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Multiple paternity in free-living root voles (Microtus oeconomus)
Behavioural Processes 82 (2009) 211–213
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Behavioural Processes journal homepage: www.elsevier.com/locate/behavproc
Short report
Multiple paternity in free-living root voles (Microtus oeconomus) Anetta Borkowska a,∗ , Zbigniew Borowski b , Kamil Krysiuk c ´wierkowa 20B, PL15-950 Białystok, Poland Institute of Biology, University of Białystok, S Department of Forest Ecology and Wildlife Management, Forest Research Institute, Braci Le´snej 3, S˛ekocin Stary, PL05-090 Raszyn, Poland c Warsaw Agricultural University, Faculty of Animal Sciences, Department of Animal Genetics and Breeding, Nowoursynowska 166, PL02-787, Warsaw, Poland a
b
a r t i c l e
i n f o
Article history: Received 5 April 2009 Received in revised form 20 May 2009 Accepted 24 May 2009 Keywords: Microsatellites Microtus oeconomus Multiple paternity Reproductive success Root vole
a b s t r a c t We used 10 microsatellite loci to determine the mating system and male reproductive success in a natural population of the root vole (Microtus oeconomus). By genotyping 21 females and their 111 offspring (5.28 ± 0.27 S.E. pups per female), we found evidence for multiple paternity in 38% of the litters sired by two or three males. Paternity was not significantly skewed away from the null expectation of equal proportions of offspring sired in any of the multiple-sired litters, and the most successful male fathered between 40% and 60% of the pups in a litter. The results indicate that promiscuity is a common mode of reproduction, consistent with the previous classification of the mating system based on the spatial structure of the root vole population. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Behavioural ecologists have long been interested in mating systems and variance of reproductive success. Highly variable molecular markers such as microsatellites are now used to reassess mating systems from the genetic point of view. Multiple paternity of litters, resulting from insemination of a female by at least two males, has been reported in rodents (e.g., Goossens et al., 1998; Bartmann and Gerlach, 2001; Dean et al., 2006). A number of genetic investigations have yielded mating system classifications diverging from earlier ones based on behavioural data. For example, the prairie vole (Microtus ochrogaster) was considered to be a monogamous species with reproductive exclusivity, territoriality, behavioural monogamy and biparental care (Getz et al., 1981). Molecular analyses, however, revealed multiple paternity in 56% of wild prairie vole litters (Solomon et al., 2004). Microtine rodents have been reported to have modes of mating systems ranging from genetic monogamy (M. pinetorum, Marfori et al., 1997) to genetic promiscuity in most vole species (e.g., Microtus pennsylvanicus, Berteaux et al., 1999; Microtus arvalis, Fink et al., 2006). In the root vole (Microtus oeconomus), both promiscuous groups (Tast, 1966) and monogamous pairs (Viitala, 1994) have been observed. However, the basic modes of spatial organisation in root vole populations are female territoriality and overlapping male home ranges (Gliwicz, 1997). According to the author, each female
∗ Corresponding author. Tel.: +48 85 745 7356; fax: +48 85 745 7302. E-mail address: [email protected] (A. Borkowska). 0376-6357/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.beproc.2009.05.003
territory is overlapped by several male ranges, and larger males have access to more territories of females. Such a spatial structure of the population suggests promiscuity rather than monogamy as the prevailing mating system. Here we investigated pattern of mating system by genotyping 21 mothers and their litters from a wild population of root voles. Information on the minimum number of fathers contributing to a given litter was used to estimate the reproductive skew among sires within litters and to determine male mating strategies. 2. Materials and methods 2.1. Sampling and microsatellite analysis The study was done in the Biebrza valley in an open sedge bog near Gugny settlement (NE Poland, 53◦ 22 N 22◦ 35 E) in 2004–2006. We used the catch-mark-release (CMR) method, with wooden livetraps placed in a 7 × 11 grid pattern at 10 m intervals. The traps were checked twice a day. A total of 21 pregnant females were caught in two breeding seasons: 2004 (n = 10) and 2005 (n = 11). The females were taken to the laboratory and housed separately in their own cages (46 cm × 29 cm × 22 cm) with wood shavings and hay as bedding and with food and water ad libitum until they weaned the young (21 litters). Mothers and their offspring were housed for 5 weeks; then samples of 20–30 hair bulbs for DNA analyses were plucked from all of them, and the voles were released in the field at the females’ catch sites. Total genomic DNA was extracted using the Genomic Mini Kit (A&A Biotechnology). All individuals were genotyped at 10
212
A. Borkowska et al. / Behavioural Processes 82 (2009) 211–213
Table 1 Allele number (A), frequency of null allele (FNull ) and exclusion probability of the locus for the first parent (ExcP1) and for the second parent with first parent assigned (ExcP2) in the population studied, for 10 microsatellite loci used to infer parentage in root vole litters. Locus
A
FNull
ExcP1
ExcP2
Ma35a Ma54a Ma66a Ma88a MAG6b MSMM2c MSMM3c MSMM5c MSMM6c MSMM7c
15 (13) 17 (11) 15 (14) 22 (20) 17 (13) 15 (13) 11 (10) 9 (8) 7 (6) 14 (12)
−0.001 −0.022 −0.008 −0.001 −0.016 −0.001 −0.022 0.025 0.019 0.002
0.592 0.610 0.575 0.687 0.636 0.541 0.505 0.373 0.404 0.622
0.746 0.759 0.731 0.815 0.778 0.704 0.675 0.557 0.586 0.767
Total
14.2 (12.0)
0.999
0.999
Number of alleles in a sample of mothers and their offspring in parentheses. All loci were in Hardy–Weinberg equilibrium. a Primer from Gauffre et al., 2007. b Primer from Jaarola et al., 2007. c Primer from Ishibashi et al., 1999.
microsatellite loci (Table 1). Forward primers were labelled with fluorescent dye (Applied Biosystems): Ma35-FAM, Ma54-PET, Ma66-VIC, Ma88-NED and MSMM6-PET in set 1; and MAG6-NED, MSMM2-PET, MSMM3-FAM, MSMM5-VIC and MSMM7-NED in set 2. Amplification of each set was carried out in a 5-L reaction volume using Qiagen Multiplex PCR Master Mix under standard multiplex PCR conditions (Qiagen) with 26 cycles. PCR products were separated using ABI3130 DNA sequencer, and microsatellite alleles were analysed using GENEMAPPER4.0 software (Applied Biosystems). The same microsatellite markers were also assayed in 110 individuals caught in the population in autumn 2006; this was done in order to estimate allele frequencies, the frequency of null alleles of the entire data set (altogether 131 field-collected voles and 111 lab-born offspring) and the exclusion probabilities for each locus and for all loci combined (Table 1) with CERVUS3.0.3 (Kalinowski et al., 2007). 2.2. Paternity analysis Paternal alleles were first identified manually, by comparison of offspring genotypes with the known maternal genotype. This method may underestimate the actual number of fathers because it assumes that there is no allele sharing among fathers or between the mother and fathers. Thus, multiple paternity was concluded only if more than two non-maternal alleles occurred in at least two loci in the litter arrays and confirmed by GERUD2.0 software (Jones, 2005) (Table 2). GERUD determines the minimum number of fathers and the number of progeny per sire, trying every possible combination of paternal genotypes until it finds one or more combinations that can explain the progeny array. We ranked the multiple solutions by likelihood, based on the allele frequencies of 10 microsatellite loci and Mendelian segregation of alleles in the progeny. Because no unique solution was found in any litters, we chose to accept the most frequent paternal contribution ratio with the greatest number of the most likely GERUD solutions. Thus, for each litter we determined the effective number of sires which takes into account the number of offspring assigned to sire and sires’ contribution to a litter (Neff et al., 2008). The reproductive skew among males was expressed as 1—(effective number of sires/actual number of sires). 2 analysis was used to test the null hypothesis that the proportion of offspring fathered by each sire is equal and that male reproductive success is random and conforms to a Poisson distribution. GERUDsim2.0 was also used to make sure that the detectability of multiple sires in a litter is high enough, and to deter-
mine the probability of correctly reconstructing paternal genotypes (Jones, 2005).
3. Results and discussion Paternity analysis and genotyping of mothers and their litters clearly indicated that the promiscuity is frequent in the root vole. Multiple paternity was detected in 8 of 21 litters (38%) both by manual comparison of mother and offspring genotypes and using GERUD software (Table 2) in two breeding seasons. Half of the multiple-sired litters were sired by two males, and the other half by three males. Multiple-sired litters averaged 5.62 ± 0.38 S.E. pups, a nonsignificant difference from the average of 5.08 ± 0.37 S.E. pups found in all other litters (Mann–Whitney test, U = 33.0, P > 0.05). The effective number of fathers (mean value 2.39 ± 0.11 S.E.) did not differ from the minimum number of fathers (2.50 ± 0.19 S.E.) in multiple-sired litters (Mann–Whitney test, U = 20.0, P > 0.05). The estimated frequencies are fairly consistent with the findings of previous studies in other voles: 22% multiple-sired litters in Myodes glareolus (Ratkiewicz and Borkowska, 2000), 50% in M. arvalis (Borkowska and Ratkiewicz, unpublished) and 56% in M. ochrogaster (Solomon et al., 2004). The microsatellite set used in our study provided considerable power to detect multiple paternity in the root vole litters. The number of alleles present at each locus ranged between 7 and 22, with a low frequency of null alleles (Table 1) which could generate high frequencies of false paternity exclusions (Dakin and Avise, 2004). Exclusion probabilities for the first parent and for the second parent with first parent assigned were high for each locus and for all loci combined. We assessed the ability to detect single, double or triple mating by simulations in GERUDsim2.0. In five independent runs of 1000 iterations and using five variable loci with the highest exclusion probabilities (Ma35, Ma54, Ma88, MAG6 and MSMM7; Table 1) and with 5 or 6 pups as litter size, one sire was detected in 100% of the simulations, two in 99.4–99.8%, and three in 60.2–89.9% of them. Therefore it seems unlikely that a mistake was made in classifying any multiple paternity cases as single paternity or in assessing the number of sires in a litter. The proportion of paternal genotypes correctly reconstructed depends on the number of offspring genotyped, and the use of additional polymorphic loci does not markedly increase the probability because it adds more paternal genotypes that must be reconstructed correctly (Jones, 2005). For this reason we set the parameters in GERUDsim2.0 at one father (with 100% of the offspring sired), two and three fathers with equal proportions of offspring sired, and two loci (Ma88 and MAG6). The probabilities of correctly reconstructing the paternal genotypes were 84.7–90.9% for one sire, 26.0–34.0% for two sires, and 6.7–10.1% for three sires, with litters of 5 or 6 pups. Because the root vole litter is relatively small, the efficacy of genotypic reconstruction is low, especially for multiple mating. However, the proportion of offspring sired by each male in a particular litter was the same despite the number and polymorphism of the loci used. In Microtine rodents, the spacing system of sexually receptive females is the most important factor determining the spatial distribution and mating strategy of males (Ims, 1987). Root vole males try both to control female and to copulate with another ones to increase their reproductive success. Males could monopolise females in their oestrus period as we found for the 62% of females single-mated in the population. In vole populations it is difficult to monopolise females when the local density of males is high (Ishibashi and Saitoh, 2008) and females actively seek fertilisation by several males (Berteaux et al., 1999). Thus, another strategy of males for increasing their reproductive success is to increase their number of mates, that is, to gain access to already mated females. Here we
A. Borkowska et al. / Behavioural Processes 82 (2009) 211–213
213
Table 2 Minimum number of fathers revealed by allele count and GERUD2.0 software, effective number of fathers, paternity proportion (most likely ratio of paternal contribution) and paternity skew estimated using GERUD for 8 multiple-sired root vole litters. Litter
L5 L7 L8 L13 L14 L16 L18 L21
NO
5 6 8 5 5 5 6 5
NA
4 (1) 4 (3) 4 (3) 3 (6) 3 (6) 4 (2) 4 (4) 5 (3)
Min. no. of fathers Allele count
GERUD
2 2 2 2 2 2 2 3
2 3 3 2 2 2 3 3
Effective no. of fathers
NT
NS
1.92 3.00 2.67 1.92 1.92 1.92 3.00 2.78
10 50 50 34 50 20 50 50
10 50 49 30 28 20 45 50
Paternity Proportion
Skew
3:2 2:2:2 4:2:2 3:2 3:2 3:2 2:2:2 2:2:1
0.04 0 0.11 0.04 0.04 0.04 0 0.07
NO : number of offspring; NA : maximum number of parental alleles, and in parentheses the number of loci with the greatest number of paternal alleles; NT : total number of GERUD solutions; NS : number of solutions that returned the same paternal contribution ratio.
found that paternity in the root vole was not significantly skewed away from the null expectation of equal proportions of offspring sired in any of the multiple-sired litters (Table 2), and the distribution of reproductive success across all sires conformed to a Poisson distribution (2 = 2.59, P > 0.05). Although there were significant differences in number of pups sired between the most successful male (2.75 ± 0.25 S.E.) and the other males (1.92 ± 0.29 S.E.) in multiple-sired litters (Wilcoxon test, Z = 2.37, P < 0.05), the most successful male sired only 40–60% of the pups in a litter. Mating with an already mated female appears to be advantageous for root vole males even if they mate second. Equal contributions to litters by several males have rarely been noted in rodents (Ratkiewicz and Borkowska, 2000), because in a competitive mating situation the effectiveness of a given insemination depends on the interaction of mating order, time interval since the previous mating, and time of insemination relative to ovulation (Huck et al., 1989). The absence of a reproductive skew among root vole males siring offspring in a multiple litter may result in changes of their copulatory behaviour in response to the risk of sperm competition, leading to, for example, a higher frequency of repeated ejaculations (Stockey and Preston, 2004). Acknowledgement This study was supported by Forest Research Institute (project no. 24-03-06) and University of Białystok (BST-117). References Bartmann, S., Gerlach, G., 2001. Multiple paternity and similar variance in reproductive success of male and female wood mice (Apodemus sylvaticus) housed in an enclosure. Ethology 107, 889–899. Berteaux, D., Bêty, J., Rengifo, E., Bergeron, J.-M., 1999. Multiple paternity in meadow voles (Microtus pennsylvanicus): investigating the role of the female. Behavioral Ecology and Sociobiology 45, 283–291. Dakin, E.E., Avise, J.C., 2004. Microsatellite null alleles in parentage analysis. Heredity 93, 504–509. Dean, M.D., Aralie, K.G., Nachman, M.W., 2006. The frequency of multiple paternity suggests that sperm competition is common in house mice (Mus domesticus). Molecular Ecology 15, 4141–4151. Fink, S., Excoffier, L., Heckel, G., 2006. Mammalian monogamy is not controlled by single gene. Proceedings of the National Academy of Sciences of the USA 103, 10956–10960.
Gauffre, B., Galan, M., Bretagnolle, V., Cosson, J.F., 2007. Polymorphic microsatellite loci and PCR multiplexing in the common vole, Microtus arvalis. Molecular Ecology Notes 7, 830–832. Getz, L.L., Carter, C.S., Gavish, L., 1981. The mating system of the prairie vole, Microtus ochrogaster: field and laboratory evidence for pair-bonding. Behavioral Ecology and Sociobiology 8, 189–194. Gliwicz, J., 1997. Space use in the root vole: basic pattern and variability. Ecography 20, 383–389. Goossens, B., Graziani, L., Waits, L.P., Farand, E., Magnolon, S., Coulon, J., Bel, M.C., Taberlet, P., Allainé, D., 1998. Extra-pair paternity in the monogamous Alpine marmot revealed by nuclear DNA microsatellite analysis. Behavioral Ecology and Sociobiology 43, 281–288. Huck, U.W., Tonias, B.A., Lisk, R.D., 1989. The effectiveness of competitive male inseminations in golden hamsters, Mesocricetus auratus, depends on an interaction of mating order, time delay between males, and the time of mating relative to ovulation. Animal Behaviour 37, 674–680. Ims, R.A., 1987. Male spacing systems in Microtine rodents. The American Naturalist 130, 475–484. Ishibashi, Y., Yoshinaga, Y., Saitoh, T., Abe, S., Iida, H., Yoshida, M.C., 1999. Polymorphic microsatellite DNA markers in the field vole Microtus montebelli. Molecular Ecology 8, 157–168. Ishibashi, Y., Saitoh, T., 2008. Effect of local density of males on the occurrence of multimale mating in gray-sided voles (Myodes rufocanus). Journal of Mammalogy 89, 388–397. Jaarola, M., Ratkiewicz, M., Ashford, R.T., Brunhoff, C., Borkowska, A., 2007. Isolation and characterization of polymorphic microsatellite loci in the field vole, Microtus agrestis, and their cross-utility in the common vole, Microtus arvalis. Molecular Ecology Notes 7, 1029–1031. Jones, A.G., 2005. GERUD2.0: a computer program for the reconstruction of parental genotypes from half-sib progeny arrays with known or unknown parents. Molecular Ecology Notes 5, 708–711. Kalinowski, S.T., Taper, M.L., Marshall, T.C., 2007. Revising how the computer program CERVUS accommodates genotyping error increases success in paternity assignment. Molecular Ecology 16, 1099–1106. Marfori, M.A., Parker, P.G., Gregg, T.G., Vandenbergh, J.G., Solomon, N.G., 1997. Using DNA fingerprinting to estimate relatedness within social groups of pine voles. Journal of Mammalogy 78, 715–724. Neff, B.D., Pitcher, T.E., Ramnarine, I.W., 2008. Inter-population variation in multiple paternity and reproductive skew in the guppy. Molecular Ecology 17, 2975–2984. Ratkiewicz, M., Borkowska, A., 2000. Multiple paternity in the bank vole (Clethrionomys glareolus): field and experimental data. Mammalian Biology 65, 6–14. Solomon, N.G., Keane, B., Knoch, L.R., Hogan, P.J., 2004. Multiple paternity in socially monogamous prairie voles (Microtus ochrogaster). Canadian Journal of Zoology 82, 1667–1671. Stockey, P., Preston, B.T., 2004. Sperm competition and diversity in rodent copulatory behaviour. Journal of Evolutionary Biology 17, 1048–1057. Tast, J., 1966. The root vole, Microtus oeconomus (Pallas), as an inhabitant of seasonally flooded land. Annales Zoologici Fennici 3, 127–171. Viitala, J., 1994. Monogamy in free living Microtus oeconomus. Annales Zoologici Fennici 31, 343–345.
Contents lists available at ScienceDirect
Behavioural Processes journal homepage: www.elsevier.com/locate/behavproc
Short report
Multiple paternity in free-living root voles (Microtus oeconomus) Anetta Borkowska a,∗ , Zbigniew Borowski b , Kamil Krysiuk c ´wierkowa 20B, PL15-950 Białystok, Poland Institute of Biology, University of Białystok, S Department of Forest Ecology and Wildlife Management, Forest Research Institute, Braci Le´snej 3, S˛ekocin Stary, PL05-090 Raszyn, Poland c Warsaw Agricultural University, Faculty of Animal Sciences, Department of Animal Genetics and Breeding, Nowoursynowska 166, PL02-787, Warsaw, Poland a
b
a r t i c l e
i n f o
Article history: Received 5 April 2009 Received in revised form 20 May 2009 Accepted 24 May 2009 Keywords: Microsatellites Microtus oeconomus Multiple paternity Reproductive success Root vole
a b s t r a c t We used 10 microsatellite loci to determine the mating system and male reproductive success in a natural population of the root vole (Microtus oeconomus). By genotyping 21 females and their 111 offspring (5.28 ± 0.27 S.E. pups per female), we found evidence for multiple paternity in 38% of the litters sired by two or three males. Paternity was not significantly skewed away from the null expectation of equal proportions of offspring sired in any of the multiple-sired litters, and the most successful male fathered between 40% and 60% of the pups in a litter. The results indicate that promiscuity is a common mode of reproduction, consistent with the previous classification of the mating system based on the spatial structure of the root vole population. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Behavioural ecologists have long been interested in mating systems and variance of reproductive success. Highly variable molecular markers such as microsatellites are now used to reassess mating systems from the genetic point of view. Multiple paternity of litters, resulting from insemination of a female by at least two males, has been reported in rodents (e.g., Goossens et al., 1998; Bartmann and Gerlach, 2001; Dean et al., 2006). A number of genetic investigations have yielded mating system classifications diverging from earlier ones based on behavioural data. For example, the prairie vole (Microtus ochrogaster) was considered to be a monogamous species with reproductive exclusivity, territoriality, behavioural monogamy and biparental care (Getz et al., 1981). Molecular analyses, however, revealed multiple paternity in 56% of wild prairie vole litters (Solomon et al., 2004). Microtine rodents have been reported to have modes of mating systems ranging from genetic monogamy (M. pinetorum, Marfori et al., 1997) to genetic promiscuity in most vole species (e.g., Microtus pennsylvanicus, Berteaux et al., 1999; Microtus arvalis, Fink et al., 2006). In the root vole (Microtus oeconomus), both promiscuous groups (Tast, 1966) and monogamous pairs (Viitala, 1994) have been observed. However, the basic modes of spatial organisation in root vole populations are female territoriality and overlapping male home ranges (Gliwicz, 1997). According to the author, each female
∗ Corresponding author. Tel.: +48 85 745 7356; fax: +48 85 745 7302. E-mail address: [email protected] (A. Borkowska). 0376-6357/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.beproc.2009.05.003
territory is overlapped by several male ranges, and larger males have access to more territories of females. Such a spatial structure of the population suggests promiscuity rather than monogamy as the prevailing mating system. Here we investigated pattern of mating system by genotyping 21 mothers and their litters from a wild population of root voles. Information on the minimum number of fathers contributing to a given litter was used to estimate the reproductive skew among sires within litters and to determine male mating strategies. 2. Materials and methods 2.1. Sampling and microsatellite analysis The study was done in the Biebrza valley in an open sedge bog near Gugny settlement (NE Poland, 53◦ 22 N 22◦ 35 E) in 2004–2006. We used the catch-mark-release (CMR) method, with wooden livetraps placed in a 7 × 11 grid pattern at 10 m intervals. The traps were checked twice a day. A total of 21 pregnant females were caught in two breeding seasons: 2004 (n = 10) and 2005 (n = 11). The females were taken to the laboratory and housed separately in their own cages (46 cm × 29 cm × 22 cm) with wood shavings and hay as bedding and with food and water ad libitum until they weaned the young (21 litters). Mothers and their offspring were housed for 5 weeks; then samples of 20–30 hair bulbs for DNA analyses were plucked from all of them, and the voles were released in the field at the females’ catch sites. Total genomic DNA was extracted using the Genomic Mini Kit (A&A Biotechnology). All individuals were genotyped at 10
212
A. Borkowska et al. / Behavioural Processes 82 (2009) 211–213
Table 1 Allele number (A), frequency of null allele (FNull ) and exclusion probability of the locus for the first parent (ExcP1) and for the second parent with first parent assigned (ExcP2) in the population studied, for 10 microsatellite loci used to infer parentage in root vole litters. Locus
A
FNull
ExcP1
ExcP2
Ma35a Ma54a Ma66a Ma88a MAG6b MSMM2c MSMM3c MSMM5c MSMM6c MSMM7c
15 (13) 17 (11) 15 (14) 22 (20) 17 (13) 15 (13) 11 (10) 9 (8) 7 (6) 14 (12)
−0.001 −0.022 −0.008 −0.001 −0.016 −0.001 −0.022 0.025 0.019 0.002
0.592 0.610 0.575 0.687 0.636 0.541 0.505 0.373 0.404 0.622
0.746 0.759 0.731 0.815 0.778 0.704 0.675 0.557 0.586 0.767
Total
14.2 (12.0)
0.999
0.999
Number of alleles in a sample of mothers and their offspring in parentheses. All loci were in Hardy–Weinberg equilibrium. a Primer from Gauffre et al., 2007. b Primer from Jaarola et al., 2007. c Primer from Ishibashi et al., 1999.
microsatellite loci (Table 1). Forward primers were labelled with fluorescent dye (Applied Biosystems): Ma35-FAM, Ma54-PET, Ma66-VIC, Ma88-NED and MSMM6-PET in set 1; and MAG6-NED, MSMM2-PET, MSMM3-FAM, MSMM5-VIC and MSMM7-NED in set 2. Amplification of each set was carried out in a 5-L reaction volume using Qiagen Multiplex PCR Master Mix under standard multiplex PCR conditions (Qiagen) with 26 cycles. PCR products were separated using ABI3130 DNA sequencer, and microsatellite alleles were analysed using GENEMAPPER4.0 software (Applied Biosystems). The same microsatellite markers were also assayed in 110 individuals caught in the population in autumn 2006; this was done in order to estimate allele frequencies, the frequency of null alleles of the entire data set (altogether 131 field-collected voles and 111 lab-born offspring) and the exclusion probabilities for each locus and for all loci combined (Table 1) with CERVUS3.0.3 (Kalinowski et al., 2007). 2.2. Paternity analysis Paternal alleles were first identified manually, by comparison of offspring genotypes with the known maternal genotype. This method may underestimate the actual number of fathers because it assumes that there is no allele sharing among fathers or between the mother and fathers. Thus, multiple paternity was concluded only if more than two non-maternal alleles occurred in at least two loci in the litter arrays and confirmed by GERUD2.0 software (Jones, 2005) (Table 2). GERUD determines the minimum number of fathers and the number of progeny per sire, trying every possible combination of paternal genotypes until it finds one or more combinations that can explain the progeny array. We ranked the multiple solutions by likelihood, based on the allele frequencies of 10 microsatellite loci and Mendelian segregation of alleles in the progeny. Because no unique solution was found in any litters, we chose to accept the most frequent paternal contribution ratio with the greatest number of the most likely GERUD solutions. Thus, for each litter we determined the effective number of sires which takes into account the number of offspring assigned to sire and sires’ contribution to a litter (Neff et al., 2008). The reproductive skew among males was expressed as 1—(effective number of sires/actual number of sires). 2 analysis was used to test the null hypothesis that the proportion of offspring fathered by each sire is equal and that male reproductive success is random and conforms to a Poisson distribution. GERUDsim2.0 was also used to make sure that the detectability of multiple sires in a litter is high enough, and to deter-
mine the probability of correctly reconstructing paternal genotypes (Jones, 2005).
3. Results and discussion Paternity analysis and genotyping of mothers and their litters clearly indicated that the promiscuity is frequent in the root vole. Multiple paternity was detected in 8 of 21 litters (38%) both by manual comparison of mother and offspring genotypes and using GERUD software (Table 2) in two breeding seasons. Half of the multiple-sired litters were sired by two males, and the other half by three males. Multiple-sired litters averaged 5.62 ± 0.38 S.E. pups, a nonsignificant difference from the average of 5.08 ± 0.37 S.E. pups found in all other litters (Mann–Whitney test, U = 33.0, P > 0.05). The effective number of fathers (mean value 2.39 ± 0.11 S.E.) did not differ from the minimum number of fathers (2.50 ± 0.19 S.E.) in multiple-sired litters (Mann–Whitney test, U = 20.0, P > 0.05). The estimated frequencies are fairly consistent with the findings of previous studies in other voles: 22% multiple-sired litters in Myodes glareolus (Ratkiewicz and Borkowska, 2000), 50% in M. arvalis (Borkowska and Ratkiewicz, unpublished) and 56% in M. ochrogaster (Solomon et al., 2004). The microsatellite set used in our study provided considerable power to detect multiple paternity in the root vole litters. The number of alleles present at each locus ranged between 7 and 22, with a low frequency of null alleles (Table 1) which could generate high frequencies of false paternity exclusions (Dakin and Avise, 2004). Exclusion probabilities for the first parent and for the second parent with first parent assigned were high for each locus and for all loci combined. We assessed the ability to detect single, double or triple mating by simulations in GERUDsim2.0. In five independent runs of 1000 iterations and using five variable loci with the highest exclusion probabilities (Ma35, Ma54, Ma88, MAG6 and MSMM7; Table 1) and with 5 or 6 pups as litter size, one sire was detected in 100% of the simulations, two in 99.4–99.8%, and three in 60.2–89.9% of them. Therefore it seems unlikely that a mistake was made in classifying any multiple paternity cases as single paternity or in assessing the number of sires in a litter. The proportion of paternal genotypes correctly reconstructed depends on the number of offspring genotyped, and the use of additional polymorphic loci does not markedly increase the probability because it adds more paternal genotypes that must be reconstructed correctly (Jones, 2005). For this reason we set the parameters in GERUDsim2.0 at one father (with 100% of the offspring sired), two and three fathers with equal proportions of offspring sired, and two loci (Ma88 and MAG6). The probabilities of correctly reconstructing the paternal genotypes were 84.7–90.9% for one sire, 26.0–34.0% for two sires, and 6.7–10.1% for three sires, with litters of 5 or 6 pups. Because the root vole litter is relatively small, the efficacy of genotypic reconstruction is low, especially for multiple mating. However, the proportion of offspring sired by each male in a particular litter was the same despite the number and polymorphism of the loci used. In Microtine rodents, the spacing system of sexually receptive females is the most important factor determining the spatial distribution and mating strategy of males (Ims, 1987). Root vole males try both to control female and to copulate with another ones to increase their reproductive success. Males could monopolise females in their oestrus period as we found for the 62% of females single-mated in the population. In vole populations it is difficult to monopolise females when the local density of males is high (Ishibashi and Saitoh, 2008) and females actively seek fertilisation by several males (Berteaux et al., 1999). Thus, another strategy of males for increasing their reproductive success is to increase their number of mates, that is, to gain access to already mated females. Here we
A. Borkowska et al. / Behavioural Processes 82 (2009) 211–213
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Table 2 Minimum number of fathers revealed by allele count and GERUD2.0 software, effective number of fathers, paternity proportion (most likely ratio of paternal contribution) and paternity skew estimated using GERUD for 8 multiple-sired root vole litters. Litter
L5 L7 L8 L13 L14 L16 L18 L21
NO
5 6 8 5 5 5 6 5
NA
4 (1) 4 (3) 4 (3) 3 (6) 3 (6) 4 (2) 4 (4) 5 (3)
Min. no. of fathers Allele count
GERUD
2 2 2 2 2 2 2 3
2 3 3 2 2 2 3 3
Effective no. of fathers
NT
NS
1.92 3.00 2.67 1.92 1.92 1.92 3.00 2.78
10 50 50 34 50 20 50 50
10 50 49 30 28 20 45 50
Paternity Proportion
Skew
3:2 2:2:2 4:2:2 3:2 3:2 3:2 2:2:2 2:2:1
0.04 0 0.11 0.04 0.04 0.04 0 0.07
NO : number of offspring; NA : maximum number of parental alleles, and in parentheses the number of loci with the greatest number of paternal alleles; NT : total number of GERUD solutions; NS : number of solutions that returned the same paternal contribution ratio.
found that paternity in the root vole was not significantly skewed away from the null expectation of equal proportions of offspring sired in any of the multiple-sired litters (Table 2), and the distribution of reproductive success across all sires conformed to a Poisson distribution (2 = 2.59, P > 0.05). Although there were significant differences in number of pups sired between the most successful male (2.75 ± 0.25 S.E.) and the other males (1.92 ± 0.29 S.E.) in multiple-sired litters (Wilcoxon test, Z = 2.37, P < 0.05), the most successful male sired only 40–60% of the pups in a litter. Mating with an already mated female appears to be advantageous for root vole males even if they mate second. Equal contributions to litters by several males have rarely been noted in rodents (Ratkiewicz and Borkowska, 2000), because in a competitive mating situation the effectiveness of a given insemination depends on the interaction of mating order, time interval since the previous mating, and time of insemination relative to ovulation (Huck et al., 1989). The absence of a reproductive skew among root vole males siring offspring in a multiple litter may result in changes of their copulatory behaviour in response to the risk of sperm competition, leading to, for example, a higher frequency of repeated ejaculations (Stockey and Preston, 2004). Acknowledgement This study was supported by Forest Research Institute (project no. 24-03-06) and University of Białystok (BST-117). References Bartmann, S., Gerlach, G., 2001. Multiple paternity and similar variance in reproductive success of male and female wood mice (Apodemus sylvaticus) housed in an enclosure. Ethology 107, 889–899. Berteaux, D., Bêty, J., Rengifo, E., Bergeron, J.-M., 1999. Multiple paternity in meadow voles (Microtus pennsylvanicus): investigating the role of the female. Behavioral Ecology and Sociobiology 45, 283–291. Dakin, E.E., Avise, J.C., 2004. Microsatellite null alleles in parentage analysis. Heredity 93, 504–509. Dean, M.D., Aralie, K.G., Nachman, M.W., 2006. The frequency of multiple paternity suggests that sperm competition is common in house mice (Mus domesticus). Molecular Ecology 15, 4141–4151. Fink, S., Excoffier, L., Heckel, G., 2006. Mammalian monogamy is not controlled by single gene. Proceedings of the National Academy of Sciences of the USA 103, 10956–10960.
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