Comparative cytogenetics on Zamenis lineatus and Elaphe quatuorlineata (Serpentes: Colubridae)

From Firenze University Press Journal: Acta Herpetologica

University of Florence
3 min readMar 17


Marcello Mezzasalma, Department of Biology, Ecology and Earth Science, University of Calabria

Elvira Brunelli, Department of Biology, Ecology and Earth Science, University of Calabria

Gaetano Odierna, Department of Biology, University of Naples Federico II

Fabio M. Guarino, Department of Biology, University of Naples Federico II

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 located 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 tive chromosome analyses can be useful to identify plesiomorphic 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 evolution, 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 variability 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 heterogamety (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 chromosome rearrangements and different morphologies and/or levels of heterochromatinization of the heteromorphic sex chromosomes have been previously used in different 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 cytogenetic analysis on E. quatuorlineata and Z. lineatus, using a combination of standard staining and banding techniques. 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 distribution of primitive and derivate chromosomal characters in the species studied and provide cytotaxonomic insights, which support the species status of Z. lineatus. We also highlight that both genera Elaphe and Zamenisshow progressive evolutionary stages of the W chromo-some, supporting the heterochromatinization model of sex chromosome diversification (see e.g., Mezzasalma et al., 2021).


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