Recombination rate variation shapes genomic variability of phylogeographic structure in a widespread North American songbird (Aves: Certhia americana).

The nonrandom distribution of chromosomal characteristics and functional elements-genomic architecture-impacts the relative strengths and impacts of population genetic processes across the genome. Due to this relationship, genomic architecture has the potential to shape variation in population genetic structure across the genome. Population genetic structure has been shown to vary across the genome in a variety of taxa, but this body of work has largely focused on pairwise population genomic comparisons between closely related taxa. Here, we used whole genome sequencing of seven phylogeographically structured populations of a North American songbird, the Brown Creeper (Certhia americana), to determine the impacts of genomic architecture on phylogeographic structure variation across the genome. Using multiple methods to infer phylogeographic structure-ordination, clustering, and phylogenetic methods-we found that recombination rate variation explained a large proportion of phylogeographic structure variation. Genomic regions with low recombination showed phylogeographic structure consistent with the genome-wide pattern. In regions with high recombination, we found strong phylogeographic structure, but with discordant patterns relative to the genome-wide pattern. In regions with high recombination rate, we found that populations with small effective population sizes evolve relatively more rapidly than larger populations, leading to discordant signatures of phylogeographic structure. These results suggest that the interplay between recombination rate variation and effective population sizes shape the relative impacts of selection and genetic drift in different parts of the genome. Overall, the combined interactions of population genetic processes, genomic architecture, and effective population sizes shape patterns of variability in phylogeographic structure across the genome of the Brown Creeper.

Regulatory and evolutionary impact of DNA methylation in two songbird species and their naturally occurring F1 hybrids.

BACKGROUND: Regulation of transcription by DNA methylation in 5'-CpG-3' context is a widespread mechanism allowing differential expression of genetically identical cells to persist throughout development. Consequently, differences in DNA methylation can reinforce variation in gene expression among cells, tissues, populations, and species. Despite a surge in studies on DNA methylation, we know little about the importance of DNA methylation in population differentiation and speciation. Here we investigate the regulatory and evolutionary impact of DNA methylation in five tissues of two Ficedula flycatcher species and their naturally occurring F1 hybrids. RESULTS: We show that the density of CpG in the promoters of genes determines the strength of the association between DNA methylation and gene expression. The impact of DNA methylation on gene expression varies among tissues with the brain showing unique patterns. Differentially expressed genes between parental species are predicted by genetic and methylation differentiation in CpG-rich promoters. However, both these factors fail to predict hybrid misexpression suggesting that promoter mismethylation is not a main determinant of hybrid misexpression in Ficedula flycatchers. Using allele-specific methylation estimates in hybrids, we also determine the genome-wide contribution of cis- and trans effects in DNA methylation differentiation. These distinct mechanisms are roughly balanced in all tissues except the brain, where trans differences predominate. CONCLUSIONS: Overall, this study provides insight on the regulatory and evolutionary impact of DNA methylation in songbirds.

A genomic basis of vocal rhythm in birds.

Vocal rhythm plays a fundamental role in sexual selection and species recognition in birds, but little is known of its genetic basis due to the confounding effect of vocal learning in model systems. Uncovering its genetic basis could facilitate identifying genes potentially important in speciation. Here we investigate the genomic underpinnings of rhythm in vocal non-learning Pogoniulus tinkerbirds using 135 individual whole genomes distributed across a southern African hybrid zone. We find rhythm speed is associated with two genes that are also known to affect human speech, Neurexin-1 and Coenzyme Q8A. Models leveraging ancestry reveal these candidate loci also impact rhythmic stability, a trait linked with motor performance which is an indicator of quality. Character displacement in rhythmic stability suggests possible reinforcement against hybridization, supported by evidence of asymmetric assortative mating in the species producing faster, more stable rhythms. Because rhythm is omnipresent in animal communication, candidate genes identified here may shape vocal rhythm across birds and other vertebrates.

Mapping seasonal migration in a songbird hybrid zone -- heritability, genetic correlations, and genomic patterns linked to speciation.

Seasonal migration is a widespread behavior relevant for adaptation and speciation, yet knowledge of its genetic basis is limited. We leveraged advances in tracking and sequencing technologies to bridge this gap in a well-characterized hybrid zone between songbirds that differ in migratory behavior. Migration requires the coordinated action of many traits, including orientation, timing, and wing morphology. We used genetic mapping to show these traits are highly heritable and genetically correlated, explaining how migration has evolved so rapidly in the past and suggesting future responses to climate change may be possible. Many of these traits mapped to the same genomic regions and small structural variants indicating the same, or tightly linked, genes underlie them. Analyses integrating transcriptomic data indicate cholinergic receptors could control multiple traits. Furthermore, analyses integrating genomic differentiation further suggested genes underlying migratory traits help maintain reproductive isolation in this hybrid zone.

Reduced hybrid survival in a migratory divide between songbirds.

Migratory divides, hybrid zones between populations that use different seasonal migration routes, are hypothesised to contribute to speciation. Specifically, relative to parental species, hybrids at divides are predicted to exhibit (1) intermediate migratory behaviour and (2) reduced fitness as a result. We provide the first direct test of the second prediction here with one of the largest existing avian tracking datasets, leveraging a divide between Swainson's thrushes where the first prediction is supported. Using detection rates as a proxy for survival, our results supported the migratory divide hypothesis with lower survival rates for hybrids than parental forms. This finding was juvenile-specific (vs. adults), suggesting selection against hybrids is stronger earlier in life. Reduced hybrid survival was not explained by selection against intermediate phenotypes or negative interactions among phenotypes. Additional work connecting specific features of migration is needed, but these patterns provide strong support for migration as an ecological driver of speciation.

Genes and gene networks underlying spatial cognition in food-caching chickadees.

Substantial progress has been made in understanding the genetic architecture of phenotypes involved in a variety of evolutionary processes. Behavioral genetics remains, however, among the least understood. We explore the genetic architecture of spatial cognitive abilities in a wild passerine bird, the mountain chickadee (Poecile gambeli). Mountain chickadees cache thousands of seeds in the fall and require specialized spatial memory to recover these caches throughout the winter. We previously showed that variation in spatial cognition has a direct effect on fitness and has a genetic basis. It remains unknown which specific genes and developmental pathways are particularly important for shaping spatial cognition. To further dissect the genetic basis of spatial cognitive abilities, we combine experimental quantification of spatial cognition in wild chickadees with whole-genome sequencing of 162 individuals, a new chromosome-scale reference genome, and species-specific gene annotation. We have identified a set of genes and developmental pathways that play a key role in creating variation in spatial cognition and found that the mechanism shaping cognitive variation is consistent with selection against mildly deleterious non-coding mutations. Although some candidate genes were organized into connected gene networks, about half do not have shared regulation, highlighting that multiple independent developmental or physiological mechanisms contribute to variation in spatial cognitive abilities. A large proportion of the candidate genes we found are associated with synaptic plasticity, an intriguing result that leads to the hypothesis that certain genetic variants create antagonism between behavioral plasticity and long-term memory, each providing distinct benefits depending on ecological context.

Genome Assembly of Pyrocephalus nanus: A Step Toward the Genetic Conservation of the Endangered Little Vermilion Flycatcher of the Galapagos Islands.

Incredibly powerful whole genome studies of conservation genetics, evolution, and biogeography become possible for non-model organisms when reference genomes are available. Here, we report the sequence and assembly of the whole genome of the little vermilion flycatcher (Pyrocephalus nanus; family Tyrannidae), which is an endemic, endangered, and declining species of the Galapagos Islands. Using PacBio HiFi reads to assemble long contigs and Hi-C reads for scaffolding, we assembled a genome of 1.07 Gb comprising 267 contigs in 152 scaffolds, scaffold N50 74 M, contig N50 17.8 M, with 98.9% assigned to candidate chromosomal sequences and 99.72% of the BUSCO passeriformes 10,844 single-copy orthologs present. In addition, we used the novel HiFiMiTie pipeline to fully assemble and verify all portions of the mitochondrial genome from HiFi reads, obtaining a mitogenome of 17,151 bases, containing 13 protein-coding genes, 22 tRNAs, 2 rRNAs, two control regions, and a unique structure of control region duplication and repeats. These genomes will be a critical tool for much-needed studies of phylogenetics, population genetics, biogeography, and conservation genetics of Pyrocephalus and related genera. This genome and other studies that use it will be able to provide recommendations for conservation management, taxonomic improvement, and to understand the evolution and diversification of this genus within the Galapagos Islands.

Dorsal and Ventral Plumage Coloration Evolve as Distinct Modules with Different Environmental Correlations.

AbstractMany animals exhibit contrast between their dorsal coloration and their ventral coloration. If selection acts differently on dorsal versus ventral coloration, ancestral covariance between these traits should break down, eventually leading to independent modules of trait evolution. Here, we compare the evolution of feather color across body regions for a clade of Australasian songbirds (Meliphagoidea). We find evidence for three modules of covarying color regions. Among these modules, ventral feathers evolve with high lability, evolving at three times the rate of dorsal plumage and 20 times the rate of flight feathers. While both dorsal plumage and ventral plumage are darker in areas with more precipitation and vegetation, we find that dorsal plumage is twice as similar to colors in satellite photos of background substrates. Overall, differential selection on ventral and dorsal colors likely maintains these as distinct modules over evolutionary timescales-a novel explanation for dorsoventral contrast in pigmentation.

Night-time neuronal activation of Cluster N in a North American songbird.

Night-migrating songbirds utilize the Earth's magnetic field to help navigate to and from their breeding sites each year. A region of the avian forebrain called Cluster N has been shown to be activated during night migratory behavior and it has been implicated in processing geomagnetic information. Previous studies with night-migratory European songbirds have shown that neuronal activity at Cluster N is higher at night than during the day. Comparable work in North American migrants has only been performed in one species of swallows, so extension of examination for Cluster N in other migratory birds is needed. In addition, it is unclear if Cluster N activation is lateralized and the full extent of its boundaries in the forebrain have yet to be described. We used sensory-driven gene expression based on ZENK and the Swainson's thrush, a night-migratory North American songbird, to fill these knowledge gaps. We found elevated levels of gene expression in night- vs. day-active thrushes and no evidence for lateralization in this region. We further examined the anatomical extent of neural activation in the forebrain using 3D reconstruction topology. Our findings demonstrate that Swainson's thrushes possess an extensive bilateral night-activated Cluster N region in the forebrain similar to other European avian species, suggesting that Cluster N is highly conserved in nocturnal migrants.

Male song structure predicts offspring recruitment to the breeding population in a migratory bird.

Bird song is a classic example of a sexually selected trait, but much of the work relating individual song components to fitness has not accounted for song typically being composed of multiple, often-correlated components, necessitating a multivariate approach. We explored the role of sexual selection in shaping the complex male song of house wrens (Troglodytes aedon) by simultaneously relating its multiple components to fitness using multivariate selection analysis, which is widely used in insect and anuran studies but not in birds. The analysis revealed significant variation in the form and strength of selection acting on song across different selection episodes, from nest-site defense to recruitment of offspring to the breeding population. Males that sang more song typically employed in close communication sired more offspring that were subsequently recruited to the breeding population than those that sang more far-communication song. However, this relationship was not consistent across earlier selection episodes, as evidenced by non-linear selection acting on these song components in other contexts. Collectively, our results present a complex picture of multivariate selection on male song structure that would not be evident using univariate approaches and suggest possible trade-offs within and among song components at different points of the breeding season.

A highly contiguous genome assembly for the Yellow Warbler (Setophaga petechia).

The Yellow Warbler (Setophaga petechia) is a small songbird in the wood-warbler family (Parulidae) that exhibits phenotypic and ecological differences across a widespread distribution and is important to California's riparian habitat conservation. Here, we present a high-quality de novo genome assembly of a vouchered female Yellow Warbler from southern California. Using HiFi long-read and Omni-C proximity sequencing technologies, we generated a 1.22 Gb assembly including 687 scaffolds with a contig N50 of 6.80 Mb, scaffold N50 of 21.18 Mb, and a BUSCO completeness score of 96.0%. This highly contiguous genome assembly provides an essential resource for understanding the history of gene flow, divergence, and local adaptation in Yellow Warblers and can inform conservation management of this charismatic bird species.

Genetic Basis and Evolution of Structural Color Polymorphism in an Australian Songbird.

Island organisms often evolve phenotypes divergent from their mainland counterparts, providing a useful system for studying adaptation under differential selection. In the white-winged fairywren (Malurus leucopterus), subspecies on two islands have a black nuptial plumage whereas the subspecies on the Australian mainland has a blue nuptial plumage. The black subspecies have a feather nanostructure that could in principle produce a blue structural color, suggesting a blue ancestor. An earlier study proposed independent evolution of melanism on the islands based on the history of subspecies divergence. However, the genetic basis of melanism and the origin of color differentiation in this group are still unknown. Here, we used whole-genome resequencing to investigate the genetic basis of melanism by comparing the blue and black M. leucopterus subspecies to identify highly divergent genomic regions. We identified a well-known pigmentation gene ASIP and four candidate genes that may contribute to feather nanostructure development. Contrary to the prediction of convergent evolution of island melanism, we detected signatures of a selective sweep in genomic regions containing ASIP and SCUBE2 not in the black subspecies but in the blue subspecies, which possesses many derived SNPs in these regions, suggesting that the mainland subspecies has re-evolved a blue plumage from a black ancestor. This proposed re-evolution was likely driven by a preexisting female preference. Our findings provide new insight into the evolution of plumage coloration in island versus continental populations, and, importantly, we identify candidate genes that likely play roles in the development and evolution of feather structural coloration.

A late burst of colour evolution in a radiation of songbirds (Passeriformes: Parulidae) suggests secondary contact drives signal divergence.

Evolutionary radiations provide important insights into species diversification, which is especially true of adaptive radiations. New World wood warblers (Parulidae) are a family of small, insectivorous, forest-dwelling passerine birds, often considered an exemplar of adaptive radiation due to their rapid diversification followed by a slowdown. However, they deviate from the expectations of an adaptive radiation scenario due to the lack of conspicuous morphological and ecological differentiation. We fitted several macroevolutionary models to trait data in 105 species of wood warblers. We tested whether morphological traits underwent an early burst of evolution (suggesting adaptation to new ecological niches in adaptive radiations) and whether song and colour underwent a diversity-dependent acceleration of trait evolutionary rate (consistent with reproductive interference driving signal evolution). Morphology and song evolved gradually under stabilizing selection, suggesting niche conservatism, with morphology possibly acting as a constraint on song evolution. In contrast, many feather colour traits underwent a diversity-dependent burst of evolution occurring late in the clade's history. We suggest that a two-step process has led to the remarkable diversification of wood warblers. First, their early diversification probably proceeded by allopatric speciation. Second, feather colour divergence likely occurred during secondary contact after range expansion. This diversification of signalling traits might have facilitated species coexistence, in combination with behavioural niche partitioning. Wood warblers seem to present characteristics of both adaptive and non-adaptive radiations.

Gene regulation and speciation in a migratory divide between songbirds.

Behavioral variation abounds in nature. This variation is important for adaptation and speciation, but its molecular basis remains elusive. Here, we use a hybrid zone between two subspecies of songbirds that differ in migration - an ecologically important and taxonomically widespread behavior---to gain insight into this topic. We measure gene expression in five brain regions. Differential expression between migratory states was dominated by circadian genes in all brain regions. The remaining patterns were largely brain-region specific. For example, expression differences between the subspecies that interact with migratory state likely help maintain reproductive isolation in this system and were documented in only three brain regions. Contrary to existing work on regulatory mechanisms underlying species-specific traits, two lines of evidence suggest that trans- (vs. cis) regulatory changes underlie these patterns - no evidence for allele-specific expression in hybrids and minimal associations between genomic differentiation and expression differences. Additional work with hybrids shows expression levels were often distinct (transgressive) from parental forms. Behavioral contrasts and functional enrichment analyses allowed us to connect these patterns to mitonuclear incompatibilities and compensatory responses to stress that could exacerbate selection on hybrids and contribute to speciation.

The role of recombination dynamics in shaping signatures of direct and indirect selection across the Ficedula flycatcher genome†.

Recombination is a central evolutionary process that reshuffles combinations of alleles along chromosomes, and consequently is expected to influence the efficacy of direct selection via Hill-Robertson interference. Additionally, the indirect effects of selection on neutral genetic diversity are expected to show a negative relationship with recombination rate, as background selection and genetic hitchhiking are stronger when recombination rate is low. However, owing to the limited availability of recombination rate estimates across divergent species, the impact of evolutionary changes in recombination rate on genomic signatures of selection remains largely unexplored. To address this question, we estimate recombination rate in two Ficedula flycatcher species, the taiga flycatcher (Ficedula albicilla) and collared flycatcher (Ficedula albicollis). We show that recombination rate is strongly correlated with signatures of indirect selection, and that evolutionary changes in recombination rate between species have observable impacts on this relationship. Conversely, signatures of direct selection on coding sequences show little to no relationship with recombination rate, even when restricted to genes where recombination rate is conserved between species. Thus, using measures of indirect and direct selection that bridge micro- and macro-evolutionary timescales, we demonstrate that the role of recombination rate and its dynamics varies for different signatures of selection.

Fine-Scale Map Reveals Highly Variable Recombination Rates Associated with Genomic Features in the Eurasian Blackcap.

Recombination is responsible for breaking up haplotypes, influencing genetic variability, and the efficacy of selection. Bird genomes lack the protein PR domain-containing protein 9, a key determinant of recombination dynamics in most metazoans. Historical recombination maps in birds show an apparent stasis in positioning recombination events. This highly conserved recombination pattern over long timescales may constrain the evolution of recombination in birds. At the same time, extensive variation in recombination rate is observed across the genome and between different species of birds. Here, we characterize the fine-scale historical recombination map of an iconic migratory songbird, the Eurasian blackcap (Sylvia atricapilla), using a linkage disequilibrium-based approach that accounts for population demography. Our results reveal variable recombination rates among and within chromosomes, which associate positively with nucleotide diversity and GC content and negatively with chromosome size. Recombination rates increased significantly at regulatory regions but not necessarily at gene bodies. CpG islands are associated strongly with recombination rates, though their specific position and local DNA methylation patterns likely influence this relationship. The association with retrotransposons varied according to specific family and location. Our results also provide evidence of heterogeneous intrachromosomal conservation of recombination maps between the blackcap and its closest sister taxon, the garden warbler. These findings highlight the considerable variability of recombination rates at different scales and the role of specific genomic features in shaping this variation. This study opens the possibility of further investigating the impact of recombination on specific population-genomic features.

Passerine sister clade comparisons reveal variable macroevolutionary outcomes of interhemispheric dispersal.

Dispersal events offer a unique window into macroevolutionary processes, especially with respect to the effects of competition on diversification. Empirical studies testing alternative predictions of competitive effects are often limited in either geographic or phylogenetic scale. Here, we tested some of these hypotheses by comparing an assemblage of 16 oscine passerine clades, representing independent dispersal events into the Western Hemisphere, to their sister clades in the Eastern Hemisphere. We also compared the diversity of this assemblage of clades to an older, incumbent passerine clade in the Western Hemisphere, the suboscines. Specifically, we tested for ecological opportunity and incumbency-mediated constraints by analysis of clade-specific morphological disparities and rates of evolution relative to dispersal history. While there was no consistent outcome of oscine dispersal and macroevolution in the Western Hemisphere relative to their Eastern Hemisphere sister groups, most clades supported a role for ecological opportunity or incumbency effects, and such effects were better explained by differences in species accumulation than by differences in rates of trait evolution or colonization timing. This general pattern was not evident when comparing the entire oscine assemblage of the Western Hemisphere to the incumbent suboscine radiation; oscines and suboscines occupy comparable regions of functional trait diversity and, despite higher rates of trait evolution in oscines, these observations were consistent with simulated null expectations. This result suggests that oscine and suboscine assemblages may have evolved in relative isolation for a significant fraction of their history.

RADseq data reveal widespread historical introgression in four familiar North American songbirds.

Population genetic structure is influenced by a combination of contemporary and historical events; however, this structure can be complicated by ongoing gene flow. While it is well known that contemporary hybridization occurs frequently among many closely related species, it often remains uncertain as to which populations are involved in introgression events, and this can be even more difficult to infer when introgression is historical. Here we use restriction-site associated DNA sequencing to look at the level of introgression among four species of songbirds in North America: the black-capped, mountain, boreal, and chestnut-backed chickadee. Samples from both sympatric and allopatric sites across the species' ranges supported limited ongoing mixing among the four species with Bayesian clustering and principal component analyses. In contrast, f4-statistics and admixture graphs revealed extensive historical introgression among geographically structured populations. Almost all historical admixture events were among populations west of the Rocky Mountains, and almost all populations west of the Rocky Mountains, excluding island and coastal populations, showed evidence of historical admixture. The inclusion of all four chickadee species proved crucial in differentiating which species were involved in hybridization events to avoid erroneous conclusions. Taken together, the results suggest a complex pattern of divergence with gene flow.

Tracking hybrid viability across life stages in a natural avian contact zone.

Hybrid inviability is an important post-zygotic reproductive barrier between species, but emerging signs of reduced viability can be difficult to study across the lifespan of natural hybrids. We use a combination of long-term monitoring, extra-pair paternity, and mitochondrial DNA identification in a natural hybrid zone of Ficedula flycatchers to detect emerging signs of intrinsic hybrid inviability across their entire lifespan. We evaluate possible evidence of Darwin's corollary to Haldane's rule, predicting asymmetries in inviability between hybrids resulting from reciprocal crosses, due to incompatible genetic factors with sex-specific inheritance patterns. We found higher hatching failure among mixed-species pairs, possibly indicating early developmental impairments associated with specific parental genetic combinations. Adult hybrids had a higher basal mortality rate than both parental species and different age-specific mortality trajectories. There were signs of differences in age-independent mortality rates between the reciprocal hybrid crosses: hybrids with a pied flycatcher mother experienced slightly increased mortality later in life. Using an exceptional dataset with many natural hybrids tracked across life stages, we provide evidence for several emerging signs of reduced hybrid viability. Incompatibilities between alleles located on autosomes and uniparentally inherited factors such as Z-linked and/or mitochondrial genes are strong candidates underlying intrinsic hybrid dysfunction in this system.

The impact of helping experience on helper life-history and fitness in a cooperatively breeding bird.

Cooperative breeding occurs when helpers provide alloparental care to the offspring of a breeding pair. One hypothesis of why helping occurs is that helpers gain valuable skills that may increase their own future reproductive success. However, research typically focuses on the effect of helping on short-term measures of reproductive success. Fewer studies have considered how helping affects long-term fitness measures. Here, we analyze how helping experience affects key breeding and fitness-related parameters in the Seychelles warbler (Acrocephalus sechellensis). Importantly, we control for females that have cobred (reproduced as a subordinate by laying an egg within a territory in which they are not a dominant breeder), as they already have experience with direct reproduction. Helping experience had no significant association with any of the metrics considered, except that helpers had an older age at first dominance. Accounting for helping experience, females that had cobred produced more adult offspring (≥1 year) after acquiring dominance and had a higher lifetime reproductive success (LRS) than females that had never cobred. Our results suggest that, in the Seychelles warbler, helping experience alone does not increase the fitness of helpers in any of the metrics considered, and highlights the importance of separating the effects of helping from cobreeding. Our findings also emphasize the importance of analyzing the effect of helping at various life-history stages, as higher short-term fitness may not translate to an overall increase in LRS.