Introduction
Ectothermic vertebrates are highly vulnerable to climate change and
chemical pollution, because several aspects of individual development,
including sex differentiation depend on environmental conditions in
numerous species of reptiles, amphibians and fish (Bókony, Kövér,
Nemesházi, Liker, & Székely, 2017; Bókony et al., 2018; Eggert, 2004;
Holleley, Sarre, O’Meally, & Georges, 2016; Orton & Routledge, 2011;
Ospina-Álvarez & Piferrer, 2008; Tamschick et al., 2016). In species
with genetic sex determination, thermal and chemical disturbances during
embryonic or larval development can cause sex reversal, meaning that
genetically female individuals become phenotypic males, or vice
versa (Eggert, 2004; Holleley et al., 2016; Ospina-Álvarez & Piferrer,
2008). Laboratory experiments show that sex-reversed individuals of some
species may have reduced reproductive success (Harris et al., 2011;
Senior, Nat Lim, & Nakagawa, 2012), and theoretical studies suggest
that sex reversals may lead to serious consequences for natural
populations, including changes in genetic variability, distorted sex
ratios, and even extinction (Bókony et al., 2017; Quinn, Sarre, Ezaz,
Marshall Graves, & Georges, 2011; Wedekind, 2017). Therefore, it is
imperative to gain information on the prevalence and fitness of
sex-reversed individuals in natural populations, to be able to assess
and forecast the effects of anthropogenic environmental changes.
For studying sex reversal, one needs to identify not only the phenotypic
sex but also the genetic sex of each individual. The latter can be
especially difficult in non-model organisms, due to lack of information
on sex-linked DNA sequences. Because of their highly conserved sex
chromosome system, universal sex-linked DNA markers have long been
available for birds and mammals (Fridolfsson & Ellegren, 1999;
Griffiths & Tiwari, 1993; Shaw, Wilson, & White, 2003), making
molecular sexing a routine in these taxa. However, in the majority of
ectothermic vertebrates, sex chromosome turnover (i.e. the swapping of
the chromosome used for genetic sex determination) is common and the sex
chromosomes of many species are homomorphic (Devlin & Nagahama, 2002;
Holleley et al., 2016; Jeffries et al., 2018; Miura, 2017).
Consequently, there is often little homologous sex-linked variation
between and sometimes even within species, making molecular sexing
challenging (Ezaz, Stiglec, Veyrunes, & Marshall Graves, 2006; Perrin,
2009; Stöck et al., 2013). Furthermore, type of sex-chromosome system
(i.e. male or female heterogamety) can differ between closely related
species or even between different populations of the same species,
especially in amphibians (Holleley et al., 2015; Rodrigues et al., 2017;
Sarre, Ezaz, & Georges, 2011).
For the above reasons, genetic sexing methods need to be developed and
validated species by species in amphibians. Recombination between the
sex chromosomes (Ezaz et al., 2006; Perrin, 2009; Stöck et al., 2013) is
expected to be reduced in the vicinity of the ‘master sex-determination
gene’ (Bachtrog, 2006; Bachtrog et al., 2014; van Doorn & Kirkpatrick,
2007), providing a preferential target for sex marker development.
Unfortunately, the master sex-determination gene remains elusive in all
but a few amphibian species (Eggert, 2004; Miura, 2017; Nakamura, 2013;
Yoshimoto et al., 2010), and the size of the non-recombining region
around it can be small. Thus, in order to find markers which make
reliable identification of the sex chromosomes possible in the species
of interest, researchers must test high numbers of loci across the
genome (Lambert, Skelly, & Ezaz, 2016; Olmstead, Lindberg-Livingston,
& Degitz, 2010; Stöck et al., 2011). Owing to these challenges,
reliable sex-linked markers only exist for a handful of amphibian
species so far (Alho, Matsuba, & Merilä, 2010; Berset-Brändli,
Jaquiéry, Dubey, & Perrin, 2006; Brelsford, Lavanchy, Sermier, Rausch,
& Perrin, 2017; Eggert, 2004; Lambert et al., 2016; Ma, Rodrigues,
Sermier, Brelsford, & Perrin, 2016; Olmstead et al., 2010; Rodrigues et
al., 2017; Stöck et al., 2011).
Due to this general lack of sex markers, we know troublingly little
about sex reversals in nature: how widespread they are, which
environmental factors they are associated with, and how they affect
individual fitness and population viability. To our knowledge, the
frequency of sex reversal in the wild has been published for only two
amphibian species so far: 9% of genetic females were phenotypically
male in a Finnish common frog (Rana temporaria ) population, while
8.5% female-to-male and 3% male-to-female sex reversal was found in
green frogs (Rana clamitans ) in the USA (Alho et al., 2010;
Lambert, Tran, Kilian, Ezaz, & Skelly, 2019).
In this study, we investigated sex reversals in the agile frog(Rana dalmatina) . This species is widespread in Europe, but its
population sizes show a decreasing tendency (Kaya et al., 2009). It
inhabits light deciduous woodlands, but also occurs near or in urbanized
areas. Similarly to most Rana species, its diploid karyotype
consists of 26 chromosomes (Spasić-Bošković, Tanić, Blagojević, &
Vujošević, 1997); its sex chromosomes were identified only recently,
showing a male-heterogametic (XX/XY) sex-determination system (Jeffries
et al., 2018). Because no molecular sexing method has been published for
agile frogs yet, first we searched for sex-linked markers using an
existing Restriction Site Associated sequencing (RADseq) dataset
(Jeffries et al., 2018) and validated them to provide a reliable genetic
sexing method for this species. Subsequently, we studied the occurrence
of sex reversals in wild agile frog populations in North-Central
Hungary, and tested if sex reversals are more common in populations
associated with anthropogenic land use. Finally, we examined if sex
reversal was associated with fitness costs by comparing fitness-related
traits between sex-reversed and normal individuals.