Genes of the major histocompatibility complex (MHC) are essential in vertebrate adaptive immunity, and they are highly diverse and duplicated in many lineages. While it is widely established that pathogen‐mediated selection maintains MHC diversity through balancing selection, the role of mate choice in shaping MHC diversity is debated. Here, we investigate female mating preferences for MHC class II (MHCII) in the bluethroat (Luscinia svecica), a passerine bird with high levels of extra‐pair paternity and extremely duplicated MHCII. We genotyped family samples with mixed brood paternity and categorized their MHCII alleles according to their functional properties in peptide binding. Our results strongly indicate that females select extra‐pair males in a nonrandom, self‐matching manner that provides offspring with an allelic repertoire size closer to the population mean, as compared to offspring sired by the social male. This is consistent with a compatible genes model for extra‐pair mate choice where the optimal allelic diversity is intermediate, not maximal. This golden mean presumably reflects a trade‐off between maximizing pathogen recognition benefits and minimizing autoimmunity costs. Our study exemplifies how mate choice can reduce the population variance in individual MHC diversity and exert strong stabilizing selection on the trait. It also supports the hypothesis that extra‐pair mating is adaptive through altered genetic constitution in offspring.

Genotyping of classical major histocompatibility complex (MHC) genes is challenging when they are hypervariable and occur in multiple copies. In this study, we used several different approaches to genotype the moderately variable MHC class I exon 3 (MHCIe3) and the highly polymorphic MHC class II exon 2 (MHCIIβe2) in the bluethroat (Luscinia svecica). Two family groups (eight individuals) were sequenced in replicates at both markers using Ion Torrent technology with both a single‐ and a dual‐indexed primer structure. Additionally, MHCIIβe2 was sequenced on Illumina MiSeq. Allele calling was conducted by modifications of the pipeline developed by Sommer et al. (BMC Genomics, 14, 2013, 542) and the software AmpliSAS. While the different genotyping strategies gave largely consistent results for MHCIe3, with a maximum of eight alleles per individual, MHCIIβe2 was remarkably complex with a maximum of 56 MHCIIβe2 alleles called for one individual. Each genotyping strategy detected on average 50%–82% of all MHCIIβe2 alleles per individual, but dropouts were largely allele‐specific and consistent within families for each strategy. The discrepancies among approaches indicate PCR biases caused by the platform‐specific primer tails. Further, AmpliSAS called fewer alleles than the modified Sommer pipeline. Our results demonstrate that allelic dropout is a significant problem when genotyping the hypervariable MHCIIβe2. As these genotyping errors are largely nonrandom and method‐specific, we caution against comparing genotypes across different genotyping strategies. Nevertheless, we conclude that high‐throughput approaches provide a major advance in the challenging task of genotyping hypervariable MHC loci, even though they may not reveal the complete allelic repertoire.

We contrast two methods for estimating the trends of bowhead whales (Balaena mysticetus) in West Greenland: (1) double platform visual aerial survey, corrected for missed sightings and the time the whales are available at the surface; and (2) a genetic capture-recapture approach based on a 14-yr-long biopsy sampling program in Disko Bay. The aerial survey covered 39,000 km 2 and resulted in 58 sightings, yielding an abundance estimate of 744 whales (CV = 0.34, 95% CI: 357-1,461). The genetic method relied on determining sex, mitochondrial haplotypes and genotypes of nine microsatellite markers. Based on samples from a total of 427 individuals, with 11 recaptures from previous years in 2013, this resulted in an estimate of 1,538 whales (CV = 0.24, 95% CI: 827-2,249). While the aerial survey is considered a snapshot of the local spring aggregation in Disko Bay, the genetic approach estimates the abundance of the source of this aggregation. As the whales in Disko Bay primarily are adult females that do not visit the bay annually, the genetic method would presumably yield higher estimates. The studies indicate that an increase in abundance observed between 1998 and 2006 has leveled off.

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Sperm morphology is highly diversified among species and at higher taxonomic levels. In birds, there is also increasing evidence of geographical differentiation in sperm traits within species, especially in those with strong sperm competition. Geographical divergences in sperm traits might imply the formation of a reproductive barrier in a speciation process. Here we study sperm morphology variation of willow warblers Phylloscopus trochilus in a geographical context in Scandinavia, across the range of two subspecies that are differentiated in certain genetic markers, morphology and migratory routes. We describe geographical patterns in genotypes (two previously described single‐nucleotide polymorphism (SNP) markers and four polymorphic microsatellites); in wing length, tarsus length and body mass; and in sperm traits of 330 male willow warblers sampled at 33 localities across Norway (58°N–69°N). Birds were on average larger and longer‐winged in the north (spp. acredula) than in the south (spp. trochilus), and showed a sigmoid change in the SNP allele frequencies and body morphology around 65°N. We found no evidence of genetic structuring in the microsatellites. There was no geographical variation in sperm traits across Norway, except that sperm heads were on average longer in the south. Sperm head length was also associated with the two SNP markers, with longer sperm heads for the southern alleles, and midpiece length was weakly associated with one of the SNP markers. Similar among‐male variances in total sperm length among the 33 sampling sites indicate uniform levels of sperm competition across Norway. We conclude that sperm morphology remains a rather undifferentiated trait between the two willow warbler subspecies in Scandinavia, which is consistent with a pattern of a shallow genetic divergence. This indicates that sperm morphology is not a reproductive barrier maintaining the narrow hybrid zone.

Genes of the major histocompatibility complex (MHC) are crucial for adaptive immunity, and theory predicts that the optimal number of alleles is intermediate rather than maximal. Support for such an intermediate optimum in MHC allele count has mainly been demonstrated in studies on parasite load and reproductive traits. In this study we tested if an intermediate number of MHC class II (MHCII) alleles based on positively selected sites was correlated with an increased skin-swelling response to two consecutive injections of phytohaemagglutinin (PHA) in bluethroat Luscinia svecica nestlings. We found that nestlings that were closer to the population mean in their number of alleles had a higher response to the first injection, supporting an intermediate, optimal number of alleles. After the second PHA injection, we found that the skin swelling was associated with MHCII allele count only at the among-brood level, and with paternity (extra-pair offspring had a larger swelling than within-pair offspring). We therefore conclude that the PHA response is partly correlated with MHCII allele count, but that additional factors must be involved in both the PHA response and in female extra-pair mate choice in this species.

Genes of the major histocompatibility complex (MHC) are crucial for adaptive immunity in jawed vertebrates, and theory predicts that there should be mate choice for optimizing MHC constitution in the offspring. In a previous study, we demonstrated a non-random female choice of extra-pair males in the bluethroat (Luscinia svecica), yielding offspring that was closer to an intermediate MHC class II (MHCII) allele count than their within-pair halfsiblings. The present study tests whether social pairs with only within-pair young (WPY) in their brood, in the same study population, had a combined MHC-constitution closer to a presumed intermediate optimum, than social pairs with extra-pair young (EPY), with a corresponding pattern in their offspring. As expected, we found that WPY from pure WPY-broods were more MHC-optimal than WPY from mixed broods, but only in broods of young (second year) males. Correspondingly, there was a tendency for social pairs with only WPY in their brood to be more MHC-compatible than social pairs with EPY in their brood, when the male was young. Older bluethroat males have considerably larger testes than young males, and their higher sperm competitiveness could help them secure paternity in their own brood, also when they are not MHC-compatible. In other words, in the sexual conflict over paternity, females may be more likely to realise their preference for a MHC-compatible mate when paired to a young male. As a possible fitness indicator, immune responsiveness to an injected antigen (PHA) was elevated for offspring closer to “the golden mean” in MHCII allele count. Significance statement This study contributes to our understanding of MHC-based mate choice in extra-pair mating systems, by showing that female bluethroats (Luscinia svecica) with an MHCII-compatible social mate tend to have no extra-pair young in their brood, but only when the social male is young. This elucidates a possible sexual conflict, in which older social males are able to override female preferences and prevent other males from gaining paternity in their brood through higher sperm production. Studying systems in which extra-pair paternity occurs offers an insight into the genetic benefits of mate choice, as extra-pair males, in contrast to social males, generally contribute only sperm. Further, the strict and thorough genotyping scheme applied in this study enabled us to demonstrate a preference for “the golden mean” in MHC-diversity in a species with one of the highest MHC class II-diversity known to date.

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