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We examined the reproductive success of 48 adult brown trout (Salmo trutta L.) which were allowed to reproduce in a stream that was controlled for the absence of other trout. Parentage analyses based on 11 microsatellites permitted us to infer reproductive success and mate choice preferences in situ. We found that pairs with intermediate major histocompatibility complex (MHC) dissimilarity mated more often than expected by chance. It appears that female choice was the driving force behind this observation because, compared with other individuals, males with intermediate MHC dissimilarity produced a larger proportion of offspring, whereas female reproductive output did not show this pattern. Hence, rather than seeking mates with maximal MHC dissimilarity, as found in several species, brown trout seemed to prefer mates of intermediate MHC difference, thus supporting an optimality-based model for MHC-dependent mate choice.
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Mate choice can include considerations of genetic compatibility (Trivers in Campbell, 1972; Tregenza & Wedell, 2000; Neff & Pitcher, 2005) and the most extensively studied genes in this context are the MHC class I and class II loci (Apanius et al., 1997; Penn & Potts, 1999; Bernatchez & Landry, 2003; Piertney & Oliver, 2006). These genes are primarily involved in vertebrate adaptive immune response regulation by encoding an essential part of the MHC molecule, presenting self- and nonself peptides to T cells at cell surfaces (Klein, 1986). Consequently, these loci are under pathogen-mediated selection (Apanius et al., 1997; Penn & Potts, 1999) and the polymorphism is the highest of all coding loci found among vertebrates (Apanius et al., 1997).
Several hypotheses have been suggested to describe the selection underlying the widespread MHC polymorphism in natural populations. The intimate role of MHC genes in the immunological framework focuses attention on the pathogen-driven selection. According to the overdominance hypothesis (Doherty & Zinkernagel, 1975), heterozygote advantage should sustain polymorphism, whereas the negative frequency-dependent selection hypothesis holds that polymorphism is preserved when rare alleles have a selective advantage (Takahata & Nei, 1990; Slade & Mccallum, 1992). When MHC polymorphism is maintained by pathogen-driven selection, reproductive behaviours resulting in matings with MHC-dissimilar mates are also favoured (Penn & Potts, 1999). The key mechanisms in such MHC-based sexual selection models are negative assortative (disassortative) mating, i.e. avoidance of MHC similar mates, and associated fitness benefits (Brown & Eklund, 1994; Penn & Potts, 1999). One benefit could be enhanced pathogen resistance in offspring, either by increasing MHC heterozygosity (Potts & Wakeland, 1990) or by presenting a ‘moving target’ for evolving pathogens (Potts & Slev, 1995; Penn & Potts, 1999). An alternative and nonexclusive hypothesis involves avoidance of inbreeding (Potts & Wakeland, 1993; Brown & Eklund, 1994), whereby MHC-based disassortative mating preferences function to prevent kin mating, thereby reducing the negative consequences of inbreeding, similar to other genetic incompatibility systems, e.g. the SI system in plants (Matton et al., 1994).
Intuitively, consideration of MHC compatibility between mates is an appealing ingredient of mate choice, given the role of MHC genes in the discrimination of self from nonself at the cellular level. It has recently been found that MHC peptide ligands are used as olfactory cues and that MHC-linked olfactory receptor genes are involved in the process of discrimination in mice and fish (Boehm & Zufall, 2006), e.g. in three-spined sticklebacks (Gasterosteus aculeatus) (Milinski et al., 2005). These findings indicate a molecular mechanism by which individuals sense compatibility through the body odour of conspecifics. The high variability of MHC loci in combination with the ability of MHC alleles to influence body odour provides the necessary ingredients for a genetically based recognition system at the organismal level (Grafen, 1990).
The first indication of MHC-based mating preference was reported 30 years ago when congenic female mice were found to prefer MHC-dissimilar mates (Yamazaki et al., 1976). Since then, MHC-based mate preferences have been further described in mice (Yamazaki et al., 1988; Potts et al., 1991; Penn & Potts, 1998) and several other vertebrates, e.g. humans (Ober et al., 1997; Jacob et al., 2002), reptiles (Olsson et al., 2003), birds (Freeman-Gallant et al., 2003; Bonneaud et al., 2006) and fish (Landry et al., 2001; Reusch et al., 2001). In some mating systems, however, there may be no room for consideration of MHC compatibility, and several studies have found no correlations between MHC characteristics and mate choice (Paterson & Pemberton, 1997; Ekblom et al., 2004; Westerdahl, 2004; Richardson et al., 2005). Moreover, some features of the adaptive immune system predict that, under certain circumstances, optimally dissimilar mates are preferable to mates of even greater dissimilarity. Although disassortative mate preferences increase pathogenic scope or reduce the risk of inbreeding, at some point the positive effects of reproducing with disparate mates will be exceeded by negative effects such as self-reactive autoimmune responses and a net loss of T cells (Nowak et al., 1992; Deboer & Perelson, 1993) or by other factors such as outbreeding depression (Bateson, 1983; Thornhill, 1993) and fitness loss caused by the disruption of co-adapted gene complexes (see Hendry et al., 2000; Neff, 2004; Bonneaud et al., 2006). This would result in a trade-off, and the optimal and preferred choice would be mates of intermediate MHC dissimilarity (Penn & Potts, 1999). The predominant finding in studies examining MHC-based mate choice, however, has been disassortative preferences, whereas conclusive empirical support for the theoretically predicted optimally based MHC-dependent mating preference has been scarce in the literature (but see Reusch et al., 2001; Bonneaud et al., 2006).
Genetically based mate choice can function to increase genetic quality in offspring by means of ‘good genes’ or ‘compatible genes’ (reviewed by Neff & Pitcher, 2005). Good genes are characterized by alleles capable of increasing fitness irrespective of the architecture of the rest of the genome, and the intrinsic additive genetic variation responds to directional selection. In such mating systems, individuals of the choosing sex will have similar mate preferences for individuals of the other sex possessing the ‘good gene’, e.g. a favourable allele. In mating systems based on ‘compatible genes’, on the other hand, the interactions of male and female genotypes are more important, and mate preference varies among individuals searching for genotypes to complement their own. Neff & Pitcher (2005) propose that mating systems are simultaneously influenced by the effects of ‘good genes’ and ‘compatible genes’ and that they alternate between states of high and low levels of additive and nonadditive genetic variation over evolutionary time scales. This means that the underlying selective mechanisms for MHC-based mate choice vary over time, and previous MHC studies in salmonid species have found features consistent with both the ‘good’ (Lohm et al., 2002) and the ‘compatible’ genes’ (Arkush et al., 2002) mating systems.
Salmonids have previously been found to discriminate conspecifics based on MHC class IIβ characteristics, for example arctic charr (Salvelinus alpinus) (Olsen et al., 1998) and Atlantic salmon (Salmo salar) (Landry et al., 2001). MHC-based mate preferences are most likely to evolve in species at risk of inbreeding (Penn & Potts, 1999) and as brown trout reproduce in their natal stream as a result of strong homing behaviour, the probability of meeting close kin at spawning sites is obvious. Brown trout is a polygamous species with a mating system mainly driven by intrasexual competition; females hold and defend territories whereas males compete for access to females (Petersson & Jarvi, 1997; Petersson et al., 1999). During reproduction females bury their eggs in gravel and the chosen male sheds sperm over them. The fertilized eggs hatch and the fry (or alevins) emerge from the gravel on their own without parental care. Hence, the mating system diminishes the scope for some confounding factors common in studies of mate choice, e.g. cryptic female choice, parental care and biased investment in zygotes sired by specific males.
The aim of this study was to investigate mate choice in relation to genetic similarity at MHC and microsatellite loci in brown trout (S. trutta). Inferences of mate choice preferences were made after examining mating patterns as an effect of dissimilarity of pairs and by investigating the effects of individual genetic properties on the reproductive success of individuals.
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The major finding in this study was that pairs with an intermediate amino acid distance at the examined MHC class IIβ locus had mated more often than expected by chance. The permutation test showed that the high mating frequency of intermediate pairs was not merely an effect of random sampling in a population with a high proportion of intermediate pairs. We used a one-tailed test as we reasoned that the hypothesis of equal variances was one sided and that the expectation of greater variance among the observed matings compared with randomly generated matings was unrealistic.
We also found that males with intermediate MHC dissimilarity had produced a larger proportion of offspring compared with males with lower and higher MHC dissimilarity. This association was significant when MHC dissimilarity between pairs was estimated using the maximal distance method. The nonlinear effect of male MHC dissimilarity was significant in the first replicate but not in the second. As can be seen in Fig. 2a–d, offspring from parents with low MHC scores were absent in the second replicate. This pattern was not found in corresponding plots with microsatellite scores (Fig. 2e,f), indicating that offspring from parents with low MHC scores were not produced or were lost during the second replicate. The higher mortality is consistent with the lower total number of offspring in the second replicate.
Males with high microsatellite dissimilarity mated more often and produced a higher proportion of offspring than other males. If these patterns resulted from female preferences for genetically dissimilar males, it would decrease the risk of producing inbred offspring and indicate an active avoidance of inbreeding. Similar results were recently reported in the closely related Atlantic salmon (S. salar), as females that mated with more dissimilar males increased the chances of producing outbred offspring when estimated at five microsatellite loci (Garant et al., 2005).
A consequence of performing mate choice studies in situ is that preferences must be estimated subsequently. We used the mating of pairs and individual reproductive output as proxies for mate choice, similar to the design of Landry et al. (2001), where sampling of offspring was also conducted some time after the actual mating. Factors not taken into account in the natural settings could have affected individual reproductive success, although using the following rationale we argue that an effect of female mate choice can be detected in the reproductive output of males and that male mate choice can likewise be detected in the reproductive output of females.
If the observed patterns in male reproductive success (Fig. 2a,c,e) were caused by some factor other than female mate choice, a corresponding pattern would most likely have been found in the reproductive success of females (Fig. 2b,d,f). As illustrated in Table 3, no factor apart from female mate choice can explain why the observed pattern of reproductive success of intermediates was only found when the offspring were sorted based on paternal dissimilarity. For example, if there had been an unknown selective advantage in offspring from parents with intermediate MHC dissimilarity (such as a higher survival rate of the eggs, larvae or fry), the observed pattern in male reproductive success would most likely have been found in the reproductive success of females as well. Similarly, if mate choice had been a random process, independent of MHC in both sexes, the high proportion of MHC intermediate males (Fig. 3a) and the high proportion of MHC intermediate females (Fig. 3c), would have resulted in a high proportion of offspring from intermediate parents in both sexes. Using the same logic, the distribution of microsatellite scores of males (Fig. 3b) and females (Fig. 3d) was similar, with a high proportion of individuals with low scores. These distributions were not reflected in the patterns of male or female reproductive success (Fig. 2e,f). Instead, the positive correlation between male reproductive success and male microsatellite dissimilarity indicates a nonrandom female mate choice with respect to microsatellite dissimilarity and a female preference for males of a general genomic dissimilarity. We therefore propose that the observed patterns of male reproductive success were the results of female mate choice.
Table 3. Potential causal factors related to the observation of higher reproductive output of MHC intermediate parents.
|Factors with the potential to explain the higher reproductive output of MHC intermediate parents||If true, pattern likely to be found in|
|Male reproductive success||Female reproductive success|
|1. MHC-dependent survival of eggs or fry||x||x|
|2. MHC-dependent behaviour of fry||x||x|
|3. Sampling biased on fry MHC properties||x||x|
|4. Large number of MHC intermediate males||x|| |
|5. Large number of MHC intermediate females|| ||x|
|6. Male MHC-based mate choice of MHC-intermediate females|| ||x|
|7. Female MHC-based mate choice of MHC-intermediate males||x|| |
The summation and the maximal distance methods for estimating individual MHC dissimilarity differ from a biological perspective in the sense that the sum of distances between individuals reflects an approximation of the alleles in potential mates, whereas the maximal distance represents the largest distance of alleles in potential mates. The two methods are similar and comparable, as both assign higher scores to more divergent individuals and the individual scores generated by the methods are strongly positively correlated (F1,46 = 54.94, P < 0.001), and the coefficient of variance of the estimators was also similar, CV 0.1047 (sum) and 0.1042 (MD).
Although MHC-based mate choice preferences have been described in several vertebrate species, the findings have been dominated by disassortative preferences and only a few examples in the literature describe preferences for optimal MHC divergent mates (e.g. Reusch et al., 2001; Bonneaud et al., 2006). It has been argued that selection for intermediate number of alleles would not maintain high polymorphism over time in natural populations without unrealistically high mutation rates (Hedrick, 2004). On the other hand, mutations would not be the only source of MHC variation in natural populations, and processes such as recombination could occur (Wegner et al., 2004). MHC-dependent mate choice does not appear to be a universal phenomenon, as certain mating systems may have no room for genetic compatibility considerations, and several studies have found no correlations between MHC characteristics and reproductive success (e.g. Paterson & Pemberton, 1997; Ekblom et al., 2004; Westerdahl, 2004; Richardson et al., 2005). However, if the purpose of MHC-based preferences is optimal differences between mates, the outcomes of mate choice studies will be variable and highly dependent on the actual MHC diversity among potential mates. It is therefore to be expected that some studies will only find a slight deviation from a random mating pattern when comparing observed mean differences between mates and random expectation in populations where most mates are of intermediate dissimilarity. Hence, optimal MHC-dependent mate choice preferences may be more common than indicated in the literature.
Most species examined for MHC-based mate choice, e.g. mice, humans and birds, have a complex MHC with several loci coding for MHC molecules with MHC class I and class II genes arranged in tightly linked haplotypes (Klein, 1986). In teleostean fish, on the other hand, the classic MHC class I and II genes are typically found in different linkage groups (Hansen et al., 1999; Sato et al., 2000; Shum et al., 2001; Stet et al., 2003) and salmonid species usually express one MHC class IIβ locus (Langefors et al., 1998; Hansen et al., 1999; Grimholt et al., 2000; Landry & Bernatchez, 2001; Shum et al., 2001). In species possessing several MHC class IIβ loci, theory predicts selective mechanisms for an optimal number of expressed alleles (Penn & Potts, 1999) but this prediction is weakened in species expressing only one MHC class IIβ locus. It is tempting to interpret the preference for males with an optimal MHC dissimilarity on the basis of the optimal outbreeding theory (Bateson, 1983). However, the general genomic dissimilarity as reflected in the 11 microsatellite loci was not correlated with the MHC class IIβ dissimilarity in the 48 breeders (anova; effect of MHC scores on microsatellite scores; F3,44 =0.08, P = 0.78). Similar patterns of independence between MHC and neutral markers have previously been described in Atlantic salmon (S. salar) (Landry & Bernatchez, 2001) and sticklebacks (G. aculeatus) (Reusch et al., 2001). Under these conditions, MHC-based mate choice cannot function to reduce general genomic inbreeding, although it may prevent fitness costs associated with homozygosity in close-kin matings (Apanius et al., 1997).
Alternatively, female avoidance of the most MHC-dissimilar males may have evolved in response to preventing outbreeding depression at the MHC where individuals with elevated MHC dissimilarity are less well adapted to the local pathogenic environment compared with other individuals. Recently, McGinnity et al. (2003) reported a decrease in local adaptation and reduced fitness in a wild population of Atlantic salmon (S. salar) during interactions with escaped farm salmon without local adaptation, thus demonstrating the importance of local adaptation for fitness in a closely related species. Research on anadromous salmonids has found high levels of population differentiation at the MHC (Langefors et al., 1998; Landry & Bernatchez, 2001; Miller et al., 2001), indicating the local adaptation of these species’ MHC. It is likely that a similar MHC differentiation exists in the brown trout, with its closely related phylogenetical, ecological and behavioural patterns. If these assumptions hold true, it would suggest that female avoidance of highly MHC-dissimilar males could be a mechanism that has evolved in response to selection to ensure local adaptation of the MHC.
In conclusion, the permutation test showed that MHC intermediate pairs had mated more often than expected by chance. This process is most likely driven by female mate choice, as males with intermediate MHC dissimilarity had a higher reproductive output than other males, an observation not paralleled in the analysis of female reproductive output. The avoidance of males with highly dissimilar MHC would drive a process of local adaptation and an MHC splitting process between populations. A possible consequence of the preference for MHC intermediate males could therefore be reproductive isolation of populations over time. Such a process would be counteracted by the female preference for males with high general genomic dissimilarity.