Higher sex-reversal rate of urban frogs in a common-garden experiment suggests adaptive microevolution

Higher sex-reversal rate of urban frogs in a common-garden experiment suggests adaptive microevolution | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 29 November 2024 V1 Latest version Share on Higher sex-reversal rate of urban frogs in a common-garden experiment suggests adaptive microevolution Authors : Veronika Bókony 0000-0002-2136-5346 [email protected] , Emese Balogh , Zsanett Mikó , Andrea Kásler , Zoltán Örkényi , and Nikolett Ujhegyi Authors Info & Affiliations https://doi.org/10.22541/au.173289954.47719156/v1 Published Evolutionary Applications Version of record Peer review timeline 298 views 132 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Ectothermic vertebrates with genotypic sex determination may adjust their sexual phenotype to early-life environmental conditions by sex reversal, with diverse theoretical consequences for population dynamics and microevolution. Environments with frequent sex-reversing effects may select for or against sex-reversal propensity depending on the relative fitness of sex-reversed individuals. Yet, empirical data on the adaptive value and evolutionary potential of rex reversal is scarce. Using a common-garden experiment with agile frogs (Rana dalmatina) that respond to larval heat stress by sex reversal, we demonstrate that sex-reversal rate is higher in sibgroups originating from anthropogenic habitats where sex-reversing heat events are more frequent, compared to populations inhabiting cooler woodlands. Furthermore, among genotypically female juveniles, phenotypic males are larger than sex-concordant females. These results suggest that sex-reversal propensity increased in anthropogenic environments by adaptive microevolution. Thus, environmental sex reversal may provide an adaptive strategy for ectothermic vertebrates to cope with challenges of the Anthropocene. Introduction Ongoing anthropogenic environmental changes are exposing life on Earth to systematic alterations in biotic and abiotic factors, and this has been accompanied by a multitude of phenotypic changes in wild populations (Alberti et al. 2017; Johnson & Munshi-South 2017; Parmesan 2006; Seress & Liker 2015; Turcotte et al. 2017). Although some of these phenotypic changes may be neutral to fitness or even maladaptive, like the phenological mismatches between trophic levels (Renner & Zohner 2018), a growing body of evidence demonstrates their adaptive value (Lambert et al. 2021; Liker 2020). The mechanisms underlying these phenotypic changes are of particular importance because different processes may enable the most efficient phenotypic adaptation under different circumstances, such as different generation times and gene-flow levels (Reed et al. 2011). Microevolution can result in local adaptation (Fig. 1AB); for example, fish populations in polluted habitats have evolved tolerance against deadly contaminants (Whitehead et al. 2012). This requires heritable variation in the phenotypic trait, although transgenerational changes may also happen by epigenetic responses to the environment (Hammond et al. 2016). Phenotypic plasticity during the individuals’ lifetime can also produce adaptive phenotypes (Fig. 1C); for example, urban animals often show reduced behavioral fear-responses to repeated human disturbance, and habituation can play an important role in this (Samia et al. 2015). Microevolution and phenotypic plasticity can also act together (Fig. 1D); for example, both processes seem to have contributed to the higher heat tolerance of water fleas in warm urban ponds (Brans et al. 2018b). Furthermore, plasticity itself can evolve (Fig. 1E), resulting in different reaction norms in different populations (Crispo et al. 2010; Matesanz & Ramírez‐Valiente 2019). An interesting form of phenotypic plasticity is environmentally sensitive sex determination. Its most well-known form is temperature-dependent sex determination (TSD), such as in crocodilians and many turtles and fish, whereby the individual’s phenotypic sex is determined by the thermal conditions experienced during a sensitive window during early development (Edmands 2021). Both theoretical models and empirical data suggest that TSD is an adaptive strategy when the early environment has sex-dependent effects on fitness prospects (Bock et al. 2023; Schwanz et al. 2016; Valenzuela 2021). However, under rapid unidirectional environmental change, TSD may become maladaptive by failing to produce one of the two sexual phenotypes altogether (Mitchell & Janzen 2010). A special kind of environmentally sensitive sex determination is environmental sex reversal, which can occur in species that exhibit genotypic sex determination. In such species, early environmental stimuli may override the effects of sex chromosomes or of a polygenic sex-determining system, resulting in discordance between phenotypic and genotypic sex (Edmands 2021). Traditionally, it was thought that sex reversal is a rare deviation from the norm or a side effect of unnatural environmental conditions, typically confined to laboratory experiments (Lambert 2015). However, a recent surge of empirical evidence indicates that sex reversal may be much more widespread than previously recognized in ectothermic vertebrates under ecologically relevant circumstances (reviews by Baroiller & D’Cotta 2016; Holleley et al. 2016; Nemesházi & Bókony 2022). Theoretically, sex reversal may be an adaptive sex-allocation strategy similarly to TSD, allowing the individual to express the sexual phenotype that best matches its environment (Geffroy & Douhard 2019). Empirically, however, we know next to nothing about the adaptive value of sex reversal in the wild (Wild et al. 2022), and the scant data from studies done on captive animals are contradictory (Bókony et al. 2021; Holleley et al. 2015; Senior et al. 2012). Environmental sex reversal is highly relevant for understanding the effects of anthropogenic environmental change, because the triggers of sex reversal coincide with the environmental factors that are being most conspicuously altered by humankind, such as thermal and chemical conditions (Baroiller & D’Cotta 2016; Holleley et al. 2016; Nemesházi & Bókony 2022). Theoretical models predict that, under environmental change, sex reversal can have complex and far-reaching consequences for population persistence and microevolution, and the outcomes depend on many characteristics of the species as well as the environment (Bókony et al. 2017; Grossen et al. 2011; Nemesházi et al. 2021; Schwanz et al. 2020). One such decisive characteristic is the relative fitness of sex-reversed individuals. If the traditional view is right, and sex reversal is a negative side effect of environmental stress, it might endanger biodiversity by facilitating population collapses in environmentally sensitive species (Nemesházi et al. 2021). However, if sex reversal is adaptive, it might help populations cope with the challenges of ongoing environmental change (Pen et al. 2010; Schwanz et al. 2013). In both cases, we can expect changes in the propensity for sex reversal in response to anthropogenic environmental change (Nemesházi et al. 2022). If sex reversal is disadvantageous, resistance to it may be adaptive in environments where sex-reversing stressors are pervasive. For example, if sex reversal is a side effect of heat stress suffered during early ontogeny, individuals that are less prone to heat-triggered sex reversal may be favored in urban habitats, where temperatures are higher and heat waves are more frequent due to the urban heat island effect (Bókony et al. 2024a; Brans et al. 2018a). On the other hand, if sex reversal is beneficial under certain conditions, it may be most selected for in environments where such conditions occur often. For example, if early-life heat stress affects males and females differently, then heat-induced sex reversal may allow individuals to choose the sexual phenotype that maximizes their fitness given their thermal experiences. This capacity for sex reversal may have the highest pay-off in environments where heat waves are common, such as urban habitats or agricultural areas where shading vegetation is missing. In contrast, sex-reversal capacity may be neutral in habitats where heat waves rarely ever happen. To test these alternative hypotheses, we studied the agile frog ( Rana dalmatina ), an emerging model system of ecologically relevant sex reversal. This species is common in Europe, although its numbers are declining; it prefers light woodlands but also occurs in various anthropogenically modified habitats (IUCN SSC Amphibian Specialist Group 2023). It has an XX/XY sex-chromosome system, and a few-days heat wave around the middle of larval development causes genotypic females to develop into phenotypic males (Mikó et al. 2021). In wild populations, 20% of phenotypically male adults are genotypic females, especially in urban and agricultural areas (Nemesházi et al. 2020). Earlier studies that compared fitness-related traits between sex-reversed and sex-concordant agile frogs found heterogeneous results (Bókony et al. 2021; Mikó et al. 2021; Nemesházi et al. 2020), but none of those studies was able to make the most relevant comparison: between heat-exposed genotypic females that developed the male vs. female phenotype (i.e. those that did vs. did not undergo sex reversal in response to the same heat stress). In the present study, we performed a common-garden experiment by raising agile frogs from eggs collected in anthropogenic habitats and natural woodlands together in captivity, and exposed half of them to a simulated heat wave to measure the rate of sex reversal. We predicted that, if the tendency for sex reversal is selected for in ”hot” environments by microevolution, individuals originating from anthropogenic habitats should be more likely to undergo sex reversal than individuals originating from woodlands when experiencing the same thermal conditions during their ontogeny. Furthermore, we measured larval development time and early-life growth, both of which can affect lifetime fitness in amphibians (Altwegg & Reyer 2003; Smith 1987). We predicted that, if sex reversal is adaptive in agile frogs, the negative effect of heat stress on fitness-related traits should be smaller in individuals that undergo sex reversal compared to those that do not respond to the heat stress by sex reversal. Material and methods Experimental procedures This study was approved by the Ethics Committee of the Plant Protection Institute and licensed by the Environment Protection and Nature Conservation Department of the Pest County Bureau of the Hungarian Government (PE-06/KTF/00754-8/2022, PE-06/KTF/00754-9/2022, PE-06/KTF/00754-10/2022, PE/EA/295-7/2018, PE/EA/00270-6/2023). We used six study sites that are described in detail in an earlier study (Bókony et al. 2024a): three ponds with <0.1% anthropogenically modified habitat within 500 m, and three ponds in three different townships with ca. 70% anthropogenically modified land cover (including urban and agricultural areas) within 500 m. Water temperatures in the tadpole developing season are significantly higher in the latter than in the former (Bókony et al. 2024a). For simplicity, we are henceforth referring to the three anthropogenic sites as ”urban”. We collected freshly spawned eggs from the six ponds on 13-14 March 2023. From each pond, we took ca. 50 embryos from each of six egg masses and transported them to our laboratory. We kept each group of siblings (henceforth ”sibgroup”) in a separate container with ca. 1 cm deep reconstituted soft water (RSW; 48 mg NaHCO 3 , 30 mg CaSO 4 × 2 H 2 O, 61 mg MgSO 4 × 7 H 2 O, 2 mg KCl added to 1 L reverse-osmosis filtered, UV-sterilized, aerated tap water). Over the course of the study, temperature in the lab was set to gradually increase from 16 °C to 22 °C (mean ± standard deviation: 19.7 ± 1.7 °C), and we adjusted the photoperiod weekly to mimic the natural dark-light cycles. When the animals reached the free-swimming state, i.e. developmental stage 25 according to Gosner (1960), we started the experiment on 28 March. We haphazardly selected 20 healthy-looking tadpoles from each sibling group (N = 720 in total) and placed them individually in 2-L plastic rearing containers filled with 1 L RSW, arranged in a randomized block design to ensure that all six populations were homogeneously distributed across the shelves in the laboratory. We changed the rearing water twice a week and fed the tadpoles ad libitum with chopped, slightly boiled spinach. The remaining tadpoles were released at their ponds of origin. Two weeks after starting the experiment, we applied heat treatment for 6 days following our previously described methods (Ujszegi et al. 2022). During this period, all tadpoles were kept in 1.7 L RSW which we changed every other day; at the same times we fed them a reduced (ca. 1/3) amount of spinach to prevent water fouling and anoxia in the heat treatment. Throughout the treatment period, the tadpole-rearing containers were kept in groups of 12 in a tray of water which was circulated by a water pump and, for the heat-treatment group, heated to 27.7 ± 0.4 °C (mean ± SD) by a thermostat aquarium heater. In the control treatment, the water was kept at room temperature, which was 18.2 ± 0.5 °C (mean ± SD) during the treatment period. From each sibling group, we randomly assigned 10 tadpoles to each treatment, resulting in 60 heat-treated and 60 control tadpoles from each pond. After the treatment, we raised the tadpoles using the same methods as during their first two weeks of larval development. When an individual initiated metamorphosis as marked by the emergence of forelimbs (development stage 42), we measured body mass (± 0.1 mg), and we replaced the rearing water with 0.1 L fresh RSW. The containers were slightly lifted on one side to ensure a dry surface inside, and covered with a transparent, perforated lid. At complete tail resorption (development stage 46), we moved the froglet into a similar, new container, lined with wet paper towels and a piece of egg carton as a shelter. The froglets were fed twice a week with small crickets ( Acheta domesticus) sprinkled with a mixture of vitamins, minerals and amino acids. Phenotypic sexing was performed when the froglets were 9-12 weeks old after finishing metamorphosis; by this time the gonads are well differentiated and easy to observe. Taking individuals in a stratified random order which ensured that age at dissection was homogeneously distributed across populations and treatments, we humanely euthanized them in a shallow water bath of 6 g/L tricaine-methanesulfonate (MS-222) buffered to neutral pH with the same amount of disodium hydrogen phosphate. To ensure death, the animals were left in the bath for at least one hour. Then we removed both feet from each individual using flame-sterilized equipment and stored the tissue samples in 96% ethanol for genotypic sexing. We dissected the animals and recorded whether they had testes or ovaries using an APOMIC SHD200 digital microscope. Because the digestive tract often contained food remains, we cut it out, measured its mass (± 0.01 g) and subtracted it from total body mass measured right before euthanasia to obtain ”net body mass” (henceforth ”body mass”). Genotypic sex was diagnosed using one foot sample per individual, following the protocol described by Nemesházi et al. (2020). In short, we extracted DNA using E.Z.N.A. Tissue DNA Kit (Omega Bio-tek) following the manufacturer’s protocol, except that digestion time was overnight. We measured DNA concentration in all samples using a NanoDrop 1000 spectrophotometer (Thermofisher Scientific). We tested all froglets for sex marker Rds3 using high-resolution melting (HRM; for melting curves see Fig. S1 in the Supplementary Material). The total HRM reaction volume was 15 μl, containing 3 μl 5× HOT FIREPol® EvaGreen® HRM Mix, without ROX (Solis BioDyne), 1 μl forward and 1 μl reverse primer (10 μM each), and 80-100 ng genomic DNA in MQ water to reach the final volume. Reactions were performed in a Roche LightCycler® 96 qPCR Instrument and the results were analyzed with the LightCycler 96 v.1.1.0.1320 qPCR software (Roche Diagnostics International LTD). When Rds3 genotype did not match phenotypic sex, the individual was tested for sex marker Rds1 using conventional PCR, and sex reversal was accepted only if both markers confirmed sex reversal. In the present study, all individuals were successfully genotyped, and there was no mismatch between the two markers. Statistical analysis We performed the analyses using R version 4.3.2 (R Core Team 2023). With the analysis of sex-reversal rate, we faced several difficulties due to the nature of the data. First, our response variable was binary, and it had zero variation in certain groups; this phenomenon is called separation in binomial models. Specifically, there were no sex-reversed individuals in 16 out of the 36 sibgroups, and in the control group from one of the three woodland sites. Second, the effects of site and sibgroup are nested in the effect of habitat type. These issues make parameter estimation uncertain with the otherwise ideal model structure where habitat type is a fixed effect and site and sibgroup are nested random effects. However, simply omitting the random effects might lead to over-estimation of the degrees of freedom. Therefore, we analysed the data in two steps. In the first step, we explored the level of non-independence in the data by testing various random-effects structures to identify which groups of data are correlated. We used the ‘glmer’ function from the ‘lme4’ package to run generalized linear mixed models with binomial error. Three of the models had random intercepts only (sibgroup, or site, or sibgroup nested in site), whereas three models additionally had random slopes to allow for the effect of treatment to vary among sibgroups, or sites, or both; the seventh model had no random effects. In every model, the fixed effects were habitat type (urban or woodland), treatment (control or heated), and their interaction. The dependent variable was phenotypic sex (i.e. we modelled the likelihood of becoming male), and the dataset was restricted to genotypic females. We used likelihood ratio tests to compare model fit between nested models. When dropping a random effect significantly reduced model fit as expressed by Akaike’s information criterion (AIC), we concluded that the data within the categories of that random effect were non-independent. In the second step, we aggregated the data by the random factor that we had identified in the first step as a source of non-independence. Specifically, we aggregated the data by sibgroup (see Results) by calculating the proportion of sex-reversed genotypic females for each sibgroup. We also did this separately for the control and the heat-treated animals. We used the function ‘glm’ for running generalized linear models with binomial error to test the fixed effect of treatment separately for urban and woodland sibgroups, and we estimated the 84% confidence intervals (CI) of these effects using the ‘emmeans’ package. We compared the two CIs to test if the treatment effect differed by habitat type (note that the lack of overlap between two 84% CIs is equivalent to a significant difference, i.e. to a 95% CI around the difference that does not include zero). Then, because the treatment effects did not differ (see Results), we tested the overall effect of habitat type on the dataset aggregated only by sibgroups (i.e. sums of control and heat-treated animals for each sex). This way, we tested three comparisons on the aggregated data; we corrected the significance values from these three tests for the false discovery rate (Pike 2011). To compare fitness-related traits between heat-treated genotypic females that did vs. did not undergo sex reversal, we analysed two dependent variables: time to metamorphosis and net body mass at dissection. For these analyses we used linear mixed-effects models, ran with the ‘lme’ function of the ‘nlme’ package to allow for heteroscedasticity. We included treatment, habitat type, and sex (XX female, XX male, or XY male) as fixed factors with all their two-way and three-way interactions, and we allowed different variance for each combination of the fixed factors using the ‘varIdent’ function. As random factor, we included sibgroup identity. For froglet mass, we added the age from the completion of metamorphosis as a numeric covariate. For both models we calculated type-2 analysis-of-deviance tables using the ‘Anova’ function of the ‘car’ package. Then we simplified each model by omitting all non-significant terms excepting sex × treatment, and from the simplified models we tested the difference between heat-treated XX females and XX males using the ‘emmeans’ function. Finally, we simplified the models further by dropping all non-significant terms to test the effects of treatment and sex without interaction. For residual diagnostics, we used the ‘DHARMa’ package for the binomial models, and standard residual plots for the linear models. These diagnostics showed that all statistical requirements were met by our data. We report mean estimates with ± standard errors (SE). The annotated script of our analyses, including R output, is available in FigShare (see Data Availability Statement). Results By the end of the experiment, sample size decreased to 438 froglets. Mortality (Table 1) was similar in the heat-treated group (36.1%) as in the control group (35%) but almost twice as high among animals that originated from urban habitats (45%) than among animals that originated from woodlands (26.1%; for further details see Fig. S2 in the Supplementary Material). Out of the froglets that survived to the end of the experiment, 225 (51.4%) were genotypically female, and 38 (16.9%) of those were sex-reversed (Table 1). Sex-reversal rate differed between sibgroups, as sibgroup was significant as a random intercept although not as a random slope (Table 2). Heat treatment significantly increased sex-reversal rate (Fig. 2) in urban sibgroups (odds ratio: 4.93 ± 2.63, p = 0.008, 84% CI: 2.33 - 10.40) as well as in woodland sibgroups (odds ratio: 3.46 ± 2.13, p = 0.043, 84% CI: 1.46 - 8.21); the overlap between the CIs indicates that the magnitude of the treatment effect did not differ significantly by habitat type. Sibgroups originating from urban habitats had significantly higher sex-reversal rates (0.23 ± 0.04) than sibgroups originating from woodlands (0.12 ± 0.03; odds ratio: 2.34 ± 0.85, p = 0.029; Fig. 2). Heat treatment resulted in longer larval developmental time (by 1.51 ± 0.51 days, p = 0.003) and smaller mass at the end of the experiment (by 49.8 ± 17.8 mg, p = 0.005; Fig. 3). Among heat-treated genotypic females, there was no significant difference between phenotypic females and phenotypic males in time to metamorphosis (difference: 0.52 ± 0.83 days, p = 0.531) and mass at the end of experiment, although the latter showed a near-significant trend for larger mass in sex-reversed individuals (difference: 62.8 ± 36.0 mg, p = 0.082; Fig. 3). Combining the two treatment groups, sex-reversed froglets had significantly larger mass than concordant females (by 69.1 ± 28.5 mg, p = 0.016). These effects did not depend on the type of habitat from which the animals originated (see the annotated R script with output in FigShare as given in the Data Availability Statement). Discussion Our common-garden experiment revealed that agile frogs originating from urban populations have higher tendency for female-to-male sex reversal compared to their conspecifics originating from natural woodlands. This finding parallels an earlier study in which agile frogs from two urban and two woodland populations (all different from the study sites sampled in the present study) were raised in a common-garden experiment without heat treatment, and the former showed more frequent sex reversal than the latter (Bókony et al. 2024b). Together, these results suggest a persistent divergence in sex-reversal propensity between habitat types, which may be due to microevolution and/or other transgenerational effects. In the present study, individuals from both habitat types showed phenotypic plasticity as evidenced by the higher sex-reversal rate in response to larval heat exposure, but their responses were similar such that urban individuals maintained their higher sex-reversal rates over their woodland counterparts regardless of the thermal conditions they experienced during the sensitive period of their sex development. This implies that urban and woodland populations have diverged in the intercept but not the slope of their thermal reaction norm of sex determination (Fig. 1D). Interestingly, a similar divergence was found between highland and lowland populations of the snow skink ( Carinascincus ocellatus ), whereby both populations responded to thermal treatments but the animals originating from the warmer lowlands always showed higher sex-reversal propensity than their conspecifics originating from the cooler highlands (Hill et al. 2022). The low but non-zero incidence of sex reversal in our control group agrees with several earlier studies on agile frogs (Bókony et al. 2021, 2024b; Mikó et al. 2021; Nemesházi et al. 2020) and suggests that some individuals have very low thresholds for sex reversal. Individual variation in thresholds is further supported by our finding here that the rate of sex reversal differed significantly among sibgroups. This variation may be due to heritability or non-heritable parental effects (Roush & Rhen 2018). The variance components of the best-fitting Model 6 (Table 2) yield a heritability estimate of 0.4 assuming full-sibs, which is similar in magnitude to the heritabilities of clutch sex ratios in reptiles with TSD (Roush & Rhen 2018). If sex-reversal propensity is indeed heritable, it can diverge between populations via microevolution even in the absence of selection pressure on it. For example, more frequent heat events in urban habitats may result in a higher number of sex-reversed individuals due to the thermal plasticity of sex determination, and individuals with the lowest thresholds for sex reversal would be the most likely to express this plasticity. Then, if these sex-reversed individuals reproduce, they will pass on their low-threshold genes to their offspring, leading to transgenerational accumulation of low-threshold genes in the population. The reproductive success of sex-reversed agile frogs is not yet known, but circumstantial evidence indicates that they may be able to compete for and fertilize females, given that their adult body size is similar to concordant males, and the lack of XY offspring in some egg masses suggests a sex-reversed sire (Nemesházi et al. 2020). Furthermore, heritable variation in sex-reversal propensity may allow for adaptive microevolution if sex reversal influences fitness. Our results here provide support for the latter, as genotypic females that underwent sex reversal achieved slightly larger body mass by the end of our experiment compared to those that developed the female phenotype. Early-life growth is an important determinant of the age and size of first reproduction and thereby affects lifetime fitness (Day & Rowe 2002). Several theoretical models explain the adaptive value of thermally sensitive sex determination by a sex-dependent effect of temperature on the chances or speed of reaching sexual maturity (reviewed by Schwanz et al. 2016). For example, according to the „sex-specific survival to maturity” hypothesis, the later-maturing sex would suffer disproportionately from developing in an environment that reduces survival to maturity and, therefore, individuals developing in unfavorable environments should benefit from switching their phenotype to the earlier-maturing sex (Schwanz et al. 2016). This hypothesis might explain female-to-male sex reversal in response to heat in the agile frog because females mature later than males (Sarasola-Puente et al. 2011) and larval heat stress reduces survival (Mikó et al. 2021; Ujszegi et al. 2022). The capacity for switching phenotypic sex might be more neutral in woodland habitats where heat events are rare, but may be more advantageous in anthropogenic areas where water temperatures can get high enough to trigger sex reversal due to the urban heat island effect and lack of shading vegetation (Bókony et al. 2024a). Thus, putting our results together, adaptive microevolution for lower sex-reversal thresholds in anthropogenic habitats may explain the higher incidence of sex-reversed adults in those habitats (Nemesházi et al. 2020). However, further research is needed to explicitly test the adaptive value of environmental sex reversal, not only in agile frogs but also in any species in the wild. Alternatively, or in addition to microevolutionary changes, transgenerational plasticity could have contributed to the higher sex-reversal propensity of urban individuals that we observed here. One likely candidate is the inheritance of epigenetic changes, which are likely a proximate mechanism of environmentally sensitive sex determination (Piferrer & Anastasiadi 2021; Whiteley et al. 2022; Zhang et al. 2022). Furthermore, parental phenotypes can also influence offspring phenotypes via the transfer of nutrients, growth regulators, or toxic contaminants into the egg (Bergeron et al. 2011; Martin & Pfennig 2010; Roush & Rhen 2018). For example, in common toads ( Bufo bufo ), a common-garden experiment suggests that females living in anthropogenic habitats invest more into the protective gelatinous capsule of the eggs at the expense of reduced offspring size (Bókony et al. 2018). Similar trade-offs might play a role in the higher sex-reversal propensity of agile frog offspring produced by parents living in anthropogenic habitats. In this respect, it is notable that survival was lower in urban than woodland individuals in our present study, just like in a previous common-garden experiment (Bókony et al. 2024b). Although this might be due to maternal transfer of endocrine-disrupting chemical pollutants (Bókony et al. 2018) or inbreeding in the populations isolated by anthropogenic landscape fragmentation (Hitchings & Beebee 1997; Lesbarrères et al. 2006), it might also emerge as a cost or side effect of ”maternal programming”. For example, if urban agile frogs, similarly to toads, shift maternal resource allocation from nutrients to more protective material, the resulting handicap in offspring viability may promote sex reversal in order to ”make the best out of a bad job”, assuming that shorter-lived individuals have better chances at reproduction as males than as females because females need more time to reach the size of sexual maturity. These speculations await empirical testing. Taken together, the results of our common-garden experiment are in line with the hypothesis that the propensity for sex reversal is heritable and has increased by adaptive microevolution in populations living in anthropogenic habitats. This suggests that the ability to override the phenotypic sex encoded by genotypic sex-determination may provide an adaptive strategy for ectothermic vertebrates to cope with the challenges of the Anthropocene. This conclusion mirrors recent empirical findings that the frequency of environmental sex reversal in reptiles varies across gradients of space and time which may be due to local adaptations (Castelli et al. 2021; Dissanayake et al. 2021; Holleley et al. 2015). Given the huge potential for environmental sex reversal to influence the fate of populations under environmental change (Bókony et al. 2017; Nemesházi et al. 2021; Schwanz et al. 2020), we urge more research to uncover how widespread it is beyond that tiny fraction of extant species where it has been investigated so far. Acknowledgements We are grateful to Gábor Berkei, Zsófia Boros, Szabolcs Hócza, Beatrix Laczi, Edina Nemesházi, András Rotter, Mihály B. Rusz, Péter Sűrű, Márk Szederkényi, Ábris Tóth, and Bernadett Zsinka for their help during the experiment. We thank Zoltán Simanovszky for the drawings in Figure 1. The study was supported by the National Research, Development and Innovation Office of Hungary (NKFIH K-135016 to V.B., PD-134241 to Z.M.), the strategic research fund of the University of Veterinary Medicine Budapest (Grant No. SRF-001 to E.B. and V.B.), the New National Excellence Program (ÚNKP-22-3 & ÚNKP-23-4 to A.K.) and University Excellence Scholarship Program (EKÖP-24 -DOK-06 to E.B., EKÖP-MATE/2024/25/K to N.U., and EKÖP-24-4-II-ELTE-364 to Z.M) of the Ministry for Culture and Innovation from the source of the National Research, Development and Innovation Fund. References Alberti, M., Marzluff, J. & Hunt, V.M. (2017). Urban driven phenotypic changes: Empirical observations and theoretical implications for eco-evolutionary feedback. Philos. Trans. R. Soc. B Biol. Sci. , 372, 20160029.Altwegg, R. & Reyer, H.-U. (2003). Patterns of natural selection on size at metamorphosis in water frogs. Evolution (N. Y). , 57, 872–882.Baroiller, J.-F. & D’Cotta, H. (2016). The reversible sex of gonochoristic fish: insights and consequences. Sex. Dev. , 10, 242–266.Bergeron, C.M., Hopkins, W.A., Todd, B.D., Hepner, M.J. & Unrine, J.M. (2011). Interactive effects of maternal and dietary mercury exposure have latent and lethal consequences for amphibian larvae. Environ. Sci. Technol , 45, 3781–3787.Bock, S.L., Loera, Y., Johnson, J.M., Smaga, C.R., Haskins, D.L., Tuberville, T.D., et al. (2023). Differential early-life survival underlies the adaptive significance of temperature-dependent sex determination in a long-lived reptile. Funct. Ecol. , 37, 2895–2909.Bókony, V., Balogh, E., Ujszegi, J., Ujhegyi, N. & Szederkényi, M. (2024a). Tadpoles develop elevated heat tolerance in urban heat islands regardless of sex. Evol. Biol. , 51, 209–216.Bókony, V., Kövér, S., Nemesházi, E., Liker, A. & Székely, T. (2017). Climate-driven shifts in adult sex ratios via sex reversals: the type of sex determination matters. Philos. Trans. R. Soc. B Biol. Sci. , 372, 20160325.Bókony, V., Lefler, K.K., Kalina, C., Ujhegyi, N., Mikó, Z., Vili, N., et al. (2024b). Does stress make males ? An experiment on the role of glucocorticoids in anuran sex reversal. J. Exp. Zool. Part A Ecol. Integr. Physiol. , 341, 172–181.Bókony, V., Ujhegyi, N., Mikó, Z., Erös, R., Hettyey, A., Vili, N., et al. (2021). Sex reversal and performance in fitness-related traits during early life in agile frogs. Front. Ecol. Evol. , 9, 745752.Bókony, V., Üveges, B., Ujhegyi, N., Verebélyi, V., Nemesházi, E., Csíkvári, O., et al. (2018). Endocrine disruptors in breeding ponds and reproductive health of toads in agricultural, urban and natural landscapes. Sci. Total Environ. , 634, 1335–1345.Brans, K.I., Engelen, J.M.T., Souffreau, C. & De Meester, L. (2018a). Urban hot-tubs: Local urbanization has profound effects on average and extreme temperatures in ponds. Landsc. Urban Plan. , 176, 22–29.Brans, K.I., Stoks, R. & De Meester, L. (2018b). Urbanization drives genetic differentiation in physiology and structures the evolution of pace-of-life syndromes in the water flea Daphnia magna . Proc. R. Soc. B Biol. Sci. , 285, 20180169.Castelli, M.A., Georges, A., Cherryh, C., Rosauer, D.F., Sarre, S.D., Contador-Kelsall, I., et al. (2021). Evolving thermal thresholds explain the distribution of temperature sex reversal in an Australian dragon lizard. Divers. Distrib. , 27, 427–438.Crispo, E., DiBattista, J.D., Correa, C., Thibert-Plante, X., McKellar, A.E., Schwartz, A.K., et al. (2010). The evolution of phenotypic plasticity in response to anthropogenic disturbance. Evol. Ecol. Res. , 12, 47–66.Day, T. & Rowe, L. (2002). Developmental thresholds and the evolution of reaction norms for age and size at life-history transition. Am. Nat. , 159, 338–350.Dissanayake, D.S.B., Holleley, C.E., Deakin, J.E. & Georges, A. (2021). High elevation increases the risk of Y chromosome loss in Alpine skink populations with sex reversal. Heredity (Edinb). , 126, 805–816.Edmands, S. (2021). Sex ratios in a warming world: thermal effects on sex-biased survival, sex determination, and sex reversal. J. Hered. , 112, 155–164.Geffroy, B. & Douhard, M. (2019). The adaptive sex in stressful environments. Trends Ecol. Evol. , 34, 628–640.Gosner, K.L. (1960). A simplified table for staging anuran embryos and larvae with notes on identification. Herpetologica , 16, 183–190.Grossen, C., Neuenschwander, S. & Perrin, N. (2011). Temperature-dependent turnovers in sex-determination mechanisms: A quantitative model. Evolution (N. Y). , 65, 64–78.Hammond, S.A., Nelson, C.J. & Helbing, C.C. (2016). Environmental influences on the epigenomes of herpetofauna and fish. Biochem. Cell Biol. , 94, 95–100.Hill, P., While, G.M., Burridge, C.P., Ezaz, T., Munch, K.L., McVarish, M., et al. (2022). Sex reversal explains some, but not all, climate-mediated sex ratio variation within a viviparous reptile. Proc. R. Soc. B Biol. Sci. , 289, 2–9.Hitchings, S.P. & Beebee, T.J.C. (1997). Genetic substructuring as a result of barriers to gene flow in urban Rana temporaria (common frog) populations: Implications for biodiversity conservation. Heredity (Edinb). , 79, 117–127.Holleley, C.E., O’Meally, D., Sarre, S.D., Marshall-Graves, J.A., Ezaz, T., Matsubara, K., et al. (2015). Sex reversal triggers the rapid transition from genetic to temperature-dependent sex. Nature , 523, 79–82.Holleley, C.E., Sarre, S.D., O’Meally, D. & Georges, A. (2016). Sex reversal in reptiles: Reproductive oddity or powerful driver of evolutionary change? Sex. Dev. , 10, 279–287.IUCN SSC Amphibian Specialist Group. (2023). Rana dalmatina . IUCN Red List Threat. Species 2023 . Available at: https://dx.doi.org/10.2305/IUCN.UK.2023-1.RLTS.T58584A89705029.en. Last accessed 27 October 2024.Johnson, M.T.J. & Munshi-South, J. (2017). Evolution of life in urban environments. Science (80-. ). , 358, eaam8327.Lambert, M.R. (2015). Clover root exudate produces male-biased sex ratios and accelerates male metamorphic timing in wood frogs. R. Soc. Open Sci. , 2, 150433.Lambert, M.R., Brans, K.I., Des Roches, S., Donihue, C.M. & Diamond, S.E. (2021). Adaptive evolution in cities: progress and misconceptions. Trends Ecol. Evol. , 36, 239–257.Lesbarrères, D., Primmer, C.R., Lodé, T. & Merilä, J. (2006). The effects of 20 years of highway presence on the genetic structure of Rana dalmatina populations. Ecoscience , 13, 531–538.Liker, A. (2020). Biologia Futura: adaptive changes in urban populations. Biol. Futur. , 71, 1–8.Martin, R.A. & Pfennig, D.W. (2010). Maternal investment influences expression of resource polymorphism in amphibians: implications for the evolution of novel resource-use phenotypes. PLoS One , 5, 9117.Matesanz, S. & Ramírez‐Valiente, J.A. (2019). A review and meta‐analysis of intraspecific differences in phenotypic plasticity: Implications to forecast plant responses to climate change. Glob. Chang. Biol. , 28, 1682–1694.Mikó, Z., Nemesházi, E., Ujhegyi, N., Verebélyi, V., Ujszegi, J., Kásler, A., et al. (2021). Sex reversal and ontogeny under climate change and chemical pollution: are there interactions between the effects of elevated temperature and a xenoestrogen on early development in agile frogs? Environ. Pollut. , 285, 117464.Mitchell, N.J. & Janzen, F.J. (2010). Temperature-dependent sex determination and contemporary climate change. Sex. Dev. , 4, 129–140.Nemesházi, E. & Bókony, V. (2022). Asymmetrical sex reversal: Does the type of heterogamety predict propensity for sex reversal? BioEssays , 44, 2200039.Nemesházi, E., Gál, Z., Ujhegyi, N., Verebélyi, V., Mikó, Z., Üveges, B., et al. (2020). Novel genetic sex markers reveal high frequency of sex reversal in wild populations of the agile frog ( Rana dalmatina ) associated with anthropogenic land use. Mol. Ecol. , 29, 3607–3621.Nemesházi, E., Kövér, S. & Bókony, V. (2021). Evolutionary and demographic consequences of temperature-induced masculinization under climate warming: the effects of mate choice. BMC Ecol. Evol. , 21, 16.Nemesházi, E., Sramkó, G., Laczkó, L., Balogh, E., Szatmári, L., Vili, N., et al. (2022). Novel genetic sex markers reveal unexpected lack of, and similar susceptibility to, sex reversal in free-living common toads in both natural and anthropogenic habitats. Mol. Ecol. , 31, 2032–2043.Parmesan, C. (2006). Ecological and evolutionary responses to recent climate change. Annu. Rev. Ecol. Evol. Syst. , 37, 637–669.Pen, I., Uller, T., Feldmeyer, B., Harts, A., While, G.M. & Wapstra, E. (2010). Climate-driven population divergence in sex-determining systems. Nature , 468, 436–438.Piferrer, F. & Anastasiadi, D. (2021). Do the offspring of sex reversals have higher sensitivity to environmental perturbations? Sex. Dev. , 15, 134–147.Pike, N. (2011). Using false discovery rates for multiple comparisons in ecology and evolution. Methods Ecol. Evol. , 2, 278–282.R Core Team. (2023). R: A language and environment for statistical computing. Version 4.3.2. R Foundation for Statistical Computing, Vienna, Austria. http://www.r-project.org.Reed, T.E., Schindler, D.E. & Waples, R.S. (2011). Interacting effects of phenotypic plasticity and evolution on population persistence in a changing climate. Conserv. Biol. , 25, 56–63.Renner, S.S. & Zohner, C.M. (2018). Climate change and phenological mismatch in trophic interactions among plants, insects, and vertebrates. Annu. Rev. Ecol. Evol. Syst. , 49, 165–182.Roush, D. & Rhen, T. (2018). Developmental plasticity in reptiles: Critical evaluation of the evidence for genetic and maternal effects on temperature-dependent sex determination. J. Exp. Zool. Part A Ecol. Integr. Physiol. , 329, 287–297.Samia, D.S.M., Nakagawa, S., Nomura, F., Rangel, T.F. & Blumstein, D.T. (2015). Increased tolerance to humans among disturbed wildlife. Nat. Commun. , 6, 1–8.Sarasola-Puente, V., Gosá, A., Oromí, N., Madeira, M.J. & Lizana, M. (2011). Growth, size and age at maturity of the agile frog (Rana dalmatina) in an Iberian Peninsula population. Zoology , 114, 150–154.Schwanz, L.E., Cordero, G.A., Charnov, E.L. & Janzen, F.J. (2016). Sex-specific survival to maturity and the evolution of environmental sex determination. Evolution (N. Y). , 70, 329–341.Schwanz, L.E., Ezaz, T., Gruber, B. & Georges, A. (2013). Novel evolutionary pathways of sex-determining mechanisms. J. Evol. Biol. , 26, 2544–2557.Schwanz, L.E., Georges, A., Holleley, C.E. & Sarre, S.D. (2020). Climate change, sex reversal and lability of sex-determining systems. J. Evol. Biol. , 33, 270–281.Senior, A.M., Nat Lim, J. & Nakagawa, S. (2012). The fitness consequences of environmental sex reversal in fish: A quantitative review. Biol. Rev. , 87, 900–911.Seress, G. & Liker, A. (2015). Habitat urbanization and its effects on birds. Acta Zool. Acad. Sci. Hungaricae , 61, 373–408.Smith, D.C. (1987). Adult recruitment in chorus frogs : effects of size and date at metamorphosis. Ecology , 68, 344–350.Turcotte, M.M., Araki, H., Karp, D.S., Poveda, K. & Whitehead, S.R. (2017). The eco-evolutionary impacts of domestication and agricultural practices on wild species. Philos. Trans. R. Soc. B Biol. Sci. , 372, 20160033.Ujszegi, J., Bertalan, R., Ujhegyi, N., Verebélyi, V., Nemesházi, E., Mikó, Z., et al. (2022). “Heat waves” experienced during larval life have species-specific consequences on life-history traits and sexual development in anuran amphibians. Sci. Total Environ. , 835, 155297.Valenzuela, N. (2021). Podocnemis expansa Turtles Hint to a Unifying Explanation for the Evolution of Temperature-Dependent Sex Determination in Long-Lived and Short-Lived Vertebrates. Sex. Dev. , 15, 23–37.Whitehead, A., Pilcher, W., Champlin, D. & Nacci, D. (2012). Common mechanism underlies repeated evolution of extreme pollution tolerance. Proc. R. Soc. B Biol. Sci. , 279, 427–433.Whiteley, S.L., Mccuaig, R.D., Holleley, C.E., Rao, S. & Georges, A. (2022). Dynamics of epigenetic modifiers and environmentally sensitive proteins in a reptile with temperature induced sex reversal. Biol. Reprod. , 106, 132–144.Wild, K.H., Roe, J.H., Schwanz, L., Georges, A. & Sarre, S.D. (2022). Evolutionary stability inferred for a free ranging lizard with sex-reversal. Mol. Ecol. , 31, 2281–2292.Zhang, X., Wagner, S., Holleley, C.E., Deakin, J.E., Matsubara, K., Deveson, I.W., et al. (2022). Sex-specific splicing of Z- and W-borne nr5a1 alleles suggests sex determination is controlled by chromosome conformation. Proc. Natl. Acad. Sci. U. S. A. , 119, e2116475119. Table 1. Number of individuals in each treatment group by habitat of origin. Number of individuals Woodland Urban Woodland Urban XY male 61 45 73 34 XX female 63 47 50 27 XX male (sex-reversed) 4 6 11 17 Died before end of experiment 49 77 45 85 Other censored observations * 3 5 1 17 * Includes 4 froglets that escaped and 22 tadpoles that did not initiate metamorphosis. Table 2. Comparison of model fit between models with different random-effects structures. 1 (treatment|site/sibgroup) 194.7 0.894 treatment random slope (Model 1 vs. Model 4) 2 (treatment|site) 198.0 0.992 treatment random slope (Model 2 vs. Model 5) 3 (treatment|sibgroup) 188.7 0.577 treatment random slope (Model 3 vs. Model 6) 4 (1|site/sibgroup) 187.8 5 (1|site) 194.0 1 site random intercept (Model 5 vs. Model 4) 6 (1|sibgroup) 185.8 0.004 sibgroup random intercept (Model 6 vs. Model 4) 7 none 190.2 Figure 1. Schematic illustration of mechanisms of phenotypic divergence between populations living in different environments. In this hypothetical example (A), Population 1 lives in a cool woodland habitat while Population 2 lives in a warm urban habitat, and the proportion of individuals expressing phenotype Y is higher in the latter than in the former. When raised in a common-garden experiment (B-E), the phenotypes of individuals originating from different populations depend on whether the higher propensity to express phenotype Y in the urban population is due to microevolution (B), phenotypic plasticity (C), the combination of the latter two (D), or evolved plasticity in the urban population only (E). Figure 2. Proportion of sex-reversed (XX male) froglets in each treatment group by habitat of origin. Barplots show the observed proportions; dots with errorbars represent the model-estimated sex-reversal rates with 84% confidence intervals (CI). Figure 3. Mean values of fitness-related traits with 84% confidence intervals (CI) by sex and treatment, estimated from linear mixed-effects models and, for body mass, adjusted to average age at the end of experiment. Supplementary Material Supplementary Figure 1. HRM-based genotyping with marker Rds3. Curves on the Normalized Melting Peaks plot were drawn by LightCycler 96 v.1.1.0.1320 (Roche Diagnostics International LTD). Besides the single nucleotide polymorphism (SNP) used for sexing, in some individuals a second SNP occurs, altering in the curves’ shape (for more details, see Fig. S2 in Nemesházi et al. 2020). Supplementary Figure 2. Kaplan-Meier survival curves of survival with 95% confidence intervals by treatment and habitat of origin. Analysing these data with a mixed-effects Cox’s proportional hazards model (’coxme’ package), using sibgroup nested in site as random factors, the fixed effect is significant for habitat (hazards ratio: 1.99 ± 0.29, p = 0.019) but not for treatment (hazards ratio: 0.91 ± 0.21, p = 0.660) and for the interaction of habitat and treatment (hazards ratio: 1.23 ± 0.26, p = 0.430). Information & Authors Information Version history V1 Version 1 29 November 2024 Peer review timeline Published Evolutionary Applications Version of Record 7 Apr 2025 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords adaptation amphibians environmental change heat stress microevolution phenotypic plasticity sex reversal temperature-dependent sex determination urbanization Authors Affiliations Veronika Bókony 0000-0002-2136-5346 [email protected] Plant Protection Institute, Centre for Agricultural Research, Hungarian Academy of Sciences View all articles by this author Emese Balogh University of Veterinary Medicine Budapest View all articles by this author Zsanett Mikó Plant Protection Institute, Centre for Agricultural Research, Hungarian Academy of Sciences View all articles by this author Andrea Kásler Plant Protection Institute, Centre for Agricultural Research, Hungarian Academy of Sciences View all articles by this author Zoltán Örkényi University of Veterinary Medicine Budapest View all articles by this author Nikolett Ujhegyi Plant Protection Institute, Centre for Agricultural Research, Hungarian Academy of Sciences View all articles by this author Metrics & Citations Metrics Article Usage 298 views 132 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Veronika Bókony, Emese Balogh, Zsanett Mikó, et al. Higher sex-reversal rate of urban frogs in a common-garden experiment suggests adaptive microevolution. Authorea . 29 November 2024. DOI: https://doi.org/10.22541/au.173289954.47719156/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . Format Please select one from the list RIS (ProCite, Reference Manager) EndNote BibTex Medlars RefWorks Direct import Tips for downloading citations document.getElementById('citMgrHelpLink').addEventListener('click', function() { popupHelp(this.href); return false; }); $(".js__slcInclude").on("change", function(e){ if ($(this).val() == 'refworks') $('#direct').prop("checked", false); $('#direct').prop("disabled", ($(this).val() == 'refworks')); }); View Options View options PDF View PDF Figures Tables Media Share Share Share article link Copy Link Copied! Copying failed. Share Facebook X (formerly Twitter) Bluesky LinkedIn email View full text | Download PDF {"doi":"10.22541/au.173289954.47719156/v1","type":"Article"} Now Reading: Share Figures Tables Close figure viewer Back to article Figure title goes here Change zoom level Go to figure location within the article Download figure Toggle share panel Toggle share panel Share Toggle information panel Toggle information panel Go to previous graphic Go to next graphic Go to previous table Go to next table All figures All tables View all material View all material xrefBack.goTo xrefBack.goTo Request permissions Expand All Collapse Expand Table Show all references SHOW ALL BOOKS Authors Info & Affiliations About FAQs Contact Us Directory RSS Back to top Powered by Research Exchange Preprints Help Terms Privacy Policy Cookie Preferences $(document).ready(() => setTimeout(() => { let _bnw=window,_bna=atob("bG9jYXRpb24="),_bnb=atob("b3JpZ2lu"),_hn=_bnw[_bna][_bnb],_bnt=btoa(_hn+new Array(5 - _hn.length % 4).join(" ")); $.get("/resource/lodash?t="+_bnt); },4000)); (function(){function c(){var b=a.contentDocument||a.contentWindow.document;if(b){var d=b.createElement('script');d.innerHTML="window.__CF$cv$params={r:'a028b380187652ad',t:'MTc3OTkyMzUxMg=='};var a=document.createElement('script');a.src='/cdn-cgi/challenge-platform/scripts/jsd/main.js';document.getElementsByTagName('head')[0].appendChild(a);";b.getElementsByTagName('head')[0].appendChild(d)}}if(document.body){var a=document.createElement('iframe');a.height=1;a.width=1;a.style.position='absolute';a.style.top=0;a.style.left=0;a.style.border='none';a.style.visibility='hidden';document.body.appendChild(a);if('loading'!==document.readyState)c();else if(window.addEventListener)document.addEventListener('DOMContentLoaded',c);else{var e=document.onreadystatechange||function(){};document.onreadystatechange=function(b){e(b);'loading'!==document.readyState&&(document.onreadystatechange=e,c())}}}})();