Comparative cytogenetics of Hemorrhois hippocrepis and Malpolon monspessulanus highlights divergent karyotypes in Colubridae and Psammophiidae (Squamata: Serpentes)

Abstract Despite the growing interest in the evolutionary cytogenetics of squamates, chromosomal data are lacking for most taxa. We performed a preliminary molecular taxonomic analysis and a comparative cytogenetic study on Hemorrhois hippocrepis and Malpolon monspessulanus. We used a combination of standard karyotyping, Chromomycin A3/Methyl green staining, C-banding, Ag-NOR staining and NOR-FISH to provide the first karyotype description of H. hippocrepis and a re-description of the karyotype of M. monspessulanus, including chromosome markers, heterochromatin patterns and sex chromosome systems. Our results show that H. hippocrepis has 2 n = 36 chromosomes, with 16 macro- and 20 microchromosomes and NORs on the 6th pair. The 4th pair represents homomorphic (metacentric) ZW sex chromosomes, but the W chromosome is completely heterochromatic. Malpolon monspessulanus has 2 n = 44 chromosomes, with 20 macro- and 24 microchromosomes, NORs on the 6th telocentric pair. The 4th pair represents the sex chromosomes (ZZ/ZW), with a W chromosome smaller than the Z and completely heterochromatic. Comparing our cytogenetic data to those available from the literature, we note the occurrence and distribution of primitive and derived chromosomal characteristics and discuss the chromosome diversification in two snake clades belonging to Colubridae and Psammophiidae, respectively. We highlight that these two families followed different chromosome diversification pathways, characterised by a highly conserved karyotype structure in Colubridae and a higher chromosome variability in Psammophiidae, mostly driven by a progressive reduction of the chromosome number by means of chromosome fusions. We also provide cytotaxonomic insights supporting the distinction between M. monspessulanus and M. insignitus.


Introduction
Karyotype changes represent distinct markers that are helpful in detecting evolutionary trends or apomorphisms in the taxa studied (Noor et al. 2001;Rieseberg 2001;Dobigny et al. 2004;Olmo 2008).
Squamates display an extraordinary diversity in chromosome number and morphology, chromosome markers of varying number and location and in the occurrence of environmental and/or genetic sex determination, with the independent evolution of simple and multiple sex chromosome systems with either male or female heterogamety Augstenová et al. 2018;Pallotta et al. 2017;Petraccioli et al. 2019;Sidhom et al. 2020). These characteristics make squamates extraordinary model organisms to explore the karyotype evolution and the diversification of sex chromosome systems in vertebrates (Olmo 2008;Gamble 2010;Rovatsos et al. 2015;Augstenová et al. 2018;). In recent years, a growing number of studies have been performed to improve the knowledge on the chromosome evolution in squamates (Srikulnath et al. 2013;Rovatsos et al. 2015;Mezzasalma et al. 2016Mezzasalma et al. , 2020Sidhom et al. 2020;Kostmann et al. 2021). However, raw karyotype data are still lacking for the majority of the squamate species currently described and the karyological diversification of many groups remains scarcely investigated or completely unexplored (Olmo & Signorino 2005;).
Here, we performed a comparative cytogenetic analysis on the horseshoe whip snake Hemorrhois hippocrepis (Linnaeus, 1758) and the Montpellier snake Malpolon monspessulanus (Hermann, 1804). These two snake species have been largely studied with morphological and molecular methods (e.g. Utiger et al. 2002;Carranza et al. 2006;Machado et al. 2021). In contrast, no chromosome data are currently known for H. hippocrepis and the only available chromosome data on M. monspessulanus were reported by Matthey (1931). We employed a combination of different staining, banding techniques and molecular cytogenetics, providing the first karyotype description of H. hippocrepis, a redescription of the karyotype of M. monspessulanus. We describe heterochromatin patterns in these two species, the occurrence and the karyotype localization of chromosome markers as well as the possible presence of differentiated sex chromosome systems. By comparing our data to those available from the literature on phylogenetically closely related species, we also discuss the occurrence and distribution of primitive and derived chromosomal characteristics and propose a scenario for the chromosome diversification of two snake clades of Colubridae and Psammophiidae, respectively.

Sampling
We analysed preserved tissues and cell suspensions of one male and one female of H. hippocrepis (from Guelmin, Morocco) and two males and two females of M. monspessulanus (from Essaouria, Morocco) hosted and preserved at the Dipartimento di Biologia Evolutiva e Comparata, Università degli Studi di Napoli Federico II, since 1972 (sample numbers CA0204-CA0209). All samples were used in a preliminary molecular analysis and the comparative cytogenetic study as described below.

Molecular analysis
We performed a preliminary molecular analysis using a fragment of about 550 bp of the mitochondrial 16S rRNA gene (16S), a widely used molecular marker in phylogenetically related species (see e.g. Nagy et al. 2003;Carranza et al. 2004;Alencar et al. 2016;Simoes et al. 2016), in order to obtain an accurate taxonomic identification of the examined samples. DNA was extracted following Sambrook et al. (1989). For PCR amplifications, we used the same primers and parameters reported in Mezzasalma et al. (2022). Amplicons were sequenced on an automated sequencer ABI 377 (Applied Biosystems, Foster City, CA, USA) using BigDye Terminator 3.1 (ABI). Chromatograms were checked and manually edited using Chromas Lite 2.6.6 and BioEdit 7.2.6.1 (Hall 1999) and compared with available homologous traits deposited in public repositories. The newly determined sequences were deposited in GenBank (accession numbers: OQ255883-OQ255888). The H. hippocrepis and M. monspessulanus samples showed no intraspecific nucleotide differences in the 16S sequence and had an intraspecific identity score of 99.5% and 99.2%, respectively, with homologous sequences of Moroccan specimens deposited in GenBank (AY643350 and AY643354).

Cytogenetic analysis
Chromosomes were obtained from tissue samples and cell suspensions using the air-drying method, as described in Mezzasalma et al. (2019). The chromosome analysis was performed with standard karyotyping (5% Giemsa solution at pH 7 for 10 min) and different chromosome staining and banding methods. C-banding was performed following Sumner (1972) and sequential C-banding + CMA 3 + DAPI according to . Active nucleolus organizing regions (NORs) were identified following the Ag-NOR staining method described by Howell and Black (1980), Chromomycin A 3 -methyl green staining (CMA 3 /MG) as described in Sahar and Latt (1980) and Fluorescence In Situ Hybridization (FISH) following Sidhom et al. (2020), using as a probe the biotinylated 18S rRNA gene of Tarentola mauritanica (Linnaeus, 1758) amplified via PCR, in order to detect both active and inactive NORs. In brief, slides were dehydrated in ascending series of ethanol and, after denaturation in 70% formamide and 2x SSC for 2 min at 80°C, incubated overnight at 40°C with the hybridization mixture (10 ng/ml biotinylated 16 dUTP probe 0.1 µg/ ml Escherichia coli DNA in 50% formamide and 2x SSC). After hybridization slides were washed twice in 1x SSC at 75°C and room temperature for 5 min, respectively, and cytochemical detection was performed using 5 µg/ml FITC-conjugated ExtrAvidin (Sigma) in 4x SSC + 1% BSA + 0.1% Tween 20, pH 7. After washing three times in 4x SSC and 0.1% Tween 20 for 10 min at 42°C, the detection 202 M. Mezzasalma et al. 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. Slides were then mounted with antifade solution (Sigma-Aldrich) and metaphase plates were scored and recorded with an optical and an epifluorescent microscope (Axioscope Zeiss) equipped with an image analysis system. Karyogram reconstruction was performed after scoring at least 10 plates per sample and chromosomes were classified following Levan et al. (1964).

Results
The horseshoe whip snake, H. hippocrepis, showed a karyotype of 2 n = 36 chromosomes, with 16 macro-and 20 microchromosomes. Among macroautosomes, pairs 1, 3, 5 and 7 are metacentric, pairs 2 and 8 are submetacentric and pair 6 is telocentric (Arm Number, AN = 50) (Figure 1 Table I). The pair 4 corresponds to the ZZ/ZW sex chromosomes. Both the Z and the W chromosome are metacentric, but clearly distinguishable after C-banding. In fact, the Z is mostly euchromatic, showing only small centromeric and telomeric heterochromatic regions (negative to both fluorochromes), while the W is largely heterochromatic and strongly positive to CMA 3 and DAPI (Figure 1(f-h)). All macroautosomes showed telomeric, centromeric and additional peritelomeric heterochromatic spots on the elements of the pair 6, corresponding to the NOR-associated heterochromatin. In fact, Ag-NOR-, CMA 3 /MG-and NOR-FISH all showed the occurrence of NORs on the pericentromeric region of the elements of the pair 6 (Figure 1(c-e), respectively), which show a secondary constriction (Figure 1(a), (b)). All microchromosomes are mostly euchromatic (Figure 1(d), (f)).
After C-banding, almost all macrochromosome pairs showed peritelomeric and telomeric heterochromatin and NOR-associated heterochromatin, CMA 3 positive, was present on the telomeres of the chromosomes of the 6 th pair (Figure 2(f-h)). The microchromosomes are almost all euchromatic (Figure 2(f-h)). The Z chromosome showed a small centromeric C-band, negative to either CMA 3 and DAPI (Figure 2(f-h)), while the W appeared fully heterochromatic and highly positive to both fluorochromes (Figure 2(f-h)).
In particular, the two snake species here studied present a combination of primitive and derived chromosome characteristics which are particularly evident when their karyotypes are compared to phylogenetically closely related species and to the ASK. Here, we discuss the cytogenetic results on H. hippocrepis and M. monspessulanus in the light of available karyological data (Matthey 1931;Gorman 1973;Branch 1980;Aprea et al. 2003;Mezzasalma et al. 2015Mezzasalma et al. , 2018Abd Allah 2010; this study) and phylogenetic information (Pyron et al. 2013;Figueroa et al. 2016;Zaher et al. 2019) on closely related species. We highlight the occurrence and localization of different chromosomal states (number, morphology, heterochromatin distribution, and NOR localization) and rearrangements that can be considered plesiomorphic, synapomorphic and apomorphic in different taxa and describe an evolutionary scenario on the karyotype diversification in two clades of Colubridae and Psammophiidae, respectively (Figures 3 and 4).

Comparative cytogenetics of Mediterranean snakes 203
Hemorrhois hippocrepis belongs to a clade of Mediterranean colubrid whip-snakes that includes eight genera and only nine species of known karyotype ( Figure 3). The available karyological data suggest that the general configuration of the ASK is highly conserved in this clade in terms of total chromosome number, number of macro-and microchromosomes and morphology (2 n = 36 with 16 macro and 20 microchromosomes) (Matthey 1931;Gorman 1973;Mezzasalma et al. 2015; Branch

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M. Mezzasalma et al. 1980; Abd Allah 2010; this study) ( Figure 3). However, in species of different genera (H. hippocrepis, Platyceps rhodorachis, P. thomasi and Hierophis viridiflavus) NORs are located on the 6 th macrochromosome pair (see Figure 3). This condition represents a synapomorphy of this clade of whip-snakes, which probably originated through a translocation of ribosomal cistrons from a microchromosome pair of the ASK. Furthermore, interspecific differences in chromosome morphology highlight the presence of several chromosome rearrangements in the group.
In the genus Hemorrhois, H. nummifer, H. hippocrepis and H. algirus differ in the number of meta-, submetacentric, subtelocentric and telocentric autosomes (Abd Allah 2010; present paper), suggesting the occurrence of multiple chromosomal inversions (Figure 3). In addition, the homomorphic (metacentric) ZW sex chromosome pair in H. hippocrepis might be considered as an earlier differentiation stage than the heteromorphic ZW pair of H. nummifer and H. algirus, where the W element resulted submetacentric (Abd Allah 2010; present paper). In fact, a different morphology of the W chromosome in closely related species generally underlines the occurrence of different steps of the progressive diversification of the sex chromosome pair. In particular, sex chromosome pairs start their diversification as homomorphic autosomes which may progressively diverge in sequence content (mostly by heterochromatinization of the Y/W chromosome), morphology and dimensions, by means of addition/deletion of heterochromatin and chromosome rearrangements (e.g. Alam et al. 2018;Mezzasalma et al. 2019;. However, in some cases the diversification of sex chromosomes may be less linear than that suggested only by morphology or differential heterocromatinization and future studies should focus on the examination of the genomic content of sex chromosomes (e.g. Kratochvíl et al. 2021;. Differently from Mediterranean whip-snakes, the psammophiid clade which includes the genera Malpolon, Mimophis and Psammophis show highly divergent karyotypes compared to the ASK ( Figure  2, 4). The clade here considered includes six species with available karyotypes (Matthey 1931;Branch 1980;Aprea et al. 2003;Abd Allah 2010; this study), displaying 2 n = 42-44 chromosomes with a variable number of biarmed and telocentric chromosomes and representing the only available chromosome data for the whole family Psammophiidae (Figure 4), which includes nine genera and more than 50 species (Uetz et al. 2022). Overall, the composition of the known Psammophiidae karyotypes is structurally similar to that of Lamprophiidae and Pseudoxyrhophiidae, sharing several characters such as similar total chromosome number, high number of telocentric elements and comparable macro-/microchromosome ratios (see Matthey 1931;Aprea et al. 2003;Abd Allah 2010;Mezzasalma et al. 2014;Augstenová et al. 2018; this study). Psammophiidae, Lamprophiidae and Pseudoxyrhophiidae probably share an ancestral condition of 2 n = 48 with 22 mostly telocentric macrochromosomes (excluding a metacentric Z sex chromosome) and 26 microchromosomes (see Aprea et al. 2003;Mezzasalma et al. 2014). Starting from the ASK, a karyotype of 2 n = 48 was probably inherited by different families of Elapoidea (see Zaher et al. 2019, for recent phylogenetic and systematic reassessment of snake taxa), Table I. Chromosome morphometric parameters of the studied samples of H. hippocrepis and M. monspessulanus. RL = Relative length (Chromosome length/total chromosome length*100); CI = Centromeric index (short arm length/chromosome length*100); m = metacentric; sm = submetacentric; t = telocentric.
In the genus Psammophis, the only two species with a known karyotype, P. schokari and P. sibilans, show the same total chromosome number (n = 22), but a different number of macro-, microchromosomes, telocentric and biarmed elements (Figure 4). The karyotype of P. schokari shows 9 telocentric and 10 microchromosome pairs and probably originated from the psammophiid ancestral karyotype through two inversions and two tandem fusions (Figure 4). In turn, the karyotype of P. sibilans, with 7 telocentric and 13 microchromosome pairs, likely originated from one inversion in the Z chromosome and two tandem fusions (Figure 4). It is noteworthy that the subtelocentric Z chromosome of P. sibilans represents one of the few cases of morphological diversification (and chromosome rearrangement) of this chromosome in snakes. In fact, the Z chromosome is highly conserved in caenophidians, generally localized on the 4 th metacentric chromosome pair and displays a similar sequence content in different taxa (Rovatsos et al. 2015;Matsubara et al. 2016;Cole & Hardy 2019;Viana et al. 2019;Singchat et al. 2020).
Similarly to the genus Psammophis, two tandem fusions in the primitive psammophiid karyotype would have likely generated the karyotype of the Malagasy Mimophis mahfalensis, which has 11 telocentric and 10 microchromosome pairs ( Figure 4).
Overall, the evidence gathered from our newly generated chromosome data and those available from the literature indicate that H. hippocrepis and M. monspessulanus have very different karyotype structures, which are highly representative of the families Colubridae and Psammophiidae. Starting from the ASK, these two families followed different pathways of chromosomal diversification, characterized by a highly conserved karyotype in Colubridae and by a progressive reduction of the chromosome number in Psammophiidae.