BackgroundIn diverse taxa, photoperiodic responses that cause seasonal physiological and behavioural shifts are controlled by genes, including the vertebrate Clock orthologues, that encode for circadian oscillator mechanisms. While the genetic network behind circadian rhythms is well described, relatively few reports exist of the phenological consequences of and selection on Clock genes in the wild. Here, we investigated variation in breeding phenology in relation to Clock genetic diversity in a long-distance migratory bird, the barn swallow (Hirundo rustica).Methodology/Principal FindingsIn a sample of 922 adult barn swallows from a single population breeding in Italy we found one very common (Q7) and three rare (Q5, Q6, Q8) length variants of a functionally significant polyglutamine repeat. Rare (2.9%) Q7/Q8 heterozygous females, but not males, bred significantly later than common (91.5%) Q7/Q7 females, consistent with the expectation that ‘long’ alleles cause late breeding, as observed in a resident population of another bird species. Because breeding date depends on arrival date from migration, present results suggest that the association between breeding date and Clock might be mediated by migration phenology. In addition, fecundity selection appears to be operating against Q7/Q8 because late migrating/breeding swallows have fewer clutches per season, and late breeding has additional negative selection effects via reduced offspring longevity. Genotype frequencies varied marginally non-significantly with age, as Q7/Q8 frequency showed a 4-fold reduction in old individuals. This result suggests negative viability selection against Q7/Q8, possibly mediated by costs of late breeding.Conclusions/SignificanceThis is the first study of migratory birds showing an association between breeding phenology and Clock genotype and suggesting that negative selection occurs on a phenologically deviant genotype. Low polymorphism at Clock may constrain microevolutionary phenological response to changing climate, and may thus contribute to the decline of barn swallow populations.

Dissecting phenotypic variance in life history traits into its genetic and environmental components is at the focus of evolutionary studies and of pivotal importance to identify the mechanisms and predict the consequences of human-driven environmental change. The timing of recurrent life history events (phenology) is under strong selection, but the study of the genes that control potential environmental canalization in phenological traits is at its infancy. Candidate genes for circadian behaviour entrained by photoperiod have been screened as potential controllers of phenological variation of breeding and moult in birds, with inconsistent results. Despite photoperiodic control of migration is well established, no study has reported on migration phenology in relation to polymorphism at candidate genes in birds. We analysed variation in spring migration dates within four trans-Saharan migratory species (Luscinia megarhynchos; Ficedula hypoleuca; Anthus trivialis; Saxicola rubetra) at a Mediterranean island in relation to Clock and Adcyap1 polymorphism. Individuals with larger number of glutamine residues in the poly-Q region of Clock gene migrated significantly later in one or, respectively, two species depending on sex and whether the within-individual mean length or the length of the longer Clock allele was considered. The results hinted at dominance of the longer Clock allele. No significant evidence for migration date to covary with Adcyap1 polymorphism emerged. This is the first evidence that migration phenology is associated with Clock in birds. This finding is important for evolutionary studies of migration and sheds light on the mechanisms that drive bird phenological changes and population trends in response to climate change.

Circannual rhythms often rely on endogenous seasonal photoperiodic timers involving ‘clock’ genes, and Clock gene polymorphism has been associated to variation in phenology in some bird species. In the long-distance migratory barn swallow Hirundo rustica, individuals bearing the rare Clock allele with the largest number of C-terminal polyglutamine repeats found in this species (Q8) show a delayed reproduction and moult later. We explored the association between Clock polymorphism and migration scheduling, as gauged by light-level geolocators, in two barn swallow populations (Switzerland; Po Plain, Italy). Genetic polymorphism was low: 91% of the 64 individuals tracked year-round were Q7/Q7 homozygotes. We compared the phenology of the rare genotypes with the phenotypic distribution of Q7/Q7 homozygotes within each population. In Switzerland, compared to Q7/Q7, two Q6/Q7 males departed earlier from the wintering grounds and arrived earlier to their colony in spring, while a single Q7/Q8 female was delayed for both phenophases. On the other hand, in the Po Plain, three Q6/Q7 individuals had a similar phenology compared to Q7/Q7. The Swiss data are suggestive for a role of genetic polymorphism at a candidate phenological gene in shaping migration traits, and support the idea that Clock polymorphism underlies phenological variation in birds.

Individuals often considerably differ in the timing of their life-cycle events, with major consequences for individual fitness, and, ultimately, for population dynamics. Phenological variation can arise from genetic effects but also from epigenetic modifications in DNA expression and translation. Here, we tested if CpG methylation at the poly-Q and 5′-UTR loci of the photoperiodic Clock gene predicted migration and breeding phenology of long-distance migratory barn swallows (Hirundo rustica) that were tracked year-round using light-level geolocators. Increasing methylation at Clock poly-Q was associated with earlier spring departure from the African wintering area, arrival date at the European breeding site, and breeding date. Higher methylation levels also predicted increased breeding success. Thus, we showed for the first time in any species that CpG methylation at a candidate gene may affect phenology and breeding performance. Methylation at Clock may be a candidate mechanism mediating phenological responses of migratory birds to ongoing climate change.

Migratory behaviour is controlled by endogenous circannual rhythms that are synchronized by external cues, such as photoperiod. Investigations on the genetic basis of circannual rhythmicity in vertebrates have highlighted that variation at candidate 'circadian clock' genes may play a major role in regulating photoperiodic responses and timing of life cycle events, such as reproduction and migration. In this comparative study of 23 trans-Saharan migratory bird species, we investigated the relationships between species-level genetic variation at two candidate genes, Clock and Adcyap1, and species' traits related to migration and geographic distribution, including timing of spring migration across the Mediterranean Sea, migration distance and breeding latitude. Consistently with previous evidence showing latitudinal clines in 'circadian clock' genotype frequencies, Clock allele size increased with breeding latitude across species. However, early- and late-migrating species had similar Clock allele size. Species migrating over longer distances, showing delayed spring migration and smaller phenotypic variance in spring migration timing, had significantly reduced Clock (but not Adcyap1) gene diversity. Phylogenetic confirmatory path analysis suggested that migration date and distance were the most important variables directly affecting Clock gene diversity. Hence, our study supports the hypothesis that Clock allele size increases poleward as a consequence of adaptation to the photoperiodic regime of the breeding areas. Moreover, we show that long-distance migration is associated with lower Clock diversity, coherently with strong stabilizing selection acting on timing of life cycle events in long-distance migratory species, likely resulting from the time constraints imposed by late spring migration.

The timing of major life-history events, such as migration and moult, is set by endogenous circadian and circannual clocks, that have been well characterized at the molecular level. Conversely, the genetic sources of variation in phenology and in other behavioral traits have been sparsely addressed. It has been proposed that inter-individual variability in the timing of seasonal events may arise from allelic polymorphism at phenological candidate genes involved in the signaling cascade of the endogenous clocks. In this study of a long-distance migratory passerine bird, the willow warbler Phylloscopus trochilus, we investigated whether allelic variation at 5 polymorphic loci of 4 candidate genes (Adcyap1, Clock, Creb1, and Npas2), predicted 2 major components of the annual schedule, namely timing of spring migration across the central Mediterranean sea and moult speed, the latter gauged from ptilochronological analyses of tail feathers moulted in the African winter quarters. We identified a novel Clock gene locus (Clock region 3) showing polyQ polymorphism, which was however not significantly associated with any phenotypic trait. Npas2 allele size predicted male (but not female) spring migration date, with males bearing longer alleles migrating significantly earlier than those bearing shorter alleles. Creb1 allele size significantly predicted male (but not female) moult speed, longer alleles being associated with faster moult. All other genotype–phenotype associations were statistically non-significant. These findings provide new evidence for a role of candidate genes in modulating the phenology of different circannual activities in long-distance migratory birds, and for the occurrence of sex-specific candidate gene effects.

Natural and sexual selection arise when individual fitness varies according to focal traits. Extra-pair paternities (EPPs) can affect the intensity of selection by influencing variance in fitness among individuals. Studies of selection require that individual fitness is estimated using proxies of lifetime reproductive success (LRS). However, estimating LRS is difficult in large, open populations where EPPs cause reallocation of biological paternity. Here, we used extensive field sampling to estimate LRS in a population of barn swallows (Hirundo rustica) to estimate selection on lifespan and ornamental traits of males. We found selection on lifespan mediated both by within- and extra-pair fertilization success and selection on tail length mediated by within- but not extra-pair fertilization success. In addition, we found selection on tail white spots via extra-pair fertilization success after controlling for selection on other traits. These results were not confounded by factors that hamper studies of LRS, including nonexhaustive sampling of offspring and biased sampling of males. Hence, natural and sexual selection mediated by LRS operates on lifespan, tail length, and size of the tail white spots in barn swallows.

On the River Ticino in northern Italy, a small number of captive Eurasian otters Lutra lutra, belonging to the European breeding programme for self-sustaining captive populations, were reintroduced in 1997, after the species had been declared locally extinct in the 1980s. We surveyed for otter signs in 2008, 2010, 2016–2017 and 2018, confirming the presence of what is probably a small population. To assess the abundance and viability of the population, we genotyped fresh spraints collected during the last two surveys, using 11 microsatellite markers, and modelled the population trend using Vortex. A minimum of six individuals were identified from 25 faecal samples. The analysis of mitochondrial DNA determined that the reintroduced otters share a transversion that is characteristic of the Asiatic subspecies Lutra lutra barang, confirming the contribution of the Asiatic subspecies to the genetic pool of the captive-bred founder population. Population size was consistent with the release of three pairs of otters and all models implied that the number of founders was too small to ensure the long-term survival of the population. Stochastic factors are therefore likely to threaten the success of this reintroduction.