Acta Herpetologica 16(1): 37-44, 2021 ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah DOI: 10.36253/a_h-10418 Sex chromosome diversification in the smooth snake Coronella austriaca (Reptilia, Serpentes) Marcello Mezzasalma1,2,*, Gaetano Odierna1 1 Department of Biology, University of Naples Federico II, Via Cinthia 26, 80126, Naples, Italy 2 CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, InBIO, Universidade do Porto, Campus Agrário de Vairão, Rua Padre Armando Quintas, No 7, 4485-661 Vairão, Portugal (current address) *Corresponding author. E-mail: m.mezzasalma@gmail.com Submitted on: 2021, 13th February; revised on: 2021, 16th April; accepted on: 2021, 25th April Editor: Fabio M. Guarino Abstract. The smooth snake Coronella austriaca is a widespread Palearctic colubrid species. The species has been the subject of several molecular and phylogeographic studies which highlighted the occurrence of distinct genetic lineages in different areas of the species distribution, but scarce cytogenetic data are currently available on the species. In this paper we present a molecular and karyological study performed with several banding, staining methods and NOR- FISH on samples of C. austriaca from different geographical areas (Italy and Greece) of the species distribution. The molecular and phylogenetic analysis unambiguously placed the studied samples in different clades with a clear geo- graphical pattern. The karyotype of the two female samples studied was composed of 2n = 36 chromosomes with 16 macro- and 20 microchromosomes and a mix of plesiomorphic and derivate chromosome features. All macrochromo- somes were biarmed with the exception of pair 5 that was telocentric. NORs were detected on a microchromosome pair. In both females, the pair 4 was heteromorphic (and completely heterochromatic after C-banding in the Italian female), representing the first report of a ZZ/ZW sex chromosome system with female heterogamety in C. austriaca. In addition, the W chromosome showed a different morphology between the two female studied (submetacentric and subtelocentric), highlighting the occurrence of a chromosomal diversification among distinct geographical areas of the species distribution and further supporting that the species contains different diverging evolutionary clades. Keywords. Karyotype, heterochromatin, FISH, sex chromosomes, squamates, snakes. INTRODUCTION The smooth snake Coronella austriaca Laurenti, 1768, is a small sized ovoviviparous colubrid with a widespread distribution across the western Palearctic (Strijbosch, 1997). The species occurs in western, central and south- ern Europe reaching as far as the Ural Mountains and the Caspian Sea, up to northern Iran and western Kazakh- stan. Coronella austriaca is absent from European and Mediterranean islands, except for Southern England, Sic- ily, the Island of Elba and the Island of Krk (Engelmann, 1993; Strijbosch, 1997; 2006). In its large distribution range, the species have a relatively uniform morphol- ogy and only two subspecies are currently recognised: C. a. acutirostris Malkmus, 1995, from the north-western Iberian Peninsula and the nominal subspecies C. a. aus- triaca which occupies the rest of the geographical range of the species. A third subspecies, C. a. fitzingeri (Bona- parte, 1840) was formerly described from southern Italy and Sicily, but it has been recently synonymised with the nominal subspecies (Speybroeck et al., 2016). The species has been the object of several molecular and phylogeographic studies (Santos et al., 2008; Llorente et al., 2012; Galarza et al., 2015; Jablonsky, 2019) and rep- 38 M. Mezzasalma, G. Odierna resents an ideal candidate to assess patterns of intraspe- cific genetic diversity among different populations as well as the occurrence of different refugial areas across its dis- tribution range. These studies evidenced that C. austriaca comprises several distinct clades showing a complex hap- lotype structure and deep genetic divergence. These evi- dences do not reflect the current taxonomy of the species and a revision is probably required to better describe its molecular and geographic intraspecific diversity. On the other hand, the available chromosome data on C. austriaca are dated and refer to the studies by Mat- they (1931) and Kobel (1967) that described the karyo- type of two males from Switzerland, but no information is currently available from other, geographically and genetically distinct clades of the species. The karyotype described by Matthey (1931) and Kobel (1967) was com- posed of 2n = 36, with 16 macrochromosomes and 20 microchromosomes. All the macrochromosomes were biarmed, excluding the elements of 5th pair that were telocentric. Furthermore, these studies were performed only with standard staining methods. No information is currently available on the sex chromosome system, location of nucleolar organizer regions (NORs) or other chromosome markers which would be useful for karyo- type comparison among different population of the spe- cies or with other phylogenetically related colubrid spe- cies. In fact, cytogenetic inferences, especially when linked to molecular data in a phylogenetic perspective, are useful tools to detect plesio- and apomorphic states and to reconstruct evolutionary trends in the studied species (Odierna et al., 1987; Mezzasalma et al., 2013, 2017 a, 2017b; Fuller et al., 2018). In this work we present the results of a karyological study performed on smooth snake samples belonging to different populations and geographical areas of the spe- cies distribution (Italy and Greece) using several standard staining and banding methods and molecular cytogenet- ics. The aim of this study was to evidence the possible presence of a chromosome sex determination system, the occurrence and location of heterochromatic regions and of NORs. We also performed a molecular and a phylo- genetic analysis on the studied samples, adding our data to those available from the literature, in order to evaluate their genetic diversity and place our chromosome data in a phylogenetic context. MATERIAL AND METHODS Sampling We studied preserved tissue and cell suspensions of two samples of C. austriaca obtained from existing collections and no aminal was collected during the realization of this study. Studied samples include one female from Italy (Picentini Moun- tains, Avellino), and one female from Greece (Peloponnese) both hosted at the Dipartimento di Biologia Evolutiva e Com- parata, Università degli Studi di Napoli Federico II, since 1972 (sample numbers CA0201- CA0202). These two samples were both used in a preliminary molecular analysis and in the kary- olgical study as described below. The samples used in this study were already used in previous analyses (Mezzasalma et al., 2014, 2016a). Molecular and phylogenetic analysis In order to properly identify the studied samples, assess their genetic diversity and establish the taxonomic affini- ties with other available sequences on the species, a molecular analysis was performed using a fragment of the mitochondrial Cytochrome b (Cytb) gene. This mtDNA gene was chosen con- sidering the number of available sequences for several popula- tions of C. austriaca (Llorente et al., 2012; Santos et al., 2008, 2012; Galarza et al., 2015; Jablonsky et al., 2019). Genomic DNA was extracted from both samples using the standard phenol-chloroform method by Sambrook et al. (1989) and then used in the PCR amplification of a fragment of about 600 bp of the Cytb. The primers used were FORWARD: 5’ AACTTCGGATCCATACTACTAA 3’ and REVERSE: 3’ TAAA- GATGTTAGGGGTGAATGA 5’, and the PCR parameters those reported by Mezzasalama et al. (2015a). PCR products were sequenced on an automated sequenc- er ABI 377 (Applied Biosystems, Foster City, CA, USA) using BIGDYE TERMINATOR v3.1 (ABI). Sequences were blasted in GenBank and chromatograms were checked and edited using CHROMAS LITE 2.1.1 and BIOEDIT 7.2.6.1 (Hall, 1999). The best-fitting substitution model (GTR+I+G) was chosen using JMODELTEST 2.1.7 (Darriba et al., 2012), under the corrected Akaike information criterion (AICc). In the phylogenetic analy- sis we used the two newly determined sequences (Accession Numbers: MW861682-MW861683) and available homologous sequences from Jablonski et al. (2019), downloaded from Gen- Bank, choosing when possible, the longest available sequences for each major clade (see Fig. 1). As outgroup we used an avail- able homologous sequence of C. girondica (AN: AF471088). Phylogenetic analysis with Bayesian inference (BI) was per- formed using MRBAYES 3.2.7 (Ronquist et al., 2012), with two parallel runs of 8 000 000 generations and four incrementally heated Markov chains (using default heating values), with a burn-in of 25% and sampling the chains every 500 generations. A 50% majority-rule consensus tree was retrieved from the post-burn-in samples and chain convergence was checked with convergence diagnostic values (average standard deviation of split frequencies >> 0.01, potential scale reduction factor). Chromosome analysis Chromosomes were obtained from tissue samples and cell suspensions using the air-drying method, as described in Mez- 39Karyological diversification in the smooth snake zasalma et al. (2019). The chromosome analysis was performed with traditional staining (5% Giemsa solution at pH 7 for 10 min) and several chromosome staining and banding techniques: Quinacrine staining, Chromomycin A3-methyl green staining (CMA/MG) (Mezzasalma et al., 2015b), C-banding (Sumner, 1972), and sequential C-banding + CMA + DAPI (Sidhom et al. 2020a, b). Karyotype reconstruction was performed after scoring at least five metaphase plate from each sample studied. Nucleolus organizing regions (NORs) were located following the Ag-NOR banding protocol reported by Howell and Black (1980) and fluorescence in situ hybridization (NOR-FISH) as described in Sidhom et al. (2020a), using as probe the PCR- amplified and biotinylated 18S rRNA gene of the gekkonid Tarentola mauritanica. The detection of FISH signals was car- ried out using ExtrAvidin FITC (Sigma Aldrich), counter- stained with propidium iodide (PI). Metaphase plates were detected and recorded using an epifluorescent microscope (Axioscope Zeiss) equipped with an image analysis system. RESULTS Molecular and phylogenetic analysis A a fragment of about 500 bp of the Cytb was suc- cessfully amplified and sequenced from the two original samples, respectively from Italy and Greece (see Sam- pling), used in the cytogenetic analysis. No interruptions of the reading frame were detected in either sequences. Nucleotide identity samples was about 99% between the newly sequenced sample from Italy (Monti Picentini) and an available sequence from Italy (AN: MH382919), and 100% between the newly determined sequence from Greece and an available sequence from Greece (AN: EU022647). In the phylogenetic analysis, the final Cytb align- ment contained 39 sequences and 1031 nucleotide posi- tions. The resulting tree (Fig. 1) retrieved all the main clades and a topology similar to that reported by Jablon- ski et al. (2019). The relative evolutionary relationships among the main clades mostly reflect an East-West Fig. 1. Phylogenetic analysis with Bayesian Inference (BI) of Cytb sequences (up to 1031 bp) of C. austriaca. Numbers at nodes represent posterior support values. Clades denomination follows Jabloski et al. (2019). Red circles = original samples used in the cytogenetic analysis. 40 M. Mezzasalma, G. Odierna scenario of geographical diversification by distance. Our tree retrieved, four major clades, comprising sev- eral smaller subclades: Iranian 1, Sicilian + Iberian 1-3 + Western 1-2, Anatolian 1-2 +Balkan + Central Euro- pean, and Iranian 2 + Transcaucasian + Eastern, respec- tively (Fig. 1). In our tree, the Easternmost major clade (excluding the only sample of the Iranian 1 clade), com- prising the Iranian 2 + Transcaucasian + Eastern sub- clades, is the outgroup of all the remaining clades. The two original samples from Italy and Greece clustered unambiguously within the Western 2 and Balkan clades, respectively (Fig. 1). Statistical support values were gen- erally high at terminal nodes while varied from high to low at deeper nodes (Fig. 1). Chromosomal analysis The Italian and Greek samples of C. austriaca showed a very similar karyotype composed of 2n = 36 chromo- somes, with 16 macrochromosomes and 20 microchro- mosomes (Fig. 2). Among macrochromosomes, pairs 1, 3, 6 and 8 were metacentric, pairs 2 and 7 were submeta- centric, and pair 5 was telocentric. The pair 4 was het- eromorphic, carrying the female sex chromosomes, ZW. In particular, the chromosome Z was always metacentric while the W resulted subtelocentric in the female from Picentini Mountains (Italy) (Fig. 2A) and submetacentric in the female from Peloponnese (Greece) (Fig. 2B). Other chromosome stainings and banding tech- niques were performed only on the Italian sample of C. austriaca, as quality and quantity of metaphase plates of the Greek individual were adequate just for its karyotype description. Quinacrine stained evenly both macro- and micro- chromosomes (Fig. 3A), while CMA/MG evidenced two microchromosomes and the telomeric regions of all macrochromosomes and the telomeric regions almost all microchromosomes (Fig. 3B). A microchromosome pair resulted specifically marked by both Ag-NOR staining and NOR-FISH, (Fig. 3C and D). After C-banding, heterochromatic bands were hardly visible, if not absent, on most macro- and microchromo- somes, with the exclusion of the W chromosome, which was completely heterochromatic (Fig. 4A), but not evi- denced with CMA or DAPI (Fig. 4B, C). DISCUSSION Our phylogenetic inference produced similar results to those obtained by Jablonski et al. (2019), retrieving all the 14 clades previously described. The main differ- ences concern the position of one of the major clades (comprising Iranian 2 + Transcaucasian + Eastern sub- Fig. 2. Giemsa stained karyotypes of C. austriaca from Italy (Picentini Mountains) (A) and Greece (Peleponnese) (B). The frame includes the ZW sex chromosome pair. Fig. 3. Metaphase plates of C. austriaca stained with Quinacrine (A), CMA/MG (B), Ag-NOR staining (C) and NOR-FISH banding (D). Arrows point at a microchrosome pair evidenced with CMA/MG, Ag-NOR staining- and NOR-FISH. Scale bar applies all images. 41Karyological diversification in the smooth snake clades) which is not involved in a basal polytomy with the other major clades but appear to be the outgroup of all the remaining clades. This results further supports the hypothesis of an East-West geographical diversification process in C. austriaca. Similar East-West differentia- tion processes are also shared by different European and Palearctic reptile taxa, including distinct major snake lin- eages of Asiatic origin (see, e.g., Utiger et al., 2002; Nagy et al., 2004; Mezzasalma et al. 2015a, 2018). However, the phylogenetic position and the evolutionary relationships of some clades (such as the Iranian 1 clade) remain to be better determined and this hypothesis should be further tested. In fact, as already highlighted by Jablonski et al. (2019), the uncertain phylogenetic position of some sub- clades as well as the low statistical support values at some nodes is probably due to a number of missing haplotypes and a more inclusive sampling with the addition of more molecular markers is probably required to better assess the evolutionary relationship of some subclades. The karyological formula of the two females of C. austriaca here studied is consistent with that previously described by Mattey (1931) and Kobel (1967) for two males from Switzerland. However, the comparison among the pair 4 of the different karyotypes allowed us to detect for the first time the ZZ/ZW sex chromosome system in C. austriaca. In particular, the metacentric element of the 4th pair was identified as the Z sex chromosome and its heteromorphic counterpart as the W chromosome. In addition, the W chromosome showed a different mor- phology among the two studied females, suggesting the occurrence of a chromosomal diversification among dis- tinct molecular clades. Overall, the karyotype of C. aus- triaca shows a mixture of plesiomorphic and derivate chromosomal characters. In particular, the chromosomal characters that can be considered plesiomorphic in colu- brids include the karyological formula, the microchro- mosomal localization of the NORs, and the pair 4 rep- resenting the ZZ/ZW sex chromosomes. A chromosome complement composed of 2n = 36 chromosomes with 16 macro- and 20 microchromosomes is displayed by both primitive (Henophidia) and advanced (Caenophidia) snake lineages and is supposed to represent the ances- tral snake karyotype (Gorman and Gress, 1970; Singh, 1972;, 1986, Oguiura et al., 2009; Mezzasalma et al., 2014, 2016b, 2019). Concerning the localization of NORs, the meth- ods here used show their presence on two microchro- mosomes. Ag-NOR banding suggests that both loci of NORs are active. In fact, Ag binds to proteins essential to nucleolar structure and therefore to the transcrip- tional activity of ribosomal cistrons during the previ- ous interphase (Howell, 1977; Jiménez et al., 1988). The occurrence of NORs on a microchromosome pair is not unusual among snakes, being exhibited in representa- tives of various families, including Colubridae (Olmo and Signorino, 2006), and it is considered a primitive condition in Squamata (Porter et al., 1991; Aprea et al., 2006). Derivate chromosome characters in the karyo- type of C. austriaca can be considered the morphology of the pair 5 (telocentric in all the studied samples), and the occurrence of an heteromorphic, completely hetero- chromatic W sex chromosome, which is also morpho- logically differentiated betwenn the two studied samples from different geographic regions. In fact, in the puta- tive ancestral snake karyotype of 2n = 36 all the macro- chromosome pairs are biarmed (meta- or submetacen- Fig. 4. Metaphase plates of C. austriaca sequentially stained with C-banding + Giemsa (A),+CMA (B), and +DAPI (C). Arrows point at the W sex chromosome. Scale bar applies to all images. 42 M. Mezzasalma, G. Odierna tric), though a telocentric morphology of the pair 5 has been observed in different colubrid species belonging to independent evolutionary lineages, such as different spe- cies of the genera Elaphe and Hierophis (see also Singh, 1972; Kobel 1967; Mezzasalma et al., 2015b). According to Singh (1972), because the fifth pair is biarmed in most of the other colubrids, a simple pericentric inversions can be assumed to explain the different morphology of this pair. The ZZ/ZW sex determination system was sup- posed to be a plesiomorphic state in snakes (Mengden, 1981; Matsubara et al., 2016), however recent evidences suggest that different sex chromosome systems evolved multiple times, independently, in different snake line- ages, including species with either female (ZZ/ZW) or male (XX/XY) heterogamety along with a discrete num- ber of species with undifferentiated sex chromosomes (Gamble at al., 2017; Mezzasalma et al., 2019). This makes the suborder Serpentes, and more in general the whole order Squamata, which also includes various taxa with temperature-dependent sex determination (Gamble, 2010; Gamble et al., 2017; Pallotta et al. 2017; Alam et al., 2018), a unique study system to analyze the evolu- tion and diversification of different mechanisms of sex determination. Nevertheless, in the family Colubridae, the fourth macrochromosome pair is usually composed of the ZW elements: the Z is metacentric and conserved in most species, while the W is often heteromorphic compared to the Z and largely heterochromatic (see e.g., Mengden, 1981; Mezzasalma et al., 2015a; Rovat- sos et al., 2015; Matsubara et al., 2016). In C. austriaca, the studied Greek and Italian samples display a different morphology of the W chromosome, resulting submeta- centric and subtelocentric, respectively. In the studied Italian female, the W chromosome is completely hetero- chromatic but not evidenced with fluorochromes (DAPI and CMA3). The lack of data on the chromatin compo- sition and distribution of the W chromosome from the Greek female here studied, does not allow us to establish if the differences between the W chromosomes of the Italian and the Greek sample also concern the hetero- chromatin pattern. Nevertheless, the different W mor- phology here found among Greek and Italian samples of C. austriaca highlight the occurrence of a karyologi- cal diversification among different clades of the species (Balkan and Western 2, see Fig. 1 and Jablonski et al. 2019), further supporting that the species contains dif- ferent diverging evolutionary lineages. From a biogeo- graphic point of view, as already documented for other Palearctic reptiles, the Quaternary climatic oscillations had an important role in shaping the current diversity of extant species, mainly through the contraction and re-expansion of their distribution ranges and the isola- tion of populations in different “refugia within refugia” (see, e.g., Ursenbacher et al., 2008; Gvoždík et al., 2010; Kindler et al., 2013; Mezzasalma et al., 2018). Unstable climatic conditions, fragmentation of the distribution range, small population size and isolation in distinct glacial refugia are also particularly favorable conditions for the fixation of chromosome mutations (see also Mez- zasalma et al., 2015a, 2017a for similar examples in dif- ferent amphibian and reptile species), which may happen in different populations independently. In conclusion, this paper provides the first record of a ZZ/ZW sex chromosome system in C. austriaca, with the occurrence of different morphologies of the W chro- mosome in different clades (Western and Balkan). More inclusive molecular and cytogenetic data from other areas of the wide distribution of C. austriaca would be useful to characterize the chromosome variability of different molecular clades the European smooth snake, helping to better assess their taxonomy. AKNOWLEDGEMENTS This research would not have been possible without the collaboration of the former Dipartimento di Biologia Evolutiva e Comparata of the Università degli Studi di Napoli Federico II which hosted several preserved cell suspensions amphibians and reptiles and provided us with the study samples. REFERENCES Alam, S.M.I., Sarre, S.D., Gleeson, D., Georges, A., Ezaz.T. (2018): Did lizards follow unique pathways in sex chromosome evolution? Genes 9: 239. Aprea, G., Gentilli, A., Zuffi, M.A.L., Odierna, G. (2006): The karyology of Vipera aspis, V. atra, V. hugyi, and Cerastes vipera. Amphibia-Reptilia 27: 113-119. Darriba, D., Taboada, G. L., Doallo, R., Posada, D. (2012): jModelTest 2: more models, new heuristics and paral- lel computing. Nat. Met. 9: 772. Engelmann, W.E. (1993): Coronella austriaca (Lauren- ti,1768) – Schilngatter, Glatt – oderHaselnatter. In: Handbuchder Reptilien und AmphibienEuropas, p. 200-245. Böhme, W., Ed., Aula-Verlag, Wiesbaden. Fuller, Z.L., Leonard, C.J., Young, R.E., Schaeffer, S.W., Phadnis, N. (2018): Ancestral polymorphisms explain the role of chromosomal inversions in speciation. PLoS Genet. 14: e1007526. Galarza, J.A., Mappes, J., Valkonen, J.K. (2015): Biogeog- raphy of the smooth snake (Coronella austriaca): ori- 43Karyological diversification in the smooth snake gin and conservation of the northernmost population. Biol.J. Linn. Soc. 114: 426-435. Gamble, T. (2010): A review of sex determining mecha- nisms in geckos (Gekkota: Squamata). Sex Dev. 4: 88-103. Gamble, T., Castoe, T.A., Nielsen, S.V., Banks J.L., Card, D.C., Schield D.R., Schuett. W.G, Booth, W. (2017): The discovery of XY sex chromosomes in a Boa and Python. Curr. Biol. 27: 2148-2153. Gorman, G.C., Gress F. (1970): Chromosome cytology of four boid snakes and a varanid lizard, with comments on the cytosystematics of primitive snakes. Herpeto- logica 26: 308-317. Gvoždík, V., Jandzik, D., Lymberakis, P., Jablonski, D., Moravec, J. (2010): Slow worm, Anguis fragilis (Reptil- ia: Anguidae) as a species complex: genetic structure reveals deep divergences. Mol. Phylogenet. Evol. 55: 460-472. Hall, T.A. (1999): BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41: 95-98. Howell, W.M. (1977): Visualization of ribosomal gene activity: silver stain proteins associated with RNA transcribed from oocyte chromosomes. Chromosoma 62: 361-367. Howell, W.M., Black, D.A., (1980): Controlled silver staining of nucleolus organizer regions with a protec- tive colloidal developer: 1-step method. Experientia 36: 1014-1015, Jablonski, D., Nagy, Z.T., Avci, A., Olgun, K., Kukushkin, O.V., Safaei-Mahroo, B., Jandzik D. (2019): Cryptic diversity in the smooth snake (Coronella austriaca). Amphibia-Reptilia 40: 179-192. Jiménez, R., Burgos, M., Diaz de la Guardia, R. (1988): A study of the silver staining significance in mitotic NORs. Heredity 60: 125-127. Kindler, C., Böhme, W., Corti, C., Gvoždík, V., Jablonski, D., Jandzik, D., Metallinou, M., Široký, P., Fritz, U. (2013): Mitochondrial phylogeography, contact zones and taxonomy of grass snakes (Natrix natrix, N. meg- alocephala). Zool. Scripta 42: 458-472. Kobel, H.R. (1967). Morphometrische Karyotyp analyse einiger Schlangenarten. Genetica 38: 1-31. Llorente, G.A., Vidal-García, M., Garriga, N., Carranza, S., Pleguezuelos, J.M., Santos, X. (2012): Lessons from a complex biogeographical scenario morphological characters match mitochondrial lineages within Ibe- rian Coronella austriaca (Reptilia, Colubridae). Biol. J. Linn. Soc. 106: 210-223. Matsubara, K., Nishida, C., Matsuda, Y., Kumazawa, Y. (2016): Sex chromosome evolution in snakes inferred from divergence patterns of two gametologous genes and chromosome distribution of sex chromosome- linked repetitive sequences. Zool. Lett. 2: 19. Matthey, R. (1931): Chromosomes de Reptiles, Sauriens, Ophidiens, Cheloniens. L’evolution de la formule chromosomiale chez les Sauriens. Rev. Suisse Zool., 38: 117-186. Mengden, G.A. (1981): Linear differentiation of the C-band pattern of the W chromosome in snakes and birds. Chromosoma 83: 275-287 Mezzasalma, M., Andreone F., Branch W. R., Glaw F., Guarino F. M., Nagy Z. T., Odierna G., Aprea G. (2014): Chromosome evolution in pseudoxyrhophiine snakes from Madagascar: a wide range of karyotypic variability. Biol. J. Linn. Soc. 112: 450-460. Mezzasalma, M., Andreone, F., Glaw, F., Petraccioli A., Odierna G., Guarino, F. M. (2016b): A karyologi- cal study of three typhlopid species with some infer- ences on chromosome evolution in blindsnakes (Scolecophidia). Zool. Anz. 264: 34-40. Mezzasalma, M., Andreone, F., Glaw F., Guarino, F. M., Odierna G., Petraccioli A., Picariello, O. (2019): Changes in heterochromatin content and ancient chromosome fusions in the endemic Malagasy boid snakes Sanzinia and Acrantophis (Squamata: Ser- pentes). Salamandra 55: 140-144. Mezzasalma, M., Andreone F., Aprea, G., Glaw, F., Odi- erna, G., Guarino, F. M. (2017a): When can chro- mosomes drive speciation? The peculiar case of the Malagasy tomato frogs (genus Dyscophus). Zool. Anz. 268: 41-46. Mezzasalma, M., Andreone, F., Aprea, G., Glaw, F., Odi- erna, G., Guarino, F.M. (2017b). Molecular phylogeny, biogeography and chromosome evolution of Malagasy dwarf geckos of the genus Lygodactylus (Squamata, Gekkonidae). Zool. Scripta 46: 42-54. Mezzasalma, M., Dall’Asta, A., Cheylan, M., Loy, A., Zuffi, M.A.L., Lymberakis, P., Tomovìc, L., Odierna, G., Guarino, F.M. (2015a): A sisters’ story: compara- tive phylogeography and taxonomy of Hierophis vir- idiflavus and H. gemonensis (Serpentes, Colubridae). Zool. Scr. 44: 495-508. Mezzasalma, M., Di Febbraro, M., Guarino, F.M., Odi- erna, G., Russo, D. (2018): Cold-blooded in the Ice Age: “refugia within refugia”, inter-and intraspecific biogeographic diversification of European whipsnakes (Squamata, Colubridae, Hierophis). Zoology 127: 84-94. Mezzasalma, M., Glaw, F., Odierna, G., Petraccioli, A., Guarino, F.M. (2015b). Karyological analyses of Pseudhymenochirus merlini and Hymenochirus boett- geri provide new insights into the chromosome evo- 44 M. Mezzasalma, G. Odierna lution in the anuran family Pipidae. Zool. Anz. 258: 47-53. Mezzasalma, M., Guarino, F.M., Aprea, G., Petraccioli, A., Crottini, A., Odierna, G. (2013): Karyological evi- dence for diversification of Italian slow worm popu- lations (Squamata, Anguidae). Comp. Cytogenet. 7: 217-227. Mezzasalma, M., Visone, V., Petraccioli, A., Odierna, G., Capriglione, T., Guarino, F.M. (2016a): Non-random accumulation of LINE1-like sequences on differenti- ated snake W chromosomes. J. Zool. 300: 67-75. Nagy, Z.T., Lawson, R., Joger, U., Wink, M. (2004): Molecular systematics of racers, whipsnakes and rela- tives (Reptilia: Colubridae) using mitochondrial and nuclear markers. J. Zool. Syst. Evol. Res. 42: 223-233. Odierna, G., Olmo, E., Cobror, O. (1987): Taxonomic Implications in Lacertid Lizards of NOR-Localization. Amphibìa-Reptilia 8: 373-382. Oguiura, N., Ferrarezzi, H., Batistic, R. F. (2009): Cytoge- netics and molecular data in snakes: a phylogenetic approach. Cytogenet. Genome Res. 127: 128-142. Olmo, E. (1986): Reptilia. In: Animal Cytogenetics, Vol. 4, pp. 1-100. John, B., Ed, Gebrüder Borntraeger, Ber- lin, Stuttgart. Olmo, E., Signorino, G. (2006): Chromorep: a rep- tile chromosomes database. Internet references: http://193.206.118. 100/professori/chromorep.pdf, 15.09.06. [accessed on 20 Jan 2021] Pallotta, M.M., Turano, M., Ronca, R., Mezzasalma, M., Petraccioli, A., Odierna, G., Capriglione, T. (2017): Brain gene expression is influenced by incubation temperature during leopard gecko (Eublepharis macu- larius) development. J. Exp. Zool. B Mol. Dev. Evol. 328: 360-370. Porter, C.A, Hamilton, M.J., Sites, J.W., Baker, R.J. (1991): Location of ribosomal DNA in chromosomes of squa- mate reptiles: Systematics and evolutionary implica- tions. Herpetologica 47: 271-280. Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D. L., Darling, A., Hohna, S., Larget, B., Liu, L., Suchard, M. A., Huelsenbeck, J. P. (2012): MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 61: 539-542. Rovatsos, M., Vukić, J., Lymberakis, P., Kratochvíl, L. (2015): Evolutionary stability of sex chromosomes in snakes. Proc. R. Soc. B. 282: 20151992. Sambrook, J., Fritsch, E.F., Maniatis, T. (1989): Molecu- lar cloning: a laboratory manual. 2nd edition. Cold Spring Harbor Lab Press, New York, 1626 pp. Santos, X., Rato, C., Carranza, S., Carretero, M.A., Ple- guezuelos, J.M. (2012): Complex phylogeography in the southern smooth snake (Coronella girondica) sup- ported by mtDNA sequences. J. Zool. Syst. Evol. Res. 50: 210-219. 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 Pleistocene range expansions. Amphibia-Reptilia 29: 35-47. Sidhom, M., Said, K., Chatti, N., Guarino, F.M., Odier- na, G., Petraccioli, A., Picariello, O., Mezzasalma, M. (2020a): Karyological characterization of the common chameleon (Chamaeleo chamaeleon) provides insights on the evolution and diversification of sex chromo- somes in Chamaeleonidae. Zoology 141: 125738. Sidhom, M., Said, K., Chatti, N., Guarino, F.M., Odier- na, G., Petraccioli, A., Picariello, O., Mezzasalma, M. (2020b): Karyological and bioinformatic data on the common chameleon Chamaeleo chamaeleon. Data in Brief 30: 105640. Singh, L. (1972): Evolution of Karyotypes in Snakes. Chromosoma 38: 185-236. Speybroeck, J., Beukema, W., Bok, B., Van Der Voort, J., Velikov, I. (2016): Field Guide to the Amphibians and Reptiles of Britain and Europe. Bloomsbury Publish- ing. Strijbosch, H. (1997): Coronella austriaca Laurenti, 1768. In: Atlas of Amphibians and Reptiles in europe, p. 344-345, Gasc, J.P., Cabela, A., Crnobrja-Isailovic, J., Dolmen,D., Grossenbachner, K., Haffner, P., Les- cure, J., Martens, H., Martínez Rica, J.P., Maurin, H., Oliveira, M.E., Sofiandou, T.S., Veith, M., Zuiderwijk, A., Eds, Societas Europea Herpetologica & Muséum Nationald’Historie Naturelle (IEGB/SPN), Paris. Sumner, A.T. (1972): A simple technique for demonstrat- ing centromeric heterochromatin. Exp. Cell Res. 75: 304-306. Ursenbacher, S., Schweiger, S., Tomović, L., Crnobrnja- Isailović, J., Fumagalli, L., Mayer, W. (2008): Molecu- lar phylogeography of the nose-horned viper (Vipera ammodytes, Linnaeus (1758)): evidence for high genetic diversity and multiple refugia in the Balkan peninsula. Mol. Phylogenet. Evol. 46: 1116-1128. Utiger, U., Helfenberger, N., Schätti, B., Schmidt, C., Ruf, M., Ziswiler, V. (2002): Molecular systematics and phylogeny of old and new world ratsnakes, Elaphe Auct., and related genera (Reptilia, Squamata, Colu- bridae). Russ. J. Herpetol. 9: 105-124.