Genomics-driven breeding for local adaptation of durum wheat is enhanced by farmers' traditional knowledge.

In the smallholder, low-input farming systems widespread in sub-Saharan Africa, farmers select and propagate crop varieties based on their traditional knowledge and experience. A data-driven integration of their knowledge into breeding pipelines may support the sustainable intensification of local farming. Here, we combine genomics with participatory research to tap into traditional knowledge in smallholder farming systems, using durum wheat (Triticum durum Desf.) in Ethiopia as a case study. We developed and genotyped a large multiparental population, called the Ethiopian NAM (EtNAM), that recombines an elite international breeding line with Ethiopian traditional varieties maintained by local farmers. A total of 1,200 EtNAM lines were evaluated for agronomic performance and farmers' appreciation in three locations in Ethiopia, finding that women and men farmers could skillfully identify the worth of wheat genotypes and their potential for local adaptation. We then trained a genomic selection (GS) model using farmer appreciation scores and found that its prediction accuracy over grain yield (GY) was higher than that of a benchmark GS model trained on GY. Finally, we used forward genetics approaches to identify marker-trait associations for agronomic traits and farmer appreciation scores. We produced genetic maps for individual EtNAM families and used them to support the characterization of genomic loci of breeding relevance with pleiotropic effects on phenology, yield, and farmer preference. Our data show that farmers' traditional knowledge can be integrated in genomics-driven breeding to support the selection of best allelic combinations for local adaptation.

The Mitogenome Relationships and Phylogeography of Barn Swallows (Hirundo rustica).

The barn swallow (Hirundo rustica) poses a number of fascinating scientific questions, including the taxonomic status of postulated subspecies. Here, we obtained and assessed the sequence variation of 411 complete mitogenomes, mainly from the European H. r. rustica, but other subspecies as well. In almost every case, we observed subspecies-specific haplogroups, which we employed together with estimated radiation times to postulate a model for the geographical and temporal worldwide spread of the species. The female barn swallow carrying the Hirundo rustica ancestral mitogenome left Africa (or its vicinity) around 280 thousand years ago (kya), and her descendants expanded first into Eurasia and then, at least 51 kya, into the Americas, from where a relatively recent (<20 kya) back migration to Asia took place. The exception to the haplogroup subspecies specificity is represented by the sedentary Levantine H. r. transitiva that extensively shares haplogroup A with the migratory European H. r. rustica and, to a lesser extent, haplogroup B with the Egyptian H. r. savignii. Our data indicate that rustica and transitiva most likely derive from a sedentary Levantine population source that split at the end of the Younger Dryas (YD) (11.7 kya). Since then, however, transitiva received genetic inputs from and admixed with both the closely related rustica and the adjacent savignii. Demographic analyses confirm this species' strong link with climate fluctuations and human activities making it an excellent indicator for monitoring and assessing the impact of current global changes on wildlife.

Evolution of an insect immune barrier through horizontal gene transfer mediated by a parasitic wasp.

Genome sequencing data have recently demonstrated that eukaryote evolution has been remarkably influenced by the acquisition of a large number of genes by horizontal gene transfer (HGT) across different kingdoms. However, in depth-studies on the physiological traits conferred by these accidental DNA acquisitions are largely lacking. Here we elucidate the functional role of Sl gasmin, a gene of a symbiotic virus of a parasitic wasp that has been transferred to an ancestor of the moth species Spodoptera littoralis and domesticated. This gene is highly expressed in circulating immune cells (haemocytes) of larval stages, where its transcription is rapidly boosted by injection of microorganisms into the body cavity. RNAi silencing of Sl gasmin generates a phenotype characterized by a precocious suppression of phagocytic activity by haemocytes, which is rescued when these immune cells are incubated in plasma samples of control larvae, containing high levels of the encoded protein. Proteomic analysis demonstrates that the protein Sl gasmin is released by haemocytes into the haemolymph, where it opsonizes the invading bacteria to promote their phagocytosis, both in vitro and in vivo. Our results show that important physiological traits do not necessarily originate from evolution of pre-existing genes, but can be acquired by HGT events, through unique pathways of symbiotic evolution. These findings indicate that insects can paradoxically acquire selective advantages with the help of their natural enemies.

Clock gene polymorphism, migratory behaviour and geographic distribution: a comparative study of trans-Saharan migratory birds.

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.

Polymorphism at the Clock gene predicts phenology of long-distance migration in birds.

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.

Targeting environmental adaptation in the monocot model Brachypodium distachyon: a multi-faceted approach.

BACKGROUND: The local environment plays a major role in the spatial distribution of plant populations. Natural plant populations have an extremely poor displacing capacity, so their continued survival in a given environment depends on how well they adapt to local pedoclimatic conditions. Genomic tools can be used to identify adaptive traits at a DNA level and to further our understanding of evolutionary processes. Here we report the use of genotyping-by-sequencing on local groups of the sequenced monocot model species Brachypodium distachyon. Exploiting population genetics, landscape genomics and genome wide association studies, we evaluate B. distachyon role as a natural probe for identifying genomic loci involved in environmental adaptation. RESULTS: Brachypodium distachyon individuals were sampled in nine locations with different ecologies and characterized with 16,697 SNPs. Variations in sequencing depth showed consistent patterns at 8,072 genomic bins, which were significantly enriched in transposable elements. We investigated the structuration and diversity of this collection, and exploited climatic data to identify loci with adaptive significance through i) two different approaches for genome wide association analyses considering climatic variation, ii) an outlier loci approach, and iii) a canonical correlation analysis on differentially sequenced bins. A linkage disequilibrium-corrected Bonferroni method was applied to filter associations. The two association methods jointly identified a set of 15 genes significantly related to environmental adaptation. The outlier loci approach revealed that 5.7% of the loci analysed were under selection. The canonical correlation analysis showed that the distribution of some differentially sequenced regions was associated to environmental variation. CONCLUSIONS: We show that the multi-faceted approach used here targeted different components of B. distachyon adaptive variation, and may lead to the discovery of genes related to environmental adaptation in natural populations. Its application to a model species with a fully sequenced genome is a modular strategy that enables the stratification of biological material and thus improves our knowledge of the functional loci determining adaptation in near-crop species. When coupled with population genetics and measures of genomic structuration, methods coming from genome wide association studies may lead to the exploitation of model species as natural probes to identify loci related to environmental adaptation.

Time trees and clock genes: a systematic review and comparative analysis of contemporary avian migration genetics.

Timing is a crucial aspect for survival and reproduction in seasonal environments leading to carefully scheduled annual programs of migration in many species. But what are the exact mechanisms through which birds (class: Aves) can keep track of time, anticipate seasonal changes, and adapt their behaviour? One proposed mechanism regulating annual behaviour is the circadian clock, controlled by a highly conserved set of genes, collectively called 'clock genes' which are well established in controlling the daily rhythmicity of physiology and behaviour. Due to diverse migration patterns observed within and among species, in a seemingly endogenously programmed manner, the field of migration genetics has sought and tested several candidate genes within the clock circuitry that may underlie the observed differences in breeding and migration behaviour. Among others, length polymorphisms within genes such as Clock and Adcyap1 have been hypothesised to play a putative role, although association and fitness studies in various species have yielded mixed results. To contextualise the existing body of data, here we conducted a systematic review of all published studies relating polymorphisms in clock genes to seasonality in a phylogenetically and taxonomically informed manner. This was complemented by a standardised comparative re-analysis of candidate gene polymorphisms of 76 bird species, of which 58 are migrants and 18 are residents, along with population genetics analyses for 40 species with available allele data. We tested genetic diversity estimates, used Mantel tests for spatial genetic analyses, and evaluated relationships between candidate gene allele length and population averages for geographic range (breeding- and non-breeding latitude), migration distance, timing of migration, taxonomic relationships, and divergence times. Our combined analysis provided evidence (i) of a putative association between Clock gene variation and autumn migration as well as a putative association between Adcyap1 gene variation and spring migration in migratory species; (ii) that these candidate genes are not diagnostic markers to distinguish migratory from sedentary birds; and (iii) of correlated variability in both genes with divergence time, potentially reflecting ancestrally inherited genotypes rather than contemporary changes driven by selection. These findings highlight a tentative association between these candidate genes and migration attributes as well as genetic constraints on evolutionary adaptation.

Birds of a feather flock together: a dataset for Clock and Adcyap1 genes from migration genetics studies.

Birds in seasonal habitats rely on intricate strategies for optimal timing of migrations. This is governed by environmental cues, including photoperiod. Genetic factors affecting intrinsic timekeeping mechanisms, such as circadian clock genes, have been explored, yielding inconsistent findings with potential lineage-dependency. To clarify this evidence, a systematic review and phylogenetic reanalysis was done. This descriptor outlines the methodology for sourcing, screening, and processing relevant literature and data. PRISMA guidelines were followed, ultimately including 66 studies, with 34 focusing on candidate genes at the genotype-phenotype interface. Studies were clustered using bibliographic coupling and citation network analysis, alongside scientometric analyses by publication year and location. Data was retrieved for allele data from databases, article supplements, and direct author communications. The dataset, version 1.0.2, encompasses data from 52 species, with 46 species for the Clock gene and 43 for the Adcyap1 gene. This dataset, featuring data from over 8000 birds, constitutes the most extensive cross-species collection for these candidate genes, used in studies investigating gene polymorphisms and seasonal bird migration.

A Chromosome-Level Reference Genome for the Black-Legged Kittiwake (Rissa tridactyla), a Declining Circumpolar Seabird.

Amidst the current biodiversity crisis, the availability of genomic resources for declining species can provide important insights into the factors driving population decline. In the early 1990s, the black-legged kittiwake (Rissa tridactyla), a pelagic gull widely distributed across the arctic, subarctic, and temperate zones, suffered a steep population decline following an abrupt warming of sea surface temperature across its distribution range and is currently listed as Vulnerable by the International Union for the Conservation of Nature. Kittiwakes have long been the focus for field studies of physiology, ecology, and ecotoxicology and are primary indicators of fluctuating ecological conditions in arctic and subarctic marine ecosystems. We present a high-quality chromosome-level reference genome and annotation for the black-legged kittiwake using a combination of Pacific Biosciences HiFi sequencing, Bionano optical maps, Hi-C reads, and RNA-Seq data. The final assembly spans 1.35 Gb across 32 chromosomes, with a scaffold N50 of 88.21 Mb and a BUSCO completeness of 97.4%. This genome assembly substantially improves the quality of a previous draft genome, showing an approximately 5× increase in contiguity and a more complete annotation. Using this new chromosome-level reference genome and three more chromosome-level assemblies of Charadriiformes, we uncover several lineage-specific chromosome fusions and fissions, but find no shared rearrangements, suggesting that interchromosomal rearrangements have been commonplace throughout the diversification of Charadriiformes. This new high-quality genome assembly will enable population genomic, transcriptomic, and phenotype-genotype association studies in a widely studied sentinel species, which may provide important insights into the impacts of global change on marine systems.

A chromosome-level reference genome and pangenome for barn swallow population genomics.

Insights into the evolution of non-model organisms are limited by the lack of reference genomes of high accuracy, completeness, and contiguity. Here, we present a chromosome-level, karyotype-validated reference genome and pangenome for the barn swallow (Hirundo rustica). We complement these resources with a reference-free multialignment of the reference genome with other bird genomes and with the most comprehensive catalog of genetic markers for the barn swallow. We identify potentially conserved and accelerated genes using the multialignment and estimate genome-wide linkage disequilibrium using the catalog. We use the pangenome to infer core and accessory genes and to detect variants using it as a reference. Overall, these resources will foster population genomics studies in the barn swallow, enable detection of candidate genes in comparative genomics studies, and help reduce bias toward a single reference genome.

Use of homologous and heterologous gene expression profiling tools to characterize transcription dynamics during apple fruit maturation and ripening.

BACKGROUND: Fruit development, maturation and ripening consists of a complex series of biochemical and physiological changes that in climacteric fruits, including apple and tomato, are coordinated by the gaseous hormone ethylene. These changes lead to final fruit quality and understanding of the functional machinery underlying these processes is of both biological and practical importance. To date many reports have been made on the analysis of gene expression in apple. In this study we focused our investigation on the role of ethylene during apple maturation, specifically comparing transcriptomics of normal ripening with changes resulting from application of the hormone receptor competitor 1-methylcyclopropene. RESULTS: To gain insight into the molecular process regulating ripening in apple, and to compare to tomato (model species for ripening studies), we utilized both homologous and heterologous (tomato) microarray to profile transcriptome dynamics of genes involved in fruit development and ripening, emphasizing those which are ethylene regulated.The use of both types of microarrays facilitated transcriptome comparison between apple and tomato (for the later using data previously published and available at the TED: tomato expression database) and highlighted genes conserved during ripening of both species, which in turn represent a foundation for further comparative genomic studies. The cross-species analysis had the secondary aim of examining the efficiency of heterologous (specifically tomato) microarray hybridization for candidate gene identification as related to the ripening process. The resulting transcriptomics data revealed coordinated gene expression during fruit ripening of a subset of ripening-related and ethylene responsive genes, further facilitating the analysis of ethylene response during fruit maturation and ripening. CONCLUSION: Our combined strategy based on microarray hybridization enabled transcriptome characterization during normal climacteric apple ripening, as well as definition of ethylene-dependent transcriptome changes. Comparison with tomato fruit maturation and ethylene responsive transcriptome activity facilitated identification of putative conserved orthologous ripening-related genes, which serve as an initial set of candidates for assessing conservation of gene activity across genomes of fruit bearing plant species.

Long walk to genomics: History and current approaches to genome sequencing and assembly.

Genomes represent the starting point of genetic studies. Since the discovery of DNA structure, scientists have devoted great efforts to determine their sequence in an exact way. In this review we provide a comprehensive historical background of the improvements in DNA sequencing technologies that have accompanied the major milestones in genome sequencing and assembly, ranging from early sequencing methods to Next-Generation Sequencing platforms. We then focus on the advantages and challenges of the current technologies and approaches, collectively known as Third Generation Sequencing. As these technical advancements have been accompanied by progress in analytical methods, we also review the bioinformatic tools currently employed in de novo genome assembly, as well as some applications of Third Generation Sequencing technologies and high-quality reference genomes.

Manual Sampling and Video Observations: An Integrated Approach to Studying Flower-Visiting Arthropods in High-Mountain Environments.

Despite the rising interest in biotic interactions in mountain ecosystems, little is known about high-altitude flower-visiting arthropods. In particular, since the research in these environment can be limited or undermined by harsh conditions and logistical difficulties, it is mandatory to develop effective approaches that maximize possibilities to gather high-quality data. Here we compared two different methods, manual sampling and video observations, to investigate the interactions between the high-mountain arthropod community and flowers of Androsace brevis (Primulaceae), a vulnerable endemic alpine species with a short flowering period occurring in early season. We manually sampled flower-visiting arthropods according to the timed-observations method and recorded their activity on video. We assessed differences and effectiveness of the two approaches to estimate flower-visiting arthropod diversity and to identify potential taxa involved in A. brevis pollination. Both methods proved to be effective and comparable in describing the diversity of flower visitors at a high taxonomic level. However, with manual sampling we were able to obtain a fine taxonomic resolution for sampled arthropods and to evaluate which taxa actually carry A. brevis pollen, while video observations were less invasive and allowed us to assess arthropod behavior and to spot rare taxa. By combining the data obtained with these two approaches we could accurately identify flower-visiting arthropods, characterize their behavior, and hypothesize a role of Hymenoptera Apoidea and Diptera Brachycera in A. brevis pollination. Therefore, we propose integrating the two approaches as a powerful instrument to unravel interactions between flowering plants and associated fauna that can provide crucial information for the conservation of vulnerable environments such as high-mountain ecosystems.

SMRT long reads and Direct Label and Stain optical maps allow the generation of a high-quality genome assembly for the European barn swallow (Hirundo rustica rustica).

Background: The barn swallow (Hirundo rustica) is a migratory bird that has been the focus of a large number of ecological, behavioral, and genetic studies. To facilitate further population genetics and genomic studies, we present a reference genome assembly for the European subspecies (H. r. rustica). Findings: As part of the Genome10K effort on generating high-quality vertebrate genomes (Vertebrate Genomes Project), we have assembled a highly contiguous genome assembly using single molecule real-time (SMRT) DNA sequencing and several Bionano optical map technologies. We compared and integrated optical maps derived from both the Nick, Label, Repair, and Stain technology and from the Direct Label and Stain (DLS) technology. As proposed by Bionano, DLS more than doubled the scaffold N50 with respect to the nickase. The dual enzyme hybrid scaffold led to a further marginal increase in scaffold N50 and an overall increase of confidence in the scaffolds. After removal of haplotigs, the final assembly is approximately 1.21 Gbp in size, with a scaffold N50 value of more than 25.95 Mbp. Conclusions: This high-quality genome assembly represents a valuable resource for future studies of population genetics and genomics in the barn swallow and for studies concerning the evolution of avian genomes. It also represents one of the very first genomes assembled by combining SMRT long-read sequencing with the new Bionano DLS technology for scaffolding. The quality of this assembly demonstrates the potential of this methodology to substantially increase the contiguity of genome assemblies.

Inter-generational resemblance of methylation levels at circadian genes and associations with phenology in the barn swallow.

Regulation of gene expression can occur via epigenetic effects as mediated by DNA methylation. The potential for epigenetic effects to be transmitted across generations, thus modulating phenotypic variation and affecting ecological and evolutionary processes, is increasingly appreciated. However, the study of variation in epigenomes and inter-generational transmission of epigenetic alterations in wild populations is at its very infancy. We studied sex- and age-related variation in DNA methylation and parent-offspring resemblance in methylation profiles in the barn swallows. We focused on a class of highly conserved 'clock' genes (clock, cry1, per2, per3, timeless) relevant in the timing of activities of major ecological importance. In addition, we considerably expanded previous analyses on the relationship between methylation at clock genes and breeding date, a key fitness trait in barn swallows. We found positive assortative mating for methylation at one clock locus. Methylation varied between the nestling and the adult stage, and according to sex. Individuals with relatively high methylation as nestlings also had high methylation levels when adults. Extensive parent-nestling resemblance in methylation levels was observed. Occurrence of extra-pair fertilizations allowed to disclose evidence hinting at a prevalence of paternal germline or sperm quality effects over common environment effects in generating father-offspring resemblance in methylation. Finally, we found an association between methylation at the clock poly-Q region, but not at other loci, and breeding date. We thus provided evidence for sex-dependent variation and the first account of parent-offspring resemblance in methylation in any wild vertebrate. We also showed that epigenetics may influence phenotypic plasticity of timing of life cycle events, thus having a major impact on fitness.

Classical genetic and quantitative trait loci analyses of heterosis in a maize hybrid between two elite inbred lines.

The exploitation of heterosis is one of the most outstanding advancements in plant breeding, although its genetic basis is not well understood yet. This research was conducted on the materials arising from the maize single cross B73 x H99 to study heterosis by procedures of classical genetic and quantitative trait loci (QTL) analyses. Materials were the basic generations, the derived 142 recombinant inbred lines (RILs), and the three testcross populations obtained by crossing the 142 RILs to each parent and their F(1). For seedling weight (SW), number of kernels per plant (NK), and grain yield (GY), heterosis was >100% and the average degree of dominance was >1. Epistasis was significant for SW and NK but not for GY. Several QTL were identified and in most cases they were in the additive-dominance range for traits with low heterosis and mostly in the dominance-overdominance range for plant height (PH), SW, NK, and GY. Only a few QTL with digenic epistasis were identified. The importance of dominance effects was confirmed by highly significant correlations between heterozygosity level and phenotypic performance, especially for GY. Some chromosome regions presented overlaps of overdominant QTL for SW, PH, NK, and GY, suggesting pleiotropic effects on overall plant vigor.

Candidate genes have sex-specific effects on timing of spring migration and moult speed in a long-distance migratory bird.

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.

Lifetime reproductive success, selection on lifespan, and multiple sexual ornaments in male European barn swallows.

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.

Methylation of the circadian Clock gene in the offspring of a free-living passerine bird increases with maternal and individual exposure to PM10.

The consequences of exposure to particulate matter (PM) have been thoroughly investigated in humans and other model species, but there is a dearth of studies of the effects of PM on physiology and life-history traits of non-human organisms living in natural or semi-natural environments. Besides toxicological relevance, PM has been recently suggested to exert epigenetic effects by altering DNA methylation patterns. Here, we investigated for the first time the association between the exposure to free-air PM10 and DNA methylation at two loci ('poly-Q exon' and '5'-UTR') of the Clock gene in blood cells of the nestlings of a synanthropic passerine bird, the barn swallow (Hirundo rustica). The Clock gene is a phylogenetically highly conserved gene playing a major role in governing circadian rhythms and circannual life cycles of animals, implying that change in its level of methylation can impact on important fitness traits. We found that methylation at both loci significantly increased with PM10 levels recorded few days before blood sampling, and also with PM10 exposure experienced by the mother during or shortly before egg laying. This study is the first where methylation at a functionally important gene has been shown to vary according to the concentration of anthropogenic pollutants in any animal species in the wild. Since early-life environmental conditions produce epigenetic effects that can transgenerationally be transmitted, DNA methylation of genes controlling photoperiodic response can have far reaching consequences for the ecology and the evolution of wild animal populations.