Journal of Zoology

Journal of Zoology. Print ISSN 0952-8369

Female-biased sex ratios in subarctic common frogs

J. S. Alho, G. Herczeg & J. Merila
Ecological Genetics Research Unit, Department of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland

Keywords arctic; CMR analysis; population decline; reproduction; sex ratio; survival. Correspondence Juha Merila, Ecological Genetics Research Unit, Department of Biological and Environmental Sciences, PO Box 65, FI-00014 University of Helsinki, Finland. Tel: +358 40 837 41 65 Email: juha.merila@helsinki. Received 2 November 2007; accepted 5 December 2007 doi:10.1111/j.1469-7998.2007.00409.x

Abstract
In vertebrates with genetic sex determination, large deviations from the 1:1 sex ratio at the population level are rare and demand an explanation. We investigated adult sex-ratio variation of common frogs Rana temporaria in two subarctic breeding ponds over a 6-year (19992004) period using capturemarkrecapture (CMR) methods. Using the same data, we also tested for the occurrence of the biannual breeding cycle in female common frogs and sought evidence for population size decline. Sex ratios were highly female biased in both populations: on average, only about 30% of breeding individuals were males. CMR analyses further suggested that the female-biased sex ratio was not explainable by higher adult mortality among males as the survival probabilities over years were similar (c. 70%) for both sexes. Alternative explanations for these highly female-biased sex ratios include sex-specic mortality at earlier life stages and environmental inuences on the sex determination system. While further studies are required to differentiate between the alternatives, the observed sex-ratio bias is among the strongest reported in any amphibian population so far, and also in the direction opposite to that usually observed (males4females). Our analyses found no support for the contention that female common frogs in northern populations may reproduce only in every second year. However, both study populations were declining during the study period.

Introduction
According to the DusingFisher (Fisher, 1930; Edwards, 2000) sex-ratio theory, the evolutionarily stable sex ratio in organisms with chromosomal sex determination is an equal number of males and females in the population. However, deviations from this stable sex ratio are possible as shown by several theoretical models predicting the evolution of skewed sex ratios (e.g. Trivers & Williard, 1973; Charnov & Bull, 1977; Frank, 1990; Pen & Weissing, 2002). For instance, Trivers & Williard (1973) pointed out that once the expected tness returns from producing males and females differ for individual females, then adjustment of the primary sex ratio with respect to individual females qualities is favored by natural selection. Sex-ratio bias may also result if abiotic environmental factors such as temperature or environmental contaminants (Hayes, 1998; Wallace, Badawy & Wallace, 1999) interfere with the sex determination system. Hence, biased sex ratios can have both adaptive and non-adaptive causes. Sex-ratio variation in amphibians has received less attention than that among other vertebrates (but see Sakisaka et al., 2000). One possible reason for this is that both primary- and population-level sex ratios among adults is difcult to determine. As to the primary sex ratio, many amphibians, such as the common frog Rana temporaria, do not have well-differentiated sex chromosomes, making the

sexing of the juveniles very difcult. Likewise, while the adult frogs can be easily sexed, estimation of the actual sex ratio of a population is complicated by the difculty of sighting adults. For instance, although there are numerous studies describing operational (and population) sex ratios of common frogs at breeding aggregations (Gibbons & McCarthy, 1984; Elmberg, 1990; Ryser, 1996), none of these studies have used capturemarkrecapture (CMR) methods to estimate the actual male and female population sizes. Because the capture probabilities among the sexes may differ, it is impossible to know how representative the sexratio estimates are. A further complication in sex-ratio estimation occurs if one of the sexes does not reproduce annually, as is the case in many ectothermic vertebrates (Bull & Shine, 1979). The aim of this study was to investigate sex-ratio variation among breeding common frogs applying CMR methods on data collected during 6 consecutive years from two breeding ponds in northern Finland. In particular, we wished to establish whether the sex ratios among breeding adults are equal, and if not, whether the skew could be explained by differential adult survival of the sexes. We also used the data to test the hypothesis that females in these northern populations are biannual breeders. In addition, given the recent concern about the global decline of amphibian populations, we looked for population size trends in these two subarctic ponds.
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c 2008 The Authors. Journal compilation c 2008 The Zoological Society of London Journal of Zoology 275 (2008) 5763

Sex-ratio bias in the common frog

J. S. Alho, G. Herczeg and J. Merila

Methods
Study species and populations
The common frog is a medium-sized anuran frog with a wide Palearctic distribution (Gasc et al., 1997). In our study ponds at Kilpisjarvi in northern Finland (69104 0 N, 20146 0 E), the breeding starts in late Mayearly June when the adults migrate to breeding ponds. Mating and egg laying is usually nished by mid-June when the rst eggs hatch. First tadpoles metamorphose in late July, but most in August and early September. In some years, the majority of tadpoles are still in the ponds in late Septemberearly October when the ponds start to freeze. Rana temporaria tadpoles are thought to be unable to overwinter, and we have never observed successfully over-wintered tadpoles in our study ponds despite attempts to nd them. As revealed by skeletochronology, metamorphosed individuals reach sexual maturity at the age of 35 years (males) or 68 years (females; ter Schure et al., 2002; Alho, 2004). The two study ponds are located in the same mountain valley with a distance of 0.5 km between them: one in the southern and the other in the northern end of the valley. The area is characterized by brooks, wet meadows, bushes, heather and small woodlands of the downy birch Betula pubescens czerepanovii. The study area is bordered by a sand ridge to the west, a parking lot to the south, a road to the east and a customs station to the north. Based on a radiotelemetry study conducted in 2002 (J. Merila, unpubl. data), adults breeding in these ponds spend their lives throughout the year in the vicinity of the ponds.

fences. Likewise, frequent and regular efforts have been made in all study years to locate frogs in suitable habitats around the fences to ensure that some individuals are not consistently missed because of being trap-shy. These efforts have revealed that the number of individuals remaining outside of the fences is small relative to that in study ponds, and that their sex ratio is similar to that within the study ponds (data not shown). All captured frogs were taken to a laboratory for measurements. Individuals were sexed according to their external secondary sexual characteristics (e.g. nuptial patches in males; general body shape differences) and the presence of eggs (females). They were marked with passive integrated transponders (PIT-tags, Trovan Ltd, Douglas, UK) and the longest toe of their right front leg was taken for future age measurements (skeletochronology) and DNA studies. For these two surgical operations, as well as for morphological measurements, the animals were anesthetized by submerging them in a solution of MS-222 (tricaine methanesulfonate, Sigma-Aldrich, Helsinki, Finland). The frogs were returned to the study area after they had recovered from anesthesia. Although we have no reason to believe that these procedures would have inuenced the survival or recapture probabilities (but see May, 2004; McCarthy & Parris, 2004), it is worth stressing that all individuals were treated similarly.

Analysis
To test our hypotheses about survival and breeding cycle and to derive population size and sex-ratio estimates, we tted a Bayesian hierarchical model (see e.g. Gelman et al., 2004) to the CMR data (see e.g. Lebreton et al., 1992; in a Bayesian context e.g. Brooks, Catchpole & Morgan, 2000; Link & Barker, 2005) using OpenBUGS version 3.0.3 (Thomas et al., 2006; http://www.mathstat.helsinki./openbugs/). The data consisted of individual capture histories and two individual covariates: population and sex. Capture histories were expressed as a matrix X with elements xi,j being binary variables indicating whether an individual i was (xi,j = 1) or was not (xi,j = 0) observed in year j. To account for the individuals that may have been present in the study area but by chance were never observed, we included in the dataset a hundred individuals with capture history consisting entirely of zeros in addition to the n individuals that were observed at least once. We then dened parameter N with a noninformative categorical prior distribution from n to n+100 to be the total number of individuals actually existing in the area during the study. This augmentation of X has been used previously by for example Fienberg, Johnson & Junker (1999) and Durban & Elston (2005). The breeding pond and sex of the unobserved individuals were treated as missing data. Because only two individuals were observed in both ponds during the study (see Results), the breeding pond was treated as a xed character and for the two individuals observed in both as missing data. It was modelled as a Bernoulli variable with a uniform prior distribution for the proportion of individuals breeding on each of the two ponds. Similarly, sex was modelled as a

Field methods
We conducted a CMR study in Kilpisjarvi from 1999 to 2004. An effort was made to capture all individuals entering or leaving the two breeding ponds: Parkki and Tulli. The ponds were surrounded by drift fences and equipped with pitfall traps. The drift fences were founded in the autumn of 1998 and erected thereafter each year in mid-May during the snowmelt before the frogs had emerged from hibernation and started their migration to the breeding ponds, and pulled down by the beginning of July, when the majority of the frogs had left the ponds. As a precautionary measure, the pitfall traps were protected against avian predation by wooden covers and metallic nets. The capture effort was constant during the study period, except in 2001, when there was a shortage of eld workers and, as a consequence, the capture rate was lower than usual (see Results). An important methodological concern in CMR studies is that all individuals are assumed to be equally available for capture. This assumption could be violated for instance if some of the frogs overwinter within the connes of the drift fences, or stay away from the ponds because of being repelled by the drift fences. We note that overwintering within the connes of the drift fence is possible only in the Parkki pond, but that this occurs rarely as indicated by yearly control catches made by hand and dip-nets within the
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J. S. Alho, G. Herczeg and J. Merila

Sex-ratio bias in the common frog

Bernoulli variable with uniform prior distributions for the overall sex ratio for each pond. For each individual i  N, we modelled its annual status as a binary variable Fi,j, indicating whether the individual was alive or not in year j. We modelled F of the rst year of the study as a Bernoulli variable with a probability dp,s,1, where p is the pond and s the sex of individual i. dp,s,1 was dened to be the chance that individuals of pond p and sex s entered the study area, that is became liable for capture, during the rst year. Note that we use the term enter here for simplicity in reality, the term encompasses here all processes whereby individuals become liable for capture be it maturation, immigration or simply the launch of the study. For subsequent years j, the status Fi,j was modelled as a Bernoulli variable with a probability jp;s; j Fi; j1 dp;s;j 1 vi;j 1

avoid capture for any reason, conditional on being captured last year. We derived sex-specic population size estimates, sexratio estimates and the Pearson productmoment correlation coefcients between total population size and year for both of our study ponds by incorporating these variables into the model and calculating them on the basis of individual status variables Fi,j. We ran three chains, each with 30 000 iterations, of which 5000 were discarded as burnin. The convergence and mixing of the chains were checked by eye.

Results
Sex ratios
The observed sex ratios in the two ponds were strongly female biased (Fig. 1). The same was true when male and female population sizes were estimated using CMR methods taking into account the variation due to capture probabilities among years and populations (Fig. 1; Table 1). Sexratio estimates differed signicantly from the 1:1 sex ratio in 11 of the 12 cases (Table 1).

where p is the pond and s the sex of individual i, jp,s, j the survival probability from year j1 to year j for individuals of pond p and sex s, dp,s, j the probability of individuals of pond p and sex s to enter the area for the rst time in year j and ni,j an indicator of whether the individual has already entered the study area in previous years. Survival probabilities jp,s, j were assigned uniform priors U(0, 1). For each four combination of p and s, entering probabilities dp,s, j were assigned a non-informative prior Dirichlet (a1, . . ., a6) with aj = 1 for all j. For the rst year of the study, the capture history of individual i, xi,1, was modelled as a Bernoulli process with a probability pp,s,1, where p is the pond and s the sex of individual i. For subsequent years j, the probability was dened as pp;s; j Fi;j 1 xi;j 1 lp; s 2

Survival analyses
A total of 299 adult individuals, 208 females and 91 males, were captured during the study period. The annual survival and capture probabilities are shown in Fig. 2. There was no evidence for differential survival between sexes the mean difference of male survival probability from female survival was 0.01 [95% condence intervals (CI) 0.10 to 0.12] in Parkki and 0.01 (95% CI 0.13 to 0.11) in Tulli, and the annual differences in both ponds were non-signicant. The mean survival probability for females in Parkki was 0.69 (95% CI 0.630.76) and 0.71 (95% CI 0.640.78) in Tulli. The mean survival probability for males in Parkki was 0.69 (95% CI 0.590.78) and 0.72 (95% CI 0.620.82) in Tulli.

where pp,s, j is the probability of capture for individuals of pond p and sex s in year j and lp,s the probability of individuals of pond p and sex s to skip the breeding season conditional on them being captured last year. Capture and skipping probabilities were assigned uniform priors U(0, 1). Note that strictly lp,s is the probability of individuals to

(a) 100
80

(b) 100
Females Males

80

Population size

60

60

40

40

20

20

0
1999 2000 2001 2002 2003 2004

0 1999 2000 2001 2002 2003 2004

Year

Year

Figure 1 Size of the male and female population in the two study ponds Tulli (a) and Parkki (b) over the 6-year study period. Box bars represent observed numbers of individuals, circles show the posterior median estimates of population sizes from CMR analysis and the error bars indicate the 95% condence intervals. CMR, capturemarkrecapture.

c 2008 The Authors. Journal compilation c 2008 The Zoological Society of London Journal of Zoology 275 (2008) 5763

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Sex-ratio bias in the common frog

J. S. Alho, G. Herczeg and J. Merila

Table 1 Sex ratio (proportion males) of adult common frogs in two study ponds in different study years as estimated from CMR data Tulli 95% CI Year 1999 2000 2001 2002 2003 2004 Posterior mean 0.24 0.20 0.21 0.36 0.41 0.43 Lower 0.21 0.17 0.16 0.30 0.36 0.35 Upper 0.31 0.23 0.29 0.45 0.46 0.51 Posterior mean 0.31 0.36 0.34 0.27 0.26 0.28 Parkki 95% CI Lower 0.29 0.33 0.30 0.23 0.21 0.24 Upper 0.35 0.41 0.39 0.32 0.33 0.32

CMR, capturemarkrecapture; CI, condence intervals.

(a) 1.0
0.8

Parkki

(b) 1.0
0.8

Tulli

Survival probability

0.6

0.6

0.4

0.4

0.2

0.2

0.0 1999 2000 2001 2002 2003 2004

0.0 1999 2000 2001 2002 2003 2004

(c) 1.0
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(d ) 1.0
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0.6

0.6

0.4

0.4

0.2

0.2

Females Males

0.0 1999 2000 2001 2002 2003 2004

0.0

1999 2000 2001 2002 2003 2004

Year

Year

Figure 2 Annual survival (a, b) and capture (c, d) probabilities for the two study ponds Tulli and Parkki with 95% condence intervals.

Female breeding cycle

To test the proposition that females in these northern breeding sites would reproduce only in every 2 years, we estimated the probability of skipping a breeding season after being captured in the previous year. This probability was 0.02 (95% CI 0.000.07) for females in Parkki and 0.17 (95% CI 0.030.31) in Tulli. For males, the probability was 0.05 (95% CI 0.000.17) in Parkki and 0.09 (95% CI 0.000.23) in Tulli. Adult individuals seem to be site tenacious: of the 299 individuals captured during the study, only two were captured in both ponds.
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Population size trends

Estimating the correlation between total population size and year for each population as part of the model, the data suggest that population size in both ponds was declining over the study period as indicated by the negative correlation between total population sizes and year (Parkki: r =0.72, Po0.001; Tulli: r =0.29, P = 0.017; Fig. 1).

Discussion
Our results demonstrate an extremely female-biased sex ratio of adult common frogs in two breeding ponds in

c 2008 The Authors. Journal compilation c 2008 The Zoological Society of London Journal of Zoology 275 (2008) 5763

J. S. Alho, G. Herczeg and J. Merila

Sex-ratio bias in the common frog

northern Finland. This bias deviates signicantly from the theoretically predicted and evolutionary stable 1:1 sex ratio (Karlin & Lessard, 1986). Although biased sex ratios are frequently observed in vertebrates, the deviations are hardly ever of this magnitude (e.g. Karlin & Lessard, 1986; but see: Richards & Nace, 1978). Consequently, the strong female bias in adult sex ratios observed in this study demands an explanation. One possible explanation for sex-ratio bias is that male frogs at the study ponds experience higher mortality rates than females. This is plausible, given the fact that males spend more time on leks (Savage, 1961) and could therefore be more exposed to predation than females. Male-biased mortality among adult amphibians due to predation (e.g. Denton & Beebee, 1993) and differential vulnerability to weather-related mortality (Verrell & Halliday, 1985) resulting in female-biased sex ratios has indeed been suggested. However, our results indicate that this is not the case: the survival rates of the two sexes were similar according to the survival analyses based on CMR data. Hence, if the sex-ratio bias among adults is to be explained by sex-specic mortality rates, this mortality has to occur before the frogs reach breeding age (males: c. 35 years; females: c. 68 years; ter Schure et al., 2002; Alho, 2004). To test this hypothesis, one would need to know the primary sex ratio. Unfortunately, despite our attempts to nd sex-specic genetic markers to be used in sex identication (Matsuba, Miura & Merila, 2008), we do not currently have means of estimating primary sex ratios with high certainty. Likewise, estimation of mortality between metamorphosis and adulthood is difcult. This is because CMR studies with juveniles are complicated by difculties in sexing, marking and capturing them. Another explanation for the female-biased sex ratio is that some environmental factor(s) is (are) interfering with the genetic sex-determination system, making most individuals develop into females. There are a number of endocrine disruptive compounds with feminizing effects (e.g. Olsson et al., 1998), and there are reports of feminization and/or sex reversals in amphibians due to these compounds (Hayes et al., 2002; Levy et al., 2004). Furthermore, the amphibian sex determination system is also known to be labile, and temperature-induced sex reversals are known to occur in some species (Witschi, 1929a,b; Hayes, 1998; Wallace et al., 1999). Of particular interest here is that there are old reports about frequent sex reversals in the common frog (Witschi, 1929b), and experimental evidence for temperature-dependent sex determination from Rana sylvatica (Witschi, 1929a), the northern American ecological equivalent of R. temporaria. However, it seems unlikely that temperature-induced sex reversals could explain female-biased sex ratios in R. temporaria because high, rather than low temperatures, are supposed to have feminizing effects. Nevertheless, given that the ambient temperatures in the northern Fennoscandia have been increasing recently (Sorvari, Korhola & Thompson, 2002; Rais & Alexanders anen son, 2003; Tuomenvirta, 2004), it would be premature to exclude this possibility. Matsuba et al. (2008) provide some preliminary but still inconclusive evidence for sex reversals in northern Finnish common frogs.

Breeding cycle
Females of many ectothermic vertebrates are known to reproduce biannually, hence skipping the opportunity to reproduce every year (Bull & Shine, 1979). This has been thought to be a life-history strategy selected for in environments where investment on growth and energy storages in alternate years will result in higher tness than annual reproduction (Bull & Shine, 1979). Some anecdotes in the herpetological literature have led to the suspicion that also R. temporaria females at high latitudes might reproduce only every second year. Our results refute this belief: using direct observations of individually marked animals, and subjecting these data to explicit statistical testing, we found no evidence for systematic skips of reproduction in two study populations. This veries the indirect evidence based on growth layer analyses by Elmberg (1991): northern common frog females are annual reproducers. To our knowledge, this is only the second time when CMR models have been used in this context. Earlier, Schmidt, Schaub & Anholt (2002) used a similar approach to show that common toad Bufo bufo females are annual breeders.

Population-size trends
During the past decades, many amphibian populations have shown clear signs of population declines (Alford & Richards, 1999; Houlahan et al., 2000; Collins & Strofer, 2003). These declines have occurred in different parts of the world, leading to a concern that has become known as the global decline of amphibian populations. From this perspective, it is interesting to note that our study populations also displayed negative population-size trends over the study period. Comparable data on other common frog populations are scarce, but Meyer, Schmidt & Grossenbacher (1998), studying three Swiss populations over the past three decades, found that one of the three populations was declining. As to the reasons for the observed declines in this study, one possible explanation is found from habitat changes that have taken place at pond Parkki. In autumn 2000, a road construction work changed the hydrodynamics of the pond slightly, and perhaps also inuenced adult survival in that population. However, the fact that the adult survival rates have remained roughly constant over the study period might argue against this explanation. Furthermore, because males recruit to the breeding population rst at an age of 3 years or more, and females even later, possible changes in reproductive success due to road construction should not be visible in the adult population until 20032004 at the earliest. However, the observed decline starts earlier. Hence, it seems more likely that the negative population-size trends are due to declines in recruitment rates predating the year when this study was initiated.

Conclusions
Our results demonstrate an extreme bias in the sex ratios of breeding adult common frogs in northern Finland. Although our analyses allow us to rule out the possibility
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c 2008 The Authors. Journal compilation c 2008 The Zoological Society of London Journal of Zoology 275 (2008) 5763

Sex-ratio bias in the common frog

J. S. Alho, G. Herczeg and J. Merila

that this bias is caused by differential mortality among sexes in adulthood, the other potential explanations remain to be investigated when means of estimating sex ratios at birth are available. The results also refute the suspicion that common frog females at high latitudes would be bi rather than annual reproducers, contributing to the basic understanding of the species reproductive biology at the northern edge of its distribution range.

Acknowledgements
We thank all those numerous people who have helped us during this study. In particular, thanks are due to Oula Kalttopa Rauni Partanen, a, Anssi Laurila, Antero Jarvinen, l Karoliina Ras Mattias Sterner, Robert OHara, Abige anen, Gonda and Fredrik Soderman. Special thanks are due to Kilpisjarvi Biological station and its staff for continuous support and help. We also thank the Finnish Forestry Research Institute for allowing us to conduct this study on their property. Our research was supported by the Swedish Natural Science Research Council, Academy of Finland, Research Foundation of the University of Helsinki, Hungarian Eotv os State Fellowship, CIMO Fellowship, Emil Aaltonen Foundation, Finnish Ministry of Education and EU LABIAT program.

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c 2008 The Authors. Journal compilation c 2008 The Zoological Society of London Journal of Zoology 275 (2008) 5763

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