Molecular Ecology (2008)

doi: 10.1111/j.1365-294X.2008.03748.x

Do male moor frogs (Rana arvalis) lek with kin? Blackwell Publishing Ltd

T. K N O P P , M . H E I M O V I RTA , H . K O K K O and J . M E R I L Ä Ecological Genetics Research Unit, Department of Biological and Environmental Sciences, PO Box 65 FIN-00014, University of Helsinki, Finland

Abstract Many amphibian species are known to form leks during breeding season, yet it has seldom been tested which evolutionary forces are likely to act on lek formation in this taxon. We tested the kin selection hypothesis for lek formation by using eight variable microsatellite loci to compare the genetic relationship of 203 males in seven Rana arvalis leks. The results indicate that moor frog males do not lek with kin: their relatedness within leks was not higher than expected by chance. Furthermore, spatially distinct leks within same water bodies could not be distinguished from each other as separate units. These results are not expected if kin selection underlie lek formation. On the basis of these results and general knowledge of anuran breeding biology, we suggest that lek formation in explosively breeding amphibians might have evolved by female choice for breeding aggregations, combined with female choice of habitat. Future work should aim at predicting aggregations based on rules of phonotaxis over different spatial scales, and empirical work should document visitation rates not only for leks of a specific size, but also for different travel distances that visiting females may have had to cover. Keywords: amphibia, kin selection, lek, Rana arvalis, relatedness Received 16 January 2008; revision accepted 21 February 2008

Introduction The aggregation of males displaying for females at a communal mating ground was originally identified in birds (Lloyd 1867), and this taxon has often been in focus when considering lekking behaviour (e.g. Trail 1985; Höglund & Lundberg 1987; Hovi et al. 1994; McDonald & Potts 1994; Petrie et al. 1999; Krakauer 2005). Although rare, lekking behaviour is taxonomically widespread (Krebs & Davies 1991; Höglund & Alatalo 1995; Sherman 1999). Besides birds, lek-like mating systems have been identified in insects (Downes 1969), mammals (e.g. Clutton-Brock et al. 1993), fishes (e.g. Nordeide & Folstad 2000) and amphibians (Wells 1977; Höglund & Alatalo 1995). According to the definition of leks (Höglund & Alatalo 1995), females only visit them to choose a male for mating. Typically, there is high mating skew among the males on a lek (Mackenzie et al. 1995; Kokko et al. 1998), so that females appear to have preferences that lead to a situation where some males gain several matings while others have to do without. Therefore, in lek evolution theories, one of the key issues has been to Correspondence: T. Knopp, Fax: +358-9-19157694; E-mail: [email protected] © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd

explain why males that might face low chances of reproduction should join a lek at all. Several theoretical models have been developed to evaluate the selective pressures acting on the evolution of leks (e.g. Bradbury & Gibson 1983; Kokko 1997; Isvaran & St Mary 2003). Two of these models known as ‘hotspot models’ postulate that to increase their mating success, males aggregate in areas favoured by females or around preferred males, respectively. On its own, the latter hypothesis relies on the chances that the less-preferred males can occasionally secure matings by being close to the preferred ones. But if subordinates establish themselves on a lek where the top male is a close relative, there is the potential for kin-selected benefits as well (Kokko & Lindström 1996). These benefits arise even if the subordinates do not get matings but large leks as a whole attract more females than smaller ones. The larger leks may be more visible or audible (passive attraction, Parker 1983), or females may actively prefer to choose among many males (Kokko 1997). The genetic structuring of leks has been investigated in some birds (Tetrao urogallus, Regnaut et al. 2006; Centrocercus urophasianus, Gibson et al. 2005; Meleagris gallopavo, Krakauer 2005; Manacus manacus, Shorey et al. 2000; Chiroxiphia lanceolata, DuVal 2007; Tetrao tetrix,

2 T. K N O P P E T A L . Höglund et al. 1999; Pavo cristatus, Petrie et al. 1999), fishes (Salmo trutta: Carlsson & Carlsson 2002; Perca fluviatili, Gerlach et al. 2001; Gasterosteus aculeatus, Peuhkuri & Seppä 1998; Poecilia reticula, Russell et al. 2004), insects (Plagiolepis pygmaea, Trontti et al. 2005; Anastrepha fraterculus, Segura et al. 2007), and a mammal species (Lontra canadensis, Blundell et al. 2004). Some of these studies support the idea that kin selection may play a role in lek evolution (Höglund et al. 1999; Petrie et al. 1999; Shorey et al. 2000; Gerlach et al. 2001; Carlsson & Carlsson 2002; Krakauer 2005; Trontti et al. 2005; Regnaut et al. 2006). To our knowledge, the genetic relationships within amphibian leks remain unexamined. Many anuran amphibian species are known to form leks (e.g. Savage 1961; Pfennig et al. 2000; Murphy 2003; Friedl & Klump 2005), although the definition of a lek may vary across species and breeding systems (Wells 1977). Most obviously, the definition of a lek differs between prolonged and explosive breeders. In prolonged breeding, anuran males aggregate around a common spawning site (i.e. lek). Within this lek, the males defend a small territory from which they call for females (Wells 1977). The females will then orientate towards a specific calling male (reviewed in Gerhardt 1994). In explosive breeders, on the other hand, breeding sites typically consist of several smaller male groupings (here referred to as leks), which together form a breeding assemblage ranging from a few to several thousand males. Females are attracted to the breeding sites by the advertisement calls of the males (Wells 1977; Stebbins & Cohen 1995). In explosive breeders, orientation towards a specific male has not been shown. However, female choice within a lek might occur indirectly through male–male competition, where the fittest male is expected to win and thus gain a mating (Telford & Van Sickle 1989; Stebbins & Cohen 1995; Hutchinson 2005). In order to address the question whether kin selection can underlie lek formation in an explosively breeding amphibian, we conducted a genetic survey on seven Rana arvalis leks situated in two populations in southern Sweden. The main aim was to study the genetic structure of the male population. First, we tested whether the relatedness among males within leks is higher than expected by chance. Second, the two populations were chosen according to a differing spatial location of leks. The aim was to examine whether a contrasting spatial configuration of leks might affect the within population lek structure and the genetic relationship of the males.

Materials and methods Study species The moor frog (Rana arvalis) is a common amphibian species inhabiting damp fields, swamps, moors and meadows

across Eurasia (Arnold 2002). In southern Sweden, it is one of the most abundant anurans (Fog et al. 1997). It is an explosive breeder, with mating taking place under a short period in early spring. Within leks male–male competition occurs, but once in amplexus, the ovipositioning appears to take place without disturbance by other males (personal observation). Females lay one clutch (approximately 400– 1600 eggs) per year, and eggs take about 80–100 days to develop into metamorphs (Fog et al. 1997). Moor frogs reach sexual maturity after their second to fourth winter (Fog et al. 1997; Arnold 2002). During lekking, some moor frog males develop a blue dorsal colouration that may vary in intensity among individuals (Arnold 2002; Sheldon et al. 2003). It has been shown that when exposed to large predators, offspring of brighter blue males have a higher survival probability than those of dull males (Sheldon et al. 2003). This indicates that male colouration could act as an honest signal of male quality. However, studies examining female choice based on male colouration in the moor frog are currently lacking.

Sampling Field work was conducted during a 2-week period in April 2004 in Scania, southern Sweden (Fig. 1). Two populations, separated by approximately 30 km, were chosen (Fig. 1). In the first population (Frihult; 55°33′N, 13°38′E), the moor frog leks were situated in agricultural moorland ponds, with interpond distances ranging from 15 to 200 m. Three leks were sampled for a total of 95 males (FRIH 1–3; Fig. 2a). The second population, Sandbymosse (55°43′N, 13°25′E), was a large marsh area (approximately 0.5 ha) consisting of shallow water with some vegetation. Within the area, leks were clearly separate units albeit connected by water. Several leks were observed but only four were sampled for a total of 108 males (SM 1–4; Fig. 2b). When sampled, the males were caught by net or by hand and kept in a storage box. After removing 3 mm of tissue (with sterilized sharp nail scissors) from one toe, the males were released to the lek of capture. All leks were sampled many times during several days. The sample was taken from a different toe in each lek, and thus, movement of males in between the leks could be monitored. The tissue samples were stored in 70% ethanol at +4 °C until the DNA was extracted.

Molecular methods DNA extraction. DNA was extracted according to the protocol described by Elphinstone et al. (2003) with slight modifications (see below). Approximately 3 mm of R. arvalis toe tissue was placed in 150 μL digestion buffer (0.4 m NaCl, 10 mm Tris-Hcl, 2 mm EDTA, 2% SDS, pH 8) together with 7 μL of proteinase K (Finnzymes) and © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd

DO MOOR FROGS LEK WITH KIN? 3 Fig. 1 Map showing the geographical location of the two study populations in Scania, southern Sweden.

incubated at 60 °C for a minimum of 2 h. After centrifugation (1 min at 5.9× g) 50 μL of supernatant fluid was transferred to a microtitre filtration plate (AcroPrep by Pall Corp.) together with 10 μL silica suspension (silica gel 60 by Merck, double-distilled water) and 140 μL binding buffer (6 m NaI, saturated with 0.2 m Na2SO4). Samples were washed twice with 200 μL of cold wash buffer (20 mm Tris pH 7.4, 1 mm EDTA, 0.1 m NaCl, 100% ethanol) and the genomic DNA was eluted in 50 μL of prewarmed (60 °C) double-distilled water. Concentration of the extracted DNA (on average 20 ng/μL) was determined using GeneQuant pro RNA/DNA Calculator. Microsatellites. Eight microsatellite loci, four of which had been designed for R. arvalis and five for other species in the family Ranidae, were used in the study (Table 1). Polymerase chain reaction (PCR) amplification was conducted in a total © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd

volume of 10 μL containing: 0.5 μm of each primer (one labelled with a fluorescent dye), 0.25 μm deoxyribonucleotides, 1.5 μm MgCl2, 1× NH4 Reaction Buffer, 0.1 U BioTaq DNA polymerase (Bioline) and 10–20 ng DNA template. PCR profiles consisted of the following steps for the separate loci: WRA 1–22, WRA 1–28 and WRA 6–8: initial denaturation for 3 min at 94°, followed by 35 cycles of 30 s at 94 °C, 30 s at 50 °C and 60 s at 72 °C, and a final extension step of 10 min at 72 °C; WRA 1–160: 3 min at 94 °C; 5 s at 94 °C, 45 s at 60 °C, 1 min at 72 °C (35 cycles) and 10 min at 72 °C; for RCIDII and Recalq: 3 min at 95 °C; 45 s at 55 °C correspondingly 50 °C, 45 s at 72 °C, 45 s at 95 °C (35 cycles); 45 s at 50 °C and 3 min 72 °C; Ca41 and Rt2Ca25: 3 min at 94 °C; 30 s at 94 °C, 30 s at 60 °C in first cycle, then lowering 0.5 °C/cycle, 30 s at 72 °C (20 cycles); 30 s at 94 °C, 30 s at 50 °C, 30 s at 72 °C (15 cycles); and 5 min at 72 °C. Genotyping was conducted by the automated

4 T. K N O P P E T A L . Table 1 Microsatellite primers used in this study Locus

Origin

Allele size

N (alleles)

WRA 1-22* WRA1-28* WRA1-160* WRA6-8* RCIDII† RECALQ† RtCa25‡ RlatCa41§

R. arvalis R. arvalis R. arvalis R. arvalis R. catesbeiana R. esculenta R. temporaria R. latastei

145–161 209–347 270–304 164–212 165–16 230–236 133–172 151–169

8 44 11 10 3 3 16 7

*Paul Arens, unpublished (Genbank Accession nos AJ419881–AJ419884). †Vos et al. (2001). ‡Knopp et al. (2007). §Garner & Tomio (2001).

Fig. 2 Maps illustrating spatial details of the two study populations. (a) Frihult. The sampled leks (black circles) are in separate ponds (light grey). A road separates FRIH 1 pond from FRIH 2 and FRIH 3. (b) Sandbymosse. The sampled leks SM 1–SM 4 are connected by water. Inter-pond distances (m), sample sizes (N) and the estimated number of egg clumps (N tot) at study leks are shown.

capillary sequencer MegaBACE. 1000 (Amersham Biosciences) and visualized using the software fragment profiler (version 1.2; Amersham Biosciences).

Data analysis Genetic diversity was quantified in terms of allele number, observed (HO) and expected (HE) heterozygosities using microsatellite toolkit (Park 2001). Probabilities for Hardy–Weinberg equilibrium per locus and population were assessed using the Markov chain method as implemented in genepop 3.2 (Raymond & Rousset 1995). Likewise, all loci were tested for linkage disequilibrium with genepop. The presence of possible null alleles was checked by using

family data (offspring with known father and mother) from earlier experiments (Räsänen et al. 2003a, b). The pattern of substructuring within and among populations was investigated by Wright 1969) F-statistics and by a Bayesian clustering method. F-statistics were estimated using Weir & Cockerham 1984) FST in the program fstat 2.9.3 (Goudet 1995). The pairwise FST estimates were calculated for the two populations and for each lek separately. Bayesian clustering was done with the software baps 3.1 (Corander et al. 2003). In this program, both individual genotypes and sample groups (here defined by leks) can be assigned into clusters (K) assuming Hardy–Weinberg and linkage equilibriums (Corander et al. 2003). To investigate the genetic structuring at lek level, the data from both populations were pooled (N = 7 leks). In the individual level analyses, the populations were considered separately (Frihult N = 95, Sandbymosse N = 108). The maximum number of groups (K) was set to two, five and 10, and for each value of K, 10 repeats of the simulation were run. To test whether the mean relatedness within leks is higher than expected in unstructured populations, we used the program identix (Belkhir et al. 2002) to simulate and resample from 1000 populations with random mating. Resampling was done at the allelic level. Identity index (the expected proportion of loci that are homozygous in the offspring of a pair of individuals) was chosen as an estimator of relatedness due to its superior performance in tests concerning fewer than 10 loci (Belkhir et al. 2002). All leks were tested separately. Finally, by using kinship (Goodnight & Queller 1999), the likelihood of two randomly drawn individuals being full-sibs (H1 rp,m = 0.5, H0 rp,m = 0.0) was calculated for all pairwise comparisons within a population, where after the ratio of probable (P < 0.05) full-sibs was calculated separately for within leks and between leks (i.e. Nfullsibs/Ntotal © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd

DO MOOR FROGS LEK WITH KIN? 5 Table 2 Basic information on genetic variation within moor frog leks Lek

N

HE

SD (HE )

HO

SD (HO)

FIS

SM 1 SM 2 SM 3 SM 4 FRIH 1 FRIH 2 FRIH 3

36 33 25 14 33 27 35

0.55 0.58 0.57 0.55 0.54 0.53 0.57

0.10 0.09 0.10 0.09 0.10 0.10 0.09

0.54 0.57 0.59 0.60 0.55 0.55 0.59

0.03 0.03 0.03 0.05 0.03 0.03 0.03

0.02 0.02 −0.04 −0.08 −0.02 −0.02 −0.04

Table 3 Genetic differentiation (FST) between the leks in the two study populations (FRIH, Frihult; SM, Sandbymossen)

FRIH 2 FRIH 3 SM 1 SM 2 SM 3 SM 4

FRIH 1

FRIH 2

FRIH 3

SM 1

SM 2

SM 3

0.017 0.000 0.019* 0.040* 0.050* 0.011

0.006 0.022* 0.032* 0.052* 0.015

0.013* 0.024* 0.033* 0.011*

0.006 0.008 −0.010

−0.005 0.001

0.011

*P < 0.05 after sequential Bonferroni correction (Rice 1989). N, sample size. HE, expected heterozygosity. SD, standard deviation. HO, observed heterozygosity.

The total number of alleles per microsatellite locus varied from three to 44 (Table 1) and average expected heterozygosities within leks ranged from 0.53 to 0.58 (Table 2). Allele counts were similar between the two populations and among all the sampled leks. No linkage disequilibrium or departure from Hardy–Weinberg equilibrium was detected for any of the loci or population (P > 0.05). In family data, no deviations from Mendelian inheritance patterns were found (results not shown). This suggests that null alleles should not be of great concern and thus all loci were included in further analyses.

system; Table 3). In fact, FST was zero between the two spatially most distant leks in Frihult (FRIH 1 and FRIH 3, Table 3). In line with previous analyses, the Bayesian clustering method (Corander et al. 2003) also identified two separate populations at lek level. All the seven leks were clustered correctly according to their population of origin but leks were not detected as being differentiated units (K = 2). At the individual level analysis, the optimal clustering was reached within Frihult at K = 7 (P = 0.95) and in Sandbymosse at K = 7 (P = 0.88). In both optimal partitionings, the clusters included individuals from several different leks, and thus, division a priori expectations remained unsupported. In relatedness analyses, the mean pairwise identity index at the seven leks ranged between 0.38 and 0.48 (average = 0.42). When compared to random expectation, none of the leks had males genetically more related than expected by chance (P = 0.22). A similar pattern is evident when considering probable full-sibs: when considering all possible pairwise comparisons of two individuals within a lek, the proportion of probable full-sibs was not higher than the proportion of probable full-sibs when the two individuals were sampled from different leks. Instead, there was a trend in the opposite direction (6.0% and 6.9%, respectively, total number of comparisons n = 3023 and n = 7220). In the analyses of known full-sibs, the observed mean relatedness (0.53) did not differ from the expected value of 0.5, indicating that our tests of relatedness for the lekking males should be reliable.

Population substructuring

Discussion

The pairwise exact test of differentiation revealed a significant (P < 0.05) subdivision between Frihult and Sandbymosse (FST = 0.025), but no differences in allele frequencies were detected among the leks within the populations (Table 3: Frihult mean FST = 0.008, Sandbymosse mean FST = 0.002, P > 0.05). Contrary to our expectations, FST among the leks in Frihult (leks in separate ponds) was not higher than among the leks in Sandbymosse (marsh

The kin-selection hypothesis evoked to explain the evolution of leks was not supported by our data from a lekking amphibian: the degree of genetic structuring within populations was low, as was the mean relatedness among males within leks too. Leks did not contain probable full-sibs more often than expected by chance (i.e. when picking two individuals from different leks). Hence, it seems that the clustering of moor frog males is random in

pairwise comparisons). Under the kin-selection hypothesis, the number of related individuals should be higher within leks than across leks (Kokko & Lindström 1996; Höglund et al. 1999). Due to the high degree of philopatry in frogs (e.g. Hitchings & Beebee 1997; Seppä & Laurila 1999; Newman & Squire 2001), the half-sib and parent–offspring relationships were disregarded from the test. The reliability of the results was tested with family data consisting of 29 offspring derived from four families.

Results Genetic variation

© 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd

6 T. K N O P P E T A L . respect to their relatedness and an explanation for the formation of leks has to be found elsewhere. Consequently, we now briefly review how theoretical explanations for lek evolution might apply to amphibians. Of the different theoretical models evaluating the evolution of leks (see Introduction), the hypotheses where lek formation is driven by a female preference of clustered males (including the kin-selection hypothesis) are based on the premise that a larger lek should be visited by females more frequently than smaller ones (Höglund & Alatalo 1995; Kokko & Lindström 1996). Experiments with birds (Alatalo et al. 1992; Hovi et al. 1994; Gibson 1996) and insects (Shelly & Greenfield 1991; Svensson & Petersson 1992) have shown that number of females visiting a breeding site indeed increases with the number of males displaying on it, and similar patterns are evident in observational data (reviewed in Hutchinson 2005). However, in an experiment with a prolonged breeding anuran (Hyla gratinosa), a reduction in the number of calling males did not decrease the females’ visitation rate on a lek (Murphy 2003). Moreover, females visited breeding sites although no males were present; a pattern observed for other anuran species as well (Wells 1977; Sinsch 1988). Hence, Murphy (2003) suggested that in H. gratinosa both sexes respond similarly to environmental variables (e.g. rainfall or temperature) and either one of the hotspot hypothesis (a female preference of breeding site or certain males) is most likely to explain lek formation in this species. Several studies on prolonged breeding species have demonstrated that females in experimental conditions display a clear preference for males with certain call characteristics (reviewed in Gerhardt 1994). This could lead to lek formation if subordinate males cluster around preferred males (the ‘hotshot’ hypothesis, see also Humfeld 2008). However, in natural populations, the advertisement calls of a specific male might be indistinguishable for females because of considerable background noise of conspecifics and other species (e.g. Ehret & Gerhardt 1980; Gerhardt 1994; Bishop et al. 1995). Consequently, despite the fact that the call characteristics of a male could be a fitness indicator, it has been suggested that this trait in itself would be unimportant for determining the male’s mating success (Friedl & Klump 2005). Instead, data often show a correlation between lek attendance and mating success (Fiske et al. 1998; Friedl & Klump 2005). There is thus strong selection pressure for males to spend as much time as possible on the lek. Assuming that attendance is a condition-dependent trait (e.g. Kokko et al. 1999; Sullivan & Kwiatkowski 2007), females that mate on leks will have a high probability of mating with a high-quality male even if choice within the lek is random (Friedl & Klump 2005). In explosive breeding species, no equivalent female choice for male characteristics has been demonstrated, and thus, the advertisement calls of explosive breeders are thought to be mainly a long-

distance call to attract females to the breeding grounds (Wells 1977). The female perspective is similar to that described above. Mating males within a lek are winners of intense direct male–male competition (Wells 1977); thus, mating on the lek makes it relatively unlikely that the sire is a lowquality male. Thus, despite a lack of correlation between lek size and female attendance, for both types of amphibian reproduction strategies the male assemblages are likely to reduce the females cost of mate search. If the male groupings simultaneously improve the chances of finding high quality males, lek evolution is in good accordance with theory: fast and easy mate search is predicted to be particularly important when the benefits being sought (genetic quality differences among males) are not necessarily large (Alatalo et al. 1998; Kokko et al. 2006; Kotiaho & Puurtinen 2007). In addition, for explosive breeders fast reproduction is highly important due to the short time given for larval development. As eggs are often laid in temporary waters that will desiccate during summer, the eggs laid later will reduce survival probability due to a precipitated development (e.g. Loman 2002). While female movements towards assembled males might explain lekking itself, it does not yet explain why explosive breeders establish several smaller male groupings (here referred to as leks) within a breeding ground. One reason could be the patchy distribution of suitable waters, combined with female reluctance to move large distances. Moor frogs lek and lay their eggs preferably in sunny patches of shallow water, often found in smaller areas within a deeper pond. Then, once a preferred habitat is saturated, it would be beneficial to establish a new lek. Alternatively (or additionally), female travel costs are important because the mating success of a specific male depends on the number of females arriving on a lek and the relationship between lek size and mating skew (Widemo & Owens 1995). The per capita mating prospects decrease on very large leks if the female visitation rate fails to increase in proportion with lek size (Hutchinson 2005). If all males in a large area have grouped together, yet females prefer fast and easy mate search, it becomes beneficial for males to form smaller groups within reasonable travel distances for females. Future work should clearly aim at predicting exactly how the rules of phonotaxis and travel costs create female visitation rates for different-sized male aggregations, together with spatial considerations of how far females and males have to travel when certain sized aggregations are formed. Variations in population density will influence female mate-location costs (Sullivan & Kwiatkowski 2007 and references therein); thus, lek formation under varying densities would be a particularly interesting study topic (see also Kokko & Rankin 2006). As with females, travelling increases energy expenditure for males too, and the risk of missing a mating opportunity © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd

DO MOOR FROGS LEK WITH KIN? 7 might increase during movement between leks (Figenschou et al. 2004). Therefore, the tendency to form small leks at intermediate distances can be influenced by male as well as female costs. Capture–recapture studies conducted earlier suggest that adult moor frogs are extremely philopatric (Haapanen 1970). In line with these results, our study recorded a low rate of interpond movements. Although disturbed, males were recaptured at the initial lek several times, while movement between leks was observed only twice. These movements occurred between two leks within the marsh area, which implies that leks connected by water might be less stable than isolated leks. However, more data on movements are required to verify this conclusion. Unfortunately, with the current data it is not possible to address the issue whether moor frog males show sitetenacity at the lek-level between years, as shown for Hyla arborea (Friedl & Klump 2005). Our failure to find genetic structuring within populations indicates that dispersal occurs between leks, but it does not distinguish between natal and breeding dispersal or otherwise identify the life-history stage in which dispersal occurs. Finally, we note that the estimation of relatedness with molecular markers is methodologically a demanding task, were the results often are dependent on the markers and estimators used (Smith et al. 2001; Blouin 2003; Csilléry et al. 2006). However, a possible underestimation of relatedness was checked with family data, which clearly showed that for the methods chosen, the loci had enough information for rather accurate conclusions. Further, our results are comparable with earlier studies focusing on kinship. Several studies have succeeded in finding support for a role of kin selection in lek formation using only a few moderately polymorphic loci (Höglund et al. 1999; Petrie et al. 1999; Gerlach et al. 2001; Höglund & Shorey 2003; Krakauer 2005). In the future, behavioural studies should combine genetic data on lek visitation rates in different spatial settings. This could be combined with models predicting visitation rates from mechanistic considerations of known phonotaxis rules (e.g. Bishop et al. 1995), or taking simple passive attraction as a null model (Parker 1983). This should shed light on the question of how moor frogs leks are formed, and how stationary leks are between the years. Repeating the exercise for a prolonged breeding amphibian would help answering the question whether lek formation obeys different rules in different breeding systems.

Acknowledgements We would like to thank Jon Loman for supporting us with the data on moor frog populations in Scania. We also thank Fredrik Söderman for all the advice and practical help given and Peder Fiske and one anonymous referee for useful comments on an earlier version of this manuscript. Our research was supported by Suomen Biologian Seura Vanamory, Societas pro Fauna & Flora © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd

Fennica, Jenni & Antti Wihuri Foundation, and the Academy of Finland (H.K., J.M.). A license for toe-clipping was admitted by Länsstyrelsen i Skåne län, record no. 521-7137-04, 1200-001.

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This study was originally the master thesis of Minttu Heimovirta. It is a part of Theresa Knopp’s PhD thesis which aims to uncover the population structure and phylogeorgraphy of Rana arvalis in northern Europe. Both M.H. and T.K. are members of the Ecological Genetic Research Unit, lead by Professor Juha Merilä. J.M. interests are in population and evolutionary quantitative genetics, as well as in animal adaptation spatial and temporal environmental heterogeneity. Professor Hanna Kokko is head of the Laboratory of Ecological and Evolutionary Dynamics, studying interactions between individual behaviour, life-history theory and population dynamics.