Multiple paternity in the common frog (Rana temporaria): genetic evidence from tadpole kin groups

Biological Journal of the Linnean Society (1998), 63: 221–232.

Multiple paternity in the common frog (Rana temporaria): genetic evidence from tadpole kin groups ¨ 1,2 ANSSI LAURILA1 AND PERTTU SEPPA 1

Division of Population Biology, Department of Ecology and Systematics, and 2Division of Genetics, Department of Biosciences, P.O. Box 17, FIN-00014 University of Helsinki, Finland Received 11 April 1997; accepted for publication 28 August 1997

Very few studies have investigated the occurrence of multiple paternity and sperm competition in amphibians. We studied genetic relatedness within kin groups of tadpoles of an aquatically breeding anuran Rana temporaria using allozymes. We collected samples from 52 naturally fertilized spawn clumps produced by single females at three breeding sites in two populations. We estimated relatedness (r) within kin groups, and compared the observed genotype distributions of the tadpoles (on average 23 individuals in each group) with the expected distributions based on single mating. Average relatedness over five polymorphic loci was 0.44 and 0.43 in the two populations, the latter being significantly smaller than that expected by single mating (0.5). The number of patrilines, calculated from relatedness estimates, was 1.3 in one population and 1.4 in the other. Genotype distributions deviated significantly from the expected in half of the kin groups and at all breeding sites. The results show that egg clutches of R. temporaria commonly contain multiply sired offspring. We suggest that communal breeding may affect paternity patterns in R. temporaria as well as in anurans in general.  1998 The Linnean Society of London

ADDITIONAL KEY WORDS:—Amphibia – Anura – allozymes – genetic relatedness – multiple paternity – sperm competition. CONTENTS

Introduction . . . . . . . . . . . . . . . . . . . . . . . 222 Material and methods . . . . . . . . . . . . . . . . . . . 223 Study object and sample collection . . . . . . . . . . . . . . 223 Electrophoresis . . . . . . . . . . . . . . . . . . . . 223 Estimation of paternity patterns from the tadpole kin groups . . . . . 223 Results . . . . . . . . . . . . . . . . . . . . . . . . 225 Genetic composition of the populations . . . . . . . . . . . . 225 Relatedness in the tadpole kin groups . . . . . . . . . . . . . 226 Genotypic composition of tadpole kin groups . . . . . . . . . . 226 Discussion . . . . . . . . . . . . . . . . . . . . . . . 227 Multiple paternity in the common frog . . . . . . . . . . . . 227 Behavioural mechanisms and multiple paternity in anurans . . . . . . 228 Acknowledgements . . . . . . . . . . . . . . . . . . . . 230 References . . . . . . . . . . . . . . . . . . . . . . . 230 Appendix . . . . . . . . . . . . . . . . . . . . . . . . 232 Correspondence to A. Laurila. E-mail: [email protected] Present address P. Seppa¨: Department of Genetics, Uppsala University, Box 7003, S-75007 Uppsala, Sweden. 0024–4066/98/020221+12 $25.00/0/bj970180

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 1998 The Linnean Society of London

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¨ A. LAURILA AND P. SEPPA INTRODUCTION

Mating with multiple partners should generally be advantageous for males due to their ability to produce gametes at a higher rate (Trivers, 1972). In females, single mating is usually enough to fertilize all the offspring in a brood (but see e.g. Robertson, 1990; Elmberg, 1991; Bourne, 1993 for exceptions), and, consequently, the benefits of multiple mating are not obvious (e.g. Hunter et al., 1993). In addition to fertility assurance, direct benefits to females include acquisition of nutrients and paternal care as well as avoidance of male harassment (see Birkhead & Parker, 1997 for a recent review). Females may also gain indirect benefits like improved genetic quality of offspring by mating with multiple partners (Birkhead & Parker, 1997). Consequently, mating with multiple partners is a potential factor driving sexual selection in both sexes (Møller, 1992; Møller & Birkhead, 1994), and may affect patterns of parental care (Trivers, 1972; Westneat & Sherman, 1993). Among vertebrates, multiple paternity within a brood has been found in several bird species (reviewed by Birkhead & Møller, 1992), mammals (e.g. Birdsall & Nash, 1973; Hanken & Sherman, 1981; Keane et al., 1994), reptiles (Stille et al., 1986, Schwartz et al., 1989; Olsson et al., 1994) and fish (Hutchings & Myers, 1988; Philipp & Gross, 1994). Among amphibians, females produce multiply sired clutches in terrestrially breeding salamanders with internal fertilization and prolonged sperm storage (Tilley & Hausman, 1976). Studies on multiple paternity have mostly concentrated on species with internal fertilization, and there has been less interest in species with external fertilization (but see e.g. Hutchings & Myers, 1988; Philipp & Gross, 1994; Shapiro et al., 1994; D’Orgeix & Turner, 1995; Levitan & Petersen, 1995). In most anurans (frogs and toads) fertilization is external. Male anurans compete strongly for mating opportunities, and numerous papers have reported mating advantage of large males (see Andersson, 1994 for review). However, none of those studies has addressed how this advantage is realized in the number of fertilized offspring. In many species, males attempt to displace each other from the female’s back and when males are unable to solve their dispute over a female, several males may be attached to her at the moment of fertilization. These multi-male breeding groups are common in some terrestrially breeding frogs (Kusano et al., 1991; Jennions & Passmore, 1993; D’Orgeix & Turner, 1995), and high fertilization success among the competing males has been reported in the treefrog Agalychnis callidryas (D’Orgeix & Turner, 1995). In aquatically breeding anurans it is not unusual to see many males fighting for a female at the spawning site, but, possibly for practical reasons, there are no observations of simultaneous sperm release by several males. In addition, it has been suggested that sperm competition may occur in communal breeding areas where amplectant pairs gather to spawn (Berger & Rybacki, 1992). To our knowledge, the study by D’Orgeix & Turner (1995) is the only one giving genetic evidence for sperm competition and multiple paternity in anurans. Studies on amphibians breeding in aquatic environment are lacking. In the present paper we report on paternity patterns in two populations of an aquatically breeding anuran, the common frog Rana temporaria. We analysed the genetic composition of tadpole groups derived from naturally fertilized egg clumps produced by single females. We chose to use allozymes as genetic markers, because they provide rapid information on population-level phenomena and a large number of individuals can be screened within a reasonable time span. We analysed paternity patterns by estimating

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relatedness among kin group members and by deriving effective mate numbers based on these estimates. We also compared the observed genotype distributions of the offspring with the expectation based on single mating. MATERIAL AND METHODS

Study object and sample collection Rana temporaria breeds in a wide range of freshwater habitats across Central and Northern Europe. Our study population breeds in rock-pools on rocky islands along the Baltic coast in southwestern Finland. The common frog is an ‘explosive’ breeder (sensu Wells, 1977). In southern Finland breeding occurs in late April and normally lasts less than 3 weeks (Elmberg, 1990; A. Laurila, pers. observ.). However, within a single rock-pool breeding occurs during a considerably shorter time, usually less than a week (A. Laurila, pers. observ.). In our study population of R. temporaria mating occurs almost exclusively at night, and the behaviour of mating individuals is susceptible to disturbance. Consequently, we were not able to carry out direct observations of mating behaviour. Because each female lays a single distinct clump of eggs per breeding season and fresh clumps can reliably be told apart from the clumps laid by other females (Savage, 1961), analyses of paternity patterns were possible. In late April 1994, we collected freshly laid R. temporaria eggs on two islands near Tva¨rminne Zoological Station, Hanko. On the island of La˚ngska¨r, a total of 121 females bred in 21 different rock-pools in 1994. In rock-pool A, we collected samples from every deposited egg clump (20 breeding females in 1994). In pool B (24 breeding females), samples were collected from nine egg clumps. On the island of Porska¨r (182 breeding females in 23 rock-pools in 1994), samples were collected from 23 egg clumps in a pool with 47 breeding females in 1994 (pool C in the Appendix). On both islands, we collected samples from the rock-pools where the numbers of breeding R. temporaria females were the highest. A sample of approximately 50 eggs was taken from each egg batch and brought to the laboratory. The eggs from each clump were then placed in separate plastic pans filled with 2 l of water. The eggs hatched in laboratory, and after reaching developmental stage 25 (Gosner, 1960), the tadpoles were preserved at −80°C until laboratory analysis. Electrophoresis On average 23 (range 20–36) offspring from each clump were analysed using horizontal starch-gel electrophoresis. Twenty-two enzyme systems were stained using standard formulations (Harris & Hopkinson, 1976). Five loci were polymorphic in one or both of the populations to estimate relatedness and the mating system reliably (see Table 1). The total number of tadpoles analyzed in 52 groups was 1189. Estimation of paternity patterns from the tadpole kin groups Paternity patterns and the general mating structure of the populations (panmixis) were examined separately in the two populations. We studied paternity patterns by

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T 1. The enzymes and loci studied, Enzyme Commission (E.C.) numbers, buffers used, and the number of alleles (nall) in each locus. Enzyme

Locus

E.C.

Buffer

nall

Mannose phosphate isomerase Phosphogluconate dehydrogenase Isocitrate dehydrogenase Aconitase Phosphoglyceratekinase

Mpi Pgd Idh Aco Pgk

5.3.1.8 1.1.1.44 1.1.1.42 4.2.1.3 2.7.2.3

I II II I II

2 2 3 2 2

Buffers: (I): Gel: 42.9mmol l−1 TRIS, 4.2mmol l−1 citrate, pH 8.4; Tray: 135mmol l−1 TRIS, 42.9mmol l−1 citrate, pH 7.1 (Varvio-Aho & Pamilo 1980). (II): Gel: 9mmol l−1mM TRIS, 3mmol l−1mM citrate, 1.2mmol l−1mM EDTA, pH 7; Tray: 135mmol l−1 TRIS, 44.5mmol l−1 citrate, 1.2mmol l−1 EDTA, pH 7 (Ayala et al., 1974).

estimating genetic relatedness within the kin groups from the genotype frequency data, and examining multilocus genotype patterns in the individual kin groups. Relatedness (r) was estimated as genotypic correlation among group members (Pamilo, 1990). This method was originally tailored for describing colony kin structures in haplo-diploid social insects (see Crozier & Pamilo, 1996), but it is perfectly applicable for diploid organisms living in social groups as well (McCauley & O’Donnell, 1984; Avise & Shapiro, 1986; Schwartz et al., 1989; Costa & Ross, 1993). Panmixis was estimated using the inbreeding coefficient F=(1−Ho/He), where Ho is the observed and He is the Hardy-Weinberg expected heterozygosity (Wright, 1951). Both relatedness and inbreeding estimates were calculated using computer software provided by P. Pamilo, and jackknifed over groups and loci to obtain standard errors (Efron & Tibshirani, 1993). These were then used to test the differences of the estimates from expected values (assuming t-distribution). For relatedness, we used r=0.5 as the expected value, which is the expected relatedness among full siblings. The expected value for the inbreeding coefficient is zero, which indicates total panmixis (e.g. Nei, 1987). Relatedness among the members of a group is determined by the number, relatedness and breeding success of the individuals in the parental generation (Ross, 1993; Queller, 1993). Therefore, relatedness not only describes the kin structure of the groups, but can also be used to characterize the breeding system of the species (the number of males and females contributing to the group). When groups of diploid organisms belong to the same generation, and mating in the population is random, genetic relatedness within the group (rf ) can be used to calculate the number of matrilines (M) and patrilines (P) that have contributed to this group: rf=0.25/M+(rM/4)[(M−1)/M]+0.25/P+(rP/4)[(P−1)/P]

(1)

(Queller, 1993), where rM is relatedness among females and rP relatedness among males that have contributed to this group. In our case, equation (1) can be considerably simplified, because the egg clumps of R. temporaria are produced by a single female (M=1). If multiple males contributing to the offspring of a single female are related (rP >0), our rf values are underestimates. However, our study populations turned out to be panmictic (see results), and multiple males contributing to the same group were therefore assumed to be unrelated (rP=0). Thus, the

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T 2. Allele frequencies of the Rana temporaria populations (estimated weighing groups equally) of the loci studied. Mpi La˚ngska¨r Porska¨r

100

F

100

Pgd F

100

Idh F

S

100

Aco S

100

Pgk S

0.548 0.677

0.452 0.323

0.891 0.990

0.109 0.010

0.497 0.597

0.205 0.142

0.299 0.262

0.632 0.730

0.368 0.270

0.936 0.873

0.064 0.127

relatedness among the tadpoles in a kin group depends only on the number of males fathering the brood. By substituting M=1, rP=0, and solving for P gives: P=1/(4rf−1)

(2)

Matrilines and patrilines in this context are genetically effective numbers. Therefore they refer to the harmonic means across groups, reflecting the genetic effects of multiple females or males contributing to the groups (Wade, 1985). Multiple paternity was also analysed directly from the genotype distributions of each offspring. Deviation from single mating by females was determined by comparing the observed genotypes in all loci to the ones expected from a single pair using v2 tests (with the Yates correction when appropriate). The parental genotypes were deduced from the offspring genotypes by always choosing the ones requiring the smallest number of fathers according to simple Mendelian rules. For example, in tadpole group C-1 in enzyme Mpi, there were ten offspring of the genotype 100/ 100, and ten of F/100 (Appendix). The deduced parental genotypes were 100/100, and F/100, although the mother being 100/100, she could have had two mating partners, 100/100 and F/F. Consequently, the estimates of paternity frequency estimated from genotype distributions were conservative.

RESULTS

Genetic composition of the populations Genotype frequencies of individual offspring are shown in the Appendix, and allele frequencies (estimated weighing groups equally) of the two populations in Table 2. Enzymes Pgd, Aco and Pgk were all bi-allelic systems. The more common allele was designated 100, and the rare allele either Fast (F) or Slow (S) depending on the relative mobility compared to the common allele. Enzyme Idh was tri-allelic, with both a fast and a slow rare allele. In enzyme Mpi, two fast alleles (with relative mobilities 105 and 107) were first separated besides the common allele (100). The fast alleles were, however, difficult to separate, and because there may have been ambiguities in scoring them, they were combined for the data analysis (as allele F). The inbreeding estimates per locus, and overall loci for the two populations are presented in Table 3. The single-locus estimates varied slightly around zero, but none deviated significantly from zero. Consequently, the population estimates were close to zero (Table 3) indicating random mating within our study populations. The La˚ngska¨r population was sampled from two distinct rock-pools; thus the zero inbreeding coefficient suggests that they belong to the same breeding population.

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T 3. Genetic relatedness (r±SE) and inbreeding (F±SE), estimated from the tadpole kin groups of Rana temporaria. tF refers to the deviation of the inbreeding coefficients from zero (one-sample (two-sided) t-tests). None of the tF-values were significant at the level P