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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 lempuraria 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. tnnpwm;l commonly contain multiply sired offspring. We suggest that communal breeding
may afiect paternity patterns in R. fanpa& as well as in anurans in general.
0 19911 The Iinnran Soriefy of London
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
Refcrences . . . . . . . . . . . . . . . . . . . . . . . 230
Appendix . . . . . . . . . . . . . . . . . . . . . . . . 232
Correspondence to A. Laurila. E-mail: anssi.laurila@helsi.fi
Present address P. Seppa: Department of Genetics, Uppsala University, Box 7003, S-75007 Uppsala, Sweden.
22 1
0024-4066/98/020221 + 12 $25.00/0/bj970180 0 1998 The Linnean Society of London
222 A. LAURIL4 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,
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.
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. taporaria
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
Tvarminne Zoological Station, Hanko. O n the island of Lgngskar, a total of 121
females bred in 2 1 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. O n the island of
Porskar (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). O n both islands, we collected samples from the rock-pools where the
numbers of breeding R. taporaria 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 1 of water. The eggs hatched in laboratory, and after reaching
developmental stage 25 (Gosner, 1960), the tadpoles were preserved at -8OoC until
laboratory analysis.
Electrophoresis
O n 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.
Paternity patterns and the general mating structure of the populations (panmixis)
were examined separately in the two populations. We studied paternity patterns by
224 A. LAURIL4 AND P. SEPPA
TABLE1. The enzymes and loci studied, Enzyme Commission (E.C.) numbers, buffers used, and the
number of alleles (no,,)in each locus.
Buffers: (I): Gel: 42.9mmol I-' TRIS, 4.2mmol I-' citrate, pH 8.4; Tray: 135mmol I-' TRIS, 42.9mmol I - '
citrate, pH 7.1 (Varvio-Ah0 & Pamilo 1980).
(11): Gel: 9mmol I-'mM TRIS, 3mmol I-'rnM citrate, 1.2rnmol I-'mM EDTA, pH 7; Tray: 135rnmol I - ' TRIS,
44.5mmol I - ' citrate, 1.2mmol I - ' EDTA, pH 7 (Ayala ef 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 -H J H J , where
H, 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 (9)can be used to calculate the
number of matrilines (A4) and patrilines (P)that have contributed to this group:
+
r/=0.25/M+ (rM/4)[(M- 1)/M] 0.25/P+ ( ~ p / 4 ) [ ( P1)/pl
- (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>O), our r/ 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=O). Thus, the
MULTIPLE PATERNITY IN RANA 72NPORARk4 225
2. Allele frequencies of the Rana temporaria populations (estimated weighing groups equally) of
TABLE
the loci studied.
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 /(4?71) (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 x2
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 ZOO/
ZOO, and ten of F/ZOO (Appendix).The deduced parental genotypes were ZOO/ZOO,
and F/ZOO,although the mother being ZOO/ZOO, she could have had two mating
partners, Z OO/ZOO and F/F. Consequently, the estimates of paternity frequency
estimated from genotype distributions were conservative.
RESULTS
TABLE
3. Genetic relatedness (r+ SE) and inbreeding (F+ SE), estimated from the tadpole kin groups
of Rana temporaria. tF refms to the deuiation of the inbreeding coeficients fmm <em (one-sample (two-sided) t-tests).
None ofthe tf-values were significant at the level -0.05.
r SE F SE b
Ungskar Mpi 0.389 0.085 -0,058 0.107 0.54
In two kin groups from LHngskr (A-54, B-23), and in one group from Porskar
(C-ll), the observed genotypes could not have been produced by a single pair
(Appendix). In addition, on LHngskr the observed genotype frequencies were
significantly different from the expected based on single mating in one or more loci
for 14 kin groups, and on Porskar for nine groups (Table 4). These figures add up
to 16 kin groups in LBngskar (55%), and 10 groups in Porskar (43%), as the
proportion of females producing kin groups fathered by more than one male. In
ten cases, the deviation from single mating in a single kin group was corroborated
by a significant value at more than one locus.
MULTIPLE PATERNITY IN RANA 7EMPORARL.4 227
DISCUSSION
number of patrilines calculated from these estimates. Second, the estimate in Porskar
was not significantly lower than the expected value for full sibs. Finally, the number
of patrilines estimated from the genotype frequencies of the offspring may have
been exaggerated, because of the large number of x2 tests. If a standard Bonferroni
correction is made for the significance levels of the tests (separately for each locus),
the values showing P<O.OOl in Table 4 remain significant. This would leave
Berger & Rybacki (1 992) suggested that, in nature, sperm competition occurs at
communal oviposition sites where amplectant pairs gather to release the gametes.
In R. temporaria, as in many other explosively breeding anurans, oviposition occurs
within a very restricted area in the breeding pond (Wells, 1977; Seale, 1982;
Waldman, 1982; A. Laurila, pers. observ.).For example, in our study pools oviposition
took place in an area of approximately 1 m2 in pools A and B and 1 .5m2 in pool
female that has chosen the best available mating partner may avoid situations
leading to multiply sired offspring. On the other hand, if multiple paternity results
from a female’s assurance strategy against reduced fertilization success and limited
mate choice opportunities, it may be more widespread among species with explosive
synchronous breeding systems. Further studies are needed to study whether paternity
patterns covary with breeding systems in anurans.
Tvarminne zoological station provided the laboratory facilities for rearing the
tadpoles. Comments by J. Elmberg, I. K. Hanski, K. Lindstrom, N. Peuhkuri, E.
Ranta and L. Sundstrom substantially improved the manuscript. This work has
been financially supported by University of Helsinki (to AL, PS), the Academy of
Finland (AL, PS), and the Emil Aaltonen Foundation (PS).
REFERENCES
Harris H, Hopkinson DA. 1976. Handbook ofengme electrophoresis in human genetics. Amsterdam: North
Holland.
Howard RD, Kluge AG. 1985. Proximate mechanisms of sexual selection in wood frogs. Evolution
39: 260-277.
Hunter FM, Petrie M, Otmnen My Birkhead T, Meller AP. 1993. Why do females copulate
repeatedly with one male? Trend3 in Ecology and Evolution 8: 2 1-26.
Hutchings JA, Myers RA. 1988. Mating success of alternative maturation phenotypes in male
APPENDIX
100 F
Mpi
F 100 f' F 100 F 100
Idh
F F S
Aco
100 100 S 100
Pgk
100 S
UngskPr:
A-1 9 12 21 13 7 2 7 1 1 7 2
A-2 9 11 20 4 7 3 8 12 10
A-3 4 8 8 21 3 3 1 1 4 7 3 1 0
A-4 9 9 3 21 1 4 5 6 4 13 4 21
A-52 8 14 3 24 12 9 17 8 12 I 1
A-53 10 10 11 9 8 1 1 5 5 7 3
A-54 20 20 4 6 4 1 3 5 10 5 20
A-55 5 15 20 1 8 I1 10 10 10 9
A-56 13 8 7 14 7 11 3 13 5 21
A-57 13 20 36 30 1 1 17 18 13 19 2
A-58 11 9 7 14 4 7 6 8 6 2 1
A-59 15 9 6 10 8 24 5 15 4 18 4
A-60 11 11 22 13 9 7 15 21
A-61 1 21 24 7 8 3 3 8 9 23
A-73 11 7 4 3 3 5 1 34 33
A-74 7 11 6 26 10 12 25 26
A-79 22 13 10 12 11 7 16 23 1
A-82 22 22 8 10 1 15 7 22
A-88 4 12 7 20 4 12 9 6 10 8 11
A-89 11 15 10 14 6 5 10 5 13 12 26
B-19 6 13 4 26 5 8 9 4 13 9 26
B-2 1 I1 1 25 19 2 9 3 17 3
B-23 6 16 23 2 2 10 8 9 8 24
B-77 5 16 11 10 11 8 2 2 11 5 21
B-81 11 12 23 5 4 8 5 22 22
B-83 2 10 11 14 10 9 2 8 1 4 11 6 24
B-84 17 1 9 10 11 4 13 7 12 7
B-85 11 11 15 8 10 13 12 9 23
B-86 13 I1 13 I1 1 2 9 1 2 23 19
Porskar:
c-1 10 10 20 3 3 4 12 8 6 4
C-2 8 13 1 23 9 14 23 23
c-3 I1 11 23 6 5 7 5 12 10 22
c-4 12 5 7 24 13 9 12 11 20 4
c-5 10 4 8 22 10 I1 8 13 14 6
C-6 18 2 20 14 6 14 20
C-7 14 6 1 21 8 1 6 1 1 1 9
C-8 5 12 7 24 9 4 8 3 12 12 24
c-9 4 15 2 20 9 12 7 13 8 4
c-10 2 13 7 24 4 5 3 3 9 1 3 2 2 3
C-11 8 11 1 21 7 10 1 2 12 9 21
C-12 8 13 22 22 7 15 11
(2-13 20 20 15 5 18 10
C-14 5 15 I 11 10 2 9 2 4 2 12 7 18 3
C-15 5 10 5 20 10 9 20 20
C-16 25 25 3 12 10 12 12 25
C-17 21 12 7 2 22 9
C-18 14 8 22 9 13 12 10 17 5
C-20 8 12 2 22 6 4 4 8 12 8 12 2
c-22 4 9 26 7 9 3 3 3 10 10 11 15
C-23 5 9 4 21 4 15 10 11 8 1
C-24 12 11 2 14 8 22 1 10
(2-25 11 10 21 5 4 21 6 3