Comparative cytogenetic analysis in Erythrolamprus snakes ( Serpentes : Dipsadidae ) from Argentina

We described the karyotypes of five snake taxa from Argentina: Erythrolamprus almadensis, E. ceii, E. poecilogyrus caesius, E. p. schotti and E. p. sublineatus, and also intergrading individuals between the last two subspecies by conventional staining, chromosome bandings and fluorescent in situ hybridization (FISH) with 28S ribosomal DNA probes. Erythrolamprus ceii and E. almadensis share a diploid chromosome number of 2n= 28, whereas in E. poecilogyrus intraspecific variations were observed: E. p. caesius has 2n= 28, E. p. schotti and E. p. sublineatus as well as in the intergrading individuals have 2n= 32. In E. almadensis and E. p. caesius, the 2 and 6 chromosome pairs respectively are heteromorphic by size, morphology and C-banding pattern. These results allow us to suggest that these chromosome pairs might be considered as the ZW sex chromosomes in these species. The present comparative cytogenetic analyzes contributes to the already remarkable karyotypic variability in Erythrolamprus genus and propose a hypothesis about potential mechanisms involved in the chromosome evolution among taxa analyzed. Furthermore, the karyotypic differences observed between E. p. caesius (2n= 28) and E. p. schotti and E. p. sublineatus (2n= 32) might play a causal role in speciation.


INTRODUCTION
The cytogenetic analysis has been useful for taxonomic studies in Serpentes with inter-and intraspecific chromosome variability as Micrurus (Gutiérrez and Bolaños 1981) and Vipera (Aprea et al. 2006).Moreover, the differences in the karyotype through the redistribution of the chromosomes have been identified as a key mechanism in the evolutionary process of speciation (White 1968, 1969, Olmo 2005).
In this cytogenetic study, we describe the karyotype of Argentinean snakes included in poecilogyrus morphological group (Cei 1993): Erythrolamprus almadensis (Wagler 1824), E. ceii (Dixon 1991) (Dixon and Markezich 1992).The cytogenetic characters described for each analyzed taxon were taxonomically informative and allowed us to provide a hypothesis about potential mechanisms involved in the chromosome evolution into the poecilogyrus group.The specimens were injected intraperitoneally with 0.1% colchicine (1ml/100g body weight) for 3 hours prior to animal dissection.We practiced euthanasia according to the method described by Beaupre et al. (2004).The intestine was dissected and swollen for 50 minutes at RT in a hypotonic solution (0.075 M KCl), cut into small fragments, and then fixed in freshly prepared fixative solution (methanol:glacial acetic acid, 3: 1) and stored at -20ºC until further use.

Cytogenetic
For mitotic chromosome preparations, twothree intestine fragments were transferred to a tube with a few drops of 60% acetic acid for a few minutes until the epithelial cells shed, and then the gut fragments were removed.Some drops of cell suspension were carefully placed onto slides, which had been previously pre-heated in a thermal bath at 45º C for maintaining a thin film of water at the time when the drops fall on the slide.Cells were spread on the slide using a heating plate at 45°C as described in Traut (1976).Then, the preparations were dehydrated in an ethanol series (70, 80, and 96%, 30 s each) and stored at -20°C until further and III = microchromosomes.Based on RL values three chromosome groups could be recognized: small (1 -2.5% of haploid set), medium (2.51 -5%) and large (> 5%).

CHROMOSOME COMPLEMENTS
Erythrolamprus ceii, E. almadensis and E. poecilogyrus caesius share a diploid chromosome number 2n = 28 (Fig. 1a-d).Based on the RL values the chromosome pairs 1 to 3 are larger, 4 to 7 pairs have medium size and 8 to 14 pairs are smaller (Fig. 1a-d; Table I).
The karyotype formula of both sexes of E. poecilogyrus schotti, E. p. sublineatus and intergrading individuals is 32 = 24+8+0, NF = 56 (Fig. 1e-g).The chromosome pairs 1, 4 -6, and 9 -16 are metacentric and the pairs 2, 3, 7, and 8 are telocentric (Fig. 1; Table I).According to the RL values, the pair 1 belongs to the large group, the pairs 2 to 9 to the medium one, and pairs 10 to 16 to the smaller (Table I).In these taxa neither a heteromorphic pair and no secondary constriction is observed on the poecilogyrus group chromosome complements.use.For conventional staining, preparations were stained with 10% Giemsa solution (pH 6.8) for 7 min at RT.
Chromosome preparations were observed in a epifluorescence microscope Leica DMLB equipped with a Leica DFC350 FX CCD camera and Leica IM50 software, version 4.0 (Leica Microsystems Imaging Solutions).Black-and-white images of chromosomes were recorded separately for each fluorescent dye.Images were pseudocolored (light blue for DAPI, green for CMA 3 , and red for Cy3) and processed with an appropriate software.
Chromosome sizes were measured using the computer application MicroMeasure version 3.3 (available at http://www.colostate.edu/Depts/Biology/Micromeasure) ( Reeves and Tear 2000).Measurements were performed on ten metaphase plates of each specimen.Relative chromosomal lengths (RL) and centromeric index (CI) were calculated and expressed as percentage of the haploid set.These data were used to describe the karyotype of each Argentinean snake according to Levan et al. (1964).The chromosomal formula was determined following to Peccinini-Seale (1981) (2n = I+II+III) being I = metacentric or submetacentric macrochromosomes, II = telocentric or subtelocentric macrochromosomes

C-AND FLUORESCENT BANDINGS
In Erythrolamprus ceii, three chromosome pairs have heterochromatic C-positive bands at centromeric regions, i.e., the chromosome pair 2, 4 and 6 (Fig. 1a).In males of E. almadensis, the chromosome pairs 1, 2, 3, 6, and 9.In female mitotic preparations, C-positive bands are present at centromeric regions of the chromosome pairs 1, 3-6, 9, and 10 (Fig. 1b, c).In the heteromorphic chromosome pair 2, the large homologous chromosome shows a C-positive band at the centromeric region, whereas the small one has a pericentromeric C-positive band in the p arm (Fig. 1c).In E. p. caesius, C-positive centromeric bands are revealed on each chromosome of pairs 1 and 2 (Fig. 1d).Besides, both sexes show differences in the C-banding pattern of the chromosome pair 6.In males, each subtelocentric homologous chromosome has C-positive centromeric bands.In females, no C-bands are detected on the metacentric chromosome, whereas a C-positive band is observed in its subtelocentric homologous (Fig. 1d).In Erythrolamprus p. schotti, the chromosome pair 1, 6 and 14 show heterochromatic C-positive bands placed on centromeric regions (Fig. 1e).No C-positive bands were detected in both E. p. sublineatus and the intergrading specimens (Fig. 1f, g).

LOCATION OF rDNA BY FISH AND Ag-NOR BANDING
At mitotic metaphases, positive Ag-NORs are observed in one of the small chromosome pairs in all species of the Erythrolamprus analyzed, i.e.Erythrolamprus ceii (Fig. 3a), E. almadensis (Fig. 3b), E. p. caesius (Fig. 3c), E. p. schotti (Fig. 3d), and E. p. sublineatus (Fig. 3e), and also in intergrading individuals (Fig. 3f).In mitotic metaphases of males of E. p. sublineatus and females of E. p. schotti, FISH experiments with the 28S rDNA probe showed a single cluster of rDNA genes located in each homologous chromosomes of one of the small pair (Fig. 3g-l).
In snakes, the karyotype 2n = 36 (16 macrochromosomes + 20 micro-chromosomes) is proposed to be an ancestral character shared throughout most families (Oguiura et al. 2009).Taking into account this hypothesis, both chromosome changes must have occurred throughout the karyotype evolution of Erythrolamprus poecilogyrus group and the different karyotypes should be considered as derivatives.The lowest diploid numbers could derive through fusion between macro and microchromosomes and/or between micro-chromosomes.Moreover, differences in chromosome morphology could be due to pericentric inversions and heterochromatin addition.In Squamata, there is a trend to reduce the number of micro-chromosome because of translocation onto macro-chromosomes, or fusion among micro-chromosomes (Olmo 2008, Uno et al. 2012).
Concerning the content, distribution and location of constitutive heterochromatin, different C-banding patterns have been previously described in snakes, i.e., large blocks at centromeric regions, and also at terminal and interstitial position in macro-chromosomes, whole heterochromatic arms ( Mengden andStock 1980, Moreno et al. 1987), only placed on microchromosomes (Singh and Majumdar 1994) and scarce heterochromatin in autosomes (Mezzasalma et al. 2014).The results obtained here revealed a low content of constitutive heterochromatin among the species of the poecilogyrus group, intra-and intraspecific differences and the enrichment of heterochromatin mainly of CG base pairs.
The karyotypes of Erythrolamprus ceii, E. almadensis and E. p. caesius (2n = 28) are similar after conventional staining, although we detected differences from the comparison of their DAPI/ CMA 3 banding patterns.Our cytogenetic    (Olmo et al. 1986).Furthermore, the intraand interspecific variation observed in the C-band patterns would appear to be unrelated to phylogeny (Odierna et al. 1985).Thus, the diversity observed in the C-banding pattern of the species and subspecies studied here constitutes evidence that supports the evolutionary trend for reptiles.
The number and location of the NORs in a pair of micro-chromosomes is frequent in Serpentes and also in Squamata (Camper and Hanks 1995, Aprea et al. 2006, Mezzasalma et al. 2014).Within Xenodontini, the location in micro-chromosomes has been reported in E. poecilogyrus schotti and three Lygophis species (Trajtengertz et al. 1995, Falcione et al. 2016).In all taxa studied here, a single NOR has been observed in one pair of small chromosomes.
In most organisms, the repeating unit of the ribosomal genes (rDNA) is often G+C-rich (Miller 1981, Sumner 2003).The presence of a CMA 3 -bright band is generally associated to NORs (Sumner 2003, Aprea et al. 2006).From the results of the analysis of fluorescent bandings in E. almadensis, the CMA 3 -bright band placed on one small chromosome pairs could represent an NOR.
In the present study, the 2 nd and 6 th chromosome pairs are assigned as sex chromosome pair (ZZ/ ZW) in both E. almadensis and E. p. caesius, respectively.In females of E. almadensis, both homologous are metacentric but differ in size.However, in males the two chromosomes are of equal size.Furthermore, the smallest metacentric chromosome of pair 2 exhibits a pericentromeric C-positive band on the p arm in females.Therefore, the presence of this heteromorphic pair in females allows us to propose that chromosomal pair 2 would be the ZW sex pair, because the smallest metacentric chromosome would be resting to females, being W chromosome.
In the female diploid chromosome complement of E. poecilogyrus caesius, the pair 6 is heteromorphic by morphology, since one of the chromosomes is metacentric and the other sub-telocentric.In contrast, male karyotype the same pair is homomorphic, both homologues being subtelocentric.Similarly, in females only the subtelocentric chromosome of this pair has a C-positive band in the p-arm, whereas in males C-positive bands are distinguished in the p-arm of both subtelocentric chromosomes.Considering to these result, we suggest that the metacentric chromosome of the pair 6 would be the W chromosome, whereas the subtelocentric chromosome the Z, because it is only present in the female complement.Pericentric inversions, amplification of pre-existent heterochromatin or the addition of new heterochromatin, and deletions of euchromatic regions would have been the mechanisms involved in the evolution of sex chromosomes of Erythrolamprus almadensis and E. p. caesius as there were proposed in Serpentes (Ohno 1967, Ray-Chaudhuri et al. 1971, Singh et al. 1976, Beçak 1983, Matsubara et al. 2006).
Cytotaxonomy plays a key role in elucidating the taxonomy and chromosomal evolution of snakes when morphological characteristics are insufficient for the resolution of taxonomic problems (Gutiérrez and Bolaños 1981).From this point of view, we provide an overview of the current cytogenetic Erythrolamprus genus.Moreover, our results show that E. p. caesius (2n = 28) has karyological specific characteristics that differentiate it from E. p. schotti and E. p. sublineatus (2n = 32).This karyotype might constitute a mechanism of reproductive isolation making it a different species: Erythrolamprus caesius.Complementarily morphological and molecular analysis should provide further information.
, E. poecilogyrus caesius (Cope 1862), E. p. schotti (Schlegel 1837) and E. p. sublineatus (Cope 1860) and the intergrading individuals between the last two subspecies through conventional staining, different chromosome banding (C, DAPI/ CMA 3 and Ag-NOR) and fluorescent in situ hybridization (FISH) of ribosomal DNA.Erythrolamprus poecilogyrus (Wied-Neuwied 1825) is considered one of the most noteworthy examples of polymorphism due to the geographic variation in color and design patterns and natural mosaic of morphometric characters and lepidosis

Figure 3 -
Figure 3 -Location of rDNA by FISH and Ag-NOR banding of Erythrolamprus poecilogyrus group species.Ag-NOR-stained metaphases of a) Erythrolamprus ceii, b) E. almadensis, c) E. p. caesius, d) E. p. schotti, e) E. p. sublineatus and f) Intergrading individuals.The black arrowheads indicate the chromosomes bearing NORs.FISH with DNAr 28S probe in male mitotic metaphase of E. p. sublineatus (g-i) and female of E. p. schotti (j-l).Chromosomes counterstained with DAPI (blue) (g, j), hybridization signals (red) (h, k) and combination of figures g, j with h and k, respectively (i-l).The white arrowheads indicate the hybridization signals and the chromosomes carriers.Scale bar= 10 μm.
analyses were performed on females and males of Erythrolamprus ceii, E. almadensis, E. poecilogyrus caesius, E. p. schotti and E. p. sublineatus and intergrading individuals (see details in Appendix).Voucher specimens are deposited in the Herpetological Collection of the National University of the Northeast (UNNEC), Corrientes, República Argentina.Erythrolamprus poecilogyrus subspecies were identified by the different color patterns according to Fernandes da Silva (2006).Individuals with intermediate patterns of coloration between E. p. schotti and E. p. sublineatus were considered as intergrading individuals.