Introduction
Studies of sex-specific biology enhance our understanding of processes driving evolution and maintenance of biodiversity, and assist in developing conservation management (Amos et al., 2014; Pavlova et al., 2013). However, lack of sex-specific genetic markers hampers such studies in many monomorphic fish species. Sex-determining systems in teleost fish are highly variable, including sequential hermaphroditism, genotypic sex-determination (with X0, XY or complex XY being more prevalent than Z0, ZW or complex ZW) and environmental dependency (Bachtrog et al., 2014; Mank, Promislow, & Avise, 2006). In fishes with genotypic sex-determination, sex chromosomes are often homomorphic, where X/Y or Z/W pairs are similar or nearly identical in gene content and size (Bachtrog, 2013; Bachtrog et al., 2014). Various genes (including amhy , amhr2 , bcar1 ,dmrt1/dmY/dm-W , gsdfY, sdY and members of the SOX: SRY -like HMG -box-containing gene family), single nucleotide polymorphisms (SNPs), inversions, or multiple loci, can all contribute to sex-determination in fish (Bao et al., 2019; Graves & Peichel, 2010; Martínez et al., 2014; Natri, Merilä, & Shikano, 2019). Moreover, new sex-determination systems can evolve relatively rapidly and frequently, resulting in intra-specific variation in sex-determination (Natri et al., 2019). Thus, identification of reliable, generally applicable sex-linked markers in fish can be challenging.
Homomorphic sex-chromosomes occur where gametologs have recombined relatively recently, and may result from recent switches in the chromosome pair used for sex determination (Bachtrog et al., 2014). In contrast, heteromorphic sex-chromosomes, where X and Y, or Z and W, are diverged and highly distinct, evolve from homomorphic sex chromosomes through suppression of recombination between sex chromosomes and subsequent degradation of Y or W (Charlesworth, Charlesworth, & Marais, 2005). This suppression often occurs progressively and stepwise along chromosomes, starting from a sex-determining gene, resulting in strata of different ages, i.e. regions of the chromosome where recombination stopped at different evolutionary times (Charlesworth et al., 2005).
Several genomic approaches can be used to detect sex chromosomes (reviewed in Palmer, Rogers, Dean, & Wright, 2019). Two are particularly applicable to wildlife, because they use DNA sequence obtainable from non-lethally-collected samples and do not require captive breeding to build linkage maps. The first approach uses different ploidy of sex chromosomes diverged through Y or W degeneration: the homogametic sex (e.g. XX females or ZZ males) will have two copies of the same sex chromosome, whereas heterogametic sex (e.g. XY males or ZW females) will have one copy of each sex chromosome. This pattern will be reflected in read-depth coverage: for example, old strata on X or Z will show half as many reads in the heterogametic sex as the homogametic one, and Y- or W- loci will be absent in the homogametic sex. But in young strata or homomorphic sex-chromosomes, having high similarity between X and Y or Z and W, the read depth will be similar between sexes and to that of autosomal regions. For these younger regions of sex-chromosomes, an approach based on differences insex-specific SNP density across genomic regions is more appropriate. With Y or W accumulating mutations faster than X or Z due to reduced recombination and weaker purifying selection, higher SNP density in young strata is expected in the heterogametic than homogametic sex (Palmer et al., 2019). In contrast, in older strata with substantial Y or W degeneration, X- and Z-linked loci will be effectively hemizygous in the heterogametic sex (halved read depth), and higher SNP density is expected in the homogametic sex. Accordingly, combining approaches based on read depth and sex-specific variability can assist in detecting sex-linked loci.
Sex-determination is not well understood in fish of the family Percichthyidae. Species from this family dominate the Australian freshwater fish fauna, and three genera occur in eastern Asia (Coreoperca and Siniperca ), and South America (Percichthys ). Many Australian species are threatened, including the endangered Macquarie perch Macquaria australasica and trout cod Maccullochella macquariensis . Although sex of an adult can be ascribed when it produces gametes, lack of sexual dimorphism and ways to determine the sex of individuals non-invasively outside breeding seasons hinders better understanding of species biology and more efficient conservation.
Shams et al. (2019) examined karyotypes for two Percichthyid species, golden perch Macq. ambigua and Murray cod Macc. peelii , and for both reported male heterogametic sex-chromosome systems (XX females/XY males) with diploid chromosome number 2n = 48. Heteromorphism was detected in sex-chromosomes of Murray cod, but homomorphism in golden perch (Shams et al., 2019). Consistent with low morphological differentiation of golden perch sex chromosomes, in its sister-species Macquarie perch (Lavoué et al., 2014), a set of >1200 genome-wide reduced-representation SNP loci did not reveal markers consistent with Y-linkage (i.e. present in males only) or strict X/Y homology, i.e. always heterozygous in males and always homozygous in females (Lutz et al., 2020). This suggests that in Macquarie perch sex-chromosomes may have young strata.
Sex determination in many fishes can be influenced by environmental factors, especially temperature (Devlin & Nagahama, 2002; Penman & Piferrer, 2008). Hatchery work suggests that environmental conditions may influence sex determination at an early stage of development in some Percichthyids (Ingram, Ho, Turchini, & Holland, 2012). Lyon et al. (2012) reported 2.5 times as many females as males in a population of trout cod stocked from hatchery-bred fish. Because sex-ratio biases in stocked fish have implications for recovery of populations through stocking, being able to determine genetic sex of fish would benefit conservation management of threatened Percichthyid species.
Here, we sequenced, assembled and annotated the genomes of two closely related sexually-monomorphic Percichthyids, Macquarie perch and golden perch. Then, using genomic datasets targeted for different resolution, we explored sex-linkage in these species. In particular, we aimed to test whether XY sex determination fully explains patterns of sex-linked genetic variation in these species, and to develop a non-lethal, affordable and rapid molecular test to determine the genetic sex of individuals. We tested for sex-linkage of SNPs obtained using reduced genomic representation DArTseq (Kilian et al., 2012) for Macquarie perch and golden perch of known sex, and examined whether putative sex-linked loci are clustered on some of the newly assembled scaffolds of respective reference genomes. Next, we obtained whole-genome resequencing (WGS) data for 50 female and 50 male Macquarie perch equally representing two relatively large populations used as sources for conservation interventions. We searched for SNPs significantly differentiated between sexes, using genotypes of four population-by-sex pools. This enabled identification of a small genomic region, showing predominantly XY patterns (one allele in females, two in males) on Macquarie perch scaffold 633. We explored individual sequence variation, identified X- and Y-linked haplotypes for this region, and developed a PCR-RFLP (polymerase chain reaction - restriction fragment length polymorphism) molecular sexing assay targeting a SNP with a Y-linked (male-specific) allele. We tested this assay in a panel of known-sex Macquarie perch individuals, and on smaller equivalent panels of golden perch, Murray cod and trout cod. We used amplicon sequencing to further explore sequence variation in this region for the four species. Our results revealed that the XY-homologous genomic region in Macquarie perch is species-specific and furthermore could be specific to populations related to those in which the test was developed. Overall, sequence variation across one small scaffold 633 region in two Macquarie perch populations generally supports XY sex-determination, but deletions in sexing region on both X and Y chromosomes, variation across species, and potentially across other genomic regions, calls for more complex sex-determination scenarios. The results and resources developed here will facilitate research on Percichthyidae, including the evolution of sex-determination. More broadly, the workflow described here could be used for developing molecular tests to sex other fish species with monomorphic sex chromosomes.