Acta Herpetologica 17(2): 115-124, 2022 ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah DOI: 10.36253/a_h-12556 Preliminary genetic characterisation of Southern Smooth Snake Coronella girondica (Serpentes, Colubridae) populations in Italy, with some considerations on their alpine distribution Matteo R. Di Nicola1, Raffaella Melfi2, Francesco P. Faraone3,*, Daniel L.N. Iversen4, Gabriele Gia- calone5, Giovanni Paolino1, Mario Lo Valvo6 1 Unit of Dermatology, IRCCS San Raffaele Hospital, Via Olgettina 60, 20132 Milan, Italy 2 Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche (STEBICEF), University of Palermo, Viale delle Scienze Ed. 16-17, 90128 Palermo, Italy 3 Viale Regione Siciliana S.E., 532, 90129 Palermo, Italy 4 Viale Giovanni Prati, 38066, Riva del Garda, Italy 5 Cooperativa Silene, Via D’Ondes Reggio, 8/a, 90127 Palermo, Italy 6 Dipartimento Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche, University of Palermo, Via Archirafi, 18, 90123 Palermo, Italy *Corresponding author. Email: paolofaraone@libero.it Submitted on: 2022, 8th January; revised on: 2022, 4th March; accepted on: 2022, 4th May Editor: Adriana Bellati Abstract. The Southern smooth snake, Coronella girondica, is a small-sized colubrid found in Northwest Africa and Southwest Europe. Mitochondrial DNA-based studies showed that the species can be split into five clades: two from Northwest Africa (one Moroccan and one Tunisian-Algerian) and three from Europe (one in the south-west of the Iberian Peninsula, one in the south-east of Spain and one in the rest of the European range). With regards to Ita- ly, to date, only two samples have been analysed both from the Province of Pisa, Tuscany, pointing at that fact that genetic characterisation of Italian populations is still lacking. Accordingly, we have increased the sampling coverage with 19 new samples from northern and central regions of Italy, including two populations, apparently disconnected from the rest of the known range, and analysed their phylogenetic relationships using a portion of the mitochondrial cytochrome b gene. Our results confirm the general phylogenetic arrangement detected in previous studies; specifi- cally for Italian populations, no variability emerged from the Apennine populations, and a slight differentiation could be shown for the Alpine and subalpine ones. This pattern can be explained assuming past spread and recent isolation of C. girondica relict populations in the Alpine region, likely during the Last Glacial Maximum. Later, during the Hol- ocene, the Italian Alps and the Po Plain went through various climatic variations and high anthropization which may have influenced C. girondica distribution through expansion and contraction processes. Keywords. Coronella girondica, Italy, distribution, relict populations. INTRODUCTION The Southern Smooth Snake Coronella girondica (Daudin, 1803) is a small-sized Colubridae with a Med- iterranean chorotype (West-Mediterranean, Sindaco et al., 2013). The species is present in Northwest Africa (north-central Morocco, northern Algeria and north- western Tunisia) and Southwest Europe (Portugal, Spain, Southern France and Peninsular Italy), where it usually lives in dry and stony Mediterranean scrub habitats (Sindaco et al., 2013; Geniez, 2018; Di Nicola et al., 2021). 116 Matteo R. Di Nicola et alii With regards to Italy, the secretive and mainly crepus- cular habits of the species (Ferri and Morimando, 2004; Razzetti and Bernini, 2011) hampered the full description of its distribution framework for a long time (Razzetti and Bonini, 2006) and for some areas its presence has even only been recently confirmed (Razzetti et al., 2000; Cap- ula et al., 2010; Rugiero et al., 2018; Di Nicola et al., 2020; Ferri and Soccini, 2020; Iversen et al., 2020). The species, originally described by Daudin (1803) as Coluber girondicus, is currently considered monotypic (Razzetti and Bernini, 2011; Santos and Pleguezuelos, 2015; Sindaco and Razzetti, 2021). Subsequently, Rhine- chis amaliae (Boettger, 1881) was described from Moroc- can specimens, on the basis of a single morphological character relating to the rostral zone (Santos and Ple- guezuelos, 2015). However, further morphological inves- tigations did not provide support for the validity of this taxon (Saint Girons, 1956; Domergue, 1962; Santos and Pleguezuelos, 2003). More recently, Santos et al. (2012a) performed a molecular study across the distribution range of the whole species using analysis of mitochon- drial genes and highlighted the presence of three major clades: one from Northwest Africa, one from the south- east of Spain and the latter occurring in the rest part of the European range. According to their calibration, the divergence among the clades occurred around 1.4-2.0 Ma, roughly coinciding with the Plio-Pleistocene transi- tion. Within two out of the three major clades, further differentiation was detected into five clades (see Santos et al., 2012b): two from Northwest Africa (one Moroccan and one Tunisian-Algerian) and three from Europe (one in the south-west of the Iberian Peninsula, one in the south-east of Spain and one in the rest of Europe). In Italy, C. girondica is distributed in the north-west- ern part of the country and extends south to the Gargano peninsula (Northern Apulia) and southern Lazio, includ- ing the region of Molise (Capula et al., 2010; Razzetti and Bernini, 2011; Rugiero et al., 2018; Di Nicola et al., 2021). Recently, two apparently isolated populations have been found in western Lombardy (Di Nicola et al., 2020) and around Lake Garda, in an area that is transitional between the Trentino-Alto Adige, Veneto and Lombardy regions (Ferri and Soccini, 2020; Iversen et al., 2020). These isolated populations are out of the geographical range of the other populations by around 70 km east and 160 km north (Fig. 1, 2). With regards to genetic characterisation of the spe- cies in Italy, only two samples have been analysed, both from the province of Pisa (GenBank accession numbers JQ837570 and JQ837571). The present work therefore describes, for the first time, the intraspecific relationships between Italian populations of C. girondica including some recently confirmed observations from Northern Ita- ly (Di Nicola et al., 2020; Ferri and Soccini, 2020; Iversen et al., 2020). MATERIALS AND METHODS Given the lack of adequate sample coverage within the Italian range of the species, a total of 19 new samples were collected from seven regions of Northern and Cen- tral Italy (Table 1; Fig. 1), including five samples from the isolated populations, respectively western Lombardy (n = 1) and Trentino-Alto Adige (n = 4). Small tissue frag- ments (tail tips, pieces of ventral scales) were removed and preserved in ethanol 96% from individuals that were found dead (most of the samples) or live specimens. For live specimens, a non-invasive scale clipping of ventral scales was performed. Coronella girondica was sampled by nocturnal active search only at two separate localities of Arco (Iversen et al., 2020) and Somma Lombardo (Di Nicola et al., 2020), while in other cases samples were recovered from dead specimen as roadkills, domestic cats or human persecution (Table 1). In order to infer the phylogenetic relationships between Italian samples and other populations, we select- ed the mitochondrial DNA cytochrome b (cyt b) gene, a marker often used to infer intra-specific diversity on many vertebrates and invertebrates species, including snakes (Carranza et al., 2006; Mezzasalma et al., 2015; Faraone et al., 2020b). Approximately 20 mg of tissue was used to extract total DNA as described in Tagliavia et al. (2016). Genomic DNA was used as a template for PCR amplification with primers CB1(F) (5’CCATC- CAACATCTCAGCATGATGAAA3’) and CB2(R) (5’CCCTCAGAATGATATTTGTCCTCA3’) (Carran- za et al., 1999). DNA bands of the expected size (~300 bps) were obtained and then sequenced with the primer CB1(F) (BMR Genomics, Padua, Italy). The resulting sequences were each around 255 nucle- otides long and were analysed and manually proof-read with the DNA sequencing software CHROMAS v. 2.6.6 (Technelysium Pty. Ltd. 1998, Queensland, Australia). The coding gene fragments of cyt b were translated into amino acids to assess the lack of premature stop codons. Later, using CLUSTAL W (Larkin et al., 2007) with default parameters, the sequences from Italian samples generated in this study were aligned with homologous sequence downloaded from GenBank (Carranza et al., 2004; Santos et al., 2008, 2012a; Carvalho et al., 2017). Four species belonging to Colubridae and Psammophii- dae families were used as outgroups (Carranza et al., 2006; Santos et al., 2012a; Faraone et al., 2020a, b). 117Genetic characterisation of Coronella girondica in Italy The phylogenetic analysis was performed with Maximum Likelihood (ML) under the Akaike Informa- tion Criterion using the “Smart Model Selection” (SMS) (Lefort et al., 2017), implemented in PHYML v. 3 (Guin- don et al., 2010). Jukes-Cantor (JC) (Jukes and Cantor, 1969) was the most appropriate evolutionary model (-Log likelihood value 1283.58), with a 0.32 gamma estimate of invariable sites and a 1.00 discrete approximation of the gamma distribution. The same model was obtained by using both all available sequences and by previously collapsing the sequences into haplotypes. Node sup- port was estimated by bootstrap (Felsenstein, 1985) with 1,000 replicates and the MEGA X software (Tamura et al., 2021) was used to implement the ML tree. The unroot- ed minimum spanning network were obtained using the median-joining algorithm (Bandelt et al., 1999) imple- mented in PopART (http://popart.otago.ac.nz/) (Leigh and Bryant, 2015). With the aim of consolidating current knowledge of C. girondica distribution in Northern Italy, biblio- graphical data from Razzetti and Bonini (2006) were recorded. Furthermore, unpublished observations from the northern Po River area were collected between 2011 and 2021 from authors’ field observations and online records. All observations that were not reported by Raz- zetti and Bonini (2006) were considered new and, subse- quently, compared with previous unconfirmed findings. In addition, Citizen Science (see Haklay et al., 2021) was also critical to the generation of distributional datasets through collaborative efforts between herpethologists around Italy and users of a Facebook group managed by two of the authors (MRDN and FPF) “Identificazione Table 1. Italian samples and observation details of Coronella girondica used in the present study. The numbers and letters reported respec- tively in the first and second column are referred to localities shown in Fig. 1 and Fig. 2. The haplotype code is shown in brackets after the GenBank accession number. Samples marked with an asterisk were previously published by Santos et al. (2012a). Fig. 1 Fig. 2 Year Locality N E Observer/Reference Accession number 1 C 2019 Arco, Trentino Alto Adige 45.9238° 10.9433° Iversen et al., 2020 OK573460 (H2) 2 C 2020 Arco, Trentino Alto Adige 45.9238° 10.9433° Iversen et al., 2020 OK573463 (H2) 3 C 2020 Arco, Trentino Alto Adige 45.9238° 10.9433° Iversen et al., 2020 OK573462 (H2) 4 C 2021 Arco, Trentino Alto Adige 45.9238° 10.9433° Iversen et al., 2020 OK573461 (H2) 5 G 2020 Somma Lombardo, Lombardy 45.6713° 8.6833° Di Nicola et al., 2020 OK573464 (H2) 6 2020 Cassinelle, Piedmont 44.5760° 8.5616° Cavanna S. pers. obs. OK573465 (H3) 7 2020 Isola del Cantone, Liguria 44.6432° 8.9664° De Cresi U. pers. obs. OK573473 (H1) 8 2018 Albenga, Liguria 44.0970° 8.2129° Graglia M. pers. obs. OK573469 (H1) 9 2018 Albenga, Liguria 44.0970° 8.2129° Graglia M. pers. obs. OK573468 (H1) 10 2019 Albenga, Liguria 44.0970° 8.2129° Graglia M. pers. obs. OK573467 (H1) 11 2020 Peagna, Liguria 44.0989° 8.2013° Graglia M. pers. obs. OK573472 (H1) 12 2020 Peagna, Liguria 44.0989° 8.2013° Graglia M. pers. obs. OK573471 (H1) 13 2020 Peagna, Liguria 44.0989° 8.2013° Graglia M. pers. obs. OK573470 (H1) 14 2020 Aurigo, Liguria 43.9953° 7.9209° Fecchio L. pers. obs. OK573466 (H1) 15 2021 Vigolzone, Emilia Romagna 44.9110° 9.6879° Gereschi V., Mazzotta M. pers. obs. OK573478 (H4) 16 2020 Foreste casentinesi, Emilia Romagna 43.8874° 11.8939° Molinari G. pers. obs. OK573475 (H1) 17 2021 Foreste casentinesi, Emilia Romagna 43.8874° 11.8939° Molinari G. pers. obs. OK573476 (H1) 18 N/A S. Giuliano Terme, Tuscany* 43.7579° 10.4434° Santos et al., 2012a JQ837570 (H1) 19 N/A S. Giuliano Terme, Tuscany* 43.7579° 10.4434° Santos et al., 2012a JQ837571 (H1) 20 2020 Piombino, Tuscany 42.9268° 10.5310° Banchi R. pers. obs. OK573474 (H1) 21 2020 Capestrano, Abruzzo 42.2867° 13,7942° D’Amico M. pers. obs. OK573477 (H1) A 2011 Rivoli Veronese, Veneto 45.5872° 10.8215° Campagnari M. pers. obs. A 2013 Caprino Veronese, Veneto 45.5898° 10.8215° Campagnari M. pers. obs. B 2020 Avio, Trentino Alto Adige 45.7383° 10.9433° Secchi M. pers. obs. D 2019 Pietramurata, Trentino Alto Adige 46.0268° 10.9420° Iversen et al., 2020 E 2020 Limone sul Garda, Lombardy 45.8108° 10.7866° Di Nicola et al., 2020 F 2020 Toscolano Maderno, Lombardy 45.6666° 10.6166° Ferri & Soccini, 2020 H 2021 Sostegno, Piedmont 45.6658° 8.2852° Zonari A. pers. obs. I 2015 Zubiena, Piedmont 45.4833° 8.0333° Ciracì A. pers. obs. 118 Matteo R. Di Nicola et alii Anfibi e Rettili”. The users of the Facebook group provid- ed locations for known European amphibians and reptiles and provided further distributional sites and samples for C. girondica found dead in various Italian regions. For each observation recorded through social networks, the authors meticulously checked the original files, the spe- cies identification and the location provided by the users through field survey of the coordinates. The new observa- tions were mapped using Adobe Photoshop CC (©1990- 2018 Adobe Systems incorporated, Release 19.1.5), together with those available from recent literature (Raz- zetti and Bonini, 2006; Di Nicola et al., 2020; Iversen et al., 2020; Ferri and Soccini, 2020; Di Nicola et al., 2021). RESULTS Overall, 94 sequences of 255 bp total length were analysed including the outgroups and the results confirm the same overall phylogenetic arrangement previously shown by Santos et al. (2012a) with five major clades (Fig. 3). All the 21 Italian samples (GenBank accession numbers JQ837570, JQ837571 and OK573460-78) fall within ‘Clade 5’ (sensu Santos et al., 2012a) which also includes sequences from France and most of Iberian Pen- insula (Fig. 3). However, the monophily of ‘Clade 5’ is not adequately supported (bootstrap = 65%) on the basis of the cyt b fragment analysed here and it is considered as an haplogroup. We found four cyt b haplotypes amongst the Italian C. girondica populations, differing by 1-2 mutation steps (Fig. 1). Most of the samples (n: 14) shared the same hap- lotype (H1), which is also shared with samples from Spain (JQ837574, JQ837587, JQ837589, JQ837607, JQ837610, JQ837635) and Portugal (JQ837569, JQ837582, JQ837595, JQ837634) (Fig. 3). In contrast, the sample from Emilia Romagna (OK573478) share the haplotype H4 with a sample from Ciudad Real, Spain (JQ837591). The follow- ing two unpublished haplotypes were detected among the northernmost Italian samples: H2 shared by all the five samples of the recently confirmed separated locali- ties in western Lombardy (OK573464) and Trentino-Alto Adige (OK573460-63) and H3 from southern Piedmont (OK573465). H2 and H3 were shown to belong to the same haplogroup (Figs. 1, 3). With the exclusion of the observations recently reported in the literature (Iversen et al., 2020; Di Nicola et al., 2020; Ferri and Soccini, 2020), three new localities (B, I, H) that were not recently confirmed (Razzetti and Bonini, 2006) have been registered, see Table 1 and Fig. 2 for details. Observation B resulted from a live adult snake observed on a low wall near Avio (province of Trento, Trentino-Alto Adige) just east of Lake Garda, as part of a cluster which includes observations recently published by Iversen et al. (2020) and Ferri and Soccini (2020) (Table 1, Fig. 2). Observations I and H corresponded to a live and a road-killed snake respectively and were both recorded in the Biella province (Piedmont) falling within the geographic range already known in the north-western regions of the Alps (Razzetti and Bonini, 2006) and the point G, a separated locality recently reported in Lom- bardy by Di Nicola et al. (2020) (Fig. 2). DISCUSSION The results presented here confirm a low genetic diversity for C. girondica within the cluster ‘Clade 5’, as shown by Santos et al. (2012a). For this haplogroup, rapid expansion process from south to north has been hypoth- esised to be likely derived from climatic warming events and followed by a bottleneck effect (Santos et al., 2012a). This pattern of recent expansion from the Iberian Penin- Fig. 1. Minumum spanning network based on the Coronella girondica cytochrome b fragment in Italy, and geographic distribu- tion of the haplotypes. Circle sizes are proportional to haplotype frequency, and each black bars represents a mutational step. Sam- ples are numbered as in Table 1. The green area in the map repre- sents the approximate range of C. girondica in Italy. 119Genetic characterisation of Coronella girondica in Italy sula towards the north-east has also been hypothesised for other snake species such as Natrix maura (Guicking et al., 2002). The genetic structure that emerges from the Italian C. girondica samples shows a lack of mitochondrial variabil- ity in the Apennine cyt b samples and a slight differen- tiation in Alpine and subalpine samples (Fig. 1, 3), which highlight that the endemic haplotypes H2 and H3 belong to the same cluster. This mtDNA pattern partially match- es with that from other species of the Italian herpetofau- na (Canestrelli et al., 2007; Canestrelli and Nascetti, 2008; Salvi et al., 2013; Chiocchio et al., 2021), including the Barred grass snake Natrix helvetica (Schultze et al., 2019) and confirms the margin of the Northern Apennines as a suture area of the Italian Peninsula (Hewitt, 2011; Chioc- chio et al., 2021). This is compatible with recent spread- ing and isolation of C. girondica in Northern Italy, likely during the last glacial maximums, which allowed a slight haplotype differentiation. Subsequently, the Italian Alps and the Po Plain went through various climatic fluctua- tions and extensive human impact during the Holocene (Colombaroli et al., 2010; Nussbaumer et al., 2011; Joan- nin et al., 2013). These factors may have modulated the distribution of C. girondica with expansion and contrac- tion processes. The H4 haplotype, found in a single Italian sample (OK573478) collected in Emilia Romagna (Table 1, Figs. 1, 3), is identical to JQ837591 from Ciudad Real, central Spain (Fig. 3). This observation is similar to other cases reported for Natrix natrix and N. helvetica (Kindler et al., 2017; Schultze et al., 2019, 2020) and could be indicative of human translocation events. However, this hypothesis still needs further investigation through testing additional markers. Recent Italian findings of C. girondica north of the Po River can be grouped into two main clusters. A North- western Alpine cluster includes some Alpine valleys of Northern Piedmont and Aosta Valley, and it is appar- ently separated from both the neighbouring French and Italian populations (Razzetti and Bonini, 2006; Sillero et al., 2014). An Eastern Alpine cluster is located around Lake Garda, and falls within the territories of Lom- bardy, Trentino-Alto Adige and Veneto (Iversen et al., 2020; Di Nicola et al., 2020; Ferri and Soccini, 2020). The populations around Lake Garda have been reported from the literature with many detailed records (De Betta, 1857; Dalla Torre, 1912) and later attested only by a few museum specimens dated up to 1977 (see Iversen et al., 2020). Other more recent information has been reported by Lorenzi and Bruno (2006) which, however, do not Fig. 2. Distribution of Coronella girondica in the Italian Alps north of the Po river. Red circle are the areas reported up to Razzetti and Bonini (2006), green landmarks represent the observations published after 2006 (Di Nicola et al., 2020; Iversen et al., 2020; Ferri and Soc- cini, 2020), yellow landmarks are the unpublished findings. The details of each point are shown in Table 1. 120 Matteo R. Di Nicola et alii Fig. 3. Maximum Likelihood (ML) tree of Coronella girondica inferred from the mitochondrial cytochrome b gene, with a detail on the hap- lotypes of the ‘Clade 5’ (sensu Santos et al., 2012a) cluster. The haplotypes that occur in Italy are marked with the same colors used in Fig. 1. The numbers at nodes are ML bootstrap percentages. With the exception of the unpublished samples (see Table 1) all the others are taken from literature (Carranza et al., 2004, 2006; Santos et al., 2008, 2012a, b; Carvalho et al., 2017; Faraone et al., 2020a, b). 121Genetic characterisation of Coronella girondica in Italy clearly contextualise their personal observations. The scarcity of findings during the twentieth century has led zoologists to consider this population close to extinction (Razzetti and Bonini, 2006) or probably extinct (Raz- zetti and Bernini, 2011). Recently, new observations led to the confirmation of the presence of C. girondica in Veneto (Novarini et al., 2017; Iversen et al., 2020), Tren- tino-Alto Adige (Iversen et al., 2020) and Lombardy (Di Nicola et al., 2020; Ferri and Soccini, 2020). New obser- vations around Lake Garda largely confirmed previous records provided by Dalla Torre (1912), including the more recent point “B” (Fig. 2), which falls within a local- ity highlighted by the author between 1896 and 1900. The observations “I” and “H” are located between the Northwestern cluster and point “G”, recently reported in Lombardy by Di Nicola et al. (2020) (Table 1, Fig. 2). Point “I” is an unpublished locality, while “H” surpris- ingly confirms a site with a single specimen (MSNM RE 1439) preserved in the Civic Natural History Museum of Milan (see also Andreone and Sindaco, 2002), dated back to 1926. The geographic position of points “H” and “I” (Fig. 2) suggests that the North-western cluster and point “G” could be connected by small, scattered and low-den- sity populations that may be detected by increasing the environmental monitoring of the species. As indicated by Bombi et al. (2009), with the exclusion of the Maritime Alps and the Northern Apennines, North- ern Italy has very low suitability values for C. girondica since only a few scattered sub-optimal patches in the area north of the Po River have been found. The results obtained here confirm this scenario, since C. girondica has a clearly fragmented distribution in the Alpine and sub- alpine areas. Furthermore, this species is mostly found in habitats within sub-Mediterranean climates, often charac- terised by xerophilous faunistic and vegetational commu- nities which are very specific compared to other habitats and surrounding territories (La Greca, 1956; Gratani and Varone, 2003; Agabiti et al., 2005). In conclusion, the results generated in this study suggest that the fragmented distribution of C. girondica north of the Po river can be mainly attributed to relict populations, based on the following points: (a) the find- ing of endemic haplotypes, compatible with a recent separation occurred during the last glacial events; (b) the few and scattered observations, highly localised in small patches with xeric Mediterranean features, suitable for the species; (c) current records largely match with histori- cal records which, on the other hand, indicate few Alpine areas without recent confirmation; (d) the post-glacial history of Northern Italy is characterised by changes in ecosystems caused by climatic fluctuations and a strong human impact, and this could have caused the expansion and contraction of C. girondica as a thermophilic snake. This hypothesis will be further investigated through more extensive sampling in the field and the analysis of a greater number of loci in order to further detail the genetic structure of the Italian C. girondica populations. A greater fieldwork effort will be necessary, especially on the Western Alps, where our results could indicate a probably incomplete C. girondica distribution, which may be slightly less fragmented than previously thought. ACKNOWLEDGEMENTS The authors would like to thank all the observers reported on Table 1 for their valuable contribution; Mat- teo Graglia, Sergio Mezzadri and Giuseppe Molinari for their precious contribution in the fieldwork, and Andrea Ciracì for his information. The authors are also grateful to Roberta Alberigo, Federica Bracaletta, Simona Cor- neti, Francesco Di Toro, Eduardo Di Trapani, Lorenzo Laddaga, Luca Miselli, Massimo Pellegrini, Silvia Pelti, Federico Pino, Marta Teves, Marina Trevisan for report- ing us C. girondica specimens during their hikes. Thanks also to Giovanni Boano, director of the Civic Natural History Museum of Carmagnola (Turin), and Stefano Scali from the Civic Natural History Museum of Milan. Finally, thanks to Enrica Calò and Lesley Dhonau for a pre-review of the manuscript and to Jean-Lou Dorne for the proofreading. The animals were handled following ministerial permits: MATTM reg. 0083124, 16/10/2020; Prot. ISPRA 46387, 12/10/2020. REFERENCES Agabiti, B., Valentinotti, R., Salvadori, C. (2005): The grasshoppers (Insecta, Orthoptera) of a sub-mediter- ranean zone of the Trentino region (North-East Italy). Studi Trent. Sci. Nat, Acta Biol. 82: 61-67. Andreone, F., Sindaco, R. (1998): Erpetologia del Pie- monte e della Valle d’Aosta, Atlante degli Anfibi e dei Rettili. Monografie del Museo Regionale di Scienze Naturali, Torino. Bandelt, HJ., Forster, P., Röhl, A. (1999): Median-joining networks for inferring intraspecific phylogenies. Mol. Biol. Evol. 16: 37-48. Boettger, O. (1881): Diagnoses Reptilium novorum Maroccanorum. Zool. Anz. 4: 570-572. Bombi, P., Luiselli, L., Capula, M., Salvi, D. (2009): Pre- dicting elusiveness: potential distribution model of the Southern smooth snake, Coronella girondica, in Italy. Acta Herpetol. 4: 7-13. 122 Matteo R. Di Nicola et alii Busack, S. D. (1986): Biogeographic analysis of the her- petofauna separated by the formation of the Strait of Gibraltar. Nat. Geogr. Res. 2: 17-36. Canestrelli, D., Nascetti, G. (2008): Phylogeography of the pool frog Rana (Pelophylax) lessonae in the Italian peninsula and Sicily: multiple refugia, glacial expan- sions and nuclear-mitochondrial discordance. J. Bio- geogr. 35: 1923-1936. Canestrelli, D., Cimmaruta, R., Nascetti, G. (2007): Phy- logeography and historical demography of the Italian treefrog, Hyla intermedia, reveals multiple refugia, population expansions and secondary contacts within peninsular Italy. Mol. Ecol. 16: 4808-4821. Capula, M., Carafa, M., De Lisio, L., Loy, A. (2010): Il Progetto Atlante degli Anfibi e Rettili del Molise. In: Atti VIII Congresso Nazionale Societas Herpetologica Italica, CH, 22-26 Settembre 2010, p. 37-49, Di Tizio, L., Di Cerbo, A.R., Di Francesco, N., Cameli, A., Eds, Ianieri Edizioni, Pescara. Carranza, S., Arnold, E.N., Thomas, R.H., Mateo, J.A., Lopez Jurado, L.F. (1999): Status Of The Extinct Giant Lacertid Lizard Gallotia simonyi simonyi (Reptilia: Lacertidae) Assessed Using mtDNA Sequences From Museum Specimens. Herpetol. J. 9: 83-86. Carranza, S., Arnold, E.N., Wade, E., Fahd, S. (2004): Phy- logeography of the false smooth snakes, Macroprotodon (Serpentes, Colubridae): mitochondrial DNA sequenc- es show European populations arrived recently from Northwest Africa. Mol. Phylogen. Evol. 33: 523-532. Carranza, S., Arnold, E.N., Pleguezuelos, J.M., (2006): Phylogeny, biogeography, and evolution of two Medi- terranean snakes, Malpolon monspessulanus and Hem- orrhois hippocrepis (Squamata, Colubridae), using mtDNA sequences. Mol. Phylogen. Evol. 40: 532-546. Carvalho, S.B., Velo-Antón, G., Tarroso, P., Paula Portela, A., Barata, M., Carranza, S., Moritz, C., Possingham, H.P. (2017): Spatial conservation prioritization of bio- diversity spanning the evolutionary continuum. Nat. Ecol. Evol. 1: 0151. Chiocchio, A., Arntzen., J.W., Martínez-Solano, I., De Vries, W., Bisconti, R., Pezzarossa, A., Maiorano, L., Canestrelli, D. (2021): Reconstructing hotspots of genetic diversity from glacial refugia and subsequent dispersal in Italian common toads (Bufo bufo). Sci. Rep. 11: 260. Colombaroli, D., Henne, P.D., Kaltenrieder, P., Gobet, E., Tinner, W. (2010): Species responses to fire, cli- mate and human impact at tree line in the Alps as evidenced by palaeo-environmental records and a dynamic simulation model. J. Ecol. 98: 1346-1357. Dalla Torre, K.W. (1912): Die Schlangen Tirols- Zoogeog- raphische Studie. Z. Ferdinand. 3: 1-68. Daudin, F.M. (1803): Histoire naturelle, générale et par- ticulière des reptiles: ouvrage faisant suite aux oeuvres de Leclerc de Buffon, et partie du cours complet d’histoire naturelle rédigée par CS Sonnini (Vol. 8). F. Dufart, Paris. De Betta, E. (1857): Erpetologia delle Provincie Venete e del Tirolo meridionale. Mem. Acc., Verona 35: 1-365. Di Nicola, M.R., Colombo, M., Manica, M., Ivers- en, D.L.N., Spada, M., Laddaga, L. (2020): Nuove osservazioni di Coronella girondica (Daudin, 1803) (Serpentes, Colubridae) per la Lombardia settentrion- ale nelle province di Varese e Brescia. Riv. Mus. Civ. Sci. Nat. “Enrico Caffi” 33: 9-14. Di Nicola, M.R., Cavigioli, L., Luiselli, L., Andreone, F. (2021): Anfibi & Rettili d’Italia. Edizione aggiornata. Edizioni Belvedere, Latina. Domergue, C.A. (1962): Observations sur le pénis des ophidiens (Deuxième note). Bull. Soc. Sci. Nat. Phys. Maroc. 42: 87-101. Faraone, F.P., Melfi, R., Di Nicola, M.R., Giacalone, G., Lo Valvo, M. (2020a): The genetic identity of the only Italian population of the genus Macroprotodon Guichenot, 1850 on the island of Lampedusa, Sicily. Vertebr. Zool. 70: 235-240. Faraone, F.P., Melfi, R., Di Nicola, M.R., Giacalone, G., Lo Valvo, M. (2020b): Phylogenetic relationships of the Italian populations of Horseshoe Whip Snake Hemor- rhois hippocrepis (Serpentes, Colubridae). Acta Herpe- tol. 15: 129-135. Felsenstein, J. (1985): Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783-791. Ferri, V., Morimando, R. (2004): Coronella girondica. In: Atlante degli Anfibi e dei Rettili della Lombardia, Pia- nura, Monografie n° 5, p. 147-148, Bernini, F., Bonini, L., Ferri, V., Gentilli, A., Razzetti, E., Scali, S., Eds., Provincia di Cremona. Ferri, V., Soccini, C. (2020): Conferma della presenza di Coronella girondica (Daudin, 1803) nel Parco Alto Garda Bresciano (Reptilia: Serpentes, Colubridae). Ann. Mus. Civ. Sci. Nat., Brescia 43: 149. Geniez, P. (2018): Snakes of Europe, North Africa and the Middle East. A photographic guide. Princeton Univer- sity Press, Princeton. Gratani, L., Varone L. (2003): Structural and functional plasticity of Quercus ilex seedlings of different prov- enances in Italy. Trees-Struct. Funct. 17: 515-521. Guicking, D., Joger, U., Wink, M. (2002): Molecular phy- logeography of the Viperine Snake Natrix maura and the Dice Snake Natrix tessellata: first results. Biota 3: 49-59. Guindon, S., Dufayard, J.F., Lefort, V., Anisimova, M., Hordijk, W., Gascuel, O. (2010): New algorithms and 123Genetic characterisation of Coronella girondica in Italy methods to estimate Maximum-Likelihood phylog- enies: assessing the performance of PhyML 3.0. Syst. Biol. 59: 307-321. Haklay, M.M., Dörler, D., Heigl, F., Manzoni, M., Heck- er, S., Vohland, K. (2021): What is citizen science? The challenges of definition. In:  The Science of Citi- zen Science, pp: 13-33, Vohland, K., Land-Zandstra, A., Ceccaroni, L., Lemmens, R., Perelló, J., Ponti, M., Samson, R., Wagenknecht, K., Eds., Springer, Amster- dam. Hewitt, G.M. (2011): Mediterranean Peninsulas - the evo- lution of hotspots. In: Biodiversity Hotspots, p. 123- 148, Zachos, F.E., Habel, J.C., Eds., Springer, Amster- dam. Iversen, D.L.N., Tabarelli De Fatis, K., Romano, A., Pedrini, P., Di Nicola, M.R. (2020): The Southern Smooth Snake, Coronella girondica (Daudin, 1803), in North-East Italy: new findings and a review of the historical distribution. Herpetol. Notes 13: 543-548. Joannin, S., Vannière, B., Galop, D, Peyron, O., Haas, J.N., Gilli, A., Chapron, E., Wirth, S.B., Anselmetti, F., Desmet M., Magny, M. (2013): Climate and vegeta- tion changes during the Lateglacial and early-middle Holocene at Lake Ledro (southern Alps, Italy). Clim. Past. 9: 913-933. Jukes, T.H., Cantor, C.R. (1969): Evolution of protein molecules. In: Mammalian Protein Metabolism, p. 21-132, Munro, H.N., Ed., Academic Press, New York. Kindler, C., Chèvre, M., Ursenbacher, S., Böhme, W., Hille, A., Jablonski, D., Vamberger, M., Fritz, U. (2017): Hybridization patterns in two contact zones of grass snakes reveal a new Central European snake species. Sci. Rep. 7: 7378. La Greca, M. (1956): Significato biogeografico di ripar- tizioni disgiunte in Ortotteri non montani d’Italia. Arch. Bot. Bioegeogr. Ital. 32: 113-129. Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., Mcgettigan, P.A., Mcwilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson, T.J., Higgins, D.G. (2007): Clustal W and Clustal X version 2.0. Bioinformatics 23: 2947-2948. Lefort, V., Longueville, J.-E, Gascuel, O. (2017): SMS: Smart Model Selection in PhyML. Mol. Biol. Evol. 34: 2422-2424. Leigh, J.W., Bryant, D. (2015): Data from: PopART: Full- feature software for haplotype network construction. Methods Ecol. Evol. 6: 1110-1116. Lorenzi, P., Bruno, S. (2006): Note erpetologiche. Ann Mus Civ Rovereto. 22: 277-286. Mezzasalma, M., dall’Asta, A., Loy, A., Cheylan, M., Lym- berakis, P., Zuffi, M.A.L., Tomović, L., Odierna, G., Guarino, F.M. (2015): A sisters’ story: comparative phylogeography and taxonomy of Hierophis viridifla- vus and H. gemonensis (Serpentes, Colubridae). Zool. Scr. 44: 495-508. Novarini, N., Zanovello, L., Bonato, L. (2017): Aggiorna- mento della distribuzione dell’erpetofauna in Veneto: prime valutazioni in base ai dati dell’ultimo decennio. In: Atti 8° Convegno Faunisti Veneti - Sedico, 21-22 ottobre 2017, p. 15-23, Bonato, L., Spada, A., Cassol, M., Eds., Boll. Mus. St. Nat., Venezia, suppl. Vol. 69. Nussbaumer, S., Steinhilber, F., Trachsel, M., Breiten- moser, P., Beer, J., Blass, A., Grosjean, M., Hafner, A., Holzhauser, H., Wanner, H., Zumbühl, H. (2011): Alpine climate during the Holocene: A comparison between records of glaciers, lake sediments and solar activity. J. Quat. Sci. 26: 703-713. Razzetti, E., Bernini, F. (2011): Coronella girondica. In: Fauna d’Italia, vol. XLV, Reptilia, p. 481-487, Corti, C., Capula, M., Luiselli, L., Sindaco, R., Razzetti, E., Eds., Calderini, Bologna. Razzetti, E., Bonini, L. (2006) Coronella girondica. In: Atlante degli anfibi e dei rettili d’Italia / Atlas of Ital- ian Amphibians and Reptiles, p. 530-535, Sindaco, R., Doria, G., Razzetti, E., Bernini F., Eds., Societas Her- petologica Italica, Edizioni Polistampa, Firenze. Razzetti, E., Bonini, L., Colombari, P. (2000): Revisione della distribuzione e nuovo limite orientale di Coro- nella girondica (Daudin, 1803) (Reptilia: Colubridae). Atti Soc. Ital. Sci. Nat. Mus. Civ. St. Nat., Milano. 141: 169-174. Rugiero, L., Luiselli, L., Capula M., (2018): Coronella girondica. In: Anfibi e Rettili del Molise - Atlante e Guida, p. 206-212, Capula, M., Di Tizio, L., De Lisio, L., Carafa, M., Brugnola L., Eds., Societas Herpetolog- ica Italica, Sezione Abruzzo e Molise, Ianieri Edizioni, Pescara. Saint Girons, H. (1956): Les serpents du Maroc. Var. Sci. Rec. Soc. Sci. Nat. Phys. Maroc. 8: 1-29. Salvi, D., Harris, J., Kaliontzopoulou, A., Carretero, M. A., Pinho C. (2013): Persistence across Pleistocene ice ages in Mediterranean and extra-Mediterranean refu- gia: phylogeographic insights from the common wall lizard. BMC Evol. Biol. 13: 147. Santos, X., Pleguezuelos, J.M. (2003): Variación mor- fológica en la culebra lisa meridional Coronella girondica (Daudin, 1803) a través de su área de dis- tribución. Rev. Esp. Herpetol. 17: 55-73. Santos, X., Pleguezuelos, J.M. (2015): Culebra lisa meridi- onal - Coronella girondica. In: Enciclopedia Virtual de los Vertebrados Españoles, Salvador, A., Marco, A., Eds, Mus. Nac. Ci. Nat., Madrid. Available on-line at: http://www.vertebradosibericos.org/reptiles/corgir. html [Accessed on 17 March 2020] 124 Matteo R. Di Nicola et alii Santos. X., Roca. J., Pleguezuelos, J.M., Donaire, D., Car- ranza, S. (2008): Biogeography and evolution of the smooth snake Coronella austriaca (Serpentes: Colubridae) in the Iberian Peninsula: evidence for Messinian refuges and Pleistocenic range expansions. Amphibia-Reptilia 29: 35-47. Santos, X., Rato, C., Carranza, S., Carretero, M. A., Ple- guezuelos, J. M. (2012a): Complex phylogeography in the Southern Smooth Snake (Coronella girondica) supported by mtDNA sequences. J. Zoolog. Syst. Evol. Res. 50: 210-219. Santos, X., Rato, C., Carranza, S., Carretero, M.A., de la Vega, J.P.G., Pleguezuelos, J.M. (2012b): Morphol- ogy matches mtDNA lineages in the southern smooth snake (Coronella girondica) from Iberia. Zoology 115: 398-404. Schultze, N., Laufer, H., Kindler, C., Fritz, U. (2019): Dis- tribution and hybridisation of barred and common grass snakes (Natrix helvetica, N. natrix) in Baden- Württemberg, South-western Germany. Herpetozoa 32: 229-236. Schultze, N., Spitzweg, C., Corti, C., Delaugerre, M., Di Nicola, M. R., Geniez, P., Lapini, L., Liuzzi, C., Lung- hi, E., Novarini, N., Fritz, U. (2020): Mitochondrial ghost lineages blur phylogeography and taxonomy of Natrix helvetica and N. natrix in Italy and Corsica. Zool. Scr. 49: 395-411. Sillero, N., Campos, J., Bonardi, A., Corti, C., Creemers, R., Crochet, P.-A., Crnobrnja Isailovic, J., Denoël, M., Ficetola, G.F., Gonçalves, J., Kuzmin, S., Lymberakis, P., de Pous, P., Rodríguez, A., Sindaco, R., Speybroe- ck, J., Toxopeus, B., Vieites, D.R., Vences, M. (2014): Updated distribution and biogeography of amphibians and reptiles of Europe. Amphibia-Reptilia 35: 1-31. Sindaco, R., Razzetti, E. (2021): An updated check-list of Italian amphibians and reptiles. Atti Soc. Ital. Sci. Nat. Mus. Civ. St. Nat., Milano 8: 35-46. Sindaco, R., Venchi, A., Grieco, C. (2013): The Reptiles of the Western Palearctic. 2. Annotated checklist and distributional atlas of the snakes of Europe, North Africa, Middle East and Central Asia, with an Update to the Volume 1. Edizioni Belvedere, Latina. Tagliavia, M., Nicosia, A., Salamone, M., Biondo, G., Ben- nici, C.D., Mazzola, S., Cuttitta, A. (2016): Develop- ment of a fast DNA extraction method for sea food and marine species identification. Food Chem. 203: 375-378. Tamura, K., Stecher, G., Kumar, S. (2021): MEGA11: Molecular Evolutionary Genetics Analysis version 11. Mol. Biol. Evol. 38: 3022-3027. Acta Herpetologica Vol. 17, n. 2 - December 2022 Firenze University Press Cryptic diversity in pygmy chameleons (Chamaeleonidae: Rhampholeon) of the Eastern Arc Mountains of Tanzania, with description of six new species Michele Menegon1,2,*, John V. Lyakurwa3,4, Simon P. Loader5, Krystal A. Tolley6,7 Preliminary genetic characterisation of Southern Smooth Snake Coronella girondica (Serpentes, Colubridae) populations in Italy, with some considerations on their alpine distribution Matteo R. Di Nicola1, Raffaella Melfi2, Francesco P. Faraone3,*, Daniel L. N. Iversen4, Gabriele Giacalone5, Giovanni Paolino1, Mario Lo Valvo6 Species diversity and distribution of amphibians and reptiles in Sardinia, Italy Claudia Corti1,2,*, Marta Biaggini1, Valeria Nulchis2, Roberto Cogoni2, Ilaria Maria Cossu2, Salvatore Frau4, Manuela Mulargia2, Enrico Lunghi2, Lara Bassu2. The Italian wall lizard, Podarcis siculus campestris, unexpected presence on Gorgona Island (Tuscan Archipelago) Marco A.L. Zuffi1,*, Alan J. Coladonato2, Gianluca Lombardo3, Antonio Torroni3, Matilde Boschetti1, Stefano Scali4, Marco Mangiacotti2, Roberto Sacchi2 Molecular analysis of recently introduced populations of the Italian wall lizard (Podarcis siculus) Oleksandra Oskyrko1,2,*, Lekshmi B. Sreelatha1,12,13, Iolanda Silva-Rocha1, Tibor Sos3,4, Sabina E. Vlad5,6,7, Dan Cogălniceanu5,6, Florina Stănescu6,7,8, Tavakkul M. Iskenderov9, Igor V. Doronin10, Duje Lisičić11, Miguel A. Carretero1,12,13 Sunny-side up: ontogenetic variation in egg mass temperatures of the wood frog Rana sylvatica Ryan Calsbeek*, Ava Calsbeek, Isabel Calsbeek Ecological niche differentiation in the Anatolian rock lizards (Genus: Anatololacerta) (Reptilia: Lacertidae) of the Anatolian Peninsula and Aegean Islands Mehmet Kürşat Şahin1,*, Kamil Candan2,3, Danae Karakasi4, Petros Lymberakis4, Nikos Poulakakis4,5,6, Yusuf Kumlutaş2,3, Elif Yıldırım2,3, Çetin Ilgaz2,3 Occupancy and probability of detection of the introduced population of Eleutherodactylus coqui in Turrialba, Costa Rica Jimmy Barrantes-Madrigal1,*, Manuel Spínola Parallada1, Gilbert Alvarado 2, Víctor J. Acosta- Chaves3,4. One site, three species, three stories: syntopy of geckoes Euleptes europaea (Gené, 1839), Hemidactylus turcicus (Linnaeus, 1758), Tarentola mauritanica (Linnaeus, 1758) in a coastal area of southern Tuscany (central Italy) Giacomo Radi1,2, Marco A.L. Zuffi1,* Comparative cytogenetics on Zamenis lineatus and Elaphe quatuorlineata (Serpentes: Colubridae) Marcello Mezzasalma1,* , Elvira Brunelli1, Gaetano Odierna2, Fabio M. Guarino2