Ever-young sex chromosomes in European tree frogs

Abstract

Non-recombining sex chromosomes are expected to undergo evolutionary decay, ending up genetically degenerated, as has happened in birds and mammals. Why are then sex chromosomes so often homomorphic in cold-blooded vertebrates? One possible explanation is a high rate of turnover events, replacing master sex-determining genes by new ones on other chromosomes. An alternative is that X-Y similarity is maintained by occasional recombination events, occurring in sex-reversed XY females. Based on mitochondrial and nuclear gene sequences, we estimated the divergence times between European tree frogs (Hyla arborea, H. intermedia, and H. molleri) to the upper Miocene, about 5.4-7.1 million years ago. Sibship analyses of microsatellite polymorphisms revealed that all three species have the same pair of sex chromosomes, with complete absence of X-Y recombination in males. Despite this, sequences of sex-linked loci show no divergence between the X and Y chromosomes. In the phylogeny, the X and Y alleles cluster according to species, not in groups of gametologs. We conclude that sex-chromosome homomorphy in these tree frogs does not result from a recent turnover but is maintained over evolutionary timescales by occasional X-Y recombination. Seemingly young sex chromosomes may thus carry old-established sex-determining genes, a result at odds with the view that sex chromosomes necessarily decay until they are replaced. This raises intriguing perspectives regarding the evolutionary dynamics of sexually antagonistic genes and the mechanisms that control X-Y recombination.

Figure 1. Expected gene genealogies under different evolutionary scenarios.

The focal gene is localized either on an autosome (green) or on a sex chromosome (red) in H. arborea (Ha), H. intermedia (Hi), or H. molleri (Hm). Arrows indicate turnovers in sex-determination systems. (a) Reference genealogy for an autosomal or mitochondrial marker. (b) In H. arborea, the marker lies on a proto sex chromosome recently derived from an autosome. Sex linkage is restricted to H. arborea, and genealogy conforms to species genealogy. (c) The marker is on ancestral sex chromosomes and thus sex-linked in all three species, but its genealogy still conforms to species genealogy due to occasional X-Y recombination. (d) The marker is on ancestral sex chromosomes and thus sex-linked in all three species, but due to absence of X-Y recombination, alleles cluster according to gametologs, not species. Within gametologs, gene genealogy conforms to species genealogy. (e) In H. arborea, the marker lies on a proto sex chromosome recently derived from an ancestral sex chromosome (dashed arrow), such that Ha_Y clusters with the ancestral Ha_X. The marker is sex linked in all three species, but in the sister group of H. arborea, alleles cluster according to gametolog, not species. Note that a similar genealogy would result from local gene conversion (see Figure 1 in [35]).

Figure 2. Maximum-likelihood phylogeny for tree frog cytochrome b lineages.

The divergence time between mtDNA cytochrome b lineages of H. arborea and sister-group species (complete sequences of ca 1000 bp, multiple samples across species geographic ranges) averages 7.1 my (2.3–15.8 my 95% HPDI). Origin of samples and GenBank accession numbers are provided in Table S5.

Figure 3. Recombination maps for sex-linked markers

. The complete absence of recombination in males (right) contrasts sharply with the high recombination rates found in females (left). Lengths are given in cM units for the consensus map, and correspondences are provided graphically for species-specific maps. In each case the map is the one with highest likelihood, except that for H. intermedia, ranking third but with a log-likelihood very close to (and not significantly lower than) the first one (−120.79 versus −119.71).

Figure 4. Gene genealogies for two sex-linked loci.

The transcription cofactor HaMed15 (left, ca. 1 kb sequences with two introns and two exons, including the marker Ha 5–22) and the non-coding Ha A-103 (right, ca. 510 bp sequences) are 93.8 cM apart on the female recombination map (Figure 3). For both markers, the X and Y alleles (marked with the same label when amplified from the same male) cluster by species, not by gametolog. Bootstrap values are higher for the non-coding Ha A-103 (≥98%) than for the highly conserved transcription cofactor HaMed15 (≤90%), and higher for H. arborea than for species from its sister group.