Abstract Despite growing recognition that female bird song is widespread, large-scale comparative tests of the evolutionary drivers of female song remain rare. Most conceptual frameworks for studying birdsong have been developed and tested primarily using male traits, leaving open the question of whether ecological, morphological, and social pressures shape song evolution in females. Here, we apply phylogenetic comparative methods to test three classic hypotheses of signal evolution—acoustic adaptation, morphological adaptation, and species recognition—using female song data from 197 species of antbirds (Thamnophilidae), a socially monogamous Neotropical clade with widespread female vocalizations. We found that habitat structure and morphological traits constrain multiple aspects of female song. Species in unexposed (i.e., closed) habitats produced songs with fewer notes, slower pacing, and lower vocal performance, consistent with the acoustic adaptation hypothesis. Larger-bodied females with larger bills produced longer songs with lower peak frequency, narrower bandwidth, and lower vocal performance, supporting the morphological adaptation hypothesis. These results indicate that female song diversification is shaped by ecological and morphological constraints. We also found that syntopic species pairs—those that overlap in both habitat and elevation—exhibited reduced vocal similarity, supporting the species recognition hypothesis. However, divergence time also explained some variation in vocal similarity between syntopic pairs, indicating that both ecological interactions and evolutionary history contribute to female song divergence. Altogether, these results provide a rare comparative test of classic signal evolution hypotheses using female song. This study provides the first macroevolutionary test of multiple hypotheses of signal evolution in female birdsong. By showing that female song is shaped by both ecological and morphological constraints and diverges slightly more among syntopic species, our results emphasize the need to integrate female traits into evolutionary models of communication. Incorporating female perspectives will be essential to building a complete understanding of signal diversity and its evolution across species.

The exponential growth of molecular sequence data over the past decade has enabled the construction of numerous clade-specific phylogenies encompassing hundreds or thousands of taxa. These independent studies often include overlapping data, presenting a unique opportunity to build macrophylogenies (phylogenies sampling >1000 taxa) for entire classes across the Tree of Life. However, the inference of large trees remains constrained by logistical, computational, and methodological challenges. The Avian Tree of Life provides an ideal model for evaluating strategies to robustly infer macrophylogenies from intersecting data sets derived from smaller studies. In this study, we leveraged a comprehensive resource of sequence capture data sets to evaluate the phylogenetic accuracy and computational costs of four methodological approaches: (1) supermatrix approaches using concatenation, including the “fast” maximum likelihood (ML) methods, (2) filtering data sets to reduce heterogeneity, (3) supertree estimation based on published phylogenomic trees, and (4) a “divide-and-conquer” strategy, wherein smaller ML trees were estimated and subsequently combined using a supertree approach. Additionally, we examined the impact of these methods on divergence time estimation using a data set that includes newly vetted fossil calibrations for the Avian Tree of Life. Our findings highlight the advantages of recently developed fast tree search approaches initiated with parsimony starting trees, which offer a reasonable compromise between computational efficiency and phylogenetic accuracy, facilitating inference of macrophylogenies.

Acoustic signal complexity varies widely in animals, from single notes to highly sophisticated vocal displays. In birds, vocal complexity can evolve as an honest signal of individual quality driven by sexual selection. However, this hypothesis is rarely explored in conjunction with alternative drivers, including competition for ecological resources (social selection) and intra-group communication, both of which may favour increased signal complexity. Using Bayesian phylogenetic models, we test whether these alternative mechanisms predict the complexity of innate songs in 1288 species of suboscine passerine birds, while accounting for ecological constraints on sound production, transmission and detection. We found that overall song complexity was reduced by sexual selection (estimated from mating systems) and declined with body size and vegetation density. Conversely, note count and song length increased in territorial species, particularly those using song to defend year-round territories during the non-breeding season. These findings challenge the common assumption that sexual selection is the main driver of increased signal complexity and highlight the role of social selection via territorial competition as a factor increasing the temporal complexity of songs. Our results suggest that signal complexity depends on social, cultural and ecological contexts, reflecting a combination of multiple inter-related drivers and constraints.

Abstract The large grackles in the subgenus Cassidix—Quiscalus mexicanus (Great-tailed Grackle), Q. major (Boat-tailed Grackle), and Q. palustris (Slender-billed Grackle)—form a clade with a complex evolutionary history of recent divergence and secondary contact. Quiscalus mexicanus is widespread from the western U.S. to Peru and encompasses 2 divergent mitochondrial lineages. Quiscalus major is found on the Atlantic and Gulf Coasts of the U.S. and shows striking geographic variation in eye color. Quiscalus palustris was endemic to central Mexico but went extinct during the early 20th century. Previous research has called into question the monophyly of Q. mexicanus and has suggested that Q. major and Q. mexicanus may hybridize in Texas and Louisiana. Additionally, the patterns of genetic structuring among subspecies within Q. mexicanus and Q. major are unclear. We resolved many of these questions by sequencing and analyzing data from nuclear ultraconserved elements (UCEs) and mitochondrial DNA collected from samples representing all 13 subspecies in the Cassidix clade. Our results show no evidence of admixture between Q. major and Q. mexicanus, suggesting that if hybrids occur in the contact zone, they are infertile or rare. Our results also support the monophyly of Q. major and Q. mexicanus, with Q. palustris likely sister to a unified Q. mexicanus, contrary to evidence from mitochondrial DNA. Finally, these data resolve intraspecific relationships in the Cassidix complex, several of which differ from current subspecies-level taxonomy, and show that geographic breaks, not eye color differences, structure genetic variation within Q. major.

Suture zones are geographic regions encompassing multiple phylogeographic breaks and contact zones involving pairs of closely related taxa (animals and plants), regardless of whether hybridization is occurring. We provide a comprehensive characterization of avian suture zones at the species and subspecies-levels in the Pan-Amazonia region, which includes the Amazonas river basin, the east slope of the tropical Andes, the Tepuis, and other adjacent regions. We grouped avian taxa into species complexes based on their relationships as defined by phylogenetic and taxonomic studies. We identified and described 1,983 taxon turnovers across 53 different suture zones, including 534 taxon turnovers at the species-level. These include areas across rivers, mountain ranges, along Amazonian rivers' floodplains (v & aacute;rzea), and elsewhere in lowland Amazonia. We also performed linear regressions to evaluate the number of taxon turnovers across major Amazonian rivers throughout their entire extension, from mouth to headwater regions. We found that the number of taxon turnovers steadily decreases as we move upstream for all major rivers. By mapping suture zones, we provide crucial data for future studies and emphasize the significant role of the Neotropical region in creating and maintaining its rich biodiversity. The Amazon is home to an incredible variety of bird species and subspecies, but why so many different birds live there has long been a mystery. Rivers and mountains often divide where animals and plants live, sometimes leading to the formation of new species. To explore this, we studied and mapped "suture zones"-places where closely related birds meet and may have overlapping distributions. Using maps of nearly 4,000 bird species and subspecies across the Amazon, we identified 53 major suture zones. Many suture zones are along large rivers like the Amazon, Negro, and Madeira, or in the Andes. We also identified 2,000 different pairs of related birds replacing each other at different locations. Our findings reinforce the role of rivers, mountains, and other geographic features as key to the Amazon's bird diversity, offering important insights for future speciation and conservation studies. Zonas de sutura s & atilde;o regi & otilde;es geogr & aacute;ficas que abrangem m & uacute;ltiplas quebras filogeogr & aacute;ficas e zonas de contato envolvendo pares de t & aacute;xons relacionados (animais e plantas), independentemente de haver hibridiza & ccedil;& atilde;o ou n & atilde;o. No presente trabalho n & oacute;s caracterizamos zonas de sutura de aves no n & iacute;vel de subesp & eacute;cies na regi & atilde;o da Pan-Amaz & ocirc;nia, que inclui a bacia do rio Amazonas, a encosta leste dos Andes tropicais, os Tepuis e outras regi & otilde;es adjacentes. Agrupamos os t & aacute;xons de aves em complexos de esp & eacute;cies com base em suas rela & ccedil;& otilde;es, conforme definido por estudos filogen & eacute;ticos e taxon & ocirc;micos. Identificamos e descrevemos 1,983 trocas de t & aacute;xons, 534 dessas entre esp & eacute;cies diferentes, em 53 diferentes zonas de sutura. Estas zonas de sutura incluem & aacute;reas atravessando rios, cadeias de montanhas, ao longo das plan & iacute;cies alag & aacute;veis dos rios amaz & ocirc;nicos (v & aacute;rzea) e em outras & aacute;reas da Amaz & ocirc;nia. Tamb & eacute;m realizamos regress & otilde;es lineares para avaliar o n & uacute;mero de quebras na distribui & ccedil;& atilde;o de taxa relacionados ao longo dos principais rios amaz & ocirc;nicos em todas as suas extens & otilde;es, da foz & agrave;s regi & otilde;es de nascente. Descobrimos que o n & uacute;mero de quebras diminui constantemente & agrave; medida que nos deslocamos a montante para todos os principais rios. Ao mapear zonas de sutura, fornecemos dados importantes para estudos futuros e enfatizamos o papel significativo da regi & atilde;o Neotropical na cria & ccedil;& atilde;o e no sustentamento da sua rica biodiversidade.

Aim Assess how local variations in bird taxon composition across lowland Amazonia are associated with environmental factors and rivers. Location Lowland Amazonia below 500 m a.s.l., South America. Time Period Contemporary. Major Taxa Studied Birds. Methods We constructed maps illustrating changes in bird taxon composition across lowland Amazonia and calculated Jaccard dissimilarity between adjacent localities. We then applied geographically weighted regression (GWR) to evaluate how variation in environmental variables and riverine features (width, water discharge, meandering, floodplain extent) explains spatial turnovers in taxon composition. We used comparative phylogenetic analyses to test whether ecological traits predict cross-river taxon turnover. Results Geographic variation in bird taxon composition was mostly associated with the presence and physical characteristics of rivers, especially river width and discharge, which predicted composition dissimilarities along their lower courses. Away from rivers, variations in forest cover, habitat heterogeneity, and temperature seasonality were the strongest variables associated with composition turnovers. Phylogenetic analyses showed that taxa inhabiting terra firme forests were disproportionately likely to exhibit cross-river replacements, whereas dispersal-related morphological traits had limited explanatory power. Main Conclusions Local Amazonian bird taxon composition is primarily associated with variation in forest cover, habitat heterogeneity, and temperature seasonality, except across major rivers. The influence of river characteristics, particularly width and discharge, on bird taxon turnover highlights the role of rivers in structuring Amazonian biodiversity at local and global scales. Because these variables operate at different scales, taxon composition turnover patterns reflect a mosaic of ecological and geomorphological processes. These findings highlight the need for conservation strategies that account for bird habitat specialisation, river dynamics, and ongoing landscape changes.

BACKGROUND:With over 10,000 recognized species, birds constitute one of the most diverse and widely distributed vertebrate groups. Although avian genomics has advanced rapidly over the past decade, substantial gaps remain across the global avifauna. Filling these gaps is essential for understanding macroevolutionary patterns, population structure, and the molecular basis of ecological and behavioral diversity. Worldwide museum collections represent invaluable resources for filling these gaps, yet the typically degraded DNA and limited quantities from historical specimens have posed significant challenges for generating high-quality genome assemblies. RESULTS:Here, the Bird Genome 10 K Project adopted low-input sequencing strategies that reduce costs while improving assembly quality compared with earlier order- and family-level genomes. Using mainly stLFR, complemented by 10X Genomics and standard next-generation sequencing, we assembled 177 avian genomes from museum specimens and tissue collections representing 161 genera, including 102 newly sequenced at the genomic level. The assemblies average ∼1.2 Gb in size, with scaffold N50 = 8.03 Mb, contig N50 = 120 kb, 93% BUSCO completeness, and Merqury Quality Value score of 56. CONCLUSIONS:These genomes greatly expand avian taxonomic coverage and demonstrate the efficiency of low-input sequencing for generating high-quality assemblies from limited and often degraded material sourced from museum specimens. This resource provides a foundation for comparative genomics, conservation genetics, and evolutionary studies across the avian tree of life.

Super black plumage has been observed across the avian phylogeny, yet few studies have examined the prevalence of super black plumage at the species and subspecies levels. One group where additional super black taxa likely exist is manakins in the genus Lepidothrix. Previous work on these sexually dichromatic taxa showed that male Lepidothrix velutina (Velvety Manakins) have super black plumage, and we observed that males of several other Lepidothrix taxa have black plumage with a velvety appearance that is often associated with super black barbule morphologies. Here, we combine spectrophotometry with scanning electron microscopy to examine the occurrence of super black plumage throughout Lepidothrix. We determine how the barbules of super black taxa differ structurally from barbules of black taxa that lack super black plumage. Our results show that 5 species and 7 subspecies of Lepidothrix have super black plumage, that barbules of back feathers are wider and closer together in super black taxa than in black taxa, and that the super black plumage trait appears to have evolved multiple times independently in the genus. We also show that olive-green immature males of super black taxa exhibit wider barbules that are closer together than their counterparts, suggesting the development of super black plumage begins before males molt into their definitive adult plumage. Super black plumage has been reported across the avian phylogeny, but few studies have examined its occurrence at the species and subspecies levels. We measured reflectance, barbule width, interbarbule distance, and barbule angle in Lepidothrix manakins to examine the prevalence and causes of super black plumage throughout the genus. We found that 5 species and 7 subspecies of Lepidothrix have developed super black plumage, that super black plumage is associated with wider barbules that are closer together, and that the development of super black feathers begins before males reach their definitive adult plumage. The relatively short time spans during which super black plumage has evolved suggest the genetic mechanisms affecting this trait may be relatively simple. We show a previously unrecognized degree of variation in the reflectance and morphology of black feathers within a single bird genus, suggesting that additional studies of the occurrence of super black barbule morphologies across the bird Tree of Life are needed. Ao longo da filogenia de aves h & aacute; v & aacute;rias descri & ccedil;& otilde;es de plumagem super negra, no entanto poucos estudos examinaram a preval & ecirc;ncia dessa plumagem no n & iacute;vel de esp & eacute;cie e subesp & eacute;cie. Um grupo em que provavelmente h & aacute; t & aacute;xons com plumagem super negra ainda n & atilde;o descrita & eacute; o g & ecirc;nero Lepidothrix. Um estudo anterior com esses t & aacute;xons mostraram que o macho de Lepidothrix velutina apresenta plumagem super negra, e n & oacute;s observamos que outros machos de t & aacute;xons de Lepidothrix possuem plumagem preta com apar & ecirc;ncia de veludo, como & eacute; frequentemente descrita a plumagem super negra. Neste trabalho n & oacute;s combinamos dados de espectrofotometria e microscopia eletr & ocirc;nica de varredura para examinar a ocorr & ecirc;ncia de plumagem super negra em todo o g & ecirc;nero Lepidothrix. N & oacute;s determinamos como as b & aacute;rbulas de pena super negra diferem estruturalmente das b & aacute;rbulas de penas quem n & atilde;o tem cor super negra. Nossos resultados mostraram que cinco esp & eacute;cies e sete subesp & eacute;cies de Lepidothrix tem plumagem super negra, que b & aacute;rbulas s & atilde;o mais grossas e pr & oacute;ximas entre si na plumagem super negra do que nas penas que n & atilde;o s & atilde;o super negra, e que a plumagem super negra parece ter evolu & iacute;do m & uacute;ltiplas vezes independentes neste g & ecirc;nero. N & oacute;s tamb & eacute;m mostramos que machos juvenis imaturos com plumagem verde oliva possuem penas com b & aacute;rbulas intermedi & aacute;rias em grossura e proximidade uma das outras quando comparada com a morfologia de penas verde oliva de f & ecirc;meas e super negra de machos adultos. Este resultado sugere que o desenvolvimento da morfologia de pena super negra come & ccedil;a antes que os machos atingem a plumagem de adultos.

Reference genome assemblies are essential infrastructure for investigating phylogeny and population/conservation genetics of wild organisms. Birds serve as model vertebrates in ecology and evolutionary biology due to their well-documented natural histories and extensive community science data. We release a set of 350 newly assembled avian genomes, which, when combined with 97 previously published genomes, represent 447 of the bird species recorded in Denmark, the Faroe Islands, and Greenland-the largest regional dataset of a vertebrate group to date. These genomes are published for various research activities. This data release advances the global effort to build comprehensive and accessible biodiversity genomic resources for the research community.

As an old group that has diversified in South America over millions of years, the tinamous (Palaeognathae: Tinamidae) are of high interest for understanding the evolution of birds and the assembly of the Neotropical biota. However, there are currently no complete species-level phylogenies of this group. Most prior work has been based on either morphological data or a small number of molecular markers, each of which has limited capability for reconstructing the tinamou phylogeny. Therefore, the interrelationships of most tinamou species are uncertain. We analyzed 80 whole genomes from a mix of historical study skins and frozen tissues, including all 46 recognized species of tinamous to (1) reconstruct their interrelationships, (2) estimate the timeframe of tinamou evolution, and (3) examine for the effects of incomplete lineage sorting (ILS) and ancestral introgression on genome evolution. We compared results for coding (BUSCO) and ultraconserved element (UCE) loci, as well as sex-linked and autosomal markers, and used fossil-calibrated tip-dating to estimate divergence times. Tinamous diverged from their sister group, the extinct Moas, 50-60 Ma, and their crown divergence occurred roughly 30-40 Ma, followed by constant diversification rates until the present. Phylogenetic reconstructions were largely robust across methods and data sets. Only one clade in the genus Crypturellus displayed substantial species-tree discordance across the different data sets. To investigate the impacts of introgression on this discordance, we quantified introgression for 100-kb non-overlapping windows across the genome, and identified pervasive genome-wide introgression. The distribution of this introgression across the genome was dependent on the assumed phylogeny applied to the f-branch model. When assuming one of these topologies in the f-branch model, patterns of introgression matched theoretical predictions about genome architecture. Overall, we present the most complete phylogeny for tinamous to date, identify an unrecognized species, and provide a case study for species-level phylogenomic analysis using whole genomes.

We use single genomes from seven tinamou species (Palaeognathae: Tinamidae) and apply a genome-architecture-aware phylogenomic approach to (1) quantify the landscape of introgression, (2) discriminate among conflicting genealogical signals in the genome, and (3) reconstruct evolutionary history. Using summary statistics and full likelihood, we detect pervasive asymmetric introgression between two sympatric Amazonian species clouding phylogenetic relationships in this group. However, these introgression results are sensitive to the assumed species tree. When assuming the Z-chromosome phylogeny as the species tree, the landscape of introgression is non-random; introgression is negatively associated with chromosome length, elevated at macrochromosome ends, and near zero for the Z-chromosome (the avian sex chromosome). A pseudo-autosomal region on the Z-chromosome, which recombines homologously with that of the W-chromosome, shows elevated introgression comparable to that of autosomes. Our results resolve the species tree of tinamous and imply a history of linked selection purging migrant alleles that are deleterious in the hybrid genome. This selection is countered in regions of the genome expected to experience elevated recombination, allowing introgressed alleles to accumulate across >20% of the genome. This work demonstrates a genome-architecture-aware approach to resolving the species tree in the face of introgression. As chromosome-level reference genomes spanning the tree of life become increasingly available, phylogenomic studies would benefit from the adoption of genome-architecture-aware approaches like ours. This is because the genomic landscapes of introgression and genealogy are largely predicted by chromosomal characteristics. ### Competing Interest Statement The authors have declared no competing interest. U.S. National Science Foundation, DEB-1855812, DEB-2203228

We examined speciation in streamertail hummingbirds (Trochilus polytmus and Trochilus scitulus), Jamaican endemic taxa that challenge the rule that bird speciation cannot progress in situ on small islands. Our analysis shows that divergent selection acting on male bill color, a sexual ornament that is red in polytmus and black in scitulus, acts as a key reproductive barrier. We conducted a population-level analysis of genomic and phenotypic patterns to determine the traits that contribute the most to speciation despite ongoing gene flow across a narrow hybrid zone. We characterized genomic patterns using 6,451 single-nucleotide polymorphisms and a segment of the mitochondrial control region. Our analyses revealed high diversity within species, and low divergence between them, consistent with a recent speciation event or extensive gene flow following secondary contact. We observed narrow clines in two phenotypic traits and several SNP loci. The cline width for male bill color is only 2.3 km, marking it as one of the narrowest phenotypic clines documented in an avian hybrid zone. The coincidence of estimated cline centers with the Rio Grande Valley suggests that this landscape feature may contribute to hybrid zone stability. However, given that streamertails are highly mobile, it is unlikely that such a narrow river acts as a physical barrier to dispersal. The limited genomic divergence across scanned regions of the genome offers little support for postmating reproductive barriers. Instead, our findings point to strong premating selection acting on bill color as the primary driver of streamertail speciation.

Boat-tailed Grackles (Quiscalus major) are marsh-dwelling blackbirds that are endemic to the eastern United States. Various aspects of their biology have been studied extensively, including their mating system, plumage and molt patterns, diet, and interspecific interactions. Boat-tailed Grackles are also interesting because they exhibit variation in their iris color that is associated with geography. However, resources that enable genomic studies of Boat-tailed Grackles and other related grackle species are few. Here, we combined Pacific Biosciences long-read, HiFi data with short-read Illumina data from a HiC library to produce haplotype-phased, chromosome-scale genome assemblies for Boat-tailed Grackles. The final version of the assembly, bQuiMaj1, includes two, contiguous haplotypes with total lengths of ~1 Gbp, N50s of ~70 Mbp, and L50s of 5-6. BUSCO and merqury analyses suggest both haplotypes are also relatively complete (95-99%) with respect to gene and k-mer content. The resulting assemblies will significantly enhance our understanding of Boat-tailed Grackle biology and physiology, as well as contribute to the growing number of genomes representing species belonging to the taxonomic family Icteridae (the New World blackbirds).

Gene flow connects populations and facilitates exchanging alleles, impacting speciation and adaptation. In Panama, lekking golden-collared and white-collared manakins (Manacus vitellinus and M. candei) interbreed in a narrow hybrid zone. Males' brilliant yellow plumage, principally controlled by the carotenoid metabolism gene BCO2, has introgressed from vitellinus into candei under sexual selection as far as the largest river in the region. Introgression is sharply limited across its lower reaches, but both color forms occur on both banks at its headwaters. Previous authors have therefore speculated that the river is a strong barrier to gene flow. In this study, we used ∼14,000 single nucleotide polymorphisms to test this hypothesis by assessing cross-river genetic differentiation and estimating gene flow. We found that, while the river clearly structured genetic variation, particularly downriver, it did not prevent extensive gene flow upriver. This result mirrors patterns observed at some of the world's largest rivers, albeit on a much smaller scale. It also implicates several alternatives to the barrier hypothesis, including that introgression is still ongoing or that selection for plumage color varies across the river-both rare phenomena to capture in nature. We recommend behavioral studies to further untangle this intriguing case of evolution in action.

The Variable Antshrike (Thamnophilus caerulescens) includes eight recognized subspecies across a broad range from Peru to eastern Brazil. Previous genetic studies suggested deep divergence among some populations, but limited geographic sampling hindered taxonomic resolution. We analysed plumage, morphology, vocalizations, and genome-wide genetic markers across the full range of T. caerulescens to assess geographic variation and population structure. We also conducted field playback experiments to test vocal differences as potential premating reproductive barriers. Our data reveal nine phenotypically distinct populations, including seven of the eight currently recognized taxa and two new taxa. Increased geographic sampling uncovered several hybrid zones, some spanning hundreds of kilometres. The geographically isolated T. c. cearensis showed substantial genetic and vocal differences from the remaining taxa, overlapping in acoustic trait space only with the distant T. c. melanchrous. In playback experiments, T. c. cearensis responded to its own and T. c. melanchrous songs but ignored others. These results suggest strong behavioural isolation and probable intrinsic postzygotic barriers between T. c. cearensis and other taxa, supporting its recognition as a separate species. In contrast, most other taxa exhibit only modest differentiation in traits linked to reproductive isolation and show weak barriers in contact zones.

The evolutionary histories of different genomic regions typically differ from each other and from the underlying species phylogeny. This makes species tree estimation challenging. Here, we examine the performance of phylogenomic methods using a well-resolved phylogeny that nevertheless contains many difficult nodes, the species tree of living birds. We compared trees generated by maximum likelihood (ML) analysis of concatenated data, gene tree summary methods, and SVDquartets. We also conduct the first empirical test of a “new” method called METAL (Metric algorithm for Estimation of Trees based on Aggregation of Loci), which is based on evolutionary distances calculated using concatenated data. We conducted this test using a novel dataset comprising more than 4,000 ultraconserved element (UCE) loci from almost all bird families and two existing UCE and intron datasets sampled from almost all avian orders. We identified “reliable clades” very likely to be present in the true avian species tree and used them to assess method performance. ML analyses of concatenated data recovered almost all reliable clades with less data and greater robustness to missing data than other methods. METAL recovered many reliable clades, but only performed well with the largest datasets. Gene tree summary methods (weighted ASTRAL and weighted ASTRID) performed well; they required less data than METAL but more data than ML concatenation. SVDquartets exhibited the worst performance of the methods tested. In addition to the methodological insights, this study provides a novel estimate of avian phylogeny with almost 99% of the currently recognized avian families. Only one of the 181 reliable clades we examined was consistently resolved differently by ML concatenation versus other methods, suggesting that it may be possible to achieve consensus on the deep phylogeny of extant birds.

Rivers frequently delimit the geographic ranges of species in the Amazon Basin. These rivers also define the boundaries between genetic clusters within many species, yet river boundaries have been documented to break down in headwater regions where rivers are narrower. To explore the evolutionary implications of headwater contact zones in Amazonia, we examined genetic variation in the Blue-capped Manakin (Lepidothrix coronata), a species previously shown to contain several genetically and phenotypically distinct populations across the western Amazon Basin. We collected restriction site-associated DNA sequence data (RADcap) for 706 individuals and found that spatial patterns of genetic structure indicate several rivers, particularly the Amazon and Ucayali, are dispersal barriers for L. coronata. We also found evidence that genetic connectivity is elevated across several headwater regions, highlighting the importance of headwater gene flow for models of Amazonian diversification. The headwater region of the Ucayali River provided a notable exception to findings of headwater gene flow by harboring non-admixed populations of L. coronata on opposite sides of a < 1-km-wide river channel with a known dynamic history, suggesting that additional prezygotic barriers may be limiting gene flow in this region.

Birds display a rainbow of eye colours, but this trait has been little studied compared with plumage coloration. Avian eye colour variation occurs at all phylogenetic scales: it can be conserved throughout whole families or vary within one species, yet the evolutionary importance of this eye colour variation is under‐studied. Here, we summarize knowledge of the causes of eye colour variation at three primary levels: mechanistic, genetic and evolutionary. Mechanistically, we show that avian iris pigments include melanin and carotenoids, which also play major roles in plumage colour, as well as purines and pteridines, which are often found as pigments in non‐avian taxa. Genetically, we survey classical breeding studies and recent genomic work on domestic birds that have identified potential ‘eye colour genes’, including one associated with pteridine pigmentation in pigeons. Finally, from an evolutionary standpoint, we present and discuss several hypotheses explaining the adaptive significance of eye colour variation. Many of these hypotheses suggest that bird eye colour plays an important role in intraspecific signalling, particularly as an indicator of age or mate quality, although the importance of eye colour may differ between species and few evolutionary hypotheses have been directly tested. We suggest that future studies of avian eye colour should consider all three levels, including broad‐scale iris pigment analyses across bird species, genome sequencing studies to identify loci associated with eye colour variation, and behavioural experiments and comparative phylogenetic analyses to test adaptive hypotheses. By examining these proximate and ultimate causes of eye colour variation in birds, we hope that our review will encourage future research to understand the ecological and evolutionary significance of this striking avian trait.