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

Abstract

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 (Bufo viridis subgroup), which had so far only been characterized in laboratory-bred hybrids. Our data support an XY system in B. balearicus, B. viridis and B. variabilis. 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 Xenopus tropicalis) to represent the sex chromosomes in diploid species of the B. viridis subgroup (B. siculus, B. shaartusiensis, B. balearicus, B. turanensis, B. variabilis, B. viridis, and probably B. boulengeri). This chromosome harbors DMRT1, 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 DMRT1, and we preliminarily reject its role as the master sex determination gene, pending future extension of this evidence to the entire DMRT1 gene. We further create a chain of evidence, which supports the hypothesis that linkage group 1 of X. tropicalis 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.

Sex reversal assessments reveal different vulnerability to endocrine disruption between deeply diverged anuran lineages

“…This model-species, in which genetic sex is governed by a female heterogametic (ZZ/ZW) chromosome system 45 , proved to be more sensitive to EE2 with a lower dose provoking sex reversals and more affected animals ( Table 1 ). On the other hand, B. viridis and H. arborea , both diverged 206 My from X. laevis and possessing male heterogametic (XX/XY) sex chromosomes 32 35 , showed higher percentages of mixed sex individuals than X. laevis .…”

“…In most previous EDC-studies, sex reversal had to be inferred by comparing sex ratios of control and exposed frogs, assuming a normal 1:1 proportion, which may have easily led to wrong conclusions about EDC-impacts on sex ratios. While all amphibian species investigated show genetic sex determination 31 , exhibiting either male (XX/XY) or female (ZZ/ZW) heterogamety, an extrapolated 96% of all species have microscopically indistinguishable sex chromosomes 32 , requiring molecular sexing methods. Although EDC-studies with molecular sexing were applied to the model Xenopus 26 33 , sex markers have become only recently available for some non-model anurans 32 34 35 36 37 38 , and have not been used in EDC-experiments.…”

The plasticizer bisphenol A affects somatic and sexual development, but differently in pipid, hylid and bufonid anurans

“…More than 200 Million years of divergence between Pipidae, Bufonidae and Hylidae, and about 100 My between the latter two anuran families (www.timetree.org), have led to the evolution of profound differences in genome size and composition with ecotoxicological relevance (Helbing, 2012), various life history and ecologies including larval feeding (Avila and Frye, 1978;Degani, 1986;Schneider and Grosse, 2009;St€ ock et al, 2009), as well as physiology (Wilbur, 1980) and sex determination pathways (e.g. Berset-Br€ andli et al, 2006;St€ ock et al, 2011aSt€ ock et al, , 2011bTamschick et al, 2015;Yoshimoto et al, 2008). Not surprisingly, these complex differences appear to cause susceptibility differences to endocrine disruption (cf.…”

“…DNAs were eluted in 200 ml AE buffer (Qiagen) and stored at À20 C. To establish genetic sex, species-specific polymerase chain reactions (PCRs) were conducted with subsequent gel electrophoresis and genotyping. Sexing of H. arborea and X. laevis was conducted as described (Tamschick et al, 2016), sexing of B. viridis involved markers C201, C223 (Tamschick et al, 2015), Cherp, DMRT1 and VLDLR (Gerchen et al, 2016). Only frogs and toads that completed metamorphosis were genetically sexed.…”

“…These ecological differences might cause variance in EDC-exposure, especially between ground-feeding and filterfeeding larvae. Importantly, these species also differ in their sex determination systems with either male heterogamety (XX/XY) in H. arborea (Berset-Br€ andli et al, 2006;St€ ock et al, 2011aSt€ ock et al, , 2011b and B. viridis (St€ ock et al, 2011a;Tamschick et al, 2015) or female heterogamety (ZZ/ZW) in X. laevis (Chang and Witschi, 1956;Yoshimoto et al, 2008).…”