mm THE ROYAL 93aS SOCIETY
Received 31 October 2001 Accepted 27 November 2001 Published online 18 March 2002
Inbreeding depression and male fitness in black grouse
Jacob Hoglund1*, Stuart B. Piertney2, Rauno V. Alatalo3, Johan Lindell1!, Arne Lundberg1 and Pekka T. Rintamaki1
1 Population Biology/Evolutionary Biology Centre, University of Uppsala, Norbyvagen 18D, SE-752 36, Uppsala, Sweden
2 Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, UK
3Department of Biological and Environmental Science, University of Jyvaskyla, PO Box 35, FIN-40351, Finland
The male lifetime lekking performance was studied, and related to inbreeding-outbreeding in a wild population of black grouse (Tetrao tetrix) in central Finland between 1989 and 1995. Inbreeding was measured as the mean heterozygosity and mean d2 of 15 microsatellite loci. We found a significantly positive relationship between mean d2 and lifetime copulation success (LCS), while the relationship between heterozygosity and LCS was close to significant. We also found that males that never obtained a lek territory had significantly lower mean heterozygosity than males that were observed on a territory at least during one mating season in their life. Furthermore, among males that were successful in obtaining a lek territory, LCS and mean d2 were highest for those males that held central territories. We suggest that inbred males have a disadvantage (or outbred males have an advantage) in the competition for territories that may explain the relationships with LCS and inbreeding. Furthermore, the fact that mean d2 was positively correlated with LCS whereas heterozygosity was not when we restricted the analysis to territorial males, suggests that mean d2 provides more information about levels of inbreeding-outbreeding than heterozygosity alone, and potentially highlights the effects of heterosis. To our knowledge, this is the first time that measures of inbreeding and lifetime fitness have been linked in a non-isolated population. This is important in establishing that the relationships found in previous studies are not artefacts of low gene flow created by limited dispersal but a general feature of wild vertebrate populations.
Keywords: inbreeding; inbreeding depression; lifetime copulation success; microsatellites; heterozygosity; mean d2
1. INTRODUCTION
The detrimental effects of inbreeding (inbreeding depression) on traits relating to individual fitness and population persistence have been investigated for several decades. Inbreeding depression reflects the consequences of increased homozygosity for alleles that affect fitness, and may be manifest either through superior performance of heterozygote over homozygote genotypes, and/or the effects of recessive deleterious alleles (Charlesworth & Charlesworth 1987, 1999). When two inbred lines are crossed it is commonly observed that fitness increases in the outbred Fj generation, an effect termed heterosis.
There is ample evidence of inbreeding depression in captive vertebrate populations from a range of taxa (see Ralls et al. 1988; Lacy et al. 1993, and references therein), but few studies have involved natural populations (Keller 1998; Crnokrak & Roff 1999). This is due, in part, to the difficulties associated with identifying parameters that reflect lifetime reproductive success (LRS) and the ability to monitor these in natural populations, and, also, an inability to establish the required pedigrees among individuals for a classical inbreeding analysis (Wright 1969).
~ Author for correspondence (jacob.hoglund@ebc.uu.se). f Present address: Centre for Biodiversity and Conservation Biology, Royal Ontario Museum, 100 Queen's Park, Toronto, Ontario, Canada M5S 2C6.
Moreover, the majority of studies on inbreeding have focused on early life-history stages. Inbreeding studies that examine only juvenile stages in captive or semi-captive populations probably underestimate the effects of inbreeding because they ignore the effects of deleterious alleles that affect adult life-history stages, and also cannot properly mimic the natural conditions that are likely to reveal conditionally deleterious alleles (see Meagher et al. 2000, and references therein).
Recent developments in DNA marker-based methods for detecting inbreeding have now made it possible to study inbreeding without pedigrees, using surrogate estimators such as heterozygosity and mean d2 (Bierne et al. 1998; Coltman et al. 1998; Coulson et al. 1998, 1999; Fisher & Mathies 1998; Pemberton et al. 1999; Saccheri et al. 1998). Such approaches have shown, for example, a negative relationship between inbreeding and male and female LRS in red deer (Cervus elaphus) (Slate et al. 2000), and a positive relationship between outbreeding and offspring survival in the horseshoe bat (Rhinolopus ferrumequinum) (Rossiter et al. 2001). In addition, the effects of inbreeding are enhanced by male-male competition in semi-natural populations of the house mouse (Mus domesticus) (Meagher et al. 2000). With the gradual accumulation of studies using genetic markers to study inbreeding effects, the predictions of inbreeding theory are confirmed: offspring born to genetically dissimilar partners seem to show above average general fitness, regardless of
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712   J. Höglund and others   Inbreeding and fitness in black grouse
whether the effects are due to the negative effects of inbreeding depression or the positive effects of heterosis (Amos et al. 2001a,6).
A common feature of previous studies that use molecular markers to examine levels of inbreeding (Coltman et al. 1999; Slate et al. 2000; Rossiter et al. 2001) is that they are carried out on island, or isolated populations, where the effects of inbreeding may be more demonstrable. This is because dispersal to, and from, isolated populations may be hampered or impossible for extended time-periods, and the effective population size is often small in isolated populations. We describe the effects of inbreeding on LRS in black grouse (Tetrao tetrix) from a population in a continuous taiga habitat where extensive dispersal of individuals is possible (Hoglund et al. 1999). Our previous study showed that, at a local scale, this population is moderately structured as a consequence of male philopa-try but that female natal dispersal is extensive (see also Willebrandt 1988), a pattern common to all grouse species studied (Jamieson & Zwickel 1983; Small & Rausch 1989; Piertney et al. 1998). Black grouse have a lek mating system whereby males gather to display and females come to mate but subsequently nest elsewhere, away from the male (Hoglund & Alatalo 1995). We propose that, assuming competitive interactions between males at a lek are designed to advertise fitness (Alatalo et al. 1991), those males that are successful in attaining a territory on a lek, and also having the highest reproductive success among territory holders, are predicted to be the fittest, possibly because of reduced inbreeding effects.
2. MATERIAL AND METHODS
(a) Study area and population
We studied a population of black grouse from a nature reserve near Petajavesi, Central Finland (62°10'N, 25°05'E) from 1989 to 1995, where hunting is prohibited. Males were caught at feeding sites during winter using traps baited with oats, or during the mating season, by cannon netting. A blood sample was taken from the wing vein for subsequent DNA analysis. Males were ringed with a metal ring for permanent identification and three colour rings for identification at a distance. We subsequently studied the lekking behaviour of males annually at their traditional lek sites during the main part of the breeding season in April and May. Briefly, we recorded the identity, territorial position, display behaviour and annual copulatory success of individual males. We defined longevity as the number of years any male was observed to be present in the study area, and male lifetime copulation success (hereafter LCS) as the observed cumulative number of copulations by any given male throughout his lifetime. Our measure of longevity is probably a conservative underestimate in cases in which males were still alive at the termination of the study. The timing of copulations in black grouse are exceptionally synchronized in that the majority of the annual copulations take place within one week, and we have been unable to detect any significant proportion of copulations taking place outside the leks (Alatalo et al. 1996; Rintamaki et al. 2001). As all males at all leks each year were observed for the entire lekking period, there was no need to standardize copulations for differences in observation period per male. A previous study using multilocus DNA fingerprinting indicated that observed copulation in most instances is a reliable measure of male reproductive success (Alatalo et al. 1996). Details of the
field procedures and the study area are available in Rintamaki et al. (2001).
(b) Micro satellite genotyping and genetic estimates of inbreeding
DNA was extracted from blood using standard phenol/ chloroform extraction procedures (Sambrook et al. 1989). Microsatellite allele length variation was determined at 15 microsatellite loci, 10 of which were specifically cloned from black grouse (BG primer pairs; Piertney & Hoglund 2001), three loci were cloned from capercaillie (Tetrao urogallus) (TUT1-3 primers; Segelbacher et al. 2000) and two loci were cloned in red grouse (Lagopus lagopus scoticus) (LLST1 and 3 primers; Piertney & Dallas 1997). PCR reaction conditions and temperature profiles followed the original publications. In all cases, PCR fragments were separated by electrophoresis on 6% denaturing polyacrylamide gels and visualized using silver staining.
The mean d2 was calculated as the squared difference in repeat units between two alleles at a locus averaged over all loci at which an individual was scored (Coulson et al. 1998; Pemberton et al. 1999; Slate et al. 2000) such that
mean d2= l/n^(ia — i^)2,
where «a and ib are the lengths in repeat units of alleles a and b at locus i, and n is the total number of loci. The mean heterozygosity was determined as the proportion of typed loci for which an individual was heterozygous while standardizing for the heterozygosity at any given locus (Coulson et al. 1998; Slate et al. 2000). We chose not to standardize the mean d2, as this enhances the contribution of less variable loci on mean d2, whilst reducing the contribution of highly variable loci. For example, if the relationship between locus-specific variance in mean d2 and number of alleles at each locus is exponential, as in black grouse, standardization for locus-specific variance overemphasizes the contribution of loci with a few alleles (J.H. and J. Dannewitz, unpublished data).
(c) Statistical analyses
The relationship between LCS and other traits described in § 2b were analysed using generalized linear models (McCullagh & Nelder 1989). In all models, we used a Poisson error structure and a log-link function because the large number of males with LCS = 0 violates the assumptions of standard linear regression. All analyses were performed using Genstat, v. 4.1 (Genstat 5 Committee 1993).
3. RESULTS
A total of 139 males were genotyped at 11.3 + 3.3 (mean+s.d.) microsatellite loci. Males with data from seven loci or less were excluded from the analysis. For all individuals, both mean d2 and mean heterozygosity were positively related to LCS, although only the former was significant (mean d2X{ = 18.81, p < 0.001; heterozygosity X2 =3.78, p = 0.052).
Assuming that male black grouse need to obtain a territory to become a member of the reproducing population, we examined whether there were any differences in the genetic variables between males that were observed as permanent territory holders during at least one reproductive season, and those that were never observed on territories.
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(a)
1.0-
0.8'
■p. 0.6'
0.4-
0.2'
0.0'
•• •
0.4
0.6
0.8
1.0
1.2
1
1.4
Ho
20 15 10 5H
0' -5' -10'
10
20 mean d2
30
-I
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Figure 1. (a) Territoriality (data points: 0, never observed on a territory; 1, observed on a territory at least once during lifetime) against mean standardized heterozygosity, Ho. The fitted relationship is the predicted outcome of the logistic regression E (Territorial I Ho) = —1.73 + 2.68 Ho. (b) Residual lifetime copulation success (when the effects of territorial centrality, longevity and attendance are controlled for) plotted against mean d2.
Table 1. Full general models of lifetime copulation success in male black grouse. In the first model (a), we used mean d2 as the genetic variable (mean deviance ratio = 59.48, d.f. = 4, 43, p < 0.001). In the second model (b), we used mean heterozygosity (mean deviance ratio = 37.79, d.f. = 4,43, p < 0.001).
(a)
attendance territorial centrality longevity mean d2
(b)
attendance territorial centrality longevity
mean heterozygosity
4.92 -3.23 3.08
4.69
4.70 -4.37
2.39 0.81
<0.001 <0.001 0.002 <0.001
<0.001 <0.001 0.017 n.s.
Table 2. Relationships between genetic variables ((a) mean d2, (b) mean heterozygosity) and other variables explaining a significant proportion of the variance in lifetime copulation success for territorial males. N = 43 in all cases.
(a)
attendance territorial centrality longevity
(b)
attendance territorial centrality longevity
1.43 -3.84 2.34
0.27 1.89 0.72
n.s. <0.001 0.019
0.785 0.059 0.474
For males that achieved territorial status in the main study area, we ran generalized multiple regression models to examine the effect of the genetic variables together with other sources of variance in LCS. Male attendance at the lek, a measure of territorial centrality, longevity and mean d2 all independently explained some of the variance in LCS, but we could not detect any significant residual effect of mean heterozygosity (figure lb, table 1). The only variables that were significantly correlated with any of the genetic variables were territorial centrality and longevity, which were significantly correlated with mean d2 (table 2). Mean d2 and heterozygosity were positively correlated but the relationship levelled off at high values of mean d2 (figure 2), such that the variance among the data was significantly better explained by a logarithmic relationship than by a linear relationship (p < 0.001 and n.s., respectively).
In this analysis, we only included males from the main part of the study area where we had complete data on male territoriality. Therefore, this is a conservative test as some of the males that were assigned as non-territorial may have dispersed out of the study area and have occupied territories where we could not observe them. There was no effect of mean d2 on the probability of territory ownership (logistic regression: F1 101 = 1.44, n.s.) but a significant effect of mean heterozygosity (logistic regression: Fljl01 = 2.51, p = 0.012, figure la).
4. DISCUSSION
We have established that, among male black grouse, an individual's position on the inbreeding-outbreeding continuum affects reproductive fitness. Overall, LCS was positively correlated with both mean d2 and heterozygosity, and those males that were successful in securing a territory on a lek at least once during their lifetime had a higher heterozygosity than males that never obtained a territory. Moreover, among territorial males, those found
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714   J. Höglund and others   Inbreeding and fitness in black grouse
1.2
1.0
K 0.8
0.6
0.4
I—
10
mean d
20
2
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Figure 2. Mean heterozygosity (Ho) plotted against mean d2.
The fitted line is the predicted relationship from
Ho = 0.97 - 1.09 x 0.14 mean d2, F2=llv= 8.14, p< 0.001.
on central, and hence more successful, territories had a higher mean d2. As increasing heterozygosity and mean d2 reflect a higher degree of outbreeding, our results indicate that more inbred individuals have reduced competitive abilities. As the majority of studies that have detected detrimental inbreeding effects have tended to focus on early life-history stages (Ralls et al. 1988; Lacy et al. 1993; Crnokrak & Roff 1999), and we did not examine fertilization success, embryo mortality or the relative success of chicks and juveniles, the importance of inbreeding in denning the overall fitness of individual black grouse may be even more pronounced.
The competitive interactions between black grouse at a lek are designed to advertise their fitness to potential mates (Alatalo etal. 1991). A demonstration that the highest reproductive success is associated with reduced inbreeding highlights that these advertisements are honest and reflect a component of overall genomic quality rather than just single-locus effects. In this case, the positive associations between mean d2, heterozygosity and LCS seem to be mediated through an effect on territoriality. Meagher et al. (2000) highlighted the reduced competitive ability of more inbred mice in agonistic interactions during territory establishment. Given the intense competition for territory acquisition that has been observed in black grouse (Hoglund & Alatalo 1995), a similar effect is entirely plausible.
One particular concern over the use of mean d2 to measure an individual's position in the inbred-outbred continuum is the lack of congruence between associations of fitness with the mean d2 and heterozygosity. Hedrick et al. (2001) examined the predictive power of both heterozygosity and mean d2 for measuring levels of inbreeding in a captive population of wolves (Cams lupus) of known pedigree. They found that the mean d2 was less predictive of the known inbreeding coefficient than heterozygosity, and hence carried no more information than heterozygosity about the levels of inbreeding or outbreeding. They
therefore state that it is unclear whether studies that find fitness associations with mean d2, especially in the absence of association with heterozygosity, are a reflection of high statistical power rather than biological reality. By contrast, Coulson et al. (1998) suggested that differences are due to the mean d2 reflecting processes associated with heterosis following population admixture, whereas heterozygosity provides an indication of the negative effects of inbreeding depression.
In black grouse, the acquisition of a territory is essential for recruitment to the adult population and subsequent reproductive potential. As we found an association between ability to get a territory and heterozygosity, this means that individuals with low heterozygosity are automatically excluded from subsequent examination of fitness effects on LCS (lower left portion of figure 2). As such, a low level of variation in heterozygosity among territory holders makes it difficult to find significant association with other fitness parameters measured subsequently. By contrast, there is still considerable variation in the mean d2 among territory holders, and hence more scope for detecting fitness associations. We feel that this is a more probable explanation for a lack of congruence between heterozygosity and mean d2 for measuring fitness associations among territory holders, rather than inferring that inbreeding depression affects fitness prior to territory acquisition, and that heterosis underpins LCS.
As with the majority of bird species, male black grouse are philopatric, whereas females display natal dispersal (Willebrandt 1988; Hoglund et al. 1999). This creates a socially induced population structure with related males and unrelated females (Sugg et al. 1996). As such, the only potential for incestuous breeding occurs if females reproduce with their sons in subsequent years. The observation that two-year-old, or older, females mate with their mate from the previous year if he is still alive (Rintamaki et al. 1995) suggests a possible inbreeding avoidance mechanism. However, as male mortality can be high and inbreeding effects were observed in this study, some females may have been forced to mate with relatives or face yet another dispersal phase. Future studies should address whether females have more subtle mechanisms for inbreeding avoidance, such as disassortative mating based on parasite resistance (Coltman et al. 1999) and immuno-competence genes (Penn & Potts 1999; Grahn 2000).
The authors thank R Ekblom, S. A. Saether and two anonymous referees for valuable comments on the manuscript, U. Bornhauser and G. Olsson for help in the laboratory and the Finnish Bird Ringing Centre, Helsinki, for their cooperation. This study was supported by grants from the following institutions: Swedish Natural Sciences Research Council (J.H. and A.L.), Carl Trygger Foundation (J.H.), Engkvist Foundation (J.H.), Emil Aaltonen Foundation (P.T.R.) and the Finnish Academy (R.V.A.).
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