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.

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.

Telomere shortening is associated with corticosterone stress response in adult barn swallows.

When vertebrates face stressful events, the hypothalamic-pituitary-adrenal (HPA) axis is activated, generating a rapid increase in circulating glucocorticoid (GC) stress hormones followed by a return to baseline levels. However, repeated activation of HPA axis may lead to increase in oxidative stress. One target of oxidative stress is telomeres, nucleoprotein complexes at the end of chromosomes that shorten at each cell division. The susceptibility of telomeres to oxidizing molecules has led to the hypothesis that increased GC levels boost telomere shortening, but studies on this link are scanty. We studied if, in barn swallows Hirundo rustica, changes in adult erythrocyte telomere length between 2 consecutive breeding seasons are related to corticosterone (CORT) (the main avian GC) stress response induced by a standard capture-restraint protocol. Within-individual telomere length did not significantly change between consecutive breeding seasons. Second-year individuals showed the highest increase in circulating CORT concentrations following restraint. Moreover, we found a decline in female stress response along the breeding season. In addition, telomere shortening covaried with the stress response: a delayed activation of the negative feedback loop terminating the stress response was associated with greater telomere attrition. Hence, among-individual variation in stress response may affect telomere dynamics.

Prenatal yolk corticosterone exposure promotes skeletal growth and induces oxidative imbalance in yellow-legged gull embryos.

Maternally derived hormones induce variation in offspring phenotype, with consequences that can carry over into post-natal life and even into adulthood. In birds, maternal egg corticosterone (CORT) is known to exert contrasting effects on offspring morphology, physiology and behaviour after hatching. However, information on the effects of CORT exposure on pre-hatching embryonic development is limited. We experimentally increased yolk CORT levels in yellow-legged gull (Larus michahellis) eggs, and assessed the effects on embryo pre-hatching development and oxidative status of brain and liver. CORT-supplemented embryos reached a larger skeletal size and liver mass compared with controls. Embryos from CORT-injected last-laid eggs showed decreased activity of the hepatic antioxidant enzymes superoxide dismutase and catalase, while intermediate-laid eggs showed increased levels of lipid peroxidation. However, elevated yolk CORT did not affect oxidative stress endpoints in the brain. Our results indicate that elevated yolk CORT levels affect prenatal embryo development by promoting skeletal growth, and induce laying sequence- and organ-specific oxidative imbalance, with potential adverse consequences during postnatal life, especially for late-hatched offspring.

Prenatal exposure to triclosan induced brain telomere shortening in a wild bird species.

Exposure to the antimicrobial agent Triclosan (TCS) induces oxidative stress in diverse organisms, including birds. However, whether TCS-induced oxidative stress effectively translates into detrimental effects is still unclear. The present study examined whether prenatal TCS exposure induces oxidative stress and telomere shortening in the brain and the liver of near-term embryos of the yellow-legged gull (Larus michahellis). Prenatal TCS exposure caused a significant overproduction of reactive oxygen species (ROS) in the brain, but no oxidative damage occurred. Telomeres of TCS-exposed embryos had brain telomeres 30 % shorter compared to controls, probably because the relatively modest antioxidant defenses of this organ during prenatal development cannot counteract the impact of the TCS-induced ROS. No telomere shortening was observed in the liver. Our results demonstrated that prenatal exposure to TCS in wild bird species can modulate the oxidative status and induce telomere shortening in the brain of the yellow-legged gull embryos.

The genome sequence of the European nightjar, Caprimulgus europaeus (Linnaeus, 1758).

We present a genome assembly from an individual female Caprimulgus europaeus (the European nightjar; Chordata; Aves; Caprimulgiformes; Caprimulgidae). The genome sequence is 1,178 megabases in span. The majority of the assembly (99.33%) is scaffolded into 37 chromosomal pseudomolecules, including the W and Z sex chromosomes.

Trace elements fingerprint of feathers differs between breeding and non-breeding areas in an Afro-Palearctic migratory bird, the barn swallow (Hirundo rustica).

Trace elements are widespread contaminants that can potentially threaten ecosystems and human health. Considering their distribution and toxicity, monitoring their presence in animals represents a priority in environmental risk assessment. Migratory birds have been suggested to be useful biomonitors for trace elements because they can provide information on contaminants even from remote areas that they may exploit during their life cycle. The aim of this study was to analyse the contamination fingerprint of trace elements of African non-breeding staging grounds and European breeding areas in a long-distance migratory passerine bird, the barn swallow (Hirundo rustica). We collected feathers grown in the African non-breeding grounds and those grown in the breeding areas of Northern Italy and measured the levels of 12 trace elements (Al, As, Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se and Zn) by DRC-ICP-MS. Multivariate analysis showed that elemental profiles of feathers grown in African non-breeding areas and in the Italian breeding ones clearly differed, with feathers grown in Africa showing higher concentrations of Al, Cu, Fe, Mn and Ni, but lower concentrations of As, Se and Zn, compared to those grown in Italy. In addition, levels of trace elements were age-dependent, with higher levels in older individuals than in younger ones. Our results add to the growing evidence that feathers of long-distance migratory birds are useful tools to monitor trace elements contamination profiles across continents.

Perinatal variation and covariation of oxidative status and telomere length in yellow-legged gull chicks.

The perinatal period is critical to survival and performance of many organisms. In birds, rapid postnatal growth and sudden exposure to aerial oxygen around hatching markedly affect the chick redox status, with potentially negative consequences on physiology mediated by oxidative stress. In addition, telomere length (TL) undergoes reduction during birds' early life, partly depending on oxidative status. However, relatively few studies have focused specifically on the changes in oxidative status and TL that occur immediately after hatching. In this study of the yellow-legged gull Larus michahellis, we found that chicks undergo a marked increase in plasma total antioxidant capacity and a marked decrease in the concentration of pro-oxidant molecules during the first days after hatching. In addition, TL in erythrocytes decreased by 1 standard deviation over the 4 days post-hatching. Body mass and tarsus length covaried with total antioxidant capacity and concentration of pro-oxidants in a complex way, that partly depended on sex and laying order, suggesting that oxidative status can affect growth. Moreover, TL positively covaried with the concentration of pro-oxidant molecules, possibly because retention of high concentrations of pro-oxidant molecules results from mechanisms of prevention of their negative effects, including reduction in TL. Thus, this study shows that chicks undergo marked variation in oxidative status, which predicts growth and subsequent TL, prompting for more studies of the perinatal changes in the critical post-hatching stages.

Predation risk affects egg mass but not egg steroid hormone concentrations in yellow-legged gulls.

Predators have both direct, consumptive effects on their prey and non-lethal effects on physiology and behavior, including reproductive decisions, with cascading effects on prey ecology and evolution. Here, we experimentally tested such non-lethal effects of exposure to increased predation risk on clutch size, egg mass, and the concentration of yolk steroid hormones in the yellow-legged gull Larus michahellis. We simulated increased predation risk by displaying stuffed predators (adult fox Vulpes vulpes, and adult buzzard Buteo buteo) to breeding adults before egg laying. The concentration of corticosterone, which has been shown to increase under exposure to maternal predation risk in other species, and of testosterone did not differ between eggs from mothers exposed to the predators and eggs from control mothers (i.e., eggs exposed to a novel object of similar size and position to the stuffed predators). The concentration of the two hormones negatively covaried. Clutch size did not vary according to experimental treatment, whereas egg mass was markedly larger in clutches from nests exposed to predators than in clutches from control nests. By increasing egg mass, mothers may reduce the risk of cooling of the eggs when incubation is impeded by predators, boost energy reserves, reduce post-natal detectability caused by food solicitation, and/or enhance development at hatching, thus increasing the chances of offspring survival. In general, our results are inconsistent with most of the few previous studies on similar non-lethal predator effects and suggest that such effects may vary among species according to ecological conditions, social behavior, and developmental mode.

Egg Testosterone Differentially Affects Telomere Length in Somatic Tissues of Yellow-Legged Gull Embryos.

Maternal decisions on egg composition have major consequences for offspring. Maternal egg androgens have diverse, often contrasting, effects depending on offspring trait and life stage, suggesting that mothers face trade-offs in egg hormone transfer. However, the effect of egg androgens on embryonic telomere length, which is a major trait potentially affecting performance, has been never investigated. We administered a physiological dose of testosterone (T) to yellow-legged gull (Larus michahellis) eggs and found that, compared to controls, telomere length shortly before hatching was reduced in the liver but unaffected in the brain, heart, and pectoralis muscle. Telomere length varied across somatic tissues, and, independent of egg treatment, it was not correlated between them, suggesting independent telomere dynamics. Thus, we showed for the first time that increased egg T can increase telomere shortening in the embryo and that maternal T allocation strategies may evolve also in response to such effect. Moreover, contrary to observations in adult birds, at the embryonic stage telomere length in one somatic tissue may not reflect telomere length in other body districts.

Prenatal independent and combined effects of yolk vitamin E and corticosterone on embryo growth and oxidative status in the yellow-legged gull.

Variation in the concentration of antioxidants and hormones of maternal origin in the eggs of birds can have a profound influence on offspring phenotype both prenatally and postnatally. Egg maternal substances can have interacting effects, but experimental studies of the consequences of the combined variation in the egg concentration of such molecules are extremely rare, particularly as far as prenatal stages are concerned. We manipulated the yolk concentration of vitamin E and corticosterone, which are, respectively, the main antioxidant and the main glucocorticoid hormone in bird eggs, both independently and simultaneously, and we tested their separate and combined effects on growth and oxidative status in the liver and in the brain of yellow-legged gull (Larus michahellis) embryos. Egg supplementation of relatively large physiological doses of corticosterone depressed embryo growth (total body mass, tarsus length and liver mass), whereas administration of vitamin E in association with corticosterone restored normal growth. Vitamin E did not affect embryo growth when administered alone. We further analysed the independent and combined effects of vitamin E and corticosterone on liver and brain total antioxidant capacity, the concentration of reactive oxygen molecules and lipid peroxidation. Vitamin E significantly reduced liver total antioxidant capacity, while corticosterone depressed brain lipid peroxidation. Prenatal exposure to vitamin E and corticosterone appears to have antagonistic effects on body growth, although vitamin E is not limiting in yellow-legged gull eggs. In combination with the results of previous experiments on the same species applying smaller experimental doses or focusing on the postnatal rather than prenatal life stages, our findings indicate that the effects of a physiological increase in the egg concentration of these substances can be life stage and dose specific, implying that generalizing prenatal effects of egg compounds may not be feasible.

Cloacal microbiomes and ecology of individual barn swallows.

Microbiomes can be considered as 'second genomes' for the host, and can deeply affect its physiology, behaviour and fitness. We investigated the cloacal microbiomes (CMs) of adult and nestling barn swallows (Hirundo rustica), a small insectivorous migratory passerine bird, in order to assess whether CM structure was related to major ecological traits of individuals. Illumina sequencing of the V5-V6 hypervariable regions of the bacterial 16S rRNA gene showed that barn swallow CMs were dominated by Proteobacteria, Firmicutes, Actinobacteria, Tenericutes and Bacteroidetes. Nestling CMs were more similar to one another than adult ones, but showed higher alpha diversity. Sibling nestlings had more similar CMs than non-sibling ones. CMs of adult males also differed from those of adult females, but pair members had more similar CMs than expected by chance. In contrast, CMs did not differ between male and female nestlings. Finally, in adults, CMs strongly different from the 'average' CM were associated to lower survival prospects of the host. The CMs of a bird species in the wild are therefore related to important traits of individuals, such as survival, suggesting that microbiomes should be included among the traits examined in ecological studies.

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.

Embryotoxic effects of in-ovo triclosan injection to the yellow-legged gull.

Triclosan (TCS) is an antimicrobial agent used in diverse personal care products that is considered as an emerging contaminant of both aquatic and terrestrial ecosystems. Although TCS aquatic ecotoxicity is well known, information on the presence and effects on terrestrial organisms is still scarce. This study was aimed at exploring the embryotoxicity of TCS to the yellow-legged gull (Larus michahellis) induced by the in-ovo injection of 150 ng TCS/g egg weight. Effects of TCS on embryo morphological traits (i.e. body mass, tarsus length and head size). Moreover, oxidative and genetic effects were assessed in the embryo liver, by measuring the amount of reactive oxygen species (ROS), the activity of antioxidant (superoxide dismutase and catalase) and detoxifying (glutathione S-transferase - GST) enzymes, the levels of lipid peroxidation and DNA fragmentation. After the injection, the concentration of TCS measured in the yolk of unincubated eggs (159 ±â€¯35 ng/g wet weight, ww) was close to the expected concentration. Triclosan was found in residual yolk (2.9 ±â€¯1.1 ng/g ww), liver (2.3 ±â€¯1.1 ng/g ww) and brain (0.2 ±â€¯0.1 ng/g ww) of embryos soon before hatching. Triclosan did not significantly affect embryo morphological traits, while it increased ROS levels and promoted GST activity, inducing the onset of both oxidative and genetic damage. This study demonstrated, for the first time in a wild euriecious bird species with mixed habits, that TCS can be maternally transferred to developing embryos, representing a potential threat for offspring.

Yolk vitamin E positively affects prenatal growth but not oxidative status in yellow-legged gull embryos.

Parental effects occur whenever the phenotype of parents or the environment that they experience influences the phenotype and fitness of their offspring. In birds, parental effects are often mediated by the size and biochemical quality of the eggs in terms of maternally transferred components. Exogenous antioxidants are key egg components that accomplish crucial physiological functions during early life. Among these, vitamin E plays a vital role during prenatal development when the intense metabolism accompanying rapid embryo growth results in overproduction of pro-oxidant molecules. Studies of captive birds have demonstrated the positive effect of vitamin E supplementation on diverse phenotypic traits of hatchling and adult individuals, but its effects on embryo phenotype has never been investigated neither in captivity nor under a natural selection regime. In the present study, we experimentally tested the effect of the in ovo supplementation of vitamin E on morphological traits and oxidative status of yellow-legged gull (Larus michahellis) embryos. The supplementation of vitamin E promoted somatic growth in embryos soon before hatching, but did not affect their oxidative status. Our results suggest that maternally transferred vitamin E concentrations are optimized to prevent imbalances of oxidative status and the consequent raise of oxidative damage in yellow-legged gull embryos during prenatal development.

Physiological increase of yolk testosterone level does not affect oxidative status and telomere length in gull hatchlings.

Conditions experienced during early-life can cause the onset of oxidative stress, resulting in pervasive effects on diverse life-history traits, including lifespan. In birds, maternally-transferred egg substances may exert positive or negative influence over the offspring phenotype. Among these, testosterone can upregulate the bioavailability of certain antioxidants but simultaneously promotes the production of pro-oxidants, leading to an oxidative stress situation, which is one of the main forces causing telomere attrition However, no study has investigated the role of this androgen on telomere dynamics in birds and little is known about the effects of yolk testosterone on oxidative status in early-life of these species. We physiologically increased the levels of yolk testosterone by in ovo injections in yellow-legged gull (Larus michahellis) to evaluate the effects induced by this androgen on hatchlings plasma total antioxidant capacity, amount of pro-oxidant molecules and telomere length at hatching. Testosterone supplementation did not increase hatchling body growth, did not result in the overproduction of pro-oxidant molecules nor a reduction of antioxidant capacity. Accordingly, telomere length at hatching was not affected by testosterone treatment, although hatchlings from the third-laid eggs showed shorter telomeres than their siblings from first- and second-laid eggs, independently of testosterone treatment. Our results suggest that injection of physiological levels of testosterone does not induce oxidative stress to hatchlings and, consequently do not affect telomere dynamics during early post-natal periods.

Independent and combined effects of egg pro- and anti-oxidants on gull chick phenotype.

Oviparous mothers transfer to their eggs components that have both independent and combined effects on offspring phenotype. The functional interaction between egg components, such as antioxidants and hormones, suggests that a change in the concentration of one component will have effects on offspring traits that depend on the concentration of other interacting components. However, the combined effects of variation in different egg components are virtually unknown. Bird eggs contain vitamin E, a major antioxidant, and also maternal corticosterone. The independent consequences of variation in the egg concentrations of these compounds for offspring phenotype are largely unknown and no study has investigated their combined effects. We manipulated the concentration of vitamin E and corticosterone in the eggs of the yellow-legged gull (Larus michahellis) by administering a physiological (2 s.d.) dose both independently and in combination. We tested for an effect on chick post-natal growth, plasma antioxidant capacity (TAC) and oxidative compounds (TOS). Separate administration of vitamin E or corticosterone caused a reduction in body mass relative to controls, whereas the combined administration of the two compounds reversed their negative effects. These results suggest that maternal egg components, such as antioxidants and steroid hormones, interact and mothers must balance their concentrations in order to achieve optimal offspring phenotype. The functional relationship between vitamin E and corticosterone is corroborated by the observation of positive covariation between these compounds.

Effect of yolk corticosterone on begging in the yellow-legged gull.

Behavioral lateralization is widespread across vertebrates. The development of lateralization is affected by both genetic and environmental factors. In birds, maternal substances in the egg can affect offspring lateralization via activational and/or organizational effects. Corticosterone affects the development of brain asymmetry, suggesting that variation in yolk corticosterone concentration may also influence post-natal behavioral lateralization, a hypothesis that has never been tested so far. In the yellow-legged gull (Larus michahellis), we increased yolk corticosterone concentration within physiological limits and analyzed the direction of lateralization of hatchlings in reverting from supine to prone position ('RTP' response) and in pecking at dummy parental bills to solicit food provisioning ('begging' response). We found that corticosterone treatment negatively affected the frequency of begging and it may cause a slight leftward lateralization. However, the direction of lateralization of the RTP response was not affected by corticosterone administration. Thus, our study shows a maternal effect mediated by corticosterone on a behavioral trait involved in parent-offspring communication during food provisioning events. The findings on lateralization are not conclusive due to the weak effect size but provide information for further ecological and evolutionary studies, investigating mechanisms underlying the development of lateralization.

Wing morphology, winter ecology, and fecundity selection: evidence for sex-dependence in barn swallows (Hirundo rustica).

Variation in wing morphology results from the combination of diverse selection pressures. Wing feather morphology within species varies with sex and ontogenetic effects, and also with ecological factors. Yet, the direction of causation for the wing morphology-ecology association remains to be elucidated. Under the 'ecology-dependence' hypothesis, wing morphology covaries with ecological conditions, because the latter affect feather molt. Alternatively, the 'habitat choice' hypothesis posits that individuals with different wing morphology choose different habitats because of the habitat-dependent advantages of a specific wing morphology. We tested these competing hypotheses in the migratory, aerially insectivorous barn swallow (Hirundo rustica). We quantified wing morphology (isometric size, pointedness, and convexity) on the same individuals during consecutive breeding seasons (i.e., before and after molt in sub-Saharan wintering areas) and located wintering areas using light-level geolocators. Wing pointedness of females but not males during 1 year negatively correlated with vegetation vigor (gauged by the Normalized Difference Vegetation Index; NDVI) in the African area where individuals spent the next winter. Partial least-squares path modelling showed that the association between wing morphology and NDVI was sex-dependent. Conversely, NDVI during wintering did not predict wing morphology in the next breeding season. Because wing morphology can have carry-over effects on subsequent performance, we investigated selection on wing traits and found strong positive fecundity selection on wing size of females. Our results suggest that female barn swallows choose their wintering habitat depending on their wing morphology. In addition, directional fecundity selection operates on females, suggesting sex-dependence of current selection on the flight apparatus.

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.