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Low level of extrapair parentage in wild zebra finches
Animal Behaviour 79 (2010) 261–264
Contents lists available at ScienceDirect
Animal Behaviour journal homepage: www.elsevier.com/locate/anbehav
Low level of extrapair parentage in wild zebra finches Simon C. Griffith a, b, *, Clare E. Holleley a, b, Mylene M. Mariette a, b, Sarah R. Pryke a, b, Nina Svedin a a b
Department of Brain, Behaviour and Evolution, Macquarie University School of Biological, Earth and Environmental Sciences, University of New South Wales
a r t i c l e i n f o Article history: Received 28 September 2009 Initial acceptance 12 October 2009 Final acceptance 16 November 2009 Available online 7 December 2009 MS. number: 09-00639 Keywords: extrapair paternity intraspecific brood parasitism sexual conflict sexual selection sperm competition Taeniopygia guttata zebra finch
The captive zebra finch, Taeniopygia guttata, has become one of the key vertebrate model systems for studying a range of behavioural, physiological and neurological phenomena. In particular, this species has played a key role in developing our understanding of sexual selection and sperm competition. In contrast with the large number of studies using domesticated zebra finches, relatively few studies have focused on free-living populations of wild zebra finches. Investigating the incidence of extrapair paternity in zebra finches in the Australian desert, we found a very low level; 1.7% of 316 offspring from four of 80 broods fathered outside the pair bond. These numbers contrast with the high levels of extrapair paternity observed in domesticated aviary populations, and suggest a low level of sperm competition and sexual selection in natural populations. Our finding of such a low rate of extrapair paternity in the wild zebra finch suggests that it is one of the most genetically monogamous of all passerine species and that has important implications for future studies of this model organism in studies of sexual selection and reproductive biology. In addition, we found that 5.4% of 316 offspring were not related to either putative parent and hatched from eggs that had been dumped by intraspecific brood parasites. Ó 2009 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
In birds, about 90% of all species form socially monogamous pair bonds, and in these species 11% of offspring, on average, are sired by an extrapair male (Griffith et al. 2002). Extrapair paternity (EPP) is an important evolutionary phenomenon in socially monogamous species because it can dramatically increase the variance in reproductive success among males within a population (Whittingham & Dunn 2005), and it can provide an important postcopulatory mechanism for selection on genetic compatibility (Griffith & Immler 2009). In addition to describing the variation in the level of EPP and accompanying sexual selection across species, research has focused on the physiological and behavioural processes that relate to the incidence of EPP. Foremost among these is sperm competition (Parker 1970), an area in which the zebra finch, Taeniopygia guttata, has been one of the most widely used model organisms because of its amenability as a captive animal. Studies of the zebra finch represent a significant proportion of all the empirical work contributing to our understanding of the mechanisms of sperm competition in birds, including the timing and frequency of pair and extrapair copulations (Birkhead et al. 1989), sperm precedence models (Birkhead et al. 1988b), fertilization success and sperm quality (Birkhead et al. 1993), sperm storage
* Correspondence: S. C. Griffith, Department of Brain, Behaviour and Evolution, Macquarie University, Sydney, New South Wales 2109, Australia. E-mail address: simon.griffi[email protected] (S.C. Griffith).
(Birkhead et al. 1989), sperm depletion and allocation (Birkhead & Fletcher 1995), sperm morphology (Birkhead et al. 2005), and female propensity to engage in extrapair copulations (Forstmeier 2007). The zebra finch has been similarly prominent in the field of sexual selection through studies of mate choice (e.g. Rutstein et al. 2007), ornamental song (e.g. Collins et al. 1994), parental care (e.g. Burley 1988), sex allocation (e.g. Burley 1981) and maternal effects (e.g. Gil et al. 1999). All of these important and influential studies have focused on domesticated birds that have been selectively bred for over 100 generations (Zann 1996), and held in conditions that are far removed from those experienced by free-living wild birds. The use of domesticated model organisms is a crucial component of modern biological research. However, to aid the interpretation of captive studies it is important to develop an understanding of the wild context from which the model organism originated. A previous study of the zebra finch population studied over a long period by R. Zann in Victoria, Australia (Birkhead et al. 1990) found a very low level of EPP and intraspecific brood parasitism. However, there are a number of currently unresolved issues arising from this study. First, the study by Birkhead et al. (1990) was based on a small sample of just 25 families situated on an irrigated farm in temperate Victoria, which bred at very low density and synchrony and may not have truly represented the species in its native environment (T. R. Birkhead, personal communication). Furthermore, Birkhead et al. (1990) reported a potential case of quasiparasitism from families in which the molecular methods used were unable to
0003-3472/$38.00 Ó 2009 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.anbehav.2009.11.031
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S.C. Griffith et al. / Animal Behaviour 79 (2010) 261–264
make an accurate discrimination of the level of relatedness to a brood parasite and the pair male simultaneously (see Griffith et al. 2004). Here therefore for the first time we investigated the level of EPP in wild populations of free-living zebra finches breeding in their natural arid zone environment in Australia. METHODS Sampling of Wild Populations Between August 2005 and December 2007, we monitored 572 breeding attempts in nestboxes at four localities at the Fowlers Gap Arid Zone Research Station in far-west New South Wales, Australia (31050 S, 142 420 E; details in Griffith et al. 2008). Nestlings were banded before fledging and a small blood sample (<20 ml) was taken from the brachial vein. Adults were caught using nestbox traps that were watched and birds removed at the time of capture. All adults were blood sampled (<20 ml) and banded with a numbered aluminium band and either a unique combination of colour bands or a transponder tag (11 mm long and 2 mm wide and 0.1 g, Trovan ID100) attached to a plastic colour band. Putative parents were either captured while feeding nestlings, or later confirmed to have revisited the same nestlings through direct observation or remote reading of the transponder tag. We selected 80 complete families for a molecular survey to represent the different years of study and the different areas within the site. To estimate the opportunity for females to engage in extrapair copulations (EPCs), for each sampled pair, we counted the other reproductively active pairs (i.e. eggs or chicks present in the nest) within 1 km of their nest during the egglaying period of the focal female. This work was conducted under the authorities of the Animal Ethics Committees at the University of New South Wales and Macquarie University, a Scientific Research Permit from the New South Wales Parks and Wildlife Service and a banding Authority of the Australian Bird & Bat banding Scheme. Molecular Methods We used six fluorescently labelled microsatellite loci (Tgu1, Tgu3, Tgu8, Tgu10, Tgu12, Tgu4) that had previously been isolated and characterized in this species (Forstmeier et al. 2007) and a sex identification locus (Griffiths et al. 1998) in two multiplex PCR reactions that were designed with the program Multiplex Manager 1.0 (Holleley & Geerts 2009). Fragment size analysis for all seven loci was conducted concurrently in a single run on a 48-Capillary 3730 DNA Analyser (Applied Biosystems, Foster City, CA, U.S.A.), and conducted using GeneMapper version 3.7 (Applied Biosystems). There was some amplification failure but individual samples were rerun to ensure that each individual was scored with at least five of the six microsatellite loci. The overall final amplification failure rate of each locus was 0.21–3.33%, which given the frequency of null alleles is possibly due to a low number of individuals carrying two null alleles at a particular locus. In the 169 presumably unrelated adults genotyped, the loci were all highly variable with an average of 34 alleles per locus (range 22–42). The combined nonexclusion probabilities calculated by CERVUS 3.0 (Kalinowski et al. 2007) for this set of markers in this population were P ¼ 0.00008 for the first parent and P < 0.00001 for the second parent. Because of the characteristics of the genetic markers in this population, all offspring could be readily assigned unambiguously to their putative parents (or excluded) based on shared or mismatched alleles.
RESULTS Of 316 offspring, 17 (5.4%) from 14 of 80 broods (17.5%) were not related to either parent and hence were the result of intraspecific brood parasitism (IBP; Tables 1, 2). In each of three broods two chicks resulted from IBP and in all three cases these two dumped chicks were unrelated to each other (with no more than one allele matching across the six multisatellite loci) and hence must have resulted from two different parasitic females. We found no case of quasiparasitism. Of the remaining 299 offspring, five (1.7%) from four of 80 broods (5.0%) were sired by an extrapair male (Table 1). Multiple mismatching loci were used to exclude all offspring resulting from EPP or IBP. Although mismatch with the putative father or mother occurred at a single locus in 26 offspring, all these mismatches can be explained by allelic dropout (i.e. the offspring or parent was a homozygote and therefore the mismatched alleles are likely to have simply failed to amplify during the molecular procedure). In addition, given the high level of allelic diversity and frequencies of the different alleles, it is implausible that another individual could have been responsible and matched the offspring by chance at five of the six loci. Furthermore, all of the offspring that mismatched their parents at multiple loci mismatched consistently at between four and six of the loci used (average number of mismatched loci ¼ 5.18). All 80 families were sampled during the main breeding season and focal birds nested in close proximity to other breeding birds with each pair having an average of 18 other actively breeding pairs within 1 km radius of their nest (with at least twice as many nonbreeding adults around), presenting ample opportunity for EPC (Table 1).
DISCUSSION Wild zebra finches breeding in the Australian desert had a very low level of EPP (1.7% offspring in 5% of broods), suggesting that the zebra finch is among the most genetically monogamous bird species surveyed to date (Griffith et al. 2002). By contrast, in a captive population of 30 pairs of ‘wild-type’ North American domestic zebra finches living in a single aviary (53 m3), Burley et al. (1996) found that 28% of 278 offspring in 37% of 126 broods were fathered by an extrapair sire, a level of EPP that would put the species in the upper quartile with respect to variation in the level of extrapair fertilization. The difference in the rate of EPP between the free-living wild birds and the captive population of domesticated birds could be caused by social or environmental factors or possibly by artificial selection imposed by aviculturists for over 100 generations. Given
Table 1 The incidence of extrapair paternity (EPP) and intraspecific brood parasitism (IBP) among families in four different areas across 3 different years with the average number of pairs actively breeding in those areas at the time of the sampled reproductive attempt Area
Year
No. of broods
No. of broods with EPP
East Mandelman West Mandelman West Mandelman Gap Hills Gap Hills Gap Hills Saloon Saloon Saloon
2005 2005 2006 2005 2006 2007 2005 2006 2007
15 7 3 12 14 19 7 2 1
0 1 1 0 1 1 0 0 0
1 0 0 3 2 4 4 0 0
80
4
14
Total
No. of broods with IBP
No. of pairs within 1 km (average) 18 23 7 25 13 40 15 7 17 18.33
S.C. Griffith et al. / Animal Behaviour 79 (2010) 261–264 Table 2 The incidence of extrapair paternity and intraspecific brood parasitism across broods of different size Brood size
Total no. sampled
No. of broods with EPP
No. of broods with IBP
1 2 3 4 5 6
1 7 24 22 15 11
0 0 2 2 0 0
1 1 1 2 5 4
that males apparently court females for EPC at a similar rate in captive and wild populations (Birkhead et al. 1988a; Burley et al. 1994), the difference in rates of EPP may simply reflect differences in female propensity to accept or reject EPC in the two contexts. Domestic females appear to be less choosy than their wild counterparts (Rutstein et al. 2007), and thus may be less likely to resist EPCs. The levels of both EPP and IBP found in our study of desert-living birds were very similar to those reported previously for a wild population on an irrigated farm in northern Victoria, Australia (Birkhead et al. 1990). In their study, Birkhead et al. (1990) found that two of 82 offspring (2.4%) were extrapair, in two of 25 broods (8%), and that 10 offspring (11%) from nine of 25 broods (36%) hatched from eggs that were the result of IBP, one of which was suggested to be the result of quasiparasitism (Birkhead et al. 1990; Griffith et al. 2004). Our study increases the number of wild families studied (from 25 to 105) and used a more ‘classic’ zebra finch population, both in terms of habitat (the arid and semiarid zone represents >80% of the zebra finch distribution) and the range of breeding densities and synchronies, than the earlier study conducted in the nonarid zone on birds breeding at low density and synchrony (Birkhead et al. 1990). The remarkably small difference (0.7%) in the level of EPP found in these two studies conducted over 1000 km and 17 years apart suggests that we can now be fairly confident that EPP in natural populations of the zebra finch is very low, accounting for only 1–2% of offspring (the combined rate of EPP from both wild studies is 1.7% (95% confidence interval 0.5–3.0). Although extrapair courtship and copulations do occur in the wild (Birkhead et al. 1988a), this low level of EPP suggests that sperm competition is likely to be limited, with social partners acquiring most fertilizations, probably because of the combined effects of female resistance and the timing and rates of extrapair and within-pair copulations (Birkhead et al. 1988a, b, 1989). The zebra finch has provided a good model for investigating the components of avian reproductive biology that were essential in the development of a mechanistic understanding of the process of sperm competition. For example, work on the captive zebra finch in Europe has helped to demonstrate the role of the sperm storage tubules in avian reproduction (Birkhead et al. 1993) and the fact that a passive loss model (of sperm from the female reproductive tract), coupled with the difference in ejaculate size between males, can explain why the last male to copulate with a female typically fertilizes the majority of the eggs (Birkhead et al. 1988b; Colegrave et al. 1995). It would not have been feasible to conduct the necessarily controlled experiments that led to such findings on wild birds, and therefore the zebra finch was probably the only passerine that could have been the focus of such a study. However, a more recent study of sperm design probably reflects a situation in which, by being an inappropriate model, the zebra finch initially caused confusion rather than clarity. In their study, Birkhead et al. (2005) used a large pedigree of 1526 individuals to investigate the source and consequence of variation among males in the morphology of their sperm (again something that would be
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difficult to achieve in an alternative avian model system). A considerable amount (relative to standard morphological traits) of intermale variation was found with respect to sperm design and these traits were all highly heritable (Birkhead et al. 2005). The paradox here is that we should not expect to find so much variation in traits that are closely associated with fitness and under such a high level of genetic control. The low level of EPP that we have demonstrated here helps to put the earlier study (Birkhead et al. 2005) of sperm design into context. As males are rarely exposed to selection on the ability of their sperm to swim faster than sperm from other competing males then it is not too surprising that lots of morphological variation in sperm design exists in populations of zebra finches. Further comparative study has indeed revealed that the level of intermale variation in sperm morphology is much lower in species with higher levels of EPP than the zebra finch (Kleven et al. 2008). By contrast, in species in which offspring are regularly produced by EPP and thus where there is a high incidence of sperm competition, the sperm produced by males across a population exhibited greater similarity of form and presumably reflected a more tightly selected optimum for competing within the female reproductive tract (Kleven et al. 2008). The zebra finch has thus provided a good model system for understanding the fundamental reproductive biology of passerine birds but may not be such a good system for understanding the way that the selection driven by EPP (which occurs at a significantly higher rate in most other birds) affects morphology, behaviour and physiology. Our findings in the wild zebra finch have important implications for the use of this species as a model system in a more general sense (than in sperm competition). In socially monogamous species, EPP can be an important driver of sexual selection and sexual conflict, because it can increase the variance in reproductive success among individuals (Whittingham & Dunn 2005) and create disparity between the evolutionary interests of the male and female, respectively. Given that wild zebra finches form pair bonds that are maintained throughout the year and apparently last until the death of one partner (Zann 1996), the low level of EPP we observed suggests that this species more closely approaches true monogamy than most other birds; thus there may be relatively little sexual conflict. If most individuals in wild populations remain genetically monogamous then most sexual selection will be on the reproductive success of pairs rather than individuals, and therefore the focus of research in this species should perhaps be placed on parenting behaviour and the ability of individuals to bond, synchronize and reproduce effectively with their partner. In contrast however, over the past couple of decades much of the research in this species has investigated conflict between the male and female partner (e.g. Burley 1988; Gil et al. 1999; Royle et al. 2002). It is important to stress that we believe that sexual conflict and sexual selection do occur in this species; indeed the study by Royle et al. (2002) is one of the clearest demonstrations of sexual conflict in any vertebrate, while many studies have demonstrated active choice of social partners (e.g. Rutstein et al. 2007). Like many of those who have worked on captive zebra finches, we agree that the zebra finch is a good model system to study because of its amenability to highly manipulative and controlled experiments. However, we suggest that caution is used when trying to infer general conclusions from studies relating to sexual selection and sexual conflict in this species. Rather than representing the ‘average’ socially monogamous bird, the zebra finch may instead represent the less interesting end of avian biodiversity in relation to sexual selection, sexual conflict and sperm competition and it is likely that there are more extreme, more complex and more interesting processes occurring in other avian species with respect to each area of study.
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Acknowledgments We thank James Brazill-Boast, Gareth Davies, Richard Merrill, Emma Pariser, Ian Stewart, Ingrid Stirnemann, Harriet Stone and Megan Taylor for assistance in the field; Wolfgang Forstmeier for providing access to unpublished primers; and Wolfgang Forstmeier and Jan Lifjeld for useful comments on the manuscript. S.C.G., C.E.H. and S.R.P. were supported by the Australian Research Council, M.M.M. by a Macquarie University Research Excellence Scholarship, and N.S. by a Postdoctoral Fellowship from the Swedish Research Council (Vetenskaprådet). References Birkhead, T. R. & Fletcher, F. 1995. Male phenotype and ejaculate quality in the zebra finch Taeniopygia guttata. Proceedings of the Royal Society B, 262, 329–334. Birkhead, T. R., Clarkson, K. & Zann, R. 1988a. Extra-pair courtship, copulation and mate guarding in wild zebra finches Taeniopygia guttata. Animal Behaviour, 36, 1853–1855. Birkhead, T. R., Pellatt, J. & Hunter, F. M. 1988b. Extra-pair copulation and sperm competition in the zebra finch. Nature, 334, 60–62. Birkhead, T. R., Hunter, F. M. & Pellatt, J. E. 1989. Sperm competition in the zebra finch, Taeniopygia guttata. Animal Behaviour, 38, 935–950. Birkhead, T. R., Burke, T., Zann, R., Hunter, F. M. & Krupa, A. P. 1990. Extra-pair paternity and intraspecific brood parasitism in wild zebra finches Taeniopygia guttata, revealed by DNA fingerprinting. Behavioral Ecology and Sociobiology, 27, 315–324. Birkhead, T. R., Pellatt, E. J. & Fletcher, F. 1993. Selection and utilization of spermatozoa in the reproductive tract of the female zebra finch Taeniopygia guttata. Journal of Reproduction and Fertility, 99, 593–600. Birkhead, T. R., Pellatt, E. J., Brekke, P., Yeates, R. & Castillo-Juarez, H. 2005. Genetic effects on sperm design in the zebra finch. Nature, 434, 383–387. Burley, N. 1981. Sex-ratio manipulation and selection for attractiveness. Science, 211, 721–722. Burley, N. 1988. The differential-allocation hypothesis: an experimental test. American Naturalist, 132, 611–628. Burley, N. T., Parker, P. G. & Lundy, K. 1996. Sexual selection and extrapair fertilization in a socially monogamous passerine, the zebra finch (Taeniopygia guttata). Behavioral Ecology, 7, 218–226. Burley, N. T., Enstrom, D. A. & Chitwood, L. 1994. Extra-pair relations in zebra finches: differential male success results from female tactics. Animal Behaviour, 48, 1031–1041.
Colegrave, N., Birkhead, T. R. & Lessells, C. M. 1995. Sperm precedence in zebra finches does not require special mechanisms of sperm competition. Proceedings of the Royal Society B, 259, 223–228. Collins, S. A., Hubbard, C. & Houtman, A. M. 1994. Female mate choice in the zebra finch: the effect of male beak color and male song. Behavioral Ecology and Sociobiology, 35, 21–25. Forstmeier, W. 2007. Do individual females differ intrinsically in their propensity to engage in extra-pair copulations? PLoS ONE, 2, e952. Forstmeier, W., Schielzeth, H., Schneider, M. & Kempenaers, B. 2007. Development of polymorphic microsatellite markers for the zebra finch (Taeniopygia guttata). Molecular Ecology Notes, 7, 1026–1028. Gil, D., Graves, J., Hazon, N. & Wells, A. 1999. Male attractiveness and differential testosterone investment in zebra finch eggs. Science, 286, 126–128. Griffith, S. C. & Immler, S. 2009. Female infidelity and genetic compatibility in birds: the role of the genetically loaded raffle in understanding the function of extrapair paternity. Journal of Avian Biology, 40, 97–101. Griffith, S. C., Owens, I. P. F. & Thuman, K. A. 2002. Extra-pair paternity in birds: a review of interspecific variation and adaptive function. Molecular Ecology, 11, 2195–2212. Griffith, S. C., Lyon, B. E. & Montgomerie, R. 2004. Quasi-parasitism in birds. Behavioral Ecology and Sociobiology, 56, 191–200. Griffith, S. C., Pryke, S. R. & Mariette, M. 2008. Use of nest-boxes by the zebra finch (Taeniopygia guttata): implications for reproductive success and reseach. Emu, 108, 311–319. Griffiths, R., Double, M. C., Orr, K. & Dawson, R. J. G. 1998. A DNA test to sex most birds. Molecular Ecology, 7, 1071–1075. Holleley, C. E. & Geerts, P. G. 2009. Multiplex Manager 1.0: a cross-platform computer program that plans out and optimizes multiplex PCR. BioTechniques, 46, 511–517. 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. Kleven, O., Laskemoen, T., Fossøy, F., Robertson, R. J. & Lifjeld, J. T. 2008. Intraspecific variation in sperm length is negatively related to sperm competition in passerine birds. Evolution, 62, 494–499. Parker, G. A. 1970. Sperm competition and its evolutionary consequences. Biological Reviews, 45, 525–567. Royle, N. J., Hartley, I. R. & Parker, G. A. 2002. Sexual conflict reduces offspring fitness in zebra finches. Nature, 416, 733–736. Rutstein, A. N., Brazill-Boast, J. & Griffith, S. C. 2007. Evaluating mate choice in the zebra finch. Animal Behaviour, 74, 1277–1284. Whittingham, L. A. & Dunn, P. O. 2005. Effects of extra-pair and within-pair reproductive success on the opportunity for selection in birds. Behavioral Ecology, 16, 138–144. Zann, R. 1996. The Zebra Finch a Synthesis of Field and Laboratory Studies. Oxford: Oxford University Press.
Contents lists available at ScienceDirect
Animal Behaviour journal homepage: www.elsevier.com/locate/anbehav
Low level of extrapair parentage in wild zebra finches Simon C. Griffith a, b, *, Clare E. Holleley a, b, Mylene M. Mariette a, b, Sarah R. Pryke a, b, Nina Svedin a a b
Department of Brain, Behaviour and Evolution, Macquarie University School of Biological, Earth and Environmental Sciences, University of New South Wales
a r t i c l e i n f o Article history: Received 28 September 2009 Initial acceptance 12 October 2009 Final acceptance 16 November 2009 Available online 7 December 2009 MS. number: 09-00639 Keywords: extrapair paternity intraspecific brood parasitism sexual conflict sexual selection sperm competition Taeniopygia guttata zebra finch
The captive zebra finch, Taeniopygia guttata, has become one of the key vertebrate model systems for studying a range of behavioural, physiological and neurological phenomena. In particular, this species has played a key role in developing our understanding of sexual selection and sperm competition. In contrast with the large number of studies using domesticated zebra finches, relatively few studies have focused on free-living populations of wild zebra finches. Investigating the incidence of extrapair paternity in zebra finches in the Australian desert, we found a very low level; 1.7% of 316 offspring from four of 80 broods fathered outside the pair bond. These numbers contrast with the high levels of extrapair paternity observed in domesticated aviary populations, and suggest a low level of sperm competition and sexual selection in natural populations. Our finding of such a low rate of extrapair paternity in the wild zebra finch suggests that it is one of the most genetically monogamous of all passerine species and that has important implications for future studies of this model organism in studies of sexual selection and reproductive biology. In addition, we found that 5.4% of 316 offspring were not related to either putative parent and hatched from eggs that had been dumped by intraspecific brood parasites. Ó 2009 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
In birds, about 90% of all species form socially monogamous pair bonds, and in these species 11% of offspring, on average, are sired by an extrapair male (Griffith et al. 2002). Extrapair paternity (EPP) is an important evolutionary phenomenon in socially monogamous species because it can dramatically increase the variance in reproductive success among males within a population (Whittingham & Dunn 2005), and it can provide an important postcopulatory mechanism for selection on genetic compatibility (Griffith & Immler 2009). In addition to describing the variation in the level of EPP and accompanying sexual selection across species, research has focused on the physiological and behavioural processes that relate to the incidence of EPP. Foremost among these is sperm competition (Parker 1970), an area in which the zebra finch, Taeniopygia guttata, has been one of the most widely used model organisms because of its amenability as a captive animal. Studies of the zebra finch represent a significant proportion of all the empirical work contributing to our understanding of the mechanisms of sperm competition in birds, including the timing and frequency of pair and extrapair copulations (Birkhead et al. 1989), sperm precedence models (Birkhead et al. 1988b), fertilization success and sperm quality (Birkhead et al. 1993), sperm storage
* Correspondence: S. C. Griffith, Department of Brain, Behaviour and Evolution, Macquarie University, Sydney, New South Wales 2109, Australia. E-mail address: simon.griffi[email protected] (S.C. Griffith).
(Birkhead et al. 1989), sperm depletion and allocation (Birkhead & Fletcher 1995), sperm morphology (Birkhead et al. 2005), and female propensity to engage in extrapair copulations (Forstmeier 2007). The zebra finch has been similarly prominent in the field of sexual selection through studies of mate choice (e.g. Rutstein et al. 2007), ornamental song (e.g. Collins et al. 1994), parental care (e.g. Burley 1988), sex allocation (e.g. Burley 1981) and maternal effects (e.g. Gil et al. 1999). All of these important and influential studies have focused on domesticated birds that have been selectively bred for over 100 generations (Zann 1996), and held in conditions that are far removed from those experienced by free-living wild birds. The use of domesticated model organisms is a crucial component of modern biological research. However, to aid the interpretation of captive studies it is important to develop an understanding of the wild context from which the model organism originated. A previous study of the zebra finch population studied over a long period by R. Zann in Victoria, Australia (Birkhead et al. 1990) found a very low level of EPP and intraspecific brood parasitism. However, there are a number of currently unresolved issues arising from this study. First, the study by Birkhead et al. (1990) was based on a small sample of just 25 families situated on an irrigated farm in temperate Victoria, which bred at very low density and synchrony and may not have truly represented the species in its native environment (T. R. Birkhead, personal communication). Furthermore, Birkhead et al. (1990) reported a potential case of quasiparasitism from families in which the molecular methods used were unable to
0003-3472/$38.00 Ó 2009 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.anbehav.2009.11.031
262
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make an accurate discrimination of the level of relatedness to a brood parasite and the pair male simultaneously (see Griffith et al. 2004). Here therefore for the first time we investigated the level of EPP in wild populations of free-living zebra finches breeding in their natural arid zone environment in Australia. METHODS Sampling of Wild Populations Between August 2005 and December 2007, we monitored 572 breeding attempts in nestboxes at four localities at the Fowlers Gap Arid Zone Research Station in far-west New South Wales, Australia (31050 S, 142 420 E; details in Griffith et al. 2008). Nestlings were banded before fledging and a small blood sample (<20 ml) was taken from the brachial vein. Adults were caught using nestbox traps that were watched and birds removed at the time of capture. All adults were blood sampled (<20 ml) and banded with a numbered aluminium band and either a unique combination of colour bands or a transponder tag (11 mm long and 2 mm wide and 0.1 g, Trovan ID100) attached to a plastic colour band. Putative parents were either captured while feeding nestlings, or later confirmed to have revisited the same nestlings through direct observation or remote reading of the transponder tag. We selected 80 complete families for a molecular survey to represent the different years of study and the different areas within the site. To estimate the opportunity for females to engage in extrapair copulations (EPCs), for each sampled pair, we counted the other reproductively active pairs (i.e. eggs or chicks present in the nest) within 1 km of their nest during the egglaying period of the focal female. This work was conducted under the authorities of the Animal Ethics Committees at the University of New South Wales and Macquarie University, a Scientific Research Permit from the New South Wales Parks and Wildlife Service and a banding Authority of the Australian Bird & Bat banding Scheme. Molecular Methods We used six fluorescently labelled microsatellite loci (Tgu1, Tgu3, Tgu8, Tgu10, Tgu12, Tgu4) that had previously been isolated and characterized in this species (Forstmeier et al. 2007) and a sex identification locus (Griffiths et al. 1998) in two multiplex PCR reactions that were designed with the program Multiplex Manager 1.0 (Holleley & Geerts 2009). Fragment size analysis for all seven loci was conducted concurrently in a single run on a 48-Capillary 3730 DNA Analyser (Applied Biosystems, Foster City, CA, U.S.A.), and conducted using GeneMapper version 3.7 (Applied Biosystems). There was some amplification failure but individual samples were rerun to ensure that each individual was scored with at least five of the six microsatellite loci. The overall final amplification failure rate of each locus was 0.21–3.33%, which given the frequency of null alleles is possibly due to a low number of individuals carrying two null alleles at a particular locus. In the 169 presumably unrelated adults genotyped, the loci were all highly variable with an average of 34 alleles per locus (range 22–42). The combined nonexclusion probabilities calculated by CERVUS 3.0 (Kalinowski et al. 2007) for this set of markers in this population were P ¼ 0.00008 for the first parent and P < 0.00001 for the second parent. Because of the characteristics of the genetic markers in this population, all offspring could be readily assigned unambiguously to their putative parents (or excluded) based on shared or mismatched alleles.
RESULTS Of 316 offspring, 17 (5.4%) from 14 of 80 broods (17.5%) were not related to either parent and hence were the result of intraspecific brood parasitism (IBP; Tables 1, 2). In each of three broods two chicks resulted from IBP and in all three cases these two dumped chicks were unrelated to each other (with no more than one allele matching across the six multisatellite loci) and hence must have resulted from two different parasitic females. We found no case of quasiparasitism. Of the remaining 299 offspring, five (1.7%) from four of 80 broods (5.0%) were sired by an extrapair male (Table 1). Multiple mismatching loci were used to exclude all offspring resulting from EPP or IBP. Although mismatch with the putative father or mother occurred at a single locus in 26 offspring, all these mismatches can be explained by allelic dropout (i.e. the offspring or parent was a homozygote and therefore the mismatched alleles are likely to have simply failed to amplify during the molecular procedure). In addition, given the high level of allelic diversity and frequencies of the different alleles, it is implausible that another individual could have been responsible and matched the offspring by chance at five of the six loci. Furthermore, all of the offspring that mismatched their parents at multiple loci mismatched consistently at between four and six of the loci used (average number of mismatched loci ¼ 5.18). All 80 families were sampled during the main breeding season and focal birds nested in close proximity to other breeding birds with each pair having an average of 18 other actively breeding pairs within 1 km radius of their nest (with at least twice as many nonbreeding adults around), presenting ample opportunity for EPC (Table 1).
DISCUSSION Wild zebra finches breeding in the Australian desert had a very low level of EPP (1.7% offspring in 5% of broods), suggesting that the zebra finch is among the most genetically monogamous bird species surveyed to date (Griffith et al. 2002). By contrast, in a captive population of 30 pairs of ‘wild-type’ North American domestic zebra finches living in a single aviary (53 m3), Burley et al. (1996) found that 28% of 278 offspring in 37% of 126 broods were fathered by an extrapair sire, a level of EPP that would put the species in the upper quartile with respect to variation in the level of extrapair fertilization. The difference in the rate of EPP between the free-living wild birds and the captive population of domesticated birds could be caused by social or environmental factors or possibly by artificial selection imposed by aviculturists for over 100 generations. Given
Table 1 The incidence of extrapair paternity (EPP) and intraspecific brood parasitism (IBP) among families in four different areas across 3 different years with the average number of pairs actively breeding in those areas at the time of the sampled reproductive attempt Area
Year
No. of broods
No. of broods with EPP
East Mandelman West Mandelman West Mandelman Gap Hills Gap Hills Gap Hills Saloon Saloon Saloon
2005 2005 2006 2005 2006 2007 2005 2006 2007
15 7 3 12 14 19 7 2 1
0 1 1 0 1 1 0 0 0
1 0 0 3 2 4 4 0 0
80
4
14
Total
No. of broods with IBP
No. of pairs within 1 km (average) 18 23 7 25 13 40 15 7 17 18.33
S.C. Griffith et al. / Animal Behaviour 79 (2010) 261–264 Table 2 The incidence of extrapair paternity and intraspecific brood parasitism across broods of different size Brood size
Total no. sampled
No. of broods with EPP
No. of broods with IBP
1 2 3 4 5 6
1 7 24 22 15 11
0 0 2 2 0 0
1 1 1 2 5 4
that males apparently court females for EPC at a similar rate in captive and wild populations (Birkhead et al. 1988a; Burley et al. 1994), the difference in rates of EPP may simply reflect differences in female propensity to accept or reject EPC in the two contexts. Domestic females appear to be less choosy than their wild counterparts (Rutstein et al. 2007), and thus may be less likely to resist EPCs. The levels of both EPP and IBP found in our study of desert-living birds were very similar to those reported previously for a wild population on an irrigated farm in northern Victoria, Australia (Birkhead et al. 1990). In their study, Birkhead et al. (1990) found that two of 82 offspring (2.4%) were extrapair, in two of 25 broods (8%), and that 10 offspring (11%) from nine of 25 broods (36%) hatched from eggs that were the result of IBP, one of which was suggested to be the result of quasiparasitism (Birkhead et al. 1990; Griffith et al. 2004). Our study increases the number of wild families studied (from 25 to 105) and used a more ‘classic’ zebra finch population, both in terms of habitat (the arid and semiarid zone represents >80% of the zebra finch distribution) and the range of breeding densities and synchronies, than the earlier study conducted in the nonarid zone on birds breeding at low density and synchrony (Birkhead et al. 1990). The remarkably small difference (0.7%) in the level of EPP found in these two studies conducted over 1000 km and 17 years apart suggests that we can now be fairly confident that EPP in natural populations of the zebra finch is very low, accounting for only 1–2% of offspring (the combined rate of EPP from both wild studies is 1.7% (95% confidence interval 0.5–3.0). Although extrapair courtship and copulations do occur in the wild (Birkhead et al. 1988a), this low level of EPP suggests that sperm competition is likely to be limited, with social partners acquiring most fertilizations, probably because of the combined effects of female resistance and the timing and rates of extrapair and within-pair copulations (Birkhead et al. 1988a, b, 1989). The zebra finch has provided a good model for investigating the components of avian reproductive biology that were essential in the development of a mechanistic understanding of the process of sperm competition. For example, work on the captive zebra finch in Europe has helped to demonstrate the role of the sperm storage tubules in avian reproduction (Birkhead et al. 1993) and the fact that a passive loss model (of sperm from the female reproductive tract), coupled with the difference in ejaculate size between males, can explain why the last male to copulate with a female typically fertilizes the majority of the eggs (Birkhead et al. 1988b; Colegrave et al. 1995). It would not have been feasible to conduct the necessarily controlled experiments that led to such findings on wild birds, and therefore the zebra finch was probably the only passerine that could have been the focus of such a study. However, a more recent study of sperm design probably reflects a situation in which, by being an inappropriate model, the zebra finch initially caused confusion rather than clarity. In their study, Birkhead et al. (2005) used a large pedigree of 1526 individuals to investigate the source and consequence of variation among males in the morphology of their sperm (again something that would be
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difficult to achieve in an alternative avian model system). A considerable amount (relative to standard morphological traits) of intermale variation was found with respect to sperm design and these traits were all highly heritable (Birkhead et al. 2005). The paradox here is that we should not expect to find so much variation in traits that are closely associated with fitness and under such a high level of genetic control. The low level of EPP that we have demonstrated here helps to put the earlier study (Birkhead et al. 2005) of sperm design into context. As males are rarely exposed to selection on the ability of their sperm to swim faster than sperm from other competing males then it is not too surprising that lots of morphological variation in sperm design exists in populations of zebra finches. Further comparative study has indeed revealed that the level of intermale variation in sperm morphology is much lower in species with higher levels of EPP than the zebra finch (Kleven et al. 2008). By contrast, in species in which offspring are regularly produced by EPP and thus where there is a high incidence of sperm competition, the sperm produced by males across a population exhibited greater similarity of form and presumably reflected a more tightly selected optimum for competing within the female reproductive tract (Kleven et al. 2008). The zebra finch has thus provided a good model system for understanding the fundamental reproductive biology of passerine birds but may not be such a good system for understanding the way that the selection driven by EPP (which occurs at a significantly higher rate in most other birds) affects morphology, behaviour and physiology. Our findings in the wild zebra finch have important implications for the use of this species as a model system in a more general sense (than in sperm competition). In socially monogamous species, EPP can be an important driver of sexual selection and sexual conflict, because it can increase the variance in reproductive success among individuals (Whittingham & Dunn 2005) and create disparity between the evolutionary interests of the male and female, respectively. Given that wild zebra finches form pair bonds that are maintained throughout the year and apparently last until the death of one partner (Zann 1996), the low level of EPP we observed suggests that this species more closely approaches true monogamy than most other birds; thus there may be relatively little sexual conflict. If most individuals in wild populations remain genetically monogamous then most sexual selection will be on the reproductive success of pairs rather than individuals, and therefore the focus of research in this species should perhaps be placed on parenting behaviour and the ability of individuals to bond, synchronize and reproduce effectively with their partner. In contrast however, over the past couple of decades much of the research in this species has investigated conflict between the male and female partner (e.g. Burley 1988; Gil et al. 1999; Royle et al. 2002). It is important to stress that we believe that sexual conflict and sexual selection do occur in this species; indeed the study by Royle et al. (2002) is one of the clearest demonstrations of sexual conflict in any vertebrate, while many studies have demonstrated active choice of social partners (e.g. Rutstein et al. 2007). Like many of those who have worked on captive zebra finches, we agree that the zebra finch is a good model system to study because of its amenability to highly manipulative and controlled experiments. However, we suggest that caution is used when trying to infer general conclusions from studies relating to sexual selection and sexual conflict in this species. Rather than representing the ‘average’ socially monogamous bird, the zebra finch may instead represent the less interesting end of avian biodiversity in relation to sexual selection, sexual conflict and sperm competition and it is likely that there are more extreme, more complex and more interesting processes occurring in other avian species with respect to each area of study.
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