoardo razzetti4 first report of bufo bufo (linnaeus, 1758) from sardinia (italy) ilaria maria cossu1, salvatore frau1, massimo delfino2,3, alice chiodi4, claudia corti1,5*, adriana bellati4 natural history and conservation of the nurse frog of the serranía del perijá allobates ignotus (dendrobatoidea: aromobatidae) in northeastern colombia hernán darío granda-rodríguez1,2, andrés camilo montes-correa3,*, juan david jiménez-bolaño3, marvin anganoy-criollo4,5 exploring body injuries in the horseshoe whip snake, hemorrhois hippocrepis juan m pleguezuelos*, esmeralda alaminos, mónica feriche how effectively do european skinks thermoregulate? evidence from chalcides ocellatus, a common but overlooked mediterranean lizard grigoris kapsalas, aris deimezis-tsikoutas, thanos georgakopoulos, ismini gkourtsouli-antoniadou, kallirroi papadaki, katerina vassaki, panayiotis pafilis thermal tolerance for two cohorts of a native and an invasive freshwater turtle species jun geng, wei dang, qiong wu, hong-liang lu* diet of a restocked population of the european pond turtle emys orbicularis in nw italy dario ottonello1, fabrizio oneto1,2, monica vignone2, anita rizzo2, sebastiano salvidio2,* helminths of the lizard colobosauroides cearensis (squamata, gymnophthalmidae) in an area of caatinga, northeastern brazil aldenir f. da silva neta*, robson w. ávila acta herpetologica 12(1): 49-54, 2017 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-18354 feeding ecology of two sympatric geckos in an urban area of northeastern brazil josé guilherme g. sousa1, adonias a. martins teixeira2,*, diêgo alves teles2, joão antônio araújo-filho2, robson waldemar ávila3 ¹ programa de pós-graduação em ecologia e recursos naturais, departamento de ciências biológicas, universidade federal do ceará, campus universitário do pici, cep 60021970, fortaleza, ceará, brazil ² programa de pós-graduação em ciências biológicas (zoologia), departamento de sistemática e ecologia – dse, centro de ciências exatas e da natureza – ccen, universidade federal da paraíba – ufpb, cidade universitária, campus i, cep 58059-900, joão pessoa, pb, brazil. *corresponding author. e-mail: adoniasteixeira01@gmail.com ³ laboratório de herpetologia, departamento de ciências biológicas, universidade regional do cariri, rua cel. antônio luiz, 1161, campus do pimenta, 63105-000, crato, brazil; and departamento de ciências biológicas, universidade federal do ceará, campus universitário do pici, cep 60021970, fortaleza, ceará, brazil submitted on 2016, 31st may; revised on 2016, 11th november; accepted on 2016, 16th december editor: sandra hochsheid abstract. the diets of two sympatric gecko species, hemidactylus mabouia and phyllopezus pollicaris, were studied from an urban area of the crato municipality, northeastern brazil. while the house gecko h. mabouia is an introduced species widely distributed in north, central, and south america, the brazilian gecko p. pollicaris is a native species distributed along the great diagonal of open formations of south america. the diets of both species were mainly composed by arthropods, diptera was the most important item for both species, corroborating others studies with lizards in urban areas. male and female adults of both h. mabouia and p. pollicaris use similar microhabitats which can explain the high sexual and interspecific trophic niche overlap. in these populations from an urban area of the crato municipality, the alien h. mabouia seems to have not negatively affected the trophic niche of the native p. pollicaris. keywords. trophic ecology, diet composition, gekkota, hemidactylus mabouia, invasive lizard, phyllopezus pollicaris, body size. introduction gekkota is a species-rich clade of lizards with a great potential to invade new habitats, frequently introduced in anthropogenically disturbed areas and/or urban habitats (hanley et al., 1998; carranza and arnold, 2006; gamble et al., 2008). its success in colonizing and establishing populations in new habitats is due in part to its great plasticity, which led to a wide distribution of geckos, especially hemidactylus, in the old and new world (rocha et al., 1994; hanley et al., 1998; vences et al., 2004; gamble et al., 2008; meshaka, 2011). the tropical house gecko hemidactylus mabouia is native to africa and has successfully colonized south, central, and north america (rocha et al., 2011). in brazil, h. mabouia is frequently found in human-altered areas, but also in pristine habitats in the amazon, atlantic forest, cerrado, and caatinga (vanzolini, 1978; vanzolini et al., 1980; araújo, 1991; rocha et al., 2000; rocha et al., 2002; rocha et al., 2011; albuquerque et al., 2013). several studies have demonstrated the role of hemidactylus spp. in niche displacement of resident geckos by means of direct exploitative competition, indirect competition, aggression, and predation (meshaka, 1995; 50 j.g.g. sousa et alii meshaka and moody, 1996; meshaka, 2000; meshaka et al., 2005; hoskin, 2011; hughes et al., 2015). buildings in brazil are colonized by many native gecko species, but the co-occurrence with h. mabouia caused exclusion by competition in some species, such as thecadactylus rapicauda (vitt and zani, 1997). however, phyllopezus pollicaris, a medium-sized gecko inhabiting boulder slabs in the dry formations of south america, is also frequently found in human habitations syntopic with h. mabouia (vanzolini et al., 1980; vitt, 1995; sousa et al., 2010; recoder et al., 2012). plasticity of feeding habits, associated with ecological and biological conditions, has been considered the main cause of h. mabouia´s invasion success (zamprogno and teixeira, 1998; bonfiglio et al., 2006). thus, diet studies can provide valuable information on the importance of prey types on the mechanism by which hemidactylus geckos interact with other lizard species (belver and avila, 2001). herein, we present data on the diets of h. mabouia and p. pollicaris and test if the invasive species has affected the native lizard, considering the trophic niche, in order to answer the following questions: i) is there sexual dimorphism between h. mabouia and p. pollicaris and between species? ii) how do the diets of these two geckos differ regarding composition? iii) what are the average niche breadths and the niche overlaps between these species? iv) is niche overlap between the studied species caused by chance? materials and methods lizard specimens were collected from march to november 2011 in human habitations in the city of crato (7°14’s and 39°24’w, datum wgs84), ceará state, northeastern brazil. the regional climate is predominantly tropical, hot, and sub-humid (average annual temperatures vary from 24 to 26 °c). the rainy period occurs between january and may, with an average annual rainfall of 1,091 mm (ipece, 2013). the lizards were collected manually, humanely killed with lethal doses of 2% lidocaine, fixed in 10% formaldehyde, and preserved in 70% ethyl alcohol. the specimens were sacrificed for another study on parasitism which has been already published (sousa et al., 2014). snout-vent length (svl), jaw width (jw), mouth length (ml), and head length (hl) of each lizard were measured using a digital caliper (± 0.01 mm; table 1). voucher specimens were deposited in the coleção herpetológica da universidade regional do cariri urca. to test for morphological variation due to the sex of each species, between adults of h. mabouia and p. pollicaris in body shape (using all morphometric variables), and between each individual variable, we performed a multivariate discriminant function analysis with residuals of morphological variables of each sampled lizard species. residuals were obtained from a simple linear regression following sousa and ávila (2015). discriminant function analysis was executed using statistica version 10.0 (statsoft, 2011). lizards were dissected in the laboratory and their stomach contents were analyzed under a stereomicroscope. prey items were identified and classified to the order level; length and width were measured with a digital caliper (precision 0.01mm). the percentage of lizards with empty stomachs was calculated and only lizards with identifiable items were used. the volume of each prey item was estimated by the ellipsoid formula: v = 4 3 π l 2 ⎛ ⎝ ⎜ ⎞ ⎠ ⎟ w 2 ⎛ ⎝ ⎜ ⎞ ⎠ ⎟ 2 , where l = length and w = width. the importance value index (i) was estimated by the following formula (powell et al., 1990): i = f%+n%+v% 3 , where f% is the relative frequency, n% is the numerical percentage, and v% is the volumetric percentage of each prey item. trophic niche breadth (both numerical and volumetrically) was calculated by the inverse of the simpson’s diversity index (simpson, 1949): b=1/ pi 2 i=1 n ∑ , where p is the numeric or volumetric proportion of prey category i and n is the number of categories (pianka, 1973). the value of niche breadth varies from 1 to n, where the lowest values represent a more specialized diet and the highest values indicate a generalist diet. differences in number and volume of prey items between males and females were tested by nonparametric mann-whitney u test using statistica version 10.0 (statsoft-inc, 2011). trophic niche overlap between sexes and species was calculated using the pianka’s overlap index (pianka, 1973): ∅ik = pijpik i=1 n ∑ (pij2)(pik2) i=1 n ∑ , where pij and pik are the rate consumption of prey category i, with j and k representing the compared species and sexes. pianka’s overlap index varies from zero (no overlap) to one (complete overlap). finally, we compared the observed niche overlap values for h. mabouia and p. pollicaris against a null model (1,000 interactions) generated by the randomization algorithm 3 (ra3) (lawlor, 1980). the use of ra3 seems adequate because it retains the niche breadth of each species, but randomizes which particular resource states are used, allowing the species to potentially use other resource states (winemiller and pianka, 1990). niche overlaps and null models were calculated using ecosimr r code for null model analysis (gotelli and ellison, 2013). differences between proportions of each prey category among the two gecko species were tested by a non-parametric 51feeding ecology of two sympatric geckos multivariate analysis of similarity (anosim), using the braycurtis similarity coefficient and 9,999 permutations in the software past 3.0 (hammer et al., 2001). a similarity percentage analysis (simper) was performed to determine which preys were responsible for dissimilarity in diets between the two gecko species. data matrix for anosim and simper were standardized (as a percentage) to minimize the discrepancy between samples. results we collected 58 h. mabouia, 10 adult males (mean ± standard deviation) (svl = 56.35 ± 9.76 mm), 21 adult females (svl = 53.69 ± 6.21 mm), and 27 juveniles (svl = 30.92 ± 5.36 mm). of the species p. pollicaris, we collected 100 specimen, 38 adult males (svl = 65.48 ± 9.06 mm), 54 adult females (svl = 64.58 ± 12.62 mm), and 8 juveniles (svl =34.54 ± 4.32 mm). of these, 18 (31.03%) h. mabouia (2 males, 7 females, and 9 juveniles) and 45 (45%) p. pollicaris had empty stomachs (12 males, 27 females, and 6 juveniles). there were no significant sexual differences in morphometric variables of both species (table 1). on the other hand, adults of p. pollicaris were significantly larger than h. mabouia in body shape, with svl and jw of p. pollicaris greater than those of h. mabouia (table 1). the diet of both species consisted of 10 arthropod prey items: diptera, hymenoptera, and coleoptera were the most important items for h. mabouia and diptera, coleoptera, and hemiptera for p. pollicaris (table 2). hemidactylus mabouia had numerical and volumetrically niche breadth values of 1.904 and 1.979, respectively, which was smaller than the niche breadth of p. pollicaris: 4.05 and 3.036 (table 2). the average number of prey items per stomach (only those individuals whose stomachs were not empty) was similar for both species: 3.86 ± 8.22 for h. mabouia and 3.47 ± 4.52 for p. pollicaris, with no significant difference (u = 1,017.5; p = 0.387). however, the average number of prey categories per stomach was 1.54 ± 0.61 and 1.24 ± 0.51 for h. mabouia and p. pollicaris, respectively, and there was a significant difference (u = 769.5; p = 0.014). males and females of p. pollicaris ingested more prey items (4.26 ± 6.02 and 3.18 ± 3.07, respectively) than those of h. mabouia (2.2 ± 2.69 and 1.68 ± 1.21), but there were no statistical differences in prey items ingested by females of p. pollicaris and h. mabouia (u= 230.5; p= 0.836) neither between males of both species (u = 32; p= 0.081). also, there were no significant differences in number (u = 117; p = 0.385) or volume (u = 111; p = 0.3) of the prey items between h. mabouia males and females, between p. pollicaris males and females (number: u = 624.50; p= 0.323; volume u = 605; p = 0.25), or in the number between adult individuals of both species (u = 995; p = 0.21). on the other hand, there was a significant difference in volume between adults of p. pollicaris (211.858 ± 435.062) and h. mabouia and (36.749 ± 141.807) (u = 697; p < 0.001). niche overlap between the sexes was 0.9159 in h. mabouia and 0.9526 in p. pollicaris; and between the two gecko species, niche overlap was 0.9094. the high niche overlap between adult individuals of both studied species could be due to chance (average overlap simulated = 0.2740; p = 0.99). there was no significant difference table 1. morphometric data (mean ± standard deviation) and results of discriminant function analysis. hm= hemidactylus mabouia; pp= phyllopezus pollicaris. m= males; f= females; body shape= pooled variables; svl= snout-vent lenght; jw= jaw width; ml= mouth lenght; hl= head lenght; λ = wilks' lambda. hm pp hm pp m f m f adults adults svl 53.4 ± 12.9 53.6 ± 6.8 65.9 ± 11.8 65.8 ± 11.5 55.1 ± 8.3 65.8 ± 11.6 jw 9.6 ± 2.2 9.6 ± 1.0 11.7 ± 2.1 11.8 ± 2.0 9.6 ± 1.3 11.7 ± 2.0 ml 10.2 ± 2.2 10.5 ± 1.2 12.0 ± 2.3 12.0 ± 2.0 10.4 ± 1.4 12.0 ± 2.1 hl 14.2 ± 2.5 14.6 ± 1.4 16.7 ± 3.0 16.8 ± 2.6 14.5 ± 1.7 16.7 ± 2.7 λ (m vs f) p λ (m vs f) p λ (hm vs pp) p body shape 0.951 0.884 0.983 0.860 0.753 <0.0000* svl 0.997 0.802 0.983 0.989 0.908 0.000* jw 0.965 0.385 0.994 0.361 0.835 0.001* ml 1.000 0.970 0.989 0.498 0.773 0.100 hl 0.996 0.775 0.986 0.636 0.756 0.563 52 j.g.g. sousa et alii between the proportions in prey use (anosim, r = 0.7037; p = 0.2035). the results of the simper analysis also showed a small dissimilarity between diet composition (32.69%), with diptera, hemiptera, and coleoptera as prey groups that contribute most to dissimilarities between the two geckos, explaining 32.2, 26.7 and 11.1% of the variation. discussion the diets of the sympatric h. mabouia and p. pollicaris in urban habitats of northeastern brazil were very similar, with both species consuming 10 prey categories. diptera, coleoptera, hemiptera, and hymenoptera were the most frequently ingested preys; diptera were the most important prey for both species. however, hymenoptera and coleoptera were the second most important prey for h. mabouia and p. pollicaris, respectively. these insects are commonly found in urban habitats, mainly close to streetlights (robinson, 2005). food composition of urban populations of lizards may differ from that of pristine habitats (hódar and pleguezuelos, 1999). moreover, the opportunistic behavior of these two gecko species, plus changes in diversity and abundance of prey, may contribute to differences between study sites (zamprogno and teixeira, 1998). vitt (1995), studying the diet of h. mabouia and p. pollicaris at caatinga habitats from exu, found that insect larvae were the most important prey for both species. in the coastal plain of the espírito santo state, zamprogno and teixeira (1998) found araneae, homoptera, and isopoda as the most important prey for h. mabouia, while apterygota, araneae, and orthoptera were the main items for p. pollicaris at cerrado habitats in the state of tocantins (recoder et al., 2012). albuquerque et al. (2013) found cannibalistic habits for h. mabouia, where h. mabouia was the most important item, followed by formicidae and hemiptera; for p. pollicaris, the most important items were coleoptera, araneae, and homoptera in periantropic environments in the state mato grosso do sul. our results corroborate the study of bonfiglio et al. (2006), in an urban area in rio grande do sul, who found that diptera were the most important item in the diets of the two species studied here. that convergence in diet composition may be due to the fact that diptera is often present at urban environments, mainly close to streetlights as mentioned above. in this case, perhaps geckos could act as a control of diptera in cities. energetic balance in a given population of lizards can be estimated by the proportion of empty stomachs, where nocturnal lizards have a higher proportion of empty stomachs (24.1%) than diurnal lizards (10.5%); the mean percentage of empty stomachs for nocturnal geckos is 21.2% (huey and pianka 1981). in the present study, h. mabouia (31.01%) and p. pollicaris (42.6%) showed higher proportions of empty stomachs than stated by huey and pianka (1981). this may be explained by the time table 2. diet composition and numerical and volumetric niche breadth of hemidactylus mabouia and phyllopezus pollicaris from an urban area of crato city, ceará state, brazil. n= total of analyzed individuals. f = frequency of occurrence, n = number of preys, v= volume (mm³) and i = relative importance index. category h. mabouia (n = 58) p. pollicaris (n = 100) f f % n n% v v % i f f % n n% v v % i araneae 4 7.02 4 2.80 28.83 2.12 3.98 8 10.96 8 3.98 364.37 1.55 5.50 blatodae 1 1.37 1 0.50 51.92 0.22 0.70 coleoptera 8 14.04 13 9.09 160.26 11.78 11.64 13 17.81 51 25.37 2605.41 11.10 18.09 diptera 24 42.11 102 71.33 936.65 68.86 60.76 26 35.62 79 39.30 11319.23 48.15 41.05 h. mabouia shed skin 1 1.75 3 2.10 10.45 0.77 1.54 vertebrate eggshell 1 1.75 1 0.70 8.83 0.65 1.03 1 1.37 3 1.49 131.19 0.56 1.14 hemiptera 8 10.96 27 13.43 6544.06 27.89 17.43 hymenoptera 10 17.54 10 6.99 173.73 12.77 12.44 6 8.22 15 7.46 1804.91 7.69 7.79 isoptera 1 1.75 1 0.70 3.00 0.22 0.89 insect larvae 5 8.77 6 4.20 18.57 1.37 4.78 4 5.48 5 2.49 265.74 1.13 3.03 lepidoptera 1 1.75 1 0.70 12.42 0.91 1.12 2 2.74 8 3.98 293.33 1.25 2.66 ortoptera 2 3.51 2 1.40 7.49 0.55 1.82 4 5.48 4 1.99 83.53 0.36 2.61 total 57 100 143 100 1360.24 100 100 73 100 201 100 23463.69 100 100 niche breadth 1.90 1.98 4.05 3.04 empty stomachs 18 45 53feeding ecology of two sympatric geckos when the lizards were captured, i.e., in the first hours of the night. the lack of sexual differences in the diets of the species we studied is probably a result of similar use of space and time, which led to the use of the same prey types for both sexes and higher dietary overlap (see rocha and anjos, 2007). dietary overlap was higher even when we paired both species (0.91), and the null model showed that high niche overlap between them is due to chance, indicating that the two species may eventually compete for prey. however, p. pollicaris presented higher prey volume and niche breadth than h. mabouia (probably due to its larger body) and despite the expected, h. mabouia was apparently not significantly affected by the trophic niche of p. pollicaris. dissimilarity between the diets of these two geckos was about 30% in the present study, but this pattern can vary according to prey availability and/or greater time of colonization by h. mabouia. these factors can make this invasive species compete more intensely, damaging the spatial and/or trophic niche of native species (see rodder et al., 2008; rocha et al., 2011). additionally, further studies are necessary to understand the role of competition for food and/or space in the coexistence of native and invader lizard species in disturbed and natural areas in brazil. acknowledgments we thank the coordenação de aperfeiçoamento de pessoal de nível superior – capes for providing a fellowship to jggs, aamt, and jaaf, the conselho nacional de desenvolvimento científico (cnpq) for a research grant to rwa (process # 303622/2015-6) and a fellowship to dat. we also thank sandra hochscheid and two anonymous reviewers for their valuable suggestions. the specimens were collected under the license of the instituto chico mendes de conservação da biodiversidade-icmbio 29613-1. references albuquerque, n.r.d., costa-urquiza, a.d.s., soares, m.p., alves, l.s., urquiza, m.v.s. 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(1998): hábitos alimentares da lagartixa de parede hemidactylus mabouia (reptilia, gekkonidae) da planície litorânea do norte do espírito santo, brasil. braz. j. biol. 58: 143-150. acta herpetologica vol. 12, n. 1 june 2017 firenze university press morphological variation and sexual dimorphism in liolaemus wiegmannii (duméril & bibron, 1837) (squamata: liolaemidae) from uruguay joaquín villamil1,*, arley camargo2, raúl maneyro1 sex does not affect tail autotomy in lacertid lizards panayiotis pafilis1,*, kostas sagonas2, grigoris kapsalas1, johannes foufopoulos3, efstratios d. valakos4 fire salamander (salamandra salamandra) males’ activity during breeding season: effects of microhabitat features and body size raoul manenti*, andrea conti, roberta pennati call variation and vocalizations of the stealthy litter frog ischnocnema abdita (anura: brachycephalidae) pedro carvalho rocha1,2,*, joão victor a. lacerda2,3, rafael félix de magalhães2,3, clarissa canedo4, bruno v. s. pimenta5, rodrigo carrara heitor6, paulo christiano de anchieta garcia2,3 feeding ecology of two sympatric geckos in an urban area of northeastern brazil josé guilherme g. sousa1, adonias a. martins teixeira2,*, diêgo alves teles2, joão antônio araújo-filho2 and robson waldemar ávila3 a pattern-based tool for long-term, large-sample capture-mark-recapture studies of fire salamanders salamandra species (amphibia: urodela: salamandridae) jeroen speybroeck1,*, koen steenhoudt2,$ skeletal variation within the darwinii group of liolaemus (iguania: liolaemidae): new characters, identification of polymorphisms and new synapomorphies for subclades linda díaz-fernández*, andrés s. quinteros, fernando lobo marking techniques in the marbled newt (triturus marmoratus): pit-tag and tracking device implant protocols hugo le chevalier1, olivier calvez2, albert martínez-silvestre3, damien picard4, sandra guérin4,5, francis isselin-nondedeu6, alexandre ribéron1, audrey trochet1,2,* evidence for directional testes asymmetry in hyla gongshanensis jindongensis qing gui wu1, wen bo liao2,* the advertisement call of pristimantis subsigillatus (anura, craugastoridae) florina stănescu1,4, rafael márquez2, paul székely3,4,*, dan cogălniceanu1,4,5 nematodes infecting anotosaura vanzolinia (squamata: gymnophthalmidae) from caatinga, northeastern brazil bruno halluan s. oliveira¹, adonias a. martins teixeira¹,*, romilda narciza m. queiroz¹, joão a. araujo-filho¹, diêgo a. teles¹, samuel v. brito2, daniel o. mesquita¹ where is my place? quick chorus structure assembly in the european tree frog michal berec a possible mutualistic interaction between vertebrates: frogs use water buffaloes as a foraging place piotr zduniak1,*, kiraz erciyas-yavuz2, piotr tryjanowski3 good vibrations: a novel method for sexing turtles donald t. mcknight1,2,*, hunter j. howell3, ethan c. hollender1, and day b. ligon1 errata to mecke, s., hartmann, l., mader, f., kieckbusch, m., kaiser, h. (2016): redescription of cyrtodactylus fumosus (müller, 1895) (reptilia: squamata: gekkonidae), with a revised identification key to the bent-toed geckos of sulawesi. acta herpetologica 11(2): acta herpetologica 13(2): 195-199, 2018 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-22592 population density, sex ratio and body size in a population of salamandra atra atra on the dolomites antonio romano1,2,*, matteo anderle1, alessandro forti1, piergiovanni partel3, paolo pedrini1 1 muse museo delle scienze, sezione di zoologia dei vertebrati, corso del lavoro e della scienza 3, 38122 trento, italy. *corresponding author. e-mail: antonioromano71@gmail.com 2 consiglio nazionale delle ricerche, istituto per i sistemi agricoli e forestali del mediterraneo, via patacca, 84, 80056 ercolano (na), italy 3 parco naturale “paneveggio pale di san martino”, villa welsperg, località castelpietra 2, 38054 tonadico (tn), italy submitted on: 2018, 31th january; revised on: 2018, 4th may; accepted on: 2018, 25th september editor: dario ottonello abstract. salamandra atra atra is the most widespread subspecies of the alpine salamander, both in italy and in the other parts of the species distribution range. however, in particular for italian populations, its ecology and demographic parameters are poorly known. we studied biometry (length, mass, body condition index) and demography (population density, sex ratio, proportion of gravid females) of this fully terrestrial salamander in the “paneveggiopale di san martino” natural park in the dolomites. we used removal methods to estimate abundance on a surface of about 1000 m2. density estimate of adults was 472 salamanders/ha, which falls within the density estimates that are available for this taxon. sexes did not differ significantly in size and body mass. body sizes of adults included the maximum sized salamander recorded in italy. there was a high rate of gravid females (50%), which were comparable in size with non-gravid females. males and non-gravid females did not show significant differences in their body condition index. keywords. amphibian, body condition, population density, salamanders. salamandra atra laurenti, 1768 is a terrestrial and viviparous salamander. the species occurs in the central and eastern alps and in the dinaric alps where some isolated populations occur (sillero et al., 2014). four subspecies are generally recognised: the nominal s. a. atra, s. a. prenjensis and the italian endemics s. a. aurorae and s. a. pasubiensis. italian populations of the nominate subspecies are generally found from 1200 to 2200 m a.s.l.; in the dolomites (italian part of the southern limestone alps), the species occurs from 650 m up to 2500 m (bonato et al., 2007). the habitat consists of mixed coniferous and deciduous forests, alpine meadows and rocky tundra-like, mainly on limestone substrates. although s. a. atra is the most widespread subspecies (also in italy), ecological, morphometric and demographic information are scarce and, for italian populations, almost completely absent (see table 3 in bonato et al., 2007). indeed, exhaustive information is available only for a population from the easternmost part of the italian alps (luiselli et al., 2001). here we reported the results of a study on a population of s. a. atra on the dolomites. we collected and analysed data on density, body size, weight, sex ratio and proportion of gravid females and we compared our results with those available in literature. the study area is located in the “paneveggio-pale di san martino” natural park (trentino alto adige region), at about 1850 m a.s.l., near the locality malga venegiotta (tonadico; 46°18’48”n, 11°48’53”e). the study area consists of coniferous woodland dominated by the european larch (larix decidua) and norway spruce (picea abies), 196 antonio romano et alii alternated to open habitats (pastures, other grasslands and rocky areas). salamanders were searched by eye during their surface activity on the ground by two researchers, during both day and night hours on 1st september 2017 at temperatures of 3-18 °c, during optimal weather condition (slightly rainy or just after rain). two sites were sampled. the first (aa) was a polygonal area of 890 m2, where salamanders were searched for one hour during three sampling sessions (first session: 6:00-7:00; second: 12:00-13:00; third session: 22:30-23:30). the site aa is an open habitat on a stony slope with south-western orientation, dominated by erica carnea and herbaceous vegetation, on the boundary of a small stream, near a trekking path. the second site (bb) was a portion (about 400 m long, 2-3 m width) of this trekking path, which have to be travelled to reach the area aa. in the site bb salamanders were sampled only once (21:30-22:30). population size was estimated in the site aa, using the software capture (otis et al., 1978; white et al., 1982) with a temporary removal experiment (white et al., 1982), with the three removal samples within 18 hours. the removal method theory assumes that population is closed and that the sampling effort is constant among the sampling sessions (white et al., 1982). we met these assumptions by sampling salamanders over a very short time period (18h) for a standardised time (1 hour). we used the generalized removal model mbh that allows for variation in capture probabilities (b allows for capture probabilities to vary by behavioural response or “capture history”; h is the individual heterogeneity effects and allows for capture probabilities to differ between individuals). salamanders collected in the first and second removal sample were stored at 4 °c in a refrigerator in individual plastic tubes for a maximum of 20 hours near the study area (about 6 km). for the evaluation of the sex ratio, proportion of gravid females, body mass and size, we pooled the salamanders sampled in the site aa and those in the site bb. salamanders were considered as “adults” when they were longer than 90 mm in total length (klewen, 1986; luiselli et al., 2001). salamander sex was assessed by direct observation of the cloacal region, which is swollen in the adult males and flat in females (klewen, 1988). females were considered “gravid females” when their pregnancy was obvious, that is the posterior part of their trunk was distinctly swollen laterally because they were at a late stage of gestation (klewen, 1988; bonato et al., 2017) and when, for females whose trunk was swollen but not so evident, our gentle palpation of their belly confirmed their pregnancy (andreone et al., 1996; 1999; luiselli et al., 2001). the “non-gravid females” included presumably both actually non-gravid ones and females with embryos in early stages of development. throughout the text, “females” include both gravid and non-gravid females. in accordance with wilson and hardy (2002), sex ratio was expressed as the proportion of males [males/ (males+females)] and deviations from evenness were assessed by two-tailed binomial tests. salamanders were photographed perpendicularly to the dorsal surface with a digital camera, near a calliper to set the scale. digital photographs of salamanders were imported into the imagej® software program (version 1.44; national institute of health, available from http://rsb.info.nih.gov/ ij/) to measure their total length, after scale setting of each picture (totl: distance from the tip of the snout to the tip of the tail; precision 0.1 mm) while body mass was measured using a digital scale (precision 0.01 g). to quantify the magnitude of sexual size dimorphism, we used the sexual dimorphism index, sdi = [(mean totl of the larger sex/mean totl of the smaller sex) − 1], arbitrarily defined as positive when females are larger and negative when males are larger (lovich and gibbons, 1992). in addition, a maximum size dimorphism index (sdimax) based on the mean totl of the largest 20% of the sample of each sex was also calculated (du toit et al, 2003; romano et al., 2009). totl and mass were combined to calculate the body condition index as scaled mass index (smi) (peig and green, 2009). the smi was calculated only for males and non-gravid females, while the proportion of gravid females on the total number of females may be used as a proxy of the fertility parameter (luiselli et al., 2001). since our biometric data did not match parametric assumptions, body size, body mass and smi of males, females and gravid females were compared using the non parametric mann-whitney-u test and kruskal-wallis anova. all salamanders captured were adults (totl > 90 mm, see klewen, 1986; tab. 1). in site aa we captured 24 individuals (6, 9 and 9 individuals in the first, second and third removal session respectively). the estimated abundance of salamanders was 42 ± 7 (estimate ± s.e.; 95% c.i. = 33-62). therefore salamander’s density, computed as the estimated abundance in 890 m2 (site aa) extrapolated to the hectare, resulted 472 individuals/ha (95% c.i = 310-633, calculated following the delta method as reported in cooch and white, 2018). in site bb, during a single sampling session (slightly rain, night), were found 27 salamanders. on the whole, pooling salamanders from sites aa and bb, we measured, weighed and sexed 51 salamanders (31 males, 20 females). our results showed a sex ratio of 0.61 which did no differ significantly from 0.5 (two tailed binomial test, p = 0.119). body size and body mass of males, non-gravid females and gravid females 197density, population structure and body size of salamandra atra are shown in tab. 1. although males are generally longer than females, sexes did not differed significantly in their mean size (mann-whitney u test: n = 51; u = 222; p = 0.09) and body mass (comparison between males and non-gravid females: n = 41; u = 129.5; p = 0.44). gravid and non-gravid females had comparable sizes (n = 20; u = 42; p = 0.58). kruskal-wallis anova, followed by mann-whitney pairwise comparisons with bonferroni correction for multiple comparisons, showed that gravid females differed significantly in body mass both from males and non-gravid females (kruskal-wallis; h = 6.69; p = 0.03; p < 0.05 in both statistically significant comparisons). the sample of salamanders we studied showed a slightly negative sdi = -0.05. conversely, the sdimax showed a positive value (+0.11), because although males are generally larger than females the largest individuals in our sample were females. males and non-gravid females did not show significant differences in their body condition index (smi) (mann-whitney u test; n = 41; u = 124, p = 0.36). information on ecology, morphometry and demographic parameters of italian populations of alpine salamanders (s. a. atra) are scarce (cf. bonato et al., 2007). most of the results we obtained corroborate available data from other populations of alpine salamanders. population density of s. a. atra seems to be highly variable among different sites, spanning from more than 3000 individuals/ha (helfer et al., 2012) to 120 ind./ha (klewen, 1986), with intermediate values (1383 ind./ha: helfer et al., 2012; 2380 ind./ha: klewen, 1986, 1988). density of the subspecies s. a. aurorae ranged from 475 to 40 ind./ ha (bonato and fracasso, 2003; romano et al., 2018a). spatial distribution of salamandra atra may be significantly aggregated also within small areas (about 1300 m2; bonato and fracasso, 2003). consequently, extrapolation of population density from very small areas to hectares should be regarded with caution because could greatly underor over-estimate the actual densities at larger scale. the highest density of s. atra (3056 ind./ha, helfer et al., 2012) was extrapolated on an hectare from a small study area (625 m2) while lower values were extrapolated by larger areas (3000 m2) and, in our opinion, they are more reliable and are closer to the actual density of this salamander. in our population the observed density of about 470 ind./ha was comparable to those in areas of maximum density of the subspecies s. a. aurorae in the venetian prealps (bonato and fracasso, 2003). in our three removal sessions we did not observe decline in number of captures which may indicates a poor capture success (e.g., rodda, 2012). in the third sampling session (slightly rainy night) we found 9 salamanders in the site aa and 27 in the site bb where no animals were detected in the first two sessions. this corroborates available information about the alpine salamander detectability which may vary with site characteristics (like deep cracks within rocks) and with survey conditions (weather) (e.g., bonato et al., 2007). body size data of the study population generally are in agreement with those available in the literature. maximum size (totl) and mass recorded in italian populations of s. a. atra were 140 mm and 11.8 g, 143 mm and 14.2 g for males and females respectively (luiselli et al., 2001; bonato et al., 2007). for the whole subspecies the maximum records are still 144 mm and 12 g, 151 mm and 15 g for males and females, respectively (bonato et al., 2007) and our data are congruent (see table 1). however, two males in our study population (140.3 mm and 143.4 mm, tab. 1) exceeded the maximum size recorded in other italian populations. as a rule, there is no significant sexual size dimorphism in alpine salamanders (bonato et al., 2007), as it was the case for our study population. the sex ratio and the proportion of gravid females (at least 50% of the females were gravid) corroborated the information reported in previous studies form other populations (luiselli et al., 2001; bonato et al., 2007). here we assumed a negligible variation in detectability between males and females. however, available information on different survey methods on s. a. aurorae showed that they provide different level of efficiency in detecting males and females (lefosse et al., 2016). the method we used (searching active salamanders on the ground during slightly rain) could provide a similar efficiency in detecttable 1. body size and body mass of salamandra atra atra from the study site in the “paneveggio-pale di san martino” natural park (trentino alto adige region, northern italy). “females” are the gravid and non-gravid females pooled together. n total length (mm) body mass (g) mean ± s.d range mean ± s.d range males 31 122.6 + 12.8 94.4-143.4 8.1 + 1.9 4.2-12.0 females 20 116.3 + 13.1 95.7-145.4 8.6 + 2.3 4.2-12.4 non-gravid females 10 114.5 + 15.2 95.7-145.4 7.5 + 2.3 4.2-11.8 gravid females 10 118.0 + 11.2 103.0-139.6 9.7 + 1.7 6.4-12.4 198 antonio romano et alii ing both sexes (lefosse et al., 2016). however s. a. aurorae shows some ecological and behavioural characteristics that are different from those of the nominate subspecies (bonato et al., 2007; lefosse et al., 2016). as general rule, in salamanders males show higher capture rates than females (lefosse et al., 2016; romano et al., 2018b) but although the sex ratio obtained by counts may overestimate one sex (generally the males) it can be congruent with the actual demographic trait of the population (romano et al., 2018b). further studies based on cmr are desirable to understand if the observed sex ratio reflects an actual ecological trait of the population or is the outcome of different capture probabilities. in our population, gravid and non-gravid adult females had comparable sizes (table 1). this finding is not in agreement with the data of other populations where gravid females were significantly larger (cf. luiselli et al., 2001) but this discrepancy could be due to our small sample size (20 females). body condition is a parameter in many ecological and monitoring studies (e.g., welsh et al., 2008; see also tab.1 in milenkaya et al., 2013). the use of condition indices in conservation needs to control for the possible confounding effects of sex, because body condition may differ between males and females (see milenkaya et al., 2013). if it differs between sexes, the individuals cannot be pooled for spatial (different populations) or temporal (the same population in different times) comparisons because results may be affected by the sex ratio. here we showed that in s. atra the sexes (males and non-gravid females) did not differ in body condition index and, therefore, they may be pooled in population studies that take into account this parameter. this contribution provides additional information on little studied aspects in the alpine salamanders in italy, such as population density, body sizes, proportion of gravid females and body condition index. furthermore we confirms that this area is suitable to more in-depth researches, which are in planning to study distribution, abundance and ecology of s. a. atra in the “paneveggiopale di san martino” natural park. acknowledgements the study was authorised by the italian ministry of environment (authorisation pnm-eu-2017-005370) and supported by the “paneveggio-pale di san martino” natural park. thanks to andrea costa for his help in the final draft. we also thank two anonymous reviewers, whose comments and suggestions greatly improved a previous version of the manuscript. references andreone, f., clima, v., de michelis, s. 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(2002): statistical analysis of sex ratios: an introduction. in: sex ratios – concepts and research methods, pp. 48-92. hardy, i.c.w., ed., cambridge university press, cambridge. acta herpetologica vol. 13, n. 1 june 2018 firenze university press acta herpetologica 12(2): 133-137, 2017 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-20572 brown anole (anolis sagrei) adhesive forces remain unaffected by partial claw clipping austin m. garner*, stephanie m. lopez, peter h. niewiarowski department of biology, program in integrated bioscience, university of akron, akron, oh 44325-3908, usa. *corresponding author. e-mail: amg149@zips.uakron.edu submitted on: 2017, 19th april; revised on: 2017, 28thjuly; accepted on: 2017, 31th july editor: fabio m. guarino abstract. morphological properties of animal locomotor systems should reflect costs and benefits associated with particular environments. lizards that possess both claws and adhesive toe pads are specialized for environments that require movements in both the horizontal and vertical planes, and on both rough and smooth substrates. although toe/claw clipping is a common technique for marking free-ranging lizards, this technique is disadvantageous to those lizards possessing adhesive toe pads. a previous study removed entire claws of anolis carolinensis and observed a significant reduction in adhesive abilities, which was likely attributed to damage of underlying tendons that play critical roles in engaging the adhesive pads. here, we report on the clinging ability of brown anoles (anolis sagrei) with partial claw removal. we found that adhesive capacities were not affected by partial removal of the claw, suggesting that partial claw removal prevents damage to the underlying tendons and that it may be a safer alternative for short-term marking of adhesive pad-bearing lizards. keywords. adhesion, anolis, claw clipping, clinging force, toe clipping. introduction substrate characteristics of the natural environments of most lizards are usually heterogeneous and complex. presumably, particular morphological properties of the locomotor systems of lizards reflect evolutionary responses to the costs and benefits associated with specializations for particular substrates. for example, several clades of lizards have evolved elaborate adhesive toe pads (while at the same time retaining claws) allowing them to cling to smooth substrates (ruibal and ernst, 1965; williams and peterson, 1982; peterson, 1983). claws perform well on rough or irregularly patterned substrates, whereas subdigital adhesive pads excel on smooth substrates (irschick et al., 1996; zani, 2000). lizards that encounter both types of substrates may benefit by having both claws and toepads, as do species in the genus anolis, allowing them to travel effectively on both smooth and rough surfaces (irschick et al., 1996; zani, 2000; crandell et al., 2014). toe or claw clipping is a widely utilized technique for marking small reptiles and amphibians (dunham et al., 1988). although this may be a convenient marking technique for terrestrial species, few studies have investigated how toe or claw clipping affects adhesive toe pad function and whole organism performance in arboreal adhesive pad-bearing lizards. bloch and irschick (2005) examined the effects of complete claw clipping on clinging abilities of the adhesive pad-bearing lizard, anolis carolinensis. after clipping toes at the distalmost portion of the adhesive toepad (effectively removing the entire claw; fig. 1a), they found that claw removal significantly decreased clinging abilities. several explanations were suggested including a change in the way the animal perceives its environment (a tactile response), and a morphological 134 austin m. garner et alii effect on the function of the toepad after the claw was removed (i.e., tendon damage; bloch and irschick, 2005). although these mechanisms are not mutually exclusive, we wanted to test the relative importance of the possibility that clinging performance would suffer due to tendon damage as a result of claw clipping. while bloch and irschick (2005) did not directly state a mechanism of tendon damage, anoles (like most tetrapods) control flexion of the digits via flexor tendons attached to the musculus flexor digitorum longus. these flexor tendons span across the entire toe, including the distal phalanges (abdala et al., 2009). when bloch and irschick (2005) removed the distalmost portion of the toe, they likely severed flexor tendons as well. given this, it is possible that the destruction of those tendons may have resulted in decreased adhesion because the anoles were not able to properly engage their adhesive system. in our study, we used partial claw clipping to leave those tendons intact and measured maximum clinging ability on smooth substrates. partial claw clipping reduces claw length and changes claw shape, but also decreases the interference between the adhesive pad and the substrate (fig. 1b). we hypothesized that partial claw clipping would not result in significantly reduced clinging abilities in anolis sagrei on smooth substrates, likely because the less destructive marking technique would leave the underlying tendons preserved. the results of this study will not only increase our understanding of how claw clipping affects whole animal adhesive performance, but may also influence marking techniques utilized by researchers studying wild populations of adhesive pad-bearing lizards that also have claws. materials and methods animals a total of 19 captured brown anoles (anolis sagrei) were used for experiments, including both adult males and females (table 1). all lizards used for this study were collected at the key west botanical gardens (key west, fl) between 11 may 2008 and 16 may 2008, and then transported back to the mote marine laboratory on summerland key, fl for clinging experiments. anoles were released back to marked capture points in the gardens within 24 hours of capture. experimental procedures clinging experiments were completed using a custom-designed, motorized rig that measured adhesive force (niewiarowski et al., 2008). the first set of trials were conducted on a sheet of glass covered with an acetate sheet (bloch and irschick, 2005). the two front feet of each anole were pulled three times in rapid succession along the substrate at a uniform speed of approximately 5 cm/s. for consistency, force measurements were done by a single individual. after the three pulls, the sheet of acetate was removed and the same anole was pulled along the sheet of glass three more times. pulls on glass were used to test the possibility that claws (intact or clipped) contributed to clinging force by ‘digging’ into the acetate sheet (bloch and irschick, 2005). maximum clinging force was recorded for each set of pulls and was defined as the force recorded at the point when both front feet of the anole were visibly slipping on the substrate. subsequent to pulls with intact claws, claws on both front feet were partially clipped using a small pair of nail scissors. unlike bloch and irschick (2005), claws were only clipped halfway to avoid potential damage to the underlying tendons (fig. 1b). after clipping, clinging ability was tested identically to tests withint act claws. smooth substrates were chosen to isolate the effect of partial claw clipping on the adhesive capability of anolis sagrei. rough or irregular substrates generally increase clinging ability of lizards with claws because claws can generate friction or mechanically interlock with surface asperities, thus possibly resulting in a bias towards higher clinging ability with claws left intact (zani, 2000; crandell et al., 2014). the order in which anoles were tested was randomized. fig. 1. diagram of an anole toe indicating the differences in claw clipping locations between bloch and irschick (2005) and the present study. the clipping location is represented by the solid line and the removed portion of the claw is represented by the dashed line. bloch and irschick (2005) removed the entire claw at the distalmost portion of the adhesive toe pad (a), possibly compromising the underlying tendons that facilitate adhesive toe pad engagement. we partially removed the claw (b) in an effort to preserve the underlying tendons. table 1. sample size, mass, and mean clinging forces of brown anoles (anolis sagrei) used in experimental trials. all values reported as mean ± se. n mass (g) intact (n) clipped (n) acetate (n) glass (n) 19 4.55 ± 0.08 0.82 ± 0.12 0.60 ± 0.08 0.59 ± 0.09 0.84 ± 0.11 135effect of claw clipping on brown anole adhesion statistical analysis we used a mixed model analysis of variance (anova) to compare maximum clinging ability as a function of substrate, claw removal, and their interaction. maximum clinging ability was the dependent variable. substrate type (glass or acrylic), claw state (intact or clipped), and their interaction were the independent variables. individual anole was modeled as a random effect. before statistical analysis, maximum clinging ability data were log-transformed in order to meet the assumptions of an anova. all statistical analyses were completed using jmp pro 12 (sas institute, inc., cary, nc, usa). results the mixed model anova using an assumption of compound symmetry (littell et al., 2000) fit the data nearly as well as the model relaxing the assumption of compound symmetry and leads to qualitatively similar conclusions, but we report the latter as it is more conservative. the effect of claw removal on maximum clinging ability of anolis sagrei revealed that neither claw state (f1,18 = 0.683, p = 0.419) nor the substrate by claw state interaction (f1,18 = 0.725, p = 0.406) had a significant effect on maximum clinging ability (fig. 2 and table 2). however, maximum clinging ability was significantly affected by substrate type, with glass producing higher maximum clinging forces than acetate (f1,18 = 4.435, p = 0.049; fig. 3 and table 2). discussion bloch and irschick (2005) reported a significant decrease in clinging forces when the claws of anolis carolinensis were completely removed. because several mechanisms were proposed to explain this effect, we examined the possibility that tendon damage may be responsible for the reduction in adhesion. to investigate this, we partially removed claws of anolis sagrei and compared their clinging abilities to anoles with fully intact claws. our results demonstrated that partial claw clipping had no significant effect on maximum clinging force, supporting the hypothesis that entire claw removal may actually damage the underlying tendons (bloch and irschick, 2005). the main difference between our study and bloch and irschick (2005) is that claws were only partially clipped, not removed, thus the underlying tendons were left intact. all claws on both front feet were clipped in our work, as opposed to removfig. 2. mean maximum clinging forces of anolis sagrei on acetate and glass substrates with fully intact or partially clipped claws. a mixed model anova indicates no significant difference in the mean maximum clinging forces of a. sagrei as a function of claw state (f1,18 = 0.683, p = 0.419) or the substrate by claw state interaction (f1,18 = 0.725, p = 0.406). error bars represent ± 1 se. table 2. results of the mixed model analysis of variance (anova). the table displays a significant difference in maximum clinging ability as a function of substrate (*p < 0.05). there is no significant difference in maximum clinging ability as a function of claw state or the interaction between substrate and claw state. effect numdf dendf f p substrate 1 18 4.435 0.049* claw state 1 18 0.683 0.419 substrate * claw state 1 18 0.725 0.406 fig. 3. mean maximum clinging forces of anolis sagrei on acetate and glass substrates. a mixed model anova found that the mean maximum clinging force of a. sagrei was significantly higher on glass compared to acetate (f1,18 = 4.435, p = 0.049). error bars represent ± 1 se. 136 austin m. garner et alii ing 2-4 claws on the front feet. yet, our recorded clinging forces are similar to those observed by bloch and irschick (2005). importantly, the difference in clinging forces between intact and partially clipped claws (~ 0.2 n or ~ 20 g) is about five times smaller in our study than what bloch and irschick (2005) found (~ 1 n or ~ 100 g). although our samples sizes are smaller than bloch and irschick (2005), we used a mixed model anova to account for the non-independence of observations attributable to individual lizards, and the small differences between treatments is likely to be biologically insignificant as well. do claws interfere with toe pads? in lizards that possess both claws and subdigital adhesive pads, it has been demonstrated that increases in claw curvature, length, and height are correlated with increases in toe pad adhesive force on smooth substrates (zani, 2000; crandell et al., 2014). while morphological variation in claws appears to influence adhesion, the mere presence of claws should result in reduced adhesive capabilities. clinging forces generated by adhesive toe pads are directly related to the numbers of individual setae that can make appropriate contact with the substrate (contact fraction) (autumn et al., 2000; autumn, 2006). contact fraction depends on many factors, including pre-loads applied by the lizard, as well as the degree and nature of roughness of the substrate (russell et al., 2007). on smooth substrates, it is intuitive to hypothesize that claws may result in decreased adhesive force because the toe pads may be more removed from the substrate resulting in decreased contact fraction. thus, one would expect the partial removal of claws to result in an increase in adhesive force on smooth substrates. surprisingly, our results reveal no significant difference in clinging ability between anoles with fully intact and partially clipped claws, at least on smooth substrates. furthermore, mahendra (1941) observed that the adhesive locomotion of hemidactylus flaviviridis on smooth substrates appeared to be unaffected by claw removal. thus, it may be that morphological or behavioral mechanisms minimize theoretical trade-offs between claws and toepads in their functional roles during adhesive locomotion. lizards that possess both claws and subdigital adhesive pads are likely able to effectively cling to both smooth and rough substrates (irschick et al., 1996; zani, 2000). while it appears that partial claw clipping does not result in a reduction in adhesive performance on smooth substrates, it is possible that claw removal results in lower clinging abilities on rough substrates, although this has yet to be tested empirically. therefore, it is interesting to consider the ecological implications of losing one of the two clinging mechanisms discussed (adhesion via van der waals interactions or clinging via claws). however, considering the heterogeneity of substrates in the natural habitat of anolis sagrei, it is difficult to gauge which mechanism of clinging would be more costly to a. sagrei if it was lost via toe/claw removal. regardless, partial claw clipping is the least invasive of the two marking techniques discussed and does not result in a reduction of adhesive performance. glass versus acetate we recorded a significantly higher clinging force on glass than on acetate when claws were left intact. two aspects of this result are relevant. first, it suggests that claws do not enhance adhesion by ‘digging’ into the acetate sheets (bloch and irschick, 2005). second, and consistent with theory underlying the mechanics of van der waals forces, substrates with higher surface energies should lead to higher clinging forces (glass is about 100 mj/m2 and acetate is about 30-40 mj/m2) (israelachvili, 1992). a potential confounding factor for future adhesion studies could be which of these substrates is more appropriate to use and which is most similar to a smooth substrate encountered by adhesive pad-bearing lizards in their natural environments. marking techniques a non-destructive, permanent marking technique would be ideal for working with reptiles, yet it is very challenging to achieve, particularly with animals that rely on adhesive functions during locomotion (paulissen and meyer, 2000). bloch and irschick (2005) addressed this problem with complete claw clipping as a permanent, relatively reliable solution, but with an undesired effect: a reduction in adhesive performance. when comparing permanence of a marking technique, complete claw clipping/removal is more effective than partial claw clipping. however, our work here suggests that partial claw clipping may serve as an excellent alternative during shortterm mark and recapture studies because no significant change in adhesive capabilities was observed. acknowledgements we thank d. pund, t. shultz, j. flinn, and m. sanchez for helping collect, measure, and weigh animals. we also thank key west botanical gardens for allowing us to utilize their grounds for animal capture and to mote 137effect of claw clipping on brown anole adhesion marine laboratories for allowing us to use their summerland key facilities to conduct our research. all experimental procedures were in accordance with the ethical guidelines published by the american society of ichthyologists and herpetologists (beaupre, 2004). all protocols were in accordance with the regulations of the university of akron iacuc, protocol #07-4g. references abdala, v., manzano, a.s., tulli, m.j., herrel, a. (2009): the tendinous patterns in the palmar surface of the lizard manus: functional consequences for grasping ability. anat. rec. 292: 842-853. autumn, k. (2006): how gecko toes stick. am. sci. 94: 124-132. autumn, k., liang, y.a., hsieh, s.t., zesch, w., chan, w.p., kenny, t.w., fearing, r., full, r.j. (2000): adhesive force of a single gecko foot-hair. nature 405: 681-685. beaupre, s.j., ed. (2004): guidelines for use of live amphibians and reptiles in field and laboratory research, 2nd edition. american society for icthyologists and herpetologists, usa. bloch, n., irschick, d.j. (2005): toe-clipping dramatically reduces clinging performance in a pad-bearing lizard (anolis carolinensis). j. herpetol. 39: 288-293. crandell, k.e., herrel, a., sasa, m., losos, j.b., autumn, k. (2014): stick or grip? co-evolution of adhesive toepads and claws in anolis lizards. zoology 117: 363369. dunham, a.e., morin, p.j., wilbur, h.m. 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(2000): the effect of toeclipping on the gecko hemidactylus turcicus. j. herpetol. 34: 282-285. peterson, j.a. (1983): the evolution of the subdigital pad in anolis. i. comparisons among the anoline genera. in: advances in herpetology and evolutionary biology: essays in honor of ernest e. williams, pp. 245283. rhodin, g.j., miyata, k., eds, museum of comparative zoology, harvard univ., cambridge, ma. ruibal, r., ernst, v. (1965): the structure of the digital setae of lizards. j. morphol. 117: 271-293. russell, a.p., johnson, m.k., delannoy, s.m. (2007): insights from studies of gecko-inspired adhesion and their impact on our understanding of the evolution of the gekkotan adhesive system. j. adhes. sci. technol. 21: 1119-1143. williams, e., peterson, j. (1982): convergent and alternative designs in the digital adhesive pads of scincid lizards. science 215: 1509-1511. zani, p. (2000): the comparative evolution of lizard claw and toe morphology and clinging performance. j. evol. biol. 13: 316-325. acta herpetologica vol. 12, n. 2 december 2017 firenze university press meristic and morphometric characters of leptopelis natalensis tadpoles (amphibia: anura: arthroleptidae) from entumeni forest reveal variation and inconsistencies with previous descriptions susan schweiger1, james harvey2, theresa s. otremba1, janina weber1, hendrik müller1,* brown anole (anolis sagrei) adhesive forces remain unaffected by partial claw clipping austin m. garner*, stephanie m. lopez, peter h. niewiarowski species and sex comparisons of karyotype and genome size in two kurixalus tree frogs (anura, rhacophoridae) shun-ping chang1,2, gwo-chin ma2,3,4, ming chen2,5,6,7,8,*, sheng-hai wu1,* non-native turtles in a peri-urban park in northern milan (lombardy, italy): species diversity and population structure claudio foglini1, roberta salvi2,* species composition and richness of anurans in cerrado urban forests from central brazil cláudia márcia marily ferreira1,*, augusto cesar de aquino ribas2, franco leandro de souza3 the life-history traits in a breeding population of darevskia valentini from turkey muammer kurnaz, alı i̇hsan eroğlu, ufuk bülbül*, halıme koç, bılal kutrup influence of desiccation threat on the metamorphic traits of the asian common toad, duttaphrynus melanostictus (anura) santosh mogali*, srinivas saidapur, bhagyashri shanbhag predation of common wall lizards: experiences from a study using scentless plasticine lizards jenő j. purger*, zsófia lanszki, dávid szép, renáta bocz reproductive timing and fecundity in the neotropical lizard enyalius perditus (squamata: leiosauridae) serena najara migliore1,2,*, henrique bartolomeu braz2,3, andré felipe barreto-lima4, selma maria almeida-santos1,2 observations on the intraspecific variation in tadpole morphology in natural ponds eudald pujol-buxó1,2,*, albert montori1, roser campeny3 and gustavo a. llorente1,2 reliable proxies for glandular secretion production in lacertid lizards simon baeckens diet of juveniles of the venomous frog aparasphenodon brunoi (amphibia: hylidae) in southeastern brazil rogério l. teixeira1, ricardo lourenço-de-moraes2, débora c. medeiros3, charles duca3, rogério c. britto4, luiz c. p. bissoli5, rodrigo b. ferreira3,* who are you? the genetic identity of some insular populations of hierophis viridiflavus s.l. from the tyrrhenian sea ignazio avella, riccardo castiglia, gabriele senczuk* acta herpetologica 12(1): 89-93, 2017 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-17902 evidence for directional testes asymmetry in hyla gongshanensis jindongensis qing gui wu1, wen bo liao2,* 1 ecological security and protection key laboratory of sichuan province, mianyang normal university, mianyang 621000, china 2 key laboratory of southwest china wildlife resources conservation (ministry of education), china west normal university, nanchong 637009, sichuan, china. *corresponding author. e-mail: liaobo_0_0@126.com submitted on 2016, 30st january; revised on 2016, 30st november; accepted on 2016, 6th december editor: giovanni scillitani abstract. the compensation hypothesis predicts that one testis may grow more for compensating for a reduced function in the other testis, thus exhibiting a directional asymmetry in testis size. in this study, we tested the prediction of the compensation hypothesis in the chinese endemic tree frog hyla gongshanensis jingdongensis in a population in kegong reserve site of yunan province in western china. for fifty-three male samplings, we found that the left testis size was significantly bigger than the right testis, which exhibited a significantly directional testis asymmetry, consistent with the evidence that mainly the left testis is functional with the right testis having a compensatory role, i.e. the left testis would increase in size if the right testis became non-functional. however, the relative testes size and the degree of testes asymmetry were not correlated with body condition in this species, suggesting that the testes asymmetry can not reflect male quality: high-quality individuals would not have more asymmetric testes. keywords. jingdong tree frog, hylidae, body condition, compensatory function, directional testes asymmetry. the compensation hypothesis predicts that one testis may grow more for compensating for a reduced function in the other testis (møller, 1994). as a result, testis size exhibits a directional asymmetry where the testis on one side, often the left, is bigger than the other side, the right one in bird because the right testis increases in size to compensate for any reduction in function of the left (møller, 1994; birkhead et al., 1997). hence, the directional testes asymmetry (dta) is assumed where the left testis size increases if the right testis serves a compensatory role in frogs due to developmental stress, such as growth season length (hettyey et al., 2005). in recent years, the testis asymmetry has been confirmed in some anurans species (hettyey et al., 2005; zhou et al., 2011; liu et al., 2011; mi et al., 2012). testis size asymmetry is widely used as a measure of male body condition (møller, 1994; hettyey et al., 2005). as a result, the degree of directional asymmetry in testes size reflects male quality: high-quality individuals have more asymmetric testes. because possibilities for energy acquisition were correlated with male quality, the energy acquisition affected the degree of directional testes asymmetry (møller, 1994). for instance, male individual with good condition develops higher degree of directional testes asymmetry than that with poor condition in the common frog, rana temporaria (hettyey et al., 2005). by contrast, the degree of testes size asymmetry is not correlated with body condition in the guenther’s frog, hylarana guentheri (liu et al., 2011). our primary aim in this study was to test the compensation hypothesis in a frog. a prerequisite for testing the hypothesis is good evidence that a particular highquality male is attractive to females (jin et al., 2016a). we studied the jingdong tree frog (hyla gongshanensis jingdongensis), a species endemic to sichuan and yunnan province in china. the species is restricted to a nar90 q. gui wu, w. bo liao row range of altitudes, ranging from 1500 to 2470 m (fei and ye, 2001). males have well-developed vocal sacs and attract their mates by their calls during the breeding period (fei and ye, 2001). besides, little information on testes size asymmetry in h. g. jingdongensis is available. here, we tested the prediction of the compensation hypothesis that the difference in size of the left and right testis arose in h. g. jingdongensis. specially, we examined whether the directional testes asymmetry is occurred in all samplings, and whether there is a positive correlation between body condition and the degree of the directional testes asymmetry. the population is located at kegong reserve site in bama snow mountain nature reserve of yuanan province, western china (27°33'n, 99°19'e; a.s.l. 2422 m). the reserve is characterized by a subtropical climate with a strong seasonality due to the high altitude which has an annual average temperature of 14-18°c (liao et al., 2015; liao et al., 2016a). all individuals were caught by hand at night from march to may in 2011. we collected a total of 53 males and brought them to the laboratory. until processing, males were kept individually in a rectangular tank (1 × 0.5 × 0 .4 m; l × w × h) with a water depth of 25 cm at room temperature. two day after collection, body size (snout–vent length, svl) of each frog was measured to the nearest 0.1 mm using a caliper and body mass was weighted to the nearest 0.1 g using an electronic balance. we used the single-pithing to sacrifice all animals and then anatomized them (liao et al., 2016b; mai et al., 2017; lüpold et al., 2017). left and right testes were removed, and weighed to the nearest 0.1 mg using an electronic balance. following the suggestion of møller and swaddle (1997), we calculated the degree of the directional testis size asymmetry as dta = (left-testis mass – right-testis mass)/0.5(left-testis mass + right-testis mass). relative testes size was calculated as the ratio of observed testes mass to that predicted by the allometric regression equation between testes mass and body size. body condition was measured as the ratio of observed body mass to that predicted by linear regression by entering male body mass as a dependent variable and body size as an independent variable. body size, body mass and testes mass were log10transformed to achieve normality. we compared the sizes of the left and right testes using a paired t-test. we performed line regressions to test the relationships between both body mass and body size and testes mass. we also analyzed the relationships between male body condition and both relative testis mass and the degree of the testis asymmetry using pearson correlation analysis. all analyses were conducted using spss 21.0 (statistical product and service solutions company, chicago, usa). testis mass was positively correlated with body mass (fig. 1a; f1, 52 = 28.679, r2 = 0.360, p < 0.001, log (testis mass (mg)) = 1.283 log (body mass (g)) +0.451). an increase in testes mass with body mass was larger than predicted by the power law (β > 1), providing an evidence for an allometric relationship. testis mass was also positively correlated with svl (fig. 1b; f1, 52 = 9.414, r2 = 0.156, p = 0.003, log (testis mass (mg)) = 2.824 log (svl (mm)) -3.470). the relative testes size was not correlated with body condition (fig. 2; pearson’s correlation coefficient: r = -0.082, n = 53, p = 0.558). the jingdong tree frog exhibited a directional testes asymmetry: the left testis (4.5 mg ± sd 2.7) was significantly bigger than the right one (3.5 mg ± sd 1.9) in 71.69% of all individuals (paired t-test: t = 3.67, df = 53, p = 0.001). left testis mass was positively correlated with right testis mass (r = 0.723, n = 53, p < 0.001). we found a non-significant correlation between the degree of the directional testes asymmetry and body condition (fig. 3; pearson’s correlation coefficient: r = -0.144, n = 53, p = 0.304). fig. 1. relationships between (a) body mass and testes mass, (b) svl and testes mass. displayed values are log-transformed data. 91testes asymmetry in a tree frog our results uncovered positive correlations between testis mass and both body mass and svl in h. g. jingdongensis in a population. furthermore, the left testis was significantly bigger than the right one, which exhibited a directional testes size asymmetry. however, body condition was non-significantly correlated with both the relative testes size and the degree of the testes asymmetry. previous studies have shown that the directional testes asymmetry is common in animal taxa (anurans: hettyey et al., 2005; liao and lu, 2010; zhou et al., 2011; liu et al., 2011; liu et al., 2012; jin et al., 2016b; chen et al., 2016; birds: wright and wright, 1944; møller, 1994; birkhead et al., 1997; mammals: yu, 1998). for h. g. jingdongensis left testis was bigger than right testis in most individuals, demonstrating a directional testes asymmetry. meanwhile, most individuals had at least one testis smaller than the median for the population, which was considered as natural selection for compensation, consistent with the compensation hypothesis (møller, 1994). furthermore, a larger left testis and a smaller right testis might be advantageous because constraints during embryonic development results in the higher efficiency in sperm production of the left testis (kempenaers et al., 2002; liao and lu, 2012). however, if a bigger sperm production on left testicle influences for the decrease on sperm production on right testicle, that otherwise would maintain the same rate of production, this condition does not represent an advantage on testes asymmetry. there is a heritability evidence of male body condition that sexual selection supports females making use of the genetic correlation between body condition and sperm traits in frogs (byrne et al., 2003). as a result, male individuals with good body condition have larger testis size than that with poor body condition to obtain mates. in the study, we found that the relative testis size was not correlated with male body condition, suggesting that males in good condition did not exhibit the higher ability of sperm competition. hettyey and roberts (2007) found that sperm-depletion after mating led to a non-significant correlation between body condition and testis mass. in our study, a non-significant correlation between body condition and relative testes size in h. g. jingdongensis might result from sperm depletion. hence, we showed a consistent hypothesis for the lack of body condition and asymmetrical directional testicles. previous studies have shown that the degree of the testes asymmetry may not be a good measure of body condition in birds (birkhead et al., 1997; birkhead et al., 1998; kimball et al., 1997; kempenaers et al., 2002). by contrast, the house sparrow and the barn swallow exhibit a positive correlation between testes asymmetry and body condition (møller, 1994). for anurans, some species exhibit a positive correlation between body condition and testes asymmetry while other species do not exhibit correlation between them (hettyey et al., 2005; zhou et al., 2011; liu et al., 2011). for instance, the degree of the testes asymmetry is significantly correlated with body mass in r. temporaria (hettyey et al., 2005). however, zhou et al. (2011) found that the degree of the testes asymmetry is negatively correlated with body size in pelophylax nigromaculata. in this study, the degree of the testes asymmetry did not co-vary with male body condition, suggesting that the degree of the directional testes asymmetry may not be a good indicator of male quality. similar results have been observed in two species of anurans (hylarana guentheri, liu et al., 2011; rana omeimontis, liu et al., 2012). although the degree of the testis asymmetry in h. g. jingdongensis did not reflect good body fig. 2. relationship between body condition and relative testes size in a h. g. jingdongensis population. fig. 3. relationship between body condition and the degree of testes asymmetry in a h. g. jingdongensis population. 92 q. gui wu, w. bo liao condition, males developed larger left testes and smaller right testes for supporting the compensation hypothesis. in conclusion, consistent with the prediction of the compensation hypothesis, our analyses of difference in left and right testes in h. g. jingdongensis showed a directional testes asymmetry, suggesting that the left testis exhibited functional and the right testis had a compensatory role. however, body condition was not significantly correlated with the degree of the testes asymmetry, suggesting that directional asymmetry in testes size may not be a good measure of male quality. acknowledgements financial support was provided by the open scientific research foundation of ecological security and protection key laboratory of sichuan province, mianyang normal university (esp1405), the project of mianyang normal university (qd2015b07 and 2015a01). the reported experiments comply with the current laws of china concerning animal experimentation, and permission to collect frogs was received from the ethical committee for animal experiments in china council on animal care (ccac) guidelines. all experiments involving the sacrifice of these live animals were approved by the animal ethics committee at mianyang normal university. references birkhead, t.r., buchanan, k.l., devoogd, t.j., pellatta, e.j, székelyd, t., catchpoleb, c.k. 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(2010): age structure and body size of the chuanxi tree frog hyla annectans chuanxiensis from two different elevations in sichuan (china). zool. anz. 248: 255-263. liao, w.b., lu, x. (2012): adult body size = f (initial size + growth rate age): explaining the proximate cause of bergman’s cline in a toad along altitudinal gradients. evol. ecol. 26: 579-590. liao, w.b., liu, w.c., merilä, j. (2015): andrew meets rensch: sexual size dimorphism and the inverse of rensch’s rule in andrew’s toad (bufo andrewsi). oecologia 177: 389-399. liao, w.b., luo, y., lou, s.l., lu, d., jehle, r. (2016a): geographic variation in life-history traits: growth season affects age structure, egg size and clutch size in andrew’s toad (bufo andrewsi). front. zool. 13: 6. liao, w.b., lou, s.l., zeng, y., kotrschal, a. (2016b): large brains, small guts: the expensive tissue hypothesis supported in anurans. am. nat. 188: 693-700. liu, w.c., huang, y, liao, y.m. 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(1994): directional selection on directional asymmetry: testes size and secondary sexual characters in birds. proc. roy. soc. b 258: 147-151. møller, a.p., swaddle, j.p. (1997): asymmetry, developmental stability, and evolution. oxford: oxford university press. wright, p.l., wright, m.h. (1944): the reproductive cycle of the male red-winged blackbird. condor 46: 46-59. yu, z.h. (1998): asymmetrical testicular weights in mammals, birds, reptiles and amphibians. internat. j. androl. 21: 53-55. zhou, c.q., mao, m., liao, w.b. (2011): testis asymmetry in the dark-spotted frog pelophylax nigromaculata. herpetol. j. 21: 181-185. acta herpetologica vol. 12, n. 1 june 2017 firenze university press morphological variation and sexual dimorphism in liolaemus wiegmannii (duméril & bibron, 1837) (squamata: liolaemidae) from uruguay joaquín villamil1,*, arley camargo2, raúl maneyro1 sex does not affect tail autotomy in lacertid lizards panayiotis pafilis1,*, kostas sagonas2, grigoris kapsalas1, johannes foufopoulos3, efstratios d. valakos4 fire salamander (salamandra salamandra) males’ activity during breeding season: effects of microhabitat features and body size raoul manenti*, andrea conti, roberta pennati call variation and vocalizations of the stealthy litter frog ischnocnema abdita (anura: brachycephalidae) pedro carvalho rocha1,2,*, joão victor a. lacerda2,3, rafael félix de magalhães2,3, clarissa canedo4, bruno v. s. pimenta5, rodrigo carrara heitor6, paulo christiano de anchieta garcia2,3 feeding ecology of two sympatric geckos in an urban area of northeastern brazil josé guilherme g. sousa1, adonias a. martins teixeira2,*, diêgo alves teles2, joão antônio araújo-filho2 and robson waldemar ávila3 a pattern-based tool for long-term, large-sample capture-mark-recapture studies of fire salamanders salamandra species (amphibia: urodela: salamandridae) jeroen speybroeck1,*, koen steenhoudt2,$ skeletal variation within the darwinii group of liolaemus (iguania: liolaemidae): new characters, identification of polymorphisms and new synapomorphies for subclades linda díaz-fernández*, andrés s. quinteros, fernando lobo marking techniques in the marbled newt (triturus marmoratus): pit-tag and tracking device implant protocols hugo le chevalier1, olivier calvez2, albert martínez-silvestre3, damien picard4, sandra guérin4,5, francis isselin-nondedeu6, alexandre ribéron1, audrey trochet1,2,* evidence for directional testes asymmetry in hyla gongshanensis jindongensis qing gui wu1, wen bo liao2,* the advertisement call of pristimantis subsigillatus (anura, craugastoridae) florina stănescu1,4, rafael márquez2, paul székely3,4,*, dan cogălniceanu1,4,5 nematodes infecting anotosaura vanzolinia (squamata: gymnophthalmidae) from caatinga, northeastern brazil bruno halluan s. oliveira¹, adonias a. martins teixeira¹,*, romilda narciza m. queiroz¹, joão a. araujo-filho¹, diêgo a. teles¹, samuel v. brito2, daniel o. mesquita¹ where is my place? quick chorus structure assembly in the european tree frog michal berec a possible mutualistic interaction between vertebrates: frogs use water buffaloes as a foraging place piotr zduniak1,*, kiraz erciyas-yavuz2, piotr tryjanowski3 good vibrations: a novel method for sexing turtles donald t. mcknight1,2,*, hunter j. howell3, ethan c. hollender1, and day b. ligon1 errata to mecke, s., hartmann, l., mader, f., kieckbusch, m., kaiser, h. (2016): redescription of cyrtodactylus fumosus (müller, 1895) (reptilia: squamata: gekkonidae), with a revised identification key to the bent-toed geckos of sulawesi. acta herpetologica 11(2): acta herpetologica 9(2): 219-225, 2014 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-14028 the compensatory effect of tail regeneration on swimming speed in larval hoplobatrachus chinensis osbeck, 1765 (anura: ranidae) after tail removal guo-hua ding, zhi-hua lin*, li wei college of ecology, lishui university, lishui, zhejiang, 323000, china. *corresponding author. e-mail: zhlin1015@126.com submitted on 2014, 31st january; revised on 2014, 9th september; accepted on 2014, 10th september editor: rocco tiberti abstract. we used hoplobatrachus chinensis tadpoles as a model species to evaluate if the locomotor costs of tail loss could be compensated by tail regeneration. different proportion (0%, 20%, 40% and 60%) of tail segment were removed in four experimental groups and the tadpoles were reared for 7 days. swimming speed was measured three times for each experimental tadpole: before tail removal, after tail removal, and 7 days from tail removal. gosner’s stage, body length, tail length and survival rate were measured for each experimental tadpole before tail removal and at 7 days from tail removal. we daily measured the length of the regenerative tail and of the remaining tail in 20% to 60% tail removal treatments. overall, our results suggest that (1) tail removal affects final tail length and swimming speed, but not body length, developmental stage and survival rate in captive h. chinensis tadpoles; (2) tadpoles with more serious tail injuries have faster tail regeneration rate; (3) swimming speed can be compensated with tail regeneration after tail removal. keywords. frog tadpole, tail removal and regeneration, burst speed, survival rate, growth and development. introduction anuran tadpoles escape from aquatic predators by sudden turn and burst speed (wilbur and semlitsch, 1990), in which the tail of tadpole plays a key role. the swimming performances of tadpoles depend on the tail size (van buskirk and mccollum, 2000a; teplitsky et al., 2005; arendt, 2010; ding et al., 2014) and on its shape (mccollum and van buskirk, 1996; van buskirk et al., 1997; doherty et al., 1998; van buskirk and mccollum, 2000b), and tail morphology influence the probability of predation (van buskirk and mccollum, 2000b; van buskirk et al., 2003). usually, the swimming performance of tadpoles can be affected only by serious tail injury (e.g. the swimming performance of hyla chrysoscelis tadpoles was significantly reduced only by more than 50% tail removal; figiel and semlitsch, 1991). tail injuries are quite common in tadpoles from natural populations, due to the predation attempts of fish, salamanders, crayfish and dragonfly larvae (van buskirk and mccollum, 2000b; van buskirk et al., 2003; wilson et al., 2005). the harm from tail removal is often relatively small when tadpoles escape from aquatic predators (wilbur and semlitsch, 1990; doherty et al., 1998; blair and wassersug, 2000) and larger tails, attracting predator strikes away from the more vulnerable body regions, could enhance the survival of tadpoles (hoff and wassersug, 2000; van buskirk et al., 2003; miner et al., 2005; laurila et al., 2008; kishida et al., 2010). this mechanism is clearer in the presence of visual aquatic predators and when tadpoles show bright-colored tails (mccollum and van buskirk, 1996; skelly, 1997; van buskirk et al., 2004). tail loss can reduce the risk of predation in tadpoles, but it can reduce their locomotor ability (van buskirk and mccollum, 2000a; maginnis, 2006; marvin, 2011, 2013) potentially affecting their survival after injury. tadpoles respond to tail removal with tail regeneration (marvin, 2011), which is a common phenomenon in 220 guo-hua ding et alii amphibians (dinsmore, 1996; vaglia et al., 1997; marvin, 2011, 2013). the chinese tiger frog (hoplobatrachus chinensis; anura: ranidae) is a medium-large size frog (up to 120 mm or more in snout-urostyle length; fei, 2009), which is widely reared in many regions of china as an edible frog (fu, 2010). the larvae are carnivorous and can be cannibalized by sibling when food is scarce (fei, 2009). we used h. chinensis tadpoles as a model species to examine: (1) the costs of tail removal in terms of survival rate, final length of body and tail, possible developmental delays, and swimming speed; (2) the tail regeneration rates among different tail removal treatments, to test if tadpoles compensate more serious injuries with faster tail regeneration; (3) the increment of swimming speed in relation to tail length and regeneration, to test if different tail regeneration rates could compensate the loss of mobility of tadpoles under more serious tail removal treatment. material and methods animal collection and rearing we collected 10 clutches of h. chinensis tadpoles, all of which were at gosner’s (1960) stage 29-35 (gs29-35) in july 2012 from the froggery at lishui university. seventy eight tadpoles were randomly collected as experimental animals by a circular net (diameter = 150 mm) at one time from the mixed population, composed by twenty tadpoles collected randomly from each clutch. all of the tadpoles were individually placed in a 10 × 5 × 5 cm (length × width × height) plastic bin containing 200 ml of dechlorinated tapwater (from an aerated 500 l tank) with spirogyra. then all the 78 bins were transferred to a room where water temperature was controlled at 30 ± 0.5°c. during our experiment the tadpoles were fed daily with commercially sold food for larval frogs (crude protein ≥ 42%, crude fiber ≤ 4%, crude ash ≤ 18% and water content ≤ 12%; ningbo techbank co., china), and water was renewed every two days. experimental treatment the tadpoles were randomly divided into four experimental groups including a 20% tail removal group (hereafter e20%; n = 18), a 40% tail removal group (hereafter e40%; n = 21) and a 60% tail removal group (hereafter e60%; n = 21), and a control group (hereafter c; n = 18). before tail removal, gosner’s stage was identified, and body length (from snout to anal front) and tail length (from anal front to tail end) were measured. after that, the swimming speed of each tadpole was recorded in a 50-cm-long straight lane (water temperature was set at 30°c) by digital video camera (sony dcr-sr42e). the video files were later examined with the software ulead videostudio 8.0 (ulead software co., canada) for the fastest swimming speed in 15 cm interval. two hours after the swimming recording, we cut off the corresponding proportion (0%, 20%, 40% and 60%) of tail length from tail tip on each tadpole by using a surgical blade according to the different experimental groups. the swimming speed measurement was repeated with the same methods after two hours from tail removal. then, the growth length of the remaining tail (calculated as the difference between its length at each day of the experiment and its length just after tail removal) and the length of the regenerative tail (from the cutting point to the tail tip) were daily measured in the three experimental groups. the duration of the experiment was seven days, which was also the maximum duration of the experiment, since at this time to see the cutting point of the tail begin to be difficult. indeed, some black spots were always visible on the remaining tail, but not on the regenerative tail, enabling us to accurately determine the cutting point of the tail, and to measure the regenerative tail length, but at the seventh day the black spots appeared again on the regenerative tail of h. chinensis tadpoles. the number of surviving tadpoles in each group was recorded, gosner’s stage was identified, and morphological traits (body length and tail length) and swimming speed were measured at 7 days from tail removal. data analyses all statistical analyses were performed with the statistica software (version 6.0, tulsa, ok, usa). we tested the data for normality (kolmogorov-smirnov test), and homogeneity of variances (bartlett’s and box’s m tests) to meet the assumption of the following parametric analyses. the growth length of the remaining tail was calculated as the difference between its length in each day of the experiment and its length just after tail removal, while the increment of swimming speed was calculated as the difference between the swimming speed at 7 days from tail removal and just after tail removal. we used g-test to examine whether survival rate of tadpoles differed among the four treatments. we used linear regression analysis to examine whether increment of swimming speed was related to regenerative tail length. we used one-way anova to examine whether the gosner’s stage, body length, tail length, swimming speed and increment of swimming speed were different among the treatments. we used repeated-measures anova to test whether the tail removal treatments, the measuring time (seven times: day 1 to 7; or three times: before, just after, and 7 days after tail removal), and their interaction had a significant effect on the growth length of the remaining tail, on the length of the regenerative tail, and on the swimming speed of tadpoles. tukey’s post hoc comparisons were performed when the results from the anova were significant. throughout this paper, values are presented as mean ± se, and the significance level was set at α = 0.05. results before tail removal, the gosner’s stage, body length and tail length were not significantly different in the four experimental groups (table 1). at 7 days from tail 221compensatory effects after tadpole tail removal removal, the gosner’s stage (f3, 72 = 1.56, p = 0.208) and body length (f3, 72 = 1.40, p = 0.250) of tadpoles were not affected, but the mean value of tail length was longer in c than in e20%, e40% and e60%, and longer in e20% and e40% than in e60% (f3, 72 = 27.55, p < 0.001) (fig. 1). the survival rate was 94.4% (17/18) in c, 100% (18/18) in e20%, 95.2% (20/21) in e40% and 100% (21/21) in e60%, and did not show significant differences among the experimental groups (g = 0.47, df = 3, p = 0.925). during the tail regeneration period, the experimental treatment did not affect the growth length of remaining tail, but the length of the regenerative tail was significantly longer in e60% than in e40% and e20%, and longer in e40% than in e20% (fig. 2). both the growth length of remaining tail and the length of the regenerative tail increased gradually during the experiment (results from tukey’s hsd) and were affected by the treatment × measuring time interaction (table 2). the swimming speed of h. chinensis tadpoles differed among the four groups and it was significantly faster in c than in e40% and e60% and faster in e20% than in e60% (results from tukey’s hsd). the swimming speed differed among the three measuring times and it was faster before tail removal and at 7 days from tail removal than after tail removal (results from tukey’s hsd). the swimming speed was also affected by the treatment × measuring time interaction (fig. 3, table 2). especially, after tail removal, the swimming speed differed significantly among the four groups (f3, 72 = 60.08, p < 0.001) and it was significantly faster in c than in all the tail removal treatments and faster in e20% and e40% than in e60% (fig. 3). at 7 days from tail removal, the swimming speed of h. chinensis tadpoles differed among the four groups (f3, 72 = 3.53, p < 0.02) and it was faster in c and e20% than in e60% (results from tukey’s hsd). the increment of swimming speed differed among the three experimental groups, which was greater in table 1. descriptive statistics (mean ± se) and range (in brackets) for gosner’s stage, body length, and tail length of h. chinensis tadpoles in the control and experimental groups before tail removal treatment (one-way anova was used to test the differences among experimental groups. ns: not significant. n: number of samples). variable control group experimental groups f df p 20% tail removal 40% tail removal 60% tail removal n 17 18 20 21 gosner’s stage 32.5 ± 0.2 (31–34) 31.8 ± 0.3 (30–35) 32.0 ± 0.5 (29–35) 32.0 ± 0.3 (29–35) 0.60 3, 72 ns body length (mm) 10.8 ± 0.3 (9.4–13.0) 10.3 ± 0.2 (8.6–12.1) 10.2 ± 0.3 (7.6–13.1) 10.4 ± 0.2 (8.6–13.2) 0.79 3, 72 ns tail length (mm) 20.3 ± 0.5 (13.9–25.2) 20.3 ± 0.3 (16.8–25.0) 20.2 ± 0.6 (16.5–25.2) 20.4 ± 0.5 (16.6–24.7) 0.10 3, 72 ns fig. 1. mean values (+ se) for gosner’s stage (a), body length (b) and tail length (c) of h. chinensis tadpoles at 7 days from tail removal. letters at the top of the bars indicate the statistical significance of differences between treatments. treatments with different letters are significantly different at the p < 0.05 level (one-way anova followed by tukey’s hsd; a > b). c: control; e20%: 20% tail removal; e40%: 40% tail removal; e60%: 60% tail removal. 222 guo-hua ding et alii e60% than in e20% and e40% (f2, 56 = 5.47, p < 0.01; fig. 4a). the linear regression between regenerative tail length and increment of swimming speed showed that the increment of swimming speed is positively related to the regenerative tail length (r = 0.49, f1, 57 = 18.17, p < 0.001; fig. 4b). discussion tail injury may affect many aspects of individual performances such as survivorship, growth, development, locomotion, foraging and escaping predation (maginnis, 2006). serious tail removal (50-75%) may not only affect the escaping ability of tadpoles, but also their survival (van buskirk et al., 2003). for example the survival rate of tadpoles with 50% tail loss level in bufo gargarizans was only 68.4%. however the same level of tail loss did not affect the survival of other species (e.g. rana zhenhaiensis tadpoles; ding et al., 2014) and our results show that also the survival rate of h. chinensis tadpoles is not affected by tail removal, suggesting that some species could be particularly resistant to tail injuries. a decrease of the growth and developmental rate of tadpoles has also been documented after experimental tail removal (e.g. rana catesbeiana tadfig. 2. mean values (+ se) for growth length of remaining tail (a) and regenerative tail length (b) in the first 7-day period. letters at the top of the bars indicate the statistical significance of differences between treatments. treatments with different letters are significantly different at the p < 0.05 level (repeated-measures anova followed by tukey’s hsd; a > b > c). c: control; e20%: 20% tail removal; e40%: 40% tail removal; e60%: 60% tail removal. table 2. effect of tail removal treatments and measuring time on growth length of the remaining tail, length of the regenerative tail and swimming speed of h. chinensis tadpoles (results from repeated-measures anova; ns: not significant). dependent variables treatment measuring time treatment × measuring time f df p f df p f df p growth length of the remaining tail 1.43 2, 56 ns 611.42 6, 336 < 0.001 3.47 12, 336 < 0.001 length of the regenerative tail 38.82 2, 56 < 0.001 411.54 6, 336 < 0.001 44.21 12, 336 < 0.001 swimming speed 14.19 3, 72 < 0.001 70.73 2, 144 ns 11.39 6, 144 < 0.001 fig. 3. mean values (+ se) for swimming speed before tail removal, after tail removal and at 7 days from tail removal in h. chinensis tadpoles. letters at the top of the bars indicate the statistical significance of differences between treatments. treatments with different letters are significantly different at the p < 0.05 level (one-way anova followed by tukey’s hsd; a > b > c). c: control; e20%: 20% tail removal; e40%: 40% tail removal; e60%: 60% tail removal. 223compensatory effects after tadpole tail removal poles; wilbur and semlitsch, 1990). on the contrary, these parameters were not affected by the tail loss in our experiment. our tadpoles were maintained in artificially safe conditions during the tail regeneration period (no stress from predators or cannibalism), probably influencing the high survival rates and unaltered developmental rates in all the experimental groups. tail regeneration is a common phenomenon in amphibians (maginnis, 2006). some anuran tadpoles were reported to lose their regenerative ability transiently in gs45-47 (beck et al., 2003), but the latest gosner’s stage of h. chinensis tadpoles was smaller than gs40 by the end of the experiment, so there was not possibility that experimental tadpoles lose their tail regenerative ability. our results revealed that different tail removal had no significant effect on the growth of remaining tail, but on tail regeneration in h. chinensis tadpoles (fig. 2). h. chinensis tadpoles can compensate more serious tail injuries with faster tail regenerative rate. similar compensatory mechanism was also been found in other amphibians species (e.g. xenopus laevis tadpoles and desmognathus quadramaculatus; tseng et al., 2007; marvin, 2011) and in other organisms (e.g. hemiptera insects can hyperregenerate seriously injured antennae; ikeda-kikue and numata, 1991). in many caudate aquatic organisms, such as tadpoles and newts, the swimming performance depends on the size and other morphological traits of tail (webb, 1984; wassersug and hoff, 1985; long and nipper, 1996; van buskirk and mccollum, 2000a). the swimming speed is usually affected only in the presence of serious (> 60 %) tail removal (e.g. hyla chrysoscelis tadpoles and desmognathus quadramaculatus; figiel and semlitsch, 1991; marvin, 2013). however, in our experiment the swimming speed of h. chinensis tadpoles progressively decreased with the proportion of tail removal increased (fig. 3), which was similar with the results in hyla versicolor, bufo gargarizans and rana zhenhaiensis tadpoles (van buskirk and mccollum, 2000a; ding et al., 2014). we conclude that tail injuries could often incur locomotor cost in h. chinensis tadpoles in natural conditions, as relatively small tail injuries can affect their mobility. tail regeneration does not only repair tail injury (maginnis, 2006), but also compensate the locomotor cost caused by tail loss (marvin, 2011). our results showed that tail regeneration in h. chinensis tadpoles could completely recover their swimming ability in a relatively short time (7 days), with the exception of the tadpoles in e60% (their swimming speed was 2.5 cm/s slower at the end of the experiment), which however showed a greater growth rate of tail and a greater increment of their swimming speed. both tail regeneration rates and the increment of swimming speed among different tail removal treatments indicate that h. chinensis tadpoles can compensate more serious tail injuries (and the related locomotor costs) with a faster tail regeneration and greater speed increment. under natural conditions, tadpoles with serious tail injuries are preferentially preyed (figiel and semlitsch, 1991), and these compensatory mechanisms are likely to be an adaptive strategy to partially reduce the costs of tail loss. fig. 4. mean values (+ se) for increment of swimming speed (a) and increment of swimming speed in relation to regenerative tail length (b) in the three experimental groups. letters at the top of the bars indicate the statistical significance of differences between treatments. treatments with different letters are significantly different at the p < 0.05 level (repeated-measures anova followed by tukey’s hsd; a > b). regression equation and coefficient are given. c: control, e20%: 20% tail removal, e40%: 40% tail removal, e60%: 60% tail removal. 224 guo-hua ding et alii acknowledgments our experimental procedures complied with the current laws on animal welfare and research in china, and were specifically approved by the animal research ethics committee of lishui university (permit no. areclu 2012-04). we thank frank feng, wen-chun shi, cun-tong zhou, xiao-nan zhang for their help during the research. this project was supported by grants from national science foundation of china (project no. 30970435 & 31270443), zhejiang provincial national science foundation of china (project no. ly13c030004), the scientific research foundation of ph.d. in lishui university (qd1301), and open project of laboratory in lishui university (2014-26-10). references arendt, j. 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(2005): escape behaviour and ultimate causes of specific induced defenses in an anuran tadpole. j. evol. biol. 18: 180-190. tseng, a.s., adams, d.s., qiu, d., koustubhan, p., levin, m. (2007): apoptosis is required during early stages of tail regeneration in xenopus laevis. dev. biol. 301: 62-69. 225compensatory effects after tadpole tail removal vaglia, j.l., babcock, s.k., reid, r.n. (1997): tail development and regeneration through the life cycle of the four-toed salamander hemidactylium scutatum. j. morphol. 233: 15-29. van buskirk, j., aschwanden, j., buckelmuller, i., reolon, s., ruttiman, s., fox, s.f. (2004): bold tail coloration protects tadpoles from dragonfly strikes. copeia 2004: 599-602. van buskirk, j., anderwald, p., lupold, s., reinhardt, l., schuler, h. (2003): the lure effect, tadpole tail shape and the target of dragonfly strikes. j. herpetol. 37: 420-424. van buskirk, j., mccollum, s.a. (2000a): influence of tail shape on tadpole swimming performance. j. exp. biol. 203: 2149-2158. van buskirk, j., mccollum, s.a. (2000b): functional mechanisms of an inducible defence in tadpoles: morphology and behaviour influence mortality risk from predation. j. evol. biol. 13: 336-347. van buskirk, j., mccollum, s.a., werner, e.e. (1997): natural selection for environmentally induced phenotypes in tadpoles. evolution 51: 1983-1992. wassersug, r.j., hoff, k. (1985): the kinematics of swimming in anuran larvae. ibid 119: 1-30. webb, p.w. (1984): body form, locomotion and foraging in aquatic vertebrates. am. zool. 24: 107-120. wilbur, h.m., semlitsch, r.d. (1990): ecological consequences of tail injury in rana tadpoles. copeia 1990: 18-24. wilson, r.s., kraft, p.g., van damme, r. (2005): predator-specific changes in the morphology and swimming performance of larval rana lessonae. funct. ecol. 19: 238-244. acta herpetologica 11(2): 161-169, 2016 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-18201 the castaway: characteristic islet features affect the ecology of the most isolated european lizard petros lymberakis1, efstratios d. valakos2, kostas sagonas2, panayiotis pafilis3,* 1 natural history museum of crete, university of crete. knossos av., p.b. 2208, 71409, irakleio, crete, greece 2 section of animal and human physiology, department of biology, national and kapodistrian university of athens, panepistimioupolis, gr-15784, greece 3 section of zoology and marine biology, department of biology, national and kapodistrian university of athens, panepistimioupolis, gr-15784, greece. * corresponding author. e-mail: ppafil@biol.uoa.gr submitted on 2016, 27th april; revised on 2016, 3rd june; accepted on 2016, 20th july editor: adriana bellati abstract. the ecological importance of islet endemics are in the front line of conservation efforts and thus the good knowledge of their biology is required. podarcis levendis is a lacertid lizard, endemic to two rocky islets in the cretan sea, greece, that was raised to specific level in 2008 and since then no data on its biology are available. here we present the first ecological information on the species, focusing on population density, tail autotomy and feeding preferences. we recorded regenerated and damaged tails in the field and estimated population density with the transect method. we also dissected museum specimens and analyzed their stomach content. regenerated tails were common and reached a considerable 71%. the latter finding could be attributed to the intense intraspecific competition due to high population density but also to the seasonal predation pressure by migratory birds. the diet of p. levendis coincides with that of other insular congenerics, including high percentages of plant material. keywords. islands, population density, intraspecific competition, feeding ecology, lacertidae, podarcis levendis. introduction deviations of island life from mainland norms have been repeatedly underlined (van valen, 1973; adler and levins, 1994). studying the constellation of these departures, island biology attracted scientific interest and became a hot spot in ecological and evolutionary studies during the last 30 years (carlquist, 1974; losos and ricklefs, 2009). herpetological research is in the frontline of this general trend. insular giants (harlow et al., 2010) and dwarfs (hedges and thomas, 2001) and the impressive adaptations of island herpetofaunas (herrel et al., 2008; stuart et al., 2014) stimulate a growing body of literature. in this framework, mediterranean islands lend themselves to understanding the particularities of insularity. among the numerous endemic reptiles and amphibians that live on the mediterranean islands, podarcis wall lizards (family lacertidae) stand out. podarcis is the largest genus of european lizards comprising 23 species that occur in europe and north africa (uetz and hošek, 2016). though most podarcis species have both mainland an insular populations, 10 of them are strictly endemic to mediterranean islands. the overall biology of these species depart in many aspects from their mainland peers, including thermal biology (grbac and bauwens, 2001; adamopoulou and valakos, 2005), feeding ecology (salvador, 1986; capula and luiselli, 1994), life history (adamopoulou and valakos, 2000; castilla and bauwens, 2000), digestive performance (pafilis et al., 2007, in press), defensive tactics (pafilis et al., 2009a; li et al., 2014), and behavior (pérez-mellado et al., 2000; traveset and riera, 2005; cooper et al., 2009; 162 petros lymberakis et alii brock et al., 2014a). podarcis islanders differ in the limits of their range. three of them live on very large islands (p. tiliguerta in corsica and sardinia, p. waglerianus in sicily and p. cretensis in crete), six on medium-sized islands (e.g., p. filfolensis in malta, p. gaigeae in skyros), while three are exclusively restricted on small islets: p. lilfordi lives on rocky islets off menorca and mallorca and the cabrera archipelago in spain, p. raffonei inhabits three rocky islets and few isolated places on vulcano island at the aeolian islands in italy, and p. levendis occurs only on two islets in the west cretan sea in greece. podarcis levendis was recognized as distinct species and separated from p. erhardii, which dominates the south and central aegean islands, only recently (lymberakis et al., 2008). the species is found on the islets pori and lagouvardos, north of antikythira island, between crete and the peloponnese (fig. 1). this very restricted and remote range had two main consequences: the complete lack of knowledge on the biology of the species since its first description, and its categorization by iucn as ‘vulnerable’. to the best of our knowledge, the only other paper referring to the pori population is the first record of lizards (under the former name, p. erhardii) on the islet (valakos et al., 1999). in order to effectively protect species that may face the threat of extinction we need to know their biology as best we can. in this study we provide the first ecological data for p. levendis. we assessed population density, body size (estimating also body condition, a proxy for fitness), frequencies of autotomized tails and feeding preferences of the pori population. we made three hypotheses. first, we predicted that the population should be dense. due to their small area, islets usually host very few (or even no) predators and, as a result, lizard densities are usually high (pérez-mellado et al., 2008). alternatively high densities could be attributed to high food abundance through ‘marine subsidies’ (polis and hurd, 1996). second, we expected high frequencies of broken tails. intraspecific competition on islets could be intense due to high population density and these antagonistic interactions may induce caudal autotomy (pafilis et al., 2008; cooper et al., 2015). furthermore pori serves as a refueling station of migratory birds that increase predation pressure during their stay on the islet. third, we hypothesized that plant material would constitute a significant part of stomach content as many microinsular lacertids demonstrate a clear shift towards herbivory (van damme, 1999). fig. 1. map of the two islets hosting p. levendis in northwest cretan sea (greece, ne mediterranean basin). 163ecology of an islet lizard materials and methods study system levendis wall lizard (podarcis levendis) is a well-built, medium sized lacertid lizard (snout vent length, svl, 72.25 ± 3.44 mm). the vegetation on the islets pori (0.317 km2, max altitude: 129 m) and lagouvardos (0.0127 km2, max altitude: 19.3 m) consists of sparse phrygana shrubs among which sarcopoterium spinosum and euphorbia dendroides are the most common while several olea europaea and pistacea lentiscus are also present. the only other terrestrial reptile living on the two islets is the kotschy’s gecko (mediodactylus kotschyi). fieldwork was conducted on pori islet in may 2010. lizards were captured by noose and then transferred to the laboratory facilities of the department of biology at the university of athens. animals were housed individually in vitreous terraria (80×30×40 cm) with sand and artificial shelters and were held at 30oc under a controlled photoperiod with fluorescent tube lighting (12 h light: 12 h dark). additional incandescent lamps (60 w) allowed animals to thermoregulate for 8 h/day. lizards had access to water ad libitum and were fed every other day with mealworms (tenebrio molitor), coated with a powder containing vitamins and minerals supplements (terravit powder, jbl gmbh & co. kg). morphological measurements for each lizard we recorded svl and body mass using a digital caliper (silverline 380244, accurate to 0.01 mm) and a digital scale (ohaus, scout-tm, accurate to 0.01g), respectively. to define body condition (bc) we included only lizards with intact or fully regenerated tail and non-gravid females. we estimated bc using the classical body mass index (mass divided by svl), a standard measure for reptiles (goodman, 2008; battles et al., 2013; damas-moreira et al., 2014), and tested for differences between sexes using a mann-whitney test. additionally, we examined the tail condition (intact or broken/regenerated) in the 22 adult lizards (16 males and 6 females) that were captured in the field and also in 44 museum specimens (18 males and 26 females) that are deposited to the herpetological collection of the natural history museum of crete (collected in january 1992). chi-square test was used to examine for differences between sexes. population density we estimated population density using the line transect method (lovich et al., 2012). eight random line transects of 100 m were walked by the same observer (lp) and all lizards seen within 4-m wide belt (2 m on either side of the survey line; total area covered per trail 400 m2) were recorded. transect surveys were made during morning hours when podarcis lizards are active and line transects were chosen to cover as many different microhabitats as possible. diet composition to assess the feeding preferences of the p. levendis we dissected 36 preserved specimens (15 males and 21 females) that were deposited to the herpetological collection of the natural history museum of crete and removed the digestive tract to examine the prey remnants. prey items were analyzed under a binocular dissecting microscope and identified to order level. we recorded the percentage of the total number of prey items found in the stomachs (%n) as well as the percentage of lizards that ate a given prey taxon (f). spearman correlation was performed to test whether f is related to %n. we also recorded the consumption of plant material (estimated as frequency of presence). we used the shannon-wiener diversity index (krebs, 1998): h’ = ∑pilnpi, to calculate the niche breadth (h’), where pi is the percentage of each prey item found in the stomachs. we conducted a t-test to search for differences between sexes regarding the diversity index. as a complementary approach to test for sexual differences in food composition and to control for the effects of the most abundant prey items, we used the jaccard similarity coefficient (jaccard, 1908) as implemented in past (hammer et al., 2001): j(a,b) = x∩y x∪y , where x and y correspond to the sets of entities that occur at a and b groups, respectively. finally, using the program ecosim 7.0 (gotelli and entsminger, 2001), we employed the pianka’s overlap index (qjk) and estimated the food niche similarity between sexes (pianka, 1975): qjk = pijpik∑ pij 2pik 2∑ , where j and k refer to the two groups under comparison and pij and pik to the proportion of the food component i in each group. results morphological measurements and population density males had significant longer svl than females (mann-whitney test; z = 2.469, p = 0.013) (table 1). in addition, males were heavier compared to females, but these differences were not statistically significant (mannwhitney test; z = 1.769, p = 0.077). finally, we found no significant differences in body condition between sexes (mann-whitney test; z = 0.589, p = 0.55) (table 1). 164 petros lymberakis et alii we examined tail condition in 66 adult lizards overall. 47 of them (71%) had regenerated or broken tails. we found no differences in the percentage of autotomized tails between males and females, either for lizards that we captured in the field (males: 75% and females: 67%; χ2 = 0.008, p = 0.275), or for museum specimens (males: 66% and females: 73%; χ2 = 0.006, p = 0.357). pori islet hosts a rather dense population of approximately 262 lizards per hectare. taking into account the area of the islet (31.74 ha) our finding regarding population density provides the first (though rough) estimation of the total population of the species on the islet, which should be less than 7,000 individuals (given that the peripheral parts of the islands close to the sea are not available to lizards). diet composition we found a significant correlation between the proportion of the number of prey taxa in the stomachs (%n) and the proportion of lizards that ate that given taxa (f) (spearman test, r = 0.99, p < 0.05) (table 2). the predominant prey groups in the diet of p. levendis were coleoptera, araneae and formicidae (table 2). we also found a considerable consumption of plant material, mostly leaves (five out of 36 lizards; 14%). food niche breath was high for both males (h’ = 1.923) and females (h’ = 2.054) and the comparison between them revealed no differences (t-test; t = 1.042, p = 0.30). we found no statistically significant differences between males and females regarding the total number of prey items found in the stomachs (mann-whitney test; z = 0.513, p = 0.608). it is worth noting that almost 20% of the specimens examined had empty stomachs. finally, our results revealed a high food niche overlap between males and females (qjk = 0.91), despite the relative low jaccard similarity index (0.67). discussion our results suggest that the particular conditions on pori islet shaped the ecological profile of p. levendis. our initial hypotheses were verified: levendis wall lizard enhances its diet with plant material and autotomizes its tail frequently. population density was high, though not as high as in other islet podarcis. body condition of lizards was remarkably high, suggesting a sufficient energy flow. population density was estimated at 262 individuals per hectare. this is a rather high value for eastern mediterranean standards where insular lizards do not form very dense populations. valakos (1990) reported 76 lizards/ha (p. erhardii, naxos island), pafilis et al. (2009b) 95-185 lizards/ha (p. gaigeae, skyros island), adamopoulou (1999) 395 lizards/ha (p. milensis, milos island) and chondropoulos and lykakis (1983) found densities that varied across islands from 118 to 247 lizards/ha (p. tauricus, numerous ionian islands). a striking deviation comes from diavates islet (off skyros) that harbors 875 lizards/ha, the denser population on east mediterranean islands (pafilis et al., 2013). lizard densities from western mediterranean islands are much higher (delaugerre and cheylan, 1992; scalerà et al., 2004), reaching even 8,000 lizards/ha (pérez-mellado et al., 2008). table 1. snout vent length (svl; mm), body mass (bm; g) and body condition (bc) of males (n = 16) and females (n = 16) of p. levendis population at pori islet. table reports means ± sd values; ranges are also given (within brackets). sexes svl bm bc males 75.25 ± 2.11 (71.0-79.0) 8.21 ± 0.78 (6.7-9.5) 0.11 ± 0.01 (0.09-0.13) females 66.00 ± 9.48 (54.0-76.0) 7.07 ± 1.46 (5.0-8.8) 0.11 ± 0.01 (0.09-0.12) table 2. diet composition of p. levendis for males and females during summer. %n refers to the percentage of prey items in the stomachs, f refers to the proportion of lizards having eaten a specific prey category while h’ to the shannon-wiener diversity index. sexes males females f %n f %n araneae 0.40 23.08 0.29 18.18 opilionidia 0.00 0.00 0.14 4.55 orthoptera 0.07 1.92 0.05 1.52 gastropoda 0.20 7.69 0.24 9.09 homoptera 0.00 0.00 0.14 4.55 diptera 0.07 1.92 0.00 0.00 isopoda 0.13 3.85 0.14 10.61 lepidoptera 0.13 3.85 0.10 3.03 coleoptera 0.67 28.85 0.48 24.24 hymenoptera 0.07 3.85 0.00 0.00 formicidae 0.20 7.69 0.43 16.67 insect larvae 0.33 17.31 0.19 7.58 plant material 0.13 0.14 total preys 52 66 specimens 15 21 prey/stomach 3.47 3.14 h’ 1.923 2.054 165ecology of an islet lizard insular populations are much denser than the mainland ones (rodda et al., 2001; buckley and roughgarden, 2006) as a result of the relaxed predation and interspecific competition on the islands (buckley and jetz, 2007; novosolov et al., 2016). this is the case for pori islet where there is not a single terrestrial predator while the only other reptile is kotshyi’s gecko (mediodactylus kotschyi), which does not compete with podarcis lizards for food or space (valakos and vlachopanos, 1989). certain birds nest on the islet, such as falcons (falco eleonorae), european shags (phalacrocorax aristotelis) and yellow-legged gulls (larus michahellis). these birds do not prey on podarcis lizards (walter, 1967; pérez-mellado et al., 2014). to the contrary, lizards seem to have developed a particular mutualism, at least with falcons, and benefit from their presence (walter, 1967; delaugerre et al., 2012). in addition, sea birds enhance islet ecosystems through ‘marine subsidies’ (polis and hurd, 1996) that fuel dense lizard populations (barrett et al., 2005). some mediterranean islets also receive this sea-derived energy and thus may support high lizard abundances (vidal et al., 2001; pafilis et al., 2011). the frequencies of autotomized tails are typically considered to reflect predation pressure (arnold, 1988). however, intraspecific competition through aggressive encounters may also end up to caudal autotomy (pafilis et al., 2008; pafilis et al., 2009b). the high lizard densities on mediterranean islets have been proven to be the major factor inducing tail shedding (cooper et al., 2015; donihue et al., 2015). predation pressure is minimal at pori, so the high population density should account for the high percentages of regenerated and broken tails in the field. the observed 71% lies within the top percentile for other islet podarcis (pérez-mellado et al., 1997; pafilis et al., 2008; pafilis et al., 2009a; brock et al., 2014a). nevertheless, we may not exclude the possibility of predation pressure resulting from bird predators during migration as the islets are on an important migratory route (hellenic ornithological society, 2016). food abundance on mediterranean islands is quite restricted (brown and pérez-mellado, 1994; blondel et al., 2010). as a result insular lacertids widen their feeding preferences and thus adopt a wider food niche breadth (pérez-mellado and corti, 1993; sagonas et al., 2015a). food scarcity is even more exacerbated on small islets (pérez-mellado and corti, 1993; castilla et al., 2008) where lizards adopt extreme feeding behaviors in order to survive (castilla and herrel, 2009; brock et al., 2014b). though our sample size was rather small, our data implied that p. levendis followed the general trophic profile of the genus feeding mainly on terrestrial invertebrates with a clear preference on insects (arnold, 1987). the predominant food groups were coleoptera, araneae and formicidae (table 2). araneae and, much more, coleoptera are the commonest prey groups in podarcis lizards (maragou et al., 1997; carretero, 2004; zuffi and giannelli, 2013). myrmecophagy, on the other hand, is a typical feeding strategy of island podarcis, especially during late spring and summer when other food resources come in short supplies (valakos, 1986; pérez-mellado and corti, 1993; valakos et al., 1997; adamopoulou et al., 1999; lo cascio and capula, 2011). ants may be less rich in terms of nutrients and energy compared to beetles, but they compensate this disadvantage with their high numbers. previous studies reported an important shift towards herbivory in most island lacertid populations (pérezmellado and corti, 1993; van damme, 1999; sagonas et al., 2015b). the percentage of plant material in the stomachs of p. levendis was not very high, though it seems to conform to this trend (14%). islet populations follow the aforementioned pattern, demonstrating a clear preference for plant consumption (lo cascio and pasta, 2006; lo cascio et al., 2006; carretero et al., 2010; pérez-cembranos et al., 2016) that in some cases may be extreme and even induce morphological changes (herrel et al., 2008; vervust et al., 2010). mediterranean islets are demanding habitats with limited food resources (ouboter, 1981; brown et al., 1992). podarcis lizards living on islets share many common characteristics imposed by the particular conditions of these habitats: low predation pressure (tsasi et al., 2009; durand et al., 2012), high population densities (pérez-mellado et al., 2008) and strong intraspecific competition (cooper et al., 2015), while they usually have high body condition (van den berg et al., 2015). podarcis levendis conforms to this general pattern. the importance of such isolated populations, some of which have developped unique adaptations (pérez-mellado et al., 2000; herrel et al., 2008; raia et al., 2010), is of top priority in conservation biology (capula et al., 2002). acknowledgements we are grateful to grigoris kapsalas for drawing the map. fieldwork and lab measurements were conducted in accordance with the greek national legislation (presidential decree 67/81). references adamopoulou, c. 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(2013): trophic niche and feeding biology of the italian wall lizard, podarcis siculus campestris (de betta, 1857) along western mediterranean coast. acta herpetol. 8: 35-39. acta herpetologica vol. 11, n. 2 december 2016 firenze university press predator-prey interactions between a recent invader, the chinese sleeper (perccottus glenii) and the european pond turtle (emys orbicularis): a case study from lithuania vytautas rakauskas1,*, rūta masiulytė1, alma pikūnienė2 effective thermoregulation in a newly established population of podarcis siculus in greece: a possible advantage for a successful invader grigoris kapsalas1, ioanna gavriilidi1, chloe adamopoulou2, johannes foufopoulos3, panayiotis pafilis1,* the unexpectedly dull tadpole of madagascar’s largest frog, mantidactylus guttulatus arne schulze1,*, roger-daniel randrianiaina2,3, bina perl3, frank glaw4, miguel vences3 thermal ecology of podarcis siculus (rafinesque-schmalz, 1810) in menorca (balearic islands, spain) zaida ortega*, abraham mencía, valentín pérez-mellado growth, longevity and age at maturity in the european whip snakes, hierophis viridiflavus and h. carbonarius sara fornasiero1, xavier bonnet2, federica dendi1, marco a.l. zuffi1,* redescription of cyrtodactylus fumosus (müller, 1895) (reptilia: squamata: gekkonidae), with a revised identification key to the bent-toed geckos of sulawesi sven mecke1,*,§, lukas hartmann1,2,§, felix mader3, max kieckbusch1, hinrich kaiser4 the castaway: characteristic islet features affect the ecology of the most isolated european lizard petros lymberakis1, efstratios d. valakos2, kostas sagonas2, panayiotis pafilis3,* sources of calcium for the agamid lizard psammophilus blanfordanus during embryonic development jyoti jee1, birendra kumar mohapatra2, sushil kumar dutta1, gunanidhi sahoo1,3,* mediodactylus kotschyi in the peloponnese peninsula, greece: distribution and habitat rachel schwarz1,*, ioanna-aikaterini gavriilidi2, yuval itescu1, simon jamison1, kostas sagonas3, shai meiri1, panayiotis pafilis2 swimming performance and thermal resistance of juvenile and adult newts acclimated to different temperatures hong-liang lu, qiong wu, jun geng, wei dang* olim palus, where once upon a time the marsh: distribution, demography, ecology and threats of amphibians in the circeo national park (central italy) antonio romano1,*, riccardo novaga2, andrea costa1 on the feeding ecology of pelophylax saharicus (boulenger 1913) from morocco zaida ortega1,*, valentín pérez-mellado1, pilar navarro2, javier lluch2 notes on the reproductive ecology of the rough-footed mud turtle (kinosternon hirtipes) in texas, usa steven g. platt1, dennis j. miller2, thomas r. rainwater3,*, jennifer l. smith4 heavy traffic, low mortality tram tracks as terrestrial habitat of newts mikołaj kaczmarski*, jan m. kaczmarek book review: sutherland, w.j., dicks, l.v., ockendon, n., smith, r.k. (eds). what works in conservation. open book publishers, cambridge, uk. http://dx.doi.org/10.11647/obp.0060 sebastiano salvidio acta herpetologica 14(1): 21-26, 2019 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-23871 tonal calls as a bioacoustic novelty in two atlantic forest species of physalaemus (anura: leptodactylidae) thiago r. de carvalho1,2,*, célio f.b. haddad1, marcos gridi-papp2 1 laboratório de herpetologia, departamento de zoologia e centro de aquicultura (caunesp). universidade estadual paulista, rio claro, sp, brazil. *corresponding author. email: thiago_decarvalho@yahoo.com.br 2 department of biological sciences, university of the pacific, stockton, ca 95211, usa submitted on: 2018, 3rd september; revised on: 2018, 19th december; accepted on: 2019, 18th january editor: raoul manenti abstract. the frog genus physalaemus has almost 50 species with vocalizations that are mostly composed of a single note. this note tends to have a broad harmonic structure or a pulsed structure. the sister species p. lateristriga and p. olfersii have pulsed advertisement calls that have been described as a noisy and long-lasting warbling sound. we provide the first account of inclusion of tonal sounds as part of the vocal repertoire of these species. pure tones can (1) be long and form the entire call; (2) form prefixes of variable length separated by silence from the advertisement call; (3) be brief and form the onset or the offset of the regular advertisement call. tonal calls may be an evolutionary novelty and they are not known from other populations of p. olfersii and p. lateristriga. identification of the mechanism of sound production and of the behavioural roles of these unique calls may help elucidate the evolution of call complexity in frogs. keywords. animal communication, bioacoustics, leiuperinae, vocal repertoire. introduction most anurans communicate using stereotyped signals with relatively simple acoustic structure (gerhardt and huber, 2002; köhler et al., 2017). various call traits can be shared among the species of a clade and yet enough variation is present, in most cases, to make the advertisement call of each species unique. advertisement call differences can form major prezygotic isolation barriers and can be used to separate morphologically cryptic species (blair, 1958; johnson, 1966). the vocal repertoires of frogs in the neotropical genus physalaemus are almost always simple with calls composed of a single note (tárano, 2001; giaretta et al., 2009). the calls of this group are of particular interest because its sister genus engystomops, which used to be included in physalaemus, contains the túngara frog (e. pustulosus; lynch, 1970; nascimento et al., 2005; lourenço et al., 2015), a model organism in behavioural and acoustic communication studies. a variety of studies has explored the evolution of an optional secondary pulsed sound (chuck) that the male túngara frog can add to the end of his regular advertisement call (whine; ryan, 1985). the laryngeal mechanism underlying the addition of the chuck has not been fully described, however, and further insight may be gained from comparisons with other species in the group. in both physalaemus and engystomops, advertisement calls tend to be long and have a pronounced harmonic structure with a smooth amplitude envelope (cannatella et al., 1998; wilczynski et al., 2001; provete et al., 2012). descending frequency modulation throughout the call is very common with most of the change concentrated at the beginning. some species, however, have pulsed calls 22 thiago r. de carvalho, célio f.b. haddad, marcos gridi-papp and lack the descending frequency modulation (bokermann, 1966; padial and köhler, 2001; weber et al., 2005b). physalaemus olfersii and p. lateristriga are examples of this latter type of advertisement call (drewery et al., 1982; giaretta et al., 2009; cassini et al., 2010). based on acoustic structure and laryngeal morphology, their call was speculated to be possibly produced in the same manner as the chuck encountered in the call of the túngara frog (drewery et al., 1982). here, we report on previously unknown call complexity in the genus physalaemus generated through the incorporation of tonal sounds. we document its occurrence in p. olfersii and p. lateristriga. we also discuss the potential role and the mechanism underlying this striking acoustic novelty based on the vocal biomechanics of the group. material and methods vocalizations were recorded in the field using analogue recorders (marantz pmd 420 with sony metal-sr 100 audio cassette) and supercardioid microphones (sennheiser me-80) positioned at an approximate distance of 2 m from the calling males. the recordings were digitized at a 44.1-khz sampling rate and 16-bit sample size. calls were recorded from two localities in the brazilian state of são paulo (south-eastern brazil): physalaemus olfersii—parque estadual da serra do mar, núcleo santa virgínia, são luiz do paraitinga (-23.332200°, -45.096828°; 979 m a.s.l.); physalaemus lateristriga—ribeirão branco (-24.358600°, -48.743000°; 840 m a.s.l.). voucher specimens and recordings are housed at the célio f. b. haddad collection (cfbh). information on sound recordings is as follows: physalaemus lateristriga—(1) cfbh 63, recorded on 4 february 1995, at 22:20 h, air temperature 19°c; (2) mgp 5011401, recorded on 14 january 2005, at 19:30 h, air temperature 19°c, water temperature 22°c; call voucher is one male of the series cfbh 16564‒16567, 16569‒16575, 16577‒16582, 16584‒16585. physalaemus olfersii—(1) mgp 4111701‒4111705, recorded on 17 november 2004. the acoustic analysis was conducted in soundruler (gridipapp, 2007), a package of matlab scripts (matlab, 2004) that allows for unbiased quantification of acoustic traits using automated procedures. call rate, however, was measured manually in audacity (audacity team, 2017). the settings used in the frequency analysis included fft size = 1024 samples, fft overlap = 90%, window type = hanning, contrast = 70%. settings for automated recognition of pulses were (in samples): pulse detection (smoothing = 250, resolution = 25); pulse delineation (smooth factor = 1, smoothing = 25, resolution = 1). settings for frequency tracking range: 1000 hz each step. critical amplitude ratio: cfbh 63 (-1; disabled); mgp 5011401 (5.0). a 500-hz high-pass filter at 48 db was applied to the sound file cfbh 63 in audacity to reduce background noise. temporal traits were measured from oscillograms and spectral traits from spectrograms. the acoustic terminology is summarized in table 1. a call-centred approach (sensu köhler et al., 2017) was used for the acoustic characterization of acoustic signals described in the present study. results tonal calls were recorded from two males of p. lateristriga that only produced tonal calls during the recording. three males of p. olfersii were also recorded making tonal calls but these were shorter and positioned as prefixes or suffixes of the regular (pulsed) advertisement calls. the two males of p. lateristriga that only produced tonal calls were recorded at the same locality and temperature but with a separation of 10 years. both exhibited an interruption within the call but it corresponded to a frequency shift in one individual and to a silent gap in the other. the tonal calls (n = 8; fig. 1a) of the first male p. lateristriga (cfbh 63) lasted 0.83‒1.40 sec (1.18 ± 0.09; min‒max, x ± sd) and were emitted at a rate of 17.8 calls/min. rise time was 8.3‒89.3% (62.4 ± 27.1) of the call’s length. the dominant frequency of the call was 1572‒1701 hz (1641.9 ± 51.2). the call started at about 1500 hz and gradually modulated up to approximately 1750 hz. an abrupt shift in frequency separated the initial part of the call from the last 10‒20% (see fig. 1a). the final section of the call had a downward frequency modulation approximately from 1300 hz to 1100 hz. table 1. acoustic terminology adopted in this study. traits description temporal traits call length (sec) time from initial 10% to final 10% of amplitude of one call rise time (%) point of maximum amplitude relative to call length call rate per minute (total number of calls 1) / time from beginning of first call to beginning of last call spectral traits dominant frequency (hz) frequency containing the greatest energy in one call. it matches the fundamental frequency in tonal calls. frequency modulation (hz) dominant frequency at 10% final minus that at initial 10% of one call 23tonal calls in physalaemus the tonal calls (n = 14; fig. 1b) of the second male p. lateristriga (mgp 5011401) lasted 0.90‒2.57 sec (2.12 ± 0.39) and were emitted at a rate of 8.84 calls/min. rise time was 33.5‒95.2% (66.1 ± 20.4) of the call’s length. the calls had a silent gap of 0.0689-0.1076 sec (0.0817 ± 0.0105) near the first third or half of the call’s length (see fig. 1b). the duration of the first part of the call varied from 0.33 to 1.05 sec (0.78 ± 0.19), while that of the second part varied from 0.22 to 1.63 sec (1.09 ± 0.46). the dominant frequency was 1787‒1830 hz (1811.9 ± 22.1). ascending frequency modulation occurred throughout the call, stabilizing in the last 25%, with a drop in frequency of only 50-100 hz in the last 10%. the dominant frequency started at 1450‒1600 hz (1470.4 ± 55.0) and ended at 1700‒1750 hz (1728.8 ± 21.4). fig. 1. (a-b) two males of physalaemeus lateristriga recorded from ribeirão branco (são paulo, brazil): (a) tonal call from the recording cfbh 63; (b) tonal call from the recording mgp 5011401. (c) male physalaemus olfersii recorded from santa virgínia (são paulo, brazil). a vocal bout from the recording mgp 4111701 depicting a short tonal call followed by a typical advertisement call (overlapped by another male calling in the background). fig. 2. waveform and 4096-fft amplitude spectrum of (a) a 20-msec section from a tonal call (recording mgp 5011401; p. lateristriga), and a 60-msec section from (b) a typical advertisement call (mgp 4111704; p. olfersii), respectively. 24 thiago r. de carvalho, célio f.b. haddad, marcos gridi-papp unlike p. lateristriga, males of p. olfersii did not produce call bouts containing only tonal calls in our recordings. three male p. olfersii (mgp 4111701‒3) emitted (fig. 1c) a tonal call shortly followed by the typical pulsed advertisement call. however, the tonal portion was consistently shorter than those described earlier for p. lateristriga. all three males of p. olfersii were recorded while calling in antiphony with neighbouring conspecific males (fig. 1c). two other recorded males of p. olfersii (mgp 4111704‒5) did not emit tonal calls, but changed to a tonal-like structure at the very final portion of a few calls: 50‒70 msec in duration and dominant frequency of 1250‒1750 hz. in both species, the tonal calls were very narrowly tuned whereas the pulsed calls had energy distributed through a wide frequency range (fig. 2). the dominant frequency was always centred near 1700 hz but in the tonal calls the amplitude of the fundamental frequency was 40 db higher than that of any other harmonic, whereas in the pulsed calls the amplitude of the dominant frequency was less than 5 db higher than that of the closest side-band. discussion the acoustic structure of the tonal calls diverged sharply from that of the pulsed advertisement calls in physalaemus lateristriga and p. olfersii. the striking contrast between these structures made p. olfersii males stand out acoustically from the chorus when they added tonal sounds to their calls. tonal calls also made p. lateristriga stand out in their diverse acoustic assemblage of tropical anurans and insects due to the rarity of tonal sounds in nature (rossing, 2007). a single other species of physalaemus (p. fernandezae) has been reported to produce tonal sounds (barrio, 1965). this species, different from p. lateristriga and p. olfersii, appears to always produce tonal-only advertisement calls and no other call types have been described. the brief acoustic description provided by barrio (1965) indicates other similarities with the calls recorded in our study. in addition to being tonal, the calls of p. fernandezae have high fundamental frequency and ascending frequency modulation. these shared characteristics are unlikely to reflect common descent because p. fernandezae is not closely related to the p. olfersii group, within which p. olfersii and p. lateristriga are sister taxa (lourenço et al., 2015). tonal calls are therefore likely to have evolved independently in these two groups and the accompanying acoustic similarities may reflect a common laryngeal mechanism for the production of tones rather than homology. the addition of a tonal prefix or suffix adds complexity to the advertisement call of p. olfersii. a complex vocal repertoire has been described for p. spiniger but it did not include tonal sounds (haddad and pombal, 1998; costa and toledo, 2013). in engystomops, some species (e. pustulosus, e. petersi) are known to optionally add sounds with a distinct acoustic structure to their advertisement call as ornaments that make the call more attractive to females (ryan and rand, 1993). the behavioural significance of the tonal calls in the p. olfersii group is unclear. all recorded individuals of p. olfersii and p. lateristriga vocalized from the ground within a few meters from the water. neither females nor other males were observed to interact with the callers during shifts between calls including or not tonal sounds. the callers themselves did not change position, orientation, or indicate any change of behavioural context when switching between emitting or not tonal sounds. in other anurans, the second most commonly reported call type is emitted during aggressive interactions (wells, 2007), but in engystomops pustulosus the second most common sound (the chuck) increases the attractiveness of the call to females (ryan, 1985). the tonal call could also be an accidental acoustic consequence of variation in the structure or mechanics of the larynx and may lack an exclusive role. the mechanistic origin of such distinctive acoustic signals is intriguing. the dominant frequency in both the pulsed advertisement calls and the tonal calls is about 1700 hz and the main difference between them is the presence or absence of pulsing. this aspect is also present in the advertisement call of e. pustulosus in which the chuck is pulsed while the whine is not. large laryngeal fibrous masses have been described in some species of leiuperine frogs including e. pustulosus and p. olfersii (ryan and drewes, 1990). such structures attached to vocal cords have proven to be necessary for the production of the chuck but not of the whine (gridi-papp et al., 2006). the onset of the chuck in e. pustulosus has been suggested to be controlled by muscles that deform the larynx (drewery et al., 1982; ryan and drewes, 1990). alternatively, a mathematical model indicates that the mechanical interactions between the fibrous mass and the vocal cords may determine the onset of the chuck with base on laryngeal airflow (kime et al., 2018). one way or the other, the lack of pulsing in the tonal calls of p. lateristriga and p. olfersii could result from halting the vibration of the laryngeal fibrous masses. without pulses, the calls of p. olfersii and p. lateristriga could be expected to exhibit a pronounced harmonic structure like those of most leiuperine frogs, but instead, they are tonal. this raises the question of whether the ton25tonal calls in physalaemus al calls are being produced by vocal cord vibration or as whistles, by oscillations of airflow as it passes between the arytenoid cartilages. some evidence in favour of a reduced role of vocal cords can be observed in the frequency modulation of the call. the p. olfersii group can present subtle ascending frequency modulation at the attack phase and descending frequency modulation during the decay phase of the call as best seen in p. feioi, p. orophilus, and p. soaresi (weber et al., 2005a; cassini et al., 2010). this matched modulation of amplitude and frequency is expected whenever the vocal cords do not block the airflow immediately before the onset and after the offset of the call (gridipapp, 2014). the tonal calls could be produced by the vocal cords, however. while not the highest among leiuperines (see comparison in provete et al., 2012: table 5), the advertisement calls of most species in the p. olfersii species group have a high dominant frequency (weber et al., 2005a; cassini et al., 2010). extensive filtering of the harmonics could concentrate all the energy in the fundamental frequency and result in tonal calls. this study shows that males of p. lateristriga produce pulsed or tonal calls and males of p. olfersii can switch between these two structures within a call. these species offer a unique opportunity for study of the acoustic mechanisms that underlie two call modes that are fixed in other species of physalaemus. furthermore, tonal calls are not known from other populations of either species, indicating that this might be an opportunity to document the evolution of a novel acoustic signal. finally, this case seems analogous to that of e. pustulosus, in which males optionally incorporate a categorically different signal into their calls. the study of this system may provide new insight into the evolution of diversity in acoustic communication. acknowledgements trc is currently supported by são paulo research foundation (fapesp) with a postdoctoral fellowship (#2017/08489-0). cfbh is grateful to fapesp grants (#2013/50741-7 and #2014/50342-8), and conselho nacional de desenvolvimento científico e tecnológico (cnpq) for a research fellowship. we thank the instituto brasileiro do meio ambiente e dos recursos naturais renováveis (ibama) for providing the collection permits. references audacity team (2017): audacity(r): free audio editor and recorder, version 2.2.1. boston, ma, usa. https://audacityteam.org. barrio, a. 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(2007): the ecology and behavior of amphibians. the university of chicago press, chicago and london. wilczynski, w., rand, a.s., ryan, m.j. (2001): evolution of calls and auditory tuning in the physalaemus pustulosus species group. brain behav. evol. 58: 137-151. acta herpetologica vol. 14, n. 1 june 2019 firenze university press uzungwa scarp nature forest reserve: a unique hotspot for reptiles in tanzania john valentine lyakurwa1,2,*, kim monroe howell2, linus kasian munishi1, anna christina treydte1,3 experience of predacious cues and accessibility to refuge minimize mortality of hylarana temporalis tadpoles santosh mogali*, bhagyashri shanbhag, srinivas saidapur tonal calls as a bioacoustic novelty in two atlantic forest species of physalaemus (anura: leptodactylidae) thiago r. de carvalho1,*, célio f.b. haddad1, marcos gridi-papp2 scientific publication of georeferenced molecular data as an adequate guide to delimit the range of korean hynobius salamanders through citizen science amaël borzée1,*, hae jun baek2,3, chang hoon lee2,3, dong yoon kim2, jae-young song4, jaehwa suh5, yikweon jang1, mi-sook min2* mirrored images but not silicone models trigger aggressive responses in male common wall lizards stefano scali1,*, roberto sacchi2, mattia falaschi1,3, alan j. coladonato2, sara pozzi2, marco a.l. zuffi4, marco mangiacotti1,2 using an in-situ infra-red camera system for sea turtle hatchling emergence monitoring fatima n. oğul1,#,*, franziska huber2,#, sinem cih1, kumsal düzgün3, ahmet e. kideyş1, korhan özkan1 descriptive osteology of an imperiled amphibian, the luristan newt (neurergus kaiseri, amphibia: salamandridae) hadi khoshnamvand1, mansoureh malekian1,*, yazdan keivany1, mazaher zamani-faradonbe1, mohsen amiri2 does color polymorphism affect the predation risk on phalotris lemniscatus (duméril, bibron and duméril, 1854) (serpentes, dipsadidae)? fernanda r. de avila1,2,*, juliano m. oliveira3, mateus de oliveira1, marcio borges-martins4, victor hugo valiati2, alexandro m. tozetti1 age structure of a population of discoglossus scovazzi camerano, 1878 (anura discoglossidae) in extreme environmental conditions (high atlas, morocco) mohamed amine samlali, abderrahim s’khifa, tahar slimani* acta herpetologica 12(2): 193-197, 2017 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-20894 observations on the intraspecific variation in tadpole morphology in natural ponds eudald pujol-buxó1,2,*, albert montori1, roser campeny3, gustavo a. llorente1,2 1 departament de biologia evolutiva, ecologia i ciències ambientals, universitat de barcelona, barcelona, spain. *corresponding author. e-mail: epujolbuxo@ub.edu 2 institut de recerca de la biodiversitat (irbio), universitat de barcelona, barcelona, spain 3 minuartia estudis ambientals, barcelona, spain submitted on: 2017, 10th july; revised on: 2017, 21st august; accepted on: 2017, 26th august editor: marco mangiacotti abstract. intraspecific morphological variation of anuran tadpoles occurs in response to several factors. causes and consequences of this variation have been largely studied hitherto in controlled environments, but data from natural habitats is clearly less abundant. here, we present a series of observations on the morphology – mainly tail depth – of three tadpole species from ne iberian peninsula across different pond typologies. according to experimental data on tadpole morphology and selective pressures along the pond permanency gradient, we should expect that tadpoles inhabiting ponds with a short hydroperiod – mainly facing desiccation risk – have shallower tail fins than tadpoles from ponds with longer hydroperiod – mainly facing predation risk. thus, we expected that the link between these complementary selective pressures – predation risk, desiccation risk – and hydroperiod could make possible to detect intraspecific variation in tadpole morphology among different typologies of natural ponds. morphological differences were found in all studied species, and variation, when present, agreed with theory: tadpoles had deeper fin tails as they were collected in ponds with a longer hydroperiod. interestingly, in most cases these morphological differences were more marked as tadpoles were larger in size. although distances among the studied ponds were generally short – posing phenotypic plasticity as the most plausible proximate mechanism – specifically designed studies would be needed to disentangle the relative role of other processes like local adaptation. keywords. alytes obstetricans, hyla meridionalis, rana temporaria, predation risk, desiccation risk, phenotypic plasticity. introduction intraspecific morphological variation of anuran tadpoles occurs in response to several factors and is created through different mechanisms. phenotypic plasticity and various processes creating population-level genetic changes (van buskirk and mccollum, 1999; pfennig and murphy, 2000; relyea, 2004; 2005) have been listed as natural sources of this variation. usually, a series of both biotic and abiotic stressors – desiccation and predation risk, tadpole competition and density – combined with the particular life history characteristics of each species, creates a set of predictable tadpole morphologies (relyea, 2004; richter-boix et al., 2006a; 2006b; 2007; touchon and warkentin, 2008; van buskirk, 2009). importantly, these morphologies have been proved to correlate with individual fitness during larval stages (johnson et al., 2008; dijk et al., 2016; pujol-buxó et al., 2017) and to influence also post-metamorphic morphology and fitness in turn (tejedo et al., 2010; johansson and richter-boix, 2013; pujol-buxó et al., 2013). causes, effects and consequences of intraspecific morphological variation in tad194 eudald pujol-buxó et alii poles have been largely studied so far, but mainly using laboratory experimental procedures or controlled garden experiments (e.g., relyea, 2004; 2005; touchon and warkentin, 2008). hence, in this field of study, morphological data of tadpoles from natural ponds is clearly less abundant (but see van buskirk, 2009; 2014; johnson et al., 2015). this data is crucial to confirm the trends observed in laboratory or garden experiments and to spur novel research questions and hypotheses. the pond permanency gradient – ranging from ephemeral pools to permanent water bodies (skelly, 1995; schneider and frost, 1996; wellborn et al., 1996) – correlates with most selective pressures acting on tadpoles in the mediterranean area. predation and pond desiccation are arguably the most important selective pressures acting on tadpole populations, and they tend to create a trade-off along the pond permanency gradient (skelly, 1995): the mean time a pond contains water each year negatively correlates with its desiccation risk, but it is also commonly linked to an increasing number or diversity of predators (smith, 1983; pearman, 1995; schneider and frost, 1996; richter-boix et al. 2006b; 2007). interestingly, as showed by laboratory experiments, both selective pressures also create opposite morphological outcomes in the tail shape of tadpoles. thus, tadpoles under predation risk display deeper tail fins to lure predators away from lethal surfaces in case of attack (van buskirk et al., 2003; johnson et al., 2008), while tadpoles under desiccation risk display shallower tails, investing more energy in the feeding and digesting structures located in the main body (vences et al., 2002; richter-boix et al., 2006a). therefore, assuming an inverse correlation between predation and desiccation risk along the pond permanency gradient, we can expect from experimental data that tadpoles inhabiting ponds with a long hydroperiod should usually display – either by phenotypic plasticity or other mechanisms – deeper tail fins than tadpoles from ponds with a short hydroperiod (smith, 1983; richter-boix et al., 2006a; 2006b; 2007; van buskirk, 2009). here, we explore this assumption re-analysing simple morphological data – tail depth and total length of tadpoles – on three european species inhabiting more than one pond typology. materials and methods we gathered available morphological data of tadpoles of three anurans inhabiting different pond typologies in two natural parks (np) located near barcelona (catalonia, spain), namely alytes obstetricans (anura, alytidae) and hyla meridionalis (anura, hylidae) from garraf np; and rana temporaria (anura, ranidae) from montseny np. data from garraf np was initially collected as part of a monitoring of the parks’ anuran populations during spring of year 1991, and data from montseny np is from a phd thesis by campeny (2001) on tadpole trophic ecology made during years 1985 and 1986. in both cases, tadpoles had been dip-netted from natural ponds along several weeks or months of spring, being the ponds in montseny np the same for both years (tables s1, s2 and s3). since all tadpoles were euthanized for other purposes within each study, they could not be possibly sampled twice. although tadpoles were measured differently in both studies – using a caliper garraf np, and using a binocular microscope in montseny np – we did not perform comparisons across species or parks, and therefore we can discard possible biases due to the measurement methods. in both cases, we assigned ponds to a certain category – ephemeral, temporary or permanent – according to criteria by richter-boix et al. (2006b) and each pond’s usual hydroperiod during the years of sampling. according to these criteria, alytes obstetricans in garraf np chooses mainly permanent water bodies as reproduction ponds, using temporary and even ephemeral ponds occasionally (montori et al., 2015), while hyla meridionalis mostly uses temporary ponds, breeding also in all pond typologies present in garraf np (montori et al., 2015). on the other hand, rana temporaria in montseny np breeds in most types of water bodies, from permanent streams to temporary or occasionally ephemeral ponds (campeny, 2001). since necessary assumptions for parametric tests were not met – mainly due to important differences in the numbers of specimens measured in each pond –, differences in tail depth (fig. s1) were analysed using non-parametric randomization tests implemented in the package lmperm (wheeler and torchiano, 2016), using 1000 randomizations in each case. tests were run separately for each species: tail depth as response variable, pond as factor and total length of the tadpole as a covariate, allowing for interactions. when there were multiple ponds to test for the same species, we used the same procedures in pairwise tests to detect statistically homogeneous groups if global differences were found. experimental data for comparison using the same measurements (in this case on discoglossus pictus and pelodytes punctatus) was re-analysed from a study on inducible defences (pujol-buxó et al., 2013). in this case we used linear mixed models instead of permutation tests – using the same model structure – to account for lack of independence, by adding a random intercept depending on tank. all statistical analyses and figures were done in r v3.2.3 (r core team, 2015). results the relationship between tail depth and total length of a. obstetricans tadpoles significantly differed in slope (that is, effects of the interaction were significant: f4,423 = 6.44, p < 0.001) and intercept (f4,423 = 21.6, p < 0.001) when testing all five ponds together. however, there were clearly two types of ponds according to posterior pairwise analysis: on one hand, a. obstetricans tadpoles from permanent ponds displayed the steepest slopes, not differing in slope among them (f1,391 = 0.01, p = 0.863) but having the pond g6 a higher intercept than pond g1 195intraspecific variation in tadpole morphology (f1,392 = 25.6, p < 0.001). on the other hand, tadpoles from temporary and ephemeral ponds showed more gentle slopes, not differing among them neither in slope (f2,32 = 0.06, p = 0.883) nor in the intercept (f2,34 = 2.65, p = 0.131) (fig. 1, both pond typologies grouped together for clarity). relationship between tail depth and total length of h. meridionalis tadpoles differed in slope (f2,286 = 36.2, p < 0.001) and intercept (f2,286 = 8.44, p = 0.039) among the three studied ponds when tested all together (fig. 1). according to pairwise tests, the slope of the ephemeral pond is significantly more gentle than the ones of permanent (f1,275 = 70.7, p < 0.001) and temporary (f1,56 = 11.69, p = 0.001) ponds. tadpoles from the permanent and temporary ponds did not differ in slope (f1,241 = 0.71, p = 0.261). differences in the intercept disappeared in pairwise analyses (all p > 0.05). differences in morphology between r. temporaria tadpoles from the temporary and permanent ponds (fig. 2) were significant in both studied years, being the slope between tail depth and total length of tadpoles always steeper in the permanent pond (f1,266 = 6.48, p = 0.003 for 1985, and f1,189 = 29.84, p < 0.001 for 1986). differences in the intercept were also found in both cases (f1,266 = 51.1, p < 0.001 for 1985, and f1,189 = 70.3, p < 0.001 for 1986). differences in experimental morphology between d. pictus tadpoles under or without predation risk from anax sp. included as well a significant interaction (f1,84 = 10.93, p = 0.001), being the slope between tail depth and total length of tadpoles steeper when a caged predator was present (fig. s1). the same applies for experimental data on p. punctatus (f1,85 = 6.29, p = 0.014), being again the slope steeper when a caged predator was present (fig. s2). discussion morphological differences among ponds were found in all studied species, and variation, when present, 20 40 60 8010 20 30 40 50 60 70 80 2 4 6 8 10 12 14 16 18 20 ta il de pt h (m m ) total length (mm) alytes obstetricans permanent pond g1 permanent pond g6 temporary and ephemeral ponds alytes obstetricans permanent pond g1 permanent pond g6 temporary and ephemeral ponds a) 10 15 20 25 1 2 3 4 5 6 7 8 ta il de pt h (m m ) total length (mm) hyla meridionalis permanent pond g6 temporary pond g2 ephemeral pond g3 hyla meridionalis permanent pond g6 temporary pond g2 ephemeral pond g3 b) fig. 1. intraspecific morphological variation among different nearby natural ponds from garraf np (for pond information see supplementary material): a) alytes obstetricans, b) hyla meridionalis. 10 20 30 40 5010 15 20 25 30 35 40 45 50 55 2 4 6 8 10 12 14 ta il de pt h (m m ) total length (mm) permanent pond m3 temporary pond m5 permanent pond m3 temporary pond m5 a) 10 20 30 40 5010 15 20 25 30 35 40 45 50 55 2 4 6 8 10 12 14 ta il de pt h (m m ) total length (mm) permanent pond m3 temporary pond m5 permanent pond m3 temporary pond m5 b) fig. 2. intraspecific morphological variation in rana temporaria from two nearby natural pools of montseny np in consecutive years (for pond information see supplementary material): a) year 1985, b) year 1986. 196 eudald pujol-buxó et alii agreed with theory: tadpoles had deeper fin tails as they were collected in ponds with a longer hydroperiod. thus, observations coincide with theoretical predictions, arguably posing the trade-off among desiccation and predation risk (skelly, 1995) as the possible underlying cause of the observed intraspecific morphological differences. unluckily, given that these observations were not originally taken to explore this hypothesis, we lack data on predator density and diversity in the studied ponds – among other potentially useful data –, making impossible to assess if the observed morphological trends are in each case rather a consequence of desiccation risk, predation risk, or both. interestingly, morphological differences among pond typologies were always expressed through a significant interaction between pond type and total length, that is, as changes in the relationship among both measures along growth (i.e., slope differences seen in fig 1 and fig 2). thus, when morphological differences are found among pond typologies, these become more exaggerated as tadpoles are larger in size, coinciding with the re-analyzed experimental data on anti-predator morphology from pujol-buxó et al. (2013), and being consistent with similar studies examining tadpole morphology along wide size ranges (relyea, 2003). morphological differences between alytes obstetricans tadpoles from the two permanent ponds, where differences were found in the intercept, represent the only exception to this pattern. the exaggeration of morphological differences with size might be consistent with previous works reporting that behavioural defences are, in relative terms, more used in the first stages of tadpole life, while morphological differences become more marked as tadpoles grow larger (relyea, 2003; pujol-buxó et al., 2017). which is the process creating the variation we observe in these ponds? the two ponds from montseny np are separated less than 1km, and the mean distance among studied ponds in the other study area (garraf np) is approximately 3.15 km (tables s1 and s2). given these distances, we cannot discard gene flow and therefore we suggest a role of phenotypic plasticity in shaping the observed morphological differences (dewitt and scheiner, 2004; van buskirk, 2009). however, another complementary option is that, even assuming moderate rates of gene flow (lind et al., 2011), after several generations of natural selection the sub-populations breeding in the different ponds have also constitutively departed in their morphology (ledón-rettig et al., 2008; lind et al., 2011; van buskirk, 2014). this could be expressed in a default production of – or a greater tendency to produce – deeptailed tadpoles in populations usually breeding in permanent ponds and shallow-tailed tadpoles in populations from temporary and ephemeral ponds. interestingly, our data of r. temporaria in different consecutive years from the same two ponds shows that although general patterns may repeat year after year, exact results – the degree of morphological divergence – may vary across years (fig. 2). thus, in both areas, neither microevolutionary processes among nearby ponds – mediated by processes like genetic accommodation (ledón‐rettig et al., 2008; wund et al., 2008) – nor a prominent role of phenotypic plasticity cannot be totally disregarded. further studies specifically designed to disentangle the relative role of these mechanisms would be needed. finally, it is necessary to highlight that, although results agreed with prediction and the number of tadpoles sampled was high in some cases, our observations are based on too few ponds to be conclusive, and other additional studies would be needed to confirm the observed pattern. acknowledgements the study was carried according to the national and local laws in force at the time, and both natural parks gave permission to capture and to euthanize. we are grateful to several reviewers for useful discussions on the manuscript. supplementary material supplementary material associated with this article can be found at <http://www.unipv.it/webshi/appendix> manuscript number 20894. references campeny, r. 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(2008): a test of the “flexible stem” model of evolution: ancestral plasticity, genetic accommodation, and morphological divergence in the threespine stickleback radiation. am. nat. 172: 449-462. acta herpetologica vol. 12, n. 2 december 2017 firenze university press meristic and morphometric characters of leptopelis natalensis tadpoles (amphibia: anura: arthroleptidae) from entumeni forest reveal variation and inconsistencies with previous descriptions susan schweiger1, james harvey2, theresa s. otremba1, janina weber1, hendrik müller1,* brown anole (anolis sagrei) adhesive forces remain unaffected by partial claw clipping austin m. garner*, stephanie m. lopez, peter h. niewiarowski species and sex comparisons of karyotype and genome size in two kurixalus tree frogs (anura, rhacophoridae) shun-ping chang1,2, gwo-chin ma2,3,4, ming chen2,5,6,7,8,*, sheng-hai wu1,* non-native turtles in a peri-urban park in northern milan (lombardy, italy): species diversity and population structure claudio foglini1, roberta salvi2,* species composition and richness of anurans in cerrado urban forests from central brazil cláudia márcia marily ferreira1,*, augusto cesar de aquino ribas2, franco leandro de souza3 the life-history traits in a breeding population of darevskia valentini from turkey muammer kurnaz, alı i̇hsan eroğlu, ufuk bülbül*, halıme koç, bılal kutrup influence of desiccation threat on the metamorphic traits of the asian common toad, duttaphrynus melanostictus (anura) santosh mogali*, srinivas saidapur, bhagyashri shanbhag predation of common wall lizards: experiences from a study using scentless plasticine lizards jenő j. purger*, zsófia lanszki, dávid szép, renáta bocz reproductive timing and fecundity in the neotropical lizard enyalius perditus (squamata: leiosauridae) serena najara migliore1,2,*, henrique bartolomeu braz2,3, andré felipe barreto-lima4, selma maria almeida-santos1,2 observations on the intraspecific variation in tadpole morphology in natural ponds eudald pujol-buxó1,2,*, albert montori1, roser campeny3 and gustavo a. llorente1,2 reliable proxies for glandular secretion production in lacertid lizards simon baeckens diet of juveniles of the venomous frog aparasphenodon brunoi (amphibia: hylidae) in southeastern brazil rogério l. teixeira1, ricardo lourenço-de-moraes2, débora c. medeiros3, charles duca3, rogério c. britto4, luiz c. p. bissoli5, rodrigo b. ferreira3,* who are you? the genetic identity of some insular populations of hierophis viridiflavus s.l. from the tyrrhenian sea ignazio avella, riccardo castiglia, gabriele senczuk* acta herpetologica 10(1): 31-37, 2015 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-14988 basal frequency of micronuclei and hematological parameters in the side-necked turtle, phrynops hilarii (duméril & bibron, 1835) maría a. latorre1,2,*,#, evelyn c. lópez gonzález1,2,#, pablo a. siroski1,2,3, gisela l. poletta1,2,4 1 “proyecto yacaré” – laboratorio de zoología aplicada: anexo vertebrados, facultad de humanidades y ciencias, unl/ministerio de aguas, servicios públicos y medio ambiente, av. aristóbulo del valle 8700, santa fe, argentina. *corresponding author. e-mail: gul_16@hotmail.com 2 consejo nacional de investigaciones científicas y técnicas, conicetav. rivadavia 1917, caba, argentina 3 instituto de ciencias veterinarias del litoral, icivet litoral-unl-conicetr.p. kreder 2805, esperanza, santa fe, argentina 4 cát. toxicol. y bioq. legal, fbcb-unl, ciudad universitaria, paraje el pozo s/n, santa fe, argentina # these authors contributed equally to this work submitted on 2014, 15th september; revised on 2014, 22nd october; accepted on 2014, 27th october editor: giovanni scillitani abstract. the present study aimed to evaluate basal frequency of micronuclei (mn) and hematological values in adult phrynops hilarii in order to propose this aquatic turtle, broadly distributed in our region, as a biological monitor for future studies of environmental pollution assessment. thirty-two adult turtles from a semi-natural environment located at the zoological experimental station (santa fe, argentina) were used. blood samples were taken and the following parameters were determined: basal frequency of mn (bfmn), total red blood cells (rbc) count, hematocrit (ht), hemoglobin (hb), total and differential white blood cells (wbc). the bfmn determined for the species was 3.56 ± 1.39, while hematological parameters showed the following reference values: 0.937  ±  0.12 x106 rbc/µl, 27062.50 ± 4565.43 wbc/mm3, hematocrit 18 ± 1.81% and hb concentration 4.80 ± 0.45 g/dl. differential wbc counts were: 76 ± 2.90% for lymphocytes, 20.12 ± 2.56% for heterophils, 1.5 ± 0.19% for monocytes, and 2.12 ± 0.61% for eosinophils, while no basophils were observed in any of the samples analyzed. no differences were observed between males and females in any of the variables analyzed. data provided in this work could be useful as reference values for future studies of natural regions where p. hilarii occurs, employing this species as a sentinel organism for genotoxic and immunotoxic evaluation of environmental pollutants. keywords. genotoxicity, immune system, hematological values, micronucleus test. introduction phrynops hilarii (duméril and bibron, 1835), commonly called side-necked turtle, belongs to the suborder pleurodira, family chelidae. this species is distributed in the east of uruguay, southern brazil, almost all of paraguay and argentina (cabrera, 1998; van dijk et al., 2012). in this country, it can be found in the provinces of buenos aires, santa fe, entre rios, corrientes, chaco, misiones, formosa, córdoba, tucuman and mendoza (richard, 1999). this species inhabits in streams and lagoons which in many cases constitute important areas of pollutants discharge, generally coming from agricultural and industrial activities. evaluation of toxics effects produced by chemicals on hematological, immunological and genetic parameters in wild species is of fundamental importance for maintaining biodiversity, as they are 32 m.a. latorre et alii good indicators of the health of populations in potential exposure to different types of xenobiotics (donald, 2004; poletta et al., 2008). the immune system (is) is an excellent indicator of the health of an organism (burns et al., 1996). its integrity is essential for the defense against infectious organisms and their toxic products and, therefore, for the survival of all individuals. the is is also very sensitive to chemicals exposure and considering the fast response of some immune parameters, many of them are used as markers of such exposure (lafuente giménez et al., 2001). in this sense, the exposure of wild or domestic animals to certain chemicals, whether acute or chronic, may affect the defense mechanisms. this information also allows the identification of associated diseases such as anemia, malnutrition, dehydration, inflammation, parasitemia, hematopoietic malignancies and disorders of hemostasis (barboza et al., 2007). white blood cells (wbc) are the cellular components of the is and are involved in a significant number of processes. certain situations may cause an increase or decrease of the values of selected blood components; in turn, these are used for the interpretation of physiological phenomena or accurate diagnosis of diseases and nutritional status (gonzález fernández, 2003). in addition, traditional hematological parameters may provide information about the general stress, especially in birds and reptiles (gross and siegel, 1983; morici et al., 1997; lance and elsey, 1999). as it has been mentioned above, many individual factors (age, sex, stress, nutritional condition, hormones concentrations, body hydration levels, etc.) as well as environmental factors (temperature, pressure, oxygen concentrations, etc.) may affect hematological values and so they can be considered as biomarkers of exposure (dessauer, 1970; aguirre et al., 1995). on the other hand, biomarkers of genotoxicity reveal alterations induced by various agents on genetic material of organisms. in particular, the mn test (schmid, 1975) detects the effects of agents which modify the structure and/ or segregation of chromosomes by the identification of acentric fragments and/ or lagging chromosomes that remain separated from the main nucleus of the daughter cells during cell division. this technique allows the detection of early biological responses, before the damage is irreversible and causes imbalances in the health of an organism (carballo and mudry, 2006). thus, the determination of the frequency of mn (fmn) is used as an indicator of the effects of various agents on the genetic material and the “machinery” associated with cell division (schmid, 1975). the basal frequency of micronuclei (bfmn) is the result of “normal” errors in the processes of replication and/ or cell division, which are not caused by the incidence of genotoxic agents. it is specific for each species and each cell population, and constitutes a reference value to determine the usefulness of a species as a sentinel or biological monitor. in reptiles, the mn test was applied to peripheral blood erythrocytes in different species (zuñiga gonzález et al., 2000; poletta et al., 2008; schaumburg et al., 2012), being a useful tool to determine the genotoxic potential of chemical (poletta et al., 2009; poletta et al., 2011; lópez gonzález et al., 2013) and physical agents (schaumburg et al., 2010). up to our knowledge, there is no report on the literature about the application of the mn test in this species, while the studies on hematologic values in phrynops hilarii are extremely scarce (pitol et al., 2007). the aim of this study was to determine the bfmn and reference hematological values in adult p. hilarii, in order to propose this aquatic turtle of great importance in our region, as a biological monitor for future studies of environmental pollution assessment. materials and methods animals all animals in this study were treated in accordance with the reference ethical framework for biomedical research: ethical principles for research with laboratory, farm, and wild animals (national scientific and technical research council, 2005), using non-invasive techniques of blood collection and minimizing stress and suffering by suitable management methods. thirty-two adults phrynops hilarii (from 0.61 to 5.46 kg, fig.  1) from a semi-natural environment at zoological experimental station (santa fe, argentina), were bled (1 ml) immediately from the external jugular vein (rogers and booth, 2004). all individuals were sexed, weighed and measured morphometrically. micronucleus test (mn) the mn test was conducted according to the technique described by poletta et al. (2008) for its application in peripheral blood erythrocytes of another reptile species, with some modifications. two smears were performed per animal, fixed with ethanol and stained with 10% giemsa solution previously centrifuged and filtered. the baseline frequency of mn (bfmn: number of cells with mn/1000 erythrocytes analyzed) was determined under an optical microscope at 1000x. hematological parameters for hematological study the following parameters were determined: rbc (red blood cells) and wbc (white blood cells) 33micronuclei and hematological parameters in phrynops hilarii counts, differential leukocyte count (dlc), ht and hb. the total leukocyte count was performed using a neubauer chamber due to the presence of nucleated erythrocytes. in this species, like in all non-mammalian vertebrates, the use of automatic counter is impossible because the presence of nucleated erythrocytes gives wrong numbers. an aliquot of blood was diluted 1:200 with 0.6% nacl (lewis et al., 2008). the samples were observed under an optical microscope at 400x. for the dlc two smears were made per animal on clean slides, fixed with ethanol for 10 min and stained with may grünwald (50%)-giemsa (10%) solutions. the samples were observed under an optical microscope at 1000x. hb concentration was determined using an autoanalyzer. statistical analysis the statistical analysis was conducted with the software spss 17.0 for windows. mean and standard error (se) of each variable analyzed were calculated from data of all animals. all variables were analyzed in normality by kolmogorov-smirnov test and homogeneity of variances by levene test. we used the mann whitney u-test to compare the bfmn and monocytes between males and females, while the rest of the variables were analyzed trough the student t-test. linear regressions were carried out to analyze the relation between reference values and weight of the animals. a p value <  0.05 was considered statistically significant. results reference values of all variables analyzed and the particular values for males and females are shown in table 1 (fig. 2). concerning hematological parameters no basophils were observed in any of the dlc analyzed and no differences were observed between males and females in any of the variables (p > 0.05, table 1). in the case of the bfmn, females showed higher values than males, but the difference was not statistically significant (p  >  0.05, table 1). no relationship was found between bfmn and weight of the animals (p > 0.05, r2 = 0.001). discussion currently, there is great interest in those studies that allow collecting information on the health status of wild populations. in the case of reptiles, hematological techniques have been evolving favorably (wilmoth, 1994; lowell, 1998), however, there are few hematology studies on the testudines freshwater turtles (metin et al., 2006; pitol et al., 2007). reference hematological values found for different species of turtles (troiano et al., 1998; montilla fuenmayor et al., 2006; cabrera et al., 2011) differ from the values reported in our study, and this can be explained by the multiple factors that influence them, such as the species, the environment where they live and their geographic distribution. in the case of rbc and wbc counts, we found higher values than those reported by other authors (troiano et al., 1998; montilla fuenmayor et al., 2006; cabrera et al., 2011; table 2). it has to be noted that the conditions and habits of the turtles were different in the mentioned works and this clearly exert a particular influence on certain hematological parameters. hematocrit of phrynops hilarii was similar to those found in other turtle species (troiano et al., 1998; montilla fuenmayor et al., 2006; cabrera et al., 2011), while hemoglobin was lower than the values reported by troiano et al. (1998) and cabrera et al. (2011) (table  2). leukocytes populations showed different results. in the case of lymphocytes, our values were higher than those reported in similar species (troiano et al., 1998, montilla fuenfig 1. adult specimens of phrynops hilarii (approximately 4-5 kg). table 1. reference values for all variables studied in p. hilarii, including males and females particular values (bfmn, baseline frequency of micronuclei; rbc, red blood cells; se, standard error; wbc, white blood cells). variables (mean ± se) males females total rbc (x106/µl) 0.937 ± 0.12 0.98 ± 0.23 0.91 ± 0.15 total wbc (x103/µl) 27.06 ± 4.56 26 ± 2.75 27 ± 7.47 hematocrit (%) 18 ± 1.81 20.17 ±0.35 16.73 ± 2.84 hemoglobin (g/dl) 4.80 ± 0.45 5.17 ± 0.16 4.58 ± 0.73 lymphocytes (%) 76 ± 2.90 77.0 ± 7.02 75.40 ± 2.94 heterophils (%) 20.12 ± 2.56 18.33 ± 5.84 21.20 ± 2.71 monocytes (%) 1.5 ± 0.19 1.33 ± 0.33 1.60 ± 0.24 eosinophils (%) 2.12 ± 0.61 1.67 ± 0.33 2.4 ± 0.98 bfmn 3.56 ± 1.39 3.08 ± 2.39 4.27 ± 1.92 34 m.a. latorre et alii mayor et al., 2006; cabrera et al., 2011) but the opposite was detected for heterophils. with respect to monocytes, our values were lower than those found in chelonia mydas (montilla fuenmayor et al., 2006) and chelonoidis chilensis chilensis (troiano et al., 1998), but higher than those of geochelone denticulata (cabrera et al., 2011). unlike, eosinophils found in phrynops hilarii were lower than those reported by troiano et al. (1998) and cabrera et al. (2011) in chelonoidis chilensis chilensis and geochelone denticulata, respectively, but higher than that of chelonia mydas (montilla fuenmayor et al., 2006) (table  2). basophils were not observed in any of the animals studied; this could be due to the small number of this cell type present in the circulation of healthy animals (work et al., 1998). as we mentioned, the influence of factors such as age, sex, reproductive status, stress, temperature, time of year, capture techniques and methods of analysis could modify reference values substantially. because of this, in order to be compared with those of other species, it is extremely important to describe in details most of the factors and conditions involved. similar to our study, some authors found no significant differences in hematological values between females fig 2. (1) micronucleated erythrocyte, (2) lymphocyte and (3) eosinophil (arrows) of phrynops hilarii stained with may grunwald giemsa (1000x). table 2. hematological parameters reported by different authors in turtles (hb, hemoglobin; ht, hematocrit; rbc, red blood cells; wbc, white blood cells). hematological parameters referencewbc (106/µl) rbc (103/ µl) ht (%) hb (g/dl) lymphocytes (%) heterophils (%) monocytes (%) eosinophils (%) basophils (%) phrynops hilarii 0.937 27.06 18 4.8 76 20.12 1.5 2.12 0 this study chelonoidis chilensis chilensis 0.70 9.2 25.2 11 26 28 5 32 0 troiano et al. (1998) chelonia mydas 0.42 6.16 29.4 __ 14.7 82.9 1.97 0.47 ___ montilla fuenmayor et al., (2006) geochelone denticulata 0.44 7.82 20.3 7.0 25.5 55.6 0.4 15.8 1.5 cabrera et al. (2011) 35micronuclei and hematological parameters in phrynops hilarii and males in caiman crocodilus (rossini, 2004), while other authors reported such differences in other reptiles species (frair, 1977; wood and ebanks, 1984; hart et al., 1991). we only found a moderate positive correlation between heterophils and weight. several studies in recent years have used the mn test as a biomarker of genotoxicity in various species of reptiles exposed to different chemicals (poletta et al., 2009; 2011; borrat et al., 2011; capriglione et al., 2011; lópez gonzález et al., 2013). primarily, it is necessary to establish basal values of mn as it is species-specific and may vary with age, sex and health status of individuals. the results of our study have determined that bfmn of p. hilarii is 3.56  ±  1.39. this value is much higher than those found by our group in other endemic reptile species from argentina. we found a bfmn of 0.87 ± 0.74 in the broad-snouted caiman (caiman latirostris; poletta et al., 2008) and of 0.95 ± 0.27 in the tegu lizard (tupinambis merianae; schaumburg et al., 2012). this finding could probably be attributed to factors intrinsic to the species, such as lifespan of circulating erythrocytes and removal time of senescent or damaged erythrocytes, or to phylogenetic distance (caliani et al., 2014). moreover, zúñiga-gonzález et al. (2000; 2001) reported bfmn in species of lizards, snakes, and crocodiles between 0.10 and 0.30/1000, while in species of turtles (macroclemys temminckii; n = 1 and kinosternon subrubrum; n = 2) the value reported was 0/1000. the low values obtained in the cited study could be due to the small number of individuals used, as no more than one or two animals were used per species. however, similar data were obtained by strunjak-perovic et al. (2010) for the snake hierophis gemonensis (bfmn  = 0.30/1000; n = 10). borrat et al. (2011) studied chelonia mydas (marine green turtle) as a biological monitor to evaluate the level of pollution in a contaminated area (n = 60). in this study, animals used as controls were rehabilitated specimens with a fmn of 9/1000 erythrocytes, while animals coming from two contaminated sites showed a fmn of 42 and 627/1000 erythrocytes, respectively. even when the control fmn was much higher than the one found in our study, it is important to highlight that animals used were rehabilitated, so comparison is not appropriate. finally, we found no relationship between bfmn and weight. these results agree with those reported in juvenile caiman latirostris (poletta et al., 2008) and adults tupinambis merianae (schaumburg et al., 2012); newborn tegu lizards showed instead a weak negative correlation between the bfmn and weight, possibly indicating a relation between poor nutritional state and deficiencies in the mechanisms of protection and/or repair of genetic damage (schaumburg et al., 2014). this study is the first report on the application of the mn test and determination of reference hematological values in phrynops hilarii, considering individual variation due to factors such as sex, age and health conditions of individuals. this represents an important contribution to the knowledge of the biological characteristics of this species and is the initial step to propose it as a sentinel organism for future studies on the biomonitoring of pollutants present in its natural habitat. acknowledgements agencia nacional de promoción científica y tecnológica (pict 2011-1349: glp), universidad nacional del litoral (caid 201150120110100189), consejo nacional de investigaciones científicas y técnicas. references aguirre, a., balazs, g., speaker, t., gross, t. 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(2001): differences in the number of micronuleated erythrocytes among young and adult animals incluiding humans. spontaneus micronuclei in 43 species. mutat. res. 494: 161-167. acta herpetologica vol. 10, n. 1 june 2015 firenze university press obituary: valery k. eremchenko (1949-2014) leo j. borkin1, tatjana dujsebayeva2, roberto sindaco3, matthias stöck4 haplotype variation in founders of the mauremys annamensis population kept in european zoos barbora somerová1, ivan rehák2,*, petr velenský2, klára palupčíková1, tomáš protiva1, daniel frynta1 reproductive ecology of sichuan digging frogs (microhylidae: kaloula rugifera) wei chen1,*, lina ren2, dujuan he2, ying wang2, david pike3 toxic effects of carbaryl on the histology of testes of bufotes variabilis (anura: bufonidae) özlem çakici basal frequency of micronuclei and hematological parameters in the side-necked turtle, phrynops hilarii (duméril & bibron, 1835) maría a. latorre 1,2,*,#; evelyn c. lópez gonzález1,2, #; pablo a. siroski1,2,3; gisela l. poletta1,2,4 into a box interiors: clutch size variation and resource allocation in the european pond turtle marco. a.l. zuffi1,*, simonetta citi2, elena foschi1, francesca marsiglia1, eva martelli1 where to “rock”? choice of retreat sites by a gecko in a semi-arid habitat andreia penado1,2, ricardo rocha3,4,*, marta sampaio3, vanessa gil3, bruno m. carreira3, rui rebelo3 age structure, growth and longevity in the common toad, rhinella arenarum, from argentina clarisa de l. bionda1,2,*, silvia kost 4, nancy e. salas1, rafael c. lajmanovich3, ulrich sinsch4, adolfo l. martino1 on a putative type specimen of pleurodema bibroni tschudi, 1838 from chile (anura: leptodactylidae) daiana paola ferraro re-description of the external morphology of phyllomedusa iheringii boulenger, 1885 larvae (anura: hylidae), with comments on the external morphology of tadpoles of the p. burmeisteri group samanta iop¹, victor mendes lipinski¹, bruno madalozzo¹, franciele pereira maragno¹, sonia zanini cechin¹, tiago gomes dos santos² book review: harold heatwole, john w. wilkinson (eds). amphibians biology. volume 11 status of conservation and decline of amphibians. eastern hemisphere. part 4 . southern europe and turkey sebastiano salvidio book review: antonio romano. atlante degli anfibi del parco nazionale del cilento vallo di diano e alburni distribuzione, biologia, ecologia e conservazione sebastiano salvidio acta herpetologica journal of the societas herpetologica italica acta herpetologica 11(2): 101-109, 2016 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-18261 predator-prey interactions between a recent invader, the chinese sleeper (perccottus glenii) and the european pond turtle (emys orbicularis): a case study from lithuania vytautas rakauskas1,*, rūta masiulytė1, alma pikūnienė2 1 life sciences centre, vilnius university, saulėtekio ave. 7, lt-10221 vilnius, lithuania. *corresponding author. e-mail: vytucio@ gmail.com 2 lithuanian zoo, radvilėnų str. 21, lt-50299 kaunas, lithuania submitted on may, 5th 2016; revised on june, 11th 2016; accepted on june, 12th 2016 editor: uwe fritz abstract. the european pond turtle, emys orbicularis, is a critically endangered species in most european countries. habitat degradation and fragmentation are considered the main reasons for the decline of e. orbicularis. however, the spread of invasive species may also contribute to the disappearance of e.  orbicularis populations. we examined the range overlap and predator-prey interactions between the invasive chinese sleeper, perccottus glenii, and e.  orbicularis through controlled experiments and in field studies. field surveys showed that both species occupied similar habitats. predator-prey experiments suggested that newly hatched turtles are resistant to p.  glenii predation. conversely, adults of e.  orbicularis consumed juvenile p.  glenii even when other food sources were available. overall, these findings suggested that e.  orbicularis is not among the potential prey organisms in the diet of the invasive p.  glenii, and that this fish does not directly contribute to the decline of e. orbicularis in europe. keywords. turtle, aquatic invasion, endangered species, inland waters, lithuania. introduction at present, there is no doubt that one of the main causes of the loss of biodiversity is the spread of introduced, invasive species. these species reduce local biodiversity through both indirect competitive and direct predatory impacts on resident native populations. the spread of invasive species is now considered to be an international problem, and local governments are actively working to reduce their spread and impacts (eu regulation no 1143/2014). however, aquatic invasive species spread is on-going, and its impacts on local communities cannot be predicted (sundseth, 2014). understanding the pattern of invasive species interactions with native species can help us to predict those effects and build better strategies for protection of endangered species. from a biodiversity management point of view, the impact of the invasive versatile predator chinese sleeper, perccottus glenii (dybowski, 1877) on populations of the endangered native european pond turtle, emys orbicularis (linnaeus, 1758) is one such case in point. perccottus glenii, a fish native to the amur river basin, eastern asia (mori, 1936; berg, 1949). this is one of the most widespread alien invasive freshwater fish in eurasia (reshetnikov, 2010; reshetnikov and ficetola, 2011). it is also a recent invader in the baltic sea region (aleksejevs and birzaks, 2011; witkowski and grabowska, 2012; reshetnikov and karyagina, 2015; pupina et al., 2015; rakauskas et al., 2016). in europe p.  glenii occurs mostly in water bodies that either have weak current or are stagnant, with well developed aquatic vegetation. such habitats include river flood plains, littoral zones 102 vytautas rakauskas et alii of lakes, and swampy water bodies (reshetnikov, 2010; reshetnikov and ficetola, 2011; pupina et al., 2015). shallow, well-vegetated and isolated lakes, ponds, drainage ditches and oxbows are important reserves of biological diversity for many groups of aquatic and semi-aquatic animals, including pond turtles. colonisation of such water bodies by p.  glenii leads to a dramatic simplification of ecosystem taxonomic structure. p.  glenii is a versatile predatory fish and represents a specific threat for macroinvertebrates and small fish and amphibians (shlyapkin and tikhonov, 2001; reshetnikov, 2003; reshetnikov, 2004; koščo et al., 2008; grabowska et al., 2009; reshetnikov, 2013). due to its generalist predatory habit, it has been assumed that p.  glenii preys upon hatchling e.  orbicularis turtles (pupina et al., 2015). adult turtles can deter predation by their size and bony shell, but hatchlings lack the adult’s size and shell strength. populations of e.  orbicularis hatchlings were assumed to be under p.  glenii predation pressure until their first or second years. this paper addresses the predator-prey interactions between p.  glenii and locally endangered e.  orbicularis. concerns about p.  glenii are based on observations that this fish (1) inhabits similar habitats as e.  orbicularis (mitrus, 2004; najbar, 2007; briggs and reshetylo, 2009; reshetnikov, 2010; reshetnikov and ficetola, 2011); (2) is an opportunistic, competitive predator that rises to the top of the food chain in invaded ecosystems (reshetnikov 2003; reshetnikov, 2004; reshetnikov, 2013); and (3) grows large enough to prey on newly hatched e.  orbicularis. this scenario, however, has not been well substantiated for interactions in european waters. to fill this knowledge gap, we performed predatorprey experiments between p.  glenii and two aquatic turtle species. in laboratory experiments we tested the capacity of p.  glenii to consume newly hatched chinese pond turtles, mauremys reevesii (gray, 1831), when it is the only available prey. it was assumed that there would be no differences in p.  glenii feeding preferences for same-sized e.  orbicularis and m.  reevesii juveniles. additionally, we tested the capacity of the adult e.  orbicularis to consume 0+ age p.  glenii when several prey sources were available. as a complement to the laboratory studies, we analyzed the overlap of recent distributions of p.  glenii and e. orbicularis species in natural lithuanian freshwaters. material and methods study area natural habitat overlap between p.  glenii and e.  orbicularis species was assessed for the inland waters of lithuania. lithuania stands within the baltic sea drainage basin, along the southeastern shore of the baltic sea. the country has a territory of 64,800 km2, which is divided by seven main river basins (kažys, 2013) (fig. 1a). there are 2,850 lakes that have surface areas larger than 0.005 km2, and 3,150 smaller lakes, with a combined area of 913.6 km2. in addition, there are 1,132 reservoirs and more than 3,000 ponds in lithuania (kažys, 2013). distribution of p. glenii and e. orbicularis the recent distribution of p.  glenii and e.  orbicularis in lithuanian inland waters was calculated from widely published (rakauskas et al., 2016) and specialty (bastytė, 2015) publications. additional data on the presence of p.  glenii within the local range of e.  orbicularis was provided by our own (to be published) surveys. the results of our surveys include data from 32 lentic water bodies, each with a surface area smaller than 0.3 km2 and each known to have been occupied by e.  orbicularis (fig. 1b). p.  glenii populations were investigated by dip net (25 × 25 cm opening, 1.5 mm mesh size) sampling in water depths of 0.3-1.3 m in areas of the vegetated littoral zone (pupina et al., 2015). from five to ten study sites were examined on each water body waving with a dip net for ten minutes at each study site. in general, if p. glenii was to be found in a body of water, it was caught within the first 5 minutes (rakauskas pers. obs.). experiment 1 (p. glenii vs. m. reevesii) predator-prey experiments (p.  glenii vs. m.  reevesii) were conducted to determine if the invasive predator p.  glenii recognize newly hatched aquatic turtles as potential prey, and if they consume them under experimental conditions. m.  reevesii was chosen as a model for e.  orbicularis as it is easy available, not protected, and is of similar size and coloration as e.  orbicularis (mitrus and zemanek, 2000; stephens and wiens, 2003; lovich et al., 2011; lin et al., 2015). previous studies have shown that e.  orbicularis juveniles are similar length (approximate 26 mm) (drobenkov, 2000; mitrus and zemanek, 2000; zinenko, 2004; najbar and mitrus, 2013) when they first reach the water, as were the m.  reevesii juveniles used in these experiments (3.0 ± 0.1 mm). e. orbicularis is less available and is protected by european and local laws (council directive 92/43/eec; rašamavičius, 2007). newly hatched (two week old) m.  reevesii were transported to the laboratory one week before the experiments from the local zoo-shop. the experiment turtles were maintained in twelve 10.6-l aquaria (22.5 cm deep and long × 21 cm wide) filled with tap water forming a closed circulation system with an ammonia filter (38.5 cm deep and wide × 49.0 cm long aquarium filled with expanded clay and jbl filter start (gmbh & co, germany)). during this period, daily rations of frozen midge larvae were provided. adults of p.  glenii were collected from small lakes using electric fishing gear (samus-725mp) in september 2015. for one week before conducting the experiments, all fish were accli103predator-prey interactions of perccottus glenii and emys orbicularis mated separately in twelve 92.4-l rectangular tanks (49.0 cm deep and long × 38.5 cm wide) filled with tap water forming a closed circulation system with an ammonia filter. during this period, daily rations of live rutilus rutilus (linnaeus, 1758) fry were provided at ~4% of the fish’s body weight. the fish were starved for 48 h before the predation experiment. in total, 12 p.  glenii and m.  reevesii individuals were used in these experiments (tables 1 and 2). predator-prey experiments were performed in the same experimental aquaria where fish were acclimatised. each experimental aquarium was filled with tap water to 44 cm depth resulting in an actual water volume of 83 l per aquarium, with water flow through each experimental aquarium of 3.0 l min-1. animals were kept under a 15 hours per day photoperiod and at a temperature of 19 ºc throughout these studies. no substrate was added into the experimental aquaria. after acclimation period, a single individual of m.  reevesii was added to each aquarium. during the experiments, turtles were fed frozen midge larvae every second day. overall, the fish were allowed to forage for one week, after which turtles were removed back to their acclimation aquaria and the conditions of all turtles in each experimental aquarium were recorded. assessment of turtle condition was based on appearance and movement. surviving turtles were crawling and no injuries were seen on their skin or carapace. these turtles were kept for three months after the experiments to confirm that they retained their normal viability and activity levels. experiment 2 (e. orbicularis vs. p. glenii) predation experiments (e.  orbicularis vs. p.  glenii) were conducted to determine if e.  orbicularis recognize p.  glenii juveniles as prey and consumed them. approximately 150 p.  glenii juveniles were collected from a small lake using a standard dip net (25 × 25 cm) in august 2015. all fish were small enough to be preyed upon by turtles and were selected for size similarity (body length of 3.3 ± 0.2 cm) to avoid cannibalism. for one week before conducting the experiments, all specimens were acclimated in six 92.4-l rectangular tanks (49.0 cm deep and long × 38.5 cm wide) filled with tap water forming a closed circulation system with an ammonia filter. daily rations of live midge larvae were provided. predator-prey experiments (e. orbicularis vs. p. glenii) were performed in august 2015. three concrete water reservoirs were used for the experiments. reservoirs were in cages under the natural outdoor conditions. cages were made from stainless steel to protect the experiment from wild birds and animals. the first cage contained two identical reservoirs of approximate 5 m2 area (2.8 m long × 1.8 m wide, with a range of 5−45º slope and 0.1−0.6 m of water depth). the second cage contained one large rectangular reservoir of approximate 10 m2 area (4.5 m long × 2.3 m wide, with a range of 5-45º slope and 0.1-0.7 m of water depth). all reservoirs were filled with tap water. woody shelters, stones and muddy gravel substrate were present in all experimental reservoirs. an average noonday temperature of table 1. percottus glenii specimens used in predator-prey experiments. values are means ± standard deviation. experiment i experiment ii first cage experiment ii second cage number of used specimens 12 27 33 range of total body length, cm 17.0-24.7 2.9-3.2 3.3-3.7 average of total body length, cm 20.3 ± 3.0 3.0 ± 0.2 3.5 ± 0.1 range of total weight, g 90.3-280.0 0.9-1.5 1.1-1.8 average of total weight, g 150.8 ± 62.1 1.2 ± 0.2 1.4 ± 0.2 range of p. glenii mouth diameter, cm 3.0-4.8 – – average of p. glenii mouth diameter, cm 3.7 ± 0.6 – – table 2. mauremys reevesii and emys orbicularis specimens used in predator-prey experiments. values are means ± standard deviation. experiment i m. reevesii experiment ii (1) e. orbicularis experiment ii (2) e. orbicularis number of specimens 12 10 8 range of carapace length, cm 2.9-3.1 9.5-12 15.2-18.1 average of carapace length, cm 3.0 ± 0.1 10.8 ± 0.8 16.9 ± 1.1 range of carapace width, cm 2.3-2.0 7.6-10.1 13.6-16.0 average of carapace width, cm 2.2 ± 0.1 8.7 ± 0.7 14.8 ± 0.9 range of plastron length, cm 2.7-2.9 7.8-11.5 12.0-18.5 average of plastron length, cm 2.8 ± 0.1 9.8 ± 1.6 15.5 ± 2.5 range of total weight, g 6.8-7.2 10.6-37.1 39.0-167.0 average of total weight, g 7.0 ± 0.1 22.2 ± 9.8 89.5 ± 42.6 104 vytautas rakauskas et alii 20.6 ± 3.2 ºc was observed. 18 e.  orbicularis adults big enough to prey on these juveniles were divided, with ten smaller individuals closed in the first experimental cage and eight larger ones in the second (table 2). after an acclimation period, 27 and 33 fish were transferred into the experimental reservoirs of the first and second cages respectively. in the first cage released individuals were equally divided (14 + 13) per each reservoir. fish in the second cage were slightly bigger compare to the fish in the first one (table 1). 60 fish were left as a control group in six acclimation tanks (ten individuals per tank) for the whole experiment period. overall, e.  orbicularis were kept with p.  glenii juveniles for two weeks. the turtles and the fish were fed once a day with dried or live insects, earth worms, snails, and small pieces of meat during the experiments. daily turtle food ratios are presented in table 3. moreover, there was lots of naturally breeding culex pipiens (linnaeus, 1758) larvae inside the reservoirs which were an alternative food source for the fish. the reservoirs were surveyed for dead fish every day during the experiments. after the experiment termination, reservoirs were pumped out, all remained fish were removed back to their acclimation aquaria and the condition of all fish in each reservoir was recorded. assessment of fish condition was based on appearance and movement. surviving fish were swimming and no external injuries were seen. results distribution of p. glenii and e. orbicularis in lithuania less than 400 e.  orbicularis survive, mostly in waters of southern part of the country (bastytė, 2015). meanwhile p.  glenii has been recorded in all of the country’s river basins (rakauskas et al., 2016). analysis of the recent distribution range of p. glenii and e. orbicularis in the inland waters showed these species overlapping on a regional scale. both species were recently reported from the nemunas river basin in southern lithuania (fig. 1a). additionally, our surveys of p. glenii presence in well known e.  orbicularis habitats revealed the small-scale overlap of these two species. both were found inhabiting one shallow water body (fig. 1b). four p.  glenii individuals of body lengths ranging from 43 to 61 mm were captured in a water body inhabited by e.  orbicularis. seven water bodies with well-known e.  orbicularis populations were occupied by carassius carassius (linnaeus, 1758). experiment 1 (p. glenii vs. m. reevesii) experiments showed that none of the tested p.  glenii adults were capable of ingesting or even injuring newly hatched m.  reevesii turtles, though tested fish appeared big enough to do so. the mouth diameter of tested fish was significantly larger than the carapace lengths of the turtles (mann-whitney u test: z = 3.2, p < 0.002; tables 1 and 2). however, only a few predation signs were observed during the first ten minutes after newly hatched turtles were offered for p. glenii predation. the largest tested fish specimens (tl > 22.0 cm and mouth diameter > 4.0 cm) repeatedly attacked turtles for the first few minutes. in these cases the prey was completely sucked in the fish mouth cavity and jaws were fully closed. however, after a few seconds the prey was expelled undamaged. overall, none of the tested turtles were injured; all were alive and healthy three months after the experiments. experiment 2 (e. orbicularis vs. p. glenii) adult e.  orbicularis were found to consume p.  glenii juveniles in caged experiments. 15 individual p.  glenii (55.6% of the cohort) were missing after 14 days in the first cage where smaller turtles were foraging. similarly, 13 p.  glenii (39.4%) were missing in a cage with larger turtles. no dead or injured fishes were found during or after the experiment. predatory behaviour was frequently observed. turtles stalked and struck at p.  glenii juveniles. cannibalism was not observed among the fish, including in control tanks, where all 60 specimens left in tanks survived the experimental period. discussion the european pond turtle, e.  orbicularis is the most widely distributed freshwater turtle species in europe (fritz, 2001, 2003). the geographic range of e.  orbicularis extends from north africa over most of europe to latvia and to lake aral in the middle east (fritz, 1998; fritz, 2001, 2003). however, the turtle is critically endangered in lithuanian, and all other european waters (council directive 92/43/eec; fritz, 2001, 2003; rašamavičius, table 3. daily turtle diet rations of various food types per specimens during the experiments. food type quantity flesh fish 10 (g) flesh meat 10 (g) dried crustacean 5 (ind.) beetle larvae (live) 5 (ind.) insects (cricket, cockroach) (live) 3 – 4 (ind.) earthworm (live) 5 – 10 (ind.) snail (live) 3 – 4 (ind.) midge larvae (live) 10 (ind.) cabbage 1 (g) 105predator-prey interactions of perccottus glenii and emys orbicularis 2007). in lithuania reproductive e.  orbicularis populations number less than 400 turtles and are found mostly in waters of the southern part of a country (fritz and günther, 1996; meeske, 2008; bastytė, 2015). habitat degradation and fragmentation have been identified as the main reasons for the decline of e. orbicularis populations (kovacs et al., 2004; fritz and chiari, 2013; bastytė, 2015). however, the recent spread of invasive aquatic species may also negatively affect e.  orbicularis populations. the introduction of exotic trachemys scripta (schoepff, 1792) has been reported to have indirect effects on e.  orbicularis populations. introduced t.  scripta competes against e.  orbicularis for habitat resources, and reduces the survival of the turtle (cadi and joly, 2004; lacomba and sancho, 2004). the invasive predatory fish micropterus salmoides (lacépède, 1802), is assumed to prey on e. orbicularis hatchlings and juveniles (lacomba and sancho, 2004; ayres and cordero, 2007). correspondingly, direct and/ or indirect interactions between invasive predatory fish p.  glenii and e.  orbicularis could be expected. both species prefer similar habitats and diets. however, the impact of p.  glenii on the natural populations of e.  orbicularis in inland european waters remains largely undocumented. habitat overlap our review of the literature on p. glenii and e. orbicularis habitat preferences indicated that both species are commonly found in shallow waters with weak or with no current, and with well-developed aquatic vegetation (mitrus, 2004; meeske et al. 2006; najbar, 2007; briggs and reshetylo, 2009; reshetnikov, 2010; reshetnikov and ficetola, 2011; pupina et al., 2015). both species usually forage in littoral zones of small shallow lakes or swampy water bodies. both species are particularly abundant in small water bodies unsuitable for most ichthyophagous fishes. in these situations, they are the top predators (meeske et al., 2006; briggs and reshetylo, 2009; bastytė, 2015; reshetnikov, 2003; grabowska et al., 2009). therefore these two species could be expected to compete against, and even attack each other in these environments. our field surveys confirmed that p.  glenii and e.  orbicularis may settle in the same water bodies, although both species are relatively rare in the inland waters of lithuania. the presence of both species in the same water body was also reported from latvian inland waters (pupina et al., 2015). however, we found p.  glenii only in one (3.1%) from all investigated water bodies settled by e. orbicularis. an increase in conflicts between these two species is expected in a future where both species expand their ranges within the inland waters of lithuania. the rapid natural spread of p.  glenii is ongoing not only in lithuanian (rakauskas et al. 2016) but also in other european waters (alexandrov et al., 2007; grabowska et al., 2010; mastitsky et al., 2010; wolter and röhr, 2010; reshetnikov and ficetola, 2011; semenchenko et al., 2011; movchan 2015). the fish pursues a profligate reproductive strategy, and exhibits resistance to adverse conditions fig. 1. (a) distribution of the european pond turtle, emys orbicularis (squares) and the chinese sleeper, perccottus glenii (triangles) in the inland waters of lithuania (followed by bastytė, 2015; rakauskas et al., 2016). different river basins are marked by different shading. (b) p. glenii presence in water bodies inhabited by e. orbicularis: presence (opened circles), absence (closed circles). 106 vytautas rakauskas et alii (reshetnikov, 2004). on the other hand, an intensive reestablishment program of e.  orbicularis populations is ongoing in lithuanian waters. up to 100 individuals are released annually to areas they formerly occupied (pikūnienė unpub. data). therefore, there is little doubt that direct and indirect interactions between these two species will increase. predator-prey interactions in small lakes unsuitable for most ichthyophagous fish, p.  glenii can grow up to 25 cm in body length (reshetnikov, 2003; pupina et al., 2015) and become a top predator (reshetnikov, 2003; koščo et al., 2008; grabowska et al., 2009). in such waters almost all links of the trophic network converge to p.  glenii. although adult p.  glenii feed primarily on fish and large insects, they occasionally prey on newts, frogs or other larger animals (shlyapkin and tikhonov, 2001; reshetnikov, 2003; reshetnikov, 2004; koščo et al., 2008; grabowska et al., 2009; reshetnikov, 2013). due to this dietary breadth, it has been assumed that large p. glenii individuals are capable of ingesting and eating e. orbicularis hatchlings (pupina et al., 2015). newly hatched pond turtles are less than 3 cm in length (najbar and mitrus, 2013) and could be attacked by p.  glenii, which has a wide gape (up to 5 cm) and forages in shallow waters. however, our experiments did not support the hypothesis that p.  glenii may directly, through the predator-prey interaction, threaten e.  orbicularis populations. none of the tested specimens of p.  glenii consumed or injured newly hatched turtles, even though they were big enough to prey on them and even though no other food was available to them. thus it was concluded that the presence of p.  glenii would not directly threaten wild e. orbicularis populations. we used a different pond turtle species m.  reevesii instead of e.  orbicularis during the experiments. the applicability of our conclusions to e.  orbicularis is supported by the similarity of the behaviour of the two turtle species in the water and the similarities in their sizes and their camouflage (mitrus and zemanek, 2000; stephens and wiens, 2003; lovich et al., 2011; lin et al., 2015). our results showed that only the largest p.  glenii specimens showed interest in preying on turtle juveniles. p.  glenii specimens shorter than 22 cm length did not even approach newly hatched turtles. we used p.  glenii specimens in a range of sizes, up to the largest found in european waters, 24.7 cm of a total body length (the biggest recorded specimens have been 25 cm in total body length; reshetnikov, 2003; pupina et al., 2015). specimens over 20 cm were found only in 11 (14.6%) of a total 75 lentic waters bodies inhabited by p.  glenii in lithuania (rakauskas unpub. data). similar results were obtained from other countries where p.  glenii was usually reported to live for about 5-7 years and reach up to 15 cm length (grabowska et al., 2011; nowak et al., 2008; grabowska et al., 2009; terlecki and palka, 2012; kutsokon et al., 2014). our findings suggest that e.  orbicularis consumption by predatory p.  glenii in natural environments where many of other food sources are available is not likely. similar results were obtained with other pond turtle and predatory fish species. britson and gutzke (1993) revealed that although turtle hatchlings of t.  scripta and chrysemys picta (schneider, 1783) were attacked by predatory fish m.  salmoides they were subsequently rejected unharmed. it was concluded that hatchling behaviour such as clawing or biting may be harmful to the gill apparatus or digestive tract of fish and thus provides defence against predation (britson and gutzke, 1993). our experiments revealed that adults of e. orbicularis preyed on p.  glenii juveniles even when other food sources were available. fish fry consumption by e.  orbicularis has been shown in other studies (kotenko, 2000; briggs and reshetylo, 2009). no cannibalism cases were observed within a control fish group during our experiments, suggesting that the fish were consumed by the turtles. our findings suggest that not only will p.  glenii not directly endanger european pond turtle populations, but that e.  orbicularis may even control p.  glenii populations where their ranges overlap. concluding remarks the decline in european pond turtle populations likely results from a complex set of factors, linked to the modern decline in biodiversity worldwide. however, this study demonstrates that one factor is probably not the threat posed to these turtles by the invasive predatory fish p.  glenii. conversely, we found that mature adults of e.  orbicularis can prey on p.  glenii juveniles. the turtle could possible come to control the invasive fish with the increase of the habitat overlap between the two species. p.  glenii may impact local e.  orbicularis populations indirectly, through depletion of available macroinvertebrate food sources in invaded water bodies (reshetnikov, 2001; reshetnikov, 2003). benthic invertebrates usually dominate in both species diet (lebboroni and chelazzi, 1991; kotenko, 2000; ottonello et al., 2005; ficetola and de bernardi, 2006; koščo et al., 2008; grabowska et al., 2009). p.  glenii may also serve as a vector for e.  orbicularis parasite transfer (pupina et al., 2015). the fish is a host for the e.  orbicularis parasitic nematode spiroxys 107predator-prey interactions of perccottus glenii and emys orbicularis contortus (rudolphi), whose paratenic hosts also include small fish, insect larvae, tadpoles and adult anura (hedrick, 1935, moravec, 1994). further investigation of the growth and physiology conditions of e.  orbicularis during the preand post-invasion periods of p.  glenii in various water bodies will be needed to sort out these multiple effects. acknowledgements we thank the environmental protection agency of lithuania, under the ministry of environment of the republic of lithuania, for the permits used to collect, breed and grow e.  orbicularis hatchlings and adults at the lithuanian zoo: permits no. (6)-a4-2084; lgf-1440; 032/2014. we also thank the lithuanian zoo for the permit to conduct predator-prey experiments within the zoo: permit no. lzs-96/2015. the zoo shop “zuvytes. com” is greatly appreciated for providing us with newly hatched m.  reevesii turtles. for the permission to catch p.  glenii we are also grateful to the environmental protection agency under the ministry of environment of the republic of lithuania, permit no. 056/2015. comments and suggestions of anonymous reviewers and steve daubert (university of california, davis) greatly improved the manuscript. references aleksejevs, e., birzaks, j. (2011): long-term changes in the ichthyofauna of latvia’s inland waters. scientific journal of riga technical university 7: 9-18. alexandrov, b., boltachev, a., kharchenko, t., lyashenko, a., son, m., tsarenko, p., zhukinsky, v. (2007): trends of aquatic alien species invasions in ukraine. aquat. invasions. 2: 215-242. ayres, c., cordero, a. 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(2004): notes on egg-laying, clutch size and hatchling feeding of emys orbicularis in the kharkiv region, ukraine. biologia, bratislava, 59/suppl. 14: 149-151. acta herpetologica vol. 11, n. 2 december 2016 firenze university press predator-prey interactions between a recent invader, the chinese sleeper (perccottus glenii) and the european pond turtle (emys orbicularis): a case study from lithuania vytautas rakauskas1,*, rūta masiulytė1, alma pikūnienė2 effective thermoregulation in a newly established population of podarcis siculus in greece: a possible advantage for a successful invader grigoris kapsalas1, ioanna gavriilidi1, chloe adamopoulou2, johannes foufopoulos3, panayiotis pafilis1,* the unexpectedly dull tadpole of madagascar’s largest frog, mantidactylus guttulatus arne schulze1,*, roger-daniel randrianiaina2,3, bina perl3, frank glaw4, miguel vences3 thermal ecology of podarcis siculus (rafinesque-schmalz, 1810) in menorca (balearic islands, spain) zaida ortega*, abraham mencía, valentín pérez-mellado growth, longevity and age at maturity in the european whip snakes, hierophis viridiflavus and h. carbonarius sara fornasiero1, xavier bonnet2, federica dendi1, marco a.l. zuffi1,* redescription of cyrtodactylus fumosus (müller, 1895) (reptilia: squamata: gekkonidae), with a revised identification key to the bent-toed geckos of sulawesi sven mecke1,*,§, lukas hartmann1,2,§, felix mader3, max kieckbusch1, hinrich kaiser4 the castaway: characteristic islet features affect the ecology of the most isolated european lizard petros lymberakis1, efstratios d. valakos2, kostas sagonas2, panayiotis pafilis3,* sources of calcium for the agamid lizard psammophilus blanfordanus during embryonic development jyoti jee1, birendra kumar mohapatra2, sushil kumar dutta1, gunanidhi sahoo1,3,* mediodactylus kotschyi in the peloponnese peninsula, greece: distribution and habitat rachel schwarz1,*, ioanna-aikaterini gavriilidi2, yuval itescu1, simon jamison1, kostas sagonas3, shai meiri1, panayiotis pafilis2 swimming performance and thermal resistance of juvenile and adult newts acclimated to different temperatures hong-liang lu, qiong wu, jun geng, wei dang* olim palus, where once upon a time the marsh: distribution, demography, ecology and threats of amphibians in the circeo national park (central italy) antonio romano1,*, riccardo novaga2, andrea costa1 on the feeding ecology of pelophylax saharicus (boulenger 1913) from morocco zaida ortega1,*, valentín pérez-mellado1, pilar navarro2, javier lluch2 notes on the reproductive ecology of the rough-footed mud turtle (kinosternon hirtipes) in texas, usa steven g. platt1, dennis j. miller2, thomas r. rainwater3,*, jennifer l. smith4 heavy traffic, low mortality tram tracks as terrestrial habitat of newts mikołaj kaczmarski*, jan m. kaczmarek book review: sutherland, w.j., dicks, l.v., ockendon, n., smith, r.k. (eds). what works in conservation. open book publishers, cambridge, uk. http://dx.doi.org/10.11647/obp.0060 sebastiano salvidio acta herpetologica 12(1): 117-121, 2017 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-19982 good vibrations: a novel method for sexing turtles donald t. mcknight1,2,*, hunter j. howell3, ethan c. hollender1, day b. ligon1 1 department of biology, missouri state university, spring field, missouri, usa. *corresponding author. e-mail: donald.mcknight@ my.jcu.edu.au 2 college of science and engineering, james cook university, townsville, queensland, australia 3 department of biological sciences, towson university, towson, maryland, usa submitted on december 19th, 2016; revised on april 18th, 2017; accepted on april 21th, 2017 editor: ernesto filippi abstract. the ability to accurately determine the sex of individuals is important for research and conservation efforts. while most species of turtle exhibit secondary sexual dimorphisms that can be used to reliably infer sex, there are some species that are very difficult to sex, and even within many dimorphic species, it is not uncommon to encounter individuals that appear to exhibit both male and female secondary sex characteristics. therefore, we tested the novel method of using a vibrator to sex turtles by stimulating male turtles to evert their penises. we tested this method on males of four species (three families) with known sexual dimorphisms: spiny softshell turtles (apalone spinifera; n = 14), western chicken turtles (deirochelys reticularia miaria; n = 17), mississippi mud turtles (kinosternon subrubrum hippocrepis; n = 10), and common musk turtles (sternotherus odoratus; n = 9). the method accurately sexed 100% of a. spinifera, 64.7% of d. r. miaria, 80.0% of k. s. hippocrepis, and 55.6% of s. odoratus. despite the low success rates in some species, there are situations in which this method will be useful for researchers working with species that are difficult to sex using external morphological characteristics. keywords. apalone, chelonia, deirochelys, kinosternon, penis, sternotherus, vibrator. the ability to accurately differentiate males and females is important for ecological studies, and for many turtle species this is a relatively simple task. turtles often exhibit a variety of secondary sexual dimorphisms in traits such as size, color, claw length, plastron shape, and pre-cloacal tail length (gibbons and lovich, 1990; readel et al., 2008). nevertheless, some species lack obvious dimorphisms, and dimorphisms may vary among populations (iverson, 1985; rowe, 1997). further, even for species that are strongly dimorphic, it is not uncommon to encounter individuals that appear to have some characteristics of males and some characteristics of females, thus making them difficult to sex (mcknight, pers. obs.). several methods to overcome these problems are available, such as measuring testosterone levels (owens et al., 1978; rostal et al., 1994), laparoscopy (wibbels et al., 1989; ligon et al., 2009), and cloacoscopy (ligon et al., 2013); however, these methods are often invasive, time-consuming, and difficult to implement in the field. recently, two methods have been published for inducing penile erections in male turtles, thus allowing males and females to be differentiated. while penile eversion is a common method for sexing squamates, it has received little attention in turtles. although this is a promising technique, the methods that have been proposed so far appear to be species-specific and have only been applied to common snapping turtles (chelydra serpentina), whose penis can be everted by gently bouncing a turtle up and down (de solla et al., 2001; dustman, 2013), and cotinga river toadhead turtles (phrynops tuberosus), whose penis can be everted by immobilizing the neck and limbs (rodrigues et al., 2014).   118 d.t. mcknight et alii vibrators may provide a more broadly applicable method of penile eversion. lefebvre et al. (2013) found that a vibrator could be used to induce ejaculation in male turtles, and ejaculation was preceded by a visible erection. therefore, this method may be valuable as a means of sexing turtles. we examined its utility on four species of freshwater turtle representing three different families. to test our method of using a vibrator to induce erections in males, we used species that can be sexed using external sexual dimorphisms such as size, color, and tail morphometrics. thus, we could test the efficiency of the method by seeing how frequently it induced an erection in individuals that were known to be males. the four species that we used were: western chicken turtles (deirochelys reticularia miaria; family: emydidae), mississippi mud turtles (kinosternon subrubrum hippocrepis; family: kinosternidae), common musk turtles (sternotherus odoratus; family: kinosternidae), and spiny softshell turtles (apalone spinifera; family: trionychidae). we captured them using hoop nets placed in ponds in southeastern oklahoma (detailed trapping methods in mcknight et al., 2015). once a male turtle was captured, we attempted to induce an erection by applying an 18 cm, variable-speed, silver bullet vibrator to its shell and tail. we vibrated turtles for 10 min or until an erection was achieved, and we recorded the amount of time that it took to induce an erection. trials were scored as “unsuccessful” if an erection had not been induced by the end of the 10-minute trial period. our preliminary trials indicated that turtles needed to be fairly relaxed and willing to extend their limbs and tails before the method would be effective. therefore, for the sake of time, we limited our trials to turtles that were already active at the time of capture. although this is a drawback of our method, most turtles quickly acclimate to being handled, and a few moments of holding a turtle is generally sufficient for it to extend its limbs (mcknight, pers. obs.). during our trials, we held turtles vertically, with their plastrons facing the researcher that was operating the vibrator. then, we gently applied the tip of the vibrator to the plastron, carapace, and tail (fig. 1). we moved it among those regions based on the turtles’ responses (i.e., if a turtle responded by tightly pulling its limbs and tail against its body, we moved to a different area). for each species, erections generally occurred when the vibrator was placed on the tail itself, but it was often necessary to first vibrate areas other than the tail, because starting with the tail generally resulted in the turtles pulling the tail tightly against the body, rather than extending it. therefore, we started with the plastron or carapace, and moved to the tail once a turtle had fully extended its tail. in general, turtles appeared to respond best when only the tip of the vibrator was touching them and when the vibrator had fresh batteries and was set on the fastest setting. also, they seemed to respond best when the tip was held firmly against them (rather than allowing it to bounce), but not be pressed hard against them. both allowing it to bounce and pressing it too hard generally resulted in turtles holding their limbs and tail tightly against the body, rather than relaxing. additionally, it was often useful to move the vibrator around in small, slow, steady circles. as a general rule, we tried to hold the vibrator against the tail whenever possible (including following the tail if the turtle is waving it from side to side), but if this caused the turtle to retract its tail, then we moved the vibrator to a different position until the tail was extended again. finally, sometimes males only protracted their penises briefly and quickly retracted them, rather than maintaining an erection. therefore, it was necessary to watch the cloaca closely. although this was the general pattern, each species responded differently, so we had to adapt our protocol based both on the species and individual responses, and it will be necessary to test different positions and techniques when trying this method on a new species. for a. spinifera it was generally not necessary to spend time on parts of the body other than the tail. they usually extended their tails immediately and would allow us to hold the vibrator against their tails. they did frequently wave their tails from side to side, forcing us to move the vibrator with the tail, but they generally did not hold the tail against the body in a stressed position. in contrast, k. s. hippocrepis, s. odoratus, and d. r. miaria usually held their tails against their bodies initial-fig. 1. a male spiny softshell turtle (apalone spinifera) being vibrated on the tail. 119a novel method for sexing turtles ly and required stimulation to other parts of their body before they would relax and extend their tails. for k. s. hippocrepis and s. odoratus, this generally involved moving the vibrator in slow, small circles on the abdominal and pectoral scutes (the diameter of the circle was only 1-2 cm). deirochelys r. miaria was similar, but it was usually necessary to vibrate slightly higher (more on the pectoral scutes than abdominal scutes). also, sometimes they responded to being vibrated on the carapace (usually on the first vertebral scute). finally, lefebvre et al. (2013) reported that, when inducing male turtles to ejaculate, vibrating turtles on their heads was often effective, however, that method generally did not work in our study. this further illustrates the differences among species and highlights the need for testing several different locations and methods when vibrating a species for the first time. in addition to the differences in the techniques necessary for stimulating the different species, our success rates also varied among species (table 1). the method was the most successful for a. spinifera (100%), followed by k. s. hippocrepis (80.0%). it was less successful for d. r. miaria (64.7%) and s. odoratus (55.6%). we compared the success rates among species using a fisher’s exact test, and this showed that there was a statistically significant difference (p = 0.026). the median time required to induce an erection also varied among the species, but it was lowest in a. spinifera and k. s. hippocrepis. because the utility of this method varied among species, it will need to be validated on a species by species basis. despite the low success rate in some species, we think that this method has potential to be useful in several situations. first, based on our success employing this technique on a. spinifera and k. s. hippocrepis, it should be useful for some species or populations that are difficult to sex. however, it will first need to validated using individuals of a known sex (such as individuals in a captive population that have been sexed by other methods). if it is successful on those known individuals, then it will provide a cheap and non-invasive way of sexing that species in the field. second, even for species that can usually be sexed via secondary sexual dimorphisms, it is not uncommon to find individuals that possess some characteristics of both males and females, thus making them difficult to sex. this method can be applied to those individuals even if it has not been validated for that species. in other words, if the method has been validated, then the outcome of vibrating the turtle can be used to assign the sex as either male or female; however, if it has not been validated for that species, inducing an erection would allow the turtle to be sexed as a male, and failure to induce an erection would simply leave the turtle with an unassigned sex code. using a vibrator in this manner had already been helpful for sexing problematic individuals in our own research (mcknight, pers. obs.). third, it is often desirable to collect or monitor several individuals of a known sex (e.g., for movement studies). this is another situation where the method can be used even for species for which vibrating has a low or undetermined success rate. for example, if a research endeavor requires ten males of a species that is difficult to sex, an investigator could simply vibrate turtles until they had ten with erect phalli. finally, in situations where a researcher is working with a species, subspecies, or population for which secondary sexual dimorphisms are unknown or questionable, this method can be used to help validate a secondary sexual dimorphism. it is often possible to identify some individuals as females by palpating turtles for the presence of eggs (zuffi et al., 1999) or employing a sonogram to look for evidence of enlarged follicles, and using the method we described to vibrate turtles will allows some males to be identified. therefore, the combination of these two methods would allow researchers to easily compare the morphometrics of known males and known females to identify secondary sexual dimorphisms. indeed, this final method has proved useful in our research. at the outset of this project, we were not certain if our populations of d. r. miaria (a subspecies that has been poorly studied) were sexually dimorphic. we had a few known females (identified by the presence of eggs or enlarged follicles), but the majority of individuals appeared to be males (with a few immature females), resulting in a strongly skewed sex ratio (8:1 m:f [adult sex ratio], 4.7:1 [including suspected immature females]). based on our extensive trapping, the sex ratio did not appear to be a trapping artifact, but it was skewed enough that we were not confident that published sexual dimorphisms were correct for this subspecies at this location (ernst and lovich, 2009). however, by using the vibrator method to confirm that a subset of the suspected males were actually males, we were able to plot regressions (fig. 2), which then allowed us to use secondary sexual dimorphisms to confidently assign sex to turtles in our populations. table 1. sample sizes and results for the species that we vibrated. both “median time” and “time range” represent the time for trials that were successful. unsuccessful trials were aborted after 600 s. deirochelys reticularia miaria kinosternon subrubrum hippocrepis sternotherus odoratus apalone spinifera n 17 10 9 14 % successful 64.7 80 55.6 100 median time (s) 145 82 112 77 time range (s) 20–580 6–332 42–162 7–150 120 d.t. mcknight et alii in conclusion, although this method may not be a silver bullet for sexing all problematic turtle species, it is reliable for some species, and it has value even for species with low or undetermined rates of efficiency. it is cheaper, easier to implement in the field, and less invasive than many of the alternative techniques, and it has already proved useful in our own research. therefore, we think that it will enhance other research projects as well. acknowledgements we would like to thank the oklahoma nature conservancy and several private land owners for granting us access to their properties and assisting us with our research. the research presented here was conducted as part of a large survey effort that was funded by an oklahoma department of wildlife conservation state wildlife grant and the delta foundation and approved by the missouri state university institutional animal care and use committee (iacuc protocol no. 10014). we conducted the research under oklahoma department of wildlife conservation scientific collecting permits #5269, 5950, and 5610 and adhered to the asih/hl/ssar guidelines for use of live amphibians and reptiles. references de solla, s.r., portelli, m., spiro, h., brooks, r.j. (2001): penis displays of snapping turtles (chelydra serpentina) in response to handling: defensive or displacement behavior. chelonian conserv. biol. 4: 187-189. dustman, e.a. (2013): sex identification in the common snapping turtle (chelydra serpentina): a new technique and evaluation of previous methods. herpetol. rev. 44: 235-238. ernst, c.h., lovich, j.e. (2009): turtles of the united states and canada, 2nd ed. johns hopkins university press, baltimore, maryland, usa. gibbons, j.w., lovich, j.e. (1990): sexual dimorphism in turtles with emphasis on the slider turtle (trachemys scripta). herpetol. monogr. 4: 1-29. iverson, j.b. (1985): geographic variation in sexual dimorphism in the mud turtle kinosternon hirtipes. copeia 1985: 388-393. lefebvre, j., carter, s., mockford, s.w. (2013): new experimental method for semen extraction in freshwater turtles. herpetol. rev. 44: 595-600. ligon, d.b., bidwell, j. lovern, m.b. (2009): temperature effects on hatchling growth and metabolic rate in the african spurred tortoise, geochelone sulcata. can. j. zool. 87: 64-72. ligon, d.b., backues, k., fillmore, b.m., thompson, d.m. (2013): ontogeny of gonad and genital morphology in juvenile alligator snapping turtles (machrochelys temminckii). herpetol. conserv. biol. 9: 146-155. mcknight, d.t., harmon, j.r., mcknight, j.l., ligon, d.b. (2015): taxonomic biases of seven methods used to survey a diverse herpetofaunal community. herpetol. conserv. biol. 10: 666-678. owens, d.w., hendrickson, j.r., lance, v., callard, l.p. (1978): a technique for determining sex of immature chelonia mydas using radioimmunoassy. herpetologica 34: 270-273. readel, a.m., dreslik, m.j., warner, j.k., banning, w.j., phillips, c.a. (2008): a quantitative method for sex identification in emydid turtles using secondary sexual characters. copeia 2008: 643-647. rodrigues, j.f.m., soares, d.de o., silvia, j.r.f. (2014): sexing freshwater turtles: penile eversion in phrynops fig. 2. secondary sexual dimorphisms in western chicken turtles (deirochelys reticularia miaria). a) prior to vibrating several males, we had identified females by the presence of eggs, but because of the skewed sex ratio and large size of the confirmed females, we were not confident that the relative precloacal tail length was reliable for this species. b) the vibrator allowed us to confirm the sex of several males, thus establishing that this is a true sexual dimorphism (precloacal tail length was not recorded for two of the vibrated males). m = males, f = females, j = juveniles, (vibr) = confirmed via the vibrator, (eggs) = confirmed via the presence of eggs or enlarged follicles. regression lines are only shown for confirmed males and confirmed females. 121a novel method for sexing turtles tuberosus (testudines: chelidae). acta herpetol. 9: 259-263. rostal, d.c., grumbles, j.s., lance, v.a., spotila, j.r. (1994): non-lethal sexing techniques for hatchling and immature desert tortoises (gopherus agassizii). herpetol. monogr. 8: 83-87. rowe, j.w. (1997): growth rate, body size, sexual dimorphism and morphometric variation in four populations of turtles (chrysemys picta bellii) from nebraska. am. midl. nat. 138: 174-188. wibbels, t., morris, y.a., owens, d.w., dienberg, g.a., noell, j., leong, j.k., king, r.e., millan, r.m. (1989): predicted sex ratios from the international kemp’s ridley sea turtle head start research project. in: proceedings of the first international symposium on kemp’s ridley sea turtle biology, conservation, and management, pp. 77-81. caillouet, c.w., jr., landry, jr. a.m., eds, texas a&m university. zuffi, m.a.l., odetti, f., meozzi, p. (1999): body size and clutch size in the european pond turtle (emys orbicularis) from central italy. j. zool. soc. lond. 247: 139-143. acta herpetologica vol. 12, n. 1 june 2017 firenze university press morphological variation and sexual dimorphism in liolaemus wiegmannii (duméril & bibron, 1837) (squamata: liolaemidae) from uruguay joaquín villamil1,*, arley camargo2, raúl maneyro1 sex does not affect tail autotomy in lacertid lizards panayiotis pafilis1,*, kostas sagonas2, grigoris kapsalas1, johannes foufopoulos3, efstratios d. valakos4 fire salamander (salamandra salamandra) males’ activity during breeding season: effects of microhabitat features and body size raoul manenti*, andrea conti, roberta pennati call variation and vocalizations of the stealthy litter frog ischnocnema abdita (anura: brachycephalidae) pedro carvalho rocha1,2,*, joão victor a. lacerda2,3, rafael félix de magalhães2,3, clarissa canedo4, bruno v. s. pimenta5, rodrigo carrara heitor6, paulo christiano de anchieta garcia2,3 feeding ecology of two sympatric geckos in an urban area of northeastern brazil josé guilherme g. sousa1, adonias a. martins teixeira2,*, diêgo alves teles2, joão antônio araújo-filho2 and robson waldemar ávila3 a pattern-based tool for long-term, large-sample capture-mark-recapture studies of fire salamanders salamandra species (amphibia: urodela: salamandridae) jeroen speybroeck1,*, koen steenhoudt2,$ skeletal variation within the darwinii group of liolaemus (iguania: liolaemidae): new characters, identification of polymorphisms and new synapomorphies for subclades linda díaz-fernández*, andrés s. quinteros, fernando lobo marking techniques in the marbled newt (triturus marmoratus): pit-tag and tracking device implant protocols hugo le chevalier1, olivier calvez2, albert martínez-silvestre3, damien picard4, sandra guérin4,5, francis isselin-nondedeu6, alexandre ribéron1, audrey trochet1,2,* evidence for directional testes asymmetry in hyla gongshanensis jindongensis qing gui wu1, wen bo liao2,* the advertisement call of pristimantis subsigillatus (anura, craugastoridae) florina stănescu1,4, rafael márquez2, paul székely3,4,*, dan cogălniceanu1,4,5 nematodes infecting anotosaura vanzolinia (squamata: gymnophthalmidae) from caatinga, northeastern brazil bruno halluan s. oliveira¹, adonias a. martins teixeira¹,*, romilda narciza m. queiroz¹, joão a. araujo-filho¹, diêgo a. teles¹, samuel v. brito2, daniel o. mesquita¹ where is my place? quick chorus structure assembly in the european tree frog michal berec a possible mutualistic interaction between vertebrates: frogs use water buffaloes as a foraging place piotr zduniak1,*, kiraz erciyas-yavuz2, piotr tryjanowski3 good vibrations: a novel method for sexing turtles donald t. mcknight1,2,*, hunter j. howell3, ethan c. hollender1, and day b. ligon1 errata to mecke, s., hartmann, l., mader, f., kieckbusch, m., kaiser, h. (2016): redescription of cyrtodactylus fumosus (müller, 1895) (reptilia: squamata: gekkonidae), with a revised identification key to the bent-toed geckos of sulawesi. acta herpetologica 11(2): acta herpetologica 11(1): 85-89, 2016 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-16491 introduction of eleutherodactylus planirostris (amphibia, anura, eleutherodactylidae) to hong kong wing ho lee1, michael wai-neng lau2, anthony lau3, ding-qi rao4, yik-hei sung5,* 1 department of life science, tunghai university, taichung 407, taiwan 2 wwf-hong kong, kwai chung, hong kong sar, china 3 school of biological sciences, university of hong kong, pokfulam road, hong kong sar, china 4 kunming institute of zoology, the chinese academy of sciences, kunming 650223, yunnan, china 5 department of biology, hong kong baptist university, kowloon tong, hong kong sar, china. *corresponding author. e-mail: yhsung@hkbu.edu.hk submitted on 2015, 13th august; revised on 2016, 7th january; accepted on 2016, 9th january editor: uwe fritz abstract. an unidentified small frog species was first encountered in hong kong special administration region (sar), china, in 2000, where the local amphibian diversity is well-studied. we herein identified this unknown frog as eleutherodactylus planirostris (greenhouse frog) using dna barcoding. we found that its distribution in hong kong is widespread (>18 localities), and breeding has been observed in multiple occasions. the populations in at least four localities persisted for over seven years. we discuss its potential negative impacts to terrestrial ecosystems in hong kong, with particular concern of its potential competition with the endemic liuixalus romeri. we call for studies to investigate the impacts of the introduced e. planirostris on the local ecosystem. screening for e. planirostris in exported plants from hong kong should be carried out. keywords. amphibians, biological invasions, greenhouse frog, liuixalus romeri. the amphibian diversity of hong kong, is well studied (lau, 1999). owing to the subtropical climate, altitudinal range (highest peak at 957m) and variable terrain it contains a rich mixture of habitats. as a result the highly urbanized hong kong is home to a surprisingly high species richness of amphibians [23 species of frogs and 1 species of salamander (chan et al., 2005)]. in 2000, a morphologically distinct frog not assigned to any of the known native species was captured by the first author in a container yard at hung shui kiu in hong kong (fig 1, table 1). subsequently, additional frogs with similar morphology have been found at 18 localities between 2002 and 2015. in asia-pacific region, e. planirostris has been recorded in guam (christy et al., 2007a) and the philippines (olson et al., 2014; sy et al., 2015; sy and saigo, 2015); to our knowledge, this is the first reported case of e. planirostris introduction and establishment in continental asia. this study was conducted in hong kong special administrative region, china. distribution data of this frog species was compiled from region-wide opportunistic surveys between 2000 and 2015. geographic locations of where this species occurred were recorded using a handheld gps unit. two adult specimens were collected from wu kau tang in 2013 for molecular analysis and were deposited at the museum of biology, sun yat-sen university (museum voucher number: sys a004514 and sys a004515). genomic dna from the two specimens was extracted using a dna extraction kit (favorprep tissue genomic dna extraction mini kit, favorgen biotech corporation, ping-tung, taiwan) following manufacturer’s protocol and a 562-bp fragment of ribosomal 16s gene was 86 wing ho lee et alii amplified using polymerase chain reaction. the resulting products were sequenced on an abi prism 3730 automated dna sequencer and the results were submitted to the program blast to identify similar species from the genbank database and program mole-blast to produce phylogenetic tree. genetic sequences have been deposited in genbank (genbank accession number km252679 and km252680). a study has since been carried out to confirm the identity of this unknown species and to document its distribution in hong kong. the unknown frog has been identified as eleutherodactylus planirostris (greenhouse frog), a direct-developing eleutherodactylid native to a number of caribbean islands (bahamas, cayman islands and cuba) (dodd, 2013). it has been accidentally introduced to many countries throughout the new world (olson et al., 2012; andrew et al., 2011; heinicke et al., 2011), and asia-pacific islands including hawaii (kraus, 1999), guam (christy et al., 2007a) and the philippines (olson et al,. 2014; sy et al., 2015; sy and saigo, 2015), most likely via the live plant trade. the sequences of the frogs collected from wu kau tang had 100% max identity with the sequences of e. planirostris collected at naples, collier, florida (genbank accession number: dq283107; fig. 2) (frost et al., 2004). four sites (aberdeen, pokfulam country park, tai lam country park and tai tong) were revisited between 2014 and 2015 and robust e. planirostris populations were observed at all four sites. e. planirostris appear to be habitat generalist in hong kong, occupying a diversity of habitats including secondary forests, shrubland, agricultural fields, near fishponds, urban parks and near human settlements such as village houses and container yards. we found 22 eggs on wet leaf litter in aberdeen in may 2006 and the eggs hatched after two weeks in captivity. egg masses were also found in pokfulam country park and tai tong in july 2007. fig. 1. locations where eleutherodactylus planirostris were found in hong kong. letters identify the localities, as presented in table 1. the grey area indicates natural range of liuixalus romeri. asterisks indicates distribution localities of e. planirostris that overlap with translocation sites of l. romeri. 87introduction of eleutherodactylus planirostris to hong kong several individuals kept in captivity from 2009 to 2011 readily ate small crickets, spiders, termites and fruit flies. they produced egg clutches containing 11-17 eggs. e. planirostris feed on invertebrates including insects, spiders and snails (global invasive species database, 2010). in hong kong, e. planirostris is sympatric with eight species of anurans (duttaphrynus melanostictus, fejervarya limnocharis, hoplobatrachus rugulosus, kaloula pulchra, liuixalus romeri, megophrys brachykolos, odorrana chloronota and polypedates megacephalus). however, little is known about the diet of e. planirostris in the wild in hong kong and the population status of terrestrial invertebrates in hong kong, thus its impacts on native invertebrate populations remain unknown. in the originally amphibian-free hawaii, introduced e. planirostris can exist in high density, where it competes for food with other insectivores (olson et al., 2012; beard et al., 2009; beard and pitt, 2005) including birds (global invasive species database 2010). the congeneric eleutherodactylus coqui is listed as one of the world’s worst invasive alien species by iucn (lowe et al., 2000). on the ecosystem level, the introduction of e. coqui can alter ecosystem functioning by distorting nutrient cycling in hawaii (sin et al., 2008), however the impacts caused by e. coqui can be outweighed by that caused by invasive plants (tuttle et al., 2009). the impact of e. planirostris’s introduction on the endemic l. romeri is of potential concern. as adult e. planirostris are smaller than most of the frog species in hong kong, we speculate that resource competition will be most intense with the similar-sized, small bodied l. romeri (lau and zhao, 2004). competition between e. planirostris and l. romeri might be significant because (1) their sizes are similar and e. planirostris consume arthropods (olson et al., 2012) that overlap broadly with the natural preys of l. romeri (lau, 1998) which indicates a potentially high trophic similarity and (2) we observed e. planirostris dwell on the forest floors in secondary forests and forest edges, where l. romeri inhabit. we found e. planirostris at tai lam country park and kadoorie farm and botanic garden, where populations of l. romeri were translocated which was a mitigation measures of the construction of the chek lap kok airport in 1990s (lau and banks, 2008). e. planirostris has yet to be found in the natural range of l. romeri, including chek lap kok, lamma island, lantau island and po toi island (lau, 1999; fig. 1). monitoring of e. planirostris should be carried out in the l. romeri range and further studies are urgently needed to understand the table 1. distribution of eleutherodactylus planirostris and the respective year of discovery in hong kong. site label locations gps coordinates year of discovery a hung shui kiu 22°25.62’n, 113°59.59’e 2000 b tai tong 22°25.25’n, 114°1.33’e 2002 c aberdeen reservoir 22°15.34’n, 114°9.59’e 2006 d pokfulam country park 22°16.03’n, 114°8.34’e 2007 e hei ling chau 22°15.41’n, 114°1.90’e 2007 f shek kong 22°25.46’n, 114°6.38’e 2007 g tai lam country park 22°24.15’n. 114°2.19’e 2007 h chai wan 22°15.53’n, 114°13.65’e 2008 i tai shue wan 22°14.19’n, 114°9.96’e 2010 j university of hong kong 22°16.88’n, 114°8.24’e 2010 k shing mun country park 22°22.83’n, 114°8.56’e 2011 l kadoorie farm and botanic garden 22°25.98’n, 114°7.06’e 2011 m nam fung road 22°15.26’n, 114°10.68’e 2012 n shek kip mei 22°19.88’n, 114°10.09’e 2012 o wu kau tang 22 30.33’n, 114 14.63’e 2012 p long valley 22°30.64’n, 114°6.768’e 2012 q wong nai chung reservoir 22°15.44’n, 114°11.75’e 2013 r tai tam country park 22°15.54’n, 114°12.11’e 2015 fig. 2. phylogenetic tree derived from partial fragments of 16s rrna genes using fast minimum evolution method produced by program mole-blast. 88 wing ho lee et alii impacts of the introduced e. planirostris to the l. romeri. our results demonstrate that e. planirostris have established in hong kong. the populations at several sites, i.e., aberdeen, pokfulam country park, tai lam country park, tai tong, persisted for at least seven years. the reproduction of e. planirostris is successful in hong kong because (1) it is a direct-developing species (i.e. no aquatic larval stage) (goin, 1947), and (2) the suitable subtropical climate of hong kong [annual mean temperature = 22.8oc; mean annual precipitation = 2214 mm (dudgeon and corlett, 2004)] that matches the climate niche of the species (rödder and lötters, 2010). numerous control methods have been carried out to control e. coqui populations in hawaii, and are expected to have similar effectiveness on e. planirostris (olson et al., 2012). it is noteworthy that control methods for e. coqui are only effective in eradicating small and isolated populations (beard and pitt, 2005). if e. planirostris is found to have significant ecological impacts in hong kong, prompt control and eradication methods should be executed before the populations become too large. it is likely that e. planirostris was introduced accidentally to hong kong via the live plant trade, which was similar to the introduction to hawaii and guam (kraus et al., 1999; christy et al., 2007b). large volume of live plants was imported to hong kong from continental united states of america in early 2000s when e. planirostris was first detected in hong kong (census and statistics department, hong kong sar, 2001). this was supported by one frog being found in a potted tillandsia cyanea plant bought from the flower market in mong kok in urban kowloon in 2011 (louis fung, per. comm.) and another frog found in an apartment on ap lei chau in 2015 that was likely to arrive together with an indoor plant. in addition, we made observations of high densities of frogs (> 30 in 200 m2) near newly renovated slopes where nursery plants were planted on. due to the extensive trade of live plants in the region, it seems it is a matter of time that e. planirostris will spread to other places that import plants from hong kong. in 2015, hong kong exported or reexported over 100,000 kg of plants or parts of plants to tropical or subtropical countries/cities, including australia, china, macau, malaysia, singapore, thailand, taiwan and vietnam, where the climate may be favorable to the colonization of e. planirostris (census and statistics department, hong kong sar, 2015). screening for e. planirostris, including adults and eggs, in exported or re-exported plants from hong kong should be carried out. the small size and variations in coloration of e. planirostris as well as morphological similarity with certain asian species (e.g., liuixalus sp. and philautus sp.) prevented identification before molecular approach was applied to confirm its identity (armstrong and ball, 2005). this delayed proper measures to be taken to control this introduced species. prompt molecular analysis to identify suspicious alien species should be encouraged. we also hope this paper will help arouse the attention on this introduced frog in asia. acknowledgments we are grateful to louis fung, billy hau, pan lau, angie ng and philip yip for providing useful information. references armstrong, k., ball, s. 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(2009): invasive litter, not an invasive insectivore, determines invertebrate communities in hawaii forests. biol. invasions 11: 845-855. acta herpetologica vol. 11, n. 1 june 2016 firenze university press feeding habits of mesoclemmys vanderhaegei (testudines: chelidae) elizângela silva brito1,*, franco leandro souza2, christine strüssmann3 morphology, ecology, and behaviour of hylarana intermedia, a western ghats frog ambika kamath1, rachakonda sreekar2,3 a new account for the endangered cerrado rocket frog allobates goianus (bokermann, 1975) (anura: aromobatidae), with comments on taxonomy and conservation thiago ribeiro de carvalho1,2,*, lucas borges martins1,2, ariovaldo antonio giaretta1 molecular phylogenetics of the pristimantis lacrimosus species group (anura: craugastoridae) with the description of a new species from colombia mauricio rivera-correa, juan m. daza* population structure and activity pattern of one species of adenomera steindachner, 1867 (anura: leptodactylidae) in northeastern brazil maria juliana borges-leite1,*, joão fabrício mota rodrigues2, patrícia de menezes gondim1, diva maria borges-nojosa1 vocal repertoire of scinax v-signatus (lutz 1968) (anura, hylidae) and comments on bioacoustical synapomorphies for scinax perpusillus species group marco antônio peixoto1,*, carla silva guimarães1, joão victor a. lacerda2, fernando leal2, pedro c. rocha2, renato neves feio1 amendment of the type locality of the endemic sicilian pond turtle emys trinacris fritz et al. 2005, with some notes on the highest altitude reached by the species (testudines, emydidae) federico marrone*, francesco sacco, vincenzo arizza, marco arculeo photo-identification in amphibian studies: a test of i3s pattern marco sannolo1,*, francesca gatti2, marco mangiacotti3,4, stefano scali3, roberto sacchi4 no evidence for the ‘expensive-tissue hypothesis’ in the dark-spotted frog, pelophylax nigromaculatus li zhao, min mao, wen bo liao* no short term effect of clinostomum complanatum (trematoda: digenea: clinostomatidae) on survival of triturus carnifex (amphibia: urodela: salamandridae) giacomo bruni1,*, claudio angelini2 yeasts in amphibians are common: isolation and the first molecular characterization from thailand srisupaph poonlaphdecha, alexis ribas* introduction of eleutherodactylus planirostris (amphibia, anura, eleutherodactylidae) to hong kong wing ho lee1, michael wai-neng lau2, anthony lau3, ding-qi rao4, yik-hei sung5,* correlation between endoscopic sex determination and gonad histology in pond sliders, trachemys scripta (reptilia: testudines: emydidae) david perpiñán1, albert martínez-silvestre2,3, ferran bargalló3, marco di giuseppe4, jorge orós5, taiana costa6,* book review: john w. wilkinson. amphibian survey and monitoring handbook. pelagic publishing franco andreone book review: susan newman. frogs amphibians and their threatened environment. discovery and expression through art k-3. frogs are green franco andreone issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah acta herpetologica 9(1): 99-102, 2014 doi: 10.13128/acta_herpetol-13606 observations on the use of tarantula burrows by the anurans leptodactylus bufonius (leptodactylidae) and rhinella major (bufonidae) in the dry chaco ecoregion of bolivia christopher m. schalk1, marco sezano2 1ecology and evolutionary biology program, department of wildlife and fisheries sciences, and biodiversity research and teaching collections, texas a&m university, college station, texas, usa. corresponding author. e-mail: cschalk@tamu.edu 2sección de herpetología, museo de historia natural noel kempff mercado, santa cruz de la sierra, bolivia submitted on 2013,21st november; revised on 2014, 31st january; accepted on 2014, 2nd february abstract. some species of anurans have been observed utilizing burrows of other animals, such as rodents and tarantulas. here we report the observations of two anuran species, leptodactylus bufonius and rhinella major, utilizing the burrows of tarantulas (acanthoscurria sp.; family theraphosidae) in the dry chaco ecoregion of bolivia. both species of anurans never co-occurred with tarantulas in the burrows and used burrows that were wider in diameter and closer to breeding ponds as compared to the total available tarantula burrows in the area. these burrows may serve as refuges from predators, especially for conspicuous, calling males. keywords. acanthoscurria, amphibian, neotropics, predation, refuge, spider, theraphosidae, tropical dry forest. a number of animals are known to utilize cavities created by other animals, such as the many cavity-nesting birds that require tree cavities created by other animals for reproduction (scott et al., 1977). previous work has documented anurans using burrows of other animals including rodents, tortoises, and tarantulas (gentry and smith, 1968; cocroft and hambler, 1989; witz et al., 1991; dundee et al., 2012; schalk, 2012). while spiders are known predators of anurans at both the larval (schulze and jansen, 2010) and post-metamorphic (toledo, 2005) life stages, surveys of tarantula (family theraphosidae) burrows in both north america and south america have found that species of frogs in the family microhylidae actually take refuge within an occupied tarantula burrow (blair, 1936; cocroft and hambler, 1989; dundee et al., 2012). a study of the skin chemistry of gastrophryne carolinensis (family microhylidae) demonstrated that they produce toxic skin secretions making them unpalatable to spiders and thus allow them to coexist with the tarantulas within their burrows (garton and mushinsky, 1979). although previous studies have focused on anuran use of burrows still occupied by the tarantula, anurans and other taxa can also utilize abandoned burrows (witz et al., 1991). during a 50-day survey in the dry chaco ecoregion of bolivia, we observed two species of anurans, leptodactylus bufonius (family leptodactylidae) and rhinella major (family bufonidae) utilizing tarantula (acanthoscurria sp., family theraphosidae) burrows. to our knowledge, this is the first instance of anuran species outside the family microhylidae using tarantula burrows. also, while anurans in the gran chaco have been observed utilizing the burrows of the vizcacha (lagostomus maximus; family chinchillidae) (cei, 1980; schalk, 2012), this is the first documentation of anurans in the gran chaco ecoregion utilizing tarantula burrows. the objectives of this study were to document if these anurans 1) overlapped temporally with tarantulas in their occupancy of a burrow, and 2) utilized a non-random subset of the available burrows based on microhabitat characteristics of the burrows. 100 c.m. schalk, m. sezano the study was conducted in an area of the bolivian gran chaco within the vicinity of yande yari (18° 41’ 30.516” s, 62° 18’ 6.9474” w), a park guard camp in the kaa-iya of the gran chaco national park, cordillera province, santa cruz department, bolivia. the climate of the chaco is seasonal, having a wet, hot summer (november–march), and a dry, cool winter (april–october). average rainfall and temperature for this area are 513 mm and 24.6 ºc, respectively (navarro and maldonado, 2002). the area is characterized by scrubby, short trees (e.g., schinopsis lorentzii and aspidosperma quebracho-blanco), while shrubs (capparis sp., acacia sp.), bromeliads, and cactii, (e.g., opuntia sp., cleistocactus baumannii and eriocereus guelichii) are common understory plants (navarro and maldonado, 2002). as part of an effort to document the reptiles and amphibians in the area, we conducted nightly time-constrained visual encounter surveys from 9 february 2012 to 29 march 2012 (schalk et al., in press). our visual encounter surveys were conducted between 20:00 h and 00:00 h along one of four trails at the camp. each night we surveyed one of the four available trails, which was randomly chosen each night. while conducting these visual encounter surveys, we also conducted a two-step survey process of the occupants of the tarantula burrows found along the trail. the first step consisted of identifying the burrow to be that of a tarantula’s by confirming it as an occupant. when we encountered a burrow, we shined our flashlight down a burrow to determine if it was occupied. when a burrow was occupied by a tarantula, we measured its diameter to the nearest mm using a ruler, and measured the distance to the nearest source of water to the nearest cm using a 50 m measuring tape. the burrow was then marked using bright flagging tied to a nearby plant or bush to assist in locating the burrow upon subsequent visits. the second step of the process consisted of revisiting the marked tarantula burrows at a later date during the nightly survey to determine if they contained an anuran as an occupant. if the burrow was found to be occupied by an anuran on a subsequent visit, we then measured the same variables previously mentioned. we measured burrow diameter to examine whether these anurans were excluded from utilizing burrows of smaller diameters due to their body size. we measured distance to a pond because we hypothesized that frogs might utilize burrows closer to breeding ponds as refuges from predators. given that calling males are more conspicuous to predators (ryan et al., 1981; schalk and morales, 2012), we hypothesized that those unoccupied tarantula burrows that were in close proximity to breeding sites may offer some form of protection to the frogs if they were approached by a predator. inherent in this hypothesis is that the frogs exhibit a preference to utilize tarantula burrows closer to ponds; therefore we assumed that the likelihood of a burrow being abandoned by a tarantula and therefore, the availability of abandoned tarantula burrows was independent relative to the distance to a pond. since the burrows occupied by frogs were previously documented to be occupied by tarantulas from the first step in the survey process, we did not include the frog-occupied burrows in our analysis of the tarantula burrows as it would be temporal pseudoreplication (hurlbert, 1984). the data failed to meet the assumptions for parametric t-tests, thus we conducted a nonparametric mann-whitney u-test comparing the burrows occupied only by tarantulas to those occupied by anurans. we found a total of 54 occupied tarantula burrows in the study area. of the 54 burrows, 46 of the burrows only contained tarantulas as occupants, while eight of the 54 burrows were found to be utilized by anurans of two species when we re-surveyed the burrows. we did not observe frogs and tarantulas utilizing the same burrow simultaneously. of the eight frog observations, six were male l. bufonius, while the other two were a male and female r. major. all of the male l. bufonius were calling from within the burrow or at the edge of the burrow and when approached, entered the burrow for refuge. the male r. major was not observed calling. during our surveys, we did witness a predation attempt in which a tarantula tried to capture a female l. bufonius. the burrows utilized by the frogs were significantly larger (median = 31.9 mm) in their diameter than those burrows in which we only found tarantulas (median = 23.7 mm; fig. 1a; mann-whitney, u = 86, p = 0.01757). the burrows used by the frogs were also significantly closer (median = 3.5 m) to a source of water as compared to the burrows used only by tarantulas (median = 12.7 m; fig. 1b; mann-whitney, u = 75, p < 0.01). all of the male l. bufonius used burrows less than four meters from the edge of a pond, whereas the male and female r. major were using burrows 5.4 m and 18.7 m from a pond, respectively. burrows often provide an amenable environment that offers shelter from harsh abiotic conditions or refuge from predators (cocroft and hambler, 1989; witz et al., 1991). in the chaco ecoregion, several anurans had been documented as being burrow associates of the vizcacha (l. maximus); the known burrow associates included l. bufonius (cei, 1980), as well as leptodactylus laticeps (cei, 1980), and physalaemus biligonigerus (schalk, 2012). the frogs utilized a subset of size classes of the total available burrows used by tarantulas in the area. specifically, they used burrows which were larger in diameter and closer to a water source. both l. bufonius and r. 101use of tarantula burrows by anurans major are moderately sized anurans (mean svl = 53 mm and 58.8 mm, respectively) and may be unable to utilize the smaller diameter burrows as a result. all six male l. bufonius using the abandoned tarantula burrows were observed calling in or close to the tarantula burrow entrance, and all sought refuge inside the burrow when approached. predation rates are often skewed towards male frogs at a breeding site as males are more conspicuous than females (ryan et al., 1981; lodé, 1996). these abandoned tarantula burrows may offer some form of protection to the more conspicuous males. in this region, documented predators of calling males include invertebrates (schalk, 2010), foxes (schalk and morales, 2012), and other anurans (schalk and montaña, 2011). the breeding biology of l. bufonius may also permit it to utilize these burrows several meters from the pond’s edge. the males of l. bufonius construct underground nest chambers over a meter from the pond’s edge where the eggs are deposited and the nest is sealed with mud; the tadpoles can persist inside until the nest is flooded by heavy rains and the tadpoles enter the nearby pond (cei, 1980; reading and jofré, 2003). rhinella major call from the pond’s edge (schalk and morales, 2012) and deposit eggs directly in the water (cei, 1980), therefore they may be unable to utilize these burrows that are several meters away from the pond as refuges during breeding bouts. the feeding experiments in cocroft and hambler’s (1989) demonstrate that leptodactylid and bufonid frogs were palatable to the tarantula xenesthis immanis. during our surveys, we observed an unsuccessful predation attempt on a female l. bufonius by a tarantula; the tarantula ambushed the l. bufonius, but it was able to escape quickly. we also never observed either species of frog and tarantulas co-occurring in the same burrow at the same time, suggesting that antagonistic interactions in the form of a predator-prey relationship may be occurring. however, without conducting feeding experiments (sensu cocroft and hambler, 1989; dundee et al., 2012), we cannot comment on the palatability of l. bufonius or r. major to the tarantulas in the area. acknowledgements this research was conducted as an agreement between the biodiversity research and teaching collections at texas a&m university and the kaa-iya of the gran chaco national park. we thank r.l. cuellar and k. rivero for permit support and the park guards c. socoré, g. depita, g. castro, and j. alupi for their assistance in the field. we also thank r. bertani for identifying the tarantula. c.g. montaña, a. schulze, and the langerhans lab at ncsu provided constructive comments on the manuscript. support for cms was provided by the national science foundation’s graduate research fellowship program and the applied biodiversity science nsf-igert program at texas a&m university (nsf-igert award # 0654377). this is publication number 1467 of the biodiversity research and teaching collections at texas a&m university. references blair, w.f. (1936): a note on the ecology of microhyla olivacea. copeia 1936: 115. cei, j.m. (1980): amphibians of argentina. mon. zool. ital. mono. 2: 1–609. cocroft, r.b., hambler, k. (1989): observations on a commensal relationship of the microhylid frog chiasmocleis ventrimaculata and the burrowing theraphosid fig. 1. boxplots of the a) diameter of the burrows, and b) the distance to a pond used by tarantulas and frogs during the survey period. an asterisk (*) indicates p < .05, and a double asterisk (**) indicates p < .01. outliers are depicted by an open circle symbol. 102 c.m. schalk, m. sezano spider xenesthis immanis in southeastern peru. biotropica 21: 2–8. dundee, h.a., shillington, c., yeary, c.m. (2012): interactions between tarantulas (aphonopelma hentzi) and narrow-mouthed toads (gastrophryne olivacea): support for a symbiotic relationship. tulane studies zool. bot. 32: 31–38. garton, j.d., mushinsky, h.r. (1979): integumentary toxicity and unpalatability as an antipredator mechanism in the narrow mouthed toad, gastrophryne carolinensis. can. j. zool. 57: 1965–1973. gentry, j.b., smith, h.m. (1968): food habits and burrow associates of peromyscus polionotus. j. mammol. 49: 562–565. hurlbert, s.h. (1984): pseudoreplication and the design of ecological field experiments. ecol. mono. 54: 187– 211. lodé, t. (1996): polecat predation on frogs and toads at breeding sites in western france. ethol. ecol. evol. 8: 115–124. navarro, g., maldonado, m. (2002): geografía ecológica de bolivia: vegetación y ambientes acuáticos. santa cruz de la sierra. centro de ecológia difusión simon i patiño. 719 pp. reading, c.j., jofré, g.m. (2003): reproduction in the nest building vizcacheras frog leptodactylus bufonius in central argentina. amphibia reptilia 24: 415-428. ryan, m. j., tuttle, m.d., taft, l.k. (1981): the costs and benefits of frog chorusing behavior. behav. ecol. soc. 8: 273–278. schalk, c.m. (2010): physalaemus biligonigerus (ncn) predation. herpetol. rev. 41: 202. schalk, c.m. (2012): physalaemus biligonigerus burrow use. herpetol. rev. 43: 123–124. schalk, c.m., montaña, c.g. (2011): ceratophrys cranwelli (cranwell’s horned frog) diet. herpetol. rev. 42: 409–410. schalk, c.m., morales, f. (2012): predation of a rhinella major (anura: bufonidae) by a pampas fox (lycalopex gymnocercus) in the bolivian gran chaco. herpetol. notes 5: 369–370. schalk, c.m., sezano, m., cuellar, r.l. in press. inventory of the reptiles and amphibians from a locality in the kaa-iya of the gran chaco national park, bolivia. kempffiana. schulze, a., jansen, m. (2010): a tadpole of trachycephalus venulosus (anura: hylidae) as prey for a fishing spider (araneae: pisauridae) in the bolivian chiquitano dry forest. herpetol. notes 3: 297–298. scott, v.e., evans, k.e., patton, d.r., stone, c.p. (1977): cavity nesting birds of north american forests. agricultural handbook 511, u.s. dept. agriculture, washington, d.c., usa. toledo, l.f. (2005): predation of juvenile and adult anurans by invertebrates: current knowledge and perspectives. herpetol. rev. 36: 395–400. witz, b.w., wilson, d.s., palmer, m.d. (1991): distribution of gopherus polyphemus and its vertebrate symbionts in three burrow categories. amer. mid. nat. 126: 152–158. acta herpetologica vol. 8, n. 2 december 2013 firenze university press journal of the societas herpetologica italica acta herpetologica acta herpetologica 10(1): 63-66, 2015 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-15155 on a putative type specimen of pleurodema bibroni tschudi, 1838 from chile (anura: leptodactylidae) daiana paola ferraro instituto nacional de limnología (inali-conicet), paraje el pozo s/nº (s3000act) santa fe, argentina, and división herpetología, museo argentino de ciencias naturales “bernardino rivadavia” (macn-conicet), ángel gallardo 470 (c1405djr) buenos aires, argentina. e-mail: dferraro@macn.gov.ar submitted on: 2014, 19th november; revised on 2015, 27th february; accepted on 2015, 2nd march editor: sebastiano salvidio abstract. the original description of the neotropical frog pleurodema bibroni was based on material collected at “monte-video” [montevideo, uruguay], but no type specimen was originally designated. in several publications, a specimen deposited at the national museum of natural history of leiden (the netherlands, rmnh 2277) and collected at valparaíso, chile, has been referred as type specimen of p. bibroni. herein, it is argued that rmnh 2277 is not a type specimen of p. bibroni; nor can it be assigned to p. bibroni, but probably to pleurodema thaul, a species with which p. bibroni was long confused. keywords. leiuperinae, pleurodema thaul, type series, uruguay. the rijksmuseum van natuurlijke historie (rmnh, currently national museum of natural history “naturalis”) was founded in 1820 in leiden, the netherlands (holthuis, 1995). the swiss naturalist j.j. von tschudi was working at the rmnh during 1837, along with the curator hermann schlegel. during his stay at the museum, tschudi described several amphibian taxa, which were published in his classification der batrachier (1838) and housed at leiden collection (gassó miracle et al., 2007). tschudi (1838) described the genus pleurodema, and included a unique species, pleuroderma bibroni (incorrect original spelling). this description was based on an unspecified number of specimens collected at “america merid. (monte-video)” [montevideo, uruguay] and transported by d’orbigny to paris (tschudi, 1838: 84). tschudi did not designate type specimens, but he included “bombinator ocellatus mus. ludg.” in the synonymy of p. bibroni. pleurodema bibroni has been long confused with pleurodema thaul (schneider, 1799), a polymorphic species described for chile, for which type specimens were not fixed (ferraro and lavilla, 2013). interestingly, these taxonomic confusions occur since their descriptions to nowadays, on the basis of both misidentified specimens (e.g. donoso-barros, 1960; ferraro and lavilla, 2013) and adn sequences (faivovich et al., 2012). the correct use of both binomia was resolved by donoso-barros (1969). the current distribution of both taxa extends to other south american countries. pleurodema thaul has a broad distribution in chile and a restricted distribution in southwestern argentina (ortiz and díaz-páez, 2006; ferraro and casagranda, 2009), while p. bibroni inhabits uruguay, rio grande do sul and paraná states at brazil, and northeastern paraguay (braun, 1973; barrio, 1977; natale and maneyro, 2008; kolenc et al., 2009, 2011, 2012; lema and martins, 2011; trein et al., 2014). several authors cited a few specimens as those possibly used by tschudi in his description of pleurodema bibroni. donoso-barros (1969) stated that the material used for the description of p. bibroni could not be traced at the museum of neuchâtel (switzerland) without additional information. hoogmoed (1985) established that syntypes included several specimens deposited at the 64 d.p. ferraro muséum national d’histoire naturelle of paris (mnhn, not traced at that moment) and one specimen housed at the rijksmuseum van natuurlijke historie (rmnh 2277) from valparaíso, chile, described as “… a specimen of pleurodema thaul, which was referred by tschudi (1838) as syn. bombinator ocellatus mus. ludg …”. posteriorly, ortiz and lescure (1989) designated the specimen mnhn 4501 as lectotype of p. bibroni, pointing out that this was the only specimen of pleurodema deposited at that time at mnhn collected by d’orbigny at the río de la plata region. finally, gassó-miracle et al. (2007) considered the specimen rmnh 2277 (assigned to p. thaul) a possible paralectotype of p. bibroni. herein, the identity of the specimen rmnh 2277 is re-evaluated on the basis of phenotypic characters. also, the assignation of the specimen rmnh 2277 as type specimen of p. bibroni, as published in several publications, is analyzed according to the rules of the “international code of zoological nomenclature” (anonymous 1999; cited below as iczn). photographs of specimens mnhn 4501 and rmnh 2277 were carefully examined, as well as specimens of p. bibroni and p. thaul housed at herpetological collections (see appendix). when possible, exo-morphological and some anatomical characters were compared. osteological preparations were made following wassersug (1976), to distinguish bone (alizarin red s) and cartilage (alcian blue). institutional abbreviations follow leviton et al. (1985) with the exception of centro nacional de investigaciones iológicas (cenai, now housed at macn collection). according with the old and hand-written catalogue of the national museum of natural history of leiden, the specimen rmnh 2277 was catalogued as bombinator ocellatus (in litt. e. dondorp, 9 september 2014), i.e. the taxon considered as synonymous of p. bibroni in the original description (tschudi, 1838). also, electronic database shows that this specimen was collected by c. gaudichaud at valparaiso, chile (in litt. r. de ruiter, 24 january 2011). specimen rmnh 2277 is today faded and poorly preserved. it is not possible to accurately determine the color pattern, but one or two spots are observed over lumbar gland, as well as dark longitudinal stripes are observed over thighs (fig. 1 a). these two character states are shared by p. bibroni and p. thaul (figs. 1 b-c). in addition, a distinguishing character between p. bibroni and p. thaul – a dark band over the upper lip present in p. thaul but absent in p. bibroni – cannot be evaluated in specimen rmnh 2277. regarding osteological characters, vomerine teeths are present in specimen rmnh 2277 and in p. thaul (figs. 1 d-e), but absent in p. bibroni (fig. 1 f). morphological observations added to locality data suggest that specimen rmnh 2277 cannot be assigned to p. bibroni because the specimen possesses vomerine teeths and was not collected in the distribution area for   fig. 1. a) specimen rmnh 2277 (chile: valparaiso), dorsal view. b) pleurodema thaul (mlp 4002, argentina: neuquén: los lagos: laguna pire). c) pleurodema bibroni (photo: f. kolenc; uruguay: rocha: barra de balizas). d) specimen rmnh 2277, detail of buccal cavity with voremine teeths. e) pleurodema thaul (macn 32114, argentina, neuquén, junín de los andes), detail of buccal cavity with voremine teeths. f) pleurodema bibroni (cenai 6205, uruguay: rocha: barra de balizas), detail of buccal cavity without voremine teeths. 65type specimen of p. bibroni p. bibroni. the poor preservation of the specimen rmnh 2277 avoid an unambiguous assignation to any chilean anuran species, but, considering the presence and shape of the lumbar gland and the locality data, the specimen rmnh 2277 could possibly belong to p. thaul. the specimen rmnh 2277, catalogued as bombinator ocellatus, was considered as syntype of p. bibroni by hoogmoed (1985). after ortiz and lescure (1989) designated mnhn 4501 as lectotype of p. bibroni, the specimen rmnh 2277 was considered as paralectotype (gassómiracle et al., 2007), apparently based on the concept that if a lectotype is designated between the syntypes, the rest of the syntypes becames paralectotypes (iczn 1999, art. 74.1.3). this statement is valid if all the specimens under consideration were effectively syntypes. hoogmoed (1985), as well as gassó-miracle et al. (2007), identified the specimen rmnh 2277 as p. thaul collected at valparaiso, chile. however, independently of its taxonomic identification and locality data (which do not match with data of the original description), i consider that the specimen rmnh 2277 is not a type specimen of p. bibroni, because it was explicitly included by tschudi (1838) as a synonym of the species under description. specimen rmnh 2277 can be considered, in turn, the type specimen of bombinator ocellatus, fixed by posterior evidence (i.e. the handwritten information of the old catalogue of the rmnh; iczn, art. 72.4.1.1). however, it is important to note that the binomen bombinator ocellatus is not an available nomen for at least two reasons: (i) its first mention was not accompanied by a description (nomen nudum), and (ii) it was first published as a junior synonym (iczn, art. 11.6) and never used as an available name. acknowledgments i am indebted to a. ohler, e. dondorp, r. de ruiter, and r. vonk for sharing information about specimens under their care. also, a. ohler kindly provided photographs of the specimen mnhn 4501, r. de ruiter and e. kruidenier of the specimen rmnh 2277, and f. kolenc the live picture of p. bibroni. j. faivovich, f. kolenc, and l. nicoli critically read the manuscript. thanks also to two anonymous reviewers that improved the ms. this research was supported by grants picts 2011/1895 and 2013/404, and conicet. references anonymous [iczn] (1999): international code of zoological nomenclature. international trust for zoological nomenclature, london. barrio, a. (1977): aportes para la elucidación del “status” taxonómico de pleurodema bibroni tschudi y pleurodema kriegi (müller) (amphibia, anura, leptodactylidae). physis 37: 311-331. braun, c.a.s. (1973): sobre a ocorrêmcia de pleurodema bibronii tschudi, 1838 no estado de rio grando so sul, brasil (anura, leptodactylidae). iheringia (zoologia) 44: 28-31. donoso-barros, r. (1969): posición nomenclatural de un leptodactylido uruguayo (amphibia-anura). bol. soc. biol. concepción 41: 161-162. faivovich, j., ferraro, d.p., basso, n.g., haddad, c.f.d., rodrigues, m.t., wheeler, w.c., lavilla, e.o. (2012): a phylogenetic analysis of pleurodema (anura: leptodactylidae: leiuperinae) based on mitochondrial and nuclear gene sequences, with comments on the evolution of anuran foam nests. cladistics 28: 460-482. ferraro, d.p., casagranda, d. (2009): geographic distribution of the genus pleurodema in argentina (anura: leiuperidae). zootaxa 2024: 33-55. ferraro, d.p., lavilla, e.o. (2013): the identity of rana lutea molina, 1782 (amphibia, anura). zootaxa 3608: 264-272. gassó miracle, m.e., van den hoek ostende, l.w., arntzen, j.w. (2007): type specimens of amphibians in the national museum of natural history, leiden, the netherlands. zootaxa 1482: 25-68. holthuis, l.b. (1995): 1820-1958: rijksmuseum van natuurlijke historie. nationaal natuurhistorisch museum, leiden. hoogmoed, m.s. (1985): in: amphibian species of the world. a taxonomic and geographical reference. frost, d.r. ed., allen press inc. and the association of systematics collections. lawrence, kansas. kolenc, f., borteiro, c., baldo, j.d., ferraro, d.p., prigioni, c. (2009): the tadpoles and advertisement calls of pleurodema bibroni tschudi and pleurodema kriegi (müller), with notes on their geographic distribution and conservation status (amphibia, anura, leiuperidae). zootaxa 1969: 1-35. kolenc, f., baldo, d., borteiro, c., marangoni, f., ferraro, d.p., faivovich, j. (2011): the identity of eupemphix fuscomaculatus steindachner, 1864 (amphibia: anura). copeia 2011: 513-522. kolenc, f., borteiro, c., gonzález, e.m., barrasso, d.a., prigioni, c.m. (2012): recent findings of the declining frog pleurodema bibroni tschudi, 1838 (anura: leiuperidae) in uruguay. herp. notes 5: 181-183. lema, t., martins, l.a. (2011): anfíbios do rio. grande do sul: catálogo, diagnoses, distribuição, iconografia. edipucrs, porto alegre. leviton, a.e., gibbs, r.h. jr., heal, e. & dawson, c.e. (1985): standards in herpetology and ichthyology: 66 d.p. ferraro part 1. standard symbolic codes for institutional resource collections in herpetology and ichthyology. copeia, 1985: 802-832. natale, g.s., maneyro, r. (2008): amphibia, anura, leiuperidae, pleurodema bibroni: rediscovery. check list 4: 47-49. ortiz, j.c., díaz-páez, h. (2006): estado de conocimiento de los anfibios de chile. gayana 70: 114-121. ortiz, j.c., lescure, j. (1989): les types d’amphibiens anoures du chili dans les collections du muséum national d’histoire naturelle de paris. bull. mus. hist. natl. paris 11: 113-122. schneider, j.g. (1799): historia amphibiorum naturalis et literarariae. fasciculus primus. continens ranas, calamitas, bufones, salamandras et hydros in genera et species descriptos notisque suis distinctos. friederici frommanni, jena. trein, f.l., lima, l.p., ulandowski, l.k.a., morato, s.a.a. (2014): pleurodema bibroni tschudi, 1838 (anura: leiuperidae): distribution extension and first record for the state of paraná, brazil. check list 10: 417-418. tschudi, j.j. (1838): classification der batrachier, mit berücksichtigung der fossilen thiere dieser abtheilung der reptilien. mem. soc. sci. nat. neuchâtel 2: 1-99. wassersug, r.j. (1976): a procedure for differential staining of cartilage and bone in whole, formalin fixed vertebrates. stain tech. 51: 131-134. appendix. specimens examined an asterisk (*) denotes cleared and stained specimens. pleurodema bibroni. – the same appendix that kolenc et al. (2011, zootaxa: 1969: 1-35). new specimens: uruguay: “monte–video” mnhn 4501 (syntype, examined by photographs); barra de valizas cenai 6204*, cenai 6205*. pleurodema cf. thaul. – chile: valparaiso rmnh 2277 (examined by photographs). pleurodema thaul. – chile: concepción cenai 4428-4430; el correntoso (chamiza): near puerto montt cenai 1952*; icalma mlp 1170; los andes fml 3309; mallín de lago todos los santos mlp 3530; pirehueico fml 3761 (specimens); quebrada el teniente fml 3310; valdivia: selvas de toltein macn 4640-4646. argentina: chubut province: esquel: parque nacional los alerces cenai 8826*; neuquén province: aluminé macn 11648*, macn 11649*; isla victoria mlp a. 1449-1450, cenai 4107*, macn 9092*; junín de los andes macn 28701*, macn 28702*, macn 28703*, macn 29214*, macn 32114*, macn 32118*; lago aluminé: la angostura mlp 1168; lago curruhé cenai 2170-2171; los lagos: laguna pire mlp 4002; río negro province: el bolsón cenai 3617*, cenai 3619*; nahuel huapi mlp 1276–1277; near puerto blest cenai 1527 (specimen 5)*, cenai 1527 (specimen 8)*; san carlos de bariloche cenai 7156, macn 31552. acta herpetologica vol. 10, n. 1 june 2015 firenze university press obituary: valery k. eremchenko (1949-2014) leo j. borkin1, tatjana dujsebayeva2, roberto sindaco3, matthias stöck4 haplotype variation in founders of the mauremys annamensis population kept in european zoos barbora somerová1, ivan rehák2,*, petr velenský2, klára palupčíková1, tomáš protiva1, daniel frynta1 reproductive ecology of sichuan digging frogs (microhylidae: kaloula rugifera) wei chen1,*, lina ren2, dujuan he2, ying wang2, david pike3 toxic effects of carbaryl on the histology of testes of bufotes variabilis (anura: bufonidae) özlem çakici basal frequency of micronuclei and hematological parameters in the side-necked turtle, phrynops hilarii (duméril & bibron, 1835) maría a. latorre 1,2,*,#; evelyn c. lópez gonzález1,2, #; pablo a. siroski1,2,3; gisela l. poletta1,2,4 into a box interiors: clutch size variation and resource allocation in the european pond turtle marco. a.l. zuffi1,*, simonetta citi2, elena foschi1, francesca marsiglia1, eva martelli1 where to “rock”? choice of retreat sites by a gecko in a semi-arid habitat andreia penado1,2, ricardo rocha3,4,*, marta sampaio3, vanessa gil3, bruno m. carreira3, rui rebelo3 age structure, growth and longevity in the common toad, rhinella arenarum, from argentina clarisa de l. bionda1,2,*, silvia kost 4, nancy e. salas1, rafael c. lajmanovich3, ulrich sinsch4, adolfo l. martino1 on a putative type specimen of pleurodema bibroni tschudi, 1838 from chile (anura: leptodactylidae) daiana paola ferraro re-description of the external morphology of phyllomedusa iheringii boulenger, 1885 larvae (anura: hylidae), with comments on the external morphology of tadpoles of the p. burmeisteri group samanta iop¹, victor mendes lipinski¹, bruno madalozzo¹, franciele pereira maragno¹, sonia zanini cechin¹, tiago gomes dos santos² book review: harold heatwole, john w. wilkinson (eds). amphibians biology. volume 11 status of conservation and decline of amphibians. eastern hemisphere. part 4 . southern europe and turkey sebastiano salvidio book review: antonio romano. atlante degli anfibi del parco nazionale del cilento vallo di diano e alburni distribuzione, biologia, ecologia e conservazione sebastiano salvidio acta herpetologica journal of the societas herpetologica italica acta herpetologica 14(1): 51-56, 2019 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-22817 descriptive osteology of an imperiled amphibian, the luristan newt (neurergus kaiseri, amphibia: salamandridae) hadi khoshnamvand1, mansoureh malekian1,*, yazdan keivany1, mazaher zamani-faradonbe1, mohsen amiri2 1 department of natural resources, isfahan university of technology, isfahan, iran. *corresponding author. email: mmalekian@cc.iut.ac.ir 2 department of basic sciences, islamic azad university of hamedan, hamedan, iran submitted on: 2018, 4th march; revised on: 2018, 19th december; accepted on: 2019, 17th april editor: raoul manenti abstract. osteological structures are important biological features which provide valuable biological and ecological information. luristan newt (neurergus kaiseri), is an endemic salamander, inhabiting the southern part of zagros mountains of iran. the current study was conducted to describe the osteological characteristics of the luristan newt which might be important in understanding the evolutionary process of newt species. the skull of n. kaiseri has a dense structure, severely ossified elements and a low amount of cartilaginous elements, only in mobile facets. vertebral number in the axial skeleton of the species equals 50. the cervical, abdominal and caudal parts of the vertebral column have two, 16 and 32 vertebrae, respectively. each hand and foot consisted of four fingers, having three or four phalanxes. the metacarpal includes seven bones and the number of metatarsus bones is eight. hands are connected to humur through ulnare and radius and then connected to scapulocoracoid. each leg includes two bones (fibula and tibia) which are connected to femur. the head of the femur articulates with the acetabulum in the pelvic bone, while the distal part of the femur articulates with the tibia. keywords. salamanderidae, newt, neurergus kaiseri, skull, descriptive osteology. introduction the vertebrate skeletal system and its elements are important in evolutionary biology. vertebrate skeletons can be regarded as combinations of apparently discrete units (namely bones) which have attracted the interest of comparative anatomists (simpson, 1944). osteological data can be used to identify different taxa and phylogenetic relationships (e.g., hill, 2005) and to understand biological features of animals such as feeding, respiration, swimming and movement (eastman, 1980; helfman et al., 2009). in addition, the skeletal structure contains biological information that can be used to distinguish species type, age, sex, size, and even environmental conditions of their habitats (helfman et al., 2009). in iranian freshwater basins live three genera of salamanders including, triturus, salamandra and neurergus, all belonged to salamandridae. the latter genus has a relatively vast geographic distribution, ranging from zagros mountains (western iran) to iraq and southern turkey (baloutch and kami, 1995). the luristan newt (neurergus kaiseri, schmidt 1952) is endemic to the southern zagros mountains of iran, with a distribution area of approximately 900 km2 (mobaraki et al., 2016). the species inhabits first order rivers and ponds in open woodlands dominated by oak trees (quercus brantii). this newt is classified as vulnerable (vu) by the iucn red list because of its small range, illegal trading, habitat loss and climate induced drought (iucn, 2018). it is also amended to the appendix i of the convention to the international trade to endangered species (cites, 2010). 52 hadi khoshnamvand et alii current knowledge on n. kaiseri is restricted to some aspects of the ecology such as distribution, including reports of new localities for the species (sharifi et al., 2013; mobaraki et al., 2014), and demography (age structure, longevity and growth patterns) of a local population (farasat and sharifi, 2015). a genetic study reported the presence of two genetically distinct clades within the luristan newt (farasat et al., 2016). sexual dimorphism in n. kaiser was evaluated using headand bodyrelated characters (khoshnamvand et al., 2018). skeleton structure of the species is yet to be described, which can provide additional data and a basis for understanding the remarkable and adaptive variation in bone-forms among newt species. osteology also helps in understanding the important taxonomic characters for identification and classification of a species. in salamandridae, a number of studies include osteological characters in the description of a new taxon (min et al., 2005; wu et al., 2009; wu et al., 2010; wake et al., 2012) or provide comparative descriptions of closely related taxa (wake and özeti, 1969; venczel, 2008; wu et al., 2012). amongst neurergus species, little information is available on descriptive osteology. akia et al., (2010) described the cranial osteology of a closely related species, neurergus microspilotus and compared it with salamandra infraimmaculata semenovi. the current study aims to describe skull characteristics of n. kaiseri and compare size, shape and connections of bones with other newts, where possible. such data provide a basis for further investigations on the subject and help to understand the biological features of iranian salamanders. material and methods field surveys were conducted in late july 2015, when the luristan newt breeding season was over to minimize disturbances to the species. the luristan newt habitats are mainly streams and springs, located at high elevations (800 to 1500 m a.s.l), separating from each other by steep and rocky mountains. further, the species is rare, occurring in low density in a limited number of sites, as many of the ponds and springs in the region have dried out due to the drought during the past few years (iucn, 2018). the vulnerable (vu) status of the species also entails sampling restrictions for capturing live individuals. therefore only deceased carcasses of n. kaiseri could be taken and used for osteological analyses. therefore, we accessed to the newt habitats by climbing the mountains and camping in the region for 10 days, with the assistance from the environmental guards. due to the water flow, which could wash away the possible carcasses, 2 mm fish nets were installed in down streams. fish nets were regularly checked during the day and removed from each site before the dark. two dead individuals were captured by the fish net at two localities in korki region, lorestan province, at the elevation of 957 and 1100 m a.s.l. respectively. both specimens were adult male with the body length of 132 and 130 mm respectively. samples were preserved in 96% ethanol in the field prior to the lab experiment. we followed the standard protocols of cleaning and staining bones (taylor and van dyke, 1985; torres and ramos, 2016). the carcasses were fixed in 500 ml of 10% neutral formalin for 4 days. before proceeding with the staining procedure, the specimens were washed thoroughly under running tap water for at least one hour to remove excess of formalin. the specimens were then placed in 1% alizarin red solution, added drop by drop to the freshly prepared 5% koh solution. about 15 ml of the staining solution was utilized, holding the specimens for three days or until the medium (koh) showed pinkish violet color. the specimen were then transferred into alkaline blue stain solution for two days. to clean bones, specimens were transferred into trypsin solution at ~37 °c, changing enzyme solution and washing the specimens in distilled water every 3 days to avoid bacterial digestion (repeated three times). the stained specimens were examined using a stereomicroscope (hp snp 120), and different skeletal elements were dissected and scanned in lateral, dorsal and ventral view, using a scanner (hp scanjet g4050). for each specimen or view, a series of images were taken to produce a single image with maximum depth of field. the final image was drawn using coreldrawx7 software. the terminology of the skeletal elements follows vassilieva et al. (2015). results the skull of n. kaiseri has a dense structure, severely ossified elements and a low amount of cartilaginous elements, only in mobile facets. the premaxillae are unpaired and nearly arched dorsally. the pars dorsalis of premaxillae are distinctly and widely separated by a midline fontanelle. a groove that starts in the top of nasal opening is separating the pars dorsalis of premaxillae and continues forward to pars dentalis. this groove further goes toward the antroventral part of the palate and form a pore in the vomer bone. the premaxilla contacts the prefrontal and nasal posteriorly, and contribute to the nasal cavity laterally. the maxilla are paired and complete the arch of the upper jaw that borders the nostril at the anterior part and meet the nasal bones medially (fig. 1). the paired nasal bones are nearly subrectangular and bordered anteriorly by the premaxillae. anteriorly, they border the nostril and laterally connected to the prefrontals. the prefrontals are relatively triangular, anteriorly connected to the pars dorsalis, anterolaterally to the nasals and posteromedially to the frontal (fig. 1). the lacrimal is a small triangular bone enclosed by maxilla, prefrontal, and nasal bones. this bone participates in forming the orbit cavity. in the right half of the head, it has a distinct boundary on both sides, but in the left half 53osteology of neurergus kaiseri of the head, this bone is fused to frontal with no boundary (fig. 1). the frontals are flattest paired bones in the skull and trapezoid that makes a posterolateral projection. a very tiny cartilage binds this projection to squamosal to form a ring. the frontals meets the prefrontal anterolaterally, premaxillae anteriorly and parietal posteriorly. the parietals are relatively flat with small curvature in the posterolateral region towards to the orbit cavity. the parietals are rather smaller than the frontals and together complete the roof of the neurocranium. two parts of parietal, having an overlap toward the foramen magnum. the squamosal is almost triangular that lying on the lateral side of exoccipital, becoming closely fixed with it to form a lateral projection. the base of the skull is ossified. the orbitosphenoid articulates with the premaxillae (pars dentalis1) and maxilla anteriorly and laterally, respectively. the orbitosphenoid is located in the orbital region and makes an optic foramen posteriorly to pass off the optic nerves into the cranial cavity. near the premaxilla, the orbitosphenoid has a medial foramen that opens into the braincase. pterygoids bear relatively triangular depression on ventral surface from basicranial to orbital foramen. the unpaired and oval-shaped parasphenoid is the largest bone in middle part of ventral view of the skull. the quadrate is a relatively small bone located in anterior side of squamosal. the exoccipital forms the posteriormost part of the skull base, encircling the foramen magnum and articulating via the occipital condyles with the first vertebra. exoccipitals have trapezoid shape ventrally and paired but never meet together at midline. exoccipitals are anteriorly in contact with the parietal and laterally with squamosal (fig 1). further, exoccipital can be seen at the top of the skull. squamosal is connected to the pterygoids bone and both pterygoids and squamosal are connected to the occipital. mandibles are paired and include mentomandibulars, dentaries and pre-articulars. the mandible is separated and symmetrical on both sides. the shape of the mandible (lower jaw) is a simple solid bony arch and dentary is the main element of the mandible (fig. 2). phalanxes are well developed in all fingers, showing a tendency toward bifurcation, and are consistently seen to have small cartilaginous tips (fig. 3). further, each hand has four fingers. the biggest finger has four phalanxes, while the other fingers have only three phalanxes. the metacarpal includes seven bones. one of them is larger than the others and is connected to the ulnare and radius. the hands connected to humur through ulnare and radius and then connected to scapulocoracoid (fig. 3). feet have five fingers and each finger has three phalanges (fig. 3). the number of metatarsus bones is eight and four of them are bigger than the others. each leg includes two bones (fibula and tibia) which are connectfig. 1. explanatory drawings of dorsal (a) and ventral (b) view of n. kaiseri skull. scale bar: 1 mm 54 hadi khoshnamvand et alii ed to femur. the head of the femur articulates with the acetabulum in the pelvic bone, while the distal part of the femur articulates with the tibia (fig. 3). vertebral number in the axial skeleton equals 50. the cervical, abdominal and caudal parts of the vertebral column have two, 16 and 32 vertebrae, respectively. also hands are connected to the body at the vertebrae number 2-3 and legs are connected at vertebrae number 16-17. discussion we described here, for the first time, the skeletal structure of n. kaiseri, an endemic and threatened newt in the southern zagros mountains of iran. the species inhabits mountainous streams at high altitudes, which makes its habitat hard to access due to the rarity and vulnerability of the species, only two deceased individuals of n. kaiseri could be obtained and used for osteological analyses. the two specimens examined here were similar (adult females with the total length of ~130 mm), which minimizes differences in ossification due to age. body length is linearly correlated with age in many salamandrid species until maximum length is reached (lima et al., 2000; üzüm, 2009). the skull of n. kaiseri is very compact and mineralized with low amounts of cartilaginous elements. within the neurergus genus, skull characteristics has only studied in n. microspilotus (akia et al., 2010). the cranial characters and the number of characters of n. kaiseri and n. micropilotus show a general similarity. posterior part of the skull in both species is wide and the snout being short and the entire skull parts are almost fully mineralized in the adults. the frontal and premaxillae of both n. kaiseri and n. microspilotus is relatively the same. however, it seems that the skull of n. kaiseri being smaller compared to n. micropilotus. other noteworthy differences between the skulls of n. kaiseri and n. microspilotus include differences in the shape and size of fig. 2. explanatory drawing of the lower jaw and hyobranchial apparatus of n. kaiseri. scale bar: 1 mm fig. 3. hand (a) and leg (b) of n. kaiseri. i-iv: digits i-iv. scale bar: 1 mm. 55osteology of neurergus kaiseri maxilla, squamosal, exooccipital and cavum internasale. the pterygoids in n. kaiseri is shorter and weakly cartilaginous, whereas in n. microspilotus is larger, narrower with a great cartilaginous part. squamosal is connected to the pterygoids bone and both pterygoids and squamosal are connected to the occipital. the premaxillae in n. kaiseri is distinct and well ossified. less ossification, however, has been observed in other salamandrids (e.g., salamandra infraimmaculata semenovi (akia et al., 2010), which may agree with the primitive position of this taxon (steinfartz et al., 2007) compared to more derived taxa such as neurergus species. skull morphology of n. kaiseri may be an adaptation for living in mountainous streams. skulls in plethodontine salamanders that live in the mountain habitats (usually at altitudes above 900 m) tend to be harder and stronger (buckley et al., 2010) compared to those inhabiting the flatter areas. maxillary bones in n. kaiseri are more flattened and thicker than n. micropilotus and nasal bones are long and elongated in the length of skull roof. naylor (1978) has suggested that the squamosal arch of newts is an anti-predator adaptation designed to add structural support to the skull and protect retracted eyes. ehmcke and clemen (2006) showed that the skull of the plethodontid salamanders is membranous and flexible. similarities are observed between the osteological structures of the hands, legs, lower jaw and hyobranchial apparatus of n. kaiseri with other newt species (ghosh et al., 1994). metacarpal establishes a connection between fingers and forearm bones which facilitate the animal movement (fabrezi and barg, 2001). in general, similarities are observed between the skeleton structure of n. kaiseri and n. micropilotus. both species are mountains species, however, there are some interspecific differences which could be related to the different total size of the adult individuals. however, there are other differences, which apparently only depend on the phylogenetic position of a species. further research with a larger sample size and including other neurergus species is required to investigate the remarkable and adaptive variation in bone-forms among these newt species. references akia, f., rastegar-pouyani, n., faizi., h. 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(2010): a new newt of the genus cynops (caudata: salamandridae) from fujian province, southeastern china. zootaxa 2346: 42-52. acta herpetologica vol. 14, n. 1 june 2019 firenze university press uzungwa scarp nature forest reserve: a unique hotspot for reptiles in tanzania john valentine lyakurwa1,2,*, kim monroe howell2, linus kasian munishi1, anna christina treydte1,3 experience of predacious cues and accessibility to refuge minimize mortality of hylarana temporalis tadpoles santosh mogali*, bhagyashri shanbhag, srinivas saidapur tonal calls as a bioacoustic novelty in two atlantic forest species of physalaemus (anura: leptodactylidae) thiago r. de carvalho1,*, célio f.b. haddad1, marcos gridi-papp2 scientific publication of georeferenced molecular data as an adequate guide to delimit the range of korean hynobius salamanders through citizen science amaël borzée1,*, hae jun baek2,3, chang hoon lee2,3, dong yoon kim2, jae-young song4, jaehwa suh5, yikweon jang1, mi-sook min2* mirrored images but not silicone models trigger aggressive responses in male common wall lizards stefano scali1,*, roberto sacchi2, mattia falaschi1,3, alan j. coladonato2, sara pozzi2, marco a.l. zuffi4, marco mangiacotti1,2 using an in-situ infra-red camera system for sea turtle hatchling emergence monitoring fatima n. oğul1,#,*, franziska huber2,#, sinem cih1, kumsal düzgün3, ahmet e. kideyş1, korhan özkan1 descriptive osteology of an imperiled amphibian, the luristan newt (neurergus kaiseri, amphibia: salamandridae) hadi khoshnamvand1, mansoureh malekian1,*, yazdan keivany1, mazaher zamani-faradonbe1, mohsen amiri2 does color polymorphism affect the predation risk on phalotris lemniscatus (duméril, bibron and duméril, 1854) (serpentes, dipsadidae)? fernanda r. de avila1,2,*, juliano m. oliveira3, mateus de oliveira1, marcio borges-martins4, victor hugo valiati2, alexandro m. tozetti1 age structure of a population of discoglossus scovazzi camerano, 1878 (anura discoglossidae) in extreme environmental conditions (high atlas, morocco) mohamed amine samlali, abderrahim s’khifa, tahar slimani* acta herpetologica 9(2): 179-186, 2014 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-14180 temporal distributions, habitat associations and behaviour of the green lizard (lacerta bilineata) and wall lizard (podarcis muralis) on roads in a fragmented landscape in western france roger meek rue georges clemenceau 7, chasnais, france. e-mail: rogermeek85@aol.com submitted on 2014, 10th march; revised on 2014, 5th september; accepted on 2014, 19th september editor: marco a.l. zuffi abstract. observations of the green lizard (lacerta bilineata) and wall lizard (podarcis muralis) on roads in western france indicated that basking close to the road edge was the predominant activity in l. bilineata but p. muralis mostly foraged. spatial locations of road mortalities in both species reflected this with the median distances from the road edge greater in p. muralis. temporal differences in road presence, based on mortality counts and those of live lizards, indicated significantly more lizards were present on roads during late summer and autumn, especially in p. muralis. a significant correlation was found between the monthly presence of live lizards and monthly road mortalities in p. muralis (r = 0.73) but not in l. bilineata (r = 0.64). numbers of l. bilineata found on roads bisecting low-density urban areas and roads bordered by hedgerows were higher than expected in relation to the occurrence of these habitats at roadsides. in p. muralis higher than expected numbers were found alongside low-density urban areas and roads bisecting woodland. generally both species were less commonly seen on roads alongside agricultural areas with no hedgerow border. keywords. lizards, roads, behaviour, habitat associations, temporal distributions. introduction habitat selection is a key aspect of animal ecology that can influence their behaviour and physiology (e.g. huey, 1991). reptiles are ideal organisms with which to understand habitat selection (smith and ballinger, 2001), as they are often numerous and adapted to a wide range of habitats and hence may provide insight into the effects of human alterations on the environment for wildlife. for example, urban and agricultural landscapes may constrain reptiles to live alongside roads due to the unsuitability of the surrounding habitat (spellerberg, 2002) and although numerous studies have now been undertaken on these effects on reptiles, most have concerned snakes. fewer works have examined lizard presence on roads perhaps in part because of detection difficulties due to their frequent small size and rapid carcass degradation (e.g. koenig et al., 2002; delgado garcia et al., 2007; lebboroni and corti, 2006; fahrig and rytwinski, 2009; meek, 2009). additionally, if lizard populations fluctuate across both temporal and spatial scales this may necessitate long-term research to gather an adequate database for effective analysis. the present paper attempts to increase understanding of lizard road presence using the results of a 9-year study of two lizard species in an agricultural landscape in western france. in western europe, two species of lizards that are frequently seen on roads are the western green lizard lacerta bilineata and wall lizard podarcis muralis (e.g. lebboroni and corti, 2006; meek, 2009). both species are heliothermic and found in a variety of habitats but p. muralis is more often associated with human habitations, including on walls and the sides of buildings in town centres (arnold and burton, 2000). both species 180 roger meek are primarily insectivorous but there are differences in lifestyle; p. muralis is essentially a foraging species whilst lacerta bilineata is a sentinel predator (verwaijen and van damme, 2008). although road mortalities have been documented (lebboroni and corti, 2006; meek, 2009) little information is available on how these lizards interact with roads, either on temporal or spatial scales or indeed their behaviour when they move onto road surfaces. the present study was prompted by observations that both species, in contrast to sympatric snakes, may attempt to use roads as a resource, for instance for securing prey and thermal resources. this paper addresses the following questions: 1) does the presence of lizards on roads change seasonally? 2) how do the lizards behave when they are on roads? 3) what are the habitat associations of the places where lizards enter or cross roads? methods surveying was carried out over a 9-year period between april 2005 and november 2013 on six roads in vendée, western france with additional observations on road behaviour made until 31 july 2014. the roads surveyed differed in width from 5 to 7 metres with one lane in each direction. the surveyed sections ranged in length from 1 to just over 6 km and totalled around 16 km. they connected the town of lucon with four villages; chasnais, st denis-du-payre, lairoux and la brettoniere-la-clay (46o27`n, 1o53`w). figure 1 shows the location of the study locality. all road surfaces were tarmac based but apart from town or village centres only the d2949 (previously the d949 see meek, 2009) between lucon and chasnais had a sidewalk. during the study period land changes along roadsides were minimal and mostly concerned woodcutting at woodland areas (for fuel), but re-growth was normally well underway the following year. new house construction was often present in villages but this had little or no impact on measurement of roadside habitats, as it mainly concerned buildings replacing buildings or new constructions that were at not actually on the roadside. surveying was between 4 and 6 times per month at around 4 day intervals by a single observer usually between 1000-1700hrs on a bicycle at a maximum speed of around 5-10 km/hour. data are based on point observations of individual lizards that were either present on roads or found as road-kill. when a live or road-killed lizard was observed or found, snout to vent length (svl) in mm, approximate location where found, proximate roadside habitat and distance from the road edge were recorded. distance from road edge is to the centre of the road irrespective of the side of the road the carcass was found. if it was suspected that a carcass had been displaced from the original point of the mortality (i.e. by being subsequently run over by other vehicles) the measurement was discarded. most measurements of live lizards were approximate (± 10mm) and derived from photographic records, which were then compared with some object in the immediate vicinity for length comparison, for instance wood debris or distances between sections of larger stone aggregate on roads. measurements of road–killed lizard svl had a maximum estimated error of 5mm depending on body condition. behaviour the behaviour records were determined using vef (visual encounter frequency; latham et al 2005) and were only of animals actually on the road surface. behaviour of each lizard was ascertained at its initial sighting. when there was any uncertainty, for example if the observer disturbed the lizard before behaviour could be determined, the observation was discarded. in general it was possible to identify three behaviours; basking, foraging and road crossing. examples are shown in fig. 2 (avery, 1979). foraging was defined as a moving lizard where the head was closer to the ground than when running. this also included making frequent direction changes and sometimes chasing and securing prey. basking was defined as a lizard sitting on the road with no significant locomotory movement often with the body flattened laterally and the limbs sprawled sideways. the flattened body and sprawled limbs can be seen particularly clearly in fig. 2c. however, these postures probably represent a combination of sentinel and/or thermoregulafig. 1. schematic diagram showing survey roads with approximate distances surveyed on each road in parenthesis. broken lines represent road segments not surveyed. map insert indicates location of the study locality.   st denis-du-payre chasnais lairoux lucon d2949 (1.2km) d44 (1.2 km) d60 continued d60 (6.1 km) d127 (1km) rue de bourneau (0.5km) un-named road (6 km) n w. france 181lizards on roads tory behaviour (figs. 2b and c). road crossings were when a lizard was observed making a complete crossing from one side of the road to the opposite side with little or no changes in direction. usually the head of the lizard was held higher off the ground than when foraging and usually involved running very fast. since crossings were sometimes made at high speeds and hence of limited duration, it opens up the possibility that the frequency of road crossings may be underrepresented due to having less chance of being observed, potentially introducing some bias in the behaviour results. the photographs in fig. 2 also illustrate the good contrast with the road surfaces in l. bilineata, but less distinct in p. muralis. estimating habitat proportions the relevant (major) habitat parameters alongside roads were identified and quantified in linear terms. the various habitats were continuous and hence assumed potentially available to both species. because of low counts in certain habitats and hence potential bias, male and female data were pooled. habitat segments were broadly defined as roads with hedgerow borders (33.6% of roadside habitat), monocultures without hedgerows (18.2%) woodland edge (16.5%), high-density urban areas – i.e. villages (25.0%) and low-density urban areas (6.7%). low-density urban areas were defined as a small number of houses or buildings (usually no more than three) with high density urban defined as villages or towns. due to crop rotation and the presence or absence of grazing animals, monocultures are transient environments and hence although their composition varied over time they were always agricultural areas. however, over the 9-year study period roadside habitat generally varied little except for some roadside woodcutting during 2013, which reduced woodland cover by around 10m along the roadside. sample effort in each habitat type was approximately proportional to habitat availability since observer movement along each road was at a reasonably constant speed. however, some bias will have resulted from pauses to gather data and from occasional searches into roadside habitats, for instance to confirm species identification. the latter were confined to around 5 times per year and not expected to introduce significant bias. statistical analysis to determine whether lizards were associated with particular roadside habitat requires selection analysis, which compares habitat use with habitat availability. statistically this requires models that will produce patterns of expected probabilities that can be compared with real data (gotelli & mcgill, 2006). a null model was constructed by determining roadside habitat occurrence defined as their linear distances along road edges using the distance-measuring tool on google earth (http://www.google.co.uk/intl/en_uk/earth/). google earth applies an average of many sampled points in order to smooth fig. 2. examples of l. bilineata and p. muralis road behaviour. foraging in l. bilineata is shown in a, basking/sentinel behaviour in b and c. d shows a road basking p. muralis to illustrate potential differences in detection on roads compared to l. bilineata. see `methods` for definitions of behaviour. 182 roger meek the data in the horizontal plane and ignores undulations in the landscape. a comparison using the trip meter of two different cars and a motorcycle indicated reasonable agreement with the google estimates; likely due to the limited distances being surveyed and relatively flat topography of the study locality. the proportions of roadside habitat types x in metres were calculated as fractions of total available roadside habitat in y in metres from x/y. the common fractions were then converted to decimal fractions and compared with the proportion of lizards found or observed alongside each of the corresponding habitat types. the null hypothesis predicts lizards will be randomly distributed when their habitat associations do not depart significantly from habitat availability and therefore no selection is involved; habitat selection requires a significant departure from the null model. the tests employed were one-dimensional χ2 goodness of fit statistics that were then subject to monte carlo randomisations of 5000 iterations. the simulated χ2 values this produced where then compared with the true χ2 values. the test for significance is the number of the simulated χ2 values that equalled or exceeded the true values should not exceed 5%. if this interval was surpassed the true values would be considered unreliable and potentially due to random chance. this is a useful test when the true χ2 is close to the 95% interval. since the reliability of the χ2 statistic is proportional to the number of values in the cells, the tests for temporal distributions, where low cells counts were present, were made using the kolmogorov-smirnov test (dmax). this test avoids cell re-binning, is robust and importantly not sensitive to cell counts. the null model employed is equality of cell counts across months and applies only to months when lizards were observed/road-killed on roads, which was from april to november. this test has also been used to examine for annual deviations of monthly road presence from the general 9-year trends. the expected probabilities were derived from the summed yearly trends after conversion to decimal fractions. the test is set at the 95% interval with deviation from the expected probabilities indicated if the 95% intervals were attained or exceeded. no departure from the null model indicates annual monthly presence was in agreement with the 9-year trend. two tailed z-tests for independent proportions were used to test for differences in behaviour and non-parametric mannwhitney u-tests for differences in carcass location on roads after testing for normality using the anderson-darling test. leven`s test (l) was used to determine homogeneity of variances with the null hypothesis σ2 1 / σ2 2 = 1 and α = 0.05. all ± values represent 1 standard deviation from the mean. results annual counts of lizards on roads were relatively low and mostly seasonal. a total of 369 lizards were recorded as mortalities or observed active on roads; 175 p. muralis (50 live and 125 road mortalities) and 194 l. bilineata (47 live and 147 mortalities). this result suggests greater road presence in l. bilineata than the more locally abundant p. muralis (see below). all size classes were observed as mortalities; l. bilineata s.v. range 41-135, mean = 97.01±18.3mm, p. muralis s.v. range 24-73, mean = 51.99±11.7mm and had comparable size ranges to live lizards on roads; l. bilineata s.v. range 55-121, mean = 102.2±11.4mm, p. muralis s.v. range 22-61, mean = 47.4±11.1mm. temporal aspects monthly road activity and mortality occurrence was irregular in both species and deviated significantly from monthly equality, whether for mortalities (p. muralis, dmax = 0.339, p <0.01; l. bilineata, dmax = 0.115, p < 0.05) or mortalities and live counts pooled (p. muralis, dmax = 0.3, p < 0.01; l. bilineata, dmax = 0.12, p < 0.01). significantly higher deviations were found in both species during august and september compared to other months and were respectively (mortalities and live) 2 and 4.5 times greater in p. muralis and 1.81 and 1.47 times in l. bilineata. correlation of monthly road presence of live lizards and monthly road mortalities was significant in p. muralis (r = 0.73, p = 0.04) but not in l. bilineata (r = 0.64, p = 0.08). a relatively high number of live l. bilineata were observed on roads in april, which was in agreement with mortality counts. can these general trends predict annual monthly road presence? the kolmogorov-smirnov tests indicated deviations in annual monthly presence from the general 9-year trends were found only in l. bilineata during 2012 when the test statistic reached the 95% interval (dmax = 0.234, p = 0.05). monthly road presence of l. bilineata in 2012 was 1.6 times less than expected during june and 1.65 and 1.36 times greater than expected during august and september respectively compared to the general trends. the distributions on which these statistics are based are shown in fig. 3. road behaviour basking / sentinel behaviour on roads was more frequent in l. bilineata (72.3%) than p. muralis (42%), with the latter spending more time foraging (51.1 versus 12.5%). the differences between species were significant (basking z = 3.013, p < 0.001; foraging z = 4.017, p < 0.0001). fewer lizards of both species were observed crossing roads and in similar frequency (p. muralis 10% versus l. bilineata 17.1%; z = 1.01, p > 0.05). the data are summarized in fig. 4. 183lizards on roads road location of mortalities and live lizards median distances of carcass location on roads were positively skewed towards the road edge in both species (p. muralis s = 0.56, l. bilineata s = 1.1) but p. muralis carcasses were found at significantly greater distances (measured in mm) from the roadside (medians ± 95% confidence intervals; p. muralis = 1240±114.5 versus l. bilineata 900±70.4mm; w = 10342, p = 0.0024). live l. bilineata were observed closer to the road edge than the location of their mortalities (w = 8689.0, p < 0.00001) with live lizard locations also having significantly smaller distributions (l = 6.4, p = 0.012). p. muralis mortalities were also found further from the road edge than live lizards (w = 9926.5, p = 0.0002) but had similar variances (l = 0.105, p = 0.74). these results are what might be expected in a lizard that was foraging across a road surface compared to one that was operating as basking/sentinel species sitting mostly at the roadside. histograms of the distributions on which these statistics are based are shown in fig. 5. roadside habitat associations both species departed significantly from the null model of a random distribution across roadside habitats. p. muralis was found in greater than expected frequency alongside low-density urban areas and woodland, whilst greater than expected numbers of l. bilineata were found on roads bisecting low-density urban areas and hedgerow bordered roads. lower than expected numbers were found alongside monocultures with no hedgerow borders in both species. table 1 gives the statistics with graphical representations of the observed frequencies against the random model of expected frequencies shown in fig. 6.   n um be rs o f in di vi du al s 0 20 40 60 80 0 20 40 60 a m j j a s o n p. muralis l. bilineata fig. 6. roadside habitat associations based on data from both live lizards and mortalities. solid histograms represent l. bilineata and open p. muralis. cross-hatched represents the expected proportions if lizards were randomly distributed alongside available roadside habitat. see text for details. fig. 3. monthly distributions of lizard mortalities (solid histograms) and live lizards (open) on roads.   basking foraging road crossing p er ce nt 0 20 40 60 80 l. bilineata, n = 47 p. muralis, n = 50 fig. 4. road behaviour of l. bilineata and p. muralis. solid and open histograms represent l. bilineata and p. muralis respectively.   n um be r of li za rd s 0 5 10 15 20 25 30 0 5 10 15 20 25 30 0 100 200 300 distance from the road edge (cm) p. muralis l. bilineata fig. 5. distances from the road edge of lizard mortalities (solid) and live lizards when first sighted (open). for live lizards road crossings are omitted. increments on the x-axis are 20cm. see text for further details.   0 10 20 30 40 50 hd urban ld urban woodland hedgerows monocultures p er ce nt f re qu en cy 184 roger meek discussion a perhaps unforeseen result of this study was the major temporal differences in lizard road presence with high numbers observed in autumn (august and september) especially in p. muralis. a compelling explanation is a comparable temporal presence of the saurophagus snake hierophis viridiflavus on roads or along roadsides (capula et al., 1997; van damme and quick, 2001). for example, over the equivalent 9 year period monthly road presence in both lizard species was correlated with the presence of hatchling h. viridiflavus on roads (based on 165 hatchling road mortalities); versus p. muralis, r = 0.95, p < 0.0001, versus l. bilineata, r = 0.69, p = 0.04. the correlation is also significant (r = 0.71, p = 0.03) between l. bilineata and adult h. viridiflavus presence (n = 122), but not with p. muralis r = 0.08, p = 0.83. it is recognised that correlation does not necessarily imply causation but this does present several possibilities; for instance that lizards may exploit the absence of visual barriers on roads to enhance snake detection. if road use enables increases in flight initiation distance (cooper et al., 2009) then the higher running speeds of most lacertid lizards compared to the fastest snakes (vanhooydonck et al., 2001; vanhooydonck and van damme, 2003; alexander, 2012) should reduce predation risk (lima and dill, 1990). lacertid lizards are additionally able to detect snake presence using chemical cues (van damme and quick, 2001) and therefore it is conceivable that when h. viridiflavus are present in numbers alongside roads, this may initiate lizard movement onto roads. interestingly, in contrast, no strong association could be found between the life cycles of the lizards and their presence on roads other than a slight increase in basking l. bilineata during early spring following emergence from hibernation (april – see fig. 3). the high autumn road presence of p. muralis did not involve juvenile lizards, which contrasts with sympatric h. viridiflavus (meek, 2009). an additional explanation is that the simplified visual environments presented by roads enhance prey detection. simultaneous surveys through 2011-2012 recorded 626 road-killed/crossing invertebrates on the six roads throughout the active months (april-november). the majority were lepidoptera (31.6%) and coleoptera (26.2%) but also gryllidae (10.6%), orthoptera (3.5%) and non-toxic/hairy lepidoptera larvae (6.1%). many of these invertebrates were road-killed or injured, which would facilitate easy prey capture. the sample shows similarities with reported diets of l. bilineata (korsos, 1984; perkins and avery, 1989; angelici et al., 1997), especially the presence of coleoptera, suggesting prey opportunities could be one of a suite of factors that attract lizards to roads. reptile presence on or alongside roads has previously been correlated with potential prey concentrations (andrews et al., 2008), which supports the patch use model (gilliam and fraser, 1987) that predicts animals should select habitat patches offering the lowest predation risk to food harvest ratio (e.g. martin and lopez, 1995; shepard, 2007) and hence is determined by the presence of predators and prey. both species were observed to use road surfaces mostly for foraging and basking with greater than expected presence on roads that bisected urban areas, woodland and hedgerows. the former agrees with non-road behaviour of both species (verwaijen and van damme, 2008) and the latter supports the notion that roadside habitat is selected in both species since non-selective animals should use habitats in proportion to their availability (alldredge et al., 1998). the greater than expected presence alongside hedgerows in l. bilineata indicates they use this habitat both for permanent occupancy and for potential connectivity between habitats. hedgerows have already been identified as important in this respect for sympatric snakes (saint girons, 1996; meek, 2015). the presence of p. muralis on roads bisecting urban environments was perhaps predictable given they are often abundant around human habitations (arnold and burton 2002), but given that this species is heliothermic, rather less anticipated was their higher than expected presence alongside woodland, which has more limited table 1. observed versus expected frequencies of habitat-associated presence of lizards on roads. columns indicate multiples of greater or less (negative values) than expected crossings in relation to habitat availability (a value of 1 indicates observed = expected). urban hd represents high-density urban areas; urban ld, low-density urban areas and mono/no hedge, monocultures with no hedgerow border. values of p specify levels of significance in χ2 tests at d.f. = 4. the p-values for monte carlo randomisation tests give probability that the true χ2 statistics were equalled or exceeded after 5000 randomisations. see text for details. hd urban ld urban wood hedgerow mono/ no hedge χ2 p monte carlo n p. muralis 1.3 2.1 1.9 -0.5 -5.8 50.2 <0.0001 <0.0001 172 l. bilineata -1.3 1.7 1.0 1.3 -5.9 23.2 <0.0001 <0.0001 187 185lizards on roads access to sunlight than other habitat types. lizards have been found to thrive in human altered habitats, including woodland, as a result of road construction (koenig et al., 2002; delgado garcia et al., 2007) with road and rail networks presenting movement pathways that enable p. muralis to colonise new areas in both europe (delgado garcia et al., 2007; gherghel et al., 2009) and north america, where is it as an introduced species (hedeen and hedeen, 1999; deichsel and gist, 2004). road corridors through woodland have previously been identified as important edge habitat enabling other heliothermic lizards to penetrate and exploit unsuitable habitat (delgado garcia et al., 2007). the mismatch between the cues for costs (e.g. mortalities from traffic) and benefits (e.g. enhanced predator detection and increased prey opportunities) for reptiles on roads has resulted in roads becoming ecological and evolutionary traps (fahrig and rytwinski, 2009). understanding the behavioural differences in road behaviour in lizards is critical if conservation measures are to be implemented to reduce mortalities. nevertheless ascertaining accurate levels of road presence is problematical. for instance, smaller size and greater rapid carcass degradation in p. muralis could potentially skew mortality data (see fig. 1). this is most likely in hatchling and juvenile p. muralis that are far more difficult to detect. a previous study found that the frequency distribution of carcass duration of both snakes and lizards on roads was skewed towards low persistence time (meek, 2009). however, if the present results are a representation of the relative differences between the two species then higher road mortalities of the locally less abundant l. bilineata is indicated. this difference has also be observed between the two species in italy (lebboroni and corti, 2006) and is probably due to prolonged basking and/ or sentinel behaviour of l. bilineata on roads, which increases the potential for a vehicle collision. when roads had paved sidewalks (as on the d2949 segment) lizard mortalities were in general lower (meek, 2009) indicating the possibility that the construction of sidewalks coupled to the implementation of speed restrictors could offer potentially simple preliminary solutions to reduce mortalities. acknowledgements comments and suggestions by dr roger avery and dr marco zuffi made valuable improvements on an earlier version of the manuscript. references alldredge, j.r., thomas, d.l., mcdonald, l.l. 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(2008): foraging mode and its flexibility in lacertid lizards from europe. j. herpetol. 42: 124-133. acta herpetologica 15(1): 47-54, 2020 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/a_h-7648 genetic characteristics of an introduced population of bombina bombina (linnaeus, 1761) (amphibia: bombinatoridae) in moselle, france jean-pierre vacher1,2,*, damien aumaître3, sylvain ursenbacher2,4 1 association bufo, musée d’histoire naturelle et d’ethnographie, 11 rue de turenne, f-68000 colmar, france 2 section of conservation biology, department of environmental sciences, university of basel, st. johanns-vorstadt 10, ch-4056 basel, switzerland 3 conservatoire d’espaces naturels de lorraine, 3 rue robert schuman, 57400 sarrebourg, france 4 info fauna – karch, university of neuchâtel, avenue de bellevaux 51, ch-2000 neuchâtel, switzerland * corresponding author. email: jpvacher@gmail.com submitted on: 2019, 16th december; revised on: 2019, 21th december; accepted on: 2019, 22th december editor: adriana bellati abstract. the fire-bellied toad bombina bombina has recently been introduced in moselle, north-eastern france, in an area where the yellow-bellied toad bombina variegata occurs naturally. both species hybridize in a wide area throughout europe where their distribution overlaps. therefore, there is a risk of introgression regarding the bombina variegata population in north-eastern france. in order to assess the status of the introduced population of bombina bombina and its origin, we investigated its genetic characteristics and structure using both mitochondrial (cytochrome b) and nuclear dna (microsatellites markers). the results demonstrated a lack of introgression in the bombina variegata population. though experiencing a bottleneck effect, the introduced bombina bombina population displays a high genetic diversity. if a propensity for expansion is found within the introduced population of bombina bombina, it could be considered as a potential invasive species in france, and thus threaten the native species. keywords. invasive species, population genetics, conservation, cytochrome b, microsatellites. introduction introduction of allochthonous species in natural habitats represents one of the aggravating factors of the current loss of biodiversity (kats and ferrer, 2003). such introductions may induce numerous effects on native species, such as ecological competition, over predation, transmission and dispersal of pathogens, and genetic introgression (mooney and cleland, 2001; strayer et al., 2006). in the context of conservation biology, it is important to understand the factors which enabled introduced species to adapt to their new environment, in order to define prevention, monitoring, and management plans for these species (strayer et al., 2006). amphibians are currently in the focus for conservation biologists as they represent the most endangered group of vertebrates worldwide (stuart et al., 2004; stuart et al., 2008). invasive alien species are a major threat to amphibians (kats and ferrer, 2003). among them, other amphibian species can have an impact on native ones. for example, they can be an important source of dispersal of the chytrid fungus batrachochytrium dendrobatidis (bd), a pathogen which affects many amphibian species around the globe and causes their decline (ficetola et al., 2008; fisher and garner, 2007; garner et al., 2006). another possible threat linked with introduced species is genetic introgression, which can, in some cases, lead to local extinctions (arntzen and thorpe, 1999; dufresnes et al., 2016; rhymer and simberloff, 1996). in france, at least six species of amphibians have been introduced: triturus carnifex, discoglossus pictus, lithobates catesbeianus, xenopus laevis, pelophylax 48 jean-pierre vacher et alii ridibundus, and p. bedriagae (duguet and melki, 2003; lescure and de massary, 2012). in 2009, an introduced population of the fire-bellied toad bombina bombina has been discovered in the eastern part of the moselle department, close to the sarre valley (vacher and pichenot, 2012) (fig. 1). in this region, the yellow-bellied toad bombina variegata occurs naturally (lescure et al., 2011; lescure and de massary, 2012; thiriet and vacher, 2010). this last species is considered as endangered in france and listed as ‘vulnerable’ on the national red list published by the french committee of the iucn (uicn france et al., 2015). the occurrence of introduced bombina bombina close to natural populations of b. variegata raises conservation concern as b. bombina could lead to the decline of b. variegata through introgression. indeed, hybridization has already been demonstrated where both species are in contact in their native range (gollmann et al., 1988; szymura, 1976; yanchukov et al., 2006). in order to characterize the status of this introduced population, and its possible interaction with the native bombina variegata, we assessed if hybridization already occurred between the native and the introduced species using genetic markers as well as the putative origin of the introduced individuals. materials and methods sampling design and laboratory methods we collected 61 dna samples from individuals morphologically assigned to bombina bombina from three localities in moselle (6.87°e, 48.92°n) and 64 samples from individuals morphologically assigned to bombina variegata from four neighbouring localities in moselle and bas-rhin in 2011 and 2012. dna samples were collected through buccal swabbing (beebee, 2008; pidancier et al., 2003). the two westernmost bombina bombina localities were 1 km distant from each other, and the third was 5 km south-east from the others. dna was extracted from the buccal swabs using the qiagen dneasy blood & tissue kit (qiagen®). as we suspected that all bombina bombina individuals would originate from the same locality, we amplified by pcr a fragment of 1200 bp of the mitochondrial cytochrome b (cytb) using l16245 and h17444 primers (hofman and szymura, 2007) from only five out of the 61 samples. amplifications were performed following hofman and szymura (2007) and sequencing were performed by macrogen (amsterdam, the netherlands). the new sequences were deposited in genbank (table 1). ten microsatellite loci specifically developed for the firebellied toad (bobomf2, bobom5f, bobom9h, bobom1a, bobomf22, bobom10f, bobom8a, bobomd2, bobomb13 and bobom11d) were amplified by pcr for all 125 samples, following the pcr conditions suggested by hauswaldt et al. (2007) and stuckas and tiedemann (2006). forward dyed primers were used in order to analyse them with an automatic sequencer (ab3130xl applied biosystem). allele lengths were then read with the software peak scanner v.1.0 (applied biosystem). data analysis we used cytb sequences from genbank to confirm taxonomic assignation of our samples. the cytb sequences obtained were first aligned automatically using the software mafft (katoh and standley, 2013), and then the alignment was checked in mega (tamura et al., 2011). we subsequently grouped our sequences with other bombina bombina cytb sequences published in a previous study on the phylogeography of the species (fijarczyk et al., 2011). after inferring the best sequence evolutionary model in partitionfinder v.1.1.1 (lanfear et al., 2012), using a bic approach, we constructed a phylogenetic tree with a maximum likelihood method in raxml v.8 (stamatakis, 2014) under the gtr+g model. we used one sequence of bombina variegata as an outgroup to root our tree. after visualizing the position of our samples in the tree, we subsequently selected the sequences that were the closest to the samples from moselle, and constructed a haplotype network using the software hapview (salzburger et al., 2011). each microsatellite locus was first examined for null allele occurrence with micro-checker v.2.2.3 (van oosterhout et al., 2004) for each population. loci showing a high probability (p > 0.05) of null alleles were discarded from the dataset. for each retained locus, we estimated allele frequency, allelic richness (ar), observed and expected heterozygosity (ho, he), and intrapopulation structuration (fis) with the packages adegenet (jombart, 2008) and hierfstat (goudet, 2005) implemented in r (r development core team, 2016). moreover, hardy-weinberg equilibrium was tested for each locus with allele randomizations (1000 permutations per test) with the package pegas (paradis, 2010) implemented in r. in addition, we evaluated the number of genetic clusters (k) using a bayesian clustering approach implemented in the software structure v.2.3.3 (pritchard et fig. 1. global distribution of bombina bombina and b. variegata and location of the newly introduced population in france. map sources: naturalearth/iucn. 49genetics of introduced bombina bombina in france al., 2000). first, we conducted the analysis for the three putative populations of b. bombina, and then for b. bombina and b. variegata populations together. we performed ten independent runs for each k, and tested between 1 and 3 for b. bombina alone, and up to seven clusters for b. bombina and b. variegata grouped together, according to the number of localities we sampled. each replicate was run for 400,000 iterations following a burn-in period of 200,000. as the three sampled populations of b. bombina were supposedly closely related, we used the admixture model with allele frequencies that were correlated among populations (falush et al., 2003). in order to identify the most likely value of k, the logarithmic probability of the data [ln p(d)] was estimated for each simulation. additionally, the value of δk, representing the second order of change, was estimated (evanno et al., 2005). finally, we tested if a bottleneck effect (significant heterozygosity excess) was detected within the population of bombina bombina with the software bottleneck v.1.2.02 (cornuet and luikart, 1996; piry et al., 1999), using a wilcoxon signed-rank test. such an effect is expected after a strong reduction in population size (hedrick et al., 1986), such as in recent introduced populations. results blast and haplotypes the blast results showed that the five samples matched with bombina bombina cytb sequences deposited in genbank. maximum likelihood inference suggests that the samples from lorraine are nested within a clade that originates from austria and czech republic (fig. 2). the haplotypes found in moselle cluster within haplogroup b3-1 (fijarczyk et al., 2011), that contains specimens from southern europe. more precisely, one individual of moselle is identical to haplotype b14 (fig. 3), that includes specimens from czech republic, slovakia, austria, croatia, serbia, hungary, and ukraine (fijarczyk et al., 2011), and the four other haplotypes retrieved from the specimens from moselle only differ from b14 by three to eight substitutions (fig. 3). genetic variation and diversity we detected an excess of homozygosity, thus the probable presence of null alleles for bobomf2, bobomf22, and bobomd2 within b. bombina populations only. therefore, these markers were discarded from the subsequent analyses for b. bombina, which were consequently conducted with seven microsatellites markers (bobom5f, bobom9h, bobom1a, bobom10f, bobom8a, bobomb13, and bobom11d). the number of alleles in b. bombina of moselle varies from three (bobom5f and bobom1a) to ten (bobom9h) fig. 2. best maximum likelihood inference obtained from raxml using ~1000 bp of cytb mtdna of bombina bombina. bootstrap values above 70 are given at each nodes. the tree is rooted on bombina variegata (not shown). the genbank accession numbers are provided, new sequences are indicated in bold. 50 jean-pierre vacher et alii fig. 3. haplotype network based on a fragment of the mitochondrial cytb in bombina bombina. the haplogroups were defined in fijarczyk et al. (2011). the five new haplotypes from the introduced specimens in moselle (this study) are represented in red. each line represents a single mutation, and the size of the circles represents the frequency of a haplotype. table 1. genbank accession numbers and additional information of the locality of the newly introduced bombina bombina in france (in bold) and the samples from genbank used in the genetic analysis. no location accession genbank reference 1 moselle, france mn784157 this study 2 moselle, france mn784158 this study 3 moselle, france mn784159 this study 4 moselle, france mn784160 this study 5 moselle, france mn784161 this study 6 hungary jf898363 fijarczyk et al., 2011 7 austria jf898357 fijarczyk et al., 2011 8 romania jf898366 fijarczyk et al., 2011 9 bulgaria jf898352 fijarczyk et al., 2011 10 hungary jf898356 fijarczyk et al., 2011 11 romania jf898353 fijarczyk et al., 2011 12 ukraine jf898347 fijarczyk et al., 2011 13 bulgaria jf898351 fijarczyk et al., 2011 14 bulgaria jf898350 fijarczyk et al., 2011 15 moldova jf898348 fijarczyk et al., 2011 16 hungary jf898364 fijarczyk et al., 2011 17 slovakia jf898345 fijarczyk et al., 2011 18 romania jf898346 fijarczyk et al., 2011 19 belarus jf898340 fijarczyk et al., 2011 20 turkey jf898362 fijarczyk et al., 2011 21 turkey jf898361 fijarczyk et al., 2011 22 ukraine jf898343 fijarczyk et al. , 2011 23 ukraine jf898342 fijarczyk et al., 2011 24 turkey jf898360 fijarczyk et al., 2011 25 moldova jf898349 fijarczyk et al., 2011 26 austria jf898358 fijarczyk et al., 2011 no location accession genbank reference 27 serbia jf898344 fijarczyk et al., 2011 28 romania jf898355 fijarczyk et al., 2011 29 moldova jf898354 fijarczyk et al., 2011 30 ukraine jf898339 fijarczyk et al., 2011 31 romania jf898365 fijarczyk et al., 2011 32 czech republic jf898359 fijarczyk et al., 2011 33 ukraine jf898341 fijarczyk et al., 2011 34 belarus jf898338 fijarczyk et al., 2011 35 russia jf898330 fijarczyk et al., 2011 36 poland jf898321 fijarczyk et al., 2011 37 poland jf898326 fijarczyk et al., 2011 38 slovakia jf898325 fijarczyk et al., 2011 39 russia jf898334 fijarczyk et al., 2011 40 ukraine jf898333 fijarczyk et al., 2011 41 russia jf898332 fijarczyk et al., 2011 42 ukraine jf898329 fijarczyk et al., 2011 43 kazakhstan jf898328 fijarczyk et al., 2011 44 poland jf898324 fijarczyk et al., 2011 45 kazakhstan jf898327 fijarczyk et al., 2011 46 ukraine jf898337 fijarczyk et al., 2011 47 russia jf898335 fijarczyk et al., 2011 48 ukraine jf898331 fijarczyk et al., 2011 49 poland jf898322 fijarczyk et al., 2011 50 russia jf898336 fijarczyk et al., 2011 51 poland jf898320 fijarczyk et al., 2011 52 poland jf898323 fijarczyk et al., 2011 fig. 4. (a) clusters from retrieved in the structure analysis from seven microsatellite markers; (b) distribution in space of the two nuclear dna clusters. the geographic coordinates are not provided on purpose. map source: global forest watch. 51genetics of introduced bombina bombina in france (table 1). the mean ar was 4.23 (calculated from 61 diploid individuals). the mean he value was 0.65, and the mean ho value was 0.66. there was no significant difference between the overall values of ho and he (bartlett’s k-squared = 0.035, df = 1, p = 0.8). the overall fis value was 0.01 and ranged from -0.05 for bobom 11d to 0.09 for bobom 5f (table 1). genetic structure of populations the analysis conducted with structure did not reveal any population differentiation between the three sampling sites of bombina bombina in moselle. in the analysis conducted with all the samples of b. bombina and b. variegata, both species formed two well-differentiated clusters, indicating a complete lack of introgression (fig. 4). bottleneck the bottleneck analysis revealed a bottleneck effect within the bombina bombina population of moselle. indeed, the wilcoxon test showed an excess of heterozygotes compared to the expected equilibrium heterozygosity under both the smm and tpm models (wilcoxon test: p = 0.007). discussion globally, the genetic diversity observed in this introduced population of b. bombina is rather high, since the mean he value of 0.65 for seven loci is similar to the ones found in 11 natural populations of bombina bombina that occur in the core of the range of the species in germany and that averages 0.70 [0.59-0.78] for six loci (dolgener et al., 2012). this could be explained by the introduction of numerous individuals, maybe through different episodes. as the biggest population was observed in a series of lakes that are used for fish farming, it is highly probable that the presence of bombina bombina in this area resulted from the transport of tadpoles caught together with young fishes from one or several close localities in central europe. such cases of translocations have been observed in brandenburg and in saxony (berger, 1996; dolgener et al., 2012), so it is very likely that the occurrence of b. bombina in moselle might result from a similar event. as tadpoles are small organisms, it is possible that hundreds, or maybe thousands of them have been introduced, therefore maintaining a high genetic diversity. still it was expected to detect a bottleneck effect within this population as it is the case with recently introduced populations (lee, 2002; puillandre et al., 2008). in comparison with other amphibian species, the fire-bellied toad does not have a high fertility rate, with around 300400 eggs per female per year (gollmann et al., 2011). therefore, the high diversity and the high number of alleles observed in some markers indicate that introduction of only a few founder individuals, as observed in other introduction of amphibians in france such as the bullfrog lithobates catesbeianus (ficetola et al., 2008), is unlikely. we could think that even though the primary source of the b. bombina population in moselle resulted from numerous tadpoles, they still represent a small fraction of a broader population located in the core area of distribution and that though a bottleneck effect could be detected, it was not sufficient enough to affect the genetic diversity of this population. the discovery of bombina bombina in lorraine is recent, certainly dating back to 2009 (vacher and pichenot, 2012). right now, its distribution is geographically restricted and does not directly overlap with that of b. variegata (fig. 4). as it can hybridize with b. variegata in the wild, a monitoring of b. bombina in the area should be set up to track the population dynamics, its dispersal behaviour, the evolution of its distribution in the area, and to determine possible concurrence with b. variegata. however, both species seem to display contrasted ecological preferences in their native habitat: b. bombina is known to prefer ponds or swamps for reproduction compared to b. variegata that favours puddles (barandun and reyer, 1997; gollmann b. et al., 2011; gollmann g. et al., 2011; kruuk and gilchrist, 1997). consequently, we might expect that ecological competition should be limited. moreover, b. bombina shows table 2. microsatellite loci used for the genetic analyses of 61 individuals of bombina bombina introduced in moselle, northeastern france. the estimations were conducted with the adegenet and hierfstat packages implemented in r. bp: base pairs; ar: allelic richness; ho: observed heterozygosity; he: expected heterozygosity; fis: intrapopulation structure index. microsatellite length (bp) alleles number ar ho he fis bobom5f 126-146 3 3 0.54 0.64 0.09 bobom9h 115-203 10 7.6 0.83 0.86 0.00 bobom1a 341-353 3 2.95 0.57 0.54 -0.04 bobom10f 207-223 4 3.97 0.75 0.73 -0.02 bobom8a 275-315 5 3.77 0.61 0.63 -0.01 bobomb13 117-147 4 3.21 0.63 0.66 0.12 bobom 11d 268-296 6 5.12 0.82 0.78 -0.05 52 jean-pierre vacher et alii higher site fidelity, suggesting lower dispersal capacities (gollmann et al., 2011). still, b. bombina seems to display a broader ecological tolerance by colonizing puddles in areas where ponds are scarce and where both species occur and hybridize (maccallum et al., 1998). therefore, a close attention to habitat components in the landscape (i.e., density of ponds and small lakes) should be integrated in a monitoring protocol to track the dynamics of this introduced population. the removal of a species at an early stage is normally the best method to avoid future competition with native species. we can consequently recommend to monitor the competition between both species of bombina in the area, and perhaps also conduct some actions to reduce or remove this introduced population. additionally, it would be necessary to scan for bd and maybe other pathogens within this introduced population, as they can represent a further threat on native amphibian populations that occur in the area such as the european tree frog hyla arborea or the common frog rana temporaria (ohst et al., 2013). acknowledgements we are grateful to laurent godé from the parc naturel régional de lorraine, julie lambrey from conservatoire d’espaces naturels de lorraine, and julian pichenot for their help on the field. we would like to thank nicolas boileau and valerie zwahlen for their help in the laboratory. a handling permit for bombina variegata was delivered to jean-pierre vacher by the préfecture du département du bas-rhin and to damien aumaître by the préfecture du département de la moselle. this study was funded by the direction régionale de l’environnement, de l’aménagement et du logement lorraine (grand est) in the scope of the national action plan for bombina variegata. we also thank two anonymous reviewers whose remarks helped enhance the manuscript. references arntzen, j.w., thorpe, r.s. 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(2006): hybridization of bombina bombina and bombina variegata (anura, discoglossidae) at a sharp ecotone in western ukraine: comparisons across transects and over time. evolution 60: 583-600. acta herpetologica vol. 15, n. 1 june 2020 firenze university press has the west been won? a field survey and a species distribution model of iberolacerta horvathi in the alps giuseppe de marchi1,*, giovanni bombieri1, bruno boz1, fausto leandri2, jacopo richard3 first record of underwater sound produced by the balkan crested newt (triturus ivanbureschi) simeon lukanov identification of biologically active fractions in the dermal secretions of the genus bombina (amphibia: anura: bombinatoridae) iryna udovychenko1,*, oleksandra oskyrko1, oleksii marushchak2, tetiana halenova1, oleksii savchuk1 don’t tread on me: an examination of the anti-predatory behavior of eastern copperheads (agkistrodon contortrix) andrew adams1,2,*, john garrison2, scott mcdaniel2, emily bueche2, hunter howell2,3 an overview of research regarding reservoirs, vectors and predators of the chytrid fungus batrachochytrium dendrobatidis jarid prahl, thomas p. wilson*, david giles, j. hill craddock genetic characteristics of an introduced population of bombina bombina (linnaeus, 1761) (amphibia: bombinatoridae) in moselle, france jean-pierre vacher1, 2,*, damien aumaître3, sylvain ursenbacher2,4 adaptive significance of the transparent body in the tadpoles of ornamented pygmy frog, microhyla ornata (anura, amphibia) santosh m. mogali*, bhagyashri a. shanbhag, srinivas k. saidapur comparison of complement system activity amongst wild and domestic animals maría s. moleón1,2,*, mark e. merchant3, hugo h. ortega4, pablo a. siroski1,2 effects of temperature and food level on plasticity of metamorphic traits in bufo gargarizans gargarizans larvae tong lei yu*, yan ting han acta herpetologica 11(1): 59-61, 2016 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-17873 amendment of the type locality of the endemic sicilian pond turtle emys trinacris fritz et al. 2005, with some notes on the highest altitude reached by the species (testudines, emydidae) federico marrone*, francesco sacco, vincenzo arizza, marco arculeo dipartimento di scienze e tecnologie biologiche, chimiche e farmaceutiche (stebicef), università di palermo, via archirafi 18, 90123 palermo, italy. *corresponding author. e-mail: federico.marrone@unipa.it submitted on 2016, 27th january; revised on 2016, 12th february; accepted on 2016, 15th february editor: sebastiano salvidio abstract. the type locality of the sicilian pond turtle emys trinacris is here amended, and its correct name and geographical coordinates are provided. the locus typicus of the species lies at 1007 m a.s.l., i.e. nearly 400 m below what previously thought. the updated altitudinal distribution range of the species, based on verified published localities only, is between 0 and 1036 m a.s.l. keywords. altitudinal limit, type locality, sicily, emydidae. the largely nearctic family emydidae is represented in the palaearctic region by two species belonging to the genus emys duméril 1805: the polytypic european pond turtle, e. orbicularis (linnaeus 1758), which is widely spread in eurasia and maghreb, and the endemic sicilian pond turtle, e. trinacris fritz et al., 2005 (stuckas et al., 2015; vamberger et al., 2015). the sicilian pond turtle was originally described based on molecular data only (fritz et al., 2005), while its morphological variability and the possible morphological or morphometric differences between e. trinacris and e. orbicularis were later investigated by several authors (e.g. fritz et al., 2006; zuffi et al., 2006; d’angelo et al., 2008; arizza et al., 2014). some data on the leech parasites occurring on e. trinacris and e. orbicularis are reported by marrone et al. (2006). the holotype of emys trinacris is an adult male collected on may 9th 1968 on the nebrodi mountain range (north-eastern sicily) by e. kramer and s. dereani, and stored in museo zoologico ‘la specola’, florence, with accession number mzuf11136 (fritz et al., 2005). in the description of the species, the name of the collection locality has been reported as “lago gian fenaro, below the pass of pizzo laminaria approximately 1400 m above sea level” (fritz et al. 2005). the same locality name for this specimen was reported by turrisi and vaccaro (1997), and di cerbo (2011), all of them directly or indirectly referring to fritz (1995) or fritz et al. (2005). conversely, referring to the same specimen stored in ‘la specola’, the locality name was reported as “gran ferraro” by zuffi et al. (2006), and the specimen was erroneously reported as a female. in the light of the unusually high altitude reported for the type locality of e. trinacris, and of the lack of other information about the type locality of this important endemic species, we attempted to gather further information on the precise location where the holotype was collected. we explored the sicilian toponyms through the ‘portale cartografico nazionale” of the italian ‘ministero dell’ambiente e della tutela del territorio e del mare’ (http://www.pcn.minambiente.it/gn/) and through an accurate study of the available cartography focusing on 60 federico marrone et alii the monti nebrodi area, but the toponyms “gian fenaro” and “pizzo laminaria” were not found in the whole sicilian territory. through the courtesy of dr. annamaria nistri, we obtained access to the original hand-written label for the e. trinacris holotype, and could check for the spelling of the locality reported on it. the label reads: “nel lago gian ferraro, sotto il passo di pizzo laminaria (monti nebrodi; sicilia) m 1400 s.l.m.” (fig. 1). in fact, on the nebrodi mountains, few hundreds of metres nw of pizzo luminaria (coord.: 14.50695 e, 37.95337 n, elevation: 1260 m a.s.l.), there is a spring (sorgente gianferraro, coord.: 14.50078 e, 37.95612 n, altitude: 1088 m a.s.l.) which feeds a pond. this last pond does not have a name on the official italian igm maps, but local shepherds call it “laghetto gianferraro” (i.e. “lakelet gianferraro”). we positively think that this pond is the locality where the specimen coded mzuf11136 in the collection of ‘la specola’ was collected, furthermore we believe that “pizzo laminaria” (which does not exist in sicily) is to be considered a misspelling of “pizzo luminaria”, and that “lago gian ferraro” is a misspelling of “lago gianferraro”. accordingly, the erroneous name of the type locality was corrected, following recommendation 74a.2 of the international code on zoological nomenclature (iczn, 1999). thus, the amended type locality of emys trinacris is: laghetto gianferraro coordinates (wgs84, decimal degrees): 14.497241 e, 37.951625 n elevation: 1007 m a.s.l. this pond occurs within the protected area “parco regionale dei nebrodi”, in the municipality of caronia (province of messina), sicily, italy. the amended type locality for emys trinacris lies at an altitude of 1007 m a.s.l., which is within the known range of the species. in fact, although existing literature reports for the species an altitudinal distribution range of 0-1200 m a.s.l. (e.g. turrisi, 2008), or 0-1400 m a.s.l., when the altitude reported in the label of the specimen mzuf11136 is included (e.g. podloucky, 1997; turrisi and vaccaro, 1997, di cerbo, 2010; mazzotti and zuffi, 2006), to our knowledge the higher occurrence locality for the species published to date is the pond urio quattrocchi (coord.: 14.395758 e, 37.901304 n), occurring at 1036 m a.s.l. on the nebrodi mountain range (municipality of mistretta); the occurrence of the species at higher altitudes is to date only anecdotal, and the isolate observations of single e. trinacris specimens in the highly popular lago maulazzo (coord.: 14.672098 e, 37.941825 n, altitude: 1448 m a.s.l.) (f.p. faraone and m. romano, pers. comm.) are likely to fig. 1. label of the holotype of emys trinacris fritz et al., 2005 stored in the museo ‘la specola’, florence. note that the code reported on the label is “111136”, while on the specimen the code “11136” is reported (cf. fig. 2b). fig. 2. holotype of emys trinacris. a: anterior view; b: posterior view. note the label with the code “11136”. 61amendment of emys trinacris type locality be ascribed to the anthropogenic introduction of observed specimens. accordingly, to date, the updated altitudinal distribution range of the species is 0-1036 m a.s.l. acknowledgments dr. a. nistri (museo ‘la specola’, florence) and dr. s. lo bianco (university of palermo, palermo) are kindly acknowledged for the help they provided in accessing and taking pictures of the label of emys trinacris holotype. prof. u. fritz (senckenberg dresden, dresden) is acknowledged for the stimulating discussion on the topic of this note. references arizza, v., russo, d., marrone, f., sacco, f., arculeo, m. 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(2006). geographic variation of sexual size dimorphism and genetics in the european pond turtle, emys orbicularis and emys trinacris, of italy. ital. j. zool. 73: 363-372. acta herpetologica vol. 11, n. 1 june 2016 firenze university press feeding habits of mesoclemmys vanderhaegei (testudines: chelidae) elizângela silva brito1,*, franco leandro souza2, christine strüssmann3 morphology, ecology, and behaviour of hylarana intermedia, a western ghats frog ambika kamath1, rachakonda sreekar2,3 a new account for the endangered cerrado rocket frog allobates goianus (bokermann, 1975) (anura: aromobatidae), with comments on taxonomy and conservation thiago ribeiro de carvalho1,2,*, lucas borges martins1,2, ariovaldo antonio giaretta1 molecular phylogenetics of the pristimantis lacrimosus species group (anura: craugastoridae) with the description of a new species from colombia mauricio rivera-correa, juan m. daza* population structure and activity pattern of one species of adenomera steindachner, 1867 (anura: leptodactylidae) in northeastern brazil maria juliana borges-leite1,*, joão fabrício mota rodrigues2, patrícia de menezes gondim1, diva maria borges-nojosa1 vocal repertoire of scinax v-signatus (lutz 1968) (anura, hylidae) and comments on bioacoustical synapomorphies for scinax perpusillus species group marco antônio peixoto1,*, carla silva guimarães1, joão victor a. lacerda2, fernando leal2, pedro c. rocha2, renato neves feio1 amendment of the type locality of the endemic sicilian pond turtle emys trinacris fritz et al. 2005, with some notes on the highest altitude reached by the species (testudines, emydidae) federico marrone*, francesco sacco, vincenzo arizza, marco arculeo photo-identification in amphibian studies: a test of i3s pattern marco sannolo1,*, francesca gatti2, marco mangiacotti3,4, stefano scali3, roberto sacchi4 no evidence for the ‘expensive-tissue hypothesis’ in the dark-spotted frog, pelophylax nigromaculatus li zhao, min mao, wen bo liao* no short term effect of clinostomum complanatum (trematoda: digenea: clinostomatidae) on survival of triturus carnifex (amphibia: urodela: salamandridae) giacomo bruni1,*, claudio angelini2 yeasts in amphibians are common: isolation and the first molecular characterization from thailand srisupaph poonlaphdecha, alexis ribas* introduction of eleutherodactylus planirostris (amphibia, anura, eleutherodactylidae) to hong kong wing ho lee1, michael wai-neng lau2, anthony lau3, ding-qi rao4, yik-hei sung5,* correlation between endoscopic sex determination and gonad histology in pond sliders, trachemys scripta (reptilia: testudines: emydidae) david perpiñán1, albert martínez-silvestre2,3, ferran bargalló3, marco di giuseppe4, jorge orós5, taiana costa6,* book review: john w. wilkinson. amphibian survey and monitoring handbook. pelagic publishing franco andreone book review: susan newman. frogs amphibians and their threatened environment. discovery and expression through art k-3. frogs are green franco andreone acta herpetologica 11(2): 197-212, 2016 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-18061 olim palus, where once upon a time the marsh: distribution, demography, ecology and threats of amphibians in the circeo national park (central italy) antonio romano1,*, riccardo novaga2, andrea costa1 1 consiglio nazionale delle ricerche, istituto di biologia agroambientale e forestale, via salaria km 29,300 i-00015 monterotondo scalo, rome, italy. * corresponding author. e-mail: antonioromano71@gmail.com 2 viale dello statuto 37, latina, italy. submitted on 2016, 27th february; revised on 2016, 27th june; accepted on 2016, 7th july editor: gentile francesco ficetola abstract. the circeo national park lies in a territory that was deeply shaped by human activity, and represents one of the few remaining patches of plain wetland habitat in central italy. in this study distribution and few demographic information of the amphibians in the park were provided. seven species and 25 bibliographic and 84 original breeding sites were recorded, and population size estimations were carried out for a population of these three species: pelophylax sinkl esculentus, bufo balearicus and rana dalmatina. for the studied populations of pool frog and green toad the operational sex ratio and the demographic effective population size was also estimated. for rana dalmatina, which is strictly associated to forest environment, a positive and significant correlation between the number of egg clutches and maximum depth of the swamps was found. the state plain forest is the most important habitat for amphibians’ conservation in the park. the occurrence of dangerous alien species was investigated and they are evaluated as the major threat for amphibians in the park, especially the crayfish procambarus clarkii in the state plain forest. index of calling survey were performed for anurans and the medians did not differ among species. the potential distribution of amphibians in the park was evaluated by building a species distribution model. finally, the absence of three species reported in literature in the 60’s of the last century (bombina pachypus, salamandrina perspicillata, rana italica) is also discussed. keywords. alien species, capture-marking-recapture, effective population size, index of calling survey, land reclamation, species distribution models, swamps. introduction until the early twenties of the past century, marshlands dominated the landscapes along the coast about 45 km southeast of rome, from anzio to terracina towns between the  tyrrhenian sea and the volsci chain, at inland distances from  15 to 25  km. the territory, which now belongs to the province of latina (latium region), was an extensive marsh at about sea level originated in an alluvial plain (e.g., linoli, 2005).  forested swamps dominated above sea level while areas below the sea level were covered by mud flats and pools. these so called “paludi pontine” (pontine marshes) were the subject of land reclamation works, performed periodically since the preroman period, initially by the italic tribe  of latins, but with scarce success. land reclamation was conducted extensively with considerable success by fascist regime, starting in the 1920s and radically changing the landscape of the area, which was converted into an extensive agricultural plain and new towns were founded and built (littoria, renamed latina, pontinia, sabaudia, aprila and pomezia are the most important ones). out of the origi198 antonio romano et alii nal 20,700 hectares of forest and swampland, about 3,200 were put under protection with the creation in 1934 of the circeo national park (cnp), which includes also other not forested areas. among terrestrial vertebrates, amphibians are the class most strictly associated to wetlands. they are an important component of biodiversity and suffer a recent worldwide decline (stuart et al., 2004; wake and vredenburg, 2008). in italy protected areas play a key role in conservation of amphibians, and act as stepping stones in the face of climate change (d’amen et al., 2011). the area where cnp falls is classified in the highest irreplaceability naturalistic values (maiorano et al., 2006). however data on the fauna before the land reclamation was available only for mammals and birds (lepri, 1935). consequently, information on which herpetological species occurred in this area before land reclamation could be only deducted by current species distribution on wider areas (bruno, 1973; bruno 1981; bologna et al., 2000), by specific works on the fauna of the cnp (carpaneto, 1986; ravenna, 2013; cinquegranelli et al., 2015), and by herpetological census of surrounding areas which were also subjected to land reclamation in the 30’s (novaga et al., 2013). in particular, the recent paper of cinquegranelli et al. (2015) provided few updated data on distribution of amphibians in the park. however cinquegranelli an co-authors surveyed only 15 sites, and information on habitat use, occupancy level and species detection probability were the main goals of their work. the aim of our study was fourfold. first, by performing an extensive survey on aquatic habitats, we provide an updated species distribution of amphibians. second, we estimated some demographic and abundance parameters. third, we evaluated habitat preferences of amphibians and we provided potential distribution information. finally, we detected relevant threats for species, population or habitats and we provided information on conservation measures. materials and methods study area the circeo national park covers 8,484 hectares of a coastal area of the central italy, and it consists of five main environments: the plain forest, four coastal lakes, the coastal dune area, the limestone massif of mount circeo (541 m asl, a promontory that marks the southwestern limit of the former  pontine marshes) and the island of zannone. the plain state forest covers about 3,190 hectares and mainly consists of deciduous woods. with many areas few meters below the sea level, most of the park does not exceed the 30 meters a.s.l. the park ranges from latitude 41°13’n to 41°24’n, and from longitude 12°50’e to 13°07’e. cnp is covered by wooded areas and semi-natural habitats for the 56%, agricultural fields cover the 18%, water bodies the 13%, artificial territories the 11% and, finally, a small portion is covered by wetlands (2%; giagnacovo et al., 2003). the climate falls in the lower mesomediterranean thermotype, upper subhumid ombrotype (blasi, 1994). mean temperature of this area is from 9.5 °c to 17.1 °c, and temperatures below zero are uncommon. precipitation is mainly concentrated in autumn and early winter  (october-december); relative humidity is high all over the year, wind is frequent with a south-western dominance (padula , 1985). distribution and species occurrence the data reported in literature, when they were provided with enough accuracy, were georeferenced. field surveys (march-september 2015) were preceded by a careful analysis of the maps produced by the istituto geografico militare (i.g.m, 1:25000) and by a analysis of satellite images to detect water bodies not reported in the maps (see romano et al., 2012). information on methods used to detect the occurrence of amphibians are reported in detail by romano et al. (2010; 2012). to describe species rarity and their diffusion we used a method that weights both diffusion (w: wide; m: medium; l: limited) and density (c: common; f: frequent; r: rare) and it consists of a graph of the relationship between the coverage (%) of the utm grid (we used a mesh of 2x2 km) and the mean number of observations for a square occupied by each species, according to the method proposed by doria and salvidio (1994) and used in other herpetological studies (turrisi and vaccaro, 2004; romano et al., 2012). in the computation of the score to build the graph of rarity and diffusion we decided to use only a subset of meshes. we included only the meshes where at least one species record occurred (i.e., 35 meshes), excluding, for example, meshes in highly urbanised areas with no data. ecology breeding aquatic sites were assigned to the following typologies: (i) ponds and marshes; (ii) slow running waters: ditches, streams and artificial channels (iii) rheocrenic springs (which were checked s up to 50 meters from they source); (iv) forest swamps (including those whose filling is partially due to limnocrenic springs); (v) artificial tanks; (vi) brackish coastal lakes. correspondence analysis (coa) was used to identify associations among amphibian species and aquatic habitats. considering that the variance of the data was homogenous (levene’s test for homogeneity, based on means, p = 0.758), the hypothesis that habitat categories may host different syntopic number of species was tested using one-way anova. correlation between the habitat availability and the number of species for each habitat type was tested using non-parametric spearman’s rank correlation. analyses concerning habitat typologies were performed both on original and bibliographic data, when the latter could be certainly assigned to a given habitat typology. ecological analyses were performed in the statistical package past (hammer et al., 2001). 199amphibians of the circeo national park population abundance estimation populations estimates were performed using four different methods: index of calling survey (ics), capture-mark-recapture  (cmr), removal sampling (res), and egg mass counts (emc). for the ics, which is the relative measure of calling density (mossman et al., 1998; weir and mossman, 2005), surveys were performed as reported in dorcas et al. (2009) and romano et al. (2012). ics may vary among 0 and 3. considering that ics provides scores as ordinal measures and with many ties, the scores were analysed using the median test as performed in statistica® ver. 5.0 (statistica package, statsoft inc., usa). differences in calling survey scores among anuran species were compared using the non parametric kruskall-wallis test and relative post hoc comparisons. using photo-identification, cmr analysis was applied on a population of bufo balearicus breeding in an artificial tank in the inner of sabaudia town; we used the software capture (otis et al. 1978) to estimate adult population size (n) analysing data from the four sampling sessions performed in a short time range (23 september – 2 october; light rain, between 9 and 11 p.m.). res was applied on a population of pelophylax sinkl. esculentus breeding in the artificial pond of the headquarter of the park (sabaudia). we used the jackknife estimator of pollock and otto (1983) as performed in the program capture to estimate n on the basis of three removal sessions performed in about three hours. both for bufo balearicus and pelophylax sinkl. esculentus populations, the operational sex ratio (calculated just on the number of males and females captured and not on population estimates), in accordance with wilson and hardy (2002), was expressed as the proportion of mature males, i.e. males/(males + females). the demographic effective population size (ne) was estimated as ne = (4*nm*nf)/( nm+nf), where n is the number of mature males (m) or females (f ) individuals (wright, 1938), which is a widely used equation to obtain demographic estimates of ne (e.g. jehle et al., 2001; schmeller and merila, 2007). emc was used in march to estimate breeding populations of rana dalmatina in seven natural ponds in forest environment; egg counts were performed in a unique sampling session per pond; as r. dalmatina is an explosive breeder (sofianidou and kyriakopoulou-sklavounou, 1983; guarino and bellini, 1993) and each female lays a single egg mass per season (nollert and nollert, 1992), consequently we considered the counts of egg masses as a good proxy of the minimum female population size (griffiths and raper, 1994; grossenbacher et al., 2002). surface areas of breeding ponds and swamps were estimated by walking the  perimeter  of each site with a gps, automatically calculating the area inside the resulting shape. where egg masses of r. dalmatina were counted, spearman  rank  correlation  was used to test the association between density of egg masses and size or maximum depth of the swamps. correlation between water surface and their maximum depth was also tested for all 15 swamps where r. dalmatina breed (correlations were performed in past; hammer et al., 2001). potential distribution the potential distribution of amphibians in the cnp was evaluated by building a species distribution model (sdm), using the algorithm of maximum entropy (maxent, phillips et al., 2006). selection of environmental data layers, to be employed for these analysis, was based on availability and  a priori  expectation of influences on amphibian population. considering that the park area runs along the coast and it is a small area, precipitations and other climatic variables were considered homogenous in that area and were not included among variables. we used the digital elevation model (dem) with a spatial resolution of 90m to obtain other topographic variables. all variables were resampled in order to match the 90m resolution of the dem. the environmental variables used were: corine land cover data 2006; landsat tree cover, representing the percentage of canopy cover of trees higher than 5 m; distance from forest swamps; distance from running waters; distance from lakes; topographic variables calculated from dem were elevation, aspect (northness or eastness), valley depth, topographic wetness index (twi; sörensen et al., 2006), topographic ruggedness index (tri; riley et al., 1999), wind exposure, direct insolation (kw/h per square meter). to build the sdm, both original and bibliographic data were pooled together. the whole data set was split in two subgroups by random selection: 70% of points were used for building the model (training), while the remaining 30% of point-data were employed to evaluate its predictive power. this procedure was repeated 10 times for each species, generating an averaged prediction of amphibians’ distribution. finally, the predictive power of the model was evaluated by calculating the area under the receiver operating characteristic curve (auc). analysis were conducted in software maxent 3.3.3k and default software settings were used, with the exception of the employment of bootstrapping procedure and number of iterations (1000). all gis processing to obtain the above mentioned layers was performed using software saga gis. threats considering that in the park there is a high-density road network and that road mortality may be considered as an additional factor contributing to the amphibian decline (puky, 2006; glista et al., 2008), roads were checked systematically. in particular we controlled the two parallel coastal road and waterfront roads (both about 25 km) after rains in spring and summer. the causes for amphibian declines are many (e.g., collins, 2010) but habitat loss and alteration, predator alien species and emerging diseases are among the leading. habitat loss and habitat alterations in progress was recorded and we searched for exotic animal species for which is well established that they are a threats for amphibians (i.e., predator fishes, crayfishes). alien species were searched in every site where amphibians surveys 200 antonio romano et alii were performed, both using visual surveys (using a binocular too) and blind dip netting (20-30 dip netting per site). information on fish was also obtained by fishermen. results distribution and species occurrence distribution was the result of both bibliographic (when they could be georeferenced with a good approximation, i.e. 500 m; n = 25) and original data (n =84). all available literature (bruno, 1973; bruno 1981; bologna et al., 2000; carpaneto, 1986; ravenna, 2013; cinquegranelli et al., 2015) is consistent in reporting the following species in the park: triturus carnifex (laurenti, 1768), lissotriton vulgaris (linnaeus, 1758), bufo balearicus boettger, 1880, bufo bufo (linnaeus, 1758), hyla intermedia boulenger, 1882, pelophylax sinkl. esculentus (linnaeus, 1758) and rana dalmatina fitzinger in bonaparte, 1838. the pool frog synklepton is formed by two entities: the parental species, p. lessonae (camerano, 1882) and its hemiclonal hybrid, the klepton p. kl. easculentus (linnaeus, 1758). our research confirmed the occurrence of all these species. conversely, the occurrence of salamandrina perspicillata (savi, 1821), bombina pachypus (bonaparte, 1838) and rana italica dubois, 1987 reported by bruno (1981) in many localities of the park was not confirmed by our samplings, consistently with other researches (carpaneto, 1986; bologna et al., 2000). species distributions are reported in figs. 1 and 2 the graph related to diffusion and density (fig. 3) showed that triturus carnifex is the species having the most critical situation in the park, with very limited diffusion and occurrence (i.e., low number of sites). no species are positioned in the upper right corner of the graph, however rana dalmatina and pelophylax sinkl. esculentus are of little concern. all the other species, considering their distribution and rarity, do not appear to be particularly threatened. for the particular situation and position in the fig. 3 of bufo balearicus, at the boundary between 4 quadrants, see discussion. ecology fig. 4 shows aquatic habitat preferences of each species. both newts occurred in a limited number of habitat typologies while the more ecologically plastic species was the tree frog that breeds in all the habitat typologies. variation in amphibian species composition among habitats is highly explained (more than 80%) by the first two axes of the coa scatter plot (fig. 5). associations were found between the amphibian species and the various aquatic habitat typologies. the results show that all the species are quite different from each other for habitat preference. few species are strictly associated to only one aquatic habitat typology: rana dalamatina, hyla intermedia and pelophylax sinkl. esculentus are closely linked to forest swamps, ponds and slow running waters respectively. bufo balearicus is associated with ponds and lakes while b. bufo seems to be associated with springs and artificial tanks. the newts display a moderate and comparable association with forest swamps, ponds and slow running waters. the number of syntopic species did not differ significantly among the six habitat categories (one-way anova, f1,5 = 0.32, p = 0.90). number of species in the different aquatic habitat typologies ranges between 3 (lakes) and 7 (ponds and slow running waters). spearman’s  rank  correlation did not detect a significant association between the habitat availability and the number of species for each habitat type (r = 0.806; p = 0.083). population abundance estimation index of calling survey (ics) was performed to several populations of b. balearicus (16 populations), h. intermedia (16), p. sinkl. esculentus (52) and b. bufo (16). for the three first species all the four ics scores were recorded (0-3), but for the latter the highest score lacked (fig. 6). the score 1 for both toads clearly exceeded other scores proportionally. medians of ics did not differ among species (χ2 = 5.678, df = 3, p = 0.128). capture-marking-recapture (cmr) was applied on a population of bufo balearicus living in a urban meadow (5800 m2) surrounding the breeding site (a concrete tank). during the four sampling sessions, we performed a total of 56 captures in which 26 different individuals were marked. closure test confirmed that the population was closed (z = 1.89; p = 0.97). the recapture rate was high and the estimated population was 27 ± 1.32 adult toads (estimate ± se; ci 95% = 27-33). considering that the operational sex ratio was extremely balanced (0.46), the estimate of effective population size (ne=25.8) was similar to that of the adult population census size (n). the n of p. sinkl. esculentus breeding in an artificial ponds (88 m2), was 60 ± 6 individuals (estimate ± se; ci 95% = 53-78). the operational sex ratio was strongly male biased (0.94) and, as a result, effective population size was much lower than n (ne=11.25). the seven swamps where egg mass counting (emc) of r. dalmatina was performed had greatly variable water surface area (mean ± sd = 4637.43 ± 5691.36 m2; range = 124-15860 m2) and maximum depth (mean 201amphibians of the circeo national park ± sd =64.28 ± 26.37 cm; range = 40-110 cm). surface area and maximum depth were not significantly correlated (r=0.654; p=0.128). correlation remained above the significance threshold even considering all the swamps (n = 15) where reproductive activity was recorded (i.e., pooling sites where egg counts was performed and these where it was not performed) (r = 0.506; p = 0.053). number and density of egg masses greatly varied among sites (fig. s7). a total of 1419 egg masses were recorded (mean ± sd = 202.71 ± 199.09 sd; range 13-604) and their density ranged from 0.01 to 0.47 egg clutches per square meter (mean ± sd = 0.14/m2 ± 0.18). a signifilissotriton vulgaris salamandrina perspicillata bombina pachypus rana italica bufo balearicus a b c d figure 1 o triturus carnifex lissotriton vulgaris bufo balearicus a b c d salamandrina perspicillata bombina pachypus rana italica o m fig. 1. distribution of amphibians in the circeo national park (central italy). a = distribution of three species reported just once in the literature but not recorded during further researches. for figs. b, c and d, circles = original data; triangles = bibliographic data. grid reports 10x10 km utm squares. dashed line: park boundary. dotted lines: surface hydrography. dotted areas: lakes. the urban area of sabaudia is showed in grey. 202 antonio romano et alii cant correlation among eggs parameters and swamps features was detected only between the number of egg masses and maximum depth of the swamps (r = -0.509, p = 0.249; r = -0.055, p = 0.919, for egg density vs. swamp size or depth respectively; r = 0.643, p = 0.109; r = 0.836, p = 0.025 for egg number vs. swamp size or depth respectively. see fig. 7). potential distribution species distribution models (sdm) were built for all seven amphibian species occurring in the park. the number of available data to be employed (both as training and test data) for model building was dependent on the species (see tab. 1 for details) and ranged between hyla intermediabufo bufo figure 2 a b c d pelophylax sinkl esculentus rana dalmatina fig. 2. distribution of amphibians in the circeo national park (central italy). circles = original data; triangles = bibliographic data. grid reports 10x10 km utm squares. dashed line: park boundary. dotted lines: surface hydrography. dotted areas: lakes. the urban area of sabaudia is showed in grey. 203amphibians of the circeo national park 11 (triturus carnifex) and 57 (pelophylax sinkl. esculentus). all models shown a high predictive power, as revealed by auc: indeed all averaged models received an auc value > 0.970 (ranging from 0.974 for bufo balearicus to 0.997 for rana dalmatina) with the exception of the models regarding triturus carnifex, which shown a slightly lower predictive power (auc = 0.947). as a rule, among all variables included in the analyses, the most important ones for the major part of the species are the distance from water bodies (both lakes and swamps), tree cover and insolation, altogether with many corine categories. the detailed results, regarding the list of variable effect, together with variable percentage contribution, for each species, is reported in tab. 1. potential distribution maps are presented as supplementary materials (figs. s1-s4) threats in the circeo national park we found three ponds and a swamps which are breeding sites of lissotriton vulgaris, pelophylax sinkl. esculentus and rana dalmatina that suffer progressive filling with soil, reduction of the depth and earlier dry up. in april 2015, remains of several r. dalmatina eggs desiccated before hatching were recorded in these ponds. the water body with highest danger of disappearing was the one in locality “cerreto fig. 3. relationship between percentage of amphibian species occurrence in utm square grids (2x2 km) and mean number of observations per utm square. tricar = triturus carnifex; lisvul = lissotriton vulgaris; bufbuf = bufo bufo; bufbal = bufo balearicus; hylint = hyla intermedia; randal = rana dalmatina; pelesc = pelophylax synkl. esculentus. figure 4 fig. 4. habitat partitioning (number of sites on the left vertical axe) of amphibians in the circeo national park (central italy). codes of species are as reported in fig. 3. fig. 5. correspondence analysis (coa) scatter plot illustrating variations of amphibians species (black dots) distribution with aquatic habitat typologies (white squares). the percentages of variation explained by each axis are given in round brackets (codes of species are as reported in fig.3). 204 antonio romano et alii fontana”, a pond with surface area of 240 m2 and maximum depth of about 20 cm. no relevant evidence of road killing on amphibians were recorded and, during about 20 surveys, just few individuals of b. balearicus were found crushed in summer, after a rainy day, in a small stretch of the seafront road (loc. bufalara, sabaudia). reproductive populations of five alien species, identified by the literature as threat to amphibians (see discussion), were detected. in ascending order of threat to amphibians they were one reptile, three fishes and a crustacean: trachmeys scripta; carassius auratus, lepomis gibbosus, gambusia sp., procambarus clarkii. on the whole they occurred at least in 14 utm mashes (2x2 km), that is 32% of the total meshes occupied by the park for at least 10% of their surface (n= 44; fig. 8). amphibian occurred in all 2x2 km utm meshes where alien species were recorded. original data concerning the distributions of these alien species are shown in supplementary materials (figs s5-s6). discussion species occurrence and their actual and potential distribution in the 84 sites with amphibians, we found seven species in the circeo national park as reported in literature (carpaneto, 1986; cinquegranelli et al., 2015). however the occurrence ratios among species we found (l. vulgaris: 17%; t. carnifex 4%; b. balearicus: 18%; b. bufo: 25%, h. intermedia: 19%; p. synkl. esculentus: 62%; fig. 6. percentage of index of calling survey (ics) scores for four anuran species in the circeo national park (code of species as in fig. 3). n = number of sites for each species. fig. 7. trend of the relationship between the number of rana dalmatina egg masses and maximum depth of seven swamps. alphanumerical codes refer to swamps: ver (piscina della verdesca), bag (piscina delle bagnature), car (piscina del carpino). correlation was statistically significant (spearman correlation, r = 0.836, p = 0.025). 0 5 10 15 20 25 trachemys scripta carassius auratus lepomis gibbosus gambusia affinis poracambarus clarkii alien species sites utm (2x2 km) % utm with amphibians (n=35) fig. 8. number of sites and utm mashes (2x2 km) where alien species were recorded in the circeo national park. 205amphibians of the circeo national park r. dalmatina: 30%) differed, in some cases, from those reported by cinquegranelli et al., 2015 (20%, 13%, 20%, 20%, 53%, 67%, 20%, respectively). these authors, performing 14 visit in each site, provide an interesting contribution testing the species detection probabilities (p), misdetection rates (mr) and minimum number of visits (nm maximum=10.3) necessary to be 95 % certain that an unrecorded species is in fact absent from a given site. we performed 1-5 samplings in each site and the species presence we recorded is, obviously, affected by species table 1. contribution to species distribution models of amphibians in of the circeo national park (central italy). auc, that may range from 0 to 1 (null-maximum predictive power of the model) is also shown. lissotriton vulgaris auc = 0.995 occurrence locations = 24 variable % contribution effect / corine categories distance from swamps 54.1 negative tree cover 13.1 positive for values > 50% corine 12.1 agricultural areas with significant portions of natural vegetation, forested areas – scrubs and herbaceous – sclerophyllous vegetation, inland water-bodies – water courses triturus carnifex auc = 0.947 occurrence locations = 11 variable % contribution effect / corine categories corine 50.3 agricultural areas with significant portions of natural vegetation, agro-forestry areas, forested areas – scrub and herbaceous – sclerophyllous vegetation distance from swamps 24.3 negative distance from lakes 14.2 negative bufo balearicus auc = 0.974 occurrence locations = 20 variable % contribution effect / corine categories corine 42.6 agricultural areas with significant portions of natural vegetation, forested areas, open spaces with little vegetation distance from lakes 36.1 negative insolation 11.2 negative bufo bufo auc = 0.993 occurrence locations = 24 variable % contribution effect / corine categories corine 25.7 forested areas distance from lakes 21.9 negative tri 20.3 positive hyla intermedia auc = 0.989 occurrence locations = 23 variable % contribution effect / corine categories tree cover 19.2 positive distance from lakes 18.7 negative corine 18 inland water-bodies wetlands, forested areas distance from swamps 17.5 negative pelophylax sinkl. esculentus auc = 0.981 occurrence locations = 57 variable % contribution effect / corine categories distance from swamps 44.4 negative tree cover 15.6 positive distance from lakes 14.4 negative rana dalmatina auc = 0.997 occurrence locations = 29 variable % contribution effect / corine categories distance from swamps 75.1 negative tree cover 10.7 positive for intermediate values 206 antonio romano et alii detection probabilities. however in the 40% of the sites sampled by cinquegranelli et al. (2015) we found at least one additional species (1-4). we think that this discrepancy among the species’ ratios could be probably due to the high difference in the number of sampling sites, to different sampling protocols, and to different years (difference in annual precipitation may affect, intuitively, amphibian species detectability) our original data showed that urodelans have a more limited diffusion and occurrence than anurans. in particular, t. carnifex has been detected only in three sites during our survey, resulting as the rarest species in the park (fig. 3). its distribution appeared associated with the deep swamps and slow running waters of the plain forest (fig. 4; tab. 1), and this datum agrees with the habitat preferences known for this species (andreone and marconi, 2006). however, data for the surrounding areas (novaga et al., 2013) indicate that vernal ponds and marshes are often colonized by both t. carnifex and l. vulgaris, suggesting that the limited distribution of newts outside the state forest might also be affected by the occurrence of alien species (fig. s5), especially p. clarkii (fig. s6). bufo balearicus was recorded in open habitat with sandy and clay soils, as typically showed by this species in other italian areas (balletto et al., 2007), around the coastal lakes (fig. 1) where retrodunal ponds and marshes in grazing lands are the most preferred breeding sites (fig 4; tab. 1). the concentration of its elective habitats along the coastal areas explains the overall limited diffusion of this toad in the park area. on the contrary, b. bufo appears widespread in different habitat typologies (fig. 2), and uses a greater variety of breeding sites (fig. 4). hyla intermedia has been detected both in open and forest habitats but the ics scores showed higher concentrations in the wooded habitats, as could be predicted for a semi-arboreal species. rana dalmatina can be considered to be the most representative species of the hygrophilous plain forest and no breeding sites were found outside the forest boundary. finally, p. sinkl. esculentus is the commonest species in the park (fig. 2 and 3), as expected for a species which is ecologically plastic and tolerant to anthropic disturbance in a highly urbanised context. during our surveys we did not find s. perspicillata, r. italica and b.pachypus. the question is whether these species actually occurred in the 60s’ as reported by bruno (1981) and became extinct in the last decades, or their records reported by this author have to be considered as erroneous. these three species are characterised by different ecological requirements, with salamandrina and r. italica strictly associated to clean running waters, and mainly shady, cool and damp areas (utzeri et al., 2004, angelini et al., 2007). conversly, bombina pachypus has a realtively wide ecological niche, but it is an heliophilous and thermophilus species tipically linked to open, lentic and shallow waters (guarino et al., 2007). while for the latter species suitbale habitat were actually identified in the park and its occurrence in past decades cannot be excluded, for the first two amphibians we did not find terrestrial and aquatic habitats matching their ecological requirements. suitable breeding sites could presumably be available before land reclamation (20s’ of the xx century) in a restricted piedmont area on the northern slope of the circeo massif (loc. “sorgente mezzomonte” and “rio torto”). ecology the full range of aquatic habitats available in the park is largely exploited by toads, tree frog and pool frog, while the two newts and rana dalmatina exhibited a narrow habitat niche (fig. 4). larger pools are generally deeper in environments similar to that of the cnp (brooks, 2005) but in the forest we studied this correlation was not significant. wet phase duration is generally correlated with both pond surface area and maximum depth (schneider and frost, 1996; brooks and hayashi, 2002) but, if these two features are considered independently, maximum water depth is generally the best predictor of hydroperiod (calhoun et al., 2003; skidds and golet, 2005). the habitat suitability index for r. dalmatina elaborated by radiguet (2012) showed that the date of drying of the pond is one of the key components to make habitats highly suitable for this frog. the positive and significant correlation we found between the number of egg clutches of r. dalmatina and maximum depth of the swamps (fig. 7) is probably related to wet phase duration. furthermore, as in other italian areas (e.g., bernini et al., 2004), also in the cnp rana dalmatina was strictly associated with swamps and eggs were preferentially spawned in water bodies with intermediate values of tree cover (tab. 1; fig. 4 and 5). distance from swamps also negatively affected the occurrence of both newt species (tab. 1; fig. 5). toads are associated with the environment surrounding the lakes (tab. 1), but the two species differed in their canopy requirements, as the green toad was associated with open spaces with little vegetation, while the common toad was associated with forested areas. population abundance estimation abundance of b. bufo, b. balearicus, h. intermedia and p. sinkl. esculentus greatly varied among sites, as emerged from the ics (fig. 6); the maximum score (3) was recorded for almost all species (2-19% of the surveyed 207amphibians of the circeo national park sites, see fig. 6), except the common toad. the question of whether index of calling survey (ics) can be considered a good proxy of actual population size is controversial (see for instance jansen, 2009 and corn et al., 2011 for articulated discussions on this topic). the reliability of ics depends on species and is higher for species with loud calls (i.e., higher detectability), as that we studied, and for tree frogs and pool frogs in particular (pellet and schmidt, 2005; tanadini and schimdt, 2011). for b. bufo, b. balearicus, h. intermedia and p. sinkl. esculentus in the circeo national park, ics could be easily used in extensive monitoring programs with relatively low effort. furthermore, monitoring data collected for ics may be elaborated using a recent class of statistic models that provide abundance estimations and that consider detectability (n-mixture models; royle, 2004; royle and link, 2005). fort three species we elaborated demographic estimates. capture-marking-recapture (cmr) method estimated about 27 green toads in a population, displaying  an even sex ratio; a population of pool frogs, using removal method, was estimated to consist of about 60 adults, with strongly male biased sex ratio. in several italian populations of b. balearicus sex ratio is typically male biased and their ne are highly variable (see table 1 in giacoma, 1999). in 26 italian populations demographic parameters are (range, mean ± s.d., 25th75th  percentile): n= 3-292; 89.46 ± 79.66, 28-144.5; sex ratio = 0.53-1, 0.82 ± 0.13, 0.73-0.94, ne = 0-254.94, 50.27 ± 61.85, 12.84-64.05; (data elaboration from synoptic table 1 in giacoma, 1999). these two anurans populations showed a contrasting situation if n and ne are considered independently. one season of data collection provides the size of effectively breeding individuals (nb) which is directly connected and derived by ne because nb times the generation time approximates ne (waples, 1990). thus both nb and ne are connected to population’s persistence probability, and may be used as indicators of a population’s viability (e.g., frankham et al., 2002). considering both the high accessibility of the two sites and the ease of the sampling, these two populations of b. balearicus and p. sinkl. esculentus could monitored in be long term to corroborate our data and to assess populations trends. rana dalmatina may be considered the most representative species of the park, because this frog is strictly associated with the swamps in the state forest which represents the residual environment of the pre-reclamation (tab. 1). although this frog has a limited distribution it is common (fig. 3) and abundant (figs 2d and 7) in its elective environment. the sex ratio in rana dalmatina is male biased in 90% of the breeding ponds (lodé et al., 2004; lodé et al., 2005; lodé 2009) ranging from about 0.8 to 3. considering this range of sex ratio, in the seven swamps we studied in the state forest, the whole population size might approximately range between 2550 and 5700 adults. threats and conservation strategies habitat loss and alteration. the cnp is among the most urbanized protected areas at national level. as a consequence, aquatic habitat loss and alteration (which are the main causes of amphibian decline at global level; see collins, 2010) associated with land development, present the greatest challenge to the persistence of these habitats and their animals. however we did not find significant evidence of aquatic habitat loss due to current anthropic pressure. the aquatic habitat characterizing the pnc are swamps and ponds with a typical semiperennial or seasonal hydroperiod. they are lentic shallow water bodies that are deep, on average, about 60 cm (see results). the main conservation problem of swamps and ponds was an habitat evolution toward dryer situations in shorter time, which is largely due to the dramatic change in the hydrological regime resulting from the past land reclamation. an excavation to increase water depth and the hydroperiod duration was planned for the pond in the locality “cerreto fontana” where three amphibian species spawn and that is exposed to a fast drying (see results). road mortality. road mortality in the cnp does not seem to be a problem for amphibians and, probably, only one situation (loc. bufalara) deserves further researches to estimate the actual impact on the green toad population. alien species. invasive alien species are among the key factors threatening biodiversity (eea, 2012). we found five alien species that, considering the available literature, may be considered as threats to amphibians (fig. 8, fig. s5-s6). the red-eared  terrapin trachemys scripta may have a large negative impact on amphibian populations (tadpoles; polo-cavia et al., 2010) in water bodies with high numbers of alien turtles. trachemys scripta seems to be more diffused in the northern part of the park (fig. s5a). its occurrence in the swamps of the state forest was never recorded, even in the past (a. romano pers. obs.). we did not find massive aggregations of trachemys, although some sites (e.g., the surroundings of the fogliano lake) support higher population density than southernmost areas. as a consequence of the apparently low density of trachemys, we think that its management, for the conservation of amphibians, is less urgent than that of other aliens species. three alien fishes were detected in small water basin too. the gold fish, carassius auratus can strongly affect 208 antonio romano et alii amphibian populations either by predation at different life stages (e.g., monello and wright, 2001) and influencing reproductive behavior (winandy and denoël, 2013). however when gold fish does not reach high demographic density, it seems that its presence is compatible with persistence of native amphibians (hartel, 2004). therefore to well understand the threat level to amphibians, the occurrence of gold fish in the park should be evaluated both in the distribution and in population size. the presence data here reported (fig. s5b), which shows a scattered and limited distribution, are the only one recorded during this study, but it should be considered that this fish is more widespread in the park (zerunian, 1984; zerunian and leone, 1996). gold fish did not occur in the water bodies of the state forest, probably because these aquatic habitats have a seasonal hydroperiod. the pumpkinseed sunfish, lepomis gibbosus, was introduced for the first time in italy in the varano lake in 1900 (central italy) and experienced an impressive increase of distribution in these last years (zerunian, 2002). due to its relatively small size l. gibbosus mainly preys upon amphibian larvae and eggs, but can severely damage also the adults (hartel et al., 2007). information about its negative impact on amphibian populations are corroborated from experiments with  controlled conditions (adams, 2000). l. gibbosus, that we recorded in several ditches, is probably widespread in this park characterised by a high connectivity among linear water bodies. it is worth to mention that l. gibbous was recorded at high density in a large concrete artificial tank (30x5 m, about 2 m depth) on the southern slope of the circeo massif, (fig. s5c), which is the area with lowest water habitat availability in the park. we found a female of bufo bufo in that tank but no breeding activity was recorded. the artificial tank is a potential habitat for at least three amphibians species, because b bufo, p. sinkl. esculentus, and h. intermedia breed in a similar tank 1.6 km away on the same slope (fig. 2). the eradication of lepomis gibbosus for these sites is planned and will be carried out in 2016 by the park. vredenburg (2004) demonstrated that removing introduced fish can enable amphibian populations to recover to pre-decline levels. the mosquitofishes of the genus gambusia were introduced into natural or artificial water environments in many parts of the world as a  biocontrol  to mosquito populations, in particular where there are (or there were) malaria infections. the effectiveness of this fish in combating malaria is still debated and this discussion is outside the scope of this paper. by the way, it was imported for the first time in italy, in pontine marshes on 1922 by g.b. grassi (sella, 1926; ronchetti, 1968) where, as the other italian areas, is widespread. the introduction of gambusia sp. strongly depresses all amphibian populations (adams, 2000; katz and ferrer, 2003; wells, 2007; segev et al., 2009). the observations in the park indicate widespread presence (fig. s5d), as the mosquitofish occurs in a least in a quarter of aquatic sites that host amphibians (fig. 8). the mosquitofish seems to be absent from the water bodies in the state forest, probably because they have of a seasonal wet regime. eradication of mosquitofish was planned and carried out in 2015, in an artificial ponds where only pool frogs breed. around this aquatic site, located in a meadow garden, six rock piles were also placed to offer additional refugia to small vertebrates. after fish removal and the placing of the artificial shelters (november 2015), on february 2016, the pond was colonised by b. bufo, but it is a potential breeding site also for tree frogs that are in the surroundings. the pond is in the area devoted to tourist and visitor reception; information panels about the performed conservation action were also placed. the last alien species we recorded, and the most dangerous one, is the red swamp crayfish, procambarus clarkii, which is an efficient predator of amphibian larvae of several european species (gherardi et al, 2001; cruz and rebelo, 2005; cruz et al., 2006a; ficetola et al., 2011). the presence of this crayfish is a deterrent for the colonization of potentially suitable aquatic habitats by amphibians (cruz et al., 2006a, b; ficetola et al., 2011). furthermore, p. clarkii is the cause of massive local extinction of amphibians, as happened for instance in a portugal natural reserve where crayfish caused the disappearance of more than 50% of amphibian species (cruz et al., 2006b), or, in italy, the extinction of rana latastei from one part of its already small distribution range (mazzotti et al., 2007). it is also a vector of the pathogen batrachochytrium dendrobatidis which is capable to depressing or to extinct of amphibian populations. in the cnp it is the alien species with more records and widest distribution (figs 8 and s6) and its occurrence is likely underestimated. procambarus clarkii is the only alien species reported within the state forest (fig. s6), so all amphibians occurring in the forest are severely threatened. eradication of p. clarkii from the site in the state forest, is an aim of the park. to achieve this goal, an integrate strategy is under consideration, using both intensive removal (hein et al., 2007) and biological control (aquiloni et al., 2010). an annual monitoring to detect further invasions into other areas of the forest was also planned. a pilot study to test the effectiveness of active removal (i.e., the less expensive method) on the state forest populations is currently in progress. an updated, large, and geo-referenced database of species distribution is the essential prerequisite for any protected area to effectively manage its resources and 209amphibians of the circeo national park to plan appropriate conservation strategies. in the cnp seven species were recorded, few of them strictly associated with particular aquatic habitats and thus with limited distribution, as rana dalamatina is. for this species the maintenance of swamps is an essential prerequisite for conservation. the debated issue about the presumed presence of salamandrina perspicillata, bombina pachypus and rana italica can reasonably be regarded as solved: these species are absent in the park and their closest sites are at least 10 km away, on the volsci chain. no reliable information are available to assess if they occurred before land reclamation or not. despite the highly urbanised territory, habitat loss and alteration seem to be limited, and few practical and rather simple actions can be made to improve the current situation. the main threats to amphibians in the park, in our opinion, are the spread of alien species. particular concern deserves the invasion of the red swamp crayfish in the state plain forest which is the area with highest level of species richness. acknowledgement this research was carried out under the project “progetto di sistema dei parchi nazionali italiani; action 6: monitoraggio delle specie  di  ambiente umido  /  acquatico”, funded by the italian ministry of environment (direttiva mattm ex cap. 1551). capture permit and manipulation of individuals were approved by the italian ministry of environment with the authorisation number pnm-2015-0016824/pnm. ester del bove (pnc) and alessandra noel (corpo forestale dello stato) greatly support this research; luigi loffredi contributed to field researches. thanks to marta biaggini for her help in the field study on green toad. the budding naturalist francesco maria romano has contributed with great enthusiasm to sampling activities. we are indebt with two anonymous reviewers who greatly improved the ms. supplementary material supplementary material associated with this article can be found at < http://www.unipv.it/webshi/appendix >. references adams, m.j. 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(eds). what works in conservation. open book publishers, cambridge, uk. http://dx.doi.org/10.11647/obp.0060 sebastiano salvidio acta herpetologica 12(1): 3-17, 2017 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-18188 morphological variation and sexual dimorphism in liolaemus wiegmannii (duméril & bibron, 1837) (squamata: liolaemidae) from uruguay joaquín villamil1,*, arley camargo2, raúl maneyro1 1 laboratorio de sistemática e historia natural de vertebrados, instituto de ecología y ciencias ambientales, facultad de ciencias, udelar, iguá 4225, montevideo, uruguay; *corresponding author. e-mail: joakorep@gmail.com 2 programa de desarrollo universitario, centro universitario de rivera, udelar, ituzaingó 667, rivera, uruguay submitted on: 2016, 20th april; revised on: 2016, 1st december; accepted on: 2017, 26th february editor: aaron m. bauer abstract. intraspecific morphological variation is a relatively common pattern among lizards, where several selective factors have been suggested as responsible for this phenomenon. for instance, geographic variation could result from natural selection along with historical processes, whereas sexual dimorphism has usually been attributed to sexual selection, natural selection, and niche segregation. liolaemus wiegmannii is a diurnal lizard distributed in the center, center-east and north-west of argentina, as well as on the shores of south-west and south uruguay. information about morphological variation in this species is almost entirely limited to differences in mid-body scales between populations in the north and center of argentina and some sex-based morphometric variation. herein, we studied the geographic and sexual morphological variation of liolaemus wiegmannii from uruguay to test the hypothesis of morphological isolation by distance and morphological structuring by geographic barriers (rivers), as well as exploring the occurrence of sexual dimorphism in morphometry and lepidosis. neither geographic distance nor rivers seem to play an important structuring role on the external morphology of liolaemus wiegmannii in uruguay. multiple multivariate analyses support the hypothesis that most of the external morphological variation is probably due to sexual dimorphism. natural and sexual selection acting on females and males, respectively, are the most plausible mechanisms underlying the dimorphism observed in this species. keywords. morphometry, lepidosis, sexual dimorphism, liolaemus, uruguay. introduction intraspecific morphological variation can arise as the result of selective pressures, phenotypic plasticity or historical factors acting at a population level. geographic variation in morphology is quite frequent among lizards and has been attributed to both genetic and non-genetic factors (ballinger, 1983; dunham et al., 1988; qualls and shine, 1998). intersexual variation is also common, and at least three hypotheses have been proposed in order to explain its causes in lizards: sexual selection (maynard smith, 1987; carothers, 1984; braña, 1996; cox et al., 2003); fecundity advantage (tinkle et al., 1970; braña, 1996; fairbairn, 1997; blanckenhorn, 2005) and trophic niche segregation (schoener, 1967; pianka and huey, 1978). sexual selection as the main factor behind sexual dimorphism predicts higher reproductive success in larger males or in those that are more attractive to females. consequently, those structures involved in male-male combat or related to female choice of male quality often become conspicuous (carothers, 1984; andersson, 1994; andersson and simmons, 2006). alternatively, the fecun4 j. villamil, a. camargo, r. maneyro dity advantage hypothesis states that natural selection will favor larger females, and therefore, predicts a proportional increase of fecundity with body size (tinkle et al., 1970; kozlowski, 1989; braña, 1996; fairbairn, 1997; zamudio, 1998; cox et al., 2003; blanckenhorn, 2005; du et al., 2005). otherwise, sexual dimorphism could also be a consequence of competition for trophic resources, where the resulting morphological differences between sexes allow a more efficient exploitation of the trophic niche (schoener, 1967; pianka and huey, 1978; herrel et al., 1999). with about 240 recognized species, liolaemus is one of the most widely distributed and species rich lizard genera worldwide (lobo et al., 2010; etheridge and frost, 2010; breitman et al., 2011, 2013; avila et al., 2013). liolaemus is distributed exclusively in south america, occurring in bolivia, paraguay, peru, chile, argentina, brazil and uruguay, spanning different environments from the andes mountains to the atacama desert and from the pacific ocean shores to the atlantic ocean coasts (lobo, 2001; avila, 2003; pincheira-donosso et al., 2008). the systematics of the genus is quite complex, with several sections, series and groups being recognized (schulte et al., 2000; espinoza et al., 2004; cruz et al., 2005; fontanella et al., 2012; olave et al., 2014). liolaemus wiegmannii (duméril and bibron, 1837) is a member of the l. wiegmannii group, a species group that has been suggested as monophyletic based on molecular and morphological data (etheridge, 2000; schulte et al., 2000; espinoza et al., 2004; avila et al., 2006; abdala, 2007; pincheiradonoso et al., 2008; but see olave et al., 2014). it is a diurnal terrestrial lizard, which occupies a variety of habitats throughout its extensive and fragmented range, although it is often found in sand dunes and sandy soils (etheridge, 2000). its distribution in argentina includes the provinces of río negro, buenos aires, la pampa, entre ríos, santa fé, córdoba, san luis, mendoza, san juan, catamarca, tucumán, jujuy and salta, whereas in uruguay, this species occurs in the sandy shores of the departments of río negro, soriano, colonia, san josé, montevideo, canelones, maldonado, and rocha, to the west of valizas creek (cei, 1986, 1993; carreira et al., 2005; carreira and maneyro, 2013; párraga, 2011, avila et al., 2013; stellatelli et al., 2014). nonetheless, numerous authors have pointed out that liolaemus wiegmannii is probably a species complex containing several disjunct populations that may represent separate species (avila, 2003; morando, 2004; avila et al., 2006; avila et al., 2009; aiassa and gorla, 2010; olave et al., 2014). beyond its systematic complexity, the morphological variation of liolaemus wiegmannii across its whole range has been poorly studied. cei (1979), and avila and martori (1996), reported some geographic variation in the number of mid body scales from argentina. etheridge (2000) published data on snout-vent length, dorsal scales, supralabials, infralabials and precloacal pores. in addition, avila et al. (2009) provided some morphological information about l. wiegmannii. however, the last two studies did not provide a geographically explicit analysis of the morphological variation. some authors have noted sexual dichromatism in liolaemus wiegmannii, with males exhibiting conspicuous orange and blue scales in the reproductive season that in general are absent in females, although, a tenuous orange coloration can be occasionally observed on females (cei, 1986, 1993; etheridge, 2000; carreira et al., 2005; avila et al., 2009). recently, cabrera et al. (2013) studied the sexual size dimorphism of several species of the liolaemus laurentii group (sensu abdala 2007), and showed that liolaemus wiegmannii has sexual differences in humerus length and axila-groin distance. however, sexual dimorphism in lepidosis and other morphometric traits have not been considered yet. in addition, because l. wiegmannii is a species complex, it is possible that the individuals analyzed by cabrera et al. (2013) from argentina could belong to a species different to the one occurring in uruguay. in this work, we studied the intraspecific morphological variation of liolaemus wiegmannii throughout its range in uruguay using meristic (lepidosis) and morphometric data. we hypothesized that the morphological variation between localities should show a geographic pattern due to isolation-by-distance. alternatively, considering the influence of rivers on the variation of terrestrial organisms (pound and jackson, 1981; gascon et al. 1998; pellegrino et al., 2005; geghring et al., 2012), three main rivers were considered as potential geographic barriers that could influence the structure morphological variation. finally, we expected to find significant differences in lepidosis and other morphological traits between sexes, in addition to those sexually-dimorphic traits found in previous studies. materials and methods morphological variation of liolaemus wiegmannii was studied through a set of eight classic morphometric and nine meristic variables (lepidosis) (table 1) from 134 specimens housed in the vertebrate collection of the faculty of sciences, university of the republic and the herpetological collection of the national museum of natural history (montevideo, uruguay) (appendix 1). the geographic coverage of the specimens includes 40 localities spanning almost the whole range of this species in uruguay (fig. 1). 5variation and dimorphism in liolaemus wiegmannii variables were chosen based on etheridge (2000), verrastro et al. (2003), and avila et al. (2009), and the lepidosis terminology followed smith (1946). morphometric measurements were taken with a digital caliper to the nearest 0.01 mm, whereas for meristic variables a stereoscopic microscope was used. sex was determined based on the shape of the cloaca, which is squaredshaped in males and rounded in females (cabrera et al., 2013). in order to remove the effect of size from morphometric variables, each variable was transformed as z =yi(svl/svli ) b (1) following lleonart et al. (2000), where z represent the transformed value of the variable y, which is the variable affected by size, represented in this case as the snout-vent length (svl). the exponent b is the slope of the linear regression between logy and logsvl. this transformation completely removes all the information related to size by scaling individuals to the same size and adjusting their shape to that they would have at the new size according to allometry (lleonart et al., 2000). to check that no size effect persisted after transformation, slopes of linear regression between each transformed variable and svl were evaluated trough a student’s t test implemented in r 3.3.0 (r core team 2016) (see appendix 2). under a successful size correction a slope of zero is expected. principal component analyses were computed separately for morphometric (excluding svl) and meristic variables through a variance-covariance matrix with the purpose of understanding the structure of the morphological variation and the contribution of each variable to the components that explain most of the variation observed. the assumption of multivariate normality was evaluated through mardia (mardia, 1970) and omnibus (dornik and hansen, 2008) tests implemented in past 3.07 (hammer et al., 2001). for testing the hypothesis of isolation by distance, a mantel test between a morphological and geographical distance matritable 1. morphometric and meristic variable abbreviations and their corresponding meanings. morphometricvariables detail meristic variables detail svl snout-vent length mbsc scales around mid-body hl head length dsc dorsal scales hw head width vsc ventral scales huml humerus length pp precloacal pores antbl forearm length lam3 subdigital lamellae of third finger fl femur length lam4 subdigital lamellae of fourth toe tibl tibia length inflab infralabial scales a-g axilla-groin distance suplab supralabial scales lorlab lorilabials scales fig. 1. (a) distribution of liolaemus wiegmannii after etheridge (2000) and avila et al. (2009). (b) localities of uruguay used for geographic analysis with permanova. a and b: west and east of rosario river (1) localities; c: east santa lucía river (2) localities; d: east maldonado stream (3) localities. 6 j. villamil, a. camargo, r. maneyro ces was conducted. the original dataset was divided into four matrices (one for each sex and class of variable, i.e. morphometric and meristic), which were independently analyzed using mahalanobis and correlation distances for morphometric and meristic data respectively and geographic distances obtained from coordinates. correlation distances were obtained with past software as 1-r, where r is the pearson’s coefficient. to test for statistical differences between sexes and geography, morphometric and meristic variables were analyzed separately through a permanova (anderson, 2001), which was computed based on mahalanobis and correlation distances respectively, and one million permutations. this analysis uses a multivariate statistic analogous to fisher’s f-ratio constructed from sums of squared distances within and between groups and provides a p value that is calculated through permutations (anderson, 2001). for testing geographic variation independently from sex, geographic arrangements were tested separately for males and females. three main rivers were considered as potential barriers (rosario river, santa lucía river, and maldonado stream), and therefore localities were grouped into four groups limited by these courses (fig. 1b). moreover, differences between localities west and east of the santa lucía river, and between all localities were also tested. sexual dimorphism was also explored through a discriminant function analysis (dfa), considering morphometric and meristic measurements separately. besides the discriminant function, mann-whitney and student’s t-test were performed to evaluate which variables show significant differences between sexes. all the statistical analyses, except student’s t-test on slopes, were implemented in past 3.07 software (hammer et al., 2001). results descriptive statistics based on the descriptive statistics for each untransformed variable, there are several sexually-dimorphic variables (table 2). considering both sexes, individuals of l. wiegmannii from uruguay exhibit a maximum snoutvent length of 60.82 mm. although males reached larger maximum sizes than females, both mean and median of females were higher than males (svl male mean: 38.84 mm; svl female mean: 41.89 mm; svl male median: 36.10 mm; svl female median: 44.61 mm) (table 2). regarding lepidosis, the ranges observed were particularly broad except for the number of lorilabial scales, which was 2 for all specimens analyzed except one (not shown in table 2). the number of scales around the mid-body (mbsc) was similar for both sexes (males: 41–58; females: 42–58), whereas the range of the number of dorsal scales (dsc) was broader in females (males: 41–59; females: 43–65). the number of ventral scales (vsc) shows overlapping ranges between sexes, although females exhibit a higher maximum value (males: 40–59; females: 45–64). the number of precloacal pores ranged between 0 and 7 for males and between 0 and 6 for females. although these intervals are quite similar, the median of males was 5 whereas that for females was 0. in addition, the frequency of males without precloacal pores (data not shown) was markedly lower than for females (males: 0.097; females: 0.325). the number of subdigital lamellae on both the third finger and the fourth toe had very similar intervals among sexes. the range (4-7) and the median (5) of supralabials were equal between sexes, whereas the infralabials differ in range (males: 5-8; females: 5-7), but have the same median (6). ordination analysis considering the transformed morphometric variables and excluding svl, the pca shows that 81% of the variance is comprised by the first three components with the first principal component (pca1) accounting for 47% of the variation. on the other hand, 84% of the meristic variance is explained by the first three components with a 50% of variation accounted for in the first principal component (table 3). for morphometric variables, the correlation coefficients show that pca1 has a very strong positive correlation with the axilla-groin distance (fig. 2a), whereas pca2 is mostly positively correlated with the tibia and femur lengths, and in a lesser way, with the head length (fig. 2b). regarding to meristic characters, pca1 has a high positive correlation with ventral and dorsal scales. to a lesser extent, pca1 shows a negative correlation with the number of precloacal pores, where the magnitude of this correlation is about half of the absolute value of the maximum correlation observed in pca1 (fig. 2c). moreover, pca2 is mainly linked with the number of scales around midbody, with which it shows a very strong positive correlation (fig. 2d). the bidimensional projection of the first two principal components shows a substantial overlap of sexes for both morphometric (fig. 3a) and meristic data (fig. 3b). however, from a morphometric point of view, it is possible to note that females tend to be located towards the positive values of pca1 and negative values of pca2, whereas males show the opposite tendency (fig. 3a). taking into account the variables that have the strongest correlation with these two components (fig. 2a and b), this tendency might reflect that females have longer axilla-groin distances than males whereas males tend to have longer femur, tibia and head lengths. for meristic variables, females tend to be located toward the positive values of the pca1 while males are principally located in the region of negative values (fig 3b). considering the 7variation and dimorphism in liolaemus wiegmannii correlation of each variable in pca1 (fig. 2c), this pattern might reflect that females have higher numbers of dorsal and ventral scales than males, whereas males have more precloacal pores than females. dispersion among pca2 axes is particularly high, so meristic data for this component do not show a conspicuous pattern among sexes (fig. 3b). dfa based on transformed morphometric variables correctly classified 72% of the analyzed individuals (75% of males and 71% of females, table 6). the discriminant function obtained (df = – 0.198zhl – 0.029zhw – 0.027zhuml – 0.028zantbl – 0.12zfl – 0.24ztl + 0.97za-g) shows that the axilla-groin distance is the most influential variable on the equation; therefore, it is the morphometric variable that best discriminates between sexes. other less important variables for morphometric sex discrimination are tibia and head length, which show an opposite pattern: the sex with longer axilla-groin distance has a shorter head and tibia, and vice versa. for meristic variables, the dfa classified correctly 85% of the specimens with similar percentages between sexes (table 4). in the discriminant function for these variables (df = –0.046mbsc + 0.67dsc + 2.87vsc – 1.40pp + 0.010lam3 + 0.39lam4 – 0.060suplab – 0.029inflab) the number of ventral scales (vsc) and the numbers of precloacal pores are the most important variables for discriminating between sexes. again, the sex with higher values for one of these variables has lower values for the other. multivariate tests multivariate normality of the entire dataset was rejected with a 95% confidence (table 5). according to the non-parametric multivariate analysis of variance (permanova) there are significant differences between sexes for both morphometric (f = 6.31; p < 0.001) and meristic (f = 54.85; p < 0.001) data, whereas this analysis rejected geographic differences for all arranges of localities considering main rivers as barriers and also between localities individually treated (table 6). mantel tests did not find significant correlations and therefore rejected the hypothesis of isolation by distance for both males (morphometric data: r = –0.09, p = 0.895; meristic data: r = –0.08, p = 0.910) and females (morphometric data: r = 0.06, p = 0.159; meristic data: r = –0.01, p = 0.557). table 2. descriptive statistics for morphometric and meristic variables considered for l. wiegmannii from uruguay. min: minimum value; max: maximum value; me: median; x – : mean; sẍ: standard error of the mean; s2n-1 : variance; sn-1: standard desviation. svl: snout-vent length; hl: head length; hw: head width; huml: humerus length; antbl: forearm length; fl: femur length; tibl: tibial length; a-g: axillagroin distance; mbsc: scales around mid-body; dsc: dorsal scales; vsc: ventral scales: pp: precloacal pores; lam3: subdigital lamellaeof third finger; lam4: subdigital lamellaeof fourth toe; suplab: supralabial scales; inflab: infralabial scales. svl hl hw huml antbl fl tibl a-g mbsc dsc vsc pp lam3 lam4 suplab inflab min. ♂ 23.21 6.07 5.38 3.37 2.25 3.75 3.2 9.3 41 41 40 0 13 16 4 5 ♀ 22.4 5.79 5.22 3.17 2.28 3.56 2.79 8.68 42 43 45 0 12 17 4 5 max. ♂ 60.82 11.34 11.07 7.9 6.68 10.06 9.17 28.74 58 59 59 7 19 24 7 8 ♀ 57.65 12.8 11.12 8.12 6.82 8.97 8.23 29.26 58 65 64 6 20 24 7 7 me ♂ 36.10 8.07 7.67 5.14 4.12 6.22 5.67 15.68 48 50 49 5 15 20 5 6 ♀ 44,82 8.45 8.38 5.66 4.66 7.25 6.50 20.02 48 50 53 0 16 20 5 6 x – ♂ 38.84 8.24 7.90 5.30 4.35 6.43 5.91 16.59 48.24 49.28 48.69 4.06 15.36 19.89 5.43 6.19 ♀ 42.05 8.41 8.35 5.62 4.61 6.75 6.07 19.47 48.16 50.40 53.51 1.71 15.38 20.54 5.33 6.15 sẍ ♂ 1.18 0.17 0.19 0.15 0.13 0.19 0.19 0.53 0.37 0.45 0.48 0.27 0.11 0.18 0.08 0.08 ♀ 1.03 0.15 0.16 0.13 0.11 0.16 0.16 0.55 0.36 0.41 0.38 0.23 0.13 0.19 0.06 0.06 s2n-1 ♂ 100.64 2.17 2.55 1.64 1.25 2.64 2.57 20.50 9.90 14.34 16.4 5.18 0.91 2.21 0.47 0.47 ♀ 87.69 1.84 2.18 1.45 0.98 2.10 2.02 24.91 10.88 13.97 11.83 4.16 1.30 2.99 0.32 0.32 sn-1 ♂ 10.03 1.47 1.60 1.28 1.12 1.62 1.60 4.53 3.15 3.79 4.05 2.28 0.95 1.49 0.69 0.17 ♀ 9.36 1.36 1.47 1.20 0.99 1.45 1.42 4.99 3.30 3.74 3.44 2.04 1.14 1.73 0.57 0.57 table 3. principal components explaining most of the morphological variation of liolaemus wiegmannii from uruguay. variables principal component eigenvalue % of variance explained morphometrics 1 1.63446 46.52 2 0.759644 21.621 3 0.45371 12.913 meristic 1 27.6334 50.226 2 10.7795 19.593 3 8.02788 14.591 8 j. villamil, a. camargo, r. maneyro univariate tests univariate tests (mann-whitney or t-test) show that those variables that are most influential for discriminating between sexes according to the dfa, also have highly significant sexual differences (p < 0.010 and p < 0.001) (i.e., axilla-groin distance; tibia length; head length; number of ventral scales, and number of precloacal pores) (table 7). in addition, there are also significant differences (p < 0.05) in snout-vent length (svl) and number of subdigital lamellae of the fourth toe (lam4). however, it is possible that an asymmetric distribution of juveniles and adults among sexes could be influencing this result for svl. unfortunately, given that data about minimum size at sexual maturity are only available for females (ramirez pinilla 1991; martori and aun 1997), the limit between juveniles and adults cannot be established for both sexes. fig. 2. correlation coefficients of each variable in the first two principal components. a: morphometrics variables in the first principal component; b: morphometric variables in the second principal component; c: meristic variables in the first principal component; d: meristic variables in the second principal component. svl: snout-vent length; hl: head length; hw: head width; huml: humerus length; antbl: forearm length; fl: femur length; tibl: tibial length; a-g: axilla-groin distance; mbsc: scales around mid-body; dsc: dorsal scales; vsc: ventral scales: pp: precloacal pores; lam3: subdigital lamellae of third finger; lam4: subdigital lamellae of fourth toe; suplab: supralabial scales; inflab: infralabial scales. 9variation and dimorphism in liolaemus wiegmannii discussion intraspecific and geographic variation considering both males and females, specimens of liolaemus wiegmannii from uruguay reach a maximum snout-vent length of 61 mm with females tending to be larger than males. this observed size is in the range of those reported in the literature for individuals from argentina (etheridge 2000; avila et al. 2009). cei (1979) and avila and martori (1996) reported geographic variation in size and number of scales around mid-body in individuals from argentina. the highest values were observed in bahía blanca (56-60), whereas the lowest were found in tucumán (46-48), while mendoza (48-50) had intermediate ranges. the range of this variable in individuals from uruguay (41-58) is markedly broader than that observed in each one of these populations, being comparable with the whole interval reported for this species (etheridge 2000; avila et al. 2009). unlike the number of scales around mid-body, information about other meristic variables in literature is scarce fig. 3. bidimensional projection of the morphological data of liolaemus wiegmannii in the first two principal components. a: morphometric variables; b: meristic variables. squares represent males whereas black full circles are females. table 4. confusion matrix of the discriminant function analysis for morphometric and meristic variables of liolaemus wiegmannii from uruguay. values in the third and fourth column represent the number of individual assigned to each sex by the discriminant function. male female total correctly assigned (%) morphometric variables male 54 18 72 75.0 female 24 58 82 70.7 total 78 76 154 meristic variables male 61 11 72 84.7 female 12 70 82 85.4 total 73 81 154 table 5. multivariate normality test results for the morphology of liolaemus wiegmannii from uruguay. all analyses were implemented in past 3.07. test parameter coefficient statistic d.f. p(normal) mardia skewness 39.06 1003 680 8.62x10-15 kurtosis 269.7 4.044 5.27x10-5 dornik & hansen omnibus 124.5 1.78x10-13 table 6. permanova results for morphometric and meristic data of liolaemus wiegmannii from uruguay considering three levels of variation. “a+b/c+d” tests differences between west and east santa lucía river localities. “a/b/c/d” evaluates differences between all the groups of localities divided by rosario river, santa lucía river and maldonado stream and “localities” considers the hypothesis of differences between each locality. groups tested data f p a+b/c+d morphometric ♂ 0.680 0.814 ♀ 0.626 0.775 meristic ♂ -0.223 0.887 ♀ 3.248 0.098 a/b/c/d morphometric ♂ 0.970 0.505 ♀ 0.825 0.705 meristic ♂ -0.064 0.861 ♀ 1.277 0.341 localities morphometric ♂ 1.359 0.088 ♀ 0.996 0.487 meristic ♂ 0.722 0.589 ♀ 1.954 0.175 10 j. villamil, a. camargo, r. maneyro and without specific geographic information, hampering the comparison with our data. nevertheless, it can be observed that lepidosis in uruguay shows broad ranges for almost all variables that overlap with the known variation across the whole distribution of the species (etheridge, 2000). most of the morphological variation observed in uruguay seems to be related to differences between sexes because most variables highly correlated with pca1 and pca2 are the same that statistically discriminate sexes. however, other variables are also highly correlated with the first two principal components (fig. 2), but do not show significant differences between sexes (table 7), suggesting that there is some morphological variation that is not explained by sexual dimorphism and accounts for the overlap seen in pca space (fig. 3). nevertheless, this additional variation could not be attributed to geographic variability, given that morphological differences among groups of localities were rejected and there was no correlation between morphological and geographic distances. the absence of geographically-structured morphological variation in uruguay contrasts with the differences in mid body scales found by cei (1979) and avila and martori (1996) among several isolated populations in argentina, which could actually represent separate species (avila, 2003; morando, 2004; avila et al., 2006; avila et al., 2009; olave et al., 2014). sexual dimorphism sexual dimorphism in l. wiegmannii from uruguay is strongly supported by several independent multiand univariate analyses, which show that males have a longer head and tibia, and present a higher number of precloacal pores than females. on the other hand, females have a longer axilla-groin distance and exhibit a higher number of ventral scales. based on seven morphometric variables, cabrera et al. (2013) found that males and females differ only in axilla-groin distance and humerus length. unlike the axilla-groin distance, the humerus length is not sexually dimorphic in specimens from uruguay. indeed, the humerus length is one of the variables with the lowest correlation coefficient with pca1 and pca2, and the least influential on the morphometric discriminant function. these discrepancies might suggest differences on the distribution of morphological variation among populations from argentina and uruguay. among liolaemus, head dimensions and axilla-groin distance are sexually dimorphic in most of the species analyzed, suggesting that it is a relatively common pattern among these lizards (villavicencio et al., 2003; verrastro, 2004; laspiur and acosta, 2007; cabrera et al., 2013; astudillo et al., 2015). taking into account the main hypotheses regarding the causes of sexual dimorphism in lizards, sexual selection might adequately explain the differences in head and table 7. morphological differences of l. wiegmannii between sexes, tested for each variable through a mann-whitney or t test, depending on the normality of each variable according to shapiro-wilk test. z indicates transformed variables following the method proposed by lleonart et al. (2000). variable shapiro-wilk test mann-whitney test t test svl* w=0.95; p=0.0050 u=2381; p=0.039 z-hl** w=0.98; p=0.015 u=2198; p=0.0064 z-hw w=0.99; p=0.55 t=0.49; p=0.62 z-huml w=0.99; p=0.18 t=0.36; p=0.72 z-antbl w=0.99; p=0.78 t=0.48; p=0.64 z-fl w=0.98; p=0.017 u=2598; p=0.20 z-tibl** w=0.99; p=0.39 t=2.99; p=0.0031 z-a-g*** w=0.99; p=0.65 t=-6.72; p=3.45x10-10 mbsc w=0.97; p=0.0024 u=2888; p=0.82 dsc w=0.97; p=0.0091 u=2513; p=0.11 vsc*** w=0.99; p=0.40 t=-7.98; p=3.30x10-13 pp*** w=0.81; p=8.24x10-13 u=1280; p=2.95x10-10 lam3 w=0.86; p=1.12x10-10 u=2817; p=0.60 lam4* w=0.95; p=5.52x10-5 u=2339; p=0.023 suplab w=0.78 ; p=6.23x10-14 u=2727; p=0.36 inflab w=0.78; p=7.96x10-14 u=2866; p=0.72 p<0.05*; p<0.01**; p<0.001***. 11variation and dimorphism in liolaemus wiegmannii tibia length and the number of precloacal pores found in this work. longer heads in males of l. wiegmannii could suggest the existence of agonistic encounters between them, where individuals with longer heads would have an advantage in combats getting greater access to females (carpenter and fergusson, 1977; carothers, 1984; anderson and vitt, 1990; herrel et al., 1999; verrastro, 2004; huyghe et al., 2005; vanhooydonck et al., 2010). in this sense, although scarcely documented, some male-male agonistic encounters have been observed among liolaemus species (halloy, 1996; martins et al., 2004; verrastro, 2004; labra et al., 2007; cabrera et al., 2013; halloy et al., 2013). additionally, males of some species of liolaemus show hierarchical structure according to the size of their home range, suggesting the occurrence of male-male interactions, and therefore a potential evidence of sexual selection (halloy and robles, 2002; frutos and belver, 2007; robles and halloy, 2009; cabrera et al., 2013). moreover, martins et al. (2004) has pointed out that the head and limbs are structures that play an important role in communication in liolaemus lizards, and consequently, it is possible that sexual differences in these traits could be related to their use in this context. head-bobbing seems to be a common behavior for signaling among liolaemus males, both in aggressive interactions and courtship (martins et al., 2004; labra et al., 2007; halloy, 2012; halloy et al., 2013; vicente and halloy, 2015). in addition, several species displaying these behaviors, also show sexual dimorphism in head size, where males are always the sex with larger heads (villavicencio et al., 2003; laspiur and acosta, 2007; cabrera et al., 2013; astudillo et al., 2015). this might suggest, at least for some species, a possible relationship between head-bobs and sexual dimorphism in head size that deserve further study. although little is known about the behavior of l. wiegmannii, achaval and olmos (2007) have mentioned that head movements are observed during courtship, suggesting the possibility that head size differences could be related to its use in courtship or for warning other males during territory defense. alternatively, sexual dimorphism in head length observed in l. wiegmannii could also be the result of trophic niche segregation. however, vanhooydonck et al. (2010) pointed out that bite force and consequently head dimension differences in male liolaemus lizards is better explained by sexual selection rather than natural selection (i.e., niche segregation). therefore, considering the phylogenetic context, head length sexual dimorphism found in l. wiegmannii is more likely to be the result of sexual selection than trophic niche segregation. even so, given that information about the intersexual diet variation in l. wiegmannii remains scarce (see aun et al., 1999), the role of niche segregation on sexual dimorphism still demands further research. several lizard species show a strong positive correlation between hindlimb length and sprint speed (snell et al., 1988; losos, 1990; miles, 1994; bauwens et al., 1995; bonine and garland, 1999). additionally, some studies have suggested that variation in sprinting ability can affect survival probabilities within populations of reptiles (christian and tracy, 1981; jayne and bennett, 1990; miles, 2004, vervust et al., 2007). in this sense, bauwens et al. (1995) pointed out that the evolution of longer hindlimbs relative to body size is one of the main factors driving the evolution of high maximum sprinting ability in lizards. in this context, considering the notorious sexual differences in coloration observed in l. wiegmannii, where males are clearly less cryptic than females and thus, probably more detectable, it is reasonable to think that predation might play a more important role as a selection pressure in males, favoring faster runners, and therefore longer tibia. alternatively, it should be taken into account that sprint speed could be also important for a more effective territory defense, where faster males are expected to defend a larger territory and/or more females through exclusion of slower rival males that still can usurp mates (husak et al., 2006, 2008; peterson and husak, 2006). consequently, if territorial defense occurs in liolaemus wiegmannii, as in other species of the genus, and if the longer tibia (and thus, hindlimb) of males provide them an advantage for faster movements around territory boundaries, sexual selection could also have favored a longer tibia in males. on the other hand, given that among liolaemus only the forelimbs are known to be implicated in communication (martins et al., 2004; halloy and castillo, 2006; halloy, 2012), it seems unlikely that differences in hindlimb dimensions are related to a communication use in this species. precloacal pores allow for the external exudation of chemical secretions by integumentary glands (valdecantos et al., 2014), which in liolaemus species have been suggested as important for stimulating copulation (rocha 1996). other authors think that these secretions play a role in territorial defense and recognition contexts similar to those in which head-bob displays are observed (labra and niemeyer, 1999; martins et al., 2004). in addition, the use of chemical secretions by some male lizards allows them to reduce the cost of territory defense (labra and niemeyer, 1999 and references therein). in this scenario, the presence of more and bigger precloacal pores in males of liolaemus wiegmannii could suggest that these might play a more important role in males than females, for instance, for territorial defense. if this is the case, it is possible that sexual selection could be underly12 j. villamil, a. camargo, r. maneyro ing the observed dimorphism in the number of precloacal pores. moreover, the presence of precloacal pores in both male and female individuals suggests that the role of these glands on communication is not only for male territorial defense. it is known that chemical recognition of females during reproductive season occurs in liolaemus tenuis, a species with precloacal pores present in both sexes (labra and niemeyer, 1999). therefore, although the dimorphism in the number of precloacal pores found in this study may have evolved for male-male defense, we cannot discard their role in male-female communication, or even both roles as the drivers of sexual selection. in many lizard species, females are under fecundity selection for larger abdomen size, suggesting that this is probably the most frequent mechanism underlying the widespread pattern of sexual dimorphism in axilla-groin distance (scharf and meiri, 2013). an increase in axillagroin distance, and therefore in abdomen size, provides to females the possibility of enlarging the space for storing eggs and, consequently, increasing their fecundity, which easily becomes a target of natural selection (tinkle et al., 1970; kozlowski, 1989; braña, 1996; fairbairn, 1997; zamudio, 1998; cox et al., 2003; blanckenhorn, 2005; du et al., 2005). based on the fecundity advantage hypothesis, it is reasonable to think that the larger abdomen found in females of l. wiegmannii is a result from fecundity selection. in addition, it is possible that the enlargement of the female’s abdomen could have led to an increase in the number of ventral scales observed in this sex. however, there is a reduction in the size of the ventral scales of females toward the cloacae, which might also contribute to the sexual differences found. finally, it is important to see that although sexual dimorphism seem to be a widespread pattern among lizards, where head (larger in males) and abdomen (larger in females) are the most frequent traits that differ between sexes, studies that test explicitly the hypotheses behind this phenomenon remain scarce (scharf and meiri, 2013). in this sense, the potential mechanisms proposed here for explaining the occurrence of sexual dimorphism in liolaemus wiegmannii, should be taken as tentative. further research is needed to explore and test explicitly the role of fecundity, behavior, and niche segregation on the sexual dimorphism observed on this species. acknowledgements we are grateful to the research and innovation agency of uruguay (anii) for financial support through the grant fce 1-2014-1-104109. we also acknowledge financial support from national system of investigators (sni-anii) and pedeciba. we thank diego arrieta and melitta meneghel for allowing us to access to the collections of the national museum of natural history (mnhn) and the faculty of sciences (university of the republic), respectively. references abdala, c.s. 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(1998): the evolution of female-biased sexual size dimorphism: a population-level comparative study in horned lizards (phrynosoma). evolution 1998: 1821-1833. 16 j. villamil, a. camargo, r. maneyro appendix 1 individuals of liolaemus wiegmannii used in the analysis and their respective localities from uruguay. zvc: vertebrate collection of the faculty of sciences, university of the republic; mnhn: collection of the museum of natural history, uruguay. *excluded from geographical analysis. locality individuals arazatí, san josé zvc 303, zvc 324, zvc 1229-1233, zvc 1223, zvc 6876, zvc 6877, mnhn 5838, mnhn 5839 artilleros, colonia mnhn 3346, mnhn 3348 balneario argentino, canelones zvc 2498 bello horizonte, canelones mnhn 5662 boca del cufré, san josé zvc 3761 boca del mauricio, san josé zvc 6869, zvc 6870, zvc 6872-6875 boca del san salvador, soriano mnhn 154 brisas del plata, colonia zvc 2105 cabo polonio, rocha zvc 1499, zvc 1863, zvc 2504, zvc 3387, zvc 1839, zvc 1932, zvc 1497, zvc 1497, zvc 1864, zvc 646, zvc 1224, zvc 6621, zvc 6625, zvc 6626, zvc 6855-6857, mnhn 3425, mnhn 3427, mnhn 5671 carrasco stream, canelones zvc 1939 carrasco, montevideo zvc 3549, zvc 5153, mnhn 149, mnhn 165, mnhn 3304 coast of rio negro river, in front of villa soriano, soriano zvc 774* cuchilla alta, canelones mnhn 3361 el pinar, canelones zvc 5050, zvc 6859 la floresta, canelones mnhn 168 la paloma, rocha zvc 914, mnhn 3127 lagomar, canelones zvc 6023, zvc 6227, zvc 3712, zvc 2824, zvc 2825, zvc 3711, zvc 3764 laguna de garzón, rocha mnhn 157, mnhn 3338, mnhn 3339 laguna de rocha zvc 5366 laguna del diario mnhn 3344 las cañas, río negro mnhn 3308* las vegas, canelones zvc 6860, zvc 6861 lomas de carmelo, colonia zvc 68786881 los titanes, canelones zvc 2435 malvin, montevideo mnhn 1064, mnhn 1065, mnhn 3349, zvc 595, zvc 876 zvc 585 manantiales, maldonado zvc 2442 médanos de solymar, canelones zvc 3570 nueva palmira, colonia zvc 1967, zvc 1968, zvc 1969 pajas blancas, montevideo zvc 4357, zvc 4361, zvc 4362, zvc 6845-6849 pando, canelones mnhn 196, mnhn 199 pinamar, canelones zvc 1342 playa pascual, san josé mnhn 2445, mnhn 3310, mnhn 3311, mnhn 3312, mnhn 3313, mnhn 3314, mnhn 3337, zvc 5152, zvc 6865-6868, zvc 6871 portezuelo, maldonado mnhn 176 punta espinillo, montevideo zvc 6850-6854 punta negra, maldonado mnhn 266, mnhn 3306, mnhn 3340, mnhn 3342, mnhn 3343, zvc 6308 rio de la plata, 3 km east of martin chico, colonia zvc 2164 san gregorio stream, san josé zvc 970 san luis, canelones zvc 3307 tigre stream, san josé zvc 1506-1508 west side of cerro de montevideo, montevideo zvc 1385 17variation and dimorphism in liolaemus wiegmannii appendix 2 results of the student’s t-test on the slopes of linear regressions between each transformed variable and snout-vent length (svl). z indicates transformed variables; hl: head length, hw: head width, huml: humerus length, antbl: forearm length, fl: femur length, tibl: tibia length, a-g: axilla-groin distance. zhl vs svl zhw vs svl zhuml vs svl zantbl vs svl zfl vs svl ztibl vs svl za-g vs svl slope 0.001163 6.53x10-5 -3.798x10-5 -0.0008418 -0.001766 -0.001699 -0.0005518 t value 0.236 0.017 -0.008 -0.222 -0.322 -0.326 -0.053 p(slope=0) 0.813 0.986 0.994 0.825 0.748 0.745 0.958 acta herpetologica vol. 12, n. 1 june 2017 firenze university press morphological variation and sexual dimorphism in liolaemus wiegmannii (duméril & bibron, 1837) (squamata: liolaemidae) from uruguay joaquín villamil1,*, arley camargo2, raúl maneyro1 sex does not affect tail autotomy in lacertid lizards panayiotis pafilis1,*, kostas sagonas2, grigoris kapsalas1, johannes foufopoulos3, efstratios d. valakos4 fire salamander (salamandra salamandra) males’ activity during breeding season: effects of microhabitat features and body size raoul manenti*, andrea conti, roberta pennati call variation and vocalizations of the stealthy litter frog ischnocnema abdita (anura: brachycephalidae) pedro carvalho rocha1,2,*, joão victor a. lacerda2,3, rafael félix de magalhães2,3, clarissa canedo4, bruno v. s. pimenta5, rodrigo carrara heitor6, paulo christiano de anchieta garcia2,3 feeding ecology of two sympatric geckos in an urban area of northeastern brazil josé guilherme g. sousa1, adonias a. martins teixeira2,*, diêgo alves teles2, joão antônio araújo-filho2 and robson waldemar ávila3 a pattern-based tool for long-term, large-sample capture-mark-recapture studies of fire salamanders salamandra species (amphibia: urodela: salamandridae) jeroen speybroeck1,*, koen steenhoudt2,$ skeletal variation within the darwinii group of liolaemus (iguania: liolaemidae): new characters, identification of polymorphisms and new synapomorphies for subclades linda díaz-fernández*, andrés s. quinteros, fernando lobo marking techniques in the marbled newt (triturus marmoratus): pit-tag and tracking device implant protocols hugo le chevalier1, olivier calvez2, albert martínez-silvestre3, damien picard4, sandra guérin4,5, francis isselin-nondedeu6, alexandre ribéron1, audrey trochet1,2,* evidence for directional testes asymmetry in hyla gongshanensis jindongensis qing gui wu1, wen bo liao2,* the advertisement call of pristimantis subsigillatus (anura, craugastoridae) florina stănescu1,4, rafael márquez2, paul székely3,4,*, dan cogălniceanu1,4,5 nematodes infecting anotosaura vanzolinia (squamata: gymnophthalmidae) from caatinga, northeastern brazil bruno halluan s. oliveira¹, adonias a. martins teixeira¹,*, romilda narciza m. queiroz¹, joão a. araujo-filho¹, diêgo a. teles¹, samuel v. brito2, daniel o. mesquita¹ where is my place? quick chorus structure assembly in the european tree frog michal berec a possible mutualistic interaction between vertebrates: frogs use water buffaloes as a foraging place piotr zduniak1,*, kiraz erciyas-yavuz2, piotr tryjanowski3 good vibrations: a novel method for sexing turtles donald t. mcknight1,2,*, hunter j. howell3, ethan c. hollender1, and day b. ligon1 errata to mecke, s., hartmann, l., mader, f., kieckbusch, m., kaiser, h. (2016): redescription of cyrtodactylus fumosus (müller, 1895) (reptilia: squamata: gekkonidae), with a revised identification key to the bent-toed geckos of sulawesi. acta herpetologica 11(2): acta herpetologica 12(2): 167-173, 2017 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-17658 the life-history traits in a breeding population of darevskia valentini from turkey muammer kurnaz, alı i̇hsan eroğlu, ufuk bülbül*, halıme koç, bılal kutrup karadeniz technical university, faculty of science, department of biology, 61080, trabzon, turkey * corresponding author. e-mail: ufukb@ktu.edu.tr submitted on: 2015, 17th december; revised on: 2017, 2nd february; accepted on 2017, 16th october editor: giovanni scillitani abstract. we investigated the age structure, body size, longevity and growth in a breeding population of darevskia valentini inhabiting highland altitude in balahor, turkey. according to the skeletochronological analysis (n= 25; 14 ♂♂, 11 ♀♀), the estimated ages ranged from 3 to 9 years (from 4 to 7 in males and from 3 to 9 in females). the maximum life span was 7 years in males and 9 years in females. the age at maturity was found to be 3 years in both sexes. the mean age and svl were not statistically different between sexes. for both sexes, we found a significant positive correlation between body size and the number of lags. the growth coefficient (k) was lower in females (0.30) than in males (0.76) while asymptotic svl was higher in females (70.06) than in males (60.55). growth rates were found to be significantly different between both sexes (females grew faster than males). however, a low level of female-biased sexual size dimorphism (ssd) was observed in the population. keywords. skeletochronology, longevity, von bertalanffy model, growth rate, ssd. introduction the valentin’s lizard, darevskia valentini (boettger, 1892) inhabits highland areas above 1800 m (baran and atatür, 1998; eiselt et al., 1992). it has three subspecies (d. v. valentini, d. v. lantzicyreni and d. v. spitzenbergerae) distributed in a large area including most of anatolia. many studies about systematic, distribution and ecology were performed on this species (darevsky, 1972; franzen, 1990; eiselt et al., 1992; mulder, 1995; sindaco et al., 2000; tarkhnishvili et al., 2013), but to date, there is no study related to age determination in turkey. age determination yields important data for ecological studies of the reptilian species. one of the most commonly used techniques to determine the individual age in reptiles is skeletochronology that many studies have proven to be a very successful and reliable method (castilla and castanet, 1986; girons et al., 1989; piantoni et al., 2006; nayak et al., 2008; kim et al., 2010; guarino, 2010). skeletochronology estimates the age using lines of arrested growth layers in the bone tissue (castanet and baez, 1991; castanet, 1994). due to its reliability and the fact that it is less time-consuming than other age-determination methods such as marking-recapture (halliday and verrell, 1988), the number of the studies using skeletochronology method has increased recently (luís et al., 2003; altunışık et al., 2013; tok et al., 2013; gül et al., 2014; üzüm et al., 2014; yakın and tok, 2015; kanat and tok, 2015; gül et al., 2015; üzüm et al., 2015; comas et al., 2016). since there are no detailed studies based on age and growth parameters of darevskia valentini, here we present data on life-history traits (age structure, body size and some growth parameters) of the species. in order to assess population dynamics of d. valentini living at altitudes between 1300-3000 m a.s.l. (tok et al., 2009), 168 muammer kurnaz et alii we chose a mountain population (2400 m a.s.l) living at mean altitude regarded as typical for the species. materials and methods field study a total of 25 individuals of valentin’s lizard, darevskia valentini (14 ♂♂, 11 ♀♀) were caught during breeding season from balahor plateau (near gümüşhane), turkey. the balahor population is located in a highland area (40°29’51’’n, 39°56’24’’e) at an altitude of 2400 m a.s.l. the habitat consists of rocky areas and open ground. the active period for lizards varies from early may to the middle of september. during the sampling period (1620 august 2015), the average air temperature in daytime was recorded as 27 °c. lizards were caught by hand. the sex of each individual was determined by direct examination of the sexual organs and secondary sex characters (e.g., dark blue spots on the margins of ventral plates and dorsal coloration of males). snout-vent length (svl) was measured to the nearest 0.01 mm using a digital caliper. we quantified sexual size dimorphism (ssd) with the lovich and gibbons (1992) index according to the following formula: sdi = (mean length of the larger sex / mean length of the smaller sex) ± 1. in this formula, we used -1 because females were larger than males and defined as positive. for each lizard, the second phalange from the longest finger of the hind limb was clipped and preserved in 10% formalin solution for subsequent histologic analyses. after registration and toe-clipping, the lizards were released back into their natural habitats. the animals were treated in accordance with the guidelines of the local ethics committee. skeletochronological analyses the procedure of skeletochronology is based on the calculation of the lines of arrested growth (lags) in transverse sections of the middle part of phalangeal diaphyses using a portion of the second phalanx from the third toe (gül et al., 2014). after removing the skin, toes were treated for 2.5 hours with 5% nitric acid for decalcification of bone tissue. all samples were then loaded in a tissue processor (leica tissue processor tp1020, germany) and all tissue samples were embedded in paraffin by a tissue embedding device (thermo shandon b64100010, usa). crosssections (15 µm) were obtained with a rotary microtome and thereafter were stained using the haematoxylin procedure. the stained sections were mounted using entellan and observed under a light microscope. age determination was estimated using skeletochronology analysis (castanet and smirina, 1990; smirina, 1994). the numbers of lags in each section were independently calculated by three observers (m. kurnaz, a.i̇. eroğlu and u. bülbül) and results were compared. possible double lines were counted as a single lag. a double line is two adjacent age rings formed during a winter season. as previously stated in the study of özdemir et al. (2012), we assessed endosteal resorption of the first lag by comparing the diameters of eroded marrow cavities with the diameters of non-eroded marrow cavities in sections from the youngest specimens. endosteal resorption did not interfere with age estimation procedures because the resorption zone never reached the first lag. the distance between two adjoining lags is a good indicator of individual growth in a given year (kleinenberg and smirina, 1969; özdemir et al., 2012). where we observed an obvious decrease in spacing between two subsequent lags, we took it to mark the age when sexual maturity was achieved (ryser, 1998; yılmaz et al., 2005; özdemir et al., 2012). uncountable cross section samples were not incorporated into our study. statistical analyses normality of the svl and age distribution for the males and females was tested with the one-sample kolmogorov-smirnov test (p ≥ 0.05). being that the variables normally distributed, we used parametric independent sample t-test to estimate significant differences (p < 0.05). pearson’s correlation was used to estimate the relationship between svl and age (p < 0.05). all statistical tests were processed with ibm spss 21.0 for windows. growth pattern estimating the growth patterns were estimated by using the von bertalanffy equation model between body size and age according to previous studies (james, 1991; wapstra et al., 2001; roitberg and smirina, 2006; guarino et al., 2010). the general form of the von bertalanffy equation used is lt = l∞ (1 e-k (t-t0)) where lt is length at age t, l∞ is a parameter depicting asymptotic maximum length, e is the base of the natural logarithm, k is a growth coefficient, and t0 is the age at hatching, which is the starting point of the growth interval under the present study. as 169age and growth of darevskia valentini applied in the study of guarino et al. (2010), we assumed the mean value provided by tayhan et al. (2011) as size at hatching (lt0 = 25.3 mm) because of the lack of incontrovertible data on the size at hatching of the populations studied, due to lack of young lizards collected during the field studies. the parameters l∞ (asymptotic svl) and k, and their asymptotic confidence intervals (ci) were estimated using the non-linear regression procedure by means of the ibm spss 21.0 software program. then, the growth rates were calculated as r = k (l∞ lt). growth curves were considered to be significantly different if the 95% confidence intervals did not overlap (james, 1991; wapstra et al., 2001). results age ranged from 4-7 years in males and 3-9 years in females (table 1). the mean age of the specimens was not different between the sexes (independent sample t-test: t = -0.802; df = 23; p = 0.431). intersexual differences in body size (length) was female-biased (sdi = 0.052). the mean svl (t = -1.143; df = 23; p = 0.431) did not differ between sexes. there was a significant positive correlation between svl and age for both males (pearson’s correlation r = 0.797; p < 0.01) and females (r = 0.836; p < 0.01). the growth pattern estimated by von bertalanffy’s showed a best fit to the relation between age and svl (fig. 1). for males, the estimated asymptotic svl was lower than the maximum svl record (svlasym ± ci: 63.89 ± 5.80 mm) while for females, it was higher than the maximum svl record (svlasym ± ci, females: 73.71 ± 5.30 mm). the growth coefficient was higher in males than in females (k ± ci, males: 0.27 ± 0.11; females: 0.19 ± 0.10). for all populations (males + females), the asymptotic svl, growth coefficient (k) and mean growth rate were calculated as 64.60 ± 4.33 mm, 0.24 ± 0.08 and 4.11 ± 0.79 mm per year, respectively. the growth curve of males was significantly different from that of females. in this population, females grow faster than males (the average growth rate was 0.97 ± 0.43 in males and 4.12 ± 0.98 in females) (fig. 2). the mean growth rates of males and females were significantly different within the population (independent sample t-test: t = -2.323; df = 9; p < 0.05). descriptive statistics of the growth rates for the balahor population are given in table 2. a growth zone and thin hematoxylinophilic outer line most likely corresponding to a winter line of arrested growth were present in sections of the phalanges in 100% of both male (n = 14) and female (n = 11) adult specimens (fig. 3). the resorption zone never reached the first lag and did not interfere with age determination. we observed double lines in 18 (72%) specimens. the oldest females and males were 9 and 7 years old, respectively (fig. 4). the age at maturation was 3 years for both sexes in the population. table 1. descriptive statistics of age and svl of the balahor population. for abbreviations, see text (n: number of samples; range: maximum and minimum values and se: standard error). characters sex n mean range se age ♂♂ 14 5.36 4-7 0.29 svl 14 59.24 53.03-67.40 1.33 age ♀♀ 11 5.82 3-9 0.54 svl 11 62.31 49.05-71.65 1.95 age ♂♂ ♀♀ 25 5.56 3-9 0.28 svl 25 60.59 49.05-71.65 1.15 fig. 1. the von bertalanffy growth curves for males (open circle, solid line), females (solid circle, grey line) and all specimens (dot line) of d. valentini. open square shows svl mean of the lizards at hatching (25.3 mm) as reported by in den bosch and bouth (1998). growth parameters are given in the text. fig. 2. relationships among the growth rates of age groups belonged to individuals of d. valentini from balahor population. 170 muammer kurnaz et alii discussion the present study provides data on the age structure and growth patterns of the valentin’s lizard from a turkish population. life history traits (e.g., mean age, age at sexual maturity and maximum longevity) could depend on genetic characteristics and be species-specific. in the present study, the mean age of the darevskia valentini was found to be 5.36 years in males and 5.82 years in females of the balahor population located in a high elevation site (2400 m). however, gül et al. (2014) reported a mean age about 1 year lower (4.3 in males and 4.8 in females) in a high altitude population (inhabiting 2137 m a.s.l.) of another rock lizard species (d. rudis). conformably, arakelyan et al. (2013) reported a lower mean age for the four parthenogenetic rock lizards (4.46 years for d. armeniaca, 4.44 years for d. unisexualis, 4.44 years for d. sapphirina and 4.06 years for d. uzzelli) living in highland habitats (between 1400 and 2000 m a.s.l.). apart from species variation, age structure may also differ among different populations of the same species. the age structure of lizards may fluctuate according to environmental parameters (e.g., climate, altitude, latitude and predation) and other conditions such as proportion and energetic costs of reproduction, activity season and hibernation period (roitberg and smirina, 2006). a different climatic condition might affect population demography by generating differences in age structure and longevity (özdemir et al., 2012). for instance, an increase in mean age with higher altitude has been reported for various lizard species in colder environments (roitberg and smirina, 2006). apart climatic conditions, other factors such as different rates of predation might affect the demography of the lizards studied. like mean age, longevity is dependent on the active period, which is in turn related to altitude, latitude and other climatic and environmental factors. since the climatic conditions in balahor were not favourable for lizard activity due to the presence of snow for at least 6.5 months, the active period of the studied population was found to be lower than other species inhabiting lowland habitats. in general, individuals from high-elevation sites have higher longevity than those from low-elevation sites (wapstra et al. 2001; roitberg and smirina, 2006; guarino et al., 2010). congruently, we found a high longevity table 2. descriptive statistic of growth rate and growth coefficient (k) of the balahor population. for abbreviations, see text (n: number of samples; range: maximum and minimum values and se: standard error). characters sex n mean range se growth rate ♂♂ 4 4.06 2.57-5.85 0.71 k 14 0.27 0.11 growth rate ♀♀ 6 4.81 2.47-8.04 0.76 k 11 0.20 0.10 growth rate ♂♂ ♀♀ 7 4.11 1.79-7.55 0.79 k 25 0.24 0.08 fig. 3. a cross section (15 µm thick) through phalange of a fiveyear-old female (63.87 mm svl) d. valentini from balahor population. for abbreviations, see text (mc: marrov cavity; eb: endosteal bone; rl: resorption line; dl: double line; erl: endosteal resting line; p: periosteal bone). fig. 4. age distributions for both males and females of d. valentini from balahor population. 171age and growth of darevskia valentini (7 years in males and 9 years in females) in the balahor population. similar to our results, the maximum longevity was found to be 8 years in d. armeniaca, d. unisexualis (arakelyan et al., 2013) and d. rudis (gül et. al., 2014) while it was found to be 6 years in d. sapphrina and d. uzzelli (arakelyan et al., 2013). in the present study, the age of both sexes was significantly correlated with their body size (svl). the adult body size depends on many factors including age at maturity and longevity (özdemir et al., 2012). in some species, the male lizards mature earlier than females (beebee and griffiths, 2000; olsson and madsen, 2001). however, age at maturity and svl were not found to be significantly different between both sexes in our study. a low level of female-biased sexual size dimorphism (ssd) was observed in the balahor population. longevity and age at first reproduction have been identified as the main determinants of ssd at an intra-specific level (liao and lu, 2010; lyapkov et al., 2010; liao et al., 2013; liao et al. 2015). congruently, age at maturity was found to be similar in both sexes. moreover, ssd in many adult lizards arises due to sexual differences in the growth rates, and the larger sex grows faster than the smaller (johnadler and cox, 2007; kolarov et al., 2010; üzüm et al., 2014). although there was not a high-level of femalebiased ssd, we found significant differences between the growth rates of both sexes. our data show that females grew faster than males and that the growth trajectories were different between sexes. the lower value of k in the von bertalanffy equation in females suggests that they attain the asymptotic body length slower than males. when considering the overall population, we found higher growth rates in our individuals. this is in accordance with the general prediction that lizards grow faster at high elevation sites. double lines are found in higher percentages in some unsuitable ecological conditions (e.g., hot climate and dry period) (jakob et al., 2002; guarino and erişmiş, 2008; özdemir et al., 2012). on the other hand, food availability may negatively affect the number of double lines (bülbül et al., 2016). since the balahor population has a cold climate and less food sources, the high percentage of double lines (72 %) observed in this steppe region suggests that the unfavorable food supply has a greater effect than climatic conditions. in conclusion, our data on the body size, age structure, longevity, and growth of d. valentini may contribute to the knowledge of the life-history traits of this species. our results show different growth rates between sexes in the balahor population, having different longevity, despite having a similar age at sexual maturity. however, long-term studies including different populations under various environmental conditions are neeeded to reveal a more comprehensive picture. acknowledgements we are grateful to zekeriya azak for his help on field studies. the study was carried out by permissions of ministry of forest and water affairs (72784983-488.04147328) and karadeniz technical university animal care and ethics committee (ktü.53488718-341/2015/23). english revision was accomplished by duncan gullick lien who is an instructor at karadeniz technical university, school of foreign languages in turkey. references altunışık, a., gül, ç., özdemir, n., tosunoğlu, m., ergül, t. 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(2005): age determination and some growth parameters of a rana ridibunda population in turkey. acta zool. hung. 51: 67-74. acta herpetologica vol. 12, n. 2 december 2017 firenze university press meristic and morphometric characters of leptopelis natalensis tadpoles (amphibia: anura: arthroleptidae) from entumeni forest reveal variation and inconsistencies with previous descriptions susan schweiger1, james harvey2, theresa s. otremba1, janina weber1, hendrik müller1,* brown anole (anolis sagrei) adhesive forces remain unaffected by partial claw clipping austin m. garner*, stephanie m. lopez, peter h. niewiarowski species and sex comparisons of karyotype and genome size in two kurixalus tree frogs (anura, rhacophoridae) shun-ping chang1,2, gwo-chin ma2,3,4, ming chen2,5,6,7,8,*, sheng-hai wu1,* non-native turtles in a peri-urban park in northern milan (lombardy, italy): species diversity and population structure claudio foglini1, roberta salvi2,* species composition and richness of anurans in cerrado urban forests from central brazil cláudia márcia marily ferreira1,*, augusto cesar de aquino ribas2, franco leandro de souza3 the life-history traits in a breeding population of darevskia valentini from turkey muammer kurnaz, alı i̇hsan eroğlu, ufuk bülbül*, halıme koç, bılal kutrup influence of desiccation threat on the metamorphic traits of the asian common toad, duttaphrynus melanostictus (anura) santosh mogali*, srinivas saidapur, bhagyashri shanbhag predation of common wall lizards: experiences from a study using scentless plasticine lizards jenő j. purger*, zsófia lanszki, dávid szép, renáta bocz reproductive timing and fecundity in the neotropical lizard enyalius perditus (squamata: leiosauridae) serena najara migliore1,2,*, henrique bartolomeu braz2,3, andré felipe barreto-lima4, selma maria almeida-santos1,2 observations on the intraspecific variation in tadpole morphology in natural ponds eudald pujol-buxó1,2,*, albert montori1, roser campeny3 and gustavo a. llorente1,2 reliable proxies for glandular secretion production in lacertid lizards simon baeckens diet of juveniles of the venomous frog aparasphenodon brunoi (amphibia: hylidae) in southeastern brazil rogério l. teixeira1, ricardo lourenço-de-moraes2, débora c. medeiros3, charles duca3, rogério c. britto4, luiz c. p. bissoli5, rodrigo b. ferreira3,* who are you? the genetic identity of some insular populations of hierophis viridiflavus s.l. from the tyrrhenian sea ignazio avella, riccardo castiglia, gabriele senczuk* acta herpetologica 14(2): 129-133, 2019 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/a_h-7751 visible implant alphanumeric (via) as a marking method in the lesser snouted treefrog scinax nasicus andrea caballero-gini1,2,3,*, diego bueno villafañe2,3, lía romero2, marcela ferreira2,3, lucas cañete4, rafaela laino2, karim musalem2,5 1 insituto de biología subtropical, universidad nacional de misiones, félix de azara 1552, cp 3300, posadas, misiones, argentina. *corresponding author. email: ancgini@gmail.com 2 centro de investigación del chaco americano, fundación manuel gondra, san josé 365, asunción, paraguay 3 instituto de investigación biológica del paraguay, del escudo, cp, asunción, paraguay 4 universidad nacional de asunción, facultad de ciencias exactas y naturales (facen), campus universitario, san lorenzo, paraguay 5 world wildlife fund, bernardino caballero 191, asunción, paraguay submitted on: 2019, february 22nd; revised on: 2019, june 3th; accepted on: 2019, june 3th editor: daniele pellitteri-rosa abstract. in this study we assessed the efficacy of visible implant alphanumeric (via) for marking adults and juveniles of the neotropical treefrog scinax nasicus. we evaluated the success of this technique in the identification of individuals and the prevalence of tags in the field. as a control, we marked the same individuals through toe-clipping. of 196 marked individuals, 57 were recaptured in a 7-month study period. only one mark was unreadable because it was located too deep in the skin. we found one case of tag expulsion and two inverted tags. almost complete regeneration of the adhesive disk was observed by the fifth month of the study in all recaptured frogs. we suggest via tagging method as suitable for s. nasicus over long term studies. even though, a hybrid method for marking (via + toe-clipping) is recommended for species with dark and/or loose skin, or large frogs. keywords. amphibian, mark-recapture, fluorescent elastomer, toe-clipping. the recognition of individuals in a population is a key aspect in many amphibian ecology and conservation studies (seber, 1982; campbell et al., 2009; clemas et al., 2009). their identification over time and space helps researchers to estimate individual and demographic parameters such as growth and mortality rates, dispersal, population size and habitat use (osbourne et al., 2011). in cases where individuals of a species do not possess characteristics that distinguish them from each other, like coloration and color patterns, and photo-identification is not suitable, it is necessary to use marking techniques (donelly et al., 1994). for amphibians, the most commonly used are toe-clipping, passive integrated transponder (pit), visible implant elastomer (vie) tags, and more recently visible implant alphanumeric (via) tags, the last two manufactured by north-west marine technology, shaw island, usa (heard et al., 2008; branelly et al., 2014). toe-clipping is the most widely used technique for marking anurans and salamanders (ferner, 2010). however, this method is highly criticized because a decrease in the probability of recapture has been detected in some amphibian species (clarke, 1972; mccarthy and parris, 2004; waddle et al., 2008). another disadvantage of toe-clipping in long term studies is the possibility of tissue regeneration, although this has been observed only in a few species (ferner, 2007; ursprung et al., 2011). pit tags consist of an electromagnetic capsule with an alphanumeric code, which is read by a scanner. insertion is done subcutaneously or in a body cavity (ferner, 130 andrea caballero-gini et alii 2010). however, loss of tags and deleterious effects related to survival have been detected using this technique (scherer et al., 2005; guimaraes et al., 2014). vie tagging is done through the subcutaneous injection of an elastomer mixed with a curing agent. this technique allows the individualization of many animals by creating different fluorescent color codes as well as placing the marks in various body locations (moosman and moosman, 2006). when comparing these three techniques, branelly et al. (2014) mention that the least efficient is the vie tagging, due to the migration, darkening and in some cases expulsion of the tag. via tags are compacted elastomers in rectangular shape with an alphanumeric code in fluorescent colors visible under uv light (heard et al., 2008). the insertion is done with an injector provided by the manufacturer, but in some cases the results are better making a previous incision (buchan et al., 2005; gower et al., 2006; heard et al., 2008; clemas et al., 2009; kaiser et al., 2009). despite being considered a reliable technique due to the capacity of individual identification, time of marking, handling of the individuals, durability of the marks and relatively low cost (haw et al., 1990; buchan et al., 2005; gower et al., 2006), this technique also has disadvantages when used in amphibians: difficulty of tag insertion in species with loose skin, variation on tag retention among species and darkening of the tag when marking heavily pigmented species (kaiser et al., 2009). additionally, tag retention was low in studies of mark-recapture of tadpoles (courtois et al., 2013). we tested the via tags in the lesser snouted treefrog scinax nasicus (cope, 1862) in order to 1) evaluate the effectiveness of this technique in the identification of individuals, 2) determine the prevalence of tags in markrecapture studies, and 3) provide some recommendations for further studies upon this species and others with similar morphology. s. nasicus is a small sized hylid frog distributed in northern and central argentina, paraguay, uruguay, eastern bolivia, and central and southern brazil (frost, 2019). the species is commonly found in open areas of the atlantic forest, cerrado, chaco, pampa, and pantanal domains (dalmolin et al., 2017). we captured individuals of s. nasicus using 173 pvc pipes as a refuge in an area of approximately 3 km2 of wetland and associated riparian forest in benjamín aceval, presidente hayes department in paraguay (-24.960522s, -57.359425w). field work was done during the months of november 2017, january, march and may 2018. the individuals were tagged both with via tags and toe-clipping. additionally, measurements of the snoutvent length (svl) were taken with a digital caliper mitutoyo absolute aos digimatic. the via tags used were of standard size (1.2 × 2.7 mm) in fluorescent green with black letters. the insertion site was the ventral region of the right thigh following buchan et al. (2005) (fig. 1). the implantation site was sterilized with ethanol 90%, then the tag was inserted under the skin using the injector provided by nmt following the manufacturer protocol and no veterinary glue was used. all marking equipment was sterilized between frogs by immersion in 90% ethanol for several minutes. toe-clipping was carried out as control following the method of martof (1953). as suggested by kinkead et al. (2006), no anesthesia was used in either both procedures. frogs showed no signs of discomfort, e.g., emitting distress calls or abnormal movements of the affected limb and/or foot. frogs were put under observation for 24 hours in plastic bags filled with air to observe presence of redness, edema or bleeding on the treated foot or at the injection site. subsequently they were released on the same pvc pipe where they were captured. we marked 196 individuals of s. nasicus (55 females, 46 males, and 95 juveniles). frogs averaged 25.9 mm (sd = 5.34 mm) of svl. we obtained a total of 57 (29%) recaptures, of which 2 individuals were recaptured three times, 14 individuals twice, and 41 individuals only once. the recapture rates in juveniles was 47%, males 16% and females 37% (fig. 2). we did not find significant differences in the recapture rates of juveniles and adults (t = 0.3487, d.f. = 4, p = 0.7449); therefore, it can be suggested that the via implants did not have negative effects on the survival of juveniles. every time the label was observed, the code could be identified without ambiguity with the help of the uv light lantern provided by the kit, in 98% of the times. in one case, the label was located too deep under the skin and could not be read properly. at the same time, by january 2018, we observed tissue regeneration in recaptured fig. 1. location of the via tag in the inner thigh of scinax nasicus. via tag was injected just below the skin. 131evaluating via tags in scinax nasicus frogs where the beginning of the growth of the adhesive discs was noticeable, and by march 2018 the discs had similar diameters to those that were not clipped (fig. 3). in general, discs presented similar shape and coloration to those not clipped and no cases of aberrant growth were observed, such as those described by hoffman et al. (2008). kaiser et al. (2009) studied the possibility of using via tags as a marking method that does not require the recapture of animals (by placing the tags on the back of a small frog species). they concluded that it is not possible to read the codes on the labels without having the animal in hand. when comparing the handling times between via tags and toe-clipping, clemas et al. (2009) found that toe-clipping took slightly less amount of initial time of handling and marking than time with the via tags, but the last one took less time during identification. despite results showed by clemas et al. (2009), by using via tags in adults and juveniles, we had success identifying individuals of s. nasicus through this methodology. regarding the second objective of this work, we observed the retention of tags on 97% of the cases. only on one occasion the label was expelled and, in another opportunity, as a result of an inflammation caused in a frog’s leg, the tag had to be removed when the animal was recaptured. also, in two cases the tags were inverted but could be easily rotated by prodding with a finger, and on one occasion a small piece of the tag broke when removed from the tag block but was not discarded. when marking individuals at the caudal end of the dorsum, kaiser et al. (2009) noted that very often the labels were turned over and migrated ventrally, but they do not mention if this affects frog’s survivor or movement capacity. they also mention that when tags were not easily detected they remarked animals, and this constituted an extra effort and a waste of resources. contrarily, heart et al. (2008) compared tag migration placing them in the thigh and dorsolateral region of the thorax, finding that tag retention was higher in the latter site. although, movement of tags in dorsal regions of the body is less common, this area tends to be more pigmented, making reading difficult (moosman and moosman, 2006). we opted for the ventral thigh location because this area is not heavily pigmented in s. nasicus, being almost translucid, thus we did not observe any noticeable migration, which may arise when the target species is large and allows movements of the tag in the interfemoral sac (clemas et al., 2009). we also consider that the use of veterinary glue is not necessary since only in one case we observed the expulsion of the tag. when we purchased the product, manufacturers mentioned that the injection needle was re-designed and that it should not be necessary to make an initial incision. however, for marking large or thick-skinned species as well as when marking a large number of individuals this may be necessary, in order to maintain the instrument sharp and in shape to not cause discomfort to the animals. the estimation of population and demographic parameters in ecological studies are based on assumptions that depend on the marking technique; these are: (1) no loss of marks; (2) no misidentification of marks; and (3) marking procedures do not alter survival or capture probabilities (seber, 1986; pollock et al., 1990). this last assumption is the most controversial because in most techniques’ negative effects on both survival and recapture probability have been observed, being toe-clipping the most deleterious technique and vie and via tagging the least, although this last one has been scarcely studied (heard et al., 2008; schmidt and schwarzkopf, 2010; sapsford et al., 2014, 2015). via tags are an interesting method to test in amphibians due to its relatively low cost, lower invasiveness when compared to other techniques such as toeclipping, and straightforward code interpretation. in our study, the rate of success of the via tag method suggests that it is suitable for s. nasicus, as it was easy, safe, rapid and effective to carry out, as well as easy to detect. moreover, the method is well advised for the species fig. 3. toe pad regeneration in scinax nasicus. a: after a week of clipping, b: after two months of clipping. fig. 2. number of capture and recapture males, females and juveniles of scinax nasicus. 132 andrea caballero-gini et alii if long term studies are made, due to the pad regeneration observed in clipped toes. still, we suggest keeping a hybrid marking method (i.e., via tags + toe-clipping) when working with other species than s. nasicus, since via tags can have a distinct rate of success depending on several factors, such as the degree of stretch in the skin, size of the leg or other body part, transparency. we also advise to take time to inspect carefully the animals to detect via tag migration or loss, so there is no resource waste and unnecessary animal stress. acknowledgements the permits to perform this study were obtained from the ministerio del ambiente y desarrollo sostenible (scientific collection permit n° 173-2017). this work is part of the project pinv15-143 financed by the consejo nacional de ciencia y tecnología (conacyt). we thank for the help received by the staff of estancia playada and the american chaco research center, also to paloma moreno and humberto sánchez for their help in fieldwork. we thank the programa nacional de incentivo a los investigadores (pronii) from conacyt. references brannelly, l.a., berger, l., skerratt, l.f. 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(2008): modeling the effect of toe clipping on treefrog survival: beyond the return rate. j. herpetol. 42: 467473. acta herpetologica vol. 14, n. 2 december 2019 firenze university press podarcis siculus latastei (bedriaga, 1879) of the western pontine islands (italy) raised to the species rank, and a brief taxonomic overview of podarcis lizards gabriele senczuk1,2,*, riccardo castiglia2, wolfgang böhme3, claudia corti1 substrate type has a limited impact on the sprint performance of a mediterranean lizard pantelis savvides1,*, eleni georgiou1, panayiotis pafilis2,3, spyros sfenthourakis1 coping with aliens: how a native gecko manages to persist on mediterranean islands despite the black rat? michel-jean delaugerre1,*, roberto sacchi2, marta biaggini3, pietro lo cascio4, ridha ouni5, claudia corti 3 pit-tags as a technique for marking fossorial reptiles: insights from a long-term field study of the amphisbaenian trogonophis wiegmanni pablo recio, gonzalo rodríguez-ruiz, jesús ortega, josé martín* occurrence of batrachochytrium dendrobatidis in the tensift region, with comments on its spreading in morocco ait el cadi redouane1, laghzaoui el-mustapha1, angelica crottini2, slimani tahar1, bosch jaime3,4, el mouden el hassan1,* hematological parameters of the bolson tortoise gopherus flavomarginatus in mexico cristina garcía-de la peña1,*, roger iván rodríguez-vivas2, jorge a. zegbe-domínguez3, luis manuel valenzuela-núñez1, césar a. meza herrera4, quetzaly siller-rodríguez1, verónica ávila-rodríguez1 ontogenetic and interspecific variation in skull morphology of two closely related species of toad, bufo bufo and b. spinosus (anura: bufonidae) giovanni sanna visible implant alphanumeric (via) as a marking method in the lesser snouted treefrog scinax nasicus andrea caballero-gini1,2,3,*, diego bueno villafañe2,3, lía romero2, marcela ferreira2,3, lucas cañete4, rafaela laino2, karim musalem2,5 morphological variation of the newly confirmed population of the javelin sand boa, eryx jaculus (linnaeus, 1758) (serpentes, erycidae) in sicily, italy francesco p. faraone1,*, salvatore russotto2, salvatore a. barra3, roberto chiara3, gabriele giacalone4, mario lo valvo3 variability in the dorsal pattern of the sardinian grass snake (natrix natrix cetti) with notes on its ecology enrico lunghi1,2,3,4,*, simone giachello5, manuela mulargia6, pier paolo dore7, roberto cogoni8, claudia corti1 estimating abundance of the stripeless tree-frog hyla meridionalis by means of replicated call counts federico crovetto, sebastiano salvidio, andrea costa* at-rich microsatellite loci development for fejervarya multistriata by illumina hiseq sequencing yan-mei wang, jing-yi chen, guo-hua ding*, zhi-hua lin acta herpetologica 13(2): 177-183, 2018 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-21972 body size, age and population structure of triturus carnifex (urodela: salamandridae) in the context of facultative paedomorphosis giacomo bruni1,*, giulia tessa2, claudio angelini3 1 vrije universiteit brussel, boulevard de la plaine 2, 1050 ixelles, bruxelles, belgium. *corresponding author. e-mail: giacomo.b90@ gmail.com 2 dipartimento di scienze della vita e biologia dei sistemi, università degli studi di torino, via accademia albertina 13, 10123 torino to, italy 3 via guglielmo marconi, 30, 04018 sezze, italy submitted on: 2017, 10th october; revised on: 2018, 10th august; accepted on: 2018, 4th may editor: raoul manenti abstract. facultative paedomorphosis occurs rarely in the genus triturus compared to other european newts such as ichthyosaura alpestris and lissotriton spp., with most of observation related to single or few individuals per site. in this paper we report about body size, age and population size of two populations of triturus carnifex with paedomorphs. since one of the populations consisted of approximately 25% of paedomorphs, this is the first study about a population of large-bodied newts with a conspicuous number of paedomorphic individuals. we found evidences for an ecological causation of paedomorphosis, as well as further support for two recent findings about paedomorphosis: the reduction of sexual size dimorphism and the female-biased sex-ratio within paedomorphs. keywords. italian crested newt, heterochrony, metamorphosis, sexual-size dimorphism, population structure. facultative paedomorphosis is a phenotypic polymorphism occurring in several caudate species (duellman and trueb, 1994; denoël et al., 2005). it consists of the retention of larval morphological features by sexually mature individuals in species which usually show a metamorphosed adult stage (denoël, et al., 2005; fig. 1). paedomorphic and metamorphic phenotypes may coexist in the same population. the genetic and physiological mechanisms involved in facultative paedomorphosis are still not entirely clear (voss et al., 2003), however it is known that this phenomenon can emerge via two different paths: progenesis and neoteny (denoël and joly, 2000). in the former, there is an early sexual maturation, while the latter consists of a retardation of the somatic development with a normal gonad maturation rate (healy, 1974). there are evolutionary advantages derived from facultative paedomorphosis which can enhance the fitness of the individual, e.g., paedomorphs do not have to bear the seasonal migration to the breeding sites and they can start to breed earlier in the season (semlitsch and gibbons, 1985), with subsequent increase in larvae survival chance (ryan and plague, 2004). in addition, in the specific case of progenesis, the precocious sexual maturation allows paedomorphs to reproduce years before the metamorphs of the same age (denoël and joly, 2000). the morphological characteristics which separate the two morphs, such as the breathing mechanisms and the feeding apparatus, also entail differences in the use of aquatic habitat and food resources (denoël et al., 2005). in fact, paedomorphs tend to stay in deep and open waters where they can prey on planktonic invertebrates, instead metamorphs prefer to stay near the surface or along the edge of the water basin, feeding on non-aquatic organisms (denoël et al., 2005). facultative paedomorphosis may lead to some limitations. for instance, paedomorphosis may preclude dis178 giacomo bruni et alii persion to other breeding sites, thus preventing gene flow and giving rise to inbreeding phenomena (whiteman, 1994). however, facultative paedomorphosis is a plastic process and paedomorphic individuals are able to metamorphose in the presence of challenging environmental conditions like desiccation (denoël, 2003). there is evidence that facultative paedomorphosis is promoted by ecological factors such as the depth of the water body, level of dissolved oxygen content (denoël and ficetola, 2014), the availability of food resources (ryan and semlitsch, 2003), the population density (harris, 1987), the hydroperiod (semlitsch et al., 1990), the temperature (sprules, 1974) and the slope of the water basin banks (denoël and ficetola 2014). on the other hand, it is adversely affected by the presence of fish (denoël et al. 2009a). denoël and ficetola (2014) have further pointed out that the presence of appropriate terrestrial habitats nearby does not increase the frequency of metamorphic newts in the population. in the salamandridae family, facultative paedomorphosis is typically revealed by the presence of feathery external gills in adult amphibians. in particular, this phenomenon appears more common in small-bodied newts like ichthyosaura alpestris (denoël et al., 2001) and various species of the genus lissotriton (denoël, 2006; bozkurt et al., 2015). in the large-bodied newts of the genus triturus, paedomorphosis is uncommon and the few reports for t. macedonicus, t. cristatus, t. pygmaeus and t. dobrogicus generally are based on a few individuals per population (kalezić et al., 1994; dolmen, 1980; ceacero et al., 2010; mester et al., 2013). in t. carnifex paedomorphosis is considered rare and very little information is available about its occurrence (piazzini et al., 2005; vanni and nistri, 2006). herein we reported new records of paedomorphosis in t. carnifex from two different sites in central italy and we aim to provide population size and life-history trait of metamorphs and paedomorphs in both sites. we studied two sites with paedomorphic individuals of triturus carnifex in tuscany, central italy: 1) an artificial, permanent pond built in 2008, 35 m a.s.l., volume of ~390 m3 and a depth of 0.9 m on average, inside the natura 2000 site sci-spa it51140011, in sesto fiorentino municipality (coded here as sf); 2) a natural permanent pool in a xerophytic wood, 655 m a.s.l., volume of about 50 m3 and a depth of 1 m on average, in anghiari municipality (coded here as an). they are also breeding sites for lissotriton vulgaris, neither are known to contain fish. the sites are located about 70 km from each other and fig. 2. ventral view of paedomorphic individuals of t. carnifex. (a) male from sf; (b) female from sf; (c) female from an with two eggs stuck to the right foot. fig. 1. paedomorphic individuals of t. carnifex. (a) male from sf; (b) female from an. scale bar refers only to picture b. 179body size, age and population structure of triturus carnifex both have water levels that are approximately constant all year round. newts were sampled by dip-netting from march 2012 to march 2013 at sf (16 capture incidents, one every 26.7 days on average,), and from july 2012 to may 2013 at an (seven capture incidents, one every 53 days on average); these capture sessions lasted about two hours and were used for the capture-mark-recapture (cmr) study. at sf two funnel traps were also placed in the pond for the whole night before the dip-netting session. newts were individually marked by photo of the ventral spot pattern, they were sexed and their life stage was recorded (paedomorphic or metamorphosed adult). we captured in total 203 metamorphs (111 males and 92 females) and 143 paedomorphs (48 males and 95 females) in sf and 141 metamorphs (66 males and 75 females) and 3 paedomorphs (all females) in an. during the last fieldwork sessions, we measured the snout-vent length (svl) of 88 newts at sf and of 46 newts at an (the individuals have been chosen randomly). paedomorphic individuals were distinguished from overwintering larvae on the basis of cloaca shape (denoël, 2016; fig. 2) and by the presence of secondary sexual characteristics like the whitish tail band in males (dorsal crest was not fully developed in any of the observed paedomorphic males and few of them had one or two small crest teeth). due to the very low number of paedomorphs captured in an, they were not be included in the analyses. during the last capture session in an and after it in sf, we surgically removed the third finger from the forelimb storing it in ethanol 70% to be used for the age assessment through scheletochronology (79 newts from sf and 46 from an; the individuals have been chosen randomly). newts were immediately released after the in-field procedure. the skeletochronological analysis was successfully carried out on 35 paedomorphs and 36 metamorphs from sf and 40 metamorphs from an, followed the standard protocol used for other large newts with some adaptations (miaud et al., 1993). muscles and skin were removed and the phalanx was washed in distilled water. phalanges were decalcified in 5% nitric acid for approximately 1.5 hours, cross-sectioned at 16 mm with a cryostat and stained with ehrlich’s haematoxylin for 15 minutes. sections were observed under a light microscope by two independent researchers and then imaged with a digital camera. we identified the lines of arrested growth (lags) which are caused by seasonal growth patterns (castanet et al., 1993). evaluation of bone resorption (castanet and smirina, 1990), and consequently of the resorption of inner lags, was carried out through osteometric analysis (guarino et al., 2004), comparing the medium diameter of sections’ external margin of younger specimens, with the medium diameter of medullar cavity of older specimens. as the first lag was partially resorbed but visible in all the samples, no additional lags were added to the count of the estimated age. age and svl data were used to compare populations and life-stages. due to the low number of paedomorphs we captured at an, we ran two different analyses. firstly, we compared metamorphs from both populations and secondly, we compared life-stages within sf. in both cases, we used a two-way anova to evaluate the effect of (1) population of origin and sex and (2) life-stage and sex on age and size. then, we ran two separate ancova analyses to evaluate the effect of the same factors listed above on individual size while accounting for age. due to significant interaction between the covariate age and the main factors, we used an independent slope design for both analyses. all the variables met the requirements of parametric statistics except age, thus we log-transformed it before of the analyses. we also compared the degree of sexual size dimorphism within the two metamorphic populations and the paedomorphs by calculating their respective size dimorphism index (sdi = [(mean svl of females/mean svl of males) 1]) (lovich and gibbons, 1992). the software statistica 7 (statsoft inc.) was used for these analyses. population size of metamorphs and paedomorphs at both sites were estimated using the capture-mark-recapture data and the superpopulation approach of the jolly-seber model (wagner et al., 2011). the same data have already been used for a survival analyses in the framework of the cormack-jolly-seber model (bruni and angelini, 2016). from this analyses it was clear that metamorphs and paedomorphs have similar survival over time in the sf populations, and that the capture probability was constant but different between the life-stages [phi(t)p(.*l)] (aicc weight = 0.998). thus, for the current analysis, we assumed the same parametrization as above for survival and capture probability and allowed to the probability of entering the population (pent) to vary by time and to be dependent or independent from life-stage. consequently, we obtained two candidate models to be fitted for obtaining the population sizes of metamorphs and paedomorphs split based on sex (four superpopulations). for an population, we obtained model uncertainty in the former analysis, thus in the current analysis we ran models with the same parametrization for survival (i.e., varying by time or constant and depending or not on sex) and capture probabilities (time varying or constant), and we allowed to pent to time vary, thus obtaining eight candidate models for obtaining the population sizes of males and females (two superpopulations). model fitting has been done via the software mark (white and burnham, 1999) and model selection 180 giacomo bruni et alii is based on the akaike information criterion corrected for small sample size (aicc). information on age and svl of the individuals belonging to sf and an populations are summarized in table 1, taking into account their life-stage and sex. table 2 reports the two-way an(c)ova tables for the effects “population”, “sex” and their interaction, based only on metamorphs from an and sf. metamorphs are larger at an, and adult females are larger than males; however, both differences depended on individual age (used as covariate), since the separate slope design did not detect any significant effects. a post-hoc test (unequal n hsd) after the separate slope design ancova showed that females from sf are larger than sf males, but significantly smaller than females from an, and they are similar to an males. table 3 reports the twoway an(c)ova tables for the effects “life stage”, “sex” and their interaction, based on paedomorphs and metamorphs from sf only. at sf, metamorphs are older and larger than paedomorphs. even after taking into account individual age, the separate slope design detected size differences between the life stages. the sdi index was always female biased: an metamorphs: 0.079, sf  metamorphs: 0.032, sf paedomorphs: 0.018. the best js model for sf [phi(t)p(.*l)pent(t*l), model weight = 1] estimated that during the study period there were: 407 metamorphosed males (95% c.i.: 287.9576.2), 336 metamorphosed females (95% c.i.: 236.1478.7), 82 paedomorphic males (95% c.i.: 65.2-103), 162 paedomorphic females (95% c.i.: 135.4-196.9). the best js model for an [phi(t)p(t)pent(t), model weight = 0.92] estimated that during the study period there were: 77 metamorphosed males (95% c.i.: 72.5-82.4) and 88 metamorphosed females (95% c.i.: 82.3-93.4). facultative paedomorphosis is a well-known phenomenon in newts and salamanders (e.g., denoël et al., 2005), however it occurs rarely in the genus triturus (dolmen, 1980; kaledzić et al., 1994; denoël et al., 2007; ceacero et al., 2010; mester et al., 2013) and especially in t. carnifex, though information is mostly based on anecdotal observations (lanza, 1983; piazzini et al., 2005; vanni and nistri, 2006). this study represents the first evidence of a population of this species with a substantial number of paedomorphic individuals. sf was built in spring 2008, and soon some larvae of the year from neighbouring temporary sites were transferred into it (these neighbour sites were going to be demolished for building). interestingly, the oldest paedomorphic newt is 6 years old, that is the same age of the pond. as lags are also visible in the larval period (lima et al., 2001) and every lag correspond to the low metabolism period (winter), some of the transferred larvae were hatched not later than autumn 2007 (in the area where sf is located oviposition may exceptionally begin in november [bruni, pers. obs.]), these newts at the time of sampling were in their 6th year of life. furthermore, the presence of metamorphosed newts far older than seven years testifies to immigration from neighbour sites. due to the hydroperiod of the ephemeral pools, none of neighbour sites hosted paedomorphic newts (the closest site with paedomorphic t. carnifex was about 2 km far from sf and it was demolished in 1997 [m. petrolo, pers. comm.]). thus, we have strong evidence of specific ecological conditions for sf that cause paedomorphosis. further support for the role played by ecology comes from the lower density at sf (2.5 individual/m3, including both metamorphs and paedomorphs) compared to an (3.3 metamorph/m3), low density is a favourable aquatic condition for paedomorphosis (harris, 1987; semlitsch, 1987). we also found that a very low number of paedotable 1. age and size (svl) of t. carnifex from populations sf and an, divided by life-stage and sex. population life-stage sex age (mean ± sd; n° and range) svl (mean ± sd; n° and range) sf metamorphs male 6.6 ± 0.7 n = 16, 4 – 15 6.2 ± 0.1 n = 18, 5.6 – 7.3 female 6.1 ± 0.4 n = 20, 3 – 11 6.4 ± 0.1 n = 26, 5.5 8 paedomorphs male 4.8 ± 0.1 n = 15, 4 – 6 5.6 ± 0.1 n = 15, 5.3 6 female 5.2 ± 0.1 n = 20, 4 – 6 5.7 ± 0.1 n= 29, 5 – 6.2 an metamorphs male 5.9 ± 0.5 n = 17, 3 – 11 6.3 ± 0.1 n = 21, 5.3 – 7 female 7 ± 0.5 n = 23, 3 – 14 6.8 ± 0.1 n = 25, 5.3 – 7.9 181body size, age and population structure of triturus carnifex morphs has been found in an, and, although both sites hosted populations of l. vulgaris, only sf hosted paedomorphic l. vulgaris (caffara et al., 2014). the importance of the environment is also evident when considering the relationship between age and size of different morphs. paedomorphic newts were younger and smaller than metamorphs of the same population; furthermore, the ancova analysis showed that the size difference was not dependent on age, but likely on different growth patterns. such patterns are consistent with the progenesis responsible for paedomorphosis (denoël and joly, 2000). further, the same authors list some environmental features of sf, namely: low elevation, absence of fish and shallow water, as favourable to progenesis. in sf, paedomorphic newts are less than half the number of metamorphs. where paedomorphs and metamorphs of the same species coexist, the proportion between morphs may differ significantly, and it depends on the interplay between environment and genotype (denoël et al., 2005). for example, denoël and ficetola (2014) found that the percentage of paedomorphs in populations of l. helveticus varied between 2% and 96%. additionally, in the context of facultative paedomorphosis, the sex-ratio may vary between populations; however, it is expected that the sex ratio is male-biased within paedomorphs (continued presence in ponds increases reproductive frequencies) and female-biased within metamorphs (egg production requires higher energy income than available in permanent ponds; whiteman, 1997; denoël et al., 2005). such pattern is confirmed in the genus triturus for t. macedonicus: kaledzić et al. (1994) found only paedomorphic males. interestingly, in sf, metamorphs males outnumbered the female counterpart, but female paedomorphs are about twice male paedomorphs. female-biased sex-ratios have been found in other paedomorphic populations (see denoël et al., 2005), and also recently obtained experimental support (mathiron et al., 2017), with paedomorphic males that metamorphose earlier in response to drying conditions. ssd is usually female-biased in t. carnifex (e.g., malmgren and tholleson, 1999; ficetola et al., 2010). interestingly, the populations we studied show low ssd (see tables). we found that metamorphs from an possessed the highest sdi index, despite an and sf metamorphs having similar body sizes overall. the lowest sdi index was found to be that of sf paedomorphs, this is consistent with the expectation that paedomorphosis reduces ssd in female-biased ssd species (denoël et al. 2009b). we wonder whether the low ssd of sf metamorphs depends on some ecological factors (e.g., sdi increases in cold climate (ficetola et al., 2010) while sf is at very low altitude), or whether such low ssd is itself due to facultative paedomorphosis. that is to say, perhaps when paedomorphs undergo metamorphosis they do not develop different sizes dependant on their sex. in order to address this question we suggest two main lines of further research; one, to investigate the growth patterns of paedomorphs following metamorphosis and two, to quantify the relative population size of paedomorphic individuals that undergo metamorphosis. table 2. an(c)ova table for the effects “population”, “sex” and their interaction (“population*sex”) on age and size (svl) of metamorphosed t. carnifex from populations sf and an. population sex population*sex anova age f1,72 = 0.01, p = 0.92 f1,72 = 0.42, p = 0.52 f1,72 = 1.74, p = 0.19 svl f1,86 = 4.25, p < 0.05 f1,86 = 11.14, p < 0.01 f1,86 = 1.25, p = 0.27 ancova (separate slope design) svl (covariate: age) f1,68 = 0.5, p = 0.48 f1,68 = 1.37, p = 0.24 f1,68 = 0.01, p = 0.98 table 3. an(c)ova table for the effects “life-stage”, “sex” and their interaction (“life-stage*sex”) on age and size (svl) of t. carnifex from populations sf. life stage sex life stage*sex anova age f1,67 = 9.73, p < 0.01 f1,67 = 0.09, p = 0.77 f1,67 = 1.36, p = 0.25 svl f1,84 = 52.03, p < 0.01 f1,84 = 3.43, p = 0.07 f1,84 = 0.87, p = 0.35 ancova (separate slope design) svl (covariate: age) f1,63 = 6, p < 0.05 f1,63 = 1.05, p = 0.31 f1,63 = 0.16, p = 0.69 182 giacomo bruni et alii acknowledgements the permits to perform this study were obtained from the italian ministry of environment, land and sea (0019536 10/09/2012 pnm-ii and 0029422 23/10/2012 pnm-ii). we want to thank elia serafini for let us know the site of anghiari, daniele nembi and simone guidotti for making it possible to create the sf site, massimiliano petrolo for his report of an extinct population with paedomorphic newts, francesco visintin for the help in make the figure collage and lewis campbell for the linguistic revision of the paper. references bozkurt, e., olgun, k., wielstra, b. 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(1997): maintenance of polymorphism promoted by sex‐specific fitness payoffs. evolution 51: 2039-2044. acta herpetologica vol. 13, n. 1 june 2018 firenze university press acta herpetologica 13(2): 147-154, 2018 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-21026 age structures and growth parameters of the levantine frog, pelophylax bedriagae, at different localities in denizli, turkey eyup başkale*, sevay ayşe ulubeli, yakup kaska pamukkale university, faculty of arts and sciences, department of biology, kınıklı campus 20100 pamukkale denizli, turkey. *corresponding author. e-mail: eyupbaskale@gmail.com submitted on: 2017, 26th july; revised on: 2018, 1st january; accepted on: 2018, 20th september editor: rocco tiberti abstract. skeletochronology is a reliable tool for assessing several parameters in amphibian populations. we used skeletochronology to determine the age structure, growth rate, age at first reproduction, and longevity of levantine frog pelophylax bedriagae populations from different localities in denizli, turkey. all examined individuals (n = 161) exhibited lines of arrested growth in the bone cross-sections. age structure and age at first reproduction were similar among localities and sexes, while longevity and growth rates showed significant variation among localities. obtained results were compared with literature data on age-related and grow parameters in pelophylax bedriagae and cognate species. keywords. skeletochronology, levantine frog, longevity, growth rate, sexual maturity. introduction turkish water frogs were long considered as a single species, and pelophylax bedriagae and pelophylax caralitanus were previously considered to be subspecies of pelophylax ridibundus (sinsch and schneider, 1999). however, both subspecies have been raised to species level, based on morphometric, molecular and bioacoustic evidence (schneider and sinsch, 1999; sinsch and schneider, 1999; jdeidi et al., 2001; plötner et al., 2001; akın et al., 2010a, 2010b). the levantine frog is a highly opportunistic amphibian, widely distributed in the eastern mediterranean region, including several greek islands close to the coast of turkey (papenfuss et al., 2009). in turkey, it is present along the aegean coast and the southern part of the anatolian highlands, from sea level to 2100 m (özeti and yılmaz, 1994). it is highly aquatic and inhabits a wide variety of running and standing water habitats, e.g., puddles, shallow ponds, large lakes, large rivers, mountain streams. the levantine frog has been listed in the least concern category of the iucn red list of threatened species because of its wide distribution and presumed large populations, but habitat loss and its commercial use as food could threaten natural populations (papenfuss et al., 2009). the levantine frog also spread outside its native range by means of several introductions (e.g., for aquaculture), threatening the genetic integrity of native water frogs (e.g., plötner et al., 2015). the aim of the present study is to describe the age structure and individual growth patterns of levantine frog populations from distinct areas of turkey. we used skeletochronology for estimating the age of amphibians and studying age-related parameters in four frog populations (e.g., mean longevity, growth rate, age at sexual maturity; castanet et al., 1993; smirina, 1994; guarino et al., 1999, 2003; esteban and sanchiz, 2000). skeletochronology can be applied on amphibian phalanges, which can be sampled without animal culling and without affecting amphibian populations and endangered species (smirina, 1994; khonsue et al., 2000; guarino and erişmiş, 2008; guarino et al., 2011; bionda et al., 2015). 148 eyup başkale et alii material and methods study sites the field study was conducted during the 2015-2017 breeding seasons at four distinct localities at different altitudes in denizli, turkey. süleymanlı lake, buldan, denizli province, turkey (38°03’n, 28°46’e; 1163 m a.s.l.) is a natural lake which covers 0.5 km2 and attains a maximum depth of 2 m. the lake is surrounded by coniferous tree (red pine pinus brutia), willow trees (salix alba) and reeds (phragmites australis). the water surface is covered by aquatic vegetation (i.e., myriophyllum spicatum). vali recep yazıcıoğlu dam, denizli, turkey (37°46’n, 29°07’ e; 328 m a.s.l) is a reservoir located in the city centre of denizli. it is fed by underground water and by the gökpınar creek. this reservoir covers approximately 2 km2 and is surrounded by sparsely distributed trees such as salix alba and juniperus sp. aquatic vegetation is absent, but some vegetation patches are present in the littoral zone. acıgöl lake, denizli, turkey (37°47’n, 29°51’e; 839 m a.s.l.) is included in a natural wetland surrounded by agricultural land. the lake covers approximately 47 km2 but it reaches approximately 100 km² if the surrounding wetlands are included. the littoral area and the water surface are covered by aquatic vegetation. ornaz valley, denizli province, turkey (37°46’n, 28°58’e; 834 m a.s.l.) is crossed by a slow-flowing stream, which starts at karcı mountain and continues to başkarcı. along the river, there are many ponds that form natural amphibian breeding areas rich in aquatic and riparian vegetation. field studies and morphometric measurements the levantine frogs were captured by 2-3 people with a dip net or by hand after sunset using flashlights. snout-vent length (svl) was measured with a dial calliper (accuracy: 0.02 mm) and body mass (w) with a precision balance (accuracy: 0.1 g). sex was determined based on secondary sexual characteristics of males, i.e. presence of metatarsal tubercules on the first finger of the front feet and vocal sac. the sexual dimorphism index (sdi) was calculated as sdi = [(mean length of the larger sex / mean length of the smaller sex) 1] according to the method of lovich and gibbons (1992). we collected bone samples only from breeding individuals, i.e. calling males, females responding to the males, and already paired individuals. we assumed that the age at first reproduction (age at maturity: am) is the lowest age recorded among the breeding individuals. the longest digit of the hind foot was cut off, fixed in 70% ethanol, and then processed according to the routine skeletochronological procedure (e.g., castanet et al., 1993; castanet and smirina 1990; smirina 1994). following tissue removal, the bones were washed in running water for 12-14 hours, decalcified for 3-5 hours in 5% nitric acid and then placed in distilled water overnight. the bones were dehydrated using graded ethanol series and then cleared in xylene, before embedding in paraffin. using a rotary microtome, we obtained 16-µm-thick cross-sections from the central region of the diaphysis. bone sections were stained with haematoxylin and eosin and analysed under a light microscope. the age of the frog was determined by two of the authors who independently counted the number of lines of arrested growth (lags) present in each of the bone sections. with regard to first lag resorption, we distinguished between “completely resorbed” (the first lag was not observable in bone cross-section), “partially resorbed” (only some parts of the first lag were visible in bone cross-section), and “not resorbed” (the first lag well visible in bone cross-section). statistical analyses length and weight data were normally distributed (kolmogorov–smirnov d test, all p > 0.05), thus allowing comparisons using parametric tests. we used a t-test for independent samples to compare the lengths and weights of male and female frogs. length–weight relationships were estimated separately for each sex and locality. growth rate was estimated according to the von bertalanffy equation (von bertalanffy, 1938): svlt = svlmax (svlmax svlmet)e-k(t-tmet) [1] where svlt is the average body length at age t; svlmax is the asymptotic maximum body length; svlmet is the body length at metamorphosis that was calculated on newly metamorphosed individuals at the end of the summer (fixed to mean 26.3±3.22 mm); k is the body growth rate coefficient (year-1); tmet is age at metamorphosis (0.3 years). the parameters svlmax and k and their asymptotic confidence intervals (ci) were estimated by nonlinear regression. all statistical analyses were conducted using spss ver. 20.0 (spss 2011). results a total of 161 individuals (80♂, 81♀) were examined. svl and body mass were significantly higher in females than in males (table 1), but not among the sampling localities (females: f3,77 = 3.324, p > 0.05; males: f3,76 = 2.326, p > 0.05). consistently the sexual dimorphism index was equal to 0.13 for all studied populations, i.e. female levantine frogs are larger and heavier than males. all the examined bones sampled from adult frogs had well-defined lags with visible layers (fig. 1). the first lag was partially resorbed in 22.3% of females and 19.2% of males and completely resorbed in 8.8% females and 6.4% males. the age structure of the levantine frogs from the different sampling sites is summarized in table 1. the average age varied between 4.6 and 6.5 years among the populations, most individuals were between 4-8 years, and a few individuals reached a maximum age of 12 years (fig. 2). the age of males and females did not show significant differences (table 1). the sex-specific age structure did not differ significantly among localities 149levantine frog age structure and growth parameters (females: f3,77 = 2.406, p > 0.05 and males: f3,76 = 2.351, p > 0.05). the age at first reproduction was two years in all localities and in both sexes. the asymptotic svl (svlmax) was estimated as 88.1±1.68 mm (ci = 85.0891.58) for males and 91.5±1.57 mm (ci = 88.62-94.58) for females, while growth coefficient (k) was estimated as 0.239 ± 0.0761 (ci = 0.196-0.255) and 0.346 ± 0.0924 (ci = 0.3145-0.7162) respectively (fig. 3). discussion our findings showed that counting lags on the phalanges of p. bedriagae was relatively simple because the growth arrest was clear during the winter and the degree of endosteal resorption was low. we did not observe double lags in any sample. the lack of double lines, which indicate a double annual growth cycle (e.g., due to aestivation period; guarino and erişmiş, 2008), or partly resorbed lags, suggests that these frogs experience an uninterrupted and regular annual bone growth cycle. as observed in other cognate species, female levantine frogs grow larger than males (sinsch and schneider, 1999; erişmiş, 2005; erişmiş and chinsamy, 2010; çiçek et al., 2011; erişmiş, 2011; gül et al., 2011; table 2). many amphibians show sexual size dimorphism, and females are larger than males in 90% of anuran species (shine, 1979). sexual size dimorphism can be explained in terms of differences in the age structure between the sexes in breeding populations (monnet and cherry 2002), or by adaptive differences in the grow rates table 1. student t tests comparisons of mean snout-vent length (svl), body mass and age in male and female levantine frogs from four localities in denizli, turkey. location parameters gender n min max mean std. error mean t df sig. vali recep yazıcıoğlu dam svl (mm) female 30 51.0 86.0 71.20 1.726 3.242 50 0.002** male 22 43.6 72.3 63.01 1.774 body mass (g) female 30 12.9 71.3 38.59 2.751 4.001 50 0.001** male 22 11.6 34.5 24.49 1.679 age female 30 2 9 5.20 0.334 1.198 50 0.237 male 22 2 9 4.59 0.382 süleymanlı lake svl (mm) female 24 57.0 104.9 76.63 2.194 3.079 47 0.003** male 25 45.0 84.2 68.01 1.760 body mass (g) female 24 12.6 94.4 46.05 3.710 2.354 47 0.023* male 25 11.6 75.9 35.29 3.075 age female 24 2 12 6.46 0.474 0.025 47 0.981 male 25 2 11 6.44 0.575 acıgöl lake svl (mm) female 15 45.0 90 71.93 2.872 2.511 30 0.018* male 17 44.8 80.7 63.09 2.127 body mass (g) female 15 11.3 84.4 37.59 4.540 1.971 30 0.058 male 17 8.7 60.4 26.98 3.095 age female 15 2 9 5.13 2.232 -0.648 30 0.522 male 17 2 9 5.59 1.734 ornaz valley svl (mm) female 14 50.4 103.0 81.14 4.635 2.168 26 0.039* male 14 50.0 96.0 69.49 2.718 body mass (g) female 14 32.4 94.5 67.39 5.462 4.011 26 0.000** male 14 24.3 74.3 41.36 3.504 age female 14 2 12 7.21 0.903 1.596 26 0.122 male 14 2 11 5.50 0.571 pooled localities svl (mm) female 81 45.0 104.7 74.31 1.337 5.027 159 0.000** male 80 43.6 94.0 65.78 1.040 body mass (g) female 81 11.3 94.4 45.33 2.243 4.889 159 0.000** male 80 8.7 75.9 31.92 1.568 age female 81 2 12 5.79 0.264 0.375 159 0.708 male 80 2 12 5.65 0.265 *p< 0.05, **p < 0.01. 150 eyup başkale et alii between sexes (as observed in p. bedriagae; present study). indeed, larger females are able to produce more and larger eggs (crump, 1974; crump and kaplan, 1979; berven, 1988; kupfer et al., 2004; haddad and prado, 2005; gunzburger, 2006) and males may prefer larger females (dittrich et al., 2018). body size parameters were comparable with previous studies performed on other populations of levantine frog (çiçek et al., 2011) and on cognate species, such as p. ridibundus (kyriakopoulou-sklavounou et al., 2008; gül et al., 2011; erişmiş, 2011; ashkavandi et al., 2012) and p. caralitanus (erişmiş and chinsamy, 2010; başkale et al., 2017; erişmiş, 2018) (table 2). the age composition of amphibian populations may depend on several genetic and environmental factors, e.g., climate, trophic resources, habitat quality, and interspecific interactions (miaud et al., 2001; morrison and hero, 2003; üzüm et al., 2011; gümüş özcan and üzüm, 2015). although our sampling habitats were quite different in terms of altitude, surface area, and habitat quality, all the studied populations had a similar age structure. however, different age structures can be retrieved in literature. çiçek et al. (2011) and i̇smail and çiçek (2017) determined that the mean age of a levantine frog population from sülüklü lake, manisa, turkey was much lower than what we observed (table 2), possibly due to anthropogenic pressures. similarly, also the age structure of the other cognate species may present high degrees of variation (table 2). compared to the present study (max longevity = 12 years), çiçek et al. (2011) and i̇smail and çiçek (2017) found a lower longevity in p. bedriagae possibly due to anthropogenic stressors. considering cognate species, the maximum ages can reach 11 years (range = 5-11 years) and age at maturity ranged from 1-4 years in different parts of their range (table 2). fig. 3. von bertalanffy growth curve (age-length relationship) for levantine frogs from different localities in denizli, turkey.fig. 1. phalangeal cross-sections (16 μm thick) of levantine frog. the white arrows show lags in the periosteal bone: (a) male: 4 lags with 65 mm svl, (b) female, 6 lags with 79.5 mm svl. eb = endosteal bone, pb= periosteal bone, mc =marrow cavity, rl=resorption line. fig. 2. age distribution of four levantine frog reproductive populations from different localities in denizli, turkey. 151levantine frog age structure and growth parameters local climatic conditions can determine the activity period of amphibians and can be a key factor controlling the activity and reproductive periods and population parameters in temperate species of anurans (stebbins table 2. body size and grow parameters in p. bedriagae and cognate species: a comparison with existing literature. am: age at sexual maturity; k: von bertalanffy growth coefficient. species locality sex n mean svl (mm) max svl (mm) mean age (years) max age (years) am (years) k references p. bedriagae vali recep yazıcıoğlu dam, denizli, turkey m 30 63.01 80.17 4.59 9 2 0.18 present study f 22 71.20 83.75 5.20 9 2 0.27 süleymanlı lake, denizli, turkey m 24 68.01 73.80 6.44 11 2 0.37 f 25 76.63 82.67 6.46 12 2 0.22 acıgöl lake, denizli, turkey m 15 63.09 70.04 5.59 9 2 0.37 f 17 71.93 80.79 5.13 9 2 0.24 ornaz valley, denizli, turkey m 14 69.49 78.64 5.9 12 2 0.19 f 14 81.14 96.41 6.8 12 2 0.33 pooled localities m 80 65.78 88.1 5.65 12 2 0.24 f 81 74.31 91.5 5.79 12 2 0.35 sülüklü lake manisa, turkey m 14 56.1 73.2 2.5 4 2 0.30 çiçek et al., 2011 f 22 64.5 92.4 2.95 5 2 m 51 59.80 120 3.45 7 2 0.22 i̇smail and çiçek, 2017 f 76 59.78 137 4.33 9 2 0.36 p. ridibundus yıldızlı stream, trabzon, turkey m 38 64.58 3.90 7 3-4 yılmaz et al., 2005 f 11 76.64 3.72 6 3-4 lake vistonis, lagos, greece m 52 69.03 93.4 2.96 5 1 0.57 kyriakopoulou-sklavounou et al., 2008 f 56 82.38 107.5 3.73 5 1 0.54 milicz ponds reserve, stawno, polland m 32 72.2 90 3.7 6 2 0.76 socha and maria, 2010 f 38 79.8 102.3 4.4 7 3 0.59 artvin (borçka, lake karagöl), turkey m 20 72.96 5.15 8 2 gül et al., 2011 f 25 63.49 4.20 7 2 dörtyol, hatay, turkey m 20 64.70 5.50 11 4 f 19 76.74 5.58 7 3 verkhne-tagil reservoir, tagil and vogulka rivers, middle urals. m ivanova et al., 2011 f 92.8 116 5.4 9 2 the reftinskii reservoir reft river, middle urals. m f 26 112.9 132 4.4 8 2 the north of lorestan province, central zagros, iran m 26 71.14 6.43 11 3 ashkavandi et al., 2012 f 14 74.05 4.5 7 3 p. caralitanus beysehir lake, turkey m 38 75.56 109 5.01 9 3-4 0.18 erişmiş and chinsamy 2010 f 51 92.05 126.24 6.01 10 3-4 0.16 m 96 90.41 111.35 5.63 9 3 erişmiş, 2018 f 73 98.29 126.50 6.33 10 3 karamık lake, turkey m 66 82.33 99.48 4.86 7 3 f 76 88.36 111.63 5.30 8 3 işıklı lake, turkey m 49 73.06 93.68 3.69 6 2 f 47 82.54 106.72 4.80 8 2 eğirdir lake, turkey m 90 86.44 110.12 5.42 8 3 f 97 94.52 120.28 6.21 10 3 152 eyup başkale et alii and cohen, 1995; kyriakopoulou-sklavounou, 2000). for example, the bergmann’s rule (bergmann, 1847) indicates that the individuals living in a cold climate tend to be larger than individuals inhabiting warmer climates (i.e. miaud et al., 2001; khonsue et al., 2001; but see ashton (2002) for several exceptions to this general role). altitude variation causes steep environmental gradients at small geographic scale (e.g., temperature and humidity), but altitude had limited effects on the parameters of our study populations. erişmiş (2018) observed that the p. caralitanus from the lake district of anatolia exposed to warmer temperatures had longer growth seasons, but smaller growth rates. our results may depend on an unusually weak link between local climatic conditions and altitude. alternatively, they could indicate that p. bedriagae could have stable population parameters across its range, e.g., gokhelashvili and tarkhnishvili (1994) stated that p. ridibundus (formerly known as rana ridibunda) from georgia showed a constant age of maturation (after two hibernations) as a species-specific trait not depending on local environmental factors. our calculated grow rate in the present study is similar to literature values for p. bedriagae (e.g., 0.30; çiçek et al., 2011) and is lower than the values reported for most european pelophylax species (table 2). previous studies on p. ridibundus have shown significant correlations between svl and age for both sexes (yılmaz et al., 2005; kyriakopoulou-sklavounou et al., 2008) as commonly reported for the genus pelophylax (erişmiş et al., 2009; esteban et al., 1996, 1999). in the present study, we found that the growth coefficients were higher in females than in males, and that the growth curves reached a plateau at an age of 3-4 years. in addition, the sizes at the same ages were highly variable among individuals, so that the size ranges of different age classes overlapped. these results show that body size is not an accurate indicator of the age of p. bedriagae, as already observed for other amphibians (halliday and verrell, 1988). acknowledgements the permissions for field work, handling the frogs and laboratory studies were issued by the animal ethics committee of pamukkale university (protocol no: pauhdek-2012/033), the ministry of forestry and water affairs, the general directorate of nature conservation and natural parks and the turkish ministry of food, agriculture and livestock. we would like to thank to pamukkale university scientific research projects unit – bap (2012fbe029) for their support during this study. references akın, ç., bilgin, c.c., beerli, p., westaway, r., ohst, t., litvinchuk, s.n., uzzell, t., bilgin, m., hotz, h., guex g-d., plötner, j. 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(2005): age determination and some growth parameters of a rana ridibunda population in turkey. acta zool. acad. sci. hung. 51: 67-74. acta herpetologica vol. 13, n. 1 june 2018 firenze university press acta herpetologica 13(1): 3-11, 2018 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-21327 species diversity and distribution of lizards in montenegro katarina ljubisavljević1,2,*, ljiljana tomović3, aleksandar urošević1, slađana gvozdenović2, vuk iković2, vernes zagora2, nenad labus4 1 department of evolutionary biology, institute for biological research “siniša stanković”, university of belgrade, bulevar despota stefana 142, 11000 belgrade, serbia 2 montenegrin ecologists society, bulevar sv. petra cetinjskog 73, 81000 podgorica, montenegro 3 institute of zoology, faculty of biology, university of belgrade, studentski trg 16, 11000 belgrade, serbia 4 biology department, faculty of science and mathematics, university of priština, lole ribara 29, 38220 kosovska mitrovica, serbia * corresponding author. e-mail: katarina.ljubisavljevic@ibiss.bg.ac.rs submitted on: 2017, 25th september; revised on: 2018, 28th march; accepted on: 2018, 1st april editor: aaron m. bauer abstract. the southern part of montenegro has been identified as an area with high diversity of herpetofauna. however, comprehensive studies of distribution and diversity patterns of reptiles on the country level are still missing. such studies are essential in designating areas of special conservation importance and nature protection planning in a milieu of increased habitat loss and degradation due to rapid urbanization and tourism development in this small mediterranean country. to make progress on this problem, we analyzed distribution and diversity patterns of the lizards in montenegro using a large database consisting of literature data and our unpublished records. we found that fifteen lizard species inhabit montenegro, and two additional species may be present. the lizards were most diverse in the maritime biogeographic region of montenegro, while low diversity was found predominantly along the state borders in the mountain-valley region. the identified pattern of lizard diversity is at least partly influenced by sampling bias. the eastern mountainous subregion had a distinct species composition compared to all other parts of the country. the east-mediterranean chorotype was the most dominant, represented by seven species. the great diversity of the lizard fauna of montenegro can be attributed to its specific topographic position with great influence of mediterranean climate, heterogeneity of biomes, complex geological history and diverse physiogeographic features. high lizard species richness in the maritime region and a unique species composition in the eastern subregion of montenegro indicate that these areas are of high conservation interest. keywords. balkan peninsula, distribution pattern, lacertilia, species richness. introduction the number of studies of herpetofaunal distribution and diversity patterns in the balkan countries has increased over the last decade (jablonski et al., 2012; cogălniceanu et al., 2013; tomović et al., 2014; sterijovski et al., 2014; uhrin et al., 2016; mizsei et al., 2017). they have been driven not only by the creation of large distributional databases resulting from intensive biodiversity surveys for conservation purposes (such as implementation of the natura 2000 network, preparation of national red lists and books of threatened species) and/ or research of particular taxa, but also by the recognition that basic biogeographical studies have gained increased significance in ecological, evolutionary and systematic research (e.g., jetz et al., 2011; zachos and habel, 2011). such studies are especially important in areas of high conservation concern such as the balkans, one of the main european centers of endemism and speciation within some herpetofaunal taxa (džukić and kalezić, 2004). 4 katarina ljubisavljević et alii the southern part of montenegro lies within the mediterranean belt, one of the globally recognized biodiversity hotspots (myers et al., 2000). it has also been identified as an area of high concentration of reptile species (crnobrnja-isailović and džukić, 1995; džukić and kalezić, 2004; sillero et al., 2014). this has been further confirmed through studies on reptile distribution, intraspecific differentiation and speciation events in this area (böhme et al., 2007; ljubisavljević et al., 2007; polović and ljubisavljević, 2010; polović and čađenović, 2014; jablonski et al., 2016). however, despite the evident importance of herpetofaunal diversity of montenegro, there have been no comprehensive and detailed studies of distribution and diversity patterns of reptile species for the country as a whole. the need for such studies is increasing due to newly described or discovered lizards in montenegro (ljubisavljević et al., 2007; vergilov et al., 2016), recent nomenclatural (sillero et al., 2014) and taxonomic changes (jablonski et al., 2016; marzahn et al., 2016) and the necessity of updating the national list of protected species and preparation of the red book of threatened species, bearing in mind the growing negative influence of habitat loss and degradation in recent years (bataković et al., 2014). in order to make progress and respond to these needs, we here present all available distribution data for lizards in montenegro and analyze their patterns of diversity. in doing so, we provide the first zoogeographical analysis of the most numerous reptile group in this country. in this way, our study presents a starting point for the consideration of the distribution and diversity of all reptiles in the territory of montenegro. material and methods study group lizards (squamata: lacertilia) comprise over 6000 species worldwide, with over 80 species in europe. thirty-six of these (belonging to six families) occur on the balkan peninsula and adjacent islands (excluding crete) (speybroeck et al., 2016). the lizards of the balkans are quite diverse in terms of morphology and lifestyle. they include small to large taxa, normal-legged (true lizards), tiny-legged (e.g., ablepharus kitaibelii) and legless forms (slow worms), diurnal to nocturnal species (geckos), and those with conspicuous or rather cryptic behavior (e.g., most skinks and slow worms). there are ground-dwelling (e.g., lacerta agilis and zootoca vivipara), saxicoulous (e.g., dalmatolacerta oxycephala and dinarolacerta spp.) to semi-arboreal lizards (e.g., green lizards) with oviparous (e.g., wall and green lizards) and viviparous reproductive modes (e.g., z. vivipara). study area montenegro is located in the west-central part of the balkan peninsula, bounded by the adriatic sea to the south and dinaric mountains to the north. the total land area of montenegro is 13,812 km2. marković (1970) divided the territory of montenegro into two main regions (maritime and mountain-valley) and six subregions (fig. 1). the maritime region is divided into mediterranean and sub-mediterranean subregions. the mediterranean subregion of montenegro is narrow coastal area (2–10 km wide), separated from the hinterland by the steep dinaric mountains (orjen, lovćen, sutorman and rumija). it is characterized by a typical mediterranean climate with hot dry summers and mild wet winters (matvejev, 1961). the sub-mediterranean part of montenegro includes valleys of the zeta river and skadar lake, hilly areas in the western and central parts of the country and is bordered by high coastal mountains to the south. the climate in this subregion is modified mediterranean with somewhat cooler and moister winters and long, dry and hot summers. the mountain-valley region is divided into central, eastern, northern and western subregions according to geographic position. the mountain-valley region is dominated by medium to high mountains intersected by deep canyons of the tara and piva rivers in the northern subregion, the morača river in the central and the valley of the lim river in the eastern subregion. the western subregion is a largely high fig. 1. geographical regions and subregions of montenegro (according to stešević and caković, 2013 and vuksanović et al., 2016). maritime region (in light grey) with mediterranean (m) and sub-mediterranean (sm) subregions. mountain-valley region (in dark grey) with western (w), central (c), eastern (e) and northern (n) subregions. 5lizards of montenegro dry karst area with scarce surface water bodies. the temperate continental climate in the mountain-valley region, characterized by warm to hot summers and very cold winters, is often modified under the influence of high altitudes (mountain climate of the alpine type with short wet summers and long, cold snowy winters) or mediterranean influence that spreads along the river canyons, gorges and valleys far inland (modified continental climate with milder winters) (stevanović and stevanović, 1995). methods the dataset of locations in montenegro where lizards were recorded consists of 1284 entries. the records were sorted by the year of observation (unpublished records), or the year of publication (literature records), and characterized as old or recent according to whether they were published/observed before or after 1990. this year was chosen in accordance with other similar studies of the balkan herpetofauna (cogălniceanu et al., 2013; mizsei et al., 2017), and because more intense and systematic surveys of herpetofauna in certain areas of the country started after 1990. the majority of our records were collected during field surveys in the last decade as results of independent projects or sub-projects on lizards (see acknowledgements). a number of unpublished records came from the field databases of dr. miloš kalezić, dr. georg džukić and late prof. dr. gojko pasuljević. all species were identified according to standard herpetological literature by visual inspection of diagnostic characters (arnold and ovenden, 2002). regarding taxonomy and current nomenclature, we followed sillero et al. (2014) and speybroeck et al. (2016). accordingly, anguis fragilis and a. graeca were treated as a species complex, because the distinction between these two taxa and their precise distribution in montenegro require further research (sillero et al., 2014; jablonski et al., 2016). the distinct lineages of lacerta viridis-bilineata complex which, according to the newest study (marzahn et al., 2016), occur in the western balkans including montenegro but have not been taxonomically evaluated, were treated as lacerta viridis complex following marzahn et al. (2016) and mizsei et al. (2017). the distributions of two species with doubtful old records (tarentola mauritanica and podarcis tauricus), the presence of which has not been confirmed during recent decades, are presented in table 1 and supplementary material, but excluded from further biogeographic analyses. the geographic distribution of every lizard species in montenegro was presented on the country map overlain by a 10 × 10 km utm (universal transverse mercator) grid (see figs. s3-s7). doubtful findings were marked with a question mark (?). all georeferences are based on the wgs84 datum. the database includes the following locality information: site name, coordinates in decimal degrees, 10 × 10 km utm square and data source type (see table s1). a general distribution map of lizards in montenegro (see fig. s2 and supplementary data file) was constructed using the free diva-gis 7.5.0 software (hijmans et  al.,  2012), based on relationship between species records and the utm 10 x 10 km grid using the grid cell code as a common attribute (cogălniceanu et al., 2013). similarities among biogeographic subregions of montenegro were evaluated using the bray-curtis dissimilarity index (bray and curtis, 1957). chorotypes were determined according to vigna taglianti et al. (1999). for the analyses and designation of centres of lizard diversity in montenegro, we used an application created in visual basic 6.1 in the program winword 2003 (niketić, 1999), using the method of walter and straka (1970), at national grid utm reference for montenegro 10 × 10 km. results the dynamics of publications on and data collection for lizards in montenegro from the end of the 19th century to 2017 are presented in fig. 2. an increase in field research and publication effort is evident from the 1990s onwards. there were many more records dated after 1990 than before 1990 (table 1). the majority of the published and unpublished data came from the last seven years, when field survey activities have intensified. the increase in published data during the nineties of the last century may be attributed to the systematic research on the herpetofauna, particularly lizards, of the skadar lake region. unpublished records from the 1970s–1980s came from the unpublished notes of the late prof. dr. pasuljević. fifteen lizard species from four families were confirmed to inhabit montenegro (table 2). the most diverse group were lacertid lizards (11 species). about 54 % of the utm 10 × 10 km grid cells that cover the surveyed territory contained only recent records of lizards, 34% contained both old and recent records, whereas only 12 % of them contained only records from surveys performed prior to 1990 (fig. s1). roughly 63% of the total national utm grid cells contained records of at least one lizard fig. 2. number of lizard records in montenegro by year of collection (unpublished data) or publication (published data). 6 katarina ljubisavljević et alii species, of which 22% were located in areas not covered by previously published surveys. consequently, 37% of the utm grid cells were without records (fig. s2). only potentially introduced species or those with doubtful records (tarentola mauritanica, podarcis tauricus, p. siculus) had more old than recent records (table 1). nearly all species, with the exception of hemidactylus turcicus, had more published than unpublished records. the species with the highest number of records was podarcis muralis (240), followed by dalmatolacerta oxycephala (139), podarcis melisellensis (137) and pseudopus apodus (134). ablepharus kitaibelii (1), dinarolacerta table 1. the list of lizard species in montenegro with number of records. old and recent records correspond to the publication/collection dates before and after 1990. (*) species with doubtful old records, the presence of which has not been confirmed in recent times. species total records published records unpublished records old records recent records hemidactylus turcicus 45 20 25 12 33 tarentola mauritanica* 1 1 0 1 0 algyroides nigropunctatus 44 30 14 4 40 dalmatolacerta oxycephala 139 99 40 39 100 dinarolacerta montenegrina 14 13 1 0 14 dinarolacerta mosorensis 123 119 4 37 86 lacerta agilis 54 36 18 7 47 lacerta viridis complex 90 58 32 9 81 lacerta trilineata 112 66 46 18 94 podarcis melisellensis 137 72 65 24 113 podarcis muralis 240 166 74 34 206 podarcis tauricus* 2 2 0 2 0 podarcis siculus 8 8 0 5 3 zootoca vivipara 22 19 3 7 15 ablepharus kitaibelii 1 1 0 0 1 anguis fragilis complex 118 70 48 22 96 pseudopus apodus 134 77 57 14 120 total 1284 857 427 235 1049 table 2. list of confirmed lizard species in montenegro with endemicity status for the balkans, marginality and range fragmentation on the territory of montenegro, extent of their occurrence in subregions, and utm squares and chorotype classification. family species marginal zone balkan endemic fragmented range subregion n of utms chorotype gekkonidae hemidactylus turcicus m,sm 14 mediteranean lacertidae algyroides nigropunctatus + m,sm,c 22 e-mediterranean dalmatolacerta oxycephala + + m,sm,c,n,w 40 e-mediterranean dinarolacerta montenegrina + + e 4 e-meditrranean dinarolacerta mosorensis + + + m,sm,c,n,w 21 e-mediterranean lacerta agilis + + c,e,n,w 21 euro-siberian lacerta viridis complex m,sm,c,e,n,w 39 s-european lacerta trilineata + m,sm 31 e-mediterranean podarcis melisellensis + m,sm,w 38 e-mediterranean podarcis muralis m,sm,c,e,n,w 70 s-european podarcis siculus + ? m 1 s-european zootoca vivipara + + e 9 euro-siberian scincidae ablepharus kitaibelii + ? e 1 e-mediterranean anguidae anguis fragilis complex m,sm,c,e,n,w 45 euro-siberian pseudopus apodus m,sm 38 turano-mediterranean 7lizards of montenegro montenegrina (14) and zootoca vivipara (22) had the lowest number of records for the confirmed and native species (table 1). almost half of the confirmed species (47%) reaches the edge of their distribution in montenegro, while 40% of them are endemic to the balkan peninsula (table 2). most of species that inhabited medium to high-altitude areas of montenegro (dinarolacerta mosorensis, lacerta agilis and z. vivipara) had fragmented ranges (table 2, figs. s4 and s5). recent discovery (a. kitaibelii) and/or low number of confirmed records (p. siculus) precluded evaluation of range fragmentation in these species (table 2). podarcis muralis was the most widely distributed lizard in montenegro, inhabiting the territory covered by 39% of the national utm grid cells, followed by p. melisellensis, p. apodus, l. viridis complex, d. oxycephala and a. fragilis complex which occurred in 21% to 25% of utm grid cells (figs. s4-s7). podarcis siculus, a. kitaibelii and d. montenegrina were the rarest species, confined to fewer than 3% of the utm cells covering the montenegrin territory (figs. s4, s6 and s7). the highest numbers of species (eight to nine) were recorded in 10 utm squares located along the adriatic coast, in the skadar lake region, the zeta river plain and around the capital of montenegro (podgorica). however, a great number of utm squares (30) contained only one lizard species, while nearly one third of utm squares that cover the territory of montenegro were without any records. most of the squares with no lizard species or low lizard diversity were located in the north-eastern border area and western part of the country (figs. 3, s1 and s2). in line with these results, species diversity was greater in the maritime than in the mountain-valley region. mediterranean and sub-mediterranean subregions were inhabited by 11 and 10 species, respectively, whereas central, eastern and western subregions had seven species each (table 2). the lowest number of species (six) was found in the northern, high mountain subregion. three taxa (l. viridis complex, p. muralis and a. fragilis complex) with both wide geographic and altitudinal ranges were distributed in all subregions. according to our cluster analysis mediterranean and sub-mediterranean subregions were the most similar to one another (fig. 4). they formed a distinct cluster due to identical composition of 10 lizard species, with only one additional species (p. siculus) found in the mediterranean subregion. northern, central and western parts of the mountain-valley region formed another cluster, differing mutually by no more than one species. the eastern subregion was the most distinct due to the presence of three species (a. kitaibelii, d. montenegrina and z. vivipara) that were not found in other subregions. the lizards of montenegro were classified into five chorotypes (table 2). the most common chorotype was east-mediterranean, represented by seven species. the euro-siberian and south-european chorotypes were repfig. 4. cluster diagram of bray-curtis similarity index of lizard fauna for biogeographic subregions in montenegro. fig. 3. diversity of lizard species in montenegro in the 10 × 10 km utm grid. colours and sizes of squares present numbers of lizard species per utm cell (left-hand side of the legend), and colours and sizes of the rectangles present numbers of utm squares with corresponding numbers of lizard species (right-hand side of the legend). 8 katarina ljubisavljević et alii resented with three species each. the rarest chorotypes were mediterranean and turano-mediterranean, represented with one species each. discussion with 15 species confirmed, montenegro is amongst the countries with the highest diversity of lizards in the balkans (e.g., petrov, 2007; krofel et al., 2009; jablonski et al., 2012; cogălniceanu et al., 2013; sillero et al., 2014; tomović et al., 2014; uhrin et al., 2016; mizsei et al., 2017). furthermore, 10 recorded species are regional endemics, having marginal ranges in montenegro or a fragmented range in it (table 2), contributing to a high potential for further evolutionary diversification of the lizard fauna (džukić and kalezić, 2004). a comparative analysis of the diversity of lacertid lizards among the balkan countries showed a clear distinction between the western and eastern countries, with montenegro grouping together with bosnia and herzegovina, croatia and slovenia within the western balkan cluster (see urošević et al., 2015). this pattern remained the same when we included all lizards (results not shown). the lizard fauna of montenegro is most similar to those of bosnia and herzegovina and croatia, mostly owing to the presence of the western balkan endemic lacertids dalmatolacerta oxycephala and dinarolacerta mosorensis in these three countries. actually, the only difference in lizard fauna between montenegro and bosnia and herzegovina is the presence of the endemic dinarolacerta montenegrina in montenegro. this points out the important impact of landscape features of the dinaric mountains in shaping biodiversity of the western balkans (e.g., redžić et al., 2011; ivković and plant, 2015). a great diversity of lizard fauna inside the smallest balkan country can be attributed to its specific geographic position with great influence of mediterranean climate, heterogeneity of biomes, complex geological history and diverse physiogeographic features ranging from high mountains in the north, through deep river canyons and valleys to the broad plain in the south (matvejev, 1961; matvejev and puncer, 1989). macroevolutionary events, such as speciation within the genus dinarolacerta (ljubisavljević et al, 2007), or division of the “mainland clade” of dalmatolacerta oxycephala (podnar et al., 2014) took place in the montenegrin mountains. furthermore, heterogeneous mountain topography and proximity to the mediterranean were important factors in shaping genetic diversity of two species complexes of lizards, the l. viridis complex and the a. fragilis complex (böhme et al., 2007; jablonski et al, 2016; marzahn et al., 2016). marzahn et al. (2016) revealed the existence of two distinct lineages of l. viridis complex in montenegro, while jablonski et al. (2016) identified contact zones between two lineages of slow worms (a. fragilis complex) in southernmost part of the country. although the presence of five chorotypes points out the variability of zoogeographical links, the predominance of mediterranean chorotypes clearly indicates a dominant influence of the mediterranean. higher lizard diversity in areas under the influence of mediterranean climate is not surprising and is evidenced in other studies of reptile diversity of the balkan countries (e.g., jablonski et al., 2012; urošević et al., 2015; mizsei et al., 2017). a great diversity of lizards, and herpetofauna in general, in the skadar lake region and coastal zone (crnobrnja-isailović and džukić, 1995; džukić and kalezić, 2004; polović and ljubisavljević, 2010; polović and čađenović, 2014; this study) indicates that these areas are of particular conservation interest, especially bearing in mind the trend of rapid urbanization and development of touristic infrastructure in recent years. although a wide range of eu policies and legislation which address specific problems evidenced in coastal environments were adopted in montenegro, their implementation has been slow and represents a significant challenge for future conservation initiatives (knežević et al., 2015). furthermore, the presence of some endemic species (d. montenegrina) and species with scarce records at the periphery of their distribution (a. kitaibelii, z. vivipara) in the eastern mountainous subregion indicate that this area should be also considered during conservation planning. observed patterns of lizard diversity in montenegro could be influenced by the elevational gradient, which is correlated with temperature and mediated by precipitation (mccain, 2010; mizsei et al., 2017), but also by the so-called “linnean shortfall” (the knowledge gap of the number of existing species) and “wallacean shortfall” (incomplete knowledge about the geographical distribution of species, which is usually related to variation in survey effort) (lomolino, 2004; hortal et al., 2015). the relatively recent discovery of d. montenegrina (ljubisavljević et al., 2007) and the possible existence of still unidentified or undescribed taxa, e.g., within l. viridis complex (böhme et al., 2007; marzahn et al., 2016), a. fragilis complex (jablonski et al., 2016) or d. oxycephala mainland clade (podnar et al., 2014), may indicate that lizard diversity is subject to the linenan shortfall. large area without lizard distributional data in the mountainvalley region suggests that this pattern may be also affected by the wallacean shortfall. for example, utm squares that cover the areas of durmitor mt., bjelasica mt. and krnovo mountain plateau have the highest number of 9lizards of montenegro species in the mountain-valley region and, at the same time, are hotspots of sampling effort. these mountains have been surveyed frequently during the course of different herpetological studies (džukić, 1991; tomović et al., 2004; polović and čađenović, 2013). nevertheless, considerably higher  sampling effort during the  recent period has contributed to reduce gaps in the knowledge of the distribution of lizards in montenegro. new records extended national ranges for many species. the ranges of l. agilis, and the l. viridis and a. fragilis complexes were extended to the east and west, while the ranges of p. melisellensis and p. muralis were extended to the central and western part of the country. new data also helped to fill in some distribution gaps for h. turcicus, algyroides nigropunctatus and d. oxycephala. among the confirmed species, only p. siculus, represented by the stenoendemic subspecies p. s. cattaroi could have anthropochorous origin (ljubisavljević et al., 2005; podnar et al., 2005). the moorish gecko (t. mauritanica), a species with a doubtful record in montenegro (bruno, 1988), is also easily transported by humans and considered to be introduced to the adriatic coast and greece (krofel et al., 2009; speybroeck et al., 2016). our results showed that distribution gaps still remain for most species, especially in the north-eastern and north-western parts of montenegro. survey efforts should be focused on lizards with high conservation concern such as dinarolacerta spp. (ljubisavljević et al., 2017). distribution gaps in areas with suitable habitats for at least some of the wide-ranging species (e.g., l. viridis complex, p. muralis, a. fragilis complex) suggest that a scattered distribution pattern is rather a result of a lack of systematic field surveys than a consequence of specific ecological demands. perhaps the best example of this is the recent discovery of a. kitaibelii, a species known from neighboring countries, in eastern montenegro (vergilov et al., 2016). other species, such as mediodactylus kotschyi and podarcis tauricus, known from border regions of northwestern albania (haxhiu, 1998; mizsei et al., 2017), are also likely to be discovered in montenegro in upcoming years, due to the presence of suitable habitats in southeastern montenegro and lack of barriers to dispersal of these species. furthermore, northern, western and northeastern areas of montenegro should be of high priority for future systematic reptile field research. acknowledgements research was funded by ministry of education, sciences and technological development of serbia (grant no. 173043) and by the rufford small grants foundation (grant nos. 14714-1, 19600-2, 19309-2). we would like to acknowledge the constructive reviews provided by shai meiri and anonymous reviewers that greatly improved interpretation of our data. we would like to thank dr. marjan niketić and dr. gordana tomović (belgrade) for help with distribution maps and biodiversity analyses. we are grateful to dr. miloš kalezić for sharing distribution data with us. field data of late professor gojko pasuljević were also included in this paper. also, many thanks to milan fatić, nenad preradović and dejan bogdanović for help in the fieldwork. supplementary materials supplementary material associated with this article can be found at <http://www.unipv.it/webshi/appendix> manuscript number 21327. references arnold, n., ovenden, d. 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(2011): biodiversity hotspots distribution and protection of conservation priority areas. springer-verlag, berlin-heidelberg. acta herpetologica vol. 13, n. 1 june 2018 firenze university press species diversity and distribution of lizards in montenegro katarina ljubisavljević1,2,*, ljiljana tomović3, aleksandar urošević1, slađana gvozdenović2, vuk iković2, vernes zagora2, and nenad labus4 the first demographic data and body size of the southern banded newt, ommatotriton vittatus (caudata: salamandridae) abdullah altunişik diet and helminth parasites of freshwater turtles mesoclemmys tuberculata, phrynops geoffroanus (pleurodira: chelidae) and kinosternon scorpioides (criptodyra: kinosternidae) in a semiarid region, northeast of brazil antonio marcos alves pereira*, samuel vieira brito, joão antonio de araujo filho, adonias aphoena martins teixeira, diêgo alves teles, daniel oliveira santana, vandeberg ferreira lima, waltécio de oliveira almeida electric circuit theory applied to alien invasions: a connectivity model predicting the balkan frog expansion in northern italy mattia falaschi1,2,*, marco mangiacotti3, roberto sacchi3, stefano scali2, edoardo razzetti4 first report of bufo bufo (linnaeus, 1758) from sardinia (italy) ilaria maria cossu1, salvatore frau1, massimo delfino2,3, alice chiodi4, claudia corti1,5*, adriana bellati4 natural history and conservation of the nurse frog of the serranía del perijá allobates ignotus (dendrobatoidea: aromobatidae) in northeastern colombia hernán darío granda-rodríguez1,2, andrés camilo montes-correa3,*, juan david jiménez-bolaño3, marvin anganoy-criollo4,5 exploring body injuries in the horseshoe whip snake, hemorrhois hippocrepis juan m pleguezuelos*, esmeralda alaminos, mónica feriche how effectively do european skinks thermoregulate? evidence from chalcides ocellatus, a common but overlooked mediterranean lizard grigoris kapsalas, aris deimezis-tsikoutas, thanos georgakopoulos, ismini gkourtsouli-antoniadou, kallirroi papadaki, katerina vassaki, panayiotis pafilis thermal tolerance for two cohorts of a native and an invasive freshwater turtle species jun geng, wei dang, qiong wu, hong-liang lu* diet of a restocked population of the european pond turtle emys orbicularis in nw italy dario ottonello1, fabrizio oneto1,2, monica vignone2, anita rizzo2, sebastiano salvidio2,* helminths of the lizard colobosauroides cearensis (squamata, gymnophthalmidae) in an area of caatinga, northeastern brazil aldenir f. da silva neta*, robson w. ávila acta herpetologica 14(2): 101-107, 2019 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/a_h-7747 pit-tags as a technique for marking fossorial reptiles: insights from a long-term field study of the amphisbaenian trogonophis wiegmanni pablo recio, gonzalo rodríguez-ruiz, jesús ortega, josé martín* dept. ecología evolutiva, museo nacional de ciencias naturales, csic, josé gutiérrez abascal 2, 28006 madrid, spain. *corresponding author. e-mail: jose.martin@mncn.csic.es submitted on: 2019, 12th february; revised on: 2019, 22nd april; accepted on: 2019, 12th may editor: aaron m. bauer abstract. many field studies of ecology or conservation require individual identification of the animals, and for this, several marking techniques have been developed. however, no specific labeling technique has been tested for fossorial reptiles, such as amphisbaenians. we describe the use of passive integrated transponder (pit) tags as a longterm labeling method of the amphisbaenian trogonophis wiegmanni. we present the details of the marking procedure and examine the benefits and drawbacks of the technique considering the fossorial environment. after marking many individuals in a long-term field study, we can ensure that the marks were easily applicable and were not lost over a period of at least four years. moreover, pit tags did not negatively affect the body condition of amphisbaenians. we conclude that pit tags are useful for doing field studies of this and similar fossorial species. keywords. amphisbaenians, trogonophis wiegmanni, pit-tagging, body condition, fossorial reptiles. introduction many field studies of ecology, behavior or conservation require individual recognition of the subjects that make up a population. being able to distinguish individuals allows the assessment of diverse ecological traits such as the size and dynamics of the population, survivorship, movements, home ranges, activity patterns, social interactions, etc. (reviewed in plummer and ferner, 2012; ferner and plummer, 2016). for that reason, labeling individuals is often necessary, and diverse tagging techniques have been developed depending on the species and/or the traits that are the object of study. ideally, these marks should allow a correct identification and be easily applicable, but without causing suffering to the animals, and they should last for at least the duration of the entire field study, but without affecting survival or behavior of the marked animals (reviewed in ferner and plummer, 2016). diverse methods of marking individuals have been described for reptiles. some are intended for short-term studies, such as external painting marks, beads, adhesive tapes, elastic bands, metal or plastic discs, buttons, etc. (gibbons and andrews, 2004; ribeiro and sousa, 2006; ferner and plummer, 2016). while others are focused on long-term studies, such as toe clipping, scale clipping, shell notching on turtles, heat/freeze branding, photo identification based on natural markings, visible implant elastomer (vie) tags and/or passive integrated transponder (pit) tags (daniel et al., 2006; hutchens et al., 2008; ekner et al., 2011; ferner and plummer, 2016). several groups of reptiles and amphibians, comprising as much as 20% of the global herpetofauna, or nearly 3,000 species, are fossorial (measey, 2006). however, as is the case with other fossorial animals, their ecology and conservation status are much less well understood than those of their epigeal relatives (copley, 2000; wolters, 2001; böhm et al., 2013). this may be explained because 102 pablo recio et alii of the difficulty of doing field studies of fossorial animals (measey, 2006; henderson et al., 2016), which includes difficulties in individually marking these animals, given their burrowing habits. although several marking techniques have been tested in fossorial caecilians (measey et al., 2001) and ambystoma salamaders (connette and semlitsch, 2012), to our knowledge, no specific labeling technique has been tested for limbless fossorial reptiles such as amphisbaenians (henderson et al., 2016). due to the morphology of most amphisbaenian species (i.e., elongated body without limbs in most species), it is obviously not possible to use many types of marking methods. further, given the fossorial habits of amphisbaenians, most external markings (painting, beads, adhesive tapes, etc.) may be incompatible with the burrowing behavior of these animals and will be quickly lost by repeated contact of the body with the soil. therefore, potential methods that could be used for long-term marking of amphisbaenians might be restricted to scale clipping, heat/freeze branding, vie tags and/or pit tags (camper and dixon, 1988; jemison et al., 1995; hutchens et al., 2008; ferner and plummer, 2016). here, we describe the use of pit tags as a labeling method for long-term field studies of the checkboard amphisbaenian trogonophis wiegmanni, kaup 1830. a pit tag is a microchip with an electromagnetic coil encased in a biocompatible glass cylinder, encoded alphanumerically in an unique way, that is implanted in the animal (gibbons and andrews, 2004). we present here the detailed marking procedure that we applied to amphisbaenians, examine the potential benefits and drawbacks of the technique, considering the peculiar characteristics of the fossorial environment, and discuss its utility for doing ecological studies of this and similar fossorial species. materials and methods study species the checkboard amphisbaenian t. wiegmanni, kaup 1830 is a representative of the family trogonophidae (gans, 2005) (fig. 1a) that inhabits arid areas from southwest morocco to northeast tunisia (bons and geniez, 1996). these amphisbaenians live all their life buried in the soil, but they are frequently found under rocks (civantos et al., 2003; martín et al., 2013a). little research has been carried out on this species, as on other amphisbaenians, but there is now a growing body of information on aspects such as its thermal biology (lópez et al., 2002), microhabitat and soil selection (civantos et al., 2003; martín et al., 2013a), reproduction (bons and saint girons, 1963), social behavior and population structure (martín et al., 2011b, c) or diet (martín et al., 2013b; baeckens et al., 2017). however, all these studies have been made by randomly sampling unmarked amphisbaenians. more detailed studies would require to individually identify the amphisbaenians that are being examined. this is important, not only because of the scientific interest in understanding the ecology and behavior of amphisbaenians, but because several conservation problems that may potentially affect their populations have been noted (martín et al., 2011a, 2015, 2017), and a detailed long-term monitoring of these populations require the ability to individually identify and follow the study subjects. field study and marking procedure we have carried out field and laboratory studies of t. wiegmanni amphisbaenians on the chafarinas islands (spain) for almost twenty years. this is an archipelago, formed by three small islands, located in the southwestern area of the mediterranean sea (35°10’n, 02°25’w), 2.5 nautical miles to the north of the moroccan coast (ras el ma, morocco) and 27 miles to the east of the spanish city of melilla. the islands have a dry, warm, mediterranean climate, and vegetation is dominated by bushy plants (suaeda, salsola, lycium and atriplex) adapted to salinity and drought. trogonophis wiegmanni is very common and is represented by very large populations on these islands (martín et al., 2011a). during the years 2015-2018, we made field campaigns twice a year, during two weeks in spring (march-april) and two weeks in autumn (september-october), to capture, mark and recapture t. wiegmanni. we delimited three study plots (surface area = 0.14 ha, 0.40 ha and 0.58 ha) on different islands, which we walked systematically and intensively during the morning and afternoon of different days. amphisbaenians were found by carefully lifting almost all rocks located inside the study plots. individuals were captured by hand, measured and immediately after marked in the field with pit tags. we used one of the smallest available pit tags (biomark minihpt8; biomark, inc., boise, idaho, usa), with a length of 8.4 mm, 1.4 mm in diameter and a weigh of 0.03 g. this weigh represents 0.6 % of the mean body mass (i.e., around 5 g) of a typical adult amphisbaenian in our population (martín et al., 2011c). we gently implanted pit tags subcutaneously in the upper right side of the body of amphisbaenians (fig. 1). for this, we made a small puncture at around 3 cm from the snout (mean svl of adult amphisbaenians is around 14 cm) using a stainless steel needle (biomark n165 needle; length = 5.1 cm, needle diameter = 1.49 mm), disinfected with alcohol before and after puncturing each individual, which was fitted to a specially designed syringe style implanter (biomark mk165 syringe). we gently lifted the skin from the underlying muscle and then inserted the transponder subcutaneously using the implanter. during the insertion of the pit tag, the needle was maintained parallel to the body to ensure that the tag remained under the skin and did not enter the coelomic cavity (fig. 1). the injection site was immediately disinfected with alcohol after the implant. according to brown (1997), losses of pit tags may occur immediately after the implant is done, while the wound is still open. to avoid this, incisions may be sealed with medical grade suture glue. however, in our case, this was not needed as the incision was 103marking fossorial reptiles very small and the tags showed no evidence of becoming displaced. further, at least in lizards, the glue may slow the healing process (le galliard et al., 2011). moreover, the long needle pushed up the tag under the skin towards the posterior part of the animal. thus, the tag was implanted at least 2 cm posterior of the small puncture point, which precluded tits loss when the amphisbaenian burrowed forward. all the marking procedure could be easily made by a single experienced researcher, holding the amphisbaenian with one hand and the implanter with the other. however, the presence of an additional researcher, who prepared the equipment and took notes, made the process easier and quicker, decreasing the manipulation time and disturbance to the animals. this marking technique is particularly appropriate for amphisbaenians, as their skin attachment is quite loose and leaves a subcutaneous space where the pit-tag is inserted. the skin is connected to the axial mass by costocutaneous and vertebrocutaneous muscles, that allow the skin to move independently from the body, mainly in rectilinear locomotion (gans, 1978; gasc, 1981; see illustrations in smalian, 1884), as those muscles are numerous and redundant, the insertion of a strange body (or even the damage of some muscle fibers) should not interfere with the normal locomotion or excavation. although amphisbaenians obviously “felt” and showed a small aversive response to the puncture with the needle, we did not observe any subsequent additional negative behavioral responses (e.g., stress, immobility, forced unnatural movements, or attempts to remove the tag) (warwick et al., 2013). amphisbaenians behaved normally when they were released at their capture points a few minutes after being captured and marked. the implant procedure very rarely resulted in a small drop of blood, but in that case the wound was cleaned with alcohol and bleeding stopped rapidly. we avoided the use of local anesthesia, because the duration of the recovery time from anesthesia could be much longer than the natural recovery from the implanting procedure. moreover, the administration of anesthesia per se is an additional procedure that requires increasing manipulation time and careful control of conditions, and it could have negative physiological side effects for small reptiles (heard, 2001; chatigny et al., 2017). a hand-held portable reader (biomark 601 reader) was used to read the individual unique code of the tag (the tags have a 134.2 khz, iso fdx-b, frequency), the code can be provided either as a hexadecimal or as a decimal number (15 digits). in the practice, the four last digits were enough for a reliable identification of individuals in each study population. the reader works in the field with aa rechargeable batteries but it may be also used in the lab with an ac power supply. to test the long-term effect of pit tags in amphisbaenians, we compared the body condition of individuals at first capture, when they were untagged, and when they were recaptured one year after being implanted with a pit tag. body condition was assessed as the residuals of an ordinary least squares linear regression of log-transformed mass (measured with an electronic balance to the nearest 0.1 g) against log-transformed total length (measured with a metallic ruler to the nearest 1 mm). to ensure that amphisbaenians had empty stomach and intestines before being weighed, we gently compressed their vents to force the expulsion of feces (used for a study of diet). the small weight of the tag was considered negligible. results and discussion in the four years of marking amphisbaenians, we have implanted pit tags in a mean of 45 + 4 amphisbaenians per study plot and campaign (3 plots and 7 campaigns of 15 days each), which so far leads to a grand total of 930 marked individuals in the four years. the number of individuals found and marked was significantly higher in spring than in autumn for a similar search effort (f1,19 = 11.82, p = 0.003). recapture rate was, however, relatively low; only around 15% of individuals found had already been marked. this is likely attributable to the difficulty of findfig. 1. an adult amphisbaenian (trogonophis wiegmanni) as it was found under a stone (left); pit tag implantation procedure (right). 104 pablo recio et alii ing the same individual on several occasions in a relatively short field campaign (i.e., each study plot is surveyed only during 3-4 days per campaign) and the high density of amphisbaenians, rather than to the fact that the marking procedure might affect survivorship or that the tags were lost and we were not be able to identify previously marked individuals. in fact, when we captured an unmarked individual, we always ensured that it had no scars at the usual injection point, which may indicate that it had been marked previously but had no tag inside. such scars are typical of marked individuals, but they have never been observed in unmarked individuals. also, we have not noted a decrease of population size, as assessed from the number of individuals usually found in a working day, which might reflect low survivorship of marked animals. moreover, nearby populations on the islands, where we sampled amphisbaenians without marking them, show similar trends to the marked populations (unpublished data). therefore, we are confident that the marking procedure is effective and it is not adversely affecting the populations. several authors have considered that pit tags are not always permanent (brown, 1997; ott and scott, 1999), while others claim permanence for more than 20 (germano and williams, 1993) or 70 years (ferner and plummer, 2016). in our study, we have recaptured individuals marked in the first year of the fieldwork after four years and we are confident that the mark will persist during the entire life of the amphisbaenian. gibbons and andrews (2004) postulated that tag migration may complicate code checking when it is not possible to find the tag, and can also lead to health problems when migrating through the digestive or urinary systems (jemison et al., 1995). this problem may be greater in fossorial burrowing animals due to the constant friction with the substrate (measey et al., 2001). in our study, although tag migration occurred in several individual amphisbaenians, in all cases, the tag had stayed just under the skin and was relocated posteriorly of the injection point, reaching a point close to the cloaca in the longest observed migrations. this movement of the tag seems to be along the subcutaneous space typical of amphisbaenians (see above) (gans, 1978; gasc, 1981). we did not detect injuries or health problems (e.g., infection, sores, bleeding, low body condition, etc) in any case. besides, we did not encounbter any problems in reading the tag, even in cases where its exact location was not easily detected at first sight, probably because the small size of t. wiegmanni allowed us to scan the entire body surface under the reader at the same time. on the other hand, long-term effects of pit tags have been described for several species. however, lobos et al. (2013) did not find significant impacts on growth rates, mating, or risk of predation when pit tagging different species of liolaemus lizards. brown (1997) also concluded that pit tags did not make any difference in survivorship nor body condition of diverse amphibians, and keck (1994) obtained similar results for growth rates and mobility in several snake species. nevertheless, females of the newt ichthyosaura alpestris laid significantly more eggs when marked, which seems to be related to a stress response (perret and joly, 2002). also, measures of corticosterone in blood have shown that the pit tag implanting procedure can be stressful for small skinks at least 14 days after the implant (langkilde and shine, 2006), but have no effects on stress five days after in common lizards (le galliard et al., 2011). our data show that pit tags do not have a negative long-term impact on the body condition of t. wiegmanni (body condition of the same individuals, initial vs. recapture: 0.05 + 0.03 vs. 0.06 + 0.04; one-way repeated measures anova: f1,96 = 0.34, p = 0.56). this lack of change of the body condition is a good indication of the absence of long-term negative effects of the pit tag on health of individuals, as it is known that natural and anthropomorphic alterations of the soil are reflected in a low body condition of these amphisbaenians (martín et al., 2015, 2017). another disadvantage associated with pit tagging may be related to the price of the reader and each transponder (gibbons and andrews, 2004). in our case, the current price of each pit tag is $2.58 (they are provided in packs of 100 units), the implanters cost $5 each (each one is useful for many markings), the needles costs $2 each (for an optimal functioning, we used a different needle for every 20 punctures), and the reader costs $595. these costs may be normally easily assumed by research or conservation projects financed by the government or other institutions. with respect to the invasiveness of the procedure, it has been recommended that it should not be used for individuals smaller than 8 cm (camper and dixon, 1988; gibbons and andrews, 2004; ferner and plummer, 2016). in our study, the small size of the pit tags allowed us to mark amphisbaenians as small as 90 mm svl without problems, the suitability for being marked depending more on the diameter of the body than on the length. these “small” amphisbaenians are second year young subadult individuals (martín et al., 2001c). only newborn individuals (svl<70 mm when they born in autumn) seem unsuitable for marking with these pit tags, considering the size of the currently available tags. this can be a problem that precludes the study of aspects of population dynamics, such as growth rates and survivorship of juveniles in their first year. however, given the low movement rate of amphisbaenians, is still possible to assess the 105marking fossorial reptiles number of individuals born in a population if we control for the geographic location of the newborn individuals found, to ensure that we do not repeat the same individual on different days. we expect that the future development of smaller pit tags will allow them to be used in all individuals. in contrast to pit tags, scale clipping and heat branding may be cheaper (winne et al., 2006; ekner et al., 2011), whereas vie tags can be seen without capturing the subject (daniel et al., 2006; hutchens et al., 2008), and none of these techniques are as invasive as pit tagging (gibbons and andrews, 2004; ferner and plummer, 2016). however, pit tags offer numerous advantages compared with the other long-term techniques potentially useful for marking amphisbaenians. pit tags are permanent and marks are unmistakable, while brands made by scale clipping or heating/freezing procedures may be confounded with natural marks or become unreadable as time passes due to external agents (winne et al., 2006; ekner et al., 2011). similarly, vie tags might be hard to detect in darkly pigmented tissues (hutchens et al., 2008; petit et al., 2012). also, pit tags are able to provide data even after the death of the marked individual, and/or can be even reused (gibbons and andrews, 2004). finally, pit-tag telemetry (i.e., detecting the radiofrequency signal of the tag at distance; e.g., connette and semlitsch, 2012; ousterhout and semlitsch, 2014) may allow the detection and relocation of fossorial animals burrowed underground without physically contacting them. however, for this technique a special, and more expensive, detector with an attached antenna is needed to carefully scan the soil surface, and the detection range for the smallest 8 mm pit tags is only 16 cm in depth increasing to 30 cm for a 12 mm pit tag (ousterhout and semlitsch, 2014). in conclusion, although further and more specific studies are needed to test the usefulness and effectiveness of pit tagging in fossorial reptiles, it seems to be a valid procedure for individual recognition in long-term field studies of amphisbaenians such as trogonophis wiegmanni, and, therefore, we suggest that it may be also applied to other similar limbless fossorial species. acknowledgments we thank ricardo montero for useful comments and the personal and facilities of the field station of the “zec islas chafarinas” (organismo autónomo de parques nacionales) for logistical support. we specially thank j.i. montoya, j. díaz, g. martínez, a. sanz, f. lópez and a. ruiz for friendship and help in the islands. legal authorization for the study was provided by the spanish national parks authority and the experimental procedures were supervised by the bioethical committee of the spanish national research council (csic). the implanting procedure was carried out by researchers holding the adequate animal experimentation accreditation. financial support was provided by the project pgc2018-093592-bi00 (mciu/aei/feder, ue) of the spanish ministerio de ciencia, innovación y universidades. references baeckens, s., garcía-roa, r., martín, j., ortega, j., huyghe, k., van damme, r. 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(2001): biodiversity of soil animals and its function. eur. j. soil biol. 37: 221-227. acta herpetologica vol. 14, n. 2 december 2019 firenze university press podarcis siculus latastei (bedriaga, 1879) of the western pontine islands (italy) raised to the species rank, and a brief taxonomic overview of podarcis lizards gabriele senczuk1,2,*, riccardo castiglia2, wolfgang böhme3, claudia corti1 substrate type has a limited impact on the sprint performance of a mediterranean lizard pantelis savvides1,*, eleni georgiou1, panayiotis pafilis2,3, spyros sfenthourakis1 coping with aliens: how a native gecko manages to persist on mediterranean islands despite the black rat? michel-jean delaugerre1,*, roberto sacchi2, marta biaggini3, pietro lo cascio4, ridha ouni5, claudia corti 3 pit-tags as a technique for marking fossorial reptiles: insights from a long-term field study of the amphisbaenian trogonophis wiegmanni pablo recio, gonzalo rodríguez-ruiz, jesús ortega, josé martín* occurrence of batrachochytrium dendrobatidis in the tensift region, with comments on its spreading in morocco ait el cadi redouane1, laghzaoui el-mustapha1, angelica crottini2, slimani tahar1, bosch jaime3,4, el mouden el hassan1,* hematological parameters of the bolson tortoise gopherus flavomarginatus in mexico cristina garcía-de la peña1,*, roger iván rodríguez-vivas2, jorge a. zegbe-domínguez3, luis manuel valenzuela-núñez1, césar a. meza herrera4, quetzaly siller-rodríguez1, verónica ávila-rodríguez1 ontogenetic and interspecific variation in skull morphology of two closely related species of toad, bufo bufo and b. spinosus (anura: bufonidae) giovanni sanna visible implant alphanumeric (via) as a marking method in the lesser snouted treefrog scinax nasicus andrea caballero-gini1,2,3,*, diego bueno villafañe2,3, lía romero2, marcela ferreira2,3, lucas cañete4, rafaela laino2, karim musalem2,5 morphological variation of the newly confirmed population of the javelin sand boa, eryx jaculus (linnaeus, 1758) (serpentes, erycidae) in sicily, italy francesco p. faraone1,*, salvatore russotto2, salvatore a. barra3, roberto chiara3, gabriele giacalone4, mario lo valvo3 variability in the dorsal pattern of the sardinian grass snake (natrix natrix cetti) with notes on its ecology enrico lunghi1,2,3,4,*, simone giachello5, manuela mulargia6, pier paolo dore7, roberto cogoni8, claudia corti1 estimating abundance of the stripeless tree-frog hyla meridionalis by means of replicated call counts federico crovetto, sebastiano salvidio, andrea costa* at-rich microsatellite loci development for fejervarya multistriata by illumina hiseq sequencing yan-mei wang, jing-yi chen, guo-hua ding*, zhi-hua lin issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-20890 acta herpetologica 12(1): 123, 2017 errata to mecke, s., hartmann, l., mader, f., kieckbusch, m., kaiser, h. (2016): redescription of cyrtodactylus fumosus (müller, 1895) (reptilia: squamata: gekkonidae), with a revised identification key to the bent-toed geckos of sulawesi. acta herpetologica 11(2): 151-160. in acta herpetologica 11(2), mecke et al. (2016) redescribed cyrtodactylus fumosus (müller, 1895) (reptilia: squamata: gekkonidae) and provided an identification key to the bent-toed geckos of sulawesi. after the publication of this article it came to our attention that some aspects of this paper have to be corrected as follows: • page 152, material and methods section, left column, lines 5 and 6: “…we recorded data for 31 eidonomic characters…” should read “…we recorded data for 30 eidonomic characters…” • page 153, table 2, definition of “precloacal pores.” in this definition we mention that we counted the number of precloacal pores situated in the precloacal groove. however, precloacal pores may not be situated in a groove. non-adult males of cyrtodactylus fumosus do possess pores, but their groove might not be fully developed (see p. mecke et al. 2016: 156) • page 156, right column, paragraph 2. the author “mumpini” is correctly spelled “mumpuni” (see also page 160, left column, first reference). • page 156, right column, paragraph 3: “…were collected at elevations 1200-1260 m…” should read “…were collected at elevations of 1200-1260 m…” • page 158, key to the species of the genus cyrtodactylus of sulawesi. as stated in our comparisons (page 153-155), the species cyrtodactylus batik, c. hitchi, c. jellesmae, and c. wallacei possess tubercles on the lateral fold. an error regarding this characteristic in the named species occurs in choice 2b of our key: “2b enlarged precloacofemoral scales; pores; precloacal groove; and tubercles on lateral fold absent.......3” should read: “2b enlarged precloacofemoral scales, pores, and precloacal groove absent; tubercles on lateral fold present...............................................................................3” acknowledgemts we thank awal riyanto (mzb), who pointed out to us the error in the identification key. acta herpetologica vol. 12, n. 1 june 2017 firenze university press morphological variation and sexual dimorphism in liolaemus wiegmannii (duméril & bibron, 1837) (squamata: liolaemidae) from uruguay joaquín villamil1,*, arley camargo2, raúl maneyro1 sex does not affect tail autotomy in lacertid lizards panayiotis pafilis1,*, kostas sagonas2, grigoris kapsalas1, johannes foufopoulos3, efstratios d. valakos4 fire salamander (salamandra salamandra) males’ activity during breeding season: effects of microhabitat features and body size raoul manenti*, andrea conti, roberta pennati call variation and vocalizations of the stealthy litter frog ischnocnema abdita (anura: brachycephalidae) pedro carvalho rocha1,2,*, joão victor a. lacerda2,3, rafael félix de magalhães2,3, clarissa canedo4, bruno v. s. pimenta5, rodrigo carrara heitor6, paulo christiano de anchieta garcia2,3 feeding ecology of two sympatric geckos in an urban area of northeastern brazil josé guilherme g. sousa1, adonias a. martins teixeira2,*, diêgo alves teles2, joão antônio araújo-filho2 and robson waldemar ávila3 a pattern-based tool for long-term, large-sample capture-mark-recapture studies of fire salamanders salamandra species (amphibia: urodela: salamandridae) jeroen speybroeck1,*, koen steenhoudt2,$ skeletal variation within the darwinii group of liolaemus (iguania: liolaemidae): new characters, identification of polymorphisms and new synapomorphies for subclades linda díaz-fernández*, andrés s. quinteros, fernando lobo marking techniques in the marbled newt (triturus marmoratus): pit-tag and tracking device implant protocols hugo le chevalier1, olivier calvez2, albert martínez-silvestre3, damien picard4, sandra guérin4,5, francis isselin-nondedeu6, alexandre ribéron1, audrey trochet1,2,* evidence for directional testes asymmetry in hyla gongshanensis jindongensis qing gui wu1, wen bo liao2,* the advertisement call of pristimantis subsigillatus (anura, craugastoridae) florina stănescu1,4, rafael márquez2, paul székely3,4,*, dan cogălniceanu1,4,5 nematodes infecting anotosaura vanzolinia (squamata: gymnophthalmidae) from caatinga, northeastern brazil bruno halluan s. oliveira¹, adonias a. martins teixeira¹,*, romilda narciza m. queiroz¹, joão a. araujo-filho¹, diêgo a. teles¹, samuel v. brito2, daniel o. mesquita¹ where is my place? quick chorus structure assembly in the european tree frog michal berec a possible mutualistic interaction between vertebrates: frogs use water buffaloes as a foraging place piotr zduniak1,*, kiraz erciyas-yavuz2, piotr tryjanowski3 good vibrations: a novel method for sexing turtles donald t. mcknight1,2,*, hunter j. howell3, ethan c. hollender1, and day b. ligon1 errata to mecke, s., hartmann, l., mader, f., kieckbusch, m., kaiser, h. (2016): redescription of cyrtodactylus fumosus (müller, 1895) (reptilia: squamata: gekkonidae), with a revised identification key to the bent-toed geckos of sulawesi. acta herpetologica 11(2): acta herpetologica 13(2): 109-115, 2018 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-22876 population ecology and home range of the mexican rough-footed mud turtle (kinosternon hirtipes murrayi) in central mexico ivette enríquez-mercado1, alejandro montiel-ugalde2, ángeles aparicio1, eder gaona murillo3, taggert butterfield2, rodrigo macip-ríos2,4,* 1 facultad de ciencias biológicas, benemérita universidad autónoma de puebla, 72530 puebla, méxico 2 escuela nacional de estudios superiores unidad morelia, universidad nacional autónoma de méxico, antigua carretera a pátzcuaro 8701, ex. hacienda de san josé la huerta, 58190 morelia, méxico. *corresponding author. e-mail: rmacip@enesmorelia.unam.mx 3 facultad de biología, universidad michoacana de san nicolás de hidalgo, 58040 morelia, méxico 4 laboratorio nacional de síntesis ecológica y conservación genética, universidad nacional autónoma de méxico, 58190 morelia, méxico submitted on: 2018, 11th march; revised on: 2018, 26th june; accepted on: 2018, 27th june editor: uwe fritz abstract. population ecology and demographic data are fundamental for species management and conservation planning. for mexican kinosternid turtles there is a need for basic natural history and population ecology data. the rough-footed mud turtle (kinosternon hirtipes murrayi) is one of the lesser-studied species, even though it is broadly distributed, occurring from western texas to central mexico. we conducted a study on the species in michoacán, mexico for two years. basic population parameters were estimated, and telemetry was used to measure home range size and movements of males and females. population size in a 1.42-hectare wetland was calculated to be 301 (± se 5.89) individuals, mainly adults. the adult sex ratio was skewed toward males (3.1:1). female home range size was larger than that of males, and males moved larger distances between relocation events. the radio-tracked individuals did not leave the water during winter months and during the dry season. habitat degradation due to eutrophication may be affecting population survivorship and recruitment. keywords. mexican rough-footed mud turtle, kinosternon hirtipes murrayi, population ecology, home range, méxico. introduction knowledge of demographic characteristics, home range size, and movement patterns are important for designing conservation and management strategies for species (gibbs and amato, 2000; primack, 2012). demographic characteristics include: population size, abundance, sex ratio, population structure, survivorship, and the contribution of these parameters to populations dynamics through time (caswell, 2001). to measure any of these population characteristics accurately requires long-term data or a high rate of recaptures to estimate these parameters accurately (lemos-espinal et al., 2005; molina-zuluaga et al., 2013). however, because turtles are long-lived organisms, collection of demographic data presents a challenge because their lifespan can reach several decades (crouse et al., 1987; edmonds and brooks, 1996; enneson and litzgus, 2008) and recaptures can be sparse (chao, 1989). on the other hand, measuring home range size and movement patterns requires more detailed studies where individuals are followed through space and time (hays, 1992; godley et al., 2002; pérez-pérez et al., 2017). typical home range and movement studies are conducted using radiotelemetry so that individuals can be located repeatedly (cochran, 1980; singer and blakenhol, 2015). when long term studies are coupled with radiotelemetry, clearer patterns of habitat use, migration, resource use, and seasonal patterns like aestivation can 110 ivette enríquez-mercado et alii be detected. despite the importance of long-term and detailed studies of turtle populations, most of the information that exists is on species within the united states (iverson, 1991; rouane et al., 2008; enneson and litzgus, 2008; lovich and ennen, 2013). outside of the us, long-term and detailed studies with turtles have been largely neglected. for example, mexico has the second most diverse turtle fauna in the world (rhodin et al., 2017), yet long-term mark-recapture and radiotelemetry studies are few to non-existent (legler and vogt, 2013). only recently, biologists have started generating this kind of data on mexican turtle species (macipríos et al., 2009; 2011; vázquez-gómez et al., 2016; pérezpérez et al., 2017). mud turtles (kinosternidae) have been a particular focus, as they are the most diverse turtle lineage in mexico (legler and vogt, 2013). one of those species, the mexican rough-footed mud turtle (kinosternon hirtipes) is broadly distributed from the big bend region in a few localities in western texas (platt and medlock, 2015) to central michoacán in the mexican transvolcanic belt (iverson, 1992). throughout this range, five k. hirtipes subspecies are recognized: k. h. hirtipes in the valley of mexico; k. h. megacephalum (extinct) in viesca, coahuila; k. h. magdalense in the magdalena river basin, michoacán; k. h. chapalense in chapala lake and zapotlán lake, jalisco; k. h. tarascense in patzcuaro lake, michoacán; and k. h. murrayi, from the big bend region of texas to the highlands of michoacán (iverson, 1981). despite their wide distribution in mexico, most ecological information on k. hirtipes is from iverson (1981; 1985), who described sexual size dimorphism, morphological differences, and basic distributional patterns among the subspecies. some information also exists on k. h. murrayi. for example, iverson et al. (1991) described the growth and reproduction of this subspecies in chihuahua, platt et al. (2016a) described their diet, platt et al. (2016b) also described the reproductive ecology in their northern distribution limit in texas, platt and medlock (2015) studied aestivation behavior, and smith et al. (2015; 2018) reported a new body size record and nesting behavior. in general, these studies demonstrate that k. h. murrayi exhibits more morphological variation than other subspecies, has wide variation in body size, and is sexually dimorphic, with males typically being larger than females (glass and hartweg, 1951; iverson, 1985). our aim was to generate additional ecological information on k. h. murrayi using capture-mark-recapture methods and radiotelemetry in a wetland near morelia, michoacán, méxico. our specific objectives were to describe basic population ecology parameters, home range, and movement patterns of k. h. murrayi in a human-modified landscape. materials and methods study site this study was carried out near morelia, michoacán, méxico in irrigation canals that come from a wetland called “la mintzita” (13°38’n, 101°16’e). “la mintzita” is a natural spring that is associated with a 57-hectare (ha) wetland recognized by ramsar (the ramsar convention of wetlands, 2014). this wetland has been managed by humans since pre-columbian time and still maintains significant biological diversity including the presence of several species of fishes (some of them endemic like zoogeneticus quitzeoensis, skiffia lermae, yuriria alta, among others), waterfowl, amphibians, reptiles, and various mammals (marin-togo and blanco-garcía, 2012). the two main irrigation canals at “la mintzita” are fed by the most northern part of the wetland. these canals are used by local people to irrigate corn fields and manage runoff during the wet season when water levels are high. the canals have a low current and flow northeast for 2 km before joining a tributary of the cuitzeo lake basin that is also known as the “rio grande” of morelia. compared to other tributaries of the “rio grande”, the irrigation canals studied here are less polluted and degraded. the total area sampled has a coverage area of 14266 m2. trapping protocol this study was conducted during two wet seasons, from may-december 2016 and june-november 2017. we placed traps for one night, at least two times per month. we used two fyke nets and 9-12 minnow traps (promar, garden, ca.). traps were baited with fresh fish and placed in the irrigation canals from approximately 17:00 h to 10:00 h the following day. for each trapping session, traps were placed in the same place in 2016, but had to be changed in 2017 due to an unusually high level of water at the beginning of the season and a dramatic increase in invasive aquatic plants, elodea sp. and eichhornia crassipes. our sampling effort for the first season (may-december 2016) was 2142 trap hours using 12 minnow traps plus two fyke nets, and 1836 trap hours in the second sampling season (june-november 2017) using 9 minnow traps plus two fyke nets. turtle measuring protocol all captured turtles were marked using the shell-notch code system from cagle (1939), then measured. we measured body mass (bm) to the nearest gram using a spring scale (1 g). morphological characters, including straight-line carapace length (cl), straight-line plastron length (pl), carapace width (cw), and carapace height (ch) were measured to the nearest 0.01 mm using dial calipers. males were identified by using secondary sexual characteristics: long and bulky tail, a developed spine at the end of the tail, a prominent notch in the hind lobe of the plastron, and had a concave plastron (iverson, 1999). females were identified by their size and absence of the characteristics used to iden111population ecology and home range of k. hirtipes tify males. females had a plastron that covered all body parts. according to iverson et al. (1991), female k. hirtipes are sexually mature at 95-100 mm cl and males of this size are hard to differentiate from females. age classes were divided into four categories for our population structure analysis: hatchlings/yearlings (less than 50 mm cl), immature (50-90 mm cl), adults (95-140 mm cl), and asymptotic adults or old adults (larger than 140 mm cl). captured adult females were brought to the laboratory for take x-ray photographs to determine reproductive status. females stayed two or three days in the laboratory and were then returned to the field. radio telemetry protocol we equipped 11 turtles (six females in 2016 and five males in 2017) with radio transmitters (models: txe-315g telenax, ciudad del carmen, quintana roo; pr 99 wildlife materials, murphysboro, il, and r1900 series from advanced telemetry systems, isanti, mn) that weighed less than 30 g. a yagi antenna and two different receivers (telenax r1000 and advanced telemetry systems r2000) were used to locate individuals. turtle relocations were recorded to the nearest 3 m with a handheld gps (garmin etrex 10). individuals were relocated at least twice per month during the study period. statistical analyses due to the low recapture rate in our data set, population size was estimated with a heterogeneity estimator (mh) following chao (1989). this analysis was done with the care1 package in r (chao and yang, 2003). a chi-squared test was used to test if the sex ratio was significantly different from 1:1 (zar, 1999). differences in body size and other morphological measurements between males and females were tested with a student’s t-test. parametric assumptions for normality and homogeneity were tested using shapiro-wilks and bartlett tests (zar, 1999). statistical analyses were run in jmp v5.0.1 (sas institute, 2002). locations were originally collected in decimal degrees, then transformed to the utm coordinate system using earth point (clark, 2018) for subsequent analysis. home range size was estimated using two methods, the minimum convex polygon (mcp) and kernel density (kd). fifty percent kernel density estimates were calculated to remove the influence of outliers and a smoothing parameter (h) was calculated using least squares cross validation (lscv). home range sizes were calculated in the adehabitat package in r (calenge, 2006; r development core team, 2008). total distances moved between relocations and estimated daily movements were calculated by hand using the location-sequenced utm coordinates. we did not compare male and female home range sizes because of unequal sample sizes. for all analyses, we used α = 0.05. results a total of 96 k. h. murrayi turtles were marked during both sampling seasons and 18 were recaptured. eighty-eight of these turtles were captured only once, nine were captured twice, two were captured three times, and two were captured four times. estimated population size using the mh model was 301 (± se 5.89) individuals (lower confidence interval (ci) = 297.7 – upper ci = 305.1). based on the area sampled (surface water coverage), turtle density was estimated as 211 turtles/ha. the 96 turtles captured included 69 males, 22 females, three immature juveniles, and two hatchlings/yearlings. the adult sex ratio was significantly biased toward males 3.1:1 (χ2 = 24, p < 0.0001). average straight-line carapace length, (± sd), for males was 118.1 (13.91) mm and 121.31 (15.37) mm for females, but they were not significantly different (t89 = 0.87, p = 0.38). females (49.60 ± 6.78 mm) had greater ch (43.12 ± 4.9 mm; t89 = 4.14, p < 0.0001), longer pl (females = 110.92 ± 15.13 mm; males = 97.52 ± 8.33, t89 = 3.96, fig. 1. population structure of the kinosternon hirtipes murrayi population at “la mintzita” wetland. grey bars are immature individuals, including hatchlings and immatures. table 1. home range area for kinosternon hirtipes males and females calculated by mcp and 50% kernel estimates. home ranges are given in hectares. turtle id locations per individual sex mcp home range 50% kernel home range 5 24 female 0.14 0.02 7 21 female 0.28 0.08 9 19 female 18.91 8.50 15 25 female 0.33 0.05 16 23 female 2.33 3.18 17 22 female 0.90 0.34 82 6 male 0.02 0.60 92 5 male 0.00 0.56 93 5 male 0.02 0.28 94 5 male 0.001 0.92 95 5 male 0.07 1.40 112 ivette enríquez-mercado et alii p = 0.0005), and greater bm than males (females = 313 ± 113.80 g; males = 237 ± 63.49 g; t89 = 2.99, p = 0.006). ten females were brought to the lab to take radiographs. only two females have eggs on oviduct. one with five (cl = 117.6 mm) and the other with six eggs (cl = 136 mm). both females were collected during september 2016. egg were measured on the radiographs. eggs length averaged 27.18 mm in length and 15.25 in width in the five-eggs clutch, and egg length averaged 28.32 mm in length and 16.50 mm in width in the six-egg clutch. relocations ranged from 25 to 19 for females and from 6 to 6 for males. combined relocations mean was 15.54 (± sd 9.08). female home range size varied from 0.14 – 18.91 ha based on the mcp estimates and 0.02 – 8.53 hectares for kd estimation (table 1). mean home range size, (± sd), for females was 3.81 (7.43) ha for mcp method and 2.02 (3.39) ha for kd. home range size for males varied from near 0 – 0.072 ha with the mcp method and 0.28 – 1.40 ha with the kd method (table 1). average home range size, (± sd), for males was 0.024 (0.028) ha with the mcp method and 0.75 (0.42) ha when estimated with kd. average distances moved, (± sd), between relocations by females was 57.76 (123.27) m and 73.83 (109.82) m for males (table 1). average estimated daily movements, (± sd), for females were 6.20 (13.09) m per day and 4.89 (7.38) m per day for males. discussion estimated population size was similar to other population estimates for kinosternids such as k. oaxacae (vazquez-gómez et al., 2017), k. integrum (macip-ríos et al., 2009), and k. sonoriense (hulse, 1982), although areas sampled varied. compared with the abundance (detectability) of data for the same subspecies presented by iverson et al. (1991) for a population in chihuahua (604 captures, but no population size information), and platt et al. (2016b) (87 marked turtles and 2.4:1 sex ratio), our data on sex ratios are generally the same. male-biased sex ratios have also been reported before in other kinosternids such as sternotherus odoratus (smith and iverson, 2002), k. sonoriense (stone et al., 2015), k. leucostomum (ceballos et al., 2016), and other populations of k. hirtipes such as those in texas (platt et al., 2016b). according to gibbons and lovich (1990) a skewed sex ratio could be caused because one sex reaches sexual maturity earlier (generally are smaller in size), which could affect population structure. the population structure observed in our study (many adults and very few hatchlings and immature individuals) also agrees with data reported in other kinosternid studies (frazer, 1991; iverson, 1991; van loben sels et al., 1997; macip-ríos et al., 2011). this may be an artifact of trapping techniques because it is hypothesized that hatchlings and yearlings have a low catchability rate, or the mesh of traps may allow them to escape (ceballos et al., 2016). however, macip-ríos et al. (2018) did capture a large proportion of hatchlings and immatures with the same traps and bait in a kinosternon creaseri population in the yucatan peninsula. also, vázquez-gómez et al. (2016) found hatchlings using the same trapping protocol in a k. oaxacae population. thus, our results may represent a close approximation to the true population structure. there are several reasons that could explain biased sex ratios and the absence of hatchlings and yearlings in population structure. according to smith and iverson, (2002), differential mortality among the sexes could influence sex ratios, while climate change and habitat degradation could change incubation temperatures that also affect sex ratio (eisenberg et al., 2017). moreover, different habitat usage could also affect these results. nevertheless, as we mentioned before, we used the same trapping protocol as in previous studies (macip-ríos et al., 2011; vázquez-gómez et al. 2016; macip-ríos et al., 2018) where we were able to capture hatchling and yearling individuals. because of this, we presume that the low capture rate of hatchling and yearlings could be attributed to low recruitment, which could be related to the malebiased sex ratio. the overall recapture rate was very low at 19%. this could indicate two things; turtles move extensively in the study area, or turtle catchability is affected by the trapping protocol. even though this trapping protocol has been successful in other mud turtle populations, the “la mintzita” population could have been affected by the dramatic changes in water levels and abundance of invasive plants. because of these changes to the habitat, we were unable to set up the traps in one of the irrigation canals during the last sample season because the thick aquatic vegetation prevented traps from sinking and there were very few places with “open water” to place the traps. there is evidence that turtles inhabit eutrophic habitats (iverson, 1999; germano, 2010); however, when we forced our traps into water full of elodea sp. and eichhornia crassipes no turtles were captured. contrary to a general pattern among kinosternids, where males are generally larger than females (ceballos and iverson, 2014), in the “la mintzita” population females appear to be larger than males. for k. hirtipes, similar sexual size dimorphism results have been reported for other populations (carmen and verde basins) and 113population ecology and home range of k. hirtipes subspecies such k. h. tarascense, k. h. magdalense, k. h. megacephalum, and k. h. chapalense (iverson, 1985). furthermore, our largest male (cl = 147.5 mm) was smaller than the 195 mm cl male reported by smith et al. (2015). our largest female was 177 mm in cl, which is smaller than record sizes previously reported (ernst and lovich, 2009). our result contrasts with data from other studies of k. hirtipes in presidio, texas (platt et al., 2016b) and the santa maria river in chihuahua (iverson et al., 1991), where males were also larger than females in cl. our data are comparable to those presented by iverson et al. (1991) who reported that females have a larger plastron than males. apparently, variation in sexual size dimorphism is common in k. hirtipes, which contrasts with other kinosternids like k. subrubrum, k. bauri (lovich and lamb, 1995) k. integrum (macip-ríos et al., 2009), k. scorpioides (forero-medina et al., 2007), and k. sonoriense (stone et al., 2015). body size differences between males and females have been interpreted as a result of sexual selection interacting with natural selection (wilbur and morin, 1988; gibbons and lovich, 1990). these differences could also be driven by minimum size required for mating and reproduction (iverson, 1985), differential survivorship, and habitat selection, which could affect growth patterns (huey, 1982). according to iverson et al. (1991), k. hirtipes males grow faster and larger than females during their first five years; during this time, males start showing secondary sexual characteristics, while females start showing their secondary sexual characteristics at from 6-8 years old. home range results indicated that k. hirtipes did not show signs of aestivation, as previously described (iverson, 1981). “la mintzita” turtles exclusively lived in aquatic habitats, with the exception of two individuals that moved to other irrigation channel at 700 m straightline distance. male and female home ranges slightly overlap, and even though home range sizes are not comparable due to unequal sample sizes, females seem to have a larger home range. larger home range size in females could be explained by the need to find suitable nesting sites (pérez-pérez et al., 2017). however, the habitat degradation previously noted could force females to move more in the landscape to find suitable food resources and nesting sites. we acknowledge that our relocations could be insufficient since home range and movement standard deviations are close or large than mean values; however, our data still provide an estimate of overall home range of this population for an understudied turtle, but more research is needed to determine differences in habitat use and movement habits between males and females. during our two-year observations in the “la mintzita” habitat, we identified habitat degradation by water reduction and saturation of elodea sp. and eichhornia crassipes, that could contribute to decline turtle abundance. females in this population are still breeding, as evidenced by the observation of eggs in x-radiographs taken from females collected in both sampling seasons. however, due to the skewed male sex ratio, the reproductive potential of the population may be limited (le galliard et al., 2005). this is a conservation concern since this k. h. murrayi population represents one of the southernmost locations of this subspecies. acknowledgments this study was conducted with permission from semarnat (permission number faut:0304). we want to thank the dgapa-papiit program for their support through grant ia200216. sergio nicasio, chesed alvarado, diana martínez, frida castillo, nora vieyra, and anahid sánchez helped with fieldwork. american turtle observatory partially funded the project with radio transmitters. mike t. jones helped to start the project by providing advice with telemetry techniques. two anonymous reviewers made important comments to improve early versions of this manuscript. references cagle, f.r. 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(1999): biostatistical analysis. prentice hall, new jersey. acta herpetologica vol. 13, n. 1 june 2018 firenze university press acta herpetologica 11(2): 127-133, 2016 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-18117 thermal ecology of podarcis siculus (rafinesque-schmalz, 1810) in menorca (balearic islands, spain) zaida ortega*, abraham mencía, valentín pérez-mellado department of animal biology, university of salamanca, campus miguel de unamuno, 37007, salamanca, spain .*corresponding author. e-mail: zaidaortega@usal.es submitted on 2016, 15th march; revised on 2016, 20th july; accepted on 2016, 25th july editor: sebastiano salvidio abstract. we studied the thermal ecology of an introduced population of the italian wall lizard, podarcis siculus, in menorca (balearic islands, spain). we measured field body temperatures of adult lizards, as well as air and substrate temperatures at their capture places, during spring and summer. we assessed the relations between body and air temperatures, and between body and substrate temperatures, for both seasons. we studied the preferred temperature range of p. siculus in a laboratory thermal gradient. in addition, we recorded the operative temperatures of the habitat of the italian wall lizard during summer. then, we calculated the three indexes of behavioural thermoregulation for summer: thermal quality of the habitat, accuracy of thermoregulation, and effectiveness of thermoregulation. as expected, our results show that italian wall lizards achieved significantly higher body temperatures during summer than during spring. body temperatures were not significantly related to air temperatures in spring, but the correlation was significant in summer. in addition, body temperatures were not significantly related to substrate temperatures for any season. the preferred temperature range of the species was similar for males and females: 28.40-31.57 °c. introduced italian wall lizards of menorca are effective thermoregulators, with an effectiveness of 0.82 during summer. keywords. thermal biology, behavioural thermoregulation, temperature, heliothermy, lacertidae, italian wall lizard, podarcis siculus. introduction thermal ecology is a central point in the biology of squamate vertebrates. their ability to exploit any resource is closely related to an effective control of their body temperature (cowles and bogert, 1974; huey, 1974; adolph and porter, 1993). themal ecology would cover two important traits: thermal sensitivity and thermoregulation (angilletta, 2009). thermal sensitivity is the dependence of physiological performance on temperature, which ranges from thermal specialists to generalists (angilletta et al., 2002; angilletta, 2009). thermoregulation is the capacity to regulate body temperatures, which ranges from thermoconformers, whose body temperatures would totally depend on ambient temperatures, to perfect thermoregulators, whose body temperatures would be constant, regardless of ambient temperatures (huey, 1974; hertz et al., 1993; sears and angilletta, 2015). lizards mainly use three mechanisms to regulate their body temperature: adjusting activity periods (hertz, 1992; adolph and porter, 1993), shuttling between different microhabitats (heath, 1970; bauwens et al., 1996), and adjusting their body posture (bauwens et al., 1996). the combination of these strategies depends on the balance between costs and benefits, which in turn depends on different biotic and abiotic factors (huey and slatkin, 1976; sears and angilletta, 2015). within lizards, lacertids generally are effective thermoregulators and mostly 128 zaida ortega et alii heliothermic, which use to move between sunny and shade microhabitats for thermoregulation (avery, 1976; van damme et al., 1990; castilla et al., 1999; ortega et al., 2016a). our aim is to study the thermal ecology of an introduced population of the italian wall lizard, podarcis siculus, in menorca (balearic islands, spain). we measured body temperatures of active lizards, as well as air and substrate temperatures of the microhabitats occupied by lizards. in order to search for seasonal effects in the thermoregulation, we compared these measures for spring and summer. we hypothesized that lizards would achieve higher body temperatures in summer than in spring, as it is usual in lacertid lizards (e.g. díaz and cabezas-díaz, 2004; ortega et al., 2014). we also measured the thermal preferences of lizards in a thermal gradient. in addition, we recorded the operative temperatures of the habitat during summer. finally, we studied the thermal quality of the habitat, the accuracy of thermoregulation, and the effectiveness of thermoregulation (hertz et al., 1993) of the italian wall lizard during summer. material and methods study species and area the italian wall lizard podarcis siculus (rafinesqueschmalz, 1810) is a robust ground-dwelling lacertid lizard. the original distribution covers italy (continental italy, sardinia, sicily and several coastal islets), corsica (france) and the east coast of the adriatic sea, from slovenia to montenegro (henle and klaver, 1986). however, p. siculus has been introduced in many mediterranean countries and in the united states (corti et al., 2004). here we studied the population of menorca (balearic islands, spain), which inhabits all kinds of habitats, from coastal dunes to forests and anthropogenic walls (pérezmellado, 1998; pérez-mellado, 2002), and would be introduced from sicily and/or sardinia (silva-rocha et al., 2012). the italian wall lizard is a heliothermic lizard, which previously reported mean temperatures range between 29 °c in spring and approximately 33 °c in summer (avery, 1978; van damme et al., 1990; foà et al., 1992; tosini et al., 1992). we studied the population of es canutells, in southern menorca (spain), an almost undisturbed mediterranean habitat of mixed woodland and scrubland (patches of pines and holm oaks, and patches of large shrubs, mainly pistacia lentiscus), spotted with large rocks. the studied population exhibited a clear sexual size dimorphism, with larger (mean svl males: 73.35 ± 1.22 mm, n = 20; mean svl females: 64.97 ± 1.00 mm, n = 9; one-way anova, f1, 27 = 18.417, p < 0.0001) and heavier (mean weight males: 10.28 ± 0.41 g, n = 20; mean weight females: 7.09 ± 0.41 g, n = 9; one-way anova, f1, 27 = 22.061, p < 0.0001) males. field sampling we recorded field temperatures of podarcis siculus between 27 may and 30 july 2013, in 12 sunny days of fieldwork (7 in spring and 5 in summer). we considered the natural seasons: the data obtained before the 21st of june have been considered as spring data, and those obtained after that date as summer data. we captured active adult lizards by noosing, during their daily activity period, from 07:00 to 17:00 h (gmt), 16 in spring (11 males and 5 females) and 15 in summer (11 males and 4 females). immediately after capture (within 30 s), we measured cloacal body temperature (tb) with a testo® 925 digital thermometer, shadowing the probe, as well as air temperature (ta) 1 cm above the capture point, and substrate temperature (ts) of the capture point. we also recorded the type of substrate, the height of the perch (in cm), and the sunlight situation (full sun, filtered sun, or full shade). finally, we measured wind speed with a kestrel® 3000 anemometer, but during field work, its variation was almost insignificant (a mean of 0.15 ms-1). so, for this study, we discarded the wind as a possible variable affecting thermal behaviour of lizards. as a null hypothesis for thermoregulation, we recorded operative temperatures (te). we recorded te during the same days of the field sampling of summer (between 16 july 2013 and 30 july 2013) in the same area of study (es canutells), in order to control for potential variations in weather conditions. we used copper models as null te models (bakken and angilletta, 2014). these models achieve similar temperatures to those of non-thermoregulating lizards. we placed one thermocouple probe into each hollow model and connected it to a data logger hobo® h8 (onset computer corporation), programmed to take a temperature record every five minutes. we randomly placed the copper models in different microhabitats and used the te hourly mean of each microhabitat for analysis, since raw te data could be autocorrelated. based in observations of the behaviour of lizards, we selected four types of microhabitats: rock, soil, grass, and logs of pistacia lentiscus; each of them was considered in the three sunlight situations (see above). preferred temperature range (ptr) we measured selected body temperatures (tsel) of p. siculus between 12 june 2013 and 14 june 2013 in a laboratory thermal gradient. we captured lizards from the same location of field sampling and immediately transported them to the laboratory in es castell (menorca, spain). there, we housed lizards on individual terraria and fed them with mealworms and crickets. water was provided ad libitum during the length of the experiment. we built the thermal gradient in a glass terrarium (100 x 60 x 60 cm) with a 150 w infrared lamp over one of the sides, obtaining a gradient between 20 to 60 ºc. then, we measured the selected temperature of a lizard each hour from 08:00 to 17:00 h (gmt) with a digital thermometer. we used 24 p. siculus adult lizards, 14 males and 10 females. we considered the 50% of the central values of selected body temperatures as the preferred temperatures range (ptr) in all analyses, as it is the more common procedure, although we also report the 80% 129thermoregulation of podarcis siculus in menorca ptr, since some authors employ this range (hertz et al., 1993; blouin-demers and nadeau, 2005). we released lizards at their capture places immediately after the experiment. data analysis to test the null hypothesis of thermoregulation, that is, if lizards use microhabitats randomly regarding temperature, we followed the protocol developed by hertz et al. (1993), and calculated their three indexes of thermoregulation. the first is the index of accuracy of thermoregulation (db − ), that is the mean of absolute values of the deviations between each tb from the preferred temperature range. thus, the values of the index of accuracy of thermoregulation are counterintuitive: higher values of db − indicate lower accuracy of thermoregulation, and vice-versa. the second is the index of thermal quality of habitat (de − ), calculated as the mean of absolute values of the deviations of each te from the preferred temperature range. accordingly, the values of the index of thermal quality of the habitat are also counterintuitive: higher values of de − indicate a lower thermal quality of the habitat, and vice-versa. the third is the index of effectiveness of thermoregulation (e), that is calculated as ε = 1 (db − / de − ). hence, values of e range from 0 to 1, meaning the higher effectiveness of thermoregulation the higher the value of e (see hertz et al., 1993). effectiveness of thermoregulation was calculated with thermo, a minitab module written by richard brown. thermo uses three kinds of input data: tb, te and tsel of the preferred temperature range, and was programed to perform bootstraps of 100 iterations, building pseudo-distributions of three kinds of output values: the arithmetic mean of the index of accuracy of thermoregulation (db − ), the arithmetic mean of the index of thermal quality of the habitat (de − ), and the arithmetic mean of the index of effectiveness of thermoregulation (e). as we measured te in summer, we only computed this protocol of study for the body temperatures of summer. we performed parametric statistics when data followed the assumptions of normality and variance homogeneity. when data did not fulfill these assumptions, even after log-transformations, we carried out non-parametric equivalent tests (sokal and rohlf, 1995; crawley, 2012). we conducted all analyses on r, version 3.1.3 (r core team, 2015), and we computed posthoc comparisons of kruskal-wallis tests with nemenyi test with the package pmcmr (pohlert, 2014). we reported mean values of variables accompanied by standard errors. significance level was α = 0.05. results selected body temperatures (tsel) were similar regarding sex (mean tsel of males: 29.84 ± 0.41 °c, n = 14; mean tsel of females: 30.15 ± 0.31 °c, n = 10; one-way anova, f1, 22 = 0.301, p = 0.589). thus, we pooled them in subsequent analyses, and considered a preferred temperature range (ptr) for this population. the 50% ptr is 28.40 31.57 °c, and the 80% ptr is 26.85-32.54 °c. body temperatures (tb) were also similar regarding sex (mean tb of males = 30.99 ± 0.53 °c, n = 22; mean tb of females = 30.23 ± 1.09 °c, n = 9; one-way anova, f1, 29 = 0.494, p = 0.488). thus, also in this case, we pooled data from males and females for subsequent analyses. body temperatures (tb) of lizards (one-way anova, f1, 29 = 7.996, p = 0.008), as well as air temperatures (oneway anova, f1, 29 = 18,704, p < 0.0001) and substrate temperatures (ts; one-way anova, f1, 29 = 8.244, p = 0.008) were significantly higher in summer than in spring (table 1). although sample size for subsets of each sex within each season is small, we checked for potential differences in tb between sexes, in order to confirm if males and females should be pooled together within each season. results show similar tb of males and females both in spring (one-way anova, f1, 15 = 0.136, p = 0.718) and in summer (one-way anova, f1, 14 = 0.267, p = 0.614). an ancova test reveals that the linear relation between tb and ta significantly changed between spring and summer (ta as a covariate; interaction season*ta: f1, 27 = 5.590, p = 0.026). thus, linear regressions must be studied separately regarding season. correlation between tb and ta was not significant in spring (r = 0.209, p = 0.438, n = 16), but was significant in summer (r = 0.756, p = 0.001, n = 15). the linear regression slope of ta on tb was also not significant (β = 0.21, p = 0.438, n = 16; r2 = 0.044; fig. 1) in spring, and was statistically significant in summer (β = -0.61, p = 0.001, n = 15; r2 = 0.571; fig. 1). however, the slope of the linear regression of ts on tb was similar for both seasons (ancova, ts as covariate; interaction season*ts: f1, 27 = 0.042, p = 0.839). the correlation coefficient was significant (r = 0.481, p = 0.003), as well as the regression coefficient (β = 0.38, p = 0.006, n = 31; r2 = 0.231; fig. 1). the available microhabitats at the study site provided different operative temperatures (kruskal-wallis test, h = 222.525, p < 0.0001, n = 528, df = 12; see table 2 and fig. 2). only grass and rock in full shade provided optimal temperatures for the thermoregulation of p. siculus (i.e., within the ptr) during all hourly periods of the day (fig. 2). the index of thermal quality of the habitat (de) showed a mean of 8.07 ± 0.05, the index of thermal accutable 1. mean ± se (sample size) body temperatures (tb), air temperatures (ta) and substrate temperatures (ts) of podarcis siculus at menorca (balearic islands, spain). temperatures are in °c. spring summer tb 29.57 ± 0.56 (16) 32.05 ± 0.68 (15) ta 25.77 ± 0.56 (16) 28.92 ± 0.45 (15) ts 27.11 ± 0.76 (16) 30.27 ± 0.80 (15) 130 zaida ortega et alii racy (db) was 1.41 ± 0.04, and the index of effectiveness of thermoregulation (e) of p. siculus in summer was 0.820 ± 0.005. discussion the preferred temperature range of p. siculus, obtained in the late spring, ranges from 28.40 to 31.70 °c. this is lower than the preferred temperature range of the endemic lacertid lizard from menorca, p. lilfordi, which showed a range between 31.78 and 35.68 °c during spring (unpublished data), and 32-36 °c during summer (pérez-mellado et al., 2013; ortega et al., 2014). this is also lower than the preferred temperature range of the third lacertid lizard present in menorca, scelarcis perspicillata, which showed a range from 33.90 to 36.10 °c during summer (ortega et al., 2016b). thus, the precision of thermoregulation obtained for the italian wall lizard was 3.3 °c, while the balearic lizard exhibited 3.9 °c, and the moroccan rock lizard 2.2 °c. the thermal preferences in a laboratory thermal gradient represent the optimal temperatures that lizards would intend to achieve in their habitats if there were no other ecological constraints than temperature (e.g., dawson, 1975; huey and bennett, fig. 1. slopes of the simple linear regressions models of body temperatures of podarcis siculus lizards (tb) against air temperatures (ta; left plot) and of the simple linear regressions of tb against substrate temperatures (ts; right plot) in spring and summer. the regression tb-ta was not significant in spring, but was is significant in summer, and the regression tb-ts was significant and had a similar slope for both seasons (see results in the text). table 2. mean values of the operative temperatures (te) of the different microhabitats studied for podarcis siculus at menorca (balearic islands, spain). temperatures are in °c. the letters between brackets match the non-significant pairs of the nemenyi post-hoc comparisons of the kruskal-wallis test (p > 0.05 in the paired comparisons). to avoid pseudoreplication, calculations are based in the hourly means of te so sample size coincides with the hours of monitoring of each microhabitat at the study site. n te se under rock (b, d) 44 37.55 0.88 rock full sun (a) 33 45.40 1.08 rock filtered sun (h, i) 44 40.64 1.12 rock full shade 44 30.78 0.28 soil full sun (a) 11 47.57 3.84 soil filtered sun (f, g, h) 44 39.98 1.19 soil full shade 44 29.30 0.33 grass full sun (a) 44 51.56 1.75 grass filtered sun (e, f, i) 44 40.59 1.38 grass full shade 44 30.37 0.29 pistacia full sun (b, c) 44 36.86 0.94 pistacia filtered sun (c, d, e, g) 44 38.48 1.20 pistacia full shade (b) 44 36.43 0.92 131thermoregulation of podarcis siculus in menorca fig. 2. operative temperatures (te) provided by the different microhabitats studied in es canutells (menorca, spain) for the italian lizard, podarcis siculus. the dotted lines comprise the preferred temperature range (ptr) of the species. 132 zaida ortega et alii 1987). the thermal preferences are closely related with thermal sensitivity of performance (angilletta et al., 2002; martin and huey, 2008). our results suggest that p. siculus would perform better at lower temperatures than the other two diurnal lacertid lizards inhabiting menorca. our results were coherent with previous studies about body temperatures of p. siculus. italian wall lizards showed mean body temperatures approximately 2 °c lower in june and july at menorca than those recorded near florence, in italy (avery, 1978). however, mean body temperatures found in summer in menorca are similar to those found in summer near pisa, (tosini et al., 1992). regarding spring thermoregulation, our data were similar to those found in corsica (france) in may: mean tb are ≈ 2 °c lower, mean ta are ≈ 2 °c higher, and mean ts are similar (van damme et al., 1990). in addition, the regression slope between body and air temperatures was very similar to the slope reported by van damme et al. (1990) for p. siculus of corsica during spring, and was also not significantly different from zero. our results also confirmed the conclusion of van damme et al. (1990) and tosini et al. (1992) about the lack of a sexual effect on body temperatures of p. siculus. mean body temperatures of the italian wall lizard were significantly higher in summer than in spring, but approximately 3 °c lower, for each season, than the body temperatures of the balearic lizard in the close islets of aire and colom (ortega et al., 2014). mean body temperatures were also approximately 2 °c lower than those of the moroccan rock lizard in menorca (ortega et al., 2016b). during summer, the italian wall lizard achieved a lower accuracy and effectiveness of thermoregulation (db − ≈ 1.41 °c; e ≈ 0.82) than the balearic lizard (db − ≈ 0.50 °c; e ≈ 0.91; ortega et al., 2014) and the moroccan rock lizard (db − ≈ 0.62 °c; e ≈ 0.88; ortega et al., 2016b). however, our data shows that the italian wall lizard is an effective thermoregulator lacertid, which seems well adapted to inhabit a wide range of microhabitats. a comparative study on the flexibility of thermal physiology and behavioural thermoregulation of p. siculus lizards and the species with which they coexist worldwide would help explain the possible causes of the remarkable ability of this species to adapt to different environments. acknowledgements we thank mario garrido and ana pérez-cembranos for their company during fieldwork, and mary trini mencía and joe mcintyre for linguistic revision. we captured lizards under the licenses of the government of the balearic islands. zaida ortega and abraham mencía had financial support from predoctoral grants of the university of salamanca. during the preparation of the manuscript, this work was supported by the research project cgl2015-68139-c2-2-p from the spanish ministry of economy and competitivity and feder european funds. all research was conducted in compliance with ethical standards and procedures of the university of salamanca. references adolph, s.c., porter, w.p. 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(1990): comparative thermal ecology of the sympatric lizards podarcis tiliguerta and podarcis sicula. acta oecol. 11: 503-512. acta herpetologica vol. 11, n. 2 december 2016 firenze university press predator-prey interactions between a recent invader, the chinese sleeper (perccottus glenii) and the european pond turtle (emys orbicularis): a case study from lithuania vytautas rakauskas1,*, rūta masiulytė1, alma pikūnienė2 effective thermoregulation in a newly established population of podarcis siculus in greece: a possible advantage for a successful invader grigoris kapsalas1, ioanna gavriilidi1, chloe adamopoulou2, johannes foufopoulos3, panayiotis pafilis1,* the unexpectedly dull tadpole of madagascar’s largest frog, mantidactylus guttulatus arne schulze1,*, roger-daniel randrianiaina2,3, bina perl3, frank glaw4, miguel vences3 thermal ecology of podarcis siculus (rafinesque-schmalz, 1810) in menorca (balearic islands, spain) zaida ortega*, abraham mencía, valentín pérez-mellado growth, longevity and age at maturity in the european whip snakes, hierophis viridiflavus and h. carbonarius sara fornasiero1, xavier bonnet2, federica dendi1, marco a.l. zuffi1,* redescription of cyrtodactylus fumosus (müller, 1895) (reptilia: squamata: gekkonidae), with a revised identification key to the bent-toed geckos of sulawesi sven mecke1,*,§, lukas hartmann1,2,§, felix mader3, max kieckbusch1, hinrich kaiser4 the castaway: characteristic islet features affect the ecology of the most isolated european lizard petros lymberakis1, efstratios d. valakos2, kostas sagonas2, panayiotis pafilis3,* sources of calcium for the agamid lizard psammophilus blanfordanus during embryonic development jyoti jee1, birendra kumar mohapatra2, sushil kumar dutta1, gunanidhi sahoo1,3,* mediodactylus kotschyi in the peloponnese peninsula, greece: distribution and habitat rachel schwarz1,*, ioanna-aikaterini gavriilidi2, yuval itescu1, simon jamison1, kostas sagonas3, shai meiri1, panayiotis pafilis2 swimming performance and thermal resistance of juvenile and adult newts acclimated to different temperatures hong-liang lu, qiong wu, jun geng, wei dang* olim palus, where once upon a time the marsh: distribution, demography, ecology and threats of amphibians in the circeo national park (central italy) antonio romano1,*, riccardo novaga2, andrea costa1 on the feeding ecology of pelophylax saharicus (boulenger 1913) from morocco zaida ortega1,*, valentín pérez-mellado1, pilar navarro2, javier lluch2 notes on the reproductive ecology of the rough-footed mud turtle (kinosternon hirtipes) in texas, usa steven g. platt1, dennis j. miller2, thomas r. rainwater3,*, jennifer l. smith4 heavy traffic, low mortality tram tracks as terrestrial habitat of newts mikołaj kaczmarski*, jan m. kaczmarek book review: sutherland, w.j., dicks, l.v., ockendon, n., smith, r.k. (eds). what works in conservation. open book publishers, cambridge, uk. http://dx.doi.org/10.11647/obp.0060 sebastiano salvidio acta herpetologica 13(1): 83-88, 2018 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-20887 thermal tolerance for two cohorts of a native and an invasive freshwater turtle species jun geng, wei dang, qiong wu, hong-liang lu* hangzhou key laboratory for animal adaptation and evolution, school of life and environmental sciences, hangzhou normal university, hangzhou 310036, zhejiang, china. *corresponding author. e-mail: honglianglu@live.cn submitted on: 2017, 3rd july; revised: on 2017, 13th november; accepted on: 2017, 20th november editor: marco sannolo abstract. the ability to tolerate environmental stress may determine invasion success of alien species. comparative data on physiological thermal tolerance between native and invasive vertebrates are quite limited. here, we assessed the difference in thermal tolerance between a native (mauremys reevesii) and an invasive (trachemys scripta elegans) freshwater turtle species. we incubated eggs of m. reevesii and t. scripta elegans from different cohorts at 29 °c, and measured the critical thermal minimum (ctmin) and maximum (ctmax) of hatchlings. our results preliminarily showed that the hatchlings of t. scripta elegans had a greater high-temperature tolerance and wider tolerance range than the hatchlings of m. reevesii; in the two-cohort system, individuals from the high-latitude cohort seemed to have greater low-temperature tolerance but similar high-temperature tolerance compared with those from the low-latitude cohort. relatively greater thermal tolerance ability for t. scripta elegans might reflect its environmental adaptability to thermal stress. keywords. mauremys reevesii, trachemys scripta elegans, critical thermal limits, thermal tolerance ability, invasion success. introduced alien species must overcome several potential challenges due to local climatic and biotic conditions to become established and successfully invade a novel environment (crowl et al., 2008; kelley, 2014). climatic constraints have been considered as the first barrier for restricting invasion success of alien species (olyarnik et al., 2009). the ability of invasive species to withstand harsh environmental conditions, such as high salinity, drought and extreme temperatures, may affect their invasive potential (lee, 2002; bates et al., 2013; kelley, 2014). it has been shown that many invasive species are often more tolerant to environmental stress than co-occurring native species (nyamukondiwa et al., 2010; lockwood and somero, 2011; zerebecki and sorte, 2011; weldon et al., 2016). one of the most important environmental factors impacting species’ distributions is temperature (johnston and bennett, 2008; gallien et al., 2010). physiological thermal tolerance for a given species is limited (angilletta, 2009). eurythermal species can maintain physiological function over a wider temperature range and are expected to have a broader distribution range than stenothermal ones (overgaard and hoffmann, 2011; gerick et al., 2014). eurythermality may contribute to the successful invasion of alien species. consequently, invasive species are expected to be eurythermal more often than native species (zerebecki and sorte, 2011; kelley, 2014). a comparison of the physiological thermal tolerance between native and invasive organisms has been made in many species (e.g., kimball et al., 2004; kolbe et al., 2010; lockwood and somero, 2011; zerebecki and sorte, 2011; yu et al., 2012; ju et al., 2013; urquhart and koetsier, 2014; davies et al., 2015; barahonasegovia et al., 2016). these studies mainly focused on invertebrate species, and some 84 jun geng et alii results were opposite to the above prediction (human and gordon, 1996; mcmahon, 2002; barahona-segovia et al., 2016). for example, two species of aquatic invasive bivalves (corbicula fluminea and dreissena polymorpha) show lower physiological thermal tolerances than the correspondent native species, because they experience less selection pressure for evolution of thermal tolerance (mcmahon, 2002). the red-eared slider turtle (trachemys scripta elegans) is native to southern united states and northern mexico, and has been introduced in many countries of africa, asia and europe as a pet, becoming invasive in many areas, and posing a serious threat to the survival of native turtle species (lowe et al., 2000; cadi and joly, 2004; pearson et al., 2015). previous studies have shown that t. scripta elegans has a number of distinct competitive advantages over native turtle species, such as a more aggressive behavior (cadi and joly, 2003; polo-cavia et al., 2010, 2011; pearson et al., 2015), wider niche breadth (polo-cavia et al., 2008; wang et al., 2013) and greater thermal inertia (polo-cavia et al., 2009). however, none of these studies has still focused on interspecies differences in physiological thermal tolerance between invasive t. scripta elegans and native turtle species. t. scripta elegans was introduced to china in the 1980s, and spread into most southern provinces over the last decades (liu et al., 2011). since its introduction, the native chinese threekeeled pond turtle, mauremys reevesii, which is one of the most common and widespread turtle species in southern china (zhao and adler, 1993), has suffered a considerable decline, and started to be displaced by t. scripta elegans in its natural habitats (liu et al., 2011). our aim here was to compare the thermal tolerance of the invasive t. scripta elegans and the native turtle species m. reevesii. for this purpose, we incubated eggs of both species from two sites at different latitudes and measured the critical thermal minimum (ctmin) and maximum (ctmax) of hatchlings. we collected a total of 80 fertilized eggs (20 eggs selected randomly from unidentified clutches (probably 6–8 clutches for m. reevesii and 5–6 clutches for t. scripta elegans) for each cohort of both cultured turtle species) that laid within a 2 to 4-day period from two private hatcheries in haikou (hainan province, southern china, 19°46’, 110°19’e, hereafter the low-latitude cohort) on may 22, and haining (zhejiang province, eastern china, 30°19’n, 120°25’e, hereafter the high-latitude cohort) on june 2 of 2016, respectively. the low-latitude site had a higher annual mean air temperature with relatively less thermal variation than the high-latitude site (fig. 1, http://data.cma.cn). in both hatcheries, cultured turtles were kept in outdoor artificial ponds (length × width × height: 20 × 15 × 1.5 m3) and thus were exposed to the local thermal environments. the eggs were randomly allocated into plastic containers (25 × 20 × 10 cm3) filled with moist vermiculite (approximately −12 kpa water potential, 1 g dried vermiculite/2 g water, du et al., 2007). the eggs were half-buried in the substrate, with the surface near the embryo exposed to air inside the container. the containers were placed into a fpq incubator (ningbo life science and technology ltd., china) and held at a temperature of 29 ± 1 °c (this temperature yields an approximate 1:1 offspring sex ratio for both species; wibbels et al., 1998; du et al., 2007, 2009). every other day, water was added into the substrate to keep the water potential of the substrate relatively constant. the containers were daily moved among the shelves to minimize any effects of thermal gradients inside the incubator. when eggs were found to have pipped, they were moved individually into glass jars. body mass of each turtle was measured once yolk sac was completely absorbed. then hatchling turtles were housed individually in plastic containers (20 × 15 × 12 cm3) with 3 cm water depth, which placed in a temperature-controlled room at 29 °c with an 11 h light: 13 h dark photoperiod. thirty-two hatchlings (8 individuals in each cohort of both species) were randomly selected to measure ctmin and ctmax following the procedures by xu et al. (2015). the hatchlings were placed into the fpq incubators that initially were set at 29 °c, and then cooled or heated at a rate of 0.3 °c/min (but more slowly, at a rate of 0.1 °c/ min, when temperatures were lower than 5 °c or higher fig. 1. monthly mean air temperatures at the two sites where the eggs of mauremys reevesii and trachemys scripta elegans were collected. 85thermal tolerance in two freshwater turtles than 35 °c). the body temperatures of hatchlings were measured using an electronic thermometer (ut-325, unitrend group ltd., china) when they lost righting response. all trials were run between 10:00 and 15:00. we first measured ctmin, and then ctmax a week later to minimize possible interactions between ctmin and ctmax measurement. the thermal tolerance range (ttr) was calculated by subtracting the ctmin from the ctmax for each individual (xu et al., 2015; gu et al., 2016). we used linear regression analysis to examine potential relationships between turtle body size (mass) and ctmin, ctmax or ttr. we used nested analysis of variance (anova) or analysis of covariance (ancova) with cohort being nested within species to determine whether there were differences in body mass, ctmin, ctmax and ttr between species and between cohorts. normality of data and assumption of homogeneity of variances was checked using kolmogorov-smirnov tests and bartlett’s test, respectively. all statistical analyses were performed using spss 18.0 for pc. the sex effect was ignored in this study because of the difficulty in determining the sex of hatchling turtles. there was no significant difference in hatchling body mass between different cohorts (f2,28 = 2.35, p = 0.114). however, t. scripta elegans hatchlings were heavier than m. reevesii hatchlings (f1,28 = 53.70, p < 0.001) (table 1). ctmin differed significantly between different cohorts (f2,28 = 7.32, p < 0.01) but not between species (f1,28 = 0.26, p = 0.612), whereas ctmax differed between species (f1,28 = 6.06, p = 0.020) but not between different cohorts (f2,28 = 0.76, p = 0.479). overall, the cohorts of both turtle species from the low-latitude site had higher mean values of ctmin than the cohorts from the highlatitude site (fig. 2a). t. scripta elegans had higher mean values of ctmax than m. reevesii in both cohorts (fig. 2b). ttr differed significantly between species (f1,28 = 4.53, p = 0.042) and between cohorts (f2,28 = 3.57, p = 0.042). ttr of t. scripta elegans was wider than that of m. reevesii; and ttr for the high-latitude cohorts was wider than that for the low-latitude cohorts (fig. 2c). linear regression analysis showed that ctmax and ttr (but not ctmin) was positively correlated with body mass of turtles (ctmin, r2 = 0.03, f1,30 = 0.86, p = 0.361; ctmax, r2 = 0.28, f1,30 = 11.81, p < 0.01; ttr, r2 = 0.22, f1,30 = 8.31, p < 0.01). both between-species (ctmax, f1,27 = 0.002, p = 0.967; ttr, f1,27 = 0.02, p = 0.887) and between-cohort (ctmax, f2,27 = 0.27, p = 0.763; ttr, f2,z27 = 2.50, p = 0.101) differences in ctmax and ttr were not significant after removing the effect of body mass of turtles. table 1. mean hatchling body mass (± se) of two turtle species (mauremys reevesii and trachemys scripta elegans) from two different cohorts. mauremys reevesii trachemys scripta elegans low-latitude cohort 6.24 ± 0.23 g 7.69 ± 0.34 g high-latitude cohort 6.17 ± 0.23 g 8.47 ± 0.19 g fig. 2. mean values (± se) for critical thermal minimum (a), critical thermal maximum (b) and thermal tolerance range (c) of hatchlings of mauremys reevesii and trachemys scripta elegans from two different cohorts. 86 jun geng et alii our study was limited by relatively small sample size (8 individuals in each cohort for both species) and by the lack of cohort replication (one cohort from each site), but it could allow us to provide a preliminary evaluation on between-species and between-cohort differences in thermal tolerance of hatchling turtles. except for slightly higher ctmax for hatchling m. reevesii, the values of ctmin and ctmax in this study fell within the ranges reported in previous studies on these turtle species (xu et al., 2015; gu et al., 2016). both m. reevesii and t. scripta elegans are semi-aquatic species. their critical thermal limits were similar to the values for other semi-aquatic turtle species, but seemed to be intermediate between primarily aquatic (ctmin: 2.7–4.8 °c for pelodiscus sinensis, wu et al., 2013; ctmax: 39–41 °c, hutchison et al., 1966) and terrestrial turtle species (ctmax: 43–44 °c, hutchison et al., 1966). despite resulting from a larger body size, higher ctmax and wider ttr for t. scripta elegans probably indicated that invasive t. scripta elegans had greater thermal tolerance to high temperature and to thermally variable habitats than native m. reevesii. such abilities may enhance invasion success when an alien species expands its geographic range and frequently faces extreme environmental conditions, such as near-critical high temperatures (dukes and mooney, 1999; zerebecki and sorte, 2011; kelley, 2014). relatively greater thermal tolerance ability has been found in most studied invasive species (lockwood and somero, 2011; yu et al., 2012; ju et al., 2013; lejeusne et al., 2014; davies et al., 2015). unfortunately, due to only one native turtle species in our study, more data from other native turtle species should be collected before drawing the conclusion that thermal tolerance ability of invasive turtles is greater than native turtles. the mechanistic bases for difference in thermal tolerance between native and invasive species may lie in  differences in the expression of some stress-related genes and thermal sensitivity of some enzymes (lockwood et al., 2010; kelley et al., 2011; zerebecki and sorte, 2011; yu et al., 2012). for example, a high level of heat-shock protein (hsp) 70 and 24 expression in invasive species is believed to contribute to enhancing their high-temperature tolerances (zerebecki and sorte, 2011). the ability to stabilize enzymatic activity at relatively high temperature may be related to high-temperature tolerance of invasive species (lockwood and somero, 2011). thermal tolerance abilities differ between lowand high-latitude populations (or cohorts) of some ectothermic species (fangue et al., 2006; yang et al., 2008; kelley et al., 2011; gaitán-espitia et al., 2013). such differences were also showed in both m. reevesii and t. scripta elegans, without considering hatchery pond effects. however, it was too early to draw a final conclusion due to the limitation in terms of lack of cohort replication. our results preliminarily suggested that high-latitude turtles had a greater low-temperature tolerance than low-latitude turtles. geographic difference in thermal tolerance might result from local adaptation to thermal environments (kelley, 2014; gaitán-espitia et al., 2014). the individuals living in thermally variable environments should have a greater ability to withstand extreme temperatures than those living in relatively stable environments (kelley, 2014). turtles from the high-latitude site would experience larger daily and seasonal thermal fluctuations than those from the low-latitude site. this might be the cause of greater thermal (especially low-temperature) tolerances for high-latitude turtles. furthermore, thermal tolerance plasticity may play a role in enhancing invasion potential of alien species (nyamukondiwa et al., 2010; tepolt and somero, 2014; davies et al., 2015). over similar ranges of acclimation temperatures, ctmin and ctmax varied by 1.9 and 2.6 °c for m. reevesii (acclimation temperatures from 17 to 33 °c, xu et al., 2015), and by 2.5 and 2.1 °c for t. scripta elegans (from 16 to 32 °c, gu et al., 2016), respectively. the greater acclimation response of ctmin in t. scripta elegans may confer a survival advantage over other native turtles when facing a novel environment. surprisingly, the magnitude of acclimation change of ctmax in t. scripta elegans seemed to be lower than in m. reevesii. it might probably reflect that the ability to adapt quickly to a lowtemperature environment is more important than to a high-temperature environment for successful invasion of species spreading to colder regions. acknowledgments we thank anonymous reviewers for their critical comments and suggestions that helped to improve the manuscript. the experimental procedures were approved by the animal welfare and ethics committee of hangzhou normal university (hznu-201605-011). this work was supported by grants from the national science foundation of zhejiang province (ly15c030006), scientific research program of hangzhou city (20150432b04), and the china scholarship council. references angilletta, m.j. 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(1993): herpetology of china. ohio: society for the study of amphibians and reptiles, u.s.a. acta herpetologica vol. 13, n. 1 june 2018 firenze university press species diversity and distribution of lizards in montenegro katarina ljubisavljević1,2,*, ljiljana tomović3, aleksandar urošević1, slađana gvozdenović2, vuk iković2, vernes zagora2, and nenad labus4 the first demographic data and body size of the southern banded newt, ommatotriton vittatus (caudata: salamandridae) abdullah altunişik diet and helminth parasites of freshwater turtles mesoclemmys tuberculata, phrynops geoffroanus (pleurodira: chelidae) and kinosternon scorpioides (criptodyra: kinosternidae) in a semiarid region, northeast of brazil antonio marcos alves pereira*, samuel vieira brito, joão antonio de araujo filho, adonias aphoena martins teixeira, diêgo alves teles, daniel oliveira santana, vandeberg ferreira lima, waltécio de oliveira almeida electric circuit theory applied to alien invasions: a connectivity model predicting the balkan frog expansion in northern italy mattia falaschi1,2,*, marco mangiacotti3, roberto sacchi3, stefano scali2, edoardo razzetti4 first report of bufo bufo (linnaeus, 1758) from sardinia (italy) ilaria maria cossu1, salvatore frau1, massimo delfino2,3, alice chiodi4, claudia corti1,5*, adriana bellati4 natural history and conservation of the nurse frog of the serranía del perijá allobates ignotus (dendrobatoidea: aromobatidae) in northeastern colombia hernán darío granda-rodríguez1,2, andrés camilo montes-correa3,*, juan david jiménez-bolaño3, marvin anganoy-criollo4,5 exploring body injuries in the horseshoe whip snake, hemorrhois hippocrepis juan m pleguezuelos*, esmeralda alaminos, mónica feriche how effectively do european skinks thermoregulate? evidence from chalcides ocellatus, a common but overlooked mediterranean lizard grigoris kapsalas, aris deimezis-tsikoutas, thanos georgakopoulos, ismini gkourtsouli-antoniadou, kallirroi papadaki, katerina vassaki, panayiotis pafilis thermal tolerance for two cohorts of a native and an invasive freshwater turtle species jun geng, wei dang, qiong wu, hong-liang lu* diet of a restocked population of the european pond turtle emys orbicularis in nw italy dario ottonello1, fabrizio oneto1,2, monica vignone2, anita rizzo2, sebastiano salvidio2,* helminths of the lizard colobosauroides cearensis (squamata, gymnophthalmidae) in an area of caatinga, northeastern brazil aldenir f. da silva neta*, robson w. ávila acta herpetologica 11(1): 21-30, 2016 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-17491 a new account for the endangered cerrado rocket frog allobates goianus (bokermann, 1975) (anura: aromobatidae), with comments on taxonomy and conservation thiago ribeiro de carvalho1,2,*, lucas borges martins1,2, ariovaldo antonio giaretta1 1 laboratório de taxonomia, sistemática e evolução de anuros neotropicais. faculdade de ciências integradas do pontal, universidade federal de uberlândia (ufu), rua 20, 1600, 38304-402, ituiutaba, minas gerais, brasil. 2 programa de pós-graduação em biologia comparada, universidade de são paulo, departamento de biologia/ffclrp. avenida dos bandeirantes, 3900, 14040-901, ribeirão preto, são paulo, brasil. *corresponding author. e-mail: thiago_decarvalho@yahoo.com.br submitted on 2015, 11th november; revised on 2016, 23rd march; accepted on 2016, 26th march editor: paolo casale abstract. we report a new distributional record of allobates goianus in niquelândia (goiás state, central brazil), and its rediscovery in minaçu since the creation of the serra da mesa dam lake in 1997. moreover, we describe the species advertisement call from niquelândia, reporting on a new calling emission pattern for the species, plus we provide the first comprehensive definition, diagnosis, and interspecific comparisons of a. goianus based on our two newly collected additional specimens since its original description in the mid-1970s. allobates goianus is compared with the 22 allobates species known to occur in brazil due to geographic proximity. morphological, color, and acoustic traits diagnose a. goianus from all analyzed congeners, except a. brunneus and a. olfersioides. our study draws attention to the importance of rediscovering a. goianus at the type locality (chapada dos veadeiros) aiming to properly address the species limits of the three brazilian allobates species (a. brunneus, a. goianus, and a. olfersioides) that are indistinguishable with regard to phenotypic traits. the conservation status of a. goianus, endemic to the cerrado of central brazil, is discussed as its natural habitats are under increased pressure due to habitat fragmentation in the cerrado. keywords. advertisement call, allobates brunneus, allobates olfersioides, habitat fragmentation, threatened species, type locality. introduction the genus allobates currently comprises 51 species distributed along south america on both sides of the andes, and central america (grant et al., 2006; frost, 2015). allobates goianus (bokermann, 1975) was described from central brazil (alto paraíso de goiás, chapada dos veadeiros, goiás state) and is regarded as a cerrado endemic species. the species was recently reported as endangered according to the latest brazilian list of threatened species (icmbio, 2014), due to habitat loss, the increasing fragmentation of habitats, and restricted extent of occurrence. besides the type locality, a. goianus has been reported from only two additional localities, both in goiás state: i) floresta nacional de silvânia, a federal protected area (bastos et al., 2003), from where its advertisement call was recently described by bastos et al. (2011), and ii) minaçu (serra da mesa region), where it was recorded in 1996 by brandão and araújo (2008), before the creation of the serra da mesa dam lake, supposedly flooding its habitats according to these authors. recent faunal inventories failed to locate a. goianus, including in the areas reported here (see oda et al., 2009; 22 thiago ribeiro de carvalho et alii morais et al., 2012; santoro and brandão, 2014). furthermore, this species has not been reported from its type locality either since its original description in the mid 1970’s (santoro and brandão, 2014). the purposes of this study were to report a new distributional record of a. goianus in central brazil, based on two male specimens and its rediscovery in minaçu since the creation of the serra da mesa dam lake in 1997, based on a call recording. furthermore, we describe the advertisement call of a. goianus from niquelândia based on a larger sample size, and report on a pattern of calling emission other than the previously described by bastos et al. (2011); also, we provide the first comprehensive definition, diagnosis, and interspecific comparisons of a. goianus based on our two newly collected specimens since its original description by bokermann (1975). materials and methods fieldwork was conducted from november to december 2014 in the serra da mesa region, municipalities of niquelândia (14°21’50’’s, 48°30’17’’w; ca. 710 m a.s.l.) and minaçu (13°45’35’’s, 48°18’31’’w; ca. 900 m a.s.l.), goiás state, central brazil (fig. 1). these localities lie 110 km southwest and 95 km northwest from the type locality of a. goianus, respectively. given that a. goianus is a brazilian cerrado endemic species, interspecific comparisons were restricted to allobates species occurring in brazil due to geographic proximity, i.e. the 18 species listed in the latest brazilian amphibian list (segalla et al., 2014): a. brunneus (cope, 1887), a. caeruleodactylus (lima and caldwell, 2001), a. conspicuus (morales, 2002), a. crombiei (morales, 2002), a. femoralis (boulenger, 1884), a. flaviventris melo-sampaio, sousa and peloso, 2013, a. fuscellus (morales, 2002), a. gasconi (morales, 2002), a. grilissimilis simões, sturaro, peloso and lima, 2013, a. hodli simões, lima and farias, 2010, a. marchesianus (melin, 1941), a. masniger (morales, 2002), a. nidicola (caldwell and lima, 2003), a. olfersioides (lutz, 1925), a. paleovarzensis lima, caldwell, biavati and montanarin, 2010, a. subfolionidificans (lima, sanchez and souza, 2007), a. sumtuosus (morales, 2002), a. vanzolinius (morales, 2002), with the addition of a. bacurau simões, 2016, a. magnussoni lima, simões and kaefer, 2014, a. myersi (pyburn, 1981), and a. tapajos lima, simões and kaefer, 2015, which were not included in this species list. new specimens are deposited in the collection of amphibians of the museu de biodiversidade do cerrado at the universidade federal de uberlândia (aag-ufu) under the following accession numbers: aag-ufu 5064–5065. the type series of a. goianus is deposited at the museu de zoologia da universidade de são paulo (mzusp) under the following accession numbers: mzusp 76652 (holotype), and mzusp 73706, 76651 (paratypes). morphological and coloration characters follow the diagnostic traits listed in kok et al. (2013). definitions and terminology are according to grant et al. (2006). snout-vent length (svl) was measured with digital calipers of 0.01 mm precision and follows duellman (1970); relative finger lengths follow grant et al. (2006). morphological and color traits were assessed under a stereomicroscope; finger lengths were measured with a micrometric piece coupled to a stereomicroscope. calls were recorded using a sennheiser k6/me66 directional microphone and a m-audio microtrack-ii digital recorder, and k6/me67 directional microphones and marantz pmd 670 or pmd 671 digital recorders. both recorders were set at a sampling rate of 48 khz and a sample size of 16 bits. calls were analyzed with raven pro 32-bit 1.5 beta version (bioacoustics research program, 2012) with the following settings: window size = 256 samples; window type = hann; 3db filter bandwidth = 270 hz; overlap = 85%; hop size = 0.79 ms; dft size = 1024 samples; grid spacing = 46.9 hz. temporal traits were measured from the oscillogram and spectral traits from the spectrogram. dominant frequency was determined from the entire call using the “peak frequency” measurement function. sound figures were generated with seewave package version 1.7.3 (sueur et al., 2008) on r platform version 3.1.0 (r core team, 2014) with the following settings: window type = hanning; window length = 512 samples (fft); overlap = 85%. definitions of call traits follow those of cocroft and ryan (1995) in most cases with terminological modifications in carvalho et al. (2015). note rate is expressed in notes/sec, call rate in calls/min. see appendix 1 for information about sound recordings and voucher specimens. results new distributional record our new distributional record of a. goianus in niquelândia is located approximately 110 km southwestward from its type locality, whereas the site where we rediscovered the species in minaçu is located approximately 95 km northwestward from the type locality (fig. 1). morphology and color patterns the type series of a. goianus is composed of three specimens: the holotype (mzusp 76652, an adult female with svl 17.4 mm); and two paratypes (mzusp 73706, a juvenile with svl 12.8 mm; and mzusp 76651, probably a sub-adult with svl 14.7 mm). due to poor preservation conditions, we decided not to seek for further evidence for sexing these specimens (vocal slits or internal morphology). color of the holotype was fainted so that coloration comparisons were restricted to general coloration of dorsum. dorsal blotch was barely discernible and ill-outlined, but even so this color trait could be associated with a lanceolate or hourglass-shaped blotch. morphological features and size of our newly two collected adult male specimens from niquelândia essentially matched the holotype description of a. goianus (boker23a new account for allobates goianus mann, 1975), so we assigned the newly collected specimens to a. goianus after the re-examination of the species holotype. given that holotype’s color traits could not be properly assessed, and also taking into account that five out of six diagnostic traits of a. goianus are color traits (see in the next paragraph), species definition, diagnosis, and interspecific comparisons were based on the two newly collected adult males from niquelândia, for which color traits were assessed both in life and in preservative. allobates goianus (figs 2-3) definition and diagnosis: the specimens are assigned to allobates by the absence of median lingual process, throat collar, and dark dermal collar, and by a diurnal calling activity. the specimens of a. goianus can be defined by the following phenotypic traits: (1) svl, 16.8 mm and 17.0 mm; (2) in life skin on dorsum shagreened to faintly granular posteriorly; (3) annulus tympanicus indistinct posterodorsally; (4) vocal sac distinct, subgular; (5) maxillary teeth absent; (6) distal tubercle on finger iv present; (7) tip of finger iv not reaching distal subarticular tubercle of finger iii when fingers are pressed together; (8) relative lengths of fingers i (3.5 mm and 3.5 mm) and ii (3.4 mm and 3.4 mm) subequal (finger i 3% longer than finger ii, n = 2) in length; (9) finger discs weakly expanded; (10) lateral fringes on fingers absent; (11) metacarpal ridge absent; (12) finger iii not swollen; (13) carpal pad absent; (14) thenar tubercle conspicuous, weakly protuberant; (15) tarsal keel tubercle-like and strongly curved at proximal end, extending from metatarsal tubercle; (16) tarsal fringe absent; (17) toe iv disc moderately expanded; (18) basal webbing only between toes ii–iii, and between toes iii– iv; (19) metatarsal fold absent; (20) weak preand postaxial fringes on toes ii–iv (preaxial ones more conspicuous); (21) dorsal coloration sorrel with a distinctive dark brown hourglass-shaped blotch; (22) dorsolateral stripe absent; (23) oblique lateral stripe as a diffuse broad area, extending from groin to about mid-body length, including light-colored, small spots, arranged irregularly; (24) ventrolateral stripe present, whitish, with no distinctive dark blotches ventrolaterally; (25) paracloacal marks present, light yellow; (26) throat coloration yellow in life, cream in preservation; (27) belly coloration cream to yellow in life, and cream in preservation; (28) iris metallic gold; (29) dorsal thigh color pattern brown with dark fig. 1. distribution map of allobates goianus (circles) and a. brunneus (triangles) in the brazilian cerrado. circles: (1) alto paraíso de goiás, chapada dos veadeiros (type locality); (2) minaçu (brandão and araújo, 2008; present study); (3) niquelândia (present study); (4) silvânia (bastos et al., 2003); triangles: (1) chapada dos guimarães [collection sites 1–3 in lima et al. (2009)], presumably the type locality [a site that lies approximately 30 miles northeast from cuiabá according to the original description: cope (1887)]; (2) other record sites in chapada dos guimarães [collection sites 4–9 in lima et al. (2009)]. brazilian state abbreviations: go (goiás), mt (mato grosso). 24 thiago ribeiro de carvalho et alii brown bands/blotches. allobates goianus is diagnosable from all compared species by the combination of character states 12, 21, 22, 24, 26, 27. comparison with brazilian congeners allobates goianus (character states of comparative species are between parentheses) can be distinguished from a. bacurau by having an hourglass-shaped blotch (absent; simões et al., 2016); from a. caeruleodactylus by having fingers and toe discs dark-colored with white speckles (blue-colored; lima and caldwell, 2001); from a. conspicuus, a. masniger, a. paleovarzensis, and a. tapajos by lacking dorsolateral stripe (present; morales, 2002; lima et al., 2010; lima et al., 2015); from a. subfolionidificans by having ventrolateral stripe (absent; lima et al., 2007); males of a. goianus can be distinguished from those of a. crombiei (transparent whitish throat; lima et al., 2012), a. flaviventris (gray or violet gray throat; melo-sampaio et al., 2013), a. grillisimilis (white to translucent throat; simões et al., 2013b), a. magnussoni (grayish violet throat; lima et al., 2014), a. marchesianus (gray throat; caldwell et al., 2002), a. nidicola (black to light gray throat; caldwell and lima, 2003), and a. sumtuosus (white to light gray throat; simões et al., 2013a) by having a yellow throat; from a. fuscellus, a. gasconi, a. sumtuosus and a. vanzolinius by having finger iii not swollen (finger iii swollen; morales, 2002); from a. femoralis (two morphotypes with reddish or black and white ventral coloration; simões et al., 2008) and a. hodli (black, white, and reddish-orange ventral coloration; simões et al., 2010) by having a cream to yellow ventral coloration. no morphological and color trait can unambiguously distinguish a. goianus from a. olfersioides (verdade and rodrigues, 2007) and a. brunneus (lima et al., 2009). fig. 2. two adult males of allobates goianus from niquelândia (goiás state, central brazil). above: aag-ufu 5064 (svl 17.0 mm); below: aag-ufu 5065 (svl 16.8 mm). fig. 3. dorsal (a) and ventral (b) views, sole of foot (c) and palm of hand (d) of an adult male of allobates goianus from niquelândia, central brazil: aag-ufu 5064 (svl 17.0 mm). 25a new account for allobates goianus advertisement call of allobates goianus from niquelândia. vocalization of a. goianus (n = 4 males; table 1) consists of a multi-note call (fig. 4a-b) composed of 2-41 notes (mean = 14.4, sd = 2.7), with duration varying from 0.3-11.8 sec (mean = 3.9, sd = 0.8), separated by intervals of 0.4-15.3 sec (mean = 2.8, sd = 1.7), and emitted at rates of 7.3-11.9 calls per minute (mean = 8.9, sd = 1.8). a few isolated notes (n = 4 in our sample) are emitted between typical multi-note calls. notes consist of one type of short, non-pulsed signal with ascendant frequency modulation, and may also have irregular amplitude modulations (fig. 4c). notes last from 30-51 msec (mean = 40.0, sd = 2.8), separated by intervals of 203-483 msec (mean = 261.0, sd = 8.7), and emitted at rates of 3.1-3.9 notes per second (mean = 3.5, sd = 0.1). the dominant frequency is always within the second harmonic, varying from 4922-5578 hz (mean = 5230.7, sd = 184.7). fundamental frequency (first harmonic) peaks from 2531-2766 hz (mean = 2630.1, sd = 56.1). see table 1 and fig. 5 for comparative acoustic traits for the male recorded in minaçu. comparison with advertisement calls of brazilian congeners the call of a. goianus can be distinguished from those of a. caeruleodactylus (x = 62 msec; lima and caldwell, 2001) and a. magnussoni (65-104 msec; lima et al., 2014) by a shorter note duration (30-51 msec); from those of a. crombiei (45-69 msec; lima et al., 2012) and a. marchesianus (119-212 msec; caldwell et al., 2002) by a longer note interval (203-483 msec), and from that of a. subfolionidificans (550-860 msec; lima et al., 2007) by a shorter note interval (203-483 msec); from those of a. femoralis (2-note or 4-note call patterns; simões et al., 2008), a. hodli (calls formed by two whistle-like notes; simões et al., 2010), and a. tapajos (2-note call pattern; lima et al., 2015) by having a multi-note call (mean = 14 notes/call, mode = 16); from that of a. flaviventris (up to 10-note calls; melo-sampaio et al., 2013) by a greater number of notes per call (up to 41-note calls); from those of a. bacurau (5890-6340 hz; simões, 2016), a. grillisimilis (5868-6651 hz; simões et al., 2013 b), and a. sumtuosus (5609-6549 hz; simões et al., 2013 a) by a lower dominant frequency (4922-5578 hz), and from those of a. masniger (4363-4694 hz; tsuji-nishikido et al., 2012), a. myersi (< 3000 hz; simões and lima, 2011), a. nidicola (3896–4413 hz; tsuji-nishikido et al., 2012), and a. paleovarzensis (4045–5093 hz, mean = 4534.2; kaefer and lima, 2012) by a higher dominant frequency (4922-5578 hz, mean = 5230.7). no acoustic trait can distinguish a. goianus (table 1) from a. brunneus (lima et al., 2009). habitat and natural history males were observed calling in rainy days on, or under moist leaf litter, or on twigs close to the ground inside riverine forests. in niquelândia, specimens were found from the border towards the inner gallery forest of approximately 50 m wide, whose abutting areas were already completely altered for pasture. in minaçu, they were found in a rocky grassland area, calling from inside a forest patch alongside a seasonal streamlet. at both sites, specimens were found calling in mid-afternoon (ca. 16:00 h on), stopping calling activity at sunset. table 1. advertisement call traits of allobates goianus from the municipalities of niquelândia and minaçu, goiás state, central brazil. n = number of recorded males (analyzed calls / analyzed notes). mean±sd (min–max). allobates goianus niquelândia n = 4 (81/82) minaçu n = 1 (3/9) call duration (sec) 3.9±0.8 (0.3–11.8) 2.6±0.4 (2.1–2.9) call interval (sec) 2.8±1.7 (0.4–15.3) 7.7±2.5 (5.9–9.4) calls/min 8.9±1.8 (7.3–11.9) 5.9 notes per call 14.4±2.7 (2–41) 8.0±1.0 (7–9) notes/sec 3.5±0.1 (3.1–3.9) 2.8±0.2 (2.5–2.9) note duration (msec) 40.0±2.8 (30–51) 38.4±2.4 (36–43) note interval (msec) 261.0±8.7 (203–483) 308.5±118.6 (239–599) fundamental frequency (hz) 2630.1±56.1 (2531–2766) 2554.7±25.1 (2531–2578) dominant frequency (hz) 5230.7±184.7 (4922–5578) 5085.9±100.2 (4922–5156) air temperature (°c) 23.0–26.7 25.0 26 thiago ribeiro de carvalho et alii fig. 4. advertisement call of allobates goianus from niquelândia, goiás, central brazil. (a) four multi-note calls (ca. 24.5 sec); (b) spectrogram and respective oscillogram of the third multi-note call from a; and (c) spectrogram and respective oscillogram of three mid-notes from b. sound file: allobates_goianusniquelandiago1ctrc_aagm (see appendix 1 for additional information). fig. 5. advertisement call of allobates goianus from minaçu, goiás, central brazil. spectrogram and respective oscillogram of one multi-note call. sound file: allobates_goianusminaçugo1lbm_aagm671 (see appendix 1 for additional information). 27a new account for allobates goianus discussion our new distributional record of a. goianus in niquelândia was expected by oda et al. (2009), due to the geographic proximity and similar anuran species compositions between this locality and minaçu in northern goiás (serra da mesa region), the latter being the locality from where a. goianus was recorded before 2001 (brandão and araújo, 2008). our sound recording of the species in minaçu confirms that the species is still present at that locality. the first description of the call of a. goianus was based on one male from silvânia (bastos et al., 2011) and did not provide a detailed description of the emission pattern, nor any sound figure with a wide time frame (see fig. 2 in bastos et al., 2011). therefore, we could not infer if the emission pattern could be assigned to a multi-note call or a continuous call. in silvânia, it was rare to find males of a. goianus in calling activity, and the calls had a continuous emission pattern (a. r. morais pers. comm.). by contrast, the advertisement call of a. goianus from both niquelândia and minaçu consists of a multi-note call, and we thus could not compare the call rates among populations. spectral call traits (fundamental and dominant frequencies, i.e. second harmonic) for the individual from silvânia had relatively lower values than those from niquelândia and minaçu (see table 1; bastos et al., 2011), whereas values for note duration (= call in bastos et al., 2011) from silvânia completely diverged (65-79 ms) from those from niquelândia and minaçu populations (combined value ranges: 30-51 ms). because of small sample sizes (n < 5 males recorded for each population), we could not investigate whether the between-population differences observed for note duration could be better explained by individual variability or the expected temperature influence on temporal traits of call (see gerhardt and huber, 2002). if a. goianus emits its calls in two different emission patterns, it falls under the same pattern that was previously described for a. brunneus by lima et al. (2009), in which conspecific males had both multi-note (trilled) and continuous emission patterns. additionally, caldwell et al. (2002) reported that two males of a. marchesianus switched from series of notes (= multi-note call) to a continuous call pattern. variation in note emission patterns has been described for other species of allobates as well (simões et al., 2008, 2013 a; simões and lima, 2011; lima et al., 2009, 2014). the behavior of switching note emission patterns might convey different messages to conspecific and heterospecific individuals (amézquita et al., 2006), and ought to be addressed within an experimental framework. we did not find any straightforward character (size, morphology, color pattern, advertisement call) that could distinguish a. goianus from a. brunneus (sensu lima et al., 2009) and a. olfersioides (sensu verdade and rodrigues, 2007). allobates goianus (hourglass-shaped blotch) might possibly be differentiated from a. olfersioides (single or multiple intercrossing xs; verdade and rodrigues, 2007) by different character states for the dorsal marks. on the other hand, these color patterns may sometimes be so variable in shape, outline, and contrasting background color to the extent that these character states could be hardly discriminated from each other in some cases. so we assume that this color trait might not represent a reliable feature for species discrimination between a. goianus and a. olfersioides. it is noteworthy that there is a large gap (ca. 750 km horizontal distance) between the distributional records for the two species (a. brunneus and a. goianus) that occur in the cerrado of central and western brazil (fig. 1), and both cerrado species are completely isolated from a. olfersioides (at least 800 km between the distributional records for a. goianus and a. olfersioides), which is restricted to the atlantic forest (see fig. 1 in verdade and rodrigues, 2007). these results shed light on the importance of rediscovering a. goianus at the type locality (chapada dos veadeiros), which would allow a formal re-description of the species, as well as a comparative analysis of the three brazilian species of allobates without a clear species-level discrimination through phenotypic traits, including a reappraisal of their species limits supplemented by genetic information. further studies are still required to assess additional population-level information about a. goianus, improving our knowledge on the conservation threats and risks of extinction of its isolated populations in central brazil. for instance, the population recorded by us in niquelândia occurs in an almost completely human-altered area, and such a forest remnant is probably maintained owing to the course of a streamlet inside the forest. nevertheless, government monitoring and oversight are limited and these areas are not free of threats. furthermore, despite being in protected areas, populations from chapada dos veadeiros and silvânia also seem to be under threat (morais et al., 2012; icmbio, 2014; santoro and brandão, 2014) due to habitat fragmentation (icmbio, 2014). therefore, this unfavorable scenario requires immediate attention, especially conservation strategies aiming to the maintenance of the few remaining forested areas and establishment of connections between them in the brazilian cerrado, which might help not only this endangered and poorly known allobates species, but also sev28 thiago ribeiro de carvalho et alii eral other forest dwellers inhabiting this region, which are currently under increased pressure mostly due to habitat fragmentation. acknowledgements financial support was provided by cnpq, fapemig, and fapesp. a research grant conceded by cnpq to aag. doctoral fellowships by fapesp to trc and cnpq to lbm. special thanks to christine strussmann for making available a comparative recording of a. brunneus, taran grant for making available type material under his care at museu de zoologia da universidade são paulo, alessandro r. morais for sharing with us relevant field information on a. goianus, leandro drummond for providing allobates comparative pictures, philippe kok and anonymous reviewers for valuable comments on earlier versions of this study. collection permit was granted by instituto chico mendes through the online platform sistema de autorização e informação em biodiversidade (icmbio/sisbio 30059-5). references amézquita, a., hödl, w., lima, a.p., castellanos, l., erdtmann, l., araújo, m.c. 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(2012): sexual signals of the amazonian frog allobates paleovarzensis: geographic variation and stereotypy of acoustic traits. behaviour 149: 15-33. kok, p.j.r., hölting, m., ernst, r. (2013): a third microendemic to iwokrama mountains of central guyana: a new “cryptic” species of allobates zimmerman and zimmerman, 1988 (anura: aromobatidae). org. divers. evol. 13: 621-638. lima, a.p., caldwell, j.p. (2001): a new amazonian species of colostethus with sky blue digits. herpetologica 57: 180-189. lima, a.p., caldwell, j.p., biavati, g., montanarin, a. (2010): a new species of allobates (anura: aromobatidae) from paleovárzea forest in amazonas, brazil. zootaxa 2337: 1-17. lima, a.p., caldwell, j.p., strüssmann, c. (2009): redescription of allobates brunneus (cope) 1887 (anura: aromobatidae: allobatinae), with a description of 29a new account for allobates goianus the tadpole, call, and reproductive behavior. zootaxa 1988: 1-16. lima, a.p., erdtmann, l.k., amézquita, a. (2012): advertisement call and colour in life of allobates crombiei (morales) “2000” [2002] (anura: aromobatidae) from the type locality (cachoeira do espelho), xingu river, brazil. zootaxa 3475: 86-88. lima, a.p., sanchez, d.e.a., souza, j.r.d. (2007): a new amazonian species of the frog genus colostethus (dendrobatidae) that lays its eggs on undersides of leaves. copeia 2007: 114-122. lima, a.p., simões, p.i., kaefer, i.l. (2014): a new species of allobates (anura: aromobatidae) from the tapajós river basin, pará state, brazil. zootaxa 3889: 355-387. lima, a.p., simões, p.i., kaefer, i.l. (2015): a new species of allobates (anura: aromobatidae) from parque nacional da amazônia, pará state, brazil. zootaxa 3980: 501-525. melo-sampaio, p.r., souza, m.b., peloso, p.l.v. (2013): a new, riparian, species of allobates zimmermann and zimmermann, 1988 (anura: aromobatidae) from southwestern amazonia. zootaxa 3716: 336-348. morais, a.r., bastos, r.p., vieira, r., signorelli, l. (2012): herpetofauna da floresta nacional de silvânia, um remanescente de cerrado no brasil central. neotrop. biol. conserv. 7: 114-121. morales, v.r. “2000” (2002): sistematica y biogeografía del grupo trilineatus (amphibia, anura, denbrobatidae, colostethus) con descripción de once nuevas especies. publ. asoc. amigos doñana 13: 1-59. oda, f.h., bastos, r.p., lima, m.a.c.s. (2009): taxocenose de anfíbios anuros no cerrado do alto tocantins, niquelândia, estado de goiás: diversidade, distribuição local e sazonalidade. biota neotrop. 9: 219-232. r core team (2014): r foundation for statistical computing, austria. http://www.r-project.org. santoro, g.r.c.c., brandão, r.a. (2014): reproductive modes, habitat use, and richness of anurans from chapada dos veadeiros, central brazil. north-west. j. zool. 10: 365-373. segalla, m.v., caramaschi, u., cruz, c.a.g., grant, t., haddad, c.f.b., langone, j.a., garcia, p.c.a. (2014): brazilian amphibians: list of species. herpetol. bras. 3: 37-48. simões, p.i. (2016): a new species of nurse-frog (aromobatidae, allobates) from the madeira river basin with a small geographic range. zootaxa 4083: 501-525. simões, p.i., kaefer, i.l., farias, i.p., lima, a.p. (2013 a): an integrative appraisal of the diagnosis and distribution of allobates sumtuosus (morales, 2002) (anura, aromobatidae). zootaxa 3746: 401-421. simões, p.i., lima, a.p. (2011): the complex advertisement calls of allobates myersi (pyburn, 1981) (anura: aromobatidae) from são gabriel da cachoeira, brazil. zootaxa 2988: 66-68. simões, p.i., lima, a.p., farias, i.p. (2010): the description of a cryptic species related to the pan-amazonian frog allobates femoralis (boulenger 1883) (anura: aromobatidae). zootaxa 2406: 1-28. simões, p.i., lima, a.p., magnusson, w.e. (2008): acoustic and morphological differentiation in the frog allobates femoralis: relationships with the upper madeira river and other potential geological barriers. biotropica 40: 607-614. simões, p.i., sturaro, m.j., peloso, p.l.v., lima, a.p. (2013 b): a new diminutive species of allobates zimmermann and zimmermann, 1988 (anura, aromobatidae) from the northwestern rio madeira-rio tapajós interfluve, amazonas, brazil. zootaxa 3609: 251-273. sueur, j., aubin t., simonis, c. (2008): seewave, a free modular tool for sound analysis and synthesis. bioacoustics 18: 213-226. tsuji-nishikido, b.m., kaefer, i.l., freitas, f.c., menin, m., lima, a.p. (2012): significant but not diagnostic: differentiation through morphology and calls in the amazonian frogs allobates nidicola and a. masniger. herpetol. j. 22: 105-114. verdade, l.k., rodrigues, m.t. (2007): taxonomic review of allobates (anura, aromobatidae) from the atlantic forest, brazil. j. herpetol. 41: 566-580. 30 thiago ribeiro de carvalho et alii appendix 1 list of sound recordings of allobates goianus from serra da mesa region (niquelândia and minaçu, goiás state: go) with the respective voucher males (aag-ufu accession numbers), and additional information. locality sound recording voucher time; date niquelândia (go) allobates_goianusniquelandiago1atrc_aagm 5064 17:49 h; 19 dec 2014 allobates_goianusniquelandiago1btrc_aagm 5064 17:52 h; 19 dec 2014 allobates_goianusniquelandiago1ctrc_aagm 5064 17:53 h; 19 dec 2014 allobates_goianusniquelandiago2trc_aagm uncollected 18:16 h; 19 dec 2014 allobates_goianusniquelandiago3lbm_aagm671 uncollected 17:20 h; 20 dec 2014 allobates_goianusniquelandiago4albm_aagm671 5065 17:40 h; 20 dec 2014 allobates_goianusniquelandiago4blbm_aagm671 5065 17:41 h; 20 dec 2014 minaçu (go) allobates_goianusminacugo1lbm_aagm671 uncollected 17:15 h; 22 nov 2014 acta herpetologica vol. 11, n. 1 june 2016 firenze university press feeding habits of mesoclemmys vanderhaegei (testudines: chelidae) elizângela silva brito1,*, franco leandro souza2, christine strüssmann3 morphology, ecology, and behaviour of hylarana intermedia, a western ghats frog ambika kamath1, rachakonda sreekar2,3 a new account for the endangered cerrado rocket frog allobates goianus (bokermann, 1975) (anura: aromobatidae), with comments on taxonomy and conservation thiago ribeiro de carvalho1,2,*, lucas borges martins1,2, ariovaldo antonio giaretta1 molecular phylogenetics of the pristimantis lacrimosus species group (anura: craugastoridae) with the description of a new species from colombia mauricio rivera-correa, juan m. daza* population structure and activity pattern of one species of adenomera steindachner, 1867 (anura: leptodactylidae) in northeastern brazil maria juliana borges-leite1,*, joão fabrício mota rodrigues2, patrícia de menezes gondim1, diva maria borges-nojosa1 vocal repertoire of scinax v-signatus (lutz 1968) (anura, hylidae) and comments on bioacoustical synapomorphies for scinax perpusillus species group marco antônio peixoto1,*, carla silva guimarães1, joão victor a. lacerda2, fernando leal2, pedro c. rocha2, renato neves feio1 amendment of the type locality of the endemic sicilian pond turtle emys trinacris fritz et al. 2005, with some notes on the highest altitude reached by the species (testudines, emydidae) federico marrone*, francesco sacco, vincenzo arizza, marco arculeo photo-identification in amphibian studies: a test of i3s pattern marco sannolo1,*, francesca gatti2, marco mangiacotti3,4, stefano scali3, roberto sacchi4 no evidence for the ‘expensive-tissue hypothesis’ in the dark-spotted frog, pelophylax nigromaculatus li zhao, min mao, wen bo liao* no short term effect of clinostomum complanatum (trematoda: digenea: clinostomatidae) on survival of triturus carnifex (amphibia: urodela: salamandridae) giacomo bruni1,*, claudio angelini2 yeasts in amphibians are common: isolation and the first molecular characterization from thailand srisupaph poonlaphdecha, alexis ribas* introduction of eleutherodactylus planirostris (amphibia, anura, eleutherodactylidae) to hong kong wing ho lee1, michael wai-neng lau2, anthony lau3, ding-qi rao4, yik-hei sung5,* correlation between endoscopic sex determination and gonad histology in pond sliders, trachemys scripta (reptilia: testudines: emydidae) david perpiñán1, albert martínez-silvestre2,3, ferran bargalló3, marco di giuseppe4, jorge orós5, taiana costa6,* book review: john w. wilkinson. amphibian survey and monitoring handbook. pelagic publishing franco andreone book review: susan newman. frogs amphibians and their threatened environment. discovery and expression through art k-3. frogs are green franco andreone acta herpetologica 13(1): 43-49, 2018 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-21867 first report of bufo bufo (linnaeus, 1758) from sardinia (italy) ilaria maria cossu1, salvatore frau1, massimo delfino2,3, alice chiodi4, claudia corti1,5,*, adriana bellati4 1 sezione sardegna shi, societas herpetologica italica, via marconi 8, 09070 seneghe, oristano, italy 2 dipartimento di scienze della terra, università di torino, via valperga caluso 35, 10125 torino, italy 3 institut català de paleontologia miquel crusafont, universitat autònoma de barcelona, edifici icp, campus de la uab s/n, 08193 cerdanyola del vallès, barcelona, spain 4 dipartimento di scienze della terra e dell’ambiente, università di pavia, via ferrata 9, 27100 pavia, italy 5 museo di storia naturale dell’università di firenze, sezione di zoologia “la specola”, via romana 17, 50125 firenze, italy *corresponding author. e-mail: claudia.corti@unif.it submitted on: 2017, 9th october; revised on: 2017, 1st november; accepted on: 2017, 6th november editor: marcello mezzasalma abstract. the common toad bufo bufo (linneaus, 1758) was found for the first time in sardinia in 2016 during herpetological surveys. the species appears to be well established in the finding area. many adults and juveniles, tadpoles at different developmental stages, and eggs have been found during repeated monitoring. in order to infer the geographic origin of the sardinian population, we amplified two mitochondrial markers (16s, cytb) and compared sequences with those available for the species across its natural range. we also screened samples for the presence of bd pathogen to assess the risk of infection mediated by the species in the area. results suggest that sardinian individuals are genetically close to the central italian populations, although they show a unique distinct haplotype. though the species should be considered allochthonous to the island, further molecular and ecological data are urgently needed to assess the genetic structure and the possible impact on the local fauna, which is largely composed by endemic taxa. particularly, possible interactions with other native amphibians like the green toad bufo balearicus (boettger, 1880), also present in the area, should be investigated, both in terms of competition for breeding sites and genetic pollution, as these species are already known to hybridize in the wild. keywords. common toad, amphibians, early detection, sardinia, mediterranean basin. introduction the common toad bufo bufo (linnaeus, 1758) is widespread in the western palearctic region, from north africa to the 68°n in finland and from the western iberian peninsula to the baikal lake in siberia (lizana, 2002; böhme et al., 2007). the only exceptions are iceland, the coldest northern parts of scandinavia, ireland and several mediterranean islands, among which malta, crete, corsica, sardinia and the balearic islands (böhme et al., 2007). until recently, the evolutionary history of this species has been controversial and so far molecular studies have revealed the existence of distinct subspecies and delimited their respective ranges (litvinchuk et al., 2008; garcia-porta et al., 2012; recuero et al., 2012; arntzen et al., 2013a,b). in europe, several distinct evolutionary lineages occur (at least 7, according to garcia-porta et al., 2012) which diversified between the late miocene and early pliocene (11.6-3.6 ma). for instance, distinct phylogroups have been detected in the italian peninsula, one in the south, including sicily, and the other in the northern44 ilaria maria cossu et alii central regions, as a result of the persistence of populations in multiple refugia during the pleistocene glaciations (according to the “refugia within refugia” hypothesis, gómez and lunt, 2007). bufo bufo has been discovered in north-central sardinia in late summer 2016. several surveys, carried out in the area of the first detection, revealed a high density of both adults and larvae. later on, adults and juveniles have been also detected far from the original detection point, suggesting the occurrence of a stable and viable population in the region. according to the composition of the sardinian amphibian community, the species could be classified as allochthonous, likely resulting from one or multiple human-mediated introductions from the mainland. therefore, in order to provide information concerning the geographic origin of the common toad found in sardinia, informative mitochondrial molecular markers were selected to compare insular haplotypes with the mainland ones. materials and methods study area the study area is located in north-central sardinia at an elevation of 400-1000 m a.s.l., in a mountain woodland region characterized by a mosaic of high mediterranean maquis, as well as coniferous and deciduous forest resulting from several anthropic interventions. artificial small lakes and ponds occur throughout the area. the small streams flowing from the above mentioned water bodies offer a suitable habitat for amphibians. the climate is characterized by mild wet winters (rarely t min goes below 0°c), when high precipitation rate is recorded. in summer, the dry season lasts in general no longer than three months. the area therefore presents suitable ecological conditions for the species. sampling and molecular analysis surveys were carried out from late summer 2016 until june 2017. toads were searched by active search (ves = visual encounter system; crump and scott, 1994) at night under rainy weather conditions to facilitate the detection of adults. several adults were observed throughout the study area as well as, at least 150, post-metamorphs on the shore of a small water body. swabs and tissues were collected in order to perform genetic characterization by molecular analysis. specifically, biological samples were obtained from one tadpole, three post-metamorphs, and two adults. tissue samples were collected only from death animals. the first buccal swab was collected on an adult on january 12th, 2017. all swabs and tissue samples (n = 6) were preserved in ethanol 96% until dna extraction. in the laboratory, genomic dna was extracted using the commercial kit genelute mammalian genomic dna miniprep kit (sigmaaldrich, saint louis, usa) and following the manufacturer’s instructions. to infer the putative geographic origin of the sampled individuals, we amplified two different diagnostic mitochondrial markers, corresponding to a portion (504 bp) of the ribosomal 16s rdna gene (16s) and a portion (722 bp) of the protein-encoding cytochrome b (cytb) region, using primer pairs already available in the literature (palumbi et al., 1991; recuero et al., 2012). the selected markers are known to be diagnostic for discriminating italian populations and lineages (recuero et al., 2012; arntzen et al., 2017). pcr products were purified with genelute gel extraction kit (sigma-aldrich, saint louis, usa) following the manufacturer’s instructions, and sequenced using the forward primer of each pair (eurofins genomics/applied genomics, ebersberg, germany). chromatograms were imported in geneious 10.0.9 (biomatters ldt.) and checked manually for insertions or deletions (indels) and ambiguous positions in protein-encoding mitochondrial gene fragments. the latter were also translated into amino acidic sequences to exclude the presence of non-functional copies of target markers (i.e., pseudogenes), which can be detected by premature stop codons or non-sense codons occurring in the coding frame. sequences were aligned for each gene independently with the online version of mafft (katoh and toh, 2008) applying parameters by default (auto strategy, gap opening penalty: 1.53, offset value: 0.0). obtained sequences were compared with all the available homologous sequences from genbank (https://www.ncbi.nlm. nih.gov/) using the basic local alignment search tool (blast, http://blast.ncbi.nlm.nih.gov/blast.cgi) and setting the nucleotide blast (nblast) algorithm with default parameters. concatenated alignment was built for including 61 homologous sequences from 22 italian populations (recuero et al., 2012; arntzen et al., 2017), then individual sequences were merged into single gene haplotypes using the on-line web tool dnacollapser 1.0 available at fabox site (http://users-birc. au.dk/biopv/php/fabox/) (please refer to table 1 for all the accession numbers and information concerning populations and haplotypes). relationships among mitochondrial dna (mtdna) haplotypes were visualized by reconstructing a statistical parsimony haplotype network, selecting the 95% connection limit as a reliable parsimony threshold as implemented in tcs 1.21 (clement et al., 2000). this procedure allowed examining the extent of haplotype sharing between the sardinian population and those from the mainland, under the statistical parsimony method (templeton et al., 1992). finally, tissues were screened for the presence of the diagnostic molecular marker (300 bp long) in order to detect the presence of the chytrid fungus batrachochytrium dendrobatidis in our samples according to annis et al. (2004). results all the biological samples collected in the field were successfully amplified, nor gap nor premature stop codon were detected in the protein-encoding cytb sequences. 45bufo bufo in sardinia fig. 1. distribution of the sardinian and other italian populations of bufo bufo analysed in this study. different regions for which mtdna data were available have been drawn using different colours. population numbers refer to table 1. bottom-left: haplotype network reconstruction of concatenated mtdnas (16s+cytb) showing phylogeographic relationships among populations and haplotypes. each circle corresponds to a distinct haplotype, and size is proportional to the number of samples sharing the same mtdna sequence. colours refer to distinct italian regions. each bar refers to a single point mutation connecting two haplotypes, each dot corresponds to missing haplotypes (not sampled or extinct). reference haplotype names have been modified from recuero et al. (2012) and arntzen et al. (2017). 46 ilaria maria cossu et alii we detected one single 16s haplotype (accession number: mg199041) and one cytb haplotype (accession number: mg199042) in all sardinian samples. the 16s haplotype yielded a 100% match with homologous sequences from latium and emilia-romagna (recuero et al., 2012). by contrast, no exact match was found in genbank for the sardinian cytb haplotype, which returned 99% match probability with homologous sequences from the same localities as 16s, and from tuscany (recuero et al., 2012; see table 1 for all the accession numbers). in the final concatenated alignment of 67 mitochondrial sequences (1226 bp), including sardinian samples and homologous sequences from the italian peninsula and sicily (recuero et al., 2012), 25 unique haplotypes were detected, corresponding to 23 populations (table 1). the table 1. list of localities and mtdna haplotypes (16s+cytb) used as reference for the sardinian common toad population, with specification of sample sizes (xn), accession numbers and reference ([1] recuero et al., 2012; [2] arntzen et al., 2017). in bold: reference sequences showing 100% (16s) and 99% (cytb) sequence similarity with the sardinian haplotypes. map locality idhaploxn accession numbers reference 16s cytb 1 north-central sardinia bbs(x6) mg199041 mg199042 this study 2 doganella, latium bb107(x4) jn647054-57 jn647367-70 [1] 3 subiaco, latium bb120(x1) bb107(x1) jn647094 jn647095 jn647407 jn647408 [1] 4 jenne, latium bb113(x1) bb107(x1) bb114(x1) jn647071 jn647072 jn647073 jn647384 jn647385 jn647386 [1] 5 rocca sinibalda, fiume turano, latium bb107(x3) jn647099-101 jn647412-14 [1] 6 canale monterano, latium bb107(x3) bb118(x1) jn647082-83/85 jn647084 jn647395-96/98 jn647397 [1] 7 barbarano, latium bb104(x2) jn647179-80 jn647365-66 [1] 8 bosco del foglino, latium bb110(x1) bb107(x1) bb111(x1) jn647064 jn647065 jn647066 jn647377 jn647378 jn647379 [1] 9 molella, fonti di locullo, latium bb107(x3) bb117(x1) jn647078-79/81 jn647080 jn647391-92/94 jn647393 [1] 10 campo tizzoro, tuscany bb121(x1) bb122(x2) jn647096 jn647097-98 jn647409 jn647410-11 [1] 11 monghidoro, emilia-romagna bb107(x2) jn647069-70 jn647382-83 [1] 12 favero, piedmont bbe2.1(x1) bbe2.2(x2) mf461242 mf461243-44 mf461270 mf461271-72 [2] 13 pian di verteglia, campania bb100(x3) bb123(x1) jn647102/04-05 jn647103 jn647415/17-18 jn647416 [1] 14 alberobello, apulia bb100(x1) jn647175 jn647361 [1] 15 tortora, c.da massadita, calabria bb115(x1) bb114(x1) bb100(x2) jn647074 jn647075 jn647076-77 jn647387 jn647388 jn647389-90 [1] 16 orsomarso, calabria bb100(x3) jn647086-87-88 jn647399-400-01 [1] 17 fagnano castello, lago paglia, calabria bb108(x1) bb109(x1) jn647058 jn647059 jn647371 jn647372 [1] 18 fiumefreddo bruzio, calabria bb103(x4) jn647060-63 jn647373-76 [1] 19 bel monte calabro marina, calabria bb103(x1) jn647178 jn647364 [1] 20 lago dell’antigola, calabria bb101(x1) bb102(x1) jn647176 jn647177 jn647362 jn647363 [1] 21 stilo, calabria bb101(x1) jn647093 jn647406 [1] 22 gambarie, calabria bb101(x1) bb112(x1) jn647068 jn647067 jn647381 jn647380 [1] 23 fiume irminio, sicily bb101(x2) bb119(x2) jn647089-92 jn647090-91 jn647402-05 jn647403-04 [1] 47bufo bufo in sardinia reconstruction of the haplotype network allowed a better resolution of phylogeographic relationships between the sardinian population and the other italian ones, as the sardinian haplotype clearly clustered together with samples from central italy (fig. 1). the most closely related haplotypes were bb107, already found in 8 localities in latium by previous authors, and bb121 recovered in the only population screened from tuscany (recuero et al., 2012) (table 1). finally, none of the samples was positive for the presence of diagnostic chytrid marker (not shown). discussion the common toad, never recorded for sardinia (sindaco et al., 2006), was found on the island in 2016. therefore, a genetic analysis was performed in order to assess the origin of the sardinian population. according to our molecular results, the low genetic variation detected at both markers (one single haplotype shared by all the samples) attests a general low genetic variation of the population, as expected when founder effect occurs after the arrival of few colonizers. the occurrence of b. bufo in sardinia could be due to direct release of captive individuals. however, considering the distribution and the high density of the individuals detected, it is also possible to assume alternative introduction routes. toads could have been introduced on the island along with plants imported from central italy in the ’70s and ’80s of the last century, but the most reliable cause could be the introduction of eggs and tadpoles along with fish stocks (particularly, the rainbow trout oncorhynchus mykiss) introduced for game fishing about 30-40 years ago. indeed, this activity is still widely carried out in the study area. according to the molecular results, central italy should be considered as the most likely source area of the sardinian common toad population. indeed, haplotypes previously recovered in this area, belonging to the native populations of the species, were close to the one found in sardinia according to both markers. nevertheless, although the 16s haplotype perfectly matches mainland sequences from several localities (mainly in central italy), the cytb haplotype of the sardinian samples is at present unique of this population, suggesting that the source population has not yet been sampled. repeated field surveys allowed to observe different life stages showing the reproductive success of the common toad in sardinia and its relatively wide distribution in the area, at present corresponding approximately to 17.000 ha (further surveys will be carried out in order to better define the distribution of the sardinian b. bufo population). all the ecological requirements of the species seem to be fulfilled in the study area (e.g., occurrence of streams and different kinds of water bodies, favorable climate, and trophic availability). the common toad in fact successfully reproduces as larval stages were detected in late winter and juveniles occurred in late spring following the reproductive ecology of the species. according to our results, none of the samples turned out to be positive to chytridiomycosis. however, we acknowledge that considering the limited number of samples screened, and in the absence of histological data, we could not rule out the occurrence of the pathogen in the whole population. nevertheless, as most of the samples were collected from adults and post-metamorphs, we are sufficiently confident that the population is not currently undergoing a massive epidemic. sardinia hosts an extremely valuable herpetofauna, six amphibian species are endemic, the sardinian mountain newt (euproctus platycephalus) and five species of cave salamanders (speleomantes flavus, s. imperialis, s. sarrabusensis and s. supramontis and atylodes genei). others could be considered sub-endemic while occurring also in corsica and on some north-tyrrhenian and ligurian islands, like the tyrrhenian painted frog (discoglossus sardus) and the tyrrhenian tree frog (hyla sarda). among the bufonids, the balearic green toad (bufo balearicus) is thought to be autochthonous to the island (poggesi et al., 1995; lanza et al., 2007). the presence of h. sarda and b. balearicus has been directly assessed during the surveys, and it is likely to assume that other amphibians like d. sardus should also be present in the area. therefore, considering the peculiarity of some sardinian faunal elements, which originate from the long lasting isolation of the island, a recent introduction of the common toad may raise questions about the possible interaction with other species, particularly bufo balearicus. however, reduced competition was observed between the two toad species because of their different ecology (sinisch et al., 1999). bufo balearicus (identified in the literature as bufo viridis or bufo gr. b. viridis) appears rather continuously throughout the paleontological sardinian record from the late pliocene (capo mannu d1 local fauna, mandriola, western sardinia, delfino et al., 2011) to the pleistocene (monte tuttavista, nuoro, abbazzi et al., 2004; grotta di dragunara, capo caccia, western sardinia, kotsakis, 1980) and holocene (grotta corbeddu, oliena, central sardinia, sondaar et al., 1984; porto leccio, trinità d’agultu, northern sardinia, delfino, 2002; grotta di su guanu, gonagosula, oliena, central sardinia, sanchiz, 1979). by contrast, fossils ascribed to bufo bufo have been reported to date only once for the neolitic site of su guanu (sanchiz, 1979), co-occurring with remains undoubtedly referable to b. balearicus. the presence of b. 48 ilaria maria cossu et alii bufo at su guanu is based on fragmentary evidence represented only by two humeri (a male and a female), two radioulnae and two femura that were partly described and figured by a competent anuran expert. the referral of the material to b. bufo, the femura in particular, could be therefore correct and was considered as such in the list of the fossil salientia published nearly two decades later by the same author (sanchiz, 1998). according to these findings, despite the most supported scenario is suggesting human-mediated translocation of b. bufo to sardinia happened in very recent times, the fact that the species already occurred in the past could not be ruled out, and the relative proximity (about 50 km) of the neolithic site of su guanu and the recently discovered population here reported is curious at least. moreover, amphibian dispersal across the sea was reported several times despite the limitations related to the osmotic stress by salt water (vences et al., 2003; measey et al., 2007). it is also known that brackish waters do not prevent common toads from swimming in open water in the north baltic sea, even allowing gene flow between islands, indicating a certain degree of salt tolerance in this species (seppa and laurila, 1999). to date, even if no evidence of negative impact of bufo bufo on the sardinian population of b. balearicus was detected, further ecological and distributional studies are needed to better detail possible interactions between the common toad and the green toad that also coexist on the mainland. however, it is likely that the common toad will not be able to spread far beyond the limits imposed by its ecological requirements, which do not allow this species to persist in the xeric habitats that characterize most of the sardinian territory (costantini et al., 2005). at present, it should be acknowledged that, although possible drawbacks could not be ruled out (e.g., f1 hybrids have been sporadically detected in the wild by bressi et al., 2000; duda, 2007), the two toads actually coexist on the mainland without interfering. moreover, the arrival of a palearctic element such as bufo bufo 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(2003): multiple overseas dispersal in amphibians. proc. roy. soc. london b 270: 2435-2442. acta herpetologica vol. 13, n. 1 june 2018 firenze university press species diversity and distribution of lizards in montenegro katarina ljubisavljević1,2,*, ljiljana tomović3, aleksandar urošević1, slađana gvozdenović2, vuk iković2, vernes zagora2, and nenad labus4 the first demographic data and body size of the southern banded newt, ommatotriton vittatus (caudata: salamandridae) abdullah altunişik diet and helminth parasites of freshwater turtles mesoclemmys tuberculata, phrynops geoffroanus (pleurodira: chelidae) and kinosternon scorpioides (criptodyra: kinosternidae) in a semiarid region, northeast of brazil antonio marcos alves pereira*, samuel vieira brito, joão antonio de araujo filho, adonias aphoena martins teixeira, diêgo alves teles, daniel oliveira santana, vandeberg ferreira lima, waltécio de oliveira almeida electric circuit theory applied to alien invasions: a connectivity model predicting the balkan frog expansion in northern italy mattia falaschi1,2,*, marco mangiacotti3, roberto sacchi3, stefano scali2, edoardo razzetti4 first report of bufo bufo (linnaeus, 1758) from sardinia (italy) ilaria maria cossu1, salvatore frau1, massimo delfino2,3, alice chiodi4, claudia corti1,5*, adriana bellati4 natural history and conservation of the nurse frog of the serranía del perijá allobates ignotus (dendrobatoidea: aromobatidae) in northeastern colombia hernán darío granda-rodríguez1,2, andrés camilo montes-correa3,*, juan david jiménez-bolaño3, marvin anganoy-criollo4,5 exploring body injuries in the horseshoe whip snake, hemorrhois hippocrepis juan m pleguezuelos*, esmeralda alaminos, mónica feriche how effectively do european skinks thermoregulate? evidence from chalcides ocellatus, a common but overlooked mediterranean lizard grigoris kapsalas, aris deimezis-tsikoutas, thanos georgakopoulos, ismini gkourtsouli-antoniadou, kallirroi papadaki, katerina vassaki, panayiotis pafilis thermal tolerance for two cohorts of a native and an invasive freshwater turtle species jun geng, wei dang, qiong wu, hong-liang lu* diet of a restocked population of the european pond turtle emys orbicularis in nw italy dario ottonello1, fabrizio oneto1,2, monica vignone2, anita rizzo2, sebastiano salvidio2,* helminths of the lizard colobosauroides cearensis (squamata, gymnophthalmidae) in an area of caatinga, northeastern brazil aldenir f. da silva neta*, robson w. ávila acta herpetologica 13(2): 201-205, 2018 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-23582 a lizard acting as carrier of the amphibian-killing chytrid batrachochytrium dendrobatidis in southern brazil mariana r. pontes1,2,*, guilherme augusto-alves1,3, carolina lambertini1,3, luís felipe toledo1 1 laboratório de história natural de anfíbios brasileiros (lahnab), departamento de biologia animal, instituto de biologia, universidade estadual de campinas, campinas, são paulo, 13083-862, brazil. *corresponding author. e-mail: maah.retuci@gmail.com 2 programa de pós-graduação em ecologia, instituto de biologia, universidade estadual de campinas (unicamp), campinas, são paulo, brazil 3 programa de pós-graduação em biologia animal, instituto de biologia, universidade estadual de campinas (unicamp), campinas, são paulo, brazil submitted on: 2018, 3th march; revised on: 2018, 10th august; accepted on: 2018, 29th september editor: dario ottonello abstract. fungal infections are causing widespread population declines and extinctions in all vertebrate classes. among them, an important fungal disease chytridiomycosis, caused by the pathogenic chytrid batrachochytrium dendrobatidis (bd). with an aquatic infectious phase,  bd  does not survive desiccation for long, but may be transported by non-amphibian carriers. such mechanism is key to understand amphibian-chytrid dynamics and may contribute to local amphibian conservation action plans. therefore, we surveyed bd in reptiles from two different brazilian rainforests, looking for possible bd carriers. we sampled 35 individuals belonging to 11 squamate families, five from the atlantic forest and 30 from the amazon. we detected bd in one adult lizard, placosoma glabellum. this lizard feeds, shelters, and breeds in the leaf-litter, and moves between atlantic forest streams. hence, it may be carrying bd from stream to stream, and also spreading the pathogen to direct-developing amphibians, which have no contact with water bodies and are more susceptible to chytridiomycosis than aquatic species. this is the first record of a non-amphibian chytrid carrier in south america. we suggest that additional field and museum samplings will contribute to understand whether bd can actually infect reptiles, and how reptile carriers can affect chytrid dynamics in the wild. keywords. amphibia, reptilia, squamata, amazon, atlantic forest, chytridiomycosis, infection dynamics, pathogen vector. infectious diseases, caused by a diversity of pathogenic agents, have become one of the most important threats to wildlife (daszak et al., 2000; daszak et al., 2013). for example, fungal infections are responsible for worldwide vertebrate population declines (blehert et al., 2009; turner et al., 2011; fisher et al., 2012; lorch et al., 2016), especially jeopardizing amphibians (berger et al., 1998; lips et al., 2006). chytridiomycosis is an amphibian infectious disease caused by the waterborne fungus batrachochytrium dendrobatidis (hereafter bd) (longcore et al., 1999). bd infects epidermal tissues of amphibian hosts, affecting essential physiological processes as osmoregulation (berger et al., 2005; van rooij et al., 2015), which may cause sub-lethal effects (e.g., bovo et al., 2016, salla et al., 2015), or death of infected hosts through asystolic cardiac arrest, as a consequence of reductions on blood plasma sodium and potassium concentrations (voyles, 2009). it has also been noticed that bd produces toxic factors that inhibits lymphocyte proliferation on hosts (fites et al., 2013). chytridiomycosis is associated with massive losses of amphibian populations all over the globe (olson and 202 mariana r. pontes et alii ronnenberg, 2014; james et al., 2015; carvalho et al., 2017). this pathogen has a biphasic life-cycle, with flagellated free-swimming zoospores, often discharged into water bodies as streams and ponds, and sessile zoosporangia with intracellular development (rosenblum et al., 2010; greenspan et al., 2012). bd does not survive desiccation due to its aquatic life-cycle (johnson et al., 2003), but may be carried by non-amphibian species (mcmahon et al., 2013), which might contribute to pathogen transmission (kilburn et al., 2011). past studies have shown that non-amphibian organisms such as algae (johnson and speare, 2003), nematodes (shapard et al., 2012), crayfishes (brannelly et al., 2015), reptiles (kilburn et al., 2011), and waterfowls (garmyn et al., 2012) also transport bd zoospores. in this sense, besides the studies with amphibian hosts it is essential to increase knowledge about other organisms that may be acting as carriers of this pathogen. thus, since there are no bd records for reptiles in south america, here in order to find additional bd potential carriers we swab-sampled reptiles from brazil’s atlantic forest and amazon, both tropical rainforests, but different in terms of bd occurrence and infection intensity (carvalho et al., 2017; lambertini et al., 2017). we swabbed reptiles collected from the states of paraná, são paulo and espírito santo in brazil’s atlantic forest, and from the state of pará in the brazilian amazon. we sampled a total of 35 individuals of 11 squamata families, five from the atlantic forest and 30 from the amazon (table 1). we swabbed live individuals throughout their body, ventral and dorsal surfaces, limbs (for lizards), and head (as in hyatt et al., 2007) to test for the presence of bd zoospores. we then extracted dna from each swab using prep-man ultra® (life technologies) and proceeded with taqman® qpcr assay for bd detection and quantification (boyle et al., 2004; lambertini et al., 2013). we considered positive results greater than or equal to one zoospore genomic equivalent (ge) (kriger et al., 2007). we detected one bd positive individual, placosoma glabellum (peters, 1870) (currently housed at museu de zoologia “prof. adão j. cardoso” universidade estadual de campinas: zuec-rep 3735), with a load of 3.14 bd zoospore ge. to confirm the positive result, we rinsed this individual in running water for 10 minutes (following the protocols of rodriguez et al., 2014 and becker et al., 2016), swabbed the lizard again and run an additional qpcr experiment. in the second run we detected a load of 1.67 zoospore ge. this is the first report of bd in a reptile from south america. this pathogen has already been detected in lizards and snakes from panama, also by qpcr detection and without histological analysis, which would confirm whether these reptiles are infected with bd or only act as carriers (kilburn et al., 2011). anyway, despite the absence of histological analyses, it is clear that reptiles, including arboreal snakes and semiaquatic or terrestrial lizards, carry bd, contributing to zoospore dispersion table 1. sampled species for batrachochytrium dendrobatidis presence. number of sampled individuals is in parentheses. species in bold was positive for bd. taxonomy follows costa and bérnils (2018). species municipality, state biome squamata: “lizards” dactyloidae norops sp.(1) belém, pará amazon gekkonidae hemidactylus mabouia (1) belém, pará amazon gymnophthalmidae leposoma scincoides (1) santa teresa, espírito santo atlantic forest placosoma glabellum (2) morretes, paraná atlantic forest phyllodactylidae thecadactylus rapicauda (2) belém, pará amazon polychrotidae polychrus sp.(1) belém, pará amazon mabuyidae copeoglossum nigropunctatum (1) belém, pará amazon sphaerodactylidae chatogekko amazonicus (1) belém, pará amazon coleodactylus sp. (1) belém, pará amazon gonatodes sp.(1) belém, pará amazon lepidoblepharis sp. (2) belém, pará amazon tropiduridae plica umbra (3) belém, pará amazon uranoscodon sp. (3) belém, pará amazon squamata: “serpentes” boidae boa constrictor (1) belém, pará amazon dipsadidae dipsas catesbyi (1) belém, pará amazon erythrolamprus sp. (1) belém, pará amazon helicops angulatus (2) acará, pará amazon imantodes cenchoa (2) belém, pará amazon imantodes lentiferus (1) acará, pará amazon leptodeira annulata (1) belém, pará amazon oxyrhopus clathratus (1) peruíbe, são paulo atlantic forest thamnodynastes sp. (1) dores do rio preto, espírito santo atlantic forest typhlopidae amerotyphlops reticulatus (4) belém, pará amazon 203lizard as a carrier of the amphibian chytrid across different habitats (kilburn et al., 2011; present study). the bd-positive lizard was collected on 9 january 2013, at the estrada da graciosa road (pr-410), in the municipality of morretes, state of paraná. this site is within the same area where three divergent bd lineages were isolated: bd-brazil/asia-2, bd-gpl and a hybrid between them (schloegel et al., 2012; jenkinson et al., 2016; o’hanlon et al., 2018). besides that, this individual lizard was collected on top of a rock inside a stream, syntopic to an endemic rheophilic frog species, cycloramphus rhyakonastes (anura: cycloramphidae) (nunes-de-almeida et al., 2016). this is the only known population of this species (silvano, 2004), and given that bd infected individuals were already recorded [with a prevalence of 55% (10 out of 18 sampled), l.f.t., unpublished data], continuous exchange of bd in this area can facilitate hybridization of the pathogen (jenkinson et al., 2016), producing some strains that could potentially be hypervirulent (greenspan et al., 2018), posing a real short-term threat to the endemic c. rhyakonastes. furthermore, placosoma glabellum is a terrestrial small-size lizard with distribution restricted to south and southeast of brazil (uetz, 2017). studies have shown that bd can survive up to seven weeks out of amphibian hosts, specifically in lake water (johnson and speare, 2003). also, it can survive up to three hours on dry bird feathers (johnson and speare, 2005). this ability to survive may lead to a greater range of bd zoospore dissemination by other organisms. in this specific case, the bd-positive lizard species could carry bd zoospores from the stream into the forest, potentially transmitting this pathogen to other water bodies, or to direct-developing anurans that lives in leaf-litter inside forested areas, and can be less tolerant to chytridiomycosis than indirectdeveloping aquatic anurans (mesquita et al., 2017). given that reptiles are more vagile than amphibians, and less restricted to water bodies, they may play an important role in bd dissemination in the wild. in this sense, we highlight the need for more studies attempting to understand how reptiles and other non-amphibian species may contribute to bd transmission dynamics in wildlife. acknowledgments we thank alexandre f. r. missassi, anat belasen, anyelet valencia-aguilar, carlos henrique l. nunes-dealmeida, clarisse betancourt-roman, david rodriguez, kelly r. zamudio, timothy y. james, and thomas s. jenkinson for field assistance. this work was funded by são paulo research foundation (fapesp #2016/25358-3) and national council for scientific and technological development  (cnpq #300896/2016-6). mrp and cl thank the coordination for the improvement of higher education personnel (capes 001) for fellowships. sampling permit was approved by instituto chico mendes de conservação da biodiversidade (icmbio 17242-3) and is in accordance with sistema nacional de gestão do patrimônio genético e do conhecimento tradicional associado (sisgen a60b4d9). references becker, c.g., rodriguez, d., lambertini, c., toledo, l.f., haddad, c.f.b. 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(2009): pathogenesis of chytridiomycosis, a cause of catastrophic amphibian decline. science 326: 582-585. acta herpetologica vol. 13, n. 1 june 2018 firenze university press acta herpetologica 15(1): 55-57, 2020 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/a_h-8871 adaptive significance of the transparent body in the tadpoles of ornamented pygmy frog, microhyla ornata (anura, amphibia) santosh m. mogali*, bhagyashri a. shanbhag, srinivas k. saidapur department of zoology, karnatak university, dharwad-580 003, karnataka state, india * corresponding author. email: santoshmogali@rediffmail.com submitted on: 2019, 21st april; revised on: 2019, 24th december; accepted on: 2020, 6th march editor: raoul manenti abstract. in southern india, during the southwest monsoon phase, the newly formed ephemeral water bodies harbour several species of tadpoles as well as some of their predators. tadpoles of microhyla ornata dwell in surface or column zones of water. they face predation threat from aquatic insect predators and carnivorous tadpoles of other anurans though they are invisible due to their transparent body form. we tested whether transparent body form of m. ornata tadpoles is a useful attribute against predation by exposing tail fin stained (with the nile blue) subjects to a naturally occurring predator (hoplobatrachus tigerinus tadpoles that detect prey using both visual and chemical cues). the study shows that susceptibility of stained m. ornata tadpoles to predation increased significantly compared to the unstained transparent individuals. we conclude that the transparent body form is of great significance in escaping predation during the larval phase of life in m. ornata. keywords. hoplobatrachus tigerinus, microhyla ornata, predation, tadpoles, transparent body most anuran amphibians are opportunistic breeders; in south india, they often breed communally in rainfilled puddles soon after the onset of southwest monsoon. therefore, tadpoles of several species coexist during their aquatic larval phase (saidapur et al., 2009). in such ephemeral ponds, the tadpoles commonly experience desiccation threat, competition for resources from homospecific and heterospecific members (mogali et al., 2011a, b, 2015, 2017). in addition, predation threat from aquatic insects and their larvae are routinely encountered by the herbivorous tadpoles in such ephemeral ponds (skelly, 1997). furthermore, the tadpoles of several species are carnivorous or omnivorous (saidapur et al., 2009). in response to coexisting predators, the prey tadpoles have evolved defence strategies that often involve utilization of refuge shelters when available, reduced movements, high burst speed when movement is necessary, species-specific habitat choices (hiragond and saidapur, 2001; saidapur et al., 2009; hossie and murray, 2010; mogali, 2018; mogali et al., 2019) and so on. within the community of anuran larvae found in the ephemeral water bodies of southern india, tadpoles of hoplobatrachus tigerinus are notorious predators and hunt actively (saidapur, 2001; saidapur et al., 2009). of the several species of anuran larvae found in southern india, the tadpoles of microhyla ornata are unique in many aspects; they are predominantly found in the surface or column zone of the pond, devoid of teeth, feed on micro-plankton, delicate, slow movers, remain still for longer time and more importantly, their body is transparent (hiragond and saidapur, 2001) and hence not easily visible. despite a highly vulnerable nature, m. ornata is highly successful and show wide distribution throughout asia and the indian subcontinent. evidently, the success of any anuran species with aquatic larval phase depends on the survival rate of 56 santosh m. mogali et alii their larvae, emergence on the land with optimum size and, subsequent reproduction. we hypothesized that transparent body of m. ornata tadpoles is a key feature in ensuring their survival and escape from predation. therefore, we conducted experiments on m. ornata tadpoles whose tail fins were stained with nile blue or left unstained and then tested for their efficiency to escape from predation using highly predacious sympatric tadpoles of h. tigerinus. six egg clutches of m. ornata were collected from rain-filled puddles/ ponds located on the karnatak university campus, dharwad (latitude 15.440407°n, longitude 74.985246°e) in june 2008. each egg clutch was placed separately in plastic tubs (42 cm diameter and 16 cm deep) containing 3 l of pond water. eggs from all clutches hatched almost synchronously at gosner stage 19 (gosner, 1960) a day after their collection. soon after hatching, the tadpoles of different clutches were mixed and reared in 3 separate glass aquariums (75 cm l × 45 cm w × 15 cm h) each with ~ 600 tadpoles in 15 l of pond water to provide plankton material for feeding. the tadpoles in the gosner stage 25-26 with comparable body size (length 12.65 ± 0.42 mm, mean ± se) were used in trials. the tadpoles of h. tigerinus (gosner stage 27-28; ~ n = 70) were also collected from the rain-filled ponds located on the university campus. they were reared individually to avoid cannibalism in plastic bowls (16 cm diameter and 7 cm deep) in 0.5 l of aged tap water. hoplobatrachus tigerinus tadpoles of gosner stage 29-30 having comparable body size (length 32.15 ± 0.49 mm mean ± se) were used in the trials. the experiment was made with two sets of tadpoles of m. ornata (n = 25 per set) chosen randomly. in the first set of experiment, tadpoles of m. ornata (n = 25) with a natural transparent body, were released into a rectangular glass tank (40 cm l × 25 cm w × 30 cm h) with 10 l of aged tap water that created a column height of 10 cm. after acclimatization for 15 min, a single h. tigerinus tadpole (starved for 24 h) was introduced into the tank and left there for 24 h to estimate the quantum of predation. after the trial period, the number of surviving prey tadpoles was recorded to compute the number of tadpoles lost due to predation. in the second set of experiment, the tail fins of prey tadpoles were applied nile blue (1 mg/ml water and filtered by using whatman filter paper no. 40) using a fine brush before the trials. the nile blue coloured tadpoles (n = 25) were left with one starved h. tigerinus tadpole for 24 h as in the first set of experiment. after completion of the trial duration, the number of tadpoles consumed by the predator was recorded. twenty-five trials were carried out for both sets of experiments. the data on the number of tadpoles consumed by the predator between the two experiments were analyzed by the mann-whitney u test. the results show that the predator consumed a significantly greater number of prey tadpoles that had blue tinge on their tail fins (u = 15.500, p < 0.001, fig. 1) compared to those that did not have any colour on the tail fins. thus, the insatiable predatory tadpoles of h. tigerinus that are active chase hunters predated a significantly smaller number of prey subjects that were transparent compared to those whose tail fins showed blue tinge due to nile blue. most previous studies on prey-predator interactions have dealt with the tadpoles residing in the substrate or benthic zones (relyea, 2001; saidapur et al., 2009; jara and perotti, 2010; mogali et al., 2011a, 2012). it may be noted that the aquatic insects and their larvae that prey on tadpoles are generally found along with anuran larvae at the substratum and these are mostly ‘sit and wait’ predators; they wait for the prey to come near before attacking them (luttbeg et al., 2008; miller et al., 2014). microhyla ornata tadpoles are principally surface/ column zone dwellers (hiragond and saidapur, 2001). therefore, one can expect the least predation of m. ornata tadpoles by the aquatic insects and their larvae. on the other hand, predatory h. tigerinus tadpoles are generally substrate dwellers but also capable of moving all over. besides they are active chase hunters which make use of both visual as well as chemical cues of the prey subjects in devouring them (hiragond and saidapur, 2001; saidapur et al., fig. 1. number of microhyla ornata (transparent or tail-fin stained) tadpoles consumed by the predator, hoplobatrachus tigerinus tadpoles in trials of 24 h. data represent mean ± se and analyzed by mann-whitney u test; n = 25 trials per group with 25 tadpoles per trial. an asterisk indicates a significant difference between the groups (p < 0.001). 57adaptive significance of transparent body in microhyla ornata 2009). the predation threat from such predators is enormous even to surface dwellers like the m. ornata tadpoles. the present findings clearly show that m. ornata tadpoles have a greater capacity to escape predation from the active and chase hunting h. tigerinus tadpoles. however, a mere blue tinge of tail fins following application of the vital dye nile blue made them highly susceptible to predation. nile blue used in this study was in micro quantities. hence, in such large quantity of water, chemical cues arising from the dye, if any, would be presumably very weak. however, the study shows that h. tigerinus tadpoles perceive even slightly coloured blue tinged m. ornata tadpoles more efficiently than that of the transparent subjects. thus, it appears that less vulnerability of this tadpole species to predation is largely due to its unique transparent body form. the present study, however, does not convey us about the perception of the type of visual cues arising after application of nile blue to prey tadpoles by h. tigerinus tadpoles. therefore, further additional and differently designed studies are needed to decipher the impact of different colours/ wavelengths to know what exactly is perceived by the predator. in conclusion, the transparent body form of m. ornata tadpoles appears to be a key feature ensuring larval survival and escape from predators. acknowledgements bas is thankful to indian national science academy (insa), new delhi for support. this research was conducted according to ethical guidelines laid down by cpcsea, new delhi under registration no. 639/02/a/ cpcsea. references gosner, k.l. (1960): a simplified table for staging anuran embryos and larvae with notes on identification. herpetologica 16: 183-190. hiragond, n.c., saidapur, s.k. (2001): microhabitat choice of tadpoles of seven anuran species. curr. herpetol. 20: 51-60. hossie, t.j., murray, d.l. (2010): you can’t run but you can hide: refuge use in frog tadpoles elicits densitydependent predation by dragonfly larvae. oecologia 163: 395-404. jara, f.g., perotti, m.g. (2010): risk of predation and behavioural response in three anuran species: influence of tadpole size and predator type. hydrobiologia 644: 313-324. luttbeg, b.j., hammond, i., sih, a. (2008): dragonfly larvae and tadpole frog space use games in varied light conditions. behav. ecol. 20: 13-21. miller, j.r.b., ament, j.m., schmitz, o.j. (2014): fear on the move: predator hunting mode predicts variation in prey mortality and plasticity in prey spatial response. j. animal ecol. 83: 214-222. mogali, s. (2018): predatory cues influence the behavioral responses and metamorphic traits of polypedates maculatus (anura: rhacophoridae). asian herpetol. res. 9: 188-194. mogali, s.m., saidapur, s.k., shanbhag, b.a. (2011a): levels of predation modulate antipredator defense behaviour and metamorphic traits in the toad bufo melanostictus. j. herpetol. 45: 428-431. mogali, s.m., saidapur, s.k., shanbhag, b.a. (2011b): receding water levels hasten metamorphosis in the frog, sphaerotheca breviceps (schneider, 1799): a laboratory study. curr. sci. 101: 1219-1222. mogali, s.m., saidapur, s.k., shanbhag, b.a. (2012): tadpoles of the bronze frog (rana temporalis) assess predation risk before evoking antipredator defense behavior. j. ethol. 30: 379-386. mogali, s.m., shanbhag, b.a., saidapur, s.k. (2015): strong food odors mask predation risk and affect evocation of defense behaviors in the tadpoles of sphaerotheca breviceps. j. ethol. 33: 41-46. mogali, s., saidapur, s., shanbhag, b. (2017): influence of desiccation threat on the metamorphic traits of the asian common toad, duttaphrynus melanostictus (anura). acta herpetol. 12: 175-180. mogali, s., shanbhag, b., saidapur, s. (2019): experience of predacious cues and accessibility to refuge minimize mortality of hylarana temporalis tadpoles. acta herpetol. 14: 15-19. relyea, r.a. (2001): morphological and behavioral plasticity of larval anurans in response to different predators. ecology 82: 523-540. saidapur, s.k. (2001): behavioral ecology of anuran tadpoles: the indian scenario. proc. indian natn. sci. acad. (pinsa) b67: 311-322. saidapur, s.k., veeranagoudar, d.k., hiragond, n.c., shanbhag, b.a. (2009): mechanism of predator-prey detection and behavioral responses in some anuran tadpoles. chemoecology 19: 21-28. skelly, d.k. (1997): tadpole communities: pond permanence and predation are powerful forces shaping the structure of tadpole communities. am. sci. 85: 36-45. acta herpetologica vol. 15, n. 1 june 2020 firenze university press has the west been won? a field survey and a species distribution model of iberolacerta horvathi in the alps giuseppe de marchi1,*, giovanni bombieri1, bruno boz1, fausto leandri2, jacopo richard3 first record of underwater sound produced by the balkan crested newt (triturus ivanbureschi) simeon lukanov identification of biologically active fractions in the dermal secretions of the genus bombina (amphibia: anura: bombinatoridae) iryna udovychenko1,*, oleksandra oskyrko1, oleksii marushchak2, tetiana halenova1, oleksii savchuk1 don’t tread on me: an examination of the anti-predatory behavior of eastern copperheads (agkistrodon contortrix) andrew adams1,2,*, john garrison2, scott mcdaniel2, emily bueche2, hunter howell2,3 an overview of research regarding reservoirs, vectors and predators of the chytrid fungus batrachochytrium dendrobatidis jarid prahl, thomas p. wilson*, david giles, j. hill craddock genetic characteristics of an introduced population of bombina bombina (linnaeus, 1761) (amphibia: bombinatoridae) in moselle, france jean-pierre vacher1, 2,*, damien aumaître3, sylvain ursenbacher2,4 adaptive significance of the transparent body in the tadpoles of ornamented pygmy frog, microhyla ornata (anura, amphibia) santosh m. mogali*, bhagyashri a. shanbhag, srinivas k. saidapur comparison of complement system activity amongst wild and domestic animals maría s. moleón1,2,*, mark e. merchant3, hugo h. ortega4, pablo a. siroski1,2 effects of temperature and food level on plasticity of metamorphic traits in bufo gargarizans gargarizans larvae tong lei yu*, yan ting han acta herpetologica 10(2): 111-120, 2015 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-17171 a new species of chameleon (sauria: chamaeleonidae: kinyongia) highlights the biological affinities between the southern highlands and eastern arc mountains of tanzania michele menegon1,*, simon p. loader2, tim r.b. davenport3, kim m. howell4, colin r. tilbury5, sophy machaga3, krystal a. tolley5,6 1 tropical biodiversity section, museo delle scienze, corso  del lavoro e della scienza 3, 38122 trento, italy. *corresponding author. e-mail: michele.menegon@muse.it 2 department of life sciences, university of roehampton, holybourne avenue, room 1053, london sw15 4jd united kingdom 3 wildlife conservation society (wcs), po box 1475, mbeya & po box 922, zanzibar, tanzania 4 department of zoology & wildlife conservation, po box 35064, university of dar es salaam, dar es salaam, tanzania 5 south african national biodiversity institute, private bag x7, claremont, cape town, south africa 6 department of botany & zoology, university of stellenbosch private bag x1, matieland, 7602, stellenbosch, south africa submitted on 2015, 10th october; revised on 2015, 25th october; accepted on 2015, 27th october editor: sebastiano salvidio abstract. a new species of chameleon is described from the livingstone and udzungwa mountains of tanzania. the new species is morphologically most similar to kinyongia vanheygeni. furthermore, a single, short rostral appendage shows the species similarity to other eastern arc endemic kinyongia species (e.g. k. uthmoelleri, k. oxyrhina, k. magomberae and k. tenuis). females of all these species lack any rostral ornamentation and are all very similar morphologically. males of the new species, on which the morphological diagnosis is based, can be distinguished from other kinyongia by a shorter rostral appendage that bifurcates at the tip. they are easily distinguished from k. vanheygeni, otherwise the most similar species, by differences in head scalation and the length and shape of the rostral appendage. the new species is associated with montane rainforest and is known from only four forest fragments of which two are in the udzungwa and two in the livingstone mountains. phylogenetically, the new species is sister to k. tenuis and k. magomberae, which together, form a clade that also contains k. oxyrhina. the disjunct distribution of the new species, in the livingstone and udzungwa mountains, stretches across the ‘makambako gap’ which is a putative biogeographical barrier separating the distinct faunas of the southern highlands and eastern arc mountains. evidence from this species however, points to potentially closer biological affinities between the livingstone and udzungwa mountains. keywords. southern highlands, tanzania, eastern afromontane, biodiversity, chamaeleonidae, east africa, new species, reptiles. introduction exploration and subsequent research in the past decades have substantially improved our understanding of the biodiversity from the eastern afromontane region (ear), which is known for its high species richness (menegon and davenport, 2008). collectively the eastern arc mountains (eam) and southern highlands of tanzania form a system of mountain blocks spanning from southern kenya, through tanzania and into malawi (lovett and wasser, 1993). because of the prevailing climatic influence from the warm indian ocean, the eam receives high orographic rainfall providing a relatively stable climate. this climate stability is thought to have reduced extinction rates for forest endemic taxa (e.g., tolley et al., 2011; loader et al., 2014). this, coupled to ele112 michele menegon et alii vated speciation rates as a result of specialization due to ecotones between forest and savanna (caro et al., 2013), has presumably resulted in high diversity and endemism across the eam (loader et al., 2015). our understanding of more general evolutionary patterns and processes for the eam, including biogeographic patterns has increased dramatically for some taxonomic groups (e.g., tolley et al., 2011; dimitrov et al., 2012; loader et al., 2014). despite this, a number of vertebrate species are discovered and described each year (e.g., rovero et al., 2014), indicating that our knowledge is far from complete in this region. a prime example are chameleons, for which new species are steadily being described (tolley and menegon, 2013), or previously named taxa are elevated from synonymy (tilbury and emmrich 1996; menegon et al., 2002; mariaux and tilbury, 2006; tilbury et al., 2006; mariaux et al., 2008, tilbury and tolley, 2009; menegon et al., 2009; greenbaum et al., 2012; branch et al., 2014) and these contributions have subsequently been utilized for revealing broader evolutionary patterns (tolley et al., 2011; tolley et al., 2013; ceccarelli et al., 2014). essentially, the scientific focus on eam region has led to a substantial increase in knowledge on the flora and fauna, but the biota of the southern highlands, which is separated from the more northern lying eam by the dry, low-lying makambako gap (lovett and wasser, 1993), is relatively poorly known. indeed, the makambako gap is considered an important turn-over region (e.g., rovero et al., 2014) and as a result, many biodiversity studies have instead focused on the eam because of its known biological wealth (e.g. newmark, 1998; stanley et al., 1999). chameleons in the genus kinyongia (tilbury et al., 2006) are a prominent group in the eam because findings have contributed to a broader understanding of species richness, endemism, and biogeography (e.g., tolley et al., 2011). twelve of the 16 described species of kinyongia occur on isolated massifs within the eam, with the remainder found to the northwest in mountainous regions of the albertine rift in democratic republic of the congo, uganda and rwanda and on isolated volcanoes, such as kilimanjaro, meru, mt. kenya (tilbury, 2010; tolley et al., 2011; greenbaum et al., 2012). many species have small distributional ranges, and are usually found on the forested slopes of just one or a few isolated massifs. their isolated and restricted distributions have provided evidence for a long history of persistence in eam, and as well as allowed inferences as to the formation and maintenance of refugial areas (tolley et al., 2011). new biological surveys in unexplored regions such as southern tanzania continually reveal the presence of new species, including matilda’s horned viper atheris matildae (menegon et al., 2011), the two chameleons kinyongia vanheygeni (necas, 2009) and k. magomberae (menegon et al. 2009), and africa’s only new genus of monkey described in the last 80 years, the kipunji rungwecebus kipunji (davenport et al., 2006). unexplored forests to the south of the eam (e.g. southern highlands) are therefore predicted to contain a host of species not yet known to science. in this study, we describe a new chameleon species in the genus kinyongia (fig. 1) that is found in afrotemperate forest from both the livingstone (southern highlands) and udzungwa (eam) mountains (fig. 2). using both morphological and molecular evidence, we determine the taxonomic placement and evolutionary relationships for this new taxon. both morphological characters and genetic markers were examined and compared to the other species of kinyongia. furthermore we examine the biogeographical implications of this new taxon given the phylogenetic hypothesis inferred from the data. materials and methods material examined the following specimens (table 1) were examined from the herpetological collections of the science museum of trento, trento, italy (mtsn and muse), the department of zoology & wildlife conservation of the university of dar es salaam, dar es salaam, tanzania and the collection of the wcs’ southern highlands conservation project (shcp), mbeya, tanzania: k. vanheygeni (muse 13523 and muse 13524 from mt. rungwe), k. tenuis (kmh 21325 and kmh 21304 from nilo forest reserve, east usambara mts.), k. oxyrhina (kmh 28277 from ukami forest, udzungwa mts.; kmh 28302 from nyumbanitu forest, udzungwa mts., mtsn 8454 and mtsn 8412 from nguru south forest reserve), k. tavetana (mtsn 8658 and mtsn 8661 from kindoroko forest reserve, north pare mts.) molecular analysis to understand the phylogenetic placement the new kinyongia species a phylogenetic analysis was carried out which included 10 individuals from the two mountain ranges, plus multiple representatives from 17 of 19 kinyongia species from published datasets (menegon et al., 2009; tolley et al., 2011; greenbaum et al., 2012). the resulting dataset consisted of 47 individuals, including the outgroup taxa (bradypodion pumilum and b. melanocephalum). dna extraction, pcr amplification, and cycle sequencing of two mitochondrial gene fragments (nd2 and 16s) were carried out following standard procedures using the following primers for nd2: l4437b and h5934 (macey et al., 1997a, b), and 16s: l2510 and h3080 (palumbi, 1996). standard pcr and sequencing were followed for this gene fragment, with pcr annealing temperature at 57˚c. all 113a new species of chameleon from tanzania new sequences were deposited in european nucleotide archive (table 2). bayesian inference was used to investigate optimal tree space using mrbayes 3.1.2 (huelsenbeck and ronquist, 2001) for the combined mitochondrial markers (321 characters, partitioned by marker: nd2, 856 bp; 16s, 465 bp), although 18 bases were excluded for 16s due to ambiguous alignment. to investigate which evolutionary model best fit the data, jmodeltest was used (posada, 2008), and the aic test indicated the same model for all markers was appropriate (gtr + g). therefore, mrbayes was run specifying six rate categories with uniform priors for the gamma distribution for each of the partitions. to ensure the results were robust for both datasets, the mcmc was run twice in parallel for 10 million and 20 million generations (four chains in each run), with trees sampled every 1000 generations. burn-in was estimated as 1 million generations (1000 trees), as determined by examination the average standard deviation of split frequencies, the convergence diagnostic (psrf values ~ 1.0) as well as the log-probabilities and the values of each parameter for stabilization (ronquist and huelsenbeck, 2003). in addition, tracer v1.4.1 (rambaut and drummond, 2007) was used to check that the effective sample size (ess) of all parameters was greater than 200 after burn-in. a 50% majority rule tree was constructed and nodes with ≥ 0.95 posterior probability considered supported. in addition to the bayesian analysis, a maximum likelihood (ml) search was run for both datasets using raxml hpc 7.2.8 (stamatakis, 2006). the datasets were partitioned as in the bayesian analysis, with a gtr+i+g model for all markers and rapid bootstrapping halted automatically (stamatakis et al., 2008) this analysis was run three times to ensure that independent ml searches produced the same topologies. we considered nodes with a bootstrap value of ≥ 70% as supported in this analysis. both bayesian and likelihood analyses were run on the cipres science gateway (miller et al., 2010; www.phylo. org/sub_sections/portal/). finally, to provide a rough indicafig. 1. kinyongia msuyae sp. nov. from livingstone mountains in life. pictures showing (upper) adult male, (lower left) close up of male head, (lower right) adult female. 114 michele menegon et alii tion of the degree of divergence between species, uncorrected p-distances were estimated in mega 5.05 for 16s which had the most complete taxon sampling (tamura et al., 2011). results molecular analysis the likelihood and bayesian searches produced the same topology and supported nodes (fig. 3). the phylogenetic analysis showed that the kinyongia sampled from the livingstone and the udzungwa mountains form a well-supported clade. the uncorrected net sequence divergence (p-distance) between this clade and other closely related species of kinyongia range from 1.43.7% (table 3), which is similar to within species values of other chameleons (e.g. menegon et al., 2009; tilbury and tolley; 2009, greenbaum et al., 2012, tolley et al., 2012; branch et al., 2014). although sequence divergence between chameleons from livingstone and udzungwa (< 1.3% for the 16s marker) are close to the lowest values found between some other kinyongia (e.g. k. tenuis and k. magomberae; table 3), additional samples from udzungwa would be needed to obtain a more accurate estimate of sequence divergence between the mountain ranges. given the genetic distinctiveness, we take the opportunity to describe the individuals from livingstone and udzungwa mountains as a new species. taxonomy kinyongia msuyae sp. nov (fig. 1; 3) holotype: adult male in the science museum of trento, mtsn 9374 collected in mdandu forest reserve, livingstone mountains in january 2011 by michele menegon, tim davenport, simon loader, sandra dürrenberger, sandra rudolf and sophy machaga type locality: mdandu forest reserve, livingstone mountains 1900 m above sea level, mbeya region, south eastern tanzania (-9.769549621; 34.78832024) k. oxyrhina k. vanheygeni k. magomberae k. tenuis k. msuyae k. uthmoelleri other kinyongia species k. cf. oxyrhina possible dispersal routes makambako gap udzungwa mts. southern highlands (livingstone mts.) mahenge mts. kilombero valley fig. 2. distribution of kinyongia species in the eastern afromontane region. inset map shows enlargement of udzungwa and southern highlands region of tanzania with possible dispersal routes of montane associated species. table 1. biometrics of the holotype and paratypes. continuous measurements given in mm. total length svl tl head length head width casque lenght casque eye snouth lenght eye diameter eye-eye gap upper labials rostral process mtsn 9374 150.74 69.56 81.18 23.54 10.12 16.68 10.78 7.02 6.61 5.38 20 4.62 mtsn 9375 142.3 65.02 77.28 23.18 10.42 15.11 10.29 8.14 6.69 5.39 20 4.4 mtsn 9898 103.77 43.84 59.93 17.55 18.21 10.58 7.98 6.39 4.88 4.52 18 3.13 mtsn 9373 129.44 57.46 71.98 19.64 9.29 13 8.04 6.58 5.38 5.37 14 no mtsn 9377 103.5 52.69 50.81 15.75 7.77 10.11 6.12 5.99 4.53 4.41 14 no mtsn 7497 124.46 61.99 62.47 21.08 11.13 13.39 8.23 7.07 7.75 5.93 13 no mtsn 9378 97.36 40.2 57.16 13.31 6.59 7.79 6.21 4.37 4.61 3.6 14 no muse 13521 141. 89 68.35 73.54 22.57 11.03 14.99 10.03 7.56 7.06 5.41 19 3.63 muse 13522 145.53 64.25 81.28 22.37 10.31 15.66 10.36 7.14 6.39 5.01 17 4.87 115a new species of chameleon from tanzania paratype: mtsn 9375, adult male, mtsn 9373, mtsn 9377, adult females; mtsn 9378, juvenile, same data as holotype. mtsn 7497 collected in sakara nyumo forest reserve (livingstone mountains) in january 2011 by michele menegon, tim davenport, simon loader and sophy machaga; mtsn 8686 (adult male) collected in kigogo forest reserve, udzungwa mountains in february 2006 by michele menegon; referred material: muse 13521 (field number cam 1013) collected in kigogo forest reserve by charles a. msuya. muse 13522 (field number kmh 28302), adult male, collected in nyumbanitu forest reserve by louis hansen. diagnosis: a small, elongated chameleon, lacking distinctive colours or pattern, with a tail longer than the snout-vent length. it has a short, bone-based rostral appendage formed by a converging, scaly elongation of the canthi rostrales, the areas bound by the two canthi is concave and covered in flattened scales. the tips of these elongations are free and they appear like a double-tipped ct498 ct500 mtsn 9373 mtsn 9374 mtsn 9375 mtsn 9377 mtsn 9378 mtsn 7497 ludewa mtsn 8686 livingstone mountains udzungwa mountains cas 168917 k. tenuis ct103 k. tenuis mtsn 8218 k. magomberae mtsn 8835 k. magomberae ct192 k. oxyrhina ct193 k. oxyrhina ct490 k. vanheygeni schp/08/r/50 k. vanheygeni schp/08/r/91 k. vanheygeni ct189 k. uluguruensis ct191 k. uluguruensis cas168852 k. matschiei ct105 k. matschiei cas168921 k. vosseleri ct104 k. vosseleri ct110 k. multituberculata ct111 k. multituberculata bm29 k. boehmei jm2946 k. boehmei ct334 k. fischeri mtsn8490 k. fischeri ct151 k. uthmoelleri ct339 k. uthmoelleri cas201593 k. adolfifriderici cas201594 k. adolfifriderici ebg2390 k. gyrolepis ebg2391 k. gyrolepis ct345 k. carpenteri ct346 k. carpenteri ct350 k. xenorhina ct351 k. xenorhina ct209 k. excubitor ct106 b. melanocephalum kt62 b. pumilum 0.05 substitutions/site ct113 k. tavetana ct207 k. tavetana mtsn8661 k. tavetana k. msuyae fig. 3. the best scoring maximum likelihood tree for kinyongia, with nodes supported by maximum likelihood (bootstrap > 70%) and bayesian (posterior probabilities > 0.95) analyses indicated by black circles. grey circle indicate support with bayesian posterior probabilities only. 116 michele menegon et alii table 2. museum, genbank and european nucleotide archive accession numbers (16s, nd2) for kinyongia used in this study (cas = california academy of sciences; mstn = science museum of trento (formerly museo tridentino di scienze naturali); pem = port elizabeth museum (bayworld). n/a: sequences not available. species locality id specimen 16s nd2 b. melanocephalum kwazulu-natal, south africa ct016 n/a ay289813 hf570475 b. pumilum western cape, south africa kt62 n/a ay756639 ay756689 k. adlofifriderici bwindi n.p., uganda cas201593 cas201593 dq923820 ef014304 k. adlofifriderici bwindi n.p., uganda cas201594 cas201594 gq221944 gq221965 k. boehmei taita hills, kenya bm29 n/a gq221942 gq221963 k. boehmei taita hills, kenya jm2946 n/a gq221948 gq221969 k. carpenteri rwenzori mtns, uganda ct345 pem r16572 dq923821 ef014305 k. carpenteri rwenzori mtns, uganda ct346 pem r16573 dq923822 ef014306 k. excubitor mount kenya, kenya ct209 pem r16571 dq923823 ef014307 k. fischeri nguru mountains, tanzania ct334 pem r16566 dq923829 ef014313 k. fischeri nguru mountains, tanzania mtsn 8490 mtsn 8490 gq221951 gq221971 k. gyrolepis lendu plateau, drc utep 20341 utep20341 jn602059 jn602049 k. gyrolepis lendu plateau, drc utep 20342 utep 20342 jn602055 jn602050 k. magomberae udzungwa mountains, tanzania mtsn 8218 mtsn 8218 gq221950 gq221970 k. magomberae magombera forest, tanzania mtsn 8492 mtsn 8492 gq221952 gq221972 k. matschiei east usambara mtns, tanzania cas 168852 cas 168852 fr716605 fr716641 k. matschiei east usambara mtns, tanzania ct105 n/a gq221946 gq221967 k. multituberculata west usambara mtns, tanzania ct110 pem r5735 dq923824 ef014308 k. multituberculata west usambara mtns, tanzania ct111 n/a gq221947 gq221968 k. msuyae livingstone mountains, tanzania ct498 n/a ln997632 k. msuyae livingstone mountains, tanzania ct500 n/a ln997633 ln997642 k. msuyae livingstone mountains, tanzania ludewa n/a ln997638 k. msuyae livingstone mountains, tanzania mtsn7497 mtsn7497 ln997637 ln997643 k. msuyae udzungwa mountains, tanzania mtsn8686 mtsn8686 ln997639 k. msuyae livingstone mountains, tanzania mtsn9373 mtsn9373 ln997634 ln997644 k. msuyae livingstone mountains, tanzania mtsn9374 mtsn9374 ln997635 ln997645 k. msuyae livingstone mountains, tanzania mtsn9375 mtsn9375 ln997636 ln997646 k. msuyae livingstone mountains, tanzania mtsn9377 mtsn9377 ln997647 k. msuyae livingstone mountains, tanzania mtsn9378 mtsn9378 ln997648 k. oxyrhina uluguru mountains, tanzania ct192 pem r16569 dq923831 ef014315 k. oxyrhina uluguru mountains, tanzania ct193 pem r16552 dq923832 ef014316 k. tavetana mount kilimanjaro, tanzania ct113 pem r5736 dq991233 fj717801 k. tavetana mount meru, tanzania ct207 pem r16563 dq923833 ef014317 k. tavetana north pare mountains, tanzania mtsn 8661 mtsn 8661 fr716615 fr716649 k. tenuis east usambara mtns, tanzania cas 168917 cas 168917 dq923834 ef014318 k. tenuis east usambara mtns, tanzania ct103 pem r5731 dq923835 ef014319 k. uluguruensis uluguru mountains, tanzania ct189 pem r16565 dq923825 ef014309 k. uluguruensis uluguru mountains, tanzania ct191 pem r16557 dq923826 ef014310 k. uthmoelleri south pare mtns, tanzania ct151 pem r16585 dq923836 ef014320 k. uthmoelleri mount hanang, tanzania ct339 n/a dq923837 ef014321 k. vanheygeni poroto mountains, tanzania ct490 ln997631 ln997649 k. vanheygeni poroto mountains, tanzania schp-08-r-50 ln997640 ln997650 k. vanheygeni poroto mountains, tanzania schp-08-r-91 ln997641 ln997651 k. vosseleri east usambara mtns, tanzania cas 168921 cas 168921 gq221943 gq221964 k. vosseleri east usambara mtns, tanzania ct104 n/a gq221945 gq221966 k. xenorhina rwenzori mtns, uganda ct350 pem r16570 dq923838 ef014322 k. xenorhina rwenzori mtns, uganda ct351 pem r15568 dq923839 ef014323 117a new species of chameleon from tanzania short horn protruding over the snout by 3 to 5 mm. the appendage is plated with subequal rounded tubercules. laterally, the appendage continues from the supra-orbital crest, formed by low peaked tubercles, becoming more serrated over the anterior rim, from where it continues forward as a scaly rostral short horn. in the males examined it extends between 3 and 4 mm beyond the anterior margin of the rostral scale. females lack any rostral appendage and have a lower casque. k. msuyae does resemble k. vanheygeni necas, 2009 and, to a lesser extent, k. uthmoelleri (müller, 1938) in size, general body and head shape and by possession of a single, bone-based rostral appendage in males. it differs from k. vanheygeni in the length of the rostral appendage being longer, formed by more than ten scales and pointing straight forward (less than ten scales and slightly pointing upward in k. vanheygeni), from k. uthmoelleri by having a horn-like longer rostral appendage (canthal scales in k. uthmoelleri males meet to form a ‘rostral wall’, or protruding in form of a very short rostral projection). kinyongia msuyae can easily be distinguished from the other known kinyongia species by the combination of the following characters: (1) presence of rostral process in males formed by the partial fusion of the canthi rostrales and protruding forward over the snout by 3 to 5 mm. (2) tail longer than svl in both sexes, and (3) gular, ventral and dorsal crest absent. description of the holotype adult male. total length 162.4 mm, svl 73.7, tail length 88.7. casque elongated, posteriorly raised, covered by flattened polygonal scales, giving it a smooth appearance. parietal crest formed by a series of low peaked tuberculated scales, temporal and orbital crests present. no occipital lobes. nostril posteriorly directed, positioned halfway between tip of snout and the anterior rim of the eye, and separated from upper labials by two to three rows of flattened scales. canthi rostrales converge above and before the nostrils in forming a single, short, rostral appendage with two tips, giving the appearance of two very short horns protruding beyond the rostral scale by 4.5 mm. the rostral process is completely ossified and covered by sub-equal, convex scales, the superior edge is serrated. upper labials 16, lower labials 15 on each side. the sides of gular region is lined with 6 shallow grooves on each side. there is no dorsal or gular crest, while the central part of the gular region has no groove. no signs of dorsal, or ventral crests. scales on body flat and homogeneous, arranged is small clusters, those on the upper part of the dorsum are more quadrangular and arranged in vertical rows. scales on limbs sub-equal, rounded, and flattened. tail longer than the snout/vent length, laterally compressed, and covered by quadrangular scales arranged in vertical rows. hemipenes: unenverted. colour in preservative: the overall colour is whitishgrey with few paler areas paratype variation: paratypes show no relevant morphological variation compared to the holotype. variation in scutellation and body proportions for the type series and referred material are shown in table 2. colour in life: k. msuyae is an overall brown to green chameleon, sometimes with broad pale transversal bands and scattered blue spots formed by single scales or clusters of several scales. females have often a larger round spot of contrasting colour on the flanks (fig. 1). the tip of the snout, rostral appendage and limbs and top of the casque are often brownish to grey. distribution: refer to figure 2. etymology: the species is named after and dedicated to charles a. msuya, a pioneer of tanzanian herpetology, who collected the first known specimen attributable to this species and has spent most of his life studying tanzanian wildlife. discussion the phylogenetic analyses, sequence divergence estimates, and morphological assessment suggest that there is a previously unknown but distinctive species of kinyongia from the livingstone and udzungwa mountains, which we describe as kinyongia msuyae. this species is sister to chameleons found in the eastern arc mountains (i.e. k. tenuis from usambara mountains, k. magomberae from udzungwa mountains, and k. oxyrhina from uluguru mountains). there was no clear morphological differentiation between the population on the udzungwa and that from livingstone mountains, despite these two mountain ranges being separated by table 3. sequence divergence (p-distances) for kinyongia within species (on diagonal) and between selected species for 16s (lower matrix). populations of k. msuyae from livingstone and udzungwa are given separately. n/a = not available. 1 2 3 4 5 1 k. msuyae (livingstone) 0.0006         2 k. msuyae (udzungwa) 0.0129 n/a       3 k. tenuis 0.0262 0.0292 0.0000     4 k. oxyrhina 0.0208 0.0235 0.0246 0.0000   5 k. magomberae 0.0269 0.0370 0.0137 0.0186 0.0000 118 michele menegon et alii the makambako gap (ca. 150 km apart). sequence divergence between chameleons from these localities less than what is normally found between species. population level differences may exist, but additional sampling would be required to confirm that hypothesis. the close relationship among populations has some important biogeographic implications. the southern highlands have long been regarded as isolated and not part of the eastern arc mountains, with the makambako gap considered inhospitable, preventing dispersal. however, recent molecular data has started to alter this view. phylogenetic analyses for the shrew, myosorex, suggest that the makambako gap is of little consequence in the historical biogeography of the genus (stanley and esseltyn, 2010). similarly, there is little morphological variation among populations of the murid rodent hylomyscus arcimontensis on either side of the makambako gap (carleton and stanley, 2005), and the newly discovered kipunji monkey (rungwecebus kipunji) has populations on both sides of the gap (jones et al., 2005; davenport et al., 2006). there are also some commonalities in the avifauna among populations on the nyika plateau, mount rungwe, and the southern udzungwas, with no evidence of the makambako gap having a biogeographic influence (stuart et al., 1993). furthermore the southern highlands might have served as a dispersal route for amphibians, connecting the udzungwa and the mahenge mts, the two southernmost mountain blocks of the eastern arc (menegon et al., 2011; loader et al., 2014). interestingly, from a biogeographic perspective, the most suitable dispersal route for forest endemics from udzungwa and mahenge mountains – both part of the eastern arc mountains does not appear to be the shortest straight line distance, which would require crossing the kilombero valley (an ancient, deep, wide valley). instead, the continuous ridge of highlands connecting the southern udzungwa, through the southern highlands/ livingstone mountains via the makambako gap and then northeast to the mahenge mountains may have remained more suitable over historical times, potentially with forested areas (see fig. 2). the description of kinyongia msuyae provides a tantalizing piece of evidence suggesting strong biogeographical affinities between the southern highlands (i.e. livingstone mountains) and the eastern arc mountains (i.e. udzungwa mountains). the makambako gap may not be a turn-over region of high significance between the eam and the southern highlands, rejecting previous claims of its biogeographical importance as a barrier. instead, it is likely that some taxa can or have crossed this barrier, or that the gap was formerly less dry, forming a corridor between udzungwa and the southern highlands. our increased understanding of the southern highlands is revealing that the region is more species rich than had been supposed, possibly similar in scale to some of the eastern arc mountain forests. acknowledgements for advice, help with fieldwork, granting national and local permits for research and export in tanzania, we thank (no particular order) tanzania commission for science and technology (costech research permit rca 2001-272; rca 2007-153, rca 2009-306-na2009-201, 2011-239-na-2011-82, 2006 and 2007-72-na2006-19), tanzania wildlife research institute (tawiri), wildlife division. we are also grateful to many people and organizations that provided assistance in the field, logistical support and advice, including sandra dürrenberger, sandra rudolf, noah mpunga, tanzania forest conservation group, and colleagues of the wildlife conservation societies southern highlands conservation programme. thanks to nicholas barbieri for the help in the lab. this work was supported by the swiss national science foundation (grant number 31003a-133067 to spl), swiss academy of sciences, freiwillige akademische gesellschaft basel, the university of basel, and the south african national biodiversity institute. mm is grateful to the gino zobele fund for research and the lipparini family for their generous support. phylogenetic analyses were run at the cyberinfrastructure for phylogenetic research science gateway v 3.3 (cipres). references branch, w.r., bayliss, j., tolley, k.a. 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(2013): largescale phylogeny of chameleons suggests african origins and eocene diversification. pr. r. soc. london, b. 280: 20130184. acta herpetologica vol. 10, n. 1 june 2015 firenze university press acta herpetologica journal of the societas herpetologica italica acta herpetologica 9(2): 227-230, 2014 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-14480 records of introduced stripe-necked terrapins (mauremys species) in italy mattia panzeri1,§, emiliano mori2,§,*, giuseppe mazza3,4, mattia menchetti3 1 department of environment, health, safety, university of insubria, via j.h. dunant 6, 1100 varese, italy 2 di.s.a.f.a., entomology and zoology, university of turin, via leonardo da vinci 44, 10095 grugliasco (turin), italy. *corresponding author. e-mail: moriemiliano@tiscali.it 3 department of biology, university of florence, via romana 17, 50125 florence, italy 4 consiglio per la ricerca e la sperimentazione in agricoltura, centro di ricerca per l’agrobiologia e la pedologia (cra-abp), via di lanciola 12/a, 50125 cascine del riccio, florence, italy § these authors equally contributed to this manuscript submitted on 2014, 29th april; revised on 2014, 5th july; accepted on 2014, 7th july editor: uwe fritz abstract. freshwater turtles belong to the most popular pets and are often introduced outside their native range. some species are highly invasive and may compete with native species. three mauremys species are naturally distributed in the mediterranean region, but none is native to italy. in this work we summarize records of mauremys spp. in italy. records exist for 9 regions, but there are no verified breeding records. reproduction could have happened in southern tuscany for m. leprosa. keywords. popular pets, invasive species, pond turtles, mauremys leprosa, inter-specific competition, native species. the introduction of invasive alien species represents one of the main causes of extinction at the global scale (e.g. vitousek et al., 1997, lowe et al., 2000, clavero et al., 2009) and it determines major economic, health and societal issues (mooney and hobbs, 2000; mazza et al., 2014; menchetti and mori, 2014). among reptiles, the highly invasive trachemys scripta elegans has been massively released worldwide with consequences of outcompeting with indigenous species (cady and joly, 2004; pupins and pupina, 2011). this problem concerns also the italian peninsula, where the only native freshwater turtles are emys orbicularis and e. trinacris, whose populations are declining mainly for habitat loss and alien competition (gariboldi and zuffi, 1994; ferri, 1995; semenzato et al., 1998; chelazzi et al., 2000). three species of the genus mauremys occur in the mediterranean region and in the near and middle east: m. leprosa, m. caspica and m. rivulata (barth et al., 2004; mantziou et al., 2004), with m. leprosa in north africa and the iberian peninsula (keller and busack, 2001; fritz et al., 2006), m. caspica in the near and middle east (wischuf and fritz, 2001; vamberger et al., 2013) and m. rivulata in the eastern mediterranean region (wischuf and busack, 2001; vamberger et al., 2014). in syntopic populations of mauremys spp. and e. orbicularis, the latter species often seems to suffer from the dominance of mauremys (see the review in fritz, 2003), thus the introduced mauremys may pose a threat to local native populations of e. orbicularis. this problem could also relate to populations of e. orbicularis and e. trinacris in italy. to evaluate the present situation, here we summarize records of alien mauremys spp. in italy, presenting a synthesis of published data and new records. published and unpublished records of mauremys spp. in italy were collected from: (i) scientific papers; (ii) general books and articles; (iii) data from an italian national mailing-list dealing with herpetology, social networks and contacts with experts in several ital228 m. panzeri et alii ian regions, and (iv) check of the zoological collections of the italian natural history museums. between december 2013 and april 2014 we also conducted an extensive survey of online aquarium and pet shops in italy to assess the presence of mauremys species in the pet trade. in addition, to confirm the morphological identification of two road-killed terrapins collected in gabellino (cf. fig. 1), about 650 bp of the mitochondrial subunit 1 of cytochrome c oxidase of one specimen were sequenced and compared with genbank sequences of mauremys according to dna barcoding guidelines (http://ibol.org). occurrences of mauremys spp. have been reported for 9/20 italian regions (fig. 1). scalera (2001) reported observations of mauremys spp., without any specific determination, in seven italian regions: piedmont, lombardy, emilia romagna, latium, abruzzi, molise and apulia. mauremys cf. sinensis was recently observed in the botanical garden of palermo (b. borri, pers. comm.). the greatest group of mauremys was introduced, with 1000-1500 individuals, in the north of ravenna (emilia romagna, romea state road), in the early 1980s (v. ferri, pers. comm.). some terrapins, maybe from this introduction, were still present in 1997 (scolo marana: d. miserocchi, pers. comm.); two more individuals have been recently removed from the city of bologna (d. scaravelli, pers. comm.). in northern italy, at least 10 mauremys were recorded in a wetland of bosco di barco (province of brescia, lombardy: v. ferri, pers. comm.); in march 2013, an adult mauremys has been observed in the lake of arignano (province of torino, piedmont: n. destefano, pers. comm.). a further maurefigure 1. records of mauremys spp. in italy. grey regions represent those including at least one record. white circles represent exact observation sites. in brackets, year of observation and number of individuals (if more than one). the black triangle represents the site of the only genetically verified specimens (m. leprosa). asterisks refer to specimens stored in natural history museums. 229distribution of non-native mauremys in italy mys was recently (march 2014) removed from an artificial pond in grugliasco, piedmont (pers. obs.). detailed records for latium were reported by bologna et al. (2000; 2007), with single individuals in the circeo national park before 1980s, in the areas of monti della tolfa (rio fiume and magnone) in the late 1990s, in the reserve of macchiatonda (2004), and in villa doria pamphilii, rome (2005). a photograph of a single adult mauremys cf. leprosa was taken in spring 2012 near florence, northern tuscany (locality “la querciola”), during spring of 2012 (g. bruni, pers. comm.). two further mauremys were caught at villa al ventaglio, florence (b. borri, pers. comm.). a group of six adult mauremys has been found by the owner of a private pond, in the surroundings of the wetland of gabellino (grosseto, southern tuscany). in october 2013, two mauremys were collected as road kills (samples currently stored in 100% ethanol at the maremma natural history museum, grosseto: l131020 and l131027) in the surroundings of the same pond, an adult female (length of carapace: 19.2 cm) and a young individual (length of carapace: 5.7 cm); the skull of a third adult was collected on the bank of an irrigation canal. dna barcoding analysis confirmed that these individuals represent mauremys leprosa (cf. photos in appendix 1). there are only two records from 13 museums/regional observatories in italy (fig. 1): an adult mauremys sp. (mzuf 20042) collected in grosseto in 1968 is stored at the natural history museum of florence “la specola” (s. vanni, pers. comm.); two specimens of m. rivulata (msnm re 3938 and msnm re 3939), collected at the beginning of 1990s along the lambro river (maybe from the same group recorded as “bosco di barco”) are stored in the natural history museum of milan (s. scali, pers. comm.). according to a screening of 54 online pet shops distributed in all italy and main rural fairs, no mauremys species was found to be offered as pet species. terrapins belonging to the genus mauremys have been observed several times in italy, but, in most cases, records refer to single individuals and without species identification. however, we cannot exclude that some turtles were confused with the common invasive slider turtle (trachemys scripta). according to our summary, most introductions of mauremys concentrate in two regions (provinces of brescia and ravenna), for which no recent data are available, with the last records dating to 1997. this suggests that no established populations exist there. if we exclude removed individuals, the most recent observations of more than one wild mauremys (late summer 2013) refer to a hilly area of northern grosseto province (gabellino). six m. leprosa were observed in a temporary pond located at the end of an irrigation canal, within an alfalfa field. we suggest that these terrapins originate from the “carapax center”, which was closed in 2009 as a result of penal actions. this centre was located about 15 km distant from the observation site and there, m. leprosa were kept (r. capecchi, pers. comm.). the record of a road-kill of a juvenile m. leprosa suggests reproduction in the wild. acknowledgements we would like to thank b. borri, g. bruni, r. capecchi, n. destefano, v. ferri, d. miserocchi, d. scaravelli and a. vannini for information. dna sequencing was carried out in collaboration with fem2-ambiente srl (piazza della scienza 2, i 20126 milano, italy). data from museums/regional observatories were kindly provided by: g. boano (museo civico di storia naturale di carmagnola), s. guioli (museo di scienze naturali di voghera – pv), s. scali (museo civico di storia naturale di milano), k. tabarelli de fatis (muse, trento), n. novarini, m. bon (museo di storia naturale di venezia), l. latella (museo di storia naturale di verona), l. lapini (museo friulano di storia naturale, udine), g. doria (museo civico di storia naturale “giacomo doria”, genova), s. vanni (museo di storia naturale “la specola”, firenze), a. sforzi (museo di storia naturale della maremma, grosseto), m. natali, a.m. paci, u. sergiacomi (osservatorio faunistico regionale, umbria), m. capula, and l. ancillotto (museo civico di zoologia, roma). thanks to p. jennings and g. petri for language revision. two anonymous reviewers greatly improved the first version of our draft. references barth, d., bernhard, d., fritzsch, g., fritz, u. 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(2001): mauremys caspica (gmelin, 1774) – kaspische bachschildkröte. in: handbuch der reptilien und amphibien europas. schildkröten (testudines) i, pp. 43-56. fritz, u., ed., aula-verlag, wiebelsheim. acta herpetologica 12(1): 95-101, 2017 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-18731 the advertisement call of pristimantis subsigillatus (anura, craugastoridae) florina stănescu1,4, rafael márquez2, paul székely3,4,*, dan cogălniceanu1,4,5 1 ovidius university constanţa, faculty of natural and agricultural sciences, al. universităţii 1, campus b, 900470 constanţa, romania 2 fonoteca zoológica, dept. de biodiversidad y biología evolutiva. museo nacional de ciencias naturales (csic), josé gutiérrez abascal 2, e-28006 madrid, spain 3 universidad técnica particular de loja, departamento de ciencias biológicas, san cayetano alto, calle marcelino champagnat s/n, loja, ecuador 4 asociación chelonia, str. pașcani, nr. 5, bucurești, 062082, romania. *corresponding author. e-mail: jpszekely@utpl.edu.ec 5 universidad nacional de loja, citiab, ciudadela universitaria la argelia, ec 110101 loja, ecuador submitted on 2016, 11th august; revised on 2016, 18th october; accepted on 2016, 26th october editor: adriana bellati abstract. we describe for the first time the advertisement call of pristimantis subsigillatus from southern ecuador. our study provides a detailed quantitative characterization of the advertisement call of p. subsigillatus, filling a gap in our knowledge of this genus, the most speciose among vertebrates. males called perched on vegetation 0.5-2.5 m above ground, always during mild rain. the advertisement call is composed of a single note with a duration of 63-80 ms, with an initial short pulse (3-10 ms) followed by a longer tonal component. call rates ranged between 4-12 calls/ min. the dominant frequency varied between 2.02-2.82 khz. keywords. advertisement call, anura, craugastoridae, acoustics. vocalizations play an important role in the biology and evolution of anurans since acoustic parameters may encode information about species, sex, fitness, reproductive availability, territoriality or distress (gerhardt and huber, 2002; wells and schwartz, 2006). acoustic parameters can be genetically and environmentally shaped, therefore deciphering the information within vocal repertoires and analysing their variability in time and space is vital to tracing and understanding evolutionary patterns (duellman and trueb, 1994; gerhardt, 1994; cocroft and ryan, 1995; wells and schwartz, 2006). four main types of calls are currently described in anurans (duellman and trueb, 1994): advertisement, reciprocation, release and distress, and their functionality is relatively well known. advertisement calls represent one of the most conspicuous and important components of anuran communication, acting as a premating isolating mechanism and thus providing valuable data for phylogenetic studies (cocroft and ryan, 1995; vences and wake, 2007). with more than 470 species, direct-developing frogs of the genus pristimantis make up for almost one third of the anuran species of ecuador (ron et al., 2011), and the genus is by far the most diverse among terrestrial vertebrates (hedges et al., 2008; frost, 2016). species of the genus pristimantis have a high phenotypic variability within populations and scant morphological differences among species (crawford and smith, 2005). this frequently causes misidentifications even in museum specimens (padial et al., 2008). advertisement calls can be a valuable tool in describing or distinguishing among species when morphological characters are not sufficient in species identification (e.g., köhler and lötters, 1999; reichle et al., 2001). 96 f. stănescu et alii pristimantis subsigillatus (fig. 1) was described by boulenger (1902) from salidero, esmeraldas province, ecuador and is considered a locally abundant species in western ecuador and south-western colombia at elevations of 100-930 m a.s.l. (lynch, 1980; lynch and duellman, 1997; frenkel et al., 2013). this species is encountered most frequently in forests, at night, perching on vegetation at heights of 0.6-10 m above the ground; males usually call during rainy nights from vegetation at 2-5 m height (lynch and duellman, 1997). the advertisement call was described by lynch (1980) as a single sharp explosive ‘’tweet” or as “a single, clear, bell-like note”. however, to the best of our knowledge, there is no quantitative characterization of these calls. our paper provides the first quantitative description of the advertisement call in p. subsigillatus. dc recorded the advertisement calls of three p. subsigillatus males from buenaventura ecological reserve, el oro province, ecuador (table 1). calls were recorded with an olympus ls-11 linear pcm recorder and a røde ntg2 condenser shotgun microphone, at 44.1 khz sampling frequency and 16-bit resolution, in wav file format. air temperature and humidity were measured with a data logger (lascar electronics, model el-usb2-lcd, accuracy: ± 0.5°c; ± 5%). the three focal males were captured following the recording sessions after being photographed. the snout-vent length (svl) was measured to the nearest 0.1 mm with dial callipers, and then the animals were anaesthetized with benzocaine, fixed with formalin 10% and preserved in 70% alcohol. the specimens were deposited in the amphibian collection of museum of zoology of the pontificia universidad católica del ecuador (specimen id is provided in table 1). following toledo et al. (2015) recordings were deposited in fonoteca zoológica www.fonozoo.com, at museo nacional de ciencias naturales (csic), madrid, spain (records id are provided in table 1). individuals were identified as p. subsigillatus based on the characters described by lynch and duellman (1997), especially as having skin on dorsum finely shagreened, that of venter areolate, without dorsolateral folds, discoidal fold prominent, tympanic membrane and annulus evident, snout subacuminate in dorsal view, truncate or protruding in profile, snout bearing a small papilla at tip, canthus rostralis relatively sharp, upper eyelid lacking tubercles, outer fingers bearing broad discs, heel lacking or bearing very small tubercle and fifth toe much longer than the third (lynch, 1980; lynch and duellman, 1997). despite the fact that identification of pristimantis species based only on morphological characters is challenging, we are confident that the recorded individuals belong to p. subsigillatus. we have conducted a thorough inventory of amphibians in the area and are familiar with the species found there (e.g., székely et al. 2016), and p. subsigillatus cannot be confounded with other species of the genus present based on morphological characters. at present, p. subsigillatus is not assigned to any species group after it was removed from the former, nonmonophyletic, pristimantis unistrigatus group (padial et al., 2014). pristimantis subsigillatus is most similar to p. nyctophylax from which it is distinguished by the absence fig. 1. male pristimantis subsigillatus calling (photo credit: josé seoane rodríguez). table 1. information regarding the recordings used in this study. air temperature = temp; humidity = h; distance from the tip of the microphone and the focal male = dist. specimen id/ fonozoo id svl (mm) coordinates altitude (m) date time (h) temp (°c) h (%) dist (cm) qcaz47284/ 9863-64 28.2 3°39’21.4”s 79°46’08.0”w 464 12 september 2014 23:00 100 qcaz62538/ 9865-66 26.1 3°39’16.9”s 79°46’34.8”w 453 08 september 2015 21:00 21 98 25 qcaz62543/ 9867-69 3°39’07.0”s 79°45’43.6”w 600 14 september 2015 18:45 21 98 50 97call of pristimantis subsigillatus of observable tubercles on the eyelids and heels, presence of small terminal papilla at the tip of the snout, presence of numerous supernumerary plantar tubercles and of the inner tarsal tubercle. additionally, the eye of p. nyctophylax is very distinctive in having orange or red sclera (lynch and duellman, 1997). also, this species occurs usually at higher elevations (1140-2100 m a.s.l.) compared to p. subsigillatus. we used raven pro 1.4 software (http://www.birds. cornell.edu/raven) to analyse 54 advertisement calls. we measured the temporal parameters from the oscillograms and the spectral parameters from spectrograms obtained through hanning function, at a window size of 1024 samples, and 50% overlap. we measured the duration, rise time proportion, dominant frequency (df ), and aggregate entropy (entropy) of the calls and component parts. we computed rise time proportion, as the ratio between the period from the onset of the sound and the moment of maximum amplitude within the analysed call, and the call duration (márquez et al., 2005). we also computed the dominant frequency modulation within a call (dfm) as the difference between the dominant frequencies of the two component parts (márquez et al,. 1996). the aggregate entropy provides a measure of the overall disorder in the sound, by analysing the distribution of energy in the spectrogram; zero entropy values characterize single pure tones, while higher entropy values correspond to greater disorder in the acoustic spectrum (charif et al., 2010). following gerhardt (1991), we computed a coefficient of within-individual variation (cv% = sd/mean x 100) in order to differentiate between static (cv% < 5) and dynamic (cv% > 12) call parameters. the three p. subsigillatus males were calling after sunset perched on leaves or branches up to 2.5 m above ground, during mild rain. in each case, several other males were calling in the background. the advertisement call was composed of a single note with two consecutive parts: an initial short pulsed part, followed by a longer tonal part with harmonics (fig. 2). call rates ranged table 2. quantitative description of p. subsigillatus advertisement calls. mean ± sd (above the line) and min-max values (below the line) are provided for all acoustic parameters analysed. n = call sample size, df = dominant frequency, dfm = dominant frequency modulation. specimen measurement duration (ms) rise time (%) df (hz) dfm (hz) entropy (u) 47284 (n = 9) call 80 ± 2.9 0.141 ± 0.177 2651.0 ± 19.9 435.5 ± 260.8 2.9 ± 0.3 77 85 0.01 0.43 2627.1 2691.7 -21.5 624.5 2.5 3.4 part 1 7 ± 1.4 0.346 ± 0.186 2215.5 ± 273.9 5.0 ± 0.1 6 10 0.10 0.71 2024.1 2670.1 4.9 5.2 part 2 73 ± 3.0 0.298 ± 0.097 2651.0 ± 19.9 2.6 ± 0.1 69 78 0.09 0.43 2627.1 2691.7 2.4 2.8 62538 (n = 15) call 63 ± 0.9 0.025 ± 0.005 2584 ± 0 140.7 ± 124.5 2.4 ± 0.5 61 – 65 0.02 0.03 2584 0 430.7 1.5 3.1 part 1 3 ± 0.5 0.443 ± 0.123 2433.3 ± 124.4 4.0 ± 0.1 3 4 0.33 0.67 2153.3 2584.0 3.6 4.2 part 2 60 ± 0.8 0.073 ± 0.006 2584 ± 0 2.2 ± 0.5 58 61 0.07 0.09 2584 1.4 2.9 62543 (n = 30) call 70 ± 3.0 0.016 ± 0.004 2822.3 ± 33.4 476.6 ± 202.3 2.6 ± 0.4 61 74 0.01 0.03 2756.2 2885.4 0 645.9 1.9 3.2 part 1 7 ± 0.7 0.152 ± 0.046 2345.7 ± 201.9 3.8 ± 0.2 6 8 0.12 0.29 2239.5 2842.4 3.3 4.1 part 2 63 ± 3.1 0.226 ± 0.163 2822.3 ± 33.4 1.9 ± 0.2 53 67 0.02 0.48 2756.2 2885.4 1.5 2.6 all (n = 54) call 70 ± 6.1 0.378 ± 0.083 2727.5 ± 112.2 376.4 ± 242.4 2.6 ± 0.4 61 85 0.01 0.43 2584.0 2885.4 -21.5 645.9 1.5 3.4 part 1 6 ± 1.9 0.265 ± 0.166 2351.1 ± 208.0 4.1 ± 0.5 3 10 0.10 0.71 2024.1 2842.4 3.3 5.2 part 2 64 ± 5.2 0.198 ± 0.150 2727.5 ± 112.2 2.1 ± 0.4 53 78 0.02 0.48 2584.0 2885.4 1.4 2.9 98 f. stănescu et alii between 4-6 calls/min (individual 47284) to 9-12 calls/ min (individuals 62538 and 62543). descriptive statistics of the analysed acoustic parameters and their intra-individual variation are presented in detail in tables 2 and 3. the first part of the note was composed of 2 to 4 pulse groups in individual 47284, while in the other two individuals it consisted of a single pulse group. in individual 62543, the pulse was bell-shaped (fig. 2c). the second part of the note was always longer than the first, and had a harmonic structure, with the fundamental harmonic corresponding to the dominant frequency. the dominant frequency was higher in the second part and therefore the calls presented an upward frequency modulation pattern. call duration, part 2 duration and dominant frequency showed low within individual variability (cv < 5%), and therefore can be considered static acoustic parameters, useful in distinguishing among individuals of the population. the first part of the call presented the highest acoustic variability. our study provides the first detailed characterization of the advertisement call of p. subsigillatus, filling a gap in our knowledge of this poorly studied group. the calls described in this study are markedly different from the calls of pristimantis eugeniae, p. nyctophylax and p. phoxocephalus which are considered similar species by lynch and duellman (1997). p. eugeniae has calls with a much higher dominant frequency (3400-3800 hz) and lack the pulsed initial part of the call, p. nyctophylax has calls with lower dominant frequencies, around 2100 hz. as for p. phoxocephalus, the recordings posted in amphibia webecuador show a high variation of dominant frequencies, from 1900 hz to more than 3000 hz, suggesting that maybe more than one taxon was included in these recordings. advertisement calls are an important tool in describing and characterizing anuran species, since they act as a premating isolating mechanism (ryan, 1988). based on detailed acoustic analyses, taxonomic relationships can be elucidated (e.g., padial et al., 2008). as the most speciose vertebrate genus, pristimantis species have been subject to taxonomic debates for a long time (e.g., hedges et al., 2008; padial et al., 2014). in august 2016, the total number of pristimantis species recognized was 480, of which 121 new species were described during the last decade only (frost, 2016). most descriptions do not include calls and are often based only on museum specimens. recent reviews based only on molecular data could not shed light on the systematics of this genus (e.g., hedges et al., 2008; padial et al., 2014). in order to facilitate taxonomic work, this large genus has been subdivided into several species groups (lynch and duellman, 1997). there is a huge variation in the call parameters of the frogs within the genus pristimantis (padial et al., 2007), and acoustic parameters could prove to be an important tool in the revision of this genus. acknowledgements the secretaría de educación superior, ciencia, tecnología e innovación, republic of ecuador (senescyt) provided funding for paul székely and dan cogălniceanu through the prometeo project. the jocotoco foundation provided access to buenaventura forest reserve. florina stănescu, paul székely and dan cogălniceanu received additional support from the synthesys project. the synthesys project is financed by the european–community–research infrastructure action under the fp7 “capacities” specific programme. partial call analyses and call deposit were funded by project tatanka (cgl2011-25062), ministerio de ciencia e innovación, table 3. within-individual coefficient of variation of the analyzed acoustic parameters. n = call sample size, df = dominant frequency, dfm = dominant frequency modulation. cv values < 5% are highlighted in grey (static parameters sensu gerhardt, 1991). specimen measurement duration rise time df dfm entropy 47284 (n = 9) call 3.6 125.5 0.8 59.9 10.3 part 1 20.0 53.8 12.4 2.0 part 2 4.9 32.6 0.8 3.8 62538 (n = 15) call 1.4 20.0 0.0 88.5 20.8 part 1 16.7 27.8 5.1 2.5 part 2 1.3 8.2 0.0 22.7 62543 (n = 30) call 4.3 26.6 1.2 42.4 15.4 part 1 10.0 30.3 8.6 5.3 part 2 4.9 72.1 1.2 10.5 99call of pristimantis subsigillatus fig. 2. oscillogram and spectrogram views of the advertisement calls of the three males of pristimantis subsigillatus: specimen 47284 (a), 62538 (b), and 62543 (c). spectrogram window size: 256 samples, 50% overlap, hop size 128 samples. 100 f. stănescu et alii fcw (cgl2011-16159-e). we are grateful to dr. santiago ron, pontificia universidad católica de ecuador for his support. collecting permits were granted to pontificia universidad católica de ecuador as no. 005-14 ic-faudnb/ma and no. 003-15 ic-fau-dnb/ma. references boulenger, g.a. 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(2005): diverse types of advertisement calls in the frogs eupsophus calcaratus and e. roseus (leptodactylidae): a quantitative comparison. herpetol. j. 15: 257-263. padial, j.m., castroviejo-fisher, s., koehler, j., domic, e., de la riva, i. (2007): systematics of the eleutherodactylus fraudator species group (anura: brachycephalidae). herpetol. monogr. 21: 213-240. padial, j.m., köhler, j., muñoz, a., de la riva, i. (2008): assessing the taxonomic status of tropical frogs through bioacoustics: geographical variation in the advertisement calls in the eleutherodactylus discoidalis species group (anura).  zool. j. linn. soc-lond. 152: 353-365. padial, j.m., grant, t., frost, d.r. (2014): molecular systematics of terraranas (anura: brachycephaloidea) with an assessment of the effects of alignment and optimality criteria. zootaxa 3825: 1-132. reichle, s., lötters, s., de la riva, i. (2001): a new species of the discoidalis group of eleutherodactylus (anura, leptodactylidae) from inner-andean dry valleys of bolivia. j. herpetol. 35: 21-26. ron, s.r., guayasamin, j.m., menendez-guerrero, p.a. (2011): biodiversity and conservation status of amphibians of ecuador. in: amphibian biology 9, status of decline of amphibians: western hemisphere: uruguay, brazil, colombia, and ecuador, pp. 129-185. heatwole, h., barrio-amorós, c.l., wilkinson, j.w., eds, surrey beatty & sons, baulkham hills. ryan, m.j. (1988): constraints and patterns in the evolution of anuran acoustic communication. in: the evolution of the amphibian auditory system, pp. 637-677. fritzch, b., ryan, m., wilczynski, w., walkowiak, w., 101call of pristimantis subsigillatus hetherington, t., eds, john wiley & sons, new york. székely, p., cogălniceanu, d., székely d., páez, n., ron, s.r. (2016): a new species of pristimantis from southern ecuador (anura, craugastoridae). zookeys 606: 77-97. toledo, l.f., tipp, c., márquez, r. (2015): the value of audiovisual archives. science 347: 484-484. vences, m., wake, d. (2007): speciation, species boundaries and phylogeography of amphibians. in: amphibian biology 7, pp. 2613-2671. heatwole, h., tyler, m.j., eds, surrey beatty & sons, chipping norton. wells, k.d., schwartz, j.j. (2006): the behavioural ecology of anuran communication. in: hearing and sound communication in amphibians, pp. 44-86. narins, p., feng, a.s., fay, r.r., eds, springer, new york. acta herpetologica vol. 12, n. 1 june 2017 firenze university press morphological variation and sexual dimorphism in liolaemus wiegmannii (duméril & bibron, 1837) (squamata: liolaemidae) from uruguay joaquín villamil1,*, arley camargo2, raúl maneyro1 sex does not affect tail autotomy in lacertid lizards panayiotis pafilis1,*, kostas sagonas2, grigoris kapsalas1, johannes foufopoulos3, efstratios d. valakos4 fire salamander (salamandra salamandra) males’ activity during breeding season: effects of microhabitat features and body size raoul manenti*, andrea conti, roberta pennati call variation and vocalizations of the stealthy litter frog ischnocnema abdita (anura: brachycephalidae) pedro carvalho rocha1,2,*, joão victor a. lacerda2,3, rafael félix de magalhães2,3, clarissa canedo4, bruno v. s. pimenta5, rodrigo carrara heitor6, paulo christiano de anchieta garcia2,3 feeding ecology of two sympatric geckos in an urban area of northeastern brazil josé guilherme g. sousa1, adonias a. martins teixeira2,*, diêgo alves teles2, joão antônio araújo-filho2 and robson waldemar ávila3 a pattern-based tool for long-term, large-sample capture-mark-recapture studies of fire salamanders salamandra species (amphibia: urodela: salamandridae) jeroen speybroeck1,*, koen steenhoudt2,$ skeletal variation within the darwinii group of liolaemus (iguania: liolaemidae): new characters, identification of polymorphisms and new synapomorphies for subclades linda díaz-fernández*, andrés s. quinteros, fernando lobo marking techniques in the marbled newt (triturus marmoratus): pit-tag and tracking device implant protocols hugo le chevalier1, olivier calvez2, albert martínez-silvestre3, damien picard4, sandra guérin4,5, francis isselin-nondedeu6, alexandre ribéron1, audrey trochet1,2,* evidence for directional testes asymmetry in hyla gongshanensis jindongensis qing gui wu1, wen bo liao2,* the advertisement call of pristimantis subsigillatus (anura, craugastoridae) florina stănescu1,4, rafael márquez2, paul székely3,4,*, dan cogălniceanu1,4,5 nematodes infecting anotosaura vanzolinia (squamata: gymnophthalmidae) from caatinga, northeastern brazil bruno halluan s. oliveira¹, adonias a. martins teixeira¹,*, romilda narciza m. queiroz¹, joão a. araujo-filho¹, diêgo a. teles¹, samuel v. brito2, daniel o. mesquita¹ where is my place? quick chorus structure assembly in the european tree frog michal berec a possible mutualistic interaction between vertebrates: frogs use water buffaloes as a foraging place piotr zduniak1,*, kiraz erciyas-yavuz2, piotr tryjanowski3 good vibrations: a novel method for sexing turtles donald t. mcknight1,2,*, hunter j. howell3, ethan c. hollender1, and day b. ligon1 errata to mecke, s., hartmann, l., mader, f., kieckbusch, m., kaiser, h. (2016): redescription of cyrtodactylus fumosus (müller, 1895) (reptilia: squamata: gekkonidae), with a revised identification key to the bent-toed geckos of sulawesi. acta herpetologica 11(2): acta herpetologica 12(2): 199-204, 2017 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-20841 reliable proxies for glandular secretion production in lacertid lizards simon baeckens laboratory of functional morphology, department of biology, university of antwerp, 2610 wilrijk, belgium department of organismic and evolutionary biology, harvard university, 02138 cambridge (ma), usa e-mail: simon.baeckens@uantwerp.be submitted on: 2017, 21st june; revised on: 2017, 26th august; accepted on: 2017, 11th october editor: marco mangiacotti abstract. the epidermal glands of lizards are considered an important source of semiochemicals involved in lizard communication. many features of the lizard epidermal gland system vary among and within species (e.g., gland number, size, and shape), and some are believed to reflect the degree of intraand interspecific variation in glandular secretion production, and by extension, the chemical signalling investment of lizards. traditionally, herpetologists estimate secretion production based on the number of glands or the size of the glands, rather than quantifying the amount of secretion produced. still, the reliability of these proxies for secretion production has never been validated. here, i explored the relationship among secretion production (in mass), pore size (surface area, diameter), and gland number in three species of lacertid lizards (acanthodactylus boskianus, timon lepidus, holaspis guentheri), and tested which proxies predicted secretion production variation best, and examined whether the same trend is true for all species. the findings of this study show that the total secretion production of lacertids is highly variable among and within species. variation in secretion production among-species (but not within-species) could partly be explained by variation in body size. while both measures of pore size were positively related with secretion production, my tests revealed the model with only pore diameter as contributing variable explaining absolute secretion production variation (both within and across species) as the best one. although gland number appeared a suboptimal estimate for secretion production in the three lacertids under study, only family-wide, multi-species comparative tests counting large within-species sample sizes can provide further insight on the matter. keywords. chemical signals; epidermal gland secretions; femoral pores, lacertidae, secretion quantity. chemical signals are essential for interand intrasexual communication in many animals, and lizards represent no exception (mason and parker, 2010). yet, the extent to which lizards utilize their chemosensory system varies greatly among species (baeckens et al., 2017a, b). this phenomenon seems also true for the signalling system of lizards, which is illustrated by the fact that merely half of all non-ophidian squamate species are equipped with epidermal glands (lizards’ leading source of socially relevant chemical signals; mayerl et al., 2015). it is even so that the number of epidermal glands that lizards possess varies among (and sometimes even within) species (martín and lópez, 2000; pincheira-donoso et al., 2008; baeckens et al., 2015). in search for the constraints and selective pressures driving this variation in chemical signalling investment, researchers have focussed on various morphological characteristics of the lizard epidermal gland system to quantify chemical signalling investment, and ultimately, to compare among lizards. based on the premise that overall secretion production (thus ‘secretion quantity’) reflects how much a particular lizard invests in and relies upon chemical signalling, herpetologists traditionally use gland number and/or the size of the gland opening (i.e., pore) as proxies for secretion quantity (alberts et al., 1992; escobar et al., 2001; pincheiradonoso et al., 2008; iraeta et al., 2011; valdecantos et al., 200 simon baeckens 2014; baeckens et al., 2015, 2017c). however, whether these features are truly reliable measures for a lizard’s total amount of secretion production (and reliable for interspecific comparisons) has never been validated. in this study, i quantify the overall secretion production (in mass) of lizards of three different lacertid species, and test which characteristics predict secretion production best (gland number, pore diameter, and/or pore surface area). in total, i obtained 32 adult male lizards (14 acanthodactylus boskianus, 12 holaspis guentheri, 6 timon lepidus) from local reptile hobbyist or through the pet trade (fantasia reptiles, belgium, license hk51101419). lizards were accommodated at the university of antwerp facility, and housed in glass terraria (100 x 40 x 50 cm). a 60-watt bulb suspended above one end of the terrarium provided light and heat so that lizards could maintain a body temperature within their preferred range. lizards had access to freshwater at all times, and were fed up to three times a week. snout-vent length (svl) was measured using digital callipers (mitutuyo, cd-15cpx, accuracy = 0.01 mm). average pore size was estimated by digitising (imagej, abramoff et al., 2004) the (1) diameter and (2) surface area of the two most proximal pores of the left femur on images obtained with a stereomicroscope (leica m165 c), and by subsequently calculating mean pore diameter and mean pore surface area per individual (fig. 1). next, i collected gland secretions of all lizards, by gently pressing with forceps around the pores until each gland was completely emptied and all secretion yielded (following baeckens et al., 2017c). secretion collection occurred within the lizards’ reproductive period in june 2014 (castilla and bauwens, 1989; schleich et al., 1996; pianka and vitt, 2003; khannoon, 2009; grimm et al., 2014), when they were active (between 10:00 and 16:00 h). secretions extracted from all glands of the left thigh were directly weighed on a microbalance (mettler toledo mt5, accuracy = 1 µg). prior to statistical analyses in spss v. 24 (chicago, il, usa), variables were log10 (svl, pore surface area, pore diameter, secretion mass) or square root (gland number) transformed to meet assumptions of normality (shapiro-wilks test: w ≥ 0.95). the results of this study show significant intraand interspecific variation in all aspects of the lizard epidermal gland system, including secretion production (table 1). while interspecific variation in secretion production could be partially explained by among-species differences in body size (with the largest species in this study, t. lepidus, producing high amount of secretion in comparison to the other two smaller lizard species), the observed within-species variation could not (table 1 and 2). further, results show that none of the epidermal gland characteristics (gland number, pore area, pore diameter) of lizards belonging to a. boskianus and h. guentheri were affected by body size (all; pearson correlation, r < 0.45, p > 0.15). the surface area and diameter of the pores of t. lepidus, however, were strongly linked with body size (resp., r = 0.94, p = 0.006; r = 0.95, p = 0.005). similar to the other species, gland number did not correlate with svl in t. lepidus (r = 0.08, p = 0.89). overall, there was no significant relationship between a lizard’s number of glands and its total secretion production (table 2). it was even so that the lizard species with the least number of glands (i.e., t. lepidus) was equipped with the largest pores, which moreover produced the largest amount of glandular secretion (table 1). pore surface area and pore diameter, on the other hand, correlated strongly with secretion mass in h. guentheri and t. lepidus but not in a. boskianus (table 2). in t. lepidus, where pore size is affected by svl, a partial correlation test (controlling for svl) revealed also a positive relationship between relative secretion mass and pore surface area, but not pore diameter. multiple regression analyses (backward stepwise elimination with gland number, pore diameter, and pore surface area) indicated the model with only pore diameter as the significant contributing independent variable explaining secretion mass variation (a. boskianus, r2 = 0.31, f1,13 = 5.35, p = 0.039; h. guentheri, r2 = 0.45, f1,11 = 8.27, p = 0.017; t. lepidus, r2 = 0.79, f1,5 = 15.32, p = 0.017). the same was true for an analogous multiple regression, but then across species encompassing all individuals (r2 = 0.74, f1,31= 83.43, p < 0.001, fig. 2). overall, the findings of this study reveal that the total glandular secretion production of lacertid lizards is highly variable among and within species. while body size fig. 1. measuring pore surface area and pore diameter of the epidermal pores of a timon lepidus lizard. 201proxies for lizard chemical signalling investment was unable to explain the observed intraspecific variation in secretion production in the species under study, a positive link between body size and secretion quantity has been observed in other lizard species, such as in the iguanid iguana iguana (alberts et al., 1992) and the lacertid podarcis muralis (baeckens et al., 2017c). latter researchers argue that since secretion production is most probably an energetically costly affair (martín and lópez, 2014; mayerl et al., 2015), it is very likely that only individuals in a good condition (which are often the largest individuals; jakob et al., 1996) can afford high secretory activity rates. these discordant findings among studies and species imply that the biosynthetic pathways that produce glandular secretion might be species-specific. an alternative explanation for not finding a link between body size and secretion production among individuals of the same species concerns the origin of the animals in this study. since pet store animals generally receive plenty of nutritious food throughout their lives in the store, it is unlikely to find biological significant variation in body condition (linked with secretion production) among individuals. notwithstanding, large-scale phylogenetical-informed comparative studies could shed light on how idiosyncratic or universal the link between body size and secretion production really is, and how they scale (allometric/isometric) among and within species. aside from quantifying (variation in) chemical signalling investment in lacertid lizards, the ultimate aim of this table 1. descriptive statistics for the morphological variables measured on male lacertid lizards of the three species used in this study. the table also shows the results of the analyses of variance (anova) testing for absolute among-species differences (followed by tukey post-hoc tests), and the results of the analyses of covariance (ancova, with svl as covariate) testing for relative differences. acanthodactylus boskianus (a) n = 14 timon lepidus (t) n = 6 holaspis guentheri (h) n = 12 interspecific comparison absolute differences relative differences mean se min max mean se min max mean se min max f2,31 p post-hoc f2,31 p svl (mm) 73.42 0.90 67.80 81.35 146.20 13.60 119.99 207.46 49.35 1.15 45.21 59.02 213.77 <0.001 t > a > h — — gland number 26.04 0.42 22.5 28.5 12.75 0.48 12.0 15.0 22.71 0.42 20.5 25.0 203.01 <0.001 a > h > t 0.66 0.527 pore surface area (mm2) 1.25 0.04 0.86 1.43 3.11 0.71 1.53 6.08 1.01 0.07 0.39 1.30 23.22 <0.001 t > a = h 1.26 0.299 pore diameter (mm) 0.49 0.02 0.37 0.66 1.25 0.28 0.61 2.36 0.38 0.02 0.17 0.50 28.26 <0.001 t > a > h 0.55 0.586 secretion mass (mg) 0.80 0.08 0.29 1.27 11.43 6.23 0.96 35.68 0.79 0.10 0.14 1.47 10.57 <0.001 t > a = h 2.45 0.106 abbreviations: a = acanthodactylus; h = holaspis; t = timon. table 2. results of pearson correlation tests (r), testing for correlations between (a) svl and gland traits, and (b) secretion mass and gland traits. also shown are the results of partial correlation tests (c), which accounted for differences in svl. variables were transformed prior to analyses to meet the assumptions of normality. bold values indicate statistical significance (p < 0.05). acanthodactylus boskianus (n = 14) timon lepidus (n = 6) holaspis guentheri (n = 12) r p r p r p (a) correlation with svl secretion mass 0.43 0.125 0.74 0.092 -0.12 0.710 gland number 0.15 0.959 0.76 0.886 0.43 0.163 pore surface area -0.01 0.985 0.94 0.006 0.39 0.211 pore diameter 0.44 0.115 0.94 0.005 0.44 0.156 (b) correlation with secretion mass gland number -0.12 0.679 -0.17 0.747 -0.32 0.314 pore surface area 0.32 0.259 0.91 0.011 0.58 0.048 pore diameter 0.56 0.039 0.89 0.017 0.67 0.017 (c) partial correlation with secretion mass gland number -0.14 0.644 -0.34 0.577 -0.30 0.375 pore surface area 0.36 0.226 0.92 0.026 0.69 0.020 pore diameter 0.45 0.121 0.85 0.068 0.81 0.002 202 simon baeckens study was to document reliable proxies, if any, for secretion production. while both measures for pore size were positively correlated with secretion mass, the findings here suggest that pore diameter was the best predictor of secretion production quantity, hence chemical signalling investment. this was true both within species and across species, advocating pore diameter as the most adequate estimate of gland productivity in interspecific comparisons. alberts and colleagues (1992) established a similar relationship in i. iguana males (with n = 10, r = 0.81, p = 0.002), although baeckens et al. (2017c) did not so in the species p. muralis. however, the latter investigators merely measured pore surface area, not pore diameter. why diameter turned out to be a better predictor of secretion quantity than surface area might be partly ascribed to the shape of the pores. the lacertids under study bared pores of a long-stretched oval form (unlike, for example, those of the teiid tupinambis merianae; chamut et al. 2009), which varied among individuals largely in length (diameter) and less in area. the lengthy oval shape of the pores might allow lizards to maximize their scent-mark area by increasing the contact zone between pores and substrate (along the proximal-distal limb axis of the limb). more research on lizard scent-marking behaviour and the functional significance of pore shape is necessary to make well-founded predictions on the matter. surprisingly, gland number came out as a poor predictor of secretion production in the three lacertids under study here. yet, several intraand interspecific comparative studies have assumed that gland number reflects species’ investment in and use of chemical communication (e.g., escobar et al., 2001; pincheira-donoso et al., 2008; iraeta et al., 2011; baeckens et al., 2015). notwithstanding, their theory cannot be considered as illogically, for a lizard with x number of glands will produce a lower amount of secretion than a hypothetical identical lizard, but with x+1 number of glands. besides, since this study only comprises three different lizard species belonging to merely one lizard family (lacertidae), it would be incorrect to generalize and label gland number as a poor proxy reflecting chemical signalling investment in lizards. clearly, only a broad, multi-species study counting large within-species sample sizes can provide further insight on the matter. while the goal of this short note was to underscore the importance of choosing the appropriate proxy for lizard secretion production, i wish to note that the findings of this study should be interpreter cautiously due to the following. firstly, i only used secretion quantity to estimate chemical signalling investment, whilst disregarding secretion ‘quality’. the chemical composition of the secretion of lizards is a mixture of proteins and lipids and is highly species-specific (mayerl et al., 2015; mangiacotti et al., 2017; baeckens et al., 2017d). one can easily imagine a trade-off between secretion quantity and ‘quality’, with lizards producing low amounts of secretion, but investing highly in, for instance, a rich or diverse chemical design with high concentrations of certain key compounds (such as described for some invertebrates; wyatt, 2014). future studies on the chemical signalling investment of lizards should, ideally, integrate both the chemical architecture of the glandular secretion and the total amount of secretion produced. secondly, this study concentrates solely on follicular epidermal gland secretions, while neglecting any other source of semiochemicals. although it is generally believed that follicular gland secretions are the leading source of semiochemicals (martín and lópez, 2014; mayerl et al., 2015), there is plenty of evidence that generation glands, faeces, cloacal secretions, and skin lipids contain socially relevant chemical stimuli too (cooper and vitt, 1984; mason and gutzke, 1990; cooper, 1995; labra, 2008; moreira et al., 2008; mouton et al., 2010). whether lizards that invest little in the production of gland secretions are investing more strongly in semiochemicals of other origins (and vice versa) is, however, uncertain, but certainly not improbable. thirdly, the use of animals obtained through the commercial pet trade, rather than using life-caught animals from the wild, -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 -0 .5 0. 0 0. 5 1. 0 1. 5 log10 (pore diameter) lo g1 0 ( se cr e� on m as s) acanthodactylus boskianus holaspis guentheri timon lepidus fig. 2. a scatterplot showing the positive relationship (r2 = 0.74, f1,31 = 83.43, p < 0.001; slope = 1.85) between pore diameter and glandular secretion production in three species of lizards. because the slopes of the three species did not significantly differ, only one regression line (covering all lizards of the three species) is shown. shaded area represents 95% confidence intervals. 203proxies for lizard chemical signalling investment might bring along a series of uncertainties concerning pre-purchase animal stress, transport, and housing conditions. yet, similar to previous experiments using pet trade lizards (obtained through the same commercial dealer as used in the current study; herrel et al., 2007, driessens et al., 2014), animals were in good condition at the onset of the experiments. overall, i am confident that these limitations did not compromise the main objective of this work, which was: quantifying intraand interspecific variation in secretion production in a small subset of lacertid lizards, and exploring the best possible morphological traits to estimate secretion production. based on the findings of this study, i advise scholars, at times when assessing secretion mass seems unfeasible (e.g., in museum specimen), to be cautiously thorough and integrate pore area, diameter, and number in any future studies scoring secretion production quantity. although gland number played out to be a suboptimal quantity-proxy in the three lacertid lizards under study, broad-scale comparative analyses should examine this in more detail. acknowledgments author thanks raoul van damme, alison heath, and katleen huyghe for co-designing this study. jan scholliers and jorrit mertens for animal care, and three anonymous reviewers for significantly improving drafts of this manuscript. all work was carried out in accordance with the university of antwerp animal welfare standard and protocols (ecd 2014-32). this work was carried out at the university of antwerp, and made possible through financial support from the university of antwerp. references abràmof, m.d., magalhães, p.j., ram, s.j. 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(2014): pheromones and animal behaviour: chemical signals and signatures. cambridge university press, cambridge. acta herpetologica vol. 12, n. 2 december 2017 firenze university press meristic and morphometric characters of leptopelis natalensis tadpoles (amphibia: anura: arthroleptidae) from entumeni forest reveal variation and inconsistencies with previous descriptions susan schweiger1, james harvey2, theresa s. otremba1, janina weber1, hendrik müller1,* brown anole (anolis sagrei) adhesive forces remain unaffected by partial claw clipping austin m. garner*, stephanie m. lopez, peter h. niewiarowski species and sex comparisons of karyotype and genome size in two kurixalus tree frogs (anura, rhacophoridae) shun-ping chang1,2, gwo-chin ma2,3,4, ming chen2,5,6,7,8,*, sheng-hai wu1,* non-native turtles in a peri-urban park in northern milan (lombardy, italy): species diversity and population structure claudio foglini1, roberta salvi2,* species composition and richness of anurans in cerrado urban forests from central brazil cláudia márcia marily ferreira1,*, augusto cesar de aquino ribas2, franco leandro de souza3 the life-history traits in a breeding population of darevskia valentini from turkey muammer kurnaz, alı i̇hsan eroğlu, ufuk bülbül*, halıme koç, bılal kutrup influence of desiccation threat on the metamorphic traits of the asian common toad, duttaphrynus melanostictus (anura) santosh mogali*, srinivas saidapur, bhagyashri shanbhag predation of common wall lizards: experiences from a study using scentless plasticine lizards jenő j. purger*, zsófia lanszki, dávid szép, renáta bocz reproductive timing and fecundity in the neotropical lizard enyalius perditus (squamata: leiosauridae) serena najara migliore1,2,*, henrique bartolomeu braz2,3, andré felipe barreto-lima4, selma maria almeida-santos1,2 observations on the intraspecific variation in tadpole morphology in natural ponds eudald pujol-buxó1,2,*, albert montori1, roser campeny3 and gustavo a. llorente1,2 reliable proxies for glandular secretion production in lacertid lizards simon baeckens diet of juveniles of the venomous frog aparasphenodon brunoi (amphibia: hylidae) in southeastern brazil rogério l. teixeira1, ricardo lourenço-de-moraes2, débora c. medeiros3, charles duca3, rogério c. britto4, luiz c. p. bissoli5, rodrigo b. ferreira3,* who are you? the genetic identity of some insular populations of hierophis viridiflavus s.l. from the tyrrhenian sea ignazio avella, riccardo castiglia, gabriele senczuk* acta herpetologica 12(1): 55-63, 2017 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-20147 a pattern-based tool for long-term, large-sample capture-markrecapture studies of fire salamanders salamandra species (amphibia: urodela: salamandridae) jeroen speybroeck1,*, koen steenhoudt2,$ 1 research institute for nature and forest, kliniekstraat 27, 1070 brussels, belgium. *corresponding author. e-mail: jeroenspeybroeck@ inbo.be, jeroen.speybroeck@hotmail.com 2 waterschaapstraat 30, 1570 galmaarden, belgium $ programming and database development was solely done by k.s. submitted on january, 11th 2017; revised on march, 12th 2017; accepted on march, 22nd 2017 editor: uwe fritz abstract. solid population studies depend on reliable capture-mark-recapture (cmr) methodology. the available methods for such studies on amphibians are often invasive, unsuitable for long-term studies, time-consuming and/ or expensive. we present a new software tool, connected to a ms access database, mandermatcher, for cmr study of fire salamanders (salamandra salamandra and related species) by means of a robust matching algorithm using 44 pattern characteristics. metadata related to standardised counts (e.g., weather and lunar variables) as well as a myriad of individual sighting variables can be entered and saved as well. application of the presented method to a database of 9,397 sighting records gathered over a period of eight years, as well as a random sample validation, demonstrate the accuracy of the applied matching algorithm. differences with other methods are discussed. the program is made freely available for download and widespread application is encouraged, especially given the contemporary context of a fungal disease threatening survival of fire salamander populations. keywords. salamandra salamandra, pattern recognition, capture-mark-recapture, software, methodology, free download introduction long-term demographic studies depend on reliable capture-mark-recapture (cmr) methodology to model population size and abundance, survival and detection rate, as well as to provide data on a myriad of life history features such as individual growth, reproductive cycles, longevity, dispersal and migration (e.g. ferner, 1979; schmidt et al., 2002; schmidt, 2004; amstrup et al., 2005). a number of methods are available for practical execution of such studies in amphibians. toe clipping as well as the use of tattoos or passive or active transponders, however, all require a certain degree of (at best temporary) damage to the subject’s body, and are thus commonly labelled as invasive and, at least potentially, damaging (e.g. davis and ovaska, 2001; le galliard et al., 2011). furthermore, given the regenerative capacity of certain taxa, namely salamanders, toe clipping becomes unsuitable for long-term studies (e.g., ferner, 1979; davis and ovaska, 2001) and may in certain cases even reduce recapture and survival rates (mccarthy et al., 2009). use of transponders sets additional limitations such as being costly and fairly time-consuming in the field (ott and scott, 1999), as well as in some cases having low retention rates in smaller individuals and species (ryan et al., 2014), while detection and recapture rates may differ 56 jeroen speybroeck, koen steenhoudt according to the size of the used tags (ousterhout and semlitsch, 2014). species with an individually varying pattern of colour spots offer the possibility of recognising animals without the need to apply invasive techniques. a number of (more or less) automated image analysis tools has been developed, such as wildid (bolger et al., 2012), amphident (drechsler et al., 2015), i³s (van tienhoven et al., 2007, with newer, improved versions having been successfully applied to the newt triturus carnifex by sannolo et al., 2016, as well as to reptiles, as reviewed by sacchi et al., 2016), aphis (moya et al., 2015) etc. while these claim to offer more robust recognition procedures and automatic pattern recognition, they usually require labour-intensive image processing steps in order to select a certain ‘area of interest’. thus, they do not necessarily guarantee faster processing than alternatives based on interpretative pattern coding. one (amphident) is even fairly expensive, especially for non-institutional users. additionally, while pattern shape becomes of greater importance, spot shapes may alter drastically given the position of the animal within the picture. as such, higher demands are set on the quality of the pictures, resulting in more time spent properly positioning live and fairly hard to immobilise animals while collecting data. the use of non-invasive recognition techniques to study fire salamander salamandra salamandra evolved throughout the last decades, making use of the typical pattern of yellow spots on a black background in this species. earlier authors used time-consuming, direct comparison of photographs to recognise individuals (e.g., feldmann, 1971; klewen, 1985; seifert, 1991). kopphamberger (1998) coded different s. salamandra pattern types into an alphanumerical code, while not ruling out occurrence of type doubles, thus causing obvious problems in larger samples. carafa and biondi (2004) elaborated on this by combining a colour typology with a software application storing data into an ms access database for the italian subspecies s. s. gigliolii, using a sample of 233 individual animals. given the limited description of the applied characteristics in their paper and the fact that the software was not made freely available, use of and detailed insight into this methodology remained unavailable to a wider audience. a different approach was presented by šukalo et al. (2013), using the number of glandular pores located within the boundaries of the yellow spots. in many subspecies of s. salamandra, however, the number, size and shape of these spots is known to change throughout an individual’s lifetime (eiselt, 1958; klewen, 1991; mutz, 1992; bogaerts, 2002). becoming stable at adulthood only (klewen, 1991), pattern changes may be common, limiting indeed the use of colour patterns for individual recognition at earlier life stages. while the degree of colour pattern alteration during early terrestrial life may vary considerably between s. salamandra subspecies (bogaerts, 2002), a relatively stable pattern is said to be reached after at least two or three years (eiselt, 1958). this leads to changes in pore count within the span of a single year in some individuals, even in animals larger than 12 cm (speybroeck, unpublished data). thus, the method of šukalo et al. (2013) seems of limited value for studies spanning more than a couple of months at most, and a methodology combining a multitude of characteristics seems preferable. as such, use of an individual-specific code which can be applied to easily collected and processed images, which is sufficiently stable over time and can be queried by an automated matching algorithm applied to a database, is of merit. we present a new software tool, connected to a ms access database, mandermatcher, for cmr study of salamandra salamandra and other spotted species of the genus by means of a robust matching algorithm using 44 pattern characteristics. we demonstrate how this tool can serve study of large sample sizes involving a high number of different individuals. material and methods salamandra salamandra and related spotted species (s. algira, s. corsica and s. infraimmaculata) are characterised by a black background colour covered with a variable amount of most commonly yellow (but in certain taxa rather orange or red) spots or stripes (thiesmeier, 2004; beukema et al., 2016b; speybroeck et al., 2016). the top of the head is usually covered with yellow on the eyes and parotoid glands, while additional spots may be present on the snout tip. the tail may be crossed by spots as well. toes may be black or yellow. this pattern layout offers potential for coding each animal’s pattern into a series of numbers. these numbers can be supplied to an algorithm that matches each pattern with pattern codes from previously collected data. pattern code the spot pattern is coded into 44 characteristics. each of those indicates the number, presence or mutual relation between the yellow spots within well-defined areas of the salamander body: head (11 characteristics), back and tail (19) and toes (14). in case of doubt, characteristics can be left blank and will not be taken into account by the matching algorithm. as patterns may vary over time, reliable use is advised for animals with a total length of more than 14 cm only. matching may work on smaller animals, depending on the stability of that animal’s pattern as well as the portion of characteristics that are left blank by the user. thus, while no reliable assessment of subadult and juvenile population size can be made with this 57a pattern-based tool for cmr studies (or any) pattern-based method, it may allow certain other studies which do not require tracing every single individual (e.g. growth analysis). in contrast to criteria used in some of the other cited methods, each of the 44 characteristics has a clear definition, allowing a limited array of entered values only and leaving little room for variation caused by interpretation. all are discussed and illustrated in the manual of the freely available program, serving as supplementary material to this paper (available for download, along with the program itself, at http://www. hylawerkgroep.be/jeroen/index.php?id=85). for demonstration to new users, a version containing already some data is also provided. the mandermatcher website also offers data exploration tools for data stored in the program's database). program a software tool, mandermatcher, was developed to allow cmr recognition and storage of other visit and sighting related data. the program was written in the vb.net (visual basic) language, using the freely available basic version of visual studio express. practical use is discussed at length in the manual (see above link). the program basically consists of three main windows. the first is the ‘visits’ window, available after launch as the left tab (fig. 1). it lists the visits and allows to enter metadata related to that visit. visits are time-restricted count events, covering e.g. a standardised transect or habitat surface. the second window is the ‘sightings’ window (fig. 2), available as the second tab, next to the visits window. the ‘pivot animal’, shown at the upper left, will be matched with the entire database of available sightings at the right on the basis of the pattern code that was entered for it. at the lower left, criteria can be specified to filter and restrict the database entries which are returned at the lower right. the as such selected entries are sorted by their resemblance to the pivot animal, with the most similar entry at the top. the third window is accessed through the buttons for editing one of the two sightings shown at either the upper left or fig. 1. ‘visits’ window for edit and creation of visits, and entry of environmental data in the program mandermatcher. 58 jeroen speybroeck, koen steenhoudt fig. 2. ‘sightings’ window for recapture recognition and database querying in the program mandermatcher. fig. 3. window for entry of individual sighting records in the program mandermatcher. 59a pattern-based tool for cmr studies the upper right. this window is used for entry of new sightings, as well as for editing of existing sightings. at the left, the cmr pattern code is entered, while the upper fields allow to store several other variables. hitting the ‘ok’ button at the right after data entry/edit leads back to the ‘sightings’ window and initiates the matching process. in the cited available methods (see introduction), a list of observations ordered by decreasing match likelihood is the endpoint of the matching process. unlike any other, however, our method offers filter criteria to reduce this list (by entering features which are considered to be stable) as an additional tool to detect recaptures. this is particularly useful for matching younger specimens, which are likely to have undergone certain pattern changes. additional variables besides cmr matching, the program offers several tools which are relevant for ecological study of salamanders. a first set involves data related to the individual sighting, based on characteristics of the animal. we refer for a full list to the provided download link and the manual of the program, and only provide a general outline here. biometric fields are available – weight and length measurements (total length and snout-vent length). the latter can be measured within the program itself if a calibration object is properly inserted into the picture. ecological aspects can be documented in the shape of discrete variables to discern patterns in e.g. activity and habitat use throughout salamander life history. several fields allow further specification of details concerning the encounter location. all individual-related data is entered through the individual sighting entry window (fig. 3). a second set of variables, which are entered through the ‘visits’ window (fig. 1), consists of metadata related to the count event (visit), including an array of environmental variables which can be registered both at start and end of the count event. all are defined and discussed in the program’s manual. database all data is saved into a ms access database, which offers ample opportunity to query the entered data and export for analysis. application the program was applied to s. s. terrestris data collected in a forest in the town of merelbeke, flanders, belgium. sampling was performed along a standardised transect of 1.3km. from march 2008 to january 2017, 169 sampling events were executed by night during activity-provoking weather conditions. all encountered post-metamorphic fire salamanders were captured, photographed and released. validation to validate the applied search algorithm, a random sample of 100 sightings of adult salamanders was drawn from the database. one-by-one visual photograph comparison was conducted. results application a total of 9,397 sighting records were collected during standardised transect sampling events. of these, fig. 4. recapture rate by cumulative number of sighting records of adult animals, by visit (circles) and for entire accumulating dataset (triangles) with non-parametric, locally weighted regression smoother (loess) and 95% confidence interval. point size relates to absolute number of encountered adults within each visit (encounter event) for both point types (circles and triangles), presented as a continuous variable. 60 jeroen speybroeck, koen steenhoudt 6,899 are sightings of animals larger than 14 cm, here treated as adults. these adult sightings consist of (re) captures of 1,477 different individual adult salamanders. the overall percentage of animals captured at least twice, as obvious from the growing, cumulative database is shown as a function of the growing total of adult individual observations by the triangles in figure 4, each data point representing a capture event. the cumulated dataset recapture rate amounts to 62.4% in the entire database at the final instance of data collection. the circles in the graph, however, not showing the percentage of recaptures in the cumulated database but the percentage within the data of a single visit, reach far higher values and, for instance, correspond to 91% of the adult individuals captured during the last visit (at the extreme right in the graph). validation the random sample comprised 89 adult individuals, of which 80 were encountered only once within the sample, 7 were encountered twice, and 2 individuals were encountered three times. the exact same result was obtained by application of the program, even though matching of these 100 randomly selected sightings was done against up to 9,397 records, thus making successful match finding more complicated than if 100 new records were to be entered into an empty database. while matching through individual picture comparison took about 4 hours, matching 100 new records one by one with an unstructured collection of 9,397 photographs would take an impractical amount of time. while the speed of using mandermatcher differs depending on the size of the database and recapture rate within the data, using the matching algorithm and finding a match or not (excluding entry of other data) takes a user which is sufficiently acquainted with the program an average of 2 minutes or less per record. the uniqueness of certain code combinations can be calculated theoretically and from our data on s. s. terrestris. as an example, we consider the toe characteristics. fourteen toe characteristics (with potential values 0 and 1) corresponds to 214 or 16,384 theoretical combinations. using our data to assess the number of actual combinations, we used a subset of 1,597 observations for which all toe characteristics were determined. these observations relate to 737 individual animals. within these 737 individuals, 488 different toe colour combinations occur, of which 52.2% are found in a single individual only. the most commonly encountered combination (i.e., all black toes) is shared by 5.4% of the individuals. discussion while the actual recapture numbers shown for the example population greatly depend on the applied count methodology and, possibly, the nature of the investigated population, they serve as illustration that with sufficient effort high recapture success can be achieved. manual validation showed no mismatches in comparison with application of the algorithm. after the program’s initial release, as substantiated by this paper, tools for exploratory analysis are under development (speybroeck and steenhoudt, in prep.). these include analysis of population dynamics through space and time and mapping features, but also analysis of pattern variability, allowing the recognition variables to serve a second purpose, apart from cmr matching. several non-invasive methods exist for cmr recognition of individuals. some may require a lot of time in the field and/or behind the desk. we have demonstrated how mandermatcher allows effective matching of flexiblebodied fire salamanders at a swift rate, requiring limited handling time per record in the field, no photograph processing at the desk and easy and fast record entry. during a high number of recapture events (169), the method was successfully applied to a dataset of 9,397 records and 1,477 adult individuals, which is clearly larger than any to which other methods were applied (e.g., 1,606 records of 1,321 individuals in drechsler et al., 2015; 852 photos of 324 individuals in sannolo et al., 2016). more importantly, the dataset spans a period of eight years. this is particularly relevant to assess how changes of pattern over time may affect recognising individual animals. using data from a single season does not offer assurance that the quality of the matching process remains sufficiently high over time. for example, sannolo et al. (2016) used data collected over a four month period of a single year. while these authors claim the ventral spot pattern of triturus carnifex to be stable over time (citing arntzen and wallis, 1999, who however do not provide proof for this statement), this seems unlikely, as drechsler et al. (2015) showed this to be incorrect for the closely related t. cristatus. only by using data from a period of time which is long enough to span a relevant portion of adult life and growth of the considered taxon (e.g., three years in drechsler et al. 2015, eight years in our study), the impact of pattern changes (whether they exist and/ or are relevant or not) on the use of the recognition algorithm can be assessed. while the dorsal pattern of adult fire salamanders is traditionally considered to remain stable over time (e.g., eiselt, 1958; klewen, 1991), changes may occur (speybroeck, unpublished data). application of mandermatcher to data collected over eight years has 61a pattern-based tool for cmr studies shown that the algorithm is sufficiently robust to prevent such changes from causing matching problems, which is likely to be an advantage of using numeric coding of the pattern, instead of image analysis tools basing matching on the extent of pigmentation. mandermatcher does not require photographs to be taken in a standard way (e.g., with the animal perfectly stretched), which is particularly time-consuming when dealing with salamanders, such as the species we studied, and newts, including the subject species of drechsler et al. (2015) and sannolo et al. (2016). finally, in contrast to all other tools, our program is a comprehensive research tool, including tools for length measurement and a myriad of variables related to each individual and each record. while the pattern coding was so far only applied to s. s. terrestris, the character definitions offer sufficient precision and resolution for use with most spotted taxa within the salamandra genus (s. s. salamandra, s. s. almanzoris, s. s. terrestris, s. s. longirostris, s. s. bejarae, s. s. morenica and most s. s. bernardezi) as well as s. algira, s. corsica and s. infraimmaculata). two exceptions, however, may exist. populations with a high portion of animals with poorly defined spots (e.g., populations of s. s. gallaica, s. s. crespoi, and part of s. s. bernardezi (i.e., populations formerly assigned to s. s. alfredschmidti but placed in synonymy by beukema et al., 2016a)) may offer coding difficulties, whereas taxa with a high portion of animals with low numbers of spots (e.g., s. s. fastuosa, s. s. gigliolii) will have identical values for a number characteristics across many individual animals. however, considering the frequency distribution of actual toe colour combinations in our data, we have demonstrated how powerful the algorithm may be. only 5.4% of the individuals share the most common combination, allowing to drastically reduce the number of individuals to be compared by using the toe characteristics alone. further use will proof applicability to other taxa/populations, and it seems likely that for some of the taxa with less welldefined dorsal spots (e.g., s. s. gallaica) toe colour may provide sufficient discerning power. the program is made freely available for use by any researcher, while the authors applaud getting in touch with them for potential collaboration, applying the same methodology across fire salamander taxa. while this method is presented for a single taxon (as was the case for the initial publication of most other methods, e.g., van tienhoven et al., 2007; drechsler et al., 2015), the presented approach has potential to be used to develop similar tools for use with other species, applying a modified code with relevant characteristic definitions. it is particularly useful for species which have a pattern that can be coded to a sufficient number of characteristics. how high this number has to be in order to allow precise matching, depends on the sample size and the frequency distribution of possible values of each characteristic. as such, using a smaller number of characteristics may be sufficient for smaller datasets. for the species at hand, population studies may serve as a crucial baseline at this point in time, given the imminent threat presented by lethal fungal infection caused by batrachochytrium salamandrivorans (martel et al., 2013, 2014). after decimating the dutch population (spitzen-van der sluijs et al., 2013), severe fungus-caused mortality has occurred at several sites in belgium and the fungus has also been detected in germany (spitzen-van der sluijs et al., 2016). thus, the availability of an easy-to-use, inexpensive cmr methodology for study of both infected and non-infected populations is of great value. acknowledgements wouter beukema provided valuable comments to an earlier draft of the manuscript. the flemish agency for nature and forest (agentschap natuur en bos, anb) is thanked for providing the necessary permits. references amstrup, s.c., mcdonald, t.l., manly, b.f.j. 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(2016): field guide to the amphibians and reptiles of britain and europe. bloomsbury publishing, london. spitzen-van der sluijs, a., martel, a., asselberghs, j., bales, e.k., beukema, w., bletz, m.c., dalbeck, l., goverse, e., kerres, a., kinet, t., kirst, k., laudelout, a., marin da fonte, l.f., nöllert, a., ohlhoff, d., sabino-pinto, j., schmidt, b.r., speybroeck, j., spikmans, f., steinfartz, s., veith, m., vences, m., wagner, n., 63a pattern-based tool for cmr studies pasmans, f., lötters, s. (2016): expanding distribution of lethal amphibian fungus batrachochytrium salamandrivorans in europe. emerg. infect. dis. 22: 1286-1288. spitzen-van der sluijs, a., spikmans, f., bosman, w., de zeeuw, m., van der meij, t., goverse, e., kik, m., pasmans, f., martel, a. (2013): rapid enigmatic decline drives the fire salamander (salamandra salamandra) to the edge of extinction in the netherlands. amphibia-reptilia 34: 233-239. šukalo, g., dorđević, s., golub, d., dmitrović, d., tomović, l. 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(2004): der feuersalamander. laurenti verlag, bielefeld. van tienhoven, a.m., den hartog, j.e., reijns, r.a., peddemors, v.m., (2007): a computer-aided program for pattern-matching of natural marks on the spotted raggedtooth shark carcharias taurus. j. appl. ecol. 44: 273-280. acta herpetologica vol. 12, n. 1 june 2017 firenze university press morphological variation and sexual dimorphism in liolaemus wiegmannii (duméril & bibron, 1837) (squamata: liolaemidae) from uruguay joaquín villamil1,*, arley camargo2, raúl maneyro1 sex does not affect tail autotomy in lacertid lizards panayiotis pafilis1,*, kostas sagonas2, grigoris kapsalas1, johannes foufopoulos3, efstratios d. valakos4 fire salamander (salamandra salamandra) males’ activity during breeding season: effects of microhabitat features and body size raoul manenti*, andrea conti, roberta pennati call variation and vocalizations of the stealthy litter frog ischnocnema abdita (anura: brachycephalidae) pedro carvalho rocha1,2,*, joão victor a. lacerda2,3, rafael félix de magalhães2,3, clarissa canedo4, bruno v. s. pimenta5, rodrigo carrara heitor6, paulo christiano de anchieta garcia2,3 feeding ecology of two sympatric geckos in an urban area of northeastern brazil josé guilherme g. sousa1, adonias a. martins teixeira2,*, diêgo alves teles2, joão antônio araújo-filho2 and robson waldemar ávila3 a pattern-based tool for long-term, large-sample capture-mark-recapture studies of fire salamanders salamandra species (amphibia: urodela: salamandridae) jeroen speybroeck1,*, koen steenhoudt2,$ skeletal variation within the darwinii group of liolaemus (iguania: liolaemidae): new characters, identification of polymorphisms and new synapomorphies for subclades linda díaz-fernández*, andrés s. quinteros, fernando lobo marking techniques in the marbled newt (triturus marmoratus): pit-tag and tracking device implant protocols hugo le chevalier1, olivier calvez2, albert martínez-silvestre3, damien picard4, sandra guérin4,5, francis isselin-nondedeu6, alexandre ribéron1, audrey trochet1,2,* evidence for directional testes asymmetry in hyla gongshanensis jindongensis qing gui wu1, wen bo liao2,* the advertisement call of pristimantis subsigillatus (anura, craugastoridae) florina stănescu1,4, rafael márquez2, paul székely3,4,*, dan cogălniceanu1,4,5 nematodes infecting anotosaura vanzolinia (squamata: gymnophthalmidae) from caatinga, northeastern brazil bruno halluan s. oliveira¹, adonias a. martins teixeira¹,*, romilda narciza m. queiroz¹, joão a. araujo-filho¹, diêgo a. teles¹, samuel v. brito2, daniel o. mesquita¹ where is my place? quick chorus structure assembly in the european tree frog michal berec a possible mutualistic interaction between vertebrates: frogs use water buffaloes as a foraging place piotr zduniak1,*, kiraz erciyas-yavuz2, piotr tryjanowski3 good vibrations: a novel method for sexing turtles donald t. mcknight1,2,*, hunter j. howell3, ethan c. hollender1, and day b. ligon1 errata to mecke, s., hartmann, l., mader, f., kieckbusch, m., kaiser, h. (2016): redescription of cyrtodactylus fumosus (müller, 1895) (reptilia: squamata: gekkonidae), with a revised identification key to the bent-toed geckos of sulawesi. acta herpetologica 11(2): acta herpetologica 11(2): 171-178, 2016 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-15109 sources of calcium for the agamid lizard psammophilus blanfordanus during embryonic development jyoti jee1, birendra kumar mohapatra2, sushil kumar dutta1, gunanidhi sahoo1,3,* 1 department of zoology, north orissa university, baripada, odisha 757 003, india. *corresponding author. e-mail: gunanidhi.nou@ gmail.com 2 institute of mineral & materials technology, bhubaneswar, odisha 751 013, india 3 current address: post graduate department of zoology, utkal university, bhubaneswar 751 004, india submitted on 2014, 26th october; revised on 2015, 14th may; accepted on 2016, 30th august editor: aaron m. bauer abstract. we determined the sources of calcium for the developing embryo and the parallel changes in eggshell structure in the indian agamid lizard psammophilus blanfordanus. the developing eggs were opened at 0 (freshly laid), 10, 20, 30, 35, 38, and 40 days of incubation and at hatching (day 41) and subjected to chemical and structural analyses. the oval and flexible-shelled eggs had undergone significant changes in size (40% increase in length, 68% increase in breadth and 315% increase in weight) from laying to hatching. the fresh eggshell contained 2.76 mg (12.51%) calcium whereas the hatched eggshell had only 1.02 mg (7.20%), or a 63% reduction from its original content. the yolk + fluids fraction provides only 0.47 mg to the 1.76 mg of calcium in the hatchling, the rest being resorbed from the eggshell during development. the fresh eggshell (62 µm thick) had a rough granular structure in its calcareous layer with near uniform rectangular/polygonal fields made up of globules of varying sizes. the membrane layer had a multilayered mat of interwoven, irregularly oriented and bifurcated, fibres of uneven thickness. the spherical globules were absent at several places in the hatched eggshell as a result of eggshell calcium utilisation by the developing embryo. hence, like that of most reptiles, the eggshell of psammophilus blanfordanus also acts as a secondary source of calcium for the developing embryo. the embryo utilizes the eggshell calcium towards the end of development. keywords. psammophilus blanfordanus, embryonic development, eggshell, calcium, ultrastructure. introduction eggshell structure of reptiles is diverse, ranging from the small, flexible, parchment-shelled eggs of most squamates to the large, rigid-shelled eggs of crocodilians. it consists of an outer, inorganic layer underlain by an organic (shell) membrane comprised of multiple layers of fibres. the structural units in the calcareous layer are also diverse (schleich and kästle, 1988). the eggshell of reptiles plays a complex, but only partially understood, role in development, particularly as a source of inorganic ions (packard et al., 1992). some major functions of the eggshell are to accommodate permeability to gas and water, provide mechanical stability, and to serve as a potential calcium reserve (schleich and kästle, 1988). the eggshells of most oviparous lepidosourians are fibrous and poorly calcified (packard et al., 1982a, b; packard and demarco, 1991; thompson et al., 2001).the inorganic (mineral) content of the shell is usually restricted to the outermost portion and is comprised mainly of calcium carbonate in the form of calcite (packard et al., 1982a, b, deeming, 1988). yolk provides a considerable amount of calcium during embryonic growth for most oviparous reptiles whereas the eggshell calcium supplements that of yolk late in the incubation period (packard et al., 1984, 1985; shad172 jyoti jee et alii rix et al., 1994; packard and clark, 1996; stewart et al., 2004; sahoo et al., 2009). as embryos of oviparous squamates depend heavily on the yolk as a source of calcium, their calcium mobilisation pattern has been proposed as the most appropriate model for functional characteristics of the common ancestor of oviparous amniotes (packard and seymour, 1997; stewart et al., 2004). the morphological changes in eggshell structure are indeed caused by the mobilisation of eggshell calcium by the developing embryo. the present work describes the eggshell calcium utilization pattern and the parallel structural changes in the eggshell of an agamid lizard, psammophilus blanfordanus, during various stages of embryonic development. material and methods five clutches of psammophilus blanfordanus eggs (n= 12-14) were collected on campus of north orissa university from nests just after oviposition in july, 2013 and brought to the laboratory. the eggs were measured, weighed and numbered. two eggs from each clutch were reserved for chemical analyses. the remaining eggs of each clutch were incubated in separate plastic boxes at ambient temperature (33° c average, range = 28–38° c) with sandy soil as the medium of incubation. the soil medium was rehydrated at regular intervals. the development of two more clutches (n = 13 and 12) were also observed in their original nests from the time of oviposition. laboratory eggs were maintained at similar hydric condition as those in the field. one egg from each clutch from both field and laboratory studies was removed on days 10, 20, 30, 35, 38, and 40 of incubation. eggs (total of eight sample days – freshly laid, 10, 20, 30, 35, 38, 40 day and hatched) were washed in tap water; egg dimensions and weight were measured and eggs were separated into shell and egg components which included yolk, albumen and embryo including the extraembryonic membranes) and subjected to chemical and structural analyses. chemical analysis determination of inorganic composition requires conversion of the solid samples to liquid form through acid digestion. the samples (eggshell and egg components: yolk, albumen and embryo) were dried to constant weight at 80 ºc in an oven and digested following the method of geisey and weiner (1978) with a slight modification. pre-weighed samples (about 1 g) were heated to 100˚c until dry with 20 ml of concentrated nitric acid, cooled and again heated until dry with 5 ml of the acid. after cooling, 10 ml of 30% hydrogen peroxide was added to the digestate and heated for 30 minutes until a clear solution was obtained. distilled water was added to reconstitute the samples that were then filtered and diluted to 100 ml. calcium estimation was done gravimetrically using calcon indicator (0.4 g solid calcon dissolved in 100 ml methanol). to 10 ml of the digestate, 20 ml distilled water, 2-3 ml naoh solution and 3-4 drops of calcon were added to make the solution alkaline. this solution was titrated against 0.02 m edta. samples with very low levels of calcium (e.g., egg components) were analyzed using an atomic absorption spectrophotometer (perkinelmer, analyst 200) with standard solutions of 1 ppm, 2 ppm, 4 ppm and 10 ppm of calcium (r = 0.995074). magnesium in the digestate was analyzed by an atomic absorption spectrophotometer and potassium by a flame photometer (systronics, model k-iii). phosphorus was determined with a spectrophotometer (varian, model – carry 100). the characteristic blue colour was developed with a phosphomolybdenum complex and ascorbic acid and the intensity of the colour was measured at 882 nm. structural analysis the structure of eggshells (fresh, 10 day, 20 day, 30 day, 35 day, 38 day, 40 day) was determined with a scanning electron microscope (jeol, jsm 35 cf) with working voltage of 5-15 kv at magnifications ranging from 100 to 1500x. the eggshell samples were air dried and broken into pieces suitable for microscopic observations. for sem study, outer surface, radial section and sub-surface samples were mounted in stubs with doublesided adhesive carbon tape and coated with gold for 2 minutes by an ion sputterer (jfc-1100). the thickness of the fiber and mineral layers was measured using the scale bar on the photomicrographs. tests for homogeneity of variance, one way anova and tamhane’s post-hoc tests were conducted for comparing the differences in eggshell calcium concentration at all eight developmental stages using spss 15.0 (spss inc., chicago) results physical characteristics the physical and chemical composition of psammophilus blanfordanus eggs from both laboratory and in-situ field samples were similar and were therefore pooled for analysis (table 1). the incubation period varied from 41-42 days (laboratory eggs) to 44 days (field eggs). at oviposition, eggs were 1.21 ± 0.46 cm long, 0.75 ± 0.27 cm in diameter, and weighted 0.393 ± 0.021 g, n =65); variation in linear dimensions and weight among clutches was not significant (one-way anova and tamhane’s post-hoc test, p < 0.05). the wet weight of eggshells ranged from 0.037 to 0.048 g (0.042 ± 0.004, n =10). the average wet weight of the egg contents was 0.351 g ± 0.019 (88.4%) whereas the eggshell formed 11.6% of the total egg weight. the fresh egg contained about 62.8% water. the total dry weight varied from 0.132 to 0.166 g (0.146 ± 0.007 g). the shell weight varied from 0.020 to 0.027 g and the egg content 173sources of calcium for psammophilus from 0.113 to 0.147 g. on dry weight basis, the eggshell composed 15% and the egg components 85% of the total egg weight. the eggs of p. blanfordanus increased in size and weight from laying to hatching (fig. 1). the eggs increased 40% in length (from 1.21 cm at laying to 1.69 cm at hatching); 68% in width (0.75 cm at laying to 1.26 cm at hatching); and 315% in weight (from 0.393 g at laying to 1.631 g at hatching) (table 1). calcium content of the eggshell and the egg components at different days of incubation are presented in table 1 and fig. 2. the amount of other minerals like magnesium (0.79 mg in fresh egg contents and 0.75 mg in hatched embryo) and potassium (2.68 mg in fresh egg contents and 2.23 mg in embryo) did not change during development. the fresh eggshell contained 2.76 mg of calcium that formed 12.51% of the total eggshell. the fresh egg components contained only 0.47 mg of calcium (0.31% of the total egg). thus, 3.23 mg calcium was present in the freshly laid egg. the calcium content of 35 day incubated eggshell (1.61 mg, 9.42%) was comparable to that at 30 days. the calcium contents of 38, 40 day developed and that of the hatched eggshell was almost comparable (one-way anova and tamhane’s post-hoc test, p < 0.05 for pair wise comparisons between all stages, table 1). table 1. changes in physical and chemical composition of psammophilus blanfordanus eggs during embryonic development (n = 7 per sample date, field and laboratory eggs combined). day of incubation wet weight (g) dry weight (g) calcium content (dry weight) eggshell egg contents including embryo total wt. shell egg component total wt. shell egg component mg % mg % freshly laid 0.393 0.042 0.351 0.146 0.022 0.124 2.76 12.51 0.47 0.31 10 day 0.526 0.044 0.482 0.145 0.022 0.123 2.19 9.95 0.47 0.31 20 day 0.603 0.051 0.602 0.144 0.021 0.123 2.04 9.71 0.55 0.41 30 day 1.120 0.269 0.851 0.122 0.020 0.102 1.73 9.63 0.59 0.43 35day 1.454 0.211 1.243 0.139 0.017 0.122 1.61 9.42 0.74 0.62 38 day 1.586 0.127 1.459 0.159 0.015 0.143 1.05 7.48 1.38 1.69 40 day 1.623 0.176 1.447 0.167 0.014 0.153 1.03 7.30 1.54 1.80 hatched 1.631 0.087 1.544 0.102 0.014 0.088 1.02 7.20 1.76 1.91 fig.1 fig.1. changes in the size of psammophilus blanfordanus eggs as a function of the day of incubation. fig. 2 fig. 2. percentage calcium in eggshell and egg contents + embryo of psammophilus blanfordanus eggs through incubation. 174 jyoti jee et alii the yolk fraction of the fresh egg contained 0.47 mg of calcium (0.3% of the egg content). the calcium content of the hatchling increased over the embryonic period. the freshly emerged hatchling contained 1.76 mg of calcium (1.91%). calcium utilisation pattern the total calcium content of the freshly laid egg was 3.23 mg of which the eggshell contained 2.76 mg and the egg component 0.47 mg. the calcium in the egg component increased with the growth of the embryo. significant alternations were observed in calcium content of the eggshell from laying to hatching (fig. 2). the eggshell calcium content decreased slowly from day 10 to day 38 (table 1). the mobilization of calcium from the eggshell was slow up to 35 day of incubation. however, a quantum jump was observed thereafter up to 40 days of incubation. a parallel gradual increase in calcium content of the egg component was also observed during development. the hatched eggshell (1.02 mg) contained about 63% less calcium than that of the fresh eggshell (2.76 mg). structural characteristics the fresh eggshell surface morphology: the outer surface of the eggshell exhibited a rough granular structure (fig. 3a). the surface was organized into regular rectangular and/or polygonal fields of almost uniform size. the fields were arranged in a parallel fashion. the boundaries of such fields appeared to be elevated. the outer surface at higher magnification (fig. 3b) also did not reveal any definite crystal structure. rounded/spherical globules of varying sizes made up the entire thickness of the calcareous layer. peripheral margins of the polygonal fields were formed by larger globules whereas the central area was formed by smaller ones. cross section: the thickness of the shell was about 62 µm (membrane layer 50 µm, calcareous layer 12 µm) (fig. 3c). the calcareous layer did not show any discernible structure in this view. figure 3d reveals the demarcations of the polygonal calcareous fields on the surface of the egg. the thick membrane layer appeared to have layers of fibers. inner surface: the inner surface of the egg shell was bounded by a very thin smooth boundary layer (fig. 3e and f). it revealed irregularly placed globules/spheres of varying sizes. the underlying fibres of the membrane layer were not visible because of a smooth covering layer. the hatched eggshell upper surface: the smooth outer surface at lower magnification (fig. 4a) revealed clear grooves arranged in a regular parallel pattern. the grooves were formed due to absence of larger globules from the boundaries of the polygonal areas that were present on the fresh eggshell. fig. 4b depicts irregular absence of smaller globules from inside the fields. the absence of globules from the outer surface exposed the underlying membrane fibers. inner surface: the inner surface with a thin shell membrane (fig. 4c) revealed the underlying fibers to be distinctly visible showing their arrangement. the membrane layer contained a mat of interwoven, irregularly oriented uneven fibrils that were bifurcated at intervals (fig. 4d-f). the fibrils were arranged into several layers. discussion the eggshells of p. blanfordanus are typical of the flexible shelled eggs of squamates (packard et al., 1982c). the calcareous layer is thin and the shell membrane contributes the majority of shell thickness. calcium carbonate crystals are present as calcite, the form of calcium carbonate found in vast majority of lacertilian eggs (packard et al., 1982a, 1982b; packard and hirsch, 1986). also, the eggs increased in size throughout incubation with uptake of water from the substrate, which is typical of flexible shelled squamate eggs in favorable hydric environments (packard et al., 1980, 1982a; andrews and sexton, 1981). some of the eggs tripled their mass during incubation. water uptake is essential for normal embryonic development and survival for many squamate embryos with flexible-shelled eggs (muth, 1980; tracy, 1980). the most important function of the egg is related to embryonic nutrition (bellairs, 1964; cook, 1968; ackerman, 1991; linville, 2010) for which it contains all the necessary materials. any loss of egg components may seriously affect embryonic development within the egg and hatchling maintenance and growth. embryos of oviparous lizards have two potential sources of calcium for developmental requirements; calcium sequestered in yolk during vitellogenesis and calcium deposited on the eggshell by oviducal secretions. packard (1994) reviewed the calcium contribution of the eggshell to the embryos in various groups of oviparous, amniotic vertebrates (snakes, lizards, etc.). according to her, the degree of calcification of the eggshell is very important as it acts as a source of embryonic calcium in these groups of animals. psammophilus blanfordanus eggshell is poor in calcium in comparison to other species (stewart and ecay, 2010) and has a thin calcareous 175sources of calcium for psammophilus fig. 3. scanning electron micrographs of freshly laid eggshells (a & b upper surface, c & d cross section, e & f inner surface) of psammophilus blanfordanus: (a) upper surface at low magnification showing regular nearly polygonal fields on a smooth surface. (b) part of calcareous layer removed to show the inner surface. (c) much of the thickness of the shell is made up of membrane fibres with a thin calcareous layer. (d) the cross section slightly tilted showing the irregular field-like arrangement on the surface of the eggshell. (e & f) the inner surface is covered by a smooth inner membrane (boundary layer). globules of varying sizes are embedded on this surface that faces the egg contents. 176 jyoti jee et alii fig. 4. scanning electron micrograph of hatched eggshell of p. blanfordanus. (a) upper surface: larger globules on the boundary of the irregular fields (see fig.3a) are absent. this has made the formation of clear grooves on the surface. (b) enlarged view of (a) smaller globules are also absent at places. (c) inner surface: the thin boundary membrane present on the inner surface exposes the inner membrane fibres. (d–f) inner surface, enlarged view showing the arrangement of ramfied and somewhat curved fibres of uneven thickness on the membrane layer. 177sources of calcium for psammophilus layer, yet it provides a considerable proportion of calcium to the developing embryo. the freshly laid egg contains 3.23 mg of calcium (2.76 mg in shell and 0.47 mg in egg component), whereas the embryo needs 1.7 mg of calcium for its complete development. the embryo initially utilized the calcium of the yolk fraction for its development. this means the embryo required 1.29 mg more calcium from sources other than yolk. at the same time the eggshell had undergone 1.74 mg reduction in its calcium content over the entire embryonic period. this suggests that the embryo has utilized this much amount of calcium from the eggshell. thus, the embryo used calcium from two sources: initially from the yolk followed by the eggshell. the calcium requirement of the embryo and loss of that of the eggshell was comparable. according to abdel-salam et al. (2006), concentration of calcium in the eggshell of reptiles is higher than that of sodium/magnesium and calcium distribution in the eggshell differs before and after hatching. in the present study, concentrations of minerals like potassium and magnesium in the egg contents of a freshly laid egg did not alter through incubation. the eggshell provides about 63% of its calcium to the developing embryo, the rest being supported by the yolk. the eggshell had undergone considerable structural changes from laying to hatching. the calcareous layer formed distinct grooves due to the absence of larger globules. besides, smaller globules were also absent exposing the underlying membrane fibres. absence of globules was caused due to the resorption of calcium by the developing embryo from the calcareous layer. however, no change was observed in the thickness of the shell membrane and in the arrangement of the membrane fibres. embryos of most oviparous reptiles obtain calcium from both yolk and eggshell, but patterns of calcium mobilization vary (packard and packard, 1984; packard, 1994). oviparous squamate reptiles generally have lightly calcified eggshells and embryos mobilize most calcium from yolk, whereas embryonic turtles, crocodilians and birds are highly dependent on calcium from the eggshell. however, in p. blanfordanus, 63% of embryonic requirement is supplied by the eggshell. thus, the eggshell composition and structure of psammophilus blanfordanus fall within the range reported for other species in this group. the role of eggshell as a calcium source in lizards is little studied. future research into this field is important to understand the complex role the eggshell played in development of the embryo. the broad similarities in chemical composition and structure of eggshells of agamid lizards (packard and demacro, 1991; osborne and thompson, 2005; stewart and ecay, 2010) suggest a conserved evolutionary strategy in eggs of lizards, but more species must be studied to confirm such a conclusion. acknowledgements gs is thankful to his students for information regarding the laying of eggs by psammophilus blanfordanus at various locations. formal permission was not required for research on the species, as the species is listed as least concern as it is widely distributed and very common throughout its range. references abdel-salam,  z.a., abdou, a.m., harith, m.a.  (2006): elemental and ultrastructural analysis of the eggshell: ca, mg, and na distribution during embryonic development via libs and sem techniques.  int. j. poultry sci. 5: 35-42. ackerman, r.a. 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(1980): on the water relations of parchmentshelled lizard (sceloporus undulatus) eggs. copeia 1980: 478-82. acta herpetologica vol. 11, n. 2 december 2016 firenze university press predator-prey interactions between a recent invader, the chinese sleeper (perccottus glenii) and the european pond turtle (emys orbicularis): a case study from lithuania vytautas rakauskas1,*, rūta masiulytė1, alma pikūnienė2 effective thermoregulation in a newly established population of podarcis siculus in greece: a possible advantage for a successful invader grigoris kapsalas1, ioanna gavriilidi1, chloe adamopoulou2, johannes foufopoulos3, panayiotis pafilis1,* the unexpectedly dull tadpole of madagascar’s largest frog, mantidactylus guttulatus arne schulze1,*, roger-daniel randrianiaina2,3, bina perl3, frank glaw4, miguel vences3 thermal ecology of podarcis siculus (rafinesque-schmalz, 1810) in menorca (balearic islands, spain) zaida ortega*, abraham mencía, valentín pérez-mellado growth, longevity and age at maturity in the european whip snakes, hierophis viridiflavus and h. carbonarius sara fornasiero1, xavier bonnet2, federica dendi1, marco a.l. zuffi1,* redescription of cyrtodactylus fumosus (müller, 1895) (reptilia: squamata: gekkonidae), with a revised identification key to the bent-toed geckos of sulawesi sven mecke1,*,§, lukas hartmann1,2,§, felix mader3, max kieckbusch1, hinrich kaiser4 the castaway: characteristic islet features affect the ecology of the most isolated european lizard petros lymberakis1, efstratios d. valakos2, kostas sagonas2, panayiotis pafilis3,* sources of calcium for the agamid lizard psammophilus blanfordanus during embryonic development jyoti jee1, birendra kumar mohapatra2, sushil kumar dutta1, gunanidhi sahoo1,3,* mediodactylus kotschyi in the peloponnese peninsula, greece: distribution and habitat rachel schwarz1,*, ioanna-aikaterini gavriilidi2, yuval itescu1, simon jamison1, kostas sagonas3, shai meiri1, panayiotis pafilis2 swimming performance and thermal resistance of juvenile and adult newts acclimated to different temperatures hong-liang lu, qiong wu, jun geng, wei dang* olim palus, where once upon a time the marsh: distribution, demography, ecology and threats of amphibians in the circeo national park (central italy) antonio romano1,*, riccardo novaga2, andrea costa1 on the feeding ecology of pelophylax saharicus (boulenger 1913) from morocco zaida ortega1,*, valentín pérez-mellado1, pilar navarro2, javier lluch2 notes on the reproductive ecology of the rough-footed mud turtle (kinosternon hirtipes) in texas, usa steven g. platt1, dennis j. miller2, thomas r. rainwater3,*, jennifer l. smith4 heavy traffic, low mortality tram tracks as terrestrial habitat of newts mikołaj kaczmarski*, jan m. kaczmarek book review: sutherland, w.j., dicks, l.v., ockendon, n., smith, r.k. (eds). what works in conservation. open book publishers, cambridge, uk. http://dx.doi.org/10.11647/obp.0060 sebastiano salvidio acta herpetologica 14(2): 153-158, 2019 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/a_h-7755 at-rich microsatellite loci development for fejervarya multistriata by illumina hiseq sequencing yan-mei wang, jing-yi chen, guo-hua ding*, zhi-hua lin adi, college of ecology, lishui university, lishui 323000, zhejiang, china. * correspondence author. email: guwoding@qq.com submitted on 2018, 22th may; revised on: 2019, 3rd june; accepted on: 2019, 23rd august editor: emilio sperone abstract. in our study, a total of 2561 sequences that contained microsatellite loci were found potentially to be used for primer design. furthermore, illumina hiseq sequencing technology identified trinucleotide repeats and at-rich repeats with the the highest proportion in our genomic dna sequence library of fejervarya multistriata. eighteen new microsatellite loci of f. multistriata were isolated and we characterize these loci genotyping 48 individuals sampled from 3 populations in lishui city, zhejiang province, china. seventeen loci were polymorphic, with the number of alleles ranging from 2 to 11 within each population. the polymorphic information content, observed and expected heterozygosity ranged 0-0.845, 0-1.0 and 0-0.871, respectively. none of the loci was observed in linkage disequilibrium. one locus (fma294) was deviated from hardy-winberg equilibrium in each population separately and combined. these informative microsatellite loci will be applicable for conservation genetic studies of f. multistriata across varying scales from inter-individual to inter-population. keywords. fejervarya multistriata, genome, microsatellite, next-generation sequencing, polymorphism. introduction microsatellite dna loci, also known as simple sequence repeats, occur at thousands of locations within the eukaryotic genome, and are highly variable and sufficient in nuclear genome (ellegren, 2004; wei et al., 2015; shao et al., 2017). therefore, microsatellite dna loci are widely applied as molecular markers in population genetics, species identifying, genetic breeding and genetic map (selkoe and toonen, 2006; abe et al., 2012; wambulwa et al., 2016; soulard et al., 2017). thanks to the development of next-generation sequencing technology, both throughput and efficiency of developing microsatellite dna has increased with a decreased cost of sequencing process. in recent years, microsatellite dna markers have been quickly developed in many species at a low cost when using the next-generation sequencing technology on illumina hiseq and ion torrent pgm platforms (yang et al., 2012; lü et al., 2013; zhang et al., 2013; sultana et al., 2014; igawa et al., 2015; song et al., 2017). fejervarya multistriata (anura: dicroglossidae) is a species of frog, which is widely found in south of the yellow river in china and some countries (regions) in southeast asia, such as northern india, vietnam and myanmar (amphibiachina, 2018). the conservation status of this species is listed as data deficient in iucn (amphibiachina, 2018). this species prefers to inhabit paddy field and still water and its ovulation remains active from april to september every year (amphibiachina, 2018). as a dominant amphibian species, f. multistriata plays an important role in farmland ecosystem, and its population density in field has decreased due to urbanization (li et al., 2016). meanwhile, environmental degradation also threatens the survival of this species (othman et al., 2009). in previous studies, mitochondrial d-loop sequences were used as molecular mark154 yan-mei wang et alii ers to study phylogeography of f. multistriata populations (zhong et al., 2008). twenty-one microsatellite loci, mainly including dinucleotide repeats, had been isolated for the species, and only approximately 24% loci had at repeats (chen et al., 2018). here, we sequentially developed 18 new microsatellite dna loci containing at-rich repeats for f. multistriata by illumina hiseq sequencing. these new at-rich microsatellite loci definitely would be useful in examining genetic diversity and protecting genetic resources of f. multistriata. material and methods forty-eight f. multistriata individuals (n = 16 for each population) used in this study were collected by hand and net from 3 localities in lishui city, zhejiang province, china, which were lanshantou (lst, 119.7607°e, 28.36366°n), baimashan (bms, 119.1337°e, 28.63823°n) and jiulongshan (jls, 118.8452°e, 28.39538°n), respectively. our experimental procedures are compliant with current laws on animal welfare and research in china, which are also specifically approved by the animal research ethics committee of lishui university (permit no. arec-lu 2017-04). genomic dna was extracted from toe muscle tissue of one male f. multistriata from lst population using the dneasy tissue kit (qiagen). the concentration of dna sample was measured by using a spectrophotometer at 260 and 280 nm and dna sample was quantified on an agarose gel. a 200-400 bp sequencing library was constructed according to the manufacturer instructions (illumina). this library was sequenced using an illumina hiseq 2500 platform with rad-tag at novogene bioinformatics technology co., ltd (beijing, china, http:// www.novogene.com/). the microsatellite primer pairs of f. multistriata were designed using primer premier 3.0 software, which was used to check against potential primer dimers, hairpin structures and the occurrence of mismatches. parameters for designing the primers were set as follows: primer length ranged from 18 bp to 24 bp with 22 as the optimum; pcr product size ranged from100-280 bp; melting temperature ranged from 50 °c to 65 °c with 55 °c as the optimum annealing temperature; gc content ranged from 30% to 70% with 50% as the optimum. finally, thirty newly designed primer pairs were selected to synthesize and initially screened for microsatellite loci using total genomic dna isolated from 6 f. multistriata individuals collected from the lst population. pcr amplification reactions were performed using a thermal cycler (t100, bio-rad, usa). the total volume of each pcr mixture was 20 μl, containing 1 μl genomic dna (100 ng/μl), 10 μl premix taq (takara, japan), 1 μl of each primer (10 μm) and 6 μl double distilled h2o. the conditions of the pcr amplification were as follows: 95 °c for 5 min, then 35 cycles at 95 °c for 30 s, ta (the optimal annealing temperatures, see table 1) for 30 s, 72 °c for 30 s, and a further extension at 72 °c for 10 min. twenty-six primer pairs were further selected due to successful amplification in the 6 individuals, and the forward primer was labeled with fam or hex fluorescent dye (sangon biotech ltd. co., shanghai, china). the pcr products were genotyped on an abi 3730 sequencer (applied biosystems) and following data were analyzed with genemarker v1.8 software. population genetic parameters for polymorphic loci such as number of alleles (na), observed heterozygosity (ho), expected heterozygosity (he), polymorphic information content (pic), p values in hardy-weinberg equilibrium (hwe) tests and linkage disequilibrium were calculated by genepop 4.0 (rousset, 2008), cervus 2.0 (marshall et al., 1998) and fstat 2.9.3.2 (goudet, 1995), respectively. results and discussion sequence data from illunima hiseq we obtained a total of 6970707900 bp and 23235693 reads in a single sequencing run on illunima hiseqtm using rad-tag. the distribution frequency of read length for this species had a single peaks at approximately 125 bp. in a total of 307793 reads with more than 125 bp, 2561 reads contained microsatellite loci (0.83%). compared to a traditional library-based approach such as magnetic beads enrichment (guo et al. 2015; chang et al. 2016), next-generation sequencing technology is a more powerful approach to develop microsatellite markers due to its efficiency and low cost. sufficient microsatellite sequences can be constructed in a genomic dna sequence library on illunima hiseqtm using radtag. this result agrees with recent studies on other anuran species (wei et al., 2015; shao et al., 2017). furthermore, microsatellite obtained rate maybe higher on illunima hiseq platform (e.g., 0.83% in our study) than on ion torrent pgm platform (e.g., 0.32–0.57% in igawa et al., 2015). frequency and distribution of microsatellite loci in the genome the length of the microsatellite loci ranged from 12 to 33 bp (15.7 ± 5.2, mean ± sd). the microsatellite dna loci included 5 motif types: dinucleotide repeats (36.20%), trinucleotide repeats (52.60%), tetranucleotide repeats (9.64%), pentanucleotide repeats (0.90%) and hexanucleotide repeats (0.66%) (fig. 1a). the frequency distribution of the 5 motif types was different significantly (g-test, g = 3085.9, df = 4, p < 0.001). the motif repeat number of microsatellite loci ranged from 4 to 16, while 97.66% of the microsatellite loci had 4-12 motif repeats, and motifs with more than 12 repeats were only with a frequency of <1.0% (fig. 1b). there were 4 dinucleotide motif types, and the main types were ac/gt (50.70%), at (35.17%) and ag/ct (13.92%) (fig. 1c), 155microsatellite loci for f. multistriata respectively. there were 10 trinucleotide motif types, and the main types were aat/att (60.65%) and agg/cct (13.51%) (fig. 1c), respectively. there were 18 tetranucleotide motif types, and the main types were aaat/ attt (43.32%), acat/atgt (11.34%), aatt (10.12%) and agat/atct (10.12%) (fig. 1c), respectively. there were 13 pentanucleotide motif types, and the main types were aaaat/atttt (39.13%) and aaatt/aattt (13.04%) (fig. 1c), respectively. there were 6 hexanucleotide motif types, and the main types were aaaaat/ attttt (58.82%) and actccg/cggagt (17.65%) (fig. 1c), respectively. the frequency of different repeat types of microsatellites in f. multistriata was different from xenopus tropicalis (xu et al., 2008), odorrana narina, hoplobatrachus tigerinus, and buergeria japonica (igawa et al., 2015). the dominant repeat type was trinucleotide in f. multistriata, but dinucleotide in the last 4 anuran species (xu et al., 2008; igawa et al., 2015). such results may be related to the different next-generation sequencing platforms used in constructing sequence library. since the library of microsatellite sequences was constructed by illunima hiseq platform for f. multistriata, however, by ion torrent pgm platform for o. narina, h. tigerinus and b. japonica (igawa et al., 2015). in addition, the results suggested that the frequency of the repeat type changed randomly for each species and was species-specific. the frequencies decreased when the repeat unit length in each repeat type motif of f. multistriata increased (fig. 1b), indicating that a relatively short repeat unit of microsatellites might be a main component in the genome of f. multistriata. our finding suggested that the ratio of the repeat motifs with an at content of approximately 56% in our f. multistriata was similar to other reported anuran species (e.g., x. tropicalis, xu et al., 2008; o. narina, h. tigerinus and b. japonica, igawa et al., 2015), suggesting that the at content could be an important repeat unit in anurans. the dominant repeat motif in the trinucleotide type of f. multistriata, (aat/att repeat) was similar to the other four reported anuran species (xu et al., 2008; igawa et al., 2015). however, other types of repeat motif were different among these species. for example, f. multistriata had a higher frequency in ac/gt and aaat/ attt repeats, but o. narina, h. tigerinus, b. japonica and x. tropicalis in at and agat/atct repeats (xu et al., 2008; igawa et al., 2015). these results implied that the accumulation rates of repeat motifs were maintained in modern anurans, but skewed in a common ancestor (igawa et al., 2015). fig. 1. characterization of microsatellite loci in fejervarya multistriata genome. (a) distribution of different repeat motif types of microsatellite loci; (b) number of different repeat motifs; (c) frequency distribution of 5 different repeat types based on different motif types. number represent number of sequence. 156 yan-mei wang et alii characterization of microsatellite loci twenty-six primer pairs were used to successfully amplify genomic dna of f. multistriata from lst population. of the 26 pairs, 18 pairs generated target bands, and the other 8 pairs generated non-target bands. finally, a total of 18 primer pairs were characterized, of which 17 were polymorphic. the genomic sequences containing a microsatellite locus, which were used to design these primers, were deposited in genbank (accession number: mg744293–mg744310). the information of primer sequences, repeat motifs, ta, na, pic and heterozygosity for each locus were shown in table 1. the na, pic and heterozygosities (ho and he) ranged from 1 to 11 (4.815 ± 2. 699, mean ± sd), 0 to 0.845 (0.549 ± 0.240, mean ± sd), 0 to 1.0 (0.452 ± 0.246, mean ± sd), 0 to 0.871 (0.571 ± 0.237, mean ± sd) within each population, respectively. no significant linkage disequilibrium was observed after bonferroni correction for multiple tests (all p > 0.05). of the 18 loci, one (fma294) deviated significantly from hwe testing each population separately and combined (all p < 0.05; table 1), which indicated that null alleles may be present in this locus (song et al., 2017). the locus was assessed to contain moderately high table 1. characterization of 18 microsatellite dna markers developed for f. multistriata. size range: size range of fragment; bp: base pair; ta,: annealing temperature of primer pairs; na: number of alleles; ho: observed heterozygosity; he: expected heterozygosity; hwe: hardyweinberg equilibrium; pic: polymorphic information content; bold: significant deviation from hwe after bonferroni correction (p < 0.05). locus (genbank #) primer sequences (5’-3’) repeat motif ta (°c) size range (bp) na ho he phwe pic fma102 mg744293 f: gcactgtagagcactggattc r: gagcgtcataggggtcaaatag (ta)16 53 129-219 13 0.4792 0.7592 1.000 0.72 fma349 mg744294 f: cactcatgttatcactctactctc r: cctcctacctcttgtactaaattg (tat)11 53 156-237 11 0.3333 0.8145 1.000 0.782 fma117 mg744295 f: acttgagtctattctattctgctg r: actgctgctctgatctctatg (ata)7 53 149-158 4 0.4167 0.5893 0.996 0.495 fma402 mg744296 f: agacattaccttaaagccatagtg r: cttctgacatgacctgttcttc (agat)6 53 189-201 4 0.4583 0.6090 0.975 0.538 fma041 mg744297 f: ccaggaggattctagtgacag r: atgaaggcaagagcaatgtac (at)12 53 152-192 12 0.3958 0.8169 1.000 0.789 fma466 mg744298 f: ggtgccactgtcttaactatcc r: agtccaatcaagtccaattcaaac (ttttta)4 53 199-205 2 0.3125 0.4086 0.975 0.323 fma294 mg744299 f: gtcctcctacctcttgtactg r: cgaatgagaaccttcacagac (ata)10 53 187-238 6 0.9375 0.6515 < 0.01 0.586 fma302 mg744300 f: tccgacctcttgaaactgtattg r: aggatcaccactaggagcatc (ata)10 53 205-259 13 0.6042 0.8680 1.000 0.845 fma355 mg744301 f: tatgaccacagtctagcatcc r: ctccagtagttatcaccttcttg (aaat)5 58 158 1 – – na 0 fma188 mg744302 f: cctcttgtgttggtgtatttctg r: ttatgcttgtgttctggtcattc (aat)8 63 209-215 3 0.6042 0.5340 0.170 0.434 fma231 mg744303 f: gctgctgcatgatagtgtctc r: tgatgtctgatggtcgtcctg (tat)8 53 155-185 10 0.7917 0.8353 0.992 0.805 fma072 mg744304 f: tgcagtagacatcggagttg r: gcctctctcatcttattaagtgg (ta)13 53 209-237 10 0.6875 0.7934 0.998 0.757 fma140 mg744305 f: ttcattgtgccaagtgtaacg r: taacaaagaggtcatcactaatcc (att)7 63 153-165 3 0.1875 0.2254 0.927 0.206 fma403 mg744306 f: gcgtggatcgttattgaagtg r: ggtgacctaatgtgaaattcctg (attt)6 63 193-197 2 0.2708 0.2945 0.858 0.249 fma116 mg744307 f: ctccctaactattgtaaagcactg r: attatagatggaagcaacaggaac (ata)7 55 165-195 8 0.5208 0.7825 1.000 0.743 fma399 mg744308 f: ttcaggctacaggcattacag r: ataagggtgttctgctaaatcaag (aata)6 55 168-228 4 0.2708 0.4476 1.000 0.416 fma269 mg744309 f: aatgcttgcagaactattcacac r: tacggcggtcctaagatgg (tat)9 55 177-192 6 0.2500 0.6732 1.000 0.616 fma139 mg744310 f: gattgatggattgatgatggactg r: aatgttcaagatggacgaattacc (atg)7 55 177-189 5 0.6042 0.6535 0.813 0.584 157microsatellite loci for f. multistriata polymorphism degree when the value of pic was larger than 0.5 (song et al., 2017). overall, 11 loci had high polymorphism degree (pic > 0.5), 6 loci had low polymorphism degree (pic < 0.5). these informative microsatellite loci will be applicable for conservation genetic studies of f. multistriata across varying scales from inter-individual to inter-population. acknowledgments we thank yao-fei yu, zhi-qiang chen for their help in field work. this study was funded by national science foundation of china (31500308), zhejiang provincial natural science foundation of china (lq16c040001), key research projects of lishui city (sh2017001), zhejiang science and technology innovation program for college students (2018r434003, 2019r434006). references abe, h., hayano, a., inoue-murayama, m. 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(2008): phylogeography of the rice frog, fejervarya multistriata (anura: ranidae), from china based on mtdna d-loop sequences. zool. sci. 25: 811-882. acta herpetologica vol. 14, n. 2 december 2019 firenze university press podarcis siculus latastei (bedriaga, 1879) of the western pontine islands (italy) raised to the species rank, and a brief taxonomic overview of podarcis lizards gabriele senczuk1,2,*, riccardo castiglia2, wolfgang böhme3, claudia corti1 substrate type has a limited impact on the sprint performance of a mediterranean lizard pantelis savvides1,*, eleni georgiou1, panayiotis pafilis2,3, spyros sfenthourakis1 coping with aliens: how a native gecko manages to persist on mediterranean islands despite the black rat? michel-jean delaugerre1,*, roberto sacchi2, marta biaggini3, pietro lo cascio4, ridha ouni5, claudia corti 3 pit-tags as a technique for marking fossorial reptiles: insights from a long-term field study of the amphisbaenian trogonophis wiegmanni pablo recio, gonzalo rodríguez-ruiz, jesús ortega, josé martín* occurrence of batrachochytrium dendrobatidis in the tensift region, with comments on its spreading in morocco ait el cadi redouane1, laghzaoui el-mustapha1, angelica crottini2, slimani tahar1, bosch jaime3,4, el mouden el hassan1,* hematological parameters of the bolson tortoise gopherus flavomarginatus in mexico cristina garcía-de la peña1,*, roger iván rodríguez-vivas2, jorge a. zegbe-domínguez3, luis manuel valenzuela-núñez1, césar a. meza herrera4, quetzaly siller-rodríguez1, verónica ávila-rodríguez1 ontogenetic and interspecific variation in skull morphology of two closely related species of toad, bufo bufo and b. spinosus (anura: bufonidae) giovanni sanna visible implant alphanumeric (via) as a marking method in the lesser snouted treefrog scinax nasicus andrea caballero-gini1,2,3,*, diego bueno villafañe2,3, lía romero2, marcela ferreira2,3, lucas cañete4, rafaela laino2, karim musalem2,5 morphological variation of the newly confirmed population of the javelin sand boa, eryx jaculus (linnaeus, 1758) (serpentes, erycidae) in sicily, italy francesco p. faraone1,*, salvatore russotto2, salvatore a. barra3, roberto chiara3, gabriele giacalone4, mario lo valvo3 variability in the dorsal pattern of the sardinian grass snake (natrix natrix cetti) with notes on its ecology enrico lunghi1,2,3,4,*, simone giachello5, manuela mulargia6, pier paolo dore7, roberto cogoni8, claudia corti1 estimating abundance of the stripeless tree-frog hyla meridionalis by means of replicated call counts federico crovetto, sebastiano salvidio, andrea costa* at-rich microsatellite loci development for fejervarya multistriata by illumina hiseq sequencing yan-mei wang, jing-yi chen, guo-hua ding*, zhi-hua lin acta herpetologica 14(1): 27-33, 2019 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-24102 scientific publication of georeferenced molecular data as an adequate guide to delimit the range of korean hynobius salamanders through citizen science amaël borzée1,*, hae jun baek2,3, chang hoon lee2,3, dong yoon kim2, jae-young song4, jae-hwa suh5, yikweon jang1, mi-sook min2,* 1 division of ecoscience, ewha womans university, 03760, seoul, republic of korea. *corresponding authors. e-mail: amaelborzee@ gmail.com; minbio@yahoo.co.kr 2 research institute for veterinary science, college of veterinary medicine, seoul national university, 08826, seoul, republic of korea 3 national institute of ecology, seocheon, 33657, south chungcheong province, republic of korea 4 national park research institute, korea national park service, wonju, 26441, gangwon province, republic of korea 5 national institute of biological resources, 22689, incheon, republic of korea submitted on: 2018, october 26th; revised on: 2019, february 20th; accepted on: 2019, march 1st editor: daniele pellitteri-rosa abstract. despite the importance of clearly assessing the distribution boundaries of species, it is not possible for scientists to acquire genetic information and conduct molecular analysis for all populations. consequently, citizen science is of increasing importance for large scale data collection. in this study, we described the range boundaries of the four hynobius species occurring in korea based on genetic identification and refined their distribution through citizen science data. the genetic identification of individuals was extracted from the literature, while the citizen science data were extracted from inaturalist through gbif. distribution boundary lines were drawn from the genetic data and consistency with citizen science datapoints was assessed through a comparative analysis with the points found beyond the established boundary lines. depending on the species, 1.43 to 25.00% of the observations extracted from the citizen science data were located beyond the boundaries suggested by the molecular analyses, with average distances ranging from 3.51 ± 2.97 to 51.47 ± 30.87 km (mean ± sd). we considered these variations negligible in the view of the whole distribution of these species. in general, the distributions extracted from inaturalist were accurate and adequately representative of the distribution of the species, with the exception of the recently split h. quelpaertensis. additionally, citizen science data highlighted the absence of gaps in the distribution of these species. in conclusion, given the good accuracy of citizen science data, we recommend the publication of molecular based data so that citizen science platforms could help define accurately the range of species for which data is missing or outdated. keywords. hynobius, range description, population presence, public engagement, inaturalist, republic of korea, north east asia, citizen science. introduction despite a newly renewed interest in citizen science over the last decades (reed, 2008; crain et al., 2014; jordan et al., 2015), citizen science has been playing a major role in the advancement of natural and ecological sciences over the last centuries (gray et al., 2017; mckinley et al., 2017), with demonstrated benefit to biodiversity conservation (newman et al., 2012, 2017; mckinley et al., 2017). because of the ubiquitous impact of human activities on biodiversity (pimm and raven, 2000; steffen et al., 2011) and the species extinction rate, which could be a thousand 28 amaël borzée et alii times higher with respect to pre-human levels (pimm et al., 1995; scheffers et al., 2012), large-scale data collection is becoming more and more urgent. since one of the strengths of citizen science is the quantity of datapoints collectable over a short period of time (lintott et al., 2010), relying on this discipline to create conservation policies for the remaining species, through ever more convenient and accurate technologies (sullivan et al., 2009; joppa et al., 2012; bowser et al., 2014) is therefore one of the potential ways to breach the wave of destruction. citizen science involvement takes several forms and it is assessed to be extremely efficient when conducted with clear protocols and objectives (shirk et al., 2012; gray et al., 2017), or combined with molecular analysis (silvertown et al., 2011). conversely, not everyone is satisfied by the quality of data collected through citizen science, as discussed by cohn (2008), conrad and hilchey (2011) and dickinson et al. (2010). it is however impossible for a single researcher to conduct large scale field surveys on the totality of a species’ range. for instance, it took four years of field work to describe the comparatively small range of the suweon treefrog (dryophytes suweonensis), a non-cryptic and highly detectable species, via a study conducted almost every day of the species’ breeding season (borzée et al., 2017). in comparison, as of 1 february 2018, the totality of the species’ range, minus a single location, is available from the citizen science website inaturalist (https://www.inaturalist.org). the species used as an example is comparatively well studied (borzée, 2018) but there are other well-studied species which geographic ranges are poorly described (jetz et al., 2012; meyer et al., 2015). additionally, ranges are dynamic and need regular updates, as they can show geographic shifts in response to climate change (chen et al., 2011). given that technologies become increasingly userfriendly and convenient for citizen science, an increase in the quality and resolution of the data uploaded is expected. for instance, it is common to upload datapoints directly from the observation site, including gps coordinates at the cm resolution, pictures and other metadata. a rising platform for the upload of observations is inaturalist (www.inaturalist.org), and the platform success in recording species is demonstrated by the presence of data for about 75% of bird species and 35% of amphibian species (as of 1 february 2018). the particularity of inaturalist is that, despite anyone being able to upload any observation, classified as “need id” if fulfilling minimum requirements, the observations are then cross-validated to obtain a “research grade”. this requires the id to be confirmed by at least two-thirds of the identifiers (i.e. anyone interested in confirming or reassigning the species/genera/clade id of the observation). therefore, the id provided does not reflect the knowledge of a single person but that of the community, and therefore of a meta-brain, including scientist expert in their field (joppa et al., 2012; he and wiggins, 2015). here, we first defined the range of the four hynobius salamander species occurring in korea through traditional molecular tools, and then refined range and presence within ranges through the platform inaturalist. the four species are hynobius leechii, h. quelpaertensis, h. yangi and h. unisacculus. the secondary purpose of this work was to highlight the accuracy of citizen science in a region where it is still comparatively under-used (roh et al., 2014). materials and methods species four of the described hynobius species are present in the republic of korea, and three of these species are endemic (min et al., 2016). hynobius leechii is widespread on the korean peninsula and north-east china, while h. quelpaertensis, h. yangi and h. unisacculus are restricted to the southern coastal area of the peninsula (yang et al., 1997, 2001, 2005; kim et al., 2003; min et al., 2016). this coastal area is also populated by three candidate species (baek et al., 2011a, 2011b), although these were not included in our analyses as the clades have not yet been given the species status. the species breed between february and may, both in natural streams and modified landscapes in the form of rice paddies. they are locally abundant species present under vegetation and litter of forested hills outside of the breeding season. molecular assessment the hynobius sequences used here were extracted from the literature (kim et al., 2003; yang et al., 2005, 2007; baek et al., 2011a, 2011b; min et al., 2016). each data point for which molecular identification was available, based on any gene sequence, was incorporated in the dataset, resulting on n = 270 for molecular-based species assignment. citizen science data prior to data download, the citizen science data on inaturalist (https://www.inaturalist.org) were curated on 15 october 2017 for obvious errors. a query for observations was created with the filters “hynobius” and “south korea”, and a few observations were flagged as “captive” when coming from zoos or private collections, based on gps coordinates. the citizen science datapoints were then downloaded through gbif. org (https://doi.org/10.15468/dl.tb0v6j; accessed 5 february 2018), filtered for hynobius observations in the republic of 29hynobius spp. ranges and citizen science korea and from inaturalist only, dated up to 15 october 2017. only the observations reaching “research grade” on inaturalist are transferred to gbif, and the research grade can only be reached when more than two-thirds of the identifiers agree on a taxon. the original download included 852 datapoints, but all the observations with known issues flagged by gbif were removed, and duplicated records were deleted. additionally, only datapoints geolocated with an accuracy of at least three decimal places (100 m resolution) were maintained. finally, to avoid spatial autocorrelation, any point within 200 m of another point from the same dataset was deleted, in correspondence to the core range of several salamander species (semlitsch, 1998; semlitsch and bodie, 2003). this selection resulted in 468 datapoints, collected between 18 april 2005 and 26 august 2017. spatial and statistical analysis the two datasets where then uploaded on arcmap 10.5 (environmental systems resource institute, redlands, california, usa) and each species was colour coded (fig. 1). based on the genetic analyses data, we drew lines joining the border localities of each species. for localities close to the seashore, border lines which guaranteed the smallest distance between the locality and the coast were drawn. given the geographically representative sampling, in relation to the low vagility of the species and of salamanders in general (semlitsch, 1998; semlitsch and bodie, 2003), we can consider the drawn lines as adequate estimates of each species’ range. in addition, since no large-scale hybrid zones are expected between the species in this study (baek et al., 2011a; min et al., 2016), we excluded the possibility of a significant misidentifications because of cytonuclear disequilibrium. once the boundary of the four species were established, here referred to as “distribution boundary lines”, we counted for each species the number of localities identified by citizen science which were external to the distribution boundary lines and measured the distance between the focal locality and the closest distribution boundary line. we did not include datapoints located between dna identified localities and the sea shore, as not all islands were genetically tested. the presence points within the species boundary lines were then investigated in gis through the distance tool for clear gaps in distribution. for this purpose, a gap was defined as one tenth of the longest diagonal crossing the range of the species, here limited to the republic of korea. the distance between datapoints and distribution boundary lines was then statistically tested for differences between species. as the data was not normally distributed for each cell of the design (observation of q-q plots), and there were no significant correlations between the four species and distance to the boundary lines (pearson correlation; r = 0.09, n = 27, p = 0.663), we used an independent-samples kruskal-wallis test to assess the relationship among the distances between datapoints and distribution boundary lines. the statistical analyses were performed with spss v21.0 (spss, inc., chicago, usa). results the dna based location map used to draw the distribution boundary lines included 270 samples (fig. 1), divided into 164 datapoints for h. leechii (fig. 2), 45 for h. quelpaertensis (fig. 3), 19 for h. unisacculus (fig. 4) and 43 for h. yangi (fig. 5). the citizen science data included 468 samples, distributed into 350 datapoints for h. leechii, 92 for h. quelpaertensis, 12 for h. unisacculus and 14 for h. yangi. there were 27 (13.28%) citizen science datapoints that were external to the distribution boundary lines: 5 for h. leechii (1.43% of datapoints), 18 for h. quelpaertensis (19.57%), 3 for h. unisacculus (25.00%) and 1 for h. yangi (7.14%). we did not find any gap in population presence that was higher than one tenth of the longest diagonal crossing the range of the species within the republic of korea. the average distance for datapoints beyond the distribution boundary lines was 37.17 ± 32.54 km (mean ± sd). h. unisacculus displayed the shortest average distance (3.51 ± 2.97; n = 3), followed by h. leechii (4.75 ± 2.99; fig. 1. distribution of hynobius spp. in the republic of korea. the map includes h. leechii, h. quelpaertensis, h. unisacculus and h. yangi data extracted from both mtdna and citizen science (inaturalist through gbif; doi.org/10.15468/dl.tb0v6j). 30 amaël borzée et alii n = 5), by h. yangi (20.67 km; n = 1) and finally by h. quelpaertensis (51.47 ± 30.87; n = 18). the independentsamples kruskal-wallis test used to assess whether the distance between citizen science datapoints and the distribution boundary lines varied between species was significant (h = 1.49, df = 3, n = 27, p = 0.009). the largest divergence between distribution boundary lines and citizen science observation was observed for h. quelpaertensis (fig. 6). discussion through the integration of citizen science-based data collection following the guides provided by molecular tools, we refined the distribution of the four korean fig. 2. distribution of hynobius leechii in the republic of korea. the map includes both mtdna and citizen science datapoints (inaturalist through gbif; doi.org/10.15468/dl.tb0v6j), with the distribution boundary lines drawn from mtdna data. fig. 3. distribution of hynobius quelpaertensis in the republic of korea. the map includes both mtdna and citizen science datapoints (inaturalist through gbif; doi.org/10.15468/dl.tb0v6j), with the distribution boundary lines drawn from mtdna data. fig. 4. distribution of hynobius unisacculus in the republic of korea. the map includes both mtdna and citizen science datapoints (inaturalist through gbif; doi.org/10.15468/dl.tb0v6j), with the distribution boundary lines drawn from mtdna data. fig. 5. distribution of hynobius yangi in the republic of korea. the map includes both mtdna and citizen science datapoints (inaturalist through gbif; doi.org/10.15468/dl.tb0v6j), with the distribution boundary lines drawn from mtdna data. 31hynobius spp. ranges and citizen science hynobius species and established the absence of gaps in their distribution. gaps in distribution may be found at higher elevations but analyses with a better accuracy would be needed to ascertain this point. additionally, the number of datapoints collected through citizen science that were external to the distribution boundary lines defined by molecular tools was very low, both in number and in average distance. in most cases, the ranges described by citizen science were comparatively conservative, with 86.72 % of sites within the distribution boundary lines. moreover, our results do not imply that citizen science points beyond the boundary lines are incorrect, as further genetic testing may narrow down the width of contact zones between species. finally, we confirm that when citizen science data collection follows clear landmarks set by molecular analyses, the results provided are clear and accurate. however, we need to emphasize that citizen science is most efficient when conducted within a specified framework (shirk et al., 2012; gray et al., 2017). our work follows the steps of other citizen science projects focused on amphibians (e.g., mossman et al., 1998; corn et al., 2000; roh et al., 2014) and we recommend the broader development of this type of projects. if specific projects were set up for the distribution of hynobius, instead of the opportunistic data collected here, even more precise results would be expected. regarding the discrepancy points between the two methods, the largest majority was located within the distribution range of h. quelpaertensis before the species was split into h. quelpaertensis and h. unisacculus (min et al., 2016). therefore, once these cases removed, only nine discrepancy points remained, highlighting the impact of recent taxonomic modifications onto natural science enthusiasts. eventually, the combination of knowledgeable nature enthusiasts and the verification method for “research grade” on inaturalist provides clear distribution patterns. interestingly, the distribution patterns drawn here from citizen science are more accurate than the ones extracted from the red list of the international union for conservation of nature (http://www.iucnredlist.org; as of february 2018). we therefore recommend the use of citizen science platforms, such as inaturalist, to assess the distribution of species that have not been assessed yet, or those in need of an updates, such as the korean hynobius species. our results however call for a resolution of the taxonomic question regarding the overlapping ranges and potential hybridisation between the different korean hynobius clades. for instance, the known subclades within h. leechii, geographically located between the distribution of h. unisacculus and h. yangi (baek et al., 2011a, 2011b; min et al., 2016) could not be used in this study. however, the taxonomic resolutions are also expected to cause confusion in the identification of hynobius individuals by nature enthusiast when and if new species are described. for this reason, the involvement of experts on citizen science platforms is essential and the resulting general education, together with the development of interests, is one of the best ways to reach conservation purposes on the long term (cooper et al., 2007; marshall et al., 2012). an important point that also needs to be raised here is that citizen science accuracy is directly related to the taxon studied (cohn, 2008; crall et al., 2011; gardiner et al., 2012). the identification of hynobius sp. based on morphology is not considered easy (kim et al., 2003), even for experienced researchers, and the molecular identification available was the most important contributing factor for the refinement of the ranges presented here. additionally, a small group of dedicated users on the citizen science platform could make a significant difference. most observations used for this study were confirmed by a group of dedicated users, allowing the observations to reach the “research grade”, or oppositely, downgraded to the genus identification because of disagreements with the original observer. these people are therefore important in their own rights for the accumulation of knowledge on species, as already observed by rotman et al. (2012) and johnston et al. (2017). acknowledgements the data collected for this project was downloaded from gbig.org, an open-access database, such as defined in the memorandum of understanding on gbif, paragraph 8. data were not downloaded from inaturalist directly as it does not provide a doi for the dataset, unlike gbif. this work was supported by a research grant from the rural development administration (pj012285) to yj, and by a grant from the national research foundation of korea (nrf) funded by the ministry of education (nrf-2016r1d1a1b03934071) to msm. references baek, h.j., lee, m.y., lee, h., min, m.s. 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(2007): survey on the habitat of gori salamander in the site of new-gori nuclear power plant no. 3 and no. 4 and the measures for conservation. korea hydro & nuclear power, republic of korea. acta herpetologica vol. 14, n. 1 june 2019 firenze university press uzungwa scarp nature forest reserve: a unique hotspot for reptiles in tanzania john valentine lyakurwa1,2,*, kim monroe howell2, linus kasian munishi1, anna christina treydte1,3 experience of predacious cues and accessibility to refuge minimize mortality of hylarana temporalis tadpoles santosh mogali*, bhagyashri shanbhag, srinivas saidapur tonal calls as a bioacoustic novelty in two atlantic forest species of physalaemus (anura: leptodactylidae) thiago r. de carvalho1,*, célio f.b. haddad1, marcos gridi-papp2 scientific publication of georeferenced molecular data as an adequate guide to delimit the range of korean hynobius salamanders through citizen science amaël borzée1,*, hae jun baek2,3, chang hoon lee2,3, dong yoon kim2, jae-young song4, jaehwa suh5, yikweon jang1, mi-sook min2* mirrored images but not silicone models trigger aggressive responses in male common wall lizards stefano scali1,*, roberto sacchi2, mattia falaschi1,3, alan j. coladonato2, sara pozzi2, marco a.l. zuffi4, marco mangiacotti1,2 using an in-situ infra-red camera system for sea turtle hatchling emergence monitoring fatima n. oğul1,#,*, franziska huber2,#, sinem cih1, kumsal düzgün3, ahmet e. kideyş1, korhan özkan1 descriptive osteology of an imperiled amphibian, the luristan newt (neurergus kaiseri, amphibia: salamandridae) hadi khoshnamvand1, mansoureh malekian1,*, yazdan keivany1, mazaher zamani-faradonbe1, mohsen amiri2 does color polymorphism affect the predation risk on phalotris lemniscatus (duméril, bibron and duméril, 1854) (serpentes, dipsadidae)? fernanda r. de avila1,2,*, juliano m. oliveira3, mateus de oliveira1, marcio borges-martins4, victor hugo valiati2, alexandro m. tozetti1 age structure of a population of discoglossus scovazzi camerano, 1878 (anura discoglossidae) in extreme environmental conditions (high atlas, morocco) mohamed amine samlali, abderrahim s’khifa, tahar slimani* acta herpetologica 15(1): 15-20, 2020 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/a_h-7863 first record of underwater sound produced by the balkan crested newt (triturus ivanbureschi) simeon lukanov institute of biodiversity and ecosystem research, bulgarian academy of sciences, tzar osvoboditel № 1, 1000 sofia, bulgaria. email: simeon_lukanov@abv.bg submitted on: 2019, 29th june; revised on: 2019, 27th september; accepted on: 2019, 14th november. editor: simon baeckens abstract. this study presents first evidence for underwater sounds produced by adult balkan crested newts (triturus ivanbureschi). recordings were made in spring of 2019 in controlled laboratory conditions using a commercially available omnidirectional hydrophone connected to a linear pcm recorder. a total of 27 animals (21 males, 6 females) were recorded under different conditions: (a) alone in an empty tank, (b) alone in a tank full of vegetation, and (c) a pair in an empty tank. results indicated that both male and female newts produced a click-like sound with a mean duration of 34 ms (± 5.31 sd; range: 27-51) and mean peak frequency of 1887 hz (± 405 sd; range: 1162-2770). not all newts tested produced sounds and there were no statistically significant differences between males and females or recordings under different conditions in terms of click number, duration and frequency parameters, with the exception of the ratio of peak frequency/bandwidth at 50% peak amplitude, which was lower for clicks produced in the vegetated tank. newt snout-vent length and body mass also had no effect on any of the studied parameters. the obtained results suggest that clicks could have a function in orientation and exploratory behaviour. keywords. clicks, conditions, environment, interaction, orientation, phase. introduction sound production plays an important role for many taxa across the animal kingdom. for amphibians, this is mainly true for anurans – the first early attempts at more detailed studies of the functions of vocal signals in anurans date from the first half of the 20th century (noble and noble, 1923), and the first comprehensive work on acoustic communication in amphibians and reptiles is by bogert (1960), which is also the first attempt to view the information of amphibian vocal signals in an evolutionary and ecological frame. in recent decades there is a growing number of publications describing the structure of anuran calls (e.g., ziegler et al., 2011; guerra et al, 2018; stanescu et al., 2018; carvalho et al., 2019), geographic variations of vocal signals in some species (e.g., amezquita et al., 2009; kaefer and lima, 2012), as well as the role of the signals as phylogeographic indicators (e.g., stöck et al., 2008; vences et al., 2013). in contrast, caudate amphibians have received very little attention in this regard. while they lack a tympanic middle ear, a number of studies have established that their underwater auditory abilities are sufficient for them to sense sound frequencies by other means, such as their mouths or lungs (e.g., hetherington and lombard, 1983; christensen et al., 2015). available data on their sound production is limited to just a few taxa, and even then, the information is mostly descriptive, with little effort to analyse its ecological function. in addition, most of the data is on north american species, leaving the rest of the world almost unstudied. even though a variety of sounds made by caudate amphibians have been described as early as the mid-twentieth century (maslin, 1950; neil, 1952), they were assumed to be mostly non-func16 simeon lukanov tional and produced by unintentional expulsion of air (bogert, 1960). on land, some caudate species are known to produce squeaking noises when stressed (“mouselike squeaking note”; maslin, 1950). although a number of authors have registered underwater hisses, clicks or squeaks in various species (maslin, 1950; gehlbach and walker, 1970; wyman and thrall, 1972; davis and brattstrom, 1975; crovo et al., 2016), their exact purpose is still unknown. maslin (1950) suggests they are unintentional, but others propose they could serve a purpose in social interactions (gehlbach and walker, 1970; davis and brattstrom, 1975; crovo et al., 2016) or orientation (gehlbach and walker, 1970). a recent study by hubáček et al. (2019) registered a high number of underwater low and mid-frequency clicks produced by ichthyosaura alpestris and lissotriton vulgaris, suggesting that newts are much more vocally active than demonstrated by currently available data. this study tested the hypothesis that the balkan crested newt triturus ivanbureschi arntzen & wielstra, 2013 was vocally active underwater. the aim was to describe the registered sounds and to present possible explanations for the potential role of the produced clicks in the species orientation or interaction. materials and methods crested newts (triturus cristatus superspecies) are a group of closely related species with medium size (mean adult snoutvent length [svl] varies between 62.5-84.4 mm for different species, review in lukanov and tzankov, 2016) and a biphasic lifestyle (aquatic and terrestrial phase) that are distributed parapatrically in the old world. the balkan crested newt is distributed in both europe and asia – its range covers the eastern balkan peninsula, including most of bulgaria, the eastern parts of greece, north macedonia and serbia, as well as northwestern turkey (speybroeck et al., 2016). in bulgaria, the species occurs across most of the country from sea level up to 1700 m elevation, inhabiting various types of lentic water bodies (stojanov et al., 2011). while other newt species (e.g., the alpine newt i. alpestris) have been observed to produce squeaks when handled out of the water or soft gulping sounds when taking air at the water surface (maslin, 1950), there is no literature data on the sound production of any crested newt species. in april 2019 a total of 27 adult newts (21 males, 6 females) were caught using funnel traps in a small natural pond overgrown with bulrush (typha sp.), yellow iris (iris pseudacorus) and common bladderwort (utricularia vulgaris) near the city of sofia, bulgaria (42°35’42”n, 23°22’5”e). all animals were housed indoors in a large (100×100×60 cm) glass tank filled with water and were fed common earthworms (lumbricus terrestris) ad libitum. ambient temperature was constant at 18 °c. experiments were conducted in a small glass tank (40×20×60 cm) with water level at 15 cm, with the hydrophone completely submerged 5 cm below the water surface and fixed in such a position as to not touch the bottom or the sides of the tank. three types of trials were conducted to test for sound production under different conditions – single newt in an empty tank (trial 1), single newt in a tank full of water vegetation (trial 2), and a pair of newts in an empty tank (trial 3). the pair in the third trial would consist of female/female, female/male or male/male. the trials were designed in order to assess whether any registered sounds played a role in orientation or social interaction. after sex determination, newts were placed in the tank individually for the first two trials or as a pair for the third trial, and after 5min of adaptation, newt sounds were recorded for the next 10 min. snout-vent length (svl) of all newts was measured with a ruler (to 0.1 cm) and body mass was weighted (to 0.01 g) using digital balance scales (durascale d2 capacity pocket scale). because of the crepuscular/nocturnal lifestyle of the study species, all trials were performed in complete darkness. no animals were used in the same trial twice (except for the six females, which were used once in female/female pairs and a second time in female/male pairs) or recorded more than once per day. vegetation used in the second trial was collected from the study pond and consisted of bulrush and common bladderwort leaves (both freshly cut and old); it covered 2/3 of the tank, with the hydrophone fixed in the other 1/3. during the tests there was no human presence in the room and newts had approximately three days to rest between trials. all experiments complied to the international requirements for ethical treatment of animals (lehner, 1996) and after they were completed, all newts were released at the site of capture. vocal activity was recorded using an olympus ls-5 linear pcm recorder and an omnidirectional aquarian h2a hydrophone (sensitivity: -180 db re: 1v/µpa, useful range: 10 hz to 100 khz). recordings were made in a wav-pcm mode with sampling frequency of 44.1 khz, 20-21.00 hz and 24-bit resolution. the recordings were processed with the open source software soundruler v. 0.9.6.0. (gridi-papp et al., 2007). the following parameters were measured: (1) number (the number of clicks produced during each recording), (2) duration (difference between the beginning and the end of a click, measured in ms from the spectrogram), (3) peak frequency (click frequency with the highest energy, measured in hz), (4) tune 50 (the ratio of peak frequency/bandwidth at 50% peak amplitude), and (5) tune 10 (the ratio of peak frequency/bandwidth at 10% peak amplitude). spectrograms used for the measurements of click duration had a hanning window type and an fft length of 512 points. all data was tested for normality using a shapiro-wilk test and the null hypothesis was rejected (p < 0.001). a mannwhitney u test was used to compare clicks produced by males and females in the solitary treatment, and a kruskal–wallis h test was used to check for differences in the measured parameters between recordings under different conditions. a spearman rank order correlation was used to test whether svl and body mass were related to the studied sound parameters. a kruskalwallis h test revealed no statistically significant differences between pairs in trial 3 (see appendix) and they were treated as one sample for the statistical analyses in the comparison between the trials. the chosen level for statistical significance 17underwater sound of t. ivanbureschi was p < 0.05. all analyses were carried out using the computer program statistica v.7.0 (statsoft, inc., 2004). results on average, newts started to produce clicks after 173 s (± 122 sd, range: 8-396) and although that latency varied widely between individuals, there were no statistically significant differences across trials (kruskal-wallis h test; h(2) = 0.148, p = 0.929) or sexes (mann-whitney u test; u = 68.50, p = 0.856). all registered clicks had a simple non-harmonic structure and their peak frequency was in the lower ranges of the spectrum (fig. 1). descriptive statistics for all measured parameters are presented in table 1. the mann-whitney u test revealed there were no statistically significant differences between sexes in all studied parameters of the registered clicks: number (u = 322.500, p = 0.582), duration (u = 621.500, p = 0.473), peak frequency (u = 449.500, p = 0.490), tune 50 (u = 401.500, p = 0.193) and tune 10 (u = 424.500, p = 0.313). results from the kruskal–wallis h test used for the comparison of the three trials were similar – there were no statistically significant differences among the trials in terms of number (h(2) = 0.443, p = 0.801), duration (h(2) = 5.319, p = 0.070) and peak frequency (h(2) = 3.910, p = 0.142) of the clicks, as well as in parameter tune 10 (h(2) = 2.829, p = 0.243). however, there was a significant difference between the parameter tune 50 from trial 2 and the other two trials h(2) = 18.067, p = 0.001; values for this parameter were significantly lower in trial 2 compared to trial 1 and trial 3 (table 1). normally clicks were not produced in series, but on three occasions (two in trial 1 and one in trial 2) there were more than one in quick succession. in two of the cases (one in trial 1 and trial 2) the duration of the intervals between the clicks was comparable to that of the clicks themselves at 69 ms and 34 ms, respectively; in the third case, there were three clicks in the space of around one second, with the intervals between them standing at 551 ms and 325 ms. there was a statistically significant difference in both svl and body mass between males and females, but there was no significant effect of svl and body mass on any of the studied parameters (table 2). it has to be noted that during handling of the animals, 10 (two females, eight males) out of the 27 newts produced both clicks and squeaks – however, attempted recordings of these sounds were too faint to analyse and therefore were discarded. fig. 1. from top to bottom: indicative oscillogram, spectrogram and amplitude spectrum of a click. table 1. measured parameters of the recorded clicks in t. ivanbureschi (number of vocalizing newts/pairs is in parenthesis). statistically significant differences between trials are in bold; when no such differences are present, a combined value for all groups is given in the last column. data is presented as mean (min-max ± sd). empty tank (trial 1), n = 27 vegetated tank (trial 2), n = 27 pair (trial 3) n = 13 (9) all groups male (13) female (4) combined (17) male (9) female (4) combined (13) total clicks 26 4 30 28 11 39 17 86 number 1.14 (0-4 ± 1.195) 1 (0-2 ± 0.632) 1.111 (0-4 ± 1.086) 1.24 (0-7 ± 2.022) 2.16 (0-8 ± 3.060) 1.444 (0-8 ± 2.258) 1.211 (0-3 ± 1.182) 1.260 (0-8 ± 1.625) duration 36 (26-51 ± 5.910) 33 (29-36 ± 2.655) 36 (26-51 ± 5.500) 35 (27-51 ± 6.910) 33 (27-39 ± 3.781) 34 (27-51 ± 6.050) 34 (30-40 ± 2.783) 34 (27-51 ± 5.314) frequency 1920 (13922656 ± 288) 2096 (11622684 ± 510) 1961 (11622684 ± 348) 1769 (13972346 ± 375) 1785 (13922340 ± 379) 1774 (13922346 ± 370) 2026 (011622770 ± 535) 1887 (11622770 ± 405) tune 50 9.919 (2.30026.622 ± 6.709) 6.894 (2.23211.214 ± 3.751) 9.221 (2.23226.622 ± 6.222) 3.985 (1.5996.779 ± 1.348) 3.628 (1.6256.002 ± 1.5508) 3.877 (1.5996.779 ± 1.398) 8.980 (2.13916.154 ± 5.146) n/a tune 10 1.906 (0.4945.189 ± 1.214) 2.322 (0.5275.378 ± 2.311) 2.002 (0.4945.378 ± 1.490) 1.224 (0.6463.257 ± 0.538) 1.122 (0.6441.499 ± 0.326) 1.193 (0.6443.257 ± 0.481) 1.861 (0.4858.389 ± 2.082) 1.606 (0.4858.389 ± 1.329) 18 simeon lukanov discussion the study demonstrated that the balkan crested newt emits sounds underwater. parameters of produced clicks highly overlapped between sexes and across the three test trials, with svl and body mass also having no visible effect. while underwater sound production has been previously documented in four urodelan families – ambystomatidae (wyman and thrall, 1972; licht, 1973), amphiumidae (crovo et al., 2016), salamandridae (davis and brattstrom, 1975; hubáček et al., 2019) and sirenidae (gehlbach and walker, 1970) – this is the first report for representatives of the crested newts species group, and the second on european salamandrid species, following the recent study of hubáček et al. (2019). wien et al. (2011) assert that sound-producing lineages of salamanders have split from their common ancestors around 60 mya, which indicates that sound production is either a shared trait among newts, or has evolved independently in several lineages. steinfartz et al. (2007) estimate that the last common ancestor of the triturus species group lived around 64 mya, which is a very similar timeline. the latter study established that the triturus assemblage included four monophyletic groups: large-bodied newts (incl. t. ivanbureschi, referred to as “t. karelinii”), smallbodied newts (incl. l. vulgaris, study species in hubáček et al., 2019), i. alpestris (study species in hubáček et al., 2019) and ommatotriton vitatus. this early separation between large-bodied crested newts and the smaller species of the assemblage offers at least partial explanation for the differences between the produced sounds. hubáček et al. (2019) established two types of clicks (low frequency and mid-high frequency), but found no difference between clicks produced by l. vulgaris and i. alpestris. their mean duration varied between 7.67 ms and 10.74 ms for the low clicks and between 10.60 ms and 12.14 ms for the mid-high clicks, which is around three to five times shorter than the clicks recorded in the present study. the low-frequency clicks had a mean peak frequency between 7.46 khz to 7.97 khz, which is still significantly higher than the average of 1.89 khz established for t. ivanbureshi. in any case, direct comparisons should be considered with caution, as some of the differences could be due to the different equipment and settings used for the recordings. nevertheless, it has to be noted that clicks in t. ivanbureschi were more similar to the ones in the american salamandrid species taricha torosa, which had duration between less than 0.1 s up to 0.4 s and frequency of 1.48 khz (davis and brattstrom, 1975). results of the present study also seem to be consistent with the authors’ observation that clicks in t. torosa were produced during exploratory behaviour, when newts were placed in an unfamiliar setting; however, additional studies are needed in order to clarify this. both species are of similar size and overall body shape (in contrast to l. vulgaris and i. alpestris, which are smaller and less robust), but at present more studies are needed in order to establish whether phenotypic similarities could result in production of similar sounds. unlike t. ivanbureschi, clicks produced by t. torosa did have a harmonic structure, with harmonics at 3.5, 5 and 6.5 khz (davis and brattstrom, 1975). while there was only one statistically significant difference between trial 2 and the other two trials (parameter tune 50), this and the relatively higher number of recorded clicks in trial 2 could implicate the role of sound in t. ivanbureschi orientation. for some anurans, call parameters such as call rate and pulse number have been established to have a highly directional effect (gerhardt, 1991) and this could also be true for newts, with the dynamic ratio of frequency/amplitude being beneficial in orientation. while data for definitive conclusions is still lacking, there are studies demonstrating that acoustic information in the form of anuran calls improves orientation in other newt species (e.g., diegorasilla and luengo 2004, 2007). the only other study on sound production in caudate amphibians that also provides data for body mass is that of hubáček et al. (2019), which also did not find any relation between weight and produced clicks, so the possible role of svl and body mass on sound production in newts remains to be clarified. the fact that some of the newts used in this study produced clicks while being handled out of the water could indicate that clicks are some form of a distress signal, or displacement activity (involuntary response) – the latter has been demonstrated for visual signals in some anuran species (furtado et al., 2016); however, since recordings of terrestable 2. measurements of svl and body mass comparison between sexes and correlations across studied parameters, with statistically significant differences in bold. data is presented as mean (min-max ± sd). svl (cm) body mass (g) males 7.75 (7.20-8.70 ± 0.43) 12.01 (7.75-20.35 ± 2.79) females 8.3 (7.90-9.10 ± 0.46) 18.57 (12.71-21.42 ± 3.04) mann-whitney u test u = 20.00, p = 0.012 u = 10.00, p = 0.002 spearman rank order correlation test number r = 0.074, p = 0.592 r = 0.199, p = 0.149 duration r = -0.218, p = 0.074 r = -0.074, p = 0.548 frequency r = 0.003, p = 0.980 r = -0.002, p = 0.990 tune 50 r = -0.045, p = 0.733 r = 0.052, p = 0.698 tune 10 r = -0.110, p = 0.408 r = -0.097, p = 0.466 19underwater sound of t. ivanbureschi trial clicks could not be analysed, it is yet unclear if they are identical to the ones registered underwater. wyman and thrall (1972) report two types of clicks for ambystoma maculatum, with mean frequency and duration around 1.5 khz/0.09 s and 0.5-6.5 khz/0.04 s, respectively. the authors state that while the behavioural significance of both sounds was unknown, the lower frequency click was only recorded during the breeding season, suggesting a possible role in reproduction. considering that april is peak breeding season for t. ivanbureschi, such a possibility cannot be excluded, but it is still not very likely, as none of the pairs in trial 3 produced significantly more clicks. however, juveniles or adult newts outside of the breeding season were not tested in the present study, so additional research is needed in order to establish the potential role of age and phase in sound production for this species. for the congeneric species of a. gracile, licht (1973) describes sounds that are very similar to those of a. maculatum (peak frequency 0.42.5 khz, duration 0.04-0.06 s) and suggests that they are associated with both defensive and aggressive behaviour. again, this seems unlikely for t. ivanbureschi, as in trial 3 there were no signs of antagonistic behaviour. while crovo et al. (2016) report both lowand highfrequency clicks in amphiuma means as having a role in social interaction, they admit that “the high-frequency clicks produced, however, were not associated with highfrequency hearing” – i.e., their behavioural significance remains unclear. low-frequency clicks were associated with communication in siren intermedia, but the authors also suggest that the acoustic signals may play a role in orientation when visual and olfactory cues are absent (gehlbach and walker, 1970). both of the abovementioned species are exclusively aquatic, while t. ivanbureschi has a pronounced biphasic lifestyle, meaning that underwater sound communication could be expected to be less complex in this species. while results for the sounds produced by t. ivanbureschi are not conclusive, they imply that sound plays a certain role in this species behaviour. it has to be said that sound production in caudate amphibians is likely to prove a more productive area for scientific research than previously thought even the few existing studies so far exhibit a large variation of results and different interpretations. crested newts in particular could be suitable study species because of their well-studied phylogeny, allowing for better inter-species comparisons. acknowledgements this work was partially supported by the bulgarian ministry of education and science under the national research programme “young scientists and postdoctoral students” approved by dcm no. 577/17.08.2018. all fieldwork and handling of animals were carried out in accordance with bulgarian ministry of environment and water permit number no. 767/24.01.2019. references amezquita, a., lima, a., jehle, r., castellano, l., ramos, o., crawford, a., gassers, h., hodl, w. 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(1972): sound production by the spotted salamander, ambystoma maculatum. herpetologica 28: 210-212. ziegler, l., arim, m., narins, p.m. (2011): linking amphibian call structure to the environment: the interplay between phenotypic flexibility and individual attributes. behav. ecol. 22: 520-526. appendix results of the kruskal-wallis h test for comparison of the studied parameters between newt pairs (female/ female, female/male, male/male), n = 13. • number of clicks: h(2) = 0.952, p = 0.621 • duration of clicks: h(2) = 4.532, p = 0.104 • peak frequency: h(2) = 0.344, p = 0.842 • tune 50: h(2) = 0.557, p = 0.757 • tune 10: h(2) = 0.524, p = 0.769 acta herpetologica vol. 15, n. 1 june 2020 firenze university press has the west been won? a field survey and a species distribution model of iberolacerta horvathi in the alps giuseppe de marchi1,*, giovanni bombieri1, bruno boz1, fausto leandri2, jacopo richard3 first record of underwater sound produced by the balkan crested newt (triturus ivanbureschi) simeon lukanov identification of biologically active fractions in the dermal secretions of the genus bombina (amphibia: anura: bombinatoridae) iryna udovychenko1,*, oleksandra oskyrko1, oleksii marushchak2, tetiana halenova1, oleksii savchuk1 don’t tread on me: an examination of the anti-predatory behavior of eastern copperheads (agkistrodon contortrix) andrew adams1,2,*, john garrison2, scott mcdaniel2, emily bueche2, hunter howell2,3 an overview of research regarding reservoirs, vectors and predators of the chytrid fungus batrachochytrium dendrobatidis jarid prahl, thomas p. wilson*, david giles, j. hill craddock genetic characteristics of an introduced population of bombina bombina (linnaeus, 1761) (amphibia: bombinatoridae) in moselle, france jean-pierre vacher1, 2,*, damien aumaître3, sylvain ursenbacher2,4 adaptive significance of the transparent body in the tadpoles of ornamented pygmy frog, microhyla ornata (anura, amphibia) santosh m. mogali*, bhagyashri a. shanbhag, srinivas k. saidapur comparison of complement system activity amongst wild and domestic animals maría s. moleón1,2,*, mark e. merchant3, hugo h. ortega4, pablo a. siroski1,2 effects of temperature and food level on plasticity of metamorphic traits in bufo gargarizans gargarizans larvae tong lei yu*, yan ting han acta herpetologica 14(1): 57-63, 2019 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-24274 does color polymorphism affect the predation risk on phalotris lemniscatus (duméril, bibron and duméril, 1854) (serpentes, dipsadidae)? fernanda r. de avila1,2,*, juliano m. oliveira3, mateus de oliveira1, marcio borges-martins4, victor hugo valiati2, alexandro m. tozetti1 1 laboratório de ecologia de vertebrados terrestres, programa de pós-graduação em biologia, universidade do vale do rio dos sinos, avenida unisinos, 950, cristo rei, cep 93022-970, são leopoldo, rs, brasil. *corresponding author. email: fernandar.avila@gmail.com 2 laboratório de genética e biologia molecular, programa de pós-graduação em biologia, universidade do vale do rio dos sinos, avenida unisinos, 950, cristo rei, cep 93022-970, são leopoldo, rs, brasil 3 laboratório de ecologia vegetal, programa de pós-graduação em biologia, universidade do vale do rio dos sinos, avenida unisinos, 950, cristo rei, cep 93022-970, são leopoldo, rs, brasil 4 laboratório de herpetologia, departamento de zoologia, instituto de biociências, universidade federal do rio grande do sul, avenida bento gonçalves, 9500, agronomia, cep 91501-970, porto alegre, rs, brasil submitted on: 2018, 3rd december; revised on: 2018, 12th april; accepted on: 2018, 2nd may editor: rocco tiberti abstract. the snake phalotris lemniscatus is a polymorphic species regarding color, which varies between light shades with a yellow predominance (pale yellow-brown) to darker shades with a red predominance (red-dark). pale yellowbrown individuals are more frequent in coastal populations while there is a tendency of increasing the frequency of red-dark morphs in inland areas. considering the variation in substrate color along the species distribution (light/ sandy on the coast to reddish and dark/argillaceous in inland areas), we raise the hypothesis that the predation rate of each morph would be lower in sites were its crypsis potential is higher. if correct, this hypothesis would reinforce the idea that the predation risk is one of the factors influencing the spatial structuring in morph frequency distributions in populations of p. lemniscatus. to test this hypothesis, we performed a field experiment using plasticine p. lemniscatus artificial models that represent two morphs: red-dark and pale yellow-brown. the models were distributed in three localities where the following substrate types predominate: light (coastal site), intermediary (lowland site) and reddish dark (highland site). our predictions were corroborated only at the coastal site, where the less cryptic morph was the most preyed one. we verified that there is a regional variation in the predation risk on different morphs. thus, the possibility that the selective pressure by predators is a relevant element in the structuring of the frequencies of different morph populations of this species cannot be completely excluded. keywords. behavior, brazil, coral-pattern, mimetism, phenotype, snake. introduction polymorphism is characterized by the presence of different phenotypes in a population (mayr, 1963) and does not only include the morphological features, but also those related to the life history and behavior of the organisms (huxley, 1955; hedrick, 2006). there are many processes behind the maintenance of different morphs in a population, and these processes are not easily identified (calsbeek and cox, 2012; deitloff et al., 2013; karpestam et al., 2016, barnett et al., 2018). questions such as “how do morphs vary their 58 fernanda r. de avila et alii appearance and abundance in a spatial scale” and “what are the habitat components that favor the existence and sympatry of two or more morphs” would be better answered from experiments performed under natural conditions (hoffman and blouin, 2000; roulin, 2004; gray and mckinnon, 2006). in squamates, particularly in snakes, some of the most well-documented types of polymorphism are the multiple color forms (color polymorphism). from an ecological view, morphs can be considered cryptic (when they maximize the animal’s camouflage; clarke, 1962; king and lawson, 1995; eizirik et al., 2003; hoffman et al., 2006), aposematic (when they highlight a warning signal; brodie and brodie, 2004; noonan and comeault, 2009) or a combination of both (brodie and brodie, 1980; wang and shaffer, 2008; barnett et al., 2018). variations in the color are recorded in other taxa and are associated with an improvement in the performance of intraspecific communication, thermoregulation or as anti-predation mechanisms (endler, 1978; pérez et al., 2017). snakes have many different and complex patterns of intraspecific color polymorphism, from systems with bright and contrasting colors to those with cryptic color sets or disruptive patterns (cox and rabosky, 2013; holmes et al., 2017; martínez-freiría et al., 2017; santos et al., 2017). undoubtedly, crypsis is an important factor that might bring higher survival chances to the morphotype, since the animal’s color matches the color of the substrate, making it difficult to be detected by vision-oriented predators (johannesson and ekendahl, 2002; venesky and anthony, 2007). these predators are expected to find and attack more easily the more conspicuous morphs in the population, according to their crypsis (stimson and berman, 1990). in southern brazil, there are consistent records of polymorphic variations spatially structured for the dumeril’s diadem snake (phalotris lemniscatus) (duméril, bibron and duméril, 1854) (fig. 1a and 1c). this species has different morphs with shades going from red-dark to pale yellow-brown (ferrarezzi, 1993; esteves, 2011; noronha, 2012). the variations seem to be restricted to these shades, with only one record of albinism (abegg, 2015) and no records of a melanistic form. the distribution of p. lemniscatus morphs is spatially structured in the following way: individuals of predominantly pale yellow-brown color occur more frequently in populations from regions of sandy substrate of the southern brazil and uruguay coasts, while the predominantly red-dark individuals occur more frequently in more continental regions of brazil and argentina (noronha, 2012). in these more continental localities, the substrates are darker due to the predominance of organic matter and clay in the soil (lema, 2002; esteves, 2011), allowing differential crypsis between the morphs. many snake predators in the extreme south of brazil are visually oriented (e.g., birds: dell’ aglio et al., 2012; santos et al., 2013), and probably respond to variations in the level of contrast between their prey and the substrate. with this premise, we expect that predation is an important selective factor for the definition of the rare (more predated) and more frequent (less predated) morphs in each population. thus, the predation rate of the morphs of p. lemniscatus should vary between regions with different substrate colors, being higher on artificial models of the red-dark type than on those of the yellow-brown type in the coastal region, and the opposite in continental regions. because predation events are generally difficult to observe in the wild, they have been largely studied using experimental approaches as a manner to observe the interactions between predator and prey (brodie, 1993; guimarães and sawaya, 2011; purger et al., 2017). the use of artificial plasticine models has been employed successfully in predation experiments with invertebrates (koh and menge, 2006), amphibians (kuchta, 2006) and reptiles (stuart-fox et al., 2002; valkonen et al., 2011; dell´aglio et al., 2012), with highlight on snakes (brodie, 1993; dell’aglio et al., 2012; farallo and forstner, 2012; santos et al., 2013; akcali et al., 2019). in the present study, we used models of p. lemniscatus to test the hypothesis that the predation rate of each morph would be lower in sites were its crypsis potential is higher. if correct, this hypothesis would reinforce the idea that predation risk is one of the factors for the spatial structuring in the distribution of morph frequencies in populations of p. lemniscatus. material and methods we distributed, in the wild, two types of artificial models of the snake: red-dark and yellow-brown (fig. 1b and 1d). their color was based on live specimens captured in the study area in order to accurately represent the color of the morphs that have the extremes of variation between the lighter and darker shades (noronha, 2012; fig. 1a and 1c). these models were manufactured with non-toxic plasticine that allowed the record and quantification of marks left by predator attacks (brodie, 1993). the models measured 30 cm in length and 1 cm in diameter, which represents the mean size of adult animals (carreira et al., 2012). the samplings were performed in localities with different natural proportions in the abundance of the morphs of p. lemniscatus, these being: 1 – coastal site (32°43’2.49”s; 52°28’29.87”w) in the municipality of rio grande (annual mean temperature of 17.5 °c, rossato, 2014), where the pale yellow-brown morph is more abundant; 2 – highland site (29°39’23.04”s; 51°23’7.40”w) in the municipality of são francisco de paula, where the red59polymorphism and predation risk in phalotris lemniscatus dark morph is more abundant (annual mean temperature of 14.5 °c; rossato, 2014; minimum temperatures frequently close to 0 °c in winter; maluf, 2000); 3 – lowland site (29°27’0.20”s; 50°34’59.83”w) in the municipality of capela de santana (annual mean temperature of 17.0 °c; rossato, 2014), where both morphs are observed in similar proportions (noronha, 2012). the sampled localities extend in extreme points of an area of approximately 12660 km² located at least at 404 km from each other (fig. 2). the prevailing substrates in each locality are light and sandy (coastal site) and dark and argillaceous (highland and lowland sites), allowing the evaluation of different contrast levels between model and background. we distributed 200 models in each locality (100 red-dark and 100 pale yellow-brown), along five transects, each 400 m long. the transects were at least 1000 m from each other and each received 40 snakes (one every 10 meters). in each transect, the two morphs were interspersed so that they had 20 red-dark and 20 pale yellow-brown models, a protocol similar to that of dell’aglio et al. (2012) and farallo and forstner (2012). we used this 1:1 ratio to avoid a possible frequency-dependent predation effect. each model received an identification code and its position was marked with a gps device to facilitate their monitoring. we also took the care to arrange all the transects in an area of similar vegetation cover (low and scarce vegetation with the prevalence of exposed soil) that would not provide any visual barrier to the predators. thus, the contrast between the models and the background happened due to the color of the artificial snake and the soil. the artificial models remained exposed in the field for 48 hours. during this time, they were inspected twice, after 24 and 48 hours since the installation. during the inspections, we recorded the presence of attack marks on the models. each artificial model that clearly showed marks of bird attack (e.g., peckings) was considered a predation event (brodie, 1993; dell’aglio et al., 2012). the models showing attack marks during the first inspection were replaced by new models. it is worth highlighting that the three localities have a similar fauna of predatory birds (fontana et al., 2008; petry and scherer, 2008; accordi and hartz, 2006), mainly of birds of prey (e.g., caracara plancus, milvago chimango), egrets (e.g., ardea alba, syrigma sibilatrix) and even some other generalist foragers (e.g., guira guira). data analysis just clearly identifiable pecking marks were considered as an attack (or predation event). we quantified only the presence and not the number of marks. thus, a model with one or more marks corresponded to one predation event. to evaluate the predation intensity of each morph, we calculated the predation rate. to do so, we divided the number of models of each morph with predation evidence (number of events) by the number of exposure hours of these models. the number of exposure hours corresponds to the total number of hours between the installation of the model and its final inspection. this calculation was done for each transect. to test the differences in predation rates of the morphs between the localities we performed an analysis of variance with randomization test (pillar and orlóci, 1996). in these analyses, we used euclidian distance matrices between the morphs, restricting the permutations within the transects (blocks) to control possible discrepancies in factors related to the predation that were not directly verified (e.g. number and species of predators). the analyses of variance were performed by means of the software multiv v.3.34b (pillar, 1997). results we recorded altogether 162 predation events, which corresponds to an overall predation rate of 1.06 events per hour. at the lowland site, 90 models were attacked (23%). this locality had a larger number of attacks directed to the fig. 1. general aspect of the specimens of phalotris lemniscatus used as a reference to the pale yellow-brown (a) and red-dark (c) patterns and their models manufactured in plasticine (b and d, respectively). the image e show some marks considered as attacks from predators. fig. 2. geographic location of the sampling sites. 60 fernanda r. de avila et alii red-dark morph (57 models; 0.22 events per hour) than to the pale yellow-brown morph (33 models; 0.13 events per hour), this difference being marginally significant (sqe = 0.02; r2 = 41%; p = 0.057; n = 10). at the coastal site, we recorded 22 predation events (11%). as for the lowland site, the coastal site also showed more attacks on the red-dark models (16 models; 0.07 events per hour) than on the pale yellow-brown models (6 models; 0.03 events per hour) (sqe = 0.005; r2 = 25%; p = 0.049; n = 10). at the highland site, there was no significant variation in the number of attacks between the different morphs (sqe = 0.0004; r2 < 1%; p = 0.735; n = 10) (fig. 3). discussion our results suggest that the morphs of phalotris lemniscatus have different levels of predation and the predation rate varies between areas. our predictions regarding the importance of crypsis were corroborated only at the coastal site, where the predation rate was higher on the red-dark models that have higher contrast (less cryptic) in relation to the substrate of the region. similar results were also observed for the morphs of the mottled rock rattlesnake (crotalus lepidus lepidus; farallo and forstner, 2012) and the sand hills mice (linnen et al., 2013), both in the united states. experiments with artificial models showed that differential crypsis and predation are the main forces of the spatial structuring of the western rattlesnake’s morphs (farallo and forstner, 2012). similarly, linnen et al. (2013) pointed out that the color of the soil offers differential camouflage opportunity to sandhill mice against owls and other raptors and is determinant to the spatial structuring of their colored morphs. however, our data show that predation does not seem to be the main factor acting on the spatial structuring of the morphs of p. lemniscatus since the naturally more uncommon morph in the lowland site was the one that suffered less predation (pale-yellow). in other words, the low frequency of the pale-yellow morph in the lowland site population does not seem to be the result of predation. it is worth highlighting that all the sampled localities have a similar predator composition. however, there is a possibility of existing regional variations in the ability of these predators in detecting prey, which would have influenced the local number of attacks on each model type. experiments showed that the capacity of predators to detect the different morphs based on motionless prey is variable and depends on their ability to generate a specific searching image according to the form, size and color of the prey (e.g., brodie, 1993; olsson, 1993; gotmark, 1994). it is reasonable to imagine that the search image established by a predator is compatible with a certain type of prey that has a higher probability of being found. thus, the naturally rare morphs in each locality may not be part of the searching image of local predators (dukas, 1998). in this case, they might not be easily detectable, even if their color shows more contrast with the background. therefore, their predation rate would remain relatively low, even if their population was experimentally increased with the introduction of artificial models (wennersten and forsman, 2009; karpestam et al., 2014). in addition, animals that attack potentially dangerous organisms (e.g., snakes) commonly reduce their exploratory behavior toward new and different prey morphs (greenberg and mettke-hofmann, 2001), thus exhibiting a neophobic behavior (avoiding a new environmental aspect) (greggor et al., 2016). although possible, this hypothesis would be fig. 3. mean predation rate (events per hour) on the different morphs (red-dark and pale yellow-brown) in the three areas (highland, lowland and coastal). 61polymorphism and predation risk in phalotris lemniscatus applicable to the lowland and highland sites, but not to the coastal site, where the less common and less cryptical models were the most preyed ones. our results point toward the possibility that other factors besides crypsis interfere on the predation rate. among those, we can mention the mimetic aposematic potential of the red morph since their bright colors are associated with poisonous or nonpalatable animals (cuthill et al., 2005; tarvin et al., 2017). the establishment of a parallel between our data and such studies seems convenient due to the slightly reddish pattern of one of the morphs of p. lemniscatus. yet, the effectiveness of the aposematism based on coral-like coloration is questionable regarding canids (tozetti et al., 2004) and birds (smith, 1969). besides the passive strategies such as crypsis and immobility (venesky and anthony, 2007), snakes show active defense strategies or behavioral displays such as escape (forsman and appelqvist, 1998; creer, 2005; allen et al, 2013). additionally, p. lemniscatus has the antipredatory behavior called “erratic movements” (tozetti et al., 2009), which would reduce the predation effectiveness after being detected by a predator (forsman and appelqvist, 1998). these behaviors may act in combination with the crypsis for the maintenance of each morph in each population. our study failed to obtain answers to the proposed questions. one of the reasons might be that the frequency of different morphs in each population may be related to other environmental factors such as thermoregulation (trullas et al., 2007). in general, ectotherms from cold environments tend to have darker colors, which favor the absorption of solar radiation (gibson and falls, 1979; clusella‐trullas et al., 2008; allen et al., 2013). considering that the highland site has the lowest temperatures in the distribution area of p. lemniscatus, there would be a positive selective pressure favorable to red-dark individuals in the highland site and to pale yellow-brown individuals in the coastal site (see bittner et al., 2002). despite the weakness of our hypothesis based on our results, we verify a regional variation on the predation risk of the different morphs of this species, which does not completely exclude the possibility that the selective pressure by predators is a relevant element in the structuring of the frequencies of different morphs in populations of this species. references abegg, a.d., entiauspe-neto, o.m., lema, t. 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(2009): does colour polymorphism enhance survival of prey populations? p. roy. soc. lond. b bio. 276: 2187-2194. acta herpetologica vol. 14, n. 1 june 2019 firenze university press uzungwa scarp nature forest reserve: a unique hotspot for reptiles in tanzania john valentine lyakurwa1,2,*, kim monroe howell2, linus kasian munishi1, anna christina treydte1,3 experience of predacious cues and accessibility to refuge minimize mortality of hylarana temporalis tadpoles santosh mogali*, bhagyashri shanbhag, srinivas saidapur tonal calls as a bioacoustic novelty in two atlantic forest species of physalaemus (anura: leptodactylidae) thiago r. de carvalho1,*, célio f.b. haddad1, marcos gridi-papp2 scientific publication of georeferenced molecular data as an adequate guide to delimit the range of korean hynobius salamanders through citizen science amaël borzée1,*, hae jun baek2,3, chang hoon lee2,3, dong yoon kim2, jae-young song4, jaehwa suh5, yikweon jang1, mi-sook min2* mirrored images but not silicone models trigger aggressive responses in male common wall lizards stefano scali1,*, roberto sacchi2, mattia falaschi1,3, alan j. coladonato2, sara pozzi2, marco a.l. zuffi4, marco mangiacotti1,2 using an in-situ infra-red camera system for sea turtle hatchling emergence monitoring fatima n. oğul1,#,*, franziska huber2,#, sinem cih1, kumsal düzgün3, ahmet e. kideyş1, korhan özkan1 descriptive osteology of an imperiled amphibian, the luristan newt (neurergus kaiseri, amphibia: salamandridae) hadi khoshnamvand1, mansoureh malekian1,*, yazdan keivany1, mazaher zamani-faradonbe1, mohsen amiri2 does color polymorphism affect the predation risk on phalotris lemniscatus (duméril, bibron and duméril, 1854) (serpentes, dipsadidae)? fernanda r. de avila1,2,*, juliano m. oliveira3, mateus de oliveira1, marcio borges-martins4, victor hugo valiati2, alexandro m. tozetti1 age structure of a population of discoglossus scovazzi camerano, 1878 (anura discoglossidae) in extreme environmental conditions (high atlas, morocco) mohamed amine samlali, abderrahim s’khifa, tahar slimani* acta herpetologica 13(2): 185-189, 2018 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-23394 the mitogenome of elaphe bimaculata (reptilia: colubridae) has never been published: a case with the complete mitochondrial genome of e. dione evgeniy simonov1,2,*, artem lisachov3, natalia oreshkova1,4, konstantin v. krutovsky1,5,6,7 1 laboratory of forest genomics, genome research and education center, siberian federal university, krasnoyarsk 660036, russia. *corresponding author. e-mail: ev.simonov@gmail.com 2 laboratory of biodiversity monitoring, tomsk state university, tomsk 634050, russia 3 institute of cytology and genetics, siberian branch of russian academy of sciences, novosibirsk 630090, russia 4 laboratory of forest genetics and selection, v.n. sukachev institute of forest, siberian branch of russian academy of sciences, krasnoyarsk 660036, russia 5 department of forest genetics and forest tree breeding, georg-august university of göttingen, büsgenweg 2, 37077 göttingen, germany 6 laboratory of population genetics, vavilov institute of general genetics, russian academy of sciences, moscow 119991, russia 7 department of ecosystem science and management, texas a&m university, college station, tx 77843-2138, usa submitted on: 2018, 14th june; revised: on 2018, 3rd august; accepted on: 2018, 3rd august editor: uwe fritz abstract. the steppes ratsnake, elaphe dione (pallas, 1773), is widely distributed across eurasia, but the systematics and phylogeography of this species remain poorly studied. sequencing of the full mitochondrial genome of this species provides a reference for its further study. here, we report the full mitochondrial genome of an e. dione specimen from krasnoyarsk krai (east siberia, russia). we found that it is highly similar to the previously reported mitochondrial genome of the sister species, e. bimaculata. both species misidentification by the authors of e. bimaculata mitogenome and the introgressive hybridization between these taxa can possibly explain this observation. keywords. colubridae, elaphe, mitogenome, phylogeny, siberia. ratsnakes of the genus elaphe make up a widely distributed colubrid group of 15 species (the reptile database: uetz et al., 2018), which inhabits a range from western europe to the russian far east and china. some closely related genera (often also referred to as “ratsnakes” or elaphe sensu lato) such as pantherophis, zamenis, gonyosoma, etc. inhabit zones with temperate, subtropical and tropical climate almost all over eurasia and north america. relatively few mitochondrial genomes of ratsnakes have been sequenced so far, excluding e. anomala (liu and zhao, 2015a), e. bimaculata (yan et al., 2014), e. carinata (ding et al., 2016), e. davidi (xu et al., 2015), and e. schrenckii (liu and zhao, 2015b). the steppes ratsnake, elaphe dione (pallas, 1773), is the most widespread species of the genus. it is present from ukraine in the west to the shores of the pacific ocean in the east, and from the 56th degree of latitude in russia in the north to iran in the south (schulz, 2013). type locality of the species is “gratscheffskoi outpost, near semijarsk, upper irtysh area, semipalatinsk district”, kazakhstan [currently grachi village, beskaragay district of east kazakhstan region] (restricted by mertens and mueller, 1928). the systematics of this species remains controversial: so far, several subspecies have been described (such as e. d. tenebrosa sobolevsky, 1929 and e. d. czerskii vedmederya et al., 2009), but none of them 186 evgeniy simonov et alii have been widely accepted. while the mitogenome of the steppes ratsnake has never been sequenced, it would provide an important resource for further studies in systematics and phylogeography of this widespread species. therefore, we sequenced and annotated the complete mitochondrial genome of e. dione specimen and reconstructed the mitogenome phylogeny with other related species of the genus. dna was sampled via non-lethal buccal swabs from e. dione collected in krasnoyarsk krai, russia (53.59°n 91.64°e) in june 2016, and extracted using standard proteinase k and phenol-chloroform methods (sambrook et al., 1989). dna quality and concentration were examined by electrophoresis in 1.5% agarose gel and qubit fluorimeter, respectively (thermo fisher scientific, usa). the dna was fragmented using an ultrasonic bioruptor sonication system (diagenode), and paired-end libraries were prepared using the truseq dna lt sample prep kit (illumina) according to the truseq dna sample preparation guide. the quality control of the prepared library was carried out on the electrophoretic system bioanalyzer 2100 (agilent technologies) using agilent dna 1000 reagents (agilent technologies). the fragment size was approximately 400 bp (with insert size 260-280 bp). the library was sequenced on the miseq illumina platform using the miseq reagent kit v3 (300-cycle, 2x150 bp) illumina kit at the laboratory of forest genomics, siberian federal university. read quality was assessed with fastqc 0.11.7 (andrews, 2010). adapter and quality trimming was performed using clc genomics workbench (clc bio, aarhus, denmark). to assemble the mitochondrial genome, reads were mapped to previously published mitogenomes of congeneric species: e. bimaculata (km065513.1) and e. schrenckii (kp888955.1). successfully mapped reads were merged into single consensus sequence representing mtdna of e. dione. all aforementioned steps were also done with clc genomics workbench. the e. dione mitochondrial genome was annotated in the mitos2 web server (http://mitos2.bioinf. uni-leipzig.de/index.py), manually checked and corrected for errors. mitochondrial genomes of e. anomala (kp900218.1), e. bimaculata (km065513.1), e. carinata (ku180459.1), e. davidi (km401547.1), and e. schrenckii (kp888955.1) were obtained from genbank to examine phylogenetic relationships between e. dione and related taxa basing on complete mtdna sequences. some members of closely related genera were used as outgroup: oocatochus rufodorsatus (kc990020.1), orthriophis taeniurus (kc990021.1), oreocryptophis porphyraceus (gq181130.1), pantherophis slowinskii (dq523162.1), and pituophis catenifer (ku833245.1). a multiple sequence alignment was produced by clustal omega (sievers et al., 2011) and trimmed with gblocks (talavera and castresana, 2007); 95% (16,631) of the original 17,330 bp alignment remained after trimming. maximum likelihood (ml) phylogenetic tree was inferred with iq-tree 1.6.1 (nguyen et al., 2015) using the tim2+f+i+g4 substitution model (selected within 286 tested models by modelfinder; kalyaanamoorthy et al., 2017) and 1,000 ultrafast bootstrap replicates (hoang et al., 2018). the uncorrected genetic distances between species were calculated in mega7 (kumar et al., 2016) with pairwise deletion of gaps/missing data. in total, 2,132,080 illumina paired-end reads were generated. we successfully retrieved 16,994 bp of sequence data of the e. dione mitochondrial genome with an average coverage of 15x (0.09% of all reads were mapped to mtdna). no differences were found between the mtdna sequences generated by mapping to e. bimaculata or e. schrenckii reference mitogenomes. the very small portions of the nd5 gene and the second d-loop region were not covered by the obtained reads. we estimated the length of the non-covered region was 178 bp. thus, the full length of the e. dione mitochondrial dna was around 17,172 bp with only ~1% not covered. the newly generated mitogenome is available under ncbi genbank accession number mh460961. the phylogenetic tree based on full mitochondrial genomes agreed with previous studies, placing members of genus elaphe into a distinct monophyletic group (utiger et al., 2002; chen et al., 2010). the uncorrected genetic distance (p-distance) between the mitogenome of e. dione and the previously published mitogenome of e. bimaculata (km065513.1) was 0.89% (for 16,989 aligned sites), while the mean distance between other elaphe species is 10.1% (table 1). thus, the mitogenome of e. dione from krasnoyarsk krai was highly similar to the recently sequenced genome of its sister species e. bimaculata. the observed distance between the two genomes was too low even for closely related species and is rather at the intraspecific level. the same result was reported by hofmann et al. (2016), when they compared 12s, nd4, cyt b, and coi sequences with this e. bimaculata mitogenome. however, the pronounced genetic difference between the two considered species has been shown previously (utiger et al., 2002) and confirmed by hofmann et al. (2016). to further clarify this situation, we extracted partial sequences of the 12s rrna mitochondrial gene from mitogenomes of both species and compared them to the 12s sequences of e. bimaculata and e. dione available in genbank. on the 12s gene tree (fig. 1b), some e. bimaculata sequenc187running title table 1. uncorrected genetic distances (%) between mitochondrial genomes of some species of the genera elaphe, orthriophus, oocatochus, pituophis, pantherophis, and oreocryptophis. species 1 2 3 4 5 6 7 8 9 10 1. elaphe dione 2. elaphe bimaculata 0.9 3. elaphe schrenckii 10.9 10.8 4. elaphe carinata 10.7 10.7 10.6 5. elaphe anomala 10.9 10.9 0.2 10.6 6. elaphe davidi 11.0 10.8 11.2 10.7 11.2 7. orthriophis taeniurus 13.7 13.6 13.1 13.7 13.1 14.1 8. oocatochus rufodorsatus 13.7 13.6 13.6 14.1 13.6 14.5 13.6 9. pituophis catenifer 14.3 14.2 13.8 14.2 13.8 14.9 14.0 13.5 10. pantherophis slowinskii 14.3 14.3 14.0 14.3 14.0 14.8 14.0 13.4 9.9 11. oreocryptophis porphyraceus 14.5 14.4 14.1 14.7 14.2 15.0 13.7 13.3 14.6 14.3 the distances between species of the genus elaphe are highlighted in bold. fig. 1. (a) maximum likelihood phylogenetic tree of the elaphe sensu lato group based on full mitochondrial genomes; (b) maximum likelihood gene tree of e. dione and e. bimaculata based on 12s rrna sequences. bootstrap values above 50 are indicated. 188 evgeniy simonov et alii es form a separate clade, distinct from the e. dione lineage. two other e. bimaculata sequences (including the one extracted from the mitogenome) clearly fall into the e. dione cluster. the e. dione sample used in our work belongs to the e. dione clade. it is evident that the mitogenome of “e. bimaculata” sequenced by yan et al. (2016) belongs to e. dione. the two species are very similar phenotypically, and their ranges overlap in china (schulz, 2013; wallach et al., 2014). thus, species misidentification by the authors of e. bimaculata mitogenome could be a possible explanation. an alternative explanation is introgressive hybridization between the species. this phenomenon is well documented in animals (including reptiles) and results in bidirectional or unidirectional introgression of mtdna (e.g. plötner et al., 2008; machado et al., 2014; ermakov et al., 2015; johnson et al., 2015). if hybridization between these two species indeed occurs in the area of their sympatry, an e. bimaculata specimen with introgressed mtdna could be accidentally used for the mitogenome sequencing. unfortunately, yan et al. (2016) did not provide any information about geographical origin or other details of the specimen they used for mtdna sequencing. by this communication, we would like to not only provide a reliable mitogenome of e. dione, but also highlight the need for careful selection and documentation of specimens intended for full mitogenome/genome sequencing to avoid further confusion. acknowledgements we thank lengxob yong (university of exeter, uk) for english editing, and two reviewers for their valuable comments on the manuscript. the study is supported by “tsu competitiveness improvement programme” grant 8.1.19.2018. no animals were killed or removed from nature for this work. according to the national guidelines (russia) no permit for animal handling or collection of non-invasive samples is necessary. references andrews, s. 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(2016): the complete mitochondrial genome of elaphe bimaculata (reptilia, serpentes, colubridae). mitochondr. dna 27: 1285-1286. acta herpetologica vol. 13, n. 1 june 2018 firenze university press acta herpetologica 12(2): 139-150, 2017 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-20742 species and sex comparisons of karyotype and genome size in two kurixalus tree frogs (anura, rhacophoridae) shun-ping chang1,2, gwo-chin ma2,3,4, ming chen2,5,6,7,8,*, sheng-hai wu1,* 1 department of life sciences, national chung hsing university, 145 xingda rd., taichung, taiwan; *corresponding author. e-mail: shwu@dragon.nchu.edu.tw 2 department of genomic medicine, changhua christian hospital, 176 zhanghua rd., changhua, taiwan; *corresponding author. e-mail: mingchenmd@gmail.com 3 institute of biochemistry, microbiology and immunology, chung shan medical university, 110 jianguo n. rd. (sec. 1), taichung, taiwan 4 department of medical laboratory science and biotechnology, central taiwan university of science and technology, 666 buzih rd., taichung, taiwan 5 department of obstetrics and gynecology, changhua christian hospital, 135 nanxiao st., changhua, taiwan 6 department of obstetrics and gynecology, college of medicine and hospital, national taiwan university, 1 roosevelt rd. (sec. 4), taipei, taiwan 7 department of medical genetics, national taiwan university hospital, 1 changde st., taipei, taiwan 8 department of life sciences, tunghai university, 1727 taiwan boulevard (sec. 4), taichung, taiwan submitted on: 2017, 6th june; revised on 2017, 22nd september; accepted on 2017, 22nd september editor: uwe fritz abstract. kurixalus is a rhacophorid genus of tree frogs that are similar in morphology but vary in reproductive behavior. we investigated the cytogenetic features and genome size using conventional g-banding, c-banding and silver-staining techniques, fluorescence in situ hybridization (fish), and flow cytometry in two representatives of kurixalus (k. eiffingeri boettger, 1895 and k. idiootocus kuramoto and wang, 1987) and compared the data between species and sex. the two kurixalus species share a diploid chromosome number 2n = 26 and fundamental number fn = 52. prominent differences between species were noted in the distribution of secondary constriction (sc)/nucleolus organizer region (nor) and dense heterochromatin. other interspecies differences including variations in the number of metacentric and submetacentric chromosomes and staining intensity of heterochromatin were also found. the cytogenetic results are consistent with the observed differences in their genome sizes. fish with telomeric motif (ttaggg)n for both species detected signals in the terminal regions. intersex comparisons revealed no differences in terms of cytogenetic features and genome size in the two species. despite the apparent highly conserved diploid chromosome number, data on the karyotype microstructure characterize the cytogenetic profile of the two kurixalus species that contribute to clarification of the chromosomal homologies and the rearrangement mechanisms occurring during the karyotype evolution of kurixalus. no heteromorphic chromosome pair in both species is consistent with the view that homomorphic sex chromosome is common in amphibians. keywords. kurixalus eiffingeri, k. idiootocus, karyotype, homomorphic chromosome, sex determination. introduction kurixalus is a rhacophorid genus of small tree frogs that occurs across east to southeast asia, including the ryukyu islands, taiwan, china, india, myanmar, cambodia, vietnam, thailand, malays, sumatra, borneo and the philippines (frost, 2017). phylogenetic data support the 140 shun-ping chang et alii monophyletic origin of kurixalus despite its broad distribution (li et al., 2013; biju et al., 2016; jiang et al., 2016). however, since these frogs are similar in morphology and overlap in body size, taxonomy is difficult and mainly based on molecular analyses (wilkinson et al., 2002; li et al., 2008; li et al., 2009; hertwig et al., 2013; yu et al., 2013; nguyen et al., 2014a; nguyen et al., 2014b; wu et al., 2016). currently, 14 species are recognized in kurixalus (frost, 2017). despite the taxonomy of kurixalus species continues to update, considerable differences in the reproductive behavior are noted among some known species. for example, k. eiffingeri boettger, 1895, a species found in taiwan and two southern islands of japan (maeda and matsui, 1989), breeds in water-filled tree holes and bamboo stumps, and displays a complex parental care including paternal care for eggs and subsequent maternal care for larvae. the tadpoles are oophagous, feeding exclusively on unfertilized eggs laid by the female frogs which return to nests (kam et al., 1997; kam et al., 2001). on the contrary, k. idiootocus kuramoto and wang, 1987, a species endemic to taiwan, breeds in soils and crevices that are frequently covered with dead leaves. eggs hatch when heavy rainfall occurs and the hollow fills up, and the tadpoles get washed into nearby waterways or pools. the tadpoles are herbivorous, subsisting on algae and plants. while much attention has been put on subjects of the reproductive behavior in kurixalus species (lin and kam, 2008; cheng wc et al., 2013; tung et al., 2015), little information is available on their genetic complements in terms of species difference and sex differentiation. it is thus interesting to investigate the cytogenetic and genomic features in members of this genus. variations in chromosome number and structure among species and/or populations can be used to estimate phylogenetic relationships (yates et al., 1979) and has been applied by various kinds of cytogenetic techniques for classifying species with similar morphology (medeiros et al., 2003; rosa et al., 2003; veiga-menoncello et al., 2003; carvalho et al., 2009). cytogenetic analyses can also reveal sex determination of targeted species. if heteromorphic sex chromosomes are present in species, genetic sex determination is inferred (hanada, 2002). even though heteromorphic chromosome pairs are not always recognized in species, different chromosome staining techniques, such as g-banding, c-banding and ag-nor staining are still widely accepted in detecting cryptic structural alterations of chromosomes (hanada, 2002; wu et al., 2007). additionally, sophisticated painting techniques, such as fluorescence in situ hybridization (fish) and comparative genomic hybridization (cgh), are also useful tools in the studies of comparative cytogenetics. this can be instanced by the use of fish with a highly conserved vertebrate telomeric motif (ttaggg)n to identify chromosome rearrangements (e.g., fusion, fission and translocation events) if the interstitial telomeric sequences (itss) were detected. the important role of the telomeric sequences in karyotypic diversity, chromosome evolution, and chromosome instability has been highlighted (bruschi et al., 2014). the cgh, a fish-based method using total dna from two taxa as probes, can differentiate chromosomes from different genomes and has been used in studies of genomewide comparison (yu et al., 2012). karyotypes in the genus kurixalus are poorly understood. available cytogenetic data are restricted to the common features, such as diploid chromosome number, in two species (kuramoto, 1977; kuramoto and wang, 1987). in this study, with the aim of providing cytogenetic information of kurixalus, the karyotypes of two representatives of kurixalus (k. eiffingeri boettger, 1895 and k. idiootocus kuramoto and wang, 1987) were characterized using g-banding, c-banding, and ag-nor staining. because karyotypic variations may involve rearrangements that are not detected by conventional karyotyping, we also included here the telomeric fish and cgh analyses in the karyotypes of both species. moreover, the genomic sizes were also examined to yield a natural link to the cytogenetic data that provide a genetic basis for comparative studies of interspecies as well as intersex differentiation in kurixalus. materials and methods sampling a total of 40 specimens of kurixalus eiffingeri (male: n = 10, female: n = 10) and k. idiootocus (male: n = 10, female: n = 10) were collected from xitou (elevation 1,092 m a.s.l.; 23°40’35.2”n, 120°47’46”e) and yuchi (elevation 634 m; 23°53’47”n, 120°54’13”e), respectively, in nantou county, taiwan (fig. 1). both of the two kurixalus species examined have been taxonomically identified, in addition to phenotypic classification, by phylogenetic analysis using the mitochondrial dna cytochrome c oxidase subunit 1 (co1) and the 16s rdna sequencing (wu et al., 2016). the reference co1 and 16s rdna sequences have been uploaded to genbank with accession numbers of dq468678 and dq468670 for k. eiffingeri, and dq468682 and dq468674 for k. idiootocus. tissues of gonad (from both sexes) and kidney were obtained and cultured according to chinchar and sinclair (1978). cytogenetic analyses for chromosome preparation, mitotic metaphase arrest was induced by adding 0.1 μg/ml colcemid 4 h before cell harvest141comparisons of karyotype and genome size in two kurixalus species ing. hypotonic kcl solution (0.075 m) was applied for 20 min, followed by fixation with fresh carnoy’s fixative (3:1 of methanol to glacial acetic acid) at 4 °c for 1 h. g-banding, c-banding and ag-nor staining were carried out according to published protocols (wang et al., 2004; wu et al., 2007). in short, for g-banding, the metaphase chromosomes were digested with trypsin at room temperature (rt) for 15 sec and subsequently stained with wright’s stain at rt for 2 min; for c-banding, the metaphase chromosomes were treated with an alkali barium hydroxide 5% ba(oh)2 at 50 °c for 15 sec, incubated in 2 x ssc solution at 60 °c for 1 h, and afterward stained with wright’s dye; for ag-nor staining, the working solution (mixture of 100 μl of the 2% gelatin colloid solution and 200 μl of the silver nitrate solution) was mixed immediately before use and pipetted onto the slides with the metaphase chromosomes at 70 °c for 2 min. after rinsing off with deionized water, the metaphase chromosomes were stained with wright’s dye at rt for 2 min. telomeric fish the physical map of telomeric sequences was detected by fluorescence in situ hybridization (fish) using all human telomeres probe (ttaggg)n (qbiogene/mp biomedicals llc, tucson, az, usa) and followed manufacturer’s manual. briefly, 10 µl of the fixed, pre-treated and homogenized samples were applied on a microscope slide and dried at 50 °c for 15 min, immersed the slide in 2x ssc for 2 min at rt without agitation, followed by a successive dehydration step with 70%, 85% and 100% ethanol respectively for 2 min and allowed to dry. the sample and probe were co-denatured by heating the slide at 75 °c for 2 min, and then hybridizations were performed at 37 °c for 12 h. the slide were immersed in 0.4 x ssc (ph 7.0) at 72 °c for 2 min and then were immersed in 2 x ssc, 0.05% tween-20 at rt (ph 7.0) for 1 min. finally, the slides were drained and applied 10µl of 0.125 µg/ml dapi antifade onto hybridization areas, and were viewed with a fluorescence microscope. comparative genomic hybridization (cgh) comparative genomic hybridization is often used for the evaluation of the differences between the chromosomal complements of different sexes (badenhorst et al., 2013; matsubara et al., 2013; rovatsos et al., 2015). the cgh between sexes was performed as previously described (yu et al., 2012), with some modifications. briefly, the genomic dna from male and female were labeled with fluorescein isothiocyanate (fitc)-12-dutp and texas red (txred)-5-dutp (perkin elmer life sciences, boston, ma, usa) respectively by nick translation kit (roche diagnostics, basel, switzerland) and co-precipitated with a 200fold excess of cot-1 dna. genomic probes were re-dissolved and kept in hybridization buffer (50% formamide, 10% dextran sulphate, and 2 x ssc). the hybridization mixture was denatured at 80 °c for 10 min, followed by preannealing at 37 °c for 60 min. the slides were incubated in a moist chamber at 37 °c for 72 h. after standard washing procedure, chromosomes were counterstained with 0.125 μg/ml dapi added to antifade reagent (abbott, illinois, usa). image capture and analysis karyotype images, fish and cgh results were analyzed for at least 10 well-spread metaphases in each sample by cytovision system (applied imaging, carlsbad, ca, usa). for karyotyping, chromosome types were classified according to the nomenclature of levan et al. (1964). genome size estimation the genome sizes were estimated by flow cytometry based on the description of matsuba and merilä (2006). blood samples from each sex of both species were collected in phosphatebuffered saline without divalent cations and stored at 4 °c. as internal references, male blood cells from lithobates catesbeianus (haploid genome size: c value = 7.37 pg) and homo sapiens (c value = 3.5 pg) were analyzed simultaneously in 1:10 mixed with surveyed cells. cells were suspended in 0.5 ml of a solution containing 25 μg propidium iodide (pi), 0.1% sodium citrate, 25 μg rnaase, and 0.1% triton x-100. samples were filtered through 30 μm mesh and kept at 4 °c in the dark for over 2 h. mean fluorescence of co-stained nuclei was quantified fig. 1. map showing sampling locations of the two kurixalus species in nantou county, taiwan. —1. yuchi (23°53’47”n, 120°54’13”e) for k. idiootocus; —2. xitou (23°40’35.2”n, 120°47’46”e) for k. eiffingeri. 142 shun-ping chang et alii on a beckman-coulter epics xl-mcl flow cytometer with an argon laser (emission at 488 nm/15 mw power), and analyzed with winmdi 2.9 software. the pi fluorescence and genome size of l. catesbeiana and h. sapiens were used as standards to calculate the unknown genome sizes in samples (vinogradov, 1998). student t-test was used to compare the difference of genome sizes between species/sexes by spss software (ibm corp., armonk, ny, usa). the bonferroni correction was applied to set the significance cut-off at α/n, with α = 0.05 and n = number of samples. results a total of 138 and 125 metaphase spreads from k. eiffingeri (individual number = 10) and k. idiootocus (individual number = 10) respectively were subjected to chromosome analyses. both species had the same haploid and diploid chromosome numbers (n = 13 and 2n = 26), and chromosomes are readily classified into two distinct size groups, large pairs of chromosomes (no. 1-5) and small pairs of chromosomes (no. 6-13). all chromosome pairs were either metacentric or submetacentric, giving a fundamental number (fn) of 52 (table 1). though karyotypes of the two species were similar, differences were noted in detailed chromosome morphology and banding pattern (fig. 2). no chromosomal polymorphism (e.g., inversions, translocations, deletions and duplications) was noted among individuals examined in each of the two species. in k. eiffingeri the chromosome pairs 2, 3, and 9 were submetacentrics and the others metacentrics while in k. idiootocus chromosome pairs 2 and 4 were submetacentrics and the others metacentrics. the karyotype formulas of k. eiffingeri and k. idiootocus were thus 10 metacentric + 3 submetacentric and 11 metacentric + 2 submetacentric, respectively. an achromatic secondary constriction (sc) was found in the long arm near the centromere of chromosome pair 8 and pair 12 in k. eiffingeri and k. idiootocus respectively (fig. 2). additionally, significant differences (anova test, p < 0.0001) in relative chromosome length were found in chromosome pairs 1-3 (longer in k. eiffingeri) and in chromosome pairs 6-9 and 11-13 (longer in k. idiootocus) (table 2). no heteromorphic chromosome pair (sex chromosomes) could be identified in each species. c-banding analysis showed that constitutive heterochromatin was located at the centromeres of all chromosomes of k. eiffingeri and k. idiootocus, but heterochromatins differed in banding intensity corresponding with blocks of constitutive heterochromatin. an additional dense heterochromatin was only found on proximal short arm of chromosome pair 8 of k. eiffingeri (fig. 3). the nucleolus organizer region (nor) was detected by silver staining on the long arm near the centromere of chromosome pair 8 in k. eiffingeri, but on the long arm near the centromere of chromosome pair 12 in k. idiootocus (fig. 2). the active nors are within the corresponding regions of scs in both species (fig. 2 and table 1). fish with telomeric probe (ttaggg)n detected strong signals on the chromosome ends of all chromosomes of k. eiffingeri and k. idiootocus (fig. 4). no (ttaggg)n signal was found at interstitial region in either species. the cgh for k. eiffingeri and k. idiootocus revealed no notable chromosome difference between sexes. most of stronger painting signals were observed in the centromeres with blocks of tandemly repeated satellite sequences, which are embedded in heterochromatic regions. a strong hybridization signal was noted in the telomeric region of the long arm of chromosome pair 3 of k. eiffingeri using either male or female probe (fig. 5). the dna c value of was estimated as 5.06 ± 0.13 pg (male: 5.02 ± 0.08 pg, female: 5.11 ± 0.16 pg) in k. eiffingeri (n = 20), and 4.18 ± 0.21 pg (male: 4.23 ± 0.22 pg, female: 4.13 ± 0.20 pg) in k. idiootocus (n = 17) (table 1). significant difference in genome size between species was evidenced (student t-test, t35 = 15.29, p < 0.0001). in contrast, no sexual difference in genome size was found in both species (student t-test, t18 = -1.597, p = 0.128 in k. eiffingeri, 10 male vs 10 female; t15 = 0.968, p = 0.349 in k. idiootocus, 9 male vs 8 female). discussion the chromosomes of frogs in the family rhacophoridae is considered to be conservative since almost all species studied so far show invariable 2n = 26 karyotypes (li, 2007). distinct diploid chromosome number in this family is extremely rare and only reported in few species (e.g., chiromantis doriae 2n = 16, in rao and yang, 1996, but 2n = 16 or 26 in tan, 1987). rhacophorid karyotypes also share other characteristics including composition of two size groups of chromosomes (i.e., large and small chromosome pairs) and predominant presence of metacentric and submetacentric chromosomes (kuramoto, 1977; blommers-schlösser, 1978; kuramoto, 1985; kuramoto and wang, 1987; tan, 1987; rao and yang, 1996; joshy and kuramoto, 2011). these features, together with the fact that very few acrocentric and telocentric chromosomes were found, have led to the suggestion that the karyotype of rhacophoridae represent a derived condition in anura (rao and yang, 1996) because the primary direction of anuran karyotype evolution has been advocated to reduce chromosome number by fusions of acrocentrics to metacentrics (morescalchi, 1980; suarez 143comparisons of karyotype and genome size in two kurixalus species ta bl e 1. s um m ar y of k ar yo ty pe a nd c hr om os om es f ea tu re s of t he t w o sp ec ie s: 2 n, d ip lo id c hr om os om e nu m be r; m , m et ac en tr ic c hr om os om es ; s m , s ub m et ac en tr ic c hr om os om es ; fn , f un da m en ta l n um be r; s c , s ec on da ry c on st ri ct io n; n o r , n uc le ol us o rg an iz er r eg io n; n sf , n o sp ec ifi c fin di ng s. 2n k ar yo ty pe fo rm ul a fn si ze g ro up o f ch ro m os om es (l ar ge + s m al l) sc n o r h et er oc hr om at in s (b y c -b an di ng ) te lo m er ic m ot if (t ta g g g )n (b y te lo m er ic f is h ) c g h c v al ue (p g; m ea n ± sd ) k . e iffi ng er i m al e 26 10 m + 3 s m 52 5 + 8 8q 8q 1. c en tr om er e of a ll ch ro m os om es 2. p ro xi m al 8 p (d en se ) te lo m er es o f a ll ch ro m os om es st ro ng er p ai nt in g si gn al o n 3q 5. 02 ± 0 .0 8 fe m al e 26 10 m + 3 s m 52 5 + 8 8q 8q 1. c en tr om er e of a ll ch ro m os om es 2. p ro xi m al 8 p (d en se ) te lo m er es o f a ll ch ro m os om es st ro ng er p ai nt in g si gn al o n 3q 5. 11 ± 0 .1 6 o ve ra ll 26 10 m + 3 s m 52 5 + 8 8q 8q 1. c en tr om er e of a ll ch ro m os om es 2. p ro xi m al 8 p (d en se ) te lo m er es o f a ll ch ro m os om es st ro ng er p ai nt in g si gn al o n 3q 5. 06 ± 0 .1 3 k . i di oo to cu s m al e 26 11 m + 2 s m 52 5 + 8 12 q 12 q c en tr om er e of a ll ch ro m os om es te lo m er es o f a ll ch ro m os om es n sf 4. 23 ± 0 .2 2 fe m al e 26 11 m + 2 s m 52 5 + 8 12 q 12 q c en tr om er e of a ll ch ro m os om es te lo m er es o f a ll ch ro m os om es n sf 4. 13 ± 0 .2 0 o ve ra ll 26 11 m + 2 s m 52 5 + 8 12 q 12 q c en tr om er e of a ll ch ro m os om es te lo m er es o f a ll ch ro m os om es n sf 4. 18 ± 0 .2 1 144 shun-ping chang et alii et al., 2013). the two kurixalus species analyzed here revealed conservative karyological features of rhacophoridae, including the modal diploid number, 5 larger and 8 smaller chromosomal pairs, but no acrocentric (either metacentric or submetacentric) chromosomes. although the diploid states of the two kurixalus species are similar, evidence of karyotype divergence through chromosomal rearrangements was found. the two kurixalus species are different in karyotype microstructure. a highly dense heterochromatin was only found in the proximal short arm of the chromosome pair 8 in k. eiffingeri, which could be a chromosome marker in karyotype of this species. besides, the long arm of chromosome pair 3 of k. eiffingeri was painted with strong fluorescence signals in karyotypes of both sexes, suggesting the region is rich in repeated sequences in this species. in both species, nors were only found in one pair of chromosomes and the nor localizations were coincided with regions of sc. however, the nor-bearing chromosome pairs differ between k. eiffingeri and k. idiootocus: one in pair 8 and the other in pair 12. extensive studies have shown that nors can differ in extent, localization and number among chromosomes from different species that make nors a useful chromosome marker in comparative cytogenetic studies (morescalchi, 1980; mahony and robinson, 1986; bruschi et al., 2014). for instance, the nor on chromosome pair 7 has been recognized as a structural distinction between z and w chromosomes in buergeria buergeri, which is the only known rhacophorid species with heteromorphic sex chromosomes (schmid et al., 1993). recently, the possible role of nors as rearrangement hotspots during evolution is also discussed (cazaux et al., 2011; britton-davidian et al., 2012). chromosomal rearrangements can be traced by chromosomal mapping of telomeric sequences, which located at the ends of eukaryotic chromosomes with an fig. 2. g-banding karyotypes of k. eiffingeri for male (a), female (b); and proposed idiogram (c), and k. idiootocus for male (d), female (e) and proposed idiogram (f). the g-banding karyotypes of female and male are similar in each of the two species. bars = 10 μm. an achromatic secondary constriction (sc; indicated by stars) was found in the long arm near the centromere of chromosome pair 8 of k. eiffingeri (g) and in the long arm near the centromere of chromosome pair 12 of k. idiootocus (h). the scs were accompanied by positive ag-nor staining (indicated by arrows) in both species (g and h). 145comparisons of karyotype and genome size in two kurixalus species table 2. arm ratio (length of long arm/short arm) and relative length (percentage of total haploid chromosome length) of chromosomes in k. eiffingeri and k. idiootocus (mean ± sd). abbreviation m and sm indicate metacentric and submetacentric respectively, and n is the number of metaphase spreads analyzed pair no. k. eiffingeri k. idiootocus anona test arm ratio attribute† relative length (%) arm ratio attribute† relative length (%) male (n = 19) female (n = 17) male (n = 19) female (n = 17) male (n = 21) female (n = 19) male (n = 21) female (n = 19) f p 1 1.26 ± 0.09 1.23 ± 0.10 m 17.00 ± 0.33 16.87 ± 0.36 1.34 ± 0.07 1.35 ± 0.11 m 15.92 ± 0.62 16.06 ± 0.48 80.3 <0.0001‡ 2 1.84 ± 0.08 1.88 ± 0.11 sm 13.34 ± 0.63 13.43 ± 0.61 1.73 ± 0.09 1.76 ± 0.11 sm 12.65 ± 0.26 12.33 ± 0.46 57.4 <0.0001‡ 3 2.64 ± 0.33 2.55 ± 0.35 sm 13.22 ± 0.54 13.13 ± 0.47 1.53 ± 0.17 1.48 ± 0.09 m 11.82 ± 0.26 12.02 ± 0.37 171.7 <0.0001‡ 4 1.50 ± 0.12 1.47 ± 0.10 m 11.46 ± 0.29 11.55 ± 0.41 1.81 ± 0.24 1.84 ± 0.17 sm 11.22 ± 0.28 11.35 ± 0.41 7.3 0.0086 5 1.38 ± 0.07 1.41 ± 0.11 m 9.61 ± 0.45 9.69 ± 0.58 1.29 ± 0.04 1.31 ± 0.11 m 9.68 ± 0.25 10.04 ± 0.47 3.9 0.0040 6 1.52 ± 0.18 1.55 ± 0.15 m 5.48 ± 0.28 5.53 ± 0.41 1.56 ± 0.13 1.54 ± 0.15 m 5.79 ± 0.22 5.71 ± 0.19 15.7 <0.0001‡ 7 1.51 ± 0.17 1.53 ± 0.14 m 5.06 ± 0.12 4.98 ± 0.21 1.41 ± 0.09 1.36 ± 0.07 m 5.46 ± 0.14 5.27 ± 0.25 61.2 <0.0001‡ 8 1.59 ± 0.17 1.56 ± 0.13 m 4.85 ± 0.22 4.97 ± 0.21 1.27 ± 0.09 1.23 ± 0.08 m 5.18 ± 0.16 5.11 ± 0.22 27.4 <0.0001‡ 9 1.82 ± 0.19 1.78 ± 0.21 sm 4.67 ± 0.11 4.74 ± 0.11 1.70 ± 0.18 1.62 ± 0.13 m 5.04 ± 0.19 5.01 ± 0.21 74.3 <0.0001‡ 10 1.40 ± 0.18 1.42 ± 0.13 m 4.53 ± 0.13 4.57 ± 0.14 1.11 ± 0.07 1.08 ± 0.06 m 4.45 ± 0.16 4.44 ± 0.22 6.9 0.0106 11 1.21 ± 0.12 1.20 ± 0.11 m 3.90 ± 0.17 3.83 ± 0.17 1.13 ± 0.11 1.09 ± 0.09 m 4.40 ± 0.16 4.37 ± 0.21 154.2 <0.0001‡ 12 1.37 ± 0.16 1.41 ± 0.13 m 3.77 ± 0.07 3.65 ± 0.14 1.41 ± 0.15 1.36 ± 0.11 m 4.30 ± 0.18 4.26 ± 0.19 234.4 <0.0001‡ 13 1.24 ± 0.11 1.22 ± 0.08 m 3.11 ± 0.24 3.06 ± 0.23 1.36 ± 0.09 1.33 ± 0.09 m 4.09 ± 0.22 4.03 ± 0.17 376.5 <0.0001‡ †levan et al. (1964). ‡statistical significance (α = 0.05/76, after bonferroni correction). fig. 3. c-banding karyotypes of k. eiffingeri for male (a) and female (b), and k. idiootocus for male (c) and female (d). constitutive heterochromatins were regularly detected on the centromeres of all chromosomes. particularly, a dense heterochromatin was also noted on the proximal region of short arm of chromosome pair 8 in k. eiffingeri. the c-banding karyotypes of female and male are similar in each of the two species. bars = 10 μm. 146 shun-ping chang et alii important function of chromosomal stability by protecting them from end-to-end fusion, degradation, and recombination (fagundes and yonenaga-yassuda, 1998; bruschi et al., 2014). the presence of interstitial telomeric sequences (itss) in chromosomes has been considered as evidence of chromosome rearrangements that occurred during the evolution of karyotypes. telomeric fish for the karyotypes of the two kurixalus species showed that the (ttaggg)n motifs are restricted to the terminal regions of chromosomes. the absence of itss cannot be explained by the absence of chromosome rearrangements because evidence of karyotypic differences between k. eiffingeri and k. idiootocus were noted. we postulate that subtle chromosome changes irrelevant to itss (e.g., intrachromosomal rearrangements) may play a role in karyotype evolution. recent genomic studies have shown that amphibians have a slow rate of interchromosomal rearrangements but intrachromosomal rearrangements of loci are not uncommon (smith and voss, 2006; sun et al., 2015). thus, the undetected chromosome changes in kurixalus may be uncovered by advanced molecular analyses. the present data demonstrated that the karyotypes of the two kurixalus species seem to be conserved, despite minor differences in chromosome morphology were observed. as a result, the interspecies differences at chromosomal level are unsatisfying to link directly with their behavioral difference in reproductive mode. in addition to cytogenetic characteristics, genome sizes of the two kurixalus species are different. the estimated dna c value of k. eiffingeri is about 1.21 times greater than that of k. idiootocus. currently, no information is available about the c values of other rhacophorid species and the few data collected in genome size database (http://www.genomesize.com/) actually belonged species in the mantellidae. in amphibians, the c values ranged from fig. 4. fluorescence in situ hybridization (fish) with telomeric probe exclusively mapped (ttaggg)n repeats to terminal regions of all chromosomes of k. eiffingeri (a) and k. idiootocus (b). no interstitial telomeric sequence (its) was detected in both species. fig. 5. comparative genomic hybridization (cgh) using male and female genomic probes (labeled by fitc and txred, respectively) for chromosomes from male and female of k. eiffingeri showed similar hybridization patterns between sexes (a). a large chromosomal fragment with stronger painting signal (indicated by an arrow) were observed on the terminal regions of long arm of chromosome pair 3, presumably rich in repeated sequences (b). similar patterns between sexes without any stronger painting signal in chromosome pairs were observed in k. idiootocus (data not showed). p, short arm; q, long arm. 147comparisons of karyotype and genome size in two kurixalus species 0.95 to 120 pg. the more than 130-fold variation in c value has rendered amphibian a taxon with the most variable genome size among vertebrates (gregory, 2003). genome size can be affected by various genetic events, such as duplication, insertion, recombination, deletion and polyploidization (gregory, 2005). difference in genome sizes thus reflects dissimilarity in genomic compositions. identification of heteromorphic chromosome pairs has been generally acknowledged as the first step in recognition of sex determination mechanism (valenzuela et al., 2003). some heteromorphic chromosome pairs differ in chromosomal appearance and are easily observable, whereas others require advanced high-resolution tools to clarify. in this study, cytogenetic analyses for the two kurixalus species revealed all chromosome pairs are homomorphism. even the cgh was executed for both sexes in each species, no recognizable sex difference in karyotypes. this is not surprising because in amphibians most species lack morphologically distinguishable sex chromosomes despite exhibiting gonochorism (distinct male and female). so far, only a few of amphibian species with heteromorphic sex chromosomes were reported (hayes, 1998; berset-brandli et al., 2006; ezaz et al., 2006). it is a little strange that, despite the lack of heteromorphism in chromosome pairs of most amphibian species, genetic sex determination is generally considered the rule for this group (nakamura, 2009). it was proposed that since sex chromosomes evolved from homomorphic autosomes through the acquisition of a sex determining gene (ohno, 1967), the reason that heteromorphic sex chromosomes could not be highlighted in amphibians was not due to their nonexistence but because the genes involved in sex determination were located on poorly discriminated regions of the chromosomes (hayes, 1998). for example, the gene for adp/atp translocase was described as a sex-linked marker in rana rugosa (miura et al., 1998) of xx/xy system and sex-specific loci in xenopus laevis (mawaribuchi et al., 2017) of zz/zw system, but none of the species exhibited notable differences in karyotypes between sexes. however, in the two kurixalus species analyzed the sexual differences in genomic size are too small to be detected, leading to inconclusive evidence if genetic differentiation exists between sexes. traditionally, a good strategy to understand the possible sex determination mode in species is to examine the sex ratio in natural populations (janzen and paukstis, 1991). balanced sex ratio is the most general explanation for species with genetic determination. the sex ratio of k. idiootocus in nature was not known, but k. eiffingeri had balanced adult sex ratio (51.2% proportion of males) (kam et al., 2000). although this provides a possible link to the genetic sex determination in k. eiffingeri, segregation distortion, such as temperature-induced differential mortality, differential fertilization, and embryo abortion, can result in biased primary (fertilization) or secondary (births) sex ratio (valenzuela et al., 2003). besides, in a few frog species the sex can be affected by temperature despite they are considered as genetic sex determination (witschi, 1914; piquet, 1930; yoshikura, 1963). basically, all above arguments must move ahead from a well-established base of sufficient cytogenetic information just like our efforts in the two kurixalus species. we considered that the loss of cytogenetic differences between sexes of the two kurixalus species does not refute the idea that these species possess genetic sex determination. however, the present data did not provide evidence to support the existence of sex-specific genetic markers in either species. it is possible that the sex chromosomes might be homomorphic, with the extensive pseudoautosomal region (roco et al., 2015), and small sex specific regions that harbor key genes from the sexdetermination pathway, such as the cases with sex specific dmrt1 gene in rana temporaria (ma et al. 2016; rodriguez et al 2017), which would need more advanced molecular approaches (e.g., genotyping by length-polymorphic markers) to exploring the genetic sex-determination. at the moment, it remains unclear if all amphibian species are of genetic sex determination, but species with diverse reproductive behaviors like kurixalus provides an interesting object to study this question. we demonstrated that the two kurixalus tree frogs have distinct karyotypes and genome sizes. cytogenetic characteristics, such as the distribution of sc/nor and staining intensity of heterochromatins that distinguished the karyotypes of the two species, were not previously reported. in either species, the finding of no differences in cytogenetic and genomic features between sexes is in line with previous reports that showed no heteromorphic chromosome pair in most rhacophorid species. moreover, our telomeric fish demonstrated no its in chromosomes of both species, suggesting subtle chromosome changes irrelevant to itss (e.g., intrachromosomal rearrangements) may play a role in their karyotype evolution. the cytogenetic data and the genomic information described in this work contribute to our understanding of the genetic features in kurixalus species and are potentially useful as taxonomic and reproductive traits for additional studies in this genus as well as in the family rhacophoridae. acknowledgements the authors are thankful to yin-ju yang, miin-yu horng, yi fu lin, chun-hsin tsai, chia-ming kuo, and 148 shun-ping chang et alii hui-shan tsai for valuable help with sample collection, and to the staff of the experimental forest of the national taiwan university at xitou for a permit to collect samples in the experimental forest and providing accommodation. the wild animals were sampled according to the taiwan animal protection act with permits from nantou county government (permit no. 10000438710) and experimental forest of the national taiwan university (permit no. 1000002098). the research was reviewed and approved by the institutional animal care and use committee of changhua christian hospital (cchae-103-007). the study was partly supported by changhua christian hospital (project no: 104-cch-irp-101) and ministry of science and technology, taiwan (project no: 104-2621-b-005-003). references badenhorst, d., stanyon, r., engstrom, t., valenzuela, n. 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(2012): use of a cytogenetic whole-genome comparison to resolve phylogenetic relationships among three species: implications for mammalian systematics and conservation biology. theriogenology 77: 1615-1623. acta herpetologica vol. 12, n. 2 december 2017 firenze university press meristic and morphometric characters of leptopelis natalensis tadpoles (amphibia: anura: arthroleptidae) from entumeni forest reveal variation and inconsistencies with previous descriptions susan schweiger1, james harvey2, theresa s. otremba1, janina weber1, hendrik müller1,* brown anole (anolis sagrei) adhesive forces remain unaffected by partial claw clipping austin m. garner*, stephanie m. lopez, peter h. niewiarowski species and sex comparisons of karyotype and genome size in two kurixalus tree frogs (anura, rhacophoridae) shun-ping chang1,2, gwo-chin ma2,3,4, ming chen2,5,6,7,8,*, sheng-hai wu1,* non-native turtles in a peri-urban park in northern milan (lombardy, italy): species diversity and population structure claudio foglini1, roberta salvi2,* species composition and richness of anurans in cerrado urban forests from central brazil cláudia márcia marily ferreira1,*, augusto cesar de aquino ribas2, franco leandro de souza3 the life-history traits in a breeding population of darevskia valentini from turkey muammer kurnaz, alı i̇hsan eroğlu, ufuk bülbül*, halıme koç, bılal kutrup influence of desiccation threat on the metamorphic traits of the asian common toad, duttaphrynus melanostictus (anura) santosh mogali*, srinivas saidapur, bhagyashri shanbhag predation of common wall lizards: experiences from a study using scentless plasticine lizards jenő j. purger*, zsófia lanszki, dávid szép, renáta bocz reproductive timing and fecundity in the neotropical lizard enyalius perditus (squamata: leiosauridae) serena najara migliore1,2,*, henrique bartolomeu braz2,3, andré felipe barreto-lima4, selma maria almeida-santos1,2 observations on the intraspecific variation in tadpole morphology in natural ponds eudald pujol-buxó1,2,*, albert montori1, roser campeny3 and gustavo a. llorente1,2 reliable proxies for glandular secretion production in lacertid lizards simon baeckens diet of juveniles of the venomous frog aparasphenodon brunoi (amphibia: hylidae) in southeastern brazil rogério l. teixeira1, ricardo lourenço-de-moraes2, débora c. medeiros3, charles duca3, rogério c. britto4, luiz c. p. bissoli5, rodrigo b. ferreira3,* who are you? the genetic identity of some insular populations of hierophis viridiflavus s.l. from the tyrrhenian sea ignazio avella, riccardo castiglia, gabriele senczuk* acta herpetologica 11(2): 213-219, 2016 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-17821 on the feeding ecology of pelophylax saharicus (boulenger 1913) from morocco zaida ortega1,*, valentín pérez-mellado1, pilar navarro2, javier lluch2 1 department of animal biology, university of salamanca, campus miguel de unamuno, 37007, salamanca, spain. *corresponding author. email: zaidaortega@usal.es 2 department of zoology, university of valencia, c/ doctor moliner, 50, 46100, burjassot, valencia, spain submitted on 2016, 14th january; reviewed on 2016, 11th april; accepted on 2016, 7th may editor: sebastiano salvidio abstract. the sahara frog is the most common amphibian found in north africa. however, the knowledge of its natural history is rather fragmentary. in the present work we studied the trophic ecology of pelophylax saharicus at some areas of morocco through the analysis of 130 gastric contents. we did not find any significant sexual dimorphism in body size of adult individuals. consumed prey show similar sizes in both sexes, while bigger frogs normally eat larger prey. as in other palearctic frogs, the diet is basically insectivorous, including terrestrial and aquatic prey. we found some differences in the diet of juveniles, with a higher proportion of flying prey, probably indicating a foraging strategy closer to ambush hunting. in the atlas region, the high consumption of slow-moving terrestrial prey, as gastropoda, stands out. only in the atlas region, the diet was similar to that described from other areas of north africa, as tunisia. keywords. trophic ecology, ranidae, green frogs, morocco, pelophylax saharicus. the sahara frog, pelophylax saharicus, inhabits a large portion of north africa, from south sahara to the mediterranean coast, through the atlas mountains (pasteur and bons, 1959; amor et al., 2010), living at altitudes of more than 2600 m.a.s.l. its distribution ranges from morocco to egypt, being the most common green frog of north africa (salvador, 1996; amor et al., 2010). the species is strictly aquatic and is found both in natural and artificial permanent ponds, even when these are slightly eutrophized (salvador, 1996). pelophylax saharicus is currently considered a full species, following bons and géniez (1996) that summarized the discussion about the controversial status of moroccan green frogs. molecular studies support the specific status of p. saharicus (plötner, 1998; frost et al., 2006; lymberakis et al., 2007; lansari et al., 2015; nicolas et al., 2015), ranging it as the sister group of pelophylax perezi, apart from the other species of the genus. both males and females reach the sexual maturity in the second year of life, and are able to live as much as six years (e.g. oromi et al., 2011). previous studies concluded that p. saharicus does not show sexual dimorphism in the size of adult animals (esteban et al., 1999). in a preliminary study of feeding ecology of palearctic frogs, smith (1951) described the diet of rana ridibunda ridibunda. then, lizana et al. (1989) compared the feeding ecology of p. perezi with other iberian amphibians and with trophic availability at an area of the central iberian peninsula. subsequent studies have addressed the feeding ecology of other pelophylax species (çiçek et al., 2006; sas et al., 2009; mollov et al., 2010; paunović et al., 2010; bogdan et al., 2012, 2013; plitsi et al., in press). a recent study assesses the effect of temperature, density and food in the growth and metamorphosis of p. saharicus tadpoles (bellakhal et al., 2014). regarding its trophic ecology, some data have been published about 214 zaida ortega et alii p. saharicus in the oases of kettana, in tunisia (hassine and nouira, 2009). here we present the first data about trophic ecology of the sahara frog in morocco. all samples used in this study came from moroccan areas within the semi-arid mediterranean zone of north africa (le houéron, 1989). frogs were captured during 1996 in three different areas of morocco: (1) the western plateau, an area of subhumid to semiarid climate and two localities were sampled: el borj and zwiat cheikh, (2) rif mountains and adjacent areas (this is the most humid area of morocco with more than 600 mm of annual rainfall), and (3) the middle atlas, with a climate of strong continental characteristics. sample sizes were 9 males, 4 females and 5 juveniles for the western plateau, 26 males, 52 females and 6 juveniles for the rif mountains, and 11 males, 16 females and 1 juvenile for the middle atlas. frogs were euthanized during the field work because they were captured to study helminthic parasites in the framework of a parasitological research (see navarro and lluch, 2006). maturity and sex of the individuals were determined by direct examination of the gonads after dissection. the analysis included 130 gastric contents. prey items were identified to family or order level. prey size was measured from intact items with a micrometric ocular. afterwards, absolute frequencies of each prey type and its percentage in the diet were calculated for each region, as well as the number of gastric contents in which such prey was present. we used spearmann rank correlation and ancova on prey size, with svl (snout-vent length) as a covariate, to study the relation between body length of frogs and the size of consumed prey for each category (adult males, adult females, and juveniles). then, we estimated and compared diet diversities using the approach proposed by pallmann et al. (2012). instead describing diet diversity through a given index as, for example, simpson or shannon indices, we converted these “raw” indices into “true” diversities. that is, regarding different measures as special cases of hill’s general definition of diversity measures (hill, 1973). to study differences in diversity between males, females and juveniles, we performed two-tailed tests for integral hill numbers of orders -1 ≤ q ≤ 3. this selection includes the transformed versions of the three following indices: the species richness index, hsr (q = 0), the shannon entropy index, hsh (q → 1) and the simpson concentration index, hsi (q = 2). all comparisons among diversities of groups were made with tukey-like contrasts employing a resampling procedure. we did 5000 bootstrap replications so as to obtain reliable p-values (westfall and young, 1993). methods described here are implemented in r package “simboot” (scherer and pallmann, 2014) and are fully described in pallmann et al. (2012). all calculations were done in r version 3.0.3 (r core team, 2014). finally, in order to visualize differences in the composition of the diet of adults of both sexes and juveniles, we conducted a discriminant function analysis. box’s m test of equality of covariance matrix was not significant, so data were suitable for discriminant analysis. only two variables (dictyoptera larvae and dermaptera larvae) failed the tolerance test, so were excluded from the analysis, the rest of the variables (table 3) were suitable for analysis (tolerance test with p > 0.05). the diet of p. saharicus was mainly insectivorous and more varied in females than in males or juveniles. but, we did not find significant differences in the diversity values of males, females and juveniles (p > 0.05 in all pairwise comparisons, table 2). diptera were the most important prey item. the diet of juvenile individuals, principally dominated by formicidae and other small hymenoptera, was less diverse than that of adult males and females. we observed a high proportion of hymenoptera in the diet of western plateau frogs, much higher than for tunisian populations (hassine and nouira, 2009). this is principally due to the massive presence of this prey in five juvenile individuals, in which we found 95.58% (65 of 68 prey items) of all sampled hymenoptera. in addition, all adult individuals of p. saharicus from the plateau ate proportionally more hymenoptera than those from the atlas or rif regions, suggesting a greater availability of such prey at the plateau. alternatively, these differences can be due to a different foraging behaviour in different areas. according to our results, there is no sexual dimorphism in adult individuals of p. saharicus (see also esteban et al., 1996; meddeb et al., 2007). svl of juveniles was 51.00 ± 2.00 mm (mean ± se, n = 8). we did not find significant differences in body size of adult males and females of p. saharicus (one-way anova of log-transformed data, f = 0.304, p = 0.583, homogeneous variances, levene test, p = 0.90; svl of adult males, mean = 88.43 ± 4.96 mm, range = 48-97 mm, n = 44; adult females, mean = 92.51 ± 4.40 mm, range = 48-223, n = 74), even if females were slightly larger than males. we measured 803 prey items (mean = 5.26 ± 0.18 mm, range = 0.5-70 mm). we found a significant correlation between frog body size (svl) and prey size (spearmann rank correlation, rs = 0.510, p < 0.001, n = 803). this correlation was also maintained within adult individuals (rs = 0.399, p < 0.001, n = 695; mean = 5.68 ± 0.20 mm, range = 0.5-70 mm). according to this result, we analysed the prey size in both genders employing the svl as the covariate. adult females ate prey of significantly larger size than adult males (ancova analysis, f = 4.816, p = 0.029, with no significant differences in regression slopes, f = 1.055, p = 0.305; mean = 5.04 215feeding ecology of pelophylax saharicus ± 0.24, n = 268 for adult males, and mean = 6.08 ± 0.29 mm, n = 427 for adult females). for the discriminant analysis, the correlations between the variables and the two discriminant axes are provided in table 3. the discriminant function is able to correctly classify the 64.5% of individuals as adult males, adult females or juveniles according to their diet, so the goodness of fit is acceptable. differences in the diet of table 1. data from the analysis of 130 stomach contents of pelophylax saharicus. fi is the absolute frequency of each type of prey item group in the sample, % fi the relative frequency of the group in the sample, p is the presence of each group (i.e. the number of stomach contents in which the group appears), and % p the percentage of the presence of the item in the sample. group total juveniles adult males adult females fi % fi p % p fi % fi p % p fi % fi p % p fi % fi p % p gastropoda 48 5 21 16.2 2 1.74 2 18.18 9 2.90 6 13.64 37 6.93 13 17.57 araneae 21 2.18 17 13.1 0 0 0 0 13 4.19 11 25 8 1.50 6 8.11 acarina 2 0.21 1 0.8 2 1.74 1 9.09 0 0 0 0 0 0 0 0 ostracoda 5 0.52 2 1.5 0 0 0 0 0 0 0 0 5 0.94 2 2.70 isopoda 7 0.73 6 4.6 0 0 0 0 1 0.32 1 2.27 6 1.12 5 6.76 crustacea 1 0.1 1 0.8 0 0 0 0 1 0.32 1 2.27 0 0 0 0 diplopoda 1 0.1 1 0.8 0 0 0 0 1 0.32 1 2.27 0 0 0 0 chilopoda 6 0.62 3 2.3 0 0 0 0 0 0 1 2.27 5 0.94 2 2.70 diplura larvae 1 0.1 1 0.8 1 0.87 1 9.09 1 0.32 0 0 0 0 0 0 thysanura 2 0.21 2 1.5 0 0 0 0 1 0.32 1 2.27 1 0.19 1 1.35 odonata 1 0.1 1 0.8 0 0 0 0 1 0.32 1 2.27 0 0 0 0 ephemeroptera 9 0.94 1 0.8 0 0 0 0 9 2.90 1 2.27 0 0 0 0 plecoptera 4 0.41 4 3.1 0 0 0 0 1 0.32 1 2.27 3 0.56 3 4.05 plecoptera larvae 6 0.62 4 2.3 0 0 0 0 2 0.64 2 4.54 4 0.75 2 2.70 orthoptera 28 2.92 21 16.2 0 0 0 0 5 1.61 5 11.36 23 4.31 16 21.62 orthoptera larvae 1 0.1 1 0.8 0 0 0 0 1 0.32 1 2.27 0 0 0 0 dictyoptera 16 1.66 5 3.8 2 1.74 1 9.09 2 0.64 2 5.54 12 2.25 2 2.70 dictyoptera larvae 1 0.1 1 0.8 0 0 0 0 1 0.32 1 2.27 0 0 0 0 dermaptera 4 0.41 4 3.1 0 0 0 0 1 0.32 1 2.27 3 0.56 3 4.05 dermaptera larvae 1 0.1 1 0.8 0 0 0 0 1 0.32 1 2.27 0 0 0 0 phasmida 7 0.73 1 0.8 0 0 0 0 0 0 0 0 7 1.31 1 1.35 embioptera 1 0.1 1 0.8 0 0 0 0 0 0 0 0 1 0.19 1 1.35 thysanoptera 4 0.41 4 3.1 2 1.74 2 18.18 0 0 0 0 2 0.37 2 2.70 homoptera 28 2.92 25 19.2 1 0.87 1 9.09 12 3.87 12 27.27 15 2.81 12 16.22 homoptera larvae 3 0.31 3 2.3 0 0 0 0 2 0.64 2 4.54 1 0.19 1 1.35 heteroptera 45 4.69 29 22.3 2 1.74 2 18.18 20 6.45 12 27.27 23 4.31 15 20.27 heteroptera larvae 1 0.1 1 0.8 0 0 0 0 1 0.32 1 2.27 0 0 0 0 diptera 245 25.52 87 66.9 18 15.65 7 63.64 97 31.29 33 75 129 24.16 47 63.51 diptera larvae 19 1.98 14 10.8 1 0.87 1 9.09 5 1.61 4 9.09 13 2.43 9 12.16 trichoptera larvae 1 0.1 1 0.8 0 0 0 0 0 0 0 0 1 0.19 1 1.35 lepidoptera 9 0.94 7 5.4 0 0 0 0 5 1.61 4 9.09 4 0.75 3 4.05 lepidoptera larvae 1 0.1 1 0.8 0 0 0 0 0 0 0 0 1 0.19 1 1.35 coleoptera 149 15.52 65 50 6 5.22 3 27.27 50 16.13 23 52.27 93 17.42 39 52.70 coleoptera larvae 29 3.02 11 8.5 6 5.22 3 27.27 2 0.64 2 4.54 21 3.93 6 8.11 hymenoptera 155 16.14 55 42.3 68 59.13 8 72.73 27 8.71 17 38.64 60 11.24 30 40.54 formicidae 74 7.71 42 32.3 2 1.74 2 18.18 30 9.68 15 34.09 42 7.86 25 33.78 undet. arthropoda 5 0.52 5 3.8 0 0 0 0 1 0.32 1 2.27 4 0.75 4 5.40 undet. larvae 18 1.87 14 10.8 2 1.74 2 18.18 7 2.26 6 13.64 9 1.68 6 8.11 birds 1 0.1 1 0.8 0 0 0 0 0 0 0 0 1 0.19 1 1.35 total 960 130 115 11 310 44 534 74 216 zaida ortega et alii males, females and juveniles are plotted in figure 1. on one hand, the discriminant axes 1 somewhat divides diet of males (negative values) from diet of females (positive values), and it is mainly positively correlated with the presence of formicidae and coleoptera, and negatively correlated with the presence of hymenoptera, larvae of diplura, acarina and tysanoptera (fig. 1, table 3). on the other hand, the discriminant axes 2 divides the diet of juveniles (negative values) from the diet of adults (positive values), and it is mainly positively correlated with the presence of orthoptera, gastropoda, larvae of isopoda, ostracoda and larvae of coleoptera, among others, and mainly negatively correlated with the presence of araneae, ephemenoptera, larvae of orthoptera larvae, diplopoda, larvae of dermaptera, or larvae of dictyoptera, among others (fig. 1, table 3). regarding the area of study, we did not detect with the discriminant analysis any clear pattern in the composition of the diet (fig. 2). the diet of juvenile individuals is clearly different, being less diverse than the diet of adults (table 2). young frogs use to hunt smaller prey than adults, mainly small hymenoptera. hirai and matsui (1999) found a significant correlation between svl and prey size of pelophylax nigromaculatus, as we observed in p. saharicus, suggesting that individuals of green frogs tend to eat larger prey as they grow. table 2. simpson’s diversity values of the diet of males, females and juveniles of p. saharicus and p-values from pairwise comparisons of hill’s numbers (see more details in the text) adult males adult females juveniles diversity values 0.8521 ± 1.80 x 10-4 0.8818 ± 5.51 x 10-5 0.6273 ± 2.19 x 10-3 hill’s numbers males-females females-juveniles juveniles-males q = 0 0.8398 0.9134 0.6960 q = 1 0.7928 0.7014 0.4258 q = 2 0.8300 0.7280 0.4742 fig. 1. values of each dimension selected in the discriminant function analysis of the diet of pelophylax saharicus are plotted for each studied frog. individuals are marked regarding age and sex in order to visualize the age and sex differences in the trophic ecology of the sahara frog. fig. 2. values of each dimension selected in the discriminant function analysis of the diet of pelophylax saharicus are plotted for each studied frog. individuals are marked regarding the area of study: the western plateau, the rif mountains, and the middle atlas. 217feeding ecology of pelophylax saharicus pelophylax saharicus has a similar feeding ecology composition that its sister taxon, p. perezi, from the iberian peninsula (lizana et al., 1989), and other species of the genus, as p. ridibundus (çiçek et al., 2006; mollov et al., 2010). diptera predominates as the main prey item of adult individuals of both species, followed in abundance by coleoptera prey (aquatic species mostly) and hymenoptera, often formicidae. the diet of p. saharicus in morocco has some differences with the diet of other species of pelophylax, as p. kurtmuelleri in greece, which actively selects arachnids over other types of prey (plitsi et al., in press). nonetheless, we lack data about availability of prey in the habitat of sahara frogs, which limits our results. thus, our results about the differences in the diet of sexes and ages should be taken with caution, since it is possible that the electability of each type of prey would be similar to their availability in the environment. therefore, future research in the diet of p. saharicus frogs, including the availability of prey in their habitats and seasonal comparisons would be useful to get deeper knowledge about the ecology of the species. furthermore, the diet of p. kurtmuelleri frogs is highly influenced by their habitat (plitsi et al., in press), and the diets of p. ridibundus and p. esculenta are also influenced by seasonality (sas et al., 2009; mollov et al., 2010) and weather conditions (bogdan et al., 2012). thus, we cannot exclude that p. saharicus could also employ a variable foraging strategy. lizana et al. (1989) observed that the females of the iberian green frog ate significantly larger prey than adult males, as we observed in p. saharicus. the ingestion of larger prey by females and their more diverse diet could be the reason of the slightly bigger parasite load of this sex. in this sense, and working with the same frogs, navarro and lluch (2006) found that females showed more diverse helminth infracommunities, even if differences with males were not statistically significant. the inclusion of a large amount of flying prey in the diet reinforces the hypothesis that p. saharicus is a sit-and-wait forager. gastropoda were only important in the rif sample, with a similar proportion as the reported for the tunisian studied population (hassine and nouira, 2009). in p. ridibunda of turkey no differences of diet regarding sex were found (çiçek et al., 2006). the consumption of formicidae is not higher in p. saharicus than in p. perezi of the iberian peninsula, and it is consistent with the diet of the tunisian population (hassine and nouira, 2009). the consumption of ants and other prey groups could be due to a foraging behaviour near the water or at more terrestrial habitats. in fact, many rivers and natural ponds of morocco scarcely have riverside edges, forcing the individuals to stand close to water shore. sas et al. (2009) found that p. esculenta of romania changes the proportion of aquatic and terrestrial preys along the year activity period, which, although unknown yet, would be also possible for p. saharicus of morocco. table 3. pooled values of within-groups correlations between the discriminating variables (the prey items) and the standardized canonical discriminant functions (the two discriminant axes). discriminating variables are ordered by absolute size of correlation within the discriminant axes 1. group discriminant axes 1 discriminant axes 2 hymenoptera -0.481* -0.019 diplura larvae -0.302* -0.043 acarina -0.302* -0.043 thysanoptera -0.281* 0.087 formicidae 0.119* -0.059 coleoptera 0.088* 0.052 plecoptera larvae 0.050* 0.030 araneae 0.144 -0.319* orthoptera 0.109 0.280* gastropoda 0.049 0.211* ephemenoptera 0.041 -0.200* orthoptera larvae 0.041 -0.200* diplopoda 0.041 -0.200* dermaptera larvaea 0.041 -0.200* dictyoptera larvaea 0.041 -0.200* crustacea 0.041 -0.200* odonata 0.041 -0.200* heteroptera larvae 0.041 -0.200* isopoda larvae 0.061 0.188* ostracoda 0.032 0.174* homoptera larvae 0.061 -0.159* lepidoptera 0.084 -0.140* coleoptera larvae -0.084 0.140* diptera 0.049 -0.139* undet. arthropoda 0.060 0.137* lepidoptera larvae 0.023 0.125* embioptera 0.023 0.125* trichoptera larvae 0.023 0.125* phasmida 0.023 0.125* bird 0.023 0.125* diptera larvae 0.076 -0.119* diplura larvae 0.040 0.110* chilopoda 0.037 0.101* homoptera 0.093 -0.095* dictyoptera -0.020 0.090* plecoptera 0.055 0.089* dermaptera 0.055 0.089* undet. larvae -0.018 -0.069* thysanura 0.045 -0.052* * largest absolute correlation between each variable and any discriminant function a this variable not used in the analysis. 218 zaida ortega et alii acknowledgements mohamed el ayadi helped during field work in morocco. angélica hernández-gonzález and maría marquínez helped during laboratory work. field work was possible thanks to collecting permits (“ordres de mission”) issued by morrocan government to faculté des sciences de l’université abdelmalek essaadi. laboratory work and data analysis were supported by the grants cgl2006-10893-co2-02 and cgl2012-39850 from the spanish ministry of science and technology and spanish ministry of economy and competitiveness. references amor, n., velo-antón, g., farjallah, s., said, k. 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(1993): resampling-based multiple testing: examples and methods for p-value adjustment. new york: john wiley & sons, inc. acta herpetologica vol. 11, n. 2 december 2016 firenze university press predator-prey interactions between a recent invader, the chinese sleeper (perccottus glenii) and the european pond turtle (emys orbicularis): a case study from lithuania vytautas rakauskas1,*, rūta masiulytė1, alma pikūnienė2 effective thermoregulation in a newly established population of podarcis siculus in greece: a possible advantage for a successful invader grigoris kapsalas1, ioanna gavriilidi1, chloe adamopoulou2, johannes foufopoulos3, panayiotis pafilis1,* the unexpectedly dull tadpole of madagascar’s largest frog, mantidactylus guttulatus arne schulze1,*, roger-daniel randrianiaina2,3, bina perl3, frank glaw4, miguel vences3 thermal ecology of podarcis siculus (rafinesque-schmalz, 1810) in menorca (balearic islands, spain) zaida ortega*, abraham mencía, valentín pérez-mellado growth, longevity and age at maturity in the european whip snakes, hierophis viridiflavus and h. carbonarius sara fornasiero1, xavier bonnet2, federica dendi1, marco a.l. zuffi1,* redescription of cyrtodactylus fumosus (müller, 1895) (reptilia: squamata: gekkonidae), with a revised identification key to the bent-toed geckos of sulawesi sven mecke1,*,§, lukas hartmann1,2,§, felix mader3, max kieckbusch1, hinrich kaiser4 the castaway: characteristic islet features affect the ecology of the most isolated european lizard petros lymberakis1, efstratios d. valakos2, kostas sagonas2, panayiotis pafilis3,* sources of calcium for the agamid lizard psammophilus blanfordanus during embryonic development jyoti jee1, birendra kumar mohapatra2, sushil kumar dutta1, gunanidhi sahoo1,3,* mediodactylus kotschyi in the peloponnese peninsula, greece: distribution and habitat rachel schwarz1,*, ioanna-aikaterini gavriilidi2, yuval itescu1, simon jamison1, kostas sagonas3, shai meiri1, panayiotis pafilis2 swimming performance and thermal resistance of juvenile and adult newts acclimated to different temperatures hong-liang lu, qiong wu, jun geng, wei dang* olim palus, where once upon a time the marsh: distribution, demography, ecology and threats of amphibians in the circeo national park (central italy) antonio romano1,*, riccardo novaga2, andrea costa1 on the feeding ecology of pelophylax saharicus (boulenger 1913) from morocco zaida ortega1,*, valentín pérez-mellado1, pilar navarro2, javier lluch2 notes on the reproductive ecology of the rough-footed mud turtle (kinosternon hirtipes) in texas, usa steven g. platt1, dennis j. miller2, thomas r. rainwater3,*, jennifer l. smith4 heavy traffic, low mortality tram tracks as terrestrial habitat of newts mikołaj kaczmarski*, jan m. kaczmarek book review: sutherland, w.j., dicks, l.v., ockendon, n., smith, r.k. (eds). what works in conservation. open book publishers, cambridge, uk. http://dx.doi.org/10.11647/obp.0060 sebastiano salvidio acta herpetologica 15(2): 87-94, 2020 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/a_h-9670 potential effects of climate change on the distribution of invasive bullfrogs lithobates catesbeianus in china li qing peng1, min tang1, jia hong liao1, hai fen qing1, zhen kun zhao1, david a. pike2, wei chen1,* 1 ecological security and protection key laboratory of sichuan province, mianyang normal university, mianyang 621000, china 2 department of biology, rhodes college, memphis tennessee 38111, usa *corresponding author. e-mail: wchen1949@163.com submitted on 2020, 4th september; revised on 2020, 10th october; accepted on 2020, 26th october editor: rocco tiberti abstract. climate plays important roles in determining the geographical distribution of species, including the invasion area of alien species. little is known, however, about the influence of climate change on the distribution area of invasive amphibian species in china. we adopted a maximum entropy model to predict the potential suitable invasive range of invasive bullfrogs lithobates catesbeianus in china under two future climate scenarios in 2050 and 2070. our results reveal that bullfrogs were mainly distributed in east and central china at present, and the suitable area for the species may decrease in future. this suggests that climate change may negatively impact this alien-invasive species. keywords. bullfrog, climate change, environmental limitations, invasive species, potential distribution, species distribution model. introduction biological invasion of alien invasive species is considered to be the second leading cause of global biodiversity loss and habitat degradation (pimentel et al., 2000; bellard et al., 2012; runyon et al., 2012), seriously threatening the health of ecosystems (hobbs and huenneke, 1992; d’antonio et al., 2004; vilà et al., 2011; espíndola et al., 2012; sorte et al., 2013) and causing significant economic losses (pimentel et al., 2000). the proliferation and outbreak of invasive species are becoming more and more serious (pyšek and hulme, 2010). the acceleration of globalization has affected the distribution of invasive species and almost no ecosystem is immune to the impact of alien species (weber and li, 2008; catford et al., 2012). china is a large country encompassing many different climatic regions, where many invasive species can find suitable habitats where to establish. investigating the potential distribution of invasive species could help to address the conservation efforts to eliminate or reduce the negative effects of biological invasions on local wildlife and ecosystems (xie et al., 2001). as in the rest of the world, climate change is affecting also china’s ecosystems (hu et al., 2012). climate change has shown enormous influence on species distribution (erasmus et al., 2002; walther et al., 2002; root et al., 2003; hari et al., 2006; guralnick, 2007). for example, climate change in the 20th century has changed the distribution of butterflies (parmesan et al., 1999), birds (thoms and lennon, 1999), amphibians (araújo et al., 2006) and mammals (hersteinsson and macdonald, 1992). climate change has attracted wide attention of governments and scientists because of its enormous influences on ecosystem functions and global environmental quality (thomas et al., 2004; kiritani, 2011). the bullfrog lithobates catesbeianus is native to eastern north america, but has been introduced throughout the world during the past two centuries (lever, 2003). 88 li qing peng et alii the species is considered as one of the most harmful and threatening invasive species, since it is relatively large and negatively affects native amphibians through competition (zhou et al., 2005), predation (kiesecker and blaustein, 1998; lowe et al., 2000) and disease transmission (hanselmann et al., 2004). knowledge of the patterns of bullfrog invasion is, therefore, extremely important for planning conservation strategies aiming to understand and reduce the impacts of their invasion. bullfrogs were introduced into china in 1959 via the aquaculture and aquarium trades (han, 1991). the species successfully established wild populations, and it is spreading locally (li and xie, 2004; wu et al., 2004). once established it is extremely difficult to eradicate (li and xie, 2004). although the distribution of the species has been simulated at a global scale (ficetola et al., 2007) to predict areas susceptible to invasion, little is known about its potential distribution in china and how future climate scenarios will influence its distribution. we therefore modeled the potential distribution of bullfrog based on current climatic models and projected the results onto future climate scenarios (2050 and 2070) under two emissions scenarios, rcp4.5 (a radiative forcing of 4.5 w/ m2 at the end of 2100) and rcp8.5 (a radiative forcing of 8.5 w/m2 at the end of 2100). our main aims were to describe the current potential distribution of the bullfrogs in china and to model its distribution under future climate change scenarios. materials and methods we collected individual records of bullfrogs in china from: 1) the relevant literature (n = 83 records); 2) the global biodiversity information facility database (gbif, http://data.gbif. org, n = 6 records); and 3) our own field investigations (n = 6 records). we used arcgis 10.2, combined with google earth, to extract the longitude and latitude coordinates and discard duplicate records (warren and seifert, 2011). all the distribution points with a spatial resolution of 30 arc-sec are buffered in gis to ensure that only one point exists within the range of 30 arc-seconds (approximately 1 km × 1 km). totally, we achieved 95 individual records of bullfrogs in china. we downloaded climate data with a spatial resolution of 30 arc-sec from the worldclim database (http//www.worldclim.org/ bioclim). we used arcgis 10.2 to unify all the factors into the same coordinate system and extent (tang and yang, 2006). as our base map, we used a 1: 4,000,000 map of china as original map from the national basic geographic information system (http://nfgis.nsdi.gov.cn). we prepared a total of 22 layers of variables (19 environmental variables and 3 topography variables), that mainly reflect seasonal variation in temperature and precipitation (hijmans et al., 2005), and topography factors (elevation, aspect and slope). we extracted their values at each distribution point and we calculated the pairwise pearson product-moment correlation coefficients. in the cases where two variables were inter-correlated to a high degree (r > 0.75, nori et al., 2011a, b), we selected the most important biologically factors (bourke et al., 2018). we selected 6 final bioclimatic variables and 3 topography variables that did not show high correlation with other variables (r < 0.75) (nori et al., 2011a, b). the final variable set included “annual mean temperature” (bio1), “mean diurnal range of temperature” (bio2; the mean of monthly maximum temperatures minus the monthly minimum temperatures), “isothermality” (bio3, mean diurnal range/(max temperature of warmest month-min temperature of coldest month)×100), “mean temperature of wettest quarter” (bio8), “annual precipitation” (bio12), and “precipitation seasonality” (bio15, coefficient of variation), elevation, aspect and slope. to estimate the influence of global climate change on the potential distribution of the species, we modeled the distribution for three different time slices: present, 2050 and 2070. the climate data was available from the worldclim data (http//www.worldclim.org/bioclim). due to the large effect of different atmosphere global circulation models (agcms) in species range projections (diniz-filho et al., 2009), we selected three different agcms (bcc-csm1-1, access1-0 and ipsl_cm4) for each time slice with each climate models involving two future emissions scenarios developed by ipcc’s fifth assessment report (rcp4.5 and rcp8.5) (http//www.worldclim.org/bioclim). the selected agcms have different equilibrium climate sensitivity values ranging from 0.9 °c to 4.8 °c. maximum entropy modeling (maxent) is a useful method to simulate the potential habitat redistribution under climate change, due to high predictive accuracy and strong stability (phillips et al., 2006; steven et al., 2006; wisz et al., 2008). we used a maximum entropy approach to model climatically suitable areas of bullfrogs in china using maxent 3.3.3e (www. cs.princeton.edu/~shapire/maxent), and we validated the model using a cross-fold approach (hijmans, 2012). we randomly selected 75% of bullfrog records for model training (bourke et al., 2017) and the remaining 25% for model testing, with a logistic output format ranging from 0 (unsuitable environmental conditions) to 1 (optimal) (values near 0.5 representative of average habitat quality; phillips and dudík, 2008). jackknife tests were run to measure variable importance (phillips et al., 2006). in addition, a bias file was included in the run to represent sampling effort to reduce the sampling bias and increasing speed (young et al., 2011). the accuracy of the model was evaluated by using the area under the receiver operating characteristic curve called auc (swets, 1988), commonly recognized as the optimal model prediction since it is unaffected by the threshold value and insensitive to incidence of species (fielding and bell, 1997). auc scores quantify the sdm’s ability to differentiate between random prediction (auc = 0.5) and perfect identification of suitable grid cells (auc = 1.0) (hanley and mcneil, 1982; phillips et al., 2006; wang et al., 2007). after converting the maxent output avg.asc into raster format, we reclassified the results of maxent with thresholds in arcgis (lu et al., 2012) and divided the suit bal environmental conditions into 4 levels based on the fitness index size (wang et al., 2007; zhai and li, 2012) with 89potential effects of climate change on the distribution of invasive bullfrogs lithobates catesbeianus in china low potential (< 0.2), moderate potential (0.2-0.4), good potential (0.4-0.6), high potential (> 0.6) (yang et al., 2013). to test for possible differences of the predicted distribution under different climate scenarios, each out of the twelve maps was compared to the current distribution map using map comparison kit software (version 3.2.3; mck, 2017) and an overall similarity index was computed between a map pair. we applied the “fuzzy numerical” algorithm as these maps were numerical (visser and de nijs, 2006; falaschi et al., 2018). results we obtained a good sdm performance with an average test auc value of 0.867, which indicated that the prediction has high credibility. analysis of variable contributions revealed that the “annual precipitation” had the highest explanative power, explaining 34.7% of the variation, followed by “mean diurnal range” (33.9%), “elevation” (20.4%) and “annual mean temperature” (3.1%), suggesting that the geographical distribution of bullfrog was most affected by these four factors. the results from maxent analysis showed at present there were many areas unsuitable for habitation by bullfrogs: inner mongolia, gansu, qinghai and tibet. overall, mainly the center, east, southeast and the southwest of china were suitable area of bullfrog survival, with a small number of suitable areas in xinjiang, ningxia, jilin, liaoning and heilongjiang (fig. 1). the auc values were above 0.8 in all of the models, indicating that the prediction results have high credibility. generally, climatically suitable areas may become narrower as the invasion begins to retract in the southeast coastal the north of the north china plain, sichuan basin and the middle and lower reaches of the yangtze river (fig. 2; table 1). only minor differences were observed in model projection onto climate change scenarios derived from bcc-csm1-1, access1-0 and ipsl_cm4 (fig. 3; table 1), and these differences and similarities were also confirmed by the fuzzy numerical comparison performed in mck: similarity maps (fig. 4) showed only slight differences between current distribution map and these future distribution maps with the similarity index rose from 0.552 to 0.773. discussion we investigated the current potential and future distribution for bullfrogs under different climate change scenarios. the results show that under the current climatic conditions, bullfrogs have a wide range of potential distribution in china, located in the center, east, southeast and southwest china, with only a small number of suitable areas in north china including xinjiang, ningxia, liaoning, jilin and helongjiang. generally, our models also revealed that global climate change is likely to shrink slightly the extent of suitable habitat under future scenarios. compared to ficetola et al. (2007), who found that bullfrogs are mainly distributed in eastern china, our study results extend its distribution area to central china, with a few locations in the west and northeast china, which may represent new invasion areas. this can be explained by the facts that some new invasion sites have been found in china recently (fei et al., 2012). the current distribution pattern of bull frogs in china can mainly be explained by precipitation and temperatures. previous study also showed that bullfrog presence seems to be positively related to precipitation (ficetola et al., 2007). the availability of water (including the presence of permanent wetlands) for breeding are commonly recorded important environmental features needed for the presence of bullfrogs (maret et al., 2006) and their tadpoles’ growth, development and metamorphosis (govindarajulu et al., 2006). in addition, mean diurnal range also influences the distribution of bullfrogs. this is also similar to the results from ficetola et al. (2007) and, indeed, bullfrog is a ‘warm-adapted species’ (bachmann, 1969; harding, 1997). besides, previous studies showed that the current distribution of bullfrogs in china is also explained by 1) the proximity to the frogfarms, from where bullfrogs can escape: most of the bullfrog farming in china is surrounded by highly suitable habitats, and the frogs can establish wild population there (wu et al., 2004; li and xie, 2004); 2) the abandonment/release of bullfrogs mainly by religious groups, which also led to the establishment of new wild population, e.g., in yunnan fig. 1. map of the suitable distribution of bullfrog in china (present). 90 li qing peng et alii and sichuan (wu et al., 2004; li and xie, 2004). as shown by the fuzzy numerical comparison performed in mck, slight differences between current and future distribution maps have been observed. also, projecting bullfrogs’ climatically suitable areas on future climate change scenarios (rcp4.5 and rcp8.0) indicated that climatically suitable areas will become narrower in china. the potential habitats of bullfrogs in china will retreat to the most suitable area including the north of the north china plain, sichuan basin and the middle and lower reaches of the yangtze river (fig. 2), where bullfrog farming is particularly common (fei et al., 2012). biological invasions are complex and the potential habitat distribution is determined by a variety of factors (li et al., 2009). in this study, we only considered the effect of the climate and terrain, but we did not consider the effect of the other factors including the vegetation cover, biotic interactions with other species, species fig. 2. maps of the potential suitable distribution of bullfrog in china in 2050 and 2070. 91potential effects of climate change on the distribution of invasive bullfrogs lithobates catesbeianus in china migration capacity, species evolutionary adaptations, and human exploitation of wild populations, on the potential distribution of the bullfrog. if these factors were fully considered, the predicted results could have been more closely related to the current distribution of species (graham and hijmans, 2006). to effectively prevent further invasions of bullfrogs in china, management policies should be more pragmatic, preventing new introductions within suitable habitats and eradicating populations when possible. based on the predictions on bullfrog potential habitats from sdms, the authorities should consider the model results to focus the management strategies on these potentially sensitive regions. in addition, authorities should tighten control of bullfrog farming to prevent their escape. in addition, frog factories could be moved to areas which are surrounded by unsuitable habitats of bullfrogs, which would reduce a lot the possibility of survival of escaped captive individuals. acknowledgments we thank litao gan, kejun hua and xuli ren for assistance in the field, and rocco tiberti, marco mangiacotti and anonymous reviewers for their kind suggestion. this study was funded by the natural sciences foundation for distinguished young scholar of sichuan (grant number 2016jq0038), key foundation of sichuan provincial department of education (grant number 18za0255) and the national sciences foundation of china (grant number 31670392). references araújo, m.b., thuiller, w., pearson, r.g. 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(2005): food comparison between tadpoles of rana catebeiana and r. chaochiaoensis from the same habitat. zool. res. 26: 89-95. acta herpetologica vol. 15, n. 2 december 2020 firenze university press estimating abundance and habitat suitability in a micro-endemic snake: the walser viper gentile francesco ficetola1,2,*, mauro fanelli3, lorenzo garizio3, mattia falaschi1, simone tenan4, samuele ghielmi5, lorenzo laddaga6, michele menegon7,8, massimo delfino3,9. potential effects of climate change on the distribution of invasive bullfrogs lithobates catesbeianus in china li qing peng1, min tang1, jia hong liao1, hai fen qing1, zhen kun zhao1, david a. pike2, wei chen1,* a bibliometric-mapping approach to identifying patterns and trends in amphibian decline research claudio angelini1,*, jon bielby2, corrado costa3 food composition of a breeding population the endemic anatolia newt, neurergus strauchii (steindachner, 1887) (caudata: salamandridae), from bingöl, eastern turkey kerim çiçek1,*, mustafa koyun2, ahmet mermer1, cemal varol tok3 stomach histology of crocodylus siamensis and gavialis gangeticus reveals analogy of archosaur “gizzards”, with implication on crocodylian gastroliths function ryuji takasaki1,2,*, yoshitsugu kobayashi3 does chronic exposure to ammonium during the pre-metamorphic stages promote hindlimb abnormality in anuran metamorphs? a comparison between natural-habitat and agrosystem frogs sonia zambrano-fernández1, francisco javier zamora-camacho2,3,*, pedro aragón2,4 confirming lessona’s brown frogs distribution sketch: rana temporaria is present on turin hills (piedmont, nw italy) davide marino1, angelica crottini2, franco andreone3,* phylogenetic relationships of the italian populations of horseshoe whip snake hemorrhois hippocrepis (serpentes, colubridae) francesco paolo faraone1, raffaella melfi2, matteo riccardo di nicola3, gabriele giacalone4, mario lo valvo5* first karyological analysis of the endemic malagasy phantom gecko matoatoa brevipes (squamata: gekkonidae) marcello mezzasalma1,2,*, fabio m. guarino3, simon p. loader1, gaetano odierna3, jeffrey w. streicher1, natalie cooper1 notes on sexual dimorphism, diet and reproduction of the false coral snake oxyrhopus rhombifer duméril, bibron & duméril, 1854 (dipsadidae: pseudoboini) from coastal plains of subtropical brazil fernando m. quintela1,*, felipe caseiro¹, daniel loebmann¹ acta herpetologica 14(2): 135-139, 2019 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/a_h-7752 morphological variation of the newly confirmed population of the javelin sand boa, eryx jaculus (linnaeus, 1758) (serpentes, erycidae) in sicily, italy francesco p. faraone1,*, salvatore russotto2, salvatore a. barra3, roberto chiara3, gabriele giacalone4, mario lo valvo3 1 viale regione siciliana s.e., 532, 90129 palermo, italy 2 contrada grassura mollaka faia, s.n., 92027 licata (ag), italy 3 dipartimento scienze e tecnologie biologiche, chimiche e farmaceutiche, university of palermo, via archirafi, 18, 90123 palermo, italy 4 cooperativa silene, via d’ondes reggio, 8/a, 90127 palermo, italy *corresponding author. email: paolofaraone@libero.it submitted on: 2019, 13th may; revised on: 2019, 20th june; accepted on: 2019, 29th june editor: dario ottonello abstract. the presence of the javelin sand boa in sicily has recently been confirmed. here the morphological characters and sexual dimorphism of the sicilian population of eryx jaculus are presented. seven meristic and six metric characters in 96 specimens from sicily were examined. the results show that tail length, snout-vent length, the distance between nostrils and the number of ventral and subcaudal scales are different between sexes. the characters found in the sicilian population of the javelin sand boa resemble those of the african population (ssp. jaculus) rather than the eurasian population (ssp. turcicus), but biomolecular studies are necessary to understand its taxonomic identity. keywords. eryx jaculus, serpentes, morphological variation, folidosis. the javelin sand boa eryx jaculus (linnaeus, 1758), a member of the family erycidae, is found in the southern balkans, middle east and north africa (sindaco et al., 2013). two morphologically defined subspecies are usually recognized for this species. the nominate subspecies is present in north africa and the ssp. turcicus (olivier, 1801) is found in the balkans and the middle east (tokar and obst, 1993; sindaco et al., 2013; geniez, 2015). according to tokar and obst (1993), the transcaucasian ssp. familiaris eichwald, 1831 could be synonymous with e. j. turcicus. morphological variations of e. jaculus have been reported both at a global (tokar, 1991) and a local scale (cattaneo, 1984, 2005, 2010; rhadi et al., 2015; zarrintab et al., 2017; eskandarzadeh et al., 2018). recently, the presence of e. jaculus has been confirmed in a small area of southern sicily, in italy (insacco et al., 2015). knowledge of the javelin sand boa in sicily is currently limited to scant preliminary information on the population’s distribution, morphology, habitat and diet (insacco et al., 2015; faraone et al., 2017a, b). in this paper the first comprehensive report of the morphological characters and sexual dimorphism in the sicilian population of e. jaculus is presented. sampling was carried out within the currently known geographic range of the javelin sand boa in south central sicily, within an area between palma di montechiaro (province of agrigento) and butera (province of caltanissetta) (see insacco et al., 2015; faraone et al., 2017b). a total of 55 adult (30 males; 25 females) and 41 juvenile (21 males; 136 francesco p. faraone et alii 17 females; 3 undetermined) specimens were examined between august 2014 and september 2018. among them, 47 specimens were collected during active searches at night, 12 specimens were rescued from abandoned cisterns and the tunnel greenhouse in which they were trapped, four were found dead inside cisterns, one had been killed by domestic cats and 32 specimens were roadkilled. sex was determined by examining external sexual features (spurs, tail shape) and, mainly in young snakes, also by cloacal popping. the reliability of external features for sexual determination was also confirmed by cloacal probing in dead specimens. in absence of detailed references, e. jaculus with a snout-vent length < 270 mm (see itescu et al., 2018; eskandarzadeh et al., 2018) were considered as juveniles. seven meristic characters were considered: the number of scales between the eyes (be), around the eye (re), posterior to the internasal (pin), between the eye and the nasal (ben), the rows of scales between the eye and the supralabial area (bes), the ventrals (vs) and the subcaudals (scds). six metric characters were also recorded: snout-vent length (svl), tail length (tl), the distance between the eyes (dbe), the distance between the posterior edge of the eye and the tip of the snout (des), the distance between the nostrils (dbn) as well as body weight (w). for bilateral characters, only the right side was recorded. biometric characters were measured using a digital caliper accurate to 0.01 mm, except for svl, which was measured with a mm ruler, and body weight (w), which was measured only on live individuals using a digital scale with 1 g precision. in order to remove the information related to size, each metric variable was transformed using the formula: z =yi (svl/ svli )b following lleonart et al. (2000), where z is the transformed value of the variable y, which is the variable affected by size, represented as the snout-vent length (svl), and b is the slope of the linear regression between logy and logsvl. the data were checked for parametric assumptions using levene’s test and the shapiro-wilk test. for each variable, parametric (independent t-test) or non-parametric (mann-whitney u test) analysis was conducted, with a level of significance at 0.05. differences between the sexes were checked by using the principal component analysis (pca), applied to the correlation matrix. body weight (w) was not included in pca, because its value can vary greatly depending on the condition of each specimen (pregnancy, decay, freshly ingested prey, etc.). svl and the characters with a p value greater than 0.05 were also excluded. color morph was classified according to the three types reported by tokar (1991) and tokar and obst (1993). the ‘discrete’ morph is characterized by a reduced dorsal pattern, with narrow transverse dorsal bars and small scattered spots on the sides. in the ‘standard’ morph, the dorsal pattern is more extensive than in the ‘discrete’ morph and the dorsal blotches are spaced by light areas of similar thickness and are not in contact with each other. the ‘negative’ morph is characterized by a greater development of the dark pattern, while the light parts are reduced to narrow suboval blotches on the back. the descriptive statistic and a comparison between sexes were made for adults (table 1). significant intersexual differences in five characters were found: the numtable 1. descriptive statistics and univariate comparisons between sexes of the sicilian population of eryx jaculus. the first five characters are in millimetres, the sixth in grams and the others are meristic. significant differences (p<0.05) are shown in bold. *= transformed values.     males females t u p mean ± sd range n mean ± sd range n svl 342.1 ± 42.4 270.0 420.0 26 400.5 ± 86.1 270.0 580.0 23 3.2 0.004 *tl 40.7 ± 6.0 26.9 53.9 26 31.4 ± 6.2 20.7 45.9 23 7.9 0.000 *des 7.1 ± 0.7 5.8 8.7 26 7.6 ± 1.2 5.9 10.5 21 1.8 0.086 *dbn 4.0 ± 0.4 3.3 4.7 26 4.2 ± 0.6 3.3 5.6 21 2.4 0.019 *dbe 7.0 ± 0.7 6.1 8.3 26 7.5 ± 1.1 5.5 10.0 21 165.0 0.194 w 36.9 ± 19.5 18 95 14 64.2 ± 49.6 15 – 204 16 76.0 0.135 vs 175.9 ± 2.8 170 182 26 181.9 ± 3.9 176 190 21 6.1 0.000 scds 25.2 ± 1.9 22 29 29 19.7 ± 1.4 17 22 23 11.8 0.000 pin 2.6 ± 0.5 2 3 30 2.6 ± 0.5 2 3 24 357.0 0.958 be 6.8 ± 0.6 6 8 30 6.8 ± 0.4 6 7 24 358.0 0.972 re 9.4 ± 0.8 8 11 29 9.0 ± 0.7 8 10 22 238.5 0.126 ben 3.0 ± 0 3 3 30 3.0 ± 0 3 3 24 bes 1.0 ± 0.2 1 2 30 1.0 ± 0 1 1 24 348.0 0.835 137morphological variation of eryx jaculus in sicily ber of ventral scales and snout-vent length have higher values in females and the number of subcaudal scales, the distance between the nostrils and the tail length have higher values in males. the result of the principal components analysis (pca) shows that 85.1% of the variance is explained by the first two components (pc1 = 58.1%, pc2 = 27.0%). the scatterplot of scores with the equiprobability ellipses at 95% (lagonegro and feoli, 1985) projected on the first two principal components (fig. 1) shows a clear separation between sexes on pc1, with males distributed within the negative values of the axis and the females within the positive values. the correlation coefficients of the variables (table 2) show that pc1 has a strong positive correlation with ventral scales (vs) and a negative correlation with subcaudal scales (scds) and tail length (tl). the examined specimens showed different color patterns (fig. 2). in most of the snakes, the ‘standard’ morph was identified, however in some individuals the dorsal dark blotches were so extended that they appeared similar to the ‘negative’ morph, yet these spots were not connected on the sides, so they were assigned to the ‘standard’ type. seven snakes could generally be attributed to the ‘negative’ phenotype, but some small parts of their back looked like the ‘standard’ morph, similar to what was observed in some e. jaculus by tokar and obst (1993) and werner (2016). young individuals were generally lighter than adults, with a lower contrast in the dark pattern. no difference in dorsal color pattern was noticed between sexes. the dorsal base color is orange, sand or yellowish. the ventral, paraventral and some adjacent scales were usually pale yellowish orange (in only one large female it was a whitish-cream color). ventral parts were adorned with small blackish spots. the orange color of the belly in a few cases extended to the subcaudal scales, which usually appeared whitish with a few scattered orange scales. the results show clear intersexual differences in the sicilian population of e. jaculus. the females usually reach a greater size in terms of snout-vent length and have a greater number of ventral scales than males, and males have a greater number of subcaudal scales, a longer tail and a greater distance between the nostrils. significant intersexual differences in ventral scales have already been observed in this species (tokar, 1991) but are not found in some asian populations (rhadi et al., 2015; eskandarzadeh et al., 2018) nor in some other eryx species (al-sadoon and al-otaibi, 2014; eskandarzadeh et al., 2013). the pattern of sexual dimorphism observed in the sicilian population is also reported in other species of snakes (marques et al., 2006, pizzatto et al., 2007, zanella and cechin, 2010, siqueira et al., 2013, manjarrez et al., 2014). larger females can provide more space for incubating eggs and embryos and more body reserves to fig. 1. scatterplot of scores with the equiprobability ellipses at 95% projected on the first two principal components between sexes of the sicilian population of eryx jaculus. fig. 2. examples of color and pattern variation in the sicilian population of eryx jaculus. (a) adult, male (b) adult, female (c) juvenile, male (d) ventral variation in three adult females. table 2. correlation coefficients of the variables for the first two factors used in the principal component analysis (pca). variables pc 1 pc 2 tl -0,81 0,52 dbn -0,40 -0,88 vs 0,81 0,16 scds -0,92 0,07 138 francesco p. faraone et alii invest in reproduction (bonnett et al., 1998) and generally, have a larger clutch size (shine, 1993) and lower mortality in their offspring (kissner and weatherhead, 2005). moreover, in various snake species the number of ventral scales corresponds to the number of dorsal vertebrae, which are generally more numerous in the larger sex (shine, 2000). on the other hand, smaller males have greater agility during the search for females and a lower metabolic cost (rivas and burghardt, 2001). a larger tail and a greater number of subcaudal scales in males are linked to the positioning of the hemipenes within the tail and produce better performance in the courtship phase (king, 1989; luiselli, 1996; shine et al., 1999). the males of sicilian e. jaculus also have a relatively larger internasal distance (dbn) than the females. sexual dimorphism in head shape is a phenomenon known in various snake species and is often linked to a different trophic niche between males and females (camilleri and shine, 1990; vincent et al., 2004). this result deserves to be investigated using appropriate tools (geometric morphometric analysis) and its relation to the dietary habits of this population, which is currently being studied, should be taken into account. the three dorsal patterns proposed by tokar (1991) should be used with caution. it is sometimes difficult to apply these patterns to specimens with mixed phenotypes (see tokar and obst, 1993) that may be locally common (werner, 2016). the subspecies of e. jaculus, proposed by tokar and obst (1993), is currently accepted (sindaco et al., 2013; werner, 2016). the criteria proposed by tokar and obst (1993) are based on an in-depth revision of the morphological variation of e. jaculus within its geographical range (tokar, 1991) and have been confirmed by recent research on a local scale (eskandarzadeh et al., 2018; rhadi et al., 2015; zarrintab et al., 2017). comparing the characters reported by tokar and obst (1993) with those of the examined specimens in this study, a higher resemblance between the sicilian population and the north african nominal subspecies, rather than with the eurasian ssp. turcicus, was detected. in the sicilian population, as well as in e. j. jaculus, there are usually seven scales between the eyes (74.7%; n = 95) (usually six in e. j. turcicus) and three postinternasal scales (64.6%; n = 96) (usually two in e. j. turcicus). moreover, the sicilian ventral scales range (170-190) overlaps with both the nominal subspecies (170-198) and ssp. turcicus (161-200). furthermore, a more frequent presence of the ‘standard’ phenotype (92.7%; n = 96) is observed with respect to the ‘negative’ and ‘discrete’ dorsal patterns, which are very common in european and asian populations respectively (see also tokar, 1991). the morphological affinities between the sicilian and african populations reported here has only a preliminary value and, obviously, a biomolecular approach is necessary in order to properly assess the phylogeographic and taxonomic structure of the javelin sand boa. acknowledgements we are grateful to agostino cantavenera, matteo di nicola, angelica rallo, alex venutelli for their help in fieldwork. we also thank edoardo razzetti for kindly providing us some interesting bibliographic references and viviana tinnirello for language revision. the temporary capture and handling have been carried out with the permits established by law (matt prot. n.2766 / t-a31, 12/01/2018 and regione siciliana prot. n, 1637, 24/01/2018). references al-sadoon, m.k., al-otaibi, f.s. 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(2000): vertebral numbers in male and female snakes: the roles of natural, sexual and fecundity selection. j. evolution. biol. 13:455-465. shine, r.g., olsson, m.m., moore, i.t., lemaster, m.p., mason, r.t. (1999): why do male snakes have longer tails than females? proc. r. soc. lond. b. biol. sci. 266: 2147-2151. sindaco, r., venchi, a., grieco, c. (2013): the reptiles of the western palearctic. 2. annotated checklist and distributional atlas of the snakes of europe, north africa. middle east and central asia, with an update to the, vol 1, edizioni belvedere, latina. siqueira, d.m., nascimento, l.p., montingelli, g.g., santos-costa, m.c. (2013): geographical variation in the reproduction and sexual dimorphism of the boddaert’s tropical racer, mastigodryas boddaerti (serpentes: colubridae). zoologia 30: 475-481. tokar, a.a. (1991): a revision of the subspecies structure of javelin sand boa, eryx jaculus (linnaeus, 1758) (reptilia, boidae). herpetological researches 1: 18-41. tokar, a., obst, f.j. (1993): eryx jaculus westliche sandboa. in: handbuch der reptilien und amphibien europas, band 3/i., schlangen (serpentes) i, pp. 35-54. böhme,w., ed., aula-verlag, wiesbaden. vincent, s.e., herrel a., irschick, d.j. (2004): sexual dimorphism in head shape and diet in the cottonmouth snake (agkistrodon piscivorus). j. zool., lond. 264: 53-59. werner, y.l. (2016): reptile life in the land of israel. edition chimaira, frankfurt am main. zanella, n., cechin, s.z. (2010): reproductive biology of echinanthera cyanopleura (serpentes: dipsadidae) in southern brazil. zoologia 27: 30-34. zarrintab, m., milto, k.d., eskandarzadeh, n., zangie, b., jahane, m., gholi kamif, h., rastegar-pouyani, n., rastegar-pouyani, e., rajabizadeh, m. (2017): taxonomy and distribution of sand boas of the genus eryx daudin, 1803 (serpentes: erycidae) in iran. zool. middle east 63: 117-129. acta herpetologica vol. 14, n. 2 december 2019 firenze university press podarcis siculus latastei (bedriaga, 1879) of the western pontine islands (italy) raised to the species rank, and a brief taxonomic overview of podarcis lizards gabriele senczuk1,2,*, riccardo castiglia2, wolfgang böhme3, claudia corti1 substrate type has a limited impact on the sprint performance of a mediterranean lizard pantelis savvides1,*, eleni georgiou1, panayiotis pafilis2,3, spyros sfenthourakis1 coping with aliens: how a native gecko manages to persist on mediterranean islands despite the black rat? michel-jean delaugerre1,*, roberto sacchi2, marta biaggini3, pietro lo cascio4, ridha ouni5, claudia corti 3 pit-tags as a technique for marking fossorial reptiles: insights from a long-term field study of the amphisbaenian trogonophis wiegmanni pablo recio, gonzalo rodríguez-ruiz, jesús ortega, josé martín* occurrence of batrachochytrium dendrobatidis in the tensift region, with comments on its spreading in morocco ait el cadi redouane1, laghzaoui el-mustapha1, angelica crottini2, slimani tahar1, bosch jaime3,4, el mouden el hassan1,* hematological parameters of the bolson tortoise gopherus flavomarginatus in mexico cristina garcía-de la peña1,*, roger iván rodríguez-vivas2, jorge a. zegbe-domínguez3, luis manuel valenzuela-núñez1, césar a. meza herrera4, quetzaly siller-rodríguez1, verónica ávila-rodríguez1 ontogenetic and interspecific variation in skull morphology of two closely related species of toad, bufo bufo and b. spinosus (anura: bufonidae) giovanni sanna visible implant alphanumeric (via) as a marking method in the lesser snouted treefrog scinax nasicus andrea caballero-gini1,2,3,*, diego bueno villafañe2,3, lía romero2, marcela ferreira2,3, lucas cañete4, rafaela laino2, karim musalem2,5 morphological variation of the newly confirmed population of the javelin sand boa, eryx jaculus (linnaeus, 1758) (serpentes, erycidae) in sicily, italy francesco p. faraone1,*, salvatore russotto2, salvatore a. barra3, roberto chiara3, gabriele giacalone4, mario lo valvo3 variability in the dorsal pattern of the sardinian grass snake (natrix natrix cetti) with notes on its ecology enrico lunghi1,2,3,4,*, simone giachello5, manuela mulargia6, pier paolo dore7, roberto cogoni8, claudia corti1 estimating abundance of the stripeless tree-frog hyla meridionalis by means of replicated call counts federico crovetto, sebastiano salvidio, andrea costa* at-rich microsatellite loci development for fejervarya multistriata by illumina hiseq sequencing yan-mei wang, jing-yi chen, guo-hua ding*, zhi-hua lin acta herpetologica 16(1): 27-36, 2021 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-9970 reptile diversity in a mediterranean wetlands landscape (alto guadalquivir region, southeastern spain): are they affected by human impacts? arancha de castro expósito1, enrique garcía-muñoz1, francisco guerrero1,2,* 1 departamento de biología animal, biología vegetal y ecología. campus de las lagunillas, s/n, 23071 jaén, spain 2 centro de estudios avanzados en ciencias de la tierra, energía y medio ambiente. campus de las lagunillas, s/n, 23071 jaén, spain * corresponding author. e-mail: fguerre@ujaen.es submitted on: 2020, october 30th; revised on: 2021, january 20th; accepted on: 2021, february 26th editor: daniele pellitteri-rosa abstract. this study was carried out to evaluate the diversity in reptile communities in wetland landscapes located in the mediterranean region. for this, the status of the reptile populations linked to different mediterranean wetlands in relation to the different types of land use established in the nearby drainage basins (500 m around the wetlands perimeter) was determined. the different types of land use were determined together with the presence/absence, abundance and size class of the different reptile species. the results showed that areas with high anthropic pressure had a lower diversity of species, as well as a less balanced community structure, that could put at risk the effective recruitment and hence the maintenance of the reptile populations in these areas. the reasons behind the decline in the reptile community are similar to those put forward for explaining the decline in amphibians in the same area. keywords. aquatic ecosystems, biodiversity crisis, mediterranean landscapes, reptiles decline. introduction species extinction is a natural process, but in recent times there has been a more general decline in biodiversity with a species extinction rate that is estimated to be higher than it might be expected (rockström et al., 2009; de vos et al., 2014). the decline in biodiversity has been reported by many authors, and different causes have been considered in order to understand the complexity of joining cause and effect, being human activities identified as a crucial threat (wake and vredenburg, 2008; pimm et al., 2014; ripple et al., 2019). in this process of loss of diversity, herpetological fauna is one of the groups with the highest risk of extinction (böhm et al., 2013; alroy, 2015). the report of the international union for conservation of nature (iucn, 2010) reflects this critical situation, indicating that 41% of the amphibians, and 28% of the reptiles evaluated are critically threatened. as with other vertebrate groups, the main reasons behind decline are climate change (foufopoulos et al., 2011), destruction and alteration of their habitats (lizana and barbadillo, 1997; wake and vredenburg, 2008), ecosystem pollution (sparling, 2003; sparling et al., 2015) or the introduction of allochthonous species (pleguezuelos, 2002), among others. in the iberian peninsula there are 56 species of reptiles (márquez and lizana, 2002). one of the most important threats to these species is the destruction of habitats, as a consequence of the mechanization of the agroecosystem since the industrial revolution (ceacero et al., 2007). the establishment of monocultures, due to intensive agriculture, has led to a homogenization of the ecosystem, characterized today by simplistic mosaic models, which has caused a reduction in biodiversity 28 a. de castro expósito, e. garcía-muñoz, f. guerrero and to the loss of natural habitats (guerrero et al., 2006; garcía-muñoz et al., 2010, 2016). th is simplifi cation of the agroecosystems has been widely linked to the excessive use of agrochemicals that are endangering many species (sparling et al., 2000; mann et al., 2009; böll et al., 2013; garcía-muñoz et al., 2019). in fact, many species of reptiles live in highly specifi c environments, have very restricted distributions and population growth to very slow rates (pleguezuelos et al., 2002). accordingly, the presence of some species of reptiles or the state of their populations will depend, among other factors, on the characteristics of the species and to what extent the habitats have been disturbed (pleguezuelos, 2002). th erefore, the aim of this research was to determine the status of the reptile populations in diff erent wetlands of the alto guadalquivir region (andalusia, south of spain), in connection to the diff erent types of land use established in the surrounding drainage basins. th e hypothesis in this study was that the wetlands in which a greater number of habitats were conserved in their drainage basins would have reptile populations with higher diversity and abundance compared to those in which intensive monoculture had been established. in order to test this hypothesis, we proceeded to (i) determine the diff erent types of land use in the drainage basins of each wetland; (ii) determine the presence/absence, abundance and size structures of the diff erent reptile species in each wetland under study; and (iii) determine how the conservation status of the wetland drainage basin infl uenced reptile communities. th e results from this research could enable us to propose conservation measures to be implemented for the reptile species present in the wetlands. materials and methods study area th e study was conducted in the alto guadalquivir (andalusia), a region located in the southeast of the iberian peninsula. th is area has a complex geological history and is ideal scenario for herpetological studies since it has both iberian-north african and baetic endemisms and species of atlantic origin (ceacero et al., 2007). th e study area is characterized by three geological units: the depression of the guadalquivir river with its tributaries, which has had a long history of anthropization, as well as two mountainous units, sierra morena to the north and baetic mountains ranges to the south and east of the province (vera, 1994). due in large part to this complex geological history, this last region displayed the highest species richness in terms of reptiles, with a total of 39 species, which is higher than the surrounding regions (salvador and pleguezuelos, 2002; ceacero et al., 2007). three types of wetlands can be classified based on the extent of the alteration of their drainage basins (garcía muñoz et al., 2010, 2016; gilbert et al., 2014, 2017): (i) those that have been greatly altered and have a very high level of human activities, situated in the valley of the guadalquivir river; (ii) those that have been partially conserved in the areas of the sierra morena and there is a low level of human activities; and (iii) those that have been conserved, such as the wetlands belonging to the baetic mountain ranges. in this study, nine wetlands representative of the three zones in the study area in the alto guadalquivir region have been investigated (fig. 1): sierra morena (ardal, tobaruela and santisteban), the guadalquivir valley (honda, brujuelocirueña and hituelo) and the baetic mountain ranges (castillo, siles and orcera). although we are aware that the number of wetlands per area is limited, the value of this study is precisely to address three diff erent areas and to be able to make comparisons between them. notwithstanding, since the selected wetlands are the most representative of each zone (garcía-muñoz et al., 2010; gilbert et al., 2014, 2015, 2017), in the future this study should be expanded to a greater number of wetlands. data collection for each area, a sampling buff er zone of 500 m from the shoreline of the wetland included in the drainage basin was established. we characterized the diff erent land uses in the area according to the habitat classifi cations of the spanish society of ornithology (seo, 2008). for each habitat category, the surface (in hectares) was obtained within qgis 2.14 soft ware environment (see table 1). a bibliographic review on the reptile species present in each wetland was also carried out (sref; pleguezuelos et al., 2002; ceacero et al., 2007; ahe database, 2016). fig. 1. geographical location of the study area in southern spain and the nine wetlands in the alto guadalquivir region. th ese wetlands are representative of the three zones presented in the study area: (i) sierra morena; (ii) the guadalquivir valley; and (iii) baetic mountain ranges. 29reptiles decline in mediterranean landscapes for field data collection (sobs), the same procedure was replicated (three times regarding each wetland) for all samplings from april to july 2016 (concerning the season in which there is the highest reptile activity on this area). different timeconstrained searches were used (see hutchens and deperno, 2009): (i) linear transects, covering all the land uses present in each drainage basin of each wetland, within the delimited area of study; (ii) standardized road searches during twilight hours; and (iii) sampling based on fixed points for 30 minutes in each one, with a more intensive search of possible refuges, such as stones, logs or abandoned farmhouses, in all the habitats that were in each wetland. all the individuals registered were identified, their abundance was counted, and each individual was classified into the three size ranges: juvenile, sub-adults and adults. the authors are aware that the detection of adults, subadults and juveniles is not the same. however, as the sampling strategy is the same and the detection level is similar between wetlands, we consider that the data obtained are totally valid at a comparative level between wetlands. for aquatic reptiles (mauremys leprosa, natrix maura and n. natrix) we chose to use a visual detection method and fixed points sampling (three fixed points on the shores of each wetland). attending to surface area of the ponds, we chose three points because the entire wetland could be observed as a whole and thus avoiding an underestimation of the aquatic species. this was made to avoid possible translocations of diseases among study wetlands, because there have been verified cases of chytridiomycosis in the wetlands of the baetic mountain ranges (bosch and gonzález-miras, 2012), and that reptiles may be potential vectors of this disease (kilburn et al., 2011). statistical analysis to evaluate the relationship between land uses and the reptile species in each wetland, multivariate analyses were carried out with the canoco program (v 5.03). first, a segmented correspondence analysis (dca: detrended correspondence analysis) was performed to calculate the gradient length. when the value of the gradient length is less than 1.5, as was our case, an rda is recommended (iturrondobeitia et al., 2004). the rda (redundancy analysis) is based on a constrainer that explains the variation in species composition by environmental variables (ramírez-gonzález, 2005). similarity percentage analysis (simper, primer v. 5.2) was subsequently used to identify the features of the species of each geographic group of wetlands (clarke and warwick, 2001): group 1 (sierra morena wetlands), group 2 (guadalquivir valley wetlands), and group 3 (baetic mountain ranges wetlands). both statistical analyses were carried out with all the species registered in the wetlands under study (stotal = sobs + sref). finally, the simpson’s dominance index (simpson, 1949) was calculated just using the observed abundance data (sobs) in the three types of wetlands previously mentioned. simpson’s index measures the probability that two individuals randomly selected from a sample will belong to the same species. diversity values can also be obtained after subtracting by one the value given in simpson’s dominance. results table 2 shows the fifteen species of reptiles that have been observed in the wetlands under study (sobs) and five more species that were within the study area following the bibliographic review (sref). the dca results showed that the three first axes explained 72.65% of the variation. the results of the multivariate rda analysis showed the relationship between land use and species (fig. 2). moreover, in the centre of the graph a group of more generalist species, such as malpolon monspessulanus, timon lepidus, psammodromus algirus or rhinechis scalaris could be seen; while other groups with more specialist species, such as vipera latastei, coronella girondica, natrix natrix or chalcides bedriagai, were located far from the centre of the graph. siles and orcera were locations in the plot that were farthest from rainfed crops, roads, olive groves and built-up land. therefore, they appeared as less degraded and more inaccessible. honda, santisteban, hituelo, tobaruela and brujuelo-cirueña wetlands were closely linked table 1. percentage land use in the study wetlands following the habitat proposed by seo (2008). habitat ardal tobaruela santisteban honda brujuelocirueña hituelo castillo siles orcera wetland surface area 4.32 1.20 3.37 10.49 4.46 3.86 0.15 0.32 0.03 olive crops area 1.128 40.77 12.03 131.36 122.26 102.30 24.67 --rainfed crops area 19.57 27.10 69.86 -12.21 -37.91 --roads and tracks length (m) 4821.1 5967.98 6745.08 4021.97 1785.43 2650.54 2584.13 1029.82 1937.59 urbanization area 22.58 17.33 8.92 --0.44 ---scrubland area -10.85 -6.87 3.33 -11.14 --holm oak and cork oak forest area 68.86 -----2.58 --pines forest and grassland area -------76.69 70.13 altitude (m a.s.l.) 400 363 637 446 458 476 780 1280 1270 30 a. de castro expósito, e. garcía-muñoz, f. guerrero to the olive groves and dry land crops, and a multitude of roads have been created in order to provide better accessibility for agricultural purposes. ardal wetland is mostly surrounded by oak forests but is also near built-up land because a few years ago a housing estate was built nearby. the results of the simper analysis (fig. 3) show the average contribution of each species in the three groups analysed. it can be seen how p. algirus, t. mauritanica, t. lepidus, r. scalaris, m. monspessulanus, n. maura and m. leprosa are the species that most contributed to the percentage of similarity within each group and among the three study groups. moreover, m. leprosa could be seen in 100% of the wetlands of groups 1 and 2, but in group 3 it could only be observed in castillo. vipera latastei is the species that most contributed to the dissimilarity in group 3 compared to the other ones (group 1-3: 13.24%, group 2-3: 10.64%). a. erythrurus is the species that most contributed to dissimilarity in group 1 (group 1-2: 13.41%; group 1-3: 7.2%) compared to the other ones, although a juvenile was also found in orcera. table 3 shows the results of the size structures in each wetland under study. in all of them an inverted pyramid has been observed, which showed there to be more adults than juveniles or sub-adults. in addition, in some wetlands, such as tobaruela, honda and brujuelo-cirueña, there did not appear to be any recruitment; while in other ones, for example, ardal, castillo, orcera and siles, the highest number of juveniles and sub-adults were found in comparison with the other wetlands under study (see table 3). the results of simpson’s dominance index (d) showed a gradient for group 1 (sierra morena; d = 0.501), with maximum dominance for group 2 (guadalquivir valley; d = 0.323) and group 3 (baetic mountain ranges; d = 0.250) with the maximum diversity value. discussion reptiles play important ecological roles contributing to the maintenance of environmental heterogeneity (kaczor and hartnett, 1990) having a keystone functions in the ecosystem structure (ashton, 2010). in fact, they tend to be strongly associated with local habitat quality and could be excellent bioindicators of the ecosystem conservation state (pianka, 1967; overmann and krajicek, 1995; crain and guillette, 1998; amaral et al., 2012). although extinction marks the end of any form of life, the speed at which a group reaches this point could table 2. presence registered in the field sampling (+, sobs), presence registered by bibliographic data (+*, sref) and absence (-) of the reptile species in the study wetlands. species ardal tobaruela santisteban honda brujuelocirueña hituelo castillo siles orcera acanthodactylus erythrurus +* +* + podarcis vaucheri +* +* + +* +* + psammodromus algirus + + + + + + + + + psammodromus hispanicus +* + + +* tarentola mauritanica + +* + + + + + + + timon lepidus + + +* + + +* + + + blanus cinereus +* + +* +* +* coronella girondica + hemorrhois hippocrepis +* +* + + +* +* malpolon monspessulanus +* + +* + + + + +* +* rhinechis scalaris +* + +* +* + +* +* +* +* chalcides striatus +* + +* +* + + chalcides bedriagai + + mauremys leprosa + + +* +* +* + + natrix maura +* +* +* +* +* +* +* +* vipera latastei +* +* +* macroprotodon brevis +* +* +* hemidactylus turcicus +* +* +* algyroides marchi +* +* natrix natrix +* richness 15 11 9 11 8 9 12 16 14 31reptiles decline in mediterranean landscapes increase due to the combination of causes and eff ects derived from the activity of other species. as expressed in the title of this paper, the current situation of reptiles in the wetlands of the south of iberia is reminiscent of the path taken by amphibians in the same study area (garcíamuñoz et al., 2010, 2016). th e simplifi cation of agroecosystems with extensive and intensive monocultures that is ever present nowadays, that impoverishes the natural ecosystem are leading to the extinction of species worldwide. in general terms, the species richness obtained is inversely related to the rise in monocultures (olive and cereal crops), both in terms of surface expansion and intensive production. as a result, the wetlands showed greater species richness in those areas (sierra morena and betic mountains) where there are fewer crops and greater inaccessibility. it can therefore be seen that wetlands related to olive groves, cereal crops, and built-up land are more accessible for humans, and, consequently, show a lower richness in reptiles. however, and eliminating the option of the existence of an intrinsic geographical gradient in the distribution of species in the study area, wetlands located in mountainous areas (baetic mountain ranges) showed greater species richness. as regards the diff erent presence of species in the wetlands investigated by the study (table 2), fi ve of them were found in all ecosystems. th ese species could be used in future research to develop diff erent biomarkers (oxidative stress, bioaccumulation, etc.) to compare the state of health of the ecosystems. however, these species could be resistance to disturbance and, hence, only indicators of severe stress. th erefore, the eff ects on the species most sensitive to disturbances (stenoic species), which have been shown to present a negative spatial response to fragmentation and disturbance of the landscape (devictor, 2008), must also be taken into account to allow early detection of degradation processes. other research carried out in the same study area showed how complex the interaction between human activity and nature was. specifically, garcía-muñoz and carretero (2013) studied the rate of water loss in two species of lizards (podarcis vaucheri and algyroides marchi) that coexisted in the same habitat but had diff erent physiological rates. minimal variations on a microecosystems level could have been related to their physiological capacities (such as their ability to lose water) that could have explained specifi c (local) extinctions (santos and cheylan, 2013; ferreira et al., 2016). th e establishment of a monoculture and the consequent ecological oversimplifi cation (e.g., loss of micro-habitats with diffig. 2. relationship of wetlands land uses and reptile species by rda. acanthodactylus erythrurus (ae), podarcis vaucheri (pv), psammodromus algirus (pa), psammodromus hispanicus (ph), tarentola mauritanica (tm), timon lepidus (tl), blanus cinereus (bc), coronella girondica (cg), hemorrhois hippocrepis (hh), malpolon monspessulanus (mm), rhinechis scalaris (rs), chalcides striatus (cs), chalcides bedriagai (cb), mauremys leprosa (ml), natrix maura (nm), vipera latastei (vl), macroprotodon brevis (mb), hemidactylus turcicus (ht), algyroides marchi (am), natrix natrix (nn). fig. 3. main species organization according to values of exclusivity. typical species to each group is represented in the exclusive zone of each circle, while species shared between two groups are represented with the union of two circles. th e species that appear in all three zones are in the intersection of the three circles (as: average similitude; ads average dissimilitude from simper analysis). 32 a. de castro expósito, e. garcía-muñoz, f. guerrero table 3. species observed (sobs) and number of individuals in the three size ranges in the studied wetlands. species ardal tobaruela santisteban honda brujuelocirueña hituelo castillo siles orcera number acanthodactylus erythrurus adults ----------subadults ----------juveniles --------1 1 podarcis vaucheri adults -----1 ---1 subadults --------1 1 juveniles ----------psammodromus algirus adults 7 21 -14 26 4 16 14 52 154 subadults --------1 1 juveniles 2 -------3 5 psammodromus hispanicus adults ----1 --37 -38 subadults ----------juveniles ----------tarentola mauritanica adults 4 -1 -5 3 2 1 1 17 subadults ---8 -----8 juveniles 1 --------1 timon lepidus adults 4 ---4 -4 5 6 23 subadults -1 -1 ---2 -4 juveniles ------1 2 2 5 blanus cinereus adults -1 -------1 subadults ----------juveniles ----------coronella girondica adults ----------subadults -------1 -1 juveniles ----------hemorrhois hippocrepis adults ----------subadults ---1 ------juveniles -----1 ---1 malpolon monspessulanus adults -3 -1 2 1 2 --9 subadults ------2 --2 juveniles ------1 --1 rhinechis scalaris adults -1 --2 ----3 subadults -2 -------2 juveniles ----------chalcides striatus adults --11 ----7 5 23 subadults -------1 1 2 juveniles --1 ----3 -4 chalcides bedriagai adults 1 ------5 -6 subadults ----------juveniles 1 --------1 mauremys leprosa adults ------20 --20 subadults -1 ---1 ---2 juveniles 1 -----1 --2 natrix maura adults 1 -12 1 2 9 2 --27 subadults -1 ----2 --3 juveniles --1 ---1 --2 total adults 17 26 24 15 40 15 44 69 64 314 subadults -5 -10 -1 2 4 2 24 juveniles 5 -2 --1 5 5 6 24 33reptiles decline in mediterranean landscapes ferent humidity ranges, as has occurred in the guadalquivir valley) could have brought about changes that gave rise to more complex extinction processes. this concept could have been linked with the facilitation theory, with abiotic facilitation to be specific (facilitation that is mediated through changes in the abiotic environment; wright et al., 2017), so that oversimplifications in the agroecosystem could lead to an insufficient number of habitats, which, in turn, would bring about an alteration in the species richness. in this respect, other studies on the state of conservation of the alto guadalquivir wetlands (gilbert et al., 2017) showed how intensive cultivation activities affected conservation of the diversity of wetlands. furthermore, oversimplification in ecosystems brought about by monocultures, not only hinders the number of species and individuals, but also reduces reptile recruitment. the disappearance of natural refuges as a consequence of the management methods inherent to monocultures (whose aim is to remove any objects from the agricultural area), the indiscriminate use of herbicides and insecticides (which eradicate natural refuges and food) and the bioaccumulation and bioamplification process (daley et al., 2014), could explain the lack of recruitment in these areas. this study was designed in order to find out what the current status of reptiles in the alto guadalquivir region was. however, more studies are necessary to determine what has led to this process of decline. in order to anticipate moves in the game of extinction, we need to consider a range of environmental strategies. the first measure proposed is linked to the theory of conservation biology (soulé, 1985; sagoff, 2013), by maintaining biodiversity, the structure and function of ecosystems, expanding protected areas in the sierra morena and the baetic mountain ranges. the second one lies within the context of the ecosystem management theory; that in the study area is related to integrated crop management to make monoculture more beneficial by recovering their biodiversity (rey-zamora et al., 2017). the latter measure increases heritage values, environmental sustainability and landscape values. moreover, it enables traditional landscape planning to be recovered. these changes in agricultural strategy have been more specifically linked to a reduction in erosion, a decrease in the use of toxic substances and to a rise in land-use heterogeneity. moreover, these actions could also promote conservation of the biodiversity of other related groups, such as amphibians (garcía-muñoz et al., 2010). in addition, the third measure for consideration could be to use biomarkers developed with the species found in all the wetlands, such as p. algirus, t. mauritanica, t. lepidus, m. monspessulanus and r. scalaris. on a final note, authors would like to indicate an issue of concern, which is the increasing popularity of leisure pursuits in areas of natural beauty where access is limited for most people. the rise in this phenomenon, without taking into account the negative effects they have on wildlife, could increase the likelihood of encountering reptiles that, apart from the aversion that these organisms arouse in some people, could lead to the re-emergence of threats to this group that have been forgotten and also give rise to new ones that have not yet been created. in the former we are referring to direct persecution of reptiles by humans (ceríaco, 2012) and in the latter the translocation of diseases such as chytriodiomycosis, which has been detected in baetic mountains (bosch and gonzález-miras, 2012). acknowledgements authors thank junta de andalucía for the permission to sample reptiles in alto guadalquivir wetlands (permits number: 61/cv716yb107/2016 sveenpp54/2016). we also thank two anonymous reviewers who have improved the final version of the manuscript. references ahe database – asociación herpetológica española (2016): www.herpetologica.es. 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(2017): the overlooked role of facilitation in biodiversity experiments. tree 32: 383-390. acta herpetologica 12(2): 175-180, 2017 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-21016 influence of desiccation threat on the metamorphic traits of the asian common toad, duttaphrynus melanostictus (anura) santosh mogali*, srinivas saidapur, bhagyashri shanbhag department of zoology, karnatak university, dharwad-580 003, karnataka state, india *corresponding author. e-mail: santoshmogali@rediffmail.com submitted on: 2017, 20th july; revised on: 2017, 6th august; accepted on 2017, 21th august editor: raoul manenti abstract. phenotypic plasticity of metamorphic traits, in response to desiccation threat, was studied in duttaphrynus melanostictus under laboratory conditions. newly hatched gosner stage 19 tadpoles were exposed to decreasing water levels (gradually or rapidly) up to the beginning of metamorphic climax (mc, gosner stage 42). the control group was reared in unchanging water levels. the tadpoles experiencing desiccation threat reached mc earlier than those reared in constant water levels and metamorphosed (gosner stage 46) at smaller body sizes. time to reach mc was comparable between the groups of tadpoles experiencing a gradual or rapid decrease in water levels but their size at the completion of metamorphosis varied. they emerged at a significantly smaller size under rapid desiccation threat compared to the gradual desiccation threat. impact on size at emergence was in proportion to the level of desiccation threat and this accelerated development and led to an early metamorphosis. the study shows the ability of d. melanostictus for developmental plasticity under adverse ecological conditions like the desiccation threat. keywords. duttaphrynus melanostictus, desiccation threat, phenotypic plasticity, metamorphic climax, metamorphic traits, toad tadpoles. introduction phenotypic plasticity is widespread in nature; it allows exploitation of different habitats and, to face unpredictable ecological conditions (relyea, 2002; pigliucci, 2005; miner et al., 2005; fusco and minelli, 2010). phenotypic plasticity is also encountered during the development of anuran amphibians that generally have complex life cycles comprising of an aquatic larval stage, and transformation into an adult-body shape after completing metamorphosis (newman, 1992; denver et al., 1998; relyea, 2002; miner et al., 2005). therefore, age and size at metamorphosis are important metamorphic traits in amphibians (wilbur and collins, 1973; wilbur, 1980; werner, 1986; smith, 1987). these traits are subject to variation in response to changes in both biological and non-biological factors present in the habitat. the biological factors that influence amphibian development include availability of food resources (travis, 1984; newman, 1998; laurila and kujasalo, 1999; enriquez-urzelai et al., 2013), predatory pressures (werner, 1986; skelly and werner, 1990; benard, 2004; mogali et al., 2011a, 2016), inter and intraspecific competitions, density (semlitsch and caldwell, 1982; newman, 1987; richter et al., 2009; mogali et al., 2016), and association with kin or non-kin (girish and saidapur, 1999, 2003). among non-biological factors, variations in water temperature (newman, 1989; hayes et al., 1993; maciel and juncá, 2009; tejedo et al., 2010) and risk of pond drying/desiccation (denver et al., 1998; brady and griffiths, 2000; székely et al., 2010; mogali et al., 2011b, 2016) are the two main factors influencing anuran metamorphosis. in transient water bodies, desiccation threat is serious and completion of metamorphosis before the ponds 176 santosh mogali et alii dry is obligatory. evidently, slow growth rates and/or prolonged larval periods in unpredictable hydroperiods of the ponds are sure to decrease the chances of tadpoles completing metamorphosis before the ponds dry (altwegg and reyer, 2003; johansson et al., 2005; wells, 2007). on the other hand, a hastened larval development can lower larval mortality, but it is invariably at the cost of growth resulting in a smaller size at metamorphosis that may have consequences in their later survival and reproductive success. smaller metamorphs have lower locomotory capacity (semlitsch et al., 1988; richterboix et al., 2006), lower tolerance to dehydration (newman and dunham, 1994), reduced resistance to parasites (goater, 1994), weaker immunity (gervasi and foufopoulos, 2008) lower juvenile survivorship (reques and tejedo, 1997; altwegg and reyer, 2003), and lower reproductive success (smith, 1987; scott, 1994). yet, when larval mortality risk increases due to pond drying an early metamorphosis may be favoured despite the costs associated with a smaller size. hence, a phenotypic plasticity involving the trade-off between certain life history traits (e.g., larval growth, duration of the larval period, size at transformation) is a useful strategy. the original models of amphibian metamorphosis that attempt to evaluate optimal size at transformation predict that in an aquatic environment when conditions are favourable for larval growth (i.e., in permanent or slowly desiccating ponds), tadpoles should delay metamorphosis and transform at a larger size (wilbur and collins, 1973; werner, 1986). but when conditions of the temporary ponds become precarious a strategy to adjust the developmental processes so as to metamorphose early and emerge on land is useful. a developmental strategy of phenotypic plasticity can reduce the exposition to risky conditions and thereby enhance the survival rate. in dharwad, many anuran species reproduce in rainfilled ephemeral water bodies formed during south-west monsoon. tadpoles living in such ponds face the perennial threat of desiccation due to the failure of intermittent monsoon showers (mogali et al., 2011b). the asian common toad, duttaphrynus melanostictus (earlier known as bufo melanostictus) breeds both in rain-filled ephemeral ponds and in cement cisterns within the parks holding water round the year (saidapur and girish, 2001). hence, d. melanostictus tadpoles offer an excellent model to study developmental plasticity in response to varying degrees of dropping levels of water (desiccation threat). therefore, the present study was designed to determine, in a laboratory set-up, the influence of gradual or rapid water depletion on the two key metamorphic traits, the larval duration, and size at emergence. we hypothesized that tadpoles facing the rapid depletion of water (high desiccation threat) would metamorphose earlier and at a smaller size than those developing in a gradual decline in water levels (low desiccation threat). importantly, the experimental design permitted us to exclude the influence of confounding factors such as food scarcity and predator pressure which generally interfere with the growth and development of these tadpoles in nature. material and methods four egg clutches of d. melanostictus were collected on 29 may, 2014 from rain-filled ponds in and around (within 2 km distance) the karnatak university campus (latitude 15.440407°n, longitude 74.985246°e). soon after collection, the eggs were brought to the laboratory and placed separately in plastic tubs (32 cm diameter and 14 cm deep) containing 5 l of aged (dechlorinated) tap water. all eggs hatched almost synchronously at gosner stage 19 (gosner, 1960) a day after their collection. soon after hatching, the tadpoles from different clutches were mixed and used for the experiment. tadpoles were picked randomly and were reared in the plastic tubs with 3 l of water until the onset of metamorphic climax stage (mc, gosner stage 42). fifteen such tubs with 20 tadpoles in each were maintained (in total 300 tadpoles, i.e., 100 tadpoles in each group). the experimental groups were as follows: i. control: tadpoles were reared in constant water levels (3 l). ii. gradual desiccation: tadpoles were reared in 3 l of water for the first 4 days and then subjected to 0.5 l decrease in water at 4 day intervals. iii. rapid desiccation: tadpoles were reared in 3 l of water for a day and from the second day onwards 0.25 l of water was reduced each day. in receding water groups when water reached 0.5 l (day 20 in group ii; day 10 in group iii) no further reduction was made. the groups ii and iii thus provided low and high desiccation threat. all tadpoles were fed on the boiled spinach ad libitum. water was changed on alternate days and fresh food was provided. the rearing tubs were placed on a flat surface in a room under natural photoperiod and temperature. the positions of tubs were randomized on an alternate day to avoid possible effects of position. the water temperature (°c) in tubs was recorded twice, daily at 10:00 h and 15:00 h. following the onset of metamorphic climax (mc, emergence of forelimbs, gosner stage 42), the subjects were transferred to small plastic tubs (19 cm diameter and 7 cm deep) covered with fine nylon mesh with a little water and, placed inclined to provide the semi-terrestrial environment to facilitate emergence. the days to reach mc were noted for each individual. after completion of metamorphosis (gosner stage 46), snout-vent length (svl in mm) and body mass (in mg) were recorded. two tadpoles in group i, and one tadpole each in groups ii and iii died during the course of the experiment. after completion of experiments, the toadlets were released near natural water bodies. data on days to reach mc, svl, body mass of toadlets, and water tem177influence of desiccation threat on toad tadpoles perature were analysed by one-way anova followed by tukey’s post-hoc test. data for each parameter were organized into frequency distribution to know the percentage of individuals falling within a particular dataset. results time taken to reach mc, and size at metamorphosis (svl, body mass) differed significantly between different groups (p < 0.001, table 1). tadpoles reared in declining water levels (groups ii and iii) reached mc earlier (p < 0.001) and metamorphosed at a smaller size (p < 0.001) than those reared in unchanging water levels (group i). further, tadpoles experiencing rapid depletion of water (group iii) metamorphosed at smaller sizes (p < 0.001) than those experiencing gradual depletion in water levels (group ii). however, number of days required for the onset of mc was comparable in both these groups (p = 0.937). the daily water temperature of various tubs fluctuated between 23-24 °c and as such did not differ significantly throughout the course of the experiments (morning: f2,86 = 0.298; p = 0.743; and afternoon hours: f2,86 = 0.281; p = 0.756). therefore, the effects of temperature, if any, were uniform across the control and experimental groups. the frequency distribution data showed that 78.78% of individuals from rapid desiccation group and 28.28% of individuals from gradual desiccation group metamorphosed (gosner stage 46) at ≤ 7.99 mm svl, but none subjected to constant water levels metamorphosed at comparable svl (fig. 1). further, 96.96% individuals in rapid desiccation group and 83.83% individuals in gradual desiccation group metamorphosed at a smaller body mass (≤ 69 mg) while only 12.24% of tadpoles reared in unchanging water levels (group i) metamorphosed at this low body mass (fig. 2). the data on table 1. snout-vent length (svl), body mass of metamorphs, and days required for the onset of metamorphic climax (mc; gosner stage 42) in duttaphrynus melanostictus reared in waters with different levels of desiccation. data represent mean ± se; n = 100 tadpoles for each group (300 tadpoles in total); dissimilar superscripts (a, b, c) indicate significant differences between the groups in the same column; significance level was set to 0.05. rearing groups svl (mm) body mass (mg) onset of mc (in days) i. control 9.42 ± 0.07a 85.89 ± 1.71a 26.00 ± 0.24a ii. gradual desiccation 8.24 ± 0.05b 58.34 ± 1.14b 24.28 ± 0.18b iii. rapid desiccation 7.62 ± 0.05c 50.00 ± 1.00c 24.18 ± 0.20b f value f = 265.328 f = 204.139 f = 24.166 p value p < 0.001 p < 0.001 p < 0.001 fig. 1. percent metamorphs of duttaphrynus melanostictus per snout-vent length class (mm) in different rearing groups fig. 2. percent metamorphs of duttaphrynus melanostictus per body mass class (mg) in different rearing groups fig. 3. percent tadpoles of duttaphrynus melanostictus per class (days) to reach metamorphic climax (mc; gosner stage 42) in different rearing groups 178 santosh mogali et alii days for onset of mc showed that 33.33% individuals reared in rapid desiccation group (with mean svl, 7.10 ± 0.06 mm and mean body mass, 40.78 ± 0.78 mg), 37.37% individuals from gradual desiccation group (with mean svl, 7.73 ± 0.05 mm and mean body mass, 47.08 ± 0.95 mg) took ≤ 23 days; while only 12.24% tadpoles reared in unchanging water levels initiated mc (with mean svl, 8.58 ± 0.03 mm and mean body mass, 64.84 ± 1.26 mg) by this same time (fig. 3). discussion amphibian metamorphosis is often characterized by the developmental phenotypic plasticity involving a trade-off between larval period and size at transformation. a risk of mortality in the larval environment increases due to two major factors like the predator pressure and desiccation threat as most anurans breed opportunistically in ephemeral ponds (laurila and kujasalo, 1999; lardner, 2000). the present study deals with one such factor, the desiccation threat, imposed by decreasing water levels in the rearing tubs. in nature, the threat of pond drying leads to early metamorphosis and transformation at a smaller, vulnerable body size (werner, 1986; rowe and ludwig, 1991; brady and griffiths, 2000; rudolf and rödel, 2007; mogali et al., 2011b). the present empirical study shows that tadpoles of d. melanostictus when subjected to low or high desiccation threat, in fact, accelerate their development and emerge on the land earlier than the control group. apparently, this is a strategy to escape mortality in the larval stage of development that occurs in water. in consistent with our predictions, the size of newly emerged toadlets was significantly small in response to the level of desiccation threat; those experiencing rapid desiccation threats emerged at a significantly smaller body size than those exposed to gradual water depletion. the former transformed at the smallest mean svl and body mass (table 1, fig. 1 and 2). we had hypothesized that tadpoles facing rapid desiccation threat may advance the onset of mc over those facing gradual desiccation threats. interestingly, there was no difference in the time taken for the onset of mc in both low or high desiccation risk groups; though these reached mc significantly early compared to the no desiccation risk group. the observations suggest that even though a relevant degree of plasticity can be adaptively present, a given species may need minimum threshold period to complete development. the present findings are however in good agreement with the view that anuran tadpoles from natural populations exhibit phenotypic plasticity in their growth and development when subjected to adverse ecological conditions (lind et al., 2008). it may be noted that some individuals in all the groups took ≤ 23 days to reach mc; others took a variable amount of time, as much as 33 days, especially in the group having no desiccation threat. these observations suggest that phenotypic plasticity response in the developmental rate may depend upon the severity of the ecological conditions and, genotypic variations among the group members. the minimum time taken to reach mc in certain individuals of different groups was 20 days; this period appears to be the minimum threshold time for onset of mc in the toad. the minimum period required to reach mc may also be species-specific and therefore differ in different species. the mechanisms proposed to explain the acceleration of metamorphosis in anuran tadpoles facing the threat of desiccation differ. elevated temperature (newman, 1992; tejedo and reques, 1994) or a decrease in food (alford and harris, 1988; newman, 1994) has been attributed to lowered growth rate with the accelerated developmental rate. yet other studies indicated that the temperature has no influence on developmental rate (loman, 1999; laurila and kujasalo, 1999; márquez-garcía et al., 2009). in the present study, there was no difference in water temperature of the rearing tubs in all groups and food provided was in excess quantity. hence, the major factor influencing accelerated metamorphosis of the toad tadpoles is the desiccation threat rather than temperature or scarcity of food availability. interestingly, individuals reaching mc by 23 days differed in their size between the groups. tadpoles in constant water level (group i) grew bigger than those reared in rapid and gradual depletion of water levels. further, individuals from gradual desiccation group were bigger than those in rapid desiccation group. these findings suggest that plasticity in the developmental rate of d. melanostictus is a normal feature and allows adjusting the growth of individuals in relation to extrinsic factors operating in their habitat, in this case, desiccation risk. attainment of a smaller size at the time of completion of metamorphosis in rapid desiccation group may be partly due to increased crowding and intraspecific competition among the group members. an earlier study on the toad tadpoles has shown that they metamorphose late and at a smaller size when raised in constant water (without any desiccation risk) but under crowded condition (saidapur and girish, 2001). the study also showed that under uncrowded conditions and in the absence of any desiccation threat the toad tadpoles extend the larval period and maximize their growth. it appears that the toad tadpoles are capable of assessing the level of desiccation risk and 179influence of desiccation threat on toad tadpoles adjust their developmental and growth rates. developmental plasticity is believed to be an attribute of the individual to generate different phenotypes depending upon the ecological conditions (pigliucci, 2005). according to theoretical models of plastic responses, natural selection will favour reaction norms that balance cost avoidance with resource acquisition; for example, the cost of maintaining a plastic response is expected to trigger the evolution of reaction norms that increase adaptation to more frequently changing environmental conditions (pigliucci, 2005). indeed, the tadpoles of the toad and several other anuran species that breed with the onset of monsoon rains in southern india, and frequently encounter unpredictable ecological challenges like the desiccation risk. therefore, observance of phenotypic plasticity in the development of the toad tadpoles in response to desiccation threat supports the above view. further, it is believed that genetic variations among the populations also play a role in the expression of plasticity (pigliucci, 2005). in our study, four parental lines were mixed creating more heterogeneity of the group members which may have actually lowered values of plastic response as the data represents an average response of pooled genotypes. therefore, further studies are needed to clarify the relationship between variation in genotype and expression of phenotypic plasticity in the toad population. acknowledgements smm is thankful to ugc’s dskpdf, new delhi and bas is thankful to insa, new delhi for support. this research was conducted according to ethical guidelines laid down by cpcsea, new delhi under registration no. 639/02/a/cpcsea. references alford, r.a., harris, r.n. 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(1973): ecological aspects of amphibian metamorphosis. science 182: 1305-1314. acta herpetologica vol. 12, n. 2 december 2017 firenze university press meristic and morphometric characters of leptopelis natalensis tadpoles (amphibia: anura: arthroleptidae) from entumeni forest reveal variation and inconsistencies with previous descriptions susan schweiger1, james harvey2, theresa s. otremba1, janina weber1, hendrik müller1,* brown anole (anolis sagrei) adhesive forces remain unaffected by partial claw clipping austin m. garner*, stephanie m. lopez, peter h. niewiarowski species and sex comparisons of karyotype and genome size in two kurixalus tree frogs (anura, rhacophoridae) shun-ping chang1,2, gwo-chin ma2,3,4, ming chen2,5,6,7,8,*, sheng-hai wu1,* non-native turtles in a peri-urban park in northern milan (lombardy, italy): species diversity and population structure claudio foglini1, roberta salvi2,* species composition and richness of anurans in cerrado urban forests from central brazil cláudia márcia marily ferreira1,*, augusto cesar de aquino ribas2, franco leandro de souza3 the life-history traits in a breeding population of darevskia valentini from turkey muammer kurnaz, alı i̇hsan eroğlu, ufuk bülbül*, halıme koç, bılal kutrup influence of desiccation threat on the metamorphic traits of the asian common toad, duttaphrynus melanostictus (anura) santosh mogali*, srinivas saidapur, bhagyashri shanbhag predation of common wall lizards: experiences from a study using scentless plasticine lizards jenő j. purger*, zsófia lanszki, dávid szép, renáta bocz reproductive timing and fecundity in the neotropical lizard enyalius perditus (squamata: leiosauridae) serena najara migliore1,2,*, henrique bartolomeu braz2,3, andré felipe barreto-lima4, selma maria almeida-santos1,2 observations on the intraspecific variation in tadpole morphology in natural ponds eudald pujol-buxó1,2,*, albert montori1, roser campeny3 and gustavo a. llorente1,2 reliable proxies for glandular secretion production in lacertid lizards simon baeckens diet of juveniles of the venomous frog aparasphenodon brunoi (amphibia: hylidae) in southeastern brazil rogério l. teixeira1, ricardo lourenço-de-moraes2, débora c. medeiros3, charles duca3, rogério c. britto4, luiz c. p. bissoli5, rodrigo b. ferreira3,* who are you? the genetic identity of some insular populations of hierophis viridiflavus s.l. from the tyrrhenian sea ignazio avella, riccardo castiglia, gabriele senczuk* acta herpetologica 15(2): 105-110, 2020 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/a_h-8370 food composition of a breeding population of the endemic anatolia newt, neurergus strauchii (steindachner, 1887) (caudata: salamandridae), from bingöl, eastern turkey kerim çiçek1,*, mustafa koyun2, ahmet mermer1, cemal varol tok3 1 zoology section, department of biology, faculty of science, ege university, 35100, izmir, turkey 2 department of biology, faculty of arts and sciences, bingöl university, 12000, bingöl, turkey 3 department of biology, faculty of arts and sciences, çanakkale onsekiz mart university, 17100, çanakkale, turkey *corresponding author. e-mail: kerim.cicek@ege.edu.tr submitted on: 2020, 19th march; revised on: 2020, 25th august; accepted on: 2020, 17th september editor: aaron m. bauer abstract. the study presents data on the food composition of a breeding population of the anatolia newt neurergus strauchii (steindachner, 1887), from bingöl, turkey. a total of 953 prey items were determined in the food contents of 46 individuals (18 males and 28 females) examined in the study. insecta (63%) and malacostraca (35%) constitute 97% of the food composition by number. while the most frequently encountered prey groups in the diet are diptera (87%), amphipoda (85%), neurergus eggs (74%), and coleoptera (37%), the comparison of food composition by volume is amphipoda (40%), neurergus eggs (41%), and diptera (10%) respectively. no significant difference in food niche was observed between the sexes. the species generally fed on aquatic, poor-flying or slow-moving invertebrates. keywords. ecology, diet, trophic niche, dipterans, amphipods, oophagy. introduction understanding the feeding relationships in amphibian communities is of fundamental interest to herpetologists and ecologists due to their key role in aquatic ecosystems (hirai and matsui, 1999). quantitative information about the role of amphibians in ecosystems is extremely important, particularly in their capacity as consumers in both aquatic and terrestrial habitats (whiles et al., 2006). furthermore, determining their feeding relationships in this trophic network is an essential step to understand the life cycle of the species and population fluctuations, as well as to develop successful conservation strategies (e.g., duellman and trueb, 1986; cogălniceanu et al., 2001; solé and rödder, 2010). the middle eastern newt genus neurergus comprises five stream-dwelling species [neurergus strauchii (steindachner 1887), n. barani öz 1994, n. crocatus cope 1862, n. kaiseri schmidt 1952, and n. microspilotus (nesterov 1916)] distributed in anatolia, iran, and iraq (leviton et al., 1992; papenfuss et al., 2009; sparreboom, 2014; rancilhac et al., 2019). the anatolia newt, n. strauchii, is endemic to eastern turkey (schmidtler and schmidtler, 1970; öz, 1994; tok et al., 2016; yıldız et al., 2018). due to its narrow area of occupancy, which is less than 2000 km2, its fragmented distribution, and habitat destruction, the species is listed as vulnerable in the iucn red list of threatened species (papenfuss et al., 2009). while n. strauchii inhabits small, cool mountain streams at altitudes between 950 and 1900 m above sea level, it overwinters on land not far from streams under stones and in burrows (schmidtler and schmidtler, 1970; başoğlu et al., 1994; bogaerts et al., 2012). being semiaquatic salamanders adapted to a high-elevation climate, 106 kerim çiçek et alii adult anatolia newt emerge from hibernation in early spring and thereafter migrate into streams that form during or after snowmelt where they will mate and breed (bogaerts et al., 2010; schneider and schneider, 2010). the species is widely studied with respect to distribution (e.g., schmidtler and schmidtler, 1970, 1975; yıldız et al., 2018) and taxonomy (e.g., steinfartz et al., 2002; pasmans et al., 2006; özdemir et al., 2009; olgun et al., 2016; rancilhac et al., 2019). unfortunately, there are still limited data on its ecology (e.g. schneider and schneider, 2010; bogaerts et al., 2006, 2010, 2012). whilst limited information is available regarding the food habits of neurerus species in captive (bogaerts et al., 2012) and natural populations (farasat and sharifi, 2014), there are no data on the food habits of n. strauchii in nature. therefore, the present study aimed to determine the food habits of a breeding population of the anatolia newt from bingöl, turkey. material and methods in the study, the ingested food items of 46 aquatic adults (18 males and 28 females) collected between mid-may and early june 2014 from soğukpınar village in genç – a district of bingöl, turkey (38°42’n, 40°27’e, 1,075 m a.s.l.) were examined. the newts were caught by hand and using fish nets in a small stream flowing into the murat river between 07.00 and 13.00. following capture, the sex of each individual was recorded and secondary sexual characters, snout-vent length (distance from the tip of the snout to the cloaca, svl) and total length (from the cloaca to the tip of the tail, tl) were measured by using a caliper with a precision of 1 mm and recorded. within an hour following capture, the stomach contents were extracted in field by flushing the stomach with pressurized water. the obtained food contents were preserved in a 70% ethanol solution for further analysis (solé and rödder, 2010; çiçek, 2011). the food contents were identified to the least inclusive possible taxonomic level. vegetal materials, sand and small pebbles were also encountered in the food contents of four individuals (frequency of occurrence, f% = 8.7%). however, we concluded that these materials had most likely been ingested accidentally during foraging and were not regarded as food. the following parameters of dietary items were recorded: frequency of occurrence (the frequency of newt stomachs containing a particular prey type, f%), numeric proportion (the number of a particular prey item type relative to all prey items, n%), and volumetric proportion (the volume of a particular prey item as a percentage of all prey items, v%). the prey volume was calculated using an ellipsoid formula (dunham, 1983): v= 4/3π (l/2) (w/2)2, where v is the prey volume, l is the length of prey item, and w is the width of prey item. to determine the relative importance of each prey category in the diet of the species, the index of relative importance (iri, pinkas et al., 1971) was calculated using the following formula: iri = (n % + v %) f %, where n % is the numerical proportion of each prey item in the diet; v % is the volumetric proportion of each prey item in the diet; and f % is the relative frequency of occurrence of prey. the higher value indicates the greater importance of a particular prey item in the overall dietary composition. trophic niche overlap was calculated using pianka’s index (o, 1973). this index ranges from 0 (no similarity) to 1 (identical). food-niche breadth was determined using shannon’s index (h, shannon, 1948). all niche calculations were made using “ecosimr vers. 1.0” package (gotelli et al., 2014) in r vers. 3.6.1 (r core team, 2019). the sexes were compared by t-test, and mann-whitney u tests were performed using the deducer statistical package (fellows, 2012) in the same version of r. mean values are provided with their standard deviations. results the average neurergus strauchii body length (svl) was 70.3 ± (sd) 2.84 mm (range = 63-75 mm) for males and 71.2 ± 3.01 mm (65-77 mm) for females. the average total length (tl) was determined as 146.8 ± 7.08 mm (134-159 mm) in males and 148.3 ± 5.77 mm (136-158 mm) in females. no statistically significant difference was observed between the sexes in terms of their sizes (svl, t = 0.983, p = 0.332; tl, t = 0.016, p = 0.987). in the food content of 46 individuals, a total of 953 prey items, with body lengths ranging from 3 to 55 mm, were determined with a median number of 18 (range = 1-50) dietary items per individual. the median number of prey items was 16 (1-50) for males and 17.5 (1-39) for females. while the number of prey items was slightly greater in females, there was no significant difference between the sexes (mann-whitney u test, z = -0.452, p ≤ 0.652). among the prey taxa shown in table 1, the highest frequency of occurrence (f% > 30%) in the food composition was observed for diptera (87%), amphipoda (85%), neurergus eggs (74%), and coleoptera (37%). diptera (46%) and amphipoda (34%) were the most abundant prey groups (n% > 20%). the greatest prey volumes (v% = 20%) were accounted for by amphipoda (40%) and neurergus eggs (41%). more active prey items such as adult diptera, coleoptera, hemiptera, lepidoptera, and orthoptera were less frequently encountered in the food contents. larval prey accounted for 36% of dietary items in number, 93% in frequency, and 10% in volume in the total food content. vegetal fragments (e.g., leaves, roots, and seeds) and inorganic material were observed in 4.4% of the newts and were considered to have been ingested accidentally during foraging. in addition, the presence of shed-skin remains was detected in the diet of two individuals (f% = 4.4%). amphipoda (iri = 6827), diptera (5386), neurergus eggs (3739) and coleoptera (318) were the most important prey categories in anatolia newts (table 1). 107food composition of a breeding population the endemic anatolia newt ta bl e 1. th e fo od c om po si tio n of 4 6 (1 8 m al es a nd 2 8 fe m al es ) n . s tr au ch ii in di vi du al s fr om e as te rn a na to lia , t ur ke y. f : f re qu en cy o f oc cu rr en ce , n : n um er ic p ro po rt io n, v : v ol um et ri c pr op or tio n, i r i: in de x of r el at iv e im po rt an ce . pr ey t ax a f (% ) n ( % ) v ( % ) ir i fe m al es m al es o ve ra ll fe m al es m al es o ve ra ll fe m al es m al es o ve ra ll m o ll u sc a : g a st r o po d a 8 (2 8. 6% ) 1 (5 .6 % ) 9 (1 9. 6% ) 10 ( 1. 7% ) 1 (0 .3 % ) 11 ( 1. 2% ) 62 ( 0. 9% ) 4 (0 .1 % ) 67 ( 0. 6% ) 29 .9 ly m na ei da e 6 (2 1. 4% ) 6 (1 3. 0% ) 7 (1 .2 % ) 7 (0 .7 % ) 48 ( 0. 7% ) 48 ( 0. 4% ) 14 .8 pl an or bi da e 2 (7 .1 % ) 1 (5 .6 % ) 3 (6 .5 % ) 3 (0 .5 % ) 1 (0 .3 % ) 4 (0 .4 % ) 15 ( 0. 2% ) 4 (0 .1 % ) 19 ( 0. 2% ) 3. 8 a n n el id a : c li t el la ta 3 (1 0. 7% ) 3 (6 .5 % ) 3 (0 .5 % ) 3 (0 .3 % ) 12 4 (1 .7 % ) 12 4 (1 .1 % ) 8. 9 lu m br ic id ae 3 (1 0. 7% ) 3 (6 .5 % ) 3 (0 .5 % ) 3 (0 .3 % ) 12 4 (1 .7 % ) 12 4 (1 .1 % ) 8. 9 a r t h r o po d a 28 ( 10 0% ) 18 ( 10 0% ) 46 ( 10 0% ) 56 7 (9 7. 8% ) 37 2 (9 9. 7% ) 93 9 (9 8. 5% ) 70 69 ( 97 .4 % ) 45 43 ( 99 .9 % ) 11 61 2 (9 8. 4% ) 55 80 1. 7 c hi lo po da 1 (5 .6 % ) 1 (2 .2 % ) 1 (0 .3 % ) 1 (0 .1 % ) 4 (0 .1 % ) 4 (< 0. 1% ) 0. 3 m a la c o st r a c a : i so po d a 2 (7 .1 % ) 2 (1 1. 1% ) 4 (8 .7 % ) 2 (0 .3 % ) 5 (1 .3 % ) 7 (0 .7 % ) 45 ( 0. 6% ) 48 ( 1. 0% ) 92 ( 0. 8% ) 13 .2 o ni sc id ae 2 (7 .1 % ) 2 (1 1. 1% ) 4 (8 .7 % ) 2 (0 .3 % ) 5 (1 .3 % ) 7 (0 .7 % ) 45 ( 0. 6% ) 48 ( 1. 0% ) 92 ( 0. 8% ) 13 .2 m a la c o st r a c a : a m ph ip o d a 26 ( 92 .9 % ) 13 ( 72 .2 % ) 39 ( 84 .8 % ) 16 3 (2 8. 1% ) 32 8 (3 4. 4% ) 25 30 ( 34 .9 % ) 47 63 ( 40 .4 % ) 68 27 .3 g am m ar id ae 26 ( 92 .9 % ) 13 ( 72 .2 % ) 39 ( 84 .8 % ) 16 3 (2 8. 1% ) 16 5 (4 4. 2% ) 32 8 (3 4. 4% ) 25 30 ( 34 .9 % ) 22 33 ( 49 .1 % ) 47 63 ( 40 .4 % ) 68 27 .3 in se c ta 26 ( 92 .9 % ) 18 ( 10 0% ) 44 ( 95 .7 % ) 40 2 (6 9. 3% ) 20 1 (5 3. 9% ) 60 3 (6 3. 3% ) 44 94 ( 61 .9 % ) 71 0. 8 (1 5. 6% )1 87 2. 8 (1 5. 9% ) 20 30 1. 5 o r t h o pt er a 1 (3 .6 % ) 1 (2 .2 % ) 1 (0 .2 % ) 1 (0 .1 % ) 6 (0 .1 % ) 6 (0 .1 % ) 0. 3 ep h em er o pt er a 4 (1 4. 3% ) 1 (5 .6 % ) 5 (1 0. 9% ) 11 ( 1. 9% ) 4 (1 .1 % ) 15 ( 1. 6% ) 24 ( 0. 3% ) 10 ( 0. 2% ) 34 ( 0. 3% ) 20 .2 h em ip t er a 3 (1 0. 7% ) 4 (2 2. 2% ) 7 (1 5. 2% ) 7 (1 .2 % ) 6 (1 .6 % ) 13 ( 1. 4% ) 21 ( 0. 3% ) 26 ( 0. 6% ) 47 ( 0. 4% ) 26 .8 n ot on ec tid ae 3 (1 0. 7% ) 4 (2 2. 2% ) 7 (1 5. 2% ) 7 (1 .2 % ) 6 (1 .6 % ) 13 ( 1. 4% ) 21 ( 0. 3% ) 26 ( 0. 6% ) 47 ( 0. 4% ) 26 .8 h y m en o pt er a 1 (5 .6 % ) 1 (2 .2 % ) 1 (0 .3 % ) 1 (0 .1 % ) 1 (< 0. 1% ) 1 (< 0. 1% ) 0. 2 fo rm ic id ae 1 (5 .6 % ) 1 (2 .2 % ) 1 (0 .3 % ) 1 (0 .1 % ) 1 (< 0. 1% ) 1 (< 0. 1% ) 0. 2 c o le o pt er a 10 ( 35 .7 % ) 7 (3 8. 9% ) 17 ( 37 .0 % ) 21 ( 3. 6% ) 15 ( 4. 0% ) 36 ( 3. 8% ) 16 7 (2 .3 % ) 40 2 (8 .8 % ) 56 9 (4 .8 % ) 31 7. 9 c ol eo pt er a su cu l 4 (1 4. 3% ) 1 (5 .6 % ) 5 (1 0. 9% ) 14 ( 2. 4% ) 4 (1 .1 % ) 18 ( 1. 9% ) 88 ( 1. 2% ) 20 ( 0. 4% ) 10 8 (0 .9 % ) 30 .5 c ol eo pt er a la rv a 6 (2 1. 4% ) 5 (2 7. 8% ) 11 ( 23 .9 % ) 7 (1 .2 % ) 10 ( 2. 7% ) 17 ( 1. 8% ) 79 ( 1. 1% ) 36 4 (8 .0 % ) 44 3 (3 .8 % ) 13 2. 4 c ar ab id ae 1 (5 .6 % ) 1 (2 .2 % ) 1 (0 .3 % ) 1 (0 .1 % ) 18 ( 0. 4% ) 18 ( 0. 2% ) 0. 6 le pi d o pt er a 3 (1 0. 7% ) 4 (2 2. 2% ) 7 (1 5. 2% ) 4 (0 .7 % ) 4 (1 .1 % ) 8 (0 .8 % ) 11 ( 0. 1% ) 20 ( 0. 4% ) 31 ( 0. 3% ) 16 .8 le pi do pt er a in de t. 1 (3 .6 % ) 2 (1 1. 1% ) 3 (6 .5 % ) 2 (0 .3 % ) 2 (0 .5 % ) 4 (0 .4 % ) 1 (< 0. 1% ) 5 (0 .1 % ) 6 (< 0. 1% ) 3. 1 le pd op te ra la rv ae ( in de t.) 2 (7 .1 % ) 2 (1 1. 1% ) 4 (8 .7 % ) 2 (0 .3 % ) 2 (0 .5 % ) 4 (0 .4 % ) 10 ( 0. 1% ) 15 ( 0. 3% ) 25 ( 0. 2% ) 5. 5 d ip t er a 24 ( 85 .7 % ) 16 ( 88 .9 % ) 40 ( 87 .0 % ) 29 8 (5 1. 4% ) 14 3 (3 8. 3% ) 44 1 (4 6. 3% ) 93 3 (1 2. 9% ) 25 1 (5 .5 % ) 11 84 ( 10 .0 % ) 53 85 .9 d ip te ra a du lt 2 (7 .1 % ) 1 (5 .6 % ) 3 (6 .5 % ) 5 (0 .9 % ) 2 (0 .5 % ) 7 (0 .7 % ) 37 ( 0. 5% ) 1 (< 0. 1% ) 38 ( 0. 3% ) 6. 9 c hi ro ni m id l ar va e (i nd et .) 18 ( 64 .3 % ) 11 ( 61 .1 % ) 29 ( 63 .0 % ) 11 6 (2 0. 0% ) 34 ( 9. 1% ) 15 0 (1 5. 7% ) 39 0 (5 .4 % ) 10 4 (2 .3 % ) 49 5 (4 .2 % ) 12 56 .4 c ul ic id ae 8 (2 8. 6% ) 6 (3 3. 3% ) 14 ( 30 .4 % ) 90 ( 15 .5 % ) 25 ( 6. 7% ) 11 5 (1 2. 1% ) 34 7 (4 .8 % ) 57 ( 1. 3% ) 40 4 (3 .4 % ) 47 1. 5 c ul ic id la rv ae 17 ( 60 .7 % ) 10 ( 55 .6 % ) 27 ( 58 .7 % ) 87 ( 15 .0 % ) 82 ( 22 .0 % ) 16 9 (1 7. 7% ) 15 9 (2 .2 % ) 89 ( 2. 0% ) 24 8 (2 .1 % ) 11 64 .1 a m ph ib ia : c a u d a ta 18 ( 64 .3 % ) 16 ( 88 .9 % ) 34 ( 73 .9 % ) 60 ( 10 .3 % ) 28 ( 7. 5% ) 88 ( 9. 2% ) 33 32 ( 45 .9 % ) 15 48 ( 34 .0 % ) 48 80 ( 41 .3 % ) 37 38 .6 n eu re rg us e gg s 18 ( 64 .3 % ) 16 ( 88 .9 % ) 34 ( 73 .9 % ) 60 ( 10 .3 % ) 28 ( 7. 5% ) 88 ( 9. 2% ) 33 32 ( 45 .9 % ) 15 48 ( 34 .0 % ) 48 80 ( 41 .3 % ) 37 38 .6 to ta l n um be r of p re y ite m s 28 18 46 58 0 37 3 95 3 72 55 45 48 11 80 3 108 kerim çiçek et alii the food niche breadth (shannon’s index) was low and quite similar between the sexes (1.62 for males and 1.34 for females). the food spectrum of the newts yielded a moderate niche breadth based on the index value. prey composition was quite similar between the sexes (pianka’s niche overlap index, 0.68), indicating the use of similar microhabitats for foraging. despite small differences in percentages, no statistically significant differences were found between the sexes with respect to food contents. discussion adult neurergus newts mainly feed on aquatic prey items in their larval stages (e.g., kutrup et al., 2005; david et al., 2009; covaciu-marcov et al., 2010; kopecký et al., 2012; farasat and mozafar, 2014). the close relative of n. strauchii, n. microspilotus predominantly feeds on benthic macroinvertebrates, mainly mycetophilidae, baetidae, corbiculidae, gammaridae, and lumbricidae (farasat and mozafar, 2014). in this study, the most frequent prey items of anatolia newts were dipterans, amphipods, coleopterans, and their own eggs, whereas the greatest prey volume was attributable to amphipods, n. strauchii eggs, and dipterans. the dietary components of anatolia newts are similar to those of other species of newts (farasat and mozafar, 2014). the utilization of amphibian eggs as food is beneficial for several reasons, one of them being the low energy consumption needed for a large volume of food (denoël and demars, 2008). the sampling was done during the breeding period of the species and there were many egg clutches in the study area. eggs were identified in the food contents of 34 individuals (74%). the consumption of amphibian eggs has also been recorded in other newt species (covaciu-marcov et al., 2010; kopecky et al., 2012; farasat and mozafar, 2014), though farasat and mozafar (2014) reported n. microspilotus to consume its own eggs at a lower rate. our observation in the field showed that the prey spectrum in the environment of the species was limited and that the easily obtained eggs could substitute for potential prey requiring greater foraging effort. it has been shown that in captivity, n. strauchii will eat a wide variety of food items offered, even dead unnatural food items, such as slices of liver and octopus (bogaerts et al., 2012). this means that they are certainly able to identify and eat non-moving foods like an egg in their natural habitats. under poor environmental conditions, this could be an effective strategy to avoid starvation. there were no sex-specific differences detected in the food composition of n. strauchii in anatolia. this also coincides with many studies on the same issue reporting that there is no difference between the sexes (e.g., covaciu-marcov et al., 2010; farasat and mozafar, 2014). this indicates that both sexes use the same habitat for foraging. however, some species (e.g. lissotriton vulgaris, lissotriton montandoni, triturus cristatus group) exhibit sex-specific differences in the feeding strategy and a certain level of trophic selectivity (covaciu-marcov et al., 2002, 2010) depending on newt size variation (david et al., 2009; covaciu-marcov et al., 2010). data suggest that amphibians exhibit two modes for foraging: sit-and-wait foraging and active foraging. while actively foraging, amphibians wander around the environment in search of prey, thus spending considerable energy in the search phase but little energy in the capture phase of foraging (vitt and caldwell, 2014). they mostly encounter and consume non-moving or slow-moving prey items (pianka, 1966) and generally use their visual and olfactory senses while foraging (vitt and caldwell, 2014). their food niche breadth is rather narrower than that of sit-and-wait predators (perry and pianka, 1997). having its diet based mainly on composed slow-moving aquatic invertebrates, the anatolia newt could be considered an active forager. as stated in the study, the anatolia newt’s food items comprise slow-moving aquatic invertebrates, particularly aquatic dipterans, amphipods, and coleopterans, as well as their own eggs. there is no significant difference in food composition between the sexes. the species is an active predator usually feeding on poor-flying or non-flying prey items. it has low food niche breadth, mostly limited to the classes insecta and malacostraca. the abundance of these prey taxa in habitats might potentially indicate streams habitable by the anatolia newt within its range. the data on the biology and ecology of the anatolia newt are still insufficient to identify the ecological pressures on populations. unfortunately, further studies are necessary to determine its ecology, habitat, and distribution pattern. acknowledgements we thank prof. dr. aaron bauer (villanova university, usa) and an anonymous referee for their valuable comments on earlier versions of the manuscript and for reviewing english style. the study protocol was approved under decision no. 2015-03/01 by the laboratory animals 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(2008): some new records of anatolia newt, neurergus strauchii (steindacher 1887) from eastern anatolia, turkey. biodicon 11: 120124. acta herpetologica vol. 15, n. 2 december 2020 firenze university press estimating abundance and habitat suitability in a micro-endemic snake: the walser viper gentile francesco ficetola1,2,*, mauro fanelli3, lorenzo garizio3, mattia falaschi1, simone tenan4, samuele ghielmi5, lorenzo laddaga6, michele menegon7,8, massimo delfino3,9. potential effects of climate change on the distribution of invasive bullfrogs lithobates catesbeianus in china li qing peng1, min tang1, jia hong liao1, hai fen qing1, zhen kun zhao1, david a. pike2, wei chen1,* a bibliometric-mapping approach to identifying patterns and trends in amphibian decline research claudio angelini1,*, jon bielby2, corrado costa3 food composition of a breeding population the endemic anatolia newt, neurergus strauchii (steindachner, 1887) (caudata: salamandridae), from bingöl, eastern turkey kerim çiçek1,*, mustafa koyun2, ahmet mermer1, cemal varol tok3 stomach histology of crocodylus siamensis and gavialis gangeticus reveals analogy of archosaur “gizzards”, with implication on crocodylian gastroliths function ryuji takasaki1,2,*, yoshitsugu kobayashi3 does chronic exposure to ammonium during the pre-metamorphic stages promote hindlimb abnormality in anuran metamorphs? a comparison between natural-habitat and agrosystem frogs sonia zambrano-fernández1, francisco javier zamora-camacho2,3,*, pedro aragón2,4 confirming lessona’s brown frogs distribution sketch: rana temporaria is present on turin hills (piedmont, nw italy) davide marino1, angelica crottini2, franco andreone3,* phylogenetic relationships of the italian populations of horseshoe whip snake hemorrhois hippocrepis (serpentes, colubridae) francesco paolo faraone1, raffaella melfi2, matteo riccardo di nicola3, gabriele giacalone4, mario lo valvo5* first karyological analysis of the endemic malagasy phantom gecko matoatoa brevipes (squamata: gekkonidae) marcello mezzasalma1,2,*, fabio m. guarino3, simon p. loader1, gaetano odierna3, jeffrey w. streicher1, natalie cooper1 notes on sexual dimorphism, diet and reproduction of the false coral snake oxyrhopus rhombifer duméril, bibron & duméril, 1854 (dipsadidae: pseudoboini) from coastal plains of subtropical brazil fernando m. quintela1,*, felipe caseiro¹, daniel loebmann¹ acta herpetologica 13(1): 13-19, 2018 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-21171 the first demographic data and body size of the southern banded newt, ommatotriton vittatus (caudata: salamandridae) abdullah altunişik recep tayyip erdoğan university, faculty of arts and sciences, biology department, rize, turkey. email: abdullah.altunisik@erdogan. edu.tr submitted on: 2017, 26th august; revised on: 2017, 28th october; accepted on: 2017, 14th november editor: fabio m. guarino abstract. in this study, the age structure and some life history traits were revealed for the first time in a population of ommatotriton vittatus, living in tarsus (mid-south part of turkey), at the western limit of the species’ range. maximum longevity was recorded to be eight years in both females and males and age at maturity was estimated as three years for males and four years for females. although mean age did not differ significantly between sexes, males had significantly larger snout-vent length (svl) than females. age and body size were positively correlated with each other for both females and males. since the populations of the southern banded newt in turkey are in decline, the present study that provides preliminary data on life history traits of this newt could be helpful for future biological studies. keywords. amphibia, age, demography, skeletochronology, life history, newt. introduction the study of growth in amphibians is important because it directly influences age at sexual maturity, timing of reproductive events, fecundity, and longevity (hemelaar, 1988; smirina, 1994; miaud and merilä, 2000; navas, 2006). therefore, knowing age structure of a population is essential for obtaining information about the life history of the species (altunışık and özdemir, 2015). although there are several methods (e.g., body size correlation, mark and recapture and skeletochronology) to estimate the age and growth of amphibians and reptiles, skeletochronology which is based on counting the lines of arrested growth (lags) visible in histological sections, has been the most preferred method by researchers in recent years (carranza et al., 2014; bionda et al. 2015; sinsch et al., 2015; altunışık et al., 2016; bülbül et al., 2016), as it yields reliable results. as previously described in many amphibian species, periosteal bone growth depends on ecological factors, e.g., changing favorable and unfavorable periods of bone growth. unlike active growth seasons, in the winter season the periosteal bone growth is reduced due to the influence of the cold climate and a different growth rate occurs, which forms the lags. the differences in body size between males and females of the same species is a key feature that leads to significant biological insights (denoël et al., 2009) and several hypotheses have been put forward to explain these variations. it is well documented that the basic factors that elicit sexual dimorphism are generally sexual selection, natural selection and fecundity selection (andersson, 1994; fairbairn, 1997; pincheira-donoso et al., 2008; liao et al., 2015; altunışık, 2017). in addition, sexual differences in age and growth rate associated with life history hypothesis may also cause the emergence of sexual size dimorphism (ssd), which is a well-known population property (young, 2005; cadeddu et al., 2012; liao et al., 2013). a member of the cosmopolitan family salamandridae, ommatotriton genus has three species, ommatotri14 abdullah altunışık ton nesterovi, ommatotriton ophryticus (northern banded newt) and ommatotriton vittatus (southern banded newt; van riemsdijk et al., 2017). here we focus on the southern banded newt, which is a medium-sized newt of 90–110 mm in total length. during the breeding period it inhabits shallow, still or slowly flowing waters with a lot of vegetation, otherwise found in wooded areas or open places with loose rocks or stones not far from water. o. vittatus ranges from mid-south of turkey, through western syrian arab republic, lebanon and northwestern jordan, south to israel (olgun et al., 2009; iucn, 2017; fig. 1). mid-south of turkey represents the western limit of the species’ range. there are two subspecies of o. vittatus, namely o. v. vittatus and o. v. ciliensis. while the distribution area of the first subspecies is turkey, syria and israel, the distribution area of the second one is within the borders of turkey (bogaerts et al., 2013). so far, demographic investigations have been confined to northern banded newt (kuzmin, 1999; tarkhnishvili and gokhelasvili, 1999; kutrup et al., 2005; özcan and üzüm, 2015) of ommatotriton genus and no data are available for the southern banded newt. the aim of this study is to determine age structure (longevity, age at sexual maturity, survivorship, adult life expectancy), mean body size and ssd of a population of o. vittatus in the southeastern turkey, at the western limit of the species’ range, using skeletochronology. material and methods study area and species the study site (25 m a.s.l.) is located in tarsus, mersin (36°54’n, 34°53’e) in the mid-south part of turkey and has a climate that is mediterranean and moderately continental. according to climatic data obtained from the meteorological station situated near the study site (tarsus meteorological station) for the years 1950-2016, the average temperature during summer was 27.16°c, and 10.9°c in the winter (www.mgm.gov.tr). a total of 40 adult o. vittatus individuals (23 males and 17 females) were caught by hand or using a dip net in the daytime during the breeding season in 2017. after anesthesia in ms-222, their snout-vent length (svl) was measured using a digital vernier caliper (500-706-11, mitutoyo, tokyo, japan) to the nearest 0.01 mm precision and the fourth toe of the left foot was clipped. in order to use toe samples in skeletochronological analysis, firstly they were fixed in 10% formalin and later preserved in 70% ethanol. after sampling, all newts were released back to their habitats. the sexes of the individuals were determined by externally visible secondary sexual characters (prominent cloaca and dorsal crest in males) (bogaerts et al., 2013). skeletochronological analysis we applied standard skeletochronology procedure that was described by castanet and smirina (1990) for laboratory protocols to assess the age structure. firstly, we washed the phalanges kept in 70% ethanol in tap water then decalcified in nitric acid (5%) from 30 to 90 min depending on the bone structure. using a freezing microtome (shandon cryostat, germany), preferentially second phalange was cross-sectioned at 12-16 μm and stained with harris hematoxylin for 10 min. the selected cross-sections from the narrow part of the phalanx diaphysis were placed in glycerin for scanning under a light microscope (olympus bx51) at 200x and 400x magnifications and their photographs (1-10 for each cross-section) were taken by a camera (pixera), which is connected to this microscope. the number of lags was assessed independently by two observers. the distance between two adjacent lags is a good indicator of individual growth in a given year (kleinenberg and smirina 1969). therefore, in this study age at sexual maturity was determined using the distance between lags. where we observed an obvious decrease in spacing between two subsequent lags, we took it to mark the age when sexual maturity was achieved (ryser, 1988; özdemir et al., 2012). data analysis the normality of data was analyzed by shapiro-wilk test and the homogeneity of variances was analyzed by levene test. after confirming that all data were normally distributed (p > 0.05), independent sample t-test was used to test if females and males differ in mean svl or in mean age. ssd was quantified with lovich and gibbons (1992) sexual dimorphism index fig. 1. distribution map of o.vittatus (iucn, 2017). 15age structure of ommatotriton vittatus sdi = (size of larger sex / size of smaller sex) ± 1 (+1 if males are larger or -1 if females are larger), and arbitrarily defined as positive when females are larger than males and negative in the converse case. survivorship rate for adult individuals was calculated from the age structure according to following formula (robson and chapman, 1961): s= t/(r+t–1), where s is the finite annual survivorship rate estimate, t = n1 + 2n2 + 3n3 + 4n4 + …. + nnn, r = σni, and ni is the number of individuals in the age group i. adult life expectancy (esp), the expected life span of the salamanders that have reached sexual maturity, was computed according to the formula of seber (1973): esp = [0.5+1/(1-s)] where s represents the survivorship rate. pearson’s correlation coefficient was computed to measure the degree of relationship between svl and age and in order to determine the correlation equation between the two variables; regression analysis was also performed. the growth patterns were estimated according to the von bertalanffy growth model, which has been used earlier in several studies on amphibians (ryser 1988; miaud et al., 2000; guarino et al., 2011). the general form of the von bertalanffy growth equation used was lt = l∞.(1 – e-k(t-t0)), where lt is length at age t, l∞ is a parameter representing asymptotic maximum size, e is the base of the natural logarithm, k is a growth coefficient, and t0 is the age at metamorphosis, which is the starting point of the growth curve in the present study. because data on the size at metamorphosis of the studied population were lacking, we used size at metamorphosis (lt0 = 21.5 mm), provided by tarkhnishvili and gokhelasvili (1999). the parameters l∞ and k were estimated using microsoft excel program. data analysis was conducted using spss 17 statistical software package (ibm spss statistics for windows). results lags recorded in phalange bones were clearly visible in all cross-sections, as it can be seen in fig. 2. according to the results of age reading obtained by direct observation in the microscope and photographs on the computer, the maximum lifespan was 8 years in both females and males. females of o. vittatus were on average 5.82 ± 0.30 years old (range: 4-8) and males had an average age of 5.65 ± 0.29 years (range: 3-8; table 1). no significant difference was detected between the sexes with regard to mean age (t = 0.407, df = 38, p > 0.05) and age distribution (kolmogorov-smirnov test, p > 0.05). as it can be seen from age distribution of the population (fig. 3), the age group with the highest frequency is six (14 individuals). skeletochronological pattern showed that males and females attained sexual maturity at age of 3 and 4, respectively. since age structure did not differ between the sexes, s and esp calculations were combined and s was found to be 0.79 and esp was 5.26 years. sdi was -0.055, indicating a male biased size dimorphism. independent sample t-test (t= -2.327, df= 38, p <0.05) showed that males had significantly larger svl than that of females. for both sexes, the maximum svl recorded was higher than the estimated asymptotic svl (svlasym: males, 48.74 mm; females, 47.28 mm). the growth coefficient was higher in males than in females (k: males, 1.80; females, 1.49). fig. 2. cross-section of a phalanx of an individual at the age of 7. white arrows show lags. m.c. = marrow cavity. table 1. descriptive statistics for the studied population. sex n age (years) svl (mm) esp (years) s mean ± se range mean ± se range females 17 5.82 ± 0.30 4-8 45.60 ± 0.79 40.38-52.30 5.26 0.79 males 23 5.65 ± 0.29 3-8 48.12 ± 0.72 41.7254.15 16 abdullah altunışık body size was positively correlated with age in both females (pearson’s correlation r = 0.589, p < 0.05; fig. 4a) and males (r = 0.648, p < 0.01; fig. 4b). discussion the first demographic data on an ommatotriton vittatus population was provided in this study. the results suggest that southern banded newt has a lower lifespan than the northern banded newt. we found that maximum age could be reached at 8 years in both males and females in tarsus population. in congeneric o. ophryticus sensu lato, maximum lifespan was recorded as 21 years for georgian populations (kuzmin, 1999; tarkhnishvili and gokhelasvili, 1999) and 10 and 16 years for the low and high altitude turkish populations, respectively (kutrup et al., 2005; özcan and üzüm, 2015). in the present study, no significant difference was detected between females and males with regard to mean age. a similar result was recorded for bahçesultan and erbaa populations of o. ophryticus sensu lato (özcan and üzüm, 2015). delayed maturity and longer lifespan are expected phenomenons for high-elevation populations, because they are exposed to colder temperatures and shorter growth seasons in comparison with low-elevation populations. in line with this trend, the studied low-altitude population of o. vittatus had lower lifespan and age at maturity than that of high-altitude populations of o. ophryticus sensu lato, formerly referred to as triturus vittatus ophryticus, (kuzmin, 1999; tarkhnishvili and gokhelasvili, 1999; kutrup et al., 2005; özcan and üzüm, 2015). it is expected that amphibians with indetermined growth should enhance not only their age at maturity but also division of resources between reproduction and growth over the years following sexual maturation (kozlowski and uchmanski, 1987). generally, amphibians reach sexual maturity when they reaches a certain size, therefore sexual maturity may vary between different species and populations (gibbons and mccarthy, 1984; hemelaar, 1986; ryser, 1988). the age at maturity, which is determined to be 3-4 years, is compatible with the two populations of o. ophryticus (kutrup et al., 2005). however, in the tosya and bahçesultan populations of o. nesterovi and erbaa populations of o. ophryticus located at higher altitudes, the age at maturity was determined as 4-6 years (özcan and üzüm, 2015). kuzmin (1999) stated that the sexual maturity was attained at 3-5 years in o. ophryticus for caucasus populations. similarly, tarkhnishvili and gokhelasvili (1999) established the earliest age at maturation in georgian o. ophryticus populations from near tbilisi as 4 years for females and 3 years for males (table 2). sexual size dimorphism is common among amphibians. more specifically, about 90% of the anurans and 61% of the urodeles indicate a female-biased ssd (shine, 1979; kupfer, 2007; reinhard et al., 2015). interestingly, our results show that males of o.vittatus did not conform to this tendency but indicate male-biased dimorphism. similar results were reported by kutrup et al. (2005), çiçek and ayaz (2011), başkale et al. (2013), özcan and üzüm (2015) and cvijanović et al. (2017) for diffig. 4. relationship between age and body size (svl). females (a) and males (b). fig. 3. age distribution of o. vittatus from tarsus population. 17age structure of ommatotriton vittatus ferent populations of o. ophryticus sensu lato. although rarely found in the literature, males of some urodele species (e.g., lissotriton vulgaris, halliday and verrell, 1988; mertensiella caucasica, üzüm, 2009; lyciasalamandra atifi, rewieved in reinhard et al., 2015) are larger than females in terms of mean body size. most of the variation in size dimorphism has been considered to result from differences in age structure between sexes (monnet and cherry, 2002; cadeddu et al., 2012; liao et al., 2013). the annual survival rate of this newt differs from the other congeneric species (o. nesterovi), because it was calculated as 0.35 for a long-term mark-recapture and skeletochronological study (başkale et al., 2013). the fact that southern banded newts have a higher annual survival rate than northern banded newt may lead to differences in the potential reproductive lifespan between these two newts. it is widely assumed that age and body size are positively correlated in amphibians (halliday and verrell, 1988). although there is no positive correlation for some species, in some cases this correlation may vary depending on sex. similar to o.nesterovi’s tosya, bahçesultan (özcan and üzüm, 2015) and sakarya (başkale et al., 2013) and o. ophryticus’s erbaa (özcan and üzüm, 2015) populations, in the studied o. vittatus population, body size of both males and females showed positive significant correlations with age. contrarily, no correlation was reported between body size and age in both populations of northern banded newt (kutrup et al., 2005). this discrepancy among the congeneric species may be explained by the decrease of growth rates after sexual maturity is achieved, as well as by the different interannual reproductive effort of individuals, resulting in different individual growth curves (kutrup et al., 2005). in conclusion, as mentioned before the relatively low longevity and age at maturity observed in southern banded newt could result from local ecological conditions (e.g., altitude, temperature, length of the activity period). it is also necessary to investigate more populations exposed to various conditions in order to evaluate more precisely the interpopulational variation of these life-history characteristics in o. vittatus. acknowledgements i am grateful to m. emin altunışık for his assistance in collecting animals from the field. this study was supported by recep tayyip erdoğan university bap under grant number 2016.53007.102.03.01. all experiments were performed in accordance with the turkish law (number of permission to capture: 72784983-488.0445006) and with permission for animal experiments of the local ethics committee of the recep tayyip erdoğan university (approval reference number: 2015/71). i kindly table 2. demographic life history traits in ommatotriton species throughout its geographical range. species location altitude (a.s.l.,m) sex mean age (years) age at maturity (years) longevity (years) mean svl (mm) references o. ophryticus different parts of georgia male 3-5 8-21 kuzmin (1999) female 3-5 8-21 o. ophryticus akhaldaba, georgia 750 male 5.53 3 8 tarkhnishvili and gokhelashvili (1999)female 6.97 4 12 o. ophryticus gürbulak, trabzon, turkey 300 male 4 2-3 10 61.7 kutrup et al. (2005) female 4 2-3 10 53.3 o. ophryticus hıdırnebi, trabzon, turkey 1300 male 8.2 4 16 59.6 kutrup et al. (2005) female 9.2 4 16 51.0 o. nesterovi* sakarya, turkey 31 male 4.92 3 8 90.79 başkale et al. (2013) female 4.90 2 6 86.06 o. nesterovi* bahçesultan, bilecik, turkey 1015 male 7.4 6 10 72.39 özcan and üzüm, 2015 female 7.4 6 10 63.40 o. nesterovi* tosya, kastamonu, turkey 1202 male 6.4 4 9 64.07 özcan and üzüm, 2015 female 7.06 5 9 55.57 o. ophryticus erbaa, tokat, turkey 1239 male 8.2 6 12 71.07 özcan and üzüm, 2015 female 8.4 5 11 60.36 o. vittatus tarsus, mersin, turkey 25 male 5.65 3 8 48.12 < female 5.82 4 8 45.60 svl, snout-venth length; *referred to as o.ophryticus 18 abdullah altunışık thank three anonymous reviewers for constructive suggestions on improving the manuscript. references altunışık, a. 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(2005): life-history variation and allometry for sexual size dimorphism in pacific salmon and trout. proc. biol. sci. 272: 167-72. acta herpetologica vol. 13, n. 1 june 2018 firenze university press species diversity and distribution of lizards in montenegro katarina ljubisavljević1,2,*, ljiljana tomović3, aleksandar urošević1, slađana gvozdenović2, vuk iković2, vernes zagora2, and nenad labus4 the first demographic data and body size of the southern banded newt, ommatotriton vittatus (caudata: salamandridae) abdullah altunişik diet and helminth parasites of freshwater turtles mesoclemmys tuberculata, phrynops geoffroanus (pleurodira: chelidae) and kinosternon scorpioides (criptodyra: kinosternidae) in a semiarid region, northeast of brazil antonio marcos alves pereira*, samuel vieira brito, joão antonio de araujo filho, adonias aphoena martins teixeira, diêgo alves teles, daniel oliveira santana, vandeberg ferreira lima, waltécio de oliveira almeida electric circuit theory applied to alien invasions: a connectivity model predicting the balkan frog expansion in northern italy mattia falaschi1,2,*, marco mangiacotti3, roberto sacchi3, stefano scali2, edoardo razzetti4 first report of bufo bufo (linnaeus, 1758) from sardinia (italy) ilaria maria cossu1, salvatore frau1, massimo delfino2,3, alice chiodi4, claudia corti1,5*, adriana bellati4 natural history and conservation of the nurse frog of the serranía del perijá allobates ignotus (dendrobatoidea: aromobatidae) in northeastern colombia hernán darío granda-rodríguez1,2, andrés camilo montes-correa3,*, juan david jiménez-bolaño3, marvin anganoy-criollo4,5 exploring body injuries in the horseshoe whip snake, hemorrhois hippocrepis juan m pleguezuelos*, esmeralda alaminos, mónica feriche how effectively do european skinks thermoregulate? evidence from chalcides ocellatus, a common but overlooked mediterranean lizard grigoris kapsalas, aris deimezis-tsikoutas, thanos georgakopoulos, ismini gkourtsouli-antoniadou, kallirroi papadaki, katerina vassaki, panayiotis pafilis thermal tolerance for two cohorts of a native and an invasive freshwater turtle species jun geng, wei dang, qiong wu, hong-liang lu* diet of a restocked population of the european pond turtle emys orbicularis in nw italy dario ottonello1, fabrizio oneto1,2, monica vignone2, anita rizzo2, sebastiano salvidio2,* helminths of the lizard colobosauroides cearensis (squamata, gymnophthalmidae) in an area of caatinga, northeastern brazil aldenir f. da silva neta*, robson w. ávila acta herpetologica 13(1): 89-93, 2018 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-22518 diet of a restocked population of the european pond turtle emys orbicularis in nw italy dario ottonello1, fabrizio oneto1,2, monica vignone2, anita rizzo2, sebastiano salvidio2,* 1 cesbin srl, corso europa 26, 16132 genova, italy 2 distav, università degli sudi di genova, corso europa 26, 16132 genova, italy. *corresponding author. e-mail: salvidio@dipteris. unige.it submitted on: 2017, 16th january; revised on: 2017, 27th february; accepted on: 2017, 6th march editor: daniele pellitteri-rosa abstract. recently several projects have been implemented for the conservation of the european turtle emys orbicularis, but few aspects of the captive-bred animals released into the wild have been described. in this note we report about the trophic habits of a small restocked population of the endemic subspecies e. o. ingauna that is now reproducing in nw italy. faecal contents from 25 individuals (10 females, 11 males and 4 juveniles) were obtained in june 2016. overall, 11 taxonomic categories of invertebrates were identified, together with seeds and plant remains. plant material was present in 24 out of 25 turtle faecal contents, suggesting that ingestion was deliberate. there were no differences between the dietary habits of females and males, and the trophic strategy of adult individuals was characterised by a relatively high specialization on dragonfly nymphae. these findings suggest that captive bred turtles are adapting well to the wild and that restocked individuals assumed an omnivorous diet, a trophic behaviour typical of other wild turtle populations living in similar habitats. keywords. captive-breeding, food habits, freshwater turtle, omnivory, restocking. in europe the populations of the pond turtle emys orbicularis (linnaues, 1758) are declining and in many countries the species is considered threatened or even locally endangered (fritz and chiari, 2013). therefore, many conservation projects have been implemented to improve the species’ status, in particular by restocking captive-breed individuals, for example in portugal (teixeira et al., 2013), france (cadi and miquet, 2004; thienpont et al., 2013) and spain (ayres et al., 2013). the outcomes of these restocking projects are monitored by means of different descriptors, and in particular by the estimation of the annual survival of captive-bred individuals released into the wild (cadi and miquet, 2004; masin et al., 2015; canessa et al., 2016) or by the extent of individual displacements and habitat use obtained by radiotracking (mignet et al., 2014). in general, however, few data are available on the ecological adaption of captive-bred animals to their new environment. to assess this topic, we focused our interest on the feeding habits of a restocked population, an issue widely studied in wild individuals (e.g., fritz, 2001; ottonello et al., 2005; zuffi et al., 2011; balzani et al., 2016). in fact, captive bred animals are usually fed with commercial pellets, or with fresh or frozen food like fishes, shrimps, chironomidae (non-biting midges) and tenebrionidae (darkling beetles) larvae. hence information about the feeding habits of captivebred individuals after their release in the wild is of fundamental interest when evaluating their adaptation to their new environment. in this context, we use as case study a small restocked population of the endemic subspecies e. o. ingauna found in liguria, nw italy (jesu et al., 2004). these individuals are part of a conservation project, that began in 1999 and is still ongoing (ficetola et al., 2013; canessa et al., 2016). restocked animals were born in an 90 dario ottonello et alii outdoor facility (i.e., the “centro emys” situated in leca di albenga, nw italy) from local genetically screened adults (manfredi et al., 2013). turtles were bred in the facility for 2-5 years until restocking; they were fed with commercial pellets in the first year, then with frozen shrimps and fish. before restocking, all animals were marked by both scute notches (servan et al., 1986) and subcutaneous pit tags, while their health status was evaluated by screening for blood and gastrointestinal pathogens in accordance to veterinarian protocols (canessa et al., 2016). turtles were sampled in two semi-permanent clay ponds in the alluvial plain of the river centa, in the province of savona (liguria, nw italy). for conservation purposes we are not giving exact localisations of the sites. in one pond, native turtles are still found, but the other pond was excavated in 2009 and hosts only restocked animals. in the surroundings of this latter site, three turtle nests with successfully hatched eggs were observed in november 2017 (dario ottonello, pers. obs.), giving the first evidence of reproduction in the wild of this restocked population. turtles were captured by unbaited fyke nets, from the 25th to the 26th of june 2016, and transported to the “centro emys”, situated less than 2 km from both sites, where they were checked for sex and their straight carapax length (scl) measured. only individuals with evident sexual characters were considered as adults (zuffi and gariboldi, 1995), while the others were considered as juveniles. faecal samples were obtained from turtles kept in individual buckets for 24h. then, samples were filtered on a sieve and prey remains stored in 70% ethanol. prey items were identified under a dissecting microscope in the laboratory by at least two observers (ar; ss; mv), while plant remains could not be assigned to species or genus because they were highly fragmented. all turtles were returned to their capture site and no mortality was observed during the study. the diet of female and male turtles was analysed by means of the frequency of occurrence (fo), defined as the proportion of faecal samples containing that category divided by the entire sample, by shannon’s diversity index (h) and by the equitability index (h/hmax), this latter expressing how prey items are distributed among prey categories (magurran and gills, 2011). therefore, we analysed differences between sexes, with and without plant material, by means of the analysis of similarity (anosim) using bray-curtis dissimilarity index (clarke, 1993). the population trophic strategy was estimated by the graphical method of amundsen et al. (1996). this method projects each prey category on a plane delimited by an x axis which is given by the fo, and an y axis represented by the prey-specific abundance (pi). prey-specific abundance is the percentage of each prey category i, considering the total number of prey items ingested only by those animals that ate that specific category (amundsen et al., 1996; ottonello et al., 2017). the distribution table 1. prey categories, number of prey items, frequency of occurrence (fo in percentage) and diversity indexes for e. o. ingauna females, males and juveniles. females (n =10) males (n = 11) adults total (n = 21) juveniles (n = 4) number fo number fo number fo number fo odonata nymphs 22 0.80 21 1.00 43 0.90 1 0.25 diptera larvae 2 0.20 9 0.27 11 0.24 10 0.50 heteroptera nymphs 1 0.10 3 0.18 4 0.14 0 ephemeroptera larvae 0 1 0.09 1 0.05 4 0.75 coleoptera adults 0 4 0.36 4 0.19 0 plecoptera larvae 0 0 0 1 0.25 imenoptera formicidae 1 0.10 0 0 1 0.25 tricoptera larvae 0 0 0 1 0.25 oligochaeta 0 1 0.09 1 0.05 0 isopoda 0 1 0.09 1 0.05 0 gastropoda 1 0.10 0 1 0.05 0 hexapoda indet. 2 0.20 1 0.09 3 0.14 2 0.50 shannon diversity (h) 0.927 1.375 1.356 equitability (h/hmax) 0.517 0.707 0.589 seeds 152 0.60 126 0.36 278 0.48 9 0.75 plant fragments 122 1.00 95 0.82 217 0.90 26 1.00 91diet of emys orbicularis in nw italy of prey categories in the plot describes the main trophic strategy of the focal population, as being specialised (upper quadrants of the graph) or generalist (lower quadrants) and with a high between (upper left quadrant) or within (lower right quadrant) population component (see fig. 3 in amundsen et al., 1996). statistical analyses and ecological indexes were obtained by means of past software (harper and ryan, 2001). during the study, 26 european pond turtles were captured: 10 females, 12 males and 4 juveniles, but no faecal contents could be obtained from one male (table 1). females were larger (mean scl = 109.26 mm ± 21.89 sd) than males (mean scl = 106.20 mm ± 7.00 sd), this difference being not significant (welch test for unequal variances t = -0.44; p = 0.67). overall, 11 invertebrate categories were found in the faecal contents of the pond turtles: 5 in females, 7 in males and 7 in juveniles (table 1). seeds and plant remains (mainly root and leaf fragments) were found in 23 out of 24 faecal contents, suggesting that ingestion was deliberate. concerning animal items, all of them were aquatic with the exception of two ants, probably captured on the water surface. the most frequent prey categories were composed by dragonfly (odonata) nymphae and fly larvae, that were mainly nonbiting midges (chironomidae). no vertebrate remains were found in the faecal contents of the study sample. shannon’s diversity and equitability indexes did not differ between females and males (table 1: shannon h, p = 0.09 and equitability h/hmax, p = 0.08, both values obtained after 1000 permutations). moreover, there were no differences in diet composition between females and males considering animal categories alone (anosim, r = 0.02, p = 0.29) or when vegetal matter was included in the analysis (anosim, r = -0.04, p = 0.74). on the basis of these results, the trophic strategy of the adult population was examined after pooling females and males together (ottonello et al., 2017). figure 1 shows that the turtle population assumed a generalist diet for all categories, with the exception of dragonfly nymphaea, that were consumed by several relatively specialised individuals. our study highlights two main findings, the first that the overall dietary habits of captive-bred female and male e. o. ingauna released in the wild were similar, the second that adult and juvenile turtles behaved as omnivorous feeders, switching between a carnivorous and herbivorous diet within the same site and within a relative short time period. concerning the absence of sex differences in diet, other studies found low variation between the adult male and female pond turtles. for example, çiçek and ayaz (2011) and ottonello et al. (2017) observed similar diets between the sexes of emys orbicularis in turkey and of emys trinacris in sicily, respectively. according to these authors, correspondence in food habits between sexes is explained by a similar use of microhabitats, an explanation that may by realistic also for the e. o. ingauna population analysed in this study. in reptiles, herbivory tends to evolve in environments where food quality and quantity fluctuate temporally and spatially (cooper and vitt, 2002), as is the case in mediterranean ponds, that are characterised by high seasonal variations in water level and water temperature. also the diet of juveniles’ captive-bred turtles appeared to contain many vegetable remains, but our small sample size hindered any quantitative comparison with the adults. another interesting result was that adult turtles seemed relatively specialised on dragonfly nymphae, a feature that could be due to the high availability of this kind of prey in the environments or to an overestimation bias due to the chitinous structure of their mouth parts, a feature that may facilitate retrieval and identification of this taxon in faecal contents. in any case, only sampling the potential prey community available to the turtles will provide information to better understand this issue. the contemporary presence of plant and animal material in the diet of e. o. ingauna is not peculiar to this population, considering that it has already been described in other european pond turtle populations living in mediterranean regions of central italy (lebboroni and chelazzi, 1991), southern france (ottonello et al., 2005), northwestern spain (ayres et al., 2010) and turkey (çiçek and ayaz, 2011). moreover, ottonello et al. (2017) found plant remains in the congeneric emys trinacris from sicily, suggesting a widespread use of vegetation as supplementary food in turtles belonging to the genus emys. fig. 1. amundsen’s plot showing the trophic strategy of the emys orbicularis ingauna restocked population. fo = frequency of occurrence, pi = prey-specific abundance (see text for definitions). 92 dario ottonello et alii ackowledgements the conservation project is authorised by the italian ministry of environment (scn/20/99/6496 of april, 1999) and turtles were captured according to permit prot. 0013862 pnm of june, 2016. we also acknowledge the contributions of two anonymous reviewers that improved the manuscript. references amundsen, p.a., gabler, h.m., staldvik, f.j. (1996): a new approach to graphical analysis of feeding strategy from stomach contents data modification of the costello (1990) method. j. fish biol. 48: 607-614. ayres, c., calvino-cancela, m., cordero-rivera, a. 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(2011): emys orbicularis (linnaeus, 1758). in: fauna d’italia reptilia, pp. 153-163. corti, c., capula, m., luiselli, l., razzetti, e., sindaco, r., eds, edizioni calderini, bologna. acta herpetologica vol. 13, n. 1 june 2018 firenze university press species diversity and distribution of lizards in montenegro katarina ljubisavljević1,2,*, ljiljana tomović3, aleksandar urošević1, slađana gvozdenović2, vuk iković2, vernes zagora2, and nenad labus4 the first demographic data and body size of the southern banded newt, ommatotriton vittatus (caudata: salamandridae) abdullah altunişik diet and helminth parasites of freshwater turtles mesoclemmys tuberculata, phrynops geoffroanus (pleurodira: chelidae) and kinosternon scorpioides (criptodyra: kinosternidae) in a semiarid region, northeast of brazil antonio marcos alves pereira*, samuel vieira brito, joão antonio de araujo filho, adonias aphoena martins teixeira, diêgo alves teles, daniel oliveira santana, vandeberg ferreira lima, waltécio de oliveira almeida electric circuit theory applied to alien invasions: a connectivity model predicting the balkan frog expansion in northern italy mattia falaschi1,2,*, marco mangiacotti3, roberto sacchi3, stefano scali2, edoardo razzetti4 first report of bufo bufo (linnaeus, 1758) from sardinia (italy) ilaria maria cossu1, salvatore frau1, massimo delfino2,3, alice chiodi4, claudia corti1,5*, adriana bellati4 natural history and conservation of the nurse frog of the serranía del perijá allobates ignotus (dendrobatoidea: aromobatidae) in northeastern colombia hernán darío granda-rodríguez1,2, andrés camilo montes-correa3,*, juan david jiménez-bolaño3, marvin anganoy-criollo4,5 exploring body injuries in the horseshoe whip snake, hemorrhois hippocrepis juan m pleguezuelos*, esmeralda alaminos, mónica feriche how effectively do european skinks thermoregulate? evidence from chalcides ocellatus, a common but overlooked mediterranean lizard grigoris kapsalas, aris deimezis-tsikoutas, thanos georgakopoulos, ismini gkourtsouli-antoniadou, kallirroi papadaki, katerina vassaki, panayiotis pafilis thermal tolerance for two cohorts of a native and an invasive freshwater turtle species jun geng, wei dang, qiong wu, hong-liang lu* diet of a restocked population of the european pond turtle emys orbicularis in nw italy dario ottonello1, fabrizio oneto1,2, monica vignone2, anita rizzo2, sebastiano salvidio2,* helminths of the lizard colobosauroides cearensis (squamata, gymnophthalmidae) in an area of caatinga, northeastern brazil aldenir f. da silva neta*, robson w. ávila acta herpetologica 13(2): 117-123, 2018 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-23189 new insights on the phylogenetic position and population genetic structure of the critically endangered karpathos marsh frog pelophylax cerigensis (amphibia: anura: ranidae) elisavet a. toli1,*, souzanna siarabi1, anastasios bounas1, panayiotis pafilis2, petros lymberakis3, konstantinos sotiropoulos1 1 molecular ecology & conservation genetics lab, department of biological applications & technology, university of ioannina, 45110, ioannina, greece. *corresponding author. e-mail etoli@cc.uoi.gr 2 section of zoology and marine biology, department of biology, national and kapodistrian university of athens, 15784, athens, greece 3 natural history museum of crete, university of crete, 71409, irakleio, greece submitted on: 2018, 24th april; revised on: 2018, 9th august; accepted on: 2018, 26th august editor: marcello mezzasalma abstract. the genus pelophylax, which currently comprises 26 species, is a well studied group due to its complex history and high diversification, although some phylogenies remain unresolved. here we assess the phylogenetic position and the population genetic structure of the critically endangered karpathos frog, pelophylax cerigensis, endemic to karpathos island. a total of 42 cytb sequences were examined including specimens from rhodes island, and amplified fragment length polymorphisms (aflps) were generated to investigate the genetic structure and connectivity of the only two known populations on karpathos. molecular analyses reveal two major waterfrog lineages in the eastern mediterranean: clade a comprises pelophylax bedriagae from the middle east and the island of cyprus, while clade b includes both p. bedriagae from the east aegean sea and p. cerigensis. specimens from karpathos and rhodes constitute a single clade, contrasting previous studies, thus indicating the occurrence of karpathos frog also to the neighboring rhodes. the aflp markers revealed low but statistically significant levels of genetic divergence between the two karpathos’ populations and similar levels of genetic diversity. our results suggest that the current taxonomy of the species should be re-evaluated. we also strongly recommend the need of conservation actions to maintain the levels of diversity in the declining population of the karpathos frog. keywords. pelophylax cerigensis, insular populations, phylogeny, cytochrome b, genetic structure, aflps, conservation. introduction the elucidation of phylogeographic history and the study of intraand inter-population diversity are inextricably linked to the paleogeographic events that took place during the pleistocene. the balkan peninsula, which is considered a pleistocenic refugium, shows high levels of biodiversity and endemism (sfenthourakis et al., 2001; michaux et al., 2004; sotiropoulos et al., 2007) and the long-term favorable environmental conditions allowed the preservation of genetically differentiated populations (canestrelli et al., 2010) resulting in the complex history of many terrestrial species (beerli et al., 1996; douris et al., 1998; poulakakis et al., 2003). the genus pelophylax fitzinger, 1843, which currently comprises 26 species, is one of the most well studied groups (lymberakis et al., 2007; plötner et al., 2012; vervust et al., 2013; litvinchuk et al., 2015) due to its 118 elisavet a. toli et alii complex history and high diversification in the balkan peninsula and east mediterranean. isolation of land areas during the pleistocene and the formation of islands in the aegean region had a great impact in the genetic differentiation of the genus pelophylax. currently, researchers suggest long-separated lineages and diverse evolutionary histories of the western palearctic water frogs (lymberakis et al., 2007; akın et al., 2010), although some phylogenies are still under question and further revision of their status is needed. among the three major lineages in the eastern mediterranean region, the balkan-anatolia (ridibunda/ bedriagae) lineage which comprises the species p. cretensis, p. epeiroticus, p. bedriagae, p. cerigensis, p. kurtmuelleri and p. ridibundus, is considered to have emerged from a widely-distributed common ancestor through vicariance events during the climatic changes in the pliocene (lymberakis et al., 2007). pelophylax cerigensis is a medium-sized, mainly insectivore water frog, endemic to karpathos island (valakos et al., 2008; pafilis et al., 2018). karpathos is the second largest island of the dodecanese archipelago (aegean sea, greece), located approximately 47 kilometers southwest of rhodes. the island is characterized by xeric habitats with dry pine and oak forests, mediterranean maquis, and phrygana. according to beerli et al. (1994), who first described the species, phylogenetic analyses based on electrophoretic data cluster the karpathos and rhodes populations together. in addition, akın et al. (2010) found a shared haplotype in both karpathos and rhodes, a result further corroborated by lymberakis et al. (2007). however, the taxonomy of the rhodes populations remains unresolved and further evidence is needed for the clarification of the species status. the karpathos frog is regarded as the most threatened frog in europe (beerli et al., 2009; temple and cox, 2009) due to its limited distribution and current population decline. habitat loss and degradation of aquatic terrains in this dry island are considered the major factors compromising the species survival whereas they could potentially affect the populations’ genetic status. therefore, studies on population genetic diversity and structure are necessary to design and implement proper management actions for the conservation of the species. due to their simplicity and cost-effectiveness as genetic markers, aflps have been widely used in population genetic studies in the past decades. several studies have examined the genetic structure through aflp markers using f-statistics (wright, 1950; meudt et al., 2007) and quantified the levels of differentiation and connectivity between populations of several species. here, we aim to evaluate the phylogenetic position of the karpathos frog within the bedriagae lineage as well as to assess the genetic structure and connectivity of the population between the two known localities in the island of karpathos. material and methods sample collection and laboratory procedures a total of 30 buccal swab samples of p. cerigensis were collected during 2016 from the two known breeding sites (argoni and nati rivers) on karpathos (table a1, fig. 1). additionally, three samples of water frogs (presumed p. bedriagae) from rhodes island (nhmc80.2.99.40, nhmc80.2.99.41, nhmc80.2.99.42) were used in the phylogenetic study. total genomic dna was extracted from buccal swabs using the nucleospin tissue kit (macherey-nagel) following the manufacturer’s protocol. a partial sequence of approximately 340 bp from the mitochondrial cytb gene was successfully amplified for 26 samples (including the three from rhodes, table a1), with the universal primers l14841 and h15149 (kocher et al., 1989). pcr amplifications were carried out in 12.5μl volume reactions containing 0.05 u taq (kapa biosystems), 1.5 mm mgcl2, 0.2 mm dntps, 0.4 μμ of each primer, 1x taq buffer (kapa biosystems) and 10-20 ng dna template, under the following conditions: an initial denaturation step at 94 °c for 5 min, followed by 35 cycles of denaturation at 94 °c for 60 sec, annealing at 47 °c for 60 sec, extension at 72 °c for 60 sec and a final extension for 7 min at 72 °c. pcr products were purified using the nucleospin extractii (macherey-nagel) cleanup kit and single strand sequencing was conducted by cemia (cellular & molecular immunological applications, larisa, greece). aflp analysis was performed according to a modified protocol from vos et al. (1995), for 28 samples of high quality dna, collected from the two breeding sites on karpathos, argoni (18 samples) and nati (10 samples). total genomic dna was digested using two restriction enzymes taqi and ecori (takara bio inc.). digestion was carried out in a final volume of 20 μl containing 1 mm taqi buffer, 0.1 mm bsa, 0.5u of ecori and 200 ng of genomic dna for 2 h at 37 °c. after the addition of 0.5u taqi samples were incubated for two more hours at 65 °c. the ligation was carried out in a final volume of 30 μl containing 1x ligase buffer (takara), 1.8 μm of each adaptor, 1u t4 dna ligase and the digested dna, and the reaction was incubated overnight at 16 °c. the digested-ligated dna fragments were diluted 25-fold to be used as templates for the pre-amplification reaction. each 50 µl pre-selective reaction contained 1x taq buffer, 1.5 mm mgcl2, 0.2 mm of each dntp, 0.3 μμ of each primer (t01p2, t02p2), 0.3 μμ of presel eco primer and 10 μl of the diluted ligation product. the pcr amplifications were carried out using the following profile: an initial denaturation step at 94 °c for 120 sec followed by 20 cycles of 30 sec at 94 °c, 60 sec at 56 °c, and 60 sec at 72 °c with a final extension step at 72 °c for 300 sec. the pre-amplification products were diluted 25-fold to be used as template for the selective amplification. the selective amplifications were performed in a total volume 25 μl containing 10 mm taq buffer (takara), 3 mm 119phylogeny and population structure of the karpathos frog mgcl2, 0.3 mm of each dntp, 0.2 μm of ecori primers, 0.5 μm of 5 selective primers (t101p2, t105p2, t106p2, t204p2 & t205p2) and 5 μl of diluted pre-amplified dna. selective amplification was carried out using a touchdown protocol with an initial denaturation step at 94 °c for 120 sec, 30 sec at 94 °c, 60 sec at 65 °c followed by 11 cycles where the annealing temperature was gradually reduced 0.7°c per cycle followed by 23 cycles of 30 sec at 94 °c, 30 sec at 56°c, and 60 sec at 72 °c. the primers used in each pre-selective and selective pcrs are presented in table a2. selective products were separated in fragment analyzer (advanced analytical technologies inc.) using the dsdna 910 reagent kit. aflp patterns were visualized and processed using the prosize 2.0 software (advanced analytical technologies) simultaneously by two persons to reduce scoring bias. scoring bias was quantified by calculating cohen’s kappa coefficient for inter-rater agreement. aflp profiles were scored according to presence/absence of peaks. genetic analyses mitochondrial sequences were edited by eye in mega v.7 (kumar et al., 2016) and aligned with clustal w (thompson et al., 1994). additionally, 15 sequences of p. bedriagae, which were retrieved from genbank, and one of p. cretensis that was used as outgroup, were added in the analyses. to visualize the relationships among the detected haplotypes, a median joining (mj) network (bandelt et al., 1999) was constructed with the software popart (leigh et al., 2015), excluding the outgroup sequence and setting the parameter ε equal to zero. additionally, a neighbor-joining (nj) tree (saitou and nei, 1987) was constructed according to the kimura-2 parameter substitution model, implemented in mega. the reliability of the nodes was assessed by 50000 bootstrap replications. between-population uncorrected sequence divergences (p-distance) were estimated using mega. additionally, haplotype and nucleotide diversity values within each recognized clade were calculated with dnasp v.5 software (librado and rojas, 2009). aflp data were used to estimate nei’s (1973) gene diversity and levels of genetic differentiation between the two populations (argoni and nati), using a bayesian approach with nonuniform prior distribution through 1000 bootstrap replicates implemented in the software aflp-surv (vekemans, 2002) which is based on the methods described by lynch and milligan (1994). to evaluate the presence of genetic substructure in the population, a principal coordinates analysis (pcoa) was fig. 1. sampling localities of p. cerigensis used in the study. shaded areas correspond to the current distribution of p. bedriagae. 120 elisavet a. toli et alii performed in r 3.4.1 (r core team, 2017) to visualize the clustering of individuals based on their aflp band patterns. individuals with missing data were excluded from the analysis. results the final dataset included 42 sequences (table a1) and the analysis of 286 bp of cytb revealed 12 haplotypes with high levels of haplotypic diversity (hd= 0.62). we detected 49 variable sites (excluding the outgroup) of which 27 were parsimony-informative. both the haplotype network (fig. 2) and the nj phylogenetic tree (fig. a2) revealed two well-defined lineages, which are in accordance with the geographical origin of the specimens. clade a (5 haplotypes, hd = 0.89, pi = 0.014) corresponds to bedriagae specimens of syria and cyprus, while clade b (7 haplotypes, hd =0.42, pi = 0.019) consists of the bedriagae specimens from turkey, the aegean islands and p. cerigensis. between clade p-distance was 6%, while p-distances between the various geographical locations ranged from 0.1 to 7.2% (table 1). specimens from the islands of karpathos and rhodes share one haplotype (b-1) while one additional haplotype (differing by one substitution) was found in rhodes (b-2). in total, 87 aflp fragments (fig. a1) were produced from all primer combinations and the mean number of segregating peaks per individual were 59. cohen’s kappa coefficient value was 0.83, suggesting that no scoring bias was present in our dataset. the results are summarized in table 2. the two breeding populations showed low but statistically significant levels of genetic differentiation, while levels of gene diversity are similar between them (table 2). in the pcoa plot, individuals from each breeding population formed two separated groups that widely overlapped (fig. 3). the first and the second axis accounted for 24% and 15.9% of the total variance, respectively. fig. 2. median joining network constructed using the 12 detected haplotypes presenting the two major waterfrog lineages (a, b). vertical lines correspond to the mutational steps observed between the different haplotypes. table 1. % p-distances (below diagonal) with the corresponding standard error (above diagonal) between the geographical locations of p. bedriagae and p. cerigensis. karpathos isl. 0.1 1.4 1.3 1.9 1.2 1.2 1.7 1.3 rhodes isl. 0.1 1.4 1.3 1.9 1.3 1.2 1.8 1.3 astypalaia isl. 4.6 4.7 0.9 1.7 0.9 0.9 1.5 1 chios isl. 3.6 3.8 1.8 1.5 0.6 0.6 1.3 0.8 cyprus 6.5 6.6 4.6 3.7 1.4 1.5 1 1.4 dadia 3.6 3.8 1.8 0.9 2.7 0.6 1.2 0.8 lesvos isl. 3.6 3.8 1.8 0.9 3.7 0.9 1.3 0.8 syria 7.1 7.2 5.2 4.2 2.8 3.3 4.1 1.3 turkey 4.1 4.2 2.3 1.3 3.2 1.3 1.3 3.9 table 2. gene diversity within the two populations of p. cerigensis. hj: nei’s gene diversity, s.e.: standard error, p %: level of polymorphism and fixation index (fst value). population sample size hj s.e. p % fst (95% ci) argoni 18 0.35 0.013 71.2 nati 10 0.38 0.011 67.8 0.04 (-0.03-0.01) fig. 3. a two-dimensional pcoa representation of the dissimilarities among individuals with 95% confidence ellipses. points represent sampled individuals from the two breeding sites (argoni, nati). 121phylogeny and population structure of the karpathos frog discussion taxonomic implications our results reveal two major water frog lineages in the eastern mediterranean: clade a comprises p. bedriagae from the middle east, while clade b includes both p. bedriagae specimens of east aegean sea and p. cerigensis specimens. the results suggest homogeneity in respect to mitochondrial dna for the specimens from karpathos and rhodes and reveal one shared haplotype between the karpathos and rhodes specimens and a second one in rhodes island. the two island populations comprise a single clade, in contrast to previous studies, where pelophylax specimens of rhodes clustered with specimens of p. bedriagae from asia minor (lymberakis et al., 2007). this does not seem to be the case in the present study, thus raising an argument concerning the taxonomic validity of the karpathos frog. however, the results of the above study were underpinned by different genetic markers, validating the need of utilizing both mitochondrial and nuclear markers in each study. the shared haplotype present in both islands in addition to the low levels of genetic divergence, support the presence of p. cerigensis in the neighboring rhodes, as proposed by akin et al. (2010). however, according to geological data karpathos and rhodes were already completely isolated during the pleistocene (beerli et al., 1996). although it is not clear when the two islands were separated from asia minor, geological records propose that karpathos was isolated earlier while rhodes was still connected to anatolia until late pliocene or early pleistocene (boger and dermitzakis, 1985; beerli et al., 1994). this disagreement with the paleogeographic history should be investigated using nuclear data from several species along with geological information, which could unravel the complex biogeographic history of the aegean archipelago. the high pairwise p-distances within clade b (3.6-4.7%) corroborate the divergent and complicated history of the genus pelophylax, which is linked with the events during the pleistocene. our results confirm the necessity of revision of the current taxonomy of the species by utilizing both mitochondrial and nuclear markers. in addition, further analysis of bioacoustic data could possibly shed light on the subject as they are proven to play an important role in anuran taxonomy (schneider and sinsch, 1999; padial et al., 2008). additionally, the use of more specimens covering the species’ distribution is highly recommended to verify its taxonomic validity. population structure and implications for conservation according to the population genetic analysis, both karpathos populations show moderate levels of genetic variation and differentiation. the aflp markers showed low but statistically significant levels of genetic divergence between the two population groups and similar levels of gene diversity. according to the pcoa analysis, individuals from argoni and nati form two largely overlapping clusters. the observed low inter-group differentiation reflects ongoing gene flow hence dispersal movements between the two localities may occur. in fact, these two localities are small brooks located in the opposite sides of the central mountain ridge and are separated by the main island road. the smallest distance between them is few hundred meters (approx. 500 m), a distance that lies within the dispersal capability of the species, thus dispersal movements along an elevational gradient could take place leading in a higher connectivity between them. further analysis with other genetic markers such as microsatellites could offer an insight using more polymorphic loci, and assess recent events of gene flow (manel et al., 2005). a primary objective of conservation actions is to maintain levels of diversity and heterozygosity, which are strongly recommended due to the observed population decline on karpathos. currently, the largest threat to populations of p. cerigensis is the loss of suitable breeding sites due to fragmentation and climatic change (beerli et al., 2009). small wetlands are unique ecosystems preserving amphibian populations and providing patches of suitable habitats and the overall pressure of climatic change is also apparent in other species inhabiting such fragile ecosystems. hence, there should be increased efforts in the conservation of these areas that provide suitable habitats for the karpathos marsh frog along with future studies in the biology and behavior of the species. acknowledgements sampling took place according to the hellenic national law (presidential decree 67/81) and under a special permit (code: 6ψt04653π8-υο3) issued by the ministry of the environment. the study was fully funded by the mohamed bin zayed species conservation fund. supplementary material supplementary material associated with this article can be found at <http://www.unipv.it/webshi/appendix> manuscript number 23189. 122 elisavet a. toli et alii references akın, ç., can bilgin, c., beerli, p., westaway, r., ohst, t., litvinchuk, s.n., uzzel, t., bilgin, m., hotz, h., guex, g.d., plotner, j. 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(1950): genetical structure of populations. nature 166: 247-249. acta herpetologica vol. 13, n. 1 june 2018 firenze university press acta herpetologica 14(2): 109-115, 2019 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/a_h-7748 occurrence of batrachochytrium dendrobatidis in the tensift region, with comments on its spreading in morocco redouane ait el cadi1, el-mustapha laghzaoui1, angelica crottini2, tahar slimani1, jaime bosch3,4, el hassan el mouden1,* 1 laboratory of biodiversity and ecosystem dynamic, faculty of sciences, semlalia, cadi ayyad university, marrakech, morocco. *corresponding author. email: elmouden@uca.ac.ma 2 cibio centro de investigação em biodiversidade e recursos genéticos, vairão, portugal 3 museo nacional de ciencias naturales, csic, madrid, spain 4 umib research unit of biodiversity (csic, uo, pa), universidad de oviedo, campus de mieres, spain submitted on: 2019, 27th february; revised on: 2019, 4th september; accepted on: 2019, 17th september editor: adriana bellati abstract. the chytrid fungus batrachochytrium dendrobatidis (bd) is a generalist pathogen that affects many amphibian species and is responsible of chytridiomycosis onset, considered as the main causes of species extinctions and populations declines worldwide. the chytrid fungal pathogen has been first described in north africa in 2011. the present work reported the first survey on bd prevalence and intensity in the tensift region of morocco. the survey has been conducted on 11 different localities by collecting skin swabs and tissue samples of 97 individuals. using a quantitative polymerase chain reaction (qpcr) protocol, low-intensity of bd infection has been detected in the area of study. in fact, the chytrid fungal pathogen has been identified in 10 individuals distributed in six of the 11 sites investigated, placing the 95% confidence interval for overall prevalence at 5.5-19.6%. the survey confirmed the occurrence of bd at both high and low altitude localities, on four species out of seven known to inhabit the region and added two additional species (pelophylax saharicus and sclerophrys mauritanica) to the list of bd susceptible amphibians in morocco. the present records extended bd distribution more than 400 km in the south of morocco, indicating that the chytrid fungal pathogen is more widespread in the country than previously thought. keywords. batrachochytrium dendrobatidis, amphibians, tensift, prevalence, intensity. introduction habitat degradation and overexploitation are the main large-scale factors causing loss of biodiversity worldwide (hoffmann et al., 2010). while these two factors are to blame for the rapid declines of many vertebrate species, amphibians continue to decline also in undisturbed habitats, due essentially to chytridiomycosis, an emerging infectious skin disease caused by the chytrid fungus batrachochytrium dendrobatidis (bd) (berger, 1998; longcore and pessier, 1999; bosch and martínez-solano, 2006; lips, 2016). bd is considered as the first wildlife fungal pathogen to have caused widespread species extinctions (skerratt et al., 2007), and has been implicated in rapid population declines and extinction of several amphibians (retallick et al., 2004; schloegel et al., 2006; skerratt et al., 2007; kolby and daszak, 2016; lips, 2018). it has been detected in about 48% of tested amphibian species (olson et al., 2013), and is recently known to be rapidly expanding its global range of distribution (fisher et al., 2009; o’hanlon et al., 2018). in north africa, bd has been detected for the first time in morocco at three (out of 51) tested localities, and was found in only three species (discoglos110 ait el cadi redouane et alii sus scovazzi, hyla meridionalis and pelobates varaldii), with a total prevalence of 6% (el mouden et al., 2011). the sites where bd has been detected are in the northern part of the country (tingitana peninsula and its surroundings) near south of spain. el mouden et al. (2011) explained this presence through a possible arrival from spain where chytrid fungus is largely distributed, and it’s responsible for severe decline of amphibian populations in mountain areas (bosch et al., 2001; bosch et al., 2007; walker et al., 2010; lips, 2016). these findings suggest further investigations in other areas were needed to collect more information on the potential extent of the spread of this fungal pathogen in morocco, and to elucidate its potential source. morocco is home to many endangered amphibian populations that are already threatened by habitat alteration and destruction, pollution, and climatic changes (fahd et al., 2015; ben hassine and nouira, 2012; escoriza and ben hassine, 2017). the aim of this study is to evaluate the chytrid fungal presence and prevalence using qpcr test for amphibians naturally occurred in tensift region, an area located around 400 km south from the localities where bd was detected for the first time in 2011. this region is characterized by a high diversity of habitats from wetlands (high atlas mountains) to arid environments (jbilets), with seven amphibians species known to inhabit the area, two of which are moroccan endemics (bons and geniez, 1996; beukema et al., 2013). materials and methods surveys were conducted between october 2013 and october 2017 in the tensift region (central morocco). the study area covers about 20,000 km2, surrounded by the southern crest of the high atlas mountains (with an altitude up to 4000 m a.s.l.) and the northern jbilet hills (separated by the haouz plain), while the coastline of essaouira extends in the east (fig. 1). the climate of the region is characterized by strong annual variability, with mean temperatures ranging between a maximum of 37.7 °c and a minimum of 4.9 °c. the rainfall is generally irregular, with occasional prolonged droughts. the mean annual rainfall varies from 800 mm in the mountain to 190 mm in the plain, with significant snowfall between december and march at high elevation (up to 2000 m a.s.l.) (alaoui haroni et al., 2009). the semi-arid environment dominates throughout the region, while the sub-humid zones appear at high altitude (up to 1500 m a.s.l.). the tensift region is characterized by a complex landscape and topography, including escarpments, floodplains, and a great variety of aquatic ecosystems (rivers, permanent ponds and a large network of mediterranean temporary ponds). this diversity of water ecosystems provides a wide availability of breeding sites for seven native amphibian species: hyla meridionalis boettger, 1874, bufotes boulengeri (lataste, 1879), sclerophrys mauritanica (schlegel, 1841), bufo spinosus daudin, 1803 (near threatened in morocco), barbarophryne brongersmai (hoogmoed, 1972) (endemic to morocco and algeria), pelophylax saharicus (boulenger, 1913) (endemic to north africa) and discoglossus scovazzii camerano, 1878 (endemic to morocco). amphibians were searched opportunistically in 11 sites (table 1; fig. 1), during the rainy season (when detectability of amphibians is higher), during nocturnal and occasional diurnal surveys. we extensively searched in all water bodies available in the area. after capture, animals were swabbed (using medical wire sterile dryswab™ mw100) and released immediately after sampling to the place of capture. to prevent the transmission of diseases by animals, each individual was handled with disposable gloves. in total, 86 individuals were swabbed. swabs were then air-dried and stored at cool temperature (4 °c) until processing. tissues samples have been collected from 11 specimens that were found dead in two sites fig. 1. map of the northern morocco showing the result of batrachochytrium dendrobatidis (bd) sampling sites realized during the present study (circles); dark circles indicate populations with bdpositive samples and open circles indicate populations with bdnegative samples. within bd positive sites, pie charts indicate the proportion of infected individuals in black and uninfected individuals in white. squares indicate infected populations as reported by el mouden et al. (2011). 111survey of batrachochytrium dendrobatidis in morocco (dam-oukaimden and dam d’ouled abbas). these 11 tissue samples were preserved in ethanol (brem et al., 2007; hyatt et al., 2007). in total, 97 amphibian specimens were sampled belonging to four families and six species. of these, 56 individuals were sampled in the high atlas mountains, 16 in the arid area of jbilets, 23 in the el gantour region and two in the haouz plain (table 1; fig. 1). dna was extracted from both swabs and tissue samples using prepman ultra reagent and extractions were diluted 1:10 before real-time pcr amplification, performed in duplicate with a cfx96 thermocycler (bio-rad), following boyle et al. (2004). each 96-well assay plate included samples, a negative control and standards of 100, 10, 1, and 0.1 bd zoospore genome equivalents in duplicate. samples were considered positives when both replicates were ≥ 0.1 and the amplification curves had the typical sigmoidal shape. when only one replicate from any sample amplified, we ran this sample a third time. if the third amplification did not result in an amplification profile, we considered sample as negative for infection. samples that showed signs of inhibition (non-sigmoidal amplification) were further diluted to 1:100 and re-analyzed. if signs of inhibition remained, the samples were excluded. results the numbers of screened and testing positive individuals from each population and sampling event, along with geographic information on the sampling site and year of capture are reported in table 1. the results show that 10 out of 97 screened individuals were bd infected (10.3%), corresponding to a 95% confidence interval of overall bd prevalence of 5.5-19.6%. across the four species that tested positive, the infection prevalence was 11.9% (95% ci: 5.5-19.6%). this study confirmed bd occurrence in both the northern and southern parts of the tensift watershed at elevations between 468 and 2625 m a.s.l (fig. 1). in the study area, bd was detected in six out of 11 investigated sites (table 1), with prevalence values ranging between 0 and 25%, and lower ge (genomic equivalent) values that varied between 0.3 and 26.8. these results indicate a significant presence of bd in the tensift region, with low infection prevalence and intensities across all sites that tested positive for bd. however, table 1. surveyed localities of the tensift region (morocco) with date and approximate coordinates and altitude (meters a.s.l.) of each site. species names are abbreviated as follows: sm, sclerophrys mauritanica; bb, bufotes boulengeri; br, barbarophryne brongersmai; ds, discoglossus scovazzi; hm, hyla meridionalis; ps, pelophylax saharicus. life history stage (lhs): ad, adult; juv, juvenile. p/s: number of individuals testing positive / number of analyzed individuals. bd load in genomic equivalents of zoospores. (*) indicate individual found dead in the field and tested positive for bd. date locality coordinates altitude species lhs p/s bd load oct 2013 sidi bouathman 31.9 -7.92 ~ 517 ps ad 0/1 nov 2013 ijoukak n’fis 31.059 -8.164 ~ 1042 ps juv 0/1 ad 1/3 26.8 dec 2013 sidi bouathman 31.9 -7.92 ~ 517 ps ad 0/3 dec 2013 dam d’ouled abbas 31.966 -8.447 ~ 468 sm* ad 1/7 0.3 dec 2013 dam-oukaimeden 31.208 -7.851 ~ 2623 ps ad 0/7 dec 2013 tighedouine 1 31.4 -7.532 ~ 1145 ps ad 0/6 avr 2014 tighedouine 2 31.423 -7.524 ~ 1066 ps ad 1/4 0.3 hm ad 1/7 0.4 avr 2014 ijoukak tizgui 30.971 -8.125 ~ 1142 ds ad 0/2 bb ad 0/1 ps ad 0/5 dec 2014 el gantour 1 32.189 -8.335 ~ 402 ps ad 0/5 dec 2014 el gantour 2 32.187 -8.329 ~ 398 ds ad 0/2 ps juv 0/1 ps ad 1/8 1.3 jan 2015 jaidate 31.87 -7.79 ~ 623 bb ad 0/9 br ad 0/3 ps ad 0/2 avr 2015 tighedouine 2 31.423 -7.524 ~ 1066 ps ad 1/1 1.7 jun 2016 tifergine-oukaimeden 31.207 -7.84 ~ 2565 ds ad 1/13 1.3 oct 2017 dam-oukaimeden 31.208 -7.851 ~ 2623 ps* ad 3/6 0.6, 3.0, 0.3 112 ait el cadi redouane et alii in october 2017, 50% prevalence was recorded in damoukaimden, with three testing positive samples out of the six screened individuals, which represents the maximum prevalence obtained in this study. it seems that bd prevalence can vary among species and elevations (table 2). consequently, 95% confidence intervals for bd prevalence for individuals grouped according to these two parameters were determined. all confidence intervals were overlapping. thus, no significant taxonomic and altitudinal difference in bd prevalence has been detected given our sample sizes. however, the obtained results tend towards a more significant prevalence at higher elevations (14.3% vs 4.9%). likewise, species infection rate varied between 0 and 14.3%. the higher percentage was observed in three species. bd was found in seven out of 53 p. saharicus across five sampling sites. in the dam-oukaimden site, where a large numbers of dead p. saharicus were found in october 2017, we detected bd on three out of six tested individuals (95% ci: 11.8-88.2%), which represents the highest prevalence recorded by species and by site during this study. for both h. meridionalis (tighedouine region; april 2014) and s. mauritanica (culinary dam of ouled abbas; december 2013), bd has been detected in one out of seven individuals, corresponding to a confidence interval for the two species of 0.40-57.9%. during our investigation at ouled abbas dam, numerous dead specimens of s. mauritanica were observed in the water. seven tissue samples were collected and screened. the results showed that one sample tested positive for bd. discussion in our study area, four out of six amphibian species have been tested as bd positive, although in comparison with other regions in marocco, they have lower infection rate. lower ge values are probably the result of unfavorable conditions, such as changes in the abiotic environment (ron, 2005; thorpe et al., 2018). it has been reported that bd is temperature sensitive, with optimal growth ranging between 17 and 25 °c (piotrowski et al., 2004; pounds et al., 2006), with higher temperature (more than 30 °c) reported as unfavorable for its development (watve, 2013). previous studies have shown that lower temperature regime resulted in extended zoospore longevity and in such conditions zoospores numbers in water bodies could be expected to be greater than in warmer water (voyles et al., 2012; thorpe et al., 2018). the higher temperatures (more than 30 °c) recorded in the study area expected to have a negative impact on the development of this fungal pathogen. additionally, optimum rainfall for bd development has been reported to range between 1500 and 2500 mm/year (thorpe et al., 2018), which are much higher values of the one observed in the tensift region. only, oukaimden approach these rainfall values, but in general remains below to optimum parameters for the development of bd. in morocco, bd surveys were previously conducted only in the northern tingitana area. el mouden et al. (2011) reported bd occurrence in three sampling sites. through this study, bd occurrence has been reported in six additional moroccan localities. considering all these data, 14.5% of the screened moroccan localities tested positive to bd. the finding that bd was recorded in two separated areas suggested a possible wider distribution across the country. similarly, bd was detected in different sites ranging from 400 to over 2600 m a.s.l, indicating a probable wide distribution also along the altitudinal gradient. sample size is still limited and should be extended to have a more robust overview of the pattern of bd presence across morocco. the report on the occurrence of bd in the tingitana peninsula (north of morocco) was potentially explained by the extensive commercial trade of products and animals across the straits of gibraltar with a possible introduction of bd from spain (el mouden et al., 2011). this hypothesis is in agreement with the explanations of o’hanlon et al. (2018) who found bd spreading out of asia and dated its emergence on the early 20th century, coinciding with the international expansion of commercial trade in amphibians for exotic pet, medical, and food purposes. the presence of bd in the tensift region can be explained by a dissemination process from the north table 2. bd prevalence with 95% clopper-pearson binomial confidence intervals for individuals grouped by species and elevation. group sample size bd prevalence (%) 95% ci family/species ranidae pelophylax saharicus 53 13.2 5.6 25.8% alytidae discoglossus scovazzi 17 5.9 0.1 28.7% bufonidae bufotes boulengeri 10 0 0 30.8 % sclerophrys mauritanica 7 14.3 0.4 57.9% barbarophryne brongersmai 3 0 0 70.8% hylidae hyla meridionalis 7 14.3 0.4 57.9% elevation (for all species) less than 700 m 41 4.9 0.6 16.2% more than 700 m 56 14.3 6.1 25.4% 113survey of batrachochytrium dendrobatidis in morocco of the country to the south, but also by a possible independent episode of introduction through other commercial routes, such as airports and harbors. the spreading of bd through the country can be carried out by potential natural vectors, including waterfowl on their feathers or feet (johnson and speare, 2005; garmyn et al., 2012; burrowes and de la riva, 2017), water (johnson and speare, 2003), and non-susceptible to chytridiomycosis amphibians acting as carrier (kolby et al., 2015). in morocco, as in most regions around the world, the detected bd belongs to the global panzootic lineage (j. bosch, unpublished data). this is a highly virulent and highly transmissible chytrid fungus, which is currently infecting more that 700 amphibian species worldwide (olson and ronnenberg, 2014; lips, 2016). we detected a mass mortality of amphibians in two localities (s. mauritanica in dam d’ouled abbas and p. saharicus in dam-oukaimden) and some of the sampled individuals tested positive for bd infection. however, in the absence of a detailed study, we have no evidence of bd-associated amphibian mortality especially because mortality of individuals generally occurred with higher infection rates than those found in the present study. other biotic and abiotic factors can be the cause of the witnessed mass mortality (e.g., croteau et al., 2008; hayes et al., 2010; relyea et al., 2012; whittaker et al., 2013; budzik et al., 2014; de wijer et al., 2018). however, it is worth noting that the two new species found infected by bd (p. saharicus and s. mauritanica) have a wide distribution and abundance in the southern mediterranean region (bons and geniez, 1996; schleich et al., 1996; mateo et al., 2013), which can make bd dissemination faster. a systematic survey to determine the impact of bd occurrence in morocco is crucial to better characterize its impact on the amphibian’s population dynamic. in the mean time, other bd positive sites located in tensfit region have been recorded and helped extending the known bd occurrence area in morocco, both at the high and the low altitudes (486-2625 m a.s.l.). in addition, having detected bd at multiple sites and in two new amphibian species contributes to the growing knowledge on the global pattern of bd distribution in morocco and north africa. acknowledgments we would like to thank “haut commissariat aux eaux et forêts et à la lutte contre la désertication (hceflcd)” for the permit to work on the field. c. monsalve-caraño helped with qpcr analyses. this research was supported by project [icgvsa] founded by the hassan ii academy of sciences and technology. the portuguese national funds through fct (foundation for science and technology) support the investigator fct grant to ac (if/00209/2014). references alaoui haroni, s., alifriqui, m., simonneaux, v. 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(2013): global declines of amphibians. in: encyclopedia of biodiversity, second edition, pp. 691-699. levin s.a., ed, academic press, cambridge. acta herpetologica vol. 14, n. 2 december 2019 firenze university press podarcis siculus latastei (bedriaga, 1879) of the western pontine islands (italy) raised to the species rank, and a brief taxonomic overview of podarcis lizards gabriele senczuk1,2,*, riccardo castiglia2, wolfgang böhme3, claudia corti1 substrate type has a limited impact on the sprint performance of a mediterranean lizard pantelis savvides1,*, eleni georgiou1, panayiotis pafilis2,3, spyros sfenthourakis1 coping with aliens: how a native gecko manages to persist on mediterranean islands despite the black rat? michel-jean delaugerre1,*, roberto sacchi2, marta biaggini3, pietro lo cascio4, ridha ouni5, claudia corti 3 pit-tags as a technique for marking fossorial reptiles: insights from a long-term field study of the amphisbaenian trogonophis wiegmanni pablo recio, gonzalo rodríguez-ruiz, jesús ortega, josé martín* occurrence of batrachochytrium dendrobatidis in the tensift region, with comments on its spreading in morocco ait el cadi redouane1, laghzaoui el-mustapha1, angelica crottini2, slimani tahar1, bosch jaime3,4, el mouden el hassan1,* hematological parameters of the bolson tortoise gopherus flavomarginatus in mexico cristina garcía-de la peña1,*, roger iván rodríguez-vivas2, jorge a. zegbe-domínguez3, luis manuel valenzuela-núñez1, césar a. meza herrera4, quetzaly siller-rodríguez1, verónica ávila-rodríguez1 ontogenetic and interspecific variation in skull morphology of two closely related species of toad, bufo bufo and b. spinosus (anura: bufonidae) giovanni sanna visible implant alphanumeric (via) as a marking method in the lesser snouted treefrog scinax nasicus andrea caballero-gini1,2,3,*, diego bueno villafañe2,3, lía romero2, marcela ferreira2,3, lucas cañete4, rafaela laino2, karim musalem2,5 morphological variation of the newly confirmed population of the javelin sand boa, eryx jaculus (linnaeus, 1758) (serpentes, erycidae) in sicily, italy francesco p. faraone1,*, salvatore russotto2, salvatore a. barra3, roberto chiara3, gabriele giacalone4, mario lo valvo3 variability in the dorsal pattern of the sardinian grass snake (natrix natrix cetti) with notes on its ecology enrico lunghi1,2,3,4,*, simone giachello5, manuela mulargia6, pier paolo dore7, roberto cogoni8, claudia corti1 estimating abundance of the stripeless tree-frog hyla meridionalis by means of replicated call counts federico crovetto, sebastiano salvidio, andrea costa* at-rich microsatellite loci development for fejervarya multistriata by illumina hiseq sequencing yan-mei wang, jing-yi chen, guo-hua ding*, zhi-hua lin acta herpetologica 12(1): 19-27, 2017 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-20245 sex does not affect tail autotomy in lacertid lizards panayiotis pafilis1,*, kostas sagonas2, grigoris kapsalas1, johannes foufopoulos3, efstratios d. valakos4 1 department of zoology and marine biology, school of biology, national and kapodistrian university of athens, panepistimioupolis, ilissia 15784, greece. *corresponding author. e-mail: ppafil@biol.uoa.gr 2 school of biological and chemical sciences, queen mary university of london, mile end road, london e1 4ns, united kingdom 3 school of natural resources and environment, dana building, 430 east university, university of michigan, ann arbor, mi 481091115, usa 4 department of animal and human physiology, school of biology, national and kapodistrian university of athens, panepistimioupolis, ilissia 15784, greece submitted on 2017, 26th january; revised on 2017, 16th february; accepted on 2017, 22nd february editor: marco mangiacotti abstract. caudal autotomy is one of the most effective and widespread defensive mechanisms among lizards. when predators grasp the tail, lizards are able to shed it from the point of the attack and further. numerous factors have been reported to affect tail-shedding performance such as temperature, age, predation pressure, intraspecific competition etc. interestingly, the impact of sex on tail loss remains greatly understudied. here, we analyzed tail autotomy performance, simulated in the lab, in 12 species of lacertid lizards belonging to five genera (algyroides, anatololacerta, hellenolacerta, ophisops, podarcis). our aim was to investigate whether sex affects caudal autotomy and/or the duration of post-autotomic tail movement. we failed to detect any effect of sex on tail loss in the species examined. also, we did not find any sexual impact on the duration of tail movement after autotomy, with a single exception. our findings suggest that autotomy serves as a defensive tactic equally in both sexes and is used in the same extent. keywords. predation, intraspecific competition, defense, greece. introduction autotomy, the self-amputation of a body limb, is rather rare among vertebrates: the behavior is restricted to reptiles (cooper and alfieri, 1993; hoare et al., 2006), salamanders (marvin, 2010; romano et al., 2010) and very few mice (seifert et al., 2012). undoubtedly, lizards are the champions of autotomy (arnold, 1984; bellairs and bryant, 1985). most families of the suborder shed their tail in response to mechanical stimuli exerted by a predator’s attack (arnold, 1987; downes and shine, 2001; bateman and fleming, 2009). the identity of the predator may vary. it could be any occasional or specialized saurophagous predator (e.g., snakes, birds, mammals) as traditional theory predicts (pianka, 1970; turner et al., 1982; cooper et al., 2004), or a conspecific, triggered by intraspecific competition, as recent literature suggests (pafilis et al., 2009b; donihue et al., 2016; itescu et al., 2017). in either case the result remains the same: the shed tail trashes vigorously, fuelled by anaerobic metabolism, to distract the predator while the tailless lizard escapes (dial and fitzpatrick, 1983; pafilis et al., 2005). many factors are known to affect caudal autotomy such as temperature, age and body shape (arnold, 1984; daniels, 1984; pafilis and valakos, 2008; fleming et al., 2013). previous studies on other aspects of autotomy did not report sexual effects on the trait, without though focusing on this particular issue (chapple and swain, 2004; pafilis et al., 2005; brock et al., 2015, but see itescu et al., 2017). however there are several clues indicat20 p. pafilis et alii ing that tail autotomy could be sexually biased (pérezmellado et al., 1997; bateman and fleming, 2009). male lizards, particularly those belonging to territorial species, expose themselves in their effort to defend their territory (kaiser and mushinsky, 1994; salvador and veiga, 2001). thus, typical ‘alien’ predators (non conspecifics) have better chances to prey on the more conspicuous males that patrol or oversee their territory, a fact that could lead to higher autotomy rates (bateman and flemming, 2011; marshall et al., 2016). also, intramale competition may increase tail loss. male lizards are much more aggressive against their peers compared to females (kwiatkowski and sullivan, 2002; lailvaux and irschick, 2007; mcevoy et al., 2013). ergo, agonistic encounters between males are more common and may end up to tail loss (cooper and vitt, 1987; bateman and fleming, 2009; cooper et al., 2015). nonetheless, a byproduct of this increased intraspecific competition could be a higher propensity of males to hold on to tails more strongly. on the other hand, females are expected to lessen their ability to autotomize because of the high energetic demands of vitellogenesis and offspring production (dial and fitzpatrick, 1981; hare and miller, 2010). species with high reproductive output (massive clutches) restrict or even completely lose autotomic abilities to offset the high costs of caudal autotomy (pafilis and valakos, 2008). also, intrafemale competition is minimal since females do not defend territories (braña, 1996; moreira et al., 2006). moreover, in some lizard families where males maintain harems, females interact frequently and do not compete (zamudio and sinervo, 2000). hence females do not attack each other and incidents of tail loss are rare (cooper et al., 2015). tail autotomy comes with many disadvantages, such as degradation of social status (fox et al., 1990; salvador et al., 1995), loss of caudal fat that many species store in their tail (roig et al., 2000; chapple and swain, 2002a; cencetti et al., 2011), alterations in locomotion (chapple and swain, 2002b; cromie and chapple, 2012; savvides et al., 2017, but see kelehear and webb, 2006), reduction of the immune function (slos et al., 2009) and impaired reproduction (fox and mccoy, 2000; chapple et al., 2002). nonetheless, in the case of intraspecific predation there is a clear advantage for the conspecific predator. by shedding and consuming a conspecific tail, males kill two birds with one stone: they may reduce their rival’s ability to mate (fox and rostker, 1982; martín and salvador, 1993) and gain an energetically rich meal (mcconnachie and whiting, 2003; cooper et al., 2015). hence, intraspecific predation comes with a strong advantage, particularly favorable beneficial for males. in this study we aimed to clarify whether sex influences caudal autotomy performance. to this end we simulated tail shedding in the lab in 12 species of lacertid lizards. first, we hypothesized that since males are more exposed to predation (interor intraspecific) due to their particular social role, they would demonstrate higher rates of tail loss. in the case of insular species though, conditions are more complicated. predation is more relaxed on the islands (macarthur and wilson, 1967; whittaker and fernándezpalacios, 2007) and this drives to higher lizard densities (rodda and dean-bradley, 2002; buckley and jetz, 2007) that trigger more intense intraspecific competition (knell, 2009; raia et al., 2010). relaxed predation advocates lower autotomy rates (traditional theory) whereas intraspecific competition suggests higher ones (recent approach), which would be even higher among males due to more frequent intraspecific agonistic encounters (mougeot et al., 2003; kokko and rankin, 2006; cooper et al., 2015). second, we expected that post-autotomy duration of tail movement between males and females would not differ, as this feature appears to be conservative among species (pafilis et al., 2005; pafilis et al., 2008). material and methods study species we examined the rates of caudal autotomy in 12 lacertid lizards assigned in five genera: algyroides (a. moreoticus and a. nigropunctatus), hellenolacerta (h. graeca), ophisops (o. elegans) and podarcis (p. cretensis, p. peloponnesiacus, p. erhardii, p. gaigeae, p. milensis, p. muralis and p. tauricus) (table 1 for sample sizes 914 individuals in total). the focal species are distributed in different locations and habitats in mainland and insular greece (fig. 1). all of them are small, diurnal insectivorous predators, with snout vent length (svl) varying from 55 up to 85 mm (valakos et al.; 2008). for each individual we recorded svl, sex and the condition of the tail (intact or regenerated). for the purposes of this study we worked exclusively with adult individuals with intact tails. captured lizards were transferred to the laboratory facilities of the department of biology at the university of athens. all animals were housed individually in vitreous terraria (18 × 32 × 15 cm) with sand and artificial shelters and were held at 30 oc under a controlled photoperiod with fluorescent tube lighting (12 h light: 12 h dark). an incandescent heat lamp (60 watts) above each terrarium allowed lizards to thermoregulate for eight hours per day. lizards were fed every other day with mealworms coated with supplementary vitamins and minerals and had access to water ad libitum. predation simulation and postautotomy tail movement prior to the experimental procedure food was withheld from lizards for two days (pafilis et al., 2009a). we simulated predation using the method proposed by pérez-mellado et al. 21no sexual effect on tail autotomy (1997). since body temperature may affect caudal autotomy (bustard, 1968; daniels, 1984), lizards were allowed to thermoregulate for two hours in a specially designed terrarium (100 × 25 × 25 cm) with two ice bags at one end and two heating lamps (100 w and 60 w) at the other end that provided a thermal gradient ranging from 10 to 50 oc (van damme et al., 1986). after achieving its preferred body temperature, each lizard was placed in a terrarium with cork substrate in order to maintain good traction during predation simulation. we used a pair of calipers to simulate the bite of a predator and grasped the tail 20 mm behind the cloaca for 15 sec. to standardize pressure, the calipers were closed to half the original diameter of the tail (pérez-mellado et al. 1997). lizards were free to react and were not restrained. if autotomy occurred, we recorded the duration of movement of the shed, thrashing tail from the moment of autotomy to complete cessation of movement (no continuous twitches for 20 sec) using a digital timekeeper. fig. 1. map of the collecting sites in mainland and insular greece, ne mediterranean basin. 22 p. pafilis et alii statistics we examined the normality of our data using the kolmogorov-smirnov and lilliefors normality tests. whenever parametric assumption was not met, data were log-transformed. t-test was used to compare the svl between two sexes. chisquare test followed by fischer exact test was used to compare autotomy performance between the two sexes in each species. we used analysis of variance (anova) to compare the duration of tail movement between the two sexes in each species. in order to eliminate the influence of svl on the duration of tail movement we repeated the above-mentioned analysis using the svl as covariate (ancova). all comparisons were conducted independently for each species. all statistical analyses were conducted in r 3.2.5 (r development core team, 2015). results the comparison of body length revealed significant sexual dimorphism for all species (table 1), with males being larger than females (the opposite pattern was revealed only in one species, podarcis muralis). chisquare test showed that rates of caudal shedding (all p > 0.05; table 2) did not differ between sexes within each species (fig. 2). the same lack of sexual differences was detected regarding the post-autotomy duration of tail movement (all p > 0.05; table 3). discussion caudal autotomy is quite common among lizards and a growing body of literature continuously provides new insights. however, the impact of sex on tail loss remains rather obscure. here, we examined the possible sexual effect by investigating aspects of tail autotomy. our results clearly refuted our first working hypothesis. we found no sexual differences in the rates of tail shedding in the 12 focal species. our analyses revealed that insular species also conformed to this pattern. in line with our second prediction, we found no difference in the duration of the post-autotomic movement between sexes. sex had no effect on caudal loss. though several differences arose from the rates of tail shedding among species unveiling striking differences (e.g., 80% in a. nigropunctatus compared to 32% in p. gaigeae), intraspecies analyses yield a uniform pattern in tail autotomy performance between males and females. contrary to our initial prediction, the higher exposure to predators and the table 1. values for snout-vent length (svl; mm) in males and females for all species and t-test results (p > 0.05, ns; p ≤ 0.05, *; p ≤ 0.01, **; p ≤ 0.001, ***). means ± standard deviation; sample size in parenthesis. species males females t-test algyroides moreoticus 48.07 ± 1.25 (22) 44.43 ± 1.85 (15) *** algyroides nigropunctatus 62.08 ± 3.13 (26) 58.29 ± 2.44 (18) *** anatololacerta oertzeni 65.18 ± 1.91 (26) 60.10 ± 2.10 (20) *** hellenolacerta graeca 80.16 ± 2.24 (23) 77.47 ± 2.31 (17) *** ophisops elegans 55.33 ± 2.18 (24) 51.18 ± 1.78 (17) *** podarcis cretensis 64.65 ± 2.11 (17) 58.34 ± 2.14 (14) *** podarcis peloponnesiacus 85.01 ± 2.23 (46) 80.49 ± 2.04 (42) *** podarcis erhardii 66.90 ± 4.69 (40) 63.89 ± 5.05 (29) * podarcis gaigeae 61.02 ± 3.44 (19) 55.74 ± 4.36 (18) *** podarcis milensis 65.22 ± 2.09 (24) 60.04 ± 2.24 (16) *** podarcis muralis 68.15 ± 2.22 (28) 70.35 ± 1.92 (13) ** podarcis tauricus 79.59 ± 2.89 (18) 75.08 ± 2.41 (14) *** table 2. differences in tail autotomy rates between males and females for all species and chi-square (χ2) test results (df = degree of freedom). species χ2 df p algyroides moreoticus 0.099 1 0.753 algyroides nigropunctatus 0.076 1 0.783 anatololacerta oertzeni 0.172 1 0.687 hellenolacerta graeca 0.360 1 0.549 ophisops elegans 0.022 1 0.881 podarcis cretensis 0.121 1 0.728 podarcis peloponnesiacus 0.022 1 0.883 podarcis erhardii 0.216 1 0.642 podarcis gaigeae 0.067 1 0.795 podarcis milensis 0.286 1 0.593 podarcis muralis 0.134 1 0.715 podarcis tauricus 0.168 1 0.682 23no sexual effect on tail autotomy different levels of intrasexual aggressiveness were not transformed into higher autotomy rates for males (fig. 2). our findings suggest that both sexes employ caudal autotomy at the same extent, at least among lacertids. the few studies that have assessed the impact of sex on tail autotomy provide contradictory results. on the one hand, itescu et al. (2017) reported that male geckos (mediodactylus kotshyi) had higher autotomy frequencies fig. 2. rates of laboratory autotomy (dark bars for males and light bars for females). black diamonds denote island species. table 3. values for the duration of post-autotomy tail movement (min) in males and females for all species and anova and ancova results (p values in parenthesis): means ± standard deviation; sample sizes are the same reported in table 1. species males females anovas ancovas algyroides moreoticus 5.19 ± 0.25 5.34 ± 0.31 f1,35 = 2.88 (0.099) f1,34 = 0.035 (0.851) algyroides nigropunctatus 5.29 ± 0.31 5.15 ± 2.44 f1,42 = 4.04 (0.051) f1,41 = 3.68 (0.062) anatololacerta oertzeni 6.15 ± 0.27 6.31 ± 0.29 f1,44 = 3.89 (0.055) f1,43 = 0.09 (0.762) hellenolacerta graeca 6.55 ± 0.22 6.69 ± 0.23 f1,38 = 3.72 (0.061) f1,37 = 2.78 (0.104) ophisops elegans 4.95 ± 0.23 5.09 ± 0.23 f1,39 = 3.43 (0.072) f1,38 = 1.1, (0.292) podarcis cretensis 5.54 ± 0.27 5.71 ± 0.27 f1,29 = 2.86 (0.101) f1,28 = 3.09 (0.089) podarcis peloponnesiacus 6.48 ± 0.35 6.34 ± 0.34 f1,86 = 3.63 (0.060) f1,85 = 1.95 (0.167) podarcis erhardii 6.14 ± 0.30 6.31 ± 0.05 f1,67 = 3.09 (0.083) f1,66 = 2.76 (0.101) podarcis gaigeae 5.71 ± 0.30 5.55 ± 0.23 f1,35 = 3.44 (0.076) f1,34 = 3.51 (0.069) podarcis milensis 6.46 ± 0.24 6.61 ± 0.25 f1,38 = 3.76 (0.060) f1,37 = 3.81 (0.060) podarcis muralis 6.23 ± 0.31 6.14 ± 0.29 f1,39 = 0.86 (0.360) f1,38 = 1.23 (0.273) podarcis tauricus 5.45 ± 0.22 5.59 ± 0.21 f1,30 = 3.32 (0.079) f1,42 = 0.56 (0.459) 24 p. pafilis et alii than females in 31 different populations. these authors attributed the higher male autotomic rates to the more intense intraspecific competition. on the other hand, fox et al. (1998) found that males of the phrynosomatid lizard uta stansburiana shed their tail less easily compared to females and retain it more strongly as they approach sexual maturity. the latter researchers ascribed the tendency of males to avoid autotomy to the significance of the tail in reproductive success (fox et al., 1998). the above indicate that phylogeny might have a distinct role on the sexual differentiation of autotomy. coming to lacertids, although aggressive interactions with conspecific are well known (castilla and van damme, 1996; salvador and veiga, 2001; cooper et al., 2015), they do not seem to account for sexual differences in tail shedding performance according to our results. at this point we have to stress out an important caveat in our study: both sampling in the field and experimental procedure in the lab took place during the nonreproductive period. reproduction triggers major shifts in lizards (bauwens and thoen, 1981; brodie, 1989). future mothers avoid exposing themselves to open areas so as to avoid predation, and adopt a more cryptic behavior (shine, 1980; karasov and anderson, 1984; braña, 1993). on the contrary, males are much more active and aggressive during the same period as they protect their territory and fight against rivals for access to females (martín and forsman, 1999; salvador and veiga, 2001; troncoso-palacios and labra, 2012). most probably, interand intraspecific predation pressure during the reproductive period would differ because of the dramatic behavioral shifts that both sexes undergo. reassessment of tail autotomy performance during this period would shed further light on the impact of reproduction (e.g., cooper et al., 2009). despite the two contradicting drivers of tail autotomy prevailing on islands (low predation and high intraspecific competition), islanders followed the same pattern with mainland species and showed no sexual differences in tail autotomy performance (fig. 2). however, island size might play a role. lizard densities are higher on islands thanks to ecological release (buckley and jetz, 2007; novosolov et al., 2016) and this applies to mediterranean lacertids as well (chondropoulos and lykakis, 1983; adamopoulou, 1999; scalera et al., 2004). the highest densities, though, have been reported from very small islets (castilla and bauwens, 1991; pérez-mellado et al., 2008; pafilis et al., 2013). intraspecific competition peaks on these islets and very often includes consumption of conspecific limbs such as tails (raia et al., 2010; donihue et al., 2016; lymberakis et al., 2016). cannibalism on these islets may be as common that lizards may change their physiology and morphology to cope with this extreme intraspecific competition (pafilis et al., 2016). such antagonistic encounters are much more common and intense among males (brock et al., 2015; cooper et al., 2015). our study was carried out on large islands that might host abundant populations (varying from 76 to 396 individuals per hectare) but certainly not as dense as those on the small mediterranean islets. also, we have to clarify that these large islands had considerably many predators, fewer though compared to the mainland (pafilis et al., 2009a). repeating the experiment on predatorfree islet populations might yield useful new insights. the duration of post-autotomy movement did not differ between sexes, in accordance with our second prediction. at this point, we have to stress out that though there was marginal significance in the duration of tail thrashing post autotomy between the sexes, this difference was eliminated by taking into account body size. shed tails thrashed between five and seven minutes (table 3), receiving values that fall within the same range with other greek lacertids (pafilis et al., 2005; pafilis et al., 2009a). our findings come to corroborate previous reports on podarcis lizards (pafilis et al., 2005; pafilis et al., 2008). duration of movement after tail shedding is very important for the successful escape from predators (dial and fitzpatrick, 1983; cooper et al., 2004). we believe that this importance is reflected in the lack of sexual differentiation in post-autotomy movement. we report that tail shedding performance and post-autotomic duration are not affected by sex. future research that would repeat the experiment during the reproductive period and include gravid females would provide further interesting results. also, experimental work on small islets would shed light on the impact of intense intramale competition on autotomy rates. acknowledgements lizards were captured during the non-reproductive period (july to october, 2000 and 2001), handled and housed in accordance with greek national law (presidential decree 67/81). at the end of the experimental procedures all animals were released in the habitats they derived from. references adamopoulou, c. 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(2000): polygyny, mateguarding, and posthumous fertilization as alternative male mating strategies. proc. natl. acad. sci. usa 97: 14427-14432. acta herpetologica vol. 12, n. 1 june 2017 firenze university press morphological variation and sexual dimorphism in liolaemus wiegmannii (duméril & bibron, 1837) (squamata: liolaemidae) from uruguay joaquín villamil1,*, arley camargo2, raúl maneyro1 sex does not affect tail autotomy in lacertid lizards panayiotis pafilis1,*, kostas sagonas2, grigoris kapsalas1, johannes foufopoulos3, efstratios d. valakos4 fire salamander (salamandra salamandra) males’ activity during breeding season: effects of microhabitat features and body size raoul manenti*, andrea conti, roberta pennati call variation and vocalizations of the stealthy litter frog ischnocnema abdita (anura: brachycephalidae) pedro carvalho rocha1,2,*, joão victor a. lacerda2,3, rafael félix de magalhães2,3, clarissa canedo4, bruno v. s. pimenta5, rodrigo carrara heitor6, paulo christiano de anchieta garcia2,3 feeding ecology of two sympatric geckos in an urban area of northeastern brazil josé guilherme g. sousa1, adonias a. martins teixeira2,*, diêgo alves teles2, joão antônio araújo-filho2 and robson waldemar ávila3 a pattern-based tool for long-term, large-sample capture-mark-recapture studies of fire salamanders salamandra species (amphibia: urodela: salamandridae) jeroen speybroeck1,*, koen steenhoudt2,$ skeletal variation within the darwinii group of liolaemus (iguania: liolaemidae): new characters, identification of polymorphisms and new synapomorphies for subclades linda díaz-fernández*, andrés s. quinteros, fernando lobo marking techniques in the marbled newt (triturus marmoratus): pit-tag and tracking device implant protocols hugo le chevalier1, olivier calvez2, albert martínez-silvestre3, damien picard4, sandra guérin4,5, francis isselin-nondedeu6, alexandre ribéron1, audrey trochet1,2,* evidence for directional testes asymmetry in hyla gongshanensis jindongensis qing gui wu1, wen bo liao2,* the advertisement call of pristimantis subsigillatus (anura, craugastoridae) florina stănescu1,4, rafael márquez2, paul székely3,4,*, dan cogălniceanu1,4,5 nematodes infecting anotosaura vanzolinia (squamata: gymnophthalmidae) from caatinga, northeastern brazil bruno halluan s. oliveira¹, adonias a. martins teixeira¹,*, romilda narciza m. queiroz¹, joão a. araujo-filho¹, diêgo a. teles¹, samuel v. brito2, daniel o. mesquita¹ where is my place? quick chorus structure assembly in the european tree frog michal berec a possible mutualistic interaction between vertebrates: frogs use water buffaloes as a foraging place piotr zduniak1,*, kiraz erciyas-yavuz2, piotr tryjanowski3 good vibrations: a novel method for sexing turtles donald t. mcknight1,2,*, hunter j. howell3, ethan c. hollender1, and day b. ligon1 errata to mecke, s., hartmann, l., mader, f., kieckbusch, m., kaiser, h. (2016): redescription of cyrtodactylus fumosus (müller, 1895) (reptilia: squamata: gekkonidae), with a revised identification key to the bent-toed geckos of sulawesi. acta herpetologica 11(2): acta herpetologica 13(2): 155-163, 2018 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-22830 short term spatial structure of a lizard (darevskia sp.) community in armenia neftalí sillero1,*, elena argaña1, susana freitas2,3, enrique garcía-muñoz2, marine arakelyan4, claudia corti5, miguel a. carretero2 1 cicge centro de investigação em ciências geo-espaciais, faculdade de ciências da universidade do porto (fcup), observatório astronómico prof. manuel de barros, alameda do monte da virgem, 4430-146, vila nova de gaia, portugal. *corresponding author. e-mail: neftali.sillero@gmail.com 2 cibio research centre in biodiversity and genetic resources, inbio, universidade do porto, campus de vairão, rua padre armando quintas, nº 7. 4485-661 vairão, vila do conde, portugal 3 department of animal and plant sciences, the university of sheffield, sheffield s10 2tn, uk 4 faculty of biology, yerevan state university, alek manoogian 1, 0025, yerevan, armenia 5 museo di storia naturale dell’università di firenze, sezione di zoologia “la specola”, via romana 17, 50125 firenze, italia submitted on: 2018, 6th march; revised on: 2018, 18th september; accepted on: 2018, 30th september editor: simon baeckens abstract. factors driving the spatial patterns of communities of sedentary organisms are still poorly understood. in this context parthenogenetic animals are useful to test the contribution of sexual and interspecific interactions on spatial patterns. as such, mixed communities of asexual and sexual species are expected to be spatially organized as a single sexual species, with sexes randomly distributed and mutually independent. during the reproductive period, we determined the instantaneous spatial structure in a community of darevskia rock lizards from armenia composed of one sexual species (d. valentini), two asexual species (d. armeniaca, d. unisexualis), and their hybrids. we also analysed the specific composition of clusters and the species segregation by habitat. we used the ripley’s k distance function to measure clustering spatial patterns, and the delaunay’s triangulation to identify the clusters and their specific composition. we estimated the spatial segregation among species by calculating the overlap between species pairs, by comparing pairwise distances from males to other males and from males to females, and by comparing the frequencies of both sexes and reproduction modes (asexual and sexual) in plant cover and height using log-linear models. species displayed a clustered spatial structure, with parthenogens (mainly d. armeniaca) or their hybrids in all clusters. females and males were concentrated in areas with medium plant cover. d. armeniaca and d. valentini were the species with the highest overlap. males were closer to males than to females. this community displays an instantaneous spatial pattern resembling a population of a single sexual species. spatial statistics offer new insights to analyse the spatial structure of species communities. keywords. armenia, darevskia, gis, local distribution patterns, spatial statistics. introduction the analysis of the short-term spatial structure within a community of species with low dispersal allows understanding how individuals share the space and modify their home ranges depending on the presence of other species (competitors, predators, prey items) or other environmental factors (temperature, shelters) (sillero and goncalves-seco, 2014; sillero and gomes, 2016). contrary to home range analyses on the spatial needs of single individuals, species distribution analysis focuses on the presence of one or more species in a particular geograph156 neftalí sillero et alii ical area characterized by certain environmental variables (mainly climate) at coarse scales (regional or continental) (sillero et al., 2014; sillero and gomes, 2016). linking both analytical levels, community spatial distributions can identify the main environmental factors with a high spatial resolution, applying techniques of species distribution studies to the same scale of home ranges but during short time periods and with a different purpose. namely, the community spatial distribution approach aims at providing a snapshot of the multispecies spatial patterns at a local scale. as such, species’ interactions can be revealed without collecting repeatedly individual data (as required for home range studies). species within a community can be distributed randomly, regularly, or in clusters (gorton et al., 1979; frost and bergmann, 2012). random distributions are typical of non-territorial or non-competing species living in habitats with abundant and widespread resources. the probability of finding an individual is the same across the study area and independent from the presence of other individuals. species can be regularly distributed when individuals avoid being mutually close (due competition or territoriality) and resources are evenly distributed. species appear clustered when the resources are irregularly distributed, hence, the probability to find either a second individual near the first or areas without individuals is higher than expected by random. clustering is the most frequent pattern observed in communities (underwood and chapman, 1996) with variable intensity (moody et al., 1997). however, distribution patterns may also shift in time. for instance, some species shift from regular to clustered distribution as population density increases (gorton et al., 1979). many potential factors may drive the spatial structure of a multispecies community, including the distribution of energy or matter sources (light, water, soil nutrients, food), availability of shelters and resting places, presence of other species (predators, competitors, parasites), mates and other conspecifics. in animals, few studies describe how populations are locally distributed (frost and bergmann, 2012; sillero and gonçalves-seco, 2014) and even less considered as well species interactions (underwood and chapman, 1996). therefore, the factors driving the distribution pattern of a community are still poorly understood. in this context, the spatial ecology of parthenogenetic (all-female) species can be particularly elucidative as it may provide insights on the contribution of sexual reproduction to species’ spatial patterns. are parthenogenetic species using the space in a similar way as their sexual relatives? how are both types of species spatially organized when they coincide? in particular, parthenogenetic lizards can provide an excellent model system for testing the effects of sexual and interspecific interactions on spatial patterns. some studies analysed the home ranges of single parthenogenetic lizards (eifler and eifler, 1998; galoyan, 2013), but only sillero et al. (2016) considered several species in sympatry and none analysed the community spatial distributions. according to the lizard literature, adult males and females often are randomly distributed, while juveniles have a regular distribution, as they are excluded to less suitable habitats (frost and bergmann, 2012; sillero and gonçalves-seco, 2014). in principle, a population of a parthenogenetic species may have a more clustered distribution. in fact, our non-systematic observations reported groups of many females basking together in the same spot, as expected from the lack of mate competition and aggregation for resources. however, following sillero et al., (2016), if a mixed community composed of asexual and sexual species behaves like a population composed by a single sexual species, we should expect a similar spatial pattern: both sexes distributed randomly (frost and bergmann, 2012). therefore, if there is no competition for limited resources but only interferences among individuals (žagar et al., 2015) of asexual and sexual species, we should expect their distributions to be mutually independent (depending only on conspecifics and resources). on the contrary, if they compete for space we should expect larger distance between heterospecifics than between conspecifics. the main aim of this work is, hence, to analyse the instantaneous spatial structure of a lizard mixed community in order to determine whether different species segregate spatially or not. here, we studied a community of lizards in true sympatry composed of several darevskia species (darevskia armeniaca, d. valentini, d. unisexualis) and their hybrids at kuchak, armenia (danielyan et al., 2008). the genus darevskia (arribas, 1999), the first vertebrate group where parthenogenesis was described (darevsky, 1967), occurs across all caucasus, adjacent regions of asia minor, northern iran and balkans. a total of 25 sexual species and seven parthenogenetic forms have been described (darevsky, 1967; fu et al., 1995; murphy et al., 1996; arnold et al., 2007; freitas et al., 2016b) although their phylogenetic relationships and taxonomy are still under discussion. in armenia, up to nine darevskia species occur along a relatively small area and frequently overlap locally (arakelyan et al., 2011). specifically, the aims of this study are: (1) to determine whether the short-term spatial structure of a community of lizards displays a clustered, random, or regular distribution. we predict that the species will not be distributed randomly or regularly along 157spatial structure of a lizard community the study area, but in clusters, largely determined by the spatial structure of the area. (2) to identify the community clusters and their specific composition. we hypothesize that lizards will form clusters of several species, since parthenogenetic female lizards are supposed to be less aggressive than sexual females (tarkhnishvili et al., 2010), and asexual populations have been reported to attain higher densities than sexual ones in similar habitats (darevsky, 1967). as we do not expect much behavioural interference between species, parthenogens will be present in almost all clusters due to their low intraspecific aggressiveness and high population density (tarkhnishvili et al., 2010). (3) to determine if habitat is a segregating factor among species, given differential habitat selection among species has been reported for armenia (arakelyan et al., 2011) with d. valentini tending to occupy meadows and grassland scattered with rocks, d. unisexualis steep rocky exposures, and d. armeniaca being intermediate in habitat use. materials and methods study area the study area was located near the village of kuchak (armenia; 44.385 n, 40.532 w, ca. 1940 m a.s.l.; fig. 1) at the foothills of mount aragats. the study area includes several longitudinal rocky outcrops alternating with grasslands and bushes (for a general view of the landscape see figure 151 in arakelyan et al., 2011). these outcrops are composed by accumulations of big rock boulders, reaching an approximate altitude of 1955 m a.s.l. in the highest point. sampling was performed around the highest outcrops (0.34 ha; fig. 1). previous surveys had identified high density of lizards in the study area. species community composition the lizard community was composed of three species: one sexual (darevskia valentini) and two asexual (d. armeniaca, d. unisexualis), as well as two hybrid forms (d. valentini/d. armeniaca, d. valentini/d. unisexualis). in this locality hybridisation between sexual and asexual species is frequent, producing both diploid and polyploid hybrids (danielyan et al., 2008). individuals were first determined at species level using external characteristics according to darevsky (1967), danielyan et al. (2008), and arakelyan et al. (2011) and then corroborated by microsatellite analysis (freitas et al., 2016a). surveys we performed intensive surveys across the study area during three consecutive days (1-3 june 2011), coinciding with the reproductive period (arakelyan et al., 2011). we concentrated sampling effort within a short time period to prevent pooling spatial locations shifted in time as expected from dispersal mediated by social interactions or with seasonal changes on home ranges (boudjemadi et al., 1999; galoyan, 2013). each survey took around 8 hours covering the whole study area (fig. 1). we surveyed all outcrops inside the study area. therefore, each zone of the study area was visited only once. lizards were also captured once to prevent pseudo-replication, we used tailremoval for parallel genetic analyses (see below) as individual mark. we recorded the position of each lizard with a professional gps unit (trimble geoexplorer, 2008 hx), with a precision of 50 cm after post-processing. data collection and lizard capturing we created a gps data dictionary with trimble gps pathfinder office software v 5.0, transferring it posteriorly to the gps unit. for all the observed lizards we recorded species, size class, sex; plant cover (0-25, 25-50, 50-75, 75-100%); and plant height (top, middle, down). two team members (ns and egm) walked randomly through the study area capturing lizards with a noose (garcía-muñoz and sillero, 2010). mac measured svl with a calliper to the nearest 0.01 mm and took pictures of each individual for confirmation of species identifications. finally, ea recorded the exact location of the individual with the gps unit as well as introduced all the data inside the gps dictionary. in the end, we released each lizard in the exact capture site. the tail tip of each lizard was collected and kept for later genetic analyses, in order to confirm species determinations (freitas et al., 2016a). global clustering analysis we applied several tests of spatial statistics to describe the distribution pattern of the lizards. first, we analysed the distance threshold of clustering for all the species together. subsequently, we grouped species by reproductive mode (parthenogens: darevskia armeniaca and d. unisexualis; and sexual individuals: d. valentini and hybrids). for this, we used the ripley’s k distance function (bivand et al., 2008; ripley, 1976; rogerson, 2001), which measures the distribution of pairwise distances among events. the ripley’s k function was calculated using the envelope function of the package “spatstat” (baddeley and turner, 2005) of the r software (r team, 2014). in addition, a complete spatial randomness (csr) point process with the same estimated intensity in the study area was simulated (999 replicates) and compared with the empirical values of ripley’s k, to check whether the empirical function is contained inside. determination of topological clusters many methods have been proposed to determine clusters in a cloud of points (bivand et al., 2008; rogerson, 2001). what is considered a cluster, depends on the size of the study 158 neftalí sillero et alii area and a threshold distance (sillero and gonçalves-seco, 2014). multiple solutions are possible to define clusters inside a cloud of points, depending on the distance threshold selected, i.e. the distance where any cluster point is farther from any point outside the cluster. in order to avoid subjective solutions, this threshold distance must be determined statistically. reliable solutions are distance functions like the k function (ripley, 1976) or the nearest neighbor index (nni; clark and evans, 1954). every pair of points separated by a distance below the threshold distance is supposed to belong to the same cluster. in our study case, we used the delaunay’s triangulation to identify spatially the clusters of lizards’ locations using the expected mean distance between neighbours provided by the nni as statistical threshold. the delaunay’s triangulation is a very well-known mathematical method, where for a given set p of discrete points in a plane, a triangulation is defined that no point in p is inside the circumcircle of any triangle in the plane. the expected distance provided by the nni determines a cluster when the mean nearest neighbour distance is lower than the expected nearest neighbour distance. we selected the delaunay triangles with lines shorter than the expected nearest neighbour distance (clark and evans, 1954). the points inside the selected delaunay triangles were considered clustered. this analysis was performed in qgis 2.0. spatial segregation we measured the spatial segregation among species in two different ways. first, we calculated the degree of overfig. 1. study area and records distribution. a: the study area was located near the village of kuchak (armenia) at the foothills of mount aragats. b: the lizard community is composed by one sexual species darevskia valentini, two parthenogens (d. armeniaca and d. unisexualis) and the hybrids between the sexual and the asexual species. 159spatial structure of a lizard community lap between species pairs. we calculated buffers with a radius equal to the expected nearest neighbour distance (clark and evans, 1954). we expect that species sharing space will have a high degree of overlap, while those spatially segregated will have a low degree of overlap. the overlaps were calculated with the intersect function of qgis 2.8. and second, we tested if males and females used differently habitats by means of loglinear models of the frequencies of both sexes and reproduction modes (asexual and sexual) in plant cover and height. results in the surveys we found a total of 149 lizards (table 1 and fig. 1): 101 darevskia armeniaca, three d. unisexualis, 24 d. valentini, and 21 triploid hybrids (two d. armeniaca – d. valentini, and 19 d. unisexualis – d. valentini). there were fewer females of d. valentini (six) in comparison with hybrid females (13; table 1 and fig. 1). however, we found 18 males of d. valentini and only eight hybrid males (table 1 and fig. 1). ripley’s k indicated that all species presented a clustered distribution, either together or grouped by reproduction mode (parthenogenetic and sexual individuals; fig. 2). we identified 11 clusters located in the north and in the south of the study area, with two clusters in the middle (fig. 3). d. armeniaca was the main species inside the clusters (table 2). the clusters were composed of one to three species, with at least one parthenogen or hybrid participating (table 2). nevertheless, d. unisexualis never participated in a cluster (table 2). the species pairs with the lowest overlap always included d. unisexualis. in fact, there was not overlap between d. unisexualis and hybrids of d. armeniaca/d. valentini. the pair of species overlapping the most was d. armeniaca and d. valentini (table 3). females and males used similar plant heights (χ2 = 0.411, p = 0.873; table 4); both used areas with intermediate plant cover more often than expected by chance (χ2 = 8.451, p = 0.014; table 4). lizards with both types of reproduction were more frequent at middle height and in areas with intermediate plant cover (respectively: χ2 = 1.946, p = 0.378; χ2 = 6.197, p = 0.045; table 4). when considering sex and reproduction mode, all individuals were associated to middle plant heights (χ2 = 2.485, p = 0.672; table 4), and used intermediate plant cover more often than expected by chance (χ2 = 9.543, p = 0.044; table 4). discussion as predicted, species were not distributed randomly or regularly in the space but presented a clustered distribution, either together or separately, as expected if resources within the study area were not randomly distributed (kwiatkowski and sullivan, 2002). indeed, since kuchak area is composed by longitudinal rock outcrops alternating with grasslands and bushes, refuges and basking sites can be considered the main constraints for lizards. in fact, other studies on lizards’ communities showed similar results (sillero and gonçalves-seco, 2014; sillero and gomes, 2016). local clusters were composed of one to three species. as predicted, parthenogens (except darevskia unisexualis) or their hybrids were present in all clusters probably due to their lower intraspecific aggressiveness (galoyan, 2013) and high abundance (tarkhnishvili et al., 2010). particularly, d. armeniaca, the most abundant species in kuchak, entered in all clusters. conversely, d. unisexualis did not enter in any cluster, likely because of its low presence in kuchak (only three individuals recorded). asexual females are supposed to be less aggressive than sexual females (tarkhnishvili et al., 2010; galoyan, 2013) and asexual populations have been reported to attain higher densities than sexual ones in similar habitats (darevsky, 1967). it is important to highlight here that d. valentini table 1. list of species detected and number of records per species, sex, and age. species female male adult subadult juvenile adult subadult juvenile d. armeniaca 96 2 3 d. unisexualis 3 d. valentini 6 17 1 hybrid d. arm -d. val 2 hybrid d. unid. val 9 1 1 6 1 1 total result 116 3 4 23 2 1 160 neftalí sillero et alii females seem to be in minority in kuchak, while hybrid females are more abundant. this pattern was also found by danielyan et al. (2008), sillero et al. (2016) and carretero et al. (2018) in the same site in different years. females and males were concentrated in areas with medium plant cover. we were not able to confirm that d. valentini occupies ground habitats, i.e. meadows and grassland as described in general for armenia (arakelyan et al., 2011). as reported previously, sexual species (as well as parthenogens) were located mainly in habitats of middle height, which do not correspond to ground habifig. 2. ripley’s k plots of all records together and grouped by reproduction modes (parthenogens: darevskia armeniaca and d. unisexualis; and sexual individuals: d. valentini and hybrids). the continuous line is the observed function of the species records; the dashed line is the theoretical function of a complete spatial randomness (csr) point process; and the grey shadow is the lower and higher limits of the csr point process after 999 replications. if the observed function is above the csr limits, the point process is considered as clustered; if it is below the limits, as regular; if it is between the limits, as random. fig. 3. distribution of clusters identified with delaunay triangulation and the expected nearest neighbour distance (see methods for more details). numbers refer to cluster numbers in table 2. table 2. number of species records per cluster. a cluster is a set of points where the distance to any point inside the cluster is farther from any other point outside the cluster. cluster distance was determined using the expected nearest neighbour distance (20 m). see methods for more details. column numbers refer to cluster numbers in figure 3. species 0 1 2 3 4 5 6 7 8 9 10 total d. armeniaca 2 3 23 2 3 4 3 3 5 1 5 54 d. unisexualis 0 d. valentini 6 1 1 1 1 10 hybrid d. arm – d. val 1 1 2 hybrid d. uni – d. val 2 1 1 2 6 total 3 3 32 3 5 4 5 3 6 3 5 72 161spatial structure of a lizard community tats (arakelyan et al., 2011). distance analyses showed d. armeniaca and d. valentini were the species with a higher degree of overlap. this was expected since the first species is present in all habitats across the area. in contrast, d. unisexualis was included only in the clusters with the lowest overlaps, probably because of its low presence in kuchak. as we stated above, the low abundance of this species was mostly responsible for its absence in clusters. these results seem to confirm that the kuchak community acts like a sexual population of a single species (sillero et al., 2016), even if strongly biased in sex ratio (carretero et al., 2018). this is corroborated by the indirect evidence from inguinal scars in females which indicate that copulation attempts between species are as frequent as within species in this community (carretero et al., 2018). the low density of d. valentini females strongly supports this conclusion. d valentini females are scarce likely due to genetic incompatibilities (haldane’s rule) and parthenogens (and hybrid females) may be replacing them even for sexual interactions (sillero et al., 2016). on the other hand, the species pair with the lowest overlap was constituted by both types of hybrids, likely because they are scarce and tend to behave like normal sexual species. local spatial segregation by habitat or competition has been also related to clustered distribution patterns (underwood and chapman, 1996), with different degrees of intensity (moody et al., 1997). habitat is the main segregating factor in several communities of reptiles (jones and droge, 1980; mellado, 1980; scali and zuffi, 1994). micro-habitat selection promotes spatial segregation in lizards (ortega and barbault, 1982): juveniles of anolis aeneus occupy open habitats instead of forests to avoid predation by a. richardi (stamps, 1983a, 1983b). spatial segregation in birds can be caused by competition between species (moody et al., 1997). plants segregate due to competition (phillips and macmahon, 1981; haase et al., 1996; getzin et al., 2006; gray and he, 2009) and habitat selection (schenk et al., 2003). overall, spatial statistics offer new insights to interpret the spatial structure of species communities. by having a better statistical support, we were able to interpret how and why species segregate locally in space. this could be completed by a habitat segregation analysis as a continuous component of the environment once geographical information of habitats will be made available for armenia. although some spatial statistic methods require data with homogeneous intensity, the clustering and overlapping analyses applied here do not require this assumption as they are topographical distance-based table 4. number of individuals per plant height (down, middle, top) and plant cover (0-25, 25-50, 50-75, 75-100%), grouped by sex as well as sex and reproduction mode. height plant cover % down middle top total 0-25 25-50 50-75 total sex female 24 74 25 123 32 86 23 123 male 6 16 4 26 1 22 3 26 total 30 90 29 149 33 90 26 149 sex/rep mode sexual female 6 10 3 19 5 12 2 19 sexual male 6 16 4 26 1 22 3 26 asexual female 18 64 22 104 27 56 21 104 total 30 90 29 149 33 90 26 149 table 3. spatial segregation by pairs of species. values are ranked from lowest to the highest degree of overlap. species pairs overlap (m2) d. unix × h d. arm 0.00 d. uni × h d. uni 1034.96 d. uni × d. val 1143.84 d. val × h d. arm 1236.06 h d. arm × h d. uni 2158.25 d. arm × h d. arm 2472.13 d. arm × d. uni 2650.51 d. val × h d. uni 8852.00 d. arm × h d. uni 11716.96 d. arm × d. val 13852.21 d. arm: darevskia armeniaca; d. uni: d. unisexualis; d. val: d. valentini; h d. arm: hybrid d. armeniaca-d. valentini; h d. uni: hybrid d. unisexualis-d. valentini. 162 neftalí sillero et alii methods. as such, they are independent of the type of intensity distribution. therefore, these methods perform better than standard ones when the sample size is low and the intensity is heterogeneous. then, studies of spatial biology can be more frequent and we will have a better insight on questions such as species segregation and habitat use of species communities. acknowledgments ns was supported by a post-doctoral grant (sfrh/ bpd/26666/2006) and research contract (if2013) from fundação para a ciência e tecnologia (fct, portugal) and mac is supported by project this research was supported by the fct projects fcomp-01-0124-feder-007062, ptdc/bia-bec/102280/2008, fcomp-010124-feder-008929, ptdc/bia-bec/101256/2008, by the project “biodiversity, ecology and global change” co-financed by north portugal regional operational programme 2007/2013 (on.2 ¨c o novo norte), under the national strategic reference framework (nsrf), through the european regional development fund (erdf) and by the project “preserving armenian biodiversity: joint portuguese ¨c armenian program for training in modern conservation biology.” of gulbenkian foundation (portugal). research was approved by yerevan state university with permit nº 0606-433. references arakelyan, m., danielyan, f., corti, c., sindaco, r., levinton, a. 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(2015): a place in the sun: does interspecific interference affect thermoregulation in coexisting lizards? behav. ecol. sociobiol. 69: 1127-1137. acta herpetologica vol. 13, n. 1 june 2018 firenze university press acta herpetologica 14(2): 71-80, 2019 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/a_h-7744 podarcis siculus latastei (bedriaga, 1879) of the western pontine islands (italy) raised to the species rank, and a brief taxonomic overview of podarcis lizards gabriele senczuk1,2,*, riccardo castiglia2, wolfgang böhme3, claudia corti1 1 museo di storia naturale dell’università di firenze, sede “la specola”, via romana 17, 50125 firenze, italy. *corresponding author. e-mail: gabriele.senczuk@uniroma1.it 2 dipartimento di biologia e biotecnologie “charles darwin”, università di roma la sapienza, via a. borelli 50, 00161 roma, italy 3 zoologisches forschungsmuseum alexander koenig, adenauerallee 160, d53113, bonn, germany submitted on: 2019, 12th march; revised on: 2019, 29th august; accepted on: 2019, 20th september editor: aaron m. bauer abstract. in recent years, great attention has been paid to many podarcis species for which the observed intra-specific variability often revealed species complexes still characterized by an unresolved relationship. when compared to other species, p. siculus underwent fewer revisions and the number of species hidden within this taxon may have been, therefore, underestimated. however, recent studies based on genetic and morphological data highlighted a marked differentiation of the populations inhabiting the western pontine archipelago. in the present work we used published genetic data (three mitochondrial and three nuclear gene fragments) from 25 podarcis species to provide a multilocus phylogeny of the genus in order to understand the degree of differentiation of the western pontine populations. in addition, we analyzed new morphometric traits (scale counts) of 151 specimens from the main islands of the pontine archipelago. the phylogenetic analysis revealed five principal podarcis groups with biogeographic consistency. the genetic distinctiveness of the podarcis populations of the western pontine islands is similar or even more ancient than those observed in numerous other pairs of podarcis sister species. in the light of these evidences we raise the western pontine lizards to specific rank; thus they should be referred to as podarcis latastei. keywords. reptilia, podarcis latastei, podarcis siculus, insular lizards, mediterranean. introduction the wall lizards belonging to the genus podarcis wagler, 1830 are among the most speciose vertebrates in europe, representing one of the most important faunistic elements of the mediterranean insular biota. originally, two opposite taxonomic viewpoints (“lumping“ and “splitting“) were – partly emotionally – discussed in the late 19th and early 20th centuries. at that time the most prominent representative of the “lumpers“ was george a. boulenger (1859-1937) who joined numerous wall lizards together under the name “lacerta muralis“ distinguishing, as some predecessors did, only “varieties“ within this species (boulenger, 1887, 1905, 1913, 1920). his main antagonist, representing the taxonomic “splitter“ faction, was ludwig von méhely (1862-1953) who considered many of boulenger’s “varieties” to be distinct species (méhely, 1907, 1909). he wrote: «like a night-mare, the so-called muralis question is burdening the mind of herpetologists» (méhely, 1907). despite modern approaches, molecular genetics included, méhely was closer to the current concept than his more influential contemporary colleague; however, the number of podarcis species is still debated. for example, 21 taxa were recognized as 72 gabriele senczuk et alii valid species by speybroeck et al. (2010), whereas other authors have suggested 23 (sindaco et al., 2013; uetz and hošek, 2016; but see psonis et al., 2017). the taxonomic wavering of the genus podarcis is mainly due to the presence of marked intra-specific variability with multiple species complexes characterized by unresolved relationships (harris and arnold, 1999; oliverio et al., 2000; harris et al., 2005; lymberakis et al., 2008). table 1 summarizes this taxonomic/nomenclatural history of the currently recognized podarcis species. in contrast to the great taxonomic attention paid to numerous podarcis species, p. siculus has undergone fewer revisions and the number of species hidden within this taxon may have been underestimated. podarcis siculus (rafinesque-schmaltz, 1810) was originally described as lacerta sicula. however, because of its distribution over a large part of italy (sicily, sardinia and numerous minor islands, islets and rocks) and dalmatia, several subspecies were described. some of them were originally assigned to “lacerta” muralis (more than 90 were listed together with their type localities by henle and klaver, 1986). this situation led some authors to hypothesize the presence of a species complex similar to those observed in other podarcis assemblages (oliverio et al., 1998, 2000; harris and sa-sousa, 2002). more recent studies based on mitochondrial (podnar et al., 2005) and nuclear (senczuk et al., 2017) markers have supported the monophyly of p. siculus and revealed surprisingly high genetic divergences between the main constituent evolutionary lineages, most comparable to those observed between many recognized podarcis species (harris et al., 2005). in addition, recent studies using molecular markers (mitochondrial and nuclear dna) and geometric morphometrics have revealed that the populations from the western pontine islands represent an evolutionarily independent unit (senczuk et al., 2018a, 2018b). the genetic distances of these populations with respect to mainland ones were extraordinary high (p-distances of 7-10% for the mtdna cytb gene), and the head morphology was clearly distinguishable with respect to the mainland and sicilian populations (senczuk et al., 2018a; 2018b). the pontine archipelago is located 40 km off the tyrrhenian coast and comprises the western pontine islands ponza, palmarola, zannone and gavi, and the eastern pontine islands ventotene and santo stefano. from the pontine archipelago, the following nominal insular intraspecific taxa have been described: lacerta muralis var. latastei = podarcis siculus latastei (bedriaga, 1879 a, b) from ponza; lacerta muralis parkeri = podarcis siculus parkeri (mertens, 1926) from santo stefano; lacerta sicula sancti-stephani = podarcis siculus sanctistephani (mertens, 1926) from santo stefano; lacerta sicula ventotenensis = podarcis siculus ventotenensis (taddei, 1949) from ventotene; lacerta sicula pasquinii = podarcis siculus pasquinii (lanza, 1952) from scoglio cappello near palmarola; lacerta sicula patrizii = podarcis siculus patrizii (lanza, 1952) from zannone; lacerta sicula lanzai = podarcis siculus lanzai (mertens, 1967) from gavi and lacerta sicula palmarolae = podarcis siculus palmarolae (mertens, 1967) from palmarola (cfr. lanza and corti, 1996; corti et al., 2010). podarcis siculus parkeri was synonymized with p. s. sanctistephani (mertens and wermuth, 1960; mertens, 1965), which is believed to have become extinct during the first decades of the last century (1914 at the latest), and replaced by p. s. siculus (mertens, 1965). henle and klaver (1986), reviewing the intraspecific taxa, followed mertens (1965) in considering p. s. ventotenensis as a synonym of the nominotypical form, and listed p. s. latastei, p. s. lanzai, p. s. pasquinii, p. s. patrizii and p. s. palmarolae as valid subspecies. these five taxa occur in the western pontine islands, which are believed to have been connected to the mainland in the pleistocene, whereas the eastern pontine islands (ventotene, santo stefano) seem never to have been, being located along the 500 m isobath (woldstedt, 1958; mertens, 1965, 1967). the deep genetic distance recently found between the eastern and the western pontine islands populations (senczuk et al., 2018a), geometric morphometrics (senczuk et al., 2018b), classical morphometric and meristic data, as well as an updated time calibrated multilocus phylogeny of podarcis (wagler, 1830), all suggest that the western pontine lizards deserve their own specific status and should be referred to as podarcis latastei (bedriaga, 1879), which we characterize and redescribe here. materials and methods molecular phylogenetics to obtain a robust and time calibrated phylogeny of podarcis as a whole, three mitochondrial (16s; cytb, and nd4) and three nuclear (mc1r, pod15b and pod55) gene fragments from 25 podarcis species, including several subspecies, were retrieved from genbank (all samples are reported in fig. 1 and table 1, localities and accession numbers are reported in appendix 1, table a1). most of the retrieved sequences for each species belong to the same individual, when not possible we selected individuals of close geographic origin. all final consensus alignments were computed for each gene separately using bioedit 7.2 (hall, 1999). coding gene fragments (cytb, nd4 and mc1r) were translated into amino acids to assess the lack of stop codons. for each alignment we used jmodeltest v.2.1.3 (darriba et al., 2012) to assess the best model of nucleotide evolution under the corrected akaike information criterion (aicc). to 73new podarcis species from western pontine islands reconstruct phylogenetic relationships we used a bayesian coalescent framework implemented in beast v.1.8 (drummond et al., 2012). to calibrate the tree in absolute time we used two vicariant calibration points: the separation between the peloponnesus (p. peloponnesiacus) and the islands of crete and pori (p. cretensis and p. levendis); and the separation between the islands of menorca and mallorca (p. lilfordi) and the pytiusic islands (p. pytiusensis). both episodes occurred following the messinian salinity crisis (msc, at about 5.2 mya) yielding the sudden separation of these landmasses (poulakakis et al., 2003; brown et al., 2008). a normal distribution using the mean in real space option (μ = 5.325; sd = 0.2) has been incorporated for each of the aforementioned nodes. we used a yule process in a linked tree partition and a lognormal relaxed model maintaining unlinked clock partitions. as substitution models we used gtr+i+g for 16s and cytb; tvm+i+g for nd4 and mc1r; hky for pod15b and hky+i for pod55. we performed three independent runs of 108 generations sampling every 104 steps. convergence was checked using the software tracer v 1.5 (rambaut and drummond, 2007) and after combining the trees using logcombiner, the final consensus tree was computed in treeannotator (drummond et al., 2012). morphology we used the measurements and scale counts published by mertens (1967) for diagnosing the subspecific taxa of p. siculus recognized by him and compared them with our own data taken from the holdings deposited in the florence museum (mzuf). we measured and counted the scales of 151 specimens (60 females and 91 males) from the main islands of the pontine archipelago preserved at the natural history museum of the university of florence (mzuf) (see table 2). specimens previously studied by lanza (1952, 1967) and used for his descriptions of p. s. patrizii and p. s. pasquinii were also included. we analyzed sex, snout-vent length (svl), and the following meristic characters: a) number of mid-body dorsal scales (dors); b) number of ventral plates counted longitudinally along the intermediate row (vent); c) number of gular scales counted along the throat mid-line from the collar to the confluence of maxillaries (gul); d) number of collar scales (coll); e), number of femoral pores on the right leg (porf). to test for significance of differences between sexes and islands, we used a two-way analysis of variance (anova). an additional two-way anova was performed to test differences between ventotene island and santo stefano island sampled in 1954 and 1966, and the western pontine and santo stefano island sampled in 1878. results and discussion the final alignment of 3117 bp included 27 taxa (supplementary information). the three independent runs fig. 1. distribution of the genus podarcis and location of the samples used for the phylogenetic analysis, as reported in table 1. geographic distribution of podarcis latastei is also reported at the top right. 74 gabriele senczuk et alii showed effective sample size (ess) for each parameter of more than 200. the phylogenetic tree obtained is shown in fig. 2. the tree topology is rather well supported (most of the nodes showed posterior probabilities higher than 0.95) and the relationships among species only partly corresponds to previous phylogenetic reconstructions. within the podarcis radiation we found five principal groups with biogeographic consistency (fig. 1-2). 1 – the podarcis hispanicus complex currently includes seven species distributed from north africa to the iberian peninsula and south-western france. all species from the p. hispanicus complex were first described as intraspecific taxa of the collective species p. muralis and later raised to species rank in order to resolve paraphyly (see table 1) (oliverio et al., 2000; sá-sousa and harris, 2002; geniez et al., 2007, 2014). our phylogenetic analysis support a similar phylogenetic relationships among species as previously reported, and suggested, albeit with moderate support (0.91), p. muralis as the sister species of all the iberian podarcis. 2 – the “erhardii” group comprises species of the balkan peninsula and the greek islands. because of a paraphyletic relationship between p. erhardii (bedriaga, 1882) and p. peloponnesiacus (bibron and bory, 1833), two new insular endemics p. cretensis (wettstein, 1952) and p. levendis (lymberakis et al., 2008) were raised to table 1. currently accepted podarcis species and their original description name and reference. the geographic localities are shown in fig. 1. loc. species (author and year of description) described as reference 1 p. hispanicus (steindachner, 1870) lacerta oxycephala var. hispanica geniez et al. 2007 2 p. carbonelli pérez-mellado, 1981 podarcis bocagei carbonelli sá-sousa and harris, 2002 3 p. bocagei (lopez-seoane, 1885) lacerta murals bocagei sá-sousa et al., 2000 5 p. liolepis (boulenger, 1905) lacerta muralis var. liolepis lacerta muralis atrata geniez et al., 2014 6 p. vaucheri (boulenger, 1905) p. hispanicus vaucheri oliverio et al., 2000 7a, 7b p. guadarramae (boscá, 1916) lacerta muralis guadarramae, podarcis hispanicus “type 1a, 1b” geniez et al., 2014 8 p. virescens (geniez et al., 2014) podarcis hispanicus “type 2” geniez et al., 2014 9 p. muralis (laurenti, 1768) lacerta muralis 10 p. lilfordi (günther, 1874) 11 p. pityusensis (boscá, 1883) lacerta muralis var. pityusensis 12 p. tiliguerta (gmelin, 1789) lacerta tiliguerta 13a, 13b p. siculus (rafinesque-schmaltz, 1810) lacerta sicula 14 p. latastei (bedriaga, 1876) 15 p. waglerianus (gistel, 1868) podarcis muralis var. wagleriana 16 p. raffoneae (mertens, 1952) lacerta sicula raffonei capula, 1994 17 p. filfolensis (bedriaga, 1876) 18 p. melisellensis (braun, 1877) 19a, 19b p. tauricus (pallas, 1814) 20 p. gaigeae (werner, 1930) lacerta taurica gaigeae 21 p. milensis (bedriaga, 1882) lacerta muralis fusca var. milensis 22 p. peloponnesiacus (bibron and bory, 1833) 23 p. erhardii (bedriaga, 1882) lacerta muralisfusca var. erhardii 24 p. cretensis (wettstein, 1952) lacerta erhardii cretensis 25 p. levendis (lymberakis et al., 2008) table 2. population number and relative sample size for both males and females for each island. *individuals collected in santo stefano island in 1954/1966. n° island females males 1 ponza 18 22 2 gavi 3 9 3 palmarola 11 21 4 zannone 6 6 5 santo stefano (1878) 5 5 santo stefano* 5 14 6 ventotene 8 12 7 scoglio cappello 4 2    tot. 60 91 75new podarcis species from western pontine islands the species rank (poulakakis et al., 2003; 2005a; lymberakis et al., 2008). 3 – the “tauricus” group includes two species p. tauricus (pallas, 1814) and p. melisellensis (braun, 1877) distributed over a large part of the balkans and two endemic insular species: p. gaigae (werner, 1930) from skyros and surrounding islands, and p. milensis (bedriaga, 1882) from milos and surrounding islands (poulakakis et al., 2005a, 2005b). however, a recent species delimitation approach (psonis et al., 2017), suggested the presence of nine species within the tauricus group: p. melisellensis, p. gaigeae, p. milensis, and six in the p. tauricus complex. based on the absence of support to the monophyly of p. tauricus, the authors proposed to raise the subspecies p. t. ionicus (lehrs, 1902) to the species rank (psonis et al., 2017). our phylogenetic analysis confirms this scenario indicating an ancient divergence between p. ionicus and p. tauricus (fig. 2). it is interesting to note that although the geographic distribution of p. filfolensis (bedriaga, 1876) would suggest a close relationship with the other two endemic species of the siculo-maltese area, p. waglerianus (gistel, fig. 2. bayesian phylogenetic tree based on multilocus data (cytb, 16s, nd4, mc1r, pod15b and pod55) using beast v. 1.8. black filled circles indicate nodes used to calibrate phylogeny (poulakakis et al., 2003; brown et al., 2008). the times of the most recent common ancestor are reported for each node as well as the posterior probability. 76 gabriele senczuk et alii 1868) and p. raffoneae (mertens, 1952), previous molecular analysis has resulted in contrasting phylogenies regarding the position of these three species (harris et al., 2005; psonis et al., 2017; salvi et al., 2017). our phylogenetic reconstruction supports a tangled evolutionary history indicating p. filfolensis as the sister species of the podarcis “tauricus” group (fig. 2). 4 – podarcis species from the western mediterranean islands include p. tiliguerta (gmelin, 1789), p. lilfordi (günther, 1874) and p. pityusensis (boscá, 1883). podarcis tiliguerta distributed in sardinia, corsica and surrounding islands, has also been argued to be a species complex showing very deep phylogeographic discontinuities (harris et al., 2005; rodriguez et al., 2017; salvi et al., 2017; senczuk et al., 2019). on the other hand, p. lilfordi and p. pityusensis from the balearic and pityusic islands showed closer phylogenetic relationship as a consequence of vicariance following the messinian salinity crisis (brown et al., 2008). the phylogenetic reconstruction reported here, confirms the close relationship of these endemic western mediterranean species. 5 – podarcis species from the italian peninsula, sicily and surrounding islands forms a monophyletic assemblage that includes p. siculus, p. waglerianus and p. raffoneae. the last of these was raised to the species rank on the basis of allozyme analysis although further studies showed relatively low genetic distances from p. waglerianus (3.3% at cytochrome b), far lower than those observed between many other podarcis species (capula, 1994; harris et al., 2005). based on our data, the lineage including p. waglerianus and p. raffoneae is sister to podarcis siculus and the lizards of the western pontine archipelago. the western pontine podarcis are separated from p. siculus by approximately 4 mya based on our results. the genetic distinctiveness of these insular populations is comparable or even greater than several other table 3. scale counts after mertens (1965) (minimum – mean – maximum) of the insular populations of p. latastei (the six left columns) and p. siculus (the two right columns). dors = no. of mid-body dorsal scales; vent = no. of ventral plates; coll = no. of collar scales; porf = no. of femoral pores on the right leg; m. = males; f. = females. ponza gavi zannone palmarola sc. cappello s. stefano 1878 s. stefano 1963 ventotene dors m 68-70.4-75 71-76.3-81 66-72.8-78 69-76.6-86 72-73.2-76 72-75.8-79 60-65.6-72 61-66.8-78 f 62-67.7-73 70-73.2-78 63-68.0-74 66-68.7-71 66-68.7-71 71-75.3-79 59-61.2-63 60-60.3-68 vent m 25-26.1-27 26-26.3-27 25-26.7-28 24-25.2-26 25-25.7-26 25-26.3-27 24-24.8-26 22-24.6-26 f 27-28.1-29 27-28.0-30 27-28.4-30 27-28.2-29 28-28.7-30 27-28.1-30 27-28.0-29 25-26.9-29 coll. m 9-10.8-12 9-10.0-11 9-10.5-12 9-10.4-13 10-10.5-11 12-12.1-13 8-9.1-11 9-10.6-12 f 10-10.7-11 9-10.2-11 9-10.5-11 10-10.7-11 11-11.0-11 10-11.0-12 7-8.2-09 8-9.8-11 porf. m 22-24.8-29 22-24.3-26 22-25.1-28 21-24.9-29 22-24.7-28 24-25.3-28 19-23.8-26 20-23.5-27 f 21-23.8-28 22-24.7-26 19-23.0-25 22-24.1-26 23-24.5-26 22-24.8-27 20-21.6-24 20-22.0-23 table 4. snout-vent length (svl) and scale counts (minimum – mean – maximum) of specimens preserved at the natural history museum of the university of florence (mzuf). dors = no. of mid-body dorsal scales; vent = no. of ventral plates; gul = no. of gular scales; coll = no. of collar scales; porf = no. of femoral pores on the right leg; m. = males; f. = females. *individuals collected in santo stefano island in 1954/1966. ponza gavi zannone palmarola sc. cappello s. stefano 1878 s. stefano* ventotene svl m. 58-68.6-78.8 70.5-73.2-78 70.4-67.1-76.2 58-67-75 62.5-65-67.5 69-76.1-81.5 67-73.5-81.6 60-70.7-77 f. 50-58.6-68.6 61-63.5-67 52.6-62.7-76.1 57.8-52-63 56.5-57.9-60 55-63.1-69 59-63.5-70 53.5-60.4-66 dors m. 67-70.1-76 73-74.3-77 69-70.1-73 68-72.4-76 72 68-71-74 59-64.1-69 62-66-70 f. 62-68.2-74 67-72-75 63-67.3-74 63-68-74 65-67-69 69-71.8-75 55-58-63 57-61.4-67 vent m. 18-19.9-21 22-23-24 19-21.3--20 18-19.3-21 20-20.5-21 20-20.3-21 17-18.9-21 17-19.2-20 f. 22-22.7-24 18-19.9-23 22-22.2-23 2122.4-24 24 19-21-23 20-21.4-23 21-21.6-23 coll m. 11-12.7-14 11-12.3-13 10-12.1-13 10-12.4-15 12-13-14 13-14-15 9-12.3-16 10-12.6-15 f. 11-13-15 11-13-15 10-11.5-13 11-12.8-14 12-12.8-13 12-13.4-15 9-11.2-12 11-12.9-16 gul m. 28-32.4--37 31-34.8-38 31-33.7-40 27-33--39 31 34-34.3-35 23-26.8-32 27-30.1-34 f. 27-31.2--35 32-33.3-35 31-33-36 27-33--36 23-24-25 33-36.4-40 23-25.4-28 25-27-29 porf m. 22-24.5-26 23-24.7-26 23-25-26 21-24.8-28 25-25.5-26 26-26.5-27 21-22.2-24 21-23.7-27 f. 21-23.9-27 22-24.4-27 21-23.3-27 21-22.8-26 23-24-25 23-27.4-30 20-20.8-21 20-22-25 77new podarcis species from western pontine islands pairs of podarcis sister species (i.e., p. bocagei/p. guadarramae, p. carbonelli/p. virescens, p. cretensis/p. levendis, p. gaigeae/p. milensis, p. tauricus/p. ionicus). our morphological analysis substantially confirms what bedriaga (1879a) and mertens (1965) already observed. indeed, we found significant differences comparing the specimens of the western pontine islands and the santo stefano island collected in 1878, with those collected in santo stefano in 1954/1966 and ventotene island (tables 3, 4 and 5; fig. a1). furthermore, we also found significant differences when considering sexes and islands as factors (tables 3, 4 and 5; fig. a1). smaller dorsal scales (resulting in higher dorsal scales counts) were already reported by bedriaga (1879a) to characterize his new taxon latastei. slight discrepancies between the scale counts taken by mertens (1967) as compared with ours (tables 3 and 4) are likely due to a different counting method, which was not precisely defined in mertens’ (1967) paper, e.g., in the number of oblique ventral rows which is dependent on whether only complete or also incomplete rows are counted. based on multiple sources of evidence from genetics (herein and senczuk et al., 2018a), morphology (herein and in senczuk et al., 2018b) we believe that this insular endemic taxon deserves specific rank and should be referred to as podarcis latastei (bedriaga, 1879). we propose to adopt as common name “lataste’s lizard” for this species, following bedriaga, (1979b). in accepting the specific status for the lizards of the western pontine islands, under the oldest name available podarcis latastei (bedriaga, 1879), type locality ponza island, we nevertheless accept the infraspecific subdivisions within the western pontine islands assigned by earlier authors to p. siculus. this means that the former subtaxa of the latter taxon, viz. patrizii, pasquinii, lanzai etc. now become subspecies of p. latastei. the various island populations of podarcis latastei in the western pontine archipelago exhibit variable color patterns. the patterned color morphs often show a tendency for longitudinal stripes to dissolve into oblique bands, thus forming a reticulate pattern with light ocelli included (fig. 4, 5 and 6). in other individuals, particularly from gavi island (fig. 5a, 5b) there is a strong tendency for a reduction of black-pigmented color pattern elements, corresponding to the “concolor” mutation that also occurs in other podarcis species. these differences in body dimensions, scalation and color pattern justify, in our opinion, the maintenance of their subspecific names, at least for conservation purposes (senczuk et al., 2018a). bedriaga (1879a) based his nomen latastei on an unknown number of individuals – “in anzahl” which means “in a certain quantity” – collected by himself on ponza island in summer 1878, plus one individual from a rock west off ponza which he called faraglioni of ponza. obviously, he kept all specimens in a cage alive during his travel and brought them via nice (nizza), france, from where he sent a part of them to f. lataste to paris, to his residential town of heidelberg, germany, where he continued to observe them in life, mainly in respect to colour change phenomena (bedriaga, 1879a). in 1879 and 1902 he sent preserved specimens to some german museums, table 5. analysis of variance (anova) for svl and meristic characters of the mzuf specimens. significant p-value at 0.05 are marked in bold. degrees of freedom (d.f.) are also reported. in the last column, anova results using the endemic insular taxon (p. latastei + the extinct p. s. sanctistephani) and introduced p. siculus as factors, are reported. svl = snout-to-vent length; dors = no. of mid-body dorsal scales; vent = no. of ventral plates; gul = no. of gular scales; coll = no. of collar scales; porf = no. of femoral pores. *individuals collected in santo stefano island in 1954/1966. sex islands s. stefano 1878 + w. pontine/ s. stefano* + ventotene d.f. 1 7 2 svl f 152.96 8.73 4.54 p <0.001 <0.001 <0.05 dors. f 40.73 27.15 57.42 p <0.001 <0.001 <0.001 vent. f 259.4 9.88 5.4 p <0.001 <0.001 <0.01 coll. f 1.74 2.9 4.44 p 0.18 <0.01 <0.05 gul. f 5.71 24.86 62.13 p <0.05 <0.001 <0.001 porf. f 15.41 10.71 57.42 p <0.001 <0.001 <0.001 fig. 3. detail of the entry of a p. latastei specimen from ponza collected by j. v. bedriaga, in the catalogue of the göttingen zoological museum. 78 gabriele senczuk et alii including frankfurt and munich as well as the zoological museum of the university of göttingen (whose herpetological holdings have been in bonn since 1977), and one specimen of his ponza lizard is still documented in the old göttingen catalogue, although unfortunately it was lost some time before 1968 (böhme, 2014, fig. 3). we failed to retrieve any of these old syntypes in any of the mentioned collections. so, there seems to be no extant type material of this taxon, and the single colour image provided by bedriaga (1879b), can be regarded as the figure of the individual that could have been chosen as a lectotype if it would be still extant (fig. 4). a neotype selection, however, seems presently to be unnecessary in this case. according to the genetic and geometric morphometric data published by senczuk et al. (2018a, b) and to our data, the wall lizards of the western pontine islands, so far classified as belonging to podarcis siculus, clearly merit their own specific status und should be treated under the oldest fig. 4. detail of plate ix. (bedriaga, 1879b) with a specimen (right) of his “lacerta muralis var. latastei (= podarcis latastei). fig. 5. examples of living representatives of the pontine islands populations. gavi: a, b, c; ponza: d, e; palmarola: f. fig. 6. two different color morphs from zannone island: a “quasi” concolor individual above and a dark reticulated individual below. 79new podarcis species from western pontine islands available name for these western pontine populations, i.e., podarcis latastei (bedriaga, 1879). because of the marked morphological differences between these populations, their former insular subspecific names (ponza: latastei, gavi: lanzai, zannone: patrizii, palmarola: palmarolae, and scoglio cappello: pasquinii) which were ranked as subspecies of p. siculus before, should be maintained but now attached as subspecific names to podarcis latastei. each of these island populations has its own characteristics and may well turn out to be a distinct conservation unit. acknowledgements we wish to thank annamaria nistri for allowing us to access to the mzuf specimens, ulla bott for providing copies of some old papers and jean-michel delaugerre for having provided the beautiful colour picture for the cover of acta herpetologiga. we also would like to thank paolo colangelo for his helpful suggestions. all animals have been handled in accordance with relevant guidelines in full compliance with specific permits released by the italian environment ministry (prot. 00017879/pnm-09/09/2012) and no lizards were killed. we thank the circeo national park for the permission to collect tissue samples from the zannone island population (n.487, 16/02/2015). supplementary material supplementary material associated with this article can be found at <http://www.unipv.it/webshi/appendix> manuscript number 25159. references bedriaga, j. von (1879a): herpetologische studien (fortsetzung). arch. f. naturges. 45: 234-339. bedriaga, j. von (1879b): mémoire sur les variétés européennes du lézard des murailles. bull. soc. zool. france 4: 194-228. böhme, w. 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(1958): das eiszeitalter. grundlinien einer geologie des quartärs, ii. europa, vorderasien und nordafrika im eiszeitalter, 2nd edition. enke, stuttgart, germany. acta herpetologica vol. 14, n. 2 december 2019 firenze university press podarcis siculus latastei (bedriaga, 1879) of the western pontine islands (italy) raised to the species rank, and a brief taxonomic overview of podarcis lizards gabriele senczuk1,2,*, riccardo castiglia2, wolfgang böhme3, claudia corti1 substrate type has a limited impact on the sprint performance of a mediterranean lizard pantelis savvides1,*, eleni georgiou1, panayiotis pafilis2,3, spyros sfenthourakis1 coping with aliens: how a native gecko manages to persist on mediterranean islands despite the black rat? michel-jean delaugerre1,*, roberto sacchi2, marta biaggini3, pietro lo cascio4, ridha ouni5, claudia corti 3 pit-tags as a technique for marking fossorial reptiles: insights from a long-term field study of the amphisbaenian trogonophis wiegmanni pablo recio, gonzalo rodríguez-ruiz, jesús ortega, josé martín* occurrence of batrachochytrium dendrobatidis in the tensift region, with comments on its spreading in morocco ait el cadi redouane1, laghzaoui el-mustapha1, angelica crottini2, slimani tahar1, bosch jaime3,4, el mouden el hassan1,* hematological parameters of the bolson tortoise gopherus flavomarginatus in mexico cristina garcía-de la peña1,*, roger iván rodríguez-vivas2, jorge a. zegbe-domínguez3, luis manuel valenzuela-núñez1, césar a. meza herrera4, quetzaly siller-rodríguez1, verónica ávila-rodríguez1 ontogenetic and interspecific variation in skull morphology of two closely related species of toad, bufo bufo and b. spinosus (anura: bufonidae) giovanni sanna visible implant alphanumeric (via) as a marking method in the lesser snouted treefrog scinax nasicus andrea caballero-gini1,2,3,*, diego bueno villafañe2,3, lía romero2, marcela ferreira2,3, lucas cañete4, rafaela laino2, karim musalem2,5 morphological variation of the newly confirmed population of the javelin sand boa, eryx jaculus (linnaeus, 1758) (serpentes, erycidae) in sicily, italy francesco p. faraone1,*, salvatore russotto2, salvatore a. barra3, roberto chiara3, gabriele giacalone4, mario lo valvo3 variability in the dorsal pattern of the sardinian grass snake (natrix natrix cetti) with notes on its ecology enrico lunghi1,2,3,4,*, simone giachello5, manuela mulargia6, pier paolo dore7, roberto cogoni8, claudia corti1 estimating abundance of the stripeless tree-frog hyla meridionalis by means of replicated call counts federico crovetto, sebastiano salvidio, andrea costa* at-rich microsatellite loci development for fejervarya multistriata by illumina hiseq sequencing yan-mei wang, jing-yi chen, guo-hua ding*, zhi-hua lin acta herpetologica 12(2): 205-208, 2017 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-20547 diet of juveniles of the venomous frog aparasphenodon brunoi (amphibia: hylidae) in southeastern brazil rogério l. teixeira1, ricardo lourenço-de-moraes2, débora c. medeiros3, charles duca3, rogério c. britto4, luiz c.p. bissoli5, rodrigo b. ferreira3,* 1 in memoriam 2 laboratório de herpetologia e comportamento animal, departamento de ecologia, universidade federal de goiás, campus samambaia, 74001-970 goiânia, go, brazil 3 laboratório de ecologia de populações e conservação, programa de pós-graduação em ecologia de ecossistemas, universidade vila velha. rua comissário josé dantas de melo, boa vista ii, 29102-920 vila velha, es, brazil. *corresponding author. e-mail: rodrigoecologia@yahoo.com.br 4 associação capixaba em defesa da água e da mata atlântica (acadama). rua lourenço roldi,  29665-000 são roque do canaã, es, brazil 5 ello ambiental. av.getúlio vargas, 29700-010 colatina, es, brazil submitted on: 2017, 17th april; revised on: 2017, 29th may; accepted on: 2017, 27st september editor: daniele pellitteri-rosa abstract. seventy juvenile individuals of aparasphenodon brunoi were collected on the low parts of tree trunks in an atlantic forest remnant. arthropods were the dominant prey found in their stomachs. coleoptera (adult and larvae) was the most important prey regarding prey frequency, number, weight, and index of relative importance. secondary preys included hymenoptera that was important regarding number of prey and hemiptera that was important regarding prey weight. trophic ontogeny was detected. the diversity of prey suggests a. brunoi is an opportunistic sit-andwait predator. keywords. anura, ecology, hylidae, predation, ontogeny, feeding pattern. studies regarding dietary aspects of animals may provide important data for a better understanding of the fundamental niche, position in the food webs, feeding time strategy, and metabolic needs of a species (christian et al., 2007; miller et al., 2010). anurans are usually considered opportunistic predators regarding their feeding habits and most of them are arthropod consumers (wells, 2007). generally, the diet of anurans is related to their snout-vent length, ability to detect and capture prey as well as prey availability in the environment (giaretta et al., 1998; ferreira et al., 2015). aparasphenodon brunoi miranda-ribeiro, 1920 is a hylid treefrog that uses bromeliads and hollows of tree trunks as diurnal refuge (i.e., bromelicolous species, sensu peixoto, 1995) and reproduce in temporary ponds (wogel et al., 2006). it is endemic to the coastal plain of brazil (i.e., restinga habitat) from são paulo to bahia states (ruas et al., 2013; frost, 2016). this species has a highly toxic secretion that may be injected by bony spines on the head (jared et al., 2015). furthermore, some studies have shown that the toxicity of a frog is determined by the compounds sequestered from the diet during early development stage (e.g., daly et al., 1994; sime et al., 2000). there is little information on the ecology of juveniles of the venomous treefrog a. brunoi, and diet studies on adults have shown that this treefrog is a generalist forager of arthropods (teixeira et al., 2002; mesquita et al., 2004). the aim of this study is to describe the diet of juveniles 206 rogério l. teixeira et alii of a. brunoi in an atlantic forest remnant from southeastern brazil. the study area is in the coastal plain of pontal do ipiranga district, municipality of linhares, espírito santo state, brazil (19°13’s, 39°43’w). the region is characterized by pastures and remnants of atlantic forest. the treefrogs were manually captured at night between september and october 2000. according to mesquita et al. (2004), individuals of this species smaller than 48 mm are considered juveniles. the individuals were anesthetized using lidocaine cream (5%) and fixed in a solution of formalin 10% for 72 hours and then washed and placed in a solution of alcohol 70%. in laboratory the specimens were measured to the snout-vent length (svl in mm), weighted (0.01 g precision) and dissected to determine the sex and to analyze the stomach content. the stomach contents were removed, spread in petri dish and identified to the lowest taxonomic level possible. preys were dried on paper towels and the following parameters for each prey category were evaluated: occurrence frequency, number, wet weight (0.0001 g precision), and index of relative importance (iri). the iri was calculated according to pinkas et al. (1971) and the percent iri (%iri) according to cortés (1997). for intraspecific comparison, iri was calculated for adults of a. brunoi using data available on teixeira et al. (2002). intact preys were measured to the total length with a caliper (0.1 mm precision). the voucher specimens were deposited at the zoological collection of the instituto nacional da mata atlântica/museu de biologia professor mello leitão (mbml), espírito santo state, brazil. the data were tested for normality using kolmogorov-smirnov test and for homogeneity using bartlett test. svl and weight were compared between sexes using the analysis of variance (anova). in this analysis, sex was the independent variable while svl and weight were the dependent variables (neter et al., 1990). cluster analysis based on euclidean distance was used to evaluate trophic ontogeny based on size classes of a. brunoi. only the weight value of the main prey was used in this analysis and the data were log transformed. the relationship between prey length and anuran svl was tested by a regression analysis. mean and standard deviation are provided. the significance level was set to 0.05. seventy juvenile individuals of a. brunoi were collected on the low parts of tree trunks (< 1m). adults were on trees canopy and were not collected. thirty-two individuals were juvenile males and 38 were juvenile females. juvenile males ranged on svl from 31.0 to 39.2 mm (mean = 34.3, sd = 2.0 mm) and in weight from 2.0 to 4.4 g (mean = 2.9, sd = 2.0 g). juvenile females ranged on svl from 32.1 to 43.6 mm (mean = 36.7, sd = 2.7 mm) and in weight from 2.1 to 6.7 g (mean = 3.6, sd = 2.7 g). the svl and weight differed significantly between table 1. prey items of juveniles of aparasphenodon brunoi from espírito santo state, southeastern brazil. f = occurrence frequency; n = number of prey; w = weight; iri = index of relative importance. prey f %f n %n w %w iri %iri insecta blattodea 2 3.77 2 3.23 120.1 3.94 27.03 0.77 coleoptera (adult) 19 35.85 21 33.87 963.5 31.64 2348.53 67.26 coleoptera (larvae) 8 15.09 8 12.90 267.9 8.80 327.45 9.38 hemiptera 3 5.66 3 4.84 946.5 31.09 203.36 5.82 homoptera 1 1.89 1 1.61 1.8 0.06 3.16 0.09 hymenoptera 7 13.21 10 16.13 127.1 4.17 268.16 7.68 isoptera 1 1.89 1 1.61 19.3 0.63 4.23 0.12 orthoptera 5 9.43 5 8.06 333.1 10.94 179.17 5.13 insect remnant 3 5.66 23.3 0.76 arachnida araneae 3 5.66 3 4.84 160.6 5.27 57.22 1.64 crustacea ostracoda 3 5.66 8 12.90 1.5 0.05 73.30 2.10 other anuran skin 5 9.43 80 2.63 total 62 100.00 3044.7 100.00 207diet of juveniles of aparasphenodon brunoi the sexes (f1,68 = 17.3, p < 0.01; f1,68 = 12.6, p < 0.01, respectively). females were on average larger and heavier than males. fifty-three (76%) individuals had food items in their stomach, representing 12 prey categories, mostly arthropods, (table 1). coleoptera (adult and larva) was the most important prey regarding prey frequency, number, weight, and iri. secondary preys included hymenoptera that was important regarding number of prey and hemiptera that was important regarding prey weight. it was detected trophic ontogeny in a. brunoi (fig. 1). specimens less than 36.9 mm had similar diet, possibly because they predated more coleoptera (fig. 2). specimens larger than 36.9 mm had similar diet by feeding mostly upon hemiptera. the length of intact prey ingested by a. brunoi varied from 2.8 to 15.9 mm (mean = 8.9, sd 3.7). there was no relationship between total length of prey and anuran svl (r² = 0.003, p > 0.05). the variety of prey categories suggests that a. brunoi is an opportunistic sit-and-wait predator. the diet of a. brunoi from another two populations had also a variety of prey categories (teixeira et al., 2002; mesquita et al., 2004). in fact, the feeding upon small arthropods has been reported in several studies of anurans (teixeira and coutinho, 2002; ferreira and teixeira, 2009; ferreira et al., 2012). the presence of ostracods, common on tank bromeliads (oliveira et al., 1994), indicates bromelicolous habit of a. brunoi at the studied fragment. apparently, juveniles of a. brunoi feed upon different prey categories compared to adults. in our study, coleoptera was the most important prey (%iri = 67.26). in teixeira et al. (2002), hymenoptera was the most important prey (%iri = 20.1) for adults of a. brunoi. coleoptera was the least important prey (%iri = 0.7) for this population of adults. in our study, a. brunoi feeds on different prey categories according to their svl. surprisingly, a. brunoi svl was not related to prey size. changes on diet correlated to anuran svl have been reported for other species (giaretta et al., 1998; teixeira and vrcibradic, 2003; ferreira et al., 2007). trophic ontogeny may be an important mechanism to avoid intraspecific competition and predation. probably the diet of the adults at tree canopy may strengthen the trophic ontogeny in our studied population. the studied remnant may provide necessary amount of prey for a. brunoi due to the high percentage of stomachs with prey. we recommend future studies to evaluate the relationship between prey items and a. brunoi toxicity. acknowledgments we thank gladstone i. almeida, aílston anastácio and josé a. p. schineider for fieldwork assistance. petrobras funded this research. ibama provided the sampling permit (07/98, 02001.003072/97-21). rlm thanks cnpq for scholarship (process 140710/2013-2; 152303/2016-2). rbf and dcm thank capes for scholarships. references christian, k., webb, j.k., schultz, t., green, b. 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(2006): the tadpole of the casque-headed frog, aparasphenodon brunoi myranda-ribeiro (anura: hylidae). south am. j. herpetol. 1: 54-60. acta herpetologica vol. 12, n. 2 december 2017 firenze university press meristic and morphometric characters of leptopelis natalensis tadpoles (amphibia: anura: arthroleptidae) from entumeni forest reveal variation and inconsistencies with previous descriptions susan schweiger1, james harvey2, theresa s. otremba1, janina weber1, hendrik müller1,* brown anole (anolis sagrei) adhesive forces remain unaffected by partial claw clipping austin m. garner*, stephanie m. lopez, peter h. niewiarowski species and sex comparisons of karyotype and genome size in two kurixalus tree frogs (anura, rhacophoridae) shun-ping chang1,2, gwo-chin ma2,3,4, ming chen2,5,6,7,8,*, sheng-hai wu1,* non-native turtles in a peri-urban park in northern milan (lombardy, italy): species diversity and population structure claudio foglini1, roberta salvi2,* species composition and richness of anurans in cerrado urban forests from central brazil cláudia márcia marily ferreira1,*, augusto cesar de aquino ribas2, franco leandro de souza3 the life-history traits in a breeding population of darevskia valentini from turkey muammer kurnaz, alı i̇hsan eroğlu, ufuk bülbül*, halıme koç, bılal kutrup influence of desiccation threat on the metamorphic traits of the asian common toad, duttaphrynus melanostictus (anura) santosh mogali*, srinivas saidapur, bhagyashri shanbhag predation of common wall lizards: experiences from a study using scentless plasticine lizards jenő j. purger*, zsófia lanszki, dávid szép, renáta bocz reproductive timing and fecundity in the neotropical lizard enyalius perditus (squamata: leiosauridae) serena najara migliore1,2,*, henrique bartolomeu braz2,3, andré felipe barreto-lima4, selma maria almeida-santos1,2 observations on the intraspecific variation in tadpole morphology in natural ponds eudald pujol-buxó1,2,*, albert montori1, roser campeny3 and gustavo a. llorente1,2 reliable proxies for glandular secretion production in lacertid lizards simon baeckens diet of juveniles of the venomous frog aparasphenodon brunoi (amphibia: hylidae) in southeastern brazil rogério l. teixeira1, ricardo lourenço-de-moraes2, débora c. medeiros3, charles duca3, rogério c. britto4, luiz c. p. bissoli5, rodrigo b. ferreira3,* who are you? the genetic identity of some insular populations of hierophis viridiflavus s.l. from the tyrrhenian sea ignazio avella, riccardo castiglia, gabriele senczuk* acta herpetologica 12(1): 103-108, 2017 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-18765 nematodes infecting anotosaura vanzolinia (squamata: gymnophthalmidae) from caatinga, northeastern brazil bruno halluan s. oliveira¹, adonias a. martins teixeira¹,*, romilda narciza m. queiroz¹, joão a. araujo-filho¹, diêgo a. teles¹, samuel v. brito2, daniel o. mesquita¹ 1 programa de pós-graduação em ciências biológicas (zoologia), departamento de sistemática e ecologia – dse, centro de ciências exatas e da natureza – ccen, universidade federal da paraíba – ufpb, cidade universitária, campus i, cep 58059-900, joão pessoa, pb, brazil. *corresponding author. e-mail: adoniasteixeira01@gmail.com 2 centro de ciências agrárias e ambientais, universidade federal do maranhão, boa vista, cep 65500-000. chapadinha, ma, brazil. submitted on 2016, 30th august; revised on 2016, 13th december; accepted on 2016, 21st december editor: paolo casale abstract. the present study investigated the composition of helminth parasites of the completely unknown lizard anotosaura vanzolinia (squamata: gymnophthalmidae) and evaluated the effects of sex, host size, and seasonality on endoparasite abundance in two areas of caatinga, northeast brazil. we collected 110 lizards between may 2013 to june 2014 and found 173 nematodes (overall prevalence: 16.3%), with 49 nematodes infecting seven adult males (prevalence: 25%), 84 nematodes infecting six adult females (prevalence: 23%), and 40 nematodes infecting five juveniles (prevalence: 8.9%), where one nematode was in the lungs and 172 were in the gastrointestinal tracts. we identified all nematodes as oswaldocruzia brasiliensis, representing a new record for the host and for gymnophthalmidade, showing overall intensity of infection ± sd of 9.6 ± 5.2. furthermore, abundance of endoparasites was related to the rainy season and sex, but not to host body size (svl). keywords. parasites, lizards, neotropical, oswaldocruzia brasiliensis, nematodes. in the last decade, there has been an increasing number of studies about endoparasites affecting reptiles. the last survey in south america recorded about 155 helminth species infecting lizards and amphisbaenians (ávila and silva, 2010). although the number of these studies in neotropical regions is still increasing, some lizard families are more deeply studied, such as leiosauridae (vrcibradic et al., 2008; barreto-lima and anjos, 2014; dorigo et al., 2014); phyllodactylidae (ávila et al., 2010a; sousa et al., 2010); teiidae (brito et al., 2014b), and tropiduridae (almeida et al., 2009; anjos et al., 2012; pereira et al., 2012). in contrast, studies about gymnophthalmidae are scarce and mainly regard new host records (ávila and silva, 2010). currently, parasitological studies are only available for ten gymnophthalmidae species in neotropical regions: cercosaura eigenmanni and c. oshaughnessyi (bursey and goldberg, 2004); alopoglossus angulatus (goldberg et al., 2007b); leposoma osvaldoi and neusticurus ecpleopus (goldberg et al., 2007a); iphisa elegans elegans (ávila et al., 2010b); bachia scolecoides and cercosaura ocellata ocellata (ávila and silva, 2011); and micrablepharus maximiliani (brito et al., 2014a). gymnophthalmid lizards, popularly known as microteiids, are small (40 to 150 mm snout-vent length) and commonly distributed in neotropical regions from south mexico to argentina, including some islands of central and south america, totalling 220 species in 48 genera (presch, 1980). in brazil, there are about 93 gymnophthalmid species, distributed in 33 genera. one of them is anotosaura, which comprises two species, a. vanzolinia and a. collaris (costa and bérnils, 2015). 104 b.h.s. oliveira et alii anotosaura vanzolinia is a small lizard restricted to semi-arid regions in northeastern brazil, locally known as caatinga, with fossorial habits, occurring in sites with accumulated leaf litter, its diet is comprised basically of microarthropods associated with sites where these lizards live (oliveira and pessanha, 2013). however, there are no reports about parasites of a. vanzolinia, possibly because a. vanzolinia is a rare lizard in caatinga. therefore, this study aims to evaluate the effect of sex, host size, and seasonality on the composition and abundance of helminths associated with the lizard anotosaura vanzolinia, using a considerable sample from caatinga, northeast of brazil. we collected anotosaura vanzolinia specimens during expeditions in the complexo aluízio campos forest park (7°16’34”s, 35°53’7”w), a caatinga area with an altitude of approximately 500 m, with shrubby vegetation represented mainly by bromeliaceae and cactaceae, a larger number of rock outcrops, and accumulated leaf litter (alves et al., 2010; silva et al., 2010) and in the são josé da mata district (7°11’2.85”s, 35°59’6.17”w), located between the arboreal formations locally known as “brejo” and caatinga, with an altitude of approximately 700 m. the site is settled between the “agreste” and “sertão”, being probably one of the last remains of the transitional arboreal vegetation of paraiba, with typical plant species from caatinga and the atlantic forest (barbosa et al., 2007). both sites are located in paraiba, northeastern brazil (fig. 1). climate is tropical, with a mean temperature of 22.9ºc; highest precipitation occurs between march and august (99 mm) and lowest between september and february (29 mm) (climate-date, 2016). we collected 110 lizards between may 2013 to june 2014 (table 1) by hand and using pitfall-traps (six sets in each area), constructed with four buckets (20 l each), totalling 24 buckets per area, arranged in three lines of three meters each, forming angles of 120° from a same central point and connected by a plastic fence fixed with staples on wooden stakes. fig. 1. collecting sites in paraíba state (a) in northeast brazil, campina grande municipality (b). study areas (c): são josé da mata district (sjm) and complexo aluízio campos forest park (cac). 105nematodes infecting the lizard a. vanzolinia from brazil we killed the lizards with a lethal injection of 2% lidocaine hydrochloridec and measured snout-vent length (svl) with digital calipers. subsequently, we sexed them, preserved them in 10% formalin, and stored them in 70% alcohol. all lizards were kept in the coleção herpetológica da universidade federal da paraíba (chufpb). in the laboratory, we removed the respiratory and gastrointestinal tracts and analyzed them in a stereomicroscope to search for endoparasites. the endoparasites found were cleared with hoyer’s solution, counted, identified, stored in 70% alcohol, and kept in the coleção de invertebrados paulo young, in universidade federal da paraíba (ufpbnem: 0001; 0002). the following infection rates were calculated according to bush et al.(1997), where parasite abundance is defined as the total number of parasites found in a sample (individual host, host population and / or host community); mean intensity of infection is the total number of parasites found in a sample, divided by the number of hosts infected with that parasite; prevalence (p%) is the number of host infected with one or more individuals of a particular parasite species divided by total host number. throughout the text, means appear ± 1 sd. to analyze the influence of svl on endoparasite abundance, we performed simple linear regression (excluding juveniles), using the statistica software, version 8.0 (statsoft, 2007). in addition, we performed a generalized linear model (glm), adopting poisson’s distribution, using the software r, package “r commander” (r core team, 2008) to evaluate whether parasite abundance was influenced by host sex, seasonality and interaction between sex and seasonality. we examined 110 anotosaura vanzolinia specimens, of which 26 were adult females (svl: 42.6 ± 2.3), 28 adult males (svl: 37.9 ± 2), and 56 juveniles (svl: 26.7 ± 5.2). we found 173 nematodes (overall prevalence: 16.3%), with 49 nematodes infecting seven adult males (prevalence: 25%), 84 nematodes infecting six adult females (prevalence: 23%), and 40 nematodes infecting five juveniles (prevalence: 8.9%), where one nematode was in the lungs and 172 were in the gastrointestinal tracts. we identified all nematodes as oswaldocruzia brasiliensis, representing a new host record and the first record for gymnophthalmidade, showing overall intensity of infection of 9.6 ± 5.2 (table 2). simple linear regression revealed a non-significant relationship between svl and endoparasite abundance (f1,12 = 4.24; r2 = 0.26; p = 0.061). we observed a significant variation between the abundance of endoparasites and sex of the host (gl = 3; z value = 2.669; p = 0.0076), where females had more parasites than males (fig. 2). in addition, abundance of endoparasites was higher in the rainy season (gl = 3; z value = -4. 318; p <0.0001) (fig. 2). however, abundance of endoparasites was not influenced by interaction sex-season (gl= 4; z value = 0.024; p = 0.981) (fig. 2). several factors can influence parasitism by helminths in reptiles (aho, 1990), especially host size (poulin and george‐nascimento, 2007), sex (galdino et al., 2014), season (brito et al., 2014), and life cycle of the parasite (araujo-filho et al., 2016). usually, larger hosts (mass and body size) have the capacity to provide shelter to a greater number of parasites, consequently, more resources for parasite development and reproduction (george‐nascimento et al., 2004). however, in the present study, we did not observe a significant relation between svl and parasite abundance in a. vanzolinia. for lizards, this hypothesis is supported mainly by tropiduridade: tropitable 1. number of lizards collected in each sampled area, months of the year and season. cac = complexo aluízio campos forest park; sjm = são josé da mata district. months locality cac sjm wet season dry season wet season dry season may 8 – 1 – jun 9 – 1 – jul 4 – 0 – aug 8 – 2 – sep – 7 –  2 oct – 9 – 6 nov – 4 – 2 dec – 7 – 1 jan – 16 – 1 feb – 7 – 1 mar 6 – 0 – apr 7 – 1 – table 2. parasitological data from population of anotosaura vanzolinia from caatinga, northeast brazil, infected by oswaldocruzia brasiliensis. p% = prevalence; sd = standard deviation; i = intestine, l = lung. host sex and maturity host sample size svl/sd (mm) p% (host infected) mean intensity of infection/ sd site infection males 28 37.9 ± 2 25 (7) 7 ± 3.8 i females 26 42.6 ± 2.3 23 (6) 14 ± 8.5 i,l juveniles 56 26.7 ± 5.2 8.9 (5) 8 ± 3.4 i total 110 16.3 (18) 9.6 ± 5.2 106 b.h.s. oliveira et alii durus torquatus (range of intensity: 22.1 ± 20.2) (ribas et al., 1998); t. hispidus (range of intensity: 8.5 ± 1) (anjos et al., 2012); t. torquatus (range of intensity: 8.2 ± 6.9) (pereira et al., 2012); and teiidae species: ameiva ameiva (range of intensity: 7.2 ± 7.3) (ribas et al., 1998); a. festiva (intensity of infestation: 21, range: 1-115) (ramírezmorales et al., 2012). nevertheless, besides body size and host mass, other factors can explain the variation in parasite abundance, such as ecology (aho, 1990), physiology (poulin and mouillot, 2004; poulin, 2007), behaviour, and phylogeny (poulin and mouillot, 2005; patterson et al., 2008; brito et al., 2014a). furthermore, the small size of a. vanzolinia (svl = 42 mm) can constrain the diversification of its parasitic fauna, hindering niche differentiation and microhabitat segregation by endoparasite competitors (kuris et al., 1980; ávila et al., 2010a) thus explaining the presence of only a single nematode species infecting the host studied. this observation was also recorded in other small lizard species, such as liolaemus lutzae (svl = 61 mm), aspronema dorsivittatum (svl = 64 mm), and phyllopezus lutzae (svl = 42 mm), which exhibit up to two endoparasites species (rocha, 1995; rocha et al., 2003; ávila et al., 2010a). we found significant differences in the abundance of nematodes in relation to host sex, where females were more parasitized than males. according to poulin (1996), physiological, morphological, and behavioral differences between males and females may explain the differences of infection rates between sexes. however, due to the absence of research that verified possible differences in the ecology between the sexes for the lizard a. vanzolinia, we can not explain what factors may have influenced the increase of the parasite abundance in females in the present study. the nematode oswaldocruzia brasiliensis has a monoxenous life cycle (lent and freitas, 1935), and congeneric species infect mainly frogs (well slimane and durettedesset, 1996), lizards (bursey et al., 2006), and snakes (durette-desset et al., 2006). according to anderson (2000), monoxenous nematodes can easily be influenced by variations in temperature and humidity. our results support this hypothesis, considering that a. vanzolinia presents a greater endoparasite abundance in the rainy season, mainly because of its fossorial habits, increasing the possibility of infection by o. brasiliensis through accidental ingestion of eggs (in the faeces of other hosts, substrate in general) or by active penetration of infective larvae (via the skin and / or mucosa).this can also explain the higher parasite abundance in females, because they lay eggs during the rainy season and in humid microhabitats to avoid egg desiccation (oliveira and pessanha, 2013). the present study revealed that the parasitic fauna of a. vanzolinia from caatinga consists of a single nematode, oswaldocruzia brasiliensis, representing the first record of infection to the host and to gymnophthalmidae. furthermore, endoparasite abundance is related to the rainy season and sex, but is not correlated with host body size (svl). acknowledgments we thank the fundação universitária de apoio ao ensino, à pesquisa e à extensão (furne) for providing collecting permits at complexo aluízio campos, mr. guilherme leão, who not only granted access to his property in são josé da mata, but also encouraged our survey, and mr. vandeberg ferreira lima for helping with statistical analyses. aamt thank coordenação de aperfeiçoamento da pessoa de nível superior – capes for fig. 2. mean abundance of nematodes between seasons for species of lizard a. vanzolinia, males (triangle) and female (circle). 107nematodes infecting the lizard a. vanzolinia from brazil a research fellowship. dat, jaaf and dom thank the conselho nacional de desenvolvimento científico e tecnológico – cnpq for a research fellowship (303610/20140).the study was approved by the instituto chico mendes de conservação da biodiversidade with the permit to collect the animals (process 36166-2). we thank proofreading-service.com ltda, for revision professional of english version of the manuscript. references aho, j.m. 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(2008): helminth parasites of two sympatric lizards, enyalius iheringii and e. perditus (leiosauridae), from an atlantic rainforest area of southeastern brazil. acta parasitol. 53: 222-225. acta herpetologica vol. 12, n. 1 june 2017 firenze university press morphological variation and sexual dimorphism in liolaemus wiegmannii (duméril & bibron, 1837) (squamata: liolaemidae) from uruguay joaquín villamil1,*, arley camargo2, raúl maneyro1 sex does not affect tail autotomy in lacertid lizards panayiotis pafilis1,*, kostas sagonas2, grigoris kapsalas1, johannes foufopoulos3, efstratios d. valakos4 fire salamander (salamandra salamandra) males’ activity during breeding season: effects of microhabitat features and body size raoul manenti*, andrea conti, roberta pennati call variation and vocalizations of the stealthy litter frog ischnocnema abdita (anura: brachycephalidae) pedro carvalho rocha1,2,*, joão victor a. lacerda2,3, rafael félix de magalhães2,3, clarissa canedo4, bruno v. s. pimenta5, rodrigo carrara heitor6, paulo christiano de anchieta garcia2,3 feeding ecology of two sympatric geckos in an urban area of northeastern brazil josé guilherme g. sousa1, adonias a. martins teixeira2,*, diêgo alves teles2, joão antônio araújo-filho2 and robson waldemar ávila3 a pattern-based tool for long-term, large-sample capture-mark-recapture studies of fire salamanders salamandra species (amphibia: urodela: salamandridae) jeroen speybroeck1,*, koen steenhoudt2,$ skeletal variation within the darwinii group of liolaemus (iguania: liolaemidae): new characters, identification of polymorphisms and new synapomorphies for subclades linda díaz-fernández*, andrés s. quinteros, fernando lobo marking techniques in the marbled newt (triturus marmoratus): pit-tag and tracking device implant protocols hugo le chevalier1, olivier calvez2, albert martínez-silvestre3, damien picard4, sandra guérin4,5, francis isselin-nondedeu6, alexandre ribéron1, audrey trochet1,2,* evidence for directional testes asymmetry in hyla gongshanensis jindongensis qing gui wu1, wen bo liao2,* the advertisement call of pristimantis subsigillatus (anura, craugastoridae) florina stănescu1,4, rafael márquez2, paul székely3,4,*, dan cogălniceanu1,4,5 nematodes infecting anotosaura vanzolinia (squamata: gymnophthalmidae) from caatinga, northeastern brazil bruno halluan s. oliveira¹, adonias a. martins teixeira¹,*, romilda narciza m. queiroz¹, joão a. araujo-filho¹, diêgo a. teles¹, samuel v. brito2, daniel o. mesquita¹ where is my place? quick chorus structure assembly in the european tree frog michal berec a possible mutualistic interaction between vertebrates: frogs use water buffaloes as a foraging place piotr zduniak1,*, kiraz erciyas-yavuz2, piotr tryjanowski3 good vibrations: a novel method for sexing turtles donald t. mcknight1,2,*, hunter j. howell3, ethan c. hollender1, and day b. ligon1 errata to mecke, s., hartmann, l., mader, f., kieckbusch, m., kaiser, h. (2016): redescription of cyrtodactylus fumosus (müller, 1895) (reptilia: squamata: gekkonidae), with a revised identification key to the bent-toed geckos of sulawesi. acta herpetologica 11(2): acta herpetologica 13(2): 191-194, 2018 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-22955 egg and clutch sizes of western chicken turtles (deirochelys reticularia miaria) donald t. mcknight1,2,*, ethan c. hollender1, hunter j. howell3, john l. carr4, kurt a. buhlmann5, day b. ligon1 1 department of biology, missouri state university, spring field, missouri, usa. *corresponding author. email: donald.mcknight@ my.jcu.edu.au 2 college of science and engineering, james cook university, townsville, queensland, australia 3 department of biology, university of miami, coral gables, florida, usa 4 department of biology, university of louisiana at monroe, louisiana, usa 5 savannah river ecology laboratory, university of georgia, aiken, south carolina, usa submitted on: 2018, 4th february; revised on: 2018, 8th august; accepted on: 2018, 20th september editor: aaron m. bauer abstract. chicken turtles (deirochelys reticularia) are generally characterized as having atypical reproductive characteristics relative to other sympatric emydids. however, the comparatively understudied western chicken turtle (d. r. miaria) has been shown to exhibit some reproductive characteristics that differ from the other subspecies. therefore, we examined clutch and egg sizes from six d. r. miaria (13 clutches) in oklahoma and compared the results to values that have been reported for the other two subspecies. females nested up to three times per year, with clutches ranging from 8-13 eggs per clutch (mean = 10.9). the eggs were 19.4-25.3 mm wide (mean = 22.2 mm). these values are greater than the means reported for the other subspecies, but the differences were not statistically significant. keywords. chelonia, emydidae, subspecies, reproduction, geographic variation. chicken turtles (deirochelys reticularia) occur in shallow swamps and ponds throughout the southeastern united states, and they exhibit several traits that are uncommon for north american turtles, the most noteworthy of which relate to their unusual reproductive patterns. the eastern chicken turtle (d. r. reticularia) is the most studied of the three recognized subspecies, and, unlike most north american turtles, females nest in the fall through early spring (gibbons and greene, 1990; buhlmann et al., 2009). eggs deposited in the ground in the fall enter embryonic diapause and resume development in spring after a period of chilling (ewert et al., 2006). additionally, when conditions become unfavourable (too cold) in the fall, females can retain eggs, overwinter with them, and lay them the following spring (buhlmann et al., 1995, 2009). this behavior is uncommon in turtles but has also been reported for the sonoran desert tortoise (gopherus morafkai) in arizona, usa (lovich et al., 2017). florida chicken turtles (d. r. chrysea) extend this strategy by nesting throughout the winter and laying up to three clutches (rarely four) per year (iverson, 1977; jackson, 1988). in contrast, the western subspecies (d. r. miaria) nests in the late spring and summer (may-july), a pattern that is more consistent with other sympatric emydids (mcknight et al., 2015a; carr and tolson, 2018). however, this subspecies is the least-studied of the three, and although current research indicates that it differs from the other subspecies in diet, seasonal activity patterns, and some reproductive patterns (mcknight et al., 2015a, b), there is still a great deal that we do not know about its life history. one of these knowledge gaps is a dearth of information about their clutch and egg sizes. currently, clutch and egg 192 donald t. mcknight et alii sizes for d. r. miaria are only reported from five clutches in louisiana (carr and tolson, 2018), three clutches in arkansas (dinkelacker and hilzinger, 2014) and one in texas (david, 1975). to expand our knowledge of this topic, we captured d. r. miaria in shallow beaver ponds and similar habitats in southeastern oklahoma from 2013-2015 using a variety of hoop nets (detailed trapping methods available in mcknight et al. 2015c). we also attached radio transmitters (ri-2b 10 g or ri-2b 15 g, holohil systems ltd., corp., ontario, canada) to the carapaces of three females and recaptured them approximately every two weeks. upon capture (via trapping or tracking) we used a portable ultrasound (echo camera ssd-500v, hitachi aloka medical, inc., tokyo, japan) to check for the presence of follicles or shelled eggs. if shelled eggs were observed, we immediately transported the turtles to a nearby veterinary clinic and used x-radiography to record the size and number of eggs (gibbons and greene, 1979). we included a coin or other object of known size to correct for magnification from the x-radiograph (graham and petokas, 1989). we counted the eggs and measured their width, but we did not measure their length, because length is only accurate when the eggs are oriented parallel with the plastron (bertolero et al., 2007), a position that cannot be confirmed in a live animal. additionally, we included data from a female that was captured in southeastern oklahoma on 16 june 2008 and transported to the tulsa zoo. shelled eggs were observed via x-radiography on 9 july 2008, and 11 eggs were laid on 20 july 2008, following induction using oxytocin (ewert and legler, 1978; a single egg was laid and crushed on the previous day). these 11 eggs were measured after being laid (including the mass and length), rather than measuring the x-radiograph (they were included with the data from the other clutches for all summary statistics). we measured and recorded clutch sizes for a total of 13 clutches from six females (two additional clutches were observed but could not be x-rayed for quantification or measurements). the clutch and egg sizes are shown in fig. 1 and compared to other studies and subspecies in table 1. the female that laid eggs at the tulsa zoo had a mean egg mass of 9.9 g (sd = 1.14; range = 8-12), mean egg length of 31.6 mm (sd = 1.43; range = 30.2-34.4), and mean egg width of 23.6 mm (sd = 0.88; range = 21.9-25.3; the width measurements are included in figure 1 and table 1). for the three transmittered females that produced multiple clutches in a year, two produced at least two clutches for each of the two years we monitored them (we were not able to x-ray one of those clutches and it was not included in our summary statistics or the total number of clutches). the third individual produced at least one clutch in the first year, and at least three clutches in the second year (the third clutch was undergoing calcification when we x-rayed it, and the shells were not sufficiently visible to reliably count or measure the eggs; therefore, it was not included in our summary statistics or total number of clutches). this is the first report of d. r. miaria producing up to three clutches in a single year. deirochelys r. chrysea is known to nest up to four times per year (typically 2-3), but d. r. reticularia and d. r. miaria have only previously been reported nesting twice per annual cycle (gibbons, 1969; gibbons and greene, 1978; ewert et al., 2006; mcknight et al., 2015a). our mean clutch and egg sizes agreed closely with the results reported by carr and tolson (2018) and dinkelacker and hilzinger (2014) for d. r. miaria in louisiana and arkansas, respectively. all three studies found large mean egg and clutch sizes relative to most reports for the other subspecies. indeed, the maximum egg widths in both our study and carr and tolson (2018) were the largest egg widths reported for any d. reticularia subspecies. to statistically compare egg and clutch sizes between d. r. miaria and d. r. reticularia, we combined our data with the clutches reported in carr and tolson (2018) and compared them with data from d. r. reticularia that were captured in south carolina between july 1995 and february 1996 (previously published in buhlmann et al., 2009). this resulted in a total of 17 clutches from nine female d. r. miaria and 25 clutches from 16 female d. r. reticularia (one clutch from carr and tolson [2018] was not included because female plastron length was not recorded). we constructed mixed-effects models in r (v3.4.1; r core fig. 1. deirochelys reticularia miaria egg widths in oklahoma. each box is the data from a single clutch, and the dashed lines separate individual females. numbers above the boxes indicate clutch size and numbers below the boxes indicate female plastron length at the time the clutch was observed. whiskers indicate the 10th and 90th percentiles and all outliers are shown. the center box (clutch size = 12, plastron length = 179) shows the data for the female that laid eggs at the tulsa zoo. all other boxes are from x-ray data. 193egg and clutch sizes of western chicken turtles team 2017) using the package lme4 (v1.1-15; bates et al. 2015) with subspecies and plastron length as fixed effect factors (including an interaction). for egg width, we used a linear model via the lmer function, and for clutch size, we used a generalized linear model with a poisson distribution via the glmer function. to avoid pseudoreplication, we included female id as a random effect factor for the model comparing clutch sizes, and for the model comparing egg widths, we include female id and clutch id as random effect factors, with clutch id nested in female id. we used quantile-quantile plots and residual plots to check the models’ assumptions, and we assessed the significance of both comparisons using the anova function in the package car (v2.1-6; fox and weisberg, 2011) with type ii sums of squares and the chi-square test statistic. we did not find a significant difference in egg width between d. r. miaria and d. r. reticularia (χ2 = 0.8715, p = 0.3505), but egg width and plastron length were correlated (χ2 = 8.1154, p = 0.0044), which is consistent with previous research on d. r. reticularia (gibbons et al., 1982; buhlmann et al., 2009). the interaction term was not significant (χ2 = 1.4155, p = 0.2341). for the comparison of clutch size, there was not a significant difference between subspecies (χ2 = 0.6655, p = 0.4146), or a significant association with plastron length (χ2 = 1.4070, p = 0.2355), or a significant interaction (χ2 = 0.2898, p = 0.5904). these results do not suggest a difference in egg or clutch sizes between the subspecies after accounting for female body size, however, our sample size was small, resulting in limited statistical power. it would be useful for future researchers to expand this sample and conduct a more robust comparison. acknowledgements we are indebted to the staff of the all animal veterinary hospital, particularly dr. dana harvey, for x-raying our turtles for us. we are also grateful to dr. kay backues at the tulsa zoo for sharing data on the turtle captured in 2008. additionally, we would like to thank jona tucker for her assistance with this research, as well as the oklahoma nature conservancy and private land owners who allowed us to conduct research on their property. this study was funded by an oklahoma department of wildlife conservation state wildlife grant and the delta foundation, and it was approved by the missouri state university institutional animal care and use committee (iacuc protocol no. 10014). kab was supported by the u.s. department of energy under award number defc09-07sr22506 to the university of georgia research foundation. we conducted the research under oklahoma department of wildlife conservation scientific collecting permits #5269, 5950, and 5610 and adhered to the asih/ hl/ssar guidelines for use of live amphibians and reptiles. table 1. clutch and egg sizes for chicken turtle (deirochelys reticularia) subspecies. mean pl = mean plastron length of females. for results from the current study, mean egg width was calculated by averaging the means per clutch, whereas the range is the maximum and minimum of any measured eggs. subspecies state mean clutch size clutch size range mean egg width (mm) egg width range (mm) # of clutches mean pl (mm) source d. r. miaria ok 10.9 9–13 22.2 19.4–25.3 13 174.8 current study d. r. miaria la 10.6 8–13 22.9 20.7–24.7 5a 186.1 carr & tolson, 2018 d. r. miaria ar 10.7 9–12 22.3 b 21.1– 23.5 b 3b 174.9 dinkelacker & hilzinger, 2014 d. r. miaria tx 8 — — 17–23 1 — david, 1975 d. r. reticularia sc 7.2 1–12 — — 57 159 gibbons et al., 1982 d. r. reticularia sc 8 — 20.8 — 15 — congdon et al., 1983 d. r. reticularia sc 8 — 20.8 — 13 160 congdon & gibbons, 1985 d. r. reticularia sc 9.8 5–17 21.8 17.4–23.4 32 177.5c buhlmann et al., 2009 d. r. chrysea fl 10.5d 8–12d 21.2 19.9–22.9 4d — iverson, 1977 d. r. chrysea fl 9.5 2–19 22.4 20.1–23.6 29 176.3 jackson, 1988 d. r. chrysea fl 10.9 6–18 — — 16 — ewert et al., 2006 a plastron length was only recorded for four females. b egg width was based on only two clutches. c webplotdigitizer (rohatgi 2017) was used to extract measurements from figure 3 of buhlmann et al. 2009. d one female contained 5 eggs and “6 ovarian follicles of ovulatory size.” based on our experience with d. r. miaria, we treated the follicles as unshelled eggs and assumed a clutch size of 11 (otherwise, the mean is 9 and range is 5–12). 194 donald t. mcknight et alii references bertolero, a., nougarède, j.p., cheylan, m., marín, a. 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(2017): webplotdigitizer, version: 4.0. available at: https://automeris.io/webplotdigitizer. acta herpetologica vol. 13, n. 1 june 2018 firenze university press acta herpetologica 15(1): 31-37, 2020 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/a_h-7757 don’t tread on me: an examination of the anti-predatory behavior of eastern copperheads (agkistrodon contortrix) andrew adams1,2,*, john garrison2, scott mcdaniel2, emily bueche2, hunter howell2,3 1 stem division, harford community college, 401 thomas run road, bel air, maryland, 21015, usa 2 susquehannock wildlife society, 1725 trappe church road, darlington, maryland, 21034, usa 3 department of biology, university of miami, 1301 memorial drive, #215 cox science center, coral gables, florida, 33146, usa * corresponding author. email: anadams@harford.edu submitted on: 2020, 13th january; revised on: 2020, 10th april; accepted on: 2020, 14th april editor: marco sannolo abstract. venomous snake species across the globe have been historically categorized as aggressive and dangerous, leading to widespread persecution and killings. despite the conservation importance of educating the public about the docile nature of these species, few studies have attempted to quantify the response of viperid species to human interactions. here we report the responses of free-ranging copperheads to a potential human encounter using a set of hierarchical behavioral trials. out of a total of 69 snakes, only two individuals feigned striking and only two attempted to bite (3% of all individuals). our results support the findings of previous studies documenting the docile nature of other viperid species and can hopefully be used to change the public perception of venomous snakes. convincing the public and policy makers that viperid species are docile is critical to long-term conservation of these species in the u.s. and around the globe. keywords. human-wildlife conflict, optimality theory, venomous species, viper, viperidae. introduction as human populations continue to grow and encroach further into uninhabited or sparsely populated areas, there is a subsequent increase in the prevalence of human-wildlife conflict (woodroffe et al., 2005; skogen et al., 2008; dickman, 2010). the outcomes of humanwildlife conflict are rarely more pronounced and potentially lethal to both parties than the interaction between humans and venomous snakes. venomous snakes have long been a source of great fear for the general public, and have been historically (and currently) mischaracterized as being aggressive and dangerous (blythe, 1979; seigel and mullin, 2009; burghardt et al., 2009; pandey, 2016). even scientific medical publications describing envenomations as late as 2002 (juckett and hancox, 2002) continued to perpetuate the myth that viperid species like the cottonmouth (agkistrodon piscivorus) are aggressive and readily attack humans. misinformation and negative public perception have led to the wholesale slaughter of venomous pit vipers across north america and europe. events such as rattlesnake and copperhead roundups in the us (adams et al., 1994; fitch, 1998; burghardt et al., 2009), and the killing of individual snakes, such as the meadow viper (vipera ursinii), the cyperian blunt-nosed viper (macrovipera lebetina lebetina), and the northern adder (vipera berus) when they are encountered in europe (edgar and bird, 2006; stumpel et al. 2015, julian and hodges, 2019) are examples of direct persecution against viperid species. this widespread persecution continues to take place in both areas, and is a serious conservation concern for many viperid species, despite these species causing very low numbers of fatalities across these two continents (chip32 andrew adams et alii paux, 2012). it is imperative that any conservation action plan seeking to protect these species will need to incorporate some type of public outreach to reduce direct persecution of these species (seigel and mullin, 2009). despite the largely negative public perception surrounding pit-vipers, papers have been published in the last two decades that clearly demonstrate the passive and even cowardly nature of other viperid species (shine et al., 2000; gibbons and dorcas, 2002; glaudas et al., 2005). if snakes are confronted by a large potential predator, the decision to no longer rely on passive defensive behaviors and strike could have a host of potentially short and long-term negative consequences for the snake (gibbons and dorcas, 2002; broom and ruxton, 2005). for cryptic species, optimality theory predicts that the most efficient strategy to both reduce energy waste and avoid potential mortality is to remain in hiding if possible and to flee immediately if detected by the predator (ydenberg and dill, 1986; broom and ruxton, 2005; mcknight and howell, 2015). like other cryptic species responding to large predators, cryptic viperid species should rely primarily on crypsis or fleeing as primary sources of predator evasion, followed only after these two tactics have failed, by striking and envenomation. when confronted and detected by a potential predator, a snake should first attempt to escape, then employ a suite of passive deterrents (e.g., musking, tail vibrating, mouth gaping), and finally commit to active defenses (biting or striking; roth and johnson, 2004). in addition to the decision-making process driven by optimality theory (see ydenberg and dill, 1986), there are a host of intrinsic and extrinsic factors that may act to mediate the chance that a snake will strike (cooper and vitt, 2002; roth and johnson, 2004). intrinsic factors such as size (hailey and davies, 1986; whitaker and shine, 1999), body temperature (layne and ford, 1984; goode and duvall, 1989), sex (scudder and burghardt, 1983), time since feeding (herzog and bailey, 1987), prior predator exposure (glaudus, 2004), and gestation may all play a role in the likelihood of striking (glaudas et al., 2005). however, studies have found contradictory results regarding the role that each of these factors may play, suggesting that the exact influence of these factors are likely species specific (roth and johnson 2004). extrinsic factors like the severity of the threat and the relative location of the snake may also impact strike likelihood (gibbons and dorcas, 2002; shine et al., 2002; glaudas et al., 2005). the eastern copperhead (agkistrodon contortrix) is perhaps the most commonly persecuted snake species in the eastern us. this wide-ranging species can be found throughout the eastern united states from massachusetts to florida, west into texas and across a wide variety of habitat types (ernst and ernst, 2003). copperheads are an ideal species for a study examining the defensive behavior of a viperid species to human presence and subsequent interaction, because they are widespread across the heavily populated areas of the mid-atlantic and southeastern us, are responsible for a large proportion (49.2%) of the reported envenomations in the us (gummin et al., 2017), and are both widely feared and heavily persecuted when located by the general public. while there were 2,048 reported copperhead envenomations in the us in 2016 (gummin et al., 2017), the overwhelming majority of these envenomations (94%) were either of a moderate or lower health-risk and there were zero reported fatalities (gummin et al., 2017). with the continued and rapid expansion of urbanized areas across the copperhead’s range, especially in the southeastern us, where copperheads are still abundant, the number of copperhead-human interactions is likely to increase in the future. therefore, an understanding of the anti-predatory behavior of copperheads may be used to dispel misinformation, inform the public about the behavior of this common and widespread venomous species, and potentially serve to mitigate the negative consequences of future human-snake encounters. the aim of the present research is to examine the anti-predatory behavior of the eastern copperhead when contacted by a potential human predator. based on optimality theory, we predict that copperheads will rely on crypsis to avoid predation and will very rarely resort to defensive anti-predatory tactics. materials and methods our study areas (n = 10) were dispersed throughout the state of maryland, a small state located in the mid-atlantic region of the us, and included a variety of habitats within each of the state’s physiographic provinces. maryland is comprised of six physiographic provinces, the atlantic continental shelf province, the coastal plain province, the piedmont plateau province, the blue ridge province, the ridge and valley province, and the appalachian plateau province (reger and cleaves, 2002). copperheads are widely distributed across maryland and occupy different habitats within these physiographic provinces (e.g., bottomland swamps, rocky stream banks, south facing slopes) across the state. the location of each site was non-random, with study sites chosen based on prior distribution records collected through the maryland amphibian and reptile atlas (cunningham and nadrowicz, 2018) or historical localities gathered from harris (1975). all encounters occurred within the state of maryland. searches were conducted during the copperhead’s active season, from 01 may 2017 to 01 november 2017, and again from 01 may 2018 to 01 november 33anti-predatory behavior of eastern copperheads 2018. the snakes were located by visually searching each study site by foot (karns, 1986; gibbons and dorcas, 2002). in general, snakes were found in areas adjacent to wintering den sites with a large amount of adjacent cover in the form of rock crevices and piles. once a snake was visually located, the body position of each individual, either coiled or extended, was recorded prior to any further approach (shine et al., 2000; glaudas et al., 2005). since body surface temperature can play a role in defensive behavior (arnold and bennett, 1984), we used a ryobi tek4 non-contact infrared digital thermometer (ryobi, chicago, il, usa) to record body temperature at three different locations on the body (head, mid-section, and cloaca) from a distance of ~2 m and then averaged these values together (garrick, 2008). ambient environmental temperature was recorded by extracting local weather data from the nearest weather station using the weather underground mobile application software (v5.11.9, twc product and technology, atlanta, ga) from the national weather service, operating under the national oceanic and atmospheric administration (2018). once located, snakes had 1) an apparatus with a boot attached placed directly adjacent to the snake to simulate a possible human interaction while actively hiking (gibbons and dorcas, 2002; shine et al., 2002). after the initial approach and first trial, the snake then had 2) the apparatus placed gently on top of it (to simulate accidental contact with a hiker; gibbons and dorcas, 2002). finally, the snake was 3) grabbed and picked up using a pair of snake tongs covered with a leather glove to simulate a human hand (gibbons and dorcas, 2002; glaudas, 2004; glaudas et al., 2005; maritz 2012). a previous study showed that a human hand elicits a strong anti-predatory response, suggesting that faux gloved hand might elicit a similar anti-predatory response (herzog et al., 1989). each stage of the test (1-3) was carried out for 20 seconds and was videotaped using a digital video camera (to allow post hoc analysis of the defensive response). during each phase, the observers recorded the defensive behavior of the snake from the anterior end. behaviors were categorized into four separate categories during each stage of the experiment (fleeing, tail vibrating, feigning a strike, and striking; gibbons and dorcas 2002) to represent escalating levels of antipredatory responses. a feigned strike was classified as a lunge forward without any discernible opening of the mouth. to test the effect of human activity, environmental temperature, snake’s body temperature, and the snake’s initial posture on anti-predatory behavior, we categorized each snake’s response across trials into one ordered value based on their most defensive response to any of the trials (no-response [0], fleeing [1], benign antipredatory response [2; tail vibrating], or defensive anti-predatory response [3; feigning a strike or striking]). to test for associations between ambient and body temperatures and behavior we used an anova. both environmental temperature and the snake’s body temperature were normally distributed (shapirowilk w test, w = 0.94 and 0.97 respectively). to test for associations between initial body condition and anti-predatory behavior we used a mann-whitney u-test. all statistical analyses were conducted in jmp pro (v14, sas institute inc., cary, nc). we did not collect and individually mark snakes since it would have been impossible to collect many of the snakes that rapidly fled into adjacent cover (e.g., deep rock crevices, den sites, heavy vegetation) in a manner that did not harm the snakes or lead to the potential envenomation of the researchers. additionally, since contact prior to the trials would have biased behavior, marking could not have been performed prior to the initiation of the trials. to help prevent “double-testing” of the same snake, integument patterns (specifically the darker “hourglass” bands that may have been thin or wide, uneven, broken on the dorsal side, etc.) were used as a basis for individual recognition and were supplemented by recordings of scale abnormalities (carlstrom and edelstam, 1946; shine et al., 1988; moon, et al., 2004). post-hoc visual photo comparison between each individual snake was conducted to remove any duplicate trials. in total, we removed one snake trial from all analysis after post-hoc comparison confirmed that it had been tested during a prior sampling period. results in total, we recorded encounters with 69 snakes across all 10 sites (fig. 1). of these 69 snakes, 15 escaped immediately upon discovery without performing any other anti-predatory behavior and were not available for any further trials. during the initial approach, one snake performed tail vibrating before fleeing, one snake performed tail vibrating and a feigned strike before fleeing, and one snake attempted a strike. after accounting for these 18 snakes, 52 snakes remained for further trials. for a summary of the responses to each of the individual trials (stepped next to (n = 52), stepped on (n = 33), and picked up (n = 14)), see fig. 2. in total across all trials, five snakes displayed tail-vibrating behavior and one exhibited a feigned strike followed by fleeing (fig. 2). across all trials, we recorded only two instances of striking (3% of all snakes). there was no relationship between snake anti-predatory behavior and either ambient temperature (anova: f3,65, p = 0.92) or snake body temperature (anova: f3,62, p = 0.45). similarly, there was no difference in anti-predatory behavior between snakes that were initially coiled or extended (u = 255, p = 0.496). thus, across all conditions snakes showed similarly low percentages of antipredatory behavior. discussion overall, our results provide evidence to support the hypothesis that copperheads respond to potential predators in a manner consistent with their cryptic patterning. specifically, copperheads are more likely to either remain in crypsis or flee in the presence of a human rather than display defensive behavior. across the various trials of 34 andrew adams et alii fig. 1. geographic distribution of the study sites across the state of maryland. each study site is represented by a pie chart with the binned behavioral responses from all individuals at that site. fig. 2. responses of copperheads to four increasing threat levels in a hierarchical anti-predatory trial (found, stepped next to, stepped on, picked up). 35anti-predatory behavior of eastern copperheads the study, 93% of the snakes fled when we approached or made physical contact with them. furthermore, a higher proportion of snakes (6%, n = 4) displayed no response to any of our interactions (including being picked up) than those snakes that struck during one of the trials (3%, n = 2). while the proportion of strikes was low, these results mirror the findings of other studies examining pit-vipers’ responses to humans and consistently demonstrate that despite the public’s perception of these species as being dangerous and aggressive, that pope (1958) was correct when claiming that snakes are “first cowards, then bluffers, and last of all, warriors.” to examine how different intrinsic and extrinsic factors influenced anti-predatory response, we analyzed the effect of the initial body posture and body temperature of each individual, and ambient environmental temperature on anti-predatory responses. while we did not collect individuals to gather morphometric data, the extremely low prevalence of snake strikes makes it highly unlikely that any effect of sex or size class on a snake’s anti-predatory response would have been detected. however, with a larger sample size, differences in anti-predatory behavior based on various intrinsic or extrinsic factors may be detected. the published literature on the anti-predatory behavior of snakes is full of conflicting results regarding the role of intrinsic and extrinsic factors on anti-predatory behaviors both between and among species (shine et al., 2000; roth and johnson, 2004). unfortunately, our work does little to elucidate the differences between these conflicting studies, except perhaps to further emphasize that differences in evolutionary history may be prohibitive when attempting to produce general models of antipredatory behavior in snakes. while for obvious safety reasons we were unable to approach the snakes with an exposed hand or forearm, other studies have also used a gloved apparatus to simulate the human hand (gibbons and dorcas, 2002; glaudas 2004; glaudas et al., 2005). while it is possible that the difference in temperature between the gloved apparatus and a human hand may have modified the anti-predatory behavior of the copperheads due to their heat-sensing capabilities, no study has yet assessed the importance of thermal cues in the modification of antipredatory behavior in free-ranging pit-vipers. since no studies have examined the effect of predator temperature on anti-predatory behavior, examining the literature on the influence of temperature on predatory behavior may be instructive. however, in the only field studies conducted to this point examining the importance of thermal cues on predatory behavior, shine and sun (2003) found that while adult snakes were more likely to strike at warmer objects, temperature was not a predictor of juvenile strikes, and schraft et al. (2018) found that absolute temperature was not an important predictor of predation attempts. the public perception of venomous snakes as aggressive and dangerous leads to a suite of problems for the conservation of viperid species (see seigel and mullin, 2009 for an overview). most notable among these issues are the large organized round-ups that may lead to localized extirpation of rattlesnakes (adams et al., 1994; fitch, 1998; burghardt et al., 2009) and the lack of resources that are made available for habitat protection or management of these species (seigel and mullin, 2009). while some studies provide cautionary tales about the potential backfiring of educational material (hoff and maple, 1982), it is clear that increasing the public’s positive perception of snakes will be a necessary component of any long-term conservation plan (seigel and mullin, 2009). more recent studies have shown that well designed education programs focusing on biodiversity conservation, the ecological role or snakes, or the use of antivenom for medicinal use can improve feelings and attitudes about snakes (murphy and xanten, 2007; markwell and cushing, 2009). as part of this public outreach and education, providing examples demonstrating the docile nature of most venomous species may convince some individuals to support legislation to prevent the organized killings that persist to this day. the results of this research provide previously unavailable information to inform the public of the docile nature of copperheads and potentially assuage fears surrounding the perceived aggressive nature of viperid species. these striking results should prove useful in convincing the proportion of the public that is still impressionable of the copperheads’ benign nature and may result in an increase in positive public perception. future conservation of imperiled viperid species may hinge on the ability of scientists to persuade policy makers and the public of the importance and docile nature of these species (seigel and mullin, 2009). this study provides further evidence that common venomous species are not aggressive and rely on striking only as a last resort. as charas (1677) noted over 300 years ago, “the viper is taken by many for an image of malice and cruelty; but in reality, she is guilty of no such thing”. acknowledgement we would like to thank m. addicks, c. auth, h. deery, b. durkin, m. gacheny, r. hamilton, j. hansen, v. lannen, r. mady, j. marlow, a. rodriguez, g. ross, k. stenta, m. szymanski, and j. wimmer for their valuable 36 andrew adams et alii assistance in the field during data collection. c. searcy, s. clements, c. mothes, d. mcknight, and r. mady assisted with manuscript revisions and provided statistical guidance. s. smith was a critical component of the permitting process. all work was conducted under maryland state department of natural resources permit # 56452, and in accordance with asih/hl/ssar guidelines for use of live amphibians and reptiles in field research. the authors declare no conflict of interest. references adams, c.e., thomas, j.k., strnadel, k.j., jester, s.l. 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(1986): the economics of fleeing from predators. adv. stud. 16: 229-249. acta herpetologica vol. 15, n. 1 june 2020 firenze university press has the west been won? a field survey and a species distribution model of iberolacerta horvathi in the alps giuseppe de marchi1,*, giovanni bombieri1, bruno boz1, fausto leandri2, jacopo richard3 first record of underwater sound produced by the balkan crested newt (triturus ivanbureschi) simeon lukanov identification of biologically active fractions in the dermal secretions of the genus bombina (amphibia: anura: bombinatoridae) iryna udovychenko1,*, oleksandra oskyrko1, oleksii marushchak2, tetiana halenova1, oleksii savchuk1 don’t tread on me: an examination of the anti-predatory behavior of eastern copperheads (agkistrodon contortrix) andrew adams1,2,*, john garrison2, scott mcdaniel2, emily bueche2, hunter howell2,3 an overview of research regarding reservoirs, vectors and predators of the chytrid fungus batrachochytrium dendrobatidis jarid prahl, thomas p. wilson*, david giles, j. hill craddock genetic characteristics of an introduced population of bombina bombina (linnaeus, 1761) (amphibia: bombinatoridae) in moselle, france jean-pierre vacher1, 2,*, damien aumaître3, sylvain ursenbacher2,4 adaptive significance of the transparent body in the tadpoles of ornamented pygmy frog, microhyla ornata (anura, amphibia) santosh m. mogali*, bhagyashri a. shanbhag, srinivas k. saidapur comparison of complement system activity amongst wild and domestic animals maría s. moleón1,2,*, mark e. merchant3, hugo h. ortega4, pablo a. siroski1,2 effects of temperature and food level on plasticity of metamorphic traits in bufo gargarizans gargarizans larvae tong lei yu*, yan ting han acta herpetologica 11(2): 179-187, 2016 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-18176 mediodactylus kotschyi in the peloponnese peninsula, greece: distribution and habitat rachel schwarz1,*, ioanna-aikaterini gavriilidi2, yuval itescu1, simon jamison1, kostas sagonas3, shai meiri1, panayiotis pafilis2 1 department of zoology, tel aviv university, tel aviv 6997801, israel. * corresponding author. e-mail: rachelschwarz13@gmail.com 2 department of zoology and marine biology, school of biology, university of athens, panepistimioupolis, ilissia, greece 3 department of human and animal physiology, school of biology, university of athens, panepistimioupolis, ilissia, greece submitted on 2016, 14th april; revised on 2016, 03th july; accepted on 2016, 20th august editor: marco sannolo abstract. the gecko mediodactylus kotschyi is considered rare in mainland greece, yet it is very abundant on the aegean islands. it has been thought to be saxicolous throughout much of its range. in a recent survey on the peloponnese peninsula, however, we encountered it mainly on trees, and with higher frequency than previously reported. we combined our observations of localities in which we detected this gecko, and places where we failed to detect it, with data about its occurrence from the literature and museum collections. we posited two hypotheses as possible causes for the apparent relative scarcity of m. kotschyi in the peloponnese: that it is associated with low precipitation and that it has an aversion to limestone rock. we predicted that m. kotschyi would be more likely to be found in arid places and where limestone is not the dominant type of rock, since it has been reported that this substrate is less suitable for this species. moreover, we predicted that geckos occurring in limestone regions would be found on trees rather than under rocks. geckos were indeed found mainly in the more arid parts of the peloponnese, but not exclusively so. we found no evidence of limestone avoidance. we suggest that, because m. kotschyi is better known as being mostly saxicolous over most of its range, and exclusively so on the greek islands, in the peloponnese the search for this species has been restricted to a single habitat type, i.e., under rocks and not on trees. it may thus inhabit more localities in the peloponnese and be more abundant there than has previously been thought. keywords. arboreality, habitat preferences, mediodactylus kotschyi, peloponnese, rock type. introduction the genus mediodactylus is predominantly asian, with only one of 13 species being found in europe. the mediterranean thin-toed gecko, mediodactylus kotschyi (steindachner, 1870) (reptilia: gekkonidae), has a discontinuous distribution incorporating southern italy, through to the balkans and the crimean peninsula, to israel and iran (arnold and ovenden, 2002; sindaco and jeremcenko, 2008). in greece, m. kotschy is ubiquitous and highly abundant on the sporades, cyclades, and south aegean islands as well as around crete, where its distribution is well documented (e.g., wettstein, 1937; beutler and gruber, 1977; beutler, 1981; chondropoulos, 1986), and its diversity is high: 13 subspecies are currently recognized from the greek islands (karandinos and paraschi, 1992; kasapidis et al., 2005; uetz and hošek, 2016). however, it is considered to be rare on the greek mainland and throughout the balkans (stojanov et al., 2011; tomovic et al., 2014), and is often absent from peloponnese species checklists (e.g., werner, 1929; cyrén, 1935; bischoff and bischoff, 1980; henle, 1989; pèrez-mellado et al., 1999). the peloponnese peninsula nonetheless forms a major part of m. kotschyi’s distribution on mainland greece 180 rachel schwarz et alii (valakos et al., 2008). it has been recorded from several locations (e.g., monemvasia, the terra typica of m. k. bibroni, beutler and gruber, 1977; sparta and kalamata, stepánek, 1937), and is thought to be widespread in the western peloponnese (valakos et al. 2008). it is nonetheless considered rare, exhibiting a low population density almost everywhere on the greek mainland (ajtić, 2014). m. kotschyi is described as being mainly saxicolous across much of its range, being found under rocks and stone piles, on dry stone walls and even on the external walls of houses and other buildings (beutler, 1981 and citations therein; musters and in den bosch, 1982; arnold and ovenden, 2002; ajtic 2014). in greece it is described as preferring dry areas with phrygana (=dwarf shrub steppe) vegetation, although it also inhabits cultivated areas (beutler, 1981). phrygana is common on the aegean islands, but is rare on mainland greece, and this has been claimed to be the main reason for its rarity on the mainland (beutler, 1981). in israel, in contrast, m. kotschyi is almost obligatorily arboreal, and its hebrew name (“עצים tree-gecko”) reflects this (werner, 1993; bar“ = ”שממית and haimovitch, 2012; and our pers. obs.). in israel, southern turkey and iraq it can be found on some common mediterranean trees such as oak, olive, fig, almond and carob, as well as on introduced species such as eucalyptus (weber, 1960; beutler, 1981; and pers. obs.). another major factor thought to influence the distribution of m. kotschyi is that of substrate. like all members of its genus, this species lacks adhesive toe pads (gamble et al., 2012). many pad-less saxicolous species are associated with rough rock surfaces (higham, 2015), such as sandstone (russell et al., 2007), perhaps because they are able to attain a secure grip on these types of rocks compared to smoother ones. beutler (1981) suggested that in the cyclades, where m. kotschyi is very abundant, the ground is comprised mainly of slate, granite, mica, marble and volcanic rocks. however, the main rock type on the greek mainland is limestone, which according to beutler is less suitable for m. kotschyi. consequently, he has claimed that the only mainland region where m. kotschyi is abundant is in the taygetos mountain range south of sparta, where the dominant rock types are shale and sandstone (beutler, 1981). the range of precipitation for mediterranean vegetation is ~250-800 mm, the lower limit of which corresponds to phrygana vegetation (aschmann, 1973), which covers the peloponnese peninsula’s coastline from south to east (mavromatis, 1978). we hypothesized that, because m. kotschyi is thought to be strongly associated with sparse phrygana, it would be more common in the eastern peloponnese, where the climate is drier due to the rain shadow cast by the central mountains (kotinizabaka, 1983; bringsoe, 1985). we further hypothesized that m. kotschyi would be rare in areas where the main type of rock is limestone (beutler, 1981), and that in wet regions, and where limestone predominates, it would be more likely to occur on trees than among rocks. material and methods to test our predictions we constructed a presence and absence distribution map of m. kotschyi, incorporating 68 locations from across the entire peloponnese. sixteen of these observations (eight presences and eight absences, table 1a, b) are derived from fieldwork we conducted in june and october 2015, including two locations from which the gecko had been previously reported (maragou et al., 2015) as well as six new localities in both phrygana habitats and tree groves. the remaining 52 locations were sourced from publications, museum records and localities surveyed independently by ks (table 2). we searched for m. kotschyi by turning over rocks and visually scanning tree trunks during daylight hours (m. kotschyi is mostly diurnal, active during all but the hottest hours of the day, beutler, 1981; valakos et al., 2008; baier et al., 2009; and pers. obs.). in each locality two to four people searched for geckos for at least 30 minutes. weather conditions were favourable for m. kotschyi activity throughout. if no individuals were observed in a locality we considered it to be absent, although we acknowledge that false absences are a possibility. we recorded the habitat and type of substrate on which the individuals were found. we recorded gps coordinates of all locations surveyed, for both presence and absence of m. kotschyi, and assembled them on a map using arcgis (esri, 2011). most literature-based locations (table 2) are provided only as verbal descriptions (usually the name of a town). we digitized the coordinates of these using google maps (2015). to determine whether a connection exists between the distribution of m. kotschyi and aridity, we assigned mean annual precipitation data (from worldclim, hijmans et al., 2005) to sampled localities (presence and absence, tables 1, 2). we also recorded rock type for all such locations using geological maps (higgins and higgins, 1996) in order to test for substrate preferences. we performed statistical tests of rock type associations only for the presence locations for which substrate data were specified. we used χ2 tests for goodness of fit to compare observations from wet and arid regions, student’s t tests were applied to test for a connection between substrate type and precipitation, and to compare climatic conditions at sites with and without geckos. we used fisher’s exact test to search for a connection between rock type and preferred substrate. all analyses were carried out using r version 3.0.1 (r development core team, 2013). results during our 2015 survey we encountered geckos in eight locations but failed to encounter them in the remaining eight (table 1). most records of m. kotschyi 181mediodactylus kotschyi in the peloponnese peninsula are from the central and eastern parts of the peninsula (fig. 1). the 800 mm isohyet divides the peloponnese into roughly equal areas (above 800 mm: 10,134.45 km2; below 800 mm: 11,113.96 km2), and thus the null expectation would be for 10:11 number of observations from arid and wet regions. nevertheless, most records of presence (77%, 46 out of 60, table 1 a) are from where annual mean precipitation is lower than 800 mm (χ2 goodness of fit test, χ2 = 14.21, n = 60, p = 0.0002). fourteen locations where presence has been recorded are from regions table 1. presence (a) and absence (b) of mediodactylus kotschyi at sites surveyed during our field work in june and october 2015 in the peloponnese. data on substrate and rock type, annual precipitation (mm) and gps coordinates were combined with the data presented in table 2 for map construction. a. presence location habitat searched substrate rock type annual precipitation (mm) no. individuals caught (rocks/ trees) latitude longitude north west of neapolis, malea peninsula, laconia sparse phrygana with carob trees and eucalypt logs on carob and olive trees, and eucalypt logs limestone 564 (2/7) 36.5631n 23.0040e neapolis, malea peninsula, laconia olive grove on olive and carob trees and on a building’s wall alluvium 548 (2/4) 36.5339n 23.0421e north west of neapoli, malea peninsula, laconia dense phrygana under a rock limestone 585 (1/0) 36.5690n 22.9890e gefira, near monemvasia, malea peninsula, laconia dense phrygana with carob trees under rocks and on carob trees limestone 543 (3/2) 36.6867n 23.0368e north west of monemvasia, malea peninsula, laconia eucalypt, almond and carob grove on eucalypt, almond and carob trees phyillites 572 (0/17) 36.7291n 22.9802e north east of geraki, laconia phrygana with carob trees bordering an olive grove on carob and olive trees alluvium 688 (0/5) 36.999n 22.722e kato vervena, arcadia olive grove on olive trees alluvium 600 (0/5) 37.4396n 22.7370e kalogria, south west of patras, achaea eucalypt and pine forest under rock piles and on eucalypt trees alluvium 766 (4/2) 38.13n 21.37e b. absence location habitat searched rock type annual precipitation (mm) latitude longitude lagokili, mani peninsula olive grove (both on trees and under rocks) limestonemarble 707 36.6502n 22.4017e south of platsa, mani peninsula, messenia stone piles limestonemarble 752 36.800n 22.318e kardamyli, mani peninsula eucalypt grove (trees only) neogene sediments 719 36.891n 22.233e west of prosilio, mani peninsula phrygana (rocks only) limestone 770 36.9134n 22.2240e south east of agii anargiri, laconia phrygana (rocks only) neogene sediments 676 37.0160n 22.6360e south west of kosmas, laconia stream bed (rocks only) limestonemarble 849 37.0800n 22.7200e east of kalogria, achaea phrygana (rocks only) limestone 793 38.1605n 21.3847e trochalia, malea peninsula, laconia eucalypt grove (trees only) alluvium 546 36.6535n 23.0241e 182 rachel schwarz et alii table 2. locations, substrate and rock type, annual precipitation (mm), sources of data, and estimated gps coordinates (longitude and latitude in decimal degrees) used for map construction. location name substrate rock type annual prec. (mm) latitude longitude source lachos, mani peninsula unspecified limestone-marble 688 36.48n 22.37e valakos et al. 2008 kokkala, mani peninsula unspecified limestone-marble 651 36.52n 22.47e valakos et al. 2008 kato kastania, malea peninsula, laconia phrygana with many rocks limestone 596 36.52n 23.11e bringsoe 1985 ano kastania, malea peninsula, laconia unspecified phyllites 606 36.537n 23.102e valakos et al. 2008 lira, malea peninsula, laconia unspecified phyllites 637 36.640n 22.964e valakos et al. 2008 loutsa, mani peninsula unspecified limestone-marble 711 36.643n 22.474e valakos et al. 2008 monemvasia, malea peninsula, laconia unspecified limestone 546 36.69n 23.05e beutler and gruber 1977 south of agios ioannis, malea peninsula, laconia unspecified limestone 557 36.726n 23.007e valakos et al. 2008 5 km north of monemvasia, malea peninsula, laconia building limestone 549 36.73n 23.02e bringsoe 1985 methoni, messenia unspecified alluvium 742 36.82n 21.704e valakos et al. 2008 kastania, kariofouni and driopi area, laconia area with stone walls limestone-marble 762 36.84n 22.35e bauer 2004; valakos et al. 2008 between saidona and kastane, mani peninsula unspecified limestone-marble 797 36.87n 22.29e bringsoe 1985; valakos et al. 2008 lakkos, mani peninsula on trees in forest neogene sediments 768 36.893n 22.259e pers. obs. kostas sagonas 2014 exochori (taygetos), mani peninsula unspecified neogene sediments 768 36.90n 22.26e werner 1937; valakos et al. 2008 mandina near kampos, mani peninsula unspecified flysch 781 36.93n 22.20e naturhistorisches museum wien kalamata, messenia unspecified alluvium 762 37.04n 22.11e stepánek 1937; valakos et al. 2008 2 km south of gargalianoi, messenia on the ground in open field peridotite and serpentinite 813 37.049n 21.634e pers. obs. kostas sagonas 2014 mystras, laconia unspecified limestonemarble 776 37.06n 22.37e beutler and gruber 1977; stepánek 1937; valakos et al. 2008 10 km west of sparta, laconia unspecified limestone-marble 913 37.064n 22.305e valakos et al. 2008 pyrgos, elis near stone terraces neogene sediments 839 37.07n 21.69e bringsoe 1985 sparta, laconia unspecified alluvium 712 37.071n 22.430e valakos et al. 2008 5 km north east of kosmas, arcadia eggs under a flat rock limestone 715 37.12n 22.78e bringsoe 1985 rouzaki, messenia on trees in forest neogene sediments 790 37.236n 21.662e pers. obs. kostas sagonas 2014 agii asomatoi, arkadia under stones in maquis flysch 671 37.332n 22.699e pers. obs. kostas sagonas 2014 tegea, arcadia unspecified alluvium 798 37.45n 22.41e naturhistorisches museum wien didima, corinthia unspecified limestone 527 37.461n 23.171e valakos et al. 2008 tripoli, arcadia unspecified limestone 807 37.507n 22.371e valakos et al. 2008 1 km south west of mainalo, arkadia under stones in maquis limestone 836 37.529n 22.299e pers. obs. kostas sagonas 2014 methanon, malea peninsula, laconia unspecified volcanic rocks 452 37.58n 23.39e naturhistorisches museum wien 183mediodactylus kotschyi in the peloponnese peninsula with > 800 mm precipitation annually (fig. 1), although only seven of these are from areas with > 850 mm precipitation annually. in seven of the eight locations (table 1a) in which we encountered the species in our 2015 fieldwork we found geckos on trees (in three of them exclusively on trees), and in five locations we encountered them under rocks (in one exclusively under rocks). absence from both microhabitats was also recorded (table 1b). thirty-two percent of gecko localities in the peloponnese are located in regions where limestone is the dominant rock type (combined data from tables 1a and 2). we found no connection between rock type and arboreality (fisher’s exact test, two on trees and nine under rocks in limestone habitats, seven on trees and 11 under rocks in other rock types, p = 0.41), or between habitat type (rocks or trees) and precipitation (trees: 649 ± 35 mm, rocks: 692.1 ± 28 mm; t-test assuming unequal variances, t = 2.1, n = 29, p = 0.34). twenty five percent of our absence findings were in localities in which limestone is the dominant rock type (although we did not try to identify the type of the rocks under which we searched for geckos), and all of them were for locations in which we searched for geckos under location name substrate rock type annual prec. (mm) latitude longitude source tiryntha, argolis peninsula unspecified alluvium 583 37.59n 22.80e beutler and gruber 1977; valakos et al. 2008 argos, argolis peninsula unspecified alluvium 603 37.632n 22.732e valakos et al. 2008 palea epidavros, argolis peninsula unspecified alluvium 502 37.635n 23.153e valakos et al. 2008 epidavros, argolis peninsula unspecified limestone 560 37.65n 23.14e beutler and gruber 1977 archea olympia, elis under stones in phrygana alluvium 781 37.651n 21.618e pers. obs. kostas sagonas 2014 inachos, corinthia unspecified alluvium 603 37.659n 22.750e valakos et al. 2008 nea epidavros, argolis peninsula unspecified peridotite and serpentinite 555 37.675n 23.126e valakos et al. 2008 nea epidavros, corinthia under stones in phrygana peridotite and serpentinite 538 37.675n 23.134e pers. obs. kostas sagonas 2014 levidi, arcadia unspecified limestone 873 37.68n 22.29e beutler and gruber 1977; valakos et al. 2008 kamenitsa, laconia unspecified limestone 833 37.72n 22.19e bringsoe 1985; valakos et al. 2008 tropaia, arcadia unspecified limestone 864 37.730n 21.954e valakos et al. 2008 3 km north east of sofiko, corinthia stone terrace limestone 639 37.81n 23.08e bringsoe 1985 9 km east of lampeia, archaia olympia 4 eggs under a flat rock limestone 927 37.85n 21.91e bringsoe 1985 ano tripotama, achaea unspecified limestone 887 37.857n 21.912e valakos et al. 2008 archea korinthos, achaea unspecified neogene and pleistocene sediments 587 37.903n 22.882e valakos et al. 2008 korinthos, achaea unspecified neogene and pleistocene sediments 566 37.936n 22.927e valakos et al. 2008 feneos, corinthia on walls alluvium 835 37.950n 22.325e koppitz 2013 kokkoni, achaea unspecified alluvium 600 37.966n 22.779e valakos et al. 2008 2.5 km east of karia, corinthia on a wall in phrygana alluvium 607 38.009n 22.442e pers. obs. kostas sagonas 2014 4 km south west of kalavrita, achaea unspecified limestone 883 38.010n 22.079e valakos et al. 2008 2 km south of kalavrita, achaea unspecified neogene sediments 865 38.018n 22.102e valakos et al. 2008 mega spilaio, achaea unspecified neogene sediments 848 38.08n 22.17e stepánek 1937 trapeza, achaea on a wall neogene sediments 728 38.172n 22.229e pers. obs. kostas sagonas 2014 184 rachel schwarz et alii rocks (table 1b). precipitation was not significantly greater in localities where geckos were not present (table 1b) than where they were encountered (table 1a; presence: 690 ± 23 mm, absence: 727 ± 32 mm; t-test assuming unequal variances, t = 2.13, n = 33, p = 0.38). discussion our hypotheses were only partially supported. geckos were indeed more common in the more arid areas of the peloponnese. however, we did not find evidence of limestone avoidance. the high frequency of occurrence of m. kotschyi on trees in the peloponnese contrasts with that on the greek islands. during fieldwork in the cyclades (once or twice from 2013 to 2015; e.g., slavenko et al., 2015) we observed m. kotschyi on a tree trunk only twice (on ano koufonisi, in may 2013, and on kimolos island, in may 2015, fig. 2). all our other observations (~ 800, from 40 islands) of this species were on and under rocks, in stone piles, on dry stone walls and on low building walls, under various objects of refuse and in abandoned stone shelters (see also arnold and ovenden, 2002; beutler, 1981; musters and in den bosch, 1982 and citations therein). during our 2015 survey in the peloponnese, most specimens (81%) were found on trees, especially on almond, olive and eucalypts (table 1a). in only three locations were specimens found under rocks (table 1a), despite searching localities with apparently suitable phrygana habitats. because our sole criterion for establishing absence was that we did not find the species following a search under what we considered to be suitable conditions for m. kotschyi, we are well aware that some absences may 1 2 annual precipitation (mm) fig. 1. presence and absence localities derived from the literature and our field observations plotted on an annual mean temperature map of the peloponnese (adopted from worldclim, hijmans et al. 2005). squares (☐) designate specimens found among rocks; triangles (∆) designate specimens found on trees; circles (o) designate specimens for which substrate was not specified; dots inside the symbols (•) represent published, museum and observational data (otherwise: our data); blue: presence; red: absence. 185mediodactylus kotschyi in the peloponnese peninsula very well be false-absences. this can only be supported (or refuted), however, by future surveys. that said, we have no reason to believe that reported absences are more likely for either the tree or for the rock microhabitat, or for different geographic locations, and thus false absences are unlikely to alter our conclusions. werner (1993) described m. kotschyi as being a “paradoxical” species. he contended that, in israel and iraq, it lives mainly on large tree trunks with exfoliating bark, such as carob, eucalypts and oak (werner, 1993), even though it lacks the characteristic adhesive toe pads of other arboreal geckos (gamble et al., 2012). m. kotschyi is nonetheless superbly camouflaged against the background pattern of tree trunks (werner, 1993; baier et al., 2009; bar and haimovitch, 2012; see also fig. 2), making it hard to dismiss the idea that it is well adapted to living on trees as well as on rocks. according to the most comprehensive phylogenies available (pyron and burbrink, 2014), the closest relatives of m. kotschyi are the arboreal mediodactylus sagittifer and the saxicolous mediodactylus heteropholis and mediodactylus heterocercus (m. kotschyi is sister to a clade containing all three). more distantly-related allies (pyron and burbrink, 2014) include members of the mostly saxicolous and terrestrial genera tenuidactylus and cyrtopodion (note that tenuidactylus caspius is described as arboreal, saxicolous and terrestrial, rogner 1997), and the mostly terrestrial bunopus, agamura and crossobamon. the ancestral state of m. kotschyi is thus most likely terrestrial or saxicolous although an arboreal ancestor cannot be ruled out. the fact that m. kotschyi is saxicolous over most of its distribution may imply that this species was originally saxicolous and later adapted to inhabit trees too. current data on the occurrence of this species in the peloponnese (tables 1 and 2) do not suggest a strong preference of m. kotschyi for a specific type of substrate, and we did not detect an aversion to limestone. the thintoed gecko does occur in places where the mean annual precipitation is greater than 850 mm, although it is probably scarce in such regions. it is certainly not obligatorily associated with phrygana, in contrast to what was previously suggested (beutler, 1981). our findings, along with our observations of this species on trees, lead us to suggest that m. kotschyi is highly flexible and adaptable in its habitat preference, which may have contributed to its successful establishment and broad range. our observations indicate that m. kotschyi is relatively abundant on trees in the peloponnese, whereas it is extremely abundant and conspicuous on and under rocks and on stone walls in the cyclades. this might have led to the general misconception that it is purely saxicolous. although it is certainly much more abundant on islands (as many lizards are, novosolov et al., 2013), we suggest that m. kotschyi is more common in the peloponnese than has previously been considered, because it was formerly sought mostly on and under rocks. acknowledgements this research was done under permit number 20305/824 from the ministry of the environment. we wish to thank oliver tallowin, anat feldman and maria novosolov for help with gis construction. we wish to thank three anonymous referees for comments on a previous version of this manuscript. this study is funded by an isf grant #1005/12 to sm. references ajtić, r. 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(1937): uber balearen eidechsenvierzehn neue reptilienrassen von den sudlichen agaischen inseln. zool. anz. 118: 79-90. acta herpetologica vol. 11, n. 2 december 2016 firenze university press predator-prey interactions between a recent invader, the chinese sleeper (perccottus glenii) and the european pond turtle (emys orbicularis): a case study from lithuania vytautas rakauskas1,*, rūta masiulytė1, alma pikūnienė2 effective thermoregulation in a newly established population of podarcis siculus in greece: a possible advantage for a successful invader grigoris kapsalas1, ioanna gavriilidi1, chloe adamopoulou2, johannes foufopoulos3, panayiotis pafilis1,* the unexpectedly dull tadpole of madagascar’s largest frog, mantidactylus guttulatus arne schulze1,*, roger-daniel randrianiaina2,3, bina perl3, frank glaw4, miguel vences3 thermal ecology of podarcis siculus (rafinesque-schmalz, 1810) in menorca (balearic islands, spain) zaida ortega*, abraham mencía, valentín pérez-mellado growth, longevity and age at maturity in the european whip snakes, hierophis viridiflavus and h. carbonarius sara fornasiero1, xavier bonnet2, federica dendi1, marco a.l. zuffi1,* redescription of cyrtodactylus fumosus (müller, 1895) (reptilia: squamata: gekkonidae), with a revised identification key to the bent-toed geckos of sulawesi sven mecke1,*,§, lukas hartmann1,2,§, felix mader3, max kieckbusch1, hinrich kaiser4 the castaway: characteristic islet features affect the ecology of the most isolated european lizard petros lymberakis1, efstratios d. valakos2, kostas sagonas2, panayiotis pafilis3,* sources of calcium for the agamid lizard psammophilus blanfordanus during embryonic development jyoti jee1, birendra kumar mohapatra2, sushil kumar dutta1, gunanidhi sahoo1,3,* mediodactylus kotschyi in the peloponnese peninsula, greece: distribution and habitat rachel schwarz1,*, ioanna-aikaterini gavriilidi2, yuval itescu1, simon jamison1, kostas sagonas3, shai meiri1, panayiotis pafilis2 swimming performance and thermal resistance of juvenile and adult newts acclimated to different temperatures hong-liang lu, qiong wu, jun geng, wei dang* olim palus, where once upon a time the marsh: distribution, demography, ecology and threats of amphibians in the circeo national park (central italy) antonio romano1,*, riccardo novaga2, andrea costa1 on the feeding ecology of pelophylax saharicus (boulenger 1913) from morocco zaida ortega1,*, valentín pérez-mellado1, pilar navarro2, javier lluch2 notes on the reproductive ecology of the rough-footed mud turtle (kinosternon hirtipes) in texas, usa steven g. platt1, dennis j. miller2, thomas r. rainwater3,*, jennifer l. smith4 heavy traffic, low mortality tram tracks as terrestrial habitat of newts mikołaj kaczmarski*, jan m. kaczmarek book review: sutherland, w.j., dicks, l.v., ockendon, n., smith, r.k. (eds). what works in conservation. open book publishers, cambridge, uk. http://dx.doi.org/10.11647/obp.0060 sebastiano salvidio acta herpetologica 14(1): 3-14, 2019 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-25008 uzungwa scarp nature forest reserve: a unique hotspot for reptiles in tanzania john valentine lyakurwa1,2,*, kim monroe howell2, linus kasian munishi1, anna christina treydte1,3 1 department of sustainable agriculture and biodiversity ecosystem management, the nelson mandela african institution of science and technology, p.o. box 447, arusha, tanzania 2 department of zoology and wildlife conservation, university of dar es salaam, p.o. box 35064, dar es salaam, tanzania 3 agroecology in the tropics and subtropics, university of hohenheim, stuttgart, germany * corresponding author. email: johnlyakurwa@udsm.ac.tz submitted on: 2018, 23rd november; revised on: 2019, 10th march; accepted on: 2019, 4th april editor: marco sannolo abstract. while knowledge of african vertebrate biodiversity has increased dramatically in recent years, the field of herpetology which encompasses many threatened and endemic species, has lagged behind, and many areas have not been adequately explored. intensive field work was conducted during the rainy season from december 2017 to april 2018 to assess reptile occurrence mostly in previously unexplored areas of the uzungwa scarp nature forest reserve (usnfr) which is part of the udzungwa mountain ranges in the eastern arc mountains (eam), and adjacent agricultural areas. bucket pitfall traps, funnel traps, night transects and opportunistic search methods were used to sample reptiles across four zones: in lowland, submontane and montane forests of the usnfr, and in neighboring farmlands. forty-five reptile species across 14 families were recorded, mostly concentrated on the lowland and submontane forests. the number of endemic and threatened species in the usnfr reaches 20 and 14 respectively, and most are found in the submontane forest. nineteen species were new records for the usnfr, five of them representing range extensions. reptile species richness, abundance and diversity differed significantly across the four zones, except between montane and farmland zones and between lowland and submontane. however, farmland zone was discordant from other zones in terms of species composition. this study adds to the importance of the eam not only in harbouring large numbers of species but also as an important hotspot for endemic and threatened reptiles. it also calls for proper land-use practices in farms adjacent to protected areas for sustainable conservation of biodiversity. keywords. eastern arc mountains, farmland, elevation, iucn threatened species. introduction in spite of the alarming trends on the loss of species (lawton and may, 1995; baillie et al., 2004; pimm et al., 2014), little has been done to assess patterns of biodiversity and threats facing reptiles in africa (meng et al., 2016). a recent assessment by meng et al. (2016) shows that 321 reptile species occur in tanzania, of which about 13% and 28% are threatened with extinction and are endemic, respectively. most of these highly fragile reptile populations are found in the eastern arc mountains (eam) (meng et al., 2016; spawls et al., 2018). the eastern arc mountains have faced a number of threats, with forest fires, agricultural encroachment, firewood collection, logging and climate change being the most important ones (burgess et al., 2002; newmark, 2002; ehardt et al., 2005; menegon and salvidio, 2005; meng et al., 2016). the mountains have lost over 70% of forest cover to agriculture within the last six decades (newmark, 1998; newmark, 2002; hall et al., 2009) and 4 john valentine lyakurwa et alii currently support a large number of people (ndangalasi et al., 2007; platts et al., 2011). the same impacts have been reported from the uzungwa scarp nature forest reserve (usnfr) (zilihona et al., 1998; menegon and salvidio, 2005; rovero et al., 2012), which encompasses the southern portion of the eam. this reserve was recently upgraded from “forest reserve” to “nature reserve” category (urt, 2017), calling out for a higher protection status due to its unique biodiversity. despite this upgrade, information regarding usnfr’s biodiversity is extremely scant. since some reptile species possess very narrow distributional ranges and depend on highly-specific habitat requirements (spawls et al., 2004; meng et al., 2016), they become more vulnerable to the ongoing anthropogenic activities than wide-ranging species. menegon and salvidio (2005) showed that elevation determined distribution patterns of reptiles in the usnfr and reported most endemic species to be restricted to higher elevations. the same high elevation areas have faced severe agricultural expansion in the usnfr (zilihona et al., 1998; ehardt et al., 2005) and little is known on how reptiles utilize the transformed areas. as some of the farms are found on high elevations, the latter generally hosting more endemic species compared to lower elevations, these reptile species might extend to the farms close to the forest edge on the plateau side. while there are several reports on reptiles of the usnfr (e.g., menegon and salvidio, 2005; lyakurwa, 2017), most of these surveys were limited to the southern part of the reserve and to our knowledge, no study has ever investigated how reptiles utilize the agricultural areas bordering the usnfr. since the previous reports examined both amphibians and reptiles simultaneously (except lyakurwa, 2017), the surveys were limited to methods which could capture both species groups. a project on uzungwa scarp hyper-endemic amphibians has revealed a number of new records in the same area, especially with respect to the distribution extension of the hyper-endemic species and of new species of nectophryoides (tonelli et al., 2017), that were mostly found in the previously unexplored areas, and has emphasized the need for detailed surveys for reptiles. this study focused on assessing reptile occurrence in the least explored areas of the usnfr and adjacent agricultural lands. our results on how endemic and threatened reptiles utilize the usnfr and the nearby areas dominated by human activities can be used for local and long-term conservation planning in this and other protected areas of tanzania. materials and methods study site this study was carried out in the uzungwa scarp nature forest reserve (usnfr) and adjacent areas. the usnfr covers the southeastern part of the udzungwa mountains and lies between 7°39’-7°51’s, and 35°51’-36°02’ e (ndangalasi, 2005). with an altitudinal range of 300 m.a.s.l to 2,068 m a.s.l it covers a total area of 207 km2 (shangali et al., 1998; ndangalasi et al., 2007; urt, 2017). it borders the chita river to the south, the kidete river to the north and the ruaha, iwolo and lukosi rivers to the west (ndangalasi, 2005). average rainfall in the usnfr is unimodal (from november to may) and ranges from 1,800 mm to 3,000 mm per year (shangali et al., 1998; ndangalasi, 2005). the average temperature varies seasonally and is estimated to range from 15 to 20 °c on the highlands and 19 to 27 °c in the lowlands (ndangalasi, 2005). the nature reserve is comprised of lowland (< 800 m a.s.l), submontane (7001,400 m a.s.l) and montane forests (> 1,400 m a.s.l), with areas of seasonally inundated grasslands and grassland with bushes (shangali et al., 1998; zilihona et al., 1988). data collection data were collected during day and night for five consecutive months in the wet season, from mid-december 2017 to the end of april 2018. selection of sampling sites was primarily based on elevation, vegetation types (shangali et al., 1998; zilihona et al., 1998) and land use type. other factors known to influence reptile abundance and distribution were also considered at each site. these factors included the amount of leaf litter, availability of rotten logs, distance from water bodies and from rock crevices, following howell (2002) and mcdiarmid et al. (2012). the study area was divided into four zones; three inside the usnfr, i.e., lowland forest, submontane forest and montane forest following shangali et al. (1998) and zilihona et al. (1998) with some slight modifications. the fourth zone was set in farmlands bordering the usnfr. these farms were located on the plateau side of the reserve (with elevation range similar to that of a montane zone) and were of interest to this study to verify if the observed pattern of endemism in the reserve would extend beyond the protected area. each zone consisted of three sites (12 sites in total), each with a radius of 1 km, and placed at least 2 km apart. data collection took place for ten days at each site (alternated between zones to reflect the timing and commonality of the season between sites throughout the data collection period), making a total of 120 days (90 and 30 days in and outside the usnfr, respectively). several methods (bucket pitfall traps with drift fences, funnel traps, night transects and opportunistic searches) were used following howell (2002) and mcdiarmid et al. (2012) in order to maximize captures. one bucket pitfall trap line (howell, 2002) consisted of a 55 m long drift fence, eleven 20-l buckets, set at an interval of 5 m and 10 double-ended funnel traps placed alternately between each bucket. two bucket pitfall trap lines were established at each site, summing up to a total of six trap lines (66 buckets, and 60 5reptiles of the uzungwa scarp and adjacent areas funnels traps) per zone. trapping was done for eight consecutive nights, in which trap monitoring was done immediately following sunrise and late afternoon, following stanley et al. (1998) and howell et al. (2012). a total of 176 bucket pitfall trap nights and 160 funnel trap nights were carried at each site leading to 2112 and 1920 bucket pitfall trap nights and funnel trap nights, respectively, for the entire study. in addition, a total of four 50 m night transects were set at each site (total of 48 transects for the entire study), encompassing a range of micro-habitats (sensu menegon et al., 2008). each transect was located, marked in advance and searched thoroughly following lyakurwa (2017). since pitfall traps and night transects alone cannot adequately sample all species of reptiles, these methods were supplemented by opportunistic searching, during which reptiles were searched for in their possible hiding/ basking places. all reptiles encountered casually or in locations apart from the 12 sampling sites but within the study area were also recorded as opportunistic encounters. species identification followed spawls et al. (2018) while threat status followed meng et al. (2016). grouping of endemic/near endemic species based on their dependency to the forest followed burgess et al. (2007). kruskal wallis test (kruskal and wallis, 1952) with dunn’s multiple comparisons was used to compare the overall reptile species abundance in the four zones while diversity was compared using hutcheson’s t-test (hutcheson, 1970). shannon wiener index was used for species diversity. species composition among the four zones and between the surveyed sites was compared using the bray-curtis similarity index (legendre, 1998; greenacre and primicerio, 2013). data were analyzed using r software version 3.5.0 and paleontological statistics software (past) version 2.17 (hammer et al., 2001). statistical significance was considered when p was less than 0.05. voucher materials were deposited at the department of zoology and wildlife conservation of the university of dar es salaam (appendix 1). results a total of 358 individual reptiles were recorded, representing 45 species in 14 families (appendix 1). thirtythree species were found in the usnfr alone, two in farmland alone, and 10 in both (appendix 1). seven species (kinyongia sp, trioceros deremensis, broadleysaurus major, crotaphopeltis tornieri, dendroaspis angusticeps, gonionotophis nyassae and lycophidion uzungwense) were single observations while three were double observations (urocotyledon wolterstorffi, trioceros tempeli and afrotyphlops nigrocandidus). most individuals were found on trees (40.3%), understorey (25.5%), underground (9.1%), dead logs (6.6%), rocks (3.3%) and in farmlands, some individuals were found on house walls (0.8%). among reptiles which were found above the ground (n = 177), 52.0% were found at 50-100 cm height, 32.2% between 100-300 cm height, and 15.8 % above 300 cm from the ground. nineteen species were new records for the usnfr, five of them representing range extensions (appendix 1) from previously known distributional ranges. this raised the number of species in the usnfr and surrounding areas to 60 species across 16 families (table 1 and appendix 1). we documented that the usnfr harbours about 21% (20 species) of reptiles that are endemic/near endemic to tanzania (appendix 1). about 69% of reptiles endemic/near endemic to eam are now confirmed to occur in the usnfr (appendix 1). a large number of these endemics were chameleons (7 species), a number which is equivalent to 29% of all tanzanian endemic chameleons. the number of species considered as globally threatened/ near threatened with extinction (near threatened, vulnerable, endangered or critically endangered) reached 14 in the usnfr (appendix 1), equivalent to 33% of all reported threatened/ near threatened reptile species in tanzania. most of these species were found in the submontane forest (appendix 1; fig. 1 and 2). except for afrotyphlops nigrocandidus, all strictly forest dependent endemic/ near endemic species were found only in the protected areas of the usnfr (appendix 1). similarly, other endemic reptiles were found in areas inside the usnfr with the exception of trioceros tempeli and t.werneri which were found both inside and outside the reserve and lycophidion uzungwense which was only found outside the reserve (appendix 1). outside the usnfr, a. nigrocandidus was found in a farm plot while t. tempeli, t.werneri and l. uzungwense were found in natural forest fragments, in fruit trees (the former two) table 1. total number of reptiles species, number of iucn threatened species, per families found in and around usnfr (sources: menegon and salvidio 2005; lyakurwa 2017, this study). nt = near threatened, vu = vulnerable, en = endangered family total endemic nt vu en agamidae 1 0 0 0 0 atractaspidae 2 0 0 0 0 chamaeleonidae 9 7 2 0 1 colubridae 12 2 1 0 1 elapidae 2 0 0 0 0 gekkonidae 8 2 0 2 0 gerrhosauridae 1 0 0 0 0 lamprophiidae 3 1 0 0 0 natricidae 1 0 0 0 0 psammophiidae 2 0 0 0 0 pseudoxyrhophiidae 2 0 0 1 0 pythonidae 1 0 0 0 0 scincidae 9 2 0 1 1 typhlopidae 1 1 0 1 0 varanidae 1 0 0 0 0 viperidae 5 1 0 3 0 6 john valentine lyakurwa et alii fig. 1. distribution of endemic, vulnerable, near threatened and endangered reptile species in the uzungwa scarp nature forest reserve and adjacent areas as assessed from december 2017 to april 2018. low 1,2,3 = lowland sites, farm1,2,3 = farmland sites, sub1,2,3 = submontane sites, mon 1,2,3= montane sites. 7reptiles of the uzungwa scarp and adjacent areas and commercial forests dominated by cupressus sp. and pinus sp. maize and bean fields were poor in reptile species, with only philothamnus hoplogaster, lygodactylus grotei and trachylepis varia being the common residents. all reptiles observed by lyakurwa (2017) were also recorded during this study, except for buhoma procterae, natriciteres variegata, python natalensis and xyelodontophis uluguruensis (appendix 1). nine species recorded by menegon and salvidio (2005) in the same area were not found during this study (appendix 1). lycophidion uzungwense, previously found inside the usnfr by menegon and salvido (2005), was only found in a natural forest patch outside the usnfr. these patches, together with commercial forests and fruit trees in agricultural lands, also proved to be important for chameleons (especially trioceros tempeli and t. werneri) (fig. 3a). kinyonfig. 2. mean number of endemic, near threaterned (nt), vulnerable (vu) and endangered (en) reptile species in the four surveyed zones of the uzungwa scarp forest nature reserve and adjacent agricultural areas. fig. 3. some of the reptile species recorded in the usnfr from december 2017 to april 2018. a male trioceros werneri on a commercial plant outside the usfnr (a), kinyongia sp (b), aparallactus sp (c), cnemaspis sp (d), male (a) and female (b) urocotyledon wolterstorffi. the above cnemaspis, aparallactus and kinyongia could not be identified with certain to species level using spawls et al. (2018). 8 john valentine lyakurwa et alii gia sp (fig. 3b) is believed to be a new species similar to kinyongia fischeri based on morphological grounds. similarly, aparallactus sp (fig. 3c) needs further studies as the currently available identification key by spawls et al. (2018) was not sufficient to identify it to species level. the genera lygodactylus, cnemaspis (fig. 3d) and urocotyledon (fig. 3e and 3f) encompass individuals with highly varying morphology and our findings likely represent more than one cryptic species in these genera. lowland and submontane forests contained a similar number of species, which decreased more than half towards montane forest and farmlands (appendix 1). overall reptile abundance differed significantly across the zones (h = 18.187, p = 0.0004). further analysis using dunn’s multiple comparison showed no significant difference between farmland and montane forest and between lowland and submontane forests (table 2). all other pairs were significantly different in overall reptile abundance (table 2). however, bray-curtis similarity index showed farmland to be the most discordant zone (fig. 4), with lowland and submontane zones being more similar in species composition. lowland, submontane and montane zones contained more forest dependent species than farmland zone (appendix 1). sites in the farmland zone were very similar in species composition than when compared with sites in the protected area (appendix 2). also, sites close to each other were more similar in species composition than distant sites, whereby some distant sites showed complete dissimilarity (appendix 2). species table 2. dunn’s multiple comparison of overall reptile abundance in the four sampled zones of the usnfr and surrounding areas comparison p value farmland vs lowland 0.001 farmland vs montane 0.467 farmland vs submontane 0.004 lowland vs montane 0.001 lowland vs submontane 0.281 montane vs submontane 0.003 fig. 4. similarity cluster among the four zones of the uzungwa scarp nature forest reserve and adjacent areas based on bray-curtis similarity index (single average link) as per the current study. table 3. hutchesons’ t test summary of species diversity for the four surveyed zones of the usnfr and the surrounding areas. comparison t value df p value farmland vs lowland 8.854 181 <0.001 farmland vs montane 1.678 124 0.096 farmland vs submontane 8.148 179 <0.001 lowland vs montane 5.971 98 <0.001 lowland vs submontane 0.912 196 0.363 montane vs submontane 5.324 97 <0.001 table 4. species richness, diversity and chao richness estimator (±se) for the four sampled zones   lowland submontane montane farmland species observed 26 24 9 11 chao estimator 32.17±5.13 43.97±17.26 9.98±2.22 17.19±7.48 shannon diversity 2.23 2.16 1.17 0.79 fig. 5. rarefaction curves for species recorded in the four sampled zones of the usnfr and surrounding areas from december 2017 to april 2018. farm= farmland (circle), low=lowland (triangle), mon=montane (square), sub=submontane (plus sign). shaded region surrounding each line represent 95 % confidence levels 9reptiles of the uzungwa scarp and adjacent areas diversity in lowland and submontane was significantly higher than farmland and montane (table 3). however, species rarefaction curves for the zones did not reach an asymptote (fig. 5). the mean (±se) number of species for chao estimator was higher than the observed species in all zones (table 4). discussion with our study, we were able to almost double the number of reptile species for the usnfr, from 33 species (menegon and salvidio, 2005) and 38 species (lyakurwa, 2017) to 60 species. such a result pinpoints the udzungwa mountains as biologically the richest mountain block in the eam in terms of herpetofauna, harboring the highest number of endemic and near endemic reptile species (34), followed by east usambara (32), uluguru (29) and nguru (19) (burgess et al., 2007). previously, in terms of herpetofauna, this mountain range was ranked after usambara and uluguru mountains (howell, 1993; burgess et al., 2007). three species out of the nine classified as globally threatened, endemic to tanzania and climate change-vulnerable by meng et al. (2016), are now confirmed to occur in the usnfr. this result highlights the importance of protecting these mountains and calls out for more long-term surveys in other parts of the udzungwa and the eam. although faunal surveys are recognized as one of the most critical steps in assessing forest biodiversity (stanley et al., 1998), little attention has been given to african herpetology (spawls et al., 2004; largen and spawls, 2010; meng et al., 2016; tolley et al., 2016). there are many areas in east africa which are yet to be explored in a herpetological context (spawls et al., 2004) and this study shows the need for detailed surveys even in previously visited areas, supporting howell (1993) and spawls et al. (2004), who showed the possibility of getting new records in most areas of east africa, due to lack of intensive surveys in most parts of the region. the overall shortage of information adds more risk to the conservation of african biodiversity (tolley et al., 2016), particularly herpetofauna and may lead to misallocated conservation priorities (pimm et al., 2014), especially in a biodiversity hotspot country like tanzania. contrary to previous studies, we found that most endemic, near endemic and iucn threatened species were concentrated in the submontane forest of the usnfr. menegon and salvidio (2005) and menegon et al. (2008) reported that the number of endemic and near endemic reptile species increases with altitude. similarly, burgess et al. (2002) reported more endemic vertebrates in montane forests of the eam and fewer in lowland, submontane and upper montane forests. a large number of endemic and threatened species in the submontane forest areas might be due to the intermediate environmental conditions in the mid-elevation zones, which accommodate both high and low elevation specialists (mccain, 2010). however, the same zone has suffered from severe forest loss in recent years (burgess et al., 2002) and it is not clear how this has been affecting reptiles. we hope that the recent upgrading of the protection status of the reserve will reduce the destruction activities that have been going on in submontane forests. farmland zone had fewer forest dependent species compared to other zones which agree with burgess et al. (2007) who reported most eam endemic species as specialists of dense forests. our findings also highlight how the type of farming (e.g commercial tree plantation and some natural vegetation around/in the farm plots) might influence reptile assemblage and shows the potential of the farms surrounding the usnfr in buffering the montane forests. some strictly forest-dependent species were found at the forest edge and can act as important indicators of ecosystem health following more studies. therefore, land-use planning is highly important, particularly in the farmlands as the endemic species were found mainly in natural forest patches, fruit trees and commercial tree plantations near the usnfr, of which the species might decline in the future without proper land management. while we have gathered data on reptiles from many more sites and over a prolonged period in the wet season compared to any other study in the udzungwa mountains, there is still a need for subsequent surveys in the area, both in the dry and wet seasons. this is especially important, as the current species accumulation curves have not yet reached an asymptote showing the possibilities of getting new records. some reptile species are highly secretive, have low population densities and/or are locally distributed (spawls et al., 2004; meng et al., 2016), making it possible to miss them when sampling only in one season. since we found only a few reptiles (especially chameleons in the genus trioceros) more than 10 m high, we recommend more efforts on sampling canopy dwellers (e.g., use of arboreal traps in future studies). similarly, sampling all zones simultaneously might provide more meaningful data, which, was not possible in our study due to logistical constraints. three to five years of consecutive trapping (mcdiarmid et al., 2012) across various seasons (stanley, 1998; howell, 2002) has been recommended in order to increase the probability of recording rare species. subsequent surveys will also enable documenting species that this study failed, adding to the con10 john valentine lyakurwa et alii servation value of not only the usnfr and udzungwa mountains but the entire eam region. this article has shown the importance of re-assessing the herpetofauna of eam, and adds to the importance of conserving these mountains. acknowledgement we would like to thank wwct and wwf (prince bernhard scheme) for the financial supports, tfs, and the usnfr management (especially yusuph tango and renatus msabo) for issuing research permits (tfs/shz/usnr/281/402/01/10/ tfs/shz/ usnr/281/402/01/10/54). we are grateful to our field assistants abdala chiponda, magnus kahise, ngwabi moto, michael mpwage, dan kinyele, ronald nganyori and various porters for their help. additional advice was given by elena tonelli, simon loader and andy bowkett on the study design. we also acknowledge village government officers and villagers from all villages surrounding the usnfr for their generosity. in addition, we thank stephen spawls and michele menegon for sharing some of their unpublished materials and support in identification of some species. references baillie, j.e.m., hilton-taylor, c., stuart, s.n. 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(1998): human activities threatening the biodiversity of uzungwa scarp forest reserve-tanzania. afr. nat. hist. 87: 319-326. 12 john valentine lyakurwa et alii appendices appendix 1. reptile species, threat category, and endemism per families recorded in uzungwa scarp nature forest reserve and surrounding areas. note; * = species that were recorded by menegon and salvidio 2005, ᵠ = recorded by menegon and salvidio but not surely in usfnr (either from general bibliography or from surrounding villages); ‡ = collected by lyakurwa 2017 and not found by this study, y = endemic, f = forest visitor, f= mainly forest, ff= strictly confined to forest. species voucher lowland submontane montane farmland threat category forest dependency endemic/ near endemic agamidae agama mossambica peters, 1854 x lc chamaeleonidae kinyongia sp jvl 1709 x nt ff y kinyongia oxyrhina (klaver & böhme, 1988) x x nt ff y rhampholeon moyeri menegon, salvidio & tilbury, 2002 x x lc ff y rieppeleon brevicaudatus (matschie, 1892) x x lc trioceros deremensis (matschie, 1892) jvl 1718 x lc ff y trioceros laterispinis (loveridge, 1932) ᵠ en f y trioceros tempeli (tornier, 1899) x x lc f y trioceros werneri (tornier, 1899) x x lc f y gekkonidae cnemaspis cf dickersonae (schmidt, 1919) jvl 1735, jvl 1733, jvl 1733 x lc cnemaspis uzungwae perret, 1986 jvl 1712 x x vu ff y hemidactylus mabouia (moreau de jonnès, 1818) jvl 1724 x lc hemidactylus platycephalus peters, 1854 jvl 1725 x lc hemidactylus sp jvl 1723 x lygodactylus capensis (smith, 1849) x x lc lygodactylus cf angularis günther, 1893 jvl 1701, x x x lc lygodactylus grotei sternfeld, 1911 x x lc urocotyledon wolterstorffi (tornier, 1900) jvl 1737, jvl 1722 x x vu ff y gerrhosauridae broadleysaurus major (duméril, 1851) jvl 1727 x lc opportunistic in lowland farms scincidae leptosiaphos kilimensis (stejneger, 1891) jvl 1707, jvl 1706 x lc melanoseps loveridgei brygoo & roux-estève, 1982 * lc melanoseps uzungwensis loveridge, 1942 jvl 1710, jvl 1711, jvl 1731, jvl 1732, jvl 1731, jvl 1732 x x en ff y mochlus afer (peters, 1854) jvl 1715, jvl 1716 x lc mochlus sp jvl 1719 x scelotes uluguruensis barbour & loveridge, 1928 * vu ff y trachylepis maculilabris (gray, 1845) jvl 1719 x x lc trachylepis striata (peters, 1844) x lc trachylepis varia (peters, 1867) x x x lc varanidae varanus niloticus (linnaeus, 1766) x lc atractaspidae aparallactus sp jvl 1729, jvl 1721 x x atractaspis aterrima günther, 1863 jvl 1708, jvl 1720 x x lc 13reptiles of the uzungwa scarp and adjacent areas species voucher lowland submontane montane farmland threat category forest dependency endemic/ near endemic colubridae boaedon fuliginosus (boie, 1827) x x x lc crotaphopeltis tornieri (werner, 1908) x lc ff y dasypeltis medici bianconi, 1859 * lc dipsadoboa werneri (boulenger, 1897) * nt ff y philothamnus hoplogaster (günther, 1863) jvl 1703 x x x x lc philothamnus macrops (boulenger, 1895) x x lc f y philothamnus punctatus peters, 1867 x lc philothamnus semivariegatus (smith, 1840) ᵠ lc telescopus semiannulatus smith, 1849 x lc thelotornis kirtlandii (hallowell, 1844) * lc thelotornis mossambicanus (bocage, 1895) x x lc xyelodontophis uluguruensis broadley & wallach, 2002 ‡ en ff y elapidae dendroaspis angusticeps (smith, 1849) x lc naja cf melanoleuca hallowell, 1857 x x lc lamprophiidae gonionotophis nyassae (günther, 1888) jvl 1724 x lc lycodonomorphus whytii (boulenger, 1897) jvl 1713 x x lc lycophidion uzungwense loveridge, 1932 x lc f y natricidae natriciteres variegata (peters, 1861) ϯ lc psammophiidae psammophis tanganicus loveridge, 1940 x lc psammophylax variabilis günther, 1893 jvl 1704, jvl 1705 x x lc pseudoxyrhophiidae buhoma procterae (loveridge, 1922) ϯ vu ff y duberria lutrix (linnaeus, 1758) x lc pythonidae python natalensis smith, 1840 ‡ lc typhlopidae afrotyphlops nigrocandidus (broadley & wallach, 2000) jvl 1702 x x vu ff y viperidae atheris barbouri loveridge, 1930 ᵠ vu f y atheris ceratophora werner, 1896 x x vu f y bitis arietans merrem, 1820 ᵠ lc bitis gabonica duméril, bibron & duméril, 1854 ᵠ vu causus defilippii (jan, 1863) ᵠ lc 14 john valentine lyakurwa et alii appendix 2. bray-curtis species similarity index summary for the 12 sites surveyed in the uzungwa scarp nature forest reserve and adjacent areas from december 2017 to april 2018. note; 0 represents no similarity (100% dissimilarity) while 1 represents 100% similarity. low=lowland, sub = submontane, mon= montane, farm= farmland. numbers in bold indicate more strongly related sites (>50%) while those italicized indicate 100% dissimilarity. farm 1 farm 2 farm 3 mon 1 mon 2 mon 3 sub 1 sub 2 sub 3 low 1 low 2 farm 2 0.5634 farm 3 0.8400 0.6575 mon 1 0.0625 0.0364 0.0588 mon 2 0.1951 0.2813 0.1861 0.3200 mon 3 0.0377 0.0264 0.0364 0.2703 0.3044 sub 1 0.0364 0.0513 0.0351 0.2051 0.2083 0.4333 sub 2 0.0615 0.0000 0.0000 0.0408 0.0000 0.0286 0.2222 sub 3 0.1200 0.0882 0.0769 0.2353 0.2791 0.2182 0.5263 0.2887 low 1 0.0377 0.1316 0.0364 0.0541 0.0435 0.0345 0.0333 0.1714 0.1091 low 2 0.0526 0.0000 0.0000 0.0000 0.0000 0.0000 0.2169 0.5591 0.2308 0.2716 low 3 0.1000 0.0317 0.0952 0.0833 0.0606 0.0444 0.1277 0.1053 0.0952 0.2667 0.1765 acta herpetologica vol. 14, n. 1 june 2019 firenze university press uzungwa scarp nature forest reserve: a unique hotspot for reptiles in tanzania john valentine lyakurwa1,2,*, kim monroe howell2, linus kasian munishi1, anna christina treydte1,3 experience of predacious cues and accessibility to refuge minimize mortality of hylarana temporalis tadpoles santosh mogali*, bhagyashri shanbhag, srinivas saidapur tonal calls as a bioacoustic novelty in two atlantic forest species of physalaemus (anura: leptodactylidae) thiago r. de carvalho1,*, célio f.b. haddad1, marcos gridi-papp2 scientific publication of georeferenced molecular data as an adequate guide to delimit the range of korean hynobius salamanders through citizen science amaël borzée1,*, hae jun baek2,3, chang hoon lee2,3, dong yoon kim2, jae-young song4, jaehwa suh5, yikweon jang1, mi-sook min2* mirrored images but not silicone models trigger aggressive responses in male common wall lizards stefano scali1,*, roberto sacchi2, mattia falaschi1,3, alan j. coladonato2, sara pozzi2, marco a.l. zuffi4, marco mangiacotti1,2 using an in-situ infra-red camera system for sea turtle hatchling emergence monitoring fatima n. oğul1,#,*, franziska huber2,#, sinem cih1, kumsal düzgün3, ahmet e. kideyş1, korhan özkan1 descriptive osteology of an imperiled amphibian, the luristan newt (neurergus kaiseri, amphibia: salamandridae) hadi khoshnamvand1, mansoureh malekian1,*, yazdan keivany1, mazaher zamani-faradonbe1, mohsen amiri2 does color polymorphism affect the predation risk on phalotris lemniscatus (duméril, bibron and duméril, 1854) (serpentes, dipsadidae)? fernanda r. de avila1,2,*, juliano m. oliveira3, mateus de oliveira1, marcio borges-martins4, victor hugo valiati2, alexandro m. tozetti1 age structure of a population of discoglossus scovazzi camerano, 1878 (anura discoglossidae) in extreme environmental conditions (high atlas, morocco) mohamed amine samlali, abderrahim s’khifa, tahar slimani* acta herpetologica 14(1): 65-68, 2019 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-24806 age structure of a population of discoglossus scovazzi camerano, 1878 (anura discoglossidae) in extreme environmental conditions (high atlas, morocco) mohamed amine samlali, abderrahim s’khifa, tahar slimani* cadi ayyad university, faculty of sciences semlalia, biodiversity and ecosystems dynamic laboratory, po box 2390, marrakech 40 000, morocco. *corresponding author. email: slimani@uca.ac.ma submitted on: 2019, 25th february; revised on: 2019, 12thapril; accepted on: 2019, 29th april editor: fabio m. guarino abstract. the age structure and growth of the moroccan painted frog (discoglossus scovazzi camerano, 1878) in a population living in the high atlas at the upper altitudinal limit of the species’ range was estimated for the first time by skeletochronology. individual age was determined by counting the lines of arrested growth (lags) from crosssections of the phalanges. both males and females reached sexual maturity at 3-4 years of age at which point the speed of osteogenesis and body growth slow down. males and females have maximum lifespans of six and five years, and average sizes of 47. 50 mm (n = 21, sd = 1.40) and 39.70 mm (n = 53, sd = 0.90) respectively. we detected a positive relationship between age and size, suggesting that the oldest individuals are always bigger and heavier. sizes corresponding to the same age class are very heterogeneous reflecting divergent conditions and growth strategies. keywords. endemic species, skeletochronology, life-history traits, extreme environment, morocco. degradation and fragmentation of natural habitats due to human activities have harmful effects on amphibian populations (collins and crump, 2009; hamer and mcdonnell, 2008, 2010; collins and fahrig, 2017). the central plateau of the oukaimeden massif in the high atlas region of morocco has been heavily impacted by overgrazing and the banks of its two main rivers are seriously degraded by the bulk removal of sand for construction (ait babahmad, 2012; ait zidan, 2018). both human activities have degraded water habitats and altered the richness and abundance of their amphibian communities. morphological abnormalities have been recorded in adults and tadpoles (ait babahmad, 2012; lansari, 2018), especially in the frog pelophylax saharicus, which is widespread in the massif. this habitat degradation could cause a demographic collapse leading to local disappearances (mckinney, 2002; löfvenhaft et al., 2004; bionda et al., 2013; babini et al., 2015; green and bailey, 2015; zhelev et al., 2017), especially in species that are relatively sensitive to human activities due to habitat specialization or specific life history (rubbo and kiesecker, 2005; hamer and mcdonnell, 2008, 2010). the moroccan painted frog (discoglossus scovazzi camerano 1878, family discoglossidae) is listed as least concern in the iucn red list of threatened species (salvador et al., 2009; başkale et al., 2018). endemic to morocco, the species is generally common, especially in sub-humid and humid areas. they are quite cryptic outside the breeding season, with adults preferring the proximity of small bodies of water such as temporary ponds, low-flow streams and sources. although the species may tolerate a slight modification of its habitat, its sensitivity to the particular conditions and the quality of its aquatic and terrestrial environment remains largely unknown (reques et al., 2013). because the demographic changes are in response to environmental conditions (sinsch et al., 2004; 66 mohamed amine samlali, abderrahim s’khifa, tahar slimani sinsch et al., 2007; spear et al., 2009), the purpose of this study is to estimate the age structure, age at first reproduction and longevity of a high mountain population of discoglossus scovazzi, by using skeletochronology. discoglossus scovazzi is an anuran of small body size known from a large number of aquatic biotopes throughout morocco from sea level up to the atlas mountains, excluding the saharan areas. it reaches its upper altitudinal limit in the high atlas at oukaimeden, over 2600 m a.s.l. (bons and geniez, 1996). this study is based on mark-recapture tracking of 74 adult individuals (21 males and 53 females) between 2016 and 2018. the study took place at the population’s breeding site, which consists of small pools of water fed by a permanent watercourse. solitary and discreet during most of the year, discoglossus scovazzi begins reproduction in early spring (ait babahmad, 2012; beukema et al., 2013; samlali, 2016). egg-laying is spread over 8 to 10 days from early april to mid-june. during this period, the water temperature is about 13 °c and the outside temperature is about 10 °c. the population is located at 2660 m of altitude in the oukaimeden massif (31°11’n, 07°50’w) about 75 km south-east of marrakech. the climate is typically mediterranean with cold winters and an average annual temperature of 9.5 °c. the average maximum temperature of the hottest month (july) is 22.2 °c and the average minimum temperature of the coldest month (january) is -3.3 °c. the average annual precipitation is circa 518 mm and snow fall mainly between november and march. the dry period lasts about four months (june-september). the vegetation consists of spiny xerophytes and a herbaceous layer, essentially graminaceous, relatively rich during rainy periods but heavily overgrazed. the population breeds in pits (diameter 6-7 m and at least one meter deep) that resulted from sand extraction and used as waterers in the bed of a permanent river (assif-n-irène). intensive use for livestock watering negatively impacts water quality and accelerates drying (ait babahmad, 2012; samlali, 2016). sampling by hand occurred from february to june. we measured the frogs from snout to cloaca with calipers and weighed them using a field balance (accuracy: 0.1 g). we determined sex by the presence of nuptial callosities and calling in males. after measurements, we collected the third toe of the left hind leg and we immediately released the frog at the place of its capture. the samples are kept in absolute alcohol. we sectioned phalanges from the toe to count lines of arrested growth (lags) following the method by castanet and smirina (1990), which is based on the detection of lags generated by the cold season (each lag is interpreted as one year of age). in order to avoid the errors of the estimation of the age induced by the medullary resorption (francillon-vieillot, 1987; fretey and le garff, 1992; tsiora and kyriakopoulou-sklavounou, 2002; guarino et al., 2003; 2008; liao and lu, 2010; liao, 2011; huang et al., 2013; bionda et al., 2015; jin et al., 2017), we selected, for each individual, diaphysis sections in which the size of the medullar cavity was at its minimum and that of the periosteal bone at its maximum (oromi et al., 2016). on the other hand, bone remodeling and the close rapprochement of the outer most lines, especially in the oldest individuals (wagner et al., 2011) could also compromise the age estimation. in these cases, at the insertion site of the phalangeal ligament allows to discern the peripheral lags and to reliably count them (bionda et al., 2018). the number of lines of arrested growth (lags) in each section is counted by three authors (m.a.s., a.s. and t.s.). since all the individuals studied were collected during the breeding season, the age at sexual maturity was revealed by the presence of lags suddenly becoming very closely adjacent, reflecting the slowing of growth after sexual maturity. all variables were first tested for normality (kolmogorov-smirnov test) and the difference between sexes was tested using anova. in addition, the relationship between size and age was analyzed by spearman correlation test. we used the significance level of alpha = 0.05 in all tests. a total of 74 reproductive adults (21 males and 53 female) were studied. individuals were collected at breeding sites during the reproduction periods in 2016, 2017 and 2018. the cross-sections show the presence of strongly stained growth lines in periosteal bone in most individuals (fig. 1). since the frogs were collected in march and april, we considered the perimeter of the bone as corresponding to an indicator lag of last winter. we clearly detected a decrease in the distance that fig. 1. transverse sections at diaphysal region of phalanx bones of discoglossus scovazzi. (a) male with 6 visible lags. (b) female with 4 visible lags (cm: medullary cavity. 67age structure of discoglossus scovazzi separating the lags suggesting that sexual maturity is reached at 3-4 years in both males and females (fig.1 a, b). at the breeding site, we sampled some females younger than three years, although three-year individuals were the most frequently observed (fig. 2). highest estimated age is six years in males and five years in females. body size varied from 34.0 to 64.0 mm in males (mean ± sd = 47.5 ± 1.4 mm, n = 21) and from 33.0 to 56.1 mm in females (39.7 ± 0.9 mm, n = 53), with a mass of 14.5 ± 5.17 g in males and 8.05 ± 3.45 g in females. the observed pattern of sexual dimorphism in body size was significant (f1, 72 = 21.4; p < 0.0001). because we did detect a significant relationship between age and body size in males (r = 0.786, p = 0.018, n = 21) and in females (r = 0.847, p = 0.003, n = 53) (fig. 3), the oldest individuals were always the largest ones. indeed, anurans often exhibit continuous growth, particularly females because of the strong relationship between fecundity and body size (lengagne et al., 2007). in most anuran species, males are smaller than females (shine, 1979). for d. scovazzi, the sexual size dimorphism is inversed (garcía-paris et al., 2004; oromi et al., 2016). the variation in size dimorphism results from differences in the age structure between sexes (monnet and cherry, 2002; liao et al., 2013a, b). in fact, in species where females exhibit smaller sizes (rhinella achalensis, rana cascadae and hylarana nigrovittata), males mature at later ages (monnet and cherry, 2002), suggesting that delayed maturity in males produces size dimorphism as stated in the rensch’s rule (liao et al., 2015). however, as in d. pictus (oromi et al., 2016), we did not find differences in the age structure between the sexes in d. scovazzi which suggests that these two species failed to support rensch’s rule. in some anuran species, increased male body size is an important determinant of male mating success (wells, 2007; liao and lu, 2011). furthermore, large males are more likely to breed than smaller ones if competition for females is intense (oromi et al., 2016). however, little information is available on the mating tactics of d. scovazzi and other factors might explain the sexual size dimorphism in this species. our study provides original data on the structure of a population at the upper altitudinal limit of this endemic species of morocco. the earliest age at which sexual maturity was attained in most individuals of both sexes is about 3 years. however, the three-year-old frogs, theoretically fewer numerous than those aged two as a result of annual mortality, are more frequent at breeding sites than younger females. as observed in brongersma’s toad in arid environments (fattah et al., 2014), this asymmetrical age structure in females of discoglossus scovazzi in the high atlas suggests that this difference in young females is due to the fact that not all the females reach sexual maturity before three years, or that some females reach sexual maturity at the age of two years. compared to its mediterranean congener, d. pictus, studied by oromi et al. (2016) in north africa, the late age at maturity in our population suggests a low growth rate in juveniles, strongly constrained by the harsh climatic conditions of this alpine environment (low temperature, snowfall, strong wind), or the modification of aquatic environments (due to excessive sand removal) and the deterioration of their physico-chemical quality (due to livestock). the low values in terms of longevity (5-6 years) and reproductive potential (about two years) recorded in this population are related to climatic conditions and ongoing changes in habitat quality. several other hypotheses could, however, be formulated, including moving larger adults /older to other sites, the low survival of larger individuals /older, or the case of an unstable population maintained by smaller / younger adults. this highlights a need monitoring. fig. 2. age distribution of the reproductive population of discoglossus scovazzi at oukaimeden in 2016-2018. fig. 3. relationship between estimated age (x-axis, in year) and body size (y-axis, snout-vent length in mm) in the studied population. 68 mohamed amine samlali, abderrahim s’khifa, tahar slimani acknowledgements this research was financed by funds from hassan ii academy of science and technics (icgvsa project, pi slimani t.). it was authorized by the high commissariat for water and forest (decision number 05/2013 hceflcd/dlcdpn/dprn/cff) morocco. we would like to thank the two anonymous reviewers who provided important comments to improve the manuscript. references ait babahmad, a. 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fernanda r. de avila1,2,*, juliano m. oliveira3, mateus de oliveira1, marcio borges-martins4, victor hugo valiati2, alexandro m. tozetti1 age structure of a population of discoglossus scovazzi camerano, 1878 (anura discoglossidae) in extreme environmental conditions (high atlas, morocco) mohamed amine samlali, abderrahim s’khifa, tahar slimani* acta herpetologica 11(2): 221-225, 2016 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-17842 notes on the reproductive ecology of the rough-footed mud turtle (kinosternon hirtipes) in texas, usa steven g. platt1, dennis j. miller2, thomas r. rainwater3,*, jennifer l. smith4 1 department of biology, box c-64, sul ross state university, alpine, tx 79832, usa 2 box 792, alpine, texas 79831, usa. present address: wildlife conservation society, myanmar program, office block c-1, aye yeik mon 1st street, hlaing township, yangon, myanmar 3 tom yawkey wildlife center & belle w. baruch institute of coastal ecology and forest science, clemson university, georgetown, south carolina 29440, usa. * corresponding author. e-mail: trrainwater@gmail.com 4 department of biology, new mexico state university-alamogordo, alamogordo, new mexico 88310, usa submitted on 2016, 20th january; revised on 2016, 25th june; accepted on 2016, 27th june editor: paolo casale abstract. kinosternon hirtipes is among the least-studied north american turtles and little is known concerning reproduction. in the united states, k. hirtipes occurs at < 10 sites within a single creek drainage in presidio county, texas where it is considered a threatened species. we investigated the reproductive ecology of one of these populations at plata wetland complex in 2007. we captured nine female k. hirtipes in wire mesh traps and hoop nets baited with fish. eggs were obtained by injecting females with oxytocin. we recovered 19 eggs from six females captured in may and june. the smallest female that produced eggs was about 7.1 years old. mean (± 1sd) clutch size, egg length, egg width, egg mass, and clutch mass were 3.1 ± 1.4 eggs, 28.7 ± 1.4 mm, 17.5 ± 0.9, 5.3 ± 0.6 g, and 17.5 ± 0.8 g, respectively. relative egg mass and relative clutch mass were 0.035 and 0.011, respectively. there was a significant, positive linear relationship between female carapace length (cl) and egg width. the relationship between cl and relative egg mass was negative, and approached statistical significance. relationships between cl and clutch size, egg length, and egg mass were not significant. reproductive attributes of k. hirtipes in texas are similar to those reported from a population in mexico. keywords. clutch size, egg size, kinosternon hirtipes, reproduction, threatened species, texas. the rough-footed mud turtle (kinosternon hirtipes) is a highly variable species comprising six recognized subspecies distributed from west texas, usa, southwards into chihuahua, mexico, and south and east on the mexican plateau to the chapala, zapotlán, san juanico, pátzcuaro, and valle de méxico basins (iverson, 1981; legler and vogt, 2013). kinosternon hirtipes murrayi is the largest subspecies (straight-line carapace length [cl] to 196 mm; smith et al., 2015) and has the most extensive distribution, occurring from west texas and chihuahua, south into northern jalisco, northern michoacan, and eastern estado de mexico (iverson, 1981; legler and vogt, 2013). the northernmost populations of k. hirtipes (sensu lato) occur in presidio county, texas where < 10 small, isolates are known from the alamito creek drainage (ernst and lovich, 2009; platt and medlock, 2015). kinosternon hirtipes is considered a threatened species by texas parks and wildlife department (2013) owing to a limited geographic distribution within the state and on-going habitat degradation. kinosternon hirtipes ranks among the least-studied north american turtles and many aspects of its natural history remain poorly known (lovich and ennen, 2013). in particular, there is a notable paucity of information on 222 steven g. platt et alii reproductive ecology. iverson et al. (1991) investigated reproduction among a population in chihuahua, mexico, and laboratory studies indicate that hatchling sex is determined by incubation temperature (ewert et al., 2004); otherwise the reproductive ecology of k. hirtipes has gone unreported. importantly, published reports of reproduction among the remnant k. hirtipes populations in texas are lacking, although such natural history data are critical for designing effective conservation measures (dayton, 2003). we here report on the reproductive ecology of one of the few known k. hirtipes populations in texas. our study was conducted at plata wetland complex (pwc; elevation = 1125 m), located on a private ranch in the alamito creek drainage approximately 56 km se of marfa in presidio county, texas. pwc consists of four livestock tanks (ponds): railroad (612 m2), crotalus (900 m2), turner one (2520 m2), and turner two (3780 m2). railroad tank is fed by an artesian spring and linked to crotalus tank by a shallow drainage ditch about 244 m long. turner one is located approximately 190 m from railroad tank and water is supplied by rainfall and a wind-driven pump. turner two is approximately 245 m from turner tank one and reliant on rainfall for water. because railroad and crotalus tanks are spring fed, water levels remain relatively stable throughout the year with a maximum depth of about 1.5 m. water depth in turner tanks one and two varies depending on seasonal rainfall (less so at turner one); maximum depth in each is about 1.2 m. the environmental characteristics of the study site are described in greater detail elsewhere (wilde and platt, 2011; platt et al., 2016). regional climate is characterized by mild winters (rarely < 0 °c) and hot summers (> 40 °c) with highly variable annual rainfall (mean ca. 370 mm) (powell, 1998). we trapped turtles at pwc from april through midjuly, and september 2007 as part of a larger population study of k. hirtipes conducted in the alamito creek drainage (2007-2010). we captured turtles using a combination of wire mesh funnel traps (1.0 m long × 50 cm diameter; mesh = 12.5 mm) and hoop nets (2.5 m long × 1.0 m diameter; mesh = 25 mm). traps and hoop nets were baited with sardines (packed in oil or water) or fresh carcasses of locally captured sunfish (lepomis). wire mesh traps were set from mid-morning to early evening (ca. 1000-2030 hr) and checked at intervals of 1-2 hours. hoop nets were deployed in mid-morning (ca. 1030) on one day and checked the following morning. each captured turtle was permanently marked by shell notching (cagle, 1939) and then measured (cl and plastron length [pl]) with dial (± 0.1 mm) or tree calipers (± 1.0 mm) depending on body size. body mass (bm) was determined with spring scales (± 1.0 g). turtles with a cl ≥ 100 mm were sexed using external secondary sexual characteristics (iverson 1985b). we were unable to reliably determine the sex of turtles below this size threshold. male and juvenile turtles were processed in the field and released, while females were returned to the lab and held in plastic wading pools for 21-24 days to insure that oviducal eggs were fully shelled. we then induced oviposition by injecting oxytocin into the pectoral muscles at a dosage of 2.0 units/100 g of body mass (ewert and legler, 1978). following the injection, each female was placed in a 38 liter tub half-filled with water and fitted with a wire-mesh grate positioned 5 cm above the bottom; this allowed eggs to fall through and prevented accidental trampling by the female (platt et al., 2008; legler and vogt, 2013). a second injection was administered within 24 hours to insure deposition of the complete clutch. most eggs were removed from the tub 10-20 minutes after deposition, although a few remained underwater for somewhat longer; all eggs were recovered < 1 hour after being deposited. eggs were then weighed on an ohaus triple beam balance (± 0.1 g), and measured (length and width) with dial calipers (± 0.1 mm). following iverson et al. (1991) we calculated relative clutch mass (rcm) [clutch mass / (gravid female body mass — clutch mass) × 100] and relative egg mass (rem) [rcm/clutch size]. female turtles were released at their respective capture sites 24-48 hours after oviposition. we used linear regression to explore relationships between female body size and clutch and egg attributes. statistical analyses were performed by program jmp (version 3.2, sas institute, cary, north carolina, usa). general statistical references are from zar (1996). mean values are presented as ± 1sd, and results were considered significant at p ≤ 0.05. we captured 87 turtles (25 males, nine females, and 53 juveniles) at pwc. we treated the females (mean cl = 142 ± 15 mm; range = 121-163 mm) captured in may-june with oxytocin, and recovered 19 eggs from six females (cl = 132-159 mm) in late june (table 1). based on von bertalanffy growth models (fabens, 1965) developed for this population (smith, 2016), the smallest female in our sample that produced eggs was estimated to be 7.1 years old. there was a significant positive linear relationship between egg width (ew) and cl (r = 0.91; f1, 5 = 20.72; p = 0.0104; fig. 1). the relationship between cl and relative egg mass (rem) was negative (rem = -0.0002cl + 0.04) and approached statistical significance (r = 0.76; f1, 5 = 5.52; p = 0.0785). the relationships between cl and clutch size (r = 0.03; p = 0.94), egg length (r = 0.02; p = 0.96), and egg mass (r = 0.68; p = 0.13) were not significant. our data provide the only information on the reproductive ecology of k. hirtipes outside of mexico (iverson 223kinosternon hirtipes reproduction et al., 1991). the clutch size (mean and range) we report for k. hirtipes in texas is almost identical to the clutch size estimated from corpora lutea counts in chihuahua, mexico (3.0 ± 0.8 eggs; range = 1-5 eggs; n = 34; iverson et al. 1991). however, it should be noted that egg retention can occur after oxytocin injections (congdon and gibbons, 1985) and without taking radiographs of females (gibbons and greene, 1979), we cannot be certain that females deposited a complete clutch. therefore, the clutch size we report is best considered a conservative estimate. that said, tucker (2007) found that clutch size in a group of female red-eared sliders (trachemys scripta elegans) treated with oxytocin was statistically equivalent to clutch size in natural nests. although iverson et al. (1991) found a positive correlation between clutch size and cl in k. hirtipes, no such relationship was evident in our study, possibly owing to the small number of females we sampled. because k. hirtipes deposits multiple clutches during a single reproductive season (iverson et al., 1991), cl may be a more suitable predictor of total annual fecundity rather than individual clutch size. the values we report for egg mass (em), egg length (el), and egg width (ew) are similar to those from a much larger sample of k. hirtipes in mexico (em = 4.8 ± 1.0 g; range = 3.7-6.5 g; n = 11; el = 28.8 ± 1.8 mm; range = 24.1-33.2 mm; n = 74; ew = 16.3 ± 0.8 mm; range = 14.6-18.5; n = 74; iverson et al., 1991). because females in our study deposited eggs directly into the water and these remained submerged for varying (but usually brief ) periods, it is possible some eggs absorbed water and increased in mass (e.g., wilgenbusch and gantz, 2000). however, any increase in mass was probably minimal as most eggs were retrieved < 20 minutes after deposition. clutch mass (cm) and relative clutch mass (rcm) were also similar to values reported by iverson et al. (1991) among k. hirtipes in mexico (cm = 14.4 ± 4.3 g; range = 6.7-29.4 g; n = 28; rcm = 0.071 ± 0.014; range = 0.048-0.105; n = 28). the negative relationship between rem and body size reported by iverson et al. (1991) was likewise evident in our data, although of marginal statistical significance. this relationship suggests a decreased investment in each egg with increasing maternal body size (iverson et al., 1991). similar to our results, iverson et al. (1991) found a significant positive relationship between female cl and mean egg width. this relationship is not unexpected if egg width is determined by the pelvic aperture diameter which in turn scales to body size (congdon and gibbons, 1987). however, evidence for pelvic aperture constraints on egg size in kinosternids is conflicting (macip-rios et al., 2013). some studies suggest egg width is determined by pelvic morphology (wilkinson and gibbons, 2005; macip-rios et al., 2012, 2013), while others found no evidence for such constraints (iverson, 1991; macipríos et al., 2009; lovich et al., 2012; macip-rios et al., 2013). larger female k. hirtipes are producing larger eggs and according to iverson et al. (1991), this increase is achieved by increases in egg width rather than length, which is probably constrained by oviduct length. the two smallest mature females with corpora lutea found by iverson et al. (1991) in mexico had cls of 99.4 and 97.4 mm, a body size very similar to the smallest females exhibiting secondary sexual characteristics in our study population. however, the smallest female from which we recovered eggs was considerably larger (cl = 132 mm). despite this difference between the two populations, growth models suggest sexual maturity is attained at approximately the same age in texas (7-8 years) and mexico (6-8 years; iverson et al., 1991). obviously given our small sample size of reproductive females, further study is necessary to fully address this question. table 1. attributes of clutches obtained from six female kinosternon hirtipes collected in presidio county, texas, usa. *see text for discussion of potential methodological biases. attribute n mean ± 1 sd range clutch size* 6 3.1 ± 1.4 1–5 egg length (mm) 19 28.7 ± 1.4 24.7–30.8 egg width (mm) 19 17.5 ± 0.9 15.0–18.7 egg mass (g)* 19 5.3 ± 0.6 3.6–6.4 mean clutch mass (g)* 6 17.5 ± 8.8 5.3–29.1 relative clutch mass* 6 0.035 ± 0.019 0.009–0.061 relative egg mass* 6 0.011 ± 0.002 0.008–0.015 carapace length (mm) 130 140 150 160 e gg w id th (m m ) 16.0 16.5 17.0 17.5 18.0 18.5 r2 = 0.83 p = 0.0104 fig. 1. relationship between carapace length and egg width in a sample (n = 6) of female kinosternon hirtipes murrayi from presidio county, texas, usa. 224 steven g. platt et alii acknowledgements this research was conducted under a scientific research permit (spr-0307-844) issued to sgp by the texas department of parks and wildlife, with the approval of the sul ross state university (srsu) institutional animal care and use committee. support for this project was provided by research enhancement grants from srsu to sgp. we thank lewis medlock for field assistance and hardrock mining company for allowing us to conduct research on their property. references cagle, f.r. 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(1996): biostatistical analysis. prentice hall, saddle river. acta herpetologica vol. 11, n. 2 december 2016 firenze university press predator-prey interactions between a recent invader, the chinese sleeper (perccottus glenii) and the european pond turtle (emys orbicularis): a case study from lithuania vytautas rakauskas1,*, rūta masiulytė1, alma pikūnienė2 effective thermoregulation in a newly established population of podarcis siculus in greece: a possible advantage for a successful invader grigoris kapsalas1, ioanna gavriilidi1, chloe adamopoulou2, johannes foufopoulos3, panayiotis pafilis1,* the unexpectedly dull tadpole of madagascar’s largest frog, mantidactylus guttulatus arne schulze1,*, roger-daniel randrianiaina2,3, bina perl3, frank glaw4, miguel vences3 thermal ecology of podarcis siculus (rafinesque-schmalz, 1810) in menorca (balearic islands, spain) zaida ortega*, abraham mencía, valentín pérez-mellado growth, longevity and age at maturity in the european whip snakes, hierophis viridiflavus and h. carbonarius sara fornasiero1, xavier bonnet2, federica dendi1, marco a.l. zuffi1,* redescription of cyrtodactylus fumosus (müller, 1895) (reptilia: squamata: gekkonidae), with a revised identification key to the bent-toed geckos of sulawesi sven mecke1,*,§, lukas hartmann1,2,§, felix mader3, max kieckbusch1, hinrich kaiser4 the castaway: characteristic islet features affect the ecology of the most isolated european lizard petros lymberakis1, efstratios d. valakos2, kostas sagonas2, panayiotis pafilis3,* sources of calcium for the agamid lizard psammophilus blanfordanus during embryonic development jyoti jee1, birendra kumar mohapatra2, sushil kumar dutta1, gunanidhi sahoo1,3,* mediodactylus kotschyi in the peloponnese peninsula, greece: distribution and habitat rachel schwarz1,*, ioanna-aikaterini gavriilidi2, yuval itescu1, simon jamison1, kostas sagonas3, shai meiri1, panayiotis pafilis2 swimming performance and thermal resistance of juvenile and adult newts acclimated to different temperatures hong-liang lu, qiong wu, jun geng, wei dang* olim palus, where once upon a time the marsh: distribution, demography, ecology and threats of amphibians in the circeo national park (central italy) antonio romano1,*, riccardo novaga2, andrea costa1 on the feeding ecology of pelophylax saharicus (boulenger 1913) from morocco zaida ortega1,*, valentín pérez-mellado1, pilar navarro2, javier lluch2 notes on the reproductive ecology of the rough-footed mud turtle (kinosternon hirtipes) in texas, usa steven g. platt1, dennis j. miller2, thomas r. rainwater3,*, jennifer l. smith4 heavy traffic, low mortality tram tracks as terrestrial habitat of newts mikołaj kaczmarski*, jan m. kaczmarek book review: sutherland, w.j., dicks, l.v., ockendon, n., smith, r.k. (eds). what works in conservation. open book publishers, cambridge, uk. http://dx.doi.org/10.11647/obp.0060 sebastiano salvidio acta herpetologica 15(2): 143-148, 2020 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/a_h-7875 notes on sexual dimorphism, diet and reproduction of the false coral snake oxyrhopus rhombifer duméril, bibron & duméril, 1854 (dipsadidae: pseudoboini) from coastal plains of subtropical brazil fernando m. quintela*, felipe caseiro, daniel loebmann laboratório de vertebrados, instituto de ciências biológicas, universidade federal do rio grande, avenida itália s/n, vila carreiros, rio grande, rio grande do sul, brazil fundação aury luiz bodanese, rua joão martins, 219d, são cristovão, chapecó, santa catarina, brazil *corresponding author. e-mail: fmquintela@yahoo.com.br submitted on: 2020, 22nd january; revised on: 2020, 8th july; accepted on: 2020, 9th july editor: stefano scali abstract. herein, we provide information on diet, sexual dimorphism and reproductive biology of the false coral snake oxyrhopus rhombifer from the southernmost brazilian coast, pampa biome, a region under influence of subtropical climatic domains. the analysis of 142 specimens revealed a marked sexual dimorphism, with significant differences for all the characters analyzed. the diet of the species is composed by small rodents and squamate reptiles, the latter consisting mainly of lizard tails, probably autotomized. males reached sexual maturity at smaller sizes (svl) than females (354 mm vs 451 mm, respectively). secondary follicles were found mainly in spring months and one single female presented 3 oviductal eggs in january (summer). our results add to the knowledge on biology of snakes in the pampa morphoclimatic domain. keywords. feeding habits, reproductive biology, pampa biome, squamata, xenodontinae. investigating the natural history of species is essential for understanding ecological processes at different levels and provides crucial information for species conservation (greene, 1993; gaiarsa et al., 2013). regarding neotropical snakes, the last decades have experienced an increment in the number of studies concerning diverse traits of natural history and sexual dimorphism, but these are still fragmented and incipient, considering the group’s high diversity (barbo et al., 2011; gaiarsa et al., 2013). the oxyrhopus genus comprises 14 species of medium-sized pseudoboines distributed from southeastern mexico to central argentina (lynch, 2009; uetz et al., 2017). most oxyrhopus species exhibit a coral snake mimetic pattern (savage and slowinski, 1992), responsible for the popular name ‘false coral snakes’. oxyrhopus rhombifer (fig. 1) is the southernmost distributed oxyrhopus, ranging from southeastern brazil to central argentina (province of buenos aires) (giraudo, 2001). there are few scattered data available o. rhombifer diet and reproductive biology (vidal, 2002; maschio et al., 2003, 2004; sawaya et al., 2003, 2008; gaiarsa et al., 2013) and no data on its sexual dimorphism. herein, we examined size at sexual maturity, female reproductive cycle, feeding habits and sexual dimorphism of o. rhombifer from southernmost brazilian coastal environments. specimens were collected along a stretch of ca. 470 km of the coastal plain of the rio grande do sul state (30°15’44”s, 50°28’03”w; 33°40’27”s, 53°30’52”w), southern brazil, between august 2008 and october 2017. sampling method consisted in active search performed by two researchers in 77 field trips with an average duration from two to 11 hours (average of four hours). 144 fernando m. quintela, felipe caseiro, daniel loebmann searches were conducted on foot and by car at low speed (20-40 km/h) during day and night periods. this region is inserted in the pampa biome, under “subtemperate humid” and “temperate humid” climatic domains according to the regional classification by maluf (2000). seasons are well-defined, with monthly average air temperature ranging from 13 °c in the winter (july) to 22 °c in the summer (january). average rainfall is 1,271 mm and rain is distributed mainly from june to october (climatedata, 2019). predominant vegetation types are shrub grassland, and other phytophysiognomies with less coverage include the coastal peat and sandy forests (restinga forests) and psamophyte formations of coastal dunes. specimens were euthanized through intraperitoneal injection of pentobarbital sodium 50 mg/ml, in accordance with guidelines from international protocols. this procedure had been authorized in our laboratory since 2006 and it is in accordance with the institutional committee for the use of animals in research (ceua-furg). collection was permitted by the brazilian environmental agency (icmbio process nº 43658-1). the snakes had to be euthanized for the analyses of the reproductive condition and digestive tract content and for the availability of specimens and tissues for the projects “taxonomic revision and systematics of oxyrhopus rhombifer (serpentes: dipsadidae)” and “male reproductive cycle of oxyrhopus rhombifer (serpentes: dipsadidae) in southern brazil”. oxyrhopus rhombifer is classified as least concern by iucn (arzamendia et al., 2019). all specimens are deposited in the herpetological collection of universidade federal do rio grande (chfurg) (appendix). sex was determined through verification of the presence or absence of hemipenes via subcaudal incision. the following measurements were taken from each specimen, with a digital caliper to the nearest 0.01 mm: snout-vent length (svl), tail length (tl), head length (hl), head width (hw), inter-ocular length (iol), internostril length (inl). we examined the existence of sexual dimorphism in the following characters: 1) svl; 2) tail proportion in relation to body length (ratio tl/svl); 3) number of ventral scales; 4) number of subcaudal scales; 5) head length in proportion to body length (ratio hl/ svl); 6) head width in proportion to head length (ratio hw/hl); 7) inter-ocular length in relation to head length (ratio iol/hl); 8) inter-nostril length in relation to head length (ratio inl/hl). all database was checked for normality through shapiro-wilk tests, which detected normal distributions only for svl, tl/svl, and number of subcaudal scales. for these characters, the existence of significance differences between males and females was examined with a student’s t test. for the other characters (hl/svl, hw/hl, iol/hl, inl/hl and number of ventral scales) we applied a mann-whitney test aiming to verify significant differences between the sexes. only mature specimens were examined for sexual dimorphism in svl, tl/svl, hl/svl, hw/hl, iol/hl and inl/ hl. mature and immature specimens were pooled for the analysis on sexual dimorphism in the number of ventral and subcaudal scales. statistical tests were performed in software past v.3.25 (significance p < 0.05). (hammer et al., 2001). a ventral incision was made from the esophageal region to around 5 mm above the cloaca and the digestive and reproductive tracts were externalized for analysis. all stomach and gut contents were removed and identified to the lowest possible taxonomic category. whenever possible, the direction of prey intake (head first or tail first) was annotated. numeric abundance (n%) of all consumed taxa was determined as the ratio between the absolute number of prey from one taxon and the sum of prey of all identified taxa (corrêa et al., 2016). the following reproductive data were recorded from females: total number of ovarian follicles; number of follicles in secondary vitellogenesis (secondary follicles are defined as enlarged follicles with yolk accumulation; diameter ≥ 9 mm, based on the scatterplot of the largest follicles of all females and on coloration [almeidasantos et al., 2014]); diameter of the largest secondary follicle; total number of eggs; occurrence of celomatic fat storage; and largest diameter of the largest egg. females were considered mature when showing at least one of the following characteristics: 1) secondary follicles; 2) oviductal eggs; and 3) folded oviducts, indicating recent oviposition. males were considered mature when presented coiled and opaque ductus deferens, indicating the presence of sperm. the female reproductive cycle was evaluated by: 1) analysis of the distribution of females fig. 1. oxyrhopus rhombifer (unvouchered) from rio grande, rio grande do sul state, southern brazil. 145notes on sexual dimorphism, diet and reproduction of the false coral snake carrying secondary follicles and oviductal eggs along the months of the year; 2) analysis of the annual profile resultant from the plot of the largest follicles and eggs of each female (mesquita et al., 2013; almeida-santos et al., 2014). fecundity was determined by the number of eggs in the oviduct (real fecundity) and the number of secondary follicles (potential fecundity) in the ovarium (mesquita et al., 2013). the correlation between female svl and potential fecundity was examined with a simple linear regression analysis (mesquita et al., 2013). a total of 141 o. rhombifer specimens (89 males, 52 mature and 37 immature; 53 females, 22 mature, 31 immature) were analyzed. females presented higher mean values of svl and number of ventral scales. males presented higher mean values for tl/svl, hl/svl, hw/ hl, iol/hl, inl/hl, and number of subcaudal scales (table 1). summary statistics for all the variables analyzed for sexual dimorphism are presented in table 1. significant differences between sexes were found for all the variables tested (table 1). twenty-four specimens contained 24 prey item each. prey was comprised of rodents (n% = 50.0) and squamata reptiles (n% = 50.0) (table 1). eight out of nine prey in which the direction of intake could be determined were head-first consumed (table 2). eleven out of 12 rodents identified were consumed by mature individuals. lizards were consumed by both mature and immature individuals. the single snake identified was consumed by an immature individual. half of the squamate prey consisted of tail fragments of scincomorpha and anguidae lizards. the smallest female exhibiting secondary follicles presented 451 mm of svl, while the smallest male exhibiting coiled/opaque ducti deferentes presented 354 mm of svl. secondary follicles were found mainly from table 1. summary statics of the variables analyzed for sexual dimorphism in oxyrhopus rhombifer from southern brazilian coast (rio grande do sul state) and results of student’t and mann-whitney tests performed on the variables. analyses on svl, tl/svl, hl/svl, hw/hl, iod/hl and ind/hl were performed in mature specimens. analyses on number of ventral and subcaudal scales were performed on both mature and immature specimens. see main text for abbreviations; n = number of specimens. variable males females test value p value svl 445 ± 59 (354-597) n = 52 616 ± 92 (451-758) n = 22 9.58 1.701e-14 tl/svl 0.276 ± 0.023 (0.226-0.358) n = 52 0.189 ± 0.012 (0.161-0.215) n = 22 16.35 5.902e-26 hl/svl 0.034 ± 0.003 (0.026-0.041) n = 49 0.032 ± 0.003 (0.027-0.039) n = 22 2.94 0.0032 hw/hl 0.557 ± 0.065 (0.478-0.779) n = 49 0.520 ± 0.061 (0.429-0.675) n = 22 2.38 0.017 iod/hl 0.0346 ± 0.036 (0.296-0.455) n = 49 0.0317 ± 0.006 (0.246-0.368) n = 22 2.56 0.010 ind/hl 0.202 ± 0.028 (0.153-0.289) n=49 0.184 ± 0.022 (0.138-0.226) n = 22 2.40 0.016 nº ventral scales 180 ± 6 (162-197) n = 78 192 ± 9 (160-212) n = 51 7.74 9.76e-15 nº subcaudal scales 65 ± 3 (58-71) n = 81 57 ± 3 (50-63) n = 50 16.06 2.19e-22 table 2. absolute number (n) and numeric abundance (n%) of prey items found in digestive tracts of oxyrhopus rhombifer from the southern brazilian coast (rio grande do sul state), frequency and percentage (between parentheses) of prey intake directions of identified taxa, namely: head first (hf ), tail first (tf ) and not determined (nd). prey n n% hf tf nd sauropsida: squamata ophiodes sp. (anguidae) 2 8.3 2 (100) aspronema dorsivittatum (scincidae) 1 4.2 1 (100) cercosaura schreibersii (gymnophthalmidae) 7 29.2 3 (42.9) 1 (14.2) 3 (42.9) lizard not identified 1 4.2 1 (100) snake not identified 1 4.2 1 (100) mammalia: rodentia deltamys kempi (cricetidae: akodontini) 1 4.2 1 (100) oligoryzomys flavescens (cricetidae: oryzomyini) 3 12.5 3 (100) small rodent not identified 8 33.3     8 (100) 146 fernando m. quintela, felipe caseiro, daniel loebmann september to november (spring). a single female presented secondary follicles in january (early summer) and another one showed secondary follicles in may (middle autumn) (fig. 2). potential fecundity varied from two to 13 (x = 7.9 ± 3.4 sd; n = 11). one single female, euthanized in january, presented eggs in the oviduct (three eggs). this same female also presented secondary and primary follicles in the ovaries, indicating the possible occurrence of more than one clutch during the cycle. this female did not present expanded oviducts, which indicates that there was no previous egg laying, and the three oviductal eggs may correspond to a single oviposition event. a greater fat storage was observed in mature females during initial and intermediate secondary vitellogenesis while low celomatic fat deposition was found in females in advanced secondary vitellogenesis. we did not find any significant, albeit positive, correlation (r = 0.07; p = 0.84) between female svl and potential fecundity. an aggregation of three similar-sized males and one larger female, intertwined, but not involved in copulation, was observed in october (early spring). our results indicated that sexual dimorphism is very well marked in o. rhombifer from southern brazilian coastal plain, considering that all the variables herein tested were significantly different between sexes. the dimorphism in body size (svl) is noteworthy, with females significantly larger and showing higher number of ventral scales. males, however, presented tail proportionally longer, higher number of subcaudal scales, head proportionally longer and wider and greater interocular and inter-nostril measurements. the larger body in females and the tail proportionally longer in males is a well-known aspect in dipsadid snakes (aguiar and di-bernardo, 2005; balestrin and di-bernardo, 2005; mesquita et al., 2013; quintela et al., 2017; quintela and loebmann, 2019a, b), including the congener oxyrhopus trigeminus (alencar et al., 2012). mature females of oxyrhopus guibei also showed svl significantly larger than mature males (pizzatto and marques, 2002). the longer body size in females in species without male-male combat is associated with fecundity so that a larger body can accommodate a larger offspring. thus, females experience the fertility selection, in which larger bodies take advantage (shine, 1994). in relation to head dimensions, studies have demonstrated that sexual dimorphism in head traits of snakes are related to differences in the maximum size of preys consumed by different sexes (shine, 1989; pearson et al., 2002; shetty and shine, 2002; vincent et al., 2004). a broader analysis of the feeding habits of the species, based on a larger sample of preyed items, could reveal if in fact males feed on larger prey. another hypothesis to be considered is the use of the head by males for the immobilization of females during mating. oxyrhopus rhombifer feeds on small mammals and squamate reptiles in the studied area, which is in agreement with data from other regions where the species occurs (vidal, 2002; maschio et al., 2003, 2004; sawaya et al., 2008; gaiarsa et al., 2013) and from other oxyrhopus species (alencar et al., 2012; gaiarsa et al., 2013). however, the high occurrence of solely lizard tail fragments in the analyzed digestive tracts (25% of all prey items, 50% of squamata items; tab. 1), possibly autotomized to avoid predation, is remarkable. caudal autotomy is a very common defensive mechanism in lizards (clause and capaldi, 2006), considering that solely tail fragments have been found in tracts of other xenodontine species such as philodryas aestiva and p. patagoniensis (quintela, pers. comm.). females attained sexual maturity at much larger sizes than males, in agreement with o. guibei (pizzatto and marques, 2002), o. trigeminus (alencar et al., 2012) and other xenodontine genera (pizzato et al., 2008; mesquita et al., 2013; rebelato et al., 2016; quintela et al., 2017, quintela and loebmann, 2019a, b). the small number of mature females in our sample, however, did not allow further analysis or conclusions about the reproductive cycle and fecundity. the occurrence of females showing secondary follicles in spring may indicate a seasonal reproductive cycle, a pattern already recorded for other xenodontines in the studied region (oliveira et al., 2011; rebelato et al., 2016; quintela et al., 2017; quintela and loebmann, 2019a, b). a marked greater accumulation of celomatic fat in females during initial and intermediate secondary vitellogenesis when compared to females in advanced secondary vitellogenesis also indicates a capital-breeding strategy (bonnet et al., 1998), where the consumption of lipidic energy sources occurs along fig. 2. monthly variation in diameter (mm) of the largest follicles (black circles) and egg (white circles) of oxyrhopus rhombifer from the southern brazilian coast (rio grande do sul state). the horizontal line indicates size from which follicles were considered as being in secondary vitellogenesis (secondary follicles). 147notes on sexual dimorphism, diet and reproduction of the false coral snake the reproductive cycle (almeida-santos et al., 2014). the only data on real fecundity (three oviductal eggs) showed a clutch size smaller than those observed in o. rhombifer from other regions (four to 17; pontes and di-bernardo, 1988; yanosky et al., 1996; gallardo and scrocchi, 2006; gaiarsa et al., 2013), but within the clutch size variation found for o. guibei (three to 20; pizzato and marques, 2002) and o. trigeminus (one to 11; alencar et al., 2012). sampling and examination on a greater number of females and histological analyses of male gonads, already in progress, will soon allow a more profound understanding of o. rhombifer reproductive biology in the southern brazilian coast. acknowledgments we thank: capes (coordenação de aperfeiçoamento de pessoal de nível superior) for the postdoctoral fellowship granted to the first author; samara arsego, omar m. entiauspe-neto, victor h. s. teixeira, ruth a. regnet, melise l. silveira, rafael a. porciuncula and franck l. silveira for help in fieldwork and laboratory procedures; rebecca alves and omar m. entiauspe-neto for language review; and icmbio/sisbio environmental agency for the permission to transportation, collection and euthanasia (proc. 43658). all procedures adopted are in accordance with guidelines from institutional committee on ethics in the use of animals for research (ceua/furg). references aguiar, l.f.s., di-bernardo, m. 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(2002): sexual divergence in diets and morphology in fijian sea snakes, laticauda colubrina (laticaudidae). aust. ecol. 27: 77-84. shine, r. (1989): ecological causes for the evolution of sexual dimorphism: a review of the evidence. q. rev. biol. 64: 419-461. shine, r. (1994): sexual dimorphism in snakes revisited. copeia 1994: 326-346. uetz, p., hosek, j., hallermann, j. (2017): the reptile database, http://www.reptile-database.org, accessed october 17, 2017. vidal, s.c. (2002): alimentación de los ofidios de uruguay. associación herpetológica española, barcelona. vincent, s.e., herrel, a., irschick, d.j. (2004): sexual dimorphism in head shape and diet in the cottonmouth snake (agkistrodon piscivorus). j. zool. 264: 53-59. yanosky, a., dixon, j., mercolli, c. (1996): ecology of a snake community of el bagual reserve, argentina. herpetol. nat. hist. 4: 97-110. appendix specimens examined and deposited in the herpetological collection of universidade federal do rio grande (chfurg). oxyrhopus rhombifer: brasil: rio grande do sul: palmares do sul (chfurg 1267, 2548); tavares (chfurg 4320, 4321); rio grande, quinta (chfurg 2015), parque marinha (chfurg 833, 4580), vila carreiros (chfurg 2540, 2963), distrito industrial (chfurg 1313, 1314, 1704, 1846, 1851, 1886, 1888, 1899, 1901, 1904, 1906, 1907, 1908, 1909, 1915, 1959, 1961, 1962, 2385, 3156, 3328, 4561, 5070, 5078, 5243), senandes (chfurg 832), área de proteção ambiental da lagoa verde (chfurg 1926, 1963, 1964, 1965, 2975, 3436), cassino (chfurg 1120, 1900, 1902, 1905, 3239, 4570, 4582, 4583, 5169, 5170, 5171, 5172, 5174, 5175, 5176, 5177, 5178, 5276, 5404, 5957, 5958, 5959, 5960, 5961, 5962, 5963, 5964), barra (chfurg 1910, 3076, 3078, 3079, 3090, 3208, 3274, 3328, 3329, 4419, 4444, 4567, 4578, 4579, 4581, 4584, 4671, 4680, 4687, 4688, 4789, 4790, 4820, 4891, 4902, 4918, 4920, 4921, 5071, 5072, 5073, 5074, 5075, 5076, 5077, 5078, 5079, 5080, 5081, 5082, 5083, 5173, 5201, 5238, 5239, 5240, 5241, 5242, 5243, 5244, 5245, 5246, 5247, 5249, 5250, 5285, 5633, 5966), estação ecológica do taim (chfurg 1500); santa vitória do palmar (chfurg 5376). acta herpetologica vol. 15, n. 2 december 2020 firenze university press estimating abundance and habitat suitability in a micro-endemic snake: the walser viper gentile francesco ficetola1,2,*, mauro fanelli3, lorenzo garizio3, mattia falaschi1, simone tenan4, samuele ghielmi5, lorenzo laddaga6, michele menegon7,8, massimo delfino3,9. potential effects of climate change on the distribution of invasive bullfrogs lithobates catesbeianus in china li qing peng1, min tang1, jia hong liao1, hai fen qing1, zhen kun zhao1, david a. pike2, wei chen1,* a bibliometric-mapping approach to identifying patterns and trends in amphibian decline research claudio angelini1,*, jon bielby2, corrado costa3 food composition of a breeding population the endemic anatolia newt, neurergus strauchii (steindachner, 1887) (caudata: salamandridae), from bingöl, eastern turkey kerim çiçek1,*, mustafa koyun2, ahmet mermer1, cemal varol tok3 stomach histology of crocodylus siamensis and gavialis gangeticus reveals analogy of archosaur “gizzards”, with implication on crocodylian gastroliths function ryuji takasaki1,2,*, yoshitsugu kobayashi3 does chronic exposure to ammonium during the pre-metamorphic stages promote hindlimb abnormality in anuran metamorphs? a comparison between natural-habitat and agrosystem frogs sonia zambrano-fernández1, francisco javier zamora-camacho2,3,*, pedro aragón2,4 confirming lessona’s brown frogs distribution sketch: rana temporaria is present on turin hills (piedmont, nw italy) davide marino1, angelica crottini2, franco andreone3,* phylogenetic relationships of the italian populations of horseshoe whip snake hemorrhois hippocrepis (serpentes, colubridae) francesco paolo faraone1, raffaella melfi2, matteo riccardo di nicola3, gabriele giacalone4, mario lo valvo5* first karyological analysis of the endemic malagasy phantom gecko matoatoa brevipes (squamata: gekkonidae) marcello mezzasalma1,2,*, fabio m. guarino3, simon p. loader1, gaetano odierna3, jeffrey w. streicher1, natalie cooper1 notes on sexual dimorphism, diet and reproduction of the false coral snake oxyrhopus rhombifer duméril, bibron & duméril, 1854 (dipsadidae: pseudoboini) from coastal plains of subtropical brazil fernando m. quintela1,*, felipe caseiro¹, daniel loebmann¹ acta herpetologica 14(1): 35-41, 2019 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-24651 mirrored images but not silicone models trigger aggressive responses in male common wall lizards stefano scali1,*, roberto sacchi2, mattia falaschi1,3, alan j. coladonato2, sara pozzi2, marco a.l. zuffi4, marco mangiacotti1,2 1 museo di storia naturale, corso venezia 55, 20121 milano, italy. *corresponding author. email: stefano.scali@comune.milano.it 2 dipartimento di scienze della terra e dell’ambiente, università degli studi di pavia, via taramelli 24, 27100 pavia, italy 3 dipartimento di scienze e politiche ambientali, università degli studi di milano, via celoria 26, 20133 milano, italy 4 museo di storia naturale, università degli studi di pisa, via roma 79, 56011 calci, italy submitted on: 4th september 2018; revised on: 23rd january 2019; accepted on: 4th april 2019 editor: uwe fritz abstract. disentangling the effects of single releasers in animal communication is a demanding task because a releaser often consists of a combination of different key stimuli. territorial communication in reptiles usually depends on visual, chemical, and acoustic stimuli, but the role of each of them depends on phylogeny. lacertids are modern lizards that rely mainly on chemical cues for their communication, but they also use aggressive displays based on visual recognition. we experimentally tested the visual stimuli that release an aggressive response in the males of a typical lacertid, the common wall lizard (podarcis muralis), testing the effects of silicone models and mirrored images in captivity. the response to models and control (a blank sheet) was not significantly different and these stimuli did not release any aggressive behaviour. on the contrary, the reflected image in a mirror caused overt aggression (i.e., bites against it) in 63% of tested individuals. the results clearly demonstrate the role of visual stimuli in territorial communication, but only as a combined effect of shape and motion, differently from other lizard families for which shape is enough to stimulate aggressive responses. mirrors can be useful tools to investigate aggression related to physiological and morphological aspects in lacertid lizards. keywords. visual stimulus, aggression releaser, mirrored image, plaster model, lacertidae. introduction animal communication systems have evolved so that individuals can make decisions based upon the behaviour, morphology, and physiology of other individuals (endler, 1993). communication depends on signal transmission between a signaller and a receiver and in order for the signal to be successful, it must be detected by the receiver against a background of other potential stimuli (fuller and endler, 2018). signals act as releasing mechanisms, intended as the totality of all parts of the nervous system that are involved in the filtering of incoming stimuli and it ensures that only the “appropriate” stimuli release a specific behaviour pattern (immelmann, 1983). the key stimulus or releaser may consist of single or complex cues and disentangling their effect to fully understand which of them releases certain behaviours could be challenging. the majority of releasers consists of a combination of motor patterns acting as a signal and a morphological structure enhancing the signal’s effect (lorenz, 1981), so a single portion of a key stimulus could be insufficient to release a particular behaviour. notably, when the behaviour pattern is simple and the risk of error is low, releasers are extremely simplified, such as the pecking at the red spot on the beak triggering the regurgitation of food by adult herring gulls (tinber36 stefano scali et alii gen, 1951). by contrast, when costs associated with errors are high, the releasers are more complex to ensure that behavioural patterns start only when they are necessary. this especially applies to aggressive behaviours, whose primary function is releasing of aggression against fellow members of the species and to avoid unnecessary fighting against heterospecific opponents (immelmann, 1983). aggressive behaviours are most common in territorial species, because individuals are forced to compete for limited resources, such as partners, food, shelters and reproductive sites, even if territoriality can be also assessed without overt aggression (brown, 1964; myrberg and thresher, 1974; van den berghe, 1974; stamps, 1977; kaufmann, 1983). among reptiles, many cases of territorialism are well documented for chelonians, crocodiles, tuataras, and, particularly, for lizards (pough et al., 2004). three main kinds of stimuli can be used for territorial communication by reptiles: visual, chemical, and acoustic. the latter is typical of species living in habitats where visual displays are ineffective and it is used only by nocturnal geckoes (marcellini, 1977), terrestrial tortoises (galeotti et al., 2005a; galeotti et al., 2005b) and crocodilians (vliet, 1989). chemical cues are effective communication tools, used by most reptiles, but particularly developed in modern species, like snakes and most scleroglossan lizards, thanks to the evolution of a complex vomeronasal system that freed the tongue from its ancestral role and allowed the transformation to a chemosensory organ (mason, 1992; schwenk, 1993; cooper, 1994). disentangling the role of intraspecific communication channels needs experiments analysing the single cues separately. for example, numerous studies have been done on iguanians (in particular on agamids and iguanids), a reptile clade that bases most of its territorial communication on visual stimuli, like posture, dewlap extension, colour patches and colour changes (yang et al., 2001; yang and wilczynski, 2002; van dyk and evans, 2008; norfolk et al., 2010; osborne et al., 2012; dunham and wilczynski, 2014; yewers et al., 2016). on the contrary, information is still lacking on the role of visual cues in lacertids, a family of scleroglossan lizards that relies mostly on chemical communication by means of femoral pores to assess territoriality (cooper, 1994; martin and lópez, 2015; mangiacotti et al., 2017; baeckens et al., 2018). visual stimuli can be tested using four kinds of methods: silicone models that mimic shape and colours of the species, the reflected image in a mirror of the subjects involved in the experiments, video playbacks showed to lizards, and direct staged encounters with another male. the latter method has been often used, but many factors can affect results, such as opponent size, residence status, and individual motivation (sacchi et al., 2009). video playbacks have been successfully used for some species (macedonia and stamps, 1994; yang et al., 2001; ord et al., 2002; van dyk and evans, 2008; frohnwieser et al., 2017), but they require a long preparation time to acquire video sequences or to prepare animated images after 3d scanning. hence, it requires a substantial a priori knowledge of the species’ stereotyped behaviours to present a complete sample set to the subjects. silicone models have been used, for example, with stellagama stellio and pseudotrapelus sinaitus (norfolk et al., 2010), platysaurus minor and p. monotropis (korner et al., 2000) and they proved to be adequate cues for territorial behaviours both for agamids and cordylids. mirrors are the most used visual stimuli in experimental designs because they are easy, cheap, and typically stimulate aggressive behaviours against the reflected image (balzarini et al., 2014). furthermore, the signal is enhanced by positive feedback, because an aggressive posture or behaviour is immediately replicated by the mirrored lizard. numerous species have been successfully tested using this methodology, particularly from families agamidae, phrynosomatidae, and dactyloidae (korzan et al., 2000; brandt, 2003; farrell and wilczynski, 2006; osborne et al., 2012; dunham and wilczynski, 2014). podarcis muralis, our model species, is a typical representative of this family. it is a small (snout to vent, svl up to 7.5 cm) and sexually dimorphic lizard, with males stouter and with bigger heads than females; males show a marked territorial behaviour (edsman, 1990; sacchi et al., 2009), as supported also by data about testosterone levels and homing behaviour in previous works (scali et al., 2013; sacchi et al., 2017). as a consequence, intraspecific communication in this species has been intensively studied as far as chemical cues are concerned (martin et al., 2008; heathcote et al., 2014; pellitteri-rosa et al., 2014; baeckens et al., 2017; mangiacotti et al., 2017, 2019), but no information is available about visual stimuli and communication during intraspecific encounters (but see zagar et al., 2015). since aggressive displays and postures of podarcis muralis have never been detailed before (but see sacchi et al., 2009 and abalos et al., 2016 for some information), we discarded playback videos and chose a dual experimental approach based on silicone models and mirrors as visual stimuli to boost and record aggressive behaviours. the specific aim of our work was to assess if visual stimuli can trigger an aggressive response in a typical lacertid lizard that bases most of its intraspecific communication on chemical cues. we did this by comparing the aggressive response to: i) a static and oversimplified visual cue (i.e., a silicone model); ii) a more complex and realistic visual stimulus combining movement, behaviour, and posture (i.e., a mirrored image). 37visual stimuli and aggression in lizards materials and methods ninety p. muralis adult males (svl > 50 mm) were captured by noosing in various localities in lombardy (northern italy) between april and june 2016, to maximize territorial response in accordance with reproductive season (corti and lo cascio, 2002; sacchi et al., 2017). the capture sites were located within 50 km each other and had similar ecological conditions, being all peripheral urban habitats, with comparable habitats, presence of predators and densities. lizards were carried to the natural history museum of milan and housed in individual plexiglas boxes (40 × 40 × 30 cm) with a refuge positioned near one box’s wall, water ad libitum and fed with three mealworms (tenebrio molitor) per/day. a sheet of absorbent paper was used as substrate, to keep the resident odour in each terrarium and thus lizards could consider it as their own territory. the vertical sides of the boxes were also covered externally with white paper sheets, to avoid external stimuli and wall reflectance. the room was exposed to a natural day/night cycle. after an acclimation period lasting between three and seven days, lizards were tested in the same terrarium where they were kept, after removing water and food. a heating lamp (zoomed repti basking spot lamp, 150 w) was turned on for 15 minutes to achieve a plateau body temperature similar for all the individuals (sannolo et al., 2014), then it was turned off and a cold led lamp (greenergy, 8 w, 600 lm) was lighted to ensure uniform lighting in the terrarium. a surveillance camera (sony super night vision camera, m020-s53-001, located near the heating lamp) was turned on and lizard behaviour was recorded for 15 minutes immediately after inserting a visual stimulus in the box. individuals were randomly assigned to the following three different stimuli (n = 30 for each treatment without replicates): i) a white paper sheet covering one side of the box, and used as control to simulate the insertion of an object by researcher’s hand; ii) a silicone model simulating a new lizard invading the resident’s territory; iii) a mirror covering one side of the box, reflecting lizard image and movements. all the stimuli were positioned inside the terrarium near the wall of the box opposite to the refuge. the lizard model was a silicon-rubber cast prepared by the museum taxidermist using a dead male p. muralis specimen that was painted brown on the back and white on the belly and throat using water-based tempera colours (fig. a1 in supplementary materials). the model was painted one month before starting the experiment to perfectly dry the paint. a pushup stance was obtained inserting an iron wire in the model to simulate a territorial posture with the anterior part of the body raised and showing throat colouration (molina borja, 1981). since difference in individual size has been proved to affect the outcome of male-male combats in p. muralis (sacchi et al., 2009) and the lizards tested against the model did not always had the same size as the model (svl = 67 mm, see table a1 in supplementary materials for lizard mean size), we performed a preliminary test to investigate the potential inhibitory effect of such a difference. latency (i.e., the time between the insertion of the model in the terrarium and the first movement of the focal lizard) was used as a proxy for the potential inhibitory effect (the longer the latency, the larger the effect) and it was regressed against the signed difference between lizard and model size (svllizard – svlmodel). the regression was not significant (one-way anova: f1,28 = 1.27; p = 0.27), so we assumed that model size did not affect lizard aggressive response. the videos were analysed in the platform boris (behavioral observation research interactive software, friard and gamba, 2016, freely available at www.boris.unito.it). all the behaviours addressed to the stimulus were scored as follows: 1) no interest (i.e., walking across the terrarium without any interactions with the stimulus); 2) interest without aggression (i.e., observing or tongue-flicking towards the stimulus); 3) interest with overt aggression (i.e., biting against the stimulus). scores for the three treatments were compared using a kruskal-wallis test and mann-whitney tests were used as posthoc tests. analyses were performed under the r rel. 3.4.2 statistical environment (r development core team, 2017) and, otherwise stated, reported values represent means and standard errors. results the higher aggression score (i.e., 3) was observed only for the mirror treatment, where 63.3% (19 out of 30) of males bit the stimulus. the highest score achieved by males in the other two treatments was 2, with 66.7% and 56.7% in control and silicone model treatments respectively. these differences were highly statistically significant (kruskal-wallis: χ² = 26.021, d.f. = 2; p < 0.001). mann-whitney post-hoc tests showed that aggression scores did not differ between control and silicone models (p = 0.44); on the opposite, both comparisons involving fig. 1. proportions of aggressive responses against the stimuli (control, plaster model, and mirror respectively) by common wall lizards (see methods for scoring details). 38 stefano scali et alii mirror were significant (p < 0.001 in both cases), being the score of the mirror treatment always higher than the other two (fig. 1). discussion our experiment demonstrated that visual stimuli are important releasers in triggering aggressive behaviours in the common wall lizard. it is often difficult to disentangle the effect of single key stimuli in a complex stimulus, but the approach used in this study allowed us to separate the effect of a simple cue, the shape of a lizard, by the composite effect of shape and motion. the result is not as trivial as it might seem, because some lizard species actively respond to simplified and motionless models by activating some territorial behaviours, demonstrating that they do not necessarily need more complex visual stimuli. this is true for some agamidae, such as the tawny dragon lizard, ctenophorus decresii, whose territorial behaviour has been intensively studied. indeed, males of this polymorphic species engage in complex displays to defend territories and they use the same behavioural patterns also against models, responding differently even to throat colours (yewers et al., 2016). also cordylids, such as platysaurus minor and p. monotropis, show overt aggression behaviours against models, demonstrating poor species recognition when models of different congenerics were proposed (korner et al., 2000). all the above examples support the hypothesis that oversimplified visual stimuli can provoke an aggressive response in these taxa. the common wall lizards were not interested in the presence of models within the enclosures and their only possible reaction was moving sometimes around and on the fake lizards and tongue-flicking at a certain distance. these behaviours were adopted even when the control stimulus (i.e., the white paper sheet) was inserted in the enclosure, so no conclusion can be inferred because they could be due to simple exploration activity. by contrast, the mirrored image always caused an alert posture, such as “freezing” in front of the mirror, repeated tongue-flicking or push-up displays, often culminating in overt aggression against the image with multiple bites or jumps. numerous authors proved that mirrors are efficient stimuli able to release aggressive behaviours for many species belonging to different families (agamidae, phrynosomatidae, and dactyloidae) (korzan et al., 2000; brandt, 2003; farrell and wilczynski, 2006; norfolk et al., 2010; osborne et al., 2012; dunham and wilczynski, 2014). interestingly, in all these cases the species belong to visually-oriented lizard clades, making such a result expectable. on the opposite, to our knowledge this is the first time that the same kind of visual stimulus (i.e., a mirrored image) releases aggressive behaviour in a lacertid lizard, which is thought to be more chemicalthan visual-oriented (but see garcia-roa et al., 2017; baeckens et al., 2018). one main objection to the reliability of our results could be the different smell between the proposed stimuli and a real lizard, but we chose to exclude chemical stimuli in our experiment to disentangle the effect of shape, movement, and chemical hints. our results confirm the observations by other authors that aggression releasers often are not single visual stimuli, such as a still image, but the combination of different stimuli, such as shape and motion (macedonia and stamps, 1994; yang et al., 2001; ord et al., 2002; yang and wilczynski, 2002; van dyk and evans, 2008; frohnwieser et al., 2017). previous research on the role of motion patterns in the visual displays of anoles demonstrated that motion is fundamental to attract the attention of lizards, particularly when a specific motion pattern is exhibited (fleishman, 1992). the response to motion patterns is used by many territorial species to signal their presence to intraspecific opponents. these lizards use dewlap extension to communicate with rivals and partners and this display is often accompanied by a stereotypical headbobbing movement. a detailed study on signal efficacy showed that motion patterns that combined high acceleration with high velocity were particularly effective. nonetheless, at a short distance, even a small-amplitude motion in the visual periphery can be perceived by a lizard, causing a shift of gaze so that the image falls on a high-resolution portion of the retina (fleishman, 1992). of course, lacertids and anoles do not share the same evolutionary history (pyron et al., 2013), so a detailed study on visual acuity and efficacy of the formers would be hoped. simple stimulus, such as shape, could be enough to trigger a territorial response in basal lizard clades, but not in modern lizards that use chemical stimuli as the main releaser in intraspecific communication. previous studies on animal communication demonstrated that the sensory system of the receiver determines which signals can be detected and that, in majority of cases, sensory systems serve multiple purposes and must be capable of detecting many types of different stimuli, such as mates, food, habitat, and opponents (fuller and endler, 2018). responding to all the stimuli could be extremely costly, so there is selection on sensory systems to efficiently capture relevant stimuli in the environment (fleishman, 1992; fuller and endler, 2018). this could explain why motionless or slow-moving shapes do not elicit aggressive responses in the common wall lizard, whereas mirrored images do. further studies will be necessary to fully understand 39visual stimuli and aggression in lizards the aggressive behaviour of the common wall lizard and the underlying releasing mechanisms, as well as to support the relationship between phylogeny, territoriality, and the complexity of visual stimuli. nevertheless, we demonstrated that mirrored images are able to activate the aggressive response also in a lacertid lizard, which sets the stage for a wider and comparative study using other species and stimuli. acknowledgements we are grateful to chiara di perna and simone facchinetti for their assistance during the experiments. ermano bianchi made the silicon casts of the lizard. this research was carried out in conformity with current italian laws (dpn/ii div/10423/pnm 26/05/2015 and dpn/ ii div/9720 12/05/2015). supplementary material supplementary material associated with this article can be found at < http://www.unipv.it/webshi/appendix > manuscript number 24651. references abalos, j., pérez i de lanuza, g., carazo, p., font, e. 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revised on: 2019, 2nd september; accepted on: 2019, 17th september editor: marco mangiacotti abstract. the sardinian grass snake (natrix natrix cetti) is a critically endangered snake endemic to sardinia (italy), for which information is still scarce. in the present work, we report information obtained from 36 observations of n. n. cetti performed in different areas of the island. three different colorations were mainly observed and darker snakes were in general males and big adults; the only juvenile found showed a complete different dorsal colouration. snakes were observed active during day-time and often far from the aquatic habitats. keywords. melanism, abundism, mimicry, predator avoidance, island. the sardinian grass snake (natrix natrix cetti) is a critically endangered snake endemic to sardinia, for which we still have very limited information (european reptile & amphibians specialist group, 1996; vanni and cimmaruta, 2010). a recent phylogenetic analysis performed on the natrix natrix complex grouped the sardinian grass snake together with the corsican subspecies (n. n. corsa) into a distinct genetic group, which was likely promoted by their isolation occurred during glaciations (fritz et al., 2012; kindler et al., 2013). according to the most recent phylogenetic study, the western grass snakes belong to a new clade called natrix helvetica (kindler et al., 2017); however, in this study no samples from sardinia have been analysed. its elusiveness could be one of the causes of the few data available on its distribution, ecology and biology (lunghi et al., 2016; lunghi et al., 2018; vanni and cimmaruta, 2010). it is known that n. n. cetti can exploit different environments, from dry rocky areas to wetlands, rivers and even caves, and that the species is relatively widespread on the island (capula et al., 1994; de pous et al., 2012; lanza 1986; mulargia et al., 2018; salvi and bombi, 2010). however, the presence of the viperine snake (natrix maura), as potential competitor, seems to affect its distribution (stefani 1983; vanni and cimmaruta 2010). adults of n. n. cetti differ morphologically from the continental subspecies because of the lack of the typical light “collar”, and the smaller size (speybroeck et al., 2016; vanni and cimmaruta, 2010). stefani (1983) reports for n. n. cetti a typical light greyish background 142 enrico lunghi et alii colour, a characteristic that should be of help in distinguishing the sardinian grass snake from the corsican one, being the latter characterised by a dark greenish background. recently, abundistic (i.e., dorsum characterized by enlarged dark stripes) and melanotic (i.e., nearly completely black coloration) individuals of n. n. cetti were observed (lunghi et al., 2016), thus increasing the variability of the dorsal colouration known for this subspecies. furthermore, as far as we know, no information exists on juveniles. here we report observations of natrix natrix cetti gathered during three years fieldwork. all captured snakes were measured, and information on dorsal pattern and habitat were recorded. the observation of one juvenile is also reported. from 2016 to 2018, we conducted herpetological field observations focusing on eastern and southern sardinia (see table 1; coordinates are not reported for species safeguard, see lunghi et al., 2019). repeated linear transects, based on ves (visual encounter survey), were carried out. the surveys were performed by day (9 a.m. – 5 p.m.) throughout the year, but the most in spring (jan-mar = 9.1%; apr-jun = 70.6%; jul-sep = 14.4%; oct-dec = 5.9%; total surveys = 153). for each snake we recorded: locality, elevation and time of the observation, habitat typology and dorsal background colour pattern (greyish, greenishbrownish, melanotic) (fig. 1). captured snakes were measured (total length) and sexed. total length was measured photographing the snakes on a plasticised millimetre paper; snakes’ length was extrapolated using the program imagej. sex was assessed combining visual inspection of the morphology of the snake (proportion of the head and tail) and number of sub-caudal scales (vanni and cimmaruta, 2010). head pattern was used for individually snake recognition (sacchi et al., 2016; vaughan, 1999). a generalized linear model (r software with nlme package; pinheiro et al., 2016; r development core team 2018) was used to assess whether snake coloration is related to sex, elevation, time of survey and total length (tl). we used colouration as dependent variable, while sex, tl, time of survey and elevation as independent variables; year and transect identity as random factors. to use colfig. 1. the four different dorsal colourations of natrix natrix cetti reported in this study: a) greyish, b) greenish-brownish, c) black, d) the juvenile. (photos a and b by m. di nicola; c by e. lunghi; d by s. giachello). 143dorsal pattern in sardinian grass snake ouration as dependent variable, we ascribed an ascending order to the different colourations, going from the lightest (greyish = 1) to the darkest (greenish = 2) (table 1). in this analysis we excluded the juvenile, the single melanotic individual, and, for the individual captured twice (see below) only the first observation. in total we performed 153 surveys on 3 different areas (barega, no. of transects = 1, total surveys = 5; monte albo, no. of transects = 11, total surveys = 102; sette fratelli, no. of transects = 2, total surveys = 46) and we observed 19 natrix natrix cetti; seventeen were adults. most of the observations (17) were performed on the sette fratelli mountain and only an adult female was captured twice. the greenish-brownish (n = 12) coloration resulted to be the most common, followed by the greyish (n = 5) and the melanotic (n = 1) (fig. 1a-c). the observed juvenile (tl = 22 cm) showed a complete different dorsal colouration: the background colour was black, the stripes white and a white collar was also present (fig. 1d). snakes were observed at an elevation between 341 and 1029 m a.s.l. two individuals were found in small temporary water bodies, one in a mine, while the others 15 in rocky areas sometimes even more than 1 km far from the nearest water body. snakes’ coloration significantly correlated only with total length (f1,8 = 8.94, p = 0.017); the darker (greenish) coloration was most frequently observed in the longer snakes (fig. 2). no significant effect was observed for other variables (sex, f1,8 = 2.00, p = 0.195; elevation, f1,8 = 2.90, p = 0.127; time of survey, f1,8 = 1.69, p = 0.229). during our study, a female with a wounded eye was captured twice, the first on may 2016 and the second in april 2017; in both occasions this female was active at about the same time (16:41 and 16:21 respectively). this individual was recaptured almost 90 m far from the site of first encounter (difference in altitude 98 m). the sardinian grass snake is one of the least studied italian species. elusiveness, rather than its potential rarity, could be the reasons for the few existing studies on this species (vanni and cimmaruta, 2010); indeed, although being made of just 19 records, our dataset is one of the richest available. the dorsal coloration of natrix natrix cetti seems to be more variable than previously reported; indeed, most of our observations highlighted the high frequency of the greenish-brownish coloration, a characteristic that should be typical of the corsican grass snake (stefani, 1983). because of our dataset paucity, we cannot provide any assumptions on the causes supporting the high pattern differentiation observed in this subspecies (but see also lunghi et al., 2016). further studies are needed to assess whether factors are influencing the observed varitable 1. data of the captured individuals of natrix natrix cetti: sex, total length (tl), dorsal background colour (1 = greyish, 2 = greenishbrownish, 3 = melanotic), elevation (m a.s.l.), time at which snakes were observed (24h), province, year, transect identity and the mountain complex. *individuals captured twice. sex tl (cm) colour elevation time province year transect mountain female 65.7 2 341 16:15 carbonia-iglesias 2017 barega1 barega female 44 1 474 14:32 cagliari 2017 cagliari3 sette fratelli female 72.1 2 515 10:30 cagliari 2016 cagliari2 sette fratelli female 74 1 534 10:08 cagliari 2018 cagliari2 sette fratelli female 53.1 1 535 11:10 cagliari 2017 cagliari2 sette fratelli female 62.3 2 539 12:00 cagliari 2017 cagliari2 sette fratelli female 61.8 2 541 14:01 cagliari 2017 cagliari2 sette fratelli male 54.3 2 633 11:02 cagliari 2017 cagliari2 sette fratelli female 59 2 634 10:36 cagliari 2017 cagliari2 sette fratelli *female 63 2 638 16:21 cagliari 2017 cagliari2 sette fratelli female 57.9 2 670 15:45 cagliari 2016 cagliari2 sette fratelli male 64.7 2 689 14:05 cagliari 2017 cagliari2 sette fratelli male 60.1 3 706 13:45 cagliari 2016 cagliari2 sette fratelli female 58.8 2 730 16:42 cagliari 2016 cagliari2 sette fratelli *female 63 2 736 16:40 cagliari 2016 cagliari2 sette fratelli female 63 1 792 15:58 cagliari 2018 cagliari2 sette fratelli male 46.4 2 830 15:50 cagliari 2017 cagliari2 sette fratelli female 59.8 2 863 13:20 cagliari 2017 cagliari3 sette fratelli male 55.5 1 1029 14:45 nuoro 2018 m_albo3 monte albo 144 enrico lunghi et alii ability and, if darker coloration may be beneficial for snakes’ longevity (castella et al., 2013; clusella trullas et al., 2007; fulgione et al., 2015; stevens et al., 2009; zuffi 2008). the juvenile highlighted ontogenetic variability in dorsal coloration in n. n. cetti. our study agrees with stefani (1983) and lanza (1986) considering n. n. cetti not strictly related to water bodies. this likely allows the snake to exploit further habitats, increasing prey availability and lowering competition with natrix maura (lunghi et al., 2018; stefani, 1983; vanni and cimmaruta, 2010). our study was carrried out in day-time (see also lanza, 1986) but we cannot exclude nocturnal activity as reported by capula et al. 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(2008): colour pattern variation in populations of the european whip snake, hierophis viridiflavus: does geography explain everything? amphibiareptilia 29: 229-233. acta herpetologica vol. 14, n. 2 december 2019 firenze university press podarcis siculus latastei (bedriaga, 1879) of the western pontine islands (italy) raised to the species rank, and a brief taxonomic overview of podarcis lizards gabriele senczuk1,2,*, riccardo castiglia2, wolfgang böhme3, claudia corti1 substrate type has a limited impact on the sprint performance of a mediterranean lizard pantelis savvides1,*, eleni georgiou1, panayiotis pafilis2,3, spyros sfenthourakis1 coping with aliens: how a native gecko manages to persist on mediterranean islands despite the black rat? michel-jean delaugerre1,*, roberto sacchi2, marta biaggini3, pietro lo cascio4, ridha ouni5, claudia corti 3 pit-tags as a technique for marking fossorial reptiles: insights from a long-term field study of the amphisbaenian trogonophis wiegmanni pablo recio, gonzalo rodríguez-ruiz, jesús ortega, josé martín* occurrence of batrachochytrium dendrobatidis in the tensift region, with comments on its spreading in morocco ait el cadi redouane1, laghzaoui el-mustapha1, angelica crottini2, slimani tahar1, bosch jaime3,4, el mouden el hassan1,* hematological parameters of the bolson tortoise gopherus flavomarginatus in mexico cristina garcía-de la peña1,*, roger iván rodríguez-vivas2, jorge a. zegbe-domínguez3, luis manuel valenzuela-núñez1, césar a. meza herrera4, quetzaly siller-rodríguez1, verónica ávila-rodríguez1 ontogenetic and interspecific variation in skull morphology of two closely related species of toad, bufo bufo and b. spinosus (anura: bufonidae) giovanni sanna visible implant alphanumeric (via) as a marking method in the lesser snouted treefrog scinax nasicus andrea caballero-gini1,2,3,*, diego bueno villafañe2,3, lía romero2, marcela ferreira2,3, lucas cañete4, rafaela laino2, karim musalem2,5 morphological variation of the newly confirmed population of the javelin sand boa, eryx jaculus (linnaeus, 1758) (serpentes, erycidae) in sicily, italy francesco p. faraone1,*, salvatore russotto2, salvatore a. barra3, roberto chiara3, gabriele giacalone4, mario lo valvo3 variability in the dorsal pattern of the sardinian grass snake (natrix natrix cetti) with notes on its ecology enrico lunghi1,2,3,4,*, simone giachello5, manuela mulargia6, pier paolo dore7, roberto cogoni8, claudia corti1 estimating abundance of the stripeless tree-frog hyla meridionalis by means of replicated call counts federico crovetto, sebastiano salvidio, andrea costa* at-rich microsatellite loci development for fejervarya multistriata by illumina hiseq sequencing yan-mei wang, jing-yi chen, guo-hua ding*, zhi-hua lin acta herpetologica 12(2): 151-156, 2017 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-20700 non-native turtles in a peri-urban park in northern milan (lombardy, italy): species diversity and population structure claudio foglini1, roberta salvi2,* 1 indipendent researcher. via l. b. alberti 8/a, i-20092 cinisello balsamo (mi), italia 2 indipendent researcher . via a. moro 1, i-24030 carvico (bg), italia. *corresponding author. e-mail: clafogli@libero.it both authors contributed equally to this work. submitted on: 2017, 30th of may; revised on: 2017, 16th of august; accepted on: 2017, 6th of october editor: simon baeckens abstract. trachemys scripta (slider turtle) and other freshwater turtle species have been traded worldwide as pets, but they are often released by owners in wild or in semi-natural habitats, especially in suburban and urbanized areas. in four artificial lakes and ponds of a regional park in the north outskirt of milan (italy, lombardy), we trapped non native turtles using basking-traps and landing nets from april to august 2013.we captured 156 trachemys scripta, 10 graptemys pseudogeographica and four pseudemys sp.; the capture-mark-recapture approach estimated an overall number of 224 individuals (95% c.i. 195-270). there was also a strong indirect support for a probable in situ reproduction, with small juveniles captured and a nest-digging female observed. key-words. invasive alien species, morphometrics, non-native-turtles, reproduction, slider turtle, trachemys scripta. introduction the global introduction of non-native amphibian and reptile species has increased exponentially trough the last 150 years (çiçek and ayaz, 2015). one animal group that is globally very popular in the pet trade is freshwater turtle, in particular subspecies and hybrids of the american slider turtle trachemys scripta. turtles belonging to this group are massively traded at the global scale (ballasina, 1995; kraus, 2009) and are usually sold when they are only few centimetres long. due to their fast growing rate these turtles are then often released into the wild or in semi natural habitats (crescente et al., 2014). trachemys scripta compete for food and basking places in wetlands where they coexist with european emys orbicularis and mauremys leprosa (cadi and joly, 2003; perez-santigosa et al., 2008; ficetola et al., 2009), and massive turtle presence can influence ecosystem functioning and aquatic communities (lindsay et al., 2013). free-living slider turtles’ populations occur in many countries of southern europe (spain: martínez-silvestre et al., 2011; france: cadi et al., 2004; italy: agosta and parolini, 1999; ferri and soccini, 2003; ficetola et al., 2003; monti, 2010) and the ongoing climate change will probably expand the areas of suitability in the future (ficetola et al., 2009). in addition, following the european ban on import of t. s. elegans in the 1997 (european commission, 1997), legal pet trade switched to other slider subspecies: mainly t. s. scripta, hybrids t. s. scripta x t. s. elegans and, but to lesser extent, t. s. troostii (bringsøe, 2006). after a new european ban, extended to all trachemys subspecies (european commission, 2001 and subsequent amendments), different species of graptemys and pseudemys were also imported (bringsøe, 2006). in this study we investigated: (1) the number and size structure of turtles; (2) the presence of different species and subspecies of several non-native turtles; (3) the possibil152 claudio foglini, roberta salvi ity of reproduction in the artificial lakes and ponds of the nord milano park (italy, lombardy, province of milan). materials and methods the study was carried out in the nord milano park (fig. 1), a 640 ha peri-urban regional park located in the north outskirts of milan (italy, lombardy). the park was created on industrial brownfield during later sixties, and first reforestation dates back to 1983. during the years, woods, artificial wetlands, small lakes and ditches were added, while many others environmental improvement measures are still in progress. the presence of several non-native turtles in the park is well known, but has never been deeply investigated. the counting sessions of this study were focused into four wetlands: three artificial lakes (cinisello, suzzani and bresso lakes) and one ditch-like site (breda ditch) (see fig. 1 legend). to avoid problems with double-counts and with low detectability (i.e., water turbidity, lack of basking sites, aquatic macrophytes cover) turtles were captured with basking traps from april to august 2013: each trap was visited every two or three days between 11:00 am and 03:00 pm, when the basking activity is greater (cadi and joly, 2000). during each visit additional turtles were captured with landing nets. parameters such as the straight line plastron length (spl), the minimum straight carapace length (sclmin) and the straight carapace width (scw) were measured with callipers (bjorndal and bolten, 1989) by the same operator. each individual was weighted and sexed using secondary sexual characteristics such as foreclaws and tail length (readel, 2008). specimens with spl less than 150 mm (gibbons, 1990) and with doubtful elements were considered unsexed. each animal was also unambiguously marked with a unique notches combination on the marginal carapace scutes. the release of invasive turtles after marking was an exceptional procedure, necessary to collect the basic knowledge for a pilot capture-recapture study; this was essential to develop and refine an effective control or eradication plan in the study area. given the fact that capture and recapture invariably occurred for each individual within the same site, a close populations scenario was assumed. this assumption is supported by the fact that trapping sites are not connected and are separated by wooded areas, lawns, footpaths and bikeways. for this reason, in order to estimate the overall number of turtles, the capture-mark-recapture (cmr) model for close populations (jhe closed population model estimation) provided by the software noremark (white, 1996) was used. fig. 1. study area (black box, not scaled). the position and a detailed view of each wetland is given by numbers: 1) cinisello lake; 2) bresso lake; 3) breda ditch; 4) suzzani lake. wetland boundaries are marked in white (modified from www.d-maps.com and geoportale regione lombardia). 153non-native turtles population in peri-urban wetland results species richness and abundance we captured and individually marked, weighted and sexed 156 slider turtles: 16 red-eared slider trachemys scripta elegans, 35 yellow-bellied slider t. scripta scripta, 7 cumberland slider t. scripta troostii and 98 t. scripta hybrids. specimens were classified as hybrids when they showed a mix of elements from parental species (seidel et al., 1999; for details see supplementary fig. s1). we also captured, measured and marked 10 false map turtle graptemys pseudogeographica (supplementary fig. s2) and cooter pseudemys sp. (supplementary fig. s3). details about sex-ratio and species repartition into each sampling site are shown in table 1, while details about morphometrics are shown in table 2. using the acquired data, the following overall population was estimated with noremark software (95 % confidence interval in parentheses): 13 (13) turtles in the cinisello lake, 84 (73-102) in the suzzani lake, 106 (90-129) in the bresso lake and 21 (19-26) in the breda ditch (supplementary table s4). trachemys scripta population structure due to the lack of data for the others turtles genera, only the population structure of trachemys scripta subspecies was analysed. among sexed specimens, in three sampled wetlands we found a shift towards a female-biased sex-ratio. in the suzzani and bresso lakes (fig. 2a and 2b, respectively), we found mainly medium-small turtles; full-grown adults are present in small number, and we found also some juveniles. small turtles were prevalent also in the breda ditch, in which we found also only two juveniles and one adult female (fig. 2c). in the cinisello lake, the small number of individuals does not allow a population description (fig. 2d). the presence of small specimens with spl between 26 and 51 millimetres was noteworthy. four were t. scripta hybrids, three of which trapped in the bresso lake and one in the breda ditch (spl 26 to 51 mm, μ 36.25 ± 10.56 sd). one t. s. elegans (spl 43 mm) was captured in the suzzani lake, and one t. s. scripta (spl 42 mm) was captured in the bresso lake (supplementary fig. s5a and s5b). three more trachemys juveniles have spl table 1. number and sex of non-native turtles captured in each pond, grouped by taxa. m=male; f=female; n.d.=unsexed. wetland t. s. elegans t. s. scripta t.s. troostii t. s. hybrids pseudemys sp. g. pseudogeographica m f n.d. m f n.d. m f n.d. m f n.d. m f n.d. m f n.d. suzzani lake 0 2 4 0 1 11 0 0 3 3 6 27 0 1 2 0 0 4 bresso lake 2 5 3 0 1 14 0 1 1 1 7 34 0 0 0 0 1 5 breda ditch 0 0 0 0 0 4 0 0 2 0 1 11 0 1 0 0 0 0 cinisello lake 0 0 0 0 0 4 1 0 0 0 0 8 0 0 0 0 0 0 table 2. morphometrics (mean ± sd and range) of captured specimens, grouped by taxa. spl = straight line plastron length; sclmin = minimum straight carapace length; weight. n may vary, as some data is missing. t. s. elegans t. s. scripta t. s. troostii t. s. hybrids pseudemys sp. g. pseudogeographica spl (mm) ± sd 143.19 ± 43.64 100.57± 32.31 112.86 ± 44.95 108.14 ± 38.57 189.00 ± 46.91 105.80 ± 40.58 n 16 35 7 97 4 10 min. – max. (mm) 43 – 210 42 – 183 50 – 168 26 – 188 123 – 223 53 – 167 sclmin (mm) ± sd 156.31 ± 45.99 111.97 ± 35.18 120.71 ± 50.45 116.67 ± 40.49 206.25 ± 47.67 119.60 ± 44.81 n 16 35 7 97 4 10 min. – max. (mm) 47 – 221 46 – 200 53 – 180 31 201 140 – 242 61 – 187 weight (g) ± sd 751.67 ± 506.78 347.06 ± 287.41 382.14 ± 274.89 407.18 ± 302.53 1437.50 ± 807.65 360.00 ± 281.66 n 15 34 7 94 4 10 min. – max. (g) 100 – 2000 50 – 1600 50 – 850 50 – 1500 400 – 2150 50 – 800 154 claudio foglini, roberta salvi inside the same range, but their scl exceed 60 mm. discussion trachemys scripta has been included in the “top 100 world’s worst invaders” as a result of their formerly massive import by the pet trade (lowe et al., 2000). it is now listed among the invasive alien species (ias) of european union concern (european commission, 2016). pseudemys and graptemys have partially replaced sliders as pets-turtle, but due to their higher price they are not so common as feral individuals (bringsøe, 2006). species composition and population status captured slider turtles were mainly t. scripta hybrids and t. s. scripta, followed by red-eared slider and just few t. s. troostii. few individuals of other species (i.e., false map turtle and cooter) were also captured. among slider turtles, the female-biased sex ratio we found is common in non-native population, because slider turtles were reared with artificially high temperature: this accelerate their development (prevot-julliard et al., 2007), but increase female development in species with temperature-dependent sex determination (godfrey et al., 2003). in suitable conditions, the unbalanced sex ratio could increase the number of recruits of non-native population (ficetola et al., 2009). according to the number and the size of small turtles captured, bresso lake was the only wetland with apparently active recruiting: this could be due either to new release of relatively young individuals (uncommon with pet turtle) or to active reproduction (see below). reproduction several mediterranean and southern-continental european areas currently have suitable climate conditions for slider turtles’ successful reproduction (tzankov et al., 2015). concerning breeding turtles in the park, before this study there was only an anecdotic report of one hatchling with eggshell remains, ran over on the side of a bikeway (mariani g., pers. com.). unfortunately, because of the advanced decomposition, it was possible to identify the specimen only as belonging to the trachemys genus. the smallest slider turtles recorded (see results) can be considered clues of probably recent breeds, because their carapace lengths were similar to those reported for newly born juveniles in ernst et al. (1994). moreover, the juvenile t. s. elegans captured was almost certainly feral, because the trade of this specie in the eu was banned since 1997 (european commission, 1997). fig. 2. number of slider turtles, classified after their straight plastron length, hosted in each wetland (completion year shown in brackets). 155non-native turtles population in peri-urban wetland although we did not record nests during this study, in july 2016 an adult female was observed during nestdigging (gelso m.r., pers.com.; supplementary fig. s6), and during the same month, in the bresso lake another juvenile of t. scripta was reported (ghislandi a. and ghislandi m., pers. com.; supplementary fig. s7). conclusions as the park is deeply engulfed in the urban matrix, no european pond turtle emys orbicularis is present, but on the other end there is a small population of smooth newt (lissotriton vulgaris), classified as near threatened (nt) in italy (rondinini et al., 2013). for all these reasons, we find unusual that the park has not yet adopted an invasive turtle control plan, even more after the ratification of the european regulation 1143/2014 and the european implementing regulation 1141/2016 (european commission, 2014; 2016). according to the evidences we found (i.e. reproductive population, number of potential breeding turtles), we hope that this work could represent a first step towards the adoption of a non-native turtles monitoring and control program in the area, even with eradication measures. due to the high level of anthropization, and the easy accessibility of wetlands in the study area, we also urged the adoption of steady actions aimed at discouraging further releases by turtle owners. acknowledgments we would like to thank nord milano park management for research opportunities, and all the employees (especially gianmario bernasconi, francesco carbone and marco siliprandi) for their generous support. we would like to thank francesco ficetola and thania manfredi for their valuable advice. many thanks also to anna and maurizio ghislandi and to maria rita gelso for some of the pictures. supplementary material supplementary material associated with this article can be found at <http://www.unipv.it/webshi/appendix> manuscript number 20700. references agosta, f., parolini, l. 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(1996): noremark: population estimation from mark-resighting surveys. wildlife soc. bull. 24: 50-52. acta herpetologica vol. 12, n. 2 december 2017 firenze university press meristic and morphometric characters of leptopelis natalensis tadpoles (amphibia: anura: arthroleptidae) from entumeni forest reveal variation and inconsistencies with previous descriptions susan schweiger1, james harvey2, theresa s. otremba1, janina weber1, hendrik müller1,* brown anole (anolis sagrei) adhesive forces remain unaffected by partial claw clipping austin m. garner*, stephanie m. lopez, peter h. niewiarowski species and sex comparisons of karyotype and genome size in two kurixalus tree frogs (anura, rhacophoridae) shun-ping chang1,2, gwo-chin ma2,3,4, ming chen2,5,6,7,8,*, sheng-hai wu1,* non-native turtles in a peri-urban park in northern milan (lombardy, italy): species diversity and population structure claudio foglini1, roberta salvi2,* species composition and richness of anurans in cerrado urban forests from central brazil cláudia márcia marily ferreira1,*, augusto cesar de aquino ribas2, franco leandro de souza3 the life-history traits in a breeding population of darevskia valentini from turkey muammer kurnaz, alı i̇hsan eroğlu, ufuk bülbül*, halıme koç, bılal kutrup influence of desiccation threat on the metamorphic traits of the asian common toad, duttaphrynus melanostictus (anura) santosh mogali*, srinivas saidapur, bhagyashri shanbhag predation of common wall lizards: experiences from a study using scentless plasticine lizards jenő j. purger*, zsófia lanszki, dávid szép, renáta bocz reproductive timing and fecundity in the neotropical lizard enyalius perditus (squamata: leiosauridae) serena najara migliore1,2,*, henrique bartolomeu braz2,3, andré felipe barreto-lima4, selma maria almeida-santos1,2 observations on the intraspecific variation in tadpole morphology in natural ponds eudald pujol-buxó1,2,*, albert montori1, roser campeny3 and gustavo a. llorente1,2 reliable proxies for glandular secretion production in lacertid lizards simon baeckens diet of juveniles of the venomous frog aparasphenodon brunoi (amphibia: hylidae) in southeastern brazil rogério l. teixeira1, ricardo lourenço-de-moraes2, débora c. medeiros3, charles duca3, rogério c. britto4, luiz c. p. bissoli5, rodrigo b. ferreira3,* who are you? the genetic identity of some insular populations of hierophis viridiflavus s.l. from the tyrrhenian sea ignazio avella, riccardo castiglia, gabriele senczuk* acta herpetologica 15(2): 137-141, 2020 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/a_h-8437 first karyological analysis of the endemic malagasy phantom gecko matoatoa brevipes (squamata: gekkonidae) marcello mezzasalma1,2,*, fabio m. guarino3, simon p. loader1, gaetano odierna3, jeffrey w. streicher1, natalie cooper1 1 department of life sciences, natural history museum, cromwell road, london, sw7 5bd, uk 2 cibio, research centre in biodiversity and genetic resources, inbio, universidade do porto, campus agrário de vairão, rua padre armando quintas, no 7, 4485-661 vairão, portugal (current address) 3 department of biology, university of naples federico ii, via cinthia 26, 80126, naples, italy *corresponding author. e-mail: m.mezzasalma@gmail.com submitted on: 2020, 10th april; revised on: 2020, 3rd june; accepted on: 2020, 15th october editor: adriana bellati abstract. the genus matoatoa includes two malagasy endemic species, m. brevipes and m. spannringi. due to their cryptic behaviour, the two species are known only from a handful of specimens and have been included in few molecular studies. here we carried out a molecular barcoding analysis using a fragment of the mitochondrial nadh dehydrogenase subunit 2 (nd2) and the first chromosomal analysis of m. brevipes. the molecular analysis confirmed the identity of the studied samples as m. brevipes. however, the level of genetic divergence (4% uncorrected p-distance) between our samples and other sequences of m. brevipes, suggests previously unrecognised diversity within the species. the karyotype of m. brevipes is composed of 2n = 34 chromosomes: the first pair is metacentric, while all the other pairs are telocentric and gradually decreasing in length (arm number, an = 36). c-banding revealed little evidence of centromeric heterochromatin, while nor-associated heterochromatin was found on the telomeres of a medium sized telocentric pair. no heteromorphic chromosome pairs were found in the karyotype of the species, suggesting that putative sex chromosomes are at an early stage of differentiation. karyological comparisons with closely related species were performed with christinus marmoratus, and representatives of the genera phelsuma, ebenavia, paroedura and uroplatus. comparisons across genera suggest that chromosome diversification in this group of geckos probably occurred by means of chromosome fusions and inversions, leading to a reduction of the chromosome number and the formation of biarmed elements in different species. keywords. chromosomes, leaf-toed geckos, matoatoa, madagascar, evolution, reptiles. madagascar is one of the hottest reptilian biodiversity hotspots, including more than 430 reptile species (glaw and vences, 2006; uetz et al., 2019). the mostly endemic reptile fauna of madagascar is still relatively poorly understood, despite significant progress in the last few decades (glaw and vences, 2006), with several new species described every year (uetz et al., 2019). a significant number of these new reptile species are known only from a few specimens, and despite researchers applying molecular barcoding techniques (nagy et al., 2012) with large numbers of comparative molecular sequences, the reptile diversity remains significantly underestimated. compared to other groups, only a small fraction of reptile species have been studied with cytogenetic methods, despite increasing evidence that species-level diversity is reflected at the karyotype level (e.g. mezzasalma et al., 2016, 2018; rovatsos et al., 2017). malagasy vertebrates display dynamic patterns of chromosome evolu138 marcello mezzasalma et alii tion including augmentation and reduction of the chromosome number and the independent diversification of sex chromosome systems (e.g. mezzasalma et al., 2017a; rovatsos et al., 2017), making the malagasy reptile fauna an exciting study system for evolutionary cytogenetics. the genus matoatoa includes two endemic malagasy gecko species, m. brevipes (mocquard, 1900) and m. spannringi nussbaum, raxworthy and pronk 1998. species of matoatoa are part of a clade including the southern african genera afrogecko, cryptactites, kolekanos and ramigekko, the australian genus christinus and the more distantly related genera afroedura, goggia, phelsuma, paroedura, ebenavia and uroplatus (heinicke et al., 2014). karyological data are currently available for christinus marmoratus and different species of phelsuma, paroedura, ebenavia and uroplatus (king and rofe, 1976; king and king, 1977; aprea et al., 1996, 2013). however, there are no available chromosome data from the genus matoatoa. here we present the results of the first karyological study on the endemic malagasy phantom gecko m. brevipes and a preliminary mitochondrial analysis to provide a taxonomic identification of the sample studied. finally, we compared our newly generated karyotype data to those from closely related gecko genera and hypothesise how karyotype evolution occurred in the group. we used a sample from a female specimen of m. brevipes collected 20 km south of tulear (the cell suspension and tissue sample are deposited in the natural history museum, london, uk, and in the department of biology, university of naples federico ii, with voucher ga436). the sample was preliminary treated with colchicine (1mg/ml; 0.1 ml/10 gr body weight) and, after two hours, tissue samples with high mitotic indices (intestine and gonads) were placed for 30 min in hypotonic solution (sodium citrate 0.50% + kcl 0.56%) and then transferred in carnoy’s fixative (methanol + glacial acetic acid, 3:1). we performed a phylogenetic analysis using a fragment of 561 bp of the mitochondrial gene nadh dehydrogenase subunit 2 (nd2). genomic dna was extracted using the standard phenol-chloroform method (sambrook et al., 1989). for the nd2 analysis we used the primers and pcr parameters reported in heinicke et al. (2014). after sequencing, the obtained chromatogram (accession number mt579309) was manually corrected and aligned with homologous nd2 segments of m. brevipes deposited in genbank using chromas lite 2.2.1 and bioedit 7.2.6.1 (hall, 1999). phylogenetic analysis was performed with bayesian inference (bi) using mrbayes 3.2.5 (ronquist et al., 2012), and maximum likelihood (ml) using mega 10.1.7 (tamura et al., 2013), with 2,000,000 generations and 1000 bootstrap replicates, respectively. in both analyses we included homologous nd2 sequences of representatives of phylogenetically closely related gecko genera: christinus alexanderi (kf666813), christinus guentheri (kf666801), cryptactites peryngueyi (kf666814), ramigekko swartgergensis (kf666793) and afrogecko porphyreus (kf666772). we used as outgroup two homologous sequences of the phylogenetically closely related kolekanos plumicaudus (kf666791, jx041304). in the cytogenetic analysis, chromosomes were prepared by the scraping + air drying method (see mezzasalma et al., 2016), and a combination of traditional staining (giemsa staining at ph7) and banding techniques (sequential c-banding + giemsa + cma3 + dapi; see mezzasalma et al., 2013, 2015, 2017b). in our phylogenetic analysis, the studied female (ga436) is the sister taxon of the other three available nd2 sequences of m. brevipes (kf666815, kf666816, and ef490777) (fig. 1). the uncorrected p-distance of ~4%, between our newly generated nd2 sequence and those available from genbank, highlights the occurrence of intraspecific diversity. in turn, the other three available nd2 sequences of the species do not show any appreciable nucleotide variability (fig. 1). potential explanations for the intraspecific divergence in m. brevipes may be related to geographical patterns of genetic diversity, however, all three nd2 sequences of the species available from genbank (kf666815, kf666816, and ef490777) are not associated with specific sampling localities (see nussbaum et al., 1998; heinike et al., 2014) and more fig. 1. phylogenetic tree with ml (1000 bootstrap replicates) and bi (2,000,000 generations), with our newly generated nd2 sequence (ga436) and available homologous sequences of matoatoa brevipes (kf666815, kf666816, ef490777). we included homologous sequences of christinus alexanderi (kf666813), christinus guentheri (kf666801), cryptactites peryngueyi (kf666814), ramigekko swartgergensis (kf666793), afrogecko porphyreus (kf666772) and kolekanos plumicaudus (kf666791, jx041304). bootstrap and bayesian posterior values are reported below and above tree branches, respectively. 139chromosome analysis of matoatoa brevipes data are needed to advance hypotheses on the intraspecific molecular diversity of m. brevipes. we found that m. brevipes has a karyotype composed of 2n = 34 chromosomes, with one metacentric pair (1st) and 16 telocentric pairs, gradually decreasing in length and without any clear distinction between macro and micro-chromosomes (fig. 2). sequential c-banding revealed a scarce heterochromatin content in the genome of m. brevipes, without any completely or largely heterochromatic chromosome (fig. 3). centromeric and telomeric heterochromatin was more easily viewed with c-banding + cma3. furthermore, the telomeric heterochromatin of two medium sized telocentric chromosomes was relatively brighter with cma3, suggesting the colocalization of the nor associated heterochromatin (fig. 3). in fact, c-banding + cma3 can be a useful technique to localize nors. unambiguous co-localization of cma3 signals after c-banding and ag-nor staining and norfish signals have been consistently documented in different papers and taxa (see e.g. pardo et al., 2001; suman and kaur, 2013). this is especially true for karyotypes showing low heterochromatin content like squamates and lacking other paired cma3+ heterochromatic blocks. karyological comparisons with closely related genera were possible with christinus marmoratus and different species of phelsuma, paroedura, ebenavia and uroplatus. christinus marmoratus has accentuated chromosome polymorphism, with different populations possessing different chromosome number (2n = 32, 34 and 36) and morphology, with or without differentiated zw sex chromosomes, and an invariable arm number (an) of 40 (king and rofe, 1976; king and king, 1977). christinus marmoratus probably includes cryptic species (kay, 2008), and the variable karyotypes found in different clades are similar to those found in other geckos of the same group in chromosome number (2n = 32-36) and the prevalence of telocentric elements, with one (as in m. brevipes and p. picta) or more bi-armed chromosomes occurring on different chromosome pairs. we superimposed the updated chromosomal data on the phylogeny of heinicke et al. (2014) to explore possible scenarios of chromosome diversification in the group (fig. 4). based on the distribution of karyotypes, we hypothesise a putative ancestral karyotype with 2n = fig. 3. c-banding + giemsa (a), cma3 (b) and dapi (c) on metaphase plates of the female specimen of m. brevipes ga436, showing low heterochromatin content and absence of completely or largely heterochromatic chromosomes. white arrows in (b) point at norassociated heterochromatic blocks. scale bar = 10 μm. fig. 2. giemsa stained karyotype of matoatoa brevipes with 2n = 34 chromosomes. fig. 4. (a) phylogenetic relationships of circum-indian ocean leaf toed geckos (redrawn from heinicke et al., 2014) superimposed with available haploid karyograms (king and rofe, 1976; aprea et al., 1996, 2013; this study). (b) karyotype evolutionary hypothesis and relative chromosome rearrangements. 140 marcello mezzasalma et alii 36 and all telocentric elements (an = 36). in fact, higher numbers of chromosomes and telocentric elements are considered primitive features of lizard karyotypes (see deakin and ezaz, 2019). furthermore, the hypothesised ancestral karyotype of 2n = 36 is the most common karyotype in the focal gecko species and is conserved in all uroplatus, ebenavia, and phelsuma species studied so far (aprea et al., 1996, 2013 and our unpublished data). starting from an ancestral 2n = 36 karyotype, multiple fusion and inversion events likely led to the formation of biarmed elements and the reduction of the chromosome number in different clades (fig. 4). in particular, one fusion between two medium sized telocentric pairs probably originated the karyotype of m. brevipes (with 2n = 34 and one metacentric pair). one inversion involving two primitive telocentric pairs may explain the karyotypic variation across species of paroedura (aprea et al., 2013; koubová et al., 2014) and a subsequent centric fusion probably shaped the karyotype of p. bastardi, reducing the chromosome number to 2n = 34 (see also aprea et al., 2013; koubová et al., 2014). in christinus marmoratus, two inversions involving pairs 7 and 18, likely formed the karyotype of 2n = 36 populations, and one and two centric fusions of telocentric elements formed the karyotype of 2n = 34 and 2n = 32 populations, respectively. similar chromosome rearrangements have been hypothesized in different phylogenetic lineages within the family gekkonidae, with distinct karyotype formulas progressively diverging from the hypothesized ancestral condition by means of independent chromosome fusions, fissions and inversions (see also trifonov et al., 2011; srikulnath et al., 2014). concerning sex determination systems, differentiated zw chromosomes are common in squamates, and have been observed in various paroedura species (koubová et al., 2014), including a complex z1z1z2z2/z1z2w sex chromosome system in p. gracilis (aprea et al., 2013). however, the female specimen of m. brevipes studied here did not possess heteromorphic or largely heterochromatic chromosomes. this suggests that the zw sex chromosomes are at an early differentiation stage, as has been observed in a 2n = 36 population of c. marmoratus (king and king, 1977). variability in sex chromosome morphology is not unexpected as squamate reptiles can have either xy or zw sex chromosomes and these conditions can evolve and differentiate rapidly, sometimes occurring within the same taxon (e.g. gamble, 2010; koubová et al., 2014; mezzasalma et al., 2019). our results indicate that the nor loci of m. brevipes are probably located on the telomeres of a medium sized telocentric chromosome pair. this contrasts with nor location in related taxa indicating a remarkable variability in their chromosomal localization in geckos. for example, nors are on the first chromosome pair in e. inunguis, on the last pair in paroedura and on different chromosome pairs in phelsuma and uroplatus (aprea et al., 1996, 2013 and our unpublished data), highlighting a high genomic mobility of these elements. in conclusion, the karyotype of m. brevipes is composed of 2n = 34 chromosomes with one metacentric and 16 telocentric pairs. comparison with the available karyotypes of closely related gecko genera allowed us to hypothesise an evolutionary scenario where a karyotype of 2n = 36 with all telocentric chromosomes was the ancestral state of the group. this ancestral state would necessitate chromosome diversification occurring by means of fusions and inversions, leading to a reduction of the chromosome number and the formation of biarmed elements. acknowledgements we thank gennaro aprea for providing us with the study sample. this project has received funding from the european union’s horizon 2020 research and innovation programme under the marie skłodowska-curie grant agreement no 797027. references aprea, g., odierna, g., capriglione, t., caputo, v., morescalchi, a., olmo, e. 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(2019): the reptile database, http://www.reptile-database.org, accessed [june 25, 2020]. acta herpetologica vol. 15, n. 2 december 2020 firenze university press estimating abundance and habitat suitability in a micro-endemic snake: the walser viper gentile francesco ficetola1,2,*, mauro fanelli3, lorenzo garizio3, mattia falaschi1, simone tenan4, samuele ghielmi5, lorenzo laddaga6, michele menegon7,8, massimo delfino3,9. potential effects of climate change on the distribution of invasive bullfrogs lithobates catesbeianus in china li qing peng1, min tang1, jia hong liao1, hai fen qing1, zhen kun zhao1, david a. pike2, wei chen1,* a bibliometric-mapping approach to identifying patterns and trends in amphibian decline research claudio angelini1,*, jon bielby2, corrado costa3 food composition of a breeding population the endemic anatolia newt, neurergus strauchii (steindachner, 1887) (caudata: salamandridae), from bingöl, eastern turkey kerim çiçek1,*, mustafa koyun2, ahmet mermer1, cemal varol tok3 stomach histology of crocodylus siamensis and gavialis gangeticus reveals analogy of archosaur “gizzards”, with implication on crocodylian gastroliths function ryuji takasaki1,2,*, yoshitsugu kobayashi3 does chronic exposure to ammonium during the pre-metamorphic stages promote hindlimb abnormality in anuran metamorphs? a comparison between natural-habitat and agrosystem frogs sonia zambrano-fernández1, francisco javier zamora-camacho2,3,*, pedro aragón2,4 confirming lessona’s brown frogs distribution sketch: rana temporaria is present on turin hills (piedmont, nw italy) davide marino1, angelica crottini2, franco andreone3,* phylogenetic relationships of the italian populations of horseshoe whip snake hemorrhois hippocrepis (serpentes, colubridae) francesco paolo faraone1, raffaella melfi2, matteo riccardo di nicola3, gabriele giacalone4, mario lo valvo5* first karyological analysis of the endemic malagasy phantom gecko matoatoa brevipes (squamata: gekkonidae) marcello mezzasalma1,2,*, fabio m. guarino3, simon p. loader1, gaetano odierna3, jeffrey w. streicher1, natalie cooper1 notes on sexual dimorphism, diet and reproduction of the false coral snake oxyrhopus rhombifer duméril, bibron & duméril, 1854 (dipsadidae: pseudoboini) from coastal plains of subtropical brazil fernando m. quintela1,*, felipe caseiro¹, daniel loebmann¹ acta herpetologica 16(1): 3-9, 2021 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-9758 microhabitat segregation of two coexisting tadpole species on emei mountain zijian sun1,2, chunlin zhao2,3, dan xu1,2, wenbo zhu2, wenbo fan2, tian zhao1,2,*, shengqi su1,* 1 college of fisheries, southwest university, chongqing 400715, china 2 cas key laboratory of mountain ecological restoration and bioresource utilization & ecological restoration biodiversity conservation key laboratory of sichuan province, chengdu institute of biology, chinese academy of sciences, chengdu 610041, china 3 key laboratory of bio-resources and eco-environment of the ministry of education, college of life sciences, sichuan university, 610064, chengdu, china *corresponding authors. e-mail: zhaotian@cib.ac.cn, sushengqi@swu.edu.cn submitted on: 2020, 19th september; revised on: 2021, 3rd march; accepted on: 2021, 8rd march editor: fabio m. guarino abstract. understanding mechanisms determining the coexistence between different species is one of the key issues in community ecology and biodiversity conservation. microhabitat segregation is a way for species to coexist, which reflects the specific habitat selection of coexisting species in a finer spatial scale. despite quantitative studies have been conducted to investigate the microhabitat segregation of coexisting species, this type of studies was not often performed on tadpoles. in this study, we assessed the habitat selection of two coexisting tadpoles (quasipaa boulengeri and leptobrachium boringii) in a stream on emei mountain, china. our results demonstrated that l. boringii and q. boulengeri tadpoles occupied different microhabitats. specifically, q. boulengeri tadpoles preferred deep, narrow, and weak acid stream segments with slow current velocity and low value of conductivity, while l. boringii tadpoles tended to occur in a wide, shallow water bodies with relatively higher ph, conductivity, and current velocity. overall, our study supported the hutchinson’s niche concept, showing that at least one dimension of niche differentiation (i.e., microhabitat) occurred between coexisting tadpole species. keywords. microhabitat selection, species coexistence, niche differentiation, environmental variables. introduction understanding mechanisms determining the coexistence between different species is one of the key issues in community ecology and biodiversity conservation (adler et al., 2010; hanane, 2015). previous studies indicated that coexisting species should occupy specific ecological niche (e.g., microhabitat niche, trophic niche; grinnell, 1917) to evade competition based on the limiting similarity theory (macarthur and levins, 1967). as ecological niche is an n-dimensional ecological space satisfying all the essential conditions that support the organisms (hutchinson, 1957), coexisting species exhibit at least one dimension of niche differentiation in the same ecosystem (caceres and machado, 2013; hanane, 2015). this phenomenon can be referred to as niche partitioning involved in several facets, such as temporal or spatial distribution, as well as the trophic habits (baker and ross, 1981; de andrade et al., 2014; schoener, 1974). therefore, hutchinson’s niche concept primarily focuses on habitats and resources utilization of sympatric species, as well as their environmental tolerances (rosenfeld, 2002). accordingly, the segregation of habitats can be considered as one of the important niche partitioning forms allowing the coexistence of species (melo et al., 2013; schoener, 1974; wei et al., 2000). for instance, 4 zijian sun et alii guo et al. (2012) indicated that two species of goby fish (rhinogobius giurinus and rhinogobius cliffordpopei) were introduced into lake erhai, china in 1961. they eventually had to adapt to a different ecological niche in order to coexist in the ecosystem. at a mesohabitat scale, habitat segregation is usually tested in physiognomically homogeneous units (heggenes and saltveit, 2007; kano et al., 2013; rezende et al., 2010). however, when focusing on species at a finer spatial scale, habitat segregation is usually assessed by measuring a set of environmental parameters (rincon, 1999). this can reflect exact habitat utilization of coexisting species in the same ecosystem, particularly in the aquatic ecosystems (jackson et al., 2001; jorgensen, 2004; leger et al., 1983; leitao et al., 2015). for instance, two turtle species coexist in freshwater streams in southwest of iberian peninsula due to their divergence in habitat selection. specifically, the european pond turtle (emys orbicularis) shows a preference for temporary, shallow, well vegetated, and sandy stream segment, while the mediterranean pond turtle (mauremys leprosa) tends to occupy deeper stream segment with more rocks (segurado and figueiredo, 2007). similar situation is also observed in two coexisting fish species in anizacate river. in this ecosystem, current velocity is the key factor that facilitates the coexistence of the stream catfish (trichomycterus corduvense) and the eel catfish (heptaterus mustelinus), who prefers the high-velocity and the low-velocity water flows, respectively (hued and bistoni, 2006). despite quantitative studies have been conducted to investigate the microhabitat segregation of coexisting species in different ecosystems (e.g., invertebrates, mammola et al., 2016; fish, horinouchi, 2008; kessler and thorp, 1993; and amphibian adults, ayala et al., 2018), empirical studies are still relatively rare in anuran tadpoles. tadpoles can exhibit plasticity in terms of functional traits (jordani et al., 2019; zhao et al., 2019), behavior (freitas et al., 2019; zhao et al., 2019), and metabolism (freitas et al., 2019; wang et al., 2019) in response to environmental change. in addition, tadpoles could influence the composition and abundance of plankton and periphyton, which has cascading effects on primary productivity in aquatic ecosystems (alford and wilbur, 1985; seale, 1980; strauss et al., 2010). more importantly, tadpoles are the larval stage of amphibian adults, which face multiple threats (e.g., habitat loss, climate change, and pollution, alford, 2011). therefore, identifying the microhabitat selection of coexisting tadpoles may bring important insights to tadpole biodiversity conservation, and suggest priorities for the improved management of mountain stream ecosystems. in the present study, we evaluated the habitat selection of two coexisting tadpoles, quasipaa boulengeri and leptobrachium boringii in a mountain stream. specifically, we first compared the difference of variables of microhabitat occupied by l. boringii and q. boulengeri tadpoles. we then explored the distribution pattern of the two tadpoles in the stream. based on previous studies (e.g., winston, 1995; xu et al., 2020), we predict that the coexisting tadpoles of two different species occupy different microhabitat. material and methods study area and species description field work was carried out in heilongjiang stream, qingyinge of emei mountain, sichuan province, china (fig. 1). the vegetation of this area is mostly composed by evergreen broadleaved forest. the elevation of this region is about 680m a.s.l., and the weather is characterized by subtropical monsoon climate. the mean annual temperature and the mean annual precipitation is about 17.29 °c and 1555.3 mm (gu and li, 2008; ling, 2005). several tadpoles belonging to different species were detected during our field work in this stream (e.g., megophrys omeimontis, megophrys minor, odorrana graminea, q. boulengeri, l. boringii, leptobrachella oshanensis, and odorrana schmackeri). the dominant species were l. boringii and q. boulengeri, which occupied 90% of the proportion of the individuals. we focused on the divergences in habitat selection occurring between these two dominant species, as it would be more physiological than that showing how non-dominant species can coexist (lyons et al., 2005; barrett et al., 2008). data collection based on the distribution of target tadpoles and accessible for the sampling, one kilometer of the segment of the stream was selected as the transect. the transect was fixed and extended through a gorge, with strong variability in its physical variables due to the complex terrain and different vegetation cover rate. we divided this transect into three parts based on the blocking of rocks (i.e., approximate 200m of the upstream transect, 450 m of the medium-stream transect, and 350 m of the downstream transect). tadpole sampling was carried out after sunset (between 20:30 and 23:00) from 22nd to 27th in august 2018. we searched at the both edges (about 1m from the bank) of the stream where the tadpoles distributed, with one part of one side being sampled per night. specifically, we divided each side of the transect into 1000 squares (1 × 1 m), and two persons searched these plots of 1 m2 intensively from downstream to upstream using torch (220 lm). once the target tadpoles were detected, the related square was recorded as one valid quadrat. after that, all the tadpoles located in the quadrat were collected by sweeping all potential microhabitats for tadpoles (i.e., water column and edge of rocks with and without vegetation) using hand nets (mesh size: 2 mm). we assumed that tadpoles likely did not have chances to move from one side to the other 5microhabitat segregation of tadpoles of the river, or to cross the rocks limiting the sections during the survey period. the two target tadpoles can be easily identified based on their external morphology. specifically, there is a light “y” symbol at the back of l. boringii tadpole, while q. boulengeri tadpole has flat head and back, thick caudal muscle, and i:4+4/1+1:ii labial tooth row (fei et al., 2012, fig. s1). collected tadpoles were kept in 500 ml plastic bottles with freshwater from the stream separately. the stage of each tadpole was determined based on gosner (1960). in each quadrat where we found tadpoles, a set of eight environmental factors were measured. details of the factors and the related measurement approaches are as follows: substrate type was divided into two groups (i.e., gravel and a mix of gravel and humus, associating with different food resources in the quadrats), water temperature was measured to the nearest 0.1 °c by a thermometer (ktj ta318, china, shenzhen), river width was measured to the nearest 0.1 m using a tape meter, the maximum depth of the quadrat was measured to the nearest 0.1 cm with a ruler, current velocity was measured to the nearness 0.1 m/s using a portable current meter (ls1206b, china, nanjing), ph, conductivity (to the nearest 0.1μs/cm) and dissolved oxygen (to the nearest 0.1 mg/l) were measured using a portable fluorescence photometer (star a, 520m 01a, thermo fisher scientific, usa). during the measurements, the researcher remained outside the stream to do not affect the recorded parameters (ferreira et al., 2015). statistical analyses shapiro-wilk test was used to test the normality of the seven environmental variables of microhabitats. in order to compare the difference of variables of microhabitat occupied by l. boringii and q. boulengeri tadpoles, we conducted student’s t tests for variables which followed a normal distribution, or mann-whitney u test for those were not normal distributed. after that, we used detrended correspondence analysis (dca) to explore the distribution pattern of the two tadpoles (linear model or single peak model). considering the dca axis lengths is less than 3 (i.e., the species distribution was fitted well with the linear model), we finally chose redundancy analyses (rda) to quantify the environmental determinants of the distribution of the two target tadpoles. all statistical analyses were performed in r version 3.6.1 (r development core team 2020) using the packages stats, spaa, and vegan. results in total, 27 quadrats were sampled and measured for environmental variables, in which 13 quadrats were occupied by l. boringii tadpoles, and 14 quadrats were occupied by q. boulengeri tadpoles. these two tadpoles were not detected at the same time in each of the sampled quadrat. we overall captured 74 individuals of l. boringii from stage 24 to 37 and 193 individuals of q. boulengeri from stage 25 to 43. student’s t test indicated that there was no significant difference of dissolved oxygen (t = -1.674, p = 0.107) in the microhabitats that were occupied by l. boringii and q. boulengeri tadpoles. mann-whitney u tests revealed that ph, conductivity, river width, water depth, and current velocity were significantly different (p < 0.05; fig. 2; substrate type cannot be tested as it was a categorical variable). specifically, l. boringii tadpoles occupied quadrats that had higher values of ph, conductivity, river width, and current velocity, but lower values of water depth. in contrast, q. boulengeri tadpoles occupied quadrats that had lower values of ph, conductivity, river width, fig. 1. geographical location of the study area. the solid line describes the range of emei mountain. 6 zijian sun et alii and current velocity, but higher values of water depth. in addition, no significant difference existed in terms of water temperature values (w = 93.5; p > 0.05). the rda model was significant when testing the environmental determinants of the distribution of l. boringii and q. boulengeri tadpoles (p = 0.001). the first two axes accounted for 65.75% of the variation (62.45% and 3.28%, respectively). our results revealed that five environmental factors including substrate type, river width, current velocity, ph, and conductivity had highly significant effects on the distribution of l. boringii tadpoles and q. boulengeri tadpoles (p = 0.001, table 1), and the influence of water depth was significant (p < 0.05, table 1). obvious dissimilarity of the distribution of l. boringii and q. boulengeri tadpoles could be observed in the rda sequence diagram (fig. 3). specifically, most of the quadrats occupied by l. boringii tadpoles were distributed in the second and third quadrant densely, which were positively associated with current velocity, river width, ph, conductivity, and substrate type. however, they were negatively correlated with water depth. in contrast, most of the quadrats occupied by q. boulengeri tadpoles exhibited opposite distribution pattern, which were positively correlated with water depth, and negatively associated with current velocity, river width, ph, conductivity, and substrate type. discussion discrepant preferences for microhabitat utilization on a small scale is often considered to be responsible for the fig. 2. comparison of environmental variables of microhabitats between l. boringii tadpoles and q. boulengeri tadpoles. “leb”: l. boringii tadpoles, “qub”: q. boulengeri tadpoles. table 1. the influence of eight environmental factors tested by rda analyses on the two tadpole species. environmental variables rda1 rda2 r2 p ph -0.918 0.397 0.524 0.001 conductivity -0.968 0.252 0.573 0.001 dissolved oxygen 0.997 0.082 0.139 0.160 water temperature -0.650 0.760 0.012 0.869 river width -0.847 0.531 0.596 0.001 water depth 0.793 -0.609 0.266 0.014 current velocity -0.737 0.676 0.600 0.001 substrate type -0.799 0.600 0.632 0.001 significant effects are indicated in bold. fig. 3. redundancy analyses of the relationships between environmental factors and the distributions of model tadpoles. the length of an environmental vector indicates the degree of correlations. only significant variables are depicted (p < 0.05), “leb”: l. boringii tadpoles, “qub”: q. boulengeri tadpoles. 7microhabitat segregation of tadpoles coexistence of sympatric species (dammhahn and goodman, 2014; dammhahn et al., 2013; escoriza et al., 2018; wei et al., 2000; yang et al., 2019). our results demonstrated that despite q. boulengeri and l. boringii tadpoles co-occurred in the same stream, microhabitat segregation existed between them. specifically, l. boringii tadpoles tended to occur in a wide, shallow water bodies with relatively higher ph, conductivity, and current velocity, which was consistent with previous study (wei et al., 2017). in contrast, q. boulengeri tadpoles were apt to occupy deep, narrow stream segments with low ph, conductivity and current velocity. therefore, our results indicated that these two tadpoles occupied totally different microhabitat in this stream. indeed, due to the poor movement ability, tadpoles’ microhabitats are largely determined by the selection of breeding habitats by breeding adults (biesterfeldt et al., 1993). therefore, the occupation of the microhabitat of the two tadpole species were in accordance with previous observation showing that female q. boulengeri spawned in puddles under the stream waterfall, while female l. boringii spawned at streams segments with more rocks and slow water flow (fei et al., 2012). it has been recognized that tadpole functional traits were correlated with their selection of microhabitats in water bodies (fatorelli et al., 2015; glos et al., 2017), as well as other factors (e.g., predatory occurrence and strategy, mogali et al., 2020). based on our previous study (xu et al., 2020), these two tadpole species have distinct phenotypic functional traits, which also reflected their adaptation of different environment in the water bodies (zhao et al., 2017). specifically, q. boulengeri tadpoles have flattened bodies and stubby tails, associating with their selection of deep and slow flowing water bodies. l. boringii tadpoles have long and muscular tails, which can be useful for them to swim when water velocity is high. these external functional traits reflect the food acquisition and locomotion of tadpoles, which are critical for them to obtain nutrients to survive, and to facilitate the movement in water bodies (schoenfuss and blob 2007). in the present study, substrate type was one of the main environmental factors determining the distribution of the two tadpoles, which probably related to their feeding preferences. that is, q. boulengeri tadpoles tended to choose a substrate with gravels which can provide them adherent algae and benthos. in contrast, l. boringii tadpoles preferred a mixture of humus and gravels where organic detritus and invertebrates are more abundant. however, more work such as stable isotope analyses are needed to verify our inferences. overall, the present study evaluated the microhabitat segregation of two sympatric tadpole species in a mountain stream. our observations supported hutchinson’s niche theory demonstrating that the existence of at least one dimension of niche differentiation between coexisting species. therefore, specific microhabitat features should be incorporated into the conservation strategies for different species. beyond our studies, future work could focus on the roles that coexisting species played in communities and ecosystems. furthermore, as mountain streams are vulnerable to anthropogenic disturbance, how increasing fishing pressure on tadpoles from tourists may affect ecosystem functioning can be also tested. acknowledgements we thank janak r. khatiwada, xiaoyi wang, shengnan yang, xuecheng guo, and chunpeng guo for their help during the field work. we also thank the handling editor fabio m. guarino and two anonymous reviewers for their helpful suggestions that greatly improve this manuscript. this work is supported by national natural science foundation of china (31700353), the second tibetan plateau scientific expedition and research program (step), grant no. 2019qzkk0501, and china biodiversity observation networks (sino bon). the animal care and use committee of chengdu institute of biology, cas provided full approval for this study (number: cib2011032201). field work was approved by the management o8ce of the emei mountain management committee (project number: ems-201304006). the raw data of this study can be found in figshare (https://figshare. com/s/2130bf68ad78958e9492). supplementary material supplementary material associated with this article can be found at <htttp://www.unipv.it/webshi/appendix> manuscript number 9758 references adler, p.b., ellner, s.p., levine, j.m. 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(2019): effects of urea on behavior and functional traits of asiatic toad (bufo gargarizans) tadpoles. aquatic ecol. 53: 9-19. acta herpetologica 14(2): 117-122, 2019 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/a_h-7749 ontogenetic and interspecific variation in skull morphology of two closely related species of toad, bufo bufo and b. spinosus (anura: bufonidae) giovanni sanna naturalis biodiversity center, p.o. box 9517, 2300 ra leiden, the netherlands department of biological, geological, and environmental sciences, university of bologna, via selmi 3, 40126 bologna, italy marine research department, senckenberg am meer, südstrand 40, 26382 wilhelmshaven, germany e-mail: giovanni.sanna3@studio.unibo.it submitted on: 2019, 31st january; revised on: 2019, 6th june; accepted on: 2019, 5th august editor: marcello mezzasalma abstract. using micro-ct and 3d landmark-based geometric morphometrics, i investigated postmetamorphic shape variation in the skull of bufo bufo and bufo spinosus, two widespread european toad species with small phenotypic differences. two ontogenetic series were compared, for a total of 58 individuals. they exhibited similar allometric growth patterns, characterised by cranial widening with relative shortening and dorsoventral compression. however, some interspecific shape divergence was observed, particularly among adults: a relatively shorter skull and a more dorsally extended snout distinguished b. spinosus from b. bufo. this disparity, which gives further support to species separation, can probably be ascribed to changes in the allometric trajectories, and seen in light of the evolutionary history of the two lineages. keywords. allometry, bufo bufo, bufo spinosus, divergence, geometric morphometrics, landmark, ontogeny, skull shape. introduction divergent traits can be expected to be found in closely related species with a broad geographical distribution, relatively to ecological and climatic variation, but developmental constraints may also play an important role in restricting or channelling phenotypic evolution (cvijanović et al., 2014; ivanović and arntzen, 2018). the common toad, bufo bufo (linnaeus, 1758), and the spined toad, bufo spinosus daudin, 1803, are members of the common toad species group of the western palaearctic (arntzen et al., 2013b). b. bufo has a wide eurasian distribution that comprises northern and eastern france, central and southern europe (including sardinia; cossu et al., 2018), and stretches northwards into scandinavia and eastwards deep into russia; b. spinosus is found in the iberian peninsula, western and southern france, and north africa, from morocco to tunisia (arntzen et al., 2013a). their lineages have diverged around 9 ma (million years ago; recuero et al., 2012), but in contrast to a deep genetic differentiation, b. bufo and b. spinosus appear phenotypically similar (arntzen et al., 2013a). whereas a few diagnostic characters were described for the external morphology, virtually nothing is known about interspecific osteological differences, neither in the postcranial nor in the cranial skeleton. such a complex structure as the skull is of particular interest in a wide range of studies, due to its fundamental biological functions and the fact that it often undergoes adaptive variation (ivanović et al., 2012). substantial changes are 118 giovanni sanna known to occur in the anuran skull after metamorphosis (ponssa and candioti, 2012). the purpose of this investigation was to highlight potential interspecific differences in the skull morphology of b. bufo and b. spinosus, in the context of postmetamorphic development, using a geometric morphometrics approach. the analysis of ontogenetic shape variation in these two species is interesting not only for a taxonomic evaluation based on morphology; it can provide insights into their morphological evolution, to be interpreted in the context of their evolutionary history and past distribution. moreover, this analysis could contribute to a better understanding of the interplay between ontogeny and morphological differentiation among anuran species. material and methods a total of 58 alcohol-stored specimens, including freshly metamorphosed, juvenile, and adult (male and female) toads, were analysed (table s1). these toads had been collected from various populations in different localities of the iberian peninsula (b. spinosus), france (b. spinosus, b. bufo), and the netherlands (b. bufo). they were subdivided into two ontogenetic series, made up of 28 bufo bufo and 30 bufo spinosus individuals, respectively. body size in the whole sample ranged from 16.0 mm to 78.0 mm sul (snout-urostyle length). mature b. spinosus individuals were larger on average (mean sul 71.5 mm) than b. bufo ones (mean sul 60.4 mm), reflecting size disparity between western european common toads and spined toads (cvetković et al., 2009). three-dimensional imaging skulls were ct-scanned with two x-ray machines: skyscan 1172 (bruker, kontich, belgium) and zeiss xradia 520 versa (carl zeiss xrm, pleasanton, ca, usa). the former was used for the smaller toads, with 0.5 mm aluminium filter, 2k resolution (2000 × 1336), pixel size of 13.17 µm, voltage of 29-54 kv, exposure time of 420-750 ms, 0.4 rotation step, averaging of four frames. voltage and exposure time were modified when scanning specimens of different sizes (which showed differences in skull density), in order to keep image quality consistent. xradia scanner was employed for adult individuals, mainly due to its larger sample holder; resolution was set at 1k (1000 × 1024), with pixel size of 34.18 µm, and voltage of 80 kv. scanning was followed by two-dimensional reconstruction of raw image data into stacks, which were processed with avizo 9 software (fei sas, france): the segmentation editor was used to segregate homogeneous volumes (corresponding to skull bones), with manual adjustment of masking. notable variation in the extent of cranial ossification, not merely restricted to small juveniles, was observed at this point. a 3d surface model of the skull was then generated, applying a variable degree of unconstrained smoothing according to ossification extent. landmarks thirty-one homologous landmark points were collected on each 3d surface model to describe overall skull shape (fig. 1), using landmark editor 3.6 (institute for data analysis and visualization, university of california, davis, 2007). fifteen points were bilateral and symmetric, while one was median. anatomical description of points is provided in table s2. incomplete skull ossification of small juveniles made it challenging, at times, to perform an accurate placement of homologous landmarks (zelditch et al., 2004). geometric morphometrics morphometric and statistical analyses based on the landmark coordinates, and qualitative observation of the associated shape changes, were carried out with morphoj 1.06 software (klingenberg, 2011). a generalized procrustes analysis (gpa), consisting of a full procrustes superimposition for object symmetry, was applied: the symmetric components of shape variation, as a measure of skull shape, and centroid size, as a measure of skull size, were computed for each individual (klingenberg, 2016). the covariance matrix of shape variables was then generated and used to perform a principal component analysis (pca), in order to explore overall patterns of shape variation. shape changes in relation to growth were evaluated with a multivariate regression of shape (symmetric components, i.e. dependent variables) on size (log-transformed centroid size, i.e. independent variable), one for each ontogenetic series (monteiro, 1999), in association with a permutation test against independence between dependent and independent variables (made up of 10⁴ randomization rounds). the angle between the two regression vectors was calculated for comparison, including a test against randomness of vector directions in shape tangent space. interspecific morphological distinction was assessed by performing a discriminant function analysis (dfa) with leaveone-out cross-validation (lachenbruch, 1967; webster and sheets, 2010) on shape data of the 25 largest individuals, 12 b. bufo and 13 b. spinosus toads comprising adults and subadults. such a subsample was selected in order to maximize interspecific differences, since the pca had previously shown shape divergence in late stages of growth (fig. 2). results of this analysis were compared with those of a dfa on the remaining individuals of the sample, namely juveniles. both analyses included a parametric t-square test against the null hypothesis of equal group means, and a permutation test for the t-square statistic with 10,000 randomization rounds. results the first two principal components (pc1, pc2) of the pca accounted for 71.9% of total shape variation. pc1 alone grouped 65.4% of variation and was positively correlated with size: therefore, the scatter plot of pc2 119skull shape variation in bufo toads vs. pc1 can be looked at as a morphospace that contains the ontogenetic shape trajectories of b. bufo and b. spinosus (fig. 2). cranial shape variation along pc1 was characterised, in the positive direction, by a broadening (at the level of the jaw joint), an overall shortening (at the level of both the exoccipital and the premaxilla), and dorsoventral compression. some interspecific variation was comprised in pc2, especially for the largest toads; positive direction shape changes along this axis were an increase in skull length, dorsoventral compression, and a slight widening. both regression analyses found a significant association between shape and size (p < 0.0001 for both), indicating allometric growth: 63.8% of shape variation in b. bufo and 68.5% of shape variation in b. spinosus were predicted by regression on size (fig. 3). the two vector directions formed an angle of 17.2° (whereas 0° denote complete correspondence, 90° maximum divergence) and were not random in the shape tangent space (p < 0.00001). the major shape changes associated with regression were corresponding between the two species and matched those related to the first principal component of the pca, thus confirming the correlation of pc1 with skull size. discriminant function analysis applied to the largest toads showed a clear distinction between the two species (fig. 4): after cross-validation, 10/12 b. bufo individuals were reassigned to their true group and 2/12 were allocated to the spined toad group, while 13/13 b. spinosus individuals were reassigned to their own group (cohen’s k = 0.84). however, the t-square test for the difference between group means was not statistically significant (t² = 281, p = 0.82). permutation produced a significant result instead (p < 0.0001). the major interspecific shape differences pointed out by the analysis concerned cranial length and height: b. spinosus exhibited a longer upper jaw (with a more posterior jaw joint), yet a shorter fig. 1. landmark positions on the digitized skulls, in dorsal (a), right lateral (b), and frontal (c) views. for the anatomical description of landmarks, see table s2. fig. 2. ontogenetic shape variation of b. bufo and b. spinosus, described by the scatter plot of the first two principal components (with 95% confidence ellipses). pc1 summarises allometric variation, while pc2 displays species divergence in late development. 120 giovanni sanna skull (due to a shorter occipital region), with a dorsally expanded snout; skull width did not show noteworthy variation. dfa on juveniles yielded similar results, with a weaker interspecific distinction (fig. 4): 13/16 b. bufo and 14/17 b. spinosus toads were reallocated to their group (k = 0.63); t-square test was not significant (t² = 790.5, p = 0.72), while permutation test was significant (p < 0.0001). discussion most of the shape variation in the whole dataset was explained by differences in size, as shown by the concordant results of principal component and regression analyses. therefore, i suggest that ontogenetic allometry (i.e., the association between morphological changes and ontogenetic growth; klingenberg, 2016) characterises skull development of both bufo bufo and bufo spinosus, mainly in the form of a considerable widening (typical of skull growth in frogs; ponssa and candioti, 2012), and a relative shortening and dorsoventral flattening. cranial allometry has already been recognized in other anuran species, both in larval (e.g., in rana sylvatica; larson, 2002) and post-metamorphic development (e.g. in rhinella marina and the leptodactylus fuscus group; birch, 1999; ponssa and candioti, 2012). allometric constraints are likely to limit phenotypic evolution of the skull, even in presence of selective pressure (simon et al., 2016). this kind of scenario can be logically applied to the current study, which found shape disparities between fig. 3. shape changes of b. bufo (a) and b. spinosus (b) in relation to size, described by the scatter plot of regression scores against logtransformed centroid size. fig. 4. interspecific distinction, described by dfa histograms in which light grey bars represent b. bufo and dark grey bars represent b. spinosus. species assignment and discriminant scores are shown before (a, c) and after (b, d) cross-validation, for the largest individuals (a, b) and juveniles (c, d). 121skull shape variation in bufo toads b. bufo and b. spinosus that are moderate if compared to the common allometric variation. nevertheless, there seems to be a shift in the ontogenetic trajectories of the two species, highlighted by both the principal component analysis (fig. 2) and the angle between regression vectors (17.2°), and reflected in the morphological distinction provided by the discriminant function analyses (fig. 4). along with both latter analyses, t-square test against equal species mean shapes yielded significant results only after permutation, which probably compensated for the relatively small sample size. interspecific divergence, however, resulted higher in the group of largest individuals: they showed greater agreement to true species membership, as described by a k coefficient of 0.84 against the 0.63 of juveniles. consequently, it might be easier to discriminate between b. bufo and b. spinosus at mature stages, rather than among young individuals. this would be in contrast with the findings from other studies on amphibians, which pointed out a decrease in interspecific disparity over ontogeny (adams and nistri, 2010; ponssa and candioti, 2012). dfa and, to some extent, pca, indicate that b. bufo toads have on average a longer skull and a more dorsally compressed snout than b. spinosus toads. this morphological divergence could theoretically be placed within either the non-allometric or the allometric regime. variation in non-allometric shape would imply some sort of relaxation of ontogenetic constraints, which seems very unlikely, as the present evidence suggests that these constraints are strong in both species. alternatively, interpreting interspecific variation as comprised in the allometric framework should better reflect the results of the analyses. allometric variation could have arisen in two ways. first, from changes in developmental timing of the ancestral shape trajectory, with a conserved shapesize relationship (ontogenetic scaling hypothesis; strelin et al., 2016); however, this also seems unlikely, because the divergent skull shapes do not correspond to different stages of the same trajectory. secondly – and more plausibly – divergent evolution of the ontogenetic programme in the two lineages may have occurred, with a conserved direction of early post-metamorphic shape trajectories. the latter hypothesis would imply that interspecific differences have arisen during the long-lasting separation of the lineages (about 9 ma), and it could even be surprising that they are not more pronounced; as a possible explanation for this moderate divergence, a recent morphological evolution, following rapid postglacial european expansion of b. bufo from its balkan refugium, cannot be excluded (recuero et al., 2012; arntzen et al., 2016). climate is thought to have an indirect influence on skull morphology, because it determines food type and availability (simon et al., 2016), and changes in these ecological factors may lead to skull shape divergence – even in closely related species. alternative climate-driven factors could also induce skull differentiation, such as reproduction sites (water pools) occurrence, and the ability of toads to detect them, which involves the olfactory capsules in the snout region (trueb, 1993; simon et al., 2016). whether such factors are related to the interspecific differences found here – some of which concern the snout, more expanded in b. spinosus – it is not possible to say. arntzen et al. (2013a) hypothesized that in european toads a larger body size, a more pronounced presence of keratinous spines on the cheek warts and a wider head shape might favour defence against predators, namely grass snakes. although b. spinosus apparently meets these requirements more than b. bufo, relative cranial width did not show significant interspecific disparity in the current study. thus, no link was found between widely divergent parotoid glands, a distinctive trait of b. spinosus, and a wider skull. nevertheless, spined toads could compensate by attaining a wider skull through their bigger size. for further assessments of drivers and extent of morphological divergence between b. bufo and b. spinosus, it would be convenient to use a larger sample, especially for adult individuals, in order to properly account for the role of sexual dimorphism and benefit from the highest disparity. different populations should be considered, since b. bufo exhibits remarkable variation across its wide range; for instance, mediterranean common toads appear to resemble spined toads in body size – large – and skin texture – thick and warty (de lange, 1973; cvetković et al., 2009; arntzen et al., 2013a). acknowledgements this study was conducted in association with naturalis biodiversity center. i would like to thank marta calvo (museo nacional de ciencias naturales) and esther dondorp (naturalis biodiversity center) for access to the material under their supervision, ana ivanović for precious suggestions on study design, rob langelaan for technical support with computed tomography, and pim arntzen and two anonymous reviewers for valuable feedback and remarks on earlier versions of the manuscript. supplementary material supplementary material associated with this article can be found at <http://www.unipv.it/webshi/appendix> manuscript number 24709. 122 giovanni sanna references adams, d.c., nistri, a. 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(2004): geometric morphometrics for biologists: a primer. elsevier/academic press, amsterdam. acta herpetologica vol. 14, n. 2 december 2019 firenze university press podarcis siculus latastei (bedriaga, 1879) of the western pontine islands (italy) raised to the species rank, and a brief taxonomic overview of podarcis lizards gabriele senczuk1,2,*, riccardo castiglia2, wolfgang böhme3, claudia corti1 substrate type has a limited impact on the sprint performance of a mediterranean lizard pantelis savvides1,*, eleni georgiou1, panayiotis pafilis2,3, spyros sfenthourakis1 coping with aliens: how a native gecko manages to persist on mediterranean islands despite the black rat? michel-jean delaugerre1,*, roberto sacchi2, marta biaggini3, pietro lo cascio4, ridha ouni5, claudia corti 3 pit-tags as a technique for marking fossorial reptiles: insights from a long-term field study of the amphisbaenian trogonophis wiegmanni pablo recio, gonzalo rodríguez-ruiz, jesús ortega, josé martín* occurrence of batrachochytrium dendrobatidis in the tensift region, with comments on its spreading in morocco ait el cadi redouane1, laghzaoui el-mustapha1, angelica crottini2, slimani tahar1, bosch jaime3,4, el mouden el hassan1,* hematological parameters of the bolson tortoise gopherus flavomarginatus in mexico cristina garcía-de la peña1,*, roger iván rodríguez-vivas2, jorge a. zegbe-domínguez3, luis manuel valenzuela-núñez1, césar a. meza herrera4, quetzaly siller-rodríguez1, verónica ávila-rodríguez1 ontogenetic and interspecific variation in skull morphology of two closely related species of toad, bufo bufo and b. spinosus (anura: bufonidae) giovanni sanna visible implant alphanumeric (via) as a marking method in the lesser snouted treefrog scinax nasicus andrea caballero-gini1,2,3,*, diego bueno villafañe2,3, lía romero2, marcela ferreira2,3, lucas cañete4, rafaela laino2, karim musalem2,5 morphological variation of the newly confirmed population of the javelin sand boa, eryx jaculus (linnaeus, 1758) (serpentes, erycidae) in sicily, italy francesco p. faraone1,*, salvatore russotto2, salvatore a. barra3, roberto chiara3, gabriele giacalone4, mario lo valvo3 variability in the dorsal pattern of the sardinian grass snake (natrix natrix cetti) with notes on its ecology enrico lunghi1,2,3,4,*, simone giachello5, manuela mulargia6, pier paolo dore7, roberto cogoni8, claudia corti1 estimating abundance of the stripeless tree-frog hyla meridionalis by means of replicated call counts federico crovetto, sebastiano salvidio, andrea costa* at-rich microsatellite loci development for fejervarya multistriata by illumina hiseq sequencing yan-mei wang, jing-yi chen, guo-hua ding*, zhi-hua lin acta herpetologica 13(2): 165-169, 2018 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-23290 discovery of an italian slow worm (anguis veronensis pollini, 1818) population on a western mediterranean island confirmed by genetic analysis julien renet1,*, daniela lucente5, michel delaugerre2, olivier gerriet3, grégory deso4, chiara abbattista5, roberta cimmaruta5 1 conservatoire d’espaces naturels de provence-alpes-côte d’azur, pôle biodiversité régionale, 96, rue droite, 04200, sisteron, france. *corresponding author. e-mail: julien.renet@cen-paca.org 2 conservatoire du littoral, résidence st marc, 2, rue juge falcone, 20200, bastia, france 3 muséum d’histoire naturelle de nice, 60, boulevard risso, 06300, nice, france 4 association herpétologique de provence alpes méditerranée, hameau du nivernais, 84100, orange, france 5 department of ecological and biological sciences, tuscia university, largo dell’università snc, 01100, viterbo, italy submitted on: 2018, 24th may; revised on: 2018, 19th august; accepted on: 2018, 13th september editor: daniele pellitteri-rosa abstract. the genus anguis is known to be mainly continental in the mediterranean area, and accordingly it has never been recorded in western mediterranean islands. here we report for the first time the presence of the slow worm in a western mediterranean island, the ile sainte-marguerite from lérins archipelago (southeastern france). the molecular analysis of nd2 and prlr genes assigned the specimens to a. veronensis pollini, 1818 and showed that they are genetically related to the mainland population from les mayons, in mainland france. keywords. anguis veronensis, lérins archipelago, haplotype network. phylogenetic classification of the genus anguis (reptilia: anguidae) has undergone deep changes over the last years. although morphologically identical, four widespread species were genetically identified from the western palearctic: a. fragilis linnaeus, 1758, a. graeca bedriaga, 1881, a. colchica (nordmann, 1840) and a. cephallonica werner, 1894 (gvoždík et al., 2010). such pattern of genetic differentiation is likely the result of geographic barriers (mainly mountain systems) leading to the isolation of allopatric populations in the area, according to the “refugia within refugia” model (jablonski et al., 2016). more recently, slow worms from the italian peninsula have been recognized as a fifth separate species (former a. fragilis becoming a. veronensis pollini, 1818) according to strong genetic divergence and morphological differentiation of the sampled specimens (gvoždík et al., 2013). while contact zones have been discovered between a. fragilis and a. veronensis in eastern italy and slovenia (gvoždík et al., 2013), the distribution limits of a. veronensis, as well as sympatric areas with a. fragilis, remain poorly known in the western part of the range, despite the relatively high number of samples genetically studied (fig. 1). in the mediterranean area, the genus anguis is known to be mainly continental with some insular populations (table 1) and accordingly it was never recorded in western mediterranean insular environments (including all tyrrhenian, baléaric and galite islands, delaugerre and cheylan, 1992; cabela, 1997; turrisi and vaccaro, 1998; galan, 2002; vanni and nistri, 2006; zanghellini, 2006; sindaco and jeremčenko, 2008; geniez and cheylan,  2012; ineich, 166 julien renet et alii 2012; silva-rocha et al., 2018). however, the presence of an island population was signaled in may, 2011 (renet and martinerie, unpublished data) on ile sainte-marguerite (ism, 43°31’25”n, 7°02’43”e), the largest (210 ha) and most forested island in the lérins archipelago, located along the southeastern french coasts (fig. 1). this discovery raises several questions concerning not only species assignment but also the origin of this population. to answer these questions, a field mission was conducted in may 2016 on ile sainte-marguerite. six individuals (3 adult females, 1 adult male and 2 subadults) were collected (under tree stumps) inside a high forest composed of aleppo pine pinus halepensis miller, 1768 and holm oak quercus ilex linnaeus, 1753 in the moat shaded at the “fort sainte-marguerite” (a historical military construction) and in small fresh adjacent valleys. the presence of numerous decaying woods and a thick layer of litter provide optimal ecological condition for this anguis population (fig. 2). tissue fragments of 1 mm in length were sampled by tail clipping and stored in eppendorf tubes filled with ethanol (96%). total genomic dna was extracted using a ctab protocol slightly modified from doyle and doyle (1987). the two genes targeted for the molecular analysis were the mitochondrial nadh dehydrogenase subunit 2 (nd2) and the nuclear prolactine receptor (prlr), to obtain results comparable with those reported in recent literature (gvoždík et al., 2013; jablonski et al., 2016). in literature, nd2 gene was sequenced within a 1428 bp long fragment comprising also transfer rna genes (gvoždík et al., 2010, 2013). to target the nd2 gene only, we specifically designed two primer pairs using the software primer3 v. 4.1.0: anf1  (5’acaaaatacttcctcacacaagca-3’) and anr1  (5’gagtatgaaagtcgyaggtagaag-3’); anf2  (5’tgagcccyataattacctcaatct-3’) and anr2 (5’gggccatrttttgtgytagtagyt-3’). for the prlr gene we used the primers prlr_f1 and prlr_r3 (townsend et al., 2008), as reported by gvoždík et al. (2013). pcr conditions were those reported in cimmaruta et al. (2015) and lucente et al. (2016). purification and sequencing reactions were outsourced to macrogen inc. (www.macrogen.com). the sequences obtained were deposited in genbank (mh316862-5). to assign the obtained sequences to a species of anguis, the recovered nd2 and prlr haplotypes were compared to the homologous sequences of a. fragilis, a. veronensis, a. colchica, a. graeca and a. cephallonica recovered in genbank through a megablast search (clark et al., 2016). alignments were performed using the software mega v. 6.0 (tamura et al., 2013). table 1. location of the currently known island populations of slow worm in the mediterranean basin. species island sea area country references anguis cephallonica kephallonia ionian greece grillitsch and cabela, 1990; mayer et al., 1991; jablonski et al., 2016 ithaki ionian greece lefkas ionian greece zakynthos ionian greece anguis graeca corfu ionian greece tóth et al., 2002; valakos et al., 2008; jablonski et al., 2016 thasos aegean greece euboea aegean greece anguis fragilis/veronensis (hybrid zone) cres adriatic croatia tóth et al., 2006; kryštufek and kletečki, 2007; gvozdík et al., 2010 krk adriatic croatia košljun adriatic croatia anguis veronensis sainte-marguerite ligurian france this study fig. 1. localization of ile sainte-marguerite (yellow star) on a distribution map reporting the localities where a. veronensis (in yellow) and a. fragilis (in red) have been genetically studied so far. redrawn from gvoždík et al. (2013). 167an island population of anguis veronensis in western mediterranean a statistical parsimony network was constructed for nuclear prlr haplotypes using tcs v. 1.21 with 95% as disconnection limit (clement et al., 2000), by including the haplotypes found in this study and those reported by gvoždík et al. (2013). the gene fragments obtained from the six individuals from ile sainte-marguerite (ism) were 662 bp long for nd2 and 544 bp for prlr. the sequences recovered for nd2 did not show stop codons and provided a single haplotype for all the six sampled specimens (mh316865). a megablast search in genbank showed that these six sequences were identical to kc881558, which belongs to a. veronensis from les mayons (mainland municipality in the nearby department of var) as reported in gvoždík et al. (2013). we found three different haplotypes for the prlr fragment (mh316862-3-4): four specimens showed the same haplotype recovered by gvoždík et al. (2013) as the most frequent in a. veronensis (pv1) in homozygosity, while the other two specimens resulted to be heterozygous. they showed two haplotypes both recorded for the first time, being pv1/pv4 and pv1/pv5 their genotypes. the haplotype network showed that the two new haplotypes are derivative from the most widespread pv1 and differ by a single mutation each (fig. 3). to our knowledge, this finding attests for the first time the presence of the slow worm on a western mediterranean island, ile sainte-marguerite from lérins archipelago (southeastern france). the molecular analysis carried out has allowed identifying the specimens as the italian species a. veronensis. this is not an unexpected result, since a. veronensis ranges not only in the italian peninsula but also in southeastern france (gvoždík et al., 2013). however, the origin of this island population was not straightforward, since possible anthropogenic dispersal could not be ruled out. the habits of a. veronensis are indeed continental and the crossing of geographic barriers as a sea channel, even if 1.3 km wide and of a few meters of depth, may be challenging for this species. for example, a recent study correlated the genetic heterogeneity of the balkan species (in particular a. graeca and a. cephallonica) to the terrain ruggedness (jablonski et al., 2016). although in the cited case the geographic barriers were represented specifically by altitudinal differences and steep slopes, slow worms are small legless lizards with semi-fossorial habits and consequently low dispersal ability (haley, 2014). despite this, the data showed that the slow worm population from ile saint-marguerite is naturally derived from the one living on the nearby mainland. indeed, the gene pool of this population is characterized by the presence of the same mitochondrial nd2 haplotype observed at les mayons (v11) and of the most common nuclear prlr haplotype (pv1). the population of ile saint-marguerite also showed two newly found prlr haplotypes, derived from pv1, so suggesting that this is likely a natural population that has been inhabiting lérins archipelago for a long period of time. it might be the remnant of a former continental population that has been severed by sea level rise that occurred 5000 to 7000 years ago (lambeck and bard, 2000). the network analysis confirmed that the prlr haplotypes of a. veronensis are not shared with other species, and occupy an internal position within the network, further supporting the idea that a. veronensis lineage is ancestral within the genus, as already reported based on fig. 3. statistical parsimony network based on prlr sequences and including the sequences both from gvoždík et al. (2013) and from this study (isle sainte-marguerite). haplotypes are named as in gvoždík et al. (2013); the newly found haplotypes pv4 and pv5 are in bold. fig. 2. habitat of anguis veronensis from sainte-marguerite island, southeastern france. 168 julien renet et alii previous molecular and karyological data (gvoždík et al., 2013; mezzasalma et al., 2013). the data presented show that small islands may host genetically variable slow worm populations, despite anguis was considered mainly a mainland distributed genus. the south-eastern france is confirmed as inhabited by a. veronensis, but a more detailed sample of the southern french coasts would allow identifying western boundaries with a. fragilis. given the age of this lineage of anguis, the species were likely already present on balkan, italian and se provence continental areas at the time islands were severed by sea level rise, after the last glacial maximum. the reason why the species is nowadays so uncommon in insular context is more likely related to climate and ecological constraints such as scarceness of fresh forested habitats and summer drought, prevailing on western mediterranean islands. acknowledgements permission for this programme was issued by order of the prefet [2016-506], according to french law. we thank dr. alexandre villers and two anonymous reviewers for providing useful comments. we also warmly thank dr. petros lymberakis for providing additional information and salomon brodier for improving the english language. references cabela, a. 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(1998): contributo alla conoscenza degli anfibi e dei rettili di sicilia. boll. accad. gioenia sci. nat. 30: 5-88. valakos, e.d., pafilis, p., sotiropoulos, k., lymberakis, p., maragou, p., foufopoulos, j. (2008): the amphibians and reptiles of greece. edition chimaira, frankfurt am main. vanni, s., nistri, a. (2006): atlante degli anfibi e dei rettili della toscana. università di firenze, museo di storia naturale, sezione di zoologia la specola e regione toscana, firenze. zanghellini, s. (2006): anguis fragilis linnaeus, 1758. in: atlante degli anfibi e dei rettili d’italia / atlas of italian amphibians and reptiles, pp. 426-429. sindaco, r., doria, g., razzetti, e., bernini, f., eds, societas herpetologica italica, edizioni polistampa, firenze. acta herpetologica vol. 13, n. 1 june 2018 firenze university press acta herpetologica 13(1): 95-100, 2018 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-21100 helminths of the lizard colobosauroides cearensis (squamata, gymnophthalmidae) in an area of caatinga, northeastern brazil aldenir f. da silva neta*, robson w. ávila programa de pós-graduação em bioprospecção molecular, departamento de química biológica, universidade regional do cariri – urca, rua coronel antônio luis, 1161, pimenta, cep 63100-000, crato, ce, brazil.*corresponding author. e-mail: aldenirferreira_@ hotmail.com submitted on: 2017, 4th august; revised on: 2017, 25th september; accepted on: 2018, 25th january editor: dario ottonello abstract. lizards are hosts to a variety of parasites, but in south america only 15% of lizard species have been studied for helminths. in the present study, the component community of helminths associated with the gymnophthalmid colobosauroides cearensis in an area of caatinga (7°22’46.08” s, 38°38’47.87”w) is reported. we examined 91 specimens from the brazilian state of ceará, and five taxa of helminths were recovered: four nematoda (parapharyngodon largitor, spauligodon sp., physaloptera sp. and oswaldocruzia sp.) and one cestoda (oochoristica sp.). parapharyngodon largitor was the most prevalent species (61%), and presented the highest mean abundance of infection (1.60 ± 0.18). lizard body size influenced the richness and abundance of helminths, while infection parameters were not related to lizard sex. keywords. parasites, nematodes, cestodes, neotropical. parasitological studies are necessary to understand host–parasite interactions and the role of parasite species within ecosystems (bittencourt and rocha, 2003; hudson, 2005). although the richness of parasites is greater than that of hosts, they are much less studied, which means a considerable portion of the biodiversity is unknown (poulin and morand, 2000; rocha et al., 2016). lizards are host to a variety of parasites, including helminths (anderson, 2000; ávila and silva, 2010; ávila et al., 2011; 2012). despite the recent increase in parasitological studies in lizards (anjos et al., 2005; ávila and silva, 2010; brito et al., 2014a; bezerra et al., 2015), the knowledge of helminths remains scarce. for example, south america harbors more than 1120 lizard species (uetz and hosek, 2016), but only 15% of these species have had their associated helminths studied (ávila and silva, 2010). the new world lizard family gymnophthalmidae includes 235 species (uetz and hosek, 2016), and less than 10% of these species have been studied regarding their parasitological aspects (ávila and silva, 2010). information about parasitism in gymnophthalmidae is punctual and usually appears in descriptions or records of new occurrences of parasites (bursey et al., 2005; ávila et al., 2011; albuquerque et al., 2012). in brazil, data on the parasitic fauna of gymnophthalmids is concentrated in studies of the amazon (baker and bain, 1981; bursey et al., 2005; albuquerque et al., 2012; ávila and silva, 2013), cerrado (ávila et al., 2011) and restinga (almeida et al., 2009). in the caatinga domain, the knowledge related to helminth communities associated with gymnophthalmids remains little explored (brito et al., 2014a). colobosauroides cearensis is a semifossorial and diurnal lizard with relictual distribution in the caatinga. to the best of our knowledge, there are no records of helminths being associated with this lizard, mainly due to its fossorial habits (cunha et al., 1991). herein, we present data on the helminth community composition of the 96 aldenir f. da silva neta, robson w. ávila lizard colobosauroides cearensis in an area of caatinga in the northeast region of brazil. we conducted this study in the municipality of mauriti (7°22’46.08”s, 38°38’47.87”w), state of ceará, northeastern brazil (fig. 1). the vegetation is characterized mainly by deciduous forest and hypoxerophytic caatinga. the local climate is hot and semiarid, with the rainy period occurring from october to april, and the mean annual precipitation ranging from 500 to 800 mm (ipece, 2015). we captured 91 colobosauroides cearensis (39 ± 7 mm svl) by hand during three to five-day fieldtrips from december 2015 to december 2016; this comprised 47 adult females (39.9 ± 7.5 mm svl), 36 adult males (42.8 ± 5.5 mm svl) and 8 juveniles (22.0 ± 0.9 mm svl). we euthanized the lizards with a lethal injection of sodium thiopental (cfmv, 2013); these were then fixed in 10% formalin, preserved in 70% alcohol and deposited in the herpetological collection of the regional university of cariri (8451, 8452, 8453, 11453–11490; 11663– 11687; 12399–12423). for each specimen, we measured the snout–vent length (svl) with a digital caliper (± 0.01 mm) and mass to the nearest gram with a pesola® spring scale (± 0.1 g). we removed all organs of the respiratory and gastrointestinal tracts and examined them individually under a stereoscope for helminths. we transferred the collected helminths to 70% ethanol. we stained the cestodes with alcoholic hydrochloric acid-carmine, which were subsequently cleared in creosote, while nematodes were diaphanized in lactophenol. for each helminth species, the prevalence, mean abundance, and mean intensity of infection (following bush et al., 1997) were estimated, where parasite mean abundance is defined as the arithfig. 1. collecting site in state of ceará (a) in northeast brazil; mauriti municipality (b); study area (c): são miguel district. 97helminths of the lizard colobosauroides cearensis metic mean of the number of individuals of a particular parasite species per host examined; mean intensity of infection is the total number of parasites found in a sample, divided by the number of hosts infected with that parasite; and prevalence (p%) is the number of hosts infected with one or more individuals of a particular parasite species divided by the total host number. throughout the text, results are reported as means ± se. we used a generalized linear model with poisson distribution (glm) to analyze the relationship between abundance and helminth species richness with host body size and sex. we used a t-test to infer intersexual differences between svl and mean helminth richness. all analyses were performed using the package rcmdr in the r platform, version 2.15.0 (r development core team, 2013). we recovered 165 helminths with an overall prevalence of 69.2%. mean overall abundance was 1.8 ± 0.2 and mean intensity of infection was 2.6 ± 1.6. the component community of helminths associated with colobosauroides cearensis comprised five taxa (table 1): one cestode, oochoristica sp., and four nematodes, parapharyngodon largitor, spauligodon sp., physaloptera sp. and oswaldocruzia sp. fifty-six individuals of c. cearensis (32 females and 24 males) were parasitized with parapharyngodon largitor (61% prevalence), with this nematode also presenting the highest mean abundance (1.60 ± 0.18). oswaldocruzia sp. were the least prevalent (1.1%) and the least abundant (0.02 ± 0.02). the relationship between lizard svl and the mean abundance of helminths was significant (z = 2.604; p = 0.009). although males (42.8 ± 5.5) were larger than females (39.9 ± 7.4; t = 2.212; p = 0.029), sex did not influence mean abundance (z = 0.935; p = 0.30). the mean richness of helminths was low (0.736 ± 0.050), with no influence of host sex (z = -0.068; p = 0.60), but the svl influenced helminth mean richness (z = 2.282; p = 0.02). intersexual differences in mean richness were not found, even when the effect of svl was removed (t = 0.267; df = 80.91; p = 0.70). moreover, no juvenile lizards were parasitized. our study provides the first parasitological record to the host colobosauroides cearensis. most taxa reported here were not identified to species level due to juvenile condition (physaloptera sp.), bad conditions of preservation (oochoristica sp. and oswaldocruzia sp.) or because the present species probably represented an undescribed species (spauligodon sp.). parapharyngodon largitor is a generalist species, since it has been reported in several lizard species (rodrigues, 1970; vicente et al., 1993; vrcibradic et al., 2002; bittencourt and rocha, 2003; anjos et al., 2005; ávila and silva, 2010; ávila et al., 2011). despite the higher prevalence, p. largitor presented low intensity of infection (2.6 ± 1.6), which is also noted for the genus parapharyngodon in other lizard species: hemidactylus mabouia (squamata: gekkonidae), phyllopezus pollicaris (squamata: phyllodactylidae), tropidurus itambere, t. torquatus, t. hispidus (squamata: tropiduridae) (rodrigues, 1987; anjos et al., 2005; pereira et al., 2011; sousa et al., 2014; araújo-filho et al., 2016). higher prevalence (61%) of p. largitor indicates success in colonization within host populations, which suggests that this species may be important in parasite community structure (bush and holmes, 1986; holmes, 1987). specimens of genus oswaldocruzia sp. are frequently found infecting the intestines of amphibians and reptiles (santos et al., 2008). oswaldocruzia sp. has direct life cycle, transmission can occur through ingestion or larvae penetration in the skin (anderson, 2000). oswaldocruzia sp. was observed parasitizing several lizards from brazil (ávila and silva, 2010), including tropidurus semitaeniatus, brasiliscincus heathi and anotosaura vanzolinia in a caatinga area (brito et al., 2014a; oliveira et al., 2017). the low prevalence and intensity of infection found here suggests accidental ingestion of eggs, which could occur through tongue-flicking behavior in substrate (menezes et al., 2004). table 1. mean abundance (ma), mean intensity of infection (mii) with range, prevalence (p) and site of infection of helminth community associated with the lizard colobosauroides cearensis in the caatinga, northeastern brazil. values are mean ± se. si: small intestine; st: stomach; li: large intestine. helminth ma mii p site of infection cestoda oochoristica sp. 0.05 ± 0.03 1.6 ± 0.7 (1-2) 3.3% si nematoda physaloptera sp. 0.09 ± 0.07 4.5 ± 3.5 (2-7) 2.2% st parapharyngodon largitor 1.60 ± 0.18 2.6 ± 1.6 (1-10) 61.0% si, li, st oswaldocruzia sp. 0.02 ± 0.02 2.0 ± 2.0 (-) 1.1% si spauligodon sp. 0.06 ± 0.04 2.0 ± 1.0 (1-3) 4.4% si, li 98 aldenir f. da silva neta, robson w. ávila the other helminth taxa found in the present study have been recorded in lizards from south america (ávila et al., 2010), which have also been reported in other gymnophthalmids: oochoristica sp. (apoglossus sp., micrablepharus maximiliani), physaloptera sp. (cercosaura argulus, bachia scolecoides), spauligodon sp. (micrablepharus maximiliani) (ávila et al., 2011; brito et al., 2014a). the infection of other gymnophthalmids by the same taxa of parasites reported here may suggest phylogenetic relationships, since phylogenetically close taxa may present similarity in the use of a niche, body shape and behavior (wiens and graham, 2005; lima et al., 2012; brito et al., 2014a). host body size influences the establishment of populations and communities of parasites (poulin, 2004; kamiya et al., 2014a; 2014b). the individual’s parasitic load (poulin and george-nascimento, 2007) is due in part to a larger ‘area’ of exploration and colonization provided by larger-sized specimens (macarthur and wilson, 1967; aho, 1990). in addition, another relevant factor is that larger individuals are older and have therefore suffered longer exposure to parasitic agents (aho, 1990). studies of lizard populations have found a positive relationship between host body size and helminth infection rates (barreto-lima et al., 2011; ávila and silva, 2013; araújo-filho et al., 2014; brito et al., 2014b). there was no variation of richness and abundance between the sexes in c. cearensis, contrary to findings for other gymnophthalmids (brito et al., 2014a). intersexual variation in parasite loads may be related to hormonal, physiological and behavioral features; for example, larger males exhibit territorial behavior and frequently engaged in combat with other individuals, which may increase stress levels while decrease their immune response, thus becoming more susceptible to parasitic agents. in addition, testosterone production could be a powerful suppressor of immune system (zuk and mckean, 1996). however, data regarding physiological and behavioral variation are lacking for c. cearensis. moreover, other studies found no relationship between the parasitic rates and the sex of the lizards (anjos et al., 2011; bezerra et al., 2015). gymnophthalmids present low helminth richness, when compared to other lizards of the superfamily teiioidea (goldberg et al., 2013; teixeira et al., 2016). small body sizes in lizards of this family could restrict available niches to colonization and habitat segregation for endoparasites. poor helminth richness compared to other lizard species in the same family was also reported in cercosaura argulus (4 spp), cercosaura ocellata (1 spp), leposoma osvaldoi (1 spp), micrablepharus maximiliani (3 spp) e anotosaura vanzolinia (1 spp) (ávila and silva, 2010; brito et al., 2014a; oliveira et al., 2017). in the present study, we found the highest helminth richness within the lizard family gymnophthalmidae, also providing new locality and host records. acknowledgements we are grateful to conselho nacional de desenvolvimento científico e tecnológico – cnpq for the research grant awarded to rwa (process 303622/2015-6); and to coordenação de aperfeiçoamento de pessoal de nível superior – capes for the master fellowship awarded to afsn. this study was approved by the animal ethics committee of the universidade regional do cariri (process no 00026/2015.2). the collection permit (52214-1) was in accordance with the instituto chico mendes de conservação da biodiversidade. references aho, j.m. 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(1996): sex differences in parasite infections: patterns and process. internation j. parasitol. 26: 1009-1024. acta herpetologica vol. 13, n. 1 june 2018 firenze university press species diversity and distribution of lizards in montenegro katarina ljubisavljević1,2,*, ljiljana tomović3, aleksandar urošević1, slađana gvozdenović2, vuk iković2, vernes zagora2, and nenad labus4 the first demographic data and body size of the southern banded newt, ommatotriton vittatus (caudata: salamandridae) abdullah altunişik diet and helminth parasites of freshwater turtles mesoclemmys tuberculata, phrynops geoffroanus (pleurodira: chelidae) and kinosternon scorpioides (criptodyra: kinosternidae) in a semiarid region, northeast of brazil antonio marcos alves pereira*, samuel vieira brito, joão antonio de araujo filho, adonias aphoena martins teixeira, diêgo alves teles, daniel oliveira santana, vandeberg ferreira lima, waltécio de oliveira almeida electric circuit theory applied to alien invasions: a connectivity model predicting the balkan frog expansion in northern italy mattia falaschi1,2,*, marco mangiacotti3, roberto sacchi3, stefano scali2, edoardo razzetti4 first report of bufo bufo (linnaeus, 1758) from sardinia (italy) ilaria maria cossu1, salvatore frau1, massimo delfino2,3, alice chiodi4, claudia corti1,5*, adriana bellati4 natural history and conservation of the nurse frog of the serranía del perijá allobates ignotus (dendrobatoidea: aromobatidae) in northeastern colombia hernán darío granda-rodríguez1,2, andrés camilo montes-correa3,*, juan david jiménez-bolaño3, marvin anganoy-criollo4,5 exploring body injuries in the horseshoe whip snake, hemorrhois hippocrepis juan m pleguezuelos*, esmeralda alaminos, mónica feriche how effectively do european skinks thermoregulate? evidence from chalcides ocellatus, a common but overlooked mediterranean lizard grigoris kapsalas, aris deimezis-tsikoutas, thanos georgakopoulos, ismini gkourtsouli-antoniadou, kallirroi papadaki, katerina vassaki, panayiotis pafilis thermal tolerance for two cohorts of a native and an invasive freshwater turtle species jun geng, wei dang, qiong wu, hong-liang lu* diet of a restocked population of the european pond turtle emys orbicularis in nw italy dario ottonello1, fabrizio oneto1,2, monica vignone2, anita rizzo2, sebastiano salvidio2,* helminths of the lizard colobosauroides cearensis (squamata, gymnophthalmidae) in an area of caatinga, northeastern brazil aldenir f. da silva neta*, robson w. ávila acta herpetologica 12(2): 181-186, 2017 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-20339 predation of common wall lizards: experiences from a study using scentless plasticine lizards jenő j. purger*, zsófia lanszki, dávid szép, renáta bocz department of ecology, institute of biology, university of pécs, ifjúság útja 6, 7624 pécs, hungary *corresponding author. e-mail:purger@gamma.ttk.pte.hu submitted on: 2017, 19th february; revised on: 2017, 18th august; accepted on: 2017, 22nd august editor: paolo casale abstract. the potential influence of predators on lacertid lizards has been studied by using models made of plasticine which shows the attack marks of predators and as such allows their identification and estimation of predation pressure. the general aim was to study predation on plasticine models of lizards and to improve methods, since the results depend on the number of plasticine models used, their spatial pattern and the duration of experiments. we estimated the density of the common wall lizard podarcis muralis population on stone walls of a vineyard in the city of pécs (hungary) in august 2015 in order to imitate the real density in our experiment with plasticine models. the density of common wall lizards was 8.2 ind. /100 m2 and accordingly we placed 25 scentless plasticine lizards on the stone walls on the first transect with 10 m distance between them, which imitates the real pattern. in the second transect 25 lizard models were placed more sparsely, the distance between them being 20 m. during four weeks the predation rate was 24% in densely spaced plasticine lizards and 40% in sparsely spaced plasticine lizards, but the difference was not significant. the daily survival rate of densely spaced lizards was 0.99 (=99.1%) and that of sparsely spaced lizard models was 0.98 (=98.25%), but this difference was not significant either. on the basis of marks left on plasticine lizards, mammal predators (e.g. beech marten) dominated, while the impact of bird predators was smaller than expected. predators attacked the head of plasticine lizards more frequently than their trunk, tail or limbs, but a significant preference of body parts was not detected. from our experience it is important to study the distribution and density of real animals, to imitate their real pattern, instead of an arbitrarily designed experiment with models. the typical scent of plasticine also could influence the results, which can be avoided by using scentless plasticine models coated with liquid rubber. we suggest the calculation of daily survival rates in order to produce results that allow the comparison of different studies. keywords. abundance, density dependence, hungary, podarcis muralis, survival rates. introduction predator-prey interactions have been in the focus of ecological and evolutionary research (e.g., cooper and blumstein, 2015). there are many difficulties in studying predation events directly in the wild, especially on small vertebrates; therefore during the last decades models made of soft materials (e.g., plasticine) have been used (bateman et al., 2016). the potential influence of predators on lacertid lizards has been also studied by using plasticine models which show the attack marks of predators and as such allow their identification and estimation of predation pressures (e.g., castilla and labra, 1998; castilla et al., 1999; diego-rasilla, 2003a,b; shepard, 2007; vervust et al., 2007, 2011; pérez-mellado, 2014; sato et al., 2014; fresnillo et al., 2015; stellatelli et al., 2015). the results of these studies mainly depend on the quality of materials and the number of plasticine models used, on 182 jenő j. purger et alii the duration of experiment and also on how often they were checked for evidences of attack (bateman et al., 2016). a further problem is that the unnatural smell of plasticine can influence the results, through modifying the behaviour of mammal predators which rely on olfactory cues (bayne and hobbson, 1999; purger et al., 2012; fresnillo et al., 2015). these models are immobile, while bird predators relying on visual cues mostly react to a moving prey (rangen et al., 2000). the many lizard species are diurnal animals and their main predators are birds (e.g., vervust et al., 2007; pérez-mellado, 2014; fresnillo et al., 2015), therefore the unnatural attractiveness of models for nocturnal mammal predators should be avoided by using scentless models (purger et al., 2012). the duration of experiments performed with plasticine lizards was mostly arbitrarily decided (e.g., diego-rasilla, 2003a,b) in dependence of the abundance of potential predators and their ability to discover and predate the models (e.g., castilla and labra, 1998). since predation events depend on the density of prey, it is important to study the pattern (e.g., distribution, density) of real animals in the particular area before starting an experiment with models, to imitate real pattern, instead of an arbitrarily designed experiment. in some studies the distance between models was only 2 m (vervust et al., 2007, 2011) or 5 m (e.g., castilla et al., 1999; diego-rasilla, 2003a,b; pérez-mellado, 2014), which suggest a high density, while in other studies the distance was at least 100 m apart from one to another (fresnillo et al., 2015) or the models were placed scattered (pérez-mellado, 2014). predation rates are influenced by different circumstances therefore the results of different studies are difficult to compare. our study was performed as an attempt to standardise predation experiments with lizard models. our specific goals were: a) to find out whether predation is dependent on lizard density; b) to estimate predation rates and the daily survival rates of scentless plasticine lizards; c) to identify the predators; d) to find out if predators prefer any parts of the prey’s body. material and methods the study was carried out at the st. nicholas hill research station (46°04'n, 18°09'e) of the institute of viticulture and oenology, university of pécs, on the southern slopes of mecsek mts., 180-240 m a.s.l., 5 km to the west from the centre of the city of pécs, hungary. this area (14 ha) has been used as a vineyard since the 1750’s. its surface is slightly undulating with stone walls between fields. the soil is ramann-type brown forest soil formed on pannonian red sandstone (teszlák et al., 2013). from the north the area is bordered by manna ashdowny oak (fraxino-quercetum) dry forest. the common wall lizard podarcis muralis (laurenti, 1768) is the most common lizard species in europe (guillaume, 1997), occurring everywhere in hungary with the exception of the great hungarian plain (puky et al., 2005). in suitable habitats such as open rocky hillsides, quarries and stone walls in urban environments with warm microclimate it can reach considerable densities (trócsányi and korsós, 2004). active individuals of the species are observable even on warmer days of winter months in the southern region of the country (trócsányi et al., 2007). it is an opportunistic species so habitat requirements are variable, it often lives in vineyards (vogrin, 1998), but their densities could be influenced by shelter, food and the effects of predators (gruschwitz and böhme, 1986). natural predators of this species are among mammals, birds and snakes which occur in their habitats (gruschwitz and böhme, 1986). we estimated the density of the wall lizard population on stone walls in the summer of 2015 (before the study) in order to imitate the real density in our experiment with plasticine models. the density was not estimated by capturing the animals. but instead lizards were counted by walking on the top of all six stonewalls (0.5-2.5 m high and ca. 0.4 m width) which divided vineyard terraces. the average length of stonewalls was 310 m (sd = 149.93) and the average of top surface area was 124 m2 (sd = 59.97). counting was performed by the same person, during six days from 24 august 2015, always beginning at 16:00 h, on one stonewall randomly selected each day. during counting the lizards escaped due to human appearance, and hid on the side of the walls, therefore they were counted only once. before the experiment the workers in the vineyards were informed about our study and they tried to not cause disturbance. for our study lizard replicas were created using non-toxic natural colour plasticine (produced by koh-i-noor hardtmuth, czech republic). the lizard models were made of plasticine with a wire axis which was used also for hanging them on the stone wall. we used plasticine lizard models whose body size cca. 15 cm (±1 cm) and shape were similar to those of adult wall lizards (diego-rasilla, 2003a,b). the basic colour of male and female is similar (arnold and ovenden, 2002), therefore the sculpted plasticine lizards were painted uniformly in taupe colour (tempera, produced by pannoncolor, hungary) based on the colour of observed and photographed lizards in the study area. then models were coated with uncoloured liquid rubber spray (plastidip®, usa) and were aired for two weeks in order to eliminate the scent of plasticine and thus allow equal chances for avian and mammal predators in their visual search (purger et al., 2012). in the morning of 7 september 2015 we placed 25 scentless plasticine lizards on the top of a stone wall in the first transect with 10 m spacing, imitating real density pattern. in the second transect 25 lizard models were sparsely spaced; the distance between them was 20 m. they were placed in an open area and were fully visible to avian predators (pérez-mellado, 2015). the study sites were homogeneous, very similar linear habitats, 50-70 m apart from each other; therefore we assume that there were no differences in predator communities. on the south facing side of stone walls 50 plasticine lizards were placed (25 densely and 25 sparsely spaced). unfortunately the majority of models placed on the vertical side of walls melted due high 183survival of plasticine lizards solar radiation during the experiment, and these two transects were not included in the analysis. we checked the condition of lizard models after their placement, on the afternoon of days 1, 3, 7, 14, 17, 21 and 28 between 16:00 h and 18:00 h. attacked models were removed during regular checking to avoid pseudoreplication. on the last checking day we gathered the remaining models. a lizard model was considered as being attacked by a predator when bill marks of birds, tooth marks of mammals were found, or if it had disappeared (e.g., castilla and labra, 1998; castilla et al., 1999; diego-rasilla, 2003a,b). we recorded which body part of the lizards (head, trunk, tail or limbs) had been damaged by predators (vervust et al., 2011). based on the marks on plasticine models, mammal predators were identified by the help of our collection of mammal skulls (fig. 1.). predation rates on lizard models arranged in the two transect were calculated as percentage of damaged (predated) models. daily survival rate is the probability that a lizard survives a single day. we used mayfield’s (1975) method (common in ornithological studies) for estimating the daily survival rate of a sample of plasticine wall lizards using exposure days (the cumulative number of days that the lizards in the sample were monitored) and the number of known losses. according to the mayfield method, the estimated daily survival was calculated as 1 [(number of lizard losses) / (total exposure days)]. in our study, for the comparison of daily survival rates the test proposed by johnson (1979) was applied, calculating with the free software „j-test” developed by k. halupka (2009). for comparing the proportions of predation causes and number of attacks on different body parts, chi-square goodness of fit for two and four categories was used (zar, 1999). a minimum tail probability level of p < 0.05 was accepted for all the statistical tests, and all p-values were two-tailed. results and discussion on the top of stone walls (total length 1860 m, surface area cca. 744 m2) 61 common wall lizards (8.2 individuals/100 m2) and five eastern green lizards lacerta viridis (0.7 ind. /100 m2) were counted, which means there was at least one lizard in every cca. 10 m2. our estimation of common wall lizard population density showed similarity to the results quoted by puky et al. (2005). these authors summarised data from literature and concluded that the territory of common wall lizard ranges between 3 and 50 m2. such great variation in lizard density is affected by a complex variety of factors; e.g., habitat diversity, availability of resources, presence of predators, competitors and human disturbances (pérez-mellado et al., 2008). trócsányi and korsós (2004, 2007) suggest that in mecsek mts. near the city of pécs the density of wall lizards on a brick wall was 36 individuals/100 m2 while there was only 6.5 individuals/100 m2 in a quarry. in comparison with these values the density of wall lizards estimated in the vineyard was low. applying of some sampling methods often resulted in underestimation of lizard density (e.g., smolensky and fitzgerald, 2010; ruiz de infante anton et al., 2013), however, these values may be useful in experiments with artificial models. during our study 24% of the densely spaced lizards and 40% of sparsely spaced lizards were damaged by predators, but based on the number of predation events the difference was not significant (χ2 with yates corrections = 0.56; df = 1; p = 0.546). density-dependent predation was not detected by using plasticine lizards. with a view to the fact that a high variability in the density of common wall lizards in different habitats is shown (trócsányi and korsós, 2004, 2007), we can say that in our experiment the imitated density of the same habitat was low in both transects, and therefore we could not detect significant differences between predation rates. despite this, we suggest taking into consideration the density of the studied species and placing the replicas accordingly in order to achieve more realistic results. the daily survival rate of densely spaced lizard models (total exposure days = 662, number of lizard losses = 6) was 0.99 (99.1%, 95% confidence intervals: 98.3799.83) and that of scarcely spaced lizards (total exposure days = 560.5, number of lizard losses = 10) was 0.98 (98.25%, 95% ci: 97.15-99.35), but this difference was not significant (z = 1.296, two tailed p = 0.195). the duration of our study (four weeks) was quite long because we had to wait for the first predation event to occur; scarce predation resulted in high daily survival rates. similar studies took few days or a week since they used high density (2-5 m) of prey models (e.g., castilla and labra, 1998; castilla et al., 1999; diego-rasilla, 2003a,b; sato et al., 2014, stellatelli et al., 2015) or even 20 days in the study with models 100 m apart from each other (fresnillo et al., 2015). our experience showed that in the case of few predators in the study area the studies should last longer, until the predation rate reach at least 30-40%, or the study should be repeated. we identified one mark of a bird, two marks of small mammals, two marks of weasel mustela nivalis, three of red fox vulpes vulpes (fig. 1.) and six bites of beech marten martes foina on the plasticine models. two of the lizard models disappeared; we suppose that they were taken by large mammals. predators did not damage two lizard models placed next to each other at the same occasion, which means that the liquid rubber obscured the plasticine smell, or maybe because the attacked lizard model was unpalatable and then the predator did not attack the other nearest. it is known that mammal predators use mainly olfactory cues during hunting (rangen et al., 2000), but in our study it seems that the smell of plasticine did not attract the mammal predators. noc184 jenő j. purger et alii turnal mammals did not identify lizard models as prey; we found droppings of beech marten three times on the same lizard model, which means that it marked its revier (seiler et al., 1994). red foxes, beech martens, weasels and cats felis catus were seen regularly in the study area and we found their traces and droppings. most of them are known for preying upon lizards (e.g., castilla et al., 1999; diego-rasilla, 2003a). we presume that in habitats with a lot of hiding places, preying upon small bodied fast moving lizards require big energy investment. according to ecological studies of mammal feeding, red fox, weasel and beech marten are reported to consume lizards periodically (occasionally) or rarely (e.g., lanszki et al., 1999; lanszki, 2003, 2012; lanszki and heltai, 2007). in our study the predation role of small mammals was not considerable, but it was not negligible either. among small mammals shrew (soricidae) species are often mentioned as wall lizard predators (gruschwitz and böhme, 1986), however in some predation studies replicas showing marks of rodents were considered as non-attacked (e.g., castilla et al., 1999). in our study avian predation rate was also very low. common kestrel falco tinnunculus and common buzzard buteo buteo, both being potential lizard predators (e.g., castilla et al., 1999; diego-rasilla, 2003a; vervust et al., 2011), were frequent in the study area. also, there were hooded crows corvus cornix and eurasian jays garrulus glandarius which, too, were identified as egg predators in our earlier study (purger et al., 2004). there is evidence that birds are able to visually recognize lizards as prey, based on their shape and colour pattern, even if the animals remain immobile (e.g., stuart-fox et al., 2003; shepard, 2007; stellatelli et al., 2015). the possible reason for less attack by avian predators in our experiment may be that models did not resemble wall lizard coloration and pattern with sufficient precision, similarly to the study of marshall et al. (2015). during our study we observed a smooth snake coronella austriaca just when preying on a wall lizard, but tooth marks of this well-known lizard predator (diego-rasilla, 2003a; amo et al., 2004) were not found on any of the lizard models. based on the low number of predation events recorded (n = 30) the only fact we could determine was that tooth marks of large mammals were found on the head of plasticine lizards more frequently than on their trunk, tail or limbs (fig. 2.), but significant preference of body parts was not detected (χ2 = 1.2; df = 3; p = 0.753). predators could grab different parts of the body with equal chance, since plasticine lizards were immobile. according to the results of experiments with lizard replicas (castilla et al., 1999; vervust et al., 2011) mammals tend to attack the head of a prey more often. from our experience in studies of daily active animals we suggest using scentless plasticine animal replicas coated with liquid rubber which eliminate unnatural plasticine smell and reduce the impact of nocturnal predators. it is important to study the recent distribution and density of real animals in the particular area, to imitate their real pattern, instead of an arbitrarily designed experiment with models. since predation rate depends mainly on the pattern of prey, the activity of members of predator community, as well as on the duration of experiments, we suggest the calculation of daily survival rates in order to produce results that allow the comparison of different studies. fig. 2. number of attacks by different predators on various body parts of plasticine wall lizard models (black bars – head, grey bars – trunk, white bars – limbs, hatched bars – tail). fig. 1. tooth prints left on plasticine models were compared with skulls and in this case red fox was identified as predator. 185survival of plasticine lizards acknowledgements we would like to thank for two anonymous reviewers, dragica purger and balázs trócsányi for useful comments on the previous draft of the manuscript, csaba fekete for technical help. references amo, l., lópez, p., martín, j. 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(1999): biostatistical analysis. prentice hall, london. acta herpetologica vol. 12, n. 2 december 2017 firenze university press meristic and morphometric characters of leptopelis natalensis tadpoles (amphibia: anura: arthroleptidae) from entumeni forest reveal variation and inconsistencies with previous descriptions susan schweiger1, james harvey2, theresa s. otremba1, janina weber1, hendrik müller1,* brown anole (anolis sagrei) adhesive forces remain unaffected by partial claw clipping austin m. garner*, stephanie m. lopez, peter h. niewiarowski species and sex comparisons of karyotype and genome size in two kurixalus tree frogs (anura, rhacophoridae) shun-ping chang1,2, gwo-chin ma2,3,4, ming chen2,5,6,7,8,*, sheng-hai wu1,* non-native turtles in a peri-urban park in northern milan (lombardy, italy): species diversity and population structure claudio foglini1, roberta salvi2,* species composition and richness of anurans in cerrado urban forests from central brazil cláudia márcia marily ferreira1,*, augusto cesar de aquino ribas2, franco leandro de souza3 the life-history traits in a breeding population of darevskia valentini from turkey muammer kurnaz, alı i̇hsan eroğlu, ufuk bülbül*, halıme koç, bılal kutrup influence of desiccation threat on the metamorphic traits of the asian common toad, duttaphrynus melanostictus (anura) santosh mogali*, srinivas saidapur, bhagyashri shanbhag predation of common wall lizards: experiences from a study using scentless plasticine lizards jenő j. purger*, zsófia lanszki, dávid szép, renáta bocz reproductive timing and fecundity in the neotropical lizard enyalius perditus (squamata: leiosauridae) serena najara migliore1,2,*, henrique bartolomeu braz2,3, andré felipe barreto-lima4, selma maria almeida-santos1,2 observations on the intraspecific variation in tadpole morphology in natural ponds eudald pujol-buxó1,2,*, albert montori1, roser campeny3 and gustavo a. llorente1,2 reliable proxies for glandular secretion production in lacertid lizards simon baeckens diet of juveniles of the venomous frog aparasphenodon brunoi (amphibia: hylidae) in southeastern brazil rogério l. teixeira1, ricardo lourenço-de-moraes2, débora c. medeiros3, charles duca3, rogério c. britto4, luiz c. p. bissoli5, rodrigo b. ferreira3,* who are you? the genetic identity of some insular populations of hierophis viridiflavus s.l. from the tyrrhenian sea ignazio avella, riccardo castiglia, gabriele senczuk* acta herpetologica 11(2): 135-149, 2016 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-18695 growth, longevity and age at maturity in the european whip snakes, hierophis viridiflavus and h. carbonarius sara fornasiero1, xavier bonnet2, federica dendi1, marco a.l. zuffi1,* 1 museo di storia naturale, università di pisa, via roma 79, i-56011 calci (pisa) italy. *corresponding authors: e-mail: marco.zuffi@ unipi.it 2 cebc, cnrs university of la rochelle (umr 7372), france submitted on 2016, 27th july; revised on 2015, 25th august; accepted on 2016, 30th august editor: uwe fritz abstract. age and size at maturity are major life history traits, because they influence lifetime fecundity. they represent the outcome from complex interactions among environmental pressures (abiotic and biotic) and individual characteristics. they are also difficult to measure in natural populations and thus they are rarely appraised, especially in reptiles due to the elusive nature of juveniles. using skeletochronology to circumvent these difficulties, this study aims to compare age structures, longevity, age-size relationships, growth curves, age and size at maturation and potential reproductive lifespan in three populations of the european whip snake (two hierophis viridiflavus, one h. carbonarius). we measured the body size and counted the skeletal growth marks on 132 specimens, accidentally killed or from museum collections (72 from nw france [chizé]; 28 from tuscan archipelago [montecristo], italy; 32 from s italy [calimera]). general patterns of age at maturity and longevity were consistent with previous studies based on recapture investigations. strong differences among populations suggest local adaptation to contrasted environmental conditions. these results suggest that skeletochronology is a useful technique that can be applied opportunistically in snakes (e.g., using road-kills) in order to collect otherwise unavailable data that are essential to address fundamental questions regarding longevity, life-history traits and to perform population viability analyses. keywords. insularity, snakes, hierophis, sexual maturity, skeletochronology. introduction iteroparous species with indeterminate growth (e.g., many fish, squamate reptiles) exhibit strong variations in most life history traits in response to environmental factors; body size is highly variable among and within populations for example (wimberger, 1992; madsen and shine, 1993; ryser, 1996; rohr, 1997; zuffi et al., 2007; warner, 2014). considering versatile life history traits, age at maturity is pivotal because fitness is particularly sensitive to changes in this trait (stearns, 1992). similarly, body size exerts strong effects on fecundity, survival, and thus on fitness (shine, 1988, 1990). age and body size at maturity are inextricably linked because they both depend on juvenile growth rate which in turns depends on the trophic and climatic conditions experienced by individuals (sinervo and adolph, 1994; webb et al., 2003). fast juvenile growth promotes early maturation, large body size, and thus likely increases individual potential reproductive lifespan (ryser, 1996; day and taylor, 1997; bronikowski and arnold, 1999). however, many examples suggest that there may be costs associated with fast growth; strong metabolic increase or reduced longevity in early-breeders for instance (beaupre and zaidan iii, 2001; blouin-demers et al., 2002). sexual maturity entails a shift in the allocation of resources used to sustain growth during juvenile phase to reproduction (and secondarily to growth in many 136 sara fornasiero et alii reptiles) during adulthood (shine and charnov, 1992; day and taylor, 1997; rohr, 1997; wapstra et al., 2001; stanford and king, 2004). there is a marked decrease of growth at maturity, although reduced growth may persist through life in many species with marked differences among individuals (congdon et al., 2003). as a result, marked intra-population differences of body size set at maturity are usually maintained in individual later ageclasses (madsen and shine, 1993; shine, 1994; zuffi et al., 2011). individuals that mature at a small size remain small for the rest of their life; thereby counter balancing the fecundity advantages of early maturation (halliday and verrell, 1988). overall, it is expected that the physiological processes that determine maturity, and thus the average age and body size of adults, should maximize lifetime reproductive success through differential adjustments in response to environmental conditions (stearns and crandall, 1981; stearns and koella, 1986; bernardo, 1993; ford and seigel, 1994; wapstra et al., 2001). studies that compare populations of the same species throughout different parts of its distribution range are valuable to understand how environmental factors shape age and size at maturity along with related lifehistory attributes (e.g., age-size relationship, longevity, population age structure; mateo and castanet, 1994; nobili and accordi, 1997; lima et al., 2000; miaud and guillaume, 2005). organisms that display important intraand inter-population variations in body size are of particular interest because these variations may correlate with growth rate and age at maturity (alcobendas and castanet, 2000; miaud et al., 2001; kutrup et al., 2005). unfortunately the relationships between growth rate, body size and age are not easily assessed in the field. indeed, long-term mark-recapture surveys must be set up to collect the raw data necessary to calculate growth rate and to examine key life history traits. but in most reptile species juveniles escape observations (pike et al., 2008; bjorndal et al., 2013). growth rate has been rarely measured accurately before sexual maturity (bonnet et al., 2011) and the exact age of monitored individuals is seldom known in the field (lagarde et al., 2001). this lack of information poses major difficulties to perform analyses. for example, for a given species a single body size for maturity is usually extracted from the literature, used to assign individuals into age categories, and then used to perform various analyses (e.g., regarding population viability, demography). possible variations caused by inter-individual, geographic, and time heterogeneity are systematically neglected. these limitations apply with force in snakes due both to the extremely elusive nature of immature individuals (that precludes estimating accurately the age of individuals) and to the marked phenotypic plasticity of these organisms (madsen and shine, 1993; bronikowski, 2000). skeletochronology offers a reliable alternative (halliday and verrell, 1988). this approach is based on counting the skeletal growth marks (sgm) that are successively deposited on the growing bone (castanet et al., 1992). although this method does not require long term population monitoring, major prerequisites are nonetheless important. continuous growth and a lack of bone remodelling are essential characteristics. the relationships between age and sgm count is accurate in species where an active season precisely alternates with a period of inactivity (e.g., hibernation); it has been validated through mark-recapture in several reptiles (lagarde et al., 2001). many snakes from temperate climates fulfil the above conditions, are spread across a wide range of habitats, and thus they represent suitable candidates to examine the influence of environmental factors on the relationships between age and body size. the aim of this study was to compare the mean age, mean longevity, the age-size relationship, growth curves, age and size at maturation of individuals sampled in three populations of two closely related species of whip snakes (two populations hierophis viridiflavus and in one population h. carbonarius; mezzasalma et al., 2015). the three populations are situated in distant parts of the distribution range of the species characterised by contrasted environmental conditions (one forested site in temperate climate zone and two sites in the mediterranean climate zone; one continental and one insular). we thus expected marked differences among populations in the traits observed. materials and methods study species and study sites the taxonomy of the european whip snake (hierophis [coluber] viridiflavus) has been recently revised (rato et al. 2009; mezzasalma et al., 2015). depending upon the study and criteria, it has been suggested to differentiate several subspecies or species. the debate is not closed because the taxonomic boundaries between species and subspecies are often tenuous. we considered that we sampled three populations and two species, h. viridiflavus and h. carbonarius, of the european whip snake; but we emphasize that we could not rule out the possibility that one species and two subspecies were sampled. thus, for conciseness (and cautiousness) we refer to three populations of the ‘european whip snake’ hereafter. the three populations studied are widely spread across the distribution range of the species: (1) forest of chizé, france (46°07’n, 00°25’w). the site is close to the northern limit of the distribution range of h. viridiflavus; (2) montecristo island, tuscan archipelago, central italy (42°19’54”n, 10°18’38”e) is 137growth, longevity and age at maturity in the european whip snakes situated more than 900 km south-easterly. an isolated population of h. viridiflavus inhabits this rocky island; and (3) surroundings of calimera, lecce (apulia, southern italy; 40°15’n, 18°16’e). this third site located more than 700km further south-easterly is in the southern distribution range of h. carbonarius. more than 1600 km separate the first and third populations whereas climatic and general environmental conditions strongly contrast among all populations. climatological data were obtained from the three meteorological stations closer to the respective study sites: niort (30 km from the forest of chizé), lecce (15 km from calimera) and gorgona island (65 km from montecristo island; elba island is closer to montecristo, but gorgona resembles much more montecristo island in overall surface, vegetation and climatic conditions). while it was possible to obtain data for lecce only for the period between 1961 and 1990, data for france and montecristo are referred to the same time span, between 1998 and 2006. average monthly temperatures (°c) and average monthly rainfalls (mm) for the over cited time intervals are reported in fig. 1. sampling snakes our samples were based on individuals accidentally killed (e.g., road-kills) and museum specimens and no snake was intentionally killed for this study. used animals derived from a quite short time range (less than 10 years), in order to avoid any bias and/or constraint due to a too large time gap that could prevent a proper analysis of age estimation. a total of 132 specimens were analysed during four years: 72 whip snakes from the forest of chizé (mainly road kills; bonnet et al. 1999a), 28 from montecristo island (museum specimens) and 32 from calimera (museum specimens). the specimens were all alcohol preserved and came from the herpetological collections of florence (museo zoologico “la specola”, università di firenze, italy), pisa (museo di storia naturale, università di pisa, italy), frankfurt (senkemberg museum frankfurt a.m., germany), and chizé lab. preliminary analyses showed no effect of sampling date or duration of stay in alcohol before examination (i.e. alcohol did not destroy sgm, at least over several years). each snake was sexed (except newborns and juveniles), and measured (svl, snout-to-vent length) to the nearest mm by stretching it along a measuring tape. snakes were considered adult on the basis of external body coloration and using minimal svl for maturity in each population (fornasiero et al., 2007). very small snakes with typical neonate colouration were considered as newborns; larger immature snakes were considered as juveniles. skeletochronological analysis sgm (skeletal growth marks) reveal temporary variations in osteogenesis rate (castanet et al., 1992). three categories of sgm can be recognised: opaque layers (zones), translucent layers (annuli) and lines of arrested growth (lag) (castanet et al., 1993). zones correspond to fast osteogenesis phases made of badly spatially structured bone matrix, rich in randomly distributed osteocytic lacunae. due to their 3d structure, zones are more opaque than other marks and appear therefore dark when observed under transmitted light (castanet et al., 1993). annuli alternate with zones and correspond to periods of slow osteogenesis. bone matrix is well structured (often being made of lamellar bone) and usually poor in osteocytes. when observed under transmitted light, annuli appear narrower and more translucent than adjacent zones (castanet fig. 1. climatic features of the study sites. a) chizé b) montecristo c) calimera. ( average min. temperature); ( average max. temperature); (• average monthly temperature); (▶ average monthly rainfall). 138 sara fornasiero et alii et al., 1993). lags correspond to a temporary arrest of local osteogenesis. they are narrow, not always visible, typically bordering annuli or, sometimes, appearing inside them (castanet et al., 1993). skeletochronology is based on the assumption that growth marks are the histological expression of temporary and periodical variation in bone growth rate. sgms may originate from endogenous rhythms, but they are influenced and synchronised by external seasonality, like the alternation of hibernation and active season in snakes from temperate climates (castanet and naulleau, 1974; castanet et al., 1993). until sexual maturity, when growth rate is high, annuli or lags are well separated by wide zones of fast-growing tissues, while the subsequent sgm appear narrower and more irregular. this pattern has been named “rapprochement” by francillon-vieillot et al. (1990). although caudal vertebrae can be used for age estimation of living specimens (waye and gregory, 1998), two flat skull bones, the supra-angular and the ectopterygoid especially, are preferred in dead individuals (hailey and davies, 1987). in road kills, pairs of ectopterygoids and supra-angulars (four bones) were removed. in museum specimens, the bones were removed from one side of the head only in order to preserve the external morphology and scalation of the head. the bones were stored in fresh water until organic tissues (e.g., ligaments) became soft and carefully cleaned. counting sgm sgm counting followed the procedure reported by castanet et al. (1993): bones were observed in toto with a binocular microscope, under transmitted light. during the reading of sgm, the bone was kept under water, in order to enhance the contrast between different growth marks. counting was performed by two different people (or by the same observer on separate occasions), always blind to sample identity. parallel readings on the same sample were compared, sgm were counted again in case of discrepancy. if the problem persisted, the sample was discarded. in case of divergence between ectopterygoid and supra-angular counting, ectopterygoid counting was retained because it provides more reliable results (hailey and davies, 1987). sample where strong bone remodelling occurred, obviating age estimation, were discarded. several specious countings were not retained in the analyses. measure of “rapprochement” ectopterygoids of adults presenting a clear sequence of sgm were photographed using a leica dc300f camera assembled with a wild heerbrugg makroskop m 420 1.25×. using an image-editing computer software, the distance from the basis of the first visible zone (corresponding to the first active season bone growth) and the basis of next zone was measured along a straight line broadly perpendicular to sgm and transecting lags (fig. 2). the thickness of each zone + annulus complex represents one year of bone growth (g) expressed as follow: 1 1 1 r rr g nn n = + + 1 2 where gn+1 represents the increment of bone per year in relation to the increment of the first year, expressed in arbitrary units, and r (rings). the analysis was restricted to pictures where the basis of subsequent zones was clearly visible. successive g were examined for the first 12 years in order to detect the expected drop associated with maturity (francillon-vieillot et al., 1990; castanet et al., 1993). the last year before a sharp decrease in mean bone growth was considered to be the onset of sexual maturity. analyses for most comparisons we used analysis of variance (anova) when the distribution of the data did not deviate from normality (kolmogorov-smirnov tests) and u-mann-whitney tests otherwise. mean age and longevity (maximum age recorded) were calculated for each population and each sex. the relationship between age and body size for each sex and each population was examined using pearson’s correlations, possible sex effect was assessed using analysis of co-variance (ancova). growth patterns were estimated using von bertalanffy’s equation, svlt = svlasymp (1 e-k(t-t0)) where t represents number of growing seasons (i.e., years), svlt stands for body length at age t, svlasymp represents the estimated asymptotic body size that can theoretically be reached, k is the growth coefficient, and t0 represents the intercept at the tempofig. 2. parameters for the measure of “rapprochement” (see text for definition). 139growth, longevity and age at maturity in the european whip snakes ral axis, thus hypothetical age at size 0. the parameters svlasymp, k and t0 and their asymptotic confidence intervals were estimated using nonlinear least-squares regressions. the von bertalanffy equation was fitted to age and size data for sexes within a population and for each population. data of newborns of undetermined gender were used to build both male and female curves in all the populations. two estimated svlasymp, k and t0 values were considered to be significantly different (at the 0.95 level) when their confidence intervals did not overlap. the potential reproductive lifespan was calculated for each sex and each population by subtracting the estimated age at maturity from the respective age of the oldest individual found (longevity). the estimated ages at maturity for each sex, within each population, were then substituted in the respective derived bertalanffy’s equation to obtain size at maturity. small sample sizes precluded performing several analyses (e.g. analyses of bone growth patterns were reliable results for chizé and montecristo populations only). results body size, age, longevity and population age structures chizé. the whole sample (n = 72) included 46 males, 24 females and 2 newborn specimens of undetermined sex. sixty-five specimens out of 72 were adults, and, among these, 43 were males and 22 females. adult body size was normally distributed (kolmogorov-smirnov test, z = 0.632, p = 0.820) and adult males were significantly larger than females (t-test, t = 1.171, df = 58, p = 0.001). it was possible to reliably estimate the age of 90.3% (65 of 72; fig. 3) of the whole sample. mean estimated population age was 12.1, ranging from 0 to 24 years; additional details are provided in table 1a. montecristo. the overall montecristo sample (n = 28) included 14 males, 13 females and 1 specimen of undetermined sex. all determined males and females in the population were adults, the only juvenile being of undetermined gender. adult body size was normally distributed (kolmogorov-smirnov test, z = 0.392, p = 0.998) and there was no significant difference in mean adult body length between the sexes (t-test, t = 0.995, df = 23, p = 0.330). it was possible to reliably estimate the age of 92.9% (26 of 28) of the whole sample. estimated mean population age was 19.4, ranging from 0 to 29 years (details in table 1b). calimera. the apulian sample (n = 32) included 21 males, 6 females and 5 newborns or juveniles of undetermined gender. all males were adults, while two out of the six females were subadults; on the whole the sample included therefore 25 adult specimens and seven subadults. adult body size was normally distributed (kolmogorov-smirnov test, z = 0.817, p = 0.517), however, considering the small number of adult females in the sample (n = 4), mean svl was compared between sexes using a mann-whitney u test. the analysis revealed that there was no significant difference in mean adult body fig. 3. examples of sgm reading on ectopterygoids, chizé sample. a) 0 yr; b) 12 yr; c) 20 yr; d) 13 yr; the arrow indicates a double zone, and white dots indicate the age in years. table 1. sample sizes, mean, minimum and maximum age values estimated for hierophis sample. values are reported in years. a) chizé n mean age (± 1sd) min. max. (longevity) whole population 65 12.06 ± 5.06 0 24 adult males 39 14.31 ± 3.11 10 24 adult females 19 11.42 ± 3.58 6 20 subadults 7 1.29 ± 1.98 0 5 b) montecristo whole population 26 19.35 ± 5.87 0 29 adult males 13 22 ± 4.58 14 29 adult females 12 18.08 ± 3.37 11 24 subadults 1 / / / c) calimera whole population 32 15.69 ± 9.31 0 33 adult males 21 19.9 ± 6.33 7 33 adult females 4 18.5 ± 3.70 14 22 subadults 7 1.43 ± 1.62 0 4 140 sara fornasiero et alii length between the sexes (u = 23.500, p = 0.201). this result could however be the consequence of the relatively small data-set of females. although sgms reading was difficult in many cases, as a consequence of bone remodelling and of “rapprochement” of outer sgms, it was possible to estimate the age in 100% of the whole sample. mean population age was 15.7, ranging from 0 to 33 years (details in table 1c). in all the three populations, age values were normally distributed (kolmogorov-smirnov test. chizé: z = 0.969, p = 0.305; montecristo: z = 0.554, p = 0.919; calimera: z = 0.607, p = 0.855) and adult males were on average significantly older than females in chizé and montecristo (t-test, t. chizé: 3.160, df = 56, p = 0.003; montecristo: 2.417, df = 23, p = 0.024), not in calimera (mann-whitney u test = 34.5, p = 0.577). in the three populations age distributions did not differ significantly between the sexes, when considering only adult specimens (kolmogorov-smirnov z test. chizé: 1.230, p = 0.097; montecristo: 1.121, p = 0.162; calimera: 0.611, p = 0.849). in chizé, exact test showed a significant tendency towards a leftshifted age distribution in adult females with respect to adult males (p = 0.033). however, in montecristo and calimera this pattern was not evident (exact test. montecristo: p = 0.093; calimera: p = 0.653). inter-population comparisons mean adult male svl differed significantly among the three populations (anova, with svl as the dependent variable and population as the factor, f2, 71 = 36.106, p < 0.001). a post-hoc test revealed that males from chizé were larger compared to the males from the two other populations (bonferroni post-hoc test, both multiple comparison p < 0.001), while males from montecristo and from apulia did not differ significantly in mean body size (bonferroni post-hoc test, p = 0.086). the same analysis was then performed on adult females (anova, with svl as the dependent variable and population as the factor, f2,32 = 6.794, p = 0.003; homogeneity of variances assumption was not met, but the anova is quite robust to violations of this assumption, zar, 1984). the bonferroni post-hoc test revealed that chizé females were larger than montecristo females (p = 0.003). mean estimated age was significantly different between adult males from the three populations (f2,70 = 19.195, p < 0.001). bonferroni post-hoc multiple comparison test revealed that males from chizé were significantly younger than males from montecristo and from apulia (both p < 0.001), while no differences were found between adult males from the two italian populations (p = 0.575). similarly, adult females from the three sites showed significant differences in mean estimated age (anova, same design, f2,32 = 16.066, p < 0.001), females from chizé being significantly younger than females from montecristo and apulia (bonferroni post-hoc test, p < 0.001 and p = 0.003 respectively). the mean age between the two italian female samples was not significantly different (p = 1). in table 2 we reported results of kolmogorov-smirnov tests on differences in age distributions among sexes and sites: both age distributions of males and females from france are significantly shifted to the left (thus to younger ages) with respect to age distributions of italian whip snakes. moreover, no significant differences in age distributions were found between the two italian populations in both sexes. relationship between age and body size body size was highly correlated with age both in the whole population (pearson correlation. chizé: r = 0.904, p < 0.001; montecristo: r = 0.876, p < 0.001; calimera: r = 0.936, p < 0.001) and considering each sex separately (pearson correlation. chizé: rmales = 0.882, pmales < 0.001, rfemales = 0.838, pfemales < 0.001; montecristo rmales = 0.792, pmales = 0.001, rfemales = 0.818, pfemales = 0.001; calimera: rmales = 0.820, pmales < 0.001; rfemales = 0.921, pfemales = 0.009). differences in mean age between the sexes were then re-analyzed taking into account this relationship. an ancova performed with age as the dependent variable, svl as the covariate and sex as the factor revealed that table 2. results of statistical comparisons of age distributions between the different populations. sex populations compared n1 n2 z asymp. sig. exact sig. males chizé montecristo 39 13 2.242 < 0.001** < 0.001** chizé calimera 39 21 2.260 < 0.001** < 0.001** montecristo calimera 13 21 0.540 0.933 0.788 females chizé montecristo 19 12 2.117 < 0.001** < 0.001** chizé calimera 19 4 1.435 0.033* 0.016* montecristo calimera 12 4 0.577 0.893 0.807 141growth, longevity and age at maturity in the european whip snakes there was no difference between the sexes in age in chizé and calimera snakes, when body size was taken into account (chizé: f1,55 = 0.216, p = 0.644; calimera: f1,23 = 0.007, p = 0.935). ancova on montecristo snakes, on the contrary, revealed a significant effect of gender on age (f1,22 = 6.603, p = 0.017). it has to be noted, however, that in all the three populations, the assumption of homogeneity of slopes was not met (p < 0.001). a t-test was then performed comparing the unstandardized residuals obtained from the linear regression of the ln-transformed estimated age on the ln-transformed svl. the result again highlighted the absence of any significant sexual difference in size-corrected age in chizé and calimera snakes (t-test. chizé: t = 0.177, df = 22.486, p = 0.861; calimera: t = −0.331, df = 24, p = 0.744). males from montecristo were on average older than females when estimated age was corrected for body size (t = 2.473, df = 23, p = 0.021). inter-population comparisons taking into account the positive correlation between age and body size, males from the three populations exhibited significant differences in estimated age (ancova, with age as the dependent variable, svl as the covariate and population as the factor, f2,69 = 89.752, p < 0.001). bonferroni post-hoc multiple comparisons were all highly significant (all p ≤ 0.001), showing that, for similar svl values, montecristo males were the oldest, while males from chizé were the youngest. however, the assumption of homogeneity of slopes was not met (p < 0.001). yet the differences among populations were marked (fig. 4a), suggesting that possible effect of violation of homogeneity assumption on our main conclusions was limited. the analysis was then performed by considering the unstandardized residuals obtained from the linear regression of the ln-transformed estimated age on the ln-transformed svl. results confirmed that population differences in mean age, among males, were not dependent on differences in mean body sizes (anova, with residuals as the dependent variable and population as the factor, f2,68 = 191.326, p < 0.001; all post-hoc multiple comparisons p < 0.001; fig. 5). within females, age was significantly different among populations, when age-size correlation was considered (ancova, with age as the dependent variable, svl as the covariate and population as the factor, f2,32 = 41.042, p < 0.001). for similar svl values, females from chizé were significantly younger than females from the other two populations (bonferroni post-hoc test, both p < 0.001), while no differences were found between italian females from montecristo and from apulia, even if there was a trend towards older island females and the small apulian sample size may have influenced the result (p = 0.074; fig. 4b). unstandardized residuals of the linear regression of the ln-transformed estimated age on the lntransformed svl were furthermore analysed, and the over mentioned result was confirmed (anova, with residuals as the dependent variable and population as the factor, f2, 33 = 34.870, p < 0.001). females from chizé were significantly younger than females from both montecristo (posthoc dunnett test, p < 0.001) and calimera (post-hoc dunnett test, p = 0.05), while the difference was not significant between females from italian populations (posthoc dunnett test, p = 0.149; fig. 5). fig. 4. relationship between age and body size in males (a) and females (b) of the three populations. (• chizé); (▶ montecristo); (o calimera). 142 sara fornasiero et alii population growth curves in the three studied populations, derived values for svlasymp, k and t0, their asymptotic confidence intervals and the coefficients of correlation of the von bertalanffy equation fitted separately on male and female data (table 3). in the three populations, modelled curves fitted well the relation between age and body size in both sexes (correlation coefficients in table 4). growth curves had similar shapes for males and females (chize: fig. 6a; montecristo: fig. 6b; calimera: fig. 6c): even if svlasymp was higher in males and k was higher in females, these differences were not significant (confidence intervals widely overlap in all considered populations). in the snakes from chizé, the estimated male svlasymp was very close to actual maximum svl observed in the field (1200 mm, n > 1,600 records; x. bonnet, personal unpubl. data), while the von bertalanffy model slightly underestimated the asymptotic body length for females (maximum svl observed in the field 1080 mm, n > 1,100 records; x. bonnet, personal unpubl. data). in the snakes from montecristo the model provided a satisfactory male asymptotic size (maximum svl observed in the field 873 mm, n = 53) but slightly overestimated it in females (maximum svl observed in the field 790 mm, n = 30). in the snakes from calimera the model slightly overestimated asymptotic sizes, both in males (maximum svl observed in the field 920 mm, n = 16) and in females (maximum svl observed in the field 825 mm, n = 4). inter-population comparisons of growth comparisons of growth parameters are reported in table 3. while the estimated k and t0 for males were table 3. coefficients of correlation and parameters of the von bertalanffy’s estimated model for male and female h. viridiflavus from the three populations considered. 95% confidence intervals are in parentheses. sex svlasymp k t0 r2 chizé males 1216.97 (1032.86-1401.10) 0.086 (0.052-0.119) −2.69 (−3.85-−1.52) 0.925 females 1012.38 (811.47-1213.28) 0.123 (0.049-0.197) −2.19 (−3.83-−0.55) 0.907 montecristo males 843.05 (686.61-999.50) 0.068 (0.025-0.110) −5.01 (−8.29-−1.75) 0.943 females 854.20 (637.19-1071.22) 0.071 (0.018-0.124) −4.72 (−7.84-−1.60) 0.953 calimera males 965.60 (843.36-1087.82) 0.073 (0.043-0.103) −4.10 (−5.85-−2.34) 0.958 females 861.67 (690.74-1032.59) 0.095 (0.028-0.162) −3.55 (−5.84-−1.27) 0.977 table 4. coefficients of correlation and parameters of the von bertalanffy’s estimated models for the three populations considered. 95% confidence intervals are in parentheses. population svlasymp k t0 r2 chizé 1178.22 (1028.90-1327.54) 0.091 (0.060-0.122) −2.55 (−3.66-−1.44) 0.901 montecristo 820.81 (731.52-910.11) 0.077 (0.047-0.107) −4.55 (−6.92-−2.17) 0.907 calimera 948.26 (842.03-1054.50) 0.076 (0.048-0.105) −3.94 (−5.56-−2.31) 0.954 fig. 5. intra population and intraspecific differences in size-corrected age. grey=males; white=females. 143growth, longevity and age at maturity in the european whip snakes comparable across populations with a strong overlap of the 95% confidence intervals, there was a limited overlap between confidence intervals for asymptotic svl between chizé and calimera (5.05%) and no overlap with montecristo (see table 3). this suggests that males from different populations attained different asymptotic body sizes following comparable growth rates. a different pattern was observed in females: k and t0 were relatively similar across populations with strongly overlapping of 95% confidence intervals, and svlasymp confidence intervals were overlapping (even if estimated values were dissimilar between populations). we note however, that the small calimera sample limited the power of this analysis. the absence of significant male-female differences for growth parameters within each sample allowed to estimate cumulative population growth curves by fitting the von bertalanffy equation to each population entire data set. derived values for svlasymp, k and t0, their asymptotic confidence intervals and the coefficients of correlation of the von bertalanffy equation fitted for each population are reported in table 4. growth curves for the three sites are plotted in fig. 6. while the growth coefficient k and the estimated t0 had similar values throughout populations, and their 95% confidence intervals strongly overlapped, there was only a negligible overlap (e.g., 2.43%) between confidence intervals of asymptotic svl of chizé population and of calimera population. there was no overlap between the french versus insular populations. moreover, the asymptotic svl estimated for the two italian populations overlapped only marginally, suggesting that, even if less pronounced, differences in growth pattern also exist between these two populations (fig. 7). these results suggested that whip snakes from the forest of chizé (h. viridiflavus) follow different growth trajectories compared to the two italian populations (h. viridiflavus, h. carbonarius) and attain larger maximal size. derived values of the two main growth parameters, svlasymp and k, for males, females and the whole sample fig. 6. population growth curves for h. viridiflavus at a) chizé; b) montecristo; h. carbonarius at c) calimera. (• males); (o females); (▶ juveniles). fig. 7. estimated von bertalanffy’s growth curves for chizé (—), montecristo (....) and calimera (-) populations. 144 sara fornasiero et alii were inversely correlated within each population; this relationship was significant for the populations of chizé and calimera, while it was not for the population of montecristo island (pearson correlation, r = -0.999, p = 0.035, r = -1, p = 0.019 and r = -0.785, p = 0.425 respectively). “rapprochement”: age and size at maturity and potential reproductive lifespan it was possible to clearly measure successive annual bone growth marks for 4 males and 6 females in chizé, for 9 males and 7 females in montecristo and for for 10 males and only 2 females in calimera. in chizé, the annual bone growth showed a sudden decrease between the seventh and the eighth year of age for males (see also fig. 8a, an example for all the populations) and between the sixth and the seventh for females (fig. 8b). it is therefore likely that males hierophis viridiflavus of this population attain sexual maturity at 7 years of age, while females become reproductive when they are one year younger, at 6 years of age. in both sexes bone growth was particularly rapid during the second active season, and then it showed a decrease, followed by a subsequent increase and a slowing down until the sharp decrease in connection with sexual maturation. from these estimated values it emerged a potential reproductive lifespan of 17 years for males and 14 for females. using the derived bertalanffy’s equation for each sex, it emerged that male size at maturity is 687.9 mm, while females reach sexual maturity at a mean size of 642.6 mm. in montecristo, annual bone growth in males showed the highest mean value during the second year of life, then it decreased but remained at more or less constant values until the eighth year, when it showed a strong decrease. therefore males hierophis viridiflavus of this population likely reach sexual maturity when they are 8 years old. females showed a bone growth pattern characterised by a decrease of growth after the second active season, followed by a subsequent increase and a slowing down until sharp decrease between the sixth and the seventh years. however, mean annual bone growth slowed down under initial values only after the eighth year. it is therefore likely that island females, as island males, attain sexual maturity at 8 years of age. substituting these values in the derived bertalanffy’s equation for each sex it emerged that male size at maturity is 495.1 mm, while females reach sexual maturity at a mean size of 507.9 mm. calculated potential reproductive lifespan for males and females were, respectively, 21 and 16 years. in calimera, unfortunately, as a consequence of bone remodelling, lags on ectopterygoids of this population were not so sharply differentiated on the bone surface and measurements of annual radius (e.g., rn,) were sometimes performed quite subjectively. the following results are therefore only indicative. male annual bone growth showed the highest mean value during the third year of life, and then it suddenly decreased. the last drop off was in correspondence of the sixth active season (when mean annual bone growth slowed down under initial values). male hierophis viridiflavus of calimera may therefore attain sexual maturity during their sixth year of life, with an estimated potential reproductive lifespan of 27 years. bone growth pattern was obtained only for two females; hence it was not possible to estimate age at maturity for females of this population. subfig. 8. annual bone growth (mean g) in males (a) and females (b) hierophis viridiflavus from chizé. lines connect mean values, bars represent ±1sd. age in years in the abscissa. 145growth, longevity and age at maturity in the european whip snakes stituting male estimated age at maturity the respective derived bertalanffy’s equation it emerged that male size at maturity is 503.7 mm. inter-population comparisons of age at maturity. only chizé and montecristo populations with reliable estimated age at maturity were considered. plotting estimated values for each sex we observed a positive trend (not significant, pearson correlation, r = 0.827, p = 0.173), between the estimated age at maturity and longevity. moreover, there was a clear positive relationship between the estimated svl at maturity and asymptotic body length (svlasymp). this correlation was statistically significant (pearson correlation, r = 0.954, p = 0.046). discussion skeletochronology enabled us to estimate the age of most of the sampled individuals, providing novel information (otherwise unavailable) in both sexes and for wide spectrum of body sizes. below we first examine broad patterns and then population divergences. broad patterns in the european whip snake, estimated size at maturity ranged from 57% to 64% of the maximal body size (in chizé males and montecristo females respectively), in accordance with previous studies in snakes (shine and charnov, 1992). the positive correlations between estimated age at maturity and longevity, or between size at maturity and bertalanffy’s asymptotic length, also conform to the general pattern described in ectotherms in general (shine and charnov, 1992) and in snakes more specifically (parker and plummer, 1987). these relationships represent trade-offs between maturation programmes, growth patterns, and costs versus benefits of large body size (shine and charnov, 1992). the congruence of our results with previous studies suggests that skeletochronology provided useful information. in the studied populations, we found a strong relationship between estimated age and body size (all r2 > 0.91). following a fast growing juvenile phase, growth rate decreased with body size. similar results have been documented in other reptile species monitored through capture-mark-recapture surveys (cmr); growth following asymptotic patterns derived from the von bertalanffy curve (dunham, 1978; james, 1991; el mouden et al., 1999; bonnet et al., 2011). however, despite a strong relationship between age and size, there was a considerable variance in age-related body size, especially among older individuals. our study confirms that svl is not a reliable estimator of age in snakes, especially after maturity and in large specimens. many idiosyncratic and environmental factors influence growth in snakes, notably after maturity, and can explain the strong inter-individual divergences of trajectories (bronikowski, 2000; madsen and shine, 2000; bonnet et al., 2002; lelièvre et al., 2013). sex is one of those important factors (koos slob and van der werff ten bosch, 1975; kuwamura et al., 1994). on average males were older and attained greater maximal longevity compared to females. in the three populations studied, male and female growth curves were not significantly different. male hierophis showed the highest values of svlasymp and the smallest values of k within each population: this could means that i) the tendency for males to reach a larger maximum size approaching this asymptote at slower rate than females is overall present in the populations considered or that ii) males grow more rapidly than females after maturity. yet, because they continue to grow they need longer time to reach their maximal size. in other words, the trend for a cessation of growth in females means that they reach earlier maximal svl, but not at a faster rate. consequently, the observed male-biased sexual size dimorphism (ssd; fornasiero et al., 2007; zuffi, 2007) was essentially attributable to a longer period of growth after maturity in males (king, 1989). sexual bimaturation (sexes maturing at different ages) can also influence ssd (shine, 1990, 1994): because growth decreases after maturity, earlier maturing sex tend to exhibit smaller mean adult sizes (e.g., lagarde et al., 2001). our results provide a partial support to this scenario. in the northern population (chizé), males reached maturity one year after females, no difference was observed in the montecristo population, and a small sample size precluded performing robust analyses in the apulian population. overall, a longer growth period after maturity in male european whip snakes may explain the larger mean body size in this sex. however, the respective contribution of possible underlying mechanisms remains unknown. many males are killed during mate searching and suffer from a high-risk mortality compared to females (bonnet et al., 1999a; meek, 2009). this sex difference in mortality should induce a female biased ssd and thus does not fit well with the above results. however, survival in emaciated females after reproduction can be very low in snakes (bonnet et al., 1999b). examining the effect of sex and age on survival is necessary to clarify these issues (bonnet et al., 2011). beside fundamental morphological differences (bonnet et al., 1998) various ecological and physiological factors may generate sexual divergences in growth rate. 146 sara fornasiero et alii during the mating season male european whip snakes engage into vigorous ritual combats and display very high testosterone levels (bonnet and naulleau, 1996). in reptiles, this androgenic steroid stimulates sexual behaviours and growth, resulting into male biased ssd (cox et al., 2009). moreover, high reproductive investment controlled by high estradiol levels during vitellogenesis exhausts body reserves and can markedly hampers growth in females (bonnet et al., 1994, 2011; van dyke and beaupre, 2011). in species exhibiting male-to-male combats, selection for large body size of males tends to overrule selection for large body size to accommodate larger clutches in females (shine, 1993, 1994). european whip snakes display all these traits; lower growth rate in females than in males is thus expected in this species but our results show no sex effect. yet, other key factors may balance growth rates between sexes. for example, males tend to be anorexic during the mating season while females forage during the whole active period (bonnet and naulleau, 1996), females possess more developed attributes to process food (bonnet et al., 1998), and thus females may assimilate greater amounts of food available for growth compared to males. sex difference in prey selection can also influence body size (zuffi et al., 2010). overall, our results regarding age and size pose more questions than offering responses; cmr studies combined with eco-physiological investigations are necessary to obtain a general understanding of the determinants of ssd in free ranging snakes. inter-population variations we found strong differences in mean adult body size, mean adult age, age distribution, longevity, growth rate, age and size at maturity among the three populations studied. these results are important to better interpret already documented inter population variations in body size and reproductive traits (fornasiero et al., 2007; zuffi et al., 2007). the most salient differences were observed between genetically close populations (h. viridiflavus [sub]species) respectively sampled in the northern and southern parts of the distribution range (chizé versus montecristo). the genetically distinct third population (h. carbonarius from apulia) was relatively more similar to the island population (montecristo). thus genetic proximity did not translate into similarities in age/size life history traits. on average, the snakes from chizé were markedly larger and younger compared to the two other populations. less pronounced differences in body size were found between specimens from montecristo and apulia. snakes from chizé reached larger asymptotic size without difference in the bertalanffy’s growth coefficients (k), and thus exhibited higher absolute growth rate than snakes from montecristo and apulia (dunham, 1978; wapstra et al., 2001; stanford and king, 2004). additionally, french specimens exhibited lower longevity compared to italian snakes. growth rate and mean body size should be higher at lower latitudes in ectotherms due to more favourable temperatures and longer activity period. on the contrary, we found a reverse trend, likely because mediterranean, dry and arid habitats of the italian populations offer less favourable trophic (zuffi, 2007) and hydric conditions compared to more productive areas typical of mild oceanic climate. in fact, during drought periods snakes remain sheltered to maintain their hydro-mineral balance (bonnet and brischoux, 2008), even entering into a prolonged estivation period (vipera aspis, m.a.l. zuffi, unpublished data). hierophis carbonarius from calimera follows an intermediate growth trajectory between the opposite extremes represented by the insular and the northern population, being however closer in this pattern to the former one. limits of the study estimating age with skeletal marks does not pose major problems to assess broad patterns for comparisons among sexes and populations because possible methodological biases will apply equally in the different groups examined. however, absolute values should be considered with caution. inferring the exact age at maturity and exact growth rates rely on a set of assumptions. our results suggest that whip snakes from montecristo island mature at an estimated age of 8 yr, and at a minimum derived body size (svl) of 495 and 507 mm in males and females respectively. but the smallest female with developed follicles from montecristo measured 598 mm in svl, leading to an estimated age of 12 yr according to our growth curves. this discrepancy might be due to insufficient sampling of reproductive females in the field (n = 18), to imprecision in the estimates (e.g., small sample size in juveniles hampered comparing linear versus nonlinear functions to select the best fitting equations between age and body size) or due to other factors (e.g., a lack of perfect correspondence between reduced bone growth and maturity). indeed, 8yr is already an elevated age for maturity for snakes, 12yr would be a remarkable value. our results in the chizé population suggest an estimated age at maturity of 7 yr for males and 6 yr for females, with a derived minimum size at maturity of 687 and 642 mm in svl respectively. the smallest reproductive female found in the forest of chizé measured 680 mm in svl (x. bonnet, unpubl. data), a value 147growth, longevity and age at maturity in the european whip snakes in accord with skeletochronology. however, using our results, it also suggests an estimated age of 7 yr for maturity, a value that does not fit well with cmr data. lelièvre et al. (2013) showed that juvenile growth rate averages 0.04 cm/day in chizé, leading to an age for maturity of 3-4 years. as above, this discrepancy might be caused by inappropriate sampling (e.g., few road-kill juveniles were found intact) or to insufficient fitting of the growth models used. further analyses based on larger number of juveniles are required to better calibrate skeletochronology to cmr data and to take into account marked interindividual differences in growth trajectories. whatever the case, the strong differences of age at maturity observed between populations likely reflect adaptation to local conditions. the markedly slow growth, delayed maturity, smaller body size and higher longevity of montecristo snakes are expected in a dry environment where the acquisition of trophic resources (see also zuffi, 2001) is more challenging than under mild oceanic climate. relative clutch size, an index of energetic investment per reproductive bout is lower in montecristo snakes, in accordance with the notion that resources availability is limited in this island (zuffi et al. 2007). populations respond by shifting a set of traits along a slow-fast gradient (stearns and koella, 1986; wapstra et al., 2001). using dead snakes, our results suggest a strong plasticity of major traits driven by trophic and climatic conditions. collecting opportunistically and examining dead snakes might be useful to address key questions. major parameters, especially before maturity, can be inferred and implemented to calibrate models that aim to examine the impact of climatic changes. indeed currently implemented mean values do not permit to encompass the wide range of variations observed among individuals and across populations. acknowledgments we thank jacques castanet, museum national d’histoire naturelle in paris for support during sf stay in paris. gunther köhler at senkenberger museum provided several montecristo samples. two anonymous referees and hervé lelièvre provided helpful comments on previous drafts and revision. references alcobendas, m., castanet, j. 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(eds). what works in conservation. open book publishers, cambridge, uk. http://dx.doi.org/10.11647/obp.0060 sebastiano salvidio acta herpetologica 13(1): 51-64, 2018 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-20693 natural history and conservation of the nurse frog of the serranía del perijá allobates ignotus (dendrobatoidea: aromobatidae) in northeastern colombia hernán darío granda-rodríguez1,2, andrés camilo montes-correa3,*, juan david jiménez-bolaño3, marvin anganoy-criollo4,5 1 programa de postgrado en áreas silvestres y conservación de la naturaleza, facultad de ciencias forestales y de la conservación de la naturaleza, universidad de chile, santiago, chile 2 programa de biología, universidad del magdalena, santa marta, colombia 3 grupo de investigación en manejo y conservación de fauna, flora y ecosistemas estratégicos neotropicales (miku), universidad del magdalena, santa marta, colombia. *corresponding author. e-mail: andresc.montes@gmail.com 4 grupo de cladística profunda y biogeografía histórica, laboratorio de anfibios, instituto de ciencias naturales, universidad nacional de colombia, bogotá d.c., colombia 5 laboratorio de anfibios, instituto de biociencias, universidad de sao paulo, brasil submitted on: 2017, 25th may; revised on: 2017, 6th september; accepted on: 2018, 1st march editor: emilio sperone abstract. we describe new findings on the geographic distribution, habitat uses, relative abundance, tadpoles, and advertisement call of allobates ignotus. data of habitat uses and relative abundance were collected during five annual fieldtrips of seven days each one, from april 2010 to june 2014, in a mining zone of canime creek basin, la jagua de ibirico, cesar department, northeast colombia. tadpoles and advertisement call were obtained in la veguita creek basin, manaure balcón del cesar, cesar. we report six new localities with presence of a. ignotus, all between 7-96 km in straight line to the type locality, from 194-1236 m a.s.l. this species is recorded for the first time in the tropical dry forest life zone and in la guajira department. four substrate categories used by a. ignotus were detected (leaf-litter, rocks, naked floor, and lower branches), and differential use was highlighted. the larval morphology of a. ignotus was described, based on 11 specimens between 25-29 stages. like other allobates tadpoles, the gap of the second teeth row is “wide”. the advertisement call of a. ignotus is composed by a series of high frequency ascending pulsed notes. possibly, the high frequency of the call is due to the acoustic disturbance generated by the streamflow noise of the creeks. in 2014, a decrease of relative abundance of a. ignotus in canime creek was detected. this, coupled to restricted distribution and loss of habitat quality are sufficient criteria to suggest the category of vulnerable for a. ignotus. keywords. advertisement call, colombian andes, colombian caribbean, geographic distribution, habitat uses, relative abundance, tadpoles, tropical dry forest. introduction the serranía del perijá is a mountain system of the northern south america, whose highest points make up the northern boundary between colombia and venezuela. because its geographic position, the serranía del perijá functioned in the past as a biological corridor between andes and caribe, showing biogeographical affinities with the sierra nevada de santa marta (= santa marta mountains) and the cordillera oriental (= colom52 hernán darío granda-rodríguez et alii bian eastern andes) (hernández-camacho et al., 1992). currently, the serranía del perijá is considered of great importance from an amphibian biodiversity approach by the high endemicity, deserving to constitute a distinct ecogeographic unit (romero-martínez and lynch, 2012). in colombia, 14 species of the genus allobates zimmerman and zimmerman, 1988 (aromobatidae) are recognized, broadly distributed in the wet and dry forests of the western and eastern lowlands of the country (acostagalvis, 2017). however, there are gaps in the knowledge of the colombian allobates about of their taxonomy and natural history (anganoy-criollo, 2012). for the species of this country, there are some notes on the abundance, habitat use, conservation and feeding ecology of allobates juanii (cáceres-andrade and urbina-cardona, 2009; astwood-romero et al., 2016), and a bit more information for allobates femoralis and allobates talamancae (v. gr. junca and eterovick, 2007; ringler et al., 2009; hopkins and lahanas, 2011; montanarin et al., 2011; keafer et al., 2012; lechelt et al., 2014), even if these data do not come from colombian populations. for all other colombian allobates, the information on their geographic distribution, habitat use, and calls, is mostly provided only by the original (re-)descriptions. allobates ignotus anganoy-criollo, 2012 (fig. 1) is an aromobatid frog, only known from its original description, endangered, and endemic to the serranía del perijá (iucn ssc amphibian specialist group, 2017). in fig. 1. specimens of allobates ignotus from canime creek (a), la frontera’s way (b), la veguita creek (c), cesar department, colombia; and quebrada nicaragua (d), la guajira department, colombia. photographs by h.d. granda-rodríguez (a), a. del portillo-mozo (b), and j.d. jiménez-bolaño (c, d). 53natural history and conservation of allobates ignotus the distribution of this species, only three localities are known (anganoy-criollo, 2012; acosta-galvis, 2017) with very few available information. as a consequence, being many aspects of the biology, ecology, and natural history of a. ignotus unknown, it is hard to determine the conservation status, threats, and measures to species protection. in this paper, we report new data on several aspects of the natural history (geographic distribution, free-swimming tadpole, advertisement call, habitat use, and abundance) and conservation status of a. ignotus. materials and methods study area all records of allobates ignotus were collected on the foothills and midlands on the western flank of the serranía del perijá. these localities have a bimodal rainfall regime, with high to moderate water deficit in dry periods (rangel-ch. and carvajal-cogollo, 2012). the first rainy peaks is from april to may and second from september to october. the two periods of low rains are from december to march and in july (rangelch. and carvajal-cogollo, 2009). the foothills of the serranía correspond to the “zonobioma tropical alternohígrico” (follow to hernández-camacho and sánchez 1992) or to the tropical dry forest life zone (sensu holdridge, 1967). the midlands of the serrania is a biome known as “orobioma de la selva subandina” (hernández-camacho and sánchez, 1992) or as moist forest, wet forest and rain forest life zones (holdridge, 1967). the latter has a low water deficit in dry periods (rangel-ch. and carvajal-cogollo, 2009). habitat use and abundance we obtained the relative abundance, habitat and microhabitat uses data in three sections of the canime creek (sector 1: 09°33’16.10’’n, 73°16’00.90’’w, 250 m a.s.l.; sector 2: 09°36’10.20’’n, 73°16’53.70’’w, 200 m a.s.l.; sector 3: 09°34’22.42”n, 73°15’52.24”w, 194 m a.s.l.), a quaternary tributary of the cesar river, in the municipality of la jagua de ibirico, cesar department, northeast colombia. surveys were conducted in a mining area, where most of the land corresponds to coal exploitation zones and deposits of waste material. grasslands, wooded savannas, and cattle pastures occupy another larger percentage of the land use. the tropical dry forest cover is established in a smaller proportion, only in the margins of streams, in mosaics of gallery forest, bushes, stubble, and deciduous forest. we performed five field trips to the canime creek, one in dry season (february 2011) and four in rainy season (april 2010, august 2012, april 2013, and june 2014), for a total of seven days for each trip. for frogs sighting, we used the visual encounter survey (ves) (crump and scott, 1994). daily, two observers conducted random walks between 09:00-12:00 h and 15:00-16:00 h, for a total sampling effort of 109 h × observer. we recorded the substrate just under the specimen at the time of sighting. we collected some voucher specimens, sacrificed on immersion to chlorobutanol solution (10%). all collected vouchers were deposited in the amphibian collection of the centro de colecciones biológicas de la universidad del magdalena, at santa marta, colombia (cbumag:anf). to confirm the species level determination, the collected voucher was directly compared to the type series of a. ignotus deposited in the amphibian collection of the instituto de ciencias naturales at universidad nacional de colombia (icn 55434, holotype; 55427-433, 55435-439, paratypes) relative abundance was calculated as the number of individuals in each sample relative to capture effort, expressed in individuals/hours × observer (ra = ind/h × obs) (lips, 1999). we performed a homogeneity chi square test (χ2) in order to reject the null hypothesis that there is no differential use of the microhabitat by a. ignotus. the expected values were calculated equitably from each substrate category detected in field, assuming that all substrates had the same probability to be occupied. free-swimming tadpoles from december 2015, we collected tadpoles in la veguita creek (10°23’12.57”n, 73°03’19.03”w, 633 m a.s.l.), la veguita village, municipality of manaure balcón del cesar, cesar department, colombia. this creek is surrounded for a secondary tropical dry forest relict, on a slope of approximately 20°, with a more or less staggered configuration of small waterfalls and pools, and abundant boulders. la veguita creek is the repository of waste waters of the municipality, showing grayish color. we found the tadpoles on the marginal puddles of the stream. these puddles had a low depth (< 20 cm) and recorded water temperature was 28 °c. the puddle bottom was very sediment, but column was transparent. tadpoles were sacrificed in an immersion in the formaldehyde solution (10%). we assigned the tadpoles to allobates ignotus because the metamorphic individuals observed in the same site have adult features (dorsolateral, oblique, and ventrolateral stripes, and color pattern) of a. ignotus. in addition, the only one dendrobatoid frog detected in the study area was this species. description was based on a set of 11 specimens (cbumag:anf 936), collected by j.d. jiménez-bolaño, j.a. rincón, and a.c. montes-correa. no other species of allobates (or any other dendrobatoid frog) were found in la veguita creek. here, we provided a description of the free-swimming tadpoles at stages 25-29 (according to gosner, 1960). no other developmental phases were available (i.e., neither back-riding tadpoles nor metamorphic tadpoles). measurements (in mm) and proportions are provided for all tadpoles. larval terminology were determined according to altig and mcdiarmid (1999). to describe the attached type of the vent tube to the ventral fin, we follow the figure 3.5 of altig and mcdiarmid (1999). description and measurement of the number of marginal papillae on each lip followed anganoy-criollo (2013) and disposition of the row of marginal papillae followed sánchez (2013). values of the gap of the second tooth row of other species of allobates were obtained from the measurements or illustra54 hernán darío granda-rodríguez et alii tions of the oral disc of free-swimming tadpoles provided in each description. measurements were taken with a manual caliper (0.01 mm) and with an ocular micrometer of a carl zeiss stemi 2000 stereomicroscope (0.001 mm). for morphological characters the following abbreviations were used: (al gap) anterior lip gap, (a-2 gap) medial gap in second anterior tooth row, (bh) body height, (bl) body length, (bw) body width, (end) eye-nostril distance, (ind) internostril distance, (iod) interorbital distance, (ljw) lower jaw sheath width, (odw) oral disc width, (p-al) papillae on the right side of anterior lip, (p-pl) papillae on posterior lip in a space of 1 mm, (sh) spiracle height at its base, (sl) spiracle length, (snd) snout-nostril distance, (soh) spiracle opening height, (ssd) snout-spiracle distance, (tal) tail length, (tmw) tail muscle width at its base, (tmh) tail muscle height at its base, (tl) total length, (ujw) upper jaw sheath width, (vt) vent tube length. advertisement call from december 2015, we collected three recordings of advertisement calls of three males in la veguita creek, village of la veguita, municipality of manaure balcón del cesar, department of cesar, colombia. digital recordings were made at a sampling rate of 44 khz and 16 bit resolution with a sony lcd-px 240 voice recorder with built-in microphone. we collected 281 sec of total recording. one of the males was preserved as a voucher specimen. calls were recorded at 16:20 pm, with a temperature of 25.4 °c under shade. calls were analyzed in praat 6.0.13 for windows (boersma and weenink, 2007). each sound file of each individual contained four to six calls, which were analyzed individually. the acoustic parameters measured were: call duration (sec), number of notes per call, note duration (sec), internote interval (sec), and rate of notes per sec (notes/sec); as well as the spectral properties, such as dominant frequency and frequency range, with respective mean and standard deviation (sd) (littlejohn, 2001; köhler et al., 2017). graphics was produced with the r package seewave (sueur et al., 2008). results geographic distribution based on museum specimens and field observations, we recorded six new localities with confirmed presence of allobates ignotus (fig. 2); five localities in cesar department (three sectors in the canime creek, 194-250 m a.s.l., la jagua de ibirico; la frontera’s way, 1236 m a.s.l., agustin codazzi; la veguita creek, 570 m a.s.l., manaure balcón del cesar) and one locality in la guajira department (nicaragua creek , 905 m a.s.l., la jagua del pilar). the new localities are between 7-96 km in straight line from the type locality, between 194-1236 m a.s.l. the life zone of these localities is tropical dry forest and the transition with the low montane wet forest. the new records widen the occurrence of the species to 605 km2, and represent the first record of a. ignotus in tropical dry forest and la guajira department. habitat uses in the three sections of the canime creek, la jagua de ibirico lowlands, we recorded 187 individuals of allobates ignotus, all restricted to low flow and shallow creeks, with abundant gallery forest of closed canopy. allobates ignotus were not recorded in the deciduous forest, bushes, stubble, or pastures adjacent to the riparian forest. in the gallery forest interior, we determined four categories of microhabitat (substrate) occupied by allobates ignotus: leaf-litter, rocky substrate (boulders-likes), fallen trunks, and lower branches (<50 cm to floor). allobates ignotus uses differentially the available substrata (χ2 = 51.65, df = 3, p < 0.001, table 1): 46.64% were recorded active on the leaf-litter; a slightly lower percentage fig. 2. geographic distribution of allobates ignotus in colombia. blue (light grey in the printed version) symbols for known localities and red (dark grey in the printed version) symbols for new records. development by lorena benitez-cubillos. table 1. frequency and homogeneity chi square test (χ2) of microhabitat uses by allobates ignotus in canime creek basin, cesar department, colombia. substrata n χ2 df p leaf-litter 76 18.301 rocks 65 7.124 fallen trunks 30 6.001 lower branches 16 20.226 total 187 51.652 3 <0.001 55natural history and conservation of allobates ignotus (34.76%) occurred on rocky substrates; fallen trunks and lower branches were used to a lesser extent (16.04% and 8.56%, respectively). free-swimming tadpoles all measurements and proportions of free living tadpoles are given in table 2. tadpoles have an ovoid body in dorsal view, wider at mid-body; depressed in lateral view (fig. 3a-c), barely higher at level of intestines (bh/ bw = 0.67-0.80; bw/bl = 0.57-0.71), shorter than half of the total length (bl/tl = 0.27-0.39). snout rounded in dorsal and lateral view (fig. 3a, c). the nostrils are rounded or slightly ovoid, positioned dorsally, and opening directed dorsolaterally; located about midway between the eye and the tip of snout; nostril 25-36% of eye diameter; inner margin with a thick fleshy ring and very low fleshy projection dorsally, sometimes no visible. the internarial distance is equivalent to 53-75% of interorbital distance. interorbital distance 43-55% of body width. eyes dorsal, directed dorsolaterally. eye diameter larger than nostril-eye distance (except a tadpole in stage 27, with eye about equal to the nostril-eye distance) and 39-51% of interorbital distance. spiracle sinistral, slightly longer than high (0.9-1.2 times) with cylindrical shape (i.e., the spiracle height is the same through of spiracle length). spiracle directed posterodorsally is located below longitudinal midline and on vertical midline of body. spiracular opening ending 63-77% of body length (from tip of snout), and is barely separated from body. vent tube short, 8-15% of body length, attached to right side of ventral fin in b-type; opening of vent tube with posterior direction and smooth edge. inner intestines were visible through translucent skin; the longitudinal axis of intestines is sinistral to body; guts in 5-10 coils in ventral view (fig. 3b). tail length of 61-73% of total length. at body-tail junction, tail muscle height from 45-66% of body height. myotomes higher than wide at body-tail junction, except in a tadtable 2. measurements (in mm) of the free-swimming tadpoles of allobates ignotus. the range (mean ± standard deviation) for linear measurements are reported; only the range for proportions. for abbreviations, see “tadpoles” section in materials and methods. measurement stage 25 (n = 2) stage 26 (n = 4) stage 27 (n = 3) stage 28-29 (n = 2) tl 8.7-16 (12.4 ± 5.2) 9.6-14.9 (12 ± 2.2) 13.9-18.2 (16.2 ± 2.2) 17.8-21 (19.4 ± 2.3) bl 3.2-4.3 (3.8 ± 0.8) 3.6-4.9 (4.3 ± 0.5) 5-6.7 (5.9 ± 0.9) 7-7.6 (7.3 ± 0.4) bw 2-2.9 (2.5 ± 0.6) 2.4-3.3 (2.9 ± 0.4) 3.4-4.2 (3.9 ± 0.4) 4.5-4.6 (4.6 ± 0.1) bh 1.6-2.2 (1.9 ± 0.4) 1.6-2.6 (2.2 ± 0.4) 2.4-3 (2.8 ± 0.3) 3.2-3.3 (3.3 ± 0.1) tal 5.5-11.7 (8.6 ± 4.4) 6-10 (7.7 ± 1.7) 8.9-11.5 (10.3 ± 1.3) 10.8-13.4 (12.1 ± 1.8) eye 0.56 0.6-0.7 (0.6 ± 0.1) 0.7-0.8 (0.7 ± 0.1) 0.8-0.9 (0.8 ± 0.1) nostril 0.2 0.2 0.2 0.2 iod 1.1-1.4 (1.3 ± 0.2) 1.3-1.6 (1.4 ± 0.1) 1.7-1.9 (1.8 ± 0.1) 2 ind 0.7-0.9 (0.8 ± 0.1) 0.8-1 (0.9 ± 0.1) 0.9-1.3 (1.2 ± 0.2) 1.4-1.5 (1.4 ± 0.1) snd 0.2-0.4 (0.3 ± 0.1) 0.4-0.7 (0.5 ± 0.2) 0.4-0.6 (0.5 ± 0.1) 0.9-1 (0.9 ± 0.04) end 0.4-0.5 (0.4 ± 0.03) 0.4-0.5 (0.4 ± 0.1) 0.5-0.7 (0.6 ± 0.1) 0.7-0.8 (0.7 ± 0.1) sl 0.6-1 (0.8 ± 0.3) 0.7-1.2 (0.9 ± 0.2) 0.8-1 (0.9 ± 0.1) 1.2-1.3 (1.3 ± 0.1) sh 0.5-1 (0.8 ± 0.3) 0.6-1.1 (0.9 ± 0.2) 0.8-1.1 (1 ± 0.1) 1.4-1.5 (1.4 ± 0.1) soh 0.1-0.3 (0.2 ± 0.1) 0.1-0.2 (0.2 ± 0.04) 0.2-0.3 (0.3 ± 0.02) 0.4 ssd 2-3.3 (2.7 ± 0.9) 2.7-3.4 (3.1 ± 0.3) 3.8-4.8 (4.3 ± 0.5) 5.3-5.5 (5.4 ± 0.1) vt 0.4 0.4-0.5 (0.4 ± 0.03) 0.6-0.9 (0.8 ± 0.1) 1.0 tmw 0.8-0.9 (0.9 ± 0.1) 0.8-1.2 (1 ± 0.2) 1.1-1.7 (1.4 ± 0.3) 1.8 tmh 1-1.3 (1.2 ± 0.2) 1-1.3 (1.1 ± 0.2) 1.5-1.8 (1.7 ± 0.2) 2-2.1 (2.1 ± 0.1) odw 1.4-2.2 (1.8 ± 0.2) 1-1.6 (1.4 ± 0.3) 1.5-2 (1.8 ± 0.3) 2.1-2.4 (2.3 ± 0.2) al gap 0.7-1 (0.8 ± 0.2) 0.8-1 (1 ± 0.1) 1.1-1.2 (1.2 ± 0.1) 1.4-1.6 (1.5 ± 0.2) ujw 0.5-0.6 (0.5 ± 0.1) 0.5-0.8 (0.6 ± 0.1) 0.7-0.9 (0.8 ± 0.1) 0.9-1.1 (1.0 ± 0.1) ljw 0.3 0.3-0.5 (0.4 ± 0.1) 0.6-0.7 (0.6 ± 0.1) 0.7-0.8 (0.7 ± 0.1) a-2 gap 0.3 0.3 0.3-0.4 (0.3 ± 0.03) 0.4 p-al 5 5-7 7-9 10-15 p-pl 23-25 13-19 14-17 16-18 56 hernán darío granda-rodríguez et alii pole in stage 26 that have myotomes as high as wide. the myotomes are straitened gradually towards the tip of the tail. at mid-tail, dorsal fin barely higher than ventral fin and myotomes higher than dorsal fin. dorsal fin originating at body-tail junction; dorsal fin lower on first anterior third of the tail, posteriorly of same height through tail length, sometimes higher at mid-tail. ventral fin of same height through all of tail length. tip of tail rounded. maximum tail height less, equal to, or higher than body height, 88-116% of body height. lateral line system symmetrical on both sides of body with supraorbital, anterior pit, infraorbital, oral, temporal infraorbital, supratemporal, dorsal, middle and ventral trunk lateral lines. lateral lines were visible from stage 27 (except for one tadpole in stage 26 with supraorbital lateral line). in one tadpoles in stage 27 only the supraorbital, infraorbital lateral, dorsal and middle trunk line are present, and in other tadpoles (stages 27-29) the lines already mentioned are observable. the stitches in all lateral lines are equidistant between each of them and predominantly rounded (see additional changes below). pale white spot(s) (cumuli of neuromasts), and pale line between eye-nostril are absent. oral disc positioned and ventrally directed, laterally emarginated, and surrounded by marginal papillae, except on anterior lip; sub-marginal papillae absent (fig. 3d, e). oral disc width 42-52% of body width. anterior lip gap 60-88% of oral disc width. marginal papillae arranged in rows varying by lip and stage from 1-2 rows (see below). papillae abundant on anterior and posterior lip, 5-7 papillae (stages 25-27), 15 (stage 28), and 10 (stage 29) on right side of anterior lip, and 23-25 papillae on entire posterior lip (stage 25, due that posterior lips is less than 1 mm wide) and from 13-19 papillae/mm on posterior lip (stages 26-29). papillae low and narrow on anterior and posterior lip, barely elongated, papilla width (at base) third quarter or two third than papilla height. tips of papillae rounded to sub-acuminate. labial tooth row formulae (ltrf) 2(2)/3; second upper row (a-2) with moderate gap, from 17-28% of oral disc width. length of first upper row (a-1) equal to second upper row (a-2). first lower row (p-1) equal to second and third lower row (p-2, p-3) (n = 4); p-1 equal to p-2 and p-3 slightly shorter than p-1 and p-2 (n = 4); or p-1 equal than p-3 and these slightly shorter than p-2 (n = 3). upper rows sub-equal (commonly) or slightly shorter (rarely) than first and second lower rows. jaw sheath keratinized and black or dark brown. upper jaw sheath (ujs) low in height, with long and thin lateral processes; ujs height 11-18% of ujs width. ujs width 37-50% of oral disc width. anterior edge of ujs well defined (commonly) or indistinct (rarely), forming a convex shape; posterior free edge of ujs barely sinuous, with low serrations; mid-ujs with very low notch (fig. 3e), 20-22% of ujs height. lateral processes without serrations. tips of serrations rounded on middle of ujs and acuminate to sub-acuminate towards ends. shelf on concealed ujs absent. lower jaw sheath (ljs) thin, v-shaped with proximal ends slightly open. only distal half of ljs pigmented black, ljs height (black pigmented area) 50-100% of ujs height. serrations on ljs low and with rounded tips. in preservative, the skin of all body of the free-swimming tadpoles is translucent; body dorsally and laterally with abundant dark brown (stages 25-27) or less brown dots (stages 28-29), distributed uniformly on body, without darkening the body, forming a dark brown region around of nostril. ventrally with dark brown dots (stages 25-27) or brown dots (stages 28-29), distributed uniformly from posterior oral disc to middle intestines, without darkening the venter. under magnification, the dark brown dots of venter are asterisk-like (stages 25-27) or rounded (stages 28-29) dots (fig. 3b). intestines yellowish brown, visible in lateral fig. 3. tadpole in stage 27 of allobates ignotus, in dorsal (a), ventral (b), and lateral (c) view. oral disc of a tadpoles in stage 26 (d) and in stage 29 (e). see the upper jaw sheath shape (d) and the lower tooth rows (e). scale bar equal to 1.0 mm (a, b, c) and 0.5 mm (d, e). photographs by m. anganoy-criollo. 57natural history and conservation of allobates ignotus and ventral views and partially in dorsal view. myotomes white and fins white translucent with brown dots covering the dorsal fin, myotomes and dorsal portion of the ventral fins, distributes uniformly (stages 25-27) and forming some weak reticulate pattern (stages 28-29). nostril white; spiracle and tube vent translucent and oral disc white with a few brown dots on anterior lip. the variation in free-swimming tadpoles (stages 25-29) (table 3) is as it follows: in dorsal view, one tadpole in s25 has the body slightly more rounded than the rest of tadpoles. the marginal papillae row number varies between stages 25 and 29. in the anterior lip, the marginal papillae are disposed in one row from stages 25 to 29. in the posterolateral side of posterior lip, the marginal papillae are arranged in one row in stage 25-26, one biseriated row in stage 27 and two row in stage 28-29. on the posterior region of posterior lip, the marginal papillae are in one row in stages 25-27 and 29, in one biseriated row in one tadpole in stage 25 and 27, and in two rows in stage 28. the lateral line system also varies between these tadpoles. the supraorbital lateral, dorsal and middle trunk lines are incomplete. the supraorbital lateral line of one tadpole in stage 26 and other in stage 27 extends from oral disc to nostril; whereas, the dorsal and middle trunk lines of the stage 27 ranges from body to mid-tail. the stitches are rounded in supraorbital, anterior pit, infraorbital, temporal infraorbital, supratemporal lateral and dorsal trunk lines, but are slightly elongate and parallel to the line in the oral lateral and middle trunk lines, although in middle trunk line the stitches are ovoid in the stage 29. the stitches are oval in ventral trunk line. advertisement call the advertisement call of allobates ignotus is composed by a series of pulsed notes (x = 32; sd = 27.44; range = 6-116 notes; n = 13 calls), with a mean duration of 8.168 sec (sd = 6.36; range = 1.35-27.53 sec; n = 13 calls). the mean duration of the note was 0.046 sec (sd = 0.009; range = 0.026-0.085 sec; n = 456 notes), emitted at a rate of 3.94 notes per sec (sd = 0.61; range= 2.32-4.46), in an internote interval of 0.211 sec (sd = 0.110; range = 0.043-1.790 sec; n = 443) (fig. 4b). the call frequency was between 4600-14000 hz, and the dominant frequency was 4942.82 hz (sd = 204.3; range = 5312-6662hz; n = 13). pulse has three distinct harmonics, 7490-7993 hz, 9784-10065 hz, and 12834-13132 hz, respectively (fig. 4a). our observations in la veguita creek indicate that a. ignotus may be a philopatric species regarding to vocalization site, since an uncollected male vocalized for four hours approximately on the same rock. when this frog perceived disturbances in the vegetation caused by our capturing attempts, it escaped quickly, but returned in a few minutes to continue vocalizing in the same place. this situation was repeated about six times. relative abundance the surveys performed from 2010 to 2014 showed an abrupt decrease in relative abundance of allobates ignotus in canime creek basin in the last year (fig. 5). in april 2010, we recorded 0.46 ind/h × obs (n = 25, rare) of this frog; while in february 2011 observed 0.55 ind/h × obs (n = 30, common); in august 2012, we obtained 0.92 ind/h × obs (n = 50, abundant); and april 2013 we calcutable 3. variation of the rows of marginal papillae of the oral disc of tadpoles of allobates ignotus through of the available stages of development. whether there was variation, this is reported as range. conventions: (a) papillae on anterior lip, (b) papillae on postero-lateral side of posterior lip, and (c) papillae on middle of posterior lip; (1) one row, (2) one biseriated row, (3) two rows of papillae, (n) number of tadpoles. stages refer to gosner (1960). stages (n) a b c 25 (2) 1 1 1-2 26 (4) 1 1 1 27 (3) 1 2 1-2 28-29 (2) 1 3 1-3 fig. 4. spectrogram (a) and oscillogram (b) of the advertisement call of allobates ignotus of la veguita creek, la veguita village, manuaure balcón del cesar, cesar department, colombia. air temperature 25.4 °c (cbumag:anf:00933). total duration = 1.70 sec, 512 bands resolution. 58 hernán darío granda-rodríguez et alii lated 1.47 ind/h × obs (n = 80, very abundant). nevertheless, in june 2014, we found 0.04 ind/h × obs (n = 2, very rare). although we did not find tadpoles or amplectant couples in any of the fieldtrips to canime creek basin, we observed and listened males vocalizing in all field expeditions in this basin, except to june 2014. discussion geographic distribution and habitat use allobates ignotus was previously known in only three localities in the low wet forest of the western foothills of the serranía del perijá, with an extent of occurrence of 364 km2 and 180 m altitudinal range (400 to 580 m a.s.l., anganoy-criollo, 2012; iucn ssc amphibian specialist group, 2017). with the records reported in the present study, we extended the distribution range of this frog by 241 km2, with a total extent of occurrence at 605 km2 and a altitudinal range from 194 m to 1236 m. this records are the first for the tropical dry forest life zone and for the department of la guajira. the lowest localities where a. ignotus was found fall within the altitudinal range (0-249 m a.s.l.) with the greatest amphibian diversity in colombia (284 species according to acosta-galvis, 2012). amphibians from dry environments require physiological, ecological, and behavioral strategies for dehydration tolerance (urbina-cardona et al., 2014). habitat selection is an essential aspect for amphibians of dry ecosystems by the occupation of humid microhabitats in dehydrating environments (bentley, 1966). little is known about habitat use and mechanism to avoid the drying of dendrobatoids frog from tropical dry forests. nevertheless, some species such as dendrobates truncatus (dendrobatidae) have relatively well described ecological and behavioral strategies that allow them to withstand drought conditions in caribbean tropical dry forest. strategies like displacement to humid refuges or permanently staying in them allow taking advantage of the temperature and humidity provided by some microhabitats available (cuentas et al., 2002). we do not know the strategies used by a. ignotus for dehydration tolerance, but this frog is active all year round (we found vocalizing males in dry and rainy season). it is possible that the occupation of rocky substrate and leaf litter by a. ignotus corresponds to some mechanism for temperature and humidity capture (feber, 1982; navas, 1996). however, this hypothesis needs additional studies that evaluate the physical and structural variables of the habitat that influence the distribution and density of the frogs (giaretta et al., 1999; van sluys et al., 2007). free-swimming tadpoles prior to grant et al. (2006), most species of allobates were grouped in the colostethus sensu lato. coloma (1995) described the larval external morphology of the species that were allocated in colostethus sensu lato. afterwards, anganoy-criollo (2013) provided larval features that are shared between the species previously included in colostethus sensu lato and now split in allobates, aromobates, mannophryne, rheobates (aromobatidae), colostethus and hyloxalus (dendrobatidae). the general morphology of the tadpoles of a. ignotus resembles well the one described in coloma (1995) and in anganoy-criollo (2013). however, both these works report that a-2 gap is short or narrow, though explicit measurements of a-2 gap are not provided in these descriptions. on the opposite, the a-2 gap of allobates ignotus appears nor short or narrow. anganoy-criollo (2013) tells about a narrow or short a-2 gap of hyloxalus subpunctatus (dendrobatidae) being 4-15% of od width, while the a-2 gap of a. ignotus is 17-28% of od width, therefore markedly wider. previous descriptions of tadpoles of other allobates also showed “wide” a-2 gap; for example, in a. brunneus (30% of odw; lima et al., 2009), a. caeruleodactylus (35% of odw; caldwell et al., 2002), a. femoralis (21-35% of odw; lescure, 1976; silverstone, 1976 ), a granti (52% of odw; kok et al., 2006), a. grillisimilis (30% of odw; simões et al., 2013), a. hodli (23% of odw; simões et al., 2010), a. kingsburgyi (38% of odw; castillo-trenn, 2004), a. magnussoni (22% of odw; lima et al., 2014), a. niputidea (although not explicitly reported in the text of the description, but evident from the drawing of the tadpole; grant et al., 2007), a. paleovarzensis (35% of odw; lima et al., 2010), a. subfolionidificans (23% of odw; lima et al., 2007), a. sumtuosus (25% of odw; simões and lima, 2012), a. fig. 5. temporal variation of relative abundance of allobates ignotus in canime creek basin, cesar department, colombia. 59natural history and conservation of allobates ignotus talamancae (35 or 31% of odw; breder, 1946; savage, 2002), and a. tapajos (25% of odw; lima et al., 2015). in dendrobatoidea, this wide a-2 gap was also found in some species of dendrobatid genus andinobates (silverstone, 1975; myers and daly, 1980; ruiz-carranza and ramírez-pinilla, 1992), ameerega (haddad and martins, 1994; twomey and brown, 2008; poelman et al., 2010), some phyllobates (siverstone, 1976; donnelly et al., 1990; savage, 2002), and ranitomeya (brown et al., 2008; von may et al., 2008; twomey and brown, 2009; perez-peña et al., 2010). the observed variability of the a-2 gap among the above genera suggests it is a potentially meaningful trait from both a taxonomic and phylogenetic perspective; therefore, an adequate characterization of a-2 gap width variability in the superfamily dendrobatoidea would be worthwhile. the systematic value of the larval morphology has been used in the phylogenetic relationships history of poison frogs (e.g., savage, 1968; silverstone, 1975, 1976; myers, 1987; grant et al., 2006; sánchez, 2013) showing that there is correspondence between larval features and dendrobatoids taxonomy (e.g., reduction in labial tooth row, oral disc without emargination, umbelliform oral disc, notch on ujs). at this moment, despite that the “wide” a-2 gap is present in several poison frog genera, the “wide” a-2 gap of the tadpoles of a. ignotus is shared with other allobates species, which supports the assignation of a. ignotus within the genus allobates, where anganoy-criollo (2012) provisionally located this species based on adult characters. molecular and phenotypical characters known for the genus allobates (grant et al., 2006, 2017) suggest that the “wide” a-2 gap may be a derived and shared character for this genus; however a phylogenetic analysis is needed to test this hypothesis. advertisement call comparisons of advertisement calls within others species of the genus allobates are limited by the reduced number of calls described. calls of most of the colombian allobates are unknown. the advertisement call of a. ignotus is composed by a sequence of pulsed notes (6-116 notes/call) with ascending modulation frequency, like to other aromobatid frogs as a. talamancae (5-15 notes/call, lechelt et al., 2014), a. humilis (7-16 notes/call, la marca et al., 2002), a. myersi (5-10 notes/call, simões and lima, 2011), a. paleovarsensis (3-21 notes/call, lima et al., 2010), and aromobates saltuensis (2 notes/call, barrio-amorós and santos, 2012), from which it differs by the greater number of notes per call. although in the genus allobates the advertisements calls are highly variable, some species converge their calls in the long trains of notes, such as a. crombiei (25-59 notes/call, lima et al., 2012), a. goianus (2-41 notes/call, carvalho et al., 2016), and a magnussoni (not specified in the call description, lima et al., 2014). some allobates can produce their advertisement calls at a dominant frequency similar to a. ignotus (4942 hz), such as allobates algorei (5065 hz, barrio-amorós and santos, 2009), a. femoralis (2853 hz), a. goianus (49465230.79 hz, bastos et al., 2011; carvalho et al., 2016), a. humilis (4200 hz, la marca et al., 2002), a. myersi (3400 hz, simões and lima, 2011), a. sumptuosus (6500 hz, kok and ernst, 2007), a. aff. brunneus (5450 hz, lötters et al., 2003); however, these species occupy distant geographic areas and are easily morphologically distinguishable from a. ignotus. on the other hand, only two aromobatid frogs geographically close to a. ignotus have advertisement calls described, a. talamancae and rheobates palmatus. the advertisement call of a. talamancae differs from that of a. ignotus by the slightly higher dominant frequency (5036 hz, lechelt et al., 2014). despite the advertisement call of r. palmatus consists of a long train of notes, this differs from the advertisement call of a. ignotus by the lower dominant frequency (2083-2700 hz, lüddecke, 1999; bernal et al., 2006). for other dendrobatoid with trans-andean distribution or geographically close to a. ignotus, such as a. niputidea (grant et al., 2007), a. wayuu (acosta-galvis et al., 1999), aromobates aff. saltuensis (anganoy-criollo, 2012, see specimens examined), aromobates totuko (rojas-runjaic et al., 2011), colostethus inguinalis (grant, 2004), and “colostethus” ruthveni complex (granda-rodríguez et al., 2014); there are no descriptions of their advertisement calls. relative abundance and conservation status habitat degradation and fragmentation are the principal factors in the amphibian decline and extinction, since they generate drastic changes on the amphibian community structure (mazerolle, 2003). some dendrobatoid frogs are very sensitive and have declined their populations due to forest fragmentation and degradation, e.g., a. juanii in the eastern foothills of the cordillera oriental of colombia (cáceres-andrade and urbina-cardona, 2009) and anomaloglossus stepheni (aromobatidae) in the central amazon (funk and mills, 2003). on the other hand, dendrobatoid frogs like epipedobates boulengeri (dendrobatidae) can tolerate structural changes on their habitat, although environment variables like microhabitat temperature are strongly associated with the relative abundance of this frog, conditioning their establishment (urbina-cardona and londoño-murcia, 2003). in the three localities reported herein, allobates ignotus showed fluctuations in their relative abundance dur60 hernán darío granda-rodríguez et alii ing the study period, and we found the lowest abundance in the last year. these areas are immersed in a zone of hydrocarbons exploitation activity. fragmentation and degradation of riparian forest of canime creek by mining activity, plus the stream flow reduction by severe droughts (recent and predicted for future, see nakaegawa and vergara, 2010), can be both short and long term threats, due to the decrease of habitat availability and quality. in addition, the influence of pollution with chemical and solid mining wastes on populations of these frogs should be assessed, since these factors are known to affect the development and survival of amphibians (rowe et al., 1996). all these potential threats can act synergistically and cause population declines in a. ignotus. although allobates ignotus was previously categorized as endangered species [en b1ab(iii), iucn ssc amphibian specialist group. 2017], this decision was based on the limited data provided by anganoy-criollo (2012). therefore, we re-evaluate the conservation status of the species with the data provided herein. following the iucn criteria (iucn, 2012), we suggest categorizing allobates ignotus as vulnerable [vu b2ab (iii)]; given to the decrease of the relative abundance in the three localities of the canime creek basin, the extent of occurrence lesser than 2000 km2 (605 km2), and loss of habitat quality by degradation of creeks and forests where this species lives. acknowledgements some specimens were collected under the project “estudios técnicos, sociales, económicos y ambientales, con su respectiva cartografía temática, correspondiente al entorno local, del complejo de páramo perijá en jurisdicción de la corporación autónoma regional del cesar (corpocesar) y la corporación autónoma regional de la guajira (corpoguajira)”. this project is excluded of permits according to the decree 1376 at 2013, in the frame of a scientific and technologic cooperation agreement between the instituto de investigaciones biológicas alexander von humboldt (iavh) and fundación prosierra. we thanks to adolfo del portillo mozo, efrain rada vargas, and miguel arévalo páez for donating us the specimens collected in la frontera’s way and manaure balcón del cesar. thanks to ruben fontalvo, josé rincón, and carlos ardila rodríguez for his great help on the field trip to manaure balcón del cesar. likewise, to alina gámez, ángel santana and family, joán zambrano, and the direction of the escuela normal superior maría inmaculada for helping us in our stay in manaure balcón del cesar. we are grateful to lorena benítez-cubillos for construction of the map and graham louis for lenguage revision. speciall mentios goes to patricio hernáez, martín caicedo, andrea cotes, carlos mario lópez orózco, santiago gonzález, maría v. león, rosana londoño, and sigmer quiroga for their invaluable contributions on the manuscript. references acosta-galvis, a.r. 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(1988): etho-taxonomie und zoogeographische artengruppenbildung bei pfeilgiftfröschen (anura: dendrobatidae). salamandra 24: 125-160. appendix examined specimens allobates ignotus. colombia: cesar: la jagua de ibirico: zumbador river (580 m a.s.l.), nueva granada village, icn 55434 (holotype), icn 55435-436 (paratypes); el indio creek (520 m a.s.l.), la victoria de san isidro village, icn 55427-433; canime creek 1 (250 m a.s.l.), cbumag:anf:00254-256; canime creek 2 (200 m a.s.l.), cbumag:anf:0069; canime creek 3 (194 m a.s.l.), cbumag:anf:00270-272. becerril: el veranero creek (400 m a.s.l.), el 11 village, icn 55437-439 (paratypes). agustín codazzi: la frontera’s way (1236 m a.s.l.), la frontera village, cbumag:anf:00937. manaure balcón del cesar: la veguita creek (633 m a.s.l.), la veguita village, cbumag:anf:00933, 00935-936. la guajira: la jauga del pilar, nicaragua creek, el plan village, cbumag:anf: 00934. acta herpetologica vol. 13, n. 1 june 2018 firenze university press species diversity and distribution of lizards in montenegro katarina ljubisavljević1,2,*, ljiljana tomović3, aleksandar urošević1, slađana gvozdenović2, vuk iković2, vernes zagora2, and nenad labus4 the first demographic data and body size of the southern banded newt, ommatotriton vittatus (caudata: salamandridae) abdullah altunişik diet and helminth parasites of freshwater turtles mesoclemmys tuberculata, phrynops geoffroanus (pleurodira: chelidae) and kinosternon scorpioides (criptodyra: kinosternidae) in a semiarid region, northeast of brazil antonio marcos alves pereira*, samuel vieira brito, joão antonio de araujo filho, adonias aphoena martins teixeira, diêgo alves teles, daniel oliveira santana, vandeberg ferreira lima, waltécio de oliveira almeida electric circuit theory applied to alien invasions: a connectivity model predicting the balkan frog expansion in northern italy mattia falaschi1,2,*, marco mangiacotti3, roberto sacchi3, stefano scali2, edoardo razzetti4 first report of bufo bufo (linnaeus, 1758) from sardinia (italy) ilaria maria cossu1, salvatore frau1, massimo delfino2,3, alice chiodi4, claudia corti1,5*, adriana bellati4 natural history and conservation of the nurse frog of the serranía del perijá allobates ignotus (dendrobatoidea: aromobatidae) in northeastern colombia hernán darío granda-rodríguez1,2, andrés camilo montes-correa3,*, juan david jiménez-bolaño3, marvin anganoy-criollo4,5 exploring body injuries in the horseshoe whip snake, hemorrhois hippocrepis juan m pleguezuelos*, esmeralda alaminos, mónica feriche how effectively do european skinks thermoregulate? evidence from chalcides ocellatus, a common but overlooked mediterranean lizard grigoris kapsalas, aris deimezis-tsikoutas, thanos georgakopoulos, ismini gkourtsouli-antoniadou, kallirroi papadaki, katerina vassaki, panayiotis pafilis thermal tolerance for two cohorts of a native and an invasive freshwater turtle species jun geng, wei dang, qiong wu, hong-liang lu* diet of a restocked population of the european pond turtle emys orbicularis in nw italy dario ottonello1, fabrizio oneto1,2, monica vignone2, anita rizzo2, sebastiano salvidio2,* helminths of the lizard colobosauroides cearensis (squamata, gymnophthalmidae) in an area of caatinga, northeastern brazil aldenir f. da silva neta*, robson w. ávila acta herpetologica 16(1): 11-25, 2021 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-8955 morphometric differentiation and sexual dimorphism in limnomedusa macroglossa (duméril & bibron, 1841) (anura: alsodidae) from uruguay valeria de olivera-lópez1,*, arley camargo2, raúl maneyro1 1 laboratorio de sistemática e historia natural de vertebrados, instituto de ecología y ciencias ambientales, facultad de ciencias, universidad de la república, iguá 4225, montevideo 11400, uruguay 2 centro universitario regional noreste, sede rivera, universidad de la república, ituzaingó 667, rivera 40000, uruguay * corresponding author. e-mail address: vdeolivera@fcien.edu.uy submitted on: 2020, 18th june; revised on: 2021, 9th march; accepted on: 2021, 21st march editor: raoul manenti abstract. intersexual morphological differences within a species occur in many traits, including body size and shape. many processes that cause geographic variability in morphology have been proposed: population structure, phenotypic plasticity (environmental effects on development), and natural and/or sexual selection. several hypotheses can explain patterns of sexual dimorphism in anurans, including natural or intra/inter-sexual selection, and differences in life history strategies between sexes. limnomedusa macroglossa is considered a habitat specialist restricted to rocky outcrops in brazil, argentina, paraguay, and uruguay. we evaluated the extent of sexual (size and shape) dimorphism in l. macroglossa from uruguay based on morphometrics and secondary sexual characteristics, while taking into account geographic variation. sexual dimorphism in body size of adults was found, but multivariate analyses did not demonstrate the existence of significant differences in shape. there were also significant differences in body size and hind leg measurements among six hydrographic basins as a result from the phenotypic plasticity correlated with local temperature, representing a clinal variation along the latitudinal gradient of uruguay. the sexual dimorphism found in body size is probably the consequence of higher growth rates and/or late sexual maturity in females, which favors larger body size for accommodating larger ovaries, and thus, higher reproductive output. keywords. sexual dimorphism, clinal variation, morphometrics, limnomedusa macroglossa, uruguay. introduction morphology is one of the main components of the phenotype that can be studied through qualitative as well as quantitative characteristics. in particular, morphology can be assessed via morphometrics to quantitatively describe, analyze and interpret morphological variation within and between species (kaliontzopoulou, 2011; rohlf, 1990). morphological quantitative traits are usually polygenically inherited and show considerable plasticity in relation to environmental factors (babik and rafinski, 2000). furthermore, plasticity can lead to geographic variability in morphology. in that sense, many processes have been proposed, such as: biogeographical barriers that partially isolate populations, effects of environmental parameters (precipitations and temperature) on growth rates, and action of sexual selection resulting in sexual dimorphism (schäuble, 2004). body size is a strongly plastic morphological trait (green, 2015) fundamental in physiological and ecological contexts. traditionally, snout-vent length (svl) has been used as the gold standard to measure body size in frogs (kupfer, 2007). among anurans, analyses of intraspecific geographical variability in morphology have often revealed extensive variation in body size (schäuble, 2004). due to the limited dispersal ability and high philopatry in frogs, it is common to find 12 v. de olivera-lópez, a. camargo, r. maneyro intraspecific differences in morphology among geographically separated populations, particularly in body size, caused by genetic divergence among isolated populations (baraquet et al., 2012; castellano et al., 2000). in addition to geographic distance, landscape features could account for spatial morphological variation. for instance, hydrographic basins could act as physical barriers promoting isolation and spatial structuring among populations as a result of changes in altitude, slope and landscape features among basins. moreover, climate and food availability may also vary geographically, leading to differences in the ability to grow, resulting in morphological variation (lovich and gibbons, 1992; hartmann, 2016). another source of intraspecific variation could be sexual dimorphism; the occurrence of morphological differences between individuals of different sex within a species, may affect several traits like body size, shape and sometimes, secondary sexual characteristics (wells, 2007). several factors can influence sexual dimorphism including female reproductive strategy, sexual selection, and competition for resources (fathinia et al., 2012). sexual dimorphism may have important consequences for animal ecology, and is a key aspect for understand the evolution of life history traits (kupfer, 2007). in particular, sexual size dimorphism (ssd) is defined as the difference in body length or mass of sexually mature males and females (fairbairn, 1997; kupfer, 2007; nali et al., 2014). several evolutionary processes have been proposed to explain patterns of sexual dimorphism in anurans. on one hand, the usually biased ssd in favor of females (shine, 1979) is hypothesized as the result of a fecundity advantage driven by natural selection: bigger females can harbor more eggs, and then produce larger clutches (arak, 1988; wells, 2007). whereas in males, natural selection operates against of bigger body sizes, because higher vulnerability of prolonged breeders to predators increase their cost of reproduction in terms of survival at small body sizes (camargo et al., 2008). on the other hand, some authors argue that sexual dimorphism is a consequence of sexual selection. in this sense, darwin envisioned that sexual selection depends on the struggle between males to access females, and recognized two mechanisms: intrasexual selection, through competition between members of the same sex (usually males) for access to mates, where large males defeat small ones in aggressive encounters and displace them from territories; and intersexual (epigamic) selection, where members of one sex (usually females) choose members of the opposite sex, by comparing traits of potential mates and select those that are more attractive (darwin, 1871; shine, 1979; woolbright, 1983; arak, 1988; lovich and gibbons, 1992). however, some authors proposed that sexual dimorphism is a function of differences in life history strategies between the sexes, as well as the result of a variety of selective forces. in this sense, ssd can be explained in terms of disparate age structure between sexes in reproductive populations; that is, females were larger because they were older than the males, which mature earlier at smaller size. in fact, monnet and cherry (2002) found that age differences between breeding males and females appear to have a major influence on the extent of dimorphism. female anuran fecundity appears to be correlated with body size in all anuran species in which this phenomenon has been investigated, and, as anurans display indeterminate growth (halliday and verrell, 1986), this could be expected to lead to faster growth rates and delayed reproduction in females (monnet and cherry, 2002). limnomedusa fitzinger 1843, is the most basal genus within the family alsodidae (frost et al., 2006; pyron and wiens, 2011). the only species of the genus, “rapids frog” limnomedusa macroglossa (duméril and bibron, 1841), is a generalist insect predator of medium to large size, with shades of brown-and-gray and conspicuous glands in the back, and an immaculate white belly (maneyro and carreira, 2012). as secondary sexual characteristics, males present a single vocal sac and dark nuptial pads on their fingers. it is a habitat specialist, with a restricted distribution in rocky outcrops of basaltic origin and superficial soils, with or without vegetation (maneyro and carreira, 2012). regarding its geographic variation in uruguay, larval dispersion appears to be connecting separate major basins via watercourses, although it is also likely that adults disperse between habitat patches by land. as a corollary, an isolation pattern by distance was established, which maintains population stability and genetic diversity in northern populations (fernández, 2016). recently, de olivera et al. (2018) found a correlation between body size and ovarian mass in populations of limnomedusa macroglossa from uruguay, suggesting a fecundity advantage for larger females since they can accommodate larger ovaries. moreover, they also reported a prolonged pattern of reproduction for this species, which is usually associated with higher levels of intra/ inter-sexual selection (wells, 2007). further, in populations from rio grande do sul state, ssd has been found, where females attain larger svl than males, and they also classified the pattern of reproduction as prolonged, although highly seasonal (kaefer et al., 2009). its geographic distribution includes the south of brazil (from paraná to rio grande do sul), the northeast of argentina (misiones and entre ríos), the southeast of paraguay (alto paraná), and almost the entire uruguayan territory (frost, 2020). however, despite being a relatively 13sexual dimorphism and clinal variation in limnomedusa macroglossa common species, geographical variation in morphology has not been investigated in l. macroglossa overall distribution. this circumstance is relevant since most of the distribution range occurs in uruguay, and thorough evaluation of the morphological variation across such distribution is necessary given its latitudinal, environmental gradient. lastly, in reference to its conservation status, is categorized nationally and globally as least concern according to the iucn criteria (silvano et al., 2004; maneyro et al., 2019). the aim of this work was to evaluate the occurrence of sexual (size and shape) dimorphism in limnomedusa macroglossa based on morphometrics and secondary sexual characteristics across populations from uruguay. we hypothesized that: (1) sexual dimorphism and minimum size at sexual maturity (mssm) are important life history traits due of their value in reproductive output of a species. besides, most anuran females have larger body sizes than males (female biased ssd) and, thus, females usually reach sexual maturity at larger sizes. (2) isolation pattern by distance triggered by hydrographic basins favor geographical differences in morphology. from which the following predictions emerge: (1.1) we expect that l. macroglossa present sexual size dimorphism with females larger than males, and in fact, with females reaching mssm at bigger sizes than males. (2.1) finally, hydrographic basins, due to environmental differences, will favor a greater morphological differentiation in l. macroglossa between than within basins. materials and methods field data collection we hand-captured 180 individuals of limnomedusa macroglossa between january 2012 and march 2015, of which 102 were juveniles, 34 mature females and 44 mature males. the individuals were collected along a latitudinal gradient of six hydrographic basins from uruguay (based on achkar et al., 2013): río uruguay (7 females and 8 males), río santa lucía (3 females and 4 males), océano atlántico (2 males), laguna merín (3 females and 1 male), río de la plata (6 females and 6 males), and río negro (11 females and 14 males) (fig. 1) (see appendices 1, 2 and 3). latitude and longitude location data of these individuals were obtained from a gps (garmin, etrex 20). in addition, 13 individuals not georeferenced (4 females and 9 males) were also used for ssd and sma analyses. lastly, juvenile individuals were used in another investigation (fernández, 2016). all collected individuals were euthanized using topic lidocaine and intraperitoneal injection of sodium pentobarbital (0.5 ml of a 0.2 g/ml solution), fixed with 10% formaline, and preserved in 70% ethanol, following the experimental protocol “euthanasia method for amphibians and reptiles in the field” approved by the institutional animal care and use committee (iacuc), faculty of science, university of the republic. individuals were euthanized with the purpose of being genetically studied by fernández (2016), therefore in this work, those individuals were reused. all the specimens are stored in the vertebrate zoology collection (zvc-b) of the faculty of sciences, university of the republic. we measured eleven morphometric variables using a digital calliper (0.01 mm precision) by a single observer for consistency (grenat et al., 2012): snout-vent length (svl), mandibular width (mw), head length (hl), inter-orbital distance (iod), eye diameter (ed), inter-narial distance (ind), eye–nostril distance (end), arm length (arml), tibia length (til), tarsus length (tal) and metatarsus length (mtl). we followed the methodology of duellman (1970) to obtain the measurements of svl, iod, ed, ind, til, and mtl, as well as napoli (2005) for end, and greene and funk (2009) for arml. finally, we measured tal as the straight length of the tarsus, mw as the straight line between oral commissures, and hl as the straight line distance from the posterior edge of the skull to the tip of the snout. all individuals were measured twice to ensure accuracy and all measurements were taken on the right side of the body (fig. 2). for each individual, sex and maturation status (juvenile/ adult) were determined by gonadal analysis. additionally, males were considered mature by the presence of nuptial pads in their fingers. finally, to infer mssm on each sex, we pooled all individuals from all basins and register the size of the adult male/ female with the lowest svl. fig. 1. maps of south america and uruguay showing basins where limnomedusa macroglossa was sampled for analyses of geographic variation in sexual dimorphism and morphometric differentiation. names of sampling basins are as follows: a = río uruguay, b = río negro, c = laguna merín, d = océano atlántico, e = río de la plata and f = río santa lucía. black triangles are males and red circles are females (based on achkar et al., 2013). 14 v. de olivera-lópez, a. camargo, r. maneyro data analysis using the morphometric variables, we tested for sexual dimorphism and quantified morphometric variation through univariate and multivariate analyses while taking into account geographic distribution. to remove allometric effects of body size in the sexual dimorphism analyzes we applied the transformation proposed by lleonart et al. (2000), which scales all individuals to same size and adjust their shape to that they would have in the new size. for all the variables we tested the normality (lilliefors’ test) and homogeneity of the variance (levene’s test) of raw and transformed data. a priori, the raw data did not reject the hypotheses of normality neither homogeneity of variances. although, with the transformed data, there were rejected. no outlier individuals were found in the analyzed sample. we performed a t-test to evaluate for a significant difference in svl between males and females. sexes were also compared through one-way permanova using euclidean similarity index. as differences in body size between sexes are not always related to svl and can involve body parts used in various behavioral contexts (kupfer, 2007), we conducted multivariate analyses. differences in shape between males and females were examined through a principal component analysis (pca) using the variance-covariance matrix, and a hierarchical cluster with unweighted pair-group average algorithm and euclidean similarity index with 9999 pseudoreplicates. svl, mtl and til variables were log-transformed to estimate standardized mayor axis (sma) regression slopes. this method estimates the line of best fit (slope) when both variables are measured with error (falster et al., 2006; warton et al., 2006). we estimated the sma relationship between svl and mtl/til. we tested for significant allometry assuming the null hypothesis that the slope was equal to 1 (isometry), performed slope comparisons between sexes, tested for shifts along the common sma slope and in elevation of slope between sexes using wald test, with 1000 iterations and critical p-value to 0.05. to evaluate morphometric variation, we analyzed differences among basins by one-way permanova (using bonferroni correction for p-values) and pca, based on raw measured variables, because our goal was also to evaluate the effect of body size, and we box plot svl, til and mtl variables according to basins. in addition, we calculated the average leg length (= til + tal + mtl) among individuals belonging to each basin. finally, we tested for significant differences in the leg length and svl among basins through t-test. the latitude vs. svl relationship was evaluated through regression analysis (using reduced major axis algorithm). in these analyses we used 35 mature males and 30 mature females because coordinate data were not available for all individuals. we used the freely available online programs past 3.21 (hammer et al., 2001), gnumeric 1.12 (the gnome project, 2018), smatr 2.0 (falster et al., 2006) and qgis 18.24 (qgis development team, 2016) for all statistical analyses performed in this work. results sexual dimorphism in total, we examined 180 specimens of which 102 were juveniles, 34 mature females and 44 mature males. we found dark nuptial pads in the first, second, and sometimes, the third fingers of all mature males (fig. 3). we found that females longer than 49.82 mm and males longer than 41.29 mm in svl were sexually mature (i.e., nuptial pads in males and fully-developed oocytes in females). taking this into account, we set the mssm in females at 49.82 ± 0.01 mm and in males at 41.29 ± 0.01 mm. significant differences in mature body size were found between sexes. mature females had an average svl (56.99 ± 4.27 mm) significantly higher than that of mature males (49.69 ± 4.73 mm) (t = 7.04, p < 0.001). fig. 2. morphometric measurements used for the analysis of sexual dimorphism in limnomedusa macroglossa (anura: alsodidae) from uruguay: svl = snout–vent length; mw = mandibular width; hl = head length; iod = inter-orbital distance; ed = eye diameter; ind = inter-narial distance; end = eye–nostril distance; arml = arm length; til = tibia length; tal = tarsus length and mtl = metatarsus length. fig. 3. male displaying dark nuptial pads above fingers of the foreleg (zvc-b 23281). 15sexual dimorphism and clinal variation in limnomedusa macroglossa furthermore, significant differences were found in means of all other variables, with females reaching larger measurements than males (table 1). when all the morphometric variables were introduced in a nonparametric permanova test, the comparison of sexes was not significant (f = 0.349, p = 0.865). the pca of size-adjusted measurements showed a total of ten components, with 69.09 % of the variance comprised by the first two components, with pc1 accounting for 54.14% and pc2 14.95% of the total variation. the bi-dimensional projection of the first two components exhibited a substantial overlap of sexes (fig. 4a). the loadings indicate that pc1 has a strong positive correlation with til (0.61) and mtl (0.58), and the lowest with ed, ind, end and iod (fig. 4b), whereas pc2 is moderately correlated with tal (0.35) and arml (0.33), while mtl stood out with a very strong negative correlation of 0.76 (fig. 4c). the dendrogram obtained, with hierarchical clustering, was a good representation of the data matrix given the obtained coefficient of cophenetic correlation (ccc = 0.74) and showed a topology of females and males in congruence with pca and permanova analyses, determining the absence of morphometric, sexual shape dimorphism. however, the low bootstrap percentages (<50%) do not indicate high support for most of the similarity relationships. morphometric differentiation since multivariate analyses performed previously did not reveal significant differences between sexes, we pooled males and females within each basin in subsequent analyses. when all the morphometric variables were analyzed through permanova test, significant differences were found among the six hydrographic basins evaluated in this work (f = 2.553, p < 0.05). the pairwise comparisons showed significant differences between río negro and río de la plata basins (table 2). the pca of original measured variables (including svl variable), showed a total of eleven components, with 96.4% of the variance comprised by the first two components, with pc1 accounting for 93.96% and pc2 2.47% of the total variation. the bi-dimensional projection of pc1 vs. pc2 showed the convex polygons grouping inditable 1. descriptive statistics of each morphological variables measured in limnomedusa macroglossa (anura: alsodidae) from uruguay. morphological differences between sexes were tested for each variable through t test. sex: ♂ = male, ♀ = female; n: sample size; min: minimum value; max: maximum value; x: mean; se: standard error; sd: standard deviation; vc: variance coefficient. variables for which significant differences were obtained are in bold. all measurements are shown in millimeters. sex n min. max. x se sd vc. t test p value svl ♂ 44 41.29 60.92 49.69 0.71 4.73 9.51 7.04 <0.001 ♀ 34 49.82 64.25 56.98 0.73 4.27 7.5 <0.001 mw ♂ 44 16.61 24.28 19.66 0.29 1.94 9.87 6.56 <0.001 ♀ 34 19.7 24.47 22.32 0.26 1.54 6.92 <0.001 hl ♂ 44 14.47 20.98 17.13 0.23 1.53 8.96 6.78 <0.001 ♀ 34 16.95 22.02 19.46 0.25 1.46 7.51 <0.001 iod ♂ 44 7.05 9.82 8.38 0.12 0.79 9.44 5.71 <0.001 ♀ 34 8.2 10.93 9.4 0.13 0.77 8.15 <0.001 ed ♂ 44 4.17 7.2 5.45 0.1 0.69 12.59 4.27 <0.001 ♀ 34 5.07 7 6.05 0.09 0.51 8.51 <0.001 ind ♂ 44 3.26 5.48 4.22 0.08 0.56 13.22 5.34 <0.001 ♀ 34 3.69 5.76 4.85 0.08 0.46 9.48 <0.001 ned ♂ 44 4.01 6.33 5.02 0.08 0.56 11.16 7.32 <0.001 ♀ 34 4.92 7.05 5.88 0.08 0.45 7.73 <0.001 arml ♂ 44 9.93 15.62 12.46 0.19 1.25 10.06 6.1 <0.001 ♀ 34 11.85 16.73 14.2 0.21 1.25 8.78 <0.001 til ♂ 44 24.7 37.72 30.53 0.49 3.26 10.68 6.43 <0.001 ♀ 34 30.21 39.86 35.02 0.47 2.76 7.89 <0.001 tal ♂ 44 13.57 18.72 16.05 0.22 1.49 9.29 6.93 <0.001 ♀ 34 15.66 20.65 18.34 0.24 1.39 7.57 <0.001 mtl ♂ 44 21.82 31.93 26.46 0.41 2.72 10.28 5.59 <0.001 ♀ 34 25.1 32.85 29.63 0.37 2.15 7.24 <0.001 16 v. de olivera-lópez, a. camargo, r. maneyro viduals from different basins with an elevated degree of overlap (fig. 5a). according to greene and funk (2009), in pca of morphological data, the first axis (pc1) is usually associated with size, and the remaining axes describe orthogonal axes of variation in shape. indeed, we found that, the first axis has a strong positive correlation with body size and a moderate correlation with a few hind leg measurements: svl (0.69), til (0.45) and mtl (0.33). meanwhile, head measurements (iod, ed, ind and end) showed the weakest correlation (fig. 5b). the second axis (shape axis) has a strong positive correlation with mtl (0.56), and a moderate correlation with mw (0.32) and til (0.31), while svl stood out with a strong negative correlation -0.67 (fig. 5c). given the considerable contribution of svl, til and mtl variables in size and shape axis of pca, we did a box plot according to the hydrographic basins in order to show the differences between them. significant differences were found between hind leg length of individuals from río de la plata and río negro basins (t = 3.533, p < 0.001), being those of río de la plata basin the longest hind leg (82.12 ± 2.21 mm, n = 12), while those of río negro basin were the shortest legs (72.55 ± 1.55 mm, n = 25) (fig. 6a,b), reaching a difference of 11,65%. regarding svl, we found a similar pattern, reaching higher values in río de la plata basin (56.31 ± 1.57 mm, n = 12) and lower ones in río negro basin (50.12 ± 1.06 mm, n = 25; t = 3.302, p < 0.002; fig. 7a), reaching a difference fig. 4. (a) scatter plot for the first two principal components obtained from a principal component analysis of eleven morphological variables measured in limnomedusa macroglossa (anura: alsodidae) from uruguay, including convex polygons grouping individuals according to their sex. red circles represent females and black crosses are males. coefficients of association of each morphometric variable with the first principal component (pc1) (b) and with the second principal component (pc2) (c). svl = snout-vent length, mw = mandibular width; hl = head length; iod = inter-orbital distance; ed = eye diameter; ind = inter-narial distance; end = eye–nostril distance; arml = arm length; til = tibia length; tal = tarsus length and mtl = metatarsus length. 17sexual dimorphism and clinal variation in limnomedusa macroglossa table 2. one-way permanova results for morphometric data of limnomedusa macroglossa from uruguay taking into account the six hydrographic basins evaluated in this work: río uruguay, río santa lucía, océano atlántico, laguna merín, río de la plata and río negro. bonferroni corrected p values are displayed. basins for which significant differences were obtained are in bold. río santa lucía río negro laguna merín río de la plata océano atlántico río uruguay 1 0.462 1 1 1 río santa lucía 1 1 1 1 río negro 1 0.024 1 laguna merín 1 1 río de la plata 1 fig. 5. (a) scatter plot for the first two principal components obtained from a principal component analysis of eleven morphological variables measured in limnomedusa macroglossa (anura: alsodidae) taking into account hydrographic basins from uruguay, including convex polygons grouping individuals according to basins. fill triangles represent males and circles are females. basins: blue = río uruguay; sky blue = río santa lucía; green = océano atlántico; violet = laguna merín; orange = río de la plata; black = río negro. coefficients of association of each morphometric variable with the first principal component (pc1) (b) and with the second principal component (pc2) (c) taking into account hydrographic basins from uruguay. svl = snout-vent length, mw = mandibular width; hl = head length; iod = interorbital distance; ed = eye diameter; ind = inter-narial distance; end = eye–nostril distance; arml = arm length; til = tibia length; tal = tarsus length and mtl = metatarsus length. 18 v. de olivera-lópez, a. camargo, r. maneyro of 11% between groups. additionally, a significant correlation between latitude and svl was found (r = 0.60, f = 4.35, p < 0.001; fig. 7b). allometric regressions we performed a sma analysis with the variables that showed the highest correlations with pc1 and pc2 in pc analyzes. we found a significant sma relationship between svl and mtl in females [b=0.98, 95% confidence interval (ci) = 0.78-1.23] and males [b=1.09, 95% ci = 0.94-1.25]. it was also significant between svl and til in females [b=1.06, 95% ci = 0.88-1.27] and males [b=1.13, 95% ci = 1.02-1.25] and between svl and mw in females [b=0.93, 95% ci = 0.75-1.16] and males [b=1.03, 95% ci = 0.90-1.18]. in males, the svl vs. til relationship showed a significant positive allometry (b=1.128). on the other hand, in all the other cases, there were no significant differences from isometry (table 3). in all cases, the test for common slope across sexes indicated that there are no significant differences in common slope between males and females. when testing for shifts along the common slope, we found significant shifts in all relationships with higher values in females (svl-mtl relationship: w = 42.952, p < 0.01; svl-til relationship: w = 47.729, p<0.01). the test for shift in elevation was only significant in the svl vs. mtl relationship in favor of males (w = 4.411, p < 0.05), but the difference in elevation was rather small and close to our resolution limit (0.01 mm). fig. 7. (a) box plot of body size (svl) of limnomedusa macroglossa (anura: alsodidae) according to hydrographic basins from uruguay, considering males and females grouped. (b) latitude-svl relationship for l. macroglossa. the line represents the regression model. red circles represent individuals of río de la plata basin and black circles are individuals of río negro basin. the line inside de boxes represents the median. svl measurements are in millimeters. fig. 6. box plots of til (a) and mtl (b) variables of limnomedusa macroglossa (anura: alsodidae) according to hydrographic basins from uruguay, considering males and females grouped. til = tibia length and mtl = metatarsus length. the line inside de boxes represents the median. all measurements are in millimeters. 19sexual dimorphism and clinal variation in limnomedusa macroglossa discussion in this study we determined the minimum size at sexual maturity (mssm) and described morphometric and intersexual differences in limnomedusa macroglossa. we showed that females and males differ in mssm, presence of dark nuptial pads in males (a sexually dimorphic characteristic) and body size, while no differences were found in body shape. nuptial pads can be observed during the breeding season in response to increases in circulating levels of androgens, but later regress during the non-breeding period, although without resembling to a female-like morphology (wells, 2007). some authors argued that well-developed nuptial pads are associated with breeding in water to prevent the female´s escape during amplexus (duellman and trueb, 1986). however, according to savage (1961), nuptial pads also allow the male to hold the female while defending her against rival males. the mssm is the size at which an individual has all the morphological and physiological conditions to begin to breed, and along with sexual dimorphism, are important life history traits to understand population changes through time. life history theory explains the variation in mssm between sexes through natural selection mechanisms, mainly related with adult mortality rates (tolosa et al., 2014). we found that females of limnomedusa macroglossa reach sexual maturity around 49.82 mm, while males reach it at a smaller size of about 41.29 mm. this difference between the sexes can be explained by sexual selection: selection for mating effort in males to defend territories, in detriment of larger males due to the high energetic expenditures and risks of mortality, and parental effort in females to produce more eggs to maximize their reproductive output, which favors females with a larger size; both processes have been pointed out as potential explanations for sexual maturation at different ages (howard, 1981). there was sexual dimorphism in size in limnomedusa macroglossa with females being larger than males, as it occurs in more than 90% of anurans species (shine, 1979). these results agree with those found in a population of l. macroglossa in southern brazil based on svl only (kaefer et al., 2009). taking into account the main hypotheses regarding the causes of sexual dimorphism in anurans, natural and/or sexual selection might adequately explain the differences in body sizes between females and males found in this work. given the available data until date (kaefer et al., 2009; de olivera et al., 2018) and our results, it seems that the preference for larger females evolved because they produce more oocytes per clutch (bionda et al., 2011) or bigger eggs (the fecundity advantage hypothesis), whereas in males, natural selection works against of bigger body sizes due to the existence of possible differential predation, since the long reproductive period exposes and makes them more vulnerable to predators (camargo et al., 2008). furthermore, intra/intersexual selection could be playing an important role in the differentiation between males and females, through malemale competition and/or female choice (darwin, 1871; shine, 1979; woolbright, 1983; arak, 1988). although, in our field work, we did not observe such behaviors, we cannot rule out their existence, since it has been reported that its a species with a prolonged reproduction pattern (kaefer et al., 2009; de olivera et al., 2018) which is usually associated with more territorial males, choosy females, and overall higher levels of sexual selection (wells, 2007). finally, the age structure in the reproductive populations may also be operating between sexes (halliday and verrell, 1986; monnet and cherry, 2002). thus, the sexual dimorphism in size found in l. macroglossa could be the result of distinct, possibly opposing, selective forces that trade-off differently in each sex. in addition to size, anurans exhibit other forms of sexual dimorphism, including: the proportions and muscular development of the forelimbs (related with clasping behavior), skin color, texture and glands (visual, tactile and chemical cues for sex recognition), fangs and tusks (related with combat), abdominal and laryngeal muscles, and lung capacities (calling behavior) (wells, 2007; bell and zamudio, 2012) and head morphology (feeding strategies) (khoshnamvand et al., 2018). no differences were found in shape between sexes, but significant differences were found among basins. some variables related with the hind legs showed the highest contributions to overall shape differentiation. a functional interpretation of the differentiation in the hindlimb length found in l. macroglossa could be that table 3. standarized major axis (sma) regression results and test of isometry for limnomedusa macroglossa. variables used in analyses were: svl = snout–vent length; til = tibia length and mtl = metatarsus length. abbreviations: a = intercept. significant regressions are in bold. variables sma regression test of isometry a r² p f p log mtl vs. log svl females -0.2471 0.575 < 0.01 0.034 0.855 males -0.4193 0.800 < 0.01 1.422 0.240 log til vs. log svl females -0.3117 0.733 < 0.01 0.370 0.548 males -0.4295 0.891 < 0.01 5.620 0.02 20 v. de olivera-lópez, a. camargo, r. maneyro leg proportions may influence locomotor performance. several experimental studies have shown how longer hindlimbs may improved locomotor performance (orizaola and laurila, 2009; drakulic et al., 2016; zamoracamacho, 2018; zamora-camacho and aragón, 2019), as well as jumping distance increases as the individual grows larger (zug, 1978). meanwhile, other studies revealed that locomotor performance is negatively affected at larger sizes (moreno-rueda et al., 2020), relatively large differences (>10%) in leg length can affect the jumping efficiency (emerson, 1978; babik and rafinski, 2000). differences in jumping ability could be occurring in l. macroglossa because our results showed differences greater than 10% in body size and leg length in individuals from río de la plata basin compared to those from the río negro basin. alternatively, differences in the hindlimb length may be the result of unequal growth and developmental rate during the larval and juvenile stages (emerson et al., 1988; babik and rafinski, 2000). because amphibians are ectotherms and depend on water, they show phenotypic responses to changes in environmental factors. in this sense, some phenotypic plasticity can be attributed to environmental factors such as the duration of the larval period and its relation to size as a function of temperature (vences et al., 2002). a general temperaturesize rule for ectotherms states that higher temperatures increase developmental rates, at the cost of smaller size (drakulic et al., 2016) and conversely, at low temperatures develop more slowly, so they metamorphose at larger body sizes (harkey and semlitsch, 1988). moreover, some studies replace the idea of temperature and relate body size to latitude, predicting that body size within species increases with latitude (lindsey, 1966; schäuble, 2004). in this study we found that individuals which had the longest legs were from río de la plata basin, which correlates with the colder climate in the studied distribution (inumet, 2020). on the other hand, the individuals which had shortest legs were found in río negro basin, where the temperature is significantly higher (inumet, 2020). so, we can expect that differences in environmental temperature during the larval period may have been responsible for the variation in the hindlimbs length in l. macroglossa. this trend has already been reported in other studies (atkinson, 1994, 1995; angilletta et al., 2004). furthermore, our results are consistent with the intraspecific version of bergmann’s rule. it relates to geographic variation in the body sizes of animals (blackburn et al., 1999) which has been briefly stated by mayr as: the smaller-sized geographic races of a species are found in the warmer parts of the range, the larger-sized races in the cooler districts (ray, 1960). in this study we report a clinal variation in the relative leg length and body size of limnomedusa macroglossa along a latitudinal gradient in uruguay. the body size dimorphism likely reflects differences in growth rates of males and females. in organisms with indeterminate growth, body size is a result of a trade-off between growth and reproduction (camargo et al., 2008). therefore, females of limnomedusa macroglossa appear to delay sexual maturity, while maintaining the same body shape and proportions as the males, reaching larger sizes at maturity, based on the combined evidence of distinct mssm and the body size shift along the common isometric slopes of males and females. this difference in size could be adaptive for females, since a larger body size would increase the volume of the abdominal cavity, being able to accommodate larger ovaries (de olivera et al., 2018) and consequently, increasing their reproductive output [the so called fecundity advantage hypothesis (darwin, 1871)]. therefore, sexual dimorphism in l. macroglossa could be determined by differential growth rate between the sexes, since the growth rates are usually asymptotic after maturation and sexes generally mature at different ages (sexual bimaturity) (monnet and cherry, 2002; kupfer, 2007; wells, 2007), or it may be the result of difference in the age distributions of males and females (howard, 1981). therefore, the sexual dimorphism found in body size is probably the consequence of higher growth rates and/or late sexual maturity in females of limnomedusa macroglossa, which favors a larger body size and larger ovaries, and consequently, higher reproductive output. conclusions in conclusion, our data on mssm and ssd of limnomedusa macroglossa from uruguay may contribute to the knowledge of the life history traits of this species. our results show that females attained sexual maturity at larger sizes than males with a marked female biased sexual size dimorphism. these traits are driven by a tradeoff between natural and sexual selection on each sex: parental effort in females does favor larger sizes to maximize their reproductive output, because bigger females can accommodate more eggs in their abdominal cavity. meanwhile, mating effort in males does not favor large sizes due to energetic expenditures and risk of mortality during the long breading season, because bigger males invest most of their energy in search and calling behavior and have high mortality rates due to predation risk. we also report a clinal variation in the relative leg length and body size of limnomedusa macroglossa along 21sexual dimorphism and clinal variation in limnomedusa macroglossa a latitudinal gradient in uruguay. individuals with longest legs and bigger body sizes were from río de la plata basin, meanwhile individuals with shortest legs and smaller body sizes were those found in río negro basin. these differences could be explained by phenotypic plasticity in age and size at metamorphosis when separate populations are exposed to different environmental conditions (ruthsatz et al., 2018). studies demonstrated a plastic response of metamorphic traits in anuran larvae to changes in environmental conditions such as temperature. with increasing temperature time to metamorphosis may be reduced and metamorphosis occurs at smaller body sizes (vences et al., 2002). then, this may be occurring in l. macroglossa, since río de la plata basin is correlated with the colder climate in the studied distribution, meanwhile río negro basin is correlated with a warmer one. all the evidence gathered in this work and its interpretations show that sexual dimorphism found in body size is likely the consequence of higher growth rates and/or late sexual maturity in females of limnomedusa macroglossa, which favors a larger body size and bigger ovaries, and consequently, higher reproductive output. examination of adult females and males, already in progress, will soon allow a more in depth understanding of l. macroglossa reproductive biology in uruguay. acknowledgements we thank ernesto elgue and claudia fernández for his help in fieldwork and pablo toriño for drawings and statistical support. this work was supported by comisión sectorial de investigación científica under grant csic i+d 2012 from university of the republic. all animals have been captured, handled and euthanized in accordance with relevant guidelines in full compliance with specific permits released by the institutional animal care and use committee (iacuc) of the faculty of sciences, university of the republic; 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(1978): anuran locomotion-structure and function, 2: jumping performance of semiaquatic, terrestrial, and arboreal frogs. smithsonian institution press, washington. appendix 1 six hydrographic basins of uruguay and its geographic location (based on achkar et al., 2013). basins latitude (s) longitude (w) río uruguay 30°5’10’’-33°54’59’’ 55°48’45’’-58°26’17’’ río santa lucía 33°42’1’’-34°50’24’’ 54°59’24’’-57°07’11’’ océano atlántico 33°39’56’’-34°58’26’’ 53°22’13’’-55°10’8’’ laguna merín 31°54’18’’-34°24’51’’ 53°02’27’’-55°22’10’’ río de la plata 33°52’17’’-34°58’26’’ 54°55’14’’-58°24’47’’ río negro 30°49’59’’-33°57’37’’ 54°9’42’’-58°25’7’’ 24 v. de olivera-lópez, a. camargo, r. maneyro appendix 2 thirty four mature females (♀) of limnomedusa macroglossa used in the analyses and their respective morphometric measurements, basin and latitude/longitude from uruguay. zvc-b: vertebrate collection of the faculty of sciences, university of the republic. svl: snout–vent length; mw: mandibular width; hl: head length; iod: inter-orbital distance; ed: eye diameter; ind: inter-narial distance; end: eye-nostril distance; arml: arm length; til: tibia length; tal: tarsus length; mtl: metatarsus length. acronym (adult ♀) svl mw hl iod ed ind end arml til tal mtl basin latitude longitude 12 60.95 23.98 20.47 10.75 7 5.76 5.88 14.2 37.59 18.73 29.81 río uruguay -30.1166667 -57.05 24 53.06 21.3 18.27 8.39 5.49 4.77 5.76 13.05 32.81 17.59 30.48 río santa lucía -34.0666667 -56.8833333 127 58.88 23.21 20.53 9.22 5.95 5.06 6.09 14.32 36.41 19.58 30 río uruguay -30.75 -56.3333333 132 56.15 21.17 18.02 8.66 6 4.76 5.75 14.34 32.44 17.4 28.55 río uruguay -30.75 -56.3333333 133 54.11 20.13 17.73 8.62 5.36 4.29 5.91 12.99 35.53 17.54 32.12 151 61.97 22.3 20.19 8.93 6.79 5.09 6.36 14.05 36.41 19.83 31.83 río negro -32.9166667 -54.9333333 153 56.55 24.12 20.36 10.22 6.62 5.15 5.77 15.14 36.56 19.36 31.83 laguna merín -34.2166667 -54.9333333 310 58.85 23.17 20.38 9.49 6.67 5.14 5.91 13.36 34.05 17.71 30.38 laguna merín -33.45 -54.5333333 317 55.34 20.79 18.85 9.01 5.83 4.35 6.03 13.09 33.2 18.38 27.91 río de la plata -34.5333333 -55.4 495 55.97 22.51 19.37 9.79 6.01 5.34 6.1 14.08 36.71 19.3 30.35 651 64.25 23.89 21.04 9.01 6.56 5.68 6.35 16.36 37.26 20.03 32.14 río santa lucía -34.5833333 -56.4833333 691 62.54 23.65 21.93 9.78 5.99 4.82 6.66 15.12 37.23 19.27 32.74 río santa lucía -34.3 -55.25 813 61.95 24.23 22.02 9.98 6.23 5.45 7.05 15.07 39.86 20.57 31.31 826 55.4 21.27 18.16 8.59 6.15 4.98 5.05 12.29 32.81 16.98 28.63 829 58.22 24.47 20.64 10.6 6.48 4.82 5.92 15.55 36.68 18.22 30.05 laguna merín -34.05 -54.7833333 996 59.34 24.34 21.63 10.68 6.72 5.15 6.3 14.11 38.53 18.96 32.66 río negro -32.9166667 -54.9333333 1189 50.7 21.48 19.06 9.36 6.24 4.29 5.43 13.72 32.48 16.63 27.55 río de la plata -34.55 -55.4 1247 58.21 23.69 18.62 9.3 6.44 4.85 5.93 13.24 34.01 17.62 30.09 río negro -31.55 -55.65 1324 62.79 23.58 21.14 10.05 5.96 5.12 6.57 16.46 37.97 20.08 32.85 río de la plata -34.8333333 -55.2666667 1414 51.05 19.83 17.55 8.23 5.96 4.54 5.6 13.26 31.57 16.03 27.47 río negro -32.5 -55.3166667 1511 53.55 22.15 19.26 8.99 5.44 4.95 6.18 13.66 33.94 16.96 28.9 río uruguay -30.95 -57.5333333 1523 54.17 22.39 19.45 9.14 6.29 5.14 6.07 14.3 34.84 18.28 29.76 río uruguay -30.9333333 -57.5 23088 51.71 21.63 18.21 9.03 5.44 4.39 5.56 13.14 33.35 18 27.64 río negro -31.0825 -55.8555556 23106 57.63 22.3 18.44 9.15 5.95 4.48 5.61 13.8 34.05 17.88 28.54 río negro -31.0466667 -55.8477778 23343 63.96 23.13 21.56 10.47 6.38 5.09 5.96 16.09 37.67 19.99 30.24 río uruguay -31.34177 -56.66407 23586 52.41 19.76 17.69 8.38 5.58 4.35 5.45 13.23 30.85 16.87 26.24 río negro -31.24676 -55.95104 23594 62.97 23.51 20.73 10.93 6.22 5.05 6.05 16.73 38.83 20.27 30.86 río de la plata -34.63791 -55.24744 23597 59.13 24.32 19.78 9.62 5.53 4.97 6.18 16.01 38.56 19.73 32.41 río de la plata -34.63791 -55.24744 23598 61.58 24.06 20.24 10.03 6.26 4.83 5.61 15.66 39.35 20.65 31.31 río de la plata -34.63791 -55.24744 23601 57.31 21.11 20.28 9.98 6.98 5.54 5.91 14.67 34.53 19.02 27.79 río uruguay -31.15043 -56.29138 23608 51.35 19.98 17.87 8.53 5.27 4.2 5.51 13.75 31.87 16.84 27.77 río negro -31.09436 -55.96907 23609 53.88 21 17.86 9.19 5.64 4.65 5.38 13.48 31.98 17.19 26.15 río negro -31.13739 -56.04582 23610 49.82 20.95 17.37 9.41 5.07 4.27 5.1 12.68 30.21 16.54 25.1 río negro -31.16724 -55.87382 23611 51.82 19.7 16.95 8.2 5.33 3.69 4.92 11.85 30.39 15.66 26.12 río negro -31.09436 -55.96907 25sexual dimorphism and clinal variation in limnomedusa macroglossa appendix 3 forty four mature males (♂) of limnomedusa macroglossa used in the analyses and their respective morphometric measurements, basin and latitude/longitude form uruguay. zvc-b: vertebrate collection of the faculty of sciences, university of the republic. svl: snout–vent length; mw: mandibular width; hl: head length; iod: inter-orbital distance; ed: eye diameter; ind: inter-narial distance; end: eyenostril distance; arml: arm length; til: tibia length; tal: tarsus length; mtl: metatarsus length. acronym (adult ♂) svl mw hl iod ed ind end arml til tal mtl basin latitude longitude 90 46.51 17.73 16.14 7.78 4.83 3.92 4.41 11.49 28.95 14.71 23.95 140 46.71 17.01 15.67 7.22 4.17 3.39 4.38 11.56 28.44 15.38 25.62 río uruguay -30.2833333 -57.1833333 329 57.13 22.88 20.3 9.41 6.11 5.22 5.57 13.81 35.57 17.97 31.82 río santa lucía -34.0666667 -56.8833333 357 52.61 21.27 17.88 8.58 6.31 4.96 4.98 13.78 31.25 16.25 29.04 río uruguay -33.4666667 -58.4 549 51.73 19.68 18.28 7.85 5.04 3.7 5.52 13.38 32.51 17.24 26.8 océano atlántico -34.8166667 -54.9166667 588 42.8 17.04 15.5 7.35 4.68 4.23 4.93 11.34 26.87 15.47 23.72 908 44.37 19 16.85 7.36 5.35 3.86 4.54 11.68 26.7 13.91 23.7 río santa lucía -34.5833333 -56.4833333 1121 50.87 19.84 17.73 9.15 5.65 4.28 5.3 12.6 31.87 14.64 27.81 1156 53.16 24.28 20.98 9.15 6.57 5.14 5.6 13.37 35.33 18.7 31.68 océano atlántico -34.7333333 -54.9833333 1195 58.65 23.76 19.77 9.82 6.07 5.48 6.33 15.62 37.72 17.12 31.93 río uruguay -33.85 -57.7333333 1245 43.22 16.99 14.73 7.99 5.56 4.25 5.07 11.38 27.78 14.87 24.29 laguna merín -33.1 -54.7 1342 45.21 18.21 16.38 7.88 4.88 4.01 5.1 11.89 29.07 15.27 26.89 río uruguay -30.9333333 -57.5 2120 60.92 22.81 19.61 9.82 7.2 5.24 5.86 13.81 36.88 18.72 30.62 río de la plata -34.8666667 -56.3666667 2124 56.47 22.36 18.85 9.45 6.55 5.14 5.58 13.17 33.2 18.02 30.36 río de la plata -34.8666667 -56.3666667 2853 44.19 17.68 14.71 7.48 5.03 3.47 4.04 10.51 24.7 13.57 21.96 río negro -31.1166667 -55.9833333 3013 47.1 18.95 16.45 7.9 5.27 3.84 4.11 12.36 28.96 14.88 24.44 río uruguay -31.8166667 -56.4166667 3444 52.17 20.43 17.24 9 6.06 4.66 5.19 12.08 31.68 16.82 27.14 3456 57.54 22.25 18.64 9.25 6.38 4.66 5.49 13.94 34.61 17.21 29.78 río de la plata -34.3333333 -57 4902 49.53 20.28 17.77 8.62 5.22 4.42 5.21 13.91 32.35 17.93 27.04 10254 51.5 21.19 17.31 8.98 5.5 4.29 5.55 12.59 32.01 16.81 28.36 10278 54.01 21.24 18.81 9.02 5.61 5.09 6.08 13.8 33.59 16.78 27.94 10279 53.21 20.67 18.29 9.78 5.9 5.05 5.99 13.94 34.98 18.38 28.82 10845 55.79 21.99 17.15 8.96 5.71 4.39 4.97 13.66 34.23 17.17 30.09 23105 45.68 18.31 16.37 7.78 5 3.56 4.18 10.8 27.58 15.04 25.23 río negro -31.0466667 -55.8477778 23341 46.66 18.33 15.81 7.43 5.27 4.06 4.52 10.82 26.64 13.76 23.12 río uruguay -31.60659 -56.43186 23358 48.55 18.87 15.91 7.86 5.54 3.81 4.64 12.15 29.37 15.63 25.83 río de la plata -34.471741 -55.529168 23583 49 18.48 16.49 8.44 5.11 4.07 5.08 12.6 29.33 15.42 27.6 río negro -31.18738 -55.9483 23587 52.19 19.95 18.14 8.46 5.89 3.93 4.99 13.95 32.14 17.52 27.27 río negro -31.23137 -56.09116 23588 49.49 19.95 16.74 8.94 5.38 4.16 5.03 12.56 29.73 16.2 27.03 río negro -31.3044 -56.05855 23589 48.9 19.61 17.14 8.4 5.69 3.83 4.64 12.12 29.64 15.61 25.81 río negro -31.30467 -56.05854 23590 41.29 17.28 14.47 7.43 4.26 3.26 4.16 11.2 25.46 14.29 22.07 río negro -31.32815 -56.17757 23591 50.04 18.75 17.04 8.95 4.64 4.11 5.11 11.92 28.91 15.88 25.02 río negro -31.69442 -56.12402 23592 47.05 17.24 15.46 7.54 5.48 3.57 4.01 10.48 28.59 14.85 25.16 río santa lucía -34.28159 -55.27949 23593 52.11 20.85 17.79 9.52 6.91 4.52 4.83 13.22 33.38 17.23 28.57 río santa lucía -34.28159 -55.27949 23595 48.21 20.16 16.79 8.05 4.98 4.21 5.17 12.63 30.95 16.81 25.31 río de la plata -34.63791 -55.24744 23596 51.53 19.58 17.17 8.37 4.94 4.25 5.19 12.33 31.21 16.74 25.11 río de la plata -34.63791 -55.24744 23599 52.37 20 17.48 8.41 5.65 4.56 5.41 13.77 32.47 17.47 26.21 río uruguay -30.67911 -56.51333 23600 55.14 21.15 19.55 9.06 6.29 4.45 5.37 14.37 33.82 18.33 27.39 río uruguay -30.67911 -56.51333 23602 45 19.81 17.11 8.35 4.91 3.84 4.94 10.97 28.12 14.49 24.95 río negro -31.19047 -55.90129 23603 48.79 18.62 16.56 8.13 5 3.75 5.12 12.3 29.26 15.55 26.65 río negro -31.19068 -55.90163 23604 48.13 19.05 16.68 8.06 4.94 3.96 5.08 11.45 29.42 15.87 25.33 río negro -31.19068 -55.90163 23605 44.46 17.86 16.14 7.79 4.96 3.74 4.52 11.45 26.73 13.78 22.39 río negro -31.18738 -55.9483 23606 43.54 16.61 15.16 7.1 4.72 3.63 4.31 11.53 25.93 14.24 22.67 río negro -31.18738 -55.9483 23607 43.11 17.04 14.79 7.05 4.66 3.81 4.6 9.93 25.46 13.86 21.82 río negro -31.13739 -56.04582 acta herpetologica 12(1): 65-77, 2017 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-18737 skeletal variation within the darwinii group of liolaemus (iguania: liolaemidae): new characters, identification of polymorphisms and new synapomorphies for subclades linda díaz-fernández*, andrés s. quinteros, fernando lobo instituto de bio y geociencias del noa, consejo nacional de investigaciones científicas y técnicas, universidad nacional de salta, av. 9 de julio 14, 4405 rosario de lerma, salta, argentina *corresponding author. e-mail: lindadiazfernandez@gmail.com submitted on: 2016, 19th august; revised on: 2016, 27h october; accepted on: 2017, 12th march editor: aaron m. bauer abstract. fifty-five skeletal characters (continuous and discrete) were analyzed for species of the l. darwinii group: l. albiceps, l. chacoensis, l. grosseorum, l. irregularis, l. koslowskyi, l. lavillai, l. ornatus, l. quilmes, plus l. inacayali (l. telsen group) and l. scapularis (l. wiegmannii group). we report polymorphic intraspecific variation that has not previously been taken into account and we describe 21 new characters that provide original information across the group. we detected several morphological synapomorphies for the darwinii group and subclades. the enclosure of meckel’s cartilage by a dentary outgrowth on lingual side of lower jaw (a synapomorphy of the subgenus liolaemus sensu stricto and of the phymaturus patagonicus group) also occurs within the l. darwinii group. the morphology of maxillary teeth with three conspicuous cusps may be a potential synapomorphy of the subgenus eulaemus. the morphology of maxillary teeth may have adaptive value. characters that were studied in other groups of lizards were informative for liolaemus. keywords. cranial skeleton, postcranial skeleton, l. boulengeri group, evolution. introduction osteological characters are used for phylogenetic analyses and reconstructions and for investigating environmental adaptations because skeletons exhibit a very wide range of morphological variation at supraspecific levels. detailed examples that illustrate their use in systematic and phylogenetic studies within iguania are found in etheridge (1965, 1967), de queiroz (1987), estes et al. (1988), etheridge and de queiroz (1988), lang (1989) and frost and etheridge (1989), among others. although this information was primarily used in phylogenetic reconstructions, it is also important for future works in studies of comparative biology, as it can document evolutionary changes of characters in the context of any hypothesized selective regime. lizards of the family liolaemidae extend from the andes through bolivia, peru and chile to the coast of tierra del fuego in argentina (donoso-barros, 1966; cei, 1986, 1993; lobo and quinteros, 2005; abdala, 2007). currently, this family has 298 species (uetz, 2016) and comprises three genera: ctenoblepharys, liolaemus, and phymaturus (etheridge, 1995; frost et al., 2001; schulte et al., 2003; espinoza et al., 2004). within the subgenus eulaemus of liolaemus, there is the species-rich l. boulengeri group (etheridge, 1995; abdala, 2007), which is characterized by having a group of enlarged scales at the back of the thigh, so it is also called the “patch group”. as a result of taxonomic and phylogenetic studies of the l. boulengeri group, numerous subgroups within it have been proposed (avila et al., 2006; abdala, 2007). one of these is the liolaemus darwinii clade, which was 66 l. díaz-fernández, a.s. quinteros, f. lobo defined by etheridge (1993) based on the presence of posterior teeth with straight-edged crowns and marked sexual dichromatism in which males exhibit a more colorful dorsal color pattern than females. this group was inferred to be monophyletic in different analyses, based on morphological and/or molecular characters (abdala, 2007; avila et al., 2006; fontanella et al., 2012; olave et al., 2014). following abdala’s total evidence hypothesis (2007), the l. darwinii group consists of two clades (l. grosseorum and l. ornatus clades) and of five species basal to these ones (l. abaucan, l. espinozai, l. koslowskyi, l. quilmes and l. uspallatensis). the l. ornatus clade comprises 6 species (l. albiceps, l. crepuscularis, l. calchaqui, l. irregularis, l. lavillai and l. ornatus), which have a wide distribution in the puna, montes de sierras y bolsones and the northern part of the ecoregion of monte de llanuras y mesetas of argentina (burkart et al., 1999). lizards in this group are distinguished by being viviparous and having a large number of precloacal pores in females. information about osteological features of liolaemid lizards appears in detailed descriptions of the head skeleton of liolaemus lutzae, l. occipitalis, and l. signifer (beurman and vieira, 1980; simoes-lopes and krause, 1988). in addition, studies of the appendicular skeleton of l. occipitalis (keller and krause, 1986) and the skeleton of l. lutzae and l. multiformis simonsii (beurman and vieira, 1980) have been published. osteological character states for liolaemus and phymaturus in the context of iguanian phylogenetic analysis at the generic level were recorded by etheridge and de queiroz (1988). lobo and abdala (2001; 2002) described the variation found in the skeleton of 24 species. they demonstrated the phylogenetic information contained in those characters, recovering main clades and subclades of liolaemus formally recognized in the literature. additional skeletal characters were reported recently by núñez et al. (2003) in the description of two new taxa (l. manueli and l. torresi). da silva and verrastro (2007) described the axial skeleton of l. arambarensis and gonzález-marín and hernando (2013) described the postcranial osteology of l. azarai. in the present contribution we report new characters and polymorphisms and we provide additional informative characters for the liolaemus darwinii group and subclades therein. we report the evolution of some characters of special interest such as the enclosure of meckel’s cartilage (a character traditionally studied in iguanian lizards), the morphology of maxillary teeth, the cartilaginous extremity of cervical rib iv, and the bladelike process on the posterior distal tibia mentioned by etheridge (1995) and lobo and abdala (2001). materials and methods a total of 30 adult specimens of the liolaemus darwinii group were studied (appendix a). the species included were l. albiceps, l. chacoensis, l. grosseorum, l. irregularis, l. koslowskyi, l. lavillai, l. ornatus, and l. quilmes. we also included skeletons of l. scapularis (representing the l. wiegmannii group) and l. inacayali (l. telsen group) for outgroup comparisons, and examined characters described in lobo and abdala (2001, 2002) for 24 taxa belonging to different groups of liolaemus. we studied specimens deposited in biological collections and collected some additional specimens of l. ornatus, l. scapularis, and l. quilmes, which were sacrificed by injection with 10% sodium pentothal. they were fixed in 10% formalin and finally preserved in 70% ethanol. they are deposited in the herpetological collection of the museo de ciencias naturales of the universidad nacional de salta (mcn), instituto de bio y geociencias del noa, and the fundación miguel lillo (fml). the skeletons were prepared following the technique of differential staining of bone and cartilage (wassersug, 1976). this allows the observation of cartilaginous structures that are not visible in the dry skeletons. measurements were taken using digital calipers, 0.01 mm precision, under a stereoscopic microscope. in the case of smaller structures, the measurements were taken using a micrometer eyepiece. nomenclature of bones, processes and foramina follow de queiroz (1982), keller and krause (1986), frost (1992), etheridge (2000), lobo and abdala (2001), lobo (2001, 2005) and torres-carvajal, (2004). in total, 55 characters, 19 continuous and 36 discrete, of the cranial and postcranial skeleton were examined. the discrete characters were coded as non-polymorphic binary, polymorphic binary, non-polymorphic multistate, and polymorphic multistate. we add our data matrix to that of abdala’s (2007) total evidence analysis and performed a new phylogenetic analysis. character evolution mapping and the optimization of new characters were performed using tnt (goloboff et al., 2003) over the resulting tree (same topology as recovered by abdala 2007; fig. 1). we follow abdala (2007) because it includes the taxa studied here (instead of fontanella et al., 2012 or olave et al., 2014; whose studies lacked many of the species of interest). data on diet were taken from the literature (aun and martori, 1998; espinoza et al., 2004; semhan et al., 2013). results all morphological variation observed in the skeletons of liolaemus is summarized in the following list of char67skeletal variation in the liolaemus darwinii group acters, indicating the state of the character in each case. variation of discrete and continuous characters is presented in tables 1 and 2, respectively. updated list of osteological characters (modified from lobo and abdala, 2002) 1. number of scleral ossicles: (0) 15; (1) 14; (2) 13. polymorphic multistate. 2. bones forming parietal foramen: (0) formed mainly by frontal bone; (1) mainly by parietal bone; (2) both bones participate approximately equally. polymorphic multistate. 3. shape of parietal foramen: (0) with regular edges; (1) with irregular edges. polymorphic binary. 4. ceratohyal process: (0) gradually widened; (1) abruptly widened; (2) hook-shaped. polymorphic multistate. 5. distal ending of ceratobranchial ii: (0) narrow; (1) widened. polymorphic binary. 6. anterior process of arytenoid: (0) reaches the level of the anterior process of the cricoid; (1) does not reach that level. polymorphic binary. 7. number of tracheal rings. continuous (table 2). 8. number of incomplete tracheal rings / total number of rings. continuous (table 2). 9. number of pterygoid teeth. continuous (table 2). 10. number of maxillary teeth. continuous (table 2). 11. number of modified anterior maxillary teeth (heterodonty): in most species the anterior-most teeth of the maxilla are conical, elongated, and exhibit only one cusp. continuous (table 2). 12. maxillary tooth morphology i (fig. 2): (0) crowns with their anterior and posterior margins divergent, expanded crowns; (1) crowns with anterior and posterior margins straight. non-polymorphic binary. 13. maxillary tooth morphology ii: (0) crowns without differentiated cusps, (1) three conspicuous cusps (all species studied here). species of the l. nigromaculatus group (subgenus liolaemus sensu stricto) were reported as having broad maxillary teeth without secondary cusps (lobo, 2001). non-polymorphic binary. 14. meckel’s groove (fig. 3): (0) open; (1) fused, meckel’s cartilage is hidden by an extensive outgrowth of the dentary bone. this last character state was described as an apomorphy of the l. chiliensis group (subtable 1. discrete osteological characters and their variation within the leiolaemus darwinii group. intraspecific variation indicated by in brackets surrounding the relevant carácter states. polymorphism in characters 1, 4, and 20, have not previously been reported. 1 2 3 4 5 6 12 13 14 15 16 18 19 20 21 22 23 33 l. albiceps [02] 0 [01] 1 0 [01] 0 1 0 1 [01] 1 0 0 1 1 [12] 0 l. chacoensis [12] [02] [01] 1 0 0 1 1 1 [01] [01] 1 0 0 [01] 0 [12] 0 l. grosseorum 2 0 0 1 0 0 1 1 1 1 [01] 1 0 0 [01] 1 [12] 0 l. inacayali [12] [01] 0 2 0 0 0 1 0 1 0 1 0 0 1 1 2 0 l. irregularis 1 0 [01] 1 0 [01] 0 1 0 1 [01] 1 0 0 [01] 1 2 0 l. koslowskyi [12] [012] [01] 1 [01] 1 0 1 1 1 [01] 1 [01] 0 [01] 1 [12] 0 l. lavillai 1 1 1 1 0 0 1 1 1 1 1 1 [01] 0 1 1 1 0 l. ornatus 1 [12] 1 1 0 0 0 1 0 1 1 1 0 0 0 1 [123] 0 l. quilmes 1 [02] 1 [01] 0 [01] 1 1 1 1 [01] 1 0 [01] [01] 1 1 0 l. scapularis 2 [02] 0 2 0 1 1 1 0 1 0 1 [01] 1 1 1 [12] 0 34 35 36 37 38 39 41 45 46 47 48 49 50 51 52 53 54 55 l. albiceps 0 1 0 [01] 0 1 [012] 0 [01] 1 0 0 0 0 [01] 0 1 0 l. chacoensis 0 0 0 [01] [01] 0 2 [01] [01] 1 1 [01] 1 0 1 1 1 [01] l. grosseorum 0 1 1 1 0 0 2 1 1 1 0 0 0 0 1 1 0 [01] l. inacayali 0 1 0 1 1 0 2 0 1 0 0 0 0 0 1 1 0 0 l. irregularis 0 1 0 0 1 1 2 1 [01] 1 0 0 0 0 [12] [01] 0 0 l. koslowskyi 0 1 0 1 1 0 0 [01] [01] 1 0 0 0 0 [01] 1 1 1 l. lavillai 0 0 0 0 1 0 2 1 [01] 1 0 0 0 0 1 0 1 0 l. ornatus 0 1 1 0 0 0 0 1 1 1 0 1 0 [01] [01] [01] 1 0 l. quilmes 0 1 1 0 0 0 [02] 1 [01] 1 1 1 [01] [01] [01] [01] 1 0 l. scapularis 0 1 1 1 0 0 2 1 [01] 1 0 0 0 0 1 [01] 0 0 68 l. díaz-fernández, a.s. quinteros, f. lobo genus liolaemus) and it is the condition exhibited by the phymaturus patagonicus group according to etheridge (1995), lobo et al. 2012 (its fig. 7d). nonpolymorphic binary. 15. cervical rib iii: this rib, when present, is very small and remains cartilaginous. (0) present; (1) absent. polymorphic binary. 16. cartilaginous extremity of cervical rib iv (fig. 4a-b): (0) bifurcated; (1) not bifurcated. polymorphic binary. 17. number of postxiphisternal elongated ribs: according to etheridge (1995), liolaemus species exhibit the most common pattern of postxiphisternal (“inscriptional ribs posterior to xiphisternals”), with their free endings bearing an elongated cartilage. continuous (table 2). 18. posterior process of the sternum: (0) present; (1) absent (all species studied here). state (0) reported only for the l. pictus group by lobo and abdala (2001). non-polymorphic binary. table 2. continuous osteological characters (see text for character descriptions). above: min-max. below: mean + standard deviation. chars liolaemus albiceps (n=4) liolaemus chacoensis (n=4) liolaemus grosseorum (n=2) liolaemus inacayali (n=1) liolaemus irregularis (n=4) liolaemus koslowskyi (n=3) liolaemus lavillai (n=2) liolaemus ornatus (n=4) liolaemus quilmes (n=5) liolaemus scapularis (n=2) 7 53-67 (61; 6) 52-52 (52; 0) 55-67 (61; 9) 52 56-75 (62; 9) 52-63 (59; 6.3) 49-60 (55; 7.8) 41-65 (55; 10.6) 47-57 (52; 5) 54-57 (56; 2.1) 8 0.2-0.3 (0.3; 0.04) 0.19-0.4 (0.3; 0.1) 0.23-0.25 (0.2; 0.01) 0.3 0.25-0.32 (0.3; 0.03) 0.16-0.27 (0.2; 0.05) 0.15-0.31 (0.2; 0.1) 0.2-0.39 (0.3; 0.08) 0.25-0.4 (0.3; 0.07) 0.17-0.26 (0.2; 0.06) 9 5-8 (7; 1.4) 0-4 (2.3; 1.7) 0-3 (1.5; 2.1) 7 5-6 (5.8; 0.7) 3-5 (4.3; 1.2) 4-4 (4; 0) 2-6 (3; 2) 1-3 (2.6; 0.9) 2-4 (3; 1.4) 10 16-18 (17; 1) 15-20 (17.6; 2) 16-18 (14; 1.4) 14 18-18 (18; 0) 16-16 (16; 0) 14-14 (14; 0) 16-16 (16; 0) 15-18 (17; 1.1) 12-14 (13; 1.4) 11 0-0 (0; 0) 0-0 (0; 0) 0-0 (0; 0) 0 0-0 (0; 0) 0-0 (0; 0) 0-0 (0; 0) 0-0 (0; 0) 1-1 (1; 1) 2-3 (2.5; 0.7) 17 3-7 (4; 2) 2-4 (3; 1.4) 2-4 (3; 1.4) 5 3-4 (3.5; 0.6) 2-3 (2.7; 0.6) 3-4 (3.5; 0.7) 4.58-5.5 (5; 0.4) 2-2 (2; 0) 3-4 (3.5; 0.7) 24 2.5-4 (3.3; 0.8) 0.3-0.4 (0.4; 0.04) 0.4 0.3 0.34-0.39 (0.38; 0.02) 0.33-0.37 (0.36; 0.02) 0.38-0.4 (0.39; 0.01) 0.36-0.39 (0.37; 0.01) 0.36-0.41 (0.39; 0.02) 0.3-0.4 (0.4; 0.04) 25 0.7-1 (0.8; 0.1) 0.6-0.8 (0.7; 0.08) 0.7-0.8 (0.7; 0.04) 0.7 0.7-0.9 (0.8; 0.06) 0.7-0.9 (0.8; 0.08) 0.8-0.81 (0.78; 0.04) 0.75-0.77 (0.76; 0.01) 0.74-0.85 (0.78; 0.05) 0.72 26 0.2-0.24 (0.21; 0.03) 0.18-0.23 (0.21; 0.02) 0.18-0.21 (0.2; 0.02) ? 0.24-0.3 (0.27; 0.03) 0.22-0.25 (0.24; 0.02) 0.24-0.27 (0.26; 0.02) 0.28-0.3 (0.29; 0.01) 0.19-0.28 (0.24; 0.04) 0.19-0.22 (0.21; 0.02) 27 1.8-3 (2.2; 0.5) 1.5-2.2 (1.8; 0.3) 2.8-3.4 (3.1; 0.5) 2.2 2-2.6 (2.3; 0.3) 1.7-3.5 (2.6; 0.9) 2.5-2.6 (2.6; 0.05) 1.9-3.8 (2.7; 0.9) 1.9-3.1 (2.3; 0.5) 1.7-2.4 (2.1; 0.5) 28 0.03-0.07 (0.05; 0.02) 0.01-0.09 (0.04; 0.04) 0.02-0.02 (0.02; 0) 0.05-0.05 (0.05; 0) 0.03-0.06 (0.05; 0.01) 0.02-0.09 (0.06; 0.04) 0.04-0.06 (0.05; 0.01) 0.04-0.08 (0.05; 0.02) 0.04-0.05 (0.05; 0.01) 0.02-0.06 (0.04; 0.02) 29 0.06-0.19 (0.13; 0.06) 0.1-0.26 (0.19; 0.07) 0.07-0.12 (0.1; 0.04) 0.19-0.19 (0.19; 0) 0.05-0.17 (0.11; 0.07) 0.06-0.19 (0.12; 0.07) 0.07-0.1 (0.09; 0.02) 0.03-0.17 (0.11; 0.06) 0.1-0.18 (0.14; 0.06) 0.05-0.2 (0.13; 0.06) 30 0.23-0.34 (0.29; 0.06) 0.26-0.33 (0.29; 0.03) 0.34-0.34 (0.34; 0) 0.27-0.27 (0.27; 0) 0.31-0.38 (0.34; 0.03) 0.28-0.34 (0.31; 0.03) 0.33-0.33 (0.33-0) 0.24-0.34 (0.28; 0.05) 0.35-0.35 (0.35; 0) 0.31-0.41 (0.35; 0.04) 31 0.04-0.11 (0.08; 0.04) 0.06-0.11 (0.09; 0.03) 0.1-0.1 (0.1; 0) 0.07-0.07 (0.07; 0) 0.05-0.11 (0.08; 0.03) 0.07-0.09 (0.08; 0.01) 0.06-0.07 (0.07; 0.01) 0.08-0.1 (0.09; 0.01) 0.06-0.06 (0.06; 0) 0.06-0.12 (0.08; 0.02) 32 0.76-0.93 (0.84; 0.09) 0.61-1.01 (0.82; 0.17) 0.76-0.79 (0.78; 0.02) 1-1 (1; 0) 0.72-1.04 (0.88; 0.14) 0.54-0.81 (0.66; 0.14) 0.83-0.89 (0.84; 0.04) 0.79-0.94 (0.86; 0.07) 0.84-0.88 (0.86; 0.03) 0.81-0.94 (0.88; 0.06) 40 8-7 (7.25;0.5) 6-3 (4.7;1.3) 5-3 (4;1.4) 6-6 (6; 0) 6-8 (6.7; 0.9) 5-12 (9;3.6) 5-8 (6.5; 2.1) 7-3 (4.7;1.7) 5-5 (5;0) 5-11 (6.6;2.5) 42 -4 (5.7;1.5) 4-4 (4;0) 5-5 (5;0) 4-4 (4;0) 4-6 (4.5; 1) 6-3 (4.67;1.63) 4-5 (4.5; 0.71) 8-7 (7.25;0.5) 7-7 (7;0) 5-4 (4.8;0.5) 43 6-6 (6; 0) 5-6 (5.7; 0.5) 6-6 (6; 0) 6-6 (6; 0) 6-6 (6; 0) 4-6 (4.7; 1.1) 6-6 (6; 0) 5-6 (5.25; 0.5) 5-6 (5.75; 0.5) 3-6 (15.4; 1.3) 44 20-22 (21; 0.8) 20-22 (20.5; 1) 18-20 (19; 1.4) 17-17 (17; 0) 22-24 (23; 1.1) 20-20 (20; 0) 18-18 (18; 0) 20-21 (20.75; 0.5) 18-20 (19; 1.4) 18-21 (19.8; 1.6) 69skeletal variation in the liolaemus darwinii group 19. clavicles: (0) without fenestra; (1) with fenestra. polymorphic binary. 20. sternal fenestra (fig. 4c-d) located in the posterior half of the sternum over the posterior half of interclavicle: (0) single; (1) divided, as described by etheridge (2000) for species of the l. wiegmannii group. polymorphic binary. 21. the posterior end of hipoischium: (0) expanded; (1) unexpanded. polymorphic binary. 22. bladelike process on posterior distal tibia: (0) absent; (1) present. the presence of the bladelike process on the posterior distal tibia was proposed as a synapomorphy of the l. montanus group by etheridge (1995) and as a synapomorphy of the subgenus eulaemus (including the l. anomalus group according to lobo et al., 2010). non-polymorphic binary. 23. caudal vertebrae without “chevron”: (0) caudal vertebra i; (1) caudal vertebrae i and ii; (2) caudal vertebrae i-iii; (3) caudal vertebrae i-iv. polymorphic multistate. 24. skull height / skull length. continuous (table 2). 25. skull width / skull length. continuous (table 2). 26. lateral rami of interclavicle / skull length. continuous (table 2). 27. diameter of major coracoid fenestra / diameter of major scapular fenestra. continuous (table 2). 28. preischial length / skull length. continuous (table 2). 29. xiphisternal rod length / skull length. continuous (table 2). 30. clavicle length / skull length. continuous (table 2). 31. maximum clavicle width / skull length. continuous (table 2). 32. sternal width / sternal length. continuous (table 2). 33. orientation of the pubis: (0) facing forward, forming an acute angle with vertebral column; (1) perpendicular to the vertebral column. state (0) only reported for l. pseudoanomalus (lobo and abdala, 2001). non-polymorphic binary. 34. membranes over coracoid fenestrae: (0) without ossification; (1) with ossification. nonpolymorphic binary. fig. 1. phylogenetic relationships recovered for the liolaemus darwinii group. fig. 2. evolution of morphology of maxillary crowns and diet within the liolaemus darwinii group. (a) character states optimized on the topology recovered, (b) maxillary teeth with straight crowns, (c) maxillary teeth with expanded crowns. dietary data for l. inacayali and l. lavillai were not found in the literature. scale = 1 mm. fig. 3. evolution of meckel’s groove within the liolaemus darwinii group. (a) character states optimized on the recovered topology, (b) meckel’s groove of lower jaw closed, (c) open meckel´s groove (c). scale = 2 mm. 70 l. díaz-fernández, a.s. quinteros, f. lobo new characters found in the present contribution 35. temporal fenestra (fig. 4e-f): (0) open (without contact between postorbital and squamosal); (1) closed (contact between postorbital and squamosal). non-polymorphic binary. 36. posterior edge of parietal (fig. 5a-b): (0) convex; (1) forming a straight margin. nonpolymorphic binary. 37. posfrontal shape (fig. 5c-d): (0) triangular; (1) elongated, not triangular. polymorphic binary. 38. premaxillary shape (fig. 5e-f): (0) nasal spine narrow and pars dentalis wide; (1) nasal spine wide and pars dentalis narrow (modified from frost, 1992). polymorphic binary. 39. otic ramus of squamosal (fig. 6a-b: (0) otic ramus located over the superior fossa of quadrate; (1) otic ramus inserted in the superior fossa of quadrate. non-polymorphic binary. 40. number of labial foramina (lateral view of maxilla). continuous (table 2). 41. disposition of labial foramina (maxilla): (0) l-shaped; (1) forming two parallel rows; (2) forming a unique series in a single line. polymorphic multistate. 42. number of mental foramina of dentary (lateral view). continuous (table 2). fig. 4. skeletal characters exhibiting variation within the liolaemus darwinii group. (a-b) cartilaginous extremity of cervical rib iv: bifurcate (a) in l. scapularis (mcn2431) and non-bifurcate (b) in l. ornatus (mcn 3545). scale = 2 mm. (c-d) sternal fenestra: single (c) in l. ornatus (mcn 3546) and divided (d) in l. quilmes (mcn 3524). scale = 2 mm. (e-f) temporal fenestra: open, without contact between postorbital and squamosal (e) in l. ornatus (mcn 3548) and closed, with contact between postorbital and squamosal (f) in l. koslowskyi (mcn 574). scale = 1mm. fig. 5. skeletal characters exhibiting variation within the liolaemus darwinii group. (a-b) posterior edge of parietal: convex (a) in l. irregularis (mcn 2443) and forming a straight margin (b) in l. quilmes (mcn 3527). scale = 1mm. (c-d) posfrontal shape triangular (c) in l. quilmes (mcn 3527) and elongated (d) in l. inacayali (mcn 500). scale = 1mm. (e-f) premaxillary shape: nasal spine narrow and pars dentalis wide (e) in l. albiceps (mcn 402) and nasal spine wide and pars dentalis narrow (f) in l. koslowskyi (mcn 573). upper arrow indicates the width of the nasal spine and bottom arrows indicate the width of the area of premaxillary teeth. scale = 2 mm. 71skeletal variation in the liolaemus darwinii group 43. number of premaxillary teeth. continuous (table 2). 44. numbers of dentary teeth. continuous (table 2). 45. lower jaw dentition (fig. 6c-d): (0) homodont; (1) heterodont. polymorphic binary. 46. first chevron shape (fig. 6e-f): chevron bones appear on anterior caudal vertebrae (hoffstetter and gasc, 1969). in liolaemus the first chevron can appear on caudal vertebra iii or iv. (0) incomplete; (1) complete. polymorphic binary. 47. length of metatarsus iv with respect to toe v (fig. 6 g-h): (0) reaches phalanx ii; (1) reaches phalanx iii (modified from arias, 2012). non-polymorphic binary. 48. length of iv metacarpal with respect to finger v: (0) reaches phalanx i; (1) reaches phalanx ii. non-polymorphic binary. 49. hipoischial fenestra: (0) absent; (1) present. polymorphic binary. 50. ischial fenestra (located close to the posterior margin of the ischium): (0) absent; (1) present. polymorphic binary. 51. number of sternal ribs: (0) three; (1) four. polymorphic binary. 52. number of branches of the xiphisternal rib: (0) three; (1) two; (2) none. polymorphic binary. fig. 6. skeletal characters exhibiting variation within the liolaemus darwinii group. (a-b) otic ramus of squamosal located outside to the superior fossa of quadrate (a) in l. irregularis (mcn 2436) and otic ramus inserted in the superior fossa of quadrate (b) in l. grosseorum (mcn 508). arrow indicates the insertion of the squamosal in the superior fossa of the quadrate. scale = 2mm. (c-d) lower jaw dentition homodont (c) in l. inacayali (mcn 500) and heterodont (d) in l. quilmes (mcn 3525). scale = 1mm. (e-f) first chevron condition: incomplete (e) in l. quilmes (mcn 3524) and complete (f) in l. albiceps (mcn 402). scale = 3mm. (g-h). length of metatarsal iv with respect to toe v: reaches phalanx iii (g) in l. irregularis (mcn 2431) and reaches phalanx ii (h) in l. inacayali (mcn 500). scale = 1 mm. fig. 7. skeletal characters exhibiting variation within the liolaemus darwinii group. (a-b) presence of open intercalated tracheal rings: present (a) in l. lavillai (mcn 4351) and absent (b) in l. grosseorum (mcn 509). scale = 1 mm. (c-d) lateral processes of the cricoid: not pronounced (c) in l. irregularis (mcn 2443) and pronounced (d) in l. albiceps (mcn 457). scale = 1mm. (e-f) sternal fenestra shape: symmetrical not widened (e) in l. albiceps (mcn 457) and wider in the posterior half (f) in l. chacoensis (mcn 599). scale = 1mm. 72 l. díaz-fernández, a.s. quinteros, f. lobo 53. open intercalated tracheal rings (fig. 7a-b): (0) present; (1) absent. polymorphic binary. 54. lateral processes of the cricoid (fig. 7c-d): (0) not pronounced; (1) pronounced. non-polymorphic binary. 55. shape of sternal fenestra (fig. 7e-f): (0) symmetrical, not widened; (1) wider in the posterior half. polymorphic binary. discussion in this contribution, twenty one new characters for the genus liolaemus were studied (characters from 35 to 55). we also report additional states for characters previously described (table 1). first, the number of scleral ossicles was previously reported by lobo and abdala (2001) as binary polymorphic (13 or 14 ossicles), whereas here we found a new state for l. albiceps (15 ossicles), we consequently coded the character as polymorphic multistate. second, the ceratohyal process was coded in lobo and abdala (2001) as non-polymorphic multistate (gradually widened, abruptly widened, and hook shaped), whereas here we found in l. quilmes a polymorphism (gradually widened and abruptly widened). third, according to lobo and abdala (2001), the sternal fenestra can be divided or single, without polymorphism. in this study we report a polymorphism in l. quilmes, which may have a divided or undivided sternal fenestra. the specimens of l. cf. quilmes included in lobo and abdala (2001) correspond to l. crepuscularis (abdala and diaz gómez, 2006), a species distinct from that included in this contribution as l. quilmes. maxillary teeth with three conspicuous cusps were found in all specimens studied. this is consistent with lobo and abdala (2001), who found this character state in all specimens of the subgenus eulaemus, whereas the species of the l. nigromaculatus group (belonging to the subgenus liolaemus sensu stricto) show the maxillary teeth without differentiated cusps. this evidence allows us to consider tricuspid maxillary teeth as a potential synapomorphy of eulaemus. the number of tracheal rings for species of the liolaemus boulengeri group reported by lobo and abdala (2001) ranges from 48 to 67. we extend this range to 41-75. a potential synapomorphy for the liolaemus darwinii group (fig. 8) involves the morphology of the terminal cartilage of cervical rib iv, which is narrow and not bifurcated in liolaemus albiceps, l. chacoensis, l. grosseorum, l. irregularis, l. koslowskyi, l. lavillai, l. ornatus, and l. quilmes (though polymorphic in the latter). lobo and abdala (2001) found this character to be polymorphic in l. koslowskyi. etheridge (1993) defined the liolaemus darwinii complex based on the “possession of maxillary teeth crowns with straight edges” as an exclusive character of this group. liolaemus scapularis (a member of the l. wiegmannii group), also shows tooth crowns with straight edges, so this character state is not exclusive to the l. darwinii complex, as etheridge (1993) proposed. moreover, our results indicate variation within this group (fig. 2a) and according to recognized relationships (abdala, 2007), this character has changed in the terminal subclade of the l. darwinii group (the l. ornatus group). therefore, it can be considered a synapomorphy of the l. ornatus group (fig. 2a-c). optimizing the character states of maxillary tooth crowns and the diet in the tree recovered, we found that an insectivorous diet and the straight-edged maxillary tooth crowns change together along the tree (fig. 2a). fig. 8. evolution of cartilaginous extremity of cervical rib iv. potential synapomorphy of the liolaemus darwinii group. recovered topology of the l. darwinii phylogeny. liolaemus crepuscularis is excluded because of the lack of information on this character state. fig. 9. evolution of bladelike process on posterior distal tibia in liolaemus. note the secondary loss in the l. anomalus group. tree modified from schulte et al. (2000). 73skeletal variation in the liolaemus darwinii group this can be interpreted as supporting a possible relationship between diet and tooth crown shape. tooth morphology can reflect ecological adaptations and exhibit derived traits which may distinguish alimentary specializations (hotton, 1965). we found that l. scapularis, l. quilmes, l. lavillai, l. grosseorum and l. chacoensis have straight crowns and are insectivorous. lobo and abdala (2001) cited straight crowns for l. crepuscularis (their l. cf. quilmes). semhan et al. (2013) reported that l. crepuscularis feeds mainly on insects, but can fluctuate to omnivorous or herbivorous diets through the year based on prey availability. all other species studied here show expanded crowns, and their diet can be characterized as omnivorous or herbivorous (l. albiceps). the only exception to this association is l. koslowskyi, which has expanded crowns and an insectivorous diet (aun and martori 1998). aun and martori (1998) do not mention the season of the study, so it is not known if this taxon can change its diet as does l. crepuscularis. phymaturus is the sister clade of liolaemus, and has a strictly herbivorous diet (lobo et al., 2010). species of phymaturus have teeth with expanded crowns (lobo and quinteros, 2005). the same phenomenon can be observed in nonliolaemid lizards. hotton (1965) found that herbivorous lizards (dipsosaurus, sauromalus, and ctenosaura) have highly cuspidate and antero-posteriorly widened teeth (similar to the expanded crowns of humans). in lizards that mainly feed on ants (phrynosoma), hotton (1965) described pointed and conical teeth, and in lizards that feed on bees and wasps (urosaurus and callisaurus), he described thin, cylindrical, sharp teeth. herrel et al. (2004) observed that lacertids with omnivorous diets show teeth with wider crowns, whereas insectivorous species had slender and pointed teeth. in agreement with these results, we found that dentition seems to vary with diet. nevertheless, further studies are needed in liolaemus in order to confirm the hypothesis of correlation between straight crowns and insectivorous diet. the exposure or not of meckel’s groove in liolaemid lizards was used by etheridge (1995) as a character in his taxonomic proposal. he proposed this character as a synapomorphy of the liolaemus chiliensis (subgenus liolaemus) group which have a fused channel, and also for the phymaturus patagonicus group within the sister genus of liolaemus (etheridge,1995; lobo and quinteros, 2005; lobo et al., 2010). lobo and abdala (2001) viewed the groove as a potential synapomorphy of the l. darwinii group. here, we found that meckel’s groove exhibits an additional change (reversal), being open in the l. ornatus group. moreover, lobo and abdala (2001) found this character state for l. crepuscularis, a basal member of the l. ornatus group. therefore, we can conclude that the open meckel’s groove can be considered a synapomorphy of the l. ornatus group (fig. 2a-c). the ancestral state would be the open meckel’s groove, already present in ctenoblepharys, the basal genus of the family liolaemidae, and preserved in the phymaturus palluma group. the hypothesis of the open meckel’s groove as the ancestral state is supported by the presence of this character state in other families related to liolaemidae (e.g., leiosauridae and opluridae according to pyron et al., 2013 and reeder et al., 2015), but exhibiting polymorphism in many iguanian families (frost and etheridge, 1989) such as leiocephalidae (etheridge, 1966) and phrynosomatidae (etheridge, 1964). the bladelike process on the posterior distal tibia was described by etheridge (1995), as a synapomorphy of the liolaemus montanus group. in his proposal, etheridge (1995) did not include the l. anomalus group inside the l. montanus group. in recent analyses (espinoza et al., 2004; abdala 2007), the l. anomalus group is inferred as more closely related to the l. boulengeri group. these two groups together are called the l. boulengeri series (included inside the l. montanus section in schulte et al., 2000). the l. anomalus group lacks this tibial process, which we consider to be a secondary loss (fig. 9). here we found that every member of the l. darwinii group studied has the bladelike process on the distal tibia. characters that were studied in other groups of lizards were informative for liolaemus, including shape of the premaxilla (frost, 1992; tropidurids); squamosalquadrate joint (modified from frost, 1992); metacarpal of iv finger reach the i or ii phalange of v finger (modified from arias, 2012; teiids); and presence of open tracheal rings (lobo and quinteros, 2005; lobo et al., 2010; phymaturus). here, we found the same variation in the shape of the premaxilla described by frost (1992) for tropidurines: (0) narrow nasal spine wide area of premaxillary tooth attachment and (1) broad nasal spinenarrow premaxillary tooth area. frost (1992) found no relationship between the width of the area of premaxillary teeth and number of teeth in it. we found similar results for the liolaemus species studied here, where the number of premaxillary teeth is constant regardless of the width of the area in which they are inserted. also, frost (1992) described two states for the squamosal-quadrate articulation. these states are related to the width of the superior fossa of the quadrate, which may be relatively small or enlarged. in the liolaemus species studied, it was observed that the superior fossa of the quadrate corresponds to the “relatively enlarged” state according to frost (1992). all species except two (l. albiceps and l. irregularis) exhibit one of the states proposed 74 l. díaz-fernández, a.s. quinteros, f. lobo by frost (1992): the otic ramus of the squamosal contacts (but is not inserted in) the posterior edge of the superior fossa of the quadrate. in contrast, l. albiceps and l. irregularis share the same state, with the otic ramus of the squamosal inserted in the medial part of the fossa (fig. 6 b), thus reinforcing the hypothesis that they are sister species. the character observed in teiids is related to the relative length of metacarpals, metatarsals and digits. arias (2012) coded a character related to the length of toe v into three states: length of toe v exceeding that of metatarsal iv, equaling that of metatarsal iv, and not reaching the length of metatarsal iv. in all liolaemus species studied here, the toe v always exceeds metatarsal iv in length, but there is variation as to which phalanx of toe v metatarsal iv reaches (fig. 6 g-h). we recorded this character differently for one of the outgroup taxa (l. inacayali, where metatarsal iv reaches phalanx ii) with respect to the l. darwinii group (in which metatarsal iv reaches phalanx iii). since we do not have samples of other members of the l. telsen group, we were not able to determine if this is a potential synapomorphy for that group. similar variation to that described above for the hindlimbs was described for the forelimbs in liolaemus. the length of metacarpal iv with respect to finger v, exhibited two states: reaches phalanx i or reaches phalanx ii. within the l. darwinii group, the character state in which finger v reaches phalanx ii occurs independently in l. quilmes and l. chacoensis. it is clear that variation between fore and hindlimbs is independent. the presence of intercalated open tracheal rings was only found in the members of the liolaemus ornatus group. therefore, this character state can be considered as a possible synapomorphy of this group. nevertheless, the intercalated open tracheal rings were also found to be polymorphic in l. scapularis and in l. quilmes, species basal to the l. ornatus group. this character state was primarily proposed for phymaturus (lobo and quinteros, 2005), but no polymorphisms were found in this genus. while newly described characters have not yet been observed in many taxa, a general idea of the polarity of the optimized character was obtained. the study of new sources of variation and the distribution of characters described here in other taxa, as well as in the other two genera of liolaemidae (phymaturus and ctenoblepharys) would allow us to hypothesize about relationships within this iguanian family. in this study we note that in the liolaemus darwinii group the most informative characters are taken from the regions of the ribs, sternal plate, pectoral girdle, snout, jaw, larynx and hyoid arches. the literature shows that the osteology has been more thoroughly studied in the families phrynosomatidae, tropiduridae and corytophanidae within iguania (reeve, 1952; etheridge, 1964; presch, 1969; etheridge and de queiroz, 1988; frost and etheridge, 1989; frost, 1992; mcguire, 1996; reeder and wiens, 1996; torrez carvajal, 2007; frost et al., 2011) and osteological characters are observed mainly from the cranial skeleton. this shows a bias in the focus of skeletal variation studies, making it difficult to determinate the levels of variation across the whole anatomy of lizards. even if the literature emphasizes the idea of the importance of using osteological characters in different phylogenetic analyses and morphological descriptions (conrad, 2008; gauthier et al., 2012; reeder et al., 2015), it is clear that there is not enough information yet to fully understand the patterns of morphological diversity across all existing iguanian families. acknowledgements we thank roberto sanchez, soledad valdecantos, alejandra paz, jessica monroig, thomas hibbard, matias quipildor, silvio salcedo, sabrina portelli, romina semhan, cristian abdala, alejandro laspiur, adolfo juarez, mario fernandez and melisa diaz fernandez for helping us with field or lab work. patricia garcia and t. hibbard improved the english style. we also thank the ministerio de ambiente y producción sustentable (secretaria de ambiente) of salta argentina and y. bonduri for assisting with scientific collection permits (resolution nº 815/13). we thank e. lavilla and s. kretzschmar (instituto de herpetología, fundación miguel lillo, tucumán argentina) for allowing us to study fml materials under their care. this study was supported by grants (fl) from conicet consejo nacional de investigaciones científicas y técnicas of argentina (pip 2841) and ciunsa consejo de investigaciones de la universidad nacional de salta, argentina (ciunsa 2036), and graduate fellowship, from consejo nacional de investigaciones científicas y técnicas (ldf). we also thank two anonymous reviewers for their careful reading of our manuscript and their insightful comments and suggestions. references abdala, c.s. 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(2001). phylogenetics of the lizard genus tropidurus (squamata: tropiduridae: tropidurinae): direct optimization, descriptive efficiency, and sensitivity analysis of congruence between molecular data and morphology. mol. phylogenet. evol. 21: 352-371. gauthier, j.a., kearney, m., maisano, j.a., rieppel, o., behlke, a.d.b. (2012). assembling the squamate tree of life: perspectives from the phenotype and the fossil record. bull. peabody mus. nat. hist. 53: 3-308. goloboff, p., farris, j., nixon, k. (2003): t.n.t.: tree analysis using new technology. available from: www.zmuc.dk/public/phylogeny. gonzález-marín, a., hernando, a. (2013): notes on the postcranial osteology of the sand lizard liolaemus azarai (squamata: liolaemidae). phyllomedusa 12: 135-141. herrel, a., vanhooydonck, b., van damme, r. (2004): omnivory in lacertid lizards: adaptive evolution or constraint? bmc evol. biol. 17: 974-984. 76 l. díaz-fernández, a.s. quinteros, f. lobo hoffstetter, r., gasc, j.p. (1969): vertebrae and ribs of modern reptiles. in: biology of the reptilia, morphology a i, pp. 201-231. gans, c., ed. academic press, london and new york. hotton, n. (1965): a survey of adaptative relationships of dentition to diet in the north american iguanidae. am. midl. nat. 53: 29-37. keller, c., krause, l. (1986): the appendicular skeleton of liolaemus occipitalis boulenger 1885 (sauria, iguanidae). rev. bras. biol. 46: 727-740. lang, m. (1989): phylogenetic and biogeographic patterns of basiliscine iguanians (reptilia: squamata: iguanidae). bonn. zool. monogr. 28: 1-172. lobo, f. (2001): a phylogenetic analysis of lizards of the liolaemus chiliensis group (iguania: tropiduridae). herpetol. j. 11: 137-150. lobo, f. (2005): las relaciones filogenéticas en el grupo chiliensis de liolaemus (iguania: liolaemidae). sumando nuevos caracteres y taxa. acta zool. lilloana 49: 67-89. lobo, f., abdala, c., valdecantos, s. (2012): morphological diversity and phylogenetic relationships within a south-american clade of iguanian lizards (liolaemidae: phymaturus). zootaxa 3315: 1-41. lobo, f., abdala, c. (2001): variación morfológica en el esqueleto de liolaemus (iguania: liolaemidae). búsqueda y descripción de caracteres. cuad. herpetol. 15:119-135. lobo, f., abdala, c. (2002): la información cladística de un set de datos morfológicos en lagartos del género liolaemus (iguania: liolaemidae). cuad. herpetol. 16: 137-150. lobo, f., quinteros, a.s. (2005): a morphological approach on the phylogenetic relationships within the genus phymaturus (iguania: liolaemidae). the description of four new species of argentina. pap. avulsos. zool. 45: 143-177. lobo, f., espinoza, r.e., quinteros, a.s. (2010): a critical review and systematic discussion of recent classification proposals for liolaemid lizard. zootaxa 2549: 1-30. mcguire, j.a. (1996): phylogenetic systematics of crotaphytid lizards (reptilia: iguania: crotaphytidae). bull. carnegie mus. nat. hist. 32: 1-143. núñez, h., navarro, j., garín, c., pincheira-donoso, d., meriggio, v. (2003): phrynosaura manueli y phrynosaura torresi, nuevas especies de lagartijas para el norte de chile (squamata: sauria). bol. mus. nac. hist. nat. 52: 67-88. olave, m., avila, l.j., sites jr., j.w., morando, m. (2014): multilocus phylogeny of the widely distributed south american lizard clade eulaemus (liolaemini, liolaemus). zool. scr. 43: 323-337. presch, w. (1969): evolutionary osteology and relationships of the horned lizard genus prynosoma (family iguanidae). copeia 1969: 250-275. pyron, r.a., burbrink, f. t., wiens, j.j. (2013): a phylogeny and revised classification of squamata, including 4161 species of lizards and snakes. bmc evol. biol. 13: 1-93. reeder, t., wiens, j.j. (1996): evolution of the lizard family phrynosomatidae as inferred from diverse types of data. herpetol. monogr. 10: 43-84. reeder, t.w., townsend, m., mulcahy, d.g., noonan, b. p., wood, p.l., sites jr., j.w., wiens, j.j. (2015): integrated analyses resolve conflicts over squamate reptile phylogeny and reveal unexpected placements for fossil taxa. plos one 10: 1-22. reeve, w.l. (1952): taxonomy and distribution of the horned lizard genus phrynosoma. univ. kans. sci. bull. 34: 816-952. schulte, j.a. ii, macey, j.r., espinoza, r.e., larson, a. (2000): phylogenetic relationships in the iguanid lizard genus liolaemus: multiple origins of viviparous reproduction and evidence for recurring andean vicariance and dispersal. biol. j. linn. soc. 69: 75-102. schulte, j.a. ii, valladares, j.p., larson, a. (2003): phylogenetic relationships within iguanidae inferred using molecular and morphological data and a phylogenetic taxonomy of iguanian lizards. herpetologica 59: 399419. semhan, v.r., halloy, m., abdala, c.s. (2013): diet and reproductive states in a high altitude neotropical lizard, liolaemus crepuscularis (iguania: liolaemidae). south am. j. herpetol. 8: 102-108. simoes-lopes, p.c.a., krause, l. (1998): osteologia do sincranio de liolaemus occipitalis boulenger, 1885 (sauria, iguanidae). rev. bras. zool. 5: 491-508. torres-carvajal, o. (2004): the abdominal skeleton of tropidurid lizards (squamata: tropiduridae). herpetologica 60: 75-83. uetz, p. (editor), the reptile database, http://www.reptile-database.org, accessed july 31, 2016. wassersug, r.j. (1976): a procedure for differential staining of cartilage and bone in whole formalin fixed vertebrates. stain technol. 51: 131-134. 77skeletal variation in the liolaemus darwinii group appendix a specimens examined. the acronyms used were mcn (museo de ciencias naturales de la universidad nacional de salta) and fml (fundación miguel lillo). liolaemus albiceps (n=4): argentina: salta: los andes: camino al acay desde estación muñano, 5-6 km, (24°18'31.6''s; 66°09'7''w) mcn 402, 407, 1452, 1453. liolaemus chacoensis (n=4): argentina. mcn 503, 504, 505, 599. no data. liolaemus grosseorum (n=2): argentina: mendoza: san rafael: orillas del embalse el nihuil, mcn 508, 509. liolaemus inacayali (n=1): argentina: rio negro: ing. jacobacci: 25 de mayo, mcn 500. liolaemus irregularis (n=4): argentina: salta: los andes: aprox. a 11km al se de san antonio de los cobres por ruta 51 (24°8'53.44''s; 66°8'21.37''w). mcn 2431, 2436, 2443, 2446. liolaemus kingii (n=1): argentina: santa cruz. mcn 565. no data. liolaemus koslowskyi (n=3): argentina: la rioja: castro barros: 6 km. e. de anillaco (28°47’s; 66°52’w). mcn 573, 574, 576. liolaemus lavillai (n=2): argentina: jujuy : extremo norte del parque nacional los cardones, oeste de la recta de tin tin. (25°05’09’’s; 66°00’00’’w). mcn 2688, 4351. liolaemus multicolor (n=1): argentina: jujuy: abra pampa: cochinoca. fml 2065. liolaemus ornatus (n=4): argentina: jujuy: castro tolay: a 7 km de s de rio las burras. mcn 3545, 3546, 3547, 3548. liolaemus pseudoanomalus (n=1): argentina: la rioja: castro barros: 6 km. e. de anillaco (28°47’s; 66°52’w). mcn 526. liolaemus quilmes (n=5): argentina: salta: cachi: cachi: a 7 km sur de palermo entre cachi adentro y palermo, (24°58’41,9’’s; 66°08’42,2’’w). mcn 3524, 3525, 3526, 3527, 3528. liolaemus scapularis (n=2): argentina: salta: cafayate: los médanos. mcn 253, 283. acta herpetologica vol. 12, n. 1 june 2017 firenze university press morphological variation and sexual dimorphism in liolaemus wiegmannii (duméril & bibron, 1837) (squamata: liolaemidae) from uruguay joaquín villamil1,*, arley camargo2, raúl maneyro1 sex does not affect tail autotomy in lacertid lizards panayiotis pafilis1,*, kostas sagonas2, grigoris kapsalas1, johannes foufopoulos3, efstratios d. valakos4 fire salamander (salamandra salamandra) males’ activity during breeding season: effects of microhabitat features and body size raoul manenti*, andrea conti, roberta pennati call variation and vocalizations of the stealthy litter frog ischnocnema abdita (anura: brachycephalidae) pedro carvalho rocha1,2,*, joão victor a. lacerda2,3, rafael félix de magalhães2,3, clarissa canedo4, bruno v. s. pimenta5, rodrigo carrara heitor6, paulo christiano de anchieta garcia2,3 feeding ecology of two sympatric geckos in an urban area of northeastern brazil josé guilherme g. sousa1, adonias a. martins teixeira2,*, diêgo alves teles2, joão antônio araújo-filho2 and robson waldemar ávila3 a pattern-based tool for long-term, large-sample capture-mark-recapture studies of fire salamanders salamandra species (amphibia: urodela: salamandridae) jeroen speybroeck1,*, koen steenhoudt2,$ skeletal variation within the darwinii group of liolaemus (iguania: liolaemidae): new characters, identification of polymorphisms and new synapomorphies for subclades linda díaz-fernández*, andrés s. quinteros, fernando lobo marking techniques in the marbled newt (triturus marmoratus): pit-tag and tracking device implant protocols hugo le chevalier1, olivier calvez2, albert martínez-silvestre3, damien picard4, sandra guérin4,5, francis isselin-nondedeu6, alexandre ribéron1, audrey trochet1,2,* evidence for directional testes asymmetry in hyla gongshanensis jindongensis qing gui wu1, wen bo liao2,* the advertisement call of pristimantis subsigillatus (anura, craugastoridae) florina stănescu1,4, rafael márquez2, paul székely3,4,*, dan cogălniceanu1,4,5 nematodes infecting anotosaura vanzolinia (squamata: gymnophthalmidae) from caatinga, northeastern brazil bruno halluan s. oliveira¹, adonias a. martins teixeira¹,*, romilda narciza m. queiroz¹, joão a. araujo-filho¹, diêgo a. teles¹, samuel v. brito2, daniel o. mesquita¹ where is my place? quick chorus structure assembly in the european tree frog michal berec a possible mutualistic interaction between vertebrates: frogs use water buffaloes as a foraging place piotr zduniak1,*, kiraz erciyas-yavuz2, piotr tryjanowski3 good vibrations: a novel method for sexing turtles donald t. mcknight1,2,*, hunter j. howell3, ethan c. hollender1, and day b. ligon1 errata to mecke, s., hartmann, l., mader, f., kieckbusch, m., kaiser, h. (2016): redescription of cyrtodactylus fumosus (müller, 1895) (reptilia: squamata: gekkonidae), with a revised identification key to the bent-toed geckos of sulawesi. acta herpetologica 11(2): acta herpetologica 14(2): 81-87, 2019 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/a_h-7745 substrate type has a limited impact on the sprint performance of a mediterranean lizard pantelis savvides1,*, eleni georgiou1, panayiotis pafilis2,3, spyros sfenthourakis1 1 dept. of biological sciences, university of cyprus, nicosia, cyprus. *corresponding author. email: savvides.pantelis@ucy.ac.cy 2 section of zoology and marine biology, dept. of biology, national and kapodistrian university of athens, greece 3 zoological museum, national and kapodistrian university of athens, greece submitted on: 2018, 7th november; revised on: 2019, 28th february; accepted on: 2019, 29th may editor: simon baeckens abstract. environmental factors may affect animal performance in diverse ways, even among different populations of a single species. here, we assess the impact of substrate type on the sprint performance (maximum speed and acceleration) of schreiber’s fringe-fingered lizard (acanthodactylus schreiberi). this species is a skillful runner that also bears micro spike-like protruding scales on its toepads (toe fringes), an adaptation for locomotion on sand. we worked with three populations living in habitats that differ in substrate type (sand, soil and rock). we measured sprint performance using a race-track with custom substrate platforms replicating the different substrate types. we formulated two hypotheses: first, we anticipated that the three populations would differ in their sprint performance due to the differences in substrate type; second, we expected that each population would perform better on its home substrate. our results generally refuted the hypothesis that sprint performance would differ on different substrate types. our results suggest that there is a restricted effect of substrate type on locomotion and indicate a multifactor interplay among alternative underlying parameters. keywords. ecophysiology, morphology, locomotion, lacertidae, cyprus. introduction sprint performance is very important for all animals, as it affects most of their daily activities. sprinting is quite common among lizards during foraging, antipredator defense and interand intraspecific competitive behavior (losos and irschick, 1996; husak et al., 2006; mcelroy et al., 2008). speed and acceleration, the main components of sprint performance, may be crucial for the overall fitness of individuals (jayne and bennett, 1990; robson and miles, 2000; miles, 2004). interactions between the ecology and morphology of species act as driving factors that exert strong selective pressures leading to optimal locomotor performance (van damme et al., 2003; husak et al., 2006). for instance, gekkonid and lacertid lizards were shown to adopt different locomotion patterns because of their distinct ecology (aerts et al., 2000). also, losos (1990) reported that locomotion parameters and morphological features evolved concordantly in 15 anolis species. the results of previous studies on the effects of substrate type on locomotion are puzzling (korff and mchenry, 2011; tulli et al., 2012; vanhooydonck et al., 2015). the texture complexity (e.g., particle size, shape and roughness) of the substrate type is also known to affect locomotor performance (brandt et al., 2015; bergmann et al., 2017). the propulsive forces applied by the toes on non-solid substrates (e.g., sand) may be reduced because of insufficient grip and friction with the substrate, therefore leading to suboptimal sprint performance (redfern et al., 2001; korff and mchenry, 2011; brandt et al., 2015). stiff, rough substrates provide more 82 pantelis savvides et alii friction that enhances grip, thus allowing higher performance (kerdok et al., 2002; van der tol et al., 2005; brandt et al., 2015; bergmann et al., 2017). morphological features that are often beneficial on certain substrate types include adaptations of the epidermis that covers the digits. for instance, the adhesive ability of many gekkonidae species depends on toe pad microarchitecture (setae) that allows them to run easily on smooth and vertical surfaces such as walls or even glass (autumn et al., 2002; autumn et al., 2005). also, some other taxa bear fringes on their toes (e.g. genera acanthodactylus, basiliscus and uma) enabling them to run fast on non-solid substrates (e.g., sand or water) without ‘sinking’, as fringes increase the amount of toe surface that comes into contact with the substrate while running (salvador, 1982; luke, 1986; carothers, 1986). in this study, we aimed to evaluate the impact of substrate type on sprint performance (maximum speed and maximum instant acceleration) in schreiber’s fringe-fingered lizard (acanthodactylus schreiberi boulenger, 1878). we worked with three cypriot populations that reside in habitats with different substrate types (soil free from rocks, rock and sand). we formulated two hypotheses. first, we anticipated that the sprint performance of the focal populations would differ due to the presence of different substrate types in the habitats and due to the different running styles required on each one (van damme et al., 1998). second, we predicted that individuals would perform better on their home substrates than those coming from other habitats (goodman et al., 2008). materials and methods study system acanthodactylus schreiberi is a medium-sized lacertid lizard (snout-vent length 73-93 mm for males and 55-76 mm for females), inhabiting various habitats all over cyprus (baier et al., 2009). even though it is considered to be mostly a sanddwelling lizard, it can be found from coastal areas to mountain pine forests (over 1,300 m a.s.l) (baier et al., 2009). the species is a skillful and swift runner that can use bipedalism while running (savvides et al., 2017). the habitats of the three focal populations vary considerably in substrate and vegetation type (fig. 1). geri (35o05’50”n, 33o26’21”e, elevation 183 m a.s.l.) is a sub-urban shrubland characterized by the presence of boxthorn (lycium ferocissimum), thorny burnet (sarcopoterium spinosum) and conehead thyme (thymbra capitata). the substrate consists mostly of solid soil without any rocks. agros (34o56’27”n, 33o00’14”e, elevation 1,348 m a.s.l.) is in a pine forest (pinus brutia) with dense shrubs and has a rocky soil as its substrate. akrotiri (34o56’27”n, 33o00’14”e, elevation 1 m a.s.l.) is a coastal dune habitat with quite sparse phrygana, where the substrate is fine-grained sand. we captured a total of 67 adult individuals of both sexes (excluding gravid females) (geri, n = 22; agros, n = 22; akrotiri, n = 23). lizards were housed in individual terraria (30 x 30 x 30 cm) in the laboratory under a constant temperature (30 oc) and controlled photoperiod (16-h light and 8-h dark), and were provided with mealworms (tenebrio molitor larvae) and fresh water ad libitum. all lizards were released at their sampling sites after the completion of the experiments (experimental lizards remained in the laboratory for two weeks). based on the substrate type of each habitat, we constructed three removable substrate platforms that were used on a custom-made wooden racetrack (240 x 12 cm2), bearing 10 cm increments on its back and clear acrylic glass on its front in order to allow video recording of each trial (fig. 1). morphological measurements snout-vent length (svl), hind limb length (hll) and hind toe length (ht) were recorded with a digital caliper (silverline 380244, accurate to 0.01 mm), before running trials. also, the length of the largest right hind toe, the total number of fringes on it and the length of the three largest fringes from base to tip were measured using stereoscopic images of their toes (n = 60, 10 males and 10 females from each population) (fig. 2), in order to test for correlation between toe length and fringe microarchitecture (length and number of fringes). sprint performance all individuals were allowed to thermoregulate for an hour in a specifically designed terrarium (van damme et al., 1986) before each trial, so as to perform at the highest level possible (irschick and losos, 1998). after this period, lizards were placed in the racetrack. we triggered motion with a brush touching the lizard’s tail base. each individual performed five trials on each substrate type in a single day. between trials on fig. 1. the three habitats studied. a. geri, b. agros, c. akrotiri and the respective platforms that were used in the laboratory to simulate the substrate of each habitat. 83substrate type and speed performance in a lizard different substrate types, lizards were left to rest for a day and were then tested on the new type (in total, 15 trials per individual within five days). trials were recorded with a video camera (olympus sh-60) viewing the racetrack from the side (covering all the distance from start to finish), at a rate of 240 frames per second. sprint performance was estimated from the video recordings (martin and avery, 1998; kaliontzopoulou et al., 2012; vanhooydonck et al., 2015; savvides et al., 2017). maximum speed was calculated for all trials, based on the number of frames needed to cover a distance of 20 cm within a known time interval (savvides et al., 2017). we chose the highest values for each individual to represent its best trial. maximum instant acceleration was calculated by digitizing the position of the lizard’s snout in every frame, for all trials, on x(movement) and y(gravity) axes (matlab dltdataviewer3; hedrick, 2008). in each frame, we estimated the displacement of the snout and converted it from pixels to meters. we filtered the curve of the instantaneous displacement of the snout over time (i.e. instantaneous velocity) using a fourth-order zero phase shift butterworth low-pass data noise filter (40hz; vba application in office excel). the time differential of the instantaneous velocity yields the instantaneous acceleration and we chose the highest value (i.e. peak) for each individual’s best trial (van wassenbergh, 2007). we only used trials during which lizards ran continuously for at least 50 cm. using the pearson product moment correlation coefficient, we found that the length of the largest hind toe correlated strongly with the length of the three longest fringes in all populations (all r values > 0.6 and p values < 0.05), so we used this measurement as a proxy of fringe size to detect possible fringe effects on sprint performance. statistical analyses we used the shapiro – wilks and levene’s tests to check for data normality and homogeneity of variance, respectively. all data were log-transformed based on the results of these tests. log-transformed data for the morphological characters were compared between sexes for each population using oneway mancova and taking svl as covariate. one-way mancova was also used to search for differences among populations in relation to the transformed data for the morphological characters (hll and ht), using again svl as a covariate. one-way manova was used to compare their sprint performance among different substrate types and populations. a post-hoc tukey test was used in order to determine the differences among populations. the friedmann test and the bonferroni correction were used to compare the performance of each individual on the three types of substrate. the effects of the logtransformed values of morphological characters (hll and ht) on sprint performance were identified independently, using linear regression. results morphological characters showed significant differences between the sexes in each population, with female individuals having relatively smaller hind limbs and hind toe length than male individuals (one-way mancova: geri, f2,18 = 9.965 roy’s largest root = 1.107; agros, f2,18 = 29.373 roy’s largest root = 3.264; akrotiri, f2,18 = 5.517 roy’s largest root = 0.613; all p values < 0.05). thus, hind limbs and hind toes length were therefore compared among populations separately for males and females (table 1), but no significant differences were found (all p values > 0.05). when comparing sprint performance among populations, we observed that the agros males (home habitat with rocky substrate) were significantly faster than the other populations (one-way manova: f6,26 = 4.072, roy’s largest root = 0.940, p = 0.005). they achieved the highest maximum speed (post hoc tukey hsd: agros vs. geri, p = 0.012; agros vs. akrotiri, p = 0.002) and maximum instant acceleration (post hoc tukey hsd: agros vs. geri, p = 0.001; agros vs. akrotiri, p = 0.002) on soil fig. 2. stereoscopic image of the toes and an example of how we measured the three largest toe fringes. table 1. mean values for morphological characters between males and females from the three populations. svl: snout-vent length, hll: hind limb length, ht: longest hind toe length. values are in cm. character geri agros akrotiri mean sd mean sd mean sd svl ♂♂ 7.10 0.43 6.90 0.64 6.35 0.40 ♀♀ 6.45 0.41 6.50 0.34 6.00 0.44 hll ♂♂ 4.60 0.23 4.65 0.14 4.49 0.43 ♀♀ 4.00 0.28 4.00 0.16 3.96 0.22 ht ♂♂ 1.29 0.14 1.24 0.12 1.22 0.10 ♀♀ 1.13 0.22 1.10 0.09 1.00 0.10 84 pantelis savvides et alii (fig. 3). we did not observe further differences in the sprint performance among other populations on the other substrates. the sprint performance of all lizard populations (for both sexes) did not differ among the three types of substrate (all p values > 0.05) (fig. 3). the length of hind limbs and toes showed no significant effects on performance among populations and substrates (all regression p values > 0.05). discussion locomotion patterns may change in response to endogenous or extraneous factors (vanhooydonck and van damme, 2003; sathe and husak, 2018). among the latter, the type of substrate is known to affect locomotion in lizards. in some cases, specific substrate types favor higher performance (e.g., solid substrates like rock), while others restrict locomotion (e.g., substrates not providing sufficient grasp, e.g., sand or mud) (vanhooydonck et al., 2005; tulli et al., 2012). in our study, we did indeed see certain differences in sprint performance among conspecific populations of schreiber’s fringe-fingered lizard. however, these differences followed a rather unclear pattern and indicated a limited effect of substrate type on locomotion. interestingly, when we analyzed sprint performance taking into account the effect of hind limb length, we did not find any significant effects for any type of substrate. also, the multiple regression analyses did not show a beneficial effect of toe fringes (using the hind toe as a proxy). hind limbs have been repeatedly reported to affect lizard locomotion (vanhooydonck et al., 2001; herrel et al., 2002; savvides et al., 2017). the absence of such effects in our study might indicate an interplay among fig. 3. mean values for speed and acceleration on each substrate within populations and for males and females. 85substrate type and speed performance in a lizard other morphological features (e.g., fore limbs, tail length, etc.) involved in locomotion in response to substrate type requirements (herrel et al., 2002). on the other hand, we cannot rule out the possibility that despite our best efforts, lizards might have underperformed and have failed to achieve their maximum performance levels. contrary to our first hypothesis regarding sprint performance on different substrates, no differences emerged from the comparison among the three populations, save the single case of the agros males (rock substrate) that were the fastest sprinters. however, subsequent analysis based on morphological features, failed to provide an underlying reason for this finding. in our initial hypothesis, we considered the three types of substrate to be of different quality. we presumed that sand would be the more challenging substrate because of its grainy texture that “sinks” under the weight of a running lizard (clemente, 2014; sathe and husak, 2015). as such, we expected that sprint performance therein would be the lowest. on the other hand, solid substrates are known to provide high traction and thus facilitate locomotion (lejeune et al., 1998; claussen et al., 2002; brandt et al., 2015; bergmann et al., 2017). apparently, these predictions were not valid in our study system, as we failed to find any significant deviations. it seems that there is no ideal substrate type for a. schreiberi locomotion and all populations are well adapted in their home habitat. according to our second hypothesis, lizards should have performed at higher levels on their home substrate. however, we did not find such a pattern in our study system. all populations performed at similar levels on all substrate types, indicating that the species conserves generalized running capabilities that allow a high level of performance on various types of substrate. the rejection of the specialized populations hypothesis, might be due to overlapping genetic pools or mixed habitat characteristics, leading the lizards in similar directions regarding their kinematics and their interactions between morphology, biomechanics and environmental factors. we also have to highlight the fact that long periods of draught in cyprus can change abruptly to rainy periods (especially during late spring and early summer). this would cause the substrate to change its properties, such as its roughness and its potential to provide grip and would thus favor the generalized pattern we observed in this study. this study will enhance the growing body of literature on saurian locomotion, as it examines some of the widely accepted principles in the field. according to our findings, the type of substrate has a limited impact on sprint performance, and at least for schreiber’s fringefingered lizard, there were no strict patterns observed. further research including more species will shed light on the fascinating interplays taking place in lizard locomotion. acknowledgments the study was carried out according to the cypriot national law on animal rights and welfare (law 55(i)/2013 for animal use on scientific experiments) and under a permit issued by the ministry of agriculture, rural development and environment (permit no: 02.15.001.003, 04.05.002.005.006). we are grateful to dr. anna-nicola chapman for her assistance. references aerts, p., van damme, r., vanhooydonck, b., zaaf, a., herrel, a. 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(2007):  https://www.uantwerpen.be/ en/staff/sam-vanwassenbergh/my-website/excel-vbatool. acta herpetologica vol. 14, n. 2 december 2019 firenze university press podarcis siculus latastei (bedriaga, 1879) of the western pontine islands (italy) raised to the species rank, and a brief taxonomic overview of podarcis lizards gabriele senczuk1,2,*, riccardo castiglia2, wolfgang böhme3, claudia corti1 substrate type has a limited impact on the sprint performance of a mediterranean lizard pantelis savvides1,*, eleni georgiou1, panayiotis pafilis2,3, spyros sfenthourakis1 coping with aliens: how a native gecko manages to persist on mediterranean islands despite the black rat? michel-jean delaugerre1,*, roberto sacchi2, marta biaggini3, pietro lo cascio4, ridha ouni5, claudia corti 3 pit-tags as a technique for marking fossorial reptiles: insights from a long-term field study of the amphisbaenian trogonophis wiegmanni pablo recio, gonzalo rodríguez-ruiz, jesús ortega, josé martín* occurrence of batrachochytrium dendrobatidis in the tensift region, with comments on its spreading in morocco ait el cadi redouane1, laghzaoui el-mustapha1, angelica crottini2, slimani tahar1, bosch jaime3,4, el mouden el hassan1,* hematological parameters of the bolson tortoise gopherus flavomarginatus in mexico cristina garcía-de la peña1,*, roger iván rodríguez-vivas2, jorge a. zegbe-domínguez3, luis manuel valenzuela-núñez1, césar a. meza herrera4, quetzaly siller-rodríguez1, verónica ávila-rodríguez1 ontogenetic and interspecific variation in skull morphology of two closely related species of toad, bufo bufo and b. spinosus (anura: bufonidae) giovanni sanna visible implant alphanumeric (via) as a marking method in the lesser snouted treefrog scinax nasicus andrea caballero-gini1,2,3,*, diego bueno villafañe2,3, lía romero2, marcela ferreira2,3, lucas cañete4, rafaela laino2, karim musalem2,5 morphological variation of the newly confirmed population of the javelin sand boa, eryx jaculus (linnaeus, 1758) (serpentes, erycidae) in sicily, italy francesco p. faraone1,*, salvatore russotto2, salvatore a. barra3, roberto chiara3, gabriele giacalone4, mario lo valvo3 variability in the dorsal pattern of the sardinian grass snake (natrix natrix cetti) with notes on its ecology enrico lunghi1,2,3,4,*, simone giachello5, manuela mulargia6, pier paolo dore7, roberto cogoni8, claudia corti1 estimating abundance of the stripeless tree-frog hyla meridionalis by means of replicated call counts federico crovetto, sebastiano salvidio, andrea costa* at-rich microsatellite loci development for fejervarya multistriata by illumina hiseq sequencing yan-mei wang, jing-yi chen, guo-hua ding*, zhi-hua lin acta herpetologica 14(1): 15-19, 2019 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-22855 experience of predacious cues and accessibility to refuge minimize mortality of hylarana temporalis tadpoles santosh mogali*, bhagyashri shanbhag, srinivas saidapur department of zoology, karnatak university, dharwad-580 003, karnataka state, india. *corresponding author. e-mail: santoshmogali@rediffmail.com submitted on: 2018, 8th march; revised on: 2018, 29thoctober; accepted on: 2018, 31st october editor: adriana bellati abstract. we explored the effect of a prior experience of predation threat and access to the refuge sites on larval mortality in hylarana temporalis with a 2 × 2 factorial design. the design included predator-naïve or predator experienced prey subjects, and presence or absence of a natural shelter. water scorpion adult individuals (laccotrephes species) provided predation threat and also an opportunity to the prey to experience a direct danger of predation. both previous experience with predators and availability of shelters affected larval survival independently and not conjointly. a prior encounter with predators enabled prey tadpoles to escape predation more effectively with a significant increase in their survival in comparison to the predator-naïve subjects. similarly, access to refuge sites significantly increased survival of predator-naïve as well as predator experienced tadpoles compared to that in the absence of shelters. clearly, ability to sense water borne predacious cues in the vicinity and use refuge sites plays a key role in escaping from predation in the bronze frog tadpoles. keywords. hylarana temporalis, laccotrephes sp., predator, refuge availability, tadpole mortality. predator-prey interactions invariably lead to an evolutionary arms race in which early detection of either party is often the key for success. progressive elimination of prey individuals from the ecosystem by the coexisting predators can have far reaching impact on their population dynamics. most anuran amphibians show a biphasic mode of life cycle involving aquatic larval phase. in the aquatic phase of life the larval mortality is often due to desiccation of the ephemeral ponds before completion of the metamorphosis, infection, and to predation by the coexisting aquatic invertebrate and vertebrate predators. therefore, adoption of phenotypic plasticity and diverse strategies become essential for their survival. many anuran larvae including hylarana temporalis are known to alter their behavior and the life-history traits under predation risk (relyea, 2007; sharma et al., 2008; saidapur et al., 2009; mogali et al., 2012, 2015, 2016; mogali, 2018). for example, h. temporalis tadpoles metamorphosed early and at a larger body size under predation threat, when provided by a caged dragonfly larva (pantala flavescens), compared to those reared under predation threat-free environment (mogali et al., 2016). they also decreased swimming activity, by remaining still for longer times, to avoid being detected and increased burst speed on encounter with the predacious cues in the vicinity (mogali et al., 2012). taking shelter under the leaf litter, aquatic vegetation and other objects that provide refuge and utilization of benthic habitat can help tadpoles by reducing the risk of being detected by predators (lima and dill, 1990; eklov and persson, 1996; hossie and murray, 2010). besides, predators may find difficult to capture a prey residing inside a refuge; at the same time, prey may have a better opportunity to assess the actual risk of predation by the predators residing in close vicinity (hemmi and zeil, 2005) and, consequently, evoke defensive behaviors. nevertheless, hiding and stay16 santosh mogali, bhagyashri shanbhag, srinivas saidapur ing still by prey involve trade-offs. these strategies may reduce encounter rates with predators but also tend to reduce time dedicated to foraging activity. indeed, often the habitats that are energetically very profitable are also the most dangerous, since the distribution of predators tends to match their prey’s resource distribution (lima, 1998). besides, the refuge may not always be the best place for feeding. for instance, inadequate time spent in feeding leads to alteration in the metamorphic traits in bufo melanostictus (mogali et al., 2011). the bronze frog (hylarana temporalis) tadpoles are found along the gently flowing streams and isolated pockets of water along sides of the streams during the post-monsoon season (october-january) in southwestern ghats of india (mogali et al., 2012). they are bottom dwellers and thrive on detritus and algal matter (hiragond and saidapur, 2001). in these water bodies visibility is low due to shadows of the dense vegetation, brownish-dark color of the benthic area that is typically covered by the leaf litter and detritus matter (veeranagoudar et al., 2004). the water bodies are also inhabited by several types of invertebrate predators that include aquatic beetles, dragonfly larvae, crabs and water scorpions that prey upon anuran tadpoles (our personal observations). during field visits we have observed water scorpions (laccotrephes sp., nepidae) actively feeding on bronze frog tadpoles. they detect their prey visually and by mechanical cues. the h. temporalis tadpoles have poor vision (veeranagoudar et al., 2004) as in most anuran tadpoles (hoff et al., 1999). however, they possess an innate ability to detect predators by chemoreception (mogali et al., 2012) and evoke antipredator behavior. further, predator-experienced h. temporalis tadpoles show enhanced defensive behaviors on subsequent encounter with predators (mogali et al., 2012). the natural habitats in which h. temporalis tadpoles live is full of detritus material and leaf litter. therefore, we hypothesized that tadpoles of the bronze frog profitably use these shelters (leaf litter) to reduce predation risk of the coexisting predators. an additional assumption was that tadpoles having prior experience of predacious cues will be more successful in escaping predation by using shelters compared to the those facing the predators for the first time (predator-naive). to test our hypotheses, tadpoles of h. temporalis were exposed to free hunting water scorpion (laccotrephes sp). the prey subjects were either predator-naïve or predator experienced. they were exposed to predator in laboratory set ups with or without the refuge sites (leaf litter) for a predetermined period to record survival/mortality of prey. eight clutches of h. temporalis were collected in november, 2013 from a stream in the western ghats near anmod village (15.430888°n, 74.373601°e), karnataka state, india. each clutch was individually reared in plastic tub (32 cm diameter and 14 cm deep) containing 5 l of aged tap (dechlorinated) water. hatching time occurred six days later, at gosner stage 19 (gosner, 1960). the tadpoles of different clutches were mixed to normalize genetic differences among the groups. after mixing, tadpoles were reared in two separate glass aquaria (90 cm l × 30 cm w × 15 cm h) each containing 25 l of aged tap water. upon reaching stage 25, tadpoles were supplied with boiled spinach ad libitum. the water scorpions (laccotrephes sp.) were collected from the same location from where the eggs of h. temporalis were obtained and were reared individually, to avoid cannibalism, in small plastic bowls (14 cm diameter and 7 cm deep) with 500 ml of aged tap water. prey tadpoles served as food for water scorpions. the tadpoles born in laboratory, and never exposed to predator, served as the predator-naïve subjects. to obtain predator-experienced tadpoles we exposed groups of 30 tadpoles to a 48 h starved predator for 8 h (09:0017:00 h). after the 8 h trial period the predator and injured tadpoles were removed from the test bowl. on an average, predator ate 3 ± 0.3 tadpoles and injured 4 ± 0.5 (x ± se) during the trial period. predator-threat experienced and uninjured tadpoles obtained from 80 trials were used in the experiments the following days, in order to assess their performance against predators on the subsequent encounter with them. the leaves of aporosa lindleyana were collected from the same sites from which h. temporalis eggs were collected. structural refuge was made using water soaked (2 days) leaves (dry mass 15 g ± 1.8; x ± se) chopped to have ~ 1 cm2 pieces. these were spread at the bottom of the testing bowls to serve as shelters/ refuge site). in all trials, prey tadpoles were of comparable sizes (length 20.48 ± 0.09 mm, width 5.31 ± 0.07 mm, and weight 62.0 ± 0.64 mg; x ± se; n = 30) and developmental stages (stages 28-30). the water scorpions used in various trials were comparable in size (length 31.0 ± 0.08 mm, width 10.11 ± 0.05 mm, and weight 622.0 ± 5.2 mg; x ± se; n = 30). the size of prey and predators was measured using a digital caliper (accuracy 0.01 mm) and weight was recorded using an electronic balance (accuracy 0.001 g). the experiment involved using a 2 × 2 factorial design with the following groups. 1. predator-naïve tadpoles were exposed to predator in the absence of refuge 2. predator-naïve tadpoles were exposed to predator in the presence of refuge 3. predator-experienced tadpoles were exposed to predator in the absence of refuge 17experience of predators and accessibility to refuges minimize larval mortality of bronze frog 4. predator-experienced tadpoles were exposed to predator in the presence of refuge each treatment consisted of 30 trials (overall 120 trials). for each trial the test tadpoles were released (n = 30) into the plastic tub (32 cm diameter and 14 cm deep) containing 3 l of aged tap water and with or without the structural refuge and allowed to familiarize themselves to the tub for 15 min. then one water scorpion starved for 48 h was introduced gently into the tub and left there. the trials were terminated after 24 h. the number of surviving tadpoles in various trials was recorded to compute the number of tadpoles lost due to predation. data were analyzed using two-way analysis of variance for assessing the overall consequence of a prior exposure to predaceous cues and the access to the refuge and their interaction on prey survival. data on number of tadpoles consumed between the two treatment groups were analyzed by independent-samples t test. two-way anova showed significant main effect of predator exposure (p < 0.001, table 1) and refuge availability (p < 0.001, table 1) but not of their interaction (p = 0.253, table 1). accessibility to the refuge significantly reduced the larval mortality in predator-naïve tadpoles when compared to that without refuge (t58 = 4.402, p < 0.001, fig. 1a). a similar trend was also evident in the case of predator-threat experienced tadpoles (t58 = 5.648, p < 0.001, fig. 1b). the larval mortality was significantly lower in predator-experienced tadpoles compared to predatornaïve tadpoles regardless of the availability (t58 = 4.144, p < 0.001) or unavailability (t58 = 4.353, p < 0.001) of refuge sites (fig. 1). the present study shows the importance of available shelters and of a prior experience with predator in evoking defensive behaviors and enhances escape predation in h. temporalis tadpoles. both these factors independently and not conjointly affect the larval survival following their encounter with water scorpion. the results reveal that accessibility to refuge sites and prior experience of predation threat are key determinants of survival from the aquatic insect predators in h. temporalis tadpoles. previous studies have shown that bronze frog tadpoles exhibit antipredator defense behaviors in response to water borne chemical cues of predator by staying away from the predator and reducing their swimming activity (mogali et al., 2012). our study revealed a heightened defense response in predator-experienced tadpoles in the subsequent encounters with the predator (mogali et al., 2012) in conformity with the observations reported on other anuran species (semlitsch and reyer, 1992; hettyey et al., 2011). the results of the present study show that predator-experienced tadpoles learn to escape predation and become less vulnerable to predator compared to the predator-naive individuals regardless of the accessibility to structural refuge. this clearly demonstrates that prior experience with predator plays a key table 1. results of two-way anova for exposure and refuge availability and their interactions. the response variable is mean number of hylarana temporalis tadpoles lost due to predator. significant values are bolded. source df ms f p exposure 1 108.300 35.382 <0.001 refuge availability 1 132.300 43.222 <0.001 exposure × refuge availability 1 4.033 1.318 0.253 fig. 1. number of predator-naïve and predator-experienced hylarana temporalis tadpoles consumed by the predator, laccotrephes sp., in relation to accessibility to refuges in trials of 24 h. an asterisk over the bar indicates a significant difference between two treatments. data represents x ± se. each trail consists of 30 tadpoles of either predator-naïve or predator-experienced. 30 trials were carried out for each treatment and total all together 120 trials. 18 santosh mogali, bhagyashri shanbhag, srinivas saidapur role in enhancing survival chances of tadpoles by escaping predation. healey and reinhardt (1995) made similar observation on coho salmon against the predacious rainbow trout. álvarez and nicieza (2006), who studied rana temporaria tadpoles, showed that 48 h association with the predator was a sufficient period of time to enhance their ability to escape predation. the present study on h. temporalis shows that even mere 8 h exposure to predatory cues is sufficient to evoke a stronger effective defense behavior on subsequent encounters with the predator. possibly, even a shorter time exposure might be sufficient for prey tadpoles to evoke their defensive behavior on following encounters with predators. however, additional studies are needed to establish the minimum period of exposure to predators that is required to condition the prey and modify their future performance of defensive behaviour. further, releasing predacious cues that remain effective for longer periods may not be in the interest of the predator. indeed, several studies showed that predaceous cues are labile in nature (peacor, 2006; sharma et al., 2008) and their half life ranges from 0.2 h to a few days (van buskirk et al., 2014). thus, it appears that persistence of predatory cues and their ability to evoke defense behaviors in prey may vary among anuran tadpoles. acknowledgements smm is thankful to ugc’s dr. ds kothari postdoctoral fellowship, new delhi. bas is thankful to indian national science academy (insa), new delhi for support. this research was conducted according to ethical guidelines laid down by cpcsea, new delhi under registration no. 639/02/a/cpcsea. references álvarez, d., nicieza, a.g. 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(2004): mechanism of food detection in the tadpoles of the bronze frog rana temporalis. acta ethol. 7: 37-41. acta herpetologica vol. 14, n. 1 june 2019 firenze university press uzungwa scarp nature forest reserve: a unique hotspot for reptiles in tanzania john valentine lyakurwa1,2,*, kim monroe howell2, linus kasian munishi1, anna christina treydte1,3 experience of predacious cues and accessibility to refuge minimize mortality of hylarana temporalis tadpoles santosh mogali*, bhagyashri shanbhag, srinivas saidapur tonal calls as a bioacoustic novelty in two atlantic forest species of physalaemus (anura: leptodactylidae) thiago r. de carvalho1,*, célio f.b. haddad1, marcos gridi-papp2 scientific publication of georeferenced molecular data as an adequate guide to delimit the range of korean hynobius salamanders through citizen science amaël borzée1,*, hae jun baek2,3, chang hoon lee2,3, dong yoon kim2, jae-young song4, jaehwa suh5, yikweon jang1, mi-sook min2* mirrored images but not silicone models trigger aggressive responses in male common wall lizards stefano scali1,*, roberto sacchi2, mattia falaschi1,3, alan j. coladonato2, sara pozzi2, marco a.l. zuffi4, marco mangiacotti1,2 using an in-situ infra-red camera system for sea turtle hatchling emergence monitoring fatima n. oğul1,#,*, franziska huber2,#, sinem cih1, kumsal düzgün3, ahmet e. kideyş1, korhan özkan1 descriptive osteology of an imperiled amphibian, the luristan newt (neurergus kaiseri, amphibia: salamandridae) hadi khoshnamvand1, mansoureh malekian1,*, yazdan keivany1, mazaher zamani-faradonbe1, mohsen amiri2 does color polymorphism affect the predation risk on phalotris lemniscatus (duméril, bibron and duméril, 1854) (serpentes, dipsadidae)? fernanda r. de avila1,2,*, juliano m. oliveira3, mateus de oliveira1, marcio borges-martins4, victor hugo valiati2, alexandro m. tozetti1 age structure of a population of discoglossus scovazzi camerano, 1878 (anura discoglossidae) in extreme environmental conditions (high atlas, morocco) mohamed amine samlali, abderrahim s’khifa, tahar slimani* acta herpetologica 13(1): 21-32, 2018 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-20323 diet and helminth parasites of freshwater turtles mesoclemmys tuberculata, phrynops geoffroanus (pleurodira: chelidae) and kinosternon scorpioides (criptodyra: kinosternidae) in a semiarid region, northeast of brazil antonio marcos alves pereira1,*, samuel vieira brito2, joão antonio de araujo filho3, adonias aphoena martins teixeira3, diêgo alves teles3, daniel oliveira santana3, vandeberg ferreira lima1, waltécio de oliveira almeida1,3 1 programa de pós-graduação em bioprospecção molecular, departamento de química biológica, universidade regional do cariri – urca, rua cel. antônio luiz 1161, campus do pimenta, 63105 –100 crato, ceará, brazil. *corresponding author. e-mail: marcosalvesp@outlook.com 2 centro de ciências agrárias e ambientais, universidade federal do maranhão – ufma, boa vista, 65500–000 chapadinha, maranhão, brazil 3 programa de pós-graduação em ciências biológicas (zoologia), departamento de sistemática e ecologia, centro de ciências exatas e da natureza , universidade federal da paraíba – ufpb, cidade universitária, campus i, 58059–900 joão pessoa, paraíba, brazil submitted on 2017, 14th february; revised on 2017, 13th november; accepted on 2017, 6th december editor: paolo casale abstract. in this study, the kinosternon scorpioides, mesoclemmys tuberculata and phrynops geoffroanus freshwater turtles collected in the cariús river, state of ceará, were analysed as to their diet composition and presence of helminths. among the 63 examined turtles 55 (87.3%) were parasitized. we found three nematoda species (physaloptera retusa, serpinema monospiculatus and spiroxys figueiredoi) and one trematoda species (gorgoderina sp.). phrynops geoffroanus had the highest indexes of prevalence (97.56%) and mean intensity of infection (33.5), followed by m. tuberculata (70% and 12.64, respectively) and k. scorpioides (50% and three, respectively). host body size was positively related to helminths abundance in both male and female chelidae species. a significant difference in helminths abundance between the sexes was found only in p. geoffroanus, where females had more parasites than males. regarding diet, the main food items ingested by m. tuberculata were odonata nymphs (aeshnidae and libellulidae), whilst p. geoffroanus feeds mainly on diptera larvae (chironomidae), odonata nymph (aeshnidae) and notonectidae, and only seeds were found in the stomach contents of k. scorpioides. here, we present the first record of s. monospiculatus parasitizing k. scorpioides, gorgoderina sp. and p. retusa were reported for the first time in p. geoffroanus, and m. tuberculata represents a new host to p. retusa and s. figueiredoi. keywords. testudines, feeding habits, helminths, nematoda, trematoda, host gender, host body size. introduction freshwater turtles are important components in the food webs of aquatic ecosystems, act in the processing of organic matter, nutrient cycling, dispersion of riparian vegetation, as well as in the maintenance of water quality (moll and moll, 2004). despite that, studies regarding feeding ecology and helminths associated with freshwater turtles are still limited (souza, 2004; anjos, 2011; moura et al., 2014). 22 maría a. latorre et aliiantonio marcos alves pereira et alii in south america, researches about the helminth diversity associated with continental turtles are mostly driven in argentina, brazil and uruguay (mascarenhas et al., 2013). in brazil, testudines is represented by 36 species and 18 genera included in eight families (costa and bérnils, 2015). nevertheless, data about helminth fauna composition of freshwater turtles are poorly known (anjos, 2011) as most studies only report species descriptions and records of new hosts. in brazil, the helminth fauna associated with kinosternon scorpioides (linnaeus, 1766) (the scorpion mud turtle), includes the nematodes serpinema magathi (sprehn, 1932) and spiroxys figueiredoi freitas and dobbin jr., 1962 (freitas and dobbin jr., 1971; viana et al., 2016), and the digenean trematodes nematophila grandis (diesing, 1839), telorchis rapidulus dobbin, 1957 and telorchis diaphanus freitas and dobbin jr., 1959 (fernandes and kohn, 2014). the occurrence of k. scorpioides, reaches from the east side of panama to northern south america and from the amazon basin to northern argentina (berry and iverson, 2011). this species has a wide habitat ecological tolerance as it can be found in weirs, lakes, streams, rivers, marshlands and other aquatic environments modified by anthropogenic activities (vanzolini et al., 1980; rueda-almonacid et al., 2007). it has an omnivore habit and a diverse diet, which includes insects and their larvae, spiders, gastropods, worms, crustaceans, fishes, frog eggs, tadpoles, adult frogs, snake scales, bird eggshells, parts of mammals, algae, fruits, nuts, seeds, flowers, and aquatic plants (vanzolini et al., 1980; ruedaalmonacid et al., 2007; berry and iverson, 2011). currently, little is known about the natural history of the tuberculate toad-headed turtle, mesoclemmys tuberculata (lüederwaldt, 1926), including its diet and helminth fauna. the nematode serpinema monospiculatus freitas and dobbin jr., 1962 is the unique parasite recorded for this turtle species (freitas and dobbin jr., 1971). this freshwater turtle is endemic to brazil (moura et al., 2014; turtle taxonomy working group, 2014) with a wide distribution in the northeast region of the country (iverson, 1992; bour and zaher, 2005; moura et al., 2014). it has been recorded mainly in the caatinga biome and in some atlantic forest and cerrado areas (silveira and valinhas, 2010; moura et al., 2015; santana et al., 2015). the species is associated with coastal ecosystems and semiarid regions, where inhabits a variety of aquatic environments, such as rivers, ponds, permanent lakes, and temporary streams (vanzolini et al. 1980; bour and zaher, 2005; santana et al., 2016). the helminths recorded in the geoffroy’s side-necked turtle, phrynops geoffroanus (schweigger, 1812) in brazil, are the nematodes s. monospiculatus, s. figueiredoi, brevimulticaecum sp. (larvae) and physaloptera sp. (larvae) (freitas and dobbin jr., 1971; silva, 2014), the digenean trematodes n. grandis, telorchis birabeni mañé-garzón and gil, 1961, prionosomoides scalaris freitas and dobbin jr., 1967, cheloniodiplostomum testudinis (dubois, 1936) and cheloniodiplostomum sp. (freitas and dobbin jr., 1967; novelli et al., 2013; silva, 2014), and the monogenean trematodes polystomoides brasiliensis vieira, novelli, sousa and souza-lima, 2008 and polystomoides sp. (vieira et al., 2008; silva, 2014). phrynops geoffroanus is a neotropical freshwater turtle that occurs in south america, in the east side of the andes, from the colombian amazon to the south of brazil, uruguay and north of argentina (pritchard and trebbau, 1984; ernst and barbour, 1989). this species inhabits ponds, streams and large rivers (medem, 1960; pritchard and trebbau, 1984), being also common in rivers and streams of urban areas (souza and abe, 2000; souza, 2004; martins et al., 2010). although this is an omnivore turtle species it could exhibit a prevailing carnivorous habit, feeding on insects, crustaceans, fishes, molluscs, annelids and carrion in addition to sewage organic matter in environments impacted by human activity (fachín-terán et al., 1995; souza and abe, 2000; souza, 2004; martins et al., 2010). thus, the current research aimed to analyse the diet and the helminth infection parameters in freshwater turtle populations in a river section in the semiarid region of northeastern brazil. this study will enrich the knowledge about helminth fauna associated with freshwater turtles and the diet of the studied species. materials and methods study site fieldwork was carried out in a stretch of the cariús river (7º05’20.0”s, 39º40’37.0”w) in the nova olinda municipality (7°05’30.0”s, 39°40’50.0”w, 445 m a.s.l.), ceará state, northeast of brazil. local climate is semiarid hot tropical with an average annual temperature varying from 24 to 26 °c. the rainy season occurs between january and may with an average annual rainfall of 682.7 mm (ipece, 2015). the study site is placed in an urban area rounded by buildings where water quality is frequently affected by household waste and sewage. besides pollution, removal of riparian forest for the development of agricultural crops and pastures probably has contributed to the river silting process. collecting and laboratory procedures the turtles were collected between october 2014 and april 2015, using fish hooks baited with pieces of chicken. 23running titlediet and helminths of mesoclemmys tuberculata, phrynops geoffroanus and kinosternon scorpioides a total of 63 freshwater turtles were collected: 41 individuals of p. geoffroanus (17 males and 24 females), 20 m. tuberculata (five males, nine females and six juveniles), and two specimens of k. scorpioides (both females). the captured individuals were transported in plastic boxes (60 liters) to the laboratório de zoologia da universidade regional do cariri (lz-urca), where they were anesthetized by administration of xylazine 2% and ketamine hydrochloride 1%, and euthanized with a lethal injection of lidocaine chlorohydrate 2%. these specimens were weighed in a digital scale (to the nearest 0.5 g) and measured with a digital caliper (precision 0.05mm) as to their straight-line carapace length (cl). the specimens’ gender was determined through a gonads analysis during the dissection process. turtles with no testicles developed or devoid of vitellogenic follicles in the ovaries and/or eggs in the oviducts, were considered juveniles. next, gastrointestinal tract, heart, liver, lungs, urinary bladder and body cavity were examined for helminths using a stereomicroscope. all helminths found were counted and preserved in 70% ethanol. nematodes were cleared in hoyer’s solution (everhart, 1957) and the trematodes were stained with hydrochloric carmine and assembled on slides temporary with hoyer’s solution. then, the helminths were analysed under an optical microscope and identified according to freitas and dobbin jr. (1971), vicente et al. (1993) and bray et al. (2008). afterwards, we calculated the prevalence, mean intensity of infection and abundance according to bush et al. (1997). helminths were deposited in the coleção parasitológica da universidade regional do cariri (urca-p 1075-1202). diet analyses the food items present in the stomach were collected during the turtles’ dissection and examined under a stereomicroscope. the specimens found as diet components were fixed in 10% formaldehyde and preserved in 70% ethanol. then, we used a specialized literature (pérez, 1988; fernandez and dominguez, 2001; mugnai et al., 2010) to identify the least possible taxonomic level (usually order or family) of each specimen. both length (l) and width (w) of the intact items were measured using a digital caliper (precision 0.01 mm) as well as their volume (v) was estimated by the ellipsoid’s formula (dunham, 1983): v = 4 3 π w 2 ' l 2 1 2 we examined the frequency (f, number of samples with a specific food item category), abundance (n, number of specimens of each category found in stomach samples) and volume (v, value calculated to each category with the ellipsoid’s formula above) of each turtle species’ diet items. we calculated the relative importance index (i) to identify the importance of each category for diet composition by the following formula (powell et al., 1990): i = f% + n% + v% 3 1 2 where f%, n% and v% are, respectively, the percent values of frequency, number and volume of each food item category. statistical analyses we used a simple linear regression (zar, 2010) to assess the relationship between the turtle body size (cl) and helminths abundance in both male and female individuals from representative species only (from which there were at least five adults of each gender). helminth abundance is defined as the total number of parasites at the individual host level regardless of whether or not the host is infected (bush et al., 1997). juvenile hosts were removed from this analysis to avoid possible ontogenetic effects bias. the general linear model (glm) with poisson distribution was used to verify whether hosts’ gender affects parasites abundance (wilson et al., 2002). since the sexual dimorphism in body size can strongly influence the abundance of helminths, we used a student’s t-test (zar, 2010) to determine whether there were significant differences in cl and body mass between the sexual genders. the normality of data was confirmed through the kolmogorov-smirnov test (zar, 2010). statistics were performed in the statistica software, version 8.0 (statsoft inc., tulsa, oklahoma, usa). results among the 63 examined turtles 55 were parasitized (87.3% overall prevalence). in general, we collected 1,520 helminths, which correspond to an overall intensity of infection of 27.63. the nematoda found were physaloptera retusa rudolphi, 1819 (physalopteridae), serpinema monospiculatus freitas and dobbin jr., 1962 (camallanidae) and spiroxys figueiredoi freitas and dobbin jr., 1962 (gnathostomatidae). gorgoderina sp. (gorgoderidae) was the single trematoda species recorded in this study (table 1). all of the recorded helminth species here have a heteroxenic life cycle. regarding the body size, m. tuberculata carapace length was positively related to helminth abundance in males (f = 59.47, r2 = 0.93, p = 0.003) and females (f = 6.28, r2 = 0.39, p = 0.03) (fig. 1, a and b). the same pattern was observed to p. geoffroanus males (f = 11.46, r2 = 0.39, p = 0.004) and females (f = 64.12, r2 = 0.73, p < 0.0001) (fig. 1, c and d). regarding the helminths abundance, we found a significant difference in the parasite load between the sexes in p. geoffroanus being females more parasitized than males (glm, wald = 148.2, df = 1, p ≤ 0.001). we found 24 maría a. latorre et aliiantonio marcos alves pereira et alii table 1. prevalence (p) (%), mean intensity of infection (mii), range (r), and sites of infection (s)* of helminths associated with freshwater turtles from cariús river, ceará state, northeast of brazil. *bc = body cavity, li = large intestine, l = lungs, si = small intestine, s = stomach. taxon / family / species mesoclemmys tuberculata overall (n= 20) phrynops geoffroanus overall (n= 41) kinosternon scorpioides overall (n= 2) p (%) mii (r) s p (%) mii (r) s p (%) mii (r) s nematoda camallanidae / serpinema monospiculatus 45 13.77 (1-32) li, si 95.12 29.12 (2-131) bc, li, l, si, s 50 3 (−) li gnathostomatidae / spiroxys figueiredoi 50 5.1 (2-9) si, s 70.73 6.41 (1-18) s − − − physalopteridae / physaloptera retusa 5 2 (-) li 7.3 1.33 (1-2) si, s − − − trematoda gorgoderidae / gorgoderina sp. − − − 9.75 3.5 (1-6) si − − − total 70 12.64 (1-37) 97.56 33.5 (3-135) 50 3 (−) fig. 1. relationship between carapace length (cl) and helminths abundance in m. tuberculata (a and b) and p. geoffroanus (c and d) adult individuals. 25running titlediet and helminths of mesoclemmys tuberculata, phrynops geoffroanus and kinosternon scorpioides no significant difference in helminths abundance between m. tuberculata males and females (glm, wald = 2.45, df = 1, p = 0.11). m. tuberculata females were significantly bigger than males regarding their carapace length (student’s t-test, t = 4.15, p = 0.001) and body mass (student’s t-test, t = 4.48, p = 0.0007). females of p. geoffroanus were also significantly bigger than males in relation to their carapace length (student’s t-test, t = 2.09, p = 0.04) and body mass (student’s t-test, t = 3.75, p = 0.0007). in the current research, all turtles with at least two individuals of s. figueiredoi presented stomach ulcers (58.73%). all individuals of s. figueiredoi were attached to the same ulcer in these turtles, but there was only one ulcer per host (fig. 2). we observed this pathology in nine specimens of m. tuberculata (45%) and 28 p. geoffroanus (68.29%). the other collected helminths were randomly distributed in the sites of infection. diet composition among the 63 analysed stomachs, only 26 presented food items (41.27%). we collected 17 samples of p. geoffroanus (15 females and two males), eight of m. tuberculata (six juveniles and two females) and one of k. scorpioides (female). this samples distribution pattern unable comparative analysis between both genders and between adults and juveniles (in the case of m. tuberculata). overall, we identified 28 categories of food items in the diet of turtles’ assemblage (table 2). we recognized 18 categories of food items in m. tuberculata diet (table 2). libellulidae (25%), aeshnidae (20%) and chironomidae (15%) were the most frequent categories, whilst libellulidae (48.53%), notonectidae (13.23%) and aeshnidae (10.29%) were numerically more important. aeshnidae (63.23%) and libellulidae (16.27%) had the greatest volumes and also the greatest relative importance index of all categories (aeshnidae, 70.30%, and libellulidae, 22.62%). we identified 17 categories of food items in the diet of p. geoffroanus (table 2). meat (23.8%), chironomidae (14.28%), leaves (11.9%) and formicidae (9.52%) were the most frequent items, whereas chironomidae (71.15%), formicidae (8.65%) and notonectidae (5.8%) were the numerically most important and aeshnidae (45.45%), chironomidae (24.7%) and notonectidae (14.15%) had the greatest volumes. regarding the relative importance of each category, chironomidae (60.5%), aeshnidae (18.56%) and notonectidae (11.55%) were the main consumed items. only seeds were found in the stomach contents of k. scorpioides (100%) (table 2). discussion both m. tuberculata and p. geoffroanus share the same three helminth species. gorgoderina sp. was found only in p. geoffroanus. also, the unique parasite species found in k. scorpioides (s. monospiculatus) was infecting all three host species. parasitological studies involving other herpetofauna groups have been noticing parasites composition overlaps between phylogenetically close host species (see brooks et al., 2006; brito et al., 2014). the phylogenetic closeness between m. tuberculata and p. geoffroanus (pleurodira: chelidae) may contribute to the occurrence of a similar helminths composition among these species. for k. scorpioides, the less diverse helminth fauna and lower diversity of food items (seeds only) here recorded, probably reflects the small sample for this species (n = 2). kinosternon scorpioides is the most widely distributed turtle species in the new world and has comparatively a vastly diverse diet along their home range (see berry and iverson, 2011). in addition, host species with large geographical ranges usually harbour a richer helminth fauna (poulin, 2004). serpinema monospiculatus and s. figueiredoi were the most prevalent parasites among the chelidae species. differences in prevalence indexes could be related to the way how helminth species are transmitted to their definitive hosts (pereira et al. 2012b) and the host’s food habits could influence this transmission as well. although there is no available information about s. monospiculatus life cycle, studies about other camallanid nematodes (espefig. 2. spiroxys figueiredoi nematodes attached to gastric mucosa of phrynops geoffroanus forming an ulcer. 26 maría a. latorre et aliiantonio marcos alves pereira et alii table 2. diet composition of mesoclemmys tuberculata, phrynops geoffroanus and kinosternon scorpioides collected in cariús river, ceará state, brazil. f = frequency of occurrence, n = number of preys, v = volume (mm3) and i = relative importance index. percentages are shown in parentheses. category mesoclemmys tuberculata (n = 8) phrynops geoffroanus (n = 17) kinosternon scorpioides (n = 1) f (%) n (%) v (%) i f (%) n (%) v (%) i f (%) n (%) v (%) i crustacea ostracoda 1 (2.38) 2 (1.92) 1.01 (0.02) 0.00 insecta coleoptera elmidae (adult) 1 (2.38) 1 (0.96) 21.19 (0.50) 0.20 elmidae (larvae) 1 (2.38) 1 (0.96) 0.62 (0.01) 0.00 gyrinidae 1 (2.38) 2 (1.92) 18.84 (0.45) 0.18 scirtidae 1 (5) 2 (2.94) 3139.73 (2.90) 0.80 diptera diptera (pupae) 1 (5) 1 (1.47) 23.55 (0.02) 0.00 1 (2.38) 1 (0.96) 13.08 (0.31) 0.13 chironomidae 3 (15) 4 (5.90) 202.16 (0.18) 0.15 6 (14.28) 74 (71.15) 1036.43 (24.70) 60.50 sciomyzidae 1 (2.38) 1 (0.96) 150.72 (3.60) 1.47 ephemeroptera baetidae 1 (2.38) 1 (0.96) 35.05 (0.83) 0.34 heteroptera belostomatidae 2 (10) 2 (2.94) 4211.05 (3.85) 2.14 corixidae 2 (10) 2 (2.94) 256.69 (0.23) 0.13 naucoridae 1 (5) 1 (1.47) nepidae 1 (5) 1 (1.47) 1714.85 (1.57) 0.43 notonectidae 2 (10) 9 (13.23) 544.52 (0.50) 0.30 2 (4.76) 6 (5.80) 593.77 (14.15) 11.55 hymenoptera (wasp) 1 (2.38) 1 (0.96) 100 (2.38) 0.97 hymenoptera formicidae 4 (9.52) 9 (8.65) 66.56 (1.60) 2.60 megaloptera corydalidae 1 (5) 2 (2.94) 4841.35 (4.43) 1.23 odonata (adult) 1 (2.38) 1 (0.96) 200 (4.76) 1.95 odonata (nymph) aeshnidae 4 (20) 7 (10.29) 69139.13 (63.23) 70.30 1 (2.38) 1 (0.96) 1907.55 (45.45) 18.56 coenagrionidae 1 (5) 1 (1.47) 427.43 (0.39) 0.10 libellulidae 5 (25) 33 (48.53) 17795.32 (16.27) 22.62 orthoptera (terrestrial) 1 (5) 1 (1.47) reptilia squamata snakes epictia sp. 1 (5) 1 (1.47) 7037.78 (6.43) 1.80 bone fragmente 1 (5) plant material leaves 5 (11.90) seeds 1 (5) 1 (1.47) 4.71 (0.00) 0.00 3 (7.14) 3 (2.88) 52.16 (1.24) 1.52 1 (100) 28 (100) 1501.96 (100) 100 unidentified material 1 (5) 2 (4.76) meat 1 (5) 10 (23.80) total numbers 68 109338.3 104 4197.01 28 1501.96 27running titlediet and helminths of mesoclemmys tuberculata, phrynops geoffroanus and kinosternon scorpioides cially s. trispinosum [leidy, 1852]) showed that freshwater copepods act as intermediate hosts, and aquatic snails, fishes, amphibians and odonates as paratenic hosts until the definitive host’s infection (moravec and vargas-vázquez, 1998; gonzález and hamann, 2007; wiles and bolek, 2015). spiroxys figueiredoi life cycle is also unknown. however, spiroxys larvae have been reported from freshwater copepods and odonata nymphs (as intermediate hosts), and fishes and amphibians, which are used as paratenic hosts (hedrick, 1935; anderson, 2000). according to other studies, m. tuberculata feeds mainly on fish (vanzolini et al., 1980) and p. geoffroanus mainly on aquatic insects, crustaceans and fish (medem, 1960; souza, 2004; martins et al., 2010). in the current research, the m. tuberculata most consumed items were odonata nymphs (aeshnidae and libellulidae), whilst p. geoffroanus feed mainly on diptera larvae (chironomidae), odonata nymph (aeshnidae) and notonectidae. thus, the high consumption of odonates (in this study) and fish could influence the serpinema and spiroxys recruitment, consequently increasing their infection indexes. gorgoderina sp. and p. retusa had the lowest registered prevalence. gorgoderina species have a complex life cycle, in which bivalve molluscs commonly act as first intermediate hosts and aquatic insect larvae and tadpoles as second intermediate hosts (coil, 1954). data on p. retusa life cycle are scarce. nevertheless, anderson (2000) reports that infection caused by physaloptera spp. on definitive hosts begins with the ingestion of insects containing infective larvae (in third stage). arthropods such as cockroaches, crickets, grasshoppers and beetles act as intermediate hosts to many physalopteras pecies larvae (e.g., p. hispida, p. maxillaris, p. preaeputilialis and p. rara) (schell, 1952; lincoln and anderson, 1975). although terrestrial insects can occasionally be consumed by freshwater turtles (souza and abe, 2000; santana, 2012), aquatic insects and its larvae are included in the most important food resources for these reptiles (souza and abe, 2000; souza, 2004; martins et al., 2010). in this study, the diet of chelidae populations was composed mostly by aquatic invertebrates, with a predominance of insect larval stages, and few terrestrial specimens were found in m. tuberculata (orthoptera and snake) and p. geoffroanus (hymenoptera and adult odonata) diet. this low frequency of terrestrial invertebrates and the absence of molluscs and tadpoles in the diet could reduce the probability of infection by physaloptera and gorgoderina species, respectively, which reflects the low prevalence and infection intensity indexes. the host body size is an important feature that frequently determines parasites species richness and abundance (poulin, 2007; poulin and leung, 2011; kamiya et al., 2014). correlations between host body size and abundance and richness of helminths are common in studies with lizards (martin et al., 2005), an approach still poorly explored for turtle parasites. several studies report a positive relationship between the host body size and abundance and diversity of parasite species (e.g., poulin, 2007; anjos et al., 2012; kamiya et al., 2014), which indicates that larger host species can provide greater space and, consequently, support a higher number of parasites (poulin, 2007). hence, our linear regression results to both chelidae species are in accordance with previous statements, since the largest turtles (both males and females) had greater helminth abundance than smaller turtles. females of p. geoffroanus were more parasitized than males. our data showed that p. geoffroanus females were significantly bigger and heavier than males. this sexual dimorphism related to body also recorded in other researches (e.g., medem, 1960; brites, 2004) must have contributed to a higher parasite load in female hosts. although m. tuberculata females were significantly bigger than males, there was no significant difference in helminth abundance between the sexual genders. further studies with larger samples of adult turtles are needed for a better understanding of the infection patterns in this chelidae species. in addition, physiological and behavioural differences related to sexual gender of the host (such as differences in habitat use, diet, reproductive behaviour, hormone levels, time of activity) may result in parasite abundance differences between males and females (aho, 1990; pereira et al., 2012b). nevertheless, such ecological aspects about most of brazilian turtle species have not been investigated in great depth (souza, 2004). regarding the available data on the helminth fauna associated with p. geoffroanus in brazil, silva (2014) developed the research with the best known turtle sampling (n = 11), in which a prevalence of 90.90% was recorded. our research recorded the major infection prevalence (97.56%). however, we did observe a relatively low helminth richness (four species). in turn, silva (2014) recorded the major helminth species richness of p. geoffroanus (10 species) in the reserva particular do patrimônio natural foz do rio aguapeí protected area, located  in a transition area (ecotone)  between cerrado and atlantic forest biomes, southeastern brazil. nevertheless, the population analysed by silva (2014) had lower mean intensity of infection (11.7) when compared to our study (33.5). variability in habitat characteristics (such as variations in climate conditions and watershed or landscape alterations) in which turtles were collected could influence the parasites abundance and diversity patterns due to less human-impacted areas usually have high hel28 maría a. latorre et aliiantonio marcos alves pereira et alii minth diversity and the hosts are parasitized by a lower number of individual parasites (mckenzie, 2007; anjos et al., 2012). thus, anthropogenic pressures on cariús river and its vicinity could lead to lower helminth diversity and higher prevalence and infection intensity indexes as recorded here. regarding the helminths we recorded, gorgoderina species are commonly found in the urinary bladder of anurans and caudates (amphibia) around the world (coil, 1954; mata-lópez et al., 2005). now considering the freshwater turtles of brazil, among 29 species 13 have been reported to harbour trematode species (fernandes and kohn, 2014). despite that, gorgoderina spp. has not been recorded in association with these reptiles. it is probably that gorgoderina adults accidentally occurred in the small intestine of p. geoffroanus because gorgoderina usually become mature inside the bladder (coil, 1954). species of the physaloptera rudolphi, 1819 are usually reported as amphibians, birds, mammals and reptiles parasites (pereira et al., 2012a). among the physaloptera spp. recorded in brazilian reptiles (p. bonnie, p. liophis, p. lutzi, p. monodens, p. obtusíssima, p. retusa and p. tupinambae), p. retusa have been found frequently in the stomach of lizards, parasitizing 36 species (ávila and silva, 2010; araujo-filho et al., 2014). in brazil, only physaloptera larvae were found in p. geoffroanus in são paulo state (silva, 2014). accounting that physaloptera infection goes through intermediate hosts (terrestrial insects which are occasionally consumed by freshwater turtles), p. retusa must has occasionally occurred in m. tuberculata (large intestine) and p. geoffroanus (stomach and small intestine). serpinema yeh, 1960 frequently parasitizes freshwater turtles (anderson, 2000). in brazil, s. monospiculatus was found in association with m. tuberculata (type host), m. nasuta (schweigger, 1812) and p. geoffroanus in the state of pernambuco (freitas and dobbin jr., 1971; vicente et al., 1993), and s. magathi was found in k. scorpioides in the states of pernambuco, pará (freitas and dobbin jr., 1971) and maranhão (viana et al., 2016). serpinema magathi was originally described as camallanus magathi by sprehn (1932) from kinosternon integrum le conte, 1854 collected in bolivia. then, caballero (1939) recorded s. magathi (=camallanus parvus) parasitizing kinosternon hirtipes wagler, 1830, in mexico (freitas and dobbin jr., 1971). in costa rica, bursey and brooks (2011) reported s. magathi as a kinosternon leucostomum (duméril and bibron, 1851) parasite. nematodes from spiroxys schneider, 1866 are usually found in the gastrointestinal tract of freshwater turtles (hedrick, 1935; berry, 1985) and parasite amphibians and reptiles (roca and garcía, 2008) just like definitive hosts such as frogs, salamanders and snakes (berry, 1985). in brazil, s. figueiredoi was found in k. scorpioides (type host) in the states of pernambuco, pará (freitas and dobbin jr., 1971) and maranhão (viana et al., 2016). in são paulo, it was also found parasitizing p. geoffroanus and pseudoboa nigra (duméril, bibron and duméril, 1854) (dipsadidae) (silva, 2014). some undescribed spiroxys specimens were recorded as mesoclemmys vanderhaegei (bour, 1973) (chelidae) parasites in the state of mato grosso do sul (ávila et al., 2010). in the state of rio grande do sul, spiroxys sp. was also found in phrynops hilarii (duméril and bibron, 1835) (chelidae) and in trachemys dorbigni (duméril and bibron, 1835) (emydidae) (bernardon et al., 2013, 2014). in the same state, s. contortus (rudolphi, 1819) was recorded in association with the acanthochelys spixii (duméril and bibron, 1835), hydromedusa tectifera cope, 1870 (chelidae) and t. dorbigni freshwater turtles (mascarenhas et al., 2013; mascarenhas and müller, 2015). in costa rica, s. figueiredoi was found in k. leucostomum and in k. scorpioides (bursey and brooks, 2011). our study reports the second occurrence of s. figueiredoi parasitizing p. geoffroanus in brazil. researches that draw forth the effects of parasitism on freshwater turtles are still scarce. in the current study, we recorded for the first time, the occurrence of erosive lesions in the gastric mucosa in m. tuberculata and p. geoffroanus individuals caused by s. figueiredoi adults. likewise, in other countries specimens of spiroxys were found causing pathological changes in their hosts. in the united states, mcallister et al. (1993) reported the involvement of s. contortus larvae in forming gastric granulomas in apalone spinifera pallida (webb, 1962)(trionychidae). in europe, miclăuş et al. (2008) found s. contortus adults causing pylorus irreversible lesions in the stomach of emys orbicularis (linnaeus, 1758) (emidydae). in addition, burke and rodgers (1982) suggest that the occurrence of gastric ulcers caused by nematode scan reduce the hosts’ appetite and stunt their growth. the high frequency of gastric ulcers in the sampled chelidae species is atypical and demands for future research efforts. among the collected helminths, s. monospiculatus is a new record to k. scorpioides. gorgoderina sp. and p. retusa were recorded for the first time in p. geoffroanus. mesoclemmys tuberculata represents a new host record for p. retusa and s. figueiredoi. acknowledgments we are grateful to coordenação de aperfeiçoamento de pessoal de nível superior (capes) for the research grant awarded to a. m. a. pereira, v. f. lima and a. a. 29running titlediet and helminths of mesoclemmys tuberculata, phrynops geoffroanus and kinosternon scorpioides m. teixeira; and to conselho nacional de desenvolvimento científico e tecnológico (cnpq) for the research grant awarded to w. o. almeida (pq-302429/2015-8), j. a. araujo filho and d. a. teles. we thank m. c. oliveira, r. g. duarte, e. g. silva, c. l. m. pinto, and n. k. s. sampaio for their invaluable cooperation during laboratory procedures. the collection and euthanasia of the turtles were approved by the instituto chico mendes de conservação da biodiversidade (icmbio, permit 454601) and are in accordance with applicable brazilian legislation for animal care. references aho, j.m. 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(2010): biostatistical analysis. pearson prentice hall, upper saddle river, new jersey. acta herpetologica vol. 13, n. 1 june 2018 firenze university press species diversity and distribution of lizards in montenegro katarina ljubisavljević1,2,*, ljiljana tomović3, aleksandar urošević1, slađana gvozdenović2, vuk iković2, vernes zagora2, and nenad labus4 the first demographic data and body size of the southern banded newt, ommatotriton vittatus (caudata: salamandridae) abdullah altunişik diet and helminth parasites of freshwater turtles mesoclemmys tuberculata, phrynops geoffroanus (pleurodira: chelidae) and kinosternon scorpioides (criptodyra: kinosternidae) in a semiarid region, northeast of brazil antonio marcos alves pereira*, samuel vieira brito, joão antonio de araujo filho, adonias aphoena martins teixeira, diêgo alves teles, daniel oliveira santana, vandeberg ferreira lima, waltécio de oliveira almeida electric circuit theory applied to alien invasions: a connectivity model predicting the balkan frog expansion in northern italy mattia falaschi1,2,*, marco mangiacotti3, roberto sacchi3, stefano scali2, edoardo razzetti4 first report of bufo bufo (linnaeus, 1758) from sardinia (italy) ilaria maria cossu1, salvatore frau1, massimo delfino2,3, alice chiodi4, claudia corti1,5*, adriana bellati4 natural history and conservation of the nurse frog of the serranía del perijá allobates ignotus (dendrobatoidea: aromobatidae) in northeastern colombia hernán darío granda-rodríguez1,2, andrés camilo montes-correa3,*, juan david jiménez-bolaño3, marvin anganoy-criollo4,5 exploring body injuries in the horseshoe whip snake, hemorrhois hippocrepis juan m pleguezuelos*, esmeralda alaminos, mónica feriche how effectively do european skinks thermoregulate? evidence from chalcides ocellatus, a common but overlooked mediterranean lizard grigoris kapsalas, aris deimezis-tsikoutas, thanos georgakopoulos, ismini gkourtsouli-antoniadou, kallirroi papadaki, katerina vassaki, panayiotis pafilis thermal tolerance for two cohorts of a native and an invasive freshwater turtle species jun geng, wei dang, qiong wu, hong-liang lu* diet of a restocked population of the european pond turtle emys orbicularis in nw italy dario ottonello1, fabrizio oneto1,2, monica vignone2, anita rizzo2, sebastiano salvidio2,* helminths of the lizard colobosauroides cearensis (squamata, gymnophthalmidae) in an area of caatinga, northeastern brazil aldenir f. da silva neta*, robson w. ávila acta herpetologica 14(2): 147-151, 2019 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/a_h-7754 estimating abundance of the stripeless tree-frog hyla meridionalis by means of replicated call counts federico crovetto, sebastiano salvidio, andrea costa* distav, university of genova, corso europa 26, i-16132 genova, italy. *corresponding author. email: andrea-costa-@hotmail.it submitted on: 2019, 16th may; revised on: 2019, 22nd july; accepted on: 2019, 16th september editor: marco mangiacotti abstract. the stripeless tree-frog hyla meridionalis reaches its eastern-most european distributional limit in nw italy, and specifically in the cinque terre national park. here for two consecutive years, we estimated tree-frog population abundance by call surveys at 24 sites. data were analysed in the framework of n-mixture open population models based on repeated counts of calling males. the results obtained by this statistical approach were effective in estimating population size together with annual recruitment and survival. the tree-frog male population size remained constant between years and site abundance was inversely related with altitude. on the bases of these findings, our application of n-mixture models to tree-frog calling males was successful and is a promising cost-effective method to obtain long-term monitoring data on this species over large geographic areas. keywords. abundance estimation, call surveys, cinque terre national park, detection probability, n-mixture models. the stripeless tree-frog hyla meridionalis boettger, 1874 is found in north-western africa (algeria, morocco and tunisa), south-western europe (portugal, spain, south france and north-western italy) and the canary and balearic islands (sillero, 2010). on a portion of its distribution range the species is considered introduced, i.e., canary and balearic islands (sillero et al., 2014), and it is also possibly introduced for other european regions (recuero et al., 2007). in italy, the stripeless tree-frog is common along the mediterranean coast of liguria (nw italy), from the province of imperia to the province of la spezia (salvidio, 2007). apart from morphometric and distributional data (salvidio, 2007), little is known about the abundance and dynamics of stripeless tree-frog populations in italy, and quantitative data on populations size should be obtained to assess the species status and its ecological requirements, in particular near the species distribution limits, where a high population fragmentation is expected (gaston, 2003). although photo-identification of stripeless tree-frogs is possible (crovetto unpublished data), the animals are arboreal and highly secretive during daytime. the use of pvc pipes may increase the probability of detection of tree-frogs (do vale et al., 2018), however, in the ctnp the majority of the species’ reproductive habitats are on private lands, and thus are not freely accessible (romano et al., 2014). therefore, the monitoring technique selected to estimate population size was based on nocturnal auditory surveys of calling males, because of the species highly distinctive mating call (schneider, 1974; márquez et al., 2005). call survey is a relatively efficient technique for evaluating the distribution and diversity of anurans (dorcas et al., 2009). therefore, calling surveys are frequently used in large-scale amphibian monitoring programmes (e.g., anthony, 2002; weir and mossman, 2005; weir et al., 2005, 2009). however, the use of call surveys for estimating population abundances and trends suffers of the same problematic issues recognized in the case of repeated counts of individuals, because the detectability of anuran calling males is < 1 (i.e., not all males are calling in the same night; schmidt and pellet, 2005. moreo148 federico crovetto, sebastiano salvidio, andrea costa ver, anuran mating call activities display high variation in response to biotic and abiotic factors, that usually remain unknown and difficult to model (royle and link, 2005; droege and eagle, 2009). in fact, using raw counts of calling males or even scores derived from abundance indexes (i.e., indexes that group calling males by classes of relative abundance; weir and mossman, 2005) without accounting for detection probability may lead to relevant bias in abundance and trend estimates (schmidt, 2004; mazerolle et al., 2007). therefore, to reliably estimate population abundance, the information derived from raw counts of calling males should always be corrected for species-specific detection probabilities (schmidt and pellet, 2005; royle and link, 2005). recently, specific modelling approaches have been proposed for estimating anuran population abundances from the count of anuran calling indexes taking into account detection probabilities (royle, 2004a; royle and link, 2005). this study aimed to estimate the abundance of stripeless tree-frog males together with some demographic parameters and ecological requirements in italy, at the eastern limit of the species distribution. moreover, we tried to establish a cost-effective monitoring protocol to provide future population trends. because of the relatively small number of tree-frog males recorded per site, we had the opportunity to apply the open population generalization of royle’s (2004b) n-mixture model (dail and madsen, 2011) to count data derived from call surveys. the eastern-most limit of the species’ range in europe is the village of riomaggiore (province of la spezia), in the cinque terre national park (ctnp), a protected area where the stripeless tree-frog reproduces in streams and in artificial water tanks used for irrigation (salvidio, 2007; romano et al., 2014). in this area water streams display short and steep courses, with relatively long summer drying periods, due to the lack of precipitations (olivari et al., 2013). among many possible land use of rural areas, agriculture is the only one in the ctnp, and from the sea level up to the hill tops vineyards and orchards are cultivated on strips of arable land, or “terraces”, sustained by dry-stone walls. irrigation is provided by means of water stored in tanks, often colonised by amphibians (olivari et al., 2013; romano et al., 2014). the survey sites were selected during both daytime and nocturnal preliminary surveys. during the day, streams and water reservoirs were inspected and selected as potential reproductive sites if adults, larvae or eggs of some amphibian species were observed. during the night, sites were located by perceiving the calls of stripeless three-frog males. in total 24 sites were surveyed in the municipalities of levanto, monterosso and riomaggiore (from west to east): 6 streams and 18 artificial water reservoirs in agricultural lands or urban settings (table 1; fig. s1). all surveys began after sundown and after hearing the first tree-frog calls. in 2017 three nocturnal surveys were performed, from the end of march to may, by two operators that counted the number of males calling at each site during a two minute period. in 2018, three nocturnal surveys were performed, from the beginning of may to the beginning of june, with the same observers and procedure of 2017. in addition in 2018, a fourth survey was performed by a single operator that tallied calling males for 4 minutes. the asynchrony and the different tonalities of calls permitted to count with confidence the minimum number of males per site that, in all cases, was ≤ 6 (table s1). all sites were surveyed during the same overcast or rainy night, but never during heavy showers that could hinder a clear hearing of frog calls. four climatic variables were obtained from the meteorological station of levanto: rainfall during the 24 h preceding the survey (rain), air temperature (temp), relative humidity (rh) and wind speed (wind), recorded during the last hour of survey. these weather variables were selected, because they are known to influence anuran calling behaviour (e.g., walls et al., 2011). finally, for each site three variables were considered: altitude above the sea level (elev), a categorical variable for the municipality of the site (city) and if the water body was a stream or an artificial site (site). repeated count data were analysed using the dailmadsen (2011) model, which is a generalization of the royle’s (2004b) n-mixture model, capable of relaxing the closure assumption by considering the population closed to immigration/births and emigration/deaths during a short period (i.e., three/four survey nights performed each year), while considering the population demographically open between years, in a robust design-similar approach. this model estimates four parameters, two of which are in common with the royle’s (2004b) n-mixture original formulation: individual detection probtable 1. continuous variables included in the n-mixture open population models (dail and madsen, 2011) used to estimate hyla meridionalis abundance, in the cinque terre national park. variable description sample size (n) mean (sd) min max elev site altitude (m) 24 35.04 (29.66) 8 83 temp air temperature (°c) 7 17.14 (3.44) 12 21 wind wind speed (m/s) 7 3.57 (1.27) 2 5 rh relative humidity (%) 7 66 (13.55) 45 85 149n-mixture models for hyla meridionalis call counts ability (p) and mean initial abundance for each site (λ). the dail-madsen (2011) model estimates two additional parameters: the recruitment rate (γ), comprehensive of births and immigrations, and the apparent survival probability (ω), comprehensive of deaths and emigrations. in our study, we built models with poisson error distribution, since negative binomial distribution could lead to identifiability issues and may produce infinite abundance estimates (barker et al., 2017; link et al., 2018). furthermore, in order to avoid truncated estimates of abundance (knape et al., 2018), we set the upper limit for integration (k) to 50 (i.e., we checked estimate stability at incremental values of k). we then began the model building procedure by fitting a global model (i.e., the most complex model on which other models are nested) and assessing the fit of this model in two ways: i) by means of a pearson chi-square test (mackenzie and bailey, 2004), using a parametric bootstrap procedure (5000 re-samplings), ii) by inspecting residuals (knape et al., 2018). in order to avoid overfitting and creating too many models, deriving from the combinations of covariates for each of the four parameters of the dail-madsen (2011) model, which can lead to uninformative and biologically unsound models, we preferred to build fewer models in a stepwise approach, considering one parameter at a time, and building biologically informative models. we proceeded modelling the detection probability, considering it to be constant, time-dependent, or to be affected by climatic variables. then we modelled the initial abundance as a function of site specific covariates (elev, city and site) or constant over sites. finally, we considered the survival to be influenced by the same site covariates as abundance, or constant over sites. for each model we considered recruitment as constant. we ranked all models with akaike’s informative criterion corrected for small samples (aicc). we conducted model selection and considered only models with ∆aicc > 2 (burnham and anderson, 2002). modelling was conducted in the r environment with package unmarked (fiske and chandler, 2011) and aiccmodavg (mazerolle, 2017). in 2017 we counted a total of 131 male frog calls during three surveys (44; 45; 42; respectively), while in 2018 we counted 129 male frogs during four surveys (33; 37; 32; 27; respectively, table s1). the global model had a good fit (goodness-of-fit, p = 0.34; c-hat overdispersion = 1.12, and visual inspection of residuals). model building procedure produced a total of 15 models (table 2). the most supported model included elevation as a covariate on the initial abundance, highlighting a negative effect of elevation (βelev = -0.331; 95% ci = -0.59 to -0.08; figure 1). the estimated mean frog abundance per site was 3.4 (95% ci = 2.5 – 4.6). individual detection probability for this model was constant, and estimated as p = 0.53 (95% ci = 0.42 – 0.63). survival probability between years was considered constant among sites and resulted ω = 0.71 (95% ci = 0.50 – 0.86). finally, the recruitment rate was constant across sites γ = 0.11 (95% ci = 0.00 – 12.10). from this best model we also obtained, as a derived parameter from the posterior distribution of the latent abundance, the total abundance of surveyed sites, which table 2. candidate n-mixture open population models (dail and madsen, 2011) used to estimate hyla meridionalis abundance, ranked by aicc. γ = recruitment rate; λ= initial site abundance; p = individual detection probability; ω = survival; aiccwt = model weights. in model list t stands for time dependence. for covariate abbreviations see table 1. model parameters aicc ∆aicc aiccwt λ(elev) p(.) ω(.) γ(.) 5 451.35 0.00 0.40 λ(elev) p(.) ω(elev) γ(.) 6 453.45 2.1 0.14 λ(city) p(.) ω(.) γ(.) 6 453.98 2.63 0.11 λ(elev) p(.) ω(city) γ(.) 6 454.95 3.61 0.07 λ(.) p(.) ω(.) γ(.) 4 454.96 3.61 0.07 λ(site) p(.) ω(.) γ(.) 5 455.05 3.70 0.06 λ(elev) p(.) ω(site) γ(.) 7 456.15 4.80 0.04 λ(.) p(temp) ω(.) γ(.) 5 456.81 5.46 0.03 λ(.) p(.) ω(site) γ(.) 5 456.93 5.59 0.02 λ(.) p(rh) ω(.) γ(.) 5 457.14 5.80 0.02 λ(.) p(wind) ω(.) γ(.) 5 475.54 6.20 0.02 λ(.) p(rain) ω(.) γ(.) 5 458.03 6.69 0.01 λ(.) p(.) ω(elev) γ(.) 5 459.15 7.80 0.01 λ(.) p(.) ω(city) γ(.) 6 459.35 8.01 0.01 λ(.) p(t) ω(.) γ(.) 11 483.07 31.73 0.00 fig. 1. effect of elevation on site specific abundance of hyla meridionalis in the cinque terre national park, with 95% confidence intervals, obtained by n-mixture open population modelling (dail and madsen, 2011). 150 federico crovetto, sebastiano salvidio, andrea costa resulted of 89 frogs in 2017 (95% ci = 81 – 147) and 64 frogs in 2018 (95% ci = 60 – 109). our study showed that n-mixture modelling applied to individual frog calls can be successfully used to estimate male population size together with demographic parameters and ecological understandings. in the ctnp, where h. meridionalis is a species of high conservation concern, the male tree-frog population size showed no significant change between years, and site abundance was negatively related with altitude (salvidio, 2007; sillero, 2010). moreover, the usefulness of n-mixture approach may be appreciated by comparing population estimated corrected by detectability to raw counts that, in the present case, underestimated the total number of males by about 45%, in both years. another important application of n-mixture population open models (dail and madsen, 2011) relies on the possibility of estimating temporal variations in inter-annual population size, this information being of interest in conservation and management programmes concerning protected species characterised by low or variable detection probabilities (ficetola et al., 2018). conversely, the major limits of our study were that the occurrence of calling males does not always assure for the presence of a breeding site, while no data on population structure (i.e., population sex ratio and proportion of juveniles) can be provided (dorcas et al., 2009). in any case, n-mixture models are cost-effective alternatives to mark-recapture and removal sampling methods (kéry and royle, 2015; kéry, 2018), and they have been used to estimate population size and temporal trends of many species in very different ecological contexts (e.g., priol et al., 2014; romano et al., 2017; kéry, 2018; costa et al., 2019). however, to our knowledge there are few applications of n-mixture modelling to anuran call counts, because of the difficulties in correctly counting calling males in large frog choruses when dozens of calls are synchronous (weir and mossman, 2005). nevertheless, when few individual males are calling at each site the application of the n-mixture modelling seems useful and can be preferred to other methods that estimate population abundance because there is no need to mark and recapture the focal individuals (royle, 2004a; royle and link, 2005). acknowledgments the constructive comments of two anonymous reviewers on a previous draft of the manuscript are appreciated. this research was funded by the cinque terre national park within the programme “azione di sistema monitoraggio delle specie di habitat umidiacquatici”. supplementary material supplementary material associated with this article can be found at <http://www.unipv.it/webshi/appendix> manuscript number 25342. references anthony, b.p. 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(2005): modeling anuran detection and site occupancy on north american amphibian monitoring program (naamp) routes in maryland. j. herpetol. 39: 627639. acta herpetologica vol. 14, n. 2 december 2019 firenze university press podarcis siculus latastei (bedriaga, 1879) of the western pontine islands (italy) raised to the species rank, and a brief taxonomic overview of podarcis lizards gabriele senczuk1,2,*, riccardo castiglia2, wolfgang böhme3, claudia corti1 substrate type has a limited impact on the sprint performance of a mediterranean lizard pantelis savvides1,*, eleni georgiou1, panayiotis pafilis2,3, spyros sfenthourakis1 coping with aliens: how a native gecko manages to persist on mediterranean islands despite the black rat? michel-jean delaugerre1,*, roberto sacchi2, marta biaggini3, pietro lo cascio4, ridha ouni5, claudia corti 3 pit-tags as a technique for marking fossorial reptiles: insights from a long-term field study of the amphisbaenian trogonophis wiegmanni pablo recio, gonzalo rodríguez-ruiz, jesús ortega, josé martín* occurrence of batrachochytrium dendrobatidis in the tensift region, with comments on its spreading in morocco ait el cadi redouane1, laghzaoui el-mustapha1, angelica crottini2, slimani tahar1, bosch jaime3,4, el mouden el hassan1,* hematological parameters of the bolson tortoise gopherus flavomarginatus in mexico cristina garcía-de la peña1,*, roger iván rodríguez-vivas2, jorge a. zegbe-domínguez3, luis manuel valenzuela-núñez1, césar a. meza herrera4, quetzaly siller-rodríguez1, verónica ávila-rodríguez1 ontogenetic and interspecific variation in skull morphology of two closely related species of toad, bufo bufo and b. spinosus (anura: bufonidae) giovanni sanna visible implant alphanumeric (via) as a marking method in the lesser snouted treefrog scinax nasicus andrea caballero-gini1,2,3,*, diego bueno villafañe2,3, lía romero2, marcela ferreira2,3, lucas cañete4, rafaela laino2, karim musalem2,5 morphological variation of the newly confirmed population of the javelin sand boa, eryx jaculus (linnaeus, 1758) (serpentes, erycidae) in sicily, italy francesco p. faraone1,*, salvatore russotto2, salvatore a. barra3, roberto chiara3, gabriele giacalone4, mario lo valvo3 variability in the dorsal pattern of the sardinian grass snake (natrix natrix cetti) with notes on its ecology enrico lunghi1,2,3,4,*, simone giachello5, manuela mulargia6, pier paolo dore7, roberto cogoni8, claudia corti1 estimating abundance of the stripeless tree-frog hyla meridionalis by means of replicated call counts federico crovetto, sebastiano salvidio, andrea costa* at-rich microsatellite loci development for fejervarya multistriata by illumina hiseq sequencing yan-mei wang, jing-yi chen, guo-hua ding*, zhi-hua lin acta herpetologica 15(1): 59-64, 2020 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/a_h-7903 comparison of complement system activity amongst wild and domestic animals maría s. moleón1,2,*, mark e. merchant3, hugo h. ortega4, pablo a. siroski1,2 1 proyecto yacaré laboratorio de zoología aplicada: anexo vertebrados (fhuc unl / maspyma) a. del valle 8700, santa fe (300), argentina 2 laboratorio de ecología molecular aplicada, instituto de ciencias veterinarias del litoral universidad nacional del litoral – consejo nacional de investigaciones científicas y técnicas, esperanza, argentina 3 department of chemistry, mcneese state university, p.o. box 90455, lake charles, la 70609, usa 4 laboratorio de biología celular y molecular (icivet-conicet-unl). r.p. kreder 2805 esperanza (3080), santa fe, argentina * corresponding author. e-mail: soledadmoleon@yahoo.com.ar submitted on: 2020, 13th february; revised on: 2020, 2nd may; accepted on: 2020, 8th may editor: daniele pellitteri-rosa abstract. multiple mechanisms have evolved for the defensive recognition of foreign components, such as microorganisms. the majority of immunological studies with vertebrates have been focused on endothermic species, and relatively little attention has been directed toward ectothermic vertebrates. we employed a colorimetric assay designed to compare plasma hemolytic activities based on the serum complement system (cs) activities amongst some representative reptiles, wild and domestic birds, and mammals. results obtained from the hemolytic assays conducted with plasma derived from all of the animal species used showed that broad-snouted caiman had the highest activity, and no differences were observed in the hemolytic activities of plasma from birds or the other reptile species. in contrast, the cs activity obtained with mammalian plasma was markedly lower than that from the other taxa. this assay has many advantages, such as the requirement of small sample volume, reproducible results, and low cost. in addition, unsensitized sheep red blood cell hemolysis can be successfully used for the evaluation of innate immune system activities in non-mammalian species; however, for mammals, it should be combined with other immunological determinates to evaluate integral innate immunocompetence. keywords. innate immunity, immunocompetence, complement system, wildlife, domestic animals. organisms are continually exposed to a multitude of pathogens through contact, ingestion, and inhalation. multiple mechanisms have evolved for the recognition of foreign antigens such as microorganisms. these strategies are the result of multiple cascade events that converge in the release of molecular signals that stimulate the recognition of foreign antigens. those mechanisms are strategically divided in two distinct, but related, systems: acquired immunity and innate immunity. the innate immune system also plays a critical role in priming and stimulating the adaptive immune response (medzhitov and janeway, 1997). the concept of innate immunity refers to the first line host defense that serves to restrain infection in the early hours after exposure to microorganisms (hoffmann et al., 1999). in turn, the adaptive immune system, more complex in nature, activates innate effector mechanisms in an antigen-specific manner as a second line of defense. the connections between the various immune components are not fully understood, but recent progress has brought us closer to an integrated view of the immune system and its function in host defense. 60 maría s. moleón et alii the majority of studies on vertebrate immunity have focused on endothermic species, and relatively little attention has been focused on ectothermic species (reviewed in juul-madsen et al., 2008; hamon and quintin, 2016). these species are not commonly studied because the features that control their growth, reproduction and general physiology are largely unknown. however, some studies have shown that the complement system, as part of the innate mechanism of fish and other poikilothermic vertebrates, is more diverse than that of higher vertebrates, and thus a broader range of antigens can be recognized (sunyer et al., 1998). crocodilians exhibit well-characterized social behaviors and responses to stressors that can trigger serious disputes between co-specific species, predators, and even conflicts with human activities. as a result, they sometimes exhibit physical trauma, serious injuries and even the loss of entire limbs. frequently, these animals live in environments, either natural or captive, containing a high concentration of potentially pathogenic microorganisms. in most cases, crocodilians tolerate these circumstances without showing signs of infection (siroski et al., 2010). the colorimetric assay used in this study detects and characterizes the serum complement system (cs, merchant et al., 2006). it is based on the disruption of sheep red blood cells (srbc) by immunological proteins circulating in plasma. these proteins recognize srbcs as foreign antigens and initiate activation of the cs cascade, which culminates in the formation of a protein complex that generates a pore (membrane attack complex, mac) in the srbc membrane and its subsequent lysis. upon lysis, released hemoglobin is quantified using a spectrophotometer and considered proportional to the cs activity. this assay is routinely used in clinical laboratories to assess cs activity (nagaki et al., 1980). to compare plasma hemolytic activity amongst different species of vertebrates, we collected blood samples from reptiles, wild and domestic birds, and mammals. blood samples from juvenile broad-snouted caiman (caiman latirostris; n = 8) were obtained from the spinal vein; from tegu lizard (salvator merianae; n = 6), lagoon  turtle (phrynops hilarii; n = 5) and painted turtle (trachemys dorbigni; n = 6) from the caudal vein. blood samples were obtained from wild pochard ducks (netta peposaca; n = 5), domestic ducks (anas domesticus; n = 5), and swan  geese (anser anser domesticus; n = 6) from the brachial vein. blood of following mammals were obtained from the jugular vein: maned wolves (chrysocyon brachyurus; n = 4), spider monkeys (ateles chamek; n = 5), and horses (equus caballus; n = 5). all blood samples were collected using heparinized syringes. all animals appeared healthy, and none were undergoing antimicrobial treatment. plasma was separated within 1 h of collection by centrifugation at 1500xg for 30 min, and stored at -18°c until analysis. the hemolytic assay was adapted and performed to evaluate the hemolytic properties of serum cs activity from different animals. as mentioned above, the srbc hemolysis assay is based on the hemolytic disruption of srbcs by means of serum immunological proteins (merchant et al., 2005; merchant and britton, 2006; merchant et al., 2009; siroski et al., 2010). fresh srbcs were obtained from heparinized whole blood collected from the jugular vein of merino sheep (ovis aries). whole blood from sheep was washed several times with phosphate-buffered saline (pbs, ph 7.4) until the supernatant was clear, and then a 2% srbc (v/v) solution was prepared (siroski et al., 2010). to make a comparison between the hemolytic properties of plasma caused by the serum cs from different animals against srbc, each sample was treated independently. validation assays were performed with the plasma of each species with and without ethylenediaminetetraacetic acid (edta) and mild heat treatment (56°c for 30 min), both considered as classical inhibitors of the cs (morgan, 2008). the tests were conducted at laboratory ambient temperature (25°c ± 2°c) by placing 0.5 ml of plasma from each animal together with 0.5 ml srbc (2% v/v in isotonic saline). after 30 min incubation, the mixture was centrifuged and 300 µl of supernatant was transferred to a well of a microtiter plate for analysis on microplate reader at 540 nm. a positive control for hemolysis was obtained by 1% srbcs solution and 0.1% (v/v) triton x-100. this mix was aggressively injected and ejected several times through a tuberculin syringe until complete hemolysis was confirmed with an optical microscope (olympus bh-2, tokyo, japan) at 400×. optical density of the supernatant was measured in a microplate reader at 540 nm, and was considered to be maximum hemolysis. the samples were performed in quadruplicate to obtain valid statistical evaluations and results were expressed as the percentage of maximum hemolysis (mh%; mean ± standard error). statistical analysis was performed using spss 16.0 software (spss for windows 2007). data were tested for normality with the kolmogorov-smirnov test, and homogeneity of variances between groups was verified by the levene test. one-way analysis of variance (anova) and tukey’s test were used to test for differences among groups, and a p value of 0.05 was considered statistically significant. the hemolytic method based on the rupture of sheep red blood cells was very effective at estimating complement activity in every species tested. plasma samples 61comparison of complement system activities from all species responded to the exposure to srbc solution (2%). results obtained from the hemolytic assays of plasma derived from ten species of vertebrates showed that c. latirostris plasma (86.38 ± 5.1) had the highest activity (p < 0.05; fig. 1). no differences were observed in the %mh generated with plasma from birds and reptiles other than the caiman. however, the tegu lizard had the second highest %mh (69 ± 5.2) after caiman. there were no significant differences between %mh of domestic duck, swam geese and wild pochard ducks (63 ± 5.6, 56 ± 8.9 and 58 ± 5.9, respectively). conversely, the %mh obtained with mammalian plasma was markedly lower than those from birds and reptiles. while the spider monkey exhibited 31 ± 2.9 activity, the maned wolf and horse had very low values of %mh (10 ± 3.9 and 10.2 ± 3, respectively). in order to eliminate the possibility that the observed hemolysis was mediated by other hemolytic mechanisms, the cs assay included 2 classical inactivators of the serum complement, mild heat treatment of the serum and ethylenediaminetetraacetic acid (edta) (morgan, 2008). untreated serum, preheated serum (56°c for 30 min), and serum treated with 50 mm edta were exposed to 2% (v/v) srbcs. data demonstrated the ability of serum to rupture srbc membranes (fig. 2). in this case, %mh values of srbcs with edta (4.4 ± 2.11) and heat (3.2 ± 2.17) were compared. the absorbance recorded indicated that there were significant reductions in the maximum hemolysis of srbcs when complement-system inhibitors were added (p < 0.001; fig. 2). the hemolytic activities found for c. latirostris in this work were similar and comparable to that values previously reported from other crocodilian species, such as alligator mississippiensis (merchant et al., 2005), crocodylus porosus and crocrodylus johnstoni (merchant and britton, 2006) and crocodylus acutus (merchant et al., 2010). these data provide evidence that the mechanisms of complement activities for diverse crocodilian species are similar. the differences between the %mh values of each species is a consequence of different habitat where they live and therefore exposure to the large variety of pathogens. the greater plasma hemolytic capacity of c. latirostris compared with that of turtle plasma had already been indicated by ferronato et al. (2009) for phrynops geoffroanus, who suggested that the higher activity detected in crocodilian species might be due to evolutionary pressure selection on this group because their environment is rich in potentially pathogenic microorganisms. the same reasoning might be extended to the species s. merianae, which showed high values, similar to those of turtles, but lower than that obtained for c. latirostris. in addition, it is interesting the fact that the values observed in these other reptilian species are lower than that observed for crocodilian species. the high complement activity of s. merianae could be associated with the aggressive behaviors displayed toward member of their own species. these conspecific aggressions may have been compensated by the evolutionary development of potent innate immunity (merchant et al., 2010). birds have been shown to have a potent nonspecific immune mechanism based on the alternative pathway of cs activity. mekchay et al. (1997) reported a significant hemolytic capacity in the plasma of birds and reported higher complement activities in wild birds than in captive birds raised under intensive commercial breeding conditions. these results are consistent with those previously detected by skeeles et al. (1988) that chickens reared for human consumption exhibited lower cs activities compared to wild turkeys. differences could be related to the fig. 2. results obtained from the evaluation of the complement system activity from broad-snouted caiman serum+srbc with edta and heat. fig. 1. results obtained from the evaluation of the complement system activity from reptiles, wild and domestic birds and mammals. * indicates significant differences. 62 maría s. moleón et alii genetic composition of these birds or reflect a difference in the environment in which the chickens were raised. however, the results from our study revealed no differences between species of domestic and wild birds. the antimicrobial activity of one species of commercial poultry (gallus gallus) was high, similar to that of reptiles, possibly resulting from a common evolutionary origin and developed in response to periodic exposure to a significant amount of pathogens (siroski et al., 2010). the highest hemolytic capacity among the plasma samples derived from mammals was detected in spider monkeys but was still much lower than that of nonmammalian species. similarly, in a study conducted to determine levels of serum hemolysis in the plasma of nine primate species against sensitized and desensitized srbc, the results were varied, but the plasma from primates mostly caused only a slight hemolysis of unsensitized srbc (ellingsworth et al., 1983). other determinations were performed with camel (camelus dromedarius) serum where lytic capacity was evaluated against desensitized erythrocytes from different species. they found that the erythrocytes in homologous species (goats, sheep, rat and bovine) were resistant to lysis, while the effect on heterologous erythrocytes was attributed to the presence of the alternative pathway (olahomukani et al., 1995). similar findings were previously reported by arya and goel (1992) in buffalo (bubalus bubalis) serum. in a study focused on the complementmediated disruption of erythrocytes from 18 species of mammals and birds, it was observed that erythrocytes were not lysed by homologous complement, with the exception of guinea pig complement, which weakly lysed homologous erythrocytes, but with only 37% lysis maximum (ish et al., 1993). the low hemolysis values of erythrocytes exhibited by mammalian serum suggests that this restriction might be involved in the regulatory principle of non-specific and role-specific factors (van dijk et al., 1983). from these studies we can assume that plasma of some mammals did not hemolyze the srbcs because they were not recognized as foreign and thus the mammalian cs was considered to recognize more limited range of antigens to trigger activation. although the main objective was to evaluate the differences between the activities of cs in the plasma of the various species studied, it is important to note that the assessment of immune function in wildlife has become an important tool for the investigation of ecological and evolutionary processes. the variety of tests that can be used in wild animals are often limited by the difficulty of capture and handling, and also difficulties of recovery or recapture, lack of specialized specific reagents, and small sample sizes of the study species (matson et al., 2005). in this case, the assay employed had many advantages, such as the requirement of small sample volume, reproducible results, and low cost for the assessment of immunocompetence. hemolysis of srbcs has been used to assess the serum complement activity of crocodilians (merchant et al., 2005; merchant and britton, 2006; merchant et al., 2010; siroski et al., 2010; merchant et al., 2013a, 2013b), varanids (merchant et al., 2012), snakes (baker and merchant, 2018), turtles (ferronato et al., 2009; baker at al., 2019), and amphibians (major et al., 2011). the results showed that the test of unsensitized srbc hemolysis can be used successfully for the evaluation of the innate immune system in a wide variety of species of reptiles and birds, but for mammals it should be utilized with other immunological determinations. these findings may reflect underlying differences in the biology and life history of each species. our study confirmed that the hemolytic method was very effective and advantageous at estimating complement activity in every species tested because small plasma samples from all species responded to the exposure to srbc solution (merchant et al., 2006). furthermore, this assay allows to be routinely used in clinical laboratories to assess cs activity. through the use of this hemolytic assay, we demonstrated a greater plasma hemolytic capacity of c. latirostris compared with other reptiles, birds and mammals. this means that the higher activity detected in crocodilian species might be due to evolutionary pressure selection on this group because their environment is rich in potentially pathogenic microorganisms and they exhibit aggressive territorial defense behaviors. due to the close phylogenetic linkage with the reptiles, we built similar deductions with the group of birds. finally, it is important to highlight that the use of hemolytic method in the current study offered a contribution to the knowledge of comparative immunology. also, it could be an appropriate tool to evaluate the role of complement in the immune function for some other wildlife species other than reptiles and for the investigation of ecological and evolutionary processes. acknowledgments we thank estación zoológica experimental la esmeralda for the samples. we thank all the proyecto yacaré team for their assistance during the experiments and for his contribution to the study. this research was evaluated and approved by the ethics committee and security (ecas)  of faculty of veterinary sciences, national university of litoral (#258/16, santa fe,  argen63comparison of complement system activities tina). all animals were handled according to the reference ethical framework for  biomedical research: ethical principles for research with laboratory, farm, and wild  animals (conicet, 2005). samples were collected from captivity wild and domestic animals, which were maintained in a registered zoo by a national resolution.  references arya, a., goel, m.c. 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(1983): a study of the sensitivity of erythrocytes to lysis by heterologous sera via the alternative complement pathway. vet. immunol. immunopathol. 4: 469-477. acta herpetologica vol. 15, n. 1 june 2020 firenze university press has the west been won? a field survey and a species distribution model of iberolacerta horvathi in the alps giuseppe de marchi1,*, giovanni bombieri1, bruno boz1, fausto leandri2, jacopo richard3 first record of underwater sound produced by the balkan crested newt (triturus ivanbureschi) simeon lukanov identification of biologically active fractions in the dermal secretions of the genus bombina (amphibia: anura: bombinatoridae) iryna udovychenko1,*, oleksandra oskyrko1, oleksii marushchak2, tetiana halenova1, oleksii savchuk1 don’t tread on me: an examination of the anti-predatory behavior of eastern copperheads (agkistrodon contortrix) andrew adams1,2,*, john garrison2, scott mcdaniel2, emily bueche2, hunter howell2,3 an overview of research regarding reservoirs, vectors and predators of the chytrid fungus batrachochytrium dendrobatidis jarid prahl, thomas p. wilson*, david giles, j. hill craddock genetic characteristics of an introduced population of bombina bombina (linnaeus, 1761) (amphibia: bombinatoridae) in moselle, france jean-pierre vacher1, 2,*, damien aumaître3, sylvain ursenbacher2,4 adaptive significance of the transparent body in the tadpoles of ornamented pygmy frog, microhyla ornata (anura, amphibia) santosh m. mogali*, bhagyashri a. shanbhag, srinivas k. saidapur comparison of complement system activity amongst wild and domestic animals maría s. moleón1,2,*, mark e. merchant3, hugo h. ortega4, pablo a. siroski1,2 effects of temperature and food level on plasticity of metamorphic traits in bufo gargarizans gargarizans larvae tong lei yu*, yan ting han acta herpetologica 16(1): 45-51, 2021 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-9824 rana temporaria on monti della laga (central italy): isolated population or wide distribution? first record in abruzzo and marche francesco di toro1,2, gianmarco minuti3, luca coppari1,4, matteo de albentiis5, paolo laghi6, dino scaravelli7, valerio ricciardi8, giacomo bruni4,9,* 1 shi sezione abruzzo-molise; c/o wwf chieti pescara, via ortona snc, chieti, italy 2 gruppo erpetologico abruzzese e molisano; c/o wwf chieti pescara, via ortona snc, chieti, italy 3 department of biology, ecology & biodiversity research unit, vrije universiteit brussels, brussels, belgium 4 shi sezione umbria-marche; perugia, italy 5 laboratory of atmospheric physics-chemistry and climatology (disputer), university “g. d’annunzio” of chieti-pescara, italy 6 museo di ecologia, meldola (fc), italy 7 department of veterinary medical sciences, alma mater studiorum università di bologna, 40064 ozzano dell’emilia (bo), italy 8 via monte fascia 9, roma, italy 9 commissione atlante shi *corresponding author. e-mail: giacomo.b90@gmail.com submitted on: 2020, 29th september; revised on: 2021, 10thmarch; accepted on: 2021, 8th april editor: marcello mezzasalma abstract. in central italy rana temporaria is only known to occur as a glacial relict on the eastern side of monti della laga (lazio). in this study we report the presence of the species in other areas of the mountain chain, with documented sightings in five distinct localities in marche and abruzzo. we use these new records, together with other occurrence data from the apennine chain, to generate a species distribution model and perform an analysis of the geological preference of the species in central italy. although the model indicates a wide area of marche and abruzzo as suitable for r. temporaria, the actual distribution of the species in northern and central apennine appears strongly associated with sandstones. therefore, we argue that the presence of this geological substrate on monti della laga, but not in surrounding karst uplands, could be among the factors explaining its isolation. our study aims at paving the way for future surveys and measures to protect these isolated populations from the threat posed by climate change. keywords. rana temporaria, relict species, species distribution model, maxent, central italy. the common frog (rana temporaria linnaeus, 1758) is a monotypic eurasian species particularly widespread in northern europe (gasc et al., 1997; dufresnes et al., 2020). adapted to cold climates, in its southern distribution the species is restricted to upland areas, reaching an altitude limit of c.a. 2800 m on the alps (tiberti and von hardenberg, 2012). the southernmost limits of its range are located on the pyrenees, in the balkan region and on the central apennines (gasc et al., 1997), where the species occurs mostly in fragmented mountain populations (bernini and razzetti, 2006). indeed, in italy r. temporaria shows a continuous distribution throughout the alps and the north-eastern apennines, becoming more localized on the mountainous areas toward central italy, where it is only known to survive as a single isolated population on the uplands of monti della laga (bernini and razzetti, 2006; stefani et al., 2012). this population is located in lazio (rieti province), on the western side of the mountain chain, about 160 km from the last known occurrence on the northern apennines (berni46 f. di toro et alii ni and razzetti, 2006; razzetti et al., 2007). the site lies between 1400 and 1600 m a.s.l. in the so called “agro nero”, an area consistent with the ecological requirements of the species, including a mosaic of meadows and beech forests with streams, small lakes (lago secco and lago selva) and seasonal ponds and puddles that are used as breeding sites (authors, pers. obs.). the population was reported in 1982 together with a sympatric population of another cold-adapted amphibian, the alpine newt ichthyosaura alpestris apuana (capula and bagnoli, 1982). therefore, r. temporaria on monti della laga is recognised as a glacial relict, which survived thanks to the favourable environmental conditions of the area (stefani et al., 2012; bartolini et al., 2014). however, when compared to the orographically continuous upland karst areas of monti sibillini (northward) and gran sasso massif (southward), monti della laga show a very distinct geology, characterized by sandstones and marls (pellegrini, 2007). this compact substrate allows rainwater and meltwater to retain longer on the surface, thus favouring the formation of perennial springs and permanent or seasonal small wetlands, both in high-altitude grasslands and in beech forests at lower altitudes. even though these geological features are common to the whole monti della laga chain, r. temporaria was not detected in other areas such as the eastern side, which belongs to marche and abruzzo regions (posillico et al., 2017; cameli et al., 2014). recently, new observations for the species were made between 980 and 1130 m a.s.l. in the western side of lazio (bruni et al., 2016). in the present study we report the first record of rana temporaria in abruzzo and marche, providing an habitat suitability analysis to evaluate its potential distribution in central italy and facilitate future research activities. the new observations were made by the authors or collected through citizen science. the individuals were photographed in situ and the coordinates and habitat information were recorded and integrated using the habitat map of the gran sasso laga national park (bagnaia et al., 2015). the coordinates were projected on the 10-km2 grid used in the italian atlas of amphibians and reptiles (bernini and razzetti, 2006) to detect the occurrence of the species in new squares. metamorphosed individuals were visually distinguished from congener species (r. dalmatina and r. italica) according to a combination of morphological features (i.e. size, body proportions, shape of the snout, warts, dorsal, ventral and upper lip colouration), whereas larval stages were identified based on body morphology, coltable 1. relevant data about the five new localities for marche and abruzzo; observation date is given as dd/mm/yyyy; locality names are followed by municipality, province and region. site date locality coordinates (latitude / longitude) altitude (m a.s.l.) utm square developmental stage; habitat, corine biotopes code (bagnaia et al., 2015) observer 1 04/06/2015 monte comunitore (arquata del tronto, ap, marche) 42.731922n 13.342084e 1607 33t uh63 tadpoles; seasonal pond in compact grasslands of the mediterranean mountains to nardus stricta and related communities (code 35.72) paolo laghi and dino scaravelli 2 22/08/2016 fosso rio della volpara (arquata del tronto, ap, marche) 42.704861n 13.369226e 1238 33t uh62 1 adult female; stream in beech forests of southern and central europe (code 41.17) giovanni rossi 3 07/07/2017 valle del castellano (valle castellana, te, abruzzo) 42.683516n 13.361997e 1772 33t uh62 1 adult (sex undetermined); stream in compact grasslands of the mediterranean mountains to nardus stricta and related communities (code 35.72) matteo de albentiis 4 01/05/2019 fosso della morricana (rocca santa maria, te, abruzzo) 42.656455n 13.397107e 1814 33t uh62 1 adult (sex undetermined); stream in mid-european montane siliceous cliffs (code 62.21), compact grasslands of the mediterranean mountains to nardus stricta and related communities (code 35.72) giancarlo tondi 5 17/07/2020 04/08/2020 sorgente “pane e cacio” (campotosto, aq, abruzzo) 42.573577n 13.396734e 1796 33t uh61 1 subadult and 1 adult female; stream in mid-european montane siliceous cliffs (code 62.21), blueberry heaths of the apennines (code 31.4a) francesco di toro and valerio ricciardi 47rana temporaria on monti della laga ouration and mouthparts (razzetti et al., 2007; ambrogio and mezzadri, 2018). in order to predict and prioritize locations for future search of r. temporaria in the area of interest, a habitat suitability map of the species was generated via maximum entropy modelling (maxent 3.4.0; phillips et al., 2006). the model focused on central and northern apennines in order to include only the apennine lineage of r. temporaria (stefani et al., 2012). the presence-only data (49 occurrence points from abruzzo, marche, lazio, toscana, emilia-romagna, piemonte and liguria) used for building the model were gathered from personal records and public databases (gbif, 2020). the complete dataset used for the analysis can be requested to the corresponding author. the environmental variables were selected among bioclimatic, topographic and ecological layers. altitude, as well as 19 bioclimatic layers averaging the period 1970-2000, were downloaded from the worldclim 2.1 database (https://www.worldclim.org). aspect, slope, and distance from water sources were calculated in qgis 3.12 (http://www.qgis.org/). ecological layers included vegetation (percent tree cover) and land cover class (https://globalmaps.github.io). all layers featured a 30 arc seconds spatial resolution and were clipped to the extent of the study area (41.5-45.0n; 8.5-14.5e). to eliminate spatial collinearity among predictors, a pearson’s correlation matrix was calculated in r 3.6.1 (r core team, 2019). for each pair of correlated variables (|r|>0.7), the one believed to be more relevant (according to the biology of r. temporaria) was retained. this resulted in the selection of the following variables: bio8 (mean temperature of the wettest quarter); bio10 (mean temperature of the warmest quarter); bio16 (precipitation of the wettest quarter); bio18 (precipitation of the warmest quarter); aspect; slope; distance from water sources; vegetation and land cover (20 classes). a total of 30 replicates were computed in maxent (default settings), each with 70% of data points randomly used for training and 30% for fig. 1. a) updated distribution of rana temporaria in central italy: agro nero (red diamond), reports from bruni et al. (2016) (blue circles), new data from the present study (yellow stars, numbers refer to table 1). b) adult individual from valle castellana (te). c) adult individual from campotosto (aq). 48 f. di toro et alii model validation. jackknife analysis was applied for estimating relative contribution of each predictor to the final model. model performance was evaluated based on average omission rate and area under curve (auc) statistics. auc is a measure of the model’s discriminatory ability between presence and background points. a model with low detectability will have auc values closer to 0.5 (indicating no greater fit than expected by chance), whereas a model with high detectability will have values closer to 1.0 (indicating perfect model fit) (elith et al., 2006). the average model prediction was used to produce the habitat suitability map for r. temporaria. the occurrence data were also used to perform a geological analysis of the substrates, intersecting the coordinates with the italian geo-lithologic map layer (http://wms.pcn.minambiente. it) in qgis. rana temporaria was found in 5 new localities (table 1), which represent the first observations of the species for marche and abruzzo (table 1; fig. 1). the species distribution model indicates that around 25% of the studied area is considered suitable for the species (fig. 2). this is mostly concentrated around the apennines, with suitability values increasing at higher altitudes. mean temperature of the warmest quarter (bio10) was by far the most important predictor (57.8% contribution), followed by mean temperature of the wettest quarter (bio8; 17.2%), precipitation of the wettest quarter (bio16; 8.0%) and precipitation of the warmest quarter (bio18; 5.8%). land cover was the most relevant ecological variable (4.7%), whether vegetation (2.6%), aspect (1.6%), distance from water (1.2%) and slope (1.1%) showed low contribution to the model. the average auc for 30 replicated runs was 0.975 ± 0.005, indicating high model performance in predicting the species occurring pattern. accordingly, test omission rate was consistently lower than what expected by chance (p < 0.001 for all replicates). results from geo-lithological analysis (fig. 3) show that 42 of 49 points (86%; tosco-emiliano and central fig. 2. species distribution modelling for rana temporaria in the northern and central apennine region based on maximum entropy algorithm (maxent 3.4.0; philips et al., 2006). warmer colours indicate higher habitat suitability. 49rana temporaria on monti della laga apennines) are located on substrates mainly composed of sandstones, such as pelitic-arenaceous hills and mountains with parental material defined by undifferentiated tertiary sedimentary rocks. the other 7 points (14%; liguria and tosco-emiliano apennines) are located on more heterogeneous substrates, mostly on calcareousmarly reliefs (limestones) and metamorphic reliefs of basic and ultrabasic rocks. the new records reveal that r. temporaria is far more widespread on monti della laga than previously known. the old and new occurrences for the species in central italy are located inside the gran sasso and monti della laga national park. besides the confirmation of the presence of r. temporaria in the 10-km square 33t uh63 (bruni et al., 2016), the record near campotosto (aq) in the 10-km square 33t uh61 represents a new national atlas square for the species (bernini and razzetti, 2006), and the new southern latitudinal limit in the italian peninsula (fig. 1). the habitat suitability map (fig. 2) shows that the uplands of the apennines harbour suitable environmenfig. 3. graphical results of geo-lithological analysis. a) presence of sandstones areas (red) in northern and central apennines and observations used for the analysis (dots). b) sandstones areas (continuous red lines) and limestones areas (dashed green lines) in central apennines. 50 f. di toro et alii tal conditions for r. temporaria. interestingly, a suitability gap is present between the northern and central apennines, reflecting the fragmented distribution of the species (bernini and razzetti, 2006). the contribution of mean temperature and precipitation of the warmest and wettest quarter on the species distribution model can be explained by the fact that these parameters influence the reproductive success r. temporaria, which breeding sites frequently consist of sun-exposed seasonal pools, especially in the study area (cammerini, 2020; authors, pers. obs.). however, according to the model, suitable environmental conditions are also present within the monti sibillini and gran sasso massif, areas where r. temporaria has never been observed. when considering geological features, it is noticeable that 86% of the occurrence records of r. temporaria on the apennines are situated on sandstone substrates, the principal rock type of monti della laga, whereas only 14% occurs on limestones (fig. 3). monti sibillini and gran sasso massif featuring mainly the latter, the formation of breeding pools and their hydroperiod might be amongst the factors explaining the absence of r. temporaria from these karst areas. pleistocene fossils of r. temporaria were found on apuan alps (bartolini et al., 2014), a karst mountain chain adjacent to the northern apennines and composed mainly of limestones, where the species does not occur nowadays (vanni and nistri, 2006). since in our model the apuan alps resulted currently suitable for the species, it is possible that r. temporaria was not able to survive warm periods at higher altitudes due to the unfavourable conditions determined by the type of substrate. since r. temporaria can be quite cryptic (e.g., marino et al., 2020), the present study highlights the need for further investigations aimed at assessing its actual distribution in central italy. in-depth research about size and structure of the (meta-) populations, gene flow among them and local environmental preferences would be pivotal to guide proper conservation measures in view of future climate change, since frigophilous species limited to isolated mountain ranges are among the most threatened of extinction (blank et al., 2013). acknowledgements we thank angelo cameli and luciano di tizio for comments and suggestions; alessandro cameriere, davide ferretti and simone forno for their help in field research; giovanni rossi, giancarlo tondi, alex borrini and matteo r. di nicola for observations and data; federico banfi for graphic help with figure 1. references ambrogio, a., mezzadri, s. 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(2006): atlante degli anfibi e dei rettili della toscana. regione toscana. acta herpetologica 14(1): 43-49, 2019 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-23941 using an in-situ infra-red camera system for sea turtle hatchling emergence monitoring fatıma n. oğul1,#,*, franziska huber2,#, sinem cihan1, kumsal düzgün3, ahmet e. kideyş1, korhan özkan1 1institute of marine sciences, middle east technical university, erdemli, mersin, turkey. *corresponding author. e-mail: fogulnua@ gmail.com 2paris lodron university of salzburg, faculty of natural sciences, salzburg, austria 3faculty of veterinary medicine, ankara university, altındağ, ankara, turkey # the two authors contributed equally submitted on: 2018, 28th october; revised on: 2019, 2nd march; accepted on: 2019, 5th april editor: paolo casale abstract. we tested for the first time the efficiency of the use of infra-red (ir) cameras for sea turtle hatchling monitoring. the cameras were installed on one green turtle (chelonia mydas) and four loggerhead turtle (caretta caretta) nests during 2014 and 2015 nesting season in the south-east mediterranean, turkey. the camera monitoring, even with the limited sample size, have successfully corroborated the previous observations and provided further insights on hatchling emergence behavior. the analysis of the camera recordings revealed that hatchlings emerged from the nests asynchronously in varying numbers of groups and different group sizes, while c. 60% hatchlings emerged during the first 5 days of emergence activity. 98.6% of hatchlings emerged at night with a peak activity between 21:00 and 00:00. the day of first emergence varied between 38 and 64 days since egg deposition, while the day of last emergence varied only between 60 and 65 days. total emergence activity continued up to maximum of 22 days, which is longer than that of previous records. overall, the present study showed that ir camera monitoring is a promising tool for sea turtle monitoring and can provide detailed insights on sea turtle hatchling behavior. keywords. loggerhead turtle, green turtle, hatchling emergence duration, infra-red camera, continuous camera monitoring, sea turtle nest monitoring, sea turtle conservation. introduction breeding success has been an essential component of sea turtle conservation (musick and limpus, 1997; hamann et al., 2010; rees et al., 2016). therefore, extensive monitoring of sea turtle breeding beaches has become an integral part of sea turtle breeding habitat management (fowler, 1979, hays et al., 2001; taskin and baran, 2001). these monitoring efforts have focused on various aspects of sea turtle breeding, such as, habitat quality, nest predation, anthropogenic effects and hatchling emergence patterns; leading to the identification of important pressures on the breeding habitats of these endangered species (kasparek et al., 2001; tomás et al., 2002), which has led to implementation of better conservation measures. sea turtle breeding beach monitoring have been almost exclusively based on regular beach patrols during the breeding period that may last for five months (henson and boettcher, 2006; medasset, 2017). these direct visual observations have important limitations in temporal resolution, feasibility and man power (garciía 44 fatıma n. oğul et alii et al., 2003). therefore, sea turtle conservation management may be benefitted from more efficient monitoring alternatives providing standardized survey data. continuous camera recordings have been used in several organism groups, such as, wild boars (huckschlag, 2008), deers (scheibe et al., 2008) and birds (pierce and pobprasert, 2013); however, they have not previously been used for sea turtle hatchling monitoring. only in florida keys beach, a live-streaming webcam was installed on a sea turtle nest in 2014 in order to raise awareness on sea turtle conservation (http://www.fla-keys.com/turtlecam/). continuous camera monitoring may provide opportunities for sea turtle breeding beach monitoring by improving monitoring efficiency as well as providing detailed insights on hatchling behavior. there are currently different monitoring and excavation protocols for different research teams and volunteer groups (henson and boettcher, 2006; medasset, 2017). for example, there is no standardized nest excavation time, although many management groups prefer to conduct an excavation within the few days of the last detected emergence. furthermore, beach patrolling during hatchling emergence period is mostly conducted in the mornings (henson and boettcher, 2006; medasset, 2017), while knowing temporal emergence patterns could facilitate the researchers to better allocate labor if encountering hatchlings is required. therefore, detailed understanding of hatchling emergence behavior specific to breeding beaches through novel technologies and standardized data may lead to a more efficient decision on excavation dates and beach patrolling schedules. furthermore, temporal patterns and group formation of sea turtle hatchling emergence might also be an important determinant of the survival of hatchlings (carr and hirth’s 1961) and therefore a more detailed understanding of hatchling behavior may also be instrumental in conservation management. overall, quantitative and standardized observations on hatchling behavior using technological monitoring tools may provide a more comprehensive understanding of sea turtle breeding ecology and conservation. the aim of this study is testing a novel method – ir camera monitoring – and assess its efficiency in sea turtle hatchling monitoring of the temporal patterns and group formations of hatchling emergence as well as hatchling behavior. material and methods study site the study was conducted at the beach of the institute of marine sciences, middle east technical university (metu ims), turkey. the beach stretches along a 1.2 km long coast and is located in a heavily urbanized area of the eastern mediterranean. the study site has restricted public access and the human activity is limited. the beach is mostly sandy and spans 15-25 m in width with insignificant tidal activity. it consists of natural sand dunes approximately 0.5-3.0 m above sea level, hosting natural coastal vegetation dominated by sand lily (pancratium maritimum, l.; cihan, 2015). the activity of breeding sea turtles from may to august and hatchlings from july to september have been monitored since 2013 using conventional beach patrols. camera monitoring system conventional infrared (ir) security cameras (balitech bl-6150) with 200 m range, 8 mm stable lens and 650 tvl resolution were installed on wooden poles placed approximately 1.5-3.0 m away and 1.0-1.5 m above the nests (fig. 1). all the cameras were connected to a digital video recorder (samsung srd-1650d) with 16 channels and 1 tb memory, placed in a cabinet that was installed c. 20 m away from the most distant nest. recordings were transferred every second day to an external 1 tb hard drive. the recordings were commenced after 51 days of egg deposition and lasted for c. 30 days. five cameras in total were installed on loggerhead (4) and green turtle (1) nests. analyses of hatchling behavior and emergence activity the video recordings were analyzed to elucidate the patterns in hatchling group size, emergence date and time. we pooled and analyzed green and loggerhead nest data together for the present analyses, although these two species significantly differ for other aspects of their breeding biology. all video recordings were analyzed with automatic screen captures at 30-second intervals. when emergence activity was detected in photos, the corresponding video clip was examined, for the exact emergence date and time, hatchling count, crawling duration, orientation, behavior as well as any predation event. emergence activity was accepted to start with the earliest time of a hatchling observed on the nest surface, end with disappearance of the last hatchling from camera view. emergence groups were categorized by the number of individuals: 1-3 as small, 4-10 as intermediate, more than 11 as large groups. individuals appeared at the same time or subsequently (less than one minute between individuals) were taken as one group even if there was a lag between their crawling activities. total days until first emergence, peak activity and last emergence were calculated from the night of nest deposition. the day when the largest emergence occurred was designated as the nest’s day of peak activity. an emergence events were designated as day or night activity according to the time of sunrise (05:50-06:23 h) and sunset (18:51-19:46 h) during study period. the emergences that occurred 10 min before sunset and 10 min after sunrise were considered as night activity to be able to account for local shading. in nest e11r, 30 hatchlings waiting on the top of the nest chamber were accidentally dug by chil45camera monitoring of sea turtle hatchlings dren just before the sunset. the surveyor immediately noticed the event, monitored the nest and the hatchlings subsequently released under monitoring. we accepted that emergence as night emergence, since the event occurred just before the sunset, and the hatchlings were ready to emerge immediately that evening. the hatchling orientation and predation events were also recorded to understand the effectiveness of beach management and efficiency of nest cages. the one side opened pyramidal shaped metal nest cages were placed on the nests’ surface as open side was directed toward sea (fig. 1). orientation of each hatchling movement relative to a seaward direction was recorded. we classified hatchling crawls within +70° of the seaward direction as seaward orientated. mortality or predation events were also recorded. a nest excavation was performed after the end of camera monitoring. the total number of eggs were estimated from the remains and compared with the counts from the video recordings. the emergence success of each nest was calculated as the ratio of hatchlings those reached to the sea and the total clutch size (miller, 1999). results the values of all the analyzed parameters for the single green turtle nest varied always within the range that was observed for the loggerhead turtle nests (table 1). accordingly, we did not discard the single green turtle nest data, instead, green turtle and loggerhead turtle nests were pooled together for the analyses. camera recordings revealed that a total of 357 hatchlings in 71 groups emerged from five nests with 42-94 hatchlings per nest. in total 62% of hatchlings emerged in large groups, 17 % of hatchlings emerged in intermediate groups and 21% of hatchlings emerged in small groups (table 1). at least one large group emergence was observed for each nest (table 1). additionally, 69 of the 72 groups (95.8%) emerged at night accounting for 352 of 357 hatchlings (98.6%; fig. 2, 3a, 3b). all of the large and intermediate groups as well as 49 of the 51 small group emergences occurred during night (fig. 2). the highest emergence fig. 1. a sample setup of camera system around a sea turtle nest. photographer: korhan özkan. table 1. number of emergence groups and hatchlings for each nest with clutch size and incubation period. cm and cc denote for green and loggerhead turtle respectively. ng and nh denote for number of groups and total number of hatchlings respectively. nest species large emergences intermediate emergences small emergences incubation duration clutch size ng nh ng nh ng nh 8r cm 2 42 3 16 13 20 53 97 e11r cc 3 58 3 13 19 23 38 103 e6r cc 1 23 2 15 4 4 64 50 e7r cc 2 64 1 5 11 22 54 104 e9r cc 1 34 2 12 4 6 54 71 total: 9 221 11 61 51 75 fig. 2. the daily temporal patterns of hatchling emergence activity. each symbol represents a different nest and the dashed line represents the approximate sunset time. the emergence activity was overwhelmingly nocturnal. 46 fatıma n. oğul et alii activity (60% of the group emergences) occurred between 21:00 and 00:00 (fig. 3a and 3b). the total number of hatchlings captured with camera recordings and the number of empty eggs found in excavations were largely consistent with an error rate between 1% and 5.8% (only 1 to 3 differences have been found per nests, table 2). total incubation period varied between 38-64 days since the egg depositions (mean = 52.6 days; table 2). the day of the peak activity varied between 47 and 64 days (mean = 54.6 days; fig. 4). the day of the last emergences was least variable among nests and varied between 60 and 65 days (mean = 63 days; fig. 4). total emergence duration between the first emergence and last emergence had a large variation, changed between 1 and 22 days (mean = 10.4 days). overall, 121 hatchlings out of 357 (33.9%) emerged during the first day (24.2 hatchlings on average per nest). 221 (61.9%) hatchlings emerged over the first 5 days following the first emergence (8.8 hatchlings per day for the fig. 3. the histogram of emergence activity by hour for number of emergence groups (a) and hatchlings (b). table 2. surveyed nests’ characteristics and nest excavation records. nest 8r e6r e7r e9r e11r species chelonia mydas caretta caretta caretta caretta caretta caretta caretta caretta egg deposition date 12/07/2014 6/06/2015 10/06/2015 16/06/2015 27/06/2015 camera installation date 1/09/2014 12/07/2015 12/07/2015 12/07/2015 12/07/2015 first emergence date 3/09/2014 9/08/2015 3/08/2015 9/08/2015 4/08/2015 last emergence date 14/09/2014 10/08/2015 14/08/2015 16/08/2015 26/08/2015 incubation duration (from egg deposition to first emergence) 53 64 54 54 38 excavation date 2/10/2014 23/08/2015 19/08/2015 21/08/2015 28/08/2015 emergence duration 11 1 11 7 22 hatched/empty eggs (from excavation) 77 41 93 49 95 camera hatchling count 78 42 91 52 94 clutch size 97 50 104 71 103 hatchlings reaching the sea 76 42 91 50 94 hatchlings predated 1 0 0 0 0 dead hatchlings (after emergence) 1 0 0 3 0 early stage embryos 9 4 2 12 2 middle stage embryos 1 0 2 1 0 late stage embryos 2 0 1 0 2 unfertilized eggs 7 4 8 6 5 fig. 4. the duration of the first emergence (fe), peak emergence (pe), last emergence (le), calculated as the total number of days since egg deposition. the least variation was observed in the day of last emergence. 47camera monitoring of sea turtle hatchlings first five days). three nests’ peak activity occurred on the first day and one nest’s peak activity occurred on the second day of emergence period. the majority of the hatchlings (88.8%) oriented successfully towards the sea. only three disorientated emergence groups (40 hatchlings) were recorded, but the predation cages re-directed them to the sea. the hatchling emergence success of the nests varied between 66% and 92% (table 2) and no dead hatchlings were found within the nest chamber during excavations. a total of five dead hatchlings were observed due to overturning and subsequent heat shock (table 2). four of the deaths happened during the day, with only one happened during the night. only one hatchling was predated by hooded crows during day. discussion the results of the present study showed that continuous camera monitoring can be an efficient tool, especially for hatchling behavior monitoring. furthermore, the analyses of the recordings corroborated the previous findings on the patterns in emergence group sizes, timings and durations, as well as provided further insights on hatchling behavior. continuous camera recordings with ir cameras provided data with very high temporal resolution on sea turtle hatchling behavior. although the present study performed on a limited number of nests, the camera monitoring documented an exceptionally long emergence activity duration of 22 days. previously, 18 days of emergence duration was reported as the longest emergence activity duration for loggerhead turtles in japan (moriya and moriya, 2011). our results suggest that longer emergence durations might be more frequent than expected and continuous video recordings may provide a more reliable estimation for the duration of hatchling emergence activity in comparison to conventional beach monitoring. continuous camera recordings also enabled us to study hatchling emergence behavior at temporal scales and with small sample sizes that are difficult to account for with conventional beach monitoring. for example, we observed a strong intra-nest asynchrony in hatchling emergences; i.e., hatchlings tended to emerge in several groups with different sizes in successive days. synchronous emergence (emerging as one large group) has been proposed to reduce the probability of hatchling predation on land (delm, 1990; heithaus, 2013; santos et al., 2016) and hatchlings might stimulate each other both during and after emergence (carr and hirth, 1961). however, several studies previously documented asynchronous emergence (peters et al., 1994; glen et al., 2005; adam et al., 2007; moriya and moriya, 2011), similar to our findings. this might also be due to a decrease in predation intensity for the hatchlings emerging in small groups (pilcher et al. 2000) or due to ambient temperature differences in the nest (adam et al., 2007). moreover, the present study demonstrated that the natural emergence activity since the egg deposition lasted between 60 and 65 days with a very limited variability among nests. however, the total emergence activity since the first hatchling emergence lasted between 1 and 22 days with a large variation. accordingly, if the natural incubation and emergence process is preferred by the local conservation managers, 65 days after nesting or 22 days after the first emergence may be waited until any excavation, if the nesting beaches are not under high predation pressures. we observed in the present study that the in-situ camera systems had considerable advantages over direct visual observations on effort, consistency and repeatability especially if the proper equipment is selected. however, we have also observed some limitations on the use of insitu camera systems. the field of view of the cameras only enabled us to monitor close vicinity of the nest and prevented us following the hatchlings to the sea, which only documented the immediate survival of the hatchlings on the nest. we also encountered hardware and recording failures mostly due to corrosion. therefore, using durable technical equipment and backing up data frequently are essential for successful in-situ camera applications. metu ims campus has limited access to public and thus it is well protected from robbery or vandalism, which enabled us to install electronic equipment freely. however, using this method in larger or remote breeding beaches would require necessary security precautions. the analyses of hatchling emergence data in the present study strongly corroborated previous observations on loggerhead and green turtle nests monitored using conventional methods. the majority (98.6%) of the hatchling events in the present study occurred nocturnally similar to previous findings (mrosovsky, 1968; witherington et al., 1990; hays et al., 1992; glen et al., 2005), probably to avoid diurnal predators and lethal daytime temperatures (glen et al., 2005). the peak emergence activity occurred between 21:00 and 00:00 h, similar to the observations in florida (23:00 and 00:00 h, witherington et al. 1990) and in greece (00:30 and 01:00 h, adam et al., 2007). accordingly, the night patrolling efforts aiming at monitoring hatchlings could be prioritized for early evening, when man power is limited. furthermore, nocturnal emergences occurred mostly (~80%) in large groups, while all diurnal emergences were in small groups (including single emergences) in the present 48 fatıma n. oğul et alii study, further corroborating previous studies (glen et al., 2005). the mean total incubation period (from egg deposition night to the first emergence) was 52.6 days (38-64 days) in the present study. this is in accord with previous observations for green turtles in northern cyprus (57.9 days; ilgaz and baran, 2001), for loggerhead turtles in turkey, greece and northern cyprus (varied between 49 and 55.2 days; ilgaz and baran, 2001; taskin and baran, 2001; margaritoulis, 2005; fuller et al., 2013). the difference between the incubation periods among different studies might be due to the differences in ambient temperature of the breeding beaches (hays et al., 1992; drake and spotilla, 2002; glen, 2005; adam et al., 2007). therefore, continuous camera recordings with temperature measurement devices might provide more accurate hatchling activity parameter estimates specific to different breeding beaches. it should also be noted that the number of the samples in the present study is limited and further studies with larger sample sizes is required for more accurate parameter estimations. the great majority of the hatchlings (98.6%) reached to the sea successfully in the present study, indicating a very high survival rate in comparison to other observations in the region (49.9% in ilgaz and baran, 2001 and 43.5 % in taskin and baran, 2001). only a single hatchling (in 357) was predated by hooded crows, which is very low predation rate (carr and hirth, 1961; tomillo et al., 2010; türkozan et al., 2011). the hatchling deaths occurred only in small group emergences in the present study. this is in accord with previous observations, where larger groups of hatchlings have been observed to be more motivated to reach the sea and more directional in their effort (carr and hirth, 1961; burger and gochfeld, 2014), while single emergences have had less chance to reach to sea than group emergences (carr and hirth, 1961). the high success rate probably reflected the efficiency of the conservation efforts at metu ims beach (i.e., artificial light and human use management). overall, the present study showed that in-situ camera systems is an alternative or complementary tool to the conventional beach monitoring for sea turtle conservation with significant advantages on labor and efficiency. furthermore, the high frequency data gathered through continuous camera monitoring even with small sample sizes provide important opportunities for studies on sea turtle hatchling behavior. acknowledgements the permits to perform this study were obtained from the directorate general for nature conservation and national parks (72784983-488.04-125729). we thank mehmet beklen, mehmet keçeci, mehmet emin polat, mehmet tolga eliuz, turan uca, bahri dinç and mehmet özalp for their technical assistance and gençay ergez for helping figures. this project was supported by dekosi̇m, bap07-01-2012-001 and bap-07-01-2016001 projects. msc student fatıma nur oğul and sinem cihan received grant from tübi̇tak 113y179, 112y394, and 11y023 projects. references adam, v., tur, c., rees, a., tomás, j. 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(1990): temporal pattern of nocturnal emergence of loggerhead turtle hatchlings from natural nests. copeia 1990: 1165-1168. acta herpetologica vol. 14, n. 1 june 2019 firenze university press uzungwa scarp nature forest reserve: a unique hotspot for reptiles in tanzania john valentine lyakurwa1,2,*, kim monroe howell2, linus kasian munishi1, anna christina treydte1,3 experience of predacious cues and accessibility to refuge minimize mortality of hylarana temporalis tadpoles santosh mogali*, bhagyashri shanbhag, srinivas saidapur tonal calls as a bioacoustic novelty in two atlantic forest species of physalaemus (anura: leptodactylidae) thiago r. de carvalho1,*, célio f.b. haddad1, marcos gridi-papp2 scientific publication of georeferenced molecular data as an adequate guide to delimit the range of korean hynobius salamanders through citizen science amaël borzée1,*, hae jun baek2,3, chang hoon lee2,3, dong yoon kim2, jae-young song4, jaehwa suh5, yikweon jang1, mi-sook min2* mirrored images but not silicone models trigger aggressive responses in male common wall lizards stefano scali1,*, roberto sacchi2, mattia falaschi1,3, alan j. coladonato2, sara pozzi2, marco a.l. zuffi4, marco mangiacotti1,2 using an in-situ infra-red camera system for sea turtle hatchling emergence monitoring fatima n. oğul1,#,*, franziska huber2,#, sinem cih1, kumsal düzgün3, ahmet e. kideyş1, korhan özkan1 descriptive osteology of an imperiled amphibian, the luristan newt (neurergus kaiseri, amphibia: salamandridae) hadi khoshnamvand1, mansoureh malekian1,*, yazdan keivany1, mazaher zamani-faradonbe1, mohsen amiri2 does color polymorphism affect the predation risk on phalotris lemniscatus (duméril, bibron and duméril, 1854) (serpentes, dipsadidae)? fernanda r. de avila1,2,*, juliano m. oliveira3, mateus de oliveira1, marcio borges-martins4, victor hugo valiati2, alexandro m. tozetti1 age structure of a population of discoglossus scovazzi camerano, 1878 (anura discoglossidae) in extreme environmental conditions (high atlas, morocco) mohamed amine samlali, abderrahim s’khifa, tahar slimani* acta herpetologica 15(2): 111-118, 2020 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/a_h-7564 stomach histology of crocodylus siamensis and gavialis gangeticus reveals analogy of archosaur “gizzards”, with implication on crocodylian gastroliths function ryuji takasaki1,2,*, yoshitsugu kobayashi3 1 department of natural history and planetary sciences, hokkaido university, kita 10, nishi 8, kita-ku, sapporo, hokkaido, 060-0810, japan 2 faculty of biosphere-geosphere science, okayama university of science, ridai-cho, kita-ku, okayama, japan 3 hokkaido university museum, hokkaido university, kita 10, nishi 8, kita-ku, sapporo, hokkaido, 060-0810, japan *corresponding author. e-mail: rtakasaki@big.ous.ac.jp submitted on: 2020, 1st december; revised on: 2020, 14th august; accepted on: 2020, 1st october editor: emilio sperone abstract. two groups of extant archosauria, crocodylia and neornithes, have two-chambered stomachs and store gastroliths inside their “gizzards”. morphological similarities of the “gizzards” lead some previous studies to assume that the presence of this structure, organ “gizzard” is synapomorphic to archosauria. however, the homology of archosaur “gizzards” had never been tested. this study provides general histological descriptions of stomachs of two crocodylian taxa, crocodylus siamensis and gavialis gangeticus, to determine the homology of crocodylian and neornithine “gizzards”. our study demonstrates that both crocodylus siamensis and gavialis gangeticus have longer, more complex glands in the fundic stomach (crocodylian “gizzard”) than in the pyloric stomach. additionally, we found that compound glands are present in the fundic stomach of crocodylus siamensis. therefore, crocodylian stomach histomorphological structures are concordant with those of other non-avian reptiles, despite the unique gross morphology. the pyloric regions of non-avian reptile stomachs are known to be homologous with the pyloric regions of mammalian stomachs as well as neornithine ventriculus (neornithine gizzard). therefore, crocodylian and neornithine “gizzards” are morphologically analogous but not homologous. the presence of pas-positive layer in the pyloric stomach of gavialis gangeticus, which resembles the koilin layer of neornithine ventriculus, further supports this interpretation. at the same time, however, the similarity in gastroliths mass/body mass ratio and the correlations between gastroliths occurrence and diet types suggest that crocodylian gastroliths might have contributed to the digestion of ingesta, even though crocodylian and neornithine “gizzards” are not homologous. keywords. histology, gizzard, gastroliths, crocodylia, stomach. introduction crocodylians present the most complex stomach known in existing members of non-avian reptiles (hereafter referred to as reptiles) (owen, 1866; richardson et al., 2002). crocodylian stomachs are composed of two distinct units: fundic and pyloric chambers. neornithes (a least inclusive clade of living birds), the closest living relatives of crocodylians, also have two-chambered stomachs: a glandular stomach (proventriculus) and a muscular stomach (ventriculus or gizzard) (ziswiler and farner, 1972; denbow, 2015). while the proventriculus excretes the mucus, pepsin, and hydrochloric acid necessary for chemical digestion, the ventriculus performs mechanical digestion of ingesta. some neornithines, mostly herbivores, consume stones and store them inside gizzards as gastroliths (geo-gastroliths, wings, 2007) to aid gastric mechanical digestion (fritz, 1937; hetland et al., 2003; jin et al., 2014). several crocodylians are also known to contain gastroliths inside their fundic stomachs (e.g., corbet, 112 ryuji takasaki, yoshitsugu kobayashi 1960; cott, 1961). since both crocodylians and neornithines have two-chambered stomachs and store gastroliths inside them, some studies refer crocodylian fundic stomach as a “gizzard” (reese, 1915; grigg and gans, 1993). based on the phylogenetic bracket of the two-chambered stomachs, together with the generality of gastroliths among archosaurs including non-avian dinosaurs, (e.g., kobayashi et al., 1999; cerda, 2008; lee et al., 2014), neornithine style muscular “gizzard” had previously been considered as a plesiomorphic feature of archosauria (varricchio, 2001; fritz et al., 2011). however, the homology of avian and crocodylian “gizzards” is considered ambiguous (schwenk and rubega, 2005). while some studies considered that crocodylian “gizzards” are homologous with neornithine gizzards (varricchio, 2001; fritz et al., 2011), some studies refute the homology (jones, 1861; huang et al., 2016). additionally, the functions of crocodylian gastroliths are still under debates (e.g., food processing, hydrostatic function, accidental intake; cott, 1961; davenport et al., 1990; taylor, 1993; wings, 2007; uriona et al., 2019). previous studies on crocodylian stomach microstructures were based only on alligator mississippiensis (eisler, 1889; reese, 1915; staley, 1925). this lack of knowledge of crocodylian stomach structures cannot allow determining the plesiomorphic status of archosaur “gizzard”. our study provides the first histomorphological information of the stomachs of crocodylus siamensis and gavialis gangeticus to test the homology of crocodylian and neornithine “gizzards”. besides, this study conducts analyses that provide new implications of the digestive function of crocodylian gastroliths. neornithine gastrolith mass is known to be correlated with a body mass (wings and sander, 2007), and the relationship is utilized as a proxy for the digestive use of dinosaur gastroliths (wings and sander, 2007; cerda, 2008; lee et al., 2014). furthermore, avian dietary habits are strongly related to the occurrence frequencies of gastroliths (best and gionfriddo, 1991; gionfriddo and best, 1996; gionfriddo and best, 1999). our study tests if crocodylian gastroliths have the same relationship as observed in neornithines to assess the digestive function of crocodylian gastroliths. the clarifications of archosaur “gizzard” homology and the crocodylian gastroliths functions are expected to contribute to better understandings of crocodylian physiology and the evolutionary history of the archosaur digestive system. material and method corpora of four juvenile individuals of captive crocodylus siamensis which were dead during winter are provided from a local farmer koike wani sohonpo co. ltd. in shizuoka prefecture of japan, and four stomachs of captive post-mortem gavialis gangeticus are provided from atagawa tropical & alligator garden in shizuoka prefecture of japan (fig. 1). all the specimens were stored frozen before sampling. small segments were sampled from the greater curvature wall, ventral wall, and pyloric wall of the stomach. the segments are fixed in 10% formalin neutral buffer solution, then dehydrated in ascending grades of ethyl alcohol, cleared with xylene, and embedded in paraffin. sections were cut at 3μm in thickness and stained with haematoxylin-eosin (he), periodic acid schiff (pas), and alcian-blue (ab) ph 2.5 for general histological observations. to avoid confusion due to different terminologies used in previous studies, this study uses the term “gizzard” for a stomach chamber that may possess gastroliths. terms fundic stomach and pyloric stomach are used for first and second chambers of the crocodylian stomach, respectively. terms proventriculus and ventriculus are used for first and second chambers of the avian stomach, respectively. crocodylian body and gastroliths weights are compiled from previous studies (corbet, 1960; cott, 1961; kennedy and brockman, 1965; brazaitis, 1969; pauwels et al., 2007). stomach contents of crocodylians are gathered from previous studies (corbet, 1960; cott, 1961; tucker et al., 1996; platt et al., 2006; wallace and leslie, 2008; platt et al., 2013). body mass and gastroliths mass are log10 transformed and occurrence frequencies of gastroliths and different food types are arcsine transformed before statistical analyses. statistical analyses are conducted using the software jmp version 14.3. results the stomach walls of all of the observed specimens are composed of 4 layers: mucosa, submucosa, musculafig. 1. stomachs of crocodylus siamensis (a) and gavialis gangeticus (b). scales: 5cm for a, 10cm for b. 113stomach histology of crocodylus siamensis and gavialis gangeticus ris externa, and serosa layers from inner to outer layers (fig. 2). the greater curvature wall is the thickest among the observed regions (fig. 1b, 2a, 2b). submucosa comprises nearly half of the stomach wall in thickness in crocodylus siamensis (~800μm), while the muscularis externa occupies more than half of the wall in thickness in gavialis gangeticus (~2000μm). the gastric folds, supported by thick submucosa, are shorter than wide in both crocodylus siamensis and gavialis gangeticus. the mucosa (~200μm in crocodylus siamensis and ~300μm in gavialis gangeticus) is thinner than submucosa and has long fundic glands in both taxa. the fundic glands are tubular and branched (fig. 2c, 2d) although postmortem damage obscures the details. the fundic glands are fig. 2. histological structures of crocodylus siamensis (a, c, e, g, i, k) and gavialis gangeticus (b, d, f, h, j, l). a-d, greater curvature wall; e-h, ventral wall; i-l, pyloric wall. abbreviations: cg, compound gland; fg, fundic gland; kl, possible koilin layer; m, mucosa; me, muscularis externa; sm, submucosa; pg, pyloric gland. scales: 1000μm for a, b, e, f, i, and j; 250μm for c, d, g, h, k, and l. 114 ryuji takasaki, yoshitsugu kobayashi mainly composed of dark oxynticopeptic cells, as previously reported in the stomachs of alligator mississippiensis (eisler, 1889; staley, 1925). there are no morphologically distinct mucous neck cells reported in most snakes (jacobson, 2007). the ventral wall is slightly thinner than the greater curvature wall (fig. 2e, 2f). the submucosa of the ventral wall is thin, and the muscularis externa comprises the largest proportion of the ventral wall. the gastric folds are well-developed in crocodylus siamensis, but it is absent in gavialis gangeticus. the mucosa is proportionally thinner than it is in the greater curvature wall, resulting in shorter fundic glands than in the greater curvature wall in both taxa. the fundic gland structures are generally the same as those in the greater curvature wall. however, gastric glands in the ventral wall are markedly larger than in the other stomach walls and form a lobule-like compound gland in crocodylus siamensis (fig. 2g). these gastric glands are separated from each other with thick connective tissue. the lobule-like compound glands could not be observed in gavialis gangeticus, partly because available stomachs are not well-preserved compared to crocodylus siamensis. the pyloric walls of the two crocodylian taxa are largely different from the greater curvature and the ventral walls in their extremely thick muscularis externa, which represents up to 80% of the stomach wall thickness (fig. 2i, 2j). on the other hand, the submucosa is reduced, unlike what was observed in the fundic stomach. muscularis mucosa is also much thicker than in the other two regions. pyloric glands are simple tubular glands and are significantly short compared to the fundic glands (fig. 2k). unfortunately, details of the pyloric glands are not available due to the impact of  postmortem damage, especially in the stomachs of gavialis gangeticus. the internal surface of the pyloric wall is locally covered by a pas-positive layer in gavialis gangeticus (fig. 2l). body mass and gastroliths mass of crocodylia (table 1) demonstrates that the average proportion of gastroliths mass relative to body mass is 0.66%. the value is slightly higher than that in neornithines (0.55%), but the difference is not statistically significant (student’s t-test, p = 0.50). regression analysis demonstrates the correlation of crocodylian body mass and gastroliths mass (fig. 3; r2 = 0.84, p < 0.001) as in neornithines (wings and sander, 2007). neither the slope nor the intercept of the regression line differs from those of neornithines (p = 0.43 and 0.73, respectively), indicating that the relationship between gastroliths mass and body mass of crocodylians are statistically indistinctive from that of neornithines. regression analyses on occurrence frequencies of gastroliths and different food types (table 2) demonstrate that occurrence frequency of gastroliths are positively correlated with those of vertebrates and negatively correlated with those of most invertebrates (table 3). the correlations are statistically significant (p < 0.05) in insecta, pisces, amphibia, and mammalia. discussion histological evaluations of crocodylus siamensis and gavialis gangeticus stomachs demonstrate that general stomach morphology is similar to each other. both taxa have long, tubular branched fundic glands and short, simple pyloric glands (fig. 2). the result is concordant with the stomach microstructure of alligator mississippiensis as reported in staley (1925), indicating that members of crocodylia share generally the same fundic and pyloric gland structures. the long, complex fundic glands and short, simple pyloric glands are also in agreement with general features of reptilian stomachs (luppa, 1977; jacobson, 2007). furthermore, the lobule-like compound fundic glands that are present in the ventral wall of crocodylus siamensis (fig. 2g) are also reported in fundic stomachs of other reptiles including caretta caretta (oppel, 1896), chamaeleon afticanus (hamdi et al., 2014), laudakia stellio (koca and gurcu, 2011), ophisops elegans (çakici and akat, 2013), and varanus niloticus (ahmed et al., 2009). therefore, the present observations demonstrate that general histomorphological structures of crocodylian stomach glands are concordant with those fig. 3. relationships of body mass to gastroliths mass in crocodylians (red diamond) and crown birds (blue circle). the red solid line represents the regression line for crocodylians and the blue dashed line represents the regression line for crown birds. 115stomach histology of crocodylus siamensis and gavialis gangeticus of other reptiles, despite the unique gross morphology of crocodylian stomach among reptilia. the general histomorphological features of reptilian fundic and pyloric glands resemble neornithine gastric glands of proventriculus and ventriculus, respectively. neornithine proventriculus contains highly branched compound glands that compose lobules, and the ventriculus contains simple tubular glands covered by the paspositive koilin layer (ziswiler and farner, 1972). through stomach muscle structure comparisons, pernkopf (1929) suggested that the reptilian pyloric stomach is homologous to the pyloric region of the mammalian stomach, which is homologous to neornithine ventriculus (smith et al., 2000). the present results suggest that the crocodylian pyloric stomach is homologous with neornithine ventriculus (neornithine gizzard), whereas the crocodylian fundic stomach (crocodylian “gizzard”) is homologous with neornithine proventriculus. since crocodylian and neornithine “gizzards” are not homologous, the previous assumption that “gizzard” is synapomorphic to archosauria (varricchio, 2001; fritz et al., 2011) is dismissed. although crocodylian and neornithine “gizzards” are not homologous, the absence of statistical difference in the body mass-gastroliths mass relationship between the two groups suggests the digestive function of crocodylian gastroliths based on previous interpretations (wings and sander, 2007). the relationships between gastroliths table 1. mean total gastroliths mass and body mass of crocodylians compiled. species mean total gastrolith mass[g] mean body mass[g] sample size relative weight of gastroliths [%] reference (gastroliths) reference (body mass) crocodile acutus 174.00 32206 2 0.54% brazaitis (1969) brazaitis (1969) alligator mississippiensis 22.00 7800 1 0.28% kennedy and brockman (1965) kennedy and brockman (1965) osteolaemus t. tetraspis (rabi oil fields) 5.54 3241 14 0.17% pauwels et al. (2007) pauwels et al. (2007) osteolaemus t. tetraspis (loango national park) 4.33 8193 8 0.05% pauwels et al. (2007) pauwels et al. (2007) crocodilus niloticus 0.5-1.0m 2.04 1524 101 0.13% cott (1961) cott (1961) crocodilus niloticus 1.0-1.5m 11.70 4518 102 0.26% cott (1961) cott (1961) crocodilus niloticus 1.5-2.0m 88.87 16540 76 0.54% cott (1961) cott (1961) crocodilus niloticus 2.0-2.5m 312.50 40900 73 0.76% cott (1961) cott (1961) crocodilus niloticus 2.5-3.0m 700.30 79390 69 0.88% cott (1961) cott (1961) crocodilus niloticus 3.0-3.5m 1321.20 131900 52 1.00% cott (1961) cott (1961) crocodilus niloticus 3.5-4.0m 1906.20 206500 16 0.92% cott (1961) cott (1961) crocodilus niloticus 4.0-4.5m 2940.40 298700 5 0.98% cott (1961) cott (1961) crocodilus niloticus 4.5-5.0m 3356.00 325500 3 1.03% cott (1961) cott (1961) crocodilus niloticus 0.3-0.5m 4.80 146 2 3.29% corbet (1960) corbet (1960) crocodilus niloticus 0.5-1.0m 2.87 1524 23 0.19% corbet (1960) corbet (1960) crocodilus niloticus 1.0-1.5m 19.79 4518 18 0.44% corbet (1960) corbet (1960) crocodilus niloticus 1.5-2.0m 66.80 16540 2 0.40% corbet (1960) corbet (1960) crocodilus niloticus 3.5-4.0m 206.50 206500 1 0.10% corbet (1960) corbet (1960) r ef er en ce c or be t ( 19 60 ) c or be t ( 19 60 ) c ot t ( 19 61 ) c ot t ( 19 61 ) c ot t ( 19 61 ) c ot t ( 19 61 ) c ot t ( 19 61 ) c ot t ( 19 61 ) c ot t ( 19 61 ) c ot t ( 19 61 ) c ot t ( 19 61 ) c ot t ( 19 61 ) w al la ce a nd l es lie ( 20 08 ) w al la ce a nd l es lie ( 20 08 ) w al la ce a nd l es lie ( 20 08 ) pl at t e t a l. (2 00 6) pl at t e t a l. (2 00 6) pl at t e t a l. (2 00 6) pl at t e t a l. (2 00 6) pl at t e t a l. (2 00 6) pl at t e t a l. (2 01 3) pl at t e t a l. (2 01 3) pl at t e t a l. (2 01 3) pl at t e t a l. (2 01 3) pl at t e t a l. (2 01 3) tu ck er e t a l. (1 99 6) tu ck er e t a l. (1 99 6) tu ck er e t a l. (1 99 6) tu ck er e t a l. (1 99 6) tu ck er e t a l. (1 99 6) tu ck er e t a l. (1 99 6) tu ck er e t a l. (1 99 6) tu ck er e t a l. (1 99 6) o cc ur re nc e fr eq ue nc ie s (% ) m am m al ia 6. 90 0. 00 0. 00 4. 93 10 .6 4 10 .8 1 9. 40 12 .4 0 21 .2 4 26 .5 3 47 .8 3 58 .3 3 3. 60 4. 20 10 .0 0 0. 00 0. 00 9. 92 3. 17 2. 08 0. 00 0. 00 3. 57 0. 00 0. 00 0. 00 2. 00 2. 00 0. 00 3. 00 0. 00 6. 00 10 .0 0 a ve s 0. 00 18 .1 8 0. 00 3. 52 2. 13 9. 91 5. 98 10 .8 5 9. 73 12 .2 4 13 .0 4 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 1. 65 4. 76 10 .4 2 0. 00 0. 00 3. 57 12 .5 0 11 .1 1 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 2. 00 r ep til ia 0. 00 0. 00 0. 00 2. 82 2. 84 7. 21 5. 98 9. 30 14 .1 6 20 .4 1 34 .7 8 41 .6 7 0. 00 4. 20 0. 00 0. 00 0. 85 6. 61 3. 17 2. 08 0. 00 6. 25 0. 00 12 .5 0 0. 00 0. 00 0. 00 0. 00 0. 00 3. 00 4. 00 8. 00 2. 00 a m ph ib ia 24 .1 4 9. 09 25 .0 0 11 .9 7 12 .7 7 0. 90 0. 85 0. 00 0. 00 0. 00 0. 00 0. 00 7. 10 4. 20 0. 00 0. 00 0. 85 5. 79 3. 17 0. 00 0. 00 0. 00 3. 57 0. 00 0. 00 3. 00 7. 00 16 .0 0 19 .0 0 15 .0 0 75 .0 0 56 .0 0 52 .0 0 pi sc es 17 .2 4 45 .4 5 0. 00 9. 86 16 .3 1 37 .8 4 45 .3 0 43 .4 1 42 .4 8 44 .9 0 13 .0 4 33 .3 3 10 .7 0 12 .5 0 80 .0 0 16 .9 0 5. 98 25 .6 2 31 .7 5 31 .2 5 5. 26 12 .5 0 25 .0 0 0. 00 11 .1 1 10 .0 0 31 .0 0 34 .0 0 21 .0 0 13 .0 0 8. 00 3. 00 0. 00 m ol lu sc a 13 .7 9 9. 09 0. 00 7. 04 17 .7 3 22 .5 2 26 .5 0 17 .8 3 20 .3 5 12 .2 4 8. 70 0. 00 0. 00 0. 00 0. 00 2. 82 5. 13 20 .6 6 41 .2 7 70 .8 3 0. 00 6. 25 3. 57 0. 00 5. 56 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 c ru st ac ea 10 .3 4 18 .1 8 0. 00 23 .9 4 28 .3 7 10 .8 1 7. 69 3. 10 5. 31 2. 04 4. 35 0. 00 0. 00 8. 30 0. 00 0. 00 7. 69 18 .1 8 14 .2 9 20 .8 3 31 .5 8 68 .7 5 89 .2 9 87 .5 0 94 .4 4 0. 00 5. 00 10 .0 0 14 .0 0 5. 00 8. 00 6. 00 10 .0 0 a ra ne id a 13 .7 9 4. 55 16 .6 7 13 .3 8 2. 13 0. 90 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 57 .1 0 41 .7 0 10 .0 0 29 .5 8 26 .5 0 6. 61 1. 59 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 48 .0 0 45 .0 0 31 .0 0 40 .0 0 41 .0 0 8. 00 11 .0 0 12 .0 0 in se ct a 96 .5 5 77 .2 7 91 .6 7 82 .3 9 58 .1 6 26 .1 3 10 .2 6 2. 33 0. 88 0. 00 0. 00 0. 00 57 .1 0 45 .8 0 20 .0 0 84 .5 1 91 .4 5 68 .6 0 34 .9 2 12 .5 0 63 .1 6 75 .0 0 14 .2 9 18 .7 5 0. 00 66 .0 0 66 .0 0 59 .0 0 42 .0 0 33 .0 0 29 .0 0 11 .0 0 12 .0 0 g as tr ol ith s 76 .6 7 92 .0 0 0. 00 50 .0 0 67 .2 4 82 .2 9 89 .6 9 10 0. 00 10 0. 00 10 0. 00 10 0. 00 10 0. 00 3. 60 20 .8 0 50 .0 0 11 .2 7 5. 98 18 .1 8 17 .4 6 14 .5 8 0. 00 0. 00 28 .5 7 31 .2 5 16 .6 7 59 .0 0 89 .0 0 82 .0 0 91 .0 0 87 .0 0 96 .0 0 94 .0 0 91 .0 0 sa m pl e # 30 25 12 14 2 14 1 11 1 11 7 12 9 11 3 49 23 12 15 1 82 53 71 11 7 12 1 63 48 19 16 28 16 18 29 62 49 43 39 24 36 42 sv l (c m ) 17 .0 -3 8. 9 39 -6 6. 3 66 .4 -1 15 .8 <1 5 15 .1 -4 0 40 .1 -6 5 65 /1 -9 0 >9 0 10 -1 9. 9 20 -2 9. 9 30 -3 9. 9 40 -4 9. 9 50 -5 9. 9 60 -6 9. 9 70 -7 9. 9 >8 0 b od y le ng th (c m ) <1 00 10 019 9 30 -5 0 50 -1 00 10 015 0 15 020 0 20 025 0 25 030 0 30 035 0 35 040 0 40 045 0 45 050 0 <3 0 30 .1 -5 0 50 .1 -1 00 10 0. 115 0 >1 50 sp ec ie s c ro co dy lu s ni lo tic us c ro co dy lu s ni lo tic us c ro co dy lu s ni lo tic us c ro co dy lu s ni lo tic us c ro co dy lu s ni lo tic us c ro co dy lu s ni lo tic us c ro co dy lu s ni lo tic us c ro co dy lu s ni lo tic us c ro co dy lu s ni lo tic us c ro co dy lu s ni lo tic us c ro co dy lu s ni lo tic us c ro co dy lu s ni lo tic us c ro co dy lu s ni lo tic us c ro co dy lu s ni lo tic us c ro co dy lu s ni lo tic us c ro co dy lu s m or el et ii c ro co dy lu s m or el et ii c ro co dy lu s m or el et ii c ro co dy lu s m or el et ii c ro co dy lu s m or el et ii c ro co dy lu s ac ut us c ro co dy lu s ac ut us c ro co dy lu s ac ut us c ro co dy lu s ac ut us c ro co dy lu s ac ut us c ro co dy lu s jo hn so ni c ro co dy lu s jo hn so ni c ro co dy lu s jo hn so ni c ro co dy lu s jo hn so ni c ro co dy lu s jo hn so ni c ro co dy lu s jo hn so ni c ro co dy lu s jo hn so ni c ro co dy lu s jo hn so ni ta bl e 2. s to m ac h co nt en ts o f c ro co dy lia ns c om pi le d. 117stomach histology of crocodylus siamensis and gavialis gangeticus occurrence frequency with dietary types (table 3) further support their digestive function. the positive correlations with vertebrate diets, although supported statistically only in mammals, may suggest that gastroliths are possibly beneficial for digesting bones. although the gastroliths might have not served as “teeth” to strongly grind ingesta as they do in herbivorous birds (moore, 1998; moore, 1999), they might have benefited digestion through ingesta mixing and facilitating stomach juice excretion (wings, 2007). these functions do not contradict with other possible gastroliths functions such as buoyancy control (taylor, 1993). therefore, the results of this study suggest a possibility that although crocodylian “gizzard” is not homologous with that of neornithines, their “gizzard” efficiently utilized gastroliths for digestion. acknowledgements we deeply appreciate dr. yasuhiro kon and dr. osamu ichii for providing the facility and instructing the histological methods. we acknowledge dr. masaya iijima, junki yoshida, and tomonori tanaka for their comments on earlier versions of the manuscript. references ahmed, y., el-hafez, a., zayed, a. 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(1972): digestion and the digestive system. avian biol. 2: 343-430. acta herpetologica vol. 15, n. 2 december 2020 firenze university press estimating abundance and habitat suitability in a micro-endemic snake: the walser viper gentile francesco ficetola1,2,*, mauro fanelli3, lorenzo garizio3, mattia falaschi1, simone tenan4, samuele ghielmi5, lorenzo laddaga6, michele menegon7,8, massimo delfino3,9. potential effects of climate change on the distribution of invasive bullfrogs lithobates catesbeianus in china li qing peng1, min tang1, jia hong liao1, hai fen qing1, zhen kun zhao1, david a. pike2, wei chen1,* a bibliometric-mapping approach to identifying patterns and trends in amphibian decline research claudio angelini1,*, jon bielby2, corrado costa3 food composition of a breeding population the endemic anatolia newt, neurergus strauchii (steindachner, 1887) (caudata: salamandridae), from bingöl, eastern turkey kerim çiçek1,*, mustafa koyun2, ahmet mermer1, cemal varol tok3 stomach histology of crocodylus siamensis and gavialis gangeticus reveals analogy of archosaur “gizzards”, with implication on crocodylian gastroliths function ryuji takasaki1,2,*, yoshitsugu kobayashi3 does chronic exposure to ammonium during the pre-metamorphic stages promote hindlimb abnormality in anuran metamorphs? a comparison between natural-habitat and agrosystem frogs sonia zambrano-fernández1, francisco javier zamora-camacho2,3,*, pedro aragón2,4 confirming lessona’s brown frogs distribution sketch: rana temporaria is present on turin hills (piedmont, nw italy) davide marino1, angelica crottini2, franco andreone3,* phylogenetic relationships of the italian populations of horseshoe whip snake hemorrhois hippocrepis (serpentes, colubridae) francesco paolo faraone1, raffaella melfi2, matteo riccardo di nicola3, gabriele giacalone4, mario lo valvo5* first karyological analysis of the endemic malagasy phantom gecko matoatoa brevipes (squamata: gekkonidae) marcello mezzasalma1,2,*, fabio m. guarino3, simon p. loader1, gaetano odierna3, jeffrey w. streicher1, natalie cooper1 notes on sexual dimorphism, diet and reproduction of the false coral snake oxyrhopus rhombifer duméril, bibron & duméril, 1854 (dipsadidae: pseudoboini) from coastal plains of subtropical brazil fernando m. quintela1,*, felipe caseiro¹, daniel loebmann¹ acta herpetologica 14(2): 123-128, 2019 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/a_h-7750 hematological parameters of the bolson tortoise gopherus flavomarginatus in mexico cristina garcía-de la peña1,*, roger iván rodríguez-vivas2, jorge a. zegbe-domínguez3, luis manuel valenzuela-núñez1, césar a. meza herrera4, quetzaly siller-rodríguez1, verónica ávila-rodríguez1 1 facultad de ciencias biológicas, universidad juárez del estado de durango, gómez palacio, durango, méxico. c.p. 35010. *corresponding author. email: cristina.g.delapena@gmail.com 2 facultad de medicina veterinaria y zootecnia, universidad autónoma de yucatán, mérida, yucatán, méxico, c.p. 97100 3 instituto nacional de investigaciones forestales, agrícolas y pecuarias, campo experimental calera, zacatecas, méxico, c.p. 98500 4 universidad autónoma chapingo-uruza, bermejillo, durango, méxico, c.p. 35230 submitted on: 2018, 21th june; revised on: 2019, 10th may; accepted on: 2019, 23rd august editor: emilio sperone abstract. we present findings of our preliminary study to determine biometry and blood chemistry values of healthy wild individuals of the critically endangered bolson tortoises (gopherus flavomarginatus) in mexico. given the absence of previously published data regarding hematology parameters for this species, these results represent an important base for additional research. hematocrit determination, stains, and cell counts were performed, as well as 18 parameters of blood chemistry. values of biometry and blood chemistry for g. flavomarginatus were similar to reference values those already reported for g. agassizii, g. polyphemus, and g. berlandieri. these similarities reflect the phylogenetic relationships among these species. however, slight differences may point to particular adaptations that each has developed to their own habitat, and so point to questions to be addressed with future research. keywords. chelonia, lymphocyte, desert, mapimí biosphere reserve. hematological analysis is a common technique for evaluating the health status of wild animals (campbell, 2004; tavares-dias et al., 2009). blood biometry and chemistry of terrestrial, semiaquatic and marine tortoises have been described already (stacy et al. 2011; campbell, 2015). however, there are still species of ecological importance whose blood information is unavailable. this is the case of gopherus flavomarginatus, a tortoise endemic to mexico and considered the largest terrestrial tortoise in north america, with a carapace length of up to 40 cm (morafka et al., 1989). this species is in danger of extinction according to the official mexican standard 059 (semarnat, 2010) and critically endangered according to the iucn red list (kiester et al., 2018). its geographical distribution is restricted to the mapimí bolson in the mexican chihuahuan desert, where it currently has protected status within the mapimí biosphere reserve (conanp, 2006). tortoises of the genus gopherus are keystone organisms for the ecosystems in which they live because, due to their feeding habits (herbivory), they perform the ecological function of seed dispersal (carlson et al., 2003), and because the burrows that they excavate are deep and provide shelter for at least 300 species of invertebrates and 60 of vertebrates (lips, 1991). due to the importance of protecting this tortoise, we conducted this preliminary study to determine biometry and blood chemistry values of healthy wild individuals. this information will serve as a basis for future hematological studies carried out on this tortoise. from may 2015 to september 2017, we captured 44 adult individuals of g. flavomarginatus (16 males and 28 females) within the mapimí biosphere reserve in mex124 cristina garcía-de la peña et alii ico (26°00’ and 26°10’n, and 104°10’ and 103°20’w). blood samples were collected from the subvertebral vein. three milliliters of blood were collected from each specimen; one milliliter was placed in a vacutainer® tube with lithium heparin as anticoagulant and the rest in a red vacutainer® tube. the tubes were stored in a cooler at a temperature of approximately 4 °c. each tortoise was determined to be healthy following the observation protocols of jacobson (2014) and usfws (2016). tortoises were then released at the site of their capture. biometric analysis of blood was performed following the protocols suggested by thrall et al. (2006) and turgeon (2012). the volume percentage of red cells (hematocrit, ht in%) was determined in the blood contained in the green vacutainer® tube, using the microhematocrit technique. the total number (tr) and percentage (pr) of red cells were obtained for each blood sample. the formula used to obtain hematocrit values was ht% = (pr/tr) × 100. hemoglobin concentration was quantified with the drabkin colorimetric method, using the spinreact® commercial kit, in a vettest® spectrophotometer. the reaction product was centrifuged (12,000 g × 5 min) to precipitate the nucleus of the cells and keep them from distorting the color to be measured (thrall et al., 2006). erythrocyte and leukocyte counts were carried out using a thoma pipette with red bead and a neubauer hemocytometer with natt and herrick’s stain (thrall et al., 2006). the erythrocyte count was carried out under 10× magnification, on both sides of the neubauer chamber, in the four corner squares and the center square within the large center square of the chamber. the following formula was applied (kemal, 2014): erythrocytes (×106 ul) = mean erythrocytes × 10000. the leukocyte count was carried out under 40x magnification in the nine large squares on both sides of the neubauer chamber. the following formula was applied: leukocytes (×103 ul) = mean leukocytes × 50 (kemal, 2014). the erythrocyte indices were calculated according to the formulas described by ball (2014) and kemal (2014): mean corpuscular volume, mcv (fl) = ht% × 10/erythrocytes (×106 ul); mean corpuscular hemoglobin, mch (pg) = (hg × 10)/erythrocytes (×106 ul); mean corpuscular hemoglobin concentration, mchc (g/dl) = (hg × 100)/ht%. two smears were prepared from each sample on glass slides using wright’s dye (analytyka®). one hundred leukocytes were counted in the body of the smear of each of the two slides. the average of both slides was obtained, and the results were expressed as the proportion of each cell type (relative differential count). to obtain the absolute differential count, the total value of leukocytes (×103 cel/ul) was multiplied by the average percentage of each type of leukocyte and the result was divided by 100 (thrall et al., 2006). the h:l ratio was obtained by dividing the percentage of heterophiles by the percentage of lymphocytes in each sample (davis et al., 2008). blood from the red vacutainer® tube was centrifuged at 12,000 g for five minutes; the serum was separated from the red cells and placed in a sterile plastic tube. no hemolysis was observed in any sample. the following parameters were analyzed: glucose, uric acid, urea, blood urea nitrogen (bun), creatinine, total proteins, albumin, globulins, cholesterol, triglycerides, alanine aminotransferase (alt), aspartate aminotransferase (ast), alkaline phosphatase (ap), chlorine, sodium, calcium, phosphorus and osmolality (mosm/kg=1.86 (na [mmol/l]) + glucose [mg/dl]/18 + bun [mg/dl]/2.8 + 9). human serum spintrol h spinreact® was used as control. these analyzes were performed in a vettest® spectrophotometer with spinreact® reagents. descriptive statistics (mean, standard error, median, standard deviation, minimum, and maximum) were obtained in past 3.14 (hammer et al., 2001). blood biometry results for g. flavomarginatus are shown in table 1 and for blood chemistry in table 2. a comparison among biometry and chemistry variables of g. flavomarginatus and other gopherus species are shown in tables 3 and 4, respectively. blood biometric and chemistry values obtained for g. flavomarginatus were similar to those reported for g. agassizii (christopher et al., 1999; dickinson et al., 2000), g. polyphemus (taylor and jacobson, 1982), and g. berlandieri (teare, 2013a) (tables 3 and 4). christopher et al. (1999) reported that the most abundant leukocytes in g. agassizii are heterophils, followed by lymphocytes, basophils, and finally eosinophils and monocytes. in our findings for g. flavomarginatus, the order of abundance of the leukocyte cells was similar to the one mentioned by duguy (1970), with lymphocytes as the most abundant leukocytes (males = 53.43%, 4.79 × 103 cel/ul; females = 44.03%, 4.20 × 103 cel/ul). this agrees with the findings of diazfigueroa (2005) for g. polyphemus, and other authors who indicate that lymphocytes are predominant in the peripheral blood of most reptile species (davis et al.2008; stacy et al., 2011). heterophils were the second most abundant leukocyte cells in g. flavomarginatus. in general, the abundance of this type of leukocyte in reptiles ranges from 30 to 45% (duguy, 1970; frye, 1991); however, in tortoises it can reach up to 50% (alleman et al., 1992; christopher et al. 1999). basophils were the third most abundant leukocytes in the blood of the bolson tortoise. alleman et al., (1992), and duguy, (1970) estimated that the percentage of basophils in healthy terrestrial and aquatic tortoises can be higher than 40%. in g. flavomargi125hematological parameters of gopherus flavomarginatus natus, the average was 17.68% for males and 21.42% for females, with wide variation between individuals (min = 6%, max = 46%). eosinophilic leukocytes were the fourth most abundant leukocytes. the seasons and type of diet can influence the amount of eosinophilic leukocytes in some species (duguy, 1970; deem et al., 2006). according to frye (1991), the percentage of eosinophils in healthy reptiles ranges from 7 to 20%; in the case of table 1. descriptive statistics of blood biometric variables from male/female gopherus flavomarginatus. se = standard error, sd = standard deviation, min = minimum, max = maximum. variable mean se median sd min max hematocit (%) 25.35/23.48 1.41/0.77 25.30/23.61 5.64/4.09 16.47/17.89 35.62/33.33 hemoglobin (g/dl) 6.53/6.59 0.40/0.26 6.56/7.15 1.62/1.41 4.05/3.89 9.67/8.95 erythrocytes (×106 cel/ul) 0.54/0.57 0.06/0.03 0.47/0.58 0.25/0.19 0.22/0.32 0.97/0.98 mcv (fl) 539.02/448.98 54.81/27.17 458.56/401.53 219.26/143.78 264.56/196.17 921.72/717.91 mch (pg) 144.12/122.92 18.96/5.79 124.11/122.53 75.86/30.67 64.66/45.95 292.86/197.78 mchc (g/dl) 26.37/28.49 1.64/1.23 25.62/28.84 6.56/6.52 17.69/16.20 39.36/44.66 leukocytes (×103 cel/ul) 8.88/9.50 0.82/0.70 8.55/8.55 3.28/3.73 5.00/2.44 17.67/21.33 heterophils (%) 25.43/29.32 1.99/1.68 26.00/29.00 7.99/8.89 14.00/13.00 39.00/45.00 eosinophils (%) 2.75/3.17 0.57/0.51 2.00/2.50 2.29/2.74 0.00/0.00 7.00/11.00 basophils (%) 17.68/21.42 2.11/1.67 15.50/18.50 8.47/8.86 6.00/8.00 29.00/46.00 lymphocytes (%) 53.43/44.03 2.99/2.06 54.00/42.50 11.99/10.92 34.00/27.00 73.00/70.00 monocytes (%) 0.68/2.03 0.17/0.40 1.00/1.00 0.70/2.16 0.00/0.00 2.00/9.00 h:l ratio 0.52/0.73 0.06/0.06 0.45/0.71 0.27/0.36 0.22/0.24 1.11/1.59 heterophils (×103 cel/ul) 2.17/2.81 0.19/0.29 2.09/2.56 0.77/1.55 0.93/0.66 3.45/7.25 eosinophils (×103 cel/ul) 0.21/0.34 0.04/0.08 0.18/0.21 0.17/0.45 0.00/0.00 0.66/2.35 basophils (×103 cel/ul) 1.61/1.95 0.28/0.16 1.45/1.80 1.14/0.85 0.30/0.39 5.12/4.16 lymphocytes (×103 cel/ul) 4.79/4.20 0.57/0.36 3.96/4.04 2.31/1.95 2.57/0.78 10.32/8.96 monocytes (×103 cel/ul) 0.07/0.18 0.02/0.03 0.06/0.09 0.09/0.20 0.00/0.00 0.35/0.85 table 2. descriptive statistics of blood chemistry variables from male/female gopherus flavomarginatus. se = standard error, sd = standard deviation, min = minimum, max = maximum. variable mean se median sd min max glucose (mg/dl) 79.19/58.34 7.97/8.28 68.33/40.47 31.90/43.82 43.32/13.34 134.67/165.30 uric acid (mg/dl) 5.63/5.58 1.07/0.71 5.66/5.96 4.28/3.76 0.27/0.24 12.45/13.48 urea (mg/dl) 25.60/31.22 0.82/0.76 25.39/31.75 3.30/7.86 20.64/14.67 32.86/51.34 bun 11.96/14.37 0.38/0.82 11.86/14.22 1.54/4.35 9.64/6.86 15.36/23.99 creatinine (mg/dl) 0.48/0.43 0.06/0.05 0.47/0.40 0.24/0.26 0.01/0.01 0.95/0.89 total protein (g/dl) 4.12/3.95 0.53/0.24 3.76/3.78 2.13/1.27 1.29/2.00 8.96/7.13 albumin (g/dl) 0.87/0.68 0.18/0.10 0.56/0.47 0.74/0.56 0.09/0.04 2.50/2.55 globulins (g/dl) 3.24/3.27 0.42/0.24 3.23/3.09 1.70/1.30 0.99/1.37 6.46/6.50 cholesterol (mg/dl) 280.55/214.94 45.28/23.50 269.50/167.43 181.13/124.36 41.46/78.32 692.27/508.20 triglycerides (mg/dl) 207.85/177.57 13.79/16.18 213.80/164.06 55.18/85.62 114.08/42.35 283.63/402.30 alt (ui/i) 7.74/8.15 1.06/1.06 7.53/6.48 4.25/5.65 2.04/1.74 15.93/22.43 ast (ui/i) 41.79/45.23 6.30/3.53 32.27/47.92 25.21/18.69 14.95/15.63 105.60/80.42 ap (ui/i) 60.30/66.53 8.08/4.58 56.91/70.05 32.32/24.24 14.11/24.86 102.60/99.32 chlorine (mmol/l) 119.90/121.64 4.24/2.98 118.90/122.31 16.97/15.81 95.68/94.53 139.62/149.14 sodium (mmol/l) 137.53/138.24 3.20/2.47 136.24/136.42 12.80/13.11 112.36/117.90 160.90/177.60 calcium (mg/dl) 11.61/13.44 0.65/0.41 12.64/13.29 2.63/2.19 7.46/10.27 15.67/18.30 phosphorus (mg/dl) 3.95/4.08 0.56/0.36 3.59/4.29 2.24/1.93 1.02/1.31 7.18/7.16 osmolality (mosm/kg) 270.50/273.87 5.92/4.73 268.60/272.56 23.70/23.85 225.43/240.81 316.39/345.66 126 cristina garcía-de la peña et alii table 3. comparison among minimum-maximum blood biometry values reported for gopherus flavomarginatus and reference values of other three species of the genus. variable g. flavomarginatus1 g.agassizii2 g. polyphemus3,4 g. berlandieri5 hematocit (%) 16.47-35.62 19.50-37.10 15.00-30.00 12.00-44.80 hemoglobin (g/dl) 3.89-9.67 4.10-9.90 4.20-8.60 erythrocytes (×106 cel/ul) 0.22-0.98 0.36-1.08 0.24-0.91 mcv (fl) 196.17-921.72 254.00-638.00 200.10-838.60 mch (pg) 45.95-292.86 74.00-186.00 mchc (g/dl) 16.20-44.66 20.00-33.00 leukocytes (×103 cel/ul) 2.44-21.33 1.49-10.92 10.00-22.00 0.00-80.650 heterophils (%) 13.00-45.00 10.00-57.00 eosinophils (%) 0.00-11.00 basophils (%) 6.00-46.00 2.00-11.00 lymphocytes (%) 27.00-73.00 32.00-79.00 monocytes (%) 0.00-9.00 3.00-13.00 h:l ratio males 0.22-1.59 heterophils (×103 cel/ul) 0.66-7.25 0.71-7.15 0.00-6.59 0.00-5.21 eosinophils (×103 cel/ul) 0.00-2.35 0.00-0.95 basophils (×103 cel/ul) 0.30-5.12 0.06-3.57 0.02-0.92 0.00-1.31 lymphocytes (×103 cel/ul) 0.78-10.32 0.63-2.74 0-4.15 0-3.12 monocytes (×103 cel/ul) 0.00-0.85 0.00-0.32 0.00-0.44 1 present study. 2 christopher et al. (1999) in summer season. 3 taylor and jacobson (1982). 4 teare (2013b). 5 teare (2013a). table 4. comparison among minimum-maximum blood chemistry values reported for gopherus flavomarginatus and reference values of other three species of the genus. variable g. flavomarginatus1 g.agassizii2 g. polyphemus3,4 g. berlandieri5 glucose (mg/dl) 13.34-165.30 65.00-186.00 55.00-128.00 9.00-157.00 uric acid (mg/dl) 0.24-13.48 1.70-9.20 0.90-8.50 0.00-8.60 urea (mg/dl) 14.67-51.34 1.00-130.00 bun 6.86-23.99 1.00-37.00 2.00-29.00 0.00-13.00 creatinine (mg/dl) 0.01-0.95 0.20-0.40 0.10-0.40 total protein (g/dl) 1.29-8.96 2.30-5.30 1.30-4.60 1.20-7.70 albumin (g/dl) 0.04-2.55 0.80-1.90 0.50-2.60 0.50-2.70 globulins (g/dl) 0.99-6.50 1.30-3.90 0.50-4.60 0.60-5.10 cholesterol (mg/dl) 41.46-692.27 33.00-381.00 19.00-150.00 triglycerides (mg/dl) 42.35-402.30 7.00-603.00 alt (ui/i) 1.74-22.43 1.00-5.00 2.00-57.00 ast (ui/i) 14.95-105.60 15.00-123.00 57.00-392.00 0.00-265.00 ap (ui/i) 14.11-102.60 25.00-114.00 11.00-71.00 chlorine (mmol/l) 94.53-149.14 101.00-138.00 35.00-128.00 89.00-122.00 sodium (mmol/l) 112.36-177.60 127.00-176.00 127.00-148.00 123.00-153.00 calcium (mg/dl) 7.46-18.30 8.60-23.90 10.00-14.00 5.00-17.40 phosphorus (mg/dl) 1.02-7.18 1.10-6.50 1.00-3.10 0.70-4.90 osmolality (mosm/kg) 225.43-345.66 252.00-352.00 1present study. 2christopher et al. (1999) in summer season. 3taylor and jacobson (1982). 4teare (2013b). 5teare (2013a). 127hematological parameters of gopherus flavomarginatus g. flavomarginatus, the average percentage found in the present study was 2.75% for males and 3.17% for females, with a total variability of 0-11%. the average percentage of monocytes in the bolson tortoise was 0.68% for males and 2.03% for females (min = 0, max = 9); these values are within the range of abundance reported for reptiles (duguy, 1970). the h:l ratio (heterophils/leukocytes) has been used as a reliable method to evaluate the exposure of vertebrates to chronic stress due to the relationship between the leukocyte profile and the adrenal response (production of cortisol). when the level of cortisol rises, the number of circulating heterophils increases, while the number of lymphocytes decreases (davis et al., 2011). davis (2009) indicated that blood biometry studies conducted with terrestrial tortoises in which the possibility of individuals being stressed was not considered (including studies with g. agassizii, g. berlandieri, and g. polyphemus), the h:l ratio was less than 2.0 (mean of 0.65, sd=0.34), which coincides with the values obtained in the present study for g. flavomarginatus. blood chemistry variables provide important information for establishing levels of hydration (bun, uric acid, osmolality), nutrition (glucose, total protein, albumin, colesterol, phosphorus), and metabolic activity (alanine aminotransferas, aspartate aminotransferase, and alkaline phosphatase activities) of desert tortoises (christopher et al., 1999). christopher et al. (1999) showed that blood chemistry values in g. agassizii change due to the effect of physiological (reproductive cycle, hibernation) and environmental factors (precipitation patterns, availability of water and food). however, we observed similar blood values between g. agassizii and g. flavomarginatus at the same times of year (summer). in general, the phylogenetic relation among g. flavomarginatus, g. agassizii, g. polyphemus, and g. berlandieri (reynoso and montellanoballesteros, 2004) may be reflecting the similarity between reported hematology values for these species, and the slight differences may point to particular adaptation conditions that each one has developed in their own habitat. our small sample size (n = 44) precluded the calculation of formal reference intervals, a process that would require a minimum of 120 individuals (geffre, et al., 2009). however, given the limited sample size available, and the lack of previously published data regarding hematology parameters in g. flavomarginatus, these results provide a useful starting point for researchers. acknowledgments to fondo sectorial de investigación para la educación sep-conacyt ciencia básica (220658) for funding this study. to magdalena rivas-garcía for her help in the field work. cristino villarreal-wislar and the mapimí biosphere reserve personnel for logistical support during the realization of this study. to cameron w. barrows (university of california, riverside) for the review of this manuscript. tortoise samples were collected under the dgvs 07249/15-16-17 permit granted by semarnat, mexico. references alleman, a.r., jacobs, e.r., raskin, r.e. 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(2012): clinical hematology: theory & procedures. 5th ed. wolters kluwer, lippincott williams & wilkins, philadelphia, usa. usfws (2016): health assessment procedures for the desert tortoise (gopherus agassizii): a handbook pertinent to translocation. u.s. fish and wildlife service, desert tortoise recovery office, reno, nevada, usa. acta herpetologica vol. 14, n. 2 december 2019 firenze university press podarcis siculus latastei (bedriaga, 1879) of the western pontine islands (italy) raised to the species rank, and a brief taxonomic overview of podarcis lizards gabriele senczuk1,2,*, riccardo castiglia2, wolfgang böhme3, claudia corti1 substrate type has a limited impact on the sprint performance of a mediterranean lizard pantelis savvides1,*, eleni georgiou1, panayiotis pafilis2,3, spyros sfenthourakis1 coping with aliens: how a native gecko manages to persist on mediterranean islands despite the black rat? michel-jean delaugerre1,*, roberto sacchi2, marta biaggini3, pietro lo cascio4, ridha ouni5, claudia corti 3 pit-tags as a technique for marking fossorial reptiles: insights from a long-term field study of the amphisbaenian trogonophis wiegmanni pablo recio, gonzalo rodríguez-ruiz, jesús ortega, josé martín* occurrence of batrachochytrium dendrobatidis in the tensift region, with comments on its spreading in morocco ait el cadi redouane1, laghzaoui el-mustapha1, angelica crottini2, slimani tahar1, bosch jaime3,4, el mouden el hassan1,* hematological parameters of the bolson tortoise gopherus flavomarginatus in mexico cristina garcía-de la peña1,*, roger iván rodríguez-vivas2, jorge a. zegbe-domínguez3, luis manuel valenzuela-núñez1, césar a. meza herrera4, quetzaly siller-rodríguez1, verónica ávila-rodríguez1 ontogenetic and interspecific variation in skull morphology of two closely related species of toad, bufo bufo and b. spinosus (anura: bufonidae) giovanni sanna visible implant alphanumeric (via) as a marking method in the lesser snouted treefrog scinax nasicus andrea caballero-gini1,2,3,*, diego bueno villafañe2,3, lía romero2, marcela ferreira2,3, lucas cañete4, rafaela laino2, karim musalem2,5 morphological variation of the newly confirmed population of the javelin sand boa, eryx jaculus (linnaeus, 1758) (serpentes, erycidae) in sicily, italy francesco p. faraone1,*, salvatore russotto2, salvatore a. barra3, roberto chiara3, gabriele giacalone4, mario lo valvo3 variability in the dorsal pattern of the sardinian grass snake (natrix natrix cetti) with notes on its ecology enrico lunghi1,2,3,4,*, simone giachello5, manuela mulargia6, pier paolo dore7, roberto cogoni8, claudia corti1 estimating abundance of the stripeless tree-frog hyla meridionalis by means of replicated call counts federico crovetto, sebastiano salvidio, andrea costa* at-rich microsatellite loci development for fejervarya multistriata by illumina hiseq sequencing yan-mei wang, jing-yi chen, guo-hua ding*, zhi-hua lin acta herpetologica 15(2): 129-135, 2020 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/a_h-9058 phylogenetic relationships of the italian populations of horseshoe whip snake hemorrhois hippocrepis (serpentes, colubridae) francesco paolo faraone1, raffaella melfi2, matteo riccardo di nicola3, gabriele giacalone4, mario lo valvo5* 1 viale regione siciliana s.e., 532, 90129 palermo, italy 2 dipartimento di scienze e tecnologie biologiche, chimiche e farmaceutiche (stebicef), university of palermo, viale delle scienze ed. 16-17, 90128 palermo, italy 3 via bobbio, 20144 milano, italy 4 cooperativa silene, via d’ondes reggio, 8/a, 90127 palermo, italy 5 dipartimento scienze e tecnologie biologiche, chimiche e farmaceutiche, university of palermo, via archirafi, 18, 90123 palermo, italy *corresponding author. e-mail: mario.lovalvo@unipa.it submitted on: 2020, 20th june; revised on: 2020, 5th september; accepted on: 2020, 1st october editor: emilio sperone abstract. hemorrhois hippocrepis is a colubrid snake with a west mediterranean distribution. it is widespread in the iberian peninsula and northwest africa. the only italian populations are found on the islands of sardinia and pantelleria. the phylogenetic relationships of these insular populations have been analysed for the first time on the basis of the mitochondrial dna cytochrome b gene. the sequences were compared with those available from the geographic range of this species. the analyses showed that the italian samples are part of a lineage that groups tunisian and east algerian samples, with which they share the same haplotype. these results strongly support the hypothesis of a recent origin of the italian populations of hemorrhois hippocrepis, probably determined by human-mediated dispersal from north africa. keywords. horseshoe whip snake, phylogeny, colubridae, cytochrome b. the horseshoe whip snake, hemorrhois hippocrepis linnaeus, 1758, is a medium to large size colubrid with a western mediterranean distribution. it is widespread across the eastern, central and southern iberian peninsula, and in the northern mesic parts of morocco, algeria, and tunisia. in italy, its presence is limited to the islands of sardinia and pantelleria (sindaco et al., 2013). the first reports of h. hippocrepis in sardinia are found in gené (1834, 1839) and refer to the southwestern part of the island. later, mertens and wermuth (1960) considered the species extinct, but subsequently, bruno and hotz (1976) rediscovered it in a territory between cagliari and oristano, and its presence there was also confirmed by puddu et al. (1988) and bruno and maugeri, (1990). hemorrhois hippocrepis in the san pietro satellite islet was reported by stefani (1971) and poggesi et al. (1995) and is still mentioned in recent references (corti et al., 2006; di nicola and mezzadri, 2018), although its presence is doubtful given the absence of corroborating observations (corti et al., 2000). more recently, h. hippocrepis appears to be present with certainty only a few localities of the cagliari province (corti et al., 2000; zuffi, 2006; di nicola and mezzadri, 2018; di nicola et al., 2019). the horseshoe whip snake has been known to inhabit the island of pantelleria (sicily) since the late 130 francesco paolo faraone et alii 1800s (doderlein, 1881; camerano, 1891; minà palumbo, 1893) and it is widespread throughout the island (cattaneo, 1985; authors’ unpublished data). the population of pantelleria has been attributed on a morphological basis to the endemic subspecies h. h. nigrescens (cattaneo, 1985), due to the melanotic color morph of adults, the large dimensions of both hatchlings and adults, and to certain distinctive pholidotic features. later, corti et al. (2000), in comparing various populations of h. hippocrepis, found no substantial morphological differences between the populations of pantelleria, sardinia and tunisia. according to some authors, the italian populations of horseshoe whip snake may have been introduced by man in historical times (bruno and hotz, 1976; zuffi, 2006; cattaneo, 2015). therefore, the ssp. nigrescens is currently considered doubtful, and h. hippocrepis is widely reported as a monotypic species (venchi and sindaco, 2006; luiselli et al., 2011; di nicola, 2019; di nicola et al., 2019), as indicated by the genetic studies of carranza et al. (2006), which however did not include samples from italian populations. in this work, we have analyzed for the first time the italian populations of h. hippocrepis from a genetic point of view, with the aim of clarifying their phylogenetic relationships and testing if their origin can be related to human mediated dispersal as we expect. in order to infer the phylogenetic relationships between the italian samples and those of the other populations, we analysed the mitochondrial dna cytochrome b (cyt b) sequence, a fragment often used for snakes in this type of analysis (carranza et al., 2004, 2006; faraone et al., 2020). tissue samples ranging from 2 to 20 mg were collected from six specimens originating from pantelleria and one from sardinia, and stored in ethanol until processing. all snakes were found as roadkills between 2013 and 2019. dna was extracted as described in tagliavia et al. (2016). 3 ml of crude lysate were used as template for pcr amplification. reactions were carried out in 30 ml in presence of 200 mm dntps and 0.15 mm primers cb1f and cb2r (respectively: 5’-ccatccaacatctcagcatgatgaaa-3’ and 5’-ccctcagaatgatatttgtcctca-3’) (korcher et al., 1989). the pcr conditions were 94 °c for 3 min, followed by 40 cycles at 94 °c for 30 s, 55 °c for 1 min, 72 °c for 1 min and by 5 min of final extension at 72 °c. pcr products were analysed by electrophoresis onto 1% agarose gel containing 0.5 μg/mlethidium bromide and sequenced with primer cb1f (bmr genomics). nucleotide sequences, each of about 320 nucleotides, were analysed and manually proofread with the software chromas v. 2.6.6 (technelysium pty. ltd. 1998, queensland, australia). coding gene fragments of cyt b were translated into amino acids to assess the lack of stop codons. cyt b sequences of h. hippocrepis were searched and downloaded from genbank (nagy et al., 2004; carranza et al., 2006; beddek et al., 2018) from the native geographic range of the species (figs 1 and 2). additionally, cyt b sequences of lytorhynchus diadema, hierophis viridiflavus, hemorrhois algirus, coronella girondica, psammophis shokari, rhagerhis moilensis and malpolon monspessulanus were downloaded and used as outgroup. nucleotide sequences were aligned using clustalw with default parameters. phylogenetic analysis was performed with the maximum likelihood (ml) method and the akaike information criterion, using “smart model selection” (sms) (lefort et al., 2017), implemented in phyml v. 3 (guindon et al., 2010). support for nodes was estimated using bootstrapping (felsenstein, 1985) with 1000 replicates. the most appropriate evolutionary model was the gtr+g+i model (-log likelihood value -1397.47), with a 0.30 estimate of invariable sites and a 0.819 discrete approximation of the gamma distribution. including the outgroups, 67 sequences of 294bp total length were analysed. all the italian samples showed the same haplotype (genbank accession numbers mt498647-mt498653), which is also shared with all the tunisian and the easternmost algerian samples. the results obtained confirm the same overall h. hippocrepis phylogenetic structure detected by previous studies on mitochondrial sequences (carranza et al., 2006; beddek et al., 2018), i.e., the existence of a western clade (subclade a) that includes the iberian peninsula, morocco, western algeria and non-native populations of the balearic islands (see silva-rocha et al., 2015), and an eastern clade (subclade b) that includes eastern algeria and tunisia. the western clade has greater haplotype diversity while the eastern clade is characterized by a single haplotype, shared by all the italian samples. the phylogenetic structure obtained also reflects the morphological variation within this species. in fact, the eastern populations (sardinia, pantelleria and tunisia) differ from the western ones (spain, portugal and morocco) in that they have a greater number of ventral scales (cattaneo, 1985; corti et al., 2000). the sharing of the same cyt b haplotype by the tunisian and italian populations of h. hippocrepis strongly supports a recent origin of the latter. this was probably determined by human-mediated fauna translocation, as previously speculated (bruno and hotz, 1976; zuffi, 2006; cattaneo, 2015) and suggested by the biogeographical and historical features of the two islands. although sardinian fossil ophidiofauna is well represented and several findings of ‘colubrinae’ are known, there are no 131genetic identity of hemorrois hippocrepis in italy fig. 1. maximum likelihood (ml) tree of hemorrhois hippocrepis inferred from the mitochondrial cytochrome b gene. the numbers at nodes are ml bootstrap values. except for the samples from pantelleria and sardinia, the remaining cyt b genbank accession numbers are from nagy et al. (2004), carranza et al. (2004, 2006) and beddek et al. (2018). 132 francesco paolo faraone et alii remains clearly attributable to h. hippocrepis (kotsakis, 1980; abbazzi et al., 2004; georgalis et al., 2019). while the only remains of terrestrial vertebrates found in pantelleria, belong to undetermined birds and artiodactyla mammals dating back to the bronze age and linked to human farming and hunting activities (wilkens, 1987). furthermore, h. hippocrepis is not listed in the overall fossil register of the italian reptiles (delfino, 2006, 2011). several current reptile species found in sardinia share close genetic affinities with tunisian populations, and this suggests they are of recent origin. the origin of the sardinian populations of natrix maura are not completely clear, due to its slight variation compared to the tunisian one (guicking et al., 2008). for other species like testudo graeca, chalcides ocellatus, and chalcides chalcides, human-mediated dispersal appears to be the most probable process involved (carranza et al., 2008; fritz et al., 2009; kornilios et al., 2010). as for pantelleria, although some studies support ancient pleistocene contacts with the tunisian mainland (pasa, 1953; bordoni, 1973), and an active colonization of some species has been hypothesized (lanza, 1973; stöck et al., 2016), the onset of catastrophic eruptive events dating back to about 45,000 years ago probably caused the extinction of most of the native fauna (agnesi and federico, 1995; massa, 1995; muscarella and baragona, 2017). this suggests a subsequent origin of the reptile community through recent passive dispersion mechanisms (cattaneo, 2015). wide cultural and commercial connections between tunisia, pantelleria and sardinia throughout history, especially around the 6th-3rd century b.c.e. under the influence of the carthaginians (bechtold, 2013), may have played a role in the human-mediated exchanges of reptile fauna across the mediterranean basin (bruno, 1985; masseti and zuffi, 2011). it may be in this context that the italian populations of h. hippocrepis originated. acknowledgements we are grateful to francesco lillo, mariagrazia graziano and sergio mezzadri for their help in fieldwork. we thank massimo delfino for his useful advices. samples were collected during several studies on the morphology and natural history of italian and sicilian reptiles, carried out with the following permits: matt prot. n. 2766/t-a31, 12/01/2018 and regione siciliana prot. n. 1637, 24/01/2018; matt permit prot. n. 12442/ pnm, 13/06/2017 and regione siciliana prot. n. 22232, 14/09/2017. fig. 2. geographic range of hemorrhois hippocrepis and distribution of all the samples included in the analysis, distinguished on the basis of the western and eastern lineages, respectively white and black dots. 133genetic identity of hemorrois hippocrepis in italy references abbazzi, l., angelone, c., arca, m., barisone, g., bedetti, c., delfino, m., kotsakis, t., marcolini, f., palombo, m.r., pavia, m., piras, p., rook, l., torre, d., tuveri, c., valli, a., wilkens, b. 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(2006): hemorrhois hippocrepis (linnaeus, 1758). in: atlante degli anfibi e dei rettili d’italia/ atlas of italian amphibians and reptiles, pp. 540-543. sindaco, r., doria, g., razzetti, e., bernini, f., eds, societas herpetologica i. acta herpetologica vol. 15, n. 2 december 2020 firenze university press estimating abundance and habitat suitability in a micro-endemic snake: the walser viper gentile francesco ficetola1,2,*, mauro fanelli3, lorenzo garizio3, mattia falaschi1, simone tenan4, samuele ghielmi5, lorenzo laddaga6, michele menegon7,8, massimo delfino3,9. potential effects of climate change on the distribution of invasive bullfrogs lithobates catesbeianus in china li qing peng1, min tang1, jia hong liao1, hai fen qing1, zhen kun zhao1, david a. pike2, wei chen1,* a bibliometric-mapping approach to identifying patterns and trends in amphibian decline research claudio angelini1,*, jon bielby2, corrado costa3 food composition of a breeding population the endemic anatolia newt, neurergus strauchii (steindachner, 1887) (caudata: salamandridae), from bingöl, eastern turkey kerim çiçek1,*, mustafa koyun2, ahmet mermer1, cemal varol tok3 stomach histology of crocodylus siamensis and gavialis gangeticus reveals analogy of archosaur “gizzards”, with implication on crocodylian gastroliths function ryuji takasaki1,2,*, yoshitsugu kobayashi3 does chronic exposure to ammonium during the pre-metamorphic stages promote hindlimb abnormality in anuran metamorphs? a comparison between natural-habitat and agrosystem frogs sonia zambrano-fernández1, francisco javier zamora-camacho2,3,*, pedro aragón2,4 confirming lessona’s brown frogs distribution sketch: rana temporaria is present on turin hills (piedmont, nw italy) davide marino1, angelica crottini2, franco andreone3,* phylogenetic relationships of the italian populations of horseshoe whip snake hemorrhois hippocrepis (serpentes, colubridae) francesco paolo faraone1, raffaella melfi2, matteo riccardo di nicola3, gabriele giacalone4, mario lo valvo5* first karyological analysis of the endemic malagasy phantom gecko matoatoa brevipes (squamata: gekkonidae) marcello mezzasalma1,2,*, fabio m. guarino3, simon p. loader1, gaetano odierna3, jeffrey w. streicher1, natalie cooper1 notes on sexual dimorphism, diet and reproduction of the false coral snake oxyrhopus rhombifer duméril, bibron & duméril, 1854 (dipsadidae: pseudoboini) from coastal plains of subtropical brazil fernando m. quintela1,*, felipe caseiro¹, daniel loebmann¹ acta herpetologica 13(2): 125-140, 2018 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-22611 batrachochytrium dendrobatidis in hungary: an overview of recent and historical occurrence judit vörös1,2, dávid herczeg3,4,*, attila fülöp5, tünde júlia gál1, ádám dán6, krisztián harmos7, jaime bosch8,9 1 department of zoology, hungarian natural history museum, 1088 budapest, hungary 2 laboratory for molecular taxonomy, hungarian natural history museum, 1083 budapest, hungary 3 lendület evolutionary ecology research group, plant protection institute, centre for agricultural research, hungarian academy of sciences, 1022 budapest, hungary. *corresponding author. e-mail herczegdavid88@gmail.com 4 institute for veterinary medical research, centre for agricultural research, hungarian academy of sciences, 1143 budapest, hungary 5 mta–de behavioural ecology research group, department of evolutionary zoology and human biology, university of debrecen, 4032 debrecen, hungary 6 molecular biology department, veterinary diagnostic directorate, national food chain safety office, 1143 budapest, hungary 7 bükk national park directorate, 3304 eger, hungary 8 museo nacional de ciencias naturales, csic, 28006 madrid, spain 9 centro de investigación, seguimiento y evaluación, parque nacional de la sierra de guadarrama, 28740 rascafría, spain 1, 2, 3, 4 these authors contributed equally to this work submitted on: 2018, 5th february; revised on: 2018, 5th may; accepted on: 2018, 28th august editor: marcello mezzasalma abstract. batrachochytrium dendrobatidis (bd) is a fungal pathogen which causes the emerging infectious disease chytridiomycosis. bd presents low host specificity and threatens amphibians worldwide, thus systematic inventory is the key in order to detect and mitigate the effects of the disease. extensive data collection was conducted in hungary in 2009-2015 from fourteen different areas. combined data – recent field sampling on sixteen taxa and the examination of archived bombina spp. specimens – from 1360 individuals were analysed with qpcr. two sentinel taxa, bombina variegata and the members of the pelophylax esculentus complex were marked to monitor the occurrence of bd in two core areas (bakony mts and hortobágy national park, respectively) of sampling. climatic variables were also examined in core areas to test their effect on prevalence and infection intensity. among the sixteen sampled amphibian taxa seven tested positive for bd and the overall prevalence in hungary was 7.46%. among the ethanol-fixed bombina spp. individuals bd was not detected. in the first core area (bakony mts) the overall prevalence in b. variegata was 10.32% and juvenile individuals showed significantly higher prevalence than adults. on the other hand there was a significant negative relationship between infection prevalence and monthly mean air temperature. finally, in the other core area (hortobágy national park) the overall prevalence in p. esculentus complex was 13.00%, and no differences were found in prevalence or infection intensity between sexes, sampling years or age classes. keywords. chytridiomycosis, emerging infectious diseases, pelophylax esculentus complex, bombina variegata, inventory, central-europe. introduction over the past decades several epidemics – caused by emerging infectious diseases – resulted in the largescale decline of numerous animal species globally (dobson and foufopoulos, 2001). one such emerging disease is chytridiomycosis in amphibians caused by the fungal pathogen batrachochytrium dendrobatidis [hereafter, bd 126 judit vörös et alii (longcore et al., 1999)]. bd is a highly generalist, waterborne pathogen which is primarily transmitted through direct contact with aquatic zoospores or infected individuals (fisher et al., 2009). bd is responsible for population declines, mass mortalities and even extinction of species, and presents one of the greatest threats to amphibians worldwide (berger et al., 1998; skerratt et al., 2007; fisher et al., 2009). bd is widespread on all continents where amphibians occur (olson et al., 2013), but the heaviest disease outbreaks were observed in the american neotropics, australia, north-america and western europe (fisher et al., 2009). in europe, the first detection of bd related mass mortalities dates back to 1997 when the first recorded population decline as a result of mass die-off after the emergence of chytridiomycosis was observed in central spain, in the guadarrama mountain national park, and targeted the common midwife toad, alytes obstetricans (bosch et al., 2001). though, as a result of the increased attention in the subsequent years, studies performed in the same region revealed that other species are highly susceptible to the disease as well (e.g. salamandra salamandra, bufo spinosus; bosch and martínez-solano, 2006; bosch et al., 2007). moreover, the evidenced strong population declines of a. obstetricans, a. muletensis and a. dickhilleni in the iberian peninsula (bosch et al., 2001; walker et al., 2010; bosch et al., 2013; doddington et al., 2013; rosa et al., 2013), and the high susceptibility of these species made the midwife toads the “flagship” species of european chytridiomycosis threat. central europe harbours several amphibian species that might be susceptible to chytridiomycosis, such as s. salamandra, b. bufo, bombina bombina or bombina variegata (baláž et al., 2014a,b). in the recent years bd infection was detected in various areas of the czech republic, as a result of a systematic inventory (civiš et al., 2012). furthermore, the presence of the fungus was recently reported in low prevalence from luxembourg (wood et al., 2009), poland (sura et al., 2010; kolenda et al., 2017), germany (ohst et al., 2013), austria (sztatecsny and glaser, 2011), slovakia (baláž et al., 2014b) and italy (federici et al., 2008; tessa et al., 2013). new data indicate that the fungus is present also in the balkans, e.g., in serbia (mali et al., 2017), albania, montenegro and macedonia (vojar et al., 2017). though, interestingly, no negative effects or bd-linked population declines have been detected from central-eastern-europe so far (vörös et al., 2014). some aspects of chytridiomycosis epizootics show environmental correlates (olson et al., 2013). bd presents a reasonably wide environmental tolerance under a variety of temperature and precipitation regimes (ron, 2005), but previous studies postulated that climate (berger et al., 2004; bosch et al., 2007; murray et al., 2009; blaustein et al., 2010; rohr et al., 2010; rödder et al., 2010) and elevation (lips et al., 2008; walker et al., 2010; becker and zamudio, 2011) can significantly influence bd outbreaks. furthermore, large intraand interspecific variations exist, especially in the prevalence (gründler et al., 2012; böll et al., 2014; spitzen-van der sluijs et al., 2014), but also in the intensity of infection (van sluys and hero, 2009; baláž et al., 2014a; spitzen-van der sluijs et al., 2014). in addition, behavioural differences influence the susceptibility to bd which is further affected by the intraspecific variability related to sex and life stages (blaustein et al., 2005, garcia et al., 2006, williams and groves, 2014). hungary is situated in the carpathian basin, a region with high amphibian diversity due to different climatic and zoogeographical influences (vörös et al., 2014). previous findings about the occurrence of bd in hungary are restricted to a few areas and species where the presence was initially detected (gál et al., 2012; baláž et al., 2014b, vörös et al., 2014, drexler et al., 2017). therefore, no large-scale distribution data on bd presence is available to date from the country. our study displays multiple goals. first, we present a general overview on the occurrence of bd in hungary summarising data collected between the years 2009-2015. the data set includes the general occurrence of bd on sixteen amphibian taxa with a special focus on the yellowbellied toad bombina variegata and water frogs belonging to the pelophylax esculentus complex. we selected these two target taxa because these species may present high levels of infection intensity in europe and so they may also act as sentinel taxa (baláž et al., 2014b); in addition, they can play a role in the spread and the persistence of the disease (baláž et al., 2014a). second, by studying b. variegata populations in hungary we assessed whether distinct phylogenetic lineages – alpine (west of the danube) and carpathian, occurring in the north hungarian range east of the danube (vörös et al., 2006) – express differences in prevalence and infection intensity. moreover, to explore the historical distribution of bd in hungary field surveys were complemented with available archived samples of bombina spp. from museum collections which comprise a dataset covering a 70 years’ time frame (1936-2005) prior to our field sampling. third, in order to further monitor bd infection levels of amphibians in hungary, we selected one population of two of the most susceptible taxa in central-eastern europe, b. variegata and the p. esculentus complex (baláž et al., 2014b), and extensively sampled these 127occurrence of bd in hungary populations for three consecutive years in two core areas. finally, we aimed to use climatic data (monthly mean precipitation and monthly mean air temperature) in these core areas to test if there is any correlation between the previously mentioned climatic variables and the occurrence of bd. materials and methods data collection altogether 1233 specimens belonging to sixteen amphibian taxa were studied in the field between 2009-2015. sampling was conducted in fourteen different regions in 45 distinct sampling points throughout hungary, covering a great variety of wetland habitats (i.e. irrigation canals, streams, marshlands, ponds, fishponds, water reservoirs and temporary wetland habitats) and elevations ranging between 84 and 734 m a.s.l. (fig. 1, table 1). bombina variegata was surveyed in five regions from transdanubia (region 1, 2, 3, 5 and 8 in table 1 and fig. 1) representing the alpine (western) genetic lineage, and in three regions from the north hungarian mountains (region 10, 12 and 13 in table 1 and fig. 1) representing the carpathian (eastern) genetic lineage, covering the distribution of the species in hungary (vörös et al., 2006). identification of the two bombina species and their hybrids was performed considering morphological characters plus genetic information provided by previous researches in hungary (vörös et al., 2006, 2007). members of the pelophylax esculentus complex were sampled in eight regions (region 1, 3, 4, 7, 8, 9, 10 and 14 in table 1 and fig. 1). age classes were characterized as tadpoles, juveniles and adults based on the external features of each species examined in the field. in those cases when we couldn’t distinguish between age and sex of an individual we discarded the sample for further analysis. additionally, 127 ethanol-fixed specimens of bombina spp., deposited in the hungarian natural history museum (budapest, hungary) and savaria museum (szombathely, hungary), collected between 1936 and 2005 from regions matching the current distribution of the species were swabbed (supplementary table s1). fig.1. map of hungary showing sampling locations of bd negative (black filled circles), bd positive (red triangles) and archived (white circles) samples. pie charts indicate bd prevalence of the fourteen studied geographic regions. numbers of regions correspond to table 1. the two core areas are marked with asterisks (region 3 and 14). drawing of bombina variegata and pelophylax ridibundus are the courtesy of márton zsoldos. 128 judit vörös et alii ta bl e 1. s um m ar y of r eg io ns , s am pl in g lo ca tio ns , c oo rd in at es a nd s am pl ed s pe ci es in o ur in ve nt or y. m td n a li ne ag es w er e in di ca te d as a lp in e (a lp ) or c ar pa th ia n (c ar p) in t he c as e of b . v ar ie ga ta . l at = l at itu de ; l on g = lo ng itu de ; n = n um be r of in di vi du al s sa m pl ed ; p re v = pr ev al en ce ; g e = g en om ic e qu iv al en ts . n r. of r eg io n a lt la t lo ng sp ec ie s m td n a lin ea ge b . va ri eg at a n po si tiv e/ sa m pl ed pr ev ( % ) pr ev 9 5% c i (% ) g e m ea n g e m ed ia n g e sd g e ra ng e 1ő rs ég 31 5. 0 46 .8 7 16 .1 3 bo m bi na v ar ie ga ta a lp 2 16 / 6 8 23 .5 3 14 .0 935 .3 8 34 .4 5 5. 01 58 .3 2 0. 20 -1 82 .7 8 26 4. 0 46 .8 7 16 .4 5 bo m bi na v ar ie ga ta a lp 7 25 3. 0 46 .8 9 16 .4 3 h yl a ar bo re a 1 25 3. 0 46 .8 9 16 .4 3 li ss ot ri to n vu lg ar is 1 25 3. 0 46 .8 9 16 .4 3 r an a ar va lis 1 25 3. 0 46 .8 9 16 .4 3 r an a da lm at in a 4 31 5. 0 46 .9 0 16 .2 4 bo m bi na v ar ie ga ta a lp 48 31 5. 0 46 .9 0 16 .2 4 ic ht hy os au ra a lp es tr is 1 26 7. 0 46 .9 1 16 .2 3 pe lo ph yl ax e sc ul en tu s 1 31 5. 0 46 .9 0 16 .2 4 r an a te m po ra ri a 2 2so pr on i m ts 49 3. 0 47 .6 5 16 .4 8 bo m bi na v ar ie ga ta a lp 14 4 / 14 28 .5 7 8. 38 -5 8. 10 2. 05 2. 40 1. 13 0. 48 -2 .9 0 3b ak on y m ts 45 5. 0 47 .0 6 17 .6 7 bo m bi na b om bi na 2 37 / 6 06 6. 11 4. 33 -8 .3 2 21 .1 5 5. 19 45 .5 8 0. 16 -2 10 .3 0 31 6. 0 47 .2 3 17 .7 4 bo m bi na v ar ie ga ta a lp 3 32 7. 0 47 .2 7 17 .6 9 bo m bi na v ar ie ga ta a lp 15 32 7. 0 47 .2 7 17 .6 9 bu fo b uf o 2 32 7. 0 47 .2 7 17 .6 9 ic ht hy os au ra a lp es tr is 12 32 7. 0 47 .2 7 17 .6 9 li ss ot ri to n vu lg ar is 19 32 7. 0 47 .2 7 17 .6 9 r an a da lm at in a 25 34 8. 0 47 .2 3 17 .6 4 bo m bi na b om bi na 2 34 8. 0 47 .2 3 17 .6 4 bo m bi na v ar ie ga ta a lp 31 0 35 6. 0 47 .2 3 17 .6 5 bu fo b uf o 61 35 6. 0 47 .2 3 17 .6 5 bu fo v ir id is 39 34 8. 0 47 .2 3 17 .6 4 li ss ot ri to n vu lg ar is 5 35 6. 0 47 .2 3 17 .6 5 pe lo ph yl ax r id ib un du s 24 34 8. 0 47 .2 3 17 .6 4 pe lo ph yl ax s p. 4 34 8. 0 47 .2 3 17 .6 4 r an a da lm at in a 83 4h an sá g 11 3. 0 47 .6 6 16 .7 4 bo m bi na b om bi na 4 3 / 33 9. 09 1. 92 -2 4. 33 0. 56 0. 16 0. 70 0. 15 -1 .3 7 11 6. 0 47 .6 3 17 .0 8 pe lo ph yl ax r id ib un du s 29 5m ec se k m ts 38 1. 0 46 .2 2 18 .3 3 bo m bi na v ar ie ga ta a lp 12 0 / 23 0. 00 0. 00 -1 4. 82 23 2. 0 46 .1 6 18 .2 4 bo m bi na v ar ie ga ta a lp 8 41 5. 0 46 .2 0 18 .3 3 bo m bi na v ar ie ga ta a lp 3 6k is ku ns ág 89 .0 46 .6 1 19 .1 2 tr itu ru s do br og ic us 13 0 / 13 0. 00 0. 00 -2 4. 71 7bu da pe st 10 0. 0 47 .1 8 18 .5 3 bo m bi na b om bi na 4 2 / 18 11 .1 1 1. 38 -3 4. 71 36 .7 7 36 .7 7 50 .1 1 1. 34 -7 2. 20 11 1. 0 47 .4 2 19 .1 4 bu fo v ir id is 4 15 6. 0 47 .5 3 19 .2 2 pe lo ph yl ax r id ib un du s 10 129occurrence of bd in hungary n r. of r eg io n a lt la t lo ng sp ec ie s m td n a lin ea ge b . va ri eg at a n po si tiv e/ sa m pl ed pr ev ( % ) pr ev 9 5% c i (% ) g e m ea n g e m ed ia n g e sd g e ra ng e 8pi lis -v is eg rá di m ts 16 8. 0 47 .7 8 19 .0 4 bo m bi na b om bi na 1 0 / 78 0. 00 0. 00 – 4 .6 2 41 8. 0 47 .7 8 19 .0 0 r an a da lm at in a 5 26 1. 0 47 .5 7 18 .9 4 bu fo b uf o 1 26 1. 0 47 .5 7 18 .9 4 sa la m an dr a sa la m an dr a 35 21 6. 0 47 .6 4 18 .7 8 bo m bi na b om bi na 2 32 9. 0 47 .7 6 18 .8 5 r an a te m po ra ri a 2 18 3. 0 47 .7 6 18 .9 1 sa la m an dr a sa la m an dr a 7 23 4. 0 47 .6 1 18 .8 8 h yl a ar bo re a 1 23 4. 0 47 .6 1 18 .8 8 pe lo ph yl ax s p. 3 20 8. 0 47 .8 5 19 .1 2 r an a te m po ra ri a 1 20 9. 0 47 .8 5 19 .1 1 sa la m an dr a sa la m an dr a 1 10 7. 0 47 .7 7 19 .0 9 h yl a ar bo re a 2 10 7. 0 47 .7 7 19 .0 9 pe lo ph yl ax s p. 4 35 8. 0 47 .7 2 19 .0 6 bo m bi na b om bi na x va ri eg at a 1 35 8. 0 47 .7 2 19 .0 6 bo m bi na v ar ie ga ta a lp 2 30 1. 0 47 .7 8 18 .9 9 pe lo ph yl ax r id ib un du s 8 30 1. 0 47 .7 8 18 .9 9 r an a te m po ra ri a 2 9g öd öl lő h ill s 22 4. 0 47 .6 3 19 .3 8 li ss ot ri to n vu lg ar is 20 0 / 56 0. 00 0. 00 -6 .3 8 15 6. 0 47 .5 3 19 .2 2 pe lo ph yl ax r id ib un du s 1 11 1. 0 47 .7 6 17 .3 4 r an a ar va lis 1 96 .0 47 .2 6 19 .2 3 r an a ar va lis 17 96 .0 47 .2 6 19 .2 3 r an a da lm at in a 3 96 .0 47 .2 6 19 .2 3 tr itu ru s do br og ic us 14 10 -m át ra m ts 49 2. 0 47 .9 0 19 .9 8 bo m bi na v ar ie ga ta c ar p 2 7 / 10 3 6. 80 2. 78 -1 3. 50 6. 93 2. 13 9. 19 0. 61 -2 3. 55 64 8. 0 47 .9 3 19 .8 9 bo m bi na v ar ie ga ta c ar p 2 64 8. 0 47 .9 3 19 .8 9 sa la m an dr a sa la m an dr a 6 59 8. 0 47 .9 0 19 .9 7 bo m bi na b om bi na 2 58 7. 0 47 .8 5 19 .9 6 bo m bi na v ar ie ga ta c ar p 3 31 6. 0 47 .9 7 19 .5 2 sa la m an dr a sa la m an dr a 1 72 0. 0 47 .9 0 19 .9 3 bo m bi na v ar ie ga ta c ar p 4 40 3. 0 47 .9 2 19 .9 7 bo m bi na b om bi na 2 30 4. 0 47 .9 3 19 .9 8 bo m bi na b om bi na x va ri eg at a 1 63 6. 0 47 .8 7 19 .9 7 bo m bi na v ar ie ga ta c ar p 32 72 7. 0 47 .8 8 20 .0 1 bu fo b uf o 1 72 7. 0 47 .8 8 20 .0 1 ic ht hy os au ra a lp es tr is 11 130 judit vörös et alii n r. of r eg io n a lt la t lo ng sp ec ie s m td n a lin ea ge b . va ri eg at a n po si tiv e/ sa m pl ed pr ev ( % ) pr ev 9 5% c i (% ) g e m ea n g e m ed ia n g e sd g e ra ng e 41 1. 0 47 .9 3 19 .9 6 pe lo ph yl ax e sc ul en tu s 1 72 7. 0 47 .8 8 20 .0 1 r an a te m po ra ri a 1 72 7. 0 47 .8 8 20 .0 1 sa la m an dr a sa la m an dr a 3 36 4. 0 47 .9 0 19 .7 4 bo m bi na b om bi na 3 36 2. 0 47 .9 3 19 .7 6 bo m bi na v ar ie ga ta c ar p 1 52 2. 0 47 .8 9 20 .1 0 bo m bi na b om bi na 6 27 4. 0 47 .9 1 20 .1 4 bo m bi na b om bi na x va ri eg at a 1 63 3. 0 47 .8 9 20 .1 1 bo m bi na v ar ie ga ta c ar p 12 63 6. 0 47 .9 3 19 .9 3 bo m bi na b om bi na x va ri eg at a 5 41 1. 0 47 .9 3 19 .9 6 bo m bi na v ar ie ga ta c ar p 2 41 1. 0 47 .9 3 19 .9 6 pe lo ph yl ax e sc ul en tu s 1 11 -b ük k m ts 24 9. 0 48 .1 2 20 .2 4 bu fo b uf o 1 1 / 9 11 .1 1 0. 28 -4 8. 25 8. 10 8. 10 n a n a 32 0. 0 48 .1 5 20 .1 0 r an a te m po ra ri a 1 44 3. 0 48 .0 4 20 .5 6 ic ht hy os au ra a lp es tr is 6 33 0. 0 48 .1 5 20 .0 8 r an a te m po ra ri a 1 12 -a gg te le k k ar st 28 6. 0 48 .5 4 20 .6 6 bo m bi na v ar ie ga ta c ar p 6 0 / 12 0. 00 0. 00 -2 6. 46 23 8. 0 48 .5 3 20 .6 4 sa la m an dr a sa la m an dr a 6 13 -z em pl én m ts 46 8. 0 48 .2 7 21 .2 9 bo m bi na v ar ie ga ta c ar p 10 6 / 22 27 .2 7 10 .7 350 .2 2 24 4. 00 10 1. 15 32 8. 43 13 .0 388 2. 54 28 1. 0 48 .4 8 21 .3 3 bo m bi na v ar ie ga ta c ar p 6 34 1. 0 48 .4 8 21 .3 2 r an a te m po ra ri a 1 34 1. 0 48 .4 8 21 .3 2 sa la m an dr a sa la m an dr a 4 44 9. 0 48 .4 0 21 .4 5 bo m bi na v ar ie ga ta c ar p 1 14 -h or to bá gy 86 .0 47 .5 7 20 .9 4 pe lo ph yl ax e sc ul en tu s 18 16 / 1 78 8. 99 5. 23 -1 4. 19 10 .4 8 1. 48 17 .9 8 0. 64 -5 7. 91 84 .0 47 .6 0 20 .8 8 pe lo ph yl ax le ss on ae 1 86 .0 47 .5 7 20 .9 4 pe lo ph yl ax r id ib un du s 2 85 .0 47 .6 2 21 .0 8 pe lo ph yl ax e sc ul en tu s 25 86 .0 47 .6 1 21 .0 7 pe lo ph yl ax r id ib un du s 56 86 .0 47 .6 3 21 .0 8 pe lo ph yl ax s p. 12 85 .0 47 .4 4 21 .1 4 pe lo ph yl ax e sc ul en tu s 20 85 .0 47 .4 4 21 .1 4 pe lo ph yl ax r id ib un du s 42   84 .0 47 .4 5 21 .1 7 pe lo ph yl ax s p.   2               to ta l 12 33 131occurrence of bd in hungary systematic sampling of sentinel taxa in two core areas core areas were selected based on the prevalence found previously or in the first year of sampling (gál et al., 2012; baláž et al., 2014b). in bakony mts, b. variegata was systematically sampled in 2010-2012. data of 2010 were published previously (gál et al., 2012), thus our analyses includes a comparison of data from 2010 and new data from 2011 and 2012. surveys were completed between march and september in 2010, april and september in 2011, may and july in 2012. the assigned locality, iharkút (see asterisk on fig. 1), is an old open bauxite mine, where human activities are common due to being a famous paleontological research site (ősi et al., 2012). in iharkút we were able to locate only two water bodies: a small lake and a nearby stream. because of the close proximity (ca. 50 meters) and the presumed connection of the two habitats, all the toads belonged to the same population. members of the p. esculentus complex were screened for bd in the hortobágy national park (hnp; see asterisk on fig. 1). hnp is the largest continuous alkaline steppe in europe covering 80.000 hectares. this natural reserve is abundant in wetland habitats like alkaline marshes, fishponds, wet grasslands and wet meadows (ecsedi, 2004). pelophylax species were sampled in three sites at hnp – nádudvar-kösély canal near the city nádudvar, a fish pond system located eastwards to hortobágy village and a marshland system at egyek-pusztakócs village – between april and october during three consecutive years (2012-2014). taxonomic identification of pelophylax esculentus complex water frog taxon identification was determined using the technique described by hauswaldt et al. (2012), and is based on allele-size polymorphism in intron-1 of the serum albumin gene (sai-1; plötner et al., 2009) named ranacr1, was identified in the serum albumin intron-1 (sai-1, with a slight modification in pcr protocol (herczeg et al., 2017). to verify sai1 fragments we sequenced representative alleles on a hitachi 3130 genetic analyzer (applied biosystems, uk). consensus sequences were compiled using bioedit version 7.0.9.0 (hall, 1999) and aligned manually. if genetic samples were not available we referred to the individuals as pelophylax sp. sampling protocol we collected bd samples following hyatt et al. (2007) by either swabbing the skin of the individuals or clipping one of the toes. according to hyatt et al. (2007) skin swabbing and toe clipping show similar performances in detectability of bd. skin swabbing was performed using two types of sterile swabs (swa90006; biolab, budapest, hungary, 5 mm diameter; and mw100-100; medical wire and equipment, wiltshire, england, 3 mm diameter). we collected each sample in a standardized way with three strokes on each side of the abdominal midline, the inner thighs, hands and feet. toe clipping was performed using sterilized scissors and toe clips were stored in 70% etoh in a freezer at -80 ˚c. skin swabs were stored dry in individually labelled vials and transferred to a freezer for longer storage throughout the field season. for both sampling procedures we used a new pair of disposable gloves per individual, and after each sampling event we sterilized all the used sampling equipment in order to avoid cross-contamination. mouthpart (oral disc) of larvae were swabbed following hyatt et al. (2007). ethanol-fixed specimens of bombina spp. were screened by skin swabbing following methodology presented above. genetic analysis of bd samples dna was extracted using prepman ultra sample preparation reagent (thermo fisher scientific, waltham, massachussetts, usa) following the recommendations of boyle et al. (2004). because of size differences between swabs (i.e. 3 mm vs. 5 mm; see above), only the top 3 mm of the larger swabs was used in all cases. extracted dna was analysed using real-time quantitative polymerase chain reaction (qpcr) following the amplification methodology of boyle et al. (2004) and hyatt et al. (2007) targeting the partial its-1 – 5.8s rrna regions. samples were run in triplicate and an internal positive control was included (taqman exogenous internal positive control reagents; 4308323; thermo fisher scientific, waltham, massachussetts, usa) to detect potential inhibitors present in the dna extractions. we considered evidence of infection if genomic equivalents (ge) were ≥ 0.1 and we considered a sample positive if all three wells returned a positive reaction. when a sample returned an equivocal result, it was re-run. if it again returned an equivocal result, it was considered negative (n = 17, 1.3% of total samples). the templates were run on a rotor-gene 6000 real-time rotary analyser (corbett life science, sydney, australia). ge were estimated from standard curves based on positive controls of 100, 10, 1, 0.1 developed from the bd isolate ia 2011, from acherito lake, spain. finally, ge values of the three positive replicates were averaged. in order to identify lineages of bd found on amphibians in hungary, 2 µl of dna extract from three individuals (one juvenile p. ridibundus plus one juvenile b. variegata from bakony mts, and one adult b. variegata from őrség) were selected as template for amplification of a partial fragment of its-1 rrna. nested pcr approach described by gaertner et al. (2009) was performed. the amplified fragments were sequenced on an applied biosystems/hitachi 3130 genetic analyser (thermo fisher scientific, waltham, massachussets, usa). sequences were aligned manually using bioedit version 7.0.9.0. (hall, 1999) and were blasted against available sequences from genbank for identification. climatic data climatic data were provided by the hungarian meteorological service (omsz). for the core areas of b. variegata and p. esculentus complex climatic data were obtained from the closest meteorological station of each sampling site: pápa city (47.29, 17.37), 135.5 m a.s.l, 21.5 km distance from iharkút (bakony mts), and kunmadaras village (47.46, 20.89), 88.8 m a.s.l. 12.5 132 judit vörös et alii km distance from egyek-pusztakócs (hnp), which is the closest sampling point to the station. we used monthly mean precipitation and monthly mean air temperature data for the period 2010-2014 to test if any relationship between climate and prevalence or infection intensity exists. statistical analyses statistical analyses were performed in r (version 3.4.4; r core team, 2018). prevalence was expressed as a discrete binomial variable (uninfected vs. infected). infection intensity was expressed through ge value. first, we calculated infection prevalence (%) of different amphibian species together with their 95% clopper-pearson confidence intervals (95% ci) as follows. prevalence values were obtained by dividing the cumulative number of positive samples with the total number of samples per species and multiplied with 100 to obtain percentile values, while 95% ci values were calculated using the r package ‘propcis’ (function ‘exactci’; scherer, 2018). in bd infected species we calculated the mean, median, sd and range of ge values as well. second, we tested whether prevalence and infection intensity differed between phylogenetic lineages of b. variegata, and in the two sentinel taxa (i.e. b. variegata and p. esculentus complex) we also tested for differences between study years, sexes and age classes. prevalence values were compared with chisquare tests, while infection intensities were compared using mood’s median test, as implemented in the r package ‘rvaidememoire’ (function ‘mood medtest’; hervé, 2018). finally, in the two sentinel taxa we tested the relationship between climatic variables and prevalence and infection intensity. we note here that the data set of the p. esculentus complex was restrained only on p. ridibundus, as the bd infection of p. esculentus was very low (i.e. two infected individuals in total) and the sample size of p. lessonae was also not representative (n = 1). the relationship between the climatic factors and infection prevalence was tested using generalized linear mixed models (glmms) with binomial error distribution term and the relationship between the climatic factors and infection intensity was analysed using linear mixed models with gaussian distribution (lmms). prevalence and infection intensity, respectively, were entered as dependent variables in the models, while the focal climatic variable (i.e., air temperature or precipitation) was set as continuous predictor. in all models sampling year was entered as a random effect to control for the interannual variations in infection prevalence or intensity. additionally, in the case of p. ridibundus, collection site id within the hnp was entered also as a random factor to account for the variations in prevalence and intensity between collection sites. to assure the adequate distribution of model residuals, for the lmms ge values were log(x+1)-transformed. prior entering into the models, log(x+1)-transformed ge values and the continuous predictors (i.e. climatic variables) were scaled to mean = 0 and sd = 1 to improve model convergence (see also schielzeth 2010). model fits were checked visually by plot diagnosis. in all cases for the statistical comparison of infection intensities only infected individuals were used. mixed models were constructed using the ‘lme4’ package for r (bates et al., 2015), and p-values for the linear mixed models were obtained using the function ‘anova’ (type iii) from the r package ‘car’ (fox and weisberg, 2011). we used a significance level of p ≤ 0.05 throughout. results bd occurrence in hungary in hungary, nine regions were infected with bd and the overall prevalence was 7.46% (95% ci: 6.05-9.07), indicating a low presence of the fungus in the country (table 1). among the sixteen sampled amphibian taxa seven were found infected with bd, including one unidentified pelophylax individual (table 2). details on prevalence and summary statistics of ge values are presented in table 2; while the geographic distribution of the sampling sites with the site-specific prevalence is shown in fig. 1. bd occurrence in bombina variegata in b. variegata the overall prevalence was 12.69% (95% ci: 9.91-15.92). details on prevalence and summary statistics of ge values for the different regions are presented in table 3. we found no significant difference between the two lineages of b. variegata in infection prevalence (nalpine = 422, ncarpathian = 82; χ2 = 0.155, df = 1, p = 0.693) and intensity (nalpine = 52, ncarpathian = 12, p = 0.750). bd was not detected among the ethanol-fixed b. variegata specimens. in bakony mts between 2010 and 2012 we sampled 310 individuals of b. variegata, among which 32 individuals were found to be infected with bd. here the overall prevalence was 10.32 % (95% ci: 7.16–14.25), and the mean, median, sd and range of ge values were 15.92, 5.09, 38.60 and 0.159–210.3, respectively. there was no significant difference in infection prevalence (n2010 = 80, n2011 = 144, n2012 = 86; χ2 = 4.980, df = 2, p = 0.082) nor in intensity between the three study years (n2010 = 13, n2011 = 14, n2012 = 5, p = 0.201), and we found no significant difference in prevalence (nmales = 113, nfemales = 90; χ2 = 0.241, df = 1, p = 0.623) and infection intensity between sexes (nmales = 8, nfemales = 2, p = 0.545). however, there was a significant difference in prevalence between the two age classes (njuveniles = 105, nadults = 204; χ2 = 11.563, df = 1, p < 0.001), with juveniles being more infected than adults (proportion of individuals infected: 19.04% versus 5.88%). differences in infection intensity between the two age classes were not significant (njuveniles = 20, nadults = 12, p = 0.273). there was significant negative relationship between infection prevalence and monthly mean air temperature (χ2 = 4.482 df = 1, p = 133occurrence of bd in hungary 0.034), and a marginally significant positive relationship between prevalence and monthly mean precipitation (χ2 = 3.611, df = 1, p = 0.057). there was no significant relationship between infection intensity and monthly mean air temperature (χ2 = 0.180, df = 1, p = 0.671). however, there was a significant positive relationship between table 2. batrachochytrium dendrobatidis (bd) infection in amphibian species sampled in hungary between the years 2009 and 2015. prev = prevalence; ge = genomic equivalents of zoospores. species positive/ sampled prev (%) prev 95% ci (%) ge mean ge median ge sd ge range order anura family bombinatoridae bombina bombina 1 / 29 3.45 0.09-17.76 16.41 16.41 na na bombina variegata 64 / 504 12.70 9.92-15.92 40.08 4.96 120.76 0.16-882.54 bombina bombina x variegata 0 / 8 0.00 0.00-36.94 family bufonidae bufo bufo 0 / 66 0.00 0.00-5.44 bufo viridis 2 / 43 4.65 0.57-15.81 36.77 36.77 50.11 1.34-72.20 family hylidae hyla arborea 0 / 4 0.00 0.00-60.24 family ranidae pelophylax esculentus 2 / 66 3.03 0.37-10.52 1.07 1.07 0.41 0.78-1.36 pelophylax lessonae 0 / 1 0.00 0.00-97.5 pelophylax ridibundus 21 / 164 12.80 8.10-18.91 20.21 1.59 41.72 0.15-164.30 pelophylax sp. 1 / 33 3.03 0.08-15.76 15.75 15.75 na na rana dalmatina 0 / 120 0.00 0.00-3.03 rana arvalis 0 / 19 0.00 0.00-17.65 rana temporaria 0 / 11 0.00 0.00-28.49 order caudata family salamandridae salamandra salamandra 0 / 63 0.00 0.00-5.69 triturus dobrogicus 0 / 27 0.00 0.00-12.77 lissotriton vulgaris 0 / 45 0.00 0.00-7.87 ichthyosaura alpestris 1 / 30 3.33 0.08-17.22 total 92 / 1233 7.46 6.05-9.07 table 3. batrachochytrium dendrobatidis (bd) detection in regions representing the surveyed local populations of b. variegata in hungary. prev = prevalence; ge = genomic equivalents of zoospores. genetic lineage region positive/ sampled prev (%) prev 95% ci (%) ge mean ge median ge sd ge range alpine őrség 16 / 57 28.07 16.97-41.54 34.45 5.01 58.32 0.20-182.78 soproni mts 4 / 14 28.57 8.39-58.10 2.05 2.40 1.13 0.48-2.90 bakony mts 32 / 328 9.76 6.77-13.49 15.93 5.09 38.61 0.16-210.30 mecsek mts 0 / 23 0.00 0.00-14.82 pilis-visegrádi mts 0 / 2 0.00 0.00-84.19 carpathian mátra mts 6 / 58 10.34 3.89-21.17 5.36 1.86 8.97 0.61-23.55 aggtelek karst 0 / 6 0.00 0.00-45.93   zemplén mts 6 / 16 37.50 15.20-64.57 244.00 101.15 328.43 13.03-882.54 total 64 / 504 12.59 9.83-15.80 134 judit vörös et alii infection intensity and monthly mean precipitation (χ2 = 4.227, df = 1, p = 0.039); though, this significant relationship disappeared after removing one outlier ge value from the data set (χ2 = 1.510, df = 1, p = 0.219). all the three sequences (i.e. sequences obtained from juvenile p. ridibundus and b. variegata from bakony mts, and one adult b. variegata from őrség) were identified as its-1 rrna of bd, belonging to the globally dispersed bd-gpl lineage (genbank accession numbers: mh745069-71). one sequence showed 100% identity with bd from cape cod (genbank accession number: fq176489.1, fq176492.1), south africa (jq5829034, 15, 37), and italy (fj010547). the second sequence was 100% identical with a sequence of bd from equador (fj232009.1), and the third sequence represented a unique haplotype. genetic distance (p-distance) among sequences ranged between 0.005-0.035. bd occurrence in pelophylax ridibundus in hortobágy between 2012 and 2014 we sampled 100 individuals of p. ridibundus, among which 13 were found to be infected with bd. here the overall prevalence was 13.00% (7.10-21.20), and the mean, median, sd and range of ge values were 11.52, 1.59, 19.63 and 0.635– 57.905, respectively. we found a significant difference in infection prevalence between years (n2012 = 35, n2013 = 48, n2014 = 17; χ2 = 27.750, df = 2, p < 0.001); all the infected individuals being captured in 2012 (prevalence: 37.14%), while no infected individuals being found in 2013-2014. we found no significant difference in prevalence (nmales = 42, nfemales = 30; χ2 = 0.002, df = 1, p = 0.958) and infection intensity between sexes (nmales = 7, nfemales = 6, p = 1.000). age classes did not differ in infection prevalence (njuveniles = 9, nadults = 72; χ2 = 0.827, df = 1, p = 0.363). infection intensities of the different age classes cannot be compared because no infected juveniles were captured. we found no significant relationship between infection prevalence and monthly mean air temperature (χ2 = 2.375, df = 1, p = 0.123), and between prevalence and monthly mean precipitation (χ2 = 0.010, df = 1, p = 0.920). since infection prevalence was relatively low in the p. esculentus complex and infected individuals were captured in the same month and year, the relationship between climatic variables and infection intensity could not be tested in this taxa. discussion low batrachochytrium dendrobatidis prevalence was experienced throughout the country (table 1, table 2), with similar or slightly lower values than in neighbouring countries e.g. czech republic (baláž et al., 2014a; 19% average at country level), austria (sztatecsny and glaser, 2011; 5.9-45% at country level) or poland (kolenda et al., 2017; 18% average at country level). overall, seven taxa carried the infection: bombina bombina, bombina variegata, bufo viridis, pelophylax ridibundus, pelophylax esculentus, pelophylax sp. and ichthyosaura alpestris. in accordance with previous studies in central europe (ohst et al., 2013; baláž et al., 2014a,b; kolenda et al., 2017), b. variegata and the members of the p. esculentus complex showed the highest prevalence and bd infection intensity in hungary. on the other hand, there was no difference in prevalence and infection intensity was detected between the two ancient phylogenetic lineages of b. variegata. bd was present in nine of the fourteen studied regions. the highest prevalence was experienced in the alpine foothills at őrség (region 1), soproni mts (region 2), and in the zemplén mts (region 13). these three regions represent the margins of the alps and carpathians (respectively) hosting populations with continuous distribution towards the higher regions. on the other hand, the remnant mountain regions, where prevalence was much lower (regions 3, 10 and 11), are geographically isolated from other higher elevations. in contrast, amphibians from five regions (regions 5, 6, 8, 9 and 12) seemed to not carry bd. this either indicates that bd has not reached these parts of the country yet, or more comprehensive sampling would be needed to locate its presence. the carpathian basin combines the characteristics of the neighbouring regions. despite the relatively small extent of hungary, the climatic elements have distinct temporal and spatial characters (mezősi, 2017). although the majority of the country has an elevation of less than 300 m a.s.l., hungary has several moderately high ranges of mountains and the highest peak located in the mátra mts at 1014 m a.s.l. (table 1, region 10). overall, our results rather supporting the relationship between the measured climatic variables and prevalence or infection intensity. we found significant relationship regarding b. variegata individuals in the bakony mts core area, where prevalence was negatively affected by monthly mean temperature. furthermore, the monthly mean precipitation positively affected the bd infection intensity. nonetheless, the robustness of the latter result is questionable, since the relationship disappeared when we excluded an outlier value from the analysis. this substantial effect of one outlier value could have on the outcomes of this analysis suggests the need for an extensive sampling in order to test whether this result is a statistical artefact or a real biological phenomenon. 135occurrence of bd in hungary to determine the time and location of the emergence or introduction of bd in different regions worldwide, it is important to study archived specimens deposited to museum collections. to examine the historical presence of the fungus in hungary we screened archived specimens of bombina spp. collected in the regions 1, 2, 3, 8, 10, 12, 13 and the kőszeg mts (archived data only) between 1936 and 2005. in total 127 specimens were analysed and all of the samples were bd negative. both for field and for museum samples we used the same detection methodology, following hyatt et al. (2007). the detection probability with qpcr is more sensitive and accurate compared to conventional pcr or histology (annis et al., 2004; boyle et al., 2004; kriger et al., 2006). there is no difference in regard of bd detectability between sample collection techniques (i.e., skin swabbing, brushing or scraping). nonetheless, preservation methodology and storage history may have influence on the results (soto-azat et al., 2009). the amphibian collection of the hungarian natural history museum is stored in ethanol, but no record is available about the mode of initial preparation. as formaldehyde is known to inhibit pcr reaction, there is therefore a slight chance that qpcr reactions failed to detect bd in our archived samples; however, this may be an unlikely possibility. although with testing archived specimens we did not find evidence on when bd might have been introduced into the country, our genetic analyses showed that the fungus found on amphibians in hungary is a member of the bd-gpl lineage. this was confirmed by a recent study tracking the origin of bd using a full genome approach, which detected bd-gpl lineage in hungary (from iharkút, bakony mts; o’hanlon et al., 2018) and is in line with previous findings reporting that this lineage has a widespread distribution in europe (farrer et al., 2007). during the surveys in the core area of bakony mts (region 3, table 1) juvenile b. variegata individuals showed a significantly higher prevalence compared to adults. the same pattern was observed for two b. variegata populations in a seven-year period study in the netherlands, which the authors explained by the less developed immune responses, or immunosupression, following the stress of metamorphosis (spitzen-van der sluijs et al., 2017). quite surprisingly, during our study, two juveniles changed infection state once (recovered from bd positive). it is a relatively common phenomenon in the field, when infected adult frogs lose and regain the infection which may be caused by overwintering tadpoles or larvae acting as reservoirs (briggs et al., 2010, spitzenvan der sluijs et al., 2017). in contrast, it is less frequent with juvenile individuals as it was experienced in our study. similar pattern was observed for epidalea calamita in spain, where juveniles changed infection state towards the end of metamorphosis, possibly mediated by the increasing water temperature in permanent ponds (bosch et al., unpublished). in iharkút (bakony mts), during our study period the environmental conditions changed unexpectedly. the lake which hosted most of the amphibian species – including b. variegata – dried out after the first season of sample collection. in the second year only four individuals of b. variegata were captured around this locality, however the rest of the specimens (n = 181) found shelter in a nearby stream unsuitable for breeding. during the third year the lake kept dry and only seven out of 87 individuals were found in or around the lake. even though there was no difference in prevalence between the three years, they showed a downward trend towards significance. already low prevalence (23%) dropped down to 11% in the second and to 5% in the third year. this trend could be associated with the differences in habitat type, as it was observed for salamandra salamandra in the guadarrama national park, spain (medina et al., 2015). here, bd infection was greater in salamander larvae from permanent ponds, while it was absent or weak in temporary water bodies and permanent streams. also, infection intensity in larval cohorts was reduced when water was flowing rather than standing. same authors suggested that increased water flow rate reduce the likelihood of successful pathogen transmission. chytridiomycosis is limited to the keratinized tissues of the host individual, therefore tadpoles and post-metamorphic amphibians are mostly affected by the disease (rachowicz and vredenburg, 2004). our dataset covered all life stages of amphibians and the presence of the infection was not detected in tadpoles of b. bufo and r. dalmatina (n = 39). on the other hand, post-metamorphic and juvenile individuals were found infected in the regions 1, 3, 10 and 13 of b. variegata and the members of the p. esculentus complex, even though all sampled individuals apparently didn’t display any clinical sign of chytridiomycosis. in central europe the p. esculentus complex is formed by two sexual species, the p. ridibundus and the p. lessonae and their interspecific mating produces the hybridogenetic p. esculentus. overall, our results in the core area of hortobágy national park showed higher bd prevalence in p. ridibundus compared to the hybrid p. esculentus (table 2) which is related to the fact that the hybrids have more effective peptide defence system against bd and have a richer peptide repertoire than both parental species (daum et al., 2012). further, contrary to what was observed in b. variegata in the bakony mts core area, we did not find differences in bd infection between 136 judit vörös et alii life stages and sexes in p. ridibundus individuals. our results fit into the general pattern showing significant variability in the effects of chytridiomycosis across europe. the marked difference in species susceptibility between amphibian species/communities of western and central-eastern europe might be determined by multiple linked factors, e.g. virulence of different bd strains (farrer et al., 2007), genotype (savage and zamudio, 2011), behaviour (williams and groves, 2014), microbial skin community compound of host species (bletz et al., 2013), or structure of amphibian communities (becker et al., 2014). in the iberian peninsula – that received the most attention due to mass amphibian mortalities caused by chytridiomycosis – infection was clustered within high-altitude areas, where environmental conditions are the most optimal for growth of bd (piotrowski et al., 2004). in contrast, hungary harbours only low-elevation mountains, where environmental conditions might be less favourable for bd-linked epidemics. differences in elevation might explain the relatively lower impact and infection values of amphibians in hungary, than it was reported for surrounding countries in central and eastern europe (e.g., austria, sztatecsny and glaser, 2011; czech republic, baláž et al., 2014a or poland, kolenda et al., 2017). since bd-related disease outbreak have been proven to be climate-driven (bosch et al., 2007), amphibians of central-eastern europe might be heavily impacted in the future due to global climate change. changes in the climate might alter bd diffusion and make it’s spreading less predictable, thus areas not yet affected by epidemics require particular attention and constant monitoring. acknowledgements we thank b. halpern, k. szabó, i. kiss, e. jáger, k. suri, r. dankovics, b. velekei, a. ősi, g. deák, cs. tóth, a. bérczes, g. magos, l. urbán, b. bándli, m. garcía-parís, p. mikulíček, c. gabor, c. serrano-laguna, zs. végvári, d. r. brooks, e. vörös, l. vörös, e. kovács and f. hock for providing samples or helping in the field. we are grateful to t. garner (zoological society of london) for important initial and then continuous help with bd research in hungary. t. papp and m. benkő (institute for veterinary medical research, budapest) kindly provided facility and reagents for qpcr. we gratefully acknowledge the excellent technical assistance of á. juhász and e. ottinger (national food chain safety office), as well as m. tuschek and v. krízsik (hnhm). savaria museum szombathely provided toad specimens for sampling. during the project jv was supported by the hungarian scientific research fund (otka k77841) and by the bolyai jános research scholarship of the hungarian academy of sciences (bo/00579/14/8). dh was supported by the european union and co-financed by the european social fund through the social renewal operational programme under the projects támop–4.2.2/b–10/1–2010–0024 and srop-4.2.2.b-15/1/konv-2015-0001. af was supported by the hungarian national research, development and innovation office (otka grant no. k112527). research permit was issued by the national inspectorate of environment, nature conservation and water management (14/3535/2/2010) and the tisza region inspectorate of environment, nature conservation and water management (4633/oh/2012). supplementary material supplementary material associated with this article can be found at <http://www.unipv.it/webshi/appendix> manuscript number 22611. references annis, s.l., dastoor, f.p., ziel, h., daszak, p., longcore, j.e. 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(2009): amphibian chytridiomycosis in luxemburg. bull. soc. nat. luxemb. 110: 109-114. acta herpetologica vol. 13, n. 1 june 2018 firenze university press acta herpetologica 15(1): 3-14, 2020 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/a_h-8448 has the west been won? a field survey and a species distribution model of iberolacerta horvathi in the alps giuseppe de marchi1,*, giovanni bombieri1, bruno boz1, fausto leandri2, jacopo richard3 1 associazione faunisti veneti, venezia (ve), italy 2 world biodiversity association onlus, c/o museo civico di storia naturale, verona (vr), italy 3 veneto agricoltura, agenzia veneta per l’innovazione nel settore primario, legnaro (pd), italy * corresponding author. email: dromasardeola@gmail.com submitted on: 2019, 2th june; revised on: 2019, 8th september; accepted on: 2019, 2nd november editor: dario ottonello abstract. the horvath’s rock lizard (iberolacerta horvathi) is a rupicolous mountain species endemic of the eastern alps and northern dinaric range. the species has its known western limit of the distribution in the veneto region of italy. it is not known whether the species is really rare in veneto or whether the area has been insufficiently surveyed. in addition, it is not known whether the westward distribution of the species is limited by a physiographic or by a climatic barrier. during the period 2016-2018, 118 sites were surveyed in the veneto and trentino-alto adige regions. four new occurrences of iberolacerta horvathi were discovered in veneto that: 1) largely fill the gap between the westernmost known site and the closest site to the east; 2) extend further west the known distribution by 9 km. in addition the species was confirmed in three already known sites. a species distribution model was developed with the software maxent, using 100 occurrences from italy, austria and slovenia. the best model shows that the distribution is explained by the asperity of their habitat, the sedimentary bedrock, the aspect, the average temperature of the coldest quarter, the rainfall seasonality and the average summer rainfall. the last variable appears as the most likely responsible for the rarefaction of the species at its western limit. in addition, the species distribution model suggest that the horvath’s rock lizard might be present in some additional mountain groups where it has so far not been found yet. keywords. horvath’s rock lizard, maxent, rainfall, temperature, roughness, aspect, bedrock, veneto. introduction the present distribution of reptiles in europe is much dependent on its climatic history, in particular to the pleistocene oscillations. after the climate improvement following the last glacial maximum (18,000 bp) many species spread from southern refugia and have colonized very large areas (joger et al., 2007). some species were able to spread from their refugia in a more limited way due to ecological constraints or to the presence of geographical barriers like the alps for species from the apennines peninsula and the pyrenees for species from the iberian peninsula (joger et al., 2007). the opposite happened to some cold adapted species, like the dinolacerta mountain lizards of the southern dinaric chain and the species of iberolacerta of the iberian peninsula: they have likely reduced their range and have become restricted to high altitude isolated mountain peaks (ortega et al., 2016). the conservation prospect of these high altitude species is likely going to worsen due to the ongoing climate change (le galliard et al., 2012). a different case is the distribution of the horvath’s rock lizard (iberolacerta horvathi), an endemic species of the northern dinaric chain and eastern alps (north4 giuseppe de marchi et alii west croatia, slovenia and adjoining northeastern italy and southern austria) (speybroeck et al., 2016), a species phylogenetically related to a group of mountain dwelling lizards of the iberian peninsula (carranza et al., 2004, crochet et al., 2004). as a matter of facts, the known presence localities in italy and austria are in areas that were glaciated during the last glacial maximum (ivyochs et al., 2009). so, the species should have colonized the eastern alps from refugia in the southeastern alps or from the dinaric chain. unfortunately, it is difficult to study how this colonization happened due to insufficient knowledge of the distribution of this species, as suggested by the recurrent discoveries of its presence in new mountain groups (e.g., žagar et al., 2014). this lack of data is likely the result of the difficult accessibility of the rocky cliffs inhabited by the species and by the possible misidentification with the similarly looking (žagar et al., 2012) and frequently syntopic common wall lizard, podarcis muralis (cabela et al., 2007). in italy, horvath’s rock lizard is well distributed in the friuli region (lapini et al., 2004; rassati, 2010; rassati, 2012) but its presence appears to be particularly scattered west of the piave river in the veneto region. the westernmost site was discovered in 1993 close to the village of listolade (lapini and dal farra, 1994; lapini et al., 2004), a site that was about 70 km away from the nearest known site. in spite of subsequent discoveries (rassati, 2010; rassati, 2012; lapini, 2016) and quite extensive surveys (tormen et al., 1998; bonato et al., 2007; bonato, 2011; cassol et al., 2017) there is still a gap of around 30 km between the site of listolade and the closest one to the east. therefore, it is not known whether the species is present in this large gap and even west of listolade. moreover, it would be interesting to discover whether the species has reached its potential distribution or whether its westward post glacial expansion is still lagging behind its potential distribution, as it still happens for some plants (svenning and skov, 2007; willner et al., 2009; dullinger et al., 2012) and animals (araújo et al 2008; pinkert et al., 2018). in addition it is not known which factors might limit or have limited the westward spread of the species. in order to fill these gaps in the knowledge of the species, the research has the following aims: 1) to improve the known distribution by surveying mountain groups west of the piave river in the trentinoalto adige and veneto regions of italy; 2) to correlate the presence of the species in italy, austria and slovenia to climatic, geological, physiographic and ecological parameters in order to estimate, through a species distribution model, whether the western limit of the distribution has likely been fixed or whether new sightings further west should be expected. material and methods field survey surveys were conducted during 94 days in the period april 2016 september 2018 in 118 sites by one to three researchers per site. 20% (n = 24) of the sites were visited more than once. surveyed areas were concentrated west of the piave river but a few sites were investigated also just east of it. due to the inaccessibility of many cliffs, investigated sites were not randomly distributed but were inevitably concentrated close to roads and mountain paths. species identification was rarely performed by catching the specimens with a long stick with a cotton loop. more commonly, species identification was performed by analysing pictures taken with a telephoto lens, an efficient technique also when lizards are on inaccessible rocky cliffs (de marchi et al., 2019). identification characters from the frequently syntopic common wall lizards are as in corti et al. (2010). the coordinates and altitude of the sites were obtained by hand held gps devices. in addition, the sites where horvath’s rock lizards were found were characterized by the bedrock obtained from the geological map of italy viewed on the geoportale nazionale (http://www.pcn.minambiente.it) and by the habitat classification obtained from the “carta della natura” of veneto and “carta della natura” of friuli-venezia giulia (1:50.000) (http://www.geoviewer.isprambiente.it/). species distribution model (sdm) in order to elaborate the potential distribution of the species in italy and in neighbouring austria and slovenia, a presence only modeling approach was chosen due to the lack of reliable absence points particularly outside our study area. a maximum entropy approach was developed with the software maxent (phillips et al., 2006; merow et al., 2013; phillips et al., 2017), which proved to be quite successful even when occurrence data were few and in presence of somehow biased samples (elith et al., 2006). as occurrence data of veneto are few and restricted to a too small area for statistical analyses, published data related to italy (lapini et al., 2004; rassati, 2010; rassati, 2012; lapini, 2016; lapini, 2017; rassati, 2017), austria (tiedeman, 1992; ortner, 2006; cabela et al., 2002; cabela et al., 2007) and slovenia (žagar, 2008a; žagar, 2008b; krofel, 2009; cafuta, 2010; žagar et al., 2011; žagar et al., 2012; osojnik et al., 2013; žagar, 2016; zauner, 2018) were collated. such a wider area helps to reduce the risk that some local climatic conditions might wrongly highlight the importance of an environmental variable that in fact is not a relevant one (phillips, 2006). unfortunately, some published occurrences lacked coordinates but have only the altitude and a general description of the collecting site: only when the description was considered sufficiently detailed to be confidently associated to a single 300 m × 300 m pixel (see later) the occurrences were used by extracting the coordinated from google-earth. as some occurrences are aggregated due to sampling bias, some occurrence localities were manually removed so that the remaining ones are at least 5westward expansion of the horvath’s rock lizard one km from the closest ones. eventually, the 100 occurrences of the horvath’s lizard were joined together with a background dataset of 10,000 points to randomly sample the analysed area (lat 45.35°n to 47.00°n, lon 11.20°e to 15.00°e). the environmental layers (all at european scale) were as follows. 19 bioclimatic variables at 30-arc seconds (about 1km) resolution derived from the monthly temperature and rainfall values for the period 1970-2000 (http://www.worldclim.org/ bioclim). slope, roughness (the degree of irregularity of the surface, calculated by the largest inter-cell difference of a central pixel and its surrounding cell, as suggested by wilson et al., 2007) and aspect (the compass direction that a slope faces with values ranging 0-360°) layers calculated with the gis software qgis (version 2.8.6) from a 25m digital elevation model (layer: dem-v1.1-e40n20) downloaded from the copernicus land monitoring service (https://land.copernicus.eu/). a 250m vegetation layer, corine land cover clc_12 (version 18_5), again downloaded from the copernicus land monitoring service. a geological layer from the 1 : 5 million international geological map of europe and adjacent areas (igme5000, asch, 2003) with bedrock types reclassified in the following five categories: crystalline (all igneous and metamorphic rocks), carbonates (limestone, dolomite or carbonates as predominant bedrocks), secondary carbonates (limestone, dolomite or carbonates locally present but not as predominant bedrocks), other sedimentary rocks (like sandstone, conglomerate et clay) and undifferentiated bedrock. all environmental layers were resampled to a resolution of 300 m using the package “raster” in r (hijmans and van etten, 2016). while maxent is partly able to balance between model fitting and model simplicity, an information approach was adopted to check whether simpler models with fewer variables performed better (warren and seifert, 2011; zeng et al., 2016). first, a reduced set of environmental variables was pre-selected, based on ecological or biological knowledge. out of the 19 available bioclimatic variables, bio1 (annual mean temperature), bio4 (temperature seasonality), bio5 (max temperature of warmest month), bio6 (min temperature of coldest month), bio10 (mean temperature of warmest quarter), bio11 (mean temperature of coldest quarter) bio12 (annual precipitation), bio 15 (precipitation seasonality), bio18 (precipitation of warmest quarter) and bio19 (precipitation of coldest quarter) were selected. then, a reiterative process of model formation and stepwise removal of the least contributing variables was utilized (zeng et al., 2016). unfortunately, most temperature related variables (bio1, bio5, bio6, bio10, bio11) were highly correlated (pearson coefficient >0.7). in a similar way most rainfall related variables (bio12, bio18, bio19) were highly correlated. therefore, maxent models were run in order to select the single most predictive temperature variable and the single most predictive rainfall variable. this was done (table 2) by running maxent models (regularization multiplier was set at 1 and regularization values were linearquadratic-product) alternatively containing only one of the correlated variables for temperature (bio1, bio5, bio6, bio10, bio11) and only one of the correlated variables for rainfall (bio12, bio18, bio19). among the alternative models the one with the lowest akaike information criterion corrected for low sample size (aicc) calculatd with the software enmtools version 1.4.4 (warren and seifert, 2011; shcheglovitova and anderson, 2013) was selected. the aicc is an estimator of the relative quality of statistical models for a given set of data, trading-off between the simplicity of the model and the goodness of fit of the model (warren and seifert, 2011). then, simpler models were constructed by stepwise removal of the variable with the lowest permutation importance (zeng et al., 2016) and the aicc of the resulting models was calculated in order to select the final set of variables. as ‘‘feature class’’ and ‘‘regularization multiplier’’ affect the performance of the model (morales et al., 2017) the model was fine tuned by constructing models with 7 levels of the regularization multiplier (0.05, 0.1, 0.5, 1, 2, 4, 10) and three regularization values (linear-quadratic, linear-quadratic-product and hinge). finally, the best model (regularization value = linear-quadratic; regularization multiplier = 0.1) was run 100 times with bootstrap resampling and the average model was calculated. the quality of this final model was tested by calculating the area under the curve (auc) of the receiver operated characteristic (roc) plots in order to discriminate a species’ model from a random model. finally, the percent contribution and permutation importance of the environmental variables to the final maxent model were measured (phillips, 2006). results the horvath’s lizard was found in four new sites (valle di san lucano, val del grisol, casoni and forra del piave) and its presence was confirmed in three sites (monte tudaio, val zemola and listolade) already known in literature (fig. 1). fig. 2 shows the habitat of the four new occurrences for horvath’s lizard and table 1 reports some characteristics of all the seven sites. the common wall lizard was observed in 60% of the investigated sites (n = 71, fig. 1) and was frequently syntopic with the horvath’s rock lizard, where this last species occurred (table 1). species distribution model. the mean temperature of coldest quarter (bio11) turned out to be a better predictor of the distribution of the horvath’s rock lizard (table 2) compared to other highly correlated temperature variables: annual mean temperature (bio1), max. temperature of warmest month (bio5), min. temperature of coldest month (bio6) and mean temperature of warmest quarter (bio10). the precipitation of warmest quarter (bio 18) turned out to be a better predictor of the distribution (table 2) compared to other highly correlated precipitation variables: annual precipitation (bio12) and precipitation of coldest quarter (bio19). 6 giuseppe de marchi et alii the balancing between the simplicity of the model and goodness of fit to the known distribution resulted in a model that did not include slope, temperature seasonality (bio4) and vegetation type (corine land cover), the variables with the lowest permutation importance. as a result, the best model was explained only by mean temperature of coldest quarter (bio11), rainfall seasonality (bio15), precipitation of warmest quarter (bio18), aspect, roughness and bedrocks (table 2). the best distribution model obtained after fine tuning (see methods and table 2) is shown in fig. 3 (with cloglog output, which is the easiest to conceptualize as it gives an estimate between 0 and 1 of probability of presence). the model fits the distribution (average training auc for the 100 bootstrap replicate runs is 0.922, standard deviation is 0.009) much better than a random model (auc = 0.5). the variables that explain the distribution of the species are in order of percent contribution (table 3): roughness (33%), bedrocks (26.5%), mean temperature of coldest quarter (19.5), aspect (8.9), rainfall of the warmest quarter (8.5%) and rainfall seasonality (3.3). rainfall of the warmest quarter decreases westward in the italian alps (fig. 4): the 13 westernmost occurrences are in areas with a rainfall (average±sd = 338 ± 37mm, n = 13) that is significantly lower (welch test = -9.67, p < 0.001) than the remaining occurrences (average ± sd = 452 ± 53mm, n = 87). discussion our field survey has discovered four new sites of horvath’s rock lizard in veneto (table 1 and fig. 1 and 2): forra del piave is close to already known sites; casoni (in the bosconero mountain group) and val grisol (in the schiara mountain group) fill the gap between the known westernmost site of horvath’s lizard and the previously known sites to the east; san lucano valley (in fig. 1. sites surveyed in 2016-2018. red dots show the newly discovered occurrences of iberolacerta horvathi, while orange dots show some confirmed occurrences. 7westward expansion of the horvath’s rock lizard pale di san martino mountains) extends further west the known distribution by 9 km. our extensive research in apparently suitable sites west of san lucano valley, in particular in the vette feltrine mountains and in the valsugana valley (fig. 1 and fig. 4), has been unable to discover additional sites. this lack of positive results suggests that the western limit of the distribution has been discovered. interestingly, the san lucano valley is also the western limit in the southern dolomite mountains of another reptile of eastern origin, the nose-horned viper (vipera ammodytes) (sindaco et al., 2006), a species that has been found to have a close habitat similarity with horvath’s rock lizard also in another part of its distribution (žagar et al., 2013). there is apparently no physiographic barrier to the spread of the lizards further to the west. as a matter of fact, a gravel bed mountain river like the cordevole river (fig. 4) should have not been a barrier to the westward spread of the species as proved by our discovery of the species just west of this river in the san lucano valley. in the past, gravel bed mountain rivers such as the piave and the cordevole (fig. 4) should have been an even less effective barrier to the spread of the species during the phase of great sedimentation in the valleys that followed the deglaciation (cordier et al., 2017) and continued up to 7000 bc in the piave valley (carton et al., 2009): in this condition, water mostly flowed deep inside the gravel bed (arscott et al., 2001) and could not stop the spread of lizards. therefore, the lack of any apparent physiographic barrier lets us suggest that an environmental gradient should make the species less abundant in veneto, where it is definitely rare, until it disappears. in general, an environmental variable might affect directly the species physiology (e.g., temperature) or might act in an indirect way by influencing fig. 2. pictures showing the habitats of the newly discovered sites of horvath’s rock lizard. 1. forra del piave, cadore; 2. casoni, canal del maè; 3. val del grisol; 4. valle di san lucano, agordino. in the last site, the first single individual was observed on the rock wall along the road; later, the presence of a more numerous population was documented in the neighbouring rocky cliffs (around 200 m away). 8 giuseppe de marchi et alii the availability of food (e.g., rainfall) or the presence of parasites, predators or competitors (austin, 2002). an important factor that might limit the abundance and the distribution of the horvath’s rock lizard may be the strong competition with the common wall lizard (podarcis muralis) (carranza et al., 2004). the two species have a similar overall body plan (žagar et al., 2012) and similar ecological preferences, for example they eat similar food (richard and lapini, 1993). however, horvath’s rock lizards appear more adapted to rocky habitats due to their flat head and body (corti et al., 2010), which might favour hiding in small crevices. they are more adapted to relatively cold habitats due to a higher potential metabolic activity (žagar et al., 2015b), even if they do not exhibit the partial development of the egg inside the female body that is found in some other small mountain lizards (ljubisavljević et al., 2012). on the contrary, common wall lizards are favoured in agonistic interactions with horvath’s rock lizards because of their larger head and consequent stronger biting force (žagar et al., 2017), for example when competing for thermoregulation spots (žagar et al., 2015a). common wall lizards may be favoured as well for their higher reproductive potential (amat, 2008). as a result, horvath’s rock lizards are confined to rocky habitats in low altitude shady gorges, at mid altitude in beech forest and to higher altitudes compared to the common wall lizards (žagar, 2016). vegetation type has not turned out to be important in our analysis for predicting the distribution of the horvath’s rock lizards likely because the corine land cover classification that is currently available for our study area has been developed only to the “third level”. this means, for example, that forests are classified only to the level of “coniferous forests” or “mixed forests”, a level that is likely not enough for a proper classification. apart from the land cover classification, our analysis of the importance of the variables that constrain the distribution of the horvath’s lizard (table 3) largely agrees with the previous results (cabela et al., 2007; žagar, 2016). table 1. characteristics of the new and confirmed occurrences of horvath’s lizard surveyed in 2016-2018. the number associated to each site is the same as in fig.1 and fig. 2. site * new + confirmed date (day, month, year) coord. (°n, °e) altitude (m) aspect bedrock (from geoportale nazionale italiano) vegetation (cnat of veneto and friuli-venezia giulia) observed specimens syntopic common wall lizard 1. old road in forra piave, santo stefano di cadore (bl), veneto * 13.08.16 08.08.17 46.53516, 12.50901. 46.53258, 12.49400 825-870 s r67: neritic and platform dolomites (medium triassic) scots pine oriental forest up to 3 yes 2. casoni, canal del maè, forno di zoldo (bl), veneto * 25.08.17 27.08.17 01.07.18 08.07.18 46.30444, 12.22972 750-850 so r58: pelagic limestones, marly limestone and marl (jurassic) thermophilic calciphile beech forest of the alps up to 8 yes 3. grisol de entro, val del grisol, longarone (bl), veneto * 25.07.18 46.27017, 12.22878 825 se r58: pelagic limestones, marly limestone and marl (jurassic) ostrya carpinifolia thicket 1 yes 4. pont, san lucano valley, taibon agordino (bl), veneto * 6.8.2018 9.8.2018 17.8.2018 46.30201, 11.91715. 46.30200, 11.91658. 46.30113, 11.91685. 12001250 ne-e r67: neritic and platform dolomites (medium triassic) calciphile fir forest of the alps and central-northern apennines 4 no 5. monte tudaio, vigo di cadore (bl), veneto + 12.08.16 46.51413 12.47563 886 s r67: neritic and platform dolomites (medium triassic) scots pine oriental forest 1 yes 6. val zemola, erto and casso (pn), friuli venezia giulia + 18.06.17 08.07.18 46.28607, 12.37178 1020 ne r56: neritic and platform limestones and sometimes dolomites (jurassic) thermophilic calciphile beech forest of the alps up to 2 no 7. climbing rock, listolade, (bl), veneto + 22.05.16 25.09.16 13.04.17 09.09.18 46.32622, 11.99672 720 so r67: neritic and platform dolomites (medium triassic) acidophile scots pine forest up to 11 yes 9westward expansion of the horvath’s rock lizard roughness turns out to be important certainly due the preference of the horvath’s lizard for steep rocky areas. however, the pixel resolution of our analyses (300 m), constrained by the imprecision of the coordinates of the occurrences, is not enough to discriminate between rocky and simply steep slopes. that’s why some areas that are likely unsuitable, like visentin mountain just south of belluno, are predicted as suitable (fig. 3 and 4). the categorical variable bedrock confirmed the importance of sedimentary rocks for the presence of the species with the highest suitability (more than 70%) in areas with the dominant bedrocks consisting of carbontable 2. aicc scores of different maxent species distribution models varying in number of variables, features and regularization multipliers (see methods for details). the best model (in bold) is the one with the lowest aicc model # variables features reg.mult aicc 1 bio1,bio4, bio12, bio15, aspect, asperity, slope, corine, bedrock lqp 1 2459.82 2 bio5,bio4, bio12, bio15, aspect, asperity, slope, corine, bedrock lqp 1 2468.09 3 bio6,bio4, bio12, bio15, aspect, asperity, slope, corine, bedrock lqp 1 2472.79 4 bio10,bio4, bio12, bio15, aspect, asperity, slope, corine, bedrock lqp 1 2465.77 5 bio11,bio4, bio12, bio15, aspect, asperity, slope, corine, bedrock lqp 1 2453.00 6 bio11,bio4, bio18, bio15, aspect, asperity, slope, corine, bedrock lqp 1 2447.56 7 bio11,bio4, bio19, bio15, aspect, asperity, slope, corine, bedrock lqp 1 2463.85 8 bio11,bio4, bio18, bio15, aspect, asperity, corine, bedrock lqp 1 2452.31 9 bio11, bio18, bio15, aspect, asperity, corine, bedrock lqp 1 2447.72 10 bio11, bio18, bio15, aspect, asperity, bedrock lqp 1 2429.10 11 bio11, bio15, aspect, asperity, bedrock lqp 1 2430.33 12 bio11, bio18, bio15, aspect, asperity, bedrock lqp 0.05 2424.50 13 bio11, bio18, bio15, aspect, asperity, bedrock lqp 0.1 2431.21 14 bio11, bio18, bio15, aspect, asperity, bedrock lqp 0.5 2424.09 15 bio11, bio18, bio15, aspect, asperity, bedrock lqp 2 2442.47 16 bio11, bio18, bio15, aspect, asperity, bedrock lqp 4 2462.02 17 bio11, bio18, bio15, aspect, asperity, bedrock lqp 10 2529.97 18 bio11, bio18, bio15, aspect, asperity, bedrock lq 0.05 2423.22 19 bio11, bio18, bio15, aspect, asperity, bedrock lq 0.1 2422.58 20 bio11, bio18, bio15, aspect, asperity, bedrock lq 0.5 2424.63 21 bio11, bio18, bio15, aspect, asperity, bedrock lq 1 2431.17 22 bio11, bio18, bio15, aspect, asperity, bedrock lq 2 2447.09 23 bio11, bio18, bio15, aspect, asperity, bedrock lq 4 2459.09 24 bio11, bio18, bio15, aspect, asperity, bedrock lq 10 2530.51 25 bio11, bio18, bio15, aspect, asperity, bedrock h 0.5 2795.60 26 bio11, bio18, bio15, aspect, asperity, bedrock h 1 2602.81 27 bio11, bio18, bio15, aspect, asperity, bedrock h 2 2529.67 28 bio11, bio18, bio15, aspect, asperity, bedrock h 4 2532.34 29 bio11, bio18, bio15, aspect, asperity, bedrock h 10 2571.21 table 3. relative contributions of the environmental variables to the best maxent model (see methods for details) for the distribution of horvath’s rock lizard. variable percent contribution (%) permutation importance (%) roughness 33.3 26.7 bedrock 26.5 16.6 temperature of the coldest quarter 19.5 38 aspect 8.9 6 rainfall of the warmest quarter 8.5 3.5 rainfall seasonality 3.3 9.2 10 giuseppe de marchi et alii ates, dolomite and limestone, and with an intermediate suitability (40%) for areas where these three types of rocks are present, even if they are not dominant. a lower suitability (about 28%) was found for other types of sedimentary bedrocks. the remaining two types of bedrocks (metamorphic-magmatic and undifferentiated) were predicted as unsuitable. this result likely stems from the need of the lizard for fissured rocks where they can hide from predators and where rock dwelling plants (important for attracting the invertebrate food) can take root. the inclusion of temperature as an important variable was similarly expected as the species is generally considered cold-adapted and able to live at higher altitudes and in shadier places compared to the frequently syntopic common wall lizard (žagar, 2016). why the temperature of the coldest quarter turned out to be the most imporfig. 3. the best species distribution model of horvath’s rock lizard in the alps and neighbouring dinaric chain based on 100 occurrences (black dots) in italy, austria and slovenia. the output is “cloglog”, which gives an estimate between 0 and 1 of the probability of presence. fig. 4. rainfall (the lighter areas the higher rainfall) during the warmest quarter. yellow dots show the 100 occurrences of horvath’s rock lizard. the light blue line is the 335 mm isohyet. the location of some areas mentioned in the discussion are added. 11westward expansion of the horvath’s rock lizard tant temperature related variable is not clear. it might be related to the duration of the snow cover and a too brief warm season for the species to thrive. instead, the importance of a physiological limit for survival at low temperatures would have probably resulted in a higher importance of the minimum winter temperature. aspect is of course important as the lizards need direct sunlight for warming up and the north aspect is unsuitable (žagar, 2008a). so, in spite of the low resolution of the occurrences that might mask the exact aspect of the cliff, this variable is a significant percent contribution (8.9%) in explaining the best model of distribution. our model shows the importance of two rainfall related variables, rainfall seasonality and average rainfall during the warmest quarter. rainfall seasonality increases westward in the eastern alps (data not shown) but why rainfall seasonality might be important for the ecology of the species is not clear yet. its ability to explain the distribution is, at any rate, very low (3.3%). the higher percent contribution of summer rainfall (8.5%) might stem from its likely importance to the growth of rupicolous plants where the horvath’s rock lizards look for food. common wall lizards are comparatively less dependent on this hunting ground as they are much more euryecious (sindaco et al., 2006). in addition, summer rainfall might be directly important as a source of drinking water as we have observed horvath’s lizards drinking in wet, dripping parts of the cliffs. interestingly summer rainfall decreases westwards in the eastern alps and the 13 westernmost occurrences (almost all in veneto) are all close to the 335 mm isohyet (fig. 4). so, the rarity of the species west of the piave river and its disappearance further west might effectively depend upon a reduced rainfall during the warmest quarter. two mountain groups, the monti del sole and the vette feltrine, appear suitable (fig. 3) and are not separated by any significant physiograghic barrier from the four westernmost occurrences (fig. 4); they should deserve additional field surveys even if a recent research (cassol et al., 2017) and our data have so far failed to confirm the species in those areas. the major limitation of our study comes from the low precision of the occurrences that did not allow a deeper analysis of the roughness and the inclusion of other factors that can be important, such as solar radiation, which can change radically within a few meters in rocky areas and is certainly important in the context of the competition with the common wall lizard. future species distribution models based on more precise occurrences (ernst, 2017) and on finer scaled climatic variables (now at 1 km resolution) can improve our understanding of the ecological niche of the species. hopefully, phylogeographic studies (cocca et al., 2016) will soon shed light on the glacial refugia where the horvath’s lizards survived before colonizing the deglaciating alps. aknowledgements we thank the italian ministry of environment for authorizing the research (prot. 0018347/pnm of 1 september 2016). we thank: michele cassol for suggesting a useful coauthorship and possible locations to be surveyed; luca lapini for encouragement and timely information on a new site he discovered; gianluca rassati for providing the unpublished coordinates of two sites; steven 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(2016): novel methods to select environmental variables in maxent: a case study using invasive crayfish. ecol. model. 341: 5-13. acta herpetologica vol. 15, n. 1 june 2020 firenze university press has the west been won? a field survey and a species distribution model of iberolacerta horvathi in the alps giuseppe de marchi1,*, giovanni bombieri1, bruno boz1, fausto leandri2, jacopo richard3 first record of underwater sound produced by the balkan crested newt (triturus ivanbureschi) simeon lukanov identification of biologically active fractions in the dermal secretions of the genus bombina (amphibia: anura: bombinatoridae) iryna udovychenko1,*, oleksandra oskyrko1, oleksii marushchak2, tetiana halenova1, oleksii savchuk1 don’t tread on me: an examination of the anti-predatory behavior of eastern copperheads (agkistrodon contortrix) andrew adams1,2,*, john garrison2, scott mcdaniel2, emily bueche2, hunter howell2,3 an overview of research regarding reservoirs, vectors and predators of the chytrid fungus batrachochytrium dendrobatidis jarid prahl, thomas p. wilson*, david giles, j. hill craddock genetic characteristics of an introduced population of bombina bombina (linnaeus, 1761) (amphibia: bombinatoridae) in moselle, france jean-pierre vacher1, 2,*, damien aumaître3, sylvain ursenbacher2,4 adaptive significance of the transparent body in the tadpoles of ornamented pygmy frog, microhyla ornata (anura, amphibia) santosh m. mogali*, bhagyashri a. shanbhag, srinivas k. saidapur comparison of complement system activity amongst wild and domestic animals maría s. moleón1,2,*, mark e. merchant3, hugo h. ortega4, pablo a. siroski1,2 effects of temperature and food level on plasticity of metamorphic traits in bufo gargarizans gargarizans larvae tong lei yu*, yan ting han acta herpetologica 15(2): 73-85, 2020 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/a_h-7771 estimating abundance and habitat suitability in a micro-endemic snake: the walser viper gentile francesco ficetola1,2,*, mauro fanelli3, lorenzo garizio3, mattia falaschi1, simone tenan4, samuele ghielmi5, lorenzo laddaga6, michele menegon7,8, massimo delfino3,9 1 dipartimento di scienze e politiche ambientali, università degli studi di milano, milano, italy 2 laboratoire d’écologie alpine, univ. grenoble alpes, univ. savoie mont blanc, cnrs, leca, grenoble, france 3 dipartimento di scienze della terra, università di torino, torino, italy 4 national research council, institute of marine sciences (cnr-ismar), venezia, italy 5 muse museo delle scienze, trento, italy 6 società di scienze naturali del verbano cusio ossola, museo di scienze naturali, collegio mellerio rosmini, domodossola italy 7 division of biology & conservation ecology, school of science & the environment, manchester metropolitan university, manchester, uk 8 pams foundation, p.o. box 16556, arusha, tanzania 9 institut català de paleontologia miquel crusafont, universitat autònoma de barcelona, cerdanyola del vallès, barcelona, spain *corresponding author. e-mail: francesco.ficetola@unimi.it submitted on: 2020, 1st january; revised on: 2020, 12th june; accepted on: 2020, 26th september editor: dario ottonello abstract. recently described species suffer lack of information that hampers setting up appropriate conservation strategies. the situation is particularly complex with micro-endemic snakes, for which detection and monitoring are particularly challenging. the walser viper vipera walser is a recently described snake inhabiting a small area of the sw italian alps. we combined information on species distribution with repeated monitoring to identify the areas most suitable for the species, and to obtain estimates of species abundance. species distribution models were used to identify the topographical, climatic, and land-cover features related to the occurrence of vipers. furthermore, repeated transects and n-mixture models were used to estimate abundance and to identify factors related to the variation of abundance. the available data suggested that the species has a disjunct range, with a northern range of ~45 km2, and a southern range of ~225 km2. distribution models suggested that vipers are associated with areas with open vegetation, altitude between 1300 and 2300 m, high precipitation, low forest cover, low slope, and southern aspect. n-mixture models confirmed very low detection probability of these vipers, and suggested that the species has a low abundance, with the highest abundance in south-facing plots. we provide the first quantitative information on habitats and abundance variation for walser vipers. the broad confidence intervals of abundance estimates exemplify the complexity of providing range-wide measures of abundance for secretive species. given the narrow range of these vipers, continuous monitoring is required to understand how they respond to ongoing environmental changes in mountainous areas. keywords. alpha-hulls, detectability, endemism, habitat suitability models, land cover, vipera walser. introduction italy is among the european countries with the highest endemism of amphibians and reptiles (sillero et al., 2014). in the last decades, the integration of new genetic and morphological data has greatly expanded our knowledge of italian biodiversity, with the identification of several reproductively isolated lineages, that have been pro74 gentile francesco ficetola et alii posed as new candidate species (e.g., cornetti et al., 2015; dufresnes et al., 2018; senczuk et al., 2019). the walser viper has been a striking addition to the italian herpetofauna. in ne piedmont, the occurrence of vipers morphologically assumed to be adders (vipera berus) has long been recognized (andreone and sindaco, 1998; sindaco et al., 2006). however, recent genetic data revealed strong genetic differences between these “adder-like” vipers and true adders; the closest relatives of these vipers belong to the cluster regrouping v. dinniki, v. kaznakovi and v. darevskii, in the caucasus area. as a consequence, these adder-like vipers have been described as a new species, the walser viper (vipera walser, ghielmi et al., 2016), a species endemic of a small area from the mountains between biella and the ossola valley, with a possible range < 500 km2 (fig. 1). recently described, micro-endemic species often suffer a tremendous lack of biological data. this is particularly problematic, because the small range inherently exposes these species to a high risk of extinction. therefore, sound biological and ecological information is required to set up appropriate monitoring and conservation programs. habitat suitability and spatial variation of abundance represent critical information to assess the conservation status of species. in the last years, advances in analytical tools have greatly improved our ability to provide sound biological information even in species for which only a limited amount of data is available (e.g., raxworthy et al., 2003; peterson et al., 2011; mazerolle, 2015). in this paper, we combine modelling and field data to improve our knowledge of the ecology of the walser viper. first, we used habitat suitability models to evaluate the relationships between viper distribution, land cover, and topographical features at a fine spatial scale. correlative species distribution models (sdm) allow to identify relationships between localities of presences and spatial variation of environmental variables, and can provide multiple key information (peterson et al., 2011). sdm can reveal how the species respond to broad-scale variation of environmental features, thus providing first information on the habitats that are more suitable for the target species (guisan and thuiller, 2005; peterson et al., 2011). these models often have a coarse spatial scale and sometimes lack accurate information on species microhabitat or on habitat features that can only be measured in the field (beck et al., 2012; potter et al., 2013; ficetola et al., 2018b). still, if reliable maps of habitats or land cover are available, it is possible to obtain relatively detailed information on species responses to major land cover categories. furthermore, sdm can produce spatially-explicit maps, that can refine information on species range. for instance, raxworthy et al. (2003) developed sdm for chameleons in madagascar, and then performed targeted surveys in areas suggested to be suitable by models. in several cases, these targeted surveys revealed new localities of occurrence, suggesting that sdm can greatly improve our knowledge on species living in remote areas, or for which information on the distribution is limited. second, we performed repeated visits in a large number of patches within the species range, to assess spatial variation of abundance. variation of abundance is a key parameter to assess the threat status of species, still, accurate measurement of abundance can require extremely extensive workload (pollock et al., 2002; dodd, 2010). in the last years, approaches have been proposed to obtain estimates of abundance from repeated counts, without marking or capturing individuals (royle and nichols, 2003; royle, 2004). the number of individuals counted at fixed sites on multiple occasions can be used to estimate the detection probability, and the size of populations can be estimated on the basis of n-mixture models (royle and nichols, 2003; royle, 2004; kéry et al., 2009; dail and madsen, 2011). despite several limitations (barker et al., 2018; link et al., 2018), such models can provide cost-effective estimates of abundance while accounting for imperfect detection (ficetola et al., 2018a; kéry, 2018), as detection probability is typically low in snakes. in addition, n-mixture models can provide insights on the factors determining spatial variation of population abundance and, if monitoring is repeated through time, they can provide reliable estimates of population trends (ficetola et al., 2018c). these data are particularly critical for recently described species, for which information on abundance is nearly absent. methods species range and distribution models in march 2019, we combined bibliographic data (ghielmi et al., 2006; ghielmi et al., 2016) with new field observations performed in 2016-2018 by the authors, and personal communications by local naturalists, to gather an exhaustive dataset of v. walser occurrences. we used the alpha-hull approach for an accurate definition of the species range. the alpha-hull approach is a procedure based on delauney triangulation that uses presence points to estimate species ranges, and can allow for the exclusion of unoccupied areas within a species range (burgman and fox, 2003). simulations showed that the alphahulls provide better approximations of species ranges compared to minimum convex polygons, particularly when the ranges have discontinuities (burgman and fox, 2003). alpha-hulls were built using the alphahull package in r (pateiro-lopez and 75abundance and habitat of vipera walser rodrjguez-casal, 2010), following the procedure detailed in ficetola et al. (2014). land cover data were obtained from the geoportal of the piedmont region (http://www.geoportale.piemonte.it/) at the resolution of 2 m. we considered four land cover categories: agricultural, pasture, natural open vegetation, and forest. as a measure of land cover, we calculated the percentage of each land cover category in 100 × 100 m cells. furthermore, we considered three topography variables: altitude (m a.s.l.), aspect (northness), and slope. as a climatic parameter, we considered summed annual precipitation, obtained through the chelsaclim dataset (karger et al., 2017). this dataset is available at a coarser resolution than the other predictors (30 arc-seconds, approx. 650 × 900 m in the study area). we therefore used the b-spline interpolation to downscale it at the 100-m resolution (see karger et al., 2017). temperature variables were not included because they were strongly collinear with altitude (r > 0.9). the pairwise correlation between the remaining environmental variables was always |r| < 0.65, suggesting limited collinearity issues (dormann et al., 2013). we used maximum entropy modelling (maxent) (phillips et al., 2006; elith et al., 2011) to build species distribution models relating the occurrence of vipers to land cover and topographical data. maxent is a presence-background approach that evaluates the suitability of a given cell on the basis of environmental features (habitat, climate, topography…) in that cell. comparative studies showed that maxent is among the most efficient approaches to build sdm (elith et al., 2006; elith et al., 2011). maxent is well suited to evaluate complex or nonlinear relationships between species and environmental features, and produces an output representing the suitability of a specific area. we run models with linear, quadratic, and hinge features. to identify the best regularization parameter (b), we build five models with increasing values of b: 1, 2, 5, and 10 (warren and seifert, 2011). the model with b = 2 showed the highest crossvalidation performance (see below), and was selected as model with highest generality (warren and seifert, 2011). we used two approaches for the validation of species distribution model. first, the model was tested using a 5-fold crossvalidation. presence records were split into five sub-sets; the model was built using 80% of data (calibration data), and we tested predictive performance on the remaining 20% (test data). we repeated this procedure five times, each time using a different test dataset (nogués-bravo, 2009). as a measure of model performance, we calculated the area under the curve of the receiver operator plot (auc), averaged over the five runs. auc is an imperfect measure of the performance of sdm (lobo et al., 2008), thus we also used a binomial test to evaluate if our models predict presence records better than expected under randomness, comparing the observed frequencies of correct and incorrect predictions. in this test, we assumed that a cell is suitable for vipers if it has suitability larger than 10th percentile training test threshold (pearson et al., 2007). second, the model was developed in late winter 2019. therefore, we used data collected during the 2019 field activities to confirm the reliability of model predictions. specifically, we used a likelihood ratio test to assess whether viper observations are more frequent in areas with high sdm suitability, compared to areas with low suitability. since we have a-priori expectation on the frequency of observations (i.e., we expect more observations in high-suitability areas), we used a one-tailed test (warren and seifert, 2011). field activities in the period 2016-2019, we established 71 fixed plots using visual encounter surveys (crump and scott, 1994). each plot was visited during one year only (range: 5-26 plots per year); plots were visited multiple times (average: 5.7 surveys per plot; range: 3-12 surveys) from late may to early october, i.e., during the period of highest activity of vipers. the size of plots ranged between 600 and 23,000 m2 (mean: 7600 m2), because of logistic and accessibility constraints. visits were performed from 7 am to 17 pm (solar time), but most of them (69%) were performed in late morning (between 8.30 and 12.00 am). plots were placed across the whole range of the species, mostly nearby areas with previous records of walser vipers. in 2019, the location of plots was selected after the development of species distribution models, in order to validate the sdm and identify eventual new locations of the viper. out of the 17 plots surveyed in 2019, three were in areas with low sdm suitability (suitability below the 10th percentile training presence threshold; range: 0.06-0.22) but nearby areas with high suitability. ten were in areas with high sdm suitability (> 0.45) and nearby localities where the species is known to be present, and four were in areas with high suitability but out of the known species range (mombarone; roughly 3 km se of the southern limit of the species range, see fig. 1b). during surveys, one to four observers carefully patrolled the plots, searching for active vipers. in other areas, the search under artificial shelters greatly improved the detection of reptiles (de leo et al., 2006; joppa et al., 2010; sewell et al., 2012). therefore, in 2019, visual encounters were integrated with the use of artificial cover objects (shelters). in each plot, we placed three corrugated bitumen 70 × 90 cm shelters that were checked during each survey. assessment of species abundance we used n-mixture models to estimate the abundance of vipers on the basis of repeated counts at plots, and to identify environmental variables related to variation of abundance. n-mixture models allow the joint estimation of animal abundance and detection probability on the basis of repeated surveys at fixed sites, without the need of capturing and marking individuals for identification (royle, 2004). some analyses highlighted that jointly estimating detection probability and abundance could be problematic, and these models are sensitive to violations of their assumptions (barker et al., 2018; duarte et al., 2018), still analyses of real-world data showed that n-mixture models can provide reliable estimates of the abundance of wild vertebrates (ficetola et al., 2018a; kéry, 2018; costa et al., 2019). we assumed that each plot, sampled during one single season, represented a closed population. we thus used a static (i.e., non-dynamic) n-mixture model formulation. the average distance between each plot and the closest one was 170 m (se 76 gentile francesco ficetola et alii = 19.6), a distance much longer than the typical movements of related species (e.g., zuffi et al., 1999; nash and griffiths, 2018). n-mixture models were fitted using a poisson error distribution, as in preliminary analyses poisson models showed a lower akaike’s information criterion (aic) (burnham and anderson, 2002) than the corresponding zero-inflated poisson models. in n-mixture models, we set the upper bound to approximate the infinite summation of the likelihood (k) at the maximum observed species abundance +100, since simulations suggested that this value provides stable and robust estimates (ficetola et al., 2018c). we considered four variables potentially influencing the detection of vipers: date (day of the year), hour, length of the survey (in minutes, log transformed), and the number of people participating in the survey. for date and hour, we also tested quadratic terms to take into account potential non-linear relationships. to identify the model best explaining variation of viper detection, we built models including all the potential combinations of independent variables, and ranked them on the basis of their aic values. the model with lowest aic value explains more variation with a limited number of parameters, and was selected as the minimum adequate model (burnham and anderson, 2002). first, we performed model selection on variables affecting detectability, then we performed model selection on variables affecting abundance. we considered six variables potentially affecting the abundance of vipers at plots: plot surface, aspect, altitude, slope, and suitability predicted by the species distribution model. sdm suitability was included because previous studies suggested that environmental suitability models also help to predict parameters such as population density and fitness-related traits (brambilla and ficetola, 2012; weber et al., 2017; lunghi et al., 2018). variables representing land cover were not included because 85% of plots were in areas with open natural vegetation, which is the most important land cover variables determining viper distribution (see results). we built models with all the possible combination of variables potentially affecting abundance, and ranked them using aic, while keeping constant the observational component of the model (i.e., including the variables best explaining variation in detection probability). the correlation coefficient between site covariates was weak (r < 0.4 for all the considered variables). n-mixture models were run with the package unmarked in r (fiske and chandler, 2011). before analyses, independent variables were scaled at mean = 0 and sd = 1 to improve model convergence. a goodness-of-fit test showed limited overdispersion and confirmed that the model was appropriate to the data (χ2 = 432.6, permutation p = 0.12, c-hat = 1.1). results distribution data, species range, and distribution model overall, we gathered 117 distribution records (fig. 1a). localities from the literature (ghielmi et al., 2006; ghielmi et al., 2016) accounted for 50% of records, while 47% of records were obtained through recent surveys by the authors; we also obtained some personal communications by local naturalists (3% of records). species records were clustered in two main areas: a northern group between the strona and the upper sesia valley, and a southern group between the lower sesia valley and the biella alps. the lack of known records between these two areas suggests the possibility of a disjunct distribution, with a northern range of approx. 45 km2, and a southern range of approx. 225 km2. the species distribution model suggested that vipers were associated to areas with open natural vegetation, altitude between 1300 and 2300 m, high annual precipitation, low forest cover, low slope, and with a southern aspect (fig. 2). the presence of agricultural land cover and pastures showed very limited importance (relative importance < 1%). the cross-validation procedure suggested excellent performance, with average auc on test data = 0.924 (sd = 0.038). using the 10th percentile training presence threshold (suitability threshold = 0.36), the model correctly predicted occurrence at 86.3% of test data, a performance significantly better than expected by chance (binomial test: p << 0.001; success rate expected under randomness = 13.5%). the model identified several patches of highly suitable habitats through the whole species range, in mountainous areas at altitude of 1300-2200 m, with gentle, south-facing slopes and covered by open vegetation. within the species range, the total suitable surface (i.e., with suitability higher than the 10th percentile threshold) was 81.9 km2. several areas with good suitability were detected outside the known range of the species, for instance, in the sw of the study area (e.g., the mombarone area) (fig. 1b). potentially suitable areas were also detected between the northern and southern populations. model validation with the 2019 data four plots surveyed in 2019 were outside the known range of v. walser, but in areas showing high suitability according to the sdm (mombarone area; fig. 1b). each of them received six surveys; we never detected vipers at these plots. thirteen plots surveyed in 2019 were inside the known range of v. walser; four were in areas with low suitability (suitability ≤ 0.2) and nine in areas with high suitability (> 0.45). vipers were never detected in the plots with low suitability, while they were detected in 56% of plots with high suitability. the possibility to obtain at least one detection was significantly higher in plots with high suitability (likelihood ratio test: χ21 = 4.96, p = 0.013), confirming that the sdm can successfully predict viper occurrences. 77abundance and habitat of vipera walser a) b) c) fig. 1. a) distribution records used to build species distribution models, and species range estimated using alpha-hulls. b) location of fixed plots where we performed repeated counts. the arrow indicates the plots located in possibly suitable sites outside the known range of the species (mombarone). c) suitability map, estimated using species distribution models. 0.37 is the 10th training presence threshold. to limit the risk of poaching, points plotted on the maps have a random spatial error of up to 2 km (lunghi et al., 2019). d) an adult male of v. walser (photo by gff). 78 gentile francesco ficetola et alii repeated counts and abundance estimations overall, we obtained 39 viper detections from 24 out of the 67 plots within the species range. vipers were detected across the whole study period (from june to early october). the best-aic model suggested that the number of detection was only affected by the length of surveys, longer surveys allowing a higher probability of detecting vipers (b ± se = 0.73 ± 0.16, z = 4.45, p < 0.001). conversely, the detection rate was unrelated to date or hour of the survey, nor to the number of observers (table 1). the detection probability of vipers was approx. 0.036 in 35-min. surveys (35 min. was the average length of surveys), but the incertitude of these estimates was large (95% ci of detection probability after 35 min: 0.007-0.16). the abundance of vipers was negatively related to the northness of plots, with higher abundances in south-facing plots (b = -0.70 ± 0.30, z = -2.35, p = 0.019). we did not detect any relationship between the remaining variables and the abundance of vipers (table 1). the estimated abundance per plot ranged between zero and five individuals; the best estimate of total abundance across the 67 plots was 175 vipers, but confidence intervals were wide (95% ci: 38-383 vipers). therefore, at the surveyed plots, the average density was 3.4 individuals / ha. discussion for recently described species, rapid biological and ecological studies are pivotal for a prompt assessment of the species status. by combining distribution records with ecological modelling and repeated surveys we provide key ecological data on the endemic, poorly known, walser viper. the application of alpha-hulls to species distribution data suggested that this viper has a disjunct range, with a northern range comprising the strona and the upper sesia valley, and a southern range between the biella alps and the lower sesia valley. alpha-hulls have an excellent capacity to reveal discontinuities in the specie range (burgman and fox, 2003), still they can be affected by the lack of biological records caused, for instance, by limited surveys. the available genetic data did not detect strong differences among walser vipers (ghielmi et al., 2016), still additional genetic or genomic studies, using highly variable or fast-evolving markers, are needed to understand the fine-scale genetic variation among the different populations. habitat suitability species distribution models showed that viper distribution is related to the interplay between topographic, climatic, and habitat parameters. first, vipers are associated with gentle, south-exposed slopes at altitudes between 1500 and 2300 m. this study was performed on a rather small geographical extent, and at this scale, the correlation between altitude and the available temperature data is almost perfect (r > 0.9), therefore altitude can be considered as a proxy of temperature. the narrow altitudinal range suggests a very narrow thermal niche, which is typical of many micro-endemic species (quintero and wiens, 2013; slatyer et al., 2013; cunningham et al., 2016). this inherently exposes micro-endemic species to a high risk of extinction, and is particularly alarming under scenarios of climate change (botts et al., 2013; slatyer et al., 2013; böhm et al., 2016). until recently, walsers vipers were assumed to be adders vipera berus (andreone and sindaco, 1998; sindaco et al., 2006). however, it should be remarked that the altitudinal range of walser’s vipers does not match the one observed for adders in the southern alps. present-day adder populations living in lombardy (approx. 80 km e of the study area) have a broad altitudinal range (550-2500 m) and 26% of records are above 2000 m (bernini et al., 2004). conversely, all available walser viper data are restricted at altitudes of 1300-2300 m, with just 4% of records above 2000 m. this suggests that walser vipers have a table 1. a) relationships between detections of vipera walser and variables potentially influencing detection. the table reports the outcome of univariable n-mixture models, in which only each of these variables was related to the viper detections. all the models with more than one variable showed higher aicc values than the one with length of survey only. b) relationships between detections of vipera walser, and variables potentially influencing abundance. the table reports the outcome of n-mixture models, in which viper detection probability was related to length of survey, and viper abundance was related to each of these variables. all the models with more than one variable showed higher aicc values than the model with aspect only (appendix 1). aicc z p a) variables potentially influencing detection date* 262.9 -0.321 0.749 hour* 261.6 -1.14 0.256 n observers 262.8 0.51 0.610 length of survey 244.4 4.48 <0.001 b) variables potentially influencing abundance altitude* 246.7 0.13 0.895 aspect 238.6 -2.35 0.019 slope 245.2 1.24 0.217 plot area 246.7 -0.22 0.830 precipitation 245.7 1.03 0.302 sdm suitability 243.5 1.66 0.097 * preliminary tests did not show any effect also for quadratic terms. 79abundance and habitat of vipera walser narrower niche than adders, still, additional studies are required to compare the niches of the two species. second, vipers were associated to areas with relatively high precipitation levels and high cover of natural, open vegetation. the walser viper range includes some of the valleys with the highest rainfall in the alps (mercalli et al., 2008). high precipitation levels can allow the existence of a relatively humid environment with open vegetation, which is the main habitat of this species (ghielmi et al., 2016). given the very small surface of suitable habitats, ensuring the long-term persistence of these environments will be essential for the long-term survival of walser vipers. altitude (m) aspect (northness) slope open vegetation coverforest cover 0 1000 2000 3000 4000 0 1 2 3 0 0.4 0.8 1.2 0 50% 100%0 50% 100% s ui ta bi lit y s ui ta bi lit y s ui ta bi lit y 0.0 0.3 0.6 0.9 0.3 0.6 0.9 0.0 0.3 0.6 0.9 0.3 0.6 0.9 0.3 0.6 0.9 annual precipitation (mm) 600 1000 1400 1800 2200 0.2 0.4 0.6 1.0 0.8 fig. 2. relationship between environmental variables and suitability, as estimated by species distribution models. the dark lines are the average response across the cross-validated runs; the confidence bands indicate ± one standard deviation. 80 gentile francesco ficetola et alii variation in abundance repeated counts and n-mixture models can provide estimates of the absolute abundance of individuals, and are particularly useful when no data from other sources are available. our models showed that, for individuals of walser viper, detection probability is low (around 4% in standard surveys), and the species generally has a limited abundance (average: 3.4 individuals / ha). a low detection probability has been often observed in snakes (luiselli et al., 2011; rodda, 2012), and density estimates are comparable to the values observed in other studies (reviewed in santini et al., 2018a). for instance, neumeyer (1987) used capture-mark-recapture to assess the abundance of vipera berus in subalpine environments, and found a density of approx. 3 individuals / ha. translating modelled abundance in density estimates is often complex. nevertheless, the home range size of the study species is likely small compared to plot size, thus the derived density probably does not suffer by temporary emigration (kery and royle, 2016). our plots covered well the whole range of the species (fig. 1b), still performing extrapolations of abundance across a species range is extremely complex (santini et al., 2018b). if we assume that the relationship between viper density and plot aspect is constant across the whole range, we can perform projections of the spatial variation of potential density (fig. 3) that, in turn, could be used to obtain rough estimates of abundance across the species range. such a projection would lead to a best unbiased prediction of 26,000 vipers, but the associated confidence intervals are extremely wide (95% intervals: 5500-130,000 vipers). these figures can be useful to obtain a first approximation of abundance when no other data are available, but must be taken with extreme caution. first, model extrapolation outside the sampled areas is always challenging, because we cannot be sure that the relationship between habitat and abundance is constant across the range. importantly, species distribution models often overestimate species distribution (guisan and rahbek, 2011). this occurs because additional factors can influence species abundance, for instance, when a given patch is unsuitable because of the presence of a limiting factor not considered in this analysis. second, the reliability of estimates obtained through n-mixture heavily depends on the verification of model assumptions (barker et al., 2018; link et al., 2018). for instance, unmodelled heterogeneity of detection probability can heavily bias total estimates of abundance (link et al., 2018), and it is unlikely that our model took into account all the potential factors affecting viper detections. third, the reliability of abundance estimates is sensitive to variation of detection probability, and low values of detection probability always challenge models that rely on unmarked individuals (ficetola et al., 2018c). unfortunately, the detection probability of vipers was extremely low. many conservation agencies require quantitative measures of abundance (e.g., iucn, 2001; stoch and genovesi, 2016), but obtaining reliable estimates, with high accuracy and limited uncertainty, can be challenging. our study showed that uncertainty can be large even for micro-endemic species for which a very large number of surveys is performed. given the low detectability, an alternative approach for the monitoring of walser vipers could rely on occupancy modelling (mackenzie et al., 2017). modelling co-occurrence in an occupancy framework or in a joint sdm framework may help improving inference on species occupancy when data for other species are available (e.g., common lizard). in addition, distribution may be better inferred using spatially and temporally replicated data in an occupancy framework, instead of presence-only data alone in a sdm framework. fig. 3. spatial variation of the density of vipera walser: best predictions of the n-mixture model, assuming that abundance is affected by slope. the map indicates the averaged density (individuals / ha); 95% confidence intervals of the estimates are available in the legend. projections have only been performed for the most suitable areas within the species range, according to the results of the species distribution model. 81abundance and habitat of vipera walser one solution would be integrating opportunistic information (i.e., presence-only data, probably the most abundant and widely distributed source of information for the species) with detection-non detection data from an occupancy design (dorazio, 2014). recommendation for monitoring and conservation for data deficient species, a key goal for the near future is certainly improving knowledge on distribution, abundance, and threats. walser vipers are rare and elusive, thus improving information on total abundance and population trends will be essential. repeated counts at fixed sites are an excellent strategy for a consistent assessment of temporal variation in abundance, and have been recommended for the monitoring of many reptiles (stoch and genovesi, 2016). however, a large number of sites and surveys are required for a correct estimate of trends in elusive species. for instance, several tens of sites, each surveyed multiple times per year, are required if we want to obtain reliable estimates of species trends with dynamic n-mixture models (ficetola et al., 2018c). the plots surveyed in this study can provide the needed baseline for the development of a long-term monitoring program. alternatively, approaches involving the capture of individuals can be used to collect individual longitudinal data simultaneously to occupancy data. capture-mark-recapture information would allow estimating population growth rate, the factors affecting it and its temporal variability. this can be done without the need to estimate population size and with the freedom to simultaneously test effects on drivers of demographic rates (e.g., survival, recruitment, and fecundity) (tenan et al., 2014). despite the importance of future monitoring, conservation biologists must identify priorities and plan management actions even if information is incomplete (soulé, 1985). our data can already be useful for the conservation planning of walser vipers. distribution data and species distribution models suggest a very restricted distribution, with a total extent of occurrence < 300 km2, and an estimated area of occupancy = 86.5 km2. a limited geographic range is a key criterion for redlist assessment. for instance, the iucn (2001) classifies as threatened under the criterion b species that have small geographic range (extent of occurrence < 5000 km2 and / or area of occupancy < 500 km2), and also satisfy at least two of these three conditions: a) severely fragmented or restricted number of locations; b) continuing decline of the species or his habitat; c) extreme demographic fluctuations. the walser viper has a very small range and is known from a limited number of locations. until now, no information is available on the decline of his habitat, and unrecorded habitat loss is possible. remote sensing data are an efficient approach to assess habitat trends through time, when ground information is lacking (tracewski et al., 2016). data from corine land cover suggest that the cover of suitable habitats within the species range has remained stable during the last decade, with approx. 124.6 km2 of natural open vegetation in 2006, and 125.6 km2 in 2018. however, during the last decades, the cover of open vegetation has decreased in several areas of the alps because of the abandonment of traditional livestock farming (falcucci et al., 2007), and this could cause habitat loss for walser vipers in the future. if future monitoring will identify declines of abundance, occupancy, or of suitable habitat, walser vipers can be classified as endangered according to the iucn redlist criteria. climatic change and the loss of open natural vegetation are the strongest threats to the biodiversity of montane environments of europe (brambilla et al., 2010; 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(1999): winter activity in a coastal population of vipera aspis (reptilia, viperidae). revue d’ecologie-la terre et la vie 54: 365-374. 85abundance and habitat of vipera walser appendix appendix s1. n-mixture models relating the detections of vipera walser to variables potentially influencing abundance. the table reports the regression coefficient of each variable included in each model; models are ranked on the basis of the corrected akaike’s information criterion. only models with weight >0.001 are shown. k: n of parameters in the model. model length of survey aspect altitude plot area precipitation slope k aicc weight 1 0.69 -0.70 4 238.6 0.312 2 0.69 -0.68 0.06 5 240.8 0.103 3 0.70 -0.70 -0.02 5 240.9 0.098 4 0.69 -0.70 -0.01 5 240.9 0.097 5 0.69 -0.70 0.00 5 240.9 0.097 6 0.70 -0.71 0.10 -0.07 6 243.2 0.032 7 0.70 -0.68 -0.02 0.06 6 243.2 0.031 8 0.69 -0.68 -0.01 0.06 6 243.2 0.031 9 0.70 -0.70 -0.01 -0.02 6 243.3 0.029 10 0.70 -0.70 -0.02 0.00 6 243.3 0.029 11 0.69 -0.70 -0.01 0.00 6 243.4 0.029 12 0.73 3 244.4 0.017 13 0.72 0.21 4 245.2 0.012 14 0.71 -0.71 -0.02 0.10 -0.07 7 245.7 0.009 15 0.74 0.17 4 245.7 0.009 16 0.70 -0.71 0.00 0.10 -0.07 7 245.7 0.009 17 0.70 -0.68 0.00 -0.02 0.06 7 245.7 0.009 18 0.70 -0.70 -0.01 -0.02 0.00 7 245.8 0.008 19 0.74 -0.04 4 246.7 0.006 20 0.72 0.03 4 246.7 0.006 21 0.72 0.06 0.17 5 247.4 0.004 22 0.73 -0.04 0.21 5 247.5 0.004 23 0.72 0.00 0.21 5 247.5 0.004 24 0.75 -0.03 0.16 5 248 0.003 25 0.74 0.02 0.17 5 248 0.003 26 0.71 -0.71 0.00 -0.02 0.10 -0.07 8 248.2 0.003 27 0.74 0.03 -0.04 5 249 0.002 28 0.73 -0.03 0.06 0.17 6 249.8 0.001 29 0.72 0.01 0.06 0.17 6 249.8 0.001 30 0.73 0.00 -0.04 0.21 6 249.9 0.001 31 0.75 0.03 -0.03 0.17 6 250.4 0.001 acta herpetologica vol. 15, n. 2 december 2020 firenze university press estimating abundance and habitat suitability in a micro-endemic snake: the walser viper gentile francesco ficetola1,2,*, mauro fanelli3, lorenzo garizio3, mattia falaschi1, simone tenan4, samuele ghielmi5, lorenzo laddaga6, michele menegon7,8, massimo delfino3,9. potential effects of climate change on the distribution of invasive bullfrogs lithobates catesbeianus in china li qing peng1, min tang1, jia hong liao1, hai fen qing1, zhen kun zhao1, david a. pike2, wei chen1,* a bibliometric-mapping approach to identifying patterns and trends in amphibian decline research claudio angelini1,*, jon bielby2, corrado costa3 food composition of a breeding population the endemic anatolia newt, neurergus strauchii (steindachner, 1887) (caudata: salamandridae), from bingöl, eastern turkey kerim çiçek1,*, mustafa koyun2, ahmet mermer1, cemal varol tok3 stomach histology of crocodylus siamensis and gavialis gangeticus reveals analogy of archosaur “gizzards”, with implication on crocodylian gastroliths function ryuji takasaki1,2,*, yoshitsugu kobayashi3 does chronic exposure to ammonium during the pre-metamorphic stages promote hindlimb abnormality in anuran metamorphs? a comparison between natural-habitat and agrosystem frogs sonia zambrano-fernández1, francisco javier zamora-camacho2,3,*, pedro aragón2,4 confirming lessona’s brown frogs distribution sketch: rana temporaria is present on turin hills (piedmont, nw italy) davide marino1, angelica crottini2, franco andreone3,* phylogenetic relationships of the italian populations of horseshoe whip snake hemorrhois hippocrepis (serpentes, colubridae) francesco paolo faraone1, raffaella melfi2, matteo riccardo di nicola3, gabriele giacalone4, mario lo valvo5* first karyological analysis of the endemic malagasy phantom gecko matoatoa brevipes (squamata: gekkonidae) marcello mezzasalma1,2,*, fabio m. guarino3, simon p. loader1, gaetano odierna3, jeffrey w. streicher1, natalie cooper1 notes on sexual dimorphism, diet and reproduction of the false coral snake oxyrhopus rhombifer duméril, bibron & duméril, 1854 (dipsadidae: pseudoboini) from coastal plains of subtropical brazil fernando m. quintela1,*, felipe caseiro¹, daniel loebmann¹ acta herpetologica 13(1): 65-73, 2018 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/acta_herpetol-22465 exploring body injuries in the horseshoe whip snake, hemorrhois hippocrepis juan m. pleguezuelos*, esmeralda alaminos, mónica feriche dep. zoología, fac. ciencias, univ. granada, e-18071 granada, spain. *corresponding author. e-mail: juanple@ugr.es submitted on: 2017, 18th september; revised on: 2017, 10th november; accepted on: 2018, 7th january editor: dario ottonello abstract. body injuries on snakes may serve as indices of predation rate, which is otherwise difficult to record by direct methods because of the usual scarcity and shy nature of these reptiles. we studied the incidence of body scarring and tail breakage in a mediterranean colubrid, hemorrhois hippocrepis, from south-eastern spain. this species exhibits two traits that favour tail breakage, a long tail and active foraging tactics. the large sample (n = 328) of individuals acquired also enabled us to determine how the incidence of these injuries varies ontogenetically, between the sexes, in relation to energy stores, and to seek for the possible existence of multiple tail breakage. overall, the incidence of tail breakage was rather low (13.1%) and similar to that of body scarring (12.7%). however, there was a significant ontogenetic shift in the incidence of tail breakage; such injuries barely appeared in immature snakes but stubtailed individuals were present in 38.7% of snakes in the upper decile of body size. in contrast to most other studies, our data show a sex difference in the incidence of these injuries, adult male rates quadrupling those of adult females for body scarring and males maintaining larger stubs, probably because of the presence of copulatory organs housed in the base of the tail. larger individuals did not have shorter tail stubs, a finding that does not support the multipletail-breakage hypothesis. our results also fail to support the idea of a cost of tail breakage or body scarring in terms of body condition. keywords. body scarring, predator-prey relationship, spain, tail breakage introduction in snakes, direct evidence of predator-prey interactions is difficult to obtain under natural conditions, because of their secretive behaviour (willis et al., 1982; gregory, 2016). many species, however, exhibit tail breakage, a defence that favours escape from predators (arnold, 1988; greene, 1988) and that can be used as indirect evidence of the rate of predation (pianka, 1970; martins, 1996). when grasped by the tail, after thrashing vigorously and rotating the body longitudinally in one direction, some snakes undergo an intervertebral tail breakage (greene, 1988; todd and wassersug, 2010). in contrast to typical saurians, the tail of snakes is not regenerated following breakage (pseudoautotomy; savage and slowinski, 1996), leaving a permanent record of the event. a possible drawback of this way of assessing predation pressure in snakes is that tail breakage could simply indicate inefficient predation, because the prey survives (schoener, 1979; medel et al., 1988; mushinsky and miller, 1993). however, some studies have found that snakes from study sites with fewer predators show a much lower frequency of tail breakage than did those from sites with far more predators (placyk and burghardt, 2005; santos et al., 2011). nonetheless, ecological factors other than predator pressure might influence the frequency of body injuries (placyk and burghardt, 2005); for example, tail breakage may also result both from encounters with dangerous prey (gregory and issac, 2005), a possibility that can be assessed only in study 66 juan m pleguezuelos et alii areas lacking snake predators (e.g., in pantelleria island; cattaneo, 2015), as well as from incomplete skin sloughing and parasitism (degenhardt and degenhardt, 1965; harkewicz, 2002). other body injuries of snakes, such as body scarring, might also indicate the rate of predatory attacks, although body scarring mostly reflects the efficiency of such attacks (placyk and burghardt, 2005; sparkman et al., 2013); that is, inefficient predators tend to inflict injuries upon their prey, but the prey tend to survive, and the opposite for efficient predators (seligmann et al., 1996). in recent decades interest has increased in analysing the rate of body damage in snakes and has provided evidence of ontogenetic and sexual variation in body damage (mendelson, 1992; fitch, 2003), correlations between injury rates and life-history traits (pleguezuelos et al., 2007), habitat-related differences (akani et al., 2002), differences in predation pressure among populations (santos et al., 2011), geographic distribution of injuries (dourado et al., 2013), and the cost of such injuries (pleguezuelos et al., 2013). the horseshoe whip snake, hemorrhois hippocrepis, a medium to large-sized colubrid distributed throughout the western mediterranean basin, exhibits two traits that favour the appearance of body damage, particularly through tail breakage: (1) it is an active forager and (2) is long tailed (pleguezuelos and feriche, 2014). in animals, foraging mode (active vs sit-and-wait; schoener, 1971) influences other natural history traits of the species, such as predation pressure (webb et al., 2003). active foraging strategies normally imply high rates of exposure to potential predators. that is, while the forager actively searches for food, it increases the risk of predation from other predators (huey and pianka, 1981; secor, 1995), and even of injuries from prey (greene, 1997). with respect to the second trait, tail breakage is known to have a higher incidence in long-tailed snakes (willis et al., 1982; mendelson, 1992; bowen, 2004), including h. hippocrepis (marco, 2002). we examined the frequency of tail breakage and body scarring in h. hippocrepis from the south-eastern iberian peninsula. the large sample of specimens also enabled us to test several hypotheses about patterns of injury acquisition: 1. the tail-breakage/body-scarring hypothesis, which predicts that fewer specimens will have scars on the body than will show damaged tails, as a consequence of attacks to the body being more lethal than attacks to the tail resulting in tail breakage (placyk and burghardt, 2005). 2. the size-related hypothesis, which predicts that the frequency of body injuries in snakes should be higher in larger and presumably older individuals, because they have been exposed to potential predators for a longer time than smaller ones have (seligmann et al., 2008; pleguezuelos et al., 2010). 3. the sex-related hypothesis has a dichotomy: i) some authors predict that the frequency of tail breakage or body scarring in snakes will be higher in males, because individuals of this sex move greater distances during the mating period than do females, being more exposed to potential predator attacks (vitt et al., 1974), and indeed, many more adult males than adult females have been recorded for this species in the study area during the mating season (pleguezuelos and feriche, 2014); ii) other studies have reported lower frequencies of tail breakage in males (willis et al., 1982; mendelson, 1992; placyk and burgahardt, 2005), which has been explained by the higher mortality and the decrease in reproductive success of the stub-tailed individuals of this sex (shine et al., 1999). we will consider also the effect of sexual dimorphism in body size in the appearance of sexual differences in tail breakage. 4. the multiple-breakage hypothesis predicts that the stumps of damaged tails will be shorter in larger and presumably older individuals than in smaller ones, as a consequence of snakes suffering multiple breakage events over life (savage and crother, 1989; slowinski and savage, 1995). 5. the cost-of-injuries hypothesis, which predicts that despite the immediate advantage of tail breakage or body scarring for the survival of the individuals, these wounds also imply negative consequences. the costs would derive from impaired locomotion (martin and avery, 1998), and consequent decreased foraging ability, reproductive output or survival (wilson, 1992; downes and shine, 2001; maginnis, 2006). the cost-of-injuries hypothesis predicts that damaged individuals are smaller (because they survive for a shorter time) and have lower body condition than undamaged ones would (pleguezuelos et al., 2013). material and methods study area we examined specimens of h. hippocrepis from an area of approximately 20 000 km2 in the south-eastern iberian peninsula (coordinates 37°15’-38°45’n; 2°30’-5°45’w); all specimens were from altitudes between 0-1100 m a.s.l. mean annual temperature averaged 16.4oc, and the average yearly rainfall was 412.1 mm (data summarized in moreno-rueda et al., 2009). the landscape is dominated by cultivation (olive orchards, cereal crops), mixed areas of evergreen forest and scrubland 67body injuries in the horseshoe whip snake (quercus ilex), and pine plantations (pinus sp.). within the study area, h. hippocrepis is preyed upon by three reptiles, four diurnal raptors and one mammal species (pleguezuelos and feriche, 2014), and predator composition is rather consistent. data on morphology and body condition the data (n = 328) came from vouchers in the collection of the university of granada (spain), collected from 1985-2015 as road kills or killed by local people. we measured snout-vent length (svl ± 1 mm) of all specimens, as well as the number of subcaudal scale pairs (sbc) in tail-damaged specimens. adult individuals were sexed by dissection. we checked for tail completeness and classified individuals as tail damaged or tail undamaged. this classification was conservative, as we considered the tail to be damaged only in individuals with a healed tip of the tail. non-predatory agents, such as incomplete skin sloughing (harkewicz, 2002), can also lead to tail loss. to minimize bias derived from these circumstances, individuals with just the apical scale missing were not considered cases of tail breakage. we also checked all individuals for the presence of body scars, again conservatively, ignoring road-killed specimens that were badly damaged (i.e., with body parts that could not be evaluated for body scarring; fitch, 2003). to analyse if our results were confounded by the provenance of most samples (road-kills), we compared the prevalence of body injuries in our data set with a small sample of animals captured alive in the same study area from 2005-2015 and released at the capture site after measurements. we tested the tail-breakage/body-scarring hypothesis for adult individuals only by comparing the frequency of those characters between groups (by a contingency table; individuals with damaged tail and those with body scarring). if this hypothesis is true, there will be more individuals with tail damaged than with body scars. the hypothesis of ontogenetic shift in tail breakage was assessed following the procedure in pleguezuelos et al. (2010). we divided body size into 10 classes of equal numbers (n = 31) and fitted the best function (among linear, exponential and polynomial, according to the r coefficient) to the relationship between frequency of tail breakage with body size (medians of each body size class). if this hypothesis is true, the rate of tail breakage will be higher in the classes of the largest (because probably older) individuals. we tested the sexual hypothesis by comparing (by a contingency table) the frequency of adult males and females (males svl > 540 mm, females svl > 680 mm; pleguezuelos and feriche, 2014) with damaged tails and body scarring. if this hypothesis is true, it would be sexual differences in the frequency of body injuries. we tested the multiple-tail-breakage hypothesis by correlation of the number of remaining sbc on svl, the latter trait acting as a crude surrogate for age, using only the subsample of snakes with damaged tail (mendelson, 1992); if this hypothesis is true, the number of sbc should diminish with svl of the individuals. because of the minor sexual dimorphism in the sbc scales (feriche et al., 1993), we pooled the two sexes. we tested the body-condition hypothesis by comparing fat-body level in injured vs. non-injured individuals (by the mann-whitney u test). in snakes, body condition is strongly influenced by fat-body reserve (bonnet et al., 2003), and body condition of our sample was estimated according to the importance of fat bodies (waye and mason, 2008). we were unable to accurately weigh some road-killed specimens or their fat bodies, so we scored fat reserves of h. hippocrepis into five visual categories, from 0-4 (description of the procedure in pleguezuelos and feriche, 1999). because the fat-body reserves in this species exhibit ontogenetic, sexual, and seasonal shifts (pleguezuelos and feriche, 1999), we excluded immature individuals from this analysis, thereby removing most of the ontogenetic effect, and then considered the two sexes separately. however, we were unable to account for the seasonal effect on fat-body level (because of sample size), although most of the individuals of the sample came from spring. if this hypothesis is true, injured individuals will exhibit worst body condition than noninjured ones. distributions of data were checked for normality prior to analyses; mean values are followed by ± one sd with alpha set at 0.05. the statistical analyses were performed using statistica 7.0. results we analysed 126 adult males, 104 adult females, and 98 immature or of undetermined sex of h. hippocrepis. the sample size for the different traits considered here varied, because of the varied preservation status of the vouchers. the tail breakage and body scarring hypothesis across the study area, 13.1% of h. hippocrepis voucher individuals had damaged tails (n = 328) and 12.7% had scars (n = 180), mostly in the body (n = 16) and a few on the tail (n = 5), with only one on the head. only eight individuals shared both injuries, tail breakage and body scars. there was no difference in the frequency of individuals with tail breakage and body scarring (2 × 2 table, χ2 = 0.36, p = 0.54). the small sample of live individuals exhibited approximately the same prevalence of tail breakage (12.5%, n = 32; χ2 = 2.35, p = 0.12) and body scarring (8.3%, n = 24; not compared statistically because of small sample size) as the sample of road-kill snakes. the size-related hypothesis stub-tailed snakes were larger than those without tail breakage (x ± sd = 880.6 ± 268.6 mm, n = 42 vs. 606.8 ± 68 juan m pleguezuelos et alii 222.9 mm, n = 266; t = 6.269, p << 0.001, test of homogeneity of variances, p = 0.149), and this result held true when the sexes were considered separately (t test, 123 males and 102 females, p = 0.01 in both comparisons). individuals with body scars were larger than unscarred specimens (x ± sd = 1005.1 ± 128.1 mm, n = 22 vs. 746.3 ± 217.9 mm, n = 162; m-w u test, z = 5.23, p << 0.001), results that again held when the sexes were considered separately (m-w u test, 95 males and 69 females, p = 0.05 in both comparisons). moreover, the frequency of tail breakage increased according a quadratic function with increasing body size; fewer than 5% of snakes in immature size classes exhibited tail breakage, and 38.7% of snakes in the body class of largest snakes (1005-1292 mm svl) exhibited tail breakage (fig. 1). because of small sample size, this analysis was not executed for the individuals with body scars. the sex hypothesis there was a difference between the sexes in body injuries only for body scarring: 20.9% of the males (n = 86) and 4.7% of the females (n = 63; 2 × 2 table, χ2 [yates corrected] = 4.95, p = 0.02) exhibited body scarring, and 27.3% of the males (n = 99) and 16.2% of the females (n = 74; 2 × 2 table, χ2 = 1.90, p = 0.16) exhibited tail breakage. moreover, there was a sexual difference in the number of sbc scales remaining in the stub of damaged tails: males had more sbc pairs (x ± sd = 63.9 ± 20.6, n = 22) and hence had a longer tail, on average, in broken tails than did females (x ± sd = 44.9 ± 22.3, n = 11; t = 2.43, p = 0.05; test of homogeneity of variances, p = 0.72). the mean number of sbc in males and females with intact tails proved very similar (feriche et al., 1993). the multiple-tail-breakage hypothesis tails of h. hippocrepis can break over almost their entire length, as we found broken tails with between 15-96 pairs of remaining sbc scales (mean number of sbc scales for individuals with intact tails from the study area, 102.8; pleguezuelos and feriche, 2014). the number of sbc scales remaining on stub-tailed individuals did not differ according to body size, whether sexes were combined (r = 0.283, n = 47, p = 0.115; fig. 2) or considered separately (males, r = 0.413, n = 22, p = 0.06; females, r = -0.386, n = 10, p = 0.27). thus, larger individuals did not have shorter tail stumps than smaller individuals, a finding that does not support the multipletail-breakage hypothesis. the cost-of-injuries hypothesis fat-body level did not differ between stub-tailed and whole-tailed snakes (x ± sd = 2.46 ± 1.26, n = 28 vs. 2.29 ± 1.26, n = 153; m-w u test, z = 0.572, p = 0.56), and this result held when the sexes were considered sepafig. 2. relation of subcaudal scale pairs to body size (snout-vent length) in hemorrhois hippocrepis from se iberian peninsula affected by tail breakage. fig. 1. fit of tail-breakage frequency (number of snakes in each size class with tail breakage) to medians of 10 custom body-size classes (snout-vent length) in hemorrhois hippocrepis from se iberian peninsula (n = 310). the criterion for the limits of the body-size classes was to have the same sample size (n = 31). the solid line represents the quadratic function estimated (y = 0.000018x2 0.0034x + 0.547; r2 = 0.972, p << 0.001). 69body injuries in the horseshoe whip snake rately (m-w u test, 86 males and 72 females, p = 0.79 in both comparisons). fat-body level did not differ between snakes with body scars and those without scars (x ± sd = 2.70 ± 1.26, n = 17, vs 2.53 ± 1.18, n = 100; m-w u test, z = 0.605, p = 0.54); because of small sample size, separate analyses were not performed for each sex. these results for h. hippocrepis fail to support a cost of tailbreakage or body-scarring in terms of body condition. discussion general analysis and the relationship between trail breakage and body scarring when a snake moves widely, it increases its risk of predation (huey and pianka, 1981; secor, 1995). hemorrhois hippocrepis is an active forager (pleguezuelos and feriche, 2014) and thus would be expected to undergo high predation risk and a high rate of body injuries (mushinsky and miller, 1993). however, the rate of tail breakage that we observed is rather low compared with other snakes (see mendelson, 1992; aubret et al., 2005; gregory and isaac, 2005; pleguezuelos et al., 2010; dourado et al., 2013, and references therein). we interpret the low rate of body injuries as reflecting the habits of this species, i.e., being a fast-moving snake that frequently dwells in vertical habitats (rocky slopes), traits propitious for escaping from predators. consistent with this, h. hippocrepis has been recorded in only 8.7% of the pellets of the main snake-eater in the study area, the shorttoed eagle, circaetus gallicus, whereas another colubrid of similar size and foraging habits but slow moving, rhinechis scalaris, was present in 39.5% of the pellets (gilsánchez and pleguezuelos, 2001). in pantelleria island (sicily channel), cattaneo (2015) reported a very high incidence of tail breakage (55.6%, n = 34) for the (probably introduced) population of h. hippocrepis. however, this result was influenced by the large body size of individuals in the sample (see below); furthermore, injuries apparently were not caused by predators, but by prey, as the adults of this snake population rely mainly on a large and dangerous prey, rattus rattus (cattaneo, 2015). efficient predators more often kill their prey than injure them, whereas inefficient predators cause numerous injuries and fewer deaths (stephens and krebs, 1986; seligmann et al., 1996). predatory attacks to the snake’s body or head may be more successful than those directed at the tail, and thus more individuals should be found with damaged tails than with body scars (stephens and krebs, 1986; placyk and burghardt, 2005). although in absolute terms, we found a similar frequency of the two injuries in h. hippocrepis when we took into account that the head plus the body represents more than 75% of the total length of this species (i.e., the tail accounts for less than 25% of the total length; feriche et al., 1993), our results support the tailbreakage/body-scarring hypothesis. despite the small fraction of total length that the tail represents, approximately the same number of individuals exhibited injuries in the tail as along the rest of the body. this result suggests that h. hippocrepis survives tail attacks better than body grasping by predators. in snakes, scarring frequency may be a better indication of predator efficiency than predator intensity (placyck and burghardt, 2005). the size-related hypothesis this hypothesis has often been supported in snakes (white et al., 1982; mendelson, 1992; seligmann et al., 2008; dourado et al., 2013). mendelson (1992) proposed that larger snakes may be attacked and survive more frequently, and willis et al. (1982) suggested that this result is the consequence of a survival cost of this trauma, because smaller and injured individuals were simply eliminated from the population. we propose that the struggling capacity of smaller snakes against predators is much weaker than in larger snakes, and that most encounters between predators and small snake individuals end in successful predation (bittner, 2003; pleguezuelos et al., 2010). however, larger h. hippocrepis would confront most of their predators with some bouts ending in body injuries but unsuccessful predation. in support of this, in our study area the snake-eater specialist, c. gallicus, does not consume snakes larger than 1190 mm svl and scarcely those larger than 1000 mm svl, presumably because the struggling capacities of larger snakes would be dangerous for the eagle (gil-sánchez and pleguezuelos, 2001). however, this speculation needs confirmation by population studies. the most cautionary interpretation of the size-related hypothesis is that larger, and probably older, specimens were exposed to potential predators for a longer period and thus had an increased cumulative likelihood of injury (schall and pianka, 1980; seligmann et al., 2008; sparkman et al., 2013). the sex hypothesis in snakes, males usually move more than females in the mating period, and this sex difference in behaviour would expose males to greater predation pressure (shine et al., 2001). in support of this hypothesis, we found that more adult males than adult females exhibit body scarring, in agreement with the sex differences in activity of h. hippocrepis as measured by the encounter rate in the 70 juan m pleguezuelos et alii field. that is, during spring and particularly during the mating period of this species in the study area (may-june; pleguezuelos and feriche, 1999), the number of males recorded was more than two-fold that of females, whereas no difference was found over the rest of the year (based on 171 records; unpub. data of the authors). male snakes searching for mates can also allow a much closer approach than snakes not seeking mates (shine et al., 2003; gregory, 2016). moreover, this result must partially be the consequence of the sexual dimorphism in body size of h. hippocrepis; males, being larger than females (pleguezuelos and feriche, 2014), will exhibit higher frequency of body scarring (see the size-related hypothesis). other authors have found more tail damage in females than in males (willis et al., 1982; slowinski and savage, 1995), and have proposed that this was because females survive tail attacks more than males do (placyck and burghardt, 2005). however, neither our study nor others would be decisive in establishing the real existence of sex differences in predation pressure, and most studies of body injuries of snakes that have considered sex as a factor have failed to find differences between males and females (zug et al., 1979; white et al., 1982; mendelson, 1992; mushinsky and miller, 1993; capula et al., 2000; aubret et al., 2005; pleguezuelos et al., 2010; dourado et al., 2013). notably, h. hippocrepis males with tail breaks retain more pairs of sbc scales, namely a longer stub (see dourado et al., 2013), than females. mendelson (1992) observed the same in coniophanes fissidens and related this finding to the copulatory organs housed in the base of the male’s tail; breakage affecting the hemipenis or retractor muscles would be more harmful than breaks that leave longer tail stubs (placyck and burghardt, 2005). this could explain the low frequency of males with very short tail stubs. the multiple-tail-breakage hypothesis evidence supporting this hypothesis has been obtained only in snake species with long tails that survive repeated attacks and injuries (henderson, 1984; savage and crother, 1989; savage and slowinski, 1996). without breakage, h. hippocrepis exhibit a rather long tail (see above), as is usual in snakes with strong climbing abilities (pizzatto et al., 2007). however, we failed to support this hypothesis for h. hippocrepis. the cost-of-injuries hypothesis in some reptiles a cost of body injuries has been observed in terms of a reduction in feeding efficiency (dial and fitzpatrick, 1981). in thamnophis sirtalis, shine et al. (1999) found differences in body size and body condition of individuals depending on tail integrity. thus, one deleterious effect of tail breakage or body scarring in h. hippocrepis could be a slower growth rate, preventing injured individuals from reaching the maximum body size within the population. however, we did not find smaller body size for injured individuals, but rather the opposite (see the size-related hypothesis). we also failed to find any difference in fat-body reserves between fulltailed and stub-tailed h. hippocrepis, suggesting than tail breakage did not affect foraging success in this snake, the same than in another snakes (downes and shine, 2001; aubret et al., 2005; maginnis, 2006; pleguezuelos et al., 2013). the usually small portion of the tail lost in h. hippocrepis presumably did not affect mobility or foraging success, even in this climbing snake. jayne and bennet (1989) found that an ablation of at least two thirds of the tail was necessary to decrease locomotion speed by only 4.5% in t. sirtalis. although there are no empirical data, the same must apply to body scarring. we conclude that the analysis of body injuries in snakes can reveal and generate hypotheses on some aspects of their natural history, otherwise difficult to discover for these rather clandestine reptiles. studying tail breakage and body scarring in h. hippocrepis, we have learnt that large individuals accumulate signs of inefficient predator attacks over their life, that males exhibit more body scars and longer stub-tails than females, and that the injuries considered here have no obvious cost for individuals bearing them. we admit that the sample for this study was gathered from a rather wide area and during a rather extensive period. however, there is no alternative way to obtain a large sample for some mediterranean and terrestrial snakes, because of their scarcity and/ or shy nature. in support of our procedure, in the same study area and during a shorter period, we found approximately the same percentage of live individuals of h. hippocrepis affected by tail breakage and body scarring; thus, there is no obvious bias in our sampling procedure. we encourage further studies on snake tail breakage and scarring, particularly studies based on large samples that can address the population-level consequences of injury (gregory, 2016). acknowledgments to the many people that during years helped in field surveys, like marta feriche, juan r. fernández-cardenete, luis garcía-cardenete, santiago honrubia, manuel moreno, and david pleguezuelos. mariano martín-vied71body injuries in the horseshoe whip snake ma helped in laboratory, and j. caro provided statistical advice. xavier santos suggested changes in an early version of the manuscript. the parque de las ciencias de granada preserved and managed the snake collection of the granada university. this study was partially funded by the regional government of the junta de andalucía by grants to the research group rnm-254. references akani, g.c., luiselli, l., wariboko, s.m., ude, l., angelici, f.m. 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(1979): variation in reproductive parameters of three neotropical snakes, coniophanes fissidens, dipsas catesbyi, and imantodes cenchoa. smiths. contrib. zool. 300: 1-18. acta herpetologica vol. 13, n. 1 june 2018 firenze university press species diversity and distribution of lizards in montenegro katarina ljubisavljević1,2,*, ljiljana tomović3, aleksandar urošević1, slađana gvozdenović2, vuk iković2, vernes zagora2, and nenad labus4 the first demographic data and body size of the southern banded newt, ommatotriton vittatus (caudata: salamandridae) abdullah altunişik diet and helminth parasites of freshwater turtles mesoclemmys tuberculata, phrynops geoffroanus (pleurodira: chelidae) and kinosternon scorpioides (criptodyra: kinosternidae) in a semiarid region, northeast of brazil antonio marcos alves pereira*, samuel vieira brito, joão antonio de araujo filho, adonias aphoena martins teixeira, diêgo alves teles, daniel oliveira santana, vandeberg ferreira lima, waltécio de oliveira almeida electric circuit theory applied to alien invasions: a connectivity model predicting the balkan frog expansion in northern italy mattia falaschi1,2,*, marco mangiacotti3, roberto sacchi3, stefano scali2, edoardo razzetti4 first report of bufo bufo (linnaeus, 1758) from sardinia (italy) ilaria maria cossu1, salvatore frau1, massimo delfino2,3, alice chiodi4, claudia corti1,5*, adriana bellati4 natural history and conservation of the nurse frog of the serranía del perijá allobates ignotus (dendrobatoidea: aromobatidae) in northeastern colombia hernán darío granda-rodríguez1,2, andrés camilo montes-correa3,*, juan david jiménez-bolaño3, marvin anganoy-criollo4,5 exploring body injuries in the horseshoe whip snake, hemorrhois hippocrepis juan m pleguezuelos*, esmeralda alaminos, mónica feriche how effectively do european skinks thermoregulate? evidence from chalcides ocellatus, a common but overlooked mediterranean lizard grigoris kapsalas, aris deimezis-tsikoutas, thanos georgakopoulos, ismini gkourtsouli-antoniadou, kallirroi papadaki, katerina vassaki, panayiotis pafilis thermal tolerance for two cohorts of a native and an invasive freshwater turtle species jun geng, wei dang, qiong wu, hong-liang lu* diet of a restocked population of the european pond turtle emys orbicularis in nw italy dario ottonello1, fabrizio oneto1,2, monica vignone2, anita rizzo2, sebastiano salvidio2,* helminths of the lizard colobosauroides cearensis (squamata, gymnophthalmidae) in an area of caatinga, northeastern brazil aldenir f. da silva neta*, robson w. ávila acta herpetologica 16(1): 53-57, 2021 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.36253/a_h-9843 flight initiation distance of urosaurus ornatus from the sierra de samalayuca, mexico julio a. lemos-espinal1,*, geoffrey r. smith2 1 laboratorio de ecología-ubipro, fes iztacala unam, avenida los barrios 1, los reyes iztacala, tlalnepantla,edo. de méxico, 54090, méxico 2 department of biology, denison university, granville, ohio 43023 usa * corresponding author. e-mail: lemos@unam.mx submitted on: 2020, 6th october; revised on: 2021, 10th january; accepted on: 2020, 12th january editor: simon baeckens abstract. in lizards, flight initiation distance (fid), the distance between a prey individual and a predator when escape begins, can be affected by numerous intrinsic and extrinsic factors, including sex, temperature, and level of conspicuousness. here we report on a study of fid in a population of ornate tree lizards, urosaurus ornatus, from the sierra de samalyuca, chihuahua, mexico which are cryptic due to their dorsal coloration blending into their background. urosaurus ornatus in our study population allowed close approaches (mean fid = 65 cm). mean fid did not differ between males and females. we also found no effect of body, air, or substrate temperature on fid. the short fid we observed may be related to the cryptic nature of u. ornatus. keywords. approach distance, anti-predator response, cryptic, lizard, sex, temperature. for many prey individuals one of the most important decisions that must be made when confronted with an approaching predator is when to begin their antipredator response, which for many prey is flight (ydenberg and dill, 1986). theoretically, this decision should reflect a balance between fleeing too early and losing fitness due to lost opportunities (e.g., foraging, thermoregulation, mating) and fleeing too late and being caught by the predator (ydenberg and dill, 1986; cooper and frederick, 2007). the distance from the predator to the prey when the prey begins to flee is the flight initiation distance (fid) and is a readily measured aspect of a prey’s behavior (ydenberg and dill, 1986; cooper and frederick, 2007). for lizards, fid can be affected by numerous intrinsic and extrinsic factors, including body and environmental temperature (e.g., smith and lemos-espinal, 2005; cooper et al., 2009; braun et al., 2010; cooper, 2011a); perch, habitat, or microhabitat characteristics (cooper, 2003b; cooper et al., 2009; morris and lattanzio, 2020), and sex (vanhooydonck et al., 2007; majláth and majláthova, 2009; salido and vicente, 2019). however, these factors do not always affect fid in lizards (e.g., temperature: martin and lópez, 2000; amo et al., 2005; smith and lemos-espinal, 2005; cooper, 2006; sex: cooper, 2003a, 2011b; cooper and pérez-mellado, 2011; kopena et al., 2015). for example, because lizards are ectotherms, temperature, whether body or environmental, can influence their locomotor performance, including sprint speed (van damme and vanhooydonck, 2001; herrel et al., 2007). thus, when temperature does affect lizard fid, the fid typically decreases with body or environmental temperature since lizards at higher temperatures can usually run faster and therefore can allow a predator to approach closer and still escape compared to lizards at lower temperatures (e.g., cooper, 2006, 2011b; cooper et al., 2009; braun et al., 2010). another aspect of a lizard that could affect its fid is the level of crypsis. in general, organisms with effective 54 j.a. lemos-espinal, g.r. smith crypsis should remain still and allow close approaches (cooper et al., 2008). compared to other aspects of lizards that might affect fid, the effects of conspicuousness are relatively understudied. however, previous work related to the conspicuousness of lizards and fid has generally found that more cryptic or more concealed lizards allow closer approaches than less cryptic or less concealed lizards (cooper, 2006; vanhooydonck et al., 2007; cooper et al., 2009; cooper and sherbrooke, 2010). here we report on a field study of fid in a population of ornate tree lizards, urosaurus ornatus, from the sierra de samalyuca, chihuahua, mexico. urosaurus ornatus are distributed from utah and colorado to northern mexico (wiens, 1993; haenel, 2007) and their populations often primarily use trees as perches (baltosser and best, 1990; smith, 1996b; james et al., 2003) but some populations are more terrestrial, using rocks as perches (herrel et al., 2001; haenel, 2018; taylor et al., 2018), including the population we studied (see also gadsden et al., 2021). urosaurus ornatus tend to be relatively small lizards (mean svl = 50 cm; smith, 1996b) that are sit-and-wait foragers (cooper et al., 2001). we examined whether body temperature and sex affect flight initiation distance. in addition, u. ornatus can adjust their dorsal coloration and reflectance to match their background (zucker, 1989; hamilton et al., 2008), making them cryptic. given their ability to blend into their background (see fig. 1), we expected that u. ornatus would allow closer approaches compared to other species (i.e., have a relatively short fid). we also predicted that temperatures, both body and environmental, would have little effect on fid since they may rely more on being still and remaining cryptic rather than relying on locomotor performance which can be affected by temperature (e.g., gilbert and miles, 2016, 2017). we studied the fid of u. ornatus on 10 and 11 november 1998 in a population at the ojo de enmedio, sierra de samalyuca, chihuahua, mexico ((31°22’48.2”n, 106°35´2.7”w, 1344 m elevation). ojo de enmedio is a small ranch located approximately 10 km northwest of the town of samalayuca (municipality of juárez, chihuahua), in the foothills of the extreme northwest of the sierra de samalayuca. the vegetation is typical xerophyte scrub of the chihuahuan desert. when we spotted a stationary and undisturbed lizard, one of us (jle) slowly and directly approached it at a constant speed. the same person always made the approach to promote a more consistent appearance and approach among lizards. we measured fid as the distance between the location where the approaching “predator” was when the lizard first fled and where the lizard was first observed (to nearest cm using a meter tape). since we walked through the study area and did not return to a specific site it is highly unlikely we repeated measuring fid on any individual. we captured all lizards and recorded their sex. we also measured body temperature (tb), air temperature (ta: 1 cm above surface at location lizard first observed), and substrate temperature (ts: on surface at location lizard first observed) to the nearest 0.1 c using a quick-reading cloacal thermometer. all lizards were captured within 1 minute of determining fid. all lizards were in full sun when first observed. we compared fid between males and females using an analysis of variance on log-transformed fids. we used linear regressions to analyze the relationships between fid and tb, ta, and ts. we used jmp pro 14 (sas institute, cary, north carolina, usa) to conduct all statistical analyses and used an a-value of 0.05 to determine statistical significance. means are given ± 1 s.e. overall mean fid was 64.7 ± 6.7 cm (n = 50). mean fid did not differ between males (68.9 ± 8.4 cm; n = 32) and females (57.4 ± 11.2 cm; n = 18; f1,48 = 0.18, p = 0.67). flight initiation distance was not affected by tb (n = 50, r2 = 0.057, p = 0.10), ta (n = 50, r2 = 0.02, p = 0.34), or ts (n = 50, r2 = 0.03, p = 0.22). urosaurus ornatus in our study population allowed close approaches by the human simulated predator (i.e., mean fid = 65 cm). indeed, in samalayuca, it is possible to capture u. ornatus directly with one’s hands rather than needing to use a lasso (j.a. lemos-espinal, pers. observ.). urosaurus ornatus in arizona also allow similarly close approaches, with a mean fid in males of 90 cm and 64 cm in females (morris and lattanzio, 2020). the fig. 1. photograph of a urosaurus ornatus on a rock substrate demonstrating its cryptic dorsal coloration. photograph was taken from a population at san josé de las piedras, municipality of ocampo, coahuila but the u. ornatus from our study site are very similar. photograph by j.a. lemos-espinal. 55flight initiation distance of u. ornatus short fid we observed, and as has been observed in other u. ornatus, may be related to the cryptic nature of u. ornatus (zucker, 1989; hamilton et al., 2008). the mean fid of urosaurus bicarinatus, a congener but not sister species of u. ornatus that occurs further south in mexico (wiens, 1993; reeder and wiens, 1996) and uses acacia trees as perches (lemos-espinal et al., 1997), in the cañón del zopilote in guerrero was 200 cm (smith and lemos-espinal, 2005), which is almost 3x greater than the fid we observed. however, in our experience, u. bicarinatus in the cañón del zopilote occurs mainly on trees, and although still cryptic, are more readily seen by humans than the u. ornatus in the sierra del samalayuca (j.a. lemos-espinal, pers. observ.). other phrynosomatid lizards from the southwestern united states and mexico have greater fids (e.g., sceloporus virgatus, 160-310 cm, smith, 1996a; cooper and avalos, 2010; s. anahuacus, 260 cm, smith and lemos-espinal, 2005; s. gadoviae, 283 cm, smith and lemos-espinal, 2005; s. jarrovii, 150290 cm, cooper and avalos, 2010; s. mucronatus, 605 cm, smith and lemos-espinal, 2005, uta stansburiana, 210 cm, keehn and feldman, 2018). our results are also consistent with other studied lizards that are cryptic. for example, when in situations in which they are more cryptic, phrynosoma modestum allowed closer approaches than when they were less cryptic, especially at lower temperatures (cooper and sherbrooke, 2010), and cryptic species of anolis allow a human to approach closer than other less cryptic species (cooper, 2006; vanhooydonck et al., 2007). we found no effect of body, air, or substrate temperature on fid, perhaps because of the relatively low overall fid we observed. it may be that the fid of cryptic species is less affected by body and environmental temperatures than other species since predator avoidance is not predicated on locomotor performance. this is also consistent with the fact that air and substrate temperature did not affect fid in u. bicarinatus (smith and lemos-espinal, 2005). however, the fid of u. ornatus in arizona decreased with increased body and perch temperature (morris and lattanzio, 2020), suggesting the situation may be more complex. indeed, whereas temperature did not affect fid, u. bicarinatus that were captured were using perches with lower tas than those that escaped (smith and lemos-espinal, 2005), indicating that the ability to escape, if not their predilection to flee, may still be related to temperature in these lizards. the lack of effect of temperature on fid has also been observed in non-cryptic lizards (e.g., martin and lópez, 2000; amo et al., 2005; smith and lemos-espinal, 2005; cooper, 2006). however, in other species of lizards, fid typically decreases with increased body or environmental temperatures (e.g., cooper, 2006, 2011a; cooper et al., 2009; braun et al., 2010), and fid in hobrookia propinqua increases with substrate temperature (cooper, 2000). the variation in the effects of temperature on fid in lizards needs more study to understand why there is a relationship in some species but not in others. the lack of a difference in approach distance between male and female u. ornatus in our population contrasts with results from another population of u. ornatus where male u. ornatus had greater fid than females (morris and lattanzio, 2020). however, our result is consistent with results from u. bicarinatus (smith and lemos-espinal, 2005). in a review of the literature, cooper (2011b) found that in general most species of lizards show no sexual dimorphism in fid. however, females in some lizards allow closer approaches than males (majláth and majláthová, 2009; vanhooydonck et al., 2017; salido and vicente, 2019). why some populations and species show sexual dimorphism in fid and others do not is not clear and warrants more direct investigation. in conclusion, the cryptic nature of u. ornatus in our population may lead to a short fid since staying still is probably better than fleeing. this supposition is supported by the lack of an effect of temperature, both body and environmental, and sex on fid in this population. however, directly assessing the effect of crypsis on fid is needed to confirm this conclusion. acknowledgments we thank two anonymous reviewers for helpful comments on the manuscript. support for this study was provided by dirección general de asuntos del personal académico – programa de apoyo a proyectos de investigación e innovación tecnológica (dgapa – papiit), through project in202021. this research conformed with all regulations in place in mexico at the time the research was conducted. references amo, l., lópez p., martin, j. 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(1989): dorsal darkening and territoriality in a wild population of the tree lizard, urosaurus ornatus. j. herpetol. 23: 389-398. acta herpetologica 15(1): 65-69, 2020 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/a_h-8119 effects of temperature and food level on plasticity of metamorphic traits in bufo gargarizans gargarizans larvae tong lei yu*, yan ting han department of biology, college of life science, xinyang normal university, sd 464000, china * corresponding author. email: yutonglei_00000@163.com submitted on: 2020, 20th february; revised on: 2020, 2nd march; accepted on: 2020, 6th may editor: marco mangiacotti abstract. many environmental factors such as temperature or food level may influence growth and mortality risks of ectothermic vertebrates in both aquatic and terrestrial habitats. in this study, plasticity in growth rates, survival, larval period, and size at metamorphosis were examined in bufo gargarizans gargarizans larvae under different combinations of temperature and food level. our results showed that larvae metamorphosed at an older age when reared at 17.3°c. a significant interaction between food level and temperature revealed that the food level has obviously affected length of larval period when tadpoles raised at 17.3 °c, but not at 27.3 or 31.3 °c. also, we found clear evidence that growth rates are influenced by both temperature and food level. interestingly, tadpoles reared at 17.3 °c had larger size at metamorphosis than those reared at other temperatures, suggesting that b. g. gargarizans larvae reared at cold temperatures have a longer developmental period but they are also larger as metamorphs than conspecifics reared at warmer temperatures. therefore, the global climate change or local manipulations of the environment may promote growth and development of b. g. gargarizans larvae, but not large size at metamorphosis. keywords. amphibia, anura, bufonidae, bufo gargarizans gargarizans, larval period, metamorphic size, phenotypic plasticity, china. in animals with complex life cycles, such as amphibians, metamorphic size and timing are important fitness components (arnold and wassersug, 1978; wilbur, 1980). many environmental factors such as temperature or food availability may influence size and age at metamorphosis (reviewed by álvarez and nicieza, 2002). generally, low temperatures resulted in a longer developmental periods, but larger metamorphic size than conspecifics reared at warmer temperatures because of differential effects on growth and differentiation (smith-gill and berven, 1979; álvarez and nicieza, 2002). however, previous studies indicated that the temperature variation of the reaction norms among species and phylogenetic groups was a puzzling problem, suggesting an interplay between phylogeny and adaptation to specific habitats (aquatic and terrestrial) (e.g., blouin, 1992; morand et al., 1997; joly et al., 2005). a steady food supply generally elicits a younger age and larger size at metamorphosis (werner, 1986). this also correlates with younger age and larger size at first reproduction, and thus potentially higher fecundity (harris, 1999; werner, 1986; semlitsch et al., 1988). this variation in metamorphic traits may have strong effects on later fitness as early metamorphosis, and large size at metamorphosis are favoured because of their positive effects on juvenile survival and adult fecundity (wells, 2007). temperature and food availability have been studied in numerous anurans (see review by angilletta and dunham, 2003), but little is known about how the interaction between temperature and food availability can affect tadpole growth and development (but see: laugen et al., 2005; castano et al., 2010; courtney jones et al., 2015; yu et al., 2015). in this study, we examined the potential 66 tong lei yu, yan ting han interactive effects of food level, and rearing temperature on the plasticity of metamorphic traits of bufo gargarizans gargarizans, including the length of larval period, survival, the size at metamorphosis, and growth rate. as model species we used bufo gargarizans gargarizans, a widely distributed toad, breeding in different aquatic habitats (yu and guo, 2013), which may offer a large variation in both temperature and resource availability during larval development. bufo gargarizans gargarizans is sexually dimorphic and is widely distributed in east asia. female toads are the larger sex, and clutch size is positively correlated with female body size (means = 9325 ± 279.05, range = 3275– 15880; yu and sharma, 2012). it is an explosive breeder with typical breeding habitats concentrated along the vegetated edges of large still water bodies and a relatively short breeding season (6-14 days; wells, 2007; yu and sharma, 2012). the tadpoles hatch after two weeks in the breeding ponds. the timing of larval development in natural ponds is about 50-55 days, when water temperature varies from 6 (at night of early–march) to 29 °c (at noon of mid–april, mean temperature less than 25 °c, yu, personal observations). rich spirogyra and pondweed grow in pond, and provide food for b. g. gargarizans tadpoles in the larval period (wei et al., 2011). during the peak period of breeding activity, we collected a total of 25 amplectant pairs in one population in shihe county (32°08’n, 114°01’e; datum = wgs84), henan, the central plains of china, in mid–february 2012. those animals were transported to laboratories close to spawning sites. we kept pairs separately in plastic cask (20 l) filled with approximately 12-15 cm of pond water until the eggs were deposited. once oviposition was completed, we collected 40 fresh eggs from each of the egg masses from 20 b. g. gargarizans females because five female toads did not lay eggs. on the same day, we put all fresh eggs into two 80 l plastic containers with an automatic aerator, and we left them there until hatching. a total of 360 tadpoles (gosner stage 25; gosner, 1960) were randomly allocated to six experimental treatments (n = 60). larvae were fed with commercial fish food (bieyanghong, biological co. ltd., hangzhou, china, protein content, pc; protein >45%, lipids >12%, algae >12%, fiber >4%, ash <10%). tadpoles were exposed to a 13l:11d photoperiod throughout the study period and the water in the containers was changed weekly. we used a 2×3 factorial design to examine the effects of food level and rearing temperature on larval growth rates and post-metamorphic performance. to evaluate the effects of food level, half of the tadpoles in each temperature treatment were placed at low mass-specific food level (25 mg food/g tadpole per day) and a half on a high food regimen (50 mg food/g tadpole per day) throughout the experiment. these food levels were chosen to be consistent with the previous study on this species by zhang et al. (2007). for each food regimen, three different temperature levels were kept across the rearing period: low (room) temperature (17.3 ± 1.33 °c; mean ± sd); middle temperature (27.3 ± 1.42 °c); high temperature (31.3 ± 0.56 °c). the “low” and “middle” temperature were chosen because they fall within the range this species experiences in the field, while “high” temperature approached the avoidance temperature (ma and long, 2005). aquarium heaters (minjiang, baolaihd–200, guangzhou, china) were used to raise the water temperature in the “middle” and “high” treatments. for each temperature treatment, 120 individual vessels with foam board (60 for each diet treatment), each of which is 300 ml, were randomly placed in two rectangular tanks (110 × 90 × 60 cm; l × w × h). to further minimize the possible effects of such heterogeneity, the positions of the 120 tadpole containers within a given temperature were reassigned at random every three days. after the first metamorph (defined as the emergence of at least one forelimb, gosner stage 42) was discovered, the six large tanks were checked daily and all metamorphs found were collected and kept individually in plastic vials (8 cm diameter) with sand and 1 mm of water until tail re-sorption was completed (gosner stage 46). four variables were measured: (1) age at complete metamorphosis (number of days from the beginning of the experiment until complete metamorphosis, gosner stage 46); (2) svl (snout-to-vent length) at complete metamorphosis (svl was measured with digital calipers to the nearest 0.01 mm; (3) growth rate was measured as the svl at complete metamorphosis divided by the age; (4) the percentage of surviving tadpoles that metamorphose. we analysed the length of larval period, svl at metamorphosis, and growth rate by using a generalized linear model (glm) with type iii mean squares and temperature, food level, and their interaction as fixed factors. we used log-linear model to test survival. if the overall glm or log-linear model results were significant, the data were analysed with anovas by using post-hoc multiple comparisons (fisher’s lsd) or a chi-square test to evaluate differences between food levels or between temperatures (spss 13.0, spss inc., 2004, chicago, il, usa). all given p-values are two-tailed, with values presented as means ± standard error. the effects of rearing temperature or food level on the length of larval period were significant (temperature, f2, 183 = 266.50, p < 0.001; food level, f1, 183 = 38.61, p < 0.001), as well as their interaction (f2, 183= 10.08, p 67effects of temperature and food level on bufo gargarizans tadpole growth < 0.001). tadpoles raised at 17.3 °c took longer to metamorphose than those raised at 27.3 and 31.3 °c (all p < 0.05, fig 1), while no difference was found between the latter temperature treatments. tadpoles feeding on high food level reached metamorphosis earlier than those raised at low food level (p < 0.001). a significant interaction revealed that tadpoles raised at 17.3 °c, high food level reached metamorphosis earlier than those raised at low food level (post-hoc tests: p < 0.001), but there were not different at 27.3 or 31.3 °c (both p > 0.05; fig. 1). the rearing temperature significantly affected svl at metamorphosis (f2, 183 = 3.68, p = 0.027, fig. 1), but food level, as well as temperature × food level interaction did not (food level: f1, 183 = 2.06, p = 0.153; interaction: f2, 183 = 1.37, p = 0.257). tadpoles reared at 17.3 °c had significantly larger svl at metamorphosis than those at 31.3 °c (p = 0.016), or marginal significantly than those at 27.3 °c (p = 0.051). the rearing temperature or food level positively influenced growth rate (temperature, f2, 183 = 113.89, p < 0.001; food level, f1, 183 = 25.72, p < 0.001, fig. 1), but the interaction between rearing temperature and food level was not significant (f2,183 = 0.37, p = 0.694). tadpoles feeding on a high food level had a greater growth rate than those raised at low food level (p < 0.05). tadpoles reared at 17.3 °c had a lower growth rate than those raised at 27.3 and 31.3 °c (both p < 0.001), but there was no difference between the latter two temperature treatments (p = 0.350). survival at metamorphosis was affected by food level (z = -2.65, p = 0.008): tadpoles feeding on a high food level had a higher survival rate than those raised at low food level. in contrast, nor the rearing temperature, nor the interaction between temperature and food level were significant (temperature: z = -0.81, p = 0.416; interaction: z =1.52, p = 0.129; fig. 1). in anuran amphibians, age and size at metamorphosis may be reduced with increasing temperature (harkey and semlitsch, 1988; newman, 1998; beck and congdon, 2000; merila et al., 2000; laugen et al., 2003; palo et al., 2003; liess et al., 2013; courtney jones et al., 2015; yu et al., 2015). our results confirmed that b. g. gargarizans metamorphosed at a younger age when reared at higher temperature. however, tadpoles reared at low temperature had larger svl at metamorphosis than those reared at other temperatures, which are consistent with previous studies (atkinson, 1994, 1996; angilletta and dunham, 2003; arendt, 2011; yu et al., 2016). temperature can affect metamorphic size in two ways. first, temperature is a major proximal factor determining growth and development, which cause large differences in size at metamorphosis (smith–gill and berven, 1979). this is the expected outcome if growth rates are more responsive to temperature than differentiation rates. second, temperature may influence the extent to which food level can affect growth and development, which in turn influence size at metamorphosis. in our study, food level had no effect on svl independently from the temperature treatment. however, cold-reared individuals had slow growth rates compared to tadpoles in warmer temperatures. this was also reflected in the age at metamorphosis, which was significantly older at low temperatures compared to higher temperatures. therefore, we suggested that b. g. gargarizans larvae reared at cold temperatures have a longer developmental period but they may also become larger as metamorphs than conspecifics reared at warmer temperatures, independently from the available food level. the food availability during the larval stage has important effects on timing of metamorphosis (leips and travis, 1994). several experimental studies have demonstrated that high food availability with a large proportion of protein can produce a two-fold effect, accelerating both growth and developmental rates (nathan and james, 1972; steinwascher and travis, 1983; pandian and marian, 1985). laugen et al. (2003) suggested the effect of high food availability for maximum growth and development rates were larger in the warm temperature treatments. in this study, at low temperature, tadpoles raised with high food level reached metamorphosis earlier than fig. 1. influences of temperature and food level on age, svl, growth rate and survival at complete metamorphosis of b. g. gargarizans (gosner stage 46; black columns = high temperature; grey columns = middle temperature; open columns = low temperature). 68 tong lei yu, yan ting han those raised at low food level, but they were not different at middle and high temperature. thus, the effects of food availability on larval growth were partially dependent on developmental temperature. acknowledgements we thank the parents and wife of tly for helping during all phases of the data collection. all animals were collected under the guidelines for animal care in china. handling and processing of frogs followed approved protocols from animal scientific procedures act 1988 by the state department of china. xinyang government of wildlife approved this project (approval number xy/0721), and gave permission for fieldwork. the study was funded by emergency management program of national natural science foundation of china (grant no. 31741019). the authors declare no conflicts of interest. references álvarez, d., nicieza, a.g. 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(2007): competitive strategies of two species of co-occuring tadpoles. chin. zool. res. 28: 41-46. acta herpetologica vol. 15, n. 1 june 2020 firenze university press has the west been won? a field survey and a species distribution model of iberolacerta horvathi in the alps giuseppe de marchi1,*, giovanni bombieri1, bruno boz1, fausto leandri2, jacopo richard3 first record of underwater sound produced by the balkan crested newt (triturus ivanbureschi) simeon lukanov identification of biologically active fractions in the dermal secretions of the genus bombina (amphibia: anura: bombinatoridae) iryna udovychenko1,*, oleksandra oskyrko1, oleksii marushchak2, tetiana halenova1, oleksii savchuk1 don’t tread on me: an examination of the anti-predatory behavior of eastern copperheads (agkistrodon contortrix) andrew adams1,2,*, john garrison2, scott mcdaniel2, emily bueche2, hunter howell2,3 an overview of research regarding reservoirs, vectors and predators of the chytrid fungus batrachochytrium dendrobatidis jarid prahl, thomas p. wilson*, david giles, j. hill craddock genetic characteristics of an introduced population of bombina bombina (linnaeus, 1761) (amphibia: bombinatoridae) in moselle, france jean-pierre vacher1, 2,*, damien aumaître3, sylvain ursenbacher2,4 adaptive significance of the transparent body in the tadpoles of ornamented pygmy frog, microhyla ornata (anura, amphibia) santosh m. mogali*, bhagyashri a. shanbhag, srinivas k. saidapur comparison of complement system activity amongst wild and domestic animals maría s. moleón1,2,*, mark e. merchant3, hugo h. ortega4, pablo a. siroski1,2 effects of temperature and food level on plasticity of metamorphic traits in bufo gargarizans gargarizans larvae tong lei yu*, yan ting han acta herpetologica 14(2): 89-100, 2019 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/a_h-7746 coping with aliens: how a native gecko manages to persist on mediterranean islands despite the black rat? michel-jean delaugerre1,*, roberto sacchi2, marta biaggini3, pietro lo cascio4, ridha ouni5, claudia corti3 1 conservatoire du littoral, résidence st marc, 2, rue juge falcone f-20200 bastia, france. *corresponding author. email: m.delaugerre@conservatoire-du-littoral.fr 2 dipartimento di scienze della terra e dell’ambiente, università degli studi di pavia, via taramelli 24, i-27100, pavia, italia 3 sistema museale di ateneo museo di storia naturale dell’università di firenze, sede “la specola”, via romana 17, i-50125 firenze, italia 4 associazione nesos, via vittorio emanuele 24, i-98055 lipari (me), italia 5 tunisia wildlife conservation society, faculté des sciences de tunis, université tunis el manar ii tu-2092, tunisia submitted on: 2019, 14th april; revised on: 2019, 16th june; accepted on: 2019, 24th june editor: uwe fritz abstract. how a native gecko manages to coexist with an alien rodent in the mediterranean since thousands of years? what kind of eco-ethological adaptations or evolutionary adjustments enables this gecko to persist? the present study explores the interaction between the endemic european leaf-toed gecko (euleptes europaea) and the alien black rat (rattus rattus). in the last 30 years, we compared 26 populations inhabiting “rat” and “rat-free” islands and islets in tunisia, sardinia, corsica and southern france. geckos’ populations can persist despite the occurrence of rats. in the presence of rats: 1) geckos’ average body size tends to decrease towards medium-sized individuals; 2) geckos shift their spatial behaviour avoiding to forage “in the open”; 3) geckos’ body condition is not affected by the presence of rats. moreover, shortly after rats’ eradication, geckos’ population structure seems to change and larger sized geckos prevail while the spatial behaviour is much more conservative. the mechanisms driving the interactions between the two species still need to be explained. rats could represent a stressor for geckos, compete for space, be pest vectors and even predators. coexistence of natives and aliens requires adaptive plasticity and evolutionary adjustments. in contexts where the risk of reinvasion is high, eradication programs need to be carefully evaluated, since the arrival of “new rats” on an island could have much more damaging effects on the insular biota than those caused by the eradicated population. keywords. behavioural shift, disturbance, ecological plasticity, evolutionary processes, predation, rat eradication, euleptes europea, rattus rattus. in memory of michel pascal (1947-2013) introduction the long term dynamics of insular biota reflect the interplay between recurrent immigration and extinction events (brown and lomolino, 2000). mainly because of human expansion on global scale, the natural phenomenon of sea dispersal has been greatly amplified favouring introductions and even biological invasions (simberloff et al., 2013). in the last centuries, with a speeding up in the last decades human-induced dispersal of species has implied: a) huge acceleration of the introductions’ rate; b) great number of species involved; c) remote origins of some propagules; d) possible genetic modifications originated from captive breeding and farming activities. insularity-associated features, such as high sensitivity to climate and sea level changes, high rate of speciation, immigration and extinction processes, naiveté of native species, make insular biota particularly fragile (moser et al., 2018). 90 michel-jean delaugerre et alii over the last decades, conservation biology has emphasized the detrimental effects of introduced species (mostly mammals) on insular ecosystems, but little attention has been paid to the understanding of the eco-ethological adaptations that enable a native species to persist despite the presence of aliens (martin et al., 2000; hoare et al., 2007; ruffino et al., 2009). in systems with novel combination of species (native and introduced ones) that do not show co-adaptation, rapid evolutionary changes may occur (berthon, 2015; mooney and cleland, 2001; strauss et al., 2006; sax et al., 2007; stuart et al., 2014). on islands, in particular, morphological, ecological and behavioural shift are more easily detectable than in more complex systems (simberloff, 1974). within the mediterranean, reptiles, notably lizards, are good tools to investigate insular evolution. these herpetological communities, which are currently found on continental islands and on many land-bridge islets, origin from the neighbouring continents or large islands before their isolation, dating back to the sea level raise of the last interglacials. trans-marine dispersal is less frequent in the mediterranean arid islands (foufopoulos and ives, 1999). most of the endemic herpetofauna still remains on some big islands (e.g., corsica, sardinia) and relative satellite islands, while on other major mediterranean islands the endemic herpetofauna has been completely replaced by human-introduced species, and endemic species persist only on some of their satellite islands (corti et al., 1999; silva-rocha et al., 2018, 2019). in the present work we investigated the interaction between the native european leaf-toed gecko euleptes europaea (gené, 1839) and the alien black rat, rattus rattus (linnaeus, 1758) on some small islands of the western mediterranean. the european leaf-toed gecko, a western mediterranean endemic species listed in appendix ii of the european habitat directive, is a very old inhabitant of this region, presumably since several million years ago (müller, 2001). this species underwent a process of historic extinction or steep demographic decline along the north-western (provence, france) and southern (tunisia) edges of its range (delaugerre et al., 2011). the black rat colonized the western mediterranean approximately 2000-2400 years ago (thibault et al., 1987; vigne and valladas, 1996; ruffino and vidal, 2010). thus, the coexistence between the two species might last since hundreds of geckos’ generations and thousands of rats’ generations, since euleptes lives longer than rats (salvidio et al., 2010 and ref. therein). according to ruffino et al. (2009), in the western mediterranean, 74% of the islands between 1 and 5 ha (and 99% of islands larger than 30 ha) are colonized by black rats. among the human introduced species, rats can dramatically impact on island biodiversity (recher and clark, 1974; atkinson, 1985; courchamp et al., 2003; bennett et al., 2005) and black rats in particular, have caused rapid extinctions on islands, as reported for new zealand and the hawaiian archipelago (towns et al., 2006; drake and hunt, 2009). alien rats can predate and compete with native species and modify islands’ food chains (traveset and richardson, 2006). the worldwide success of the black rat as colonizer is due to its ability to exploit a large range of habitats and resources (jones et al., 2008), as well as to shift to seasonal resources (caut et al., 2008). this plasticity is crucial to survive in poor insular ecosystems, characterized by strong resource variation. on small islets e. europaea and r. rattus are often the only sedentary vertebrates. they are both nocturnal, good climbers on rocky substrates, and they both forage on rocky outcrops and on low vegetation. rat eradication programs are often focused on one or few emblematic species (capizzi et al., 2010), without a comprehensive understanding of insular assemblages. investigating the possible interactions between the european leaf-toed gecko and the black rat the present work could provide useful data for long-term insular conservation plans. in particular, we explored the effects of the black rat on the leaf-toad gecko population structure (analysed on the basis of the relative proportion of size classes), body condition and habitat use, comparing populations living on islands colonized by rats and islands free of rats. because we expect rats to interfere with geckos’ habitat use, feeding habits and thermoregulation, geckos living on islands on which also rats live should show: 1) population structure with smaller-sized individuals resulting from a shorter lifespan; 2) poor body conditions; 3) modification of spatial behaviour by minimizing or even avoiding foraging in open spaces. material and methods ecological or evolutionary responses of native species to aliens cannot be easily distinguished from the pre-existing ecological differences (strauss et al., 2006). however, even if each micro insular population differs from the others (e.g., due to age of isolation, presence of competitors and/or predators, etc.), any detectable trend can provide strong correlative evidences. study species euleptes europaea is a small gecko (average snout-vent length, hereafter svl, 38 mm; average adult weight 1.2 g; hatchling weight 0.25 g) endemic to the western mediterranean, mostly found on islands in rocky habitats. it is strictly noctur91native gecko and black rat on mediterranean islands nal, spending daytime in narrow rocky crevices (opening 2-4 mm wide) where dorsal and ventral parts of the body are in contact with the rock. these crevices are also used for egg laying (salvidio et al., 2010). achieving its sexual maturity at the age of 3 years (salvidio and delaugerre, 2003), it might live 6 to 8 years in the wild and its maximum longevity in captivity attains 21 years (f. molle in mertens, 1970). it feeds on flying and ground dwelling invertebrates. on islets, densities are higher than on the mainland and population size ranges from several hundreds to only few dozen adults. this gecko is able to live on tiny islets (hundreds of square meters) characterized by the presence of few vascular plants, where it represents the only sedentary vertebrate (delaugerre and cheylan, 1992). body size greatly varies on islets with trends towards gigantism, dwarfism and variation of the sexual size difference (delaugerre and cheylan, 1992; salvidio et al., 2010). rattus rattus (average weight 170 g), is a good climber, both on rocks and trees, but not a long lasting swimmer and therefore considered with poor marine dispersal capacities (≤ 500 m) (cheylan, 1988; ruffino et al., 2009). in the mediterranean, rats feed mainly on plants, avoiding or rarely eating halophilous and nitrophilous ones (cheylan, 1988; cassaing et al., 2007). black rats and euleptes europaea are both nocturnal and share the same habitat. study sites and data collection we focused on islands characterized by simple ecosystems, where strong interactions between the target species are more likely to occur, namely small islands characterised by seasonal shortage of food availability for rats, and higher probability of encounters between the two species. we selected 26 islands and islets (table 1) throughout most of the leaf-toed gecko range (fig. 1). islands were classified as “rat” vs “rat-free”, depending on the presence or absence of rats; islands where rats were eradicated were considered as “rat-free”. the presence of rats was detected thanks to at least one of the following evidences: direct sightings, fresh and/or old faeces, remains of chewed olive seeds or plants, rats’ nests, rats’ urine scent. past presence/absence of rats on islands was investigated through literature and observations made by locals. nine islands (the largest ones) were inhabited by rats probably since their early colonization (abdelkrim et al, 2009), whereas 13 islands (the smallest and most remote ones, without edible plants) were presumably never inhabited by rats. on lavezzu island (corsica) we collected data before and after rat eradication (performed in 2000): the island was considered as “rat” before the eradication and as “rat-free” after eradication (table 1). five islands with “transient” rat populations (i.e., small islets close to a colonization source but lacking resources to support a permanent rat population) were treated as “rat”, because rat frequency on these islands is unknown. sampling methods observations were carried out in spring, summer and autumn from july 1983 until august 2016. sampling sessions (n = 47) consisted of 1 up to 6 nights per island (table 1). active geckos were searched using battery-powered lamps starting one or two hours after dusk and lasted until dawn. total sampling effort achieved more than 380 hours (table 1). geckos behaviour was classified as follow: “in the open” when found on bare rocks, on the ground or on plants; “under cover” when found hidden by the vegetation at the base of rocks (fig. 2). distance from the ground (height of the first sight) was also measured. geckos were carefully caught by hand and temporarily stored in bags; sex and adulthood was determined according to delaugerre and dubois (1985). snout-vent length (svl) of 1795 individuals was measured to the nearest 0.01 mm using a digital calliper, weight (w) of 424 adults was recorded to the nearest 0.01g using a digital scale. geckos were released in the area of original sighting. body condition index (bci) was calculated as from bonnet and naulleau (1994). spatial behaviour of 1012 geckos (i.e., “in the open” or “under cover”), recorded for 24 sampling sessions carried out on 17 islets, was assessed for one hour “catch per-unit-effort” (cpue). statistical analyses population structure. in order to assess whether the populations structure of e. europaea was affected by the presence of rats, we compared gecko’s size between islands with and without rats. since leaf-toed geckos’ size varies among islands independently of rats, we firstly normalized body sizes by dividing each measure by the greatest observed value in order to have all values in the 0-1 range (legendre and legendre, 2012). each individual was classified depending on its inclusion in the quartile of the distribution of the normalized body size as “small” (1st and 2nd quartiles), “medium” (3rd quartile), and “large” (4th quartile). geckos’ population structure was assessed by computing the proportion of individuals per size class. different population structures were computed for spring and autumn. sample size included 1871 individuals from 24 islets (mean values ± se: 78 ± 16); islands with less than 10 individuals measured (san bainzu, piana and porro) were excluded. data were analysed using permutational multivariate analysis of variance, permanova (anderson, 2001; mcardle and anderson, 2001) on the basis of euclidean distances among islands’ population structures. the permanova allows the multivariate information to be partitioned according to the full experimental design without any a priori assumption regarding the distributions of the original variables. the predictors were rat occurrence (yes/ no), season (spring/autumn), number of reptile species, number of sampling sessions (to account for repeated measures within an island), and island size. p-values were obtained by permutation, and the number of permutations was set to 9999. body condition. the effect of rat occurrence on the body condition of geckos was assessed using a linear mixed model in which the bci was the dependent variable; rat occurrence, sex, and season were the fixed effects, whereas the island was the random factor accounting for repeated measuring. spatial behaviour. linear mixed models were also used to investigate the effect of rat occurrence on the habitat use by geckos. in a first analysis we checked if leaf-toed geckos avoided 92 michel-jean delaugerre et alii table 1. map identification numbers (see figure 1) of the study islands, geographical region, surface, presence (“rat”) or absence (“rat-free”) of the black rat, and number of reptile species. for each island we also indicate sampling dates (and number of sampling nights), sampling effort (in minutes), number of geckos measured (n biometry), number of geckos for which activity data were recorded (n activity). n° islet/island region surface (ha) rat status n sp reptiles year (month): n nights sampling effort (min) n biometry n activity 1 gallo n_tunisia 8.9 rat 2 2008 (05):3 1320 49 / rat 2010 (07):1 270 / 30 2 toro sardinia 13.22 rat free 3 2015 (06):1 195 34 45 3 carpa sardinia 0.4 rat 2 2011 (09):1 430 50 67 4 porco sardinia 4.8 rat 4 2012 (05):1 285 44 55 5 spargiotto sardinia 11.13 rat free 3 2014 (05):1 360 66 79 6* lavezzu corsica 62.73 rat 3 1986 (08):1 390 50 / rat free 2010 (06):3 550 40 41 rat free 2011 (06):3 427 67 41 rat free 2012 (06):2 720 66 / 7 porraggia grande corsica 1.25 rat free 2 1985 (08):1 600 50 / 8 porraggia piccola corsica 0.61 rat free 2 1986 (08):1 410 59 / 9 sperduto grande corsica 0.92 rat free 1 1984 (10):2 785 40 / rat free 1986 (08):1 330 81 / rat free 2011 (06):1 435 43 43 10 toro grande corsica 1.62 rat free 2 1986 (08):1 360 59 / rat free 2005 (04):1 200 50 / rat free 2012 (07):1 140 / 52 rat free 2014 (07):1 250 48 59 11 1st islet ne of toro piccolo corsica 0.11 rat free 1 1986 (08):1 240 15 / 12* vacca corsica 0.65 rat free 2 1985 (08):3 1395 95 / rat free 2012 (07):1 165 8 15 13 roscana corsica 0.2 rat free 1 1986 (08):1 720 94 / rat free 2008 (10):3 2258 125 / rat free 2012 (09):2 824 122 / 14 locca corsica 0.79 rat 2 2010 (04):1 90 / 10 15 mezzumare corsica 35.66 rat 3 2012 (08):1 213 15 18 16 a botte corsica 0.52 rat free 1 2010 (09):3 630 35 43 rat free 2011 (06):6 990 / 62 17 gargalu corsica 20.06 rat 4 1985 (04):2 1060 50 / rat 1990 (07):2 505 19 / 18 palazzu corsica 0.47 transient 1 1986 (08):1 225 26 / 19 palazzinu corsica 0.1 transient 1 1985 (07):2 490 34 / 20 porri corsica 0.27 rat free 1 1983 (07):2 590 32 / rat free 1986 (07):1 335 69 / 21 brocciu corsica 1.11 transient 1 2012 (06):1 132 30 60 22 giraglia corsica 10.35 rat free 4 2000 (09):1 360 38 / rat free 2012 (08):2 406 33 33 rat free 2012 (10):1 230 / 50 rat free 2014 (07):1 230 / 19 rat free 2014 (10):1 190 36 38 rat free 2015 (08):1 220 / 32 23 saint ferreol provence 0.45 rat 2 2016 (05):1 700 23 40 24 la tradelière provence 1.05 rat 2 2016 (05):1 460 23 37 25 rascas provence 0.76 transient 2 1985 (09):1 290 50 / 26* gabinière provence 3.42 transient 2 2003 (10):1 345 69 / transient 2016 (05):1 187 37 43 * on lavezzu island (n. 6) rats were eradicated in 2000; on the toro islets (n. 10 and 11) in 1992, after 2-4 years of presence on the islets; on vacca (n. 12) in january 2011, after having been detected in july 2010; on gabinière (n. 26) rats were present in 1937, eradicated in 1966, rats re-invaded the island in 2010 and were eradicated again in 2014. the islets san bainzu, piana and porro, all inhabited by rats, were escluded from the analysis because less than 10 geckoes were measured. 93native gecko and black rat on mediterranean islands “in the open” microhabitats on islands with rats: the catch per unit effort (cpue) was the dependent variable, while rat occurrence, habitat type (“in the open” vs “under cover”), island size and the number of reptile species were the fixed predictors. we also added the rat occurrence × habitat type interaction to account for differential habitat use between “rat” and “rat-free” islands. we could not include in the model the season as all but three islands were sampled in spring, and the island entered the model as random effect to control for double measuring within island (i.e., cpue in open and close microhabitat). sample size included 16 islands for which we collected more than 10 individuals (n = 1001, mean values ± se: 62 ± 10) for computing cpue indexes for both “in the open” and “under cover” microhabitats. in a second analysis we checked if the height above the ground of the first sighting differed in islands with or without rats, using rat occurrence, season, number of reptile species, island size, and the rat occurrence × season as fixed predictors. as in the previous analysis, the islands entered the model as random effect, and geckos observed “under cover” were excluded. the sample for this last analysis included 469 geckos from 14 islands. analyses were performed using the package lme4 (bates et al., 2014) in r ver. 3.2.4 (r core team, 2018), and otherwise stated, data reported are means ± standard error. results population structure the permanova showed that the population structure significantly varied in response to rat occurrence, season, number of reptile species living on the island, but was invariant in respect to the sampling effort (table 2). in particular, a) the structure of geckos’ populations living on the islands with rats showed a larger proportion of medium-sized individuals and a lower proportion of large-sized geckos compared to populations occurring on “rat-free” islands (fig. 3a); b) irrespective of rat occurrence, smaller geckos were frequent in spring, while larger ones prevailed in autumn, and the relative abundance of medium-sized individuals did not vary between seasons (fig. 3b); c) the greater was the number of reptile species on the island, the smaller was the frequency of larger geckos and the higher the number of medium-sized individuals (fig. 3c). moreover, large geckos were more frequent on islets rather than on islands (fig. 3d). body condition body condition of the leaf-toed geckos did not differ between “rat” and “rat-free” islands (0.0292 ± 0.0005 fig. 2. vegetation (mostly lotus cytisoides) covering the base of the granite boulders inhabited by euleptes europaea on spargiotto islet (maddalena archipelago). may 2012. fig. 1. geographic range of euleptes europaea and studied populations. numbers refers to table 1. table 2. results of permutational anova for the variability of the population structure of the leaf-toed geckos in response to rat occurrence, season and number of reptile species after having been controlled for island size and number of sampling years. p-values are computed with 9999 permutations. variable df f r2 p rats 1,24 3.916 0.08 0.0374 islet size 1,24 3.775 0.08 0.0374 season 1,24 10.69 0.23 0.0010 n. reptiles 1,24 3.975 0.08 0.0336 n. replicates 1,24 0.0033 <0.01 0.9958 94 michel-jean delaugerre et alii and 0.0285 ± 0.0002 respectively, f1,5 = 0.0053, p = 0.94), either between males and females (0.0282 ± 0.0003 and 0.0292 ± 0.0004 respectively, f1,413 = 2.545, p = 0.11), or season (autumn: 0.0269 ± 0.0004; spring: 0.0295 ± 0.0003, f1,5 = 0.608, p = 0.46) (table 3). by contrast, the random effect (island) was highly significant (l-ratio χ² = 24.0, d.f. = 1, p < 0.001), and the effect (σ = 0.0021) accounted for 32% of the total variance, suggesting that body condition is highly dependent on the island features. spatial behaviour leaf-toed geckos were more active on “rat-free” islands than on “rat” islands (values of cpue being 7.85 ± 2.35 and 6.33 ± 2.30, respectively), but this difference was significantly depending on the habitat (rat occurrence × habitat type interaction: f1,24 = 11.99, p = 0.0019, table 4). geckos on “rat-free” islands were active “in the open” rather than “under cover” (p = 0.057), while the opposite occurred on “rat” islands (p = 0.0091, fig. 4), suggesting that the leaf-toed geckos actually avoid insecure microhabitats in presence of rats. all other predictors were not significant (see table 4 for details). the random effect of islands (l-ratio χ² = 2.92, d.f. = 1, p = 0.09) was not significant, suggesting that cpue was not affected by island features other than rat occurrence and habitat. by contrast, the height from the ground where the geckoes were observed “in the open” was not affected by the rat occurrence (“rat-free” islands: 47.6 ± 2.6; “rat” islands: 32.6 ± 6.5), either by season (autumn: 51.7 ± 5.0; spring: 44.3 ± 2.7) or by their interaction (table 4). similarly, the effects of both island size and the number of reptile species were also negligible (see table 4 for statistics). we report some additional observation on the spatial behaviour. before rat eradication, on lavezzu island in 1982, on 112 sightings ≈85% of geckos were active under plant cover; in 1986 (n = 50) the proportion was ≈80-85% (md pers. obs.). ten years after rat eradication, the percentage of geckos active under plant cover was lower but still high: 67% in 2010 (n = 41), 68% in 2011 (n = 41). on the remote vacca islet, rats were detected for the first time in 2010 and eradicated by the bouches de bonifacio natural reserve in less than one year (o. bonnenfant pers. com.). we do not know the activity pattern before eradication, but in july 2012, 87% of geckos were observed in the open. analogously, few miles away, fig. 3. variation of the population structure of the european leaftoed gecko on 22 mediterranean islands in response to (a) rat occurrence (present in yellow, absent in green), (b) season (spring in yellow, autumn in green), (c) number of reptile species living on the islands (1 sp. in red, 2 spp. in yellow, 3 spp. in green, 4 spp. in blue), and (d) island size (islet: < 10,000 m² in yellow, island: > 10,000 m² in green). bars = 95% confidence interval. table 3. results of the linear mixed model for the variability of the body condition index of male and female leaf-toed geckos in response to rat occurrence. only fixed effects are reported. predictor df f p rat occurrence 1,6 0.0053 0.94 season 1,6 0.608 0.46 sex 1,5 2.545 0.11 table 4. results of the linear mixed model for the variability of the activity of leaf-toed geckos in response to rat occurrence (df have been calculated using the satterthwaite approximation). only fixed effects are reported. predictor df f p open vs close habitats rat occurrence 1,12.7 0.781 0.39 habitat type 1,24.9 1.344 0.26 n. reptile species 1,8.5 0.629 0.45 island size 1,8.3 0.802 0.39 rat occurrence × habitat type 1,24.9 11.99 0.0019 height above ground rat occurrence 1,14.9 1.068 0.32 season 1,22.3 0.659 0.42 n. reptile species 1,2 0.142 0.74 island size 1,4.4 2.168 0.21 rat occurrence × season 1,19.2 2.446 0.13 95native gecko and black rat on mediterranean islands rats invaded the toro islands where they have stayed for about 4 years, until 1992, when eradication occurred (thibault, 1992). in april 2005 (n = 50) ≈90%, in july 2012 (n = 52) 96% and in july 2014 (n = 59) 93% geckos were observed in the open. discussion two out of the three predictions on how the black rat may influence the activity and demography of euleptes europaea were confirmed by our results: a) in the presence of rats smaller sized geckos prevail, and b) geckos are less active “in the open”; c) by contrast, no significant effect on body condition was found. transition to populations characterized by smaller sized individuals could be caused by selective predation on larger geckos. on ohınau islet in new zealand, juveniles of the giant gecko hoplodactylus duvaucelii are more vulnerable to predation by rattus exulans (hoare et al., 2007), likely because adult geckos are similar in size to the pacific rat. conversely, a 170 g black rat could easily predate european leaf-toed geckos of any size (from 0.3 to 2 g). if this would be the case, a random predation of any size class could ultimately also result in a shortage of the larger sized geckos. in the mediterranean, despite some detailed studies on this topic (cassaing et al., 2005; pérez-mellado et al., 2008; petralia et al., 2010; ruffino et al., 2011), direct predation of rattus rattus on sauria was never observed, although predation on lizards has been supposed to occur in other regions (caut et al., 2008; gasc et al., 2010); ascertained (harper and bunbury, 2015; thibault et al., 2016; clapperton et al. 2019) or observed for other rat species (towns et al., 2006). abundance of lizards (and tuataras) has been related to the presence of rats but the mechanisms still deserve to be explained and likely could involve competitive processes less obvious than predation (towns et al., 2006). other vertebrates or invertebrates living on the studied islands could interact with the leaf-toed gecko as possible competitors and/or predators such as: the ant crematogaster scutellaris, competing for rock crevices and predating hatching geckos (delaugerre, 1981); introduced cows, grazing on vegetation used by geckos foraging “under cover”, e.g., on lavezzu (delaugerre and brunstein, 1987); the bird monticola solitarius and the ant tapinoma erraticum predating e. europaea respectively on lavezzu (delaugerre and cheylan, 1992) and porraggia grande islands (delaugerre and brunstein, 1987) and perhaps the western whip snake hierophis viridiflavus, that on giraglia island has been observed to have nocturnal habits (delaugerre, 2013). however 73% of the studied islands, host just one or two reptile species (table 1), the second species being typically a podarcis (diurnal) lizard, that is unlikely a predator of the leaf-toed gecko. on the remaining 27% islands, the presence of three or four (just on three islands) reptile species is equally distributed between rat and rat-free islands. alternatively, rats may affect gecko’s population structure by interfering with their growth process. survival of geckos can be reduced by increased physiological stress, which ultimately may cause physiological shifts in life history strategy and demographic fitness components (rödl et al., 2007; trompeter and langkilde, 2011; narayan et al., 2013). rats could act as stressors for geckos just roaming around. moreover, geckos could be subjected to infections carried by black rats (prenter et al., 2004), often infested by the adult nematode mastophorus muris (cassaing et al., 2005). according to lafferty et al. (2010), native geckos of the central pacific islands might serve as paratenic hosts of m. muris; this stage being the most incline in affecting host fitness (kuris, 2003). rats might also interact with geckos in a more indirect way, for instance inducing vegetation changes. lotus cytisoides, for example, is a plant that provides high quality shelters for geckos and for a lot of invertebrates (geckos’ preys) and it is consumed by rats (cassaing et al., 2005; ruffino et al., 2011). behavioural shift in microhabitat use might be costly for geckos, since they would be forced to forage under plant cover with consequently limited access to bare rock surfaces more favourable for nocturnal thermoregulation (thigmothermy). fig. 4. variation of the foraging mode of the european leaf-toed geckos, “in the open” (open) and “under cover” (close) in response to the presence of rats. the relative activity of geckoes was obtained from sightings of one hour of “catch per-unit-effort” (cpue) on 15 islets. bars = 95% confidence interval. 96 michel-jean delaugerre et alii information gathered on lavezzu before and after rat eradication seems to confirm an influence of the presence of rats on geckos’ population structure. before rat removal, as observed for other islands with rats, geckos’ size class structure was skewed towards medium-sized individuals. a decade after eradication (three geckos generations) we observed an upward trend towards larger sized individuals, even if the sample size of some size classes is too small to perform a statistical test. rats seem to change geckos’ population structure without affecting body condition of individuals. indeed, geckos’ body condition was not influenced by the presence of rats, sex or season. however, we found a strong significant random effect of islands, suggesting that body condition in both males and females strongly depends on some island features rather than on the presence of rats. activity, assessed by the catch per unit effort, did not vary just according to rat occurrence, but also depending on the microhabitat. in the presence of rats, geckos avoided to forage “in the open” and were more active “under cover”, probably to avoid disturbance and/or predation risk induced by the ground-dwelling rodents (whitaker, 1973; hoare et al., 2007). on lavezzu island, the “under cover” was the most common mode both before and after rat eradication, even if the percentages of geckos active “under cover” decreased ten years after eradication. this pattern (even if related to one island) may suggest a certain persistence of an avoidance behaviour likely resulted from a prolonged coexistence with rodents. though in geckos as well as in most reptiles, young individuals cannot learn through parental care or imitation, the acquisition of a novel behaviour might take long time. unlike how observed for the new zealand hoplodactylus duvaucelii who recovered its arboreal habitat six months after rat removal (hoare et al., 2007), lavezzu euleptes gecko did not show a similar behavioural plasticity. dealing with antipredator behaviours on islands blumstein (2002) reported that «experience dependant behaviours change rapidly following isolation» (and the loss of predator), «whereas more hard wired behaviours may persist for many generations.» does euleptes geckos have acquired an avoidance behaviour over the last 2000 years while coexisting with black rats? or, does this behaviour date back to the pleistocene when geckos coexisted with mammals nowadays extinct (e.g., the tyrrhenian field rat, rhagamys orthodon, m. masseti and g. cheylan (pers. comm.)? however, geckos’ behavioural changes due to the presence of rats seem to be very variable (hoare et al., 2007; krebs et al., 2015). as suggested by our results, geckos may benefit from rat eradication in most contexts but a lot of caution should be paid when undertaking rat eradications on islands that likely can be reinvaded (harris et al., 2011; savidge et al., 2012). once settled on an island, a rat population may repel new invading rats through aggressive interactions, as observed by granjon and cheylan (1989) and abdelkrim et al. (2009) who found that the lavezzu population was founded by a single colonization event without further gene flow. hence, eradication might entice new invaders to settle in a competition-free ecosystem. moreover, island “old-resident” rat populations are adapted to exploit seasonal resources adjusting home range, demography and intraspecific interactions (cheylan, 1988; clark, 1980; cassaing et al., 2007; ruffino et al., 2011; pisanu et al., 2011). new invading rats (e.g., mainland rats), not familiar with the island conditions, might severely affect the local biota. observations carried out on the corsican toro islands 2-4 years after rat invasion, besides the well-known detrimental effects on the cory’s shearwater and on the pallid swift, revealed that rats preyed almost to extinction silene vellutina (caryophyllaceae) (thibault, 1992), a rare endemic plant that elsewhere was not (or only marginally) consumed by “oldresident” rats. since several eradication actions have ‘failed’, and reinvasions occurred (cheylan and granjon, 1987; howald et al., 2007; russell et al., 2010; savidge et al., 2012; sposimo et al., 2012; ragionieri et al., 2013), the urge to learn from failures led to the proposal to adopt the metapopulational (russell et al., 2008) or the eradication units approach (robertson and gemmell, 2004) by considering first islands’ assemblages and relative reinvasion sources in order to properly guide eradication programs. native island species must cope with an increasing number of biological invasions; understanding the mechanisms of invasions, as well as the interaction between native species and long coexisting aliens, may be crucial to adopt proper conservation actions. acknowledgments we would like to thank: joanne monks (born hoare), who kindly granted us to borrow part of the title and structure of the paper (hoare et al., 2007); initiative petites iles de méditerranée (pim), apal (tunisia), cen corse, réserves naturelles des bouches de bonifacio, de scandola et des iles de la pointe du cap corse, parc naturel régional de la corse, parco nazionale dell’ arcipelago de la maddalena, parc national de port-cros; the colleagues and friends who helped during field work: a. abiadh, ch.-h. bianconi, v. bosc, d. brunstein, m.h. casalonga, g. deso, y. donno, a. gaio, o. gerriet, f. grita, n. nègre-santucci, o. patrimonio, j. renet. 97native gecko and black rat on mediterranean islands our study was improved by valuable discussions with gilles cheylan, marc cheylan, marco masseti, michel pascal and jean-claude thibault. permits to handle protected species were issued by the dreal (corse and paca) and by the ministero dell’ambiente e della tutela del territorio e del mare (u. prot. dpn 0010637b 16/05/2009, 0017564 12/08/2010, 0044068 04/12/2012 and 0001805 04/02/2015) and the parco nazionale dell’arcipelago di la maddalena. the access to giraglia island was authorized by the préfet de haute-corse. references abdelkrim, j., pascal, m., samadi, s. 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(1973): lizard populations on islands with and without polynesian rats, rattus exulans (peale). proc. new zeal. ecol.soc. 20: 121-130. acta herpetologica vol. 14, n. 2 december 2019 firenze university press podarcis siculus latastei (bedriaga, 1879) of the western pontine islands (italy) raised to the species rank, and a brief taxonomic overview of podarcis lizards gabriele senczuk1,2,*, riccardo castiglia2, wolfgang böhme3, claudia corti1 substrate type has a limited impact on the sprint performance of a mediterranean lizard pantelis savvides1,*, eleni georgiou1, panayiotis pafilis2,3, spyros sfenthourakis1 coping with aliens: how a native gecko manages to persist on mediterranean islands despite the black rat? michel-jean delaugerre1,*, roberto sacchi2, marta biaggini3, pietro lo cascio4, ridha ouni5, claudia corti 3 pit-tags as a technique for marking fossorial reptiles: insights from a long-term field study of the amphisbaenian trogonophis wiegmanni pablo recio, gonzalo rodríguez-ruiz, jesús ortega, josé martín* occurrence of batrachochytrium dendrobatidis in the tensift region, with comments on its spreading in morocco ait el cadi redouane1, laghzaoui el-mustapha1, angelica crottini2, slimani tahar1, bosch jaime3,4, el mouden el hassan1,* hematological parameters of the bolson tortoise gopherus flavomarginatus in mexico cristina garcía-de la peña1,*, roger iván rodríguez-vivas2, jorge a. zegbe-domínguez3, luis manuel valenzuela-núñez1, césar a. meza herrera4, quetzaly siller-rodríguez1, verónica ávila-rodríguez1 ontogenetic and interspecific variation in skull morphology of two closely related species of toad, bufo bufo and b. spinosus (anura: bufonidae) giovanni sanna visible implant alphanumeric (via) as a marking method in the lesser snouted treefrog scinax nasicus andrea caballero-gini1,2,3,*, diego bueno villafañe2,3, lía romero2, marcela ferreira2,3, lucas cañete4, rafaela laino2, karim musalem2,5 morphological variation of the newly confirmed population of the javelin sand boa, eryx jaculus (linnaeus, 1758) (serpentes, erycidae) in sicily, italy francesco p. faraone1,*, salvatore russotto2, salvatore a. barra3, roberto chiara3, gabriele giacalone4, mario lo valvo3 variability in the dorsal pattern of the sardinian grass snake (natrix natrix cetti) with notes on its ecology enrico lunghi1,2,3,4,*, simone giachello5, manuela mulargia6, pier paolo dore7, roberto cogoni8, claudia corti1 estimating abundance of the stripeless tree-frog hyla meridionalis by means of replicated call counts federico crovetto, sebastiano salvidio, andrea costa* at-rich microsatellite loci development for fejervarya multistriata by illumina hiseq sequencing yan-mei wang, jing-yi chen, guo-hua ding*, zhi-hua lin acta herpetologica 15(2): 125-128, 2020 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/a_h-9206 confirming lessona’s brown frogs distribution sketch: rana temporaria is present on turin hills (piedmont, nw italy) davide marino1, angelica crottini2, franco andreone3,* 1 associazione natura invisibile, via avigliana, 24, i-10138 torino, italy 2 cibio, research centre in biodiversity and genetic resources, inbio, universidade do porto, campus agrário de vairão, rua padre armando quintas, no. 7, 4485-661 vairão, portugal 3 museo regionale di scienze naturali, via g. giolitti, 36, i-10123 torino, italy *corresponding author. e-mail: franco.andreone@gmail.com submitted on: 2020, 1st july; revised on: 2020, 13th july; accepted on: 2020, 30th july editor: stefano scali abstract. the presence of rana temporaria on turin hills (piedmont nw italy) has been confirmed through morphological and molecular analyses. breeding individuals of this species were found at two sites and assessed by either morpho-chromatic and genetics. this new finding represents an interesting confirmation of ancient record reported in 1877 by the renowned naturalist michele lessona, and highlights that the species is likely quite cryptical and secretive and has a distribution wider than formerly presumed. keywords. common frog, rana temporaria, new findings, turin hills, nw italy. the common frog rana temporaria shows a conspicuous wide geographical and elevational distribution, being present in most of europe and in the northern and central regions of western asia, from northern spain, france, united kingdom and the scandinavian peninsula and russia (urals and adjacent western siberia) to northern kazakhstan (lanza, 1983; kuzmin, 1999; lanza et al., 2009; sillero et al., 2014), from sea level to around 2846 m a.s.l. (vences et al., 2003, 2013; maurino and doglio, 2010; tiberti and von hardenberg, 2012; di nicola et al., 2019). in southern europe this species is usually associated to montane habitats, being absent from southern and central iberia, most of southern italy and caucasus, and shows a patchy distribution in the balkans and in the mediterranean islands. in italy it is found on the alpine and northern apennine reliefs, with a fragmented and irregular distribution in arezzo, florence and forlí-cesena provinces, and a relict population on the monti della laga (rieti province, ne latium) (capula and bagnoli, 1983; razzetti et al., 2007). in piedmont (nw italy) r. temporaria is quite common on the alps, on northern apennines and on southern hilly reliefs, known as “langhe“, with scattered, findings at low altitudes (andreone et al, 1988; andreone and sindaco, 1989, 1999). in a pioneer contribution on anuran distribution, the renowned naturalist, writer, lecturer, minister and darwin translator michele lessona published a colour map (realised by his son-in-law l. camerano) with the inferred distribution of r. temporaria in piedmont and aosta valley known at that time. in this map the species was also reported on the hill system bordering the town of turin, known as “collina di torino” (lessona, 1877). no precise localities or toponyms were provided together with this map, although lessona provided considerations on abundances, human uses and life history traits on this species in piedmont. although lessona was turin zoological museum’s director (camerano, 126 davide marino, angelica crottini, franco andreone 1894), no preserved specimen of this species from turin hills is currently present in the historical herpetological collection of turin university (now hosted by the museo regionale di scienze naturali: gavetti and andreone, 1993), and we did not find any record of r. temporaria from turin hills on the historical catalogues or quoted by previous authors, such as camerano (1884) and tortonese (1953). until now r. temporaria was not reported for turin hills, neither during the realization of the herpetological distribution atlas of piedmont and aosta valley (andreone and sindaco, 1999), nor in the italian atlas (sindaco et al., 2006), and this record was considered anecdotal. this assumption was contradicted by recent observations, reported in this paper. during a survey carried out on the 8th march 2017 to confirm the presence of the alpine newt (ichthyosaura alpestris apuana) on turin hills (marino, 2018), one of us (dm) reported a single brown frog at valsalice (altitude: 315 m a.s.l.; coordinates: 45°03’04.5”n, 7°42’46.6”e). this individual corresponded in morphology and colouration to a typical r. temporaria. this record was promptly reported on inaturalist, and further confirmed by the herpetological online community. this individual (a male) was quite large (around 60 mm snout-vent length), had a relatively short snout and hindlimbs, that, adpressed along body barely reached the eye (fig. 1). the colouration of this individual was brownish, with sparse dark spots on the back, intense purple shading under the throat and missed the yellowish belly and groin shadings typical of the agile frog r. dalmatina¸ the other brown frog confirmed in his area. a few days later another female and some males were found around an artificial pond, where typical eggsclumps were also found. on the 29th march this species was also found at at reaglie (altitude: 380 m a.s.l.; coordinates: 45°02’44.9”n, 7°44’55.1”e), about 10 km away from valsalice (fig. 2). to further confirm the species identity we analysed the tissue samples of two adults, one tadpole and one egg. we also took the tissue samples of two adults r. dalmatina of reaglie for comparison. total genomic dna was extracted from these samples using proteinase k digestion (10 mg/ml concentration) followed by a standard salt extraction protocol (bruford et al., 1992). we sequenced a fragment of ca. 550 bp of the 3’ terminus of fig. 2. the distribution of rana temporaria in piedmont and aosta valley (nw italy) based upon current data. dark and light gray show alps, prealps and inner hills. the transverse (green) bars represent the known distribution. the (red) stars mark the confirmed sites recently found on the hill system near turin. fig. 1. rana temporaria, male from turin hills, piedmont (nw italy) (a: dorsolateral view; b: ventral view) (photographs by f. andreone). 127new findings of rana temporaria in turin hills the mitochondrial rrnl gene. polymerase chain reactions (pcr) were performed in a final volume of 25 μl using 0.75 μl each of 10 pmol primer, 0.4 μl of total dntp 10 mm (promega), 0.1 μl of 5 u/ml gotaq (promega), 5 μl 5x green gotaq reaction buffer (promega) and 4 μl of mgcl2 25mm (promega). for primers and cycling protocols, see crottini et al. (2011). successfully amplified pcr products were treated to inactivate remaining primers and dntps. purified pcr templates were sequenced using dyelabelled dideoxy terminator cycle sequencing on an abi3730xl at macrogen inc. sequences were checked by eye, edited (when necessary), aligned using the bioedit sequence alignment editor (version 7.0.5.3; hall 1999) and compared to the genbank dataset. all newly determined sequences have been deposited in genbank (mt459788-mt459793). all these observations support the presence of r. temporaria on the hill system around turin and confirm old lessona’s ancient maps, providing a significant novelty in terms of species distribution in piedmont. it remains somehow surprising that no records of r. temporaria were reported since lessona’s contribution (including the lack of museum specimens and photographs): we believe this absence might be due to the species overall similarity with the agile frog r. dalmatina, and to a deficit of field research during suitable periods and in suitable sites. in fact, r. temporaria apparently prefers stream systems (in particular in late winter), which were not so frequently surveyed on the turin hills. finally, we cannot exclude that in the 19th century the species was more widespred and common than today, and since then this species has reduced its distribution, as it is already known for pelobates fuscus and zootoca carniolica, two species which survived with patchy populations at a few sites along the po river (andreone and sindaco, 1999). the presence of r. temporaria in the hill system of turin may represent a vestige of an ancient and more widespread distribution range for the species, as already supposed for the alpine newt (andreone and sindaco, 1987; marino 2018). further research efforts should be devoted to better characterize the distribution of this species around the hill system of the city of turin, and between the city and “langhe”, to valorise all remnant populations of this species. acknowledgements we thanks the authorities and friends who accompanied us in the field: g. tonelli, m. dadda and the park guards of “parco del po e collina torinese“. the portuguese national funds through fct – foundation for science and technology – supported the investigador fct (if) grant to ac (if/00209/2014). this work is also funded by national funds through fct within the if/00209/2014/cp1256/ct0011 exploratory research project. l. cavigioli greatly helped with the realization of the distribution map. permits to carry out research were issued by regione piemonte (det. n. 234 of 21/6/2018). finally, we thank the two reviewers who provided useful comments to a previous version version of this contribution. references andreone, f., delmastro, g.b., boano, g. 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(2013): radically different phylogeographies and patterns of genetic variation in two european brown frogs, genus rana. mol. phylogenetics evol. 68: 657-670. acta herpetologica vol. 15, n. 2 december 2020 firenze university press estimating abundance and habitat suitability in a micro-endemic snake: the walser viper gentile francesco ficetola1,2,*, mauro fanelli3, lorenzo garizio3, mattia falaschi1, simone tenan4, samuele ghielmi5, lorenzo laddaga6, michele menegon7,8, massimo delfino3,9. potential effects of climate change on the distribution of invasive bullfrogs lithobates catesbeianus in china li qing peng1, min tang1, jia hong liao1, hai fen qing1, zhen kun zhao1, david a. pike2, wei chen1,* a bibliometric-mapping approach to identifying patterns and trends in amphibian decline research claudio angelini1,*, jon bielby2, corrado costa3 food composition of a breeding population the endemic anatolia newt, neurergus strauchii (steindachner, 1887) (caudata: salamandridae), from bingöl, eastern turkey kerim çiçek1,*, mustafa koyun2, ahmet mermer1, cemal varol tok3 stomach histology of crocodylus siamensis and gavialis gangeticus reveals analogy of archosaur “gizzards”, with implication on crocodylian gastroliths function ryuji takasaki1,2,*, yoshitsugu kobayashi3 does chronic exposure to ammonium during the pre-metamorphic stages promote hindlimb abnormality in anuran metamorphs? a comparison between natural-habitat and agrosystem frogs sonia zambrano-fernández1, francisco javier zamora-camacho2,3,*, pedro aragón2,4 confirming lessona’s brown frogs distribution sketch: rana temporaria is present on turin hills (piedmont, nw italy) davide marino1, angelica crottini2, franco andreone3,* phylogenetic relationships of the italian populations of horseshoe whip snake hemorrhois hippocrepis (serpentes, colubridae) francesco paolo faraone1, raffaella melfi2, matteo riccardo di nicola3, gabriele giacalone4, mario lo valvo5* first karyological analysis of the endemic malagasy phantom gecko matoatoa brevipes (squamata: gekkonidae) marcello mezzasalma1,2,*, fabio m. guarino3, simon p. loader1, gaetano odierna3, jeffrey w. streicher1, natalie cooper1 notes on sexual dimorphism, diet and reproduction of the false coral snake oxyrhopus rhombifer duméril, bibron & duméril, 1854 (dipsadidae: pseudoboini) from coastal plains of subtropical brazil fernando m. quintela1,*, felipe caseiro¹, daniel loebmann¹ acta herpetologica 15(1): 39-45, 2020 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/a_h-8744 an overview of research regarding reservoirs, vectors and predators of the chytrid fungus batrachochytrium dendrobatidis jarid prahl, thomas p. wilson*, david giles, j. hill craddock department of biology, geology and environmental science, university of tennessee: chattanooga, chattanooga, tennessee, usa * corresponding author. email: thomas-wilson@utc.edu submitted on: 2019, 8th september; revised on: 2020, 13thjanuary; accepted on: 2020, 15th january editor: fabio m. guarino abstract. this review presents an overview of research from 1998-2018 regarding interactions of batrachochytrium dendrobatidis with both potential hosts and predators. to this end, 23 different studies collected from the web of science database along with two external journals were utilized, encompassing numerous taxonomic groups. numerous groups of animals were identified as potential vectors for the fungus, with crayfish and reptiles standing as the most prominent and consistent non-amphibian hosts warranting their inclusion in any future broadscale distribution surveys. an important area for future research. additionally, daphnia were noted to serve as predators of the zoospores when exposed in mesocosm scenarios, reducing infection levels in corresponding tadpoles. caecilians have also been observed to be carriers of bd, though the level as to which the chytrid impacts these organisms needs to be further researched. in total, this review indicates that future research needs to begin including freshwater crustaceans, caecilians and reptiles in field studies for presence/absence, while a broader range of taxa need to be tested to see whether they serve as vectors or hosts in natural scenarios. keywords. batrachochytrium dendrobatidis, crayfish, reptiles, daphnia, fish, caecilians, alternate hosts, review. introduction since its identification in the late 1990s, amphibian declines have been heavily attributed to a parasitic fungus called batrachochytrium dendrobatidis (longcore et al., 1999) as it spreads around the globe (longcore et al., 1999). a member of the fungal division chytridiomycota, the zoospores encyst in an amphibian’s skin, often resulting in deleterious effects, posing especially difficult to eradicate to its ability to persist in areas for years despite an apparent lack in hosts and easy spread via multiple vectors. while anthropogenic actions and naturally resistant amphibian species have long been considered potential vectors for batrachochytrium dendrobatidis (bd), interest has steadily grown over the past decade in whether non-amphibians could serve as either a vector for the disease or potentially as alternative hosts from which the fungus could complete its lifecycle (mcmahon et al., 2013). as the zoospores are specifically attracted to highly keratinized cells, such as those in the frog’s skin, early studies proposed and investigated whether the fungus would be attracted to keratin from non-amphibious sources such as snakeskin and crayfish with varying results (longcore et al., 1999; piotrowski et al., 2004; rowley et al., 2006). since then, research into the topic has been few and far between, with the subjects ranging from traditional aquatic species to those who may serve as unconventional vectors such as reptiles. material and methods in this review, we sought to provide an overview regarding research into the roles that taxa outside of the clade batrachia 40 jarid prahl et alii (frogs and salamanders) play as potential hosts or predators of bd. to select studies for inclusion in this review, we combined the web of science database along with the journals herpetological review and herpetological conservation & biology to find most of the studies/reports. when searching the database, each major taxa group investigated, combined with the chytrid, were used as keywords, with new taxa being included every time they were first eluded to in a previous study. while it is possible that some studies could have been overlooked due to differences in keywords or not being present in the database, this search method covered a broad range of taxonomic groups and serves as a solid base for this research. results these studies have been conducted in a range of fashions in both the lab and the field on animals ranging from crayfish, to reptiles and even nematodes, with several such individuals found capable of carrying the fungus. all investigated species were selected due to the presence of keratin either on surface structures or internally (such as the gastrointestinal tract), while also being associated with freshwater environments where they could easily contract the pathogen. while macroinvertebrates were the initial focus of this form of study, reptiles have become increasingly more researched. potential reservoirs when it comes to invertebrates, crustaceans have been the most prominently studied for a potential relationship with bd, with two separate approaches for two distinct groups. decapods (crayfish and shrimp) were first investigated as carriers for bd in 2006 in a study on both caridina zebra (a shrimp) and cherax quadricarinatus (queensland red claw) in field studies and in lab infection studies that initially reported the presence of the chytrid in both species before being retracted a year later with false positives being attributed to an error in the transcription of raw data (rowley et al., 2006; 2007). despite this early controversy, a later study by mcmahon et al. (2013) confirmed the presence of zoospores on both the carapace and in the gastro-intestinal tract of three crayfish species; orconectes virilis (blue crayfish), procambarus alleni (blue crayfish) and procambarus clarkii (louisiana crayfish) both in captivity and in the wild. this model study presents a few notable findings, most importantly that tadpoles exposed to infected crayfish were highly susceptible to having the pathogen transmitted to them in captivity (mcmahon et al., 2013). discharge tubules being observed in the gastrointestinal tract of both captive and wild-caught crayfish also indicates that they can serve as reservoir hosts for bd (mcmahon et al., 2013). interestingly, these crayfish were found to be both carriers and victims of bd, with gill recession occurring in individuals who were exposed to either bd filtrates or the zoospores themselves (mcmahon et al., 2013). the finding that in the natural systems where crayfish tested positive for bd, all the amphibians were negative, also helps to indicate that the crayfish may act as true alternate hosts in a broader cycle (mcmahon et al., 2013). some of these findings were corroborated by another study, that identified bd in both wild and farmed populations at 3.31% and 6% prevalence respectively across various procambarus species in louisiana (brannelly et al., 2015). unfortunately, this study was not conducted with a concurrent amphibian survey to assess a potential relationship between infection rates of the crayfish and the amphibians, limiting the ecological importance of this study. additionally, the close quarters of the farmed populations would easily result in cross-contamination that might have inflated the number of positives found in these populations. inflated or not, the finding of bdpositive (bd+) individuals pose an interesting potential for disease spread as fishermen will often purchase live crayfish as bait, potentially introducing contaminated individuals to bd-negative (bd-) sites. this potential vector needs to be further explored on a larger scale, as the various methods of transmission must be understood if we are to curb the spread of this disease. these findings of crayfish positivity were not universal, however, as the study by betancourt-roman et al. was performed in a similar fashion but found no evidence of gill recession in procambarus alleni and a fairly low prevalence rate (5%) on sampled carapaces (betancourt-roman et al., 2016). however, this study was only performed with nine exposed individuals in the lab (small compared to the previous two studies) and the restriction of sampling to the carapace, excluding the gi tract, could have potential resulted in false negatives (betancourt-roman et al., 2016). research involving crustaceans was not limited to crayfish, as a study by paulraj et al. (2016) investigated the possibility of macrobrachium rosenbergii (giant freshwater prawn) serving as a vector for bd, due to the prominent commercial species being heavily impacted by fungal infections. a field survey was conducted across 15 culture ponds in south india over the course of four years, with infection being determined through visual inspection and subsequent swabbing (paulraj et al., 2016). physical signs of infection noted by the researchers included: dullness of color, abnormal protrusion of scales, tufts of fungal growth, lethargic movement and an overall spongy appearance (paulraj et al., 2016). this 41bd reservoir, vectors and predators study mirrored previous research on crustaceans with a bd prevalence of 17.4-38.8% being detected across the sampled farming ponds with prevalence noted to be lowest in the months of april, may, june, and highest in october, november and december (paulraj et al., 2016). these findings continue to support the idea that the infection may transfer from amphibians in the spring to crustaceans in the fall, with this specific scenario needing further study. another study investigating the presence of bd on the island of jamaica included two sesarmid land crabs and 12 crayfish (unidentified) in their survey, with one of each being bd+ (holmes et al., 2014). neither of these studies investigated presence within the gastrointestinal tract (which was shown by previous studies to have higher levels in crayfish compared to carapace swabs) and did not directly observe discharge tubules, but the potential that they mirror other decapods means that these species should be investigated in a manner similar to mcmahon et al. (2013). certain fish have also been shown to function as reservoirs in the laboratory, with samples taken from danio rerio displaying various stages of development, including discharged mature zoosporangia, indicating the ability to reproduce off the larvae (liew et al., 2017). infected individuals were found to suffer from fin erosion at the 72-hour mark and later, along with skin blisters, the disruption of muscle striations, and an increased level of degeneration with these same symptoms occurring in larvae exposed to only filtered supernatants (liew et al., 2017). this damage seems to be similar in nature to the negative impacts of bd on crayfish, with the same symptoms in filtered treatments indicating the presence of some enzyme causing the damage rather than just encystation. the ability for the chytrid to clearly encyst and replicate successfully in d. rerio larvae further illustrates why the view of potential hosts for bd needs to be expanded and indicates that this is a knowledge gap that should be focused on more extensively. potential vectors reptiles ranging from squamates to those in the clade aves have been investigated the most, with the first study being undertaken in 2012 (garmyn et al., 2012). waterfowl have been of interest due to their consistent exposure to water systems such as ponds and wetlands where they would be likely to encounter bd+ environments and serve as vectors between various sites. all these studies focused on sampling the toe scales of various individuals either through swabs alone or through toe clippings. all living species that have been sampled across two studies (garmyn et al., 2012; hanlon et al., 2017) were found to have individuals positive for the chytrid, with 14.9% of branta canadensis, 19.5% of anser anser domesticus, 45% of anas strepera, 50% anas carolinensis and 62% of anas platyrhynchos being bd+. in the laboratory it was also found that spores would adhere and encyst to toe scales after 24-hours while desiccation tests found that spores remained viable after 30 minutes, indicating that the range of transmission would be based off an animal’s flight speed over thirty minutes (garmyn et al., 2012). furthermore, they were also able to have zoospores produce zoosporangia from day 4-14 after initial exposure (garmyn et al., 2012). notably, in the study by garmin et al (2012) the feet of sampled waterfowl were rinsed to remove transient spores differently from the study by hanlon et al (2017) and this could have been the reason for increased levels of positive individuals. the fungus has also been detected from museum specimens of waterfowl, but this appears to be more variable depending on the sampling technique. when comparing specimens from both the la paz museum and seville museum, 13 individuals were found to be bd+ when performing tissue sampling while only one of those same individuals was positive from swabs (burrowes and de la riva, 2017). this study also found that the oldest positive sample dated back to 1982 from an anas flavirostris (yellow-billed teal) and a plegadis ridgwayi (puna ibis) collected from ulla dam, bolivia (burrows and de la riva, 2017). other species positive for bd include anas georgica (yellow-billed pintail), rollandia rolland (whitetufted grebe), fulica ardesiaca (andean coot), fulica gigantea (giant coot), lophonetta specularioides (crested duck) and spatula puna (puna teal), having been collected from various sites across bolivia (burrows and de la riva, 2017). squamate reptiles have also been investigated as potential vectors by testing for the fungus on the ventral surface of multiple species and comparing its presence to nearby amphibian populations. anolis lionotus and a. humilis were found to be positive for bd, with a prevalence of 9% and 32% respectively among sampled individuals and an overall prevalence of 16% in survey sites in panama (kilburn et al., 2011). three snake species were also positive for the chytrid: imantodes cenchoa (blunthead tree snake), nothopsis rugosus (rough coffee snake) and pliocercus euryzonus (cope’s false coral snake), with one individual of each species being tested and a total prevalence of 38% among all sampled snakes (kilburn et al., 2011). habitat may have had an influence on which species tested positive for the fungus with the three species of snakes as a. humilis routinely encountering wet leaf litter that could retain spores, while a. lionotus is specifically noted as being semi-aquatic. it is possi42 jarid prahl et alii ble that the snakes may have acquired zoospores through predation upon infected amphibians, while bd+ reptiles may serve as vectors for the spread of this disease by direct contact or indirect environmental spreading. this study is markedly contrasted by a similar study conducted in australia, which investigated whether physignathus lesueurii (eastern water dragon) juveniles were positive for bd by conducting opportunistic sampling in murray upper national park (phillott et al., 2009). while all of the p. leseuerii were found to be negative, concurrently sampled amphibians in the area were found to be positive for bd showing that the fungus is present in the region (phillott et al., 2009). the reason infection was not able to set in on these agamids may be due to prolonged periods of desiccation or to the natural cutaneous microfauna present, but neither of these factors were specifically investigated. the potential for fish to be vectors has also been investigated in two studies in a laboratory setting, including poecilia reticulata (ornamental guppies) and gambusia holbrooki (eastern mosquitofish) and danio rerio (mcmahon et al., 2013; liew et al., 2017). while both d. rerio and p. reticulata had high levels of bd shortly after initial exposure, the guppies showed a consistent decrease in the genomic equivalents of bd dna and the mosquitofish showed no evidence of infection (liew et al., 2017). the previously mentioned colonization of d. rerio in combination with these findings shows that bd’s relationship to fish can vary extensively across taxa, with fish serving as both potential hosts and/or vectors with more extensive research being required to better shape how fish relate to bd ecology. bacteria were also shown to have a significant effect in mitigating infection of the larvae in exposed zebrafish coinciding with research regarding amphibian microbial communities having an impact on susceptibilities (liew et al., 2017). forming a profile of the three fish species’ bacterial communities would allow comparison between susceptible and resistant amphibians and may explain why infection was able to persist in d. rerio larvae and produce mature zoosporangia while infection waned in the p. reticulata and failed in g. hoolbrooki. another important note is that if the mosquitofish reacted to bd in a similar manner to the guppies, the difference in time before sampling between these two studies could be responsible for the differences in susceptibility through false negative readings. abiotic factors may also be potential vectors of bd zoospores as explored in a study based in honduras that investigated zoospore presence in raindrops. out of the 20 rainfall events that kolby et al. (2015) sampled in this study, one was found to have a bd+ sample with an extremely low density as the study ensured collection sheets were emplaced in clearings to prevent throughfall of contaminated water from the forest canopy, these zoospores were at least from drops that were windblown off contaminated amphibians and may have even been directly carried by the precipitation (kolby et al., 2015). this study proposes a potentially game-changing method of transportation if the zoospores can truly be transported through precipitation and definitely requires follow on research to verify these findings and expand upon their possible implications. predators taxa of the order cladocera (water fleas) and various other micro-invertebrates have been investigated as predators of the fungus and whether they might be effective as biological controls. individuals of the genus daphnia have been shown to have a clearly negative impact on zoospore concentrations, reducing genomic equivalents to nearly undetectable levels in a mesocosm experiment combining infected tadpoles with more than ten daphnia (hamilton et al., 2012). infected rana aurora (northern red-legged frog) tadpoles were used, with daphnia being noted to have an indirect positive effect on tadpole biomass alongside a negative impact on overall tadpole survivability (hamilton et al., 2012). to verify that zoospore presence in daphnia intestines was due to predation, another study exposed starved individuals to the zoospores and compared treatments with living and dead individuals, finding significantly higher concentrations in the intestines of living daphnia (buck et al., 2011). while these studies show evidence that members of daphnia would be beneficial in fighting the chytrid, the reduced scope of these makes it hard to draw any major generalizations, especially regarding natural systems. outside of daphnia, microfauna presence in general has been observed to be negatively correlated with zoospore prevalence in natural systems with lab experiments indicating that a portion of this impact comes from the microfauna predating upon the zoospores (schmeller et al., 2014). one study noted that higher prevalence of microfauna was a significant differentiator between low and high prevalence sites (schmeller et al., 2014), while another noted the presence of a more diverse rotifer and micro-arthropod community in bromeliads had a more negative impact on bd prevalence than the protist diversity found in the surrounding streams (blooi et al., 2017). this information coupled with the studies on daphnia illustrate why it is important to consider bd’s role in the ecological community outside of simply being a parasite if we desire to truly understand its distribution pattern along with what may help curb its spread. 43bd reservoir, vectors and predators caecilians despite being amphibians, the finding that b. dendrobatidis can infect caecilians is fairly recent and can potentially provide information regarding the ecology of the fungus that has been long overlooked. while their secluded nature and primarily subterranean lifestyle make them unlikely to serve as vectors, it is interesting to note that this has not precluded them from encountering this fungus. furthermore, it also continues to demonstrate how broadly ranging this pathogen’s potential hosts are, though the full impact of chytridiomycosis on members of order gymnophiona are hard to determine due to the difficulties of studying members of this taxa. the first study to investigate caecilians for bd specifically sampled both african and south american species, including nearly 200 individuals upon initial capture and an additional 31 being tested after being kept in captivity for two years (gower et al., 2013). while small in terms of geographic coverage, this study was quite broad regarding taxa diversity, including representatives from 10% of known species and at least one species from 12/34 known genera and 8/10 known families (gower et al., 2013). while none of the south american species were positive for the chytrid, all african genera tested had at least two positive individuals except schistometopum, showing a broad range of infectivity (gower et al., 2013). unfortunately, however, improper sampling techniques initially including handling without gloves could have led to false positives and inflated the present findings. another recent study also performed in africa confirmed the prevalence of bd in four species not reviewed in the gower study, providing confirmation of the pathogen’s presence in african species of caecilians (thorpe et al., 2018). however, this study had small sample sizes making it difficult to extrapolate the range of this disease across the broader population. a third study was also conducted in africa as a broad survey of caecilians and anurans for the fungus and found it to be present on various caecilians but gave no specific identifications regarding species sampled and which were positive for bd (hydeman et al., 2017). overall, members of gymnophiona should be investigated more thoroughly to determine the presence of bd across the order worldwide, though this may be difficult as a result of their isolated and subterranean nature. unclear relationships investigation of invertebrates was not limited to crustaceans, with the nematode species caenorhabditis elegans also being the subject of investigation in two separate studies due to their habitat overlap with amphibians/crayfish and the similarity of function between their tissues. both studies utilized heat-killed bd as controls and determined mortality by counting both the living and dead individuals present in each treatment (shapard et al., 2012; betancourt-roman et al., 2016). these two studies had contradictory results, however, as shapard et al. noted an increase in mortality from control to infected treatments, while betancourt-roman et al. saw no significant differences between the two groups. while these separate experiments were run as similarly as possible, the betancourt-roman study noted that differing lab conditions made the study hard to replicate and that double the concentration of zoospores was used. in the shapard study, the use of staining dye to mark the zoospores displayed an apparent attachment of bd to the external cuticle of the nematodes, with the cuticle frequently becoming breached afterward (shapard et al., 2012). due to the effects of zoospores on the cuticle, it is probable that the zoospores release proteolytic enzymes that serve to degrade the collagen forming the cuticle, however this enzyme was not isolated. this interaction between nematodes and bd warrants further examination, due both to the contradictory findings of these two studies and the potential for use as either vectors or hosts (as indicated by observed encystation in the laboratory). the potential of other parasites to influence the prevalence of bd in amphibians has also been investigated across many field sites and multiple anuran species. a negative correlation with both parasite abundance and diversity was noted in bd infected rana aurora tadpoles (nieto et al., 2007). echinostoma sp. specifically were negatively correlated with bd presence across various amphibians while ribeiroia ondatrae was positively correlated with bd presence at the site level (stutz et al., 2018). this aspect of disease interaction has been largely underresearched and warrants further study to better understand the overall ecology of the chytrid fungus. discussion when looking at this research it seems clear that non-amphibian hosts pose a potentially important role in both transmission and ecology of batrachochytrium dendrobatidis across numerous taxonomic groups. the presence of bd zoospores on the toe scales of waterfowl could allow for the fungus to be spread across a wide range, while the potential for zoospores to be dispersed via rainfall poses an even wider avenue for distribution. while birds are potentially capable of transporting bd the longest distance, the detected presence 44 jarid prahl et alii on some squamate and fish species raises the potential these taxa could serve to distribute among large, interconnected water systems and wetlands. the presence of zoospores in and on crayfish could also allow for a wider spread of the fungus through anthropogenic methods, especially as bd has been observed successfully replicating within their gastro-intestinal tract. fishing especially could allow for the fungus to be spread easily and much more rapidly than it would naturally, as infected crayfish can be transported long distances to potentially isolated water systems where the zoospores can then become introduced via fecal matter or upon predation of the crayfish. the finding that crayfish can be positive for the disease while amphibians in the same system are negative could also help explain how bd has managed to persist in systems after the initial extirpation of frogs. predation of the zoospores by various microfauna such as daphnia presents an alternate end of the spectrum by potentially helping to explain how and why certain areas have been more impacted by the presence of bd. repeatedly, lab studies would also observe that an enzyme produced by the zoospores had a negative impact on nonamphibian species, with gill recession in crayfish, fin erosion in fish and cuticle breaching in nematodes being tied to this enzyme. while none of these studies identified the enzyme, the fact that filtrate has the same deleterious effects as zoospore presence could mean that negative impacts experienced by non-amphibian hosts/ vectors are caused primarily by the enzyme, but future research on the subject is needed. conclusion based on these findings, it is clear that an important concern for bd field surveys is the incorporation of other known hosts outside of amphibians. the most likely taxa to be carrying the fungus besides anurans and caudatans seems to be crustaceans such as crayfish species within the genus procambarus which reside in largely the same habitats as anuran species and have been observed to have full lifecycles being conducted through the chytrid. birds could be a potentially important factor when trying to model the chytrid’s spread across broad areas, and should definitely be investigated further to observe whether they are truly viable vectors for the zoospores. this is especially true for waterfowls due to their ability to fly long distances in the thirty minutes required for the zoospores to become susceptible to desiccation, posing a major potential threat to the prevention of this disease’s spread. transportation via rainfall poses a similar potential problem and should continue to be investigated to better determine the prevalence of this phenomenon and how it may impact distribution. similarly, predation of zoospores by microfauna needs further research, potentially utilizing a mesocosm approach, to test what taxa have the largest impacts and how this interaction relates to how bd fits into the community at a larger scale. finally, it is important to continue investigating other taxa to help broaden the list of potential hosts, with a special focus on other crustaceans/macroinvertebrates and semiaquatic reptiles along with caecilian taxa when performing studies in south america or africa. acknowledgments we would like to thank the members of team salamander at the university of tennessee: chattanooga for their continuous support throughout this process, who helped improve the study via valuable discussions and support. references betancourt-roman, c.m., o’neil, c.c., james, t.y. 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(2012): batrachochytrium dendrobatidis can infect and cause mortality in the nematode caenorhabditis elegans. mycopathologia 173: 121-126. stutz, w.e., blaustein, a.r., briggs, c.j., hoverman, j.t., rohr, j.r., johnson, p.t.j. (2018): using multiresponse models to investigate pathogen coinfections across scales: insights from emerging diseases of amphibians. meth. in ecol. evol. 9: 1109-1120. thorpe, c.j., lewis, t.r., fisher, m.c., wierzbicki, c.j., kulkarni, s., pryce, d., davies, l., watve, a., knight, m.e. (2018): climate structuring of batrachochytrium dendrobatidis infection in the threatened amphibians of the northern western ghats, india. roy. soc. open sci. 5: 180211. acta herpetologica vol. 15, n. 1 june 2020 firenze university press has the west been won? a field survey and a species distribution model of iberolacerta horvathi in the alps giuseppe de marchi1,*, giovanni bombieri1, bruno boz1, fausto leandri2, jacopo richard3 first record of underwater sound produced by the balkan crested newt (triturus ivanbureschi) simeon lukanov identification of biologically active fractions in the dermal secretions of the genus bombina (amphibia: anura: bombinatoridae) iryna udovychenko1,*, oleksandra oskyrko1, oleksii marushchak2, tetiana halenova1, oleksii savchuk1 don’t tread on me: an examination of the anti-predatory behavior of eastern copperheads (agkistrodon contortrix) andrew adams1,2,*, john garrison2, scott mcdaniel2, emily bueche2, hunter howell2,3 an overview of research regarding reservoirs, vectors and predators of the chytrid fungus batrachochytrium dendrobatidis jarid prahl, thomas p. wilson*, david giles, j. hill craddock genetic characteristics of an introduced population of bombina bombina (linnaeus, 1761) (amphibia: bombinatoridae) in moselle, france jean-pierre vacher1, 2,*, damien aumaître3, sylvain ursenbacher2,4 adaptive significance of the transparent body in the tadpoles of ornamented pygmy frog, microhyla ornata (anura, amphibia) santosh m. mogali*, bhagyashri a. shanbhag, srinivas k. saidapur comparison of complement system activity amongst wild and domestic animals maría s. moleón1,2,*, mark e. merchant3, hugo h. ortega4, pablo a. siroski1,2 effects of temperature and food level on plasticity of metamorphic traits in bufo gargarizans gargarizans larvae tong lei yu*, yan ting han acta herpetologica 15(1): 21-29, 2020 issn 1827-9635 (print) © firenze university press issn 1827-9643 (online) www.fupress.com/ah doi: 10.13128/a_h-7858 identification of biologically active fractions in the dermal secretions of the genus bombina (amphibia: anura: bombinatoridae) iryna udovychenko1,*, oleksandra oskyrko1, oleksii marushchak2, tetiana halenova1, oleksii savchuk1 1 educational and scientific centre “institute of biology and medicine” taras shevchenko national university of kyiv, ukraine, 03127, hlushkova avenue, 2 2 i. i. schmalhausen institute of zoology of national academy of sciences of ukraine, kyiv, 01030, b. khmelnytskogo str. 15 * corresponding author. e-mail irisha.nikolaeva@gmail.com submitted on: 2019, 30th may; revised on: 2019, 4th november; accepted on: 2019, 20th november editor: marcello mezzasalma abstract. amphibian skin secretions have long been considered a convenient and useful natural source of bioactive compounds, but a comprehensive study of the effects of their dermal secretions on diverse parameters of the hemostasis system has not yet been carried out. this study aimed at identifying biologically active fractions in the skin secretions of b. bombina, b. variegata, and their hybrid – b.bombina × b.variegata, and to clarify whether their components can modify certain parameters of the hemostasis system. for the skin secretion analysis, we performed ion-exchange chromatography, electrophoresis, and zymography assays. plasma coagulation tests, chromogenic assays, and platelet aggregation assays were also conducted in vitro. as a result of the fractionation, a number of fractions were identified, where the proteins with miscellaneous molecular weights were revealed. the data also suggested that some fractions have proteolytic enzymes with gelatinolytic, fibrinogenolytic and collagenolytic activities. the proteins present in the fraction #5 of b. variegata and #5 of the hybrid secretions are characterized by the ability to prolong the clotting plug formation in the aptt. proteins capable of inducing platelet aggregation in the rabbit prp are present in the fraction #3 of b. variegata secretions. the ability of dermal components to activate plasma proenzymes is indicative of the fact that the non-protein components of fraction #9 of b. variegata and fraction #7 of hybrid secretions initiated the appearance of thrombin and activated protein c in plasma. keywords. amphibian skin secretions, hemostasis system, fractionation. introduction recently, a great deal of emphasis has been placed on the study of the effects of biologically active compounds derived from the sources of natural origin (dias et al., 2012; veeresham, 2012). these agents have found their applications in the treatment of pathological conditions, diagnosis of diseases, and have served as valuable tools in laboratory studies. moreover, the heightened need of society for new potent medicines with strong efficiency and high safety profile, along with the increased price for drugs based on synthetic compounds, places importance on the use of natural raw material to search for various biologically active substances. amphibian skin secretions are enriched with complex cocktails of bioactive molecules spanning a wide spectrum of biological actions (erspamer and melchiorri, 1980; daly, 1995; conlon and leprince, 2010). although recently there has been a spate of interest concerning the potential therapeutic effects of the biologically active compounds derived from amphibians’ dermal secretions (mor et al., 1994; bevins and zasloff, 1990), most of these substances are not yet widely implemented in medical and pharmaceutical industry, as the mechanism of their action and possible side effects remain insufficiently studied (könig et al., 2015). the results of our previous study showed that the crude skin secretions of ten species of amphibians: b. 22 iryna udovychenko et alii bombina, b. variegata, b. bufo, b. viridis, r. temporaria, p. ridibundus, p. esculentus, p. fuscus, s. salamandra, and the hybrid of b. bombina and b. variegata, had a pronounced protease activity with wide substrate specificity (nikolaieva et al., 2018). in fact, it was proved that these dermal venoms can be a potential source of proteolytic enzymes. in the other research we demonstrated that the whole skin secretions affect the parameters of clotting plug formation (udovychenko et al., 2018). it was shown that the b. bombina, b. variegata and their hybrid, r. temporaria, and p. ridibundus crude skin secretions prolonged the time of fibrin clot formation in the aptt. in contrast, the components of b. viridis, p. esculentus, p. fuscus, and s. salamandra prolonged the tt. another research of ours showed the presence of biologically active compounds in the p. fuscus crude skin secretions that affect some parameters of the hemostasis system (udovychenko et al., 2019). our findings confirm that amphibian skin secretions are a complex material which contains a variety of biologically active substances that differ in their biological effects. so, to identify the possible mechanism of action or/and to define the components present in the whole skin secretions, fractionation of the source material is required. several research projects have been conducted concerning the study of the effects of skin secretion components of the toads that belong to the genus bombina. a multitude of antimicrobial peptides from their dermal secretions have been discovered (simmaco et al., 1998). for example, peptides called bombenins were isolated and found to possess antimicrobial activities, which have not been detected in other amphibian genera (simmaco et al., 1991). in addition, bombenins are among the most studied amphibian constituents, and numerous studies have been published describing the various pharmacological activities of bombesin and its homologues (gonzalez et al., 2008). the in vitro antitumor assay conducted by zhou et al. (2018) showed that the bombinin-like peptide and bombinin h type peptide possessed obvious antiproliferative activity on three human hepatoma cells (hep g2/sk-hep-1/huh7) at the nontoxic doses. this indicates that the peptide family of bombinins could be a potential source of drug candidates for anti-infection and anticancer therapy. a few studies also report on the antidiabetic activity of the compounds derived from the bombina skin secretions: several insulin-releasing peptides have been isolated (marenah et. al., 2004). the mechanism underlying their insulinotropic actions suggests possible involvement of a camp dependent g-protein insensitive pathway. secretary glands of some representatives of genus bombina also produce antimicrobial peptides called maximins. according to lai et al. (2002), maximin 3 possesses a significant anti-hiv activity. furthermore, maximins tend to have a potent antimicrobial activity, cytotoxicity against tumor cells and spermicidal action. although considerable amount of research has been conducted to study the composition of the skin secretions of the bombina toads, and many biological effects of the venom constituents have been revealed, it still remains unclear whether the components of their dermal secretions affect the functioning of the hemostasis system. moreover, considering the huge amount of research that has been conducted based on the study of the similar effects of the reptile venoms (meier and stocker, 1991; markland, 1998; manjunatha, 2006), such studies in the context of the toad skin secretions are a highly promising area for investigations. in view of these facts, the aim of the present study was to identify the biologically active fractions in the dermal secretions of the genus bombina and to study their effects on some parameters of the hemostasis system. this work will provide a better understanding of the role of the proteins and enzymes present in the skin secretion of these species and might give a background for further potential medical and pharmaceutical applications of the components of amphibian skin secretions. materials and methods collection of amphibian skin secretions adult specimens of bombina bombina (n = 20, females = 2), bombina variegata (n = 15, females = 1), and their hybrid – b.bombina × b.variegata (n = 5, females = 0) were collected in kyiv, transcarpathian and khmelnitsky regions of ukraine, respectively. amphibians were authenticated by the department of zoology and ecology of taras shevchenko national university of kyiv, ukraine. skin secretions were collected as follows: frogs were put into a petri dish. after mechanically stimulated with fingers for 1-2 min, the frog skin surface was seen to exude copious secretions. skin secretions were collected by washing the dorsal region of each frog with ultra-pure water. water suspensions of skin secretions were centrifuged at 2500 g for 15 min to remove debris. the supernatants were lyophilized (telstar lyoquest) and kept at 4 °c till use. fractionation lyophilized skin secretions samples of b. bombina, b. variegata, and their hybrid (0.2 g) were dissolved in 1 ml 0.05 m tris-hcl buffer (ph 7.4), containing 0.2 m nacl and centrifuged at 10,000 g for 5 min. protein concentration in the supernatant was assayed by bradford (1976) method, using bovine serum albumin as a standard. chromatographic assays were performed using a system for liquid chromatography (biorad, 23bioactive fractions from the bombina skin secretions usa). the samples were applied to a superdex 200 pg (ge healthcare, hiloadtm 16/60) gel filtration column (flow rate 1 ml / min) equilibrated with 0.05 m tris-hcl buffer (ph 7.4), containing 0.2 m nacl. the elution was performed with the same buffer. the appearance of peaks was monitored, and the corresponding fractions were collected in the plastic tubes. the absorbance of the eluate was monitored at 280 nm. sds-polyacrylamide gel electrophoresis flow-through fractions were concentrated as follows: the aliquots of the fractions were mixed with 25% trichloroacetic acid (1:1) and centrifuged for 5 min at 10,000 g. the precipitate was diluted with 1 ml of acetone and centrifuged. the procedure was repeated twice. the precipitate was then incubated at 37 oc for 15 minutes. after that, the precipitate was mixed (1:1) with the standard sds-page sample buffer (62.5 mm tris-hcl, ph 6.8, 2% sds, 5% sucrose, and 0.002% bromophenol blue) without heating. sds-polyacrylamide gel electrophoresis (sds-page) was performed as reported (laemmli, 1970), using 4% (w/v) stacking gel and 15% (w/v) separating gel. sds-page was conducted using mini-protean tetra system (bio rad, usa) at 19 ma for stacking and 36 ma for separating gels. fraction samples were applied in 15 µl volume per well. gels were stained with 2.5% coomassie brilliant blue g-250 in 10% (v/v) ethanol, 10% (v/v) acetic acid, 15 % (v/v) isopropanol and the background of the gel was destained with 7% (v/v) acetic acid for 30 min. apparent molecular weights of proteins were estimated using protein calibration mixture (bio rad, usa) containing myosin, β-galactosidase, phosphorylase b, serum albumin, ovalbumin (mr 97; 66; 45; 31; 21; 14 kda). zymography the aliquots of the flow-through fractions were mixed with β-mercaptoethanol solution (9:1) and sample buffer (1:1) (0.01 m tris-hcl buffer, ph 6.8, 2% sodium dodecyl sulfate, 10% sucrose and 0.01% bromophenol blue) without heating. zymography was performed according to the method suggested by ostapchenko et al. (2011). separating gel (12% w/v) was polymerized in the presence of gelatin (1 mg/ml), fibrinogen (1 mg/ml) or collagen (1 mg/ml). fraction samples were applied in 15 µl volume per well. after sds-page, gels were incubated for 30 min at room temperature on a rotary shaker in 2.5% triton x-100. the gels were then washed with distilled water to remove triton x-100 and incubated in 50 mm tris-hcl (ph 7.4) at room temperature for 12 hours. gels were stained with 2.5% coomassie brilliant blue g-250 in 10% (v/v) ethanol, 10% (v/v) acetic acid, 15 % (v/v) isopropanol for 30 min. preparation of platelet-rich plasma and platelet-poor plasma platelet-rich plasma (prp) and platelet-poor plasma were obtained following the standard protocol. all procedures were carried out at room temperature. healthy adult rabbits were supplied by the vivarium of taras shevchenko national university of kyiv, ukraine. the blood was collected from the ear artery into a polyethylene tube with 3.8% sodium citrate (9:1). prp was obtained by centrifugation of stabilized blood at 300 g for 10 min. the supernatant (prp) was carefully separated and used further in the aggregation assay. platelet-poor plasma was prepared by further centrifugation of the remaining stabilized blood at 1,500 g for 30 min. plasma coagulation tests activated partial thromboplastin time, thrombin time and prothrombin time were measured in vitro to assess the effects of the components of flow-through fractions of skin secretions on plasma clotting function. the tests were conducted using the coagulation analyzer (rayto rt-2201c, china) and the standard set of reagents (renam, russian federation). to measure the activated partial thromboplastin time (aptt), 45 μl of rabbit plasma was mixed with 5 μl of fraction sample and 50 μl aptt reagent in the coagulometric cuvette. after 3 min of incubation at 37°c the 50 μl of 0.025 m cacl2 was added and the time necessary for the clotting plug formation was recorded. for the in vitro tt assay, the 45 μl of plasma and 5 μl of fraction sample were incubated in the coagulometric cuvette at 37°c for 2 min. clotting time was immediately recorded after the addition of 100 μl thrombin (final activity 3 u/ml). the prothrombin time (pt) was assessed by mixing 45 μl of plasma and 5 μl of fraction sample in the coagulometric cuvette at 37°c for 2 min. clotting time was immediately recorded after the addition of 100 μl of thromboplastin-calcium mixture. all coagulation assays were performed in triplicate using plasma from three different rabbits. plasma incubated with other components and equal amounts of 0.05 m tris-hcl buffer (ph 7.4), containing 0.2 m nacl instead of fractions samples, was used as controls. chromogenic assays the effects of flow-through fractions of the studied skin secretions on key hemostasis enzymes were assayed using p-nitroaniline chromogenic substrates: thrombin specific substrate phe-pip-arg-pna (s-2238), plasmin specific substrate val-leu-lys-pna (s-2251), factor xa specific substrate ile-glugly-arg-pna (s-2222) and activated protein c specific substrate pyroglu-pro-arg-pna (s-2366) (renam, russian federation). the direct proteolytic activity of the components of the fractions and their ability to activate plasma proenzymes was measured in vitro. assays were performed in 0.05 m tris-hcl buffer, ph 7.4 in a total volume 250 μl. to analyze the direct activity, the fractions samples with 20 μg of total protein in 0.05 m tris-hcl buffer were mixed with the corresponding chromogenic substrates. to study the ability to activate plasma, proenzymes 20 μl of plasma was additionally added to the incubation medium. the formation of p-nitroaniline was monitored at equal intervals of time at 405 nm. the amount of p-nitroaniline, which was hydrolyzed from the substrate, was calculated by 24 iryna udovychenko et alii using molar extinction coefficient of 10,000 m-1 x cm-1 for free p-nitroaniline. platelet aggregation assay platelet aggregation assay was undertaken within the first 3 h after blood sampling using photo-optical aggregometer at-02 (medtech, russia). before the assessment, the number of platelets in prp was determined (230-250 × 103 cells/μl). the effects of the flow-through fractions of the studied skin secretions on platelet functions were performed in vitro. briefly, 380 μl prp and 20 μl of the samples were incubated at 37°c in the aggregometer cuvette and the aggregation process was monitored for 5 min. the maximum degree of aggregation was recorded and compared with the degree of aggregation in response to one of the platelet physiological inducers – adp (sigma, usa) in the final concentration of 5 × 10-6 m. calculation of the results totallab 2.04 program was used to analyze the resultant electrophorograms and zymograms. all experiments were performed in parallels and repeated at least three times each using the blood of different rabbits. the statistical analysis was performed using statsoft statistica version 7.0 for windows. the data were analyzed for statistical significance of difference by student’s t-test. differences were considered to be statistically significant when p < 0.05. results fractionation of the whole skin secretions the supernatants of skin secretions of b. bombina, b. variegata and their hybrid were fractionated into several fractions by superdex 200 pg column as illustrated in fig. 1. the purification was followed by determination of various activities for each fraction. sds-polyacrylamide gel electrophoresis and zymography sds-page analysis was applied to get information about the protein composition of the flow-through fractions of the studied skin secretions (table 1 and figs. s1, s2, s3, s4). the results of protein separation revealed the presence of proteins with molecular weights ranging from 3 to 128 kda. zymography with gelatin, fibrinogen and collagen as substrates was conducted to evaluate the proteolytic potential of the flow-through fractions of the studied skin secretions. key results are presented in table 1. it was revealed that some fractions have proteolytic enzymes with gelatinolytic, fibrinogenolytic, and collagenolytic activities. aptt, pt, and tt in vitro assays table 2 summarizes the effects of the flow-through fractions of the studied skin secretions on the time of clotfig. 1. fractionation of b. bombina (a), b. variegata (b) and their hybrid (c) whole skin secretions. 25bioactive fractions from the bombina skin secretions ting plug formation, which corresponds to the plasma coagulation tests results (aptt, tt and pt). as shown by the data, the whole skin secretions of all the studied amphibians prolonged aptt clotting time: b. bombina – to 62.7 ± 0.5 s, b. variegata – to 73.6 ± 0.5 s, and their hybrid – to more than 90 s, compared to the control values – 21.35 ± 1.2 s. fraction #6 of b. variegata skin secretions prolonged aptt to 56.9 ± 1.1 s, and fraction #5 of hybrid skin secretions prolonged aptt to 74.1 ± 0.8 s vs 21.35 ± 1.2 s in control. pt and tt assays showed no changes in the time of clotting plug formation while incubating with all studied whole skin secretions and flow-through fractions. chromogenic assays the specific proteolytic activity of the components of flow-through fractions of the studied skin secretions was determined by the ability to hydrolyze the amide bond in the synthetic chromogenic substrates specific to thrombin (s-2238), plasmin (s-2251), factor xa (s-2222) and protein c (s-2366). table 3 demonstrates the results of this assay. the ability of the studied fractions to activate plasma proenzymes was determined in vitro. while performing this experiment, rabbit blood plasma was additionally introduced into the incubation medium with the appropriate substrate and the skin secretion fraction sample. table 3 indicates the emergence of activated thrombin, plasmin, and factor x in plasma while incubating with b. bombina whole skin secretions. only one fraction of this amphibia – #1 – initiated the appearance of factor xa in plasma. fraction #9 of b. variegatа skin secretions activated prothrombin, factor x, and protein c in plasma, whereas its whole secretions had no effects on plasma proenzymes. the hybrid b. bombina × variegatа whole skin secretions and fraction 8 initiated the emergence of active prothrombin and protein c in plasma. platelet aggregation assay this part of the research was aimed at investigating the potential effects of the flow-through fractions of the studied skin secretions on the process of platelet aggregation. fraction #3 of b. variegata skin secretions induced platelet aggregation. as illustrated by fig. 2, the degree of aggregation in the final concentration of 250 μg of table 1. molecular weights (mw) of proteins and the presence of proteolytic enzymes with certain substrate specificity in the flow-through fractions of skin secretions of studied amphibian species. the sign “+” the manifestation of proteolytic activity, and the sign “-” the absence. fraction # sds-page mw, kda zymography gelatin collagen fibrinogen b. b om bi na 1 103; 78; 46; 31 + + + 2 41 + 3 74; 56; 44; 37; 33; 29; 22; 16; 13; 12; 11 + + 4 55; 48; 39; 36; 33; 27; 24; 21; 18; 12 + + 5 54 + 6 55 + b. v ar ie ga ta 1 76; 43; 39; 27; 3 + 2 141; 129; 118; 101; 79; 57; 51; 49; 43; 39; 31; 27; 22; 17; 16; 3 + + 3 121; 109; 79; 42; 39; 27; 23; 22; 3 + + 4 124; 103; 80; 61; 48; 42; 36; 34; 29; 26; 22; 19; 17; 13; 9; 3 + + 5 16; 3 + 6 16; 3 7 3 h yb ri d 1 100; 53; 43; 29; 22 2 128; 108; 81; 53; 43; 39; 30; 27; 25; 23 + + + 3 93; 65; 47; 44; 36; 32; 30; 28; 24; 23 + + 4 120; 88; 51; 42; 36; 31; 27; 24; 1 + + + 5 21; 9 + + + 6 8 + 7 8 26 iryna udovychenko et alii total protein in 1 ml of prp were 73% and 65%, respectively, which corresponds to the degree of aggregation in response to the action of 5x10-6 m adp. even though the components of whole skin secretions of hybrid b. bombina × variegatа induced platelet aggregation in the rabbit prp with the degree of aggregation 52%, the fraction that might have this effect was unidentified. this might be the result of the dilution of the fractions while performing the chromatography assay. both the whole skin secretions and flow-through fractions of b. bombina had no effect on the process of platelet aggregation. discussion to adequately undertake the fractionation, a chromatographic carrier superdex 200 pg (ge healthcare, hiloadtm 16/60) gel filtration column was used in our research. the prerequisite for the use of this carrier was its wide zone of separation (from 3 to 70 kda), high stability, and inactivity to form nonspecific interactions with the fraction samples. as a result of fractionation, six protein fractions were identified in the b. bombina dermal secretions, seven protein fractions were determined in the b. variegata skin secretions, and eight protein fractions were defined in the hybrid (b. bombina × b. variegata) whole secretions. the data on sds-page assay suggests the presence of the mixture of the proteins with similar molecular weights in each identified fraction. a review of the biological effects of the fractions was conducted and compared to those of the whole skin secretions. results of our research into the proteolytic potential indicate the presence of enzymes with gelatinase activity in all b. bombina (bb) fractions, whereas the proteolytic enzymes with collagenolytic activity were determined in fractions #1, #3, and #4 of these amphibians. the fibrinogenolytic enzymes were detected only in the bb#1 fraction. the data indicate the presence of enzymes with gelatinase activity in the fractions #1, #2, #3, #4, and #5 of b. variegata (bv) skin secretions. fractions bv#2, bv#3, and bv#4 are characterized by the presence of fibrinogenolytic enzymes. no proteolytic potential was indicated in the b. variegata skin secretion fractions while conducting zymography assay with collagen as a substrate. the results of the study on the proteolytic potential of the hybrid fractions (bh) indicate the presence of gelatinolytic enzymes in the fractions #2, #4, #5, #6, and #7, collagenolytic enzymes in the fractions #2, #3, #4, #5, and #7, and the presence of fibrinogenolytic enzymes in fractions #2, #3, #4, and #5. the data clarify the relationship between the emergence of specific type of activity and the release of high molecular weight proteins while performing the sds-page assay. to assess the effects of the components of the fractions on plasma clotting function, aptt, pt, and tt were measured. the data indicate that the b. variegata and hybrid whole skin secretions prolonged the clotting plug formation in the aptt. the proteins that are responsible for the manifestation of this effect were identified in the fraction bv#5 and bh#5. thus, according to the results, table 2. the clotting time of rabbit blood plasma (sec) in the coagulation tests after incubation with the flow-through fractions of studied skin secretions. aptt pt tt control 21.35 ± 1.2 6.75 ± 0.1 30.3 ± 2.2 b. b om bi na whole secretions 62.7 ± 0.5* 7.1 ± 0.2 31.9 ± 0.4 1 20.3 ± 0.6 7.5 ± 0.3 32.6 ± 0.2 2 21.3 ± 1.2 7.2 ± 0.2 32.8 ± 0.5 3 20.7 ± 0.5 7.4 ± 0.5 32.2 ± 0.2 4 22.5 ± 0.6 7.5 ± 0.3 32.4 ± 0.5 5 20.8 ± 0.8 5.7 ± 1.2 33.2 ± 0.9 6 21.7 ± 1.0 7.2 ± 0.4 32.7 ± 0.7 b. v ar ie ga ta whole secretions 73.6 ± 0.5* 6.8 ± 0.2 32.1 ± 0.2 1 23.6 ± 0.6 6.9 ± 0.4 29.7 ± 0.5 2 19.3 ± 1.5 7.4 ± 0.3 29.0 ± 0.7 3 25.6 ± 0.3 7.4 ± 0.2 29.8 ± 0.1 4 28.1 ± 0.8 8.4 ± 1.2 32.2 ± 0.9 5 56.9 ± 1.1* 6.9 ± 0.1 32.2 ± 0.7 6 29.8 ± 0.6 7.5 ± 0.4 32.4 ± 0.7 7 33.4 ± 0.3 6.9 ± 0.2 31.5 ± 0.2 h yb ri d whole secretions >90* 6.7 ± 0.3 31.7 ± 0.5 1 18.3 ± 0.6 7.5 ± 0.4 31.3 ± 0.5 2 18.6 ± 1.2 7.4 ± 0.5 31.3 ± 0.6 3 19.4 ± 0.3 6.8 ± 0.4 30.1 ± 0.4 4 19.2 ± 0.4 6.8 ± 0.2 32.2 ± 0.5 5 74.1 ± 0.8* 7.2 ± 1.1 32.2 ± 0.2 6 17.1 ± 1.1 6.6 ± 0.2 29.7 ± 0.5 7 18.7 ± 0.4 7.0 ± 0.6 31.0 ± 0.6 8 18.4 ± 0.8 5.8 ± 0.5 31.6 ± 0.5 * – p ≤ 005 the difference is comparable to the control. fig. 2. whole skin secretions and fraction #3 of b. variegata induce platelet aggregation in rabbit prp. 27bioactive fractions from the bombina skin secretions the bv#5 fraction were characterized by the presence of two proteins with the molecular weight 16 kda and 3 kda and had a gelatinolytic activity. fraction bh#5 was characterized by the presence of proteins with molecular weight 21 kda and 9 kda and had a pronounced protease activity with wide substrate specificity. these results may be attributed to the presence of the inhibitors of certain factors of coagulation hemostasis that are present in the fractions of skin secretions, or, to the degradation of the factors of coagulation hemostasis by the active components of crude skin secretions. another test – pt – is used to evaluate the functioning of the coagulation factors v, vii, and x and the time necessary to generate fibrin after activation of factor vii in the extrinsic coagulation pathway (azevedo et al., 2007). tt measures the time required for fibrinogen to form fibrin strands in the presence of thrombin. this test only reveals disturbances in the final stages of coagulation (koch and biber, 2007). although the data shows no potential effect of the components of the flow through fractions on these coagulation tests, mention should be made of the specificity of action. the data on the dermal components ability to activate plasma proenzymes indicate that the whole secretions of the hybrid initiated the appearance of thrombin and activated protein c in plasma. this effect may be attributed to the action of the components of fraction bh#7. although the fraction bv#9 initiated the appearance of thrombin and activated protein c in plasma, no effect was observed under the action of the whole secretions of this amphibian. according to the sds-page assay, the components responsible for the manifestation of these effects have a low molecular weight, e.g., fraction bv#9 – 3 kda, or, are non-protein nature and have little interest in the context of studying of the effects of the amphibian skin secretions. these results have failed to support our hypothesis that the appearance of thrombin and activated protein c may be due to the presence of active forms of proteolytic enzymes in the crude skin table 3. the amount of hydrolyzed p-na (nmol) from the chromogenic substrates under the action of whole skin secretions and the flowthrough fractions samples of studied amphibian species. s-2238 – thrombin specific substrate; s-2251 – plasmin specific substrate; s-2222 – factor xa specific substrate; s-2366 – activated protein c specific substrate. the sign “-” the absence of the effect. proteolytic activity plasma enzymes activity s-2238 s-2251 s-2222 s-2366 trombin plasmin factor xa protein ca b. b om bi na whole secretions 0 0 3.73 ± 0.07 23.76 ± 0.71 14.48 ± 0.72 3.81 ± 0.32 8.61 ± 0.45 1 9.32 ± 0.18 2.21 ± 0.04 3.62 ± 0.07 9.11 ± 0.07 1.09 ± 0.12 2 13.79 ± 0.27 2.06 ± 0.04 5.98 ± 0.17 24.63 ± 0.62 3 13.52 ± 0.07 6.53 ± 0.19 16.97 ± 0.34 25.32 ± 0.23 4 1.03 ± 0.39 11.62 ± 0.31 17.26 ± 0.51 22.63 ± 0.43 5 4.61 ± 0.09 4.73 ± 0.05 6 5.85 ± 0.18 6.04 ± 0.17 b. v ar ie ga ta whole secretions 3.41 ± 0.13 0.73 ± 0.02 1.95 ± 0.08 1.47 ± 0.07 0.35 ± 0.02 1 1.52 ± 0.08 2 0.78 ± 0.03 3 6.01 ± 0.17 1.51 ± 0.07 3.57 ± 0.12 4 9.49 ± 0.09 3.76 ± 0.12 4.42 ± 0.15 6.67 ± 0.16 5 1.97 ± 0.08 6 7 7.62 ± 0.23 2.91 ± 0.13 11.21 ± 0.31 h yb ri d whole secretions 3.75 ± 0.18 0.75 ± 0.05 1.76 ± 0.12 1.60 ± 0.09 6.11 ± 0.12 9.71 ± 0.35 1 2.37 ± 0.12 2.31 ± 0.04 2 2.31 ± 0.09 3 2.37 ± 0.11 2.27 ± 0.07 4 6.82 ± 0.34 1.78 ± 0.15 3.78 ± 0.25 5 6 7 8 6.34 ± 0,15 10.89 ± 0,43 28 iryna udovychenko et alii secretions that can directly activate the cleavage of the corresponding proenzymes of thrombin and protein c or affect the cofactors which can promote a cascade of reactions that result in the formation of zymogens, as the data of sds-page shows no proteolytic activity in the active fractions bv#7 and bv#9. the data indicate that the whole skin secretions of b. bombina initiated the appearance of thrombin, plasmin, and factor xa in plasma, whereas the results have failed to define the fractions that were responsible for these effects. we can assume that such effects of the whole b. bombina skin secretions might be the result of the sequential activity of the proteins, when two proteins from different fractions initiated the appearance of thrombin. or this may also be due to the action of non-protein components or might be related to the high dilution of the fractions as a result of gel filtration chromatography. the results of the study on the platelet aggregation capability suggest that the b. variegata whole skin secretions induced platelet aggregation in the rabbit prp. the proteins that were responsible for this effect are concentrated in the fraction bv#3. mention should be made of the fact that the effect of the whole secretion and the protein fraction corresponds to the effect of adp, which is known as one of the physiological inducers of platelet aggregation. previous results of sds-page assay indicate the presence of several proteins in the fraction bv#3 with various molecular weights: 121 kda, 109 kda, 79 kda, 42 kda, 39 kda, 27 kda, 23 kda, 22 kda, and 3 kda. moreover, presence was also indicated of proteolytic enzymes in this fraction with specificity to gelatin and fibrinogen. the proteins present in fraction bv#3 could be a prominent source of inducers of platelet aggregation. although inducers that can modulate platelets function are not relevant in the treatment of hemostasis system disorders, they could be a useful tool for studying platelets functions and their signaling pathways. despite the activity of the components of the hybrid b. bombina × variegatа whole skin secretions to induce platelet aggregation (data is not shown), the results have failed to identify the fractions that were responsible for this effect. this may be due to the significant dilution of the fractions resulting from gel filtration chromatography. in conclusion, the results of our study demonstrate that the studied amphibian skin secretions of the genus bombina are a potential source of bioactive constituents that can affect different stages of the hemostasis system. it was defined that certain flow-through fractions have active molecules that demonstrate some specific effects. it was established that these molecules are proteins by nature and some of them have pronounced proteolytic activities. furthermore, the data showed that the proteins with the ability to prolong the clotting plug formation in the aptt in vitro assay are present in the fraction #5 of b. variegata and #5 of the hybrid skin secretions. the ability of dermal components to activate plasma proenzymes indicates that the non-protein components of fraction bv#9 and fraction bh#7 skin secretions initiated the appearance of thrombin and activated protein c in plasma. the proteins capable of inducing platelet aggregation in the rabbit prp are present in the fraction #3 of b. variegata skin secretions. our research has made a contribution into the study of the genus bombina dermal secretions, in particular, the effect of the venom constituents on the hemostasis system and the nature of the active components; however, the mechanism of their action still requires additional research. such findings may be used by biomedical researchers as a source of potential novel drug leads or pharmacological agents with a direct therapeutic effect and can rapidly provide a basis for related scientific studies such as those involved in systematic or the evolution of species. acknowledgments this research was supported by the ministry of education and science of ukraine [№ 0116u002527, 20162018]. i would like to express my deep gratitude to the dr olexiy savchuk, my research supervisor, for his patient guidance, enthusiastic encouragement and useful critiques of this research work. my grateful thanks are also extended to phd tetiana halenova for her help in doing the data analysis, and for her useful and constructive recommendations on this research. i would also like to thank mr olexiy marushchak and mrs oleksandra oskyrko for their help in collecting the amphibians and their skin secretions, and for the providing useful information about the amphibians used in the research. all animal procedures followed the european directive 2010/63/eu (ec, 2010) on protecting animals used for experimental and other scientific purposes. all manipulations were approved by the ethical committee of educational and scientific center institute of biology and medicine, taras shevchenko national university of kyiv, ukraine. all animal experiments were approved by the animal care committee of taras shevchenko national university of kyiv, ukraine. supplementary material supplementary material associated with this article can be found at 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