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Gene Duplication, Translocation, and Molecular Evolution of Dmrt1 and Related Sex-Determining Genes in Anurans.
Shinde SS
,
Veltsos P
,
Ma WJ
.
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Sex determination, the developmental process that directs embryos toward male or female fates, is controlled by master sex-determining genes whose origins and evolutionary dynamics remain poorly understood outside of a few model systems. In contrast to the highly differentiated sex chromosomes of mammals, birds, and Drosophila, most anurans (frogs and toads) maintain homomorphic sex chromosomes that exhibit a rapid turnover, even among closely related species. Master sex-determining genes evolve via gene duplication or via allelic diversification, and sex chromosome turnover is driven by gene translocation or novel mutations in the existing genes involved in the sexual developmental pathway. To uncover the mechanisms underlying the emergence of master sex-determining genes and sex chromosome turnover, we analyzed 53 published anuran genomes and one caecilian genome (>200 Mya divergence) and available transcriptomes. We asked how often master sex-determining genes arise by gene duplication, whether and how often gene translocation associates with sex chromosome turnover, and if master sex-determining genes evolve under positive selection. We find that chromosome-level synteny is remarkably conserved, with only a few fusions or fissions and no evidence for translocation of four candidate master sex-determining genes (Dmrt1, Foxl2, Bod1l, and Sox3). Only Dmrt1 duplicated in 3 out of 50 species (excluding tetraploid Xenopus), and it showed strong testis-biased expression in all 8 species with available gonadal expression data. While Dmrt1 has evolved under purifying selection, Dmrt1 duplicates exhibit elevated nonsynonymous substitution rates and tendency towards positive selection. Lineage-specific amino acid changes were observed in the conserved DM domain of Dmrt1. These results demonstrate that, in anurans, master sex-determining genes rarely arise via gene duplication, and more likely evolve via allelic diversification. Sex chromosome turnover is not associated with gene translocation and is more likely driven by mutations on genes involved in sexual developmental pathways. All candidate sex-determining genes were under strong purifying selection, with the exception of duplications which are linked to positive selection. Our results suggest future research on anuran sex determination and sex chromosome evolution should focus on identifying allelic diversification and novel mutations on genes involved in sexual developmental pathways.
Figure 1. Genome-wide synteny across 12 anuran genomes with chromosome-level genome assemblies and genome annotation. (A) Phylogenetic tree obtained from timetree.org, with known sex chromosome or candidate master sex-determining genes indicated next to each species in parentheses. Colors are consistent with those of homologous chromosomes shown in the tree. (B) Chromosome-level synteny. Chromosomes are numbered and ordered by descending size (chromosome 1 is the largest, chromosome 14 is the smallest). (C) Example of chromosome 7, which is involved in sex determination in both X. tropicalis and Hyla sarda [86,87], which shows it has undergone fissions and fusions in various anuran lineages.
Figure 2. Dmrt1 coding sequence structure and detected duplicated copies across 54 amphibia. The phylogenetic tree was conducted using multiple-loci alignment from Portik et al. (2023) [66]. Dmrt1 exon duplications are color coded (2 copies in green, 3 copies in grey). Known sex chromosomes or candidate master sex-determining genes are indicated next to each species in parentheses. Dmrt1 (the original copy) expression is plotted when data from both sexes with at least one tissue are available. The color gradient indicates expression in female (red) and male (blue) tissues. Color intensity indicates the scale of gene expression; the four expression bins are <5 FPKM, 5–10 FPKM, 10–25 FPKM, and >25 FPKM.
Figure 3. Gene order of the four candidate master sex-determining genes across the 12 anurans, and known sex chromosomes or candidate master sex-determining genes are indicated next to each species in parentheses. (A) Dmrt1, (B) Foxl2, (C) Sox3, and (D) Bod1l across 12 anurans with chromosome genome assemblies. The top row shows the focal gene, with its immediate upstream and downstream 5 genes based on the reference genome of X. tropicalis. Boxes with arrows indicate gene inversions, while paired tilted lines represent multiple genes with inverted orientations. Vertical blue boxes mark unique, uncharacterized LOC genes that are species specific, and pink lines denote other species-specific genes. Genes missing from the genome assembly are enclosed in dotted boxes. A detailed list of inversions and species-specific genes can be found in Tables S8–S11.
Figure 4. (A) The phylogenetic tree of 12 studied anurans alongside graphs of chromosome-level synteny for the micro-synteny region of (B) Dmrt1 and Bod1l, (C) Foxl2, and (D) Sox3. Known sex chromosomes or candidate master sex-determining genes are indicated next to each species in parentheses.
Figure 5. Plot of dN and dS values of Dmrt1 and its duplicated copies for 53 anurans, including the status of sex chromosomes reported in the literature among 53 anurans. (A) Spearman’s correlation between dN and dS values. (B) Boxplot of dN values, (C) Boxplot of dS values across duplication and non-duplication status, with the latter also divided into autosomal, sex chromosome, and unknown. chrDup—chromosome and duplication status; Au—autosome; SC—sex chromosome; U—unknown; nonDUP—non-duplicated copy; Dup—duplication.
Figure 6. The protein sequence alignment and protein similarity analysis for the DM domain of Dmrt1 across 53 anurans. In the top panel, colors indicate amino acid identity, while the size of each amino acid letter reflects sequence similarity across anurans, with larger letters denoting higher similarity. Sequence similarity was scored with an average of 0.96 (out of 1, with 1 representing 100% similarity without mutation), indicating high sequence similarity. Green shading highlights the Ranidae lineage with a lineage-specific amino acid mutation combination, and purple shading possibly indicates retention of ancestral amino acid in the basal lineages at position 42. Red star indicates certain amino acid mutation sites are under positive selection (position 64 in H. sarda, and various sites in two Dmrt1 duplicated copies, DmW of X. laevis and Pelodytes ibericus).
