Sex chromosome evolution in snakes inferred from divergence patterns of two gametologous genes and chromosome distribution of sex chromosome-linked repetitive sequences

Abstract

Background: The discovery of differentially organized sex chromosome systems suggests that heteromorphic sex chromosomes evolved from a pair of homologous chromosomes. Whereas karyotypes are highly conserved in alethinophidian snakes, the degeneration status of the W chromosomes varies among species. The Z and W chromosomes are morphologically homomorphic in henophidian species, whereas in snakes belonging to caenophidian families the W chromosomes are highly degenerated. Snakes therefore are excellent animal models in which to study sex chromosome evolution. Herein, we investigated the differentiation processes for snake sex chromosomes using both coding and repetitive sequences. We analyzed phylogenetic relationships of CTNNB1 and WAC genes, localized to the centromeric and telomeric regions, respectively, of the long arms on snake sex chromosomes, and chromosome distribution of sex chromosome-linked repetitive sequences in several henophidian and caenophidian species.

Results: Partial or full-length coding sequences of CTNNB1 and WAC were identified for Z homologs of henophidian species from Tropidophiidae, Boidae, Cylindrophiidae, Xenopeltidae, and Pythonidae, and for Z and W homologs of caenophidian species from Acrochordidae, Viperidae, Elapidae, and Colubridae. Female-specific sequences for the two genes were not found in the henophidian (boid and pythonid) species examined. Phylogenetic trees constructed using each gene showed that the Z and W homologs of the caenophidian species cluster separately. The repetitive sequence isolated from the W chromosome heterochromatin of the colubrid Elaphe quadrivirgata and a microsatellite motif (AGAT)8 were strongly hybridized with W chromosomes of the viperid and colubrid species examined.

Conclusion: Our phylogenetic analyses suggest that the cessation of recombination between the Z and W homologs of CTNNB1 and WAC predated the diversification of the caenophidian families. As the repetitive sequences on the W chromosomes were shared among viperid and colubrid species, heterochromatinization of the proto-W chromosome appears to have occurred before the splitting of these two groups. These results collectively suggest that differentiation of the proto-Z and proto-W chromosomes extended to wide regions on the sex chromosomes in the common ancestor of caenophidian families during a relatively short period.

Keywords: Evolution; Gametolog; Heterochromatin; Phylogeny; Repetitive sequences; Snake; W chromosome; Z chromosome.

Fig. 1

Phylogenetic relationships between snake families. Phylogeny, divergence time and classification are based on Vidal et al. [63], Pyron et al. [49], and Uetz and Hošek [65]

Fig. 2

Comparison of partial nucleotide and amino acid sequences of CTNNB1 and WAC genes. Nucleotide and amino acid sequences are aligned between the homologs of CTNNB1 (a) and WAC (b) genes in five tetrapod species: E. quadrivirgata, A. carolinensis, G. gallus, H. sapiens and X. tropicalis. Numbers on the alignments indicate nucleotide positions from the translation initiation sites. Arrowheads in b indicate two predicted translational initiation sites

Fig. 3

Molecular phylogenetic trees of CTNNB1 genes. Maximum-likelihood trees of CTNNB1 genes were constructed with the long alignment for 20 tetrapod species (a) and the short alignment for 26 squamate species (b). Bootstrap values (>50 %) are shown on each node. Classification is shown on the right side of species. Blue and pink bars in b show clades of Z and W homologs of caenophidian species, respectively

Fig. 4

Molecular phylogenetic trees of WAC genes. Maximum-likelihood trees of WAC genes were constructed with the long alignment for 21 tetrapod species (a) and the short alignment for 21 squamate species (b). Bootstrap values (> 50 %) are shown on each node. Classification is shown on the right side of species. Blue and pink bars in b show clades of Z and W homologs of caenophidian species, respectively

Fig. 5

FISH of three repetitive sequences in snakes. FITC-labeled E. quadrivirgata BamHI-4 repeat was hybridized to PI-stained metaphase spreads of B. constrictor (a), R. tigrinus (b), and B. arietans (c). E. quadrivirgata BglI-15 repeat was hybridized to metaphase spreads of R. tigrinus (d), P. flavoviridis (e), and B. arietans (f). The (AGAT)₈ microsatellite motif was hybridized to metaphase spreads of E. quadrivirgata (g), R. tigrinus (h), and P. flavoviridis (i). Arrowheads indicate hybridization signals on sex chromosomes

Fig. 6

Evolution of snake sex chromosomes. The timing of evolutionary events on snake sex chromosomes inferred by this study is shown on the cladogram [49, 63]. Horizontal lines between Z and W chromosomes stand for the presence of recombination between the homologs on the chromosomes. Chromosome region with dark gray color stand for amplification of EQU-BglI-15 and (AGAT)_n repeats on the W chromosomes in caenophidian species. Note that morphologies of Z and W chromosomes and locations of the EQU-BamHI-4 repeat, CTNNB1 and WAC genes in acrochordid species are not yet identified and that chromosomal locations of the two genes are also not yet identified in viperid species