Dense sampling of bird diversity increases power of comparative genomics.

Whole-genome sequencing projects are increasingly populating the tree of life and characterizing biodiversity1-4. Sparse taxon sampling has previously been proposed to confound phylogenetic inference5, and captures only a fraction of the genomic diversity. Here we report a substantial step towards the dense representation of avian phylogenetic and molecular diversity, by analysing 363 genomes from 92.4% of bird families-including 267 newly sequenced genomes produced for phase II of the Bird 10,000 Genomes (B10K) Project. We use this comparative genome dataset in combination with a pipeline that leverages a reference-free whole-genome alignment to identify orthologous regions in greater numbers than has previously been possible and to recognize genomic novelties in particular bird lineages. The densely sampled alignment provides a single-base-pair map of selection, has more than doubled the fraction of bases that are confidently predicted to be under conservation and reveals extensive patterns of weak selection in predominantly non-coding DNA. Our results demonstrate that increasing the diversity of genomes used in comparative studies can reveal more shared and lineage-specific variation, and improve the investigation of genomic characteristics. We anticipate that this genomic resource will offer new perspectives on evolutionary processes in cross-species comparative analyses and assist in efforts to conserve species.

Genomic islands of differentiation in a rapid avian radiation have been driven by recent selective sweeps.

Numerous studies of emerging species have identified genomic "islands" of elevated differentiation against a background of relative homogeneity. The causes of these islands remain unclear, however, with some signs pointing toward "speciation genes" that locally restrict gene flow and others suggesting selective sweeps that have occurred within nascent species after speciation. Here, we examine this question through the lens of genome sequence data for five species of southern capuchino seedeaters, finch-like birds from South America that have undergone a species radiation during the last ∼50,000 generations. By applying newly developed statistical methods for ancestral recombination graph inference and machine-learning methods for the prediction of selective sweeps, we show that previously identified islands of differentiation in these birds appear to be generally associated with relatively recent, species-specific selective sweeps, most of which are predicted to be soft sweeps acting on standing genetic variation. Many of these sweeps coincide with genes associated with melanin-based variation in plumage, suggesting a prominent role for sexual selection. At the same time, a few loci also exhibit indications of possible selection against gene flow. These observations shed light on the complex manner in which natural selection shapes genome sequences during speciation.

Concerted variation in melanogenesis genes underlies emergent patterning of plumage in capuchino seedeaters.

Coloration traits are central to animal communication; they often govern mate choice, promote reproductive isolation and catalyse speciation. Specific genetic changes can cause variation in coloration, yet far less is known about how overall coloration patterns-which involve combinations of multiple colour patches across the body-can arise and are genomically controlled. We performed genome-wide association analyses to link genomic changes to variation in melanin (eumelanin and pheomelanin) concentration in feathers from different body parts in the capuchino seedeaters, an avian radiation with diverse colour patterns despite remarkably low genetic differentiation across species. Cross-species colour variation in each plumage patch is associated with unique combinations of variants at a few genomic regions, which include mostly non-coding (presumably regulatory) areas close to known pigmentation genes. Genotype-phenotype associations can vary depending on patch colour and are stronger for eumelanin pigmentation, suggesting eumelanin production is tightly regulated. Although some genes are involved in colour variation in multiple patches, in some cases, the SNPs associated with colour changes in different patches segregate spatially. These results suggest that coloration patterning in capuchinos is generated by the modular combination of variants that regulate multiple melanogenesis genes, a mechanism that may have promoted this rapid radiation.

Selection on VPS13A linked to migration in a songbird.

Animal migration demands an interconnected suite of adaptations for individuals to navigate over long distances. This trait complex is crucial for small birds whose migratory behaviors-such as directionality-are more likely innate, rather than being learned as in many longer-lived birds. Identifying causal genes has been a central goal of migration ecology, and this endeavor has been furthered by genome-scale comparisons. However, even the most successful studies of migration genetics have achieved low-resolution associations, identifying large chromosomal regions that encompass hundreds of genes, one or more of which might be causal. Here we leverage the genomic similarity among golden-winged (Vermivora chrysoptera) and blue-winged (V. cyanoptera) warblers to identify a single gene-vacuolar protein sorting 13A (VPS13A)-that is associated with distinct differences in migration to Central American (CA) or South American (SA) wintering areas. We find reduced sequence variation in this gene region for SA wintering birds, and show this is the likely result of natural selection on this locus. In humans, variants of VPS13A are linked to the neurodegenerative disorder chorea-acanthocytosis. This association provides one of the strongest gene-level associations with avian migration differences.

Extensive hybridization reveals multiple coloration genes underlying a complex plumage phenotype.

Coloration is an important target of both natural and sexual selection. Discovering the genetic basis of colour differences can help us to understand how this visually striking phenotype evolves. Hybridizing taxa with both clear colour differences and shallow genomic divergences are unusually tractable for associating coloration phenotypes with their causal genotypes. Here, we leverage the extensive admixture between two common North American woodpeckers-yellow-shafted and red-shafted flickers-to identify the genomic bases of six distinct plumage patches involving both melanin and carotenoid pigments. Comparisons between flickers across approximately 7.25 million genome-wide SNPs show that these two forms differ at only a small proportion of the genome (mean FST = 0.008). Within the few highly differentiated genomic regions, we identify 368 SNPs significantly associated with four of the six plumage patches. These SNPs are linked to multiple genes known to be involved in melanin and carotenoid pigmentation. For example, a gene (CYP2J19) known to cause yellow to red colour transitions in other birds is strongly associated with the yellow versus red differences in the wing and tail feathers of these flickers. Additionally, our analyses suggest novel links between known melanin genes and carotenoid coloration. Our finding of patch-specific control of plumage coloration adds to the growing body of literature suggesting colour diversity in animals could be created through selection acting on novel combinations of coloration genes.

Environmental correlates of genetic variation in the invasive European starling in North America.

Populations of invasive species that colonize and spread in novel environments may differentiate both through demographic processes and local selection. European starlings (Sturnus vulgaris) were introduced to New York in 1890 and subsequently spread throughout North America, becoming one of the most widespread and numerous bird species on the continent. Genome-wide comparisons across starling individuals and populations can identify demographic and/or selective factors that facilitated this rapid and successful expansion. We investigated patterns of genomic diversity and differentiation using reduced-representation genome sequencing of 17 winter-season sampling sites. Consistent with this species' high dispersal rate and rapid expansion history, we found low geographical differentiation and few FST outliers even at a continental scale. Despite starting from a founding population of ~180 individuals, North American starlings show only a moderate genetic bottleneck, and models suggest a dramatic increase in effective population size since introduction. In genotype-environment associations we found that ~200 single-nucleotide polymorphisms are correlated with temperature and/or precipitation against a background of negligible genome- and range-wide divergence. Given this evidence, we suggest that local adaptation in North American starlings may have evolved rapidly even in this wide-ranging and evolutionarily young system. This survey of genomic signatures of expansion in North American starlings is the most comprehensive to date and complements ongoing studies of world-wide local adaptation in these highly dispersive and invasive birds.

Genomic phylogeography of the White-crowned Manakin Pseudopipra pipra (Aves: Pipridae) illuminates a continental-scale radiation out of the Andes.

The complex landscape history of the Neotropics has generated opportunities for population isolation and diversification that place this region among the most species-rich in the world. Detailed phylogeographic studies are required to uncover the biogeographic histories of Neotropical taxa, to identify evolutionary correlates of diversity, and to reveal patterns of genetic connectivity, disjunction, and potential differentiation among lineages from different areas of endemism. The White-crowned Manakin (Pseudopipra pipra) is a small suboscine passerine bird that is broadly distributed through the subtropical rainforests of Central America, the lower montane cloud forests of the Andes from Colombia to central Peru, the lowlands of Amazonia and the Guianas, and the Atlantic forest of southeast Brazil. Pseudopipra is currently recognized as a single, polytypic biological species. We studied the effect of the Neotropical landscape on genetic and phenotypic differentiation within this species using genomic data derived from double digest restriction site associated DNA sequencing (ddRAD), and mitochondrial DNA. Most of the genetic breakpoints we identify among populations coincide with physical barriers to gene flow previously associated with avian areas of endemism. The phylogenetic relationships among these populations imply a novel pattern of Andean origination for this group, with subsequent diversification into the Amazonian lowlands. Our analysis of genomic admixture and gene flow reveals a complex history of introgression between some western Amazonian populations. These reticulate processes confound our application of standard concatenated and coalescent phylogenetic methods and raise the question of whether a lineage in the western Napo area of endemism should be considered a hybrid species. Lastly, analysis of variation in vocal and plumage phenotypes in the context of our phylogeny supports the hypothesis that Pseudopipra is a species-complex composed of at least 8, and perhaps up to 17 distinct species which have arisen in the last ~2.5 Ma.

Genomic differentiation and local adaptation on a microgeographic scale in a resident songbird.

Elucidating forces capable of driving species diversification in the face of gene flow remains a key goal in evolutionary biology. Song sparrows, Melospiza melodia, occur as 25 subspecies in diverse habitats across North America, are among the continent's most widespread vertebrate species, and are exemplary of many highly variable species for which the conservation of locally adapted populations may be critical to their range-wide persistence. We focus here on six morphologically distinct subspecies resident in the San Francisco Bay region, including three salt-marsh endemics and three residents in upland and riparian habitats adjacent to the Bay. We used reduced-representation sequencing to generate 2,773 SNPs to explore genetic differentiation, spatial population structure, and demographic history. Clustering separated individuals from each of the six subspecies, indicating subtle differentiation at microgeographic scales. Evidence of limited gene flow and low nucleotide diversity across all six subspecies further supports a hypothesis of isolation among locally adapted populations. We suggest that natural selection for genotypes adapted to salt marsh environments and changes in demography over the past century have acted in concert to drive the patterns of diversification reported here. Our results offer evidence of microgeographic specialization in a highly polytypic bird species long discussed as a model of sympatric speciation and rapid adaptation, and they support the hypothesis that conserving locally adapted populations may be critical to the range-wide persistence of similarly highly variable species.

A test of the riverine barrier hypothesis in the largest subtropical river basin in the Neotropics.

The riverine barrier hypothesis proposes that large rivers represent geographical barriers to gene flow for terrestrial organisms, leading to population differentiation and ultimately allopatric speciation. Here we assess for the first time if the subtropical Paraná-Paraguay River system in the Del Plata basin, second in size among South American drainages, acts as a barrier to gene flow for birds. We analysed the degree of mitochondrial and nuclear genomic differentiation in seven species with known subspecies divided by the Paraná-Paraguay River axis. Only one species showed genetic differentiation concordant with the current river channel, but another five species have an east/west genetic split broadly coincident with the Paraná River's dynamic palaeochannel, suggesting this fluvial axis has had a past role in shaping present-day genetic structure. Moreover, dating analyses show that these splits have been asynchronous, with species responding differently to the riverine barrier. Comparisons informed by the geological history of the Paraná River and its influence on the ecological and climatic differences among ecoregions in the study area further bolster the finding that responses to this geographical barrier have been species-specific.

Phylogeographic variation within the Buff-browed Foliage-gleaner (Aves: Furnariidae: Syndactyla rufosuperciliata) supports an Andean-Atlantic forests connection via the Cerrado.

We evaluated whether the Andean and the Atlantic forests acted as refugia during the Quaternary, and tested biogeographic hypotheses about the regions involved in the connectivity between those biomes (through the Chaco or the Cerrado). To achieve these goals we selected the Buff-browed Foliage-gleaner Syndactyla rufosuperciliata (Aves, Furnariidae) as a study system, a taxon distributed between the Andean and Atlantic forest. We first explored the historical connectivity between regions through niche modeling. We subsequently used DNA sequences (n = 71 individuals) and genomic analyses (ddRADseq, n = 33 individuals) to evaluate population genetic structure and gene flow within this species. Finally, we performed population model selection using Approximate Bayesian Computation. Our findings indicate that the Andean and the Atlantic forests acted as refugia, and that the populations of the focal species from both regions contacted through the Cerrado region, thus suggesting that the historical dynamics of Andean and Atlantic forests are important for the evolution of forest birds in the region. The results are in agreement with studies of other organisms and may indicate a more general pattern of connectivity among biomes in the Neotropics. Finally, we recommend recognizing both the Andean and the Altantic forests lineages of S. rufosuperciliata as independent species.

Correlated patterns of genetic diversity and differentiation across an avian family.

Comparative studies of closely related taxa can provide insights into the evolutionary forces that shape genome evolution and the prevalence of convergent molecular evolution. We investigated patterns of genetic diversity and differentiation in stonechats (genus Saxicola), a widely distributed avian species complex with phenotypic variation in plumage, morphology and migratory behaviour, to ask whether similar genomic regions have become differentiated in independent, but closely related, taxa. We used whole-genome pooled sequencing of 262 individuals from five taxa and found that levels of genetic diversity and divergence are strongly correlated among different stonechat taxa. We then asked whether these patterns remain correlated at deeper evolutionary scales and found that homologous genomic regions have become differentiated in stonechats and the closely related Ficedula flycatchers. Such correlation across a range of evolutionary divergence and among phylogenetically independent comparisons suggests that similar processes may be driving the differentiation of these independently evolving lineages, which in turn may be the result of intrinsic properties of particular genomic regions (e.g. areas of low recombination). Consequently, studies employing genome scans to search for areas important for reproductive isolation or adaptation should account for corresponding regions of differentiation, as these regions may not necessarily represent speciation islands or evidence of local adaptation.

Subspecies delineation amid phenotypic, geographic and genetic discordance in a songbird.

Understanding the processes that drive divergence within and among species is a long-standing goal in evolutionary biology. Traditional approaches to assessing differentiation rely on phenotypes to identify intra- and interspecific variation, but many species express subtle morphological gradients in which boundaries among forms are unclear. This intraspecific variation may be driven by differential adaptation to local conditions and may thereby reflect the evolutionary potential within a species. Here, we combine genetic and morphological data to evaluate intraspecific variation within the Nelson's (Ammodramus nelsoni) and salt marsh (Ammodramus caudacutus) sparrow complex, a group with populations that span considerable geographic distributions and a habitat gradient. We evaluated genetic structure among and within five putative subspecies of A. nelsoni and A. caudacutus using a reduced-representation sequencing approach to generate a panel of 1929 SNPs among 69 individuals. Although we detected morphological differences among some groups, individuals sorted along a continuous phenotypic gradient. In contrast, the genetic data identified three distinct clusters corresponding to populations that inhabit coastal salt marsh, interior freshwater marsh and coastal brackish-water marsh habitats. These patterns support the current species-level recognition but do not match the subspecies-level taxonomy within each species-a finding which may have important conservation implications. We identified loci exhibiting patterns of elevated divergence among and within these species, indicating a role for local selective pressures in driving patterns of differentiation across the complex. We conclude that this evidence for adaptive variation among subspecies warrants the consideration of evolutionary potential and genetic novelty when identifying conservation units for this group.

Distinguishing noise from signal in patterns of genomic divergence in a highly polymorphic avian radiation.

Recently diverged taxa provide the opportunity to search for the genetic basis of the phenotypes that distinguish them. Genomic scans aim to identify loci that are diverged with respect to an otherwise weakly differentiated genetic background. These loci are candidates for being past targets of selection because they behave differently from the rest of the genome that has either not yet differentiated or that may cross species barriers through introgressive hybridization. Here we use a reduced-representation genomic approach to explore divergence among six species of southern capuchino seedeaters, a group of recently radiated sympatric passerine birds in the genus Sporophila. For the first time in these taxa, we discovered a small proportion of markers that appeared differentiated among species. However, when assessing the significance of these signatures of divergence, we found that similar patterns can also be recovered from random grouping of individuals representing different species. A detailed demographic inference indicates that genetic differences among Sporophila species could be the consequence of neutral processes, which include a very large ancestral effective population size that accentuates the effects of incomplete lineage sorting. As these neutral phenomena can generate genomic scan patterns that mimic those of markers involved in speciation and phenotypic differentiation, they highlight the need for caution when ascertaining and interpreting differentiated markers between species, especially when large numbers of markers are surveyed. Our study provides new insights into the demography of the southern capuchino radiation and proposes controls to distinguish signal from noise in similar genomic scans.

Analysis and visualization of complex macroevolutionary dynamics: an example from Australian scincid lizards.

The correlation between species diversification and morphological evolution has long been of interest in evolutionary biology. We investigated the relationship between these processes during the radiation of 250+scincid lizards that constitute Australia's most species-rich clade of terrestrial vertebrates. We generated a time-calibrated phylogenetic tree for the group that was more than 85% complete at the species level and collected multivariate morphometric data for 183 species. We reconstructed the dynamics of species diversification and trait evolution using a Bayesian statistical framework (BAMM) that simultaneously accounts for variation in evolutionary rates through time and among lineages. We extended the BAMM model to accommodate time-dependent phenotypic evolution, and we describe several new methods for summarizing and visualizing macroevolutionary rate heterogeneity on phylogenetic trees. Two major clades (Lerista, Ctenotus; >90 spp. each) are associated with high rates of species diversification relative to the background rate across Australian sphenomorphine skinks. The Lerista clade is characterized by relatively high lability of body form and has undergone repeated instances of limb reduction, but Ctenotus is characterized by an extreme deceleration in the rate of body shape evolution. We estimate that rates of phenotypic evolution decreased by more than an order of magnitude in the common ancestor of the Ctenotus clade. These results provide evidence for a modal shift in phenotypic evolutionary dynamics and demonstrate that major axes of morphological variation can be decoupled from species diversification. More generally, the Bayesian framework described here can be used to identify and characterize complex mixtures of dynamic processes on phylogenetic trees. [Bayesian; diversification; evolvability; lizard; macroevolution, punctuated equilibrium, speciation.].

Reproductive skew and selection on female ornamentation in social species.

Male animals are typically more elaborately ornamented than females. Classic sexual selection theory notes that because sperm are cheaper to produce than eggs, and because males generally compete more intensely for reproductive opportunities and invest less in parental care than females, males can obtain greater fitness benefits from mating multiply. Therefore, sexual selection typically results in male-biased sex differences in secondary sexual characters. This generality has recently been questioned, because in cooperatively breeding vertebrates, the strength of selection on traits used in intrasexual competition for access to mates (sexual selection) or other resources linked to reproduction (social selection) is similar in males and females. Because selection is acting with comparable intensity in both sexes in cooperatively breeding species, the degree of sexual dimorphism in traits used in intrasexual competition should be reduced in cooperative breeders. Here we use the socially diverse African starlings (Sturnidae) to demonstrate that the degree of sexual dimorphism in plumage and body size is reduced in cooperatively breeding species as a result of increased selection on females for traits that increase access to reproductive opportunities, other resources, or higher social status. In cooperative breeders such as these, where there is unequal sharing of reproduction (reproductive skew) among females, and where female dominance rank influences access to mates and other resources, intrasexual competition among females may be intense and ultimately select for female trait elaboration. Selection is thereby acting with different intensities on males and females in cooperatively versus non-cooperatively breeding species, and female-female interactions in group-living vertebrates will have important consequences for the evolution of female morphological, physiological and behavioural traits.

Phylogenetic disassembly of species boundaries in a widespread group of Australian skinks (Scincidae: Ctenotus).

Scincid lizards in the genus Ctenotus represent one of Australia's most species-rich vertebrate clades, with more than 100 recognized species. Formal diagnoses of many species have relied on qualitative assessments of adult color pattern, but the validity of many such species has not been tested in a phylogenetic framework. We used mitochondrial and nuclear DNA to perform the first phylogenetic analysis of species in the Ctenotus inornatus group, a complex of at least 11 nominal forms that are distributed widely across the Australian continent. Mitochondrial and nuclear gene phylogenies support the presence of multiple species in the group, but these clades largely fail to match species boundaries as currently defined. Multivariate analyses of color pattern indicate that extreme intraspecific morphological variation in this character has created a significant impediment to understanding taxonomic diversity in the group. Our results suggest that nearly all species in the C. inornatus group require substantial taxonomic revision, and several geographically widespread forms ("C. saxatilis" and "C. robustus") appear to be polyphyletic taxa drawn from phenotypically similar but genetically distinct lineages. We describe one new species and provide redescriptions for three additional species. We synonymize names applied to a number of genetically incoherent or otherwise poorly-defined forms. The results of our study highlight an acute need for population genetic studies of species boundaries in Australian skinks, many of which are recognized by morphological traits that vary greatly within and between populations.

Phylogenetics and diversification of tanagers (Passeriformes: Thraupidae), the largest radiation of Neotropical songbirds.

Thraupidae is the second largest family of birds and represents about 4% of all avian species and 12% of the Neotropical avifauna. Species in this family display a wide range of plumage colors and patterns, foraging behaviors, vocalizations, ecotypes, and habitat preferences. The lack of a complete phylogeny for tanagers has hindered the study of this evolutionary diversity. Here, we present a comprehensive, species-level phylogeny for tanagers using six molecular markers. Our analyses identified 13 major clades of tanagers that we designate as subfamilies. In addition, two species are recognized as distinct branches on the tanager tree. Our topologies disagree in many places with previous estimates of relationships within tanagers, and many long-recognized genera are not monophyletic in our analyses. Our trees identify several cases of convergent evolution in plumage ornaments and bill morphology, and two cases of social mimicry. The phylogeny produced by this study provides a robust framework for studying macroevolutionary patterns and character evolution. We use our new phylogeny to study diversification processes, and find that tanagers show a background model of exponentially declining diversification rates. Thus, the evolution of tanagers began with an initial burst of diversification followed by a rate slowdown. In addition to this background model, two later, clade-specific rate shifts are supported, one increase for Darwin's finches and another increase for some species of Sporophila. The rate of diversification within these two groups is exceptional, even when compared to the overall rapid rate of diversification found within tanagers. This study provides the first robust assessment of diversification rates for the Darwin's finches in the context of the larger group within which they evolved.

A comprehensive species-level molecular phylogeny of the New World blackbirds (Icteridae).

The New World blackbirds (Icteridae) are among the best known songbirds, serving as a model clade in comparative studies of morphological, ecological, and behavioral trait evolution. Despite wide interest in the group, as yet no analysis of blackbird relationships has achieved comprehensive species-level sampling or found robust support for most intergeneric relationships. Using mitochondrial gene sequences from all ∼108 currently recognized species and six additional distinct lineages, together with strategic sampling of four nuclear loci and whole mitochondrial genomes, we were able to resolve most relationships with high confidence. Our phylogeny is consistent with the strongly-supported results of past studies, but it also contains many novel inferences of relationship, including unexpected placement of some newly-sampled taxa, resolution of relationships among major clades within Icteridae, and resolution of genus-level relationships within the largest of those clades, the grackles and allies. We suggest taxonomic revisions based on our results, including restoration of Cacicus melanicterus to the monotypic Cassiculus, merging the monotypic Ocyalus and Clypicterus into Cacicus, restoration of Dives atroviolaceus to the monotypic Ptiloxena, and naming Curaeus forbesi to a new genus, Anumara. Our hypothesis of blackbird phylogeny provides a foundation for ongoing and future evolutionary analyses of the group.

A comprehensive multilocus assessment of sparrow (Aves: Passerellidae) relationships.

The New World sparrows (Emberizidae) are among the best known of songbird groups and have long-been recognized as one of the prominent components of the New World nine-primaried oscine assemblage. Despite receiving much attention from taxonomists over the years, and only recently using molecular methods, was a "core" sparrow clade established allowing the reconstruction of a phylogenetic hypothesis that includes the full sampling of sparrow species diversity. In this paper, we use mitochondrial DNA gene sequences from all 129 putative species of sparrow and four additional (nuclear) loci for a subset of these taxa to resolve both generic and species level relationships. Hypotheses derived from our mitochondrial (2184 base pairs) and nuclear (5705 base pairs) DNA data sets were generally in agreement with respect to clade constituency but differed somewhat with respect to among-clade relationships. Sparrow diversity is defined predominantly by eight well-supported clades that indicate a lack of monophyly for at least three currently recognized genera. Ammodramus is polyphyletic and requires the naming of two additional genera. Spizella is also polyphyletic with Tree Sparrow (Spizella arborea) as a taxonomic "outlier". Pselliophorus is embedded within a larger Atlapetes assemblage and should be merged with that group. This new hypothesis of sparrow relationships will form the basis for future comparative analyses of variation within songbirds.