Discussion
We identified three loci carrying sex-linked SNPs in agile frog
populations in Hungary. Based on a genome sequence assembly of a closely
related species, the common frog, we assume that the sex markers
reported here cover a suitably large region of the sex chromosomes (Rds1
and Rds3 being at 112 million nucleotides from each other). Furthermore,
genetic sex based on Rds3 corresponded to the sexual phenotype in 95%
of all laboratory-raised individuals, and all discrepancies were found
to be likely cases of sex reversal (as discussed in more detail below).
Therefore, we conclude that parallel usage of the best performing
markers Rds1 and Rds3 is suitable for molecular sexing in the
North-Central Hungarian populations, yielding at least 95% confidence
for individual sexing (allowing for the possibility that the 5%
mismatch between Rds3 genotype and phenotypic sex had been caused by
recombination; Ezaz et al., 2006; Perrin, 2009; Stöck et al., 2013) and
good statistical power for comparing populations or experimental groups.
Because amphibian sex determination can vary even within species (Miura,
2017; Rodrigues, Merilä, Patrelle, & Perrin, 2014), the reliability of
our sex markers should be tested before applying them in other,
especially distant populations (Lambert et al., 2016; Rodrigues et al.,
2014). However, genetic diversity of the agile frog is in general very
low across Europe (Vences et al., 2013), suggesting that our markers may
be sex-linked in other agile frog populations as well. Thus, our genetic
sexing method enables further studies on environment-induced sex
reversal in this declining species, potentially throughout its
distribution range.
According to our markers, 6 out of 125 laboratory-raised froglets were
genetically females (XX) with male phenotype (testes), despite being
raised under controlled conditions with presumably no sex-reversing
effects. There are several potential explanations to consider for these
mismatches. First, phenotypic sex might have been erroneously
categorized; however, we can exclude this possibility because the
phenotype based on gonad morphology was corroborated by histology in the
mismatching individuals. Second, the presence of sex races could result
in false assumption of sex reversal; for example, in the common frog,
some individuals develop ovaries first that turn to testes later
Rodrigues et al. (2015). This would cause overestimation of the
proportion of XY females, which we did not find in our study at all.
Third, the mismatches may have been due to recombination (Ezaz et al.,
2006; Perrin, 2009; Stöck et al., 2013); however, 4 out of the 6
concerned froglets had XY siblings in our sample, suggesting that both
Rds3 and Rds1 genotypes of chromosome Y were normal in their families.
Furthermore, all mismatching individuals showed some signs of poor
condition, and we are not aware of any reason why recombination would be
associated with the deficits we detected. The fourth interpretation is
that the mismatching individuals were indeed sex-reversed, which we
consider most likely. Recent studies suggest that sex reversal may be a
natural phenomenon in ectothermic vertebrates (Holleley et al., 2016;
Lambert, 2015; Lambert et al., 2019), due to dosage-dependent sex
determination where stochastic variation in gene expression levels may
lead to sex reversal (Perrin, 2016). Alternatively, but not mutually
exclusively, sex reversals may result not only from random variation but
also from stressful stimuli, as experiments with fishes showed that
various forms of physiological stress can induce sex reversal, and
”stress hormones” (activated by the hypothalamus-pituitary-interrenal
glands axis) mediate this process (Castañeda Cortés et al., 2019;
Fernandino, Hattori, Moreno Acosta, Strüssmann, & Somoza, 2013).
Therefore, we suspect that a few of our lab-raised animals experienced
relatively high levels of physiological stress despite the generally
favourable lab conditions, and this led to sex reversal. Their
developmental abnormalities may have been either the cause or the
consequence of the stress that ultimately caused their sex reversal; in
either case, our findings suggest that sex reversal can be associated
with reduced health and poor fitness prospects. For example, enlarged
spleen may indicate infections (Hadidi, Glenney, Welch, Silverstein, &
Wiens, 2008), and small body mass predicts low chances of surviving the
winter hibernation (Üveges et al., 2016) and low future reproductive
success (Reading & Clarke, 1995; Vági & Hettyey, 2016).
Despite the above findings suggesting that sex-reversed individuals
might have poor viability in nature, we found a relatively high number
of sex-reversed adults in free-living agile frog populations.
Genetically XX phenotypic males made up ca. 20% of phenotypic males,
and ca. 35% of genetic females, although the latter rate of
female-to-male sex reversal is probably overestimated because we had
relatively low capture success (small sample size) for females. These
numbers are relatively high compared to those reported for natural
populations of two other frog species (Alho et al., 2010; Lambert et
al., 2019). Interestingly, we found no difference in body mass between
sex-reversed and normal adult males, despite the fact that some of the
sex-reversed juveniles in the lab had seriously reduced body mass. This
suggests that those sex-reversed individuals that survive to adulthood
in nature may be able to mate, because male body size influences success
in competition for mates (Vági & Hettyey, 2016). Their reproduction
might still fail, however, if sex reversal reduces fertility, as
reported in fish (Senior et al., 2012) and indicated by some of our
findings with the lab-raised froglets, i.e. three sex-reversed juveniles
had small testes and two of them had testicular oogonia (intersex).
However, other findings of our study suggest that at least some of the
sex-reversed individuals may be fertile. First, four out of six
sex-reversed froglets showed normal testicular histology, and three of
them had relatively large testes. Second, we found one family that was
likely to be sired by an XX male: 12 laboratory-raised animals that were
randomly chosen as eggs from a single clutch were all XX individuals,
which would have a very low chance of happening merely by accidental
sampling if the clutch had the theoretically expected 1:1 sex ratio (ca.
0.0002 probability). Sex-reversed individuals were found to be fertile
in some ectothermic vertebrates (Devlin & Nagahama, 2002; Edmunds,
McCarthy, & Ramsdell, 2000; Holleley et al., 2015), and in common frogs
XX males appear to be fertile and as successful in mating as XY males
(Alho et al., 2010; Veltsos et al., 2019). If sex-reversed individuals
do reproduce in nature, the biased sex ratios of their progeny may lead
to changes in the population sex ratio, sex-chromosome frequencies, and
ultimately the sex-determination system (Bókony et al., 2017; Quinn et
al., 2011; Wedekind, 2017). Furthermore, the offspring of sex-reversed
individuals may themselves be more susceptible to sex reversal, as
suggested by empirical results from lab experiments (Holleley et al.,
2015; Shao et al., 2014).
We found higher female-to-male sex-reversal frequency in breeding
populations exposed to anthropogenic land use. However, due to the
availability of agile frog populations, our capture sites with different
levels of anthropogenic land use were unequally distributed such that
most sites West/South of the river Danube had little anthropogenic
influence whereas most sites East/North of the Danube were highly
anthropogenic (Figure S5). Also, the lab-raised animals that we used for
validating the markers originated from three western populations (Table
1). Therefore we cannot exclude the possibility that the differences we
observed in genotype-phenotype mismatches among the free-living
populations were due to phylogenetic correlation, i.e. an inherited
tendency for more frequent sex reversal in populations East/North of the
Danube, or different patterns of linkage disequilibrium between our
markers and the master sex-determination gene in these populations (e.g.
higher recombination rate in the eastern populations). For example, in
common frogs, sex-chromosome differentiation in Switzerland is mainly
explained by a major alpine ridge separating the populations (Phillips,
Rodrigues, Jansen van Rensburg, & Perrin, 2020). Alternatively, a
mutation on chromosome Y at Rds3 (e.g. at the primer binding site,
resulting in null allele) or a more complicated sex-determination system
present on the East side could cause spatial genetic population
structure (Oike et al., 2017; Rodrigues et al., 2014). These alternative
explanations could be ruled out using phylogeographic information based
on neutral autosomal loci in our populations, or by sexing lab-raised
froglets from the eastern populations as well, but unfortunately such
data are not available. However, in the region we studied, there are no
high mountains or other likely geographical barriers to gene flow
between these populations, because rivers like the Danube are not
expected to be significant barriers for migration in species like the
agile frog (Decout, Manel, Miaud, & Luque, 2012). Further, our study
sites lie relatively close to each other, mostly within ca. 40 km;
genetic structure at such small spatial scale in agile frogs is more
likely the outcome of habitat fragmentation than isolation by distance
(Lesbarrères et al. 2006, Sarasola-Puente et al. 2012). Highways pose
migration barriers for agile frogs (Lesbarrères et al. 2006,
Sarasola-Puente et al. 2012); however, the distribution of main roads
and highways between our study sites is more likely to reduce migration
along a North-South cline than separating East from West Figure S5).
Taken together, we have little reason to expect an East-West population
differentiation in our study. Our only population East/North from the
Danube that had low anthropogenic land cover (pond ”B” in Figure S5) had
high frequency of XX males; however, this pond was created from a closed
quarry and was subjected to reconstruction works about a decade ago. In
our study of chemical pollutants in anuran habitats in 2017, we found
the highest concentration of phthalates in this latter pond (Bókony et
al., 2018). Therefore, we believe that our results reflect a genuine
effect of anthropogenic environmental change on sex-reversal
frequencies.
Our results suggest that both urbanization and agriculture may
contribute to the observed relationship between sex-reversal frequency
and anthropogenic land use. Both kinds of anthropogenic habitats are
polluted by various chemicals, many of which have demonstrated
sex-reversing effects (Eggert, 2004; Hayes et al., 2002; Kloas, Lutz, &
Einspanier, 1999; Nakamura, 2013; Reeder et al., 1998; Tamschick et al.,
2016). Our result that sex reversal occurred even in the least
anthropogenic habitats concurs with our earlier finding that those
habitats are not devoid of chemical pollutants either (Bókony et al.,
2018; see also Figure S5). Furthermore, the increased female-to-male
sex-reversal rate that we found in urban agile frog populations may as
well be due to the urban heat island effect which makes urban ponds
warmer than rural ponds (Brans, Engelen, Souffreau, & De Meester,
2018), given that high temperature during larval development is a known
inducer of sex reversal (Bókony et al., 2017; Chardard, Penrad-Mobayed,
Chesnel, Pieau, & Dournon, 2004; Lambert et al., 2018). This variety of
chemical, thermal, and potentially other stressors might complicate the
relationship between sex-reversal rate and anthropogenic land use. In
line with this, no correlation was found between sex-reversal frequency
and urbanization along a forest-suburban gradient in green frogs
(Rana clamitans ; Lambert et al., 2019), although the frequency of
testicular oocytes was found to increase with urban land cover (Skelly,
Bolden, & Dion, 2010). Similarly, several but not all studies found a
positive association between agricultural land use and amphibian
intersex (Orton & Tyler, 2015), laryngeal demasculinization (Zlotnik,
Gridi-Papp, & Bernal, 2019) and reduced spermatogenesis (McCoy, Amato,
Guillette, & St. Mary, 2017). These reports together with our results
emphasise the need for further studies on sex-reversal frequency and its
causes in wild populations of vertebrates with environmentally
susceptible sex determination. Adult sex ratio has shifted towards males
over the last decades in some amphibian species (Bókony et al., 2017),
and this skew might be a consequence of sex reversals becoming more
common due to anthropogenic environmental changes of land use and
climate. The high frequency of female-to-male sex-reversal we found in
this study suggests male-biased sex ratios and consequently reduced
effective population sizes that might especially affect populations
living in anthropogenic environments. Thus, we urgently need data on the
survival and reproduction of sex-reversed individuals and their
demographic effects on natural populations; developing novel sex markers
for non-model species will be a key step in this endeavour.