Supplementary Material for: Differentiation of Sex Chromosomes and Karyotype Characterisation in the Dragonsnake <b><i>Xenodermus javanicus</i></b> (Squamata: Xenodermatidae)

Highly differentiated heteromorphic ZZ/ZW sex chromosomes with a heterochromatic W are a basic principle among advanced snakes of the lineage Colubroidea, while other snake lineages generally lack these characteristics. For the first time, we cytogenetically examined the dragonsnake, <i>Xenodermus javanicus</i>, a member of the family Xenodermatidae, which is phylogenetically nested between snake lineages with and without differentiated sex chromosomes. Although most snakes have a karyotype with a stable chromosomal number of 2n = 36, the dragonsnake has an unusual, derived karyotype with 2n = 32 chromosomes. We found that heteromorphic ZZ/ZW sex chromosomes with a heterochromatic W are present in the dragonsnake, which suggests that the emergence of a highly differentiated W sex chromosome within snakes predates the split of Xenodermatidae and the clade including families Pareatidae, Viperidae, Homalopsidae, Lamprophiidae, Elapidae, and Colubridae. Although accumulations of interstitial telomeric sequences have not been previously reported in snakes, by using FISH with a telomeric probe we discovered them in 6 pairs of autosomes as well as in the W sex chromosome of the dragonsnake. Similarly to advanced snakes, the sex chromosomes of the dragonsnake have a significant accumulation of repeats containing a (GATA)_n sequence. The results facilitate the dating of the differentiation of sex chromosomes within snakes back to the split between Xenodermatidae and other advanced snakes, i.e. around 40-75 mya

Supplementary Material for: Mixed-Up Sex Chromosomes: Identification of Sex Chromosomes in the X1X1X2X2/X1X2Y System of the Legless Lizards of the Genus Lialis (Squamata: Gekkota: Pygopodidae)

<p>Geckos in general show extensive variability in sex determining systems, but only male heterogamety has been demonstrated in the members of their legless family Pygopodidae. In the pioneering study published more than 45 years ago, multiple sex chromosomes of the type X₁X₁X₂X₂/X₁X₂Y were described in Burton's legless lizard <i>(Lialis</i><i>burtonis)</i> based on conventional cytogenetic techniques. We conducted cytogenetic analyses including comparative genomic hybridization and fluorescence in situ hybridization (FISH) with selected cytogenetic markers in this species and the previously cytogenetically unstudied Papua snake lizard <i>(Lialis jicari)</i> to better understand the nature of these sex chromosomes and their differentiation. Both species possess male heterogamety with an X₁X₁X₂X₂/X₁X₂Y sex chromosome system; however, the Y and one of the X chromosomes are not small chromosomes as previously reported in <i>L. burtonis</i>, but the largest macrochromosomal pair in the karyotype. The Y chromosomes in both species have large heterochromatic blocks with extensive accumulations of GATA and AC microsatellite motifs. FISH with telomeric probe revealed an exclusively terminal position of telomeric sequences in <i>L. jicari</i> (2n = 42 chromosomes in females), but extensive interstitial signals, potentially remnants of chromosomal fusions, in <i>L.</i><i>burtonis</i> (2n = 34 in females). Our study shows that even largely differentiated and heteromorphic sex chromosomes might be misidentified by conventional cytogenetic analyses and that the application of more sensitive cytogenetic techniques for the identification of sex chromosomes is beneficial even in the classical examples of multiple sex chromosomes.</p><br

Stable Cretaceous sex chromosomes enable molecular sexing in softshell turtles (Testudines: Trionychidae)

Turtles demonstrate variability in sex determination ranging from environmental sex determination (ESD) to highly differentiated sex chromosomes. However, the evolutionary dynamics of sex determining systems in this group is not well known. Differentiated ZZ/ZW sex chromosomes were identified in two species of the softshell turtles (Trionychidae) from the subfamily Trionychinae and Z-specific genes were identified in a single species. We tested Z-specificity of a subset of these genes by quantitative PCR comparing copy gene numbers in male and female genomes in 10 species covering the phylogenetic diversity of trionychids. We demonstrated that differentiated ZZ/ZW sex chromosomes are conserved across the whole family and that they were already present in the common ancestor of the extant trionychids. As the sister lineage, Carettochelys insculpta, possess ESD, we can date the origin of the sex chromosomes in trionychids between 200 Mya (split of Trionychidae and Carettochelyidae) and 120 Mya (basal splitting of the recent trionychids). The results support the evolutionary stability of differentiated sex chromosomes in some lineages of ectothermic vertebrates. Moreover, our approach determining sex-linkage of protein coding genes can be used as a reliable technique of molecular sexing across trionychids useful for effective breeding strategy in conservation projects of endangered species