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Because of their peculiar genomic and chromosomal characteristics, reptiles are extraordinary model organisms to study karyotype and sex chromosome evolution, but despite the growing interest in their evolutionary cytogenetics, 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 karyotyping, C-banding, sequential C-banding + CMA3 + DAPI and Ag-NOR staining. On E. quatuorlineata, we also performed 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 species 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 chromosome as already shown in different reptile taxa.

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 geographical 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 macrochromosomes 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.

The genus Matoatoa includes two Malagasy endemic species, M. brevipes and M. spannringi. Due to their cryptic behaviour, the two species are known only from a handful of specimens and have been included in few molecular studies. Here we carried out a molecular barcoding analysis using a fragment of the mitochondrial NADH dehydrogenase subunit 2 (ND2) and the first chromosomal analysis of M. brevipes. The molecular analysis confirmed the identity of the studied samples as M. brevipes. However, the level of genetic divergence (4% uncorrected p-distance) between our samples and other sequences of M. brevipes, suggests previously unrecognised diversity within the species. The karyotype of M. brevipes is composed of 2n = 34 chromosomes: the first pair is metacentric, while all the other pairs are telocentric and gradually decreasing in length (Arm Number, AN = 36). C-banding revealed little evidence of centromeric heterochromatin, while NOR-associated heterochromatin was found on the telomeres of a medium sized telocentric pair. No heteromorphic chromosome pairs were found in the karyotype of the species, suggesting that putative sex chromosomes are at an early stage of differentiation. Karyological comparisons with closely related species were performed with Christinus marmoratus, and representatives of the genera Phelsuma, Ebenavia, Paroedura and Uroplatus. Comparisons across genera suggest that chromosome diversification in this group of geckos probably occurred by means of chromosome fusions and inversions, leading to a reduction of the chromosome number and the formation of biarmed elements in different species.