Inbreeding avoidance under different null models

Transcription

1 Journal of Animal Ecology 2009, 78, doi: /j x Inbreeding avoidance under different null models Blackwell Publishing Ltd of random mating in the great tit Marta Szulkin 1 *, Przemyslaw Zelazowski 2, George Nicholson 3 and Ben C. Sheldon 1 1 Edward Grey Institute, Department of Zoology, University of Oxford, Oxford OX1 3PS, UK; 2 Environmental Change Institute, Oxford University Centre for the Environment, Oxford OX1 3QY, UK; and 3 Department of Statistics, University of Oxford, Oxford OX1 3TG, UK Summary 1. In populations where inbreeding causes a substantial decrease in fitness, selection is expected to favour the evolution of inbreeding avoidance behaviours. Elsewhere we have documented substantial inbreeding depression and the importance of dispersal in avoiding inbreeding in a long-term population study of the great tit Parus major in Wytham (UK). In this study, we ask whether individuals from this population actively avoid mating with kin. 2. We generated four contrasting models of random mate choice that assumed varying levels of mate availability in each year of the data set. This allowed us to compare observed and simulated distributions and frequencies of inbreeding coefficients from 41 years of breeding data. 3. We found no evidence that birds avoid mating with related partners. Our results show that birds breed more often with relatives than expected under null models of mate choice that lack population structure, but not when compared to scenarios where birds were mated with their nearest neighbours. Pedigree-derived F IS values were positive for all scenarios of random mating, confirming the lack of inbreeding avoidance in this population. 4. These results imply the existence of spatial genetic structure where related individuals occur closer together than nonrelated individuals while breeding, and suggest that the relatedness between breeding individuals of the opposite sex decreases with distance. Thus, while dispersal from the natal site decreases the number of relatives around an individual, it does not completely homogenize genetic structure. 5. We show that brother sister pairs are observed more often than under any scenario of random mating, suggesting that not only birds do not avoid mating with kin, but also that the apparently maladaptive choice of mating with a sibling is made more often than expected. 6. Our results provide no evidence to suggest that individuals actively avoid kin. In fact, some types of inbreeding occur more often than expected, despite the substantial fitness costs. The observed lack of inbreeding avoidance is in agreement with other studies of non-cooperatively breeding passerine birds, although the higher than expected frequency of sibling mating remains a puzzling result. Key-words: F ST, F IT, random mating simulation, incest Introduction Mate choice may be a key factor in an individual s ability to raise high-quality offspring. A high-quality partner may lead to fitter offspring by passing on its own genes to them, but offspring fitness might also depend on the interplay between *Corresponding author: marta.szulkin@zoo.ox.ac.uk maternal and paternal alleles (Tregenza & Wedell 2000). Maternal and paternal genetic contributions to the offspring s phenotype may be additive or non-additive; in the latter case, allelic effects depend on the genetic background (Lynch & Walsh 1998). A case of genetic interaction with sometimes marked effects on fitness occurs when related individuals mate: inbreeding. The likelihood of inheriting the same allele from each parent increases when parents are related, and inbreeding can thus have important fitness 2009 The Authors. Journal compilation 2009 British Ecological Society

2 Inbreeding avoidance and random mating 779 consequences by increasing the likelihood of both parents passing the same copy of a deleterious recessive allele to the offspring (Charlesworth & Charlesworth 1999). At the population level, the decrease in fitness mediated by the expression of deleterious recessive alleles, caused by inbreeding, is defined as inbreeding depression. It is expected that inbreeding avoidance should evolve in response to inbreeding depression, and there is plenty of evidence suggesting that many animal taxa indeed avoid mating with relatives (see Pusey & Wolf 1996 for a review). But avoiding inbreeding can also incur costs, such as experienced through dispersal (Bensch et al. 1998; Pärt 1995), or because of the loss of breeding opportunities (Koenig, Stanback & Haydock 1999; Kokko & Ekman 2002). Moreover, there may also be scope for kin-selected benefits of incestuous mating. For example, the study of Kleven et al. (2005) on barn swallows suggests that in some cases, animals may prefer to mate with genetically similar, rather than dissimilar partners. Whether selection favours the avoidance of inbreeding depends on the strength of inbreeding depression, but also on the life-history dependent costs of foregoing breeding opportunities for males and females (Kokko & Ots 2006). In the Wytham great tit population, the recruitment success of offspring from broods where close relatives mate is reduced by 40% (Szulkin et al. 2007). Given this substantial inbreeding depression, and the fact that great tits exhibit bi-parental care, selection for the evolution of inbreeding avoidance seems likely (Kokko & Ots 2006). Two types of inbreeding avoidance can be distinguished, which are not mutually exclusive. One is to have passive avoidance mechanisms, such as dispersal, which act to minimize the likelihood of encountering kin (Greenwood, Harvey & Perrins 1978; Szulkin & Sheldon 2008a). The term passive refers to the fact that these mechanisms could operate without assessing the relatedness of a potential partner. Alternatively, active ways of avoiding inbreeding could be used, for example by increasing rates of extra-pair paternity when mating with genetically similar individuals (Eimes et al. 2005), or by divorcing after breeding with a related partner (Szulkin & Sheldon 2008b). In addition to divorce and extra-pair paternity in response to inbreeding, actively choosing not to mate with kin in the first place could be a powerful means of inbreeding avoidance. This would presumably require some sort of kin recognition mechanism to operate (Krebs & Davies 1993). Active kin recognition has been found among several cooperatively breeding passerines, such as in the long tailed tit (Aegithalos caudatus, Sharp et al. 2005) and the Seychelles warbler (Acrocephalus sechellensis, Komdeur, Richardson & Burke 2004); kin recognition in these species is not only relevant to inbreeding avoidance, but also to maximizing inclusive fitness in cooperative societies (Komdeur & Hatchwell 1999). In noncooperative species, however, evidence for deviations from random-mating, such as might be generated by active avoidance of mating with kin, is scarce. Previous studies have suggested that mating occurs at random with respect to kinship in a Dutch population of the great tit (Parus major, van Tienderen & van Noordwijk 1988), the medium ground finch (Geospiza fortis, Gibbs & Grant 1989), the song sparrow (Melospiza melodia, Keller & Arcese 1998) and the great reed warbler (Acrocephalus arundinaceus, Hansson et al. 2007), suggesting that either other inbreeding avoidance mechanisms are operating in those populations, or perhaps that the costs of inbreeding avoidance are greater than the effects of inbreeding depression itself. All but one of these studies (Hansson et al. 2007) were carried out on island populations, where the relative importance of different means of inbreeding avoidance may differ from those encountered in large continental populations connected by high migration rates to surrounding populations. Furthermore, as selection for inbreeding avoidance is a result of the various costs and benefits of inbreeding itself (e.g. Kokko & Ots 2006), the avoidance, or preference for mating with relatives may vary across species. Thus, investigating mating preferences in relation to inbreeding in other populations and species is needed to understand situations where avoidance (or preference) of inbreeding is expected. Pärt (1996) stressed the complexities of defining an appropriate null model for testing inbreeding avoidance, arguing that inbreeding levels generated by null models of inbreeding may not be independent of what would be inbreeding avoidance behaviour (such as dispersal). Thus, null models of inbreeding avoidance against which observed levels of inbreeding are tested may already carry the signature of inbreeding avoidance. It is therefore important to investigate an array of null models, which differ in terms of their initial assumptions regarding mate choice. We showed previously that dispersal considerably reduces the likelihood of mating with kin in our study population (Szulkin & Sheldon 2008a). However, this finding does not reveal the extent to which birds avoid mating with kin given restrictions on mate availability generated by dispersal. In general, it is thus not known whether observed levels of inbreeding differ from models of random mating that take into account various levels of mate availability. As yet, it is unknown whether any relatedness-based cues for mate choice are used in this population, allowing an individual to avoid inbreeding when confronted with a potential mate that is also a relative. In a long-term study of a great tit population at Wytham, near Oxford, we have previously shown that inbreeding causes substantial fitness costs (Szulkin et al. 2007). Furthermore, birds do not avoid inbreeding by divorcing (Szulkin & Sheldon 2008b), and there is no evidence that they increase their rate of extra-pair paternity when mating with kin. If anything, rates of extra-pair paternity were lower in broods where f relative to outbred broods, but this was based on a very small sample of inbred broods (n = 5); Szulkin (2007). The aim of the present study was to ask whether related birds mate more or less often than expected by chance based on various scenarios of random mating in a finite population. To do so, we used four different scenarios of random mating characterized by different constraints on mate availability to compare simulated and observed levels of inbreeding. If birds actively avoid mating with kin, we expect

3 780 M. Szulkin et al. to find lower observed population-wide levels of inbreeding than those simulated by scenarios of random mating where general rules define hypothetical matings between members of opposite sex in the population. Materials and methods STUDY POPULATION The great tit P. major is a cavity-nesting passerine bird breeding preferentially in nestboxes when provided, and a study of this species at Wytham, Oxfordshire, UK (1 20 W, N) has been running since 1947; since 1964, the nestbox distribution throughout Wytham as well as the breeding fieldwork protocol has remained constant. The study site is a semi-deciduous forest of c. 385 ha with in excess of 1000 nestboxes scattered throughout the forest at variable densities. Nestboxes are checked at weekly intervals at the start of each breeding season (April), parents are caught while feeding young and both parents and offspring are ringed, with ring numbers recorded in the case of already ringed parents. Immigration rates in the population are quite high, as on average, 40% of males and 47% of females breeding in any year within Wytham are born outside the woods (McCleery et al. 2004). Because parental identity can only be identified when birds have hatched and reached the mid-nestling period, not all individuals that attempt to breed are identified either because the nest fails before parents can be trapped, or because parents cannot be caught despite trapping efforts. Overall, 65% of all males and 80% of all females that started breeding in the woodland during the study period analysed here were identified. The great majority of great tit pairs lay only a single clutch of eggs each year in this population; we excluded cases of second clutches for our analyses, as we find it more justified to only use pairs observed during the period of their first brood as the core data set of available mates. More details regarding data set restriction relative to second clutches can be found online (Appendix S1). RANDOM MATING SCENARIOS We compared in parallel five different data sets of mating patterns: the first described the observed pattern of mating occurring in the field during 41 consecutive breeding seasons ( ), henceforth referred to as observed values. Following earlier work (van Tienderen & van Noordwijk 1988; Gibbs & Grant 1989; Keller & Arcese 1998), we generated four further scenarios of possible mate choice using different sets of assumptions about mate availability, in which each year of the 41-year long data set was analysed separately. The different scenarios of simulated mating events are described as follows: 1. All mates scenario: in each of the 41 years, we generated all possible pairings between all males and females known to be breeding in that year in the population. The sample size of all possible pairings was substantial in all cases, but varied from year to year. 2. New mates scenario: only birds breeding for the first time, those that divorced (mate from previous year recaptured) or that were widowed (mate from previous year not known to be alive) were included as birds available for mating in any given year. This mating scenario assumes that already paired birds may not be available as potential breeding mates to other members of the population. Birds that mated with an unknown partner were included in this category of available mates, as the probability p that a bird would mate twice with the same male was relatively small: P = (1 a) * b, where a is the divorce rate (Szulkin & Sheldon 2008b), and b is the adult survival rate in the population. Hence, P = (1 0 32) * 0 45 = At a yearly level, the data set of all possible pairings in the new mates scenario was on average 17 3% smaller than the data set generated in the all mates scenario. 3. Nearest neighbour focal male scenario, and 4. Nearest neighbour focal female scenario: here we paired the male or female of any given pair to a known breeder of the opposite sex breeding in the nearest nestbox relative to the focal pair. This scenario takes into account constraints in local mate availability and possibly spatial genetic structure in the population, assuming that neighbours are potential mating partners while mate choice occurs at the onset of the breeding season (Keller & Arcese 1998). Nearest neighbour (also referred to as NN ) data was extracted from global positioning system (GPS)-mapped locations of nestboxes (see Wilkin et al. 2006). We also investigated an additional mating scenario, namely that of matching breeding pairs and their nearest neighbours of opposite sex using a data set that would be restricted solely to those birds that were known not to have retained their last year s mate (e.g. birds from the New mates data set). However, the results obtained were qualitatively equivalent to those generated by the Nearest neighbour focal male, focal female scenarios above, and are therefore not presented here. PEDIGREE BUILDING To estimate relatedness in the population, a social pedigree was used. Thus, social fathers are assumed to be genetic fathers, although it is known that extra-pair fertilization may invalidate this assumption. Extra-pair fertilization in Wytham great tits is estimated to be in the order of 14 18% (Blakey 1994; S. C. Patrick 2008). Given that the occurrence of inbreeding is fairly low, extra-pair paternity will, if anything, lead to the false categorization of an outbred individual (sired by an extra-pair male) as inbred. As fitness effects of inbreeding are not investigated in this study, the only source of concern is that the relatedness between breeding individuals may be lowered in the situation where extra-pair males sired one (or both) of the two relatives breeding together; their relatedness through the maternal line, however, remains unchanged. Furthermore, as breeding between kin involved relatively few pedigree links ( f ), we believe that in the context of our study, bias caused by using a social rather than genetic pedigree is negligible. The methodology followed for pedigree building differed slightly to that used in previous studies of inbreeding in the Wytham great tit population (see Szulkin et al for more details), as the standard procedure used previously for pedigree building was to include all ringed nestlings and their parents in one data set from which inbreeding coefficients were derived. In this study, we used a standard pedigree up to the year in which mating patterns were investigated. Hence, the pedigree was built using information on all ringed nestlings and their parents recorded in Wytham and its vicinity from 1958 up to the particular year before random mating analyses were carried out. In cases where one or both of the breeding parents could not be identified, the breeding event was included in the pedigree, but the unknown parent(s) were assigned a unique identification number specific to the breeding event in which it took part. This allowed us to identify siblings even when both parents were unknown. Whenever biological parents could not be identified (e.g. cross-fostering with no identification of cross-fostered young), the breeding event was excluded from the pedigree, but any offspring from the brood that recruited were included and assumed to have unknown, and unrelated, parents.

4 Inbreeding avoidance and random mating 781 For each year where mating patterns were investigated, we used observed and simulated pairs and grafted them to the existing pedigree by attributing to each pair one dummy offspring. For example, to generate inbreeding coefficients for 1975 we added either observed or simulated pairs to the observed pedigree from 1958 to This allowed us to include observed pairs whose broods failed, and which would not have been included in our standard pedigree, as well as to generate inbreeding coefficients of pairings present in the different mating scenarios (see below). This procedure of combining the true pedigree with observed or simulated pairings was repeated for each of the 41 years of the data set. We used the software pedigree viewer (available at: edu.au/~bkinghor/pedigree.htm) to generate inbreeding coefficients for each of the 41 years separately, where observed values of inbreeding, all mates and new mates scenarios, and scenarios where the male and the female of a pair were mated to their nearest neighbour of opposite sex were run in five separate analyses. The relatedness between mating partners is equivalent to twice the value of their offspring s inbreeding coefficient. In other words, parental relatedness translates directly into the inbreeding coefficient of their offspring. Hence, we defined any pair of individuals as inbreeding whenever they were related and produced offspring with a particular inbreeding coefficient. To investigate the frequencies of different types of inbreeding, we categorized inbreeding coefficients into the following inbreeding classes: any breeding pair whose offspring had an inbreeding coefficient of f < was classified as outbred, and the breeding partners were henceforth defined as unrelated. We further created inbreeding classes, defined as f = for f < , f = for f < 0 125, f = for f < 0 25, and finally f = 0 25 for f 0 25, resulting from matings between first-order relatives. The data set of observed values consisted of breeding events where both parents had a known identity, and each breeding attempt was given an inbreeding coefficient, reflecting offspring inbreeding. Its size varied depending on the type of random mating simulation it was compared with. The data sets of observed values in the all mates and nearest neighbours scenarios were very similar (n = 6733 and n = 6688), and the slight discrepancy resulted from the fact that 37 nestboxes, mostly recorded in the very early years of the study, did not have their location recorded by GPS in the winter of 2004 (see Wilkin et al. 2006); consequently it was not possible to establish the nearest neighbour for those nestboxes. The data set of observed breeding events in the new mates scenario consisted of 5992 matings. F IS VALUES: TESTING FOR INBREEDING AVOIDANCE Pedigree-based inbreeding coefficients f can be readily used to estimate the degree of inbreeding avoidance (F IS ) relative to random expectations using Wright s F statistics (Wright 1965), where the following relationship is applied: F IS = (F IT F ST )/(1 F ST ), where F IT is equivalent to the observed mean population inbreeding coefficient, while F ST reflects inbreeding values generated from null models of random mating, and is equivalent to the correlation between random gametes within the pedigreed population relative to gametes of the total of all possible populations (Wright 1965; Keller & Arcese 1998). F IS ranges between 1 and +1, and is negative when F ST > F IT. Thus, there is evidence for inbreeding avoidance when F IS negative, as it reflects the situation where random mating scenarios generate higher levels of inbreeding than those observed in the population. We calculated values of F IS all mates, new mates, NN focal male, NN focal female, which generated four different values of F ST. For graphical purposes, we averaged yearly F IS values over eight time periods. To test whether F IS values differ significantly from the null assumption of a mean of zero, we used all 41 years of data as independent data points in two-tailed one-sample t-tests. FREQUENCIES OF DIFFERENT LEVELS AND TYPES OF INBREEDING We used maximum likelihood chi-squared tests to compare the size of particular classes of inbreeding observed in the population with those generated by the four scenarios of random mating. We asked whether the frequency of outbred and inbred matings differed in the observed and simulated data sets, and in particular whether any differences could still be observed when the highest class of inbreeding ( f = 0 25), which may involve individuals familiar with each other, was excluded from the data set. We further investigated differences in the frequencies of different types of inbreeding classes ( f = , f = , f = 0 125, f = 0 25) between observed and simulated data sets. Finally, we tested for differences in different types of close inbreeding ( f = 0 25), and asked whether the proportions of brother sister, mother son and father daughter matings differed between observed values and those generated by the different mating scenarios. Analyses were carried out in r (version 2 3 1), genstat (version 10), matlab (version R2007a), Excel and Access (Microsoft Office 2003). INBREEDING LEVELS IN OBSERVED AND SIMULATED DATA SETS We tested for differences in overall inbreeding levels between observed values and those generated by four scenarios of random mating at a yearly level (n 1 = n 2 = 41) using a nonparametric Wilcoxon matched-pair test. However, years are not independent of each other in terms of potential inbreeding levels, as c. 45% of adults survive to the following breeding year. We therefore additionally pooled data from individual years into eight time periods, which given the short life span of the species should reduce the dependence in inbreeding levels when the eight groups are compared (n 1 = n 2 = 8). The first period consisted of seven consecutive years ( ) where the sample sizes per year were relatively low relative to the rest of the study period. Six 5-year long periods followed, ending with a 4-year long period spanning from 2001 to Results THE DATA SET We used a data set of 8654 great tit breeding events occurring between 1964 and 2004 in Wytham Woods where the identity of at least one of the two parents was known. We further restricted this data set to cases in which both parents were known and where the inbreeding coefficient of the offspring of such pairs (equivalent to the relatedness between breeding partners) could be calculated; this data set constitutes the observed values (n = 6733, n = 5992, n = 6688 in the case of all mates, new mates and nearest neighbour scenarios, respectively). The overall inbreeding level for this population was f = , which is very similar to the overall population inbreeding level of f = calculated in Szulkin et al.

5 782 M. Szulkin et al. Fig. 1. Yearly average inbreeding coefficients in a great tit population studied in Wytham near Oxford from 1964 to 2004, in comparison to four mating scenarios. (a) all mates and new mates scenarios: black lines refer to observed values of inbreeding in all mates (continuous line) and new mates (dashed line) scenarios; green and orange lines refer to inbreeding values simulated in all mates and new mates scenarios, respectively (see text for details of these models). (b) Nearest Neighbour scenarios. The black line refers to observed values of inbreeding. Light blue and pink lines correspond to inbreeding values generated from data sets where males and females were mated to their nearest neighbour of opposite sex. (2007). Factors responsible for the small difference between the two data sets are: (i) broods where both parents were known, but no young were ringed (i.e. failed broods) were not included in the pedigree used in Szulkin et al. (2007); (ii) in this study, second broods are excluded from our analyses, and perhaps most importantly (iii) we did not exclude from this study breeding events where inbreeding coefficients were known, but where interference to the nest in the form of experimental manipulations and predation was made. Excluding manipulations was necessary in prior analyses as these concerned fitness consequences of inbreeding (Szulkin et al. 2007). OBSERVED AND SIMULATED LEVELS OF INBREEDING There was a continuous increase in the number of breeding events in the population (F 1,39 = 29 16, P < 0 001), with the population increasing from a mean of 229 (63 4 SD) pairs in the period to 397 pairs (53 6 SD) in the period However, no similar trend was found for inbreeding levels (F 1,39 = 0 5, P = 0 485), which fluctuated substantially from year to year. Figure 1 presents the overall inbreeding levels recorded in the observed data set, as well as inbreeding levels generated by different scenarios of random mating. We recorded 2 8 and 2 6 times higher inbreeding levels in the observed data set relative to all mates and new mates scenarios, respectively (Fig. 1a). These differences were significant when compared both between time periods or years (Table 1). In contrast to scenarios where birds from the entire population were used for mate choice simulations, we recorded rather similar values of inbreeding when observed and nearest neighbour inbreeding values were compared. Inbreeding levels in the observed data set were only 1 2 and 1 3 times higher relative to NN focal male and NN focal female scenarios, respectively, and no significant difference in inbreeding levels were noted between observed values and nearest neighbour simulations, either at a time period or yearly level (Fig. 1b, Table 2). PEDIGREE-DERIVED MEASURE OF INBREEDING AVOIDANCE F IS F IS, reflecting the degree of inbreeding avoidance in the study population, was positive irrespective of the mating scenario investigated when averaged over the entire duration of the study; it reached overall values of , , and , when all mates, new mates, NN focal male and NN focal female random-mating scenarios were used as base reference to estimate F ST, respectively. Furthermore, these positive F IS values were significantly different from zero when all mates and new mates were used as null models of random mating (F IS all mates : t 40 = 6 44, P < 0 001; F IS new mates : t 40 = 5 40, P < 0 001; F IS NN focal male : t 40 = 0 93, P = 0 356; F IS NN focal female : t 40 = 1 83, P = 0 075). Changes in F IS over the eight time periods are shown in Fig. 2. Overall, these results do not provide any evidence for inbreeding avoidance occurring in the population. If anything, our results suggest that great tits mate with relatives more often than expected under random mating. CLASSES OF INBREEDING AND TYPES OF CLOSE INBREEDING IN OBSERVED AND SIMULATED DATA SETS We further explored differences in the frequencies of different inbreeding classes in observed and simulated data sets of

6 Inbreeding avoidance and random mating 783 Table 1. Differences in observed inbreeding levels versus those generated by all mates and new mates mating scenarios in a long-term study of the great tit All mates scenario New mates scenario Observed values Simulated values Observed values Simulated values Years Mean f n Mean f n Mean f n Mean f n No. of breeding events 1 74% 0 79% 1 59% 0 76% where f Wilcoxon matched-pair test for each year: T + = 57, n 1 = n 2 = 41, P < T + = 95, n 1 = n 2 = 41, P < for each time period: T + = 1, n 1 = n 2 = 8, P = T + = 0, n 1 = n 2 = 8, P = Table 2. Differences in the observed inbreeding level versus those generated when males and females are paired with their nearest neighbour of opposite sex in great tits Observed values Male mated to nearest neighbour female Female mated to nearest neighbour male Years Mean f n Mean f n Mean f n No. of breeding events where f % 1 51% 1 45% Wilcoxon matched-pair test for each year: T + = 378, n 1 = n 2 = 41, P = T + = 267, n 1 = n 2 = 41, P = for each time period: T + = 7, n 1 = n 2 = 8, P = T + = 4, n 1 = n 2 = 8, P = mating events. The frequencies of outbred versus inbred ( f ) matings did not differ when the true values and nearest neighbour scenarios were compared (Table 3). In contrast, there were marked differences when observed inbreeding levels were compared with all mates and new mates scenarios (Table 3), suggesting that birds breed more often with related kin than expected under these null models (Table 3; Fig. 3). Interestingly, while these differences are partly driven by the proportion of close inbreeding events ( f = 0 25, Fig. 4), which affects the sample size of breeding events where f , inbreeding versus outbreeding frequencies in all mates and new mates scenarios remained significantly different from observed values even when the inbreeding class of f = 0 25 was excluded from our analyses (Table 3). The proportions of different inbreeding classes where f varied from one mating scenario to another (Fig. 4), and while observed values did not differ in frequencies of inbreeding classes from those generated under the nearest neighbour scenarios, they significantly deviated from values generated by all mates and new mates scenarios (Table 3). Compared to observed values, all mates and new mates scenarios produced an excess of pairings with f = and a deficit

7 784 M. Szulkin et al. When the closest inbreeding class (f = 0 25) was considered, observed frequencies of brother sister, father daughter and mother son matings deviated significantly from all scenarios but one ( new mates ; Table 4, Fig. 5). Hence, any inference about the randomness of mating with relatives depends on the mating scenario taken as reference. However, in most cases where significant deviations were recorded between observed and simulated values, the direction of the deviation was that inbreeding occurred more often between full-sibs than expected at random (Fig. 5). Discussion Fig. 2. F IS values in a long-term study of the great tit, based on differences between observed inbreeding values and those simulated in four scenarios of random mating, calculated over eight time periods of the study. The thick black and grey and thin black and grey lines represent F IS all mates, F IS new mates, F IS NN focal female, F IS NN focal male, respectively. of f = 0 25 pairs (χ 2 = 23 92, P < 0 001; χ 2 = 17 28, P < 0 001; χ 2 = 0 49, P = 485; χ 2 = 1 55, P = for all mates, new mates, NN focal male, NN focal female, respectively; d.f. = 1). Hence, close inbreeding among potentially familiar kin is more common than expected under pure random mating, but inbreeding among more distant relatives is less common than expected under random mating. We investigated four models of random mating, and compared generated levels of inbreeding to values observed in the population over 41 years of great tit breeding attempts. Our results showed that observed inbreeding levels are times higher than what would be expected on the basis of mate relatedness had mating been at random at a population level. However, overall inbreeding levels did not differ from inbreeding levels generated by scenarios of nearest neighbour mating; in fact, observed inbreeding levels and those generated had males and females mated with their nearest neighbour are strikingly similar (Fig. 1b). We believe that the nearest-neighbour scenario is the most realistic of those investigated, as it encompasses territoriality, spatial variation in relatedness and within population variation in density. Independent of the strengths and weaknesses of the four mating scenarios, observed inbreeding levels were usually higher than those generated by the four simulations, which suggests little evidence for active avoidance of mating with Inbred versus outbred matings Frequencies of four inbreeding classes ( f ) χ 2 (d.f. = 1) P χ 2 (d.f. = 3) P All mates < <0 001 when f = 0 25 excluded: New mates < <0 001 when f = 0 25 excluded: male mated to NN female when f = 0 25 excluded: female mated to NN male when f = 0 25 excluded: Table 3. Comparison of observed and simulated frequencies of inbred ( f ) versus outbred matings in great tits, and between four inbreeding classes (f = , f = 06125, f = 0 125, f = 0 25). Values indicated beneath each mating scenario indicate test statistics and P values for the same test, but when the inbreeding class of f = 0 25 is excluded from the category of non-null inbreeding (f ). NN stands for nearest neighbour Table 4. The frequencies of different types of close inbreeding (brother sister, mother son and father daughter matings) differ when observed values and those derived from different scenarios of random mating are compared with each other. NN stands for nearest neighbour scenario (d.f. = 2) All mates New mates NN focal male NN focal female Observed Values χ 2 = χ 2 = 4 23 χ 2 = 7 30 χ 2 = P = P = P = P = 0 003

8 Inbreeding avoidance and random mating 785 Fig. 3. Proportion of events where f (relative to all breeding events) in observed breeding events and in simulated mating scenarios for years 1964 to The different mating scenarios are presented as follows: (a) all mates, (b) new mates, (c) male mated to nearest neighbour female and female mated to nearest neighbour male. Fig. 4. Proportions of different inbreeding classes in observed and simulated data sets when f Because the frequencies of all inbreeding classes are substantially higher in all mates and new mates scenarios than in observed and NN simulations, we scaled these values to percentages for graphical purposes only. Thus, 100% represents the total number of inbreeding events where f in each of the simulated data sets and in the observed data. Black bars represent observed inbreeding (left hand black bar: observed values from all mates and nearest neighbour data sets; right hand black bar: observed values from the new mates data set). Grey bars indicate inbreeding generated in nearest neighbour scenarios (plain grey: males mated to their nearest female neighbour; dashed grey: females mated to their nearest male neighbour). White bars: inbreeding values generated by all mates and new mates scenarios (white and dashed white bars, respectively). Fig. 5. Distribution of different types of close inbreeding ( f = 0 25) in (a) all mates, (b) new mates and (c) nearest neighbour scenarios. Black bars refer to observed values, grey bars summarize simulated results. (c) grey bars: simulated NN focal male; dashed grey bars: simulated NN focal female. B S: full-sib mating; M S: mother son mating; F D: father daughter mating.

9 786 M. Szulkin et al. kin. While recognizing kin and avoiding breeding with recognized kin may play a major role in cooperatively breeding species (Komdeur & Hatchwell 1999), our result is in agreement with previous studies showing no active inbreeding avoidance in bird taxa that are not cooperative breeders, such as great frigatebirds, blue tits, ground finches, reed warblers, song sparrows, and great tits (van Tienderen & van Noordwijk 1988; Gibbs & Grant 1989; Keller & Arcese 1998; Cohen & Dearborn 2004; Foerster et al. 2006; Hansson et al. 2007; respectively). While it is clear that the all mates and new mates random mating scenarios generate lower levels of inbreeding than observed in the wild (Fig. 1a, Fig. 3a,b), the contribution of the different classes of close inbreeding towards an overall inbreeding value in observed and simulated data sets is of interest: a lower than expected frequency of low inbreeding ( f = ), and a higher than expected frequency of high inbreeding ( f = 0 25) was found in the actual matings relative to simulated matings (Fig. 4). The explanation for this is not clear, but may hint at a degree of avoidance of relatives that varies with relatedness, or a different process affecting the formation of pairs between close relatives. Our results highlight a phenomenon that has not often been given much consideration in other studies investigating mating patterns, which is that birds may mate with kin more often than expected at random. This pattern is visible whenever we compare overall inbreeding (although not significantly so in nearest neighbour scenarios), or the frequency of null vs. non-null inbreeding events, since observed values were always higher relative to all scenarios of random mating. Although this trend is largely caused by siblings mating more often than expected by chance, we find the results noteworthy, as close relatives should be most easily recognizable as kin, either through phenotype matching or associative learning (Komdeur & Hatchwell 1999). Further, all siblings that bred together had fledged from the same nest (Szulkin & Sheldon 2008b) and thus had the opportunity to learn that they were relatives. Great tit and long-tailed tit siblings often disperse in similar directions (Matthysen, van de Casteele & Adriaensen 2005; Sharp et al. 2008, respectively), and spotted sandpiper and great reed warblers siblings return together to breeding grounds more often than expected by chance (Alberico, Reed & Oring 1992; Hansson, Bensch & Hasselquist 2003); similar results have been found for lizard dispersal (Massot & Clobert 2000). Such nonrandom dispersal of siblings may thus increase the likelihood of incestuous mating if no other inbreeding avoidance mechanism but dispersal is used. Importantly, the nearest neighbour scenario incorporates dispersal in its assumptions regarding mating opportunities; this suggests that even when dispersal behaviour is taken into account, brother sister matings occur more often than expected. The elevated frequency of mating between close relatives found in our study might suggest some kin-selected benefits resulting from inbreeding (Hamilton 1964; Kleven et al. 2005); however, given the substantial fitness costs caused by inbreeding and the low occurrence of such matings (Szulkin et al. 2007), it is doubtful whether there could be strong selection for the evolution of such mating tactics in the great tit. Instead, we suggest that for some sib pairs, social bonds may be particularly strong, and that unusually strong social bonds may lead to sexual bonds: this may act as an important mechanism of inbreeding in naturally outbred populations. Further support for this hypothesis comes from the observations that in this population, birds mating with close kin ( f = 0 25) have lower rates of extra-pair paternity (Szulkin 2007) and divorce (Szulkin & Sheldon 2008b) than outbreeding pairs. It is thus possible that unusually strong bonds between sibs may develop while in the nest, or soon after, which can be further strengthened by paired dispersal, as observed in this population (T. Wilkin, personal communication), as well as in other species (Alberico et al. 1992; Hansson et al. 2003; Shutler et al. 2004; Matthysen et al. 2005). Other studies have also suggested that siblings raised in the same nest breed with each other more often than expected at random (Keller & Arcese 1998; Shutler et al. 2004). The nearest neighbour scenario generated inbreeding levels similar to those found in observed pairings, but considerably higher than under population-wide random mating. Thus, more closely related birds breed closer together. Our results therefore imply underlying genetic structure in the breeding population, with relatedness between birds of opposite sex decreasing with increasing distance. Although rare, the use of pedigree-based estimations to detect population genetic structure is increasing (e.g. Garant et al. 2005; Postma & van Noordwijk 2005), and highlights the extent to which dispersal can potentially create genetic structuring on small spatial scales (Garant et al. 2005; Shapiro et al. 2006). To our knowledge, this is the first study not to use genetic markers to infer the existence of a fine-scale genetic structure where relatedness between individuals of the opposite sex decreases with distance. Further work on the variability of relatedness patterns on small geographical distances would thus be of great interest, and shed further light on the importance of dispersal in the genetic structuring of populations. Interestingly, three other studies comparing random mating scenarios with observed values of inbreeding did not find such discrepancies between random mating scenarios when all mates and nearest neighbour scenarios were compared (van Tienderen & van Noordwijk 1988; Gibbs & Grant 1989; Keller & Arcese 1998), suggesting that dispersal did not play as important a role in shaping relatedness patterns in these populations. Each of these populations inhabited relatively small islands, in which dispersal behaviour may conceivably be different from that in a large meta-population. Two not mutally ways through which such structure could be attained in our study are that (i) dispersal direction is nonrandom with respect to relatedness, or (ii) dispersal distances were not large enough to equalize the degree of relatedness with a prospective mate independently of where it is settled. Indeed, higher levels of inbreeding generated by the nearest neighbour random mating scenarios, relative to the population-wide scenarios can only be achieved if the average dispersal distance for great tits in this population is lower than the average distance a bird can move within the population. While nonrandom dispersal

10 Inbreeding avoidance and random mating 787 cannot be discarded as a factor causing genetic structuring of the population, limited dispersal may act as the predominant force in shaping the relatedness structure of the population. The median distance between nestboxes in Wytham is 1555 m (arithmetic mean = 1495 m). In contrast, the median value for natal dispersal in males and females is 528 m and 788 m, respectively (Szulkin & Sheldon 2008a), which suggests that natal dispersal is insufficient to homogenize population genetic structure. A similar pattern of relatedness between individuals that is decreasing with distance is now believed to be a relatively common phenomena, although it is usually described within only the less dispersive sex (e.g. Coltman, Pilkington & Pemberton 2003; Double et al. 2005; Nussey et al. 2005). Both this study and one on blue tits (Foerster et al. 2006) focus on relatedness between members of the opposite sex, and indeed find small-scale genetic structuring and relatedness decreasing with distance. The unexpectedly high number of close inbreeding events between siblings remains unexplained, and further work on associations between siblings in their first winter, and modes of settlement may shed some light on these matings. While it is unknown to what extent great tits recognize kin, our results suggest clearly that overall, they do not actively avoid mating with kin. It is theoretically possible that they do not avoid inbreeding at all, as the cost of avoiding kin can be higher than the costs of inbreeding (Kokko & Ots 2006); however this seems unlikely given that close inbreeding can reduce fitness up to 40% (Szulkin et al. 2007), while the number of relatives in a large, outbred population is limited. 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