Supplementary Material for: Sex Chromosome Conservation, <b><i>DMRT1</i></b> Phylogeny and Gonad Morphology in Diploid Palearctic Green Toads (<b><i>Bufo viridis </i></b>Subgroup)

Due to the prevailing sex chromosome homomorphy and large genome size, the knowledge on sex determination systems, sex chromosomes and sex-determining genes in amphibians remains scarce. Using 3 cross-amplifying sex-linked microsatellite markers, we uncover sex determination systems and sex chromosomes in purebred, diploid Palearctic green toads (<i>Bufo viridis</i> subgroup), which had so far only been characterized in laboratory-bred hybrids. Our data support an XY system in <i>B. balearicus</i>, <i>B. viridis</i> and <i>B. variabilis</i>. While females show recombination, it is strongly suppressed (or not detectable) in males. Markers corroborate the largest chromosome pair 1 (homologous to linkage group 1 of <i>Xenopus tropicalis</i>) to represent the sex chromosomes in diploid species of the <i>B. viridis</i> subgroup <i>(B. siculus, B. shaartusiensis, B. balearicus, B. turanensis, B. variabilis, B. viridis</i>, and probably <i>B. boulengeri)</i>. This chromosome harbors <i>DMRT1</i>, a key gene of the sexual pathway in deeply divergent animal taxa. However, our phylogenetic analysis of a 600-bp fragment of that gene in diploid green toad taxa reveals that X and Y alleles cluster by species and not by gametolog. This suggests that XY-sequence similarity stems from occasional XY recombination within <i>DMRT1</i>, and we preliminarily reject its role as the master sex determination gene, pending future extension of this evidence to the entire <i>DMRT1 </i>gene. We further create a chain of evidence, which supports the hypothesis that linkage group 1 of <i>X. tropicalis</i> appears to be maintained as the largest chromosome (1), and thus is homologous in anuran karyotype evolution from pipid to hylid, bufonid and ranid anurans.