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.