Discussion
Using two newly assembled genomes, reduced-representation sequencing
(DArTseq) and whole-genome resequencing (WGS) for panels of individuals
of known sex, and amplicon sequencing, we aimed to identify sex-linked
genomic regions and develop and test molecular sexing markers in twoMacquaria perch sister-species along with two confamilial
species. One of these species, golden perch, is inferred by karyology to
have XY sex-determination and homomorphic sex chromosomes, and the
confamilial Murray cod to have XY and heteromorphic chromosomes (Shams
et al., 2019). We did not uncover DArTseq SNPs consistent with Y-linkage
or XY-gametology in either golden perch or Macquarie perch, but found
loci polymorphic only in one sex (sometimes males, sometimes females),
scattered through different scaffolds. This suggests that regions with
old strata (with long-term lack of recombination), if present on
sex-chromosomes of these species, are short. Finding an order of
magnitude more sex-linked DArT loci in golden perch than in Macquarie
perch, none of which were on the golden perch scaffold VMKM01003747
homologous to Macquarie perch scaffold 633, indicates that these species
have independently-evolved sex-determination systems, with the Macquarie
perch one being more recent. Alternatively, Macquarie perch could have
lost sex-linked variation during the Pleistocene bottleneck involved in
colonization of the Murray Darling Basin from the coastal range or
during recent range contraction and population isolation (Pavlova, Gan,
et al., 2017). DArTseq has been used to find sex-linked loci and infer
sex-determination systems, including in catfish Clarias
gariepinus with homomorphic sex-chromosomes, where both XY and ZW
sex-determination were inferred based on the presence of moderately
male- and moderately female-linked loci (Nguyen et al., 2021).
Exploring male and female population pools of WGS data for Macquarie
perch with CMH tests guided us to focus on scaffold 633 region
90152-94017, enriched with sex-linked loci, including XY-gametologous
region 93182-94017, encompassing five SNPs and two indels in the 146-bp
sexing region 93182-93327, and an additional SNP at 94017. Female pools
had a single allele and male pools had two approximately equally
represented alleles for these six SNPs. Individual WGS genotypes showed
all females to be homozygous and 70% of males heterozygous at these
loci. Rare past or ongoing recombination within this XY-gametologous
region was suggested by 30% of males being homozygous for X- or Y-
alleles for up to seven of these eight loci. Comparisons of male and
female read depth for sex-linked loci and separately for sex-linked
alleles (Figs. 1, 2) suggested that some females and males might be
haploid for part of this XY-gametologous region (i.e. through a deletion
on one of the X-variants). Whereas similar read depth for males and
females for the first and last loci in this region (93182 and 94017)
implies diploidy of these SNP loci in both sexes, for intervening loci
93229, 93299, 93315 and 93327 most females appeared haploid (X0) for
this region: females had consistently lower depth than males (Fig. 1),
and similar depth for X-alleles as each of the X- and Y- alleles in
males (Fig. 2). Lower average X-allele depth in males than the Y-allele
suggests that some males could be haploid for Y (i.e. Y0).
Upstream of the XY-gametologous
region 93182-94017 was the region 90152-92914 containing 13 SNPs bearing
recent-Y-specific polymorphism, which might be a more recently emerging
region of sex-difference. Patterns of appearance of these
recent-Y-specific polymorphisms in males (Fig. C1) suggest that multiple
occasionally recombining Y-haplotypes are segregating in Macquarie
perch. The XY-gametologous region itself included another
recent-Y-specific SNP 93879, suggesting occasional recombination between
X and Y haplotypes (Fig. C1). The lower read depth for 93879 in males
compared to females suggested Y-specific deletions at this locus (Figs.
1 and 2). Manta analysis did not detect any structural variants that
would support X- or Y- deletion polymorphism consistent with these
patterns (Fig. D1), however our read depth coverage
(~10-20x) was much lower than the ~50x
used in original tests of Manta (Chen et al., 2016), potentially
underpowering our analysis.
Overall, in the panel of 22 loci with sex-biased patterns of
heterozygosity in scaffold 633 region 90152-94017 (8 XY-gametologs and
14 loci bearing recent-Y-specific polymorphism; Fig. C1), screened in
100 individuals from Dartmouth and Yarra, all females were homozygous
for X-alleles, and Y-alleles were present in 100% of males for 94017,
98% for 93229, 93299 and 93327, 96% of for 93182, 93184, 93278 and
93315, and in 14%-76% for the other loci. This suggests a working
hypothesis of sex-determination in which only individuals homozygous at
the X-alleles at all 22 of these loci develop a female phenotype,
whereas presence of Y-alleles at some of the loci leads to a male
phenotype. However, presence of the Y-allele at 93229 in 36% of King
Parrot Creek and Abercrombie females is not consistent with this
mechanism based on the Dartmouth and Yarra populations (Fig. B4). This
single deviation from the suggested model call for other mechanisms of
producing a female phenotype.
Tests of our molecular sexing protocol, that targeted the Y-allele of
93229 in the 146-bp sexing region of Macquarie perch scaffold 633, in
other populations of Macquarie perch, in golden perch, and in more
distant relatives from the family Percichthyidae (Murray cod and trout
cod) showed that this sexing region is Macquarie perch-specific, and
could even be limited to a subset of the species. This Y-allele was
present in all but one male out of 50 tested, hence the test should
correctly identify Dartmouth and Yarra individuals as males (based on
two or three bands on a gel) or females (based on one band), except
~2% of males may be missexed as females. The same test
is not expected to work well in other populations of Macquarie perch,
because 20% of putative females from King Parrot Creek and 60% from
Abercrombie had the male-specific allele. Unless these individuals are
feminized (egg-producing) males, female homozygosity for the reference
allele at 93299 might be limited to some populations. Unique variation
at these loci in amplicon sequencing for two coastal individuals of
unknown sex further suggests that sex-specific variation in Macquarie
perch could be specific to some populations. Intraspecific variation in
chromosomal locations of sex-determining loci occurs in other
vertebrates (Kijas et al., 2018a; Lubieniecki et al., 2015; Rodrigues,
Merilä, Patrelle, & Perrin, 2014). In the spotted snow skinkNiveoscincus ocellatus , highland and lowland populations, which
differ in their degree of influence of environmental temperature on sex,
shared- and population-specific sex-linked loci were detected,
suggesting a different degree of sex-chromosome differentiation between
populations (Hill, Burridge, Ezaz, & Wapstra, 2018). Different
sex-determination systems can evolve rapidly in populations of the same
species in response to loss of sex-chromosomes by drift. For example,
natural populations of zebrafish (Danio rerio ) have a monogenic
ZW sex-determination system, whereas polygenic sex-determination systems
of different laboratory strains have been inferred (Anderson et al.,
2012; Bradley et al., 2011; Liew et al., 2012) which evolved de-novo
after loss of W-alleles (Wilson et al., 2014).
Understanding the nature of homo/hemizygosity at the XY-gametologous
region of scaffold 633 was obscured here by the drop in read depth for
the sex-linked region compared to the genome-average, including zero
coverage around position 93400 for 59% of individuals regardless of
their overall sequence depth (Fig. 1). This lower depth might be linked
to difficulty of sequencing through a low-complexity region, including
the (GT)n microsatellite that ends around 93193 (Fig.
B1). Accumulation of repetitive sequences was previously reported for
golden perch and Murray cod sex-chromosomes (Shams et al., 2019). Given
its high read-depth coverage, amplicon sequencing may provide a more
reliable way of genotyping genomic regions that contain low-complexity
fragments. Despite the locally reduced read depth in WGS, WGS-based
genotypes were identical to amplicon-based genotypes for the four
individuals analysed with both techniques. Apparently real Y-allele
homozygotes were observed even for males with reasonable WGS depth,
including a male for which amplicon sequencing was done. Low depth also
did not preclude detecting sex-specific and population-specific patterns
of depth coverage (Fig. 2). Thus, an X0 or Y0 genotypes for some
XY-homologous loci in some individuals likely represents a biological
phenomenon. It is unlikely that the drop in depth for the scaffold 633
sexing region was due to failed mapping due to repetitive regions: BLAST
search of the 146-bp sexing region did not find highly similar regions
in other Macquarie perch scaffolds.
The scaffold 633 sex-linked region 90152-94017 was not annotated to a
known gene, but was located ~39 Kb upstream of gene ofSOX transcription factor family, and 41 Kb downstream of a
protein with Dbl homology (DH) domain superfamily, associated
with guanyl-nucleotide exchange factor activity and protein binding
(interacting selectively with any protein or protein complex).SOX genes regulate fate of stem and progenitor cells during
development (Sarkar & Hochedlinger, 2013). Three genes in particular,SOX9 , SOX8 (Koopman, 2005) and SOX3 , a progenitor
of the SRY gene in mammals (Graves, 2013), have a role in male
sex determination in different species, with their expression inducing
testes development. Thus, it is possible that SOX gene g10286,
modulated by the variation within the sex-linked region 90152-94017,
controls gonadal development in Macquarie perch.
Alternatively, polygenic sex-determination in Macquarie perch is
possible, given the many loci detected as putatively female- and
male-linked by the CMH tests (at p<1e-5; Supplementary
Material S3). In sea bass Dicentrarchus labrax at least four loci
are associated with sex determination (Palaiokostas et al., 2015). Also,
both XY and ZW sex-determining systems are present on different
chromosomes in some Metriaclima cichlids, with epistasis between
genotypes at two loci controlling sex (Ser, Roberts, & Kocher, 2010).
Three alleles (X, Y, and W) segregate at the sex determination locus and
interact in platyfish Xiphophorus maculatus (Schultheis, Böhne,
Schartl, Volff, & Galiana-Arnoux, 2009). Future studies are needed to
demonstrate whether sequence variation at the sex-linked region on
Macquarie perch scaffold 633 has mechanistic links with development of
female or male phenotypes. Acquiring WGS data for known-sex samples from
other populations will be crucial for improving our understanding of
sex-determination in this species. Sex-determination in Macquariaappears to be complex and dynamic, potentially involving multiple sites
or genomic regions and different mechanisms in quite closely-related
lineages, including within species.