Acta Herpetologica 17(2): 197-203, 2022 ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah DOI: 10.36253/a_h-13467 Comparative cytogenetics on Zamenis lineatus and Elaphe quatuorlineata (Serpentes: Colubridae) Marcello Mezzasalma1,*, Elvira Brunelli1, Gaetano Odierna2, Fabio M. Guarino2 1 Department of Biology, Ecology and Earth Science, University of Calabria, Via P. Bucci 4/B, 87036 Rende, Italy 2 Department of Biology, University of Naples Federico II, Via Cinthia 26, 80126, Naples, Italy *Corresponding author. Email: m.mezzasalma@gmail.com Submitted on: 2022, 28th July; revised on: 2022, 21st October; accepted on: 2022, 5th November Editor: Andrea Villa Abstract. Because of their peculiar genomic and chromosomal characteristics, reptiles are extraordinary model organ- isms to study karyotype and sex chromosome evolution, but despite the growing interest in their evolutionary cytoge- netics, only a small fraction of species have a known karyotype. We performed a comparative cytogenetic analysis on Elaphe quatuorlineata and Zamenis lineatus, using classic and molecular techniques. We provide the karyotype of these two species and an assessment of their chromosomal features. Chromosome analysis was performed with standard kar- yotyping, C-banding, sequential C-banding + CMA3 + DAPI and Ag-NOR staining. On E. quatuorlineata, we also per- formed CMA3-methyl green staining and Fluorescence in situ Hybridization mapping NOR loci (NOR-FISH). Elaphe quatuorlineata and Z. lineatus show a very similar karyotype of 2n = 36, with 8 macro- and 10 microchromosome pairs, but differ in the morphology of the pair 8, which resulted submetacentric in the former and metacentric in the latter species. By comparing our data to those available from the literature on congeneric species, we analysed the occurrence of primitive and derivate chromosomal characters and provide cytotaxonomic insights, which further support the spe- cies status of Z. lineatus. In both species, the 4th pair was identified as the sex chromosome pair (ZZ/ZW) and NORs were localized on a microchromosome pair. We finally highlight in both genera Elaphe and Zamenis different stages of heterochromatinization of the W chromosome, in agreement with the progressive diversification model of sex chromo- some as already shown in different reptile taxa. Keywords. Chromosome, evolution, karyotype, NORs, squamates, snakes. INTRODUCTION Classic cytogenetic through differential staining and banding of chromosomes permits to describe and compare karyotypes, whereas the molecular cytogenetic approach, employing Fluorescence in situ Hybridiza- tion (FISH) with specific probes, allows the detection of particular sequences present in genomes (Matsuda et al., 2005; Dumas and Sineo, 2014), including repetitive DNA sequences (Scardino et al., 2020a). Among those repeti- tive DNA elements are the ribosomal DNA (rDNA), encoding rRNA. These elements have been successfully used as markers for comparative cytogenetic studies and phylogenetic analyses. The rDNA is organized into 2 fam- ilies: 5S (minor) and 45S (major) rDNA. The latter com- prises the genes for 18S, 5.8S, and 28S rRNA and is locat- ed in the so-called nucleolus organizer regions (NORs). The NORs can be identified either by silver staining, which detects only transcriptionally active loci, or more accurately, by FISH, which permits the identification of both active and inactive NORs. The location of the rDNA loci in the karyotype may show a species-specific pattern, so rDNA loci are often used for complex karyotype char- acterizations (Scardino et al., 2020a). Indeed, compara- 198 Marcello Mezzasalma et alii tive chromosome analyses can be useful to identify ple- siomorphic and apomorphic states and the occurrence of different evolutionary lineages (Deakin and Ezaz, 2014; Damas et al., 2018; Scardino et al., 2020b). Chromosome rearrangements may precede or follow molecular evolu- tion, directly promoting cladogenesis or resulting from phylogenetic diversification (Noor et al., 2001; Rieseberg, 2001). In either case, they represent discrete evolutionary markers able to detect different evolutionary trends or apomorphisms in the taxa studied (Dobigny et al., 2004; Olmo, 2008; Dumas et al., 2015). Squamate reptiles, due to their peculiar genomic and chromosomal characteristics, are exceptional model organisms in the study of karyotype evolution and sex chromosome diversification of vertebrates (Olmo, 2008; Alam et al., 2018). Squamates display a remarkable vari- ability in chromosome number and morphology, number and location of different chromosome markers and the occurrence of environmental genetic sex determination, with the independent evolution of simple and multiple sex chromosome systems with either male or female het- erogamety (Olmo, 2008; Pallotta et al., 2017; Deakin and Ezaz, 2019; Sidhom et al., 2020; Mezzasalma et al., 2021 a). The cytogenetic approach used for the study of chro- mosome rearrangements and different morphologies and/ or levels of heterochromatinization of the heteromorphic sex chromosomes have been previously used in differ- ent phylogenetically closely-related European squamate taxa such as the snakes of the genus Hierophis (Fitzinger, 1843), Anguis fragilis Linnaeus, 1758, and A. veronen- sis Pollini, 1818, and geographically distinct populations of Coronella austriaca Laurenti, 1768 (Mezzasalma et al., 2013, 2015, 2018b; Mezzasalma and Odierna, 2021). However, despite the growing interest in the evolutionary cytogenetics of squamate reptiles, only a small fraction of the described squamate species have a known karyotype (Olmo and Signorino, 2006; Mezzasalma et al., 2021), leaving most of their chromosomal diversity still unex- plored. This is also true for some peculiar Mediterrane- an reptile species such as the European four-lined snake Elaphe quatuorlineata (Bonnaterre, 1790) and the Italian Aesculapian snake Zamenis lineatus (Camerano, 1891). In this work, we performed a comparative cytoge- netic analysis on E. quatuorlineata and Z. lineatus, using a combination of standard staining and banding tech- niques. We provide the first karyotype description of these two species and an assessment of their chromo- somal features. By comparing our data to those available from the literature on phylogenetically closely-related species, we evidence and discuss the occurrence and dis- tribution of primitive and derivate chromosomal char- acters in the species studied and provide cytotaxonomic insights, which support the species status of Z. lineatus. We also highlight that both genera Elaphe and Zamenis show progressive evolutionary stages of the W chromo- some, supporting the heterochromatinization model of sex chromosome diversification (see e.g., Mezzasalma et al., 2021). MATERIAL AND METHODS We analysed two samples (one male and one female) of Z. lineatus and one female of E quatuorlineata from Piedimonte Matese, Campania, Italy. Specimens were anesthetized on ice and, after taking a 0.5 ml of blood aliquot from the caudal vein, they were released in the capture site. Chromosomes were obtained from blood cultures following Odierna et al. (2004). Namely, blood aliquots were incubated for four days at 30 °C in 5 ml of lymphocyte medium culture (3.8 ml of DMEM, 0.5 ml sterile distilled water, 0.5 newborn calf serum, 0.1 ml antibiotics, 0.1 ml PHA). Chromosome harvesting was performed by adding 0.1 ml of Colcemid (10 μg/ml) and two hours later the cells were collected by centrifugation (1000 rpm/min), incubated for 30 min in 5 ml of hypo- tonic solution (KCl 0.075 M) and fixed in methanolacetic liquid (methyl alcohol + acetic acid, 3:1). Slides were pre- pared using the air-drying method, as described in Mez- zasalma et al. (2019). The cytogenetic analysis was per- formed with traditional karyotyping (5% Giemsa solution at pH 7 for 10 min) and additional chromosome stain- ing and banding techniques; in particular, C-banding was performed following Sumner (1972) and sequential C-banding + CMA3 + DAPI according to Sidhom et al. (2020), which highligh CG and AT-rich regions, respec- tively. Nucleolus organizing regions (NORs) were identi- fied following the Ag-NOR staining method described by Howell and Black (1980). Given quantity and quality of metaphase plates, on E. quatuorlineata we also performed Chromomycin A3-methyl green staining (CMA/MG) (a staining method usefulf to hightlight CG-rich chromo- some regions) as described by Sahar and Latt (1980) and Fluorescence in situ Hybridization (NOR-FISH) follow- ing Mezzasalma et al. (2018a), using as probe the PCR- amplified and biotinylated 18S rRNA gene of the gekko- nid Tarentola mauritanica (Linnaeus, 1758). In brief, after denaturation in 70% formamide and 2x SSC for 2 min at 80 °C, slides were incubated overnight at 40 °C with the hybridization mixture (10 ng/ml biotinylated 16 dUTP probe 0.1 µm/ml Escherichia coli DNA in 50% forma- mide and 2x SSC). After washing in 2x SSC, cytochemi- cal detection was performed using 5 µm/ml FITC-con- jugated ExtrAvidin (Sigma) in 4x SSC + 1% BSA + 0.1% 199Karyology of European colubrids Tween 20, pH 7. After washing three times in 4x SSC and 0.1% Tween 20 for 10 min at 42 °C, the detection of FISH signals was performed with ExtrAvidin FITC (Sigma Aldrich) counterstained with propidium iodide (PI) (200 ng/ml) in 2x SSC, pH 7, for 2 min at room temperature. Metaphase plates were scored and recorded with an opti- cal and an epifluorescent microscope (Axioscope Zeiss) equipped with an image analysis system. Karyotype reconstruction was performed after scoring at least five metaphase plates from each sample studied and chromo- somes were classified according to Levan et al. (1964). RESULTS The karyotypes of E. quatuorlineata and Z. linea- tus are both composed of 2n = 36 chromosomes, with 8 macrochromosome pairs and 10 microchromosome pairs (Fig. 1A, 2A). The two species also show the same chro- mosome morphology with the exception of the chromo- some pair 8, which resulted submetacentric in E quator- lineata and metacentric in Z. lineatus (Table 1, Fig. 2A). Arm number (AN) resulted = 50 in both colubrids. Mor- phometric parameters of each macrochromosome pair of both species studied are reported in Table 1. C-banding and Ag-NOR revealed in both species the occurrence of NOR loci on a microchromosome pair, as confirmed by NOR-FISH in E. quatuorlineata (Fig. 1B-D). C-banding showed heterochromatin content on autosomes, mostly concerning telomeric and centromer- ic regions in both E. quatuorlineata (Fig. 1E-G) and Z. lineatus (Fig. 2B-D). Furthermore, in the female samples of both species, one element of the 4th macrochromo- some pair resulted to be largely heterochromatic, allow- ing us to identify this pair as a homomorphic ZW sex chromosome pair (Fig. 1E-G, 2B-D). This W chromo- some resulted highly positive with both CMA3 and DAPI in E. quatuorlineata (Fig. 1E-G), whereas it was clearly evident with DAPI and less evident with CMA3 in Z. lin- eatus (Fig. 2B-D). Fig. 1. Karyotype and metaphase plates of E. quatuorlineata stained with Giemsa (A), Ag-NOR (B), CMA3/MG (C), NOR-FISH (D), C-banding + Giemsa (E), + CMA3 (F), + DAPI (G). * = loci of NORs. Fig. 2. Karyotype and metaphase plates of Z. lineatus stained with Giemsa (A), Ag-NOR (B), C-banding + CMA3 (C), + DAPI (D). * = loci of NORs. Table 1. Chromosome morphometric parameters. Chr. = Chro- mosome number; RL = Relative length (Chromosome length/total karyotype length*100); CI = Centromeric index (short arm length/ chromosome length*100); m = metacentric; sm = submetacentric; t = telocentric. Chr. Elaphe quatuorlineata Zamenis lineatus RL CI RL CI 1 19.4 ± 0.8 44.9 ± 3.4 (m) 19.3 ± 1.0 48.4 ± 3.4 (m) 2 16.5 ± 0.5 34.8 ± 4.0 (sm) 16.1 ± 0.7 35.2 ± 4.0 (sm) 3 11.1 ± 0.5 48.4± 3.8 (m) 10.8 ± 0.4 43.2 ± 3,8 (m) 4(Z) 7.7 ± 0.6 45.9 ± 2.8 (m) 8.3 ± 0.6 45.4 ± 2.8 (m) 4(W) 7.6 ± 0.4 45.1 ± 3.4 (m) 8.4 ± 0,3 44.9 ± 3.4 (m) 5 7.5 ± 0.7 48.3 ± 4.3 (m) 7.9 ± 0.5 42.8 ± 4.3 (m) 6 6.0 ± 0.4 0.0 ± 3.0 (t) 7.1 ± 0.6 0.0 ± 3.0 (t) 7 5.6 ± 0.7 36.0 ± 3.1 (sm) 6.3 ± 0.5 36.3 ± 3.1(sm) 8 5.3 ± 0.4 36.9 ± 3.3 (sm) 4.4 ± 0.6 40.1 ± 3.3 (m) 9-18 20.6 ± 1.1 19.8 ± 1.3 200 Marcello Mezzasalma et alii DISCUSSION Our results show that the karyotypes of E. quatuor- lineata and Z. lineatus have the same diploid number (2n = 36) and a similar general structure, but a different mor- phology of chromosome pair 8. In order to highlight the occurrence of simplesio- morphic, sinapomorphic and apomorphic states and add data for the reconstruction of the chromosomal evolu- tion in the genera Elaphe and Zamenis, we compared the newly generated karyotypes to those available from the literature on congeneric species as well as with that of the hypothesized Ancestral Snake Karyotype (ASK) (see Kobel, 1967; Bianchi et al., 1969; Singh, 1972; Itoh et al., 1970; De Smet, 1978; Augstenová et al., 2017; Rovatsos et al., 2018; Cole and Hardy, 2019) (Fig. 3). This comparison permits to show that E. quatuor- lineata and Z. lineatus, as well as most congeneric spe- cies with a known karyotype, have different chromosomal characters which are considered simplesiomorphisms and found in the hypothesized ASK (see Kobel, 1967; Bianchi et al., 1969; Singh, 1972; Itoh et al., 1970; De Smet, 1978; Augstenová et al., 2017; Rovatsos et al., 2018; Cole and Hardy, 2019; this paper). These shared ancestral char- acters include: diploid number, number of macro- and microchromosome pairs, the general morphology of several macrochromosome pairs and the localization of NOR loci on a microchromosome pair (see also Cole and Hardy, 2019). All this evidence permits to confirm that Elaphe and Zamenis are karyologically very conservative, but for the morphology and sequence content of the W chromosome, which are variable among different taxa (see also Augstenová et al., 2017; Cole and Hardy, 2019; Mezzasalma and Odierna, 2021). Nevertheless, the karyotypes of E. quatuorlineata and Z. lineatus also possess some peculiar derivate features, which characterize their respective karyotype from those of phylogenetically related species. In Elaphe, autosomal Fig. 3. Original karyograms of Z. lineatus and E. quatuorlineata compared with the Ancestral Snake Karyotype (ASK) and available litera- ture data on congeneric species (Kobel, 1967; De Smet, 1978; Itoh et al., 1970; Augstenová et al., 2017; Rovatsos et al., 2018; Cole and Hardy, 2019; Mezzasalma and Odierna, 2021). sc = secondary constriction, (I) = chromosome inversion. Red chromosomes = NOR-bearing pair. Black regions/chromosomes = heterochromatin. Red arrows indicate progressive steps of sex chromosome diversification. 201Karyology of European colubrids rearrangements from the hypothesized ancestral snake karyotype involve a putative inversion of the 8th pair in E. quatuorlineata and in E. bimaculata that can be consid- ered a sinapomorphism, and in the second species, also an inversion of the 7th pair, as previously showed (see Fig. 3) (Itoh et al., 1970; Rovatsos et al., 2018), which can be considered an apomorphism. Furthermore a translocation of loci of NORs on the 2nd chromosome pair in E. climacophora and E. quadri- virgata, evidenced by a secondary constriction (see Itoh et al., 1970), represent a further apomorphism, which is not present in the species here analyzed. Zamenis lineatus shows a metacentric 8th chromosome pair, which prob- ably originated by means of a pericentromeric inversion as previously showed also in Z. situla (Augstenová et al. 2017), thus representing a sinapomorphism linking the two species. It should also be noted that in the previously described karyotypes of Z. longissimus (Kobel 1967; De Smet, 1978), a different macrochromosome number (8 and 9, respectively) is reported, without any changes in the total chromosome count (2n = 36). The additional macrochromosome pair reported by De Smet (1978) is probably due to the amplification of NOR-linked hetero- cromatin of the NOR microchromosomes bearing pair, but more focused analyses are needed to confirm the occurrence of intraspecific chromosomal variability in Z. longissimus. Progressive steps of the configuration of the hetero- gametic W chromosome are important events in reptiles and are clearly visible in many species, supporting the general heterochromatinization hypothesis of sex chro- mosome diversification (Augstenová et al., 2017; Alam et al., 2018; Cole and Hardy 2019; Mezzasalma et al., 2020). In fact, it is widely accepted that heteromorphic sex chro- mosome pairs begin their morphological and molecu- lar diversification starting from a homomorphic state (Gamble et al., 2014; Mezzasalma et al., 2021). From this condition, two alternative pathways are known to poten- tially lead to a fully differentiated sex chromosome pair: a progressive heterochromatinization of the heterogamet- ic chromosome or the insurgence of an inversion in the homomorphic proto-W chromosome (Wright et al., 2016; Natri et al., 2019; Mezzasalma et al. 2021). In either cases, the progressive diversification of the W element eventu- ally leads to its evolutionary isolation (loss of recombi- nation) and degeneration, finally reaching the size of a microchromosome (Marshall Graves, 2016; Mezzasalma et al., 2016; Wright et al., 2016). Progressive steps of the configuration of the het- erogametic W chromosome are visible in the species here analysed and in the phylogenetically closely-related taxa Elaphe and Zamenis (Fig. 3) (see also Kobel, 1967; De Smet, 1978; Itoh et al. 1970; Augstenová et al. 2017; Rovatsos et al., 2018; Mezzasalma and Odierna 2021). In particular, the W chromosome appears at a relatively ear- lier stage of diversification in E. quatuorlineata, in which it resulted largely heterochromatic, but homomorphic to the Z (this paper). More advanced diversification stages are represented by the W elements of E. bimaculata and E. climacophora, in which the morphology of the W chromosomes progressively diverged from the Z, reach- ing a telocentric configuration in E. quadrivirgata (Fig. 3) (see also Itoh et al., 1970; Rovatsos et al., 2018). The W chromosome is homomorphic but largely het- erochomatic in Z. lineatus, representing an initial diversi- fication step from the Z element (this paper). A progres- sive addition of heterochromatin may produce a hetero- gametic chromosome, which appears sensibily larger than the Z: a condition similar to that reported in Z. situla (Augstenová et al., 2017) (Fig. 3). Furthermore, it is possible to highlight that the dif- ferences in the morphology of the W chromosome and of the 8th and 9th chromosome pairs found between Z. lineatus and Z. longissimus (Kobel 1967; De Smet, 1978; this paper) are in agreement with their species status, originally proposed using a combination of morphologi- cal and molecular data (Lenk and Wüster, 1999; Utiger et al., 2002). This evidence underlines that in squamate reptiles the cytotaxonomic approach is a useful tool for charac- terizing closely-related lineages as already shown in oth- er squamate taxa (Mezzasalma et al., 2013, 2015, 2018b; Mezzasalma and Odierna, 2021). ACKNOWLEDGEMENTS Sampling was carried out under the authorization of the 01/06/2000 n. SCN/2D/2000/9213 from the Italian Ministry of Environment. REFERENCES Alam, S.M., Sarre, S.D., Gleeson, D., Georges, A., Ezaz, T. (2018): Did lizards follow unique pathways in sex chromosome evolution? Genes 9: 239. 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Herpetol. 9: 105-124. Wright, A.E., Dean, R., Zimmer, F., Mank, J.E. (2016): How to make a sex chromosome. Nat. Commun. 7: 12087. 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